An anti-b7h3 nanobody and preparation method and use thereof

Abstract

The application provides an anti-B7H3 nanobody and a preparation method and application thereof, and belongs to the field of biological medicines. The nanobody has high affinity with B7H3 protein, and the immunological risk caused by heterogeneity is reduced to the maximum, the nanobody is more conducive to penetrating the blood-brain barrier, is more likely to reach the inside of a tumor to play a therapeutic effect, and has a wide application prospect in the preparation of medicines for preventing and / or treating tumors.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to an anti-B7H3 nanobody and a preparation method and use thereof. BACKGROUND

[0002] B7H3 (also known as CD276) is a type I transmembrane protein, belonging to the B7 immune costimulatory and coinhibitory family. Current studies show that it has a dual role, which can act as a costimulatory molecule and a coinhibitory molecule, and the specific role depends on the immune cells and microenvironment conditions involved.

[0003] In humans, B7H3 is composed of an extracellular region, a transmembrane region and a short intracellular region, without a known signal motif. There are two subtypes of B7H3: 2IgB7H3 is composed of a pair of immunoglobulin variable region (IgV) and immunoglobulin constant region (IgC) extracellular domains, and 4IgB7H3 contains two pairs of identical IgV and IgC extracellular domains, the latter being the main subtype of human cells. The exact function and receptor of B7H3 have not been elucidated. In non-malignant tissues, B7H3 plays an important role in adaptive immunity by regulating T cell function.

[0004] Although early studies support the view that B7H3 is a costimulatory molecule necessary for activating T cells, recent studies show that B7H3 has a major inhibitory role in adaptive immunity, inhibiting T cell activation, proliferation and release of effector cytokines, mainly IFN-γ and IL-2. In mice, B7H3 seems to have an anti-tumor function mediated by CD8+ T cell and NK cell activation, possibly through binding to the Trem-like transcript 2 (TLT-2) receptor on CD8+ T cells. Surprisingly, in humans, only three studies show a correlation between high expression of B7H3 and improved prognosis. On the contrary, there is overwhelming evidence that B7H3 has a pro-tumor effect. In fact, a large amount of preclinical and clinical evidence shows that B7H3 inhibits the immune response to tumor antigens through various mechanisms, including reducing the density of immune cell tumor infiltration, inhibiting NK cell-mediated tumor cell lysis, increasing regulatory T cell infiltration, reducing the release of effector cytokines and inhibiting the expression of major transcription factors; the above mechanisms lead to invasive cancer biology and metastatic potential, ultimately poor prognosis. B7H3 also seems to promote cancer progression through non-immune functions, such as by increasing the migration, invasion and metastatic potential of cancer cells, by enhancing chemotherapy resistance, and by promoting the pro-tumor cancer cell metabolic profile. Therefore, it is of great significance to study tumor treatment strategies targeting B7H3 molecules.

[0005] Enoblituzumab is a humanized IgG1κ monoclonal antibody that recognizes the human B7H3 protein. However, enoblituzumab has a molecular weight of 150 kDa, which is relatively large and hinders its ability to penetrate the blood-brain barrier. To address this issue, there is an urgent need to develop a humanized nanobody against B7H3. Summary of the Invention

[0006] The purpose of this invention is to provide an anti-B7H3 nanobody, its preparation method, and its uses.

[0007] This invention provides an anti-B7H3 nanobody comprising complementarity-determining regions CDR1-CDR3, wherein the amino acid sequence of CDR1 is RTLSNYA, the amino acid sequence of CDR2 is LRRIGDYIDSA, and the amino acid sequence of CDR3 is AAGPTINKDEYSYPGATGG.

[0008] Furthermore, the anti-B7H3 nanobody also includes four framework regions FR1-FR4 alternately linked with complementarity-determining regions CDR1-CDR3. The amino acid sequence of FR1 is QLQLVESGGGLVQAG ASLRLSCAASG, the amino acid sequence of FR2 is MGWFRQAPGKEREFVGV, the amino acid sequence of FR3 is DSADSVKGRFTFSRDNAKNTLYLYMTSLKPEDTAVYYC, and the amino acid sequence of FR4 is WGQGTQVTVSS.

[0009] Furthermore, the amino acid sequence of the anti-B7H3 nanobody is as follows:

[0010] QLQLVESGGGLVQAGASLRLSCAASGRTLSNYAMGWFRQAPGKER EFVGVLRRIGDYIDSADSADSVKGRFTSFSRDNAKNTLYLYMTSLKPEDTA VYYCAAGPTINKDEYSYPGATGGWGQGTQVTVSS.

[0011] The present invention also provides a nucleotide molecule, the nucleotide sequence of which is shown in SEQ ID NO:9.

[0012] The present invention also provides an expression vector comprising the above-described nucleotide molecules.

[0013] The present invention also provides a host cell comprising the above-described expression vector.

[0014] The present invention also provides a method for preparing the above-mentioned anti-B7H3 nanobody, the method comprising the following steps:

[0015] (1) Connect the above-mentioned nucleotide molecule to an expression vector to obtain a positive plasmid;

[0016] (2) Transform the host cell with the positive plasmid, and induce the expression of the nanobody.

[0017] The present application also provides the use of the above-mentioned nanobody in the preparation of a medicament for preventing and / or treating tumors.

[0018] Further, the tumor is glioma, melanoma, renal cell carcinoma, pancreatic cancer, breast cancer, lung cancer, prostate cancer, cholangiocarcinoma, head and neck squamous cell carcinoma, or cervical cancer.

[0019] As known to those skilled in the art, the tumors that can be treated by the anti-B7H3 antibody include glioma, malignant melanoma, renal cell carcinoma, pancreatic cancer, breast cancer, non-small cell lung cancer, advanced prostate cancer, cholangiocarcinoma, advanced head and neck squamous cell carcinoma, advanced cervical cancer, etc.

[0020] Further, the medicament for preventing and / or treating tumors is a preparation prepared by taking the above-mentioned nanobody as an active ingredient and adding pharmaceutically acceptable adjuvants.

[0021] The present application has the following beneficial effects:

[0022] The anti-B7H3 nanobody of the present application has high affinity with the B7H3 recombinant protein, and has a broad application prospect in the preparation of a medicament for preventing and / or treating tumors.

[0023] The anti-B7H3 nanobody of the present application has a molecular weight as low as 15KDa, and such an antibody is an anti-B7H3 antibody, which maximally reduces the immunological risk caused by heterogeneity, is more conducive to penetrating the blood-brain barrier, and is more likely to reach the inside of the tumor to exert a therapeutic effect.

[0024] Obviously, according to the above-mentioned content of the present application, according to the ordinary technical knowledge and common means in the art, other various forms of modifications, substitutions or changes can be made without departing from the above-mentioned basic technical idea of the present application.

[0025] The above-mentioned content of the present application will be further described in detail through the specific embodiments in the form of examples. However, this should not be understood as limiting the scope of the above-mentioned subject matter of the present application to the following examples. Any technology realized based on the above-mentioned content of the present application belongs to the scope of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The results of the affinity detection of the anti-B7H3 nanobody and B7H3.

[0027] Figure 2Cell killing effect of Enoblituzumab antibody

[0028] Figure 3 Cell killing effect of anti-B7H3 nanobody 8F1 DETAILED DESCRIPTION

[0029] The raw materials and equipment used in the present application are known products, which are obtained by purchasing commercially available products.

[0030] Example 1: Preparation of anti-B7H3 nanobody

[0031] 1. Construction of expression vector

[0032] (1) Enzymatic digestion of the backbone vector: The tool vector pcDNA3.1-x-IgG1 was digested with BamH I / EcoR I enzymes (purchased from Yixing Bio), and the double enzyme-digested plasmid system is shown in Table 1. After 37°C double enzyme digestion for 5h, the vector was recovered using a PCR product recovery kit (Cycle-Pure Kit PCR product purification reagent OMEGA D6492-01).

[0033] Table 1 Double enzyme-digested plasmid system of BamH I / EcoR I

[0034] Ingredient Amount pcDNA3.1-x-IgG1 plasmid 10 μg 10 x buffer 10 μl BamHI 2.5 μl EcoRI 2.5 μl ddH2O up to 100 μl

[0035] (2) Homologous recombination: The nucleotide fragment for recombinant expression was diluted 20 times with ddH2O, and 1 μL of the diluted sample was used for homologous recombination with the above-mentioned enzyme-digested vector (recombination enzyme, NovoRec Plus one step PCR Cloning Kit, Coastal Protein, item number NR005-01B). The homologous recombination system is shown in Table 2. The homologous recombination system was reacted in a PCR instrument at 50°C for 30 min. 100 μl of TOP10 competent cells were added to each homologous recombination product, and transformation was performed (placed on ice for 20 min, 42°C heat shock for 90 s, immediately placed on ice for 2 min, added with 800 μl of LB medium, 37°C, 220 rpm incubation for 25-45 min. Coated in Amp-resistant plate medium, 37°C plate incubator incubation overnight).

[0036] Table 2 Homologous recombination reaction system

[0037] Ingredient Amount pcDNA3.1-x-IgG1 (BamHI / EcoRI) enzyme cut vector large fragment 100 ng cut gel recovery PCR product 50 ng 5 x buffer 2 μl recombinant enzyme 0.5 μl ddH2O up to 10 μl

[0038] The sequence of the nucleotide fragment for recombinant expression is as follows:

[0039] CAGCTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGGCGGGCGCGAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCCGCACCCTGAGCAACTATGCGATGGGCTGGTTTCGCCAGGCGCCGGGCAAAGAACGCGAATTTGTGGGCGTGCTGCGCCGCATTGGCGATTATATTGATAGCGCGGATAGCGCGGATAGCGTGAAAGGCCGCTTTACCTTTAGCCGCGATAACGCGAAAAACACCCTGTATCTGTATATGACCAGCCTGAAACCGGAAGATACCGCGGTGTATTATTGCGCGGCGGGCCCGACCATTAACAAAGATGAATATAGCTATCCGGGCGCGACCGGCGGCTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGCTAA (SEQ ID NO: 9).

[0040] (3) E. coli liquid PCR identification: single colony of E. coli was picked from the plate and inoculated into 200 μΐ of LB medium (1 L solution: 10 g of tryptone (Oxoid), 5 g of yeast extract (Oxoid), 10 g of sodium chloride) and incubated at 37 °C, 220 rpm for 3 h. 1 μΐ of the bacterial liquid was used as a template for liquid PCR identification. The identified positive clones were sent for sequencing. The PCR product was electrophoresed, gel recovered (Gel Extraction Kit, OMEGA, Cat: D2500-01), and then subjected to homologous recombination again. After liquid PCR identification, the identified positive clones were sent for sequencing.

[0041] 2. Expression and purification of anti-B7H3 nanobody in Hek293F cells

[0042] (1) Antibody expression

[0043] The strain was inoculated into 20 ml of LB medium containing ampicillin and incubated in a 37 °C incubator overnight. The plasmid was extracted using a plasmid extraction kit (Plasmid Miniprep Kit II, Bovogen Cat: BW-PD1213). The Hek293 cells were subcultured to maintain good cell growth, with a viability of more than 95%. The Hek293 cell density was adjusted to 2.5 x 10 6The 50 μg expression plasmid was added to 1 ml OPM-293CD03 culture medium (Shanghai Oupaimai Biotech Co., Ltd.) and mixed, 150 μg PEI was added to 1 ml OPM culture medium and mixed, the two were mixed and shaken, and then left to stand at room temperature for 30 min before being added to 50 ml Hek293 cells. The cells were cultured in a CO2 shaker, 5% OPM culture medium was added as a feed on the second day, and the culture was continued until the seventh day. The cells were harvested by centrifugation at 10,000 rpm for 20 min, and the cell culture supernatant was used for protein purification.

[0044] (2) Antibody purification

[0045] The cell culture supernatant was filtered through a 0.22 μm filter membrane, loaded onto a protein A column, and the protein was eluted with 0.1 M Gly-Hcl at pH 3.0. The protein was neutralized by rapidly adding 10% volume of 1 M Tris-Hcl at pH 8.8. The protein concentration was detected by measuring A280 using a microspectrophotometer. The 8F1 protein concentration was 0.31 mg / ml, the total volume was 15 ml, and the purity was not less than 95%.

[0046] The amino acid sequence of the target protein 8F1 is shown in SEQ ID NO: 8, which includes the complementarity determining regions CDR1-CDR3, which are separated by four framework regions FR1, FR2, FR3 and FR4.

[0047] Table 3 Amino acid sequence of target protein

[0048]

[0049]

[0050] The activity of the anti-B7H3 nanobody of the present application is demonstrated by the following experimental examples.

[0051] Experimental Example 1: Detection of the affinity of anti-B7H3 nanobody to B7H3

[0052] 1. Experimental method

[0053] The enzyme-labeled plate was coated with 5 pg / ml B7H3-His antigen, and coated overnight at 4°C. Discard the coating solution, wash with PBS for 2 times, and block at 37°C for 2 h. Add the anti-B7H3 nanobodies 8F1 of the application and the known monoclonal antibody Enoblituzumab (Enoblituzumab) from 500 nM with 3 times dilution, respectively, and incubate at 37°C for 1 h. Wash with 0.1% PBST for 5 times (about 2-3 min for each time), and add the horseradish peroxidase-labeled Fc secondary antibody (Goat Anti-Human IgG-Fc Secondary Antibody (HRP) brand, configured with 5% skim milk at 1:10000), and incubate at 37°C for 45 min. Detect and compare the experimental results of 8F1 antibody and Enoblituzumab antibody, and calculate EC 50 (half maximal effective concentration).

[0054] 2. Experimental results

[0055] Table 4 Affinity ELISA test results

[0056] Name 8F1 Enoblituzumab EC 50 (nM) 0.4701 0.3264

[0057] The affinity ELISA test results are shown in Figure 1 and Table 4, it can be seen that the anti-B7H3 nanobodies 8F1 of the application can target B7H3 protein.

[0058] And the anti-B7H3 nanobodies 8F1 of the application have an affinity for B7H3 protein comparable to the control antibody Enoblituzumab.

[0059] Experimental Example 2: Cell killing experiment of anti-B7H3 nanobodies

[0060] 1. Experimental method

[0061] First, the target cells U-118MG (human brain astrocytoma) were labeled with Calcein-AM (BioLegend, 425201 / 10x50 pg), and after incubating the cells at 37°C for 30 min, centrifuged and resuspended. The anti-B7H3 nanobodies 8F1 (SEQ ID NO: 8) of the application and the control antibody Enoblituzumab were added to each well at a concentration of 100 nM, followed by the addition of 1.8x10 6 NK cells, 1.5x10 4Target tumor cells (NK cells: target tumor cells = 12:1), co-incubation for 4h, all solutions in each well were transferred to a centrifuge tube, cells were washed with 1xPBS, and adherent cells were digested using trypsin. All collected cells were centrifuged at 1200rpm for 5min. The cell pellet was washed with 1xPBS, centrifuged at 1200rpm for 5min, a total of 2 times. 5μL of APC Annexin V staining solution was added; 5μL of 7-AAD viability staining solution was added at the same time; gently invert the cells up and down, after centrifugation, incubate at room temperature (25℃) in the dark for 15min; after incubation, wash the cells with 1xPBS, centrifuge at 1200rpm for 5min at room temperature, discard the supernatant, resuspend the cells with 1xPBS. Before flow cytometry, store the cells on ice and avoid light.

[0062] 2. Experimental results

[0063] The cell killing experiment results of the Enoblituzumab antibody are as shown in Figure 2 The cell killing experiment results of the anti-B7H3 nanobody 8F1 are as shown in Figure 3 It can be seen that the anti-B7H3 nanobody 8F1 antibody of the present application can effectively kill tumor cells, and the killing effect is better than that of the control antibody Enoblituzumab.

[0064] In summary, the present application provides an anti-B7H3 nanobody and a preparation method and use thereof. The anti-B7H3 nanobody has high affinity to the B7H3 antigen and better tumor cell killing ability than the known antibody molecule Enoblituzumab. The anti-B7H3 nanobody provided by the present application is a nanobody itself, which maximally reduces the immune risk caused by heterogeneity, is more conducive to penetrating the blood-brain barrier, is easier to reach the inside of the tumor to play a therapeutic effect, and has a broad application prospect in the preparation of drugs for preventing and / or treating tumors.

Claims

1. An anti-B7H3 Nanobody, characterized in that, It comprises complementary determining regions CDR1-CDR3, the amino acid sequence of CDR1 is RTLSNYA, the amino acid sequence of CDR2 is LRRIGDYIDSA, and the amino acid sequence of CDR3 is AAGPTINKDEYSYPGATGG.

2. The anti-B7H3 Nanobody of claim 1, wherein, It also comprises four framework regions FR1-FR4 alternately connected with the complementary determining regions CDR1-CDR3, the amino acid sequence of FR1 is QLQLVESGGGLVQAGASLRLSCAASG, the amino acid sequence of FR2 is MGWFRQAPGKEREFVGV, the amino acid sequence of FR3 is DSADSVKGRFTFSRDNAKNTLYLYMTSLKPEDTAVYYC, and the amino acid sequence of FR4 is WGQGTQVTVSS.

3. The anti-B7H3 Nanobody of claim 2, wherein, The amino acid sequence thereof is: QLQLVESGGGLVQAGASLRLSCAASGRTLSNYAMGWFRQAPGKEREFVGVLRRIGDYIDSADSADSVKGRFTFSRDNAKNTLYLYMTSLKPEDTAVYYCAAGPTINKDEYSYPGATGGWGQGTQVTVSS.

4. A polynucleotide molecule, characterized in that, The nucleotide sequence thereof is shown as SEQ ID NO:

9.

5. An expression vector, characterized by, It comprises the polynucleotide molecule of claim 4.

6. A host cell, characterized in that, It comprises the expression vector of claim 5.

7. A method of preparing the anti-B7H3 Nanobody according to any one of claims 1-3, characterized in that, The method comprises the following steps: (1) connecting the polynucleotide molecule of claim 4 into an expression vector to obtain a positive plasmid; (2) transforming a host cell with the positive plasmid and inducing expression of the anti-B7H3 nanobody.

8. Use of the nanobody of any one of claims 1-3 in the preparation of a medicament for preventing and / or treating a tumor, the tumor being a glioma, a melanoma, a renal cell carcinoma, a pancreatic cancer, a breast cancer, a lung cancer, a prostate cancer, or a head and neck squamous cell carcinoma.

9. A medicament, characterized by comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof. It is a preparation prepared by taking the nanobody of any one of claims 1-3 as an active ingredient and adding a pharmaceutically acceptable adjuvant.

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