Anti-chicken ctla-4 monoclonal antibody and application thereof

Abstract

The application discloses an anti-chicken cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) monoclonal antibody and application thereof. The anti-chicken CTLA-4 monoclonal antibody is produced by a hybridoma cell strain CTLA-4-3D7 with a preservation number of CCTCC NO: C2022207, and the monoclonal antibody can specifically recognize CTLA-4 protein expressed in a pCAGGS-CTLA-4-Flag eukaryotic expression system and a Bac-to-Bac system, has good binding activity with chicken PBMC, has a binding rate of about 16% with CD3 + The anti-chicken CTLA-4 monoclonal antibody of the application can interfere with the CTLA-4 negative regulation of the T lymphocyte activation process, thereby enhancing the anti-virus effect of the body and improving the immune effect of a vaccine, and has important application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a monoclonal antibody against chicken cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and its application. Background Technology

[0002] Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4 or CD152) is a member of the immunoglobulin-associated receptor family, primarily expressed on activated T lymphocytes. It is a negative regulator of T-cell immune responses and plays a crucial role in maintaining immune homeostasis. CTLA-4 expression and function are intrinsically linked to T-cell activation. While the CD28 / B7 pathway provides a positive stimulating signal for T-cell activation, competitive inhibition of CTLA-4 effectively weakens T-cell activation. Unlike PD-1 and PD-L1 antibody inhibitors, which directly block the tumor PD-1 signaling pathway, CTLA-4 antibody inhibitors exert an immunomodulatory effect, primarily by enhancing the killing activity of immune cells. In human cancer research, hybridoma technology has been successfully used to prepare CTLA-4 monoclonal antibodies for the treatment of solid tumors. The combination of CTLA-4 antibodies with therapeutic vaccines shows great potential in the treatment of cancer, viruses, and chronic infections. Walsh et al. found that CTLA-4 significantly amplified during Schistosoma mansoni infection in mice, which helps it evade the host's immune attack. In his research establishing a mouse model of schistosomiasis japonicus infection, Tang discovered that anti-CTLA-4 antibodies can act as adjuvants, enhancing the immunoprotective effect of GAPDH vaccines by inducing a Th1-type immune response. Lina Liu, in her in vivo studies of triple-negative breast cancer, found that the combined use of anti-CTLA-4 antibodies and mRNA vaccines significantly enhanced the body's anti-tumor immune response compared to using antibodies or vaccines alone. These results indicate that anti-CTLA-4 monoclonal antibodies can serve as effective adjuvants to enhance the immunoprotective effect of vaccines.

[0003] Currently, some avian pathogens reduce the body's immunity by suppressing the function of lymphoid organs or lymphocytes, thereby enhancing their pathogenicity. Therefore, these viruses may have a certain impact on suppressing T-cell immune responses. Viral immunosuppressive diseases, as common infectious diseases in poultry, not only cause immunosuppression but also induce other secondary diseases. Common avian immunosuppressive diseases include infectious bursal disease, Marek's disease, avian leukosis, and reticuloendotheliosis. These diseases are mainly caused by damage to immune organs and immune cells, resulting in temporary or permanent impairment of the immune response in poultry. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the purpose of this invention is to provide an anti-chicken CTLA-4 monoclonal antibody and its applications.

[0005] The specific technical solution of the present invention is as follows:

[0006] The first aspect of this invention provides a hybridoma cell line CTLA-4-3D7, which is an anti-chicken cytotoxic T lymphocyte-associated antigen 4 monoclonal antibody with accession number CCTCC NO:C2022207. This hybridoma cell line is classified and named hybridoma cell line CTLA-4-3D7, and was deposited at the China Center for Type Culture Collection on June 29, 2022.

[0007] The second aspect of the present invention provides a monoclonal antibody against chicken cytotoxic T lymphocyte-associated antigen 4, produced by the hybridoma cell line CTLA-4-3D7 with accession number CCTCC NO:C2022207.

[0008] The third aspect of the present invention provides the use of the monoclonal antibody in the preparation of any of the products shown in (1)-(9) below:

[0009] (1) Products that are anti-chicken cytotoxic T lymphocyte-associated antigen 4;

[0010] (2) Products that relieve the inhibition of T lymphocyte activation by chicken cytotoxic T lymphocyte-associated antigen 4;

[0011] (3) Products that relieve the suppression of the body's immunity by chicken cytotoxic T lymphocyte-associated antigen 4;

[0012] (4) Products that activate chicken T lymphocytes;

[0013] (5) Products that improve the immune level of chickens;

[0014] (6) Products for detecting chicken cytotoxic T lymphocyte-associated antigen 4;

[0015] (7) Products that specifically bind to chicken cytotoxic T lymphocyte-associated antigen 4;

[0016] (8) Products for the prevention and / or treatment of viral immunosuppressive diseases in chickens;

[0017] (9) An adjunct product to enhance the immune effect of vaccines against viral immunosuppressive diseases in chickens.

[0018] In the above applications, the diseases include Marek's disease, infectious anemia, infectious bursal disease, avian adenovirus disease, avian influenza, and Newcastle disease.

[0019] In the above applications, when the disease is Newcastle disease in chickens, the vaccine is preferably the Newcastle disease LaSota vaccine strain.

[0020] A fourth aspect of the present invention provides a vaccine composition, characterized in that the vaccine composition comprises a vaccine against a viral immunosuppressive disease in chickens and the monoclonal antibody.

[0021] In the above composition, the diseases include Marek's disease, infectious anemia, infectious bursal disease, avian adenovirus disease, avian influenza, and Newcastle disease.

[0022] In the above composition, when the disease is Newcastle disease in chickens, the vaccine is preferably the Newcastle disease LaSota vaccine strain.

[0023] Furthermore, the method of applying the vaccine composition is as follows: chickens are simultaneously immunized with the vaccine and injected with monoclonal antibodies, followed by a booster immunization with monoclonal antibodies.

[0024] Furthermore, the injection dose of the monoclonal antibody is 1 mg / kg.

[0025] The beneficial effects of this invention are as follows:

[0026] 1. This invention provides a monoclonal antibody against chicken cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4-3D7), produced from the hybridoma cell line CTLA-4-3D7 with accession number CCTCC NO: C2022207. This monoclonal antibody specifically recognizes CTLA-4 protein expressed in the pCAGGS-CTLA-4-Flag eukaryotic expression system and the Bac-to-Bac system. FCM and IFA analyses show that this monoclonal antibody exhibits good binding activity to chicken PBMCs and CD3+. + The binding rate of T lymphocytes was approximately 16%, significantly higher than that of hybridoma cell lines CTLA-4-5A4 and CTLA-4-6A12, which produced monoclonal antibodies against CD3. + The binding rate of T lymphocytes has achieved unexpected technical results.

[0027] 2. The monoclonal antibody against chicken cytotoxic T-lymphocyte-associated antigen 4 of this invention enhances the body's antiviral activity and improves the immune efficacy of vaccines by interfering with the negative regulation of T-lymphocyte activation by CTLA-4. It can be used to prepare products that relieve the inhibition of T-lymphocyte activation by chicken cytotoxic T-lymphocyte-associated antigen 4, products that relieve the inhibition of the body's immunity by chicken cytotoxic T-lymphocyte-associated antigen 4, products that activate chicken T-lymphocytes, products that improve the immune level of chickens, products that prevent and / or treat chicken viral immunosuppressive diseases, and adjuvant products that enhance the immune efficacy of vaccines for chicken viral immunosuppressive diseases.

[0028] 3. The monoclonal antibody against chicken cytotoxic T lymphocyte-associated antigen 4 of the present invention, when used in combination with the Newcastle disease LaSota vaccine strain, can significantly improve the immunization effect of the Newcastle disease LaSota vaccine strain. Attached Figure Description

[0029] Figure 1 PCR amplification of the CTLA-4 gene;

[0030] Figure 2 Sequence alignment results of plasmid pFastBacHTA-CTLA-4;

[0031] Figure 3 : Sequence alignment results of plasmid rBacmid-CTLA-4;

[0032] Figure 4 IFA identification of recombinant baculovirus rBac-CTLA-4;

[0033] Figure 5 : Western blot detection of recombinant baculovirus rBac-CTLA-4; M: pre-stained protein marker; 1-5: Sf9 cells infected with rBac-CTLA-4; 6: Sf9 cells infected with wild-type virus;

[0034] Figure 6 IFA identification of CTLA-4 monoclonal antibodies; A: Anti-chicken CTLA-4 polyclonal antibody serum; B: ICR mouse serum; C: mAb-CTLA-4-3D7; D: mAb-CTLA-4-5A4; E: mAb-CTLA-4-6A12;

[0035] Figure 7 : Monoclonal antibody binding analysis to PBMCs; A: Isotype IgG antibody control; B: mAb-CTLA-4-3D7; C: mAb-CTLA-4-5A4; D: mAb-CTLA-4-6A12;

[0036] Figure 8 mAb-CTLA-4-3D7 and CD3 + T-cell binding analysis; A: Isotype IgG antibody control; B: mAb-CTLA-4-3D7;

[0037] Figure 9 NDV-specific antibody level detection;

[0038] Figure 10 : Differential analysis of immune cytokines;

[0039] Preservation Instructions

[0040] The hybridoma cell line CTLA-4-3D7, classified and named as hybridoma cell line CTLA-4-3D7, was deposited on June 29, 2022, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: C2022207. Detailed Implementation

[0041] To better understand the present invention, it is now further described with reference to the following embodiments and accompanying drawings. The embodiments are for illustrative purposes only and do not limit the invention in any way. In the embodiments, all original reagents and materials are commercially available, and experimental methods not specifically specified are conventional methods and conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0042] Example 1

[0043] I. Amplification of the chicken CTLA-4 gene

[0044] Lymphocytes were isolated from chicken peripheral blood. RNA was extracted and reverse transcribed. Using cDNA as a template, the CDS region sequence of the chicken CTLA-4 gene (nucleotide sequence as SEQ ID NO.1, amino acid sequence as SEQ ID NO.2) was amplified by PCR using designed specific primers (see Table 1). After 1% agarose gel electrophoresis, a 618 bp band of the CTLA-4 target gene, consistent with the expected sequence, was successfully obtained. Figure 1 ).

[0045] Table 1. Chicken CTLA-4 amplification primers

[0046]

[0047] II. Recombinant Baculovirus System for Systemic Expression of Chicken CTLA-4

[0048] The CTLA-4 gene was inserted into a plasmid using a bac-to-bac system to obtain pFastBacHTA-CTLA-4. The correctly sequenced pFastBacHTA-CTLA-4 (sequence alignment results are shown below) Figure 2 Transformed into DH10Bac competent cells, the recombinant baculovirus shuttle plasmid rBacmid-CTLA-4 was obtained (sequence alignment results are shown below). Figure 3The recombinant baculovirus rBac-CTLA-4 was obtained by transfecting Sf9 insect cells. Sf9 insect cells in the logarithmic growth phase were then infected with the obtained recombinant baculovirus rBac-CTLA-4. When obvious cytopathic effects appeared, IFA and Western blot experiments were performed, and CTLA-4 protein expression was detected using 6×His Tag Antibody. Both IFA and Western blot results showed that Sf9 cells infected with rBac-CTLA-4 exhibited bright green fluorescence compared to Sf9 cells infected with wild-type virus. Figure 4 Furthermore, the lysate of infected cells contained a specific target band of approximately 26 kDa, consistent with the expected size of the CTLA-4 target fragment. Figure 5 The results showed that a recombinant baculovirus capable of efficiently expressing the CTLA-4 protein in Sf9 insect cells was successfully prepared.

[0049] III. Preparation of Immunogens

[0050] Recombinant baculovirus rBac-CTLA-4 was used to infect Sf9 insect cells to efficiently express CTLA-4 protein. Sf9 insect cell suspensions that produced lesions were collected, and the cells were disrupted using an ultrasonic cell disruptor. The mixture was then mixed with adjuvant and used to directly immunize mice.

[0051] IV. Establishment of hybridoma cell lines and preparation of monoclonal antibodies

[0052] 1. Using the protein expressed by recombinant baculovirus rBac-CTLA-4 in Sf9 insect cells as an immunogen, 6-8 week old Balb / c mice were intraperitoneally immunized with Freund's adjuvant;

[0053] 2. After the third immunization, serum antibodies in immunized mice were detected using immunofluorescence.

[0054] 3. Take mice with high antibody titers and use conventional hybridoma technology to fuse spleen cells and myeloma cells SP2 / 0 from the immunized mice;

[0055] 4. Transfect the constructed pCAGGS-CTLA-4-Flag plasmid into DF-1 cells, and screen positive hybridoma cells using the IFA method;

[0056] 5. Positive wells were marked and retested. Through three consecutive subclonings, three hybridoma cell lines stably secreting anti-chicken CTLA-4 monoclonal antibodies were finally obtained, named CTLA-4-3D7; CTLA-4-5A4; and CTLA-4-6A12, respectively. Hybridoma cell line CTLA-4-3D7 was deposited at the China Center for Type Culture Collection (CCTCC) on June 29, 2022, with accession number CCTCC NO: C2022207.

[0057] Monoclonal antibodies prepared from hybridoma cell lines CTLA-4-3D7, CTLA-4-5A4, and CTLA-4-6A12 are designated as mAb-CTLA-4-3D7, mAb-CTLA-4-5A4, and mAb-CTLA-4-6A12, respectively. Subclass identification is shown in Table 2.

[0058] Table 2. Subclass identification of monoclonal antibodies

[0059]

[0060] Example 2

[0061] I. IFA Identification of CTLA-4 Monoclonal Antibodies

[0062] The amplified CTLA-4 gene was inserted into the pCAGGS vector to construct the pCAGGS-CTLA-4-Flag eukaryotic expression plasmid. The plasmid DNA was transfected into DF1 cells using a commercially available transfection reagent for transient expression, and the cells were fixed with ethanol and acetone as the detection antigen. Monoclonal antibodies were reacted with DF-1 cells transfected with pCAGGS-CTLA-4-Flag and Sf9 cells infected with recombinant baculovirus, respectively. Immunofluorescence detection was performed using anti-mouse IgG FITC-labeled antibodies. Results showed that all three monoclonal antibodies effectively recognized the CTLA-4 protein in both the pCAGGS-CTLA-4-Flag eukaryotic expression system and the baculovirus expression system. Figure 6 ).

[0063] II. Binding analysis of CTLA-4 monoclonal antibody to natural protein

[0064] Since CTLA-4 is mainly expressed on activated T lymphocytes and is weakly expressed in resting T cells, it is considered a weak expression marker. ConA 10 μg / mL was added to isolated and cultured chicken PBMCs at a density of 5 × 10⁻⁶ cells / mL. 6 The cells were incubated at 37°C for 72 h in a 5% CO2 incubator at a concentration of 1 / mL, and then subjected to FCM analysis using three monoclonal antibodies.

[0065] The FCM method was used to analyze the binding of monoclonal antibodies to PBMCs. Results showed that, using IgG isotype antibodies as a control, all three monoclonal antibodies could bind to ConA-stimulated chicken PBMCs, and mAb-CTLA-4-3D7 exhibited clear positive and negative population peaks. Figure 7 ).

[0066] To further verify whether monoclonal antibodies can bind to T lymphocytes, T lymphocytes were labeled with Mouse Anti-Chicken CD3 antibody by flow cytometry, with mouse IgG isotype antibody as a negative control. The binding of the three monoclonal antibodies to CD3 was analyzed by FCM. + The binding activity of T lymphocytes. The specific steps are as follows:

[0067] (1) Prepare PBMCs stimulated by ConA. After 72 h, collect the cell suspension in a 2 mL centrifuge tube, centrifuge at 0.4 rcf for 5 min at 4 °C, and discard the supernatant.

[0068] (2) Add 500 μL of mAb-CTLA-4-3D7 ascites fluid (1:100 dilution), incubate at room temperature for 45 min, centrifuge at 0.4 rcf for 5 min at 4℃, and wash twice with PBS.

[0069] (3) Add 500 μL of goat anti-mouse IgG-FITC secondary antibody (1:100 dilution), incubate at room temperature for 30 min in the dark, centrifuge at 0.4 rcf for 5 min at 4℃, and wash twice with PBS.

[0070] (4) Add 100 μL of Mouse Anti-Chicken CD3 flow cytometry antibody (diluted with 0.5% BSA 1:400), incubate at room temperature in the dark for 30 min, centrifuge at 0.4 rcf for 5 min at 4℃, and wash twice with PBS.

[0071] (5) Add 1 mL of 0.5% to 1% paraformaldehyde to each tube to fix the cells.

[0072] (6) The experiment also requires setting up untreated PBMCs as blank controls, PBMCs incubated with IgG isotype antibodies as negative controls, and FITC and CD3 single-staining tubes for adjustment and compensation.

[0073] (7) Detection of mAb-CTLA-4-3D7 and CD3 using CytoFlex S flow cytometer + The binding status of T lymphocytes.

[0074] The results showed that mouse IgG isotype antibodies did not interact with CD3. + T lymphocytes bind to each other, while mAb-CTLA-4-3D7 can bind to some CD3+ cells. + T lymphocytes bind to CD3, which accounts for approximately [a percentage] of CD3. + 16% of T lymphocytes ( Figure 8 ).

[0075] Example 3: Effect of anti-chicken CTLA-4 monoclonal antibody on the immunization efficacy of LaSota strain vaccine

[0076] I. Grouping and Treatment of Laboratory Animals

[0077] Fifteen 7-day-old SPF chicks were randomly divided into three groups: NC, LaSota, and LaSota+mAb-CTLA-4-3D7, with five chicks in each group. After one week of acclimatization, the chicks were immunized. The NC group was fed normally and received a saline injection as a control. The LaSota group received the LaSota vaccine at 2 weeks of age, diluted with sterile saline or distilled water according to the dosage indicated on the label, and administered via nasal or ocular drops. The LaSota+mAb-CTLA-4-3D7 group received the LaSota vaccine at 2 weeks of age and simultaneously received mAb-CTLA-4-3D7 at a dose of 1 mg / kg, as per the study by Wolchok JD et al., with a booster immunization at the second week. Differences in serum antibody levels and immune strength among the three groups were assessed experimentally. The specific procedures are shown in Table 3 below.

[0078] Table 3 Grouping and Treatment of Experimental Animals

[0079]

[0080] II. NDV-specific antibody levels

[0081] Chickens were immunized with LaSota live attenuated vaccine and mAb-CTLA-4-3D7. Blood samples were collected from the wing veins on days 7, 14, 21, and 28 to obtain chicken serum. Hemagglutination inhibition assays were performed, and the levels of anti-NDV specific antibodies in the serum of the NC group, LaSota group, and LaSota+mAb-CTLA-4-3D7 group were detected using the HI method. Antibody titers were expressed as log2 (base 2). Results showed that antibody levels in the LaSota group and the LaSota+mAb-CTLA-4-3D7 group were consistently higher than those in the NC group from day 7 after immunization. The antibody level in the LaSota+mAb-CTLA-4-3D7 group was higher than that in the LaSota group every week, with a significant difference at day 21 (*P<0.05) and a highly significant difference at day 28 (**P<0.01). Figure 9 ).

[0082] III. Differential Analysis of Immune Cytokines

[0083] Anti-CTLA-4 mAb works by binding to the CTLA-4 receptor on T lymphocytes, blocking or inhibiting the CTLA-4 / B7 signaling pathway and upregulating the body's immune response. IFN-γ and IL-2 are Th1-type cytokines, mainly playing a role in cellular immunity; IL-5 is a Th2-type cytokine, playing a role in humoral immunity. To evaluate the effects of Anti-CTLA-4 mAb on the avian immune system, cytokine levels in PBMCs of experimental animals were detected by qPCR on day 14 after the first immunization, and the expression levels of IFN-γ, IL-2, and IL-5 in chicken PBMCs of the three groups were analyzed. The results showed that there was no significant difference in the levels of IFN-γ, IL-2, and IL-5 mRNA in chicken PBMCs between the NC group and the LaSota group; compared with the NC group and the LaSota group, the levels of IFN-γ, IL-2, and IL-5 mRNA in chicken PBMCs of the LaSota+Anti-CTLA-4 mAb group showed an increasing trend, but this was not statistically significant. Figure 10 ).

[0084] IV. Lymphocyte Transformation Experiment

[0085] MTT can be reduced by certain dehydrogenases in mitochondria to form a crystalline, dark purple product called formazan, which can be completely dissolved in the presence of a specific solvent. The OD value can then be measured using a microplate reader. 570nm The absorbance was measured to determine cell proliferation activity. The MTT assay was used for lymphocyte transformation. After immunization, chicken PBMCs were stimulated with ConA in vitro for 48 hours. The proliferation activity of chicken lymphocytes in different groups was then measured, and lymphocyte proliferation curves were plotted to evaluate the immune levels of the three groups of experimental animals. The results showed that the lymphocyte proliferation activity in the LaSota+mAb-CTLA-4-3D7 group and the LaSota group was higher than that in the NC group; and the proliferation activity of chicken lymphocytes in the LaSota+mAb-CTLA-4-3D7 group was higher than that in the LaSota group. This indicates that mAb-CTLA-4-3D7 enhanced the activity of chicken lymphocytes under LaSota vaccine immunization conditions.

[0086] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. The hybridoma cell line CTLA-4-3D7, with accession number CCTCC NO:C2022207, which produces monoclonal antibodies against chicken cytotoxic T lymphocyte-associated antigen 4.

2. A monoclonal antibody against chicken cytotoxic T lymphocyte-associated antigen 4, produced by the hybridoma cell line CTLA-4-3D7 with accession number CCTCC NO:C2022207.

3. The use of the monoclonal antibody according to claim 2 in the preparation of any of the products shown in (1)-(8) below: (1) Products containing anti-chicken cytotoxic T lymphocyte-associated antigen 4; (2) Products that relieve the inhibition of T lymphocyte activation by chicken cytotoxic T lymphocyte-associated antigen 4; (3) Products that relieve the suppression of the body's immunity by chicken cytotoxic T lymphocyte-associated antigen 4; (4) Products that activate chicken T lymphocytes; (5) Products that improve the immune level of chickens; (6) Products for detecting chicken cytotoxic T lymphocyte-associated antigen 4; (7) Products that specifically bind to chicken cytotoxic T lymphocyte-associated antigen 4; (8) Adjunctive products that enhance the immune effect of Newcastle disease vaccines in chickens.

4. The application according to claim 3, characterized in that, The vaccine in question is the Newcastle disease vaccine strain LaSota.

5. A vaccine composition, characterized in that, The vaccine composition comprises a vaccine against Newcastle disease in chickens and the monoclonal antibody as described in claim 2.

6. The vaccine composition according to claim 5, characterized in that, The vaccine in question is the Newcastle disease vaccine strain LaSota.

7. The vaccine composition according to claim 5, characterized in that, The method of applying the vaccine composition is as follows: chickens are simultaneously immunized with the vaccine and injected with monoclonal antibodies, followed by a booster immunization with monoclonal antibodies.

8. The vaccine composition according to claim 5, characterized in that, The injection dose of the monoclonal antibody is 1 mg / kg.

Images