A nanobody and use thereof in the preparation of a medicament for preventing and / or treating a tumor

Abstract

The application provides an anti-B7H3 nanobody and application thereof in preparation of a medicine for preventing and / or treating tumors, 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 tumors to play a therapeutic effect, and has a wide application prospect in preparation of the medicine for preventing and / or treating tumors.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to an anti-B7H3 nanobody and its use in the preparation of drugs for the prevention and / or treatment of tumors. Background Technology

[0002] B7 homolog 3 (B7H3), also known as CD276, is a member of the immunoglobulin superfamily and is a type I transmembrane glycoprotein containing two Ig-like C2 (immunoglobulin-like) domains and two Ig-like V (immunoprotein-like) domains. B7H3 participates in regulating T cell-mediated immune responses. It also plays a protective role in tumor cells by inhibiting natural killer-mediated cell lysis and serves as a biomarker for detecting neuroblastoma cells. Furthermore, B7H3 is involved in the development of acute and chronic transplant rejection and the regulation of mucosal surface lymphocyte activity. It also plays a crucial role in providing a suitable immune environment for the placenta and fetus throughout pregnancy.

[0003] B7H3 is not expressed in monocytes, granulocytes, or normal human tissues, but it is expressed in some dendritic cells, T cells, natural killer (NK) cells, and B cells, making it a newly discovered immune checkpoint in recent years. This target can not only promote immune escape of tumor cells by inhibiting the immune system, but also promote cancer progression through non-immune pathways such as tumor metastasis, drug resistance, and angiogenesis. Studies have found that B7H3 is abnormally highly expressed in various tumor tissues, including non-small cell lung cancer, pancreatic cancer, primary liver cancer, colorectal cancer, breast cancer, prostate cancer, laryngeal cancer, and melanoma. It is also expressed on the surface of stromal cells, fibroblasts, and epithelial cells in the tumor microenvironment (TME). Overexpression of B7H3 in tumor tissues is often associated with poor prognosis and shorter overall survival and progression-free survival.

[0004] 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

[0005] The purpose of this invention is to provide an anti-B7H3 nanobody and an anti-tumor drug using the anti-B7H3 nanobody as the active ingredient.

[0006] The present invention provides a nucleotide molecule whose nucleotide sequence is shown in SEQ ID NO:9.

[0007] The present invention also provides an expression vector comprising a nucleotide molecule with a nucleotide sequence as shown in SEQ ID NO:9.

[0008] The present invention also provides a host cell comprising the above-described expression vector.

[0009] The present invention also provides a nanobody comprising complementarity-determining regions CDR1-CDR3, wherein the amino acid sequence of CDR1 is FTFSRYG, the amino acid sequence of CDR2 is IYSDGST, and the amino acid sequence of CDR3 is TRGTGGSHEADSGSTRWG.

[0010] Furthermore, the nanobody also includes four framework regions FR1-FR4 alternately linked with complementarity-determining regions CDR1-CDR3. The amino acid sequence of FR1 is EVQVVESGGGLAQPGGSLRLSC AASG, the amino acid sequence of FR2 is MGWARQVPGKGLEWVSG, the amino acid sequence of FR3 is FYARSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC, and the amino acid sequence of FR4 is RGQGTQVTVSS.

[0011] Furthermore, the amino acid sequence of the nanobody is shown in SEQ ID NO:8.

[0012] The present invention also provides a method for preparing the above-mentioned nanobody, the method comprising the following steps:

[0013] (1) A positive plasmid is obtained by ligating a nucleotide molecule with a nucleotide sequence as shown in SEQ ID NO:9 into an expression vector;

[0014] (2) Transform host cells with positive plasmids to induce the expression of nanobodies.

[0015] The present invention also provides the use of the above-mentioned nanobodies in the preparation of medicaments for the prevention and / or treatment of tumors.

[0016] Furthermore, the tumor is gastric cancer, glioma, melanoma, renal cell carcinoma, pancreatic cancer, breast cancer, lung cancer, prostate cancer, bile duct cancer, head and neck squamous cell carcinoma, or cervical cancer.

[0017] Furthermore, the tumor is a tumor.

[0018] The present invention also provides a drug for the prevention and / or treatment of tumors, which is a formulation prepared by using the above-mentioned nanobody as the active ingredient and pharmaceutically acceptable excipients.

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

[0020] The present invention has achieved the following beneficial effects:

[0021] The anti-B7H3 nanobody of the present invention has a high affinity for the B7H3 protein and has broad application prospects in the preparation of drugs for the prevention and / or treatment of tumors.

[0022] The anti-B7H3 nanobody of the present invention has a molecular weight of approximately 15 kDa, which minimizes the immune risks caused by heterogeneity, makes it more conducive to penetrating the blood-brain barrier, and makes it easier to reach the tumor to exert a therapeutic effect.

[0023] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0024] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0025] Figure 1 The results show the affinity assay between nanobody 16B9 and B7H3.

[0026] Figure 2 The results are obtained by kinetic biomembrane interferometry detection of nanobodies 16B9 and B7H3.

[0027] Figure 3 The results are obtained by kinetic biomembrane interference assay for the full-length antibody Enoblituzumab and B7H3. Detailed Implementation

[0028] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0029] Example 1: Preparation of anti-B7H3 nanobodies

[0030] 1. Constructing expression carriers

[0031] (1) Restriction vector digestion: The tool vector pcDNA3.1-x-IgG1 was digested with BamHI / EcoRI enzymes (purchased from Yisheng Biotechnology). The double digestion system is shown in Table 1. After double digestion at 37℃ for 5 h, the vector was recovered using a PCR product recovery kit (Cycle-PureKit PCR product purification kit OMEGA D6492-01).

[0032] Table 1. BamHI / EcoRI double enzyme digestion plasmid system

[0033] Element Dosage pcDNA3.1-x-IgG1 plasmid 10ug 10×buffer 10μl BamHI 2.5μl EcoRI 2.5μl <![CDATA[ddH2O]]> Add to 100 μl

[0034] (2) Homologous recombination: The nucleotide fragments used for recombinant expression were diluted 20-fold with ddH2O. 1 μL of the diluted sample was used for homologous recombination with the vector recovered by the above enzyme digestion (recombinase, NovoRec Plus one step PCR Cloning Kit, nearshore protein catalog number NR005-01B). The homologous recombination system is shown in Table 2. The homologous recombination system was reacted at 50℃ for 30 min on a PCR instrument. 100 μl of TOP10 competent cells were added to each tube of homologous recombination product for transformation (placed on ice for 20 min, heat-shocked at 42℃ for 90 s, immediately placed on ice for 2 min, added 800 μl of LB medium, and cultured at 37℃ and 220 rpm for 25-45 min. Plated on Amp-resistant plates and incubated overnight at 37℃).

[0035] Table 2 Homologous recombination reaction system

[0036] Element Dosage pcDNA3.1-x-IgG1 (BamHI / EcoRI) digestion of large vector fragment 100ng Gel extraction and recovery of PCR products 50ng 5×buffer 2μl Recombinase 0.5μl <![CDATA[ddH2O]]> Add to 10 μl

[0037] The sequence of the nucleotide fragment used for recombinant expression is as follows:

[0038] GAAGTGCAGGTGGTGGAAAGCGGCGGCGGCCTGGCGCAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCTTTACCTTTAGCCGCTATGGCATGGGCTGGGCGCGCCAGGTGCCGGGCAAAGGCCTGGAATGGGTGAGCGGCATTTATAGCGATGGCAGCACCTTTTATGCGCGCAGCGTG AAAGGCCCTTTACCATTAGCCGCGATAACGCGAAAAACACCGTGTATCTGCAGATGAACAGCCTGAAACCGGAAGATACCGCGGTGTATTATTGCACCCGCGGCACCGGCGGCAGCCATGAAGCGGATAGCGGCAGCACCCGCTGGGGCCGCGCCAGGGCACCCAGGTGACCGTGAGCAGCTAA(SEQ ID NO:9).

[0039] (3) Identification of Escherichia coli culture by PCR: Single colonies of Escherichia coli were picked from the plate and cultured in 200 μL of LLB medium (1L solution: 10g tryptone (Oxoid), 5g yeast extract (Oxoid), 10g sodium chloride) at 37℃ and 220rpm for 3h. 1 μL of the culture was used as a template for culture PCR identification. Positive clones were selected for sequencing. The PCR products were electrophoretically excised and recovered (Gel Extraction Kit OMEGA, catalog number D2500-01). Homologous recombination was performed again. After culture PCR identification, positive clones were selected for sequencing.

[0040] 2. Expression and purification of anti-B7H3 nanobodies in Hek293F cells

[0041] (1) Antibody expression

[0042] The bacterial strain was inoculated into 20 ml of LB medium containing ampicillin and incubated overnight at 37°C. Plasmids were extracted using a plasmid extraction kit (Plasmid Miniprep Kit II, BevoMed Cat: BW-PD1213). Hek293 cells were passaged to maintain good cell growth with a viability greater than 95%. The Hek293 cell density was adjusted to 2.5 × 10⁻⁶ cells / year at transfection. 6cells / ml. Take 50 μg of expression plasmid and add it to 1 ml of OPM-293CD03 medium (Shanghai Aopumai Biotechnology Co., Ltd.) and mix well. Take 150 μg of PEI and add it to 1 ml of OPM medium and mix well. After mixing the two, shake well and let stand at room temperature for 30 min. Then add it to 50 ml of Hek293 cells and culture in a CO2 shaker. On the second day, add 5% final volume of OPM medium and continue to culture until day 7. Centrifuge at 10000 rpm for 20 min to harvest the cell culture supernatant for protein purification.

[0043] (2) Antibody purification

[0044] Cell expression supernatant was filtered through a 0.22 μm filter membrane and then loaded onto a Protein A column (Chutian Microsphere Biotechnology (Changsha) Co., Ltd., TH-protein A, Y5001). Protein was eluted with 0.1 M Gly-HCl at pH 3.0, and 10% (v / v) of 1 M Tris-HCl at pH 8.8 was quickly added for neutralization. Protein concentration was determined using the A280 method with a micro-spectrophotometer. The concentration of 16B9 protein was measured to be 1 mg / ml, with a purity of not less than 95%.

[0045] The amino acid sequence of the target protein 16B9 is shown in SEQ ID NO:8. It includes complementarity-determining regions CDR1-CDR3, which are separated by four frame regions FR1, FR2, FR3 and FR4.

[0046] Table 3. Amino acid sequence of the target protein

[0047]

[0048] The following experimental examples demonstrate the activity of the anti-B7H3 nanobody of the present invention.

[0049] Experimental Example 1: Affinity Detection of Anti-B7H3 Nanobody with B7H3

[0050] 1. Experimental Methods

[0051] The microplate was coated with 5 μg / ml B7H3-His antigen and incubated overnight at 4°C. The coating solution was discarded, and the plate was washed twice with PBS and blocked at 37°C for 2 h. The anti-B7H3 nanobody 16B9 and the known monoclonal antibody Enoblituzumab were added in 3-fold serial dilutions starting at 500 nM and incubated at 37°C for 1 h. The plate was washed five times with 0.1% PBST (incubated for approximately 2-3 min each time), and horseradish peroxidase-conjugated Fc secondary antibody (GoatAnti-Human IgG-Fc Secondary Antibody (HRP), prepared with 5% skim milk powder at a 1:10000 ratio) was added and incubated at 37°C for 45 min. The experimental results of the 16B9 antibody and the Enoblituzumab antibody were detected and compared, and the EC50 was calculated. 50 (half-maximal effect concentration).

[0052] 2. Experimental Results

[0053] Table 4. Results of Affinity ELISA Test

[0054] name 16B9 Enoblituzumab <![CDATA[EC 50 (nM)]]> 0.2775 0.6139

[0055] Affinity ELISA test results as follows Figure 1 As shown in Table 4, it can be seen that the anti-B7H3 nanobody 16B9 of the present invention can target the B7H3 protein.

[0056] Furthermore, the anti-B7H3 nanobody 16B9 of this invention has a better affinity for B7H3 than the control antibody Enoblituzumab.

[0057] Experimental Example 2: Biomembrane Interferometry Detection of B7H3 Kinetics by Anti-B7H3 Nanobody

[0058] 1. Experimental Methods

[0059] Add 200 μl / well of equilibration buffer (PBS solution + 0.02% Tween-20 + 0.2% BSA, pH 7.2-7.4, 0.22 μm filtered) to column 1 of a 96-well plate and set the probe equilibration time to 300 s. Dilute the 16B9 antibody to 100 nM with equilibration buffer and add 200 μl / well to column 2 of the 96-well plate, setting the probe loading time to 120 s. Add 200 μl / well of equilibration buffer to column 3 of the 96-well plate and set the probe equilibration time to 300 s. Dilute the antigen to five concentrations (200, 100, 50, 25, 12.5, 0 nM) with equilibration buffer and add 200 μl / well to column 4 of the 96-well plate, setting the probe binding time to 60 s. Dissociation: Add 200 μl / well of equilibration buffer to column 5 of the 96-well plate and set the probe dissociation time to 300 s.

[0060] The biomembrane interference method for B7H3 kinetics using the 16B9 antibody was followed, with the only difference being that the 16B9 antibody was replaced with Enoblituzumab, and the affinity of Enoblituzumab for B7H3-His was detected.

[0061] 2. Experimental Results

[0062] Table 5. Affinity of anti-B7H3 nanobody 16B9 with B7H3-His

[0063]

[0064] Table 6. Affinity of Enoblituzumab with B7H3-His

[0065]

[0066] The results of the biomembrane interference (BLI) assay for the anti-B7H3 nanobody 16B9 are as follows: Figure 2 Table 5 shows the results of the biomembrane interference (BLI) assay for Enoblituzumab antibody. Figure 3 As shown in Table 6, it can be seen that the 16B9 antibody of the present invention has a high affinity for the B7H3 protein and is superior to Enoblituzumab.

[0067] The above results indicate that the anti-B7H3 nanobody 16B9 of the present invention has a better affinity for B7H3 than the control antibody Enoblituzumab.

[0068] In summary, this invention provides an anti-B7H3 nanobody and its use in the preparation of drugs for the prevention and / or treatment of tumors. This nanobody exhibits a high affinity for B7H3, superior to the known antibody molecule Enoblituzumab. The anti-B7H3 nanobody provided by this invention is itself a nanobody, while minimizing the immune risks associated with heterologous origin, making it more conducive to penetrating the blood-brain barrier and more easily reaching the tumor to exert its therapeutic effect. It has broad application prospects in the preparation of drugs for the prevention and / or treatment of tumors.

Claims

1. A nucleotide molecule, characterized in that, Its nucleotide sequence is shown in SEQ ID NO:

9.

2. An expression carrier, characterized in that, It comprises the nucleotide molecule as described in claim 1.

3. A host cell, characterized in that, It comprises the expression vector as described in claim 2.

4. A nanobody, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

8.

5. A method for preparing the nanobody of claim 4, characterized in that, The method includes the following steps: (1) The nucleotide molecule of claim 1 is ligated into an expression vector to obtain a positive plasmid; (2) Transform host cells with positive plasmids to induce the expression of nanobodies.

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