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

Abstract

This invention relates to a novel, highly specific and sensitive soluble FMS-like tyrosine kinase-1 (sFlt-1) antibody. This antibody can be used to detect preeclampsia and / or to monitor disease progression and treatment efficacy related to angiogenesis, providing not only more precise treatment options for patients but also reliable evidence for basic scientific research on biological processes such as angiogenesis, inflammatory responses, and tumor growth.

Description

Technical Field

[0001] This invention relates to the field of immunoassay, and more specifically to an anti-FMS-like tyrosine kinase-1 monoclonal antibody or its antigen-binding fragment, a kit comprising the antibody, and related applications. Background Technology

[0002] Preeclampsia is a pregnancy complication characterized by high blood pressure, edema, and proteinuria. It affects approximately 5-10% of pregnant women and leads to significant maternal and fetal morbidity and mortality. Symptoms of preeclampsia typically appear after the 20th week of pregnancy and are primarily detected through screening for high blood pressure and proteinuria in the mother; 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, particularly in high levels in trophoblast cells, which are crucial for placental formation. 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 both VEGF and PlGF, but instead of promoting endothelial cell proliferation, it inhibits angiogenesis and is an important biomarker for diagnosing preeclampsia.

[0004] Immunoassay techniques, such as enzyme-linked immunosorbent assay (ELISA), have been used to measure unbound and sFlt-1-bound VEGF in liquid samples. Nevertheless, new materials, methods, and kits are still needed to accurately determine sFlt-1 concentrations in test samples, particularly in the diagnosis of preeclampsia and cardiovascular disease. Furthermore, new treatment options and approaches are required for patients requiring sFlt-1 antagonist therapy or prophylaxis, such as those at risk of preeclampsia or cardiovascular disease, or those already diagnosed with these conditions.

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

[0006] The inventors immunized mice with a soluble FMS-like tyrosine kinase-1 (sFlt-1) immunogen, fused mouse spleen cells with myeloma cells, and screened hybridoma cell lines that specifically bind to the sFlt-1 immunogen using ELISA. Recombinant antibodies were then constructed to obtain recombinant anti-sFlt-1 antibodies. Thus, the present invention was achieved.

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

[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 in 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 an anti-FMS-like tyrosine kinase-1 monoclonal antibody or an antigen-binding fragment thereof, as described in the first aspect.

[0015] In a third aspect, the present invention provides a carrier 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 the use of the anti-FMS-like tyrosine kinase-1 monoclonal antibody or its antigen-binding fragment as described in the first aspect in the preparation of a diagnostic agent for the diagnosis of preeclampsia.

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

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

[0020] In summary, this invention provides a highly specific and sensitive soluble FMS-like tyrosine kinase-1 (sFlt-1) antibody. This antibody can not only be used to diagnose preeclampsia but also help monitor the progression and treatment efficacy of certain angiogenesis-related diseases. By detecting sFlt-1 levels, personalized medicine can be achieved, providing patients with more precise treatment plans. In the field of drug development, the anti-sFlt-1 antibody of this invention can serve as a research tool, helping researchers understand the impact of drugs on sFlt-1 levels, thereby evaluating the efficacy and safety of drugs. Furthermore, the anti-sFlt-1 antibody of this invention also has wide applications in basic scientific research on biological processes such as angiogenesis, inflammatory responses, and tumor growth. Attached Figure Description

[0021] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below.

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

[0023] Figure 2 The results of testing 53 human serum clinical samples using the antibody combination (S10+S52) of the present invention are shown, along with the results using Roche's sFlt-1 detection kit. Regression analysis of the test results. Detailed Implementation

[0024] The present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the following description is merely illustrative and is not intended to limit the scope of the invention; the scope of protection of the invention is defined by the appended claims. Furthermore, those skilled in the art will understand that modifications can be made to the technical solutions of the present invention without departing from its spirit and intent. 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 pertains. Before a detailed description of the invention, the following definitions are provided to better understand it.

[0026] In cases where numerical ranges are provided, such as concentration ranges, percentage ranges, or ratio ranges, it should be understood that, unless the context explicitly specifies otherwise, all intermediate values ​​between the upper and lower limits of the range, up to one-tenth of the lower limit unit, and any other values ​​or intermediate values ​​within the range are included in the subject matter. 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, limited by any specific excluded limit values ​​within the range. Where the range includes one or two limit values, the range excluding any one or both of those included limit values ​​is also included in the subject matter.

[0027] In the context of this invention, many embodiments use the expressions "comprising," "including," or "basically / mainly composed of." The expressions "comprising," "including," or "basically / mainly composed of" are generally understood as open-ended expressions, indicating that they include not only the elements, components, parts, or method steps specifically listed after the expression, but also other elements, components, parts, or method steps. However, in this document, the expressions "comprising," "including," or "basically / mainly composed of" can also be understood as closed-ended expressions in certain situations, indicating that they only include the elements, components, parts, or method steps specifically listed after the expression, and do not include any other elements, components, parts, or method steps. In this case, the expression is equivalent to the expression "composed of."

[0028] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of a "light" (L) chain and a "heavy" (H) chain). Antibody light chains can be classified as κ and λ light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes can be defined accordingly as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by "J" regions (hinge regions) of approximately 12 or more amino acids, and the heavy chain also contains "D" regions of approximately 3 or more amino acids. Each heavy chain is further divided by a heavy chain variable region (V... H ) and heavy chain constant region (C H The heavy chain constant region consists of three structural domains (C). H1 C H2 and C H3 It consists of ) light chains. Each light chain is composed of a light chain variable region (V L ) and light chain constant region (C L It consists of a light chain constant region composed of a structural domain C. L Composition. The constant region of an antibody mediates the binding of immunoglobulins 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. V H and V L The region can be further subdivided into highly variable regions (called complementarity-determining regions (CDRs)), interspersed with more conservative regions called framework regions (FRs). For each heavy or light chain, its variable region contains three CDRs: CDR1, CDR2, and CDR3. Therefore, each V H and V L It consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable region (V) of each heavy chain / light chain pair... H and V L Each of these forms an antigen-binding site.

[0029] The rules for allocating amino acids to various regions or domains have been defined in several publications: 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 / 3D structure-DB and IMGT / Domain GapAlign: 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 precise boundaries of CDRs have been defined differently depending on the system. The Kabat system not only provides a definitive residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining three CDRs, which are referred to as Kabat CDRs. Chothia discovered that certain sub-regions within Kabat system CDRs, despite significant diversity at the amino acid sequence level, have nearly identical peptide backbone conformations; these sub-regions are referred to as Chothia CDRs, which have overlapping boundaries with Kabat CDRs. These overlapping boundaries are further described by Padlan and MacCallum. CDR boundary definitions may not strictly adhere to the aforementioned systems, such as the AbM definition. In this document, 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" refers to an antibody or a fragment of an antibody from a group of highly homologous antibody molecules, i.e., a group of identical antibody molecules except for the possibility of spontaneous natural mutations. The antibody molecules can be immunoglobulins, whether they are natural immunoglobulins or partially or wholly obtained through synthetic methods. The antibody molecules may also include all polypeptides or proteins having an antibody-binding domain, and antibody fragments having an antibody domain 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, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies that typically recognize different epitopes on an antigen. Monoclonal antibodies can usually be obtained using the hybridoma technique 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 it can also be obtained using recombinant DNA technology (see US Patent 4,816,567). As used herein, the terms “monoclonal antibody” and “monoclonal antibody” have the same meaning and are used interchangeably; the terms “polyclonal antibody” and “polyclonal antibody” have the same meaning and are used interchangeably; the terms “peptide” and “protein” have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented as Ala or A. Glycine can be represented by Gly or G, valine by Val or V, leucine by Leu or L, isoleucine by Ile or I, proline by Pro or P, phenylalanine by Phe or F, tyrosine by Tyr or Y, tryptophan by Trp or W, serine by Ser or S, threonine by Thr or T, cysteine ​​by Cys or C, methionine by Met or M, asparagine by Asn or N, glutamine by Gln or Q, aspartic acid by Asp or D, glutamic acid by Glu or E, lysine by Lys or K, arginine by Arg or R, and histidine by His or H.

[0032] As used herein, the term "recombinant antibody" refers to an antibody obtained by cloning an antibody gene into an expression vector using molecular biology techniques and then transfecting that expression vector into a suitable host cell line for expression. The encoding gene of a recombinant antibody may or may not be identical to the encoding gene of a naturally derived antibody. For example, the complete encoding gene of an 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 immunizing the animal. Alternatively, the gene encoding the variable region (including the heavy chain variable region and the light chain variable region) of an antibody obtained by immunizing an animal can be cloned together with the gene encoding the constant region of an antibody from another species (e.g., human) into an expression vector for expression, thereby obtaining an antibody comprising heavy chain and light chain variable region sequences from one species and constant region sequences from another species, such as an antibody having mouse heavy chain and light chain variable regions linked to human constant regions. This type of antibody is commonly referred to in the art as a "chimeric antibody."

[0033] As used herein, the term "antigen-binding fragment" refers to a fragment of an antibody capable of binding an antigen, and antibody analogues, which typically includes at least a portion of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody. The antigen-binding fragment retains at least some of the binding activity of the parent antibody. Typically, when activity is expressed on a molar basis, the antigen-binding fragment retains at least 10% of the parent antibody's binding activity. Specifically, the antigen-binding fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the parent antibody's binding affinity 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. A "Fab fragment" consists of a light chain, a CH1 region of a heavy chain, and a variable region. The heavy chain of the Fab molecule cannot form disulfide bonds with another heavy chain molecule. The “Fab’ fragment” contains a portion of one light chain and one heavy chain (including the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains); thus, interchain disulfide bonds can be formed between the two heavy chains of two Fab’ fragments to form the F(ab’)2 molecule. The “Fv region” contains variable regions from both the heavy and light chains, but lacks constant regions.

[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 the antigen it targets.

[0035] In this invention, PCR amplification of the nucleotide sequence encoding the antibody was also performed using primer pairs. In the primer sequences, some sites involve only a single base, such as any one of adenine (A), guanine (G), cytosine (C), and thymine (T), while other sites involve combinations of two, three, or four bases. In these cases, these bases are called degenerate bases, primarily determined based on the degeneracy of the codon. Degenerate bases can be represented by the letters R, Y, M, K, S, W, H, B, V, D, and N, where 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 previously stated, the present invention aims to provide a monoclonal antibody against FMS-like tyrosine kinase-1. As previously stated, the inventors immunized mice with sFlt-1, fused mouse spleen cells with myeloma cells, and screened hybridoma cell lines capable of specifically binding to soluble FMS-like tyrosine kinase-1 using ELISA.

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

[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 in 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 one specific implementation, 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 includes an amino acid sequence defined by the heavy chain complementarity-determining region V as shown in SEQ ID NO:1-3. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes an amino acid sequence defined by the light chain complementarity-determining region V as shown in SEQ ID NO:4-6. L CDR1, V L CDR2 and V L CDR3.

[0046] In yet another specific embodiment, the heavy chain variable region includes an amino acid sequence defined by the heavy chain complementarity-determining region V as shown in SEQ ID NO:7-9. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes an amino acid sequence defined by the light chain complementarity-determining region V as shown in SEQ ID NO:10-12. L CDR1, V LCDR2 and V L CDR3.

[0047] In yet another specific embodiment, the heavy chain variable region includes an amino acid sequence defined by the heavy chain complementarity-determining region V as shown in SEQ ID NO:13-15. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes an amino acid sequence defined by the light chain complementarity-determining region V as shown in SEQ ID NO:16-18. L CDR1, V L CDR2 and V L CDR3.

[0048] In yet another specific embodiment, the heavy chain variable region includes an amino acid sequence defined by the heavy chain complementarity-determining region V as shown in SEQ ID NO:19-21. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes an amino acid sequence defined by the light chain complementarity-determining region V as shown in SEQ ID NO:22-24. L CDR1, V L CDR2 and V L CDR3.

[0049] In yet another specific embodiment, the heavy chain variable region includes a heavy chain complementarity-determining region V, whose amino acid sequence is defined by the heavy chain complementarity-determining region shown in 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 an amino acid sequence defined by the light chain complementarity-determining region V as shown in 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 includes an amino acid sequence defined by the heavy chain complementarity-determining region V as shown in SEQ ID NO:29-31. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes an amino acid sequence defined by the light chain complementarity-determining region V as shown in SEQ ID NO:32-24. LCDR1, V L CDR2 and V L CDR3.

[0051] In one specific embodiment, the antibody is a complete antibody comprising a variable region and a constant region. For the antibodies of the present invention, any frame 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 original amino acid sequence of the source FR or constant region, or it can be a different amino acid sequence obtained by substituting, deleting, adding, and / or inserting one or more amino acids into 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 its major portion, wherein the CDR or CDR group is located in relation to the naturally occurring V region encoded by the rearranged immunoglobulin gene. H and V L At the corresponding position of the CDR or CDR group of the antibody variable domain.

[0052] In one specific implementation, the heavy chain variable region further includes heavy chain frame regions HFR1, HFR2, HFR3, and HFR4, which are related to V H CDR1, V H CDR2 and V H CDR3 progresses from the amino terminus to the carboxyl terminus according to HFR1, V H CDR1, HFR2, V H CDR2, HFR3, V H The order of CDR3 and HFR4.

[0053] In a further specific embodiment, the heavy chain frame regions HFR1, HFR2, HFR3, and HFR4 each have a sequence represented by SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:38, or a sequence having a similarity of 80% or more, 85% or more, 90% or more, or even 99% or more, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or more, 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 implementation, the light chain variable region further includes light chain framework regions LFR1, LFR2, LFR3, and LFR4, which are related to V L CDR1, V L CDR2 and V L CDR3 progresses from the amino terminus to the carboxyl terminus according to LFR1, V L CDR1, LFR2, V L CDR2, LFR3, V L The order of CDR3 and LFR4.

[0058] In a further specific embodiment, the light chain framework regions LFR1, LFR2, LFR3, and LFR4 each have a sequence represented by SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, and SEQ ID NO:42, or a sequence having a similarity of 80% or more, 85% or more, 90% or more, or even 99% or more, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or more, 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 one specific implementation, 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, which can be selected by those skilled in the art as needed, and there is no particular limitation herein.

[0064] In yet another specific implementation, the species source of the constant region sequence can be 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 an anti-FMS-like tyrosine kinase-1 monoclonal antibody or an antigen-binding fragment thereof, as described in 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 entirely within their capabilities to determine its nucleic acid coding sequence. Furthermore, to obtain a monoclonal antibody via recombinant methods, the nucleic acid molecule can be cloned into a vector, and the vector can be further introduced into expression cells to express the antibody protein.

[0067] In a third aspect, the present invention provides a carrier 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 one specific implementation, 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 expressed cells are prepared by introducing the aforementioned nucleic acid molecules or the aforementioned vectors into host cells using molecular biology methods well known to those skilled in the art.

[0072] As previously described, the inventors immunized mice with the sFlt-1 immunogen, fused mouse spleen cells with myeloma cells, and screened hybridoma cell lines that specifically bind to sFlt-1 using ELISA. After screening for monoclonal cell lines secreting the target antibody, the heavy and light chain variable region cDNAs were recovered from the cell lines by reverse transcription-PCR, and suitable immunoglobulin constant regions (e.g., human constant regions) were selected. The heavy and light chain variable region cDNAs and the constant region cDNA were then transferred into host cells such as COS or CHO cells, thereby obtaining expression cells expressing the target antibody of the present invention.

[0073] Using the above-mentioned monoclonal antibody and recombinant DNA technologies, other antibodies or chimeric molecules that retain the specificity of the original antibody can be generated. These technologies may include introducing DNA encoding the variable region or complementarity-determining region (CDR) of an antibody immunoglobulin into a eukaryotic expression vector that includes the constant region or constant region plus frame region of different immunoglobulins, or both into a suitable eukaryotic expression vector, and then introducing the eukaryotic expression vector into expression cells such as CHO host cells, thereby obtaining various recombinant anti-sFlt-1 antibodies.

[0074] In one specific implementation, the expressing cells can be mammalian cells, such as Chinese hamster ovary cells, hamster kidney cells, monkey kidney cells, mouse thymoma cells, and human embryonic kidney cells. In a more specific embodiment, the expressing cells may be, for example, monkey kidney cells transformed with SV40 (COS-7, ATCC CRL1651), human embryonic kidney cells (HEK293 or subcloned HEK293 cells for growth in suspension culture, Graham et al., 1977, J. Gen Virol. 36: 59), juvenile 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 testicular supporting cells (TM4, Mather, 1980, Biol. Reprod. 23: 243-251), monkey kidney cells (CV-1, ATCC CCL70), and African green monkey kidney cells (VERO-76, ATCC). CRL-1587, human cervical cancer cells (HELA, ATCCCCL2), canine kidney cells (MDCK, ATCCCCCL34), buffalo rat hepatocytes (BRL3A, ATCCCCRL1442), human lung cells (W138, ATCCCCCL75), human hepatocytes (HepG2, HB8065), mouse mammary tumors (MMT060562, ATCCCCCL51), TR1 cells (Mather et al., 1982, Annals NYAcad. Sci. 383:44-68), MRC5 cells, FS4 cells, etc., but not limited to these.

[0075] In a fifth aspect, the present invention provides the use of the anti-FMS-like tyrosine kinase-1 monoclonal antibody or its antigen-binding fragment as described in the first aspect in the preparation of a diagnostic agent for the diagnosis of preeclampsia.

[0076] In one specific implementation, the anti-FMS-like tyrosine kinase-1 monoclonal antibody diagnoses preeclampsia by specifically binding to soluble FMS-like tyrosine kinase-1.

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

[0078] In a further specific implementation, the ELISA detection can be a direct method, an indirect method, a sandwich method, or 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, immunochromatography based on colored latex microspheres, time-resolved fluorescent microsphere immunochromatography, magnetic microsphere immunochromatography, and quantum dot immunochromatography.

[0080] In the detection method of this invention, the anti-FMS-like tyrosine kinase-1 monoclonal antibody or its antigen-binding fragment can be used as a labeling antibody. For example, the monoclonal antibody or its antigen-binding fragment can bind to 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 this invention can also be used as a coating antibody. For example, the monoclonal antibody or its antigen-binding fragment can bind to a solid phase, such as a solid support. There are no particular limitations on the solid support used in the detection method of this invention; it 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 the use of the anti-FMS-like tyrosine kinase-1 monoclonal antibody or an antigen-binding fragment thereof as described in the first aspect in the preparation of a formulation for monitoring the progression and efficacy of angiogenesis-related diseases.

[0082] In one specific implementation, the angiogenesis-related diseases may be gestational hypertension, cardiovascular disease, perinatal cardiomyopathy, bronchial dysplasia, but are not limited to these.

[0083] In yet another specific implementation, the monitoring is performed using immunochromatography, enzyme-linked immunosorbent assay (ELISA), chemiluminescence, or electrochemiluminescence. Similarly, in monitoring, the anti-FMS-like tyrosine kinase-1 monoclonal antibody or its antigen-binding fragment of the present invention can be used as a labeling antibody, for example, binding to nanoparticles, magnetic beads, microspheres, enzymes, fluorescent dyes, biotin, streptavidin, quantum dots, colloidal gold, etc.; it can also be used as a coating antibody, for example, binding to a solid phase such as a solid support.

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

[0085] In one specific implementation, the angiogenesis-related diseases may be gestational hypertension, cardiovascular disease, perinatal cardiomyopathy, bronchial dysplasia, but are not limited to these.

[0086] Example

[0087] The following examples illustrate the preparation method and characterization of the antibodies of the present invention. Unless otherwise specified, all experimental methods used are conventional methods, and all experimental materials used in the following examples were purchased from conventional reagent stores. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0088] It should be noted that the terminology used in this specification is for the purpose of describing specific embodiments only and is not intended to limit the invention. The foregoing summary section and the following detailed description are for illustrative purposes only and are not intended to limit the invention in any way. The scope of the invention is defined by the appended claims without departing from its spirit and intent.

[0089] Example 1: Obtaining hybridomas by immunizing mice with recombinant human sFlt-1 protein

[0090] Female BALB / c mice were immunized four times with sFlt-1 immunogen (Heavy Chain Biotechnology HP132-1), with each immunization spaced two weeks apart. The immunization dose was 100 μg per mouse. The first immunization used Freund's complete adjuvant (Sigma-Aldrich), mixed with an equal volume of the immunogen and injected subcutaneously at multiple sites on the back. The subsequent 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, a booster immunization (100 μg per mouse) was administered intraperitoneally with unadjuvanted immunogen.

[0091] Example 2: Mouse serum screening

[0092] Serum samples were collected from mice 7-10 days after the last immunization, and the reactivity to sFlt-1 was measured using an indirect ELISA method 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, mice were sacrificed and their spleens were removed to prepare a single-cell suspension. Mouse myeloma cells (SP2 / 0) were fused with the above-mentioned mouse spleen cells at a ratio of 1:5 and thoroughly mixed. Preheated PEG1500 was added, and the mixture was gently shaken. The cells were washed with preheated serum-free RPMI-1640 medium and then resuspended in HAT selective medium. The cell suspension was seeded at 200 μL / well into 96-well plates and cultured at 37°C and 5% CO2. After 4 to 7 days of culture, the cells were cultured in HT medium. When the fused cells grew to 1 / 10 to 1 / 5 of the bottom area of ​​the wells in the 96-well plate, the cell supernatant was collected for screening to detect antibody production.

[0095] Example 4: Hybridoma Screening and Selection

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

[0097] The OD values ​​of all wells in the ELISA plate were measured at a wavelength of 450 nm using an ELISA reader. 450nm Value. When the OD of negative serum 450nm ≤0.1, to measure the absorbance OD of the orifice. 450nm The value is the negative pore OD. 450nm A positive result is defined as a result that is more than 2.1 times higher than the threshold.

[0098] After counting and sampling cells from positive wells, the cells were diluted to 100 cells / 10 mL of culture medium. The diluted cell suspension was seeded at 100 μL / well into 96-well cell culture plates and incubated at 37°C in a 5% CO2 incubator. After 6-7 days, clonal cell formation was observed under a microscope. Individual clonal growth wells were marked, and the cell supernatant was collected for a second round of ELISA testing. Positive wells were selected for further limiting dilution. A third round of ELISA testing was performed 5-6 days after limiting dilution, again selecting positive wells for limiting dilution, until the entire 96-well plate showed a positive ELISA result. 115 hybridoma cell lines were obtained, transferred to culture flasks for expansion, and 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 10cm culture dishes using RPMI-1640 culture medium containing 15% serum. The cells were expanded to approximately 4 × 10⁻⁶ cells / year. 7Centrifuge at 800 rpm for 5 min, discard the supernatant and transfer the cells to a 2 L roller flask. Add serum-free culture medium to bring the cell density to approximately 3 × 10⁶ cells / plate. 5 Cells / mL, cultured in roller bottles. Continue culturing for 1-2 weeks, until the cell death rate reaches 80%-90% (at which point the cell density is approximately 1×10⁻⁶). 6 -2×10 6 Collect the cell suspension (cells / mL), centrifuge at 6000 rpm for 20 min, collect the supernatant, and purify the supernatant using Protein A immunochromatography.

[0101] Example 6: Screening and Detection of Antibodies Using a Double Antibody Sandwich Method

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

[0103] Mix the fluorescent microspheres by pipetting, and measure 50 μL (0.5 mg) into a 2 ml round-bottom centrifuge tube. Add 500 μL of washing buffer, mix by pipetting, and centrifuge at 16000 rpm for 10 min at low temperature to remove the supernatant. Prepare 5 μL (0.1 mg) each of EDC and NHS solutions using activation buffer. Resuspend the fluorescent microspheres in 190 μL of activation buffer and mix by pipetting. Add the above EDC and NHS solutions to make 200 μL systems respectively, mix by pipetting, and sonicate in a water bath for 5 min. Incubate at 37℃ and 200 rpm for 30 min, then centrifuge at 16000 rpm for 10 min at low temperature to remove the supernatant. Resuspend and wash once with 300 μL and 500 μL of activation buffer, respectively. Add labeling solution to the activated and washed fluorescent microspheres to a total volume of 200 μL and resuspend the microspheres. Add 0.05 mg of labeled antibody, mix by pipetting, sonicate in a water bath for 5 min, and then incubate at 37°C with shaking at 16000 rpm for 3 h. Add 200 μL of half-diluted 0.125% blocking buffer, mix by pipetting, sonicate in a water bath for 5 min, and then incubate at 37°C with shaking at 100 rpm for 30 min.

[0104] Add 400 μL of half-diluted 0.063% blocking buffer, mix thoroughly by pipetting, sonicate in a water bath for 5 min, and then incubate at 37°C with constant temperature shaking at 100 rpm for 30 min. Centrifuge at 14000 rpm for 10 min at low temperature to remove the supernatant, and resuspend in 100 μL of preservation buffer (final microsphere concentration 5 mg / mL, final antibody concentration 0.5 mg / mL).

[0105] Dilute the coated antibody to a final concentration of 1.0 mg / mL using 10 mM PBS buffer (pH 7.4) and 2% sucrose. Treat the Sartorius 140 NC membrane and dry at 37°C. Dilute the labeled antibody-microspheres to an appropriate concentration with preservation solution and spray them onto the labeling pad along with the quality control antibody. Set the gold spraying parameters of the membrane to 2 μL / cm and spray the labeled antibody onto the conjugate pad. Dry at 37°C for 4 hours.

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

[0107] Table 1: Immunochromatographic results of double-antibody sandwich fluorescent microspheres

[0108]

[0109] The screening results are shown in Table 1 above. Anti-sFlt-1 monoclonal antibodies S20 and S67, used as coating antibodies, can form sandwich structures with various other antibodies on microspheres. Among them, anti-sFlt-1 monoclonal antibodies S10, S42, S69, and S52, used as labeled antibodies, sandwiched with the two coating antibodies, exhibited high T / C values ​​and signal-to-noise ratios, indicating high detection sensitivity. Antibody S11, as a labeled antibody, had a low T / C value, and antibody S69, as a labeled antibody, had a low signal-to-noise ratio, both showing poor sensitivity. Therefore, six clones—S10, S20, S42, S67, S69, and S52—were selected from 115 monoclonal antibodies.

[0110] Example 7: Paired Screening Using Chemiluminescent Microsphere Immunoassay

[0111] Preparation of experimental materials:

[0112] Calibrators: The quality control protein (Heavy Chain Bio HP132-1) was diluted with buffer (50mM Tris + 1.5% BSA + 0.05% Tween 20 + 0.9% NaCl + 0.05% PC300, pH 7.4) to prepare calibrators with concentrations of 0, 160 pg / mL, 800 pg / mL, 4000 pg / mL, 20000 pg / mL, and 100000 pg / mL.

[0113] Take 1 mg of S52 antibody and label it with acridine ester to obtain acridine ester-labeled S52 monoclonal antibody, wherein the concentration of acridine ester-labeled S52 antibody in the working solution is 0.3 μg / mL.

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

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

[0116] Chemiluminescent microsphere immunoassay was performed using an automated analyzer. The specific method involved contacting the antigen protein with reagents on the microspheres coated with biotin-labeled S10 antibody. The reaction was carried out at 37°C on the analyzer. The microspheres were then rinsed to remove unbound antigen protein. Acridinium ester-conjugated S52 antibody was then added, and the mixture was incubated on the analyzer to form a sandwich. After rinsing to remove unbound conjugates, a luminescent reagent was added to initiate the chemiluminescent reaction. Measurements were taken in relative light units (RLUs). A standard curve was established using calibrators at multiple concentrations and baseline sera, and the detection system was calibrated.

[0117] Establishment of the standard curve: Add 30 μL of standards (concentrations of 0, 160 pg / mL, 800 pg / mL, 4000 pg / mL, 20000 pg / mL, and 100000 pg / mL) sequentially to the detection system, along with 50 μL of biotin-labeled S10 antibody pre-coupled magnetic beads (antibody and streptavidin magnetic beads 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, perform magnetic separation and washing. Add luminescent substrate solution A (0.10 mol / L H2O2 solution) and solution B (0.15 mol / L NaOH solution) to detect luminescence. Plot a standard curve of calibrator concentration versus luminescence intensity. Figure 1 As shown.

[0118] Consistency assessment:

[0119] Detection method: The concentration of sFlt-1 in the test sample was detected using the same method as the method described above for establishing the standard curve, except that the standard was replaced with the test sample, and the concentration of sFlt-1 in the test sample was calculated by using the emission signal obtained by the instrument through the standard curve described above.

[0120] According to the above detection method, the inventors used the antibody combination (S10 / S52) of the present invention to test 53 human serum clinical samples, and also used Roche's sFlt-1 detection kit. These 53 human serum samples were tested, and the results were compared with those obtained by applying the antibody combination of the present invention using regression analysis. The results are shown in Table 2 below. Figure 2The serum sFlt-1 concentration measured using the antibody combination (S10 / S52) of this invention is plotted on the ordinate, while the result measured by Roche's sFlt-1 detection kit is plotted on the abscissa. The results show a correlation equation of y = 1.0045x - 4.2413, with a coefficient of determination r² of 0.9988. Statistical analysis indicates a good correlation between the antibody combination (S10 / S52) of this invention and the clinical sample values ​​measured by Roche's kit.

[0121] Specificity test: Using the antibody combination (S10 / S52) of the present invention to test 300 clinical negative samples, one false positive result was detected, with a specificity of 99.67%.

[0122] Further pairings were performed as follows: microspheres coupled with monoclonal antibody S52 and acridine ester-labeled monoclonal antibody S69 (S52 / S69 pairing); microspheres coupled with monoclonal antibody S52 and acridine ester-labeled monoclonal antibody S20 (S52 / S20 pairing); microspheres coupled with monoclonal antibody S67 and acridine ester-labeled monoclonal antibody S10 (S67 / S10 pairing); microspheres coupled with monoclonal antibody S20 and acridine ester-labeled monoclonal antibody S42 (S20 / S42 pairing); microspheres coupled with monoclonal antibody S67 and acridine ester-labeled monoclonal antibody S42 (S67 / S42 pairing). The results, expressed as luminescence intensity (RLU), are listed in Table 2 below. The results show good linearity for the following pairings, and these pairings can also be used for the determination of sFlt-1.

[0123] Table 2: Results of Chemiluminescent Microsphere Immunoassay

[0124]

[0125] Example 8: Sequencing

[0126] This embodiment describes sequencing of the anti-sFlt-1 monoclonal antibody. Total mRNA was extracted from hybridoma cells using commercial reagents (RNAM5 extraction kit, purchased from Beijing Jumei Biotechnology Co., Ltd.) according to the manufacturer's 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 MuIgκVL3′-1. The amplified VH and VL PCR products were cloned into a commercial vector (pTOPO-Blunt Cloning kit, Beijing Jumei), and 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. The complementarity-determining region sequences of the heavy chain and the light chain are shown in Table 3 below (based on the Chothia numbering system).

[0128] Table 3: Complementary determinant region sequences of heavy and light chains

[0129]

[0130] Example 9: Preparation of recombinant antibodies

[0131] Combined with the antibody V listed in Table 3 above L CDR and V H The CDR gene, and the exemplary V shown in SEQ ID NO:35-38 H FR1-V H FR4 and V shown in SEQ ID NO:39-42 L FR1-V L FR4 was used to construct a eukaryotic expression plasmid for recombinant antibodies using molecular cloning methods. The eukaryotic expression plasmid was electroporated into CHO host cells, and after electroporation, the cells were cultured in pressure selection medium (50 μM MSX) for 20 days. The supernatant was then collected for ELISA detection (using HRP-labeled goat anti-mouse IgG as the secondary antibody for screening, using the same method as above) to screen for cell lines that stably expressed recombinant antibodies.

[0132] Recombinant antibodies s10, s67, s42, s69, s20, and s52 were obtained after purification. The affinity of the recombinant antibodies was determined using enzyme-linked immunosorbent assay (ELISA), and the antibody affinities are 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] As shown in Table 4, the six monoclonal antibodies obtained through recombination all showed affinity for the S-Flt protein in the nM range.

Claims

1. A monoclonal antibody against FMS-like tyrosine kinase-1 or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, said heavy chain variable region comprising a heavy chain complementarity-determining region V. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementarity determination region V. L CDR1, V L CDR2 and V L CDR3; where: The heavy chain variable region includes an amino acid sequence defined by the heavy chain complementarity-determining region V as shown in SEQ ID NO: 29-31. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes amino acid sequences defined by the light chain complementarity-determining regions V as shown in SEQ ID NO: 32-34. L CDR1, V L CDR2 and V L CDR3.

2. The anti-FMS-like tyrosine kinase-1 monoclonal antibody or its antigen-binding fragment according to claim 1, wherein, The heavy chain variable region further includes frame regions HFR1, HFR2, HFR3, and HFR4, which are related to V H CDR1, V H CDR2 and V H CDR3 progresses 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, wherein HFR1, HFR2, HFR3, and HFR4 respectively have sequences shown by SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38 or sequences having 80% or more, 85% or more, 90% or more, or 95% or more identical to the sequences shown by SEQ ID NO:

38. The light chain variable region further includes frame regions LFR1, LFR2, LFR3, and LFR4, which are associated with V L CDR1, V L CDR2 and V L CDR3 progresses from the amino terminus to the carboxyl terminus according to LFR1, V L CDR1, LFR2, V L CDR2, LFR3, V L The sequence of CDR3 and LFR4, wherein LFR1, LFR2, LFR3 and LFR4 respectively have the sequence shown by SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42 or a sequence that has more than 80%, more than 85%, more than 90% or more identical to the sequence.

3. A pair of antibodies comprising the anti-FMS-like tyrosine kinase-1 monoclonal antibody or its antigen-binding fragment as described in claim 1, and another anti-FMS-like tyrosine kinase-1 monoclonal antibody or its antigen-binding fragment for pairing use. The heavy chain variable region of the other anti-FMS-like tyrosine kinase-1 monoclonal antibody or its antigen-binding fragment includes an amino acid sequence determined by the heavy chain complementarity-determining region V as shown in SEQ ID NO: 1-3. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes an amino acid sequence defined by the light chain complementarity-determining region V as shown in SEQ ID NO: 4-6. L CDR1, V L CDR2 and V L CDR3.

4. A pair of antibodies comprising an anti-FMS-like tyrosine kinase-1 monoclonal antibody according to claim 1 or an antigen-binding fragment thereof, and another anti-FMS-like tyrosine kinase-1 monoclonal antibody or an antigen-binding fragment thereof for pairing use. The heavy chain variable region of the other anti-FMS-like tyrosine kinase-1 monoclonal antibody or its antigen-binding fragment includes an amino acid sequence defined by the heavy chain complementarity-determining region V as shown in SEQ ID NO: 19-21. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes amino acid sequences defined by the light chain complementarity-determining regions V as shown in SEQ ID NO: 22-24. L CDR1, V L CDR2 and V L CDR3.

5. A pair of antibodies comprising an anti-FMS-like tyrosine kinase-1 monoclonal antibody according to claim 1 or an antigen-binding fragment thereof, and another anti-FMS-like tyrosine kinase-1 monoclonal antibody or an antigen-binding fragment thereof for pairing use. The heavy chain variable region of the other anti-FMS-like tyrosine kinase-1 monoclonal antibody or its antigen-binding fragment includes an amino acid sequence defined by the heavy chain complementarity-determining region V shown in 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 amino acid sequences defined by the light chain complementarity-determining regions V shown in SEQ ID NO: 16, SEQ ID NO: 27, and SEQ ID NO: 28, respectively. L CDR1, V L CDR2 and V L CDR3.

6. The paired antibody combination according to any one of claims 3 to 5, wherein, The heavy chain variable region further includes frame regions HFR1, HFR2, HFR3, and HFR4, which are related to V H CDR1, V H CDR2 and V H CDR3 progresses 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, wherein HFR1, HFR2, HFR3, and HFR4 respectively have sequences shown by SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38 or sequences having 80% or more, 85% or more, 90% or more, or 95% or more identical to the sequences shown by SEQ ID NO:

38. The light chain variable region further includes frame regions LFR1, LFR2, LFR3, and LFR4, which are associated with V L CDR1, V L CDR2 and V L CDR3 progresses from the amino terminus to the carboxyl terminus according to LFR1, V L CDR1, LFR2, V L CDR2, LFR3, V L The sequence of CDR3 and LFR4, wherein LFR1, LFR2, LFR3 and LFR4 respectively have the sequence shown by SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42 or a sequence that is 80% or more, 85% or more, 90% or more or 95% or more identical to the sequence shown.

7. A nucleic acid molecule encoding an anti-FMS-like tyrosine kinase-1 monoclonal antibody as described in claim 1 or 2, or an antigen-binding fragment thereof, or any of the paired antibody combinations described in any one of claims 3 to 6.

8. A vector comprising the nucleic acid molecule of claim 7.

9. The vector according to claim 8, wherein the vector is a plasmid vector.

10. The vector according to claim 8, wherein the vector is any one of pEE12, pCAGGS, pTOPO, pcDNA, pTT, pTT3, pEFBOS, pBV, pJV and pBJ.

11. The vector according to claim 10, wherein the vector is pCDNA3.

1.

12. An expression cell comprising the nucleic acid molecule of claim 7 or the vector of any one of claims 9 to 11.

13. The expression cell according to claim 12, wherein the expression cell is a mammalian cell.

14. The expression cell according to claim 12, wherein the expression cell is selected from Chinese hamster ovary cells, hamster kidney cells, monkey kidney cells, mouse thymoma cells, and human embryonic kidney cells.

15. Use of the anti-FMS-like tyrosine kinase-1 monoclonal antibody or its antigen-binding fragment according to claim 1 or 2, or the paired antibody combination according to any one of claims 3 to 6, in the preparation of a diagnostic agent for the diagnosis of preeclampsia.

16. Use of the anti-FMS-like tyrosine kinase-1 monoclonal antibody or its antigen-binding fragment according to claim 1 or 2, or the paired antibody combination according to any one of claims 3 to 6, in the preparation of a reagent for detecting FMS-like tyrosine kinase-1.

17. The use according to claim 15 or 16, wherein, The diagnosis or detection is performed using immunochromatography, enzyme-linked antibody assay (ELISA), chemiluminescence immunoassay, or electrochemiluminescence immunoassay.

18. A kit for diagnosing preeclampsia and / or detecting FMS-like tyrosine kinase-1, comprising: The anti-FMS-like tyrosine kinase-1 monoclonal antibody or its antigen-binding fragment as described in claim 1 or 2, or the paired antibody combination as described in any one of claims 3 to 6; and instructions for use.

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