Determination of protein sequence of anti-human CD16 mouse monoclonal antibody and rabbit monoclonal antibody and establishment of stably transfected cell strain

Abstract

This invention provides an anti-CD16 antibody, the amino acid sequence of which is selected from SEQ ID NO. 1-3 in the heavy chain CDR region and from SEQ ID NO. 4-6 in the light chain CDR region. This invention belongs to the field of antibodies. It provides a protein sequence of an anti-human CD16 mouse monoclonal antibody, and designs a gene expression base sequence based on the sequenced protein sequence. A stable CHO cell line for recombinant expression of this gene is established. The purified antibody can specifically recognize the human CD16 antigen, and the antibody expression yield is much higher than that of antibodies prepared using traditional hybridoma cells. Furthermore, by replacing mouse IgG1 with rabbit IgG while retaining the variable region, a stable CHO cell line for recombinant expression of the rabbit monoclonal antibody is established. The purified antibody maintains its specific recognition of the human CD16 antigen.

Description

Technical Field

[0001] This invention belongs to the field of antibodies, specifically relating to the determination of protein sequences of anti-human CD16 mouse monoclonal antibody and rabbit monoclonal antibody and the establishment of stable cell lines. Background Technology

[0002] CD16, also known as FcγRIII, is a glycoprotein with a molecular weight of 50,000-70,000 Daltons, belonging to the Ig superfamily. The IgG FcR family (FcγR) consists of six receptors: FcγRI (CD64), FcγRIIa (CD32a), FcγRIIb (CD32b), FcγRIIc (CD32c), FcγRIIIa (CD16a), and FcγRIIIb (CD16b). CD16a is primarily responsible for triggering NK cell-mediated ADCC. CD16 is widely expressed on NK cells, neutrophils, mononuclear cells, and macrophages, but not on basophils. Anti-CD16 monoclonal antibodies can activate NK cell antibody-dependent cell-mediated cytotoxicity.

[0003] CD16-triggered ADCC and phagocytosis mediated by NK cells and monocytes / macrophages, respectively, are the main immune-dependent mechanisms by which monoclonal antibodies exert their tumor-killing effects and achieve therapeutic efficacy. [1] Researchers plan to regulate the anti-tumor response of monoclonal antibodies by activating or inhibiting the CD16 receptor on NK cells. This research is expected to be applied to the precise control of cell immunotherapy.

[0004] Researchers have found that intramuscular injection of influenza vaccines leads to cross-linking of antibody-antigen immune complexes, downregulating CD16 expression on NK cells, limiting NK cell degranulation, and inhibiting the ability of NK cells to respond to exogenous cytokines. [2] In this process, researchers also demonstrated that the ADAM17 protease class can inhibit CD16 shedding, thereby enhancing the cytotoxic function of NK cells. This research opens up potential applications for CD16 in NK cell immunotherapy.

[0005] CD16 is a classic target and a candidate site for many drug developments. Clinical trials mainly involve kidney transplantation and treatment of end-stage renal disease, treatment of COVID-19 infection, treatment of solid tumors, treatment of cardiovascular diseases, and treatment of respiratory infections.

[0006] Improving the targeting specificity of NK cells for solid tumors, and providing more effective adjuvant therapy strategies for cancer patients, is a key focus in the development of NK cell drugs. Research on the mechanism of action of CD16, its synergistic molecules, and its relationship with signaling pathways has provided new directions for the development of NK cell immunotherapy.

[0007] When preparing antibodies using hybridomas, the instability of the hybridoma leads to the easy loss of antibody genes. Furthermore, hybridoma preparation technology does not identify the gene and protein sequences of the target antibody, hindering subsequent production, preparation, and research. Genetic engineering methods can effectively solve these problems. Through genetic engineering, the rejection response of the human body to antibodies can be reduced or even eliminated. Genetically engineered antibodies have smaller molecular weights, which can partially reduce their murine origin, making them more effective at penetrating blood vessel walls and reaching the core of lesions. A necessary condition for antibody preparation via genetic engineering is the accurate determination of the antibody sequence. Therefore, developing monoclonal antibodies more suitable for CD16 detection is a more appropriate research direction at present.

[0008] [1]Capuano C,Pighi C,Battella S,et al.Harnessing CD16-Mediated NKCell Functions to Enhance Therapeutic Efficacy of Tumor-TargetingmAbs.Cancers(Basel).2021May 20;13(10):2500.doi:10.3390 / cancers13102500.

[0009] [2]Goodier MR, Lusa C, Sherratt S, et al. Sustained Immune Complex-Mediated Reduction in CD16 Expression after Vaccination Regulates NK CellFunction. Front Immunol. 2016Sep 26; 7:384.doi:10.3389 / fimmu.2016.00384. Summary of the Invention

[0010] To address the aforementioned issues, this invention discloses for the first time the protein sequence of an anti-human CD16 mouse monoclonal antibody. By designing the gene and protein sequence, a stable CHO cell line transfecting this gene was established for recombinant expression. Verification showed that the expressed and purified antibody specifically recognizes the human CD16 antigen, and the antibody yield is significantly higher than that produced using traditional hybridoma cells. Furthermore, by replacing mouse IgG1 with rabbit IgG while retaining the variable region, a stable CHO cell line transfecting the rabbit monoclonal antibody was established for recombinant expression. Verification showed that the expressed and purified antibody maintains its specific recognition of the human CD16 antigen.

[0011] the term:

[0012] In this invention, CDRs (complementarity-determining regions) are called complementarity-determining regions or complementarity-determining clusters. They are located in the hypervariable region of immunoglobulins, which are antigen-binding sites of antibodies and are structurally complementary to the antigenic determinants. They generally include CDR1, CDR2, and CDR3.

[0013] In this invention, the variable region refers to the region of the immunoglobulin light chain and heavy chain near the N-terminus where the amino acid sequence changes significantly.

[0014] In this invention, the heavy chain refers to two longer, relatively larger identical heavy chains (H chains) in the antibody; the light chain refers to two shorter, relatively smaller identical light chains (L chains) in the antibody.

[0015] In this invention, similarity refers to the consistency of the amino acid sequence of homologous proteins and the proportion of substituted amino acids.

[0016] In this invention, monoclonal antibody refers to a highly homogeneous antibody produced by a single B cell clone that targets only a specific antigenic epitope.

[0017] In this invention, murine antibodies refer to antibodies secreted by murine hybrid fusion cells obtained by fusing B cells from immunized mice with myeloma cells.

[0018] On the one hand, the present invention provides an anti-CD16 antibody.

[0019] The anti-CD16 antibody comprises a heavy chain and a light chain, wherein the heavy chain includes a heavy chain variable region, and the light chain includes a light chain variable region; the heavy chain variable region includes HCDR1, HCDR2, and HCDR3, and the light chain variable region includes LCDR1, LCDR2, and LCDR3, wherein:

[0020] (1) HCDR1 is the amino acid sequence shown in SEQ ID NO.1;

[0021] (2) HCDR2 is the amino acid sequence shown in SEQ ID NO.2;

[0022] (3) HCDR3 is the amino acid sequence shown in SEQ ID NO.3;

[0023] (4) LCDR1 is the amino acid sequence shown in SEQ ID NO.4;

[0024] (5) LCDR2 is the amino acid sequence shown in SEQ ID NO.5;

[0025] (6) LCDR3 is the amino acid sequence shown in SEQ ID NO.6.

[0026] Specifically, the amino acid sequence of the variable region of the heavy chain of the anti-CD16 antibody is SEQ ID NO.7, or a sequence having more than 80% sequence similarity to SEQ ID NO.7; the amino acid sequence of the variable region of the light chain of the anti-CD16 antibody is selected from SEQ ID NO.8, or a sequence having more than 80% sequence similarity to SEQ ID NO.8.

[0027] Preferably, the amino acid sequence of the heavy chain variable region of the anti-CD16 antibody is SEQ ID NO.7, and the amino acid sequence of the light chain variable region is SEQ ID NO.8.

[0028] Specifically, the heavy chain amino acid sequence of the anti-CD16 antibody is SEQ ID NO.9, or a sequence having more than 65% sequence similarity to SEQ ID NO.9; the heavy chain amino acid sequence of the anti-CD16 antibody is SEQ ID NO.11, or a sequence having more than 65% sequence similarity to SEQ ID NO.11.

[0029] The light chain amino acid sequence of the anti-CD16 antibody is SEQ ID NO.10, or a sequence having more than 65% sequence similarity to SEQ ID NO.10, and the light chain amino acid sequence of the anti-CD16 antibody is SEQ ID NO.12, or a sequence having more than 65% sequence similarity to SEQ ID NO.12.

[0030] Preferably, the heavy chain amino acid sequence of the anti-CD16 antibody is SEQ ID NO.9 or SEQ ID NO.11, and the light chain amino acid sequence is SEQ ID NO.10 or SEQ ID NO.12.

[0031] Specifically, the anti-CD16 antibody is a monoclonal antibody.

[0032] Preferably, the anti-CD16 antibody is a murine antibody.

[0033] On the other hand, the present invention provides a nucleotide sequence expressing the aforementioned anti-CD16 antibody.

[0034] The nucleotide sequence can be a sequence optimized by codon degeneracy, and can be used to encode the aforementioned anti-CD16 antibody.

[0035] Preferably, the nucleotide sequences corresponding to the heavy chains SEQ ID NO.9 and SEQ ID NO.11 are shown in SEQ ID NO.14 and SEQ ID NO.15, respectively, and the nucleotide sequences corresponding to the light chains SEQ ID NO.10 and SEQ ID NO.12 are shown in SEQ ID NO.16 and SEQ ID NO.17, respectively.

[0036] In another aspect, the present invention provides an expression vector comprising the aforementioned nucleotide sequence.

[0037] The expression vector can be a plasmid, bacteriophage, or virus.

[0038] In another aspect, the present invention provides cells expressing the aforementioned anti-CD16 antibody or nucleotide sequence or expression vector.

[0039] The cells described above can efficiently express anti-CD16 antibodies.

[0040] Specifically, the cells may be HEK293 or CHO.

[0041] In another aspect, the present invention provides a method for preparing the aforementioned anti-CD16 antibody, wherein the method includes culturing the aforementioned cells.

[0042] The method may also include an antibody purification step.

[0043] The antibody purification steps can be precipitation, broad-spectrum affinity purification, antigen-specific purification, or ion exchange.

[0044] In another aspect, the present invention provides a method for fluorescently labeling the aforementioned anti-CD16 antibody, the method comprising labeling the anti-CD16 antibody with a macromolecular dye or a small molecule dye.

[0045] Preferably, the macromolecular dye is APC, and the small molecule dye is FITC.

[0046] In another aspect, the present invention provides the application of the aforementioned anti-CD16 antibody, nucleotide sequence, expression vector, or cell in CD16 detection, wherein the application is not for disease diagnosis or treatment.

[0047] The application described is achieved through antibody-antigen binding.

[0048] The application could be the detection of CD16 in NK lymphocytes.

[0049] In another aspect, the present invention provides the application of the aforementioned anti-CD16 antibody, nucleotide sequence, expression vector, or cell in the preparation of a CD16 detection kit.

[0050] Specifically, the kit also includes other reagents for CD16 detection, such as buffer solutions and sample processing agents.

[0051] Specifically, the kit may also include molecules for fluorescently labeled antibodies.

[0052] More specifically, the molecule can be a macromolecular dye or a small molecule dye.

[0053] Preferably, the macromolecular dye is APC, and the small molecule dye is FITC.

[0054] In another aspect, the present invention provides a CD16 detection method, wherein the detection method uses the aforementioned anti-CD16 antibody for detection.

[0055] Specifically, the detection method may also include a sample pretreatment step.

[0056] The preprocessing step involves sample collection and processing.

[0057] Preferably, the detection method is not a disease diagnosis or treatment method.

[0058] In another aspect, the present invention provides a CD16 detection kit, wherein the kit includes the aforementioned anti-CD16 antibody, nucleotide sequence, expression vector, or cells.

[0059] Specifically, the kit also includes other reagents for CD16 detection, such as buffer solutions and sample processing agents.

[0060] Specifically, the kit may also include molecules for fluorescently labeled antibodies.

[0061] More specifically, the molecule can be a macromolecular dye or a small molecule dye.

[0062] Preferably, the macromolecular dye is APC, and the small molecule dye is FITC.

[0063] The technical effects achieved by this invention are as follows: This invention discloses a protein sequence of an anti-human CD16 mouse monoclonal antibody, and designs a gene expression base sequence based on the sequenced protein sequence. A stable CHO cell line for recombinant expression of this gene was established. Verification showed that the expressed and purified antibody can specifically recognize the human CD16 antigen, and the antibody expression yield is far higher than that of antibodies prepared using traditional hybridoma cells. Furthermore, by replacing mouse IgG1 with rabbit IgG while retaining the variable region, a stable CHO cell line for recombinant expression of the rabbit monoclonal antibody was established. Verification showed that the expressed and purified antibody can maintain specific recognition of the human CD16 antigen. Attached Figure Description

[0064] Figure 1 This is an SDS gel electrophoresis image of anti-human CD16 antibody.

[0065] Figure 2 Flow cytometry results for Biolegend mouse anti-human CD16 [B73.1]-FITC purchased from outside the company.

[0066] Figure 3 Flow cytometry results for the self-made mouse anti-human CD16 [ZXCloneHCD16-04]-FITC.

[0067] Figure 4 The flow cytometry result of the self-made rabbit anti-human CD16 [ZXCloneHCD16-05]-FITC.

[0068] Figure 5 Flow cytometry analysis of purchased Biolegend mouse anti-human CD16 [B73.1]-APC.

[0069] Figure 6 Flow cytometry analysis of the self-made mouse anti-human CD16 [ZXCloneHCD16-04]-APC.

[0070] Figure 7 Flow cytometry analysis of self-made rabbit anti-human CD16 [ZXCloneHCD16-05]-APC.

[0071] Figure 8 The image shows the potency of Biolegend mouse anti-human CD16 [B73.1] purchased from outside the company.

[0072] Figure 9 The potency test chart of the self-made mouse anti-human CD16 [ZXCloneHCD16-04].

[0073] Figure 10 The potency test chart of the self-made rabbit anti-human CD16 [ZXCloneHCD16-05].

[0074] Figure 11 This is a specificity diagram of the purchased Biolegend mouse anti-human CD16 [B73.1] antibody.

[0075] Figure 12 The specificity diagram of the self-made mouse anti-human CD16 [ZXCloneHCD16-04] antibody.

[0076] Figure 13 The specificity diagram of the self-made rabbit anti-human CD16 [ZXCloneHCD16-05] antibody.

[0077] Figure 14This is a specificity diagram of the control CD16[B73.1] antibody. Detailed Implementation

[0078] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0079] Example 1: Preparation and Detection of Antibodies

[0080] Two antibodies were prepared: a self-made mouse anti-human CD16 [ZXCloneHCD16-04] and a self-made rabbit anti-human CD16 [ZXCloneHCD16-05].

[0081] The heavy chain amino acid sequence of the self-made mouse anti-human CD16 [ZXCloneHCD16-04] is SEQ ID NO. 9, and the light chain amino acid sequence is SEQ ID NO. 10; the heavy chain amino acid sequence of the self-made rabbit anti-human CD16 [ZXCloneHCD16-05] is SEQ ID NO. 11, and the light chain amino acid sequence is SEQ ID NO. 12. Nucleotide sequences for expressing the corresponding antibodies were designed using conventional methods. The nucleotide sequences corresponding to the heavy chain SEQ ID NO. 9 and SEQ ID NO. 11 are shown in SEQ ID NO. 14 and SEQ ID NO. 15, respectively, and the nucleotide sequences corresponding to the light chain SEQ ID NO. 10 and SEQ ID NO. 12 are shown in SEQ ID NO. 16 and SEQ ID NO. 17, respectively. After cloning the nucleotide sequences into a stable CHO transgenic cell line, stable antibody expression was performed, and the constructed vector was further introduced into CHO cells.

[0082] Transfection and screening:

[0083] (1) CHO cells were cultured in CHO CD medium at a concentration of 2 × 10⁶. 5 Inoculate cells / mL with 4 mM glutamine. Build up cell density to 4 × 10⁶ cells / mL. 6 Cells were collected at a density of 1 × 10⁶ cells / mL with a viability greater than 95%. Cells were collected by centrifugation, resuspended in electroporation buffer, and adjusted to a density of 1 × 10⁶ cells / mL. 7 cells / mL.

[0084] (2) Take a sterile EP tube, add 0.8 mL of cell suspension and 5 μg of plasmid, mix gently, incubate for 15 min, and then incubate on ice for 5 min. Set the electroporator parameters as follows: voltage 280V, pulse count 3, pulse duration 5 ms, pulse interval 0.784 s, and electroporation cuvette inner diameter 4 mm. Add 0.5 mL of the cell and plasmid mixture to each electroporation cuvette, place it in the electroporator, and start the electroporation program. After completion, quickly aspirate the cells from the electroporation cuvette and transfer them to a 125 mL Erlenmeyer flask containing 20 mL of CHO CD medium. Repeat the above steps to electroporate the second electroporation cuvette. Gently mix the cells in the shake flask, then tighten the cap and incubate in a shaker at 37°C.

[0085] (3) After 48 hours, transfer the cells to centrifuge tubes and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant and resuspend the cells in 20 mL of CHO CD medium containing 2, 5, and 20 μM MSX. Calculate cell density and viability. The cell density should be around 1.2 × 10⁻⁶ cells / mL. 6 Approximately [number] cells / mL.

[0086] (4) Transfer the cells to new shake flasks. Starting from day seven, observe cell density and viability every two days. Cell density should be increased from an initial 1.2 × 10⁻⁶. 6 The cell density decreases from 10 to 15 days, reaching its lowest point between days 5 and 7. The cell density then begins to increase. When the cell density reaches 1 × 10⁻⁶, the density gradually decreases. 6 After reaching a cell density of 3 × 10⁶ cells / mL, the cells were passaged to a cell density of 3 × 10⁶ cells / mL using CD OPM medium containing 50 μM MSX. 5 cells / mL, continue culturing.

[0087] (5) When the cell density increases to 4×10 6 After reaching a cell / mL level, cells were expanded or passaged using CD OPM medium containing 50 μM MSX, with a minimum seeding density of 3 × 10⁻⁶ cells / mL. 5 When the number of cells / mL reaches a certain level, preparations for cryopreservation or large-scale culture can begin.

[0088] (6) Antibody collection method: Antibody purification was performed using protein A affinity chromatography.

[0089] Take 10 μg of purchased Biolegend mouse anti-human CD16 [B73.1], 10 μg of self-prepared mouse anti-human CD16 [ZXCloneHCD16-04] (prepared in Example 1), and 10 μg of self-prepared rabbit anti-human CD16 [ZXCloneHCD16-05], mix them with the loading buffer (antibody to loading buffer volume ratio 4:1), incubate at 100℃ for 30 min, and centrifuge at 10000 rpm for 1 min at room temperature to obtain the sample to be tested. Take out the pre-cast gel and install it in the electrophoresis tank, inject the electrophoresis buffer, remove the comb, and load the sample. Among them, 10 μL of protein marker is loaded and 5 μg of sample to be tested is loaded; electrophoresis settings: voltage 80V, electrophoresis time 90 min, electrophoresis is stopped when the bromophenol blue indicator band reaches the bottom; take out the gel and stain it with Coomassie brilliant blue for 30 min, then place it in destaining solution for 1 h, repeat 5 times, and then take pictures.

[0090] The results showed that the anti-human CD16 antibody prepared in Example 1 had intact heavy and light chains, clear bands, and a purity of over 95%. (See attached figures.) Figure 1 .

[0091] Example 2 Antibody Labeling

[0092] 1. FITC mark

[0093] (1) Take 0.2 mg of self-made mouse anti-human CD16 [ZXCloneHCD16-04] and 0.2 mg of self-made rabbit anti-human CD16 [ZXCloneHCD16-05] into ultrafiltration tubes respectively, wash the antibodies three times with 1 mM PBS. Centrifuge the ultrafiltration tubes at 4000g for 2 min inverted position, collect the concentrated antibody, and dilute it to 10 mg / mL with 1 mM PBS.

[0094] (2) Take small molecule dye FITC (according to n) (摩尔质量) Antibody: n (摩尔质量) Add FITC (1:15 with dye) to the monoclonal antibody reaction solution and mix well.

[0095] (3) React at room temperature of 25℃ for 45 minutes in the dark on a shaker.

[0096] (4) Transfer the reaction solution to an ultrafiltration centrifuge tube, wash the antibody 5 times with 1mM PBS. Centrifuge the ultrafiltration tube inverted at 4000g for 2min to collect the concentrated antibody, and dilute the antibody with 1mM PBS to a final concentration of 0.5mg / mL.

[0097] The labeled antibodies were named as self-made mouse anti-human CD16 [ZXCloneHCD16-04]-FITC and self-made rabbit anti-human CD16 [ZXCloneHCD16-05]-FITC.

[0098] 2. APC tag

[0099] According to the method described in the patent "Method and Kit for Labeling Fluorescent Proteins and / or Conjugate Proteins with Monoclonal Antibodies (CN202010972671.X)," self-made mouse anti-human antibodies were labeled respectively.

[0100] CD16[ZXCloneHCD16-04] and self-made rabbit anti-human CD16[ZXCloneHCD16-05] were labeled with antibodies named self-made mouse anti-human CD16[ZXCloneHCD16-04]-APC and self-made rabbit anti-human CD16[ZXCloneHCD16-05]-APC.

[0101] 3. Detection of the effect of labeled fluorescent antibody

[0102] (1) Add 100 μL of cell quality control material (manufacturer: Beckman Coulter, catalog number: 6607077) to a 1.5 mL centrifuge tube.

[0103] (2) Take 1 μL of the fluorescently labeled antibody to prepare mouse anti-human CD16 [ZXCloneHCD16-04]-FITC, prepare rabbit anti-human CD16 [ZXCloneHCD16-05]-FITC, and prepare mouse anti-human...

[0104] CD16[ZXCloneHCD16-04]-APC and self-made rabbit anti-human CD16[ZXCloneHCD16-05]-APC were added to the samples respectively; 1 μL of the control fluorescent antibody Biolegend mouse anti-human CD16[B73.1]-FITC (manufacturer: Biolegend, catalog number: 360702) and Biolegend mouse anti-human CD16[B73.1]-APC (manufacturer: Biolegend, catalog number: 360702) were added to the samples respectively.

[0105] (3) All the above samples were reacted in the dark for 15 minutes.

[0106] (4) Add 1 mL of hemolysin and continue the reaction for 15 minutes. The hemolysin used in Zhengxi flow cytometry is selected. Hemolysin: Zhejiang Medical Device Registration No. 20200133.

[0107] (5) Centrifuge at 4000 rpm for 2 min, discard the supernatant, and add 500 μL PBS to resuspend the sample to be tested.

[0108] (6) Flow cytometry (Beckman DxFlex flow cytometer) was used to detect the proportion of positive cells in the sample, the clustering of the chromatogram, and other indicators, so as to determine whether the self-made mouse anti-human CD16 [ZXCloneHCD16-04] and self-made rabbit anti-human CD16 [ZXCloneHCD16-05] could be labeled with the fluorescent dyes FITC and APC, and whether the labeled fluorescent antibody was correctly used to detect the target antigen.

[0109] Both FITC and APC successfully labeled self-made mouse anti-human CD16 [ZXCloneHCD16-04] and self-made rabbit anti-human CD16 [ZXCloneHCD16-05], with proportions similar to those of the purchased Biolegend mouse anti-human CD16 [B73.1]. (See [link to FITC and APC data]). Figures 2-7 .

[0110] Example 3 Antibody titer

[0111] Antibody titer detection methods:

[0112] The antibody used in this embodiment is:

[0113] Antibody 1: Positive control, purchased Biolegend mouse anti-human CD16 [B73.1] (manufacturer: Biolegend, catalog number: 360702);

[0114] Antibody 2: Self-made mouse anti-human CD16 [ZXCloneHCD16-04];

[0115] Antibody 3: Self-made rabbit anti-human CD16 [ZXCloneHCD16-05].

[0116] First, antibodies 1, 2, and 3, at known concentrations, were diluted to 1 mg / mL. Then, they were further diluted with PBS at ratios of 1:100, 1:1000, 1:3000, 1:6000, 1:12000, 1:24000, 1:48000, 1:60000, and 1:96000. Antibodies 2 and 3 served as the experimental group, and antibody 1 served as the control group.

[0117] The experimental group consisted of two subgroups and a control group. Each subgroup received 100 μL of healthy human blood (company reference material). 1 μL of commercially available LCD16-APC flow cytometry antibody was added to each subgroup. After incubation in the dark for 15 minutes, 1 mL of hemolysin (Zhengxi flow cytometry hemolysin) was added to each of the three experimental groups. Hemolysin (Zhejiang Medical Device Registration No. 20200133), continue the reaction for 15 min. Centrifuge at 4000 rpm for 2 min, and discard the supernatant.

[0118] The experimental group was treated with 100 μL of antibody 2 and antibody 3 diluted according to the specified ratio, while the control group was treated with 100 μL of antibody 1 diluted according to the specified ratio. After mixing, the mixture was incubated at room temperature in the dark for 30 min. The sample was then resuspended in 900 μL of PBS and centrifuged at 4000 rpm for 2 min. The supernatant was discarded, and the sample was resuspended in 500 μL of PBS. The sample was then analyzed by flow cytometry.

[0119] The affinities of the three antibodies are as follows:

[0120] Antibody 1: Biolegend mouse anti-human CD16 [B73.1] purchased externally, with an affinity (kd) of 1324.6 (1 mg / mL);

[0121] Antibody 2: Self-made mouse anti-human CD16 [ZXCloneHCD16-04], with an affinity (kd) of 395.4 (1 mg / mL);

[0122] Antibody 3: Self-made rabbit anti-human CD16 [ZXCloneHCD16-05], with an affinity (kd) of 405.2 (1 mg / mL).

[0123] The results showed that the affinity of the self-made mouse anti-human CD16 [ZXCloneHCD16-04] and the self-made rabbit anti-human CD16 [ZXCloneHCD16-05] was stronger than that of the purchased Biolegend mouse anti-human CD16 [B73.1].

[0124] Example 4 Antibody Specificity

[0125] The antibody used in this embodiment is:

[0126] Antibody (1): Anti-human CD16 antibody [ZXCloneHCD16-04] prepared in Example 1;

[0127] Antibody (2): Anti-human CD16 antibody [ZXCloneHCD16-05] prepared in Example 1.

[0128] Pure antibiotics purchased from external sources:

[0129] Antibody (3): Biolegend mouse anti-human CD16 [B73.1] purchased externally (manufacturer: Biolegend, catalog number: 360702)

[0130] Commercially available fluorescent antibodies used for detection:

[0131] Fluorescent antibody (4): Biolegend mouse anti-human CD16 [B73.1]-FITC purchased from outside (manufacturer: Biolegend, catalog number: 360702).

[0132] Commercially available fluorescent antibodies for co-staining:

[0133] Fluorescent antibody (5): CD3-APC (manufacturer: Longyang Zhengxi Biotechnology Co., Ltd., catalog number: 110610272).

[0134] Antibody-specific detection methods:

[0135] Add 100 μL of cell quality control material (Beckman Coulter, catalog number: 6607077) and 1 mL of hemolysin to 1.5 mL centrifuge tubes, and react for 15 minutes. The hemolysin used is the Zhengxi flow cytometer hemolysin. Hemolysin: Zhejiang Medical Device Registration No. 20200133. Centrifuge at 4000 rpm for 2 min, discard the supernatant, and resuspend the cells in 100 μL PBS.

[0136] The experiment was divided into a pure antibody test group and a positive control group.

[0137] Experimental group: 3 μL of anti-human CD16 antibody [ZXCloneHCD16-04] and CD16 antibody [ZXCloneHCD16-05] were added respectively. After reacting in the dark for 15 minutes, the samples were resuspended in 500 μL of PBS, centrifuged at 4000 rpm for 2 min, the supernatant was discarded, and the samples were resuspended again in 100 μL of PBS. The following were added to the two samples blocked with pure anti-CD16 [ZXCloneHCD16-04] respectively.

[0138] CD16[ZXCloneHCD16-04]-FITC was added to two samples blocked with pure anti-CD16[ZXCloneHCD16-05].

[0139] Control group: 1 μL of CD16[ZXCloneHCD16-04]-FITC was added to each resuspended sample; 1 μL of CD16[ZXCloneHCD16-05]-FITC was added to each resuspended sample.

[0140] Both the control and experimental groups were treated with 1 μL of co-stained antibody CD3-APC.

[0141] After reacting all samples in the dark for 15 minutes, resuspend the samples in 500 μL of PBS, centrifuge at 4000 rpm for 2 min, discard the supernatant, and then resuspend the samples in 500 μL of PBS.

[0142] Flow cytometry (Agilent NovoCyte flow cytometer) was used to detect indicators such as the proportion of positive cells in the sample and the clustering of cell patterns, in order to determine whether ZXCloneHCD16-04 and ZXCloneHCD16-05 could specifically recognize the CD16 target antigen.

[0143] Depend on Figures 11-14 It was observed that the experimental group showed CD3-APC signal but no signals from CD16[ZXCloneHCD16-04]-FITC and CD16[ZXCloneHCD16-05]-FITC fluorescent antibodies. The control group, however, showed signals from both CD3 and CD16 fluorescent antibodies. This demonstrates that the CD16 antibody prepared from CHO / HEK293 can specifically recognize the CD16 target antigen.

Claims

1. An anti-CD16 antibody, characterized in that, It includes a heavy chain and a light chain, wherein the heavy chain includes a heavy chain variable region, and the light chain includes a light chain variable region; the heavy chain variable region includes HCDR1, HCDR2, and HCDR3, and the light chain variable region includes LCDR1, LCDR2, and LCDR3, wherein: (1) HCDR1 is the amino acid sequence shown in SEQ ID NO.1; (2) HCDR2 is the amino acid sequence shown in SEQ ID NO.2; (3) HCDR3 is the amino acid sequence shown in SEQ ID NO.3; (4) LCDR1 is the amino acid sequence shown in SEQ ID NO.4; (5) LCDR2 is the amino acid sequence shown in SEQ ID NO.5; (6) LCDR3 is the amino acid sequence shown in SEQ ID NO.

6.

2. The anti-CD16 antibody according to claim 1, characterized in that, The amino acid sequence of the variable region of the heavy chain of the anti-CD16 antibody is SEQ ID NO.7 or a sequence having more than 80% sequence similarity to SEQ ID NO.7; the amino group of the variable region of the light chain of the anti-CD16 antibody is SEQ ID NO.8 or a sequence having more than 80% sequence similarity to SEQ ID NO.8, or the amino group of the variable region of the light chain of the anti-CD16 antibody is SEQ ID NO.13 or a sequence having more than 80% sequence similarity to SEQ ID NO.

13.

3. The anti-CD16 antibody according to claim 1, characterized in that, The heavy chain amino acid sequence of the anti-CD16 antibody is SEQ ID NO.9, or a sequence with more than 65% sequence similarity to SEQ ID NO.9; Alternatively, the heavy chain amino acid sequence of the anti-CD16 antibody may be SEQ ID NO.11, or a sequence having more than 65% sequence similarity to SEQ ID NO.11; The light chain amino acid sequence of the anti-CD16 antibody is SEQ ID NO.10, or a sequence having more than 65% sequence similarity to SEQ ID NO.10; Alternatively, the light chain amino acid sequence of the anti-CD16 antibody may be SEQ ID NO.12, or a sequence having more than 65% sequence similarity to SEQ ID NO.

12.

4. The anti-CD16 antibody according to any one of claims 1-3, characterized in that, It is a monoclonal antibody.

5. The anti-CD16 antibody according to claim 4, characterized in that, These are murine or rabbit-derived antibodies.

6. A polynucleotide encoding the anti-CD16 antibody according to any one of claims 1-5; characterized in that, The nucleotide sequence encoding the heavy chain SEQ ID NO. 9 is shown in SEQ ID NO. 14, or the nucleotide sequence encoding the heavy chain SEQ ID NO. 11 is shown in SEQ ID NO. 15; the nucleotide sequence encoding the light chain SEQ ID NO. 10 is shown in SEQ ID NO. 16, or the nucleotide sequence encoding the light chain SEQ ID NO. 12 is shown in SEQ ID NO.

17.

7. An expression vector comprising the polynucleotide of claim 6.

8. Cells comprising the anti-CD16 antibody of any one of claims 1-5, the polynucleotide of claim 6, or the expression vector of claim 7.

9. A method for detecting CD16, characterized in that, The detection is performed using the anti-CD16 antibody according to any one of claims 1-5, wherein the detection method is for purposes other than disease diagnosis and treatment.

10. A CD16 detection kit, characterized in that, Includes the anti-CD16 antibody according to any one of claims 1-5, the polynucleotide according to claim 6, the expression vector according to claim 7, or the cell according to claim 8.

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