CN111978379B - A polypeptide with binding affinity to human melanoma antigen A3 protein and use thereof - Google Patents

Abstract

The invention relates to a polypeptide with binding affinity to human melanoma antigen MAGE-A3 and application thereof. For the first time, a polypeptide having binding affinity for MAGE-A3 protein is disclosed; 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.

Description

A polypeptide with binding affinity to human melanoma antigen A3 protein and use thereof

technical field

The present invention relates to the field of biomedicine, and more particularly, the present invention relates to a polypeptide having binding affinity to human melanoma antigen A3 protein (MAGE-A3) and its application.

Background technique

Cancer is a frequently-occurring and common disease in the world. The mortality rate of malignant tumors ranks first among all diseases. Among them, the morbidity and mortality of lung cancer ranks first in the world, and gastric cancer ranks second in the death of malignant tumors. , breast cancer is the leading cause of death in women. At present, the incidence of lung cancer in my country has shown an explosive growth trend, while the incidence of gastric cancer continues to rise every year, and shows a significant trend of younger people. The MAGE gene was first discovered in melanoma cell lines and is a large family consisting of six subfamilies A, B, C, D, E and F. The MAGE-A3 gene is not only expressed in malignant tumors dominated by melanoma In tissues, it is also expressed in a large proportion in various tumors of various tissue types, such as lung cancer, breast cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, osteosarcoma, leukemia, bladder cancer, brain tumor and ovarian tumor. In particular, gastrointestinal tumors such as gastric cancer, esophageal cancer, colorectal cancer, etc. can be highly expressed. MAGE-A3 is not expressed in normal tissue cells except testis and placenta, because the inherent physiological barrier of testis and placenta can avoid autoimmune damage caused by the recognition of MAGE-A3. Therefore, MAGE-A3 is currently considered to be an ideal target antigen for tumor-specific immunotherapy and diagnosis.

Targeted therapy is the most promising method and strategy in current tumor treatment, mainly targeting epidermal growth factor receptor (EGFR) and tumor angiogenesis, such as EGFR monoclonal antibody (HER2 monoclonal antibody), small molecule compounds Tyrosine kinase antagonists (Herceptin, Herceptin and cetuximab, etc.), bevacizumab (rhuMAb-VEGF) and sunitinib, etc., by specifically blocking tumor cell signaling or by Blocking the receptor prevents tumor angiogenesis, thereby inhibiting tumor cell growth or promoting tumor cell apoptosis. However, targeted therapy based on antibody molecules still has limitations in its application, such as poor permeability, high cost, strong immunogenicity and serious side effects, which need further research and improvement. In particular, the toxic effects of toxic side effects have become a major obstacle to the development of therapeutic antibodies against cancer, resulting in liver, kidney and nervous system toxicity and reduced function. Such as cardiotoxic effects associated with the HER2-targeting antibody trastuzumab (Herceptin). Radioimmunotherapy with isotope-labeled antibodies can also lead to bone marrow suppression, among others.

Based on the above description, there is still an urgent need in the art to study new drugs or new methods for targeted treatment of MAGE-A3 expression-positive related tumor diseases, so as to improve the current clinical situation.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a polypeptide with binding affinity to human melanoma antigen A3 protein and its use.

The first aspect of the present invention provides a polypeptide with binding affinity to human melanoma antigen A3 protein, the polypeptide is relative to the amino acid sequence of the Z segment of Staphylococcus A protein, and the sequence is shown in SEQ ID NO: 1. Of polypeptides with binding affinity to human melanoma antigen A3 protein:

The 9th amino acid is mutated to P or T;

The 10th amino acid is mutated to P or L;

The 11th amino acid is mutated to C or I;

The 13th amino acid is mutated to R or L;

The 14th amino acid is mutated to W or F;

The 17th amino acid is mutated to M or S;

The 18th amino acid is mutated to A or F;

The 24th amino acid is mutated to R or P;

The 25th amino acid is mutated to Q or A;

The 27th amino acid is mutated to H or V;

The 28th amino acid is mutated to L or G;

The 32nd amino acid is mutated to P;

The 35th amino acid is mutated to L or G;

Amino acid 43 is mutated to E.

In a preferred embodiment, the amino acid sequence of the polypeptide with binding affinity to human melanoma antigen A3 protein is shown in any of SEQ ID NOs: 2-3.

In another preferred embodiment, the KD value of the interaction between the polypeptide having binding affinity to human melanoma antigen A3 protein and human melanoma antigen A3 protein is 1.03×10 ^-5 M to 3.28×10 ^-6 M.

In another aspect of the present invention, there is provided a targeting molecule targeting human melanoma antigen A3 protein, the targeting molecule comprising any of the aforementioned polypeptides, and a conjugate connected to the polypeptide The conjugate is preferably conjugated with the polypeptide, and the conjugate includes but is not limited to: cysteine residue, polypeptide tag, drug that inhibits human melanoma, or detectable label, detectable label Including but not limited to fluorescent labels, enzymes, biotin or radioisotopes.

In a preferred example, the conjugate is a peptide, and the conjugate forms a fusion polypeptide with the polypeptide having binding affinity to human melanoma antigen A3 protein.

In another preferred example, the polypeptide tags include but are not limited to: His tags, preferably 6×His, Myc tags, GST tags, and Flag tags.

In another preferred example, the drugs for inhibiting human melanoma include but are not limited to: toxins; preferably, the toxins are toxins that have the effect of inhibiting human melanoma antigen A3 protein-positive tumors, such as diphtheria toxin, ricin Cannatoxin, Pseudomonas aeruginosa exotoxin, or a functional fragment of the toxin; and, the tumor is a human melanoma antigen A3 protein-positive tumor.

In another preferred embodiment, the toxin is Pseudomonas aeruginosa exotoxin A, or the functional fragment of the toxin is the active fragment PE38KDEL of Pseudomonas aeruginosa exotoxin A. Preferably, the Pseudomonas aeruginosa exotoxin A or its functional fragment is linked to the carboxyl terminus of the polypeptide having binding affinity to the human melanoma antigen A3 protein, preferably the C terminus of the polypeptide.

In another preferred embodiment, the conjugate is linked with the polypeptide having binding affinity to human melanoma antigen A3 protein by a flexible peptide, and the flexible peptide includes but is not limited to: (Gly4Ser)3.

In another aspect of the present invention, there is provided an isolated polynucleotide encoding any of the aforementioned polypeptides having binding affinity to human melanoma antigen A3 protein.

In another aspect of the present invention, a polynucleotide is provided, which encodes the targeting molecule targeting human melanoma antigen A3 protein, and wherein the conjugate is a peptide.

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

In another aspect of the present invention, a host cell is provided, the host cell comprises the recombinant vector, or the polynucleotide is contained or integrated into the genome.

In another aspect of the present invention, there is provided a method for preparing any of the aforementioned polypeptides having binding affinity to human melanoma antigen A3 protein, the method comprising: (1) culturing the cells to express the (2) separating and purifying the polypeptide obtained in (1);

In another aspect of the present invention, there is provided the use of a polypeptide having binding affinity to human melanoma antigen A3 protein, for preparing a detection reagent for detecting human melanoma antigen A3 protein, or for preparing a detection reagent for diagnosing human melanoma The application of the diagnostic reagent for antigen A3 protein expression positive tumor, the polypeptide is obtained after 12-20 amino acid changes with the amino acid sequence of Staphylococcus A protein Z segment as the backbone.

In a preferred example, relative to the amino acid sequence of the Z segment of Staphylococcus protein A (SEQ ID NO: 1), the polypeptide with binding affinity to the human melanoma antigen A3 protein is listed in Nos. 9-11, 13-14, Amino acid mutations occurred at positions 17-18, 24-25, 27-28, 32, 35, and 43.

In another preferred example, relative to the amino acid sequence of the Z segment of Staphylococcus protein A, the sequence is shown in SEQ ID NO: 1, and the polypeptide with binding affinity to human melanoma antigen A3 protein is:

The 9th amino acid is mutated to P or T;

The 10th amino acid is mutated to P or L;

The 11th amino acid is mutated to C or I;

The 13th amino acid is mutated to R or L;

The 14th amino acid is mutated to W or F;

The 17th amino acid is mutated to M or S;

The 18th amino acid is mutated to A or F;

The 24th amino acid is mutated to R or P;

The 25th amino acid is mutated to Q or A;

The 27th amino acid is mutated to H or V;

The 28th amino acid is mutated to L or G;

The 32nd amino acid is mutated to P;

The 35th amino acid is mutated to L or G;

Amino acid 43 is mutated to E.

In another preferred embodiment, the amino acid sequence of the polypeptide having binding affinity to human melanoma antigen A3 protein is shown in any of SEQ ID NOs: 2-3.

In another aspect of the present invention, there is provided the use of the polypeptide having binding affinity to the human melanoma antigen A3 protein or the targeting molecule targeting the human melanoma antigen A3 protein,

The conjugate is a drug for inhibiting human melanoma antigen A3 protein-positive tumors, and is used to prepare a drug for treating human melanoma antigen A3-positive tumors;

Or the conjugate is a polypeptide tag or a detectable marker, which is used to prepare a detection reagent for detecting human melanoma antigen A3 protein or a diagnostic reagent for diagnosing a tumor that expresses positive human melanoma antigen A3 protein.

In a preferred example, in the targeting molecule targeting human melanoma antigen A3 protein, the conjugate is an anti-tumor drug (such as a toxin), and the The binding affinity polypeptide or the targeting molecule targeting the human melanoma antigen A3 protein is used to treat the human melanoma antigen A3 protein-positive tumor.

In another preferred embodiment, the human melanoma antigen A3 protein-positive tumors include: lung cancer, breast cancer, hepatocellular carcinoma, osteosarcoma, leukemia, bladder cancer, brain tumor, ovarian tumor, and digestive tract tumors such as gastric cancer , esophageal cancer, colorectal cancer and other cervical cancer, head and neck tumors or external genital tumors, etc.

In another aspect of the present invention, there is provided a pharmaceutical composition, which comprises: the aforementioned polypeptide having binding affinity to human melanoma antigen A3 protein or the aforementioned targeting ability to target human melanoma antigen A3 protein molecule; and a pharmaceutically acceptable carrier.

In another aspect of the present invention, there is provided a kit for diagnosing or treating a human melanoma antigen A3 protein expression-positive tumor, the kit comprising: said human melanoma antigen A3 protein with binding affinity The polypeptide, or the targeting molecule targeting human melanoma antigen A3 protein, or the pharmaceutical composition.

In a preferred embodiment, the polypeptide having binding affinity to human melanoma antigen A3 protein or the targeting molecule targeting human melanoma antigen A3 protein is in an effective amount.

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

The present invention will be further introduced below with reference to the accompanying drawings and specific embodiments of the description.

Description of drawings

Figure 1. Alignment of Z _MAGE-A3 and Zwt sequences. The modified amino acid sites in the Z _MAGE-A3 polypeptide of the present invention have been underlined in the figure (SEQ ID NO: 2-3).

Figure 2. The recombinant plasmid construction diagram of a fusion polypeptide produced in Example 1 (A) and the composition diagram of the amino acid sequence of the full-length protein (B).

Z _MAGE-A3 represents a MAGE-A3 binding domain having any one of the sequences selected from SEQ ID NOs: 2-3, 6xHis represents six histidine tags, HM represents NdeI (CATATG) translated amino acids, and LE represents XhoI (CTCGAG ) translated amino acids.

Figure 3. Electrophoresis of recombinant plasmids of pET21a(+)/affibody.

M: DNA marker; 1-3 are recombinant plasmids of pET21a(+)/Z _MAGE-A3 172, pET21a(+)/Z _MAGE-A3 770, and pET21a(+)/Zwt, respectively.

Figure 4. Identification of prokaryotic expression of MAGE-A3 recombinant protein and analysis of preparation of rabbit serum antibodies

A: SDS-PAGE electrophoresis analysis of prokaryotic expression of MAGE-A3 recombinant protein M Marker, 1. E. coli Rosetta strain transfected with pET21(+) empty vector; 2. E. coli Rosetta transfected with pET21a(+)/MAGE-A3 coli Rosetta strain was not induced; 3-6. After induction of E. coli Rosetta strain transfected with pET21a(+)/MAGE-A3;

B: SDS-PAGE electrophoresis analysis of MAGE-A3 recombinant purified protein M Marker, 1. After purification of MAGE-A3 protein;

C: Western Blot analysis of MAGE-A3 protein: M.Marker, 1. E. coli Rosetta strain transfected with pET21(+) empty vector; 2. E. coli Rosetta strain transfected with pET21a(+)/MAGE-A3 after induction (The primary antibody is mouse His-tag monoclonal antibody);

D: Western Blot analysis of MAGE-A3 protein: M. Marker, 1. E. coli Rosetta strain transfected with pET21(+) empty vector; 2. E. coli Rosetta strain transfected with pET21a(+)/MAGE-A3 after induction (The primary antibody is the prepared rabbit anti-MAGE-A3 serum antibody);

E: The response of rabbit serum antibody after MAGE-A3 protein immunization;

F: Antibody titers of rabbit serum after MAGE-A3 protein immunization.

Figure 5. SDS-PAGE electrophoresis analysis of prokaryotic expression (A) and purification (B) of Affibody recombinant protein

A: M: protein marker; 1-2 are BL21(DE3) strain and BL21(DE3) strain transfected with pET21(+) empty vector, 3-5 are pET21a(+)/Z _MAGE-A3 172, pET21a respectively (+)/Z _MAGE-A3 770 and BL21(DE3) strains transfected with recombinant plasmids of pET21a(+)/Zwt.

B: M: protein marker; 1-3 are Z _MAGE-A3 172, Z _MAGE-A3 770, and Zwt affibody recombinant fusion proteins after purification, respectively.

Figure 6. SPR detection of Z _MAGE-A3 binding peptides on the ProteOn XPR36 instrument

A, B, C, Z _MAGE-A3 172, Z _MAGE-A3 770 and Zwt protein affinity analysis with target protein MAGE-A3, respectively.

Figure 7. RT-PCR detection of MAGE-A3 expression in human melanoma and gastric cancer cell lines.

Figure 8. Identification of the affinity of Z _MAGE-A3 binding polypeptide and MAGE-A3 native protein by cell immunofluorescence co-localization method.

A: Indirect immunofluorescence co-localization detection of Z _MAGE-A3 172-binding polypeptide; B: indirect immunofluorescence co-localization detection of Z _MAGE-A3 770 protein;

Figure 9. SDS-PAGE electrophoresis and fluorescence analysis of Z _MAGE-A3- binding polypeptides labeled with Dylight755.

A: Z _MAGE-A3 172 polypeptide and Z _wt SDS-PAGE electrophoresis analysis; B: Dylight755-labeled Z _MAGE-A3 172 polypeptide and Zwt SDS-PAGE electrophoresis analysis of fluorescence. M: Marker, 1-2. Z _MAGE-A3 172 purified protein labeled with Dylight755; 3. Z _MAGE-A3 172 purified protein labeled with Dylight755; 4. Zwt purified protein labeled with Dylight755.

Figure 10. Imaging analysis of the biodistribution and tumor targeting of the Dylight755-labeled Z _MAGE-A3 172 polypeptide in tumor-bearing nude mice. A: Fluorescence imaging and kidney distribution of Z _MAGE-A3 172 polypeptide labeled with Dylight755 at each time point in healthy nude mice, and B: The ratio of fluorescence signal intensity between kidney and muscle tissue; C: Z _MAGE-A3 172 polypeptide labeled with Dylight755 Fluorescence imaging of tumor tissue at each time point in A-375, SGC-7901, and MGC-803 cell tumor-bearing nude mice, and D: the ratio of fluorescence signal intensity of tumor and muscle tissue.

Figure 11. Imaging analysis of Dylight755-labeled Z _{MAGE -A3} 172 polypeptide in various organs and tumor tissues of tumor-bearing nude mice. Fluorescence distribution in the main tissues and organs of nude mice; A2-C2 is the average fluorescence signal intensity of the corresponding tumor tissue and each organ.

Figure 12. Identification of Z _MAGE-A3 affitoxin protein by SDS-PAGE electrophoresis and WB identification

A: Construction diagram of pET21a(+)/Z _MAGE-A3 affitoxin recombinant plasmid;

B: SDS-PAGE electropherogram of Z _MAGE-A3 affitoxin prokaryotic expression protein. M: Marker; 1, BL21(DE3) strain; 2, BL21(DE3) transformed with pET21a(+) vector; 3-5 are recombinant pET21a(+)/MAGE-A3 affitoxin172, Z _MAGE-A3 affitoxin770, and Zwt affitoxin, respectively Plasmid-transfected BL21(DE3) strain;

C: SDS-PAGE electropherogram of Z _MAGE-A3 affitoxin purified protein. M: Marker; 1-3 are Z _MAGE-A3 affitoxin172, Z _MAGE-A3 affitoxin770 and Zwt affitoxin;

D, E, F: Primary antibodies were His monoclonal antibody, rabbit anti-Zwt polyclonal antibody and mouse anti-PE38KDEL polyclonal antibody, respectively. M: Marker; 1-3: Z _MAGE-A3 affitoxin172, Z _MAGE-A3 affitoxin770, Zwt affitoxin, respectively.

Figure 13. Cell immunofluorescence co-localization identification of Z _MAGE-A3 affitoxin and MAGE-A3 native protein affinity. A: Z _MAGE-A3 affitoxin172 protein; B: Z _MAGE-A3 affitoxin770 protein

Figure 14. The effect of Z _MAGE-A3 affitoxin172 on tumor cell growth

A, B: IC50 analysis of Z _MAGE-A3 affitoxin172 on A-375 and SGC-7910 tumor cells; C, D:

Z _MAGE-A3 affitoxin172 inhibits the growth of A-375 and SGC-7910 tumor cells; E: Z MAGE- _{A3 affitoxin172} inhibits the growth of A-375, SGC-7910 and MGC-803 tumor cells

Figure 15. Antitumor effect of Z _MAGE-A3 affitoxin172 on Balb/c tumor-bearing nude mice

Balb/c nude mice were inoculated with A375 cells (A), SGC7901 (B) and MGC-803 (C), respectively, and when the tumors grew to 100-200 mm3 in size, Z _MAGE-A3 affitoxin172 at a concentration of 4 mg/kg was injected into the tail vein. , Z _MAGE-A3 172 affibody and Zwt affitoxin, PE38KDEL control protein and PBS were injected every 3 days for a total of 10 times, as indicated by the arrows in the figure. After 36 days of observation, the tumor tissues were dissected, and the tumors of each group of tumor-bearing nude mice were photographed, measured and weighed. A1-A3: the tumor volume measured in each experimental group of A375, SGC7901 and MGC-803 tumor-bearing nude mice at each time period; B1-B3: photographed observation after dissection of the three tumors in each experimental group; C1-C3 : Comparison of anatomical tumor mass in each experimental group of three tumors.

Detailed ways

The present invention will be specifically described below through the examples, which are only used to further illustrate the present invention, and should not be construed as limiting the scope of protection of the present invention. Technical engineers in this field can make some non-essential improvements to the present invention according to the content of the above invention. Adjustment.

As used herein, the "polypeptide with binding affinity to MAGE-A3" refers to a polypeptide obtained after 12-20 amino acid variations using the amino acid sequence of the Z segment of Staphylococcus protein A as a backbone, and the polypeptide can be specific Sexually binds MAGE-A3 with little or no nonspecific binding.

As used herein, the "polypeptide of the present invention", "polypeptide having binding affinity to MAGE-A3", "MAGE-A3 binding polypeptide", "Z _MAGE-A3 affibody polypeptide", "Z _MAGE-A3 affibody" , "Z _MAGE-A3 ", "affibody protein", "affibody recombinant protein", "Z _MAGE-A3 recombinant protein" can be used interchangeably; SPAZ and Zwt can be used interchangeably.

As used herein, the "targeting molecule" refers to a molecule that can target MAGE-A3 obtained by linking the polypeptide with binding affinity to MAGE-A3 of the present invention with other functional conjugates. The conjugate can be a cysteine residue, a polypeptide tag, a drug that inhibits human melanoma antigen A3, an enzyme or a detectable marker, and the like.

As used herein, the "fusion polypeptide" is a subordinate concept of the "targeting molecule", which refers to the polypeptide of the present invention with binding affinity to MAGE-A3 and other functional peptides (such as toxin proteins or functional peptides) Molecules that can target MAGE-A3 obtained after ligation.

The inventors selected MAGE-A3 as the target antigen. The inventors used the Z domain (Zwt, SEQ ID NO: 1) of Staphylococcus protein A as a scaffold, and randomly mutated its surface amino acid residues to mimic the antibody binding site, and constructed a mutation library by phage display technology. MAGE- A3 was used as the target antigen for affinity screening of the library. After a lot of screening work, a polypeptide with high affinity for MAGE-A3 was finally obtained.

The polypeptide of the present invention takes the amino acid sequence of the Z domain of Staphylococcus A protein as the backbone, and is obtained by mutating 14-20 (preferably 14) amino acids. As a preferred mode of the present invention, relative to the amino acid sequence of the Z segment of Staphylococcus A protein (SEQ ID NO: 1), the polypeptides of the present invention are at 9-11, 13-14, 17-18, 24-25, 27- Amino acid mutations at positions 28, 32, 35, and 43 occurred. More preferably, the polypeptide of the present invention has the amino acid sequence shown in any one of SEQ ID NOs: 2-3, as shown in Table 1.

The invention also encompasses polypeptides formed by adding additional amino acid residues to either or both ends of the amino acid sequence of the MAGE-A3 binding polypeptide. These additional amino acid residues may play a role in the binding of the polypeptide to MAGE-A3, but may also be used for other purposes, such as one or more involved in the production, purification, stabilization, conjugation or detection of the polypeptide. These additional amino acid residues may include one or more amino acid residues added for chemical coupling purposes. For example, it is added at the first or last position of the polypeptide chain, that is, a cysteine residue is added at the N or C terminus, etc. Such additional amino acid residues may also include a "tag" for polypeptide purification or detection, such as a hexahistidine peptide ( _His6 ) tag that interacts with a labeled antibody, or a "myc" tag or a "flag" tag . In addition, other alternatives well known to those skilled in the art are also included in the present invention.

Said "additional amino acid residues" may also constitute one or more polypeptide domains with desired functions, such as the same binding function as the first, MAGE-A3 binding domain, or other binding functions, or a An enzymatic function, or a fluorescent function, or a combination thereof.

The present invention also includes a polypeptide modified to increase its stability under alkaline conditions based on the MAGE-A3 binding polypeptide. This stabilization involves site-directed substitution of any aspartame residues present in the unmodified sequence with amino acid residues that are less sensitive to basic conditions. Since affinity chromatography columns are subjected to frequent strong base treatments for elution between different reactions, this reduced sensitivity to bases facilitates the use of the polypeptides of the present invention as affinity chromatographers in affinity chromatography. Ligands that extend the life of affinity chromatography matrices.

The present invention also includes polypeptides obtained by other modifications on the basis of the MAGE-A3 binding polypeptides of the present 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 modifications can be accomplished by exposing the polypeptide to enzymes that perform glycosylation, such as mammalian glycosylases or deglycosylases. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize their solubility properties.

The MAGE-A3 binding polypeptide of the present invention can be linked with a conjugate to form a functional targeting molecule, which can be linked or adsorbed through chemical bonds (including peptide bonds); the chemical bonds are covalent bonds or non-covalent bonds. covalent bond. As a preferred way, the fusion polypeptide is formed by linking through peptide bonds. The MAGE-A3 binding polypeptide and the conjugate can be directly connected, or connected through a polypeptide linker (connecting peptide). The linker includes, for example, 1-30 amino acids; preferably 1-20 amino acids. The setting of the linker peptide does not substantially affect the activity of each polypeptide in the fusion protein. Preferably, a flexible peptide (Gly4Ser)3 can be used for attachment. Other linker peptides well known to those skilled in the art can also be used in the present invention.

In a "heterologous" fusion polypeptide, where the MAGE-A3 binding polypeptide constitutes the first domain or first moiety, and the second and other moieties have functions other than binding to MAGE-A3, these are expected to result in Also within the scope of the present invention. The second and other portions of the fusion polypeptide may comprise binding domains with affinity for other target molecules than MAGE-A3. This binding domain may also be related to the SPA domain, but with substitution mutations at 1 to about 20 positions. The result is a fusion polypeptide having at least one MAGE-A3 binding domain and at least one domain with affinity for the other target molecule. This extends the application of the polypeptides of the invention, such as as therapeutic agents or as capture, detection or separation reagents.

Other options for the second and other moieties of the fusion polypeptides of the invention include one or more moieties for therapeutic applications. In therapeutic applications, other molecules can also be covalently or non-covalently coupled to the polypeptides of the invention by other means. As a preferred embodiment of the present invention, the modified Pseudomonas aeruginosa exotoxin PE38KDEL is linked to the C-terminus of the MAGE-A3 binding polypeptide through a flexible peptide to form a fusion protein. Non-limiting examples include enzymes for "ADEPT" (antibody-directed enzyme prodrug therapy) with polypeptide-directed effector enzymes (eg, carboxypeptidases) of the invention; including those used to recruit the immune system Proteins of cells and other components; including cytokines such as IL-2, IFNγ, IL-12, TNFα, IP10; 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, etc. At the same time, in order to more conveniently incorporate radionuclides (such as ⁶⁸ Ga, ⁷⁶ Br, ¹¹¹ In, ⁹⁹ Tc, ¹²⁴ I, ¹²⁵ I) for diagnosis or radionuclides (such as ⁹⁰ Y, ¹³¹ I, ²¹¹ At) for use in diagnosis For therapy, the additional amino acids listed above (especially the hexahistidine tag and cysteine) can be considered for the purpose of coupling radioisotope chelators to the polypeptide sequence.

The present invention also covers connecting a detectable label (such as fluorescent label, biotin or radioisotope) to the MAGE-A3 binding polypeptide, so that the detection and expression of MAGE-A3 can be realized based on the specificity of the polypeptide of the present invention. The purpose of positive tumors.

装置进行检测的一种多肽特性。MAGE-A3结合亲和力可以通过一个实验进行检测，其中将MAGE-A3固定在该装置的感应芯片上，然后将含有待测多肽的样品通过该芯片。或者，也可以将待检测的多肽固定在该装置的感应芯片上，然后将含有MAGE-A3 的样品通过该芯片。本领域技术人员可以利用所获得的感应图像建立多肽的MAGE-A3结合亲和力的至少一种定性测量方法。如果需要定量测量方法，例如为了建立相互作用间的某个KD 值，也可以使用表面等离子共振方法。例如，结合值可以利用

2000装置(Biocore AB) 进行测定。MAGE-A3固定于该装置的感应芯片上，而亲和力待检测的多肽样品通过连续稀释制备并以随机顺序进行注射。然后可从结果中计算KD值。在本发明的实施例中，所述的多肽的KD值达到1.03×10^-5M至3.28×10^-6M。"MAGE-A3 binding affinity" means that the binding affinity can be obtained, for example, by utilizing surface plasmon resonance (surface plasmon resonance) techniques such as

A characteristic of a polypeptide that the device detects. MAGE-A3 binding affinity can be tested by an experiment in which MAGE-A3 is immobilized on a sensor chip of the device and a sample containing the polypeptide to be tested is then passed through the chip. Alternatively, the polypeptide to be detected can also be immobilized on the sensor chip of the device, and then the sample containing MAGE-A3 can be passed through the chip. One skilled in the art can use the obtained sensory images to establish at least one qualitative measure of the MAGE-A3 binding affinity of the polypeptide. Surface plasmon resonance methods can also be used if quantitative measurements are required, eg to establish a certain KD value between interactions. For example, the combined value can be used with

2000 apparatus (Biocore AB). MAGE-A3 was immobilized on the sensor chip of the device, and samples of polypeptides whose affinity was to be tested were prepared by serial dilution and injected in random order. KD values can then be calculated from the results. In the embodiment of the present invention, the KD value of the polypeptide reaches 1.03×10 ^-5 M to 3.28×10 ^-6 M.

The present invention also provides isolated nucleic acids encoding the MAGE-A3 binding polypeptides or targeting molecules or fusion polypeptides of the present invention, and may also be their complementary strands. Said nucleic acid can be synthesized artificially in full sequence, or can be obtained separately by PCR amplification.

The present invention also provides a vector comprising a nucleic acid molecule encoding said nucleic acid molecule. The vector may also contain expression control sequences 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 to" refers to the condition that 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 can be used, such as some vectors for cloning and expression in bacterial, fungal, yeast and mammalian cells, as described in Pouwels et al., Cloning Vectors: A Laboratory Manual.

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

Methods of producing the MAGE-A3 binding polypeptides or targeting molecules or fusion polypeptides of the present invention are also encompassed by the present invention. The method includes culturing recombinant cells containing nucleic acid encoding the corresponding polypeptide to obtain the product polypeptide. The polypeptides prepared above can be purified to a substantially homogeneous nature, eg, as a single band on SDS-PAGE electrophoresis.

Based on the information of the polypeptide to be expressed and the current technical level of recombinant protein expression, combined with the content disclosed in the present invention, those skilled in the art can easily prepare the polypeptide of the present invention. For example, a plasmid expressing the unmodified Z domain can be used as starting material. Using known techniques, desired substitution mutations can be introduced into this plasmid to obtain expression vectors of the present invention.

When chemical polypeptide synthesis methods are used to prepare the polypeptides or targeting molecules or fusion proteins of the present invention, any naturally occurring amino acid residues in the above polypeptides can be replaced by any corresponding, non-naturally occurring amino acid residues or derivatives thereof replaced, as long as the function of the product polypeptide is not substantially impaired.

The present invention also relates to the application of the MAGE-A3 binding polypeptide or targeting molecule or fusion polypeptide in different aspects, including application in therapy, diagnosis and/or detection.

The MAGE-A3 binding polypeptides of the present invention can be used as a surrogate for MAGE-A3 antibodies in various applications.

As a non-limiting example, it can be used to treat diseases characterized by MAGE-A3 expression, such as tumors (eg, melanoma, lung cancer, gastric cancer), and the like. By binding to intracellular MAGE-A3 to inhibit cell signaling, it is used for in vivo and in vitro diagnosis of related diseases. The polypeptide of the present invention can be used as a detection reagent, a capture reagent or a separation reagent, and can also be used directly as a therapeutic agent or a means to target other therapeutic agents to the MAGE-A3 protein. In vitro methods of using the polypeptides of the invention can be performed in various ways, such as microtiter plates, protein arrays, biosensor surfaces, tissue sections, and the like. Modifications and/or additions to the polypeptides of the present invention may be made without departing from the scope of the present invention in order to make them suitable for specific uses.

These modifications and additions are described in detail below, which may include additional amino acids included 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 MAGE-A3 are also encompassed by the present invention.

The MAGE-A3 binding properties of the polypeptides of the present invention and the stability of using the polypeptides to produce targeting molecules (including fusion proteins) and/or labeled binding molecules mean that the polypeptides can also be used to target other active substances to tumor sites, These tumors included cells that expressed MAGE-A3. Accordingly, another aspect of the present invention provides the use of a MAGE-A3 binding polypeptide as described herein in conjugation with a substance having anticancer activity to deliver the substance to cells expressing MAGE-A3 to produce a target Cell damage or apoptosis.

The agent for this anticancer activity may be a protein that is fused or chemically coupled to a MAGE-A3 binding polypeptide, such as an effector enzyme selected for use in "ADEPT" (antibody-directed enzyme prodrug therapy) applications; for recruitment Proteins of effector cells and other components of the immune system; cytokines, such as IL-2, IFNγ, IL-12, TNFαa, IP 10, etc.; procoagulant factors, such as tissue factor, von Willebrand factor, etc.; toxins, such as castor bean Toxin A, Pseudomonas exotoxin, calcheamicin, maytansinoids, etc. Alternatively, the active substance may also be a cytotoxic drug, such as auristatin analogs or doxorubicin or radioisotopes (such as ⁹⁰ Y, ¹³¹ I, ²¹¹ At, etc.), which can bind directly to MAGE-A3 binding polypeptides, Or bind to the MAGE-A3 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 directing a substance having anticancer activity to MAGE-A3 expressing cells in vivo comprising administering to a patient a conjugate of the active substance described herein to a MAGE-A3 binding polypeptide thing. Such conjugates have been appropriately described above.

The present invention also includes the detection of MAGE-A3 protein in a sample using the polypeptide that binds to MAGE-A3.

For example, such assays can be used to diagnose disease conditions characterized by MAGE-A3 expression. Detection of the presence of MAGE-A3 can be performed in vivo or in vitro. The preferred option for in vivo diagnosis is the use of positron emission tomography, PET. The sample to be tested can be, for example, a biological fluid sample or a tissue sample. The current common method is to use an antibody against MAGE-A3, which can be applied to the MAGE-A3-binding polypeptide of the present invention. This method is a histochemical method to detect the presence of MAGE-A3 to identify fresh, Expression of MAGE-A3 protein in frozen or formalin-fixed, paraffin-embedded tissue samples. To detect MAGE-A3,

The polypeptides of the invention can also be used as part of fusion proteins in which the other domains are reporter enzymes or luciferases. Alternatively, it may also be labeled with one or more fluorescent agents and/or radioisotopes, optionally by a chelating agent. Suitable radioisotopes ^include68Ga , ^76Br , ^111In , ^99Tc , ^124I , ^125I , and the like.

The present invention also includes applying the MAGE-A3 binding polypeptide to detect MAGE-A3 in biological liquid samples. This method includes the steps of: (1) providing a biological fluid sample of the patient to be tested, (2) subjecting the MAGE-A3 binding polypeptides described herein under conditions that enable the polypeptides to bind to any MAGE-A3 present in the sample is added to the sample under the following conditions to (3) remove unbound polypeptides, and (4) detect bound polypeptides. The amount of bound polypeptide detected correlates to the amount of MAGE-A3 present in the sample. In step (2), the MAGE-A3 binding polypeptide can be added to the sample in any suitable form, including, for example, when the MAGE-A3 binding polypeptide is immobilized on a solid support, by which the sample is contacted , or the MAGE-A3 binding polypeptide is present in solution.

Other applications of the described MAGE-A3 binding polypeptide also include: a method for detecting MAGE-A3 in a sample, comprising the following steps: (1) providing a tissue sample suspected of containing MAGE-A3, such as frozen section or using formalin forest-embedded tissue sections, (2) adding a MAGE-A3-binding polypeptide of the invention to the sample under suitable conditions that favor binding of the polypeptide to any MAGE-A3 present in the sample, ( 3) Unbound polypeptides are removed, and (4) bound polypeptides are detected. The amount of bound polypeptide detected correlates to the amount of MAGE-A3 present in the sample.

The present invention also provides a kit for diagnosing the expression of MAGE-A3 in tissue samples, comprising the MAGE-A3 binding polypeptide of the present invention fused with a reporter enzyme (such as alkaline phosphatase or horseradish peroxidase), a detection enzyme Active reagents, and positive and negative control tissue sections.

The present invention also provides a kit for diagnosing the expression of MAGE-A3 in a tissue sample, comprising a MAGE-A3 binding polypeptide of the present invention fused to a marker (such as a flag marker or myc marker) detected by an antibody, a marker specific for the marker Antibodies, secondary antibodies specific for primary antibodies and conjugated to reporter enzymes, reagents to detect enzyme activity, and positive and negative control tissue sections.

One area of diagnostic application is the detection of cancer cells or their aggregates in vivo. The present invention provides a kit for performing such a diagnosis, the kit comprising a MAGE-A3 binding polypeptide of the present invention labeled with a chelate, a diagnostic radioisotope (non-limiting examples are ⁶⁸ Ga, ⁷⁶ Br, ¹¹¹ In, ⁹⁹ Tc, ¹²⁴ I and ¹²⁵ I, etc.), and reagents for analyzing incorporation efficiency.

As mentioned above, the present invention encompasses the use of the MAGE-A3 binding polypeptides of the present invention to target active substances to MAGE-A3 expressing cells such as certain types of cancer cells. The present invention also provides a kit for this purpose comprising a MAGE-A3 binding polypeptide of the present invention labeled with a chelate, a therapeutic radioisotope (non-limiting examples are ⁹⁰ Y, ¹³¹ ) I, ²¹¹ At), and reagents for analysis of incorporation efficiency.

The present invention also provides a pharmaceutical composition, comprising: an effective amount of the polypeptide having binding affinity to human melanin antigen A3 protein or a targeting molecule targeting human melanin antigen A3 protein in an effective amount, and a pharmaceutically acceptable accepted vector.

As used herein, a "pharmaceutically acceptable" ingredient is one that is suitable for use in humans and/or mammals without undue adverse side effects (eg, toxicity), ie, has 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 pharmaceutical carriers which are not themselves essential active ingredients and which are not unduly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. A full description of pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991). Pharmaceutically acceptable carriers in the compositions may contain liquids such as water, saline, glycerol 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 composition can be formulated into various dosage forms suitable for mammalian administration, including but not limited to: injections, capsules, tablets, emulsions, and suppositories.

When in use, a safe and effective amount of the polypeptide or targeting molecule with binding affinity to human melanin antigen A3 protein of the present invention is administered to a mammal (such as a human), wherein the safe and effective amount is usually at least about 1 microgram /kg body weight, and in most cases no more than about 10 mg/kg body weight, preferably the dose is from about 1 microgram/kg body weight to about 1 mg/kg body weight. Of course, the specific dosage should also take into account the route of administration, the patient's health and other factors, which are all within the skill of the skilled physician.

The present invention will be further described below in conjunction with specific embodiments.

Example 1. Library construction and screening study of MAGE-A3 binding polypeptides

A random combinatorial library of phage-displayed MAGE-A3 binding polypeptides, ie, a library of many different SPA domain-related polypeptides, was constructed, MAGE-A3 binding polypeptides were screened from the library, and their affinity was identified.

1. Construction and identification of random combinatorial phage display library of MAGE-A3 binding polypeptides

According to the amino acid sequence and structure of wild-type SPA-Z (Nilsson B et al., Protein Eng. 1987; 1(2): 107-113), random primers were designed for the coding sequences corresponding to the three helical regions of SPA-Z, and PCR amplification was used. An SPA coding sequence which can lead to random amino acid mutation was obtained and named SPA-N. According to the conventional method of molecular cloning, the SPA-N coding sequence was cloned into the pCANTAB5E vector through the Sfi I and NotI sites to construct the pCANTAB5E/SPA-N recombinant plasmid, which was transformed into competent E.coli TG1 cells and coated with 2YT-A plates. Incubate overnight at 37°C. That is, the primary library, marked as the primary library of affibody for standby. Twenty-eight monoclonal colonies grown on the plate were randomly selected, and the extracted plasmid was identified as positive clones by double digestion with Sfi I and Not I, and their randomness was sequenced and analyzed.

Results: According to the sequencing results, among the 20 clones sent for sequencing, 16 clones were sequenced, of which 15 clones were successfully connected, and the randomness was completely different, so the recombination rate was 15/20=75%; the diversity was 15/ 15=100%. Take the bacterial liquid cultured after the above transformation, dilute it with 2×YT medium (1:10, 1:10 ² ......), spread the SOB-AG plate, count the number of single colonies on the plate, and calculate the storage capacity. By increasing the number of ligation and transformation times to accumulate capacity, the number of clones reached 2.4×10 ⁶ Z protein variants (affibody molecules) after multiple ligation and transformation. Positions 25, 27, 28, 32, 35 and 43 have random amino acid residues.

2. Screening and titer determination of MAGE-A3 binding peptides

The purified MAGE-A3 protein was coated on a 96-well microtiter plate, blocked, incubated with phage library (primary library), then added E.coli TG1 at 37°C, and incubated with gentle shaking; take 100 μl, use 2*YT medium Do gradient dilution, take 100 μl of the dilution solution and apply it to SOB-AG plate, overnight at 30°C, count the number of infected colonies bound by phage, and calculate the titer of MAGE-A3 bound phage; the results show that colonies are visible on the plate, and the titer is 10 ³ ; After the first round of panning was completed, 10 ¹⁰ of the helper phage M13KO7 and kanamycin were added to another part of the bacterial solution and cultured overnight. After centrifugation, the supernatant was taken and filtered through a 0.22 μm filter to obtain the phage library after MAGE-A3 molecular affinity screening. For the first-level library. Repeat the above 4 rounds of enrichment screening to obtain phage libraries after MAGE-A3 molecular affinity screening respectively, which are secondary, and the titers are 1×10 ⁶ respectively; on the basis of the secondary library, repeat the above 4 rounds of enrichment screening , which is a tertiary library with a titer of 1×10 ⁸ . At the same time, a blank control without phage was set for simultaneous screening.

3. Preparation and ELISA identification of MAGE-A3 binding polypeptide monoclonal phage

ELISA was used to screen phage expressing MAGE-A3 binding affibody molecules. MAGE-A3 protein was coated on a 96-well microtiter plate at 20 μg/well, overnight at 4°C; washed with PBS, blocked with 2% nonfat dry milk for 2 h; washed, and the phage obtained after four rounds of screening and an equal volume of 3% nonfat dry milk were taken Mix well, 200μl/well, 37°C, 2h. Wash, add 1:10000 diluted HRP/anti-M13 enzyme-labeled secondary antibody (rabbit anti-M13, Abcam#ab6188), 200μl/well, 37°C, 1h; wash, add OPD color developing solution 200μl/well, 37°C, 15min; 50μl/well of 2M H2SO4 to stop the reaction; set up a microplate reader (ELx800 ^™ , BIO-TEK, Winooski, USA) to read the OD490 value.

The antigen-binding affibody molecules were selected in four rounds of panning. After these four rounds of selection, the MAGE-A3 binding activity was further analyzed by phage ELISA. The ELISA with an OD490 higher than 0.5 was used as the selection criterion to identify the coding MAGE-A3-binding polypeptide phages, 100 clones above this ELISA signal value were selected, and DNA sequence analysis was performed with another 12 randomly selected clones without ELISA value.

4. Sequence detection and screening of MAGE-A3 affinity molecules

A total of 100 single clones were sent to Shanghai Sangong Company for sequencing, and the sequencing primer was CATATGGTTGACAACAAATT CAACAAAGAA (SEQ ID NO: 8). The sequencing results were analyzed by DNA STAR software to further analyze the randomness and diversity of the three helical regions of the standard sequences SPA-Z and SPA-N. As a result, 68 clones with completely correct sequences were obtained, some of which were completely repeated, and 45 clones with completely correct sequences were obtained after the repeated sequences were merged.

According to the DNA sequencing results, among the 45 correctly sequenced clones, the DNA of the two monoclonal phages with the strongest binding activity to the MAGE-A3 protein (that is, the monoclonal phages presenting the MAGE-A3 affinity molecule) was selected. Sequences (respectively Z _MAGE-A3 172 and Z _MAGE-A3 770) were used as targets for research. The amino acid sequences are SEQ ID NOs: 2 and 3 as shown in Figure 1, and their coding sequences are as SEQ ID NOs: 4 and 5. The next step was for molecular cloning, expression and functional detection of MAGE-A3 binding affibodies.

Example 2. Construction of MAGE-A3-binding polypeptide recombinant plasmid and prokaryotic protein expression and purification

The 2 clones with higher ELISA readings (Z _MAGE-A3 172, Z _MAGE-A3 770 in Figure 1) were selected as before, and Zwt was used as a negative control for the MAGE-A3 binding polypeptide. In order to test the function of the screened affibody molecules, construct recombinant plasmids, express and identify prokaryotic proteins, and prepare purified proteins.

1. Construction and identification of recombinant plasmids of pET21a(+)/affibody

斜体和下划线表示Ned I 酶切位点)，下游引物

斜体和下划线表示XhoⅠ酶切位点)；以筛选的测序正确四级库单克隆affibody Z_MAGE-A3172、 Z_MAGE-A3770为模板，通过PCR扩增affibody目的基因(SEQ ID NO:4、5)，同时将affibody Zwt (SEQ ID NO:1)经原核密码子优化后全序列(SEQ ID NO:6)合成作为阴性对照。将PCR扩增的目的基因经NdeⅠ和XhoⅠ克隆至pET21a(+)载体，构建pET21a(+)/Z_MAGE-A3的重组质粒，并经测序鉴定(图2，图3)。Design PCR primers with reference to the affibody gene sequence (GenBank: GY324633.1), upstream primers

Italics and underlines indicate Ned I restriction sites), downstream primers

Italics and underline indicate XhoI restriction site); using the screened quaternary library monoclonal affibody Z _MAGE-A3 172 and Z _MAGE-A3 770 as templates, the affibody target gene (SEQ ID NO: 4, SEQ ID NO: 4, 5), meanwhile, the full sequence (SEQ ID NO:6) of affibody Zwt (SEQ ID NO:1) after prokaryotic codon optimization was synthesized as a negative control. The target gene amplified by PCR was cloned into pET21a(+) vector by NdeI and XhoI to construct a recombinant plasmid of pET21a(+)/Z _MAGE-A3 , which was identified by sequencing (Figure 2, Figure 3).

2. Prokaryotic protein production

The recombinant plasmid was transformed into Escherichia coli (E.coli) BL21 (DE3), cultured at 37°C for 16h; 0.8mM isopropylthio-β-D-thiogalactopyranoside (IPTG) (Mercke & Co., Ltd.) was added. Company, Germany) IPTG induced and cultured for 6 h to express Z _MAGE-A3 affibody and Zwt affibody proteins with His tag. The recombinant protein expressed after induction was purified by Ni-NTA Agarose (QIAGEN, USA) affinity chromatography and identified by SDS-PAGE analysis. As a result, the pET21a(+)/Z _MAGE-A3 recombinant plasmid was successfully constructed by molecular biology techniques, and the purified Z _MAGE-A3 172, Z _MAGE-A3 770, and Zwt affibody recombinant fusion proteins were prepared by the prokaryotic expression system. It was confirmed by SDS-PAGE electrophoresis analysis (Figure 5) that the molecular mass of the band with Coomassie brilliant blue staining was about 7.8 kDa, which was consistent with the molecular mass of the expected Z _MAGE-A3 affibody polypeptide. The present invention selects the pET21a(+) vector, and in the design, the initiation enzyme site of its multiple cloning site is NdeI (CATATG), and its codon ATG is the amino acid (M) initiation codon of the translation of the target protein, so that The protein expressed by the prokaryotic expression system is the full-length target protein Z _MAGE-A3 without the carrier protein fragment, which avoids the interference of the carrier protein on the experimental results.

Example 3. Binding of Z _MAGE-A3 affibody polypeptide to MAGE-A3 recombinant protein

In order to identify the specificity of Z _MAGE-A3 affibody polypeptide binding to MAGE-A3 protein, the screened Z _MAGE-A3 172, Z _MAGE-A3 770 affibody molecules and their control Zwt affibody and the target protein MAGE were analyzed by surface plasmon resonance (SPR). - Specific binding of A3.

1. Preparation of MAGE-A3 protein

The pET21a(+)/MAGE-A3 recombinant plasmid constructed and preserved in the laboratory was transformed into Escherichia coli BL21(DE3), and the recombinant protein was expressed after induction with IPTG. The protein was purified and prepared by Ni-NTA affinity chromatography, and routinely immunized. Serum antibodies were prepared from Japanese white rabbits. As a result, SDS-PAGE electrophoresis showed that an obvious protein band appeared at a relative molecular mass (Mr) of about 48kDa, which was consistent with the expected size of the protein Mr (Fig. 4A). The recombinant pET32a(+)/MAGE-A3 stored in the laboratory The plasmid was transformed into E. coli BL21 (DE3). After purification by affinity chromatography, a relatively single protein band appeared at the position of Mr 48kDa by SDS-PAGE analysis (Fig. 4B). Western blot analysis was performed with mouse anti-6×His mAb as the primary antibody and prepared rabbit serum antibody, respectively, and a signal reaction band appeared at Mr 48kDa (Fig. 4C, D), indicating that this band was MAGE-A3 protein. The ELISA test showed that a high-titer antibody response appeared after the rabbit MAGE-A3 protein was immunized, indicating that a high-titer specific rabbit serum antibody was successfully prepared (Fig. 4E, F).

2. Sensor analysis of Z _MAGE-A3 polypeptides

The affinity analysis of the interaction between MAGE-A3 protein and Z _MAGE-A3 polypeptide was performed on the ProteOn XPR36 system instrument (Bio-Rad Company), that is, the above-mentioned His-tagged Z _MAGE-A3 was analyzed by surface plasmon resonance (SPR) 172. Interaction between Z _MAGE-A3 770 and Zwt affibody molecules (as a control) and MAGE-A3. According to the operation manual, the MAGE-A3 recombinant protein was immobilized on a different flow cell by coupling to a GLH chip, and the affinity determination with the screening polypeptide was performed. The 6th flow cell surface was activated and deactivated to serve as a blank control for injection. The affibody molecules were diluted in 5 different gradient concentrations to bind to the MAGE-A3 recombinant protein, that is, the concentrations of Z _MAGE-A3 172, Z _MAGE-A3 770 and Zwtaffibody molecules were all 2.0nM, 4.0nM, 8.0nM, 16.0nM, 32.0 nM. All analyses were performed at 25°C at a flow rate of 30 μl/min, with a volume of 200 μl of injected specimens and injected in random order at a flow rate of 30 μl/min, followed by washing with 100 mM HCl (BIO-RAD) for 6 min (dissociation) using ProteOn ManagerTM software (BIO-RAD) 1 : 1 Langmuir binding model analysis of binding curves (sensograms).

The range of results showed that as the concentration of Z _MAGE-A3 affinity molecules increased, its ability to interact with the target protein MAGE-A3 increased. Analyzing the results of repeated detection, the average KD value of the affinity equilibrium dissociation constant was 1.03×10 ^-5 mol/L and 3.28×10 ^-6 mol/L for Z _MAGE-A3 172, Z _MAGE-A3 770 and Zwt affibodies, respectively. and 3.91×10 ^-1 mol/L. Z _MAGE-A3 172 and Z _MAGE-A3 770 protein molecules differ by 10,000 to 100,000-fold in KD values of dissociation constants compared to Zwt affibody molecules. Both Z _MAGE-A3 172 and Z _MAGE-A3 770 obtained through screening can bind to the MAGE-A3 recombinant protein with an affinity reaching the nmol/L level. At the same time, the wild-type Zwt affibody molecule and the MAGE-A3 recombinant protein There is almost no binding force. It shows that the two affibody molecules screened out have high specific affinity with the MAGE-A3 recombinant protein, and the Z _MAGE-A3 172, Z _MAGE-A3 770 affibody molecules and the MAGE-A3 recombinant protein expressed by prokaryotic induction have biological activities.

Therefore, the Z _MAGE-A3 172 and Z _MAGE-A3 770 protein molecules of the present invention and the MAGE-A3 target protein molecules have the ability to bind and recognize each other. The affinity between Z _MAGE-A3 172 and Z _MAGE-A3 770 molecules and MAGE-A3 target proteins was verified from the protein level.

Example 4. Binding of Z _MAGE-A3 affibody polypeptide to cells expressing MAGE-A3 protein

In order to further verify the affinity of the screened Z _MAGE-A3 affibody polypeptide with the MAGE-A3 target protein, tumor cells expressing MAGE-A3 were used as the research objects, namely human melanoma cell line A-375 and human gastric cancer cell line SGC-7901, The MAGE-A3 negative human gastric cancer cell line MGC-803 was used as a control to further verify the binding between the Z _MAGE-A3 affibody molecule and the MAGE-A3 protein molecule.

1. Validation of MAGE-A3 expression in tumor cell lines

A-375 cells, SGC-7901 cells and MGC-803 cells were cultured in RPMI 1640 medium (10% fetal bovine serum, 2.05 mM L-glutamine and 100 IU/ml penicillin and 100 μg/ml streptomycin), respectively. The expression of MAGE-A3 was detected by QRT-PCR, that is, the cells were cultured in an incubator containing 5% CO2 at 37°C for 24 hours, the cultured cells were collected, the total RNA of each cell line was extracted and reverse transcribed, and the reverse transcription products were subjected to PCR The target gene MAGE-A3 was obtained by amplification. Design and synthesize PCR primers: the upstream primer is: 5'-ATGAGCTCATGCCTCT TGAGCAGAGGAG-3', and the downstream primer is: 5'-GAGAGAGGGGGAAGAGTGA-3'. The results of QRT-PCR amplification are shown in Figure 7. It showed that human melanoma A-375 cell line and gastric cancer SGC-7901 cell line were MAGE-A3 positive cell lines, while gastric cancer MGC-803 cell line was MAGE-A3 low or no expression cell line.

2. Cell Immunofluorescence Colocalization Analysis and Identification

Put the sterilized coverslips into a six-well plate, adjust the number of A-375, SGC-7901 and MGC-803 cells that have been cultured for 24 hours to 1×10 ⁶ /well, and incubate them at 37°C with 5% CO2 for 24 hours to a single well. layer cells. Add Z _MAGE-A3 to a final concentration of 50 μg/ml

Affibody polypeptides were cultured in the above-mentioned medium containing 10% FBS, 5% CO2 at 37°C for 2 h, the culture medium was aspirated, and washed with pre-cooled PBS; monolayer cells were fixed with 4% paraformaldehyde for 15 min, washed 3 times with PBST, and added with 0.3% Triton X-100 was punched for 10 min, washed with 1640 medium containing 10% FBS, blocked at 37°C for 1 h, washed; added mouse anti-His monoclonal antibody (ABR, USA, 1:2000) and placed at 37°C for 30 min. MAGE-A3 rabbit serum antibody (1:2000) was added, overnight at 4°C, and after washing, FITC-labeled goat anti-rabbit IgG secondary antibody (Shanghai Lianke Biotechnology Co., China) and dylight 594 goat anti-mouse IgG secondary antibody (Shanghai Lianke Biotechnology Co., Ltd., China) were added. Linktech Biotechnology Company, China), incubated at 37C for 1 h, and washed with PBST. Add 30 μl/well of Hoechst (Lianke Biotechnology, China) to stain nuclei for 5 minutes, wash and blot dry, cover with a cover glass and cover with buffered glycerol, observe and film with a confocal fluorescence microscope (Leica TCS SP2microscope, Germany) (400× ).

The results were observed under a fluorescence microscope (Fig. 8A, B), A-375 cells and SGC-7901 cells treated with Z _MAGE-A3 172 and Z _MAGE-A3 770 proteins were treated with MAGE-A3 rabbit serum antibodies (recognizing cells). After incubation with the expressed natural MAGE-A3 protein), multiple green dot-like or clump-like strong fluorescent signals were seen in the cytoplasm, while the above-mentioned His-tag monoclonal antibody (recognizing the His-tag on the Z _MAGE-A3 protein) was incubated. Multiple red dot-like or clump-like strong fluorescent signals can be seen in the cytoplasm of the cells. The superposition of the two shows that the Z _MAGE-A3 172 and Z _MAGE-A3 770 proteins have the same recognition sites as the MAGE-A3 rabbit serum antibodies. It is a yellow dot-like or clump-like fluorescent signal, but no obvious fluorescent signal is seen in the human gastric cancer cell line MGC-803. It is shown that Z _MAGE-A3 172 and Z _MAGE-A3 770 recombinant proteins can specifically recognize the naturally expressed MAGE-A3 protein in cells, which is consistent with the recognition position of MAGE-A3 rabbit serum antibody.

It can be seen that the screened Z _MAGE-A3 172 and Z _MAGE-A3 770 proteins can specifically recognize the naturally expressed MAGE-A3 protein in cells. A3 has specific binding affinity.

Example 5. Biodistribution and tumor targeting properties of Z _MAGE-A3 affibody polypeptides in tumor-bearing nude mice

In the experiment of this example, Z _MAGE-A3 172 affibody polypeptide was selected as the detection object, and the Z _MAGE -A3 172 affibody polypeptide was labeled with near-infrared fluorescent dye DyLight755 NHS Ester (Thermo Fisher Company, USA, Cat. No. 62278), and injected To study the biodistribution of Z _MAGE-A3 172 affibody polypeptides in mice bearing A-375, SGC-7901 and MGC-803 cells transplanted with tumors, and image the nude mice to study the biodistribution of labeled polypeptides and tumor targets to characteristics.

1. Preparation of Animal Tumor Models

Select 6-7 week old BALB/c-nu mice (purchased from Shanghai Slack Laboratory Animal Co., Ltd., certificate

SCXK (Shanghai) 2012-0002), weighing 15-18g). After the A-375, SGC-7901 and MGC-803 in the logarithmic growth phase were digested with EDTA (trypsin), the cells were pipetted and collected with a cell culture medium containing 10% serum, and centrifuged at 1000 rpm at room temperature for 3 min. The centrifuged cells were resuspended in serum-free culture medium and counted, prepared at 1×10 ⁶ /ml, and 0.2 ml was injected subcutaneously on the back near the right forearm to inoculate nude mice. The mental state, activity, reaction, diet, body weight, appearance and touch of the subcutaneously inoculated area of the mice were observed every 3 days, and the size and diameter of the tumor were measured with an electronic vernier caliper.

The results showed that obvious tumor growth was observed in nude mice subcutaneously inoculated with the above cells, and all 40 nude mice developed tumors. After 2 weeks, the experiment was started when the tumors reached a maximum diameter of about 300-500 ^mm3 .

2. Near-infrared fluorescent dye Dylight755 (Dy755) labeling and identification

Z _MAGE-A3 172 and the control protein Zwt affibody were labeled with Dylight755 according to the instructions and identified. That is, 91 μg DyLight 755NHS-Ester dye was dissolved in 273 μl DMF organic solvent, and then added to the dialyzed Z _MAGE-A3 172 affibody protein (300 μg/ml, 1 ml in total) solution, protected from light, and reacted at 4°C for 1 h. The resulting solution was dialyzed in the dark, and the dialysate (phosphate buffer, pH _7.2-7.4 ) was replaced every half hour. SDS-PAGE electrophoresis was used for identification. Take 1 μg of Dy755-unlabeled Z _MAGE-A3 172 and Dy755-labeled Z _MAGE-A3 172 (Dy755-Z _MAGE-A3 172), dilute to 10 μl with phosphate buffer, and add them to 10 μl protein loading buffer, respectively. Electrophoresis was performed on ice in a dark condition, and the gel was placed in an in vivo imager (CRi Maesro 2.10), the excitation light filter was 671-705nm, and the emission light filter was 750longpass, using 8bit and 2×2 mode, using 730 -950nm wavelength interval 10nm each exposure 5000ms to collect image information, and use Maesro 25 software for image processing and analysis. The identified Dy755-Z _MAGE-A3 172 was dispensed into brown centrifuge tubes and stored at -20°C for later use.

The results showed that the labeled protein Dy755-Z _MAGE-A3 172 was analyzed by SDS-PAGE electrophoresis, and a single staining band appeared at the relative molecular mass of 7.8 kDa (Fig. 9A); after scanning with a small animal in vivo imager, Dylight755 labeled Z A single fluorescent band appeared at the corresponding position of the _MAGE-A3 172 affibody polypeptide, but no band appeared in Z _MAGE-A3 172 unlabeled with Dylight755 dye (Fig. 9B). It showed that the near-infrared fluorescent dye Dylight755 was successfully labeled with Z _MAGE-A3 172affibody polypeptide.

3. Biodistribution and tumor targeting properties of Z _MAGE-A3 172 affibody polypeptide in tumor-bearing nude mice

(1) Biodistribution of Dy755-Z _MAGE-A3 172 in healthy nude mice

In order to analyze the metabolism of Dy755-Z _MAGE-A3 172 in normal nude mice, anesthesia was induced by intraperitoneal injection of 10% chloral hydrate, and 50 μg/100 μL of Dy755-Z _MAGE-A3 172 protein was injected through the tail vein after it entered a state of deep anesthesia. , placed in a small animal in vivo imager (CRi Maesro 2.10) for imaging, and maintained anesthesia with 0.8-1.0 μl/g chloral hydrate to ensure that the mice were in deep anesthesia during the imaging process. Before injection and 5min after injection , 30min, 1h, 2h, 4h, 6h, 8h, 24h, 48h and 72h continuous imaging observation. The excitation light filter for imaging is 671-705nm, and the emission light filter is 750longpass, using 8bit and 2×2 modes, 730-950nm wavelength interval 10nm, and each exposure 5000ms to collect image information, and use Maesro software for image processing and analysis, respectively. Display the background autofluorescence and target fluorescence signals, then measure their fluorescence values, set the background autofluorescence to black and the target fluorescence signal to red, and finally superimpose the two colors. The resulting data were processed with GraphPad software.

The results showed that the largest fluorescence signal appeared in the bilateral kidneys, and the ratio of it to the fluorescence signal at the leg muscles was analyzed, that is, the kidney/normal tissue ratio (K/N ratio=[the background signal of the kidney ROI/the tissue of the normal ROI ( The results showed that Dy755-Z _MAGE-A3 172 was mainly distributed in the kidney, and the fluorescence signal in the kidney gradually increased after injection, and reached a peak at 8 hours, then gradually weakened, and the fluorescence signal completely disappeared at 72 hours It showed that the Dy755-Z _MAGE-A3 172 protein was mainly distributed in the kidneys of normal nude mice, that is, excreted through the kidneys, and was completely excreted within 72 hours (Fig. 10A, B).

(2) Tumor targeting properties of Z _MAGE-A3 172 affibody polypeptide in tumor-bearing nude mice

When the tumor of nude mice grows to 300-500mm ³ , the nude mice are taken out of the SPF barrier system, and anesthesia is induced by intraperitoneal injection of 10% chloral hydrate, and 50 μg of Dy755-Z _MAGE-A3 is injected through the tail vein after they enter a state of deep anesthesia. 172 fluorescent protein, placed in a small animal in vivo imager for imaging, and maintained anesthesia with 0.8-1.0 μl/g chloral hydrate to ensure that the mice were in deep anesthesia during the imaging process. Before injection and 5min, 30min after injection , 1h, 2h, 4h, 6h, 8h, 12h, 24h, 48h and 72h were continuously imaged to observe Figures 11A1-C1. Three nude mice from each group were sacrificed and dissected 10 hours after the injection, and tissues and organs such as tumor tissue, liver, spleen, kidney, and brain were collected and scanned by an imaging system.

As a result, 30 min after the A-375 and SGC-7901 tumor-bearing nude mice were injected with 50 μg of Dy755-Z _MAGE-A3 172 fluorescent protein in the tail vein, obvious fluorescent signal appeared at the tumor site, and the fluorescent signal was the most complete from 1 h to 8 h, and then gradually decreased. , the signal intensity was the most obvious at about 6h and 8h, respectively, corresponding to the size of the tumor. After 12h, the fluorescence imaging of the tumor became significantly smaller, and there was still fluorescence imaging at 24h. No obvious fluorescence signal was detected in the MGC-803 control group during the observation period. After intravenous injection of Dy755-Z _MAGE-A3 172 fluorescent protein in the kidneys, the strongest fluorescent signal was seen from 30 min to 24 h to 48 h, and gradually disappeared to 24 h to 72 h (Fig. 10A, B). Mice were dissected when 50 μg of Dy755-Z _MAGE-A3 172 fluorescent protein was injected into the tail vein for 10 hours, and the important tissues and organs such as tumor, kidney, lung, spleen, liver and muscle of the experimental group and control group were taken for imaging, as shown in Figure 11. The distribution and intensity of fluorescence signals in tumor, kidney, brain, gastrointestinal, lung, muscle and skin tissues were basically consistent with the results of in vivo imaging, and the fluorescence intensity of tumor tissue was significantly different from that of other organs (p< 0.05), while the fluorescence intensity of tumor tissue in MGC-803 tumor-bearing mice was not significantly different from that of other organs (p>0.05). The tumor-bearing mice were in good condition during the observation period, and no obvious toxic reaction was observed.

The results show that the labeled Dy755-Z _MAGE-A3 172 polypeptide has the characteristics of targeting and binding to MAGE-A3 positive tumors without serious toxic and side effects.

Example 6. Protective effect of Z _MAGE-A3 affitoxin protein on MAGE-A3 positive cell tumor-bearing nude mice

In this example, the targeting effect of Z _MAGE-A3 172 was used to carry the active fragment PE38KDEL of the modified Pseudomonas exotoxin A (PEA) with cytotoxic effect as a cytotoxic molecule. The Z _MAGE-A3 /PE38KDEL prokaryotic expression vector was constructed, and the Z _MAGE-A3 /PE38KDEL protein, namely affitoxin 172, was prepared and purified by the prokaryotic expression system, and the specific binding of affitoxin to the target protein MAGE-A3 and the targeting effect were carried out. and validation, and validation of the inhibitory effect of MAGE-A3 expression-positive tumor cells in vivo.

(1) Preparation and identification of Z _MAGE-A3 affitoxin protein

Z _MAGE-A3 172 affibody was selected as the targeting molecule of MAGE-A3, the flexible peptide (Gly4Ser) 3 was used to connect PE38 (KDEL) at its C-terminus, and the C-terminus of the flexible peptide was connected to the prokaryotic codon-optimized PE38 (KDEL). Full sequence (SEQ ID NO: 7), constituting the Z _MAGE-A3 affitoxin molecule. The full sequence DNA of PE38(KDEL) was synthesized and cloned into pET21a(+) vector through EcoRI and XhoI sites to construct pET21a(+)/PE38(KDEL) vector, Z _{MAGE- A3} 172 was cloned by NedI and EcoRI was cloned into the pET21a(+)/PE38(KDEL) vector recombinant plasmid (Fig. 12A) and identified by sequencing. It was further transformed into E. coli BL21(DE3), and the recombinant protein was expressed after induction with IPTG, and the Ni-NTA affinity layer was used. It was purified by analytical method and identified by SDS-PAGE and Western Blot analysis. At the same time, Zwt affitoxin was constructed and prokaryotically expressed as a control protein.

As a result, pET21a(+)/Z _MAGE-A3 affitoxin 172 and pET21a(+)/Zwt affitoxin recombinant plasmids were successfully constructed; the recombinant plasmids were expressed in prokaryotic expression system and purified Z _MAGE-A3 affitoxin172 and Zwtaffitoxin recombinant proteins were obtained, SDS _-PAGE electrophoresis showed that a clear band appeared at the relative molecular mass (Mr) of about 48kDa, which was consistent with the expected theoretical value (Fig. 12B, C); The C-terminus of Z _MAGE-A3 affitoxin) contains a His-tag, so after purification by affinity chromatography, it was further identified by WesternBlot analysis. The results (Figure 12D, E, F) showed that His-tag mouse mAb, rabbit anti-Zwt (SPA-Z) polyclonal antibody and rabbit PE38KDEL polyclonal antibody were used as primary antibodies, and a single band appeared at the molecular weight of about 48kDa, suggesting that Z _MAGE-A3 affitoxin172 recombinant protein could be detected by His-tag, SPA-Z polyclonal antibody and rabbit polyclonal antibody. PE38KDEL serum antibody specifically recognizes and binds. It showed that Z _MAGE-A3 affitoxin 172 and Zwt affitoxin recombinant purified protein were successfully prepared.

(2) Binding properties of Z _MAGE-A3 affitoxin protein to cell-expressed target proteins

In order to verify the binding properties of Z _MAGE-A3 affitoxin 172 protein to MAGE-A3, the cell co-localization immunofluorescence method was used for further verification. A-375 and SGC-7901 tumor cell lines with positive MAGE-A3 expression were selected as target cells, and MGC-803 was selected as negative control cells. The method is the same as in Example 4. Briefly, the number of cells was adjusted and cultured to monolayer cells, Z _MAGE-A3 affibody was added at a final concentration of 50 μg/ml and incubated for 2 h, washed with pre-cooled PBS, fixed, punched, blocked for 1 h, and washed; mouse anti-His was added respectively. Monoclonal antibody (1:2000) was placed at 37°C for 1 h, MAGE-A3 rabbit serum antibody (1:2000) was added, overnight at 4°C, and FITC-labeled goat anti-rabbit IgG secondary antibody and dylight 594 goat anti-mouse IgG secondary antibody were added after washing. Antibody, incubated at 37°C for 1 h, and washed with PBST. The nucleus was stained with Hoechst for 5 minutes, washed, blotted dry, mounted, observed under a confocal fluorescence microscope and filmed.

The results were observed under a fluorescence microscope (Figure 13), A-375 cells and SGC-7901 cells treated with Z _MAGE-A3 affitoxin172 protein, and MAGE-A3 rabbit serum antibody (recognizing the natural MAGE-A3 protein expressed by cells) After incubation, multiple green dot-like or clump-like strong fluorescent signals can be seen in the cytoplasm, while multiple red cytoplasmic signals can be seen in the cytoplasm of cells incubated with His-tag mAb (recognizing the His-tag on Z _{MAGE- A3} affitoxin172 protein). The superimposed results show that the Z _MAGE-A3 affitoxin172 protein and the MAGE-A3 rabbit serum antibody recognize or bind to the same site, which is a yellow dot or agglomerate fluorescent signal. The human gastric cancer cell line MGC-803 showed no obvious fluorescence signal. It was shown that the Z _MAGE-A3 affitoxin172 recombinant protein can specifically recognize the naturally expressed MAGE-A3 protein in cells, which is consistent with the recognition position of the MAGE-A3 rabbit serum antibody.

It was shown that all Z _MAGE-A3 affitoxin172 proteins can specifically recognize the naturally expressed MAGE-A3 protein in cells, and it was verified from the cellular level that it has specific binding affinity with cell-expressed MAGE-A3.

(3) In vitro cell growth inhibition effect of Z _MAGE-A3 affitoxin protein

In order to study the inhibitory effect of Z _MAGE-A3 affitoxin on cell growth in vitro, Z _MAGE-A3 affitoxin172 protein was selected as the research object. Similarly, A-375 and SGC-7901 tumor cell lines with positive MAGE-A3 expression were selected as target cells. The method is the same as in Example 6. That is, the cell suspension was prepared and counted and seeded in a 96-well cell culture plate. After culturing for 24 hours, Z _MAGE-A3 affitoxin172 protein was added to set 0.160 μmol/L, 0.312 μmol/L, 0.625 μmol/L, 1.250 μmol/L, 2.500 μmol/L, respectively. μmol/L, 5.000 μmol/L, 10.000 μmol/L, 20.000 μmol/L concentration groups, and Zwt affibody as the control group, the CCK-8 kit was used to detect the cell viability. At the same time 1h, 3h, 6h, 12h, 24h, 48h and 72h after adding the sample, the cell viability was detected. Three replicate wells were set in each group, and the experiment was repeated three times. Calculate the IC50 value of cell growth and survival and the survival rate of cells in each time period.

The results are shown in Figure 14 (A, B). The IC50 values of A-375 and SGC-7901 cell growth and survival were calculated by GraphPad primer 5.0 software, which were 0.229±0.085 μM and 1.300±0.084 μM, respectively. Figure 14 (C, D, E) shows that Z MAGE- _{A3 affitoxin} 172 has a strong inhibitory effect on the growth of MAGE-A3-positive A-375 and SGC-7901, while MAGE-A3-negative MGC-803 There was no obvious inhibitory effect on cell growth. Figure 14 shows that Z _MAGE-A3 affitoxin172 acts on A-375 and SGC-7901 cells to 72h, and the cell viability is significantly different from that of MGC-803 cells

(p<0.05), and there was also a significant difference (p<0.05) between the survival rates of A-375 and SGC-7901 cells.

The above results show that Z _MAGE-A3 affitoxin 172 has the property of inhibiting the growth of A-375 and SGC-7901 cells, which further verifies that Z _MAGE-A3 affitoxin 172 has in vitro MAGE-A3 targeting binding specificity.

(4) LD50 analysis of Z _MAGE-A3 affitoxin protein

In order to study the acute toxicity of Z _MAGE-A3 affitoxin172 protein, BALB/c female mice were selected as experimental subjects, and the median lethal dose was calculated. Select 4w-year-old BALB/c female mice, 18-20g, and divide them into 8 dose groups, with 4-7 mice in each group, see Table 1. The tail vein of each mouse was administered only once, and 200 μl of the corresponding dose of Z _MAGE-A3 affitoxin172 protein was injected. Observation indicators after administration: appearance, physical signs, body weight and the time of death of mice in each dose group. The observation was continued until 14 days, and the LD50 was calculated by the Graphpad Primer 5.0 method, and the detection was repeated three times. Results The LD50 of Z _MAGE-A3 affitoxin172 protein caused the half death of mice: 16.007±2.899mg/kg.

Table 1. Acute toxic effects of Z _MAGE-A3 affitoxin172 protein

Dosage (mg/kg) 30 25 20 15 10 5 2.5 0 death rate (1) 4/4 4/4 6/7 5/7 4/7 2/7 1/7 0/7 Mortality (2) 5/5 3/5 5/7 4/7 2/7 1/7 0/7 0/7 Mortality (3) 5/5 5/5 4/5 3/5 1/5 1/5 0/5 0/5

(5) Protective effect of Z _MAGE-A3 affitoxin protein

In order to detect the protective effect of Z _MAGE-A3 affitoxin172 protein on the tumorigenic effect of tumor cells, in this example, MAGE-A3 positive human melanoma A-375 and human gastric cancer cells SGC-7901 cells and MAGE-A3 negative expression were used. The tumor-bearing model of BALB/c nude mice was prepared from gastric cancer MGC-803 cells. The specific method was the same as that in Example 5, that is, 4-5 weeks old BALB/c-nu mice were selected, weighing 12-15 g, and raised in the experiment of Wenzhou Medical University. In the animal center barrier system (SPF grade), three tumor cell tumor-bearing nude mice were divided into 5 groups, namely Z _MAGE-A3 affitoxin172, Z _MAGE-A3 172, PE38KDEL, Zwtaffitoxin and PBS control group. 5 to 7 in each group. The above three kinds of cells cultured for 48h to the logarithmic growth phase were prepared into the required number of cells, i.e. 1×10 ⁶ /ml, and 0.1ml was injected subcutaneously on the back of nude mice near the left forearm. 4mg/kg) diluted in 0.1ml of PBS, after the tumor cells in nude mice, when the tumor volume grows to about ^50-100mm3 , 0.1ml of the corresponding protein is injected through the tail vein according to the group, and only 0.1ml of the PBS group is injected PBS. First, each tumor-bearing nude mouse was injected continuously for 3 days (once a day), and then once every other day, for a total of 10 times. The life and mental state of the nude mice were observed every 3 days, the tumor size was measured and photographed and recorded until 36 days. Tumor-bearing mice were sacrificed. The tumor tissue and various important organs of nude mice were taken out, and the tumor tissue was weighed, then soaked in 4% paraformaldehyde and stored at room temperature for use in subsequent experiments.

The results are shown in Figure 15 (A1-A2), Z _MAGE-A3 affitoxin172 protein has a strong protective effect on the tumorigenic effect of MAGE-A3 positive A-375 and SGC-7901 tumor cells. In -7901 tumor-bearing nude mice, the tumor volume of Z _MAGE-A3 affitoxin172 protein group was significantly different from that of each control group on the 12th day (p<0.01). Between groups, the difference became significantly larger until day 36. For MAGE-A3 negative MGC-803 tumor cells, there was no difference between Z _MAGE-A3 affitoxin172, Zwt affitoxin protein and PBS groups (p>0.05) (Fig. 15A3). After the observation, the mice were sacrificed, and the tumor tissue was dissected and separated, photographed (Fig. 15B1-B3) and weighed (Fig. 15C1-C3). The results were consistent with the above-mentioned results of tumor in vivo measurement.

The above results show that Z _MAGE-A3 affitoxin172 protein has the effect of protecting or inhibiting the growth of tumor cells, that is, Z _MAGE-A3 affitoxin172 protein has a tumor suppressing effect on MAGE-A3 targeting. And the side effects are not obvious.

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Claims (12)

1. A polypeptide with binding affinity to human melanoma antigen A3 protein is characterized in that the amino acid sequence of the polypeptide with binding affinity to human melanoma antigen A3 protein is shown as any one of SEQ ID NO 2-3.

2. The polypeptide having binding affinity for human melanoma antigen A3 protein according to claim 1 wherein the polypeptide has a KD of 1.03X 10 for interacting with human melanoma antigen A3 protein^-5M to 3.28X 10^-6M。

3. A targeting molecule for targeting human melanoma antigen a3 protein, said targeting molecule comprising the polypeptide of any one of claims 1-2 and a conjugate linked to said polypeptide, said conjugate comprising: a cysteine residue; a polypeptide tag; substances with anti-cancer activity; or a detectable label comprising a fluorescent label, an enzyme, biotin, or a radioisotope.

4. The targeting molecule of claim 3 which targets the human melanoma antigen A3 protein wherein said conjugate comprises an anti-cancer active agent comprising an effector enzyme for ADEPT applications; proteins for recruiting effector cells of the immune system; toxin: ricin A, pseudomonas exotoxin, calcheamicin, maytansinoids; an auristatin or doxorubicin or a radioisotope.

5. An isolated polynucleotide encoding the polypeptide having binding affinity for human melanoma antigen a3 protein according to any one of claims 1-2.

6. A recombinant vector comprising the polynucleotide of claim 5.

7. A host cell comprising the recombinant vector of claim 6, or comprising or having integrated into its genome the polynucleotide of claim 5.

8. Use of a polypeptide having binding affinity for human melanoma antigen A3 protein, for the preparation of a test agent for the detection of human melanoma antigen A3 protein, or for the preparation of a diagnostic agent for the diagnosis of tumors positive for human melanoma antigen A3 protein expression, said polypeptide comprising the polypeptide having binding affinity for human melanoma antigen A3 protein of any one of claims 1-2.

9. The use of the targeting molecule of claim 3 for targeting human melanoma antigen A3 protein,

the conjugate is an anticancer active drug and is used for preparing a drug for treating positive tumors expressed by human melanoma antigen A3;

or the conjugate is a polypeptide label or a detectable marker, and the detectable marker comprises a fluorescent label, an enzyme, biotin or a radioactive isotope, and is used for preparing a detection reagent for detecting the human melanoma antigen A3 protein or a diagnostic reagent for diagnosing tumors positive for the expression of the human melanoma antigen A3 protein.

10. A pharmaceutical composition, comprising: the polypeptide having binding affinity to human melanoma antigen A3 protein according to any one of claims 1-2 or the targeting molecule targeting human melanoma antigen A3 protein according to claim 3; and a pharmaceutically acceptable carrier.

11. A kit for diagnosing a tumor positive for human melanoma antigen a3 protein expression, comprising: a polypeptide having binding affinity for human melanoma antigen a3 protein according to any one of claims 1-2;

the targeting molecule of claim 3 that targets human melanoma antigen A3 protein, wherein the conjugate is a polypeptide tag or a detectable label comprising a fluorescent label, an enzyme, biotin, or a radioisotope;

or the pharmaceutical composition of claim 10, wherein the conjugate is a polypeptide tag or a detectable label comprising a fluorescent label, an enzyme, biotin, or a radioisotope.

12. A kit for treating a tumor positive for human melanoma antigen a3 protein expression, comprising: the targeting molecule of claim 3, which targets human melanoma antigen A3 protein, wherein the conjugate of the targeting molecule is an anticancer active drug;

or the targeting molecule of claim 4 targeting human melanoma antigen A3 protein;

the pharmaceutical composition of claim 10, wherein the conjugate of the targeting molecule is an anticancer active drug.

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