A medicament for treating advanced tumors and fibrotic diseases

Abstract

The application provides a medicine for treating advanced tumors and fibrosis diseases, and belongs to the field of biological medicine. The medicine is a preparation prepared by taking a humanized nanobody as an active ingredient and adding pharmaceutically acceptable adjuvants. The humanized nanobody can block the combination of TGF beta and its receptor while keeping the affinity with TGF beta, and the immunological risk caused by heterogeneity is reduced to the maximum, the blood-brain barrier is more favorable to penetrate, and the humanized nanobody is more easy to reach the inside of tumors to play a therapeutic effect, so that the humanized nanobody has a wide application prospect in the preparation of the medicine for treating advanced tumors and fibrosis diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a drug for treating advanced tumors and fibrotic diseases. Background Technology

[0002] Transforming growth factor (TGF)β, primarily derived from macrophages, epithelial cells, and fibroblasts, is a group of multifunctional cytokines that regulate cell growth and differentiation. It comprises three structurally and functionally similar members: TGFβ1, TGFβ2, and TGFβ3. TGFβ was first isolated by Delarco and Todolo from serum-free culture medium of 3T3 cells transformed with murine sarcoma virus. It is essentially a group of polypeptides that induce the expression of a transformed phenotype in non-tumor cells. Its structure consists of two identical or similar 12.5 kDa subunits linked by disulfide bonds as homodimeric or heterodimeric basic proteins.

[0003] During the development of malignant tumors, abnormal expression of growth factors and cytokines occurs, which is closely related to tumor occurrence and prognosis. In vitro experiments have shown that TGFβ can induce cell cycle arrest in normal cells and some malignantly transformed epithelial cells by inhibiting cyclin-dependent kinases. TGFβ can also exert a growth-inhibiting effect through inactivation mutations, downregulation of its corresponding receptors, or mutations in elements of the TGFβ signaling pathway. In advanced tumor development, TGFβ tends to promote tumor progression.

[0004] Fibrotic diseases are characterized by the aggregation of large numbers of fibroblasts, extracellular matrix (ECG) deposition, and tissue destruction caused by inflammation and damage. Pulmonary fibrosis encompasses a wide range of diseases with diverse etiologies, including idiopathic pulmonary fibrosis (IPF), sarcoidosis, pneumoconiosis, allergic pneumonia, drug- and radiation-induced fibrosis, and fibrotic alveolitis associated with collagen vascular disease. Their morbidity, mortality, and severity vary, but a common characteristic is the lack of specific treatments. Current treatments for pulmonary fibrosis are limited to non-specific anti-inflammatory drugs, immunosuppressants, and glucocorticoids, with unsatisfactory efficacy. Studies have found increased TGFβ gene expression and protein products in various animal models of pulmonary fibrosis induced by bleomycin, silica, asbestos, and radiation. Similarly, TGFβ1 mRNA and protein expression are increased in human pulmonary fibrosis. When TGFβ protein is overexpressed in the lungs, it can directly lead to and induce fibrosis of myofibroblasts. The study also found that anti-TGFβ antibodies can bind to TGFβ, preventing TGFβ from binding to various types of TGFβR on the surface of its target cells and thus exerting its effective anti-fibrotic effect.

[0005] Therefore, developing drugs that can inhibit the binding of TGFβ to its receptor is of great significance for the treatment of advanced tumors and fibrotic diseases. Summary of the Invention

[0006] The purpose of this invention is to provide a humanized nanobody against TGFβ, and the use of the humanized nanobody against TGFβ in the preparation of a medicament for treating advanced tumors and fibrotic diseases.

[0007] The present invention provides a nucleotide molecule whose nucleotide sequence is shown in SEQ ID NO:9.

[0008] The present invention also provides an expression vector comprising a nucleotide molecule with a nucleotide sequence as shown in SEQ ID NO:9.

[0009] The present invention also provides a host cell comprising the above-described expression vector.

[0010] The present invention also provides a humanized nanobody comprising complementarity-determining regions CDR1-CDR3, wherein the amino acid sequence of CDR1 is FTFSGYA, the amino acid sequence of CDR2 is ITPIAHT, and the amino acid sequence of CDR3 is ADLHRMYTY.

[0011] 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.

[0012] Furthermore, the amino acid sequence of the humanized nanobody is as follows:

[0013] QVQLVESGGGLVQPGGSLRLSCAASGFTFSGYALGWFRQAPGQEREAVAAITPIAHTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCADLHRMYTYWGQGTLVTVSS.

[0014] The present invention also provides a method for preparing the above-mentioned humanized nanobody, the method comprising the following steps:

[0015] (1) A positive plasmid is obtained by ligating a nucleotide molecule with a nucleotide sequence as shown in SEQ ID NO:9 into an expression vector;

[0016] (2) Transform the positive plasmid into the host cell to induce the expression of humanized nanobodies.

[0017] The present invention also provides a drug formulation prepared by using the above-mentioned humanized nanobody as the active ingredient and adding scientifically acceptable excipients.

[0018] The present invention also provides the use of the above-mentioned humanized nanobody in the preparation of a drug for treating fibrotic diseases.

[0019] Furthermore, the fibrotic disease is pulmonary fibrosis.

[0020] As is well known to those skilled in the art, TGFβ tends to promote tumor progression in advanced stages of tumor development. Advanced tumors that can be treated with anti-TGFβ antibodies include: gastric cancer, glioma, melanoma, renal cell carcinoma, pancreatic cancer, breast cancer, lung cancer, prostate cancer, cholangiocarcinoma, head and neck squamous cell carcinoma, and cervical cancer.

[0021] The present invention also provides the use of the above-mentioned humanized nanobody in the preparation of a drug for treating advanced tumors.

[0022] 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.

[0023] 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 treating advanced tumors and fibrotic diseases.

[0024] The anti-TGFβ humanized nanobody of the present invention has a molecular weight of approximately 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.

[0025] 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.

[0026] 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

[0027] Figure 1 Affinity detection of humanized nanobodies with TGFβ1.

[0028] Figure 2Affinity detection of humanized nanobodies with TGFβ2.

[0029] Figure 3 Affinity detection of humanized nanobodies with TGFβ3. Detailed Implementation

[0030] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0031] Example 1: Preparation of humanized nanobodies

[0032] 1. Constructing expression carriers

[0033] (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).

[0034] (2) Homologous recombination: The nucleotide fragments used for recombination expression were 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).

[0035] The sequence of the nucleotide fragment used for recombinant expression is as follows:

[0036] CCAGGCTACTTTATGCTTCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTGAATTCAGGAGGAATTTAAAATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTGGCCCAGGCGGCCCAGGTGCAGCTGGTTGAAAGTGGCGGTGGCCTGGTGCAGCCGGGTGGTTCACTGCGTCTGAGTTGTGCCGCAAGCGGTTTTACCTTTAGCGGTTATGCCCTGGGCTGGTTTCGTCAGGCCCCTGGTCAGGAACGCGAAGCAGTTGCAGCCATTACCCCGATTGCACATACCTATTATGCAGATAGCGTTAAAGGCCGCTTTACCATTAGCCGCGATAATAGCAAAAATACCCTGTATCTGCAGATGAATAGTCTGCGCGCCGAAGATACCGCAGTTTATTATTGTGCAGATCTTCATAGGATGTATACCTATTGGGGTCAGGGCACCCTGGTTACCGTTAGCAGTGCGCACCACAGCGAAGACCCCCATGGCCAGGCCGGCCAGCACCATCACCATCACCATGGCGCATACCCGTACGACGTTCCGGACTACGCTTCTTAGGAGGGTGGTGGCTCTGAGGGTGGCGGTTCTGAGGGTGGCGGCTCTGAGGGAGGCGGTTCCGGTGGTGGCTCTGGTTCCGGTGATTTTGATTATGAAAAGATGGCAAACGCTAATAAGGGGGCTATGACCGAAAATGCCGATGAAAACGTGCTACAGTCTGACGCTAAAGGCAAACTTGATTCTGTCGCTACTGATTACGGTGCTGCTATCGATGGTTTCATTGGTGACGTTTCCGGCCTTGCTAATGGTAATGGTGCTACTGGTGATTTTGCTGGCTCTAATTCC(SEQ ID NO:9)。

[0037] (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.

[0038] 2. Expression and purification of humanized nanobody Hek293F in cells

[0039] (1) Antibody expression

[0040] 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.

[0041] (2) Antibody purification

[0042] 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.

[0043] 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 RS112) was collected and the protein purity was determined to be no less than 95%.

[0044] The amino acid sequence of the target protein RS112 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.

[0045] Table 1. Amino acid sequence of the target protein

[0046]

[0047] The following experimental examples demonstrate the activity of the humanized nanobody of the present invention.

[0048] Experimental Example 1: Affinity Detection of Humanized Nanobodies with TGFβ1 / 2 / 3

[0049] 1. Experimental Methods

[0050] TGFβ1 / 2 / 3 antigens were coated onto ELISA plates at a concentration of 1 μg / ml. Humanized nanobody RS112 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.

[0051] 2. Experimental Results

[0052] Affinity ELISA test results as follows Figure 1-3 As shown, the RS112 antibody of the present invention can simultaneously target the three TGFβ1 / 2 / 3 isoforms. The affinity of the RS112 antibody of the present invention for TGFβ1, TGFβ2, and TGFβ3 is comparable to that of the control antibody Fresolimumab.

[0053] Experimental Example 2: Detection of the blocking effect of humanized nanobodies on TGFβ1 and TGFβR2

[0054] 1. Experimental Methods

[0055] 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.

[0056] 2. Experimental Results

[0057] The results of the competitive ELISA assay show that the RS112 antibody can efficiently block the binding of TGFβ1 to its receptor TGFβR2 (Table 2), with an effect comparable to that of the control antibody Fresolimumab.

[0058] Table 2. Blocking effect of RS112 on the binding of TGFβ1 and TGFβR2

[0059] RS112 0.5105 Fresolimumab 0.5724 PBS 0.9194

[0060] In summary, this invention provides a drug for treating advanced tumors and fibrotic diseases. This drug is a formulation prepared using the humanized nanobody of this invention as the active ingredient, along with chemically acceptable excipients. This humanized nanobody maintains affinity for TGFβ while blocking the binding of TGFβ to its receptor, and minimizes the immune risks associated with heterologous origin. It is also more likely to penetrate the blood-brain barrier and reach the tumor interior to exert its therapeutic effect, demonstrating broad application prospects in the preparation of drugs for treating advanced tumors and fibrotic diseases.

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, 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 FTFSGYA, the amino acid sequence of CDR2 is ITPIAHT, the amino acid sequence of CDR3 is ADLHRMYTY, 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 according to claim 4, characterized in that, Its amino acid sequence is: QVQLVESGGGLVQPGGSLRLSCAASGFTFSGYALGWFRQAPGQEREAVAAITPIAHTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCADLHRMYTYWGQGTLVTVSS.

6. A method for preparing the humanized nanobody according to any one of claims 4-5, characterized in that, The method includes the following steps: (1) A positive plasmid is obtained by ligating a nucleotide molecule with the nucleotide sequence shown in SEQ ID NO:9 into an expression vector; (2) Transform the positive plasmid into the host cell to induce the expression of humanized nanobodies.

7. A drug, characterized in that, It is a formulation prepared using the humanized nanobody as the active ingredient as described in any one of claims 4-5, plus pharmaceutically acceptable excipients.

8. Use of the humanized nanobody according to any one of claims 4-5 in the preparation of a medicament for treating advanced tumors, wherein the tumor is melanoma, renal cell carcinoma, or breast cancer.

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