CN117229399A - Fully human monoclonal antibody capable of specifically recognizing CLEC12A receptor - Google Patents

Abstract

The application provides a fully human antibody specifically recognizing and combining CLEC12A, which is characterized in that the fully human antibody is recombinant immunoglobulin, the structural form is scFv-Fc, scFv refers to a single-chain antibody, the fully human antibody comprises a heavy chain variable region and a light chain variable region, and the amino acid sequence of the heavy chain variable region is SEQ ID NO:3, the amino acid sequence of the light chain variable region is SEQ ID NO:2, fc refers to the constant region. The sequence of the fully human antibody specifically recognizing CLEC12A is SEQ ID NO:4. the antibody can specifically recognize and bind to a human CLEC12A receptor, and can be used as a detection diagnosis and therapeutic antibody of the CLEC12A receptor.

Description

A fully human monoclonal antibody that specifically recognizes CLEC12A receptor

Technical field

The invention belongs to the field of biomedicine technology and relates to a monoclonal antibody against CLEC12A receptor and a preparation method thereof. Specifically, the present invention discloses a monoclonal antibody that can specifically recognize the CLEC12A receptor.

Background technique

CLEC12A (C-type lectin domain family 12 member A), also known as CLL-1, MICL, DCAL-2, KLRL-1 and CD371. CLEC12A is mainly expressed on myeloid cells, including macrophages, granulocytes and DC cell lines [1]. CLEC12A has an immunoreceptor tyrosine-based inhibition motif (ITIM) intracellularly and a C-type lectin like domain (CTLD) extracellularly. CLEC12A plays an important role in acute myeloid leukemia. A large number of studies have shown that CLEC12A is selectively and highly expressed on the surface of acute myeloid leukemia cells, while its expression is almost undetectable on the surface of normal hematopoietic cells. Therefore, CLEC12A has become a molecular marker for detecting acute myeloid leukemia cells and a new immunotherapy target for the targeted treatment of acute myeloid leukemia. In addition, CLEC12A is also a marker of cancer stem cells in myelodysplastic syndrome. At present, the most important treatment method for acute myeloid leukemia is chemotherapy, but the treatment effect is not ideal, it is easy to relapse, and it has serious side effects. Currently, the use of biotechnology to treat acute myeloid leukemia is relatively hot, such as using targeted CLEC12A antibody therapy or designing CLEC12ACAR-T cell therapy to treat acute myeloid leukemia. These treatments are currently under research or clinical trials. This patent provides new and effective therapeutic antibodies for acute myeloid leukemia by screening out antibodies targeting CLEC12A [2,3,4,5].

references:

1.Han Y M, Zhang M H, Li N, et al. KLRL1, a novel killer cell lectinlikereceptor, inhibits natural killer cell cytotoxicity[J]. Blood, 2004, 104(9): 2858-2866.

2.MarshallA S J, Willment J A, Pyz E, et al. Human MICL (CLEC12A) is differentially glycosylated and is down-regulated following cellular activation [J]. European Journal of Immunology, 2006, 36(8): 2159-2169.

3.Marshall A S J, Willment J A, Lin H H, et al. Identification and characterization of a novel human Myeloid inhibitory c-type lectin-likereceptor (MICL) that is predominantly expressed on granulocytes and monocytes[J]. Journal of Biological Chemistry, 2004, 279(15):14792-14802.

4.Bakker A B, Van Den Oudenrijn S, Bakker A Q, et al.C-type lectin-likemolecule-1: a novel myeloid cell surface marker associated with acute myeloidleukemia[J].Cancer Res, 2004, 64(22):8443 -50.

5.Leong S R,Sukumaran S,Hristopoulos M,et al.An anti-CD3/anti-CLL-1bispecific antibody for the treatment of acute myeloid leukemia[J].Blood,2017,129(5):609-618.

Contents of the invention

The purpose of the present invention is to provide a monoclonal antibody or antibody fragment that can specifically recognize CLEC12A and a preparation method thereof, and the antibody or antibody fragment can recognize and bind to CLEC12A.

In order to achieve the above purpose, the present invention provides a fully human monoclonal antibody that specifically recognizes CLEC12A, which is characterized in that it is a recombinant immunoglobulin with a structure of scFv-Fc. scFv refers to a single-chain antibody, including heavy Chain variable region and light chain variable region, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is SEQ ID NO: 1, Fc refers to the constant region. The sequence of the fully human antibody that specifically recognizes CLEC12A is SEQ ID NO: 3.

The heavy chain variable region amino acid sequence includes SEQ ID NO: 2 or an amino acid sequence having at least 80% homology with the amino acids in SEQ ID NO: 2.

The amino acid sequence of the light chain variable region includes SEQ ID NO: 1 or an amino acid sequence having at least 80% homology with the amino acids in SEQ ID NO: 1.

The genetically engineered antibody includes one of Fab fragment, F(ab') ₂ fragment, F(ab)' fragment, Fv fragment and scFv fragment, a combination of two or more of the above fragments, or one of the above fragments At least one derivative formed with other proteins or peptide chains.

The heavy chain CDR1 region sequence of the genetically engineered antibody is TYAMN, the heavy chain CDR2 region sequence is SIGSSGDHTNYADSVKG, the heavy chain CDR3 region sequence is YGSYCSTNTCYRHFKS, the light chain CDR1 region sequence is RASQGIRNDLG, the light chain CDR2 region sequence is AASSLQS, and the light chain CDR3 The zone sequence is LQNNSYPRT.

The sequence of one or a few amino acid mutations within the CDR1, CDR2, and CDR3 sequences of the heavy chain and light chain of the genetically engineered antibody. A few amino acid mutations generally refer to random amino acid mutations within 3 of the antibody CDR region.

The monoclonal antibody provided by the invention is a single-chain antibody, which is obtained by screening a fully human single-chain antibody phage library and is named Anti-CLEC12A-Fc. After detection, the single-chain antibody selectively binds to CLEC12A. Western blotting using this antibody alone can specifically detect the human CLEC12A extracellular fragment.

Description of drawings

Figure 1. Phage display panning results;

Figure 2. Phage ELISA result chart;

Figure 3. Electrophoresis diagram of expression vector pcDNA3.4-scFv-Fc;

Figure 4. Anti-CLEC12A antibody (IF4) immunoblotting results.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the invention and are not intended to limit the scope of the invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Example 1. Expression and purification of CLEC12A extracellular segment antigen

1. Materials used:

(1) Human CLEC12A gene ORF full-length cDNA (purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd., product number HG13556-G). (2) The eukaryotic expression vector pTT5-6xHis is self-prepared by the company. (3) BamH1 and XbaI restriction endonucleases were purchased from NEB Company. (4) FreeStyle culture medium was purchased from Thermo Fisher Scientific. (5) Plasmid extraction kit was purchased from Qiagen. (6)Ni-NTA-Agarose was purchased from Qiagen Company. (7) PEI transfection reagent (Shanghai Beyotime). (8) Nanodrop (Thermo Fisher, model Nano200).

2. Experimental method:

1) Use human CLEC12A gene cDNA as a template to amplify the CTLD domain of CLEC12A extracellular segment (CLEC12A-CTLD, Pro132-Val255) by PCR. The amplified gene fragment was digested with restriction endonucleases BamH1 and XbaI and then ligated into the pcDNA3.4-6xHis vector. Verify that the vector construction is correct by sequencing.

2) Mix the PEI transfection reagent and the eukaryotic antigen expression vector plasmid obtained above at a volume/mass ratio of 30 μl: 15 μg, add 30 ml of 293F suspended cells, shake at 37°C and 200 rpm for 96 hours, and centrifuge. The supernatant was collected and affinity purified using Ni-NTA-Agarose beads. The CLEC12A-CTLD antigen was purified by gel chromatography on a protein purifier. Finally, the concentration of the antigen was detected by UV absorption, and its purity was detected by SDS-PAGE ( As shown in Figure 1).

Example 2. Construction and screening of fully human single-chain antibody library

1. Materials used:

(1) High-adsorption enzyme-linked immunoreaction microplate was purchased from Corning Company. (2) Nanodrop (Thermo Fisher, model Nano200). (3) The magnetic beads of Ni-Charged Magbeads were purchased from Nanjing Genscript Company, product number L00295. (4) GelRed nucleic acid dye was purchased from Thermo Fisher Company. (5) Anti-M13 HRP antibody was purchased from Thermo Fisher Scientific. (6) M13 helper phage was purchased from Life Technologies, catalog number 18311019. (7) XL1-Blue bacteria were purchased from Agilent Technologies, catalog number 200228. (8) Developer ABTS solution was purchased from Thermo Fisher Company, product number 002024. (9) Plasmid extraction kit was purchased from Qiagen. (10) SfiI restriction endonuclease was purchased from NEB Company. (11) The pFUSE-IgG1 expression vector was purchased from Invivogen. (12) The pComb3 vector was purchased from Addgene (Plasmid#63888). (13) SOC culture medium was purchased from Sangon Company.

2. Experimental method:

1) Construction of a fully human single-chain antibody library: Design primers to amplify the heavy chain variable region and light chain variable region of the antibody, and use Linker to connect the heavy chain variable region and light chain variable region through overlap extension PCR. Up, obtain the full-length PCR product, use SfiI restriction endonuclease to digest the PCR product and pComb3 phagemid vector, electrotransform the ligated transformation product into XL1-blue competent cells, and then add 3 mL of SOC to the competent cells. Culture medium, after culturing for 1 hour at 30°C, add ampicillin with a final cell culture condition concentration of 50 μg/mL and 20 mL of 10 μg/mL tetracycline solution, and culture with shaking at 37°C for 2 hours. Then add 50 μL of M13 helper phage at a concentration of 1013/mL and incubate at room temperature for 1 hour, shaking gently every 10 minutes. Incubate at 37°C with shaking for 2 hours, then add cananamycin with a final concentration of 70 μg/mL, and incubate at 30°C overnight. Collect the supernatant by centrifugation, add PEG-8000/sodium chloride solution with a concentration of 10% (PEG is polyethylene glycol), place it in an ice bath for 1 hour, centrifuge at 8000 rpm, 4°C, discard the supernatant, and use 2 mL with a concentration of 1 % BSA (Albumin from bovine serum, bovine serum albumin) in PBS solution (Phosphate Buffer Solution, phosphate buffer solution), fully dissolve the precipitate, and centrifuge to collect the supernatant, which is a fully human single-chain antibody library.

2) Single-chain antibody screening: Mix 200 μL of the phage antibody library expressing fully human single-chain antibodies (containing phage 1×1012/mL) with 5 μg of CLEC12A-CTLD antigen, incubate at room temperature for 30 min, and then add 50 μL of nickel affinity magnetic beads. , the phages bound to the antigen were captured by nickel affinity magnetic beads, the unbound phages were removed after rinsing with PBS solution containing 0.05% Tween-20, and the phages stably bound to the magnetic beads were eluted with glycine (pH 2.2) solution. stand-by.

Inoculate 200mL of XL1-Blue bacteria (E. coli strain). After the OD (absorption value) reaches 0.6, add the above-eluted phage and let it stand with XL1-Blue bacteria at 37°C for 30 minutes. The bacterial solution is spread on ampicillin-resistant plates. on the next day, the bacteria on the ampicillin-resistant plate were eluted and collected the next day, and then infected with 1×10 ¹² pfu/mL (plaque forming unit) M13 helper phage. After amplification, the next round of screening was performed, and the above was repeated. 3 rounds of screening steps.

Dilute the XL1-Blue bacterial liquid that infected the phage in the third round with several different concentration gradients, and then spread these bacterial liquids on 15cm diameter LB solid culture medium plates with ampicillin resistance, on each plate. There are 100-500 clones, monoclonal antibodies are picked, and the final round of phage library after panning is verified by phage enzyme-linked immunoreaction.

3) Phage enzyme-linked immunoreaction: Inoculate the XL1-Blue monoclonal bacteria transfected with the phage into a 2 mL 96-well bacterial deep-well culture plate (purchased from Corning Company), and add 500 μL of SB containing ampicillin and tetracycline resistance ( SuperBroth, Sangon Bioengineering (Shanghai) Co., Ltd., Catalog No. A507026) culture medium, shaking at 37°C for 4-6 hours at 200 rpm. After detecting that the OD600 (absorbance value at 600 nm wavelength) value is close to 0.6, add 1 μL of helper phage , shake overnight at 30°C, and centrifuge at 3000g the next day to take the supernatant for later use.

Take an enzyme-linked immunosorbent microplate and coat it with antigen (including control antigen BSA) overnight at 4°C. The next day, elute with PBST and then block with PBST containing 5% skimmed milk powder (0.05% PBS with Tween-20 added). Add the phage supernatant prepared in the previous step, incubate at room temperature for 2 hours, then add Anti-M13 HRP antibody and incubate for 30 minutes, then wash 3 times with PBST, and add 50 μLABTS developing solution.

3) Experimental results: 96 monoclones were selected from the phage library obtained after the third round of screening for enzyme-linked immunoreaction verification, and the monoclones with an enzyme-linked immunoreaction reading value that was more than twice that of the control antigen were finally determined as positive monoclones. , Sequencing analysis of positive clones, and through comparison analysis, it was determined that three different antibody sequences were enriched. As shown in Figure 2, the IF4 sequence obtained is the antibody amino acid sequence protected by this patent application (SEQ ID NO: 1).

Example 3. Single-chain antibody Anti-CLEC12A-Fc protein expression, purification and immunoblotting verification

1. Materials used:

(1) The pFUSE expression vector pFUSE-hIGg1-Fc2 was purchased from Invivogen Company, product number pfuse-hg1fc2. (2) HiTrapProteinA HP columns were purchased from GE. (3) The protein purifier was purchased from GE Company. (4) Plasmid extraction kit was purchased from Qiagen. (5) SfiI restriction endonuclease was purchased from NEB Company. (6) PVDF membrane was purchased from Shanghai Biyuntian Company. (7) Nanodrop (Thermo Fisher, model Nano200). (7) ECL developer was purchased from Thermo Fisher Scientific. (8) Rabbit Anti-Human (Fc) HRP secondary antibody was purchased from Luoyang Biotong Experimental Materials Center.

2. Experimental method:

1) Digest the Ab3 positive sequence (i.e. SEQ ID NO: 1) obtained in Example 2 from phagemid and insert it into the pFUSE-hIGg1-FC2 expression vector (Invivogen Company, please refer to the instructions for the operating steps), and obtain the Fc segment. The eukaryotic antibody expression plasmid of the single-chain antibody Anti-CLEC12A-Fc is shown in Figure 3. Use 293Fectin transfection reagent (Invitrogen Company, Cat. No. 12347500) and the eukaryotic antibody expression vector plasmid obtained above to mix according to the volume mass ratio of 30 μl: 15 μg. After adding 30 ml of 293F suspended cells, shake at 37°C and 200rpm. After 72 hours, centrifuge and collect the supernatant, and use HiTrap ProteinAHP columns to purify the antibody protein (Anti-CLEC12A-Fc) on a protein purifier. Finally, the antibody concentration was determined using Nanodrop.

2) Take 2ug of antigen CLEC12A-CTLD and 2ug of negative control protein BSA and run SDS-PAGE. After transferring to the membrane, block with PBST containing 5% skim milk powder (0.05% PBS with Tween-20 added). Wash with PBST 3 times and then add 10ng/ Incubate with ulAnti-CLEC12A-Fc antibody for 1 hour, wash with PBST three times, add 1:2000 RabbitAnti-Human(Fc) HRP secondary antibody and incubate for one hour, wash with PBST for three times, add ECL developing solution and take photos for storage.

3) Experimental results: The purity of the single-chain antibody Anti-CLEC12A-Fc was detected by SDS-PAGE. The immunoblotting results are shown in Figure 4. The antibody Anti-CLEC12A-Fc (SEQ ID NO: 3) protected by this patent application can specifically recognize the extracellular fragment of CLEC12A protein.

Claims (6)

1. A fully human monoclonal antibody that specifically recognizes and binds to the CLEC12A receptor, characterized in that it is a recombinant immunoglobulin with a structure of scFv-Fc. scFv refers to a single-chain antibody, including the heavy chain variable region. And the light chain variable region, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 2, the amino acid sequence of the light chain variable region is SEQ ID NO: 1, Fc refers to the constant region. 2. The fully human monoclonal antibody that specifically binds to the CLEC12A receptor according to claim 1, wherein its sequence is SEQ ID NO: 3. 3. A genetically engineered antibody that specifically recognizes CLEC12A, characterized in that the heavy chain CDR1 region sequence of the genetically engineered antibody is TYAMN, the heavy chain CDR2 region sequence is SIGSSGDHTNYADSVKG, the heavy chain CDR3 region sequence is YGSYCSTNTCYRHFKS, and the light chain CDR3 region sequence is YGSYCSTNTCYRHFKS. The sequence of the CDR1 region is RASQGIRNDLG, the sequence of the light chain CDR2 region is AASSLQS, and the sequence of the light chain CDR3 region is LQNNSYPRT. . 4. The genetically engineered antibody that specifically recognizes CLEC12A as claimed in claim 3, characterized in that the genetically engineered antibody comprises Fab fragment, F(ab') ₂ fragment, F(ab)' fragment, Fv fragment and One of the scFv fragments, a combination of two or more of the above fragments, or a derivative of at least one of the above fragments and other proteins or peptide chains. 5. The fully human monoclonal antibody specifically binding to the CLEC12A receptor according to any one of claims 1-2 or the genetically engineered antibody according to any one of claims 3-4 is prepared for use in detecting CLEC12A-related Application of immunoblotting reagents. 6. An immunoblotting reagent for detecting CLEC12A-related immunoblotting reagents, which contains the fully human monoclonal antibody specifically binding to the CLEC12A receptor according to any one of claims 1-2 or any one of claims 3-4 The genetically engineered antibody described in the item is used as the active ingredient.