CN117106082A - Humanized nanometer antibody against TGF beta - Google Patents
Humanized nanometer antibody against TGF beta Download PDFInfo
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Classifications
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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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- 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
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- 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]
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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- C07K2317/567—Framework region [FR]
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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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
Abstract
The invention provides an anti-TGF beta humanized nanometer antibody, and belongs to the technical field of biology. The humanized nanometer antibody comprises complementarity determining regions CDR1-CDR3, wherein the amino acid sequence of the CDR1 is shown as SEQ ID NO. 1, the amino acid sequence of the CDR2 is shown as SEQ ID NO. 2, and the amino acid sequence of the CDR3 is shown as SEQ ID NO. 3. The humanized nano antibody can block the binding of TGF beta and a receptor thereof while keeping affinity with the TGF beta, and has wide application prospect in preparing medicaments for preventing and/or treating tumors. The humanized nanometer antibody has low molecular weight, reduces immune risk caused by heterology, is more beneficial to penetrating the blood brain barrier, and is easier to reach the inside of tumor to exert therapeutic effect.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an anti-TGF beta humanized nano antibody, a preparation method and application thereof.
Background
TGF beta (transforming growth factor, transforming growth factor beta) is a multifunctional cytokine, and the TGF beta and its corresponding receptor, and the channel formed by intracellular signal transduction molecules can influence the occurrence and development of diseases, regulate the transcription of genes, control the cell cycle, and influence the proliferation, differentiation, adhesion, metastasis and apoptosis of cells.
Members of the TGF-beta superfamily, including TGF-beta 1, TGF-beta 2, TGF-beta 3, have been found to be highly expressed in many tumors. Studies have also found that when tumors develop to advanced stages, tumor cells can either inhibit the tgfβ pathway or exert their own tgfβ -tolerating effects, at which time tgfβ can promote tumor cell infiltration and metastasis. The development of anti-tgfβ antibodies that target tgfβ signaling pathways is therefore of great interest for tumor therapy.
Intensive research has been conducted worldwide based on immune-related TGF- β/TGF- βr pathways. However, the main source of monoclonal antibodies against tgfβ in current clinical studies is murine antibody engineered antibodies, such as Fresolimumab (non-sappan monoclonal antibody), which is a human anti-tgfβ monoclonal antibody obtained by Genzyme company based on chimeric engineering of 1D11 murine antibodies, and can target three subtypes tgfβ1, tgfβ2, tgfβ3 simultaneously. Fresolimumab is a single chain antibody with a molecular weight of 150kDa, which is more detrimental to penetration of the blood brain barrier, and since it is a humanized murine antibody, there is a certain risk of immunity. In order to solve the above problems, there is a need to develop a humanized nanobody against tgfβ.
Disclosure of Invention
The invention aims to provide an anti-TGF beta humanized nano antibody, a preparation method and application thereof.
The invention provides a humanized nanometer antibody, which comprises complementarity determining regions CDR1-CDR3, wherein the amino acid sequence of the CDR1 is shown as SEQ ID NO. 1, the amino acid sequence of the CDR2 is shown as SEQ ID NO. 2, and the amino acid sequence of the CDR3 is shown as SEQ ID NO. 3.
Further, it also comprises four framework regions FR1-FR4 alternately connected with CDR1-CDR3 of the complementarity determining region, the amino acid sequence of FR1 is shown as SEQ ID NO. 4, the amino acid sequence of FR2 is shown as SEQ ID NO. 5, the amino acid sequence of FR3 is shown as SEQ ID NO. 6, and the amino acid sequence of FR4 is shown as SEQ ID NO. 7.
Further, the amino acid sequence of the polypeptide is shown as SEQ ID NO. 8.
The invention also provides a nucleotide molecule, the nucleotide sequence of which is shown as SEQ ID NO. 9.
The invention also provides an expression vector comprising the nucleotide molecule.
The invention also provides a host cell comprising the expression vector.
The invention also provides a method for preparing the humanized nano-antibody, which comprises the following steps:
(1) Ligating the nucleotide molecules into an expression vector to obtain a positive plasmid;
(2) The positive plasmid is transformed into host cell to induce the expression of humanized nanometer antibody.
The invention also provides application of the humanized nano antibody in preparing an anti-TGF beta antibody.
Further, the TGF-beta is TGF-beta 1, TGF2 or TGF-beta 3;
further, the anti-TGF-beta antibody is a medicament for preventing and/or treating tumors.
Further, the tumor is glioma, melanoma, renal cell carcinoma, pancreatic cancer, breast cancer, lung cancer, prostate cancer, cholangiocarcinoma, head and neck squamous cell carcinoma, or cervical cancer.
Further, 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.
Tumors that can be treated by anti-tgfβ antibodies include, as known to those of skill in the art: 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, advanced cervical cancer, etc.
The anti-TGF-beta humanized nano-antibody can block the binding of TGF-beta and a receptor thereof while keeping affinity with TGF-beta, and has wide application prospect in preparing medicaments for preventing and/or treating tumors.
The molecular weight of the anti-TGF beta humanized nano antibody is as low as 15kDa, and the antibody is humanized, so that the immune risk brought by heterology is reduced to the greatest extent, the anti-TGF beta humanized nano antibody is more beneficial to penetrating the blood brain barrier and is easier to reach the inside of a tumor to exert the therapeutic effect.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Drawings
FIG. 1 affinity detection of humanized nanobodies with TGF-beta 1.
FIG. 2 affinity detection of humanized nanobodies with TGF-beta 2.
FIG. 3 affinity detection of humanized nanobodies with TGF-beta 3.
Detailed Description
The raw materials and equipment used in the invention are all known products and are obtained by purchasing commercial products.
Example 1: preparation of humanized nanobody
1. Construction of expression vectors
(1) And (3) enzyme cutting of a skeleton carrier: the tool vector pcDNA3.1-x-IgG1 was digested with BamHI/EcoRI, double digested at 37℃for 5h, and the vector was recovered using a PCR product recovery Kit (Cycle-Pure Kit PCR product purification Kit OMEGA D6492-01).
(2) Homologous recombination: nucleotide fragments for recombinant expression Using ddH 2 O was diluted 20-fold, and 1ul of the diluted sample was subjected to homologous recombination with the vector recovered by the above cleavage (recombinase, novoRec Plus one step PCR Cloning Kit, offshore protein accession No. NR 005-01B).
The nucleotide fragment sequences for recombinant expression were: CCCTTTATGCTTCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTGAATTCAGGAGGAATTTAAAATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTGGCCCAGGCGGCCCAGGTGCAGCTGGTTGAAAGTGGCGGTGGCCTGGTGCAGCCGGGTGGTTCACTGCGTCTGAGTTGTGCCGCAAGCGGTCTGACCTTTAAGAGGAATGATCTGGGCTGGTTTCGTCAGGCCCCTGGTCAGGAACGCGAAGCAGTTGCAGTCATTCCGGCAGGTGGTTATACCTATTATGCAGATAGCGTTAAAGGCCGCTTTACCATTAGCCGCGATAATAGCAAAAATACCCTGTATCTGCAGATGAATAGTCTGCGCGCCGAAGATACCGCAGTTTATTATTGTGCAGCGTTTACTGGGCATTATGGCTATTGGGGTCAGGGCACCCTGGTTACCGTTAGCAGTGCGCACCACAGCGAAGACCCCCATGGCCAGGCCGGCCAGCACCATCACCATCACCATGGCGCATACCCGTACGACGTTCCGGACTACGCTTCTTAGGAGGGTGGTGGCTCTGAGGGTGGCGGTTCTGAGGGTGGCGGCTCTGAGGGAGGCGGTTCCGGTGGTGGCTCTGGTTCCGGTGATTTTGATTATGAAAAGATGGCAAACGCTAATAAGGGGGCTATGACCGAAAATGCCGATGAAAACGTGCTACAGTCTGACGCTAAAGGCAAACTTGATTCTGTCGCTACTGATTACGGTGCTGCTATCGATGGTTTCATTGGTGACGTTTCCGGCCTTGCTAATGGTAATGGTGCTACTGGTGATTTTGCTGGCTCTAATTCCCAAATGGCTCAAGTCGGTGACGGTGATAATTCACCTTTAATGAATAATTTCCGTCAATATTTACCTTCCCTCCCTCAATCGGTTGAATGTCGCCCTTTTGTCTTTGG (SEQ ID NO: 9).
(3) E.coli bacterial liquid PCR identification: the colony of the monoclonal escherichia coli is selected from a flat plate and cultured for 3 hours at 37 ℃ with 220rpm of a 200ul LB culture medium, 1ul of bacterial liquid is taken as a template, bacterial liquid PCR identification is carried out, positive clones selected and identified are sent to sequencing, after electrophoresis corner cut of a PCR product is recovered (Gel Extraction Kit gel recovery kit OMEGA, product number D2500-01), homologous recombination is carried out again, and after bacterial liquid PCR identification, positive clones selected and identified are sent to sequencing.
2. Expression and purification of humanized nanobody Hek293F cells
(1) Antibody expression
The strain was inoculated into 20ml of LB medium containing ampicillin, cultured overnight in an incubator at 37℃and the plasmid was extracted using a plasmid extraction kit (Plasmid Miniprep Kit II, double Warew medical Cat: BW-PD 1213). The passage Hek293 cells keep good growth state, the activity rate is more than 95 percent, and the density of the Hek293 cells is adjusted to be 2.5X106 cells/ml during transfection. Adding 50ug of expression plasmid into 1ml of OPM culture medium, mixing, adding 150ug of PEI into 1ml of OPM culture medium, mixing, shaking, standing at room temperature for 30min, adding into 50ml of Hek293 cells, adding into CO 2 Shake cultivation, adding 5% OPM culture medium feed at final volume the next day, continuing cultivation until 7 th day, centrifuging at 10000rmp for 20min, and collecting cell culture supernatant for protein purification.
(2) Antibody purification
protein a gravity column purification of antibody proteins: the gravity column was removed from the refrigerator, one column volume was rinsed with ultrapure water, one column volume was rinsed with 0.1M NaOH, and 3 column volumes were rinsed with PBS buffer.
The cell supernatant after centrifugation was all loaded onto a gravity column, after washing 3 column volumes with PBS buffer, 800ul of 0.1M glycine hydrochloride (Gly-HCl) was added for elution, the elution was repeated 2 times, the target protein (designated as RS 16) was collected, the protein concentration was determined to be 0.31mg/ml, the total volume was 15ml, and the purity was not lower than 95%.
The amino acid sequence of the target protein RS16 is shown in SEQ ID NO. 8, and includes complementarity determining regions CDR1-CDR3 separated by four framework regions FR1, FR2, FR3 and FR 4.
TABLE 1 amino acid sequence of target protein
The activity of the humanized nanobody of the invention is demonstrated by experimental examples below.
Experimental example 1: affinity detection of humanized nanobody and TGF beta 1/2/3
1. Experimental method
TGF beta 1/2/3 antigen is coated on an ELISA plate at a concentration of 1ug/ml, humanized nano antibody RS16 is added after 5-fold dilution from 50ug/ml, and anti-Fc secondary antibody is adopted to detect the binding condition of the antibody and TGF beta 1/2/3. A known monoclonal antibody Fresolimumab (non-sappan monoclonal antibody) which can simultaneously target three subtypes of TGF beta 1/2/3 is used as a control antibody.
2. Experimental results
The results of the affinity ELISA assays are shown in FIGS. 1-3, and it can be seen that the RS16 antibodies of the invention are capable of targeting three subtypes of TGF-beta 1/2/3 simultaneously.
The inventive RS16 antibodies had an affinity for tgfβ1 comparable to the control antibody Fresolimumab (fig. 1), an affinity for tgfβ2 higher than the control antibody Fresolimumab (fig. 2), and an affinity for tgfβ3 higher than the control antibody Fresolimumab (fig. 3).
Experimental example 2: detection of blocking effect of humanized nano antibody on TGF beta 1/2/3 and TGF beta R2
1. Experimental method
TGF beta R2-FC-His binding to TGF beta 1/2/3 was first detected by ELISA and TGF beta R2-FC-His at the appropriate concentration (OD 450 at this concentration is preferably around 1) was selected as the concentration to compete with the antibody for the assay. HRP-labeled anti-His secondary antibody is used for detection, 50ul PBS and 50ug/ml TGF beta R2-FC-His with equal volume are used as control, and if the OD450 of the experimental group is obviously weaker than that of the control group, the experimental group can be regarded as competition.
2. Experimental results
From the results of the competitive ELISA assay, RS16 antibodies were able to block the binding of tgfβ1 to its receptor tgfβr2 with high efficiency (table 2), and the blocking effect was superior to that of the control antibody Fresolimumab; the RS16 antibody can effectively block the binding of TGF beta 2 and a receptor TGF beta R2 (table 3), and the blocking effect is superior to that of a control antibody Fresolimumab; RS16 antibodies were able to block the binding of tgfβ3 to its receptor tgfβr2 with high efficiency (table 4) and the blocking effect was superior to that of the control antibody Fresolimumab.
TABLE 2 blocking effect of RS16 on TGF-beta 1 binding to TGF-beta R2
| Experimental group | OD value (first experiment) | OD value (second experiment) |
| RS16 | 0.42 | 0.1836 |
| Fresolimumab | 0.635 | 0.2915 |
| PBS | 1.0489 | 0.4773 |
TABLE 3 blocking of TGF-beta 2 binding to TGF-beta R2 by RS16
| Experimental group | OD value |
| RS16 | 0.61 |
| Fresolimumab | 0.6775 |
| PBS | 1.6598 |
TABLE 4 blocking effect of RS16 on TGF-beta 3 binding to TGF-beta R2
| Experimental group | OD value (first experiment) | OD value (second experiment) |
| RS16 | 0.3637 | 0.119 |
| Fresolimumab | 0.908 | 0.4476 |
| PBS | 2.2758 | 0.6691 |
In summary, the invention provides an anti-TGF-beta humanized nano-antibody, a preparation method and application thereof. The humanized nano antibody can block the binding of TGF beta and a receptor thereof while keeping affinity with the TGF beta, and has wide application prospect in preparing medicaments for preventing and/or treating tumors.
Claims (10)
1. A humanized nanometer antibody is characterized by comprising complementarity determining regions CDR1-CDR3, wherein the amino acid sequence of the CDR1 is shown as SEQ ID NO. 1, the amino acid sequence of the CDR2 is shown as SEQ ID NO. 2, and the amino acid sequence of the CDR3 is shown as SEQ ID NO. 3.
2. The humanized nanobody of claim 1, further comprising four framework regions FR1-FR4 alternately linked to CDR1-CDR3 of the complementarity determining region, the amino acid sequence of FR1 being shown in SEQ ID No. 4, the amino acid sequence of FR2 being shown in SEQ ID No. 5, the amino acid sequence of FR3 being shown in SEQ ID No. 6, the amino acid sequence of FR4 being shown in SEQ ID No. 7.
3. The humanized nanobody according to claim 2, wherein the amino acid sequence thereof is shown in SEQ ID No. 8.
4. A nucleotide molecule is characterized in that the nucleotide sequence of the nucleotide molecule is shown as SEQ ID NO. 9.
5. An expression vector comprising the nucleotide molecule of claim 4.
6. A host cell comprising the expression vector of claim 5.
7. A method of making the humanized nanobody of any of claims 1-3, comprising the steps of:
(1) Ligating the nucleotide molecule of claim 4 into an expression vector to obtain a positive plasmid;
(2) The positive plasmid is transformed into host cell to induce the expression of humanized nanometer antibody.
8. Use of the humanized nanobody of any of claims 1-3 in the preparation of an anti-tgfβ antibody.
9. The use according to claim 8, wherein the tgfβ is tgfβ1, tgf2 or tgfβ3.
10. The use according to claim 8, wherein the anti-tgfβ antibody is a medicament for the prevention and/or treatment of a tumor, preferably glioma, melanoma, renal cell carcinoma, pancreatic cancer, breast cancer, lung cancer, prostate cancer, cholangiocarcinoma, squamous cell carcinoma of the head and neck or cervical cancer.
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