Anti-gastrin-releasing peptide precursor antibody and application thereof

Abstract

The invention discloses an antibody for resisting a gastrin-releasing peptide precursor and application of the antibody, and relates to the field of antibodies. The anti-gastrin-releasing peptide precursor antibody disclosed by the invention comprises a heavy chain complementarity determining region and a light chain complementarity determining region, provides an important raw material source for detection of a gastrin-releasing peptide precursor, and has good affinity or activity.

Description

Technical Field

[0001] The present invention relates to the technical field of antibodies, and in particular, to an antibody against progastrin-releasing peptide and its application. Background Art

[0002] 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-cell interaction. ProGRP (pro-gastrin-releasing peptide) is the precursor of gastrin-releasing peptide, and there are three variants in total. Their amino acid sequence lengths are 115aa, 118aa, and 125aa respectively, which are called subtype 1, subtype 2, and subtype 3, and the content ratio is 60:5:35. In the human body, the GRP gene encodes a preproprotein of 148 amino acids. As the signal peptide dissociates, its 148 preproproteins will be further decomposed into gastrin-releasing peptide of 27 amino acids and ProGRP of 68 amino acids. Compared with the very short half-life of GRP in the blood, the half-life of ProGRP is moderate and can reflect the level of GRP and the expression of the GRP gene.

[0003] Fetal and neonatal bronchial epithelial endocrine cells are rich in GRP content, while in adults, it is only present in the nerve fibers of the brain, gastrointestinal tract, and a small part of the pulmonary neuroendocrine cells, and the level is relatively low. Except for renal insufficiency, pulmonary neuroendocrine tumors, and medullary thyroid carcinoma, ProGRP rarely increases in patients with other malignant tumors or benign diseases.

[0004] ProGRP has high sensitivity and specificity for the diagnosis of small cell lung cancer (SCLC). Its level can reflect the treatment effect and judge whether the disease progresses and regresses. ProGRP has a good role in monitoring the recurrence of SCLC patients. In the prognosis evaluation of patients, the ProGRP level has a high correlation with prognostic factors, and prognostic evaluation and prediction of the curative effect can be carried out earlier.

[0005] Due to factors such as economic cost, low-dose spiral CT is not suitable for large-scale lung cancer population screening. The detection of tumor markers meets the clinical needs of rapidity, non-invasiveness, and economy. Since it became a marker for SCLC in 1989, various detection methods such as enzyme-linked immunosorbent assay (1995), chemiluminescence immunoassay, electrochemiluminescence assay (2013), and time-resolved immunofluorometric assay have been established for ProGRP detection in the past few decades. Different methods have their own advantages and disadvantages, but all require antibodies against ProGRP. High-quality antibodies are crucial for clinical detection of ProGRP.

[0006] Therefore, there is a strong demand in the art for anti-ProGRP antibodies with good performance. Summary of the Invention

[0007] The present application provides an antibody against progastrin-releasing peptide, which provides an important raw material source for the detection of progastrin-releasing peptide and has good activity or affinity.

[0008] To achieve the above object, according to one aspect of the present invention, there is provided an antibody against progastrin-releasing peptide, the antibody comprising three complementary determining regions of a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 20 and three complementary determining regions of any one of the light chain variable regions having amino acid sequences SEQ ID NO: 22, 23, 24, 25.

[0009] To achieve the above object, according to a second aspect of the present invention, there is provided an antibody against progastrin-releasing peptide, the antibody comprising the following complementary determining regions:

[0010] HCDR1, which comprises the amino acid sequence shown in SEQ ID NO: 1, or consists of the same;

[0011] HCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 2, or consists of the same;

[0012] HCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 3, or consists of the same;

[0013] LCDR1, which comprises the amino acid sequence shown in SEQ ID NO: 4, or consists of the same;

[0014] LCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 5, or consists of the same;

[0015] LCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 6, or consists of the same.

[0016] To achieve the above object, according to a third aspect of the present invention, there is provided an antibody against progastrin-releasing peptide, comprising a heavy chain variable region and / or a light chain variable region, the amino acid sequence of the heavy chain variable region being as shown in SEQ ID NO: 20; the amino acid sequence of the light chain variable region being as shown in any one of SEQ ID NO: 22, 23, 24, 25.

[0017] To achieve the above object, according to the fourth aspect of the present invention, there is provided an antibody against progastrin-releasing peptide precursor, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 21; the amino acid sequence of the light chain is as shown in any one of SEQ ID NO: 26, 27, 28, 29.

[0018] To achieve the above object, according to the fifth aspect of the present invention, there is provided an antibody conjugate, which comprises the above antibody.

[0019] To achieve the above object, according to the sixth aspect of the present invention, there is provided a reagent or a kit, which comprises the above antibody or the above antibody conjugate.

[0020] To achieve the above object, according to the seventh aspect of the present invention, there is provided a use of the above antibody and antibody conjugate in the preparation of a product for detecting progastrin-releasing peptide precursor.

[0021] 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0023] Figure 1 Results of reducing SDS-PAGE of Anti-ProGRP 8D12 Rmb1 to Anti-ProGRP 8D12 Rmb4. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0024] In the first aspect, an embodiment of the present invention provides an antibody against progastrin-releasing peptide precursor, which comprises three complementary determining regions of a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 20 and three complementary determining regions of a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NO: 22, 23, 24, 25.

[0025] It should be noted that HCDR1, HCDR2, and HCDR3 are amino acid sequences identical to HCDR1, HCDR2, and HCDR3 of the same heavy chain variable region defined in the antibody described in the first aspect, and LCDR1, LCDR2, and LCDR3 are amino acid sequences identical to LCDR1, LCDR2, and LCDR3 of the same light chain variable region defined in the antibody described in the first aspect.

[0026] For example, the aforementioned 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: 20; the aforementioned 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: 22.

[0027] In the present invention, the term "antibody" is used in the broadest sense and may include full-length monoclonal antibodies, bispecific, multispecific antibodies, chimeric antibodies, or antigen-binding fragments, as long as they exhibit the required biological activity.

[0028] The above-mentioned antigen-binding fragments generally have the same binding specificity as the antibody from which they are derived. Those skilled in the art can easily understand from the content described in the present invention that the above-mentioned antigen-binding fragments can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction to cleave disulfide bonds. Based on the disclosure of the structure of the complete antibody in the present invention, those skilled in the art can easily obtain the above-mentioned antigen-binding fragments.

[0029] The above-mentioned antigen-binding fragments can also be obtained by recombinant genetic techniques known to those skilled in the art or by synthesis using, for example, an automated peptide synthesizer, such as those sold by Applied BioSystems and the like.

[0030] In the present invention, the terms "complementary determining region", "CDR", or "CDRs" refer to the highly variable regions of the heavy and light chains of immunoglobulins, referring to regions containing one or more or even all of the major amino acid residues that contribute to the binding of an antibody or antigen-binding fragment to its recognized antigen or epitope. In the specific embodiments of the present invention, CDRs refer to the highly variable regions of the heavy and light chains of the antibody.

[0031] In the present invention, the heavy chain complementary determining regions are represented by HCDR and include HCDR1, HCDR2, and HCDR3; the light chain complementary determining regions are represented by LCDR and include LCDR1, LCDR2, and LCDR3.

[0032] The methods for defining CDRs are well-known in the art, and the methods for defining CDRs include: Kabat definition, Chothia definition, IMGT definition, Contact definition, and AbM definition. As described herein, the "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). For the "Chothia definition", see Chothia et al., J Mol Biol 196: 901-917 (1987). There are other methods for defining CDRs that may not strictly follow one of the above schemes, but will still overlap at least a part of the CDR region defined by Kabat. Although they may be shortened or lengthened according to the prediction or experimental results of specific residues or groups of residues. The exemplary defined CDRs are listed in Table 1 below, and the definitions in different literatures are slightly different. Given the amino acid sequence of the variable region of a given antibody, those skilled in the art can routinely determine which residues contain a specific CDR. It should be noted that the CDRs defined by other methods not limited to those in Table 1 also fall within the scope of protection of the present disclosure.

[0033] Table 1: CDR Definitions 1

[0034] CDR Kabat AbM2 IMGT Chothia HCDR1 <![CDATA[H31~H35 3 > <![CDATA[H26~H35 3 > <![CDATA[H26~H33..5 5 > <![CDATA[H26~H32..34 4 > 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

[0035] 1 The numbers of all CDR definitions in Table 1 are based on the Kabat numbering system (see below). The amino acid numbers on the heavy chain are represented by "H + number", and the amino acid numbers on the light chain are represented by "L + number". Those of ordinary skill in the art can clearly correspond the Kabat numbering system to any variable region sequence without relying on any experimental data outside the sequence itself. As described herein, the "Kabat numbering" refers to the numbering system described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0036] 2 As used in Table 1, "AbM" with a lowercase "b" refers to the CDRs defined by the "AbM" antibody modeling software of Oxford Molecular.

[0037] 3If neither H35A nor H35B is present, then CDR-H1 ends at position 35; if only H35A is present, then CDR-H1 ends at position 35A; if both H35A and H35B are present, then CDR-H1 ends at position 35B.

[0038] 4 If neither H35A nor H35B is present, then CDR-H1 ends at position 32; if only H35A is present, then CDR-H1 ends at position 33; if both H35A and H35B are present, then CDR-H1 ends at position 34.

[0039] 5 If neither H35A nor H35B is present, then CDR-H1 ends at position 33; if only H35A is present, then CDR-H1 ends at position 34; if both H35A and H35B are present, then CDR-H1 ends at position 35.

[0040] According to an embodiment of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one system or a combination of multiple systems among Kabat, Chothia, IMGT, AbM or Contact.

[0041] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system.

[0042] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Chothia system.

[0043] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT system.

[0044] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the AbM system.

[0045] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Contact system.

[0046] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a combination of the Kabat, Chothia, IMGT, AbM or Contact systems.

[0047] In a second aspect, an embodiment of the present invention provides an antibody against progastrin-releasing peptide, the antibody comprising the following complementarity-determining regions:

[0048] HCDR1, which comprises the amino acid sequence shown in SEQ ID NO:1, or consists of the same.

[0049] HCDR2, which comprises the amino acid sequence shown in SEQ ID NO:2, or consists of the same.

[0050] HCDR3, which comprises the amino acid sequence shown in SEQ ID NO:3, or consists of the same.

[0051] LCDR1, which comprises the amino acid sequence shown in SEQ ID NO:4, or consists of the same.

[0052] LCDR2, which comprises the amino acid sequence shown in SEQ ID NO:5, or consists of the same.

[0053] LCDR3, which comprises the amino acid sequence shown in SEQ ID NO:6, or consists of the same.

[0054] According to the embodiments of the present invention, the HCDRs and LCDRs are defined by the Kabat system.

[0055] In the present invention, the "framework region" or "FR" region includes the heavy-chain framework region and the light-chain framework region, which refers to the regions other than the CDRs in the variable regions of the heavy chain and the light chain of the antibody; wherein, the heavy-chain framework region can be further subdivided into adjacent regions separated by CDRs, including the HFR1, HFR2, HFR3 and HFR4 framework regions; the light-chain framework region can be further subdivided into adjacent regions separated by CDRs, including the LFR1, LFR2, LFR3 and LFR4 framework regions.

[0056] In the present invention, the variable region of the heavy chain is obtained by connecting the CDRs and FRs numbered below in the following combined arrangement: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the variable region of the light chain is obtained by connecting the CDRs and FRs numbered below in the following combined arrangement: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0057] In an alternative embodiment, the antibody according to the first or second aspect further has HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4.

[0058] In an alternative embodiment, HFR1 comprises / is as set forth in SEQ ID NO:7 or an amino acid sequence having at least 80% identity thereto;

[0059] HFR2 comprises / is as set forth in SEQ ID NO:8 or an amino acid sequence having at least 80% identity thereto;

[0060] HFR3 comprises / is as set forth in SEQ ID NO:9 or an amino acid sequence having at least 80% identity thereto;

[0061] HFR4 comprises / is as set forth in SEQ ID NO:10 or an amino acid sequence having at least 80% identity thereto;

[0062] LFR1 comprises / is as set forth in SEQ ID NO:11 or an amino acid sequence having at least 80% identity thereto;

[0063] LFR2 comprises / is as set forth in SEQ ID NO:12 or an amino acid sequence having at least 80% identity thereto;

[0064] LFR3 comprises / is as set forth in SEQ ID NO:13 or an amino acid sequence having at least 80% identity thereto; and

[0065] LFR4 comprises / is as set forth in SEQ ID NO:14 or an amino acid sequence having at least 80% identity thereto.

[0066] It should be noted that in other embodiments, the amino acid sequences of the respective framework regions of the antibody against gastrin-releasing peptide precursor 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 with the corresponding framework regions (SEQ ID NO:7, 8, 9, 10, 11, 12, 13 or 14).

[0067] In an alternative embodiment, LFR1 comprises / is the amino acid sequence as set forth in SEQ ID NO:17.

[0068] In an alternative embodiment, LFR2 comprises / is the amino acid sequence as set forth in SEQ ID NO:18.

[0069] In an alternative embodiment, the LFR3 comprises the amino acid sequence as shown in SEQ ID NO: 19.

[0070] In an alternative embodiment, the antibody binds to progastrin-releasing peptide with an affinity of KD < 7.07×10 -9 M.

[0071] In an alternative embodiment, the antibody binds to progastrin-releasing peptide with an affinity of KD ≤ 10 -8 M, KD ≤ 10 -9 M, KD ≤ 10 -10 M, KD ≤ 10 -11 M or KD ≤ 10 -12 M.

[0072] In an alternative embodiment, the antibody binds to progastrin-releasing peptide with an affinity of KD ≤ 6.52×10 -10 M.

[0073] There are many methods for measuring antibody affinity (KD), which can be classified into thermodynamic detection methods, kinetic detection methods, and dynamic equilibrium detection methods according to the detection principle. Among them, common thermodynamic detection methods include isothermal titration calorimetry (ITC); common kinetic detection methods include surface plasmon resonance (SPR) and biolayer interferometry (BLI); common dynamic equilibrium detection methods include enzyme-linked immunosorbent assay (ELISA), etc.

[0074] In an alternative embodiment, the KD is measured by a kinetic detection method; optionally, surface plasmon resonance, for example, by using a biosensor system such as system.

[0075] In a third aspect, an embodiment of the present invention provides an antibody against progastrin-releasing peptide, comprising a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 20, and the amino acid sequence of the light chain variable region is any one of SEQ ID NO: 22, 23, 24, and 25.

[0076] In an alternative embodiment, the antibodies described in the first, second, and third aspects above further comprise a constant region.

[0077] In an alternative embodiment, the constant region includes a heavy chain constant region and / or a light chain constant region.

[0078] In an alternative embodiment, 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.

[0079] In an alternative embodiment, the heavy chain constant region comprises CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM, and / or CH4 of IgM.

[0080] In an alternative embodiment, the IgG is selected from IgG1, IgG2, IgG3, or IgG4.

[0081] In an alternative embodiment, the light chain constant region is selected from the kappa or lambda light chain constant regions.

[0082] In an alternative embodiment, the species origin of the constant region is bovine, equine, dairy cow, porcine, ovine, rat, mouse, dog, camel, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, fighting cock, or human.

[0083] In an alternative embodiment, the species origin of the constant region is mouse.

[0084] In the present disclosure, the division of variable and constant region sequences follows the IMGT division method, see Lefranc, the international ImMunoGeneTics database. Nucl. Acids Res., 29(1):207 - 209(2001). DOI:10.1093 / nar / 29.1.207. PMID:11125093. and Martinez - Jean C. and Bosc N. or Ehrenmann, Patrice Duroux, Chantal Ginestoux, Gene table: house mouse (Mus musculus) IGHC, IMGT Repertoire. the internationalImMunoGenetics information http: / / www.imgt.org . Created: 16 / 03 / 2011. Version: 17 / 01 / 2020. or Ehrenmann, Patrice Duroux, Chantal Ginestoux, Gene table: house mouse (Mus musculus) IGLC, IMGT Repertoire. the international ImMunoGenetics information http: / / www.imgt.org.Created: 16 / 03 / 2011. Version: 17 / 01 / 2020. There will be differences in some amino acids between the variable regions divided by different methods and the C-terminus of the variable regions or the N-terminus of the constant regions divided by IMGT. The variable regions or constant regions divided by other methods well-known in the art are also within the protection scope of the present invention.

[0085] In an alternative embodiment, the heavy chain constant region sequence (CH) is as shown in SEQ ID NO: 15, and the light chain constant region (CL) sequence is as shown in SEQ ID NO: 16.

[0086] It should be noted that 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 with the above constant regions (SEQ ID NO: 15 or 16).

[0087] In an alternative embodiment, the antibody includes any one of F(ab)2, F(ab’)2, Fab’, Fab, Fv and scFv.

[0088] In a fourth aspect, the present invention provides an antibody against progastrin-releasing peptide, including a heavy chain and / or a light chain. The amino acid sequence of the heavy chain is as shown in SEQ ID NO: 21, and the amino acid sequence of the light chain is any one of SEQ ID NO: 26, 27, 28, 29.

[0089] In a fifth aspect, the present invention provides an antibody conjugate, and the antibody conjugate includes the above antibody.

[0090] In an alternative embodiment, the above antibody conjugate further includes biotin or a biotin derivative conjugated to the antibody.

[0091] In an alternative embodiment, the antibody conjugate further includes a marker or a purification tag conjugated to the antibody.

[0092] In an alternative embodiment, the above marker refers to a class of substances with characteristics such as luminescence, color development, radioactivity, etc. that can be directly observed by the naked eye or detected or detected by an instrument. Qualitative or quantitative detection of the corresponding target can be achieved through this characteristic.

[0093] In an alternative embodiment, the marker includes but is not limited to fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based markers.

[0094] In actual use, those skilled in the art can select appropriate markers according to the detection conditions or actual needs. No matter which marker is used, it falls within the protection scope of the present invention.

[0095] In an alternative embodiment, the fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (such as, but not limited to, fluorescein isothiocyanate (FITC), hydroxy fluorescein (FAM), tetrachloro fluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (such as, but not limited to, rhodamine B isothiocyanate (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (such as, but not limited to, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, etc. or their analogs), Alexa series dyes and their derivatives (such as, but not limited to, Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 633, 647, 680, 700, 750, etc. or their analogs), and protein dyes and their derivatives (such as, but not limited to, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (PerCP), etc.).

[0096] In an alternative embodiment, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0097] In an alternative embodiment, the radioisotopes include, but are 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.

[0098] In an alternative embodiment, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean luciferin and its derivatives, ruthenium bipyridine and its derivatives, acridinium ester and its derivatives, dioxetane and its derivatives, rosamide and its derivatives, and peroxyoxalate and its derivatives.

[0099] In alternative embodiments, the nanoparticle-based markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0100] In alternative embodiments, the colloids include, but are not limited to, colloidal metals, colloidal carbon, disperse dyes, dye-labeled microspheres, and latex.

[0101] In alternative embodiments, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0102] In alternative embodiments, the colloidal metal is colloidal gold.

[0103] In alternative embodiments, the above antibody conjugate further includes a solid-phase carrier conjugated to the antibody.

[0104] In alternative embodiments, the solid-phase carrier is selected from microspheres, plates, and membranes.

[0105] In alternative embodiments, the solid-phase carrier includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic particles, microtiter plates, glass, capillary tubes, nylon, and nitrocellulose membranes.

[0106] In a sixth aspect, the present invention provides a reagent or kit, which includes the above antibody or the above antibody conjugate.

[0107] As described above, the antibodies in some embodiments or examples of the present invention can effectively bind to progastrin-releasing peptide. Therefore, a reagent or kit containing the progastrin-releasing peptide antibody can effectively qualitatively or quantitatively detect progastrin-releasing peptide. Using the reagent or kit provided by the present invention, for example, it can be used for detections such as immunoblotting and immunoprecipitation that involve the specific binding performance of progastrin-releasing peptide and its antibody. As described above, the antibodies in some embodiments or examples of the present invention have higher binding activity or affinity with progastrin-releasing peptide. Therefore, the reagent or kit containing the antibody has higher detection sensitivity or specificity.

[0108] In a seventh aspect, the present invention provides a method for detecting progastrin-releasing peptide, including: a) contacting the above antibody, antibody conjugate, reagent, or kit with progastrin-releasing peptide in a test sample 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 which indicates the presence of the antigen in the test sample;

[0109] In alternative embodiments, the immune complex further includes a second antibody that binds to the antibody.

[0110] In an alternative embodiment, the immune complex further comprises a second antibody that binds to pro-gastrin-releasing peptide.

[0111] In an eighth aspect, the present invention provides the use of the above-mentioned anti-pro-gastrin-releasing peptide antibody and antibody conjugate in the preparation of a product for detecting pro-gastrin-releasing peptide.

[0112] It should be noted that the products of the present invention include, but are not limited to, reagents, reagent kits, test strips or reagent plates.

[0113] In a ninth aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody.

[0114] In a tenth aspect, the present invention provides a vector containing the above-mentioned nucleic acid molecule.

[0115] In an eleventh aspect, the present invention provides a cell containing the above-mentioned vector.

[0116] In a twelfth aspect, the present invention provides a method for preparing an anti-pro-gastrin-releasing peptide antibody, which comprises culturing the cells as described above.

[0117] Based on the disclosure of the amino acid sequence of the anti-pro-gastrin-releasing peptide antibody in the present invention, those skilled in the art can easily conceive of using genetic engineering techniques or other techniques (chemical synthesis, recombinant expression) to prepare the anti-pro-gastrin-releasing peptide antibody. For example, the antibody can be isolated and purified from the culture product of recombinant cells capable of recombinantly expressing the antibody described in any one of the above. This is easily achievable for those skilled in the art. Based on this, regardless of the technique used to prepare the anti-pro-gastrin-releasing peptide antibody of the present invention, it falls within the protection scope of the present invention.

[0118] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments not specified by the manufacturer are all conventional products that can be obtained through commercial purchase.

[0119] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those 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, the techniques employed or considered herein are standard methods. The materials, methods and examples are illustrative only and not restrictive.

[0120] Unless otherwise indicated, the 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 capabilities of those of ordinary skill in the art. Such techniques are fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, second 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 (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); and Current Protocols in Immunology (J.E. Coligan et al., eds., 1991), each of which is hereby expressly incorporated by reference.

[0121] The features and properties of the present invention will be further described in detail below in conjunction with the examples.

[0122] Example 1 Antibody Discovery of Monoclonal Antibodies

[0123] 1. Animal Immunization

[0124] Mix the ProGRP antigen (from Fapon Biotech) with complete Freund's adjuvant in equal volume to obtain an oily emulsion. Subcutaneously inject the emulsion into BALB / c mice at multiple points at a dose of 0.2 ml per mouse. After 14 days, immunize the mice intraperitoneally with the same antigen and adjuvant. Immunize until the fourth injection, collect tail blood for titer detection, and the titer meets the fusion requirements. Three days before fusion, mix the same dose of antigen with an equal volume of 0.9% sodium chloride injection and inject intraperitoneally for booster immunization.

[0125] 2. Preparation of hybridoma cell line

[0126] On the third day after the booster immunization of the mice, take out the spleen under sterile conditions. Mix mouse tumor cells and immunized spleen cells at a cell number ratio of 1:10, fuse and culture. On the sixth day of culture, change the HT culture medium twice. On the seventh day after fusion, take the cell supernatant for antibody detection to screen hybridoma cell strains secreting specific antibodies. A total of 1 strain was obtained and named Anti-ProGRP.

[0127] Example 2 Preparation of Anti-ProGRP 8D12 monoclonal antibody

[0128] In this example, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. The MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART TM RACE cDNA Amplification Kit was purchased from Takara. The pMD-18T vector was purchased from Takara. The plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were completed by Invitrogen.

[0129] (1) Preparation of antibody gene

[0130] Extract mRNA from the Anti-ProGRP hybridoma cell strain, obtain a DNA product by RT-PCR method, perform an A-addition reaction on the product with rTaq DNA polymerase, then insert it into the pMD-18T vector, and transform it into DH5α competent cells. After colonies grow, take 4 clones each of the Heavy Chain and Light Chain gene clones and send them to a gene sequencing company for sequencing.

[0131] (2) Sequence analysis of the variable region gene of Anti-ProGRP 8D12 antibody

[0132] The gene sequences obtained from the above sequencing were analyzed in the Kabat antibody database, and the VNTI 11.5 software was used for analysis to determine that the genes amplified by the heavy chain and light chain primer pairs were correct. Among the gene fragments amplified by the Light Chain, the VL gene sequence was 321 bp, and there was a 57-bp leader peptide sequence in front of it; among the gene fragments amplified by the Heavy Chain primer pair, the VH gene sequence was 342 bp, belonging to the VH1 gene family, and there was a 57-bp leader peptide sequence in front of it.

[0133] (3) Construction of recombinant antibody expression plasmid

[0134] pcDNA TM 3.4 The vector pcDNA3.4A was the constructed recombinant antibody eukaryotic expression vector. Multiple cloning sites such as HindIII, BamHI, and EcoRI had been introduced into this expression vector, and it was named the pcDNA3.4A expression vector, hereinafter simply referred to as the 3.4A expression vector; according to the antibody variable region gene sequencing results in the above pMD-18T, specific primers for the VL and VH genes of this antibody were designed, with HindIII and EcoRI restriction sites and protective bases at both ends. A 0.70-kb Light Chain gene fragment and a 1.37-kb Heavy Chain gene fragment were amplified by PCR.

[0135] The Heavy Chain and Light Chain gene fragments were respectively digested with HindIII / EcoRI double enzymes, and the 3.4A vector was digested with HindIII / EcoRI double enzymes. After purifying and recovering the fragments and the vector, the Heavy Chain gene and the Light Chain gene were respectively ligated into the 3.4A expression vector to obtain the recombinant expression plasmids of the Heavy Chain and the Light Chain.

[0136] 2. Production of recombinant antibody

[0137] Resuscitate HEK293 cells in advance, subculture them to a 200-ml system until the cell density reaches 3 - 5×10 6 cells / ml. Select the antibody concentration and cells when the cell density reaches this level, and the cell viability > 95%; centrifuge and wash the cells, resuspend them with the medium, and at the same time adjust the cell density to 2.9×10 6Wash the cells at cells / ml, resuspend them with the culture medium, and at the same time, use it as the cell diluent. Prepare plasmid DNA and transfection reagent diluents with the culture medium respectively. Add the transfection reagent diluent to the plasmid DNA diluent, mix well and let it stand at room temperature for 15 min; slowly add this mixture to the cell diluent within 1 min, mix well, sample and count, record and observe the viability of the cells after transfection, and place it in a constant temperature incubator at 35 °C for culture, with a rotation speed of 120 rmp and a CO2 content of 8%. After 13 days, collect the samples by centrifugation. Affinity purify the centrifuged supernatant with a protein A affinity chromatography column. Take 6 μg of the purified antibody for reducing SDS-PAGE, and the electrophoresis pattern is shown in the figure. After reducing SDS-PAGE, two bands are shown, one with Mr of 50 KD (heavy chain) and the other with Mr of 28 KD (light chain).

[0138] The obtained antibody was named Anti-ProGRP 8D12Rmb1, and the mutant antibody was obtained by mutating Anti-ProGRP 8D12Rmb1. The sequences of the heavy chain (H) and light chain (L) of the above antibodies are shown in the following table:

[0139] Table 2 Antibody sequences

[0140] Antibody Name Heavy Chain Light Chain Anti - ProGRP 8D12Rmb1 SEQ ID NO:21 SEQ ID NO:26 Anti - ProGRP 8D12Rmb2 SEQ ID NO:21 SEQ ID NO:27 Anti - ProGRP 8D12Rmb3 SEQ ID NO:21 SEQ ID NO:28 Anti - ProGRP 8D12Rmb4 SEQ ID NO:21 SEQ ID NO:29

[0141] Example 3 Performance detection of the antibody

[0142] 1. Affinity analysis

[0143] Dilute the purified antibody in advance, and at the same time perform gradient dilution on the ProGRP recombinant antigen (from PhyGenesis); use the CM5 chip that has been pre-coupled with goat anti-mouse IgG to test the binding and dissociation curves of the antigen and antibody on the Biacore 8K+ device, and the instrument automatically fits to obtain the affinity constant, binding rate, and dissociation rate. (KD represents the equilibrium dissociation constant, that is, the affinity constant; ka represents the binding rate; kd represents the dissociation rate)

[0144] Table 3 Affinity data

[0145] Sample Name KD ka kd Control 7.07E-09 5.12E+04 3.62E-04 Anti - ProGRP 8D12Rmb1 2.53E-10 3.24E+05 8.21E-05 Anti - ProGRP 8D12Rmb2 2.70E-10 3.12E+05 8.41E-05 Anti - ProGRP 8D12Rmb3 5.42E-10 7.56E+05 4.10E-04 Anti - ProGRP 8D12Rmb4 6.52E-10 8.14E+05 5.31E-04

[0146] 2. Activity identification

[0147] Dilute the ProGRP recombinant antigen (self-developed) with coating solution (main component: NaHCO₃) to 3 μg / ml, add 100 μL per well, and incubate overnight at 4°C; the next day, wash twice with washing solution (main components: Na₂HPO₄ + NaCl), and pat dry; add blocking solution (20% BSA + 80% PBS), 120 μL per well, incubate at 37°C for 1 h, and pat dry; add the diluted purified antibody and control antibody, 100 μL per well, incubate at 37°C for 30 min; wash 5 times with the washing solution, and pat dry; add goat anti-mouse IgG-HRP, 100 μL per well, incubate at 37°C for 30 min; wash 5 times with the washing solution, and pat dry; add chromogenic solution A (50 μL per well), add chromogenic solution B (50 μL per well), incubate for 10 min; add stop solution, 50 μL per well; read the OD value at 450 nm (reference 630 nm) on the microplate reader.

[0148] Note: Solution A (main components: citric acid + sodium acetate + acetanilide + urea peroxide); Solution B (main components: citric acid + EDTA·2Na + TMB + concentrated HCl); Stop solution (EDTA·2Na + concentrated H₂SO₄)

[0149] Table 4 Activity data

[0150] Concentration (ng / ml) 31.25 15.63 7.81 3.91 1.95 0 Control 1.402 0.993 0.648 0.318 0.109 0.075 Anti - ProGRP 8D12Rmb1 1.818 1.655 1.382 1.03 0.594 0.084 Anti - ProGRP 8D12Rmb2 1.551 1.388 0.997 0.582 0.348 0.086 Anti - ProGRP 8D12Rmb3 2.196 1.828 1.144 0.689 0.421 0.036 Anti - ProGRP 8D12Rmb4 1.577 0.955 0.533 0.291 0.176 0.031

[0151] 3. Stability assessment

[0152] Place the above-mentioned antibody at 4°C (refrigerator), -80°C (refrigerator), and 37°C (incubator) for 21 days. Take samples on the 7th, 14th, and 21st days for status observation, and conduct activity detection on the 21st-day samples. The results show that no obvious protein status changes are observed after the antibody is placed for 21 days under the three assessment conditions, and the activity does not show a downward trend with the increase of the assessment temperature, indicating that the above-mentioned antibody is stable. Table 5 below shows the OD results of the enzyme immunoassay activity detection of the antibody Anti-ProGRP 8D12Rmb2 after 21 days of assessment.

[0153] Table 5 Stability data

[0154] Sample Concentration (ng / ml) 15.63 7.81 0.00 Sample at 4°C for 21 days 1.352 0.932 0.022 Sample at - 80°C for 21 days 1.347 0.943 0.026 Sample at 37°C for 21 days 1.352 0.945 0.027

[0155] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

[0156] Some of the amino acid sequences involved in this application are shown in Table 6 as follows:

[0157]

[0158]

Claims

1. A gastrin-releasing peptide precursor antibody, the antibody comprising three complementarity-determining regions of a heavy-chain variable region having the amino acid sequence shown in SEQ ID NO: 20 and three complementarity-determining regions of a light-chain variable region having any one of the amino acid sequences SEQ ID NO: 22, 23, 24, 25.

2. The antibody according to claim 1, wherein The complementarity-determining regions of the variable regions are defined by any one system or a combination of multiple systems of Kabat, Chothia, IMGT, AbM or Contact.

3. An antibody against progastrin-releasing peptide, characterized in that, The antibody comprises the following complementarity-determining regions: HCDR1, which comprises the amino acid sequence shown in SEQ ID NO: 1, or consists of the same; HCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 2, or consists of the same; HCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 3, or consists of the same; LCDR1, which comprises the amino acid sequence shown in SEQ ID NO: 4, or consists of the same; LCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 5, or consists of the same; LCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 6, or consists of the same; Optionally, the HFR1 comprises SEQ ID NO: 7 or an amino acid sequence having at least 80% identity therewith; The HFR2 comprises SEQ ID NO: 8 or an amino acid sequence having at least 80% identity therewith; The HFR3 comprises SEQ ID NO: 9 or an amino acid sequence having at least 80% identity therewith; The HFR4 comprises SEQ ID NO: 10 or an amino acid sequence having at least 80% identity therewith; The LFR1 comprises SEQ ID NO: 11 or an amino acid sequence having at least 80% identity therewith; The LFR2 comprises SEQ ID NO: 12 or an amino acid sequence having at least 80% identity therewith; The LFR3 comprises SEQ ID NO: 13 or an amino acid sequence having at least 80% identity therewith; and The LFR4 comprises SEQ ID NO: 14 or an amino acid sequence having at least 80% identity therewith; Optionally, the antibody binds to gastrin-releasing peptide precursor with an affinity of KD < 7.07×10 -9 M.

4. An anti-pro-gastrin-releasing peptide antibody, comprising a heavy chain variable region and / or a light chain variable region, characterized in that, The amino acid sequence of the heavy-chain variable region is as shown in SEQ ID NO: 20; the amino acid sequence of the light-chain variable region is as shown in any one of SEQ ID NO: 22, 23, 24, 25; Optionally, the antibody further comprises a constant region; Optionally, the constant region comprises a heavy-chain constant region and / or a light-chain constant region; Optionally, the heavy-chain constant region is selected from the heavy-chain constant regions 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, the hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM; Optionally, the species origin of the constant region is bovine, equine, porcine, ovine, caprine, rat, mouse, dog, camel, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human; Optionally, the species origin of the constant region is mouse; Optionally, the heavy chain constant region sequence is as shown in SEQ ID NO: 15 or has at least 80% identity thereto; Optionally, the light chain constant region sequence is as shown in SEQ ID NO: 16 or has at least 80% identity thereto; Optionally, the antibody comprises any one of F(ab’)2, Fab’, Fab, Fv, and scFv.

5. A progastrin-releasing peptide precursor antibody, comprising a heavy chain and / or a light chain, characterized in that, The amino acid sequence of the heavy chain is as shown in SEQ ID NO: 21; the amino acid sequence of the light chain is as shown in any one of SEQ ID NO: 26, 27, 28, and 29.

6. An antibody conjugate, characterized in that, The antibody conjugate comprises the antibody according to any one of claims 1 to 5; Optionally, the antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody; Optionally, the antibody conjugate further comprises a label or a purification tag conjugated to the antibody; Optionally, the label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels; Optionally, the antibody conjugate further comprises a solid-phase carrier conjugated to the antibody.

7. A reagent or kit, characterized in that, The reagent or kit comprises the antibody according to any one of claims 1 to 5 or the antibody conjugate according to claim 6.

8. Use of the antibody according to any one of claims 1-5 or the antibody conjugate according to claim 6 in the preparation of a product for detecting progastrin-releasing peptide; Optionally, the use comprises: a) contacting the antibody according to any one of claims 1-5, the antibody conjugate according to claim 6, or the reagent or kit according to claim 7 with progastrin-releasing peptide in a test sample under conditions sufficient to effect an antibody / antigen binding reaction to form an immune complex; and b) detecting the presence of the immune complex, the presence of which indicates the presence of the antigen in the test sample; Optionally, the immune complex further comprises a second antibody that binds to the antibody; Optionally, the immune complex further comprises a second antibody that binds to progastrin-releasing peptide.

9. A nucleic acid, a vector, a cell, or a method for preparing the antibody according to any one of claims 1-5, wherein the nucleic acid encodes the antibody according to any one of claims 1 to 5; the vector contains the nucleic acid encoding the antibody according to any one of claims 1 to 5; the cell contains the above nucleic acid or vector; the method comprises the above cell.

Images