Anti-CD19 nano antibody as well as preparation method and application thereof

Abstract

The invention relates to an anti-CD19 nano antibody as well as a preparation method and application thereof, and belongs to the technical field of biology. The amino acid sequence of the anti-CD19 nano antibody is one of SEQ ID NO. 1-SEQ ID NO. 2, the anti-CD19 nano antibody is composed of a heavy chain, and the heavy chain comprises three complementary determining regions, namely CDR1, CDR2 and CDR3. The nano antibody targeting CD19 is obtained through an alpaca nano antibody immune library and a phage display technology, the nano antibody can specifically recognize Human CD19 recombinant protein, HEK 293 hCD19 overexpression cells and Raji cells (human Burkitts lymphoma cells), and the nano antibody shows a good killing effect in a killing test of antibody-mediated NK cells on the Raji cells, and can be used for preparing the nano antibody targeting CD19. The method has application prospects in preparation of tumor treatment drugs and tumor detection.

Description

Technical Field

[0001] This invention relates to an anti-CD19 nanobody, its preparation method and application, belonging to the field of biotechnology. Technical Background

[0002] CD19 antigen is a transmembrane glycoprotein with a molecular weight of approximately 95 kDa, belonging to the immunoglobulin superfamily. As a type I transmembrane protein, it possesses a single transmembrane domain, a cytoplasmic C-terminus, and an extracellular N-terminus. CD19 is a biomarker for normal B cells, tumor B cells, and follicular dendritic cells. It plays a crucial role in B cell proliferation, differentiation, activation, and antibody production, and promotes B cell receptor (BCR) signal transduction. Existing research indicates that CD19 plays a key role in maintaining the balance between humoral, antigen-induced responses, and tolerance induction. Based on this, CD19 monoclonal antibodies have been explored for lymphoma treatment, making it a highly promising antigen target for tumor therapy.

[0003] Nanobodies are the smallest known antigen-binding antibody molecules, with a molecular weight of approximately 15 kDa. Compared to traditional antibodies, nanobodies offer advantages such as small relative molecular mass, high affinity, high stability, good solubility, low immunogenicity, strong penetration, simple humanization, and the ability to be expressed in large quantities in *E. coli*. The development of anti-human CD19 nanobodies using phage display technology shows promising application prospects. Summary of the Invention

[0004] The purpose of this invention is to provide an anti-CD19 nanobody that can specifically recognize recombinant Human CD19 protein, HEK 293 hCD19 overexpressing cells, and Raji cells (human Burkitts lymphoma cells), and shows good killing effect in antibody-mediated NK cell killing assays against Raji cells. The invention also provides a method for preparing the antibody and its applications.

[0005] The technical solution of this invention to solve its technical problem is as follows:

[0006] This invention provides an anti-CD19 nanobody comprising heavy chain complementarity-determining regions CDR1, CDR2, and CDR3, wherein:

[0007] i. The amino acid sequence of CDR1 is shown in SEQ ID NO:5; the amino acid sequence of CDR2 is shown in SEQ ID NO:6; the amino acid sequence of CDR3 is shown in SEQ ID NO:7; or

[0008] ii. The amino acid sequence of CDR1 is shown in SEQ ID NO:5; the amino acid sequence of CDR2 is shown in SEQ ID NO:8; and the amino acid sequence of CDR3 is shown in SEQ ID NO:9.

[0009] Preferably, the anti-CD19 nanobody comprises a heavy chain variable region having at least 85%, 90%, 95%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, and retaining the ability to bind to human CD19 antigen.

[0010] Preferably, the amino acid sequence of the heavy chain variable region of the anti-CD19 nanobody is as shown in SEQ ID NO:1 or SEQ ID NO:2.

[0011] This invention also provides:

[0012] The nucleic acid encoding the anti-CD19 nanobody described above.

[0013] Furthermore, the sequence of the nucleic acid is shown in SEQ ID NO:3 or SEQ ID NO:4.

[0014] This invention also provides:

[0015] An expression vector containing the nucleic acid described above.

[0016] This invention also provides:

[0017] A host cell containing the expression vector described above.

[0018] This invention also provides:

[0019] A method for preparing an anti-CD19 nanobody includes the following steps:

[0020] Alpacas were immunized with CD19 / His protein antigen and CD19 / hFC protein antigen using MnJ(β) colloidal manganese adjuvant, and their serum titers were measured. Peripheral blood monoclonal antibodies (PBMCs) were isolated from immunized alpacas with titers meeting the library construction criteria. These PBMCs were used to construct an alpaca nanobody immunotherapy library. The constructed antibody library was packaged with bacteriophages. Liquid chromatography was used for the first and third rounds of screening, while cell panning was used for the second and fourth rounds. After four rounds of screening, anti-CD19 bacteriophages were enriched, and single clones were further selected for screening. Positive clones were subjected to first-generation sequencing. Sequencing analysis was used to select the correct antibody sequence for expression in the mammalian system. After purification, anti-CD19 nanobodies were obtained.

[0021] This invention also provides:

[0022] A pharmaceutical composition comprising the aforementioned anti-CD19 nanobody.

[0023] This invention also provides:

[0024] The application of the anti-CD19 nanobody described above in the preparation of reagents or kits for in vitro detection of CD19 protein.

[0025] Compared with the prior art, the present invention has the following technical effects:

[0026] This invention obtained a CD19-targeting nanobody using phage display technology. This antibody can specifically recognize recombinant Human CD19 protein, HEK 293 hCD19 overexpressing cells, and Raji cells (human Burkitts lymphoma cells). It also showed good killing effect in antibody-mediated NK cell killing of Raji cells, and has the potential for application in the preparation of tumor therapeutic drugs and tumor detection. Attached Figure Description

[0027] Figure 1 The results of serum titer detection in Example 1;

[0028] Figure 2 The image shows the FACS screening results using HEK 293 hCD19 overexpressing cells as antigens in Example 2.

[0029] Figure 3 This is the result of ELISA screening using CD19 / His protein as an antigen in Example 2;

[0030] Figure 4 This is the result of FACS screening of 48 monoclonal antibodies using Raji cells as an antigen in Example 2.

[0031] Figure 5 This is a map of the antibody-mammal system expression vector in Example 3;

[0032] Figure 6 The SDS-PAGE of the purified antibody in Example 3 shows that A is G045-CD19-CP1R4-P1-B9, B is G045-CD19-CP1R4-P1-G6, R is the reducing condition, NR is the non-reducing condition, and M is the marker.

[0033] Figure 7 This refers to the binding of the purified antibody in Example 4 to HEK 293 hCD19 cells;

[0034] Figure 8 This refers to the binding of the purified antibody to control cells HEK 293 GFP in Example 4;

[0035] Figure 9 This refers to the binding of the purified antibody to Raji cells (human Burkitts lymphoma cells) in Example 4.

[0036] Figure 10 This refers to the binding of the purified antibody to the CD19 / His protein in Example 4.

[0037] Figure 11 The SPR affinity assay for the purified antibody and CD19 / His protein in Example 5 is shown, where A is G045-CD19-CP1R4-P1-B9, B is G045-CD19-CP1R4-P1-G6, C is the positive control antibody Inebilizumab, and D is the negative control antibody Anti-HEL IgG1 hFc.

[0038] Figure 12 The results are from the experiment in Example 6 on the killing of Raji cells by purified antibody-mediated NK cells. Detailed Implementation

[0039] The present invention will be further explained below with reference to specific embodiments. However, it should be noted that the following embodiments are only used to explain the present invention and cannot be used to limit the present invention. All technical solutions that are the same as or similar to the present invention are within the protection scope of the present invention. Where specific techniques or conditions are not specified in this embodiment, they shall be operated in accordance with conventional technical methods and instrument manuals in the art; where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0040] Example 1: Alpaca Immunity and Serum Titer Detection

[0041] For the initial immunization, 200 μg of CD19 / hFc protein antigen (Biointron, B22593505) was used, along with 5 times the antigen mass of MnJ(β) colloidal manganese adjuvant (Qimeng Biotechnology, MS0001). After mixing, the mixture was allowed to stand for 3-5 minutes, and sterile PBS was added to make the final volume 1 mL. The mixture was then drawn into a syringe and injected subcutaneously. Two booster immunizations of 200 μg CD19 / hFc protein antigen and two booster immunizations of CD19 / His protein antigen (Biointron, B22400303) antigen were performed. 2 mL of blood was collected, serum was separated, and serum titer was measured.

[0042] The procedure for ELISA detection of serum titer is as follows: The plate was coated with CD19 / His protein, incubated, washed, and then blocked with blocking buffer containing 0.1% BSA and 0.3% Casein sodium salt for 1 h, followed by washing. Serum was serially diluted 2-fold, incubated for 1 h, and then thoroughly washed. Goat Anti-Alpaca IgG (H+L), HRP, and Pab (Jackson, 128-035-160) were used as secondary antibodies, and incubated for 1 h, followed by another thorough wash. 100 μL of TMB chromogenic buffer (Beyotime, PO209) was added to each well, and the reaction was stopped after 3-5 min in the dark at room temperature. The titer was read using a microplate reader at 450 nm. After the fourth booster immunization, the serum titer reached a maximum of 1:256000, as shown in the results. Figure 1 As shown, NS represents pre-immunization negative serum, TB3 represents serum after the third booster immunization, and TB4 represents serum after the fourth booster immunization. This data confirms that the immunization protocol using MnJ(β) colloidal manganese adjuvant employed in this application can effectively stimulate alpacas to produce a strong antigen-specific humoral immune response.

[0043] Example 2: Screening anti-CD19 nanobodies from an alpaca nanobody immunotherapy library

[0044] Anti-CD19 nanobodies were selected from an alpaca nanobody immune library using phage display technology.

[0045] The first and third rounds used liquid phase panning, while the second and fourth rounds used cell panning. After four rounds of pressure panning, the bacterial culture obtained from the fourth round of amplification was plated and cultured. The next day, 94 clones were selected from one plate for screening.

[0046] The FACS screening procedure for monoclonal phages was as follows: HEK293 hCD19 and HEK293 GFP were mixed 1:1, and 1.5E+ 5 cells / well were seeded into 96-well V plates; another 1.5E+ 5 Raji tumor cells were also seeded into 96-well V plates at the same rate. Then, 100 μL of monoclonal phage supernatant was added to each 96-well V plate to resuspend the cells. Positive and negative controls were set up: purified antibody Inebilizumab (Abinvivo, B21631116) was used as a positive control, and Anti-HEL IgG1hFc (Biointron, B117901) was used as a negative control. After incubation for 0.5 h, cells were thoroughly washed. THE™ DYKDDDDK Tag Antibody (Thermo, A-21281) was then added to the sample wells, and Goat Anti-hIgG (FcγSpecific) pAb [Alexa Fluor 647] (Jackson, 109-605-190) was added to the control wells. After another 0.5 h incubation, cells were thoroughly washed and resuspended in 100 μL / well PBS buffer, followed by flow cytometry analysis. Positive clone determination: Sample / Negative control Median APC-H (≥3). Results are as follows. Figure 2 and Figure 3 As shown, among the 94 clones, 48 ​​bound to HEK 293 hCD19 overexpressing cells and 19 bound to Raji cells (human Burkitts lymphoma cells).

[0047] The ELISA screening process for monoclonal phages was as follows: hCD19 / His protein and blocking buffer (3% non-fat powdered milk in PBS) were coated separately onto an ELISA plate (Corning, 3590). After incubation, the plate was washed thoroughly, and then incubated with blocking buffer (3% non-fat powdered milk in PBS) for 1 h. After washing thoroughly, positive monoclonal phage samples obtained through FACS screening were added. Purified antibody Inebilizumab (Abinvivo, B21631116) was used as a positive control, and Anti-HEL IgG1 hFc (Biointron, B117901) was used as a negative control. The plate was incubated for 1 h. After washing thoroughly again, Mouseanti-M13 mAb HRP (Sino Biolo, 11973-MM05T-H) was added as a secondary antibody. The control secondary antibody used was Goat Anti-Human IgG-Fc, HRP (Sigma, A0170). The plate was incubated for 1 h and then washed thoroughly again. Add 100 μL of TMB chromogenic buffer (Beyotime, P0209) to each well and incubate at room temperature in the dark for 3-5 min, then terminate the reaction. Read the results using a microplate reader at 450 nm. Criteria for positive clones: CD19 > 3 × NC and Milk < 3 × NC, CD19 / Milk > 2.5. Detection results are as follows: Figure 4 As shown, 60 out of 94 monoclonal antibodies were positive clones that bound the CD19 / His recombinant protein.

[0048] Based on the combined results of screening with three antigens, 18 clones were identified that could simultaneously bind to Huamn CD19 recombinant protein, HEK 293 hCD19 overexpressing cells, and Raji cells (human Burkitts lymphoma cells). Sequencing analysis of these 18 positive clones yielded 6 unique sequences. The dominant monoclonal clones were G045-CD19-CP1R4-P1-B9 and G045-CD19-CP1R4-P1-G6.

[0049] The amino acid sequence of G045-CD19-CP1R4-P1-B9 is SEQ ID NO:1, the amino acid sequence of CDR1 is SEQ ID NO:5, the amino acid sequence of CDR2 is SEQ ID NO:6, the amino acid sequence of CDR3 is SEQ ID NO:7, and the corresponding nucleotide sequence is SEQ ID NO:3.

[0050] The amino acid sequence of G045-CD19-CP1R4-P1-G6 is shown in SEQ ID NO:2, the amino acid sequence of CDR1 is shown in SEQ ID NO:5, the amino acid sequence of CDR2 is shown in SEQ ID NO:8, the amino acid sequence of CDR3 is shown in SEQ ID NO:9, and their corresponding nucleotide sequences are shown in SEQ ID NO:4.

[0051] Example 3: Expression and purification of anti-CD19 nanobodies in a mammalian system

[0052] The mammalian system expression vector pcDNA3.4 was used to construct the G045-CD19-CP1R4-P1-B9 and G045-CD19-CP1R4-P1-G6 nanobodies in Example 2. (See diagram below.) Figure 5 Then, plasmids were prepared using this plasmid. CHO-K1 cells were selected as the host cells for antibody expression, with an expression volume of 40 mL. The supernatant after expression was purified using a Protein A affinity chromatography column. High-purity expressed antibody was obtained. Its purity was determined by SDS-PAGE, and the results are shown below. Figure 6 As shown, the antibody purity all reached over 95%, indicating high purity.

[0053] Example 4: Detection of the binding ability of anti-CD19 nanobody to three antigens

[0054] The FACS assay for antibody binding to HEK 293 hCD19 and Raji cells was performed as follows: HEK293 hCD19 and HEK293 GFP were mixed 1:1, and 1.5E+ cells / well were seeded into 96-well V plates. Separately, 1.5E+ Raji tumor cells were seeded into 96-well V plates. Anti-CD19 nanobodies (G045-CD19-CP1R4-P1-B9, G045-CD19-CP1R4-P1-G6) and control antibodies were diluted to 100 nM as the initial concentration, and serially diluted 3-fold, with the last well being a blank. 100 μL / well was incubated for 0.5 h and then thoroughly washed. Goat Anti-hIgG (Fcγ Specific) pAb [Alexa Fluor 647] (Jackson, 109-605-190) was added as a secondary antibody, and the plates were incubated for 0.5 h and then thoroughly washed. Cells were resuspended in 100 μL / well PBS buffer and analyzed by flow cytometry. The positive control was Inebilizumab (Abinvivo, B21631116), and the negative control was Anti-HEL IgG1 hFc (Biointron, B117901).

[0055] The results of G045-CD19-CP1R4-P1-B9, G045-CD19-CP1R4-P1-G6 binding to HEK 293 hCD19 cells are as follows: Figure 7 As shown, the results of binding with control cells HEK 293 GFP are as follows: Figure 8 As shown: G045-CD19-CP1R4-P1-B9 can bind to HEK 293 hCD19 with an EC50 of 3.09 nM, but does not bind to control cells HEK 293 GFP; G045-CD19-CP1R4-P1-G6 can bind to HEK 293 hCD19 with an EC50 of 3.23 nM, but does not bind to control cells HEK 293 GFP. The results of binding with Raji tumor cells (human Burkitts lymphoma cells) are as follows... Figure 9 As shown, both G045-CD19-CP1R4-P1-B9 and G045-CD19-CP1R4-P1-G6 can bind to Raji tumor cells, with EC50 values ​​of 4.651 nM and 5.767 nM, respectively.

[0056] The ELISA detection procedure for antibody-CD19 / His protein binding is as follows: hCD19 / His recombinant protein was coated onto an ELISA plate (Corning, 3590), incubated, and then thoroughly washed. Blocking buffer (3% non-fat powdered milk in PBS) was added, and the plate was incubated for 1 h, followed by thorough washing. Purified anti-CD19 nanobodies (G045-CD19-CP1R4-P1-B9, G045-CD19-CP1R4-P1-G6) and control antibodies were diluted to 100 nM as the initial concentration for each well, and serially diluted 3-fold. The last well was a blank. 100 μL / well was used, and the plates were incubated for 1 h, followed by thorough washing. Secondary antibody Goat Anti-Human IgG-Fc, HRP (Sigma, A0170) was used, and the plates were incubated for 1 h, followed by another thorough washing. Add 100 μL of TMB chromogenic buffer (Beyotime, P0209) to each well, and incubate at room temperature in the dark for 3-5 minutes, then terminate the reaction. Read the results using a microplate reader at a wavelength of 450 nm. The detection results are as follows: Figure 10 As shown, both G045-CD19-CP1R4-P1-B9 and G045-CD19-CP1R4-P1-G6 can bind to the CD19 / His protein, with EC50 values ​​of 1.61 nM and 4.87 nM, respectively.

[0057] Example 5: SPR affinity assay of anti-CD19 nanobodies with hCD19 / His

[0058] Purified antibodies of 2 μg / mL G045-CD19-CP1R4-P1-B9 or G045-CD19-CP1R4-P1-G6 were injected into the experimental channels at a flow rate of 10 μL / min for 60 s, with a capture volume of approximately 145-2000 RU. The hCD19 / His recombinant protein was diluted 2-fold starting at 200 nM using HBS-EP+Buffer. The diluted hCD19 / His recombinant protein was then injected sequentially into the experimental and reference channels at a flow rate of 30 μL / min, with binding and dissociation times recorded. Both binding and dissociation steps were performed in the running buffer. Protein A Chip (Cytiva, 29127556) was regenerated with 10 mM Gly-HCl (pH=1.5) at a flow rate of 30 μL / min for 30 s to wash away undissociated analytes. The KD value of the samples was calculated using Biacore 8K analysis software. The reference channel (Fc1) is used for background subtraction. The fitting model is 1:1, and the results are as follows: Figure 11 As shown: The antibodies G045-CD19-CP1R4-P1-B9 and G045-CD19-CP1R4-P1-G6 both showed binding activity to the hCD19 / His recombinant protein, with KD values ​​of 1.85E-08M and 2.82E-8M, respectively.

[0059] Example 6: Assay of NK cell killing of Raji cells mediated by anti-CD19 nanobody

[0060] Raji cells were collected and labeled with CFSE (Thermo, 35445). Raji cells were washed and resuspended in RPMI 1640 (Gibco, 11875093) complete medium at a concentration of 4E+5 / mL. NK cells were isolated from fresh PBMCs using the Human NK Cell Isolation Kit (Miltenyi, 130-092-657) and resuspended in RPMI 1640 complete medium at different densities. IL-2 was prepared using RPMI 1640 complete medium at 20 ng / mL (4-fold) to prepare the detection antibody at a maximum concentration of 400 nM. Raji cells (2E+4 50 μL) and NK cells (1E+5 50 μL) were distributed in 96-well plates (Corning, 3799). 50 μL of IL-2 (final concentration 5 ng / mL) and 50 μL of detection antibody were added to each well. Incubate at 37°C in a 5% CO2 incubator for 6 h. Remove the culture dishes, wash cells with DPBS (Biosera, LM-S2041 / 500), and then stain with eBioscience™ Fixable Viability Dye eFluor™ 780 (Thermo, 65-0865-14). Analyze dead cells (CFSE) by flow cytometry. + FVD780 + Positive controls were V6 Anti-Human CD19 (Inebilizumab) (Abinvivo, B21631116) and Anti-Human CD19 (Tafasitamab) (Abinvivo, B21569401), and negative controls were Anti-HEL IgG1 hFc (Biointron, B117901). Results are as follows. Figure 12 As shown, both G045-CD19-CP1R4-P1-B9 and G045-CD19-CP1R4-P1-G6 exhibit good lethality.

[0061] Finally, it should be noted that the above embodiments are merely illustrative of the principles, performance, and effects of the present invention, and are not intended to limit the present invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. An anti-CD19 nanobody, characterized in that, It includes heavy chain complementarity determining regions CDR1, CDR2, and CDR3, where: i. The amino acid sequence of CDR1 is shown in SEQ ID NO:5; the amino acid sequence of CDR2 is shown in SEQ ID NO:6; the amino acid sequence of CDR3 is shown in SEQ ID NO:7; or ii. The amino acid sequence of CDR1 is shown in SEQ ID NO:5; the amino acid sequence of CDR2 is shown in SEQ ID NO:8; and the amino acid sequence of CDR3 is shown in SEQ ID NO:

9.

2. The anti-CD19 nanobody according to claim 1, characterized in that, The anti-CD19 nanobody comprises a heavy chain variable region having at least 85%, 90%, 95%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, and retaining the ability to bind to the human CD19 antigen.

3. The anti-CD19 nanobody according to claim 1 or 2, characterized in that, The amino acid sequence of the heavy chain variable region of the anti-CD19 nanobody is shown in SEQ ID NO:1 or SEQ ID NO:

2.

4. The nucleic acid encoding the anti-CD19 nanobody according to any one of claims 1 to 3.

5. The nucleic acid according to claim 4, characterized in that, The sequence of the nucleic acid is shown in SEQ ID NO:3 or SEQ ID NO:

4.

6. An expression carrier, characterized in that, The expression vector contains the nucleic acid as described in claim 5.

7. A host cell, characterized in that, The host cell contains the expression vector as described in claim 6.

8. A method for preparing an anti-CD19 nanobody, characterized in that, Includes the following steps: Alpacas were immunized with CD19 / His protein antigen and CD19 / hFC protein antigen using MnJ(β) colloidal manganese adjuvant, and their serum titers were measured. Peripheral blood monoclonal antibodies (PBMCs) were isolated from immunized alpacas with titers meeting the library construction criteria. These PBMCs were used to construct an alpaca nanobody immunotherapy library. The constructed antibody library was packaged with bacteriophages. Liquid chromatography was used for the first and third rounds of screening, while cell panning was used for the second and fourth rounds. After four rounds of screening, anti-CD19 bacteriophages were enriched, and single clones were further selected for screening. Positive clones were subjected to first-generation sequencing. Sequencing analysis was used to select the correct antibody sequence for expression in the mammalian system. After purification, anti-CD19 nanobodies were obtained.

9. A pharmaceutical composition, characterized in that, It includes the anti-CD19 nanobody according to any one of claims 1 to 3.

10. The use of the anti-CD19 nanobody as described in any one of claims 1 to 3 in the preparation of reagents or kits for in vitro detection of CD19 protein.

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