Anti-CD14 anti-IL-15 super agonist fusion protein as well as preparation method and application thereof

Abstract

The invention discloses an anti-CD14 anti-IL-15 super agonist fusion protein as well as a preparation method and application thereof. Specifically, the fusion protein provided by the invention comprises a first structural unit, a second structural unit and a third structural unit, the first structural unit is an anti-CD14 antibody; the second structural unit is an IL-15R alpha fragment containing a sushi structural domain; the third structural unit is a mutant of IL-15; wherein the C end of the first structural unit is connected to the second structural unit through a covalent bond, and the second structural unit is connected to the N end of the third structural unit through a linker. The fusion protein disclosed by the invention has a remarkably enhanced anti-tumor effect.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and specifically, to an anti-CD14 antibody-IL-15 superagonist fusion protein, its preparation method and application. Background Art

[0002] Bladder cancer is one of the common urogenital system malignancies globally, with a relatively high incidence and recurrence rate. The current clinical standard platinum-based combination chemotherapy regimen has problems such as high toxicity, easy recurrence, and poor efficacy, and immunotherapy drugs have gradually become a new direction for the treatment of bladder cancer.

[0003] The IL-15 superagonist has good anti-tumor prospects due to its functions of stimulating the proliferation and activation of T cells and NK cells, inducing the synthesis of immunoglobulin by B cells, and supporting the differentiation of cytotoxic effector cells.

[0004] However, since the IL-15 superagonist can effectively activate CD8 + T and NK cells at very low concentrations, once it spreads throughout the body, it is likely to produce off-target effects, thereby triggering a severe cytokine release syndrome.

[0005] Therefore, more and more IL-15 superagonists appear in the form of immune-cytokine fusion proteins, which can effectively achieve the enrichment of cytokines at the tumor site by utilizing the targeting property of antibodies, thereby reducing the dosage and lowering the drug toxicity and side effects. There have been reports on fusing IL-15 and PD-L1 monoclonal antibody to construct an antibody-cytokine bifunctional fusion protein (CN116023503B), but this fusion protein lacks specificity for advanced high-grade bladder cancer. However, most bladder cancer patients have entered the advanced stage at the time of diagnosis, and then systemic metastasis occurs.

[0006] Therefore, there is an urgent need in the art to find more effective tumor targets and obtain more effective fusion proteins, so as to effectively achieve the radical cure of advanced high-grade bladder cancer. Summary of the Invention

[0007] The present invention provides a fusion protein that can be effectively used for the radical cure of advanced high-grade bladder cancer.

[0008] In the first aspect of the present invention, a fusion protein is provided, comprising: a first structural unit, a second structural unit, and a third structural unit; The first structural unit is an anti-CD14 antibody; The second structural unit is an IL-15Rα fragment containing a sushi domain; The third structural unit is a mutant of IL-15; Among them, the C-terminus of the first structural unit is connected to the second structural unit through a first linker or a covalent bond, and the second structural unit is connected to the N-terminus of the third structural unit through a second linker.

[0009] In another preferred example, the C-terminus of the first structural unit is connected to the N-terminus of the second structural unit through a covalent bond, and the C-terminus of the second structural unit is connected to the N-terminus of the third structural unit through a second linker.

[0010] In another preferred example, the N-terminus of the IL-15Rα fragment is connected to the C-terminus of the heavy chain of the anti-CD14 antibody through a first linker or a covalent bond, and the C-terminus of the IL-15Rα fragment is connected to the N-terminus of the mutant of IL-15 through a second linker.

[0011] In another preferred example, the anti-CD14 antibody is an anti-CD14 IgG1 antibody.

[0012] In another preferred example, the amino acid sequence of the heavy chain variable region of the anti-CD14 antibody is as shown in SEQ ID NO: 6, and the amino acid sequence of its light chain variable region is as shown in SEQ ID NO: 10.

[0013] In another preferred example, the amino acid sequence of the heavy chain of the anti-CD14 antibody is as shown in SEQ ID NO: 4, and the amino acid sequence of its light chain is as shown in SEQ ID NO: 8.

[0014] In another preferred example, the amino acid sequence of the IL-15Rα fragment is as shown in SEQ ID NO: 14.

[0015] In another preferred example, the IL-15 is a human IL-15 molecule or a mutant of the human IL-15 molecule.

[0016] In another preferred example, the mutant of IL-15 is IL-15N72D.

[0017] In another preferred example, the amino acid sequence of the IL-15 is as shown in SEQ ID NO: 18.

[0018] In another preferred example, the amino acid sequence of the second linker is as shown in SEQ ID NO: 16.

[0019] In the second aspect of the present invention, a nucleotide molecule is provided, and the nucleotide molecule encodes the fusion protein described in the first aspect of the present invention.

[0020] In another preferred example, the nucleotide molecule is DNA or cDNA.

[0021] In the third aspect of the present invention, there is provided an expression vector, characterized in that the expression vector contains the nucleotide molecule described in the second aspect of the present invention.

[0022] In another preferred embodiment, the vector includes: bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0023] In another preferred embodiment, the vector is a eukaryotic expression vector.

[0024] In the fourth aspect of the present invention, there is provided a host cell, which contains the expression vector described in the third aspect of the present invention, or the nucleotide molecule described in the second aspect of the present invention is integrated into the genome, or expresses the fusion protein described in the first aspect of the present invention.

[0025] In another preferred embodiment, the cell is a eukaryotic cell or a prokaryotic cell.

[0026] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0027] In another preferred embodiment, the host cell is selected from the group consisting of: Escherichia coli, yeast cells, mammalian cells.

[0028] In another preferred embodiment, the prokaryotic cell is Escherichia coli.

[0029] In the fifth aspect of the present invention, there is provided an immunoconjugate, which contains: (a) the fusion protein described in the first aspect of the present invention; and (b) a conjugate moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

[0030] In another preferred embodiment, the conjugate moiety is selected from: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes, radionuclides, biological toxins, cytokines, antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, virus particles, liposomes, magnetic nanoparticles, prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticles, etc.

[0031] In the sixth aspect of the present invention, there is provided a pharmaceutical composition, which contains: (i) The fusion protein as described in the first aspect of the present invention, the nucleotide molecule as described in the second aspect of the present invention, the expression vector as described in the third aspect of the present invention, the host cell as described in the fourth aspect of the present invention, or the immunoconjugate as described in the fifth aspect of the present invention; and (ii) A pharmaceutically acceptable carrier.

[0032] In another preferred example, the pharmaceutical composition is an injectable dosage form.

[0033] In another preferred example, the pharmaceutical composition is used for preparing a medicament for treating tumors, and the tumors are CD14-positive tumors or tumors with overexpression of CD14.

[0034] In another preferred example, the pharmaceutical composition further contains a second active ingredient, and the second active ingredient is an anti-tumor drug.

[0035] In another preferred example, the second active ingredient is selected from the group consisting of: chemotherapeutic drugs, targeted drugs, immunostimulants, antibody-drug conjugates, polypeptide drugs, nucleic acid drugs.

[0036] In another preferred example, the second active ingredient is atezolizumab.

[0037] In the seventh aspect of the present invention, there is provided the use of the fusion protein as described in the first aspect of the present invention, the nucleotide molecule as described in the second aspect of the present invention, the expression vector as described in the third aspect of the present invention, the host cell as described in the fourth aspect of the present invention, the immunoconjugate as described in the fifth aspect of the present invention, or the pharmaceutical composition as described in the sixth aspect of the present invention, for preparing a medicament for treating tumors; and the tumors are tumors with high expression of CD14.

[0038] In another preferred example, the tumors with high expression of CD14 are selected from the group consisting of: bladder cancer, breast cancer, non-Hodgkin lymphoma, ovarian cancer, non-small cell lung cancer, hepatocellular carcinoma, and laryngeal cancer, or a combination thereof.

[0039] In another preferred example, the bladder cancer includes advanced high-grade bladder cancer.

[0040] In another preferred example, the medicament further contains other anti-tumor drugs.

[0041] In another preferred example, the anti-tumor drugs are selected from the group consisting of: chemotherapeutic drugs, targeted drugs, immunostimulants, antibody-drug conjugates, polypeptide drugs, nucleic acid drugs, or a combination thereof.

[0042] In the eighth aspect of the present invention, there is provided a method for treating a tumor, comprising the step of administering to a subject in need thereof a therapeutically effective amount of the fusion protein as described in the first aspect of the present invention, the host cell as described in the fourth aspect of the present invention, the immunoconjugate as described in the fifth aspect of the present invention, or the pharmaceutical composition as described in the sixth aspect of the present invention, or a combination thereof.

[0043] In another preferred embodiment, the tumor is a CD14-positive tumor.

[0044] In another preferred embodiment, the tumor is a tumor with high CD14 expression.

[0045] In another preferred embodiment, the tumors with high CD14 expression are selected from the group consisting of: bladder cancer, breast cancer, non-Hodgkin lymphoma, ovarian cancer, non-small cell lung cancer, hepatocellular carcinoma, and laryngeal cancer, or a combination thereof.

[0046] In another preferred embodiment, the bladder cancer includes advanced high-grade bladder cancer.

[0047] In another preferred embodiment, the method further comprises treating the subject with another method for treating a disease.

[0048] In another preferred embodiment, the other method for treating a disease is selected from the group consisting of: surgery, radiotherapy, chemotherapy, gene therapy, DNA therapy, virus therapy, RNA therapy, adjuvant therapy, immunotherapy, or a combination thereof.

[0049] In the tenth aspect of the present invention, there is provided a method for preparing the fusion protein as described in the first aspect of the present invention, which is characterized by comprising the steps of: (a) Culturing the host cell as described in the fourth aspect of the present invention under expression conditions to express the fusion protein; (b) Separating and purifying the fusion protein obtained in (a).

[0050] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Shows the structural diagrams of the anti-CD14-IL-15 superagonist fusion protein and its control molecules.

[0052] Figure 2Shows the SDS-PAGE results of the anti-CD14-IL-15 superagonist fusion protein and its control molecule. (A) Reduced and non-reduced bands of anti-CD14; (B) Reduced and non-reduced bands of Fc-IL-15; (C) Reduced and non-reduced bands of anti-CD14-IL-15. Among them, R is the abbreviation of reduced, meaning reduced; N-R is the abbreviation of Non-reduced, meaning non-reduced; M is the abbreviation of MW, which is the protein molecular weight standard (kDa).

[0053] Figure 3 Shows the SEC-HPLC purity verification results of the anti-CD14-IL-15 superagonist fusion protein and its control molecule. (A-B) UV absorption of the anti-CD14-IL-15 solution at wavelengths of 280 nm and 214 nm; (C-D) UV absorption of the Fc-IL-15 solution at wavelengths of 280 nm and 214 nm; (E-F) UV absorption of the anti-CD14 solution at wavelengths of 280 nm and 214 nm.

[0054] Figure 4 Shows the affinity detection of anti-CD14 and anti-CD14-IL-15 with human CD14 protein. (A) Affinity detection of anti-CD14 with human CD14 protein; (B) Affinity detection of anti-CD14-IL-15 with human CD14 protein.

[0055] Figure 5 Shows the flow cytometry results of the expression levels of human CD14 on the surfaces of T24 cells, empty control T24 cells, and hCD14-overexpressing T24 cells (hCD14-OE T24).

[0056] Figure 6 Shows the verification of the binding activity at the cellular level of the anti-CD14-IL-15 superagonist fusion protein and its control molecule.

[0057] Figure 7 Shows the verification of the proliferation activities of the anti-CD14-IL-15 superagonist fusion protein and its control molecule on mouse lymphoblastoid CTLL2 cells and human megakaryocytic leukemia cells.

[0058] Figure 8 Shows CD8 + T, CD4 + T, NK and NKT cell gating strategies in human PBMCs.

[0059] Figure 9 Shows the Treg cell gating strategy in human PBMCs.

[0060] Figure 10 A - E in it show the verification of the proliferation activities of the anti - CD14 - IL - 15 super - agonist fusion protein and its control molecules on PBMCs and various immune cells.

[0061] Figure 11 It shows the verification of the promotion of NK cell killing activity by the anti - CD14 - IL - 15 super - agonist fusion protein and its control molecules.

[0062] Figure 12 It shows the efficacy evaluation of the T24 tumor subcutaneous ectopic transplantation tumor model. (A) Tumor growth curve; (B) Tumor anatomical diagram; (C) Tumor weight; (D) Mouse body weight change curve; (E) H&E staining results of heart, liver, spleen, lung, and kidney tissues and organs.

[0063] Figure 13 It shows the results of flow cytometry detection of human CD14 expression levels in bladder cancer PDX tissues. (A) Gating strategy; (B) Staining results of BV421 - Isotype control antibody; (C) Staining results of BV421 - anti - human CD14 antibody.

[0064] Figure 14 It shows the efficacy evaluation of the bladder cancer PDX subcutaneous transplantation tumor model. (A) Tumor growth curve; (B) Tumor anatomical diagram; (C) Tumor weight. Detailed implementation manners

[0065] Through extensive and in - depth research and a large number of screenings, the present inventors have first constructed an anti - CD14 antibody - IL - 15 super - agonist fusion protein, which can target CD14 - highly expressed cancer cells (such as bladder cancer cells) and promote the proliferation and activation of CD8 + T cells and NK cells nearby, thereby effectively treating CD14 - highly expressed cancers (such as advanced high - grade bladder cancer). On this basis, the present invention has been completed.

[0066] Terms To make the present disclosure easier to understand, certain terms are first defined. As used in this application, unless otherwise clearly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0067] The term "about" may refer to a value or a composition within an acceptable error range of a specific value or composition determined by a person of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. For example, as used herein, the expression "about 100" includes all values between 99 and 101.

[0068] As used herein, the terms "comprising" or "including" can be open-ended, semi-closed, and closed. In other words, the terms also include "consisting essentially of" or "consisting of".

[0069] As used herein, unless otherwise specified, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the range and, where appropriate, fractional values thereof (e.g., one-tenth and one-hundredth of an integer).

[0070] As used herein, the term "and / or" relates to and encompasses any and all possible combinations of one or more of the related listed items.

[0071] As used herein, the components of the term "pharmaceutically acceptable carrier" refer to substances that are suitable for humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), that is, substances with a reasonable benefit / risk ratio.

[0072] As used herein, the term "therapeutically effective amount" refers to an amount that produces a function or activity in humans and / or animals and is acceptable to humans and / or animals. Those of ordinary skill in the art should understand that the "therapeutically effective amount" may vary depending on factors such as the form of the pharmaceutical composition, the route of administration, the excipients of the drug used, the severity of the disease, and co-administration with other drugs.

[0073] Antibody-cytokine: A fusion protein composed of a targeting antibody and a cytokine, which brings the cytokine to a specific site through the antibody.

[0074] IL-15 superagonist: A protein dimer formed by non-covalent or covalent binding of IL-15Rα (or a fragment of IL-15Rα containing the sushi domain) or the sushi domain of IL-15Rα with IL-15 or a mutant of IL-15 (e.g., IL-15N72D).

[0075] Off-target effect: That is, the off tumor on target effect, which triggers unnecessary effects at non-tumor sites, thereby causing serious side effects.

[0076] Cytokine release syndrome: Due to the overactivation of immune cells, various cytokines are released, which have an adverse effect on the body.

[0077] CD14 Human monocyte differentiation antigen CD14 is a glycoprotein with a molecular weight of 14 kDa and exists in a membrane-anchored form on the surface of monocytes and macrophages. It is an LPS receptor that mediates the activation of the TLR-MyD88-NF-κB pathway, thereby triggering innate immune responses. In recent years, a large number of studies have shown that CD14 is highly expressed in advanced high-grade bladder cancer cells.

[0078] Advanced high-grade bladder cancer Advanced high-grade bladder cancer refers to urothelial carcinoma of the bladder with a histological grade of WHO G3 (high grade), which is characterized by: significant atypia of cancer cells (imbalance of nuclear-cytoplasmic ratio, active mitotic figures); complete loss of cell polarity and lack of normal stratification structure.

[0079] IL-15 and IL-15Rα Interleukin-15 (IL-15) is a key immunomodulatory cytokine, mainly produced by dendritic cells, monocytes and stromal cells, and enhances anti-tumor and antiviral immune responses by activating NK cells (natural killer cells) and CD8⁺ T cells (cytotoxic T cells).

[0080] IL-15Rα (interleukin-15 receptor alpha chain) is the specific alpha subunit responsible for high-affinity binding of IL-15 in the IL-15 receptor complex (IL-15R), and its key function depends on the sushi domain. The sushi domain is located in the extracellular region of IL-15Rα (amino acids 31–95 at the N-terminus) and can recognize IL-15 with high affinity and specificity.

[0081] The fusion protein of the present invention As used herein, the terms "the fusion protein of the present invention", "the anti-CD14 antibody-IL-15 superagonist fusion protein of the present invention", "the superagonist fusion protein of the present invention", and "anti-CD14-IL15" are used interchangeably and all refer to the fusion protein described in the first aspect of the present invention.

[0082] The fusion protein of the present invention comprises the following protein elements: (i) An anti-CD14 antibody; (ii) An IL-15Rα fragment containing a sushi domain; [[ID=2y7]](iii) A mutant of IL-15.

[0083] The anti-CD14 antibody in the fusion protein of the present invention can be any antibody or antigen-binding fragment thereof having specific binding affinity for CD14.

[0084] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having one light chain (L chain) and one heavy chain (H chain). In a general sense, the heavy chain can be understood as the polypeptide chain with a larger molecular weight in the antibody, and the light chain is the polypeptide chain with a smaller molecular weight in the antibody. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3), each light chain consists of a light chain variable region (VL) and a light chain constant region (CL), the light chain constant region consists of one domain CL, and the VH and VL regions can also be further divided into regions with high variability (referred to as complementarity determining regions (CDRs)). The variable regions (VH and VL) of each heavy chain / light chain pair form the antibody binding site. The term "antibody" is not limited by any specific method for generating antibodies. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be antibodies of different isotypes, such as IgG or its mutants, IgA1, IgA2, IgD, IgE, or IgM antibodies. In some embodiments of the present application, the antibody that binds to CD14 is a full-length immunoglobulin, including IgG1, IgG2, IgG3, and IgG4; preferably IgG1.

[0085] As used herein, "antigen-binding fragment" refers to a full-length antibody, Fab fragment, Fab' fragment, F(ab')2 fragment, or single Fv fragment having antigen-binding activity. An Fv antibody contains the variable region of the antibody heavy chain and the variable region of the light chain, but no constant region, and is the smallest antibody fragment with all antigen-binding sites. Generally, an Fv antibody also contains a polypeptide linker between the VH and VL domains and can form the structure required for antigen binding.

[0086] In one embodiment, the anti-CD14 antibody is a full-length antibody.

[0087] In a preferred embodiment, the anti-CD14 antibody is specifically a human IgG1 full-length immunoglobulin that binds to CD14. The amino acid sequence of the heavy chain of the human IgG1 full-length immunoglobulin is as shown in SEQ ID NO: 4, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 8.

[0088] In the present invention, the full-length antibody of the present invention also includes its conservative variants, which refer to polypeptides formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids having similar or close properties compared to the amino acid sequence of the full-length antibody of the present invention. [[ID=!4]]

[0089] As used herein, unless otherwise specified, Fc refers to the Fc fragment of human immunoglobulin. The term "immunoglobulin Fc region" refers to the constant region of an immunoglobulin chain, particularly the carboxyl terminus of the immunoglobulin heavy chain constant region or a portion thereof. For example, the immunoglobulin Fc region may include a combination of two or more domains of heavy chain CH1, CH2, and CH3 and the immunoglobulin hinge region. In a preferred embodiment, the Fc region of the immunoglobulin used includes at least one immunoglobulin hinge region, one CH2 domain, and one CH3 domain, preferably lacking the CH1 domain.

[0090] As used herein, the terms "mutant of IL-15", "IL-15 mutant", and "IL-15 mutant protein" all refer to mutants based on IL-15.

[0091] In one embodiment, the IL-15 mutant protein of the present invention has the following mutations relative to the wild-type IL-15 protein: N72D, i.e., IL-15N72D, but is not limited thereto. The IL-15 mutant protein of the present invention (or the fusion protein of the present invention containing the IL-15 mutant protein) may also include any other mutations as long as the resulting mutant has significantly enhanced IL2Rβ affinity and significantly improved biological activity relative to the wild-type IL-15 protein.

[0092] In a preferred embodiment, the amino acid sequence of the IL-15 mutant protein is as shown in SEQ ID NO: 18.

[0093] In one embodiment, the structure of the fusion protein of the present invention is as Figure 1 shown in the first molecular schematic diagram, and the amino acid sequence includes the heavy chain amino acids of the anti-CD14 antibody as shown in SEQ ID NO: 4, the light chain amino acids of the anti-CD14 antibody, the IL-15Rα fragment containing the sushi domain as shown in SEQ ID NO: 14, the linker as shown in SEQ ID NO: 16, and the mutant of IL-15 (IL-15N72D) as shown in SEQ ID NO: 18. Among them, the N-terminus of the IL-15Rα fragment containing the sushi domain is covalently bound to the C-terminus of the heavy chain of the anti-CD14 antibody, and its C-terminus is connected to the N-terminus of the mutant of IL-15 through a linker.

[0094] The fusion proteins of the present invention also include variant forms of the above-mentioned fusion proteins. These variant forms include (but are not limited to): deletion, insertion and / or substitution of 1-5 (usually 1-3, more preferably 1) amino acids, addition or deletion of one or several (usually within 5, preferably within 3, more preferably within 1) amino acids at the C-terminus and / or N-terminus, or addition of an amino acid fragment with a smaller amino acid side chain as a linker (such as glycine, serine, etc.) at the N-terminus or C-terminus of the protein. For example, in the art, when substituting amino acids with similar or comparable properties, the function of the protein is usually not changed. Also, for example, adding or deleting one or several amino acids at the C-terminus and / or N-terminus usually does not change the structure and function of the protein. In addition, the term also includes monomeric and polymeric forms of the polypeptides of the present invention. The term also includes linear and non-linear polypeptides (such as cyclic peptides).

[0095] The present invention also includes active fragments, derivatives and analogs of the above-mentioned fusion proteins. As used herein, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially retain the function or activity of the fusion proteins of the present invention.

[0096] The polypeptide fragments, derivatives or analogs of the present invention can be (i) polypeptides in which one or several conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, or (ii) polypeptides having substituents in one or more amino acid residues, or (iii) polypeptides formed by fusing the polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (a fusion protein formed by fusing with a leader sequence, a secretion sequence or a tag sequence such as 6His). According to the teachings herein, these fragments, derivatives and analogs are within the scope well known to those skilled in the art.

[0097] A preferred class of active derivatives refers to polypeptides formed by replacing at most 5, preferably at most 3, more preferably at most 1 amino acid with an amino acid having similar or comparable properties compared to the amino acid sequence of the present invention. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table A.

[0098] Table A

[0099] The present invention also provides analogs of the fusion protein of the present invention. These analogs may differ from the polypeptides of the present invention in terms of amino acid sequence, or in modified forms that do not affect the sequence, or both. Analogs also include analogs having residues different from natural L-amino acids (such as D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (such as β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

[0100] In addition, the fusion protein of the present invention can also be modified. Modification (usually without changing the primary structure) forms include: chemically derivatized forms of polypeptides in vivo or in vitro, such as acetylation or carboxylation. Modification also includes glycosylation, such as polypeptides that are glycosylated during the synthesis and processing of polypeptides or in further processing steps. Such modification can be accomplished by exposing the polypeptide to enzymes that perform glycosylation (such as mammalian glycosylating enzymes or deglycosylating enzymes). Modification forms also include sequences having phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that are modified to improve their proteolytic resistance or optimize their solubility.

[0101] The term "polynucleotide of the present invention" can be a polynucleotide encoding the fusion protein of the present invention, or a polynucleotide that also includes additional coding and / or non-coding sequences.

[0102] The present invention also relates to variants of the above polynucleotides, which encode fragments, analogs and derivatives of polypeptides or fusion proteins having the same amino acid sequence as the present invention. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, allelic variants are alternative forms of a polynucleotide that may be substitutions, deletions or insertions of one or more nucleotides, but do not substantially change the function of the encoded fusion protein.

[0103] The present invention also relates to polynucleotides that hybridize with the above sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions (or stringent conditions). In the present invention, "stringent conditions" refer to: (1) hybridization and washing at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) adding a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90% or more, preferably 95% or more.

[0104] The fusion protein and polynucleotide of the present invention are preferably provided in isolated form, and more preferably, purified to homogeneity.

[0105] The full-length sequence of the polynucleotide of the present invention can generally be obtained by PCR amplification, recombination or artificial synthesis methods. For the PCR amplification method, primers can be designed according to the nucleotide sequences disclosed in the present invention, especially the open reading frame sequences, and a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art can be used as a template for amplification to obtain the relevant sequences. When the sequence is relatively long, it is often necessary to perform PCR amplification two or more times, and then splice the amplified fragments together in the correct order.

[0106] Once the relevant sequence is obtained, the relevant sequence can be obtained in large quantities by recombination methods. This is usually to clone it into a vector, then transfer it into cells, and then isolate the relevant sequence from the proliferated host cells by conventional methods.

[0107] In addition, artificial synthesis methods can also be used to synthesize the relevant sequences, especially when the fragment length is relatively short. Usually, a very long fragment can be obtained by first synthesizing multiple small fragments and then ligating them.

[0108] Currently, it is already possible to completely obtain the DNA sequence encoding the protein (or its fragment, or its derivative) of the present invention by chemical synthesis. Then this DNA sequence can be introduced into various existing DNA molecules (such as vectors) and cells known in the art.

[0109] The method of applying PCR technology to amplify DNA / RNA is preferably used to obtain the polynucleotide of the present invention. Especially when it is difficult to obtain full-length cDNA from the library, the RACE method (rapid amplification of cDNA ends) can be preferably used. The primers for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein and can be synthesized by conventional methods. The amplified DNA / RNA fragments can be separated and purified by conventional methods such as gel electrophoresis.

[0110] Expression vector The present invention also relates to a vector containing the polynucleotide of the present invention, a host cell genetically engineered with the vector of the present invention or the coding sequence of the fusion protein of the present invention, and a method for producing the polypeptide of the present invention by recombinant technology.

[0111] In the present invention, the polynucleotide sequence encoding the fusion protein can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. Any plasmid and vector can be used as long as it can replicate and be stable in the host. An important feature of the expression vector is usually that it contains an origin of replication, a promoter, a marker gene and translation control elements.

[0112] In the method for preparing the fusion protein of the present invention, any suitable vector can be used, and it can be selected from one of pET, pDR1, pcDNA3.1(+), pcDNA3.1 / ZEO(+), pDHFR. The expression vector includes a fusion DNA sequence linked with appropriate transcriptional and translational regulatory sequences.

[0113] Both eukaryotic / prokaryotic host cells can be used for the expression of the fusion protein of the present invention. Eukaryotic host cells are preferably mammalian or insect host cell culture systems, preferably cells such as COS, CHO, NS0, sf9 and sf21, etc.; prokaryotic host cells are preferably one of DH5a, BL21(DE3), TG1.

[0114] Methods well known to those skilled in the art can be used to construct an expression vector containing the DNA sequence encoding the fusion protein of the present invention and appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombinant technology, etc. The said DNA sequence can be effectively linked to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of Escherichia coli; the λ phage PL promoter; eukaryotic promoters include the CMV immediate-early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the LTRs of retroviruses and some other known promoters that can control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0115] In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase, neomycin resistance and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0116] A vector containing the above appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell so that it can express the protein.

[0117] The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast, plant cells (such as ginseng cells).

[0118] When the polynucleotide of the present invention is expressed in a higher eukaryotic cell, the transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting factors of DNA, usually about 10 to 300 base pairs, which act on the promoter to enhance gene transcription. Examples include the 100 to 270 base pair SV40 enhancer on the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancer, etc.

[0119] Those of ordinary skill in the art are well aware of how to select appropriate vectors, promoters, enhancers and host cells.

[0120] Transformation of host cells with recombinant DNA can be carried out by conventional techniques well known to those skilled in the art. When the host is a prokaryote such as Escherichia coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, and the steps used are well known in the art. Another method is to use MgCl2. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0121] The obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. The culture is carried out under conditions suitable for the growth of the host cell. When the host cell grows to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature shift or chemical induction), and the cells are cultured for a further period of time.

[0122] The recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted extracellularly. If desired, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic lysis, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

[0123] The method of affinity chromatography can be used to separate and purify a class of fusion proteins disclosed in the present invention. According to the characteristics of the affinity column used, conventional methods such as high-salt buffer, changing pH, etc. can be used to elute the fusion proteins bound to the affinity column.

[0124] By using the above method, the fusion protein can be purified into a substantially homogeneous substance, such as a single band on SDS-PAGE electrophoresis.

[0125] Pharmaceutical composition In the present invention, a pharmaceutical composition containing the fusion protein or its immunoconjugate of the present invention is also provided.

[0126] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the fusion protein (or its conjugate) of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and their combinations. The pharmaceutical preparation should be matched with the administration method. The pharmaceutical composition of the present invention can be made into an injection form, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, such as about 10 micrograms / kg body weight to about 50 milligrams / kg body weight per day. In addition, the polypeptide of the present invention can also be used together with other therapeutic agents. The fusion protein or its immunoconjugate can form a pharmaceutical preparation together with pharmaceutically acceptable excipients to exert its efficacy more stably. These preparations can ensure the structural integrity of the amino acid core sequence of the fusion protein of the present invention, and at the same time protect the multiple functional groups of the protein from degradation (including but not limited to aggregation, deamination or oxidation). The preparations can be in various forms. Generally, for liquid preparations, they can be stored stably at least for one year at 2°C - 8°C, and for freeze-dried preparations, they can remain stable at 30°C for at least six months. Here, the preparations can be suspensions, aqueous injections, freeze-dried preparations, etc. commonly used in the pharmaceutical field, preferably aqueous injections or freeze-dried preparations.

[0127] For the pharmaceutical composition of the present invention (such as aqueous injection or freeze-dried preparation), pharmaceutically acceptable excipients thereof include one or a combination of surfactants, solution stabilizers, isotonicity regulators, and buffers. The surfactants include nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (Tween 20 or 80); poloxamer (such as poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; tetradecyl, linoleyl, or octadecyl sarcosine; Pluronics; MONAQUATTM, etc. The addition amount thereof should minimize the granulation tendency of the protein. The solution stabilizer can be saccharides, including reducing sugars and non-reducing sugars, amino acids including monosodium glutamate or histidine, and alcohols including trihydric alcohols, higher polyhydric alcohols, propylene glycol, polyethylene glycol, or a combination thereof. The addition amount of the solution stabilizer should enable the finally formed preparation to be considered stable by those skilled in the art within a stable time. The isotonicity regulator can be one of sodium chloride and mannitol, and the buffer can be one of TRIS, histidine buffer, and phosphate buffer.

[0128] When using the pharmaceutical composition, a safe and effective amount of the fusion protein or its immunoconjugate of the present invention is administered to a mammal, wherein the safe and effective amount is usually at least about 50 micrograms per kilogram of body weight, and in most cases does not exceed about 100 milligrams per kilogram of body weight. Preferably, the dose is about 100 micrograms per kilogram of body weight - about 50 milligrams per kilogram of body weight. Of course, the specific dose should also consider factors such as the administration route and the patient's health condition, which are all within the scope of the skills of a skilled physician. Typically, generally, the total administration amount cannot exceed a certain range. For example, the dose for intravenous injection is 10 to 3000 mg / day / 50 kg, preferably 100 to 1000 mg / day / 50 kg.

[0129] The fusion protein of the present invention and the pharmaceutical preparation containing the same can be used as an anti-tumor drug for tumor treatment. The anti-tumor drug referred to in the present invention means a drug having the effect of inhibiting and / or treating tumors, which can include the delay in the development of symptoms associated with tumor growth and / or the reduction in the severity of these symptoms. It further includes the alleviation of existing symptoms associated with tumor growth and the prevention of the appearance of other symptoms, and also reduces or prevents metastasis.

[0130] The above-mentioned fusion protein and its pharmaceutical preparation can also be administered in combination with other anti-tumor drugs for the treatment of tumors. These anti-tumor drugs for combined administration include but are not limited to: 1. Cytotoxic drugs (1) Drugs that act on the chemical structure of DNA: alkylating agents such as nitrogen mustards, nitrosoureas, and methylsulfonates; platinum compounds such as cisplatin, carboplatin, and oxaliplatin; mitomycin (MMC); (2) Drugs that affect nucleic acid synthesis: dihydrofolate reductase inhibitors such as methotrexate (MTX) and Alimta; thymidine synthase inhibitors such as fluorouracils (5FU, FT-207, capecitabine); purine nucleoside synthase inhibitors such as 6-mercaptopurine (6-MP) and 6-TG; ribonucleotide reductase inhibitors such as hydroxyurea (HU); DNA polymerase inhibitors such as cytarabine (Ara-C) and Gemz; (3) Drugs that act on nucleic acid transcription : Drugs that selectively act on DNA templates, inhibit DNA-dependent RNA polymerase, and thus inhibit RNA synthesis, such as: actinomycin D, daunorubicin, doxorubicin, epirubicin, aclarubicin, mithramycin, etc.; (4) Drugs that mainly act on microtubule synthesis: paclitaxel, taxotere, vinblastine, vinorelbine, podophyllotoxin, homoharringtonine; (5) Other cytotoxic drugs: asparaginase mainly inhibits protein synthesis; 2. Hormonal antiestrogens: tamoxifen, droloxifene, exemestane, etc.; aromatase inhibitors: aminoglutethimide, lantron, letrozole, arimide, etc.; anti-androgen: flutamide RH-LH agonists / antagonists: Noradrena, Enanton, etc.; 3. Biological response modifiers: interferons that mainly inhibit tumors through the body's immune function; other interleukins except IL-2; thymosin peptides; 4. Monoclonal antibodies: MabThera; Cetuximab (C225); Herceptin (Trastuzumab); Bevacizumab (Avastin); Yervoy (Ipilimumab); Pembrolizumab (Keytruda); Atezolizumab (Tecentriq); 5. Others include some drugs whose mechanisms are currently unknown and require further research; cell differentiation inducers such as retinoids; apoptosis inducers.

[0131] The main advantages of the present invention include: (a) The superagonist fusion protein of the present invention has better tumor treatment effect than the existing superagonist fusion protein (Fc-IL-15).

[0132] (b) This invention is the first to construct a superagonist fusion protein targeting human CD14, which is more targeted for cancers with high CD14 expression, especially advanced high-grade bladder cancer.

[0133] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are by weight.

[0134] Example 1 Preparation of Fusion Protein 1.1 Construction of an expression vector encoding the fusion protein A nucleotide sequence encoding an anti-CD14 antibody-IL-15 fusion protein or its two control molecules, an anti-CD14 antibody and an Fc-IL-15 fusion protein, was prepared by gene synthesis and PCR techniques, as Figure 1 shown.

[0135] Anti-CD14 antibody-IL-15 fusion protein: successively includes an anti-CD14 antibody (including a heavy chain with an amino acid sequence as shown in SEQ ID NO: 4 and a light chain with an amino acid sequence as shown in SEQ ID NO: 8), an IL-15Rα sushi domain (amino acid sequence as shown in SEQ ID NO: 14), and a mutant of IL-15 (IL-15N72D) (amino acid sequence as shown in SEQ ID NO: 18). Among them, the C-terminus of the anti-CD14 antibody heavy chain is covalently linked to the IL-15Rα sushi domain, and the IL-15Rα sushi domain is linked to the mutant of IL-15 through a linker (amino acid sequence as shown in SEQ ID NO: 16).

[0136] Control molecule anti-CD14 antibody: includes a heavy chain with an amino acid sequence as shown in SEQ ID NO: 4 and a light chain with an amino acid sequence as shown in SEQ ID NO: 8.

[0137] Control molecule Fc-IL-15 fusion protein: successively includes an Fc segment (amino acid sequence as shown in SEQ ID NO: 12), an IL-15Rα sushi domain (amino acid sequence as shown in SEQ ID NO: 14), a linker (amino acid sequence as shown in SEQ ID NO: 16), and a mutant of IL-15 (IL-15N72D) (amino acid sequence as shown in SEQ ID NO: 18).

[0138] The obtained fragment was inserted between the Hind III and BamHI digestion sites of the pcDNA3.4 plasmid by homologous recombination. The homologous recombination product was transformed into the DH5α strain to obtain the expression vector. The strain with correct vector construction was verified by sequencing. After culturing to the logarithmic growth phase, glycerol with a final concentration of 20% was added and stored at -80 °C for long term.

[0139] 1.2 Plasmid amplification The bacteria of the expression vector pcDNA3.4 were cultured with an inoculation volume expansion of 1:100. The plasmid was extracted by the alkaline lysis method using the EndoFree Maxi Plasmid Kit (product number: GDP117) provided by TIANGEN BIOTECH(BEIJING) CO.,LTD. The plasmid was filtered through a sterile filter membrane and stored at -20 °C.

[0140] 1.3 Cell culture and transient transfection to express the fusion protein 1) Cell preparation Human HEK293F cells were cultured in suspension. The culture medium formulation was a 1:1 volume mixture of OPM-293 CD03 Medium (OPM) and SMM 293-TII complete medium (Sino Biological). The suspension was passaged into fresh medium at a density of 1×10 6 / mL and cultured in suspension at 125 rpm in a 37 °C constant temperature shaker (5% CO2). Transfection was carried out when the cell density reached 3×10 6 / mL and the cell viability was over 95%.

[0141] 2) Plasmid transfection The plasmid was taken according to the mass of 1.5 μg plasmid / mL cell suspension, with the mass ratio of light chain plasmid to heavy chain plasmid being 2:1. The volume of PEI was 3 times the mass of the plasmid, and they were respectively diluted to 30 μg / mL and 90 μL / mL with SMM 293-TII complete medium. After mixing, they were left standing at room temperature for 15 min, and then gently added to the cell suspension with a pipette. The cells were cultured in a shaker, and feeding was added 24 h after transfection, and the culture continued for 6 days.

[0142] 1.4 Purification of the fusion protein 1) Sample preparation: The cell suspension was centrifuged at 2000 rpm for 5 minutes, and the precipitate was discarded to remove the cells; the harvested supernatant was centrifuged at 8000 rpm for 20 minutes, and the precipitate was discarded to remove cell debris. After harvesting the supernatant, it was filtered through a 0.45 μm filter membrane for use; 2) The protein purification system was rinsed with 20% ethanol, and the Protein A column (column volume 5 mL, provided by Lanxiao Technology) was rinsed and equilibrated with 10 column volumes of ultrapure water and the loading buffer PBS respectively; 3) Use the peristaltic pump on the AKTA protein purifier for sample loading at a flow rate of 3 mL / minute; after sample loading, continue to wash the column with PBS to wash away the unbound excess protein until the UV absorption value drops below 5.0.

[0143] 4) Use Tris-glycine buffer with pH = 3.5 as the eluent to elute the fusion protein bound to the Protein A column.

[0144] 5) The eluted solution is then passed through a molecular sieve and eluted with PBS as the mobile phase.

[0145] 6) Collect the eluate, place it in a 50 kDa ultrafiltration tube, centrifuge at 4000 rpm, and replace the previous liquid with PBS solution containing 10% trehalose.

[0146] 7) Pass the protein solution through a 0.22 μm sterile filter to remove pyrogens. After measuring the protein concentration with a Nanodrop, store it frozen in an ultra-low temperature freezer at -80 °C.

[0147] Example 2 Verification of the Molecular Weight of the Fusion Protein Mix the protein with reducing or non-reducing loading buffer and denature it in a metal bath at 100 °C for 5 - 10 minutes. Use a 1.5 mm 10-well 4% - 20% gradient precast SDS-PAGE gel. Load 10 μg of protein per well and run the gel on a Tianneng electrophoresis apparatus at 180 V until the markers are completely separated. Collect the SDS-PAGE gel, rinse it twice with ultrapure water, and then stain and decolorize it with an integrated elution Coomassie brilliant blue. Automatically expose the SDS-PAGE gel with a developer, as shown in Figure 2 A - C in.

[0148] Results and Analysis: Figure 2 In the reducing SDS-PAGE electrophoresis gel in A, the apparent molecular weights of the heavy and light chains of the anti-CD14 monoclonal antibody are respectively in the range of 25 - 35 kDa and 50 - 70 kDa, and the non-reducing electrophoresis gel shows that the apparent molecular weight of the anti-CD14 intact molecule is slightly larger than its theoretical molecular weight (145 kDa), indicating that this molecule is glycosylated.

[0149] Figure 2 In B in, the non-reducing band of the Fc-IL-15 molecule is around 100 kDa, which is basically consistent with the theoretical molecular weight, indicating that there is basically no glycosylation.

[0150] Figure 2 In C in, the light chain band of the anti-CD14-IL-15 molecule is in the same position as the light chain band of the anti-CD14 monoclonal antibody. The non-reducing band of anti-CD14-IL-15 is around 235 kDa, higher than the theoretical molecular weight. CombiningFigure 2 B in FIG. 1 shows that the glycosylation site of anti-CD14-IL-15 is located in the Fab portion of the anti-CD14 monoclonal antibody.

[0151] The reducing and non-reducing theoretical molecular weights of anti-CD14 monoclonal antibody, Fc-IL-15 molecule, and anti-CD14-IL-15 molecule are shown in Table 1.

[0152] Table 1

[0153] Example 3 Verification of fusion protein purity The mobile phase consisted of a 150 mM PB solution containing 5% isopropanol. The sample concentration was 1.0 mg / mL. The molecular sieve column was an SRT-C SEC-300 (7.8 × 300 mm, 5 µm) at a flow rate of 1.0 mL / min. The column temperature was 35°C and the sample flow rate was 1.0 mL / min. The fusion protein purity was monitored using an Agilent high-performance liquid chromatograph at dual wavelengths of 280 nm and 214 nm. The monomer content of the bifunctional fusion protein was estimated by peak area normalization.

[0154] Results and analysis: Figure 3 As shown in AF and Table 2-7, the main peak areas of the UV absorption peaks of the anti-CD14-IL-15 bifunctional fusion protein and its two control molecules at a wavelength of 280 nm are close to 100%, with no obvious aggregation and degradation, and the UV absorption peak at a wavelength of 214 nm is almost consistent with the absorption peak at 280 nm, indicating that there are no substances in the protein solution that interfere with the detection, thereby increasing the reliability of the test results.

[0155] Table 2 Detection results of anti-CD14-IL-15 solution at 280nm wavelength

[0156] Table 3 Detection results of anti-CD14-IL-15 solution at 214 nm wavelength

[0157] Table 4 Detection results of Fc-IL-15 solution at 280nm wavelength

[0158] Table 5 Detection results of Fc-IL-15 solution at 214 nm wavelength

[0159] Table 6 Detection results of anti-CD14 solution at 280nm wavelength

[0160] Table 7 Detection results of anti-CD14 solution at a wavelength of 214 nm

[0161] Example 4 Verification of the affinity of the fusion protein for human CD14 First, the anti-His antibody was conjugated to the CM5 chip. His-tagged recombinant human CD14 protein (MCE, catalog number: HY-P7786) was captured in the system, and at the same time, the anti-CD14 monoclonal antibody and the anti-CD14-IL-15 fusion protein were serially diluted 2-fold in the test buffer and then flowed through the chip. The binding time was set to 120 s, the dissociation time was 480 s, and during the detection process, the surface of the sensor chip was regenerated with Gly-HCl at pH = 1.5 to remove the bound protein. The detection results were analyzed using Biacore software.

[0162] Next, the present invention used the SPR method to detect the binding kinetics and affinity constants of the anti-CD14 monoclonal antibody and the anti-CD14-IL-15 fusion protein for hCD14.

[0163] The results are shown in Figure 4 A and B and Tables 8-9. The anti-CD14-IL-15 fusion protein has a high affinity for the hCD14 protein comparable to that of the anti-CD14 antibody, and the K D values all reach the pM level, and both molecules show kinetic characteristics of fast binding and slow dissociation to the hCD14 antigen.

[0164] Among them, the binding rate and dissociation rate of Fc-IL-15 are faster than those of the anti-CD14-IL-15 fusion protein. This is because the molecular weight of the fusion protein is larger, and the steric hindrance affects the binding of the molecule to the antigen. And the non-specific binding of the fusion protein to the antigen is stronger, resulting in a slower dissociation rate.

[0165] The results of the SPR of the binding of anti-CD14 to human CD14 protein

[0166] Table 9 SPR results of the binding of anti-CD14-IL-15 to human CD14 protein

[0167] Example 5 Construction of a T24 human bladder transitional cell carcinoma cell line stably expressing human CD14 The human bladder transitional cell carcinoma cell line T24 was obtained from the Cell Bank of the Chinese Academy of Sciences Committee on Type Culture Collection and cultured in RPMI 1640 / 10% FBS medium.

[0168] The human CD14 expression gene was cloned into the expression vector pcSLenti-EF1-EGFP-F2A-Puro-CMV- MCS-WPRE (Heyuan Biotechnology Co., Ltd.). The recombinant plasmid was transfected into HEK293T cells using Lipofectmaine 3000 (Invitrogen). After 6 hours of transfection, the original medium was discarded and replaced with fresh medium. After 48 hours, the cell supernatant was collected and added with polybrene (sigma) at a final concentration of 5 μg / mL to infect T24 cells for another 72 hours. Then, puromycin (Sigma) at a final concentration of 2 μg / mL was added to obtain a stable transfected cell pool. The monoclonal stable cell line T24 / CD14 with high CD14 expression was screened from the stable transfected cell pool using a flow cytometer (BD Biosciences). The expression level of CD14 on the cell surface was detected by flow cytometry (PE-anti-human CD14, Abcam, ab91143).

[0169] The results are shown in Figure 5 A-C below. Flow cytometry analysis of the lentivirus overexpression stable transfected cell line showed that compared with wild-type T24 cells and the empty transfected cell line, the hCD14 level on the surface of hCD14-OE T24 cells had a two-order-of-magnitude shift, with a narrower peak shape and uniform distribution, indicating that the hCD14 overexpression cell line was successfully constructed and could be used for subsequent experiments.

[0170] Example 6 Verification of the binding level of the fusion protein to the human CD14-overexpressing T24 human bladder transitional cell carcinoma cell line The human CD14-overexpressing T24 human bladder transitional cell carcinoma cell line was cultured in RPMI 1640 / 10% FBS medium containing 2 μg / mL puromycin. At a density of 2×10 per well 5Add CD14-overexpressing T24 cells with a certain cell quantity to a U-bottom 96-well cell culture plate. After centrifuging at 400 g for 5 min, discard the supernatant. Resuspend the cells with 100 μL of human IgG1 isotype control molecule, anti-CD14, or anti-CD14-IL-15 molecule solution at corresponding concentrations respectively. Set 3 replicates for each concentration and incubate at 4 °C for 40 minutes. Wash away the unbound molecules with PBS and repeat the washing 3 times. Add 100 μL of ABflo® 647 rabbit anti-human IgG (Fc) mAb (Abclonal: A22505) antibody suspension to each well to resuspend the cells, incubate at 4 °C for 30 minutes, wash away the unbound molecules with PBS and repeat the washing 2 times, and then detect the mean fluorescence intensity of each well by flow cytometry (SONY SA3800).

[0171] The results are as Figure 6 shown. Both the anti-CD14 antibody and the anti-CD14-IL-15 fusion protein have good binding activities for hCD14-OE T24 cells. The human IgG1 isotype control antibody has almost no binding to the hCD14-OE cell line, which can exclude the non-specific binding caused by the Fc segment.

[0172] The binding activity of the anti-CD14-IL-15 fusion protein is slightly higher than that of the anti-CD14 monoclonal antibody, which may be due to the enhanced non-specific binding ability to the cell surface after the fusion protein is linked with the IL-15 superagonist.

[0173] Example 7 Verification of the Biological Activity of the Fusion Protein 7.1 Evaluation of the Proliferation Activity of CTLL2 Cells Mouse lymphoblastoid cell line CTLL2 is cultured in RPMI 1640 / 10% FBS medium containing 100 U / mL IL-2 (Novo protein). Take an appropriate amount of CTLL2 cells, wash them 2 times with blank medium without IL-2, and then resuspend the cells with blank medium to 2×10 5 cells / mL. Add the cell suspension to a 9-6 well plate at a volume of 50 μL per well and culture it in a constant temperature CO2 cell incubator for 4 hours of starvation culture. Then add 50 μL of Fc-IL-15 or anti-CD14-IL-15 fusion protein diluted with gradient concentrations and culture it in a constant temperature CO2 cell incubator for 3 days. Then use an enhanced cck-8 kit (Meilunbio) to detect the cell proliferation, and set 5 replicates for each concentration.

[0174] The results are as Figure 7As shown in A, both Fc-IL-15 and anti-CD14-IL-15 have good proliferative activity on CTLL2, and the proliferative activity of anti-CD14-IL-15 on CTLL2 is slightly stronger than that of Fc-IL-15. It is speculated that this is due to the fact that the Fab part of the anti-CD14 antibody can enhance the non-specific binding of the anti-CD14-IL-15 fusion protein to CTLL2.

[0175] 7.2 Evaluation of Mo7e cell proliferative activity Human megakaryocytic leukemia cells M07e were cultured in RPMI 1640 / 10% FBS medium containing 10 ng / mL GM-CSF (Abclonal). An appropriate amount of M07e cells was taken, washed twice with blank medium without GM-CSF, and then resuspended in blank medium to 4×10 5 cells / mL. The cell suspension was added to a 96-well plate at a volume of 50 μL per well and cultured in a constant temperature CO2 cell incubator for 4 hours of starvation culture. Then, 50 μL of Fc-IL-15 or anti-CD14-IL-15 fusion protein diluted with gradient concentrations was added, and after culturing in a constant temperature CO2 cell incubator for 4 days, the cell proliferation was detected using an enhanced cck-8 kit (Meilunbio), with 5 replicates set for each concentration.

[0176] The results are as Figure 7 shown in B. Both Fc-IL-15 and anti-CD14-IL-15 have good proliferative activity on M07e, and the proliferative activity of anti-CD14-IL-15 on M07e is slightly stronger than that of Fc-IL-15. It is speculated that this is due to the fact that the Fab part of the anti-CD14 antibody can enhance the non-specific binding of the anti-CD14-IL-15 fusion protein to M07e.

[0177] 7.3 Evaluation of PBMCs proliferative activity Take 2.5×10 7 PBMCs cells (Hycell), adjust the cell density to 1×10 6 cells / mL with RPMI 1640 / 10% FBS medium, add an appropriate amount of Human CD3 / CD28 T cell activation magnetic beads (Biolegend) and activate for 3 days, then separate the magnetic beads with a MojoSort™ Magnet magnet. One part of them is at 1×10 per well 6Cells were seeded in a flat-bottom 96-well plate at a density of [number of cells] / mL, 50 μL per well. Gradient-diluted Fc-IL-15 and anti-CD14-IL-15 were added respectively. On the second day, cell proliferation was detected using the cck-8 kit (Meilunbio), and 4 replicates were set for each concentration.

[0178] PBS, 1 nM Fc-IL-15, 1 nM anti-CD14-IL-15 or 1 nM IL-2 were added to another part of activated PBMCs. Flow cytometry (Beckman) was used to detect the proportions of CD8 + T, CD4 + T, NK, NKT, Treg and other immune cells on day 0, day 3 and day 5. All flow antibodies used in this experiment were purchased from Biolegend.

[0179] The results were as Figure 10 shown in A of [[reference]]. Both Fc-IL-15 and anti-CD14-IL-15 had good proliferative activity on CD3 / CD28-activated human PBMCs, and the proliferative activity of anti-CD14-IL-15 on PBMCs was slightly stronger than that of Fc-IL-15. It was speculated that the Fab part of the anti-CD14 antibody could enhance the non-specific binding of the anti-CD14-IL-15 fusion protein to T cells and NK cells in human PBMCs.

[0180] According to the gating strategy as Figures 8 - 9 shown, the proportions of various immune cells in PBMCs were analyzed.

[0181] As Figure 10 shown in B-F of [[reference]], Fc-IL-15 and anti-CD14-IL-15 mainly acted on CD8 + T, NK and NKT cells. In addition, on Day 5, the proportion of Treg in the IL-2 group was significantly higher than that in the PBS control group, while the proportion of Treg in the IL-15 superagonist group was significantly lower than that in the PBS group, indicating that the molecule of the present invention did not have a preference for Treg.

[0182] 7.4 NK cell cytotoxicity assessment NK cells were isolated from PBMCs using the MojoSort™ Human NK Cell Isolation Kit (Biolegend) and pre-activated with 1 nM anti-CD14, Fc-IL-15, anti-CD14-Fc-IL-15 for 72 hours respectively. One day before cytotoxicity assessment, T24 cells were seeded at 1×10 4Cells were seeded at a density of [number of cells] per well in a 96-well plate. After 20 hours, NK cells were co-incubated with tumor cells at the corresponding ratio. Four hours later, cell cytotoxicity was detected using a LDH assay kit (Beyotime, C0016).

[0183] The results are as Figure 11 shown, indicating that IL-15 can enhance the tumor cytotoxicity of NK cells.

[0184] Example 8 Evaluation of the Pharmacodynamic Effect of a Subcutaneous Ectopic Xenograft Tumor Model of a Human Tumor Cell Line A subcutaneous ectopic xenograft tumor model was constructed using the human CD14-overexpressing T24 cell line established in Example 5. NCG mice (6-8 weeks old, Jiangsu Jicui Yakang Biotechnology Co., Ltd.) were introduced into a SPF-class animal room one week in advance and allowed to acclimatize for one week. Each mouse was inoculated unilaterally on the right costal region with [number of tumor cells] tumor cells. When the tumor volume reached [volume range] mm 6 , PBMCs were infused via the tail vein at a dose of [number of cells] per mouse. Twenty-four hours later, 100 μL of PBS or an equimolar concentration of anti-CD14 (2.27 mg / kg), Fc-IL-15 (1.55 mg / kg), anti-CD14+Fc-IL-15 (2.27 mg / kg + 1.55 mg / kg), or anti-CD14-IL-15 (3 mg / kg) was injected intraperitoneally. Thereafter, drugs were administered at a frequency of once every three days, and the changes in tumor volume and mouse body weight were monitored regularly. 3 6

[0185] The subcutaneous tumor volume was measured by recording the major and minor axes using an electronic vernier caliper. The calculation formula was: major axis × minor axis 2 * 1 / 2.

[0186] The experiment was terminated when the average tumor volume in the PBS group reached [volume] mm 3 . After the experiment, the mice were sacrificed by cervical dislocation. Organs and tissues such as the heart, liver, spleen, lungs, kidneys, and tumors were collected. The tumor size was recorded and photographed, and each tissue was preserved in a universal tissue fixative for H&E staining to observe for any histopathological changes.

[0187] The results are as Figure 12 shown in A-E, comparing the anti-tumor effects of PBS, anti-CD14, Fc-IL-15, anti-CD14+Fc-IL-15, and anti-CD14-IL-15 in the hCD14-overexpressing T24 tumor model.

[0188] ​​Among them, the anti - CD14 antibody group and the Fc - IL - 15 group had better anti - tumor effects than the PBS group, and the combined use group of anti - CD14 and Fc - IL - 15 (i.e., the combination) had significantly better effects than the PBS group, but was still weaker than the anti - CD14 - IL - 15 group.

[0189] In addition, Figure 12 As shown by the body weight change curve shown in D of , the body weight of the mice in the combined use group decreased significantly, while the body weight of the mice in other groups did not change significantly. And the mortality rate of the mice shown in Table 10 showed that the mortality rate of the combined use group reached 50%, while the mortality rate of the fusion protein group was only 20%. The above results all indicate that the safety of the fusion protein is better than that of the combined drug use.

[0190] Table 10 Number of dead mice and mortality rate in each group

[0191] To sum up, both anti - CD14 and Fc - IL - 15 have certain anti - tumor effects, and the anti - tumor effect is enhanced after their combined use. However, due to the lack of tumor - targeted accumulation of Fc - IL - 15, the efficacy of the combined use group is not as good as that of the fusion protein (anti - CD14 - IL - 15) group, and the toxic and side effects are relatively large.

[0192] Example 9 Construction of a subcutaneous xenograft model of bladder cancer PDX Fresh transurethral resection specimens of patients with advanced malignant bladder cancer were obtained from the Department of Urology, Xuzhou Central Hospital. After washing with normal saline, they were transported to the sterile operation room of the SPF - level animal house of the School of Pharmacy, Fudan University at low temperature in RPMI 1640 medium without serum. After removing the surrounding fat and necrotic parts of the tissue with sterile ophthalmic scissors, they were transplanted subcutaneously into the right rib of 6 - 8 - week - old NSG mice (Shanghai Southern Model). The tumor growth was observed regularly. When the tumor volume reached 1000 mm 3 ³, sub - passage was carried out. After the tissue could grow stably in the mice for 3 generations, it could be used for efficacy evaluation.

[0193] Flow cytometry was used to detect the CD14 expression level in the tissue. The specific method was as follows: Take 150 mg of tumor tissue, cut it into minced meat with ophthalmic scissors, and digest it in a mixed enzyme solution of hyaluronidase type IV (Solarbio), collagenase (Yeasen) and DNase I (Solarbio) for 90 minutes, then pass through a 70 - μm sieve, and terminate the digestion and wash once with FACS (PBS + 2% FBS) to obtain a single - cell suspension. After removing the red blood cells in the single - cell suspension with red blood cell lysate (Beyotime), count the single cells and adjust the cell density to 1×10 7cells / mL. Take 100 μL of cell suspension, add BV421-Isotype isotype control (Biolegend, Catalog: 400157) and BV421 anti-human CD14 flow antibody (Biolegend, Catalog: 367143) respectively, incubate at room temperature for 25 minutes, wash away the unbound excess antibody, and then detect by flow cytometry (Beckman).

[0194] The results are as Figure 13 shown in A-C of

[0195] Example 10 Pharmacodynamic Evaluation of Bladder Cancer PDX Subcutaneous Tumor Model After dissecting the PDX tissue from the mouse, use ophthalmic scissors to cut the tissue into small pieces of about 40-50 mm 3 in size, and transplant them subcutaneously into the right rib of 6-week-old NCG mice.

[0196] Starting from the third day after transplantation, inject 3×10 6 PBMCs into the tail vein of each mouse, and start intraperitoneal injection of drugs the next day after PBMCs infusion. The drug concentration and dosing frequency are the same as in Example 8.

[0197] When the tumor volume reaches 3000 mm 3 , it is the humanitarian endpoint. After the experiment, euthanize the mice by cervical dislocation, dissect out the tumor tissue, take pictures and record the weight of the tumor tissue.

[0198] The results are as Figure 14 shown in A-C of

[0199] The sequence information of the present invention is shown in Table B.

[0200] Table B

[0201] All the documents mentioned in the present invention are cited in this application as references, just as if each document is cited separately as a reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A fusion protein, characterized in that, Comprising: A first structural unit, a second structural unit and a third structural unit; The first structural unit is an anti-CD14 antibody; The second structural unit is an IL-15Rα fragment containing a sushi domain; The third structural unit is a mutant of IL-15; Wherein, the C-terminus of the first structural unit is covalently linked to the second structural unit, and the second structural unit is linked to the N-terminus of the third structural unit through a linker; Wherein, the amino acid sequence of the heavy chain of the anti-CD14 antibody is as shown in SEQ ID NO: 4, and the amino acid sequence of its light chain is as shown in SEQ ID NO: 8; the amino acid sequence of the IL-15Rα fragment is as shown in SEQ ID NO: 14; the mutant of IL-15 is IL-15N72D, and the amino acid sequence of the IL-15N72D is as shown in SEQ ID NO:

18.

2. The fusion protein according to claim 1, wherein, The amino acid sequence of the linker is as shown in SEQ ID NO:

16.

3. A nucleotide molecule, characterized in that, The nucleotide molecule encodes the fusion protein according to claim 1.

4. An expression vector, characterized in that, The expression vector contains the nucleotide molecule according to claim 3.

5. A host cell, characterized in that, The host cell contains the expression vector according to claim 4, or the nucleotide molecule according to claim 3 is integrated into the genome, or the fusion protein according to claim 1 is expressed.

6. An immunoconjugate, characterized in that, The immunoconjugate contains: (a) The fusion protein according to claim 1; And (b) A conjugate moiety selected from the group consisting of: a detectable label, a drug, or a combination thereof.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains: (i) The fusion protein according to claim 1, the nucleotide molecule according to claim 3, the expression vector according to claim 4, the host cell according to claim 5, or the immunoconjugate according to claim 6; and (ii) A pharmaceutically acceptable carrier.

8. Use of the fusion protein according to claim 1, the nucleotide molecule according to claim 3, the expression vector according to claim 4, the host cell according to claim 5, the immunoconjugate according to claim 6, or the pharmaceutical composition according to claim 7, characterized in that, For the preparation of a drug for treating tumors; and the tumor is a CD14-high-expressing tumor.

9. The use according to claim 8, characterized in that, The CD14-high-expressing tumors are selected from the group consisting of: bladder cancer, breast cancer, non-Hodgkin lymphoma, ovarian cancer, non-small cell lung cancer, hepatocellular carcinoma, laryngeal cancer, or a combination thereof.

10. A method for preparing the fusion protein according to claim 1, characterized in that, Including the steps of: (a) Culturing the host cell according to claim 5 under expression conditions to express the fusion protein; (b) Separating and purifying the fusion protein obtained in (a).

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