Anti-B7H3 nano antibody as well as preparation method and application thereof

Abstract

The invention provides an anti-B7H3 nano antibody as well as a preparation method and application thereof, 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 treatment effect, and has a wide application prospect in preparation of medicines 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 a preparation method and use thereof. Background Art

[0002] B7H3 (also known as CD276) is a type I transmembrane protein that belongs to the B7 immune co-stimulatory and co-inhibitory family. Current studies have shown that it has a dual role, acting as both a co-stimulatory molecule and a co-inhibitory molecule, and its specific role depends on the immune cells involved and the microenvironmental conditions.

[0003] In humans, B7H3 consists of an extracellular region, a transmembrane region, and a short intracellular region with no known signal motifs. There are two isoforms of B7H3: 2IgB7H3 consists of a pair of immunoglobulin variable region (IgV)-like and immunoglobulin constant region (IgC)-like extracellular domains, and 4IgB7H3 contains two identical pairs of IgV-like and IgC-like extracellular domains, the latter being the major isoform in human cells. The exact function and receptors of B7H3 have not yet been elucidated. In non-malignant tissues, B7H3 plays an important role in adaptive immunity by regulating T cell function.

[0004] Although early studies supported the view that B7H3 acts as a co-stimulatory molecule required for T cell activation, more recent studies have shown that B7H3 has a primarily inhibitory role in adaptive immunity, inhibiting T cell activation, proliferation, and release of effector cytokines, primarily IFN-γ and IL-2. In mice, B7H3 appears to have antitumor functions mediated by CD8+ T cell and NK cell activation, likely through binding to the Trem-like transcript 2 (TLT-2) receptor on CD8+ T cells. Surprisingly, in humans, only three studies have shown a correlation between high B7H3 expression and improved prognosis. Instead, there is overwhelming evidence that B7H3 has a protumor effect. Indeed, a large body of preclinical and clinical evidence suggests that B7H3 suppresses tumor antigen-specific immune responses through multiple 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 aggressive cancer biology and metastatic potential, and ultimately poor prognosis. B7H3 also appears 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 a 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 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

[0006] The object of the present invention is to provide an anti-B7H3 nanobody and a preparation method and use thereof.

[0007] The present invention provides an anti-B7H3 nanobody, which comprises complementary 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 connected to the complementary 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:

[0010] QLQLVESGGGLVQAGASLRLSCAASGRTLSNYAMGWFRQAPGKER EFVGVLRRIGDYIDSADSADSVKGRFTFSRDNAKNTLYLYMTSLKPEDTA VYYCAAGPTINKDEYSYPGATGGWGQGTQVTVSS.

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

[0012] The present invention also provides an expression vector, which comprises the above-mentioned nucleotide molecule.

[0013] The present invention also provides a host cell, which comprises the above 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) connecting the above-mentioned nucleotide molecule to an expression vector to obtain a positive plasmid;

[0016] (2) Transform host cells with positive plasmids to induce the expression of nanobodies.

[0017] The present invention also provides the use of the above-mentioned nano-antibody in preparing a drug for preventing and / or treating tumors.

[0018] Furthermore, the tumor is 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.

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

[0020] Furthermore, the drug for preventing and / or treating tumors is a preparation prepared with the aforementioned nano-antibody as an active ingredient and pharmaceutically acceptable excipients.

[0021] The present invention has achieved the following beneficial effects:

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

[0023] The anti-B7H3 nanoantibody of the present invention has a molecular weight as low as 15KDa. This antibody is an anti-B7H3 antibody, which reduces the immune risk caused by heterogeneity to the greatest extent, is more conducive to penetrating the blood-brain barrier, and is easier to reach the inside of the tumor to exert a therapeutic effect.

[0024] 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.

[0025] 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

[0026] Figure 1 This is the affinity test result of anti-B7H3 nanobody and B7H3.

[0027] Figure 2The cell killing effect of Enoblituzumab antibody.

[0028] Figure 3 This is the cell killing effect of the anti-B7H3 nanobody 8F1. DETAILED DESCRIPTION

[0029] The raw materials and equipment used in the present invention are all 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) 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).

[0033] Table 1 BamH I / EcoR I double restriction enzyme digestion plasmid system

[0034] Element Dosage pcDNA3.1-x-IgG1 plasmid 10 μg 10×buffer 10μl B H 2.5μl EcoRI 2.5μl <![CDATA[ddH2O]]> Add to 100 μl

[0035] (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).

[0036] Table 2 Homologous recombination reaction system

[0037] 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

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

[0039] CAGCTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGGGGCGCGAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCCGCACCCTGAGCAACTATGCGATGGGCTGGTTTCGCCAGGCGCCGGGCAAAGAACGCGAATTTGTGGGCGTGCTGCGCCGCATTGGCGATTATATTGATAGCGCGGATAGCGCGGATAG CGTGAAAGGCCGCTTTACCTTTAGCCGCGATAACGCGAAAAACACCCTGTATCTGTATATGACCAGCCTGAAACCGGAAGATACCGCGGTGTATTATTGCGCGGCGGGCCCGACCATTAACAAAGATGAATATAGCTATCCGGGCGCGACCGGCGGCTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGCTAA(SEQ ID NO:9).

[0040] (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.

[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 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.

[0044] (2) Antibody purification

[0045] The cell expression supernatant was filtered with a 0.22 μm filter membrane and loaded onto a protein A column. 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 detected by measuring A280 with a micro-spectrophotometer. The 8F1 protein concentration was determined to be 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 complementarity determining regions CDR1-CDR3, and the complementarity determining regions 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 invention is demonstrated by experimental examples below.

[0051] Experimental Example 1: Affinity detection of anti-B7H3 nanobody and B7H3

[0052] 1. Experimental Methods

[0053] 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 8F1 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 8F1 antibody and Enoblituzumab antibody, and calculate EC 50 (half maximal effect concentration).

[0054] 2. Experimental results

[0055] Table 4 Affinity ELISA test results

[0056] name 8F1 Enoblituzumab <![CDATA[EC 50 (nM)]]> 0.4701 0.3264

[0057] Affinity ELISA test results Figure 1 As shown in Table 4, it can be seen that the anti-B7H3 nanobody 8F1 of the present invention is able to target the B7H3 protein.

[0058] Furthermore, the anti-B7H3 nanobody 8F1 of the present invention has an affinity for B7H3 protein comparable to that of the control antibody Enoblituzumab.

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

[0060] 1. Experimental Methods

[0061] Calcein-AM (BioLegend, 425201 / 10x50μg) was first used to label the target cells U-118MG (human brain astroglioblastoma), and the cells were incubated at 37°C for 30 minutes and then centrifuged and resuspended. The anti-B7H3 nanobody 8F1 (SEQ ID NO: 8) of the present invention and the control antibody Enoblituzumab were added to each well at a concentration of 100nM, followed by the addition of 1.8×10 6 NK cells, 1.5×10 4Target tumor cells (NK cells: target tumor cells = 12:1), co-incubated for 4 hours, all solutions in each well were transferred to a centrifuge tube, cells were washed with 1×PBS, and adherent cells were digested with trypsin. All collected cells were centrifuged at 1200rpm for 5 minutes. The cell pellet was washed with 1×PBS and centrifuged at 1200rpm for 5 minutes, and washed twice in total. 5μL APCAnnexinV dye solution was added; 5μL 7-AAD viability dye solution was added at the same time; the cells were gently inverted upside down, and after a brief centrifugation, they were incubated in the dark at room temperature (25°C) for 15 minutes; after the incubation, the cells were washed with 1×PBS, centrifuged at room temperature for 5 minutes at 1200rpm, the supernatant was discarded, and the cells were resuspended with 1×PBS. Before flow cytometry, the cells were kept on ice and protected from light.

[0062] 2. Experimental results

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

[0064] In summary, the present invention provides an anti-B7H3 nanobody and its preparation method and use. The anti-B7H3 nanobody has a high affinity with the B7H3 antigen and has a better ability to kill tumor cells than 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. An anti-B7H3 nanobody, characterized in that: It includes 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 according to claim 1, 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 QLQLVESGGGLVQAGASLRLSCAASG, the amino acid sequence of FR2 is MGWFR QAPGKEREFVGV, the amino acid sequence of FR3 is DSADSVKGRFTFSRDNAKNTLYLYMTSLKPEDTAVYYC, and the amino acid sequence of FR4 is WGQGTQVTVSS.

3. The anti-B7H3 nanobody according to claim 2, characterized in that Its amino acid sequence is: QLQLVESGGGLVQAGASLRLSCAASGRTLSNYAMGWFRQAPGKER EFVGVLRRIGDYIDSADSADSVKGRFTSFSRDNAKNTLYLYMTSLKPEDTA VYYCAAGPTINKDEYSYPGATGGWGQGTQVTVSS.

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

9.

5. An expression vector, characterized in that: It comprises the nucleotide molecule according to claim 4.

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

7. A method for preparing the anti-B7H3 nanobody according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: (1) connecting the nucleotide molecule described in claim 4 to an expression vector to obtain a positive plasmid; (2) Transform host cells with the positive plasmid to induce the expression of anti-B7H3 nanobody.

8. Use of the Nanobody according to any one of claims 1 to 3 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 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.

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