An anti-CD127 antibody and its preparation method and application

Abstract

The present invention provides an anti-CD127 antibody, wherein the amino acid sequence of the heavy chain CDR region is selected from SEQ ID NOs. 1-3, and the amino acid sequence of the light chain CDR region is selected from SEQ ID NOs. 4-6. The present invention belongs to the field of antibodies. The novel anti-CD127 antibody provided by the present invention has good binding ability to CD127 and can be used to prepare genetically engineered cells expressing the anti-CD127 antibody, thereby achieving large-scale production and providing more convenient conditions for the detection of CD127.

Description

Technical Field

[0001] The present invention belongs to the field of antibodies, and specifically relates to an anti-CD127 antibody and a preparation method and application thereof. Background Art

[0002] CD127 is the receptor for IL-7 and plays an important role in the development of T lymphocytes, the maintenance of T cell homeostasis, and the differentiation and survival of memory T cells. It is also a useful marker for identifying memory and effector T cells.

[0003] Existing technologies have publicly studied monocytes in various inflammatory tissues in the human body through techniques such as tissue sections and single-cell transcriptome sequencing. It has been found that a significant number of monocytes with high CD127 expression are present in the lungs of COVID-19 patients and in the peripheral blood and joint tissues of rheumatoid arthritis patients. It has also been revealed that differences in CD127 expression levels between monocyte subpopulations indicate varying degrees of monocyte inflammatory phenotypes, with subpopulations with high CD127 expression exhibiting low expression of multiple pro-inflammatory factors. [1]

[0004] Given the multiple roles of CD127, its detection is extremely important. Fluorescently labeled antibodies are often used to detect CD127 in existing technologies, and developing better monoclonal antibodies against CD127 has become particularly valuable and has promising applications.

[0005] The Chinese patent application number 201911235422.6 discloses an anti-CD127 antibody, a cell line secreting the antibody, and its preparation method and application, which belong to the field of biomedicine and immunochemistry technology. The antibody or the antigen-binding fragment of the antibody specifically binds to CD127 and specifically recognizes the epitope sequence of CD127. The prepared anti-hCD127 monoclonal antibody and the monoclonal antibody hybridoma cell line CD127-7E7 that secretes the monoclonal antibody can specifically recognize human CD127 and have good affinity. It can not only be used for qualitative or quantitative detection of CD127 protein; it can also determine the epitope or functional domain of CD127 protein, so it has good practical application value. However, when preparing antibodies using the hybridoma method, the antibody gene is easily lost due to the instability of the hybridoma. In addition, the hybridoma preparation technology will not identify the gene sequence and protein sequence of the target antibody, which is not conducive to the subsequent production, preparation and research and development of the target antibody.

[0006] The currently commercialized CD127 antibody with clone number A019D5 also has the above problems and there is room for further research.

[0007] Genetic engineering methods can effectively solve the problems in the hybridoma method. The necessary condition for preparing antibodies through genetic engineering methods is to clarify the antibody sequence. Therefore, discovering monoclonal antibodies that are more suitable for CD127 detection is currently a more appropriate research and development direction.

[0008] [1]Zhang B, Zhang Y, Xiong L, et al. CD127 imprints functional heterogeneity to diversify monocyte responses in inflammatory diseases[J]. Journal of Experimental Medicine, 2022, 219(2):-. Summary of the Invention

[0009] In order to solve the above problems, the present invention provides a specific amino acid sequence of an anti-CD127 antibody, through which genetically engineered cells expressing anti-CD127 antibodies can be obtained using existing technologies, thereby improving the yield and production stability of CD127 antibodies.

[0010] In one aspect, the present invention provides an anti-CD127 antibody.

[0011] The anti-CD127 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region, and the light chain comprises a light chain variable region; the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein:

[0012] (1) HCDR1 is the amino acid sequence shown in SEQ ID NO. 1, or any one or more amino acid sequences having at least 85% sequence similarity to SEQ ID NO. 1;

[0013] (2) HCDR2 is the amino acid sequence shown in SEQ ID NO. 2, or any one or more amino acid sequences having at least 85% sequence similarity to SEQ ID NO. 2;

[0014] (3) HCDR3 is the amino acid sequence shown in SEQ ID NO. 3, or any one or more amino acid sequences having at least 85% sequence similarity to SEQ ID NO. 3;

[0015] (4) LCDR1 is the amino acid sequence shown in SEQ ID NO. 4, or any one or more amino acid sequences having at least 85% sequence similarity to SEQ ID NO. 4;

[0016] (5) LCDR2 is the amino acid sequence shown in SEQ ID NO. 5, or any one or more amino acid sequences having at least 85% sequence similarity to SEQ ID NO. 5;

[0017] (6) LCDR3 is the amino acid sequence shown in SEQ ID NO. 6, or any one or more amino acid sequences having at least 85% sequence similarity to SEQ ID NO. 6.

[0018] The anti-CD127 antibody is a monoclonal antibody.

[0019] Preferably, the anti-CD127 antibody is a murine antibody or a rabbit antibody.

[0020] Preferably, the amino acid sequence of HCDR1 of the anti-CD127 antibody is SEQ ID NO.1, the amino acid sequence of HCDR2 is SEQ ID NO.2, and the amino acid sequence of HCDR3 is SEQ ID NO.3.

[0021] Preferably, the amino acid sequence of the anti-CD127 antibody LCDR1 is SEQ ID NO.4, the amino acid sequence of LCDR2 is SEQ ID NO.5, and the amino acid sequence of LCDR3 is SEQ ID NO.6.

[0022] Preferably, the amino acid sequence of the heavy chain variable region of the anti-CD127 antibody is SEQ ID NO. 7, or a sequence having at least 80% sequence similarity to SEQ ID NO. 7.

[0023] Preferably, the amino acid sequence of the light chain variable region of the anti-CD127 antibody is selected from SEQ ID NO.8, or SEQ ID NO.9, or a sequence having at least 80% sequence similarity to SEQ ID NO.8, or a sequence having at least 80% sequence similarity to SEQ ID NO.9.

[0024] Further preferably, the amino acid sequence of the heavy chain variable region of the anti-CD127 antibody is SEQ ID NO.7, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO.8 or SEQ ID NO.9.

[0025] Preferably, the anti-CD127 antibody heavy chain amino acid sequence is SEQ ID NO.10, or a sequence having at least 65% sequence similarity to SEQ ID NO.10, or SEQ ID NO.12, or a sequence having at least 65% sequence similarity to SEQ ID NO.12; the anti-CD127 antibody light chain amino acid sequence is SEQ ID NO.11, or a sequence having at least 65% sequence similarity to SEQ ID NO.11; or SEQ ID NO.13, or a sequence having at least 65% sequence similarity to SEQ ID NO.13.

[0026] Preferably, the anti-CD127 antibody heavy chain amino acid sequence is SEQ ID NO.10, and the light chain amino acid sequence is SEQ ID NO.11.

[0027] Preferably, the heavy chain amino acid sequence of the anti-CD127 antibody is SEQ ID NO.12, and the light chain amino acid sequence is SEQ ID NO.13.

[0028] In another aspect, the present invention provides a nucleotide sequence for expressing the aforementioned anti-CD127 antibody.

[0029] In another aspect, the present invention provides an expression vector comprising the aforementioned nucleotide sequence.

[0030] In another aspect, the present invention provides a cell expressing the aforementioned anti-CD127 antibody.

[0031] Specifically, the cells include the aforementioned expression vector.

[0032] Preferably, the cells may be HEK293 or CHO.

[0033] In another aspect, the present invention provides a method for preparing the aforementioned anti-CD127 antibody.

[0034] The method comprises culturing the aforementioned cells.

[0035] In another aspect, the present invention provides a method for fluorescently labeling the aforementioned anti-CD127 antibody.

[0036] The method includes labeling the anti-CD127 antibody with a macromolecular dye or a small molecule dye.

[0037] Preferably, the macromolecular dye is APC, and the small molecule dye is FITC.

[0038] In another aspect, the present invention provides the use of the aforementioned anti-CD127 antibody or nucleotide sequence or expression vector or cell in CD127 detection.

[0039] The application is not for disease diagnosis or treatment.

[0040] The application is achieved through antibody-antigen binding.

[0041] In another aspect, the present invention provides the use of the aforementioned anti-CD127 antibody or nucleotide sequence or expression vector or cell in the preparation of a CD127 detection kit.

[0042] The kit includes the aforementioned anti-CD127 antibody or nucleotide sequence or expression vector or cell.

[0043] The kit also includes other reagents for CD127 detection, such as buffer and sample treatment agent.

[0044] The kit may also include molecules for fluorescently labeling antibodies.

[0045] The molecule can be a macromolecular dye or a small molecule dye.

[0046] Preferably, the macromolecular dye is APC, and the small molecule dye is FITC.

[0047] In another aspect, the present invention provides a method for detecting CD127.

[0048] The detection method is performed by using the aforementioned anti-CD127 antibody.

[0049] The detection method also includes a sample pre-treatment step.

[0050] The detection method is not a method for diagnosing or treating a disease.

[0051] In another aspect, the present invention provides a CD127 detection kit.

[0052] The kit includes the aforementioned anti-CD127 antibody or nucleotide sequence or expression vector or cell.

[0053] The kit also includes other reagents for CD127 detection, such as buffer and sample treatment agent.

[0054] The kit may also include molecules for fluorescently labeling antibodies.

[0055] The molecule can be a macromolecular dye or a small molecule dye.

[0056] Preferably, the macromolecular dye is APC, and the small molecule dye is FITC.

[0057] Beneficial effects of the present invention:

[0058] The present invention provides a new anti-CD127 antibody that has good binding ability with CD127 and can be used to prepare cells expressing the anti-CD127 antibody, thereby achieving large-scale production and providing more convenient conditions for the detection of CD127. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 This is the SDS gel electrophoresis diagram of anti-human CD127 antibody.

[0060] Figure 2 The flow cytometry results are for the FITC-labeled control CD127 [A019D5] antibody from Biolegend.

[0061] Figure 3 FITC-labeled Flow cytometry results.

[0062] Figure 4 FITC-labeled Flow cytometry results.

[0063] Figure 5 The figure shows the flow cytometry results of the APC-labeled control CD127 [A019D5] from Biolegend.

[0064] Figure 6 APC marker Flow cytometry results.

[0065] Figure 7 APC marker Flow cytometry results.

[0066] Figure 8 The results show the affinity test of the commercial Biolegend control CD127 [A019D5] antibody.

[0067] Figure 9 for Affinity test results.

[0068] Figure 10 for Affinity test results. DETAILED DESCRIPTION

[0069] The present invention will be further described in detail below with reference to specific examples. The following examples are not intended to limit the present invention but are merely intended to illustrate the present invention. The experimental methods used in the following examples are generally based on conventional conditions unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0070] Example 1 Preparation and detection of antibodies

[0071] Two antibodies were prepared, one is anti-human CD127 mouse antibody, numbered The other is anti-human CD127 rabbit antibody, numbered

[0072] The heavy chain amino acid sequence is SEQ ID NO.10, and the light chain amino acid sequence is SEQ ID NO.11. The heavy chain amino acid sequence is SEQ ID NO.12, and the light chain amino acid sequence is SEQ ID NO.13. The nucleotide sequence for expressing the corresponding antibody was designed using conventional methods, as follows:

[0073]

[0074] The nucleotide sequence was cloned into a CHO stable strain for antibody stable expression, and the constructed vector was further introduced into CHO cells.

[0075] Transfection and screening:

[0076] (1) CHO cells were cultured in CHO CD medium, 2×10 5 cells / mL, add 4mM glutamine. The cell density reaches 4×10 6 cells / mL, with a viability greater than 95%, the cells were collected by centrifugation and resuspended in electroporation buffer to adjust the density to 1×10 7 cells / mL.

[0077] (2) Take a sterile EP tube, add 0.8 mL of cell suspension, add 5 μg of plasmid, mix gently, incubate for 15 minutes, and place on ice for 5 minutes. The parameters of the electroporator are voltage 280V, pulse number 3 times, pulse time 5ms, pulse interval 0.784s, and inner diameter of the electroporator cup 4mm. Add 0.5 mL of cell and plasmid mixture to each electroporator cup, place it in the electroporator and start the electroporation program. After completion, quickly aspirate the cells in the electroporator cup and transfer them to a 125 mL conical flask containing 20 mL of CHO CD culture medium. Repeat the above steps to electroporate the second electroporator cup. Gently mix the cells in the shaker bottle, then cover it tightly and place it in a 37°C shaker for culture.

[0078] (3) After 48 h, transfer the cells to a centrifuge tube and centrifuge at 1000 rpm for 5 min. Remove the supernatant and resuspend the cells in 20 mL of CHO CD medium containing 2, 5, or 20 μM MSX. Calculate the cell density and activity. 6 cells / mL.

[0079] (4) Transfer the cells to a new shake flask. Starting from the seventh day, observe the cell density and activity every two days. The cell density increased from 1.2×10 6 cells / mL decreased, reaching the lowest density on the 5th to 7th day, and the cell density began to increase from the 10th to 15th day. 6 cells / mL, the cells were passaged to a cell density of 3×10 cells / mL using CD OPM medium containing 50 μM MSX. 5 cells / mL and continue culturing.

[0080] (5) When the cell density increases to 4×10 6 cells / mL, the cells were expanded or passaged using CD OPM medium containing 50 μM MSX, and the minimum seeding density was still 3×10 5 cells / mL, you can start preparing for freezing or expanding culture.

[0081] (6) Antibody collection method: Antibody purification was performed using protein A affinity chromatography.

[0082] Take the commercial anti-human CD127 antibody A019D5 and the anti-human CD127 antibody prepared in Example 1 Anti-human CD127 antibody 10 μg of each antibody was mixed with loading buffer (the volume ratio of each antibody to loading buffer was 4:1), incubated in a 100°C water bath for 30 minutes, and centrifuged at 10,000 rpm for 1 minute at room temperature to obtain the test sample. The precast gel was removed and installed in an electrophoresis tank. Running buffer was added, and the sample was loaded with a comb. 10 μL of protein marker and 10 μg of test sample were loaded. The electrophoresis instrument settings were: voltage 80 V, run time 90 minutes, and end when the bromophenol blue indicator band reached the bottom. The gel was removed and stained with Coomassie Brilliant Blue for 30 minutes. Destaining was performed in destaining buffer for 1 hour. Repeat five times before photographing.

[0083] The results showed that the heavy and light chains of the anti-human CD127 antibody prepared in Example 1 were intact, the bands were clear, and the purity was above 95%. Figure 1 .

[0084] Example 2 Antibody Labeling

[0085] Use FITC (fluorescein isothiocyanate) or APC labeling and

[0086]

[0087] 1. FITC labeling

[0088] (1) Take and 0.2 mg of each was placed in an ultrafiltration tube and washed three times with PBS containing a final concentration of 1 mM EDTA. The ultrafiltration tube was centrifuged inverted at 4000 g for 2 minutes to collect the concentrated antibody and dilute it to 10 mg / mL with PBS containing a final concentration of 1 mM EDTA.

[0089] (2) Add the small molecule dye FITC (add the dye at a molar ratio of nantibody:nFITC = 1:15) to the monoclonal antibody reaction solution and mix well.

[0090] (3) Incubate at 25°C in a shaker in the dark for 45 minutes.

[0091] (4) Transfer the reaction solution to an ultrafiltration centrifuge tube and wash the antibody five times with PBS containing a final concentration of 1 mM EDTA. Centrifuge the ultrafiltration tube upside down at 4000 g for 2 min to collect the concentrated antibody, which was then diluted with PBS containing a final concentration of 1 mM EDTA to a final concentration of 0.5 mg / mL.

[0092] The labeled antibody is recorded as and

[0093]

[0094] 2. APC labeling

[0095] According to the method described in the patent "Fluorescent protein and / or coupled protein monoclonal antibody labeling method and kit (CN202010972671.X)", the and

[0096]

[0097] The labeled antibody is recorded as and

[0098]

[0099] 3. Detection of the effect of labeled fluorescent antibodies

[0100] Add 100 μL of commercial quality control blood (manufacturer: Beckman Coulter, Inc; catalog number: 6607077) to a 1.5 mL centrifuge tube, take 1 μL of fluorescent-labeled antibody and add it to the quality control blood, and react in the dark for 15 minutes.

[0101] Add 1 mL of hemolysin and continue the reaction for 15 minutes. The hemolysin used was the hemolysin for Zhengxi flow cytometry (Zhengxi Biotechnology Co., Ltd. Hemolysin: Zhejiang Huxiebei No. 20200133).

[0102] Centrifuge at 4000 rpm for 2 min, discard the supernatant, and add 500 uL PBS to resuspend the sample to be tested.

[0103] Flow cytometry (Beckman DxFlex flow cytometer) detects the proportion of positive cells in the sample, the clustering of the graph, and other indicators to determine and Whether it can be labeled with fluorescent dyes FITC and APC, and whether the labeled fluorescent antibodies can detect the target antigen correctly.

[0104] Both FITC and APC were successfully labeled and The ratio is similar to that of the commercial Biolegend control anti-human CD127 antibody A019D5, see Figure 2-Figure 7 .

[0105] Example 3 Antibody Titer

[0106] The antibodies used in this example are:

[0107] Antibody (1): positive control, commercial anti-human CD127 antibody A019D5;

[0108] Antibody (2): Anti-human CD127 antibody obtained in Example 1

[0109] Antibody (3): Anti-human CD127 antibody obtained in Example 1

[0110] Antibody titer detection method:

[0111] First, the known concentrations of antibodies (1), (2), and (3) were diluted to 1 mg / mL, and then diluted again with PBS at a ratio of 1:100, 1:1000, 1:3000, 1:6000, 1:12000, 1:24000, 1:48000, 1:60000, and 1:96000. The antibody was used as the experimental group, and the anti-human CD127 [A019D5] antibody was used as the control group.

[0112] The experiment was divided into two groups. The control group was divided into two groups. 100 μL of commercial quality control blood (manufacturer: Beckman Coulter, Inc; catalog number: 6607077) was taken from each group. 1 μL of anti-human CD127-PE commercial flow cytometry fluorescent antibody (manufacturer: Biolegend; catalog number: 986002) was added to each group. After incubation in the dark for 15 minutes, 1 mL of hemolysin (hemolysin for Zhengxi flow cytometer, Zhengxi Biotechnology Co., Ltd.) was added to each of the three experimental groups. Hemolysin: Zhejiang Hu Ji Bei No. 20200133), the reaction was continued for 15 min. Centrifuged at 4000 rpm for 2 min, and the supernatant was discarded.

[0113] The experimental groups were added with 100 μL of diluted or For the control group, 100 μL of commercially available anti-human CD127 antibody A019D5 diluted proportionally was added. After mixing, the mixture was incubated at room temperature in the dark for 30 minutes. The sample was then resuspended in 900 μL of PBS and centrifuged at 4000 rpm for 2 minutes. The supernatant was discarded, and the sample to be tested was resuspended in 500 μL of PBS and analyzed by flow cytometry.

[0114] Test results are shown in Figure 8-10 :

[0115] Figure 8 This is a titer test chart for anti-human CD127 antibody A019D5. The calculated titer is 978.87 ng / mL.

[0116] Figure 9 Anti-human CD127 antibody Potency test chart, the calculated titer is 407.99 ng / mL.

[0117] Figure 10 Anti-human CD127 antibody Potency test chart, the calculated titer is 507.42ng / mL.

Claims

1. An anti-CD127 antibody, characterized in that comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region, and the light chain comprises a light chain variable region; the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein: (1) The amino acid sequence of HCDR1 is shown in SEQ ID NO. 1; (2) The amino acid sequence of HCDR2 is shown in SEQ ID NO. 2; (3) The amino acid sequence of HCDR3 is shown in SEQ ID NO. 3; (4) The amino acid sequence of LCDR1 is shown in SEQ ID NO. 4; (5) The amino acid sequence of LCDR2 is shown in SEQ ID NO. 5; (6) The amino acid sequence of LCDR3 is shown in SEQ ID NO.

6.

2. The anti-CD127 antibody according to claim 1, wherein It is a monoclonal antibody.

3. The anti-CD127 antibody according to claim 2, characterized in that It is a mouse or rabbit antibody.

4. The anti-CD127 antibody according to claim 3, characterized in that The amino acid sequence of the heavy chain variable region of the anti-CD127 antibody is SEQ ID NO.7 or a sequence having at least 80% sequence similarity to SEQ ID NO.7; the amino acid sequence of the light chain variable region of the anti-CD127 antibody is selected from SEQ ID NO.8, or SEQ ID NO.9, or a sequence having at least 80% sequence similarity to SEQ ID NO.8, or a sequence having at least 80% sequence similarity to SEQ ID NO.

9.

5. The anti-CD127 antibody according to claim 4, characterized in that The anti-CD127 antibody heavy chain amino acid sequence is SEQ ID NO.10, or a sequence having at least 65% sequence similarity to SEQ ID NO.10, or SEQ ID NO.12, or a sequence having at least 65% sequence similarity to SEQ ID NO.12; the anti-CD127 antibody light chain amino acid sequence is SEQ ID NO.11, or a sequence having at least 65% sequence similarity to SEQ ID NO.11; or SEQ ID NO.13, or a sequence having at least 65% sequence similarity to SEQ ID NO.

13.

6. A nucleic acid, characterized in that The nucleic acid encodes the anti-CD127 antibody according to any one of claims 1 to 5.

7. An expression vector, characterized in that Comprising the nucleic acid of claim 6.

8. A cell expressing the anti-CD127 antibody according to any one of claims 1 to 5.

9. The cell according to claim 8, characterized in that Comprising the expression vector according to claim 7.

10. The cell according to claim 9, characterized in that for HEK293 or CHO.

11. A method for preparing the anti-CD127 antibody according to any one of claims 1 to 5, characterized in that: Comprising culturing the cell according to any one of claims 8 to 10.

12. A method for fluorescently labeling the anti-CD127 antibody according to any one of claims 1 to 5, characterized in that: This includes labeling anti-CD127 antibodies with large molecule dyes or small molecule dyes.

13. The method according to claim 12, wherein: The macromolecular dye is APC, and the small molecule dye is FITC.

14. Use of the anti-CD127 antibody according to any one of claims 1 to 5, the nucleic acid according to claim 6, the expression vector according to claim 7, or the cell according to any one of claims 8 to 10 in CD127 detection, characterized in that: The application described is not an application for disease diagnosis or treatment.

15. Use of the anti-CD127 antibody according to any one of claims 1 to 5, the nucleic acid according to claim 6, the expression vector according to claim 7, or the cell according to any one of claims 8 to 10 in the preparation of a CD127 detection kit.

16. A CD127 detection method, characterized in that: The detection is performed using the anti-CD127 antibody according to any one of claims 1 to 5, wherein the method is not a disease diagnosis or treatment method.

17. A CD127 detection kit, characterized in that The method comprises the anti-CD127 antibody according to any one of claims 1 to 5, the nucleic acid according to claim 6, the expression vector according to claim 7, or the cell according to any one of claims 8 to 10.

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