WO2022166802A1 - Anti-human cd271 monoclonal antibody - Google Patents

Abstract

Provided are a hybridoma cell line CD271T-2 that secretes anti-human CD271 monoclonal antibody and an anti-human CD271 monoclonal antibody secreted by same, and the hybridoma cell line having an accession number of CGMCC 21494. Also provided is an anti-human CD271 engineered antibody having LCDRs 1-3 and HCDRs 1-3 as shown in SEQ ID NOs.1-6, respectively. Also provided is a use of the cell line or antibody.

Description

Anti-human CD271 monoclonal antibody and its use technical field

The invention belongs to the field of biotechnology, and in particular relates to an anti-human CD271 monoclonal antibody and use thereof.

Background technique

CD271 is a low-affinity nerve growth factor receptor (LNGFR), a member of the tumor necrosis factor receptor superfamily with a relative molecular mass of 75kD, also known as p75NTR, which contains 3 regions: cysteine-rich The extra-membrane domain, the transmembrane domain and the intracellular domain consisting of 155 amino acid residues. CD271 is not only expressed in the nervous system, but also closely related to the development, differentiation and survival of neural cells. It is also a suitable marker molecule for enriching mesenchymal stem cells. Compared with traditional sorting methods, CD271 has high specificity, high purity, and colony formation. The advantages of strong ability and so on.

Mesenchymal stem cells (MSCs) are a class of adult stem cells with self-renewal and multi-directional differentiation potential, which have broad application prospects in regenerative medicine research. Clinical trials of mesenchymal stem cells mainly include tissue damage repair, such as repair of bone, cartilage, and joint damage, and treatment of heart, liver, spinal cord injury, and nervous system diseases. However, the use of MSCs for targeted tissue repair has always been limited by the colonization and proliferation of stem cells in damaged sites. Therefore, in the treatment of tissue injury sites or neurological diseases, the removal or enrichment of specific cells in the graft has become the most critical issue in the current transplantation field. CD271, CD105, CD133, etc., as surface markers of mesenchymal stem cells, can effectively isolate or enrich mesenchymal stem cells. Under specific induction conditions in vivo or in vitro, they can differentiate into cartilage, muscle, tendon, etc., to achieve tissue repair. the goal of. On the other hand, CD271 is also one of the markers of various cancer stem cells (CSC). As a surface marker molecule for MSCs and CSCs, CD271 has better specificity for stem cells than CD44, CD105 and other molecules. Its monoclonal antibody can not only be used for the screening and identification of MSCs, but also for anti-tumor research. It has important Research and application value. However, there is still a lack of anti-CD271 monoclonal antibodies with broad recognition ability for mesenchymal stem cells and cancer stem cells.

SUMMARY OF THE INVENTION

In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide an anti-human CD271 monoclonal antibody and use thereof.

The extracellular structure of native CD271 includes four cysteine-rich CRD structures (CRD1-4) and a linker region (stalk region). After the expression of CD271, the CRD region is hydrolyzed by protease to be shed, so the binding of antibodies targeting the CRD region to CD271-positive cells is affected, and even the expression of CD271 cannot be detected at a certain stage of cell metabolism. The stalk region near the cell membrane has no glycosylation, phosphorylation modification or advanced spatial structure, and the structure is stable. The antibody that recognizes this region has good versatility in theory. Therefore, in order to obtain a monoclonal antibody that can stably recognize human CD271, we selected the stalk region as the target to develop a new type of anti-CD271 monoclonal antibody to provide new tools for research in this field.

In order to achieve the above purpose and other related purposes, the present invention adopts the following technical solutions:

The first aspect of the present invention provides a hybridoma cell line CD271T-2 which is anti-secreting anti-human CD271 monoclonal antibody, and the hybridoma cell line has a deposit number of CGMCC 21494. It is preserved in the China Ordinary Microbial Culture Collection and Management Center, and the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is January 25, 2021. The classification was named as the hybridoma cell line CD271T-2.

The second aspect of the present invention provides an anti-human CD271 monoclonal antibody, the anti-human CD271 monoclonal antibody is secreted by the CD271T-2 hybridoma cell line.

The anti-human CD271 monoclonal antibody has good binding affinity to human mesenchymal stem cells.

Optionally, the antibody is a murine monoclonal antibody belonging to the IgM subtype.

A third aspect of the present invention provides an engineered antibody against human CD271, the engineered antibody against human CD271 includes a light chain and a heavy chain, the light chain includes LCDR1, LCDR2 and LCDR3, and the amino acid sequence of LCDR1 is as shown in SEQ ID As shown in NO.1, the amino acid sequence of LCDR2 is shown in SEQ ID NO.2, and the amino acid sequence of LCDR3 is shown in SEQ ID NO.3; the heavy chain includes HCDR1, HCDR2 and HCDR3, and the HCDR1 The amino acid sequence of HCDR2 is shown in SEQ ID NO.4, the amino acid sequence of HCDR2 is shown in SEQ ID NO.5, and the amino acid sequence of HCDR3 is shown in SEQ ID NO.6.

Specifically, the light chain complementarity determining region LCDR1 has a length of 10aa, and the amino acid sequence is shown in SEQ ID NO.1, specifically: QSVDYDGDSY.

Light chain complementarity determining region LCDR2, length: 3aa, amino acid sequence as shown in SEQ ID NO.2, specifically: AAS.

Light chain complementarity determining region LCDR3, length: 9aa, amino acid sequence as shown in SEQ ID NO.3, specifically: QQSNEDPFT.

Heavy chain complementarity determining region HCDR1, length: 10aa, amino acid sequence as shown in SEQ ID NO.4, specifically: GFSLSTSGMG.

Heavy chain complementarity determining region HCDR2, length: 7aa, amino acid sequence as shown in SEQ ID NO.5, specifically: IWWDDDK.

Heavy chain complementarity determining region HCDR3, length: 13aa, amino acid sequence as shown in SEQ ID NO.6, specifically: ARRDYGNYYAMDY.

Further, the amino acid sequence of the light chain variable region of the engineered antibody against human CD271 is as shown in SEQ ID NO.7; or it is to perform certain substitutions, additions and/or deletions of one or more of SEQ ID NO.7 Amino acids obtain amino acid sequences with equivalent functions. The amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.8, or an amino acid sequence with an equivalent function is obtained by performing certain substitutions, additions and/or deletions of one or several amino acids to SEQ ID NO.8.

Specifically, the sequence shown in SEQ ID NO.7 is specifically:

The sequence shown in SEQ ID NO.8 is specifically:

Optionally, the engineered antibody can be a monoclonal antibody.

Optionally, the monoclonal antibody is of murine origin.

In one embodiment, the monoclonal antibody subclass is IgM.

Optionally, the antibody is produced by the CD271T-2 hybridoma cell line through reverse transcription-polymerase chain synthesis reaction.

The fourth aspect of the present invention provides a polynucleotide encoding the heavy chain variable region and/or light chain variable region or full-length amino acids of the aforementioned anti-human CD271 antibody.

The heavy chain variable region and/or light chain variable region encoding the aforementioned anti-human CD271 antibody are shown in SEQ ID NO.9-SEQ ID NO.14.

Specifically, the light chain complementarity determining region LCDR1, DNA, length: 30 bp, and the nucleotide sequence is shown in SEQ ID NO. 9, specifically: CAAAGTGTTGATTATGATGGTGATAGTTAT.

Light chain complementarity determining region LCDR2, DNA, length: 9bp, nucleotide sequence as shown in SEQ ID NO.10, specifically: GCTGCATCC.

Light chain complementarity determining region LCDR3, DNA, length: 27bp, nucleotide sequence as shown in SEQ ID NO.11, specifically: CAGCAAAGTAATGAGGATCCATTCACG.

Heavy chain complementarity determining region HCDR1, DNA, length: 30bp, nucleotide sequence shown in SEQ ID NO. 12, specifically: GGGTTTTCACTGAGCACTTCTGGTATGGGT.

Heavy chain complementarity determining region HCDR2, DNA, length: 21bp, nucleotide sequence shown in SEQ ID NO.13, specifically: ATTTGGTGGGATGATGATGATAAG.

Heavy chain complementarity determining region HCDR3, DNA, length: 39 bp, nucleotide sequence as shown in SEQ ID NO. 14, specifically: GCTCGAAGGGACTATGGTAACTACTATGCTATGGACTAC.

Further, the nucleotide sequence encoding the light chain variable region of the aforementioned anti-human CD271 antibody is shown in SEQ ID NO.15, and the nucleotide sequence encoding the heavy chain variable region of the aforementioned anti-human CD271 antibody is as shown in SEQ ID NO. NO.16 shows.

Specifically, the sequence shown in SEQ ID NO.15:

Sequence shown in SEQ ID NO.16:

A fifth aspect of the present invention provides a polynucleotide encoding the recombination sequence of the light chain variable region and the heavy chain variable region of the Fab segment of the aforementioned anti-CD271 antibody, which can obtain a single chain with a smaller molecular weight Antibody.

A fifth aspect of the present invention provides a construct comprising the aforementioned polynucleotide.

The construct may be constructed by inserting the isolated polynucleotide into a multiple cloning site of an expression vector.

The expression vector in the present invention generally refers to various commercially available expression vectors well known in the art, such as bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenovirus, retrovirus or other vectors. Specific expression vectors that can be used include but are not limited to: pET series expression vectors, pGEX series expression vectors, pcDNA series expression vectors, and the like.

The host cell of the present invention contains the aforementioned construct or the aforementioned polynucleotide of foreign origin integrated into the genome. Any cell suitable for the expression of the expression vector can be used as a host cell, for example, the host cell can be a prokaryotic cell, such as a bacterial cell, etc.; or a lower eukaryotic cell, such as a yeast cell, etc.; or a higher eukaryotic cell , such as mammalian cells. Specifically, for example, cells of yeast, insects, plants and the like can be used. Preferably, the host cell is a eukaryotic cell, and a mammalian host cell line that does not produce antibodies can be used. Specific cell lines that can be used include but are not limited to: Chinese hamster ovary cells (CHO), baby hamster kidney cells (BHK, ATCC CCL 10), young mouse Sertoli cells (Sertoli cells), monkey kidney cells (COS cells), monkey kidney CVI cells transformed by SV40 (COS-7, ATCC CRL 1651), Human embryonic kidney cells (HEK-293), monkey kidney cells (CVI, ATCC CCL-70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical cancer cells (HELA, ATCC CCL-2) and so on.

Appropriate conditions for expressing the antibody should be known to those skilled in the art, and those skilled in the art can select a suitable medium according to experience, and culture under conditions suitable for the growth of host cells. After the host cells have grown to an appropriate cell density, the promoter of choice is induced by a suitable method (eg, temperature switching or chemical induction), and the cells are cultured for an additional period of time. The recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell. If desired, recombinant proteins can be isolated and purified by various isolation methods utilizing their physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic disruption, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

A sixth aspect of the present invention provides a host cell comprising the aforementioned construct or the aforementioned exogenous polynucleotide integrated into the genome.

The seventh aspect of the present invention provides the aforementioned cell line, or the aforementioned anti-human CD271 monoclonal antibody, or the aforementioned engineered antibody against human CD271, or the aforementioned polynucleotide, or the aforementioned construct, or the aforementioned host of the antibody Use of cells, selected from any of the following:

a) cells expressing CD271, including but not limited to mesenchymal stem cells, tumor stem cells, and nervous system cells, and their in vitro detection, identification, isolation, enrichment, and targeting;

b) Mediating mesenchymal stem cells to target tissue repair;

c) Detection and tracing of stem cells, tumor stem cells and cells of the nervous system;

d) preparation of nervous system targeting drugs;

e) preparing a tumor therapeutic drug;

f) Coupling with other small chemical molecules, biological macromolecules, radioactive molecules, nanomaterials, etc., for various purposes in the above a)-e).

Compared with the prior art, the present invention has the following beneficial effects:

The monoclonal antibody secreted by the hybridoma cell line of the present invention has the advantages of high titer, good specificity and strong affinity with natural antigen. The engineered antibody against human CD271 is produced by CD271T-2 hybridoma through reverse transcription-polymerase chain synthesis reaction. The antibody can specifically bind to human CD271 molecule and has a certain affinity for human mesenchymal stem cells in vitro. CD271 is expressed on the surface of mesenchymal stem cells, tumor stem cells and cells of nervous system-related diseases, and is a potential target for mediating mesenchymal stem cells or targeting nervous system-related cells and tumors. Therefore, the anti-human CD271 monoclonal antibody is a medical antibody with great potential.

The anti-human CD271 antibody of the present invention has high specificity and affinity, and can be coupled with magnetic nanomaterials to prepare immunomagnetic beads for sorting mesenchymal stem cells, and can also be combined with other targets to design bispecificity Antibodies, or used in combination with other chemical molecules, radioactive molecules, polypeptides or proteins, nanomaterials, etc., are used for the treatment of certain tissue repair diseases and nervous system or tumor-related diseases.

The preservation information of strains of the present invention is as follows:

Cell line name: hybridoma cell line CD271T-2;

The deposit number is: CGMCC 21494;

Deposit date: January 25, 2021;

Name of preservation unit: China General Microorganism Culture Preservation and Management Center;

Abbreviation of depositary unit: CGMCC;

The address of the preservation unit: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

Description of drawings

Figure 1 shows the detection of serum titer of mice after anti-human CD271 immunization by ELISA: the coating antigen is full-length CD271 protein antigen, the antigen concentration is 4 μg/mL, and the loading serum is diluted from 1:2000 to 1:256000 dilution, 1 The serum titers of No. 2, No. 3, No. 4 and No. 5 mice were all above 1:128000, and the 5 mice had high reactivity to the antigen;

Figure 2 is the SDS-PAGE detection chart of mouse CD271T-2 ascites purified antibody: in the non-reducing condition, the ascites antibody band is relatively single, and in the reducing condition, the light chain and heavy chain of the antibody are at 26kDa and 53kDa, respectively;

Figure 3 shows the ELISA identification of purified antibody from mouse ascites: the coated antigen is full-length CD271 protein antigen, the antigen concentration is 2 μg/mL, each well is 100 μL, and the concentration gradient of loading antibody is 0.625 μg/mL, 1.25 μg/mL, 2.5 μg /mL, 5μg/mL, 10μg/mL, 20μg/mL, 40μg/mL, 100μL per well, the results show that CD271T-2 antibody can recognize and bind human CD271 antigen;

Figure 4 is the antibody affinity map after verification and purification by ForteBio;

Figure 5 is the PCR result of the heavy and light chain variable region gene cloning of the anti-human CD271 antibody of the present invention: Figure 5A is the gene cloning PCR result of the heavy and light chain variable region of the antibody of the CD271T-2 hybridoma, Figure 5B is the CD271T- 2 PCR results of hybridoma antibody heavy and light chain variable region chimeric ScFv gene cloning;

Figure 6 is the anti-human CD271 monoclonal antibody to detect the expression of CD271 on the surface of mesenchymal stem cells: Figure 6A is the positive control antibody of APC Goat anti human CD271 from Biolegend, the immunogen comes from melanoma cells, which highly express CD271 on the cell surface molecule, this antibody has a slight migration compared with the negative control; Figure 6B shows the negative control antibody of Rabbit anti human CD271 derived from Abcam, the immunogen comes from the 350-450 amino acid region in the human CD271 cell membrane, and compared with the positive control, there is no bias Figure 6C shows the purified antibody of CD271T-2 ascites. Compared with the positive and negative controls, the degree of deviation is higher, indicating that the prepared anti-human CD271 monoclonal antibody has very high affinity and specificity, and the proportion of positive cells superior to the commercially available APC mouse anti human CD271 control antibody.

Detailed ways

As used herein, the term "antibody" refers to an immunoglobulin molecule generally composed of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain . In a general sense, a heavy chain can be understood as a polypeptide chain with a larger molecular weight in an antibody, and a light chain refers to a polypeptide chain with a smaller molecular weight in an antibody. Light chains can be classified as kappa and lambda light chains. Heavy chains can generally be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH1, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from amino terminus to carboxy terminus. The variable regions (VH and VL) of each heavy/light chain pair, respectively, form the antibody binding site. The assignment of amino acids to regions or domains follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia (1989) Definition of Nature 342:878-883. In particular, the heavy chain may also contain more than 3 CDRs, eg, 6, 9, or 12. For example, in the diabody of the present invention, the heavy chain may be a ScFv in which the C-terminus of the heavy chain of an IgG antibody is linked to another antibody, in which case the heavy chain contains 9 CDRs. The term "antibody" is not limited by any particular method of producing an antibody. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies and polyclonal antibodies. Antibodies can be of different isotypes, eg, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.

In order to make the invention purpose, technical solution and beneficial technical effect of the present invention clearer, the present invention will be described in further detail below in conjunction with the embodiments. Those who are familiar with this technology can easily understand other advantages and effects of the present invention from the content disclosed in this specification. .

Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific specific embodiments; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments, It is not intended to limit the protection scope of the present invention. In the following examples, the test methods without specific conditions are usually in accordance with conventional conditions or in accordance with the conditions suggested by various manufacturers.

When numerical ranges are given in the examples, it is to be understood that, unless otherwise indicated herein, both endpoints of each numerical range and any number between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, equipment and materials used in the embodiments, according to the mastery of the prior art by those skilled in the art and the description of the present invention, the methods, equipment and materials described in the embodiments of the present invention can also be used Any methods, devices and materials similar or equivalent to those of the prior art can be used to implement the present invention.

Unless otherwise specified, the experimental methods, detection methods and preparation methods disclosed in the present invention all adopt the conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields in the technical field. conventional technology.

The "CD271-T2" used in the present invention refers to a cell line when used together with a hybridoma cell line; when used alone or together with a monoclonal antibody, antibody, etc., it refers to the monoclonal antibody or engineered antibody of the present invention.

[Example 1] Preparation of anti-human CD271 hybridoma cell line and monoclonal antibody

1. Mouse Immunization

The amino acid sequence of the immunization antigen is shown in SEQ ID NO.17, specifically:

1) Preparation of primary immunization antigen: Take a 1 mL sterile EP tube, dilute 100 μg of immunogen with PBS to 100 μL and put it in the EP tube; shake up Freund’s complete adjuvant well to mix the precipitated bifidobacteria evenly; take 100 μL of Complete adjuvant was added to a 5 mL sterile EP tube, and then the diluted immunogen was added to make the volume ratio of antigen and adjuvant 1:1, and the centrifuge tube was placed in an ice box; Place the centrifuge tube in an ice box for sonication. The ultrasonic conditions were the total power of 200W, the total time of 10min, the working time of 5 seconds, and the interval time of 6 seconds. Observe the emulsification of the solution in real time; inhale the emulsified antigen with a 1 ml disposable syringe, and place it in an ice box for later use.

2) Antigen preparation for secondary immunity and tertiary immunity: take a 1 mL sterile EP tube, dilute 100 μg of immunogen with PBS to 100 μL and put it in the EP tube; shake the incomplete Freund’s adjuvant well to mix the precipitated bifidobacteria evenly ; Take 100 μL of incomplete Freund’s adjuvant and add it to a 5 mL sterile EP tube, then add the diluted immunogen to make the volume ratio of antigen and adjuvant 1:1, and place the centrifuge tube in an ice box; prepare Good sonicator, put the centrifuge tube in the ice box to sonicate. Ultrasonic conditions were total power 200W, total time 10min, working time 5s, and interval time 6s. Observe the emulsification of the solution in real time; inhale the emulsified antigen with a 1 ml disposable syringe, and place it in an ice box for later use.

3) Preparation of booster immunization antigen: Take a 1 mL sterile EP tube, dilute 200 μg of the immunogen with PBS to 400 μL, inhale with a 1 mL disposable syringe, and place it in an ice box for later use.

4) Mouse immunization method

The immunizing antigen was inhaled with a 1 ml syringe, divided into 2-3 points, each point of about 0.1 mL, and subcutaneously injected into the back (or abdomen) of the mice; blood was collected on the third day after the three immunizations for antibody titer ELISA detection, the specific results are shown in the figure 1. Immunization can be boosted when the titer reaches 1:100000.

2. Preparation of Feeder Cells

Feeder cells can promote the growth and reproduction of single or a few scattered cells.

1) Prepare sterilized surgical instruments and glass dishes, 96-well plate, 5mL disposable syringe, 300mL 70% alcohol, several Balb/c mice, serum-free DMEM medium, and sterilized PBS.

2) Balb/c mice were killed by pulling their necks and immersed in 70% alcohol for 10 min.

3) Remove the excess alcohol from the mouse and place it on a sterilized glass plate.

4) Cut and separate the skin of the mouse abdomen (do not cut the muscle layer). Draw 5mL of DMEM serum-free medium with a 5mL syringe, inject it into the abdomen of the mouse, gently massage and shake the body of the mouse, so that the macrophages are fully mixed into the medium. Carefully suck back the culture medium with a syringe and put it into a 15mL centrifuge tube.

5) Repeat the above method 1-2 times to obtain as many macrophages as possible.

6) Centrifuge at 1500 rpm for 10 min, and discard the supernatant. Prepared with culture medium suspension, about 10 ⁶ peritoneal cells can be obtained from one adult Balb/c mouse, and 3-4 96-well plates can be plated.

3. PEG-mediated fusion of rat spleen B cells and mouse myeloma cells

1) Cells: Sp2/0-Ag14, medium: DMEM+10% FBS, passaged 48 hours before fusion, and the density should be about 80% of the bottom of the bottle covered after 48 hours.

2) Mice Balb/c mice were killed by tail clipping and neck pulling, and immersed in 70% alcohol for 10 min.

3) Cut the left upper abdomen to the left back of the mouse, isolate the spleen, remove the connective tissue on the spleen, put it into a clean plate, and add a small amount of DMEM basal medium (serum-free).

4) Cut the mouse spleen into 4-5 small sections, grind the top of the plug with a 5mL syringe, add a small amount of DMEM while grinding, and flush the ground spleen cells into a plate.

5) Transfer all the cells in the dish into a 15mL centrifuge tube, add an appropriate amount of DMEM basal medium, mix well and centrifuge at 200g for 5min.

6) Wash twice with PBS, centrifuge, 200g, 5min.

7) All Sp2/0-Ag14 cells were collected, washed with PBS and counted.

8) Mix 10 ⁸ spleen cells and 10 ⁷ myeloma cells in a 50 mL centrifuge tube (the number of myeloma cells can be increased after harvesting the cells), add 30-40 mL of serum-free DMEM, mix well, and centrifuge at 1500 rpm for 3 min , go to the supernatant.

9) Tap the bottom of the centrifuge tube to fully disperse the sediment.

10) Put the cells in a 37°C water bath, and slowly drop PEG in the following method.

11) Slowly drop 1 mL of PEG solution along the tube wall with a dropper for 1 min, gently shake while adding, 100 g, centrifuge for 2 min;

12) Drop 4.5 mL of DMEM basal medium (preheated, serum-free) in the same way for 3 min, and shake gently while adding;

13) Drop into 5mL DMEM basal medium (preheated, serum-free) in the same way for a duration of 2min, and gently shake for 30s after adding;

14) Slowly add 45mL DMEM basal medium (pre-warmed, serum-free),

15) Centrifuge at 100g for 5min, remove the supernatant, and tap the bottom.

16) Add HAT-containing DMEM complete medium (containing 20% FBS), gently resuspend, count cells with trypan blue, 100 μL/well, and seed into 5 96-well plates.

17) Culture in a 37°C, 5% CO ₂ incubator, do not shake the culture plate during incubation.

18) Perform ELISA identification 11-13 days after fusion, and measure according to cell growth, do not change the medium for 2-3 days before the measurement, so that the amount of antibody in the supernatant is fully accumulated.

4. Limiting Dilution Screening for Single Clones

1) Take out the cells from the antibody-positive wells, and prepare a cell suspension with HT medium. The samples were sampled for trypan blue staining and counted.

2) Disperse 320 cells in 6.4 mL of HT medium, mix well, and add 100 μL per well to columns 1-4 of a 96-well plate, so that there are 5 cells in each well; Add 3.2 mL of HT medium, mix well, add 100 μL to each well in 5-8 columns, so that there are 2.5 cells in each well; take 1 mL of the remaining cell fluid, add 2 mL of medium, and after thorough mixing, add 100 μL to each well. Add wells 9-12 columns to make 0.625 cells per well.

3) Put the 96-well plate into a _CO2 incubator and cultivate at 37°C.

4) On the fifth day, the growth of the cells was observed under a microscope, and the wells of the cells were recorded.

5) After the clones are propagated in large numbers, when 1/3 to 1/2 of the bottom of the well is covered, the antibody in the culture medium is measured.

6) In the well of 0.625 cells per well, select the cells in the well with the highest Elisa positive and good growth status, and do the next round of limiting dilution; at the same time, select another well of the same situation and transfer it to the 24-well plate containing feeder cells. Cryopreservation after amplification of one well.

7) After three rounds of limiting dilution, the cells that have been screened and meet the conditions for establishment of the strain will be expanded, cultured and frozen.

5. Preparation method of mouse ascites produced by hybridoma cells

1) 7-10 days before cell injection, each nude mouse was intraperitoneally injected with 0.5 mL of autoclaved pristane (paraffin oil).

2) The recovered and established cell lines were cultured and passaged, and the supernatant was tested by ELISA. When it was determined to be positive, the cells were collected by centrifugation and stained with trypan blue; 5×10 ⁵ cells per Balb/c were resuspended in 0.2 mL of PBS. .

3) Female Balb/c mice were injected intraperitoneally for 8 weeks.

4) About 1-2 weeks, ascites begins to appear. Ascites can be drawn when the animal is the size of a pregnant female. With a 5mL syringe, ascites was drawn from the periphery of the abdominal cavity, about 2-3mL per animal.

5) Centrifuge the ascites at 3000 rpm (1500 g) for 10 minutes, and store the supernatant at -70°C after aliquoting.

6) Under the condition of keeping the nude mice healthy, the extraction can be repeated at intervals of 1-3 days according to the situation of ascites.

[Example 2] Purification and identification of mouse ascites and verification of the affinity of purified antibodies by ForteBio

1. Mab Select ^SureTM purification of mouse ascites

1) The mouse ascites was centrifuged immediately after collection, and the supernatant was frozen at -80°C. Before purification, thaw at room temperature or 4 degrees, centrifuge to take the supernatant, add 1/10 volume of 1M Tris-HCl, 0.5M NaCl, pH 8.0 to adjust the pH value, filter at 0.22μm to be the sample to be loaded on the column, and sample S;

2) The column is equilibrated with 10CV of 1M Tris-HCl, 0.5M NaCl, pH 8.0;

3) Load sample, collect all flow through, and sample FL;

4) Mab Select Sure ^TM was washed with 10 CV of 100 mM Tris-HCl, 50 mM NaCl, pH 8.0;

5) Mab Select Sure ^TM was washed with 10 CV of 10 mM Tris-HCl, 5 mM NaCl, pH 8.0;

6) Pre-add 0.1CV of 1M Tris-HCl pH 9.0 to the collection tube, add 0.5CV of 100mM NaCt/HCt pH3.0 each time during elution, and mix immediately; elute Elute 1, 2, 3... tubes, divide Semi-sampling to prepare samples.

2. Identification of purified antibodies by SDS-PAGE and ELISA

1) SDS-PAGE identification

The purified ascites antibody CD271T-2 was analyzed by SDS-PAGE protein electrophoresis, and the specific results are shown in Figure 2.

2) ELISA identification

The protein in all collection tubes was collected, and the protein concentration was determined after ultrafiltration and centrifugation. The CD271T-1 and CD271T-2 were identified by ELISA. The substrate was CD271 full-length protein. The primary antibody was mouse ascites purified antibody, and the secondary antibody was Goat Anti Mouse. IgG(H+L)-HRP, the specific results are shown in Figure 3.

3. ForteBio verifies antibody affinity after purification

The full-length antigen CD271 was immobilized with proA sensor, and 30, 50, 70, 90, 110, 130nM CD271T-1, CD271T-2 antibody affinity was detected. Cure for 5 minutes (cure height is about 1 nm), combine for 5 minutes, dissociate for 5 minutes (dissociation fitting for the first 1 minute). Regenerate for 5s, neutralize for 15s, and cycle three times. All antigen-antibody dilutions were PBS, regeneration buffer was pH 1.5 10 mM glycine buffer, equilibration eluent was 0.02% Tween PBS, and neutralization was 0.05% Tween PBS. The concentration of the solidified substance (ie, antigen) was 10 μg/mL. The curing height is around 1nm. The specific parameters are shown in Table 1, and the graph is shown in Figure 4.

Table 1

	KD(M)	Kon(1/Ms)	Kdis(1/s)	Full X 2	Full R 2
CD271T-2	3.37× 10-9	6.69×10 4	2.25× 10-4	4.4542	0.98420

[Example 3] Anti-human CD271 heavy and light chain variable region gene cloning and construction of chimeric antibody 2

1. RNA extraction: using Trizol one-step method, take about 10 ⁶ hybridoma cells CD271T-2, add Trizol, extract RNA according to Kangwei Century Ultrapure RNA Kit CW0581S kit, and identify RNA integrity by nucleic acid electrophoresis; hybridoma cells CD271T- 2 has a deposit number of CGMCC 21494. It is preserved in the China General Microorganism Culture Collection and Management Center, and the preservation address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is January 25, 2021. The classification is named as hybridoma cell line CD271T-2;

2. Reverse transcription into cDNA (20 μL): take 1 μL of Oligo dT Primer (50 μM), 1 μL of dNTP Mixture (10 nM), 5 μg of template RNA, add water to 10 μL, incubate at 65°C for 5 min, and quickly cool on ice; add the above denaturation reaction solution 10μL, 5x primerscript II Buffer 4μL, RNase Inhibitor (40U/μL) 0.5μL, PrimeScirpt II RTase (200U/μL) 1μL, add water to 20μL, react at 42°C for 60min, and inactivate at 70°C for 15min;

3. PCR amplification of heavy and light chain variable region genes of anti-CD271 antibody: PCR amplification of light and heavy chain variable region genes was performed in Suzhou Hongxun Biotechnology Co., Ltd. The PCR results are shown in Figure 5A&5B;

4. Construction of sequencing vector: PMD19-T vector was purchased from takara, the heavy and light chain variable region gene PCR products were recovered, and after ligation with T vector, DH5α was transformed, and positive clones were screened at 100 μg/mL ampicillin concentration, and sent for sequencing. Alignment with several conserved framework amino acids possessed by protein databases. The obtained monoclonal antibody comprises a light chain and a heavy chain, the light chain comprises LCDR1, LCDR2 and LCDR3, the amino acid sequence of the LCDR1 is as shown in SEQ ID NO.1, and the amino acid sequence of the LCDR2 is as shown in SEQ ID NO.2, The amino acid sequence of the LCDR3 is shown in SEQ ID NO.3; the heavy chain includes HCDR1, HCDR2 and HCDR3, the amino acid sequence of the HCDR1 is shown in SEQ ID NO.4, and the amino acid sequence of the HCDR2 is shown in SEQ ID As shown in NO.5, the amino acid sequence of the HCDR3 is shown in SEQ ID NO.6. The amino acid sequence of the light chain variable region is shown in SEQ ID NO.7. The heavy chain variable region amino acid sequence is shown in SEQ ID NO.8. The nucleotide sequence encoding the light chain complementarity determining region LCDR1 is shown in SEQ ID NO.9; the nucleotide sequence encoding the light chain complementary determining region LCDR2 is shown in SEQ ID NO.10; the nucleotide sequence encoding the light chain complementarity determining region LCDR3 is shown in SEQ ID NO.10. The nucleotide sequence is shown in SEQ ID NO.11; the nucleotide sequence encoding the heavy chain complementarity determining region HCDR1 is shown in SEQ ID NO.12; the nucleotide sequence encoding the heavy chain complementarity determining region HCDR2 is shown in SEQ ID NO. 13; the nucleotide sequence encoding the heavy chain complementarity determining region HCDR3 is shown in SEQ ID NO.14. The nucleotide sequence of the light chain variable region is shown in SEQ ID NO.15, and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO.16.

[Example 4] Affinity expression identification of anti-human CD271 ascites antibody and Hu-MSCs

Properly growing Hu-MSC cells were collected by trypsin digestion and centrifugation, resuspended and washed with FACS (PBS+2% FBS), centrifuged at 1000 rpm for 3 min, and the supernatant was discarded; primary antibody was added according to Table 3, and incubated at room temperature for 30 min in the dark. Resuspend and wash with FACS (PBS+2%FBS), centrifuge at 1000rpm for 3 min, repeat 3 times, discard the supernatant, add secondary antibody according to Table 1, protect from light at room temperature for 30min, resuspend and wash with FACS (PBS+2%FBS) , centrifuged at 1000 rpm for 3 min, repeated 3 times, added 300 μL of FACS to resuspend the cells, and detected by flow cytometry. The specific results are shown in Figure 6. The figure shows that the positive antibody of APC mouse anti human CD271 derived from Biolegend, the immunogen is derived from melanoma cells, and the CD271 molecule is highly expressed on the cell surface. Compared with the negative control, this antibody has a slight migration (Figure 6A); derived from Abcam Rabbit anti human CD271 positive control antibody, the immunogen is derived from the 350-450 amino acid region in the human CD271 cell membrane, and there is no offset compared with the negative control (Fig. 6B); the ascites purified antibody of CD271T-2 was compared with the positive and negative pairs. Compared with the control, the degree of offset is higher, indicating that the prepared anti-human CD271 monoclonal antibody has very high affinity and specificity (Fig. 6C).

Table 2

The above are only preferred embodiments of the present invention, and are not intended to limit the present invention in any form or substance. It should be pointed out that for those skilled in the art, without departing from the method of the present invention, the Several improvements and supplements can be made, and these improvements and supplements should also be regarded as the protection scope of the present invention. All those skilled in the art, without departing from the spirit and scope of the present invention, can utilize the above-disclosed technical content to make some changes, modifications and equivalent changes of evolution, all belong to the present invention. Equivalent embodiments; at the same time, any modification, modification and evolution of any equivalent changes made to the above embodiments according to the essential technology of the present invention still fall within the scope of the technical solutions of the present invention.

Claims (11)

1. A hybridoma cell line CD271T-2 secreting anti-human CD271 monoclonal antibody, the hybridoma cell line has the deposit number of CGMCC 21494.
2. An anti-human CD271 monoclonal antibody, characterized in that the anti-human CD271 monoclonal antibody is secreted by the CD271T-2 hybridoma cell line of claim 1.
3. A kind of engineering antibody of anti-human CD271 is characterized in that, the engineering antibody of described anti-human CD271 comprises light chain and heavy chain, and described light chain complementarity determining region comprises LCDR1, LCDR2 and LCDR3, and the aminoacid sequence of described LCDR1 is as SEQ ID NO.1, the amino acid sequence of LCDR2 is shown in SEQ ID NO.2, and the amino acid sequence of LCDR3 is shown in SEQ ID NO.3; the heavy chain complementarity determining region includes HCDR1, HCDR2 and HCDR3 , the amino acid sequence of the HCDR1 is shown in SEQ ID NO.4, the amino acid sequence of the HCDR2 is shown in SEQ ID NO.5, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO.6.
4. The engineered antibody against human CD271 according to claim 3, characterized in that it comprises any one or more of the following features: (1) the amino acid sequence of the light chain variable region of the engineered antibody against human CD271 is as follows: As shown in SEQ ID NO.7; or to perform certain substitution, addition and/or deletion of one or several amino acids to SEQ ID NO.7 to obtain an amino acid sequence with equivalent functions; (2) The heavy chain variable region amino acid sequence is as follows: As shown in SEQ ID NO.8, or by performing certain substitutions, additions and/or deletions of one or several amino acids to SEQ ID NO.8 to obtain an amino acid sequence with equivalent functions.
5. The engineered antibody against human CD271 according to any one of claims 3 to 4, wherein the antibody is produced by a CD271T-2 hybridoma cell line through reverse transcription-polymerase chain synthesis reaction.
6. A polynucleotide encoding the heavy chain variable region and/or light chain variable region or full-length amino acids of the anti-human CD271 antibody according to any one of claims 3-5.
7. The polynucleotide of claim 6, further comprising any one or more of the following features: (1) the nucleotide sequence encoding the light chain complementarity determining region LCDR1 is as shown in SEQ ID NO.9 The nucleotide sequence encoding the light chain complementarity determining region LCDR2 is shown in SEQ ID NO.10; the nucleotide sequence encoding the light chain complementarity determining region LCDR3 is shown in SEQ ID NO.11; (2) encoding the heavy chain The nucleotide sequence of the complementarity determining region HCDR1 is shown in SEQ ID NO.12; the nucleotide sequence encoding the heavy chain complementarity determining region HCDR2 is shown in SEQ ID NO.13; the nucleotide sequence encoding the heavy chain complementarity determining region HCDR3 is shown in SEQ ID NO.13 The sequence is shown in SEQ ID NO.14.
8. A polynucleotide, characterized in that the polynucleotide encodes the recombination sequence of the light chain variable region and the heavy chain variable region of the Fab segment of the anti-CD271 antibody according to any one of claims 3-5, capable of Obtain a single-chain antibody with a smaller molecular weight.
9. A construct comprising the polynucleotide of any one of claims 6-8.
10. A host cell comprising the construct of claim 9 or the exogenous polynucleotide of any one of claims 6-8 integrated into the genome.
11. The cell line according to claim 1, or the anti-human CD271 monoclonal antibody according to claim 2, or the engineered antibody against human CD271 according to any one of claims 3-5, or claims 6-8 The polynucleotide described in any one, or the construct described in claim 9, or the purposes of the host cell of the described antibody of claim 10, is selected from following any one:

    a) cells expressing CD271, including but not limited to mesenchymal stem cells, tumor stem cells, and nervous system cells, and their in vitro detection, identification, isolation, enrichment, and targeting;

    b) Mediating mesenchymal stem cells to target tissue repair;

    c) Detection and tracing of stem cells, tumor stem cells and cells of the nervous system;

    d) preparation of nervous system targeting drugs;

    e) preparing a tumor therapeutic drug;

    f) Coupling with other small chemical molecules, biological macromolecules, radioactive molecules, nanomaterials, etc., for various purposes of the above a)-e).

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