CN117343162A

CN117343162A - Fc monomer polypeptide, fusion polypeptide thereof and application

Info

Publication number: CN117343162A
Application number: CN202311127859.4A
Authority: CN
Inventors: 袁继行; 李洁
Original assignee: Second Military Medical University SMMU
Current assignee: Second Military Medical University SMMU
Priority date: 2023-09-01
Filing date: 2023-09-01
Publication date: 2024-01-05

Abstract

The invention belongs to the technical field of biology, and particularly relates to an Fc monomer polypeptide, a fusion polypeptide thereof and application thereof. The invention provides an Fc monomer polypeptide, which comprises the following amino acid site substitution compared with the amino acid sequence shown in SEQ ID NO. 1: 14 th bit, 15 th bit, 32 th bit, 34 th bit, 36 th bit, 102 th bit. The fusion polypeptide formed by the Fc monomer polypeptide and the ERMAP extracellular segment can obviously promote the phagocytosis of Kupffer cells on tumor cells, effectively inhibit liver metastasis of tumors, and has wide application prospects in prevention and/or treatment of tumor metastasis.

Description

Fc monomer polypeptide, fusion polypeptide thereof and application

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an Fc monomer polypeptide, a fusion polypeptide thereof and application thereof.

Background

Since the success of the first generation monoclonal antibodies in 1975, therapeutic macromolecular drugs (antibodies and polypeptides) have entered clinical research and marketed in batches, and targeted therapy with therapeutic monoclonal antibodies has become one of the effective approaches to combat cancer, viral infections, and immune diseases. With the advancement of biotechnology, in order to better exert therapeutic effects of macromolecular drugs, researchers have conducted a great deal of research to explore the link between the structural properties of antibodies and their functions, one of the key areas being to modulate the interactions of antibodies with the immune system through Fc engineering.

Macromolecular drugs (particularly antibodies) generally have two methods of killing or inducing death of target cells, one is to rely on the specificity of Fab fragments in antibodies to recognize the relevant antigens on the surface of target cells and to interfere with the regulation of their relevant signaling pathways; the other is to further enhance the therapeutic effect of the antibody by the related action of the Fc region of the antibody after binding to Fc gamma receptor (FcgammaR) or other ligand on the surface of immune cells.

The Fc fragment may bind fcγr and complement protein C1q to mediate some cell killing. Fcγrs play an important role as one of the regulatory factors of immune responses, for B cell activation, dendritic cell maturation and some other effector responses including antibody dependent cell-mediated phagocytosis (ADCP), antibody dependent cell-mediated killing (ADCC), whereas IgG-Fc binding to C1q can initiate assembly of complement cascade proteins leading to complement-mediated cytotoxicity (CDC).

The design of Fc-FcgammaRs interactions is a highly efficient strategy to modulate the pharmacokinetic profile of macromolecular drugs and optimize their efficacy and ease of administration. The major applications of Fc and fcγrs site engineering are: prolonged half-life of antibody, igG clearance, antigen clearance, etc. Therefore, engineering of Fc fragments has now become a hotspot for improving therapeutic effects of macromolecular drugs, wherein the strength of binding force between Fc and the two is one of the key points of Fc engineering.

There are two currently mainstream Fc engineering approaches:

one is sugar engineering. The current glycoengineering-related engineering of the Fc region mostly aggregates on enhancing the affinity of Fc for fcγriiia. Many studies on fucose engineering have been performed and the removal of fucose has led to an increase in the binding of IgG-Fc to fcγra Asn162 to further increase the affinity of the Fc-terminus for fcγriiia and to increase ADCC/ADCP.

The other is site-directed mutagenesis. The affinity of the Fc region to its receptor can also be improved to some extent by site-directed mutagenesis, as with the goal of Fc engineering strategies by altering glycosylation. For example: chinese patent application 201380018995.8 discloses an Fc region variant of an antibody, which successfully achieves a polypeptide with improved stability compared to the parent polypeptide by mutating at least one amino acid of the loop portion of the Fc region of the antibody; further combining a plurality of amino acid variations at the loop site, and comparing the combination with a parent polypeptide, thereby successfully obtaining a polypeptide having improved thermostability and maintained or enhanced binding activity to fcγr; and polypeptides having increased thermostability with reduced binding activity to fcγr; it has also been successfully obtained a polypeptide having not only improved thermostability but also regulated binding activity to fcγr, and reduced content of associates. Chinese patent application 202180050612.X discloses a polypeptide comprising a modified Fc region of IgG, wherein the modified Fc region is non-naturally occurring, comprises at least one amino acid mutation compared to the Fc region of wild-type or naturally occurring IgG, the polypeptide having substantially no binding activity to wild-type or naturally occurring fcγ receptor, the polypeptide being capable of binding to a non-naturally occurring fcγ receptor comprising at least one amino acid mutation compared to the wild-type or naturally occurring fcγ receptor.

There remains a need in the art for more Fc regions/fragments that enhance the therapeutic effects of macromolecular drugs.

Disclosure of Invention

Tumor metastasis is the last, most damaging stage in cancer progression and is also the leading cause of cancer death (Warburg effect and its effect on tumor metastasis. Zhongguo Fei Ai Za Zhi, 179-183 (2015)). The liver is the largest organ of human body, and the unique double blood supply characteristics, abundant blood flow and nutrient substances become fertile soil for tumor cells to contact and grow along with the blood circulation process, so the liver becomes the target organ with the highest tumor metastasis rate, and especially the liver metastasis generated by digestive tract tumor is most common, including liver cancer, colorectal cancer, pancreatic cancer, gastric cancer and the like. In addition, tumors such as melanoma and ovarian Cancer often develop liver metastases (Brodt, P.Role of the Microenvironment in Liver Metastasis: from Pre-to Prometastatic Nichs.Clin Cancer Res 22,5971-5982 (2016)). The liver tissue microenvironment plays an important role in the tumor liver metastasis process, so there is a need to find new therapeutic approaches from the nature of the tumor cell interactions with the liver tissue microenvironment.

Tumor metastasis involves multiple steps: primary tumor cells locally infiltrate, enter the blood, survive in the blood circulation, colonize and amplify target organs, and finally form metastases (valastean, S. & Weinberg, r.a. tumor metassis: molecular Insights and Evolving paramigms. Cell 147,275-292 (2011)). Although a large number of tumor cells can be introduced into blood, very few tumor cells actually reach target organogenesis and form metastases (Chaffer, C.L. & Weinberg, R.A. A. Perspective on Cancer Cell Metastasis.science 331,1559-1564 (2011); gao, H.the BMP Inhibitor Coco Reactivates Breast Cancer Cells at Lung Metastatic sites.cell 150,764-779 (2012)). Therefore, the survival of the colonization after the tumor cells are transferred to the target organ and the subsequent steps are very critical for the success or failure of the tumor transfer, and the interaction of the tumor cells and the microenvironment of liver tissue has important influence on the colonization, survival and growth of the liver.

Current anti-tumor immunotherapy is mostly focused on the combination therapy of acquired immune systems, especially T cells, such as immune checkpoint inhibitors (Immune checkpoint inhibitors, ici) for example, altlizumab (PD-L1 antibodies) and bevacizumab (vascular endothelial growth factor antibodies) for advanced clinical treatment of HCC, and significantly prolongs the overall and progression free survival of patients compared to sorafenib (Finn, r.s. et al, atezuzumab plus Bevacizumab in Unresectable Hepatocellular carnoma. N Engl Med 382,1894-1905 (2020), finn.s. et al, imbraave150: updated Overall Survival (OS) data from a global, randomized, open-label phase III study of atezolizumab (atezo) + bevacizumab (bev) versu rafenib (sor) in peptides) with unresectable hepatocellular carcinoma (HCC 39,267-267 (2021)), but also resists many conditions of such treatment by sance (a.69-95-Nature, 24-17.c. et al, 17-93-95.20 (17), and (20-35, 20-17, 20).

Thus, there is a need in the art to explore new therapeutic strategies. Natural immunity is the first line of defense against tumor immunity, in which macrophages play an important role. In the process of tumor metastasis to liver, the tumor reaches the liver blood sinus along with blood circulation and meets with Kupffer cells so as to be phagocytosed and cleared, and the tumor cells are relied on to transmit an "eat me" signal to the Kupffer cells, and classical "eat me" signals which are reported at present comprise: fcγ receptor, calreticulin-membrane glycoprotein sugar chain, SLAMF7-MAC1 axis.

Earlier work by the inventors found a tumor suppressor that mediates the interaction of tumor cells with the liver-resident macrophages-Kupffer cells and can promote phagocytosis of tumor cells by the Kupffer cells: erythrocyte membrane associated proteins (ERMAP, erythroblast Membrane Associated Protein). ERMAP is a single transmembrane glycoprotein, the N-terminal extracellular segment mainly comprising an immunoglobulin-like variable region domain (Ig-like V-type), involved in mediating cell-cell recognition, adhesion and cell membrane fusion; the C-terminal intracellular segment contains primarily a B30.2/SPRY domain, which mediates downstream signal transduction through protein-protein interactions. ERMAP belongs to the butyl philin-like (BTNL) protein family, which is an important member of the immunoglobulin superfamily, similar to the Butyrophilin (BTN) and B7 (e.g., ICOS, PD-1, PD-L1, etc.) families.

However, polypeptide drugs often have short half-life, lack long-acting property and poor stability. Accordingly, the present invention contemplates fusion of the engineered Fc fragment with the extracellular segment of ERMAP to form a fusion polypeptide to extend its half-life and stability in vivo, thereby enhancing its therapeutic effect.

In order to achieve the technical purpose, the invention provides the following technical scheme:

in one aspect, the invention provides an Fc monomeric polypeptide (sFc) comprising amino acid site substitutions compared to the amino acid sequence as shown in SEQ ID NO: 1:

14 th bit, 15 th bit, 32 th bit, 34 th bit, 36 th bit, 102 th bit.

The amino acid sequence of the original IgG1-Fc fragment of the control is shown in SEQ ID NO: 1:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

in one embodiment, the Fc monomeric polypeptide may be an IgG Fc fragment.

In a preferred embodiment, the Fc monomeric polypeptide may be an IgG1 Fc fragment, an IgG2 Fc fragment, an IgG3Fc fragment, an IgG4 Fc fragment.

In a preferred embodiment, the Fc monomeric polypeptide may be an IgG1 Fc fragment.

In one embodiment, the amino acid position substitution comprises L14F, L15Q, M32Y, S34T, T36E, K102Q.

In a preferred embodiment, the amino acid sequence of the Fc monomeric polypeptide is as shown in SEQ ID NO. 2:

DKTHTCPPCPAPEFQGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCQVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

in one embodiment, the Fc monomer polypeptide has reduced affinity for fcγ receptor (fcγr) that reduces antibody dependent cell-mediated cytotoxicity (ADCC).

In a further aspect, the invention provides a fusion polypeptide comprising an extracellular segment of ERMAP or a variant thereof, and the Fc monomer polypeptide.

In one embodiment, the extracellular segment of ERMAP or a variant thereof is linked directly or via a linker (linker) to the Fc monomer polypeptide.

In one embodiment, the Fc monomer polypeptide is directly linked to the C-terminus of the extracellular segment of ERMAP or variant thereof.

In a preferred embodiment, the linker is a peptide linker.

In one embodiment, the ERMAP is a human ERMAP (hERMAP) or a murine ERMAP.

In a preferred embodiment, the ERMAP is hERMAP, the extracellular portion of which has the amino acid sequence shown in SEQ ID No. 3:

HAGDAGKFHVALLGGTAELLCPLSLWPGTVPKEVRWLRSPFPQRSQAVHIFRDGKDQDEDLMPEYKGRTVLVRDAQEGSVTLQILDVRLEDQGSYRCLIQVGNLSKEDTVILQVAAPSVGSLSPSA。

the nucleotide sequence of hERMAP extracellular segment is shown in SEQ ID NO. 5:

CACGCAGGGGATGCCGGCAAGTTCCACGTGGCCCTACTAGGGGGCACAGCCGAGCTGCTCTGCCCTCTCTCCCTCTGGCCCGGGACGGTACCCAAGGAGGTGAGGTGGCTGCGGTCCCCATTCCCGCAGCGCTCCCAGGCTGTTCACATATTCCGGGATGGGAAGGACCAGGATGAAGATCTGATGCCGGAATATAAGGGGAGGACGGTGCTAGTGAGAGATGCCCAAGAGGGAAGTGTCACTCTGCAGATCCTTGACGTGCGCCTTGAGGACCAAGGGTCTTACCGATGTCTGATCCAAGTTGGAAATCTGAGTAAAGAGGACACCGTGATCCTGCAGGTTGCAGCCCCATCTGTGGGGAGTCTCTCCCCCTCAGCA。

in a preferred embodiment, the ERMAP is a murine ERMAP having an extracellular domain with an amino acid sequence as shown in SEQ ID NO. 4:

DAGKVYIAPLRDTANLPCPLFLWPNMVLSEMRWYRPGHLPRTQAVHVFRDGQDRDEDLMPEYKGRTALVRDAHKESYILQISNVRLEDRGLYQCQVWVGNSSREDNVTLQVAVLGSDPYIHVKGYDAGWIELLCQSVGWFPKPWTEWRDTTGRALLSLSEVHSLDENGLFRTAVSSRIRDNALGNVSCTIHNEALGQEKTTAMIIGAPERGSLSSPA。

the nucleotide sequence of the extracellular section of the murine ERMAP is shown as SEQ ID NO. 6:

GATGCTGGCAAGGTCTACATAGCCCCTCTTAGGGACACAGCCAACTTGCCCTGCCCTCTCTTCCTCTGGCCTAACATGGTACTCAGCGAGATGAGGTGGTATCGGCCCGGACACCTACCTCGCACCCAGGCTGTCCACGTGTTCCGGGACGGGCAGGACAGAGATGAAGACCTGATGCCAGAATATAAGGGCAGGACGGCACTGGTGAGGGATGCCCACAAGGAAAGCTACATCCTGCAAATCAGTAATGTGAGGCTCGAGGACCGAGGGCTATACCAGTGCCAGGTCTGGGTCGGAAACTCAAGTCGAGAAGACAACGTGACCCTGCAGGTGGCAGTTTTAGGTTCTGATCCCTACATCCACGTGAAGGGCTATGATGCTGGGTGGATCGAGCTGCTGTGTCAATCAGTGGGATGGTTCCCAAAGCCGTGGACTGAGTGGAGAGACACTACGGGTAGAGCGCTACTCTCCCTTTCAGAGGTTCACTCCCTGGATGAAAATGGGCTGTTCCGAACAGCAGTGTCCAGCAGAATCAGGGACAATGCCCTGGGAAATGTGTCTTGCACTATCCATAATGAGGCCCTTGGCCAAGAGAAGACGACGGCCATGATTATTGGAGCCCCAGAACGAGGAAGTCTGTCCTCTCCAGCG。

in one embodiment, the variant of the extracellular portion of ERMAP has at least 85%, 90%, 95%, 98%, 99% sequence identity with the amino acid sequence shown as SEQ ID NO 3 or 4.

In yet another aspect, the invention provides an isolated nucleic acid molecule encoding the fusion polypeptide.

In yet another aspect, the invention provides a nucleic acid delivery vector comprising the nucleic acid molecule.

In one embodiment, the nucleic acid delivery vector comprises a nucleic acid delivery vector derived from an adenovirus, adeno-associated virus, lentivirus, or other acceptable nucleic acid delivery vector.

Those skilled in the art will recognize that any vector that stably expresses a soluble, active fusion polypeptide can be used in the present invention.

In yet another aspect, the invention provides a host cell comprising the nucleic acid delivery vector.

In yet another aspect, the invention provides a pharmaceutical composition comprising said fusion polypeptide, said nucleic acid molecule, said nucleic acid delivery vector, said host cell;

optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, carrier or adjuvant.

In one embodiment, the pharmaceutically acceptable carrier, carrier or adjuvant includes, but is not limited to, solvents, diluents, disintegrants, precipitation inhibitors, surfactants, glidants, binders, lubricants, dispersants, suspending agents, isotonic agents, thickening agents, emulsifiers, preservatives, stabilizers, hydration agents, emulsification accelerators, buffers, absorbents, colorants, flavoring agents, sweeteners, ion exchangers, mold release agents, coating agents, flavoring agents, or antioxidants.

In a further aspect, the invention provides the use of said fusion polypeptide, said nucleic acid molecule, said nucleic acid delivery vector, said host cell or said pharmaceutical composition for the preparation of a medicament for the prevention and/or treatment of tumor metastasis.

In one embodiment, the tumor metastasis can be tumor lung metastasis or liver metastasis.

In one embodiment, the tumor includes, but is not limited to, liver cancer, melanoma, glioma, colon adenocarcinoma, pancreatic cancer, colon cancer, gastrointestinal cancer, prostate cancer, bladder cancer, ovarian cancer, lung cancer, renal cell carcinoma, nasopharyngeal carcinoma, renal cancer, breast cancer, hematological cancer, or head and neck cancer.

The Fc monomer polypeptide is obtained by carrying out site-directed mutagenesis on a human IgG1 Fc fragment. The Fc monomeric polypeptide is linked to ERMAP to form fusion polypeptides hERRMAP-sFc and ERMAP-sFc. Compared with the prior art, the fusion polypeptide has at least the following beneficial effects:

(1) Compared with an Fc control group, the fusion polypeptide obviously promotes the phagocytosis of Kupffer cells in an in vitro experiment, and the phagocytosis efficiency is improved by about 40% -100%.

(2) Compared with the Fc control group, the fusion polypeptide remarkably inhibits liver metastasis of tumors;

(3) Compared with the common ERMAP-Fc fusion protein, the phagocytosis of the fusion polypeptide to the Kupffer cells is improved by about 70 percent.

Drawings

FIG. 1 shows a flow chart of the Kupffer cell fraction of Hepa1-6 cells phagocytosed with CFSE fluorescent label under different treatments (sFc and ERMAP-sFc).

FIG. 2 shows statistical graphs of the results of flow assays of the Kupffer cell proportion of Hepa1-6 cells phagocytosed with CFSE fluorescent label under different treatments (sFc and ERMAP-sFc).

FIG. 3 shows the phagocytosis/efficiency of Hepa1-6 cells by Kupffer cells based on fluorescence microscopy under different treatments (sFc and ERMAP-sFc).

FIG. 4 shows a flow chart of the Kupffer cell fraction of SNU-398 cells phagocytosed with CFSE fluorescent markers under different treatments (sFc and hERMAP-sFc).

FIG. 5 shows statistical graphs of the results of flow assays of the Kupffer cell fraction of SNU-398 cells phagocytosed with CFSE fluorescent markers under different treatments (sFc and hERMAP-sFc).

FIG. 6 shows the phagocytosis rate/efficiency of Kupffer cells on SNU-398 cells based on fluorescence microscopy under different treatments (sFc and hERMAP-sFc).

FIG. 7 shows a flow chart of the Kupffer cell fraction of B16F10 cells phagocytosed with CFSE fluorescent label under different treatments (sFc and ERMAP-sFc).

FIG. 8 shows statistical graphs of the results of flow assays of the Kupffer cell fraction of B16F10 cells phagocytosed with CFSE fluorescent label under different treatments (sFc and ERMAP-sFc).

FIG. 9 shows the phagocytosis/efficiency of B16F10 cells by Kupffer cells based on fluorescence microscopy under different treatments (sFc and ERMAP-sFc).

FIG. 10 shows a flow chart of the Kupffer cell fraction of CT26 cells phagocytosed with CFSE fluorescent label under different treatments (sFc and ERMAP-sFc).

FIG. 11 shows statistical graphs of the results of flow assays of the Kupffer cell fraction of C phagocytosed CFSE fluorescent labeled B16F10 cells under different treatments (sFc and ERMAP-sFc).

FIG. 12 shows the phagocytosis/efficiency of Kupffer cells on CT26 cells detected based on fluorescence microscopy under different treatments (sFc and ERMAP-sFc).

FIG. 13 shows the fluorescence intensity of luciferases at liver sites of mice after spleen injection of Hepa1-6 cells under different treatment (sFc and ERMAP-sFc).

FIG. 14 shows the fluorescence intensity profile of the liver sites of mice after spleen injection of Hepa1-6 cells under different treatment (sFc and ERMAP-sFc).

FIG. 15 shows the end point results of liver transfer experiments following spleen injection of Hepa1-6 cells under different treatment (sFc and ERMAP-sFc).

FIG. 16 shows statistics of end point results of liver transfer experiments after spleen injection of Hepa1-6 cells under different treatment (sFc and ERMAP-sFc).

FIG. 17 shows the end point results of liver metastasis experiments after spleen injection of SNU-398 cells under different treatment (sFc and ERMAP-sFc).

FIG. 18 shows statistical graphs of end-point results of liver metastasis experiments after spleen injection of SNU-398 cells under different treatment (sFc and ERMAP-sFc).

Fig. 19 shows the end point results of liver transfer experiments after spleen injection of B16F10 cells under different treatment (sFc and ERMAP-sFc).

FIG. 20 shows statistical graphs of liver metastasis end-point results after spleen injection of B16F10 cells under different treatment (sFc and ERMAP-sFc).

Fig. 21 shows the end point results of liver metastasis experiments after spleen injection of CT26 cells under different treatment (sFc and ERMAP-sFc).

Figure 22 shows statistics of end point results of liver transfer experiments after spleen injection of CT26 cells under different treatment (sFc and ERMAP-sFc).

FIG. 23 shows a flow chart of the Kupffer cell fraction of CFSE fluorescently labeled c cells phagocytosed under different treatments (ERMAP-Fc and ERMAP-sFc)

FIG. 24 shows statistical graphs of the results of flow assays of the Kupffer cell proportion of Hepa1-6 cells phagocytosed with CFSE fluorescent label under different treatments (ERMAP-Fc and ERMAP-sFc).

Detailed Description

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly used in the art to which this invention belongs. For the purposes of explaining the present specification, the following definitions will apply, and terms used in the singular will also include the plural and vice versa, as appropriate.

The terms "a" and "an" as used herein include plural referents unless the context clearly dictates otherwise. For example, reference to "a cell" includes a plurality of such cells, equivalents thereof known to those skilled in the art, and so forth.

The term "about" as used herein means a range of + -20% of the numerical values thereafter. In some embodiments, the term "about" means a range of ±10% of the numerical value following that. In some embodiments, the term "about" means a range of ±5% of the numerical value following that.

The term "fusion polypeptide" as used herein refers to a protein in which two or more proteins or fragments thereof are co-linearly linked by respective peptide backbones using genetic expression of the polynucleotide encoding the protein or protein synthesis methods. Preferably, the polypeptides or fragments thereof are of different origin. In some embodiments, the fusion polypeptide comprises peptide fragments from different sources, e.g., from ERMAP, ERMAP variants, igG1 Fc variants.

As used herein, having "at least 95% sequence identity" with a sequence, as compared to a sequence, means having at least 95%, at least 96%, to 97%, at least 98% or at least 99% sequence identity with the sequence.

The term "affinity" or "binding affinity" as used herein refers to the strength of the sum of the non-covalent interactions between a single binding site of a molecule and its binding ligand. The binding affinity can generally be determined by dissociation constants (K _d ) Shows the dissociation constant (K _d ) Is the dissociation rate (k) _d ) And binding rate (k) _a ) Ratio of (K) _D ＝k _d /k _a . Affinity can be measured by conventional methods known in the art, such as Surface Plasmon Resonance (SPR).

The term "substitution" as used herein with respect to amino acids refers to the replacement of at least one amino acid residue in an amino acid sequence with another, different "replacement" amino acid residue.

The fusion polypeptides of the present disclosure or fragments thereof may comprise conservative amino acid substitutions at one or more amino acid residues, e.g., at essential or non-essential amino acid residues. A "conservative amino acid substitution" is a substitution of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, amino acid, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), branched side chains (e.g., threonine, amino acid, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, in this context, an essential or non-essential amino acid residue in a fusion polypeptide or linker is preferably replaced with another amino acid residue from the same side chain family. In certain embodiments, the amino acid segments may be replaced with segments that are structurally similar and differ in the order and/or composition of the side chain family members. Alternatively, in certain embodiments, mutations may be introduced randomly along all or a portion of the coding sequence, such as by saturation mutagenesis, and the resulting mutants may be incorporated into fusion polypeptides of the invention and screened for their ability to bind to a desired target.

Immunoglobulins exist in five major classes, igA, igD, igE, igG and IgM, and these major classes can also be

Further subclasses (isotypes) such as IgGl, igG2, igG3, igG4, igAl and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ and μ, respectively. CL lengths of different types (k or λ) of immunoglobulins are substantially identical, but CH lengths of different classes of immunoglobulins are different, e.g. IgG, igA and IgD include CH1, CH2 and CH3, while IgM and IgE include CHL, CH2, CH3 and CH4. Hinge region (hinge region) is located between CH1 and CH2, is rich in proline, and is easy to stretch and bend, so that the distance between antigen binding sites is changed, and the antibody can be favorably bound to antigen epitopes located at different positions. The chain region is easy to hydrolyze by papain, pepsin and the like, and different hydrolysis fragments are generated. Papain hydrolyzes the electric site near the N-terminus of two heavy chains disulfide-linked in the longer chain region, splitting Ig into two identical Fab and one Fc fragments. The Fab fragment is an antigen binding fragment (fragment antigenbinding, fab), consisting of the VH and CH1 domains of one complete light and heavy chain.

The term "antibody", "immunoglobulin" or "Ig" Fc region as used herein refers to the constant domain or constant region (CH) at the C-terminus of an immunoglobulin heavy chain or fragment thereof, which is a crystallizable fragment (fragment crystallizable, fc) consisting of the CH2 and CH3 domains of Ig. Herein, the term "Fc region" includes wild-type Fc regions and variant Fc regions. The wild-type Fc region represents an amino acid sequence identical to that of a naturally occurring Fc region. SEQ ID NO. 1 shows the Fc region of wild-type human IgGl. The term "variant (human) Fc region" means an amino acid sequence that differs from the amino acid sequence of a wild-type (human) Fc region by at least one amino acid mutation. In some embodiments, the variant Fc region has at least one amino acid mutation, e.g., a1 to 10 amino acid mutation, as compared to the wild-type Fc region. In some embodiments, the variant Fc region has 1 to 5 amino acid mutations compared to the wild-type Fc region. In some embodiments, the Fc region or variant Fc region further comprises a hinge region (hinge region) of an immunoglobulin.

The numerical ranges used herein should be understood to have enumerated all numbers within the range. For example, a range of 1 to 20 should be understood to include any number, combination of numbers, or subrange from the following group: 1.2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

The term "linker" as used herein refers to a (peptide) linker of natural and/or synthetic origin, consisting of linear amino acids. The domains in the bispecific fusion polypeptides of the invention may be linked by linkers, wherein each linker is fused and/or otherwise linked (e.g., via a peptide bond) to at least two polypeptides or domains. In some embodiments, the amino acid sequences of all linkers present in a bispecific fusion polypeptide of the invention are identical. In other embodiments, the amino acid sequences of at least two linkers present in a bispecific fusion polypeptide of the invention are different. The linker should have a length suitable for linking two or more monomer domains in this way, the linker being able to ensure that the different domains to which it is linked are correctly folded and properly presented, thereby functioning as a biological activity thereof. In various embodiments, the linker has a flexible conformation. Suitable flexible linkers include, for example, those having glycine, glutamine, and/or serine residues. In some embodiments, the amino acid residues in the linker may be arranged in small repeat units of up to 5 amino acids, e.g., having an amino acid sequence as shown by GGGSGG or GGGSGGG.

"percent (%) sequence identity" with respect to a reference amino acid sequence refers to the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference amino acid sequence after aligning the sequences and introducing gaps (as needed) to obtain the maximum percent sequence identity, but does not consider any conservative substitutions as part of the sequence identity. To determine the percent amino acid sequence identity, the alignment can be performed in a variety of ways within the skill in the art, for example using BLAST, ALIGN or Megalign (DNASTAR) software. One skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the entire length of the sequences being compared.

The term "nucleic acid delivery vector" or "expression vector" as used herein refers to a nucleic acid sequence which, when the above-described isolated nucleic acid molecule is linked to a vector, can be linked directly or indirectly to regulatory elements on the vector, so long as the regulatory elements are capable of regulating translation and expression of the nucleic acid molecule, and the like. These regulatory elements may of course be derived directly from the vector itself or may be exogenous, i.e.not derived from the vector itself. That is, the nucleic acid molecule is operably linked to a regulatory element. "operably linked" herein refers to linking the exogenous gene to the vector such that regulatory elements within the vector, such as transcription regulatory sequences and translation regulatory sequences, and the like, are capable of performing their intended functions of regulating transcription and translation of the exogenous gene. Of course, the polynucleotides encoding the heavy and light chains of the antibody may be inserted separately into different vectors, typically into the same vector. The usual vectors may be, for example, plasmids, phages and the like.

As used herein, a "host cell" may be a eukaryotic cell and/or a prokaryotic cell. The skilled artisan can select cell types as desired, and any cell that can express a nucleic acid delivery vector can be used. The host cell may comprise an expression vector. The expression vectors may be introduced into host cells to obtain recombinant cells, which are then used to express the fusion polypeptides provided by the present disclosure. The recombinant cell is cultured to obtain the corresponding fusion polypeptide. These eukaryotic cells which can be used may be, for example, CHO cells, HEK293T, pichia pastoris and the like, prokaryotic cells such as e.coli cells and the like.

The term "pharmaceutically acceptable carrier, vehicle or adjuvant" as used herein refers to ingredients of the pharmaceutical formulation that are non-toxic to the subject other than the active ingredient. Any solvent, dispersion medium, coating, antibacterial, antifungal, isotonic and absorption delaying agent, and the like, which are physiologically compatible, may be included. Specific examples may be one or more of water, brine, phosphate buffered saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In many cases, isotonic agents, for example, sugars, polyalcohols (e.g., mannitol, sorbitol), sodium chloride, and the like, may be included in the pharmaceutical composition. Of course, the pharmaceutically acceptable carrier may also include minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, for extending the shelf life or efficacy of the antibody.

The term "treating" as used herein refers to alleviating and/or ameliorating a disorder and/or a disease, symptom or lesion associated therewith, as well as preventing exacerbation of the symptoms of the disorder. Desirable therapeutic effects include, but are not limited to, preventing occurrence or recurrence of a disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, amelioration or palliation of the symptoms, remission or prognosis of the improvement. It should be understood that treating a disease or condition does not require complete elimination of the disease, lesion or condition associated therewith.

The term "effective amount" as used herein refers to an amount that is effective over the necessary dosage and period of time in order to achieve the desired therapeutic or prophylactic effect.

The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. All reagents or equipment were commercially available as conventional products without the manufacturer's attention. Numerous specific details are set forth in the following description in order to provide a better understanding of the invention. The specific embodiments described herein are for purposes of illustration only and are not to be construed as limiting the invention in any way. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the present invention. Such structures and techniques are also described in a number of publications, such as the molecular cloning laboratory guidelines (fourth edition) (Cold spring harbor laboratory Press), ausubel, F.M et al, current Protocols in Molecular Biology, greene Publishing Assoc, and Wiley-lnterscience.

The invention relates to an experimental material:

coli (Escherichia coli) DH5 a was purchased from baori doctor technology (beijing);

human embryonic kidney cells HEK293 were purchased from the cell bank of the chinese sciences;

the silenced IgG1-sFc expression vector was engineered from the pFUSE-hIgG1-Fc2 vector from InvivoGen;

protein A antibodies or Protein A/G antibody-coupled agarose or magnetic beads were purchased from Semer Feishr technology (China);

coomassie brilliant blue staining solution and BCA detection kit were purchased from bi yun technology limited;

the homologous recombination cloning kit is purchased from offshore protein technology limited company;

reverse transcription kit was purchased from south Beijing nuowuzan biotechnology Co., ltd;

the experimental mice are male C57BL/6 mice, 6-8 weeks old, 18-22g in body mass, no specific pathogen level, and conventionally raised and purchased from Shanghai Laike laboratory animal Limited liability company;

the Hepa1-6 cell line, SNU-198 cell line, B16F10 cell line, CT26 cell line were purchased from the national academy of sciences cell bank;

Anti-F4/80 magnetic beads were purchased from Methawk and Biotechnology Co., ltd;

luciferase substrate was purchased from Gold Biotech;

cultured cells were purchased from Semer Feier technology (China) Inc. with DMEM, RPMI-1640, IMDM medium, CFSE dye, anti-F4/80 flow antibody, eFluor 670 and Fc receptor blocking agent.

The primers according to the present invention are shown in Table 1:

table 1PCR primer sequences:

example 1 preparation of hERMAP-sFc and ERMAP-sFc

1.1 preparation of amplified templates

In this example, total RNA of mouse liver tissue and human liver cancer cells was extracted and reverse transcribed to obtain cDNA. The method comprises the following specific steps:

the liver tissue of the mouse is quickly frozen in liquid nitrogen, 1mL of Trizol extract is added, and the tissue is crushed under the low temperature condition to extract the total RNA. Total RNA was extracted by direct lysis of SNU-398 cells with 90% confluency in a 6cm dish with 1mL Trizol.

The extracted total RNA is used as a template, genomic DNA is removed according to the specification of a reverse transcription (Reverse transcription, RT) kit, and various components required by the reverse transcription reaction are added for RT reaction, wherein the conditions are as follows: 15min at 37 ℃; and 5s at 85 ℃.

1.2 construction of hERMAP-sFc and ERMAP-sFc expression vectors

1) Cloning of ERMAP/Ermap extracellular segment Gene fragment

cDNA obtained from mouse liver tissue cells is used as a template, ERMAP-sFc-N/ERMAP-sFc-C is used as a primer, and an ERMAP extracellular segment gene is amplified by a PCR method. The hERMAP extracellular gene was amplified by PCR using cDNA obtained from SNU-398 cells as a template and hERMAP-sFc-N/hERMAP-sFc-C as primers.

PCR reaction procedure: (1) 98 ℃ for 10s; (2) 55 ℃ for 5s; (3) 72 ℃ for 30s; (4) extending at 72 ℃ for 10min; wherein (1) - (3) are conducted for 35 cycles.

The PCR product was separated by 1% agarose gel electrophoresis, recovered according to the DNA gel cutting recovery purification kit and the purity and concentration of the recovered fragment were checked.

2) Construction of hERMAP-sFc and ERMAP-sFc expression vectors

Cloning the purified hERMAP/ERMAP extracellular gene fragment to a silent IgG1-sFc expression vector by a homologous recombination method, transforming into an escherichia coli competent cell DH5 alpha, screening by using bleomycin (Zeocin) to obtain a resistant escherichia coli monoclonal, extracting a plasmid, and carrying out sequencing identification to finally obtain the correct hERMAP/ERMAP-sFc expression plasmid.

1.3 eukaryotic purification of hERMAP-sFc and ERMAP-sFc

Transferring the hERMAP/ERMAP-sFc expression plasmids into human embryo kidney cell HEK293 cell strains by a transfection mode respectively, collecting cell culture supernatants after 48 hours, and purifying the fusion-expressed hERMAP/ERMAP-sFc Protein by Protein A antibody or Protein A/G antibody coupled agarose or magnetic beads. Coomassie brilliant blue staining was used to identify purity and concentration was determined using BCA assay kit.

1.4 analysis of results

(1) Construction and identification of hERMAP-sFc and ERMAP-sFc expression vectors

The ERMAP extracellular segment gene fragment with homologous recombination sequence with the carrier is obtained by PCR amplification using the designed primer, and after the PCR product is subjected to 1% agarose gel electrophoresis, an obvious DNA band appears about 700bp, which is consistent with the experimental design. The hERMAP extracellular gene segment with homologous recombination sequence with IgG1-sFc carrier is obtained by PCR amplification with designed primer, and after 1% agarose gel electrophoresis, the PCR product is found to have a distinct DNA band about 400bp, which is consistent with experimental design. The PCR product is purified, cloned into IgG1-sFc expression vector plasmid which is linearized by EcoR I and Bgl II by using homologous recombinase, escherichia coli DH5 alpha is transformed, monoclonal with Zeocin resistance is selected next day, after amplification culture, recombinant plasmid is extracted, the obtained plasmid is entrusted to Shanghai Jie Liu Biotechnology Co., ltd for DNA sequencing analysis, and the result shows that the inserted DNA sequence in the positive recombinant plasmid is consistent with experimental design and the reading frame is correct. Indicating that hERMAP/ERMAP-sFc recombinant protein expression vector plasmid has been successfully constructed.

(2) Expression identification of hERMAP-sFc and ERMAP-sFc recombinant protein expression vectors

HEK393T cells are paved 24h before transfection, and when the HEK393T cells grow to have the fusion degree of 80 percent, hERMAP/ERMAP-sFc recombinant protein expression vector plasmids with correct sequence are transfected into HEK293T cell strains. After 48h, the culture supernatant was collected, and the fusion expressed hERRMAP/ERMAP-sFc Protein was purified by Protein A antibody or Protein A/G antibody-coupled agarose or magnetic beads, eluted with 100mM Glycine-HCl solution (pH 3.0) and dialyzed against PBS buffer. Protein bands of interest were found to occur between about 50-70kDa in molecular weight using 10% SDS-PAGE separation protein binding Coomassie Brilliant blue staining analysis with BCA assay protein concentration of 2mg/mL.

Example 2 in vitro functional analysis of hERMAP-sFc and ERMAP-sFc recombinant proteins

2.1Kupffer cell harvesting

Mouse livers were perfused in situ with collagenase IV at a concentration of 0.04%, and liver parenchymal cells were removed by centrifugation at 40 Xg, and liver non-parenchymal cells were collected and centrifuged using a 50%/25% Percoll density gradient. After centrifugation, cells in the middle layer were collected and subjected to cell sorting according to the instructions using anti-F4/80 magnetic beads.

2.2 construction of in vitro Co-culture System for studying Kupffer cell phagocytosis

(1) Flow detection of Kupffer cell phagocytosis

Tumor cells (Hepa 1-6, SNU-198, B16F10, CT 26) were labeled with CFSE, and Kupffer cells were pretreated with 10. Mu.g/mL Fc receptor blocking agent, and the tumor cells and Kupffer cells were treated with 4:1 ratio in IMDM medium supplemented with hERMAP/ERMAP-sFc or control sFc at a final concentration of 10. Mu.g/mL at 5% CO ₂ The cells were incubated at 37℃for 2 hours. By anti-F4The Kupffer cells in the co-culture system are labeled by the/80 flow antibody, and the ratio of CFSE positivity in the F4/80 positive cells is detected by the flow cytometer.

(2) Fluorescent microscope for observing Kupffer cell phagocytosis

Tumor cells (Hepa 1-6, SNU-198, B16F10, CT 26) were labeled with CFSE, kupffer cells were labeled with eFluor 670, and were pretreated with 10. Mu.g/mL Fc receptor blocking agent, and tumor cells and Kupffer cells were treated with 4:1 ratio in IMDM medium supplemented with hERMAP/ERMAP-sFc or control sFc at a final concentration of 10. Mu.g/mL, in slide-mounted dishes at 5% CO ₂ After culturing at 37℃for 2 hours, cells on the slide glass were fixed with 4% paraformaldehyde and then blocked, and observed with a fluorescence microscope.

2.3 analysis of results

Through the method of in-situ perfusion of mouse liver, 0.04% collagenase IV is digested for 20 minutes at 37 ℃, after the parenchymal cells are removed by centrifugation, the liver non-parenchymal cells are successfully separated through Percoll density gradient centrifugation, and Kupffer cells with the purity of more than 90% are obtained after anti-F4/80 magnetic bead separation. CFSE at final concentration of 5. Mu.M was used at 37℃with 5% CO ₂ Tumor cells were labeled in the incubator for 10 minutes, the reaction was stopped with complete medium, and after thorough washing with PBS, the cells were observed under a fluorescence microscope to be successfully labeled as 488nm laser-excitable green fluorescence.

Kupffer cell phagocytosis was detected using flow-through: kupffer cells were pretreated with 10. Mu.g/ml Fc receptor blocking agent at 4℃for 30 min before co-culturing with tumor cells (Hepa 1-6, SNU-198, B16F10, CT 26), respectively. Will be 1X 10 ⁵ Tumor cells of (C) and 0.25X10 respectively ⁵ Kupffer cells were co-cultured for 2 hours. Then, kupffer cells were stained with anti-F4/80 flow antibody (1:100) at 4℃for 30 minutes, washed 2 times with flow staining buffer, and detected on-machine. As a result, it was found that hERMAP-sFc and ERMAP-sFc significantly promoted phagocytosis of four tumor cells by Kupffer cells by about 40% -100% compared to sFc control group (FIGS. 1-2, 4-5, 7-8, and 10-11).

Using fluorescenceThe Kupffer cell phagocytosis was examined microscopically: eFluor 670 was used at a final concentration of 5. Mu.M at 37℃with 5% CO ₂ Kupffer cells were labeled in the incubator for 10 minutes, the reaction was stopped with complete medium, and washed thoroughly with PBS for co-culture experiments. Kupffer cells were pretreated with 10. Mu.g/mL Fc receptor blocking agent at 4℃for 30 min before co-culturing with tumor cells (Hepa 1-6, SNU-198, B16F10, CT 26), respectively. Will be 1X 10 ⁵ Tumor cells of (C) and 0.25X10) ⁵ The Kupffer cells were co-cultured in a slide-mounted petri dish for 2 hours, the cell samples on the slide were fixed with 4% paraformaldehyde, the slides were blocked, and the positive proportion of CFSE in eFluor 670 positive cells was observed by fluorescence microscopy. As a result, compared with sFc control group, hERMAP/ERMAP-sFc can significantly promote the phagocytosis of Kupffer cells to four tumor cells (Hepa 1-6, SNU-198, B16F10, CT 26), and the phagocytosis efficiency is improved by about 40% -100% (FIG. 3, FIG. 6, FIG. 9, FIG. 12).

Example 3 in vivo functional analysis of hERMAP-sFc and ERMAP-sFc

200 mug/mouse hERMAP-sFc and ERMAP-sFc recombinant proteins and control group sFc were intraperitoneally injected to nude mice one day before spleen injection, and once every two days after liver transfer by spleen injection, and the control was reached the experimental end point. The experimental group and the control group corresponding to each tumor cell are 6 mice.

In spleen injection liver transfer experiments, each mouse was injected 5×10 ⁶ Hepa1-6 cells, liver was photographed at experimental endpoint-28 days, and HE tissue staining was performed. The change of the Luciferase fluorescence value of the liver part in the animals on days 0,1,3 and 28 was monitored in real time. The results showed that the Luciferase fluorescence curves were significantly different between the ERMAP-sFc recombinant protein group and the experimental control group, and the HE results showed that the tumor cell area in liver tissue was reduced by about 45% -100% in the ERMAP-sFc group (fig. 13-16).

In spleen injection liver transfer experiments, each mouse was injected 2×10 ⁶ SNU-398 cells, liver was photographed at experimental endpoint-49 days and HE tissue stained. The results showed that tumor metastases decreased by about 60% -100% in the hERMAP-sFc group (fig. 17-18).

In spleen injection liver transfer experiments, each mouse was injected 1×10 ⁶ B16F10, livers were photographed and HE tissue stained after-14 days from the end of the experiment. The results showed a significant decrease in tumor metastasis of about 40% -100% in the ERMAP-sFc group (fig. 19-20).

In spleen injection liver transfer experiments, each mouse was injected 2×10 ⁶ CT26, taking liver after experimental end-28 days, taking liver for photographing, and performing HE tissue staining. The results showed a significant decrease in tumor cell area in liver tissue of about 40% -100% in the ERMAP-sFc group (fig. 21-22).

Example 4 difference in influence of ERMAP-sFc and ERMAP-Fc on phagocytosis in vivo

One day before spleen injection, 200 mug/mouse ERMAP-sFc or control ERMAP-Fc was injected intraperitoneally into the nude mice, and the experimental group and the control group were five mice. In spleen injection liver transfer experiments, each mouse was injected 5×10 ⁶ Hepa1-6 cells, perfusing the mice after 12 hours to isolate liver non-parenchymal cells, and flow cytometry detecting the proportion of CFSE positives in F4/80 positive cells after flow staining with F4/80 antibody. The results show that the ratio of CFSE positivity in the F4/80 positive cells of the ERMAP-sFc group is about 60% -100% higher than that of the ERMAP-Fc group (figures 23-24), thereby proving that the ERMAP-sFc can promote the phagocytosis of tumor cells by Kupffer cells in an in vivo environment.

Finally, it should be noted that the above description is only for illustrating the technical solution of the present invention, and not for limiting the scope of the present invention, and that the simple modification and equivalent substitution of the technical solution of the present invention can be made by those skilled in the art without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. An Fc monomer polypeptide comprising amino acid site substitutions compared to the amino acid sequence set forth in SEQ ID No. 1:

14 th bit, 15 th bit, 32 th bit, 34 th bit, 36 th bit, 102 th bit.

2. The Fc monomer polypeptide of claim 1, wherein said amino acid position substitution comprises L14F, L15Q, M32Y, S34T, T36E, K102Q.

3. The Fc monomer polypeptide of claim 2, wherein the amino acid sequence of said Fc monomer polypeptide is set forth in SEQ ID No. 2.

4. A fusion polypeptide comprising an extracellular segment of ERMAP or a variant thereof, and an Fc monomer polypeptide according to any one of claims 1-3.

5. The fusion polypeptide of claim 4, wherein the extracellular segment of ERMAP or variant thereof is linked directly or via a linker to the Fc monomer polypeptide.

6. The fusion polypeptide of claim 5, wherein the Fc monomer polypeptide is directly linked to the C-terminus of the extracellular segment of ERMAP or variant thereof.

7. The fusion polypeptide of any one of claims 4-6, wherein the ERMAP is a human ERMAP or a murine ERMAP.

8. The fusion polypeptide of claim 7, wherein the ERMAP is a human ERMAP, and the extracellular portion thereof has an amino acid sequence as shown in SEQ ID No. 3.

9. The fusion polypeptide of claim 7, wherein the ERMAP is murine ERMAP and the extracellular portion has the amino acid sequence shown in SEQ ID No. 4.

10. The fusion polypeptide of claim 8 or 9, wherein the variant of the extracellular portion of ERMAP has at least 85%, 90%, 95%, 98%, 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 3 or 4.

11. An isolated nucleic acid molecule encoding a fusion polypeptide according to any one of claims 4-10.

12. A nucleic acid delivery vector, characterized in that it comprises a nucleic acid molecule according to claim 11.

13. A host cell comprising the nucleic acid delivery vector of claim 12.

14. A pharmaceutical composition, characterized in that it comprises a fusion polypeptide according to any one of claims 4-10, a nucleic acid molecule according to claim 11, a nucleic acid delivery vector according to claim 12 or a host cell according to claim 13;

15. Use of a fusion polypeptide according to any one of claims 4-10, a nucleic acid molecule according to claim 11, a nucleic acid delivery vector according to claim 12, a host cell according to claim 13 or a pharmaceutical composition according to claim 14 for the preparation of a medicament for the prevention and/or treatment of tumor metastasis.

16. The use according to claim 15, wherein the tumour comprises liver cancer, melanoma, glioma, colon adenocarcinoma, pancreatic cancer, colon cancer, gastrointestinal cancer, prostate cancer, bladder cancer, ovarian cancer, lung cancer, renal cell carcinoma, nasopharyngeal carcinoma, renal cancer, breast cancer, hematological cancer or head and neck cancer.