WO2022121368A1 - Antibody against gastrin-releasing peptide, detection reagent and reagent kit thereof - Google Patents

Abstract

Provided are an antibody against a gastrin-releasing peptide, a detection reagent and a reagent kit thereof, relating to the technical field of antibodies. The antibody against a gastrin-releasing peptide of the present disclosure comprises a heavy chain complementarity-determining region and a light chain complementarity-determining region. The antibody of the present disclosure has better specificity and sensitivity to gastrin-releasing peptides, and can be used for detecting gastrin-releasing peptides and for the diagnosis or assisting diagnosis of related diseases having a gastrin-releasing peptide as a marker.

Description

Anti-gastrin-releasing peptide antibodies, detection reagents and kits

CROSS-REFERENCE TO RELATED APPLICATIONS

This disclosure claims the priority of the Chinese patent application with the application number CN202011446251.4 and titled "Anti-Gastrin-releasing Peptide Antibody, Detection Reagent and Kit" filed with the China Patent Office on December 8, 2020, all of which are The contents are incorporated by reference in this disclosure.

technical field

The present disclosure relates to the technical field of antibodies, and in particular, to an antibody against gastrin-releasing peptide, a detection reagent and a kit.

Background technique

Gastrin-releasing peptide (GRP) is a regulatory molecule that can stimulate gastric G cells to secrete gastrin, participate in smooth muscle cell contraction, and promote cell-to-cell interactions. ProGRP (pro-gastrin-releasing peptide) is the precursor of gastrin-releasing peptide, there are 3 variants, its amino acid sequence length is 115aa, 118aa and 125aa, respectively called subtype 1, subtype 2 and subtype 3. The content ratio is 60:5:35. In the human body, the GRP gene encodes a pre-proprotein with 148 amino acids. With the dissociation of the signal peptide, its 148 pre-protein will be further decomposed to generate a 27-amino acid gastrin-releasing peptide and a 68-amino acid ProGRP. Compared with the short half-life of GRP in blood, the half-life of ProGRP is moderate, which can reflect the level of GRP and the expression of GRP gene.

Fetal and neonatal bronchial epithelial endocrine cells are abundant in GRP, while adults are only present in brain, gastrointestinal nerve fibers and a small part of lung neuroendocrine cells, and the level is low. In addition to renal insufficiency, pulmonary neuroendocrine tumors, and medullary thyroid carcinoma, ProGRP is rarely elevated in patients with other malignancies or benign diseases.

ProGRP has high sensitivity and specificity for the diagnosis of small cell lung cancer (SCLC), and its level can reflect the effect of treatment and determine whether the disease progresses and regresses. ProGRP has a good role in monitoring the recurrence of SCLC patients. The level of ProGRP has a high correlation with prognostic factors in the prognosis evaluation of patients, which can be used to evaluate the prognosis earlier and predict the efficacy.

Considering economic cost and other factors, low-dose helical CT is not suitable for large-scale lung cancer screening. The detection of tumor markers meets the needs of clinical rapid, non-invasive and economical. Since it became a marker of SCLC in 1989, ProGRP detection has established various detection methods such as enzyme-linked immunosorbent assay (1995), chemiluminescence immunoassay, electrochemiluminescence assay (2013), time-resolved immunofluorescence assay, etc. . Different methods have their own advantages and disadvantages, but all require specific antibodies against ProGRP. High-quality antibodies are essential for clinical detection of GRP. At present, there are few sources of monoclonal antibodies against GRP, and the sensitivity, affinity and specificity are all defective.

SUMMARY OF THE INVENTION

The present disclosure provides an anti-gastrin-releasing peptide antibody or a functional fragment thereof, the antibody or functional fragment thereof comprising the following complementarity determining regions:

CDR-VH1: G-Y-X1-F-X2-D-Y-N-M-X3; where: X1 is R or K; X2 is S or T; X3 is N or D;

CDR-VH2: D-X1-N-P-X2-S-D-X3-T-X4-Y-N-Q-K-F-K-G; where: X1 is L or I; X2 is N or D; X3 is G, A or V; X4 is I or F;

CDR-VH3: X1-T-W-X2-H; wherein: X1 is S or T; X2 is I, V or L;

CDR-VL1: S-A-X1-Q-G-X2-S-N-Y-X3-S; wherein: X1 is S or T; X2 is I, V or L; X3 is I, V or L;

CDR-VL2: X1-T-S-R-X2-Y-S; where: X1 is H or Y; X2 is I, V or L;

CDR-VL3: X1-Q-Y-S-K-X2-P-W; wherein: X1 is Q or H; X2 is I, V or L.

In some embodiments, in CDR-VH1, X1 is R; in CDR-VH2, X1 is I; in CDR-VH3, X1 is S; in CDR-VL1, X1 is S; in CDR-VL3, X1 is H .

optional, in CDR-VH1, X2 is S; optional, in CDR-VH1, X2 is T; optional, in CDR-VH1, X3 is N; optional, in CDR-VH1, X3 is D; optional, in CDR-VH2, X2 is N; optional, in CDR-VH2, X2 is D; optional, in CDR-VH2, X3 is G; optional, in CDR-VH2, X3 is A; optional, in CDR-VH2, X3 is V; optional, in CDR-VH2, X4 is I; optional, in CDR-VH2, X4 is F; optional, in CDR-VH3 in CDR-VH3, X2 is I; optional, in CDR-VH3, X2 is V; optional, in CDR-VH3, X2 is L; optional, in CDR-VL1, X2 is I; optional, in CDR - VL1, X2 is V; optional, CDR-VL1, X2 is L; optional, CDR-VL1, X3 is I; optional, CDR-VL1, X3 is V; optional , in CDR-VL1, X3 is L; optional, in CDR-VL2, X1 is H; optional, in CDR-VL2, X1 is Y; optional, in CDR-VL2, X2 is I; optional, in CDR-VL2, X2 is V; optional, in CDR-VL2, X2 is L; optional, in CDR-VL3, X2 is I; optional, in CDR-VL3, X2 is V ; Optionally, in CDR-VL3, X2 is L.

In some embodiments, each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following mutation combinations 1-47:

In some embodiments, the antibody or its functional fragment binds to the gastrin-releasing peptide antigen with an affinity of K _D ≤ 3×10 ^-7 mol/L; optionally, K _D ≤ 7.7×10 ^-9 mol /L.

In some embodiments, in CDR-VH1, X1 is K; in CDR-VH2, X1 is L; in CDR-VH3, X1 is T; in CDR-VL1, X1 is T; in CDR-VL3, X1 is Q .

In some embodiments, each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following mutation combinations 48-53:

In some embodiments, the antibody comprises the light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L of the sequence shown in SEQ ID NO: 1-4, and/or the sequence of Heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H shown in SEQ ID NOs: 5-8.

Optionally, the antibody further comprises a constant region;

Optionally, the constant region is selected from the constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD;

Optionally, the species source of the constant region is bovine, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose , turkey, cockfight, or man;

Optionally, the constant region is derived from a mouse;

Optionally, the light chain constant region sequence of the constant region is shown in SEQ ID NO:9, and the heavy chain constant region sequence of the constant region is shown in SEQ ID NO:10.

In some embodiments, the functional fragment is selected from any one of VHH, F(ab')2, Fab', Fab, Fv and scFv of the antibody.

In some embodiments, the antibody comprises light chain framework regions FR1-L, FR2-L, FR3-L and FR1-L, FR2-L, FR3-L, and FR4-L, and/or, heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H, FR2-H, FR3-H, and FR4-H having at least 80% homology with sequences of SEQ ID NOs: 5, 6, 7, 8, respectively H.

The present disclosure provides a reagent or kit for detecting gastrin-releasing peptide, which includes the antibody or a functional fragment thereof.

In some embodiments, the antibody or functional fragment thereof is labeled with a detectable label.

Optionally, the detectable label is selected from fluorescent dyes, enzymes that catalyze the coloration of substrates, radioisotopes, chemiluminescent reagents and nanoparticle labels;

Optionally, the fluorescent dye is selected from fluorescein dyes and their derivatives, rhodamine dyes and their derivatives, Cy series dyes and their derivatives, Alexa series dyes and their derivatives, and protein dyes and their derivatives. thing;

Optionally, the enzyme that catalyzes the coloration of the substrate is selected from horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase and glucose 6-phosphate deoxygenase;

Optionally, the radioisotope is selected from ²¹² Bi, ¹³¹ I, ¹¹¹ In, ⁹⁰ Y, ¹⁸⁶ Re, ²¹¹ At, ¹²⁵ I, ¹⁸⁸ Re, ¹⁵³ Sm, ²¹³ Bi, ³² P, ⁹⁴ mTc, ⁹⁹ mTc, ²⁰³ Pb, ⁶⁷ Ga, ⁶⁸ Ga, ⁴³ Sc, ⁴⁷ Sc, ¹¹⁰ mIn, ⁹⁷ Ru, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁸ Cu, ⁸⁶ Y, ⁸⁸ Y, ¹²¹ Sn, ¹⁶¹ Tb, ¹⁶⁶ Ho, ¹⁰⁵ Rh, ¹⁷⁷ Lu, ¹⁷² Lu and ¹⁸ F;

Optionally, the chemiluminescent reagent is selected from luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine esters and its derivatives, dioxanes. Ethane and its derivatives, Lofenine and its derivatives, and peroxyoxalate and its derivatives;

Optionally, the nanoparticle-based marker is selected from nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles and rare earth complex nanoparticles;

Optionally, the colloid is selected from colloidal metals, disperse dyes, dye-labeled microspheres and latex;

Optionally, the colloidal metal is selected from colloidal gold, colloidal silver and colloidal selenium.

The present disclosure provides the use of the antibody or its functional fragment in the preparation of a reagent or a kit for diagnosing or assisting the diagnosis of related diseases marked by gastrin-releasing peptide.

The present disclosure provides a nucleic acid molecule encoding the antibody or functional fragment thereof.

The present disclosure provides a vector containing a nucleic acid fragment encoding the antibody or functional fragment thereof.

The present disclosure provides a recombinant cell containing the vector.

The present disclosure provides the use of the antibody or functional fragment thereof or the reagent or kit in detecting gastrin-releasing peptide.

The present disclosure provides the use of the antibody or functional fragment thereof or the reagent or kit for detecting gastrin-releasing peptide.

The present disclosure provides a method for detecting gastrin-releasing peptide, comprising:

A) contacting the antibody or functional fragment thereof with a sample from the subject under conditions sufficient for the binding reaction to occur to effect the binding reaction; and

B) Detection of immune complexes produced by the binding reaction.

The present disclosure provides the use of the antibody or its functional fragment or the reagent or kit in the diagnosis, prognostic evaluation and efficacy prediction of small cell lung cancer.

The present disclosure provides a method for diagnosing a disease associated with gastrin-releasing peptide as a marker in a subject, comprising:

A) contacting the antibody or functional fragment thereof with a sample from the subject under conditions sufficient for a binding reaction to occur to effect a binding reaction; and

B) Detection of immune complexes produced by the binding reaction.

In some embodiments, the associated disease marked by gastrin-releasing peptide is renal insufficiency, pulmonary neuroendocrine tumor, and medullary thyroid carcinoma. Optionally, the related disease marked by gastrin-releasing peptide is small cell lung cancer.

The present disclosure provides a method for preparing the antibody or a functional fragment thereof, comprising: culturing the recombinant cell, and separating and purifying the antibody or the functional fragment thereof from the culture product.

Description of drawings

In order to illustrate the technical solutions of the embodiments of the present disclosure more clearly, the following briefly introduces the accompanying drawings that need to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present disclosure, and therefore do not It should be regarded as a limitation of the scope, and for those of ordinary skill in the art, other related drawings can also be obtained according to these drawings without any creative effort.

FIG. 1 shows the results of reducing SDS-PAGE of the anti-gastrin-releasing peptide antibody of Example 1. FIG.

Implementation

In order to make the objectives, technical solutions and advantages of the embodiments of the present disclosure more clear, the technical solutions in the embodiments of the present disclosure will be described clearly and completely below. If the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be purchased from the market.

Unless otherwise defined, 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 the formulations or unit doses herein, some methods and materials are now described. Unless otherwise stated, techniques employed or considered herein are standard methods. The materials, methods and examples are illustrative only and not restrictive.

Unless otherwise indicated, the practice of this disclosure will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the purview of those skilled in the art. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis ( M.J.Gait, ed., 1984); "Animal Cell Culture" (R.I. Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); " Handbook of Experimental Immunology (eds. D.M.Weir and C.C.Blackwell); Gene Transfer Vectors for Mammalian Cells (eds. J.M. Miller and M.P. Calos, 1987); Contemporary Molecular Biology Methods (Current Protocols in Molecular Biology)" (F.M. Ausubel et al., ed., 1987); "PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction)" (Mullis et al., ed., 1994); Current Protocols in Immunology" (J.E. Coligan et al., ed., 1991), each of which is expressly incorporated herein by reference.

Definition of Terms

As used herein, the term "complementarity determining region" generally refers to: an intact or complete antibody comprises two heavy chains and two light chains; each heavy chain contains variable (VH) and CH regions; each light The chain contains a variable region (VL) and a constant region (CL); the antibody has a "Y" shape with the stem of the Y consisting of the second and third constant regions of two heavy chains held together by disulfide bonds Each arm of Y comprises the variable region and the first constant region of a single heavy chain combined with the variable region and constant region of a single light chain; the variable region of the light chain and the variable region of the heavy chain The variable regions are responsible for antigen binding; the variable regions in both chains typically contain three hypervariable regions, called complementarity determining regions.

As used herein, when used to refer to an antibody, the term "functional fragment" is intended to refer to a portion of an antibody comprising a heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment was derived. These functional fragments may include, for example, Fd, Fv, Fab, F(ab'), F(ab)2, F(ab')2, single chain Fv (scFv), diabodies, triple chain Antibodies (triabodies), tetrabodies (tetrabodies) and minibodies (minibodies). Other functional fragments may include, for example, heavy or light chain polypeptides, variable region polypeptides or CDR polypeptides or portions thereof, so long as these functional fragments retain binding activity.

As used herein, the term "constant region" refers to a relatively stable region of the light and heavy chains of an antibody molecule near the C-terminal amino acid sequence.

As used herein, the term "variable region" refers to the region of the light and heavy chains of an antibody molecule that varies greatly in amino acid sequence near the N-terminus.

As used herein, the term "naked anti-stability" refers to the change in activity of an antibody, when unlabeled and modified, after storage at a specified temperature and time.

The present disclosure provides an anti-gastrin-releasing peptide antibody, a detection reagent and a kit, the antibody has good specificity and sensitivity to gastrin-releasing peptide, and can be used for detecting gastrin-releasing peptide and for Diagnosis or auxiliary diagnosis of related diseases for which gastrin-releasing peptide is a marker.

One embodiment of the present disclosure provides an anti-gastrin-releasing peptide antibody or a functional fragment thereof, the antibody or functional fragment thereof has the following complementarity determining regions:

CDR-VH1: G-Y-X1-F-X2-D-Y-N-M-X3; where: X1 is R or K; X2 is S or T; X3 is N or D;

CDR-VH2: D-X1-N-P-X2-S-D-X3-T-X4-Y-N-Q-K-F-K-G; where: X1 is L or I; X2 is N or D; X3 is G, A or V; X4 is I or F;

CDR-VH3: X1-T-W-X2-H; wherein: X1 is S or T; X2 is I, V or L;

CDR-VL1: S-A-X1-Q-G-X2-S-N-Y-X3-S; wherein: X1 is S or T; X2 is I, V or L; X3 is I, V or L;

CDR-VL2: X1-T-S-R-X2-Y-S; where: X1 is H or Y; X2 is I, V or L;

CDR-VL3: X1-Q-Y-S-K-X2-P-W; wherein: X1 is Q or H; X2 is I, V or L.

The present disclosure provides an antibody or a functional fragment thereof having the above complementarity determining region structure, the antibody can specifically bind to gastrin-releasing peptide, and has a good affinity for the gastrin-releasing peptide antigen, and the antibody is used to detect gastrin release Peptides with good specificity and sensitivity. It can be used for the detection of gastrin-releasing peptide and the diagnosis or auxiliary diagnosis of related diseases with gastrin-releasing peptide as a marker. The present disclosure provides a richer selection of antibodies for the detection of gastrin-releasing peptides.

In alternative embodiments, in CDR-VH1, X1 is R; in CDR-VH2, X1 is I; in CDR-VH3, X1 is S; in CDR-VL1, X1 is S; in CDR-VL3, X1 is H.

The inventors of the present disclosure found that when the above-mentioned mutation sites in each complementarity-determining region are the above-mentioned amino acid residues, the antibody exhibits better affinity for gastrin-releasing peptide.

In an alternative embodiment, in CDR-VH1, X2 is S.

In an alternative embodiment, in CDR-VH1, X2 is T.

In an alternative embodiment, X3 is N in CDR-VH1.

In an alternative embodiment, X3 is D in CDR-VH1.

In an alternative embodiment, X2 is N in CDR-VH2.

In an alternative embodiment, X2 is D in the CDR-VH2.

In an alternative embodiment, in the CDR-VH2, X3 is G.

In an alternative embodiment, in the CDR-VH2, X3 is A.

In an alternative embodiment, in CDR-VH2, X3 is V.

In an alternative embodiment, in the CDR-VH2, X4 is 1.

In an alternative embodiment, in CDR-VH2, X4 is F.

In an alternative embodiment, in the CDR-VH3, X2 is 1.

In an alternative embodiment, in CDR-VH3, X2 is V.

In an alternative embodiment, in the CDR-VH3, X2 is L.

In an alternative embodiment, in CDR-VL1, X2 is 1.

In an alternative embodiment, in CDR-VL1, X2 is V.

In an alternative embodiment, in CDR-VL1, X2 is L.

In an alternative embodiment, in CDR-VL1, X3 is 1.

In an alternative embodiment, in CDR-VL1, X3 is V.

In an alternative embodiment, in CDR-VL1, X3 is L.

In an alternative embodiment, in CDR-VL2, X1 is H.

In an alternative embodiment, in CDR-VL2, X1 is Y.

In an alternative embodiment, in CDR-VL2, X2 is 1.

In an alternative embodiment, in CDR-VL2, X2 is V.

In an alternative embodiment, in CDR-VL2, X2 is L.

In an alternative embodiment, in CDR-VL3, X2 is 1.

In an alternative embodiment, in CDR-VL3, X2 is V.

In an alternative embodiment, in CDR-VL3, X2 is L.

In an alternative embodiment, each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following mutation combinations 1-47:

In an alternative embodiment, the antibody or functional fragment thereof binds to gastrin-releasing peptide with an affinity of K _D ≤ 3×10 ⁻⁷ mol/L.

In an optional embodiment, K _D ≤2×10 ^-7 mol/L, K _D ≤1×10 ^-7 mol/L, K _D ≤9×10 ^-8 mol/L, K _D ≤8×10 ^-8 mol/L, K _D ≤7×10 ^-8 mol/L, K _D ≤6×10 ^-8 mol/L, K _D ≤5×10 ^-8 mol/L, K _D ≤4×10 ^-8 mol/L, K _D ≤3×10 ^-8 mol/L, K _D ≤2×10 ^-8 mol/L, K _D ≤1×10 ^-8 mol/L, K _D ≤9×10 ^-9 mol/ L, K _D ≤8×10 ^-9 mol/L, K _D ≤7×10 ^-9 mol/L, K _D ≤6×10 ^-9 mol/L, K _D ≤5×10 ^-9 mol/L, K _D ≤4×10 ^-9 mol/L, K _D ≤3×10 ^-9 mol/L, K _D ≤2×10 ^-9 mol/L or K _D ≤1×10 ^-9 mol/L.

In an alternative embodiment, K _D ≤ 7.7×10 ⁻⁹ mol/L.

In an alternative embodiment, 1.22×10 ⁻⁹ mol/L≦K _D ≦7.73×10 ⁻⁹ mol/L.

The detection of K _D is performed with reference to the methods in the embodiments of the present disclosure.

In alternative embodiments, in CDR-VH1, X1 is K; in CDR-VH2, X1 is L; in CDR-VH3, X1 is T; in CDR-VL1, X1 is T; in CDR-VL3, X1 is Q.

In an alternative embodiment, each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following mutation combinations 48-53:

In an alternative embodiment, the antibody comprises the light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L, and/or the sequences of the light chain framework regions shown in SEQ ID NOs: 1-4 in sequence. The heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H shown in SEQ ID NOs: 5-8 in sequence.

In general, the variable region (VH) of the heavy chain and the variable region (VL) of the light chain can be obtained by linking the following numbered CDRs and FRs in the following combinations: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

It should be noted that, in other embodiments, the amino acid sequence of each framework region of the antibody or its functional fragment provided by the present disclosure may be the same as the above-mentioned corresponding framework region (SEQ ID NO: 1, 2, 3, 4, 5, 6 , 7 or 8) can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology.

In some embodiments, the antibody comprises light chain framework regions FR1-L, FR2-L, FR3-L and FR1-L, FR2-L, FR3-L, and FR4-L, and/or, heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H, FR2-H, FR3-H, and FR4-H having at least 80% homology with sequences of SEQ ID NOs: 5, 6, 7, 8, respectively H.

In alternative embodiments, the antibody further comprises a constant region.

In an alternative embodiment, the constant region is selected from the constant region of any one of IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD.

In an alternative embodiment, the species source of the constant region is mammalian or poultry. In alternative embodiments, mammals include cows, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, or humans. In alternative embodiments, poultry animals include chickens, ducks, geese, turkeys or fighting cocks.

In an alternative embodiment, the constant region is derived from a mouse.

In an alternative embodiment, the light chain constant region sequence of the constant region is shown in SEQ ID NO:9, and the heavy chain constant region sequence of the constant region is shown in SEQ ID NO:10.

In an alternative embodiment, the functional fragment is selected from any one of VHH, F(ab')2, Fab', Fab, Fv and scFv of the antibody.

Functional fragments of the above-described antibodies generally have the same binding specificity as the antibody from which they were derived. Those skilled in the art can easily understand from the content described in the present disclosure that the functional fragments of the above-mentioned antibodies can be cleaved by, for example, methods including but not limited to enzymatic digestion (including but not limited to pepsin or papain) and/or by chemical reduction Sulfur bond method. On the basis of the structure of the complete antibody provided in the present disclosure, those skilled in the art can easily obtain the above-mentioned functional fragments.

Functional fragments of the above-described antibodies can also be obtained 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, including but not limited to, synthetically obtained.

An embodiment of the present disclosure provides a reagent or kit for detecting gastrin-releasing peptide, which comprises the antibody or functional fragment thereof according to any one of the above.

In an optional embodiment, the antibody or functional fragment thereof in the above reagent or kit is labeled with a detectable label.

As used herein, "detectable label" refers to a class of substances having properties that can be directly observed by the naked eye or detected or detected by instruments, such as luminescence, coloration, radioactivity, etc., by which the corresponding Qualitative or quantitative detection of a target.

In alternative embodiments, the detectable labels include, but are not limited to, fluorescent dyes, enzymes that catalyze color development of substrates, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

In the actual use process, those skilled in the art can select an appropriate marker according to detection conditions or actual needs, and no matter what marker is used, it all falls within the protection scope of the present disclosure.

In an optional embodiment, the fluorescent dyes include, but are not limited to, fluorescein dyes and derivatives thereof (for example, including but not limited to fluorescein isothiocyanate (FITC) hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or its analogs), rhodamine dyes and derivatives thereof (such as, but not limited to, red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or the like compounds), Cy series dyes and their derivatives (such as but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (such as including But not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but Not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydinoxanthin-chlorophyll protein (preCP), etc.).

In an optional embodiment, the enzymes that catalyze the coloration of the substrate include but are not limited to horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase enzyme and glucose 6-phosphate deoxygenase.

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

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, acridine esters and its derivatives Derivatives, Dioxetane and its Derivatives, Lopine 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.

One embodiment of the present disclosure provides the use of the antibody or its functional fragment as described above in the preparation of a reagent or a kit for diagnosing or assisting the diagnosis of related diseases marked by gastrin-releasing peptide.

The antibodies or functional fragments thereof provided by the present disclosure can be used for the diagnosis or auxiliary diagnosis of any related diseases marked by gastrin-releasing peptide (such as renal insufficiency, pulmonary neuroendocrine tumors, and medullary thyroid cancer, etc.).

One embodiment of the present disclosure provides a nucleic acid molecule encoding the above-mentioned antibody or a functional fragment thereof.

One embodiment of the present disclosure provides a vector containing the above-mentioned nucleic acid molecule.

One embodiment of the present disclosure provides a recombinant cell containing the above-mentioned vector.

One embodiment of the present disclosure provides the use of the above-mentioned antibody or functional fragment thereof, or the above-mentioned reagent or kit in detecting gastrin-releasing peptide.

One embodiment of the present disclosure provides the use of the above-mentioned antibody or functional fragment thereof, or the above-mentioned reagent or kit for detecting gastrin-releasing peptide.

One embodiment of the present disclosure provides a method for detecting gastrin-releasing peptide, comprising:

A) contacting the above-described antibody or functional fragment thereof with a sample from the subject under conditions sufficient for the binding reaction to occur to effect the binding reaction; and

B) Detection of immune complexes produced by the binding reaction.

One embodiment of the present disclosure provides use of the above-mentioned antibody or functional fragment thereof, or the above-mentioned reagent or kit in the diagnosis, prognosis evaluation and efficacy prediction of small cell lung cancer.

One embodiment of the present disclosure provides a method for diagnosing a disease associated with gastrin-releasing peptide as a marker in a subject, comprising:

A) contacting the above-described antibody or functional fragment thereof with a sample from the subject under conditions sufficient for the binding reaction to occur to effect the binding reaction; and

B) Detection of immune complexes produced by the binding reaction.

In an optional embodiment, the related diseases marked by gastrin-releasing peptide include, but are not limited to, renal insufficiency, pulmonary neuroendocrine tumors and medullary thyroid cancer. In an optional embodiment, the related disease marked by gastrin-releasing peptide is small cell lung cancer.

One embodiment of the present disclosure provides a method for preparing an antibody or a functional fragment thereof, comprising: culturing the above-mentioned recombinant cells, and separating and purifying the antibody or functional fragment thereof from the culture product.

On the basis of the amino acid sequences of antibodies or functional fragments thereof provided in the present disclosure, those skilled in the art can easily conceive of using genetic engineering techniques or other techniques (chemical synthesis, hybridoma cells) to prepare the antibodies or functional fragments thereof, such as It is easy for those skilled in the art to separate and purify the antibody or its functional fragment from the culture product of recombinant cells capable of recombinantly expressing the antibody or its functional fragment as described above. Based on this, No matter what technology is used to prepare the antibody or its functional fragment of the present disclosure, it falls within the protection scope of the present disclosure.

Example

The features and properties of the present disclosure will be further described in detail below with reference to the embodiments.

In the examples, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara Company. MagExtractor-RNA extraction kit was purchased from TOYOBO Company. BD SMART ^™ RACE cDNA Amplification Kit was purchased from Takara Company. The pMD-18T vector was purchased from Takara Company. Plasmid extraction kit was purchased from Tiangen Company. Primer synthesis and gene sequencing were performed by Invitrogen.

Example 1

1 Construction of recombinant plasmids

(1) Antibody gene preparation

The mRNA was extracted from the hybridoma cell line secreting anti-gastrin-releasing peptide antibody, and the DNA product was obtained by RT-PCR. The product was added to the pMD-18T vector after adding A with DNA polymerase, and transformed into DH5α-sensing In the living cells, after the colonies were grown, 4 clones of the heavy chain (Heavy Chain) and the 4 light chain (Light Chain) gene clones were taken and sent to a gene sequencing company for sequencing.

(2) Sequence analysis of antibody variable region genes

The gene sequence obtained by the above sequencing was placed in the IMGT antibody database for analysis, and the VNTI11.5 software was used for analysis to confirm that the genes amplified by the heavy chain and light chain primers were correct, and the genes amplified by the light chain were correct. In the fragment, the VL gene sequence is 324bp, belonging to the VkII gene family, and there is a 57bp leader peptide sequence in front of it; in the gene fragment amplified by the heavy chain primer pair, the VH gene sequence is 336bp, belonging to the VH1 gene family, there is 57bp in front of it. leader peptide sequence.

(3) Construction of recombinant antibody expression plasmid

^pcDNATM 3.4

vector is the constructed recombinant antibody eukaryotic expression vector. The expression vector has introduced polyclonal restriction sites such as HindIII, BamHI and EcoRI, and is named pcDNA3.4A expression vector, hereinafter referred to as 3.4A expression vector; according to the above pMD-18T Based on the results of gene sequencing of the variable region of the antibody, the VL and VH gene-specific primers of the antibody were designed, with HindIII and EcoRI restriction sites and protective bases on both ends, respectively, and the light chain of 0.73KB was amplified by PCR amplification method. Gene fragment and 1.43kb heavy chain gene fragment.

The heavy chain and light chain gene fragments were digested with HindIII/EcoRI double enzymes respectively, and the 3.4A vector was digested with HindIII/EcoRI double enzymes. Recombinant expression plasmids for heavy and light chains were obtained.

2 Screening of stable cell lines

(1) The recombinant antibody expression plasmid was transiently transfected into CHO cells to determine the activity of the expression plasmid

The plasmid was diluted with ultrapure water to 40ug/100μL, adjusted to 1.43×10 ⁷ cells/ml of CHO cells in a centrifuge tube, 100μL of plasmid was mixed with 700μL of cells, transferred to an electroporation cup, electroporated, and the samples were counted on the 3rd, 5th, and 7th days , on the 7th day, samples were collected for testing.

The coating solution (the main component NaHCO ₃ ) diluted the GRP antigen to 1 μg/ml, 100 μL per well, overnight at 4°C; the next day, the washing solution (the main component PBS) was washed twice and patted dry; the blocking solution (20% BSA+) was added 80% PBS), 120μL per well, 37°C, 1h, pat dry; add diluted cell supernatant, 100μL/well, 37°C, 30min (partial supernatant for 1h); wash 5 times with washing solution, pat dry; add Goat anti-mouse IgG-HRP, 100 μL per well, 37°C, 30 min; wash 5 times with washing solution, pat dry; add chromogenic solution A (50 μL/well, containing citric acid + sodium acetate + acetanilide + carbamide peroxide) , add chromogenic solution B (50 μL/well, containing citric acid+EDTA·2Na+TMB+concentrated HCl) for 10min; add stop solution (50 μL/well, containing EDTA·2Na+concentrated H ₂ SO ₄ ); on the microplate reader The OD value was read at 450 nm (referenced to 630 nm). The results showed that the reaction OD was still greater than 1.0 after the cell supernatant was diluted 1000 times, and the reaction OD of the unadded cell supernatant was less than 0.1, indicating that the antibody produced by the transient transfection of the plasmid was active against the GRP antigen.

(2) Linearization of recombinant antibody expression plasmid

Prepare the following reagents: Buffer 50μL, DNA 100μg/tube, PuvI enzyme 10μL, sterile water to make up to 500μL, and digest overnight in a 37°C water bath; first use an equal volume of phenol/chloroform/isoamyl alcohol (lower layer) 25:24:1 , and then extracted with chloroform (aqueous phase) in turn; 0.1 times the volume (aqueous phase) of 3M sodium acetate and 2 times the volume of ethanol were precipitated on ice, rinsed with 70% ethanol, and the organic solvent was removed. The bacteria water was re-thawed, and finally the concentration was determined.

(3) Stable transfection of recombinant antibody expression plasmid, pressurized screening of stable cell lines

The plasmid was diluted with ultrapure water to 40ug/100μL, adjusted to 1.43×10 ⁷ cells/ml of CHO cells in a centrifuge tube, 100μL of plasmid was mixed with 700μL of cells, transferred to electroporation cup, electroporated, and counted the next day; 25umol/L MSX 96 Wells were pressurized for about 25 days.

Observe the cloned wells marked with cells under a microscope, and record the degree of confluence; take the culture supernatant and send samples for detection; select cell lines with high antibody concentration and relative concentration and transfer them to 24 wells, and transfer them to 6 wells in about 3 days; preserve seeds after 3 days Batch culture, adjust the cell density to 0.5×10 ⁶ cells/ml, 2.2 ml for batch culture, cell density 0.3×10 ⁶ cells/ml, 2 ml for seed preservation; 7-day 6-well batch culture supernatant is sent for sample detection, selection The cell lines with smaller antibody concentration and cell diameter were transferred to TPP for seed preservation and passage.

3 Recombinant Antibody Production

(1) Cell expansion

After the cells were recovered, they were first cultured in a 125ml shake flask, the inoculation volume was 30ml, and the medium was 100% Dynamis medium. After culturing for 72 hours, inoculate and expand the culture at an inoculation density of 500,000 cells/ml, and the expansion volume is calculated according to the production demand, and the medium is 100% Dynamis medium. After that, the culture was expanded every 72h. When the cell volume meets the production requirements, the seeding density is strictly controlled to be about 500,000 cells/ml for production.

(2) Shake flask production and purification

Shaking flask parameters: rotating speed 120r/min, temperature 37°C, carbon dioxide 8%. Feed feeding: start feeding every day after culturing in the shake flask to 72h, HyCloneTM Cell BoostTM Feed 7a is fed with 3% of the initial culture volume every day, and Feed 7b is fed with 1/1,000 of the initial culture volume every day. Supplement until the 12th day (feeding on the 12th day). Glucose was supplemented with 3 g/L on the sixth day. Samples were collected on the 13th day. Affinity purification was performed using a protein A affinity chromatography column. Take 4 μg of purified antibody for reducing SDS-PAGE, and 4 μg foreign control antibody as control. The electropherogram is shown in Figure 1 below. After reducing SDS-PAGE, two bands are displayed, and one Mr is 50KD (heavy chain, SEQ ID NO: 14), the other Mr is 28KD (light chain, SEQ ID NO: 13).

Example 2

Antibody performance testing

(1) Activity detection of the antibody of Example 1 and its mutants

The antibody (WT) sequence of Example 1 was analyzed, and its heavy chain variable region was shown in SEQ ID NO: 12, wherein the amino acid sequence of each complementarity determining region on the heavy chain variable region was as follows:

CDR-VH1: G-Y-K(X1)-F-S(X2)-D-Y-N-M-D(X3);

CDR-VH2: D-L(X1)-N-P-N(X2)-S-D-A(X3)-T-I(X4)-Y-N-Q-K-F-K-G;

CDR-VH3: T(X1)-T-W-I(X2)-H;

Its light chain variable region is shown in SEQ ID NO:11, wherein, the amino acid sequence of each complementarity determining region on the light chain variable region is as follows:

CDR1-VL: S-A-T(X1)-Q-G-V(X2)-S-N-Y-I(X3)-S;

CDR-VL2: H(X1)-T-S-R-V(X2)-Y-S;

CDR-VL3: Q(X1)-Q-Y-S-K-I(X2)-P-W.

On the basis of the anti-gastrin-releasing peptide antibody (WT) of Example 1, mutations were performed in the complementarity determining regions at sites related to antibody activity, wherein X1, X2, X3, and X4 were all mutation sites. See Table 1 below.

Table 1 Mutation sites related to antibody activity

Detection of antibody binding activity in Table 1:

Coating solution (main component NaHCO ₃ ) diluted GRP antigen to 1 μg/ml for microplate coating, 100 μl per well, overnight at 4°C; the next day, washing solution (main component PBS) washed twice and patted dry; added blocking solution (20%BSA+80%PBS), 120μl per well, 37℃, 1h, pat dry; add diluted RP monoclonal antibody, 100μl/well, 37℃, 30min-60min; wash 5 times with washing solution, pat dry dry; add goat anti-mouse IgG-HRP, 100 μl per well, 37°C, 30 min; wash 5 times with washing solution (PBS), pat dry; add chromogenic solution A (50 μl/well, containing 2.1 g/L citric acid, 12.25g/L citric acid, 0.07g/L acetanilide and 0.5g/L carbamide peroxide), add color developing solution B (50μl/well, containing 1.05g/L citric acid, 0.186g/LEDTA 2Na, 0.45 g/L TMB and 0.2ml/L concentrated HCl), 10min; add stop solution (50μl/well, containing 0.75g/EDTA·2Na and 10.2ml/L concentrated H ₂ SO ₄ ); 450nm on the microplate reader (refer to 630nm ) to read the OD value. The results are shown in Table 2 below.

Table 2 Activity data of WT antibody and its mutants

Antibody concentration (ng/ml)	37.50	18.75	9.38	4.69	2.34	0.00
WT	1.311	0.924	0.410	0.220	0.023	0.123
Mutation 1	2.027	1.376	0.791	0.448	0.222	0.019
Mutation 2	1.978	1.326	0.740	0.493	0.199	0.019
Mutation 3	1.951	1.36	0.681	0.479	0.182	0.017
Mutation 4	0.246	0.125	-	-	-	-
Mutation 5	0.283	0.193	-	-	-	-
Mutation 6	0.279	0.182	-	-	-	-

According to the results in Table 2, the mutant 4-mutation 6 antibodies had almost no binding activity, while the WT and mutant 1-mutation 3 antibodies had better binding activities, and the mutant 1 antibody had the highest binding activity.

(2) Affinity detection of antibodies and their mutants

(a) On the basis of mutation 1, other sites are mutated, and the sequence of each mutation is shown in Table 3 below.

Table 3 Mutation sites related to antibody affinity

Affinity analysis

Using the AMC sensor, the purified antibody was diluted to 10ug/mL with PBST, and the GRP antigen (0.699mg/mL) was serially diluted with PBST: 1.8μg/mL, 0.9μg/mL, 0.45μg/mL, 0.23μg/mL , 0.11 μg/mL and 0.056 μg/mL.

Running process: equilibrate in buffer 1 (PBST) for 60s, immobilize antibody in antibody solution for 300s, incubate in buffer 2 (PBST) for 180s, bind in antigen solution for 420s, dissociate in buffer 2 for 1200s, use 10mM pH 1.69 GLY solution and buffer 3 to regenerate the sensor and output data. The results are shown in Table 4 below. K _D is the equilibrium dissociation constant or affinity; kon is the association rate; kdis is the dissociation rate.

Table 4 Affinity detection data

	K D (M)	kon(1/Ms)	kdis(1/s)
Mutation 1	1.75E-09	1.15E+04	2.01E-05
Mutation 1-1	1.51E-09	1.78E+04	2.69E-05
Mutation 1-2	2.36E-09	1.42E+04	3.35E-05
Mutation 1-3	1.97E-09	1.58E+04	3.12E-05
Mutation 1-4	2.66E-09	1.19E+04	3.16E-05
Mutation 1-5	1.39E-09	1.87E+04	2.60E-05
Mutation 1-6	1.22E-09	1.81E+04	2.21E-05
Mutation 1-7	1.87E-09	1.05E+04	1.96E-05
Mutation 1-8	1.73E-09	1.95E+04	3.38E-05
Mutation 1-9	1.75E-09	1.08E+04	1.89E-05

Mutation 1-10	1.23E-09	2.34E+04	2.88E-05
Mutation 1-11	3.41E-09	1.14E+04	3.89E-05
Mutation 1-12	1.71E-09	1.22E+04	2.09E-05
Mutation 1-13	3.61E-09	1.07E+04	3.86E-05
Mutation 1-14	2.46E-09	1.85E+04	4.55E-05
Mutation 1-15	1.92E-09	1.03E+04	1.98E-05
Mutation 1-16	3.55E-09	1.30E+04	4.61E-05
Mutation 1-17	5.33E-09	5.40E+03	2.88E-05
Mutation 1-18	1.30E-09	1.74E+04	2.26E-05
Mutation 1-19	1.68E-09	1.22E+06	2.05E-03
Mutation 1-20	1.39E-09	2.33E+04	3.25E-05
Mutation 1-21	2.19E-09	2.03E+04	4.45E-05
Mutation 1-22	4.68E-09	6.80E+03	3.18E-05
Mutation 1-23	2.35E-09	1.44E+04	3.39E-05
Mutation 1-24	1.94E-09	1.36E+04	2.64E-05
Mutation 1-25	1.76E-09	1.20E+04	2.11E-05
Mutation 1-26	1.69E-09	2.04E+04	3.44E-05
Mutation 1-27	1.48E-09	1.82E+04	2.71E-05
Mutation 1-28	2.48E-09	1.21E+04	3.00E-05
Mutation 1-29	2.08E-09	2.38E+04	4.95E-05
Mutation 1-30	4.93E-09	9.20E+03	4.54E-05
Mutation 1-31	1.68E-09	1.48E+04	2.49E-05
Mutation 1-32	5.90E-09	5.80E+03	3.42E-05
Mutation 1-33	3.66E-09	1.08E+04	3.95E-05
Mutation 1-34	1.29E-09	1.67E+04	2.16E-05
Mutation 1-35	2.34E-09	1.46E+04	3.41E-05
Mutation 1-36	1.35E-09	1.67E+04	2.25E-05
Mutation 1-37	4.61E-09	8.40E+03	3.87E-05
Mutation 1-38	3.46E-09	1.33E+04	4.60E-05
Mutation 1-39	2.02E-09	1.09E+04	2.20E-05
Mutation 1-40	7.73E-09	5.60E+03	4.33E-05
Mutation 1-41	2.03E-09	1.34E+04	2.72E-05
Mutation 1-42	3.29E-09	8.30E+03	2.73E-05
Mutation 1-43	3.17E-09	1.51E+04	4.79E-05
Mutation 1-44	1.59E-09	1.33E+06	2.12E-03
Mutation 1-45	3.82E-09	1.00E+04	3.82E-05
Mutation 1-46	2.38E-09	2.06E+04	4.91E-05

From the results in Table 4, it can be seen that mutation 1 and its series of mutants have high affinity for GRP antigen, indicating that on the basis of mutation 1, the antibodies mutated according to the mutation method in Table 3 have high affinity. .

(b) On the basis of WT, other sites were mutated, and the affinity of each mutant was detected. The sequence of each mutation is shown in Table 5 below, and the corresponding affinity data is shown in Table 6.

Table 5 Mutations using WT as backbone

Table 6 Affinity test results of WT antibody and its mutants

	K D (M)	kon(1/Ms)	kdis(1/s)
WT	1.53E-07	3.60E+04	5.50E-03
WT 1	3.06E-07	1.93E+04	5.91E-03
WT 2	1.57E-07	3.93E+04	6.18E-03
WT 3	1.13E-07	3.56E+04	4.03E-03
WT 4	1.14E-07	3.96E+04	4.52E-03
WT 5	1.18E-07	3.03E+04	3.59E-03

According to the results in Table 6, it can be seen that WT and its series of mutants also have good affinity, indicating that on the basis of WT, the antibodies obtained by mutation according to the mutation method in Table 5 also have good affinity.

(3) Bare resistance stability assessment

The above antibodies were placed at 4°C (refrigerator), -80°C (refrigerator), and 37°C (incubator) for 21 days, and the 7-day, 14-day, and 21-day samples were taken for state observation, and the 21-day sample was tested for activity. , the results showed that under the above three test conditions, the antibody was placed for 21 days without obvious changes in protein status, and the activity did not show a downward trend with the increase of the test temperature, indicating that the above antibodies were stable. The following table 7 mutation 1 antibody is the OD result of the enzyme immunoassay activity detection for 21 days.

Table 7

Sample concentration (ng/ml)	30	8	0
4°C, 21-day samples	1.853	0.666	0.026
-80℃, 21 days sample	1.783	0.637	0.023
37°C, 21-day sample	1.842	0.562	0.027

(4) Application performance evaluation

The above antibodies were paired in a double-antibody sandwich method, detected on a chemiluminescence platform, and paired with another ProGRP antibody (available from Philips Biotech). The detection linear range of mutation 1 and its series of antibodies is 3-5000pg/ml, the detection correlation under different antigen concentrations can reach more than 0.96, and the sensitivity can reach 1.3-1.5pg/mL, with good linearity and sensitivity.

The above descriptions are only optional embodiments of the present disclosure, and are not intended to limit the present disclosure. For those skilled in the art, the present disclosure may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present disclosure should be included within the protection scope of the present disclosure.

Industrial Applicability

The present disclosure provides an anti-gastrin-releasing peptide antibody, a detection reagent and a kit, the antibody has good specificity and sensitivity to gastrin-releasing peptide, and can be used for detecting gastrin-releasing peptide and for Gastrin-releasing peptide is used as a marker for the diagnosis or auxiliary diagnosis of related diseases, and has a wide application prospect and high market value. In the case that the specific amino acid sequence is disclosed in this patent, those skilled in the art can easily produce the antibody or its functional fragment in this patent through chemical synthesis or recombination.

Claims (19)

1. An anti-gastrin-releasing peptide antibody or functional fragment thereof, characterized in that the antibody or functional fragment thereof comprises the following complementarity determining regions:

   CDR-VH1: G-Y-X1-F-X2-D-Y-N-M-X3; where: X1 is R or K; X2 is S or T; X3 is N or D;

   CDR-VH2: D-X1-N-P-X2-S-D-X3-T-X4-Y-N-Q-K-F-K-G; where: X1 is L or I; X2 is N or D; X3 is G, A or V; X4 is I or F;

   CDR-VH3: X1-T-W-X2-H; wherein: X1 is S or T; X2 is I, V or L;

   CDR-VL1: S-A-X1-Q-G-X2-S-N-Y-X3-S; wherein: X1 is S or T; X2 is I, V or L; X3 is I, V or L;

   CDR-VL2: X1-T-S-R-X2-Y-S; where: X1 is H or Y; X2 is I, V or L;

   CDR-VL3: X1-Q-Y-S-K-X2-P-W; wherein: X1 is Q or H; X2 is I, V or L.
2. The anti-gastrin-releasing peptide antibody or its functional fragment according to claim 1, wherein,

   In CDR-VH1, X1 is R;

   In CDR-VH2, X1 is I;

   In CDR-VH3, X1 is S;

   In CDR-VL1, X1 is S;

   In CDR-VL3, X1 is H;

   Preferably, in CDR-VH1, X2 is S;

   Preferably, in CDR-VH1, X2 is T;

   Preferably, in CDR-VH1, X3 is N;

   Preferably, in CDR-VH1, X3 is D;

   Preferably, in CDR-VH2, X2 is N;

   Preferably, in CDR-VH2, X2 is D;

   Preferably, in CDR-VH2, X3 is G;

   Preferably, in CDR-VH2, X3 is A;

   Preferably, in CDR-VH2, X3 is V;

   Preferably, in CDR-VH2, X4 is 1;

   Preferably, in CDR-VH2, X4 is F;

   Preferably, in CDR-VH3, X2 is 1;

   Preferably, in CDR-VH3, X2 is V;

   Preferably, in CDR-VH3, X2 is L;

   Preferably, in CDR-VL1, X2 is 1;

   Preferably, in CDR-VL1, X2 is V;

   Preferably, in CDR-VL1, X2 is L;

   Preferably, in CDR-VL1, X3 is 1;

   Preferably, in CDR-VL1, X3 is V;

   Preferably, in CDR-VL1, X3 is L;

   Preferably, in CDR-VL2, X1 is H;

   Preferably, in CDR-VL2, X1 is Y;

   Preferably, in CDR-VL2, X2 is 1;

   Preferably, in CDR-VL2, X2 is V;

   Preferably, in CDR-VL2, X2 is L;

   Preferably, in CDR-VL3, X2 is 1;

   Preferably, in CDR-VL3, X2 is V;

   Preferably, in CDR-VL3, X2 is L;

   Preferably, each complementarity determining region of the antibody or its functional fragment is selected from any one of the following mutation combinations 1-47:

   

   
3. The anti-gastrin-releasing peptide antibody or its functional fragment according to claim 2, wherein the antibody or its functional fragment and the gastrin-releasing peptide antigen have K _D ≤3×10 ^-7 mol /L affinity binding; preferably, K _D ≤7.7×10 ^-9 mol/L.
4. The anti-gastrin-releasing peptide antibody or its functional fragment according to claim 1, wherein,

   In CDR-VH1, X1 is K;

   In CDR-VH2, X1 is L;

   In CDR-VH3, X1 is T;

   In CDR-VL1, X1 is T;

   In CDR-VL3, X1 is Q;

   Preferably, each complementarity determining region of the antibody or its functional fragment is selected from any one of the following mutation combinations 48-53:

   
5. The anti-gastrin-releasing peptide antibody or its functional fragment according to any one of claims 1-4, wherein the antibody comprises a light chain backbone whose sequence is sequentially shown in SEQ ID NOs: 1-4 Regions FR1-L, FR2-L, FR3-L and FR4-L, and/or the heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H;

   Preferably, the antibody further comprises a constant region;

   Preferably, the constant region is selected from the constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD;

   Preferably, the species source of the constant region is bovine, horse, dairy cattle, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkeys, cockfights or people;

   Preferably, the constant region is derived from mice;

   Preferably, the light chain constant region sequence of the constant region is shown in SEQ ID NO:9, and the heavy chain constant region sequence of the constant region is shown in SEQ ID NO:10;

   Preferably, the functional fragment is selected from any one of VHH, F(ab')2, Fab', Fab, Fv and scFv of the antibody.
6. The anti-gastrin-releasing peptide antibody or its functional fragment according to any one of claims 1-4, wherein the antibody comprises sequences with sequence SEQ ID NOs: 1, 2, 3, and 4 in sequence, respectively. Light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L having at least 80% homology, and/or, respectively, have at least 80 % homology to the heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H.
7. A reagent or kit for detecting gastrin-releasing peptide, characterized in that it comprises the antibody or its functional fragment according to any one of claims 1-6.
8. The reagent or kit according to claim 7, wherein the antibody or its functional fragment is labeled with a detectable marker;

   Preferably, the detectable label is selected from fluorescent dyes, enzymes that catalyze the color development of substrates, radioisotopes, chemiluminescent reagents and nanoparticle labels;

   Preferably, the fluorescent dye is selected from fluorescein dyes and their derivatives, rhodamine dyes and their derivatives, Cy series dyes and their derivatives, Alexa series dyes and their derivatives, and protein dyes and their derivatives ;

   Preferably, the enzyme that catalyzes the coloration of the substrate is selected from horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase and glucose 6-phosphate deoxygenase enzymes;

   Preferably, the radioisotope is selected from ²¹² Bi, ¹³¹ I, ¹¹¹ In, ⁹⁰ Y, ¹⁸⁶ Re, ²¹¹ At, ¹²⁵ I, ¹⁸⁸ Re, ¹⁵³ Sm, ²¹³ Bi, ³² P, ⁹⁴ mTc, ⁹⁹ mTc, ²⁰³ Pb , ⁶⁷ Ga, ⁶⁸ Ga, ⁴³ Sc, ⁴⁷ Sc, ¹¹⁰ mIn, ⁹⁷ Ru, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁸ Cu, ⁸⁶ Y, ⁸⁸ Y, ¹²¹ Sn, ¹⁶¹ Tb, ¹⁶⁶ Ho, ¹⁰⁵ Rh, ¹⁷⁷ Lu, ¹⁷² Lu and ¹⁸ F;

   Preferably, the chemiluminescent reagent is selected from luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine esters and its derivatives, dioxetane Alkane and its derivatives, Lofenine and its derivatives, and peroxyoxalate and its derivatives;

   Preferably, the nanoparticle marker is selected from nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles and rare earth complex nanoparticles;

   Preferably, the colloid is selected from colloidal metals, disperse dyes, dye-labeled microspheres and latex;

   Preferably, the colloidal metal is selected from colloidal gold, colloidal silver and colloidal selenium.
9. Use of the antibody or its functional fragment according to any one of claims 1 to 6 in the preparation of a reagent or a kit for diagnosing or assisting in the diagnosis of related diseases marked by gastrin-releasing peptide.
10. A nucleic acid molecule encoding the antibody or functional fragment thereof of any one of claims 1-6.
11. A vector, characterized in that it contains a nucleic acid fragment encoding the antibody or functional fragment thereof according to any one of claims 1-6.
12. A recombinant cell, characterized in that it contains the vector of claim 11 .
13. Use of the antibody or functional fragment thereof according to any one of claims 1 to 6 or the reagent or kit according to claim 7 or 8 in detecting gastrin-releasing peptide.
14. Use of the antibody or functional fragment thereof according to any one of claims 1 to 6, or the reagent or kit according to claim 7 or 8, for detecting gastrin-releasing peptide.
15. A method of detecting gastrin-releasing peptide comprising:

    A) contacting the antibody or functional fragment thereof of any one of claims 1-6 with a sample from a subject for a binding reaction under conditions sufficient for the binding reaction to occur; and

    B) Detection of immune complexes produced by the binding reaction.
16. Use of the antibody or functional fragment thereof according to any one of claims 1 to 6 or the reagent or kit according to claim 7 or 8 in the diagnosis, prognosis evaluation and efficacy prediction of small cell lung cancer.
17. A method of diagnosing a disease associated with gastrin-releasing peptide as a marker in a subject, comprising:

    A) contacting the antibody or functional fragment thereof of any one of claims 1-6 with a sample from the subject under conditions sufficient for the binding reaction to occur to effect the binding reaction; and

    B) Detection of immune complexes produced by the binding reaction.
18. The method according to claim 17, wherein the related diseases marked by gastrin-releasing peptide are renal insufficiency, pulmonary neuroendocrine tumor and medullary thyroid cancer, preferably, the gastrin-releasing peptide A related disease that uses peptides as markers is small cell lung cancer.
19. A method for preparing the antibody or its functional fragment according to any one of claims 1-6, characterized in that it comprises: culturing the recombinant cell according to claim 12, and separating and purifying the culture product to obtain the Antibodies or functional fragments thereof.

Images