CN117230019B - Monoclonal antibody against cyclopylin A and application thereof in treating psoriasis - Google Patents

Abstract

The invention discloses a monoclonal antibody of cyclopylin A and application thereof in treating psoriasis. The invention provides a hybridoma SQHA4 secreting anti-CypA monoclonal antibody, and the preservation number of the hybridoma SQHA4 is CGMCC No.45048. Also provides the anti-CypA monoclonal antibody secreted by the hybridoma SQHA4. The experiments of the invention prove that the invention provides 1 hybridoma cell strain SQHA4 capable of secreting anti-CypA monoclonal antibody, the hybridoma cell strain can secrete monoclonal antibody SQHA4, and the monoclonal antibody SQHA4 can specifically recognize CypA and inhibit psoriasis-like inflammatory reaction.

Description

Monoclonal antibody against cyclopylin A and application thereof in treating psoriasis

Technical Field

The invention belongs to the field of biological medicine, and relates to a monoclonal antibody for resisting cyclopylin A and application thereof in treating psoriasis.

Background

Psoriasis is an autoimmune mediated inflammatory skin disease, is influenced by genetic and environmental factors, presents clinical characteristics of skin thickening, erythema and white scales, and the like, and causes great burden on patients in physical, psychological and economic aspects. Psoriasis is generally thought to be caused by a cascade of immune cells (e.g., dendritic cells, T cells, macrophages, etc.) that are abnormally activated and release inflammatory factors (IL-6, IL-17, IL-20, IL-22, etc.) and then attack skin cells, including keratinocytes, and ultimately lead to thickening of the epidermis acantha layer, abnormal skin keratinization, inflammatory cell infiltration, and neovascularization.

The treatment of psoriasis has also been a problem afflicting the medical community due to the complexity and uncertainty of the pathogenesis. Currently available therapeutic agents are mainly biological agents, transdermal hormone delivery and traditional oral drugs (e.g. methotrexate). These drugs have difficulty meeting the requirements of economy, safety and good medication compliance at the same time, so it is of practical significance to develop new therapeutic drug strategies to overcome the above drawbacks.

Cyclopylin a (Cyclophilin a, cypA) is a multifunctional immunomodulatory protein that is widely expressed in eukaryotic cells. Intracellular CypA is involved in the regulation of expression of various cytokines during inflammatory activation, and is considered to be one of the hubs for inflammatory regulation. Intracellular CypA can be secreted extracellularly under cell stress, and can bind to its known receptor CD147 and other unknown receptors, further activating downstream inflammatory signals.

Disclosure of Invention

The invention aims to provide a monoclonal antibody against cyclopylin A and application thereof in treating psoriasis.

In a first aspect, the invention provides a hybridoma cell strain SQHA4 secreting an anti-CypA monoclonal antibody (abbreviated as CypA monoclonal antibody or anti-CypA monoclonal antibody), and the preservation number of the hybridoma cell strain is CGMCC No.45048.

In a second aspect, the invention provides an anti-CypA monoclonal antibody secreted by the hybridoma cell line SQHA4 of the first aspect.

In a third aspect, the invention provides the use of an anti-CypA monoclonal antibody according to the second aspect in any one of the following;

1) Preparing a product for treating psoriasis;

2) Preparing a product for reducing inflammation caused by psoriasis;

3) Preparing a product for alleviating the condition of psoriasis;

4) Preparing a product that inhibits psoriasis-like inflammatory response;

5) The product for detecting the cyclophilin A is prepared.

The inhibition of inflammatory response is embodied in a reductionIL1B、TNFA、IL6、IL23、CCL2、IL8、Il17And/or reduced inflammatory cell infiltration.

In a fourth aspect, the invention provides a product comprising an anti-CypA monoclonal antibody according to the second aspect.

The product described above has at least one of the following functions:

1) Treating psoriasis;

2) Reducing inflammation caused by psoriasis;

3) The psoriasis disease state is relieved;

4) Inhibiting psoriasis-like inflammatory response;

5) Detecting the cyclophilin A.

In the above, the product is a kit.

In a fifth aspect, the present invention provides a method for detecting cyclophilin a comprising the steps of: and detecting whether the sample to be detected contains the cyclophilin A by taking the anti-CypA monoclonal antibody of the second aspect as an antibody.

Experiments prove that the invention provides a hybridoma cell strain capable of secreting anti-CypA monoclonal antibody, the hybridoma cell strain can secrete monoclonal antibody (SQHA 4), and the monoclonal antibody (SQHA 4) can specifically recognize CypA and inhibit psoriasis-like inflammatory reaction.

Preservation description

Strain name: SQHA4

Classification naming: mouse hybridoma cells

Preservation mechanism: china general microbiological culture Collection center (China Committee for culture Collection of microorganisms)

The preservation organization is abbreviated as: CGMCC

Address: beijing city, chaoyang area, north Chenxi Lu No. 1 and 3

Preservation date: 2022, 2 and 24 days

Accession numbers of the preservation center: CGMCC No.45048

Drawings

FIG. 1 shows ELISA for detecting the potency of SQHA4 mab.

FIG. 2 shows the SQHA4 assay of CypA expression in cell lines and mouse tissues.

FIG. 3 is the effect of SQHA4 on Hacat cell inflammatory factor expression.

Figure 4 is the effect of SQHA4 treatment on psoriasis mouse pathology.

FIG. 5 is the effect of SQHA4 treatment on psoriasis mouse cytokine expression.

Detailed Description

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.

The following examples facilitate a better understanding of the present invention, but are not intended to limit the same.

The quantitative tests in the following examples were all set up in triplicate and the results averaged.

Male DBA/1 mice were purchased from Beijing Vietnam Lihua laboratory animal Limited; male DBA/1 mice are hereinafter referred to as mice.

The CypA mab QH01 in the example is QH01 in CN 113248617B (grant date 2021.10.01; application number 202110682376.5), and the preservation number of the hybridoma cell strain secreting the CypA mab QH01 is CGMCC No.21909.

PBS (pH 7.2-7.4) in the examples below was purchased from Beijing Soy Bao technology Co., ltd., product number P1020.

The CypA protein in the examples below was purchased from peri coast protein technologies Inc., under the designation CR15.

The reagents used for the ELISA method were as follows:

coating solution (ph=9.6) was purchased from beijing solibao technologies limited under the trade designation C1050.

Washing solution (pH 7.2-7.4) was purchased from Beijing Soy Bao technology Co., ltd., product number P1031.

The sealing liquid is a washing liquid containing (mass volume percentage, g: ml) 2% of skimmed milk powder;

the color development liquid is obtained by mixing 1%A volume percent liquid and 10% B volume percent liquid, wherein the A volume percent liquid is: 1% TMB in DMSO; and (2) liquid B: containing 0.1% CH ₄ N ₂ O·H ₂ O ₂ Is added to the solution.

Example 1 preparation of anti-CypA mab

1. Acquisition of hybridoma cell line secreting anti-CypA monoclonal antibody

1. Immunized mice

6 SPF-class BALB/c female mice are immunized with CypA protein, and the initial immunization is performed by intramuscular injection, wherein the immunization amount is 20 mug protein per mouse; subsequent 6 booster immunizations were then performed in the same manner and dose, each one at one week intervals; mice were immunized by intraperitoneal impact with 50 μg CypA protein one week after the boost was completed, and after one week the mice were bled from their orbits and serum antibody titers were determined.

2. Screening SQHA4

1) ELISA detects serum titers of 6 immunized mice, and mice with higher titers are screened for hybridoma fusion.

2) Cell fusion experiments: mouse spleen cells were fused with SP2/0 cells by PEG method. The fused cells were subjected to selection culture using a semisolid medium (containing HAT).

3) Picking a monoclonal: 10 plates×93 cells were selected and cultured in 96-well cell culture plates (previously plated with thymocytes, 100 μl/well).

4) Primary screening of monoclonal cells: the monoclonal cell supernatant from the 96-well plates was discarded in total, 200. Mu.L/well, and 20% fresh bovine IMDM medium (containing HT) was added for the first screening.

5) Secondary screening of monoclonal cells: and (3) coating the recombinant CypA protein, and performing secondary screening on the selected clone by adopting an ELISA method to obtain a positive hybridoma cell strain.

6) Three screens of monoclonal cells: and (3) respectively coating the positive cell strain obtained by the secondary screening with recombinant CypA protein and "tag protein", and performing the third screening by adopting an ELISA method to finally obtain the hybridoma cell strain SQHA4 secreting the CypA monoclonal antibody (SQHA 4).

The SQHA4 secreting the SQHA4 is preserved in China general microbiological culture Collection center (CGMCC) for 24 days in 2 months of 2022, the address is 1 th yard 3 of North Xiylu in the Beijing area, the preservation number is CGMCC No.45048, and the classification is named as mouse hybridoma.

2. Preparation of anti-CypA monoclonal antibody by ascites

Injecting the SQHA4 secreting hybridoma cell strain SQHA4 obtained above into the abdominal cavity of BALB/c mouse with a cell concentration of 1.5X10 ⁶ And/mouse. And collecting ascites after 10-14 days.

The antibody in ascites was purified using a Protein G-Sepharose4B adsorption chromatography column to give anti-CypA mab SQHA4, which was collected in 0.1M glycine-hcl buffer (ph=2.7), then replaced in PBS and stored for subsequent experiments.

ELISA method for detecting titer of anti-CypA monoclonal antibody SQHA4

1. Diluting the CypA protein with a coating solution to a final concentration of 2ug/ml,100 ul/well, and washing with a washing solution for 3 times at 4 ℃ overnight;

2. sealing the sealing liquid, incubating for 2 hours at 37 ℃ at 200 ul/hole, and then washing 3 times by using a washing liquid;

3. adding primary antibody (anti-CypA monoclonal antibody SQHA4 or CypA monoclonal antibody QH 01) or PBS (Blank), performing 4-fold gradient dilution on the primary hole 10 mug/mL, performing 8-point, end hole Blank, performing 100 mug/well incubation for 1h at 25 ℃, and washing 3 times by using a washing solution;

4. adding HRP-labeled goat anti-mouse IgG secondary antibody (available from Aibotag biotechnology Co., ltd., catalog number: AS 003) diluted 10000 times with PBS, incubating at 25deg.C for 1 hr, taking out, and washing with washing solution for 3 times;

5. adding 100 mu L/hole of color development liquid, and the color development time is about 10 min;

6. adding 50 mu L of stop solution into each hole for stopping;

7. measuring absorbance values at two wavelengths (450 nm,630 nm), and recording and storing data;

as shown in FIG. 1, the half-effective concentration (EC 50) of anti-CypA mab SQHA4 was 0.1547. Mu.g/mL and the EC50 of QH01 was 2.415. Mu.g/mL.

Example 2 anti-CypA mab SQHA4 subtype identification

The subtype of anti-CypA mab SQHA4 was identified using a mouse monoclonal antibody subtype identification kit (available from proteontech under the accession number PK 20002). Reference is made to the description of the steps.

The analysis results are shown in Table 1, wherein the heavy chain of anti-CypA mab SQHA4 is IgG1 and the light chain is kappa.

Example 3 detection of specific recognition of CypA in cell lines and mouse tissues by anti-CypA mab SQHA4

Lysis Buffer (150 mM NaCl,20 mM HEPES,1 mM EDTA,1% Triton100, 10% glycerol) containing Protease Inhibitor Cocktail (Roche, #5871, applied in amounts referred to the instructions) was used to lyse human lung cancer cell line (a 549), human keratinocyte cell line (Hacat) and male DBA/1 mouse lung and skin tissues, respectively, and the lysates were collected.

An appropriate amount of 5 XSDS-PAGE protein loading buffer was added to each lysate and heated at 98℃for 15 min. Conventional SDS-PAGE electrophoresis and Western blot analysis were performed using anti-CypA monoclonal antibody SQHA4 and anti-GAPDH antibody (Santa Cruz Co., cat. No. sc-47724) from ascites fluid prepared in example 1 II as primary antibodies and HRP-labeled goat anti-mouse monoclonal antibody (Jackson, cat. No. 115035003) as secondary antibodies.

The specific recognition results of anti-CypA monoclonal antibody SQHA4 on CypA in various tissues of cells and mice are shown in FIG. 2, and it can be seen that anti-CypA monoclonal antibody SQHA4 can specifically recognize CypA protein (molecular weight about 18 kDa) in different tissues of cell lines and mice.

Example 4 Effect of anti-CypA mab SQHA4 on Hacat cell inflammatory factor expression

(1) Collecting logarithmic phase Hacat cells, regulating cell suspension concentration with DMEM medium, adding 100uL per well, and plating to adjust density of the cells to be tested to 10 ³ -10 ⁴ cells/well, 5% CO2, 37℃incubation, cell monolayer confluence.

(2) Cells were divided into four groups:

negative control group (PBS group): incubating the cells obtained in (1) above in DMEM;

m5+pbs group: incubating the cells obtained in the step (1) in a DMEM medium containing M5;

m5+qh01 group: incubating the cells obtained in 1 above in DMEM medium containing M5 and QH01 mab, wherein QH01 is anti-CypA mab in patent CN 113248617A;

m5+sqha4 group: placing the cells obtained in the step 1 in a DMEM medium containing M5 and anti-CypA monoclonal antibody SQHA4 from ascites prepared in the step 1 II for incubation;

m5 is a combination of IL-17A, IL-22, oncostatin M, IL-1α and TNF- α, each cytokine having a final concentration of 2.5ng/mL in the medium; the final concentrations of the SQHA4 monoclonal antibody and the QH01 monoclonal antibody in the culture medium are 50nM; m5 is a combination of factors that induce psoriasis patterns, with the aim of mimicking psoriasis patterns.

3 duplicate wells were placed in each group and incubated at 37℃for 24 or 72 hours with 5% CO 2.

(3) Adding 500 mu LTRIZOL into each hole, extracting total mRNA according to TRIZOL method, reverse transcribing to obtain cDNA, performing qPCR detection, and incubating for 24 hrIL1B、TNFA、IL6、IL8、IL23、CCL2Six genes (inflammatory factors), sample detection incubated for 72hKRT6AndKRT1genes (psoriasis-related factors).

qPCR reaction procedure described above: (1) 30 s at 95 ℃; (2) 30 s at 95 ℃; (3) 60 ℃ 60 s; (2) (3) cycle 40 times. The fold change was calculated using the 2- ΔΔCT method.

As shown in FIG. 3, SQHA4 was found to be effective in loweringKRT6(FIG. 3A),IL1B(FIG. 3C),TNFA(FIG. 3D),IL6(FIG. 3E),IL8(FIG. 3F),IL23(FIG. 3G),CCL2(FIG. 3H) expression, as can be seen in FIG. 3B, SQHA4 can be increasedKRT1Expression of the gene (FIG. 3B). The effect of SQHA4 is better than that of QH01 as a whole.

Example 5 therapeutic Effect of SQHA4 on IMQ-induced psoriasis mice

1. Construction of mice grouping and IMQ-induced mice pneumonia model

(1) Grouping mice

The Balb/c mice of 6-8 weeks of age are randomly grouped into 4 groups, namely a blank control group (PBS), a model group (IMQ+PBS), a treatment control group (IMQ+QH201) and a treatment group (IMQ+SQH24), wherein 6 groups (weight of 18+/-3 g) are selected.

(2) Construction of a treatment model for psoriasis in mice

C57BL/6 mice were anesthetized by intraperitoneal injection of aphtha at 200. Mu.L/10. 10 g body weight, and after anesthesia, BALB/C mice were shaved on the back and dehaired (approximately 3 cm. Times.3 cm in area), and the following groups were performed:

blank control group: topical daily administration of an equal amount of petrolatum ointment to the IMQ of the model group on the depilated skin and ears;

model group: 5% imiquimod cream (IMQ, used to simulate psoriasis) was topically applied daily to the depilated skin and ears, and an equal amount of PBS was intraperitoneally injected from day 2 post-depilated.

Treatment control group: daily topical application of 5% imiquimod cream (IMQ) to the skin and ears following depilation, 200 μl of 10mg/kg anti-CypA mab QH01 solution (solvent PBS) was intraperitoneally injected from day 2 following depilation;

treatment group: a5% imiquimod cream (IMQ) was topically applied daily to the depilated skin and ears, and 200. Mu.L of 10mg/kg anti-CypA mab SQHA4 solution (in PBS solvent) was intraperitoneally injected from day 2 post-depilated.

The following test was performed after molding for 6 days (the first application of IMQ was designated as day 0).

2. Influence of anti-CypA mab SQHA4 treatment on psoriasis mouse pathology

(1) Photograph observation, PASI score and measurement of ear thickness in mice, PASI score mainly included erythema score, scale score, infiltration score and total score, see table 3.

(2) HE pathology: the skin tissue of the mice is taken and put into 4% paraformaldehyde to be fixed for more than 16 hours, and the mice are dehydrated, waxed, embedded and sliced conventionally and dyed conventionally by HE.

As shown in fig. 4, the experimental results are shown in fig. 4, wherein a is the phenotype of each group of mice in 6 days of model building, B is the score of PSAI, and C is the ear thickness result, and it can be seen that the erythema, the scale and the infiltration of the backs of the mice in the anti-CypA monoclonal antibody SQHA4 treatment group are significantly improved compared with the treatment control group (fig. 4A), the score of PSAI is significantly reduced compared with the treatment control group (fig. 4B), the ear thickness is significantly thinned compared with the treatment control group (fig. 4C), the HE pathology shows that the spiny layer of the treatment group is significantly reduced compared with the treatment control group, the infiltration of inflammatory cells is significantly reduced compared with the treatment control group (fig. 4A), and the disease condition of the psoriatic mice is effectively relieved compared with the treatment control group.

3. Effect of SQHA4 treatment on psoriasis mouse cytokine expression

Collecting the skin tissue of each group of mice after molding for 6 days, adding liquid nitrogen for grinding, adding 500 mu LTRIZOL, extracting total mRNA according to TRIZOL method, reverse transcription to obtain cDNA, and performing qPCR detectionKrt6、Krt1、Ppia、Il1b、Tnfa、Il6、 IL23、Ccl2、Il17Expression of these eight genes (primers are shown in Table 4 below).

The qPCR reaction procedure described above was as follows: (1) 30 s at 95 ℃; (2) 30 s at 95 ℃; (3) 60 ℃ at 60 DEG Cs; (2) (3) cycle 40 times. With 2 ^-ΔΔCT The method calculates the change times.

The experimental results are shown in FIG. 5, A-I are respectivelyKrt6、Krt1、Ppia、Il1b、Tnfa、Il6、IL23、Ccl2、 Il17Expression level of SQHA4 in skin tissue of mice in the treatment groupKrt6(FIG. 5A)、Ppia(FIG. 5C)、Il1b(FIG. 5D)、Tnfa(FIG. 5E)、Il6(FIG. 5F)、IL23(FIG. 5G)、Ccl2(FIG. 5H)、Il17The expression (FIG. 5I) is significantly reduced compared to the treatment control group,Krt1the expression of (c) was significantly elevated compared to the treated control group (fig. 5B), suggesting that SQHA4 treatment may reduce inflammation in psoriatic mice. The effect of SQHA4 as a whole was better than that of the treatment control group.

Claims (3)

1. Hybridoma cell strain SQHA4 secreting anti-CypA monoclonal antibody, and the preservation number is CGMCC No.45048.

2. Use of an anti-CypA monoclonal antibody secreted by the hybridoma cell line SQHA4 of claim 1 in any one of the following;

1) Preparing a product for treating psoriasis;

2) And preparing a product for relieving psoriasis.

3. The use according to claim 2, characterized in that: the product is a medicine.