Antibody targeting CD16b and application thereof

Abstract

The invention relates to an antibody targeting CD16b. The antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises three complementary determining regions represented by amino acid sequences of SEQ ID NO: 1-3; the light chain variable region comprises three complementarity determining regions represented by amino acid sequences of SEQ ID NO: 5-7. According to the CD16b-targeting antibody provided by the invention, ELISA (Enzyme-Linked Immunosorbent Assay) and FACS (Form Amplification and Consumption Standard) detection prove that the antibody is specifically combined with CD16b but not combined with CD16a, so that the problem that most of the existing CD16-targeting antibodies cannot distinguish CD16a and CD16b subtypes is solved; in addition, since the antibody can bind to neutrophil, the antibody is a powerful tool for studying neutrophil and CD16b.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to an antibody targeting CD16b and an application thereof. Background Art

[0002] Fcγ receptor (FcγR) is a receptor for the Fc fragment of IgG antibodies, and there are three types: FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). Among them, FcγRI has a high affinity for the ligand Fcγ and is mainly expressed on monocytes and neutrophils; FcγRII binds to Fcγ with low affinity, which can promote the phagocytosis / endocytosis of immune complexes and B cell activation; FcγRIII is expressed on macrophages, NK cells, bone marrow precursor cells and neutrophils, and can mediate antibody-dependent cellular cytotoxicity (ADCC).

[0003] CD16 (FcγRIII) is a transmembrane glycoprotein, which is mainly divided into two subtypes: CD16a (FcγRIIIa) and CD16b (FcγRIIIb). CD16 molecules are widely expressed in NK cells, neutrophils, polymorphonuclear leukocytes, mononuclear cells and macrophages, but not in basophils. They have 96% sequence similarity in the extracellular Fcγ binding region. CD16a is mainly present in macrophages, monocytes and NK cells. It is a transmembrane receptor that can recognize and bind to the Fc segment of IgG, generate cytokines or produce antibody-dependent cytotoxicity, and is the main cytotoxic receptor. CD16b is mainly expressed on neutrophils, connected to the outer layer of the plasma membrane through a glycosyl-phosphatidylinositol anchor (GPI), and mediates the exocytosis of granule proteins. CD16b has no intracellular domain and cannot induce cell signaling by itself, but works synergistically with other FcγRs to promote the phagocytosis of antibody-coated microorganisms by neutrophils.

[0004] Antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by natural killer (NK) cells is an effective cytotoxic mechanism that can very effectively destroy stressed cells, such as virus-infected or tumor-transformed cells. CD16a is the main Fc receptor (FcR) on NK cells. ADCC through CD16a is one of the main mechanisms for NK cells to exert effector functions. Therefore, there is currently extensive basic research and clinical exploration of CD16a. For example, by engineering the Fc part of the antibody, its affinity for CD16a is enhanced, thereby improving ADCC. For example: in the Fc of the HER2 antibody margetuximab, some amino acids were replaced (F243L / R292P / Y300L / V305I / P396L); in the Fc of the CD20 antibody obinutuzumab, the fucose modification was performed. These amino acid replacements or glycosylation modifications can significantly increase the affinity of Fc and CD16a, improve ADCC, etc. Antibodies targeting CD16a can be used as part of NK cell engagers and applied to bispecific killer cell engagers (BiKE) or trispecific killer cell engagers (TRiKE), etc., to target tumor antigens together with CD16a, thereby enhancing the ADCC effect of NK cells. For example, AFM13 can bind to CD30 and CD16a bivalently; AFM24 can bind to CD16a on NK cells and EGFR on tumor cells. These molecules have good tumor killing effects.

[0005] However, there are relatively few studies on CD16b, and researchers speculate that its role in different types of cancer may have some commonalities and differences. As part of the innate immune system, neutrophils can recognize and bind to antibodies on the surface of cancer cells through CD16b, thereby participating in immune surveillance and clearance. CD16b may affect neutrophil-mediated ADCC by competing with CD16a for the Fc part on the antibody. This may limit the ability of neutrophils to kill cancer cells, especially in solid tumors. Neutrophils may release inflammatory mediators in the tumor microenvironment, and CD16b may be involved in regulating these inflammatory responses, thereby affecting the development and metastasis of tumors. It may affect the interaction between neutrophils and other immune cells (such as natural killer cells, dendritic cells, etc.), thereby affecting tumor immune responses. At the same time, the genetic polymorphism of CD16b may affect its expression and function in different individuals, which may be related to the differences in the response of different cancer patients to immunotherapy. Due to the role of CD16b in neutrophil-mediated immune responses, it may become a potential target for cancer treatment, especially in enhancing existing immunotherapy strategies. It is important to note that the specific role of CD16b in different types of cancer may vary depending on the tumor biology, microenvironment, and host immune status. Therefore, the role of CD16b in specific types of cancer may require more research to clarify.

[0006] More and more studies have found that neutrophils play an important role in many diseases, and have been confirmed to play an important role in the occurrence, development and treatment of cancer, and have good potential in the field of tumor treatment. However, due to the high similarity of the extracellular Fcγ binding sequence of CD16a and CD16b, the similarity is as high as 96% (such as Figure 1 ), most of the current antibodies targeting CD16 cannot distinguish between CD16a and CD16b subtypes. Therefore, the development of antibodies that specifically bind to the CD16b subtype without recognizing CD16a is of great significance for studying the functions of neutrophils and CD16b. Summary of the invention

[0007] 1. Technical issues to be resolved

[0008] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides an antibody that targets CD16b but does not bind to CD16a, thereby solving the problem that most current antibodies targeting CD16 cannot distinguish between CD16a and CD16b subtypes.

[0009] (II) Technical solution

[0010] In a first aspect, the present invention provides an antibody targeting CD16b, comprising: a heavy chain variable region (V H ) and light chain variable region (V L);

[0011] The heavy chain variable region includes a complementary determining region CDR-H1 represented by the amino acid sequence of SEQ ID NO: 1, a complementary determining region CDR-H2 represented by the amino acid sequence of SEQ ID NO: 2, and a complementary determining region CDR-H3 represented by the amino acid sequence of SEQ ID NO: 3;

[0012] The light chain variable region includes a complementarity determining region CDR-L1 represented by the amino acid sequence of SEQ ID NO:5, a complementarity determining region CDR-L2 represented by the amino acid sequence of SEQ ID NO:6, and a complementarity determining region CDR-L3 represented by the amino acid sequence of SEQ ID NO:7.

[0013] According to a preferred embodiment of the present invention, the heavy chain variable region (V H ) has the amino acid sequence shown in SEQ ID NO: 4; the light chain variable region (V L ) has the amino acid sequence shown in SEQ ID NO:8.

[0014] According to a preferred embodiment of the present invention, the antibody has an amino acid sequence as shown in SEQ ID NO:9.

[0015] In a second aspect, the present invention provides a polynucleotide encoding an antibody targeting CD16b.

[0016] According to a preferred embodiment of the present invention, the polynucleotide includes a gene fragment encoding a heavy chain variable region and a gene fragment encoding a light chain variable region; the gene fragment encoding the heavy chain variable region includes the gene fragment represented by SEQ ID NO:10-12; the gene fragment encoding the light chain variable region includes the gene fragment represented by SEQ ID NO:13-15.

[0017] According to a preferred embodiment of the present invention, the polynucleotide encoding the antibody targeting CD16b has a base sequence shown in SEQ ID NO:16.

[0018] In a third aspect, the present invention provides an expression vector loaded with the above-mentioned polynucleotide.

[0019] In a fourth aspect, the present invention provides a host cell transformed by the expression vector.

[0020] In a fifth aspect, the present invention provides a method for preparing an antibody targeting CD16b, by synthesizing a polynucleotide encoding the antibody targeting CD16b, inserting it into a vector to construct an expression vector, transferring the expression vector into a competent cell, obtaining a successfully transformed host cell, and culturing the host cell to separate and purify the antibody targeting CD16b from the host cell. The host cell may be a prokaryotic cell, such as Escherichia coli, Bacillus subtilis, Streptomyces, and Pseudomonas, or a eukaryotic cell, such as yeast, or a higher eukaryotic cell, such as an insect cell.

[0021] In a fifth aspect, the present invention provides a detection kit or an antibody chip or an antibody probe, which comprises the antibody targeting CD16b as described in any one of the above items.

[0022] (III) Beneficial effects

[0023] The present invention uses phage display technology, takes human CD16b as the target and human CD16a as the control, and screens an antibody that specifically binds to CD16b from the HSA (TM) fully human natural antibody library. The affinity of the antibody to CD16b is about 1.62-2.09nM. ELISA is used to detect CD16a ECD and CD16b ECD (ECD, extracellular domain), and FACS is used to detect CD16a and CD16b stable cell lines, which confirms that the antibody specifically binds to CD16b but not CD16a. The affinity of the antibody to CD16b is determined by BLI technology to be about 2×10 -9 mol / L. Finally, FACS experiments confirmed that the antibody could bind to neutrophils. Therefore, the fully humanized antibody specifically binding to human CD16b provided by the present invention is of great significance for the study of neutrophil immune function and the development of therapeutic antibodies.

[0024] The antibody targeting CD16b proposed in the present invention solves the problem that most current antibodies targeting CD16 cannot distinguish between CD16a and CD16b subtypes; in addition, since the antibody can bind to neutrophils, the antibody is a powerful tool for studying neutrophils and CD16b. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The homology of the amino acid sequences of CD16a and CD16b ECD is shown.

[0026] Figure 2 The figure shows the binding of 12H6 to the target protein CD16b and the control protein CD16a detected by single phage ELISA.

[0027] Figure 3 The results of SDS-PAGE detection of eukaryotic expressed 12H6 whole antibody are shown.

[0028] Figure 4 The ELISA method was used to measure the binding activity of 12H6 antibody to CD16b ECD recombinant protein.

[0029] Figure 5 The specific binding activity of 12H6 antibody to CD16b protein on the surface of stably transfected cells was determined by FACS method.

[0030] Figure 6 The binding curve and EC of 12H6 antibody to cell surface CD16b protein determined by FACS method are shown. 50 .

[0031] Figure 7 The binding curve and EC of 12H6 antibody to neutrophils measured by FACS are shown. 50 . DETAILED DESCRIPTION

[0032] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples described. The experimental methods in the following examples that do not specify specific conditions are all performed according to conventional techniques of molecular biology, biochemistry and immunology, or according to the conditions recommended by the manufacturer. The conventional techniques are fully explained in the following works: Molecular Cloning Laboratory Guide, Fourth Edition (MRGreen et al., 2017); Animal Cell Culture, Seventh Edition (R.lan Freshney, 2019); Current Protocols in Immunology, E John Coligan, 2018, etc.

[0033] In order to make the present disclosure more easily understood, some terms are defined before the embodiments:

[0034] The term "CD16" refers to the Fc fragment type III receptor of immunoglobulin G (FcγRIII), which is a receptor present on the surface of macrophages, monocytes, NK cells and neutrophils that can bind to IgG Fc with low affinity. Human CD16 has been identified as two forms, FcγRIIIa (CD16a) and FcγRIIIb (CD16b). CD16a is mainly expressed in macrophages, monocytes and NK cells, and is divided into two variants, CD16a-F176 and CD16a-V176; CD16b is mainly expressed in neutrophils and is a GPI-anchored glycoprotein, which is also divided into two variants, CD16b-NA1 and CD16b-NA2.

[0035] The term "antibody" refers to a polypeptide that contains sufficient sequence from the variable region of an immunoglobulin heavy chain and / or sufficient sequence from the variable region of an immunoglobulin light chain to be able to specifically bind to an antigen. The term "antibody" herein encompasses various forms and structures, as long as they exhibit the desired antigen-binding activity, including but not limited to: scFv, Fab, Fab', F(ab')2, etc. A complete antibody contains two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The heavy chain consists of a heavy chain variable region (abbreviated as V H ) and the heavy chain constant region (including three domains CH1, CH2 and CH3), and the light chain consists of the light chain variable region (abbreviated as V L ) and a light chain constant region (abbreviated as CL). Both VH and VL are composed of three complementary determining regions (CDRs) and four framework regions (FRs), and the order from the amino terminus to the carboxyl terminus is FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0036] The "CDR" disclosed herein is annotated and defined by the Kabat numbering system, and the tool websites used include the abYsis website (www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi) and the IMGT website (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi#results). The variable regions of the heavy and light chains contain binding domains that can interact with antigens. Antibody heavy chains can be divided into five categories according to the sequence of the constant region (CH), namely IgG, IgM, IgA, IgE and IgD, among which IgG can be divided into five subclasses, namely IgG1, IgG2, IgG3 and IgG4. Antibody light chains are divided into two types: kappa and lambda.

[0037] The term "Fc fragment" refers to the crystallizable fragment (Fc) domain of an antibody, which is located in the C segment of the antibody heavy chain and is composed of CH2 and CH3. The Fc segment of an antibody can extend the half-life of the antibody by binding to the neonatal Fc receptor (FcRn), and can also specifically bind to the Fc receptor in the body to exert corresponding biological functions, such as antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDCC) and antibody-dependent cellular phagocytosis (ADCP). In addition, the Fc segment can also specifically bind to proteinA to facilitate the purification of recombinant proteins. "Fc variant" refers to changes in the structure or function of Fc caused by the presence of one or more amino acid substitutions, insertions or deletions at appropriate sites on Fc. The Fc variant used in the present invention is a mutation introduced into the humanIgG1 Fc fragment at three sites (Leu234Phe, Leu235Glu and Asp265Ala, numbering according to the Kabat EU index), which is intended to silence the interaction between Fc and Fc receptor (FcR).

[0038] The term "single-chain Fv antibody" (or "scFv antibody") refers to an antibody fragment comprising the VH and VL domains of an antibody, which domains are present in one polypeptide chain. Generally speaking, the Fv polypeptide comprises an additional polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. A "Fab' fragment" contains a light chain and a portion of a heavy chain comprising the VH domain and the CH1 domain and the region between the CH1 and CH2 domains, whereby an interchain disulfide bond can be formed between the two heavy chains of the two Fab' fragments to form a F(ab')2 molecule.

[0039] The term "fully human antibody" refers to an antibody whose amino acid sequence corresponds to the amino acid sequence of an antibody produced by a human, and / or an antibody that has been prepared using any of the techniques for preparing human antibodies disclosed herein. This definition specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0040] The term "recombinant expression vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is connected, a vector capable of self-replicating nucleic acid structure, and can transcribe mRNA and translate into protein after being introduced into a host cell. The term "host cell" refers to a mammalian cell containing a recombinant expression vector described herein. The recombinant expression vector can be transfected into a host cell by a variety of techniques, for example, protoplast fusion, calcium phosphate precipitation, electroporation, gene gun, lipofection or other conventional techniques. Methods and conditions for culturing the transfected cells produced and for purifying antibody molecules are known to those skilled in the art and can be based on methods known in the present specification and prior art.

[0041] The term "specific binding" refers to the antibody binding to a specific antigen epitope with a higher affinity than to other antigen epitopes. It is usually expressed as an equilibrium dissociation constant (KD), which is generally expressed as 1×10 -8 The KD of M or less can be used to bind to the antigen epitope. Standard procedures such as surface plasmon resonance (SPR) or biomembrane interferometry (BLI) can be used to measure the binding and dissociation curves of the antibody and antigen, and the association rate (Kon) and dissociation rate (Koff) can be fitted, and finally the KD value (Koff / Kon) can be calculated.

[0042] The term "EC50", also known as half maximal effective concentration, refers to the concentration of an antibody that elicits a response halfway between baseline and maximum after a specified exposure time.

[0043] The recombinant proteins used in the present invention are all purchased from ACROBiosystems, and the protein catalog number information is as follows:

[0044] Bio-CD16a(F176),Biotinylated Human Fc gamma RIIIA / CD16a(F176)Protein,Avitag TM ,His Tag,Acro,CDA-H82E8;

[0045] Bio-CD16a(V176),Biotinylated Human Fc gamma RIIIA / CD16a(V176)Protein,Avitag TM ,His Tag,Acro,CDA-H82E9

[0046] Bio-CD16b(NA1),Biotinylated Human Fc gamma RIIIB / CD16b(NA1)Protein,His,Avitag TM ,Acro,CDB-H82E4

[0047] Bio-CD16b(NA2),Biotinylated Human Fc gamma RIIIB / CD16b(NA2)Protein,His,Avitag TM ,Acro,CDB-H82Ea

[0048] CD16a(V176),Human Fc gamma RIIIA / CD16a(V176)Protein,His Tag,Acro,CD8-H52H4

[0049] like Figure 1 As shown, the homology of the amino acid sequences of CD16a and CD16b ECD is shown. The amino acid sequence alignment results of the two target proteins (humanCD16b-NA1 and human CD16b-NA2) and two control proteins (human CD16a-F176 and human CD16a-V176) are shown in Figure 1. Figure 1 As shown in the figure, the two proteins CD16b and CD16a have only two amino acid residues at position 131 (G / D) and position 142 (Y / H) that differ in their primary structure (amino acid sequence), and their homology is very high, which makes it very difficult to screen antibodies that specifically bind to the CD16b subtype but do not recognize CD16a.

[0050] Example 1

[0051] In this example, a monoclonal antibody (named 12H6) was obtained by panning phage-displayed antibodies, and the antibody was sequenced to determine the amino acid sequences of the antibody and the three complementary determining regions of the heavy chain variable region and the three complementary determining regions of the light chain variable region. The steps of this example are as follows:

[0052] (1) Amplification of phage display antibody library

[0053] In this example, phage display technology was used to screen for human CD16b extracellular region protein (CD16b ECD)-specific antibodies from the HSA(TM) fully human natural antibody library.

[0054] First, the HSA(TM) fully human natural antibody phage display library was prepared. 9 cells / ml) of HSA(TM) fully human natural antibody library was inoculated into 400 mL of 2×YT medium, and 100 μg / mL of ampicillin and 2% glucose were added. The culture was shaken at 37°C and 200 rpm. When the cell density reached OD600 nm = 0.5, 10 12pfu of M13KO7 helper phage (purchased from Invitrogen), let it stand for infection for 60 minutes, collect the bacterial precipitate by centrifugation (3000×g, 15min, 4°C), resuspend the precipitate in 400mL 2×YT medium, add 100μg / mL of ampicillin and 50μg / mL of kanamycin, and culture overnight at 37°C and 200rpm. The next day, collect the supernatant by centrifugation (8,000×g, 20min, 4°C), add 1 / 4 volume of PEG8000 (20% w / v) / NaCl (2.5M) solution to the supernatant, and incubate in an ice-water bath for 1 hour to precipitate phage particles. Collect the phage precipitate by centrifugation (8,000×g, 20min, 4°C) and dissolve it in PBS.

[0055] Take 10 μL of phage solution and dilute it to 990 μL PBS (recorded as 10 -2 ), and then take 10 μL from it and add it to the second tube of 990 μL PBS and mix thoroughly (recorded as 10 -4 ), and so on to dilute to 10 -8 , take 100 μL from it and put it into the second tube with 900 μL PBS (recorded as 10 -9 ), and then take 100 μL from it and put it into the second tube of 900 μL PBS (recorded as 10 -10 ). -8 , 10 -9 and 10 -10 10 μL of phage solution was taken from the gradient to infect 100 μL of E. coli TG1 bacterial solution at the mid-log phase (OD600 nm = 0.5), and then coated on 2×YT / ampicillin resistance plates. After overnight culture at 32°C, the number of clones was counted and the titer of phage was calculated. In this case, the titer of the prepared HSA(TM) fully human natural antibody phage display library was 3.2×10 13 cfu / mL.

[0056] (2) Screening of phage display antibody libraries

[0057] The HSA(TM) fully human natural antibody phage display library was enriched for the CD16b target in the first round. First, Biotinylated HumanFc gamma RIIIB / CD16b(NA2)Protein (Acro, CDB-H82Ea) was captured with Pierce NeutrAvidin Coated Plates (thermo Fisher, 15128), and then the phage display library was added. After incubation at room temperature for 1 hour, the plate was washed with PBST (containing 0.1% Tween-20) buffer to remove non-specific binding or weak binding phages. Phages with strong binding ability were eluted from the magnetic beads with glycine-hydrochloric acid (pH 2.2), neutralized with Tris neutralizing solution (pH 9.1), and used to infect Escherichia coli TG1 in the middle of logarithmic growth, and used for the next round of screening.

[0058] In order to enrich antibodies that specifically bind to CD16b, a negative screening was performed on the first round of screening products against CD16a protein to remove phages that bind to CD16a protein in the enriched products. TM Bio-CD16a (F176) was captured by Streptavidin Coated Plates (thermo Fisher, 15121), and then the phage display library was added. After incubation at room temperature for 1 hour, the solution in the plate was collected (at this time, the antibody binding to CD16a was captured by the CD16a protein fixed on the plate). Then, the CD16b target was enriched in the second round according to the first round method.

[0059] In order to further increase the clearance of phage bound to CD16a protein, starting from the third round, in addition to negative screening and clearing of CD16a protein before screening, non-biotinylated CD16a protein was also added during the screening process for competition. The pre-cleared phage was incubated with bio-CD16b (NA2) and CD16a (V176) at room temperature for 2 hours at the same time, and then incubated with streptavidin magnetic beads MyOne C1 blocked with blocking solution 2% (w / v) BSA at room temperature for 30 minutes. After washing the magnetic beads with PBST (containing 0.1% Tween-20) buffer, the bound phage was eluted from the magnetic beads with glycine-hydrochloric acid (pH 2.2), neutralized and used to infect Escherichia coli TG1 in the middle of logarithmic growth, and used for the next round of screening. This embodiment carried out a total of 5 rounds of enrichment screening.

[0060] Monoclonal screening was performed on the screening products of the fifth round. 96 clones were randomly selected from the clones obtained in the fifth round of screening and inoculated into 96-well deep-well plates (300 μl 2×YT, 100 μg / mL ampicillin, 2% glucose) and cultured overnight at 37°C and 200 rpm. The next day, 20 μL of the culture was transferred to a new 96-well deep-well plate (500 μl 2×YT, 100 μg / mL ampicillin, 0.1% glucose). After culturing for 1.5 h at 37°C and 200 rpm, 50 μL of M13KO7 (titer of 1×10 11 pfu / ml) Helper phage, cultured at 37°C and 150rpm for 30min, and then added kanamycin with a final concentration of 50μg / mL to each well. Cultured overnight at 37°C and 200rpm, and finally, the supernatant was collected by centrifugation (5,000×g, 30min, 4°C) for subsequent phage ELISA verification.

[0061] Monoclonal phage ELISA to identify clones that specifically bind to CD16b: Streptavidin (SA, 2 μg / mL) was coated on a 96-well MediSorp ELISA plate (purchased from Nunc), and after blocking with PBST containing 2% BSA (w / v), Biotinylated Human Fc gamma RIIIB / CD16b (NA2) Protein and Biotinylated Human Fc gamma RIIIA / CD16a (F176) Protein were captured, respectively, and SA was used as a negative control. Then, the monoclonal phage supernatant solution was added at 100 μL / well, incubated at 37°C for 1 hour, washed, and then incubated with M13 antibody superoxide dismutase conjugate (purchased from GE Healthcare). Finally, 50 μl of TMB substrate was added to each well for color development, and after adding the stop solution, the extinction value was measured at 450 nm using an enzyme-linked immunosorbent assay (Bio-Rad). Combined with sequencing analysis, it was finally observed that one clone 12H6 had a strong binding signal to CD16b and did not bind to CD16a and SA ( Figure 2 ).

[0062] Example 2

[0063] In order to characterize the anti-CD16b antibody 12H6, this experiment used a eukaryotic transient expression system to prepare the complete 12H6 antibody. For this purpose, the V of the 12H6 antibody was first obtained by PCR amplification. H and V LThe fragments were fused with human IgG1CH and human CLλ to construct expression vectors pcDNA3.1(-)-12H6-H and pcDNA3.1(-)-12H6-L, respectively. In order to prevent the binding of IgG1 Fc to FcγR from interfering with subsequent detection, IgG1 CH was mutated at three sites (Leu234Phe, Leu235Glu and Asp265Ala). 50 μg of each of the two expression plasmids was mixed with a transfection reagent (Gibco, A14524) and then added dropwise to 100 mL of Expi293F cell (Gibco, A14527) culture and cultured at 37°C and 8% CO2 for 5-7 days. The expressed cell suspension was centrifuged to collect the culture supernatant, which was filtered through a 0.22 μm filter membrane and loaded onto a PrePack iProtein A Purose 4FF (Qianchun, ZA41201-02) affinity chromatography column. The column was washed with 1×PBS pH 7.4 buffer to remove impurities, and then eluted with elution buffer (0.1M Glycine, pH 2.3) to obtain the 12H6 whole antibody. The eluted antibody was neutralized to neutrality using 1M Tris-HCl pH 8.0, and then the sample buffer was replaced with 1×PBS pH 7.4 by dialysis. Finally, the purified 12H6 whole antibody was detected by SDS-PAGE (amino acid sequence and coding gene are shown in Table 1-2), indicating that its purity was above 95%. The experimental results are shown in Figure 3 By comparing with the antibody sequences registered in the antibody gene library, it was confirmed to be a new antibody gene.

[0064] Table 1: 12H6 IgG antibody amino acid sequence and coding gene sequence and numbering

[0065]

[0066] Table 2: Amino acid sequence and DNA sequence of 12H6 scFv antibody fragment

[0067]

[0068] Note: The underlined amino acid sequences or gene fragments indicate the complementarity determining regions.

[0069] Example 3

[0070] This example uses antibody concentration gradient ELISA to detect the binding activity of 12H6 antibody to two CD16a variants (F176 and V176) and two CD16b variants (NA1 and NA2) ECD recombinant proteins. The specific experimental operation is as follows:

[0071] Streptavidin (SA, 2 μg / mL) was coated on a 96-well MediSorp ELISA plate (purchased from Nunc). After blocking with PBST containing 2% BSA (w / v), four recombinant proteins (1 μg / mL) were captured: bio-CD16b (NA1), bio-CD16b (NA2), bio-CD16a (F176) and bio-CD16a (V176), and SA was used as a negative control. Then, gradient concentrations of 12H6 purified antibody were added, incubated at 37°C for 1 hour, washed, and then incubated with HRP-F(ab')2Fragment Goat Anti-Human IgG (H+L) (Jackson ImmunoResearch, 109-036-088). Finally, 50 μl of TMB substrate was added to each well for color development. After adding the stop solution, the extinction value was measured at 450 nm using an enzyme-linked immunosorbent assay (Bio-Rad). The specific experimental results are as follows. Figure 4 As shown, the results showed that the 12H6 antibody could specifically bind to CD16b (NA1) and CD16b (NA2), but not to CD16a (F176) and CD16a (V176).

[0072] Example 4

[0073] In this example, the binding affinity of 12H6 antibody to two CD16b variants (NA1 and NA2) ECD recombinant proteins was determined by the Octet RED96e system. The specific experimental operation is as follows:

[0074] Prepare BLI detection buffer: 1×PBS pH 7.4, 0.02% Tween-20, 0.2% BSA. Dilute the antibody to be tested to 3μg / mL with the above buffer, and gradient dilute CD16b protein (200nM, 100nM, 50nM, 25nM and 12.5nM). Add 200μL of BLI detection buffer to each well of the first column A1-F1 of the black 96-well plate; add 200μL of diluted 12H6 antibody solution to each well of the second column A2-F2; add 200μL of BLI detection buffer to each well of the third column A3-F3; add 5 gradient concentrations of CD16b protein in the fourth column A4-E4 in turn, and add BLI detection buffer to F4, with a sample volume of 200μL per well; add 200μL of BLI detection buffer to each well of the fifth column A5-F5.

[0075] The 6 ProA probes were loaded onto the Octet BLI instrument. They were first placed in the first column of a black 96-well plate to activate and equilibrate the probes for 180 seconds, then transferred to the second column to capture the 12H6 antibody. When the Binding signal reached 1 nm, the probes were quickly transferred to the third column to equilibrate the probes for 60 seconds. The probes were then transferred to the fourth column to incubate the CD16b target protein for Association determination, and the time was set to 60 seconds. Finally, they were transferred to the BLI detection buffer in the fifth column to determine Dissociation. The detection data was exported, and the Association and Dissociation curves were fitted using ForteBio Analysis 6.4 software, and Kon (binding rate constant), Koff (dissociation rate constant) and KD value (dissociation equilibrium constant) were calculated; see Table 3.

[0076] Table 3: BLI method to determine the affinity of 12H6 antibody for two CD16b ECD proteins

[0077] Antigen KD(M) Kon(1 / Ms) Koff(1 / s) CD16b(NA1) 1.62E-09 3.16E+05 5.12E-04 CD16b(NA2) 2.09E-09 3.05E+05 6.37E-04

[0078] Example 5

[0079] This example uses the FACS method to detect the binding activity of candidate antibodies to CD16 on the surface of stably transfected cell lines. The experimental process is as follows:

[0080] (1) Construction of CD16b and CD16a stable transfection cell lines

[0081] Lentivirus packaging: The DNA sequences of full-length human CD16A F158, CD16A V158, CD16B NA1 and CD16B NA2 were inserted into the lentivirus core plasmid pWPT to construct four plasmids pWPT-CD16A F158, pWPT-CD16AV158, pWPT-CD16BNA1 and pWPT-CD16B NA2. 293T cells were inoculated in a cell culture plate and cultured in a 37°C, 5% CO2 incubator. When the cell density reached 70% to 80%, they could be used for transfection. The four plasmids were mixed with two psPAX2 and pMD2G, respectively, and mixed with transfection reagents to prepare four transfection complexes. The above four transfection complexes were used to transfect 293T cells, cultured at 37°C, 5% CO2 for 6 to 8 hours, and then replaced with fresh culture medium. After that, the culture was continued for 48 hours, the supernatant was collected, and the four lentivirus particles were concentrated and purified.

[0082] Lentivirus infection of CHO-K1 cells: CHO-K1 cells were inoculated into cell culture plates and cultured overnight at 37°C and 5% CO2. The next day, the four lentivirus clones were used to infect CHO-K1 cells. After 24 hours of culture at 37°C and 5% CO2, fresh complete culture medium was replaced and cultured for another 48 hours. Cells were collected and single cell suspensions were prepared. After counting, 1.0×106 cells were taken for each transformation, washed twice with FACS buffer (1×PBS pH 7.4, 1% FBS), and resuspended in 100 μL FACS buffer. 3 μg CD16 Monoclonal Antibody (3G8)-FITC (Thermo, MHCD1601) was added and incubated at 4°C for 1 hour. The cells were washed again with FACS buffer and resuspended in 0.5 mL FACS buffer. The cells that could be stained by the antibody were sorted into a 96-well cell culture plate as single cells by flow sorter (SONY SH800) and cultured at 37°C and 5% CO2. Finally, these monoclonal cells were identified by flow analyzer (Agilent, NovoCyte) to obtain 4 stable cell lines.

[0083] (2) FACS method to determine the binding activity of 12H6 antibody to stably transfected cells

[0084] Four stable cells (CHO-K1 / CD16A F158, CHO-K1 / CD16A V158, CHO-K1 / CD16B NA1 and CHO-K1 / CD16B NA2) were cultured in a cell culture dish, and CHO-K1 cells were cultured as a negative control. When the cells grew to 70% confluence, the cell suspension was collected, the cells were washed with FACS buffer, and resuspended to 2.0×106 cells / mL, and then added to a V-bottom 96-well plate at 100 μL / well (each cell was laid in 3 columns, a total of 15 columns). 12H6 antibody was gradiently diluted with FACS buffer (150nM, 50nM, 16.67nM, 5.56nM, 1.85nM, 0.62nM, 0.21nM, 0.07nM, a total of 8 gradients), and added to the above-mentioned well plate at 100 μL / well, and the 8 gradients were added to rows AH in sequence. After the cells were evenly blown, they were incubated at 4°C for 1 hour. After the incubation, the cells were centrifuged at 300×g for 5 minutes, the supernatant was discarded, 200 μL of FACS buffer was added to the corresponding wells, the cells were resuspended, and the supernatant was discarded. This was repeated twice. FITC-labeled F(ab')2Fragment Goat Anti-Human IgG(H+L) secondary antibody (Jackson, 109-096-088) was added to each well and incubated at 4°C for 1 hour. After the incubation, the cells were centrifuged at 300×g for 5 minutes, the supernatant was discarded, 200 μL of FACS buffer was added to the corresponding wells, the cells were resuspended, and the supernatant was discarded. This was repeated twice. Finally, the cells were resuspended in 200 μL of FACS buffer and loaded onto a flow cytometer for detection.

[0085] The results are shown in Figure 5 and Figure 6 The results showed that under the same conditions, the candidate antibody could bind to the CD16b protein on the surface of CHO-K1 / CD16B NA1 and CHO-K1 / CD16B NA2 stable cell lines, but not to the CD16a protein on the surface of CHO-K1 / CD16AF158 and CHO-K1 / CD16A V158 cells.

[0086] Example 6

[0087] This example measures the binding activity of 12H6 antibody to human neutrophils by FACS. To this end, human neutrophils were first isolated and purified from human peripheral blood using a neutrophil isolation kit (TBD, LZS11131). The specific implementation method is as follows:

[0088] Take a 50mL sterile centrifuge tube, add 20mL of neutrophil separation solution, and then carefully add 15mL of fresh anticoagulated blood sample, 600×g, 20℃ centrifuge for 30min, at this time, two layers of ring-shaped milky white cells will appear in the centrifuge tube, the upper layer of cells is the mononuclear cell layer, and the lower layer of cells is the neutrophil layer. Transfer the neutrophil layer to a new centrifuge tube, add 10mL of washing solution (provided in the kit) to mix the cells, centrifuge at 250×g, 20℃ for 10min, discard the supernatant, and wash the cells again with 10mL of washing solution. Finally, resuspend the cells with 1×PBS pH 7.4 and measure the concentration of neutrophils by a cell counter.

[0089] The isolated neutrophils were washed with FACS buffer and resuspended to 5.0 × 10 6 cells / mL, and then added to a V-bottom 96-well plate at 100 μL / well. Detection was performed using the FACS method described in Example 5.2. The results are shown in Figure 7 The results showed that the 12H6 antibody had good binding activity to human neutrophils (EC50 = 15.37 nM).

[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An antibody targeting CD16b, characterized in that: include: A heavy chain variable region and a light chain variable region; the heavy chain variable region includes a complementary determining region CDR-H1 represented by the amino acid sequence of SEQ ID NO: 1, a complementary determining region CDR-H2 represented by the amino acid sequence of SEQ ID NO: 2, and a complementary determining region CDR-H3 represented by the amino acid sequence of SEQ ID NO: 3; The light chain variable region includes a complementarity determining region CDR-L1 represented by the amino acid sequence of SEQ ID NO:5, a complementarity determining region CDR-L2 represented by the amino acid sequence of SEQ ID NO:6, and a complementarity determining region CDR-L3 represented by the amino acid sequence of SEQ ID NO:

7.

2. The antibody targeting CD16b according to claim 1, characterized in that The heavy chain variable region has the amino acid sequence shown in SEQ ID NO:4; the light chain variable region has the amino acid sequence shown in SEQ ID NO:

8.

3. The antibody targeting CD16b according to claim 1, characterized in that The antibody has the amino acid sequence shown in SEQ ID NO:

9.

4. A polynucleotide encoding the antibody targeting CD16b according to any one of claims 1 to 3.

5. The polynucleotide according to claim 4, characterized in that The polynucleotide includes a gene segment encoding a heavy chain variable region and a gene segment encoding a light chain variable region; the gene segment encoding the heavy chain variable region includes the gene segment represented by SEQ ID NO: 10-12; the gene segment encoding the light chain variable region includes the gene segment represented by SEQ ID NO: 13-15.

6. The polynucleotide according to claim 4, characterized in that The polynucleotide encoding the antibody targeting CD16b has the base sequence shown in SEQ ID NO:

16.

7. An expression vector loaded with the polynucleotide according to any one of claims 4 to 6. A host cell transformed by the expression vector according to claim 7.

9. A method for preparing an antibody targeting CD16b, characterized in that: The method is obtained by culturing the host cell according to claim 8 and isolating and purifying the host cell from the host cell lysate.

10. A detection kit or an antibody chip or an antibody probe, comprising the antibody targeting CD16b according to any one of claims 1 to 3.

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