Soluble FMS-like tyrosine kinase-1 antibody and related detection method

Abstract

The invention relates to a novel soluble fms-like tyrosine kinase-1 (sFlt-1) antibody with high specificity and high sensitivity. The antibody can be used for detecting preeclampsia and / or monitoring progression and curative effect of diseases related to angiogenesis, not only can provide a more accurate treatment scheme for patients, but also can provide reliable basis for basic scientific research of biological processes such as angiogenesis, inflammatory response and tumor growth.

Description

Technical Field

[0001] The present invention relates to the field of immune detection, and in particular to an anti-FMS-like tyrosine kinase-1 monoclonal antibody or an antigen-binding fragment thereof, a kit comprising the antibody and related applications. Background Art

[0002] Preeclampsia is a pregnancy complication characterized by hypertension, edema, and proteinuria, which affects approximately 5-10% of pregnant women and causes substantial maternal and fetal morbidity and mortality. Symptoms of preeclampsia usually appear after the 20th week of pregnancy and are mainly detected by screening maternal blood pressure and urine protein, however, this screening method is ineffective for early diagnosis.

[0003] FMS-like tyrosine kinase-1 (Flt-1) is a transmembrane receptor expressed in endothelial cells, especially in trophoblast cells, which are essential for the formation of the placenta. Vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) are factors that bind to Flt-1 and promote endothelial cell proliferation and angiogenesis during placental development. sFlt-1 is a soluble form of Flt-1 that can bind to VEGF and PlGF, but does not promote endothelial cell proliferation, but inhibits angiogenesis, and is an important biomarker for the diagnosis of preeclampsia.

[0004] Immunoassay techniques, such as enzyme-linked immunosorbent assay (ELISA), have been used to measure unbound and sFlt-1-bound VEGF in fluid samples. Nevertheless, there is a need to develop new materials, methods, and kits to accurately determine sFlt-1 concentrations in test samples, especially for the diagnosis of preeclampsia and cardiovascular disease. In addition, new treatment options and methods are needed for patients who need sFlt-1 antagonists for treatment or prevention, such as patients at risk for preeclampsia or cardiovascular disease, or patients who already have these diseases.

[0005] The present invention aims to provide a highly specific and sensitive soluble FMS-like tyrosine kinase-1 (sFlt-1) antibody to meet these needs, to further improve the accuracy of detection and the feasibility of clinical application, and to optimize the performance of the antibody, including the affinity, specificity, production cost and convenience of clinical detection of the antibody. Summary of the invention

[0006] The inventors immunized mice with soluble FMS-like tyrosine kinase-1 (sFlt-1) immunogens, fused the spleen cells of the mice with myeloma cells, screened hybridoma cell lines that specifically bind to the sFlt-1 immunogens by ELISA, constructed recombinant antibodies, and obtained recombinant anti-sFlt-1 antibodies. Thus, the present invention was realized.

[0007] Therefore, in the first aspect, the present invention provides an anti-FMS-like tyrosine kinase-1 monoclonal antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain complementary determining region V H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementarity determining region V L CDR1, V L CDR2 and V L CDR3; of which:

[0008] V H The amino acid sequence of CDR1 is shown in SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 13, SEQ ID NO: 19 or SEQ ID NO: 29;

[0009] V H The amino acid sequence of CDR2 is shown in SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:25 or SEQ ID NO:30;

[0010] V H The amino acid sequence of CDR3 is shown in SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:21, SEQ ID NO:26 or SEQ ID NO:31;

[0011] V L The amino acid sequence of CDR1 is shown in SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:22 or SEQ ID NO:32;

[0012] V L The amino acid sequence of CDR2 is shown by SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:27 or SEQ ID NO:33; and

[0013] V LThe amino acid sequence of CDR3 is shown in SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:28 or SEQ ID NO:34.

[0014] In a second aspect, the present invention provides a nucleic acid molecule encoding the anti-FMS-like tyrosine kinase-1 monoclonal antibody or antigen-binding fragment thereof of the first aspect.

[0015] In a third aspect, the present invention provides a vector comprising the nucleic acid molecule of the second aspect.

[0016] In a fourth aspect, the present invention provides an expression cell comprising the nucleic acid molecule of the second aspect or the vector of the third aspect.

[0017] In a fifth aspect, the present invention provides use of the anti-FMS-like tyrosine kinase-1 monoclonal antibody or antigen-binding fragment thereof according to the first aspect in the preparation of a diagnostic agent for diagnosing pre-eclampsia.

[0018] In a sixth aspect, the present invention provides use of the anti-FMS-like tyrosine kinase-1 monoclonal antibody or antigen-binding fragment thereof according to the first aspect in the preparation of a preparation for monitoring the progression and therapeutic efficacy of angiogenesis-related diseases.

[0019] In a seventh aspect, the present invention provides a kit for detecting pre-eclampsia and / or monitoring the progression and therapeutic efficacy of diseases related to angiogenesis, comprising: the anti-FMS-like tyrosine kinase-1 monoclonal antibody or its antigen-binding fragment described in the first aspect; and instructions for use.

[0020] In summary, the present invention provides a highly specific and sensitive soluble FMS-like tyrosine kinase-1 (sFlt-1) antibody. The antibody can not only be used to diagnose preeclampsia, but also help monitor the progression and treatment effects of certain angiogenesis-related diseases. By detecting sFlt-1 levels, it can help achieve personalized medicine and provide patients with more accurate treatment plans. In the field of drug research and development, the anti-sFlt-1 antibody of the present invention can be used as a research tool to help researchers understand the effects of drugs on sFlt-1 levels, and then evaluate the efficacy and safety of drugs. In addition, the anti-sFlt-1 antibody of the present invention is also widely used in basic scientific research on biological processes such as angiogenesis, inflammatory response and tumor growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the specific implementation of the present invention or the technical solution in the prior art, the drawings required for use in the specific implementation or the description of the prior art are briefly introduced below.

[0022] Figure 1 The standard curve obtained by performing chemiluminescent microsphere immunoassay using the recombinant monoclonal antibody S10 of the present invention as the coating antibody and S52 as the labeling antibody is shown.

[0023] Figure 2 The results of the antibody combination (S10+S52) of the present invention on 53 human serum clinical samples are shown in Figure 1. Perform regression analysis of the test results. DETAILED DESCRIPTION

[0024] Hereinafter, the present invention will be described in detail with reference to the accompanying drawings. It should be understood that the following description is only used to illustrate the present invention by way of example, and is not intended to limit the scope of the present invention. The scope of protection of the present invention shall be subject to the attached claims. In addition, those skilled in the art will understand that the technical solution of the present invention may be modified without departing from the spirit and purpose of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0025] 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 the subject matter described herein belongs. Before describing the present invention in detail, the following definitions are provided for a better understanding of the present invention.

[0026] Where a numerical range is provided, such as a concentration range, a percentage range, or a ratio range, it is understood that each intervening value to one-tenth of the unit of the lower limit between the upper and lower limits of the range and any other stated or intervening values ​​in the stated range are included in the subject matter unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges, and such embodiments are also included in the subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or two limits, ranges excluding either or both of those included limits are also included in the subject matter.

[0027] In the context of the present invention, many embodiments use the expressions "comprising", "including" or "consisting essentially / mainly of..." The expressions "comprising", "including" or "consisting essentially / mainly of..." can usually be understood as open-ended expressions, indicating that it includes not only the elements, components, assemblies, method steps, etc. specifically listed after the expression, but also other elements, components, assemblies, method steps. In addition, in this article, the expressions "comprising", "including" or "consisting essentially / mainly of..." can also be understood as closed expressions in some cases, indicating that it only includes the elements, components, assemblies, method steps specifically listed after the expression, but does not include any other elements, components, assemblies, method steps. At this time, the expression is equivalent to the expression "consisting of..."

[0028] As used herein, the term "antibody" refers to an immunoglobulin molecule that is typically composed of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and the isotype of the antibody can be defined accordingly as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region (hinge region) of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (V H ) and the heavy chain constant region (C H ). The heavy chain constant region consists of three domains (C H1 , C H2 and C H3 Each light chain consists of a light chain variable region (V L ) and the light chain constant region (C L ). The light chain constant region consists of a domain C L The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. H and V L The variable regions can be further subdivided into regions of high variability, called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). For each heavy or light chain, its variable region each contains three CDRs, namely CDR1, CDR2 and CDR3. H and V L It consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable region (V H and V L ) respectively form the antigen binding site.

[0029] The rules for assigning amino acids to various regions or domains are defined in many papers: Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda Md (1987 and 1991)); Chothia & Lesk J. Mol. Biol. 1987; 196: 901-917; Chothia et al., Nature 1989; 342: 878-883; Ehrenmann, Francois, Quentin Kaas, and Marie-Paule Lefranc. "IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF." Nucleic acids research 2009; 38(suppl_1): D301-D307.

[0030] The exact boundaries of CDR have been defined differently according to different systems. The Kabat system not only provides a clear residue numbering system that can be applied to any variable region of an antibody, but also provides precise residue boundaries that define three CDRs, which are called Kabat CDRs; Chothia found that certain sub-portions within the Kabat system CDRs, despite having great diversity at the amino acid sequence level, have almost identical peptide main chain conformations, and these sub-portions are called Chothia CDRs, and Chothia CDRs have boundaries that overlap with Kabat CDRs. The above-mentioned overlapping boundaries are described by Padlan and MacCallum, and the CDR boundary definition may not strictly follow the above-mentioned system, such as the AbM definition. In this article, CDRs can be defined according to any of these systems, although the preferred embodiment uses the antibody numbering system of Chothia et al. to define CDRs.

[0031] As used herein, the term "monoclonal antibody" or "monoclonal antibody" refers to an antibody or a fragment of an antibody from a group of highly homologous antibody molecules, that is, a group of identical antibody molecules except for natural mutations that may occur spontaneously. The antibody molecule can be an immunoglobulin, whether it is a natural immunoglobulin or an immunoglobulin obtained partially or completely by a synthetic method. The antibody molecule can also include all polypeptides or proteins with an antibody binding domain. Antibody fragments with antibody domains are molecules such as Fab, scFv, Fv, dAb, Fd, and bifunctional antibodies. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies are relative to monoclonal antibodies, and they usually contain at least two or more different antibodies, and these different antibodies usually recognize different epitopes on the antigen. Monoclonal antibodies can usually be obtained using the hybridoma technology first reported by Kohler et al. ( G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity [J]. Nature, 1975; 256 (5517): 495), but can also be obtained by recombinant DNA technology (see US Patent 4,816,567). As used herein, the terms "monoclonal antibody" and "monoclonal antibody" have the same meaning and can be used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and can be used interchangeably; the terms "polypeptide" and "protein" have the same meaning and can be used interchangeably. And in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by Ala or A. Glycine can be represented by Gly or G, valine can be represented by Val or V, leucine can be represented by Leu or L, isoleucine can be represented by Ile or I, proline can be represented by Pro or P, phenylalanine can be represented by Phe or F, tyrosine can be represented by Tyr or Y, tryptophan can be represented by Trp or W, serine can be represented by Ser or S, threonine can be represented by Thr or T, cysteine ​​can be represented by Cys or C, methionine can be represented by Met or M, asparagine can be represented by Asn or N, glutamine can be represented by Gln or Q, aspartic acid can be represented by Asp or D, glutamic acid can be represented by Glu or E, lysine can be represented by Lys or K, arginine can be represented by Arg or R, and histidine can be represented by His or H.

[0032] As used herein, the term "recombinant antibody" means that the antibody gene is cloned into an expression vector by molecular biological techniques and then the expression vector is transfected into a suitable host cell line for expression. The recombinant antibody encoding gene may be identical or non-identical to the antibody encoding gene of natural origin. For example, the complete encoding gene of the antibody obtained by immunizing an animal can be cloned into an expression vector for expression, thereby obtaining an antibody identical to the antibody obtained by the immunizing animal, or the gene encoding the variable region (including heavy chain variable region and light chain variable region) of the antibody obtained by the immunizing animal can be cloned into an expression vector together with the gene encoding the constant region of the antibody from another species (such as human) source for expression, thereby obtaining an antibody including heavy chain and light chain variable region sequences from one species and constant region sequences from another species, such as an antibody with mouse heavy chain and light chain variable regions connected to a human constant region. The antibody is generally referred to as a "chimeric antibody" in the art.

[0033] As used herein, the term "antigen binding fragment" refers to fragments and antibody analogs from antibodies that are capable of binding to antigens, which generally include at least part of the antigen binding region or variable region (e.g., one or more CDRs) of the parental antibody. The antigen binding fragment retains at least some of the binding activity of the parental antibody. Typically, when the activity is expressed on a molar basis, the antigen binding fragment retains at least 10% of the parental binding activity. Specifically, the antigen binding fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parental antibody to the target. Examples of antigen binding fragments include, but are not limited to: Fab, Fab', F(ab')2, Fv fragments, linear antibodies, single-chain antibodies, nanobodies, domain antibodies, and multispecific antibodies. Among them, the "Fab fragment" consists of a light chain, a heavy chain CH1, and a variable region. The heavy chain of the Fab molecule cannot form a disulfide bond with another heavy chain molecule. A "Fab' fragment" contains one light chain and part of one heavy chain (including the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains); thus, an interchain disulfide bond can be formed between the two heavy chains of the two Fab' fragments to form a F(ab')2 molecule. An "Fv region" contains the variable regions from both the heavy and light chains, but lacks the constant region.

[0034] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its antigen.

[0035] In the present invention, primers are also used to carry out PCR amplification of the nucleotide sequence encoding the antibody. In the primer sequence, some sites only involve a single base, such as any one of adenine (A), guanine (G), cytosine (C) and thymine (T), while some sites involve a combination of two, three or four bases, in which case these bases are referred to as degenerate bases of each other, which are mainly determined based on the degeneracy of the codons. Degenerate bases can be represented by the letters R, Y, M, K, S, W, H, B, V, D, and N, wherein R represents A / G, Y represents C / T, M represents A / C, K represents G / T, S represents C / G, W represents A / T, H represents A / T / C, B represents G / T / C, V represents G / A / C, D represents G / A / T, and N represents A / T / C / G.

[0036] As mentioned above, the present invention aims to provide an anti-FMS-like tyrosine kinase-1 monoclonal antibody. As mentioned above, the inventors immunized mice with sFlt-1, fused the spleen cells of the mice with myeloma cells, and screened out hybridoma cell lines that can specifically bind to soluble FMS-like tyrosine kinase-1 by ELISA.

[0037] Therefore, in the first aspect, the present invention provides an anti-FMS-like tyrosine kinase-1 monoclonal antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain complementary determining region V H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementarity determining region V L CDR1, V L CDR2 and V L CDR3; of which:

[0038] V H The amino acid sequence of CDR1 is shown in SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 13, SEQ ID NO: 19 or SEQ ID NO: 29;

[0039] V H The amino acid sequence of CDR2 is shown in SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:25 or SEQ ID NO:30;

[0040] V HThe amino acid sequence of CDR3 is shown in SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:21, SEQ ID NO:26 or SEQ ID NO:31;

[0041] V L The amino acid sequence of CDR1 is shown in SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:22 or SEQ ID NO:32;

[0042] V L The amino acid sequence of CDR2 is shown by SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:27 or SEQ ID NO:33; and

[0043] V L The amino acid sequence of CDR3 is shown in SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:28 or SEQ ID NO:34.

[0044] In a specific embodiment, the anti-FMS-like tyrosine kinase-1 monoclonal antibody specifically binds to soluble FMS-like tyrosine kinase-1.

[0045] In yet another specific embodiment, the heavy chain variable region comprises a heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 1-3, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 4-6 respectively L CDR1, V L CDR2 and V L CDR3.

[0046] In yet another specific embodiment, the heavy chain variable region comprises a heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 7-9, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 10-12 respectively. L CDR1, V LCDR2 and V L CDR3.

[0047] In yet another specific embodiment, the heavy chain variable region comprises a heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 13-15, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 16-18, respectively. L CDR1, V L CDR2 and V L CDR3.

[0048] In yet another specific embodiment, the heavy chain variable region comprises a heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 19-21, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 22-24 respectively. L CDR1, V L CDR2 and V L CDR3.

[0049] In yet another specific embodiment, the heavy chain variable region comprises a heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 25, and SEQ ID NO: 26, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 27 and SEQ ID NO: 28, respectively. L CDR1, V L CDR2 and V L CDR3.

[0050] In yet another specific embodiment, the heavy chain variable region comprises a heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 29-31, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 32-24 respectively. LCDR1, V L CDR2 and V L CDR3.

[0051] In a specific embodiment, the antibody is a complete antibody comprising a variable region and a constant region. For the antibodies of the present invention, any framework region (FR) and any constant region can be used. The amino acid sequence of the FR or constant region used in the antibodies of the present invention can be the amino acid sequence of the original FR or constant region as a source, or it can be a different amino acid sequence obtained by performing one or more amino acid substitutions, deletions, additions and / or insertions on the amino acid sequence of the original FR or constant region. The structure used to support the CDR or CDR group of the present invention generally belongs to the antibody heavy chain or light chain sequence or a major part thereof, wherein the CDR or CDR group is located in the naturally occurring V sequence encoded by the rearranged immunoglobulin gene. H and V L The CDR or CDR group of the antibody variable domain corresponds to the position of the antibody variable domain.

[0052] In a specific embodiment, the heavy chain variable region further comprises heavy chain framework regions HFR1, HFR2, HFR3 and HFR4, H CDR1, V H CDR2 and V H CDR3 is arranged from the amino terminus to the carboxyl terminus according to HFR1, V H CDR1、HFR2、V H CDR2、HFR3、V H The sequence of CDR3 and HFR4 is arranged.

[0053] In a further specific embodiment, the heavy chain framework regions HFR1, HFR2, HFR3 and HFR4 have the sequences shown in SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38, or sequences having more than 80%, more than 85%, more than 90% or more than 95%, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity thereto, respectively, wherein the sequences of HFR1, HFR2, HFR3 and HFR4 are as follows:

[0054] SEQ ID NO:35(HFR1):EVQLQQSGPEVVKPGASVKISCKASSEQ ID NO:36(HFR2):FMNWVMQSHGKSLEWIGRI

[0055] SEQ ID NO:37(HFR3):TFYNQKFKGKATLTVDKSSSTAHM ELRSLASEDSAVYYCAR

[0056] SEQ ID NO: 38 (HFR4): WGRGTTVTVSS.

[0057] In yet another specific embodiment, the light chain variable region further comprises light chain framework regions LFR1, LFR2, LFR3 and LFR4, which are closely related to the V L CDR1, V L CDR2 and V L CDR3 is arranged from amino terminus to carboxyl terminus according to LFR1, V L CDR1, LFR2, V L CDR2, LFR3, V L The sequence of CDR3 and LFR4 is arranged.

[0058] In a further specific embodiment, the light chain framework regions LFR1, LFR2, LFR3 and LFR4 have the sequences shown in SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:42, or sequences having more than 80%, more than 85%, more than 90% or more than 95%, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity thereto, respectively, wherein the sequences of LFR1, LFR2, LFR3 and LFR4 are as follows:

[0059] SEQ ID NO:39(LFR1): DAVMTQIPLSLPVSLGDQVSISC

[0060] SEQ ID NO:40(LFR2):WYLQKPGQSPQLLIY

[0061] SEQ ID NO:41(LFR3):GIPARFSGSGSGTDFTLNIHPVEEED AATYYC

[0062] SEQ ID NO:42(LFR4): FGSGTKLEIK.

[0063] In a specific embodiment, the antibody further includes a constant region sequence, such as but not limited to a constant region sequence selected from any one of IgG, IgA, IgM, IgE and IgD. Those skilled in the art can make a selection based on needs, and this document does not specifically limit this.

[0064] In yet another specific embodiment, the constant region sequence may be from rat, mouse, rabbit, goat, sheep, horse, dog, cow, pig, chicken, duck, goose or human, but is not limited thereto.

[0065] In a second aspect, the present invention provides a nucleic acid molecule encoding the anti-FMS-like tyrosine kinase-1 monoclonal antibody or antigen-binding fragment thereof of the first aspect.

[0066] For those skilled in the art, knowing the amino acid sequence of a protein such as the anti-FMS-like tyrosine kinase-1 monoclonal antibody of the present invention, it is well within their capabilities to determine its nucleic acid coding sequence. In addition, in order to obtain a monoclonal antibody by recombinant means, the nucleic acid molecule can be cloned into a vector, and the vector can be further introduced into an expression cell to express the antibody protein using the expression cell.

[0067] In a third aspect, the present invention provides a vector comprising the nucleic acid molecule of the second aspect of the present invention.

[0068] In a preferred embodiment, the vector may be a plasmid vector, such as pEE12, pCAGGS, pTOPO, pcDNA, pTT, pTT3, pEFBOS, pBV, pJV and pBJ, but is not limited thereto.

[0069] In a specific embodiment, the vector is a pTOPO vector.

[0070] In a fourth aspect, the present invention provides an expression cell comprising the nucleic acid molecule of the second aspect or the vector of the third aspect.

[0071] The expression cell is prepared by introducing the above-mentioned nucleic acid molecule or the above-mentioned vector into a host cell by molecular biological methods well known to those skilled in the art.

[0072] As mentioned above, the inventors immunized mice with sFlt-1 immunogen, took the spleen cells of the mice and fused them with myeloma cells, and screened out hybridoma cell lines that can specifically bind to sFlt-1 by the ELISA method. After screening out the monoclonal cell lines that secrete the target antibody, the heavy chain and light chain variable region cDNAs can be recovered from the cell lines by reverse transcription-PCR, and the appropriate immunoglobulin constant region (such as human constant region) can be selected, and then the heavy chain and light chain variable region cDNAs and constant region cDNAs are transferred into host cells such as COS or CHO cells, thereby obtaining the expression cells expressing the target antibody of the present invention.

[0073] The above-mentioned monoclonal antibodies and recombinant DNA techniques can be used to produce other antibodies or chimeric molecules that retain the specificity of the original antibodies. These techniques may include introducing DNA encoding the variable region or complementarity determining region (CDR) of the antibody immunoglobulin into a eukaryotic expression vector comprising the constant region of different immunoglobulins or the constant region plus a framework region, or introducing both into a suitable eukaryotic expression vector, and then introducing the eukaryotic expression vector into an expression cell such as a CHO host cell, thereby obtaining each recombinant anti-sFlt-1 antibody.

[0074] In a specific embodiment, the expression cell can be a mammalian cell, such as a Chinese hamster ovary cell, a mini-hamster kidney cell, a monkey kidney cell, a mouse thymoma cell, or a human embryonic kidney cell. In a more specific embodiment, the expressing cells can be, for example, monkey kidney cells transformed by SV40 (COS-7, ATCC CRL1651), human embryonic kidney cells (HEK293 or HEK293 cells subcloned for growth in suspension culture, Graham et al., 1977, J. Gen Virol. 36:59), baby hamster kidney cells (BHK, ATCC CCL10), Chinese hamster ovary cells / -DHFR1 (CHO, Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216; e.g., DG44), mouse thymoma cells (NSO), mouse Sertoli cells (TM4, Mather, 1980, Biol. Reprod. 23:243-251), monkey kidney cells (CV-1, ATCC CCL70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical cancer cells (HELA, ATCC CCL2), canine kidney cells (MDCK, ATCC CCL34), buffalo rat liver cells (BRL3A, ATCC CRL1442), human lung cells (W138, ATCC CCL75), human liver cells (HepG2, HB8065), mouse breast tumor (MMT060562, ATCC CCL51), TR1 cells (Mather et al., 1982, Annals NY Acad. Sci. 383:44-68), MRC5 cells, FS4 cells, etc., but not limited thereto.

[0075] In a fifth aspect, the present invention provides use of the anti-FMS-like tyrosine kinase-1 monoclonal antibody or antigen-binding fragment thereof according to the first aspect in the preparation of a diagnostic agent for diagnosing pre-eclampsia.

[0076] In a specific embodiment, the anti-FMS-like tyrosine kinase-1 monoclonal antibody diagnoses pre-eclampsia by specifically binding to soluble FMS-like tyrosine kinase-1.

[0077] In yet another specific embodiment, the diagnosis is performed by immunochromatography, enzyme-linked antibody assay (ELISA), chemiluminescence, or electrochemiluminescence.

[0078] In a further specific embodiment, the ELISA detection can be a direct method, an indirect method, a sandwich method and a competitive method.

[0079] In a preferred embodiment, the detection is performed by immunochromatography, which includes but is not limited to fluorescent microsphere immunochromatography, colloidal gold immunochromatography, colored latex microsphere-based immunochromatography, time-resolved fluorescent microsphere immunochromatography, magnetic microsphere immunochromatography and quantum dot immunochromatography.

[0080] In the detection of the present invention, the anti-FMS-like tyrosine kinase-1 monoclonal antibody or its antigen-binding fragment can be used as a labeled antibody. For example, the monoclonal antibody or its antigen-binding fragment can be combined with nanoparticles, magnetic beads, microspheres, enzymes, fluorescent dyes, biotin, streptavidin, quantum dots, colloidal gold, etc. The anti-FMS-like tyrosine kinase-1 monoclonal antibody or its antigen-binding fragment of the present invention can also be used as a coated antibody. For example, the monoclonal antibody or its antigen-binding fragment can be combined with a solid phase such as a solid support. There is no particular limitation on the solid support used in the detection method of the present invention, which can be a porous or non-porous material, such as nanoparticles, magnetic beads, latex microspheres, fluorescent microspheres, microtiter plates, nitrocellulose membranes, microfluidic chips, etc.

[0081] In a sixth aspect, the present invention provides use of the anti-FMS-like tyrosine kinase-1 monoclonal antibody or antigen-binding fragment thereof according to the first aspect in the preparation of a preparation for monitoring the progression and therapeutic efficacy of angiogenesis-related diseases.

[0082] In a specific embodiment, the disease associated with angiogenesis can be pregnancy-induced hypertension, cardiovascular disease, perinatal cardiomyopathy, bronchial dysplasia, but is not limited thereto.

[0083] In another specific embodiment, the monitoring is performed by immunochromatography, enzyme-labeled antibody method (ELISA), chemiluminescence method, electrochemiluminescence method. Similarly, in monitoring, the anti-FMS-like tyrosine kinase-1 monoclonal antibody or antigen-binding fragment thereof of the present invention can be used as a labeled antibody, for example, combined with nanoparticles, magnetic beads, microspheres, enzymes, fluorescent dyes, biotin, streptavidin, quantum dots, colloidal gold, etc.; it can also be used as a coated antibody, for example, combined with a solid phase such as a solid support.

[0084] In a seventh aspect, the present invention provides a kit for detecting pre-eclampsia and / or monitoring the progression and therapeutic efficacy of diseases related to angiogenesis, comprising: the anti-FMS-like tyrosine kinase-1 monoclonal antibody or its antigen-binding fragment described in the first aspect; and instructions for use.

[0085] In a specific embodiment, the disease associated with angiogenesis can be pregnancy-induced hypertension, cardiovascular disease, perinatal cardiomyopathy, bronchial dysplasia, but is not limited thereto.

[0086] Example

[0087] In the following examples, the preparation method of the antibody of the present invention and the characterization of related properties are shown. Unless otherwise specified, the test methods used are all conventional methods, and unless otherwise specified, the test materials used in the following examples are all purchased from conventional reagent stores. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by technicians in the technical field of the present invention.

[0088] It should be noted that the terms used in the specification of the present invention are only for the purpose of describing specific embodiments, and are not intended to limit the present invention. The above summary of the invention and the detailed description below are only for the purpose of specifically explaining the present invention, and are not intended to limit the present invention in any way. Without departing from the spirit and purpose of the present invention, the scope of the present invention is determined by the attached claims.

[0089] Example 1: Immunizing mice with recombinant human sFlt-1 protein to obtain hybridomas

[0090] Female BALB / c mice were immunized 4 times with sFlt-1 immunogen (heavy chain biopharmaceutical HP132-1), with an interval of 2 weeks between each immunization, and the immunization dose was 100 μg / mouse. The first immunization used Freund's complete adjuvant (Sigma-Aldrich), mixed with an equal volume of the immunogen and injected subcutaneously at multiple points on the back. The next three immunizations used Freund's incomplete adjuvant (Sigma-Aldrich) mixed with an equal volume of the immunogen and injected intraperitoneally. Three days before the cell fusion experiment, the immunogen protein without adjuvant was injected intraperitoneally to boost immunization (100 μg / mouse).

[0091] Example 2: Mouse serum screening

[0092] Serum samples were collected 7-10 days after the last immunization of mice, and the reactivity to sFlt-1 was determined by indirect ELISA in 96-well plates. Mice with serum antibody titers higher than 1:10000 were selected for cell fusion experiments.

[0093] Example 3: Establishment of hybridoma cells

[0094] On the day of fusion, the mice were killed and their spleens were removed to prepare a single cell suspension. Mouse myeloma cells (SP2 / 0) were fused with the above mouse spleen cells at a ratio of 1:5 and mixed thoroughly. Preheated PEG1500 was added, and the cells were gently shaken. The cells were washed with preheated serum-free RPMI-1640 medium, and then resuspended with HAT selective medium. The cell suspension was plated into a 96-well culture plate at 200 μL / well, and the cells were cultured at 37°C and 5% CO2. After culturing for 4 to 7 days, HT medium was used for culture. When the fused cells grew to 1 / 10-1 / 5 of the bottom area of ​​the wells of the 96-well plate, the cell supernatant was taken for screening to detect antibody production.

[0095] Example 4: Hybridoma Screening and Selection

[0096] The anti-sFlt-1 antibodies in the hybridoma supernatant samples were analyzed by indirect ELISA. The 96-well plate was coated with a coating buffer containing the immunogen (0.05 mol / L, pH 9.6, PBS) at 4°C overnight, with an immunogen concentration of 1 μg / ml, 100 μL / well; the coating buffer was discarded, washed three times with phosphate buffered saline (PBST), and patted dry; the plate was washed with 2% BSA was blocked with PBST at 150 μL / well, incubated at 37°C for 2 h, washed 3 times with PBST, and patted dry; the supernatant of fused cells, 1:1000 diluted positive serum (as positive control) and 1:1000 diluted negative serum (as negative control) were added to the corresponding wells at 100 μL / well, incubated at 37°C for 1 h, washed 3 times with PBST, and patted dry; 1:6000 diluted horseradish peroxidase (HRP) labeled goat anti-mouse IgG (purchased from Sigma) was added at 100 μL / well, incubated at 37°C for 1 h, washed 3 times with PBST, and patted dry; tetramethylbenzidine (3,3',5,5'-Tetramethylbenzidine, TMB) substrate was added at 100 μL / well, and color was developed at room temperature in the dark for 10 min; 50 μL of 2 mol / L sulfuric acid was added to each well to terminate the reaction.

[0097] Detect the OD of all wells in the ELISA plate at a wavelength of 450 nm on the ELISA reader. 450nm When the OD of negative serum 450nm ≤0.1, to measure the absorbance value OD of the well 450nm The value is the OD of the negative well 450nm The criterion for judging is 2.1 times or more of the positive value.

[0098] After sampling and counting the cells in the positive wells, dilute them to 100 cells / 10mL culture medium, plate the diluted cell suspension to a 96-well cell culture plate at 100μL / well, and culture in a 37°C, 5% CO2 cell culture incubator. After 6-7 days, the formation of cloned cells can be observed under a microscope, and the single clone growth wells are marked. The cell supernatant is taken out, and the second round of ELISA detection is performed, and the positive wells are selected for limiting dilution again. The third round of ELISA detection is performed 5-6 days after the limiting dilution, and the positive wells are selected again for limiting dilution until the ELISA determination of the whole 96-well plate is positive. 115 hybridoma cell lines were obtained, transferred to culture flasks for expansion, and the above cell lines were stored in liquid nitrogen.

[0099] Example 5: Preparation and purification of monoclonal antibodies on cells

[0100] The 115 hybridoma cell lines obtained in Example 4 were cultured in RPMI-1640 medium containing 15% serum in a 10 cm culture dish. When the culture was expanded to about 4×10 7When the cells reached 3 × 10 / dish, centrifuge at 800 rpm for 5 min, discard the supernatant and transfer the cells to a 2 L spinner bottle. Serum-free medium was added to make the cell density about 3 × 10 5 After 1-2 weeks of continuous culture, when the cell death rate reaches 80%-90% (the cell density is about 1×10 6 -2×10 6 The cell suspension was collected and centrifuged at 6000 rpm for 20 min. The supernatant was purified by Protein A immunochromatography.

[0101] Example 6: Screening and detecting antibodies using the double antibody sandwich method

[0102] The 115 monoclonal antibodies obtained in Example 5 were screened for pairing using fluorescent microsphere immunochromatography (FICA).

[0103] Mix the fluorescent microspheres by blowing and beating, take 50μL (0.5mg), add to a 2ml round-bottom centrifuge tube, add 500μL of cleaning solution, mix by blowing and beating, centrifuge at low temperature 16000r / min for 10min to remove the supernatant. Prepare 5μL (0.1mg) of EDC solution and NHS solution respectively with activation solution. Take 190μL activation solution to resuspend the fluorescent microspheres and mix by blowing and beating. Add the above EDC solution and NHS solution to 200μL system respectively, mix by blowing and beating, and ultrasonicate in a water bath for 5min. Incubate at 37℃, 200r / min for 30min, centrifuge at low temperature 16000r / min for 10min to remove the supernatant, resuspend with 300μL and 500μL activation solution respectively, and centrifuge and wash once. Add labeling solution to the activated and washed fluorescent microspheres to a total volume of 200μL, and resuspend the microspheres. Add 0.05 mg of labeled antibody, mix by pipetting, sonicate in a water bath for 5 min, and oscillate at 37°C at 16000 r / min*3 h. Add 200 μL of 0.125% blocking solution diluted in half, mix by pipetting, sonicate in a water bath for 5 min, and oscillate at 37°C at 100 r / min for 30 min.

[0104] Then add 400 μL of 0.063% blocking solution diluted in half and mix by pipetting. After water bath sonication for 5 minutes, oscillate at 37°C, 100 r / min for 30 minutes. Centrifuge at 14000 r / min*10 minutes to remove the supernatant, and add 100 μL of preservation solution to resuspend (microsphere final concentration 5 mg / mL, antibody final concentration 0.5 mg / mL).

[0105] Use 10mM, pH 7.4 PBS buffer, 2% sucrose, dilute the coated antibody to a final concentration of 1.0mg / mL, treat the Sartorius 140 NC membrane, and dry at 37°C. Dilute the labeled antibody-microspheres to an appropriate concentration with the preservation solution and spray them on the labeling pad together with the quality control antibody. Set the spraying parameters of the film sprayer to 2μL / cm, spray the labeled antibody on the conjugate pad, and dry at 37°C for 4h.

[0106] Assembly test: The labeled fluorescent microspheres, coated NC membrane, and sample pad were assembled into a double antibody sandwich fluorescent microsphere immunochromatography rapid test card. 100uL 10ng / mL quality control (heavy chain bio HP132-1) was added to each test card. After standing at room temperature for 15 minutes, the ratio of the T line signal to the C line signal (T / C value) was determined using a fluorescence meter. The sample diluent was used as a negative control. The ratio of the quality control T / C value to the sample diluent T / C value was the signal-to-noise ratio.

[0107] Table 1: Double antibody sandwich fluorescent microsphere immunochromatography results

[0108]

[0109] The screening results are shown in Table 1 above. Anti-sFlt-1 monoclonal antibodies S20 and S67 are used as coating antibodies, which can be coated on microspheres to form a sandwich with other antibodies. Among them, anti-sFlt-1 monoclonal antibodies S10, S42, S69 and S52 antibodies are used as labeling antibodies, sandwiched with two coating antibodies, with high T / C values ​​and signal-to-noise ratios, and high detection sensitivity. S11 antibody has a low T / C value as a labeling antibody, and S69 antibody has a low signal-to-noise ratio as a labeling antibody, both showing poor sensitivity. Thus, six clones, S10, S20, S42, S67, S69 and S52, were screened out from 115 monoclonal antibodies.

[0110] Example 7: Chemiluminescent microsphere immunoassay for paired screening

[0111] Preparation of experimental materials:

[0112] Calibrator: The quality control protein (heavy chain bio HP132-1) was diluted with buffer (50 mM Tris + 1.5% BSA + 0.05% Tween20 + 0.9% NaCL + 0.05% PC300, PH7.4) to a concentration of 0, 160 pg / mL, 800 pg / mL, 4000 pg / mL, 20000 pg / mL, and 100000 pg / mL.

[0113] 1 mg of S52 antibody was taken and labeled with acridinium ester to obtain acridinium ester-labeled S52 monoclonal antibody, wherein the concentration of acridinium ester-labeled S52 antibody in the working solution was 0.3 μg / mL.

[0114] The biotin-labeled S10 antibody was taken to obtain a biotin-labeled S10 antibody (S10-biotin) with a concentration of 0.5 μg / mL.

[0115] The magnetic separation reagent was streptavidin microspheres (magnetic beads 0.2 mg / mL JSR MS160 / CA, streptavidin, heavy chain bio HP155-2).

[0116] The chemiluminescent microsphere immunoassay was performed using an automated analyzer, and the specific method was as follows: the antigen protein was contacted with the reagent of the microspheres, which were coated with biotin-labeled S10 antibodies and reacted at 37°C on the analyzer, followed by washing the microspheres to remove unbound antigen protein, and then adding acridinium ester-coupled S52 antibodies and incubating on the analyzer to form a sandwich. The unbound conjugate was then washed away, and the luminescent reagent was added to initiate the chemiluminescent reaction, which was measured in relative light units (RLUs). The standard curve was established using multiple concentrations of calibrators and fixed-value serum, and the detection system was calibrated.

[0117] Establishment of standard curve: 30 μL of standard (with concentrations of 0, 160 pg / mL, 800 pg / mL, 4000 pg / mL, 20000 pg / mL, and 100000 pg / mL, respectively) were added to the detection system in sequence, along with 50 μL of magnetic beads pre-coupled with biotin-labeled S10 antibody (the antibody and streptavidin magnetic beads were pre-coupled at a mass ratio of 1:600), and 50 μL of acridinium ester-labeled S52 antibody. After incubation at 37°C for 5 min, magnetic separation and washing were performed, and luminescent substrate solution A (solution A is a 0.10 mol / L H2O2 solution) and solution B (solution B is a 0.15 mol / L NaOH solution) were added to detect luminescence, and a standard curve of calibrator concentration relative to luminescence intensity was drawn, as shown in FIG. Figure 1 shown.

[0118] Consistency evaluation:

[0119] Detection method: The concentration of sFlt-1 in the test sample is detected by the same method as the above-mentioned standard curve establishment, wherein the standard is replaced by the test sample, and the luminescent signal detected by the instrument is used to calculate the concentration of sFlt-1 in the test sample through the above-mentioned standard curve.

[0120] According to the above detection method, the inventors used the above antibody combination (S10 / S52) of the present invention to detect 53 human serum clinical samples, and also used the sFlt-1 detection kit of Roche The 53 human serum samples were tested, and the test results were subjected to regression analysis with the test results obtained by administering the antibody combination of the present invention. The results are shown in Tables 2 and Figure 2, wherein the serum sFlt-1 concentration measured by the antibody combination (S10 / S52) of the present invention is the ordinate, and the result measured by Roche's sFlt-1 detection kit is the abscissa. The results show that the correlation equation is y=1.0045x-4.2413, and the determination coefficient r2 is 0.9988. The statistical processing results show that the antibody combination (S10 / S52) of the present invention has a good correlation with the clinical sample values ​​measured by Roche's kit.

[0121] Specificity detection: 300 clinical negative samples were tested using the antibody combination (S10 / S52) of the present invention, and 1 false positive result was detected, with a specificity of 99.67%.

[0122] The following pairings were further performed: microsphere-coupled monoclonal antibody S52, acridinium ester-labeled monoclonal antibody S69 (S52 / S69 pairing); microsphere-coupled monoclonal antibody S52, acridinium ester-labeled S20 (S52 / S20 pairing); microsphere-coupled monoclonal antibody S67, acridinium ester-labeled monoclonal antibody S10 (S67 / S10 pairing); microsphere-coupled monoclonal antibody S20, acridinium ester-labeled monoclonal antibody S42 (S20 / S42 pairing); microsphere-coupled monoclonal antibody S67, acridinium ester-labeled monoclonal antibody S42 (S67 / S42 pairing). The results are expressed in luminescence intensity (RLU) and are listed in Table 2 below. The results show that the following pairings have a good linear relationship and these pairings can also be used for the determination of sFlt-1.

[0123] Table 2: Chemiluminescent microsphere immunoassay results

[0124]

[0125] Example 8: Sequencing

[0126] This example describes sequencing of anti-sFlt-1 monoclonal antibodies. Total mRNA was extracted from hybridoma cells using commercial reagents (RNAM5 extraction kit (purchased from Beijing Polymer Biotech Co., Ltd.) according to the instructions. IgG heavy chain cDNA and kappa light chain cDNA were synthesized from the extracted mRNA using commercial mouse Ig primers MuIgGVH5'-A and MuIgGVH3'-2 and MuIgκVL5'-A and MuIgκVL 3'-1. The amplified VH and VL PCR products were cloned into a commercial vector (pTOPO-Blunt Cloning kit, Beijing Polymer Biotech), and the transformants were transformed into E. coli according to the manufacturer's instructions. Plasmids isolated from multiple transformed E. coli colonies were sequenced to identify the VH and VL gene sequences.

[0127] The antibody heavy chain variable region gene sequences and light chain variable region gene sequences of the hybridoma cell lines obtained by sequencing were analyzed, and the heavy chain complementary determining region sequences and light chain complementary determining region sequences are shown in Table 3 below (based on the Chothia numbering system).

[0128] Table 3: Complementarity determining region sequences of heavy and light chains

[0129]

[0130] Example 9: Preparation of recombinant antibodies

[0131] Combined with the antibodies listed in Table 3 above L CDR and V H The genes of CDR, and the exemplary V H FR1-V H FR4 and V shown in SEQ ID NO:39-42 L FR1-V L FR4, a eukaryotic expression plasmid of a recombinant antibody was constructed by molecular cloning. The eukaryotic expression plasmid was electroporated into CHO host cells, and after electroporation, it was added to a pressure screening medium (50 μM MSX) and cultured for 20 days. The supernatant was taken for ELISA detection (using HRP-labeled goat anti-mouse IgG as the secondary antibody for screening, the method is the same as above), and the cell line with stable expression of the recombinant antibody was screened.

[0132] After purification, recombinant antibodies s10, s67, s42, s69, s20, and s52 were obtained. The affinity of the recombinant antibodies was determined using an enzyme-linked immunosorbent assay. The antibody affinity is shown in Table 4 below, in nM.

[0133] Table 4: Antibody affinity test results of recombinant antibodies

[0134] Antibody nM s10 0.345 s67 0.423 s42 0.512 s69 0.456 s20 0.389 s52 0.378

[0135] From the results in Table 4, it can be seen that the affinity of the six monoclonal antibodies obtained by recombinant methods to S-Flt protein is at the nM level.

Claims

1. An anti-FMS-like tyrosine kinase-1 monoclonal antibody or an antigen-binding fragment thereof, the antibody or the antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain complementary determining region V H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementarity determining region V L CDR1, V L CDR2 and V L CDR3; of which: V H The amino acid sequence of CDR1 is shown in SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 13, SEQ ID NO: 19 or SEQ ID NO: 29; V H The amino acid sequence of CDR2 is shown in SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:25 or SEQ ID NO:30; V H The amino acid sequence of CDR3 is shown in SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:21, SEQ ID NO:26 or SEQ ID NO:31; V L The amino acid sequence of CDR1 is shown in SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:22 or SEQ ID NO:32; V L The amino acid sequence of CDR2 is shown by SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:27 or SEQ ID NO:33; and V L The amino acid sequence of CDR3 is shown in SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:28 or SEQ ID NO:

34.

2. The anti-FMS-like tyrosine kinase-1 monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein 1) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 1-3, H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 4-6 respectively. L CDR1, V L CDR2 and V L CDR3; 2) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 7-9, H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 10-12 respectively. L CDR1, V L CDR2 and V L CDR3; 3) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 13-15, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 16-18 respectively. L CDR1, V L CDR2 and V L CDR3; 4) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 19-21, H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 22-24 respectively. L CDR1, V L CDR2 and V L CDR3; 5) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 25 and SEQ ID NO: 26, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 27 and SEQ ID NO: 28, respectively. L CDR1, V L CDR2 and V L CDR3; or 6) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 29-31, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 32-24 respectively. L CDR1, V L CDR2 and V L CDR3.

3. The anti-FMS-like tyrosine kinase-1 monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein: The heavy chain variable region also includes framework regions HFR1, HFR2, HFR3 and HFR4, which are closely related to the V H CDR1, V H CDR2 and V H CDR3 is arranged from the amino terminus to the carboxyl terminus according to HFR1, V H CDR1、HFR2、V H CDR2、HFR3、V H The light chain variable region further comprises framework regions LFR1, LFR2, LFR3 and LFR4, wherein the HFR1, HFR2, HFR3 and HFR4 have the sequences shown in SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38, respectively, or sequences having 80% or more, 85% or more, 90% or more identity with the sequences, and the light chain variable region further comprises framework regions LFR1, LFR2, LFR3 and LFR4, which are identical to V L CDR1, V L CDR2 and V L CDR3 is arranged from amino terminus to carboxyl terminus according to LFR1, V L CDR1, LFR2, V L CDR2, LFR3, V L The invention relates to a method for treating a chromatin-containing protein comprising: a first polypeptide ... 4 . A nucleic acid molecule encoding the anti-FMS-like tyrosine kinase-1 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 .

5. A vector comprising the nucleic acid molecule according to claim 4; preferably, the vector is a plasmid vector, such as any one of pEE12, pCAGGS, pTOPO, pcDNA such as pCDNA3.1, pTT, pTT3, pEFBOS, pBV, pJV and pBJ.

6. An expression cell comprising the nucleic acid molecule of claim 4 or the vector of claim 5, preferably, the expression cell is a mammalian cell, for example, selected from Chinese hamster ovary cells, mini-hamster kidney cells, monkey kidney cells, mouse thymoma cells, and human embryonic kidney cells.

7. Use of the anti-FMS-like tyrosine kinase-1 monoclonal antibody or the antigen-binding fragment thereof according to any one of claims 1 to 3 in the preparation of a diagnostic agent for diagnosing pre-eclampsia.

8. Use of the anti-FMS-like tyrosine kinase-1 monoclonal antibody or its antigen-binding fragment according to any one of claims 1 to 3 in the preparation of a preparation for monitoring the progression and therapeutic efficacy of angiogenesis-related diseases, preferably, the angiogenesis-related diseases are pregnancy-induced hypertension, cardiovascular disease, perinatal cardiomyopathy, and bronchial dysplasia.

9. The use according to claim 7 or 8, wherein The diagnosis or monitoring is performed by immunochromatography, enzyme-linked antibody assay (ELISA), chemiluminescence, or electrochemiluminescence.

10. A kit for detecting pre-eclampsia and / or monitoring the progression and therapeutic efficacy of angiogenesis-related diseases, comprising: An anti-FMS-like tyrosine kinase-1 monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 3; and instructions for use.

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