CN117069851A

CN117069851A - anti-CK-MM antibodies or functional fragments thereof and uses thereof

Info

Publication number: CN117069851A
Application number: CN202311018160.4A
Authority: CN
Inventors: 边颖; 姬成东; 罗嘉; 吕秉翠; 汤定斌; 赵忠颢; 许路来
Original assignee: Chongqing Aishengsi Bioengineering Co ltd
Current assignee: Chongqing Aishengsi Bioengineering Co ltd
Priority date: 2023-08-14
Filing date: 2023-08-14
Publication date: 2023-11-17

Abstract

The invention relates to the field of biotechnology, in particular to an anti-CK-MM antibody or a functional fragment thereof and application thereof, and discloses an anti-CK-MM antibody or a functional fragment thereof which has better binding specificity and affinity to M subunits in natural CK-MB and does not cross B subunits, has a good detection effect on the rise of CK-MB in clinical samples, and provides more choices for immunodetection by making a plurality of effective mutations.

Description

anti-CK-MM antibodies or functional fragments thereof and uses thereof

Technical Field

The invention relates to the field of biotechnology, in particular to an anti-CK-MM antibody or a functional fragment thereof and application thereof.

Background

Acute Myocardial Infarction (AMI) is one of the most common causes of death worldwide. About 10% of patients who seek medical attention in the emergency room due to chest pain are diagnosed with heart attacks. (Does the patient with chest pain have a coronary heart diseaseDiagnostic value of single symptoms and signs- -a meta-analysis. Croat Med J.2012 Oct;53 (5): 432-41.). The overall risk assessment of chest pain patients to eliminate adverse outcomes is significant for reducing morbidity and mortality, improving the quality of life of the patient, and reducing national health expenditures. Acute myocardial infarction may be caused by heart ischemic disease and coronary artery disease. Myocardial infarction conditions occur when the atherosclerotic plaque ruptures fragments or thrombus partially or totally obstructs the coronary arteries, resulting in limited blood supply to the heart. Diagnostic criteria for acute myocardial infarction were established by the European cardiology Association (ESC) and the American cardiology Association (ACC). (Thygesen K, alpert JS, jaffe AS, et al Fourth universal definition of myocardial infusion. Eur Heart J.2018). Accordingly, a patient diagnosed with myocardial infarction needs to meet two of the following conditions, typical chest pain symptoms, an increase or decrease in a characteristic myocardial marker (e.g., CK-MB isozymes, cTnI or cTnT), and a typical electrocardiographic change.

Creatine Kinase (CK) exists in various forms in body fluids and tissues of the animal body, including different isozymes called CK-MM, CK-BB, CK-MB, and mitochondrial CK and some CK-MM and CK-MB variants with very short half-lives. These forms of creatine kinase are known to include 2 CK subunits, the B subunit and the M subunit. Circulating CK-MB content levels are currently considered to be relatively reliable indicators of myocardial damage. When the myocardial cells rich in CK-MB undergo necrosis, the content of CK-MB in the blood circulation is increased.

When a patient faces Acute Myocardial Infarction (AMI) which is life threatening, the ability to accurately and rapidly provide CK-MB detection results is critical for immediate treatment. It is estimated that to make a diagnosis early in acute myocardial infarction, it is necessary to accurately detect an increase in the concentration of CK-MB in blood of 1-2 ng/mL. In general, when the assay sensitivity of a detection method for CK-MB is increased to a level sufficient to detect such a low level of increase in CK-MB, the detection specificity of the method tends to be lowered due to interference of CK isozymes other than CK-MB, or other nonspecific action effects. Because of the inaccuracy of the measurement of low levels of elevated circulating CK-MB, the limits of detection specificity and sensitivity of the detection reagent directly determine whether an accurate diagnosis of acute myocardial infarction can be achieved.

There are a number of kits currently available on the market for the detection of CK-MB based on different detection methodologies. Comprising the following steps: chemiluminescent methods (magnetic particle chemiluminescence or capillary chemiluminescence immunoassay, etc.), immunochromatography (quantum dot-based, immunofluorescence, etc.), immunosuppression methods, etc., but there are few high-quality antibodies that can be used for accurate quantitative detection of CK-MB.

Disclosure of Invention

The invention discovers that the interference to CK-MB is negligible in actual detection, but the interference of CK-MM directly leads to inaccurate detection results, so the invention aims to provide an anti-CK-MM antibody or a functional fragment thereof, which can be combined with the CK-MM with high specificity to deactivate the CK-MM, thereby avoiding the interference of the CK-MM to the CK-MB detection, realizing accurate quantification of the CK-MB and finally being applied to an immunodetection product capable of rapidly and accurately detecting the CK-MB.

The method comprises the following steps:

the anti-CK-MM antibody or functional fragment thereof has the following complementarity determining regions:

CDR-VH1: S-D-X1-X2-T, wherein X1 is Y or P and X2 is F or W;

CDR-VH2: Y-I-X1-Y-S-G-I-T-Y-X2-N-P-X3-L-K-S, wherein X1 is R or S, X2 is Y or W, and X3 is S or R;

CDR-VH3: Y-Y-D-X1-A-M-X2-Y, wherein X1 is Y or I and X2 is E or N;

CDR-VL1: K-A-S-X1-D-I-N-X2-Y-L-S, wherein X1 is Q or K and X2 is S or H;

CDR-VL2: R-A-X1-R-L-X2-D, wherein X1 is R or N and X2 is T or V;

CDR-VL3: L-X1-Y-D-E-F-X2-W-T, wherein X1 is Q and X2 is P or W.

Further, X2 of CDR-VH1 of the complementarity determining region is W, X1 of CDR-VH2 is S, X2 of CDR-VH3 is N, X1 of CDR-VL1 is Q, X2 of CDR-VL2 is V, and X1 of CDR-VL3 is Q.

The CDR session division of the invention adopts a Kabat algorithm.

"CDR" is used herein to refer to a "complementarity determining region" within an antibody variable sequence. The variable regions of the heavy and light chains each have 3 CDRs, starting from the N-terminus of the heavy or light chain.

The antigen binding site may include six CDRs (CDR-VH 1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2 and CDR-VL3 in the present invention). Polypeptides comprising individual CDRs (e.g., CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2 and CDR-VL 3) may be referred to as "molecular recognition units". Crystallographic analysis of antigen-antibody complexes has demonstrated that the amino acid residues of CDRs form a broad contact with the bound antigen, with the broadest antigen contact being with the heavy chain CDR3. Thus, the molecular recognition unit may be primarily responsible for the specificity of the antigen binding site. Generally, CDR residues are directly and most substantially involved in influencing antigen binding.

In alternative embodiments, X1 in said CDR-VH1 is Y;

in alternative embodiments, X1 in said CDR-VH1 is P;

in alternative embodiments, X2 in said CDR-VH1 is F;

in alternative embodiments, X2 in said CDR-VH1 is W;

in alternative embodiments, X1 in said CDR-VH2 is R;

in alternative embodiments, X1 in said CDR-VH2 is S;

in alternative embodiments, X1 in said CDR-VH2 is N;

in alternative embodiments, X2 in said CDR-VH2 is Y;

in alternative embodiments, X2 in said CDR-VH2 is W;

in alternative embodiments, X3 in said CDR-VH2 is R;

in alternative embodiments, X3 in said CDR-VH2 is S;

in alternative embodiments, X1 in said CDR-VH3 is Y;

in alternative embodiments, X1 in said CDR-VH3 is I;

in alternative embodiments, X2 in said CDR-VH3 is E;

in alternative embodiments, X2 in said CDR-VH3 is N;

in alternative embodiments, X1 in the CDR-VL1 is Q;

in alternative embodiments, X1 in the CDR-VL1 is K;

in alternative embodiments, X2 in the CDR-VL1 is S;

in alternative embodiments, X2 in the CDR-VL1 is H;

in alternative embodiments, X1 in the CDR-VL2 is R;

in alternative embodiments, X1 in the CDR-VL2 is N;

in alternative embodiments, X2 in the CDR-VL2 is S;

in alternative embodiments, X2 in the CDR-VL2 is T;

in alternative embodiments, X2 in the CDR-VL2 is V;

in alternative embodiments, X1 in the CDR-VL3 is Q;

in alternative embodiments, X2 in the CDR-VL3 is P;

in alternative embodiments, X2 in the CDR-VL3 is W;

preferably, each complementarity determining region is selected from any one of the following combinations of mutations:

further, the antibody comprises light chain framework regions FR1-L, FR2-L, FR-L and FR4-L and heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H; the heavy chain framework region FR1-H, FR2-H, FR3-H and FR4-H are sequentially selected from SEQ ID NO. 1-4; the light chain skeleton region FR1-L, FR2-L, FR3-L and FR4-L are sequentially selected from SEQ ID NO. 5-8.

Further, the antibody further comprises a constant region.

Preferably, the constant region is selected from the group consisting of the constant region of any one of IgG1, igG2, igG3, igG4, igA, igM, igE and IgD, preferably, the constant region is derived from a species selected from the group consisting of cow, horse, cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, nugget or human.

Preferably, the functional fragment is selected from any one of VHH, F (ab ') 2, fab', fab, fv and scFv of the antibody.

In another aspect, the invention discloses a kit comprising the anti-CK-MM antibody or functional fragment thereof.

In another aspect, the invention discloses the use of said anti-CK-MM antibodies or functional fragments thereof in immunoassays.

In another aspect, the invention discloses the anti-interference effect of the anti-CK-MM antibody or functional fragment thereof in a CK-MB detection kit.

The term "anti-interference" as used herein means that interference of CK-MM is eliminated in the detection of CK-MB.

The beneficial effects are that:

the antibody disclosed by the invention has better binding specificity and affinity to M subunit in natural CK-MB, does not cross B subunit, and has a good detection effect on the rise of CK-MB in clinical samples by using the prepared creatine kinase MB isozyme detection kit (an immunosuppression method).

Drawings

Fig. 1: SDS-PAGE electrophoresis detection result of CKMM-D antibody;

fig. 2: comparison of results of clinical samples tested using the biochemical reagents prepared with the antibodies of the present invention.

Detailed Description

Example 1 preparation of immunogens

The natural CK-MM and CK-MB antigens were prepared by purification from human skeletal and cardiac muscles according to published methods (Purification of five creatine kinase-MM variants from human heart and skeletal. Mu. Biochim Biophys acta.1984 Nov 9;790 (3): 230-7.). Natural CK-BB is purchased from meridian bioscience company. The CK-MM subunit and CK-BB subunit are cleaved into monomers by 6.5M guanidine hydrochloride. The CK-MM subunit and the CK-BB subunit were mixed according to 1:1, removing guanidine hydrochloride by dialysis, and converging dissociated subunits to synthesize dimer during dialysis. The dialysate used was 10mM Tris buffer, pH 8.0, containing 100mM beta-mercaptoethanol. The CK-MB polymer in polymerized form is prepared by further purification by ion exchange chromatography.

Example 2 mouse immunization and potency detection

5 SPF-class female BALB/c mice of 6-8 weeks old were selected, and the native CK-MB protein was mixed with an equal volume of Freund's complete adjuvant and emulsified. The emulsified antigen is injected into mice by sole injection, back subcutaneous injection or intraperitoneal injection, and each mouse is injected with 25 mug of natural CK-MB antigen protein. Two weeks after the primary immunization was completed, the antigen protein was mixed and emulsified with Freund's incomplete adjuvant, and again, plantar injection, dorsal subcutaneous injection, or intraperitoneal injection was used, with each mouse still being injected with 25. Mu.g of antigen protein. The supernatant was collected by tail vein blood sampling, centrifuged and the serum titer was detected by ELISA. Immunization was performed every two weeks and serum titers were measured. After 2 immunizations, mice with serum titer reactive OD450 still above 1.0 after hundred thousand times dilution were screened by ELISA, boosted by spleen injection of the adjuvant-free CK-MB protein, spleen was picked up 3-4 days later, and lymph nodes were isolated for cell fusion.

EXAMPLE 3 cell fusion and Positive hybridoma cell screening and subcloning

In order to screen antibodies that specifically bind to the M subunit of native CK-MB, an indirect ELISA method and a capture free ELISA method were used for antibody screening. In the indirect ELISA screening method, CK-MM/BB/MB 3 antigens are diluted to 1ug/mL by a coating buffer (20 mM sodium bicarbonate, pH 9.6), polyvinyl chloride ELISA 96-well plates are added in an amount of 100 ul/well, the supernatant of each antibody to be detected is added to the well plates to incubate with the antigen, and finally incubation and development are carried out by using HRP-labeled goat anti-mouse secondary antibodies. Antibody clones were selected that were strongly reactive to native CK-MB, reactive to CK-MM, but non-reactive to CK-BB. In the capture free ELISA screening method, goat anti-mouse polyclonal antibody was diluted with coating solution at a concentration of 1ug/mL and then added to a polyvinyl chloride ELISA 96-well plate. Then adding each antibody to be detected for capturing, further adding three antigens of CK-MM/BB/MB marked by biotin, finally using avidin coupled with HRP for incubation and color development, screening and selecting antibody clone which has strong reactivity to natural CK-MB, has reactivity to CK-MM and does not react to CK-BB. In the present invention, a hybridoma cell numbered CKMM-D was obtained by screening.

EXAMPLE 4 production purification and electrophoretic detection of monoclonal antibodies

Two groups of 6-8 week BALB/c mice were selected and injected intraperitoneally with 500. Mu.L paraffin oil to suppress the immune response in the mice. One week after injection, a group of mice was intraperitoneally injected with 0.5ml of CKMM-D hybridoma cells, the number of cells being about 1X 10 ⁶ And each. Ascites collection was started after two weeks. The collected ascites is subjected to ammonium sulfate precipitation and protein A affinity column purification to obtain the target antibody, which is named as CKMM-D antibody. The result of SDS-PAGE electrophoresis of CKMM-D antibody is shown in FIG. 1. Wherein, the reduction treatment mode of the antibody is that 20ug CKMM-antibody is taken, 10ul of reduced protein loading buffer solution (Biyundian P0015L) is added, pure water is used for preparing 50ul of total volume, and the antibody protein is fully denatured by heating treatment for 10min at 100 ℃. The non-reduction treatment of the antibody was carried out by taking 20ug of CKMM-antibody, adding 10ul of non-reduced protein loading buffer (Biyundian P0016N), adding pure water to 50ul total volume, mixing, and loading directly.

Example 5 detection of reactivity of CKMM-D antibodies to Natural CKMB, CKMM and CKBB

Native CKMB, CKMM, CKBB protein was diluted 1ug/mL with coating solution (0.05M pH=9.5 carbonate buffer), 100. Mu.L/well was added to the ELISA plate and coated overnight at 4 ℃. In the detection, the antibody to be detected is diluted to 100ng/mL, incubated with antigen at 37 ℃, developed by using goat anti-mouse polyclonal antibody marked by HRP, and the absorbance of OD450 is detected, and the result is shown in the following table 1.

Table 1 potency assay

Antibody CKMM-D is capable of specifically recognizing CK-MB and CK-MM, but does not react with CK-BB. Meanwhile, at a lower antibody concentration, that is, the antibody still has a signal at 1ng/mL, the sensitivity of the strain antibody is higher, and the strain antibody is suitable for subsequent experiments.

Example 6 validation of antibody Performance

The CKMM-D antibody of example 3 has a heavy chain variable region shown in SEQ ID NO:9, wherein each complementarity determining region on the heavy chain variable region has the amino acid sequence as follows:

CDR-VH1：S-D-P(X1)-F(X2)-T

CDR-VH2：Y-I-R(X1)-Y-S-G-I-T-Y-W(X2)-N-P-S(X3)-L-K-S

CDR-VH3：Y-Y-D-Y(X1)-A-M-E(X2)-Y

the light chain variable region is shown as SEQ ID NO. 10, wherein the amino acid sequence of each complementarity determining region on the light chain variable region is as follows:

CDR-VL1：K-A-S-K(X1)-D-I-N-S(X2)-Y-L-S

CDR-VL2：R-A-R(X1)-R-L-T(X2)-D

CDR-VL3：L-Q(X1)-Y-D-E-F-W(X2)-W-T

on the basis of CKMM-D antibodies, mutations are made in the complementarity determining regions at sites related to the activity of the antibodies, wherein X1, X2 and X3 are mutation sites.

TABLE 2 mutation sites related to antibody Activity

TABLE 3 data from antibody Activity analysis

From the above table, the activity effect of mutation 4 is optimal, so that mutation 4 is taken as a framework sequence, other mutation sites with better affinity are screened, and partial results are as follows:

TABLE 4 mutation sites related to antibody affinity

The absorbance of OD450 was measured for each mutant antibody using CK-MB protein 100ng/ml, with the following results:

table 5 affinity testing of antibody mutations

Mutation 4-1	1.1469	Mutations 4-11	1.3679	Mutation 4-21	1.4281
						Mutation 4-2	1.1769	Mutations 4-12	1.5276	Mutation 4-22	1.4981
Mutation 4-3	1.4735	Mutations 4-13	1.3242	Mutation 4-23	1.2818
						Mutation 4-4	1.7266	Mutations 4-14	1.1623	Mutation 4-24	1.0423
Mutation 4-5	1.4356	Mutations 4-15	1.0603	Mutations 4-25	1.6652
						Mutations 4-6	1.6384	Mutations 4-16	1.7199	Mutations 4-26	1.3661
Mutations 4-7	1.4301	Mutations 4-17	1.2829	Mutations 4-27	1.1062
						Mutations 4-8	1.1095	Mutations 4-18	1.0257	Mutations 4-28	1.3824
Mutations 4-9	1.3424	Mutations 4-19	1.0626	Mutations 4-29	1.4526
						Mutations 4-10	1.1916	Mutation 4-20	1.5131

It can be seen that the affinity of each mutation is higher.

Example 7 evaluation of antibody stability

ELISA plates were coated with 1ug/mL goat anti-mouse and the stability of each treated antibody was evaluated using 100ng/mL biotin CK-MB protein. The antibodies were thermally accelerated in a defined buffer (20 mM citric acid, 0.1M NaCl, pH 5.5) at 37℃for 7 days, and the accelerated antibodies were evaluated by ELISA indirect method and compared with 4℃to identify the long-term stability of the antibodies. In addition, by repeating freeze thawing 5 times at-20 ℃, the test results are shown as deviations of the value of 4 ℃ from the value after acceleration, and the measurement results are as follows:

TABLE 6 stability study

From the above, mutation 4 to mutation 4-29 can be stably stored at 4 ℃, and the property is still stable after accelerating for 0.5 month at 37 ℃ (slight reactivity reduction does not affect the reagent performance), so that the performance of the reagent used for opening the bottle is ensured, and the accuracy and stability of the detection result are ensured.

Example 8 antibodies for creatine kinase MB isozymes (immunosuppression) Biochemical reagents

The biochemical creatine kinase MB isozyme detection kit (an immunosuppression method) is the most common CK-MB detection methodology in the market. The principle is based on the omission of CK-BB, and the complete inhibition of M subunit by anti-CK-MM antibody. The method has the advantages of rapidness, simplicity, time saving, wide application, large market occupation ratio and the like. Thus, the CK-MM antibody is an important component of a biochemical creatine kinase MB isozyme detection kit (an immunosuppression method). The R1 reagent containing the following components is prepared according to a given scheme: creatine phosphate, D-glucose, adenine nucleotide (AMP), adenosine Diphosphate (ADP), nicotinamide adenine dinucleotide phosphate (NADP+), hexokinase (HXK), imidazole acetic acid buffer, anti-human CK-MM antibodies. The R2 reagent containing the following components is prepared according to a given scheme: glucose-6-phosphate dehydrogenase (G6 PDH), 3- (cyclohexylamine) -2-hydroxy-1-propanesulfonic acid (CAPSO) buffer. The CKMM-D antibodies developed using the present invention were added to the interior of the mixed reagent to inhibit M subunit activity, and then the remaining B subunit activity was determined. In the embodiment, the activity of the B subunit is measured at the wavelength of 340nm (405 nm is taken as the detection auxiliary wavelength), and the CK-MB activity is obtained by performing corresponding operation on the detection result. The test adopts a full-automatic EXC 800 biochemical analyzer which is independently developed by the Zhongyuan Huiji for detection and calculation. From the detection results shown in fig. 2, it was confirmed that the antibody of the present invention has excellent clinical detection performance.

TABLE 7 detection of clinical sample results using the biochemical reagents of the invention

From the detection results shown in fig. 2 and table 7, it is determined that the consistency of the detection results and the clinical results of the reagent of the present invention is high, which indicates that the antibody of the present invention has excellent clinical detection performance.

Although embodiments of the present invention have been disclosed above, it is not limited to the details and embodiments shown and described, it is well suited to various fields of use for which the invention would be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the specific details and illustrations shown and described herein, without departing from the general concepts defined in the claims and their equivalents.

Claims

1. An anti-CKMM antibody or functional fragment thereof, wherein said CKMM-D antibody or functional fragment thereof has the following complementarity determining regions:

CDR-VH1: S-D-X1-X2-T, wherein X1 is Y or P and X2 is F or W;

CDR-VH3: Y-Y-D-X1-A-M-X2-Y, wherein X1 is Y or I and X2 is E or N;

CDR-VL1: K-A-S-X1-D-I-N-X2-Y-L-S, wherein X1 is Q or K and X2 is S or H;

CDR-VL2: R-A-X1-R-L-X2-D, wherein X1 is R or N and X2 is T or V;

CDR-VL3: L-X1-Y-D-E-F-X2-W-T, wherein X1 is Q and X2 is P or W.

2. The anti-CK-MM antibody or functional fragment thereof according to claim 1, wherein X2 of CDR-VH1 of the complementarity determining region is W, X1 of CDR-VH2 is S, X2 of CDR-VH3 is N, X1 of CDR-VL1 is Q, X2 of CDR-VL2 is V, and X1 of CDR-VL3 is Q.

3. The anti-CK-MM antibody or functional fragment thereof according to claim 1, wherein in an alternative embodiment, X1 in the CDR-VH1 is Y;

or X1 in said CDR-VH1 is P;

or X2 in said CDR-VH1 is F;

or X2 in said CDR-VH1 is W;

or X1 in said CDR-VH2 is R;

or X1 in said CDR-VH2 is S;

or X1 in said CDR-VH2 is N;

or X2 in said CDR-VH2 is Y;

or X2 in said CDR-VH2 is W;

or X3 in said CDR-VH2 is R;

or X3 in said CDR-VH2 is S;

or X1 in said CDR-VH3 is Y;

or X1 in said CDR-VH3 is I;

or X2 in said CDR-VH3 is E;

or, X2 in said CDR-VH3 is N;

or X1 in said CDR-VL1 is Q;

or X1 in said CDR-VL1 is K;

or X2 in said CDR-VL1 is S;

or X2 in said CDR-VL1 is H;

or X1 in said CDR-VL2 is R;

or X1 in said CDR-VL2 is N;

or X2 in said CDR-VL2 is S;

or X2 in said CDR-VL2 is T;

or X2 in said CDR-VL2 is V;

or X1 in said CDR-VL3 is Q;

or X2 in said CDR-VL3 is P;

or X2 in said CDR-VL3 is W.

4. The anti-CK-MM antibody or functional fragment thereof according to any one of claims 1-3, wherein each complementarity determining region is selected from any one of the following combinations of mutations:

5. the anti-CK-MM antibody or functional fragment thereof according to any one of claims 1-4, comprising the light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L and the heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H; the heavy chain framework region FR1-H, FR2-H, FR3-H and FR4-H are sequentially selected from SEQ ID NOs 1-4; the light chain skeleton region FR1-L, FR2-L, FR3-L and FR4-L are sequentially selected from SEQ ID NO. 5-8.

6. The anti-CK-MM antibody or functional fragment thereof according to claim 5, further comprising a constant region;

7. The anti-CK-MM antibody or functional fragment thereof according to claim 6, wherein the functional fragment is selected from any one of VHH, F (ab ') 2, fab', fab, fv and scFv of the antibody.

8. A kit comprising the anti-CK-MM antibody or functional fragment thereof of claims 1-7.

9. Use of an anti-CK-MM antibody or functional fragment thereof according to claims 1-7 for the preparation of an immunoassay kit.

10. anti-CK-MM antibodies or functional fragments thereof of claims 1-7 in a CK-MB detection kit.