Disclosure of Invention
In order to solve the technical problems, the invention provides an antibody for specifically detecting IL-21 and application thereof.
The invention uses HEK293 cell recombinant expressed human IL-21 protein as immunogen to carry out mouse immunity, and obtains hybridoma cell strain for expressing mouse antibody through cell fusion and maternal clone screening and cell subcloning. Experiments prove that the antibody can specifically identify human IL-21 protein, and different matrixes cannot interfere. Further, the invention obtains the amino acid sequence of the antibody and the nucleotide sequence of the encoding gene thereof through hybridoma sequencing.
Based on the findings, the invention provides the following technical scheme:
in a first aspect, the invention provides an antibody or antigen-binding fragment thereof that specifically detects IL-21, said antibody being selected from the following (1) and/or (2):
(1) The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 1. 2 and 3; the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO: 4. 5, 6.
SEQ ID NO：1: SYNMN；
SEQ ID NO：2: YIDPYNGGSGYNQKFKG；
SEQ ID NO：3: GDYDGGYYGMDY；
SEQ ID NO：4: RASQSVGTSIH；
SEQ ID NO：5: YASESIS；
SEQ ID NO：6: QQSNGWPFT。
(2) The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 7. 8 and 9; the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO: 10. 11, 12.
SEQ ID NO：7: DAWMN；
SEQ ID NO：8: EIRTKTSNHAAYYAASVKG；
SEQ ID NO：9: FDY；
SEQ ID NO：10: RASKNINKHLA；
SEQ ID NO：11: SGSTLQS；
SEQ ID NO：12: QQHNEYPYT。
Preferably, the antibody is selected from the following (1) and/or (2):
(1) Antibody 3G6D7
The amino acid sequence of the heavy chain variable region is:
MEWRWIFLFLLSGTTGVHSEIQLQQSGPELVKPGASVKVSCKASGYGFTSYNMNWVKQSHGKSLEWIGYIDPYNGGSGYNQKFKGKATLTVDKSSSTAYMHLISLTSDDSAVYYCARGDYDGGYYGMDYWGQGTSVTVSS（SEQ ID NO：13）；
the amino acid sequence of the light chain variable region is:
MVSTPQFLVFLLFWIPASRSDILLTQSPAILSVSPGESVSFSCRASQSVGTSIHWFQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLIINSVESEDIADYYCQQSNGWPFTFGSGTKLEIK（SEQ ID NO：14）。
(2) Antibodies 13A6G6
The amino acid sequence of the heavy chain variable region is:
MYLGLNYVFIVFLLNGVQSEVKLEESGGGLVQPGGSTKLSCAASGFTFSDAWMNWVRQSPEKGLEWIAEIRTKTSNHAAYYAASVKGRFTISRDDSKSSVYLQMNSLRPEDTGIYYCTRFDYWGQGTTLTVSS（SEQ ID NO：15）；
the amino acid sequence of the light chain variable region is:
MRFQVQVLGLLLLWISGAQCDVQITQSPSYLAASPGETITINCRASKNINKHLAWYHDKPGKTNKLLIYSGSTLQSGTPSRFSGSGSGTDFTLTISSLEPEDFAVYYCQQHNEYPYTFGGGTKLAIK（SEQ ID NO：16）。
preferably, the antibody or antigen binding fragment thereof described above is Fab, fab ', F (ab')2 Fd, fv, dAb, complementarity determining region fragments, single chain antibodies, monoclonal antibodies, animal-derived antibodies, chimeric antibodies, humanized antibodies, bispecific antibodies or multispecific antibodies.
In some embodiments of the invention, an antibody composition is provided, consisting of 3G6D7 and 13A6G6. The two antibodies can be used as paired antibodies of IL-21 and respectively used as coating antibodies and detection antibodies in double-antibody sandwich ELISA. Wherein the detection antibody may also carry a detectable label.
In some embodiments of the invention, an antibody composition is provided, consisting of 3G6D7 and 13A6G6. Wherein, 3G6D7 is used as a coating antibody in double-antibody sandwich ELISA, and 13A6G6 biotin is marked and then used as a detection antibody.
The antibody composition provided by the invention can be used for rapidly detecting the content of the human IL-21 by adopting a double-antibody sandwich ELISA method, and has the advantages of simplicity in operation, high sensitivity, high specificity, high accuracy and high precision.
In a second aspect, the invention provides a nucleic acid molecule encoding an antibody or antigen binding fragment thereof as described above.
Based on the amino acid sequences of the above antibodies or antigen binding fragments thereof, the skilled artisan can obtain nucleotide sequences of nucleic acid molecules encoding the above antibodies or antigen binding fragments thereof. Because of the degeneracy of the codons, the nucleotide sequences of the nucleic acid molecules encoding the antibodies or antigen binding fragments thereof are not unique, and all nucleic acid molecules capable of encoding the production of the antibodies or antigen binding fragments thereof are within the scope of the invention.
In a third aspect, the invention provides a biological material comprising a nucleic acid molecule as described above. Such biological materials include, but are not limited to, recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineering bacteria, or transgenic cell lines.
Preferably, the biological material is an expression cassette, vector or host cell.
The above-mentioned expression cassette can be obtained by ligating a transcription or translation regulatory element such as a promoter upstream of the nucleic acid molecule and/or ligating a transcription or translation regulatory element such as a terminator downstream thereof.
Such vectors include, but are not limited to, plasmid vectors, phage vectors, viral vectors, artificial chromosome vectors, and the like.
The host cells include microbial cells, insect cells, or other animal cells.
In a fourth aspect, the present invention provides an antibody conjugate obtained by coupling the antibody or antigen binding fragment thereof to a label selected from one or more of an enzyme label, a biotin label, a fluorescent dye label, a chemiluminescent dye label, and a radioactive label.
In a fifth aspect, the invention provides the use of any of the following (1) - (5) for said antibody or antigen binding fragment thereof or said nucleic acid molecule or said biological material or said antibody conjugate or said antibody composition:
(1) Use in the preparation of a product for detecting the presence or level of IL-21 in a sample;
(2) Use of IL-21 for detecting the presence or level of IL-21 in a sample for non-diagnostic and therapeutic purposes;
(3) Use in the manufacture of a product for detecting immune cell function or therapeutic effect of CAR-T cell therapy;
(4) Use in the preparation of a product for neutralising IL-21 activity in a sample;
(5) Use in the manufacture of a medicament for neutralising IL-21 activity in vivo.
Preferably, the application described in the above (1) or (2) includes: the antibodies or antigen-binding fragments thereof of the invention are prepared as products for detecting the presence or level of IL-21 in a sample, which are used to detect the presence or level of IL-21 in a sample. For ease of detection, the antibodies or antigen-binding fragments thereof of the invention also preferably comprise a detectable label.
The invention also provides methods of detecting the presence or level of IL-21 in a sample, comprising detecting the presence or level of IL-21 in a sample using an antibody or antigen-binding fragment thereof of the invention. The above-described methods may be for diagnostic purposes (e.g., the sample is a sample from a patient, including blood samples, etc.), or for non-diagnostic and therapeutic purposes (e.g., the sample is a culture cell supernatant, etc., not a sample from a patient).
As a method for detecting IL-21 by using the antibody or antigen-binding fragment thereof of the present invention, detection methods such as enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, immunochromatography and the like can be used.
In some embodiments of the invention, IL-21 is detected in a double antibody sandwich method using an antibody or antigen binding fragment thereof of the invention. The double antibody sandwich method is used for detecting IL-21 by using one antibody or antigen binding fragment thereof provided by the invention as a coated antibody, using another antibody or antigen binding fragment thereof provided by the invention as a detection antibody, or using one antibody or antigen binding fragment thereof provided by the invention as a coated antibody, using other known anti-IL-21 antibodies as detection antibodies, or using other known anti-IL-21 antibodies as coated antibodies, and using one antibody or antigen binding fragment thereof provided by the invention as a detection antibody.
In the above (3), the detection of the immune cell function or the therapeutic effect of the CAR-T cell therapy is specifically to determine the immune cell function or the therapeutic effect of the CAR-T cell therapy by detecting the level of IL-21 in the subject. Wherein the immune cells include T cells, NK cells, CAR-T cells, and the like.
In the above (4) and (5), the activity of neutralizing IL-21 is specifically binding to IL-21 in a sample by using the antibody or antigen-binding fragment thereof provided by the present invention, thereby neutralizing the IL-21 activity.
In the above (5), the medicament is used for preventing or treating a disease associated with IL-21 level or a disease mediated by IL-21, including but not limited to tumors, autoimmune diseases and inflammation, immunological pathology and tissue damage caused by parasites, and the like.
Preferably, the IL-21 of the invention is human IL-21.
In a sixth aspect, the invention provides an IL-21 detection kit comprising an antibody or antigen-binding fragment thereof as described above, or comprising an antibody conjugate as described above, or comprising an antibody composition as described above.
In some embodiments of the invention, an ELISA detection kit for IL-21 is provided, said kit comprising an antibody or antigen-binding fragment thereof as described above, or an antibody composition as described above. The kit may also contain other reagents for ELISA detection including, but not limited to, IL-21 standard, PBST wash, blocking solution, chromogenic solution, stop solution, and the like.
In a seventh aspect, the invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described above.
The above pharmaceutical compositions are useful for preventing or treating diseases associated with IL-21 levels or mediated by IL-21, including but not limited to tumors, autoimmune diseases and inflammation, parasite-induced immunological pathologies and tissue damage, and the like. The antibodies or antigen binding fragments thereof provided herein may be used as the sole active ingredient of the pharmaceutical compositions described above, or in combination with other pharmaceutically active ingredients. The pharmaceutical composition may further comprise pharmaceutically acceptable excipients.
In an eighth aspect, the invention provides a method of detecting IL-21 for non-diagnostic and therapeutic purposes, the method comprising: and detecting whether IL-21 and/or the existence level thereof exist in the sample to be detected by using a double-antibody sandwich ELISA method, wherein the coated antibody and the detection antibody in the double-antibody sandwich ELISA method are respectively 3G6D7 and 13A6G6.
Based on the technical scheme, the invention has at least the following advantages and beneficial effects:
the antibody 3G6D7 or the antibody 13A6G6 provided by the invention has special space epitope, and shows high affinity and high specificity to IL-21. The antibody 3G6D7 and the antibody 13A6G6 provided by the invention are jointly used for paired antibodies of IL-21, the two antibodies bind different space epitopes, the content of IL-21 in a culture medium and serum can be specifically detected by a double antibody sandwich method, the affinity is high, and other cytokines (such as IL-2, IL-4, IL-6, IL-7, IL-10, IL-15, GM-CSF, TNF-alpha and the like) are not bound, so that the paired antibodies are ideal antibody combinations for detecting the content of IL-21 protein, can be used for researching and developing or producing T cells, NK cells and IL-21 residues in a CAR-T cell process, and are beneficial to accelerating the research and development process of CGT therapy.
Detailed Description
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention. Unless otherwise indicated, all the experimental procedures used in the examples were conventional; the materials, reagents and the like used are all commercially available.
EXAMPLE 1 preparation of specific human IL-21 antibodies
1. Immunization of mice: human IL-21 protein (available from Acrobiosystems) was used as an immunogen to immunize mice with human IL-21 protein. After the immunization, the immune response level was determined by detecting serum of the immunized animal by ELISA. After the conventional immunization is completed, if the immunized animal can reach the immune response level against the immunogen, cell fusion is performed.
2. Screening: the supernatant of the fused cells was screened by ELISA to select cells positive for the specific binding of human IL-21 protein and devoid of binding to IL-2, IL-4, IL-6, IL-7, IL-10, IL-15, GM-CSF, TNF-alpha protein, etc.
3. Cloning and expanding culture: positive master clone cells were transferred to 24 well plates for expansion culture. Supernatants were collected from each of the expanded clones and tested by ELISA.
4. Subcloning: subcloning positive parent clone by limiting dilution method and subcloning screening by ELISA method.
5. Hybridoma cell antibody gene sequencing: total RNA of the hybridoma cells is extracted, and the RNA is reversely transcribed into cDNA through an RT-PCR reaction. Cloning antibody light chain and heavy chain sequences, constructing the antibody light chain and heavy chain sequences on a T vector, and then carrying out DNA sequencing analysis to obtain antibody gene sequences.
6. Antibody production and purification: and (3) transfecting the antibody gene sequence obtained in the step (5) into HEK293 cells, performing amplification culture, purifying the antibody by adopting a protein A/G affinity chromatography method, and storing the purified antibody in Phosphate Buffer (PBS) by adopting a dialysis method.
EXAMPLE 2 supernatant specific analysis of human IL-21 fused cells
In this example, 10 different cell supernatants were obtained after cell fusion according to example 1, and the human IL-21 antibody was specifically analyzed by ELISA as follows:
1. human IL-21, IL-2, IL-4, IL-6, IL-7, IL-10, IL-15, GM-CSF, TNF-alpha protein was diluted to 1. Mu.g/mL with PBS and added to wells of a microplate with 100. Mu.L per well. The plates were sealed with a plate and left to stand overnight at 4 ℃.
2. The wells were discarded, the ELISA plates were dried, washed with PBST wash solution, 300. Mu.L/Kong Jinpao, and the ELISA plates were dried and washed 3 times.
3. mu.L of blocking agent (PBST wash containing 5% BSA) was added to each well, membrane-sealed with a plate, incubated at 37℃and then washed.
4. The human IL-21 antibody cell supernatant described above was added to the ELISA plate at 100. Mu.L per well. And (5) sealing the plates by using sealing plates, placing the plates at 37 ℃ for incubation, and then cleaning.
5. HRP-Anti-Mouse IgG was diluted to 0.05. Mu.g/mL with sample dilution, 100. Mu.L was added to each well, membrane sealed with sealing plate, incubated at 37℃and washed.
6. The wells were then filled with 100. Mu.L of color development solution, and the wells were covered with a plate membrane, and incubated at 37℃in the absence of light.
7. 50 mu L of stop solution is added into each hole, and the ELISA plate is gently shaken until color development is uniform.
8. The absorbance values of 450 nm and 630nm were read with a microplate reader, and the OD630 value was subtracted with the OD450 to obtain absorbance values (OD values). The absorbance (OD) of each monoclonal antibody is shown in table 1.
TABLE 1 ELISA detection of different antibodies OD values
9. Clones that bound strongly to human IL-21 protein, and that did not bind to IL-2, IL-4, IL-6, IL-7, IL-10, IL-15, GM-CSF, TNF-alpha protein were selected for subcloning. The experimental results showed that of the 8 antibodies, clone numbers 3G6D7, 13A6G6 showed high specificity and no cross-reaction with other mutants.
EXAMPLE 3 analytical identification and functional analysis of antibodies specific for human IL-21
In this example, the human IL-7 specific antibodies screened in example 2 were analyzed for sensitivity by the double antibody sandwich Elisa method and the checkerboard method by methods known in the art, and finally two antibodies IL-21 specific antibodies (Clone: 3G6D7, 13A6G 6) with higher sensitivity were screened out for analytical identification and functional analysis, as follows:
SDS-PAGE identification result (figure 1) shows that the molecular weight of two bands of 3G6D7, 13A6G6 clone number antibody reduction electrophoresis is about 27kDa and 50kDa respectively, and the purity is more than 99%.
The quantitative detection experimental result (figure 2) shows that the antibody of clone numbers 3G6D7 and 13A6G6 can quantitatively detect the human IL-21 by adopting a double antibody sandwich method, so that the protein content in a matrix is obtained, the calculation formula is y= (5.24+0.0089)/[ 1+ (X/110.92) ] -0.97] -0.0089, wherein y is an OD value, the OD value of a sample is brought into the formula, and X is calculated, namely the concentration value of the sample.
The results of quantitative detection of human IL-21 (Table 2) show that quantitative detection of human IL-21 was performed using antibodies to clone numbers 3G6D7, 13A6G6, without interference from the matrix.
TABLE 2 double antibody sandwich assay for recovery of IL-21 in different dilution gradient matrices
The above examples merely represent a few embodiments of the present invention, which facilitate a specific and detailed understanding of the technical solutions of the present invention, but are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention.