Monoclonal antibody for treating overactive bladder and application thereof

Abstract

The invention relates to the field of antibodies, in particular to a monoclonal antibody for treating overactive bladder and application of the monoclonal antibody. The monoclonal antibody specifically recognizes and is combined with an adrenergic receptor beta3 (ADR beta3), the monoclonal antibody is obtained through a hybridoma technology, and the M13 monoclonal antibody with high affinity is successfully screened out. The M13 antibody promotes the increase of cAMP concentration in smooth muscle cells and activates a PKA pathway by activating an ADR beta3 receptor, thereby promoting the relaxation of bladder smooth muscles, increasing the bladder capacity and reducing the non-autonomous contraction of detrusor muscles. Animal experiments prove that the M13 monoclonal antibody can effectively improve the urination frequency and urine volume of a mouse OAB model, and has a good clinical application prospect. The monoclonal antibody and the preparation method thereof can be widely applied to the field of treatment of overactive bladder.

Description

Technical Field

[0001] The present invention relates to the field of antibodies, and in particular to a monoclonal antibody for treating overactive bladder and an application thereof. Background Art

[0002] Overactive bladder (OAB) is a common urinary tract dysfunction characterized by symptoms such as urinary urgency, frequency, increased nocturia, and urge incontinence, which severely impacts patients' quality of life and social function. The pathogenesis of OAB is not yet fully understood, but it is closely related to involuntary contractions of the bladder detrusor muscle, neural dysregulation, and smooth muscle dysfunction. Commonly used clinical treatments include anticholinergics and β3-adrenergic receptor (ADRβ3) agonists. However, existing drugs have significant side effects and limited efficacy, necessitating the development of new treatments.

[0003] Adrenergic receptor β3 (ADRβ3) is a type of G protein-coupled receptor that is abundantly expressed in bladder cholinergic nerves and detrusor smooth muscle cells. Studies have shown that activating ADRβ3 can directly inhibit the excitability of bladder smooth muscle cells and, by inhibiting the release of acetylcholine from cholinergic nerve endings, suppress involuntary detrusor contractions and increase bladder capacity. Although ADRβ3 agonists (such as mirabegron) are currently available on the market, their effectiveness in some patients is limited, and long-term use poses potential safety risks. Therefore, the development of specific biologics targeting ADRβ3 has become a research priority.

[0004] Monoclonal antibodies are widely used in the treatment of various diseases due to their strong targeting, long-lasting efficacy, and low side effects. Targeting the pathological mechanism of OAB, anti-ADRβ3 monoclonal antibodies obtained through hybridoma technology screening not only achieve highly specific binding to ADRβ3 but also, by activating its downstream signaling pathways (such as the cAMP / PKA pathway), further promote bladder smooth muscle relaxation and improve involuntary detrusor contractions, demonstrating significant therapeutic potential. To date, there have been no publicly reported clinical applications or patent portfolios for agonist anti-ADRβ3 monoclonal antibodies. This invention has significant innovative significance and clinical value. Summary of the Invention

[0005] The present invention aims to provide a novel treatment for overactive bladder (OAB), specifically an agonist anti-adrenergic receptor β3 (ADRβ3) monoclonal antibody and its use in the treatment of OAB. The present invention's technical solution can effectively improve bladder function in OAB patients, alleviating symptoms such as urinary urgency, frequency, nocturia, and urge incontinence, and has significant clinical application prospects.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] Using hybridoma technology, mice were immunized with recombinantly expressed ADRβ3 to screen and identify a monoclonal antibody, M13, that specifically recognizes and activates ADRβ3. This antibody contains the following variable region sequences: heavy chain CDR1 is FRQGSGGSLY, CDR2 is EITGSGGSIYSSLKYT, and CDR3 is GAGTSGYT; light chain CDR1 is RASQDINYLH, CDR2 is WILQSLLNSGVPYTF, and CDR3 is QQYDVPYS. This antibody demonstrated excellent affinity and agonistic effects in both in vitro and animal studies, significantly outperforming traditional drugs.

[0008] The monoclonal antibody M13 provided by the present invention can specifically bind to human ADRβ3, activate the intracellular cAMP signaling pathway, promote protein kinase A (PKA) phosphorylation, and then induce bladder smooth muscle relaxation, effectively increase bladder capacity, reduce involuntary contractions of the detrusor muscle, and improve OAB symptoms.

[0009] In a mouse overactive bladder model, after intraperitoneal injection of the antibody M13 of the present invention, the frequency of urination and urine volume of the animals were significantly reduced, demonstrating good biological activity and therapeutic effect.

[0010] The present invention also proposes the use of the above-mentioned monoclonal antibody in the preparation of a drug for treating overactive bladder, providing a new, safe and effective biological preparation solution for the treatment of OAB, and solving the technical problems of limited efficacy of existing therapies and obvious side effects of long-term use.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] Provides an anti-ADRβ3 monoclonal antibody M13 with a clear structure and high specificity, which can effectively stimulate ADRβ3 and improve bladder function;

[0013] This antibody can be used as a potential biological agent for OAB, showing good safety and efficacy in animal experiments and has the potential for clinical translation;

[0014] Compared with existing drugs such as anticholinergics and small molecule agonists, monoclonal antibodies have the advantages of strong targeting, long-lasting effects and few side effects, and can meet unmet clinical needs.

[0015] In summary, the present invention provides an innovative monoclonal antibody and its use in the treatment of overactive bladder, which fills the current technological gap in the relevant field and has important innovative value and industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 ELISA detection of serum antibody titers of different mice. Figure 1A For the results of immunizing animals; Figure 1B The result of cell fusion.

[0017] Figure 2 The binding activity of antibodies from different mouse sources was detected by ELISA.

[0018] Figure 3 SDS-PAGE of monoclonal antibody M13.

[0019] Figure 4 Statistics of urination intervals in mice.

[0020] Figure 5 Statistics of single urine output of mice.

[0021] Figure 6 Statistics of mouse urination pressure.

[0022] Figure 7 The interval between urinations in mice.

[0023] Figure 8 Single urine output of mice.

[0024] Figure 9 Mice urination pressure. DETAILED DESCRIPTION

[0025] In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0026] Example 1. Preparation of monoclonal antibodies

[0027] (1) Immunized animals

[0028] Balb / c mice were immunized with recombinant adrenergic receptor β3 (ADRβ3) expressed in E. coli. 50 μg of ADRβ3 was added dropwise to an equal volume of complete Freund's adjuvant for emulsification to create a fully emulsified emulsion, which was then injected subcutaneously at multiple sites, with 100 μl per well. Two weeks after the primary immunization, a secondary immunization was performed subcutaneously using an emulsion prepared with 50 μg of ADRβ3 and an equal volume of incomplete Freund's adjuvant. Two weeks after the secondary immunization, a third immunization was performed using the same procedure. Seven days after the third immunization, a small amount of serum was collected for antibody titer determination by ELISA. Recombinant ADRβ3 protein was added to a microplate at a concentration of 1 μg / ml in coating buffer (CBS) at 100 μl / well. The plate was coated overnight at 4°C and blocked the next day. Serum to be tested was added. The test serum was diluted 10-fold in DPBS containing 1% BSA, followed by a 5-fold serial dilution. Mouse blank serum was used as a negative control. The secondary antibody was HRP-labeled goat anti-mouse IgG diluted 1:3000 in DPBS containing 1% BSA. Figure 1A As shown, 1-5 are different mouse individuals, and the horizontal axis is the Log of the dilution multiple of the serum to be tested.

[0029] Cell fusion

[0030] Two weeks after the three immunizations, the mice were killed and the spleen cells were isolated. The mouse myeloma cells SP2 / 0 and the isolated spleen cells were mixed at a ratio of 1:10 and centrifuged. The supernatant was discarded, the tube wall was tapped to loosen the precipitate slightly, and 1 ml of preheated 45% PEG4000 was slowly added while shaking the centrifuge tube. After standing for 90 seconds, centrifugation was carried out, the supernatant was discarded, and the cells were resuspended in HAT selection medium containing 20% ​​fetal bovine serum. The cell suspension was diluted and added to a 96-well plate at 100ul / well. After each spleen was separated, it could be diluted to 4 96-well plates. After culturing in the incubator for 5 days, HT medium was replaced, and RPMI1640 basal medium was replaced after 2 weeks. When the hybridoma cells covered 10% of the bottom of the well, the supernatant was taken for ELISA to detect the affinity of the specific antibody. The experimental method was consistent with the serum titer determination, and the results were as follows. Figure 1B As shown, the horizontal axis is the dilution multiple of the supernatant.

[0031] Hybridoma cell cloning

[0032] Select hybridoma cells with high antibody affinity and obtain monoclones using the limiting dilution method. Gently blow off the hybridoma cells to be cloned from the culture well, count them, and dilute them to 5 cells / ml with basal culture medium. Add the diluted cell suspension at 100ul / well and culture in a 37℃ 5% CO2 incubator. Monoclonal formation can be seen after 8 days. Take the supernatant and test the antibody activity by ELISA. The antigen is recombinant ADRβ3 protein, the secondary antibody is goat-anti-mouse IgG-HRP, and the negative control is blank culture medium. The results are as follows: Figure 2 As shown, the horizontal axis represents the logarithmic dilution of the supernatant. The supernatants of monoclonal clones 1 and 4 both exhibited strong antigen-binding activity. The clones with the highest activity were expanded and cryopreserved in a cryopreservation solution of 10% DMSO + 90% fetal bovine serum.

[0033] Example 2: Purification of monoclonal antibodies

[0034] Monoclonal clone #1, which exhibits high antibody affinity and excellent stability, was selected for expansion and expanded to two T75 flasks. The resulting monoclonal antibody was designated M13. After 7 days, approximately 30 ml of supernatant was collected and centrifuged at 8000 rpm for 10 minutes at 4°C. The supernatant was collected in a new 50 ml centrifuge tube and filtered through a 0.22 μm filter. After filtration, the pH was measured and adjusted to 7.0-7.5 using neutralization buffer. 500 μl of Protein G magnetic beads were added to the supernatant, mixed thoroughly, and vortexed overnight at 4°C. The next day, the supernatant was centrifuged at 1500 rpm at 4°C for 5 minutes and the supernatant discarded. The beads were resuspended in 1 ml of cold DPBS and transferred to a chromatography column. The column was washed three times with 10 ml of DPBS. 3 ml of elution buffer was added to the column, any remaining DPBS was discarded, and the lower end of the column was plugged and incubated for 5-10 minutes. The elution was repeated once after the eluate flowed out. Add 600 μl of neutralization buffer to the collected eluate and mix thoroughly. Transfer the eluate into a 30 kDa ultrafiltration tube and centrifuge at 4000 rpm and 4°C to concentrate the protein solution. After the volume of the protein solution is reduced to 600 μl, add 5 ml of DPBS and centrifuge again. The centrifugation conditions are the same as above. Repeat 3 times until the buffer is completely replaced with DPBS. Use a spectrophotometer to determine the protein concentration of M13. Take 3 μg of M13 protein for SDS-PAGE identification, use 8-12% PAGE gel, and the spotting conditions are 120V for 40 min. The results are as follows Figure 3 As shown. M is a protein marker; lane 1 is the non-reduced state, and lane 2 is the reduced state. The molecular weight of the M13 protein after initial purification with Protein G conforms to the theoretical molecular weight of mouse IgG. In the non-reduced state, there are minor aggregates, but no chain dropouts or mispairing of the light and heavy chains. DTT can open all disulfide bonds, resulting in single-molecule heavy and light chains.

[0035] Example 3: Obtaining the variable region sequence of a monoclonal antibody

[0036] Candidate hybridoma clones were lysed with Trizol and total RNA was extracted. This was used as a template for first-strand cDNA synthesis. PCR amplification was then performed using the first-strand cDNA as a template using antibody variable region-specific primers to obtain nucleic acids for the light and heavy chain variable regions of the antibody corresponding to the hybridoma cells. The nucleic acids were then subjected to agarose gel electrophoresis, excised, and recovered, followed by Sanger sequencing to obtain the antibody variable region sequences. The sequences of the antibody heavy and light chain variable regions are shown in Table 1.

[0037] Table 1 Amino acid sequences of monoclonal antibodies

[0038]

[0039]

[0040] Example 4: Establishment of an Overactive Bladder Mouse Model

[0041] Mice were fasted for 6 hours before surgery and anesthetized with an intraperitoneal injection. After being fully anesthetized, they were fixed in the supine position under a dissecting microscope. The lower abdomen was prepared and disinfected with iodine. An epidural catheter was inserted into the bladder through the urethral orifice to support the bladder and urethra. The skin and subcutaneous tissue were incised in the center of the mouse's lower abdomen, and the rectus abdominis muscle was separated. The bladder was located, and a puncture of approximately 1 mm x 1 mm was made at the top of the bladder. A polyethylene microcatheter (PE-10) was inserted. The junction between the bladder and urethra was freed, and the proximal urethra was isolated. The proximal urethra was ligated with 5-0 surgical sutures. The bladder was repositioned, and the catheter was passed subcutaneously through the back of the neck and secured. The incision was sutured layer by layer, and the abdominal cavity was closed. Animals were housed individually in a clean and dry environment. The control group only underwent wound preparation without ligation. Postoperatively, all groups received 40,000 IU of penicillin for 3 days. Four weeks after surgery, the mice were weighed and the number of urinations and urine volume within 12 hours were counted using the urine dot method on paper.

[0042] Table 2 Overactive bladder mouse model

[0043] Group Weight g Number of spontaneous urinations within 12 hours Spontaneous urine output within 12 hours μl control group 22.35±1.98 4.98±1.23 1288±101.9 Model Group 23.01±1.76 7.02±1.08 1476±96.5

[0044] The results in Table 2 showed that there was no significant difference in body weight between the model group and the control group. The model group urinated significantly more frequently, and the urine volume was significantly greater than that of the control group.

[0045] The urodynamics of mice was tested using an animal bladder function tester. PE-10 was connected to the pressure sensor port through a 22-gauge needle and a short section of PE-50 adapter tube. The other port of the pressure sensor was connected to a micro-injection pump. 0.9% sodium chloride solution was injected into the bladder at a rate of 0.3 m / min, and the single urine output was measured by a balance under the mouse cage. The infusion volume, intravesical pressure, and urine output were recorded during a single bladder filling-urination period. The bladder volume was measured by the volume of normal saline injected into the bladder before each urination. The urination pressure was the peak intravesical pressure during urination. Statistical results were analyzed using t-test, see Figure 4-6 .

[0046] The results show that Figure 4 The urination interval of mice was statistically analyzed. The urination interval of the model group was significantly shorter than that of the control group (p<0.001). Figure 5 The vertical axis represents the single urine output of mice. The single urine output of the model group was significantly lower than that of the control group (p<0.001). Figure 6 The vertical axis represents the urination pressure statistics of mice. Compared with the control group, the urination pressure of the OAB model group increased significantly (p<0.05).

[0047] Example 5: Effect of Monoclonal Antibodies on Overactive Bladder

[0048] The mice with successful modeling were given M13 once a day at 10 mg / kg each time for intraperitoneal injection, and the positive control group was given tolterodine 0.5 mg / kg each time for gavage once a day. The mice in the model group were given 200 ul DPBS for intraperitoneal injection once a day. After the same surgical procedure as in Example 4, a PE-10 catheter was placed in the mouse bladder for mouse urodynamic testing. The statistical data were analyzed using One-Way AVOVA, and the results are shown in Table 3. After 7 days, the number of urinations within 12 hours and the urine volume within 12 hours of the mice were counted, as shown in Table 3. Figure 7-9 .

[0049] Table 3 M13 for the treatment of overactive bladder

[0050] Group Weight g Urinating frequency Urine volume μl Model Group 22.08±1.22 6.89±1.52 1493±141.3 M13 treatment group 22.58±1.05 5.02±1.18 1386±99.4 Positive control group 21.89±0.98 4.97±1.30 1296±150.2

[0051] The results in Table 3 showed that both the M13 treatment group and the positive control group could effectively reduce the frequency and volume of spontaneous urination in OAB mice within 12 h.

[0052] according to Figure 7 Compared with the blank control group, M13 can significantly increase the urination interval of OAB model mice (p<0.001). After M13 treatment, the single urine volume of mice increased significantly (p<0.05). The experimental results are as follows Figure 8 As shown. Similarly, Figure 9The experimental results showed that M13 treatment could significantly reduce the urination pressure of mice (p<0.05).

[0053] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A monoclonal antibody for treating overactive bladder, characterized in that The monoclonal antibody is an antibody that specifically binds to adrenergic receptor β3 (ADRβ3) and has the function of stimulating ADRβ3.

2. The monoclonal antibody according to claim 1, characterized in that The antibody is an M13 clone and comprises the following amino acid sequences: the heavy chain variable region amino acid sequences are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; the light chain variable region amino acid sequences are shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO:

6.

3. A pharmaceutical composition containing the monoclonal antibody according to claim 1 or 2, characterized in that: The composition can be used to treat overactive bladder and comprises a pharmaceutically acceptable carrier or auxiliary material.

4. Use of the monoclonal antibody according to any one of claims 1-2 or the pharmaceutical composition according to claim 3 in the preparation of a medicament for improving overactive bladder.

5. The use according to claim 4, characterized in that The overactive bladder is a urinary system disease characterized by frequent urination, urgency or nocturia caused by involuntary contraction of the detrusor muscle.

Images