Nanometer antibody and application thereof in preparation of medicine for preventing and / or treating tumors

Abstract

The invention provides an anti-B7H3 nano antibody and application thereof in preparation of medicines for preventing and / or treating tumors, and belongs to the field of biological medicines. The nano antibody has high affinity with the B7H3 protein, reduces the immune risk caused by heterology to the greatest extent, is more beneficial to penetrating the blood brain barrier, is easier to reach the interior of a tumor to play a therapeutic effect, and has a wide application prospect in preparation of drugs for preventing and / or treating the tumor.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to an anti-B7H3 nano antibody and use thereof in preparing a drug for preventing and / or treating tumors. Background Art

[0002] B7 homolog 3 (B7H3), a member of the immunoglobulin superfamily, also known as CD276, is a type I transmembrane glycoprotein that contains two Ig-like C2-type (immunoglobulin-like) domains and two Ig-type V-type (immunoprotein-like) domains. B7H3 can participate in the regulation of T cell-mediated immune responses. B7H3 also plays a protective role in tumor cells by inhibiting natural killer-mediated cell lysis and serves as a marker for the detection of neuroblastoma cells. In addition, B7H3 is involved in the development of acute and chronic transplant rejection and the regulation of lymphocyte activity at mucosal surfaces. It can also play a key role in providing a suitable immune environment for the placenta and fetus throughout pregnancy.

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

[0004] Enoblituzumab is a humanized IgG1κ monoclonal antibody that can recognize human B7H3 protein. However, the molecular weight of Enoblituzumab is 150KDa, which is not conducive to penetrating the blood-brain barrier. In order to solve the above problems, it is urgent to develop a humanized nanobody against B7H3. Summary of the invention

[0005] The purpose of the present invention is to provide an anti-B7H3 nanobody and an anti-tumor drug using the anti-B7H3 nanobody as an 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, which comprises a nucleotide molecule with a nucleotide sequence as shown in SEQ ID NO:9.

[0008] The present invention also provides a host cell, which comprises the above expression vector.

[0009] The present invention also provides a nanobody, which includes complementary determining regions CDR1-CDR3, 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 connected to the complementary 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) connecting the nucleotide molecule with the nucleotide sequence as shown in SEQ ID NO: 9 to an expression vector to obtain a positive plasmid;

[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 nano-antibody in preparing a drug for preventing and / or treating 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] Further, the tumor is a tumor.

[0018] The present invention also provides a drug for preventing and / or treating tumors, which is a preparation prepared with the above-mentioned nano antibody as an active ingredient and pharmaceutically acceptable excipients.

[0019] As is well 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, bile duct cancer, head and neck squamous cell carcinoma, cervical cancer, etc.

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

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

[0022] The anti-B7H3 nanoantibody of the present invention has a molecular weight of about 15 KDa, which reduces the immune risk caused by heterogeneity to the greatest extent, 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.

[0023] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0024] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the affinity test result of nano antibody 16B9 and B7H3.

[0026] Figure 2 This is the result of kinetic biomembrane interferometry detection of nanoantibody 16B9 and B7H3.

[0027] Figure 3 This is the result of the kinetic biomembrane interferometry test between the full-length antibody Enoblituzumab and B7H3. DETAILED DESCRIPTION

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

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

[0030] 1. Construction of expression vector

[0031] (1) Enzyme digestion of backbone vector: The tool vector pcDNA3.1-x-IgG1 was digested with BamH I / EcoR I enzymes (purchased from Yisheng Biotechnology). The double enzyme digestion plasmid system is shown in Table 1. After double enzyme digestion at 37°C for 5 h, the vector was recovered using a PCR product recovery kit (Cycle-PureKitPCR product purification kit OMEGA D6492-01).

[0032] Table 1 BamH I / EcoR I double restriction enzyme digestion plasmid system

[0033] Element Dosage pcDNA3.1-x-IgG1 plasmid 10ug 10×buffer 10μl B H 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 times with ddH2O, and 1 μL of the diluted sample was taken for homologous recombination with the vector recovered by the above enzyme digestion (recombinase, NovoRec Plus one step PCR CloningKit nearshore protein product number NR005-01B). The homologous recombination system is shown in Table 2. The homologous recombination system was reacted on a PCR instrument at 50°C for 30 min. 100 μL TOP10 competent cells were added to each tube of homologous recombination product for transformation (placed on ice for 20 min, heat shocked at 42°C for 90 s, immediately placed on ice for 2 min, added with 800 μL LB medium, and cultured at 37°C, 220 rpm for 25-45 min. Spread on Amp-resistant plate culture medium and cultured in a plate incubator at 37°C overnight).

[0035] Table 2 Homologous recombination reaction system

[0036] Element Dosage pcDNA3.1-x-IgG1 (BamHI / EcoRI) digested vector large fragment 100ng Gel excision 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:

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

[0039] (3) PCR identification of E. coli culture medium: single clone E. coli colonies were picked from the plate and placed in 200 μL LB medium (1 L solution: 10 g tryptone (Oxoid), 5 g yeast extract (Oxoid), 10 g sodium chloride), cultured at 37°C, 220 rpm for 3 h, 1 μL of the culture medium was taken as a template for culture medium PCR identification, and the identified positive clones were selected for sequencing. After the PCR products were recovered by gel electrophoresis (Gel Extraction Kit OMEGA, Cat. No. D2500-01), homologous recombination was performed again. After culture medium PCR identification, the identified positive clones were selected for sequencing.

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

[0041] (1) Antibody expression

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

[0043] (2) Antibody purification

[0044] The cell expression supernatant was filtered with a 0.22 μm filter membrane and loaded onto a protein A column (Truth Microsphere Biotechnology (Changsha) Co., Ltd., TH-protein A, Y5001). The protein was eluted with 0.1 M Gly-Hcl at pH 3.0 and quickly neutralized by adding 10% volume of 1 M Tris-Hcl at pH 8.8. The protein concentration was measured using a micro-spectrophotometer to measure A280. The 16B9 protein concentration was determined to be 1 mg / ml and the purity was not less than 95%.

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

[0046] Table 3 Amino acid sequence of target protein

[0047]

[0048] The activity of the anti-B7H3 nanobody of the present invention is demonstrated by experimental examples below.

[0049] Experimental Example 1: Affinity detection of anti-B7H3 nanobody and B7H3

[0050] 1. Experimental Methods

[0051] Coat the ELISA plate with 5 μg / ml B7H3-His antigen at 4°C overnight. Discard the coating solution, wash twice with PBS, and block at 37°C for 2h. Add the anti-B7H3 nanoantibody 16B9 of the present invention and the known monoclonal antibody Enoblituzumab (Ibutol) at a 3-fold dilution starting from 500nM, respectively, and incubate at 37°C for 1h. Wash 5 times with 0.1% PBST (standing for about 2-3min each time), add horseradish peroxidase-labeled Fc secondary antibody (GoatAnti-Human IgG-Fc Secondary Antibody (HRP) brand, prepared with 5% skim milk powder at 1:10000), and incubate at 37°C for 45min. Detect and compare the experimental results of 16B9 antibody and Enoblituzumab antibody, and calculate EC 50 (half maximal effect concentration).

[0052] 2. Experimental results

[0053] Table 4 Affinity ELISA test results

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

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

[0056] Furthermore, the affinity of the anti-B7H3 nanobody 16B9 of the present invention to B7H3 is superior to that of the control antibody Enoblituzumab.

[0057] Experimental Example 2: Biomembrane interferometry detection of B7H3 dynamics by anti-B7H3 nanobodies

[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 filter) to the first column of the 96-well detection plate, and set the probe to equilibrate for 300 s. Dilute the 16B9 antibody to 100 nM with equilibration buffer, add 200 μl / well to the second column of the 96-well detection plate, and set the probe to load the antibody for 120 s. Add 200 μl / well of equilibration buffer to the third column of the 96-well detection plate, and set the probe to equilibrate for 300 s. Dilute the antigen to 5 concentrations (200, 100, 50, 25, 12.5, 0 nM) with equilibration buffer, and then add it to the fourth column of the 96-well detection plate in sequence, 200 μl / well, and set the probe to bind for 60 s. Dissociation: Add 200 μl / well of equilibration buffer to the fifth column of the 96-well detection plate, and set the probe to dissociate for 300 s.

[0060] Referring to the above-mentioned biomembrane interferometry steps for the kinetics of B7H3 with 16B9 antibody, the only difference is that 16B9 antibody is replaced with Enoblituzumab, and the affinity of Enoblituzumab to B7H3-His is detected.

[0061] 2. Experimental results

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

[0063]

[0064] Table 6 Affinity of Enoblituzumab and B7H3-His

[0065]

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

[0067] The above results indicate that the affinity of the anti-B7H3 nanobody 16B9 of the present invention to B7H3 is superior to that of the control antibody Enoblituzumab.

[0068] In summary, the present invention provides an anti-B7H3 nanobody and its use in the preparation of drugs for preventing and / or treating tumors. The nanobody has a high affinity with B7H3 and is superior to the known antibody molecule Enoblituzumab. The anti-B7H3 nanobody provided by the present invention is a nanobody itself, and at the same time, the immune risk caused by heterogeneity is reduced to the greatest extent, and it is more conducive to penetrating the blood-brain barrier, and it is easier to reach the inside of the tumor to exert a therapeutic effect, and has broad application prospects in the preparation of drugs for preventing and / or treating tumors.

Claims

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

9.

2. An expression vector, characterized in that: It comprises the nucleotide molecule according to claim 1.

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

4. A nanobody, characterized in that It includes complementary determining regions CDR1-CDR3, 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.

5. The Nanobody according to claim 4, characterized in that It also includes four framework regions FR1-FR4 alternately connected to the complementary determining regions CDR1-CDR3, the amino acid sequence of FR1 is EVQVVESGGGLAQPGGSLRLSCAASG, the amino acid sequence of FR2 is MGWARQVP GKGLEWVSG, and the amino acid sequence of FR3 is FYARSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC.

6. The Nanobody according to claim 5, characterized in that Its amino acid sequence is shown in SEQ ID NO:

8.

7. A method for preparing the nanobody according to any one of claims 4 to 6, characterized in that: The method comprises the following steps: (1) connecting the nucleotide molecule described in claim 1 to an expression vector to obtain a positive plasmid; (2) Transform host cells with positive plasmids to induce the expression of nanobodies.

8. Use of the Nanobody according to any one of claims 4 to 6 in the preparation of a medicament for preventing and / or treating tumors.

9. The use according to claim 8, characterized in that 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.

10. A drug for preventing and / or treating tumors, characterized in that: The nanometer antibody is prepared by using the nanometer antibody as an active ingredient and adding pharmaceutically acceptable excipients.

Images