Anti-tgf beta humanized nanobodies for the prevention and / or treatment of tumors

Abstract

The application provides an anti-TGF beta humanized nanobody for preventing and / or treating tumors, and belongs to the technical field of biotechnology.The humanized nanobody can block the combination of TGF beta and its receptor while keeping the affinity with TGF beta, and has a wide application prospect in the preparation of drugs for preventing and / or treating tumors.The humanized nanobody has a low molecular weight, reduces the immunological risk caused by heterogeneity, is more conducive to penetrating the blood-brain barrier, and is easier to reach the inside of tumors to exert a therapeutic effect.The humanized nanobody can improve the tumor treatment effect when used in combination with a PD-L1 antibody.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a humanized anti-TGFβ nanobody for the prevention and / or treatment of tumors, its preparation method, and its uses. Background Technology

[0002] Numerous studies have shown that the development and progression of cancer are inextricably linked to abnormal transduction of multiple intracellular signaling pathways. The transforming growth factor beta (TGFβ) signaling pathway is one of them.

[0003] TGFβ can inhibit the expression of the proto-oncogene c-myc. However, during tumor development, with the introduction of mutations or changes in epigenetic modifications, cancer cells gradually become resistant to the inhibitory effect of TGFβ signaling, ultimately leading to tumor progression. Current research indicates that in advanced tumor stages, TGFβ tends to promote tumor deterioration.

[0004] Recent studies have found that increased TGFβ in the tumor microenvironment is associated with immune escape. Increased TGFβ increases T cell rejection and blocks the infiltration of TH1 effector T cells. Tauriello et al. found that TGFβ blockade made a mouse model of liver metastases more sensitive to PD1 / PDL1 therapy. Similarly, Mariathasan et al. reported that the combination of TGFβ blockade and PD-L1 antibody downregulates the TGFβ signaling pathway in stromal cells, promoting T cell infiltration into the tumor and activating a strong anti-tumor immune response. Therefore, developing anti-TGFβ antibodies targeting the TGFβ signaling pathway is of great significance for tumor therapy. Summary of the Invention

[0005] The purpose of this invention is to provide a humanized nanobody against TGFβ, its preparation method, and its uses.

[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 the above-described nucleotide molecules.

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

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

[0010] Furthermore, the humanized nanobody also includes four framework regions FR1-FR4 alternately linked with complementarity-determining regions CDR1-CDR3. The amino acid sequence of FR1 is QVQLVESGGGLVQPGGSLRLSCAASG, the amino acid sequence of FR2 is LGWFRQAPGQEREAVAA, the amino acid sequence of FR3 is YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC, and the amino acid sequence of FR4 is WGQGTLVTVSS.

[0011] Furthermore, the amino acid sequence of the humanized nanobody is as follows:

[0012] QVQLVESGGGLVQPGGSLRLSCAASGRDLSRYALGWFRQAPGQEREAVAA ITSAGRSYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAAKSSNRP VFRDDYWGQGTLVTVSS.

[0013] The present invention also provides a method for preparing the above-mentioned humanized nanobody, the method comprising the following steps:

[0014] (1) The above nucleotide molecules are ligated into an expression vector to obtain a positive plasmid;

[0015] (2) Transform the positive plasmid into the host cell to induce the expression of humanized nanobodies.

[0016] The present invention also provides the use of the above-mentioned humanized nanobody in the preparation of anti-TGFβ antibody; preferably, the TGFβ is TGFβ1, TGF2 or TGFβ3.

[0017] Furthermore, the anti-TGFβ antibody is a drug for the prevention and / or treatment of tumors, preferably 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.

[0018] The present invention also provides the use of the above-mentioned humanized nanobody in combination with PD-L1 antibody in the preparation of medicaments for the prevention and / or treatment of tumors.

[0019] As is known to those skilled in the art, tumors that can be treated with anti-TGFβ antibodies 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, and advanced cervical cancer.

[0020] The anti-TGFβ humanized nanobody of the present invention can block the binding of TGFβ to its receptor while maintaining affinity for TGFβ, and has broad application prospects in the preparation of drugs for the prevention and / or treatment of tumors.

[0021] The anti-TGFβ humanized nanobody of the present invention has a molecular weight as low as 15 kDa. This antibody is a humanized antibody, which minimizes the immune risks caused by heterogeneity, making it more conducive to penetrating the blood-brain barrier and more likely to reach the tumor to exert a therapeutic effect.

[0022] 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.

[0023] 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

[0024] Figure 1 Affinity detection of humanized nanobodies with TGFβ1.

[0025] Figure 2 Affinity detection of humanized nanobodies with TGFβ2.

[0026] Figure 3 Affinity detection of humanized nanobodies with TGFβ3. Detailed Implementation

[0027] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0028] Example 1: Preparation of humanized nanobodies

[0029] 1. Constructing expression carriers

[0030] (1) Restriction vector digestion: The tool vector pcDNA3.1-x-IgG1 was digested with BamHI / EcoRI. After double digestion at 37℃ for 5 h, the vector was recovered using a PCR product recovery kit (Cycle-Pure Kit PCR Product Purification Kit OMEGA D6492-01).

[0031] (2) Homologous recombination: The nucleotide fragment used for recombination expression was diluted 20 times with ddH2O. 1 μL of the diluted sample was then used for homologous recombination with the vector recovered by the above enzyme digestion (recombinase, NovoRec Plus one step PCR CloningKit nearshore protein catalog number NR005-01B).

[0032] The sequence of the nucleotide fragment used for recombinant expression is as follows:

[0033] CCAGGGCTTACCTTTATGCTTCGGCTCGTATGTTGTGTGGAATTGTG

[0034] AGCGGATAACAATTGAATTCAGGAGGAATTTAAAATGAAAAAGACAG

[0035] CTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTGGCCCAGG

[0036] CGGCCCAGGTGCAGCTGGTTGAAAGTGGCGGTGGCCTGGTGCAGCCG

[0037] GGTGGTTCACTGCGTCTGAGTTTGTGCCGCAAGCGGTCGTGATCTGAGT

[0038] AGGTATGCACTGGGCTGGTTTCGTCAGGCCCCTGGTCAGGAACGCGAA

[0039] GCAGTTGCAGCCATTACCAGTGCAGGTCGTAGCTATTATGCAGATAGC

[0040] GTTAAAGGCCGCTTTACCATTAGCCGCGATAATAGCAAAAATACCCTG

[0041] TATCTGCAGATGAATAGTCTGCGCGCCGAAGATACCGCAGTTTATTAT

[0042] TGTGCAGCAGCCAAGTCTTCTAATCGTCCTGTGTTTAGGGATGATTATT

[0043] GGGGTCAGGGCACCCTGGTTACCGTTAGCAGTGCGCACCACAGCGAA

[0044] GACCCCCATGGCCAGGCCGGCCAGCACCATCACCATCACCATGGCGC

[0045] ATACCCGTACGACGTTCCGGACTACGCTTCTTAGGAGGGGTGGTGGCTC

[0046] TGAGGGTGGCGGTTCTGAGGGTGGCGGCTCTGAGGGAGGCGGTTCCG

[0047] GTGGTGGCTCTGGTTCCGGTGATTTTGATTATGAAAAGATGGCAAACG

[0048] CTAATAAGGGGGCTATGACCGAAAATGCCGATGAAAACGTGCTACAG

[0049] TCTGACGCTAAAGGCAAACTTGATTCTGTCGCTACTGATTACGGTGCT

[0050] GCTATCGATGGTTTCATTGGTGACGTTTCCGGCCTTGCTAATGGTAATGGTGCTACTGGT (SEQ ID NO: 9).

[0051] (3) Escherichia coli culture PCR identification: Single colony of Escherichia coli was picked from the plate and cultured in 200 μL LB medium at 37°C and 220 rpm for 3 h. 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 removed (Gel Extraction Kit OMEGA, catalog number D2500-01) and homologous recombination was performed again. After culture PCR identification, positive clones were selected for sequencing.

[0052] 2. Expression and purification of humanized nanobody Hek293F in cells

[0053] (1) Antibody expression

[0054] 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, Bevo Medical 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 × 10⁶ cells / ml at transfection. 50 μg of expression plasmid was added to 1 ml of OPM medium and mixed well. 150 μg of PEI was added to 1 ml of OPM medium and mixed well. After mixing, the mixture was incubated at room temperature for 30 min and then added to 50 ml of Hek293 cells. The cells were incubated on a CO₂ shaker. On the second day, 5% OPM medium was added as a final feed, and the cells were cultured until day 7. The cell culture supernatant was harvested by centrifugation at 10,000 rpm for 20 min and used for protein purification.

[0055] (2) Antibody purification

[0056] Protein A gravity column purification of antibody protein: Remove the gravity column from the refrigerator, rinse with one column volume of ultrapure water, rinse with one column volume of 0.1M NaOH, and rinse with 3 column volumes of PBS buffer.

[0057] All the cell supernatant after centrifugation was loaded onto a gravity column. After washing with PBS buffer for 3 column volumes, 800 μL of 0.1 M glycine hydrochloride (Gly-HCl) was added for elution. The elution was repeated twice. The target protein (named RS108) was collected and the protein concentration was determined to be 0.31 mg / mL. The total volume was 15 mL and the purity was not less than 95%.

[0058] The amino acid sequence of the target protein RS108 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.

[0059] Table 1. Amino acid sequence of the target protein

[0060]

[0061] The following experimental examples demonstrate the activity of the humanized nanobody of the present invention.

[0062] Experimental Example 1: Affinity Detection of Humanized Nanobodies with TGFβ1 / 2 / 3

[0063] 1. Experimental Methods

[0064] TGFβ1 / 2 / 3 antigens were coated onto ELISA plates at a concentration of 1 μg / ml. Humanized nanobody RS108 was added after a 5-fold serial dilution starting at 50 μg / ml. The binding of the antibodies to TGFβ1 / 2 / 3 was detected using an anti-Fc secondary antibody. Fresolimumab (non-hematoxylin and eosin), a known monoclonal antibody that can simultaneously target all three TGFβ1 / 2 / 3 isoforms, was used as a control antibody.

[0065] 2. Experimental Results

[0066] Affinity ELISA test results as follows Figure 1-3 As shown, the RS108 antibody of the present invention can simultaneously target three subtypes of TGFβ1 / 2 / 3.

[0067] The RS108 antibody of this invention has an affinity for TGFβ1 comparable to that of the control antibody Fresolimumab. Figure 1 It has a higher affinity for TGFβ2 than the control antibody Fresolimumab. Figure 2 It has a higher affinity for TGFβ3 than the control antibody Fresolimumab. Figure 3 ).

[0068] Experimental Example 2: Detection of the blocking effect of humanized nanobodies on TGFβ1 and TGFβR2

[0069] 1. Experimental Methods

[0070] First, the binding of TGFβR2-FC-His to TGFβ1 was detected using ELISA. A suitable concentration of TGFβR2-FC-His (ideally with an OD450 of around 1) was selected as the experimental concentration to compete with the antibody. HRP-labeled anti-His secondary antibody was used for detection. Simultaneously, 50 μL of PBS and an equal volume of 50 μg / mL TGFβR2-FC-His were used as controls. If the OD450 of the experimental group was significantly weaker than that of the control group, it could be considered a competitive assay.

[0071] 2. Experimental Results

[0072] The results of the competitive ELISA assay show that the RS108 antibody can efficiently block the binding of TGFβ1 to its receptor TGFβR2 (Table 2), and the blocking effect is better than that of the control antibody Fresolimumab.

[0073] Table 2. Blocking effect of RS108 on the binding of TGFβ1 and TGFβR2

[0074] RS108 0.4851 0.2263 Fresolimumab 0.5724 0.2915 PBS 0.9194 0.4773

[0075] In summary, this invention provides a humanized nanobody against TGFβ, its preparation method, and its uses. This humanized nanobody maintains affinity for TGFβ while blocking the binding of TGFβ to its receptor, and 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 humanized nanobody against TGFβ, characterized in that, It includes complementarity-determining regions CDR1-CDR3 and four framework regions FR1-FR4 alternately linked with CDR1-CDR3. The amino acid sequence of CDR1 is RDLSRYA, the amino acid sequence of CDR2 is ITSAGRS, the amino acid sequence of CDR3 is AAAKSSNRPVFRDDY, the amino acid sequence of FR1 is QVQLVESGGGLVQPGGSLRLSCAASG, the amino acid sequence of FR2 is LGWFRQAPGQEREAVAA, the amino acid sequence of FR3 is YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC, and the amino acid sequence of FR4 is WGQGTLVTVSS.

5. The humanized nanobody against TGFβ according to claim 4, characterized in that, Its amino acid sequence is: QVQLVESGGGLVQPGGSLRLSCAASGRDLSRYALGWFRQAPGQEREAVAAITSAGRSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAAKSSNRPVFRDDYWGQGTLVTVSS.

6. A method for preparing the humanized anti-TGFβ nanobody according to any one of claims 4-5, 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 the positive plasmid into the host cell to induce the expression of humanized nanobodies.

7. Use of the humanized anti-TGFβ nanobody according to any one of claims 4-5 in the preparation of a medicament for treating tumors, wherein the tumor is glioma, melanoma, or renal cell carcinoma.

8. Use of the humanized anti-TGFβ nanobody according to any one of claims 4-5 in combination with a PD-L1 antibody in the preparation of a medicament for treating tumors, wherein the tumor is glioma, melanoma, or renal cell carcinoma.

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