CN111234019B - anti-CTLA-4 nano antibody, pharmaceutical composition and application thereof - Google Patents
anti-CTLA-4 nano antibody, pharmaceutical composition and application thereof Download PDFInfo
- Publication number
- CN111234019B CN111234019B CN202010048833.0A CN202010048833A CN111234019B CN 111234019 B CN111234019 B CN 111234019B CN 202010048833 A CN202010048833 A CN 202010048833A CN 111234019 B CN111234019 B CN 111234019B
- Authority
- CN
- China
- Prior art keywords
- ctla
- nanobody
- cancer
- nucleic acid
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
-
- 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
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- 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®
Abstract
The invention relates to an anti-CTLA-4 nano antibody, a pharmaceutical composition and application thereof. The anti-CTLA-4 nanobody is selected from at least one of the following: (1) has CDR1 shown in KYIYSNYC, CDR2 shown in IYTGGSNT and CDR3 shown in AATSRRWCSSLEKQVFGY; (2) compared to (1), at least one conservative amino acid substitution. The provided anti-CTLA-4 nano antibody has strong binding capacity with CTLA-4 extracellular domain, and can be used for diagnosis and treatment of tumor.
Description
Technical Field
The invention relates to the technical field of medicinal biology and biological pharmacy, in particular to an anti-CTLA-4 nano antibody, a pharmaceutical composition and application thereof.
Background
Immune checkpoints (immune checkpoint) primarily regulate T cell-mediated immune responses during specific immune processes in the body. When an organism is invaded by a tumor, the tumor cells inhibit the T cells to play a function by abnormally up-regulating the expression of the inhibitory co-stimulatory molecules and the ligands thereof, thereby being beneficial to the growth and escape of the tumor cells. Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is a leukocyte differentiation antigen and is a transmembrane inhibitory co-receptor on T cells. CTLA-4 induces T cell anergy in conjunction with T cell stimulation in conjunction with the binding of B7 family molecules, and participates in the down regulation of immune response. Moreover, CTLA-4 is also found in regulatory T cells (Tregs) and contributes to their suppressive function. Ligands for CTLA-4 are present at highly expressed levels in many solid tumors. These ligands, when bound to CTLA-4 on T cells, down-regulate the immune response of T cells, thereby allowing tumor cells to escape the recognition and killing effects of the immune system. Therefore, the CTLA-4 is an important target point for tumor immunotherapy. At present, anti-CTLA-4 antibodies are mostly used for tumor therapy, and the antibodies are all traditional antibody molecules with large molecular weight and need to be expressed and produced in animal cells. Therefore, it has disadvantages of high production cost and immunogenicity in vivo.
The micromolecule nano antibodies have the characteristics of small molecular mass, small volume, no Fc segment and the like, so that the micromolecule nano antibodies are superior to the traditional antibodies in performance in many aspects, have low immunogenicity, and are not easy to generate autoimmune reaction; has strong tissue permeability; easy binding of cryptic epitopes; can be produced by microorganisms, and has the advantages of low production cost and the like.
Then the anti-CTLA-4 nanobody needs further improvement.
Disclosure of Invention
The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. Therefore, an object of the present invention is to provide an anti-CTL-4 nanobody and its use.
We extract camel peripheral blood to construct phage library, and screen them to obtain anti-CTLA-4 nano antibody, and express it in colibacillus and yeast. The obtained anti-CTLA-4 nano antibody has small molecular weight, strong specificity and strong binding capacity with CTLA-4 extracellular domain, thus being applicable to diagnosis and treatment of CTLA-4 high-expression tumors.
Therefore, the invention provides the following technical scheme:
in a first aspect of the invention, the invention provides an anti-CTLA-4 nanobody selected from at least one of the following: (1) has CDR1 shown in KYIYSNYC, CDR2 shown in IYTGGSNT and CDR3 shown in AATSRRWCSSLEKQVFGY; (2) compared to (1), at least one conservative amino acid substitution. The provided anti-CTLA-4 nano antibody has small molecular weight, strong specificity and strong binding capacity with CTLA-4 extracellular domain, and can be used for diagnosis and treatment of tumors, in particular for diagnosis and treatment of CTLA-4 high-expression tumors.
According to an embodiment of the present invention, the nanobody described above has an amino acid sequence as shown below: (a) 1, SEQ ID NO; (b) has at least one conservative amino acid substitution as compared to (a).
In a second aspect of the invention, the invention provides a nucleic acid comprising a nucleic acid sequence encoding a nanobody according to the first aspect of the invention.
According to an embodiment of the present invention, the above-mentioned nucleic acid may further comprise the following technical features:
according to an embodiment of the invention, the nucleic acid comprises the nucleic acid sequence as shown below: 2, a nucleic acid sequence shown as SEQ ID NO; a sequence having at least 95% homology or more, preferably 98% or more, more preferably 99% or more, with the nucleic acid sequence shown in SEQ ID NO. 2.
In a third aspect of the invention, the invention provides an expression vector comprising a nucleic acid according to the second aspect of the invention.
According to an embodiment of the invention, the expression vector further comprises a regulatory element operably linked to the nucleic acid.
According to an embodiment of the present invention, the regulatory element includes at least one selected from the group consisting of a promoter, an enhancer and a terminator.
In a fourth aspect, the present invention provides a recombinant cell comprising an expression vector according to the third aspect of the present invention.
In a fifth aspect of the invention, the invention provides a method for producing an anti-CTLA-4 nanobody, comprising culturing the recombinant cell according to the fourth aspect of the invention, so as to obtain the anti-CTLA-4 nanobody.
In a sixth aspect of the present invention, the present invention provides a use of a nanobody in the preparation of a medicament for treating tumor, wherein the nanobody is the nanobody according to the first aspect of the present invention.
According to an embodiment of the invention, the tumor comprises at least one selected from the group consisting of prostate cancer, malignant melanoma and lung cancer. According to embodiments of the invention, the lung cancer may be non-small cell lung cancer and small cell lung cancer.
In a seventh aspect of the invention, the invention provides a pharmaceutical composition comprising the nanobody of the first aspect of the invention and a pharmaceutically acceptable carrier.
In an eighth aspect of the present invention, the present invention provides a kit for detecting CTLA-4, the kit comprising the nanobody of the first aspect of the present invention.
In a ninth aspect of the invention, the invention provides a Car-T cell comprising a nanobody according to the first aspect of the invention. The provided Car-T cells containing the anti-CTLA-4 nano antibody are used for cell transfusion to a patient and can be used for treating tumors.
Drawings
FIG. 1 is a photograph of colonies subjected to three rounds of panning provided in accordance with an embodiment of the present invention.
Figure 2 is the expression and purification results of three anti-CTLA-4 nanobodies provided by an embodiment of the present invention.
Figure 3 is the specific detection results of three anti-CTLA-4 nano antibodies provided according to embodiments of the present invention.
Figure 4 is the results of thermal stability assays of three anti-CTLA-4 nanobodies provided in accordance with embodiments of the present invention.
FIG. 5 is a graph showing the results of the CTLA-4 binding efficiency of three anti-CTLA-4 nanobodies on human malignant melanoma cells A375.
Detailed Description
The following detailed description of embodiments of the invention is intended to be illustrative, and is not to be construed as limiting the invention.
Also, some terms herein are explained and illustrated for the sake of convenience for those skilled in the art. These explanations and illustrations are intended only to facilitate understanding and should not be construed as limiting the scope of the present invention.
Herein, the term "antibody" refers to an immunoglobulin molecule capable of binding to a specific antigen. Typically, an antibody structure comprises two light chains of relatively light molecular weight and two heavy chains of relatively heavy molecular weight, the heavy (H) and light (L) chains being linked by a disulfide bond to form a tetrapeptide chain molecule. Among them, the amino-terminal (N-terminal) amino acid sequence of a peptide chain varies widely, called variable region (V region), which determines recognition of an antibody and specific binding to an antigen; the carboxy terminus (C-terminus) is relatively stable with little variation, referred to as the constant region (C-region), which generally confers important biological properties such as antibody chain binding, secretion, transplacental mobility, complement binding, and Fc receptor binding. The variable regions of heavy and light chains are commonly referred to as VH and VL.
Certain regions in the variable region, which have a higher degree of variation in amino acid composition and arrangement order, are called Hypervariable regions (HVRs), which are the sites where antigens and antibodies bind and are therefore also called complementarity-determining regions (CDRs). The heavy chain variable region and the light chain variable region both have three CDR regions. For convenience of description, the CDR regions located on the heavy chain are also referred to as heavy chain hypervariable regions, and the CDR regions located on the light chain are also referred to as light chain hypervariable regions.
The peptide variable region has relatively small changes in the amino acid composition and arrangement sequence except for the hypervariable region and is called a Framework Region (FR). There are 4 framework regions in VH and VL, denoted FR1, FR2, FR3 and FR4, respectively.
Nano antibodies (a single domain antibodies), also known as VHH (variable domain of heavy chain of heavy chain antibody), which earlier existed in camelids and lacked light chain, were cloned to obtain single domain antibodies consisting of only heavy chain variable regions, known as VHH. Compared with common antibodies, the nano antibody has the advantages of small molecular weight, simple structure, easy gene modification, small volume, good antigen specificity, strong tissue penetration, high stability and wide application prospect in the aspects of diagnosis and treatment of diseases.
In the absence of a light chain, nanobodies each have three CDRs, designated CDR1, CDR2, and CDR3, which characterize the antigen recognition and binding specificity of the nanobody. To this end, in one aspect of the present invention, the present invention provides an anti-CTL-4 nanobody, which includes at least one selected from the group consisting of: (1) has CDR1 shown in KYIYSNYC, CDR2 shown in IYTGGSNT and CDR3 shown in AATSRRWCSSLEKQVFGY; (2) compared to (1), at least one conservative amino acid substitution.
These conservative amino acid substitutions may occur at CDR1, at CDR2, at CDR3, or at CDR1, CDR2, or CDR 3. Of course, these conservative amino acid substitutions may be one amino acid substitution, two amino acid substitutions or three amino acid substitutions.
Herein, the "conservative amino acid substitution" refers to the change of some amino acid or some amino acids in the nanobody, without damaging the overall conformation and function of the nanobody. These conservative amino acid substitutions include the replacement of one amino acid with another having similar properties (e.g., polarity, hydrogen bond potential, acidity, basicity, hydrophobicity, presence of an aromatic group, etc.). Amino acids with similar properties are well known to those skilled in the art. For example, arginine, histidine and lysine are hydrophilic-basic amino acids and may be interchanged. Similarly, isoleucine (a hydrophobic amino acid) may be replaced with leucine, methionine, or valine. This change has little or no effect on the apparent molecular weight or isoelectric point of the nanobody. For another example, some natural amino acids may be replaced with unnatural amino acids, such as amino acids in the D configuration, or β or γ amino acids.
According to an embodiment of the present invention, the nanobody provided has an amino acid sequence as shown below: (a) 1, SEQ ID NO; (b) has at least one conservative amino acid substitution as compared to (a).
Wherein the amino acid sequence shown in SEQ ID NO. 1 is as follows:
MKYLLPTAAAGLLLLAAQPAMAQVQLQESGGGSVQAGGSLRLSCAATKYIYSNYCMGWFRQAPGKEREGVAAIYTGGSNTYYADSVKGRFTISHDDAKSTVYLQMNSAKPEDTAMYYCAATSRRWCSSLEKQVFGYWGQGTQVTVSSAAAYPYDVPDYGS(SEQ ID NO:1)。
the anti-CTLA-4 nano antibody provided by the invention has thermal stability. For example, at 37 degrees Celsius for 3 hours, the activity is at least 80% retained. For another example, the activity is retained by at least 40% after 3 hours at 60 ℃. Compared with the anti-CTLA-4 monoclonal antibody, the anti-CTLA-4 nano antibody provided by the invention has better stability, thereby providing convenience for storage, transportation and the like of the nano antibody. And the provided anti-CTLA-4 antibody can be used for detection under special environments. In addition, the provided anti-CTLA-4 nanobody can also be used in other non-medicinal aspects, such as being applied to shampoo and the like.
In another aspect of the present invention, the present invention also provides a nucleic acid comprising a nucleic acid sequence encoding the anti-CTL-4 nanobody described above. The nucleic acid, which is commonly referred to as a DNA molecule, may be an RNA molecule, and may be contained in any suitable vector, such as a plasmid, artificial chromosome, phage, or viral vector, and the like. According to an embodiment of the invention, the nucleotide sequence encoding the sequence shown in SEQ ID NO. 1 is shown in SEQ ID NO. 2. According to an embodiment of the present invention, the nucleic acid sequence provided may also be a sequence having at least 90% homology or more, such as at least 95% homology or more, preferably 98% homology or more, more preferably 99% homology or more, with the nucleic acid sequence shown in SEQ ID NO. 2.
The nucleic acid sequence shown in SEQ ID NO. 2 is as follows:
ATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCGGCCCAGCCGGCCATGGCCCAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTGCAGCCACTAAATATATCTACAGTAATTACTGCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGCGAGGGGGTCGCAGCTATTTATACTGGTGGCAGTAACACATACTATGCCGACTCCGTGAAGGGCCGCTTCACCATCTCCCATGACGACGCCAAGTCTACGGTGTATCTGCAAATGAACAGCGCGAAACCTGAAGATACTGCCATGTACTACTGTGCGGCGACGAGTAGGCGCTGGTGTTCATCACTGGAAAAACAAGTCTTTGGTTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGCGGCCGCATACCCGTACGACGTTCCGGACTACGGTTCCTAA(SEQ ID NO:2)。
in another aspect of the invention, the invention provides an expression vector comprising the nucleic acid described above. An expression vector, also commonly referred to in the art as a cloning vector or vector, as used herein refers to a vector into which a DNA sequence or an RNA sequence may be introduced into a host cell, which may be used for transformation into the host cell and to facilitate expression of the nucleic acid sequence, e.g., to facilitate transcription and translation. Such expression vectors may contain regulatory elements, such as promoters, enhancers, terminators, and the like, for causing or directing expression of the polypeptide. Examples of promoters and activators of expression vectors for animal cells include SV40 early promoter and activator and the like. Suitable vectors may be plasmids, such as some plasmids containing an origin of replication or integrative plasmids, e.g. pUC, pcDNA, pBR, etc. Useful viral vectors include, but are not limited to, adenovirus, retrovirus, herpes virus, and AAV vectors. Such viral vectors can be produced by techniques well known to those skilled in the art, for example, by transient or stable transfection of the virus. In viral transfection, the transfected cells that can be used may be PA317 cells, PsiCRIP cells, GPenv + cells, 293 cells, etc.
In another aspect of the present invention, the present invention provides a recombinant cell comprising the expression vector described above. The provided recombinant cells can be obtained by transforming an expression vector into a host cell. For example, a foreign gene or DNA sequence or RNA sequence can be introduced into a host cell via an expression vector to allow the host cell to express the introduced gene or sequence to produce a desired substance, which is typically a protein encoded by the gene or introduced sequence. Commonly used host cells include, but are not limited to, E.coli host cells (often with plasmid vectors for introduction), insect host cells (often with baculovirus vectors for introduction), and mammalian host cells. For example, prokaryotic cells (e.g., bacteria) and eukaryotic cells (e.g., yeast cells, mammalian cells, insect cells, plant cells, etc.) may also be included. Specific mammalian host cells may be Vero cells, CHO cells, 3T3 cells, COS cells, etc.
The anti-CTLA-4 nanobody can be obtained by culturing the recombinant cells provided above under appropriate conditions. In culturing the recombinant cells, any production technique known to those skilled in the art, such as any chemical, biological, genetic or enzymatic technique, may be employed, either alone or in combination. The obtained anti-CTLA-4 nanobody may be separated and purified by conventional protein purification methods, for example, hydroxyapatite chromatography, gel electrophoresis, affinity dialysis or chromatography.
The anti-CTLA-4 nanobody provided by the present invention can be used for treating diseases characterized by over-expression of CTLA-4, such as tumors, including but not limited to prostate cancer, malignant melanoma and lung cancer (small cell and non-small cell), etc. For example, ovarian cancer, colon cancer, rectal cancer, renal cancer, bladder cancer, breast cancer, liver cancer, lymph cancer, hematological malignancy, head and neck cancer, gastric cancer, glioma, nasopharyngeal carcinoma, laryngeal carcinoma, cervical cancer, uterine body tumor, osteosarcoma, and the like can also be cited.
In yet another aspect of the present invention, the present invention provides a pharmaceutical composition comprising the above anti-CTLA-4 nanobody and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable auxiliary ingredient, and may include, for example, any solvent, dispersion medium, coating, antibacterial or antifungal agent, isotonic or absorption delaying agent, and the like. The function and use of these pharmaceutically acceptable carriers are well known to those skilled in the art. According to the functions and the application of the pharmaceutically acceptable carriers, the carrier can be compounded with the anti-CTLA-4 nano antibody by a person skilled in the art to obtain a corresponding pharmaceutical composition which is used in the fields of pharmacy or disease treatment. The pharmaceutical compositions provided may be in various dosage forms, e.g., may be administered orally, by inhalation, parenterally (particularly by intravenous injection), and the like in suitable forms. When administered parenterally, the anti-CTLA-4 nanobodies may be provided in the form of injectable solutes and suspensions packaged in vials. The anti-CTLA-4 nanobody may be generally mixed with buffers, stabilizers, preservatives, solubilizing agents, isotonizing agents, suspending agents and the like to obtain a form for parenteral administration. The mixed substances are sterilized and then packaged in the form of intravenous injection. Useful buffers may be organophosphates. Useful suspending agents may be methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, gum arabic and/or sodium carboxymethylcellulose. Further, usable stabilizers may be sodium sulfite and sodium metabisulfite, and usable preservatives may be sodium p-hydroxybenzoate, sorbic acid, cresol, chlorocresol and the like.
The pharmaceutical compositions of the present invention may also be administered in combination therapy, i.e. in combination with other agents. For example, combination therapy may comprise the anti-CTLA-4 nanobody of the present invention in combination with at least one other anti-neoplastic agent. For example, the anti-CTLA-4 nanobodies provided by the present invention may be used in combination with antibodies targeting other tumor-specific antigens. Such antibodies targeting other tumor-specific antigens include, but are not limited to, anti-EGFR antibodies, anti-HER 2 antibodies, anti-VEGFa antibodies, and the like, which are preferably monoclonal antibodies. The anti-CTLA-4 nano antibody provided by the invention can also be combined with other tumor immunotherapy means or tumor targeting micromolecule medicines. Such other tumor immunotherapies include, but are not limited to, therapeutic antibodies directed against tumor immunomodulatory molecules, such as OX40, PDL1/PD1, CD137, or CAR-T therapeutic modalities, among others.
The provided pharmaceutical composition can also be used in combination with other tumor treatment means, such as radiotherapy, chemotherapy, surgery and the like, or before or after radiotherapy, chemotherapy or surgery.
The dosage of the anti-CTLA-4 nanobody in the pharmaceutical composition may range from 0.0001 to 100mg/kg, more typically from 0.01 to 20mg/kg of the subject's body weight. For example, the dose may be 0.2mg/kg body weight, 0.5mg/kg body weight, 1mg/kg body weight, 3mg/kg body weight, 5mg/kg body weight, 8mg/kg body weight, 10mg/kg body weight, 15mg/kg body weight, 20.2mg/kg body weight, or 30mg/kg body weight, or in the range of 1-30mg/kg body weight. For treatment or administration, weekly administration, biweekly administration, every three weeks, every four weeks, etc., may be employed.
The provided anti-CTLA-4 nanobody can also be used as a part of a kit for detecting CTLA-4. The provided kit can contain conventional reagents which are commonly used in the field and used for antigen detection besides the anti-CTLA-4 nano antibody.
In another aspect of the present invention, the present invention also provides a method for preventing and/or treating cancer, the method comprising administering to a subject an effective amount of a pharmaceutical composition provided as above or an effective amount of a nanobody, the nanobody being provided as above. An "effective amount" as referred to herein may preferably be such that cell growth or tumor growth in the subject is inhibited by at least 10%, preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%. An effective amount of the pharmaceutical composition or an effective amount of the nanobody is capable of reducing tumor size or otherwise alleviating a symptom of the subject, e.g., achieving or extending progression-free survival of a tumor patient, etc.
The anti-CTLA-4 nanobody provided by the method can be applied to the research and development of biological medicines, clinical diagnosis, tumor research and treatment and immunological research and treatment. For example, it can be applied not only to the above-mentioned respective products such as pharmaceutical compositions, kits, etc., but also to the modification into the form of multivalent antibodies or multispecific nanobodies in combination with techniques commonly used in the art. Can also be applied to the preparation of Car-T cells. These Car-T cells can be obtained by a commonly used means of genetic engineering, and thus can be used for reinfusion into a patient to treat tumors.
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1 preparation of phage Nanobody library
(1) Two blood of bactrian camel without any immune antigen are taken, each blood is taken 100mL, the collected blood is mixed with physiological saline with equal volume, the diluted blood is slowly added to the surface of lymphocyte separation liquid, and the mixture is centrifuged at 2000rpm for 20min at room temperature. The second layer of lymphocytes was aspirated, five times the volume of PBS was added, centrifuged at 2000rpm for 20min at room temperature, and repeated three times. The collected lymphocytes were added to 1mL of Trizol (purchased from Invitrogen, cat # 15596026) reagent, mixed repeatedly, and centrifuged at 12000rpm at 4 ℃ for 15 min. Then, 0.2mL of chloroform was added to the centrifuged supernatant, and after mixing well, the mixture was left to stand for 3min and centrifuged at 12000rpm at 4 ℃ for 15 min. Sucking the uppermost layer of water phase, mixing with isopropanol of equal volume, standing for 10min, and centrifuging at 12000rpm and 4 deg.C for 10 min. The supernatant was removed, 1mL of pre-cooled 75% ethanol was added, the precipitate was washed and centrifuged at 12000rpm at 4 ℃ for 10 min. Then 100. mu.L of DEPC was added to dissolve in water.
(2) The RNA was reverse transcribed into DNA using the AMV First Strand cDNA Synthesis Kit (purchased from NEB under the accession number E6550S).
The first step system conditions were as follows:
the system conditions of the second step are as follows:
(3) the VHH gene was amplified by PCR using the KAPA HiFi HotStart ReadyMix PCR Kit (purchased from KAPA under the product number KK 2611). Furthermore, since camelid antibodies have two templates, the C-termini of which are slightly different, two R-primers are used to obtain more comprehensive antibodies.
Reaction conditions are as follows:
F-primer:5′
-GAGGAGGAGGAGGAGGTGGCCCAGGCGGCCCAGGTSMARCTGCAGSAGTCWGG-3' (SEQ ID NO: 3). Wherein S represents the base G or C, W represents the base A or T, M represents the base A or C, and R represents the base G or A.
R-primer1:5′
-GAGGAGGAGGAGGAGGTGGCCCAGGCGGCCGGAGCTGGGGTCTTCGCTGTGGTGCG-3′(SEQ ID NO:4)。
R-primer2:5′
-GAGGAGGAGGAGGAGGTGGCCCAGGCGGCCTGGTTGTGGTTTTGGTGTCTTGGGTT-3′(SEQ ID NO:5)。
(4) The pComb-3X vector (available from Allle Biotechnology and Pharmaceuticals) and the VHH gene were digested with Sfi I (available from Takara, Cat. 1244A) endonuclease using the following reaction system and conditions:
(5) the pComb-3X vector and VHH gene were ligated with T4 DNA Ligase. The reaction system conditions were as follows:
(6) mu.L of the ligation product was mixed with 90. mu.L of TG1 competent cells, and the mixture was transferred to a cuvette at 1900V for 5ms, and then 1mL of SOC medium (available from Sangon Biotech, cat. No. A507009) was added and incubated at 37 ℃ for 1 hour at 250rpm in a shaker. 10mL of SOC medium was added and the culture was continued for 1 hour. To this was added 2mL of VCSM13 helper phage (purchased from Biovector) while adding 200mL of SOC medium containing ampicillin (Amp, purchased from Sangon Biotech, cat # A610028) resistance at a final concentration of 10. mu.g/mL, and the culture was continued for 2 h. Kanamycin (Kana, available from Sangon Biotech, cat. No. A600286) was then added to the cells to a final concentration of 10. mu.g/mL for overnight incubation.
(7) The overnight-cultured TG1 strain was centrifuged at 3000g for 15min at 4 ℃. The centrifuged supernatant was mixed with PEG6000 (purchased from Biosharp, cat # BY0027)/NaCl (purchased from Macklin, cat # S805275) at a ratio of 6:1, and allowed to stand on ice for 30 min. 2200 Xg centrifugation for 30min, the precipitation and 2mL PBS solution heavy suspension. Then, the mixture was centrifuged at 13200rpm for 5min at 4 ℃. The supernatant was then filtered through a 0.22 μm filter (purchased from Merck Millipore, cat # SLGP033RB) for the elutriation experiments.
Example 2 phage Nanobody library panning
The phage nanocompania obtained in example 1 was panned, comprising the following steps:
(1) with NaHCO3The coating (purchased from Sigma, cat # s6297) diluted CTLA-4 (purchased from Cassia, cat # 90213-C08H) protein, 20. mu.g CTLA-4 protein per well was conjugated to the plate, overnight at 4 ℃ and a negative control was set.
(2) The next day, the unconjugated target protein in the ELISA plate was aspirated, 300. mu.L of 0.1% PBST was added, the mixture was allowed to stand for 3min (plate washing was performed once), 2% skimmed milk powder was added to the ELISA plate, the plate was sealed at 37 ℃ for 2h, then 300. mu.L of 0.1% PBST solution was used to wash the plate once, and the mixture was allowed to stand for 3 min.
(3) And (3) uniformly mixing 300 mu L of the prepared nano antibody phage library with 2% skimmed milk powder with the same volume, adding 100 mu L of the mixed solution into each hole, and incubating for 1h at 37 ℃.
(8) The plate was washed 5-15 times with 300 μ L of 0.1% PBST solution per well and allowed to stand 3min each time (plate washing times increased correspondingly according to the number of washing rounds, final washing, and halved concentration of conjugated antigen) for three rounds of panning experiments.
(4) mu.L of 100mM triethylamine (TES, available from aladdin) solution was added to each well and incubated at room temperature for 10 min. And continuously infecting the TG1 bacterial strain with the dissociated phage in the supernatant, and continuously enriching in the process of multiple rounds of panning so as to screen out the nano antibody sequence with good specificity and high affinity.
FIG. 1 shows a colony map of three rounds of panning, in which 1.9X 10 in FIG. 15CFU represents the number of colonies obtained by the first round of panning, 3.9X 104CFU represents the number of colonies obtained by the second round of panning, 1.6X 106CFU represents the number of colonies obtained after the third round of panning. The results show that the volume of the pool after three rounds of elutriation is gradually increased.
EXAMPLE 3 phage Nanobody library screening of anti-CTLA-4 Nanobodies
The method for screening the anti-CTLA-4 nano antibody by using the elutriated phage nano antibody library comprises the following steps:
(1) different single colonies on the final round of plates randomly selected for panning were inoculated into 96-well deep-well plates (purchased from NUNC under item number 95040452) containing 1 mL/well of 2 xyt medium (purchased from Sangon Biotech under item number a507016) and labeled on the plates. After incubation at 37 ℃ for 3h at 150rpm, 1mM IPTG (from Solarbio under the reference I8070) was added and induced overnight at 28 ℃ at 150 rpm.
(2) With NaHCO3CTLA-4 protein was diluted with coating solution, 100ng CTLA-4 protein was conjugated to the plate, 4 ℃ overnight, and the corresponding negative control was set.
(3) The next day, wash the plate with 0.1% PBST, stand for 3min, add 2% skimmed milk powder to the elisa plate, block for 2h at 37 ℃, wash the plate once with 0.1% PBST solution, and centrifuge the overnight induced deep well plate for 30min at 4 ℃, 4000 rpm. The supernatant was aspirated off, and the pellet from each well was resuspended in 200. mu.L TES solution, incubated at 4 ℃ and 150rpm for 2 h.
(4) Add 300. mu.L TES/4 solution to each well and incubate at 4 ℃ for 2h at 150 rpm. The deep-well plate was then placed at 4 ℃ and 4000rpm and centrifuged for 30 min. The supernatant was added to the corresponding wells containing the target protein and negative control wells at 100. mu.L, respectively, and incubated at 37 ℃ for 1 h.
(5) mu.L of 0.1% PBST solution was added to each well and the plate was washed three times, each time for 3min, unbound antibody was removed, 100. mu.L of 1:5000 diluted anti-HA-HRP (purchased from GNI, GNI4310-HA-S) was added to each well and incubated at 37 ℃ for 1 h.
(6) The plate was washed three times with 300. mu.L of 0.1% PBST solution per well and left for 3min each time to remove unbound antibody, 100. mu.L of TMB (purchased from England, Huzhou, TMB-S-004) developer was added per well, incubated at 37 ℃ in the dark for 10min, and 100. mu.L of 2.29% sulfuric acid (purchased from Guangzhou chemical Co., Ltd.) was added per well to terminate the reaction.
(7) The value of OD450nm wavelength was measured by a microplate reader, and the antigen well was twice the absorbance of the blank well, and was considered positive.
(8) Positive transformants were inoculated into 10mL LB medium (purchased from Sangon Biotech, cat # A507002), incubated at 37 ℃ for 8h at 150rpm, and the inoculum was collected and sequenced (Sangon Biotech).
And according to the sequencing result, performing sequence comparison by using BLAST, regarding the sequences with the complementary determining regions with similar sequences as one strain, and finally screening to obtain the specific anti-CTLA-4 nano antibody. For the convenience of expression, the nanobody obtained by screening was named E3 nanobody. Through determination, the amino acid sequence of the E3 nano antibody is shown as SEQ ID NO. 1, and the nucleic acid sequence of the sequence shown as the coding SEQ ID NO. 1 is shown as SEQ ID NO. 2.
Example 4 expression purification of anti-CTLA-4 Nanobodies
(1) The above positive transformant plasmid was electrically transferred to WK6 strain (purchased from Biovector NTCC plasmid vector cell Gene Collection) at 1800V, spread on Amp-resistant LB plate, cultured overnight, and on the next day, a single colony on the plate was picked up and inoculated into 10mL of LB medium containing Amp resistance, and cultured at 37 ℃ and 220rpm for 8 hours.
(2) The above-mentioned bacterial suspension was inoculated into 330mL of Amp-resistant TB medium (obtained from ELITE-MEDIA, cat. No. M201-02) and cultured at 37 ℃ and 220rpm until OD becomes 0.7-0.8. Then, the mixture was induced overnight at 220rpm at 28 ℃ with a final concentration of 1mM IPTG.
(3) The next day, the cells were collected, resuspended in PBS and centrifuged. Finally, the ratio of the mass ratio of the thalli to the volume of TES solution is 1:10, and disrupting the cells by osmotic pressure. Centrifuging at 10000rpm for 30min, and collecting supernatant extract. The bacterial debris was removed by filtration through a 0.45 μm filter.
(4) Nickel column affinity chromatography was performed using an AKTA purifier (available from GE) by first equilibrating a nickel column (available from GE under code number 10230759) with PBS solution at a flow rate of 1mL/min until the UV280 value remained unchanged, loading at a flow rate of 1mL/min, equilibrating the nickel column with PBS solution at a flow rate of 1mL/min until the UV280 value remained unchanged, and eluting the hybrid protein and the protein of interest with 20mM, 100mM, 150mM, 200mM, 250mM, and 500mM imidazole at a flow rate of 1mL/min, respectively.
(5) The electrophoresis was performed using a pre-made gel (available from invitrogen under the trade name EC6025BOX), and 20 μ L of the prepared sample was added to the pre-made gel, and the sample was electrophoresed to the separation gel at 80V for 30min, and then electrophoresed at 120V until the indicator reached the bottom. The gel was soaked in Coomassie Brilliant blue solution (purchased from Solarbio, catalog number P1305) for 1h, and then decolorized with decolorizing solution (purchased from Solarbio, catalog number P1305) until clear protein bands could be seen, finally purified E3 nano antibody was obtained, the purity was 95% by analysis of Image J software, and FIG. 2 shows the expression purification results of specific anti-CTLA-4 nano antibody.
Example 5 detection of binding of anti-CTLA-4 Nanobodies to antigens
The anti-CTLA-4 nanobody obtained in example 4 was detected by the following procedure:
(1) CTLA-4, PD-1 (purchased from Yiqiao Shenzhou, with the product number of 10377-H02H), PD-L1 (purchased from Yi qiao Shenzhou, with the product number of 10084-HNAH), CD28 (purchased from Yi qiao Shenzhou, with the product number of 11524-HCCH), and CD80 (purchased from Yi qiao Shenzhou, with the product number of 10698-HCCH) were diluted to 1 μ g/mL, added to the plate, incubated at 100 ng/well and incubated at 4 ℃ overnight. A control was also set.
(2) Washing the plate with 300 μ L0.1% PBST, washing the plate once, standing for 3min, adding 300 μ L5% skimmed milk powder into each well, and sealing at 37 deg.C for 1 h.
(3) The plate was washed once with 300. mu.L of 0.1% PBST, left to stand for 3min, and the positive control anti-CTLA-4mAb (purchased from abcam, cat. No. ab237712), blank control PBS and panel Nanobody NbH3 were added to the microplate, 100. mu.L per well, 100 ng/well, incubated at 37 ℃ for 1 h.
(4) mu.L of 0.1% PBST solution was added to each well and the plate was washed three times, each time for 3min, to remove unbound antibody, 100. mu.L of 1:5000 diluted Mouse anti-HA mAb (purchased from proteintech, cat # 66006-2-Ig) was added to each well and incubated at 37 ℃ for 1 h.
(5) The plate was washed three times with 300 μ L of 0.1% PBST solution per well, left for 3min each time to remove unbound antibody, and 100 μ L of 1: HRP-Goat Anti-Mouse IgG (H + L) (purchased from proteintech under the code SA00001-1) at 5000 dilution was incubated at 37 ℃ for 1H.
(6) And adding 300 mu L of 0.1% PBST solution into each hole, washing the plate for five times, standing for 3min each time, adding 100 mu L of TMB solution into each hole, and incubating for 10min in a dark place at 37 ℃.
(7) And adding 100 mu L of 2.29% sulfuric acid into each hole of the ELISA plate to terminate the reaction, measuring OD values at 450nm and 630nm by using an ELISA reader, and calculating the difference value between OD450 and OD630 to obtain the OD value.
FIG. 3 shows the result of the analysis of the binding ability of anti-CTLA-4 nanobody to antigen, and the result shows that the E3 nanobody can specifically bind to CTLA-4.
Example 6 detection of thermal stability of anti-CTLA-4 Nanobodies
The thermal stability of the obtained anti-CTLA-4 nano antibody is detected by the following method:
(1) with NaHCO3CTLA-4 protein was diluted with coating solution and coupled to a microplate at 50 ng/well CTLA-4 protein overnight at 4 ℃ and corresponding negative controls were set.
(2) Washing the plate with 300 μ L0.1% PBST, washing the plate once, standing for 3min, adding 300 μ L5% skimmed milk powder into each well, and sealing at 37 deg.C for 2 h.
(3) The control group of commercially available anti-CTLA-4 antibody (obtained from Ipilimumab, product number 477202-00-9) and the experimental group of E3 nanometer antibody (concentration 500ng/mL) were placed at 25 deg.C, 37 deg.C, 60 deg.C, 90 deg.C for 0min, 10min, 30min, 60min, 120min and 180min, respectively. Immediately thereafter, the plate was placed on ice. Add 100. mu.L/well to the microplate and incubate at 37 ℃ for 1 h.
(4) The subsequent steps are as in reference example 5.
FIG. 4 shows the result of testing the thermal stability of the anti-CTLA-4 nanobody, and the experimental result shows that the E3 nanobody has good thermal stability. Generally, a typical protein will lose activity immediately at 60 degrees celsius, even at 37 degrees celsius, while the E3 nm antibody has little change in activity at 37 degrees celsius compared to 25 degrees celsius. Although some activity is lost under the condition of 60 ℃, some activity is still remained, and the result shows that the E3 nano antibody has good thermal stability.
Example 7 binding of anti-CTLA-4 Nanobody to CTLA-4 molecules on the cell membrane of human malignant melanoma cells A375
(1) A single cell suspension was obtained by digesting a human malignant melanoma cell A375 cell (purchased from Biotechnology Co., Ltd., bio-72958, Baiohobowei, Beijing) at the logarithmic growth phase with 0.25% trypsin (purchased from Gibco, cat # 25200-072), and then terminating the digestion with a DMEM medium (purchased from Gibco, cat # C11995500BT) containing 10% FBS (purchased from Gibco, cat # 10270106). Centrifuge at 1000rpm for 5min, discard the supernatant and wash twice with 1 × PBS.
(2) Then diluting the E3 nano antibody to 50 mu g/mL by using an antibody diluent, wherein each experimental group is 1X 106100 μ L of the diluted nanobody was added to each A375 cell,positive controls were run at 1: anti-CTLA-4mAb was diluted at a ratio of 100 and added to 1X 106In each A375 cell, a negative control was also set. Incubate at 37 ℃ for 1 h.
(3) Centrifuge at 1000rpm for 5min, resuspend with 1mL PBS solution, repeat three times. The experimental group diluted Mouse anti-His antibody (which was the provided E3 nanobody with His tag attached) at a ratio of 1:100, and the positive control group added 1:100 dilution of Mouse anti-CTLA-4mAb, 100 u L antibody heavy suspension cells. Incubate at 37 ℃ for 45 min.
(4) Centrifuge at 1000rpm for 5min, resuspend with 1mL PBS solution, repeat three times. Mixing the raw materials in a ratio of 1: the flow antibody, Goat anti-mouse IgG (FITC), was diluted at a ratio of 100, and 100. mu.L of the diluted flow antibody was added to each group, and the mixture was protected from light at 37 ℃ for 30 min. Centrifuge at 1000rpm for 5min, resuspend with 1mL PBS solution, repeat three times. Finally, resuspend with 500. mu.L PBS. Detection was performed with a flow cytometer (purchased from BD, FACSCalibur). The results were analyzed by Flowjo 7.6.1.
FIG. 5 is a graph showing the binding efficiency of anti-CTLA-4 nanobody to CTLA-4 molecules on the cell membrane of A375, and the results show that the E3 nanobody has strong binding ability with specificity to CTLA-4 of A375 cells. Through the specific combination, the provided E3 nano antibody can be used for treating diseases characterized by over-expression of CTLA-4.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or to implicitly indicate the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
SEQUENCE LISTING
<110> mountain Buddha Teng biological science and technology Limited company, river-south university
<120> anti-CTLA-4 nano antibody, pharmaceutical composition and application thereof
<130> PIDC3196493
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 160
<212> PRT
<213> Artificial Sequence
<220>
<223> anti-CTLA-4 nanobody
<400> 1
Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala
1 5 10 15
Ala Gln Pro Ala Met Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly
20 25 30
Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Thr Lys
35 40 45
Tyr Ile Tyr Ser Asn Tyr Cys Met Gly Trp Phe Arg Gln Ala Pro Gly
50 55 60
Lys Glu Arg Glu Gly Val Ala Ala Ile Tyr Thr Gly Gly Ser Asn Thr
65 70 75 80
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser His Asp Asp
85 90 95
Ala Lys Ser Thr Val Tyr Leu Gln Met Asn Ser Ala Lys Pro Glu Asp
100 105 110
Thr Ala Met Tyr Tyr Cys Ala Ala Thr Ser Arg Arg Trp Cys Ser Ser
115 120 125
Leu Glu Lys Gln Val Phe Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr
130 135 140
Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser
145 150 155 160
<210> 2
<211> 483
<212> DNA
<213> Artificial Sequence
<220>
<223> nucleic acid sequence encoding anti-CTLA-4 nanobody
<400> 2
atgaaatacc tattgcctac ggcagccgct ggattgttat tactcgcggc ccagccggcc 60
atggcccagg tgcagctgca ggagtctgga ggaggctcgg tgcaggctgg agggtctctg 120
agactctcct gtgcagccac taaatatatc tacagtaatt actgcatggg ctggttccgc 180
caggctccag ggaaggagcg cgagggggtc gcagctattt atactggtgg cagtaacaca 240
tactatgccg actccgtgaa gggccgcttc accatctccc atgacgacgc caagtctacg 300
gtgtatctgc aaatgaacag cgcgaaacct gaagatactg ccatgtacta ctgtgcggcg 360
acgagtaggc gctggtgttc atcactggaa aaacaagtct ttggttactg gggccagggg 420
acccaggtca ccgtctcctc agcggccgca tacccgtacg acgttccgga ctacggttcc 480
taa 483
<210> 3
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> F-primer
<400> 3
gaggaggagg aggaggtggc ccaggcggcc caggtsmarc tgcagsagtc wgg 53
<210> 4
<211> 56
<212> DNA
<213> Artificial Sequence
<220>
<223> R-primer 1
<400> 4
gaggaggagg aggaggtggc ccaggcggcc ggagctgggg tcttcgctgt ggtgcg 56
<210> 5
<211> 56
<212> DNA
<213> Artificial Sequence
<220>
<223> R-primer 2
<400> 5
gaggaggagg aggaggtggc ccaggcggcc tggttgtggt tttggtgtct tgggtt 56
Claims (14)
1. An anti-CTLA-4 nanobody, which has CDR1 shown in KYIYSNYC, CDR2 shown in IYTGGSNT and CDR3 shown in AATSRRWCSSLEKQVFGY.
2. The nanobody of claim 1, wherein the nanobody has an amino acid sequence as shown below:
1, and the amino acid sequence shown in SEQ ID NO.
3. A nucleic acid comprising a nucleic acid sequence encoding the nanobody of claim 1.
4. The nucleic acid of claim 3, wherein the nucleic acid comprises the nucleic acid sequence set forth as follows:
2, SEQ ID NO.
5. An expression vector comprising the nucleic acid of claim 3 or 4.
6. The expression vector of claim 5, further comprising a regulatory element operably linked to the nucleic acid.
7. The expression vector of claim 6, wherein the regulatory element comprises at least one selected from the group consisting of a promoter, an enhancer, and a terminator.
8. A recombinant cell comprising the expression vector of any one of claims 5 to 7.
9. A method of producing anti-CTLA-4 nanobody, comprising culturing the recombinant cell of claim 8 to obtain the anti-CTLA-4 nanobody.
10. Use of the nanobody of claim 1 or 2 in the preparation of a medicament for the treatment of tumors selected from at least one of prostate cancer, malignant melanoma, lung cancer, ovarian cancer, colon cancer, rectal cancer, kidney cancer, bladder cancer, breast cancer, liver cancer, lymphatic cancer, hematological malignancies, head and neck cancer, stomach cancer, glioma, nasopharyngeal carcinoma, laryngeal cancer, cervical cancer, uterine body tumor and osteosarcoma.
11. Use according to claim 10, wherein the lung cancer is selected from small cell lung cancer and non-small cell lung cancer.
12. A pharmaceutical composition comprising the nanobody of claim 1 or 2 and a pharmaceutically acceptable carrier.
13. A kit for detecting CTLA-4, comprising the nanobody of claim 1 or 2.
14. A Car-T cell, comprising the nanobody of claim 1 or 2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010048833.0A CN111234019B (en) | 2020-01-16 | 2020-01-16 | anti-CTLA-4 nano antibody, pharmaceutical composition and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010048833.0A CN111234019B (en) | 2020-01-16 | 2020-01-16 | anti-CTLA-4 nano antibody, pharmaceutical composition and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111234019A CN111234019A (en) | 2020-06-05 |
| CN111234019B true CN111234019B (en) | 2022-06-24 |
Family
ID=70868678
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010048833.0A Active CN111234019B (en) | 2020-01-16 | 2020-01-16 | anti-CTLA-4 nano antibody, pharmaceutical composition and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111234019B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114560942B (en) * | 2022-01-24 | 2023-05-19 | 宁夏医科大学 | anti-CTLA-4 nanobody, coding gene, recombinant nanobody, recombinant vector, recombinant bacterium and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106220732A (en) * | 2015-07-20 | 2016-12-14 | 广西医科大学 | Nano antibody Nb16 of anti-CTLA 4 and preparation method and application |
| CN108299561A (en) * | 2018-01-02 | 2018-07-20 | 暨南大学 | A kind of PD-1 nano antibodies and its cloning expression method and application |
| CN110256563A (en) * | 2019-07-05 | 2019-09-20 | 石河子大学 | CTLA-4 nano antibody, preparation method and applications |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI746473B (en) * | 2015-11-02 | 2021-11-21 | 美商辛分子醫藥有限公司 | Single domain antibodies directed against intracellular antigens |
-
2020
- 2020-01-16 CN CN202010048833.0A patent/CN111234019B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106220732A (en) * | 2015-07-20 | 2016-12-14 | 广西医科大学 | Nano antibody Nb16 of anti-CTLA 4 and preparation method and application |
| CN108299561A (en) * | 2018-01-02 | 2018-07-20 | 暨南大学 | A kind of PD-1 nano antibodies and its cloning expression method and application |
| CN110256563A (en) * | 2019-07-05 | 2019-09-20 | 石河子大学 | CTLA-4 nano antibody, preparation method and applications |
Non-Patent Citations (4)
| Title |
|---|
| A nanobody against CTLA-4 increases the anti-tumor effects of specific CD8+ T cells;Mo F. 等;《第十七届国际免疫学大会会议论文集》;20191031;第1809页 * |
| immunoglobulin heavy chain variable region, partial [Camelus bactrianus];Li,J.W. 等;《Genbank Database》;20120425;ACCESSION NO. AEX07309 * |
| 免疫检查点抑制剂在肿瘤治疗中的应用;艾斌;《医学与哲学:临床决策论坛版》;20150228;第36卷(第2B期);第18-21页 * |
| 重组人CTLA-4胞外段的表达纯化及其纳米抗体的筛选与功能鉴定;吴利文;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20210215(第2期);E059-234 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111234019A (en) | 2020-06-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109096396B (en) | anti-PD-L1 humanized nano antibody and application thereof | |
| CN110144009B (en) | CD47 single domain antibodies and uses thereof | |
| CN107216389B (en) | anti-PD-L1 nano antibody and coding sequence and application thereof | |
| US11292841B2 (en) | Anti-PD-1 nano-antibody and application thereof | |
| US20220002418A1 (en) | Anti-pd-l1/vegf bifunctional antibody and use thereof | |
| CN110144010B9 (en) | Blocking type PD-L1 camel source single domain antibody and application thereof | |
| CN107955071B (en) | Human anti-human CD47 antibody and coding gene and application thereof | |
| WO2018233575A1 (en) | Cd47-blocking nanobody and use thereof | |
| CN112457404B (en) | Anti-human EGFR nano antibody and application | |
| US9574012B2 (en) | AGR2 blocking antibody and use thereof | |
| EP2921503B1 (en) | Human anti-human epidemic growth factor receptor antibody and encoding gene and application thereof | |
| CN112574311B (en) | Antibody with double MIC binding activity and application thereof | |
| CN110144011A (en) | For the single domain antibody of T lymphocyte immunoglobulin Mucin 3 | |
| CN111171151B (en) | anti-EGFR (epidermal growth factor receptor) nano antibody and application thereof | |
| CN114478778A (en) | anti-Tim-3 nano antibody and application thereof | |
| CN111234019B (en) | anti-CTLA-4 nano antibody, pharmaceutical composition and application thereof | |
| CN111153997B (en) | anti-CTLA-4 nano antibody and application thereof in tumor treatment | |
| CN111378040B (en) | Antibody for detecting multiple malignant tumor cells and application thereof | |
| CN111153998B (en) | anti-CTLA-4 nano antibody and application thereof | |
| CN110885377A (en) | anti-CD 47/VEGF bispecific antibody and application thereof | |
| CN109879966A (en) | Humanization design and expression verifying based on source of mouse CD19 antibody | |
| WO2020253393A1 (en) | Covalent multi-specific antibody | |
| CN110872356B (en) | Bispecific antibodies and methods of use thereof | |
| CN113754770A (en) | Antibody specifically binding to human CTLA4, and medicine and kit containing antibody | |
| CN110016074B (en) | MAGE-A3 humanized T cell receptor |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |