TW201741338A - A monoclonal antibody inhibiting immunosuppressive functions of pathogens, antigen-binding fragments thereof, and hybridomas producing such antibody - Google Patents

Abstract

The present invention relates to a monoclonal that inhibits immunosuppressive functions of pathogens, antigen-binding fragments thereof, and hybridomas producing such antibody. The monoclonal antibody or antigen-binding fragments thereof bind to a peptide consisting an amino acid sequence represented by or similar to MEKVGKDGVITVE (SEQ ID NO: 1). The present invention also discloses use of the invented monoclonal antibody or antigen-binding fragments thereof, and method of preparation for such hybridomas.

Description

Monoclonal antibody with immunosuppressive function inhibiting pathogen, antigenic knot Fragment, and fusion tumor producing the same

The present invention relates to a monoclonal antibody having an immunosuppressive function for inhibiting pathogenic agents, an antigen-binding fragment thereof, and a fusion tumor producing the same, particularly for a function of blocking an immunosuppressive substance secreted by a pathogenic agent, A monoclonal antibody, an antigen-binding fragment thereof, and a fusion tumor producing the same, which enhance host immunity.

Helicobacter Pylori ( H. pylori ) is a Gram-negative bacterium that has been infected in half of the world. Chronic inflammation caused by H. pylori can lead to several outcomes ranging from peptic ulcers to gastric cancer, depending on the extent and extent of gastric inflammation caused. Although the host mainly produces a mucosal immune response that tends to Th1 type, it is unable to achieve a degree sufficient to protect the host from H. pylori infection, resulting in the formation of a chronic infection, and will develop into a gastric cancer lesion in some patients. Previous studies have demonstrated that H. pylori lysates inhibit the proliferation of T cells that induce mitogens. It is shown that there is a factor associated with immunosuppressive activity in the lysate. These factors can attenuate the activity of T cells, and their mechanism of action is independent of the bacterial virulence factors CagA and VacA. Researchers have proposed several mechanisms to explain why H. pylori can directly or indirectly inhibit the immune response of T-cells. Including: Helicobacter pylori inhibits T cell proliferation and receptor expression on T cells by arginase; H. pylori stimulates release of immunosuppressive hormone TGF-β; H. pylori interferes with VacA The expression of the antigen without a change chain; H. pylori negatively regulates the function of DCs to phosphorylate intracellular proteins by CagA; or H. pylori inhibits the phagocytic function of macrophages via VirB7 and VirB11. and many more.

Although the above various mechanisms may be justified, the current industry believes that regulatory T cells (regulatory T-cells) are the main regulatory factors that inhibit T cell activity and balance inflammation and bacterial persistent infection. It was reported in 2003 that CD4 ⁺ CD25 ⁺ T cells are associated with H. pylori-induced immunosuppression and its parasitism. Further studies have shown that host Treg cells are the primary key to protecting H. pylori-infected hosts from excessive gastric inflammation and disease symptoms, but also promote bacterial parasites on the stomach and duodenal mucosa. In addition, the co-stimulatory factor B7-H1 exhibited by gastric epithelial cells in patients also promotes the development of gastric epithelial cells of CD4 ⁺ CD25 ⁺ FoxP3 ⁺ Treg cells after H. pylori infection. This means that this pathogen will promote the induction of host Treg cells. Subsequent studies examined the function of these Helicobacter pylori-induced Treg cells. The results showed that this Treg cell can inhibit the action of H. pylori-specific affected T cells and disable them. In addition, gastritis caused by Helicobacter pylori often coincides with the infiltration of FoxP3 ⁺ Treg cells, and the degree of bacterial parasitism is related to the degree of expression of TGF-β in the mucosa. The above various studies have shown that the host Treg response induced by H. pylori is an important regulatory factor for the host H. pylori immune response and the pathogenic mechanism of H. pylori-associated diseases.

H. pylori heat shock protein 60 (HpHSP60) can induce the secretion of proinflammatory cytokines and TGF-β1 by monocytes. It has been reported that HpHSP60 is expressed in the cell wall of bacteria together with urease, and can be used as an adhesion molecule of gastric Helicobacter pylori to gastric epithelial cells. In addition, studies have found that administration of anti-HpHSP60 antibodies can interfere with the growth of H. pylori. Therefore, HpHSP60 is not only an important factor affecting the viability of H. pylori, but also provides the Helicobacter pylori required for human gastric parasitism. However, many studies have also shown that HpHSP60 is an immunogen that strongly stimulates the production of pro-inflammatory cytokines such as TNF-α, IL-8 and IL-6. These cytokines cause an inflammatory response at the site of infection, and this HpHSP60-induced inflammatory response promotes tumor malignancy, including vascular proliferation and cancer cell migration. HpHSP60 is also an important virulence factor for human Helicobacter pylori infection in human hosts.

Due to the complex relationship between HpHSP60 and Treg cells, the study of the relationship between the two has become an important issue in this industry. However, past studies have focused on the inflammatory response induced by HpHSP60. Little is known about the relationship between HpHSP60 and host immunosuppression.

U.S. Patent No. 6,403,099 is directed to a conjugated compound formed by a heat-stressed protein and a polysaccharide or oligosaccharide. The compound induces the formation of an anti-polysaccharide antibody and can be used as a vaccine for humans and animals. Wherein, the heat-stressing protein comprises H. pylori heat-stressing protein.

According to the present invention, several pathogens produce a function of inhibiting host immunity. This function is provided in particular by immunosuppressive substances secreted or produced by the pathogen. By shutting down the action of the immunosuppressive substance, the immunosuppressive function of the pathogen can be turned off. Since the immunosuppressive function is turned off, the host's own immune function is not inhibited. The purpose of reducing or even eliminating the pathogen can be achieved by the uninhibited immune function.

The inventors of the present invention have found a novel monoclonal antibody which has a significant inhibitory function on the immunosuppressive function or substance of the pathogenic agent. Based on the above findings, the monoclonal antibodies of the present invention, antigen-binding fragments thereof, and fusion tumors of the antibodies are produced, and a method for producing the same.

Accordingly, it is an object of the present invention to provide a novel monoclonal antibody. This monoclonal antibody has a significant immunosuppressive function of inhibiting pathogenic agents.

The object of the present invention is also to provide an antigen-binding fragment of the monoclonal antibody, and a fusion tumor producing the same.

The object of the present invention is also to provide such a monoclonal antibody, an antigen-binding fragment thereof, and a method for producing a fusion tumor producing the same.

The object of the present invention is also to provide the use of the monoclonal antibodies, antigen-binding fragments thereof, and fusion tumors producing the antibodies.

According to one aspect of the invention, the monoclonal antibody or antigen-binding fragment thereof binds to a peptide consisting of an amino acid sequence represented by MEKVGKDGVITVE (SEQ ID NO: 1) or a similar one thereof.

The invention also provides a fusion tumor capable of producing the monoclonal antibody or antigen-binding fragment thereof.

The monoclonal antibody of the present invention has a significant inhibitory effect on the immunosuppressive activity of a specific pathogen, and can effectively utilize the immunosuppressive phenomenon caused by inhibition of pathogenic agents. In an embodiment of the invention, the immunosuppressive function of the pathogen is provided by an immunosuppressive substance secreted or produced by the pathogenic agent. The monoclonal antibody or antigen-binding fragment thereof of the present invention mainly removes the pathogen by inhibiting the immunosuppressive function of the pathogen and activating the host immune response.

According to another aspect of the present invention, there is provided a method for producing a monoclonal antibody or an antigen-binding fragment thereof, and a fusion tumor, which comprises: using an amino acid represented by MEKVGKDGVITVE (SEQ ID NO: 1) or similar thereto a peptide composed of a sequence as an antigen, causing a mammal to immunologically react with the antigen; obtaining immune cells of a mammal immunized against the antigen, and fusing the immune cells with a mammalian myeloma cell; The fusion tumor is subjected to colonization to obtain a fusion tumor of the present invention. The method of the present invention may further comprise the steps of producing the antibody of the present invention with the fusion tumor, and recovering the antibody produced by the fusion tumor.

In a preferred embodiment of the invention, the immune cells are preferably spleen cells.

The monoclonal antibody or antigen-binding fragment thereof of the present invention can be used as it is, or can be used as a pharmaceutical composition together with a pharmaceutically acceptable additive or the like. According to an embodiment of the present invention, there is provided a pharmaceutical composition comprising the monoclonal antibody of the present invention or an antigen-binding fragment thereof. According to another aspect of the present invention, the pharmaceutical composition uses a functional inhibitor as an immunosuppressive substance. Furthermore, the invention also provides the use of a monoclonal antibody of the invention, which comprises the use in the manufacture of a pharmaceutical composition.

Figure 1 shows the experimental results of the effect of HpHSP60 on the proliferation of PBMC.

Figure 2 shows the results of an experiment in which HpHSP60 affects T cell proliferation in PBMC.

Figure 3 shows the results of an experiment to determine the effect of HpHSP60 on the cell cycle of PBMC.

Figure 4 shows the results of in vitro induction of Treg cells by HpHSP60.

Figure 5 shows the results of an experiment to test that HpHSP60 promotes Treg proliferation.

Figure 6 shows the results of an experiment in which HpHSP60-inducible Treg cells inhibit T cell proliferation.

Figure 7 shows the results of an experiment in which H. pylori is inhibited by HSP60 in a living animal to inhibit cell growth.

Figure 8 also shows the results of an experiment in which H. pylori is inhibited by HSP60 in a living animal to inhibit cell growth.

Figure 9 shows the results of an experiment in which Treg was inhibited by inhibition of HSP60 in a living animal.

Figure 10 shows the results of detection of HpHSP60 with the sequence of activity that induces Treg cells.

Figure 11 shows the results of the data of the experimental results of Figure 10.

Figure 12 shows the results of an experiment exploring the immune mechanism of the anti-HpHSP60 antibody.

Figure 13 shows the results of detection of the expression of Treg cells in the gastric mucosa by an anti-HpHSP60 antibody.

Figure 14 shows the results of detection of the expression of IL-10 in the gastric mucosa by an anti-HpHSP60 antibody.

Figure 15 shows the results of an experiment to detect a fragment of HpHSP60 recognizable by the LHP-1 (9E4) antibody.

Figure 16 shows the results of an experiment to further detect the HpHSP60 fragment recognizable by the LHP-1 (9E4) antibody.

While not wishing to be bound or limited by any theory, in accordance with the present invention, several pathogens may produce or secrete an immunosuppressive substance to inhibit the host's ability to immunosist, as a result of causing pathogenic hyperplasia to cause host disease. Such pathogens include Helicobacter pylori and other similar bacteria, such as Arcobacter suis , Tannerella forsythia , Porphyromonas gingivalis , Aggregatibacter actinomycetemcomitans , Helicobacter felis and the like. The present inventors have found that a heat-stressing protein is one of such immunosuppressive substances. According to an embodiment of the present invention, H. pylori heat-stressing protein (HpHSP60) can stimulate the production of immunosuppressive hormones IL-10 and TGF-β by acting with monocytes, thereby inducing proliferation of Treg cells, resulting in a host. Immunosuppression, resulting in a chronic infection against H. pylori.

The invention further develops a new method for improving the immunity of the host, and uses the functional inhibitor which can shut down the action of the immunosuppressive substance to close the action of the immunosuppressive substance, effectively inhibit the proliferation of Treg cells, and eliminate the phenomenon of immunosuppression.

The inventors of the present invention have found that a novel monoclonal antibody has a significant inhibitory function on the immunosuppressive function or substance of the pathogenic agent. The monoclonal antibody or antigen-binding fragment thereof is suitable as the functional inhibitor. The functional inhibitor recognizes the immunosuppressive substance or a portion thereof and blocks the action of the immunosuppressive substance.

Deposit

The original storage date of the fusionoma hybridoma 9E4 of the present invention is January 8, 2015, and is deposited at the Bioresource Conservation and Research Center of the Food Industry Research Institute (Address: No. 331, Food Road, Hsinchu City, Taiwan), and the registration number is BCRC 960493.

Individual antibody and fusion tumor

The monoclonal antibody or antigen-binding fragment thereof of the present invention binds to a peptide composed of an amino acid sequence represented by MEKVGKDGVITVE (SEQ ID NO: 1) or similar, and can effectively inhibit the immunosuppression caused by pathogenic agents. . The immunosuppressive function of the pathogen is provided by an immunosuppressive substance secreted or produced by the pathogenic agent. The monoclonal antibody or antigen-binding fragment thereof of the present invention mainly removes the pathogen by inhibiting the immunosuppressive function of the pathogen and activating the host immune response. The active monoclonal antibody or antigen-binding fragment thereof exhibits a remarkable inhibitory ability against the immunosuppressive function caused by the pathogen, and is an unprecedented phenomenon.

According to one embodiment of the invention, the antibody or antigen-binding fragment thereof is a peptide consisting of an amino acid sequence corresponding to or similar to MEKVGKDGVITVE (SEQ ID NO: 1).

The antibody or antigen-binding fragment thereof of the invention may contain a heavy chain and/or a light chain. The N-terminus of each of the light chain and the heavy chain may have a variable region, and in each variable region, four framework regions (FR) and three complementarity determining regions (CDRs) may be alternately included.

In one embodiment of the present invention, the light chain variable region of the antibody or antigen-binding fragment thereof may comprise: CDR1 consisting of amino acid sequence represented by ASQSVDYDGDVFL (SEQ ID NO: 2), by YAASN (SEQ ID NO: 3) CDR2 composed of the amino acid sequence represented by the amino acid sequence and CDR3 composed of the amino acid sequence represented by QSNEVPWT (SEQ ID NO: 4). In a preferred embodiment, the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 6 to No. 131.

In some embodiments of the invention, the heavy chain variable region of the antibody or antigen-binding fragment thereof may comprise: CDR1 consisting of the amino acid sequence represented by SGFTFSSFG (SEQ ID NO: 7), by ISNGGS (SEQ ID NO: 8) The CDR2 composed of the amino acid sequence represented by the amino acid sequence and the CDR3 composed of the amino acid sequence represented by QGLRRRGAMDY (SEQ ID NO: 9). In a preferred embodiment, the heavy chain variable region is an amino acid sequence represented by SEQ ID NO: 11 to No. 139.

In another preferred embodiment of the invention, the antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region. Wherein the light chain variable region comprises: a CDR1 composed of an amino acid sequence represented by ASQSVDYDGDVFL (SEQ ID NO: 2), a CDR2 composed of an amino acid sequence represented by YAASN (SEQ ID NO: 3), and CDR3 consisting of the amino acid sequence represented by QSNEVPWT (SEQ ID NO: 4); and the heavy chain variable region comprises: CDR1 consisting of amino acid sequence represented by SGFTFSSFG (SEQ ID NO: 7) by ISNGGS ( The CDR2 composed of the amino acid sequence represented by SEQ ID NO: 8) and the CDR3 composed of the amino acid sequence represented by QGLRRRGAMDY (SEQ ID NO: 9).

In a further preferred embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises SEQ ID NO: 6 No. 21 ~ The amino acid sequence represented by No. 131; and the heavy chain variable region contains the amino acid sequence represented by No. 20 to No. 139 of SEQ ID NO: 11.

According to an embodiment of the invention, the monoclonal antibody is preferably a chimeric antibody, a humanized antibody or a fully human type antibody.

In a preferred embodiment of the invention, the antigen binding fragment can be Fab, Fab', (Fab') 2, Fv or scFv. The whole antibody immunoglobulin can be IgG1, IgG2, IgG4, IgA, IgE or IgD.

The invention also provides a fusion tumor for producing a monoclonal antibody or antigen-binding fragment thereof of the invention. In a preferred embodiment of the invention, the fusion tumor is hybridoma 9E4.

The monoclonal antibodies, antigen-binding fragments thereof, and fusion tumors of the present invention can be produced by the following methods. Using a peptide consisting of an amino acid sequence represented by MEKVGKDGVITVE (SEQ ID NO: 1) or similar thereto as an antigen, a mammal is subjected to an immune reaction against the antigen; and a mammalian shaped cell (immunized) for the antigen is obtained (immunization) The cells are fused with a mammalian myeloma cell; the resulting fusion tumor is subjected to colonization to obtain a fusion tumor of the present invention. The method of the present invention may further comprise the steps of producing the antibody of the present invention with the fusion tumor, and recovering the antibody produced by the fusion tumor.

Among them, the method of immunizing a mammal can employ a administration method known in the art. Suitable methods include: intraperitoneal injection, intrasplenic injection, intramuscular injection, subcutaneous injection, intradermal injection, oral administration, transmucosal administration, transdermal administration, and the like. Among them, intraperitoneal injection, intrasplenic injection, etc. are more suitable. The administration interval of the antigen can be appropriately determined depending on the amount of the antigen to be administered, and the type of the mammal, for example, several times per month.

The immunologically treated mammalian species is not particularly limited. However, it is advisable to consider the suitability of the myeloma cells used for cell fusion and the like. Suitable mammals include mice, rats, hamsters and the like. Among them, mice are more suitable.

The immune cells are more suitable for spleen cells, but are not technically limited.

The cells of the immune cells are fused with the myeloma cells, and any known method can be utilized. For example, the method proposed by Milstein et al. (Methods Enzymol., 73, 3-46, 1981) is applicable. The method mainly comprises: mixing the immune cells with the myeloma cells in the medium in the presence of a fusion promoter. In the process of cell fusion, the medium should be added in an appropriate amount and repeatedly separated by centrifugation to obtain a fusion tumor.

Suitable media for cell fusion include: RPMI-1640 medium, MEM medium, and the like. These media are often used in cell fusion. In the fusion process, serum supplements such as bovine fetal serum can be appropriately added.

Generally, the cell fusion temperature is preferably 25 to 37 ° C, preferably 30 to 37 ° C. The mixing ratio of myeloma cells to immune cells should be about 1:1~1:10.

Suitable fusion promoters may include polyethylene glycol (PEG), Sendai virus (HVJ), and the like. Among them, PEG is more suitable. If PEG is used, its molecular weight can also be appropriately selected, for example, PEG with an average molecular weight of about 1,000 to 6,000. Furthermore, the concentration of PEG in the medium can be about 30 to 60% (w/v).

In the above method of the present invention, the screening of the fusion tumor may comprise the step of culturing the fusion tumor obtained by cell fusion in a medium. The medium is preferably a selective medium such as a commercially available medium such as HAT medium. Screening was performed using limiting dilution. For the screening, for example, the antibody valence of the peptide composed of the amino acid sequence represented by MEKVGKDGVITVE (SEQ ID NO: 1) can be used as an index. The culture time in the medium is sufficient to cause the cells other than the target fusion tumor to die, usually from several days to several weeks. The fusion tumor of the present invention obtained in the above manner can provide subculture in a conventional medium, and can also be stored in liquid nitrogen for a long period of time.

In the method, the step of recovering the monoclonal antibody or the antibody-binding fragment thereof of the present invention comprises: culturing the fusion tumor by a known method; and culturing the supernatant to obtain a monoclonal antibody. Another method comprises the steps of: administering a fusion tumor to a mammal adapted to the fusion tumor, proliferating the fusion tumor; and obtaining a monoclonal antibody from the ascites of the mammal. Among them, a method of obtaining a monoclonal antibody from a culture supernatant can obtain a high-purity antibody side. A method of obtaining a monoclonal antibody from the ascites of a mammal can be used to produce antibodies in large quantities. People in this industry can choose to use according to their purpose.

The monoclonal antibody or antibody-binding fragment thereof obtained by the above procedure can be further purified. The purification method may include any known methods such as salting out, colloidal filtration, affinity chromatography, and the like.

The monoclonal antibody or antigen-binding fragment thereof of the present invention can exhibit a remarkable activity of inhibiting immunosuppressive function. In use, the monoclonal antibody or antigen-binding fragment thereof of the present invention may be used as it is, or may be used as a pharmaceutical composition together with a pharmaceutically acceptable additive. The pharmaceutical composition of the present invention contains an effective amount of the monoclonal antibody of the present invention or an antigen-binding fragment thereof. The composition of the pharmaceutical composition It is used as a functional inhibitor to trigger an immunosuppressive phenomenon as a pathogen. The invention also provides the use of a monoclonal antibody of the invention, including for the manufacture of a pharmaceutical composition.

The pharmaceutical composition of the present invention comprises a functional inhibitor composition of an immunosuppressive function, which comprises the method of dissolving the monoclonal antibody or antigen-binding fragment thereof of the present invention in physiological saline for injection, distilled water for injection, buffer solution for injection, and the like. Buffering the liquid; and modulating the composition and the like. Other additives may be included in the functional inhibitor composition of the immunosuppressive function of the present invention. Suitable additives include: solvents, dissolution aids, preservatives, stabilizers, emulsifiers, suspending agents, painless agents, isotonic agents, buffers, excipients, tackifiers, colorants, and conventional carriers. For example, various ribosomes, polyamino acid carriers, synthetic polymer compounds, natural polymer compounds, and the like.

According to the present invention, there is also provided a method for inhibiting the immunosuppressive phenomenon caused by heat-stressed protein 60 secreted by a pathogenic source such as Helicobacter pylori or other similar bacteria. By administering the monoclonal antibody or the antigen-binding fragment thereof of the present invention to a living host, the immunosuppressive phenomenon caused by the heat-stressing protein 60 of the pathogenic source can be inhibited, and the disease can be eliminated by activating the host's own immune system. The effect of the source.

In this method, the monoclonal antibody or antigen-binding fragment thereof of the present invention can be administered systemically or locally to the host. The administration method includes any known methods such as drip, intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, oral administration, transmucosal administration, transdermal administration and the like.

The effective amount of the monoclonal antibody or antigen-binding fragment of the present invention is not limited in any way, and the person skilled in the art can appropriately determine the type, nature, sex, age, and the like of the host.

Hereinafter, the monoclonal antibodies of the present invention having an immunosuppressive function for inhibiting pathogenic agents, antigen-binding fragments thereof, and fusion tumors producing the same will be described by way of examples and with reference to the accompanying drawings. However, it should be understood that the scope of the invention is not limited by the scope of the embodiments. For example, although this example is exemplified by the pathogenic mechanism of Helicobacter pylori and a functional inhibitor of H. pylori heat-stressing protein 60 (HpHSP60), for example, Helicobacter felis (which can cause chronic enteritis in humans) and Arcobactersuis bacteria (causing The heat-stressed protein amino acid sequence of human periodontal disease also contains the same fragment as HpHSP60, such as HSP 60 101-200. The methods and uses of the present invention are applicable to such and other pathogenic bacteria, as well as other pathogens having the same pathogenic mechanisms.

Example 1: Cell culture, separation of PBMC from T-cells

Human peripheral blood mononuclear cells (PBMC) supplied by healthy donors were separated by density gradient centrifugation using Ficoll-Paque Plus (trademark, GE Healthcare, Uppsala, Sweden), and resuspended in 10% fetal bovine serum and 1% penicillin-streptomycin in RPMI-1640 medium. For removal of monocytes, PBMCs were cultured in 10-cm culture dishes at overnight density of 10 ⁶ /mL to allow monocytes to attach. The suspended cells were collected after centrifugation at 1500 rpm for 15 minutes. All T cells obtained from the PBMC were subjected to negative screening separation using a magnetic sorting device (Miltenyi Biotec, Inc., Massachusetts, USA). Briefly, PBMCs were co-cultured with a mixture of non-T cell antibodies (the antibody mixture was grafted onto Biotin), followed by reaction with microbeads containing anti-Biotin antibodies, and non-T cells-Biotin-anti using a magnetic base. - Biotin-magnetic bead complex adsorption, T cells are collected after the bank is washed by the manufacturer's levee.

Example 2: Effect of HpHSP60 on proliferation of PBMC

The cell proliferation was analyzed by cell proliferation assay using PBMC activated with CD3 mAb, different doses of HpHSP60, rGFP and boiled HpHSP60. 0.2 ml of each group of cells was seeded at each well at a concentration of 1 × 10 ⁶ cells/ml in each well of a 96-well microplate pretreated with anti-CD3 monoclonal antibody. Cell proliferation was measured by MTT assay after 96 hours. Figure 1 shows the experimental results. The values shown in the figure are the proliferation index. The cell proliferation index was calculated as follows: Hyperplasia index (100%) = (OD ₅₉₅ value of cells treated with anti-CD3 + HpHSP60) / (OD ₅₉₅ value of cells treated with anti-CD3-) x 100%. Compared with the control group not added with HpHSP60, if there was a significant difference, it was expressed by * (P < 0.05) (n = 15).

Figure 1 shows that (◆) T cell proliferation is inhibited after HpHSP60 is added to human peripheral mononuclear cells (PBMC). (■) rGFP is an experimental system for controlling histones and has no effect on T cell proliferation. It can be seen from the control group that not all proteins can inhibit T cell proliferation. (▲) Boiled HpHSP60 indicates that only the HpHSP60 sequence is boiled to lose protein structure, which denatures the protein. The experimental results show that the HpHSP60 sequence after boiling does not affect T cell proliferation.

Example 3: Effect of HpHSP60 on T cell proliferation in PBMC

PBMCs containing T-cells or T-free cells were treated with anti-CD3 monoclonal antibody, with or without HpHSP60 (200 ng), and the number of cells was calculated after staining with CD3 surface marker. When CD3 surface marker staining was performed, the collected cells were stained with CD3 monoclonal antibody (OKT3). This was followed by staining with IgG FITC fluorescent secondary antibody (Biolegend, Inc., California, USA). For intracellular staining of FoxP3, cells were collected and stained with CD4 FITC fluorescent monoclonal antibody (Biolegend, Inc., California, USA). The treated cells were fixed and the cell membrane was holed. Intracellular staining was then performed using FoxP3-PE monoclonal antibody (BD Biosciences, Inc., Massachusetts, USA) according to the manufacturer's specifications. Cell cycle analysis was then performed: cells were fixed with 70% ethanol after 72 hours. Further, DNA staining buffer (5% Triton-X 100, 0.1 mg/ml of RNase A and 4 μg/ml of PI) was stained for 30 minutes at room temperature, after which the change in DNA content was examined. Fluorescence analysis was performed using a FACS flow cytometer (Becton Dickinson, Heidelberg, Germany) and a CELLQUEST Pro program (Becton Dickinson, Heidelberg, Germany).

The experimental results are shown in Figure 2. The numerical proliferation index (multiples) in Figure 2 = (number of T cells or non-T cells in the anti-CD3/HpHSP60 treated group) / (number of T cells or non-T cells in the untreated control group). If there is a significant difference, it is represented by * (P < 0.05), (N = 4). The experimental results show that HpHSP60 can inhibit the proliferation of T cells in PBMC. In the figure, (□) shows the part of T cells in PBMC; (■) shows the part of non-T cells in PBMC. From the results, it was found that HpHSP60 is inhibited against T cells.

Example 4: Determination of the effect of HpHSP60 on cell cycle

PBMC cells were taken, and the percentages of subG1, G1, S and G2/M phases were observed by PBMC cells themselves, CD3-activated PBMC cells, and Anti-CD3+HpHSP60-treated PBMC. The results are shown in the histogram of Fig. 3. The figure shows the results of three replicate experiments.

Figure 3 shows that HpHSP60 inhibits T cell proliferation rather than causing T cell death. In the figure, Cell alone in Fig. 3A shows that normal T cells are in a dormant phase (G0/G1) without CD3 activation. Anti-CD3 of Figure 3B shows that after CD3 activation, T cells begin to grow and grow, forming a typical cell cycle pattern. The Anti-CD3+HpHSP60 of Figure 3C showed no change compared to Figure 3B, and the ratio in the Sub G0/G1 phase (the DNA fragment representing cell death) did not increase compared to the Anti-CD3 group. The experimental results show that the effect of HpHSP60 is to inhibit the growth of T cells, rather than causing T cell death.

Example 5: In vitro induction of Treg cells by HpHSP60

For PBMC cells treated with HpHSP60, the ratio of CD4 ⁺ FoxP3 ⁺ cells was measured over time. Compared with the control group treated only with anti-CD3, if there is a significant difference, it is represented by * (P < 0.05) (n = 5). The results are shown in Figure 4.

Since CD4 and FoxP3 are markers of Treg cells, the effect of HpHSP60 on the growth of Treg cells can be directly identified from Figure 4. In the figure, (●) cell alone indicates the original growth of Treg cells. (■) anti-CD3 indicates the growth of Treg cells after activation with CD3. (▲) Anti-CD3+HpHSP60 indicates that Treg cells proliferate significantly. The experimental results show that HpHSP60 can promote the proliferation of Treg cells.

Example 6: HpHSP60 promotes Treg cell proliferation

Following Example 5, after 72 hours of cell collection, total RNA was isolated. The amount of FoxP3 mRNA expression was determined using real-time PCR. Compared with the control group treated with the anti-CD3 antibody, if there is a significant difference, it is represented by * (P < 0.05), (n = 4). The results are shown in Figure 5.

Since FoxP3 is a marker for Treg cells, the amount of FoxP3 expression is also increased when Treg cells are activated. The figure shows that the mRNA expression results showed that the expression of FoxP3 was significantly increased after the addition of HpHSP60, further demonstrating that the addition of HpHSP60 promoted Treg proliferation.

Example 7: Effect of HpHSP60-inducible Treg cells on inhibition of T cell proliferation

The effect of HpHSP60-induced Treg cells on T cell proliferation was measured by functional assay. The results obtained are shown in the histogram of FIG. The numbers in the figure indicate the percentage of proliferating cells. This figure represents the result of three repetitions.

The experimental results show that when Treg cells increase, they will inhibit T cell activation. It was demonstrated that the addition of PBMC to HpHSP60 inhibited the activation of T cells due to proliferation of Treg cells.

Example 8: Preparation of anti-HpHSP60 serum (anti-HpHSP60 serum) and HpHSP60 monoclonal antibody

H. pylori heat-stressing protein 60 (HpHSP60) was injected into mice to cause an immunological reaction. After multiple doses of HpHSP60 (boost), mice developed resistance Antibody to HpHSP60. After the blood of the mouse was collected, the serum was taken to obtain a serum containing an anti-HpHSP60 antibody, which was called anti-HpHSP60 serum. The antibody obtained in this step is a polyclonal antibody.

The mouse spleen cells are fused with bone marrow cancer cells to form a hybridoma. Further screening, specific antibodies were picked using enzyme immunoassay (ELISA).

The obtained cell strain was diluted and re-aliquoted into a 96-well cell culture dish, and it was calculated that only one cell per cell was allowed to grow into a colony, and the specific antibody was again screened by ELISA. A monoclonal antibody was obtained.

The protein sequence of the antibody variant region is then analyzed. As a result, the sequence of the light chain variant region was found to be the amino acid sequence of Sequence Listing Nos. 2-6, and the sequence of the variant region of the seed chain was as the amino acid sequence of Sequence Listing Nos. 7-11.

Example 9: Effect of H. pylori inhibiting cell growth due to inhibition of HSP60 in vivo

C3H/HeN mice were purchased from the National Laboratory Animal Breeding Research Center in Taipei, Taiwan, and kept free of pathogen isolation. All food, water and cage supplies are disinfected prior to use. Five-week-old male mice were intravenously injected with 0.1 ml of anti-HSP60 serum (anti-HpHSP60 serum) obtained in Example 8 before inoculation of H. pylori. After 24 hours of antiserum administration, the mice were infected with 0.5 mL of live Helicobacter pylori (a strain with an ATCC number of 15,415, about ¹⁰⁹ colony forming units). The BHI liquid medium was fed twice by a period of 3 days via oral tube feeding. After confirming infection with H. pylori, the mice were intravenously injected with 0.1 ml of the anti-HSP60 serum obtained in Example 8, once every 3 days.

After the 8th week of infection with H. pylori, all mice were sacrificed under sterile conditions. Cut the entire stomach along a small bend. Each stomach is divided into two equal longitudinal samples, each sample Contains the stomach and antrum. The gastric tissue was ground and the Helicobacter pylori in the stomach tissue was cultured, and the performance of FoxP3 was analyzed by immunohistochemical staining to evaluate the state of Helicobacter pylori parasitization in the stomach. The results of the evaluation are expressed as mean ± SEM. Statistical significance was assessed using the one-tailed Student's t-test method. P < 0.05 was considered to be a significant difference. The results are shown in Figures 7, 8, and 9.

Figures 7 and 8 show that anti-HpHSP60 serum significantly reduced the number of colonies of H. pylori obtained from its gastric tissue lysate after 8 weeks of vaccination with H. pylori in mice. To investigate the mechanism by which antibodies reduce the parasitic activity of H. pylori in the stomach, the amount of Treg cells in the stomach tissue infected with H. pylori was evaluated. Figure 9 shows that treatment with anti-HpHSP60 serum significantly reduced the amount of Treg cells expressed in the gastric mucosa. The above results indicate that chronic infection of H. pylori is associated with HpHSP60, and inhibition of HpHSP60 can reduce the parasitic amount of H. pylori and also reduce the production of Treg cells.

Example 10: HpHSP60 has an active sequence for inducing Treg cells

In order to find out the active sequence of HpHSP60-inducible regulatory T cells, monoclonal antibodies against HpHSP60 were prepared, including monoclonal antibodies containing the entire sequence of HpHSP60 and fragments thereof. According to the method of Example 9, after the mice were treated with anti-HpHSP60 serum for 24 hours, the mice were fed with H. pylori and confirmed to be infected with H. pylori, the mice were intravenously injected with 0.1 ml, and PBS was administered once every 3 days. Serum, anti-HSP60 serum, LHP-1 (9E4) monoclonal antibody and LHP-2 (5A8) monoclonal antibody. After 8 weeks, the mice were sacrificed, and the stomach wall of the mice was ground, and the gastric homogenate was further cultured on the Helicobacter pylori isolation medium (EYE agar) to confirm the parasite of H. pylori in the stomach. The results are shown in Figure 10.

The red dot in Fig. 10 is a Helicobacter pylori colony, which shows that the anti-HSP60 serum has an effect of inhibiting the growth of H. pylori, but the IHP-1 (9E4) antibody can effectively eliminate H. pylori completely.

The number of colored colonies in the Helicobacter pylori isolation medium plate was calculated, and the number of Helicobacter pylori colonies (CFU) was determined. If there is a significant difference, it is represented by * (P < 0.05). The results are shown in Figure 11. Figure 11 shows that H. pylori can be completely eliminated after administration of the LHP-1 (9E4) antibody.

Example 11: Immune mechanism of anti-HpHSP60 antibody

To understand the immune mechanism of the anti-HpHSP60 antibody, for the mouse of Example 10, the urease activity of H. pylori was measured at 2, 3, and 8 weeks after inoculation of H. pylori to determine the Helicobacter pylori secretase. Urease B activity. Urease activity was normalized to the urease activity of control mice (not infected with H. pylori). The results are shown in Figure 12. The figure shows that the LHP-1 (9E4) antibody achieves an effect of inhibiting the growth of H. pylori and even eradicating H. pylori by inhibiting HpHSP60.

The LHP-1 (9E4) antibody is an amino acid sequence located at HpHSP60 101-200 as an antigen. The original storage date of the fusionoma hybridoma 9E4 containing this antibody was deposited on January 8, 2015 and deposited at the Center for Biological Resource Conservation and Research of the Food Industry Research Institute under the registration number BCRC 960493.

Example 12: Evaluation of anti-HpHSP60 antibody on the expression of Treg cells in gastric mucosa

The results of the evaluation of the anti-HpHSP60 antibody on the expression of Treg cells in the gastric mucosa were confirmed. The mouse stomach of Example 10 was fixed with 10% formalin and embedded in paraffin. Tissue sections were stained with H&E, followed by immunohistochemical staining of FoxP3. The results are shown in Fig. 13 (original 200 μm, magnification 100 times). The graph shows the amount of Treg cells present in the gastric wall sections of the sacrificed mice at week 8. Treg expression was not found in the gastric mucosa of mice treated with LHP-1 (9E4).

Example 13: Evaluation of the performance of IL-10 in gastric mucosa by anti-HpHSP60 antibody

The mouse stomach of Example 10 was fixed with 10% formalin and embedded in paraffin. Tissue sections were stained with H&E followed by immunohistochemical staining of IL-10. The results are shown in Figure 13 (original 200 μm, magnification 100 times). The figure shows the amount of IL-10 cells expressed in the gastric wall sections of the sacrificed mice at week 8. It was shown that IL-10 expression was not found in the gastric mucosa of mice treated with LHP-1 (9E4).

Example 14: HHPSP60 fragment recognizable by LHP-1 (9E4) antibody

In order to clarify the HpHSP60 fragment recognizable by the 8LHP-1 (9E4) antibody, the different fragments of HpHSP60 were identified by the 8LHP-1 (9E4) antibody. The results are shown in Figure 14. The black dot portion of the figure represents a fragment of HpHSP60 that can be recognized by the LHP-1 antibody. The identified fragments included the following, and IgK was used as the positive control group of this experiment, since most of the mouse monoclonal antibodies were kappa species: Whole table HpHSP60 1-547 full length.

1-200 table HpHSP60 1-200 fragment

101-200 table HpHSP60 101-200 fragment

1-250 table HpHSP60 1-250 fragment

200-300 table HpHSP60 200-300 fragment

300-547 table HpHSP60 300-547 fragment

From the results, it was found that the LHP-1 antibody recognizes that the fragment of HpHSP60 is 101-200. The amino acid sequence of the fragment is: EGLRNITAGANPIEVKRGMDKAAEAIINELKKASKKVGGKEEITQVATISANSDHNIGKLIADAMEKVGKDGVITVEEAKGIEDELDVVEGMQFDRGYLS

Example 15: LHP-1 (9E4) antibody recognizable HpHSP60 fragment further defined

The sequence recognized by the LHP-1 (9E3) antibody was reduced to 31 amino acid sequences by the method of Example 14. The results are shown in Figure 15. In the figure, the black dot portion represents a fragment in which HHPSP60 can be recognized by the LHP-1 (9E3) antibody. The figure shows that Δ[1] is a fragment of HpHSP60 134-200. Observed black spots, It is identifiable. While Δ[5] is a fragment of HpHSP60 101-168, no black spots were observed and were unrecognizable. This experiment can be inferred that the LHP-1 (9E3) antibody recognizes that the sequence of HpHSP60 is a 169-200 fragment. The amino acid sequence of this fragment is: KDGVITVEEAKGIEDELDVVEGMQFDRGYLS

【Biomaterial Storage】

TW Republic of China, Food Industry Development Institute, 2015/01/08, BCRC960493.

<110> Shuo Ying Sheng Medical Co., Ltd.

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Claims (17)

A monoclonal antibody or antigen-binding fragment thereof, which is a monoclonal antibody or antigen-binding fragment thereof which binds to a peptide consisting of an amino acid sequence represented by MEKVGKDGVITVE (SEQ ID NO: 1) or a similar amino acid sequence thereof. The monoclonal antibody or antigen-binding fragment thereof according to the first aspect of the patent application corresponds to a peptide composed of an amino acid sequence represented by or similar to MEKVGKDGVITVE (SEQ ID NO: 1). The monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the light chain variable region comprises the CDR1, YAASN (SEQ ID NO: 3) composed of the amino acid sequence represented by ASQSVDYDGDVFL (SEQ ID NO: 2). The CDR2 composed of the amino acid sequence represented by the amino acid sequence and the CDR3 composed of the amino acid sequence represented by QSNEVPWT (SEQ ID NO: 4). The monoclonal antibody or antigen-binding fragment thereof according to the third aspect of the patent application, wherein the light chain variable region comprises the amino acid sequence represented by No. 21 to No. 131 of SEQ ID NO: 6. The monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the heavy chain variable region comprises CDR1 and ISNGGS (SEQ ID NO: 8) composed of an amino acid sequence represented by SGFTFSSFG (SEQ ID NO: 7). The CDR3 represented by the amino acid sequence represented by the amino acid sequence and the amino acid sequence represented by QGLRRRGAMDY (SEQ ID NO: 9). The monoclonal antibody or antigen-binding fragment thereof according to claim 5, wherein the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 12 to No. 139. A use of a monoclonal antibody or antigen-binding fragment thereof as claimed in claim 1 or 2 for the inhibition of pathogenic function. The use of the scope of claim 7 wherein the pathogenic function comprises a function of inhibiting host immunity. The monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the monoclonal anti-system is selected from one of a chimera, a humanized antibody, or a human antibody. A monoclonal antibody or antigen-binding fragment thereof, as claimed in claim 1 or 2, is produced by a fusion tumor of the accession number BCRC 960493. The monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antigen-binding fragment is Fab, Fab', (Fab') 2, Fv or scFv. The monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the whole antibody immunoglobulin is IgG1, IgG2, IgG4, IgA, IgE or IgD. A fusion tumor with the registration number BCRC 960493. A fusion tumor for producing a monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2. A use of a monoclonal antibody or antigen-binding fragment thereof as claimed in claim 1 or 2 for the manufacture of a pharmaceutical composition. A method for producing a fusion tumor, comprising the steps of: using a peptide consisting of an amino acid sequence represented by MEKVGKDGVITVE (SEQ ID NO: 1) as an antigen, causing a mammal to immunologically react with the antigen; obtaining immunity against the antigen A mammalian shaped cell (immune cell), which is fused with a mammalian myeloma cell; and the resulting fusion tumor is selected for fusion to obtain a fusion tumor. A method for producing a monoclonal antibody, comprising: producing a antibody by using a fusion tumor prepared by the method of claim 16 and recovering the antibody produced by the fusion tumor.