CN117820488A

CN117820488A - Antibody pair, biological material and application of antibody pair specific to MMP-9

Info

Publication number: CN117820488A
Application number: CN202410029690.7A
Authority: CN
Inventors: 田颖; 陶勇
Original assignee: Beijing Chaoyang Hospital
Current assignee: Beijing Chaoyang Hospital
Priority date: 2024-01-09
Filing date: 2024-01-09
Publication date: 2024-04-05

Abstract

The invention discloses an antibody pair specific to MMP-9, a biological material and application thereof, belonging to the field of biomedicine. The antibody pair comprises a first antibody chrysM 22 and a second antibody chrysM 37, wherein CDRs of complementarity determining regions of a heavy chain H22 and a light chain K22 of the first antibody chrysM 22 are shown in SEQ ID NO. 01-06; the CDRs of the complementarity determining regions of the heavy chain H37 and the light chain K37 of the second antibody chrM37 are shown in SEQ ID NO. 07-12. The novel anti-MMP-9 antibody pair can realize efficient recognition and inhibition of MMP-9 and provide a novel means for detection and treatment of diseases. By introducing highly specific antibody pairs, it is expected that the activity of MMP-9 can be accurately identified and regulated at the molecular level, providing a more effective and safer solution for disease treatment and patient health.

Description

Antibody pair, biological material and application of antibody pair specific to MMP-9

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to an antibody pair specific to MMP-9, a biological material and application thereof.

Background

Matrix metalloproteinase 9 (Matrix Metalloproteinase, MMP-9) is an enzyme which plays a key role in physiological and pathological conditions, participates in degradation of extracellular matrix and tissue remodeling, and has important regulation and control effects on physiological processes such as cell migration, angiogenesis, immune response and the like. However, in certain disease states, aberrant activation and overexpression of MMP-9 is closely associated with the occurrence and progression of a variety of diseases, such as chronic inflammation, neovascular, tumor infiltration metastasis, cardiovascular disease, and the like.

Against this background, the development of a highly potent MMP-9 antibody has become an urgent need for detection and treatment of related diseases. Antibodies, particularly antibody pairs (antibodies), play a critical role in the field of disease antigen detection. For example, in enzyme-linked immunosorbent assays (ELISA), the use of antibody pairs can increase the sensitivity and specificity of the assay, as the "capture" and "detection" antibodies act synergistically to greatly enhance the recognition and signal amplification of a particular antigen. The double-insurance mechanism can obviously reduce the occurrence of false positive results, improve the reliability of detection results and reduce the requirement on sample purity so as to accurately measure the antigen concentration in complex biological samples. In addition, antibodies may also have neutralizing effects on antigens, and by neutralizing specific pathogenic proteins, such as MMP-9, these antibodies may prevent or reduce pathogenic antigens, thereby preventing further disease progression.

In view of this, the present application is specifically proposed.

Disclosure of Invention

The invention aims to provide an antibody pair, a biological material and application thereof specific to MMP-9, so as to realize efficient recognition and inhibition of MMP-9 and provide a means for detecting and treating diseases caused by abnormal activation and over-expression of MMP-9.

The invention is realized by the following technical scheme:

in a first aspect, the present application provides an antibody pair specific for MMP-9, the antibody pair comprising a first antibody chrM22 and a second antibody chrM37;

the complementarity determining region CDRs of the heavy chain H22 of the first antibody chrM22 are as follows:

CDR1 as shown in SEQ ID No.01,

CDR2 as shown in SEQ ID No.02,

CDR3 as shown in SEQ ID No. 03;

the complementarity determining region CDRs of the light chain K22 of the first antibody chrM22 are as follows:

CDR1 as shown in SEQ ID No.04,

CDR2 as shown in SEQ ID NO.05,

CDR3 as shown in SEQ ID No. 06;

the complementarity determining region CDRs of heavy chain H37 of second antibody chrM37 are as follows:

CDR1 as shown in SEQ ID NO.07,

CDR2 as shown in SEQ ID No.08,

CDR3 as shown in SEQ ID No. 09;

the complementarity determining region CDRs of light chain K37 of second antibody chrM37 are as follows:

CDR1 as shown in SEQ ID No.10,

CDR2 as shown in SEQ ID No.11,

CDR3 as shown in SEQ ID No. 12.

Further, the amino acid sequence of the heavy chain H22 of the first antibody chrM22 is shown as SEQ ID NO.13, and the amino acid sequence of the light chain K22 of the first antibody chrM22 is shown as SEQ ID NO. 14.

Further, the amino acid sequence of the heavy chain H37 of the second antibody chrM37 is shown as SEQ ID NO.15, and the amino acid sequence of the light chain K37 of the second antibody chrM37 is shown as SEQ ID NO. 16.

In a second aspect, the present application provides a biological material associated with the antibody pair described above, the biological material being any one of the following:

(a) A nucleic acid molecule encoding the antibody pair of any one of claims 1-3;

(b) An expression cassette comprising the nucleic acid molecule of (a);

(c) A recombinant vector comprising the nucleic acid molecule of (a) or the expression cassette of (b);

(d) A recombinant cell comprising the nucleic acid molecule of (a), the expression cassette of (b) or the recombinant vector of (c).

In this application, a "nucleotide molecule" may be DNA, such as cDNA or recombinant DNA, or RNA, such as mRNA or hnRNA, etc.

An "expression cassette" comprises a polynucleotide sequence encoding a polypeptide (antibody pair) to be expressed and sequences controlling its expression, such as a promoter and optionally an enhancer sequence, including any combination of cis-acting transcriptional control units. Sequences that control gene expression (i.e., transcription thereof and translation of the transcription product) are often referred to as regulatory units. Most of the regulatory units are located upstream of and operably linked to the coding sequence of the gene. The expression cassette may also contain a downstream 3' untranslated region comprising a polyadenylation site.

The vector of the "recombinant vector" may be a plasmid, phage or virus. The "recombinant cell" may be a recombinant eukaryotic cell or a recombinant prokaryotic cell, wherein the host cell of the "recombinant eukaryotic cell" may be a yeast or mammalian cell, etc.; the host cell of the "recombinant prokaryotic cell" may be a bacterium, an alga, or the like.

Further, the above-mentioned nucleic acid molecule comprises:

the base sequence of the heavy chain H22 of the first antibody chrM22 is shown in SEQ ID NO. 17;

the base sequence of the light chain K22 of the first antibody chrM22 is shown in SEQ ID NO. 18.

Further, the above nucleic acid molecule comprises:

the base sequence of the heavy chain H37 of the second antibody chrM37 is shown in SEQ ID NO. 19;

the base sequence of the light chain K37 of the second antibody chrM37 is shown in SEQ ID NO. 20.

In a third aspect, the present application provides the use of an antibody pair as described above, comprising in particular any one of the following:

(1) An application of the antibody in preparing tumor inhibitors, inflammatory disease medicines or autoimmune disease medicines.

(2) The application of the antibody in preparing a detection reagent of matrix metalloproteinase 9.

(3) Use of an antibody as described above for the preparation of an inhibitor of matrix metalloproteinase 9 or a product that binds to matrix metalloproteinase 9.

The invention has the following effects:

the application realizes the efficient recognition and inhibition of MMP-9 by developing a novel anti-MMP-9 antibody pair and provides a novel means for detecting and treating diseases. The preparation method of the anti-MMP-9 antibody and the application thereof in the detection and treatment of diseases provide innovative solutions for the technical development and the drug development in the related fields. By introducing highly specific antibody pairs, it is expected that the activity of MMP-9 can be accurately identified and regulated at the molecular level, providing a more effective and safer solution for disease treatment and patient health.

Drawings

FIG. 1 is a graph showing the results of serum immunotiter assays for immunized animals in example 2;

FIG. 2 is a graph showing the binding kinetics test of two antibodies in example 2; wherein, a. Chrm22 binding kinetics test; testing of binding kinetics of chrM 37.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to the following examples, which are to be construed as merely illustrative and not limitative of the scope of the invention, but are not intended to limit the scope of the invention to the specific conditions set forth in the examples, either as conventional or manufacturer-suggested, nor are reagents or apparatus employed to identify manufacturers as conventional products available for commercial purchase.

The following describes specific embodiments of the present invention in detail. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.

Example 1

The embodiment provides a construction of MMP-9 expression vector and stable transfer cell strain, comprising:

construction of MMP-9 expression vectors

The nucleotide sequence of the CDS region encoding human MMP-9 was obtained from NCBI database and the synthesis of the full-length gene sequence was performed. Specifically, the synthesized gene sequence was inserted into the mammalian cell expression vector pTT5 (supplied by Shandonoxinan Biotechnology Co., ltd.) through XbaI and NotI cleavage sites. The sequence is correct through sequencing verification, and the plasmid of pTT5-MMP9 is successfully constructed.

Next, the constructed pTT5-MMP9 plasmid was cultured in a liquid LB medium. Finally, plasmid DNA was extracted from the cultured strain according to the procedure of handbook using the plasmid large extraction kit provided by shanghinoxin biotechnology limited.

Expression and purification of MMP-9 antigen

293F cells in the logarithmic growth phase were selected to grow well and their numbers were controlled at 2X 10 ⁶ The density of cells/ml is below. At 37℃and 5% CO ₂ Suspension culture at 120rpm/min until cell density reaches 2×10 ⁶ Cells/ml.

Next, pTT5-MMP9 plasmid was transfected using PEI transfection reagent: cells were seeded in 6-well plates the day before transfection at a cell density of 70% -80%. DNA-PEI nucleic acid-transfection reagent complexes were formulated using 100. Mu.L of serum-free medium. mu.L of DNA-PEI nucleic acid-PEI complex was added directly to the cells, the plates were shaken and gently mixed.

Cell supernatants from day 7 post-transfection cultures were collected, centrifuged and filtered using a 0.45 μm filter, protein a medium purified for antibodies and the antibodies were replaced by dialysis into PBS ph7.2 buffer. Protein concentration and purity were determined by Nanodrop absorbance measurements and purity was checked by sodium dodecyl sulfate gel electrophoresis and coomassie staining, which showed that: MMP-9 protein content in the solution is high.

Example 2

This example provides the preparation of an MMP-9 monoclonal antibody pair

The preparation principle is as follows: antigen-specific B cells are isolated from immune animal tissues or peripheral blood, igG heavy chain and light chain variable region genes are amplified from single antibody secretion B cells by single cell PCR technology, and then monoclonal antibodies with biological activity are obtained by expression in mammalian cells.

2-1 immunization of animals

New Zealand white rabbits were selected as immunized animals. In the initial immunization phase, 200. Mu.g of antigen (MMP-9 protein) was diluted to 500. Mu.L with PBS and emulsified in a Freund's complete adjuvant mixture of equal volume, and then immunized by back multipoint injection. Two weeks later, booster immunization was performed, at which time the antigen dose was reduced to 100 μg, and mixed with an equal volume of Freund's complete adjuvant to emulsify, and then the immune response was further boosted by back multipoint injection. After five immunizations are completed, immune serum titers are detected by an ELISA conventional method, the specific detection results are shown in figure 1, and the immune titers are 1:100000, and rabbit No.1 slightly higher than rabbit No. 2.

Rabbit No.1 was selected and was subjected to impact immunization by intraperitoneal injection of 50. Mu.g of MMP-9 protein 3 days before antibody screening, and the impact immunization antigen of this time was emulsified without adjuvant, but PBS was used as a buffer. Three days later, the spleens of rabbits were removed for further study.

2-2 spleen cell separation

First, a sterilized operation was performed to obtain rabbit spleen, which was then placed in a sterilized cell culture dish. Spleens were rinsed with DPBS liquid containing 100U/ml penicillin and 100ug/ml streptomycin. Subsequently, the spleen was minced with surgical scissors, and the mixture was gently ground with a syringe core to convert the mixture into a single-cell state. Thereafter, the cell suspension was filtered with a 100 μm cell filter to collect single cell filtrate. Next, the filtrate was centrifuged at 1200rpm for 3 minutes, and the supernatant was discarded. Finally, cells were resuspended using RPMI-1640 medium containing 5% fetal bovine serum, ready for subsequent experiments.

B cell culture and identification

Memory B cells that bind to biotin-labeled MMP-9 were isolated by means of magnetic bead sorting using Anti-Biotin MicroBeads of methoprene by co-incubation with rabbit lymphocytes that have been successfully immunized with biotin-labeled MMP-9. The method comprises the following steps:

the spleen cells were resuspended in DMEM and the cells were filtered using a cell sieve to remove cell aggregates and adjust the cell density to 1×10 ⁸ Per ml, adding Biotinylated antigen, incubating for 1 hour on ice, re-suspending cells with DMEM after completion, centrifuging for 10min at 4 ℃, adding Anti-BiotinMicroBead, mixing well, incubating for 20min on ice, and eluting positive cells by a magnetic field. These B cells were then single-cell cultured in 96-well cell culture plates using a specific lymphocyte medium (supplied by Shandor Dinoxin Biotechnology Co., ltd.) at 37℃and 5% CO ₂ Is incubated under ambient conditions. After 14 days of incubation, the clone supernatants were assayed for binding activity at the protein level by ELISA. In the results, clones with binding activity 5 times or more greater than the background value were judged positive, and 64 positive clones were obtained in total.

2-4 cloning of Gene encoding Rabbit monoclonal antibody

B cell positive clones were collected, and two positive clones with the highest binding activity were selected, total RNA was extracted with RNAiso Plus and reverse transcribed into cDNA (condition: 45 ℃ C., 1 hour), and the light chain variable region and heavy chain variable region sequences were amplified by PCR method (condition: 95 ℃ denaturation-60 ℃ C., annealing-72 ℃ C., extension-75 ℃ C., termination, 30 cycles), and sequenced on PTT5 expression vectors containing the corresponding heavy chain constant region and light chain constant region, to obtain the correct sequences. Sequencing results were analyzed using VBASE2 (http:// www.vbase2.org/vbscAb. Php) to obtain antibody chrM22 and chrM37 light and heavy chain variable region sequences.

The relevant sequences of antibodies chrM22 and chrM37 are shown in tables 1 and 2.

TABLE 1 heavy and light chain sequences of antibody chrM22

TABLE 2 heavy and light chain sequences of antibody chrM37

2-5 expression purification of monoclonal antibodies

Collecting 293F cells in a logarithmic growth phase with good growth state, inoculating the 293F cells into a 250mL cell culture flask, adding 50mL culture medium for culture, and performing PEI cotransfection on 30 mug of light chain expression plasmid and heavy chain expression plasmid respectively, wherein the transfection process comprises the following steps: cells were seeded in 6-well plates one day prior to transfection, at the time of transfection

The cell density is 70% -80%. DNA-PEI nucleic acid-transfection reagent complexes were formulated using 100. Mu.L of serum-free medium. mu.L of DNA-PEI nucleic acid-PEI complex was added directly to the cells, the plates were shaken and gently mixed.

Cell supernatants from day 7 post-transfection cultures were collected, centrifuged and filtered using a 0.45 μm filter, protein a medium purified for antibodies and the antibodies were replaced by dialysis into PBS ph7.2 buffer. Antibody concentration and purity were determined by Nanodrop absorbance measurements and purity was checked by sodium dodecyl sulfate gel electrophoresis and coomassie staining.

The results are shown in Table 3:

TABLE 3 concentration and purity of the two antibodies

As can be seen from Table 3, antibodies chrysM 22 and chrysM 37 were both expressed, and the amount of chrysM 37 expressed was higher.

2-6 antibody affinity detection

Affinity detection of antibodies was performed with MMP-9 protein. Specifically, the antibodies were diluted to 10 μg/ml with 1 XKB buffer (0.05% BSA,0.02% Tween 20, PBS), 96well flat bottom plate,black (Greiner, cat No. 655209), 1 XKB buffer was added to dilute the target protein to 100nM, 1 XKB buffer was used as the baseline to subtract background, anti-hIgG Fc Capture (AHC) biosenstors (Sartorius, cat.18-5060), and the on-machine hoted 96 was tested for affinity constant KD, the results are shown in Table 4 and FIG. 2:

TABLE 4 results of binding kinetics test for two antibodies

Sample ID

Protein

Conc.(nM)

Response

KD(M)

Kon(1/Ms)

Kdis(l/s)

Full R^2

CLG4B

chrM22

100

0.3909

4.48E-08

4.38E+04

1.96E-03

0.9996

CLG4B

chrM37

100

0.6813

3.46E-09

4.78E+05

1.65E-03

0.9953

CLG4B

Bfr

100

0.0228

1.09E-08

1.75E+05

1.91E-03

0.7889

As can be seen from table 4 and fig. 2: in MMP-9 protein solution with concentration of 100nM, the binding force of chrM22 and chrM37 was measured, and dissociation constants (KD) were 4.48E-08 and 3.46E-09 moles (M), respectively; binding rates (Kon) were 4.38e+04 and 4.78e+05, respectively; the signal responses (responses) were monitored as 0.3909 and 0.6813, respectively, each higher than the negative control. Both chrM22 and chrM37 were shown to bind MMP-9, with the binding force of chrM37 being higher.

2-7 antibody epitope analysis

Antibody epitopes were analyzed by sandwick using the double anti-Sandwich epitope competition method:

one antibody (chrM 22) was immobilized on the sensor and then sequentially interacted with antigen and a second antibody (chrM 37), the second antibody binding signal was detected to determine if both antibodies recognized the same epitope. Specifically, the antibody is grabbed by an AHC sensor (the primary antibody is diluted to 15 μg/mL) to saturation, then a column of blocking antibodies is added (for saturation of the AHC sensor), then a buffer well is added, then 100nM target protein is grabbed, finally a secondary antibody diluted to 15 μg/mL is grabbed, the response signal of the secondary antibody is detected, and the obvious signal is the recognition of different epitopes (simple judgment standard, if the response >0.1nM, the signal is considered).

The results are shown in Table 5:

table 5. Results of analysis of binding epitope of chrM22 to chrM37

AB#	chrM22	chrM37
			chrM22	0.016	0.249
chrM37	0.168	-0.026

As can be seen from Table 5, the two antibodies, chrM22, are not associated with the binding epitope of chrM37 and can be used as antibody pairs in the detection and treatment of MMP-9 related diseases.

Finally, it should be noted that: the foregoing description is only of the preferred embodiments of the invention and is not intended to limit the scope of the invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An antibody pair specific for MMP-9, wherein said antibody pair comprises a first antibody chrM22 and a second antibody chrM37;

CDR1 as shown in SEQ ID No.01,

CDR2 as shown in SEQ ID No.02,

CDR3 as shown in SEQ ID No. 03;

CDR1 as shown in SEQ ID No.04,

CDR2 as shown in SEQ ID NO.05,

CDR3 as shown in SEQ ID No. 06;

the complementarity determining region CDRs of the heavy chain H37 of the second antibody chrM37 are as follows:

CDR1 as shown in SEQ ID NO.07,

CDR2 as shown in SEQ ID No.08,

CDR3 as shown in SEQ ID No. 09;

the complementarity determining region CDRs of light chain K37 of the second antibody chrM37 are as follows:

CDR1 as shown in SEQ ID No.10,

CDR2 as shown in SEQ ID No.11,

CDR3 as shown in SEQ ID No. 12.

2. The pair of antibodies specific for MMP-9 of claim 1, wherein the amino acid sequence of heavy chain H22 of the first antibody chrM22 is shown in SEQ ID No.13 and the amino acid sequence of light chain K22 of the first antibody chrM22 is shown in SEQ ID No. 14.

3. The pair of antibodies specific for MMP-9 of claim 1, wherein the amino acid sequence of heavy chain H37 of said second antibody chrM37 is shown in SEQ ID No.15 and the amino acid sequence of light chain K37 of said second antibody chrM37 is shown in SEQ ID No. 16.

4. A biological material associated with the antibody pair of any one of claims 1-3, wherein the biological material is any one of the following:

(b) An expression cassette comprising the nucleic acid molecule of (a);

5. The antibody pair-related biological material of claim 4, wherein the nucleic acid molecule comprises:

the base sequence of the light chain K22 of the first antibody chrM22 is shown as SEQ ID NO. 18.

6. The antibody pair-related biological material of claim 4, wherein the nucleic acid molecule comprises:

7. Use of an antibody according to any one of claims 1-3 for the preparation of a tumor suppressor, an inflammatory disease drug or an autoimmune disease drug.

8. Use of an antibody according to any one of claims 1-3 for the preparation of a detection reagent for matrix metalloproteinase 9.

9. Use of an antibody according to any one of claims 1-3 for the preparation of an inhibitor of matrix metalloproteinase 9 or a product that binds to matrix metalloproteinase 9.