WO2023198155A1 - Antibody for treating skin and mucosal inflammation and formulation thereof - Google Patents

Antibody for treating skin and mucosal inflammation and formulation thereof Download PDF

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WO2023198155A1
WO2023198155A1 PCT/CN2023/088120 CN2023088120W WO2023198155A1 WO 2023198155 A1 WO2023198155 A1 WO 2023198155A1 CN 2023088120 W CN2023088120 W CN 2023088120W WO 2023198155 A1 WO2023198155 A1 WO 2023198155A1
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antibody
seq
sequence
cdr
chain variable
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PCT/CN2023/088120
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French (fr)
Chinese (zh)
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石韦
武建朝
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上海韦青医药科技咨询有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/02—Drugs for genital or sexual disorders; Contraceptives for disorders of the vagina
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons

Definitions

  • the invention belongs to the field of medical biotechnology, and specifically relates to an antibody and its preparation for treating skin and mucosal inflammation.
  • Skin and mucous membranes are the first barrier between the human body and the external environment. Factors that attack the skin or mucous membranes include chemical factors, microorganisms, hot and cold stimulation, ionizing radiation, and mechanical stimulation. These exogenous and endogenous damaging factors can cause various damaging lesions of the skin or mucous membranes. Therefore, a series of complex reactions occur locally and systemically in the skin or mucous membranes to localize and eliminate the damaging factors, and remove and absorb necrosis. Tissues and cells, and repairing damage, this is the body's defensive response - inflammation. The inflammatory response is regulated by a variety of inflammatory factors such as TNFa, IL-6, IL-5, IL-1, IL-8, etc. Abnormal inflammation brings a series of allergic reactions to the skin and mucous membranes of the body, such as erythema, edema, burning, Pain, itching, etc.
  • TNFa is the earliest and most important inflammatory mediator in the inflammatory response process. Its biological activity is mainly achieved by transmitting signals through specific receptors on the cell membrane.
  • TNFa receptors There are two types of TNFa receptors, namely TNF-RI and TNF-RII. Both TNF receptors are glycoproteins. Amino acid sequence analysis shows that the amino acid sequences of the extracellular regions of the two receptors are highly similar, but the intracellular regions are completely different. Differences play different roles.
  • TNF-RI has a wide range of effects, including killer cell activity, antiviral activity, induction of the expression of inflammatory factors such as Mn-SOD, ICAM-1, IL-6, and IL-5, promotion of fibroblast proliferation, and cell programming.
  • TNF-RII mainly transmits the proliferation signals of lymphocytes such as thymocytes and NK. It increases the TNF-RI-induced effect by promoting TNF ⁇ to bind to TNF-RI, and also increases the expression of ICAM-1.
  • TNF-RI is mainly distributed in epidermal keratinocytes, mucosal epithelial cells and dendritic cells in the upper dermis, while in In skin lesions, in addition to the above distribution, TNF-RI is also distributed around the blood vessels in the parakeratotic stratum corneum and upper dermis.
  • TNF-RII is distributed in eccrine ducts and dermal dendritic cells in normal skin, while in skin lesions, it is distributed in upper dermal blood vessels and perivascular infiltrating cells.
  • TNFa is involved in pain sensitization: inflammatory tissue sites and DRG neurons release a large amount of MMP-9, which can act on the TNFa precursor on the DRG primary sensory neurons to activate it and bind to the TNFR1 receptor, thereby causing TRPV1 channels The expression and sensitivity are increased and are involved in the occurrence and development of inflammatory pain.
  • IL-5 is an inflammatory factor secreted by cells such as eosinophils, NK cells, TC2CD8+T cells, mast cells, CD45+CD4+T cells, ⁇ -delta T cells, and IL-1 ⁇ -activated endothelial cells.
  • IL-5 performs many functions on eosinophils. These include downregulation of Mac-1, upregulation of IgA and IgG receptors, stimulation of secretion of lipid mediators (leukotriene C4 and PAF) and induction of granule release.
  • IL-5 also promotes eosinophil growth and differentiation; however, the exact role of IL-5 is unclear; it may act in an auxiliary manner.
  • IL-5 acts on eosinophils and mast cells and is involved in airway mucosal inflammation.
  • IL-6 can be synthesized by a variety of cells, including activated T cells and B cells, monocytes-macrophages, endothelial cells, epithelial cells, and fibroblasts.
  • IL-6 is a heterodimer composed of two glycoprotein chains ⁇ . The ⁇ chain lacks an intracellular region and can only bind to IL-6 with low affinity. The formed complex quickly binds to the high affinity ⁇ chain and transmits information into the cell through the ⁇ chain.
  • IL-6 acts on many target cells, including macrophages, hepatocytes, resting T cells, activated B cells and plasma cells; its functions: 1 Promote the expression of IL-2R on the surface of T cells, enhance IL-1 and Mitogenic effects of TNF on TH cells.
  • 2A as a stem cell stimulating factor, it induces the synthesis of acute phase response proteins in acute inflammatory reactions caused by infection or trauma, among which the increase of amyloid A and C-reactive protein is particularly obvious.
  • 3 Promote B cell proliferation, differentiation and production of antibodies; malignant B cells of multiple myeloma can both produce IL-6 and respond to IL-6, suggesting that IL-6 may serve as an autocrine growth factor for these cells.
  • 4IL-6 can also effectively promote cachexia induced by TNF and IL-1; promote glucocorticoid synthesis; stimulate osteoclast activity and keratinocyte growth; and can also promote bone marrow hematopoietic function.
  • IL-6 cannot stimulate corresponding cells to secrete other cytokines, and its autocrine effect on immune cells is relatively weak at physiological concentrations. It is suggested that its main immunological function is to enhance the effects of other cytokines. Yang Yan et al. reported itch-related inflammatory factor receptors. IL-2 and IL-6 in the leukocyte lysates of patients with allergic dermatitis are both itching substances. For example, when IL-2 is used to treat cancer, it will induce itching in the body. Both nerve cells and Schwann cells express IL-6 receptors, and the immunoreactivity of IL-6 on nerve fibers in patients with positive skin patch tests and prurigo nodularis is higher than that in normal people. In addition, studies have shown that IL-8 also has an itching effect, but the specific mechanism is still unclear.
  • the antibacterial polyclonal antibody contains antibodies to a variety of inflammatory factors such as TNFa, IL-6, IL-5, etc.
  • inflammatory factor antibody gel alone or in combination can quickly relieve mucosal and skin inflammation, such as oral ulcers, vaginitis, etc.
  • the permeability of the inflammatory mucosa and skin increases, and the inflammatory factor antibodies work locally without causing systemic immune suppression, which is safer and better.
  • the present invention first relates to a group of monoclonal antibodies directed against inflammatory factors.
  • the inflammatory factors are human interleukin 5 (IL-5) or human tumor necrosis factor (TNFa).
  • the monoclonal antibodies are:
  • the monoclonal antibody against human interleukin 5 has the VH amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 6; the (VL) amino acid sequence of the light chain variable region as shown in SEQ ID NO: As shown in 8;
  • VH amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2; the (VL) amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4 .
  • the anti-human interleukin 5 (IL-5) monoclonal antibody is 59C4, and the VH amino acid sequence of the heavy chain variable region of the 59C4 antibody is shown in SEQ ID NO: 6; the ( VL) amino acid sequence is shown in SEQ ID NO:8.
  • the CDR classification of the heavy chain variable region VH of the 59C4 antibody is as follows:
  • the CDR-H1 sequence is shown in SEQ ID NO.20, SEQ ID NO.20: DYYIN
  • the CDR-H2 sequence is shown in SEQ ID NO.21, SEQ ID NO.21: EIYPGSGNTYYNEKFKG
  • the CDR-H3 sequence is shown in SEQ ID NO.22, SEQ ID NO.22: LTYYGSSYDYFDY
  • the CDR division of the light chain variable region VL of the 59C4 antibody is as follows:
  • the CDR-L1 sequence is shown in SEQ ID NO.23, SEQ ID NO.23: ENIYSN
  • the CDR-L2 sequence is shown in SEQ ID NO.24, SEQ ID NO.24: AAT
  • the CDR-L3 sequence is shown in SEQ ID NO.25, SEQ ID NO.25: QHFWNTPYT
  • the anti-human tumor necrosis factor (TNFa) monoclonal antibody is T10E8, and the VH amino acid sequence of the heavy chain variable region of the T10E8 antibody is shown in SEQ ID NO: 2; the (VL) amino acid of the light chain variable region The sequence is shown as SEQ ID NO:4;
  • the CDR classification of the heavy chain variable region VH of the T10E8 antibody is as follows:
  • the CDR-H1 sequence is shown in SEQ ID NO.14, SEQ ID NO.14: SYTLS
  • the CDR-H2 sequence is shown in SEQ ID NO.15, SEQ ID NO.15: YISSGGSNTYYPDTVKG
  • the CDR-H3 sequence is shown in SEQ ID NO.16, SEQ ID NO.16: QGYYRYVLYAMDY
  • the CDR division of the light chain variable region VL of the T10E8 antibody is as follows:
  • the CDR-L1 sequence is shown in SEQ ID NO.17, SEQ ID NO.17: SASSSVSHMH
  • the CDR-L2 sequence is shown in SEQ ID NO.18, SEQ ID NO.18: DTSKLAS
  • the CDR-L3 sequence is shown in SEQ ID NO.19, SEQ ID NO.19: QQWSSNPIT.
  • the monoclonal antibody is a humanized modified monoclonal antibody, which includes the heavy chain and light chain variable regions of the 59C4 antibody, or the heavy chain and light chain variable regions of the T10E8 antibody;
  • the 59C4 humanized antibody is: a humanized antibody containing the heavy chain and light chain variable regions of the 59C4 antibody and the human antibody IgG1 constant region (Fc);
  • the T10E8 humanized antibody is a humanized antibody that includes the heavy chain and light chain variable regions of the T10E8 antibody and the human antibody IgG1 constant region (Fc).
  • the invention also relates to nucleotide fragments encoding said monoclonal antibodies.
  • the present invention also relates to nucleotide fragments encoding the monoclonal antibody light chain variable region and heavy chain variable region, preferably,
  • sequence of the nucleotide fragment encoding the heavy chain variable region VH of the 59C4 antibody is shown in SEQ ID NO: 5, and the sequence of the nucleotide fragment encoding the light chain variable region VL of the 59C4 antibody is shown in SEQ ID NO. NO:7 shown;
  • sequence of the nucleotide fragment encoding the heavy chain variable region VH of the T10E8 antibody is shown in SEQ ID NO: 1
  • sequence of the nucleotide fragment encoding the light chain variable region VL of the T10E8 antibody is shown in SEQ ID NO. Shown in NO:3.
  • the present invention also relates to an antibody pharmaceutical preparation composition containing the monoclonal antibody.
  • the antibody pharmaceutical preparation composition contains a therapeutically effective amount of the monoclonal antibody and necessary pharmaceutical excipients.
  • the antibody pharmaceutical preparation composition is an antibody gel preparation, which contains: carbomer, distilled water, PBS, glycerol, ethyl paraben, 2 mg of antibody, and necessary pH regulator.
  • the antibody gel preparation contains: 12 grams of carbomer 940#, 300 ml of distilled water, 40-50 ml of 10mM PBS, 40 grams of glycerol, 4 ml of 1% ethyl hydroxyphenyl ester, 2 mg of antibody, and the pH adjustment
  • the agent is 1N sodium hydroxide.
  • the present invention also relates to a method for preparing the antibody gel preparation, which method includes the following steps:
  • the sufficient swelling at low temperature is swelling at 4°C.
  • the method is:
  • the present invention also relates to the application of the monoclonal antibody, the antibody pharmaceutical preparation composition, and the antibody gel preparation in the preparation of drugs for treating skin and/or mucosal inflammation;
  • the skin and/or mucosal inflammation is oral mucosal inflammation or vaginal inflammation.
  • the treatment of inflammation is to relieve inflammatory reactions caused by the release of inflammatory factors, such as erythema, edema, burning, pain, itching, etc.; the inflammatory factors are TNFa and/or IL-5.
  • inflammatory factors such as erythema, edema, burning, pain, itching, etc.
  • the inflammatory factors are TNFa and/or IL-5.
  • TNFa has a significant pro-apoptotic effect on L929, and the IC50 value of T10E8 antibody that inhibits the effect of TNFa is 180ng/ml.
  • IMDM incomplete medium serum-free medium IMDM (Hyclone, SH30228.01)
  • HAT complete medium IMDM containing 20% fetal calf serum plus 2% HAT (sigma, H0262),
  • Mouse fibroblast L929 Jiangsu KGI Biotechnology Co., Ltd., KG087
  • TF-1 cells a cell line for laboratory use, gifted by Teacher Yin from the School of Pharmacy of Xuzhou Medical University.
  • Actinomycin D hy-17559, MedChemExpress.
  • Carbomer 940# presented by Jiangsu Xidian Pharmaceutical Excipients Co., Ltd.
  • Inflammatory factors for immunity were purchased from Acrobiosystems, and their antigen information is as follows:
  • Recombinant human tumor necrosis factor, rhTNFa is a human tumor necrosis factor expressed in HEK293 cells. It contains the amino acid sequence Val77-Leu233 fragment (Accession#P01375-1), which induces WEH1-13VAR cytotoxicity. The half effective dose is 0.007-0.012ng/ ml (activity data see Human TNF-alpha Protein,Tag Free,low endotoxin(active trimer)(MALS verified) -ACROBiosystems ).
  • Recombinant human interleukin 5, rhIL-5a, human interleukin 5 expressed using HEK293 cells contains the amino acid sequence Ile20-Ser134 fragment (Accession#P05113-1). Immunosolid-phase IL-5 receptor ELISA analysis results show that this cell The factor can bind to the IL-5 receptor (activity data see Human IL-5 Protein, His Tag-ACROBiosystems ).
  • Each inflammatory factor was mixed in equal proportions according to the following formula, and 6-week-old BABL/c mice were immunized.
  • step (3) The third immunization is performed two weeks after the second immunization.
  • the immunization method is the same as step (2).
  • mice Ten days after the third immunization, collect the tail vein blood of the mice, centrifuge and collect the serum to measure the titer (detected by ELISA method). Mice with a titer of more than 100,000 are ready for fusion. Three days before fusion, take 25ug of antigen ( Without adjuvant), the injection volume is 200ul/animal.
  • mice were killed by neck dissection, soaked in 75% alcohol and then aseptically separated from the spleens of the mice on a clean bench, and the adhering tissues outside the spleen were separated and removed.
  • TNFa tumor necrosis factor
  • TNFa mediates L929 cell apoptosis by binding to the TNF receptor on the cell membrane. Especially in the presence of actinomycin D, this pro-apoptotic effect is more obvious, so we used the MTT method to measure L929 cell apoptosis. The rate indirectly reflects the activity of TNFa.
  • the cell apoptosis rate is positively correlated with the amount of TNF ⁇ added.
  • TNFa and anti-TNFa antibodies are added simultaneously during the culture of L929 cells.
  • the anti-TNFa antibodies specifically bind to the receptor binding site on TNFa. Since the receptor binding site on TNFa is occupied by the anti-TNFa antibody, TNFa can no longer bind to its receptor, and TNFa interrupts the pro-apoptotic pathway mediated by its receptor, thereby achieving the anti-TNFa antibody's ability to inhibit TNFa from promoting apoptosis of L929 cells. effect.
  • the neutralizing effect of anti-TNFa antibodies on TNFa activity was measured by detecting the inhibitory rate of antibodies against TNF ⁇ -promoted cell apoptosis.
  • MTT cell proliferation and toxicity detection kit (Beyotime Biotechnology, Cat. No. C0009) to determine the number of apoptotic cells. Add 10 ⁇ l MTT reagent to each well and incubate in a cell culture incubator for 8 hours. There will be varying degrees of dark purple pine needles at the bottom of the 96-well plate. So Formazan appears. Add 100 ⁇ l/well of Formazan dissolution solution and wait for 6 hours in a cell culture incubator until Formazan is completely dissolved. Use a microplate reader to measure the absorbance at 595 nm.
  • TNFa had a significant pro-apoptotic effect on L929, and adding the antibody TNFa antibody T10E8 to the culture system inhibited the apoptosis of L929 cells.
  • the IC50 value of T10E8 antibody for inhibiting TNFa is 180ng/ml ( Figure 1)
  • interleukin 5 can promote the proliferation of human erythroid leukemia tumor cells TF-1 (premyeloid cells). After adding anti-IL-5 antibodies to the culture system, the antibodies that could significantly inhibit the proliferation of TF-1 showed better neutralizing activity than the control antibodies.
  • TF-1 cells in complete culture medium RPMI-1640 (PM150110) + 2ng/ml rhGM-CSF + 10% FBS (164210-500) + 1% P/S (PB180120)
  • RPMI-1640 PM150110
  • FBS 164210-500
  • P/S 1% P/S
  • the anti-IL5 antibody 59C4 prepared in Example 1 was diluted with basal medium (RPMI1640) in freshly passaged TF-1 cells (initial concentration is 50ug/ml, 3 times gradient dilution, 10 dilutions degree) and 0.5ng/ml interleukin-5, premix and react for 2 hours at room temperature, add it to a 96-well plate where TF-1 has been passaged for 6-8 hours, set duplicate wells, and set control antibody wells.
  • basal medium RPMI1640
  • IL-5 promotes TF-1 cell proliferation
  • half effective concentration IC50 of 59C4 antibody to inhibit IL-5 activity is 800ng/ml ( Figure 2).
  • the nucleotide sequence of the antibody heavy chain variable region VH encoding gene is shown in SEQ ID NO:1, and the encoded heavy chain variable region VH amino acid sequence is shown in SEQ ID NO:2;
  • the nucleotide sequence of the gene encoding the antibody light chain variable region VL is shown in SEQ ID NO:3, and the amino acid sequence of the encoded light chain variable region VL is shown in SEQ ID NO:4;
  • the coding gene sequence of the anti-IL-5 antibody 59C4 monoclonal antibody was determined. The results are:
  • the nucleotide sequence of the antibody heavy chain variable region VH encoding gene is shown in SEQ ID NO:5, and the encoded heavy chain variable region VH amino acid sequence is shown in SEQ ID NO:6;
  • the nucleotide sequence of the gene encoding the antibody light chain variable region VL is shown in SEQ ID NO:7, and the amino acid sequence of the encoded light chain variable region VL is shown in SEQ ID NO:8.
  • amino acid sequence of the kappa chain constant region of the human antibody IgG1 constant region is:
  • amino acid sequence of the heavy chain constant region is:
  • Antibody gel composition 12 grams of carbomer 940#, 300 ml of distilled water, 40-50 ml of 10mM PBS, 10-15 ml of 1N sodium hydroxide, 40 grams of medicinal glycerin, 4 ml of 1% ethyl hydroxyphenyl ester, and 2 mg of antibody.
  • the prepared antibody gel can be packaged aseptically.
  • vaginitis There were 10 patients with vaginitis, excluding senile vaginitis and juvenile vaginitis. Patients use antibody gel two to three times a day for 5 days. The patient's itching symptoms were significantly reduced 15 minutes after applying the antibody gel, and the effect lasted for more than 4 hours.

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Abstract

An antibody for treating skin and mucosal inflammation and a formulation thereof. The antibody is an antibody against an inflammatory factor, the inflammatory factor being human interleukin 5 (IL-5) or a human tumor necrosis factor (TNFa). The antibody is a monoclonal antibody against human interleukin 5 (IL-5), comprising a heavy chain variable region (VH) with an amino acid sequence set forth in SEQ ID NO: 6 and a light chain variable region (VL) with an amino acid sequence set forth in SEQ ID NO: 8. A monoclonal antibody against a human tumor necrosis factor (TNFa), comprising a heavy chain variable region (VH) with an amino acid sequence set forth in SEQ ID NO: 2 and a light chain variable region (VL) with an amino acid sequence set forth in SEQ ID NO: 4.

Description

用于治疗皮肤及粘膜炎症的抗体及其制剂Antibodies and preparations thereof for treating skin and mucous membrane inflammation 技术领域Technical field
本发明属于医药生物技术领域，具体的，涉及一种用于治疗皮肤及粘膜炎症的抗体及其制剂。The invention belongs to the field of medical biotechnology, and specifically relates to an antibody and its preparation for treating skin and mucosal inflammation.
背景技术Background technique
皮肤及粘膜是人体和外界环境之间的第一道屏障。攻击皮肤或粘膜的因素包括化学因素、微生物、冷热刺激、电离辐射以及机械性刺激等。这些外源性和内源性损伤因子可引起皮肤或粘膜各种各样的损伤性病变，因此皮肤或粘膜局部和全身则发生一系列复杂的反应，以局限和消灭损伤因子，清除和吸收坏死组织、细胞，并修复损伤，这就是机体的防御性反应——炎症(inflammation)。炎症反应受多种炎症因子如TNFa，IL-6，IL-5，IL-1，IL-8等调节，异常的炎症给机体皮肤粘膜带来一系列过敏反应，如红斑、水肿、灼烧、疼痛、瘙痒等。Skin and mucous membranes are the first barrier between the human body and the external environment. Factors that attack the skin or mucous membranes include chemical factors, microorganisms, hot and cold stimulation, ionizing radiation, and mechanical stimulation. These exogenous and endogenous damaging factors can cause various damaging lesions of the skin or mucous membranes. Therefore, a series of complex reactions occur locally and systemically in the skin or mucous membranes to localize and eliminate the damaging factors, and remove and absorb necrosis. Tissues and cells, and repairing damage, this is the body's defensive response - inflammation. The inflammatory response is regulated by a variety of inflammatory factors such as TNFa, IL-6, IL-5, IL-1, IL-8, etc. Abnormal inflammation brings a series of allergic reactions to the skin and mucous membranes of the body, such as erythema, edema, burning, Pain, itching, etc.
TNFa是炎症反应过程中出现最早、最重要的炎性介质，其生物学活性主要是通过细胞膜上的特异性受体传递信号而实现的。TNFa受体有两种，即TNF-RI和TNF-RII，该两种TNF受体均为糖蛋白，氨基酸顺序分析显示两类受体胞外区域的氨基酸序列高度相似，但是胞内区域则完全不同发挥着不同的作用。TNF-RI引起的作用非常广泛，它包括杀伤细胞活性、抗病毒活性、诱导Mn-SOD、ICAM-1及IL-6，IL-5等炎症因子的表达、促进成纤维细胞增殖以及细胞程序化死亡、激活NF-KB等多种生物学活性物质的信号传递。TNF-RII主要传递胸腺细胞和NK等淋巴细胞的增殖信号，通过促进TNFa结合TNF-RI而增加TNF-RI诱导的作用，同时也增加ICAM-1的表达。TNFa is the earliest and most important inflammatory mediator in the inflammatory response process. Its biological activity is mainly achieved by transmitting signals through specific receptors on the cell membrane. There are two types of TNFa receptors, namely TNF-RI and TNF-RII. Both TNF receptors are glycoproteins. Amino acid sequence analysis shows that the amino acid sequences of the extracellular regions of the two receptors are highly similar, but the intracellular regions are completely different. Differences play different roles. TNF-RI has a wide range of effects, including killer cell activity, antiviral activity, induction of the expression of inflammatory factors such as Mn-SOD, ICAM-1, IL-6, and IL-5, promotion of fibroblast proliferation, and cell programming. Signal transmission of various biologically active substances such as death and activation of NF-KB. TNF-RII mainly transmits the proliferation signals of lymphocytes such as thymocytes and NK. It increases the TNF-RI-induced effect by promoting TNFα to bind to TNF-RI, and also increases the expression of ICAM-1.
TNFa的两个受体在皮肤粘膜中表达完全不同：在正常的皮肤和非皮肤损伤组织，TNF-RI主要分布在表皮角质形成细胞、粘膜上皮细胞及真皮上层的树突状细胞中，而在皮肤损伤处，TNF-RI除了上述分布外，还分布在角化不全的角质层及真皮上层的血管周围。TNF-RII在正常皮肤中分布于小汗腺导管及真皮树突状细胞，而在皮肤损伤处，则分布于真皮上层血管及血管周围浸润的细胞中。有文献报道TNFa参与痛觉敏化：炎症组织部位和DRG神经元释放大量的MMP-9，可作用于DRG初级感觉神经元上的TNFa前体使之活化并与TNFR1受体结合，继而使TRPV1通道的表达和敏感性增加，参与炎症性痛的发生和发展。The two receptors of TNFa are expressed completely differently in skin and mucous membranes: in normal skin and non-skin damaged tissues, TNF-RI is mainly distributed in epidermal keratinocytes, mucosal epithelial cells and dendritic cells in the upper dermis, while in In skin lesions, in addition to the above distribution, TNF-RI is also distributed around the blood vessels in the parakeratotic stratum corneum and upper dermis. TNF-RII is distributed in eccrine ducts and dermal dendritic cells in normal skin, while in skin lesions, it is distributed in upper dermal blood vessels and perivascular infiltrating cells. There are reports in the literature that TNFa is involved in pain sensitization: inflammatory tissue sites and DRG neurons release a large amount of MMP-9, which can act on the TNFa precursor on the DRG primary sensory neurons to activate it and bind to the TNFR1 receptor, thereby causing TRPV1 channels The expression and sensitivity are increased and are involved in the occurrence and development of inflammatory pain.
IL-5是由嗜酸性粒细胞、NK细胞、TC2CD8+T细胞、肥大细胞、CD45+CD4+T细胞、γ-delta T细胞和IL-1β活化的内皮细胞等细胞分泌的一种炎症因子。IL-5在嗜酸性粒细胞上执行许多功能。这些包括Mac-1的下调，IgA和IgG受体的上调，刺激脂质介质(白三烯C4和PAF)的分泌和诱导颗粒释放。IL-5还能促进嗜酸性粒细胞的生长和分化，然而，IL-5的确切作用尚不清楚；它可以以一种辅助的方式起作用。最后，人们对IL-5和CC趋化因子eotaxin之间的相互作用非常感兴趣。研究表明，炎症诱导和局部产生的IL-5和eotaxin可能协同作用于骨髓。IL-5作用于嗜酸性粒细胞和肥大细胞，参与气道粘膜炎症。IL-5 is an inflammatory factor secreted by cells such as eosinophils, NK cells, TC2CD8+T cells, mast cells, CD45+CD4+T cells, γ-delta T cells, and IL-1β-activated endothelial cells. IL-5 performs many functions on eosinophils. These include downregulation of Mac-1, upregulation of IgA and IgG receptors, stimulation of secretion of lipid mediators (leukotriene C4 and PAF) and induction of granule release. IL-5 also promotes eosinophil growth and differentiation; however, the exact role of IL-5 is unclear; it may act in an auxiliary manner. Finally, there is considerable interest in the interaction between IL-5 and the CC chemokine eotaxin. Studies suggest that inflammation-induced and locally produced IL-5 and eotaxin may act synergistically in the bone marrow. IL-5 acts on eosinophils and mast cells and is involved in airway mucosal inflammation.
IL-6可由多种细胞合成，包括活化的T细胞和B细胞、单核－巨噬细胞、内皮细胞、上皮细胞以及成纤维细胞等。IL-6由αβ两条糖蛋白链组成异源二聚体。α链缺少胞内区，只能以低亲合性与IL-6结合，所形成的复合物迅即与高亲和性的β链结合，通过β链向细胞内传递信息。IL-6作用的靶细胞很多，包括巨噬细胞、肝细胞、静止的T细胞、活化的B细胞和浆细胞等；其作用：①促进T细胞表面IL-2R的表达，增强IL-1和TNF对TH细胞的致有丝***作用。②作为干细胞刺激因子，在感染或外伤引起的急性炎症反应中诱导急性期反应中诱导急性期反应蛋白的合成，其中以淀粉状蛋白A和C-反应蛋白增加尤为明显。③促进B细胞增殖、分化并产生抗体；多发性骨髓瘤的恶变B细胞既能产生IL-6，又能对IL-6发生应答，提示IL-6可能作为这些细胞的自分泌性生长因子。④IL-6还能有效地促进TNF和IL-1诱导的恶病质；促进糖皮质激素合成；刺激破骨细胞活性和角质细胞生长；还能促进骨髓造血的功能。IL-6不能刺激相应细胞分泌其它细胞因子，在生理浓度下对免疫细胞的自分泌作用亦比较弱， 提示其主要免疫学功能是加强其它细胞因子的效果。杨雁等人报道痒觉相关炎症因子受体。过敏性皮炎患者白细胞裂解物中的IL-2和IL-6均是致痒物质，如利用IL-2治疗癌症的同时会诱导机体产生瘙痒。神经细胞和施旺细胞(Schwann cells)均表达IL-6受体，而且皮肤斑贴试验阳性及结节性痒疹患者神经纤维上IL-6的免疫反应性比正常人高。另外研究表明IL-8也有致痒作用，但具体机制尚不清楚.IL-6 can be synthesized by a variety of cells, including activated T cells and B cells, monocytes-macrophages, endothelial cells, epithelial cells, and fibroblasts. IL-6 is a heterodimer composed of two glycoprotein chains αβ. The α chain lacks an intracellular region and can only bind to IL-6 with low affinity. The formed complex quickly binds to the high affinity β chain and transmits information into the cell through the β chain. IL-6 acts on many target cells, including macrophages, hepatocytes, resting T cells, activated B cells and plasma cells; its functions: ① Promote the expression of IL-2R on the surface of T cells, enhance IL-1 and Mitogenic effects of TNF on TH cells. ②As a stem cell stimulating factor, it induces the synthesis of acute phase response proteins in acute inflammatory reactions caused by infection or trauma, among which the increase of amyloid A and C-reactive protein is particularly obvious. ③ Promote B cell proliferation, differentiation and production of antibodies; malignant B cells of multiple myeloma can both produce IL-6 and respond to IL-6, suggesting that IL-6 may serve as an autocrine growth factor for these cells. ④IL-6 can also effectively promote cachexia induced by TNF and IL-1; promote glucocorticoid synthesis; stimulate osteoclast activity and keratinocyte growth; and can also promote bone marrow hematopoietic function. IL-6 cannot stimulate corresponding cells to secrete other cytokines, and its autocrine effect on immune cells is relatively weak at physiological concentrations. It is suggested that its main immunological function is to enhance the effects of other cytokines. Yang Yan et al. reported itch-related inflammatory factor receptors. IL-2 and IL-6 in the leukocyte lysates of patients with allergic dermatitis are both itching substances. For example, when IL-2 is used to treat cancer, it will induce itching in the body. Both nerve cells and Schwann cells express IL-6 receptors, and the immunoreactivity of IL-6 on nerve fibers in patients with positive skin patch tests and prurigo nodularis is higher than that in normal people. In addition, studies have shown that IL-8 also has an itching effect, but the specific mechanism is still unclear.
我们在开发一种抗菌多克隆抗体治疗感染性皮肤病的过程中发现，抗菌多克隆抗体中含有多种炎症因子如TNFa、IL-6、IL-5等的抗体。通过开发相应炎症因子中和抗体，实验证实了单独或联合局部应用炎症因子抗体凝胶可以快速缓解粘膜皮肤炎症，如口腔溃疡、***炎等。炎症性粘膜皮肤通透性增加，炎症因子抗体在局部发挥作用，且不会导致机体全身的免疫抑制，安全更好。In the process of developing an antibacterial polyclonal antibody to treat infectious skin diseases, we found that the antibacterial polyclonal antibody contains antibodies to a variety of inflammatory factors such as TNFa, IL-6, IL-5, etc. By developing corresponding inflammatory factor neutralizing antibodies, experiments have confirmed that topical application of inflammatory factor antibody gel alone or in combination can quickly relieve mucosal and skin inflammation, such as oral ulcers, vaginitis, etc. The permeability of the inflammatory mucosa and skin increases, and the inflammatory factor antibodies work locally without causing systemic immune suppression, which is safer and better.
基于此，提出本发明。Based on this, the present invention is proposed.
发明内容Contents of the invention
本发明首先涉及一组针对炎症因子的单克隆抗体，所述的炎症因子为人白细胞介素5(IL-5)或人肿瘤坏死因子(TNFa)，所述的单克隆抗体为：The present invention first relates to a group of monoclonal antibodies directed against inflammatory factors. The inflammatory factors are human interleukin 5 (IL-5) or human tumor necrosis factor (TNFa). The monoclonal antibodies are:
抗人白细胞介素5(IL-5)的单克隆抗体，其重链可变区VH氨基酸序列如SEQ ID NO:6所示；轻链可变区的(VL)氨基酸序列如SEQ ID NO:8所示；
The monoclonal antibody against human interleukin 5 (IL-5) has the VH amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 6; the (VL) amino acid sequence of the light chain variable region as shown in SEQ ID NO: As shown in 8;
抗人肿瘤坏死因子(TNFa)的单克隆抗体，其重链可变区VH氨基酸序列如SEQ ID NO:2所示；轻链可变区的(VL)氨基酸序列如SEQ ID NO:4所示。
Monoclonal antibody against human tumor necrosis factor (TNFa), the VH amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2; the (VL) amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4 .
优选的，Preferably,
所述的抗人白细胞介素5(IL-5)的单克隆抗体为59C4，所述59C4抗体的重链可变区VH氨基酸序列如SEQ ID NO:6所示；轻链可变区的(VL)氨基酸序列如SEQ ID NO:8所示。The anti-human interleukin 5 (IL-5) monoclonal antibody is 59C4, and the VH amino acid sequence of the heavy chain variable region of the 59C4 antibody is shown in SEQ ID NO: 6; the ( VL) amino acid sequence is shown in SEQ ID NO:8.
按照kabat规则对59C4抗体的重链可变区VH进行CDR划分如下：The CDR classification of the heavy chain variable region VH of the 59C4 antibody is as follows:
CDR-H1序列如SEQ ID NO.20所示，SEQ ID NO.20：DYYINThe CDR-H1 sequence is shown in SEQ ID NO.20, SEQ ID NO.20: DYYIN
CDR-H2序列如SEQ ID NO.21所示，SEQ ID NO.21：EIYPGSGNTYYNEKFKGThe CDR-H2 sequence is shown in SEQ ID NO.21, SEQ ID NO.21: EIYPGSGNTYYNEKFKG
CDR-H3序列如SEQ ID NO.22所示，SEQ ID NO.22：LTYYGSSYDYFDYThe CDR-H3 sequence is shown in SEQ ID NO.22, SEQ ID NO.22: LTYYGSSYDYFDY
按照kabat规则对59C4抗体的轻链可变区VL进行CDR划分如下：According to the kabat rules, the CDR division of the light chain variable region VL of the 59C4 antibody is as follows:
CDR-L1序列如SEQ ID NO.23所示，SEQ ID NO.23：ENIYSNThe CDR-L1 sequence is shown in SEQ ID NO.23, SEQ ID NO.23: ENIYSN
CDR-L2序列如SEQ ID NO.24所示，SEQ ID NO.24：AATThe CDR-L2 sequence is shown in SEQ ID NO.24, SEQ ID NO.24: AAT
CDR-L3序列如SEQ ID NO.25所示，SEQ ID NO.25：QHFWNTPYTThe CDR-L3 sequence is shown in SEQ ID NO.25, SEQ ID NO.25: QHFWNTPYT
所述的抗人肿瘤坏死因子(TNFa)的单克隆抗体为T10E8，所述T10E8抗体的重链可变区VH氨基酸序列如SEQ ID NO:2所示；轻链可变区的(VL)氨基酸序列如SEQ ID NO:4所示；The anti-human tumor necrosis factor (TNFa) monoclonal antibody is T10E8, and the VH amino acid sequence of the heavy chain variable region of the T10E8 antibody is shown in SEQ ID NO: 2; the (VL) amino acid of the light chain variable region The sequence is shown as SEQ ID NO:4;
按照kabat规则对T10E8抗体的重链可变区VH进行CDR划分如下： The CDR classification of the heavy chain variable region VH of the T10E8 antibody is as follows:
CDR-H1序列如SEQ ID NO.14所示，SEQ ID NO.14：SYTLSThe CDR-H1 sequence is shown in SEQ ID NO.14, SEQ ID NO.14: SYTLS
CDR-H2序列如SEQ ID NO.15所示，SEQ ID NO.15：YISSGGSNTYYPDTVKGThe CDR-H2 sequence is shown in SEQ ID NO.15, SEQ ID NO.15: YISSGGSNTYYPDTVKG
CDR-H3序列如SEQ ID NO.16所示，SEQ ID NO.16：QGYYRYVLYAMDYThe CDR-H3 sequence is shown in SEQ ID NO.16, SEQ ID NO.16: QGYYRYVLYAMDY
按照kabat规则对T10E8抗体的轻链可变区VL进行CDR划分如下：The CDR division of the light chain variable region VL of the T10E8 antibody is as follows:
CDR-L1序列如SEQ ID NO.17所示，SEQ ID NO.17：SASSSVSHMHThe CDR-L1 sequence is shown in SEQ ID NO.17, SEQ ID NO.17: SASSSVSHMH
CDR-L2序列如SEQ ID NO.18所示，SEQ ID NO.18：DTSKLASThe CDR-L2 sequence is shown in SEQ ID NO.18, SEQ ID NO.18: DTSKLAS
CDR-L3序列如SEQ ID NO.19所示，SEQ ID NO.19：QQWSSNPIT。The CDR-L3 sequence is shown in SEQ ID NO.19, SEQ ID NO.19: QQWSSNPIT.
进一步的，所述的单克隆抗体为人源化改造的单克隆抗体，其包含所述59C4抗体的重链及轻链可变区，或所述T10E8抗体的重链及轻链可变区；Further, the monoclonal antibody is a humanized modified monoclonal antibody, which includes the heavy chain and light chain variable regions of the 59C4 antibody, or the heavy chain and light chain variable regions of the T10E8 antibody;
优选的，Preferably,
所述的59C4人源化抗体为：包含59C4抗体的重链及轻链可变区和人抗体IgG1恒定区(Fc)的人源化抗体；The 59C4 humanized antibody is: a humanized antibody containing the heavy chain and light chain variable regions of the 59C4 antibody and the human antibody IgG1 constant region (Fc);
所述的T10E8人源化抗体为：包含T10E8抗体的重链及轻链可变区和人抗体IgG1恒定区(Fc)的人源化抗体。The T10E8 humanized antibody is a humanized antibody that includes the heavy chain and light chain variable regions of the T10E8 antibody and the human antibody IgG1 constant region (Fc).
本发明还涉及编码所述的单克隆抗体的核苷酸片段。The invention also relates to nucleotide fragments encoding said monoclonal antibodies.
本发明还涉及编码所述的单克隆抗体轻链可变区和重链可变区的核苷酸片段，优选的，The present invention also relates to nucleotide fragments encoding the monoclonal antibody light chain variable region and heavy chain variable region, preferably,
编码所述59C4抗体的重链可变区VH的核苷酸片段的序列如SEQ ID NO:5所示，编码所述59C4抗体的轻链可变区VL的核苷酸片段的序列如SEQ ID NO:7所示；
The sequence of the nucleotide fragment encoding the heavy chain variable region VH of the 59C4 antibody is shown in SEQ ID NO: 5, and the sequence of the nucleotide fragment encoding the light chain variable region VL of the 59C4 antibody is shown in SEQ ID NO. NO:7 shown;
编码所述T10E8抗体的重链可变区VH的核苷酸片段的序列如SEQ ID NO:1所示，编码所述T10E8抗体的轻链可变区VL的核苷酸片段的序列如SEQ ID NO:3所示。

The sequence of the nucleotide fragment encoding the heavy chain variable region VH of the T10E8 antibody is shown in SEQ ID NO: 1, and the sequence of the nucleotide fragment encoding the light chain variable region VL of the T10E8 antibody is shown in SEQ ID NO. Shown in NO:3.

本发明还涉及包含所述单克隆抗体的抗体药物制剂组合物，所述的抗体药物制剂组合物包含治疗有效量的所述单克隆抗体以及必要的药用辅料。The present invention also relates to an antibody pharmaceutical preparation composition containing the monoclonal antibody. The antibody pharmaceutical preparation composition contains a therapeutically effective amount of the monoclonal antibody and necessary pharmaceutical excipients.
优选的，所述的抗体药物制剂组合物为抗体凝胶制剂，其中包含：卡波姆、蒸馏水、PBS、甘油、羟苯乙酯，抗体2mg，以及必要的pH调节剂。Preferably, the antibody pharmaceutical preparation composition is an antibody gel preparation, which contains: carbomer, distilled water, PBS, glycerol, ethyl paraben, 2 mg of antibody, and necessary pH regulator.
优选的，所述的抗体凝胶制剂中包含：卡波姆940#12克、蒸馏水300ml、10mM PBS 40～50ml、甘油40克、1％羟苯乙酯4ml，抗体2mg，所述的pH调节剂为1N氢氧化钠。Preferably, the antibody gel preparation contains: 12 grams of carbomer 940#, 300 ml of distilled water, 40-50 ml of 10mM PBS, 40 grams of glycerol, 4 ml of 1% ethyl hydroxyphenyl ester, 2 mg of antibody, and the pH adjustment The agent is 1N sodium hydroxide.
本发明还涉及所述的抗体凝胶制剂的制备方法，所述方法包括如下步骤：The present invention also relates to a method for preparing the antibody gel preparation, which method includes the following steps:
(1)将卡波姆加入纯化水中搅拌充分；(1) Add carbomer to purified water and stir thoroughly;
(2)低温充分溶胀卡波姆溶液使其溶解完全；(2) Fully swell the carbomer solution at low temperature to completely dissolve it;
(3)调节溶液pH至7.0-7.4，优选为7.2；(3) Adjust the pH of the solution to 7.0-7.4, preferably 7.2;
(4)加入甘油、1％的羟苯乙酯，搅拌均匀；(4) Add glycerol and 1% ethyl hydroxyphenyl ester and stir evenly;
(5)抗体用蒸馏水溶解后，再用PBS缓冲溶液稀释后加入到上述步骤(4)制得的溶剂基质中，搅拌均匀，低温充分溶胀即得所述抗体凝胶制剂；(5) After the antibody is dissolved in distilled water, dilute it with PBS buffer solution and then add it to the solvent matrix prepared in the above step (4), stir evenly, and fully swell at low temperature to obtain the antibody gel preparation;
(6)无菌分装。(6) Aseptic packaging.
优选的，所述的低温充分溶胀为4℃下溶胀。Preferably, the sufficient swelling at low temperature is swelling at 4°C.
更优选的，所述的方法为：More preferably, the method is:
(1)称量卡波姆12克，加入到300ml纯化水中，室温搅拌30min至没有明显的白色大块状物为止；(1) Weigh 12 grams of carbomer, add it to 300 ml of purified water, and stir at room temperature for 30 minutes until there are no obvious white lumps;
(2)低温4℃溶胀过夜至卡波姆溶解完全，看不到白色块状物及白色粉末；(2) Swell at low temperature 4°C overnight until the carbomer is completely dissolved and no white lumps or white powder can be seen;
(3)用10-15mlNaOH调节pH至7.2；(3) Adjust pH to 7.2 with 10-15ml NaOH;
(4)加入40克甘油、1％的羟苯乙酯4ml，搅拌均匀；(4) Add 40 grams of glycerin and 4 ml of 1% ethyl hydroxyphenyl ester, and stir evenly;
(5)抗体冻干粉用1ml蒸馏水溶解后，再用10mM磷酸盐缓冲溶液稀释至体积40～50ml，将抗体的稀释液加入到上述步骤(4)制得的溶剂基质中，搅拌均匀，4℃溶胀2小时即得所述抗体凝胶制剂；优选的，磷酸盐缓冲溶液的体积与步骤(3)使用的氢氧化钠溶液的体积合计55ml；(5) After the antibody freeze-dried powder is dissolved in 1 ml of distilled water, dilute it with 10 mM phosphate buffer solution to a volume of 40-50 ml. Add the antibody dilution to the solvent matrix prepared in the above step (4), stir evenly, 4 The antibody gel preparation is obtained by swelling at ℃ for 2 hours; preferably, the volume of the phosphate buffer solution and the volume of the sodium hydroxide solution used in step (3) total 55 ml;
(6)无菌分装。(6) Aseptic packaging.
本发明还涉及所述的单克隆抗体、所述的抗体药物制剂组合物、抗体凝胶制剂在制备治疗皮肤和/或粘膜炎症的药物中的应用；The present invention also relates to the application of the monoclonal antibody, the antibody pharmaceutical preparation composition, and the antibody gel preparation in the preparation of drugs for treating skin and/or mucosal inflammation;
优选的，所述的皮肤和/或粘膜炎症为口腔黏膜炎症或***炎症。Preferably, the skin and/or mucosal inflammation is oral mucosal inflammation or vaginal inflammation.
优选的，所述的治疗炎症为缓解由炎症因子释放导致的炎症反应，如红斑、水肿、灼烧、疼痛、瘙痒等；所述的炎症因子为TNFa和/或IL-5。Preferably, the treatment of inflammation is to relieve inflammatory reactions caused by the release of inflammatory factors, such as erythema, edema, burning, pain, itching, etc.; the inflammatory factors are TNFa and/or IL-5.
附图说明Description of the drawings
图1、TNFa对L929有明显的促凋亡作用，而T10E8抗体抑制TNFa作用的IC50值为180ng/ml。Figure 1. TNFa has a significant pro-apoptotic effect on L929, and the IC50 value of T10E8 antibody that inhibits the effect of TNFa is 180ng/ml.
图2、0.5ng/ml IL-5促TF-1细胞增殖，而59C4抗体抑制IL-5活性的半数有效浓度IC50为800ng/ml。Figure 2. 0.5ng/ml IL-5 promotes TF-1 cell proliferation, while the half effective concentration IC50 of 59C4 antibody inhibits IL-5 activity is 800ng/ml.
图3、重组抗体(人源化)细胞培养上清proteinA纯化后产物电泳结果。Figure 3. Product electrophoresis results after purification of proteinA from cell culture supernatant of recombinant antibody (humanized).
具体实施方式Detailed ways
材料及试剂Materials and reagents
BABL/c小鼠：8周龄北京维通利华实验动物技术有限公司BABL/c mice: 8 weeks old Beijing Weitonglihua Experimental Animal Technology Co., Ltd.
IMDM不完全培养基：不含有血清的培养基IMDM(Hyclone，SH30228.01) IMDM incomplete medium: serum-free medium IMDM (Hyclone, SH30228.01)
HAT完全培养基：在含有20％胎牛血清的IMDM中添加2％的HAT(sigma，H0262)，HAT complete medium: IMDM containing 20% fetal calf serum plus 2% HAT (sigma, H0262),
小鼠成纤维细胞L929：江苏凯基生物技术有限公司，KG087Mouse fibroblast L929: Jiangsu KGI Biotechnology Co., Ltd., KG087
TF-1细胞：实验室自用细胞系，源自徐州医科大学药学院印老师馈赠。TF-1 cells: a cell line for laboratory use, gifted by Teacher Yin from the School of Pharmacy of Xuzhou Medical University.
放线菌素D：hy-17559，MedChemExpress.Actinomycin D: hy-17559, MedChemExpress.
卡波姆940#：江苏西典药用辅料有限公司赠送Carbomer 940#: presented by Jiangsu Xidian Pharmaceutical Excipients Co., Ltd.
实施例1炎症因子免疫原的准备Example 1 Preparation of inflammatory factor immunogen
免疫用炎症因子购自Acrobiosystems公司，其抗原信息如下：Inflammatory factors for immunity were purchased from Acrobiosystems, and their antigen information is as follows:
重组人肿瘤坏死因子抗原氨基酸序列：
Recombinant human tumor necrosis factor antigen amino acid sequence:
重组人白介素5抗原氨基酸序列：
Recombinant human interleukin 5 antigen amino acid sequence:
重组人肿瘤坏死因子，rhTNFa，利用HEK293细胞表达的人肿瘤坏死因子，含有氨基酸序列Val77-Leu233片段(Accession#P01375-1)，其诱导WEH1-13VAR细胞毒性，半数有效剂量在0.007-0.012ng/ml(活性数据参见 Human TNF-alpha Protein,Tag Free,low endotoxin(active trimer)(MALS verified) -ACROBiosystems)。Recombinant human tumor necrosis factor, rhTNFa, is a human tumor necrosis factor expressed in HEK293 cells. It contains the amino acid sequence Val77-Leu233 fragment (Accession#P01375-1), which induces WEH1-13VAR cytotoxicity. The half effective dose is 0.007-0.012ng/ ml (activity data see Human TNF-alpha Protein,Tag Free,low endotoxin(active trimer)(MALS verified) -ACROBiosystems ).
重组人白介素5，rhIL-5a，利用HEK293细胞表达的人白细胞介素5，含有氨基酸序列Ile20-Ser134片段(Accession#P05113-1)，免疫固相IL-5受体ELISA分析结果显示，该细胞因子可以与IL-5受体结合(活性数据参见Human IL-5 Protein,His Tag-ACROBiosystems)。Recombinant human interleukin 5, rhIL-5a, human interleukin 5 expressed using HEK293 cells, contains the amino acid sequence Ile20-Ser134 fragment (Accession#P05113-1). Immunosolid-phase IL-5 receptor ELISA analysis results show that this cell The factor can bind to the IL-5 receptor (activity data see Human IL-5 Protein, His Tag-ACROBiosystems ).
实施例2抗炎症因子单克隆抗体制备Example 2 Preparation of anti-inflammatory factor monoclonal antibodies
2.1小鼠免疫2.1 Mouse immunization
每种炎症因子按照下述配方等比例混合后，免疫6周龄BABL/c小鼠。Each inflammatory factor was mixed in equal proportions according to the following formula, and 6-week-old BABL/c mice were immunized.
(1)初次免疫：25ug抗原加等体积弗氏佐剂，佐剂完全乳化后，皮下及腹腔注射小鼠，注射体积200ul/只。(1) Initial immunization: 25ug of antigen plus an equal volume of Freund's adjuvant. After the adjuvant is completely emulsified, the mice are injected subcutaneously and intraperitoneally, with an injection volume of 200ul/mouse.
(2)初次免疫三周后第二次免疫：25ug抗原加等体积弗氏佐剂不完全佐剂，佐剂完全乳化后，腹腔注射小鼠，注射体积200ul/只。(2) The second immunization three weeks after the initial immunization: 25ug of antigen plus an equal volume of incomplete Freund's adjuvant. After the adjuvant is completely emulsified, the mouse is injected intraperitoneally with an injection volume of 200ul/mouse.
(3)二次免疫两周后第三次免疫，免疫方法同步骤(2)。(3) The third immunization is performed two weeks after the second immunization. The immunization method is the same as step (2).
(4)第三次免疫10天后，采小鼠尾静脉血，离心取血清测效价(用ELISA方法检测)效价达十万以上的小鼠准备融合，在融合前三天取抗原25ug(不加佐剂)加强免疫，注射体积200ul/只。(4) Ten days after the third immunization, collect the tail vein blood of the mice, centrifuge and collect the serum to measure the titer (detected by ELISA method). Mice with a titer of more than 100,000 are ready for fusion. Three days before fusion, take 25ug of antigen ( Without adjuvant), the injection volume is 200ul/animal.
2.2细胞融合2.2 Cell fusion
(1)将培养至对数生长期的小鼠骨髓瘤F0细胞上清倒掉，加入IMDM不完全培养基吹打细胞。(1) Discard the supernatant of mouse myeloma F0 cells cultured to the logarithmic growth phase, add IMDM incomplete medium and pipet the cells.
(2)离心收集细胞，用40毫升IMDM将细胞悬起计数活细胞数量。(2) Collect cells by centrifugation, suspend the cells with 40 ml IMDM and count the number of viable cells.
(3)摘小鼠的眼球取血。(3) Remove the eyeballs of mice to collect blood.
(4)将小鼠断颈处死，放入75％酒精浸泡后于洁净台内无菌分离小鼠脾脏，分离去除脾脏外粘连的组织。(4) The mice were killed by neck dissection, soaked in 75% alcohol and then aseptically separated from the spleens of the mice on a clean bench, and the adhering tissues outside the spleen were separated and removed.
(5)磨砂玻片研磨小鼠脾脏，IMDM悬浮脾细胞，经300目筛网于离心管内收集细胞悬液，然后离心，1200pm，5min。 (5) Grind the mouse spleen with frosted glass, suspend the spleen cells in IMDM, collect the cell suspension in a centrifuge tube through a 300-mesh mesh, and then centrifuge at 1200pm for 5 minutes.
(6)取适量F0细胞和离心得到的脾细胞汇合悬浮，离心收集细胞。(6) Take an appropriate amount of F0 cells and the spleen cells obtained by centrifugation, merge and suspend them, and centrifuge to collect the cells.
(7)弃上清后(除去液体残留)，使沉淀细胞松散均匀，置37℃水浴中预热。(7) After discarding the supernatant (removing liquid residue), make the precipitated cells loose and even, and preheat them in a 37°C water bath.
(8)用移液枪吸取1ml 50％PEG1500缓慢滴加入到上述细胞团中,且在1分钟30秒加完，边加边轻轻搅拌，滴完后静置1min30s。(8) Use a pipette to slowly add 1ml of 50% PEG1500 to the above cell mass. Add it to the cell mass in 1 minute and 30 seconds. Stir gently while adding. After the addition, let it stand for 1 minute and 30 seconds.
(9)用10ml吸管逐滴先慢后快加40ml预热至37℃的IMDM培养基，然后离心收集细胞，用弯头滴管取含HAT的完全培养基(完全培养基为含有20％胎牛血清的IMDM，实施例2涉及，下同)加入装有细胞的离心管中，轻轻用弯头滴管将细胞悬起，接种96孔板，200ul/孔，然后于培养箱中培养。(9) Use a 10 ml pipette to add 40 ml of IMDM medium preheated to 37°C drop by drop first slowly and then quickly, then centrifuge to collect the cells, and use an elbow dropper to take out the complete medium containing HAT (complete medium contains 20% fetal IMDM of bovine serum (referred to in Example 2, the same below) was added to the centrifuge tube containing cells, gently suspended the cells with an elbow dropper, inoculated into a 96-well plate, 200ul/well, and then cultured in an incubator.
(10)第三天半定量换含HAT的完全培养，第六天换成含有HT的完全培养基，第十天后根据细胞生长状态，吸取细胞上清进行ELISA检测，阳性克隆细胞用有限稀释法实施3-5轮亚克隆培养。(10) On the third day, semi-quantitatively replace the complete culture medium containing HAT, and on the sixth day, replace it with the complete culture medium containing HT. After the tenth day, according to the cell growth status, aspirate the cell supernatant for ELISA detection, and use the limiting dilution method for positive clone cells. Perform 3-5 rounds of subcloning culture.
(11)混合抗原阳性的单克隆，再对单一抗原ELISA反应测定，确定其特异抗原反应活性。(11) Mix the antigen-positive monoclones, and then measure the single antigen ELISA reaction to determine its specific antigen reactivity.
实施例3抗人肿瘤坏死因子(TNFa)抗体中和活性试验Example 3 Anti-human tumor necrosis factor (TNFa) antibody neutralizing activity test
在肿瘤坏死因子TNFa生物学活性测定方法(MATTHEWS,N.,and NEALE,M.L.In:Lymphokines and Interferons,a Practical Approach,pp.221–225.IRLPress，1987)基础上设计如下方法：The following method was designed based on the biological activity determination method of tumor necrosis factor TNFa (MATTHEWS, N., and NEALE, M.L. In: Lymphokines and Interferons, a Practical Approach, pp. 221–225. IRL Press, 1987):
TNFa通过与细胞膜上的TNF受体结合，介导L929细胞凋亡，尤其是在放线菌素D存在的条件，这种促凋亡作用更加明显，所以我们用MTT法通过测定L929细胞凋亡率来间接反应TNFa的活性。TNFa mediates L929 cell apoptosis by binding to the TNF receptor on the cell membrane. Especially in the presence of actinomycin D, this pro-apoptotic effect is more obvious, so we used the MTT method to measure L929 cell apoptosis. The rate indirectly reflects the activity of TNFa.
在培养的L929细胞中添加TNFa，细胞的凋亡率与TNFa的加入量成正相关。When TNFα is added to cultured L929 cells, the cell apoptosis rate is positively correlated with the amount of TNFα added.
中和试验是在L929细胞的培养过程中同时加入TNFa和抗TNFa抗体，抗TNFa抗体特异性的结合在TNFa上的受体结合位点。由于TNFa上受体结合位点被抗TNFa抗体占据，TNFa不能再与其受体的结合，TNFa通过其受体介导的促凋亡通路中断，从而实现抗TNFa抗体抑制TNFa促L929细胞凋亡的作用。In the neutralization test, TNFa and anti-TNFa antibodies are added simultaneously during the culture of L929 cells. The anti-TNFa antibodies specifically bind to the receptor binding site on TNFa. Since the receptor binding site on TNFa is occupied by the anti-TNFa antibody, TNFa can no longer bind to its receptor, and TNFa interrupts the pro-apoptotic pathway mediated by its receptor, thereby achieving the anti-TNFa antibody's ability to inhibit TNFa from promoting apoptosis of L929 cells. effect.
通过检测抗体对TNFa促细胞凋亡的抑制率，来衡量抗TNFa抗体对TNFa活性的中和作用。The neutralizing effect of anti-TNFa antibodies on TNFa activity was measured by detecting the inhibitory rate of antibodies against TNFα-promoted cell apoptosis.
(1)L929小鼠成纤维细胞用完全培养基(含10％FBS、1mM谷氨酰胺、100U的青-链霉素的高糖DMEM(Hyclone,Cat No.SH30022.01B))，于饱和湿度，37℃，5％CO2条件下培养。(1) Complete culture medium for L929 mouse fibroblasts (high-glucose DMEM (Hyclone, Cat No. SH30022.01B) containing 10% FBS, 1mM glutamine, and 100U penicillin-streptomycin) at saturated humidity , cultured at 37°C, 5% CO2 .
(2)新鲜传代L929细胞贴壁后，向96孔板中加入终浓度为1μg/ml放线菌素D、0.12ng/ml TNFa(此浓度不低于10倍ED50)，同时加入用完全培养基梯度稀释的由实施例1制备得到的抗人TNFa抗体T10E8。(2) After freshly passaged L929 cells adhere to the wall, add actinomycin D at a final concentration of 1μg/ml and 0.12ng/ml TNFa (this concentration is not less than 10 times ED50) into the 96-well plate, and at the same time add complete culture medium The anti-human TNFa antibody T10E8 prepared in Example 1 was diluted serially.
(3)培养20小时后。用MTT细胞增殖和毒性检测试剂盒(碧云天生物，货号C0009)测定凋亡细胞数，于每孔加入10μl MTT试剂，细胞培养箱内孵育8小时，96孔板底部有不同程度的深紫色松针样Formazan出现。加入Formazan溶解液100μl/well，于细胞培养箱内6个小时待Formazan完全溶解后，用酶标仪测定595nm的吸光度。(3) After 20 hours of culture. Use MTT cell proliferation and toxicity detection kit (Beyotime Biotechnology, Cat. No. C0009) to determine the number of apoptotic cells. Add 10 μl MTT reagent to each well and incubate in a cell culture incubator for 8 hours. There will be varying degrees of dark purple pine needles at the bottom of the 96-well plate. So Formazan appears. Add 100 μl/well of Formazan dissolution solution and wait for 6 hours in a cell culture incubator until Formazan is completely dissolved. Use a microplate reader to measure the absorbance at 595 nm.
(4)绘制抗人TNFa抗体T10E8剂量对OD值的曲线，计算抗体ND50值。(4) Draw the curve of anti-human TNFa antibody T10E8 dose versus OD value, and calculate the antibody ND50 value.
结果显示，0.12ng/ml的TNFa对L929有明显的促凋亡作用，且在培养体系中加入抗体TNFa抗体T10E8，L929细胞的凋亡受到抑制。T10E8抗体抑制TNFa作用的IC50值为180ng/ml(图1)The results showed that 0.12ng/ml TNFa had a significant pro-apoptotic effect on L929, and adding the antibody TNFa antibody T10E8 to the culture system inhibited the apoptosis of L929 cells. The IC50 value of T10E8 antibody for inhibiting TNFa is 180ng/ml (Figure 1)
实施例4抗人白细胞介素5(IL-5)抗体中和活性试验Example 4 Anti-human interleukin 5 (IL-5) antibody neutralizing activity test
在GM-CSF存在条件下，白细胞介素5可以促进人红细胞白血病瘤细胞TF-1(前髓样细胞)增殖。在培养体系中加入抗IL-5抗体后，与对照组抗体相比，能够显著抑制TF-1增殖的抗体则表现出更好的中和活性。In the presence of GM-CSF, interleukin 5 can promote the proliferation of human erythroid leukemia tumor cells TF-1 (premyeloid cells). After adding anti-IL-5 antibodies to the culture system, the antibodies that could significantly inhibit the proliferation of TF-1 showed better neutralizing activity than the control antibodies.
(1)TF-1细胞于完全培养基(RPMI-1640(PM150110)+2ng/ml rhGM-CSF+10％FBS(164210-500)+1％P/S(PB180120))中，37℃，空气95％，CO25％条件下生长。(1) TF-1 cells in complete culture medium (RPMI-1640 (PM150110) + 2ng/ml rhGM-CSF + 10% FBS (164210-500) + 1% P/S (PB180120)), 37°C, air Grow under 95% CO 2 5% conditions.
(2)在新鲜传代的TF-1细胞中用基础培养基(RPMI1640)倍比稀释的实施例1制备得到的抗IL5抗体59C4(起始浓度为50ug/ml，3倍梯度稀释，10个稀释度)与0.5ng/ml的白介素5在室温条件下预混反应2小时，加入到TF-1传代6-8个小时的96孔板中，设置重复孔，设置对照抗体孔。 (2) The anti-IL5 antibody 59C4 prepared in Example 1 was diluted with basal medium (RPMI1640) in freshly passaged TF-1 cells (initial concentration is 50ug/ml, 3 times gradient dilution, 10 dilutions degree) and 0.5ng/ml interleukin-5, premix and react for 2 hours at room temperature, add it to a 96-well plate where TF-1 has been passaged for 6-8 hours, set duplicate wells, and set control antibody wells.
(3)然后于37℃培养箱中培养24小时，加入MTT试剂20ul/孔，放置5h后，加入酸性裂解液100ul/孔，吹打使其完全溶解，放入酶标仪，测定595nm的吸光度。(3) Then incubate in a 37°C incubator for 24 hours, add 20ul/well of MTT reagent, leave it for 5 hours, add 100ul/well of acidic lysis solution, pipet to dissolve completely, put it into a microplate reader, and measure the absorbance at 595nm.
(4)绘制59C4抗体剂量对OD值的曲线，计算抗体IC50值(4) Draw a curve of 59C4 antibody dose versus OD value, and calculate the antibody IC50 value
结果显示：含有GM-CSF的TF-1细胞培养***中，只有添加了IL-5后细胞才能快速增殖。实验结果证实，59C4抗体对TF-1增殖抑制作用是特异性中和了IL-5活性，细胞增殖受到了抑制。The results showed that in the TF-1 cell culture system containing GM-CSF, cells could proliferate rapidly only after adding IL-5. Experimental results confirmed that the 59C4 antibody's inhibitory effect on TF-1 proliferation specifically neutralized IL-5 activity and inhibited cell proliferation.
0.5ng/ml IL-5促TF-1细胞增殖，59C4抗体抑制IL-5活性的半数有效浓度IC50为800ng/ml(图2)。0.5ng/ml IL-5 promotes TF-1 cell proliferation, and the half effective concentration IC50 of 59C4 antibody to inhibit IL-5 activity is 800ng/ml (Figure 2).
实施例5抗炎症因子抗体可变区序列测定Example 5 Determination of variable region sequence of anti-inflammatory factor antibodies
对抗TNFa抗体T10E8单克隆抗体的编码基因测序，其结果为：Sequencing of the coding gene for the anti-TNFa antibody T10E8 monoclonal antibody, the results are:
抗体重链可变区VH编码基因的核苷酸序列如SEQ ID NO:1所示，其编码的重链可变区VH氨基酸序列如SEQ ID NO:2所示；The nucleotide sequence of the antibody heavy chain variable region VH encoding gene is shown in SEQ ID NO:1, and the encoded heavy chain variable region VH amino acid sequence is shown in SEQ ID NO:2;
抗体轻链可变区VL编码基因的核苷酸序列如SEQ ID NO:3所示，其编码的轻链可变区VL氨基酸序列如SEQ ID NO:4所示；The nucleotide sequence of the gene encoding the antibody light chain variable region VL is shown in SEQ ID NO:3, and the amino acid sequence of the encoded light chain variable region VL is shown in SEQ ID NO:4;
对抗IL-5抗体59C4单克隆抗体的编码基因序列测定，其结果为：The coding gene sequence of the anti-IL-5 antibody 59C4 monoclonal antibody was determined. The results are:
抗体重链可变区VH编码基因的核苷酸序列如SEQ ID NO:5所示，其编码的重链可变区VH氨基酸序列如SEQ ID NO:6所示；The nucleotide sequence of the antibody heavy chain variable region VH encoding gene is shown in SEQ ID NO:5, and the encoded heavy chain variable region VH amino acid sequence is shown in SEQ ID NO:6;
抗体轻链可变区VL编码基因的核苷酸序列如SEQ ID NO:7所示，其编码的轻链可变区VL氨基酸序列如SEQ ID NO:8所示。The nucleotide sequence of the gene encoding the antibody light chain variable region VL is shown in SEQ ID NO:7, and the amino acid sequence of the encoded light chain variable region VL is shown in SEQ ID NO:8.
实施例6抗体人源化改造及重组抗体制备Example 6 Antibody humanization and preparation of recombinant antibodies
将抗体T10E8、59C4可变区基因连接到人抗体IgG1恒定区片段上，具体步骤为：Connect the antibody T10E8 and 59C4 variable region genes to the human antibody IgG1 constant region fragment. The specific steps are:
(1)构建重组抗体表达质粒，质粒瞬转CHO细胞48小时后，根据细胞上清中抗体表达情况，转染细胞传代。(1) Construct a recombinant antibody expression plasmid. After the plasmid is transiently transfected into CHO cells for 48 hours, the transfected cells are passaged according to the expression of antibodies in the cell supernatant.
(2)MTX加压筛选，逐步提高MTX浓度，筛选稳定高产细胞株。MTX初始浓度为10nM。(2) MTX pressure screening, gradually increase the MTX concentration, and select stable and high-producing cell lines. The initial concentration of MTX was 10 nM.
(3)细胞传代5代，没有转染目的质粒的细胞，在MTX筛选下，生长缓慢，随着传代次数的增加，逐渐消亡。(3) Cells were passaged for 5 generations. Cells that were not transfected with the target plasmid grew slowly under MTX selection and gradually died as the number of passages increased.
(4)逐步将MTX浓度30nm、100nm、300nm，每个浓度下传代5代，根据上清抗体表达情况，选择高表达的克隆继续扩大培养。(4) Gradually increase the MTX concentration to 30nm, 100nm, and 300nm, and passage it for 5 generations at each concentration. Based on the expression of supernatant antibodies, select clones with high expression and continue to expand the culture.
(5)稳定表达重组抗体细胞无血清培养，用proteinA纯化重组抗体，BCA定量，SDS-PAGE电泳鉴定纯度(见图3)，细胞生物学实验测试抗体中和活性(方法同实施例3、4，人源化抗体T10E8、59C4的IC50分别是200ng/ml、750ng/ml)。(5) Serum-free culture of cells stably expressing the recombinant antibody, purification of the recombinant antibody with proteinA, quantification of BCA, identification of purity by SDS-PAGE electrophoresis (see Figure 3), and cell biology experiments to test the neutralizing activity of the antibody (the method is the same as in Examples 3 and 4 , the IC50 of humanized antibodies T10E8 and 59C4 are 200ng/ml and 750ng/ml respectively).
其中，人抗体IgG1恒定区的kappa链恒定区氨基酸序列为：
Among them, the amino acid sequence of the kappa chain constant region of the human antibody IgG1 constant region is:
重链恒定区的氨基酸序列为：
The amino acid sequence of the heavy chain constant region is:
实施例7抗体凝胶剂制备工艺 Example 7 Preparation process of antibody gel
抗体凝胶组成：卡波姆940#12克、蒸馏水300ml、10mM PBS 40～50ml、1N氢氧化钠10-15ml、药用甘油40克、1％羟苯乙酯4ml，抗体2mg。Antibody gel composition: 12 grams of carbomer 940#, 300 ml of distilled water, 40-50 ml of 10mM PBS, 10-15 ml of 1N sodium hydroxide, 40 grams of medicinal glycerin, 4 ml of 1% ethyl hydroxyphenyl ester, and 2 mg of antibody.
制备工艺：Preparation Process:
(1)称量卡波姆12克，加入到300ml纯化水/蒸馏水中，边加边搅拌，室温搅拌半小时，没有明显的未经溶解的白色大块状物为止。(1) Weigh 12 grams of carbomer, add it to 300 ml of purified water/distilled water, stir while adding, and stir at room temperature for half an hour until there are no obvious undissolved white chunks.
(2)低温4度，溶胀过夜(不低于12小时)，卡波姆溶解完全，看不到白色块状物及白色粉末。(2) Low temperature 4 degrees, swell overnight (not less than 12 hours), carbomer is completely dissolved, and no white lumps or white powder can be seen.
(3)用10-15mlNaOH调节pH，边加边搅拌，调节pH为7.0-7.4(最优为7.2)。(3) Adjust the pH with 10-15 ml NaOH, stir while adding, and adjust the pH to 7.0-7.4 (optimum is 7.2).
(4)加入40克药用甘油、1％的羟苯乙酯4ml，搅拌均匀。(4) Add 40 grams of medicinal glycerin and 4 ml of 1% ethyl hydroxyphenyl ester, and stir evenly.
(5)抗体冻干粉用1ml蒸馏水溶解后，再用10mM磷酸盐缓冲溶液稀释至体积40～50ml(根据氢氧化钠的用量调整稀释液的体积，稀释液体积与氢氧化钠的体积合计55ml)，将加入抗体的稀释液加入到上述步骤(4)制得的基质中，搅拌均匀，4度2小时溶胀即得。(5) Dissolve the antibody lyophilized powder in 1 ml of distilled water, and then dilute it with 10 mM phosphate buffer solution to a volume of 40-50 ml (adjust the volume of the diluent according to the amount of sodium hydroxide, the total volume of the diluent and the volume of sodium hydroxide is 55 ml ), add the diluent containing the antibody to the matrix prepared in the above step (4), stir evenly, and swell at 4°C for 2 hours.
(6)制成的抗体凝胶无菌分装即可。(6) The prepared antibody gel can be packaged aseptically.
实施例8志愿者使用效果Example 8 Volunteer use effect
口腔溃疡患者10例，病程不得超过2天，年龄18-65岁，未服用相关治疗药物，排除癌性及全身疾病引起的溃疡、糖尿病患者等。患者使用适量抗体凝胶于溃疡处，能覆盖溃疡创面为为宜，每日三-四次。使用抗体凝胶后患者主诉溃疡疼痛明显减轻，且溃疡患处康复时间也有不同程度的缩短；There were 10 patients with oral ulcers, the duration of the disease should not exceed 2 days, the age was 18-65 years old, and they did not take relevant treatment drugs. Patients with ulcers caused by cancer and systemic diseases, and patients with diabetes were excluded. Patients should apply an appropriate amount of antibody gel to the ulcer, which can cover the ulcer wound, three to four times a day. After using the antibody gel, the patient complained that ulcer pain was significantly reduced, and the recovery time of the ulcer affected area was also shortened to varying degrees;
***炎患者10例，排除老年性***炎和幼女性***炎。患者使用抗体凝胶，每天两-三次，疗程5天。患者应用抗体凝胶15分钟后瘙痒症状明显减轻，且作用持续4个小时以上。There were 10 patients with vaginitis, excluding senile vaginitis and juvenile vaginitis. Patients use antibody gel two to three times a day for 5 days. The patient's itching symptoms were significantly reduced 15 minutes after applying the antibody gel, and the effect lasted for more than 4 hours.
以上数据都证实，我们开发的炎症因子抗体凝胶，于局部作用皮肤粘膜炎症部位，可以快速缓解炎症引起的患者不适，缩短炎症反应过程。The above data all confirm that the inflammatory factor antibody gel we developed can quickly relieve the patient's discomfort caused by inflammation and shorten the inflammatory reaction process by locally acting on the inflammatory sites of skin and mucous membranes.
最后需要说明的是，以上实施例仅用于帮助本领域技术人员理解本发明的实质，不用于限定本发明的保护范围。 Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention, and are not used to limit the protection scope of the present invention.

Claims (10)

  1. 一组针对炎症因子的单克隆抗体，所述的炎症因子为人白细胞介素5(IL-5)或人肿瘤坏死因子(TNFa)，所述的单克隆抗体为：A group of monoclonal antibodies directed against inflammatory factors. The inflammatory factors are human interleukin 5 (IL-5) or human tumor necrosis factor (TNFa). The monoclonal antibodies are:
    抗人白细胞介素5(IL-5)的单克隆抗体，其重链可变区VH氨基酸序列如SEQ ID NO:6所示；轻链可变区的(VL)氨基酸序列如SEQ ID NO:8所示；The monoclonal antibody against human interleukin 5 (IL-5) has the VH amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 6; the (VL) amino acid sequence of the light chain variable region as SEQ ID NO: As shown in 8;
    抗人肿瘤坏死因子(TNFa)的单克隆抗体，其重链可变区VH氨基酸序列如SEQ ID NO:2所示；轻链可变区的(VL)氨基酸序列如SEQ ID NO:4所示。The monoclonal antibody against human tumor necrosis factor (TNFa), the VH amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:2; the (VL) amino acid sequence of the light chain variable region is shown in SEQ ID NO:4 .
  2. 一组针对炎症因子的单克隆抗体，所述的炎症因子为人白细胞介素5(IL-5)或人肿瘤坏死因子(TNFa)，其特征在于，A group of monoclonal antibodies directed against inflammatory factors, the inflammatory factors being human interleukin 5 (IL-5) or human tumor necrosis factor (TNFa), characterized by:
    所述的抗人白细胞介素5(IL-5)的单克隆抗体中，In the anti-human interleukin 5 (IL-5) monoclonal antibody,
    重链的CDR为；The CDRs of the heavy chain are;
    CDR-H1序列如SEQ ID NO.20所示，The CDR-H1 sequence is shown in SEQ ID NO.20,
    CDR-H2序列如SEQ ID NO.21所示，The CDR-H2 sequence is shown in SEQ ID NO.21,
    CDR-H3序列如SEQ ID NO.22所示；The CDR-H3 sequence is shown in SEQ ID NO.22;
    轻链的CDR为：The CDR of the light chain is:
    CDR-L1序列如SEQ ID NO.23所示The CDR-L1 sequence is shown in SEQ ID NO.23
    CDR-L2序列如SEQ ID NO.24所示The CDR-L2 sequence is shown in SEQ ID NO.24
    CDR-L3序列如SEQ ID NO.25所示；The CDR-L3 sequence is shown in SEQ ID NO.25;
    所述的抗人肿瘤坏死因子(TNFa)的单克隆抗体中，Among the monoclonal antibodies against human tumor necrosis factor (TNFa),
    重链的CDR为；轻链的CDR为：The CDR of the heavy chain is; the CDR of the light chain is:
    CDR-H1序列如SEQ ID NO.14所示，The CDR-H1 sequence is shown in SEQ ID NO.14,
    CDR-H2序列如SEQ ID NO.15所示，The CDR-H2 sequence is shown in SEQ ID NO.15,
    CDR-H3序列如SEQ ID NO.16所示，The CDR-H3 sequence is shown in SEQ ID NO.16,
    轻链的CDR为：The CDR of the light chain is:
    CDR-L1序列如SEQ ID NO.17所示，The CDR-L1 sequence is shown in SEQ ID NO.17,
    CDR-L2序列如SEQ ID NO.18所示，The CDR-L2 sequence is shown in SEQ ID NO.18,
    CDR-L3序列如SEQ ID NO.19所示。The CDR-L3 sequence is shown in SEQ ID NO.19.
  3. 根据权利要求1或2所述的单克隆抗体，其特征在于，所述的抗人白细胞介素5(IL-5)的单克隆抗体为59C4抗体；所述的抗人肿瘤坏死因子(TNFa)的单克隆抗体为T10E8抗体；The monoclonal antibody according to claim 1 or 2, characterized in that the anti-human interleukin 5 (IL-5) monoclonal antibody is 59C4 antibody; the anti-human tumor necrosis factor (TNFa) The monoclonal antibody is T10E8 antibody;
    优选的，Preferably,
    所述的单克隆抗体为人源化改造的单克隆抗体，其包含所述59C4抗体的重链及轻链可变区，或所述T10E8抗体的重链及轻链可变区；The monoclonal antibody is a humanized modified monoclonal antibody, which includes the heavy chain and light chain variable regions of the 59C4 antibody, or the heavy chain and light chain variable regions of the T10E8 antibody;
    更优选的，More preferably,
    59C4人源化抗体为：包含59C4抗体的重链及轻链可变区和人抗体IgG1恒定区(Fc)的人源化抗体；The 59C4 humanized antibody is: a humanized antibody containing the heavy chain and light chain variable regions of the 59C4 antibody and the human antibody IgG1 constant region (Fc);
    T10E8人源化抗体为：包含T10E8抗体的重链及轻链可变区和人抗体IgG1恒定区(Fc)的人源化抗体。The T10E8 humanized antibody is a humanized antibody that contains the heavy chain and light chain variable regions of the T10E8 antibody and the human antibody IgG1 constant region (Fc).
  4. 编码权利要求1-3任一所述的单克隆抗体的核苷酸片段。A nucleotide fragment encoding the monoclonal antibody of any one of claims 1-3.
  5. 根据权利要求4所述的核苷酸片段，其特征在于，The nucleotide fragment according to claim 4, characterized in that,
    编码抗人白细胞介素5(IL-5)的单克隆抗体的重链可变区VH的核苷酸片段的序列如SEQ ID NO:5所示，编码抗人白细胞介素5(IL-5)的单克隆抗体的轻链可变区VL的核苷酸片段的序列如SEQ ID NO:7所示； The sequence of the nucleotide fragment of the heavy chain variable region VH of the monoclonal antibody encoding anti-human interleukin 5 (IL-5) is shown in SEQ ID NO: 5, encoding anti-human interleukin 5 (IL-5 ) The sequence of the nucleotide fragment of the light chain variable region VL of the monoclonal antibody is shown in SEQ ID NO: 7;
    编码抗人肿瘤坏死因子(TNFa)的单克隆抗体的重链可变区VH的核苷酸片段的序列如SEQ ID NO:1所示，编码抗人肿瘤坏死因子(TNFa)的单克隆抗体的轻链可变区VL的核苷酸片段的序列如SEQ ID NO:3所示。The sequence of the nucleotide fragment encoding the heavy chain variable region VH of the monoclonal antibody against human tumor necrosis factor (TNFa) is shown in SEQ ID NO: 1. The sequence of the monoclonal antibody encoding the anti-human tumor necrosis factor (TNFa) The sequence of the nucleotide fragment of the light chain variable region VL is shown in SEQ ID NO: 3.
  6. 包含权利要求1-3任一所述的单克隆抗体的抗体药物制剂组合物，所述的抗体药物制剂组合物包含治疗有效量的所述单克隆抗体以及必要的药用辅料；An antibody pharmaceutical preparation composition comprising the monoclonal antibody according to any one of claims 1 to 3, wherein the antibody pharmaceutical preparation composition contains a therapeutically effective amount of the monoclonal antibody and necessary pharmaceutical excipients;
    优选的，所述的抗体药物制剂组合物为抗体凝胶制剂，其中包含：卡波姆、蒸馏水、PBS、甘油、羟苯乙酯，抗体2mg，以及必要的pH调节剂。Preferably, the antibody pharmaceutical preparation composition is an antibody gel preparation, which contains: carbomer, distilled water, PBS, glycerol, ethyl paraben, 2 mg of antibody, and necessary pH regulator.
    更优选的，所述的抗体凝胶制剂中包含：卡波姆940#12克、蒸馏水300ml、10mM PBS 40～50ml、甘油40克、1％羟苯乙酯4ml，抗体2mg，所述的pH调节剂为1N氢氧化钠。More preferably, the antibody gel preparation contains: 12 grams of carbomer 940#, 300 ml of distilled water, 40-50 ml of 10mM PBS, 40 grams of glycerol, 4 ml of 1% ethyl hydroxyphenyl ester, 2 mg of antibody, and the pH The regulator is 1N sodium hydroxide.
  7. 包含权利要求1-4任一所述的单克隆抗体的抗体凝胶制剂的制备方法，所述方法包括如下步骤：A method for preparing an antibody gel preparation containing the monoclonal antibody according to any one of claims 1 to 4, the method comprising the following steps:
    (1)将卡波姆加入纯化水中搅拌充分；(1) Add carbomer to purified water and stir thoroughly;
    (2)低温充分溶胀卡波姆溶液使其溶解完全；(2) Fully swell the carbomer solution at low temperature to completely dissolve it;
    (3)调节溶液pH至7.0-7.4，优选为7.2；(3) Adjust the pH of the solution to 7.0-7.4, preferably 7.2;
    (4)加入甘油、1％的羟苯乙酯，搅拌均匀；(4) Add glycerol and 1% ethyl hydroxyphenyl ester and stir evenly;
    (5)抗体用蒸馏水溶解后，再用PBS缓冲溶液稀释后加入到上述步骤(4)制得的溶剂基质中，搅拌均匀，低温充分溶胀即得所述抗体凝胶制剂；(5) After the antibody is dissolved in distilled water, dilute it with PBS buffer solution and then add it to the solvent matrix prepared in the above step (4), stir evenly, and fully swell at low temperature to obtain the antibody gel preparation;
    (6)无菌分装；(6) Sterile packaging;
    优选的，所述的低温充分溶胀为4℃下溶胀。Preferably, the sufficient swelling at low temperature is swelling at 4°C.
  8. 根据权利要求7所述的方法，其特征在于，所述的方法包括如下步骤：The method according to claim 7, characterized in that the method includes the following steps:
    (1)称量卡波姆12克，加入到300ml纯化水中，室温搅拌30min至没有明显的白色大块状物为止；(1) Weigh 12 grams of carbomer, add it to 300 ml of purified water, and stir at room temperature for 30 minutes until there are no obvious white lumps;
    (2)低温4℃溶胀过夜至卡波姆溶解完全，看不到白色块状物及白色粉末；(2) Swell at low temperature 4°C overnight until the carbomer is completely dissolved and no white lumps or white powder can be seen;
    (3)用10-15ml NaOH调节pH至7.2；(3) Use 10-15ml NaOH to adjust the pH to 7.2;
    (4)加入40克甘油、1％的羟苯乙酯4ml，搅拌均匀；(4) Add 40 grams of glycerin and 4 ml of 1% ethyl hydroxyphenyl ester, and stir evenly;
    (5)抗体冻干粉用1ml蒸馏水溶解后，再用10mM磷酸盐缓冲溶液稀释至体积40～50ml，将抗体的稀释液加入到上述步骤(4)制得的溶剂基质中，搅拌均匀，4℃溶胀2小时即得所述抗体凝胶制剂；优选的，磷酸盐缓冲溶液的体积与步骤(3)使用的氢氧化钠溶液的体积合计55ml；(5) After the antibody freeze-dried powder is dissolved in 1 ml of distilled water, dilute it with 10 mM phosphate buffer solution to a volume of 40-50 ml. Add the antibody dilution to the solvent matrix prepared in the above step (4), stir evenly, 4 The antibody gel preparation is obtained by swelling at ℃ for 2 hours; preferably, the volume of the phosphate buffer solution and the volume of the sodium hydroxide solution used in step (3) total 55 ml;
    (6)无菌分装。(6) Aseptic packaging.
  9. 权利要求1-3任一所述的单克隆抗体、权利要求6所述的抗体药物制剂组合物、抗体凝胶制剂在制备治疗皮肤和/或粘膜炎症的药物中的应用；The use of the monoclonal antibody according to any one of claims 1 to 3, the antibody pharmaceutical preparation composition and antibody gel preparation according to claim 6 in the preparation of drugs for the treatment of skin and/or mucosal inflammation;
    优选的，所述的皮肤和/或粘膜炎症为口腔黏膜炎症或***炎症。Preferably, the skin and/or mucosal inflammation is oral mucosal inflammation or vaginal inflammation.
  10. 根据权利要求9所述的应用，其特征在于，所述的治疗炎症为缓解由炎症因子释放导致的炎症反应，如红斑、水肿、灼烧、疼痛、瘙痒等；所述的炎症因子为TNFa和/或IL-5。 The application according to claim 9, characterized in that the treatment of inflammation is to alleviate inflammatory reactions caused by the release of inflammatory factors, such as erythema, edema, burning, pain, itching, etc.; the inflammatory factors are TNFa and /or IL-5.
PCT/CN2023/088120 2022-04-15 2023-04-13 Antibody for treating skin and mucosal inflammation and formulation thereof WO2023198155A1 (en)

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