阅读说明：本技术 一种对沙眼衣原体momp具有结合亲和力的多肽及其应用 (Polypeptide with binding affinity to chlamydia trachomatis MOMP and application thereof ) 是由 朱珊丽 张丽芳 董海艳 陈俊 石威 李文姝 李明洋 于 2020-06-09 设计创作，主要内容包括：本发明涉及一种对沙眼衣原体主要外膜蛋白特异性结合的多肽及其应用,首次揭示了一种对沙眼衣原体主要外膜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 bound to major outer membrane protein of chlamydia trachomatis and application thereof, and discloses a polypeptide with binding affinity to major outer membrane protein of chlamydia trachomatis 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 major outer membrane protein of chlamydia trachomatis, wherein: the polypeptide is represented by SEQ ID NO: 1 as skeleton, and carrying out 12-20 amino acid variations to obtain the polypeptide.

2. A polypeptide having binding affinity for the major outer membrane protein of chlamydia trachomatis according to claim 1, wherein the polypeptide having binding affinity for the major outer membrane protein of chlamydia trachomatis is represented by the amino acid sequence as set forth in SEQ ID NO: 1, amino acid mutations occur at positions 9-11, 13-14, 17-18, 24-25, 27-28, 32 and 35 of the amino acid sequence of the Z segment of staphylococcal protein A.

3. A polypeptide having binding affinity for the major outer membrane protein of chlamydia trachomatis according to claim 2, wherein the polypeptide has binding affinity for the major outer membrane protein of chlamydia trachomatis relative to the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of fragment Z of staphylococcal protein a, said polypeptide having binding affinity for the major outer membrane protein of chlamydia trachomatis:

the 9 th amino acid is mutated into H;

the 10 th amino acid is mutated into L;

the 11 th amino acid is mutated into A;

the 13 th amino acid is mutated into L;

the 14 th amino acid is mutated into M;

the 17 th amino acid is mutated into W;

the 18 th amino acid is mutated into T;

the 24 th amino acid is mutated into R;

the 25 th amino acid is mutated into H;

the 27 th amino acid is mutated into V;

the 28 th amino acid is mutated into H;

the 32 th amino acid is mutated into H;

the 35 th amino acid is mutated into R.

4. A polypeptide having binding affinity for the major outer membrane protein of chlamydia trachomatis according to claim 3, wherein the amino acid sequence of said polypeptide is selected from the group consisting of: SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.

5. The polypeptide having binding affinity for the major outer membrane protein of Chlamydia trachomatis according to claim 4, wherein the polypeptide interacts with the major outer membrane protein of Chlamydia trachomatis with a KD value of 9.86 × 10^-7M。

6. A targeting molecule for targeting the major outer membrane protein of chlamydia trachomatis, said targeting molecule comprising the polypeptide of any one of claims 1 to 5, and a conjugate linked to said polypeptide, said conjugate comprising: a cysteine residue, a polypeptide tag, a detectable label, or an agent that inhibits chlamydia trachomatis.

7. An isolated polynucleotide encoding the polypeptide having binding affinity for the major outer membrane protein of chlamydia trachomatis according to claim 4, having the sequence of SEQ ID NO: 3, respectively.

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 chlamydia trachomatis targeting molecule of claim 6, wherein the conjugate is a medicament of the major outer membrane protein of chlamydia trachomatis for the preparation of a medicament for the treatment of a chlamydia trachomatis infectious disease;

or the conjugate is a polypeptide label or a detectable marker, and is used for preparing a detection reagent for detecting the chlamydia trachomatis infection or preparing a diagnostic reagent for diagnosing the chlamydia trachomatis infection disease.

11. A pharmaceutical composition, comprising: a polypeptide having binding affinity for the major outer membrane protein of chlamydia trachomatis according to any one of claims 1 to 5 or a targeting molecule targeting the major outer membrane protein of chlamydia trachomatis according to any one of claim 4; and a pharmaceutically acceptable carrier.

12. A kit for diagnosing a disease caused by chlamydia trachomatis infection, said kit comprising: the targeting molecule of claim 6 for targeting the major outer membrane protein of Chlamydia trachomatis, 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 a disease caused by chlamydia trachomatis infection, said kit comprising: a polypeptide having binding affinity for the major outer membrane protein of Chlamydia trachomatis according to any one of claims 1 to 5, or a targeting molecule targeting the major outer membrane protein of Chlamydia trachomatis according to any one of claims 6, or a pharmaceutical combination according to any one of claims 11.

Technical Field

The invention relates to the field of biomedicine, in particular to a polypeptide with binding affinity to Major Outer Membrane Protein (MOMP) of chlamydia trachomatis and application thereof.

Background

Chlamydia trachomatis (Ct) is one of the main pathogens of sexually transmitted diseases, mainly causes male urethritis and female cervicitis, is often infected in a mixed manner with other pathogens, infects women through sexual transmission to cause poor pregnancy such as abortion, dead fetus and the like, and is closely related to the occurrence of cervical cancer. In recent years, STDs caused by Ct in China are in a continuously rising trend, and have great harm to human health. For Ct infectious diseases, an effective control method is not yet available. As a class of strictly intracellular parasitic, prokaryotic cell-type microorganisms with a unique developmental cycle, Ct exists extracellularly as an infectious pathogen, and exists intracellularly as a promoiety with reproductive properties. The division and the propagation of the primordium generate a large amount of protomers, and the mature protomers are released from the cells to infect new susceptible cells, and then are propagated and converted into primordium in cycles. Ct has now identified 19 serogroups, of which serotypes D, E, F, G and H are the major urogenital infections, and of which serotype E is the most common type reported at home and abroad. The Major Outer Membrane Protein (MOMP) is a high-content and wide-function transmembrane protein existing on the surfaces of Ct protomer and initiator, plays an important role in mediating the Ct to adhere to host cells, contains a plurality of antigen epitopes, and can be used as an important immunogen to stimulate the immune response of an organism. MOMP contains more cysteine, and can form a complex three-dimensional structure and polymer through intrachain and interchain disulfide bonds, so that the preparation of MOMP with complete structure and natural conformation is very difficult, and the development of a MOMP-based chlamydia vaccine is greatly limited. Researches show that MOMP VD2 and VD4 regions positioned outside a cell membrane contain a plurality of B cell epitopes and T cell epitopes, so the structural domain can be used as an important target for researching Ct vaccines, targeted therapy and diagnostic preparations.

At present, with the generation of the problem of persistent infection of pathogens and the development of breakthrough research brought by the molecular targeted therapy of tumors, scholars at home and abroad have attempted to switch the research on the treatment of infectious diseases to the molecular targeted therapy. Targeted therapies represented by monoclonal antibodies (mabs) have brought new promise for the treatment of infectious diseases and tumors, such as palivizumab for the prevention of respiratory syncytial virus infection in infants and young children, resisituzumab for the treatment of inhalation anthrax, trastuzumab (herceptin) for the treatment of metastatic breast cancer, etc., which have been approved by the FDA. However, the mAb-based targeted therapy still has its application limitations, such as weak tissue permeability, large toxic and side effects, high cost, etc., which seriously affect the broad application of the mAb in targeted therapy.

Based on the above description, there is still a need in the art to develop new methods for targeted treatment of Ct infection to improve the current clinical situation.

Disclosure of Invention

The invention aims to provide a polypeptide with binding affinity to Ct MOMP and application thereof.

In a first aspect of the present invention, there is provided a polypeptide having binding affinity for Ct MOMP, which is obtained by performing 12 to 20 (preferably 13 to 16, such as 14 or 15) amino acid variations on an amino acid sequence of Z segment (Z domain) of Staphylococcal Protein A (SPA) as a backbone.

In a preferred embodiment, the polypeptide having binding affinity for Ct MOMP is mutated at positions 9-11, 13-14, 17-18, 24-25, 27-28, 32, 35, 43 with respect to the amino acid sequence of Z fragment of staphylococcal protein A (SEQ ID NO: 1).

In another preferred embodiment, the polypeptide having binding affinity for Ct MOMP has, relative to the amino acid sequence of stretch Z of staphylococcal protein a:

the 9 th amino acid is mutated into H;

the 10 th amino acid is mutated into L;

the 11 th amino acid is mutated into A;

the 13 th amino acid is mutated into L;

the 14 th amino acid is mutated into M;

the 17 th amino acid is mutated into W;

the 18 th amino acid is mutated into T;

the 24 th amino acid is mutated into R;

the 25 th amino acid is mutated into H;

the 27 th amino acid is mutated into V;

the 28 th amino acid is mutated into H;

the 32 th amino acid is mutated into H;

the 35 th amino acid is mutated into R;

the 43 th amino acid was mutated to E.

In another preferred embodiment, the amino acid sequence of the polypeptide having binding affinity for Ct MOMP is as shown in seq id NO: 2, respectively.

In another preferred embodiment, the polypeptide having binding affinity for Ct MOMP has a KD value of 9.86 × 10 for interaction with Ct MOMP protein^-7M。

In another aspect of the present invention, there is provided a targeting molecule for targeting Ct MOMP, the targeting molecule comprising a polypeptide as described in any of the above, and a conjugate linked (or conjugated) to the polypeptide, the conjugate including (but not limited to): cysteine residues, polypeptide tags, drugs that inhibit Ct, or detectable labels (e.g., fluorescent labels, enzymes, biotin, or radioisotopes).

In a preferred embodiment, the conjugate is a peptide, and the conjugate and the polypeptide having binding affinity for Ct MOMP form a fusion polypeptide.

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

In another preferred embodiment, the conjugate is linked to the polypeptide having binding affinity for Ct MOMP in flexible peptides including (but not limited to): (Gly4Ser) 3.

In another aspect of the invention, there is provided an isolated polynucleotide encoding a polypeptide having binding affinity for Ct MOMP as described above.

In another aspect of the invention, there is provided a polynucleotide encoding said Ct MOMP targeting molecule, and wherein said conjugate is a peptide.

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 invention, there is provided a method of preparing a polypeptide having binding affinity for Ct MOMP as described in any one of the preceding, the method comprising: (1) culturing said cell, thereby expressing said polypeptide having binding affinity for Ct MOMP; (2) and (3) separating and purifying the polypeptide obtained in the step (1).

In another aspect of the invention, there is provided a use of the polypeptide having binding affinity for Ct MOMP or the Ct MOMP-targeting molecule for preparing a medicament for treating Ct MOMP protein expression positive cells; or

The kit is used for preparing a detection reagent for detecting Ct MOMP; or

Used for preparing a diagnostic reagent for diagnosing Ct infection.

In a preferred embodiment, in the targeting molecule targeting Ct MOMP, the conjugate is an anti-Ct drug (e.g., toxin), the polypeptide having binding affinity for Ct MOMP, or the targeting molecule of Ct MOMP is used for treating Ct genital tract infection.

In another preferred embodiment, in the targeting molecule targeting Ct MOMP, the conjugate is a detectable label (such as a fluorescent label or an enzyme), the polypeptide having binding affinity for Ct MOMP, or the targeting molecule targeting Ct MOMP is used for Ct MOMP expression positive cells.

In another preferred example, the Ct MOMP expression positive cells include: urogenital tract infected epithelial cells, and the like.

In another aspect of the present invention, there is provided a pharmaceutical composition comprising: any one of the polypeptides having binding affinity for Ct MOMP described above or any one of the targeting molecules targeting Ct MOMP described above; and a pharmaceutically acceptable carrier.

In another aspect of the present invention, there is provided a kit for diagnosing or treating Ct MOMP expression positive cells, the kit comprising: any of the aforementioned polypeptides having binding affinity for Ct MOMP, or any of the aforementioned targeting molecules targeting Ct MOMP, or the pharmaceutical composition.

In a preferred embodiment, said polypeptide having binding affinity for Ct MOMP or said Ct MOMP targeting molecule is in an effective amount.

In another aspect of the invention, there is provided a method for treating Ct infection, comprising administering to a subject in need thereof the polypeptide having binding affinity for Ct MOMP, or the targeting molecule targeting Ct MOMP.

In another aspect of the invention, there is provided a method for diagnosing Ct MOMP expression positive cells, comprising administering to a subject in need of treatment a targeted molecule of said targeted Ct MOMP; in the targeting molecule, the conjugate is a detectable label (e.g., a fluorescent label or an enzyme).

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, Z_{Ct MOMP}And comparison of Zwt sequences. The polypeptide Z of the invention_{Ct MOMP}The modified amino acid position in (A) is indicated in bold font (SEQ ID NO: 2).

FIG. 2, Z produced in example 1_{Ct MOMP}A polypeptide recombinant plasmid construction diagram and a composition schematic diagram of recombinant protein.

(A)Z_{Ct MOMP}Constructing a diagram of the recombinant plasmid; (B) constructing a Zwt recombinant plasmid map; (C) and (D) are each Z_{Ct MOMP}And Zwt schematic composition of prokaryotic expression of full-length recombinant protein, Z_{Ct MOMP}Represents a polypeptide having an amino acid sequence selected from SEQ ID NOs: 2, Zwt represents an amino acid sequence selected from SEQ ID NO: 1, 6xHis for a six histidine tag, HM for ndei (catatg) translated amino acids, LE for xhoi (ctcgag) translated amino acids.

FIG. 3, pET21a (+)/Z_{Ct MOMP}Electrophoretogram of the recombinant plasmid (1).

A is pET21a (+)/Z_{Ct MOMP}. M1: 1kb DNAmarker; 1: pET21a (+) vector plasmid; 2: pET21a (+)/Z_{Ct MOMP}An Affibody recombinant plasmid; 3: pET21a (+)/Z_{Ct MOMP}Affibody/NdeI+XhoI；4：Z_{Ct MOMP}Affibody DNA fragments; m2: DL2000 DNAmarker. B is pET21a (+)/Zwt. M1: 1kb DNA marker; 1: pET21a (+) vector plasmid; 2: pET21a (+)/Zwt recombinant plasmid; 3: pET21a (+)/Zwt/NdeI + XhoI; 4: a Zwt DNA fragment; m2: DL2000 DNA marker.

FIG. 4, Z_{Ct MOMP}The recombinant protein is subjected to prokaryotic expression and purification of SDS-PAGE (A) and Westernblot (B) for analysis.

M: a protein Marker; 1-2 are each purified Z_{Ct MOMP}And Zwt. In the Westernblot experiment, the primary antibody is Histag monoclonal antibody.

FIG. 5, MOMP recombinant protein prokaryotic expression identification and rabbit serum antibody preparation analysis.

(A) SDS-PAGE analysis of MOMP protein, M: a protein Marker; 1: coli.bl21(DE3) strain; pET21a (+)/MOMP transformed E.coli.BL21(DE3) strain; pET21a (+)/MOMP transformed E.coli.BL21(DE3) strain prior to IPTG induction; pET21a (+)/MOMP transformed E.coli.BL21(DE3) strain after IPTG induction; 5: purifying MOMP protein; (B) western blot analysis of MOMP recombinant purified protein, wherein the primary antibody is a his-tag monoclonal antibody, 1: purified MOMP recombinant protein; 2: a carrier protein; (C) the titer of the mouse serum antibody after the MOMP recombinant protein immunization is shown.

FIG. 6, culture and identification of Ct.

(A) After HeLa229 cells were infected with Ct, cytoplasmic inclusion bodies (containing large amounts of protomers and primordia) were visualized by iodine staining and Giemsa staining, respectively. (B) And detecting the Ct inclusion body by an indirect immunofluorescence method. The rabbit anti-MOMP polyclonal antibody is a primary antibody, FITC-goat anti-rabbit IgG is a secondary antibody, and DAPI stains cell nucleus.

FIG. 7, Z_{Ct MOMP}affinity of affibody and MOMP recombinant proteins was determined by SPR on a ProteOn XPR36 instrument.

A to B are each Z_{Ct MOMP}And the affinity analysis of the Zwt protein and the target protein MOMP protein.

FIG. 8, Z_{Ct MOMP}Western blot identification of binding of affibodies to MOMP proteins.

A to B are each Z_{Ct MOMP}Western blot detection of binding of affibody and Zwt proteins to MOMP protein in HeLa229 cells infected with Ct. The internal reference is GAPDH.

FIG. 9, Z_{Ct MOMP}Immunoprecipitation identification of binding of affibodies to MOMP proteins.

A to B are each Z_{Ct MOMP}Western blot detection of binding of affibody and Zwt proteins to MOMP protein in HeLa229 cells infected with Ct. The internal references are the heavy and light chains of immunoglobulins.

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 Ct MOMP" refers to a polypeptide obtained by 12-20 amino acid variations using the amino acid sequence of Z fragment of staphylococcal protein A as a backbone, and which is capable of specifically binding Ct MOMP with little or no non-specific binding.

As used herein, the terms "polypeptide of the invention", "polypeptide having binding affinity for Ct MOMP", "Ct MOMP-binding polypeptide", "Z_{Ct MOMP}affibody polypeptides "," Z_{Ct MOMP}affibody”、“Z_{Ct MOMP}: 461, "affibody protein", "affibody recombinant protein", "Z_{Ct MOMP}Recombinant protein "may be used interchangeably; ct MOMP and MOMP can be used interchangeably; SPAZ and Zwt may be used interchangeably.

As used herein, the "targeting molecule" refers to a molecule that can target Ct MOMP obtained by linking the polypeptide having binding affinity for Ct MOMP of the present invention to other functional conjugates. The conjugate can be cysteine residue, polypeptide label, drug for inhibiting Ct, enzyme or detectable marker, etc.

As used herein, the "fusion polypeptide" is a subordinate concept of the "targeting molecule" and refers to a molecule that can target Ct MOMP and is obtained by linking the polypeptide having binding affinity for Ct MOMP of the present invention to other functional peptides (e.g., toxin protein or functional protein fragment).

The inventors selected Ct MOMP as the target antigen. The inventor takes a Z structural domain (Zwt, SEQ ID NO: 1) of staphylococcal protein A as a scaffold, carries out random mutation on a surface amino acid residue simulation antibody binding site, constructs a mutant library by a phage display technology, carries out affinity screening on the library by taking Ct MOMP as a target antigen, and finally obtains a polypeptide with high affinity for the Ct MOMP 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, 43 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 the amino acid sequence of SEQ ID NO: 2, as shown in figure 1.

The invention also encompasses polypeptides formed by adding additional amino acid residues at either or both ends of the amino acid sequence of the Ct MOMP binding polypeptide. These additional amino acid residues may function when the polypeptide binds to Ct MOMPBut may also be used for other purposes as well, such as involving one or more of 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, Ct MOMP binding domain, or other binding function, or an enzymatic function, or a fluorescent function, or a combination thereof.

The invention also includes polypeptides modified to increase their stability under alkaline conditions based on the Ct MOMP binding polypeptides. 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 includes polypeptides obtained by other modifications based on the Ct MOMP binding polypeptides of the invention. 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 Ct MOMP 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 Ct MOMP binding polypeptide and the conjugate may be linked directly or via a polypeptide linker (linker 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.

In "heterologous" fusion polypeptides, where the Ct MOMP binding polypeptide constitutes a first domain or first moiety, and the second and other moieties have other functions than binding to the Ct MOMP, these contemplated results are also within the scope of the invention. The second and further portions of the fusion polypeptide may comprise a binding domain having affinity for other target molecules than Ct MOMP. 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 Ct MOMP binding domain and at least one domain with affinity for the 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 Ct MOMP binding polypeptide, to form a fusion protein. Non-limiting examplesIncluded are enzymes that direct effector enzymes (e.g., carboxypeptidase) for "ADEPT" (antibody-mediated enzyme prodrug therapy); proteins including proteins to recruit effector cells and other components of the immune system; including cytokines such as IL-2, IFN γ, IL-12, TNFa, IP 10; including procoagulant factors such as tissue factor, von Willebrand factor; including toxins such as ricin A, 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 purpose of detecting Ct MOMP positive cells based on the specificity of the polypeptide of the invention by connecting a detectable marker (such as a fluorescent marker, biotin or a radioactive isotope) to the Ct MOMP binding polypeptide.

"Ct MOMP binding affinity" means that binding affinity can be determined, for example, by using a surface plasmon resonance (surf plasmon resonance) technique such asA polypeptide property detected by the device. Ct MOMP binding affinity can be detected by an experiment in which Ct MOMP protein is immobilized on the sensor 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 the sample containing the Ct MOMP may be passed through the chip. One skilled in the art can use the obtained sensor image to establish at least one qualitative measure of Ct MOMP binding affinity of the polypeptide. If quantitative measurement methods are required, for example to establish a certain KD-value between the interactions, a table can also be usedSurface plasmon resonance method. For example, the binding value may utilize

Ct MOMP protein was immobilized on the sensor chip of the device, and a sample of the polypeptide whose affinity is to be detected was prepared by serial dilution and injected in random order^-6M。。

The invention also provides an isolated nucleic acid encoding a Ct MOMP binding polypeptide or targeting molecule or fusion polypeptide of the invention, as well as the 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 of producing Ct MOMP binding polypeptides or targeting molecules or fusion polypeptides of the invention are also encompassed by 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 applications of the Ct MOMP binding polypeptide or the targeting molecule or the fusion polypeptide in different aspects, including treatment, diagnosis and/or detection.

The Ct MOMP binding polypeptide can be used as a substitute of a Ct MOMP antibody in different applications.

As a non-limiting example, it may be used to treat diseases characterized by Ct MOMP expression, such as urogenital chlamydia trachomatis infection, and the like. By binding to intracellular Ct MOMP, 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 preparation or a means for targeting other therapeutic preparations to Ct MOMP protein. 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, fragments of the polypeptide that retain the ability to bind Ct MOMP are also encompassed by the invention.

The Ct MOMP binding properties of the polypeptides of the invention and the stability of the binding molecules for producing targeting molecules (including fusion proteins) and/or labels with the polypeptides mean that the polypeptides can also be used to target other active substances to sites of chlamydial infection, including cells expressing Ct MOMP. Thus, another aspect of the invention provides the use of a CtMOMP binding polypeptide as described herein conjugated to a substance having anti-cancer activity to transport the substance to cells expressing Ct MOMP, resulting in damage or apoptosis of the target cell.

Such an anti-cancer active substance may be a protein fused or coupled to a Ct MOMP binding polypeptide by a chemical bond, such as a protein selected from effector enzymes for "ADEPT" (antibody-directed enzyme promoter therapy) applications, proteins for recruiting effector cells and other components of the immune system, cytokines such as IL-2, IFN γ, IL-12, TNF α, IP10, etc., procoagulant factors such as tissue factor, von Willebrand factor, etc., toxins such as ricin A, Pseudomonas exotoxin, calcheamicin, maytansinoids, etc⁹⁰y、¹³¹I、²¹¹At, etc.), such an isotope may bind directly to the Ct MOMP binding polypeptide or may bind to the Ct MOMP binding polypeptide through 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-Ct activity to Ct MOMP expressing cells in vivo comprising administering to a patient a conjugate of the active substance described herein and a Ct MOMP binding polypeptide. Such conjugates have been described appropriately hereinbefore.

The invention also includes the use of the polypeptide that binds to Ct MOMP to detect Ct MOMP in a sample.

For example, such assays can be used to diagnose disease conditions characterized by expression of Ct MOMP. Detection of the presence of Ct MOMP can be performed in vivo or in vitro. The preferred choice for in vivo diagnosis is the use of Westernblot, ELISA, etc. The sample to be tested may for example be a biological fluid sample or a tissue sample. The current general method is to use antibodies aiming at CtMOMP, and the method can be applicable to the polypeptide which is combined with Ct MOMP and is detected by methods such as Westernblot, ELISA and the like and is used for identifying the expression of CtMOMP in fresh and frozen genital tract secretion 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 Ct MOMP binding polypeptide in the detection of Ct MOMP in biological liquid samples. This method comprises the steps of: (1) providing a sample of biological fluid from a patient to be tested, (2) adding a Ct MOMP binding polypeptide as described herein to the sample under conditions that allow the polypeptide to bind to any Ct MOMP present in the sample, (3) removing unbound polypeptide, and (4) detecting bound polypeptide. The amount of bound polypeptide detected correlates with the amount of CtMOMP present in the sample. In step (2), the Ct MOMP binding polypeptide may be added to the sample in any suitable form, including, for example, when the Ct MOMP binding polypeptide is immobilized on a solid support, by which the sample is contacted, or the Ct MOMP binding polypeptide is present in solution.

Other applications of the Ct MOMP binding polypeptide include: the method for detecting Ct MOMP in the sample comprises the following steps: (1) providing a tissue sample, such as a frozen section, suspected of containing Ct MOMP, (2) adding a Ct MOMP binding polypeptide of the invention to the sample under suitable conditions conducive to binding of the polypeptide to any Ct MOMP present in the sample, (3) removing unbound polypeptide, and (4) detecting bound polypeptide. The amount of bound polypeptide detected correlates with the amount of Ct MOMP present in the sample.

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

The invention also provides a kit for diagnosing Ct MOMP expression in a tissue sample, which comprises the Ct MOMP 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 positive and negative control tissue sections.

One area of diagnostic application is the detection of Ct-infected cells or aggregates thereof in vivo. The invention provides a kit for performing such a diagnosis, comprising a Ct MOMP binding polypeptide of the invention labelled with a chelator, a diagnostic radioisotope (a non-limiting example being⁶⁸Ga、⁷⁶Br、¹¹¹In、⁹⁹Tc、¹²⁴I and¹²⁵i, etc.), and reagents for assaying incorporation efficiency.

As noted above, the invention encompasses the use of Ct MOMP binding polypeptides of the invention to target active substances to cells expressing Ct MOMP, such as urogenital mucosal cells. The invention also provides a kit for this purpose comprising a Ct MOMP 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 Ct MOMP protein or the targeting molecule of the targeting Ct MOMP 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 of the invention having binding affinity for Ct MOMP protein is administered to a mammal (e.g., a human), wherein the safe and effective amount is typically at least about 1 microgram per kilogram of body weight, and in most cases no more than 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.