An antibody that specifically recognizes CD14 and its application in nanomagnetic bead sorting

Abstract

The present invention relates to the field of antibody technology, and more particularly to an antibody that specifically recognizes CD14 and its application in nanomagnetic bead sorting. The anti-CD14 antibody of the present invention can specifically recognize and bind to human CD14 without cross-reacting with other cell surface antigens such as CD8, CD11, CD68, CD195, etc.; the antibody has a high affinity for human CD14 and can be used as a detection antibody for CD14, and can also be used as a CD14 detection antibody. + Cell sorting antibodies achieve high purity CD14 + Efficient preparation of cells or for depletion of CD14 in CAR-T cell products + Cells can be used to improve the purity and quality of CAR-T cells, and have good application prospects in immune cell preparation and quality control.

Description

Technical Field

[0001] The present invention relates to the technical field of antibodies, and in particular to an antibody that specifically recognizes CD14 and an application thereof in nanomagnetic bead sorting. Background Art

[0002] Monocytes are derived from hematopoietic stem cells in the bone marrow and are the largest white blood cells. Monocytes are the main cellular defense system responsible for eliminating pathogenic bacteria and parasites. They can also transfer phagocytic antigenic determinants to lymphocytes to induce specific immune responses of lymphocytes. They also have the ability to recognize and kill tumor cells. Therefore, monocytes are an important part of the body's defense system. At the same time, monocytes are also the precursors of macrophages and dendritic cells, which are important immune cells in the body. Therefore, monocytes have extremely important research value in tumor immunoregulation and cancer treatment. Obtaining a sufficient number of high-purity target cells is the premise and basis of cell research, so it is necessary to develop efficient and specific monocyte sorting methods.

[0003] In addition, monocytes are also the largest blood cells in the blood, with strong adhesion and the ability to phagocytize foreign matter. During the CAR-T production process, the adhesion of monocytes will interfere with the CAR-T cell production process. During the sorting and activation stage, the adhesion of monocytes may lead to a decrease in T cell purity, affecting the activation effect of the activation reagent on T cells, thereby affecting the efficiency of viral transduction, resulting in an extension of the production cycle and an increase in production costs, thereby affecting the final cell therapy effect. Therefore, effectively removing monocytes and improving the purity of CAR-T cells play a key role in ensuring the stability and safety of the CRA-T process.

[0004] CD14 is strongly expressed on most monocytes and macrophages, but not or weakly expressed on other immune cells. Therefore, this property can be used to specifically recognize CD14 and to target CD14. + Monocytes or macrophages are sorted, purified or specifically removed to isolate high-purity CD14 + The purpose of removing CD14 cells or their interference is of great significance in immunotherapy. + The sorting purity and efficiency of monocyte or macrophage sorting methods are low, mainly due to the lack of high-affinity and high-specificity anti-CD14 antibodies. Summary of the invention

[0005] The invention provides an antibody capable of specifically recognizing CD14 and application thereof in nanomagnetic bead sorting.

[0006] In the present invention, CD14 refers to membrane-bound CD14 (mCD14), which is a glycoprotein mainly distributed on the cell surface of monocytes and macrophages.

[0007] Specifically, the present invention provides the technical solution described below.

[0008] In a first aspect, the present invention provides an anti-CD14 antibody or an antigen-binding fragment thereof, wherein the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the antibody or the antigen-binding fragment thereof are shown in SEQ ID NOs: 1, 2, and 3, respectively; and the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NOs: 4, 5, and 6, respectively.

[0009] The above-mentioned antibody or antigen-binding fragment thereof can specifically recognize and bind to human CD14, thereby achieving CD14 + It can efficiently bind to cells (including monocytes, macrophages, etc.), but cannot bind to cell surface antibodies such as CD8, CD11, CD68, CD195, etc.; at the same time, the above antibodies have high affinity for human CD14 and can be used to sort and detect CD14 + cell.

[0010] Preferably, the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof is as shown in SEQ ID NO.7 or has at least 80% homology with the sequence shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.8 or has at least 80% homology with the sequence shown in SEQ ID NO.8.

[0011] Preferably, the above-mentioned antibody or antigen-binding fragment thereof is a monoclonal antibody, Fab, Fab', F(ab')2 or a single-chain antibody.

[0012] In some embodiments of the present invention, an antibody with clone number 11G10A9 is provided, wherein the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region are shown in SEQ ID NOs: 1, 2, and 3, respectively; the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NOs: 4, 5, and 6, respectively; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8.

[0013] In a second aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody or antigen-binding fragment thereof.

[0014] According to the amino acid sequence of the antibody or its antigen-binding fragment provided above, those skilled in the art can obtain the nucleotide sequence of the nucleic acid molecule encoding the above antibody or its antigen-binding fragment. Due to the degeneracy of codons, the nucleotide sequence of the nucleic acid molecule encoding an antibody or its antigen-binding fragment is not unique, and all nucleic acid molecules capable of encoding the above antibody or its antigen-binding fragment are within the scope of protection of the present invention.

[0015] In a third aspect, the present invention provides a biological material containing the above-mentioned nucleic acid molecule, wherein the biological material is an expression cassette, a vector or a host cell.

[0016] The above expression cassette can be obtained by connecting the nucleic acid molecule with transcription and / or translation regulatory elements. The transcription and translation regulatory elements include but are not limited to promoters, terminators, etc.

[0017] The above-mentioned vectors include but are not limited to plasmid vectors, phage vectors, virus vectors, artificial chromosome vectors and the like.

[0018] The host cells include microbial cells (eg, bacteria such as Escherichia coli or fungi such as yeast), insect cells or other animal cells.

[0019] In a fourth aspect, the present invention provides an antibody conjugate, which is obtained by conjugating the above-mentioned antibody or antigen-binding fragment thereof with a marker, wherein the marker is selected from one or more of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, and radioactive labeling.

[0020] In a fifth aspect, the present invention provides a detection reagent comprising the above-mentioned antibody or antigen-binding fragment thereof, or comprising the antibody conjugate.

[0021] Preferably, the detection reagent is CD14 + Cell detection or sorting reagents. The reagents may be flow cytometry detection or sorting reagents, or other immunoassay reagents, such as enzyme-linked immunosorbent assay (ELISA) kits, chemiluminescent immunoassay kits, radioimmunoassay kits, fluorescent immunoassay kits, or immunochromatography kits.

[0022] In a sixth aspect, the present invention provides any of the following uses of the above-mentioned antibody or antigen-binding fragment thereof or the antibody conjugate or the detection reagent:

[0023] (1) detecting the presence or level of CD14 in a sample;

[0024] (2) Detection of CD14 + the presence or level of cells in a sample;

[0025] (3) Flow cytometry sorting of CD14+ cell;

[0026] (4) Nanomagnetic bead separation of CD14 + cell;

[0027] (5) Purification of CAR-T cells;

[0028] (6) Quality control of CAR-T cell therapy products.

[0029] In the applications of (1) and (2) above, the antibody or antigen-binding fragment thereof provided by the present invention is used to detect CD14 or cells expressing CD14. The detection may be for non-disease diagnosis and treatment purposes, and the sample to be tested may be an isolated or cultured cell sample or a prepared protein sample. The detection method may be flow cytometry, enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, immunochromatography, etc.

[0030] In the applications of (3) and (4) above, the antibody or antigen-binding fragment thereof provided by the present invention is used to specifically bind to CD14, thereby sorting CD14 + The sorting method includes but is not limited to magnetic bead method, flow cytometry sorting method, etc.

[0031] In the application of (5) above, the antibody or antigen-binding fragment thereof provided by the present invention specifically binds to CD14 to remove CD14. + cells, and then purify CAR-T cells.

[0032] In the application of (6) above, the quality control comprises detecting CD14 contained in CAR-T cells using the antibody or antigen-binding fragment thereof. + CD14 + Cell removal, thereby controlling the quality of CAR-T cells.

[0033] In a seventh aspect, the present invention provides any of the following uses of the above-mentioned antibody or antigen-binding fragment thereof, the nucleic acid molecule, the biomaterial, the antibody conjugate, or the detection reagent:

[0034] (1) Preparing a product for detecting the presence or level of CD14 in a sample;

[0035] (2) Preparation for detection of CD14 + the presence of cells in a sample or the product of their levels;

[0036] (3) Preparation for flow cytometry sorting of CD14 + Cell products;

[0037] (4) Preparation of nanomagnetic beads for CD14 sorting+ Cell products;

[0038] (5) Preparing products for purifying CAR-T cells.

[0039] In the applications described in (1) to (5) above, the product may be a detection reagent or a kit, which includes the antibody or antigen-binding fragment thereof of the present invention or the antibody conjugate thereof, and may also include a second antibody carrying a detectable label to detect the antibody or antigen-binding fragment thereof of the present invention.

[0040] In the present invention, the CD14 is preferably human CD14.

[0041] In an eighth aspect, the present invention provides a product comprising the antibody or antigen-binding fragment thereof or the antibody conjugate.

[0042] The above-mentioned product can be a detection reagent, a kit or a pharmaceutical composition.

[0043] The beneficial effects of the present invention include at least: the anti-CD14 antibody of the present invention can specifically recognize and bind to human CD14 without cross-reacting with other cell surface antigens such as CD8, CD11, CD68, CD195, etc.; the antibody has a high affinity for human CD14 and can be used as a detection antibody for CD14, and can also be used as a CD14 detection antibody. + Cell sorting antibodies achieve high purity CD14 + Efficient preparation of cells or for removal of CD14 in CAR-T cell products + Cells can be used to improve the purity and quality of CAR-T cells, and have good application prospects in immune cell preparation and quality control.

[0044] The anti-CD14 antibody of the present invention is verified by cell sorting to be effective for CD14 + The cell enrichment is high, and the cells obtained by sorting are of high purity, which is an ideal CD14 + Cell sorting antibody reagents for CD14 + Cell sorting can also be used for CD14 in the CAR-T preparation process + The removal of monocytes ensures the purity of T cells, the stability and safety of the CRA-T process, and helps accelerate the development of CGT therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0046] Figure 1 The figure is a SDS-PAGE test result of the antibody in Example 3 of the present invention; wherein M is a protein marker, and R is the 11G10A9 clone number antibody under reducing conditions.

[0047] Figure 2 This is a graph showing the affinity BLI identification results of the antibody in Example 3 of the present invention.

[0048] Figure 3 The FACS detection of CD14 using anti-CD14 antibody in Example 3 of the present invention + Purity of sorted cells. DETAILED DESCRIPTION

[0049] In a specific embodiment of the present invention, an antibody is provided that binds to human CD14 antigen with high affinity and high specificity. The antibody can specifically bind to CD14 on the cell surface, thereby enriching CD14. + cell.

[0050] Specifically, the present invention uses human CD14 protein recombinantly expressed by HEK293 cells as an immunogen to immunize Balb / c mice, obtains hybridoma cell lines that stably express antibodies through cell fusion, screening, and subcloning, and screens suitable cell lines for antibody production and expression through activity analysis methods. It was found through experimental verification that the antibody can specifically recognize human CD14 protein, has no cross-reactivity with other CD antigens, and can achieve CD14 + Cell sorting. Furthermore, the present invention obtains the amino acid sequence of the antibody and the nucleotide sequence of its encoding gene by hybridoma sequencing. Flow cytometry is used to analyze CD14 + Assay for monocyte sorting.

[0051] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0052] Example 1 Preparation of antibodies that specifically bind to human CD14

[0053] 1. Preparation of human CD14 protein immunogen: The expression plasmid that can express human CD14 protein was constructed by gene synthesis, and then the expression plasmid was transfected into HEK293 cells using Invitrogen Lipofectamine 2000 transfection reagent. The culture supernatant was harvested after 72 hours, and then purified human CD14 protein was obtained by affinity chromatography. The protein was subjected to conventional physical and chemical analysis and biological activity analysis to ensure the reliability of the immunogen.

[0054] 2. Mouse immunization: Use the human CD14 protein prepared in step 1 as an immunogen to immunize 10 Balb / c mice, and use Freund's adjuvant to maintain the natural conformation of the protein. After the immunization, use the ELISA method to detect the serum of the immunized animals to determine the level of immune response. After the routine immunization, if the immunized animals can reach the level of immune response to the immunogen (OD value>1.0, titer reaches 1:10000), cell fusion can be performed.

[0055] 3. Cell fusion and plating: Use electrofusion method to carry out cell fusion. All fused cells are plated in 96-well plates.

[0056] 4. Screening: Use ELISA to screen the supernatant of the fused cells and select cells that are positive for specific binding to human CD14 protein and have no binding to proteins such as CD8, CD11b, CD11c, CD68 and CD195.

[0057] 5. Clone expansion and cryopreservation: Transfer positive mother clone cells to 24-well plates for expansion and culture, up to 10 strains. Collect 2 mL of supernatant from each expanded clone for indirect ELISA detection. Cryopreserve these specific positive clone cells to avoid clone loss.

[0058] 6. Subcloning: Subclone the positive mother clones using the limiting dilution method to ensure that these positive mother clones are derived from a single mother clone cell, and screen the subclones using the indirect ELISA method. Based on the confirmation of antigen recognition, select 2 stable subclone cell lines from each mother clone for cryopreservation. Collect 5 mL of supernatant from each subclone before cryopreservation, and perform subtype identification and preservation on all subclones.

[0059] 7. Hybridoma cell antibody gene sequencing: Extract total RNA from hybridoma cells, reverse transcribe RNA into cDNA through RT-PCR reaction. Clone antibody light chain and heavy chain sequences, construct antibody light chain and heavy chain sequences onto T vectors, and then perform DNA sequencing analysis to obtain antibody gene sequences.

[0060] 8. Antibody production and purification: The antibody gene sequence obtained in step 7 is transfected into CHO cells and cultured. The antibody is purified by protein A / G affinity chromatography and stored in phosphate buffered saline (PBS) by dialysis.

[0061] Example 2 Specificity analysis of supernatant of human CD14 fusion cells

[0062] According to the method in Example 1 above, 6 different monoclonal antibodies (clone numbers are 11G10A9, 18F2F9, 11H5G7, 20F9E3, 16B9D7 and 5C9B7) were obtained by screening, and the specificity of the above monoclonal antibodies was analyzed by enzyme-linked immunosorbent assay. The analysis method is as follows:

[0063] 1. Dilute CD8, CD11b, CD11c, CD14, CD68, and CD195 proteins to 1 μg / mL with PBS and add 100 μL to each well of the ELISA plate. Seal the plate with a sealing film and place at 4°C overnight (about 16 hours).

[0064] 2. Discard the liquid in the wells, pat the ELISA plate dry, wash the plate with PBST washing solution, 300μL / well, soak for 30s, pat the ELISA plate dry, and proceed to the next wash. Wash the plate 3 times in total.

[0065] 3. Add 100 μL of blocking agent (PBST containing 5% BSA) to each well, seal the plate with sealing film, and incubate at 37°C for 1.5 hours.

[0066] 4. Repeat step 2 and wash the plate 3 times.

[0067] 5. Dilute the cell supernatants corresponding to the above 6 different monoclonal antibodies at 1:100 and add them to the ELISA plate, 100 μL per well. Seal the plate with a sealing film and incubate at 37°C for 1.0 h.

[0068] 6. Repeat step 2 and wash the plate 3 times.

[0069] 7. Dilute HRP-Anti-Mouse IgG to 0.05 μg / mL with sample diluent, add 100 μL to each well, seal the plate with sealing film, and incubate at 37°C for 1.0 h.

[0070] 8. Repeat step 2 and wash the plate 3 times.

[0071] 9. Add 100 μL of color development solution to each well, seal the plate with a sealing film, and incubate at 37°C in the dark for 20 minutes.

[0072] 10. Add 50 μL of stop solution to each well and gently shake the ELISA plate until the color is evenly developed.

[0073] 11. Use a microplate reader to read the absorbance at 450 nm and 630 nm. 450 Deduction OD 630 The absorbance value (OD value) was obtained.

[0074] 12. Select clones that strongly bind to human CD14 protein and do not bind to CD8, CD11b, CD11c, CD68 and CD195 proteins. The selected clones are 11G10A9, 18F2F9, 11H5G7, 20F9E3, 16B9D7 and 5C9B7. The OD value results of the antibodies of each clone are shown in Table 1.

[0075] Table 1 ELISA detection OD values ​​of different antibodies

[0076]

[0077] Example 3 Identification and functional analysis of anti-human CD14 antibodies

[0078] In this example, the human CD14-specific antibody (clone number: 11G10A9) screened in Example 2 was identified and its function analyzed using methods known in the art, as follows:

[0079] 1. SDS-PAGE identification results ( Figure 1 ) showed that the 11G10A9 clone antibody obtained two bands after reduction electrophoresis, with molecular weights of approximately 50 kDa and 25 kDa, respectively, and a purity greater than 99%.

[0080] 2. Anti-human CD14 antibody BLI affinity detection map ( Figure 2 ) showed that the 11G10A9 clone antibody bound to the human CD14 protein with a high affinity, and its affinity (KD) was at the level of 9.25E-11.

[0081] 3. Sorting CD14 using anti-human CD14 antibody + Cell FACS detection results ( Figure 3 ) showed that the use of 11G10A9 clone number antibody for CD14 + Cell sorting, FACS detection, CD14 + The purity of the cells was 98.68%, indicating that the 11G10A9 clone antibody has a high specificity for human CD14 and can be used to isolate high-purity CD14 + Cells, excellent in the isolation of monocytes.

[0082] 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 make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An anti-CD14 antibody or an antigen-binding fragment thereof, characterized in that: The amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the antibody or its antigen-binding fragment are shown in SEQ ID NOs: 1, 2, and 3, respectively; the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NOs: 4, 5, and 6, respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

8.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The antibody or antigen-binding fragment thereof is a monoclonal antibody, Fab, Fab', F(ab')2 or a single-chain antibody.

4. A nucleic acid molecule, characterized in that It encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

5. Biomaterial, characterized in that It contains the nucleic acid molecule according to claim 4, and the biological material is an expression cassette, a vector or a host cell.

6. An antibody conjugate, characterized in that: The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 is coupled with a label, and the label is selected from one or more of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, and radioactive labeling.

7. A detection reagent, characterized in that: It comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the antibody conjugate according to claim 6.

8. Any of the following uses of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the antibody conjugate according to claim 6, or the detection reagent according to claim 7: (1) Detection of the presence or level of CD14 in a sample for purposes other than disease diagnosis and treatment; (2) Detection of CD14 for non-disease diagnosis and treatment purposes + the presence or level of cells in a sample; (3) Flow cytometry sorting of CD14 for non-disease diagnosis and treatment purposes + cell; (4) Nanomagnetic bead separation of CD14 for non-disease diagnosis and treatment purposes + cell; (5) Purification of CAR-T cells; (6) Quality control of CAR-T cell therapy products.

9. Any of the following uses of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the biomaterial according to claim 5, the antibody conjugate according to claim 6, or the detection reagent according to claim 7: (1) Preparing a product for detecting the presence or level of CD14 in a sample; (2) Preparation for detection of CD14 + the presence of cells in a sample or the product of their levels; (3) Preparation for flow cytometry sorting of CD14 + Cell products; (4) Preparation of nanomagnetic beads for CD14 sorting + Cell products; (5) Preparing products for purifying CAR-T cells.

10. A product, characterized in that It comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the antibody conjugate according to claim 6.

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