WO2024131864A1

WO2024131864A1 - Il-10 monomer fusion protein

Info

Publication number: WO2024131864A1
Application number: PCT/CN2023/140374
Authority: WO
Inventors: 张映培; 霍永庭; 芦迪
Original assignee: 广东菲鹏制药股份有限公司
Priority date: 2022-12-21
Filing date: 2023-12-20
Publication date: 2024-06-27
Also published as: CN118221829A

Abstract

The present invention relates to the field of biotechnology. Provided are an IL-10 monomer fusion protein and the use thereof. The fusion protein of the present invention has targeting capability, has improved safety, and can be used for effectively treating or preventing various tumors or inflammatory diseases.

Description

An IL-10 monomer fusion protein

CROSS-REFERENCE TO RELATED APPLICATIONS

The present disclosure claims priority to a Chinese patent application with application number “202211646452.8” and title “A IL-10 monomer fusion protein” filed with the Chinese Patent Office on December 21, 2022, the entire contents of which are incorporated by reference into the present disclosure.

Technical Field

The present disclosure relates to the field of biomedicine technology, and in particular to an IL-10 monomer fusion protein.

Background technique

Interleukin 10 (IL-10, or IL10), also known as human cytokine synthesis inhibitory factor (CSIF), is an anti-inflammatory cytokine and a homodimeric secretion. Currently, there are safety issues with IL-10 protein in clinical practice. A small amount of naturally dimerized IL-10 molecules bind with high affinity to its receptor IL-10Rα, and then further bind to IL-10Rβ to form a hexameric complex and activate downstream signals, causing biological functional responses.

Current clinical IL-10 drug molecules lack targeting, are easily off-target, and produce side effects; and these fusion protein molecules often have a normal IL-10 dimer structure. IL-10 dimers bind to IL-10Rα with high affinity, which may affect the targeting effect of the antibody end, thereby causing the design idea to fail. Moreover, if the activity of the antibody end requires a large dose, the side effects of the IL-10 end will still be a thorny problem.

The IL-10 monomers are symmetrically or asymmetrically connected to different parts of the antibody molecule. The steric hindrance of the antibody's own structure can isolate the IL-10 monomers. When the IL-10 monomer fusion protein is in a free state, even if the IL-10 monomer binds to IL-10Rα, it cannot cause a functional response. Only when the antibody end binds to the target cell can it aggregate the adjacent IL10 monomer-IL10Rα complex through the aggregation effect, and further bind to IL-10Rβ to form a complex to generate downstream signals and trigger biological functions. However, the fusion protein of IL-10 monomers connected to different parts of the antibody molecule still has the problem of in vivo toxicity.

Therefore, it is still necessary to actively explore IL-10 drugs with strong targeting and few side effects. This research direction is of great significance for the prevention or treatment of tumors or inflammatory diseases.

Summary of the invention

The purpose of the present disclosure is to provide an IL-10 monomer fusion protein.

In a first aspect of the present disclosure, the present disclosure provides a fusion protein comprising: an antibody, and an interleukin monomer or a variant thereof inserted into the antibody;

In some specific embodiments, the antibody is an immunoglobulin having two identical light chains and two identical heavy chains, wherein the light chain comprises a VL region and a CL region, and the heavy chain comprises a VH region, a CH1 region, a CH2 region, and a CH3 region; and the position at which the interleukin monomer or a variant thereof is inserted into the antibody is as follows (i) or (ii):

(i) the interleukin monomer or variant thereof is inserted between the CH2 region and the CH3 region of one of the heavy chains of the antibody;

(ii) The interleukin monomer or variant thereof is inserted between the VL region and the CL region of one of the light chains of the antibody.

In a second aspect of the present disclosure, the present disclosure provides a multispecific binding molecule, which includes the aforementioned fusion protein.

In the third aspect of the present disclosure, the present disclosure provides a nucleic acid encoding the aforementioned fusion protein or the aforementioned multi-specific binding molecule.

In a fourth aspect of the present disclosure, the present disclosure provides an expression vector comprising the aforementioned nucleic acid.

In a fifth aspect of the present disclosure, the present disclosure provides a recombinant cell, which carries the aforementioned nucleic acid, the aforementioned expression vector, and expresses the aforementioned fusion protein or the aforementioned multi-specific binding molecule.

In the sixth aspect of the present disclosure, the present disclosure provides the use of the aforementioned fusion protein, or the aforementioned multi-specific binding molecule, the aforementioned nucleic acid, the aforementioned expression vector or the aforementioned recombinant cell in the preparation of a drug, wherein the aforementioned drug is used to treat or prevent tumors or inflammatory diseases.

In the seventh aspect of the present disclosure, the present disclosure provides a pharmaceutical composition, which comprises the aforementioned fusion protein, or the aforementioned multi-specific binding molecule, the aforementioned nucleic acid, the aforementioned expression vector or the aforementioned recombinant cell; optionally, the above-mentioned pharmaceutical composition also includes a pharmaceutically acceptable carrier.

In an eighth aspect of the present disclosure, the present disclosure provides a method for treating, preventing or diagnosing a tumor or inflammatory disease, disorder or condition, comprising administering a therapeutically effective amount of the aforementioned pharmaceutical composition to a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present disclosure and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

Figure 1 is a schematic diagram of the binding mode of natural IL-10 molecules and IL-10 monomers to IL-10 receptors according to an embodiment of the present disclosure, wherein A represents a schematic diagram of the binding mode of natural IL-10 molecules to IL-10 receptors, R1 represents IL-10 receptor α (IL-10Rα), and R2 represents IL-10 receptor β (IL-10Rβ); B represents a schematic diagram of the binding mode of IL-10 monomers to IL-10 receptors, R1 represents IL-10 receptor α (IL-10Rα), and IL-10M1 represents IL-10 monomers.

FIG2 is a schematic diagram of the binding of antibodies to antigens on target cells and IL-10 monomers to IL-10 receptors in the fusion protein according to an embodiment of the present disclosure. The affinity of IL-10 monomers is relatively weak and the targeting effect is poor. When the antibody end is targeted to the tumor surface, the local enrichment of the antibody can aggregate the IL-10 monomers together to form a dimer IL-10(M)*2 to exert its effect. Among them, IL-10R1 represents IL-10 receptor α (IL-10Rα), and IL-10R2 represents represents IL-10 receptor β (IL-10Rβ), Her2 represents human epidermal growth factor receptor, which is an example of an antibody-targeted antigen, T-cell represents T cell, and Tumor represents tumor.

FIG3 is a schematic diagram of the positional structure of the IL-10 monomer inserted into the antibody molecule according to an embodiment of the present disclosure (taking the insertion of the IL-10 monomer between the CH2 region and the CH3 region of one of the heavy chains of the antibody as an example), wherein A represents a schematic diagram of the structure of inserting an IL-10 monomer between the CH2 region and the CH3 region of the heavy chain containing the Knob domain of the antibody, and the resulting fusion protein molecule is named R1738; B represents a schematic diagram of the structure of inserting an IL-10 monomer at the C-terminus of the CH3 region of the heavy chain containing the Knob domain of the antibody, and the resulting fusion protein molecule is named R1740; C represents a schematic diagram of the structure of inserting an IL-10 monomer at the C-terminus of the CH3 region of the heavy chain containing the Knob domain of the antibody, and the resulting fusion protein molecule is named R1740; A schematic diagram of the structure in which an IL-10 monomer is inserted into the C-terminus of the VL region of the light chain containing the Obscurin domain, and the resulting fusion protein molecule is named R1737; D represents a schematic diagram of the structure in which an IL-10 monomer is inserted into the C-terminus of the CL region of the light chain containing the Obscurin domain of the antibody, and the resulting fusion protein molecule is named R1739; wherein, IL-10 in the figure represents an IL-10 monomer, KIH represents a KIH heavy chain anti-mismatch structure, Obs domain represents Obscurin domain, Tit represents Titin domain, VL represents a light chain variable region, and VH represents a heavy chain variable region.

Figure 4 is a schematic diagram of the structure of the control molecules (R0987, R0989, R0674, R0862, R1049, R0579, R1187) in the embodiments of the present disclosure, wherein IL-10 in the figure represents an IL-10 monomer, VL represents a light chain variable region, and VH represents a heavy chain variable region.

Figure 5 is a graph showing the SDS-PAGE detection results of the fusion proteins R1737, R1738, R1739, and R1740 according to the embodiments of the present disclosure, wherein R refers to reducing SDS-PAGE (reducing SDS-PAGE) and NR refers to non-reducing SDS-PAGE (No-reducing SDS-PAGE).

Figure 6 is a graph showing the result of detecting the binding activity of the IL-10 monomer end to IL10Rα by flow cytometry according to the fusion protein of an embodiment of the present disclosure, wherein the abscissa (Ab conc.Log(nM)) represents the antibody concentration (nM), and the ordinate MFI PE represents the mean fluorescence intensity.

Figure 7 is a graph showing the result of detecting the binding activity of the antibody end to mPD-1 by flow cytometry according to the fusion protein of the embodiment of the present disclosure, wherein the horizontal axis (Ab conc.Log(nM)) represents the antibody concentration (nM), and the vertical axis MFI PE represents the mean fluorescence intensity.

Figure 8 is a graph showing the result of detecting the IL-10 monomer end binding activity using the reporter gene method for the fusion protein according to an embodiment of the present disclosure, wherein B is an enlarged view of R1737, R1738, R1739 and R0862 in A, the abscissa (Ab conc.Log (μg/mL)) represents the antibody concentration (μg/mL), and the ordinate (Lum) represents the luminescence fluorescence intensity; the solid graph represents the signal before enrichment, and the hollow graph represents the signal of the enrichment system.

FIG. 9 shows the results of the activity test of each IL10 fusion protein binding to IL10Rα by ELISA.

FIG. 10 is the result of detecting the activity of each IL10 fusion protein binding to IL10Rβ by ELISA.

FIG. 11 shows the results of the activity test of each IL10 fusion protein binding to IL10Rα/IL10Rβ by ELISA.

DETAILED DESCRIPTION OF THE INVENTION

In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise defined in the present disclosure, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present disclosure belongs. Unless otherwise indicated, the methods disclosed herein using kits and reagents obtained from commercial sources are generally performed according to the protocols and/or parameters defined by the manufacturer.

The articles "a", "an", and "the" in this disclosure include plural references unless the context clearly indicates otherwise. For example, "an antibody" refers to one antibody or more than one antibody.

In the present disclosure, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of the present disclosure, "plurality" means at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

In the present disclosure, the term "fusion protein" refers to a fusion polypeptide molecule comprising two or more different proteins, wherein the components of the fusion protein are directly linked by peptide bonds or linked to each other by a connecting peptide.

In the present disclosure, the terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of the corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

In the present disclosure, the term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., an alpha carbon bound to a hydrogen, carboxyl group, amino group, and R group), such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Amino acid analogs typically have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but function in a manner similar to that of a naturally occurring amino acid.

In the present disclosure, the term "connector peptide" refers to a peptide segment composed of amino acids, such as glycine and/or serine residues, used alone or in combination to connect the various domains in an antibody. In certain embodiments, the connecting peptide can be about 1 to about 100 amino acids long, for example, about 1 to 50 amino acids long. In the present disclosure, "first connecting peptide", "second connecting peptide", "third connecting peptide", "fourth connecting peptide", "fifth connecting peptide" represent different connecting peptides.

In some specific embodiments, the above-mentioned connecting peptide is a flexible connecting peptide.

In some specific embodiments, the above-mentioned flexible connecting peptide amino acid sequence includes but is not limited to (GS)n, (GGGGS)nG or (GGGGSSG)n, wherein n is a positive integer equal to or greater than 1, for example, n is a positive integer between 1-10.

In some specific embodiments, the amino acid sequence of the flexible connecting peptide can be GSGSGSGS (SEQ ID NO:6), GGGGSG (SEQ ID NO:12), GSGSGSGSGGGGSSG (SEQ ID NO:13), GGGGSSG (SEQ ID NO:16), or GGGGSGGGGSGGGGSGGGGSG (SEQ ID NO:17).

In the present disclosure, the term "spacer peptide" refers to a peptide that is inserted into a specific protein and used to change the structure and/or function of the protein. In this sense, it is different from the connecting peptide that connects other fusion partners, but the connecting peptide can be used as a spacer peptide.

In some specific embodiments, the spacer peptide has an amino acid sequence as shown in SEQ ID NO:4.

In the present disclosure, the term "antibody" is an immunoglobulin molecule that can specifically bind to an antigen, including two light chains with a relatively light molecular weight and two heavy chains with a relatively heavy molecular weight, and the heavy chain (H chain) and the light chain (L chain) are connected by a disulfide bond to form a tetrapeptide chain molecule. Among them, the amino acid sequence of the amino terminal (N terminal) of the peptide chain varies greatly, which is called the variable region (V region), and the carboxyl terminal (C terminal) is relatively stable and varies little, which is called the constant region (C region). The V regions of the L chain and the H chain are respectively called VL and VH, the C region of the L chain is CL, and the C region of the H chain includes the CH1 region, the CH2 region and the CH3 region, and the CH1 region and the CH2 region are generally connected by a hinge region.

In the present disclosure, the term "hinge" refers to the region between the CH1 region and the CH2 region of an immunoglobulin heavy chain, which includes inter-H chain disulfide bonds, is rich in proline, does not form an α-helix, is prone to stretching and a certain degree of distortion, and is conducive to the complementary binding between the antigen binding site of the antibody and the antigen epitope.

In the present disclosure, the term "interleukin (IL)" refers to a group of cytokines with complex immunomodulatory functions. In addition, they are also involved in a variety of physiological and pathological reactions of the body, such as playing an important role in inflammatory reactions. At least 38 interleukins have been discovered, named IL-1 to IL-38.

In the present disclosure, the term "monomer" refers to one of the single polypeptide chains of an aggregate. For example, for an interleukin monomer, it refers to one of the two peptide chains of certain interleukins that usually exist in the form of dimers.

In the present disclosure, the term "dimer" refers to a class of substances that appear in a pair. Interleukins with a dimer structure in the natural state include but are not limited to IL-10 (Kang Mingchao, Lu Yingchun, et al. Interleukin-10 polypeptide conjugates, dimers and uses thereof), IL-5 (Clutterbuck EJ, Hirst EM, Sanderson CJ. Human interleukin-5 (IL-5) regulates the production of eosinophils in human bone marrow cultures: comparison and interaction with IL-1, IL-3, IL-6, and GMCSF. Blood. 1989 May 1; 73(6):1504-12. PMID:2653458.), IL-12 (Song et al., 2003). Ge, Yuan Mei, Lu Shibi. Research progress on clinical application of interleukin-12[J]. Chinese Journal of Oncology Prevention and Treatment, 2007, 14(001):75-79.), IL-23(Tan Guang, Wang Zhongyu. Research progress on interleukin-23[J]. International Journal of Laboratory Medicine, 2006, 27(10):3.), IL-25(Meng Jie, Li Xiaomei, Xu Bei. Research progress on interleukin-25 in immune inflammatory diseases[J] . Chinese Journal of Rheumatology, 2012, 16(11):3.), IL-27 (Li Yang, Xia Likun. Research progress on the biological functions of interleukin-27 and its antiviral effects [J]. International Journal of Immunology, 2015, 38(2):5.) and IL-35 (Zhang Junfeng, Tian Zhikang, Xue Qingjie, et al. Research progress on the mechanism of action of IL-35 in tumors [J]. Chinese Journal of Immunology, 2020, 36(7):5.).

The native IL-5 monomer has the amino acid sequence shown below:

In the present disclosure, the terms "IL-10" and "IL10" are used interchangeably. The IL-10 molecule in the natural state usually exists in a dimer structure (including two natural IL-10 monomers, and a single natural IL-10 monomer has an amino acid sequence as shown in SEQ ID NO: 3). The binding mode of the natural IL-10 molecule to the IL-10 receptor (IL-10R) is shown in Figure 1-A. At present, clinical IL-10 drugs lack targeting, and drug molecules are easily off-target, which not only makes it difficult to exert the efficacy but also easily produces side effects. The activity of IL-10 monomer on IL-10Rα is 10 times weaker than that of natural IL-10 dimer (reference for specific experimental data: J Biol Chem. 2000 May 5; 275(18):13552-7.doi:10.1074/jbc.275.18.13552.), and it is almost impossible to mediate further binding with IL-10Rβ, so it is difficult to activate downstream signals to cause biological functional responses. The binding mode of IL-10 monomer to IL-10 receptor is shown in Figure 1-B. In some specific embodiments of the present disclosure, an IL-10 monomer is inserted into a specific position of one of the light chains or heavy chains of an antibody to form an "asymmetric structure" to prepare an asymmetric IL-10 monomer fusion protein. According to the IL-10 monomer fusion protein designed in the present disclosure, in the absence of target cell aggregation, the IL-10 monomer has only very weak binding with IL-10Rα, and there is even no IL-10 downstream signal. Only after the antibody end binds to the target cell can it aggregate the adjacent IL-10 monomer-IL-10Rα complex through the aggregation effect, and further bind to IL-10Rβ to form a complex to generate downstream signals and trigger biological functions (taking the insertion of IL-10 monomer between the CH2 region and CH3 region of one of the heavy chains of the antibody as an example, its mechanism of action is shown in Figure 2). Compared with the IL-10 monomer fusion protein, the asymmetric IL-10 monomer fusion protein has only one IL-10 monomer, so the asymmetric IL-10 monomer fusion protein does not have the same dosage limit as the normal IL-10 molecule or its antibody fusion protein. The asymmetric IL-10 monomer fusion protein can have a larger dosage, which can also provide a wider space for the fused antibody end (the clinical doses of different antibodies vary. If the dose limit of IL-10 is too low, it will seriously limit the antibody target selection of the asymmetric IL-10 monomer fusion protein) to function.

In some specific embodiments, IL-5, IL-12, IL-23, IL-25, IL-27 and IL-35 can also be applied to the fusion protein of the present disclosure to achieve similar effects as IL-10. That is, an interleukin monomer is inserted into a specific position of one of the heavy chains or light chains of the antibody, and before the antibody binds to the target cell, these interleukin monomers cannot activate downstream signals; after the antibody binds to the target cell, the interleukin monomers aggregate to form dimers and bind to the corresponding receptors, thereby exerting biological functions.

In the present disclosure, the term "variant" encompasses both naturally occurring variants and non-naturally occurring variants, and generally refers to one or more compounds having a native polypeptide sequence and structure, which have one or more amino acid additions, substitutions (conservative in nature) and/or deletions relative to the native molecule. Interleukin "variant" and "modified" interleukin are used interchangeably and refer to an equivalent term for an interleukin amino acid sequence that has one or more amino acid additions, substitutions (conservative in nature) and/or deletions relative to a native interleukin molecule or has at least 95% (e.g., 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence of a native interleukin.

In the present disclosure, the term sequence "identity" refers to the degree (percentage) to which the amino acids/nucleic acids of the two sequences are identical at equivalent positions when the two sequences are optimally aligned (introducing gaps, if necessary, to obtain the maximum sequence identity percentage, and not considering any conservative substitutions as part of the sequence identity). To determine the percentage of sequence identity, the alignment can be achieved by techniques known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring the alignment, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.

In the present disclosure, the term "IL-10 variant" refers to an IL-10 amino acid sequence that has at least 95% (e.g., 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence of wild-type IL-10 (SEQ ID NO: 3).

In some specific embodiments, the interleukin monomer or variant thereof is inserted into the antibody at position (i); the heavy chain of the antibody comprises a modification that prevents heavy chain mispairing.

Since the four polypeptide chains of the antibody in the invention include two heavy chains and light chains with different structures, after inserting an interleukin monomer or a variant thereof into the heavy chain or light chain, when the fusion protein disclosed in the present invention is prepared by genetic engineering technology, the four polypeptide chains can randomly generate a variety of possible structural combinations during the assembly process, but only one is the target product (i.e., the correctly paired product), and the remaining products are invalid products or even by-products (i.e., incorrectly paired products) generated by mismatching. Therefore, in order to improve the correct assembly of antibodies, reduce mismatching between antibody chains, and prevent mismatching and the formation of inactive molecules, when the interleukin monomer or a variant thereof is inserted into one of the heavy chains of the above-mentioned antibody, a modification that prevents mismatching of the heavy chains, i.e., a modification that prevents pairing of two identical heavy chains, can be introduced into the two heavy chains of the antibody.

In the present disclosure, the term "modification to prevent mispairing of heavy chains" refers to some designs or modifications used by those skilled in the art to prevent mispairing of heavy chains, including but not limited to KIH (Knob-In-Hole), SEEDbodies (trand-exchange engineered domain), cFAE (arm exchange), XmAb, leucine zipper technology (LUZ-Y), etc. Among them, KIH technology refers to a mutation in the CH3 region of one of the heavy chains of an antibody to form a protruding "knob"-like structure (Knob), and a mutation in the CH3 region of another heavy chain to form a concave "hole"-like structure (Hole). The knob-hole design is conducive to the correct assembly of the antibody heavy chain. The heavy chain containing the Knob is paired with the heavy chain containing the Hole, the two heavy chains containing the Knob are not paired with each other, and the two heavy chains containing the Hole are not paired with each other.

In some specific embodiments, the CH3 region of one heavy chain of the antibody contains a Knob domain, and the CH3 region of the other heavy chain of the antibody contains a Hole domain, and the Knob domain and the Hole domain pair to form a heavy chain anti-mismatching structure.

In some specific embodiments, according to the EU numbering system, the CH3 region of the heavy chain containing the Knob domain comprises the amino acid mutations: T366W, K392D and K409D, and the CH3 region of the heavy chain containing the Hole domain comprises the amino acid mutations: L368R, D399K and Y407A.

In the present disclosure, the term mutation "AxxB" means that the amino acid A at position xx is mutated to the amino acid B. For example, "T366W" means that the threonine (Thr; T) at position 366 is mutated to tryptophan (Trp; W).

In some specific embodiments, the above interleukin monomer or variant thereof is inserted into a heavy chain containing a Knob domain.

In some specific embodiments, the interleukin monomer or variant thereof is connected to the heavy chain containing the Knob domain of the antibody via a first connecting peptide.

In some specific embodiments, the N-terminus of the interleukin monomer or its variant is connected to the C-terminus of the CH2 region of the heavy chain containing the Knob domain of the antibody through a first connecting peptide, and the C-terminus of the interleukin monomer or its variant is connected to the N-terminus of the CH3 region of the heavy chain containing the Knob domain of the antibody through a first connecting peptide.

In some specific embodiments, the structure of the heavy chain containing the Knob domain of the above-mentioned antibody from N-terminus to C-terminus is as follows: VH-CH1-hinge-CH2-L1-Int-L1-CH3 (Knob); wherein L1 represents: the first connecting peptide, and Int represents: an interleukin monomer or a variant thereof.

In some specific embodiments, the structure of the heavy chain containing the Hole domain of the above-mentioned antibody from N-terminus to C-terminus is as follows: VH-CH1-hinge-CH2-CH3 (Hole).

In some specific embodiments, the structure of the light chain of the above antibody from N-terminus to C-terminus is as follows: VL-CL.

In some specific embodiments, the above-mentioned first connecting peptide has an amino acid sequence shown in SEQ ID NO:6.

In some specific embodiments, the interleukin monomer or variant thereof is inserted into the position of the antibody (ii); the antibody comprises a design for preventing light chain mispairing and a design for preventing heavy chain mispairing.

When the fusion protein of the present invention is prepared by genetic engineering technology, if the interleukin monomer or its variant is inserted into one of the light chains of the above-mentioned antibody, a modification that prevents light chain mispairing and heavy chain mispairing can be introduced into the antibody: that is, two identical light chains containing the interleukin monomer or its variant are prevented from pairing with two heavy chains, and two identical light chains not containing the interleukin monomer or its variant are prevented from pairing with two heavy chains.

In the present disclosure, the term "modification to prevent light chain mispairing" refers to some designs or modifications used by those skilled in the art to prevent light chain mispairing, including but not limited to Titin-Obscurin, CrossMab, etc. Among them, the core design of Titin-Obscurin is the introduction of a pair of interacting proteins, namely Titin (titin) and Obscurin (obscurin), replacing CH1 and CL on one side of the antibody with Titin and Obscurin, respectively, to avoid light chain mispairing.

In some specific embodiments, the CL region of one light chain of the above-mentioned antibody is replaced by an Obscurin domain, the CH1 region of one heavy chain of the above-mentioned antibody is replaced by a Titin domain, and the Obscurin domain is paired with the Titin domain to form a light chain anti-mispairing structure; the CH3 region of the heavy chain containing the Titin domain of the above-mentioned antibody contains a Knob domain, and the CH3 region of the other heavy chain of the above-mentioned antibody contains a Hole domain, and the Knob domain is paired with the Hole domain to form a heavy chain anti-mispairing structure.

In some specific embodiments, the Obscurin domain has an amino acid sequence as shown in SEQ ID NO: 10, and the Titin domain has an amino acid sequence as shown in SEQ ID NO: 11;

In some specific embodiments, according to the EU numbering system, the CH3 region of the heavy chain containing the Knob domain comprises the mutations: T366W, K392D and K409D, and the CH3 region of the heavy chain containing the Hole domain comprises the mutations: L368R, D399K and Y407A.

In some specific embodiments, the above interleukin monomer or variant thereof is inserted into a light chain containing an obscurin domain.

In some specific embodiments, the interleukin monomer or variant thereof is connected to the Obscurin domain of the light chain of the antibody via a third connecting peptide; and the Titin domain of the antibody is connected to the VH region via a second connecting peptide.

In some specific embodiments, the N-terminus of the interleukin monomer or variant thereof is connected to the C-terminus of the VL region of the light chain containing the Obscurin domain of the above-mentioned antibody via a first connecting peptide, and the C-terminus of the interleukin monomer or variant thereof is connected to the N-terminus of the Obscurin domain of the light chain of the above-mentioned antibody via a third connecting peptide;

The N-terminus of the titin domain is connected to the C-terminus of VH via a second connecting peptide;

In some specific embodiments, the structure of the light chain containing the Obscurin domain of the above-mentioned antibody from N-terminus to C-terminus is as follows: VL-L1-Int-L3-Obs, wherein L1 represents: the first connecting peptide, L3 represents: the third connecting peptide, Int represents: an interleukin monomer or a variant thereof, and Obs represents: an Obscurin domain.

In some specific embodiments, the structure of the light chain of the above antibody without the Obscurin domain from N-terminus to C-terminus is as follows: VL-CL.

In some specific embodiments, the structure of the heavy chain containing the Titin domain and the Knob domain of the above-mentioned antibody from N-terminus to C-terminus is as follows: VH-L2-Tit-CH2-CH3, wherein L2 represents: the second connecting peptide, and Tit represents: the Titin domain.

In some specific embodiments, the structure of the heavy chain containing the Hole domain of the above-mentioned antibody from N-terminus to C-terminus is as follows: VH-CH1-hinge-CH2-CH3.

In some specific embodiments, the second connecting peptide has an amino acid sequence as shown in SEQ ID NO:12, and the third connecting peptide has an amino acid sequence as shown in SEQ ID NO:13.

In some specific embodiments, the above interleukin is selected from IL-10 monomer, IL-5 monomer, IL-12 monomer, IL-23 monomer, IL-25 monomer, IL-27 monomer, and IL-35 monomer.

In some specific embodiments, the interleukin monomer is an IL-10 monomer.

In some specific embodiments, the IL-10 monomer is a natural IL-10 monomer or a modified IL-10 monomer.

In some specific embodiments, the above-mentioned natural IL-10 monomer has an amino acid sequence as shown in SEQ ID NO:3.

In some specific embodiments, the modified IL-10 monomer has a spacer peptide inserted between two amino acid sites in the natural IL-10 monomer molecule sequence;

In some specific embodiments, the modified IL-10 monomer has a spacer peptide inserted between the 116N and 117K sites of the natural IL-10 monomer molecule sequence;

In some specific embodiments, the spacer peptide comprises the amino acid sequence shown in SEQ ID NO:4;

In some specific embodiments, the modified IL-10 monomer lacks the first two amino acids at the N-terminus of the natural IL-10 monomer molecule sequence;

In some specific embodiments, the modified IL-10 monomer has an amino acid sequence as shown in SEQ ID NO:5.

In some specific embodiments, the antibody in the above fusion protein specifically binds to immune checkpoints and/or tumor antigens.

In the present disclosure, the term "immune checkpoint" refers to a class of immunosuppressive molecules that are expressed on immune cells and can regulate the degree of immune activation. They play an important role in preventing the occurrence of autoimmunity. Overexpression, overfunction or poor immunosuppression of immune checkpoint molecules can lead to abnormal immune function of the body.

In some specific embodiments, the above-mentioned immune checkpoint molecules are selected from PD-1, CD47, TIGIT, CD137, CD134, KIR, LAG-3, PD-L1, CTLA-4, B7.1, B7H3, CCRY, OX-40 and CD40.

In the present disclosure, the term "tumor antigen" refers to an antigenic polypeptide or protein derived from or associated with a tumor or cancer disease, typically derived from a tumor/cancer cell, optionally a mammalian tumor/cancer cell, and may be located in or on the surface of a tumor cell derived from a mammal, optionally derived from a human, such as a systemic or solid tumor. "Tumor antigens" typically include tumor-specific antigens (TSAs) and tumor-associated antigens (TAAs). TSAs are typically caused by tumor-specific mutations and are specifically expressed by tumor cells. TAAs are typically presented by tumor cells and "normal" (healthy, non-tumor) cells.

In some specific embodiments, the above tumor antigen is selected from GUCY2C, MSLN, Claudin18.2, GPC3, EGFR, HER2, CEA, GD2, EGFRⅧ, MUC1, PRLR, CLCA1, MUC12, GPR35, CR1L, MUC17, TMPRSS11B, MUC21, TMPRSS1IE, CD207, SLC30A8, CFC1, SLC12A3, SSTR1, GPR27, FZD10, TSHR, SIGLEC15, SLC6A3, KISSIR, QRFPR, GPR119, CLDN6, UPK2, ADAM12, S LC45A3, ACPP, MUC21, MUC16, MS4A12, ALPP, EphA2, FAP, IL13-Ra2, PSMA, ROR1, VEGFR-Ⅱ, FR-a, EpCAM, EGFRⅦ, tMUC1, PSCA, FCER2, GPR18, FCRLA, CXCR5, FCRL3, FCRL2, HTR3A, CLEC17A, TRPMI, SLC45A2, SLC24A5, DPEP3, KCNK16, LIM2, KCNV2, SLC26A4, CD171, Glypican-3, IL-13, CD79a/b and MAGEA4, etc.

In some specific embodiments, the above-mentioned antibodies specifically bind to PD-1. In the present disclosure, the term "PD-1" refers to programmed death receptor 1, which belongs to the immunoglobulin superfamily and acts as a co-inhibitory receptor to negatively regulate the immune system. In the present disclosure, "PD-1" and "PD1" are used interchangeably. PD-1 is a member of the CD28/CTLA-4 family and has two known ligands, including PD-L1 and PD-L2. PD-1 and PD-L1 bind to initiate programmed death of T cells, allowing tumor cells to obtain immune escape. Immunomodulation targeting PD-1 and PD-L1 is of great significance for anti-tumor, anti-infection, anti-autoimmune disease and organ transplant survival. The representative amino acid sequence of human PD-1 is disclosed in NCBI Accession No.: NP 005009.2, and the representative nucleic acid sequence encoding human PD-1 is shown in NCBI Accession No.: NM 005018.2.

In the present disclosure, the term "multispecific binding molecule" refers to a multispecific molecule capable of binding to two or more different target antigens or epitopes.

In the third aspect of the present disclosure, the present disclosure provides a nucleic acid encoding the aforementioned fusion protein.

In the present disclosure, the term "nucleic acid" is used interchangeably with the term "polynucleotide" herein, and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form, covering nucleic acids containing known nucleotide analogs or modified backbone residues or connections, the nucleic acids are synthetic, naturally occurring and non-naturally occurring, have similar binding properties to reference nucleic acids, and are metabolized in a manner similar to reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). Nucleic acids encoding polypeptides or fusion proteins refer to one or more nucleic acid molecules encoding polypeptides or fusion proteins, including such one or more nucleic acid molecules in a single vector or separate vectors, and such one or more nucleic acid molecules present in one or more positions in a host cell. Unless otherwise indicated, a specific nucleic acid sequence also implicitly covers conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as explicitly indicated sequences.

In a fourth aspect of the present disclosure, the present disclosure provides an expression vector containing the aforementioned nucleic acid.

In the present disclosure, the term "vector" refers to a vehicle into which a genetic element (e.g., the aforementioned nucleic acid molecule) can be operatively inserted and the genetic element can be expressed, such as producing a protein, RNA or DNA encoded by the genetic element, or replicating the genetic element. The vector can be used to transform, transduce or transfect a host cell so that the genetic element it carries is expressed in the host cell. For example, vectors include: plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC), phages such as lambda phage or M13 phage, and animal viruses, etc. The vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site. The vector may also include components that assist it in entering the cell, including, but not limited to, viral particles, liposomes or protein shells. The vector may be an expression vector or a cloning vector. In some embodiments, the vectors (e.g., expression vectors) provided by the present disclosure contain a nucleic acid sequence encoding a fusion protein as described in the present disclosure, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selection marker.

In a fifth aspect of the present disclosure, the present disclosure provides a recombinant cell, which carries the aforementioned nucleic acid, the aforementioned expression vector, expresses the aforementioned fusion protein, or expresses the aforementioned multi-specific binding molecule.

In the present disclosure, the term "recombinant cell" refers to a cell into which an exogenous polynucleotide and/or vector can be or has been introduced. The recombinant cell contains the vector, which can be introduced into a mammalian cell to construct a recombinant cell, and then the recombinant cell is used to express the antibody or antigen-binding fragment provided by the present disclosure. The recombinant cell is cultured to obtain the corresponding antibody or fusion protein. The mammalian cell that can be used can be a CHO cell, etc.

In the sixth aspect of the present disclosure, the present disclosure provides the use of the aforementioned fusion protein, the aforementioned multi-specific binding molecule, the aforementioned nucleic acid, the aforementioned expression vector or the aforementioned recombinant cell in the preparation of a drug, wherein the aforementioned drug is used to treat, prevent or diagnose tumors or inflammatory diseases.

In this disclosure, the term "treatment or prevention" refers to clinical intervention that attempts to alter the natural course of the individual being treated, and can be performed for prevention or during the course of clinical pathology. The desired effects of treatment or prevention include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, alleviating/reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and regression or improved prognosis.

In the present disclosure, the term "diagnosis" refers to the identification, disclosure, identification and/or definition of the location of the pathogenic process of a pathological state, disease or condition. In some specific embodiments, the pharmaceutical composition of the present disclosure, when administered to a subject or contacted with a sample from a subject, helps diagnose cancer, tumor formation or condition.

In this disclosure, the terms "cancer" and "tumor" are used interchangeably and refer to an abnormal mass of cells in a multicellular organism caused by uncontrolled and progressive excessive cell division. Uncontrolled growth can cause these cells to invade, enter, and even destroy adjacent tissues. Cancer cells can also spread to other locations, which can lead to the formation of metastases.

In this disclosure, the term "inflammatory disease" refers to a disease or condition characterized by abnormal inflammation (eg, increased levels of inflammation compared to a control, such as a healthy person not suffering from the disease). Examples of inflammatory diseases include, but are not limited to, autoimmune diseases, arthritis, rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), myasthenia gravis, juvenile onset diabetes, type 1 diabetes, Guillain-Barré syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, ankylosing spondylitis, psoriasis, Sjögren's syndrome, vasculitis, glomerulonephritis, autoimmune thyroiditis, Behcet's disease, Crohn's disease, ulcerative colitis, bullous pemphigoid, sarcoidosis, ichthyosis, Graves' ophthalmopathy, inflammatory bowel disease, Addison's disease, vitiligo, asthma, allergic asthma, acne vulgaris, celiac disease, chronic prostatitis, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, ischemia-reperfusion injury, stroke, sarcoidosis, transplant rejection, interstitial cystitis, atherosclerosis, scleroderma, and atopic dermatitis, among others.

In a seventh aspect of the present disclosure, the present disclosure provides a pharmaceutical composition comprising the aforementioned fusion protein, the aforementioned multispecific binding molecule, the aforementioned nucleic acid, the aforementioned expression vector or the aforementioned recombinant cell.

In the present disclosure, the term "pharmaceutical composition" is in a form that permits the biological activity of the active ingredient to be effective, and does not contain additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

In some specific embodiments, the above-mentioned pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

In the present disclosure, the term "pharmaceutically acceptable carrier" refers to ingredients in pharmaceutical formulations that are different from the active ingredient and are non-toxic to the subject, including but not limited to buffers, excipients, stabilizers, preservatives, or any physiologically compatible solvents, etc.

In the present disclosure, the term "subject" or "patient" refers to a mammalian subject or patient. Unless otherwise indicated, the terms "patient" or "subject" are used interchangeably herein. Exemplary subjects include, but are not limited to, humans, monkeys, dogs, cats, mice, rats, cattle, horses, camels, birds, goats, and sheep. In certain embodiments, the subject is a human. In some embodiments, the subject is a human suspected of having cancer, an autoimmune disease or condition, and/or an infection.

In the present disclosure, the term "administering" or "administering", as it applies to an animal, a human, an experimental subject, a cell, a tissue, an organ or a biological fluid, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent or composition with the animal, human, subject, cell, tissue, organ or biological fluid.

In the present disclosure, the term "effective amount" generally refers to an amount sufficient to reduce the severity and/or frequency of symptoms, eliminate these symptoms and/or potential causes, prevent the occurrence of symptoms and/or their potential causes, and/or improve or ameliorate damage (e.g., lung disease) caused by or associated with a disease state. In some embodiments, an effective amount is a therapeutically effective amount or a prophylactically effective amount.

In the present disclosure, the term "therapeutically effective amount" is an amount sufficient to treat a disease state or symptom, particularly a state or symptom associated with the disease state, or otherwise prevent, hinder, delay or reverse the progression of the disease state or any other undesirable symptoms associated in any way with the disease.

In the present disclosure, the term "prophylactically effective amount" is an amount that, when administered to a subject, will have a predetermined preventive effect, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the likelihood of the onset (or recurrence) of the disease state or associated symptoms. Complete treatment or prevention may not occur after administration of one dose, but may occur after administration of a series of doses. Thus, a therapeutically or prophylactically effective amount may be administered in one or more administrations.

In the present disclosure, the terms "therapeutically effective amount" and "prophylactically effective amount" may vary depending on a variety of factors, such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improved health status of the patient.

Detailed ways

In order to make the purpose, technical scheme and advantages of the embodiments of the present disclosure clearer, the technical scheme in the embodiments of the present disclosure will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

Unless otherwise indicated, the practice of the present disclosure will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. Such techniques are fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, Second Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Animal Cell Culture (R.I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwe ll ed.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos ed., 1987); Current Protocols in Molecular Biology (F. M. Ausubel et al. ed., 1987); PCR: The Polymerase Chain Reaction (Mullis et al. ed., 1994); and Current Protocols in Immunology (J. E. Coligan et al. ed., 1991), each of which is expressly incorporated herein by reference.

The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

Example 1

Method for constructing IL-10 monomer fusion protein

In this embodiment, IL-10 monomers are inserted into different positions of antibody molecules (this embodiment takes anti-PD-1 antibody as an example, and the heavy chain and light chain amino acid sequences of the anti-PD-1 antibody used in the present disclosure are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively) to obtain a fusion protein, wherein the specific experimental operations for the construction of IL-10 monomer molecules mainly refer to the "Molecular Cloning Experiment Guide".

The amino acid sequence of the natural IL-10 monomer molecule (i.e., IL-10WT) is shown in SEQ ID NO:3, and a spacer peptide with an amino acid sequence as shown in SEQ ID NO:4 is inserted between the two sites 116N and 117K of the natural IL-10 monomer molecule sequence, and the first two amino acids at the N-terminus of the natural IL-10 monomer are removed, thereby forming the modified IL-10 monomer (i.e., IL-10M) of the embodiment of the present disclosure, whose sequence is shown in SEQ ID NO:5.

Amino acid sequence of the heavy chain of anti-PD-1 antibody:

Amino acid sequence of the light chain of the anti-PD-1 antibody:

Amino acid sequence of natural IL-10 monomer molecule:

Amino acid sequence of the spacer peptide:

The amino acid sequence of the modified IL-10 monomer (hereinafter also referred to as "IL-10 monomer" or "IL-10M") of this example is:

Construction of exemplary fusion protein R1738

According to EU numbering, three amino acid sites of CH3 of one heavy chain of the antibody were mutated: T366W, K392D and K409D to form a Knob domain, and three amino acid sites of CH3 of the other heavy chain were mutated: L368R, D399K and Y407A to form a Hole domain. The Knob domain and the Hole domain are paired to form a KIH heavy chain anti-mismatching structure. The two light chains of the antibody are the same and no modification is made.

The N-terminus of the IL-10 monomer is connected to the C-terminus of the CH2 region of the heavy chain containing the Knob domain of the antibody through a peptide having an amino acid sequence as shown in SEQ ID NO:6 (i.e., the first connecting peptide, L1, linker1), and the C-terminus of the IL-10 monomer is connected to the N-terminus of the CH3 region of the heavy chain containing the Knob domain of the antibody through the first connecting peptide. The resulting exemplary fusion protein R1738 has an amino acid sequence as shown in SEQ ID NO:7 (heavy chain containing the Knob domain), SEQ ID NO:8 (heavy chain containing the Hole domain) and SEQ ID NO:9 (light chain), and the specific structure of the fusion protein is shown in Figure 3A (the insertion position of the IL-10 monomer is shown in the figure).

Amino acid sequence of the first connecting peptide:

The structure of the heavy chain containing the Knob domain in the fusion protein R1738 from N-terminus to C-terminus is as follows: VH-CH1-hinge-CH2-L1-IL10-L1-CH3, where L1 represents: the first connecting peptide

The structure of the heavy chain containing the Hole domain in the fusion protein R1738 from N-terminus to C-terminus is as follows: VH-CH1-hinge-CH2-CH3

The structure of the light chain of fusion protein R1738 from N-terminus to C-terminus is as follows: VL-CL

Construction of exemplary fusion protein R1737

The CL region of one light chain of the antibody is replaced by an Obscurin domain having an amino acid sequence as shown in SEQ ID NO:10, the CH1 region of one heavy chain of the antibody is replaced by a Titin domain having an amino acid sequence as shown in SEQ ID NO:11, the N-terminus of the Titin domain is connected to the C-terminus of the VH via a peptide segment having an amino acid sequence as shown in SEQ ID NO:12 (i.e., the second connecting peptide, L2), and the Obscurin domain and the Titin domain are paired to form a light chain anti-mismatch structure; according to EU numbering, three amino acid sites of the CH3 of the heavy chain containing the Knob domain in the antibody are mutated as T366W, K392D and K409D to form a Knob domain, and three amino acid sites of the CH3 of the other heavy chain are mutated as L368R, D399K and Y407A to form a Hole domain, and the Knob domain and the Hole domain are paired to form a KIH heavy chain anti-mismatch structure.

The N-terminus of the IL-10 monomer is connected to the C-terminus of the VL region of the light chain containing the Obscurin domain of the antibody through the first connecting peptide, and the C-terminus of the IL-10 monomer is connected to the N-terminus of the Obscurin domain of the antibody through a peptide segment having an amino acid sequence as shown in SEQ ID NO: 13 (i.e., the third connecting peptide, L3). The resulting exemplary fusion protein R1737 has an amino acid sequence as shown in SEQ ID NO: 14 (light chain containing the Obscurin domain) and SEQ ID NO: 9 (light chain without the Obscurin domain), SEQ ID NO: 15 (heavy chain containing the Titin domain and the Knob domain), and SEQ ID NO: 8 (heavy chain containing the Hole domain). The specific structure of the fusion protein is shown in Figure 3C (the insertion position of the IL-10 monomer is shown in the figure).

Amino acid sequence of the obscurin domain:

Amino acid sequence of titin domain:

Amino acid sequence of the second connecting peptide:

Amino acid sequence of the third connecting peptide:

The structure of the light chain containing the Obscurin domain in the fusion protein R1737 from the N-terminus to the C-terminus is as follows: VL-L1-IL10-L3-Obs, where L1 represents: the first connecting peptide, L3 represents: the third connecting peptide, and Obs represents: the Obscurin domain

The structure of the light chain without the obscurin domain in the fusion protein R1737 from N-terminus to C-terminus is as follows: VL-CL

The structure of the heavy chain containing the titin domain and the knob domain in the fusion protein R1737 from the N-terminus to the C-terminus is as follows: VH-L2-Tit-hinge-CH2-CH3, where L2 represents: the third connecting peptide, and Tit represents: the titin domain

The structure of the heavy chain containing the Hole domain in the fusion protein R1737 from N-terminus to C-terminus is as follows: VH-CH1-hinge-CH2-CH3

Construction of control fusion protein R1739

The CL region of one light chain of the antibody is replaced by an Obscurin domain having an amino acid sequence as shown in SEQ ID NO: 10, and the N-terminus of the Obscurin domain is connected to the C-terminus of VL via a peptide segment having an amino acid sequence as shown in SEQ ID NO: 16 (i.e., the fourth connecting peptide, L4), and the CH1 region of one heavy chain of the antibody is replaced by a Titin domain having an amino acid sequence as shown in SEQ ID NO: 11, and the N-terminus of the Titin domain is connected to the C-terminus of VH via a second connecting peptide segment. The Obscurin domain is connected by a connecting peptide, and the Titin domain is paired with the light chain anti-mismatching structure; according to EU numbering, the three amino acid sites of CH3 of the heavy chain containing the Knob domain in the antibody are mutated: T366W, K392D and K409D to form the Knob domain, and the three amino acid sites of CH3 of the other heavy chain are mutated: L368R, D399K and Y407A to form the Hole domain, and the Knob domain is paired with the Hole domain to form the KIH heavy chain anti-mismatching structure.

The N-terminus of the IL-10 monomer was connected to the C-terminus of the Obscurin domain of the light chain of the antibody through a peptide having an amino acid sequence as shown in SEQ ID NO: 17 (i.e., the fifth connecting peptide, L5). The resulting control fusion protein R1739 has an amino acid sequence as shown in SEQ ID NO: 18 (light chain containing the Obscurin domain), SEQ ID NO: 9 (light chain without the Obscurin domain), SEQ ID NO: 15 (heavy chain containing the Knob domain and the Titin domain), and SEQ ID NO: 8 (heavy chain containing the Hole domain). The specific structure of the fusion protein is shown in FIG3D (the insertion position of the IL-10 monomer is shown in the figure).

Amino acid sequence of the fourth connecting peptide:

Amino acid sequence of the fifth connecting peptide:

The structure of the light chain containing the Obscurin domain in the fusion protein R1739 from the N-terminus to the C-terminus is as follows: VL-L4-Obs-L5-IL10, wherein L4 represents: the fourth connecting peptide, Obs represents: the Obscurin domain, and L5 represents: the fifth connecting peptide

The structure of the light chain without the obscurin domain in the fusion protein R1739 from N-terminus to C-terminus is as follows: VL-CL

The structure of the heavy chain containing the titin domain and the knob domain in the fusion protein R1739 from the N-terminus to the C-terminus is as follows: VH-L2-Tit-hinge-CH2-CH3, where Tit represents: titin domain

The structure of the heavy chain containing the Hole domain in the fusion protein R1739 from N-terminus to C-terminus is as follows: VH-CH1-hinge-CH2-CH3

Construction of control fusion protein R1740

The N-terminus of the IL-10 monomer is connected to the C-terminus of the CH3 region of the heavy chain containing the Knob domain of the antibody through the fifth connecting peptide. The resulting exemplary fusion protein R1740 has an amino acid sequence as shown in SEQ ID NO: 19 (heavy chain containing the Knob domain), SEQ ID NO: 8 (heavy chain containing the Hole domain) and SEQ ID NO: 9 (light chain), and the specific structure of the fusion protein is shown in Figure 3B (the insertion position of the IL-10 monomer is shown in the figure).

The structure of the heavy chain containing the Knob domain in the fusion protein R1740 from the N-terminus to the C-terminus is as follows: VH-CH1-hinge-CH2-CH3-L5-IL10, where L5 represents: the fifth connecting peptide

The structure of the heavy chain containing the Hole domain in the fusion protein R1740 from N-terminus to C-terminus is as follows: VH-CH1-hinge-CH2-CH3

The structure of the light chain of fusion protein R1740 from N-terminus to C-terminus is as follows: VL-CL

Example 2

Preparation of asymmetric IL10 monomer fusion protein

The plasmid containing the target gene is constructed and prepared by conventional methods. The plasmid containing the target gene is introduced into the Expi293 host cell after forming a cationic complex with the transfection reagent PEI. During the time when the plasmid is in the cell, the exogenous gene on the plasmid is transcribed and translated in the cell, thereby obtaining the fusion protein. The specific experimental operation is as follows:

Expi293 host cells were cultured at 37°C, 8% carbon dioxide, and 130rpm. Before transfection, 2E6 cells were inoculated into a 1L shake flask by cell counting. The culture system was about 300mL. Prepare transfection complexes for transfection: First, add 750μg of target plasmid to a 50mL centrifuge tube containing 15mL Opti-MEM reagent, mix gently, and mark it as tube A; add 1.5mg of transfection reagent PEI to a 50mL centrifuge tube containing 15mL Opti-MEM reagent, mix gently, and incubate at room temperature for 5min, marked as tube B; add the PEI dilution in tube B dropwise to the DNA dilution in tube A, mix gently, and incubate at room temperature for 15min. After the incubation, add the PEI-target plasmid complex to Expi293 cells and continue to culture in a 37°C shaker. Collect samples after culturing to D5-D10.

The transient cell expression solution was centrifuged at 9000rpm/20min, the supernatant was collected, and then sterilized and filtered through a 0.22μm filter membrane. ProA affinity chromatography was used for purification. The process is as follows: using an AKTA avant 150 chromatography device, the chromatography column (such as MabSelectSuRe LX, GE) was equilibrated with at least 5CV equilibration buffer (10mM PBS), and the sample was loaded onto the chromatography column so that the target protein was adsorbed on the chromatography column while other impurities penetrated and separated. After the sample is loaded, the chromatography column is rinsed again with at least 5CV equilibration buffer (10mM PBS), and then the target protein is eluted with elution buffer (20mM NaAc, pH=3.4). Neutralization buffer (1M Tris, pH=8.0) was pre-added to the collection tube. The volume of neutralization buffer added depends on the estimated content of the eluted sample, and generally 10% of the elution volume is added.

R0987 has two identical light chains and two identical heavy chains, and an IL-10 monomer is inserted between the VL region and the CL region of each of the two light chains. Its structural schematic diagram is shown in FIG4 , wherein the amino acid sequence of one of the light chains is shown in SEQ ID NO: 21:

The amino acid sequence of one heavy chain is shown in SEQ ID NO: 20:

R0989 has two identical heavy chains, with an IL-10 monomer inserted between the CH2 region and the CH3 region of each heavy chain. Its structural schematic diagram is shown in FIG4 , wherein the amino acid sequence of one heavy chain sequence is shown in SEQ ID NO: 22:

R0987 has two identical light chains, one of which has an amino acid sequence as shown in SEQ ID NO: 23:

The expression data of some fusion proteins are shown in Table 1. After one-step affinity purification, the samples were tested by SEC-HPLC, and the target purity was above 60%. The samples were tested by SDS-PAGE, showing the correct distribution of light and heavy chains, which was consistent with the results of SEC-HPLC test. The electrophoresis diagram is shown in Figure 5. It was concluded that the R1737 and R1738 fusion proteins showed good production properties.

Table 1

Example 3

In vitro activity of IL-10 monomer fusion protein

3.1 Flow cytometry to detect the binding activity of the antibody end and IL-10 monomer end of the IL-10 monomer fusion protein

The fusion protein molecule, R0674 (IL10-terminal positive control molecule, composed of an Fc region and IL10WT fused at its C-terminus, which includes two identical peptide chains, one of which is as follows: Hinge-CH2-CH3-linker-IL10WT, whose structure is shown in FIG4 , whose amino acid sequence is shown in SEQ ID NO: 24, and whose light chain amino acid sequence is shown in SEQ ID NO: 25), R1049 (whose structure is shown in FIG4 , anti-PD-1 monoclonal antibody control, whose heavy chain amino acid sequence is shown in SEQ ID NO: 26, and whose light chain amino acid sequence is shown in SEQ ID NO: 27), and R0862 (whose structure is shown in FIG4 , i.e., Isotype, negative control antibody; whose heavy chain amino acid sequence and light chain amino acid sequence are shown in SEQ ID NO: 28 and 29) were diluted to an initial concentration of 200 nM, a volume of 180 μL, 3-fold gradient dilution (60 μL sample + 120 μL dilution buffer), and 10 concentration gradient points. CHO-mPD1 cells (CHO cells expressing mouse PD-1) or CHO-hIL10R cells The cells (CHO cells expressing human IL10Rα) were centrifuged at 350g/5min, the supernatant was discarded, the cell density was adjusted to 2E+06 with 3% BSA PBS buffer, and 100μL/tube was evenly distributed into 96-well V-shaped plates; the above-diluted molecules were added to the cells, 100μL/well, and incubated at 2-8 degrees for 0.5h; the 96-well plate was taken out, centrifuged at 350g for 5min, the supernatant was carefully removed, and 200μL/well of 3% BSA PBS buffer was added, and centrifuged at 350g for 5min again, and the supernatant was carefully removed; PE fluorescent secondary antibody (1:500 dilution) was prepared with PBS buffer containing 3% BSA, and 100μL/well was added to the corresponding 96-well plate, the cells were resuspended, and incubated at 2-8 degrees for 30min; the 96-well plate was taken out, centrifuged at 250g for 5min, the supernatant was carefully removed, and PBS buffer containing 3% BSA was added 200 μL/well, centrifuge again at 350g for 5 min, carefully remove the supernatant; resuspend with 100 μL/well of 1xPBS, and detect by FACS.

The amino acid sequence of one of the peptide chains of R0674:

One of the heavy chain sequences of R1049:

One of the light chain sequences of R1049:

One of the heavy chain sequences of R0862:

One of the light chain sequences of R0862:

The results of the IL-10 monomer fusion protein binding activity to IL-10Rα are shown in Figure 6. The IL-10 end binding activity of R1738 is decreased compared with R0989 and R1740; the IL-10 end binding activity of R1737 is decreased compared with R0987 and R1739.

The results of the binding activity test of the antibody end of the IL-10 monomer fusion protein are shown in Figure 7. The mPD1 end binding activity of the asymmetric IL10 monomer fusion protein molecule R1737 did not change much compared with R0987 and R1739, and the mPD1 end binding activity of the asymmetric IL10 monomer fusion protein molecule R1738 did not change much compared with R0989 and R1740.

3.2 Detection of reporter gene activity of IL10 end of fusion protein

The fusion proteins, R0674, R0579 (IL-10-positive antibody fusion protein control, in which the IL-10 is free of the first two amino acids of natural IL-10 and no spacer peptide is inserted, and its structure is shown in Figure 4. Its heavy chain amino acid sequence is shown in SEQ ID NO:30, and its light chain amino acid sequence is shown in SEQ ID NO:31), and R1187 (non-targeted IL10-positive control molecule, which is formed by fusion of non-PD1 targeting antibody and IL10M, including two identical heavy chains and two identical light chains, and the C-termini of the two light chains are fused with IL10M, and its structure is shown in Figure 4. Its heavy chain amino acid sequence, light chain amino acid sequence, Sequence as shown in SEQ ID NO:32, 33) was diluted to an initial concentration of 50nM, volume 360μL, 3-fold gradient dilution (120μL sample + 240μL dilution buffer), 6 concentration gradient points; Cell preparation: Take out the cultured HEK293-hIL10 reporter gene cells (Jiman Biotechnology Company, product number GM-C07927; this cell line stably expresses the IL10R and STAT3 signaling pathway reporter gene system, and the use of IL10 protein stimulation can activate the increase of cell luciferase expression) and observe under a microscope. The cells have normal cell adhesion, transparent particles, and cells with moderate density can be used as effector cells in the experiment.

The above cells were digested with TE, centrifuged at 350g/4min after digestion, and the supernatant was removed. The cells were resuspended with 1% FBS-PBS and washed again. The supernatant was discarded, and the cells were finally resuspended with culture medium. After cell counting, the cell density was adjusted to 5E5/mL, and 50 μL was plated per well, and then placed in a 37°C incubator for adhesion treatment for 6 hours. The target cells R0326Fc-118 cells (stable expression of PD1, which plays an enrichment role in this system) were centrifuged at 350g/5min, and the supernatant was discarded. The cell density was adjusted to 1E+06 with 3% BSA PBS buffer; the reaction system was prepared in a flat-bottom plate ( 1. Enrichment system: According to the experimental design, the diluted fusion protein was first mixed with the target cells R0326Fc-118 in an equal volume at a volume of 25μL/well, and incubated for about 30min. Then, the target cells R0326 and Ab were carefully transferred into the adherent HEK293-hIL10 reporter gene cells; 100ΜI Assay Buffer was added to the Medium only wells, and 25μL Assay Buffer was added to the Cell only wells. The final volume of all wells was 100μL, The edge wells were sealed with 200 μL of sterile water, and the 96-well plate was continued to be cultured in the incubator for 16 hours; 2. Non-enrichment system: no target cells (R0326Fc-118) were added, that is, the corresponding antibody was directly added to the cells that had been treated for adhesion; other operations were the same as the enrichment system). The Bright-LumiTM firefly luciferase detection reagent was thawed in advance and equilibrated to room temperature. After the cells were cultured to the time point, the cell culture plate was taken out and equilibrated to room temperature for 10 minutes (not more than 30 minutes). 100 μL of Bright-LumiTM firefly luciferase detection reagent was added to each well and incubated at room temperature for 5-10 minutes. 100 μL of the system after the reaction was completed was taken from each well to the 96-well all-white plate; the multi-function microplate reader was used to detect the signal in chemiluminescence mode.

One of the heavy chain sequences of R0579:

One of the light chain sequences of R0579:

The structure of the heavy chain of R1187 from N-terminus to C-terminus is VH-CH1-Hinge-CH2-CH3, and its amino acid sequence is as follows:

The structure of the light chain of R1187 from N-terminus to C-terminus is VL-CL-L5-IL10, and its amino acid sequence is as follows:

The detection results are shown in Figure 8. There is no significant change in the reporter gene signal value of the control molecule before and after enrichment; while the reporter gene signal values of R1738 and R1737 are both improved in the enrichment system with target cells; among them, the asymmetric structure IL-10 monomer fusion protein R1738 has no signal value in the system without antibody target cells, and the signal value is significantly improved in the enrichment system containing antibody target cells, indicating that the R1738 and R1737 fusion protein molecules provided in the embodiments of the present disclosure will not produce the biological activity of IL-10 in the absence of antibody target cells, have the characteristics of low side effects, and can have a larger dosage.

Example 4

ELISA was used to detect the binding activity of each IL10 fusion protein with IL10Rα

Antigen IL10Rα (IL10Rα-his) was diluted to 1 μg/mL (diluent: 50 mM CB), added to a 96-well ELISA plate at 100 μL/well, sealed with a sealing film, and left to stand overnight in a 4°C refrigerator; the next day, the plate was washed three times with 1×PBST and patted dry, and assay buffer (1% BSA) was added at 200 μL/well, and blocked at room temperature for 1.5 h; the plate was washed once with 1×PBST and patted dry, and the antibody to be tested (starting at 60 nM, 3-fold dilution at 11 concentration points) was added at 100 μL/well, and the plate was then washed ... Seal the plate with sealing film and incubate at room temperature with shaking (500 rpm) for 1.5 h; wash the plate with 1×PBST 5 times and pat dry, add GAH-IgG Fc HRP (1:10K) diluted in assay buffer at 100 μL/well, seal the plate with sealing film, and incubate at room temperature for 40 min; wash the plate with 1×PBST 6 times and pat dry, add TMB colorimetric solution at 100 μL/well, incubate at room temperature for 5-10 min; add stop solution at 100 μL/well, read the plate at 450 nm & 630 nm, and analyze the data with SoftMax Pro.

The results of binding activity detection are shown in Figure 9 ("Span" in the figure indicates the difference between the high platform and the low platform. The larger the value, the larger the window and the higher the detection sensitivity); it can be seen that the binding activity of R1737, R1738, and R1739 to IL10Rα is significantly lower than that of the control molecules (R0987, R0989, and R0991) before modification. This indicates that the insertion of IL10 monomers into the positions shown by R1737, R1738, and R1739 weakens the binding activity of IL10 monomers to IL10Rα.

Example 5

ELISA was used to detect the binding activity of each IL10 fusion protein with IL10Rβ

The antibody His-Tag Mouse mAb was diluted to 1 μg/mL (diluent: 50 mM CB), added to a 96-well ELISA plate at 100 μL/well, sealed with a sealing film, and left to stand overnight in a 4°C refrigerator; the next day, the plate was washed 3 times with 1×PBST and patted dry, and assay buffer (1% BSA) was added at 200 μL/well, and blocked at room temperature for 1.5 h; the plate was washed once with 1×PBST and patted dry, and antigen R2050 (IL10β-his, 1 μg/mL) diluted in assay buffer was added at 100 μL/well, sealed with a sealing film, and incubated at room temperature with shaking (500 rpm) for 1 h; the plate was washed 5 times with 1×PBST and patted dry, and assay buffer was added at 100 μL/well. The antibody to be tested was diluted with the assay buffer (starting at 60 nM, 3-fold dilution to 11 concentration points), sealed with a sealing film, and incubated at room temperature with shaking (500 rpm) for 1.5 h; the plate was washed 5 times with 1× PBST and patted dry, and 100 μL/well of the diluted antibody was added with the assay buffer GAH-IgG Fc HRP (1:10K), sealed with sealing film, incubated at room temperature for 40 minutes; washed the strips with 1×PBST 6 times and patted dry, added TMB colorimetric solution at 100μL/well, incubated at room temperature for 20-30 minutes; added stop solution at 100μL/well, read the plate at 450nm&630nm, and analyzed the data with SoftMax Pro.

The results of the binding activity test are shown in Figure 10; it can be seen that the binding activity of R1737, R1738, and R1739 to IL10Rβ is significantly lower than that of the control molecules (R0987 and R0989) before modification, indicating that the insertion of IL10 monomers into the positions indicated by R1737 and R1738 weakens the binding activity of IL10 monomers to IL10Rβ.

Example 6

PD1 ELISA was used to detect the binding activity of each IL10 fusion protein with IL10Rα/IL10Rβ

Antigen R2051-E7 (IL10Rα/IL10Rβ-hFc) was diluted to 1 μg/mL (diluent: 50 mM CB), added to a 96-well ELISA plate at 100 μL/well, sealed with a sealing film, and allowed to stand overnight in a 4°C refrigerator; the next day, the plate was washed three times with 1×PBST and patted dry, and assay buffer (1% BSA) was added at 200 μL/well, and blocked at room temperature for 1.5 h; the plate was washed once with 1×PBST and patted dry, and the antibody to be tested diluted in assay buffer (60 nM starting point, 3-fold dilution to 11 concentration points) was added at 100 μL/well, sealed with a sealing film, and incubated at room temperature with shaking (500 rpm) for 1.5 h; the plate was washed with 1×PBS The plates were washed 5 times with 1×PBST and patted dry, and mPD1-His (1μg/mL) diluted in assay buffer was added at 100μL/well, the plates were sealed with a sealing film, and incubated at room temperature for 1h; the plates were washed 5 times with 1×PBST and patted dry, and anti-his HRP (1:5K) diluted in assay buffer was added at 100μL/well, the plates were sealed with a sealing film, and incubated at room temperature for 40min; the plates were washed 6 times with 1×PBST and patted dry, and TMB colorimetric solution was added at 100μL/well, and incubated at room temperature for 10-15min; stop solution was added at 100μL/well, the plates were read at 450nm & 630nm, and data were analyzed with SoftMax Pro.

The results of the binding activity test are shown in Figure 11; it can be seen that the binding activity of R1737 and R1738 to IL10Rα/IL10Rβ is significantly lower than that of the control molecules (R0987 and R0989) before modification, indicating that the insertion of IL10 monomers into the positions shown by R1737 and R1738 weakens the binding activity of IL10 monomers to IL10Rα/IL10Rβ.

The above description is only an optional embodiment of the present disclosure and is not intended to limit the present disclosure. For those skilled in the art, the present disclosure may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present disclosure shall be included in the protection scope of the present disclosure.

Claims

A fusion protein, characterized in that it comprises: an antibody, and an interleukin monomer or a variant thereof inserted into the antibody;

The antibody is an immunoglobulin having two identical light chains and two identical heavy chains, wherein the light chain comprises a VL region and a CL region, and the heavy chain comprises a VH region, a CH1 region, a CH2 region, and a CH3 region; the position where the interleukin monomer or a variant thereof is inserted into the antibody is as follows (i) or (ii):

(i) the interleukin monomer or variant thereof is inserted between the CH2 region and the CH3 region of one of the heavy chains of the antibody;

(ii) the interleukin monomer or variant thereof is inserted between the VL region and the CL region of one of the light chains of the antibody.
The fusion protein according to claim 1, characterized in that the position where the interleukin monomer or its variant is inserted into the antibody is (i); and the heavy chain of the antibody comprises a modification that prevents heavy chain mispairing.
The fusion protein according to claim 2 is characterized in that the CH3 region of one heavy chain of the antibody contains a Knob domain, the CH3 region of the other heavy chain of the antibody contains a Hole domain, and the Knob domain and the Hole domain are paired to form a heavy chain anti-mispairing structure.
The fusion protein according to claim 3 is characterized in that, according to the EU numbering system, the CH3 region of the heavy chain containing the Knob domain comprises the amino acid mutations: T366W, K392D and K409D, and the CH3 region of the heavy chain containing the Hole domain comprises the amino acid mutations: L368R, D399K and Y407A.
The fusion protein according to claim 4, characterized in that the interleukin monomer or its variant is inserted into a heavy chain containing a Knob domain;

Optionally, the interleukin monomer or variant thereof is connected to the heavy chain containing the Knob domain of the antibody via a first connecting peptide.
The fusion protein according to any one of claims 3 to 5, characterized in that the N-terminus of the interleukin monomer or variant thereof is connected to the C-terminus of the CH2 region of the heavy chain containing the Knob domain of the antibody via a first connecting peptide, and the C-terminus of the interleukin monomer or variant thereof is connected to the N-terminus of the CH3 region of the heavy chain containing the Knob domain of the antibody via a first connecting peptide;

Optionally, the structure of the heavy chain containing the Knob domain of the antibody from N-terminus to C-terminus is as follows: VH-CH1-hinge-CH2-L1-Int-L1-CH3, wherein L1 represents: the first connecting peptide, and Int represents: an interleukin monomer or a variant thereof;

The structure of the heavy chain containing the Hole domain of the antibody from N-terminus to C-terminus is as follows: VH-CH1-hinge-CH2-CH3;

The structure of the light chain of the antibody from N-terminus to C-terminus is as follows: VL-CL.
The fusion protein according to claim 5 or 6 is characterized in that the first connecting peptide has an amino acid sequence shown in SEQ ID NO:6.
The fusion protein according to claim 1, characterized in that the position where the interleukin monomer or its variant is inserted into the antibody is (ii); and the antibody comprises a modification that prevents light chain mispairing and a modification that prevents heavy chain mispairing.
The fusion protein according to claim 8, characterized in that the CL region of one light chain of the antibody is replaced by the Obscurin domain, the CH1 region of one heavy chain of the antibody is replaced by the Titin domain, and the Obscurin domain is paired with the Titin domain to form a light chain anti-mismatching structure;

The CH3 region of the heavy chain containing the titin domain of the antibody contains a Knob domain, and the CH3 region of the other heavy chain of the antibody contains a Hole domain. The Knob domain is paired with the Hole domain to form a heavy chain anti-mismatching structure.
The fusion protein according to claim 9, characterized in that the Obscurin domain has an amino acid sequence as shown in SEQ ID NO: 10, and the Titin domain has an amino acid sequence as shown in SEQ ID NO: 11;

Optionally, according to the EU numbering system, the CH3 region of the heavy chain containing the Knob domain comprises the amino acid mutations: T366W, K392D and K409D, and the CH3 region of the heavy chain containing the Hole domain comprises the amino acid mutations: L368R, D399K and Y407A.
The fusion protein according to claim 9 or 10, characterized in that the interleukin monomer or variant thereof is inserted into a light chain containing an obscurin domain;

Optionally, the interleukin monomer or variant thereof is connected to the Obscurin domain of the light chain of the antibody via a third connecting peptide; and the Titin domain of the antibody is connected to the VH region via a second connecting peptide.
The fusion protein according to any one of claims 9 to 11, characterized in that the N terminus of the interleukin monomer or variant thereof is connected to the C terminus of the VL region of the light chain containing the Obscurin domain of the antibody via a first connecting peptide, and the C terminus of the interleukin monomer or variant thereof is connected to the N terminus of the Obscurin domain of the light chain of the antibody via a third connecting peptide; the N terminus of the Titin domain is connected to the C terminus of the VH via a second connecting peptide;

Optionally, the structure of the Obscurin domain-containing light chain of the antibody from N-terminus to C-terminus is as follows: VL-L1-Int-L3-Obs, wherein L1 represents: the first connecting peptide, L3 represents: the third connecting peptide, Int represents: an interleukin monomer or a variant thereof, and Obs represents: an Obscurin domain;

The structure of the light chain of the antibody without the Obscurin domain from N-terminus to C-terminus is as follows: VL-CL;

The structure of the heavy chain of the antibody containing the titin domain and the knob domain from the N-terminus to the C-terminus is as follows: VH-L2-Tit-CH2-CH3, wherein L2 represents: the second connecting peptide, and Tit represents: the titin domain;

The structure of the heavy chain containing the Hole domain of the antibody from N-terminus to C-terminus is as follows: VH-CH1-hinge-CH2-CH3.
The fusion protein according to claim 11 or 12 is characterized in that the second connecting peptide has an amino acid sequence as shown in SEQ ID NO: 12, and the third connecting peptide has an amino acid sequence as shown in SEQ ID NO: 13.
The fusion protein according to any one of claims 1 to 13, characterized in that the interleukin monomer is selected from IL-10 monomer, IL-5 monomer, IL-12 monomer, IL-23 monomer, IL-25 monomer, IL-27 monomer, and IL-35 monomer;

Preferably, the interleukin monomer is an IL-10 monomer.
The fusion protein according to claim 14, characterized in that the IL-10 monomer is a natural IL-10 monomer or a modified IL-10 monomer;

Preferably, the natural IL-10 monomer has an amino acid sequence as shown in SEQ ID NO: 3;

Preferably, the modified IL-10 monomer has a spacer peptide inserted between two amino acid sites in the natural IL-10 monomer molecule sequence;

Preferably, the modified IL-10 monomer has a spacer peptide inserted between the two sites 116N and 117K of the natural IL-10 monomer molecular sequence;

Preferably, the spacer peptide comprises the amino acid sequence shown in SEQ ID NO:4;

Preferably, the modified IL-10 monomer lacks the first two amino acids at the N-terminus of the natural IL-10 monomer molecule sequence;

Preferably, the modified IL-10 monomer has an amino acid sequence as shown in SEQ ID NO:5.
The fusion protein according to any one of claims 1 to 15, characterized in that the antibody specifically binds to immune checkpoints and/or tumor antigens;

Preferably, the immune checkpoint molecule is selected from PD-1, CD47, TIGIT, CD137, CD134, KIR, LAG-3, PD-L1, CTLA-4, B7.1, B7H3, CCRY, OX-40 and CD40;

Preferably, the tumor antigen is selected from GUCY2C, MSLN, Claudin18.2, GPC3, EGFR, HER2, CEA, GD2, EGFRⅧ, MUC1, PRLR, CLCA1, MUC12, GPR35, CR1L, MUC17, TMPRSS11B, MUC21, TMPRSS1IE, CD207, SLC30A8, CFC1, SLC12A3, SSTR1, GPR27, FZD10, TSHR, SIGLEC15, SLC6A3, KISSIR, QRFPR, GPR119, CLDN6, UPK2, ADAM12, SLC45 A3, ACPP, MUC21, MUC16, MS4A12, ALPP, EphA2, FAP, IL13-Ra2, PSMA, ROR1, VEGFR-Ⅱ, FR-a, EpCAM, EGFRⅦ, tMUC1, PSCA, FCER2, GPR18, FCRLA, CXCR5, FCRL3, FCRL2, HTR3A, CLEC17A, TRPMI, SLC45A2, SLC24A5, DPEP3, KCNK16, LIM2, KCNV2, SLC26A4, CD171, Glypican-3, IL-13, CD79a/b, and MAGEA4;

Preferably, the antibody specifically binds to PD-1.
A multispecific binding molecule, characterized in that the multispecific binding molecule comprises the fusion protein according to any one of claims 1-16.
A nucleic acid, characterized in that the nucleic acid encodes the fusion protein according to any one of claims 1 to 16 or the multispecific binding molecule according to claim 17.
An expression vector, characterized in that the expression vector comprises the nucleic acid according to claim 18.
A recombinant cell, characterized in that the recombinant cell carries the nucleic acid of claim 18, the expression vector of claim 19, expresses the fusion protein of any one of claims 1-16, or expresses the multi-specific binding molecule of claim 17.
Use of the fusion protein of any one of claims 1 to 16, the multispecific binding molecule of claim 17, the nucleic acid of claim 18, the expression vector of claim 19 or the recombinant cell of claim 20 in the preparation of a medicament for treating or preventing a tumor or an inflammatory disease.
A pharmaceutical composition, characterized in that it comprises the fusion protein of any one of claims 1 to 16, the multispecific binding molecule of claim 17, the nucleic acid of claim 18, the expression vector of claim 19 or the recombinant cell of claim 20;

Optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
A method for treating, preventing or diagnosing a tumor or inflammatory disease, disorder or condition, comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition of claim 22.