CN116003602A - Single-domain antibody targeting human HER2 and application thereof - Google Patents

Abstract

The invention discloses a single domain antibody targeting human HER2 and application thereof. The invention uses HER2 protein to immunize alpaca and then screens to obtain a single domain antibody C2-42 with high specificity, and the sequence is shown in SEQ ID No. 1. The single domain antibody C2-42 disclosed by the invention has high specificity and higher affinity, is simple in structure, is convenient to produce and store at normal temperature, improves the accuracy of detecting the HER2 receptor, and provides an alternative choice for efficiently and quickly detecting the HER2 receptor.

Description

Single-domain antibody targeting human HER2 and application thereof

Technical Field

The invention relates to a single domain antibody targeting human HER2 and application thereof, belonging to the technical field of biological medicine.

Background

HER2, which is fully known as human epidermal growth factor receptor-2, also known as ErbB2 or Neu, is a Receptor Tyrosine Kinase (RTK) belonging to the epidermal growth factor receptor (EGFR/ErbB) family. The gene encoding HER2 is located on human chromosome 17q21, belongs to protooncogenes, and is a transmembrane protein of 185kD, p185 for short, which consists of 1255 amino acids and comprises four extracellular domains and one transmembrane domain. HER2 proteins bind to their respective ligands, mainly by forming heterodimers with other members of the HER family, including EGFR (HERl/erbBI), HER3/erbB3, HER4/erbB4, thereby activating downstream signaling pathways, ligands which bind directly to them not currently being found.

The HER2 protein mediated signal transduction pathway mainly comprises Ras/Raf/MAPK/PI3K and other signal pathways. After HER2 binds to the ligand, tyrosine kinase activity is activated primarily by causing dimerization of the receptor and autophosphorylation of the cytoplasmic tyrosine kinase domain; it has a special open structure that can activate itself without the participation of specific ligands and thus is able to form homo/heterodimers with itself or other members of the HER family, and HER2 is also the first partner of HER family member heterodimers, which participate in forming heterodimers with a much stronger activity than other heterodimers.

HER2 is highly expressed in various cancers, and currently known patients with various types of cancers such as breast cancer, gastric cancer, lung cancer, endometrial cancer and the like can detect the expression level of HER 2. The detection rate of HER2 high expression in breast cancer is about 15% -25%, the detection rate of gastric cancer is about 20%, the detection rate of lung cancer is about 2.5%, and the detection rate of endometrial cancer can reach 18% -80%.

Overexpression of HER2 may lead to sustained enhanced activation of receptor tyrosine kinases leading to a series of downstream cascade reactions such as MAPK, PI3K-PKB/Akt, STAT, etc. HER 2-mediated signaling pathways may also regulate expression of tumor-associated genes, thereby promoting tumor invasion and metastasis. Overexpression of HER2 plays an important role in the occurrence and progression of certain invasive cancers, particularly invasive breast cancers, and thus HER2 has become an important biomarker and therapeutic target for breast cancer.

Monoclonal antibodies Trastuzumab (Herceptin/Herceptin), pertuzumab (Pertuzumab, patett/Perjeta), lapatinib (Lapatinib, telmisartan/Tykerb), lenatinib (Neratinib, nerlynx), pyrroltinib (Pyrotinib) are antibody drugs specifically targeting HER2, primarily for the treatment of HER2 positive invasive or metastatic breast cancer patients, inhibiting dimerization of HER2 with other HER family members by targeting binding HER2, thereby inhibiting tumor growth; lapatinib, lenatinib, and pyrroltinib can also target to inhibit HER1 (EGFR/ErbB 1) while targeting to inhibit HER2, by reducing autophosphorylation of EGFR and HER2, inhibit downstream MAPK and AKT signaling pathways, thereby inhibiting proliferation of EGFR or HER2 overexpressing tumor cells.

Most of the traditional antibodies can only be expressed in a mammal expression system, the production process is complex, the cost is high, the antibody stability is poor, the immunogenicity is high, the tissue penetrating power is low, and the comprehensive factors often lead to poor therapeutic effect of the antibody. Compared with the traditional antibody, the nanometer single-domain antibody has a plurality of advantages, can be expressed in a mammalian expression system, escherichia coli and an insect system, and the produced antibody has higher water solubility and strong stability. The nanometer single domain antibody is the smallest antibody structure mode with complete antibody activity, the molecular weight is usually 1/10 of that of the conventional antibody, which determines that the nanometer single domain antibody has higher tissue penetrating capacity, shorter half-life period, and can easily penetrate the blood brain barrier, and the serum clearance speed is higher, so the nanometer single domain antibody overcomes the defects of the conventional antibody, and the nanometer single domain antibody is more suitable for carrying radioactive isotopes. Nano-single domain antibodies can rapidly and specifically penetrate tumor tissue binding targets, whereas non-binding nanobodies can rapidly remove from the blood and reduce the radiation dose of the body. Compared with the traditional antibody, the nanometer single domain antibody has more advantages as a tracer and a targeted internal radiotherapy drug.

However, currently, targeting antibody drugs against HER2 lack truly effective nano-single domain antibodies, and thus development of suitable antibody drugs targeting HER2 is of great significance.

Disclosure of Invention

The purpose of the invention is that: aiming at the problems and defects of the prior HER2 targeting monoclonal antibody, the nanometer single domain antibody for HER2 targeting is provided, overcomes the defects of the traditional antibody, 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.

In order to achieve the above object, the present invention provides a single domain antibody targeting human HER2 receptor, the amino acid sequence of which is shown in SEQ ID No. 1.

The invention also provides a nucleic acid for encoding the single domain antibody.

The invention also provides an expression vector containing the nucleic acid.

Preferably, the expression vector is a eukaryotic expression vector or a prokaryotic expression vector.

Preferably, the eukaryotic expression vector is selected from any one of pFastBacDual, pCMV, p-NEO-BAN vector, pEGFP enhanced taenium fluorescent protein expression vector, pEGFT-action 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.

The invention also provides a host cell of the nucleic acid or the expression vector.

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

The invention also provides application of the single domain antibody, the nucleic acid or the expression vector in preparation of a kit for detecting human HER 2.

The invention also provides application of the host cell in preparing a kit for detecting human HER 2.

The invention also provides a kit for detecting human HER2, which comprises the single domain antibody.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides a single domain antibody C2-42 of specificity targeting HER2, which is obtained by screening after HER2 protein is immunized with alpaca, has higher affinity due to only heavy chains of the antibody, has simple structure, is convenient to produce and store and can be stored for a longer time at normal temperature, and the single domain antibody C2-42 disclosed by the invention has good targeting effect and provides an alternative selection object for efficient and rapid diagnosis of HER 2.

Drawings

FIG. 1 is a single domain antibody C2-42 nickel column affinity chromatography; wherein C2-42 (VHH+ tag): 26.8kDa, tag: 13.35kDa, C2-42:13.48kDa;

FIG. 2 is a flow-out diagram of the single domain antibody C2-42 after concentration by molecular sieve;

FIG. 3 is a graph showing West Blotting effects of the single domain antibody C2-42 on several different cells;

FIG. 4 is a graph showing the effect of OctetRed affinity assay on single domain antibody C2-42.

Detailed Description

In order to make the invention more comprehensible, preferred embodiments accompanied with figures are described in detail below.

Each of the reagents and the like used in the examples were obtained commercially as usual.

EXAMPLE 1 monoclonal antibody screening

The alpaca is immunized by using HER2 protein for 5-6 times, RNA inversion is extracted after blood collection, an alpaca immune library is constructed, 32 antibodies are screened from the immune library by using the HER2 protein, the antibody sequence is constructed on a Gal10-pSumo carrier for expression (20 ℃,180rpm, IPTG=0.2 mM), the purification is carried out by using a Ni column, the protein obtained by purification is further separated and purified by using a molecular sieve, a protein Buffer is replaced by PBS by using a desalting column, and then WB, ELISA and BioRed verification are carried out, and finally, the antibody C2-42 with higher specificity and affinity is screened.

Wherein the amino acid sequence of the single domain antibody C2-42 (SEQ ID No. 1) is as follows:

SQLQLVESGGGLVQAGGSLRLSCAASERTFSMDTMGWFRQAPGKEREF VATMRWTAGRTYYAESVKGRFTISGDNARNTVYLQMSNLKTEDTAVYYCAA TRRGGVVGIYIREYDYWGQGTQVTVSS。

the CDR (Complementarity Determining Region) single domain antibodies are underlined and numbered according to IMGT rules.

The specific steps of antibody screening are as follows:

(1) Artificially synthesizing a coding gene according to a human HER2 protein gene sequence, heterologously expressing HER2 protein, and purifying to obtain HER2 protein for later use;

(2) Immunization of alpaca with 5mg/kg of HER2, 5-6 consecutive immunizations, each at 24h intervals; extracting RNA by blood collection;

(3) Constructing a library by reverse transcription of the RNA obtained in the step (2), and expressing by using an escherichia coli expression system; after fermentation culture, collecting thalli, crushing, purifying and recycling target protein;

(4) Antibody binding capacity was screened.

Wherein, in the step (3), the following operations are included:

1) The Escherichia coli expression system carries His Tag, cDNA obtained by reverse transcription is inserted into an expression vector carrying 6 XHis Tag and Sumo, competent Escherichia coli is transformed, and positive transformants are screened.

2) Positive transformants were cultured to induce expression: when the bacterial liquid grows to od=0.6, IPTG (20 ℃,180 rpm) with a final concentration of 20nM is added, and the bacterial liquid is collected after induced expression for 16-20 hours.

3) The precipitated cells were collected by centrifugation, and after the cells were crushed, the target protein was recovered by purification with a Ni column, and the purification was identified by a PAGE gel, and the results are shown in FIG. 1. Further purification using molecular sieves gave pure single domain antibody C2-42, the results are shown in FIG. 2. Co-screening resulted in 32 single domain antibodies, other poorly effective single domain antibodies were not shown.

The step (4) comprises the following steps:

west Blotting was performed on the 32 single domain antibodies obtained using SKBR3 (HER 2 positive cells), BT549 (HER 2 negative cells), constructed HER2 stable strain, p95HER2 (constructed p95HER2 stable strain), primary antibody (purified protein C1 mg/ml, diluted 1:150), and secondary antibody (Rabbit anti-cam VHH-HRP (Genscrip) (1:5000)), respectively, to determine the specificity and sensitivity of the antibodies. As a result, as shown in fig. 3, it can be seen from fig. 3 that the single domain antibody C2-42 obtained by screening was only reactive to SKBR3 and HER2, but did not bind to truncated HER2 and other proteins. From this, C2-42 was found to have higher specificity for the HER2 full-length protein.

The sensitivity effect is shown in fig. 4 and table 1.

OctetRed affinity assay data of tables 1C2-42

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

KD: equilibrium dissociation constants represent the intensity of affinity, with smaller KD values and greater affinities;

kdis: dissociation rate constants represent the rate of product degradation per unit time;

kon: a binding rate constant representing the rate of product formation per unit time;

RMax: the maximum analyte concentration that can be obtained;

conc. (nM): the concentration tested;

FullR 2: the closer to 1, the better the regression fit.

From Table 1, it can be seen that C2-42 has a higher affinity for HER2, i.e., both have a high sensitivity.

The above-described embodiments are only preferred embodiments of the present invention, and are not intended to be limiting in any way and in nature, and it should be noted that several modifications and additions may be made to those skilled in the art without departing from the invention, which modifications and additions are also intended to be construed as within the scope of the invention.

Claims (10)

1. A single domain antibody targeting the human HER2 receptor, wherein 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 of claim 3, wherein the vector is a eukaryotic expression vector or a prokaryotic expression vector.

5. The expression vector of claim 4, wherein the eukaryotic expression vector is selected from any one of pFastBacDual, pCMV, p-NEO-BAN vector, pEGFP-enhanced taeniform fluorescent protein expression vector, pEGFT-action-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 comprising the nucleic acid of claim 2 or the expression vector of any one of claims 3-5.

7. The host cell of claim 6, wherein the host cell is a eukaryotic cell 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 of claim 1, the nucleic acid of claim 2, or the expression vector of any one of claims 3-5 in the preparation of a kit for detecting human HER 2.

9. Use of the host cell of claim 6 for the preparation of a kit for detecting human HER 2.

10. A kit for detecting human HER2, comprising the single domain antibody of claim 1.

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