CN117327186A - Bispecific antibodies that bind MMP3 proteins and uses thereof - Google Patents
Bispecific antibodies that bind MMP3 proteins and uses thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
- G01N2800/105—Osteoarthritis, e.g. cartilage alteration, hypertrophy of bone
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/24—Immunology or allergic disorders
Abstract
The invention discloses a bispecific antibody or a functional fragment thereof which binds to MMP3 protein, comprising: a first functional domain that specifically recognizes and binds a matrix metalloproteinase 3MMP3 protein, said first functional domain comprising an Fv region of an anti-MMP 3 specific antibody; and a second functional domain that specifically recognizes and binds to an MMP3 protein, the second functional domain comprising an scFv region of an anti-MMP 3 specific antibody. The invention also discloses application of the bispecific antibody of MMP3 protein in preparing medicines or kits for early screening, therapeutic effect monitoring and prognosis and recurrence risk assessment of rheumatoid arthritis. The MMP3 bispecific antibody provided by the invention has higher sensitivity and wider detection range, and can be used for different clinical diagnosis requirements.
Description
Technical Field
The invention relates to the technical field of immunology. More specifically, the invention relates to bispecific antibodies that bind to MMP3 proteins and uses thereof.
Background
Matrix metalloproteinase 3 (Matrix Metallopeptidase, MMP 3), also known as matrix lysin 1, is one of the major members of the matrix metalloproteinase family. MMP3, a member of matrix metalloproteinases, is able to degrade many of the components of the extracellular matrix and basement membrane, thereby participating in the development of tumors. MMPs play a very important role in the invasion and metastasis process of tumors. MMP is expressed in very little in normal steady state tissues, and its release, activation and inhibition are strictly controlled, but in tumor tissues homeostasis is broken, representing infiltration and metastasis of tumor cells. In the tumor infiltration process, tumor cells first react with basement membrane surface receptors such as fibronectin, then secrete MMP and other degrading enzymes to degrade the basement membrane and stroma, and finally tumor cells grow along the basement membrane defects and stroma voids.
Rheumatoid Arthritis (RA) is a common autoimmune disease that is mainly manifested by chronic inflammation of the synovium of the joints. While articular cartilage is irreversibly destroyed within 2 years of the onset of RA patients, MMP3 is considered as the most important protease causing cartilage degradation, and in RA early stage, the concentration of MMP3 in serum of RA patients is obviously higher than that of normal people, so that the method can be used as an important index for early diagnosis of RA. Meanwhile, the concentration of MMP3 in serum is closely related to cartilage injury and bone erosion, and the pathological progress of RA can be judged by grasping the dynamic change of the concentration of MMP 3.
Bispecific antibodies (Bispecific Antibodies, bsAb) are single molecule antibodies that have both antigen binding sites of two antibodies, and can bind to two different epitopes, either on one or on different antigens, whereas natural antibodies can recognize only one epitope. Bispecific antibodies are widely used in clinical diagnosis, imaging, disease prevention, and tumor immunotherapy. Currently, there are three main methods for the preparation of bispecific antibodies: somatic hybridoma (Somatic Hybridization), chemical coupling (Chemical Conjugation), and gene recombination (Recombinant Bispecific Antibody Molecules), which have been most rapidly developed and are most commonly used. Gene recombination methods are divided into four strategies: based on immunoglobulin variable regions alone, fusion of a second antigen binding site on IgG or IgG fragments, formation of heterodimers using asymmetric immunoglobulin constant regions and formation of heterodimer structures using non-immunoglobulin sequences.
The clinically usual detection methods for MMP3 are: enzyme-linked immunosorbent assay (ELISA), latex Particle Enhanced Turbidimetry (PETIA), latex agglutination assay (LTIA) and the like. The ELISA detection method needs to consume a great deal of labor cost, has long operation process time and cannot carry out high-flux sample detection. The use of PETIA or LTIA, while overcoming the problem of high throughput sample detection, currently available latex turbidimetric detection reagents in the market have low sensitivity to low value range regions, thereby affecting the result judgment.
Disclosure of Invention
It is an object of the present invention to address at least the above problems and/or disadvantages and to provide at least the advantages described below.
It is yet another object of the present invention to provide an MMP3 bispecific antibody and its use.
For this purpose, the technical scheme provided by the invention is as follows:
in a first aspect, a bispecific antibody or functional fragment thereof that binds an MMP3 protein, comprising:
a first functional domain that specifically recognizes and binds a matrix metalloproteinase 3MMP3 protein, said first functional domain comprising an Fv region of an anti-MMP 3 specific antibody; and
a second functional domain that specifically recognizes and binds to an MMP3 protein, the second functional domain comprising an scFv region of an anti-MMP 3 specific antibody.
Preferably, in the bispecific antibody or functional fragment thereof binding MMP3 protein, the amino acid sequences of the heavy chain variable regions VH CDR1, CDR2 and CDR3 of the first functional domain Fv are shown in SEQ ID NO. 1, 2 and 3, respectively, the amino acid sequences of the light chain variable regions VL CDR1, CDR2 and CDR3 of the first functional domain Fv are shown in SEQ ID NO. 4, 5 and 6, respectively,
the amino acid sequences of the VH CDR1, CDR2 and CDR3 of the second functional domain scFv are shown in SEQ ID NO. 7, 8 and 9 respectively, and the amino acid sequences of the VL CDR1, CDR2 and CDR3 of the light chain of the second functional domain scFv are shown in SEQ ID NO. 10, 11 and 12 respectively.
Preferably, in the bispecific antibody or the functional fragment thereof binding to MMP3 protein, the amino acid sequence of the heavy chain variable region VH of the first functional domain Fv is shown in SEQ ID NO. 13, and the amino acid sequence of the light chain variable region VL of the first functional domain Fv is shown in SEQ ID NO. 14;
the amino acid sequence of the heavy chain variable region VH of the second functional domain scFv is shown as SEQ ID NO. 15, and the amino acid sequence of the light chain variable region VL of the second functional domain scFv is shown as SEQ ID NO. 16.
Preferably, in the bispecific antibody or the functional fragment thereof which binds MMP3 protein, the amino acid sequence of the second functional domain scFv is shown as SEQ ID NO. 17,
linker intermediate the heavy chain variable region and the light chain variable region of the first domain and the second domain is (G) 4 S)n。
Preferably, in the bispecific antibody or functional fragment thereof that binds MMP3 protein, n is 3.
In a second aspect, a gene encoding a bispecific antibody or functional fragment thereof of any one of the MMP3 proteins.
In a third aspect, a recombinant vector comprising the gene.
In a fourth aspect, a composition for rapid diagnosis of rheumatoid arthritis comprising a bispecific antibody or functional fragment thereof of any one of the MMP3 proteins.
In a fifth aspect, the kit comprises a bispecific antibody or functional fragment thereof of any one of the MMP3 proteins, said kit being a latex-enhanced or chemiluminescent immunoassay rapid detection of MMP3 in vitro.
In a sixth aspect, the use of a bispecific antibody to an MMP3 protein of any of the claims in the manufacture of a medicament or kit for early screening, monitoring therapeutic efficacy and prognosis of rheumatoid arthritis and risk of recurrence.
The invention at least comprises the following beneficial effects:
according to the invention, two monoclonal antibodies with high affinity to MMP3 protein are screened out, then the MMP3 bispecific antibody is obtained through a genetic engineering means, and compared with two MMP3 monoclonal antibody combined kits, the kit containing the gene recombinant MMP3 bispecific antibody is higher in sensitivity and wider in detection range, and can be used for different clinical diagnosis requirements.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 is a schematic structural diagram of an anti-MMP 3 bispecific antibody according to an embodiment of the present invention.
FIG. 2 is a calibration graph of MMP3 assay kit prepared from MMP3 bispecific antibodies according to an embodiment of the present invention, prepared with calibrator dilutions of MMP3 antigen into a series of calibrators at concentrations of 0ng/mL,50ng/mL,150ng/mL,400ng/mL,1000ng/mL,2000ng/mL, respectively.
FIG. 3 is a graph showing the sample compliance rate of an immunonephelometric platform of an MMP3 antigen assay kit in accordance with an embodiment of the present invention.
Detailed Description
The present invention is described in further detail below to enable those skilled in the art to practice the invention by reference to the specification.
The invention is described below with reference to specific examples. As will be appreciated by those skilled in the art; the experimental methods in the following examples are all conventional methods unless otherwise specified; the raw materials, reagents, materials and the like used in the following examples were commercially available products unless otherwise specified.
The invention relates to an anti-MMP 3 bispecific antibody combined with matrix metalloproteinase 3 and application thereof, an MMP3 latex enhanced immunoturbidimetry detection kit for clinical diagnosis comprising the anti-MMP 3 bispecific antibody, a nucleic acid encoding the antibody or antigen binding fragment thereof, a carrier carrying the nucleic acid, and a method for preparing the MMP3 bispecific antibody or antigen binding fragment thereof by using the carrier and host cells.
The invention provides a bispecific antibody or a functional fragment thereof for binding MMP3 protein, which is formed by fusing a normal mouse IgG1kappa antibody with a scFv region of a second strain antibody at the C end of a heavy chain, and comprises the following components:
a first functional domain that specifically recognizes and binds a matrix metalloproteinase 3MMP3 protein, said first functional domain comprising an Fv region of an anti-MMP 3 specific antibody; and
a second functional domain that specifically recognizes and binds to an MMP3 protein, the second functional domain comprising an scFv region of an anti-MMP 3 specific antibody.
In one embodiment of the present invention, the amino acid sequences of the heavy chain variable regions VH CDR1, CDR2 and CDR3 of the first functional domain Fv are shown in SEQ ID NO. 1, 2 and 3, respectively, the amino acid sequences of the light chain variable regions VLCDR1, CDR2 and CDR3 of the first functional domain Fv are shown in SEQ ID NO. 4, 5 and 6, respectively,
the amino acid sequences of the VH CDR1, CDR2 and CDR3 of the second functional domain scFv are shown in SEQ ID NO. 7, 8 and 9 respectively, and the amino acid sequences of the VL CDR1, CDR2 and CDR3 of the light chain of the second functional domain scFv are shown in SEQ ID NO. 10, 11 and 12 respectively.
| Sequence number | Amino acid sequence |
| SEQ ID NO:1 | GFTFSSYT |
| SEQ ID NO:2 | IYPGDGST |
| SEQ ID NO:3 | ARDYYYGTFDV |
| SEQ ID NO:4 | KSISNKY |
| SEQ ID NO:5 | GTS |
| SEQ ID NO:6 | QQYNEYPYT |
| SEQ ID NO:7 | GYTFRNYW |
| SEQ ID NO:8 | ISSGGTST |
| SEQ ID NO:9 | TRRVGGYGYFDY |
| SEQ ID NO:10 | QNINVW |
| SEQ ID NO:11 | LTS |
| SEQ ID NO:12 | QQYSGYPHVYT |
In one technical scheme of the invention, the amino acid sequence of the heavy chain variable region VH of the first functional domain Fv is shown in SEQ ID NO. 13, and the amino acid sequence of the light chain variable region VL of the first functional domain Fv is shown in SEQ ID NO. 14;
SEQ ID NO. 13 as follows
DVQLQESGPGLVKPSQSLSLTCTVTGFTFSSYTWNWIRQFPGNKLEWMGYIYPGDGSTKYNEKFKGKATLTADTSSNTAYMQLNSLTSDNSAVYFCARDYYYGTFDVWGQGTTLTVSS
SEQ ID NO:14
QIVLSQSPPILSASPGEKVIMTCRASKSISNKYMHWYKQKPGSSPKPWIHGTSRLYSGVPSRFSGSGSGTDYSLTISNLKQEDFATYFCQQYNEYPYTFGAGTKLELK the amino acid sequence of the heavy chain variable region VH of the second functional domain scFv is shown in SEQ ID NO. 15, and the amino acid sequence of the light chain variable region VL of the second functional domain scFv is shown in SEQ ID NO. 16.
SEQ ID NO:15
QVQLKESGPGLVAPSQSLSITCTVSGYTFRNYWVGWVRQPPGKGLEWLGVISSGGTSTSYPDSVRGRFTISRDNAKSTLYLQMSSLRSDDTAIYYCTRRVGGYGYFDYWGQGTLVTVSA
SEQ ID NO:16
SIVMTQTPKFLLVSVGDRVTITCKASQNINVWVAWYQQKPGQSPKLLIYLTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIAIYYCQQYSGYPHVYTFGSGTKLEIK
In one of the technical schemes of the invention, in the bispecific antibody or the functional fragment thereof which binds MMP3 protein, the amino acid sequence of the second functional domain scFv is shown as SEQ ID NO. 17,
SEQ ID NO:17：
QVQLKESGPGLVAPSQSLSITCTVSGYTFRNYWVGWVRQPPGKGLEWLGVISSGGTSTSYPDSVRGRFTISRDNAKSTLYLQMSSLRSDDTAIYYCTRRVGGYGYFDYWGQGTLVTVSAGGGGSGGGGSGGGGSSIVMTQTPKFLLVSVGDRVTITCKASQNINVWVAWYQQKPGQSPKLLIYLTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIAIYYCQQYSGYPHVYTFGSGTKLEI
linker intermediate the heavy chain variable region and the light chain variable region of the first domain and the second domain is (G) 4 S)n。
In one embodiment of the invention, n is 3 in the bispecific antibody or functional fragment thereof that binds to MMP3 protein.
The invention also provides genes encoding bispecific antibodies or functional fragments thereof of any of the MMP3 proteins.
The invention also provides a recombinant vector comprising the gene.
The invention also provides a composition for rapid diagnosis of rheumatoid arthritis comprising a bispecific antibody or functional fragment thereof of any one of the MMP3 proteins.
The invention also provides a kit comprising the bispecific antibody or functional fragment thereof of any one of the MMP3 proteins, which is a kit for rapid in vitro detection of MMP3 by latex-enhanced or chemiluminescent immunoassay.
The invention also provides the application of the bispecific antibody of MMP3 protein in preparing medicines or kits for early screening, therapeutic effect monitoring, prognosis and recurrence risk assessment of rheumatoid arthritis.
In one embodiment of the invention, the invention provides a bispecific antibody, or variant thereof, or functional fragment thereof, that binds to an MMP3 protein, comprising:
a. a first functional domain that specifically recognizes and binds an MMP3 protein, comprising an Fv region of an anti-MMP 3 specific antibody; and
b. a second functional domain that specifically recognizes and binds to an MMP3 protein, comprising an scFv region of an anti-MMP 3 specific antibody.
Preferably, the functional domain that specifically recognizes and binds MMP3 protein consists of a normal-structure mouse IgG1kappa antibody fused to the scFv region of the second antibody at the C-terminus of the heavy chain.
Preferably, the anti-MMP 3 protein bispecific antibody, or variant or functional fragment thereof, is a chimeric, humanized or fully humanized antibody.
Preferably, the amino acid sequence of the heavy chain variable region that specifically recognizes and binds to the first domain of MMP3 protein is shown in SEQ ID NO. 13; the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 14.
Preferably, the amino acid sequence of the heavy chain variable region that specifically recognizes and binds to the second domain of MMP3 protein is shown in SEQ ID NO. 15; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 16; the complete sequence of the second domain is shown in SEQ ID NO. 17.
The bispecific antibodies, or variants thereof, or functional fragments thereof, of the present invention may be full length antibodies, or fusion proteins comprising only two antigen domain binding portions, such as single chain antibodies (scFv), or both.
One skilled in the art may replace, add and/or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids to the sequences of the invention to obtain variants of the sequences of the antibodies or functional fragments thereof without substantially affecting the affinity of the antibodies. They are all considered to be included within the scope of the present invention.
For a better understanding of the technical solution of the present invention, the following examples are now provided for illustration: example 1: expression and purification of recombinant protein human MMP3
Cloning human MMP3 gene sequence to pcDNA3.4 expression vector to construct eukaryotic expression plasmid, transiently converting the expression plasmid into HEK293 cell, and purifying through His tag affinity to obtain high purity MMP3 antigen protein. The purified eukaryotic MMP3 antigen protein is identified by SDS-PAGE electrophoresis to have correct protein band size, and the purity reaches more than 95 percent, and can be used for preparing monoclonal antibodies.
Example 2 acquisition of bispecific antibodies against human MMP3
Immunization of animals
1.5mg/ml of human MMP3 antigen (1 ml) was uniformly mixed with an equal volume of Freund's complete adjuvant and emulsified, and 50. Mu.g of antigen protein was injected into each mouse, followed by subcutaneous multipoint injection into 8 male BALB/C mice for 5-7 weeks. After 1 week, 1ml of MMP3 protein was homogeneously mixed with an equal volume of Freund's incomplete adjuvant and emulsified, and the mice were subjected to back subcutaneous multipoint injection. The above steps were repeated at week 3. The above steps were repeated at week 4. Three days after the fourth injection, 20. Mu.l of blood was collected from the broken rat tail tip, left standing at room temperature for 2.5 hours, centrifuged at 25℃and 12000rpm for 15 minutes, and the supernatant was collected and assayed for titer by ELISA. Mice with excellent tail blood titers were selected for cell fusion by isolating lymphocytes.
Hybridoma preparation
Mice were euthanized by dislocation, withSterilizing the whole mouse with 75% alcohol, aseptically collecting spleen cells of the mouse in a super clean bench, grinding to obtain cell suspension, and mixing 1×10 8 Spleen cells and 4X 10 7 Cell fusion of individual myeloma (SP 2/0), culturing in HAT selective medium for 5-10 days with 50% PEG as fusion agent, and culturing in HT medium under 5% CO 2 ，37℃。
Positive clone screening and epitope identification
Clone 2 strain of monoclonal hybridoma cells secreting antibodies that bind to MMP3 antigen was screened by ELISA reaction. The surface position is identified by a sandwich method, and the first antibody is captured and fixed on the surface of the ELISA plate. MMP3 antigen was then injected and bound to the first antibody. HRP-labeled secondary antibody was then reinjected. The second antibody either binds to the MMP3 antigen, creating a "sandwich" binding, or is blocked by the first antibody from binding to the MMP3 antigen. The results show that the second antibody can bind to MMP3 antigen in the presence of the first antibody, they have different epitopes and therefore belong to different bins and therefore are 2 different antibodies.
Example 3 structural design of bispecific antibodies that bind MMP3
The invention provides a structural model of a bispecific antibody capable of simultaneously and specifically binding two different epitopes of MMP3 protein, the structure of a main example of the antibody is shown in figure 1, and the anti-MMP 3 specific antibody consists of a normal mouse IgG1kappa antibody fused with a scFv region of a second antibody at the C end of a heavy chain.
EXAMPLE 4 construction of anti-MMP 3 bispecific antibody expression vectors
The complete light chain MMP3-LC coding gene is connected to pEE12.4 through HindIII/EcoRI cloning site to obtain pEE12.4-MMP3-LC; the complete heavy chain (MMP 3-HC-scFv) encoding gene was ligated to pEE6.4 through HindIII/EcoRI cloning site, resulting in pEE6.4-MMP3-HC-scFv; finally, the expression cassette obtained by double digestion of pEE6.4-MMP3-HC-scFv with BamHI/NotI is cloned into pEE12.4-MMP3-LC to obtain a single vector for expressing the anti-MMP 3 bispecific antibody, which is named pEE12.4-MMP3-BsAb.
Example 5 preparation of bispecific antibodies against MMP3
Expression of MMP3 bispecific antibodies using HEK293 host cells
HEK293 cell Density was diluted to 1X 10 6 Shake flask placed in 5% CO per ml 2 And culturing in a constant-temperature shaking table at 37 ℃ and 120rpm for 24 hours, and then carrying out transfection. Two 50ml sterile centrifuge tubes were prepared, 10ml PBS and 200. Mu.g sterile plasmid pEE12.4-MMP3-BsAb were added to one of the tubes, and gently mixed by blowing; then adding 1ml of transfection reagent PEI 40000, and gently beating and mixing; standing for 15 minutes at room temperature to prepare a plasmid-carrier compound; from CO 2 Taking out cells in a shaker, adding the prepared plasmid-carrier complex while shaking, and returning to CO 2 Shake culturing in a constant temperature shaking table. After 24 hours of transfection, 1.8ml of 293 cell protein expression enhancer VPA was added, and the amount of antibody expression was measured on day 6 after transfection.
Antibody purification
The cell culture was centrifuged at 5000rpm for 40min, and the supernatant was collected and filtered off with a 0.22 μm filter. Purifying with protein A affinity chromatographic column, desalting, replacing buffer solution, and packaging.
Example 6 comparison of performance of MMP3 bispecific antibodies and two MMP3 monoclonal antibodies in combination kits
MMP3 bispecific antibody kit calibrator configuration: diluting MMP3 antigen with calibrator buffer, and sequentially preparing calibrator concentration points: 0ng/mL,50ng/mL,150ng/mL,400ng/mL,1000ng/mL,2000ng/mL.
The detection principle of the MMP3 assay kit is as follows:
the kit adopts a latex enhanced immunoturbidimetry, and the basic principle is that an MMP3 bispecific antibody or two MMP3 monoclonal antibodies are crosslinked on latex particles, and undergo antigen-antibody combination reaction with MMP3 in a sample to be detected to form a large antigen-antibody complex, so that turbidity is increased, and MMP3 concentration is in a certain proportion with the formed turbidity. The MMP3 antigen content in the sample can be calculated by comparing the detected turbidity with a standard curve at 570nm wavelength, and the calibration curve is shown in figure 2.
Compared with two MMP3 monoclonal antibody combined kits, the MMP3 determination kit containing the MMP3 bispecific antibody has higher sensitivity and wider detection linearity, and can be used for different clinical diagnosis requirements.
The application method of the MMP3 antigen assay kit comprises the following steps:
MMP3 antigen kit is tested by using a full-automatic biochemical analyzer 7180, wherein parameters are as follows, 3 μl of sample is firstly added, then 150 μl of reagent R1 is added, incubation is carried out for 5min at 37 ℃,50 μl of reagent R2 is added, absorbance A1 is read after 5min, and the difference DeltaA=A1-A0 (initial value) of absorbance is calculated; and (3) performing multi-point calibration by using a matched calibrator to obtain a calibration curve, and performing linear fitting, wherein the concentration (unit ng/mL) of the sample can be calculated on the calibration curve through the detected absorbance difference value.
Example 7MMP3 antigen detection kit Performance
The performance of the antibody as a core raw material of an in vitro diagnostic reagent directly influences the quality of in vitro diagnostic reagent products. Sample compliance is one of the most important indicators to verify the performance of antibody raw materials, and is the most interesting part of in vitro diagnostic reagent companies when screening raw materials.
The sample compliance rate, which may also be referred to as a clinical contrast analysis, represents the degree of agreement between the measured value of the diagnostic kit for the clinically assigned sample and the theoretical value of the clinically assigned sample. The sample coincidence rate of a new antibody raw material is checked, an in-vitro diagnosis kit (a colloidal gold platform, an immune turbidimetry platform and the like) is generally prepared by taking the antibody as the raw material, and the kit is used for detecting an actual clinical specimen and carrying out statistics and analysis on a detection result so as to comprehensively check and evaluate. The performance of the antibody raw material can be indirectly deduced through the relationship between the detection result of the standard comparison research, analysis and summarization kit and the theoretical value of the clinical sample. The closer the results, the better the agreement, and the better the performance of the antibody.
MMP3 samples are 25 clinical samples assigned by a Siemens immunonephelometry platform collected from a three-dimensional hospital, and the concentrations of the clinical samples are respectively as follows: 6.6ng/ml, 6.7ng/ml, 8.7ng/ml, 8.9ng/ml, 9.2ng/ml, 17.3ng/ml, 19.1ng/ml, 21.1ng/ml, 33.4ng/ml, 50.6ng/ml, 163.7ng/ml, 226ng/ml, 401ng/ml, 508ng/ml, 698ng/ml, 791ng/ml, 943ng/ml, 1009ng/ml, 1157ng/ml, 1260.4ng/ml, 1493ng/ml, 1638ng/ml, 1742ng/ml, 1867ng/ml, 2135ng/ml.
Drawing a scatter diagram by taking an actual measurement value as an ordinate and a theoretical value as an abscissa, fitting a linear regression curve by a computer, and obtaining R 2 . Sample compliance R for MMP3 antigen detection kit as shown in fig. 3 2 >99％。
The number of modules and the scale of processing described herein are intended to simplify the description of the present invention. Applications, modifications and variations of the present invention will be readily apparent to those skilled in the art.
Although embodiments of the present invention have been disclosed above, it is not limited to the details and embodiments shown, it is well suited to various fields of use for which the invention is suited, and further modifications may be readily made by one skilled in the art, and the invention is therefore not to be limited to the particular details and examples shown and described herein, without departing from the general concepts defined by the claims and the equivalents thereof.
Claims (10)
1. A bispecific antibody or functional fragment thereof that binds to MMP3 protein, comprising:
a first functional domain that specifically recognizes and binds a matrix metalloproteinase 3MMP3 protein, said first functional domain comprising an Fv region of an anti-MMP 3 specific antibody; and
a second functional domain that specifically recognizes and binds to an MMP3 protein, the second functional domain comprising an scFv region of an anti-MMP 3 specific antibody.
2. The bispecific antibody or functional fragment thereof that binds MMP3 protein of claim 1, wherein the amino acid sequences of the heavy chain variable regions VH CDR1, CDR2 and CDR3 of the first functional domain Fv are set forth in SEQ ID NOS 1, 2 and 3, respectively, and the amino acid sequences of the light chain variable regions VL CDR1, CDR2 and CDR3 of the first functional domain Fv are set forth in SEQ ID NOS 4, 5 and 6, respectively,
the amino acid sequences of the VH CDR1, CDR2 and CDR3 of the second functional domain scFv are shown in SEQ ID NO. 7, 8 and 9 respectively, and the amino acid sequences of the VL CDR1, CDR2 and CDR3 of the light chain of the second functional domain scFv are shown in SEQ ID NO. 10, 11 and 12 respectively.
3. The bispecific antibody or functional fragment thereof that binds to MMP3 protein of claim 1, wherein the amino acid sequence of the heavy chain variable region VH of said first functional domain Fv is set forth in SEQ ID No. 13 and the amino acid sequence of the light chain variable region VL of said first functional domain Fv is set forth in SEQ ID No. 14;
the amino acid sequence of the heavy chain variable region VH of the second functional domain scFv is shown as SEQ ID NO. 15, and the amino acid sequence of the light chain variable region VL of the second functional domain scFv is shown as SEQ ID NO. 16.
4. The bispecific antibody or functional fragment thereof that binds MMP3 protein of claim 1, wherein the amino acid sequence of said second functional domain scFv is shown in SEQ ID NO. 17,
linker intermediate the heavy chain variable region and the light chain variable region of the first domain and the second domain is (G) 4 S)n。
5. The bispecific antibody or functional fragment thereof that binds to MMP3 protein of claim 3, wherein n is 3.
6. A gene encoding a bispecific antibody of the MMP3 protein of any of claims 1 to 5 or a functional fragment thereof.
7. A recombinant vector comprising the gene of claim 6.
8. A composition for rapid diagnosis of rheumatoid arthritis comprising a bispecific antibody or functional fragment thereof of the MMP3 protein of any of claims 1 to 5.
9. Kit comprising a bispecific antibody or a functional fragment thereof of an MMP3 protein according to any of claims 1 to 5, said kit being a kit for rapid in vitro detection of MMP3 by latex-enhanced or chemiluminescent immunoassay.
10. Use of a bispecific antibody to MMP3 protein according to any of claims 1 to 5 for the preparation of a medicament or kit for early screening, monitoring therapeutic efficacy and prognosis of rheumatoid arthritis and risk assessment of recurrence.
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| WO2013130078A1 (en) * | 2012-02-29 | 2013-09-06 | Gilead Biologics, Inc. | Antibodies to matrix metalloproteinase 9 |
| EP2985296A1 (en) * | 2014-08-13 | 2016-02-17 | Calypso Biotech SA | Antibodies specific for MMP9 |
| WO2023015170A2 (en) * | 2021-08-02 | 2023-02-09 | Tavotek Biotech (Suzhou) Ltd | Anti-cd38 antibodies, anti-cd3 antibodies, and bispecific antibodies, and uses thereof |
| CN115925963A (en) * | 2022-08-16 | 2023-04-07 | 北京达成生物科技有限公司 | Bispecific antibodies that bind HBP |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013130078A1 (en) * | 2012-02-29 | 2013-09-06 | Gilead Biologics, Inc. | Antibodies to matrix metalloproteinase 9 |
| EP2985296A1 (en) * | 2014-08-13 | 2016-02-17 | Calypso Biotech SA | Antibodies specific for MMP9 |
| WO2023015170A2 (en) * | 2021-08-02 | 2023-02-09 | Tavotek Biotech (Suzhou) Ltd | Anti-cd38 antibodies, anti-cd3 antibodies, and bispecific antibodies, and uses thereof |
| CN115925963A (en) * | 2022-08-16 | 2023-04-07 | 北京达成生物科技有限公司 | Bispecific antibodies that bind HBP |
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