Humanized nanobody against TGFβ

Abstract

A humanized nanobody against TGFβ, which belongs to the field of biotechnology. The humanized nanobody comprises complementarity determining regions CDR1-CDR3, wherein CDR1 has an amino acid sequence as shown in SEQ ID NO: 1, CDR2 has an amino acid sequence as shown in SEQ ID NO: 2, and CDR3 has an amino acid sequence as shown in SEQ ID NO: 3. The humanized nanobody can simultaneously maintain the affinity for TGFβ and block the binding of TGFβ to the receptor thereof, and the humanized nanobody has broad application prospects in the preparation of a drug for preventing and / or treating tumors. The humanized nanobody has a low molecular weight, so that the immunity risk caused by heterogeneity is reduced, it is more conducive to penetrating the blood-brain barrier, and it can reach the interior of a tumor easier to exert a therapeutic effect.

Description

A humanized nanobody against TGFβ Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a humanized anti-TGFβ nanobody and a preparation method and use thereof. Background Art

[0002] TGFβ (transforming growth factor β) is a multifunctional cytokine. The pathway composed of TGFβ and its corresponding receptors and intracellular signal transduction molecules can affect the occurrence and development of diseases, regulate gene transcription, control the cell cycle, and affect cell proliferation, differentiation, adhesion, metastasis and apoptosis.

[0003] Members of the TGFβ superfamily include TGFβ1, TGFβ2, and TGFβ3. Studies have found that they are highly expressed in many tumors. Studies have also shown that when tumors reach advanced stages, tumor cells can inhibit the TGFβ pathway or develop TGFβ resistance. At this stage, TGFβ can promote tumor cell infiltration and metastasis. Therefore, the development of anti-TGFβ antibodies targeting the TGFβ signaling pathway is of great significance for cancer treatment.

[0004] Based on the immune-related TGF-β / TGF-βR pathway, in-depth research has been conducted globally. However, the main source of monoclonal antibodies targeting TGFβ in current clinical research is antibodies modified from mouse antibodies, such as Fresolimumab (Fresolimin), which is a humanized anti-TGFβ monoclonal antibody obtained by Genzyme based on the 1D11 mouse antibody through chimeric modification. It can simultaneously target three subtypes of TGFβ1, TGFβ2, and TGFβ3. Fresolimumab is a single-chain antibody with a molecular weight of 150KDa. Its large molecular weight is not conducive to penetrating the blood-brain barrier, and because this antibody is a humanized mouse antibody, there is a certain immune risk. In order to solve the above problems, it is urgent to develop a humanized anti-TGFβ nanoantibody.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to provide a humanized anti-TGFβ nanobody and its preparation method and use.

[0007] The present invention provides a humanized nanobody, which includes complementary determining regions CDR1-CDR3, the amino acid sequence of CDR1 is shown in SEQ ID NO: 1, the amino acid sequence of CDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 3.

[0008] Furthermore, it also includes four framework regions FR1-FR4 alternately connected to the complementarity determining regions CDR1-CDR3, the amino acid sequence of FR1 is shown in SEQ ID NO:4, the amino acid sequence of FR2 is shown in SEQ ID NO:5, the amino acid sequence of FR3 is shown in SEQ ID NO:6, and the amino acid sequence of FR4 is shown in SEQ ID NO:7.

[0009] Furthermore, its amino acid sequence is shown in SEQ ID NO:8.

[0010] The present invention also provides a nucleotide molecule, the nucleotide sequence of which is shown in SEQ ID NO:9.

[0011] The present invention also provides an expression vector comprising the above-mentioned nucleotide molecule.

[0012] The present invention also provides a host cell comprising the above expression vector.

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

[0014] (1) ligating the above-mentioned nucleotide molecule into an expression vector to obtain a positive plasmid;

[0015] (2) The positive plasmid is transformed into host cells 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 antibodies.

[0017] Furthermore, the TGFβ is TGFβ1, TGF2 or TGFβ3;

[0018] Furthermore, the anti-TGFβ antibody is a drug for preventing and / or treating tumors.

[0019] 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.

[0020] Furthermore, the melanoma is malignant melanoma, the lung cancer is non-small cell lung cancer, the prostate cancer is advanced prostate cancer, the head and neck squamous cell carcinoma is advanced head and neck squamous cell carcinoma, and the cervical cancer is advanced cervical cancer.

[0021] As is well 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, bile duct cancer, advanced head and neck squamous cell carcinoma, advanced cervical cancer, etc.

[0022] The anti-TGFβ humanized nanobody of the present invention can block the binding of TGFβ to its receptor while maintaining affinity with TGFβ, and has broad application prospects in the preparation of drugs for preventing and / or treating tumors.

[0023] The anti-TGFβ humanized nanoantibody of the present invention has a molecular weight as low as 15KDa. This antibody is a humanized antibody, which minimizes the immune risk brought by heterogeneity, is more conducive to penetrating the blood-brain barrier, and is more likely to reach the interior of the tumor to exert a therapeutic effect.

[0024] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0025] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1. Affinity detection of humanized nanobody and TGFβ1.

[0027] Figure 2. Affinity detection of humanized nanobody and TGFβ2.

[0028] Figure 3. Affinity detection of humanized nanobody and TGFβ3. DETAILED DESCRIPTION

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

[0030] Example 1: Preparation of humanized nanobodies

[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. After double digestion at 37°C for 5 h, the vector was recovered using a PCR product recovery kit (Cycle-Pure Kit PCR Product Purification Kit OMEGA D6492-01).

[0033] (2) Homologous recombination: Dilute the nucleotide fragment for recombinant expression 20 times with ddH2O, take 1 μl of the diluted sample and perform homologous recombination with the vector recovered by the above enzyme digestion (recombinase, NovoRec Plus one-step PCR Cloning Kit, nearshore protein product number NR005-01B).

[0034] The sequence of the nucleotide fragment used for recombinant expression is:

[0035] (3) PCR identification of E. coli culture medium: Single-clone E. coli colonies were picked from the plate and cultured in 200 μl LB medium at 37°C, 220 rpm for 3 h. 1 μl of culture medium was used as a template for culture medium PCR identification. The identified positive clones were sent for sequencing. The PCR products were recovered by electrophoresis (Gel Extraction Kit, OMEGA, Cat. No. D2500-01) and homologous recombination was performed again. After culture medium PCR identification, the identified positive clones were sent for sequencing.

[0036] 2. Expression and purification of humanized nanobodies in Hek293F cells

[0037] (1) Antibody expression

[0038] The strain was inoculated into 20 ml of LB medium supplemented with ampicillin and cultured overnight at 37°C. Plasmids were extracted using a Plasmid Miniprep Kit II, BioPharma, Cat: BW-PD1213. Hek293 cells were passaged to maintain good growth and viability greater than 95%. During transfection, the cell density was adjusted to 2.5 × 106 cells / ml. 50 μg of expression plasmid was added to 1 ml of OPM medium and mixed thoroughly. 150 μg of PEI was added to 1 ml of OPM medium and mixed thoroughly. The mixture was shaken and allowed to stand at room temperature for 30 minutes before being added to 50 ml of Hek293 cells. The cells were cultured in a CO2 shaker. On the second day, 5% of the final volume of OPM medium was added as a feed. Culture was continued until day 7. The cell culture supernatant was harvested by centrifugation at 10,000 rpm for 20 minutes for protein purification.

[0039] (2) Antibody purification

[0040] Protein A gravity column purification of antibody protein: Take out the gravity column from the refrigerator and rinse with one column volume of ultrapure water, one column volume of 0.1M NaOH, and three column volumes of PBS buffer.

[0041] The cell supernatant after centrifugation was loaded onto a gravity column. After washing with 3 column volumes of PBS buffer, 800 μl of 0.1 M glycine hydrochloride (Gly-HCl) was added for elution. The elution was repeated twice to collect the target protein (named RS16). The protein concentration was determined to be 0.31 mg / ml in a total volume of 15 ml, with a purity of not less than 95%.

[0042] The amino acid sequence of the target protein RS16 is shown in SEQ ID NO: 8, which includes complementarity determining regions CDR1-CDR3, which are separated by four framework regions FR1, FR2, FR3 and FR4.

[0043] Table 1. Amino acid sequences of target proteins

[0044] The following experimental examples demonstrate the activity of the humanized Nanobodies of the present invention.

[0045] Experimental Example 1: Affinity detection of humanized nanobodies and TGFβ1 / 2 / 3

[0046] 1. Experimental Methods

[0047] TGFβ1 / 2 / 3 antigens were coated onto an ELISA plate at a concentration of 1 μg / ml. Humanized nanobody RS16 was added at a 5-fold dilution starting at 50 μg / ml. Binding to TGFβ1 / 2 / 3 was detected using an anti-Fc secondary antibody. Fresolimumab, a known monoclonal antibody that simultaneously targets all three isoforms of TGFβ1 / 2 / 3, was used as a control antibody.

[0048] 2. Experimental Results

[0049] The affinity ELISA test results are shown in Figures 1-3. It can be seen that the RS16 antibody of the present invention can simultaneously target the three subtypes of TGFβ1 / 2 / 3.

[0050] The affinity of the RS16 antibody of the present invention for TGFβ1 is comparable to that of the control antibody Fresolimumab ( FIG. 1 ), the affinity for TGFβ2 is higher than that of the control antibody Fresolimumab ( FIG. 2 ), and the affinity for TGFβ3 is higher than that of the control antibody Fresolimumab ( FIG. 3 ).

[0051] Experimental Example 2: Detection of the blocking effect of humanized nanoantibodies on TGFβ1 / 2 / 3 and TGFβR2

[0052] 1. Experimental Methods

[0053] First, use ELISA to detect the binding of TGFβR2-FC-His to TGFβ1 / 2 / 3. Select an appropriate concentration of TGFβR2-FC-His (optimally, an OD450 of approximately 1) for competition with the antibody. Detect with an HRP-labeled anti-His secondary antibody. Simultaneously, use 50 μl of PBS and an equal volume of 50 μg / ml TGFβR2-FC-His as controls. If the OD450 of the experimental group is significantly lower than that of the control group, it is considered competitive.

[0054] 2. Experimental Results

[0055] The results of the competitive ELISA test showed that the RS16 antibody could effectively block the binding of TGFβ1 to its receptor TGFβR2 (Table 2), and the blocking effect was better than that of the control antibody Fresolimumab; the RS16 antibody could effectively block the binding of TGFβ2 to its receptor TGFβR2 (Table 3), and the blocking effect was better than that of the control antibody Fresolimumab; the RS16 antibody could effectively block the binding of TGFβ3 to its receptor TGFβR2 (Table 4), and the blocking effect was better than that of the control antibody Fresolimumab.

[0056] Table 2. Blocking effect of RS16 on the binding of TGFβ1 to TGFβR2

[0057] Table 3. Blocking effect of RS16 on the binding of TGFβ2 to TGFβR2

[0058] Table 4. Blocking effect of RS16 on the binding of TGFβ3 to TGFβR2

[0059] In summary, the present invention provides a humanized anti-TGFβ nanobody, its preparation method, and use. This humanized nanobody 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.

Claims

1. A humanized nanobody, characterized in that It includes complementary determining regions CDR1-CDR3, the amino acid sequence of CDR1 is shown in SEQ ID NO:1, the amino acid sequence of CDR2 is shown in SEQ ID NO:2, and the amino acid sequence of CDR3 is shown in SEQ ID NO:

3.

2. The humanized 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 shown in SEQ ID NO:4, the amino acid sequence of FR2 is shown in SEQ ID NO:5, the amino acid sequence of FR3 is shown in SEQ ID NO:6, and the amino acid sequence of FR4 is shown in SEQ ID NO:

7.

3. The humanized Nanobody according to claim 2, characterized in that Its amino acid sequence is shown in SEQ ID NO:

8.

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 humanized 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 the positive plasmid into host cells to induce the expression of humanized nanobodies.

8. Use of the humanized nanobody according to any one of claims 1 to 3 in the preparation of anti-TGFβ antibodies.

9. The use according to claim 8, characterized in that The TGFβ is TGFβ1, TGF2 or TGFβ3.

10. The use according to claim 8, characterized in that The anti-TGFβ antibody is a drug for preventing and / or treating tumors, and the tumor is 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.

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