A single-domain antibody targeting human HER2 and its application

Abstract

The present invention discloses a single-domain antibody targeting human HER2 and its applications. The present invention screened and obtained a highly specific single-domain antibody, C2-42, by immunizing alpacas with HER2 protein. The sequence of the antibody is shown in SEQ ID No. 1. The single-domain antibody C2-42 disclosed in the present invention has high specificity and affinity, a simple structure, and is easy to produce and store at room temperature. It improves the accuracy of HER2 receptor detection and provides an alternative for efficient and rapid HER2 receptor detection.

Description

Technical Field

[0001] The present invention relates to a single-domain antibody targeting human HER2 and applications thereof, belonging to the technical field of biomedicine. Background Art

[0002] HER2, short for human epidermal growth factor receptor-2, also known as ErbB2 or Neu, is a receptor tyrosine kinase (RTK) that belongs to the epidermal growth factor receptor (EGFR / ErbB) family. The gene encoding HER2 is located on human chromosome 17q21 and is a proto-oncogene. Its product is a 185kD transmembrane protein, abbreviated as p185, which is composed of 1255 amino acids and contains four extracellular domains and one transmembrane domain. The HER2 protein primarily binds to its respective ligands by forming heterodimers with other members of the HER family (including EGFR (HER1 / erbBI), HER3 / erbB3, and 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 the HER2 protein primarily include Ras / Raf / MAPK / PI3K and Akt signaling pathways. Upon ligand binding, HER2 activates tyrosine kinase activity primarily by causing receptor dimerization and autophosphorylation of the cytoplasmic tyrosine kinase domain. HER2 possesses a unique open structure that allows for self-activation without the involvement of a specific ligand, enabling it to form homodimers or heterodimers with itself or other HER family members. HER2 is also the preferred partner for heterodimers of HER family members, and the heterodimers it forms are often more active than those formed with other heterodimers.

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

[0005] HER2 overexpression can lead to sustained, enhanced activation of receptor tyrosine kinases, triggering a series of downstream cascades, such as MAPK, PI3K-PKB / Akt, and STAT. HER2-mediated signaling pathways can also regulate the expression of tumor-related genes, thereby promoting tumor invasion and metastasis. HER2 overexpression plays a crucial role in the development and progression of certain invasive cancers, particularly invasive breast cancer. Therefore, HER2 has become an important biomarker 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 primarily used to treat patients with HER2-positive invasive or metastatic breast cancer. By targeting 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 simultaneously 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] Traditional antibodies can mostly only be expressed in mammalian expression systems, resulting in complex and costly production processes, poor antibody stability, high immunogenicity, and low tissue penetration. These factors often lead to poor therapeutic efficacy. Compared to traditional antibodies, nanobody single-domain antibodies offer numerous advantages. They can be expressed in mammalian expression systems, E. coli, and insect systems, and the resulting antibodies are generally highly water-soluble and stable. Nanobody single-domain antibodies are the smallest known antibody structures with full antibody activity. Their molecular weight is typically one-tenth that of conventional antibodies, resulting in high tissue penetration and a short half-life. They can easily cross the blood-brain barrier and are rapidly cleared from serum. Therefore, nanobody single-domain antibodies overcome many of the shortcomings of traditional antibodies, making them more suitable for carrying radioactive isotopes. Nanobody single-domain antibodies can quickly and specifically penetrate tumor tissue to bind to their targets, while non-binding nanobodies are rapidly eliminated from the bloodstream, reducing the body's radiation dose. Compared to traditional antibodies, nanobody single-domain antibodies offer numerous advantages as tracers and targeted internal radiotherapy agents.

[0008] However, there is currently a lack of effective nano-single-domain antibodies for HER2-targeted antibody drugs, so it is of great significance to develop suitable antibody drugs targeting HER2. Summary of the Invention

[0009] The purpose of the present invention is to address the problems and defects of current monoclonal antibodies targeting HER2 and propose a nano single-domain antibody targeting HER2, which overcomes the shortcomings of traditional antibodies and has 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.

[0010] To achieve the above objectives, the present invention provides a single-domain antibody targeting human HER2 receptor, the amino acid sequence of the single-domain antibody is shown in SEQ ID No. 1.

[0011] The present invention also provides a nucleic acid encoding the above-mentioned single-domain antibody.

[0012] The present invention also provides an expression vector containing the nucleic acid.

[0013] Preferably, the expression vector is a eukaryotic expression vector or a prokaryotic expression vector.

[0014] Preferably, the eukaryotic expression vector is selected from any one of pFastBacDual, pCMV, p-NEO-BAN vector, pEGFP enhanced chromatic fluorescent protein expression vector, pEGFT-Actin enhanced green fluorescent protein / human actin expression vector, pSV2 expression vector and CMV4 expression vector; the prokaryotic expression vector is selected from any one of pBAD vector, T7 vector and pET expression vector.

[0015] The present invention also provides a host cell containing the above nucleic acid or the above expression vector.

[0016] Preferably, the host cell is a eukaryotic cell or a prokaryotic cell, the eukaryotic cell is selected from any one of CHO, COS and HEK-293; the prokaryotic cell is selected from Escherichia coli or Corynebacterium glutamicum.

[0017] The present invention also provides the use of the above-mentioned single-domain antibody, nucleic acid, or expression vector in preparing a kit for detecting human HER2.

[0018] The present invention also provides use of the host cells in preparing a kit for detecting human HER2.

[0019] The present invention also provides a kit for detecting human HER2, wherein the kit comprises the above-mentioned single domain antibody.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention provides a single-domain antibody C2-42 that specifically targets HER2. The single-domain antibody is obtained by screening after immunizing alpacas with HER2 protein. Since it only contains the heavy chain of the antibody, it has a high affinity, and its structure is simple, which is convenient for production and storage, and can be stored for a longer time at room temperature. In addition, the single-domain antibody C2-42 disclosed by the present invention has a good targeting effect, providing an alternative option for the efficient and rapid diagnosis of HER2. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1This is the electrophoresis of nickel column affinity chromatography of the single-domain antibody C2-42; wherein, C2-42 (VHH+tag): 26.8 kDa, tag: 13.35 kDa, C2-42: 13.48 kDa;

[0023] Figure 2 This is the elution diagram of the single domain antibody C2-42 after concentration by molecular sieve;

[0024] Figure 3 This is a diagram showing the Western Blotting effect of the single-domain antibody C2-42 on several different cells;

[0025] Figure 4 This is a diagram showing the OctetRed affinity identification effect of the single-domain antibody C2-42. DETAILED DESCRIPTION

[0026] To make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0027] All reagents used in the examples were purchased from conventional commercial channels.

[0028] Example 1 Monoclonal Antibody Screening

[0029] Alpacas were immunized 5-6 times with HER2 protein. After blood collection, RNA was extracted and reversed to construct an alpaca immune library. 32 antibodies were screened from the immune library using HER2 protein. The antibody sequences were constructed and expressed on a Gal10-pSumo vector (20°C, 180 rpm, IPTG = 0.2 mM) and purified using a Ni column. The purified protein was further separated and purified using molecular sieves, and the protein buffer was replaced with PBS using a desalting column. Subsequently, WB, ELISA, and BioRed verification were performed, and finally, the antibody C2-42 with high specificity and affinity was screened out.

[0030] The amino acid sequence of the single-domain antibody C2-42 (SEQ ID No. 1) is shown below:

[0031] SQLQLVESGGGLVQAGGSLRLSCAAS ERTFSMDT MGWFRQAPGKEREF VAT MRWTAGRT YYAESVKGRFTISGDNARNTVYLQMSNLKTEDTAVYYC AA TRRGGVVGIYIREYDY WGQGTQVTVSS.

[0032] The underlined CDRs (Complementarity Determining Regions) of single-domain antibodies are numbered according to IMGT rules.

[0033] The specific steps of antibody screening are as follows:

[0034] (1) Artificially synthesizing the coding gene based on the human HER2 protein gene sequence, heterologously expressing the HER2 protein, and purifying the HER2 protein for future use;

[0035] (2) Immunize alpacas with 5 mg / kg of HER2 for 5-6 consecutive times, with an interval of 24 hours between each immunization; collect blood for RNA extraction;

[0036] (3) Reverse transcription of the RNA obtained in step (2) above to construct a library, and expression using an E. coli expression system; after fermentation and culture, the cells are collected, crushed, and purified to recover the target protein;

[0037] (4) Screening of antibody binding ability.

[0038] Wherein, step (3) includes the following operations:

[0039] 1) The E. coli expression system comes with a His tag. Insert the cDNA obtained by reverse transcription into an expression vector carrying a 6×His tag and Sumo, transform competent E. coli, and screen for positive transformants.

[0040] 2) Cultivate the positive transformants to induce expression: When the bacterial solution grows to OD = 0.6, add IPTG with a final concentration of 20 nM (20°C, 180 rpm), induce expression for 16-20 hours, and then harvest the bacteria.

[0041] 3) Collect the precipitated cells by centrifugation, crush the cells and purify the target protein using Ni column, and identify and purify the protein using PAGE gel. The results are as follows: Figure 1 Further molecular sieve purification was used to obtain pure single domain antibody C2-42. The results are shown in Figure 2 A total of 32 single-domain antibodies were screened, and the effects of other single-domain antibodies with poor effects are not shown.

[0042] Step (4) includes the following steps:

[0043] Western blotting was performed on the 32 single-domain antibodies obtained using SKBR3 (HER2-positive cells), BT549 (HER2-negative cells), constructed HER2 stable cells, p95HER2 (constructed p95HER2 stable cells), primary antibody (purified C protein 1 mg / ml, diluted 1:150), and secondary antibody (Rabbit anti-Camelid VHH-HRP (Genscrip) (1:5000)) to determine the specificity and sensitivity of the antibodies. The results are shown in Figure 2. Figure 3 As shown by Figure 3As can be seen, the single-domain antibody C2-42 obtained by screening reacts only to SKBR3 and HER2, but does not bind to truncated HER2 and other proteins. This shows that C2-42 has high specificity for the full-length HER2 protein.

[0044] Sensitivity effects such as Figure 4 and as shown in Table 1.

[0045] Table 1 OctetRed affinity identification data of C2-42

[0046] Loading Samples Sample name KD(M) kdis(1 / s) kon(1 / Ms) RMax Full R^2 Conc.(nM) Protein C C2-42 1.479E-07 0.00205 13860 0.2436 0.9121 1000

[0047] KD: Equilibrium dissociation constant, representing the strength of affinity. The smaller the KD value, the greater the affinity.

[0048] Kdis: dissociation rate constant, representing the degradation rate of the product per unit time;

[0049] Kon: Binding rate constant, representing the rate of product formation per unit time;

[0050] RMax: maximum analyte concentration that can be obtained;

[0051] Conc. (nM): concentration tested;

[0052] FullR^2: The closer it is to 1, the better the regression fit.

[0053] As shown in Table 1, C2-42 has a high affinity for HER2, that is, both have high sensitivity.

[0054] The above embodiments are only preferred embodiments of the present invention and are not intended to limit the present invention in any form or substance. It should be noted that ordinary technicians in this technical field can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. A single domain antibody targeting human HER2 receptor, characterized in that The amino acid sequence of the single-domain antibody is shown in SEQ ID No.

1.

2. A nucleic acid encoding the single domain antibody of claim 1.

3. An expression vector comprising the nucleic acid of claim 2.

4. The expression vector according to claim 3, characterized in that The vector is a eukaryotic expression vector or a prokaryotic expression vector.

5. The expression vector according to claim 4, characterized in that The eukaryotic expression vector is selected from any one of pFastBacDual, pCMV, p-NEO-BAN vector, pEGFP enhanced chromatic fluorescent protein expression vector, pEGFT-Actin enhanced green fluorescent protein / human actin expression vector, pSV2 expression vector and CMV4 expression vector; the prokaryotic expression vector is selected from any one of pBAD vector, T7 vector and pET expression vector.

6. A host cell containing the nucleic acid of claim 2 or the expression vector of any one of claims 3 to 5.

7. The host cell according to claim 6, characterized in that The host cell is a eukaryotic cell or a prokaryotic cell, the eukaryotic cell is selected from any one of CHO, COS and HEK-293; the prokaryotic cell is selected from Escherichia coli or Corynebacterium glutamicum.

8. Use of the single domain antibody according to claim 1, the nucleic acid according to claim 2, or the expression vector according to any one of claims 3 to 5 in the preparation of a kit for detecting human HER2.

9. Use of the host cell according to claim 6 in preparing a kit for detecting human HER2.

10. A kit for detecting human HER2, characterized in that: The kit comprises the single domain antibody according to claim 1.

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