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

Abstract

The present invention provides a nano - monoclonal antibody IDM2 - 27 targeting HER2, its preparation method and applications. The complementarity - determining regions (CDRs) of the nano - monoclonal antibody include CDR1, CDR2 and CDR3. Among them, the amino acid sequences of CDR1, CDR2 and CDR3 are GFTFSSYS, INSDGSST and GKGGLHYSDYE respectively. The nano - monoclonal antibody provided by the present invention has high specificity for human HER2 protein, strong affinity, is easy to produce and has high stability, and is suitable for various cancer diagnosis and treatment research targeting HER2.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly relates to a nano monoclonal antibody IDM2-27 targeting HER2, a preparation method thereof, and an application thereof. Background Art

[0002] HER2, also known as human epidermal growth factor receptor-2, ErbB2 or Neu, is a receptor tyrosine kinase (RTK) and belongs to the epidermal growth factor receptor (EGFR / ErbB) family. The gene encoding HER2 is located on human chromosome 17q21, belonging to a proto-oncogene, and its product is a transmembrane protein of 185 kD, abbreviated as p185, which is composed of 1,255 amino acids and contains four extracellular domains and a transmembrane domain. HER2 protein mainly binds to its respective ligand by forming heterodimers with other members of the HER family (including EGFR (HER1 / erbB1), HER3 / erbB3, HER4 / erbB4), thereby activating downstream signaling pathways. Currently, 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 and other signaling pathways. After HER2 binds to the ligand, it mainly activates the activity of tyrosine kinase by causing dimerization of the receptor and autophosphorylation of the tyrosine kinase region in the cytoplasm; it has a special open structure and can activate itself without the participation of a specific ligand, so it can form homo / heterodimers with itself or other members of the HER family. In addition, HER2 is the preferred partner of heterodimers of HER family members, and the activity of the heterodimers formed by it is often stronger than that of other heterodimers.

[0004] HER2 is highly expressed in a variety of cancers. Currently, patients with various types of cancers such as breast cancer, gastric cancer, lung cancer, and endometrial cancer are all tested for the expression level of HER2. The detection rate of high HER2 expression in breast cancer is about 15% - 25%, about 20% in gastric cancer, about 2.5% in lung cancer, and can reach 18% - 80% in endometrial cancer.

[0005] Overexpression of HER2 may lead to continuous enhanced activation of receptor tyrosine kinase, thereby causing a series of downstream cascade reactions, such as MAPK, PI3K-PKB / Akt, STAT, etc. The signaling pathway mediated by Her2 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] Monoclonal antibodies such as trastuzumab (Herceptin), pertuzumab (Perjeta), lapatinib (Tykerb), neratinib (Nerlynx), and pyrotinib are antibody drugs that specifically target HER2 and are mainly used for the treatment of 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 also 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 overexpressing 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 ability 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 insect expression systems. The antibodies produced usually have high water solubility and strong stability. Nanobodies are the smallest antibody structural mode with complete antibody activity known so far. Their molecular weight is usually 1 / 10 of that of conventional antibodies, which determines their high tissue penetration ability and short half-life. They can easily penetrate the blood-brain barrier, and the serum clearance rate is relatively fast. 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 radiation dose to the body. Compared with traditional antibodies, nanobodies have more advantages as tracers and targeted internal radiotherapy drugs.

[0008] However, there is currently a lack of practical and effective nanobodies for HER2-targeted antibody drugs. Therefore, it is of great research and application significance to develop suitable HER2-targeted antibody drugs, and new and effective specific nanobody drugs targeting Her2 should be actively developed. Summary of the Invention

[0009] To overcome the deficiencies in the prior art, the present invention provides a nano - monoclonal antibody IDM2 - 27 targeting HER2, its preparation method and applications. Nanobodies overcome the disadvantages of traditional antibodies, having characteristics such as low molecular weight, strong stability, good solubility, easy expression, low immunogenicity, strong permeability and targeting, and low production cost and easy expression and purification. Therefore, nanobodies targeting HER2 will undoubtedly have a wider range of applicability. Low immunogenicity and strong targeting mean that the same dose of antibody will have a lower burden on the body and a higher therapeutic effect; the tissue permeability of nanobodies indicates that HER2 nanobodies may still be applicable to patients with advanced brain metastases of HER2 - positive breast cancer or gastric cancer, etc.

[0010] To achieve the above - mentioned objectives, the present invention adopts the following technical solutions:

[0011] In the first aspect of the present invention, a nano - monoclonal antibody targeting HER2 is provided, and its complementarity - determining regions CDR include CDR1, CDR2 and CDR3; wherein, the amino acid sequences of CDR1, CDR2 and CDR3 are GFTFSSYS, INSDGSST and GKGGLHYSDYE respectively.

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

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

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

[0015] In the fourth aspect of the present invention, a nucleic acid construct is provided, 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] In the fifth aspect of the present invention, a host cell is provided, 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] In the sixth aspect of the present invention, a method for preparing the above - mentioned nano - monoclonal antibody or chimeric antigen receptor is provided, including: culturing the above - mentioned host cell under suitable conditions, and optionally purifying the nano - monoclonal antibody or chimeric antigen receptor from the culture.

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

[0019] The eighth aspect of the present invention is to provide the use of the above-mentioned nanomonomoclonal antibody, chimeric antigen receptor, nucleic acid molecule, nucleic acid construct, nucleic acid molecule, nucleic acid construct or host cell in the preparation of a drug for preventing or treating tumors or a kit for diagnosing cancer or predicting the therapeutic 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 nanomonomoclonal antibody, nucleic acid molecule, nucleic acid construct, nucleic acid molecule, nucleic acid construct or host cell; preferably, the kit further comprises a reagent for detecting the binding of HER2 to the nanomonomoclonal antibody; more preferably, the reagent for detecting the binding is a detectable label that can be linked to the nanomonomoclonal antibody.

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

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

[0023] Figure 1 Shows the results of identifying the purified eluted target protein with PAGE gel. Among them, the size of IDM2-27 (VHH+6×His Tag) is 26.1 kDa, the size of 6×His Tag is 13.35 kDa, and the size of IDM2-27 antibody is 12.79 kDa; Break whole: the whole bacteria after breaking the bacterial liquid; Break precipitate / supernatant: the precipitate / supernatant after centrifugation of the broken bacterial liquid; NiB: the target protein eluted by NiB adsorption purification (indicated by the red arrow); Ft: the miscellaneous protein eluted by protease digestion; Cut NiB: the target protein after protease digestion and NiB adsorption purification (indicated by the red arrow).

[0024] Figure 2 Shows the chromatogram identification results after concentration and purification of IDM2-27 protein.

[0025] Figure 3 Shows the Western Blot results of identifying HER2-positive cells with IDM2-27 antibody.

[0026] Figure 4 Shows the binding and dissociation ability of IDM2-27 antibody and HER2 extracellular recombinant protein; among them, A3, B3, C3, D3, E3, F3, G3 and H3 represent the probe numbers of the detection plate. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] The present invention will be described in detail and specifically below through specific embodiments and accompanying drawings to better understand the present invention. However, the following embodiments do not limit the scope of the present invention.

[0028] In the examples, the methods are conventional methods unless otherwise specified, and the reagents used are conventional commercially available reagents or reagents prepared by conventional methods unless otherwise specified.

[0029] Example 1 Preparation and Screening of Nanobodies

[0030] Alpacas were immunized with the extracellular recombinant protein of HER2 (Sino Biological: 110004-H08H) 5-6 times (the primary immunization was with protein + Freund's incomplete adjuvant, and the subsequent immunizations were all with protein + Freund's complete adjuvant). Immunization injections were carried out again one week after each immunization. After collecting peripheral blood, RNA was extracted and reverse transcribed to construct an alpaca immune library.

[0031] Methods and steps for constructing a phage library from PBMC after antigen immunization of alpacas:

[0032] 1. Total RNA extraction

[0033] 1.1 Take out the alpaca PBMC samples from the -80°C refrigerator, aliquot 0.5 ml / tube, add 100 μl of chloroform to each tube, shake vigorously on a shaker for 15 s, and then let it stand at room temperature for 5 min;

[0034] 1.2 Pre-cool the centrifuge to 4°C and centrifuge at 12000g for 15 min. After centrifugation, layering occurs. Carefully transfer the uppermost transparent layer to a new 1.5 ml centrifuge tube without RNase and DNase using a pipette. Add 250 μl of isopropanol to each tube, mix well by inverting up and down several times, and then let it stand at room temperature for 10 min;

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

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

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

[0038] 1.6 Keep the centrifuge tube open and let it dry at room temperature for 10 min. Add 13 μl of DEPC-treated water to each tube to dissolve, and incubate at 55°C for 10 min to ensure complete dissolution of RNA;

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

[0040] 2. RNA reverse transcription

[0041] The sample was immediately reverse-transcribed using a reverse transcription kit (TAKARA: RR037A) after total RNA extraction to reduce RNA degradation.

[0042] 2.1 Divide the above total RNA sample into two parts. Use the Oligo dT Primer in the kit as the primer for one part, and use the Random 6-mers in the kit as the primer for the other part. Prepare the reaction solution in a 1.5 ml centrifuge tube according to the instructions in the manual.

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

[0044] 2.3 Prepare the reaction solution according to the instructions in the manual. After gently mixing, place the centrifuge tube containing the reaction solution into a metal heater, react at 45 °C for 60 min, then react at 75 °C for 15 min, and cool it on ice. This is the cDNA after reverse transcription of total RNA, and it is stored at -20 °C.

[0045] 2.3 PCR amplification

[0046] Using the cDNA as a template, use Ex Hot Start Version to amplify the nanobody fragment through two rounds of PCR, and construct it into a phage to build a phage display library. Then, 32 antibodies were screened from the immune library using the HER2 protein.

[0047] 3. Construct the antibody sequence onto the pSumo vector (Youbao Biotechnology: VT2052) for expression (20 °C, 180 rpm, IPTG = 0.2 mM), and purify it using an Ni column. After the purified protein is further separated and purified by a molecular sieve, replace the protein purification buffer with 1×PBS buffer using a desalting column. A specific antibody IDM2-27 with relatively high affinity was screened out. The specific experimental steps are as follows:

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

[0049] 1) Construct it onto the Gal10-pSumo expression vector with a 6×His Tag and Sumo.

[0050] 2) Expression conditions: Dilute the bacterial solution 1:1000 and transfer it to a 2 L culture flask (containing 1 L of LB medium with ampicillin resistance), and culture it in a constant temperature shaker at 37 °C (200 rpm) for 3 - 4 h; when the OD of the bacterial solution reaches 0.6, add IPTG with a final concentration of 20 nM, and place it in a constant temperature incubator at 20 °C (180 rpm) for 16 - 20 hours after induction of expression.

[0051] 3) Centrifuge at 5000 rpm for 15 min to collect the precipitated bacteria. After disrupting the bacteria with an ultrasonic disruptor, ultracentrifuge (16000 rpm, 1 h) to collect the protein supernatant, and then use an Ni column to purify and recover the target protein.

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

[0053]

[0054]

[0055] Ni column purification process:

[0056] ddH 2 O → NiA equilibration (30 mL) → protein supernatant → NiC washing (50 mL) → NiB elution of the target protein (50 mL).

[0057] 4) After concentrating the protein with a concentrator, further separate and purify the concentrated protein on an AKTA instrument using Superdex TM 75.

[0058] 4. Cell WB identification antibody

[0059] For antibody identification, Western Blot was initially performed using HER2-positive cells (SKBR3 cells, HER2 and p95HER2 stable transfected strains stably constructed from 293T cells) and HER2-negative cells (BT549 cells). Among them, SKBR3, 293T, and BT549 were purchased from the Cell Bank of the Chinese Academy of Sciences' Committee for Type Culture Collection; primary antibody: the purified antibody (1 mg / ml) was diluted 1:300; secondary antibody: Rabbit anti-Camelid VHH-HRP (Genscrip) (1:5000); HER2 protein size: 185 kDa.

[0060] After the above screening and identification, the antibody IDM2-27 with higher specificity and affinity was obtained. Among them, the identification and purification results of the IDM2-27 protein are as Figure 1 and 2 shown, and the results identified by Western Blot are as Figure 3 shown.

[0061] The sequence information of the antibody IDM2-27 is as follows:

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

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

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

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

[0066] SQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMSWVRQAPGKGREWVAF

[0067] INSDGSSTTYADSVEGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCGKGGLH

[0068] YSDYERGQGTQVTVSS

[0069] Nucleotide sequence encoding antibody IDM2-27 (SEQ ID NO: 5): TCCCAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATTCCATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCGAGAGTGGGTGGCATTTATTAACAGTGATGGTAGTAGCACCACCTATGCAGACTCCGTGGAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGGAAAAGGGGGGCTTCACTATAGCGACTATGAACGAGGCCAGGGGACCCAGGTCACCGTCTCCTCA

[0070] Example 2 Affinity Identification

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

[0072] Table 1 Affinity Identification Results

[0073] Sample KD(M) kdis(1 / s) Kon(1 / Ms) RMax Full R2 Conc.(nM) IDM2-27 1.097E-08 0.0001935 17640 0.2294 0.8248 1000

[0074] From the above results, it can be seen that IDM2-27 has a relatively high Kon value and a low KD with the extracellular recombinant protein of HER2, indicating that the two have a strong binding ability.

[0075] The specific embodiments of the present invention have been 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 modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, equivalent transformations and modifications made without departing from the spirit and scope of the present invention should all be covered within 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 GFTFSSYS, INSDGSST and GKGGLHYSDYE 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 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 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 nano monoclonal 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 6, or the host cell according to claim 7 in the preparation of a drug for preventing or treating HER2-positive tumors or a kit for diagnosing HER2-positive cancer or predicting the treatment effect of HER2-positive 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 6, or the host cell as described in claim 7.

13. The kit for detecting HER2 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 for detecting HER2 according to claim 13, characterized in that: The reagent for detecting the binding is a detectable label that can be linked to the nanomonoclonal antibody.

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