A nano monoclonal antibody targeting HER2 and its preparation method and application

Abstract

The present invention provides a HER2-targeting nanomonoclonal antibody IDM3-59 and a preparation method and application thereof, wherein the complementary determining region CDR of the nanomonoclonal antibody includes CDR1, CDR2 and CDR3; wherein the amino acid sequences of CDR1, CDR2 and CDR3 are GSIFSTYN, LTRDGVA and KTLPLTRDWGSAD, respectively. The nanomonoclonal antibody provided by the present invention has high specificity for human HER2 protein, strong affinity, and is easy to produce and has high stability, and is suitable for various cancer diagnosis and treatment studies targeting HER2.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a HER2-targeting nano monoclonal antibody IDM3-59 and a preparation method and application thereof. Background Art

[0002] Kinase (RTK), belongs to the epidermal growth factor receptor (EGFR / ErbB) family. The gene encoding HER2 is located on human chromosome 17q21, which is a proto-oncogene. The product is a 185kD transmembrane protein, referred to as p185, which is composed of 1255 amino acids and contains four extracellular domains and one transmembrane domain. HER2 protein mainly binds to its respective ligands by forming heterodimers with other members of the HER family (including EGFR (HERl / erbBI), HER3 / erbB3, HER4 / erbB4), thereby activating downstream signaling pathways. At present, no ligand that can directly bind to it has been found.

[0003] The signal transduction pathways mediated by HER2 protein mainly include Ras / Raf / MAPK / PI3K and Akt signaling pathways. After HER2 binds to the ligand, it activates the activity of tyrosine kinase mainly by causing receptor dimerization and autophosphorylation of the tyrosine kinase region in the cytoplasm; it has a special open structure and can activate itself without the participation of specific ligands, so it can form homologous / heterodimers with itself or other members of the HER family. In addition, HER2 is the preferred partner of HER family members' heterodimers, and the activity of the heterodimers formed with it is often stronger than that of other heterodimers.

[0004] HER2 is highly expressed in many cancers. Currently, patients with various types of cancer, such as breast cancer, gastric cancer, lung cancer, and endometrial cancer, will undergo HER2 expression level testing. The detection rate of HER2 high expression in breast cancer is about 15% to 25%, in gastric cancer is about 20%, in lung cancer is about 2.5%, and in endometrial cancer, the detection rate can reach 18% to 80%.

[0005] Overexpression of HER2 may lead to the sustained enhanced activation of receptor tyrosine kinases, thus causing a series of downstream cascade reactions, such as MAPK, PI3K-PKB / Akt, STAT, etc. Her2-mediated signaling pathways can also regulate the expression of tumor-related genes, thereby promoting tumor invasion and metastasis. Overexpression of Her2 plays an important role in the occurrence and development of certain invasive cancers, especially invasive breast cancer. Therefore, Her2 has become an important biological marker and therapeutic target for breast cancer.

[0006] The monoclonal antibodies trastuzumab (Herceptin), pertuzumab (Perjeta), lapatinib (Tykerb), neratinib (Nerlynx), and pyrotinib are antibody drugs that specifically target HER2 and are mainly used to treat patients with HER2-positive invasive or metastatic breast cancer. By targeting and binding to HER2, they inhibit the dimerization of HER2 with other HER family members, thereby inhibiting tumor growth. Lapatinib, neratinib, and pyrotinib can target and inhibit HER1 (EGFR / ErbB1) while targeting and inhibiting HER2. By reducing the autophosphorylation of EGFR and HER2, they inhibit the downstream MAPK and AKT signaling pathways, thereby inhibiting the proliferation of tumor cells that overexpress EGFR or HER2.

[0007] Most traditional antibodies can only be expressed in mammalian expression systems, the production process is complex and costly, the antibody stability is poor, the immunogenicity is high and the tissue penetration is low. These combined factors often lead to poor therapeutic effects of antibodies. Compared with traditional antibodies, nanobodies (also known as VHH single-domain antibodies) have many advantages. They can be expressed in mammalian expression systems, Escherichia coli and Kunzhong expression systems, and the antibodies produced are usually highly water-soluble and stable. Nanobodies are the smallest antibody structure pattern known to have complete antibody activity. Their molecular weight is usually 1 / 10 of that of conventional antibodies, which determines that they have high tissue penetration ability and a short half-life. They can easily penetrate the blood-brain barrier and have a fast serum clearance rate. Therefore, nanobodies overcome many shortcomings of traditional antibodies, which also makes them more suitable for carrying radioactive isotopes. Nanobodies can quickly and specifically penetrate tumor tissues to bind to targets, while non-binding nanobodies can be quickly removed from the blood and reduce the body's radiation dose. Compared with traditional antibodies, nanobodies have more advantages as tracers and targeted internal radiotherapy drugs.

[0008] However, there is currently a lack of effective nano-antibodies for HER2-targeted antibody drugs. Therefore, the development of suitable antibody drugs targeting HER2 has important research and application significance, and new and effective specific nano-body drugs targeting Her2 should be actively developed. Summary of the invention

[0009] In order to overcome the defects in the prior art, the present invention provides a HER2-targeting nano monoclonal antibody IDM3-59 and its preparation method and application. Nano antibodies overcome the shortcomings of traditional antibodies, and have the characteristics of low molecular weight, strong stability, good solubility, easy expression, low immunogenicity, strong permeability and targeting, low production cost and easy expression and purification. Therefore, nano antibodies targeting HER2 will undoubtedly have a wider range of applicability. Low immunogenicity and strong targeting mean that the same dose of antibodies will have a lower body burden and higher therapeutic effect; nano antibody tissue permeability indicates that HER2 nano antibodies may still be applicable to patients with advanced brain metastases of HER2-positive breast cancer or gastric cancer.

[0010] To achieve the above object, the present invention adopts the following technical solution:

[0011] The first aspect of the present invention is to provide a nano monoclonal antibody targeting HER2, wherein the complementary determining region CDR includes CDR1, CDR2 and CDR3; wherein the amino acid sequences of CDR1, CDR2 and CDR3 are GSIFSTYN, LTRDGVA and KTLPLTRDWGSAD, respectively.

[0012] Furthermore, the amino acid sequence of the above-mentioned nano monoclonal antibody is SEQ ID NO:4.

[0013] The second aspect of the present invention is to provide a chimeric antigen receptor, which includes the above-mentioned nano monoclonal antibody.

[0014] The third aspect of the present invention is a nucleic acid molecule encoding the above-mentioned nano monoclonal antibody or chimeric antigen receptor, and its nucleotide sequence is shown in SEQ ID NO:5.

[0015] The fourth aspect of the present invention is to provide a nucleic acid construct, which includes the above-mentioned nucleic acid molecule; preferably, the nucleic acid construct is a cloning vector, an expression vector or an integration vector.

[0016] The fifth aspect of the present invention is to provide a host cell, which expresses and / or secretes the above-mentioned nano monoclonal antibody or chimeric antigen receptor, or includes the above-mentioned nucleic acid molecule or nucleic acid construct.

[0017] The sixth aspect of the present invention is to provide a method for preparing the above-mentioned nano monoclonal antibody or chimeric antigen receptor, comprising: culturing the above-mentioned host cell under suitable conditions, and optionally purifying the nano monoclonal antibody or chimeric antigen receptor from the culture.

[0018] The seventh aspect of the present invention is to provide a pharmaceutical composition, which includes the above-mentioned nano monoclonal antibody, chimeric antigen receptor, nucleic acid molecule, nucleic acid construct, nucleic acid molecule, nucleic acid construct or host cell; preferably, it also includes a pharmaceutically acceptable carrier or excipient.

[0019] The eighth aspect of the present invention is to provide the use of the above-mentioned nano monoclonal antibodies, chimeric antigen receptors, nucleic acid molecules, nucleic acid constructs, nucleic acid molecules, nucleic acid constructs or host cells in the preparation of drugs for preventing or treating tumors or kits for diagnosing cancer or predicting the effect of cancer treatment.

[0020] The ninth aspect of the present invention is to provide a kit for detecting HER2, which comprises the above-mentioned nano monoclonal antibody, nucleic acid molecule, nucleic acid construct or host cell; preferably, the kit also comprises a reagent for detecting the binding of HER2 to the nano monoclonal antibody; more preferably, the reagent for detecting the binding is a detectable marker that can be connected to the nano monoclonal antibody.

[0021] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0022] The nano monoclonal antibody provided by the present invention has high specificity and strong affinity to human HER2 protein, is easy to produce and has high stability, and is suitable for various cancer diagnosis and treatment studies targeting HER2. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The results of the eluted target protein being identified and purified using PAGE gel are shown, among which the size of IDM3-59 (VHH+6×HisTag) is 26.8kDa, the size of 6×His Tag is 13.35kDa, and the size of IDM3-59 antibody is 13.46kDa; whole bacteria: whole bacteria after bacterial liquid is broken; precipitate / supernatant: precipitate / supernatant after bacterial liquid is broken and centrifuged; loading FT: effluent after protein loading (to confirm whether the protein is successfully adsorbed); NiB washing: eluted target protein by NiB adsorption and purification (indicated by the red arrow); cutting Ft: eluted impurities by protease cutting; cutting NiC: eluted effluent by NiC after protease cutting (to remove impurities); cutting NiB: purified target protein by NiB adsorption after protease cutting (indicated by the red arrow).

[0024] Figure 2 The chromatogram identification results after IDM3-59 protein concentration and purification are shown.

[0025] Figure 3 Shown are the results of Western Blot using IDM3-59 antibody to identify HER2-positive cells.

[0026] Figure 4 The binding and dissociation abilities of IDM3-59 antibody and HER2 extracellular recombinant protein are shown; among them, A3, B3, C3, D3, E3, F3, G3 and H3 represent the probe numbers of the detection plate. DETAILED DESCRIPTION

[0027] The present invention is described in detail and specifically below through specific embodiments and drawings to provide a better understanding of the present invention, but the following embodiments do not limit the scope of the present invention.

[0028] The methods in the examples are conventional methods unless otherwise specified, and the reagents used are conventional commercial reagents or reagents prepared according to conventional methods unless otherwise specified.

[0029] Example 1 Preparation and screening of nanobodies

[0030] Alpacas were immunized 5-6 times with HER2 extracellular recombinant protein (Sino Biological: 110004-H08H) (the first immunization was protein + Freund's incomplete adjuvant, and the subsequent immunizations were protein + Freund's complete adjuvant), and each immunization was repeated one week after each immunization. Peripheral blood was collected, RNA was extracted, reverse transcribed, and an alpaca immune library was constructed.

[0031] Method and steps for constructing phage library from PBMC after antigen immunization of alpaca:

[0032] 1. Total RNA Extraction

[0033] 1.1 Take the alpaca PBMC sample out of the -80℃ freezer and dispense it into 0.5ml / tube. Add 100μl chloroform to each tube, shake vigorously on a shaker for 15s, and then leave it at room temperature for 5min.

[0034] 1.2 Precool the centrifuge to 4°C and centrifuge at 12000g for 15 minutes. After centrifugation, stratification will occur. Use a pipette to carefully transfer the top transparent layer to a new 1.5ml centrifuge tube free of RNase and DNase. Add 250μl of isopropanol to each tube, mix it upside down several times, and then let it stand at room temperature for 10 minutes.

[0035] 1.3 Centrifuge at 12000g for 10 min, remove the supernatant and keep the precipitate;

[0036] 1.4 Add 1 ml of 75% ethanol to each tube and invert several times to suspend the precipitate;

[0037] 1.5 Centrifuge at 7500g for 5 min, remove the supernatant and keep the precipitate;

[0038] 1.6 Keep the centrifuge tube open and place it at room temperature to dry for 10 minutes. Add 13 μl of DEPC-treated water to each tube to dissolve it and incubate it at 55°C for 10 minutes to ensure that the RNA is completely dissolved.

[0039] 1.7 Carefully pipette into the same centrifuge tube, which is the total RNA extracted from PBMC.

[0040] 2. RNA Reverse Transcription

[0041] After total RNA extraction, samples were immediately reverse transcribed using a reverse transcription kit (TAKARA: RR037A) to reduce RNA degradation.

[0042] 2.1 Divide the total RNA sample into two parts, one using the Oligo dT Primer in the kit as primer, and the other using the Random 6-mers in the kit as primer. Prepare the reaction solution in a 1.5 ml centrifuge tube according to the proportion specified in the instruction manual.

[0043] 2.2 Place the centrifuge tube containing the above reaction solution into a metal heater and react at 65°C for 5 minutes to denature the RNA. After the reaction is completed, place it on ice to cool quickly.

[0044] 2.3 Prepare the reaction solution according to the proportion in the instruction manual, mix slowly, put the centrifuge tube containing the reaction solution into a metal heater, react at 45℃ for 60min, then at 75℃ for 15min, cool on ice, this is the cDNA after reverse transcription of total RNA, store at -20℃.

[0045] 2.3 PCR amplification

[0046] Using cDNA as template, using Ex The Hot Start Version amplifies the nanoantibody fragment through two rounds of PCR and constructs it into phage to construct a phage display library. Then, 32 antibodies are screened from the immune library using HER2 protein.

[0047] 3. The antibody sequence was constructed into the pSumo vector (Ubao Bio: VT2052) for expression (20°C, 180rpm, IPTG = 0.2mM), and purified with a Ni column. The purified protein was further separated and purified by molecular sieves, and the protein purification buffer was replaced with 1× PBS buffer using a desalting column to screen out an antibody IDM3-59 with high specificity and affinity. The specific experimental steps are as follows:

[0048] (1) Antibody expression and purification (E. coli expression system)

[0049] 1) Constructed into Gal10-pSumo expression vector with 6×His Tag and Sumo.

[0050] 2) Expression conditions: The bacterial solution was expanded into a 2L culture flask (containing 1L of ampicillin-resistant LB medium) at a ratio of 1:1000 and cultured in a 37°C constant temperature shaker (200 rpm) for 3-4 hours; when the bacterial solution grew to OD = 0.6, IPTG was added at a final concentration of 20 nM and placed in a 20°C constant temperature incubator (180 rpm) to induce expression for 16-20 hours.

[0051] 3) The precipitated bacteria were collected by centrifugation at 5000 rpm for 15 min, and the bacteria were disrupted by ultrasonic disruptor and then ultracentrifuged (16000 rpm, 1 h) to collect the protein supernatant, and then purified and recovered by Ni column.

[0052] The composition of the purification buffer is as follows:

[0053]

[0054]

[0055] Ni column purification process:

[0056] ddH2O→NiA balance (30mL)→protein supernatant→NiC wash (50mL)→NiB elution of target protein (50mL).

[0057] 4) After the protein is concentrated using a concentrator tube, use Superdex on the AKTA instrument TM The concentrated proteins were further separated and purified.

[0058] 4. Cell WB identification antibody

[0059] Antibody identification: HER2 positive cells (SKBR3 cells, HER2 and p95HER2 stable transfection lines constructed by 293T cells) and HER2 negative cells (BT549 cells) were used for preliminary identification by Western Blot. Among them: SKBR3, 293T and BT549 were purchased from the cell bank of the Committee for the Preservation of Typical Cultures of the Chinese Academy of Sciences; primary antibody: purified antibody (1 mg / ml), diluted at 1:150; secondary antibody: Rabbit anti-Camelid VHH-HRP (Genscrip) (1:5000); HER2 protein size: 185 kDa.

[0060] After the above screening and identification, an antibody IDM3-59 with high specificity and affinity was obtained. Among them, the identification and purification results of IDM3-59 protein are as follows Figure 1 and 2 The results of Western Blot identification are shown in Figure 3 shown.

[0061] The sequence information of antibody IDM3-59 is as follows:

[0062] CDR1 amino acid sequence (SEQ ID NO: 1): GSIFSTYN

[0063] CDR2 amino acid sequence (SEQ ID NO: 2): LTRDGVA

[0064] CDR3 amino acid sequence (SEQ ID NO: 3): KTLPLTRDWGSAD

[0065] Full-length amino acid sequence (SEQ ID NO:4):

[0066] SQLQLVESGGGLVQVGGSLRLTCAASGSIFSTYNMAWYRQAPGKQREMVA

[0067] ALTRDGVAHYAHSVEGRFTISRDNAKNTMYLQMTSLKPEDTAAYYCKTLPL

[0068] TRDWGSADWGRGTQVTVSS

[0069] Nucleotide sequence encoding antibody IDM3-59 (SEQ ID NO:5):TCCCAGTTGCAGCTCGTGGAGTCTGGGGGAGGATTGGTGCAGGCGGGGGCCTCTCTGAGACTCTCCTGTGCAGCGTCTGAACGCACCCTCAGTAGATATGTCATGGCGTGGTACCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAGCAATTACCTGGAGTAGTGGTAGGACAACCTAT GCAGACTCCCTGAAGGGCCGATTCACCATCCCAGAGACAACGATAGGAACACGCTTGTATCTGCAAATGATCAGTTTGAAAGCTGAGGACACGGGCACTTATTATTGTGCAGCCGGCGACCCATTTAGTGGCTACGAAGGAGGGGGGTTTGGCTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCA

[0070] Example 2 Affinity Identification

[0071] The ForteBio Octet red 96e system was used to identify and analyze the binding dissociation constants of HER2 extracellular recombinant protein (Sino Biological: 110004-H08H) and concentrated and purified antibody protein. The identification results are shown in Table 1 and Figure 4 .

[0072] Table 1 Affinity identification results

[0073] sample KD(M) kdis(1 / s) Kon(1 / Ms) RMax Full R2 Conc.(nM) IDM3-59 4.25E-07 0.0006101 1436 0.9018 0.9947 1000

[0074] From the above results, it can be seen that IDM3-59 and HER2 extracellular recombinant protein have higher Kon values ​​and lower KD, indicating that the two have strong binding ability.

[0075] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modification and substitution of the present invention are also within the scope of the present invention. Therefore, the equalization and modification made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. A nano monoclonal antibody targeting HER2, characterized in that: The complementary determining region CDR of the nano monoclonal antibody includes CDR1, CDR2 and CDR3; wherein the amino acid sequences of CDR1, CDR2 and CDR3 are GSIFSTYN, LTRDGVA and KTLPLTRDWGSAD respectively.

2. The nano monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the nano monoclonal antibody is shown in SEQ ID NO:

4.

3. A chimeric antigen receptor, characterized in that Comprising the nano monoclonal antibody as described in claim 1 or 2.

4. A nucleic acid molecule, characterized in that It encodes the nanomonoclonal antibody as described in claim 1 or 2 or the chimeric antigen receptor as described in claim 3, and its nucleotide sequence is shown in SEQ ID NO:

5.

5. A nucleic acid construct, characterized in that Comprising the nucleic acid molecule as described in claim 4.

6. The nucleic acid construct according to claim 5, wherein The nucleic acid construct is a cloning vector, an expression vector or an integration vector.

7. A host cell, characterized in that It expresses and / or secretes the nanomonoclonal antibody as described in claim 1 or 2 or the chimeric antigen receptor as described in claim 3, or comprises the nucleic acid molecule as described in claim 4 or the nucleic acid construct as described in claim 5 or claim 6.

8. A method for preparing the nano monoclonal antibody according to claim 1 or 2 or the chimeric antigen receptor according to claim 3, characterized in that: include: Under suitable conditions, the host cell of claim 7 is cultured, and the nano monoclonal antibody or chimeric antigen receptor is optionally purified from the culture.

9. A pharmaceutical composition, characterized in that It comprises the nano monoclonal antibody as described in claim 1 or 2, the chimeric antigen receptor as described in claim 3, the nucleic acid molecule as described in claim 4, the nucleic acid construct as described in claim 5 or claim 6, or the host cell as described in claim 7.

10. The pharmaceutical composition according to claim 9, characterized in that Also included are pharmaceutically acceptable carriers or excipients.

11. Use of the nanomonoclonal antibody according to claim 1 or 2, the chimeric antigen receptor according to claim 3, the nucleic acid molecule according to claim 4, the nucleic acid construct according to claim 5 or claim 6, or the host cell according to claim 7 in the preparation of a drug for treating HER2-positive breast cancer or gastric cancer.

12. A kit for detecting HER2, characterized in that: It comprises the nano monoclonal antibody as described in claim 1 or 2, the nucleic acid molecule as described in claim 4, the nucleic acid construct as described in claim 5 or claim 6, or the host cell as described in claim 7.

13. The kit according to claim 12, characterized in that The kit also includes a reagent for detecting the binding of HER2 to the nano monoclonal antibody.

14. The kit according to claim 13, wherein The reagent for detecting the binding is a detectable label that can be linked to the nano monoclonal antibody.

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