CN119529089A

CN119529089A - Antibody against growth stimulation expression gene 2 protein and its application

Info

Publication number: CN119529089A
Application number: CN202411192571.XA
Authority: CN
Inventors: 钟冬梅; 孟媛; 梁碧; 唐丽娜; 曹慧方
Original assignee: Fapon Biotech Inc
Current assignee: Fapon Biotech Inc
Priority date: 2023-08-28
Filing date: 2024-08-27
Publication date: 2025-02-28

Abstract

本发明公开了一种抗生长刺激表达基因2蛋白的抗体及其应用，涉及抗体领域。本发明公开的抗生长刺激表达基因2蛋白抗体包括重链互补决定区和轻链互补决定区，该抗体为生长刺激表达基因2蛋白的检测提供了重要的原料来源，且具有良好的活性和灵敏度。The present invention discloses an antibody against growth stimulating expression gene 2 protein and its application, and relates to the field of antibodies. The antibody against growth stimulating expression gene 2 protein disclosed in the present invention comprises a heavy chain complementary determining region and a light chain complementary determining region, and the antibody provides an important raw material source for the detection of growth stimulating expression gene 2 protein and has good activity and sensitivity.

Description

Antibody for resisting growth stimulus expression gene 2 protein and application thereof

Cross Reference to Related Applications

The present disclosure claims priority from chinese patent application serial No. 202311095438.8, entitled "an antibody against growth-stimulating expressed gene 2 protein and its use" filed 28 at 2023, 08, the entire contents of which are incorporated herein by reference.

Technical Field

The invention relates to the technical field of antibodies, in particular to an anti-growth stimulation expression gene 2 protein antibody and application thereof.

Background

The growth stimulatory gene 2 protein (ST 2) is one of the members of the interleukin 1 (IL-1) receptor superfamily, also known as the T1, IL1RL1 or Fit1 gene and is located on chromosome 2q12 in humans, about 40kd, the transcription products of the ST2 gene have 4 subtypes, two of which are most important due to being regulated by different promoters, the first being transmembrane ST2, consisting of 3 extracellular immunoglobulin G domains, one transmembrane domain and one intracellular domain, being members of the interleukin-1 receptor family, and the other being soluble ST2, which is free flowing in the blood due to the lack of transmembrane and intracellular domains, so that it can be detected in serum.

Vascular Disease (CVD) is the leading cause of death worldwide, brain Natriuretic Peptide (BNP) and brain natriuretic peptide precursor NT-proBNP are the most well known markers of Heart Failure (HF), troponin markers improve the diagnosis of acute and chronic coronary artery disease, however, single biomarkers can only reflect a unilateral pathology of heart failure, but also be affected by pulmonary arterial hypertension, etc. due to renal function, age, BMI, etc., reducing the accuracy of these biomarkers. With the increasing awareness of ST2 acting on the cardiovascular system, one began to consider the assessment of plasma ST2 levels as a new marker of cardiovascular events, particularly of indices and clinical conditions associated with heart failure and ischemic heart disease, and ST2 was not affected by indices of renal function, age, body mass, etc. ST2 is also considered as a possible biomarker for patients with acute heart failure and associated asthma. Studies also prove that ST2 plays a role in predicting mortality, and the ST2 can be combined with other biomarkers to serve as a prognosis biomarker, can also be used for monitoring the pharmacological response of heart failure, has a correlation with the recommended therapeutic drugs for heart failure, and can provide reference for clinical selection of the therapeutic drugs and the scheme.

At present, the main methods for detecting the growth-stimulated expression gene 2 protein in China include an enzyme-linked immunosorbent assay (ELISA), a magnetic particle chemiluminescence method, a gold-labeled method and the like, and different detection methods all need antibodies aiming at the growth-stimulated expression gene 2 protein.

Thus, there is a strong need for anti-growth stimulation expressed gene 2 protein antibodies with good performance for those skilled in the art.

Disclosure of Invention

The application provides an antibody or antigen binding fragment thereof for resisting growth-stimulated expression gene 2 protein, which provides an important raw material source for detecting the growth-stimulated expression gene 2 protein and has good activity and sensitivity.

In order to achieve the above object, according to one aspect of the present invention, there is provided an antibody or antigen-binding fragment thereof for anti-growth stimulatory expression of a gene 2 protein, said antibody or antigen-binding fragment thereof comprising three complementarity determining regions having a heavy chain variable region set forth in any one of SEQ ID NOS: 22, 23, 24, 25 and three complementarity determining regions having a light chain variable region set forth in any one of SEQ ID NOS: 30, 31, 32.

In order to achieve the above object, according to a second aspect of the present invention, there is provided an antibody or an antigen-binding fragment thereof against a growth-stimulating expression gene 2 protein, the antibody or antigen-binding fragment thereof comprising the following complementarity determining regions:

HCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO. 1;

HCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID No. 2;

HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID No. 3, 17 or 18;

LCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO. 4;

LCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO. 5, and

LCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO. 6 or 19.

In order to achieve the above object, according to a third aspect of the present invention, there is provided an antibody or an antigen-binding fragment thereof against growth-stimulating expression of gene 2 protein, comprising a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NOS: 22, 23, 24, 25 and/or a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NOS: 30, 31, 32.

In order to achieve the above object, according to a fourth aspect of the present invention, there is provided an antibody or an antigen-binding fragment thereof for anti-growth-stimulating expression of gene 2 protein, comprising a heavy chain having an amino acid sequence as shown in any one of SEQ ID NOS 26, 27, 28, 29 and/or a light chain having an amino acid sequence as shown in any one of SEQ ID NOS 33, 34, 35.

In order to achieve the above object, according to a fifth aspect of the present invention, there is provided an antibody conjugate comprising the above antibody or antigen-binding fragment thereof.

In order to achieve the above object, according to a sixth aspect of the present invention, there is provided a reagent or kit comprising the above antibody or antigen-binding fragment thereof or the above antibody conjugate.

In order to achieve the above object, according to a seventh aspect of the present invention, there is provided a method for detecting a growth-stimulating expressed gene 2 protein, comprising a) contacting an antibody or antigen-binding fragment thereof, antibody conjugate, or reagent or kit as described above with a growth-stimulating expressed gene 2 protein in a sample to be detected under conditions sufficient for an antibody/antigen binding reaction to occur to form an immune complex, and b) detecting the presence of said immune complex, the presence of said complex being indicative of the presence of said antigen in said test sample.

In order to achieve the above object, according to an eighth aspect of the present invention, there is provided the use of the above antibody or antigen binding fragment, antibody conjugate, reagent or kit thereof for the preparation of a product for detecting growth-stimulating expression of gene 2 protein.

In order to achieve the above object, the present invention also provides a nucleic acid, a vector, a cell and a method for preparing the above antibody or antigen-binding fragment thereof.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the results of reducing SDS-PAGE of Anti-ST2 10F9 Rmb1 to Anti-ST2 10F9 Rmb8.

Detailed Description

In a first aspect, embodiments of the present invention provide an antibody or antigen-binding fragment thereof that is resistant to growth-stimulating expression of a gene 2 protein, the antibody or antigen-binding fragment thereof comprising three complementarity determining regions having a heavy chain variable region having amino acid sequences as set forth in any one of SEQ ID NOS: 22, 23, 24, 25 and three complementarity determining regions having a light chain variable region having amino acid sequences as set forth in any one of SEQ ID NOS: 30, 31, 32.

The HCDR1, HCDR2 and HCDR3 are amino acid sequences identical to the HCDR1, HCDR2 and HCDR3 of the same heavy chain variable region defined in the antibody or antigen-binding fragment thereof according to the first aspect, and the LCDR1, LCDR2 and LCDR3 are amino acid sequences identical to the LCDR1, LCDR2 and LCDR3 of the same light chain variable region defined in the antibody or antigen-binding fragment thereof according to the first aspect.

For example, the HCDR1, HCDR2 and HCDR3 are amino acid sequences identical to HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in SEQ ID NO. 22, and the LCDR1, LCDR2 and LCDR3 are amino acid sequences identical to LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in SEQ ID NO. 30.

In the present invention, the term "antibody" is used in the broadest sense and may include full length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies so long as they exhibit the desired biological activity.

In the present invention, the terms "complementarity determining regions", "CDRs" or "CDRs" refer to the highly variable regions of the heavy and light chains of immunoglobulins, and refer to regions comprising one or more or even all of the major amino acid residues responsible for the binding of an antibody or antigen-binding fragment to the antigen or epitope recognized by it. In a specific embodiment of the invention, CDRs refer to the highly variable regions of the heavy and light chains of the antibody.

In the present invention, the heavy chain complementarity determining region is represented by HCDR and includes HCDR1, HCDR2 and HCDR3, and the light chain complementarity determining region is represented by LCDR and includes LCDR1, LCDR2 and LCDR3.

CDR definition methods are well known in the art and include Kabat definition, chothia definition, IMGT definition, contact definition and AbM definition. As used herein, "Kabat definition" refers to the definition system described by Kabat et al, U.S. Dept. Of HEALTH AND Human Services, "Sequence of Proteins of Immunological Interest" (1983). "Chothia definition" see Chothia et al, J Mol Biol 196:901-917 (1987). Still other CDR definition methods may not strictly follow one of the above schemes, but still overlap at least a portion of the Kabat-defined CDR regions, although they may be shortened or lengthened depending on the predicted or experimental outcome of a particular residue or group of residues. Exemplary defined CDRs are listed in table 1 below, with slightly different definitions in the different documents. Given the variable region amino acid sequence of an antibody, one of skill in the art can routinely determine which residues comprise a particular CDR. It should be noted that CDRs defined by other methods not limited to table 1 are also within the scope of the disclosure.

TABLE 1 CDR definition ¹

¹ The numbering of all CDR definitions in Table 1 is according to the Kabat numbering system (see below), with the amino acid numbers on the heavy chain being indicated by "H+ numbers" and the amino acid numbers on the light chain being indicated by "L+ numbers". The Kabat numbering system can be specifically mapped to any variable region sequence by one of ordinary skill in the art without relying on any experimental data outside of the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described by Kabat et al, U.S. Dept. Of HEALTH AND HumanServices, "Sequence of Proteins ofImmunological Interest" (1983).

² The "AbM" as used in table 1 has a lower case "b" referring to CDRs defined by the "AbM" antibody modeling software of Oxford Molecular.

³ CDR-H1 ends at position 35 if both H35A and H35B are absent, CDR-H1 ends at position 35A if only H35A is present, and CDR-H1 ends at position 35B if both H35A and H35B are present.

⁴ CDR-H1 ends at bit 32 if both H35A and H35B are absent, at bit 33 if only H35A is present, and at bit 34 if both H35A and H35B are present.

⁵ CDR-H1 ends at position 33 if both H35A and H35B are absent, CDR-H1 ends at position 34 if only H35A is present, and CDR-H1 ends at position 35 if both H35A and H35B are present.

According to an embodiment of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one system or combination of systems Kabat, chothia, IMGT, abM or contacts.

In some alternative embodiments of the invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a Kabat system.

In some alternative embodiments of the invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a Chothia system.

In some alternative embodiments of the invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by an IMGT system.

In some alternative embodiments of the invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by an AbM system.

In some alternative embodiments of the invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a Contact system.

In some alternative embodiments of the invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by Kabat, chothia, IMGT, abM or Contact system combinations.

According to an embodiment of the present invention, the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 defined by the Kabat, chothia, abM or IMGT system correspond to the following Kabat numbering positions:

CDR	Kabat	AbM	IMGT	Chothia
					HCDR1	H31~H35A	H26~H35A	H26~H34	H26~H33
HCDR2	H50~H65	H50~H58	H51~H57	H52~H56
					HCDR3	H95~H102	H95~H102	H93~H102	H95~H102
LCDR1	L24~L34	L24~L34	L27~L32	L24~L34
					LCDR2	L50~L56	L50~L56	L50~L51	L50~L56
LCDR3	L89~L97	L89~L97	L89~L97	L89~L97

In a second aspect, embodiments of the present invention provide an antibody or antigen-binding fragment thereof that is resistant to growth-stimulating expression of a gene 2 protein, the antibody or antigen-binding fragment thereof comprising the complementarity determining regions:

According to an embodiment of the invention, HCDRs and LCDRs are defined by the Kabat system.

In alternative embodiments, the antibody or antigen binding fragment thereof comprises CDR sequences of any one of the following numbered combinations:

Numbering device

HCDR1

HCDR2

HCDR3

LCDR1

LCDR2

LCDR3

1

SEQ ID NO:1

SEQ ID NO:2

SEQ ID NO:3

SEQ ID NO:4

SEQ ID NO:5

SEQ ID NO:6

2

SEQ ID NO:1

SEQ ID NO:2

SEQ ID NO:17

SEQ ID NO:4

SEQ ID NO:5

SEQ ID NO:6

3

SEQ ID NO:1

SEQ ID NO:2

SEQ ID NO:18

SEQ ID NO:4

SEQ ID NO:5

SEQ ID NO:6

4

SEQ ID NO:1

SEQ ID NO:2

SEQ ID NO:3

SEQ ID NO:4

SEQ ID NO:5

SEQ ID NO:19

In the present invention, a "framework region" or "FR" region includes a heavy chain framework region and a light chain framework region, which refers to regions of an antibody heavy chain variable region and a light chain variable region other than CDRs, wherein the heavy chain framework region can be further subdivided into contiguous regions separated by CDRs, including HFR1, HFR2, HFR3, and HFR4 framework regions, and the light chain framework region can be further subdivided into contiguous regions separated by CDRs, including LFR1, LFR2, LFR3, and LFR4 framework regions.

In the present invention, the heavy chain variable region is obtained by ligating the CDRs numbered from HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4 with the FRs in a combinatorial arrangement, and the light chain variable region is obtained by ligating the CDRs numbered from LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4 with the FRs in a combinatorial arrangement.

In an alternative embodiment, the antibody or antigen binding fragment thereof of the first or second aspect further has at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR 4;

the HFR1 comprises/has an amino acid sequence as shown in SEQ ID NO 7 or having at least 80% identity thereto;

the HFR2 comprises/has an amino acid sequence as shown in SEQ ID NO 8 or having at least 80% identity thereto;

the HFR3 comprises/has as SEQ ID NO 9 or an amino acid sequence having at least 80% identity thereto;

the HFR4 comprises/has an amino acid sequence as shown in SEQ ID NO 10 or having at least 80% identity thereto;

the LFR1 comprises/is as SEQ ID No. 11 or an amino acid sequence having at least 80% identity thereto;

the LFR2 comprises/is as SEQ ID No. 12 or an amino acid sequence having at least 80% identity thereto;

the LFR3 comprises/is as SEQ ID No. 13 or an amino acid sequence having at least 80% identity thereto;

the LFR4 comprises/is as set forth in SEQ ID No. 14 or an amino acid sequence having at least 80% identity thereto.

In other embodiments, the amino acid sequence of each framework region of the antibody or antigen binding fragment thereof for anti-growth stimulatory expression of gene 2 protein provided by the present invention may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the corresponding framework region (SEQ ID NO:7, 8, 9, 10, 11, 12, 13 or 14) described above.

In an alternative embodiment, the HFR1 comprises/is the amino acid sequence shown in SEQ ID NO. 20.

In an alternative embodiment, the LFR3 comprises/is an amino acid sequence as shown in SEQ ID NO. 21.

In a third aspect, embodiments of the present invention provide an antibody or antigen-binding fragment thereof for anti-growth stimulatory expression of a gene 2 protein, comprising a heavy chain variable region having an amino acid sequence as set forth in any one of SEQ ID NOS 22, 23, 24, 25 and/or a light chain variable region having an amino acid sequence as set forth in any one of SEQ ID NOS 30, 31, 32.

In an alternative embodiment, the heavy chain variable region and the light chain variable region of the first aspect or the third aspect above are selected from any combination of:

Combination of two or more kinds of materials	Heavy chain variable region	Light chain variable region
			1	SEQ ID NO:22	SEQ ID NO:30
2	SEQ ID NO:23	SEQ ID NO:30
			3	SEQ ID NO:24	SEQ ID NO:30
4	SEQ ID NO:25	SEQ ID NO:30
			5	SEQ ID NO:22	SEQ ID NO:32
6	SEQ ID NO:22	SEQ ID NO:31
			7	SEQ ID NO:25	SEQ ID NO:32
8	SEQ ID NO:25	SEQ ID NO:31

。

In an alternative embodiment, the antibody or antigen binding fragment thereof of the first, second, or third aspects above further comprises a constant region.

In alternative embodiments, the constant region comprises a heavy chain constant region and/or a light chain constant region.

In alternative embodiments, the heavy chain constant region is selected from the group consisting of the heavy chain constant region of any one of IgG, igA, igM, igE, igD or a combination of multiple constant region segments.

In alternative embodiments, the heavy chain constant region comprises CH1 of IgG, hinge region of IgG, CH2 of IgM, CH3 of IgM, and/or CH4 of IgM.

In alternative embodiments, the IgG is selected from IgG1, igG2, igG3, or IgG4.

In alternative embodiments, the light chain constant region is selected from kappa-type or lambda-type light chain constant regions.

In alternative embodiments, the constant region is of a species derived from a cow, horse, cow, pig, sheep, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, chicken, or human.

In an alternative embodiment, the constant region is derived from rabbit species.

In an alternative embodiment, the heavy chain constant region sequence (CH) is shown in SEQ ID NO. 15 and the light chain constant region (CL) sequence is shown in SEQ ID NO. 16.

In other embodiments, the constant region sequence may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the constant region (SEQ ID NO:15 or 16) described above.

In alternative embodiments, the antigen binding fragment is selected from any one of F (ab) 2, F (ab ') 2, fab', fab, fv, and scFv of the antibody.

The antigen binding fragments of the above antibodies typically have the same binding specificity as the antibody from which they were derived. It will be readily appreciated by those skilled in the art from the teachings herein that antigen binding fragments of the above antibodies may be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by methods of chemical reduction cleavage of disulfide bonds. The antigen binding fragments described above are readily available to those skilled in the art based on the disclosure of the structure of the intact antibodies.

The antigen binding fragments of the above antibodies may also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, automated peptide synthesizers such as those sold by Applied BioSystems and the like.

In a fourth aspect, the invention provides an antibody or antigen binding fragment thereof for resisting growth stimulation expression of gene 2 protein, comprising a heavy chain and/or a light chain, wherein the amino acid sequence of the heavy chain is shown as any one of SEQ ID NO 26, 27, 28 and 29, and the amino acid sequence of the light chain is shown as any one of SEQ ID NO 33, 34 and 35.

In an alternative embodiment, the antibody or antigen binding fragment thereof of the first, second, third or fourth aspects above, comprises a heavy chain and a light chain in any one of the following combinations:

Combination of two or more kinds of materials	Heavy chain	Light chain
			1	SEQ ID NO:26	SEQ ID NO:33
2	SEQ ID NO:27	SEQ ID NO:33
			3	SEQ ID NO:28	SEQ ID NO:33
4	SEQ ID NO:29	SEQ ID NO:33
			5	SEQ ID NO:26	SEQ ID NO:35
6	SEQ ID NO:26	SEQ ID NO:34
			7	SEQ ID NO:29	SEQ ID NO:35
8	SEQ ID NO:29	SEQ ID NO:34

In a fifth aspect, the invention provides an antibody conjugate comprising an antibody or antigen binding fragment thereof as described above.

In an alternative embodiment, the above antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody or antigen binding fragment thereof.

In alternative embodiments, the antibody conjugate further comprises a label conjugated to the antibody or antigen binding fragment thereof.

In an alternative embodiment, the above-mentioned marker refers to a substance having a property such as luminescence, color development, radioactivity, etc., which can be directly observed by naked eyes or detected by an instrument, by which qualitative or quantitative detection of the corresponding target can be achieved.

In alternative embodiments, the labels include, but are not limited to, fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

In the actual use process, a person skilled in the art can select a suitable marker according to the detection conditions or actual needs, and no matter what marker is used, the marker belongs to the protection scope of the invention.

In alternative embodiments, the fluorescent dyes include, but are not limited to, fluorescein-based dyes and derivatives thereof (including, but not limited to, fluorescein Isothiocyanate (FITC) hydroxy-light (FAM), tetrachlorolight (TET), and the like, or analogs thereof), rhodamine-based dyes and derivatives thereof (including, but not limited to, red Rhodamine (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), and the like, or analogs thereof), cy-based dyes and derivatives thereof (including, but not limited to, cy2, cy3B, cy3.5, cy5, cy5.5, cy3, and the like, or analogs thereof), alexa-based dyes and derivatives thereof (including, but not limited to, alexa fluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, and the like, or analogs thereof), and protein-based dyes and derivatives thereof (including, but not limited to, for example, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polyazosin (preCP), and the like).

In alternative embodiments, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose 6-phosphate deoxygenase.

In alternative embodiments, the radioisotope includes, but is not limited to 212Bi、131I、111In、90Y、186Re、211At、125I、188Re、153Sm、213Bi、32P、94mTc、99mTc、203Pb、67Ga、68Ga、43Sc、47Sc、110mIn、97Ru、62Cu、64Cu、67Cu、68Cu、86Y、88Y、121Sn、161Tb、166Ho、105Rh、177Lu、172Lu and 18F.

In alternative embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and its derivatives, dioxane and its derivatives, lomustine and its derivatives, and peroxyoxalate and its derivatives.

In alternative embodiments, the nanoparticle-based labels include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

In alternative embodiments, the colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

In alternative embodiments, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

In an alternative embodiment, the colloidal metal is colloidal gold.

In an alternative embodiment, the above antibody conjugate further comprises a solid support coupled to the antibody or antigen binding fragment thereof.

In alternative embodiments, the solid support includes, but is not limited to, magnetic microspheres, plastic microparticles, microplates, glass, capillaries, nylon, and nitrocellulose membranes.

In a sixth aspect, the invention provides a reagent or kit comprising an antibody or antigen binding fragment thereof as described above or an antibody conjugate as described above.

As previously described, the antibodies or antigen-binding fragments thereof of some embodiments or examples of the invention are effective to bind to a growth-stimulatory expressed gene 2 protein, and therefore, reagents or kits comprising the growth-stimulatory expressed gene 2 protein antibodies or antigen-binding fragments thereof are effective to perform qualitative or quantitative detection of the growth-stimulatory expressed gene 2 protein. The reagent or the kit provided by the invention can be used for detecting the specific binding performance of the gene 2 protein expressed by growth stimulation and the antibody thereof, such as immunoblotting, immunoprecipitation and the like. As previously mentioned, the antibodies or antigen binding fragments thereof in some embodiments or examples of the invention have a higher binding activity or affinity to the growth-stimulating expressed gene 2 protein, and therefore the reagents or kits comprising the antibodies or antigen binding fragments thereof have a higher detection sensitivity or specificity.

In a seventh aspect, the invention provides a method of detecting a growth-stimulating expressed gene 2 protein comprising a) contacting an antibody or antigen-binding fragment, antibody conjugate, reagent or kit as described above with a growth-stimulating expressed gene 2 protein in a sample to be detected under conditions sufficient for an antibody/antigen binding reaction to occur to form an immune complex, and b) detecting the presence of the immune complex, the presence of the complex being indicative of the presence of the antigen in the test sample;

In an alternative embodiment, the immune complex further comprises a second antibody that binds to the antibody or antigen binding fragment thereof.

In an alternative embodiment, the immune complex further comprises a second antibody that binds to a growth-stimulating expressed gene 2 protein.

In an eighth aspect, the present invention provides the use of an anti-growth-stimulatory gene 2 protein antibody or antigen binding fragment thereof, an antibody conjugate as described above or a reagent or kit as described above for the preparation of a product for detecting growth-stimulatory expressed gene 2 protein.

In a ninth aspect, the present invention provides a nucleic acid molecule encoding an antibody or antigen binding fragment thereof as described above.

In a tenth aspect, the present invention provides a vector comprising the nucleic acid molecule described above.

In an eleventh aspect, the present invention provides a cell comprising the vector described above.

In a twelfth aspect, the present invention provides a method for preparing an anti-growth stimulatory gene 2 protein antibody or antigen binding fragment thereof, comprising culturing a cell as described above.

Based on the present invention discloses the amino acid sequence of the anti-growth stimulatory expressed gene 2 protein antibody or antigen binding fragment thereof, those skilled in the art will readily recognize that the anti-growth stimulatory expressed gene 2 protein antibody or antigen binding fragment thereof may be prepared by genetic engineering techniques or other techniques (chemical synthesis, recombinant expression), e.g., isolated and purified from a culture product of recombinant cells capable of recombinantly expressing the antibody or antigen binding fragment thereof as described in any of the above, which are readily accomplished by those skilled in the art, and therefore, whatever technique is used to prepare the anti-growth stimulatory expressed gene 2 protein antibody or antigen binding fragment thereof of the present invention, are within the scope of the present invention.

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of formulations or unit doses herein, some methods and materials are now described. Unless otherwise indicated, techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not intended to be limiting.

Unless otherwise indicated, practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ability of a person skilled in the art. Such techniques are well explained in the literature, e.g., in the molecular cloning laboratory Manual (Molecular Cloning: ALaboratory Manual), second edition (Sambrook et al, 1989), oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait et al, 1984), animal cell Culture (ANIMAL CELL Culture) (R.I. Freshney, 1987), enzymatic methods (Methods in Enzymology) (academic Press Co., ltd. (ACADEMIC PRESS, inc.), experimental immunology Manual (Handbook of Experimental Immunology) (D.M.Weir and C.Blackwell, inc.), mammalian cell gene transfer Vectors (GENE TRANSFER Vectors for MAMMALIAN CELLS) (J.M.Miller and M.P.Calos, 1987), contemporary molecular biology methods (F.M.Ausubel et al, 1987), polymerase chain reactions (28) (J.M.Weir. And C.Blackwell, inc.), PCR methods (J.34.J.37, J.J.37, J.F.37) and PCR methods (J.37, J.F.37, J.J.F.37, J.J.J.J.F.37, J.J.J.J.J.J.J.J.F.J.J.J.J.J.F.J.J.J.L).

The features and capabilities of the present invention are described in further detail below in connection with the examples.

Example 1 antibody preparation

1. Rabbit monoclonal antibody discovery against ST2 recombinant proteins

The rabbit monoclonal antibody of the anti-ST 2 recombinant protein can obtain an antibody sequence by a hybridoma technology, a phage display technology or a single B cell cloning technology, and the specific implementation method of the phage display technology is as follows:

(1) Immunization of rabbits

The emulsified preparation of ST2 recombinant protein prepared by incomplete Freund's adjuvant is used for stimulating 4-6 week old New Zealand white rabbits by subcutaneous injection to generate immune response, collecting serum before and after immunization on 0,14,28,42 and 69 days respectively for potency detection, selecting rabbits with proper potency, picking spleen of the rabbits, and preparing spleen cell suspension.

(2) Construction of phage libraries

RNA in rabbit spleen cells is extracted and reversely transcribed into cDNA, a specially designed rabbit antibody gene amplification primer is adopted, the cDNA is used as a template to amplify VH and VL gene fragments respectively, and then the VH and VL gene fragments are sequentially inserted and connected into a phage vector V02 in an enzyme digestion connection mode. Finally, electrically transforming the connected phage plasmid into TG1 competent cells, selecting monoclonal colony for PCR identification the next day, performing antibody gene sequencing, evaluating the quality of phage library, and screening phage library after passing.

(3) Screening of phage libraries

Phage library TG1 bacterial liquid is inoculated into a shake flask, when the bacterial liquid is cultured to a proper bacterial liquid concentration (OD 600 is 0.8-1.0), auxiliary phage is added for infection for 1 hour, and then the culture is continued overnight. And collecting bacterial liquid in the next day, centrifuging, taking supernatant, and purifying by adopting a salting-out precipitation method to obtain the displayed phage library. And (3) panning the phage library for 3-4 rounds by adopting a magnetic bead panning method, then selecting a monoclonal phage infection colony to express an antibody supernatant, and then screening and identifying the monoclonal phage antibody expression supernatant by adopting an ELISA screening method to obtain the monoclonal phage resisting the ST2 recombinant protein.

(4) Phage antibody gene sequencing:

and (3) carrying out antibody gene sequencing on the monoclonal phage resisting the ST2 recombinant protein, and removing repeated and invalid sequences through sequence analysis to obtain a unique rabbit monoclonal antibody sequence. And (5) carrying out recombinant expression verification on the obtained sequence.

2. Construction of expression plasmid

pcDNA^TM3.4Vector is constructed eukaryotic expression vector of recombinant antibody, which is modified to introduce polyclonal enzyme cutting site, 3.4A expression vector, VL and VH gene specific primers are designed separately based on the obtained variable region gene, and the two ends have restriction enzyme cutting site and protecting base, and the LIGHT CHAIN gene segment of 0.71Kb and the HEAVY CHAIN gene segment of 1.41Kb are amplified via PCR amplification process.

The HEAVY CHAIN and LIGHT CHAIN gene fragments are respectively subjected to double enzyme digestion by adopting restriction enzymes, the 3.4A vector is subjected to double enzyme digestion by adopting restriction enzymes, and the HEAVY CHAIN gene and LIGHT CHAIN gene after the fragments and the vector are purified and recovered are respectively connected with the 3.4A expression vector to respectively obtain recombinant expression plasmids HEAVY CHAIN and LIGHT CHAIN.

3. Sample preparation of recombinant antibodies

Recovering HEK293 cells in advance, subculturing to a 200ml system to enable the cell density to reach 3-5×10 ⁶ cells/ml and the cell activity to be more than 95%, centrifugally cleaning the cells, re-dissolving the cells by using a culture medium, and adjusting the cell density to 2.9×10 ⁶ cells/ml at the same time to be used as a cell diluent. The medium was used to prepare dilutions of plasmid DNA and transfection reagent, respectively. Adding transfection reagent diluent into plasmid DNA diluent, mixing uniformly, standing at room temperature for 15min, slowly adding the mixture into cell diluent within 1min, mixing uniformly, sampling, counting, recording and observing activity of transfected cells, culturing in a 35 ℃ constant temperature incubator at a rotating speed 120rmp and a CO ₂ content of 8%, and centrifuging for 13 days to collect samples. The supernatant was affinity purified using a proteona affinity column. 6. Mu.g of purified antibody was subjected to reducing SDS-PAGE, and the electrophoresed pattern was as shown. Two bands were shown after reducing SDS-PAGE, 1 Mr was 50KD (heavy chain) and the other Mr was 28KD (light chain).

The obtained antibody was designated as Anti-ST2 10F9Rmb1, and the Anti-ST2 10F9Rmb1 was subjected to mutation to obtain a mutant antibody, the sequences of the heavy chain (H) and the light chain (L) of which are shown in the following table:

TABLE 2 antibody sequences

Antibody name	Heavy chain	Light chain
			Anti-ST2 10F9Rmb1	SEQ ID NO:26	SEQ ID NO:33
Anti-ST2 10F9Rmb2	SEQ ID NO:27	SEQ ID NO:33
			Anti-ST2 10F9Rmb3	SEQ ID NO:28	SEQ ID NO:33
Anti-ST2 10F9Rmb4	SEQ ID NO:29	SEQ ID NO:33
			Anti-ST2 10F9Rmb5	SEQ ID NO:26	SEQ ID NO:35
Anti-ST2 10F9Rmb6	SEQ ID NO:26	SEQ ID NO:34
			Anti-ST2 10F9Rmb7	SEQ ID NO:29	SEQ ID NO:35
Anti-ST2 10F9Rmb8	SEQ ID NO:29	SEQ ID NO:34

Example 2 detection of Performance of antibodies

1. Activity assay

The coating solution (main component NaHCO ₃) diluted ST2 recombinant antigen (from Phpeng organism) to 1ug/ml, 100uL per well, 4 ℃ overnight, the next day, washing solution (main component Na ₂HPO₄ +NaCl) washed 2 times, beating dry, adding blocking solution (20% BSA+80% PBS) per well 120uL,37 ℃ for 1h, beating dry, adding diluted purified and control antibodies, 100uL per well, 37 ℃ for 30min, washing solution washed 5 times, beating dry, adding goat anti-rabbit IgG-HRP, 100uL per well, 37 ℃ for 30min, washing solution washed 5 times, beating dry, adding developing solution A (50 uL per well), adding developing solution B (50 uL per well), 10min, adding stop solution, 50uL per well, reading OD value at 450nm (reference 630 nm) on an microplate reader.

Remarks are liquid A (main component citric acid+sodium acetate+acetanilide+carbamide peroxide), liquid B (main component citric acid+EDTA.2Na+TMB+concentrated HCl), and stop solution (EDTA.2Na+concentrated H ₂SO₄)

TABLE 3 Activity data

Concentration (ng/ml)	3.13	1.56	0.78	0.39	0.2	0
							Control	1.203	0.834	0.419	0.292	0.039	0.012
Anti-ST2 10F9Rmb1	1.717	1.137	0.716	0.406	0.239	0.016
							Anti-ST2 10F9Rmb2	1.687	1.189	0.762	0.427	0.245	0.017
Anti-ST2 10F9Rmb3	1.498	0.934	0.598	0.325	0.194	0.011
							Anti-ST2 10F9Rmb4	1.647	1.086	0.686	0.385	0.24	0.018
Anti-ST2 10F9Rmb5	1.566	0.998	0.633	0.357	0.212	0.016
							Anti-ST2 10F9Rmb6	1.613	1.03	0.671	0.369	0.211	0.008
Anti-ST2 10F9Rmb7	1.745	1.201	0.752	0.424	0.262	0.019
							Anti-ST2 10F9Rmb8	1.757	1.071	0.641	0.492	0.275	0.018

2. Stability assessment

Placing the antibody at 4 ℃, -80 ℃ (refrigerator) and 37 ℃ (incubator) for 21 days, taking 7 days, 14 days and 21 days for state observation, and detecting the activity of the 21 days, wherein the results show that no obvious protein state change is seen in the antibody placed for 21 days under three examination conditions, and the activity is not in a descending trend along with the increase of the examination temperature, thus indicating that the antibody is stable. Table 5 below shows the results of the detection of OD by the antibody Anti-ST2 10F9Rmb7 on evaluation of the enzyme-free activity for 21 days.

TABLE 4 stability data

Sample concentration (ng/ml)	1.56	0.78	0.00
				4 ℃ 21 Day sample	1.271	0.779	0.011
-80 ℃,21 Day sample	1.238	0.735	0.011
				37 ℃ 21 Day sample	1.226	0.748	0.012

3. Chemiluminescent platform performance detection

3.1 Antibody coating procedure

10Mg/mL carboxyl magnetic beads, MES buffer was washed 3 times. The MES buffer was used to resuspend the beads, EDC was added at a final concentration of 1mg/mL, and the mixture was mixed on a 25℃shaker and reacted for 30min. The MES buffer was used to resuspend the beads, antibody Anti-ST2-A (from the Phpeng organism) was added at a final concentration of 0.2mg/mL, and the solution was used as the working solution for the magnetic particles, mixed well by shaking at 25℃and reacted for 120min. And storing the mixture in a Tris buffer solution at a temperature of 2-8 ℃.

3.2 Antibody labelling procedure

3Mg/ml of antibody Anti-ST2 10F9Rmb1、Anti-ST2 10F9Rmb2、Anti-ST2 10F9Rmb3、Anti-ST210F9Rmb4、Anti-ST2 10F9Rmb5、Anti-ST2 10F9Rmb6、Anti-ST2 10F9Rmb7、Anti-ST2 10F9Rmb8、 control antibody Anti-ST2-B was replaced with Zeba desalting column (10K MWCO) to PBS (100 mM PB,50mM sodium chloride, pH 8.0), respectively. Acridinium esters were formulated in 4mM solution in DMSO. 10eq of acridine ester was added to each of the Anti-cTnI-B, anti-cTnI-11F13-Rmb antibody solutions and reacted at 25℃for 2 hours. Desalting to remove excessive reagent, and taking the acridinium ester modified antibody as an acridinium ester working solution, and storing at 4 ℃ for standby.

3.3 Detection procedure

The detection is carried out on a full-automatic chemiluminescence immunoassay instrument of Kaishan i2910 model, a double-antibody sandwich method is adopted, namely, 20 mu L of sample (quality control product containing ST2 with different concentrations), 50 mu L of magnetic particle working solution and 50 mu L of acridine ester working solution are sequentially added into the instrument, the mixture is uniformly mixed and incubated for 10 minutes, the reaction mixture is washed after incubation, and a pre-excitation solution and an excitation solution are added to detect the relative luminous intensity (RLU).

3.4 Detection results

The detection data of the calibrants with different concentrations are shown in Table 5, and the results show that the detection binding activity and the sensitivity of the antibodies Anti-ST210F9Rmb 1-Anti-ST 210F9Rmb are superior to those of the control antibody on the chemiluminescent platform.

TABLE 5 chemiluminescent platform Performance data

The partial amino acid sequences involved in the present application are shown in Table 6:

TABLE 6 amino acid sequence listing

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. 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 or antigen-binding fragment thereof that is resistant to growth stimulation of expression of a gene 2 protein, said antibody or antigen-binding fragment thereof comprising three complementarity determining regions having a heavy chain variable region having an amino acid sequence set forth in any one of SEQ ID NOS: 22, 23, 24, 25 and three complementarity determining regions having a light chain variable region having an amino acid sequence set forth in any one of SEQ ID NOS: 30, 31, 32.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the complementarity determining regions of the variable regions are defined by any one or a combination of systems Kabat, chothia, IMGT, abM or Contact.

3. An antibody or antigen-binding fragment thereof that is resistant to growth stimulation of expression of a gene 2 protein, wherein the antibody or antigen-binding fragment thereof comprises the following complementarity determining regions:

LCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO. 6 or 19;

Optionally, the antibody or antigen binding fragment thereof comprises CDR sequences of any one of the following numbered combinations:

Numbering device HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 1 SEQ ID NO:1 SEQ ID NO:2 SEQ ID NO:3 SEQ ID NO:4 SEQ ID NO:5 SEQ ID NO:6 2 SEQ ID NO:1 SEQ ID NO:2 SEQ ID NO:17 SEQ ID NO:4 SEQ ID NO:5 SEQ ID NO:6 3 SEQ ID NO:1 SEQ ID NO:2 SEQ ID NO:18 SEQ ID NO:4 SEQ ID NO:5 SEQ ID NO:6 4 SEQ ID NO:1 SEQ ID NO:2 SEQ ID NO:3 SEQ ID NO:4 SEQ ID NO:5 SEQ ID NO:19

;

Optionally, the antibody or antigen binding fragment thereof further has at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR 4;

The HFR1 comprises the amino acid sequence of SEQ ID NO. 7 or at least 80% identity thereto;

The HFR2 comprises the amino acid sequence of SEQ ID NO 8 or at least 80% identity thereto;

the HFR3 comprises the amino acid sequence of SEQ ID NO 9 or at least 80% identity thereto;

the HFR4 comprises the amino acid sequence of SEQ ID NO 10 or at least 80% identity thereto;

the LFR1 comprises SEQ ID No. 11 or an amino acid sequence having at least 80% identity thereto;

The LFR2 comprises the amino acid sequence of SEQ ID No. 12 or at least 80% identity thereto;

the LFR3 comprises SEQ ID No. 13 or an amino acid sequence having at least 80% identity thereto;

the LFR4 comprises the amino acid sequence of SEQ ID No. 14 or at least 80% identity thereto.

4. An anti-growth stimulation expression gene 2 protein antibody or antigen binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, and is characterized in that the amino acid sequence of the heavy chain variable region is shown as any one of SEQ ID NO. 22, 23, 24 and 25, and the amino acid sequence of the light chain variable region is shown as any one of SEQ ID NO. 30, 31 and 32;

alternatively, the combination of heavy chain variable region and light chain variable region is selected from any one of the following combinations:

;

Optionally, the antibody or antigen binding fragment thereof further comprises a constant region;

Optionally, the constant region comprises a heavy chain constant region and/or a light chain constant region;

alternatively, the heavy chain constant region is selected from the heavy chain constant region of any one of IgG, igA, igM, igE, igD or a combination of multiple constant region segments;

optionally, the heavy chain constant region comprises CH1 of IgG, hinge region of IgG, CH2 of IgM, CH3 of IgM, and/or CH4 of IgM;

Alternatively, the constant region is of bovine, equine, porcine, ovine, caprine, rat, mouse, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose, or human origin;

optionally, the constant region is derived from a species of rabbit;

Alternatively, the heavy chain constant region sequence is as shown in SEQ ID NO. 15 or at least 80% identical thereto;

alternatively, the light chain constant region sequence is as shown in SEQ ID NO. 16 or at least 80% identical thereto;

Alternatively, the antigen binding fragment is selected from any one of F (ab ') 2, fab', fab, fv, and scFv of the antibody.

5. An anti-growth stimulation expression gene 2 protein antibody or antigen binding fragment thereof comprises a heavy chain and/or a light chain, and is characterized in that the amino acid sequence of the heavy chain is shown as any one of SEQ ID NO 26, 27, 28 and 29, and the amino acid sequence of the light chain is shown as any one of SEQ ID NO 33, 34 and 35.

6. An antibody conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 5;

optionally, the antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody or antigen binding fragment thereof;

optionally, the antibody conjugate further comprises a label conjugated to the antibody or antigen binding fragment thereof;

optionally, the label is selected from the group consisting of fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels;

Optionally, the antibody conjugate further comprises a solid support coupled to the antibody or antigen binding fragment thereof.

7. A reagent or kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 5 or the antibody conjugate of claim 6.

8. A method for detecting growth-stimulated expression of a gene 2 protein, comprising:

a) Contacting the antibody or antigen binding fragment thereof of any one of claims 1-5, the antibody conjugate of claim 6, or the reagent or kit of claim 7 with a growth-stimulating expressed gene 2 protein in a sample to be tested under conditions sufficient for an antibody/antigen binding reaction to form an immune complex, and

B) Detecting the presence of the immune complex, the presence of the complex being indicative of the presence of the antigen in the test sample;

optionally, the immune complex further comprises a second antibody that binds to the antibody or antigen binding fragment thereof;

Optionally, the immune complex further comprises a second antibody that binds to a growth-stimulating expressed gene 2 protein.

9. Use of the antibody or antigen binding fragment thereof of any one of claims 1-5, the antibody conjugate of claim 6, or the reagent or kit of claim 7 in the preparation of a product for detecting growth-stimulating expression of gene 2 protein.

10. A nucleic acid encoding the antibody or antigen binding fragment thereof according to any one of claims 1 to 5, a vector comprising a nucleic acid encoding the antibody or antigen binding fragment thereof according to any one of claims 1 to 5, a cell comprising the nucleic acid or vector, or a method of producing the antibody or antigen binding fragment thereof according to any one of claims 1 to 5.