Antibodies binding to her2, pharmaceutical compositions and uses thereof

Abstract

The application belongs to the field of biological medicine, and particularly relates to a HER2 antibody, a pharmaceutical composition and application thereof. The application provides an antibody binding to HER2, which comprises a first antigen binding part and a second antigen binding part, the first antigen binding part comprises an anti-HER2 nanobody, and the sequence of the anti-HER2 nanobody comprises an amino acid sequence as shown in any one of SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8, and the second antigen binding part comprises trastuzumab or pertuzumab. The antibody provided by the application has the effect of inhibiting HER2 related tumors, the bispecific antibody in the antibody can specifically recognize different epitopes of HER2, inhibit non-ligand dependent and ligand dependent HER2 positive tumor cell proliferation, has obvious inhibiting effect on HER2 positive tumors and HER2 low expression tumors resistant to trastuzumab, and provides a new scheme for diagnosis and treatment of HER2 related tumors.

Description

Technical Field

[0001] This application belongs to the field of biomedicine, specifically involving HER2 antibodies, pharmaceutical compositions and their applications. Background Technology

[0002] Human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase located on the cell membrane, belonging to the epidermal growth factor receptor family. When HER2 expression is abnormally increased, it forms heterodimers with other family members (such as EGFR, HER3, and HER4), triggering autophosphorylation of tyrosine residues within the cell. This activates multiple intracellular signaling pathways, promoting cell proliferation and tumor formation. Studies have shown that approximately 20% of breast cancer patients exhibit increased HER2 expression, leading to the activation of related signaling pathways. HER2 overexpression is also observed in various other solid tumors, such as gastric and gastroesophageal junction cancer (20%), salivary gland cancer (17%-44%), ovarian cancer (27%), endometrial cancer (18%-80%), cervical cancer (21%), non-small cell lung cancer (2%), cholangiocarcinoma (20%), pancreatic cancer (26%), colon cancer (5%), bladder cancer (12.4%), and prostate cancer (10%). According to 2020 global cancer statistics, breast cancer has approximately 2.26 million new cases and 685,000 deaths annually, making it the most common cancer worldwide, surpassing lung cancer. Based on this, it is estimated that there are over 400,000 HER2-positive breast cancer patients. If patients with other HER2-positive solid tumors are also included, the total number is even larger, placing a heavy burden on society and the healthcare system. Therefore, developing new drugs targeting HER2-positive tumors is of paramount importance for improving patient prognosis and reducing the disease burden.

[0003] HER2-targeted therapy is an effective treatment strategy for HER2-positive tumors, with two marketed monoclonal antibodies—trastuzumab and pertuzumab—remaining key drugs in this therapeutic field. Trastuzumab is the first humanized monoclonal antibody approved by the FDA for the treatment of HER2-positive breast cancer. It binds to the fourth extracellular domain (ECD IV) of HER2, preventing ligand-independent heterodimerization of HER2 and thus inhibiting the activation of the downstream PI3K / AKT / mTOR signaling pathway, effectively controlling the growth of HER2-positive tumor cells. Furthermore, trastuzumab can kill tumor cells through antibody-dependent cell-mediated cytotoxicity (ADCC). Pertuzumab, on the other hand, specifically binds to the second extracellular domain (ECD II) of HER2, inhibiting ligand-dependent heterodimerization of HER2 and thus interfering with the proliferation of HER2-positive tumor cells. In animal models and clinical trials, the combination of trastuzumab and pertuzumab has shown more significant anti-tumor effects than either drug alone, and therefore their combination therapy has been approved by the FDA for the treatment of HER2-positive metastatic breast cancer.

[0004] However, some HER2-related tumors are currently resistant to trastuzumab, and there is a lack of specific antibodies in the current technology that have a better inhibitory effect on tumor cells than trastuzumab and pertuzumab. Summary of the Invention

[0005] Based on the deficiencies of the prior art, this application synthesizes a specific antibody targeting HER2, which has the effect of inhibiting the proliferation of HER2-positive tumors. This includes a bispecific antibody targeting different epitopes of HER2, which can recognize different epitopes of the HER2 protein. It can promote the internalization of the HER2 receptor and simultaneously inhibit the proliferation of HER2-positive tumor cells that are neither ligand-dependent nor ligand-dependent. It has an inhibitory effect on HER2-positive tumors and HER2-low-expressing tumors that are resistant to trastuzumab, and its therapeutic effect is superior to that of the existing combination of trastuzumab and pertuzumab.

[0006] The first aspect of this application provides an antibody that binds to HER2, comprising a first antigen-binding portion and a second antigen-binding portion, wherein the first antigen-binding portion comprises an anti-HER2 nanobody, the anti-HER2 nanobody comprising an amino acid sequence as shown in any one of SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8, and the second antigen-binding portion comprises trastuzumab or pertuzumab.

[0007] Preferably, the antibody is a bispecific antibody.

[0008] A second aspect of this application provides a chimeric antigen receptor comprising the antibody as described in the first aspect above.

[0009] A third aspect of this application provides a polynucleotide that encodes an antibody as described in the first aspect above.

[0010] A fourth aspect of this application provides a vector comprising the polynucleotides described in the third aspect above.

[0011] The fifth aspect of this application provides a host cell containing a vector as described in the fourth aspect above, or a genome integrated with polynucleotides as described in the third aspect above.

[0012] The sixth aspect of this application provides an antibody-drug conjugate comprising an antibody and a therapeutic agent as described in the first aspect above.

[0013] A seventh aspect of this application provides a pharmaceutical composition comprising an effective amount of an antibody as described in the first aspect above, or a chimeric antigen receptor as described in the second aspect above, or a polynucleotide as described in the third aspect above, or a carrier as described in the fourth aspect above, or a host cell as described in the fifth aspect above, or an antibody-drug conjugate as described in the sixth aspect above.

[0014] The eighth aspect of this application provides a kit for detecting HER2-related tumors, comprising an effective amount of the antibody and detection reagent as described in the first aspect above.

[0015] The ninth aspect of this application provides the use of the antibody of the first aspect, or the chimeric antigen receptor of the second aspect, or the polynucleotide of the third aspect, or the carrier of the fourth aspect, or the host cell of the fifth aspect, or the antibody-drug conjugate of the sixth aspect, or the pharmaceutical composition of the seventh aspect, or the kit of the eighth aspect, wherein the use is selected from any one or more of the following:

[0016] 1) To prepare products for the prevention and / or treatment of HER2-related tumors;

[0017] 2) Prepare products for diagnosing HER2-related tumors;

[0018] 3) Prepare products that promote the internalization of HER2 receptors on the cell surface.

[0019] The beneficial effects of this application are as follows:

[0020] The antibody described in this application is easily expressed in eukaryotic systems, has a simple purification method, and exhibits good uniformity and high purity, thus reducing production and usage costs. The antibody prepared in this application inhibits the proliferation of HER2-positive tumor cells, including a bispecific antibody that specifically recognizes different epitopes of HER2. It can simultaneously inhibit the proliferation of both ligand-independent and ligand-dependent HER2-positive tumor cells, showing significant inhibitory effects against HER2-positive tumors and trastuzumab-resistant HER2-low-expressing tumors. This inhibitory effect is significantly superior to trastuzumab and pertuzumab alone, and also significantly superior to the currently optimal combination of trastuzumab and pertuzumab used clinically. The bispecific antibody in this application can effectively promote HER2 receptor internalization and can be used to prepare antibody-drug conjugates. This application provides a new approach for the diagnosis and treatment of HER2-related tumors. Attached Figure Description

[0021] Figure 1 The purity of the 32 HER2 antibodies prepared in Example 1 is shown, indicating that the purity of all HER2 antibodies exceeds 95%.

[0022] Figure 2 The cell viability of HER2-positive tumor cells NCI-N87 after treatment with 32 HER2 antibodies in Example 3 is shown, indicating that 7 of the bispecific antibodies have extremely strong inhibitory ability on cell proliferation.

[0023] Figure 3 The cell viability of HER2-positive tumor cell line BT-474 and trastuzumab-resistant breast cancer cell line MCF-7 after treatment with the seven bispecific antibodies in Example 3 is shown. This indicates that A2G5-Bs-1, A2G5-Bs-5, A9B5-Bs-5, and A9B5-Bs-7 have significant inhibitory effects on HER2-positive tumor cell line BT-474, and A9B5-Bs-5 and A9B5-Bs-7 have significant inhibitory effects on trastuzumab-resistant tumor cell line MCF-7.

[0024] Figure 4 The results of the binding analysis of A9B5 and HER2 in Example 4 are shown.

[0025] Figure 5 The results show the binding analysis of the bispecific antibody to HER2 in Example 4.

[0026] Figure 6 The results of bispecific antibodies A9B5-Bs-5 and A9B5-Bs-7 promoting HER2 receptor internalization in Example 5 demonstrate that bispecific antibodies A9B5-Bs-5 and A9B5-Bs-7 can promote the internalization of HER2 receptors on the cell surface.

[0027] Figure 7The cell viability of HER2-positive tumor cell lines NCI-N87 and BT-474 after treatment with bispecific antibodies A9B5-Bs-5 and A9B5-Bs-7 in Example 6 without ligand stimulation is shown, demonstrating the ligand-dependent inhibitory ability of bispecific antibodies A9B5-Bs-5 and A9B5-Bs-7 on the non-ligand-dependent proliferation of HER2-positive tumor cells NCI-N87 and BT-474.

[0028] Figure 8 The cell viability of HER2-positive tumor cell line NCI-N87 after treatment with bispecific antibodies A9B5-Bs-5 and A9B5-Bs-7 under ligand stimulation in Example 6 is shown, illustrating the inhibitory ability of bispecific antibodies A9B5-Bs-5 and A9B5-Bs-7 on the ligand-dependent proliferation of HER2-positive tumor cells NCI-N87.

[0029] Figure 9 The cell viability of HER2-positive tumor cell line BT-474 after treatment with bispecific antibodies A9B5-Bs-5 and A9B5-Bs-7 under ligand stimulation in Example 6 is shown, illustrating the ligand-dependent inhibitory ability of bispecific antibodies A9B5-Bs-5 and A9B5-Bs-7 on the ligand-dependent proliferation of HER2-positive tumor cell line BT-474. Detailed Implementation

[0030] The following specific examples illustrate the implementation of this application. Those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this specification. This application can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of this application.

[0031] Before further describing the specific embodiments of this application, it should be understood that the scope of protection of this application is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of this application is for describing specific embodiments and not for limiting the scope of protection of this application; in the specification and claims of this application, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0032] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in this application, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description in this application, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this application may be used to implement this application.

[0033] This application synthesizes an antibody targeting HER2 that can inhibit the proliferation of HER2-positive tumor cells. The binding ability of the antibody to HER2 epitopes was measured. Some antibodies are bispecific antibodies that can recognize different epitopes of the HER2 protein. They can promote HER2 receptor internalization and simultaneously inhibit the proliferation of HER2-positive tumor cells that are neither ligand-dependent nor ligand-dependent. They have an inhibitory effect on HER2-positive tumors and HER2-low-expressing tumors that are resistant to trastuzumab, and their therapeutic effect is superior to that of existing treatments using a combination of trastuzumab and pertuzumab.

[0034] This application first provides an antibody that binds to HER2, including a first antigen-binding portion and a second antigen-binding portion. The first antigen-binding portion includes an anti-HER2 nanobody, which includes an amino acid sequence as shown in any one of SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8. The second antigen-binding portion includes trastuzumab or pertuzumab.

[0035] The antigen-binding region of an antibody refers to the region in an antibody molecule responsible for recognizing and binding to a specific antigenic epitope.

[0036] The nanobody refers to an antibody domain consisting of only one heavy chain variable region (VHH), without light chains, and possessing antigen-binding ability.

[0037] In a specific embodiment of this application, the anti-HER2 nanobody may be selected from A2G5, A9B5, H2F5, and G1E4.

[0038] The amino acid sequence of the anti-HER2 nanobody A2G5 is shown in SEQ ID NO.5.

[0039] The amino acid sequence of the anti-HER2 nanobody A9B5 is shown in SEQ ID NO.6.

[0040] The amino acid sequence of the anti-HER2 nanobody H2F5 is shown in SEQ ID NO.7.

[0041] The amino acid sequence of the anti-HER2 nanobody G1E4 is shown in SEQ ID NO.8.

[0042] In a specific embodiment of this application, the second antigen-binding portion includes trastuzumab or pertuzumab.

[0043] The amino acid sequence of the heavy chain of the trastuzumab is shown in SEQ ID NO.1.

[0044] The amino acid sequence of the light chain of the trastuzumab is shown in SEQ ID NO.2.

[0045] The amino acid sequence of the heavy chain of the pertuzumab is shown in SEQ ID NO.3.

[0046] The amino acid sequence of the light chain of the pertuzumab is shown in SEQ ID NO.4.

[0047] In specific embodiments of this application, the first antigen-binding portion and the second antigen-binding portion can be directly fused, or the first antigen-binding portion can be fused to the second antigen-binding portion via a flexible peptide segment. A flexible peptide segment refers to a highly flexible peptide chain region in a protein structure. Unlike other parts of a protein, these regions do not have a fixed three-dimensional structure but can move freely and change conformation.

[0048] In a specific embodiment of this application, the flexible peptide chain has 5 to 15 amino acid residues, wherein the amino acid residues are serine residues and / or glycine residues.

[0049] In a specific embodiment of this application, the amino acid sequence of the flexible peptide chain is shown in SEQ ID NO.9.

[0050] In specific embodiments of this application, the fusion position of the anti-HER2 nanobody with trastuzumab or pertuzumab is not limited. For example, it can be the heavy chain or light chain of trastuzumab or pertuzumab, or the N-terminus or C-terminus of its heavy chain or light chain. Specifically, the anti-HER2 nanobody can be fused with the N-terminus of the heavy chain of trastuzumab, the C-terminus of the heavy chain of trastuzumab, the N-terminus of the heavy chain of pertuzumab, the C-terminus of the heavy chain of pertuzumab, the N-terminus of the light chain of trastuzumab, the C-terminus of the light chain of trastuzumab, or the C-terminus of the light chain of pertuzumab.

[0051] In a specific embodiment of this application, 32 antibodies capable of binding to HER2 were constructed and named A2G5-Bs-1, A2G5-Bs-2, A2G5-Bs-3, A2G5-Bs-4, A2G5-Bs-5, A2G5-Bs-6, A2G5-Bs-7, A2G5-Bs-8, A9B5-Bs-1, A9B5-Bs-2, A9B5-Bs-3, A9B5-Bs-4, A9B5-Bs-5, A9B5-Bs-6, and A9B5-Bs-5, respectively. -Bs-7, A9B5-Bs-8, H2F5-Bs-1, H2F5-Bs-2, H2F5-Bs-3, H2F5-Bs-4, H2F5-Bs-5, H2F5-Bs-6, H2F5-Bs-7, H2F5 -Bs-8, G1E4-Bs-1, G1E4-Bs-2, G1E4-Bs-3, G1E4-Bs-4, G1E4-Bs-5, G1E4-Bs-6, G1E4-Bs-7, G1E4-Bs-8, as follows:

[0052] In a specific embodiment of this application, when the anti-HER2 nanobody is A2G5, the anti-HER2 nanobody A2G5 is fused with the N-terminus of the heavy chain of trastuzumab to form the heavy chain of the antibody. The amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO.10. The light chain of trastuzumab is used as the light chain of the antibody. The amino acid sequence of the light chain of the antibody is shown in SEQ ID NO.2. The antibody is A2G5-Bs-1.

[0053] In a specific embodiment of this application, when the anti-HER2 nanobody is A2G5, the anti-HER2 nanobody A2G5 is fused with the C-terminus of the heavy chain of trastuzumab to form the heavy chain of the antibody. The amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO.11. The light chain of trastuzumab is used as the light chain of the antibody. The amino acid sequence of the light chain of the antibody is shown in SEQ ID NO.2. The antibody is A2G5-Bs-2.

[0054] In a specific embodiment of this application, when the anti-HER2 nanobody is A2G5, the anti-HER2 nanobody A2G5 is fused with the N-terminus of the heavy chain of pertuzumab to form the heavy chain of the antibody. The amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO.12. The light chain of pertuzumab is used as the light chain of the antibody. The amino acid sequence of the light chain of the antibody is shown in SEQ ID NO.4. The antibody is A2G5-Bs-3.

[0055] In a specific embodiment of this application, when the anti-HER2 nanobody is A2G5, the anti-HER2 nanobody A2G5 is fused with the C-terminus of the heavy chain of pertuzumab to form the heavy chain of the antibody. The amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO.13. The light chain of pertuzumab is used as the light chain of the antibody. The amino acid sequence of the light chain of the antibody is shown in SEQ ID NO.4. The antibody is A2G5-Bs-4.

[0056] In a specific embodiment of this application, when the anti-HER2 nanobody is A2G5, the heavy chain of trastuzumab is used as the heavy chain of the antibody, and the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO.1, the anti-HER2 nanobody A2G5 is fused with the N-terminus of the light chain of trastuzumab to form the light chain of the antibody, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO.14; the antibody is A2G5-Bs-5.

[0057] In a specific embodiment of this application, when the anti-HER2 nanobody is A2G5, the heavy chain of trastuzumab is used as the heavy chain of the antibody, and the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO.1, the anti-HER2 nanobody A2G5 is fused with the C-terminus of the light chain of trastuzumab to form the light chain of the antibody, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO.15; the antibody is A2G5-Bs-6.

[0058] In a specific embodiment of this application, when the anti-HER2 nanobody is A2G5, the heavy chain of pertuzumab is used as the heavy chain of the antibody, and the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO.3, the anti-HER2 nanobody A2G5 is fused with the N-terminus of the light chain of pertuzumab to form the light chain of the antibody, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO.16; the antibody is A2G5-Bs-7.

[0059] In a specific embodiment of this application, when the anti-HER2 nanobody is A2G5, the heavy chain of pertuzumab is used as the heavy chain of the antibody, and the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO.3, the anti-HER2 nanobody A2G5 is fused with the C-terminus of the light chain of pertuzumab to form the light chain of the antibody, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO.17; the antibody is A2G5-Bs-8.

[0060] In a specific embodiment of this application, when the anti-HER2 nanobody is A9B5, the anti-HER2 nanobody A9B5 is fused with the N-terminus of the heavy chain of trastuzumab to form the heavy chain of the antibody. The amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO.18. The light chain of trastuzumab is used as the light chain of the antibody. The amino acid sequence of the light chain of the antibody is shown in SEQ ID NO.2. The antibody is A9B5-Bs-1.

[0061] In a specific embodiment of this application, when the anti-HER2 nanobody is A9B5, the anti-HER2 nanobody A9B5 is fused with the C-terminus of the heavy chain of trastuzumab to form the heavy chain of the antibody. The amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO.19. The light chain of trastuzumab is used as the light chain of the antibody. The amino acid sequence of the light chain of the antibody is shown in SEQ ID NO.2. The antibody is A9B5-Bs-2.

[0062] In a specific embodiment of this application, when the anti-HER2 nanobody is A9B5, the anti-HER2 nanobody A9B5 is fused with the N-terminus of the heavy chain of pertuzumab to form the heavy chain of the antibody. The amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO.20. The light chain of pertuzumab is used as the light chain of the antibody. The amino acid sequence of the light chain of the antibody is shown in SEQ ID NO.4. The antibody is A9B5-Bs-3.

[0063] In a specific embodiment of this application, when the anti-HER2 nanobody is A9B5, the anti-HER2 nanobody A9B5 is fused with the C-terminus of the heavy chain of pertuzumab to form the heavy chain of the antibody. The amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO.21. The light chain of pertuzumab is used as the light chain of the antibody. The amino acid sequence of the light chain of the antibody is shown in SEQ ID NO.4. The antibody is A9B5-Bs-4.

[0064] In a specific embodiment of this application, when the anti-HER2 nanobody is A9B5, the heavy chain of trastuzumab is used as the heavy chain of the antibody, and the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO.1, the anti-HER2 nanobody A9B5 is fused with the N-terminus of the light chain of trastuzumab to form the light chain of the antibody, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO.22; the antibody is A9B5-Bs-5.

[0065] In a specific embodiment of this application, when the anti-HER2 nanobody is A9B5, the heavy chain of trastuzumab is used as the heavy chain of the antibody, and the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO.1, the anti-HER2 nanobody A9B5 is fused with the C-terminus of the light chain of trastuzumab to form the light chain of the antibody, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO.23; the antibody is A9B5-Bs-6.

[0066] In a specific embodiment of this application, when the anti-HER2 nanobody is A9B5, the heavy chain of pertuzumab is used as the heavy chain of the antibody, and the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO.3, the anti-HER2 nanobody A9B5 is fused with the N-terminus of the light chain of pertuzumab to form the light chain of the antibody, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO.24; the antibody is A9B5-Bs-7.

[0067] In a specific embodiment of this application, when the anti-HER2 nanobody is A9B5, the heavy chain of pertuzumab is used as the heavy chain of the antibody, and the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO.3, the anti-HER2 nanobody A9B5 is fused with the C-terminus of the light chain of pertuzumab to form the light chain of the antibody, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO.25; the antibody is A9B5-Bs-8.

[0068] In a specific embodiment of this application, when the anti-HER2 nanobody is H2F5, the anti-HER2 nanobody H2F5 is fused with the N-terminus of the heavy chain of trastuzumab to form the heavy chain of the antibody. The amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO.26. The light chain of trastuzumab is used as the light chain of the antibody. The amino acid sequence of the light chain of the antibody is shown in SEQ ID NO.2. The antibody is H2F5-Bs-1.

[0069] In a specific embodiment of this application, when the anti-HER2 nanobody is H2F5, the anti-HER2 nanobody H2F5 is fused with the C-terminus of the heavy chain of trastuzumab to form the heavy chain of the antibody. The amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO.27. The light chain of trastuzumab is used as the light chain of the antibody. The amino acid sequence of the light chain of the antibody is shown in SEQ ID NO.2. The antibody is H2F5-Bs-2.

[0070] In a specific embodiment of this application, when the anti-HER2 nanobody is H2F5, the anti-HER2 nanobody H2F5 is fused with the N-terminus of the heavy chain of pertuzumab to form the heavy chain of the antibody. The amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO.28. The light chain of pertuzumab is used as the light chain of the antibody. The amino acid sequence of the light chain of the antibody is shown in SEQ ID NO.4. The antibody is H2F5-Bs-3.

[0071] In a specific embodiment of this application, when the anti-HER2 nanobody is H2F5, the anti-HER2 nanobody H2F5 is fused with the C-terminus of the heavy chain of pertuzumab to form the heavy chain of the antibody. The amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO.29. The light chain of pertuzumab is used as the light chain of the antibody. The amino acid sequence of the light chain of the antibody is shown in SEQ ID NO.4. The antibody is H2F5-Bs-4.

[0072] In a specific embodiment of this application, when the anti-HER2 nanobody is H2F5, the heavy chain of trastuzumab is used as the heavy chain of the antibody, and the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO.1, the anti-HER2 nanobody H2F5 is fused with the N-terminus of the light chain of trastuzumab to form the light chain of the antibody, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO.30; the antibody is H2F5-Bs-5.

[0073] In a specific embodiment of this application, when the anti-HER2 nanobody is H2F5, the heavy chain of trastuzumab is used as the heavy chain of the antibody, and the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO.1, the anti-HER2 nanobody H2F5 is fused with the C-terminus of the light chain of trastuzumab to form the light chain of the antibody, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO.31; the antibody is H2F5-Bs-6.

[0074] In a specific embodiment of this application, when the anti-HER2 nanobody is H2F5, the heavy chain of pertuzumab is used as the heavy chain of the antibody, and the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO.3, the anti-HER2 nanobody H2F5 is fused with the N-terminus of the light chain of pertuzumab to form the light chain of the antibody, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO.32; the antibody is H2F5-Bs-7.

[0075] In a specific embodiment of this application, when the anti-HER2 nanobody is H2F5, the heavy chain of pertuzumab is used as the heavy chain of the antibody, and the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO.3, the anti-HER2 nanobody H2F5 is fused with the C-terminus of the light chain of pertuzumab to form the light chain of the antibody, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO.33; the antibody is H2F5-Bs-8.

[0076] In a specific embodiment of this application, when the anti-HER2 nanobody is G1E4, the anti-HER2 nanobody G1E4 is fused with the N-terminus of the heavy chain of trastuzumab to form the heavy chain of the antibody. The amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO.34. The light chain of trastuzumab is used as the light chain of the antibody. The amino acid sequence of the light chain of the antibody is shown in SEQ ID NO.2. The antibody is G1E4-Bs-1.

[0077] In a specific embodiment of this application, when the anti-HER2 nanobody is G1E4, the anti-HER2 nanobody G1E4 is fused with the C-terminus of the heavy chain of trastuzumab to form the heavy chain of the antibody. The amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO.35. The light chain of trastuzumab is used as the light chain of the antibody. The amino acid sequence of the light chain of the antibody is shown in SEQ ID NO.2. The antibody is G1E4-Bs-2.

[0078] In a specific embodiment of this application, when the anti-HER2 nanobody is G1E4, the anti-HER2 nanobody G1E4 is fused with the N-terminus of the heavy chain of pertuzumab to form the heavy chain of the antibody. The amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO.36. The light chain of pertuzumab is used as the light chain of the antibody. The amino acid sequence of the light chain of the antibody is shown in SEQ ID NO.4. The antibody is G1E4-Bs-3.

[0079] In a specific embodiment of this application, when the anti-HER2 nanobody is G1E4, the anti-HER2 nanobody G1E4 is fused with the C-terminus of the heavy chain of pertuzumab to form the heavy chain of the antibody. The amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO.37. The light chain of pertuzumab is used as the light chain of the antibody. The amino acid sequence of the light chain of the antibody is shown in SEQ ID NO.4. The antibody is G1E4-Bs-4.

[0080] In a specific embodiment of this application, when the anti-HER2 nanobody is G1E4, the heavy chain of trastuzumab is used as the heavy chain of the antibody, and the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO.1, the anti-HER2 nanobody G1E4 is fused with the N-terminus of the light chain of trastuzumab to form the light chain of the antibody, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO.38; the antibody is G1E4-Bs-5.

[0081] In a specific embodiment of this application, when the anti-HER2 nanobody is G1E4, the heavy chain of trastuzumab is used as the heavy chain of the antibody, and the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO.1, the anti-HER2 nanobody G1E4 is fused with the C-terminus of the light chain of trastuzumab to form the light chain of the antibody, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO.39; the antibody is G1E4-Bs-6.

[0082] In a specific embodiment of this application, when the anti-HER2 nanobody is G1E4, the heavy chain of pertuzumab is used as the heavy chain of the antibody, and the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO.3, the anti-HER2 nanobody G1E4 is fused with the N-terminus of the light chain of pertuzumab to form the light chain of the antibody, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO.40; the antibody is G1E4-Bs-7.

[0083] In a specific embodiment of this application, when the anti-HER2 nanobody is G1E4, the heavy chain of pertuzumab is used as the heavy chain of the antibody, and the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO.3, the anti-HER2 nanobody G1E4 is fused with the C-terminus of the light chain of pertuzumab to form the light chain of the antibody, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO.41; the antibody is G1E4-Bs-8.

[0084] In the embodiments of this application, among the 32 HER2 antibodies constructed above, A2G5-Bs-1, A2G5-Bs-2, A2G5-Bs-3, A2G5-Bs-4, A2G5-Bs-5, A2G5-Bs-6, A2G5-Bs-7, A2G5-Bs-8, A9B5-Bs-1, A9B5-Bs-2, and A9B5-Bs-3... A9B5-Bs-4, A9B5-Bs-5, A9B5-Bs-6, A9B5-Bs-7, A9B5-Bs-8, H2F5-Bs-1, H2F5-Bs-3, H2F5-Bs-4, H2F5-Bs-7, G1E4-Bs-2, G1E4-Bs-4, and G1E4-Bs-7 have the effect of inhibiting HER2-positive tumor cells.

[0085] In a specific embodiment of this application, the antibody is a bispecific antibody, which refers to an antibody that can simultaneously bind to two different targets or two different epitopes of the same target.

[0086] In a specific embodiment of this application, the bispecific antibody includes a biepisode antibody that can bind to different epitopes of the same antigen, such as different epitopes of the HER2 antigen.

[0087] In a specific embodiment of this application, the binding ability of the 32 antibodies constructed above to different epitopes of HER2 was tested. The antibodies A2G5-Bs-1, A2G5-Bs-2, A2G5-Bs-3, A2G5-Bs-4, A2G5-Bs-5, A2G5-Bs-6, A2G5-Bs-7, A2G5-Bs-8, A9B5-Bs-1, A9B5-Bs-2, and A9B5-Bs-2 were tested. B5-Bs-3, A9B5-Bs-4, A9B5-Bs-5, A9B5-Bs-6, A9B5-Bs-7, A9B5-Bs-8, H2F5-Bs-1, H2F5-Bs-2, H2F5-Bs-5, H2F5-Bs-6, G1E4-Bs-3, G1E4-Bs-4, G1E4-Bs-7, and G1E4-Bs-8 are bispecific antibodies.

[0088] In the specific embodiments of this application, among the above-mentioned bispecific antibodies, the bispecific antibodies A2G5-Bs-1, A2G5-Bs-2, A2G5-Bs-3, A2G5-Bs-4, A2G5-Bs-5, A2G5-Bs-6, A2G5-Bs-7, A2G5-Bs-8, A9B5-Bs-1, A9B5-Bs-2, A9B5-Bs-3, A9B5-Bs-4, A9B5-Bs-5, A9B5-Bs-6, A9B5-Bs-7, A9B5-Bs-8, H2F5-Bs-1, G1E4-Bs-4, and G1E4-Bs-7 have the effect of inhibiting HER2-positive tumor cells.

[0089] In a preferred embodiment of this application, among the bispecific antibodies that inhibit HER2-positive tumor cells, A2G5-Bs-1, A2G5-Bs-5, A2G5-Bs-6, A9B5-Bs-1, A9B5-Bs-5, A9B5-Bs-6, and A9B5-Bs-7 exhibit extremely strong inhibitory abilities against the proliferation of HER2-positive tumor cells, resulting in a HER2-positive tumor cell viability of less than 40%. Their inhibitory abilities are all stronger than trastuzumab (T) and the combined use of trastuzumab and pertuzumab (T+P).

[0090] In a preferred embodiment of this application, the bispecific antibodies A9B5-Bs-5 and A9B5-Bs-7 exhibit significantly better inhibitory activity against HER2-low-expressing tumor cells resistant to trastuzumab than the combined use of trastuzumab and pertuzumab (T+P).

[0091] The antibody of this application can also be modified into a chimeric antigen receptor, i.e., CAR. Therefore, this application also provides a chimeric antigen receptor, which includes the antibody described above. Specifically, the chimeric antigen receptor includes an antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, and the antigen-binding domain includes the antibody as described above.

[0092] The CAR also includes a transmembrane domain that connects the extracellular antigen recognition portion of the CAR to the intracellular signal transduction portion. In specific embodiments of this application, the transmembrane domain may be the transmembrane region of CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD20, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, DAP10, DAP12, PD-1, or the α, β, or ζ chain of the T cell receptor.

[0093] CAR also includes intracellular signal transduction domains. In specific embodiments of this application, the intracellular signal transduction domains sequentially include at least one co-stimulatory signal transduction domain and a primary signal transduction domain from the N-terminus to the C-terminus. In specific embodiments of this application, the primary signal transduction domain may be an intracellular signal transduction domain of CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD22, CD79a, DAP10, CD79b, or CD66d, etc. The co-stimulatory signal transduction domain may be an intracellular signal transduction domain of CD137(4-1BB), CD27, CD28, ICOS, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, etc., or a combination thereof.

[0094] CAR also includes a hinge region located between the C-terminus of the antigen-binding domain and the N-terminus of the transmembrane domain outside the cell. In specific embodiments of this application, the hinge region may be the hinge region of CD8α, CD28, IgG1 or IgG4.

[0095] CAR also includes a signal peptide located at the N-terminus of the chimeric antigen receptor polypeptide. In specific embodiments of this application, the signal peptide may be derived from HLA-A, CD8α, CD33, Igκ, IL-2, or GM-CSFRα, etc.

[0096] In a specific embodiment of this application, the chimeric antigen receptor CAR comprises, from its N-terminus to its C-terminus, the signal peptide, the antigen-binding domain, the hinge region, the transmembrane domain, the co-stimulatory signal transduction domain, and the primary signal transduction domain.

[0097] This application also provides a polynucleotide encoding the aforementioned antibody or the aforementioned chimeric antigen receptor.

[0098] The preparation method of the polynucleotide is an existing technology, and conventional methods can be selected according to specific circumstances. For example, it can be prepared by automated DNA synthesis, or it can be prepared by recombinant DNA technology, or it can be isolated from a suitable natural source.

[0099] Polynucleotides are polymers of nucleotides typically linked from one deoxyribose or ribose to another. There are no size limitations for polynucleotides in this application, and they may include polynucleotides containing modifications, particularly modified nucleotides. In some embodiments, polynucleotides may be RNA, DNA, or cDNA, etc.

[0100] In a specific embodiment of this application, the polynucleotide can be obtained artificially based on the amino acid sequence of the antibody.

[0101] In a specific embodiment of this application, the nucleotide sequence encoding the heavy chain of trastuzumab is shown in SEQ ID NO.42.

[0102] In a specific embodiment of this application, the nucleotide sequence encoding the light chain of trastuzumab is shown in SEQ ID NO.43.

[0103] In a specific embodiment of this application, the nucleotide sequence encoding the heavy chain of pertuzumab is shown in SEQ ID NO.44.

[0104] In a specific embodiment of this application, the nucleotide sequence encoding the light chain of pertuzumab is shown in SEQ ID NO.45.

[0105] In a specific embodiment of this application, the nucleotide sequence encoding the anti-HER2 nanobody A2G5 is shown in SEQ ID NO. 46.

[0106] In a specific embodiment of this application, the nucleotide sequence encoding the anti-HER2 nanobody A9B5 is shown in SEQ ID NO. 47.

[0107] In a specific embodiment of this application, the nucleotide sequence encoding the anti-HER2 nanobody H2F5 is shown in SEQ ID NO. 48.

[0108] In a specific embodiment of this application, the nucleotide sequence encoding the anti-HER2 nanobody G1E4 is shown in SEQ ID NO.49.

[0109] In a specific embodiment of this application, the nucleotide sequence encoding the flexible peptide is shown in SEQ ID NO.50.

[0110] Based on the nucleotide sequences encoding the heavy or light chains of trastuzumab or pertuzumab and the nucleotide sequences of anti-HER2 nanobodies, and according to the fusion mode of the anti-HER2 nanobodies and trastuzumab or pertuzumab, it is possible to synthesize polynucleotides encoding 32 HER2-binding antibodies of this application.

[0111] For example, in a specific embodiment of this application, the polynucleotide sequence includes the nucleotide sequences shown in SEQ ID NO. 51 and 43, which respectively encode the heavy chain and light chain of antibody A2G5-Bs-1.

[0112] For example, in a specific embodiment of this application, the polynucleotide sequence includes the nucleotide sequences shown in SEQ ID NO.42 and 52, which respectively encode the heavy chain and light chain of antibody A2G5-Bs-5.

[0113] For example, in a specific embodiment of this application, the polynucleotide sequence includes the nucleotide sequences shown in SEQ ID NO.42 and 53, which respectively encode the heavy chain and light chain of antibody A2G5-Bs-6.

[0114] For example, in a specific embodiment of this application, the polynucleotide sequence includes the nucleotide sequences shown in SEQ ID NO. 54 and 43, which respectively encode the heavy chain and light chain of antibody A9B5-Bs-1.

[0115] For example, in a specific embodiment of this application, the polynucleotide sequence includes the nucleotide sequences shown in SEQ ID NO.42 and 55, which respectively encode the heavy chain and light chain of antibody A9B5-Bs-5.

[0116] For example, in a specific embodiment of this application, the polynucleotide sequence includes the nucleotide sequences shown in SEQ ID NO.42 and 56, which respectively encode the heavy chain and light chain of antibody A9B5-Bs-6.

[0117] For example, in a specific embodiment of this application, the polynucleotide sequence includes the nucleotide sequences shown in SEQ ID NO.44 and 57, which respectively encode the heavy chain and light chain of antibody A9B5-Bs-7.

[0118] Based on the antibody-based recombinant expression method, this application also provides a vector comprising the aforementioned polynucleotides.

[0119] In this application, "vector" refers to a polynucleotide capable of carrying at least one polynucleotide fragment. The vector can exist in a circular or linear (linearized) form and includes vector fragments, or it can be an artificial chromosome or similar polynucleotide containing a transferable exogenous nucleic acid fragment. The vector may contain at least one expression cassette containing a regulatory sequence for the proper expression of the polynucleotide incorporated therein. The polynucleotide to be introduced into the cell (e.g., a polynucleotide encoding a target product or a selectable marker) can be inserted into the expression cassette of the vector for expression therefrom. The vector can also integrate exogenous DNA sequences into the host cell's genome, thereby achieving stable expression of the exogenous gene. It typically contains homologous arms or specific DNA sequences that can undergo homologous recombination with specific locations in the host cell's genome. The vector can be selected from DNA vectors, RNA vectors, plasmids, transposon vectors, CRISPR / Cas9 vectors, or viral vectors, etc. In a specific embodiment of this application, the vector is a pSCSTa expression vector, obtained by cloning the aforementioned polynucleotide into the pSCSTa expression vector.

[0120] Based on the above-described polynucleotides and vectors, this application may also provide a host cell containing the above-described vectors or having the above-described polynucleotides integrated into its genome.

[0121] The host cell can be selected from bacterial cells, fungal cells, insect cells, plant cells, mammalian cells, etc. Specifically, it can be selected from Escherichia coli, Streptomyces, Salmonella typhimurium, yeast, filamentous fungi, Drosophila S2 or Sf9 cells, CHO cells, COS cells, HEK293F cells, Bowes melanoma cells, NSO cells, BHK cells, PER.C6 cells, etc. For example, in a specific embodiment of this application, the host cell is Expi 293 cells. Further, the host cell is obtained by transiently transfecting Expi 293 cells with the above-mentioned pSCSTa expression vector. The transient transfection method can be selected according to the actual situation, such as calcium phosphate precipitation, liposome transfection, PEI method, microinjection, gene gun method, electroporation, electron bombardment method, and virus-mediated transformation method.

[0122] In specific embodiments of this application, the host cell may also be an immune cell, such as a T cell, to prepare CAR-T cells.

[0123] This application also provides a method for preparing an antibody or a chimeric antigen receptor, comprising culturing a host cell containing the aforementioned vector or a host cell whose genome has integrated exogenous polynucleotides, under conditions that allow the expression of the antigen-binding protein or the chimeric antigen receptor, and recovering the antibody or chimeric antigen receptor from the cultured host cell culture.

[0124] Specifically, the conditions that allow the expression of the anti-antigen binding protein mentioned above may refer to effective transfection or transduction methods, favorable promoter conditions, stable gene copies, suitable culture conditions, optimized post-translational modifications, etc.

[0125] Specifically, the effective transfection or transduction methods mentioned above can include microinjection, gene gun, electroporation, virus-mediated transformation, electron bombardment, calcium phosphate precipitation, liposome transfection, PEI, and other methods.

[0126] Specifically, the recovered antibody or its antigen-binding fragment can be selected conventionally according to the actual situation. For example, the antibody described in this application can be an antibody obtained by purification with Protein A.

[0127] Because the antibody of this application can promote the internalization of the HER2 receptor on the cell surface, this application also provides an antibody-drug conjugate, comprising the above-mentioned antibody or therapeutic agent. The antibody-drug conjugate links a biologically active small molecule drug to a monoclonal antibody via a chemical chain, with the monoclonal antibody acting as a carrier to target and deliver the small molecule drug to target cells. Its general structural formula can be A-(LU)n, where: A is an antibody or its antigen-binding fragment; U is a therapeutic agent; L is a linker; and n is an integer selected from 1 to 8.

[0128] The therapeutic agents refer to small molecule drugs with cytotoxicity that are covalently coupled to antibodies via linkers. These therapeutic agents are the core components of ADCs and are responsible for exerting anti-tumor effects. Examples include microtubule inhibitors, DNA damaging agents, topoisomerase inhibitors, apoptosis inducers, immunomodulators, and protein degraders. Microtubule inhibitors can be, for example, maytansine derivatives (DM1, DM3, DM4), monomethylolpropionate E (MMAE), paclitaxel, and eribulin. DNA damaging agents can be, for example, calicheamicin, duocarmysin, atrazocin-like drugs (PBD), camptothecins, and camptothecin derivatives (SN-3). Topoisomerase inhibitors can be, for example, DXd (Deruxtecan), SN-38, and Exatecan (DX-895). Immunomodulators can be, for example, TLR agonists, STING agonists, and BTK inhibitors.

[0129] The antibody-drug conjugate also includes a linker that conjugates the antibody or its antigen-binding fragment to the therapeutic agent. The linker can be a cleavable linker or a non-cleavable linker. Cleavable linkers can include acid-cleavable linkers, reducible linkers, enzyme-cleavable linkers, etc., while non-cleavable linkers are divided into two categories: thioether or maleimide hexanoyl (MC).

[0130] This application also provides a pharmaceutical composition comprising an effective amount of the above-described antibody, or the above-described chimeric antigen receptor, or the above-described polynucleotide, or the above-described carrier, or the above-described host cell, or the above-described antibody-drug conjugate.

[0131] The effective amount refers to the amount of a pharmaceutical compound or composition that causes a measurable clinical, biological, or pharmaceutical change or response in a biomarker, cell, tissue, system, or patient.

[0132] The form of the pharmaceutical composition is not limited and can be in various forms such as solid, liquid, gel, semi-liquid, or aerosol.

[0133] The pharmaceutical composition may also include pharmaceutically acceptable carriers or excipients. Pharmaceutically acceptable carriers or excipients refer to those auxiliary materials that do not affect the efficacy and safety of the main pharmaceutical ingredient, are non-toxic in themselves, do not produce adverse, allergic or other adverse reactions, and should be compatible with the above-mentioned effective amount of the main ingredient, that is, can be mixed with it without significantly reducing the effect of the above-mentioned effective amount of the main ingredient under normal circumstances. Specific examples of substances that can serve as pharmaceutically acceptable carriers or excipients include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, ethylcellulose, and methylcellulose; tragacanth gum powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; colorants; flavoring agents; tableting agents, stabilizers, antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; and phosphate buffers, etc. The dosage form of the pharmaceutical composition is not limited, and suitable carriers or excipients can be selected according to the dosage form.

[0134] In specific embodiments of this application, the antibody, chimeric antigen receptor, and antibody-drug conjugate in the pharmaceutical composition can be a single active ingredient, or they can be combined with one or more other active ingredients that have therapeutic effects on HER2-related tumors to form a combination formulation. The efficacy and safety of the components do not conflict. The content of each component in the combination formulation is typically a safe and effective amount, which can be adjusted based on actual usage (e.g., patient weight, application type, disease condition, severity).

[0135] The application also provides a kit containing an effective amount of the aforementioned antibody and detection reagent for diagnosing whether a patient has a HER2-related tumor.

[0136] In a specific embodiment of this application, the detection reagent is selected from one or more of enzyme-linked immunosorbent assay (ELISA) reagents, cell immunofluorescence assay reagents, flow cytometry reagents, isotope tracers, contrast agents, magnetic nanoparticles, or imaging agents.

[0137] This application also provides the use of the above-described antibody, or the above-described chimeric antigen receptor, or the above-described polynucleotide, or the above-described carrier, or the above-described host cell, or the above-described antibody-drug conjugate, or the above-described pharmaceutical composition, or the above-described kit, wherein the use is selected from any one or more of the following:

[0138] 1) Prepare products for the prevention and / or treatment of HER2-related tumors; 2) Prepare products for the diagnosis of HER2-related tumors; 3) Prepare products for the internalization of HER2 receptors on cell surfaces.

[0139] The product may include an effective dose of the above-mentioned ingredients as the main active ingredient, or may be prepared from the above-mentioned ingredients.

[0140] The products include, but are not limited to, pharmaceuticals, pharmaceutical compositions, combinations of pharmaceuticals, reagents, reagent kits, food, and health products.

[0141] The dosage form or state of the product is not limited. For example, when the product is a drug, a drug composition, or a combination of drugs, the dosage form can be one or more of the following: solution, injection, spray, nasal drops, aerosol, powder inhaler, tablet, capsule, and granule; when the product is food or health product, the state can be liquid or solid.

[0142] The product may also include pharmaceutically acceptable carriers or excipients, as described above. For example, when the product is a drug, a combination of drugs, or a pharmaceutical composition, it may be for oral administration, and suitable dosage forms for oral administration include solid dosage forms and liquid formulations. Solid dosage forms include capsules, tablets, pills, powders, and granules. In these solid dosage forms, one or more of at least one conventional inert excipient, filler or solubilizer, binder, humectant, disintegrant, slow solvent, adsorbent, and lubricant are obtained according to the prior art. In capsules, tablets, and pills, the dosage form may also contain a buffer. Solid dosage forms such as tablets, sugar pills, capsules, pills, and granules may be prepared using coatings and shell materials, such as casings and other materials known in the art. They may contain opacifying agents, and the release of the active compound or compound in such a composition may be delayed in a portion of the digestive tract. Examples of encapsulating components that may be used are polymeric substances and waxes. If necessary, the active compound may also be formed into microcapsules with one or more of the excipients described above. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers, emulsifiers, and other auxiliaries.

[0143] For example, when the product is a drug, a combination of drugs, or a drug composition, it can be administered via the gastrointestinal tract or non-gastrointestinal route, such as by injection, spray, nasal drops, eye drops, osmosis, absorption, or physical or chemically mediated methods into the body, including intramuscular, intradermal, subcutaneous, intravenous, and mucosal tissues; or by being mixed with or encapsulated by other substances before being administered into the body. Preferably, it is administered via the gastrointestinal tract. Therefore, preferably, the drug composition further includes antioxidants, buffers, antibacterial agents, and solutes that make the formulation isotonic with the subject's blood, as well as a sterile suspension containing both aqueous and non-aqueous components, which may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives.

[0144] This application also provides a method for treating HER2-related tumors, comprising administering to a subject an effective amount of the aforementioned bispecific antibody, or the aforementioned chimeric antigen receptor, or the aforementioned antibody-drug conjugate.

[0145] In this application, the HER2-related tumors are selected from HER2-positive tumors or HER2-low-expressing tumors; the HER2-positive tumors refer to tumor cells with high HER2 protein expression levels, which are usually determined by immunohistochemistry (IHC) detection of HER2 (3+) or IHC (2+) and in situ hybridization (ISH) results showing HER2 gene amplification; the HER2-low-expressing tumors refer to tumor cells with low HER2 protein expression levels, but higher than HER2-negative levels. For example, breast cancer with IHC results of (1+) or IHC (2+) and ISH results showing no HER2 gene amplification is defined as HER2-low-expressing tumors.

[0146] In specific embodiments of this application, the HER2-positive tumor may be, for example, gastric cancer or breast ductal carcinoma.

[0147] In a specific embodiment of this application, the HER2-low-expressing tumor can be HER2-low-expressing breast cancer that is resistant to trastuzumab, but the bispecific antibody of this application has a significant inhibitory effect.

[0148] In this application, the HER2-positive tumors include HER2-positive tumors with non-ligand-dependent proliferation and HER2-positive tumors with ligand-dependent proliferation. The non-ligand-dependent HER2-positive tumors typically involve mutations or amplification of the HER2 gene, leading to overexpression or structural alterations of the HER2 protein, enabling the HER2 signaling pathway to be activated even without ligand binding. This activation is independent of the binding of HER2 to its ligand, but rather due to the continuous activation of the signaling pathway caused by changes in HER2 itself. The proliferation of the ligand-dependent HER2-positive tumors depends on the binding of the HER2 receptor to its ligand. HER2 (human epidermal growth factor receptor 2) belongs to the HER family, including HER1 (EGFR), HER2, HER3, and HER4. HER2 itself does not have a known ligand, but it can bind to other HER family members to form heterodimers, activating downstream signaling pathways such as the PI3K-AKT and RAS-MAPK (ERK) pathways, thereby regulating cell proliferation, differentiation, migration, and apoptosis. In some cases, HER2 can be activated by binding to the ligands of other HER family members; this activation is called ligand-dependent activation. For example, HER3, upon binding to its ligand, can form a heterodimer with HER2, thereby activating the HER2 signaling pathway. The bispecific antibody of this application can inhibit the proliferation of both ligand-independent and ligand-dependent HER2-positive tumor cells.

[0149] In this application, the HER2-related tumors are selected from gastric cancer, breast ductal carcinoma, breast cancer, gastroesophageal junction cancer, salivary gland cancer, ovarian cancer, endometrial cancer, cervical cancer, non-small cell lung cancer, bile duct cancer, pancreatic cancer, colon cancer, bladder cancer, and prostate cancer.

[0150] In this application, the application targets of the above-mentioned products, pharmaceutical compositions, kits, and methods can be mammals, such as but not limited to humans, primates, livestock (e.g., sheep, cattle, horses, donkeys, pigs), pets (e.g., dogs, cats), laboratory test animals (e.g., mice, rabbits, rats, guinea pigs, hamsters), or captured wild animals (e.g., foxes, deer); preferably, the target is a primate; more preferably, the target is a human.

[0151] In this application, the above-mentioned products, pharmaceutical compositions, and methods can also be used in combination with other methods for treating HER2-related tumors, such as HER2-targeted drug therapy, chemotherapy drug therapy, and surgical treatment.

[0152] The present application is further illustrated below by way of examples, but these examples do not limit the scope of the application. Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this application all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. These techniques have been well described in existing literature. Unless otherwise specified, the instruments, reagents, and materials used in the examples can all be obtained through conventional means.

[0153] Example 1: Construction of HER2 Bispecific Antibody

[0154] 1.1 Sequence Acquisition

[0155] The heavy and light chain amino acid sequences of trastuzumab (T) and pertuzumab (P) were obtained from the IMGT (https: / / www.imgt.org) database. The heavy chain gene (heavy chain amino acid sequence of trastuzumab is shown in SEQ ID NO.1, nucleotide sequence of SEQ ID NO.42) and light chain gene (heavy chain amino acid sequence of trastuzumab is shown in SEQ ID NO.2, nucleotide sequence of SEQ ID NO.43) and the heavy chain gene (heavy chain amino acid sequence of pertuzumab is shown in SEQ ID NO.3, nucleotide sequence of SEQ ID NO.44) and light chain gene (heavy chain amino acid sequence of pertuzumab is shown in SEQ ID NO.4, nucleotide sequence of SEQ ID NO.45) of pertuzumab were obtained by artificial synthesis.

[0156] The amino acid sequence of the anti-HER2 nanobody A2G5 is shown in SEQ ID NO.5, and the nucleotide sequence is shown in SEQ ID NO.46; the amino acid sequence of A9B5 is shown in SEQ ID NO.6, and the nucleotide sequence is shown in SEQ ID NO.47; the amino acid sequence of H2F5 is shown in SEQ ID NO.7, and the nucleotide sequence is shown in SEQ ID NO.48; the amino acid sequence of G1E4 is shown in SEQ ID NO.8, and the nucleotide sequence is shown in SEQ ID NO.49.

[0157] 1.2 Construction of antibody expression vector and preparation of purified antibody

[0158] Eight different conformations of HER2 biepisode antibodies were designed by fusing anti-HER2 nanobodies to the N-terminus or C-terminus of the heavy or light chains of trastuzumab and pertuzumab using a linker sequence of a flexible peptide chain (the amino acid sequence is shown in SEQ ID NO. 9, and the nucleotide sequence is shown in SEQ ID NO. 50). The heavy and light chains of trastuzumab were TH and TL, respectively, while those of pertuzumab were PH and PL, respectively. The fusion heavy chain formed by fusing nanobodies with the N-terminus of TH is TH-N; the fusion heavy chain formed by fusing nanobodies with the C-terminus of TH is TH-C; the fusion heavy chain formed by fusing nanobodies with the N-terminus of PH is PH-N; the fusion heavy chain formed by fusing nanobodies with the C-terminus of PH is PH-C; the fusion light chain formed by fusing nanobodies with the N-terminus of TL is TL-N; the fusion light chain formed by fusing nanobodies with the C-terminus of TL is TL-C; the fusion light chain formed by fusing nanobodies with the N-terminus of PL is PL-N; and the fusion light chain formed by fusing nanobodies with the C-terminus of PL is PL-C.

[0159] By combining the aforementioned fused heavy chains with natural light chains, or by combining the aforementioned fused light chains with natural heavy chains, a series of bispecific antibodies were formed: TH-N and TL formed bispecific antibody Bs-1, TH-C and TL formed bispecific antibody Bs-2, PH-N and PL formed bispecific antibody Bs-3, PH-C and PL formed bispecific antibody Bs-4, TL-N and TH formed bispecific antibody Bs-5, TL-C and TH formed bispecific antibody Bs-6, PL-N and PH formed bispecific antibody Bs-7, and PL-C and PH formed bispecific antibody Bs-8. By constructing HER2 antibodies from four high-affinity nanobodies in the above manner, 32 HER2 antibodies were obtained.

[0160] Based on the nucleotide sequences of the heavy or light chains of trastuzumab or pertuzumab and the nucleotide sequences of anti-HER2 nanobodies, and according to the fusion mode of anti-HER2 nanobodies and trastuzumab or pertuzumab, genes encoding 32 antibodies that bind to HER2 were synthesized.

[0161] The heavy and light chain genes were cloned into the pSCSTa expression vector (Table 1). The expression vector construction was verified to be correct. After transient transfection of Expi 293 cells, the supernatant was collected and purified using Protein A to obtain 32 different HER2 antibodies with high purity. SDS-PAGE results showed that the purity of all HER2 antibodies exceeded 95% (see Table 1). Figure 1 ).

[0162] Table 1. Antibody sequences

[0163]

[0164]

[0165] Example 2: Bi-epitope binding ability of HER2 antibody

[0166] The binding affinity of the 32 HER2 antibodies constructed in Example 1 to two different epitopes of the HER2 antigen was determined using enzyme-linked immunosorbent assay (ELISA). Four antigens, hHER2-ECD, hHER2-ECD-D1, hHER2-ECD-D2, hHER2-ECD-D3, and hHER2-ECD-D4 (the preparation of the four antigens is based on patent CN 118420767 A; due to the extremely low expression level of hHER2(ECD)-D3, the other three chimeric proteins were used to detect the nanobody recognition epitopes) were coated onto the antibodies and reacted at room temperature for 1 hour. After washing 12 times with PBST, Anti-huFc-HRP (diluted 1:8000) was added and reacted at room temperature for 30 minutes. After washing 15 times with PBST, chromogenic buffer was added and reacted at room temperature for 3 minutes, followed by the addition of stop buffer. Finally, the wavelength (OD450) was read at 450 nm using a microplate reader. The EC50 was calculated after fitting the reaction curve using Graphpad Prism. 50 The binding efficiencies are shown in Table 2. The results indicate that all bispecific antibodies showed binding efficiencies with hHER2-ECD comparable to trastuzumab (T) and pertuzumab (P), all at the nanomolar level. Regarding epitope binding, A2G5-Bs-1, A2G5-Bs-2, A2G5-Bs-5, A2G5-Bs-6, A9B5-Bs-1, A9B5-Bs-2, A9B5-Bs-5, and A9B5-Bs-6 could simultaneously bind to epitopes on ECD I and ECD IV; A2G5-Bs-3, A2G5-Bs-4, A2G5-Bs-7, A2G5-Bs-8, A9B5-Bs-3, A9B5-Bs-4, A9B5-Bs-7, and A9B5-Bs-8 could simultaneously bind to ECDI and ECD. Epitopes on ECD II; H2F5-Bs-1, H2F5-Bs-2, H2F5-Bs-5, H2F5-Bs-6, G1E4-Bs-3, G1E4-Bs-4, G1E4-Bs-7, and G1E4-Bs-8 can bind to epitopes on both ECD II and ECD IV, making them bispecific antibodies that can bind to different epitopes of HER2.

[0167] Table 2 shows the binding efficacy of HER2 antibodies to different epitopes.

[0168]

[0169]

[0170] Example 3: HER2 bispecific antibody effectively inhibits the proliferation of HER2-positive tumor cells.

[0171] The inhibitory effect of antibodies on tumor cells was detected using the CCK-8 assay. First, HER2-positive NCI-N87 gastric cancer cell line was digested with trypsin. Cell clumps were carefully dissected to form single cells, and cell counts were performed. Cells were then seeded into 96-well cell culture plates at 4000 cells per well. Cells were incubated overnight in a constant temperature incubator. The next day, the original culture medium was discarded, and the corresponding concentration of antibody diluted completely in the original medium was added to each well. Positive control (trastuzumab, trastuzumab, and pertuzumab combined), negative control (irrelevant antibody IgG), and blank control were set up, with three replicates for each antibody and concentration. After 4 days, the antibody-containing culture medium was discarded, and 100 μL of pre-diluted 10% CCK8 solution was added to each well. Cells were incubated in a constant temperature incubator for 1.5 h. The absorbance of each well at 450 nm was measured using a microplate reader, and the cell viability after antibody treatment was calculated, thus reflecting the inhibitory effect of the antibody on cell proliferation. The calculation formula is: Cell viability (%) = [(As-Ab) / (Ac-Ab)] × 100%. Where As represents the experimental well (containing cell culture medium, CCK-8, and the test substance); Ac represents the control well (containing cell culture medium, CCK-8, and no test substance); and Ab represents the blank well (containing no cells, test substance, or culture medium and CCK-8). Cell viability after each antibody treatment is shown below. Figure 2 , Antibodies A2G5-Bs-1, A2G5-Bs-2, A2G5-Bs-3, A2G5-Bs-4, A2G5-Bs-5, A2G5-Bs-6, A2G5-Bs-7, A2G5-Bs-8, A9B5-Bs-1, A9B5-Bs-2, A 9B5-Bs-3, A9B5-Bs-4, A9B5-Bs-5, A9B5-Bs-6, A9B5-Bs-7, A9B5-Bs-8, H2F5-Bs-1, H2F5-Bs-3, H2F5-Bs-4, H2F5-Bs-7, G1E4-B s-2, G1E4-Bs-4, and G1E4-Bs-7 all have the effect of inhibiting HER2-positive tumor cells. It is obvious that the bispecific antibodies A2G5-Bs-1, A2G5-Bs-5, A2G5-Bs-6, A9B5-Bs-1, A9B5-Bs-5, A9B5-Bs-6, and A9B5-Bs-7 have extremely strong inhibitory ability on cell proliferation (cell viability is less than 40%). Their inhibitory ability is stronger than that of trastuzumab (T) and the combination of trastuzumab and pertuzumab (T+P), indicating that bispecific antibodies have the effect of treating HER2-related tumors.

[0172] The inhibitory effects of the seven bispecific antibodies on the HER2-positive BT-474 human breast ductal carcinoma cell line and the trastuzumab-resistant MCF-7 breast cancer cell line were detected using the same CCK-8 assay as described above. Cell viability after antibody treatment is shown in the figure. Figure 3 The significant differences are shown in Table 3. The results showed that, against the HER2-positive tumor cell line BT-474 human breast ductal carcinoma, the inhibitory effects of bispecific antibodies A2G5-Bs-1, A2G5-Bs-5, A9B5-Bs-5, and A9B5-Bs-7 were significantly better than the combined use of trastuzumab and pertuzumab (T+P). Against the trastuzumab-resistant MCF-7 breast cancer cell line, the inhibitory effects of bispecific antibodies A9B5-Bs-5 and A9B5-Bs-7 were significantly better than the combined use of trastuzumab and pertuzumab (T+P). In summary, the bispecific antibodies A9B5-Bs-5 and A9B5-Bs-7 exhibited particularly strong inhibitory effects in various cell lines and were significantly superior to the currently best clinical regimen of "trastuzumab and pertuzumab combined use," indicating that bispecific antibodies have a therapeutic effect on HER2-related tumors. Therefore, further research on these two bispecific antibodies is warranted.

[0173] Table 3 Cell viability

[0174]

[0175]

[0176] Example 4: Analysis of the binding mode between HER2 bispecific antibody and HER2 receptor

[0177] First, the binding site between the bispecific antibody and HER2 was analyzed using the artificial intelligence analysis tool Alphafold (https: / / golgi.sandbox.google.com / ). The amino acid sequences of the nanobody A9B5 and the extracellular fragment of HER2 were input into Alphafold, and the resulting A9B5-HER2 complex structure PDB file was imported into PyMOL and ChimeraX software for analysis. The analysis results are as follows: Figure 4 As shown, A9B5 can interact with five amino acid sites (E188, S192, L193, K206, and D212) of the extracellular domain of HER2. Further, the complex structures of trastuzumab and HER2 (1N8Z) and pertuzumab and HER2 (1S78) were aligned with the complex structure of A9B5 and HER2, respectively. The distance between the C-terminus of A9B5 and the N-terminus of the light chain of trastuzumab or pertuzumab was measured to determine the binding mode of the bispecific antibody to HER2. The analytical results are as follows: Figure 5As shown, the distance between the C-terminus of A9B5 and the N-terminus of the trastuzumab light chain is... The distance between the C-terminus of A9B5 and the N-terminus of the pertuzum light chain is angstroms. Both are greater than the maximum distance of the flexible peptide chain. Therefore, it can be inferred that the A9B5 and IgG portions of the bispecific antibody cannot bind simultaneously to the same HER2 molecule; they can only bind to two different HER2 molecules, i.e., trans binding. Thus, the binding mode of A9B5-Bs-5 and A9B5-Bs-7 to the HER2 receptor is trans binding.

[0178] Example 5: HER2 bispecific antibody promotes HER2 receptor internalization

[0179] The trans-binding of bispecific antibodies to HER2 may mediate the formation of large antigen-antibody complex aggregates, thereby promoting HER2 receptor internalization and inhibiting tumor cell growth. Next, laser confocal microscopy was used to examine whether the bispecific antibodies A9B5-Bs-5 and A9B5-Bs-7 could promote the internalization of HER2 receptors on the cell surface. Two different cell lines, NCI-N87 gastric cancer cells and MCF-7 breast cancer cells, were thoroughly smeared and incubated with antibodies for 1 hour. After washing three times with PBS, complete cell culture medium was added, and the cells were cultured for another 4 hours. After fixation with paraformaldehyde, the cells were permeabilized and blocked. Then, they were reacted with anti-human LAMP-1 antibody for 2 hours, washed three times with PBS, and reacted with secondary antibody for 1 hour. After staining the cell nuclei with DAPI, the internalization of HER2 after bispecific antibody binding was observed under laser confocal microscopy. The experimental group consisted of bispecific antibodies A9B5-Bs-5 and A9B5-Bs-7, the control group consisted of trastuzumab (T), pertuzumab (P), and a combination of trastuzumab and pertuzumab (T+P), and the blank control consisted of irrelevant antibody IgG. The results showed that ( Figure 6In the HER2-overexpressing cell line NCI-N87, the green signal in the control group was mainly at the cell edge, indicating that the control group antibody could not effectively promote HER2 receptor internalization. In contrast, A9B5-Bs-5 and A9B5-Bs-7 showed strong green signals inside the cells, indicating that they significantly promoted HER2 receptor internalization on the NCI-N87 cell surface. In the trastuzum-resistant HER2-low-expressing breast cancer cell line MCF-7, the control group showed only a weak green signal at the cell edge, indicating that the control group antibody could not promote HER2 receptor internalization on the cell surface. However, A9B5-Bs-5 and A9B5-Bs-7 showed green signals inside the cells, indicating that they promoted HER2 receptor internalization on the MCF-7 breast cancer cell line. These findings suggest that bispecific antibodies can promote HER2 receptor internalization and can be used to prepare antibody-drug conjugates.

[0180] Example 6: HER2 bispecific antibody can simultaneously inhibit the proliferation of ligand-independent and ligand-dependent HER2-positive tumor cells.

[0181] 6.1 Detection of the inhibitory effect of bispecific antibody on ligand-dependent proliferation of HER2-positive tumor cells using the CCK-8 assay.

[0182] The same CCK-8 assay as in Example 3 was used to detect HER2-positive tumor cells in two cell lines (NCI-N87 gastric cancer cell line and BT-474 breast ductal carcinoma cell line). The experimental groups used bispecific antibodies A9B5-Bs-5 and A9B5-Bs-7, the control groups used trastuzumab (T), pertuzumab (P), and a combination of trastuzumab and pertuzumab (T+P), and the blank control was an irrelevant antibody IgG. Cell viability after antibody treatment is shown below. Figure 7 The significant differences in antibody inhibitory capacity at a concentration of 150 nM are shown in Table 4. The results showed that A9B5-Bs-5 and A9B5-Bs-7 had significantly better inhibitory capacity on NCI-N87 cells than any control antibody; A9B5-Bs-5 and A9B5-Bs-7 had significantly better inhibitory capacity on BT-474 cells than trastuzumab (T) and pertuzumab (P), and were comparable to the combination of trastuzumab and pertuzumab (T+P).

[0183] Table 4. Inhibitory effect of antibodies on ligand-independent proliferation of HER2-positive tumor cells.

[0184]

[0185] 6.2 CCK-8 assay to detect the inhibitory effect of bispecific antibodies on ligand-dependent proliferation of HER2-positive tumor cells

[0186] Ligand-dependent proliferation is one of the drug resistance mechanisms of HER2-positive tumor cells, and trastuzumab and pertuzumab have weak inhibitory effects on ligand-dependent proliferation. Following the procedure in 5.1, NCI-N87 gastric cancer cell lines and BT-474 breast ductal carcinoma cell lines, after antibody incubation, were stimulated with HRG and EGF ligands, respectively. The effectiveness of the bispecific antibodies A9B5-Bs-5 and A9B5-Bs-7 in inhibiting tumor proliferation under continuous ligand stimulation was examined. Control groups included trastuzumab (T), pertuzumab (P), and a combination of trastuzumab and pertuzumab (T+P). The viability of NCI-N87 cells after antibody treatment is shown below. Figure 8 The significant differences in antibody inhibitory activity at 150 nM concentrations are shown in Table 5. The results showed that, under any ligand stimulation condition, the inhibitory activity of A9B5-Bs-7 was significantly better than that of any control group. Under EGF stimulation, the inhibitory activity of A9B5-Bs-5 was significantly better than that of any control group, while under HRG stimulation, the inhibitory activity of A9B5-Bs-5 was significantly better than that of trastuzumab (T) and pertuzumab (P), and comparable to that of trastuzumab and pertuzumab combined (T+P). Although the inhibitory activity of A9B5-Bs-5 at high concentrations (150 nM) was comparable to that of T+P, the EC50 of A9B5-Bs-5 was significantly lower. 50 The value (0.83 nM) is significantly lower than the EC50 of T+P. 50 The concentration (6.59 nM) indicates that A9B5-Bs-5's inhibitory ability at other concentrations is still superior to T+P. In summary, the bispecific antibodies A9B5-Bs-5 and A9B5-Bs-7 exhibit significantly better ligand-dependent NCI-N87 proliferation activity than existing therapeutic antibodies.

[0187] Table 5. Inhibitory effect of antibodies on ligand-dependent proliferation of HER2-positive tumor cells NCI-N87.

[0188]

[0189] The cell viability of BT-474 breast ductal carcinoma cell line after antibody treatment is as follows: Figure 9 The significant differences in antibody inhibitory activity at a concentration of 150 nM are shown in Table 6. The results showed that, under any ligand stimulation condition, the bispecific antibodies A9B5-Bs-5 and A9B5-Bs-7 exhibited superior inhibitory activity against BT-474 breast ductal carcinoma cell line cells compared to any control group.

[0190] Table 6. Inhibitory effect of antibodies on ligand-dependent proliferation of HER2-positive tumor cells BT-474.

[0191]

[0192] In summary, the anti-HER2 bispecific antibodies A9B5-Bs-5 and A9B5-Bs-7 discovered in this invention have unique dual epitope recognition capabilities, and their inhibitory ability against HER2-positive tumor cells is significantly superior to that of existing therapeutic antibodies.

[0193] The sequences involved in this application:

[0194] Heavy chain amino acid sequence of trastuzumab, SEQ ID NO.1: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVR QAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0195] Trastuzumab light chain amino acid sequence, SEQ ID NO.2: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQ KPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0196] The amino acid sequence of the heavy chain of pertuzumab, SEQ ID NO.3: EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWV RQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0197] The amino acid sequence of the light chain of pertuzumab, SEQ ID NO.4: DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQ QKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0198] The amino acid sequence of the anti-HER2 nanobody A2G5, SEQ ID NO.5: DVQLVESGGGSVQAGGSLRLSCAASGISN GLSHLGWFRQAPGKEREGVAAYYYDYSTFYYADSVKGRFTVSLDNAENTVYLQMNSLKPEDTALYYCA ATPAYVIGRTDHGLNYWGQGTQVTVSS

[0199] The amino acid sequence of the anti-HER2 nanobody A9B5, SEQ ID NO. 6: DVQLVESGGGSVQAGGSLRLSCAASGVF RSYYTLGWFRQAPGKEREGVAAVSPAGGTPYYADSVKGRFTVSLDNAENTVYLQMNSLKPEDTALYYC AAGGNEYVGDTVRVANYWGQGTQVTVSS

[0200] The amino acid sequence of the anti-HER2 nanobody H2F5, SEQ ID NO.7: DVQLVESGGGSVQAGGSLRLSCAASGDLY QSEYLGWFRQAPGKEREGVAAYDLHRSVKYYADSVKGRFTVSLDNAENTVYLQMNSLKPEDTALYYCA AYSTMYGGSASYVVNYWGQGTQVTVSS

[0201] The amino acid sequence of the anti-HER2 nanobody G1E4, SEQ ID NO. 8: DVQLVESGGGSVQAGGSLRLSCAASGFRI SQGYLGWFRQAPGKEREGVAAVSPAGGTPYYADSVKGRFTVSLDNAENTVYLQMNSLKPEDTALYYCA ATFIHDFHGRQVYQPNYWGQGTQVTVSS

[0202] Flexible peptide, SEQ ID NO.9: GGGGSGGGGSGGGGS

[0203] Heavy chain amino acid sequence of antibody A2G5-Bs-1, SEQ ID NO.10: DVQLVESGGGSVQAGGSLRLSCAASGISNGL SHLGWFRQAPGKEREGVAAYYYDYSTFYYADSVKGRFTVSLDNAENTVYLQMNSLKPEDTALYYCAATPAYVIGRTDHGLNYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0204] The amino acid sequence of the heavy chain of antibody A2G5-Bs-2, SEQ ID NO.11: EVQLVESGGGLVQPGGSLRLSCAASGFNIKD TYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSDVQLVESGGGSVQAGGSLRLSCAASGISNGLSHLGWFRQAPGKEREGVAAYYYDYSTFYYADSVKGRFTVSLDNAENTVYLQMNSLKPEDTALYYCAATPAYVIGRTDHGLNYWGQGTQVTVSS

[0205] Heavy chain amino acid sequence of antibody A2G5-Bs-3, SEQ ID NO.12: DVQLVESGGGSVQAGGSLRLSCAASGISNGL SHLGWFRQAPGKEREGVAAYYYDYSTFYYADSVKGRFTVSLDNAENTVYLQMNSLKPEDTALYYCAATPAYVIGRTDHGLNYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0206] The amino acid sequence of the heavy chain of antibody A2G5-Bs-4, SEQ ID NO.13: EVQLVESGGGLVQPGGSLRLSCAASGFTFTD YTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSDVQLVESGGGSVQAGGSLRLSCAASGISNGLSHLGWFRQAPGKEREGVAAYYYDYSTFYYADSVKGRFTVSLDNAENTVYLQMNSLKPEDTALYYCAATPAYVIGRTDHGLNYWGQGTQVTVSS

[0207] Light chain amino acid sequence of antibody A2G5-Bs-5, SEQ ID NO.14: DVQLVESGGGSVQAGGSLRLSCAASGISNGL SHLGWFRQAPGKEREGVAAYYYDYSTFYYADSVKGRFTVSLDNAENTVYLQMNSLKPEDTALYYCAATPAYVIGRTDHGLNYWGQGTQVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0208] Light chain amino acid sequence of antibody A2G5-Bs-6, SEQ ID NO.15: DIQMTQSPSSLSASVGDRVTITCRASQDVNT AVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSDVQLVESGGGSVQAGGSLRLSCAASGISNGLSHLGWFRQAPGKEREGVAAYYYDYSTFYYADSVKGRFTVSLDNAENTVYLQMNSLKPEDTALYYCAATPAYVIGRTDHGLNYWGQGTQVTVSS

[0209] Light chain amino acid sequence of antibody A2G5-Bs-7, SEQ ID NO.16: DVQLVESGGGSVQAGGSLRLSCAASGISNGL SHLGWFRQAPGKEREGVAAYYYDYSTFYYADSVKGRFTVSLDNAENTVYLQMNSLKPEDTALYYCAATPAYVIGRTDHGLNYWGQGTQVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0210] Light chain amino acid sequence of antibody A2G5-Bs-8, SEQ ID NO.17: DIQMTQSPSSLSASVGDRVTITCKASQDVS IGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSDVQLVESGGGSVQAGGSLRLSCAASGISNGLSHLGWFRQAPGKEREGVAAYYYDYSTFYYADSVKGRFTVSLDNAENTVYLQMNSLKPEDTALYYCAATPAYVIGRTDHGLNYWGQGTQVTVSS

[0211] Heavy chain amino acid sequence of antibody A9B5 - Bs - 1, SEQ ID NO.18: DVQLVESGGGSVQAGGSLRLSCAASGVFRS YYTLGWFRQAPGKEREGVAAVSPAGGTPYYADSVKGRFTVSLDNAENTVYLQMNSLKPEDTALYYCAAGGNEYVGDTVRVANYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0212] Heavy chain amino acid sequence of antibody A9B5-Bs-2, SEQ ID NO.19: EVQLVESGGGLVQPGGSLRLSCAASGFNIKD TYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSDVQLVESGGGSVQAGGSLRLSCAASGVFRSYYTLGWFRQAPGKEREGVAAVSPAGGTPYYADSVKGRFTVSLDNAENTVYLQMNSLKPEDTALYYCAAGGNEYVGDTVRVANYWGQGTQVTVSS

[0213] Heavy chain amino acid sequence of antibody A9B5-Bs-3, SEQ ID NO.20: DVQLVESGGGSVQAGGSLRLSCAASGVFRS YYTLGWFRQAPGKEREGVAAVSPAGGTPYYADSVKGRFTVSLDNAENTVYLQMNSLKPEDTALYYCAAGGNEYVGDTVRVANYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0214] Heavy chain amino acid sequence of antibody A9B5 - Bs - 4, SEQ ID NO.21: EVQLVESGGGLVQPGGSLRLSCAASGFTFTD YTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSDVQLVESGGGSVQAGGSLRLSCAASGVFRSYYTLGWFRQAPGKEREGVAAVSPAGGTPYYADSVKGRFTVSLDNAENTVYLQMNSLKPEDTALYYCAAGGNEYVGDTVRVANYWGQGTQVTVSS

[0215] The light chain amino acid sequence of antibody A9B5-Bs-5, SEQ ID NO.22: DVQLVESGGGSVQAGGSLRLSCAASGVFRS YYTLGWFRQAPGKEREGVAAVSPAGGTPYYADSVKGRFTVSLDNAENTVYLQMNSLKPEDTALYYCAAGGNEYVGDTVRVANYWGQGTQVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0216] The light chain amino acid sequence of antibody A9B5-Bs-6, SEQ ID NO.23: DIQMTQSPSSLSASVGDRVTITCRASQDVNT AVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSDVQLVESGGGSVQAGGSLRLSCAASGVFRSYYTLGWFRQAPGKEREGVAAVSPAGGTPYYADSVKGRFTVSLDNAENTVYLQMNSLKPEDTALYYCAAGGNEYVGDTVRVANYWGQGTQVTVSS

[0217] Light chain amino acid sequence of antibody A9B5-Bs-7, SEQ ID NO.24: DVQLVESGGGSVQAGGSLRLSCAASGVFRS YYTLGWFRQAPGKEREGVAAVSPAGGTPYYADSVKGRFTVSLDNAENTVYLQMNSLKPEDTALYYCAAGGNEYVGDTVRVANYWGQGTQVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0218] Light chain amino acid sequence of antibody A9B5-Bs-8, SEQ ID NO.25: DIQMTQSPSSLSASVGDRVTITCKASQDVSIG VAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSDVQLVESGGGSVQAGGSLRLSCAASGVFRSYYTLGWFRQAPGKEREGVAAVSPAGGTPYYADSVKGRFTVSLDNAENTVYLQMNSLKPEDTALYYCAAGGNEYVGDTVRVANYWGQGTQVTVSS

[0219] Heavy chain amino acid sequence of antibody H2F5-Bs-1, SEQ ID NO.26: DVQLVESGGGSVQAGGSLRLSCAASGDLYQ SEYLGWFRQAPGKEREGVAAYDLHRSVKYYADSVKGRFTVSLDNAENTVYLQMNSLKPEDTALYYCAAYSTMYGGSASYVVNYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0220] Heavy chain amino acid sequence of antibody H2F5-Bs-2, SEQ ID NO.27: EVQLVESGGGLVQPGGSLRLSCAASGFNIKD TYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSDVQLVESGGGSVQAGGSLRLSCAASGDLYQSEYLGWFRQAPGKEREGVAAYDLHRSVKYYADSVKGRFTVSLDNAENTVYLQMNSLKPEDTALYYCAAYSTMYGGSASYVVNYWGQGTQVTVSS

[0221] Heavy chain amino acid sequence of antibody H2F5-Bs-3, SEQ ID NO.28: DVQLVESGGGSVQAGGSLRLSCAASGDLYQ SEYLGWFRQAPGKEREGVAAYDLHRSVKYYADSVKGRFTVSLDNAENTVYLQMNSLKPEDTALYYCAAYSTMYGGSASYVVNYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0222] Heavy chain amino acid sequence of antibody H2F5-Bs-4, SEQ ID NO.29: EVQLVESGGGLVQPGGSLRLSCAASGFTFTD YTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSDVQLVESGGGSVQAGGSLRLSCAASGDLYQSEYLGWFRQAPGKEREGVAAYDLHRSVKYYADSVKGRFTVSLDNAENTVYLQMNSLKPEDTALYYCAAYSTMYGGSASYVVNYWGQGTQVTVSS

[0223] Light chain amino acid sequence of antibody H2F5-Bs-5, SEQ ID NO.30: DVQLVESGGGSVQAGGSLRLSCAASGDLYQ SEYLGWFRQAPGKEREGVAAYDLHRSVKYYADSVKGRFTVSLDNAENTVYLQMNSLKPEDTALYYCAA YSTMYGGSASYVVNYWGQGTQVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0224] Light chain amino acid sequence of antibody H2F5-Bs-6, SEQ ID NO.31: DIQMTQSPSSLSASVGDRVTITCRASQDVNTA VAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSDVQLVESGGGSVQAGGSLRLSCAASGDLYQSEYLGWFRQAPGKEREGVAAYDLHRSVKYYADSVKGRFTVSLDNAENTVYLQMNSLKPEDTALYYCAAYSTMYGGSASYVVNYWGQGTQVTVSS

[0225] Light chain amino acid sequence of antibody H2F5-Bs-7, SEQ ID NO.32: DVQLVESGGGSVQAGGSLRLSCAASGDLYQ SEYLGWFRQAPGKEREGVAAYDLHRSVKYYADSVKGRFTVSLDNAENTVYLQMNSLKPEDTALYYCAAYSTMYGGSASYVVNYWGQGTQVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0226] Light chain amino acid sequence of antibody H2F5-Bs-8, SEQ ID NO.33: DIQMTQSPSSLSASVGDRVTITCKASQDVSIG VAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSDVQLVESGGGSVQAGGSLRLSCAASGDLYQSEYLGWFRQAPGKEREGVAAYDLHRSVKYYADSVKGRFTVSLDNAENTVYLQMNSLKPEDTALYYCAAYSTMYGGSASYVVNYWGQGTQVTVSS

[0227] Heavy chain amino acid sequence of antibody G1E4-Bs-1, SEQ ID NO.34: DVQLVESGGGSVQAGGSLRLSCAASGFRISQ GYLGWFRQAPGKEREGVAAVSPAGGTPYYADSVKGRFTVSLDNAENTVYLQMNSLKPEDTALYYCAATFIHDFHGRQVYQPNYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0228] Heavy chain amino acid sequence of antibody G1E4-Bs-2, SEQ ID NO.35: EVQLVESGGGLVQPGGSLRLSCAASGFNIKD TYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSDVQLVESGGGSVQAGGSLRLSCAASGFRISQGYLGWFRQAPGKEREGVAAVSPAGGTPYYADSVKGRFTVSLDNAENTVYLQMNSLKPEDTALYYCAATFIHDFHGRQVYQPNYWGQGTQVTVSS

[0229] The amino acid sequence of the heavy chain of antibody G1E4-Bs-3, SEQ ID NO.36: DVQLVESGGGSVQAGGSLRLSCAASGFRISQ GYLGWFRQAPGKEREGVAAVSPAGGTPYYADSVKGRFTVSLDNAENTVYLQMNSLKPEDTALYYCAATFIHDFHGRQVYQPNYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFT FTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0230] Heavy chain amino acid sequence of antibody G1E4-Bs-4, SEQ ID NO.37: EVQLVESGGGLVQPGGSLRLSCAASGFTFTD YTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSDVQLVESGGGSVQAGGSLRLSCAASGFRISQGYLGWFRQAPGKEREGVAAVSPAGGTPYYADSVKGRFTVSLDNAENTVYLQMNSLKPEDTALYYCAATFIHDFHGRQVYQPNYWGQGTQVTVSS

[0231] Light chain amino acid sequence of antibody G1E4-Bs-5, SEQ ID NO.38: DVQLVESGGGSVQAGGSLRLSCAASGFRISQ GYLGWFRQAPGKEREGVAAVSPAGGTPYYADSVKGRFTVSLDNAENTVYLQMNSLKPEDTALYYCAATFIHDFHGRQVYQPNYWGQGTQVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0232] Light chain amino acid sequence of antibody G1E4-Bs-6, SEQ ID NO.39: DIQMTQSPSSLSASVGDRVTITCRASQDVNTA VAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSDVQLVESGGGSVQAGGSLRLSCAASGFRISQGYLGWFRQAPGKEREGVAAVSPAGGTPYYADSVKGRFTVSLDNAENTVYLQMNSLKPEDTALYYCAATFIHDFHGRQVYQPNYWGQGTQVTVSS

[0233] Light chain amino acid sequence of antibody G1E4-Bs-7, SEQ ID NO.40: DVQLVESGGGSVQAGGSLRLSCAASGFRISQ GYLGWFRQAPGKEREGVAAVSPAGGTPYYADSVKGRFTVSLDNAENTVYLQMNSLKPEDTALYYCAATFIHDFHGRQVYQPNYWGQGTQVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0234] Light chain amino acid sequence of antibody G1E4-Bs-8, SEQ ID NO.41: DIQMTQSPSSLSASVGDRVTITCKASQDVSIG VAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSDVQLVESGGGSVQAGGSLRLSCAASGFRISQGYLGWFRQAPGKEREGVAAVSPAGGTPYYADSVKGRFTVSLDNAENTVYLQMNSLKPEDTALYYCAATFIHDFHGRQVYQPNYWGQGTQVTVSS

[0235]

[0236] Nucleotide sequence of the light chain of trastuzumab, SEQ ID NO.43: GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCC GCCTCTGTGGGCGATAGGGTCACCATCACCTGCCGTGCCAGTCAGGATGTGAACACCGCGGTAGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATTTACAGCGCGAGCTTTCTGTATAGCGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAACATTATACCACCCCGCCGACGTTCGGACAGGGTACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAAAAACATAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0237]

[0238] The nucleotide sequence of the light chain of pertuzumab, SEQ ID NO.45: GACATTCAGATGACACAGAGCCCTAGCAGCCTGAGC GCTAGCGTGGGCGACAGAGTGACCATCACCTGCAAGGCTAGCCAAGACGTGAGCATCGGCGTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACAGCGCTAGCTACAGATACACCGGCGTGCCTAGCAGATTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGTCAGCAGTACTACATCTACCCCTACACCTTCGGCCAAGGCACCAAGGTGGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAAAAACATAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0239] Nucleotide sequence of anti-HER2 nanobody A2G5, SEQ ID NO.46: GATGTTCAGCTGGTTGAAAGCGGTGGC GGTAGTGTGCAAGCCGGTGGTAGTCTGCGTCTGAGCTGTGCAGCAAGTGGAATTAGTAATGGCCTGAGCCACCTAGGCTGGTTTCGTCAAGCCCCGGGCAAAGAACGCGAAGGAGTTGCTGCATATTATTATGATTATAGCACCTTCTACTACGCCGATAGCGTGAAAGGCCGCTTCACCGTTAGTCTGGATAACGCGGAGAATACGGTGTATCTGCAGATGAACAGTCTGAAACCGGAAGACACCGCGCTGTACTATTGTGCGGCCACCCCGGCGTATGTTATTGGCCGTACCGATCATGGTCTGAACTACTGGGGCCAAGGCACGCAAGTTACCGTTAGCAGC

[0240] Nucleotide sequence of anti-HER2 nanobody A9B5, SEQ ID NO.47: GATGTTCAGCTGGTTGAAAGCGGTGGC GGTAGTGTGCAAGCCGGTGGTAGTCTGCGTCTGAGCTGTGCAGCAAGTGGAGTTTTTCGTAGCTATTATACCCTAGGCTGGTTTCGTCAAGCCCCGGGCAAAGAACGCGAAGGAGTTGCTGCAGTTAGTCCAGCCGGTGGTACCCCATACTACGCCGATAGCGTGAAAGGCCGCTTCACCGTTAGTCTGGATAACGCGGAGAATACGGTGTATCTGCAGATGAACAGTCTGAAACCGGAAGACACCGCGCTGTACTATTGTGCGGCCGGAGGTAATGAATATGTTGGTGATACCGTTCGAGTAGCAAACTACTGGGGCCAAGGCACGCAAGTTACCGTTAGCAGC

[0241] Nucleotide sequence of anti-HER2 nanobody H2F5, SEQ ID NO.48: GATGTTCAGCTGGTTGAAAGCGGTGGC GGTAGTGTGCAAGCCGGTGGTAGTCTGCGTCTGAGCTGTGCAGCAAGTGGAGATCTGTATCAGAGCGAATATCTAGGCTGGTTTCGTCAAGCCCCGGGCAAAGAACGCGAAGGAGTTGCTGCATATGATTTACATCGTAGTGTTAAATACTACGCCGATAGCGTGAAAGGCCGCTTCACCGTTAGTCTGGATAACGCGGAGAATACGGTGTATCTGCAGATGAACAGTCTGAAACCGGAAGACACCGCGCTGTACTATTGTGCGGCCTATAGCACCATGTATGGTGGTTCAGCCAGCTATGTAGTAAACTACTGGGGCCAAGGCACGCAAGTTACCGTTAGCAGC

[0242] Nucleotide sequence of anti-HER2 nanobody G1E4, SEQ ID NO.49: GATGTTCAGCTGGTTGAAAGCGGTGGC GGTAGTGTGCAAGCCGGTGGTAGTCTGCGTCTGAGCTGTGCAGCAAGTGGATTTCGTATAAGCCAGGGTTATCTAGGCTGGTTTCGTCAAGCCCCGGGCAAAGAACGCGAAGGAGTTGCTGCAGTTAGTCCAGCCGGTGGTACCCCATACTACGCCGATAGCGTGAAAGGCCGCTTCACCGTTAGTCTGGATAACGCGGAGAATACGGTGTATCTGCAGATGAACAGTCTGAAACCGGAAGACACCGCGCTGTACTATTGTGCGGCCACCTTTATTCATGACTTTCATGGTCGACAGGTTTATCAGCCGAACTACTGGGGCCAAGGCACGCAAGTTACCGTTAGCAGC

[0243] Nucleotide sequence of flexible peptide segment, SEQ ID NO.50: GGTGGAGGCGGTAGTGGAGGCGGTGGTTCAGGCGGA GGCGGATCT

[0244]

[0245]

[0246]

[0247]

[0248] The light chain nucleotide sequence of antibody A9B5-Bs-5, SEQ ID NO. 55: GATGTTCAGCTGGTTGAAAGCGGTGGCGG TAGTGTGCAAGCCGGTGGTAGTCTGCGTCTGAGCTGTGCAGCAAGTGGAGTTTTTCGTAGCTATTATACCCTAGGCTGGTTTCGTCAAGCCCCGGGCAAAGAACGCGAAGGAGTTGCTGCAGTTAGTCCAGCCGGTGGTACCCCATACTACGCCGATAGCGTGAAAGGCCGCTTCACCGTTAGTCTGGATAACGCGGAGAATACGGTGTATCTGCAGATGAACAGTCTGAAACCGGAAGACACCGCGCTGTACTATTGTGCGGCCGGAGGTAATGAATATGTTGGTGATACCGTTCGAGTAGCAAACTACTGGGGCCAAGGCACGCAAGTTACCGTTAGCAGCGGTGGAGGCGGTAGTGGAGGCGGTGGTTCAGGCGGAGGCGGATCTGATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACCATCACCTGCCGTGCCAGTCAGGATGTGAACACCGCGGTAGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATTTACAGCGCGAGCTTTCTGTATAGCGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAACATTATACCACCCCGCCGACGTTCGGACAGGGTACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAAAAACATAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0249]

[0250] Light chain nucleotide sequence of antibody A9B5 - Bs - 7, SEQ ID NO.57: GATGTTCAGCTGGTTGAAAGCGGTGGCGG TAGTGTGCAAGCCGGTGGTAGTCTGCGTCTGAGCTGTGCAGCAAGTGGAGTTTTTCGTAGCTATTATACCCTAGGCTGGTTTCGTCAAGCCCCGGGCAAAGAACGCGAAGGAGTTGCTGCAGTTAGTCCAGCCGGTGGTACCCCATACTACGCCGATAGCGTGAAAGGCCGCTTCACCGTTAGTCTGGATAACGCGGAGAATACGGTGTATCTGCAGATGAACAGTCTGAAACCGGAAGACACCGCGCTGTACTATTGTGCGGCCGGAGGTAATGAATATGTTGGTGATACCGTTCGAGTAGCAAACTACTGGGGCCAAGGCACGCAAGTTACCGTTAGCAGCGGTGGAGGCGGTAGTGGAGGCGGTGGTTCAGGCGGAGGCGGATCTGACATTCAGATGACACAGAGCCCTAGCAGCCTGAGCGCTAGCGTGGGCGACAGAGTGACCATCACCTGCAAGGCTAGCCAAGACGTGAGCATCGGCGTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACAGCGCTAGCTACAGATACACCGGCGTGCCTAGCAGATTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGTCAGCAGTACTACATCTACCCCTACACCTTCGGCCAAGGCACCAAGGTGGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAAAAACATAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0251] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. An antibody that binds to HER2, comprising a first antigen-binding portion and a second antigen-binding portion fused together, the first antigen-binding portion comprising an anti-HER2 nanobody, the sequence of the anti-HER2 nanobody comprising an amino acid sequence as shown in any one of SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8, and the second antigen-binding portion comprising trastuzumab or pertuzumab.

2. The antibody according to claim 1, characterized in that, The heavy chain of the trastuzumab comprises the amino acid sequence shown in SEQ ID NO.1, the light chain of the trastuzumab comprises the amino acid sequence shown in SEQ ID NO.2, the heavy chain of the pertuzumab comprises the amino acid sequence shown in SEQ ID NO.3, and the light chain of the pertuzumab comprises the amino acid sequence shown in SEQ ID NO.

4.

3. The antibody according to claim 2, characterized in that, The first antigen-binding portion and the second antigen-binding portion of the antibody are fused by a flexible peptide segment; preferably, the flexible peptide segment has 5 to 15 amino acid residues; more preferably, the amino acid residues are serine residues and / or glycine residues; even more preferably, the sequence of the flexible peptide segment includes the amino acid sequence shown in SEQ ID NO. 9; Preferably, the antibody has any of the following characteristics: 1) The anti-HER2 nanobody is fused to the N-terminus of the heavy chain of the trastuzumab; 2) The anti-HER2 nanobody is fused to the C-terminus of the heavy chain of the trastuzumab; 3) The anti-HER2 nanobody is fused to the N-terminus of the heavy chain of the pertuzumab; 4) The anti-HER2 nanobody is fused to the C-terminus of the heavy chain of the pertuzumab; 5) The anti-HER2 nanobody is fused to the N-terminus of the light chain of the trastuzumab; 6) The anti-HER2 nanobody is fused to the C-terminus of the light chain of the trastuzumab; 7) The anti-HER2 nanobody is fused to the N-terminus of the light chain of the pertuzumab; 8) The anti-HER2 nanobody is fused to the C-terminus of the light chain of the pertuzumab.

4. The antibody according to claim 3, characterized in that, The antibody has any of the following characteristics: a1) The anti-HER2 nanobody is fused to the N-terminus of the heavy chain of trastuzumab as the heavy chain of the antibody, and the light chain of trastuzumab is used as the light chain of the antibody. The sequence of the heavy chain of the antibody is shown in SEQ ID NO.10, and the sequence of the light chain of the antibody is shown in SEQ ID NO.

2. a2) The anti-HER2 nanobody is fused to the C-terminus of the heavy chain of trastuzumab as the heavy chain of the antibody, and the light chain of trastuzumab is used as the light chain of the antibody. The sequence of the heavy chain of the antibody is shown in SEQ ID NO.11, and the sequence of the light chain of the antibody is shown in SEQ ID NO.

2. a3) The anti-HER2 nanobody is fused to the N-terminus of the heavy chain of the pertuzumab as the heavy chain of the antibody, and the light chain of the pertuzumab is used as the light chain of the antibody. The sequence of the heavy chain of the antibody is shown in SEQ ID NO.12, and the sequence of the light chain of the antibody is shown in SEQ ID NO.

4. a4) The anti-HER2 nanobody is fused to the C-terminus of the heavy chain of the pertuzumab as the heavy chain of the antibody, and the light chain of the pertuzumab is used as the light chain of the antibody. The sequence of the heavy chain of the antibody is shown in SEQ ID NO.13, and the sequence of the light chain of the antibody is shown in SEQ ID NO.

4. a5) The anti-HER2 nanobody is fused to the N-terminus of the light chain of trastuzumab as the light chain of the antibody, and the heavy chain of trastuzumab is used as the heavy chain of the antibody. The sequence of the heavy chain of the antibody is shown in SEQ ID NO.1, and the sequence of the light chain of the antibody is shown in SEQ ID NO.

14. a6) The anti-HER2 nanobody is fused to the C-terminus of the light chain of the trastuzumab as the light chain of the antibody, and the heavy chain of the trastuzumab is used as the heavy chain of the antibody. The sequence of the heavy chain of the antibody is shown in SEQ ID NO.1, and the sequence of the light chain of the antibody is shown in SEQ ID NO.

15. a7) The anti-HER2 nanobody is fused to the N-terminus of the light chain of the pertuzumab as the light chain of the antibody, and the heavy chain of the pertuzumab is used as the heavy chain of the antibody. The sequence of the heavy chain of the antibody is shown in SEQ ID NO.3, and the sequence of the light chain of the antibody is shown in SEQ ID NO.

16. a8) The anti-HER2 nanobody is fused to the C-terminus of the light chain of the pertuzumab as the light chain of the antibody, and the heavy chain of the pertuzumab is used as the heavy chain of the antibody. The sequence of the heavy chain of the antibody is shown in SEQ ID NO.3, and the sequence of the light chain of the antibody is shown in SEQ ID NO.

17. b1) The anti-HER2 nanobody is fused to the N-terminus of the heavy chain of trastuzumab as the heavy chain of the antibody, and the light chain of trastuzumab is used as the light chain of the antibody. The sequence of the heavy chain of the antibody is shown in SEQ ID NO.18, and the light chain of the antibody includes the sequence shown in SEQ ID NO.

2. b2) The anti-HER2 nanobody is fused to the C-terminus of the heavy chain of trastuzumab as the heavy chain of the antibody, and the light chain of trastuzumab is used as the light chain of the antibody. The sequence of the heavy chain of the antibody is shown in SEQ ID NO.19, and the light chain of the antibody includes the sequence shown in SEQ ID NO.

2. b3) The anti-HER2 nanobody is fused to the N-terminus of the heavy chain of the pertuzumab as the heavy chain of the antibody, and the light chain of the pertuzumab is used as the light chain of the antibody. The sequence of the heavy chain of the antibody is shown in SEQ ID NO.20, and the sequence of the light chain of the antibody is shown in SEQ ID NO.

4. b4) The anti-HER2 nanobody is fused to the C-terminus of the heavy chain of the pertuzumab as the heavy chain of the antibody, and the light chain of the pertuzumab is used as the light chain of the antibody. The sequence of the heavy chain of the antibody is shown in SEQ ID NO.21, and the sequence of the light chain of the antibody is shown in SEQ ID NO.

4. b5) The anti-HER2 nanobody is fused to the N-terminus of the light chain of the trastuzumab as the light chain of the antibody, and the heavy chain of the trastuzumab is used as the heavy chain of the antibody. The sequence of the heavy chain of the antibody is shown in SEQ ID NO.1, and the sequence of the light chain of the antibody is shown in SEQ ID NO.

22. b6) The anti-HER2 nanobody is fused to the C-terminus of the light chain of the trastuzumab as the light chain of the antibody, and the heavy chain of the trastuzumab is used as the heavy chain of the antibody. The sequence of the heavy chain of the antibody is shown in SEQ ID NO.1, and the sequence of the light chain of the antibody is shown in SEQ ID NO.

23. b7) The anti-HER2 nanobody is fused to the N-terminus of the light chain of the pertuzumab as the light chain of the antibody, and the heavy chain of the pertuzumab is used as the heavy chain of the antibody. The sequence of the heavy chain of the antibody is shown in SEQ ID NO.3, and the sequence of the light chain of the antibody is shown in SEQ ID NO.

24. b8) The anti-HER2 nanobody is fused to the C-terminus of the light chain of the pertuzumab as the light chain of the antibody, and the heavy chain of the pertuzumab is used as the heavy chain of the antibody. The sequence of the heavy chain of the antibody is shown in SEQ ID NO.3, and the sequence of the light chain of the antibody is shown in SEQ ID NO.

25. c1) The anti-HER2 nanobody is fused to the N-terminus of the heavy chain of trastuzumab as the heavy chain of the antibody, and the light chain of trastuzumab is used as the light chain of the antibody. The sequence of the heavy chain of the antibody is shown in SEQ ID NO.26, and the sequence of the light chain of the antibody is shown in SEQ ID NO.

2. c2) The anti-HER2 nanobody is fused to the N-terminus of the heavy chain of the pertuzumab as the heavy chain of the antibody, and the light chain of the pertuzumab is used as the light chain of the antibody. The sequence of the heavy chain of the antibody is shown in SEQ ID NO.28, and the sequence of the light chain of the antibody is shown in SEQ ID NO.

4. c3) The anti-HER2 nanobody is fused to the C-terminus of the heavy chain of the pertuzumab as the heavy chain of the antibody, and the light chain of the pertuzumab is used as the light chain of the antibody. The sequence of the heavy chain of the antibody is shown in SEQ ID NO.29, and the sequence of the light chain of the antibody is shown in SEQ ID NO.

4. c4) The anti-HER2 nanobody is fused to the N-terminus of the light chain of the pertuzumab as the light chain of the antibody, and the heavy chain of the pertuzumab is used as the heavy chain of the antibody. The sequence of the heavy chain of the antibody is shown in SEQ ID NO.3, and the sequence of the light chain of the antibody is shown in SEQ ID NO.

32. d1) The anti-HER2 nanobody is fused to the C-terminus of the heavy chain of trastuzumab as the heavy chain of the antibody, and the light chain of trastuzumab is used as the light chain of the antibody. The sequence of the heavy chain of the antibody is shown in SEQ ID NO.35, and the sequence of the light chain of the antibody is shown in SEQ ID NO.

2. d2) The anti-HER2 nanobody is fused to the C-terminus of the heavy chain of the pertuzumab as the heavy chain of the antibody, and the light chain of the pertuzumab is used as the light chain of the antibody. The sequence of the heavy chain of the antibody is shown in SEQ ID NO.37, and the sequence of the light chain of the antibody is shown in SEQ ID NO.

4. d3) The anti-HER2 nanobody is fused to the N-terminus of the light chain of the pertuzumab as the light chain of the antibody, and the heavy chain of the pertuzumab is used as the heavy chain of the antibody. The sequence of the heavy chain of the antibody is shown in SEQ ID NO.3, and the sequence of the light chain of the antibody is shown in SEQ ID NO.

40. Preferably, the antibody is a bispecific antibody; More preferably, the bispecific antibody is selected from any one of the antibodies described in a1), a2), a3), a4), a5), a6), a7), a8), b1), b2), b3), b4), b5), b6), b7), b8), c1), d2), and d3); More preferably, the bispecific antibody is selected from any one of the antibodies described in a1), a5), a6), b1), b5), b6), and b7); More preferably, the bispecific antibody is selected from any one of the antibodies described in b5) and b7).

5. A chimeric antigen receptor comprising the antibody as described in any one of claims 1 to 4.

6. A polynucleotide encoding an antibody as described in any one of claims 1 to 4, or a chimeric antigen receptor as described in claim 5.

7. A vector comprising the polynucleotide as described in claim 6.

8. A host cell containing the vector as described in claim 7, or having the polynucleotide as described in claim 6 integrated into its genome.

9. An antibody-drug conjugate comprising the antibody and therapeutic agent as described in any one of claims 1 to 4.

10. A pharmaceutical composition comprising an effective amount of an antibody as claimed in any one of claims 1 to 4, or a chimeric antigen receptor as claimed in claim 5, or a polynucleotide as claimed in claim 6, or a carrier as claimed in claim 7, or a host cell as claimed in claim 8, or an antibody-drug conjugate as claimed in claim 9.

11. A kit for detecting HER2-related tumors, comprising an effective amount of the antibody and detection reagent as described in any one of claims 1 to 4; preferably, the detection reagent is selected from one or more of enzyme-linked immunosorbent assay (ELISA), cell immunofluorescence assay, flow cytometry assay, isotope tracer, contrast agent, magnetic nanoparticles, or imaging agent.

12. Use of the antibody as described in any one of claims 1 to 4, or the chimeric antigen receptor as described in claim 5, or the polynucleotide as described in claim 6, or the vector as described in claim 7, or the host cell as described in claim 8, or the antibody-drug conjugate as described in claim 9, or the pharmaceutical composition as described in claim 10, or the kit as described in claim 11, wherein the use is selected from any one or more of the following: 1) To prepare products for the prevention and / or treatment of HER2-related tumors; 2) Prepare products for diagnosing HER2-related tumors; 3) Prepare products that promote the internalization of HER2 receptors on the cell surface; Preferably, the HER2-related tumor is selected from HER2-positive tumors or HER2-low expression tumors; Preferably, the HER2-positive tumor is selected from ligand-dependent HER2-positive tumors and non-ligand-dependent HER2-positive tumors. Preferably, the HER2-related tumors are selected from gastric cancer, breast ductal carcinoma, breast cancer, gastroesophageal junction cancer, salivary gland cancer, ovarian cancer, endometrial cancer, cervical cancer, non-small cell lung cancer, bile duct cancer, pancreatic cancer, colon cancer, bladder cancer, and prostate cancer.

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