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WO2025055383A1 - HUMANIZED NANOBODY AGAINST TGFβ - Google Patents

HUMANIZED NANOBODY AGAINST TGFβ Download PDF

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Publication number
WO2025055383A1
WO2025055383A1 PCT/CN2024/095207 CN2024095207W WO2025055383A1 WO 2025055383 A1 WO2025055383 A1 WO 2025055383A1 CN 2024095207 W CN2024095207 W CN 2024095207W WO 2025055383 A1 WO2025055383 A1 WO 2025055383A1
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seq
amino acid
acid sequence
humanized
tgfβ
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刘�东
余鼎
梁洪
付道兴
钟沅沅
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Chengdu Rongsheng Pharmaceuticals Co Ltd
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Chengdu Rongsheng Pharmaceuticals Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0684—Cells of the urinary tract or kidneys
    • C12N5/0686—Kidney cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2510/00—Genetically modified cells
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the present invention belongs to the field of biotechnology, and specifically relates to a humanized anti-TGF ⁇ nano antibody and a preparation method and use thereof.
  • TGF ⁇ transforming growth factor
  • TGF ⁇ transforming growth factor
  • the pathway composed of TGF ⁇ 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.
  • TGF ⁇ TGF ⁇ 1, TGF ⁇ 2, and TGF ⁇ 3. Studies have found that they are highly expressed in many tumors. Studies have also found that when the tumor develops to the late stage, tumor cells can inhibit the TGF ⁇ pathway or make themselves tolerant to TGF ⁇ . At this time, TGF ⁇ can promote tumor cell infiltration and metastasis. Therefore, the development of anti-TGF ⁇ antibodies targeting the TGF ⁇ signaling pathway is of great significance for tumor treatment.
  • Fresolimumab non-sumetumomab
  • Fresolimumab is a single-chain antibody with a molecular weight of 150KDa.
  • the purpose of the present invention is to provide a humanized anti-TGF ⁇ nanobody and a preparation method and use thereof.
  • the present invention provides a humanized nanoantibody, 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.
  • 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
  • the amino acid sequence of FR4 is shown in SEQ ID NO:7.
  • the present invention also provides a nucleotide molecule, whose nucleotide sequence is shown in SEQ ID NO:9.
  • the present invention also provides an expression vector, which comprises the above-mentioned nucleotide molecule.
  • the present invention also provides a host cell, which comprises the above expression vector.
  • the present invention also provides a method for preparing the above-mentioned humanized Nanobody, the method comprising the following steps:
  • the present invention also provides the use of the humanized nanobody in preparing anti-TGF ⁇ antibodies.
  • TGF ⁇ is TGF ⁇ 1, TGF2 or TGF ⁇ 3;
  • the anti-TGF ⁇ antibody is a drug for preventing and/or treating tumors.
  • 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.
  • 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
  • the cervical cancer is advanced cervical cancer.
  • tumors that can be treated with anti-TGF ⁇ 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.
  • the anti-TGF ⁇ humanized nanobody of the present invention can block the binding of TGF ⁇ to its receptor while maintaining affinity with TGF ⁇ , and has broad application prospects in the preparation of drugs for preventing and/or treating tumors.
  • the anti-TGF ⁇ humanized nanoantibody of the present invention has a molecular weight as low as 15KDa.
  • This antibody is a humanized 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.
  • the raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.
  • sequence of the nucleotide fragment used for recombinant expression is:
  • the strain was inoculated into 20 ml of LB medium containing ampicillin, cultured overnight at 37°C incubator, and the plasmid was extracted using a plasmid extraction kit (Plasmid Miniprep Kit II, Biomedical Cat: BW-PD1213).
  • the Hek293 cells were passaged to maintain a good cell growth state, with a viability greater than 95%.
  • the Hek293 cell density was adjusted to 2.5 ⁇ 106cells/ml during transfection.
  • 50ug of expression plasmid was added to 1ml of OPM medium and mixed, and 150ug of PEI was added to 1ml of OPM medium and mixed. The two were mixed and shaken, and then added to 50ml of Hek293 cells after standing at room temperature for 30 minutes.
  • the cells were cultured in a CO2 shaker, and 5% of the final volume of OPM medium was added the next day. The culture was continued until the 7th day, and the cell culture supernatant was harvested by centrifugation at 10000rmp for 20 minutes for protein purification.
  • Protein A gravity column purification of antibody protein Take out the gravity column from the refrigerator, rinse one column volume with ultrapure water, rinse one column volume with 0.1M NaOH, and rinse 3 column volumes with PBS buffer.
  • the cell supernatant after centrifugation was loaded onto the gravity column. After washing with 3 column volumes of PBS buffer, 800ul of 0.1M 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 0.31mg/ml, the total volume was 15ml, and the purity was not less than 95%.
  • the amino acid sequence of the target protein RS16 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.
  • TGF ⁇ 1/2/3 antigens were coated on the ELISA plate at a concentration of 1ug/ml, and humanized nanoantibody RS16 was added after 5-fold dilution starting from 50ug/ml.
  • Anti-Fc secondary antibody was used to detect the binding of antibody to TGF ⁇ 1/2/3.
  • Fresolimumab non-sumetumomab
  • a known monoclonal antibody that can simultaneously target the three subtypes of TGF ⁇ 1/2/3 was used as a control antibody.
  • the affinity of the RS16 antibody of the present invention to TGF ⁇ 1 is comparable to that of the control antibody Fresolimumab ( FIG. 1 ), the affinity to TGF ⁇ 2 is higher than that of the control antibody Fresolimumab ( FIG. 2 ), and the affinity to TGF ⁇ 3 is higher than that of the control antibody Fresolimumab ( FIG. 3 ).
  • RS16 antibody can effectively block the binding of TGF ⁇ 1 to its receptor TGF ⁇ R2 (Table 2), and the blocking effect is better than that of the control antibody Fresolimumab; RS16 antibody can effectively block the binding of TGF ⁇ 2 to its receptor TGF ⁇ R2 (Table 3), and the blocking effect is better than that of the control antibody Fresolimumab; RS16 antibody can effectively block the binding of TGF ⁇ 3 to its receptor TGF ⁇ R2 (Table 4), and the blocking effect is better than that of the control antibody Fresolimumab.
  • the present invention provides a humanized anti-TGF ⁇ nanobody and its preparation method and use.
  • the humanized nanobody can block the binding of TGF ⁇ to its receptor while maintaining affinity with TGF ⁇ , and has broad application prospects in the preparation of drugs for preventing and/or treating tumors.

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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

一种抗TGFβ的人源化纳米抗体A humanized nanobody against TGFβ 技术领域Technical Field

本发明属于生物技术领域，具体涉及一种抗TGFβ的人源化纳米抗体及其制备方法和用途。The present invention belongs to the field of biotechnology, and specifically relates to a humanized anti-TGFβ nano antibody and a preparation method and use thereof.

背景技术Background Art

TGFβ(transforming growth factor，转化生长因子β)是一类多功能的细胞因子，TGFβ与其相应的受体、细胞内信号转导分子组成的通路，能影响疾病发生和发展，调节基因的转录，控制细胞周期，影响细胞增殖、分化、黏附、转移和凋亡。TGFβ (transforming growth factor) 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.

TGFβ超家族的成员包括TGFβ1、TGFβ2、TGFβ3，研究发现，它们在许多肿瘤中高表达。研究还发现，当肿瘤发展到晚期时，肿瘤细胞可以通过抑制TGFβ通路或使自身产生TGFβ耐受作用，此时的TGFβ能够促进肿瘤细胞浸润和转移。因此开发靶向TGFβ信号通路的抗TGFβ抗体对肿瘤治疗具有重要意义。Members of the TGFβ superfamily include TGFβ1, TGFβ2, and TGFβ3. Studies have found that they are highly expressed in many tumors. Studies have also found that when the tumor develops to the late stage, tumor cells can inhibit the TGFβ pathway or make themselves tolerant to TGFβ. At this time, 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 tumor treatment.

基于免疫相关的TGF-β/TGF-βR通路，全球现已进行了深入的研究。但是，目前临床研究中针对TGFβ的单抗药物的主要来源是经鼠源抗体改造的抗体，例如Fresolimumab(非苏木单抗)，它是Genzyme公司基于1D11鼠源抗体经嵌合改造获得的人源性抗TGFβ的单克隆抗体，可同时靶TGFβ1、TGFβ2、TGFβ3三种亚型。Fresolimumab是一种分子量为150KDa的单链抗体，其分子量较大不利于穿透血脑屏障，且由于该抗体是一种人源化鼠抗，存在一定的免疫风险。为了解决上述问题，亟需开发出一种抗TGFβ的人源化纳米抗体。Based on the immune-related TGF-β/TGF-βR pathway, in-depth research has been conducted around the world. However, the main source of monoclonal antibody drugs targeting TGFβ in clinical research is antibodies modified from mouse antibodies, such as Fresolimumab (non-sumetumomab), 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 the 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 nanoantibody against TGFβ.

发明内容Summary of the invention

本发明的目的在于提供一种抗TGFβ的人源化纳米抗体及其制备方法和用途。The purpose of the present invention is to provide a humanized anti-TGFβ nanobody and a preparation method and use thereof.

本发明提供了一种人源化纳米抗体，它包括互补决定区CDR1-CDR3，CDR1的氨基酸序列如SEQ ID NO:1所示，CDR2的氨基酸序列如SEQ ID NO:2所示，CDR3的氨基酸序列如SEQ ID NO:3所示。The present invention provides a humanized nanoantibody, 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.

进一步地，它还包括与互补决定区CDR1-CDR3交替连接的四个框架区FR1-FR4，FR1的氨基酸序列如SEQ ID NO:4所示，FR2的氨基酸序列如SEQ ID NO:5所示，FR3的氨基酸序列如SEQ ID NO:6所示，FR4的氨基酸序列如SEQ ID NO:7所示。Furthermore, 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.

进一步地，它的氨基酸序列如SEQ ID NO:8所示。Furthermore, its amino acid sequence is shown in SEQ ID NO:8.

本发明还提供了一种核苷酸分子，它的核苷酸序列如SEQ ID NO:9所示。The present invention also provides a nucleotide molecule, whose nucleotide sequence is shown in SEQ ID NO:9.

本发明还提供了一种表达载体，它包含上述的核苷酸分子。The present invention also provides an expression vector, which comprises the above-mentioned nucleotide molecule.

本发明还提供了一种宿主细胞，它包含上述的表达载体。The present invention also provides a host cell, which comprises the above expression vector.

本发明还提供了一种制备上述人源化纳米抗体的方法，所述方法包括以 下步骤：The present invention also provides a method for preparing the above-mentioned humanized Nanobody, the method comprising the following steps:

(1)将上述的核苷酸分子连接至表达载体中获得阳性质粒；(1) connecting the above-mentioned nucleotide molecule to an expression vector to obtain a positive plasmid;

(2)将阳性质粒转化宿主细胞，诱导表达人源化纳米抗体。(2) Transform the positive plasmid into host cells to induce the expression of humanized nanobodies.

本发明还提供了上述人源化纳米抗体在制备抗TGFβ抗体中的用途。The present invention also provides the use of the humanized nanobody in preparing anti-TGFβ antibodies.

进一步地，所述TGFβ为TGFβ1、TGF2或TGFβ3；Further, the TGFβ is TGFβ1, TGF2 or TGFβ3;

进一步地，所述抗TGFβ抗体为预防和/或治疗肿瘤的药物。Furthermore, the anti-TGFβ antibody is a drug for preventing and/or treating tumors.

进一步地，所述肿瘤为胶质瘤、黑色素瘤、肾细胞癌、胰腺癌、乳腺癌、肺癌、前列腺癌、胆管癌、头颈部鳞状细胞癌或子宫颈癌。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.

进一步地，所述黑色素瘤为恶性黑色素瘤，所述肺癌为非小细胞肺癌，所述前列腺癌为晚期前列腺癌，所述头颈部鳞状细胞癌为晚期头颈部鳞状细胞癌，所述子宫颈癌为晚期子宫颈癌。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.

本领域技术人员公知的，抗TGFβ抗体可以治疗的肿瘤包括：胶质瘤、恶性黑色素瘤、肾细胞癌、胰腺癌、乳腺癌、非小细胞肺癌、晚期前列腺癌、胆管癌、晚期头颈部鳞状细胞癌、晚期子宫颈癌等。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.

本发明的抗TGFβ人源化纳米抗体在保持与TGFβ亲和力的同时能阻断TGFβ与其受体的结合，在制备预防和/或治疗肿瘤的药物中具有广阔的应用前景。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.

本发明的抗TGFβ人源化纳米抗体分子量低至15KDa，这种抗体是人源化抗体，其最大程度的降低了异源性带来的免疫风险，更有利于穿透血脑屏障，更易到达肿瘤内部发挥治疗效果。The anti-TGFβ humanized nanoantibody of the present invention has a molecular weight as low as 15KDa. This antibody is a humanized 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.

显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。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.

以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。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.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

图1.人源化纳米抗体与TGFβ1亲和力检测。Figure 1. Affinity detection of humanized nanoantibodies and TGFβ1.

图2.人源化纳米抗体与TGFβ2亲和力检测。Figure 2. Affinity detection of humanized nanoantibodies and TGFβ2.

图3.人源化纳米抗体与TGFβ3亲和力检测。Figure 3. Affinity detection of humanized nanobody and TGFβ3.

具体实施方式DETAILED DESCRIPTION

本发明所用原料与设备均为已知产品，通过购买市售产品所得。The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

实施例1：人源化纳米抗体的制备Example 1: Preparation of humanized nanobodies

1.构建表达载体1. Construction of expression vector

(1)骨架载体酶切：工具载体pcDNA3.1-x-IgG1进行BamH I/EcoR I酶切，37℃双酶切5h后采用PCR产物回收试剂盒(Cycle-Pure Kit PCR产物纯化试剂盒OMEGA D6492-01)回收载体。(1) Backbone vector digestion: 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 OMEGA D6492-01).

(2)同源重组：将用于重组表达的核苷酸片段使用ddH2O稀释20倍，取1ul稀释后的样品与上述酶切回收的载体进行同源重组(重组酶，NovoRec  Plus one step PCR Cloning Kit近岸蛋白货号NR005-01B)。(2) Homologous recombination: The nucleotide fragments used for recombinant expression were diluted 20 times with ddH 2 O, and 1 ul of the diluted sample was taken for homologous recombination with the vector recovered by the above enzyme digestion (recombinase, NovoRec Plus one step PCR Cloning Kit Nearshore Protein Catalog No. NR005-01B).

用于重组表达的核苷酸片段的序列为：
The sequence of the nucleotide fragment used for recombinant expression is:

(3)大肠杆菌菌液PCR鉴定：从平板上挑取单克隆大肠杆菌菌落于200ul LB培养基37℃,220rpm培养3h，取1ul菌液作为模板，进行菌液PCR鉴定，选择鉴定的阳性克隆送去测序，将PCR产物电泳切角回收(Gel Extraction Kit胶回收试剂盒OMEGA，货号D2500-01)后，再次进行同源重组，菌液PCR鉴定后，选择鉴定的阳性克隆送去测序。(3) Identification of E. coli culture fluid by PCR: Single clone E. coli colonies were picked from the plate and cultured in 200ul LB medium at 37℃, 220rpm for 3h. 1ul of the culture fluid was taken as a template for PCR identification. The positive clones were selected for sequencing. The PCR products were recovered by electrophoresis (Gel Extraction Kit OMEGA, Cat. No. D2500-01) and then homologous recombination was performed again. After PCR identification of the culture fluid, the positive clones were selected for sequencing.

2.人源化纳米抗体Hek293F细胞表达与纯化2. Expression and purification of humanized nanobodies in Hek293F cells

(1)抗体表达(1) Antibody expression

将菌种接种到20ml含有氨苄青霉素的LB培养基中，37℃培养箱培养过夜，采用质粒提取试剂盒(Plasmid Miniprep Kit II，倍沃医学Cat:BW-PD1213)抽提质粒。传代Hek293细胞保持细胞良好生长状态，活率大于95％，转染时调整Hek293细胞密度为2.5×106cells/ml。取50ug表达质粒加入到1ml OPM培养基中混匀、取150ug PEI加入到1ml OPM培养基中混匀，上述两者混合后摇匀，室温静置30min后加入至50ml Hek293细胞中，于CO2摇床培养，第二天加入5％终体积的OPM培养基补料，继续培养至第7天，10000rmp离心20min收获细胞培养上清，用于蛋白纯化。The strain was inoculated into 20 ml of LB medium containing ampicillin, cultured overnight at 37°C incubator, and the plasmid was extracted using a plasmid extraction kit (Plasmid Miniprep Kit II, Biomedical Cat: BW-PD1213). The Hek293 cells were passaged to maintain a good cell growth state, with a viability greater than 95%. The Hek293 cell density was adjusted to 2.5×106cells/ml during transfection. 50ug of expression plasmid was added to 1ml of OPM medium and mixed, and 150ug of PEI was added to 1ml of OPM medium and mixed. The two were mixed and shaken, and then added to 50ml of Hek293 cells after standing at room temperature for 30 minutes. The cells were cultured in a CO2 shaker, and 5% of the final volume of OPM medium was added the next day. The culture was continued until the 7th day, and the cell culture supernatant was harvested by centrifugation at 10000rmp for 20 minutes for protein purification.

(2)抗体纯化(2) Antibody purification

protein A重力柱纯化抗体蛋白：从冰箱中取出重力柱，用超纯水冲洗一个柱体积，0.1M NaOH冲洗一个柱体积，PBS缓冲液冲洗3个柱体积。 Protein A gravity column purification of antibody protein: Take out the gravity column from the refrigerator, rinse one column volume with ultrapure water, rinse one column volume with 0.1M NaOH, and rinse 3 column volumes with PBS buffer.

将离心后的细胞上清全部上样至重力柱，PBS缓冲液冲洗3个柱体积后，加入800ul的0.1M甘氨酸盐酸盐(Gly-HCl)洗脱，重复洗脱2次，收集目标蛋白(命名为RS16)，测定蛋白浓度为0.31mg/ml，总体积为15ml，纯度不低于95％。The cell supernatant after centrifugation was loaded onto the gravity column. After washing with 3 column volumes of PBS buffer, 800ul of 0.1M 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 0.31mg/ml, the total volume was 15ml, and the purity was not less than 95%.

目标蛋白RS16的氨基酸序列如SEQ ID NO:8所示，它包括互补决定区CDR1-CDR3，互补决定区被四个框架区FR1、FR2、FR3和FR4所分隔。The amino acid sequence of the target protein RS16 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.

表1.目标蛋白的氨基酸序列
Table 1. Amino acid sequences of target proteins

以下通过实验例证明本发明人源化纳米抗体的活性。The following experimental examples demonstrate the activity of the humanized Nanobodies of the present invention.

实验例1：人源化纳米抗体与TGFβ1/2/3的亲和力检测Experimental Example 1: Affinity detection of humanized nanobodies and TGFβ1/2/3

1.实验方法1. Experimental Methods

TGFβ1/2/3抗原以1ug/ml浓度包被酶标板，人源化纳米抗体RS16从50ug/ml开始5倍比稀释后加入，采用anti-Fc二抗检测抗体与TGFβ1/2/3结合情况。以可同时靶向TGFβ1/2/3三种亚型的已知单克隆抗体Fresolimumab(非苏木单抗)作为对照抗体。TGFβ1/2/3 antigens were coated on the ELISA plate at a concentration of 1ug/ml, and humanized nanoantibody RS16 was added after 5-fold dilution starting from 50ug/ml. Anti-Fc secondary antibody was used to detect the binding of antibody to TGFβ1/2/3. Fresolimumab (non-sumetumomab), a known monoclonal antibody that can simultaneously target the three subtypes of TGFβ1/2/3, was used as a control antibody.

2.实验结果2. Experimental results

亲和力ELISA试验结果如图1-3所示，可以看出，本发明RS16抗体能够同时靶向TGFβ1/2/3三种亚型。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 three subtypes of TGFβ1/2/3.

本发明RS16抗体对TGFβ1的亲和力与对照抗体Fresolimumab相当(图1)，对TGFβ2的亲和力高于对照抗体Fresolimumab(图2)，对TGFβ3的亲和力高于对照抗体Fresolimumab(图3)。The affinity of the RS16 antibody of the present invention to TGFβ1 is comparable to that of the control antibody Fresolimumab ( FIG. 1 ), the affinity to TGFβ2 is higher than that of the control antibody Fresolimumab ( FIG. 2 ), and the affinity to TGFβ3 is higher than that of the control antibody Fresolimumab ( FIG. 3 ).

实验例2：人源化纳米抗体对TGFβ1/2/3与TGFβR2阻断作用检测Experimental Example 2: Detection of the blocking effect of humanized nanobodies on TGFβ1/2/3 and TGFβR2

1.实验方法1. Experimental Methods

先用ELISA检测TGFβR2-FC-His与TGFβ1/2/3结合情况，选择合适浓度(该浓度处OD450以1左右为宜)的TGFβR2-FC-His作为与抗体竞争实验浓度。采用HRP标记的anti-His二抗检测，同时以50ul PBS和等体积的50ug/ml  TGFβR2-FC-His做为对照，若实验组OD450明显弱于对照组，则可视为竞争。First, use ELISA to detect the binding of TGFβR2-FC-His to TGFβ1/2/3, and select the appropriate concentration (OD450 is about 1 at this concentration) of TGFβR2-FC-His as the concentration for competition with the antibody. Use HRP-labeled anti-His secondary antibody for detection, and at the same time, use 50ul PBS and an equal volume of 50ug/ml TGFβR2-FC-His was used as a control. If the OD450 of the experimental group was significantly weaker than that of the control group, it could be considered as competition.

2.实验结果2. Experimental results

从竞争性ELISA试验结果可以看出，RS16抗体能够高效地阻断TGFβ1与其受体TGFβR2的结合(表2)，且阻断效果优于对照抗体Fresolimumab；RS16抗体能够高效地阻断TGFβ2与其受体TGFβR2的结合(表3)，且阻断效果优于对照抗体Fresolimumab；RS16抗体能够高效地阻断TGFβ3与其受体TGFβR2的结合(表4)，且阻断效果优于对照抗体Fresolimumab。From the results of competitive ELISA tests, it can be seen that RS16 antibody can effectively block the binding of TGFβ1 to its receptor TGFβR2 (Table 2), and the blocking effect is better than that of the control antibody Fresolimumab; RS16 antibody can effectively block the binding of TGFβ2 to its receptor TGFβR2 (Table 3), and the blocking effect is better than that of the control antibody Fresolimumab; RS16 antibody can effectively block the binding of TGFβ3 to its receptor TGFβR2 (Table 4), and the blocking effect is better than that of the control antibody Fresolimumab.

表2.RS16对TGFβ1与TGFβR2结合的阻断作用
Table 2. Blocking effect of RS16 on the binding of TGFβ1 to TGFβR2

表3.RS16对TGFβ2与TGFβR2结合的阻断作用
Table 3. Blocking effect of RS16 on the binding of TGFβ2 to TGFβR2

表4.RS16对TGFβ3与TGFβR2结合的阻断作用
Table 4. Blocking effect of RS16 on the binding of TGFβ3 to TGFβR2

综上，本发明提供了一种抗TGFβ的人源化纳米抗体及其制备方法和用途。该人源化纳米抗体在保持与TGFβ亲和力的同时能阻断TGFβ与其受体的结合，在制备预防和/或治疗肿瘤的药物中具有广阔的应用前景。 In summary, the present invention provides a humanized anti-TGFβ nanobody and its preparation method and use. The humanized nanobody 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.

Claims (10)

一种人源化纳米抗体，其特征在于，它包括互补决定区CDR1-CDR3，CDR1的氨基酸序列如SEQ ID NO:1所示，CDR2的氨基酸序列如SEQ ID NO:2所示，CDR3的氨基酸序列如SEQ ID NO:3所示。A humanized nanoantibody, 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. 根据权利要求1所述的人源化纳米抗体，其特征在于，它还包括与互补决定区CDR1-CDR3交替连接的四个框架区FR1-FR4，FR1的氨基酸序列如SEQ ID NO:4所示，FR2的氨基酸序列如SEQ ID NO:5所示，FR3的氨基酸序列如SEQ ID NO:6所示，FR4的氨基酸序列如SEQ ID NO:7所示。The humanized nanoantibody according to claim 1 is 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. 根据权利要求2所述的人源化纳米抗体，其特征在于，它的氨基酸序列如SEQ ID NO:8所示。The humanized nanoantibody according to claim 2 is characterized in that its amino acid sequence is shown in SEQ ID NO:8. 一种核苷酸分子，其特征在于，它的核苷酸序列如SEQ ID NO:9所示。A nucleotide molecule, characterized in that its nucleotide sequence is as shown in SEQ ID NO:9. 一种表达载体，其特征在于，它包含权利要求4所述的核苷酸分子。An expression vector, characterized in that it comprises the nucleotide molecule according to claim 4. 一种宿主细胞，其特征在于，它包含权利要求5所述的表达载体。A host cell, characterized in that it comprises the expression vector according to claim 5. 一种制备权利要求1-3任一项所述人源化纳米抗体的方法，其特征在于，所述方法包括以下步骤：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)将权利要求4所述的核苷酸分子连接至表达载体中获得阳性质粒；(1) connecting the nucleotide molecule described in claim 4 to an expression vector to obtain a positive plasmid; (2)将阳性质粒转化宿主细胞，诱导表达人源化纳米抗体。(2) Transform the positive plasmid into host cells to induce the expression of humanized nanobodies. 权利要求1-3任一项所述人源化纳米抗体在制备抗TGFβ抗体中的用途。Use of the humanized nanobody according to any one of claims 1 to 3 in the preparation of anti-TGFβ antibodies. 根据权利要求8所述的用途，其特征在于，所述TGFβ为TGFβ1、TGF2或TGFβ3。The use according to claim 8, characterized in that the TGFβ is TGFβ1, TGFβ2 or TGFβ3. 根据权利要求8所述的用途，其特征在于，所述抗TGFβ抗体为预防和/或治疗肿瘤的药物，所述肿瘤优选为胶质瘤、黑色素瘤、肾细胞癌、胰腺癌、乳腺癌、肺癌、前列腺癌、胆管癌、头颈部鳞状细胞癌或子宫颈癌。 The use according to claim 8 is 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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