阅读说明：本技术 一种对eb病毒lmp1c端蛋白特异性结合的多肽及其应用 (Polypeptide specifically bound to EB virus LMP1C terminal protein and application thereof ) 是由 张丽芳 陈俊 朱珊丽 蒋朋飞 薛向阳 于 2019-09-05 设计创作，主要内容包括：本发明涉及一种对EB病毒LMP1 C端蛋白特异性结合的多肽及其应用。首次揭示了一种对EB病毒LMP1 C端蛋白具有结合亲和力的多肽；本发明还提供了该多肽的在诊断检测中的应用,并作为靶向载体在药物或分子靶向试剂的诊断或治疗用途。(The invention relates to a polypeptide specifically binding to EB virus LMP 1C-terminal protein and application thereof. The polypeptide with binding affinity to the EB virus LMP 1C-terminal protein is disclosed for the first time; the invention also provides the application of the polypeptide in diagnostic detection, and the polypeptide can be used as a targeting carrier in the diagnosis or treatment of drugs or molecular targeting agents.)

1. A polypeptide having binding affinity for the C-terminal protein of epstein barr virus LMP1, comprising: the polypeptide is obtained by taking the amino acid sequence of the staphylococcus A protein Z segment as shown in SEQ ID NO. 1 as a framework and carrying out 12-20 amino acid variations.

2. The polypeptide having binding affinity for the C-terminal protein of epstein barr virus LMP1 of claim 1, wherein the polypeptide having binding affinity for the C-terminal protein of epstein barr virus LMP1 has amino acid mutations at positions 9-11, 13-14, 17-18, 24-25, 27-28, 32, 35 of the amino acid sequence of stretch Z of staphylococcal protein a as set forth in SEQ ID No. 1.

3. The polypeptide having binding affinity for the C-terminal protein of epstein barr virus LMP1 of claim 2, wherein said polypeptide is a polypeptide having binding affinity for the C-terminal protein of epstein barr virus LMP1, said polypeptide having binding affinity for the C-terminal protein of epstein barr virus LMP1, relative to the amino acid sequence of staphylococcal protein a segment Z as set forth in SEQ ID NO: 1:

the 9 th amino acid is mutated into R or L;

the 10 th amino acid is mutated into S or W;

the 11 th amino acid is mutated into W, D or V;

the 13 th amino acid is mutated into L, M or T;

the 14 th amino acid is mutated into R, M or S;

the 17 th amino acid is mutated to P, L or R;

the 18 th amino acid is mutated into T, G or Y;

the 24 th amino acid is mutated into P or A;

the 25 th amino acid is mutated into G or Q;

the 27 th amino acid is mutated into L, H or A;

the 28 th amino acid is mutated into Q, R or V;

the 32 th amino acid is mutated into L, R or A;

the amino acid mutation at position 35 is A, G or L.

4. The polypeptide of claim 3, having binding affinity for the C-terminal protein of epstein-barr virus LMP1, wherein the amino acid sequence of said polypeptide is selected from the group consisting of: any one of SEQ ID NO 2-4.

5. The polypeptide having binding affinity for the C-terminal protein of Epstein-Barr virus LMP1 according to claim 4, wherein the polypeptide has a KD of 1x10 for its interaction with the C-terminal protein of Epstein-Barr virus LMP1^-4M to 1X10^-8M。

6. A targeting molecule for targeting the C-terminal protein of epstein barr virus LMP1, wherein said targeting molecule comprises the polypeptide of any one of claims 1-5, and a conjugate linked to said polypeptide, said conjugate comprising: a cysteine residue, a polypeptide tag, a detectable label, or a drug that inhibits epstein-barr virus LMP 1.

7. An isolated polynucleotide encoding the polypeptide having binding affinity for epstein barr virus LMP 1C-terminal protein according to claim 4, wherein the polynucleotide sequence is as shown in SEQ ID NOs 5, 6 and 7.

8. A recombinant vector comprising the polynucleotide of claim 7.

9. A host cell comprising the recombinant vector of claim 8, or comprising a genome into which the polynucleotide of claim 7 has been integrated.

10. The use of the targeted EB virus targeting molecule according to claim 6, wherein,

the conjugate is a medicine for inhibiting EB virus LMP1, and is used for preparing medicines for treating EB virus infection diseases or EB virus LMP 1C-terminal protein expression positive tumors;

or the conjugate is a polypeptide label or a detectable marker, and is used for preparing a detection reagent for detecting EB virus infection or a diagnostic reagent for diagnosing EB virus infection diseases or EB virus LMP 1C-terminal protein expression positive tumors.

11. A pharmaceutical composition, comprising: the polypeptide having binding affinity for epstein barr virus LMP 1C-terminal protein of any of claims 1-5 or the targeting molecule targeting epstein barr virus LMP 1C-terminal protein of claim 4; and a pharmaceutically acceptable carrier.

12. A kit for diagnosing EB virus infection diseases or tumors positive to EB virus LMP1 protein expression, which is characterized by comprising: the EB virus LMP 1C-terminal protein targeting molecule according to claim 6, wherein the targeting molecule is a polypeptide tag or a detectable label, and a detection reagent for detecting the polypeptide tag or the detectable label.

13. A kit for treating EB virus infection diseases or tumors positive to EB virus LMP 1C-terminal protein expression, which is characterized by comprising: the polypeptide having binding affinity for the C-terminal protein of epstein barr virus LMP1 of any one of claims 1-5, or the targeting molecule targeting the C-terminal protein of epstein barr virus LMP1 of claim 6, or the pharmaceutical combination of claim 11.

Technical Field

The invention relates to the field of biomedicine, in particular to a polypeptide specifically binding to EB virus LMP1C terminal protein and application thereof.

Background

EB virus (Epstein-Barr virus, EBV) belongs to human herpesvirus, and is generally infected in people, EB virus is not only a pathogen of infectious mononucleosis, and is closely related to nasopharyngeal carcinoma (NPC), oral gland tumor, lymphoma, Hodgkin's disease, gastric cancer, B cell lymphoma after organ transplantation, AIDS-related lymphoma and the like, about 80% of cases of nasopharyngeal carcinoma (NPC) all over the world are counted, particularly, a high-incidence region in south China is provided, but no effective vaccine and a specific prevention and treatment method are available so far, research suggests that almost 100% of undifferentiated and low-differentiation NPC patients are latently infected with the virus, genome of the EB virus and corresponding protein expressed by the virus can be detected in nasopharyngeal carcinoma tissues, latent infection of the virus mainly expresses six nuclear proteins (EBNA) and latent membrane protein (EBNA) 1 and 2. LMP1 are latent transformation genes, can induce the transformation of the EBB virus (EBNA) 1 and the LMP 2. the LMP)1 and 2. the LMP 3611, the protein (Latenserb) which is a protein, can be used for inducing the malignant cell transformation gene of the EBB virus, and further, the tumor cell proliferation of a receptor gene, such as a receptor gene, a receptor for promoting the proliferation of tumor, a receptor, such as a receptor for inhibiting protein activating protein, a receptor for tumor, a receptor for inhibiting cell proliferation, a receptor for tumor, a receptor for inhibiting cell proliferation, such as a receptor for tumor, a receptor for inhibiting protein for tumor, a protein for inhibiting cell proliferation, a protein for inhibiting cell proliferation, a protein for inhibiting cell proliferation, a protein for inhibiting protein for tumor growth, a protein for inhibiting protein for tumor, a protein for inhibiting protein for tumor growth, a protein for inhibiting protein for tumor growth, a protein for tumor, a protein for inhibiting protein for tumor growth, a.

Targeted therapy is currently the most promising approach and strategy in tumor therapy. The Epidermal Growth Factor Receptor (EGFR) and tumor angiogenesis are used as targets for treatment, such as an EGFR monoclonal antibody (herceptin), a HER2 monoclonal antibody (cetuximab), bevacizumab, a small molecule compound tyrosine kinase antagonist (sunitinib) and the like, and the tumor angiogenesis is prevented by specifically blocking the signal conduction of tumor cells or blocking receptors, so that the growth of the tumor cells is inhibited or the apoptosis of the tumor cells is promoted. However, the targeted therapy based on antibody molecules still has the limitations of its application, such as poor permeability, high cost, strong immunogenicity, and serious toxic and side effects. The toxic effects of toxic side effects in particular have been a major obstacle to the development of antibodies directed to the treatment of tumors, producing liver, kidney and nervous system toxicity that reduces their function. Radioimmunotherapy with isotopically labeled antibodies also results in bone marrow suppression and the like.

Based on the above description, there is still a need in the art to develop new drugs or new methods for targeted therapy of epstein-barr virus infection and tumors associated therewith to improve the current clinical situation.

Disclosure of Invention

The invention aims to provide a polypeptide specifically binding to EB virus LMP1C terminal protein and application thereof.

In the first aspect of the invention, the polypeptide with binding affinity to EB virus LMP1C terminal protein is obtained by performing 12-20 amino acid variations by using an amino acid sequence of staphylococcus A protein Z segment as a framework, wherein the amino acid sequence is shown as SEQ ID NO: 1.

In another preferred embodiment, the polypeptide having binding affinity for EB virus LMP1C terminal protein has amino acid mutation at positions 9-11, 13-14, 17-18, 24-25, 27-28, 32, 35 of the amino acid sequence of staphylococcal protein A Z segment as shown in SEQ ID NO: 1.

In another preferred embodiment, the polypeptide having binding affinity for EB virus LMP1C terminal protein has amino acid mutations at positions 9-11, 13-14, 17-18, 24-25, 27-28, 32, 35 of the amino acid sequence of staphylococcal protein A Z segment as shown in SEQ ID NO:1, including

The 9 th amino acid is mutated into R or L;

the 10 th amino acid is mutated into S or W;

the 11 th amino acid is mutated into W, D or V;

the 13 th amino acid is mutated into L, M or T;

the 14 th amino acid is mutated into R, M or S;

the 17 th amino acid is mutated to P, L or R;

the 18 th amino acid is mutated into T, G or Y;

the 24 th amino acid is mutated into P or A;

the 25 th amino acid is mutated into G or Q;

the 27 th amino acid is mutated into L, H or A;

the 28 th amino acid is mutated into Q, R or V;

the 32 th amino acid is mutated into L, R or A;

the amino acid mutation at position 35 is A, G or L.

In another preferred embodiment, the amino acid sequence of the polypeptide specifically binding to the EB virus LMP1C terminal protein is shown as any one of SEQ ID NO: 2-4.

In another preferred embodiment, the polypeptide specifically binding to EB virus LMP1C terminal protein and EB virus LMP1C terminal protein have a KD value of 1 × 10^-4M to 1X10^-8M。

In another aspect of the present invention, there is provided a targeting molecule targeting the LMP1C terminal protein of epstein barr virus, wherein the targeting molecule comprises a polypeptide as described in any of the above, and a conjugate linked (or conjugated) to the polypeptide, wherein the conjugate includes (but is not limited to): a cysteine residue; a polypeptide tag; drugs that inhibit the EB virus; substances with anti-cancer activity; or detectable labels including, but not limited to: fluorescent labels, enzymes, biotin or radioisotopes.

In a preferred embodiment, the anti-cancer active agents include, but are not limited to: the polypeptide used in the present invention directs effector enzymes: a carboxypeptidase; proteins for recruiting effector cells and other components of the immune system: IL-2, IFN gamma, IL-12, TNF alpha, IP 10; procoagulant factor, tissue factor, von willebrand factor; a toxin; cytotoxic drugs: an auristatin analog, doxorubicin, a radioisotope.

In another preferred example, the drugs inhibiting epstein-barr virus include, but are not limited to: diphtheria toxin, ricin, pseudomonas aeruginosa exotoxin or a functional fragment of said diphtheria toxin, ricin, pseudomonas aeruginosa exotoxin, calcheamicin, maytansinoid;

in another preferred embodiment, the enzymes include, but are not limited to: alkaline phosphatase or horseradish peroxidase.

In another preferred embodiment, the conjugate is a peptide, and the conjugate and the polypeptide with the binding affinity to the EB virus LMP1C terminal protein form a fusion polypeptide.

In another preferred embodiment, the conjugate is linked to the polypeptide specifically binding to the LMP1C teloprotein as a flexible peptide, including (but not limited to): (Gly4Ser) 3.

In another preferred embodiment, the polypeptide tags include, but are not limited to: his tag (e.g., 6 × His), Myc tag, GST tag, Flag tag.

In another aspect of the invention, there is provided an isolated polynucleotide encoding any of the aforementioned polypeptides having binding affinity for the LMP1C terminal protein, the polynucleotide sequence being represented by seq id nos 5, 6 and 7.

In another aspect of the invention, there is provided a recombinant vector comprising said polynucleotide.

In another aspect of the invention, there is provided a host cell comprising said recombinant vector, or comprising or having integrated into its genome said polynucleotide.

In another aspect of the present invention, there is provided a method for preparing a polypeptide having binding affinity for epstein barr virus LMP1C, as defined in any one of the above, comprising: (1) culturing said cell, thereby expressing said polypeptide having binding affinity for LMP1C teloprotein; (2) and (3) separating and purifying the polypeptide obtained in the step (1).

In another aspect of the invention, the polypeptide having binding affinity to LMP1C terminal protein or the targeting molecule targeting LMP1C terminal protein is provided, wherein in the targeting molecule targeting epstein barr virus LMP1C terminal protein, the conjugate is an antitumor drug, and the polypeptide having binding affinity to epstein barr virus LMP1C terminal protein or the targeting molecule targeting epstein barr virus LMP1C terminal protein is used for treating epstein barr virus LMP1C terminal protein expression positive tumors.

In another aspect of the present invention, there is provided a use of the polypeptide having a binding affinity to LMP1C terminal protein or the targeting molecule targeting LMP1C terminal protein, wherein in the targeting molecule targeting epstein barr virus LMP1C terminal protein, the conjugate is a detectable label, a fluorescent label, an enzyme, biotin, or a radioisotope, and the polypeptide having a binding affinity to epstein barr virus LMP1C terminal protein or the targeting molecule targeting epstein barr virus LMP1C terminal protein is a diagnostic agent for diagnosing epsm virus infection diseases or epsm virus LMP1C terminal protein expression positive tumors.

In another preferred example, the tumors positive for expression of EB virus LMP1C terminal protein comprise: nasopharyngeal carcinoma, oral gland tumor, lymphoma, Hodgkin's disease, gastric cancer, B cell lymphoma after organ transplantation, AIDS-related lymphoma, etc.

In another aspect of the present invention, there is provided a pharmaceutical composition comprising: the polypeptide with binding affinity to EB virus LMP1C terminal protein or the targeting molecule targeting EB virus LMP1C terminal protein; and a pharmaceutically acceptable carrier.

In another aspect of the present invention, there is provided a kit for diagnosing tumors positive for expression of LMP1C teloprotein of epstein barr virus, said kit comprising: the targeting molecule of the targeting EB virus LMP1C terminal protein, the targeting molecule of the targeting EB virus LMP1C terminal protein, the targeting molecule is coupled with a polypeptide label or a detectable marker, and a detection reagent for detecting the polypeptide label or the detectable marker.

In another aspect of the present invention, there is provided a kit for treating tumors positive for expression of LMP1C teloprotein of epstein barr virus, said kit comprising: the polypeptide with binding affinity to EB virus LMP1C terminal protein; or the targeting molecule targeting EB virus LMP1C terminal protein, wherein the targeting molecule is coupled with the drug inhibiting EB virus or the substance with anticancer activity; or a combination of said agents.

In a preferred embodiment, the polypeptide having binding affinity for epstein barr virus LMP1C terminal protein or the targeting molecule targeting epstein barr virus LMP1C terminal protein is in an effective amount.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

The invention is further described with reference to the drawings and the detailed description.

Drawings

FIG. 1, each Z_LMP1And comparison of Zwt sequences, polypeptide Z of the invention_{LMP1C terminal protein}The modified amino acid position(s) in (b) is/are underlined in the figure (SEQ ID NO: 2-4);

FIG. 2, example Z_EBVLMP1114Z generated_LMP1Constructing a diagram of the polypeptide recombinant plasmid;

FIG. 3, pET21a (+)/Z_LMP1Electrophoretogram of recombinant plasmid of affibody;

A-C are respectively pET21a (+)/Z_EBVLMP1:15、pET21a(+)/Z _EBVLMP1114 and pET21a (+)/Z _EBVLMP1277, electrophoresis of the recombinant plasmid. M1:1 kbDNAmarker; 1: pET21a (+) vector plasmid; 2: pET21a (+)/Z_{LMP1C terminal protein}ffibody recombinant plasmids; 3: pET21a (+)/Z_LMP1affibody/NdeI+XhoI；4：Z_LMP1affibody dna fragments; m2: DL2000 DNAmarker;

FIG. 4, Z_LMP1Prokaryotic expression of affibody recombinant protein (A) and SDS-PAGE electrophoretic analysis of purification (B);

(A) m: protein marker; 1-2 BL21(DE3) and BL21(DE3) transfected with pET21(+) empty vector, respectively, and 3-6 pET21a (+)/Z_EBVLMP1:15、pET21a(+)/Z_EBVLMP1114 and pET21a (+)/Z_EBVLMP1277 and pET21a (+)/Zwt, BL21(DE3) strains transfected with recombinant plasmids.

(B) M: protein marker; 1-4 are each purified Z_EBVLMP1:15、Z_EBVLMP1:114、Z _EBVLMP1277 and Zwtaffibody recombinant proteins;

FIG. 5, prokaryotic expression identification of recombinant protein at EB virus LMP1C end and analysis of rabbit serum antibody preparation

(A) And (3) performing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis on LMP1C end recombinant purified protein of the EB virus, wherein M: a protein Marker; 1: purified EB virus LMP1C terminal protein recombinant protein; 2: purified GST carrier protein; (B) and (3) performing Westernblot analysis on the LMP1C end recombinant purified protein of the EB virus, wherein the primary antibody is a his-tag monoclonal antibody, 1: purified EB virus LMP1C end recombinant protein; 2: purified GST carrier protein; (C) the reaction of EB virus LMP1C end recombinant protein immune rabbit serum antibody; (D) the titer of the rabbit serum antibody after EB virus LMP1C end recombinant protein immunization;

FIG. 6, Z_LMP1SPR detection of affinity of affibody polypeptide and EB virus LMP1C end recombinant protein on ProteOnXPR36 instrument

A to D are each Z_EBVLMP1:15、Z_EBVLMP1:114、Z _EBVLMP1277 and Zwt protein and target protein EB virus LMP1C end recombinant protein affinity analysis;

FIG. 7, Z_LMP1Cellular immunofluorescence method identification of binding of affibody polypeptide and EB virus LMP1C terminal protein natural protein

A to D are each Z_EBVLMP1:15、Z_EBVLMP1:114、Z _EBVLMP1277 and Zwt protein combined with natural protein indirect immunofluorescence detection;

FIG. 8, Z_EBVLMP1:15、Z_EBVLMP1:114、Z_EBVLMP1Immunohistochemical analysis (400-fold microscopic image) of the recombinant protein 277 to LMP1 in tumor tissues of nude mice;

FIG. 9, Z_EBVLMP1:15、Z_EBVLMP1:114、Z _EBVLMP1277 immunohistochemical analysis of the recombinant protein for LMP1 in nasopharyngeal carcinoma tumor tissues (400-fold microscopic picture).

Detailed Description

The invention is described in detail below with reference to examples, which are intended to be illustrative only and not to be construed as limiting the scope of the invention, and many insubstantial modifications and variations of the invention can be made by an engineer skilled in the art based on the teachings of the invention.

As used herein, the term "polypeptide having binding affinity for EB virus LMP1C end protein" refers to a polypeptide obtained by performing 12-20 amino acid variations with the amino acid sequence of staphylococcal protein A Z segment as the backbone, and the polypeptide is capable of specifically binding EB virus LMP1C end protein with little or no non-specific binding.

As used herein, the "polypeptide of the present invention", "polypeptide having binding affinity for EB virus LMP1C telogen", "EB virus LMP1C telogen binding polypeptide", "EB virus LMP1C telogen binding polypeptide", "Z_{LMP1C terminal protein}affibody polypeptides "," Z_{LMP1C terminal protein}affibody”、“Z_{LMP1C terminal protein}"," affibody protein "," affibody recombinant protein "," Z_{LMP1C terminal protein}Recombinant protein "may be used interchangeably; EB virus LMP1C end protein and LMP1C end protein can be used interchangeably; z_{EBVLMP1C terminal protein}And Z_{LMP1C terminal protein}Can be used interchangeably; SPAZ and Zwt may be used interchangeably.

As used herein, the "targeting molecule" refers to a molecule which can target EB virus LMP1C terminal protein and is obtained by linking the polypeptide having binding affinity to EB virus LMP1C terminal protein (EBVLMP1 (187-386 aa) protein) of the present invention with other functional conjugates. The conjugate can be cysteine residue, polypeptide label, drug for inhibiting EB virus LMP1C terminal protein, enzyme or detectable marker, etc.

As used herein, the "fusion polypeptide" is a subset of the "targeting molecule" and refers to a molecule obtained by linking the polypeptide of the present invention having binding affinity for epstein barr virus LMP1C terminal protein with other functional peptides (e.g., toxin proteins or functional protein fragments) and capable of targeting to the cytoplasmic domain of epstein barr virus LMP1C terminal protein.

The inventors selected EB virus LMP1C terminal protein as target antigen. The invention uses Z structure domain (Zwt, SEQ ID NO:1) of staphylococcal protein A as a bracket, carries out random mutation on the surface amino acid residue simulation antibody binding site, constructs a mutant library by phage display technology, carries out affinity screening on the library by using EB virus LMP1C terminal protein as a target antigen, and finally obtains polypeptide with high affinity for EB virus LMP1C terminal protein through a large amount of screening work.

The polypeptide of the invention is obtained by taking the amino acid sequence of the Z structural domain of the staphylococcal protein A as a framework and carrying out 14-20 (preferably 14) amino acid variations. As a preferred mode of the present invention, the polypeptide of the present invention has amino acid mutations at positions 9-11, 13-14, 17-18, 24-25, 27-28, 32, 35 relative to the amino acid sequence of Z fragment of staphylococcal protein A (SEQ ID NO: 1). More preferably, the polypeptide of the invention has an amino acid sequence as shown in any one of SEQ ID NOs 2-4, as shown in FIG. 1.

The invention also covers the polypeptide formed by adding additional amino acid residues at either end or both ends of the amino acid sequence of the EB virus LMP1C terminal protein binding polypeptide. These additional amino acid residues may function when the polypeptide binds to the epstein-barr virus LMP1C terminal protein, but may also be used for other purposes as well, such as one or more of those relating to the production, purification, stabilization, coupling or detection of the polypeptide. These additional amino acid residues may include one or more amino acid residues added for chemical coupling purposes. Such as the first or last addition of a cysteine residue at the N-or C-terminus of the polypeptide chain. Such additional amino acid residues may also include a "tag" for polypeptide purification or detection, such as a hexa-histidine peptide (His) that interacts with a tag antibody₆) A tag, either a "myc" tag or a "flag" tag. In addition, other alternatives known to those skilled in the art are also encompassed by the present invention.

The "additional amino acid residues" may also constitute one or more polypeptide domains with the desired function, such as the same binding function as the first, epstein barr 1C terminal protein binding domain, or other binding function, or an enzymatic function, or a fluorescent function, or a combination thereof.

The invention also comprises a polypeptide which is modified on the basis of the EB virus LMP1C terminal protein binding polypeptide and further increases the stability of the EB virus LMP1C terminal protein binding polypeptide under alkaline conditions. This stability includes site-directed substitution of any asparagine residue present in the unmodified sequence with an amino acid residue that is less sensitive to basic conditions. This property of reduced sensitivity to alkali, which is advantageous for using the polypeptides of the invention as affinity ligands in affinity chromatography, enables a prolonged lifetime of the affinity chromatography matrix, since the affinity chromatography column is subjected to frequent strong alkali treatments for elution between different reactions.

The invention also comprises polypeptides obtained by performing other modifications on the EB virus LMP1C terminal protein binding polypeptide. These modified (usually without altering primary structure) forms include: chemically derivatized forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications in the synthesis and processing of the polypeptide or in further processing steps. Such modification may be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylase. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides modified to increase their resistance to proteolysis or to optimize solubility.

The EB virus LMP1C terminal protein binding polypeptide can be connected with a conjugate to form a functional targeting molecule, and the connection can be connected or adsorbed through chemical bonds (including peptide bonds); the chemical bond is a covalent bond or a non-covalent bond. Preferably, the linkage is by peptide bond, thereby forming a fusion polypeptide. The EB virus LMP1C terminal protein binding polypeptide and the conjugate can be directly connected or connected through a polypeptide linker (connecting peptide). The linker comprises, for example, 1-30 amino acids; preferably 1-20 amino acids. The arrangement of the linker peptide does not substantially affect the activity of each polypeptide in the fusion protein. Preferably, the linkage may be performed using a flexible peptide (Gly4Ser) 3. Other linker peptides well known to those skilled in the art may also be used in the present invention.

It is contemplated that in a "heterologous" fusion polypeptide, where the epstein barr virus LMP1C terminal protein binding polypeptide constitutes a first domain or first moiety, and the second and other moieties have other functions than binding epstein barr virus LMP1C terminal protein, such results are also within the scope of the present invention. The second and further portions of the fusion polypeptide may comprise a binding domain having affinity for a target molecule other than the epstein-barr virus LMP1C end protein. Such binding domains may also be associated with the SPA domain, but have substitution mutations at 1 to about 20 positions. The result is a fusion polypeptide having at least one epstein barr virus LMP1C end protein binding domain and at least one domain with affinity for said other target molecule. This extends the utility of the polypeptides of the invention, e.g., as therapeutic agents or as capture, detection or isolation reagents.

Other options for the second and further portions of the fusion polypeptides of the invention include one or more portions for therapeutic use. In therapeutic applications, other molecules may be covalently or non-covalently coupled to the polypeptides of the invention by other means, such as by linking engineered P.aeruginosa exotoxin PE38KDEL or granzyme (GrB), etc., via a flexible peptide to the C-terminus of the LMP 1C-terminal protein binding polypeptide to form a fusion protein. Non-limiting examples include enzymes that direct effector enzymes (e.g., carboxypeptidase) for "ADEPT" (antibody-mediated enzyme prodrug therapy) with the polypeptides of the invention; proteins including proteins to recruit effector cells and other components of the immune system; including cytokines such as IL-2, IFN γ, IL-12, TNF α, IP 10; including procoagulant factors such as tissue factor, von Willebrand factor; including toxins such as ricin, calcheamicin, maytansinoids; including toxic small molecules such as auristatin analogs, doxorubicin, and the like. At the same time, for more convenient incorporation of radionuclides (e.g. for facilitating incorporation⁶⁸Ga、⁷⁶Br、¹¹¹In、⁹⁹Tc、¹²⁴I、¹²⁵I) For diagnosis or radionuclides (e.g. of the type⁹⁰Y、¹³¹I、²¹¹At) for therapeutic use, the additional amino acids listed above (in particular hexa-histidine tag and cysteine) may be considered, with the aim of coupling the chelator of radioisotopes to the polypeptide sequence.

The invention also covers the connection of a detectable marker (such as a fluorescent marker, biotin or a radioactive isotope) on the EB virus LMP1C terminal protein binding polypeptide, so that the aim of detecting and expressing positive tumors of EB virus LMP1C terminal protein can be realized based on the specificity of the polypeptide.

"EB virus LMP1C terminal protein (EBVLMP1C terminal protein) binding affinity" means that binding affinity can be determined, for example, by using surface plasmon resonance (surface plasmon resonance) techniques such as

A polypeptide property detected by the device. Binding affinity of EB virus LMP1C terminal protein can be detected by an experiment in which EB virus LMP1C terminal protein is immobilized on an inductive chip of the device, and then a sample containing the polypeptide to be detected is passed through the chip. Alternatively, the polypeptide to be detected may be immobilized on a sensor chip of the device, and then a sample containing the LMP 1C-terminal protein of EB virus is passed through the chip. The person skilled in the art can use the acquired sensor image to establish at least one qualitative measurement of the binding affinity of epstein barr virus LMP1C telogen of the polypeptide. Surface plasmon resonance methods can also be used if quantitative measurement methods are required, for example in order to establish a certain KD value between interactions. For example, the binding value may utilize

The assay was performed on a 2000 apparatus (BiocoreAB). EB virus LMP1C end protein is fixed on the induction chip of the device, and the polypeptide sample to be detected by affinity is prepared by serial dilution and injected in random order. KD values can then be calculated from the results. In the embodiment of the invention, the KD value of the polypeptide reaches 1.70X 10^-5M to 7.52X 10^-7M。

The invention also provides an isolated nucleic acid encoding the EB virus LMP1C terminal protein binding polypeptide or targeting molecule or fusion polypeptide of the invention, and also a complementary strand thereof. The nucleic acid can be artificially synthesized in a complete sequence, and can also be obtained by a PCR amplification method respectively.

The invention also provides vectors comprising the nucleic acid molecules encoding the same. The vector may further comprise an expression control sequence operably linked to the sequence of the nucleic acid molecule to facilitate expression of the fusion protein. As used herein, "operably linked" or "operably linked" refers to a condition in which certain portions of a linear DNA sequence are capable of affecting the activity of other portions of the same linear DNA sequence. For example, a promoter is operably linked to a coding sequence if it controls the transcription of the coding sequence.

In the present invention, any suitable vector may be used, such as some vectors for cloning and expression of bacterial, fungal, yeast and mammalian cells, e.g., Pouwels et al, cloning vectors: as described in laboratory manuals.

In addition, recombinant cells containing the nucleic acid sequences are also encompassed by the present invention. The term "host cell" includes prokaryotic and eukaryotic cells. Commonly used prokaryotic host cells include E.coli, Bacillus subtilis, and the like; coli cells (e.coli), such as e.coli HMS174(DE3), or BL21(DE3), may be mentioned, for example. Commonly used eukaryotic host cells include yeast cells, insect cells, and mammalian cells.

Methods for producing EB virus LMP1C end protein binding polypeptides or targeting molecules or fusion polypeptides of the invention are also included in the invention. The method includes culturing a recombinant cell containing a nucleic acid encoding a corresponding polypeptide to obtain a product polypeptide. The polypeptide prepared as described above may be purified to substantially homogeneous properties, for example, as a single band on SDS-PAGE.

Based on the information to be expressed and the current state of the art for recombinant expression of proteins, the skilled artisan, in conjunction with the present disclosure, can readily prepare the polypeptides of the invention. For example, a plasmid expressing an unmodified Z domain may be used as starting material. The desired substitution mutations can be introduced into this plasmid using known techniques to obtain the expression vectors of the invention.

When chemical polypeptide synthesis methods are used to prepare the polypeptides or targeting molecules or fusion proteins of the invention, any naturally occurring amino acid residues in the above polypeptides may be substituted with any corresponding, non-naturally occurring amino acid residue or derivative thereof, provided that the function of the product polypeptide is not substantially impaired.

The invention also relates to application of the EB virus LMP1C terminal protein binding polypeptide or targeting molecule or fusion polypeptide in different aspects, including application in treatment, diagnosis and/or detection.

The EB virus LMP1C terminal protein binding polypeptide can be used as a substitute of EB virus LMP1C terminal protein antibody in different applications.

As a non-limiting example, it can be used for treating diseases characterized by the expression of the EB virus LMP1C terminal protein, such as tumors (e.g. nasopharyngeal carcinoma) and the like. The protein can inhibit cell signal transduction by combining with the end protein of the intracellular EB virus LMP1C, and can be used for in vivo and in vitro diagnosis of related diseases. The polypeptide of the invention can be used as a detection reagent, a capture reagent or a separation reagent, and can also be directly used as a therapeutic agent or a means for targeting other therapeutic agents to the LMP1C terminal protein of EB virus. Methods of using the polypeptides of the invention in vitro can be performed in different ways, such as microtiter plates, protein arrays, biosensor surfaces, and tissue sections, among others. In order to adapt the polypeptides of the invention for specific uses, modifications and/or additions may be made to the polypeptides of the invention without departing from the scope of the invention.

These modifications and additions are described in detail below, which may include additional amino acids contained in the same polypeptide chain, or labels and/or therapeutic agents that chemically modify or otherwise bind to the polypeptides of the invention. In addition, the invention also covers the fragment of the polypeptide which retains the capability of binding EB virus LMP1C terminal protein.

The cytoplasmic domain binding property of the EB virus LMP1C terminal protein of the polypeptide and the stability of the polypeptide in producing targeting molecules (including fusion protein) and/or labeled binding molecules means that the polypeptide can also be used for targeting other active substances to tumor sites, including cells expressing EB virus LMP1C terminal protein. Accordingly, another aspect of the present invention provides the use of an epstein-barr virus LMP 1C-terminal protein binding polypeptide as described herein coupled to a substance having anti-cancer activity, to deliver said substance to cells expressing epstein-barr virus LMP 1C-terminal protein, resulting in damage or apoptosis of target cells.

Such an anti-cancer active substance may be a protein fused or coupled by chemical bond to the LMP1C telopectin binding polypeptide of EB virus, such as an effector enzyme selected from the group consisting of those used for "ADEPT" (antibody-directed transmembrane therapeutic) applications; proteins for recruiting effector cells and other components of the immune system; cytokines such as IL-2, IFN γ, IL-12, TNF α a, IP10, and the like; procoagulant factors such as tissue factor, von Willebrand factor, and the like; toxins such as ricin, pseudomonas exotoxin, calcheamicin, maytansinoids, and the like. Alternatively, the active substance may be a cytotoxic drug, such as an auristatin analogue or doxorubicin or a radioisotope (e.g., as in the case of a drug therapy)⁹⁰Y、¹³¹I、²¹¹At, etc.), such an isotope may bind directly to the EB virus LMP1C telogen-binding polypeptide or may bind to the EB virus LMP1C telogen-binding polypeptide via a chelating agent, such as the well-known chelating agents DOTA or DTPA.

In a related aspect, the invention also provides a method of targeting a substance having anti-cancer activity to cells expressing epstein barr virus LMP1C telogen in vivo, comprising administering to a patient a conjugate of said active substance described herein and an epstein barr virus LMP1C telogen binding polypeptide. Such conjugates have been described appropriately hereinbefore.

The invention also comprises the polypeptide combined with the EB virus LMP1C terminal protein for detecting the EB virus LMP1C terminal protein in a sample.

For example, such assays may be used to diagnose disease conditions characterized by expression of the epstein barr virus LMP1C telogen. The detection of the existence of the EB virus LMP1C terminal protein can be carried out in vivo or in vitro. A preferred option for in vivo diagnosis is the use of positron emission tomography, PET. The sample to be tested may for example be a biological fluid sample or a tissue sample. The current general method is to use an antibody aiming at EB virus LMP1C terminal protein, which can be applied to the polypeptide combined with EB virus LMP1C terminal protein of the invention, and the method is a histochemical method for detecting the existence of EB virus LMP1C terminal protein and is used for identifying the expression of EB virus LMP1C terminal protein in fresh, frozen or formalin-fixed paraffin-embedded tissue samples.

The polypeptides of the invention can also be used as part of a fusion protein, wherein the other domain is a reporter enzyme or a fluorescent enzyme. Alternatively, it may be labeled with one or more fluorescent agents and/or radioisotopes, optionally labeled with a chelator. Suitable radioisotopes include⁶⁸Ga、⁷⁶Br、¹¹¹In、⁹⁹Tc、¹²⁴I and¹²⁵i, and the like.

The invention also comprises the application of the EB virus LMP1C terminal protein binding polypeptide in detecting EB virus LMP1C terminal protein in a biological liquid sample. This method comprises the steps of: (1) providing a sample of biological fluid from a subject being tested, (2) adding an epstein barr virus LMP1C terminal protein binding polypeptide as described herein to the sample under conditions which allow binding of said polypeptide to any epstein barr virus LMP1C terminal protein present in the sample, (3) removing unbound polypeptide, and (4) detecting bound polypeptide. The amount of bound polypeptide detected correlates with the amount of epstein barr virus LMP1C end protein present in the sample. In step (2), the epstein-barr virus LMP1C telopeptide binding polypeptide may be added to the sample in any suitable form, including, for example, when the epstein-barr virus LMP1C telopeptide binding polypeptide is immobilized on a solid support through which the sample is contacted, or the epstein-barr virus LMP1C telopeptide binding polypeptide is present in solution.

The other applications of the EB virus LMP1C terminal protein binding polypeptide further comprise: the method for detecting EB virus LMP1C terminal protein in the sample comprises the following steps: (1) providing a tissue sample suspected of containing epstein barr virus LMP1C end protein, such as a frozen section or a formalin-embedded tissue section, (2) adding an epstein barr virus LMP1C end protein binding polypeptide of the invention to the sample under suitable conditions conducive to binding of the polypeptide to any epstein barr virus LMP1C end proteins present in the sample, (3) removing unbound polypeptide, and (4) detecting bound polypeptide. The amount of bound polypeptide detected correlates with the amount of epstein barr virus LMP1C end protein present in the sample.

The invention also provides a kit for diagnosing the expression of EB virus LMP1C terminal protein in a tissue sample, which comprises EB virus LMP1C terminal protein binding polypeptide fused with a reporter enzyme (such as alkaline phosphatase or horseradish peroxidase), a reagent for detecting the activity of the enzyme, and/or a positive control tissue section and/or a negative control tissue section.

The invention also provides a kit for diagnosing EB virus LMP1C terminal protein expression in a tissue sample, which comprises EB virus LMP1C terminal protein binding polypeptide fused with a marker (such as a flag marker or a myc marker) detected by an antibody, a primary antibody specific to the marker, a secondary antibody specific to the primary antibody and coupled with a reporter enzyme, a reagent for detecting enzyme activity, and/or a positive control tissue section and/or a negative control tissue section. One area of diagnostic application is the detection of cancer cells or aggregates thereof in vivo. The present invention provides a kit for performing such a diagnosis, comprising an EB virus LMP1C telopeptide binding polypeptide of the present invention labeled with a chelator, a diagnostic radioisotope (a non-limiting example of which is⁶⁸Ga、⁷⁶Br、¹¹¹In、⁹⁹Tc、¹²⁴I and¹²⁵i, etc.), and reagents for assaying incorporation efficiency.

As described above, the present invention encompasses the use of the epstein-barr virus LMP 1C-terminal protein binding polypeptides of the present invention to target active substances to cells expressing epstein-barr virus LMP 1C-terminal proteins, such as certain types of cancer cells. The invention also provides a kit for this purpose comprising an EB virus LMP1C teloprotein binding polypeptide of the invention labeled with a chelator, a therapeutic radioisotope (a non-limiting example being⁹⁰Y、¹³¹I、²¹¹At), and reagents for assaying incorporation efficiency.

The present invention also provides a pharmaceutical composition comprising: the effective amount of the polypeptide with binding affinity to EB virus LMP1C terminal protein or targeting molecule of targeting EB virus LMP1C terminal protein, and a pharmaceutically acceptable carrier.

As used herein, a "pharmaceutically acceptable" component is one that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity), i.e., with a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents. The term refers to such pharmaceutical carriers: they are not essential active ingredients per se and are not unduly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. Sufficient details regarding pharmaceutically acceptable carriers can be found in Remington's pharmaceutical sciences (mackpub.co., n.j.1991). Pharmaceutically acceptable carriers in the compositions may contain liquids such as water, saline, glycerin and sorbitol. In addition, auxiliary substances, such as lubricants, glidants, wetting or emulsifying agents, pH buffering substances and stabilizers, such as albumin and the like, may also be present in these carriers.

The compositions may be formulated into a variety of dosage forms suitable for mammalian administration including, but not limited to: injection, capsule, tablet, emulsion, and suppository.

In use, a safe and effective amount of a polypeptide or targeting molecule having binding affinity for epstein barr virus LMP1C telogen of the present invention is administered to a mammal (e.g., a human), wherein the safe and effective amount is generally at least about 1 microgram per kilogram of body weight, and in most cases does not exceed about 10 milligrams per kilogram of body weight, preferably the dose is from about 1 microgram per kilogram of body weight to about 1 milligram per kilogram of body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

The invention will be further illustrated with reference to the following specific examples.