CN111978379A - Polypeptide with binding affinity to human melanoma antigen A3 protein and application thereof - Google Patents

Polypeptide with binding affinity to human melanoma antigen A3 protein and application thereof Download PDF

Info

Publication number
CN111978379A
CN111978379A CN201910439094.5A CN201910439094A CN111978379A CN 111978379 A CN111978379 A CN 111978379A CN 201910439094 A CN201910439094 A CN 201910439094A CN 111978379 A CN111978379 A CN 111978379A
Authority
CN
China
Prior art keywords
protein
mage
polypeptide
human melanoma
melanoma antigen
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201910439094.5A
Other languages
Chinese (zh)
Other versions
CN111978379B (en
Inventor
张丽芳
朱珊丽
薛向阳
蒋朋飞
陈俊
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Wenzhou Medical University
Original Assignee
Wenzhou Medical University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Wenzhou Medical University filed Critical Wenzhou Medical University
Priority to CN201910439094.5A priority Critical patent/CN111978379B/en
Publication of CN111978379A publication Critical patent/CN111978379A/en
Application granted granted Critical
Publication of CN111978379B publication Critical patent/CN111978379B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6415—Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/5743—Specifically defined cancers of skin, e.g. melanoma
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/305—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F)
    • G01N2333/31—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F) from Staphylococcus (G)

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

Polypeptide with binding affinity to human melanoma antigen A3 protein and application thereof
Technical Field
The invention relates to the field of biological medicines, in particular to a polypeptide with binding affinity to human melanoma antigen A3 protein (MAGE-A3) and application thereof.
Background
Tumors are frequently encountered and commonly seen worldwide, the mortality rate of malignant tumors is the first of all diseases, wherein the morbidity rate and the mortality rate of lung cancer are the first of all diseases worldwide, the gastric cancer is the second of all diseases, and breast cancer is the first factor of female death. At present, the incidence rate of lung cancer in China already shows a tendency of explosive growth, while the incidence rate of stomach cancer per year continuously rises and shows a remarkable trend of youthful appearance. The MAGE gene is firstly found in a melanoma cell line and is a large family consisting of A, B, C, D, E and F six subfamilies, wherein the MAGE-A3 gene is not only expressed in malignant tumor tissues mainly comprising melanoma, but also expressed in various tumors of various tissue types in a large proportion, such as lung cancer, breast cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, osteosarcoma, leukemia, bladder cancer, brain tumor, ovarian tumor and the like, and particularly digestive tract tumors such as gastric cancer, esophagus cancer, colorectal cancer and the like can be highly expressed. MAGE-A3 is not expressed in normal tissue cells except testis and placenta tissues, and autoimmune damage caused by the recognition of MAGE-A3 can be avoided due to the intrinsic physiological barriers of testis and placenta tissues. 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 tumor therapy at present, and mainly uses Epidermal Growth Factor Receptor (EGFR) and tumor angiogenesis as targets for therapy, such as EGFR monoclonal antibody (HER2 monoclonal antibody), small molecule compound tyrosine kinase antagonists (Herceptin, Herceptin and cetuximab, etc.), bevacizumab (rhuMAb-VEGF) and sunitinib, etc., to inhibit tumor cell growth or promote tumor cell apoptosis by specifically blocking signal transduction of tumor cells or blocking tumor angiogenesis through receptor blocking. However, the targeted therapy based on antibody molecules still has the limitations of its application, such as poor permeability, high cost, strong immunogenicity, and serious toxic and side effects. The toxic effects of toxic side effects in particular have been a major obstacle to the development of therapeutic antibodies against cancer, producing liver, kidney and nervous system toxicity that reduces its function. Such as cardiotoxic effects associated with HER 2-targeting antibody trastuzumab (herceptin). Radioimmunotherapy with isotopically labeled antibodies also results in bone marrow suppression and the like.
Based on the above description, there is still a need in the art to develop new drugs or new methods for targeted therapy of tumor diseases positively associated with MAGE-a3 expression, so as to improve the current clinical situation.
Disclosure of Invention
The invention aims to provide a polypeptide with binding affinity to human melanoma antigen A3 protein and application thereof.
In a first aspect of the present invention, there is provided a polypeptide having binding affinity to human melanoma antigen A3 protein, wherein the sequence of the polypeptide is represented by SEQ ID NO:1, relative to the amino acid sequence of staphylococcus protein a Z segment, and the polypeptide having binding affinity to human melanoma antigen A3 protein is:
the 9 th amino acid is mutated into P or T;
the 10 th amino acid is mutated into P or L;
the 11 th amino acid is mutated into C or I;
the 13 th amino acid is mutated into R or L;
the 14 th amino acid is mutated into W or F;
the 17 th amino acid is mutated into M or S;
the 18 th amino acid is mutated into A or F;
the 24 th amino acid is mutated into R or P;
the 25 th amino acid is mutated into Q or A;
the 27 th amino acid is mutated into H or V;
the 28 th amino acid is mutated into L or G;
the 32 th amino acid is mutated into P;
the 35 th amino acid is mutated into L or G;
the 43 th amino acid was mutated to E.
In a preferred embodiment, the amino acid sequence of the polypeptide having binding affinity to human melanoma antigen A3 protein is shown in any one of SEQ ID NO 2-3.
In another preferred embodiment, the polypeptide having binding affinity for human melanoma antigen A3 protein has a KD of 1.03X 10 for interacting with human melanoma antigen A3 protein -5M to 3.28X 10-6M。
In another aspect of the present invention, there is provided a targeting molecule for targeting human melanoma antigen a3 protein, the targeting molecule comprising a polypeptide as described in any one of the above, and a conjugate linked to the polypeptide, the conjugate being preferentially conjugated to the polypeptide, the conjugate including but not limited to: cysteine residues, polypeptide tags, drugs that inhibit human melanoma, or detectable labels including, but not limited to, such as fluorescent labels, enzymes, biotin, or radioisotopes.
In a preferred embodiment, the conjugate is a peptide, and the conjugate and the polypeptide having binding affinity to human melanoma antigen a3 protein form a fusion polypeptide.
In another preferred embodiment, the polypeptide tags include, but are not limited to: his tag, preferably 6 XHis, Myc tag, GST tag, Flag tag.
In another preferred embodiment, the drugs for inhibiting human melanoma include, but are not limited to: a toxin; preferably, the toxin is a toxin having an effect of inhibiting a human melanoma antigen a3 protein positive tumor, such as diphtheria toxin, ricin, pseudomonas aeruginosa exotoxin or a functional fragment of the toxin; and the tumor is positive tumor of human melanoma antigen A3 protein.
In another preferred embodiment, the toxin is pseudomonas aeruginosa exotoxin a, or the functional fragment of the toxin is an active fragment of pseudomonas aeruginosa exotoxin a PE38 KDEL. Preferably, the pseudomonas aeruginosa exotoxin a or functional fragment thereof is linked to the carboxy terminus of the polypeptide having binding affinity for human melanoma antigen a3 protein, preferably to the C terminus of the polypeptide.
In another preferred embodiment, said conjugate is linked to said polypeptide having binding affinity to human melanoma antigen a3 protein as a flexible peptide, 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 human melanoma antigen a3 protein as described in any one of the preceding paragraphs.
In another aspect of the invention, there is provided a polynucleotide encoding said targeting molecule targeting human melanoma antigen a3 protein, 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 present invention, there is provided a method of preparing a polypeptide having binding affinity for human melanoma antigen a3 protein as described in any one of the preceding claims, the method comprising: (1) culturing said cell, thereby expressing said polypeptide having binding affinity for human melanoma antigen a3 protein; (2) and (3) separating and purifying the polypeptide obtained in the step (1).
In another aspect of the present invention, there is provided a use of a polypeptide having a binding affinity to human melanoma antigen A3 protein, for preparing a detection reagent for detecting human melanoma antigen A3 protein, or for preparing a diagnostic reagent for diagnosing a positive tumor expressed by human melanoma antigen A3 protein, wherein the polypeptide is obtained by mutating 12 to 20 amino acids of an amino acid sequence of a Z-segment of staphylococcal protein a as a backbone.
In a preferred embodiment, the polypeptide having binding affinity for human melanoma antigen A3 protein 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-stretch of staphylococcal protein A (SEQ ID NO: 1).
In another preferred embodiment, the sequence of the polypeptide having binding affinity to human melanoma antigen A3 protein is shown as SEQ ID NO:1, relative to the amino acid sequence of Z fragment of staphylococcal protein A:
The 9 th amino acid is mutated into P or T;
the 10 th amino acid is mutated into P or L;
the 11 th amino acid is mutated into C or I;
the 13 th amino acid is mutated into R or L;
the 14 th amino acid is mutated into W or F;
the 17 th amino acid is mutated into M or S;
the 18 th amino acid is mutated into A or F;
the 24 th amino acid is mutated into R or P;
the 25 th amino acid is mutated into Q or A;
the 27 th amino acid is mutated into H or V;
the 28 th amino acid is mutated into L or G;
the 32 th amino acid is mutated into P;
the 35 th amino acid is mutated into L or G;
the 43 th amino acid was 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 one of SEQ ID NO 2-3.
In another aspect of the present invention, there is provided a use of the polypeptide having binding affinity to human melanoma antigen A3 protein or the targeting molecule targeting human melanoma antigen A3 protein,
the conjugate is a medicine for inhibiting positive tumors expressed by human melanoma antigen A3 protein, and is used for preparing a medicine for treating positive tumors expressed by human melanoma antigen A3;
or the conjugate is a polypeptide label or a detectable marker, and is used for preparing a detection reagent for detecting the human melanoma antigen A3 protein or a diagnostic reagent for diagnosing human melanoma antigen A3 protein expression positive tumors.
In a preferred embodiment, in the targeting molecule targeting human melanoma antigen A3 protein, the conjugate is an anti-tumor drug (such as a toxin), and the polypeptide having binding affinity to human melanoma antigen A3 protein or the targeting molecule targeting human melanoma antigen A3 protein is used for treating a tumor positive for expression of human melanoma antigen A3 protein.
In another preferred embodiment, the tumors positive for human melanoma antigen a3 protein expression include: lung cancer, breast cancer, hepatocellular carcinoma, osteosarcoma, leukemia, bladder cancer, brain tumor, ovarian tumor, digestive tract tumor such as gastric cancer, esophageal cancer, colorectal cancer, cervical cancer, head and neck tumor or external genitalia tumor.
In another aspect of the present invention, there is provided a pharmaceutical composition comprising: the polypeptide having binding affinity to human melanoma antigen A3 protein or the targeting molecule targeting human melanoma antigen A3 protein; and a pharmaceutically acceptable carrier.
In another aspect of the present invention, there is provided a kit for diagnosing or treating a tumor positive for human melanoma antigen a3 protein expression, the kit comprising: the polypeptide having binding affinity to human melanoma antigen A3 protein, 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 in view of the disclosure herein.
The invention is further described with reference to the drawings and the detailed description.
Drawings
FIG. 1, each ZMAGE-A3And comparison of Zwt sequences. Z of the inventionMAGE-A3The amino acid sites in the polypeptide that are modified are underlined in the figure (SEQ ID NOS: 2-3))。
FIG. 2, a recombinant plasmid construction map (A) of one fusion polypeptide produced in example 1 and a full-length protein amino acid sequence composition map (B).
ZMAGE-A3Represents a polypeptide having an amino acid sequence selected from SEQ ID NOs: 2-3, 6xHis for a six histidine tag, HM for ndei (catatg) translated amino acids, LE for xhoi (ctcgag) translated amino acids.
FIG. 3 shows the electrophoretogram of the recombinant plasmid pET21a (+)/affibody.
M: DNA marker; 1-3 are respectively pET21a (+)/Z MAGE-A3172、pET21a(+)/Z MAGE-A3770. And pET21a (+)/Zwt.
FIG. 4, MAGE-A3 recombinant protein prokaryotic expression identification and analysis of rabbit serum antibody preparation
A: performing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis on the prokaryotic expression of the MAGE-A3 recombinant protein to analyze an E.coli Rosetta strain transfected by an empty vector pET21 (1. pET 21); coli Rosetta strain transfected with pET21a (+)/MAGE-A3 was not induced; 3-6. after induction of E.coli Rosetta strain transfected with pET21a (+)/MAGE-A3;
b: performing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis on the MAGE-A3 recombinant purified protein to obtain M Marker, and purifying MAGE-A3 protein;
c: western Blot analysis of MAGE-A3 protein: marker, 1.pET21(+) empty vector transfected e.coli Rosetta strain; coli Rosetta strain after induction (primary antibody was murine His-tag mab) transfected with pET21a (+)/MAGE-A3;
d: western Blot analysis of MAGE-A3 protein: marker, 1.pET21(+) empty vector transfected e.coli Rosetta strain; coli Rosetta strain after induction (primary antibody was rabbit anti-MAGE-A3 serum antibody prepared) transfected with pET21a (+)/MAGE-A3;
e, reaction of rabbit serum antibodies after MAGE-A3 protein immunization;
f, the titer of the rabbit serum antibody after MAGE-A3 protein immunization.
FIG. 5 SDS-PAGE electrophoretic analysis of Affinibody recombinant protein prokaryotic expression (A) and purification (B)
A: m: protein marker; 1-2 transfection of BL21(DE3) strain and pET21(+) empty vectorBL21(DE3) strain (3-5) pET21a (+)/Z, respectively MAGE-A3172、pET21a(+)/Z MAGE-A3770 and pET21a (+)/Zwt, and BL21(DE3) strain transfected with the recombinant plasmid.
B: m: protein marker; 1 to 3 are respectively purified Z MAGE-A3172，Z MAGE-A3770, and a Zwt affibody recombinant fusion protein.
FIG. 6, ZMAGE-A3SPR detection of binding Polypeptides on the ProteOn XPR36 Instrument
A. B, C, each is Z MAGE-A3172、Z MAGE-A3770 and Zwt protein to the target protein MAGE-A3.
FIG. 7 and RT-PCR detection of MAGE-A3 expression in human melanoma and gastric cancer cell lines.
FIG. 8, ZMAGE-A3And (3) identifying the cell immunofluorescence co-localization method of the affinity of the binding polypeptide and the MAGE-A3 natural protein.
A：Z MAGE-A3172 indirect immunofluorescence co-localization detection of the bound polypeptide; b: z MAGE-A3770 protein is subjected to indirect immunofluorescence co-localization detection;
FIG. 9, Z marked by Dylight755MAGE-A3And performing SDS-PAGE electrophoresis and fluorescence analysis on the combined polypeptide.
A: is Z MAGE-A3172 polypeptide and ZwtSDS-PAGE electrophoretic analysis; b: z marked by Dylight755 MAGE-A3172 polypeptides and Zwt SDS-PAGE electrophoresis. M Marker, 1-2.Dylight755 unlabeled Z MAGE-A3172 purifying the protein; dylight755 marked Z MAGE-A3172 purifying the protein; 4. purified protein was a Zwt labeled Dylight 755.
FIG. 10, Z marked by Dylight755 MAGE-A3172 in tumor-bearing nude mice, and tumor-targeted imaging analysis. Dylight 755-labeled Z MAGE-A3172 fluorescence imaging and kidney distribution of the polypeptide at each time point in the healthy nude mouse, and B ratio of fluorescence signal intensity of kidney to muscle tissue; c Dylight 755-labeled Z MAGE-A3172 fluorescence imaging of tumor tissues in nude mice loaded with A-375, SGC-7901, and MGC-803 cells at each time point of the polypeptide, and D: tumorTumor to muscle tissue fluorescence signal intensity ratio.
FIG. 11, Z marked by Dylight755 MAGE-A3172 imaging analysis of the polypeptide in each organ and tumor tissue of tumor-bearing nude mice, A1-C1 is Z marked by Dylight755 injectionMAGE-A3After 24 hours, the fluorescence distribution in each main tissue organ of the tumor-loaded nude mouse is carried out after the polypeptide 172; A2-C2 represents the intensity of the mean fluorescence signal of the tumor tissue and organs.
FIG. 12, ZMAGE-A3SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic identification and WB (white-cell-binding) identification of affitoxin protein
A：pET21a(+)/ZMAGE-A3Constructing a diagram of the affitoxin recombinant plasmid;
B：ZMAGE-A3SDS-PAGE electrophoresis chart of affitoxin prokaryotic expression protein. M is Marker; 1, BL21(DE3) strain; 2, BL21(DE3) transformed with pET21a (+) vector; 3-5 are respectively pET21a (+)/MAGE-A3affitoxin172, ZMAGE-A3affitoxin770, and Zwt affitoxin recombinant plasmid transfected BL21(DE3) strain;
C：ZMAGE-A3SDS-PAGE electrophoresis chart of Affitoxin purified protein. M is Marker; 1 to 3 are each Z MAGE-A3affitoxin172,ZMAGE-A3affitoxin770 and Zwt affitoxin;
D. e, F: the primary antibodies are respectively His monoclonal antibody, rabbit anti-Zwt polyclonal antibody and mouse anti-PE 38KDEL polyclonal antibody. M is Marker; 1-3 are respectively ZMAGE-A3affitoxin172，ZMAGE-A3affitoxin770，Zwt affitoxin。
FIG. 13, ZMAGE-A3Cellular immunofluorescence co-localization identification of affinity of affitoxin with MAGE-a3 native protein. A: zMAGE-A3affitoxin172 protein; b: zMAGE-A3Affitoxin770 protein
FIG. 14, ZMAGE-A3Effect of affitoxin172 on tumor cell growth
A、B:ZMAGE-A3IC50 analysis of Affitoxin172 on A-375 and SGC-7910 tumor cells; C. d:
ZMAGE-A3affitoxin172 has an inhibitory effect on the growth of A-375 and SGC-7910 tumor cells; e is ZMAGE- A3Affitoxin172 inhibits the growth of A-375, SGC-7910 and MGC-803 tumor cells
FIG. 15, ZMAGE-A3Effect of affitoxin172 on tumor inhibition of Balb/c tumor-bearing nude mice
Balb/C nude mice are inoculated with A375 cells (A), SGC7901(B) and MGC-803(C) respectively, and are injected with Z with the concentration of 4mg/kg in tail vein when tumors grow to 100-200 mm3MAGE-A3affitoxin172、Z MAGE-A3172 affibody and Zwt affitoxin, PE38KDEL control protein and PBS were injected every 3 days for 10 times as indicated by the arrows. After 36 days of observation, tumor tissue was dissected and tumors from each group of tumor-bearing nude mice were photographed, volumetrically measured and weighed, respectively. A1-A3 shows the tumor volume size measured by each experimental group of nude mice with A375, SGC7901 and MGC-803 at each time period; B1-B3, which is the photograph observation after the three tumors of each experimental group are dissected; C1-C3 anatomical tumor mass comparisons for each experimental group of three tumors.
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 "a polypeptide having binding affinity for MAGE-A3" refers to a polypeptide obtained by 12-20 amino acid mutation using the amino acid sequence of the Z-fragment of staphylococcal protein A as the backbone, and which is capable of specifically binding to MAGE-A3 with little or no non-specific binding.
As used herein, a "polypeptide of the invention", "polypeptide having binding affinity for MAGE-A3", "MAGE-A3 binding polypeptide", "ZMAGE-A3affibody polypeptides "," ZMAGE-A3affibody”、“ZMAGE-A3"," affibody protein "," affibody recombinant protein "," ZMAGE-A3Recombinant protein "may be used interchangeably; SPAZ and Zwt may be used interchangeably.
As used herein, a "targeting molecule" refers to a molecule that targets MAGE-A3, obtained by linking a polypeptide of the invention having binding affinity for MAGE-A3 to another functional conjugate. The conjugate can be cysteine residue, polypeptide label, drug for inhibiting human melanoma antigen A3, enzyme or detectable marker, etc.
As used herein, the term "fusion polypeptide" is a subset of the term "targeting molecule" and refers to a molecule that targets MAGE-A3 and is obtained by linking a polypeptide of the invention that has binding affinity for MAGE-A3 to another functional peptide (e.g., a toxin protein or functional protein fragment).
The inventors selected MAGE-a3 as the target antigen. The inventor takes a Z domain (Zwt, SEQ ID NO:1) of staphylococcal protein A as a scaffold, carries out random mutation on the surface amino acid residue simulation antibody binding site, constructs a mutation library by a phage display technology, carries out affinity screening on the library by taking MAGE-A3 as a target antigen, and finally obtains the polypeptide with high affinity to MAGE-A3 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 an amino acid sequence as shown in any one of SEQ ID NO 2-3, as shown in Table 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 MAGE-a3 binding polypeptide. These additional amino acid residues may function when the polypeptide binds to MAGE-a3, but may also be used for other purposes as well, such as one or more of those relating to the production, purification, stabilization, coupling or detection of the polypeptide. These additional amino acid residues may include one or more amino acid residues added for chemical coupling purposes. Such as the first or last addition of a cysteine residue at the N-or C-terminus of the polypeptide chain. Such additional amino acid residues may also be includedIncluding a "tag" for polypeptide purification or detection, e.g., a hexa-histidine peptide (His) interacting 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 having the desired function, such as the same binding function as the first, MAGE-a3 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 MAGE-a3 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 encompasses polypeptides obtained by other modifications based on the MAGE-A3-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 MAGE-A3 binding polypeptide of the invention can be linked to a conjugate to form a functional targeting molecule, and the linkage can be through chemical bonds (including peptide bonds) or adsorption; 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 MAGE-a3 binding polypeptide may be linked to the conjugate 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.
It is contemplated that in "heterologous" fusion polypeptides, where the MAGE-A3 binding polypeptide constitutes a first domain or first moiety, and the second and other moieties have other functions in addition to binding to MAGE-A3, such results are also within the scope of the invention. The second and further portions of the fusion polypeptide may comprise binding domains having affinity for other target molecules than MAGE-A3. 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 MAGE-A3 binding domain and at least one domain having 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 also be coupled covalently or non-covalently to the polypeptides of the invention by other methods. As a preferred embodiment of the invention, the engineered Pseudomonas aeruginosa exotoxin PE38KDEL is linked to the C-terminus of the MAGE-A3 binding polypeptide by a flexible peptide to form a fusion protein. Non-limiting examples include enzymes that direct effector enzymes (e.g., carboxypeptidase) for "ADEPT" (antibody-mediated enzyme prodrug therapy) using the polypeptides of the invention; proteins including proteins to recruit effector cells and other components of the immune system; including cytokines such as IL-2, IFN γ, IL-12, TNF α, IP 10; including procoagulant factors such as tissue factor, von Willebrand factor;including toxins such as ricin 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 connection of a detectable label (such as a fluorescent label, biotin or a radioactive isotope) to the MAGE-A3 binding polypeptide, so that the aim of detecting MAGE-A3-expressing positive tumors can be fulfilled based on the specificity of the polypeptide.
"MAGE-A3 binding affinity" means that binding affinity can be measured, for example, by using surface plasmon resonance (surface plasmon resonance) techniques such as
Figure RE-GDA0002138646800000072
A polypeptide property detected by the device. The binding affinity of MAGE-A3 can be detected by an assay in which MAGE-A3 is immobilized on a sensor chip of the device and a sample containing the polypeptide to be detected is passed over the chip. Alternatively, the polypeptide to be detected may be immobilised on a sensor chip of the device and the sample containing MAGE-A3 passed through the chip. One skilled in the art can use the sensed images obtained 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 measurement methods are required, for example in order to establish a certain KD value between interactions. For example, the binding value may utilize
Figure RE-GDA0002138646800000071
The assay was performed on a 2000 apparatus (Biocore AB). MAGE-A3 was immobilized on the sensor chip of the device, and the polypeptide sample whose affinity was to be detected was prepared by serial dilution and injected in random order And (4) shooting. KD values can then be calculated from the results. In the embodiment of the invention, the KD value of the polypeptide reaches 1.03X 10-5M to 3.28X 10-6M。
The invention also provides isolated nucleic acids encoding the MAGE-a3 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; for example, E.coli cells (E. coli), such as E.coli HMS174(DE3), or BL21(DE3) can be used. 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 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 the use of the MAGE-a3 binding or targeting molecule or fusion polypeptide in various applications, including therapy, diagnosis and/or detection.
The MAGE-A3 binding polypeptides of the invention can be used as a substitute for MAGE-A3 antibodies in different applications.
As a non-limiting example, it may be used to treat diseases characterized by expression of MAGE-a3, such as tumors (e.g., melanoma, lung cancer, gastric cancer), and the like. By binding to intracellular MAGE-a3 to inhibit cell signaling, for in vivo and in vitro diagnosis of related diseases. The polypeptides of the 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 as a means to target other therapeutic agents to the MAGE-a3 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 which retain the ability to bind MAGE-A3 are also encompassed by the invention.
The MAGE-A3 binding properties of the polypeptides of the invention and the stability of the use of the polypeptides to produce targeting molecules (including fusion proteins) and/or labeled binding molecules means that the polypeptides can also be used to target other active substances to the site of tumors, including cells expressing MAGE-A3. Thus, another aspect of the invention provides the use of a MAGE-A3 binding polypeptide as described herein conjugated to a substance having anti-cancer activity, to deliver the substance to cells expressing MAGE-A3, resulting in damage or apoptosis of the target cell.
Such an anti-cancer active substance may be a protein fused or coupled to a MAGE-a3 binding polypeptide by a chemical bond, such as an effector enzyme selected for "ADEPT" (anti-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 a, IP 10, etc.; procoagulant factors such as tissue factor, von Willebrand factor, and the like; toxins such as ricin A, Pseudomonas exotoxin, calcheamicin, maytansinoids, and the like. Alternatively, the active substance may be a cytotoxic drug, such as an auristatin analogue or doxorubicin or a radioisotope (e.g., as in the case of a drug therapy) 90Y、131I、211At, etc.), such isotopes may be bound directly to a MAGE-A3 binding polypeptide or to a MAGE-A3 binding polypeptide by 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 an agent with anti-cancer activity to cells expressing MAGE-A3 in vivo comprising administering to a patient a conjugate of said agent and a MAGE-A3 binding polypeptide as described herein. Such conjugates have been described appropriately hereinbefore.
The invention also includes the use of the polypeptide that binds to MAGE-A3 for detecting MAGE-A3 protein in a sample.
For example, such assays can be used to diagnose disease conditions characterized by expression of MAGE-A3. Detection of the presence of MAGE-a3 can be performed in vivo or in vitro. A preferred option for in vivo diagnosis is the use of positron emission tomography, PET. The sample to be tested may for example be a biological fluid sample or a tissue sample. The current general approach is to use antibodies against MAGE-A3, which can be adapted to the MAGE-A3 binding polypeptides of the invention, and histochemical methods for detecting the presence of MAGE-A3, which are used to identify expression of MAGE-A3 protein in fresh, frozen or formalin-fixed, paraffin-embedded tissue samples. In order to detect MAGE-a3,
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 includes the use of the MAGE-A3 binding polypeptides in the detection of MAGE-A3 in biological fluid samples. This method comprises the steps of: (1) providing a sample of biological fluid from a patient to be tested, (2) adding a MAGE-A3 binding polypeptide as described herein to the sample under conditions which allow the polypeptide to bind to any MAGE-A3 present in the sample, (3) removing unbound polypeptide, and (4) detecting bound polypeptide. The amount of bound polypeptide detected correlates with the amount of MAGE-a3 present in the sample. In step (2), the MAGE-A3 binding polypeptide may be added to the sample in any suitable form, including, for example, when the MAGE-A3 binding polypeptide is immobilised on a solid support by which the sample is contacted, or the MAGE-A3 binding polypeptide is present in solution.
Other uses of the MAGE-a3 binding polypeptides include: a method for detecting MAGE-a3 in a sample, comprising the steps of: (1) providing a tissue sample suspected of containing MAGE-A3, such as a frozen section or a formalin-embedded tissue section, (2) adding a MAGE-A3 binding polypeptide of the invention to the sample under suitable conditions conducive to binding of the polypeptide to any MAGE-A3 present in the sample, (3) removing unbound polypeptide, and (4) detecting bound polypeptide. The amount of bound polypeptide detected correlates with the amount of MAGE-a3 present in the sample.
The invention also provides a kit for diagnosing the expression of MAGE-A3 in a tissue sample comprising a MAGE-A3 binding polypeptide of the invention fused to a reporter enzyme (e.g., alkaline phosphatase or horseradish peroxidase), reagents for detecting enzyme activity, and positive and negative control tissue sections.
The invention also provides a kit for diagnosing the expression of MAGE-A3 in a tissue sample, comprising a MAGE-A3 binding polypeptide of the invention fused to a marker, such as a flag marker or myc marker, detected by an antibody, a primary antibody specific for the marker, a secondary antibody specific for the primary antibody and coupled to a reporter enzyme, reagents for detecting enzyme activity, and positive and negative control tissue sections.
One area of diagnostic application is the detection of cancer cells or aggregates thereof in vivo. The invention provides a kit for performing such a diagnosis comprising a MAGE-A3 binding polypeptide of the invention labelled with a chelator, a diagnostic radioisotope (a non-limiting example of which is68Ga、76Br、111In、99Tc、124I and125i, etc.), and reagents for assaying incorporation efficiency.
As noted above, the invention encompasses the use of MAGE-A3 binding polypeptides of the invention to target active substances to cells expressing MAGE-A3, such as certain types of cancer cells. The invention also provides a kit for this purpose comprising a MAGE-A3-binding polypeptide of the invention labeled with a chelator, a therapeutic radioisotope (a non-limiting example being 90Y、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 the human melanin antigen A3 protein or the targeting molecule targeting the human melanin antigen A3 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 (Mack pub. 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 human melanin antigen a3 protein is administered to a mammal (e.g., a human), wherein the safe and effective amount is generally at least about 1 microgram/kg of body weight, and in most cases no more than about 10 mg/kg of body weight, preferably the dose is from about 1 microgram/kg of body weight to about 1 mg/kg 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.
Example 1 library construction and screening Studies of MAGE-A3 binding Polypeptides
Random combinatorial libraries of phage display MAGE-A3 binding polypeptides, i.e., libraries of many different SPA domain-associated polypeptides, were constructed, from which MAGE-A3 binding polypeptides were screened and their affinity identified.
1. Construction and characterization of random combinatorial phage display libraries 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 are designed for the corresponding coding sequences of the three helical structural regions, and the SPA coding sequence capable of causing random amino acid mutation is obtained by PCR amplification and named SPA-N. According to the conventional molecular cloning method, the SPA-N coding sequence is cloned to a pCANTAB5E vector through Sfi I and Not I sites to construct a pCANTAB5E/SPA-N recombinant plasmid, the recombinant plasmid is transformed into a competent E.coli TG1 cell, a 2YT-A plate is coated, and the cell is cultured overnight at 37 ℃. Namely the primary library, marked as affibody primary library for standby. Randomly picking 28 single clone colonies growing on the plate, identifying the extracted plasmid as positive clone by Sfi I and Not I double enzyme digestion, sequencing and analyzing the randomness.
As a result: according to the sequencing result, 16 clones with the sequencing result are sent to 20 sequenced clones, wherein 15 clones are successfully connected, the randomness is completely different, and the recombination rate is 15/20-75%; the diversity is 15/15-100%. Taking the above-mentioned bacterial liquid cultured after transformation, diluting with 2 XYT culture solution at a multiple ratio (1:10 )2… …), coating SOB-AG plates, counting the number of single colonies on the plates, and calculating the library capacity. Accumulating the storage capacity by increasing the number of times of connection transformation, and making the number of clones reach 2.4 multiplied by 10 after multiple connection transformation6Z protein variants (affibody molecules) having random amino acid residues at positions 9, 10, 11, 13, 14, 17, 18, 24, 25, 27, 28, 32, 35 and 43.
2. Screening and titer determination of MAGE-A3-binding polypeptides
Coating a 96-well enzyme label plate with the purified MAGE-A3 protein, sealing, adding a phage library (primary library) for incubation, adding E.coli TG 137 ℃, and incubating by gentle shaking; taking 100 ul, diluting with 2 × YT culture medium in gradient proportion, taking 100 ul of the diluted solution, coating an SOB-AG plate, standing overnight at 30 ℃, counting the number of colonies infected by the combined phage, and calculating the titer of the combined phage of MAGE-A3; results visible colonies on the plates, titer 103(ii) a At this time, the first round of elutriation is completed, and 10 is added to the other part of the bacterial liquid 10The helper phage M13KO7 and kanamycin are cultured overnight, after centrifugation, the supernatant is taken and filtered by a 0.22 mu M filter membrane, and the phage library after MAGE-A3 molecular affinity screening is obtained and is a first-level library. Repeating the 4 rounds of enrichment screening to respectively obtain the phage library after MAGE-A3 molecular affinity screening, which is IIGrade, titer 1X 10 respectively6(ii) a Repeating the above 4 rounds of enrichment screening on the basis of the secondary library to obtain a tertiary library with a titer of 1 × 108. Meanwhile, a blank control without phage is set for synchronous screening.
3. Preparation of MAGE-A3-conjugated polypeptide monoclonal phage and ELISA identification
The ELISA was used to screen for phages expressing MAGE-A3 binding to affibody molecules. Coating a 96-well enzyme label plate with MAGE-A3 protein at 20 mu g/well, and standing overnight at 4 ℃; washing with PBS, and sealing with 2% skimmed milk powder for 2 h; washing, mixing phage obtained after four rounds of screening with equal volume of 3% skimmed milk powder, 200 μ l/hole, 37 deg.C, 2 h. Washing, adding HRP/anti-M13 enzyme-labeled secondary antibody (rabbit anti-M13, Abcam # ab6188) diluted at 1:10000, 200 μ l/hole, 37 ℃, 1 h; washing, adding OPD color development liquid 200 μ l/hole, 37 deg.C, 15 min; 2M H2SO 450 μ l/well, terminating the reaction; microplate reader (ELx 800)TMBIO-TEK, Winooski, USA) read the OD490 values.
Antigen-binding affibody molecules were selected in four rounds of panning cycles, through which further phage ELISA assays were performed to analyze their MAGE-A3 binding activity, using ELISA values above 0.5 OD490 as selection criteria, identifying phages encoding MAGE-A3 binding polypeptides, selecting 100 clones above this ELISA signal value, and performing DNA sequence analysis with 12 additional randomly selected clones without ELISA value.
4. Sequence detection and screening of MAGE-A3 affibody molecules
A total of 100 single clones were sent to Shanghai, China for sequencing with a sequencing primer of CATATGGTTGACAACAAATT CA ACAAAGAA (SEQ ID NO: 8). Sequencing results the standard sequences SPA-Z and SPA-N were further analyzed for randomness and diversity in their three helical regions by DNA STAR software analysis. As a result, 68 clones with completely correct sequences were obtained, some of the clones had completely repeated sequences, and 45 clones with completely correct sequences were obtained after the sequences were merged.
Analysis was performed based on DNA sequencing results, and of the 45 clones sequenced correctly as described above, 2 monoclonal phages (i.e., those exhibiting MAGE-A3 affibody molecules) with the strongest binding activity to the MAGE-A3 protein were selectedDNA sequence (Z each) MAGE-A3172、ZMAGE-A3770) The target is researched, the amino acid sequences are SEQ ID NO. 2 and 3 as shown in figure 1, and the coding sequences are SEQ ID NO. 4 and 5. And the subsequent molecular cloning, expression and function detection of MAGE-A3 binding affinity body.
Example 2 construction of MAGE-A3 recombinant plasmid in combination with polypeptide and expression and purification of prokaryotic protein
2 clones with higher ELISA readings (Z in FIG. 1) were selected as beforeMAGE-A3172、ZMAGE-A3770) And Zwt as a negative control for MAGE-A3 binding polypeptide. In order to carry out functional detection on the screened affibody molecules, recombinant plasmid construction, prokaryotic protein expression and identification are carried out on the affibody molecules, and purified proteins are prepared.
Construction and characterization of recombinant plasmid pET21a (+)/affibody
PCR primers were designed with reference to the affibody gene sequence (GenBank: GY324633.1), the upstream primer
Figure RE-GDA0002138646800000111
Figure RE-GDA0002138646800000112
In italics and underlined Ned I cleavage site), downstream primer
Figure RE-GDA0002138646800000113
Italics and underlined are Xho I cleavage sites); monoclonal affibody Z of the correct four-stage library by sequencing of the Screen MAGE-A3172、 Z MAGE-A3770 as a template, amplifying affibody target genes (SEQ ID NO:4, 5) by PCR, and synthesizing the complete sequence (SEQ ID NO:6) of affibody Zwt (SEQ ID NO:1) after prokaryotic codon optimization as a negative control. The PCR-amplified target gene was cloned into pET21a (+) vector by Nde I and Xho I to construct pET21a (+)/Z MAGE-A3The recombinant plasmid of (4), and sequencing identification (FIG. 2, FIG. 3).
2. Prokaryotic protein production
The recombinant plasmid is transformed into Escherichia coli (E.coli) BL21(DE3) and cultured for 16h at 37 ℃; adding 0.8mM isopropylThiosyl-beta-D-thiogalactopyranoside (IPTG) (Merck, Germany) IPTG Induction culture 6h expression of His-tagged ZMAGE-A3affibody and Zwt affibody proteins. The recombinant protein expressed after induction was purified by affinity chromatography using a nickel chelate affinity chromatography colloid (Ni-NTA Agarose) (QIAGEN, USA) and identified by SDS-PAGE analysis. As a result, pET21a (+)/Z was successfully constructed using molecular biology techniquesMAGE-A3Recombinant plasmid and prokaryotic expression system is adopted to prepare purified Z MAGE-A3172、Z MAGE-A3770. And Zwt affibody recombinant fusion protein, which was confirmed by SDS-PAGE electrophoretic analysis (FIG. 5) to show a Coomassie brilliant blue-stained band having a molecular mass of about 7.8kDa, corresponding to the expected ZMAGE-A3The affibody polypeptides are of uniform molecular mass and size. The invention selects pET21a (+) vector, the starting enzyme site of its multiple cloning site is NdeI (CATATG) in design, its codon ATG is the amino acid (M) starting codon for target protein translation, thus the protein expressed by prokaryotic expression system is the full-length target protein Z MAGE-A3And the carrier protein fragment is not contained, so that the interference of the carrier protein on the experimental result is avoided.
Example 3, ZMAGE-A3Binding of affibody polypeptides to MAGE-A3 recombinant proteins
To identify ZMAGE-A3Specificity of binding of affibody polypeptides to MAGE-A3 protein, analysis of the screened Z by Surface Plasmon Resonance (SPR) technique MAGE-A3172、ZMAGE-A3770affibody molecules and their control Zwt affibody bind specifically to the target protein MAGE-A3.
Preparation of MAGE-A3 protein
The pET21a (+)/MAGE-A3 recombinant plasmid constructed and stored in a laboratory is transformed into escherichia coli BL21(DE3), recombinant protein is expressed after IPTG induction, the protein is prepared by purification of Ni-NTA affinity chromatography, and serum antibody is prepared by immunizing Japanese big ear white rabbits conventionally. As a result, SDS-PAGE showed that a distinct protein band appeared at a position of about 48kDa in relative molecular mass (Mr), corresponding to the expected size of the protein Mr (FIG. 4A), E.coli BL21(DE3) was transformed with the laboratory-stored pET32a (+)/MAGE-A3 recombinant plasmid. After purification on an affinity column, a single protein band at the Mr 48kDa position was observed by SDS-PAGE (FIG. 4B). Western blot analysis with mouse anti-6 XHis mAb and prepared rabbit serum antibody shows that the signal reaction band appears at Mr 48kDa (FIG. 4C, D), indicating that the band is MAGE-A3 protein. ELISA detection shows that high-titer antibody reaction occurs after rabbit MAGE-A3 protein immunization, which indicates that high-titer specific rabbit serum antibody is successfully prepared (FIG. 4E, F).
2.ZMAGE-A3Sensor analysis of polypeptides
MAGE-A3 protein and Z were carried out in a ProteOn XPR36 system instrument (Bio-Rad Co., Ltd.)MAGE-A3Affinity analysis of the interaction between the polypeptides, i.e., analysis of the His-tagged Z by Surface Plasmon Resonance (SPR)MAGE-A3172、Z MAGE-A3770 and Zwt affibody molecules (as controls) with MAGE-A3. The affinity assay for the screened polypeptides was performed by immobilizing the MAGE-a3 recombinant protein by coupling to a GLH chip on different flow cells according to the operating manual. The 6 th flow cell surface was activated and deactivated to serve as a blank at the time of injection. Affibody molecules were separately diluted at 5 different gradient concentrations to bind to MAGE-A3 recombinant protein, i.e.ZMAGE-A3172、Z MAGE-A3770 and Zwt affibody molecules were each present at 2.0nM, 4.0nM, 8.0nM, 16.0nM, 32.0 nM. All analyses were performed at 25 ℃ with a flow rate of 30. mu.l/min, a volume of injected specimens of 200. mu.l, and random sequential injections at a flow rate of 30. mu.l/min, followed by a wash with 100mM HCl (BIO-RAD) for 6min (dissociation), using a 1: the 1 langmuir binding model analyzes the binding curve (sensorgram).
Result range with ZMAGE-A3The concentration of the affibody molecule increases, and the capability of interacting with the target protein MAGE-A3 is enhanced. Analysis of the repeated results, the mean value of the dissociation constant KD of the affinity equilibrium, Z MAGE-A3172、Z MAGE-A3770 and Zwt affibody molecules are 1.03X 10, respectively-5mol/L、3.28×10-6mol/L and 3.91X 10-1mol/L。ZMAGE-A3172 and Z MAGE-A3770 protein molecule compared to Zwt affibody molecule dissociation constantKD values differ by a factor of 10000 to 100000. Z obtained by screeningMAGE-A3172、Z MAGE-A3770 were able to bind to the MAGE-A3 recombinant protein with an affinity of the order of nmol/L, while the wild-type Zwt affibody molecule and the MAGE-A3 recombinant protein had little binding. Shows that the screened 2 affibody molecules have higher specific affinity with MAGE-A3 recombinant protein, and simultaneously shows that Z expressed by prokaryotic induction MAGE-A3172、ZMAGE-A3Both the 770affibody molecule and the MAGE-A3 recombinant protein are biologically active.
Thus, Z of the invention MAGE-A3172、ZMAGE-A3The 770 protein molecule and the MAGE-A3 target protein molecule have the capability of mutually combining and recognizing. Verification of Z from the protein level MAGE-A3172、Z MAGE-A3770 affinity between the molecule and the MAGE-a3 target protein.
Example 4, ZMAGE-A3Binding of affibody polypeptides to cells expressing MAGE-A3 protein
Z screened for further validationMAGE-A3The affinity of affibody polypeptides to the MAGE-A3 target protein was further verified by using tumor cells expressing MAGE-A3 as the study subjects, namely human melanoma cell line A-375 and human gastric cancer cell line SGC-7901, and MAGE-A3 expression negative human gastric cancer cell line MGC-803 as a control MAGE-A3Binding between affibody molecules and MAGE-a3 protein molecules.
1. Verification of tumor cell line MAGE-A3 expression
A-375 cells, SGC-7901 cells and MGC-803 cells were cultured in RPMI 1640 medium (10% fetal bovine serum, 2.05mM L-glutamine and 100IU/ml penicillin and 100. mu.g/ml streptomycin), respectively. Carrying out MAGE-A3 expression detection by QRT-PCR, namely culturing cells in an incubator containing 5% C02 at 37 ℃ for 24h, collecting the cultured cells, respectively extracting total RNA of each cell line, carrying out reverse transcription, and carrying out PCR amplification on a reverse transcription product to obtain a target gene MAGE-A3. Design and synthesis of PCR primers: the upstream primer is as follows: 5'-ATGAGCTCATGCCTCT TGAGCAGAGGAG-3', the downstream primer is: 5'-GAGA GAGGGGGAAGAGTGA-3' are provided. The QRT-PCR amplification results are shown in FIG. 7. The results show that the human melanoma A-375 cell strain and the gastric cancer SGC-7901 cell strain are MAGE-A3 positive expression cell strains, and the gastric cancer MGC-803 cell strain is MAGE-A3 low expression or non-expression cell strains.
2. Cellular immunofluorescence co-localization analysis and identification
The sterilized coverslip was placed in a six-well plate, and the number of A-375, SGC-7901 and MGC-803 cells cultured for 24 hours was adjusted to 1X 106Perwell, incubate at 5% CO 237 ℃ for 24h to monolayer cells. Adding a final concentration of 50. mu.g/ml Z MAGE-A3
Culturing affibody polypeptide in the culture medium containing 10% FBS at 5% CO 237 deg.C for 2h, sucking out the culture solution, and washing with precooled PBS; fixing monolayer cells with 4% paraformaldehyde for 15min, washing with PBST for 3 times, adding 0.3% Triton X-100, perforating for 10min, adding 1640 culture medium containing 10% FBS, sealing at 37 deg.C for 1 hr, and washing; the antibody was incubated at 37 ℃ for 30min with a mouse anti-His monoclonal antibody (ABR, 1:2000) at 37 ℃ and a MAGE-A3 rabbit serum antibody (1:2000) overnight at 4 ℃ after washing, followed by FITC-labeled goat anti-rabbit IgG secondary antibody (Shanghai Unico Biotech, China) and dylight 594 goat anti-mouse IgG secondary antibody (Shanghai Unico Biotech, China) for 1h at 37 ℃ and PBST washing. Add Hoechst (Unico Biotechnology Co., Ltd., China) 30. mu.l/well for staining for 5 minutes, wash, blot, cover slide and seal with buffered glycerol, observe with confocal fluorescence microscope (Leica TCS SP2microscope Germany) and photograph (400X).
The results were observed under a fluorescent microscope (FIGS. 8A, B), over Z MAGE-A3172、Z MAGE-A3770 protein treated A-375 cells and SGC-7901 cells, which are incubated by MAGE-A3 rabbit serum antibody (natural MAGE-A3 protein expressed by recognition cells), a plurality of green punctate or lump strong fluorescence signals can be seen in cytoplasm, and the cells are mixed with His-tag monoclonal antibody (recognition Z-tag monoclonal antibody) MAGE-A3His tag on protein) a plurality of red spot-like or lump-like strong fluorescence signals were observed in the cytoplasm of the cells incubated with the His tag, and the result of the superposition of the two signals showed that Z isMAGE-A3172、ZMAGE-A3The 770 protein is consistent with the MAGE-A3 rabbit serum antibody recognition site, and shows yellow punctiform or lump fluorescent signals, while no obvious fluorescent signal is seen in the human gastric cancer cell strain MGC-803. Shows that Z isMAGE-A3172、Z MAGE-A3770 recombinant protein can specifically recognize the naturally expressed MAGE-A3 protein in cells, and is consistent with the recognition position of MAGE-A3 rabbit serum antibody.
Thus, the Z thus selected was found to beMAGE-A3172、Z MAGE-A3770 protein can specifically recognize MAGE-A3 protein naturally expressed in cells, and the affinity of the screened Affinibody protein with the specific binding of MAGE-A3 expressed by the cells is verified from the cellular level.
Example 5, ZMAGE-A3Biodistribution and tumor targeting properties of affibody polypeptides in tumor-bearing nude mice
In the experiment of this example, Z was selectedMAGE-A3172 affibody polypeptide as an object of detection, Z-labeled with near infrared fluorescent dye Dylight755 NHS Ester (Thermo Fisher Co., U.S. A., Cat. No. 62278)MAGE-A3172 affibody polypeptides, and injected into mice bearing A-375, SGC-7901 and MGC-803 cell-transplanted tumors, and subjected to Z MAGE-A3172 affibody polypeptide biodistribution studies and imaging localization of nude mice to study biodistribution and tumor targeting properties of the marker polypeptides.
1. Preparation of animal tumor model
Selecting 6-7 week old BALB/c-nu mice (purchased from Shanghai Slek laboratory animals, Inc., Certification
SCXK (Shanghai) 2012-0002) with a weight of 15-18 g). Digesting A-375, SGC-7901 and MGC-803 which are cultured to logarithmic growth phase and have good growth state by EDTA (pancreatin), blowing and collecting by using a cell culture solution containing 10% of serum, centrifuging at normal temperature of 1000rpm for 3min, resuspending the centrifugal cells by using a culture solution containing no serum, counting, and preparing into 1 × 1060.2ml of the solution was injected subcutaneously into the back near the right front arm to inoculate nude mice. The mental state, physical activity, reaction, diet, body weight and appearance and touch of the subcutaneous inoculation area of the mice were observed every 3 days, and the tumor size and diameter were measured with an electronic vernier caliper.
The results show that the subcutaneous inoculation of the cells in the nude mice can show obvious tumor growth, and 40 nude mice can fully generate tumors. After 2 weeks, the maximum tumor diameter reached about 300-3The experiment was started.
2. Near infrared fluorescent dye Dylight755(Dy755) marking and identifying
According to the specification step pair Z MAGE-A3172 and the control protein Zwt affibody, Dylight755 was labeled and identified. That is, 91. mu.g of Dylight755 NHS-Ester dye was dissolved in 273. mu.l of DMF organic solvent, and dialyzed Z was added MAGE-A3172 affibody protein (300 mu g/ml, 1ml) solution is reacted for 1h under the condition of keeping out of the sun and 4 ℃, the reacted solution is dialyzed in the sun, the dialyzate (phosphate buffer solution, pH7.2-7.4) is replaced every half hour, and Dy755-Z is collected after 2hMAGE-A3172 fluorescent protein, the concentration of the sampled protein is 100 mug/ml, and the protein is identified by SDS-PAGE electrophoresis. Respectively taking Z not marked by Dy755 MAGE-A3172 and Dy 755-labeled ZMAGE-A3172(Dy755-ZMAGE-A3172)1 mu g, diluting to 10 mu l by phosphate buffer solution, respectively adding into 10 mu l of protein loading buffer, carrying out electrophoresis under the condition of ice and avoiding light, placing the gel into a living body imager (CRi Maesro 2.10), wherein the excitation light filter is 671-. Identified Dy755-Z MAGE-A3172 min of the suspension is filled into a brown centrifuge tube, and the suspension is stored at the temperature of minus 20 ℃ for standby.
The result shows that the marker protein Dy755-Z MAGE-A3172, a single stained band at a relative molecular mass of 7.8 kDa, as analyzed by SDS-PAGE (FIG. 9A); scanned by a small animal living body imager, and Z is marked by Dylight755MAGE-A3A single fluorescent band appeared in the corresponding position of the 172 affibody polypeptides, but the Dylight755 dye did not mark Z MAGE-A3172 no banding occurs (fig. 9B). Indicating that the near-infrared fluorescent dye Dylight755 marks ZMAGE-A3The 172 affibody polypeptides were successful.
3.ZMAGE-A3Biodistribution and tumor targeting properties of 172 affibody polypeptides in tumor-bearing nude mice
(1)Dy755-Z MAGE-A3172 biodistribution in healthy nude mice
For analysis of Dy755-Z MAGE-A3172 metabolizing in normal nude mice, using 10% chloral hydrate to induce anesthesia by intraperitoneal injection, and after the mice enter a deep anesthesia state, injecting 50 mu g/100 mu L Dy755-Z by tail vein MAGE-A3172 protein, which is placed in a small animal living body imaging instrument (CRi Maesro 2.10) for imaging, and 0.8-1.0 μ l/g chloral hydrate is used for maintaining the anesthesia state so as to ensure that the mice are under deep anesthesia in the imaging process, and the continuous imaging observation is carried out for 5min, 30min, 1h, 2h, 4h, 6h, 8h, 24h, 48h and 72h before and after the injection. The imaging excitation light filter is 671-705nm, the emission light filter is 750 longpass, 8bit and 2 x 2 modes are adopted, image information is collected at the wavelength interval of 730-950nm by exposure of 5000ms at 10nm each time, image processing and analysis are carried out by Maesro software, background autofluorescence and target fluorescence signals are respectively displayed, then the fluorescence value is measured, the background autofluorescence is set to be black, the target fluorescence signal is set to be red, and finally the two colors are superposed. The data obtained were processed using GraphPad software.
As a result, the maximal fluorescence signal appeared in the bilateral kidneys, and the ratio of the fluorescence signal to that of the muscle in the leg was analyzed, i.e., the kidney/normal tissue ratio (K/N ratio) (background signal of kidney ROI/tissue (muscle) background signal of normal ROI)]}. The results show that Dy755-Z MAGE-A3172 are predominantly distributed in the kidney, and the fluorescence signal of the kidney increases gradually after injection and peaks at 8 hours, then decreases gradually, and disappears completely at 72 hours. It shows that Dy755-Z MAGE-A3172 protein was mainly distributed in the kidney of normal nude mice, i.e., excreted transrenally, and was completely excreted within 72 hours (fig. 10A, B).
(2)ZMAGE-A3Tumor targeting properties of 172 affibody polypeptides in tumor-bearing nude mice
The tumor of the nude mouse grows to 300-3Taking the nude mouse out of SPF barrier system, performing intraperitoneal injection induced anesthesia with 10% chloral hydrate, and performing tail vein injection with 50 μ g Dy755-Z after the nude mouse enters deep anesthesia state MAGE-A3172 fluorescent protein, imaging in a small animal living body imaging instrument, and maintaining the anesthesia state by using 0.8-1.0 mul/g chloral hydrate to ensure that the mouse is in the imaging processDeep anesthesia, with sequential imaging of 5min, 30min, 1h, 2h, 4h, 6h, 8h, 12h, 24h, 48h and 72h before and after injection, observed in fig. 11a 1-C1. Three nude mice in each group were sacrificed and dissected 10h after injection, and tissues and organs such as tumor tissue, liver, spleen, kidney, and brain were taken and scanned in an imaging system.
As a result, 50. mu.g Dy755-Z was injected into tail vein of A-375 and SGC-7901 tumor-bearing nude miceMAGE-A3After the fluorescent protein 172 is used for 30min, an obvious fluorescent signal appears at a tumor part, the fluorescent signal is complete within 1h-8h and then gradually reduced until the signal intensity is obvious about 6h and 8h respectively, the signal intensity corresponds to the size of the tumor, the fluorescent imaging of the tumor is obviously reduced after 12h, and the fluorescent imaging still exists after 24 h. No significant fluorescent signal was detected during the observation period of the MGC-803 control group. Dy755-Z of kidney after intravenous injectionMAGE-A3The strongest fluorescence signal can be seen from 30min after the fluorescence protein 172 for 24-48 h, and disappears gradually from 24h to 72h (FIG. 10A, B). 50 mu g of Dy755-Z is injected into tail veinMAGE-A3The mouse was dissected at 10h for 172 fluorescent protein, and important tissues and organs such as tumor, kidney, lung, spleen, liver and muscle were imaged in the experimental group and the control group, as shown in FIG. 11, the distribution and intensity of the fluorescence signals in the tissues such as tumor, kidney, brain, gastrointestinal tract, lung, muscle and skin were substantially identical to the results of in vivo imaging, and the fluorescence intensity of the tumor tissue was significantly different from that of other organs (p is the fluorescence intensity of the tumor tissue)<0.05), but the fluorescence intensity of the tumor tissue of the MGC-803 tumor-bearing mouse has no significant difference with the fluorescence intensity of other organs (p) >0.05). The tumor-bearing mice had good status during observation period, and no obvious toxic reaction was observed.
The result shows that the marker Dy755-Z MAGE-A3172 polypeptide has the characteristic of targeting and combining MAGE-A3 expression positive tumor, and has no serious toxic and side effect.
Example 6, ZMAGE-A3Protection effect of afutoxin protein on MAGE-A3 positive cell tumor-bearing nude mice
In the present embodiment, Z is utilizedMAGE-A3172, carrying the activity of a modified cytotoxic exotoxin A (PEA) of Pseudomonas aeruginosaFragment PE38KDEL as cytotoxic molecule. Construction of ZMAGE-A3The Z is prepared and purified by a prokaryotic expression system and a prokaryotic expression vector of/PE 38KDELMAGE-A3the/PE 38KDEL protein, namely affitoxin 172, carries out and verifies the specific binding and targeting effect of affitoxin and a target protein MAGE-A3, and verifies the inhibiting effect of tumor cells expressing positive MAGE-A3 in vivo.
(1)ZMAGE-A3Preparation and identification of affitoxin proteins
Selection of Z MAGE-A3172 affibody as the targeting molecule of MAGE-A3, using flexible peptide (Gly4Ser)3 to connect PE38(KDEL) at its C-terminal, and connecting the C-terminal of the flexible peptide to the complete sequence (SEQ ID NO:7) of PE38(KDEL) optimized by prokaryotic codon, to form ZMAGE-A3Affitoxin molecules. The complete sequence DNA of PE38(KDEL) is synthesized and cloned to pET21a (+) vector through EcoRI and XhoI sites to construct pET21a (+)/PE38(KDEL) vector, and Z is cloned by molecular cloning method MAGE- A3172 was cloned into pET21a (+)/PE38(KDEL) vector recombinant plasmid (FIG. 12A) by NedI and EcoRI and sequenced and identified, further transformed into Escherichia coli BL21(DE3), induced by IPTG to express recombinant protein, purified by Ni-NTA affinity chromatography and identified by SDS-PAGE and Western Blot analysis. And constructing and prokaryotic expressing Zwt affitoxin as a control protein.
As a result, pET21a (+)/Z was successfully constructedMAGE-A3Affitoxin172 and pET21a (+)/Zwt Affitoxin recombinant plasmids; the recombinant plasmid is expressed in a prokaryotic expression system and purified Z is obtainedMAGE-A3 Affiborin 172 and Zwt Affiborin recombinant proteins, SDS-PAGE showed a clear band at a position of about 48kDa relative to the molecular mass (Mr), consistent with the expected theoretical size (FIG. 12B, C); due to the recombination of pET21a (+)/ZMAGE-A3the/PE 38KDEL protein (i.e. Z)MAGE-A3affitoxin) contains His tag at C-terminal, so after purification by affinity chromatography column, further identification by Western Blot analysis, the result (FIG. 12D, E, F) shows that single bands appear at the molecular mass of about 48kDa of His-tag murine monoclonal antibody, rabbit anti-Zwt (SPA-Z) polyclonal antibody and rabbit PE38KDEL polyclonal antibody as primary antibodies, suggesting that ZMAGE-A3Affitoxin172 recombinant proteins can be usedHis-tag, SPA-Z polyclonal antibody and rabbit PE38KDEL serum antibody are specifically identified and combined. Shows that Z is MAGE-A3Affitoxin172 and Zwt affitoxin recombinant purified protein are successfully prepared.
(2)ZMAGE-A3Binding characteristics of affitoxin protein and cell expression target protein
To verify ZMAGE-A3The binding property of afutoxin 172 protein and MAGE-A3 is further verified by adopting a cell co-localization immunofluorescence method. Tumor cell strains A-375 and SGC-7901 with positive MAGE-A3 expression are selected as target cells, and MGC-803 is selected as negative control cells. The procedure is as in example 4. Briefly, cells were cultured in adjusted numbers to monolayer cells and a final concentration of 50. mu.g/ml Z was addedMAGE-A3Incubating affibody for 2h, washing with precooled PBS, fixing, perforating, sealing for 1h, and washing; adding mouse anti-His monoclonal antibody (1:2000) and placing at 37 ℃ for 1h, adding MAGE-A3 rabbit serum antibody (1:2000), standing overnight at 4 ℃, washing, adding FITC labeled goat anti-rabbit IgG secondary antibody and dylight 594 goat anti-mouse IgG secondary antibody, incubating at 37 ℃ for 1h, and washing with PBST. Adding Hoechst to stain the nucleus for 5 minutes, washing, sucking, sealing, observing by a confocal fluorescence microscope, and shooting.
The results were observed under a fluorescent microscope (FIG. 13), over ZMAGE-A3A-375 cells and SGC-7901 cells treated by Affitoxin172 protein can see a plurality of green punctate or blocky strong fluorescence signals in cytoplasm after being incubated with MAGE-A3 rabbit serum antibody (recognizing natural MAGE-A3 protein expressed by cells), and can be combined with His-tag monoclonal antibody (recognizing Z-tag monoclonal antibody) MAGE- A3His tag on affitoxin172 protein) multiple red punctate or clumpy strong fluorescent signals were visible in the cytoplasm of the incubated cells, and the results of the two superimposed showed that ZMAGE-A3The affitoxin172 protein is consistent with the MAGE-A3 rabbit serum antibody recognition or binding site, and is represented as a yellow punctiform or lump fluorescent signal, while no obvious fluorescent signal is seen in the human gastric cancer cell strain MGC-803. Shows that Z isMAGE-A3The Affitoxin172 recombinant protein can specifically recognize the naturally expressed MAGE-A3 protein in cells and is consistent with the recognition position of MAGE-A3 rabbit serum antibody.
Shows that Z isMAGE-A3Affitoxin172 protein homospecificThe MAGE-A3 protein naturally expressed in the cells was identified and its affinity for specific binding to MAGE-A3 expressed by the cells was verified at the cellular level.
(3)ZMAGE-A3In vitro cell growth inhibition of affitoxin protein
To study ZMAGE-A3Affitoxin in vitro cell growth inhibition, selection of ZMAGE-A3affitoxin172 protein was the subject of study. Tumor cell lines A-375 and SGC-7901 which express MAGE-A3 positive are also selected as target cells. The procedure is as in example 6. Namely preparing cell suspension, counting and inoculating the cell suspension to a 96-well cell culture plate, culturing for 24h, and adding ZMAGE-A3Affitoxin172 protein, sets concentration groups of 0.160 mu mol/L, 0.312 mu mol/L, 0.625 mu mol/L, 1.250 mu mol/L, 2.500 mu mol/L, 5.000 mu mol/L, 10.000 mu mol/L and 20.000 mu mol/L respectively, and uses Zwt affibody as a control group and adopts CCK-8 kit to detect the survival rate of cells. And the survival rate of the cells is detected at 1h, 3h, 6h, 12h, 24h, 48h and 72h after sample addition. Each group was set with 3 replicate wells and 3 replicate experiments were performed. The IC50 values for cell growth survival and the survival rate of the cells at each time period were calculated.
The results are shown in FIG. 14(A, B), and the IC50 values of cell growth survival of A-375 and SGC-7901 were calculated by GraphPad primer 5.0 software as 0.229. + -. 0.085. mu.M and 1.300. + -. 0.084. mu.M, respectively. FIG. 14(C, D, E) shows that ZMAGE- A3Affitoxin172 has a strong inhibitory effect on the growth of A-375 and SGC-7901 with positive expression of MAGE-A3, and has no obvious inhibitory effect on the growth of MGC-803 with negative expression of MAGE-A3. FIG. 14 shows that ZMAGE-A3Affitoxin172 acts on A-375 and SGC-7901 cells for 72h, and the cell survival rates of the Affitoxin172 are remarkably different from those of MGC-803 cells
(p <0.05), and the comparison between the survival rates of A-375 and SGC-7901 cells has a significant difference (p < 0.05).
The above results show that ZMAGE-A3Affitoxin172 has the characteristic of inhibiting the growth of cells A-375 and SGC-7901, and further verifies that ZMAGE-A3Affitoxin172 has in vitro MAGE-A3 targeted binding specificity.
(4)ZMAGE-A3LD50 analysis of median lethal dose of affitoxin proteins
To study ZMAGE-A3acute toxicity of Affitoxin172 protein, BALB/c female mouse was selected as the experimental subject, and half of the lethal dose was calculated. 4 w-old BALB/c female mice, 18-20g, were selected and divided into 8 dose groups of 4-7 mice each, see Table 1. Each mouse was given only once via the tail vein and 200. mu.l of the corresponding dose of Z was injected MAGE-A3affitoxin172 protein. Observation indexes after administration are as follows: appearance, signs, body weight and death time of mice in each dose group were observed for 14 days and LD50 was calculated by Graphpad Primer 5.0 method and the test was repeated three times. Results ZMAGE-A3LD50, which is responsible for half of the deaths of mice by the affitoxin172 protein, is: 16.007 + -2.899 mg/kg.
TABLE 1.ZMAGE-A3acute toxic effect of affitoxin172 protein
Dosage (mg/kg) 30 25 20 15 10 5 2.5 0
Mortality rate (1) 4/4 4/4 6/7 5/7 4/7 2/7 1/7 0/7
Mortality rate (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)ZMAGE-A3Protective action of affitoxin proteins
To detect ZMAGE-A3The present example, which uses MAGE-A3 positive expression human melanoma A-375 and human gastric cancer cell SGC-7901 cell and MAGE-A3 negative expression human gastric cancer MGC-803 cell to prepare BALB/c nude mouse tumor-bearing modelThe concrete method is the same as example 5, i.e. selecting BALB/c-nu mice of 4-5 weeks old, weighing 12-15g, feeding in center barrier system (SPF grade) of experimental animals of Wenzhou university of medical science, and dividing the three tumor cell tumor-bearing nude mice into 5 groups, i.e. ZMAGE-A3affitoxin172、Z MAGE-A3172. PE38KDEL, Zwt affitoxin and PBS control group. 5 to 7 pieces per group. The three cells cultured for 48h to logarithmic phase are prepared into the required number of cells, namely 1x1060.1ml is taken to be injected into the back of the nude mouse near the left forearm in a subcutaneous way, meanwhile, each histone (4 mg/kg) is diluted into 0.1ml of PBS, and after tumor cells of the nude mouse, the tumor volume is 50-100 mm 3On the left and right, the corresponding protein was injected in an amount of 0.1ml through the tail vein, and only 0.1ml of PBS was injected in the PBS group. Each tumor-bearing nude mouse was injected first for 3 consecutive days (1 time per day), followed by 1 injection every 1 day for 10 times. The nude mice were observed every 3 days for life, mental state, etc., and the tumor size was measured and recorded by photographing for 36 days. Tumor-bearing mice were sacrificed. The tumor tissue and each important organ of the nude mouse are taken out, weighed, soaked in 4% paraformaldehyde and stored at room temperature for subsequent experiments.
The results are shown in FIG. 15(A1-A2), ZMAGE-A3The affitoxin172 protein has strong protective effect on the tumorigenic effect of MAGE-A3 positive A-375 and SGC-7901 tumor cells, and Z is in A-375 and SGC-7901 tumor-bearing nude miceMAGE-A3The volume size of affitoxin172 proteome tumor is significantly different from that of each control group at day 12 (p)<0.01), the tumor volume size varied significantly between groups over time, up to 36 days. And for MAGE-A3 negative MGC-803 tumor cells, ZMAGE-A3No differences between the affitoxin172, Zwt affitoxin proteins and PBS groups (p)>0.05) (fig. 15a 3). Mice were sacrificed at the end of the observation, tumor tissues were dissected and photographed (FIG. 15B1-B3) and weighed (FIG. 15C1-C3), and the results were consistent with the results of the in vivo volume measurements of the tumors described above.
The above results show that ZMAGE-A3Affitoxin172 protein has the function of protecting or inhibiting the growth of tumor cells, namely ZMAGE-A3affitoxin172The protein has targeted tumor inhibition effect on MAGE-A3. And has no obvious toxic and side effects.
Sequence listing
<110> Wenzhou university of medical science
<120> a polypeptide having binding affinity to human melanoma antigen A3 protein and use thereof
<130> 12
<160> 10
<170> PatentIn version 3.5
<210> 1
<211> 58
<212> PRT
<213> Staphylococcus aureus
<220>
<221> MISC_FEATURE
<222> (1)..(58)
<400> 1
Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile
1 5 10 15
Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln
20 25 30
Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala
35 40 45
Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys
50 55
<210> 2
<211> 58
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (1)..(58)
<400> 2
Val Asp Asn Lys Phe Asn Lys Glu Pro Pro Cys Ala Arg Trp Glu Ile
1 5 10 15
Met Ala Leu Pro Asn Leu Asn Arg Gln Gln His Leu Ala Phe Ile Gln
20 25 30
Ser Leu Leu Asp Asp Pro Ser Gln Ser Ala Glu Leu Leu Ala Glu Ala
35 40 45
Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys
50 55
<210> 3
<211> 58
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (1)..(58)
<400> 3
Val Asp Asn Lys Phe Asn Lys Glu Thr Leu Ile Ala Leu Phe Glu Ile
1 5 10 15
Ser Phe Leu Pro Asn Leu Asn Pro Ala Gln Val Gly Ala Phe Ile Pro
20 25 30
Ser Leu Gly Asp Asp Pro Ser Gln Ser Ala Glu Leu Leu Ala Glu Ala
35 40 45
Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys
50 55
<210> 4
<211> 174
<212> DNA
<213> Artificial sequence
<220>
<221> misc_feature
<222> (1)..(174)
<400> 4
gttgacaaca aattcaacaa agaacccccg tgcgctaggt gggaaatcat ggcgctgccg 60
aacctgaacc gacaacagca cctcgctttc atccaatctc tgctcgacga cccgtctcag 120
tctgctgagc tcctggctga agctaaaaaa ctgaacgacg ctcaggctcc gaaa 174
<210> 5
<211> 174
<212> DNA
<213> Artificial sequence
<220>
<221> misc_feature
<222> (1)..(174)
<400> 5
gttgacaaca aattcaacaa agaaacgttg atcgctctct tcgaaatctc gttcctgccg 60
aacctgaacc ccgcccaggt tggagctttc atcccctctc tgggggacga cccgtctcag 120
tctgctgagc tcctggctga agctaaaaaa ctgaacgacg ctcaggctcc gaaa 174
<210> 6
<211> 174
<212> DNA
<213> Artificial sequence
<220>
<221> misc_feature
<222> (1)..(174)
<400> 6
gttgacaaca aattcaacaa agaacagcag aacgctttct acgaaatcct gcacctgccg 60
aacctgaacg aagaacagcg taacgctttc atccagtctc tgaaagacga cccgtctcag 120
tctgctaacc tgctggctga agctaaaaaa ctgaacgacg ctcaggctcc gaaa 174
<210> 7
<211> 1029
<212> DNA
<213> Polynucleotide
<220>
<221> misc_feature
<222> (1)..(1029)
<400> 7
ggtggttctc tggctgctct gaccgctcac caggcttgcc acctgccgct ggaaaccttc 60
acccgtcacc gtcagccgcg tggttgggaa cagctggaac agtgcggtta cccggttcag 120
cgtctggttg ctctgtacct ggctgctcgt ctgtcttgga accaggttga ccaggttatc 180
cgtaacgctc tggcttctcc gggttctggt ggtgacctgg gtgaagctat ccgtgaacag 240
ccggaacagg ctcgtctggc tctgaccctg gctgctgctg aatctgaacg tttcgttcgt 300
cagggtaccg gtaacgacga agctggtgct gctggtccgg ctgactctgg tgacgctctg 360
ctggaacgta actacccgac cggtgctgaa tttctgggta acggtggtga cgtttctttc 420
tctacccgtg gtacccagaa ctggaccgtt gaacgtctgc tgcaggctca ccgtcagctg 480
gaagaacgtg gttacgtttt cgttggttac cacggtacct tcctggaagc tgctcagtct 540
atcgttttcg gtggtgttcg tgctcgttct caggacctgg acgctatctg gaggggcttc 600
tacatcgctg gcgacccgga actggcttac ggttacgctc aggaccagga accggacgcg 660
cgtggtaggt tctgcaacgg tgcgctcctg cgtgtctacg tcccgcgttg gaacctgccg 720
ggtttctacc gtaccggtct gaccctggct gctccggaag ctgctggtga agttgaacgt 780
ctgatcggtc acccgctgcc gctgcgtctg gacgctatca ccggtccgga agaagaaggt 840
ggtcgtctgg aaaccatcct gggttggccg ctggctgaac gtaccgttgt tatcccgtct 900
gctatcccga ccgacccgcg taacgttggt ggtgacctgg acccgtcttc tatcccggac 960
aaagaacagg ctatctctgc tctgccggac tacgcttctc agccgggtaa accgccgaaa 1020
gacgaactg 1029
<210> 8
<211> 30
<212> DNA
<213> Artificial sequence
<220>
<221> misc_feature
<222> (1)..(30)
<400> 8
catatggttg acaacaaatt caacaaagaa 30
<210> 9
<211> 38
<212> DNA
<213> Artificial sequence
<220>
<221> misc_feature
<222> (1)..(38)
<400> 9
gggaattcca tatggttgac aacaaattca acaaagaa 38
<210> 10
<211> 28
<212> DNA
<213> Artificial sequence
<220>
<221> misc_feature
<222> (1)..(28)
<400> 10
ccggaattcc gtttcggagc ctgagcgt 28

Claims (14)

1. A polypeptide having binding affinity to human melanoma antigen A3 protein, wherein the sequence is shown in SEQ ID NO 1 relative to the amino acid sequence of staphylococcal protein A Z segment, and the polypeptide:
the 9 th amino acid is mutated into P or T;
the 10 th amino acid is mutated into P or L;
the 11 th amino acid is mutated into C or I;
the 13 th amino acid is mutated into R or L;
the 14 th amino acid is mutated into W or F;
the 17 th amino acid is mutated into M or S;
the 18 th amino acid is mutated into A or F;
the 24 th amino acid is mutated into R or P;
the 25 th amino acid is mutated into Q or A;
the 27 th amino acid is mutated into H or V;
The 28 th amino acid is mutated into L or G;
the 32 th amino acid is mutated into P;
the 35 th amino acid is mutated into L or G;
the 43 th amino acid was mutated to E.
2. The polypeptide having binding affinity for human melanoma antigen A3 protein according to claim 1, wherein the amino acid sequence of the polypeptide having binding affinity for human melanoma antigen A3 protein is as set forth in any one of SEQ ID NOs 2-3.
3. The polypeptide having binding affinity for human melanoma antigen A3 protein according to claim 1 or claim 2 wherein the polypeptide has a KD of 1.03X 10 for interaction with human melanoma antigen A3 protein-5M to 3.28X 10-6M。
4. A targeting molecule for targeting human melanoma antigen a3 protein, said targeting molecule comprising the polypeptide of any one of claims 1-3 and a conjugate linked to said polypeptide, said conjugate comprising: cysteine residue, polypeptide label, medicine for inhibiting human melanoma antigen A3 protein expression positive tumor, or detectable marker.
5. The targeting molecule of claim 4 for targeting human melanoma antigen A3 protein, wherein said drug for inhibiting a tumor positive for human melanoma antigen A3 protein expression comprises: a toxin; the toxin is toxin with tumor inhibiting effect, such as diphtheria toxin, ricin, pseudomonas exotoxin or functional fragment of the toxin; and the tumor is a tumor with positive expression of human melanoma antigen A3 protein.
6. An isolated polynucleotide encoding the polypeptide having binding affinity for human melanoma antigen a3 protein according to any one of claims 1-3.
7. A recombinant vector comprising the polynucleotide of claim 6.
8. A host cell comprising the recombinant vector of claim 7, or comprising or having integrated into its genome the polynucleotide of claim 6.
9. An application of a polypeptide with binding affinity to human melanoma antigen A3 protein is characterized in that the polypeptide is used for preparing a detection reagent for detecting the human melanoma antigen A3 protein or a diagnostic reagent for diagnosing positive tumors expressed by the human melanoma antigen A3 protein, the polypeptide takes an amino acid sequence of a staphylococcus A protein Z segment as a framework, the sequence of the amino acid sequence is shown as SEQ ID NO:1, and the polypeptide is obtained after 12-20 amino acid variations are carried out.
10. The use of the polypeptide having binding affinity for human melanoma antigen A3 protein according to claim 9, wherein the polypeptide having binding affinity for human melanoma antigen A3 protein 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 staphylococcal protein A Z segment as shown in SEQ ID NO. 1.
11. The use of a polypeptide having binding affinity for human melanoma antigen A3 protein according to claim 10, wherein the polypeptide comprises the polypeptide having binding affinity for human melanoma antigen A3 protein according to any one of claims 1-3.
12. The use of the targeting molecule of the human melanoma antigen A3 protein targeted according to claim 4 or 5, wherein the conjugate is a medicament for inhibiting the expression of human melanoma antigen A3 protein positive tumors, and is used for preparing a medicament for treating human melanoma antigen A3 expression positive tumors;
or the conjugate is a polypeptide label or a detectable marker, and is used for preparing a detection reagent for detecting the human melanoma antigen A3 protein or a diagnostic reagent for diagnosing human melanoma antigen A3 protein expression positive tumors.
13. A pharmaceutical composition, comprising: a polypeptide having binding affinity to human melanoma antigen A3 protein according to any one of claims 1-3 or a targeting molecule targeting human melanoma antigen A3 protein according to any one of claims 4 or 5; and a pharmaceutically acceptable carrier.
14. A kit for diagnosing or treating a tumor positive for human melanoma antigen A3 protein expression, said kit comprising: the polypeptide having binding affinity to human melanoma antigen A3 protein according to any one of claims 1-3, or the targeting molecule for human melanoma antigen A3 protein according to any one of claims 4 or 5, or the pharmaceutical composition according to claim 13.
CN201910439094.5A 2019-05-24 2019-05-24 Polypeptide with binding affinity to human melanoma antigen A3 protein and application thereof Active CN111978379B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910439094.5A CN111978379B (en) 2019-05-24 2019-05-24 Polypeptide with binding affinity to human melanoma antigen A3 protein and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910439094.5A CN111978379B (en) 2019-05-24 2019-05-24 Polypeptide with binding affinity to human melanoma antigen A3 protein and application thereof

Publications (2)

Publication Number Publication Date
CN111978379A true CN111978379A (en) 2020-11-24
CN111978379B CN111978379B (en) 2022-05-03

Family

ID=73437533

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910439094.5A Active CN111978379B (en) 2019-05-24 2019-05-24 Polypeptide with binding affinity to human melanoma antigen A3 protein and application thereof

Country Status (1)

Country Link
CN (1) CN111978379B (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115260290A (en) * 2022-06-23 2022-11-01 浙江大学 Melanoma-targeted polypeptide and application thereof

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1351063A (en) * 2000-10-26 2002-05-29 上海博德基因开发有限公司 Polypeptide-human melanoma antigen gene expression associated protein 15.18 and polynucleotide for coding it
US20030049723A1 (en) * 2001-05-18 2003-03-13 Yi Zhang MAGE-A3 peptides presented by HLA class II molecules
WO2012054825A1 (en) * 2010-10-22 2012-04-26 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Anti-mage-a3 t cell receptors and related materials and methods of use
CN102863515A (en) * 2011-07-05 2013-01-09 温州医学院 B-cell epitope of human melanoma antigen MAGE-A3 and application thereof
CN105820219A (en) * 2016-01-27 2016-08-03 温州医科大学 Polypeptide with binding affinity for HPV18 E7 protein and application thereof
CN105859846A (en) * 2015-01-20 2016-08-17 温州医科大学 Polypeptide having binding affinity to HPV16 E7 and application thereof

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1351063A (en) * 2000-10-26 2002-05-29 上海博德基因开发有限公司 Polypeptide-human melanoma antigen gene expression associated protein 15.18 and polynucleotide for coding it
US20030049723A1 (en) * 2001-05-18 2003-03-13 Yi Zhang MAGE-A3 peptides presented by HLA class II molecules
WO2012054825A1 (en) * 2010-10-22 2012-04-26 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Anti-mage-a3 t cell receptors and related materials and methods of use
CN102863515A (en) * 2011-07-05 2013-01-09 温州医学院 B-cell epitope of human melanoma antigen MAGE-A3 and application thereof
CN105859846A (en) * 2015-01-20 2016-08-17 温州医科大学 Polypeptide having binding affinity to HPV16 E7 and application thereof
CN105820219A (en) * 2016-01-27 2016-08-03 温州医科大学 Polypeptide with binding affinity for HPV18 E7 protein and application thereof

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CHEN XIE等: "Melanoma associated antigen (MAGE)-A3 promotes cell proliferation and chemotherapeutic drug resistance in gastric cancer", 《CELL ONCOL.》 *
FREDRIK Y FREJD等: "Affibody molecules as engineered protein drugs", 《EXPERIMENTAL & MOLECULAR MEDICINE》 *
LI ZHANG等: "HER2-targeting recombinant protein with truncated Pseudomonas Exotoxin: A translocation domain efficiently kills breast cancer cells", 《CANCER BIOLOGY & THERAPY》 *
XIAN SHEN等: "Novel immunodominant epitopes derived from MAGE-A3 and its significance in serological diagnosis of gastric cancer", 《J CANCER RES CLIN ONCOL》 *
李英彬等: "MAGEA1和MAGEA3基因在胃癌中的表达及应用", 《内蒙古医学杂志》 *
邢飞跃等: "葡萄球菌A蛋白体内抗肿瘤机理的实验研究", 《肿瘤防治研究》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115260290A (en) * 2022-06-23 2022-11-01 浙江大学 Melanoma-targeted polypeptide and application thereof
CN115260290B (en) * 2022-06-23 2023-10-31 浙江大学 Polypeptide targeting melanoma and application thereof

Also Published As

Publication number Publication date
CN111978379B (en) 2022-05-03

Similar Documents

Publication Publication Date Title
JP7127008B6 (en) Novel PD-L1 binding polypeptides for imaging
US11466085B2 (en) Anti-PD-L1 nanobody, coding sequence and use thereof
CN110144009B (en) CD47 single domain antibodies and uses thereof
CN109096395B (en) Blocking type CD47 nano antibody and application thereof
US10156559B2 (en) Lipocalin fusion partners
US9107965B2 (en) Polypeptides
Sánchez-Martín et al. The multicompartmental p32/gClqR as a new target for antibody-based tumor targeting strategies
CN109485726B (en) Application of radiolabeled anti-nano antibody in prognosis and diagnosis of cancer
CN110128513B (en) Polypeptide with binding affinity to extracellular domain of EB virus LMP2 protein C-terminal envelope and application thereof
WO2011071279A2 (en) Bpb-based cargo delivery system
CN105859846B (en) There is the polypeptide and application thereof of binding affinity to HPV16 E7
CN111978379B (en) Polypeptide with binding affinity to human melanoma antigen A3 protein and application thereof
CN105820219B (en) There is polypeptide and its application of binding affinity to HPV18 E7 albumen
CN110642928B (en) Polypeptide specifically bound to EB virus LMP1C terminal protein and application thereof
CN111153967B (en) Polypeptide specifically binding to HPV16E5 protein and application thereof
CN114395037B (en) Nanometer antibody of targeted calreticulin and application thereof
WO2023076881A1 (en) Single domain antibodies targeting the s2 subunit of sars-cov-2 spike protein
KR102557303B1 (en) A novel calreticulin-specific binding protein having human fibronectin Ⅲ domain scaffold and use thereof
CN113173978B (en) Polypeptide with binding affinity to HPV16E6 protein and application thereof
JP2023507020A (en) Anti-her2 polypeptide derivatives as novel diagnostic molecular probes
CN114989270A (en) Polypeptide with binding affinity to human CEA and application thereof
CN111748021B (en) Polypeptide with binding affinity to chlamydia trachomatis MOMP and application thereof
CN102127147B (en) Mimotope of epidermal growth factor receptor (EGFR) and use thereof
CN116813778A (en) D4 nano antibody targeting midkine MDK, derivative and application thereof
US20230040008A1 (en) Anti-her2 polypeptides derivatives as new diagnostic molecular probes

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant