Canine CD19 monoclonal antibody sequence based on rabbit source as well as preparation method and application thereof

Abstract

The invention discloses a rabbit-source-based canine CD19 monoclonal antibody sequence and a preparation method and application thereof, and relates to the technical field of biology, a complementary determining region CDR of a VHH chain in the antibody is selected from one or more of the following groups: single antibody variable region sequences CDR-H1, CDR-H2 and CDR-H3, the CDR-H1 comprises an amino acid sequence shown in any one of SEQ ID NO: 1-6, the CDR-H2 comprises an amino acid sequence shown in any one of SEQ ID NO: 3, and the CDR-H3 comprises an amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. The CDR-H2 comprises an amino acid sequence as shown in any one of the sequences SEQ ID NO: 7 to 12, and the CDR-H3 comprises an amino acid sequence as shown in any one of the sequences SEQ ID NO: 13 to 18. The rabbit-source-based canine CD19 monoclonal antibody sequence, the extraction method thereof and the monoclonal antibody development method have the characteristics of short time consumption, high throughput, high positive rate and the like, have good antigen-antibody binding activity on cell and molecular levels, and provide support for ELISA (enzyme-linked immuno sorbent assay), flow type detection and the like of canine CD19 protein. According to the method, a single B lymphocyte is taken as a research main body, and the single B lymphocyte is sorted by utilizing flow cytometry.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a rabbit-derived canine CD19 monoclonal antibody sequence, a preparation method thereof, and an application thereof. Background Art

[0002] CD19 protein is a type I transmembrane glycoprotein on the surface of B cells, with a relative molecular weight of 95 kDa, belonging to the immunoglobulin superfamily. It is a biomarker for B-cell lymphoma and follicular dendritic cells and is the best target for B-cell malignancies, including acute lymphoblastic leukemia and lymphoma. CD19 is an important member of the multi-molecular complex on the surface of mature B cells and functions together with the complement receptor CD21, CD81, and CD225. Abnormal expression of canine CD19 can lead to various B-cell related diseases. Therefore, CD19-related antibodies play a leading role in the detection and treatment of canine B-cell tumor diseases.

[0003] It is known from the literature that existing canine CD19 antibodies are obtained by the hybridoma method. The period for obtaining antibodies by hybridoma is relatively long. Excluding the animal immunization time, it takes 4 - 6 months to obtain antibodies, and the fusion efficiency of splenocytes and myeloma cells is low. Only a small part of B cells in the whole B-cell population complete fusion, and most are lost, which is not suitable for comprehensively screening a large antibody library.

[0004] Moreover, there are significant differences between batches of hybridoma cells. Monoclonal antibodies produced by the hybridoma technology are mostly murine-derived. Adoptive therapy of autologous T cells genetically modified to express CD19-specific chimeric antigen receptor (CD19 CAR-T cells) has been proven to be very effective for human B-cell malignancies, but currently there is no available canine CAR-T cell therapy in clinical practice. Therefore, in view of this situation, it is urgent to develop antibodies targeting the canine CD19 target, which has great clinical value. Summary of the Invention

[0005] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art and to be able to solve the problems raised in the background art.

[0006] On the one hand, the present invention provides an antibody sequence targeting canine CD19 protein, which can be used as a candidate sequence for the development of antibody drugs for diagnosing, preventing, and / or treating canine B-cell malignancies. Moreover, by using eukaryotic expression of CD19 protein and combining with in vitro binding experiments, it is beneficial to obtain antibodies with both excellent affinity and specificity.

[0007] On the second hand, the present invention provides a preparation method of a canine CD19 antibody (as follows)

[0008] To achieve the above object, the present invention provides the following technical solution: An anti-canine CD19 antibody, wherein the complementarity-determining region (CDR) of the VHH chain in the antibody is one or more selected from the following group:

[0009] It includes a single antibody variable region sequence CDR-H1, CDR-H2, CDR-H3. Among them, the CDR-H1 contains the amino acid sequence shown in any one of SEQ ID NO: 1-6, the CDR-H2 contains the amino acid sequence shown in any one of SEQ ID NO: 7-12, and the CDR-H3 series contains the amino acid sequence shown in any one of SEQ ID NO: 13-18.

[0010] Such as CDR1 shown in (1) SEQ ID NO: 1, CDR2 shown in SEQ ID NO: 7, and CDR3 shown in SEQ ID NO: 13; and

[0011] (2) CDR1 shown in SEQ ID NO: 2, CDR2 shown in SEQ ID NO: 8, and CDR3 shown in SEQ ID NO: 14.

[0012] A method for extracting the sequence of a rabbit-derived canine CD19 monoclonal antibody;

[0013] The method includes the following steps:

[0014] S1: Determination of animal immune titer;

[0015] S2: Isolation of rabbit PBMC;

[0016] S3: Cell staining;

[0017] S4: Sorting of memory B cells (Memory B Cell MBC) by flow cytometry;

[0018] S5: Single-cell lysis;

[0019] S6: PCR amplification;

[0020] S7: Sequence determination;

[0021] S8: Plasmid construction;

[0022] S9: Overexpression of the antibody single chain in the CHO cell line;

[0023] S10: Purification of the antibody by affinity chromatography.

[0024] Preferably, the nucleotide sequence containing the amino acid sequence encoding canine CD19 with a His tag was cloned onto the vector pCDNA3.4, and the plasmid was prepared according to established standard molecular biology methods. The canine CD19 protein eluted from the nickel ion affinity chromatography column was collected and verified by SDS-PAGE.

[0025] Preferably, the obtained canine CD19 protein was used to immunize rabbits. For the primary immunization, a suspension was prepared by thoroughly mixing complete Freund's adjuvant and the antigen in a volume ratio of 1:1, and multiple immunological detections were carried out at different stages.

[0026] Preferably, after the serum titer reached the required level, single cell suspensions could be prepared and preliminarily screened with the help of B cell surface markers.

[0027] Preferably, the amplified antibody heavy chain target plasmid was ligated onto pcDNA3.4 containing rabbit IgG Fc, and the antibody light chain gene was ligated onto blank pcDNA3.4 to construct a dual plasmid, followed by transient transfection and expression.

[0028] Preferably, the paired light and heavy chain well plate plasmids were synchronously transfected into single cells in a 96-well plate for cell culture.

[0029] Preferably, the obtained HAUC19.1 antibody sequence was then subjected to transient transfection and expression in CHO cells.

[0030] Preferably, the transfected cell culture medium was centrifuged, filtered through a 0.45 μm filter membrane after centrifugation, and then the antibody was purified according to the nickel column instruction manual.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0032] (1) The rabbit-derived canine CD19 monoclonal antibody sequence and its extraction method, the monoclonal antibody development method has the characteristics of short time consumption, high throughput, high positive rate, etc., and has good antigen-antibody binding activity at the cellular and molecular levels, providing support for the detection of canine CD19 protein by ELISA, flow cytometry, etc. The present invention uses a single B lymphocyte as the research subject and uses flow cytometry to sort single B cells. Utilizing the characteristic that each B cell can only produce one specific antibody, B cells secreting specific antibodies are directly screened out, and the genes expressing the antibody heavy chain and light chain are cloned and expressed in vitro to obtain monoclonal antibodies. Since both VH and VL are amplified from a single cell, it ensures the natural homologous pairing and development of natural antibodies, ensuring that the obtained antibodies have higher affinity, stability and specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The present invention will be further described below with reference to the drawings and embodiments:

[0034] Figure 1 It is a schematic structural diagram of the purification result of the canine CD19 protein of the present invention;

[0035] Figure 2 It is a schematic structural diagram of the ELISA titer detection result of immunized rabbits of the present invention;

[0036] Figure 3 It is a schematic structural diagram of the flow cytometry detection result of the antibody of the present invention;

[0037] Figure 4 It is a schematic diagram of the Western blot detection result of the antibody of the present invention;

[0038] Figure 5 It is a schematic diagram of the immunohistochemical result of the antibody of the present invention;

[0039] Figure 6 It is a schematic diagram of the result of the clinical case of the antibody of the present invention

[0040] Figure 7 It is a schematic structural diagram of a method for extracting the sequence of a rabbit-derived canine CD19 monoclonal antibody of the present invention. Detailed implementation manners

[0041] This part will describe in detail the specific embodiments of the present invention. The preferred embodiments of the present invention are shown in the drawings. The function of the drawings is to supplement the description in the text part of the specification, enabling people to intuitively and vividly understand each technical feature and the overall technical solution of the present invention, but it cannot be understood as a limitation on the protection scope of the present invention.

[0042] In the description of the present invention, it should be understood that for orientation descriptions, such as up, down, front, back, left, right, etc., the orientation or positional relationship indicated is based on the orientation or positional relationship shown in the drawings. It is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention.

[0043] In the description of the present invention, "greater than", "less than", "exceeding", etc. are understood as not including the number itself, and "above", "below", "within", etc. are understood as including the number itself. If there is a description of first and second, it is only for the purpose of distinguishing technical features and cannot be understood as indicating or implying relative importance or implicitly indicating the quantity of the indicated technical features or implicitly indicating the sequence relationship of the indicated technical features.

[0044] In the description of the present invention, unless otherwise clearly defined, terms such as "set", "installed", "connected", etc. should be understood in a broad sense, and those skilled in the art can reasonably determine the specific meanings of the above terms in the present invention in combination with the specific content of the technical solution.

[0045] Please refer to Figure 1-4 , the present invention provides a technical solution: a rabbit-derived canine CD19 monoclonal antibody sequence, its preparation method and application, including the amino acid sequence in Table 1

[0046]

[0047]

[0048] Step 1: Preparation of canine CD19 protein

[0049] Clone the nucleotide sequence containing the amino acid sequence encoding canine CD19 and carrying a His tag into the vector pCDNA3.4, and prepare the plasmid according to the established standard molecular biology methods.

[0050] Using the prepared plasmid, dilute the psPAX2 antibody plasmid, PMD2G plasmid and the synthetic plasmid into the culture medium, and add Lipofectamine 2000, and incubate at room temperature for 15 minutes;

[0051] Add the above mixture to HEK293E cells for transient transfection, and place it in an incubator at 110 rpm, 37 °C, and 5% CO2 for culture;

[0052] 4 - 6 days after transfection, centrifuge, collect the cells, dissolve them with 50 mM Tris, 300 mM NaCl, 8 M Urea, 20 mM imidazole, pH 8.0 buffer, and purify them with a Ni-IDA column;

[0053] Collect the canine CD19 protein eluted from the nickel ion affinity chromatography column, concentrate it and perform SDS-PAGE verification, and the results are as Figure 1 shown.

[0054] Step 2: Animal immunization

[0055] Use the canine CD19 protein obtained in Step 1 to immunize rabbits. For the primary immunization, prepare a suspension by mixing complete Freund's adjuvant and the antigen in a volume ratio of 1:1, and perform the primary immunization. On the 14th day, perform the second immunization with the canine CD19 protein. On the 7th day after the second immunization, perform the third immunization. Three days later, collect blood to detect the titer of the rabbit serum.

[0056] The serum titer was detected by indirect ELISA, and the detection process was as follows: The enzyme-linked immunosorbent assay (ELISA) plate was coated with 2 μg / mL canine CD19 protein and incubated overnight at 4 °C; washed with 0.05% PBST, blocked with 0.1% BSA at room temperature for 1 hour; washed with 0.05% PBST, and serums before and after mouse immunization diluted with a 2-fold gradient were added respectively. The first 3 wells were diluted 1000, 2000, and 4000 times, and incubated at room temperature for 2 hours; washed with 0.05% PBST, added HRP-labeled goat anti-rabbit IgG, and incubated at room temperature for 30 minutes; washed with 0.05% PBST, added TMB chromogenic solution, reacted at 37 °C for 15 minutes, and then added the termination solution to terminate the reaction, and the OD450 was read.

[0057] The results were as Figure 2 shown. The titer of the antiserum after the second immunization was ≥512K. It can be seen that this antigen can induce rabbits to produce high-titer antiserum specifically against canine CD19 protein.

[0058] Step 3: Sorting memory B cells (Memory B Cell MBC) by flow cytometry

[0059] After the serum titer reached as shown in Step 2, single-cell suspension preparation could be carried out, and preliminary screening was carried out with the help of B cell surface markers.

[0060] Different fluorescent labels were used for the immune protein and the secondary antibody against IgG. If the MBC surface could display IgG antibodies that specifically bind to the antigen, the cell would carry two fluorescent labels, and the target MBC could be sorted out one by one from the cell suspension by flow cytometry and placed in a 96-well plate.

[0061] Step 4: High-throughput expression and detection of cell supernatant

[0062] Single specific single B cells in the 96-well plate were lysed at room temperature. The 5′-end and 3′-end of cDNA were obtained based on reverse transcription PCR, and oligo-dT primers were used to reverse transcribe the mRNA of the heavy and light chain variable regions into cDNA templates. Subsequently, after RT-PCR and two rounds of nested PCR, the full sequence of the antibody light chain and the Fab region fragment of the heavy chain were obtained respectively.

[0063] The amplified antibody heavy chain target plasmid was ligated to pcDNA3.4 containing rabbit IgGFc, and the antibody light chain gene was ligated to blank pcDNA3.4 to construct a dual plasmid. Subsequently, transient transfection and expression were carried out. The transient transfection and expression were as follows: Cells were seeded one day before transfection to ensure that the cells grew to 3.0 - 5.0×10 6cells / mL, with a viability of 98%. Dilute the cell suspension with the medium and plate the cells in a 96-well plate, 400 μL per well. Plate 100 μL of the transfection buffer into the 96-well plate, then add the plasmid to the transfection buffer and pipette to mix evenly. Next, add Lipofectamine 2000 to the buffer and pipette to mix evenly. Incubate at 37 °C for 10 min, then add it to the cells in the 96-well plate and continue the culture.

[0064] After 20 h of transfection, add 25 μL of feeding to each well. After 5 days of expression, centrifuge at 4000 rpm for 10 min and collect the supernatant.

[0065] High-throughput expression: Synchronously transfect the paired light and heavy chain plasmid plates into the cells of a single well in a 96-well plate, perform cell culture, let the cells grow for 5 d, collect the cell supernatant, and detect the cell supernatant by ELISA. The method is the same as that for serum ELISA detection.

[0066] Step Five: Sequence the variable region of the HAUC19.1 antibody, as described in Step Four above.

[0067] Step Six: Antibody transfection and expression

[0068] According to the obtained HAUC19.1 antibody sequence, subsequent transient transfection and expression in CHO cells are carried out, as in the above high-throughput expression.

[0069] Step Seven: Antibody purification

[0070] Centrifuge the cell culture medium 6 days after transfection, filter it using a 0.45 μm filter membrane after centrifugation, and then purify the antibody according to the nickel column instruction manual. The purity of the obtained antibody > 90%.

[0071] Step Eight: Verification by flow cytometry

[0072] Use the purified antibody as the primary antibody, dilute it at 1 μg / μL and bind it to the target cells PBMC. After reacting at 4 °C for 20 minutes, wash it twice with PBS containing 0.1% FBS, then add the secondary antibody goat anti-rabbit AF488. After reacting at 4 °C for 20 minutes, wash it twice with PBS containing 0.1% FBS, then resuspend it with 350 μL of PBS containing 0.1% FBS and filter it, and then detect it on a flow cytometer (see Figure 3 ). In summary, the results show that the antibody can recognize canine B cells well in the experimental results, and it can also prove that the antibody can be applied to flow cytometry, which is helpful for the diagnosis and treatment of canine B cell-related diseases.

[0073] Step Nine: Western blot

[0074] After SDS-PAGE electrophoresis, the proteins in the gel were transferred to a 0.45-μm PVDF membrane. The purified antibody was used as the primary antibody and incubated overnight at 4°C. The HRP-labeled goat anti-rabbit IgG was used as the secondary antibody and incubated for 2 h. The enhanced chemiluminescence solution was used for color development (see Figure 4 ).

[0075] Step 10: Immunohistochemical verification

[0076] Canine spleen sections were prepared and pretreated according to the relevant steps of the immunohistochemistry kit. The purified antibody was used as the primary antibody, diluted 3000-fold, and incubated overnight at 4°C. PBST was used for at least 5 washes. The corresponding goat anti-rabbit secondary antibody was added, and after reacting at 37°C for 30 min, it was washed. Subsequently, the subsequent treatment was carried out according to the instructions of the immunohistochemistry kit. After sealing the slices, they were dried for about 24 h and then read under the microscope. The experimental results are as shown in Figure 5 . In the experimental results, it was clearly observed that there were a large number of B cells in the splenic corpuscles, further verifying that this antibody can be applied to the diagnosis of canine B-cell-related diseases.

[0077] Step 11: Clinical cases

[0078] In the present invention, a rabbit anti-canine CD19 monoclonal antibody with high specificity and affinity was successfully prepared, and the purity of this monoclonal antibody is very high and can be applied to clinical diagnosis. In this case, the blood and tissue fluid of two lymphoma-affected dogs were collected and tested. The experimental results are as shown in Figure 6 . It was shown that both Dog 1 and Dog 2 were diagnosed with B-cell lymphoma. Dog 1: 5 years old, female, Corgi; Dog 2: 14 years old, female, Bichon Frise.

[0079] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those of ordinary skill in the art in the technical field, various changes can be made without departing from the purpose of the present invention.

Claims

1. An anti-canine CD19 antibody, characterized in that: The complementary determining region (CDR) region of the VHH chain in the antibody is one or more selected from the following groups: Comprising single antibody variable region sequences CDR-H1, CDR-H2, and CDR-H3, wherein the CDR-H1 comprises an amino acid sequence according to any one of SEQ ID NOs: 1 to 6, the CDR-H2 comprises an amino acid sequence according to any one of SEQ ID NOs: 7 to 12, and the CDR-H3 series comprises an amino acid sequence according to any one of SEQ ID NOs: 13 to 18; (1) CDR1 shown in SEQ ID NO: 1, CDR2 shown in SEQ ID NO: 7, and CDR3 shown in SEQ ID NO: 13; and (2) CDR1 shown in SEQ ID NO:2, CDR2 shown in SEQ ID NO:8, and CDR3 shown in SEQ ID NO:

14.

2. A CD19 (canine) antibody, characterized in that: The antibodies include one or more of those described in claim 1, and the antibodies exclude their use in the preparation of canine CAR-T and bispecific antibodies.

3. A CD19 (canine) antibody, characterized in that: The antibody comprises one or more antibodies as claimed in claim 1, and the antibody is excluded from the preparation of flow cytometry antibodies or is directly used as a primary antibody in flow cytometry, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry (IHC).

4. A CD19 (canine) antibody, characterized in that: The antibodies include one or more of those described in claim 1, and the antibodies exclude their direct or modified use in dogs, such as antibody-drug combination use for tumors or autoimmune diseases.

5. A CD19 (canine) antibody, characterized in that: The antibody includes one or more of the antibodies as claimed in claim 1, wherein the antibody is used to screen B cells in vitro by magnetic beads or the like, and is used to infect canine B cells when genetically modified in vivo or in vitro by viruses, liposomes or the like.

6. A method for extracting canine CD19 monoclonal antibody sequences based on rabbit sources; Features: The steps include: S1: Animal immune titer determination; S2: Isolation of rabbit PBMCs; S3: cell staining; S4: Memory B Cell MBC sorting by flow cytometry; S5: single cell lysis; S6: PCR amplification; S7: Sequence determination; S8: plasmid construction; S9: Antibody single chain overexpressed in CHO cell line; S10: Purification of antibodies by affinity chromatography.

7. The method for extracting canine CD19 monoclonal antibody sequences based on rabbit sources according to claim 6, characterized in that: The nucleotide sequence containing the amino acid sequence encoding canine CD19 and having a His tag was cloned into the vector pCDNA3.4, and the plasmid was prepared according to the established standard molecular biology method, and the canine CD19 protein eluted from the nickel ion affinity chromatography column was collected and verified by SDS-PAGE.

8. The method for extracting canine CD19 monoclonal antibody sequences based on rabbit sources according to claim 6, characterized in that: The obtained canine CD19 protein is used to immunize rabbits, and complete Freund's adjuvant is used for the initial immunization and the antigen is mixed in a volume ratio of 1:1 to prepare a suspension, and multiple immune tests are performed at different stages. After the serum titer is reached, a single cell suspension can be prepared, and preliminary screening is performed using B cell surface markers.

9. The method for extracting canine CD19 monoclonal antibody sequences based on rabbit sources according to claim 6, characterized in that: The amplified antibody heavy chain target plasmid was connected to pcDNA3.4 containing rabbit IgGFc, and the antibody light chain gene was connected to a blank pcDNA3.4 to construct a double plasmid. The obtained antibody sequence was named HAUC19.1, and then transiently expressed in CHO cells.

10. The method for extracting canine CD19 monoclonal antibody sequence based on rabbit source according to claim 6, characterized in that: The transfected cell culture fluid was centrifuged and filtered using a 0.45 μm filter membrane, and then the antibody was purified according to the nickel column instruction manual.

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