CN111748036B

CN111748036B - Human-mouse chimeric monoclonal antibody

Info

Publication number: CN111748036B
Application number: CN202010597777.6A
Authority: CN
Inventors: 魏世辉; 李洪恩; 杨沫; 宋宏鲁; 孙明明; 周欢粉
Original assignee: Chinese PLA General Hospital
Current assignee: Chinese PLA General Hospital
Priority date: 2020-06-28
Filing date: 2020-06-28
Publication date: 2022-03-29
Anticipated expiration: 2040-06-28
Also published as: CN111748036A

Abstract

The application provides a human-mouse chimeric monoclonal antibody aiming at AQP4 antigen, which comprises a mouse-derived heavy chain variable region VH, a mouse-derived light chain variable region VL and a human-derived antibody Fc region, wherein the nucleic acid sequence of the mouse-derived heavy chain variable region VH is shown as SEQ ID NO:1, and the amino acid sequence of the mouse heavy chain variable region VH is shown as SEQ ID NO: 2, and the nucleic acid sequence of the murine light chain variable region VL is shown as SEQ ID NO: 3, and the amino acid sequence of the murine light chain variable region VL is shown as SEQ ID NO: 4. The invention provides a heavy chain and light chain variable region sequence of a human-mouse chimeric blocking type AQP4 specific monoclonal antibody, enriches the types of antibodies, lays a certain foundation for the treatment of NMO diseases, and the antibody is a blocking type AQP4 specific antibody, can be combined with AQP4 on astrocytes in a central nervous system, blocks the combination of a pathogenic AQP4 antibody and AQP4 on astrocytes, and protects astrocytes in the central nervous system; the monoclonal antibody has good specificity and high affinity.

Description

Human-mouse chimeric monoclonal antibody

Technical Field

The invention relates to the technical field of biology, in particular to a human-mouse chimeric monoclonal antibody.

Background

Neuromyelitis optica (NMO) is a demyelinating disease that involves the optic nerve and spinal cord. The earliest report of the disease was seen in 1872 and was originally thought to be a single-course central nervous system disorder. In Asian countries, there are many reports on NMO, and only in China, there are over 30 ten thousand NMO patients.

A significant portion of NMO spinal cord damage occurs after visual deterioration, which can be spaced days, weeks, months or even years, eventually leading to paraplegia, sensory and sphincter dysfunction, and possibly respiratory muscle paralysis in the worst case. Since the recurrent NMO has many common characteristics with the recurrent optic neuritis in the initial clinical presentation, it is easily misdiagnosed as the latter, which delays the treatment time on the one hand, and on the other hand, the treatment means of optic neuritis aggravates the condition of NMO, causing more serious consequences.

NMO specificity is due to its specific pathogenesis, in which the aquaporin4 (AQP 4) signaling pathway is involved. Further research shows that some pathogenic antibodies of AQP4 can specifically bind to aquaporin 4(aquaporin4, AQP4) on the astrocyte end foot in the Central Nervous System (CNS) to induce the body to generate NMO, while blocking type AQP4 specific antibodies can effectively block the pathogenic mechanism of pathogenic AQP4 antibodies after binding to AQP4 on the astrocyte, so as to achieve the effect of treating NMO diseases. At present, research on aquaporin 4(aquaporin4, AQP4) blocking type antibody drugs is in a blank stage, and related humanized antibody drugs are few, so that research on the blocking type AQP4 specific antibody and research on potential therapeutic effects of the blocking type AQP4 specific antibody are of great significance.

Disclosure of Invention

The present application provides a human-mouse chimeric monoclonal antibody to solve the above-mentioned problems.

The technical scheme adopted by the application is as follows: a human-mouse chimeric monoclonal antibody is characterized by comprising a mouse-derived heavy chain variable region VH, a mouse-derived light chain variable region VL and a human-derived antibody Fc region, wherein the nucleic acid sequence of the mouse-derived heavy chain variable region VH is shown as SEQ ID NO:1, and the amino acid sequence of the mouse heavy chain variable region VH is shown as SEQ ID NO: 2, and the nucleic acid sequence of the murine light chain variable region VL is shown as SEQ ID NO: 3, and the amino acid sequence of the murine light chain variable region VL is shown as SEQ ID NO: 4.

Further, the murine heavy chain variable region VH has three hypervariable regions CDR1, CDR2, CDR3, and the amino acid sequences of CDR1, CDR2, CDR3 are respectively shown in SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: shown at 10.

Further, the murine light chain variable region VL has three hypervariable regions CDR4, CDR5, CDR6, and the amino acid sequences of CDR4, CDR5, CDR6 are respectively as shown in SEQ ID NO: 12. SEQ ID NO: 14. SEQ ID NO: shown at 16.

The application has the advantages and positive effects that: the invention provides a heavy chain and light chain variable region sequence of a human-mouse chimeric blocking type AQP4 specific monoclonal antibody, enriches the types of antibodies, lays a certain foundation for the treatment of NMO diseases, and the antibody is a blocking type AQP4 specific antibody, can be combined with AQP4 on astrocytes in a central nervous system, blocks the combination of a pathogenic AQP4 antibody and AQP4 on astrocytes, and protects astrocytes in the central nervous system; the monoclonal antibody has good specificity and high affinity.

In addition to the technical problems addressed by the present application, the technical features constituting the technical solutions, and the advantages brought by the technical features of the technical solutions described above, other technical problems solved by the present application, other technical features included in the technical solutions, and advantages brought by the technical features will be further described in detail below with reference to the accompanying drawings.

Drawings

FIG. 1 is a flow chart of the construction and in vitro culture of eukaryotic expression vectors for heavy and light chains of the murine chimeric antibody of the present applicant;

FIG. 2 is a cell immunofluorescence assay of hybridoma cell lines screened in example III of the present application;

FIG. 3 is a graph showing the affinity assay of AQP4 full-length protein and human murine chimeric antibody obtained in the present application, wherein the curves from bottom to top are the human murine chimeric antibody added with different concentration gradients (0nM,7.5nM,15nM,30nM,60nM,120nM and 240 nM).

Detailed Description

The present application will be described in further detail with reference to examples. It is to be understood that the specific embodiments described herein are merely illustrative of the relevant invention and not restrictive of the invention.

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present application will be described in detail with reference to examples.

The first embodiment is as follows: preparation of monoclonal antibody hybridoma cells

(I) immunizing mice

A coupled polypeptide obtained by coupling a specific sequence 137-155 amino acid sequence from AQP4 and KLH is taken as an immunogen to immunize 5 BALB/c mice for 5 times at an interval of 14 days each time, and after 7 days of 5-time immunization, the immune serum titer of the mice is measured, and 2 mice with high serum titer are selected for later use.

(II) preparation of splenocytes

1. Washing the immunized mouse with high serum titer with running water, and placing in 75% ethanol solution for 5-10 min.

2. Taking out the spleen of the mouse in a sterile manner, placing the spleen in a sterile metal filter screen of 200 meshes, shearing the spleen by using a sterile surgical scissors, and grinding to obtain a single cell suspension.

3. Transferring the suspension into a 50ml centrifuge tube by using a pipette gun, adding the incomplete IMDM culture solution to 50ml, centrifuging at 1500r/min for 5min, and removing the supernatant.

4. Adding 1ml of sterile water by using a pipette, blowing and beating cell sediment by using the pipette, adding the IMDM culture solution to 50ml, centrifuging for 5min at 1500r/min, discarding the supernatant, and washing twice (1500r/min, 5min) by using the incomplete IMDM culture solution for later use.

(II) fusion and selection

1. Collecting myeloma cells SP2/0 which grow well and are in a logarithmic growth phase, centrifuging for 5min at 2000r/min, discarding the supernatant, suspending the cells by using incomplete IMDM culture solution, counting the required number of the cells, adding the incomplete IMDM culture solution to 50ml, centrifuging for 5min at 2000r/min, and suspending the cells by adding the incomplete IMDM culture solution for later use.

2. SP2/0 cells and spleen cells were mixed at a ratio of 1:2 in a 50m1 centrifuge tube, washed once with incomplete IMDM medium, and centrifuged at 1500r/min for 5 min. Abandoning the supernatant, completely absorbing the residual liquid by using a pipette, and slightly tapping the bottom of the centrifuge tube on the palm to loosen and mix the cells.

3. Placing the centrifugal tube in a 37 ℃ heat-preservation water bath cup for preheating, sucking 50% PEG solution of lml subjected to 37 ℃ preheating, finishing adding in lmin at a constant speed, and gently shaking the centrifugal tube while adding.

4. After resting at 37 ℃ for 90s, 15ml of serum-free IMDM medium was gently added along the vessel wall within 2-4min, starting drop by drop, taking care not to agitate the cells as much as possible. (lmin plus lml,3min plus 3m1, finally 10ml slowly). (Uniform dropping)

5. Centrifugation was carried out at 1500r/min for 5 minutes, and the supernatant was discarded (the tube was tilted, and the supernatant was aspirated).

6. Resuspending the precipitated cells with IMDM complete culture solution, mixing, adding dropwise into 96-well flat-bottom cell culture plate (100 ul/well), culturing at 37 deg.C in 5% C02 incubator,

7. on day 2 after the fusion, 100. mu.l of HAT medium was added to each well, and then HAT medium was changed for 2 to 3 days, and then HT medium was used for 10 to 14 days.

8. The cloning growth condition in a 96-well plate is observed every day during the fusion period, when hybridoma cells are fully distributed in the area of the bottom 1/10 of the well, specific antibodies can be detected, and a required hybridoma cell line is screened by adopting an ELISA method, which comprises the following steps:

(1) coating antigen: BSA (bovine serum albumin) -coupled AQP 4-derived specific polypeptide (amino acid sequence 137-155) was coated at 400 ng/well overnight at 2-8 ℃.

(2) Washing: the plate was washed 3 times with 0.01M PBST wash and patted dry.

(3) And (3) sealing: add blocking solution (3% calf serum) 200. mu.l, block for 2 hours at 37 ℃ and pour out the liquid in the wells and pat it dry.

(4) Sample adding: 100. mu.l of cell culture supernatant was added to the antigen-coated microplate, and SP2/0 myeloma cell supernatant was used as a negative control. The sample and the negative serum were serially diluted 10-fold with a diluent (PBS), and 100. mu.l/well was applied to the closed microplate and reacted at 37 ℃ for 1 hour.

(5) Washing: the plate was washed 3 times with 0.01M PBST wash and patted dry.

(6) Adding a secondary antibody: a secondary HRP-labeled goat anti-mouse IgG antibody was added and reacted at 37 ℃ for 1 hour.

(7) Washing: the plate was washed 5 times with 0.01M PBST wash and patted dry.

(8) Color development: adding 50 mul/hole of each of the solution A and the solution B.

(9) And (4) terminating: to be developedAdding stop solution (stop solution is 2M H)₂SO₄Solution) 50. mu.l per well.

(10) Screening: screening out the OD of the sample₄₅₀And the nm value is more than 3.5.

Table 1 shows the positive clone cell lines selected

10. Subcloning of hybridoma cells by limiting dilution method

(1) The above hybridoma cells were counted, diluted to 1-10 cells/ml with HT medium containing 20% serum to 100. mu.l per well, and added to a 96-well plate at 37 ℃ with 5% CO₂Wet culturing in incubator for 7-10 days.

(2) And detecting the activity of the antibody in time when macroscopic cloning appears. And (4) observing under an inverted microscope, marking holes with only single clone growing, taking supernatant for ELISA antibody detection, and screening positive clone cell strains with OD450nm values larger than 3.5.

TABLE 2 Positive clone cell lines selected by the first subcloning

Clone number	OD₄₅₀nm	Clone number	OD₄₅₀nm
				1#	3.6125	5#	3.6056
2#	3.8795	6#	3.5614
				3#	3.7852	7#	3.7792
4#	3.9082	8#	3.8789

(3) And transferring the positive monoclonal cell strain into a 24-well plate for amplification culture.

(4) A second subcloning was performed to further purify the monoclonal hybridoma cells, and the procedure was the same as in step (1).

TABLE 3 second subcloned cell line OD_450nmValue of

Clone number	OD_450nm	Clone number	OD_450nm
				103	2.8894	502	2.7953
202	2.8954	601	2.6859
				301	2.9812	702	2.8723
402	3.8682	802	2.6955

Example two: preparation of monoclonal antibody ascites

Each of the monoclonal hybridoma cell lines obtained in example one was subjected to expansion culture, and the cells were collected for mass production of monoclonal antibodies. The preparation of the monoclonal antibody ascites is carried out as follows:

(1) selecting female BALB/c mice born by birth, injecting 500ul of sterilized paraffin into the abdominal cavity, and stimulating immune cells to promote the proliferation of hybridoma cells;

(2) observing the state of the mice, and after 7-10 days, the weight of each mouse is about 1 multiplied by 10⁶Injecting the amount of each cell into the prepared monoclonal positive cells, observing the state of the mouse in time, expanding the abdomen of the mouse 7 days later, and removing cell components and other precipitates by centrifuging at 4 ℃ for 10min at 2000r/min if the skin is tense when the mouse touches the mouse, and collecting the supernatant of the abdomen;

(3) and purifying the supernatant of the abdomen water by Protein A affinity chromatography to obtain the antibody of the corresponding hybridoma cell strain.

Example three: ascites monoclonal antibody ELISA detection

Carrying out ELISA detection on the ascites monoclonal antibodies of different hybridoma cell strains obtained in the second embodiment, wherein the operation steps are as follows:

(1) wrapping a plate: phosphate buffer (PBS, 0.01M, pH7.4) diluted KLH coupled AQP4 polypeptide antigen to 3 μ g/ml, each 100ul add 96 well enzyme label plate, 4 degrees C incubation for 16 hours, spin coating liquid, PBST buffer solution washing plate once, dry.

(2) Blocking solution (0.01M PBS containing 10% bovine serum, pH7.4) is added into 150ul of each well for blocking, incubation is carried out at 37 ℃ for 2 hours, and the solution is discarded and patted dry for later use.

(3) Adding an antibody: diluting the monoclonal antibody obtained in the second embodiment to 1000 times according to a 10-fold dilution method, performing gradient dilution to 1:106 dilution by adopting a multiple-ratio dilution method, adding 100ul of each dilution into an ELISA plate, and making a plurality of holes; incubate at 37 ℃ for 60 minutes.

(4) The monoclonal antibody serum was spun off and the plates were washed 3 times with PBST buffer, 30 seconds apart each time, and patted dry.

(5) Adding goat anti-mouse secondary antibody marked by HRP, incubating at 37 ℃ for 60 minutes in each hole with 100 ul;

(6) PBST (0.01M, PBS + 0.05% Tween 20) buffer wash plate 3 times, each 30 seconds apart, beat dry.

(7) Adding equivalent chromogenic substrate A/B solution, incubating at 37 ℃ for 10-15 minutes in each hole with 50 ul;

(8) 2M H was added to 50ul per well₂SO₄The reaction was terminated by the stop solution.

(9) Reading: the wavelength of the microplate reader used was 450 nm.

(10) Screening out OD₄₅₀The results of the positive hybridoma cell strains with nm values of more than 3.5 are shown in Table 4.

TABLE 4 ascites monoclonal antibody OD_450nmValue of

Clone number	OD_450nm	Clone number	OD_450nm
				3-301	3.5939	8-802	3.6868
4-402	4.0917

Example four: cellular immunofluorescence assay

And (4) carrying out cell slide on each hybridoma cell strain screened in the third example.

1. Soaking the cell-crawled slide in PBS 3 times for 3min each time in the culture plate;

2. fixing the slide with 4% paraformaldehyde for 15min, and washing the slide with PBS for 3 times, each for 3 min;

3. 0.5% Triton X-100 (prepared with PBS) and permeating for 20min at room temperature;

4. soaking and washing the slide with PBS for 3 times, each time for 3min, sucking dry PBS with absorbent paper, dripping normal goat serum on the slide, and sealing at room temperature for 30 min;

5. absorbing sealing liquid by absorbent paper, not washing, dripping enough diluted primary antibody on each glass slide, putting the glass slide into a wet box, and incubating overnight at 4 ℃;

6. adding a fluorescent secondary antibody: soaking PBST in the climbing sheet for 3 times (3 min each time), sucking the excessive liquid on the climbing sheet with absorbent paper, dripping diluted fluorescent secondary antibody, incubating at 20-37 deg.C for 1h in a wet box, and soaking PBST in the climbing sheet for 3 times (3 min each time); all subsequent processing steps were performed as dark as possible from the addition of the fluorescent secondary antibody.

7. Counterstaining the nucleus: dripping DAPI, incubating for 5min in dark, staining the specimen for nucleus, and washing off excessive DAPI 5min × 4 times by PBST;

8. the liquid on the slide is blotted by absorbent paper, the slide is sealed by using a sealing liquid containing an anti-fluorescence quenching agent, and then the collected images are observed under a fluorescence microscope, as shown in figure 2, wherein, figure a is a fluorescence image of 4-402 hybridoma cell lines, figure b is a fluorescence image of 8-802 hybridoma cell lines, and figure c is a fluorescence image of 3-301 hybridoma cell lines.

As can be seen from FIG. 2, the 4-402 hybridoma cell line had the strongest fluorescence intensity, i.e., the highest viability of the cell, and was the best hybridoma cell.

Example five: sequencing of monoclonal antibodies

Extracting total RNA from the 4-402 hybridoma cell strain screened in the fourth embodiment, synthesizing first-strand cDNA by using RNA as a template through a cDNA synthesis reverse transcription kit, amplifying a hybridoma cell monoclonal antibody heavy chain variable region (VH) gene and a hybridoma cell monoclonal antibody light chain variable region ((VL) gene to perform T/A cloning on a hybridoma cell variable region PCR product sequence by using the cDNA as a template, selecting a positive colony to perform sequencing, and performing amino acid translation analysis on a sequencing structure.

The results showed that the amino acid sequences of CDR1, CDR2 and CDR3 of the single-antibody heavy chain variable region of 4-402 hybridoma cells are shown in SEQ ID NOS: 6,8 and 10, respectively, and the corresponding encoding nucleic acid sequences are shown in SEQ ID NOS: 5,7 and 9, respectively. In addition, the partial heavy chain variable region gene containing the variable region and the coded amino acid sequence are shown as SEQ ID NO:1 and 2.

4-402 hybridoma monoclonal antibody light chain variable region CDRI, CDR2 and CDR3 amino acid sequences are respectively listed in SEQ ID NO; 12,14 and 16, and the corresponding coding nucleic acid sequences are set forth in SEQ ID NOs 11,13 and 15, respectively. In addition, the partial light chain variable region gene containing the variable region and the coded amino acid sequence are shown as SEQ ID N0:3 and 4.

Example six: preparation of human murine chimeric antibody

1. The heavy chain variable region (VH) and light chain variable region ((VL) genes of the 4-402 hybridoma monoclonal antibody obtained in the fifth example were ligated with the double-digested pretreated cloning vectors pFUSE-hIgG1-Fc1 and pFUSE2-CLIg-hk, respectively, and the ligation product was transformed into competent bacterium DH5a, and since the expression vector carries the kanamycin (Kana +) resistance gene, the transformant was applied to Kana-resistant LB solid medium and cultured overnight at 37 ℃.

2. And (3) the bacteria to be plated grow dispersed colonies, selecting the colonies with clear edges and good growth, and sequencing.

3. And (3) comparing the sequencing result in the step (2) with that in the fifth example, selecting the transformation bacteria with the same sequencing result twice, and extracting the endotoxin-free expression plasmid after amplification culture.

4. Co-transfecting the Expression vector containing the target monoclonal antibody heavy chain and light chain variable region genes obtained in the step 3 into a FreeStyle 293F cell which is a eukaryotic Expression cell strain, and performing suspension culture by adopting a Gibco FreeStyle 293 Expression Medium serum-free culture Medium.

5. After the transfected FreeStyle 293F cells are continuously cultured for 4d, impurities in the supernatant are removed by centrifugation, the supernatant is filtered and sterilized by a 0.22um filter, and finally, the supernatant is collected and purified to obtain the human-mouse chimeric antibody.

Example seven: ELISA detection of human murine chimeric antibodies

Coating a 96-well plate by using an anti-human Fc antibody, adding the human-mouse chimeric antibody obtained in the sixth example, and detecting by using an anti-human Fc-HRP antibody, wherein the specific detection method comprises the following steps:

(1) wrapping a plate: the recombinant protein antigen was diluted to 3. mu.g/ml with phosphate buffer (0.05M, pH7.4), an ELISA plate was added to 100. mu.l of each well, incubated at 4 ℃ for 16 hours, the coating solution was removed, and the plate was washed once with PBST buffer and patted dry.

(2) Adding 150ul of sealing solution into each hole for sealing, incubating at 37 ℃, performing 2 hours, discarding the solution, and patting dry for later use.

(3) Adding an antibody: diluting the human-mouse chimeric antibody to 1000 times according to a 10-time dilution method, performing gradient dilution to 1:106 dilution by adopting a multiple dilution method, adding 100ul of each dilution into an enzyme label plate, and making a plurality of holes; incubate at 37 ℃ for 60 minutes. (4) Antibody serum was spun off and the plates were washed 3 times with PBST buffer, 30 sec intervals and patted dry.

(5) Adding goat anti-human Fc secondary antibody marked by HRP, incubating at 37 ℃ for 60 minutes in each hole with 100 ul;

(6) the plates were washed 3 times with PBST buffer, 30 sec intervals and blotted dry.

(8) stop solution was added to 50ul per well to stop the reaction.

(9) Reading: the wavelength of the microplate reader used was 450 nm.

In this example, a positive control of the human antibody and a negative control of the medium were set.

The test results are shown in table 5:

table 5: ELISA OD₄₅₀Value of

Human-mouse chimeric monoclonal antibody	Positive control 1	Negative control (Medium)
			2.695	2.563	0.055
2.517	2.453	0.052
			2.449	2.395	0.051

As shown in Table 5, the OD of the human-murine chimeric antibody₄₅₀OD of more than 3 times of negative control₄₅₀Values indicate successful expression of the human murine chimeric antibody.

Example eight: human murine chimeric antibody affinity assay

1. The program, Application Wizards, was opened, surface preference-Immobilization was selected, CMSChip and Amine ligation were selected.

2. Selecting a specific Flow Rate and Injection Time;

3. a Run sensory gram was selected using the full-length protein AQP4 as the coating antigen, starting in the second pass. (flow 1 is a control) control channels were not coated with protein.

4. The monoclonal antibody flow rate was set at 5ul/min, the Manual Inject input volume was selected at 50ul, and the solution placement position was 5ug/ml antibody, the loading volume was selected at 5ul, and the level of binding was determined.

5. After the amount of antibody solution required was determined, the chip was regenerated. The flow rate was adjusted to 50 ul/min. Quick inject was selected and injected with lOmM Glycine-HCl, pH 1.7,2 min. The flow rate was then adjusted back to 5 ul/minx.

6. After all procedures were completed, the affinity constant KD was generated by software kinetic analysis.

The results are shown in FIG. 3, and it is clear from FIG. 3 that the KD coefficient of the human-mouse chimeric antibody is 1.16X 10^-10It was shown to have higher affinity.

The embodiments of the present application have been described in detail, but the description is only for the preferred embodiments of the present application and should not be construed as limiting the scope of the application. All equivalent changes and modifications made within the scope of the present application shall fall within the scope of the present application.

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Claims

1. A human-murine chimeric monoclonal antibody against AQP4 antigen, comprising a murine heavy chain variable region VH, a murine light chain variable region VL, and a human antibody Fc region, wherein the nucleic acid sequence of the murine heavy chain variable region VH is as shown in SEQ ID NO:1, and the amino acid sequence of the mouse heavy chain variable region VH is shown as SEQ ID NO: 2, and the nucleic acid sequence of the murine light chain variable region VL is shown as SEQ ID NO: 3, and the amino acid sequence of the murine light chain variable region VL is shown as SEQ ID NO: 4.

2. The human murine chimeric monoclonal antibody to AQP4 antigen of claim 1, wherein the murine heavy chain variable region VH has the amino acid sequences of three hypervariable regions CDR1, CDR2, CDR3, CDR1, CDR2, CDR3, respectively as set forth in SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: shown at 10.

3. The human murine chimeric monoclonal antibody against AQP4 antigen as claimed in claim 2, wherein the murine light chain variable region VL has the amino acid sequences of three hypervariable regions CDR4, CDR5, CDR6, CDR4, CDR5, CDR6 as set forth in SEQ ID NO: 12. SEQ ID NO: 14. SEQ ID NO: shown at 16.