Anti-HER2 nano-antibody polymer, anti-HER2 nano-antibody fusion protein, and pharmaceutical composition and application of anti-HER2 nano-antibody polymer and anti-HER2 nano-antibody fusion protein
By developing anti-HER2 nanoantibodies multimers and fusion proteins, the problem of lack of dual (multi) specific nanoantibodies targeting HER2 in the prior art was solved, effectively inhibiting and diagnosis of HER2-positive tumors was achieved, and production was reduced cost.
Patent Information
- Application Number
- CN202310959359.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-08-01
- Publication Date
- 2025-02-14
AI Technical Summary
The prior art lacks bi(multiple) specific nano-antibody targeting HER2, and cannot effectively inhibit the growth of HER2-positive tumors.
Anti-HER2 nanoantibody multimer and anti-HER2 nanoantibody fusion proteins were developed to form a pharmaceutical composition with bi(multi) specific antibody characteristics by combining multiple anti-HER2 nanoantibody monomers and Fc fragments.
It has achieved efficient identification and binding of different epitopes of HER2 protein, effectively inhibited the proliferation of HER2-positive tumor cells, and reduced the production and use costs.
Smart Images
Figure CN119431597A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine, and specifically relates to anti-HER2 nano antibody polymers, anti-HER2 nano antibody fusion proteins, and pharmaceutical compositions and applications thereof. Background Art
[0002] Human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase located on the cell membrane surface and is a member of the epidermal growth factor receptor family. When overexpressed, HER2 heterodimerizes with other family members (EGFR, HER3, HER4), leading to autophosphorylation of tyrosine residues in the intracellular domain, initiating multiple intracellular signaling pathways leading to cell proliferation and tumorigenesis. A large number of studies have shown that about 20% of breast cancers have activation of related signaling pathways caused by HER2 overexpression; in addition, HER2 is also overexpressed in a variety of solid tumors, including 20% of gastric cancer and gastroesophageal junction cancer, 17%-44% of salivary gland cancer, 27% of ovarian cancer, 18-80% of uterine cancer, 21% of cervical cancer, 2% of non-small cell lung cancer, 20% of bile duct cancer, 26% of pancreatic cancer, 5% of colon cancer, 12.4% of bladder cancer and 10% of prostate cancer. According to the 2020 Global Cancer Statistics, there are approximately 2.26 million new cases of breast cancer and approximately 685,000 deaths each year, surpassing lung cancer to become the world's leading cancer. Based on this estimate, there are more than 400,000 cases of HER2-positive breast cancer alone. Together with other types of HER2-positive solid tumors, the total number of patients with HER2-positive tumors is huge, and the disease burden is heavy. Therefore, it is of great significance to continue to develop new drugs for HER2-positive tumors.
[0003] HER2 targeted therapy is an effective means of treating HER2-positive tumors. Trastuzumab and Pertuzumab, two monoclonal antibodies that have been on the market for many years, are still the cornerstone drugs of HER2 targeted therapy. Trastuzumab is the first humanized monoclonal antibody drug approved by the U.S. Food and Drug Administration (FDA) that targets HER2 for the treatment of HER2-positive breast cancer. It binds to the fourth part (ECDⅣ) of the HER2 extracellular domain (ECD) and interferes with the activation of the downstream phosphatidylinositol 3-kinase (PI3K) / protein kinase B (AKT) / mammalian target of rapamycin (mTOR) signaling pathway by blocking non-ligand-dependent HER2 heterodimerization, thereby effectively inhibiting the proliferation of HER2-positive cells. Trastuzumab can also activate antibody-dependent cell-mediated cytotoxicity (ADCC) to kill tumor cells. Pertuzumab specifically binds to the dimerization-related domain ECDⅡ of HER2 and interferes with the proliferation of HER2-positive tumors by inhibiting ligand-dependent HER2 heterodimerization. Because the combination of trastuzumab and pertuzumab has shown significantly better anti-tumor effects than single drugs in animal models and clinical trials, the combination of trastuzumab and pertuzumab has been approved by the FDA for the treatment of HER2-positive metastatic breast cancer.
[0004] Compared with traditional monoclonal antibodies, bispecific (multispecific) antibodies can specifically bind to two or more different antigens or epitopes at the same time, have better targeting and safety, achieve multi-target synergistic treatment on a single antibody molecule, and obtain more ideal therapeutic effects for complex diseases such as tumors, central nervous system diseases, and autoimmune diseases. Nanobodies, also known as single-domain antibodies (sdAbs), are a new type of antibody discovered in recent years. It is essentially a single heavy chain variable region (Variable domain of heavy chain of heavy-chain antibody, VHH) that specifically binds to antigens. One of the development directions is to construct bispecific (multispecific) antibodies based on nanobodies.
[0005] However, there is currently a lack of bi-(multi-)specific nanobodies targeting HER2 that can inhibit the growth of HER2-positive tumors in the art. Summary of the invention
[0006] In view of the shortcomings of the prior art described above, the object of the present invention is to provide anti-HER2 nanobody polymers, anti-HER2 nanobody fusion proteins and pharmaceutical compositions and uses thereof, so as to solve the problem in the prior art of lack of bi(multi)specific nanobodies targeting HER2 and capable of inhibiting the growth of HER2-positive tumors.
[0007] To achieve the above-mentioned purpose and other related purposes, the main purpose of the present invention is to provide an anti-HER2 nano antibody multimer, wherein the anti-HER2 nano antibody multimer comprises no less than two anti-HER2 nano antibody monomers, and the no less than two anti-HER2 nano antibody monomers recognize different epitopes of HER2. The anti-HER2 nano antibody multimer has the characteristics of a bi(multi) specific antibody, can recognize different epitopes of HER2 protein, and inhibit tumor cell proliferation.
[0008] Anti-HER2 nanoantibodies refer to antibodies that lack light chains and contain only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions. Their VHH structure can specifically bind to the HER2 antigen epitope and has structural stability and antigen-binding activity comparable to the original heavy chain antibody. It is the smallest unit known to bind to the target antigen.
[0009] Preferably, the at least two anti-HER2 nanobody monomers are A9F5 and H2F5, the amino acid sequence of the anti-HER2 nanobody monomer A9F5 is shown in SEQ ID NO.1, and the amino acid sequence of the anti-HER2 nanobody monomer H2F5 is shown in SEQ ID NO.2.
[0010] As certain embodiments of the present invention, the anti-HER2 nanobody monomer comprises a connecting peptide between A9F5 and H2F5. Preferably, the connecting peptide is a flexible peptide segment.
[0011] Preferably, the connecting peptide is as shown in SEQ ID NO. 3, 8, 9, 10, or 11. As certain embodiments of the present invention, the anti-HER2 nanobody monomer A9F5 is located at the N-terminus of the anti-HER2 nanobody multimer, or the anti-HER2 nanobody monomer H2F5 is located at the N-terminus of the anti-HER2 nanobody multimer.
[0012] The present invention also provides an anti-HER2 nano antibody fusion protein, which comprises the anti-HER2 nano antibody polymer and a crystallizable fragment Fc of human immunoglobulin gamma1 (IgG1).
[0013] Fc is composed of the second and third domains in the heavy chain constant region of the antibody. The Fc region can bind to Fc receptors on the surface of immune cells, thereby mediating a variety of immune responses, such as antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis, complement activation, etc.
[0014] As certain embodiments of the present invention, the anti-HER2 nanoantibody fusion protein is an anti-HER2 nanoantibody fusion protein AH-Fc, HA-Fc, H-Fc-A, or A-Fc-H; the amino acid sequence of the anti-HER2 nanoantibody fusion protein AH-Fc is shown in SEQ ID NO.4, the amino acid sequence of the anti-HER2 nanoantibody fusion protein HA-Fc is shown in SEQ ID NO.5, the amino acid sequence of the anti-HER2 nanoantibody fusion protein H-Fc-A is shown in SEQ ID NO.6, and the amino acid sequence of the anti-HER2 nanoantibody fusion protein A-Fc-H is shown in SEQ ID NO.7.
[0015] The present invention also provides a construct, which comprises a nucleic acid encoding the anti-HER2 nanobody fusion protein.
[0016] Preferably, the construct is a construct of a pSCSTa vector.
[0017] The present invention also provides a host cell, wherein the host cell comprises the construct or the genome of the host cell is recombined with the nucleic acid encoding the anti-HER2 nanobody fusion protein.
[0018] Preferably, the host cell is Expi 293 cell.
[0019] The present invention also provides a pharmaceutical composition, which comprises an effective amount of the anti-HER2 nanobody fusion protein AH-Fc, HA-Fc, H-Fc-A, or A-Fc-H, and a pharmaceutically acceptable carrier, excipient, and / or stabilizer.
[0020] Preferably, the stabilizer comprises a hydroxy compound, a sugar, an amino acid, a polymer, and / or a protein.
[0021] The present invention also provides a pharmaceutical composition, which comprises an effective amount of the anti-HER2 nanoantibody fusion protein AH-Fc and an effective amount of trastuzumab; or an effective amount of the anti-HER2 nanoantibody fusion protein AH-Fc and an effective amount of pertuzumab.
[0022] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, and / or stabilizer.
[0023] Preferably, the stabilizer comprises a hydroxy compound, a sugar, an amino acid, a polymer, and / or a protein.
[0024] The present invention also provides a diagnostic reagent, which comprises an effective amount of the anti-HER2 nanobody polymer or an effective amount of the anti-HER2 nanobody fusion protein.
[0025] The present invention also provides the use of the anti-HER2 nano antibody polymer, the anti-HER2 nano antibody fusion protein, the expression vector, the host cell, and the pharmaceutical composition in the preparation of drugs for diagnosing or targeting HER2-positive tumors.
[0026] Preferably, the HER2-positive tumors to be treated include gastric cancer and gastroesophageal junction cancer, salivary gland cancer, ovarian cancer, uterine cancer, cervical cancer, non-small cell lung cancer, bile duct cancer, pancreatic cancer, colon cancer, bladder cancer, prostate cancer and breast cancer.
[0027] As described above, the anti-HER2 nanobody multimer, anti-HER2 nanobody fusion protein, and pharmaceutical composition and use thereof of the present invention have the following beneficial effects:
[0028] 1. The anti-HER2 nanobody multimer and anti-HER2 nanobody fusion protein described in the present invention are easily expressed in a eukaryotic system, have a simple purification method, and have good uniformity and high purity, thereby reducing production and use costs.
[0029] 2. The anti-HER2 nanoantibody polymers described in the present invention can specifically recognize different epitopes of the HER2 protein and can be used for the diagnosis and treatment of HER2-positive tumors.
[0030] 3. The anti-HER2 nanobody multimer and anti-HER2 nanobody fusion protein described in the present invention can effectively promote the degradation of HER2 receptors and can be used to prepare therapeutic drugs for HER2-positive tumors.
[0031] 4. The anti-HER2 nanobody multimer and anti-HER2 nanobody fusion protein described in the present invention can effectively inhibit the proliferation of HER2-positive tumor cells and can be used to prepare therapeutic drugs for HER2-positive tumors.
[0032] 5. The anti-HER2 nanobody polymer and anti-HER2 nanobody fusion protein described in the present invention can effectively inhibit the drug resistance pathway of HER2-positive tumor cells and provide candidate drugs for HER2-positive drug-resistant tumors. BRIEF DESCRIPTION OF THE DRAWINGS
[0033] Figure 1Shown is the electrophoresis result of the purity of fusion proteins AH-Fc and HA-Fc determined by SDS-PAGE electrophoresis in Example 1 of the present invention;
[0034] Figure 2 Shown are the AH-Fc (2A) and HA-Fc (2B) spectra of fusion protein detected by high performance liquid chromatography in Example 2 of the present invention;
[0035] Figure 3 It shows that the protein imprint detection of anti-HER2 nano antibody fusion protein promotes HER2 receptor degradation in Example 3 of the present invention;
[0036] Figure 4 Shown is the inhibition of HER2 positive tumor cell proliferation by the anti-HER2 nanobody fusion proteins AH-Fc and HA-Fc in Example 4 of the present invention, and the combined use of trastuzumab and pertuzumab in the control group (T+P);
[0037] Figure 5 It shows the inhibition of HER2 positive tumor cell proliferation by the anti-HER2 nano antibody fusion proteins AH-Fc and HA-Fc, and the combined use of trastuzumab and pertuzumab in the control group (T+P) under the stimulation of the ligands HRG and EGF in Example 4 of the present invention;
[0038] Figure 6 It shows the inhibition of HER2 positive tumor cell proliferation by the anti-HER2 nano antibody fusion protein AH-Fc combined with trastuzumab (AH-Fc+T), or combined with pertuzumab (AH-Fc+P) and the control group trastuzumab and pertuzumab combined with pertuzumab (T+P) under the stimulation of ligands HRG and EGF in Example 4 of the present invention;
[0039] Figure 7 Shown is the internalization activity of the anti-HER2 nanobody fusion protein of Example 5 of the present invention binding to cell surface HER2, (AH-Fc) or the internalization rate mediated by combined use with trastuzumab (AH-Fc+T). DETAILED DESCRIPTION
[0040] The present invention is further described below in conjunction with specific embodiments, and it should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention. The embodiments of the present invention are described by specific specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied by other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.
[0041] It should also be understood that the terms used in the examples of the present invention are intended to describe specific embodiments rather than to limit the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms. When the examples give numerical ranges, it should be understood that unless otherwise specified in the present invention, both endpoints of each numerical range and any value between the two endpoints can be selected.
[0042] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, any methods, equipment, and materials of the prior art that are similar or equivalent to the methods, equipment, and materials in the embodiments of the present invention may be used to implement the present invention, based on the prior art mastery of those skilled in the art and the description of the present invention.
[0043] In addition, unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the field of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. These techniques are well described in the literature, see Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; these series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATINSTRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, Chromatin (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 304, Chromatin (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999. BIOLOGY, Vol. 119, Chromatin Protocols (PB Becker, ed.) Humana Press, Totowa, 1999, etc.
[0044] Example 1 Preparation of anti-HER2 nanobody fusion protein
[0045] The anti-HER2 nano antibody fusion protein of this embodiment comprises an anti-HER2 nano antibody polymer and Fc. The anti-HER2 nano antibody polymer consists of two nano antibody monomers and a connecting peptide. The two nano antibody monomers are A9F5 and H2F5 (hereinafter referred to as A and H, respectively) that recognize different epitopes of HER2, A9F5 recognizes amino acids 23 to 217 of hHER2 (ECD), and H2F5 recognizes amino acids 218 to 342 of hHER2 (ECD). Therefore, the anti-HER2 nano antibody fusion protein has two forms, AH-Fc and HA-Fc. The amino acid sequence of AH-Fc is shown in SEQ ID NO.4, and the amino acid sequence of HA-Fc is shown in SEQ ID NO.5. Among them, the heavy chain variable region (crystallizable fragment) of Fc human immunoglobulin gamma1 (IgG1), the same below.
[0046] After the DNA sequence of the anti-HER2 nanobody fusion protein was synthesized, it was cloned into the pSCSTa vector. The plasmid was amplified in the DH5α competent state, and the plasmid was extracted using the AXYGEN endotoxin-free plasmid medium extraction kit (Corning Life Sciences, AP-MD-EP-25). After the plasmid and the transfection reagent PEI were mixed evenly in a ratio of 1:3, they were kept in the dark for 20 minutes, and then added dropwise to the suspended cultured Expi 293 cells to complete the transfection. 16 hours after transfection, 5% feed was added to the cells, and the cells were continued to be cultured in a carbon dioxide shaking incubator for 3-4 days. The Expi 293 cell supernatant was collected using a centrifuge, and the above fusion protein was purified using a protein purification device (Cytiva, AKTA Pure 25) and a ProteinA affinity chromatography column (Borgron Biotechnology, AT ProteinA Diamond Plus). The purified protein was replaced with PBS buffer using an ultrafiltration concentrator (PALL, 3k), and the protein concentration was determined using a BCA kit (Yazyme, ZJ101). The purity of the fusion protein was then determined by SDS-PAGE to be more than 95%. The electrophoresis results are shown in Figure 1 .
[0047] Example 2 Homogeneity determination of anti-HER2 nanobody fusion protein
[0048] The retention time of the fusion protein was detected by high performance liquid chromatography (Waters, ARC), the buffer was PBS, the flow rate was 1 mL / min, and the sampling time was 30 min. The retention time of AH-Fc was 7.682 min (the high performance liquid chromatography is shown in Figure 2 A), the retention time of HA-Fc is 7.750 min (its HPLC chromatogram is shown in Figure 2 B), their retention times are basically the same, and the peak shape is single, there are no aggregates of surface fusion proteins, and the uniformity is good.
[0049] Example 3 Anti-HER2 Nanobody Fusion Protein Promotes HER2 Receptor Degradation
[0050] NCI-N87 cells were plated in 6-well cell culture plates, with 10 6 cells. After overnight culture, the cells were incubated with 100 nM / L fusion protein at 37°C for 24 h. After washing with PBS, the cells were collected with a scraper, and then 200 μL NP-40 lysis buffer was added to lyse the cells. After centrifugation, the supernatant was removed, and the total protein of the cell lysate was quantified using a BCA kit (Ya enzyme, ZJ101) and then subjected to protein blotting detection. The control group was trastuzumab (abbreviated as T, which is a trastuzumab sample prepared by the inventor, and its amino acid sequence is consistent with HERCEPTIN), and an irrelevant IgG monoclonal antibody (abbreviated as IgG) was used as a blank control. The band results of Western blotting are shown in Figure 3 Above, Figure 3 The following figure shows Figure 3 The above figure shows the quantitative values of Western blotting band intensity. Figure 3 It can be seen that AH-Fc can significantly promote the degradation of HER2 receptor, and HA-Fc has little effect on the level of HER2 receptor protein.
[0051] Example 4 Inhibitory effect of anti-HER2 nanobody fusion protein on HER2-positive tumor cells
[0052] 4.1 CCK-8 assay to detect the inhibitory ability of antibodies on tumor cells
[0053] Trypsinize NCI-N87 cells, carefully blow the cell mass to make it a single cell, count the cells, and then plate them in a 96-well cell culture plate, 4000 cells per well. Grow overnight in a constant temperature incubator, discard the original culture medium the next day, add the corresponding concentration of anti-HER2 nano antibody fusion protein diluted in complete culture medium to each well, set up positive, negative and blank controls, and set two wells for each antibody and concentration. After 4 days, discard the culture medium containing the fusion protein, add 100 μL of pre-diluted 10% v / v CCK8 solution to each well, incubate in a constant temperature cell culture incubator for 1 hour, use an enzyme marker to detect the absorbance of each well at a wavelength of 450nm, and calculate the inhibition rate of the fusion protein on NCI-N87 cells.
[0054] The calculation formula is: Inhibition rate (%) = [(Ac-As) / (Ac-Ab)] × 100%. Where As represents the experimental well (culture medium containing cells, CCK-8, and the substance to be tested); Ac: control well (culture medium containing cells, CCK-8, and no substance to be tested); Ab: blank well (culture medium without cells and the substance to be tested, CCK-8). Graphpad Prism software was used to fit the inhibition rate curve and calculate the IC 50 The drug control group of this experiment was a combination of trastuzumab and pertuzumab (abbreviated as P, which is a pertuzumab sample prepared by the inventor and has the same amino acid sequence as PERJETA) (T+P).
[0055] The results show (see Figure 4 ), anti-HER2 nanoantibody fusion proteins AH-Fc and HA-Fc can inhibit the proliferation of HER2-positive tumor cells, and their IC 50 The values were 1.045nM / L and 7.564nM / L, respectively. Among them, the IC values of trastuzumab and pertuzumab combined (T+P) were compared. 50 The value was 2.021nM / L, and the inhibitory ability of AH-Fc was slightly better than that of T+P.
[0056] 4.2 Detection of the inhibitory ability of fusion protein on tumor cells under ligand stimulation
[0057] According to the method of 4.1, NCI-N87 cells after drug addition (respectively anti-HER2 nano antibody fusion protein AH-Fc, HA-Fc and control group trastuzumab and pertuzumab combined use (T+P)) were stimulated with HRG and EGF ligands respectively to detect whether the anti-HER2 nano antibody fusion protein of the present invention can still effectively inhibit tumor proliferation under the condition of continuous ligand stimulation. The control group was trastuzumab and pertuzumab combined use (T+P) and blank control antibody (IgG). The results show (see Figure 5 ), even under the condition of continuous ligand stimulation, AH-Fc and HA-Fc can effectively inhibit the proliferation of HER2-positive tumor cells, and the inhibitory ability is not weaker than the combination of trastuzumab and pertuzumab. Among them, under the stimulation of EGF ligand, the inhibitory ability of AH-Fc is significantly better than the combination of trastuzumab and pertuzumab.
[0058] 4.3 Detection of the inhibitory ability of fusion protein combined with trastuzumab or pertuzumab on tumor cells under ligand stimulation
[0059] According to the method of 4.1, the NCI-N87 cells after drug addition were stimulated with HRG and EGF ligands respectively to detect whether the anti-HER2 nano antibody fusion protein of the present invention combined with trastuzumab (AH-Fc+T) or pertuzumab (AH-Fc+P) can produce stronger inhibition ability under the condition of continuous ligand stimulation. The control group was trastuzumab and pertuzumab combined (T+P) and blank control antibody IgG. The inhibition of fusion protein is shown in Figure 6 , and the significant difference analysis is shown in Table 1. The results showed that under any treatment method, the inhibitory ability of AH-Fc+T and AH-Fc+P was significantly better than that of T+P; under any treatment method, the inhibitory ability of AH-Fc+T was better than that of AH-Fc+P. In general, AH-Fc+T has a better ability to inhibit tumors, indicating that the combined use of AH-Fc and T that recognize different HER2 epitopes produces a synergistic enhancement effect.
[0060] Table 1
[0061]
[0062]
[0063] Example 5 Internalization activity of anti-HER2 nanobody fusion protein binding to cell surface HER2
[0064] 5.1 Two HER2-highly expressing breast cancer cell lines, BT474 and JIMT-1 (resistant to trastuzumab), were used to detect the internalization activity mediated by the anti-HER2 nanobody fusion protein of the present invention alone (AH-Fc) or in combination with trastuzumab (AH-Fc+T).
[0065] 5.2 After digestion of BT474 and JIMT-1 cells, wash twice with pre-cooled FACS buffer and plate the cells in a U-shaped plate with 10 5 cells. The antibody to be tested was incubated with the cells on ice for 1 hour, and then washed 3 times with FACS buffer to remove non-specific binding antibodies. Each cell was divided into 2 groups: one group was placed on ice as the control group; the other group was incubated in an incubator at 37°C for different times (0.5h and 4h) as the experimental group. After incubation, the cells were washed twice with FACS buffer. 100μL of 2% paraformaldehyde solution was added to each well, incubated at 4°C for 20min, and washed 3 times with FACS buffer. 100μL of PE-labeled-anti-huFc (dilution 1:500) was added to each well, incubated at 4°C for 30min, washed 3 times with FACS buffer, and then the cells were resuspended and detected by flow cytometry.
[0066] 5.3 Internalization rate (%) = 100% × (MFI value change of experimental group) / (MFI value of control group after background subtraction). The results show ( Figure 7 ), whether it is the HER2 high-expressing cell line BT4T4 or the trastuzumab-resistant cell line JIMT-1, the anti-HER2 nanoantibody fusion protein AH-Fc of the present invention can be mediated internalization after binding to cell surface HER2, and the internalization rate after binding to the cell for 4 hours is significantly higher than that of trastuzumab (T), pertuzumab (P), and trastuzumab and pertuzumab combined use (T+P) (P values are all less than 0.0001); at the same time, AH-Fc or combined use with trastuzumab (AH-Fc+T) shows stronger internalization activity than AH-Fc.
[0067] The above is only a preferred embodiment of the present invention, and is not any formal or substantial limitation of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention. Any technician familiar with this profession, without departing from the spirit and scope of the present invention, can make some changes, modifications and evolutions of the technical content disclosed above, which are equivalent embodiments of the present invention; at the same time, any changes, modifications and evolutions of any equivalent changes made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.
Claims
1. An anti-HER2 nanobody polymer, characterized in that: The anti-HER2 nanoantibody multimer comprises at least two anti-HER2 nanoantibody monomers, and the at least two anti-HER2 nanoantibody monomers recognize different epitopes of HER2; preferably, the at least two anti-HER2 nanoantibody monomers are A9F5 and H2F5, and the amino acid sequence of the anti-HER2 nanoantibody monomer A9F5 is shown in SEQ ID NO.1, and the amino acid sequence of the anti-HER2 nanoantibody monomer H2F5 is shown in SEQ ID NO.
2.
2. The anti-HER2 nanobody multimer according to claim 1, characterized in that The anti-HER2 nano antibody monomers A9F5 and H2F5 contain a connecting peptide; preferably, the connecting peptide is a flexible peptide segment; more preferably, the connecting peptide is as shown in SEQ ID NO.
34.
3. The anti-HER2 nanobody multimer according to claim 1, characterized in that The anti-HER2 nano antibody monomer A9F5 is located at the N-terminus of the anti-HER2 nano antibody multimer, or the anti-HER2 nano antibody monomer H2F5 is located at the N-terminus of the anti-HER2 nano antibody multimer.
4. An anti-HER2 nanobody fusion protein, characterized in that: The anti-HER2 nanobody fusion protein comprises the anti-HER2 nanobody polymer according to any one of claims 1 to 3 and the crystallizable fragment Fc of human immunoglobulin gamma1 (IgG1).
5. The anti-HER2 nanobody fusion protein according to claim 4, characterized in that The anti-HER2 nanobody fusion protein is an anti-HER2 nanobody fusion protein AH-Fc, HA-Fc, H-Fc-A, or A-Fc-H; The amino acid sequence of the anti-HER2 nanoantibody fusion protein AH-Fc is shown in SEQ ID NO.4, the amino acid sequence of the anti-HER2 nanoantibody fusion protein HA-Fc is shown in SEQ ID NO.5, the amino acid sequence of the anti-HER2 nanoantibody fusion protein H-Fc-A is shown in SEQ ID NO.6, and the amino acid sequence of the anti-HER2 nanoantibody fusion protein A-Fc-H is shown in SEQ ID NO.
7.
6. A construct, characterized in that The construct comprises a nucleic acid encoding the anti-HER2 nanobody fusion protein according to any one of claims 4 to 5; preferably, the construct is a construct of a pSCSTa vector.
7. A host cell, characterized in that The host cell comprises the construct according to claim 6 or the genome of which is recombined with the nucleic acid encoding the anti-HER2 nanobody fusion protein according to claim 6; preferably, the host cell is an Expi 293 cell.
8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises an effective amount of the anti-HER2 nanobody fusion protein AH-Fc, HA-Fc, H-Fc-A, or A-Fc-H according to claim 5, and a pharmaceutically acceptable carrier, excipient, and / or stabilizer; Preferably, the stabilizer comprises a hydroxy compound, a sugar, an amino acid, a polymer, and / or a protein.
9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises an effective amount of the anti-HER2 nanobody fusion protein AH-Fc according to claim 5 and an effective amount of trastuzumab; or An effective amount of the anti-HER2 nanobody fusion protein AH-Fc according to claim 5 and an effective amount of Pertuzumab; preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, and / or stabilizer; Preferably, the stabilizer comprises a hydroxy compound, a sugar, an amino acid, a polymer, and / or a protein.
10. A diagnostic reagent, characterized in that The diagnostic reagent comprises an effective amount of the anti-HER2 nanobody multimer according to any one of claims 1 to 3, or an effective amount of the anti-HER2 nanobody fusion protein according to any one of claims 4 to 5.
11. Use of the anti-HER2 nanobody multimer according to claims 1 to 4, the anti-HER2 nanobody fusion protein according to any one of claims 4 to 5, the expression vector according to claim 6, the host cell according to claim 7, and the pharmaceutical composition according to any one of claims 8 to 9 in the preparation of a drug for diagnosis or targeted treatment of HER2-positive tumors; Preferably, the HER2-positive tumors to be treated include gastric cancer and gastroesophageal junction cancer, salivary gland cancer, ovarian cancer, uterine cancer, cervical cancer, non-small cell lung cancer, bile duct cancer, pancreatic cancer, colon cancer, bladder cancer, prostate cancer and breast cancer.