CN111500668A - Method for determining biological activity of human I L-36/I L36R/I L1 RAcP pathway inhibitor - Google Patents

Method for determining biological activity of human I L-36/I L36R/I L1 RAcP pathway inhibitor Download PDF

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CN111500668A
CN111500668A CN202010599988.3A CN202010599988A CN111500668A CN 111500668 A CN111500668 A CN 111500668A CN 202010599988 A CN202010599988 A CN 202010599988A CN 111500668 A CN111500668 A CN 111500668A
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racp
monoclonal antibody
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CN111500668B (en
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熊新辉
王晋
张弢
王骊淳
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Shanghai Puming Biotechnology Co ltd
Nanjing Noah New Biotechnology Co ltd
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Nanjing Noah New Biotechnology Co ltd
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Abstract

The invention provides a method for determining the biological activity of a human I L-36/I L36R/I L01 RAcP pathway inhibitor, which comprises using cells stably expressing I L136R, I L1 RacP and NF- κ B reporter genes as effector cells, incubating the effector cells after mixing with I L-36 and human I L-36/I L36R/I L1 RAcP pathway inhibitors, determining the biological activity of the human I L-36/I L36R/I L1 RAcP pathway inhibitor according to the measured signal value of the reporter genes.

Description

Method for determining biological activity of human I L-36/I L36R/I L1 RAcP pathway inhibitor
Technical Field
The invention relates to the field of biological activity detection of biological medicines, in particular to a method for quickly, accurately and quantitatively determining the biological activity of an I L-36/I L36R/I L1 RAcP pathway inhibitor.
Background
The determination of biological activity of monoclonal antibodies at the cellular level plays an important role in the discovery and development of monoclonal antibody drugs, and cell-based biological activity determination methods are mostly adopted in the current biological activity detection methods, and comprise a cell proliferation inhibition method, a cytotoxicity method, a complement-dependent cytotoxicity method, an antibody-dependent cell-mediated cytotoxicity method, a cell E L ISA method and a reporter gene method.
Interleukin 36 cytokine (Interleukin-36, I-36) subfamily was discovered by a series of analyses of homologs encoding I-1 to the human genome sequence, and later discovered by in vitro cell experiments using various constructed animal models, four cytokines, I7-1F, I8-1F, I9-1F, I-1F, and I6-1F, were identified as I0-36R, I7-1F, I8-1F, I9-1F, and I-1F, respectively, and thus were sequentially renamed as I1-36 3, I2-36 5, I3-36 γ, and I4-36 Ra, collectively referred to as I5-36, and I7-1F is designated as I9-38, wherein I-36 Ra (I0-1F) was identified as an I2-1 member of the I1-36 family that is an antagonist of I1-36 R.I 3-36, I4-36 and I4-36 γ, and I4-36R, and I7-36R as an intracellular signal produced by the intracellular receptor (Rrpr) and was also induced by the intracellular signal produced by the intracellular receptor (Rrpr) as I1-36-6, I7-36-7-36-2 receptor.
I-36, I1-36 and I3-36 γ are mainly expressed in keratinocytes, bronchial epithelial cells, monocyte-macrophages and T lymphocytes, which are molecules with various biological activities, mainly play a role in physiological or pathological processes such as inflammatory processes, immune responses and the like in humans, more recent studies show that they are closely linked to the development and development of certain tumors in humans, I8-36R 5 and I9-36 γ mRNA overexpression in human keratinocytes stimulated by I4-1/TNF-0, increased I-36 mRNA and protein expression (Ichii et al, 1 ab. invertebrate, 90) (2010, 459. Eseik) are also observed in chronic pustular disease, increased I-36 mRNA and protein expression (Ichii et al, 2011 ab. invertebrate, 90) (3, 2010, 459.). myeloid cells (BMDC) and CD + T cells express strong I-12-psoriatic acid kinase-mediated inflammatory diseases such as psoriasis-12, psoriasis-induced by mouse epithelial factor-12, psoriasis-like, (I-12) and other psoriasis-12, psoriasis-like, such as psoriasis-13, psoriasis-12, psoriasis-4, psoriasis-12, psoriasis-like, (psoriasis-12), and other psoriasis-like psoriasis-4, psoriasis-12).
Thus, therapeutic blockade of the I L-36/I L36R/I L01 RAcP pathway could help overcome the hyperimmune response, and in order to develop more I L1-36/I L36R/I L1 RAcP pathway inhibitors and to perform quality control in the production of I L-36/I L36R/I L1 RAcP pathway inhibitors (e.g., monoclonal antibodies), it is necessary to develop methods for measuring the biological activity of I L-36/I L36R/I L1 RAcP pathway inhibitors.
The biological activity of the currently available I L-36/I L36R/I L1 RAcP pathway inhibitors (such as I L36R monoclonal antibody and I L1 RAcP monoclonal antibody) is determined by the following methods:
(1) human primary epidermal keratinocyte pnfkb/cytokine release: human primary epidermal keratinocytes were plated at 30000 cells/well in 96-well plates and incubated at 37 ℃ with 5% CO2Incubated overnight. One plate was used for analysis of pnfkb and the other plate was used for cytokine release. Then 5% CO at 37 ℃2The plates were incubated overnight, ligand (I L36 α, β or γ) and antibody were diluted at the desired concentration of 4 × in serum-starved (SS) medium antagonist (antibody) was added to the cells followed by ligand for pNF κ B at 37 ℃ with 5% CO2Next, NCI cells +/-ligand and antagonist were incubated for 1 hour then media was aspirated and cells were lysed in 100 μ L/well complete lysis buffer on ice for 30min then lysate was centrifuged at 2500RPM for 20min at 4 ℃ and transferred to MSD E L ISA plates and analyzed for pnnfkb according to manufacturer's protocol 18 hours to 24 hours post stimulation for cytokine release supernatant was transferred to MSD E L ISA plates and analyzed for cytokines according to manufacturer's protocol.
(2) NCI/ADR-RES cells pNF κ B/cytokine release assay: NCI/ADR-RES cells were plated at 45000 cells/well on RPMI medium containing 0.25% serum in 96-well plates. One plate was used for analysis of pnfkb and the other plate was used for cytokine release. The procedure was then the same as in "(1) human primary epidermal keratinocyte pNF κ B/cytokine release".
(3) Human primary dermal fibroblast pnfkb/cytokine release assay: human primary dermal fibroblasts were plated at 30000 cells/well in 96-well plates and incubated at 37 ℃ with 5% CO2Incubated overnight. One plate was used for analysis of pnfkb and the other plate was used for cytokine release. The procedure was then the same as in "(1) human primary epidermal keratinocyte pNF κ B/cytokine release".
(4) Human primary intestinal epithelial cell pNF kappa B/cytokine release assay: human primary intestinal epithelial cells were plated at 30000 cells/well in medium in 96-well plates and incubated overnight at 37 ℃ under 5% CO 2. One plate was used for analysis of pnfkb and the other plate was used for cytokine release. The procedure was then the same as in "(1) human primary epidermal keratinocyte pNF κ B/cytokine release".
(5) Human primary intestinal myofibroblast pnfkb/cytokine release assay: human primary intestinal myofibroblasts were plated at 30000 cells/well in medium in 96-well plates and incubated overnight at 37 ℃ under 5% CO 2. One plate was used for analysis of pnfkb and the other plate was used for cytokine release. The procedure was then the same as in "(1) human primary epidermal keratinocyte pNF κ B/cytokine release".
(6) Human primary proximal tubular cell pnfkb/cytokine release assay: human primary proximal tubular cells were plated at 30000 cells/well in culture medium in 96-well plates and incubated overnight at 37 ℃ under 5% CO 2. One plate was used for analysis of pnfkb and the other plate was used for cytokine release. The procedure was then the same as in "(1) human primary epidermal keratinocyte pNF κ B/cytokine release".
The six traditional methods for measuring the biological activity of the I L-36/I L36R/I L1 RAcP pathway inhibitor all require long-time cell culture and detection of the cell factor MSD E L ISA, have the disadvantages of complicated steps, large variability, high cost, long time, poor durability and repeatability of the method, and are not suitable for high-throughput screening experiments.
Thus, there remains a need in the art for novel assays for the determination of the biological activity of I L-36/I L36R/I L1 RAcP pathway inhibitors.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a novel biological activity determination method of an I L-36/I L36R/I L1 RAcP pathway inhibitor, in particular an I L36R or I L1 RAcP monoclonal antibody.
The technical scheme of the invention is as follows:
in one aspect, the invention provides a method for determining the biological activity of a human I L-36/I L36R/I L01 RAcP pathway inhibitor, said method comprising using cells expressing I L136R, I L21 RAcP and NF- κ B reporter genes as effector cells, incubating after mixing with I L3-36 and a human I L4-36/I L536R/I L61 RAcP pathway inhibitor, determining the biological activity of said human I L-36/I L36R/I L1 RAcP pathway inhibitor based on the measured signal values of said reporter genes, wherein said human I L-36/I L36R/I L1 RAcP pathway inhibitor is an I L36R monoclonal antibody and/or an I L1 RAcP monoclonal antibody.
According to the specific embodiment of the invention, the effector cell is the human peripheral blood leukemia T cell Jurkat/I L36R/I L1 RAcP/NF-kB-25 #. the cell is preserved in the China general microbiological culture Collection center (CGMCC for short, the address: West Lu No. 1 of North Chen of the Inward region of Beijing, institute of microbiology, China institute of sciences) at 25/5.2020, and the preservation number is CGMCC 19943.
Preferably, I L-36 adopted in the method of the invention is I L-36 β, further preferably, I L-36 is human I L-36 β.
Preferably, the I L36R monoclonal antibody is selected from one or more of ANB019 and sposolimab (BI-655130), and the I L1 RAcP monoclonal antibody is selected from one or more of BI-5041 and MAB-16-0030.
In particular, the method of the invention may comprise the steps of:
(1) diluting I L-36 β to 100pg/m L-20 μ g/m L by using a culture medium, diluting I L-36R monoclonal antibody and/or I L1 RAcP monoclonal antibody to 1pg/m L-1 mg/m L by using the culture medium as initial concentration, diluting the initial concentration at an equal ratio of 1:2-1:5, and mixing the obtained serial dilutions with dilutions of I L-36 β at a volume ratio of 1: 1;
(2) allowing the effector cells to grow in the culture medium at 4 × 105-40×105Diluting the cells/m L, adding the diluted cells/m L into the mixed solution obtained in the step (1) respectively according to the volume ratio of 1:1, mixing and incubating for 2-24 h;
(3) and (3) adding a chemiluminescence substrate of the reporter gene into the mixed solution obtained in the step (2), and fitting a four-parameter curve according to the obtained relative chemiluminescence unit value (R L U) to determine the biological activity of the antibody.
According to a particular embodiment of the invention, the persons I L-36 β used in the present invention are available from ACROBIOSES under the trade designation I L8-H5115.
Preferably, the NF- κ B reporter gene employed in the present invention is NF- κ B-luciferase.
Preferably, in the method of the present invention, Jurkat cells, i.e., human peripheral blood leukemia T cells Jurkat/I L36R/I L1 RAcP/NF- κ B-25#, are transfected with a vector comprising a NF- κ B-luciferase nucleic acid sequence, a vector comprising an I L36R nucleic acid sequence, and a vector comprising an I L1 RAcP nucleic acid sequence.
According to an embodiment of the present invention, the constructing includes, taking 2 × 106Jurkat cells, about 10 mug of the vector in total, were transfected with the Jurkat cells under conditions of 1400v voltage, 10ms time to shock, and 3 times number of shocks. Then stable cell strain screening is carried out in an antibiotic pressure screening mode, and monoclonal cell plating is carried out to obtain stable monoclonal cell strain.
Preferably, in step (1) of the method of the invention, I L-36 β is diluted to 50-150ng/m L, preferably 100ng/m L, using culture medium.
Preferably, in step (1) of the method of the present invention, the I L-36R monoclonal antibody and/or the I L1 RAcP monoclonal antibody is diluted to 40-50. mu.g/m L as a starting concentration using a culture medium, and then diluted at an equal ratio of 1: 4.
Preferably, in step (2) of the method of the invention, the effector cells are cultured in medium at 20 × 105The cells/m L were diluted and added to the mixture obtained in step (1) at a volume ratio of 1: 1.
Preferably, in step (2) of the method of the invention, the mixing is followed by incubation for 6 h.
According to a particular embodiment of the invention, in steps (1) and (2) of the process of the invention, the medium is 1640+10% FBS.
Preferably, in step (3) of the method of the present invention, the luciferase kit is used to detect chemiluminescence values, and a four-parameter curve is fitted to the relative chemiluminescence unit values obtained to determine the biological activity of the antibody, wherein the luciferase kit may be the Bio-L ite luciferase kit from Novonopraz, the Bio-Glo luciferase kit from Promega, the L uciferase luciferase reporter kit from Biovision, the luciferase reporter gene from Pegyun, and other kits based on luciferase luminescence detection, and the reporter gene detection is performed as described herein.
Preferably, the post-mixing incubation described in step (2) is performed in a 96-well plate, and the relative chemiluminescent unit values read in step (3) are then performed on a microplate reader.
According to a particular embodiment of the invention, the method of the invention comprises the following steps:
(1) i L-36 β is diluted to 100ng/m L by using a culture medium 1640+10% FBS, an I L-36R monoclonal antibody and/or an I L1 RAcP monoclonal antibody is diluted to 40-50 mu g/m L by using a culture medium 1460+10% FBS to serve as an initial concentration, the dilution is carried out in an equal proportion of 1:4, then the obtained serial dilutions are respectively mixed with a dilution of I L-36 β in a volume ratio of 1:1, and the mixture is paved into a 96-well plate at a rate of 50 mu L/well;
(2) the human peripheral blood leukemia T cell Jurkat/I L36R/I L1 RAcP/NF-kB-25 # provided by the invention is cultured in the culture medium at 20 × 105The cells/m L were diluted and added to the 96-well plate of step (1) at 50. mu. L/well and 5% CO at 37 ℃ in a medium2Performing medium incubation for 6 h;
(3) adding luciferase chemiluminescence substrate of 100 mu L/well into the 96-well plate in the step (2), reading relative chemiluminescence unit values on a microplate reader by using chemiluminescence, and fitting a four-parameter curve according to the measured relative chemiluminescence unit values to determine the biological activity of the antibody.
On the other hand, the constructed Jurkat/I L36R/I L1R cP/NF-kB-25 # of the Jurkat human leukemia T lymphocyte is preserved in the China general microbiological culture Collection center at 25/5 of 2020 with the preservation number of CGMCC 19943.
In a further aspect, the invention provides application of Jurkat human leukemia T lymphocyte Jurkat/I L36R/I L1 RAcP/NF-kB-25 # as an effector cell in detecting biological activity of a human I L-36/I L36R/I L1 RAcP pathway inhibitor, wherein the Jurkat human leukemia T lymphocyte Jurkat/I L36R/I L1 RAcP/NF-kB-25 # is preserved in China general microbiological culture Collection center at 25 months of 2020 in 5 years, and the preservation number is CGMCC number 19943.
Preferably, the I L36R monoclonal antibody is selected from one or more of ANB019 and sporolinmab, and the I L1 RAcP monoclonal antibody is selected from one or more of BI-5041 and MAB-16-0030.
In still another aspect, the invention also provides application of the method in quality control of production of a human I L-36/I L36R/I L1 RAcP pathway inhibitor, wherein the human I L-36/I L36R/I L1 RAcP pathway inhibitor is an I L-36R monoclonal antibody and/or an I L1 RAcP monoclonal antibody.
Preferably, the I L36R monoclonal antibody is selected from one or more of ANB019 and sporolinmab, and the I L1 RAcP monoclonal antibody is selected from one or more of BI-5041 and MAB-16-0030.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Further, some terms are explained as follows.
The term "effector cell" as used herein refers to a cell that naturally or artificially (e.g., by transfecting a vector comprising a nucleic acid sequence encoding a protein of interest into the cell) expresses an NF- κ B reporter the effector cells of the present invention function as effector cells in the I L-36/I L36R/I L1 RAcP pathway, or mimic effector cells in the I L-36/I L36R/I L1 RAcP pathway.
The term "Jurkat/I L36R/I L1 RAcP/NF-. kappa.B cells" as used herein refers to Jurkat cells that naturally or artificially (e.g., by transfecting a vector containing a nucleic acid sequence encoding a protein of interest into the cell) express I L36R protein, I L1 RAcP protein and NF-. kappa.B-luciferase.
As used herein, the term "I L-36/I L R/I L RACP pathway inhibitor" refers to an inhibitor capable of inhibiting signal transduction of the I L1-36/I L R/I L RACP pathway by binding to I L R or I L RACP, thereby blocking the interaction between I L-36 and I L736R, I L RACP, such that signal transduction of the I L-36/I L R/I L RACP pathway is inhibited I L-36/I L R/I L RACP pathway inhibitor used in the present invention includes, but is not limited to, I L R monoclonal antibody, I L RACP monoclonal antibody, small molecule inhibitor, and the like.
The term "I L-36" as used herein refers to Interleukin 36 (Interleukin 36).
The term "I L36R" as used herein refers to the I L-36 receptor (I L-36 receptor, I L-36R, also known as I L-1 Rrp2 or I L-1R L2).
The term "NF-. kappa.B" as used herein refers to the nuclear factor-. kappa.B (nuclear factor-kappa.B), which is a very critical transcription factor in the signal transduction pathway mediated by I L-36.
The term "NF-. kappa.B responsive element" as used herein refers to a base sequence to which NF-. kappa.B binds in the nucleus as a transcription factor.
The term "NF-. kappa.B reporter gene" as used herein refers to a gene of NF-. kappa.B response element operatively linked to a detectable moiety (e.g., GFP, eGFP, luciferase, FITC, quantum dots, etc.) such that its expression can be quantified.
The term "biological activity" as used herein refers to the ability of a biological product to achieve a defined biological effect based on its specific ability or potential to assess the corresponding activity of the biological product using the biological effect of a particular cell line (target cell).
The term "four-parameter curve" as used herein refers to a curve fitted according to the four-parameter equation Y = Bottom + (Top-Bottom)/(1+10^ ((L ogEC50-X) × HillSlope)), which gives four parameters Top, Bottom, EC50, HillSlope, etc.
To determine the biological activity of I L-36/I L R/I L01 RAcP pathway inhibitors, it was first necessary to establish a cell stably expressing I L136R, I L RacP and NF-. kappa.B reporter genes, and to ensure that the cell meets the required accessories for I L-36/I L R/I L RAcP pathway signaling, to obtain the effector cells in the method of the present invention, the inventors of the present application selected various host cells (including Jurkat cells, HEK293 cells, CHO cells, Hela cells, HUVEC cells, HMEC cells, HMC-1 cells, L AD cells, and KU812 cells employed in Z L72019105129384, etc.) conventionally used in the art for expressing exogenous genes as effector cells for the method of the present invention to perform tests as effector cells, and found that even if nucleic acids encoding I L R, I L1 RacP and NF-. kappa.B reporter genes are transfected into these host cells, respectively, the I6336R/I6858R/I3636 receptor pathway inhibitors can be successfully determined as I-36 RAcP pathway inhibitors, whereby the effector cells of the I5836 cells can be successfully assayed as host cells satisfying the I-36I-K293 reporter genes and the I-36 r19 r17 rK reporter genes, and the I-3 r5 r19 rIII receptor signaling pathways, the host cells.
In the case of Jurkat cells as host cells, by comparing differences of response signal-to-back ratios, diplopore CV and the like of the Jurkat cells of different strains successfully transfected with I L36R, I L1 RacP and NF-kB reporter genes to human I L0-36 β, an effector cell strain Jurkat/I L36R/I L1 RAcP/NF-kB-25 #, which is most suitable for determining the biological activity of the I L1-36/I L236R/I L31 RAcP pathway inhibitor, is obtained, and based on the differences, parameters such as appropriate effector cell concentration, I L-36 concentration, incubation time of the effector cells with the I L-36, I L-36/I L36R/I L1 RAcP pathway inhibitor and the like, which are suitable for determination are further determined.
Compared with the prior art, the method provided by the invention does not need any primary tissue source cells or other components of any person, can detect results at different times after adding detection liquid, has stable chromogenic result and more controllable quality, has short experimental period and simple and convenient operation, avoids cell pollution and errors caused by long-time incubation and multi-step operation, and ensures that the measured biological activity is related to clinical curative effect and conforms to the technical requirements related to NMPA.
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Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 shows the transfection efficiency of Jurkat cells under different electroporation conditions, wherein 1A to 1C are the results of electroporation conditions 1 to 3, respectively.
FIG. 2 is the response curves of different Jurkat/hI L36R/I L1 RAcP/NF-kB clones under stimulation of I L36 β, and 2A to 2C in the figure are the results of Jurkat/I L36R/I L1 RAcP/NF-kB-25 #, Jurkat/I L36R/I L1 RAcP/NF-kB-33 #, Jurkat/I L36R/I L1 RAcP/NF-kB-55 #, respectively.
FIG. 3 is a result of the I L36R antibody inhibiting the activation activity of Jurkat/hI L36R/I L1 Racp/NF-. kappa.B-25 # effector cells at different cell densities.
FIG. 4 is the result of the inhibition of the activation activity of the antibody I L36R on Jurkat/hI L36R/I L1 Racp/NF-. kappa.B-25 # effector cells at different incubation times.
FIG. 5 is a result of the I L36R antibody inhibiting the activating activity of I L36 β on Jurkat/hI L36R/I L1 Racp/NF- κ B-25# cells.
FIG. 6 is a result of the inhibition of the activation activity of Jurkat/hI L36R/I L1 Racp/NF- κ B-25# effector cells by the I L1 RAcP antibody at various cell densities.
FIG. 7 is the result of the I L1 RAcP antibody inhibiting the activation activity of Jurkat/hI L36R/I L1 Racp/NF- κ B-25# effector cells at different incubation times.
FIG. 8 is a result of the inhibition of the activating activity of I L36 β against Jurkat/I L36R/I L1 RAcP/NF- κ B-25# cells by the I L1 RAcP antibody.
FIG. 9 shows the results of comparison of the differences in biological activities of the I L36R monoclonal antibody measured by different Jurkat/hI L36R/I L1 RAcP/NF-. kappa.B clones in the method established in the present invention, wherein 9A to 9C are the results of Jurkat/I L36R/I L1 RAcP/NF-. kappa.B-25 #, Jurkat/I L36R/I L1 RAcP/NF-. kappa.B-33 #, Jurkat/I L36R/I L1 RAcP/NF-. kappa.B-55 #, respectively.
FIG. 10 shows the results of comparison of the differences in biological activities of the I L1 RAcP monoclonal antibody measured by different Jurkat/hI L36R/I L1R/NF-kB clones in the method established by the present invention, wherein 10A to 10C are the results of Jurkat/I L36R/I L1 RAcP/NF-kB-25 #, Jurkat/I L36R/I L1 RAcP/NF-kB-33 #, Jurkat/I L36R/I L1 RAcP/NF-kB-55 #, respectively.
FIG. 11 is a result of the inhibitory activity of antibodies against different antigens against I L36 β on Jurkat/I L36R/I L1 RAcP/NF- κ B-25# cells.
FIG. 12 is a result of the I L36R antibody inhibiting the activation activity of I L36 β on different kinds of effector cells containing I L36R, I L1 RAcP and NF- κ B genes.
Detailed Description
The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way.
The experimental procedures in the following examples are conventional unless otherwise specified. The raw materials and reagent materials used in the following examples are all commercially available products unless otherwise specified.
Preservation of biological materials:
jurkat human leukemia T lymphocyte Jurkat/I L36R/I L1 RAcP/NF-kappa B-25#, constructed according to the procedure described in example 2, was deposited in China general microbiological culture Collection center (CGMCC for short, address: Beijing City, Chaoyang district, North Cheng Xilu No. 1, institute of microbiology, China academy of sciences), 25 days in 2020, 5 months and 25 days, and the preservation number is CGMCC No. 19943.
preparation of pcDNA3.1(+)/I L36R plasmid:
the pcDNA3.1(+) plasmid (Thermo, cat. No. V79020) was digested with EcoRI and HindIII, and the I L R gene (NCBI accession No. NM-003854.2) was enzymatically ligated to the pcDNA3.1(+) plasmid to form the pcDNA3.1(+)/I L R plasmid.
preparation of pcDNA3.1(+)/I L1 RAcP plasmid:
the pcDNA3.1(+) plasmid (Thermo, cat. No. V79020) was digested with EcoRI and HindIII, and the I L1 RAcP gene (NCBI accession No. NM-002182.2) was enzymatically constructed into the pcDNA3.1(+) plasmid to form the pcDNA3.1(+)/I L36R plasmid.
Amino acid sequence of spesolimab (BI-655130):
heavy chain: 1, SEQ ID NO; light chain: 2, SEQ ID NO.
MAB-16-0030 amino acid sequence:
heavy chain: 3, SEQ ID NO; light chain: SEQ ID NO 4.
Example 1 electroporation transfection conditions for Jurkat cells
Jurkat cells are suspended human peripheral blood leukemia T cells, transfection difficulty is high, and chemical transfection method is difficult to transfect plasmids into cells, so selection is realized by electric shock transfection method.
(1) Electroporation transfection Condition 1 taking 2 × 106Jurkat cells (cell bank of Chinese academy of sciences), 10. mu.g pDSred plasmid (Clontech, 632406) (red fluorescent plasmid for identifying transfection efficiency), shock voltage 1100v, shock time 50ms, shock frequency 1;
(2) electroporation transfection Condition 2 taking 2 × 106Jurkat cells, 10 mu g pDSRed plasmid, 1300v of electric shock voltage, 20ms of electric shock time and 2 times of electric shock times;
(3) electroporation transfection Condition 3 taking 2 × 106Jurkat cells, 10. mu.g pDSRed plasmid, shock voltage 1400v, shock time 10ms, 3 times shock times.
A comparison of red fluorescent protein expression for the three transfection conditions is shown in FIG. 1. As shown in FIG. 1, the electroporation condition 3 can achieve transfection efficiency of about 50% and higher activity, and is more suitable for plasmid transfection than other conditions.
EXAMPLE 2 preparation of effector cells expressing Jurkat/I L36R/I L1 RAcP/NF-. kappa.B
3.33. mu.g of pG L4.32.32 [ luc 2P/NF-. kappa.B-RE/Hygro ] (Promega, Inc. (Beijing) Biotechnology Co., Ltd.), 3.33. mu.g of pcDNA3.1(+)/I L36R plasmid and 3.33. mu.g of pcDNA3.1(+)/I L1 RAcP were transfected into Jurkat cells (cell Bank, national academy of sciences) using the electroporation transfection condition 3 selected in example 1, and Jurkat/I L36R/I L1 RAcP/NF-. kappa.B effector cells were obtained by culturing in a medium containing 0.1mg/m L hygromycin B, 0.1mg/m L G418 and 0.05mg/m L Zeocin.
Recombinant human I L-36 β (ACROBIOSES, Cat. I β 08-H5115) was diluted to 200ng/m β 1, 66.66ng/m β 2, 22.22ng/m β 3, 7.40ng/m L, 2.46ng/m L, 0.82ng/m L, 0.26ng/m L, etc. using 1640+10% FBS medium, added to a 96-well plate (Corning, cat 3610) at 50. mu. L/well, and then different Jurkat/I L36R/I L1 RAcP/NF-kappa B effector clones were added to the above 96-well plate at 37 ℃ and L/100000/well at 50. mu. L/well,5% CO2After 6 h of incubation, luciferase (luciferase) expression was measured at 100. mu. L/well in Bio-L ite chromogenic reagent (Novozam, cat. DD 1201-02).
Through screening, the signal-to-back ratios of three clones responding to human I L-36 β can reach more than 10 times, and are respectively Jurkat/I β 036R/I β RAcP/NF-kB-25 #, Jurkat/I β 236R/I β RAcP/NF-kB-33 #, Jurkat/I β R/I L RAcP/NF-kB-55 #, the results of the clone measurement curves of the three different Jurkat/I L R/I L1 RAcP/NF-kB effector cells are shown in figure 2, the results of the parameter analysis of the activity curves of the three different clones are shown in table 1, the results of the parameter analysis of the three different clones are shown in table 1, the results of the three clones in table 1 and figure 2 are shown, the highest signal-to-back ratio of the three clones to I L-36R/I L RAcP/NF-kB effector cells is shown in table 1 and figure 2, and the highest signal-to-back ratio of the three strains of Jurkat/I3836R/I4636R-rK B-55-25 and NO-80 is shown in figure 2.
The differences between the three clones are compared on the basis of establishing a method for measuring the biological activity of the I L-36/I L36R/I L1 RAcP pathway inhibitor, and an effector cell strain which is most suitable for measuring the biological activity of the I L-36/I L36R/I L1 RAcP pathway inhibitor is preferably selected.
TABLE 1 comparison of different Jurkat/I L36R/I L1 RAcP/NF-. kappa.B effector cell cloning curve parameters
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。
Example 3 optimization and establishment of the method for determining the biological Activity of the monoclonal antibody I L36R
Jurkat/I L36R/I L1 RAcP/NF-kappa B-25# selected in example 2 was selected as an effector cell strain for optimization and establishment of the biological activity assay method for the I L36R monoclonal antibody.
(1) Optimizing the cell density:
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i L36 β preparation, I L36 β of 0.2mg/m L is diluted to 100ng/m L by using 1640+10% FBS culture medium;
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the preparation method comprises the steps of diluting the I L-36R monoclonal antibody BI-655130 to 50000ng/m L, 12500ng/m L1, 3125ng/m L2, 781.25ng/m L3, 195.31ng/m L, 48.82ng/m L, 12.20ng/m L, 3.02ng/m L and the like by using 1640+10% FBS culture medium, mixing the I L36L 0 and different concentrations of BI-655130 according to the ratio of 100 mu L to 100 mu L in a ratio of 1:1, uniformly mixing, and adding the mixture into a 96-well plate according to the ratio of 50 mu L/well;
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effector cell suspension preparation by diluting Jurkat/I L36R/I L1 RAcP/NF-kappa B-25# Effector cells to 4 × 105Pieces/m L, 1 × 106Pieces/m L, 2 × 106Uniformly spreading the powder/m L and 50 μ L/well into the above 96-well plate, i.e. 20000/well, 50000/well, 100000/well, at 37 deg.C and 5% CO2Incubating in an incubator for 6 hours;
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the detection method comprises the steps of detecting chemiluminescence values by using a luciferase kit, adding Bio-L ite developer (Novozam, cat, DD 1201-02) according to 100 mu L/hole, measuring the expression of luciferase (luciferase), reading relative chemiluminescence unit values by using chemiluminescence on an enzyme labeling instrument, fitting a four-parameter curve according to the measured relative chemiluminescence unit values to determine the biological activity of the antibody, and optimizing the method to finally optimize the cell density to be 2 × 106Specific parameters/m L can be seen in Table 2 and FIG. 3.
TABLE 2 comparison of the signal-to-back ratios at different cell densities
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。
(2) And (3) optimizing the incubation time:
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i L36 β preparation, I L36 β of 0.2mg/m L is diluted to 100ng/m L by using 1640+10% FBS culture medium;
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the preparation method comprises the steps of diluting the I L-36R monoclonal antibody BI-655130 to 50000ng/m L, 12500ng/m L1, 3125ng/m L2, 781.25ng/m L3, 195.31ng/m L, 48.82ng/m L, 12.20ng/m L, 3.02ng/m L and the like by using 1640+10% FBS culture medium, mixing the I L36L 0 and different concentrations of BI-655130 according to the ratio of 100 mu L to 100 mu L in a ratio of 1:1, uniformly mixing, and adding the mixture into a 96-well plate according to the ratio of 50 mu L/well;
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effector cell suspension preparation by diluting Jurkat/I L36R/I L1 RAcP/NF-kappa B-25# Effector cells to 2 × 106Pieces/m L, and uniformly spread into the above 96-well plate according to 50 μ L/well, at 37 deg.C and 5% CO2Incubating in an incubator for 6, 16 and 24 hours;
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the detection method comprises the steps of detecting chemiluminescence values by using a luciferase kit, adding a Bio-L ite color developing agent (Novozam, cat, DD 1201-02) according to 100 mu L per hole, measuring the expression of luciferase (luciferase), reading relative chemiluminescence unit values by using chemiluminescence on an enzyme labeling instrument, fitting a four-parameter curve according to the measured relative chemiluminescence unit values to determine the biological activity of the antibody, optimizing the method, and finally preferably setting the incubation time to be 6 hours, wherein specific parameters can be seen in a table 3 and a figure 4.
TABLE 3 comparison of the signal-to-back ratios at different incubation times
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。
Through the optimization of the cell density and the incubation time, the biological activity of the finally established I L36R monoclonal antibody is determined as follows:
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IL36 β formulation I L36 β at 0.2mg/m L was diluted to 100ng/m L using 1640+10% FBS medium;
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the preparation method comprises the steps of diluting the I L-36R monoclonal antibody BI-655130 to 50000ng/m L, 12500ng/m L1, 3125ng/m L2, 781.25ng/m L3, 195.31ng/m L, 48.82ng/m L, 12.20ng/m L, 3.02ng/m L and the like by using 1640+10% FBS culture medium, mixing the I L36L 0 and different concentrations of BI-655130 according to the ratio of 100 mu L to 100 mu L in a ratio of 1:1, uniformly mixing, and adding the mixture into a 96-well plate according to the ratio of 50 mu L/well;
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effector cell suspension preparation by diluting Jurkat/I L36R/I L1 RAcP/NF-kappa B-25# Effector cells to 2 × 106Pieces/m L, and uniformly spread into the above 96-well plate according to 50 μ L/well, at 37 deg.C and 5% CO2Incubating in an incubator for 6 hours;
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the detection method comprises the steps of detecting chemiluminescence values by using a luciferase kit, adding a Bio-L ite chromogenic reagent (Novozam, cat, DD 1201-02) according to 100 mu L/hole, measuring the expression of luciferase (luciferase), reading relative chemiluminescence unit values by using chemiluminescence on an enzyme labeling instrument, and fitting a four-parameter curve according to the measured relative chemiluminescence unit values to determine the biological activity of the antibody, wherein the biological activity of the I L36R monoclonal antibody determined by the method can be shown in figure 5, and the negative control in the figure is that only I L36 β with the same concentration is added, no antibody is added, and the positive control is that only PBS is added.
Example 4 optimization and establishment of the method for determining the biological Activity of the I L1 RAcP monoclonal antibody
Jurkat/I L36R/I L1 RAcP/NF- κ B-25# selected in example 2 was selected as the effector cell strain used in the method for optimizing and establishing the biological activity assay for the I L1 RAcP monoclonal antibody.
(1) Optimizing the cell density:
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i L36 β preparation, I L36 β of 0.2mg/m L is diluted to 100ng/m L by using 1640+10% FBS culture medium;
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the preparation of the medicine comprises the steps of diluting I L1 RAcP monoclonal antibody MAB-16-0030 to 40000ng/m L, 10000ng/m L1, 2500ng/m L2, 625ng/m L3, 156.25ng/m L, 39.06ng/m L, 9.76ng/m L and 2.44ng/m L by using 1640+10% FBS culture medium, mixing the I L36L 0 and MAB-16-0030 with different concentrations according to 100 mu L and 100 mu L at a ratio of 1:1, uniformly mixing, and adding the mixture into a 96-well plate according to 50 mu L/well;
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effector cell suspension preparation by diluting Jurkat/I L36R/I L1 RAcP/NF-kappa B-25# Effector cells to 4 × 105Pieces/m L, 1 × 106Pieces/m L, 2 × 106Uniformly spreading the powder/m L and 50 μ L/well into the above 96-well plate, i.e. 20000/well, 50000/well, 100000/well, at 37 deg.C and 5% CO2Incubating in an incubator for 6 hours;
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the detection method comprises the steps of detecting chemiluminescence values by using a luciferase kit, adding Bio-L ite developer (Novozam, cat, DD 1201-02) according to 100 mu L/hole, measuring the expression of luciferase (luciferase), reading relative chemiluminescence unit values by using chemiluminescence on an enzyme labeling instrument, fitting a four-parameter curve according to the measured relative chemiluminescence unit values to determine the biological activity of the antibody, and optimizing the method to finally optimize the cell density to be 2 × 106Specific parameters/m L can be seen in Table 4 and FIG. 6.
TABLE 4 comparison of the signal-to-back ratios at different cell densities
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。
(2) And (3) optimizing the incubation time:
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i L36 β preparation, I L36 β of 0.2mg/m L is diluted to 100ng/m L by using 1640+10% FBS culture medium;
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the preparation of the medicine comprises the steps of diluting I L1 RAcP monoclonal antibody MAB-16-0030 to 40000ng/m L, 10000ng/m L1, 2500ng/m L2, 625ng/m L3, 156.25ng/m L, 39.06ng/m L, 9.76ng/m L and 2.44ng/m L by using 1640+10% FBS culture medium, mixing the I L36L 0 and MAB-16-0030 with different concentrations according to 100 mu L and 100 mu L at a ratio of 1:1, uniformly mixing, and adding the mixture into a 96-well plate according to 50 mu L/well;
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effector cell suspension preparation by diluting Jurkat/I L36R/I L1 RAcP/NF-kappa B-25# Effector cells to 2 × 106Pieces/m L, and uniformly spread into the above 96-well plate according to 50 μ L/well, at 37 deg.C and 5% CO2Incubating in an incubator for 6, 16 and 24 hours;
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the detection method comprises the steps of detecting chemiluminescence values by using a luciferase kit, adding a Bio-L ite color developing agent (Novozam, cat, DD 1201-02) according to 100 mu L per hole, measuring the expression of luciferase (luciferase), reading relative chemiluminescence unit values by using chemiluminescence on an enzyme labeling instrument, fitting a four-parameter curve according to the measured relative chemiluminescence unit values to determine the biological activity of the antibody, optimizing the method, and finally preferably setting the incubation time to be 6 hours, wherein specific parameters can be seen in a table 5 and a figure 7.
TABLE 5 comparison of the signal-to-back ratios at different incubation times
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。
The biological activity of the finally established I L1 RAcP monoclonal antibody was determined by the above cell density and incubation time optimization method as follows:
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i L36 β preparation, I L36 β of 0.2mg/m L is diluted to 100ng/m L by using 1640+10% FBS culture medium;
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the preparation of the medicine comprises the steps of diluting I L1 RAcP monoclonal antibody MAB-16-0030 to 40000ng/m L, 10000ng/m L1, 2500ng/m L2, 625ng/m L3, 156.25ng/m L, 39.06ng/m L, 9.76ng/m L and 2.44ng/m L by using 1640+10% FBS culture medium, mixing the I L36L 0 and MAB-16-0030 with different concentrations according to 100 mu L and 100 mu L at a ratio of 1:1, uniformly mixing, and adding the mixture into a 96-well plate according to 50 mu L/well;
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effector cell suspension preparation by diluting Jurkat/I L36R/I L1 RAcP/NF-kappa B-25# Effector cells to 2 × 106Pieces/m L, and uniformly spread into the above 96-well plate according to 50 μ L/well, at 37 deg.C and 5% CO2Incubating in an incubator for 6 hours;
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the detection method comprises the steps of detecting chemiluminescence values by using a luciferase kit, adding Bio-L ite developer (Novozam, cat, DD 1201-02) into a 100 mu L/hole, measuring the expression of luciferase (luciferase), reading relative chemiluminescence unit values by using chemiluminescence on an enzyme labeling instrument, and fitting a four-parameter curve according to the measured relative chemiluminescence unit values to determine the biological activity of the antibody, wherein the biological activity of the I L1 RAcP monoclonal antibody determined by the method can be shown in figure 8, and the negative control in the figure is that only I L36 β with the same concentration is added, no antibody is added, and the positive control is that only PBS is added.
Example 5 comparison of the biological Activity of the I L36R monoclonal antibody with different Jurkat/I L36R/I L1 RAcP/NF- κ B Effector cells
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I L36 β preparation, I L36 β of 0.2mg/m L is diluted to 100ng/m L by using 1640+10% FBS culture medium;
Figure 884108DEST_PATH_IMAGE003
the preparation method comprises the steps of diluting the I L-36R monoclonal antibody BI-655130 to 50000ng/m L, 12500ng/m L1, 3125ng/m L2, 781.25ng/m L3, 195.31ng/m L, 48.82ng/m L, 12.20ng/m L, 3.02ng/m L and the like by using 1640+10% FBS culture medium, mixing the I L36L 0 and different concentrations of BI-655130 according to the ratio of 100 mu L to 100 mu L in a ratio of 1:1, uniformly mixing, and adding the mixture into a 96-well plate according to the ratio of 50 mu L/well;
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preparing effector cell suspension by diluting three different effector cells such as Jurkat/I L36R/I L1 RAcP/NF-kB-25 #, Jurkat/I L36R/I L1 RAcP/NF-kB-33 #, Jurkat/I L36R/I L1 RAcP/NF-kB-55 # to 2 × 106Pieces/m L, and uniformly spread into the above 96-well plate according to 50 μ L/well, at 37 deg.C and 5% CO2Incubating in an incubator for 6 hours;
Figure 938838DEST_PATH_IMAGE005
the detection method comprises the steps of detecting chemiluminescence values by using a luciferase kit, adding Bio-L ite developer (Novozam, cat, DD 1201-02) according to 100 mu L per well, measuring expression of luciferase (luciferase), reading relative chemiluminescence unit values by using chemiluminescence on an enzyme labeling instrument, determining biological activity of the antibody by fitting a four-parameter curve according to the measured relative chemiluminescence unit values, and comparing the difference of the measured activity among different effector cells, wherein the Jurkat/I L36R/I L1 RAcP/NF-kappa B-25# clone is the optimal clone on an experimental window and a multi-well CV for measuring the biological activity of the I L36R monoclonal antibody, as can be seen in Table 6 and FIG. 9.
TABLE 6 comparison of parameters for determination of biological Activity of the I L36R monoclonal antibody for different Jurkat/I L36R/I L1 RAcP/NF-. kappa.B effector cell clones
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。
Example 6 comparison of the biological Activity of different Jurkat/I L36R/I L1 RAcP/NF-. kappa.B Effector cells for the determination of the I L1 RAcP monoclonal antibody
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I L36 β preparation, I L36 β of 0.2mg/m L is diluted to 100ng/m L by using 1640+10% FBS culture medium;
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the preparation of the medicine comprises the steps of diluting I L1 RAcP monoclonal antibody MAB-16-0030 to 40000ng/m L, 10000ng/m L1, 2500ng/m L2, 625ng/m L3, 156.25ng/m L, 39.06ng/m L, 9.76ng/m L and 2.44ng/m L by using 1640+10% FBS culture medium, mixing the I L36L 0 and MAB-16-0030 with different concentrations according to 100 mu L and 100 mu L at a ratio of 1:1, uniformly mixing, and adding the mixture into a 96-well plate according to 50 mu L/well;
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preparing effector cell suspension by diluting three different effector cells such as Jurkat/I L36R/I L1 RAcP/NF-kB-25 #, Jurkat/I L36R/I L1 RAcP/NF-kB-33 #, Jurkat/I L36R/I L1 RAcP/NF-kB-55 # to 2 × 106Pieces/m L, and uniformly spread into the above 96-well plate according to 50 μ L/well, at 37 deg.C and 5% CO2Incubating in an incubator for 6 hours;
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the detection method comprises the steps of detecting chemiluminescence values by using a luciferase kit, adding a Bio-L ite developer (Novozam, cat, DD 1201-02) according to 100 mu L/hole, and measuring the expression of luciferase (luciferase), reading the relative reaction by using chemiluminescence on an enzyme labeling instrumentThe biological activities of the antibodies were determined by fitting a four-parameter curve to the relative measured chemiluminescence unit values, and the differences in measured activities between different effector cells were compared, as shown in Table 7 and FIG. 10. from Table 7 and FIG. 10, the Jurkat/I L36R/I L1 RAcP/NF-. kappa.B-25 # clone was the best clone over the experimental window and the replicate well CV for the biological activity of the I L1 RAcP monoclonal antibody.
TABLE 7 comparison of biological Activity parameters of different Jurkat/I L36R/I L1 RAcP/NF-. kappa.B Effector cell clones determined for I L1 RAcP monoclonal antibody
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。
Example 7 specificity assay for Activity assay Using Jurkat/I L36R/I L1 RAcP/NF-. kappa.B-25 # Effector cells
① I L36 β preparation, I L36 β of 0.2mg/m L is diluted to 100ng/m L by using 1640+10% FBS culture medium;
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the preparation method comprises the following steps of using 1640+10% FBS culture medium to respectively dilute three medicaments such as I L-36R monoclonal antibody BI-655130, CNTO7160 (ST 2 monoclonal antibody) and Nivolumab (PD-1 monoclonal antibody) to concentrations such as 50000ng/m L, 12500ng/m L1, 3125ng/m L2, 781.25ng/m L3, 195.31ng/m L, 48.82ng/m L, 12.20ng/m L and 3.02ng/m L, mixing the I L36L 0 and antibodies with different concentrations according to 100 mu L and 100 mu L in a ratio of 1:1, and adding the mixture into a 96-well plate according to 50 mu L/well;
③ Effector cell suspension preparation, Jurkat/I L36R/I L1 RAcP/NF-kappa B-25# Effector cells are all diluted to 2 × 106L per m, uniformly spreading the mixture into the 96-well plate according to the ratio of 50 mu L per well, and incubating the mixture in a 5% CO2 incubator at 37 ℃ for 6 hours;
④ detection method comprises using luciferase kit to detect chemiluminescence value, adding Bio-L ite developer (Nonunza, cat, DD 1201-02) according to 100 mu L/hole to measure expression of luciferase (luciferase), reading relative chemiluminescence unit value on an enzyme labeling instrument by using chemiluminescence, determining biological activity of the antibody by fitting a four-parameter curve according to the measured relative chemiluminescence unit value, and comparing activity difference measured among different drugs, as shown in figure 11. Jurkat/I L36R/I L1 RAcP/NF-kB-25 # effector cell has specificity to I L36/I6336R/I L36R/I L1 RAcP pathway inhibitor.
Example 8 construction of Effector cells comprising genes I L36R, I L1 RAcP, NF-. kappa.B and the like Using different cell types and BI-655130 Activity assay
3.33. mu.g of pG L4.32.32 [ luc 2P/NF-. kappa.B-RE/Hygro ] were transfected with electric shock]Plasmid, 3.33. mu.g of pcDNA3.1(+)/I L36R plasmid and 3.33. mu.g of pcDNA3.1(+)/I L1 RAcP were transfected into 2 × 106The specific shock transfection conditions of the cells such as KU812, HEK293, CHO, Hela, HUVEC and the like are that KU812 is 1000V, 30ms and 1 time, HEK293 is 1245V, 10ms and 3 times, CHO is 1260V, 10ms and 3 times, Hela is 1005V, 35ms and 2 times, HUVEC is 1200V, 40ms and 1 time, and then KU812/I L R/I L RAcP/NF-kappa B, HEK293/I L R/I L1 RAcP/NF-kappa B, CHO/I L R/I L RAcP/NF-kappa 4636/I L R/I L RAcP/I39B, Hela/I36R/I L RAcP/B, HUVEC/I3934/38 RAcP 3985 effect is obtained by culturing in a culture medium containing 0.1mg/m L hygromycin B, 0.0.0.1 mg/m L/I418 and 0.0 Zeocin.
① I L36 β preparation, I L36 β of 0.2mg/m L is diluted to 100ng/m L by using 1640+10% FBS culture medium;
l4 pharmaceutical preparation, I L-36R monoclonal antibody BI-655130 is diluted to 50000ng/m L, 12500ng/m L1, 3125ng/m L2, 781.25ng/m L3, 195.31ng/m L, 48.82ng/m L, 12.20ng/m L and 3.02ng/m L by using 1640+10% FBS culture medium, the I L36L 0 and different concentrations BI-655130 are mixed and mixed uniformly according to a ratio of 1:1 of 100 mu L and 100 mu L, and added into a 96-well plate according to a ratio of 50 mu L/well;
l1 Effector cell suspension is prepared by diluting KU812/I L36R/I L1 RAcP/NF-kappa B, HEK293/I L36R/I L1 RAcP/NF-kappa B, CHO/I L36R/I L1 RAcP/NF-kappa B, Hela/I L36R/I L1 RAcP/NF-kappa B, HUVEC/I L36R/I L1 RAcP/NF-kappa B equivalent cells to 2L 0106L per m, uniformly spreading the mixture into the 96-well plate according to the ratio of 50 mu L per well, and incubating the mixture in a 5% CO2 incubator at 37 ℃ for 6 hours;
l5 detection method, using luciferase kit to detect chemiluminescence value, adding Bio-L ite developer (Nonunoprazan, cat 1201-02) according to 100 mu L/well to determine luciferase (luciferase) expression, using chemiluminescence to read relative chemiluminescence unit value on an enzyme labeling instrument, and fitting a four-parameter curve to the relative chemiluminescence unit value to determine antibody biological activity, the determination result is shown in FIG. 12. from FIG. 12, it is known that KU812/I L036R/I L11 RAcP/NF- κ B, HEK293/I L236R/I L31 RAcP/NF- κ B, CHO/I L436R/I L1 RAcP/NF- κ B, Hela/I L36R/I L1 RAcP/NF- κ B, HUVEC/I L36R/I L1 RAcP/NF- κ B equivalent cell response to I L36/I L36R/I L R/I L1R 2I inhibitor.
The above description of the specific embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various changes and modifications according to the present invention without departing from the spirit of the present invention, which is defined by the scope of the appended claims.
SEQUENCE LISTING
<110> Nanjing Norway New Biotechnology Ltd
Shanghai Puming Biotech Co., Ltd
<120> a method for determining the biological activity of a human I L-36/I L36R/I L1 RAcP pathway inhibitor
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<170>PatentIn version 3.3
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Claims (17)

1. A method for determining the biological activity of a human I L-36/I L36R/I L01 RAcP pathway inhibitor comprises the steps of using cells which stably express I L136R, I L21 RacP and NF-kB reporter genes as effector cells, mixing the effector cells with I L3-36 and a human I L4-36/I L536R/I L61 RAcP pathway inhibitor, incubating, and determining the biological activity of the human I L7-36/I L836R/I L1 cP RAcP pathway inhibitor according to the measured signal value of the reporter genes, wherein the human I L-36/I L36R/I L1 RAcP pathway inhibitor is I L-36R monoclonal antibody and/or I L1R monoclonal antibody, the effector cells are human peripheral blood leukemia Jurkat/I L36R/I L1R/I L RAcP B-25, the biological preservation number of CGMCC No. 19943, the biological preservation number of China general microbiological culture collection No. 2020, and the preservation number of CGMCC 19943.
2. The method of claim 1, wherein I L-36 is I L-36 β.
3. The method of claim 1 or 2, wherein the I L36R monoclonal antibody is selected from one or more of ANB019 and spesolimab and the I L1 RAcP monoclonal antibody is selected from one or more of BI-5041 and MAB-16-0030.
4. The method according to claim 1 or 2, characterized in that the method comprises:
(1) diluting I L-36 β to 100pg/m L-20 μ g/m L by using a culture medium, diluting I L-36R monoclonal antibody and/or I L1 RAcP monoclonal antibody to 1pg/m L-1 mg/m L by using the culture medium as initial concentration, diluting the initial concentration at an equal ratio of 1:2-1:5, and mixing the obtained serial dilutions with dilutions of I L-36 β at a volume ratio of 1: 1;
(2) allowing the effector cells to grow in the culture medium at 4 × 105-40×105Diluting the cells/m L, adding the diluted cells/m L into the mixed solution obtained in the step (1) respectively according to the volume ratio of 1:1, mixing and incubating for 2-24 h;
(3) and (3) adding a chemiluminescence substrate of the reporter gene into the mixed solution obtained in the step (2), and fitting a four-parameter curve according to the obtained relative chemiluminescence unit value (R L U) to determine the biological activity of the antibody.
5. The method according to claim 4, wherein in step (1), I L-36 β is diluted to 50-150ng/m L using a culture medium.
6. The method according to claim 5, wherein in step (1), I L-36 β is diluted to 100ng/m L using culture medium.
7. The method according to claim 4, wherein in the step (1), the I L-36R monoclonal antibody and/or the I L1 RAcP monoclonal antibody is diluted to 40-50 μ g/m L as a starting concentration using a culture medium, and further diluted at an equal ratio of 1: 4.
8. The method of claim 4, wherein in step (2), the effector cells are cultured in a medium at 20 × 10 ™5The cells/m L were diluted and added to the mixture obtained in step (1) at a volume ratio of 1: 1.
9. The method of claim 4, wherein in step (2), the mixture is incubated for 6 hours.
10. The method according to claim 4, wherein the medium in step (1) and step (2) is 1640+10% FBS.
11. The method according to claim 4, wherein in step (3), the chemiluminescence values are detected using a luciferase kit, and the biological activity of the antibody is determined by fitting a four-parameter curve to the relative chemiluminescence unit values obtained.
12. The method of claim 4, wherein the post-mixing incubation of step (2) is performed in a 96-well plate, and the relative chemiluminescent unit values of step (3) are read on a microplate reader.
13. Human peripheral blood leukemia T cell Jurkat/I L36R/I L1 RAcP/NF-kB-25 #, which is preserved in China general microbiological culture Collection center (CGMCC) at 25.5.2020 with the preservation number of CGMCC 19943.
14. The application of human peripheral blood leukemia T cell Jurkat/I L36R/I L1 RAcP/NF-kB-25 # as effector cells in detecting the biological activity of a human I L0-36/I L136R/I L1 RAcP pathway inhibitor is characterized in that the human I L-36/I L36R/I L1 RAcP pathway inhibitor is an I L-36R monoclonal antibody and/or an I L1 RAcP monoclonal antibody, the human peripheral blood leukemia T cell Jurkat/I L36R/I L1 RAcP/NF-kB-25 # is preserved in China general microbiological culture Collection center (CGMCC) at 25/5.2020 with the preservation number of CGMCC 19943.
15. The use of claim 14, wherein the I L36R monoclonal antibody is selected from the group consisting of one or more of ANB019 and spesolimab and the I L1 RAcP monoclonal antibody is selected from the group consisting of one or more of BI-5041 and MAB-16-0030.
16. Use of the method of any one of claims 1 to 12 for quality control of production of a human I L-36/I L36R/I L1 RAcP pathway inhibitor, said human I L-36/I L36R/I L1 RAcP pathway inhibitor being a I L-36R monoclonal antibody and/or a I L1 RAcP monoclonal antibody.
17. The use of claim 16, wherein the I L36R monoclonal antibody is selected from the group consisting of one or more of ANB019 and spesolimab and the I L1 RAcP monoclonal antibody is selected from the group consisting of one or more of BI-5041 and MAB-16-0030.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022166977A1 (en) * 2021-02-08 2022-08-11 上海普铭生物科技有限公司 Anti-human il-36r antibody and application thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108026172A (en) * 2015-06-26 2018-05-11 Mab发现公司 Monoclonal anti-I L-1 RACP antibodies

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108026172A (en) * 2015-06-26 2018-05-11 Mab发现公司 Monoclonal anti-I L-1 RACP antibodies

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HIROSHI UMEHARA: "Tributylhexadecylphosphonium Bromide, a Novel Nuclear Factor of Activated T Cells Signaling Inhibitor, Blocks Interleukin-2 Induction Associated with Inhibition of p70 Ribosomal Protein S6 Kinase Phosphorylation", 《BIOL. PHARM. BULL.》 *
LIPING DING: "IL-36 cytokines in autoimmunity and inflammatory disease", 《ONCOTARGET》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022166977A1 (en) * 2021-02-08 2022-08-11 上海普铭生物科技有限公司 Anti-human il-36r antibody and application thereof

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