Fgf18 neutralizing antibodies, methods of making and using the same, nucleic acid molecules, vectors, cells, pharmaceuticals and combination compositions

Abstract

The application relates to the technical field of biological medicine, in particular to an FGF18 neutralizing antibody and a preparation method and application thereof, a nucleic acid molecule, a carrier, a cell, a medicine and a combined drug composition. The FGF18 neutralizing antibody has HCDR1 to HCDR3 with an amino acid sequence as shown in SEQ ID NO. 5 to SEQ ID NO. 7 and LCDR1 to LCDR3 with an amino acid sequence as shown in SEQ ID NO. 8 to SEQ ID NO. 10. The application provides an FGF18 neutralizing antibody which can combine FGF18 so as to effectively inhibit the activation of an FGF18 / FGFR3 channel, and achieve the effect of inhibiting the migration and growth of tumor cells. Moreover, the neutralizing antibody is combined with a PD-1 inhibitor, and the tumor inhibition effect is better.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to an FGF18 neutralizing antibody, a preparation method and application thereof, a nucleic acid molecule, a vector, a cell, a drug and a combined drug composition. BACKGROUND

[0002] Fibroblast growth factors (FGFs) are a class of bioactive substances, which were originally isolated from brain and pituitary tissue extracts and can promote the growth of fibroblasts. FGFs and their receptors (FGFRs) drive key developmental signaling pathways that are responsible for many functions, including cell proliferation, survival and migration. Therefore, FGFs and FGFRs often play a role in cancer cell progression and have been shown to be oncogenic in many cancers. So far, 22 FGFs and 4 FGFRs have been identified in mammals. According to their molecular structure, 22 fibroblast growth factors can be divided into 7 subgroups, of which FGF8, FGF17 and FGF18 belong to the FGF8 subfamily.

[0003] Fibroblast growth factor 18 (FGF18) as a member of the FGFs family is a pleiotropic growth factor that can stimulate or inhibit the proliferation of various cells in different tissues, such as primary osteoblasts, primary chondrocytes, hair follicle cells, smooth muscle cells, liver, etc. FGF18 is a potent mitogen for a variety of cells, and its function is not limited to tissue and organ metabolism and development, but also plays an important role in the development of cancer in organs or systems such as digestion and reproduction. For example, Shimokawa et al. suggested that increased expression of FGF18 can lead to the occurrence of colorectal tumors. Professor Wei's team predicted that FGF18 overexpression is an independent marker for poor prognosis of ovarian cancer and identified FGF18 as a serum biomarker for ovarian cancer, which is significantly associated with the prognosis and progression of ovarian cancer. FGF18 promotes the growth and tissue progression of lung cancer cells through the FGFR 1-ERK / p38 pathway. FGF18 regulates breast cancer cell growth and tissue progression through Akt-GSK3β-mediated β-catenin signaling. FGF18 has become a key regulatory factor in tumor progression and is involved in controlling various physiological processes of tumors in various systems. Therefore, providing antibodies targeting FGF18 is crucial for the clinical treatment of tumors. SUMMARY

[0004] Based on this, one or more embodiments of the present application provide an FGF18 neutralizing antibody, a preparation method and application thereof, a nucleic acid molecule, a vector, a cell, a drug and a combined drug composition. The technical solutions include the following:

[0005] One or more embodiments of this application provide an FGF18 neutralizing antibody, the FGF18 neutralizing antibody having amino acid sequences as shown in SEQ ID NO.5 to SEQ ID NO.7, HCDR1 to HCDR3, and amino acid sequences as shown in SEQ ID NO.8 to SEQ ID NO.10, LCDR1 to LCDR3.

[0006] In some specific embodiments of this application, the species sources of the heavy chain variable region and the light chain variable region backbone regions of the FGF18 neutralizing antibody are each independently of rat, human, cattle, horse, pig, sheep, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck or goose.

[0007] In some specific embodiments of this application, the amino acid sequence of the heavy chain variable region of the FGF18 neutralizing antibody is shown as positions 19 to 132 in SEQ ID NO.4; or / and, the amino acid sequence of the light chain variable region of the FGF18 neutralizing antibody is shown as positions 21 to 132 in SEQ ID NO.2.

[0008] In some specific embodiments of this application, the sequence of the constant region of the FGF18 neutralizing antibody is selected from the sequence of any one of the constant regions of IgG, IgA, IgM, IgE and IgD; or / and, the species source of the light chain constant region and the heavy chain constant region of the FGF18 neutralizing antibody is independently mouse, human, cattle, horse, pig, sheep, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck or goose.

[0009] In some specific embodiments of this application, the amino acid sequence of the heavy chain of the FGF18 neutralizing antibody is shown as positions 19 to 462 in SEQ ID NO.4; or / and, the amino acid sequence of the light chain of the FGF18 neutralizing antibody is shown as positions 21 to 239 in SEQ ID NO.2.

[0010] One or more embodiments of this application also provide a nucleic acid molecule that encodes the FGF18 neutralizing antibody.

[0011] In some specific embodiments of this application, the nucleotide sequence of the nucleic acid molecule is as shown in any one of SEQ ID NO.1, positions 61 to 396 of SEQ ID NO.1, positions 61 to 717 of SEQ ID NO.1, SEQ ID NO.3, positions 61 to 402 of SEQ ID NO.3, and positions 61 to 1386 of SEQ ID NO.3.

[0012] One or more embodiments of this application also provide a carrier comprising the nucleic acid molecule.

[0013] In some specific embodiments of this application, the vector includes one or more of mammalian cell viruses, bacterial plasmids, bacteriophages, and yeast plasmids.

[0014] One or more embodiments of this application also provide a cell, said cell comprising the nucleic acid molecule or the carrier described herein.

[0015] In some specific embodiments of this application, the cells are CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, or HEK293 cells.

[0016] One or more embodiments of this application also provide a method for constructing the cell, the method comprising the step of introducing the nucleic acid molecule or the vector into the cell to be modified.

[0017] One or more embodiments of this application also provide a method for preparing the FGF18 neutralizing antibody, the method comprising: culturing the cells and isolating the FGF18 neutralizing antibody from the resulting culture.

[0018] One or more embodiments of this application also provide the application of the FGF18 neutralizing antibody in the preparation of FGF18 detection products or drugs; FGF18 has a promoting effect on the disease treated by the drug.

[0019] In some specific embodiments of this application, the detection product is a diagnostic reagent, test strip, test plate, or reagent kit; the disease treated by the drug includes cancer.

[0020] In some specific embodiments of this application, the cancer includes any of the following: leukemia, colorectal tumor, ovarian cancer, lung cancer, endometrial cancer, and breast cancer.

[0021] One or more embodiments of this application also provide an FGF18 detection product, wherein the FGF18 detection product includes the FGF18 neutralizing antibody.

[0022] In some specific embodiments of this application, the FGF18 detection product is a diagnostic reagent, test strip, test plate, or kit.

[0023] One or more embodiments of this application also provide a medicament comprising the FGF18 neutralizing antibody and pharmaceutically acceptable excipients.

[0024] One or more embodiments of this application also provide a combination drug composition, the combination drug composition comprising:

[0025] (1) the FGF18 neutralizing antibody or the drug; and,

[0026] (2) PD-1 inhibitors.

[0027] In some specific embodiments of this application, the PD-1 inhibitor includes Pembrolizumab.

[0028] In some specific embodiments of this application, the mass ratio of the FGF18 neutralizing antibody to the PD-1 inhibitor is (0.5-1.5):(0.5-1.5).

[0029] One or more embodiments of this application also provide a method for detecting FGF18 in a sample, wherein the detection method uses the FGF18 neutralizing antibody or the FGF18 detection product to detect FGF18 in the sample to be tested.

[0030] Compared to traditional technologies, the beneficial effects of this application include:

[0031] This application provides an FGF18 neutralizing antibody that can bind to FGF18 and effectively inhibit the activation of the FGF18 / FGFR3 pathway, thereby inhibiting tumor cell migration and growth. Furthermore, the tumor-suppressing effect is even better when this neutralizing antibody is used in combination with a PD-1 inhibitor. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 The image shows the electrophoresis results for the purified antibody.

[0034] Figure 2 The figure shows the results of the CCK8 assay in Experiment 1, which detected the inhibitory effect of FGF18 neutralizing antibody on the promotion of FGF18 proliferation in AML cells.

[0035] Figure 3 This is a graph showing the results of Western blotting in Experiment 2, which detected the inhibition of downstream channel gene expression by the FGF18 neutralizing antibody.

[0036] Figure 4 The figure shows the results of FGF18 neutralizing antibody inhibiting tumor progression in AML mice in Experiment 3.

[0037] Figure 5The figure shows the results of treating acute myeloid leukemia in Experiment 4 using IgG, FGF18 neutralizing antibody, PD-1 inhibitor, and a combination of FGF18 neutralizing antibody and PD-1 inhibitor.

[0038] Figure 6 This is the result of verifying the safety of FGF18 neutralizing antibody therapy for tumors in vivo in Experiment 5. Detailed Implementation

[0039] The present application will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0040] 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 application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0041] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0042] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0043] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0044] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0045] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0046] In this document, terms such as “preferred,” “better,” “more suitable,” and “ideal” are merely used to describe implementation methods or examples that achieve better results, and should be understood not to limit the scope of protection of this application.

[0047] In this application, terms such as "further," "even more," and "particularly" are used for descriptive purposes and to indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0048] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0049] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0050] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0051] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0052] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0053] In this application, %(w / w) and wt% both represent weight percentage, %(v / v) refers to volume percentage, and %(w / v) refers to mass-volume percentage.

[0054] A first aspect of this application provides an FGF18 neutralizing antibody, the FGF18 neutralizing antibody having amino acid sequences as shown in SEQ ID NO.5 to SEQ ID NO.7, namely HCDR1 to HCDR3, and amino acid sequences as shown in SEQ ID NO.8 to SEQ ID NO.10, namely LCDR1 to LCDR3.

[0055] The FGF18 neutralizing antibody of this application may have the aforementioned CDRs, or a derivative fragment having the aforementioned CDRs. The derivative fragment is formed by replacing amino acids at no more than six sites relative to its corresponding CDR (“conservative modification” or “conservative substitution”), retaining the biological activity consistent with its corresponding complementarity-determining region. For example, the derivative fragment may replace one amino acid with another, or one amino acid with multiple amino acids (e.g., two), at sites 1, 2, 3, 4, 5, or 6 of its corresponding complementarity-determining region.

[0056] In the CDRs provided in this application, the derived fragments (conserved variants) refer to polypeptides formed by replacing one, two, or three amino acids with amino acids of similar or related properties compared to the amino acid sequence of the antibody in this application. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0057] Table A

[0058]

[0059] "Conservative modification" or "conservative substitution" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation, and rigidity), allowing for frequent alterations without changing the protein's biological activity. Those skilled in the art will recognize that, in general, the substitution of a single amino acid in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson et al. (1987), Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th edition)). Furthermore, substitutions of structurally or functionally similar amino acids are unlikely to disrupt biological activity.

[0060] The three-letter and single-letter codes for amino acids used in this application are as described in J.biol.chem, 243, p3558 (1968).

[0061] The neutralizing antibody in this application may be an antibody, an antigen-binding fragment of an antibody, or a small modular immunodrug. Further, it may be a monoclonal antibody, an F(ab')2 fragment, a Fab' fragment, a Fab fragment, an Fv fragment, a scFv fragment, a biclonal antibody, a multispecific antibody, a microantibody, a chelated recombinant antibody, an endogenous antibody, a nanobody, a domain-binding immunoglobulin fusion protein, or a small modular immunodrug.

[0062] The "antibody" mentioned in this application refers to immunoglobulins. A complete antibody is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the heavy chain of immunoglobulins differ, thus their antigenicity also differs. Based on this, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, based on differences in the amino acid composition of its hinge region and the number and position of disulfide bonds in its heavy chain, it can be further divided into different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chains are classified as κ chains or λ chains based on differences in their constant regions. Each of the five classes of Ig can have either a κ chain or a λ chain.

[0063] The sequence of approximately 110 amino acids near the N-terminus of both the antibody heavy and light chains varies considerably and is known as the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are called the constant region. The variable region includes three hypervariable regions (HVR) and four relatively conserved backbone regions (FR). The three hypervariable regions determine the antibody's specificity and are also called complementarity-determining regions (CDR). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

[0064] The antibody in this application may be a murine antibody, a chimeric antibody, or a humanized antibody.

[0065] In this application, the term "mouse antibody" refers to a monoclonal antibody against FGF18 prepared in accordance with the knowledge and skills in the art. Preparation involves using FGF18 or its epitopes as the antigen injection target, followed by isolation of hybridomas expressing antibodies with the desired sequence or functional characteristics. In some examples, the said mouse anti-FGF18 antibody or its antigen-binding fragment may further include a light chain constant region of a mouse κ, λ chain or its variants, or further include a heavy chain constant region of mouse IgG1, IgG2, IgG3 or its variants.

[0066] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody. It can reduce the immune response induced by murine antibodies. To create a chimeric antibody, a hybridoma that secretes murine-specific monoclonal antibodies must first be established. Then, the variable region gene is cloned from the murine hybridoma cells. Next, the constant region gene of the human antibody is cloned as needed. The murine variable region gene and the human constant region gene are then linked to form a chimeric gene, which is inserted into an expression vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic system.

[0067] The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody produced by grafting a non-human species' CDR sequence into the variable region framework of a human antibody, i.e., a human antibody framework sequence of different types. This overcomes the heterologous response induced by chimeric antibodies carrying a large number of heterologous protein components. Such framework sequences can be obtained from public DNA databases containing germline antibody gene sequences or from publicly available references. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available at www.mrccpe.com.ac.uk / vbase) and in Kabat, E.A. et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition. To avoid a decrease in activity along with a decrease in immunogenicity, minimal reverse or reversion mutations can be performed on the aforementioned human antibody variable region framework sequence to maintain activity. The humanized antibodies in this application also include humanized antibodies further derived from yeast exhibiting affinity maturation mutations of CDR.

[0068] In this application, the antibody or its antigen-binding fragment may further include a light chain constant region of human or mouse κ, λ chains or variants thereof, or further include a heavy chain constant region of human or mouse IgG1, IgG2, IgG3, IgG4 or variants thereof; preferably, it includes a heavy chain constant region of human IgG1, IgG2 or IgG4, or uses an IgG1, IgG2 or IgG4 variant with an amino acid mutation (e.g., L234A and / or L235A mutation, and / or S228P mutation).

[0069] The "variants" of the heavy chain constant region and light chain constant region of human antibodies described in this application refer to prior art variants of the heavy chain constant region or light chain constant region derived from humans that do not alter the structure and function of the antibody variable region. Exemplary variants include IgG1, IgG2, IgG3, or IgG4 heavy chain constant region variants that involve site-specific modifications and amino acid substitutions in the heavy chain constant region; specific substitutions include prior art known YTE mutations, L234A and / or L235A mutations, S228P mutations, and / or mutations that obtain a knock-in-hole structure (giving the antibody heavy chain a knock-Fc and hole-Fc combination), which have been shown to give antibodies new properties without altering the function of the antibody variable region.

[0070] The terms "human antibody" (HuMAb), "human-derived antibody," "fully human antibody," and "completely human antibody" are used interchangeably. A human antibody can be derived from a genetically modified organism (GMO) that is "engineered" to produce specific human antibodies in response to antigenic stimulation, and can be produced by any method known in the art. In some techniques, human heavy and light chain locus elements are introduced into cell lines derived from embryonic stem cell lines, where endogenous heavy and light chain loci are targeted and disrupted. The GMO can synthesize human antibodies specific to human antigens, and can be used to produce hybridomas that secrete human antibodies. A human antibody can also be an antibody in which the heavy and light chains are encoded by nucleotide sequences derived from one or more human DNA sources. Completely human antibodies can also be constructed using gene or chromosome transfection methods and phage display technology, or from in vitro activated B cells, all of which are known in the art.

[0071] The terms “full-length antibody,” “intact antibody,” “complete antibody,” and “all antibody” are used interchangeably herein to refer to an antibody in substantially its complete form, as distinguished from the antigen-binding fragment as defined below. This term specifically refers to antibodies containing constant regions in both the light and heavy chains. The term “antibody” in this application includes “full-length antibodies” and their antigen-binding fragments.

[0072] The full-length antibody of this application includes full-length antibodies formed by linking light chain variable regions with light chain constant regions and heavy chain variable regions with heavy chain constant regions in the light and heavy chain variable region combinations in the table below. Those skilled in the art can select light chain constant regions and heavy chain constant regions from different antibody sources according to actual needs, such as light chain constant regions and heavy chain constant regions derived from human antibodies.

[0073] The term "antigen-binding fragment" or "functional fragment" of an antibody refers to one or more fragments that retain the ability to specifically bind to an antigen. It has been shown that fragments of full-length antibodies can be used for antigen-binding function. Examples of binding fragments included in the term "antigen-binding fragment" of an antibody include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments connected by disulfide bridges on the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VH and VL domains on a single arm of the antibody; (v) dsFv, an antigen-binding fragment formed by interchain disulfide bonds between VH and VL; and (vi) bispecific, bispecific, and multispecific antibodies containing fragments such as scFv, dsFv, and Fab. Furthermore, although the two domains VL and VH of the Fv fragment are linked by a synthetic linker, enabling it to produce a single protein chain (referred to as a single-chain Fv (scFv) where the VL and VH regions pair to form a monovalent molecule; see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single-chain antibodies are also included in the term "antigen-binding fragment" of antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and are screened for functionality in the same manner as for intact antibodies. Antigen-binding moieties can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0074] Fab is an antibody fragment with antigen-binding activity obtained by treating IgG antibody molecules with an enzyme of the same activity as papain.

[0075] F(ab')2 is an antibody fragment with antigen-binding activity obtained by digesting IgG with an enzyme of the same activity as pepsin.

[0076] Fab' is an antibody fragment with antigen-binding activity obtained by cleaving the above-mentioned F(ab')2.

[0077] Furthermore, the Fab' can be produced by inserting DNA encoding the Fab' fragment into an expression vector and then introducing the vector into a host.

[0078] The terms "single-chain antibody," "single-chain Fv," or "scFv" refer to molecules that contain a variable domain (or region; VH) of the antibody heavy chain and a variable domain (or region; VL) of the antibody light chain linked by a linker. Such scFv molecules can have a generic structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Other connectors that may be used in this application are described in the following literature, for example, but not limited to: Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Alfthan et al. (1995), Protein Eng. 8:725-731; Choi et al. (2001), Eur. J. Immunol. 31:94-106; Hu et al. (1996), Cancer Res. 56:3055-3061; Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.

[0079] A biantibody is an antibody fragment in which scFv or Fab is dimerized, and it is an antibody fragment with bivalent antigen-binding activity. In bivalent antigen-binding activity, the two antigens can be the same or different.

[0080] Bispecific antibodies and multispecific antibodies are antibodies that can bind to two or more antigens or antigenic determinants.

[0081] dsFv is obtained by linking polypeptides in which one amino acid residue in each VH and VL is replaced by a cysteine ​​residue via disulfide bonds between cysteine ​​residues. The amino acid residues to be replaced by cysteine ​​residues can be selected based on the prediction of the antibody's three-dimensional structure using known methods (e.g., Protein Engineering, 7, 697 (1994)).

[0082] The term "amino acid difference" or "amino acid mutation" refers to an alteration or mutation of amino acids in a variant protein or polypeptide compared to the original protein or polypeptide. This includes the insertion, deletion, or substitution of one, two, three, or more amino acids in the original protein or polypeptide.

[0083] The term "antibody framework" or "FR region" refers to a portion of the variable domain VL or VH that serves as a scaffold for the antigen-binding loop (CDR) of that variable domain. Essentially, it is a variable domain without a CDR.

[0084] The terms "complementarity-determining region," "CDR," or "hypervariant region" refer to one of the six hypervariable regions within the variable domain of an antibody that primarily facilitate antigen binding. Typically, each heavy chain variable region contains three CDRs (HCDR1, HCDR2, HCDR3), and each light chain variable region contains three CDRs (LCDR1, LCDR2, LCDR3). The amino acid sequence boundaries of CDRs can be determined using any of a variety of well-known schemes, including the “Kabat” numbering rule (see Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD), the “Chothia” numbering rule (see Al-Lazikani et al., (1997) JMB 27 3: 927-948), and the ImMuno GenTics (IMGT) numbering rule (Lefranc M.P., Immunologist, 7, 132-136 (1999); Lefranc, M.P. et al., Dev. Comp. Immunol). , 27, 55-77 (2003), etc. For example, for the classical format, following Kabat rules, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Following Chothia rules, the CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), 50-56 (HCDR2), and 89-97 (LCDR3). R2) and 91-96 (LCDR3). By combining the CDR definitions of Kabat and Chothia, the CDR consists of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL. Following the IMGT rules, the CDR amino acid residues in VH are approximately numbered 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), and the CDR amino acid residues in VL are approximately numbered 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3).Following the IMGT rules, the CDR region of an antibody can be determined using the IMGT / DomainGap Align procedure.

[0085] The term "epitope" or "antigenic determinant" refers to a site on an antigen that is bound by an immunoglobulin or antibody (e.g., a specific site on a uPARAP molecule). Epitopes typically consist of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a distinctive spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996).

[0086] The terms "specific binding," "selective binding," "selective binding," and "specific binding" refer to the binding of an antibody to an epitope on a pre-defined antigen. Typically, antibodies bind with an affinity (KD) of approximately less than 10⁻⁸ M, such as approximately less than 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, 10⁻¹² M, or even less.

[0087] The term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. Typically, the antibodies in this application bind to antigens or their epitopes with a dissociation equilibrium constant (KD) of less than about 10⁻⁷ M, for example, less than about 10⁻⁸ M or 10⁻⁹ M. For example, in this application, the affinity of the antibody for cell surface antigens is determined using the FACS method to determine the KD value.

[0088] In the presence of CDRs, this application does not specifically limit the species origin of the skeletal regions of the variable regions and light chain variable regions to which the neutralizing antibody is located, and each can be independently selected from, but is not limited to: rats, humans, cattle, horses, pigs, sheep, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese.

[0089] In some examples of this application, the FGF18 neutralizing antibody has a heavy chain variable region as shown in amino acid sequence SEQ ID NO: 4, positions 19 to 132, or its heavy chain variable region has a sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9%) identical to the sequence shown in SEQ ID NO: 4, positions 19 to 132.

[0090] In some examples of this application, the amino acid sequence of the light chain variable region of the FGF18 neutralizing antibody is as shown in positions 21 to 132 of SEQ ID NO.2, or the light chain variable region has a sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9%) identical to the sequence shown in positions 21 to 132 of SEQ ID NO.2.

[0091] The FGF18 neutralizing antibody of this application also includes an antibody constant region. This application does not specifically limit the sequence or species origin of the antibody constant region. The sequence of the antibody constant region is selected from the sequences of any one of the constant regions of IgG, IgA, IgM, IgE, and IgD. The species origin of the light chain constant region and the heavy chain constant region are independently mouse, human, cattle, horse, pig, sheep, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, or goose.

[0092] Optionally, the type of the light chain constant region in the antibody constant region is κ or λ.

[0093] "Identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. When positions in two compared sequences are occupied by the same base or amino acid monomer subunit—for example, if every position in two DNA molecules is occupied by adenine—then the molecules are homologous at that position. The percentage of identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared multiplied by 100. For example, in optimal sequence alignment, if 6 out of 10 positions in two sequences match or are homologous, then the two sequences are 60% homologous; if 95 out of 100 positions in two sequences match or are homologous, then the two sequences are 95% homologous. Typically, comparisons are made when aligning two sequences to give the maximum percentage of identity. For example, comparisons can be performed using the BLAST algorithm, where the algorithm's parameters are chosen to give the maximum match between the sequences over the entire length of each reference sequence. The following references relate to the BLAST algorithm commonly used in sequence analysis: BLAST ALGORITHMS: Altschul, S.F. et al., (1990) J. Mol. Biol. 215:403-410; Gish, W. et al., (1993) Nature Genet. 3:266-272; Madden, T.L. et al., (1996) Meth. Enzymol. 266:131-141; Altschul, S.F. et al., (1997) Nucleic Acids Res. 25:3389-3402; Zhang, J. et al., (1997) Genome Res. 7:649-656. Other common BLAST algorithms, such as those provided by NCBI BLAST, are also well-known to those skilled in the art.

[0094] In some examples of this application, the amino acid sequence of the heavy chain of the FGF18 neutralizing antibody is as shown in positions 19 to 462 of SEQ ID NO.4, or has a sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9%) identical to the sequence shown in positions 19 to 462 of SEQ ID NO.4.

[0095] In some examples of this application, the amino acid sequence of the light chain of the FGF18 neutralizing antibody is as shown in positions 21 to 239 of SEQ ID NO.2, or has a sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9%) identical to the sequence shown in positions 21 to 239 of SEQ ID NO.2.

[0096] A second aspect of this application provides a nucleic acid molecule that encodes the FGF18 neutralizing antibody.

[0097] In some examples of this application, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.1, positions 61 to 396, 61 to 717 of SEQ ID NO.1, SEQ ID NO.3, positions 61 to 402 and 61 to 1386 of SEQ ID NO.3, or any of the positions shown in SEQ ID NO.1, SEQ ID NO.1, SEQ ID NO.1, SEQ ID NO.1, SEQ ID NO.1, SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.3, SEQ ID NO.3, and SEQ ID NO.1, SEQ ID NO.3 ... The sequences shown in NO.3 from the 61st to the 1386th position have at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9%) identity.

[0098] As used herein, the term "nucleic acid molecule" refers to DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, preferably double-stranded DNA or single-stranded mRNA or modified mRNA. Nucleic acids are "effectively linked" when placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to said coding sequence.

[0099] The nucleic acid molecules in this application primarily refer to isolated nucleic acid molecules. "Isolated" means that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth media. Generally, the term "isolated" is not intended to mean the complete absence of these materials or the absence of water, buffers, or salts, unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the compounds described herein.

[0100] A third aspect of this application provides a carrier comprising the aforementioned nucleic acid molecule.

[0101] The term "vector," also known as "expression vector," refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid," which refers to a circular double-stranded DNA loop to which an additional DNA segment can be linked. In another embodiment, the vector is a viral vector, in which an additional DNA segment can be linked to a viral genome. The vectors disclosed herein are capable of autonomous replication in host cells upon introduction (e.g., bacterial vectors with bacterial origins of replication and augmented mammalian vectors) or can be integrated into the host cell's genome after introduction, thereby replicating along with the host genome (e.g., non-augmented mammalian vectors). In general, vectors may be selected from, but are not limited to, mammalian cell viruses, bacterial plasmids, bacteriophages, yeast plasmids, or combinations thereof.

[0102] A fourth aspect of this application provides a cell comprising the aforementioned nucleic acid molecule or the aforementioned carrier.

[0103] The term "cell," also known as "host cell," refers to a cell into which an expression vector has been introduced. Host cells can include bacterial, microbial, plant, or animal cells. Easily transformable bacteria include members of the Enterobacteriaceae family, such as strains of *Escherichia coli* or *Salmonella*; members of the Bacillaceae family, such as *Bacillus subtilis*; *Pneumococcus*; *Streptococcus*; and *Haemophilus influenzae*. Suitable microorganisms include *Saccharomyces cerevisiae* and *Pichia pastoris*. Suitable animal host cell lines include CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, or HEK293 cells.

[0104] The terms “cell,” “cell line,” and “cell culture” used herein are used interchangeably, and all such names include progeny. Therefore, “transformant” and “transformed cell” include primary test cells and cultures derived from them, regardless of passage number. It should also be understood that, due to intentional or unintentional mutations, all progeny cannot be exactly identical in DNA content. This includes mutant progeny with the same function or biological activity as those screened from the original transformed cells. Where different names are intended, the context will be clear.

[0105] A fifth aspect of this application provides a method for constructing the cell described above, the method comprising the step of introducing the nucleic acid molecule or the vector into the cell to be modified.

[0106] Import methods can include, but are not limited to, transfection.

[0107] The term “transfection” refers to the process of introducing nucleic acids into eukaryotic cells, particularly mammalian cells. Protocols and techniques used for transfection include, but are not limited to, lipid transfection and chemical and physical methods such as electroporation. Many transfection techniques are well known in the art and are disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, ibid.; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197.

[0108] A sixth aspect of this application provides a method for preparing the FGF18 neutralizing antibody, the method comprising: culturing the cells and isolating the FGF18 neutralizing antibody from the resulting culture.

[0109] The antibody or antigen-binding fragments of this application can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. Recombinant immunoglobulin expression vectors can stably transfect host cells. As a more preferred prior art, mammalian expression systems lead to glycosylation of the antibody, particularly at the highly conserved N-terminal site in the Fc region. Stable clones are obtained by expressing specific binding proteins, and positive clones are scaled up in a bioreactor to produce antibodies. The culture medium secreting the antibody can be purified using conventional techniques, such as using an A or G Sepharose FF column with adjusted buffer. Non-specifically bound components are washed away. The bound antibody is then eluted using a pH gradient, and the antibody fragments are detected by SDS-PAGE and collected. The antibody can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieving or ion exchange. The resulting product should be immediately frozen, such as at -70°C, or lyophilized.

[0110] Methods for producing and purifying antibodies and antigen-binding fragments are well-known in the prior art, such as those described in Cold Spring Harbor's Guide to Antibody Experimentation, Chapters 5-8 and 15. For example, mice can be immunized with human antigens or fragments thereof, and the resulting antibodies can be renatured, purified, and sequenced using conventional methods. Antigen-binding fragments can also be prepared using conventional methods. The antibodies or antigen-binding fragments described in this invention utilize genetic engineering methods to add one or more human FR regions to non-human CDR regions. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website http: / / imgt.cines.fr by comparing with the IMGT Human Antibody Variable Region Germplasm Database and MOE software, or from the journal Immunoglobulins, 2001 ISBN012441351.

[0111] A seventh aspect of this application provides the use of the FGF18 neutralizing antibody in the preparation of an FGF18 detection product or drug; FGF18 has a promoting effect on the disease treated by the drug.

[0112] This application does not specifically limit the types of FGF18 detection products, such as diagnostic reagents, test strips, test plates, or kits.

[0113] This application does not specifically limit the types of diseases, including but not limited to cancer. For example, leukemia, colorectal cancer, ovarian cancer, lung cancer, and breast cancer.

[0114] Leukemia is classified into acute leukemia (such as acute myeloid leukemia and acute lymphoblastic leukemia) and chronic leukemia (such as chronic myeloid leukemia and chronic lymphocytic leukemia) based on the speed of its progression. Acute myeloid leukemia (AML) is a malignant disease associated with abnormal growth of myeloid hematopoietic stem cells / progenitor cells. Data shows that the incidence of AML gradually increases with age, and the 5-year survival rate of adult AML is only 25%-30%, with even worse prognoses for elderly patients and high-risk subtypes. Currently, the standard treatment for AML mainly includes induction chemotherapy, consolidation chemotherapy, and hematopoietic stem cell transplantation (HSCT). In recent years, the application of targeted drugs (such as FLT3 inhibitors, IDH inhibitors, and BCL-2 inhibitors) has improved the prognosis of some patients, but still faces significant challenges, including high relapse rates and drug resistance, treatment toxicity, and low coverage of targeted therapy. To improve the survival rate of AML, new treatment strategies are urgently needed, such as cytokine targeting, immunotherapy, and epigenetic regulation. However, there are no reports on FGF18 in AML research, its functional studies in AML are limited, and there has been no further in-depth research on its application in AML treatment.

[0115] Regarding antibody therapy, experiments related to blocking FGF18 or its receptor have been reported. For example, the fibroblast growth factor receptor 1 (FGFR1) Fc fusion protein (FP-1039) inhibited the growth of five lung cancer cell lines. It also inhibited tumor formation in subcutaneous tumor models of five lung cancer cell lines. FGF18 expression levels can be inhibited by the tissue-selective estrogen receptor modulator bazedoxifene (BZA), which reduces the proliferative capacity of endometrial cancer. However, there are currently no reports of FGF18 neutralizing antibodies having therapeutic effects on cancer. Therefore, existing technologies need further improvement and development. In view of the above-mentioned shortcomings of existing technologies, in order to explore whether FGF18 can be used as a drug target for AML in the field of tumor treatment, this application prepared a neutralizing antibody against FGF18. FGF18 neutralizing antibodies can block tumor signaling pathways, activate immune responses, and can also be used in combination with other treatment methods to improve overall efficacy. Therefore, FGF18 neutralizing antibodies show good application prospects in AML treatment. One objective of this application is to provide a novel application of FGF18 neutralizing antibodies, specifically their use in the preparation of drugs for treating acute myeloid leukemia (AML). This aims to address the low cure rate of existing AML treatments and the lack of application of FGF18 neutralizing antibodies in cancer therapy. This application discovers that the recombinant FGF18 cytokine signaling pathway can promote the development of AML and provides an FGF18 neutralizing antibody that can effectively inhibit the development of AML. The combined use of a PD-1 inhibitor and an FGF18 neutralizing antibody significantly enhances the inhibitory effect on AML compared to PD-1 inhibitor monotherapy, demonstrating clinical conversion value.

[0116] An eighth aspect of this application provides an FGF18 detection product comprising the aforementioned FGF18 neutralizing antibody.

[0117] Optionally, the detection product is a diagnostic reagent, test strip, test plate, or reagent kit.

[0118] This application involves a neutralizing antibody in the test product that competitively binds to FGF18 in the test sample.

[0119] When the term "competition" is used in the context of competing antigen-binding proteins for the same epitope, it refers to competition between antigen-binding proteins, which is determined by an assay in which the antigen-binding protein to be detected (e.g., an antibody or a functional fragment thereof) prevents or inhibits (e.g., reduces) the specific binding of a reference antigen-binding protein (e.g., a ligand or a reference antibody) to a common antigen (e.g., antigen X or a fragment thereof). Numerous types of competitive binding assays can be used to determine whether one antigen-binding protein competes with another. These assays include: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, for example, Stahli et al., 1983, Methods in Enzymology 9: 242-253); solid-phase direct biotin-avidin EIA (see, for example, Kirkland et al., 1986, J. Immunol. 137: 3614-3619), solid-phase direct labeling assay, solid-phase direct labeling sandwich assay (see, for example, Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct labeling RIA with I-125 label (see, for example, Morel et al., 1988, Molec. Immunol. 25: 7-15); solid-phase direct biotin-avidin EIA. (See, for example, Cheung et al., 1990, Virology 176: 546-552); and directly labeled RIAs (Moldenhauer et al., 1990, Scand J Immunol. 32: 77-82). Typically, the assay involves using a purified antigen on a solid surface or cell bound to either an unlabeled detection antigen-binding protein or a labeled reference antigen-binding protein. Competitive inhibition is measured by measuring the amount of label bound to the solid surface or cell in the presence of the detected antigen-binding protein. Typically, the detected antigen-binding protein is present in excess. Antigen-binding proteins identified by competitive assays (competitive antigen-binding proteins) include: antigen-binding proteins that bind to the same epitope as the reference antigen-binding protein; and antigen-binding proteins that bind to a neighboring epitope sufficiently close to the reference antigen-binding protein's binding epitope, the two epitopes being spatially mutually hindered from binding. Further details regarding methods for determining competitive binding are provided in the examples herein. Typically, when competing antigen-binding proteins are present in excess, they will inhibit (e.g., reduce) at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more of the specific binding of reference antigen-binding proteins to the common antigen.In some cases, the binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0120] A ninth aspect of this application provides a medicament comprising the aforementioned FGF18 neutralizing antibody and a pharmaceutically acceptable carrier.

[0121] The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in a formulation for delivering antibody or antigen-binding fragments. Carriers can be anti-adhesion agents, adhesives, coatings, disintegrants, fillers or diluents, preservatives (such as antioxidants, antibacterial agents, or antifungal agents), sweeteners, absorption delay agents, wetting agents, emulsifiers, buffers, etc. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), dextrose, vegetable oils (such as olive oil), saline, buffer solutions, buffered saline, and isotonic agents such as sugars, polyols, sorbitol, and sodium chloride.

[0122] This application includes a pharmaceutical agent for treating diseases associated with positive cells of a target antigen (e.g., FGF18), said pharmaceutical agent comprising the FGF18 antibody of this application or an antigen-binding fragment thereof as an active ingredient.

[0123] "Therapeutic use," as described herein, of FGF18-guided ADCs for the delivery of active agents (e.g., therapeutic agents or cytotoxic agents) to cells expressing FGF18, and thus for the treatment of a range of diseases and conditions characterized by FGF18 expression, particularly FGF18 overexpression. This application provides pharmaceutical compositions comprising an effective amount of an anti-FGF18 ADC as described herein, and pharmaceutically acceptable buffers, diluents, carriers, adjuvants, or excipients.

[0124] A tenth aspect of this application provides a combination drug composition, the combination drug composition comprising:

[0125] (1) the FGF18 neutralizing antibody or the drug; and,

[0126] (2) PD-1 inhibitors.

[0127] This application does not specifically limit the types of PD-1 inhibitors. In some examples of this application, the PD-1 inhibitors include Pembrolizumab.

[0128] This application does not impose any particular limitation on the amount of PD-1 inhibitor and FGF18 neutralizing antibody. In some examples of this application, the mass ratio of the FGF18 neutralizing antibody to the PD-1 inhibitor is (0.5-1.5):(0.5-1.5), for example, 0.5:0.5, 0.5:1, 0.5:1.5, 1:0.5, 1:1.5, 1.5:0.5, 1.5:1.

[0129] The eleventh aspect of this application provides a method for detecting FGF18 in a sample, which uses the FGF18 neutralizing antibody or the FGF18 detection product to detect FGF18 in the sample to be tested.

[0130] This application relates to methods for immunodetection or determination of target antigens (e.g., FGF18), reagents for immunodetection or determination of target antigens (e.g., FGF18), methods for immunodetection or determination of cells expressing target antigens (e.g., FGF18), and diagnostic agents for diagnosing diseases associated with target antigen (e.g., FGF18) positive cells, comprising an antibody or antibody fragment that specifically recognizes the target antigen (e.g., human FGF18) and binds to the amino acid sequence or its three-dimensional structure in the extracellular region as an active ingredient.

[0131] In this application, the method for detecting or determining the amount of a target antigen (e.g., FGF18) can be any known method. For example, it includes immunoassay or assay methods. Immunoassay or assay methods are methods that use labeled antigens or antibodies to detect or determine the amount of antibody or antigen. Examples of immunoassay or assay methods include radiolabeled antibody immunoassays (RIA), enzyme immunoassays (EIA or ELISA), fluorescence immunoassays (FIA), luminescent immunoassays, Western blotting, physicochemical methods, etc.

[0132] To detect cells expressing peptides, known immunoassay methods can be used, with immunoprecipitation, fluorescent cell staining, and immunohistochemical staining being preferred. Alternatively, fluorescent antibody staining using the FMAT8100HTS (Applied Biosystem) can be employed.

[0133] The application does not impose any particular restrictions on the test sample used to detect or measure the target antigen (e.g., FGF18), as long as it has the potential to contain cells expressing the target antigen (e.g., FGF18), such as tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid, or culture medium.

[0134] Depending on the required diagnostic method, diagnostic reagents containing the antibody or antibody fragment of this application may also contain reagents for performing antigen-antibody reactions or for detecting the reaction. Reagents for performing antigen-antibody reactions include buffers, salts, etc. Reagents for detection include those commonly used in immunoassay or assay methods, such as labeled second antibodies that recognize the monoclonal antibody, its antibody fragment, or conjugates, and substrates corresponding to the labeled antibodies.

[0135] A twelfth aspect of this application provides a treatment method for a disease, comprising administering to a subject an effective amount of the aforementioned FGF18 antibody, or the aforementioned combination drug composition.

[0136] "Disease" is defined in the seventh aspect.

[0137] "Administration," "giving," and "treatment," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administration," "giving," and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and cells, as well as contact between a reagent and a fluid, wherein the fluid is in contact with the cells. "Administration," "giving," and "treatment" also mean the treatment of, for example, cells, by means of a reagent, diagnostic agent, conjugate composition, or by means of another cell in vitro and ex vivo. "Treatment," when applied to humans, veterinary, or research subjects, refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic applications.

[0138] "Treatment" means administering an oral or topical therapeutic agent, such as a composition containing any of the antibodies or antigen-binding fragments of this application, to a patient who has symptoms of one or more diseases, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, a therapeutic agent is administered in a treated patient or population in an amount that effectively relieves symptoms of one or more diseases to induce the regression of such symptoms or inhibit their progression to any clinically measured extent. The amount of a therapeutic agent that effectively relieves any specific disease symptom (also referred to as a "therapeuticly effective amount") can vary depending on a variety of factors, such as the patient's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect in the patient. Whether the disease symptoms have been relieved can be evaluated using any clinical test that a physician or other healthcare professional typically uses to assess the severity or progression of the symptoms. Although the embodiments of this application (e.g., treatment methods or products) may be ineffective in alleviating symptoms of each target disease, they should reduce symptoms of the target disease in a statistically significant number of patients, as determined by any statistical test known in the art, such as the Student t-test, chi-square test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test.

[0139] "Effective amount" or "effective dose" means the amount of a drug, compound, or pharmaceutical composition necessary to achieve any one or more beneficial or desired therapeutic outcome. For prophylactic use, beneficial or desired outcomes include eliminating or reducing risk, mitigating severity, or delaying the onset of a condition, including the condition itself, its complications, and the biochemical, histological, and / or behavioral symptoms of intermediate pathological phenotypes present during the development of the condition. For therapeutic use, beneficial or desired outcomes include clinical outcomes such as reducing the incidence of various target antigen-related conditions of this application or improving one or more symptoms of said conditions, reducing the dosage of other agents required to treat the condition, enhancing the efficacy of another agent, and / or delaying the progression of the target antigen-related condition in a patient.

[0140] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0141] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0142] This application reveals that FGF18 activates FGFRs through extracellular binding, inducing receptor dimerization and trans-autophosphorylation of the receptor's intracellular kinase domain. The neutralizing antibody provided in this application, targeting FGF18, effectively inhibits the activation of the FGF18 / FGFR3 pathway, thereby suppressing the migration and growth of acute myeloid leukemia (AML) cells. This application demonstrates that FGF18 neutralizing antibodies can effectively delay the progression of AML and exhibit significant synergistic therapeutic effects when used in combination with PD-1 inhibitors. Therefore, FGF18 neutralizing antibodies can be used in the preparation of drugs for treating AML (cancer).

[0143] This application provides a detailed description of the technical solutions in its embodiments.

[0144] 1. Preparation process of FGF18 monoclonal antibody

[0145] (1) Animal immunization

[0146] Female BALB / c mice (6-8 weeks old) were immunized with recombinant h-FGF-18 protein antigen (purchased from Sinocare, Cat: 13206-H08H). Each mouse received a single immunization dose of 50 mg. For the first immunization, the immunogen was emulsified with an equal volume of complete Freund's adjuvant and injected subcutaneously at multiple sites in the abdomen. Every two weeks, the same dose of immunogen was emulsified with an equal volume of incomplete Freund's adjuvant and injected subcutaneously at multiple sites in the abdomen. One week after each immunization, 50-60 mL of blood was collected from the mouse orbital venous plexus, incubated overnight at 4°C, and then centrifuged at 4000 rpm for 15 min at 4°C. The supernatant serum was separated, and serum titer was detected using indirect ELISA. A titer of OD450-Blank greater than 1.0 at a 1:8000 dilution was considered acceptable. Animals with high ELISA titers were selected from those meeting the titer criteria for a follow-up immunization. Three to four days after the follow-up immunization, the mouse spleen was harvested for hybridoma fusion.

[0147] (2) Cell fusion

[0148] Spleen cells from immunized mice were mixed with mouse myeloma cells at a 1:1 ratio and fused using electrofusion to obtain hybridoma cells. The cells were changed twice, once on day 6 and again on day 8 post-fusion using HAT selective medium. Approximately day 10 post-fusion, the supernatant from the master clone stage cells was collected for screening and testing.

[0149] (3) Hybridoma cell screening

[0150] This includes ELISA binding screening and ELISA competitive screening. Binding screening: Indirect ELISA is used to detect binding in the supernatant of hybridoma cell culture. Competitive screening: During the hybridoma culture stage, the antibody concentration and affinity in the cell supernatant are unknown. Therefore, during the master cloning stage, the following two competitive ELISA methods are used simultaneously to competitively genotype the specific hybridoma cell supernatant. A competitive inhibition rate below 30% is initially identified as a non-competitive antibody; a competitive inhibition rate above 50% is initially identified as a competitive antibody. Because there are many influencing factors during the master cloning stage, and the competitive genotyping may change during the subcloning stage, the number of different types of candidate clones will be appropriately increased based on the actual test results. During the master cloning stage, the cell competition typing evaluation method is as follows: 1) Select master clones that are non-competitive in both ELISA competitive detection methods and classify them as non-competitive cells for subclone screening; 2) Prefer master clones that are competitive in both ELISA competitive detection methods and classify them as competitive cells for subclone screening; 3) If the number of clones that meet condition 2) is too small, select master clones that are competitive in one of the ELISA competitive detection methods and classify them as competitive cells for subclone screening.

[0151] In the final fusion master clone stage, a total of 260 positive clones were obtained that bound to the antigen protein and were able to block the binding of the immunogen to the receptor protein. One master clone cell was selected for the first limiting dilution.

[0152] (4) Limiting dilution and subcloning screening

[0153] Using HAT selective medium, each positive hybridoma cell line obtained through screening was seeded at a density of 0.75 cells per well in half of a 96-well cell culture plate and incubated statically at 37°C with 5% CO2. Approximately day 7 after culture, the supernatant was collected for indirect ELISA testing to confirm positive binding of the test antibody to the antigen. ELISA-specifically binding positive monoclonal cells were selected and subjected to 1-2 rounds of limiting dilution until stable, positive monoclonal cells were obtained. The master clone ELISA screening results are shown in Table 1.

[0154] Table 1. ELISA results of master clone supernatant

[0155]

[0156] The master clone cells were subcloned twice using a limiting dilution method. The relevant results of the two subcloning ELISA tests are shown in Tables 2 and 3.

[0157] Table 2. Results of a single subclonal supernatant ELISA assay

[0158]

[0159] Table 3. Results of ELISA detection of secondary subcloning supernatant

[0160]

[0161] In summary, a hybridoma cell strain that binds to the antigen protein and can block the binding of the immunogen to the receptor protein was obtained.

[0162] (5) Hybridoma cell culture

[0163] Transfer 1 mL of hybridoma cells into a 100 mL culture flask. When the cells have multiplied to a certain density, discard the supernatant and replace it with MHYBII medium (Beijing Yiqiao Shenzhou Technology Co., Ltd.). Continue culturing for 7-9 days before harvesting.

[0164] (6) Purification of FGF18 monoclonal antibody

[0165] The collected hybridoma cell supernatant was centrifuged using a benchtop centrifuge at 1000g for 20 min. The supernatant was then collected and filtered through a 0.45 μM filter. The hybridoma cell culture supernatant was purified using protein A affinity purification to harvest mouse monoclonal antibodies. Antibody concentration and purity were determined by spotting the sample with the corresponding buffer solution using a micro-spectrophotometer. A baseline stability was indicated when the absorbance value was within ±0.015 at 280 nm. The purified antibody samples were then spotted sequentially, and the absorbance values ​​were recorded. The concentration of the sample (mg / mL) was calculated by dividing the data by the IgG extinction coefficient (1.414). The results are shown in Table 4.

[0166] The purity of the purified antibody was verified using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This method separates proteins in the sample based on their molecular weight in the electrophoresis gel, thus verifying the purity of the target antibody. The purity of the purified antibody is shown in Table 4, and the electrophoresis results are shown in [Table 4]. Figure 1 .

[0167] Table 4. Final concentration and purity of purified antibody

[0168]

[0169] 2. Screening for FGF18 monoclonal antibodies

[0170] (1) ELISA combined detection

[0171] The binding of purified mouse monoclonal antibody to the immunogen was detected using an indirect ELISA method. The specific procedure is as follows:

[0172] (1-1) Coating: Recombinant h-FGF-18 protein antigen was coated at concentrations of 0.1 μg / mL, 1 μg / mL, and 100 μL / well, and the coating was incubated overnight at 4°C;

[0173] (1-2) Sealing: Shake off the liquid inside the plate and pat dry, add 2% BSA, 300 μL / well, seal and incubate at room temperature for 1 h;

[0174] (1-3) Washing: 300 μL / well washing solution, wash the plate twice, and pat dry after the last wash;

[0175] (1-4) Antibody dilution and loading: Dilute FGF18 monoclonal antibody to 1 μg / mL, add 100 μL to each well of the corresponding plate, mix well, and react at room temperature for 2 h;

[0176] (1-5) Washing: 300 μL / well washing solution, wash the plate 3 times, and pat dry after the last wash;

[0177] (1-6) Secondary antibody incubation: Dilute Rabbit Anti-Mouse IgG F(ab)2 / HRP secondary antibody to the working concentration, 100 μL / well, mix well, and incubate at room temperature for 1 h;

[0178] (1-7) Washing: 300 μL / well washing solution, wash the plate 3 times, and pat dry after the last wash;

[0179] (1-8) Color development: Mix solution A and solution B at a ratio of 1:1, add 200 μL to each well, and incubate at room temperature in the dark for 20 min;

[0180] (1-9) Termination and detection: Add 50 μL of stop solution to each well and immediately measure the OD value at a wavelength of 450 nm.

[0181] (2) Competitive detection of ELISA

[0182] (2-1) Coating: Coating with recombinant h-FGF-18 protein antigen at a concentration of 0.6 μg / mL, 100 μL / well, overnight at 4℃;

[0183] (2-2) Sealing: Shake off the liquid inside the plate and pat dry. Add 2% BSA, 300 μL / well, seal and incubate at room temperature for 1 h.

[0184] (2-3) Washing: 300 μL / well washing solution, wash the plate twice, and pat dry after the last wash;

[0185] (2-4) Sample dilution and loading: 50 μL of each of sample 1 and sample 2 were added to the corresponding well plates, mixed thoroughly, and reacted at room temperature for 2 h;

[0186] Sample 1: Purified antibody was diluted to 40 μg / mL and 4 μg / mL, respectively; the control group was used as the sample diluent.

[0187] Sample 2: Protein 16044-H02H diluted to 5 μg / mL;

[0188] (2-5) Washing: 300 μL / well washing solution, wash the plate 3 times, and pat dry after the last wash;

[0189] (2-6) Secondary antibody incubation: Dilute Mouse Anti-Human IgG Fc Antibody (50B4A9) [HRP], mAb secondary antibody to the working concentration, 100 μL / well, mix well, and incubate at room temperature for 1 h;

[0190] (2-7) Washing: 300 μL / well washing solution, wash the plate 3 times, and pat dry after the last wash;

[0191] (2-8) Color development: Mix solution A and solution B at a ratio of 1:1, add 200 μL to each well, and incubate at room temperature in the dark;

[0192] (2-9) Termination and detection: When the OD value of the control group is between 1.0 and 1.5, add 50 μL of stop solution to each well and immediately measure the OD value at a wavelength of 450 nm.

[0193] The purified antibodies were subjected to ELISA binding assays, as shown in Table 5. The results showed that the FGF18 monoclonal antibodies could bind to the FGF18 antigen and all exhibited good binding activity. See Table 5. The purified antibodies were then subjected to competitive ELISA assays, and the competing clone SM01 was named FGF18 monoclonal antibody A, as shown in Tables 6 and 7.

[0194] Table 5. ELISA binding detection results of purified antibody

[0195]

[0196] Table 6. Results of ELISA competitive detection of purified antibodies

[0197]

[0198] Table 7

[0199]

[0200] 3. Antibody gene sequencing of FGF18 monoclonal antibody A hybridoma cells.

[0201] Antibody genes were extracted from hybridoma cells to obtain the heavy and light chain gene sequences of the antibodies. The experimental method is as follows:

[0202] (1) RNA was extracted after hybridoma cell lysis;

[0203] (2) Reverse transcription: Using the extracted RNA as a template, cDNA was obtained in two steps using the Yiqiao Technology Reverse Transcription Kit (by first opening the secondary structure of RNA and then allowing primers to bind RNA and polymerize cDNA to obtain cDNA).

[0204] (3) Using cDNA obtained by reverse transcription as a template, the amplified fragment is amplified by PCR and inserted into an expression vector or commercial vector. The plasmid containing the correct sequence is obtained by sequencing.

[0205] The nucleotide and amino acid sequences of the heavy and light chains of FGF18 monoclonal antibody A in this application are shown below.

[0206] The full-length nucleotide sequence of the A light chain of the FGF18 monoclonal antibody is shown in SEQ ID NO.1 (720bp):

[0207] ATGATGAGTCCTGCCCAGTTCCTGTTTCTGTTAGTGCTCTGGATTCGGGAAACCAACGGTGATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATTTTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCTCAGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAA。

[0208] The full-length amino acid sequence of the light chain of monoclonal antibody A against FGF18 is shown as SEQ ID NO.2 (239aa) below:

[0209] MMSPAQFLFLLVLWIRETNGDVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTFLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFP QTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

[0210] The full-length nucleotide sequence of the A heavy chain of the FGF18 monoclonal antibody is shown in SEQ ID NO.3 (1389bp):

[0211]

[0212] The full-length amino acid sequence of the heavy chain of FGF18 monoclonal antibody A is shown in SEQ ID NO.4 (462aa):

[0213] MMVLSLLYLLTALPGILSEVQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWNWIRKFPGNKLEYMGYISYSGSTYYNPSLKSRISITRDTSKNQYYLQLNSVTTEDTATYYCAR WGGSHYWGQGTTLTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEP RGPTIKPCPPCKCPAPNLLGGPSVFIFPPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPI ERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK.

[0214] SEQ ID NO. 5 (HCDR1): GDSITSGY.

[0215] SEQ ID NO. 6 (HCDR2): ISYSGST.

[0216] SEQ ID NO. 7 (HCDR3): ARWGGSHY.

[0217] SEQ ID NO.8 (LCDR1): QSLLDSDGKTF.

[0218] SEQ ID NO.9 (LCDR2): LVS.

[0219] SEQ ID NO. 10 (LCDR3): WQGTHFPQT.

[0220] 4. Validation of the efficacy of FGF18 monoclonal antibody A (same as the FGF18 neutralizing antibody mentioned below)

[0221] Experiment 1: In vitro experimental verification that FGF18 monoclonal antibody A can inhibit the promoting effect of FGF18 on AML cells.

[0222] Cell culture conditions involved in the experiment: After resuscitation, AML cell lines (MLL-AF9 and C1498 cells) were cultured in RPMI-1640 medium containing 10% FBS (fetal bovine serum) and penicillin (100 U / mL penicillin and 100 g / mL streptomycin) under normal conditions (37°C, 5% CO2). Cells were passaged when they reached 3 / 4 of their growth density to maintain logarithmic cell growth.

[0223] Proliferation experiments were performed on AML cells: AML cells in logarithmic growth phase with trypan blue viability ≥90% were centrifuged (300g, 5min), washed once with calcium- and magnesium-free PBS, and resuspended in pre-warmed RPMI-1640 medium (containing 10% FBS and 1% penicillin and streptomycin). Cells were counted and the concentration adjusted, and 1×10⁶ cells were seeded per well. 3 Cells were added to 96-well clear plates, with 90 μL of cell suspension added to each well. Cell-free culture medium was added to the edge wells to reduce evaporation. After seeding, the cells were incubated at 37°C and 5% CO2 for 3 hours to restore their condition. AML cells were first treated with FGF18 (20 ng / mL) for 8 hours, followed by the addition of FGF18 neutralizing antibody to a final concentration of 10 μg / mL and IgG control antibody to a final concentration of 10 μg / mL. The cells were incubated at 37°C and 5% CO2, with detection time points set at days 1, 3, 5, and 7. At each time point, 10 μL of CCK-8 reagent was added to each well, gently mixed, and incubated in the dark for 3 hours. The absorbance was read at 450 nm using a microplate reader, with cell-free blank wells included for background correction. The data were normalized to 100% for the blank control group to calculate the relative proliferation rate. At least three technical replicates and three independent biological replicates were performed. Results are as follows: Figure 2 The results showed that the ability of FGF18 to promote cell proliferation could be effectively inhibited by the addition of FGF18 neutralizing antibodies.

[0224] In summary, FGF18 can significantly promote the proliferation and colony formation of AML cells, while FGF18 neutralizing antibodies can effectively inhibit these oncogenic functions.

[0225] Experiment 2: Detection of expression of FGF18 / FGFR3 downstream pathway-related proteins using Western blotting.

[0226] (1) Cell culture: After resuscitating C1498 cells, they were placed in RPMI-1640 medium containing 10% FBS and double antibiotics (100U / mL penicillin and 100µg / mL streptomycin) and cultured at 37℃ and 5% CO2. When the cells reached about 3 / 4 of the growth density, they were passaged and logarithmic growth was maintained.

[0227] (2) Serum starvation: Before the experiment, cells were incubated in serum-free RPMI-1640 (containing 0.1% FBS) for 8 h. Cells were pre-incubated at 37°C with FGF18 neutralizing antibody or IgG control antibody for 30 min; FGF18 was added to the target concentration (10 µg / mL), and stimulated at 37°C for 15 min; cells were then immediately placed on ice and washed twice with PBS to terminate the reaction.

[0228] (3) Protein extraction and quantification: Add 200 µL of pre-cooled RIPA lysis buffer (containing protease and phosphatase inhibitors); lyse on ice for 30 min. 12000×g, 4℃, 15 min, collect the supernatant. Determine protein concentration by BCA method, with a uniform loading amount of 20 µg / lane.

[0229] (4) SDS-PAGE and transfer: Add 4× loading buffer, heat at 95℃ for 5 min, and cool on ice. Separate with 10% SDS-PAGE until the bromophenol blue front appears. Activate the PVDF membrane with methanol and then perform wet transfer (120V, 90 min).

[0230] (5) Blocking and antibody incubation: Blocking: 5% BSA / TBST, room temperature for 1 hour. Primary antibody (4°C overnight) and recommended dilutions: P-PI3K 1:1000; PI3K 1:1000; P-AKT (Ser473) 1:1000; AKT 1:1000; P-mTOR (Ser2448) 1:1000; mTOR 1:1000; C-Myc 1:1000; GAPDH 1:3000.

[0231] (6) Washing membrane: TBST 3×10min.

[0232] (7) Secondary antibody: HRP-labeled secondary antibody 1:5000, room temperature 1h; then wash with TBST 3×10min.

[0233] (8) Color development and imaging: Add ECL luminescent liquid and expose using a chemiluminescence imaging system.

[0234] The results are as follows Figure 3 As shown, the FGF18 neutralizing antibody significantly reduced the expression of proteins related to downstream signaling pathways. This indicates that the FGF18 neutralizing antibody has good neutralizing activity and effectively blocks the transduction of FGF18 signaling.

[0235] Experiment 3: In vivo experiments demonstrated that FGF18 neutralizing antibody A can inhibit the growth of AML.

[0236] An AML mouse model was constructed using MLL-AF9 cells. 2 × 10⁶ cells were used. 5MLL-AF9 cells were suspended in 100 µL of sterile PBS and injected intravenously into 6-week-old C57BL / 6 mice. The day of injection was recorded as Day 1. Animals were randomly divided into three groups: control group (injected with PBS only), FGF18 neutralizing antibody group (administered with FGF18 neutralizing antibody), and IgG control antibody group (administered with IgG control antibody). Dosage regimen: Administered every other day starting from Day 7 until the experimental endpoint (i.e., administration on days 7, 9, 11, 13, 15, 17, 19, and 21). The recommended dose was 100 μg / mouse / dose, administered intraperitoneally, with PBS as the solvent. The primary endpoint was the percentage of peripheral blood leukemia cells on day 21.

[0237] The results showed that the tumor burden in mice injected with FGF18 neutralizing antibodies was significantly reduced ( Figure 4 ).

[0238] Experiment 4: In vivo experiments demonstrated that the FGF18 neutralizing antibody synergistically inhibited the growth of AML with the PD-1 monoclonal antibody.

[0239] Construct an AML mouse model, using 2×10 5 One MLL-AF9 cell was suspended in 100 mL of PBS and injected intravenously into 6-week-old C57 BL / 6 mice using a needle. The day of injection was marked as day 1. Starting from day 7, different treatments were applied to the mouse models of each component:

[0240] (1) Control group: injected with PBS;

[0241] (2) Group 1 was given a PD-1 inhibitor, 100 μg / animal / time; and an IgG control antibody, 100 μg / animal / time, was given at the same time.

[0242] (3) Group 2 was given FGF18 neutralizing antibody, 100 μg / animal / time;

[0243] (4) Group 3: PD-1 inhibitor, 100 μg / animal / time; FGF18 neutralizing antibody, 100 μg / animal / time.

[0244] For the treatment group, the drug was administered every two days for a total of 8 doses from day 7 to day 21 (i.e., on days 7, 9, 11, 13, 15, 17, 19, and 21).

[0245] Tumor burden in bone marrow and spleen was detected by flow cytometry. Figure 5 ).

[0246] Experiment 5: FGF18 neutralizing antibody therapy for tumors demonstrated good safety.

[0247] Following the procedure in Experiment 4 above, an AML mouse model was constructed. Starting from day 7, different treatments were applied to the mouse models of each component:

[0248] (1) Control group: injected with PBS;

[0249] (2) Group 1 was given FGF18 neutralizing antibody, 100 μg / animal / time;

[0250] (3) Group 2 was given IgG control antibody, 100 μg / animal / time.

[0251] Mice were administered the drug every two days for a total of 3 weeks (on days 7, 9, 11, 13, 15, 17, 19, and 21). IHC analysis was used to assess the normal function of vital organs (liver and lungs) in mice after injection of FGF18 neutralizing antibodies. The results are as follows: Figure 6 As shown, no significant histological changes were observed in the aforementioned normal vital organs when treated with FGF18 neutralizing antibodies, demonstrating that FGF18 neutralizing antibodies have good safety in vivo.

[0252] In experiments 1 to 5 above:

[0253] FGF18: Beijing Yiqiao Shenzhou Technology Co., Ltd., Cat:50177-M08H.

[0254] IgG control antibody: mouse IgG1 isotype Bio X Cell, BE0083.

[0255] PD-1 inhibitors: Pembrolizumab, MedChemExpress, HY-P9902A.

[0256] The above experimental results indicate that neutralizing antibodies against FGF18 can specifically block the FGF18 / FGFR3 signaling axis and inhibit the growth of leukemia tumors; the therapeutic effect is more significant when FGF18 neutralizing antibodies are used in combination with PD1 inhibitors and downstream pathway inhibitors.

[0257] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0258] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. An FGF18 neutralizing antibody, characterized in that, The FGF18 neutralizing antibody has amino acid sequences as shown in SEQ ID NO.5 to SEQ ID NO.7, namely HCDR1 to HCDR3, and amino acid sequences as shown in SEQ ID NO.8 to SEQ ID NO.10, namely LCDR1 to LCDR3.

2. The FGF18 neutralizing antibody according to claim 1, characterized in that, The species origin of the heavy chain variable region and light chain variable region backbone of the FGF18 neutralizing antibody are each independently of rat, human, cattle, horse, pig, sheep, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck or goose.

3. The FGF18 neutralizing antibody according to claim 2, characterized in that, The amino acid sequence of the heavy chain variable region of the FGF18 neutralizing antibody is shown as positions 19 to 132 in SEQ ID NO.4; or / and, the amino acid sequence of the light chain variable region of the FGF18 neutralizing antibody is shown as positions 21 to 132 in SEQ ID NO.

2.

4. The FGF18 neutralizing antibody according to any one of claims 1 to 3, characterized in that, The sequence of the constant region of the FGF18 neutralizing antibody is selected from the sequence of any one of the constant regions of IgG, IgA, IgM, IgE and IgD; and / or the species source of the light chain constant region and the heavy chain constant region of the FGF18 neutralizing antibody is independently mouse, human, cattle, horse, pig, sheep, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck or goose.

5. The FGF18 neutralizing antibody according to claim 4, characterized in that, The amino acid sequence of the heavy chain of the FGF18 neutralizing antibody is shown from position 19 to position 462 in SEQ ID NO.4; or / and, the amino acid sequence of the light chain of the FGF18 neutralizing antibody is shown from position 21 to position 239 in SEQ ID NO.

2.

6. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the FGF18 neutralizing antibody as described in any one of claims 1 to 5.

7. A carrier, characterized in that, It includes the nucleic acid molecule as described in claim 6.

8. A cell, characterized in that, The cell comprises the nucleic acid molecule of claim 6 or the vector of claim 7.

9. The method for constructing cells according to claim 8, characterized in that, The construction method includes the step of introducing the nucleic acid molecule of claim 6 or the vector of claim 7 into the cell to be modified.

10. The method for preparing the FGF18 neutralizing antibody according to any one of claims 1 to 5, characterized in that, The preparation method includes: culturing the cells of claim 8, and isolating FGF18 neutralizing antibodies from the resulting culture.

11. The use of the FGF18 neutralizing antibody according to any one of claims 1 to 5 in the preparation of a detection product or a drug; wherein the detection product detects tumors, and the drug treats tumors, wherein the tumor is leukemia, colorectal cancer, ovarian cancer, lung cancer, or breast cancer.

12. The FGF18 testing product is characterized by, The FGF18 detection product includes the FGF18 neutralizing antibody as described in any one of claims 1 to 5.

13. A drug, characterized in that, It includes the FGF18 neutralizing antibody as described in any one of claims 1 to 5 and pharmaceutically acceptable excipients.

14. A combination drug composition, characterized in that, The combined drug composition includes: (1) The FGF18 neutralizing antibody according to any one of claims 1 to 5 or the drug according to claim 13; and, (2) PD-1 inhibitors.

15. The combination drug composition according to claim 14, characterized in that, The PD-1 inhibitors include Pembrolizumab.

16. A method for detecting FGF18 in samples for non-diagnostic purposes, characterized in that, The detection method uses the FGF18 neutralizing antibody as described in any one of claims 1 to 5 or the FGF18 detection product as described in claim 12 to detect FGF18 in the sample to be tested.

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