WO2023016419A1 - 重组灵芝免疫调节蛋白新突变体及其应用 - Google Patents

重组灵芝免疫调节蛋白新突变体及其应用 Download PDF

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WO2023016419A1
WO2023016419A1 PCT/CN2022/110916 CN2022110916W WO2023016419A1 WO 2023016419 A1 WO2023016419 A1 WO 2023016419A1 CN 2022110916 W CN2022110916 W CN 2022110916W WO 2023016419 A1 WO2023016419 A1 WO 2023016419A1
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recombinant protein
seq
rlz
amino acid
mutant
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PCT/CN2022/110916
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English (en)
French (fr)
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张欣
宋公明
贾伟
张喜田
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长春钻智制药有限公司
上海钻智生物科技有限公司
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Priority to EP22855400.2A priority Critical patent/EP4386008A1/en
Priority to AU2022325354A priority patent/AU2022325354A1/en
Priority to KR1020247003992A priority patent/KR20240029777A/ko
Priority to CA3223071A priority patent/CA3223071A1/en
Priority to CN202280003272.XA priority patent/CN116057071B/zh
Priority to US18/069,265 priority patent/US20230212233A1/en
Publication of WO2023016419A1 publication Critical patent/WO2023016419A1/zh

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    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/375—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from Basidiomycetes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
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    • C12N2800/00—Nucleic acids vectors
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/84—Pichia

Definitions

  • the present application relates to the field of biomedicine, in particular to a mutant (rLZ-8 mutant) of recombinant Ganoderma lucidum immunoregulatory protein and its application in treating tumors.
  • LZ-8 Ganoderma lucidum immunomodulatory protein
  • rLZ-8 Recombinant Ganoderma lucidum immunoregulatory protein
  • rLZ-8 can not only rapidly and efficiently induce apoptosis of various tumor cells in vitro, but also effectively kill tumor cells in mouse tumor models in vivo, and maintain or increase the level of white blood cells.
  • the role of rLZ-8 in the treatment of tissue fibrosis, focal cerebral ischemia, thrombocytopenia, osteoporosis and heart failure has also been published and authorized.
  • the recombinant Ganoderma lucidum immunoregulatory protein mutant (rLZ-8 mutant) is obtained by mutating one or several amino acids of rLZ-8.
  • Mechanism studies have found that the rLZ-8 mutant can bind to the epidermal growth factor receptor (EGFR) on the surface of human cells and be internalized into cells, and remain in cells because they cannot be degraded. Blocking the cell membrane cycle through intensive internalization eventually leads to cell shrinkage, rupture and death.
  • the results of tumor cell killing experiments showed that the rLZ-8 mutant had a better tumor killing effect than rLZ-8.
  • Biacore research results show that the rLZ-8 mutant has a better affinity with EGFR; the results of a variety of human cancer cell orthotopic transplantation tumor model mice show that the rLZ-8 mutant can prolong the survival of mice and inhibit tumor growth. Both are better than rLZ-8.
  • the application provides a recombinant protein, which comprises a spacer peptide and a recombinant Ganoderma lucidum immunomodulatory protein mutant (rLZ-8 mutant), wherein compared with the amino acid sequence shown in SEQ ID NO: 10, the rLZ
  • the amino acid sequence of the -8 mutant contains at least one amino acid mutation.
  • the recombinant protein of the present application has at least one characteristic selected from the following group: (1) higher expression and/or secretion (for example, in yeast expression system); (2) higher safety, meeting The safety requirements of the finished drug; for example, it has passed the preclinical safety assessment, and/or, obtained the clinical trial license (3) it is easy to enter the cell through internalization; and (4) it inhibits tumor growth and has a strong ability to kill tumor cells.
  • the present application provides a kind of recombinant protein, described recombinant protein comprises spacer peptide and recombinant Ganoderma lucidum immunomodulatory protein mutant (rLZ-8 mutant), wherein compared with the amino acid sequence shown in SEQ ID NO: 10, The amino acid sequence of the rLZ-8 mutant contains at least one amino acid mutation.
  • the rLZ-8 mutant comprises the amino acid sequence shown in any one of SEQ ID NO: 13 and 16-21.
  • the rLZ-8 mutant comprises the amino acid sequence shown in SEQ ID NO: 13.
  • the spacer peptide comprises 1-5 EAs.
  • the spacer peptide comprises 2 EAs.
  • the spacer peptide comprises the amino acid sequence shown in any one of SEQ ID NO: 8-9.
  • the spacer peptide comprises the amino acid sequence shown in SEQ ID NO: 8.
  • the spacer peptide is located at the N-terminus of the rLZ-8 or a mutant thereof.
  • the recombinant protein comprises the amino acid sequence shown in any one of SEQ ID NO: 14, 15, 22-33.
  • the recombinant protein comprises the amino acid sequence shown in SEQ ID NO: 14.
  • the present application provides an isolated nucleic acid molecule encoding the recombinant protein.
  • the present application provides a vector comprising the isolated nucleic acid molecule.
  • the present application provides a cell comprising said isolated nucleic acid molecule and/or said vector.
  • the cells are eukaryotic cells.
  • the cells are yeast cells.
  • the present application provides a polypeptide, which comprises the recombinant protein.
  • the present application provides a method for preparing the recombinant protein, the method comprising culturing the cell under the condition of expressing the recombinant protein.
  • the present application provides a pharmaceutical composition, the pharmaceutical composition comprising the recombinant protein, the isolated nucleic acid molecule, the carrier, the polypeptide and/or the cell , and optionally a pharmaceutically acceptable carrier.
  • the present application provides a kit, the kit comprising the recombinant protein, the isolated nucleic acid molecule, the vector, the cell, the polypeptide and/or the said pharmaceutical composition.
  • the present application provides a drug delivery device, the recombinant protein, the isolated nucleic acid molecule, the carrier, the cell, the polypeptide and/or The pharmaceutical composition.
  • the present application provides a method for preventing, alleviating or treating tumors, the method comprising administering the recombinant protein, the isolated nucleic acid molecule, the vector, The cell, the polypeptide and/or the pharmaceutical composition.
  • the present application provides the use of the recombinant protein, the isolated nucleic acid molecule, the carrier, the cell, the polypeptide and/or the pharmaceutical composition in the preparation of a drug purposes, the medicament is used for preventing, alleviating or treating tumors.
  • the present application provides the recombinant protein, the isolated nucleic acid molecule, the vector, the cell, the polypeptide and/or the pharmaceutical composition, which are used to prevent , Relieve or treat tumors.
  • the tumor comprises a non-solid tumor.
  • the tumor comprises a solid tumor.
  • the tumor comprises a tumor with EGFR expression and/or aberrant EGFR expression.
  • the tumor comprises a tumor associated with an EGFR mutation. In certain embodiments, the tumor comprises a tumor associated with a BRAF mutation. In certain embodiments, the tumor comprises a tumor associated with a KRAS mutation. In certain embodiments, the tumor comprises a tumor associated with a HER2 mutation.
  • the tumor comprises colorectal cancer, lung cancer, liver cancer, breast cancer, gastric cancer, renal cancer, bladder cancer, neuroblastoma, ovarian cancer, epithelial squamous cell carcinoma, and/or pancreatic cancer.
  • Figure 1 shows the results of multiple yeast strains expressing rLZ-8 (SEQ ID NO: 10), wherein, M: marker; S: EAEA+rLZ-8 mutant (SEQ ID NO: 14) working reference product ( 1mg/ml); 1-11: No. 1-11 rLZ-8 (SEQ ID NO: 10) strain.
  • Figure 2 shows the results of multiple yeast strains expressing the rLZ-8 mutant (SEQ ID NO: 13), wherein, M: marker; S: EAEA+rLZ-8 mutant (SEQ ID NO: 14) working reference product (1mg/ml); 1-11: No. 1-11 rLZ-8 mutant (SEQ ID NO: 13) strain.
  • Figure 3 shows the results of multiple yeast strains expressing EAEA+rLZ-8 (SEQ ID NO:11) recombinant protein, wherein, M: marker; S: rLZ-8 (SEQ ID NO:10) working reference product (0.5 mg/ml); 1-6: No. 1-6 EAEA+rLZ-8 (SEQ ID NO: 11) strain.
  • Figure 4 shows the results of multiple yeast strains expressing EAEA+rLZ-8 mutant (SEQ ID NO:14) recombinant protein, wherein, M: marker; S: rLZ-8 (SEQ ID NO:10) standard (250mg/L); 1-6: No. 1-6 EAEA+rLZ-8 mutant (SEQ ID NO: 14) strain.
  • Figure 5 shows the results of multiple yeast strains expressing EEAEAEAEPK+rLZ-8 (SEQ ID NO:12) recombinant protein, wherein, M: marker; S: EAEA+rLZ-8 mutant (SEQ ID NO:14) Working reference product (1 mg/ml); 1-11: No. 1-11 EEAEAEAEPK+rLZ-8 (SEQ ID NO: 12) strain.
  • Figure 6 shows the results of multiple yeast strains expressing the EEAEAEAEPK+rLZ-8 mutant (SEQ ID NO: 15) recombinant protein, wherein, M: marker; S: EAEA+r LZ-8 mutant (SEQ ID NO: 14) Working reference product (1 mg/ml); 1-11: No. 1-11 EEAEAEAEPK+rLZ-8 mutant (SEQ ID NO: 15) strain.
  • Figure 7 shows the comparison of the expression results of recombinant proteins of rLZ-8 or its mutants linked by different spacer peptides, wherein, M: marker; S: EAEA+r LZ-8 mutant (SEQ ID NO: 14) work Reference product (1mg/ml); 1: rLZ-8 mutant (SEQ ID NO:13); 2: EAEA+rLZ-8 mutant (SEQ ID NO:14); 3: EEAEAEAEPK+rLZ-8 mutant (SEQ ID NO: 15); 4: rLZ-8 (SEQ ID NO: 10); 5: EAEA+rLZ-8 (SEQ ID NO: 11); 6: EEAEAEAEPK+rLZ-8 (SEQ ID NO: 12) .
  • M marker
  • S EAEA+r LZ-8 mutant (SEQ ID NO: 14) work Reference product (1mg/ml)
  • Figure 8 shows the dose-response curve of the recombinant protein shown in SEQ ID NO: 14 acting on the CR20035B organoid line.
  • Figure 9 shows the dose-response curve of the recombinant protein shown in SEQ ID NO: 14 acting on the CR5043B organoid line.
  • Figure 10 shows the dose-response curve of the recombinant protein shown in SEQ ID NO: 14 acting on the CR5082B organoid line.
  • Figure 11 shows the dose-response curve of the recombinant protein shown in SEQ ID NO: 14 acting on the CR3099B organoid line.
  • Figure 12 shows the dose-response curve of the recombinant protein shown in SEQ ID NO: 14 acting on the LU5162B organoid line.
  • Figure 13 shows the dose-response curve of the recombinant protein shown in SEQ ID NO: 14 acting on the LU11624B organoid line.
  • Figure 14 shows the dose-response curve of the recombinant protein shown in SEQ ID NO: 14 acting on the LU1235B organoid line.
  • Figure 15 shows the dose-response curve of the recombinant protein shown in SEQ ID NO: 14 acting on the LI6677B organoid line.
  • Figure 16 shows the dose-response curve of the recombinant protein shown in SEQ ID NO: 14 acting on the LI6669B organoid line.
  • Figure 17 shows the dose-response curve of the recombinant protein shown in SEQ ID NO: 14 acting on the BR9457B organoid line.
  • Figure 18 shows the dose-response curve of the recombinant protein shown in SEQ ID NO: 14 acting on the BR9466B organoid line.
  • Figure 19 shows the dose-response curve of the recombinant protein shown in SEQ ID NO: 14 acting on the GA6833B organoid line.
  • Figure 20 shows the dose-response curve of the recombinant protein shown in SEQ ID NO: 14 acting on the GA2434B organoid line.
  • Figure 21 shows the dose-response curve of the recombinant protein shown in SEQ ID NO: 14 acting on the PA20078B organoid line.
  • Figure 22 shows the dose-response curve of the recombinant protein shown in SEQ ID NO: 14 acting on the PA0787B organoid line.
  • Figure 23 shows the changes in the survival rates of mice in each treatment group and control group in the LI6669 human liver cancer orthotopic transplantation tumor model.
  • Figure 24 shows the killing effect of the rLZ-8 mutant recombinant protein on the lung cancer cell line A549.
  • Figure 25 shows the comparison of the expression results of recombinant proteins of rLZ-8 or its mutants linked by different spacer peptides, wherein, M: marker; 1: rLZ-8 mutant 4; 2: EAEA+rLZ-8 mutant 4 (SEQ ID NO:25); 3: EEAEAEAEPK+rLZ-8 mutant 4 (SEQ ID NO:31); 4: rLZ-8 mutant 5; 5: EAEA+rLZ-8 mutant 5 (SEQ ID NO :26); 6:EEAEAEAEPK+rLZ-8 mutant 5 (SEQ ID NO:32); 7:rLZ-8 mutant 6; 8:EAEA+rLZ-8 mutant 6 (SEQ ID NO:27); 9 : EEAEAEAEPK+rLZ-8 mutant 6 (SEQ ID NO:33).
  • the term "recombinant protein” generally refers to proteins produced using recombinant techniques.
  • Recombinant proteins may include (1) semi-synthetic or synthetic polypeptides produced by the combined expression of DNA molecules from different sources linked by recombinant DNA technology; (2) polypeptides of semi-synthetic or synthetic origin that are not associated with their natural counterparts (3) a polypeptide of semi-synthetic or synthetic origin that is linked to a polypeptide other than the one to which it is linked in nature; or (4) a polypeptide of semi-synthetic or synthetic origin that does not exist in nature.
  • the recombinant protein may refer to a polypeptide formed by linking the spacer peptide of the present application and the recombinant Ganoderma lucidum immunoregulatory protein mutant (rLZ-8 mutant) of the present application.
  • the term "spacer peptide” generally refers to any oligopeptide or polypeptide that functions to link any domains.
  • the spacer peptide is located at the N-terminal of the recombinant Ganoderma lucidum immunoregulatory protein (rLZ-8).
  • a spacer peptide can be located between the leader peptide and recombinant Ganoderma lucidum immunomodulatory protein (rLZ-8).
  • a spacer peptide can be part of a signal peptide.
  • a spacer peptide can facilitate secretion of the protein to which it is attached.
  • Ganoderma lucidum immunomodulatory protein may also be referred to as LZ-8, LZ8, Lingzhi-8 or Ganoderma lucidum protein 8, which generally refers to an immunomodulatory protein derived from Ganoderma lucidum.
  • LZ-8 also includes allelic variants, splice variants, derivative variants, substitution variants, deletion variants and/or insertion variants (including addition of N-terminal methionine), fusion polypeptides and interspecies homologues thereof .
  • the LZ-8 may refer to an immunomodulatory protein derived from Ganoderma lucidum.
  • the term "recombinant Ganoderma lucidum immunomodulatory protein (rLZ-8)” generally refers to LZ-8 obtained by recombinant methods.
  • the amino acid sequences of LZ-8 and rLZ-8 can be found in UniProtKB accession number P14945.
  • the LZ-8 and rLZ-8 may comprise the amino acid sequence shown in SEQ ID NO:10.
  • mutant generally refers to a sequence that differs from a reference sequence by containing one or more differences.
  • the reference sequence may be the amino acid sequence of recombinant Ganoderma lucidum immunoregulatory protein.
  • the differences may be deletions, insertions or preferably substitutions of amino acids.
  • a mutant may have the same or a different functional role than the reference sequence.
  • amino acid mutation generally covers substitutions, deletions, insertions and modifications of one or more amino acids.
  • EA generally refers to a peptide fragment in which glutamic acid (E) and alanine (A) are linked by a peptide bond.
  • isolated generally refers to biological material (eg, viruses, nucleic acids, or proteins) that is substantially free from components that normally accompany or interact with it in its naturally occurring environment.
  • isolated nucleic acid molecule generally refers to DNA or RNA of genomic, mRNA, cDNA or synthetic origin, or some combination thereof, which is not associated with all or part of a polynucleotide found in nature, Or linked to a polynucleotide to which it is not linked in nature.
  • the term "vector” generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers an inserted nucleic acid molecule into and/or between host cells.
  • the vectors may include vectors mainly used for inserting DNA or RNA into cells, vectors mainly used for replicating DNA or RNA, and vectors mainly used for expression of transcription and/or translation of DNA or RNA.
  • the carrier also includes a carrier having various functions as described above.
  • the vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, the vector can produce the desired expression product by culturing an appropriate host cell containing the vector.
  • the term "cell” generally refers to individual cells, cell lines or cell culture.
  • the cells may include progeny of a single host cell. Due to natural, accidental or deliberate mutations, the progeny cells may not necessarily be completely identical in shape or genome to the original parent cells, but it is sufficient to be able to express the antibodies or antigen-binding fragments thereof described in this application.
  • the cells can be obtained by transfecting cells in vitro with the vectors described in this application.
  • the cells can be prokaryotic cells (such as Escherichia coli) or eukaryotic cells (such as yeast cells, such as COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NSO cells or myeloma cells).
  • the cells may be mammalian cells.
  • the mammalian cells may be CHO-K1 cells.
  • the term "recombinant cell” generally refers to a cell into which a recombinant expression vector has been introduced.
  • the recombinant host cells include not only certain specific cells, but also the progeny of these cells.
  • kit generally refers to a packaged product comprising components for administering the recombinant protein of the present application to treat diseases related to EGFR expression and/or EGFR abnormal expression.
  • the components of the kit may be contained in separate vials (ie, a kit with separate parts), or provided within a single vial.
  • Kits can include reagents such as buffers, protein stabilization reagents, signal generating systems (eg, fluorescent signal generating systems), antibodies, control proteins, and test containers.
  • the kit can also comprise instructions for carrying out the methods.
  • the term "administration device” includes: (i) an infusion module for administering a pharmaceutical composition comprising an active ingredient to a subject; (ii) an infusion module for infusion A pharmaceutical composition, which contains an active ingredient selected from the group consisting of recombinant proteins, nucleic acid molecules, cells, vectors or combinations thereof; and (iii) an optional drug efficacy monitoring module.
  • tumor generally refers to all neoplastic cell growth and proliferation, whether malignant or benign, and to all precancerous and cancerous cells and tissues.
  • Tumors can include solid tumors and/or non-solid tumors (eg, hematological tumors, lymphomas).
  • the term "pharmaceutically acceptable carrier” generally includes pharmaceutically acceptable carriers, excipients or stabilizers that are nontoxic to cells or mammals to which they are exposed at the dosages and concentrations employed. of.
  • the physiologically acceptable carrier is a pH buffered aqueous solution.
  • physiologically acceptable carriers may include buffers, antioxidants, low molecular weight (less than about 10 residues) polypeptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, salt-forming counterions such as sodium; and/or nonionic surfactants.
  • treatment generally refers to a clinical intervention intended to alter the natural course of the disease in the individual being treated, and may be for prevention or during the course of a clinical disease. Desirable effects of treatment include, but are not limited to, prevention of occurrence or recurrence of disease, alleviation of symptoms, attenuation of any direct or indirect pathological consequences of disease, prevention of metastasis, reduction of rate of disease progression, amelioration or palliation of disease state, and palliation or improved prognosis.
  • the recombinant vectors of the present application can be used to delay disease development or slow down disease progression.
  • administration generally refers to administering a dose of a compound (e.g., an anticancer therapeutic) or a pharmaceutical composition (e.g., a pharmaceutical composition comprising an anticancer therapeutic) to a subject (e.g., a patient)
  • a pharmaceutical composition e.g., a pharmaceutical composition comprising an anticancer therapeutic
  • Administration can be by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration.
  • Parenteral infusion includes, for example, intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration.
  • the present application may also include functional variants, derivatives, analogs, homologues and fragments thereof.
  • a variant of any given sequence is one in which a particular sequence of residues (whether amino acid or nucleotide residues) has been modified such that the polypeptide or polynucleotide substantially retains at least one A sequence of endogenous functions.
  • Variant sequences may be obtained by addition, deletion, substitution, modification, substitution and/or variation of at least one amino acid residue and/or nucleotide residue present in a naturally occurring protein and/or polynucleotide, so long as the The original functional activity is sufficient.
  • the term “derivative” generally refers to the polypeptide or polynucleotide of the present application including any substitution, variation, modification, substitution, deletion and and/or added, so long as the resulting polypeptide or polynucleotide substantially retains at least one of its endogenous functions.
  • the term “analogue” generally refers to polypeptides or polynucleotides, including any mimetic of polypeptides or polynucleotides, that is, having at least one endogenous function of the polypeptide or polynucleotide simulated by the mimetic of chemical compounds.
  • amino acid substitutions e.g., at least 1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 20) amino acid substitutions can be made so long as the modified sequence remains substantially as desired. activity or ability. Amino acid substitutions may involve the use of non-naturally occurring analogs.
  • proteins or polypeptides used in this application may also have deletions, insertions, or substitutions of amino acid residues that produce silent changes and result in functionally equivalent proteins.
  • Deliberate amino acid substitutions can be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathic nature of the residues, so long as endogenous function is preserved.
  • negatively charged amino acids include aspartic acid and glutamic acid
  • positively charged amino acids include lysine and arginine
  • amino acids with uncharged polar headgroups with similar hydrophilicity values include aspartic acid.
  • Paragine, Glutamine, Serine, Threonine and Tyrosine are examples of amino acid residues that produce silent changes and result in functionally equivalent proteins.
  • homologue generally refers to an amino acid sequence or a nucleotide sequence having certain homology with a wild-type amino acid sequence and a wild-type nucleotide sequence.
  • the term “homology” may be equated with sequence "identity”.
  • homologous sequences may include amino acid sequences that may be at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the subject sequence .
  • a homologue will comprise the same active site, etc., as the subject amino acid sequence.
  • Homology can be considered in terms of similarity (ie, amino acid residues having similar chemical properties/functions), or can be expressed in terms of sequence homology.
  • any one of the SEQ ID NOs of the mentioned amino acid sequence or nucleotide sequence has a sequence with a percentage homology, which means that the sequence has the percentage homology on the entire length of the mentioned SEQ ID NO. source sequence.
  • sequence alignment can be performed by various means known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software and the like. Those skilled in the art can determine appropriate parameters for alignment, including any algorithms needed to achieve optimal alignment across the full-length sequences being compared.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the application provides a kind of recombinant protein, described recombinant protein comprises spacer peptide and the mutant of recombinant Ganoderma lucidum immunomodulatory protein (rLZ-8), wherein said rLZ-8 can comprise the mutant shown in SEQ ID NO:10 amino acid sequence.
  • the rLZ-8 may comprise at least 80% (for example, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%) of the amino acid sequence shown in SEQ ID NO: 10, Amino acid sequences of at least 98%, at least 99% or more) homology.
  • the present application provides a kind of recombinant protein, described recombinant protein comprises spacer peptide and recombinant Ganoderma lucidum immunomodulatory protein mutant (rLZ-8 mutant), wherein compared with the amino acid sequence shown in SEQ ID NO: 10,
  • the amino acid sequence of the rLZ-8 mutant may contain at least one amino acid mutation.
  • the recombinant protein comprises a spacer peptide and a recombinant Ganoderma lucidum immunoregulatory protein mutant (rLZ-8 mutant), wherein compared with the amino acid sequence shown in SEQ ID NO: 10, the rLZ-8 mutant
  • the amino acid sequence may contain a single amino acid mutation.
  • the recombinant protein comprises a spacer peptide and a recombinant Ganoderma lucidum immunoregulatory protein mutant (rLZ-8 mutant), wherein compared with the amino acid sequence shown in SEQ ID NO: 10, the rLZ-8 mutant
  • the amino acid sequence can contain two amino acid mutations.
  • the recombinant protein comprises a spacer peptide and a recombinant Ganoderma lucidum immunoregulatory protein mutant (rLZ-8 mutant), wherein compared with the amino acid sequence shown in SEQ ID NO: 10, the rLZ-8 mutant
  • the amino acid sequence of may comprise one amino acid mutation, said one amino acid mutation may be located at an amino acid position selected from the group consisting of: R9, L17, D20, D70, K46 and K74.
  • the one amino acid mutation may be at an amino acid position selected from the group consisting of: D70, L17, K74, and K46.
  • the amino acid mutation at L17 may be L17K.
  • the amino acid mutation at D70 may be D70K.
  • amino acid mutation at K46 may be K46E.
  • amino acid mutation at K74 may be K74E.
  • the amino acid mutation at D20 may be D20H.
  • amino acid mutation at R9 may be R9A.
  • the recombinant protein comprises a spacer peptide and a recombinant Ganoderma lucidum immunoregulatory protein mutant (rLZ-8 mutant), wherein compared with the amino acid sequence shown in SEQ ID NO: 10, the rLZ-8 mutant
  • the amino acid sequence of may comprise one amino acid mutation, and the one amino acid mutation may be selected from the group consisting of R9A, L17K, D20H, K46E, D70K and K74E. As another example, the one amino acid mutation may be selected from the group consisting of L17K, K46E, D70K and K74E.
  • the recombinant protein comprises a spacer peptide and a recombinant Ganoderma lucidum immunoregulatory protein mutant (rLZ-8 mutant), wherein compared with the amino acid sequence shown in SEQ ID NO: 10, the rLZ-8 mutant
  • the amino acid sequence of can comprise one amino acid mutation, and the one amino acid mutation can be selected from the group consisting of L17K, D70K and K46E.
  • the recombinant protein comprises a spacer peptide and a recombinant Ganoderma lucidum immunoregulatory protein mutant (rLZ-8 mutant), wherein compared with the amino acid sequence shown in SEQ ID NO: 10, the rLZ-8 mutant
  • the amino acid sequence of may comprise two amino acid mutations, and the two amino acid mutations may be selected from the group consisting of K46E and K74E.
  • the two amino acid mutations may be selected from the group consisting of L17K and D70K.
  • the rLZ-8 mutant may comprise the amino acid sequence shown in any one of SEQ ID NO: 13 and 16-21.
  • the rLZ-8 mutant may comprise at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) homologous amino acid sequences.
  • the rLZ-8 mutant may comprise the amino acid sequence shown in SEQ ID NO: 13.
  • the rLZ-8 mutant may comprise at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) homologous amino acid sequences.
  • the amino acid sequence of the rLZ-8 mutant can be as shown in SEQ ID NO: 13.
  • the recombinant protein may comprise a spacer peptide and rLZ-8, and the rLZ-8 may comprise the amino acid sequence shown in SEQ ID NO: 10, and the spacer peptide may comprise 1-5 EAs.
  • the spacer peptide may comprise 2 EAs.
  • the recombinant protein may comprise a spacer peptide and a rLZ-8 mutant, and the rLZ-8 mutant may comprise the amino acid sequence shown in any one of SEQ ID NO: 13 and 16-21, the The spacer peptide may contain 1-5 EAs.
  • the spacer peptide can comprise 2 EAs.
  • the recombinant protein may comprise a spacer peptide and rLZ-8 mutant, and the rLZ-8 mutant may comprise the amino acid sequence shown in SEQ ID NO: 13, and the spacer peptide may comprise SEQ ID NO : The amino acid sequence shown in 8.
  • the recombinant protein may comprise the amino acid sequence shown in SEQ ID NO:14.
  • the recombinant protein may comprise a spacer peptide and rLZ-8 mutant, and the rLZ-8 mutant may comprise the amino acid sequence shown in SEQ ID NO: 16, and the spacer peptide may comprise SEQ ID NO : The amino acid sequence shown in 8.
  • the recombinant protein may comprise the amino acid sequence shown in SEQ ID NO:22.
  • the recombinant protein may comprise a spacer peptide and rLZ-8 mutant, and the rLZ-8 mutant may comprise the amino acid sequence shown in SEQ ID NO: 17, and the spacer peptide may comprise SEQ ID NO : The amino acid sequence shown in 8.
  • the recombinant protein may comprise the amino acid sequence shown in SEQ ID NO:23.
  • the recombinant protein may comprise a spacer peptide and rLZ-8 mutant, and the rLZ-8 mutant may comprise the amino acid sequence shown in SEQ ID NO: 18, and the spacer peptide may comprise SEQ ID NO : The amino acid sequence shown in 8.
  • the recombinant protein may comprise the amino acid sequence shown in SEQ ID NO:24.
  • the recombinant protein may comprise a spacer peptide and rLZ-8 mutant, and the rLZ-8 mutant may comprise the amino acid sequence shown in SEQ ID NO: 19, and the spacer peptide may comprise SEQ ID NO : The amino acid sequence shown in 8.
  • the recombinant protein may comprise the amino acid sequence shown in SEQ ID NO:25.
  • the recombinant protein may comprise a spacer peptide and rLZ-8 mutant, and the rLZ-8 mutant may comprise the amino acid sequence shown in SEQ ID NO: 20, and the spacer peptide may comprise SEQ ID NO : The amino acid sequence shown in 8.
  • the recombinant protein may comprise the amino acid sequence shown in SEQ ID NO:26.
  • the recombinant protein may comprise a spacer peptide and rLZ-8 mutant, and the rLZ-8 mutant may comprise the amino acid sequence shown in SEQ ID NO: 21, and the spacer peptide may comprise SEQ ID NO : The amino acid sequence shown in 8.
  • the recombinant protein may comprise the amino acid sequence shown in SEQ ID NO:27.
  • the recombinant protein may comprise a spacer peptide and rLZ-8 mutant, and the rLZ-8 mutant may comprise the amino acid sequence shown in SEQ ID NO: 13, and the spacer peptide may comprise SEQ ID NO : Amino acid sequence shown in 9.
  • the recombinant protein may comprise the amino acid sequence shown in SEQ ID NO: 15.
  • the recombinant protein may comprise a spacer peptide and rLZ-8 mutant, and the rLZ-8 mutant may comprise the amino acid sequence shown in SEQ ID NO: 16, and the spacer peptide may comprise SEQ ID NO : Amino acid sequence shown in 9.
  • the recombinant protein may comprise the amino acid sequence shown in SEQ ID NO:28.
  • the recombinant protein may comprise a spacer peptide and rLZ-8 mutant, and the rLZ-8 mutant may comprise the amino acid sequence shown in SEQ ID NO: 17, and the spacer peptide may comprise SEQ ID NO : The amino acid sequence shown in 9.
  • the recombinant protein may comprise the amino acid sequence shown in SEQ ID NO:29.
  • the recombinant protein may comprise a spacer peptide and rLZ-8 mutant, and the rLZ-8 mutant may comprise the amino acid sequence shown in SEQ ID NO: 18, and the spacer peptide may comprise SEQ ID NO : Amino acid sequence shown in 9.
  • the recombinant protein may comprise the amino acid sequence shown in SEQ ID NO:30.
  • the recombinant protein may comprise a spacer peptide and rLZ-8 mutant, and the rLZ-8 mutant may comprise the amino acid sequence shown in SEQ ID NO: 19, and the spacer peptide may comprise SEQ ID NO : Amino acid sequence shown in 9.
  • the recombinant protein may comprise the amino acid sequence shown in SEQ ID NO:31.
  • the recombinant protein may comprise a spacer peptide and rLZ-8 mutant, and the rLZ-8 mutant may comprise the amino acid sequence shown in SEQ ID NO: 20, and the spacer peptide may comprise SEQ ID NO : Amino acid sequence shown in 9.
  • the recombinant protein may comprise the amino acid sequence shown in SEQ ID NO:32.
  • the recombinant protein may comprise a spacer peptide and rLZ-8 mutant, and the rLZ-8 mutant may comprise the amino acid sequence shown in SEQ ID NO: 21, and the spacer peptide may comprise SEQ ID NO : Amino acid sequence shown in 9.
  • the recombinant protein may comprise the amino acid sequence shown in SEQ ID NO:33.
  • the spacer peptide may be located at the N-terminal of the rLZ-8 or its mutant.
  • the recombinant protein may include the spacer peptide and the rLZ-8 mutant sequentially from the N-terminal to the C-terminal.
  • the recombinant protein may comprise a spacer peptide and rLZ-8, and the rLZ-8 may comprise the amino acid sequence shown in SEQ ID NO: 10, and the spacer peptide may comprise the amino acid sequence shown in SEQ ID NO: 8 amino acid sequence.
  • the recombinant protein may comprise a spacer peptide and rLZ-8, and the rLZ-8 may comprise the amino acid sequence shown in SEQ ID NO: 10, and the spacer peptide may comprise the amino acid sequence shown in SEQ ID NO: 9 amino acid sequence.
  • the spacer peptide may be located at the N-terminal of rLZ-8.
  • the recombinant protein may include the spacer peptide and the rLZ-8 sequentially from the N-terminus to the C-terminus.
  • the recombinant protein may comprise the amino acid sequence shown in any one of SEQ ID NO: 14, 15 and 22-33.
  • the recombinant protein may comprise an amino acid sequence having at least 80% (for example, at least 85%, at least 90%, at least 95%) of the amino acid sequence shown in any one of SEQ ID NO: 14, 15, and 22-33. , at least 96%, at least 97%, at least 98%, at least 99% or higher) homologous amino acid sequences.
  • the recombinant protein may comprise the amino acid sequence shown in SEQ ID NO: 14.
  • the recombinant protein may comprise at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, Amino acid sequences of at least 99% or greater) homology.
  • the present application provides a polypeptide, which comprises the recombinant protein.
  • the recombinant protein can be covalently or non-covalently linked to other proteins or polypeptides.
  • the present application provides one or more nucleic acid molecules, which can encode the recombinant protein and/or polypeptide described in the present application.
  • the nucleic acid molecules described herein can be isolated. For example, it may be produced or synthesized by (i) amplified in vitro, such as by polymerase chain reaction (PCR) amplification, (ii) recombinantly produced by cloning, (iii) purified (iv) synthetic, for example by chemical synthesis.
  • the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA techniques.
  • the present application provides a vector, which may comprise the nucleic acid molecule described in the present application.
  • other genes may be included in the vector, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions.
  • the vector may also contain expression control elements that permit proper expression of the coding region in an appropriate host.
  • control elements are well known to those skilled in the art, and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation, and the like.
  • Such vectors may include, for example, plasmids, cosmids, viruses, phages, or other vectors commonly used in, for example, genetic engineering.
  • the vector is an expression vector.
  • the vector is a vector suitable for expression in eukaryotic cells (eg, yeast cells).
  • the present application provides a cell, which may comprise the nucleic acid molecule or the vector described in the present application.
  • each or each cell may comprise one or more of the nucleic acid molecules or vectors described herein.
  • each or each cell may comprise a plurality (eg, 2 or more) or a plurality (eg, 2 or more) of the nucleic acid molecules or vectors described herein.
  • the vectors described herein can be introduced into the host cells, such as eukaryotic cells, such as cells from plants, fungal or yeast cells, and the like.
  • the vectors described in this application can be introduced into the host cells by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection and the like.
  • the present application provides a method for preparing the recombinant protein, the method comprising culturing the cell under the condition of expressing the recombinant protein.
  • the method can be achieved by using an appropriate medium, appropriate temperature and incubation time, and the like.
  • the present application provides a pharmaceutical composition, the pharmaceutical composition comprising the recombinant protein, the isolated nucleic acid molecule, the carrier, the polypeptide and/or the cell , and optionally a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier is nontoxic to recipients at the dosages and concentrations employed, and may include buffers, antioxidants, preservatives, low molecular weight (less than about 10 residues) polypeptides, proteins, hydrophilic polymers, amino acids, carbohydrates, salt-forming counterions, metal complexes, and/or nonionic surfactants.
  • the pharmaceutical compositions herein may also contain more than one active compound, generally those active compounds with complementary activities that do not adversely affect each other.
  • the type and effective amount of such drug depends, for example, on the amount and type of antagonist present in the formulation, as well as the clinical parameters of the subject.
  • the pharmaceutical composition described in the present application may contain the prophylactically and/or therapeutically effective amount of the recombinant protein and/or polypeptide.
  • the prophylactically and/or therapeutically effective amount is the dose required to be able to prevent and/or treat (at least partially treat) the disease or condition and/or any complications thereof in a subject suffering from or at risk of developing.
  • the present application provides a kit, the kit comprising the recombinant protein, the isolated nucleic acid molecule, the vector, the cell, the polypeptide and/or the said pharmaceutical composition. It can include the antigen binding protein described in the application, the carrier, the nucleic acid molecule, the cell, the immunoconjugate, and/or the pharmaceutical composition described in the application in a single common container, and can optionally be combined with one or more A combination of therapeutic agents, optionally formulated together in a kit.
  • the present application provides a drug delivery device, the recombinant protein, the isolated nucleic acid molecule, the carrier, the cell, the polypeptide and/or The pharmaceutical composition.
  • the present application provides a method for preventing, alleviating or treating tumors, the method comprising administering the recombinant protein, the isolated nucleic acid molecule, the vector, The cell, the polypeptide and/or the pharmaceutical composition.
  • the present application provides the use of the recombinant protein, the isolated nucleic acid molecule, the carrier, the cell, the polypeptide and/or the pharmaceutical composition in the preparation of a drug purposes, the medicament is used for preventing, alleviating or treating tumors.
  • the present application provides the recombinant protein, the isolated nucleic acid molecule, the vector, the cell, the polypeptide and/or the pharmaceutical composition, which are used to prevent , Relieve or treat tumors.
  • the tumor can include solid tumors and/or non-solid tumors (eg, hematological tumors).
  • the tumor can be a tumor that expresses EGFR.
  • the tumor can be a tumor associated with abnormal expression of EGFR.
  • the tumor can be a tumor with EGFR overexpression.
  • the tumors can include tumors associated with EGFR mutations.
  • the tumor can include a tumor associated with a BRAF mutation.
  • the tumor can include a tumor associated with a KRAS mutation.
  • the possible tumors include tumors associated with HER2 mutations.
  • the tumor may be selected from one or more of the group consisting of colorectal cancer, lung cancer, liver cancer, breast cancer, gastric cancer, kidney cancer, bladder cancer, neuroblastoma, ovarian cancer, epithelial squamous cell carcinoma, and pancreatic cancer. cancer.
  • rLZ-8 amino acid sequence as shown in SEQ ID NO: 10
  • rLZ-8 mutant amino acid sequence as shown in SEQ ID NO: 13
  • forward primer F and reverse Primer R was used to amplify rLZ-8 and rLZ-8 mutant gene fragments by PCR.
  • the obtained gene fragment does not contain the DNA sequence of EAEA, but has Xho I and Xba I restriction sites at the 5' end and 3' respectively.
  • forward primer F and reverse primer R are as follows:
  • Forward primer F CCGCTCGAGAAAAGAATGTCTGATACTGCTTTGATCTTCA (SEQ ID NO: 1)
  • Reverse primer R GCTCTAGACTAGTTCCATTGAGCGATA (SEQ ID NO: 2)
  • Example 2 The expression vector construction of rLZ-8 and rLZ-8 mutants with EAEA sequence
  • EAEA+rLZ-8 amino acid sequence is shown in SEQ ID NO:11
  • EAEA+rLZ -8 mutant amino acid sequence shown in SEQ ID NO: 14
  • Xho I and Xba I restriction enzyme sites were added to the 5'- and 3'-ends of the optimized DNA sequence, respectively, and the DNA sequence was artificially synthesized and then loaded into the pUC57 cloning vector, namely pUC57-EAEA +rLZ-8 and pUC57-EAEA+rLZ-8 mutants.
  • EAEA+rLZ-8 amino acid sequence as shown in SEQ ID NO: 11
  • forward primer F3 and forward primer R3 were used to amplify the rLZ-8 gene fragment by PCR.
  • the obtained gene fragment contains the DNA sequence of EAEA, and has Xho I and Xba I restriction sites at the 5' end and 3' respectively.
  • forward primer F3 The information of forward primer F3 and forward primer R3 is as follows:
  • Forward primer F3 CCGCTCGAGAAAAGAGAGGCTGAAGCT (SEQ ID NO: 3)
  • Reverse primer R3 GCTCTAGATCACTAGTTCCATTG (SEQ ID NO: 4)
  • the forward primer M13F and the reverse primer M13R were used to amplify the rLZ-8 mutant gene fragment by PCR.
  • the obtained gene fragment contains the DNA sequence of EAEA, and has Xho I and Xba I restriction sites at the 5' end and 3' respectively.
  • forward primer M13F and reverse primer M13R are as follows:
  • Reverse primer M13R AGCGGATAACAATTTCACACAGG (SEQ ID NO: 6)
  • EAEA+rLZ-8 and EAEA+rLZ-8 mutant gene fragments were digested with Xho I/Xba I and connected to an empty expression vector using T4 DNA ligase to obtain a recombinant expression vector.
  • rLZ-8 mutant 1 amino acid sequence shown in SEQ ID NO: 16
  • rLZ-8 mutant 2 amino acid sequence shown in SEQ ID NO: 17
  • rLZ-8 mutant 3 amino acid sequence shown in SEQ ID NO: 18
  • rLZ-8 mutant 4 amino acid sequence shown in SEQ ID NO: 19
  • rLZ-8 mutant 5 amino acid sequence The sequence is shown in SEQ ID NO: 20
  • the recombinant protein of rLZ-8 mutant 6 (amino acid sequence is shown in SEQ ID NO: 21).
  • Example 3 The expression vector construction of rLZ-8 and rLZ-8 mutants with EEAEAEAEPK sequence
  • EEAEAEAEPK+rLZ-8DNA amino acid sequence as shown in SEQ ID NO:12
  • EEAEAEAEPK+rLZ-8 mutant amino acid sequence as shown in SEQ ID NO:15
  • forward primer F2 The EEAEAEAEPK+rLZ-8 and EEAEAEAEPK+rLZ-8 mutant gene fragments were amplified by PCR with the reverse primer R.
  • the obtained gene fragment contains the DNA sequence of EEAEAEAEPK, and has Xho I and Xba I restriction sites at the 5' end and 3' respectively.
  • Forward primer F2 and reverse primer R information are as follows:
  • Reverse primer R GCTCTAGACTAGTTCCATTGAGCGATA (SEQ ID NO: 2)
  • rLZ-8 mutant 1 amino acid sequence shown in SEQ ID NO: 16
  • rLZ-8 mutant 2 amino acid sequence shown in SEQ ID NO: 17
  • rLZ-8 mutant 3 amino acid sequence shown in SEQ ID NO: 18
  • rLZ-8 mutant 4 amino acid sequence shown in SEQ ID NO: 19
  • rLZ-8 mutant 5 amino acid sequence The sequence is shown in SEQ ID NO: 20
  • the recombinant protein of rLZ-8 mutant 6 (amino acid sequence is shown in SEQ ID NO: 21).
  • Competent cell preparation (1) Competent cell preparation:
  • Bacterial solution was placed in an ice bath for 30 minutes, and divided into four 50mL centrifuge tubes in a sterile ultra-clean bench, 40mL in each tube. Centrifuge at 1500g for 5min at 4°C. Discard the supernatant, collect the bacteria, fully resuspend the cells with 160 mL of pre-cooled sterilized double distilled water, centrifuge at 1500 g, 4 °C for 5 min.
  • the recombinant plasmid was linearized with BspHI to facilitate transformation into yeast cells.
  • FIG. 1 For rLZ-8 (SEQ ID NO: 10), No. 3 clone has higher expression level (Fig. 1); For rLZ-8 mutant (SEQ ID NO: 13), No. 10 clone has higher expression level (Fig. 2 ); for EAEA+rLZ-8 (SEQ ID NO:11), No. 6 clone has higher expression level (Fig. 3); for EAEA+rLZ-8 mutant (SEQ ID NO:14), No. 7 clone has higher expression High expression level (Fig. 4); For EEAEAEAEPK+rLZ-8 (SEQ ID NO:12), No. 7 clone has higher expression level (Fig. 5); For EEAEAEAEPK+rLZ-8 mutant (SEQ ID NO:15) , No. 2 clone has a higher expression level (Fig. 6).
  • Example 7 The expression of the target protein in various expression vectors constructed in Examples 1-3 was compared according to the method in Example 5, and the results are shown in FIG. 7 .
  • EAEA can promote the expression and secretion of rLZ-8 (SEQ ID NO: 10) and rLZ-8 mutant (SEQ ID NO: 13), and the promoting effect on rLZ-8 mutant (SEQ ID NO: 13) is superior in rLZ-8 (SEQ ID NO: 10) (lanes 1, 2, 4 and 5 of Figure 7).
  • EEAEAEAEPK can promote the expression and secretion of rLZ-8 (SEQ ID NO:10) and rLZ-8 mutant (SEQ ID NO:13), and the promotion effect of EEAEAEAEPK on rLZ-8 (SEQ ID NO:10) is better than rLZ-8 mutant (SEQ ID NO: 13) (lanes 1, 3, 4 and 6 of Figure 7).
  • EAEA and EEAEAEAEPK have the effect of improving protein expression and secretion, and the two have different promoting effects on the expression and secretion of different proteins.
  • EAEA has the best promoting effect on the rLZ-8 mutant (SEQ ID NO: 13) ( Figure 7 Lanes 1, 2 and 3), while EEAEAEAEPK had the best promoting effect on rLZ-8 (SEQ ID NO: 10) (lanes 4, 5 and 6 in Figure 7).
  • Example 5 the expression of the target protein (based on rLZ-8 mutant 4 to rLZ-8 mutant 6) in the nine expression vectors constructed in Examples 1-3 was compared, and the results are shown in FIG. 25 .
  • EAEA has the effect of improving protein expression and secretion, and has the effect on rLZ-8 mutant 4 (SEQ ID NO:19), rLZ-8 mutant 5 (SEQ ID NO:20) and rLZ-8 mutant 6 (SEQ ID NO:20) ID NO:21) has the best promoting effect.
  • This embodiment measures EAEA+rLZ-8 (SEQ ID NO:11), EAEA+rLZ-8 mutant (SEQ ID NO:14), EEAEAEAEPK+rLZ-8 (SEQ ID NO:12), EEAEAEAEPK+rLZ-8 N-terminal amino acid sequences of mutant (SEQ ID NO:15), rLZ-8 (SEQ ID NO:10) and rLZ-8 mutant (SEQ ID NO:13) proteins.
  • the peptides produced by hydrolysis are separated by reverse chromatography, and the amino acids of each peptide are determined by mass spectrometry to obtain the N-terminal amino acid sequence. Specific steps are as follows:
  • test protein Take 50 ⁇ g of the test protein, dilute it to 0.5 ⁇ g/ ⁇ l with 0.1M ammonium bicarbonate solution, add 0.5 ⁇ g trypsin, mix well and incubate at 37°C for 20h. After the incubation, add an equal volume of 0.1% formic acid solution or place at 4°C to terminate the enzymatic hydrolysis reaction; centrifuge the above solution at 4°C, 12000rpm for 2min, and take the supernatant for analysis and detection.
  • the above dilution was added to a 96-well plate containing A549 cells, 100 ⁇ L/well, and three parallel wells were set for each concentration group, and marked.
  • Set a group of cell control group that is, cells only 100 ⁇ L/well, 6 parallel wells; put it into a CO 2 incubator with a CO 2 concentration of 5% and a temperature of 37° C. for 48 hours.
  • amino acid sequence of the spacer sequence of the recombinant protein used in the examples is shown in SEQ ID NO: 8
  • the amino acid sequence of the rLZ-8 mutant is shown in SEQ ID NO: 13
  • the amino acid sequence of the recombinant protein is shown in SEQ ID NO: 14 shown.
  • the recombinant protein shown in SEQ ID NO: 14 has the effect on EGFR wild type (CR20035B, Figure 8), KRAS mutation (CR5043B, Figure 9), BRAF mutation (CR5082B, Figure 10) Colorectal cancer cells with simultaneous mutations of EGFR and KRAS (CR3099B, Figure 11) all have inhibitory effects.
  • the recombinant protein shown in SEQ ID NO: 14 has an effect on EGFR wild type (LU1235B, Figure 14), KRAS mutation (LU5162B, Figure 12) and BRAF mutation (LU11624B, Figure 13) lung cancer cells have inhibitory effect.
  • the recombinant protein shown in SEQ ID NO: 14 has an inhibitory effect on gastric cancer cells with EGFR wild type (GA6833B, Figure 19) and BRAF mutation (GA2434B, Figure 20).
  • Example 10 Recombinant protein inhibits tumor growth in vivo (PDX model)
  • i.p. represents intraperitoneal injection
  • i.v. represents tail vein injection
  • p.o. represents oral gavage
  • the main observation indicators of this experiment are:
  • T/C Relative tumor proliferation rate
  • T/C% TTW/CTW ⁇ 100% (TTW: average tumor weight at the end of the experiment in the treatment group; CTW: average tumor weight at the end of the experiment in the control group).
  • TGI% (1-T/C) ⁇ 100%
  • T and C are the relative tumor volume (RTV) or tumor weight (TW) of the treatment group and the control group at a specific time point, respectively).
  • LI6669 was established from a liver cancer focus in an Asian patient Orthotopic transplantation model. This model has a certain tendency of the tumor to rupture.
  • liver cancer orthotopic transplantation model LI6669 tumor-bearing mice were harvested from tumor tissues, cut into 2-3mm diameter tumor masses and inoculated on the upper left lobe of the liver in Balb/c nude mice.
  • mice When the average tumor volume of the tumor-bearing mice reached about 50-150 mm 3 , the mice were randomly divided into groups according to Table 3 in "3. Experimental Design" based on body weight. The day of grouping was set as Day 0, and the administration began on Day 1. The day of grouping was defined as day 0.
  • mice were grouped based on body weight using StudyDirectorTM (version number 3.1.399.19, supplier Studylog System, Inc., S. San Francisco, CA, USA).
  • routine monitoring includes tumor growth and the effect of treatment on the normal behavior of animals.
  • the specific content includes the activity of experimental animals, feeding and drinking conditions, weight gain or loss, eyes, coat and other abnormalities.
  • the clinical symptoms observed during the test were recorded in the raw data. After the start of dosing, the body weight of the mice was measured twice a week. Observe and record mice requiring euthanasia and corresponding survival periods.
  • StudyDirectorTM version number 3.1.399.19, supplier Studylog System, Inc.
  • the original data was directly imported into the software after being measured by a balance and a vernier caliper, and the data Any changes will be recorded in this software.
  • the entire process of drug administration, tumor measurement and body weight weighing is carried out in a biological safety cabinet or ultra-clean workbench.
  • mice Median survival period and p-value of mice in each treatment group and control group at the end point of treatment in LI6669 human liver cancer orthotopic transplantation tumor model
  • the inhibitory effect of Erbitux on the proliferation of the A431 cell line was used as a positive control to explore the inhibitory effect of the recombinant protein shown in SEQ ID NO: 14 on the proliferation of various cancer cell lines.
  • the Promega Cell Titer-Glo kit was used to detect cell viability.
  • the reagent contains recombinant luciferase and luciferin, and the luciferase catalyzes the oxidation reaction of the substrate and releases a luminescence signal. This oxidation reaction relies on the ATP released after the living cells are lysed, and the luminescent signal generated by the reaction can indirectly reflect the number of living cells.
  • the results are shown in Table 10.
  • the concentration of the recombinant protein shown in SEQ ID NO: 14 was 0.1 mg/mL, and it was effective for 5637, HCI-H292, Calu-1, ACHN, AsPC-1, OVCAR8, SK-N-AS , Bel-7402, SK-HEP-1, MDA-MB-468, A431, HEP G2, MDA-MB-453, HLE, A549 and SNU-398 all have certain growth inhibitory effects.
  • test product i.e. the recombinant protein shown in SEQ ID NO: 14
  • concentration of the preparation used was 2.5 mg/mL.
  • Red blood cells collected from a healthy person were prepared into a 2% (v/v) suspension with sodium chloride injection.
  • test product with a preparation concentration of 2.5 mg/mL has no hemolytic effect on human erythrocytes in vitro, and does not cause erythrocyte aggregation.
  • Bama miniature pigs (20/sex) were randomly divided into 4 groups, 5/sex in each group.
  • the 1st group is given placebo as adjuvant control, and the 2nd, 3rd, 4th groups are given test article (recombinant protein shown in SEQ ID NO:14) 0.5mg/kg, 1.5mg/kg, 5.0mg/kg respectively.
  • Intravenous infusion once a day, for 28 consecutive days. The dosage volume is 2mL/kg, and the infusion rate is 30mL/kg/h.
  • the first 3 animals/sex/group were euthanized 4 weeks after administration (D29), and the remaining 2 animals of each sex in each group were euthanized 4 weeks after the last administration (D57).
  • a single intravenous injection of the test product at doses of 0.5, 2, and 8 mg/kg has no significant effect on the respiratory system function of SD rats.
  • test product has no significant effect on the central nervous system function of SD rats at doses of 0.5, 2, and 8 mg/kg.
  • the heart rate increased 0.5h-6h after administration of the test product at 3mg/kg. 2h-4h QTcB interval is shortened. 0.5h-6h T wave voltage rises.
  • ECG indicators QRS duration, QRS voltage, ST segment voltage, Tp-e interval, P wave width
  • blood pressure indicators systolic blood pressure, diastolic blood pressure, mean arterial pressure, pulse pressure difference
  • test product has no potential toxic target organs and no irreversible toxicity within the effective dosage range; it has no significant effect on the central nervous system, respiratory system and cardiovascular system.

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Abstract

涉及一种重组蛋白，所述重组蛋白包含间隔肽和重组灵芝免疫调节蛋白突变体(rLZ-8突变体)，其中与SEQ ID NO：10所示的氨基酸序列相比，所述rLZ-8突变体的氨基酸序列包含至少一个氨基酸突变。还涉及所述重组蛋白在治疗疾病中的应用。

Description

重组灵芝免疫调节蛋白新突变体及其应用 技术领域
本申请涉及生物医药领域，具体的涉及一种重组灵芝免疫调节蛋白的突变体(rLZ-8突变体)，以及其在***中的应用。
背景技术
灵芝免疫调节蛋白(LZ-8)是在1989年，Kino等人从赤灵芝菌丝体提取物中分离纯化所得。提取获得的LZ-8含有1.3％的多糖，在体外具有促有丝***活性；在体内具有免疫调节活性。同时，LZ-8对绵羊红细胞具有凝集作用，但对人红细胞(A、B、AB和O型)并没有凝集作用。在体内研究发现，如果反复给药，LZ-8可防止小鼠***性过敏反应的产生。
重组灵芝免疫调节蛋白(rLZ-8)是通过基因重组技术获得的。与天然的灵芝免疫调节蛋白相比，氨基酸序列相同、活性相似但并不含有多糖。研究显示，rLZ-8不仅在体外能快速高效诱导多种肿瘤细胞凋亡，而且在体内小鼠肿瘤模型也能有效杀死肿瘤细胞，并能保持或升高白细胞的水平。除此之外，rLZ-8在治疗组织纤维化、局灶性脑缺血、血小板减少症、骨质疏松和心力衰竭等方面的作用也已有相关专利公布授权。
重组灵芝免疫调节蛋白突变体(rLZ-8突变体)是通过对rLZ-8某个或者某几个氨基酸突变而来的。机制研究发现rLZ-8突变体可以与人体细胞表面的表皮生长因子受体(EGFR)结合内化进入细胞，由于不能被降解而滞留在细胞内。通过高强度内化进而阻滞细胞膜循环最终使细胞皱缩、破裂和死亡。肿瘤细胞杀伤实验结果表明，rLZ-8突变体比rLZ-8具有更好的肿瘤杀伤效果。Biacore研究结果表明，rLZ-8突变体与EGFR具有更好的亲和力；多种人源癌细胞原位移植瘤模型小鼠实验结果表明，rLZ-8突变体对延长小鼠生存期、抑制肿瘤生长均优于rLZ-8。
然而，虽然rLZ-8和rLZ-8突变体都表现出了肿瘤杀伤作用，但都面临低蛋白表达水平，产业化难的挑战。另一方面，蛋白分子对于人类是否安全也是药品开发需要考量的重点。
发明内容
本申请提供了一种重组蛋白，所述重组蛋白包含间隔肽和重组灵芝免疫调节蛋白突变体(rLZ-8突变体)，其中与SEQ ID NO：10所示的氨基酸序列相比，所述rLZ-8突变体的氨基酸序列包含至少一个氨基酸突变。本申请的重组蛋白具有选自下组的至少一种特点：(1) 较高的表达量和/或分泌量(例如，在酵母表达***中)；(2)具有较高的安全性，满足成药的安全性要求；例如，已通过临床前安全评估，和/或，获得临床试验许可(3)容易通过内化进入细胞；和(4)抑制肿瘤生长，杀伤肿瘤细胞能力强。
一方面，本申请提供了一种重组蛋白，所述重组蛋白包含间隔肽和重组灵芝免疫调节蛋白突变体(rLZ-8突变体)，其中与SEQ ID NO：10所示的氨基酸序列相比，所述rLZ-8突变体的氨基酸序列包含至少一个氨基酸突变。
在某些实施方式中，所述rLZ-8突变体包含SEQ ID NO：13和16-21中任一项所示的氨基酸序列。
在某些实施方式中，所述rLZ-8突变体包含SEQ ID NO：13所示的氨基酸序列。
在某些实施方式中，所述间隔肽包含1-5个EA。
在某些实施方式中，所述间隔肽包含2个EA。
在某些实施方式中，所述间隔肽包含SEQ ID NO：8-9中任一项所示的氨基酸序列。
在某些实施方式中，所述间隔肽包含SEQ ID NO：8所示的氨基酸序列。
在某些实施方式中，所述间隔肽位于所述rLZ-8或其突变体的N端。
在某些实施方式中，所述重组蛋白包含SEQ ID NO：14、15、22-33中任一项所示的氨基酸序列。
在某些实施方式中，所述重组蛋白包含SEQ ID NO：14所示的氨基酸序列。
另一方面，本申请提供了一种分离的核酸分子，所述分离的核酸分子编码所述重组蛋白。
另一方面，本申请提供了一种载体，所述载体包含所述分离的核酸分子。
另一方面，本申请提供了一种细胞，所述细胞包含所述分离的核酸分子和/或所述载体。
在某些实施方式中，所述细胞为真核细胞。
在某些实施方式中，所述细胞为酵母细胞。
另一方面，本申请提供了一种多肽，所述多肽包含所述的重组蛋白。
另一方面，本申请提供了一种制备所述的重组蛋白的方法，所述方法包括在使得所述的重组蛋白表达的条件下，培养所述的细胞。
另一方面，本申请提供了一种药物组合物，所述药物组合物包含所述的重组蛋白、所述的分离的核酸分子、所述的载体、所述的多肽和/或所述的细胞，以及任选地药学上可接受的载体。
另一方面，本申请提供了一种试剂盒，所述试剂盒包含所述的重组蛋白、所述的分离的核酸分子、所述的载体、所述的细胞、所述的多肽和/或所述的药物组合物。
另一方面，本申请提供了一种给药装置，所述给药装置所述的重组蛋白、所述的分离的核酸分子、所述的载体、所述的细胞、所述的多肽和/或所述的药物组合物。
另一方面，本申请提供了一种预防、缓解或***的方法，所述方法包括向有需要的受试者施用所述的重组蛋白、所述的分离的核酸分子、所述的载体、所述的细胞、所述的多肽和/或所述的药物组合物。
另一方面，本申请提供了所述的重组蛋白、所述的分离的核酸分子、所述的载体、所述的细胞、所述的多肽和/或所述的药物组合物在制备药物中的用途，所述药物用于预防、缓解或***。
另一方面，本申请提供了所述的重组蛋白、所述的分离的核酸分子、所述的载体、所述的细胞、所述的多肽和/或所述的药物组合物，其用于预防、缓解或***。
在某些实施方式中，所述肿瘤包括非实体瘤。
在某些实施方式中，所述肿瘤包括实体瘤。
在某些实施方式中，所述肿瘤包括具有EGFR表达和/或EGFR表达异常的肿瘤。
在某些实施方式中，所述肿瘤包括与EGFR突变相关的肿瘤。在某些实施方式中，所述肿瘤包括与BRAF突变相关的肿瘤。在某些实施方式中，所述肿瘤包括与KRAS突变相关的肿瘤。在某些实施方式中，所述肿瘤包括与HER2突变相关的肿瘤。
在某些实施方式中，所述肿瘤包括结直肠癌、肺癌、肝癌、乳腺癌、胃癌、肾癌、膀胱癌、神经母细胞瘤、卵巢癌、上皮鳞癌和/或胰腺癌。本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1显示的是多个酵母菌株表达rLZ-8(SEQ ID NO:10)的结果，其中，M：marker；S：EAEA+rLZ-8突变体(SEQ ID NO:14)工作参比品(1mg/ml)；1-11：1-11号rLZ-8(SEQ ID NO:10)菌株。
图2显示的是多个酵母菌株表达rLZ-8突变体(SEQ ID NO:13)的结果，其中，M： marker；S：EAEA+rLZ-8突变体(SEQ ID NO:14)工作参比品(1mg/ml)；1-11：1-11号rLZ-8突变体(SEQ ID NO:13)菌株。
图3显示的是多个酵母菌株表达EAEA+rLZ-8(SEQ ID NO:11)重组蛋白的结果，其中，M：marker；S：r LZ-8(SEQ ID NO:10)工作参比品(0.5mg/ml)；1-6：1-6号EAEA+rLZ-8(SEQ ID NO:11)菌株。
图4显示的是多个酵母菌株表达EAEA+rLZ-8突变体(SEQ ID NO:14)重组蛋白的结果，其中，M：marker；S：r LZ-8(SEQ ID NO:10)标准品(250mg/L)；1-6：1-6号EAEA+rLZ-8突变体(SEQ ID NO:14)菌株。
图5显示的是多个酵母菌株表达EEAEAEAEPK+rLZ-8(SEQ ID NO:12)重组蛋白的结果，其中，M：marker；S：EAEA+r LZ-8突变体(SEQ ID NO:14)工作参比品(1mg/ml)；1-11：1-11号EEAEAEAEPK+rLZ-8(SEQ ID NO:12)菌株。
图6显示的是多个酵母菌株表达EEAEAEAEPK+rLZ-8突变体(SEQ ID NO:15)重组蛋白的结果，其中，M：marker；S：EAEA+r LZ-8突变体(SEQ ID NO:14)工作参比品(1mg/ml)；1-11：1-11号EEAEAEAEPK+rLZ-8突变体(SEQ ID NO:15)菌株。
图7显示的是不同间隔肽连接的rLZ-8或其突变体的重组蛋白的表达结果的比较，其中，M：marker；S：EAEA+r LZ-8突变体(SEQ ID NO:14)工作参比品(1mg/ml)；1：rLZ-8突变体(SEQ ID NO:13)；2：EAEA+rLZ-8突变体(SEQ ID NO:14)；3：EEAEAEAEPK+rLZ-8突变体(SEQ ID NO:15)；4：rLZ-8(SEQ ID NO:10)；5：EAEA+rLZ-8(SEQ ID NO:11)；6：EEAEAEAEPK+rLZ-8(SEQ ID NO:12)。
图8显示的是SEQ ID NO:14所示重组蛋白作用于CR20035B类器官系的剂量反应曲线。
图9显示的是SEQ ID NO:14所示重组蛋白作用于CR5043B类器官系的剂量反应曲线。
图10显示的是SEQ ID NO:14所示重组蛋白作用于CR5082B类器官系的剂量反应曲线。
图11显示的是SEQ ID NO:14所示重组蛋白作用于CR3099B类器官系的剂量反应曲线。
图12显示的是SEQ ID NO:14所示重组蛋白作用于LU5162B类器官系的剂量反应曲线。
图13显示的是SEQ ID NO:14所示重组蛋白作用于LU11624B类器官系的剂量反应曲线。
图14显示的是SEQ ID NO:14所示重组蛋白作用于LU1235B类器官系的剂量反应曲线。
图15显示的是SEQ ID NO:14所示重组蛋白作用于LI6677B类器官系的剂量反应曲线。
图16显示的是SEQ ID NO:14所示重组蛋白作用于LI6669B类器官系的剂量反应曲线。
图17显示的是SEQ ID NO:14所示重组蛋白作用于BR9457B类器官系的剂量反应曲线。
图18显示的是SEQ ID NO:14所示重组蛋白作用于BR9466B类器官系的剂量反应曲线。
图19显示的是SEQ ID NO:14所示重组蛋白作用于GA6833B类器官系的剂量反应曲线。
图20显示的是SEQ ID NO:14所示重组蛋白作用于GA2434B类器官系的剂量反应曲线。
图21显示的是SEQ ID NO:14所示重组蛋白作用于PA20078B类器官系的剂量反应曲线。
图22显示的是SEQ ID NO:14所示重组蛋白作用于PA0787B类器官系的剂量反应曲线。
图23显示的是LI6669人肝癌原位移植肿瘤模型中各治疗组和对照组小鼠生存率变化。
图24显示的是rLZ-8突变体的重组蛋白的对肺癌细胞系A549的杀伤作用。
图25显示的是不同间隔肽连接的rLZ-8或其突变体的重组蛋白的表达结果的比较，其中，M：marker；1：rLZ-8突变体4；2：EAEA+rLZ-8突变体4(SEQ ID NO:25)；3：EEAEAEAEPK+rLZ-8突变体4(SEQ ID NO:31)；4：rLZ-8突变体5；5：EAEA+rLZ-8突变体5(SEQ ID NO:26)；6：EEAEAEAEPK+rLZ-8突变体5(SEQ ID NO:32)；7：rLZ-8突变体6；8：EAEA+rLZ-8突变体6(SEQ ID NO:27)；9：EEAEAEAEPK+rLZ-8突变体6(SEQ ID NO:33)。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，本领域技术人员可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“重组蛋白”通常是指使用重组技术产生的蛋白质。重组蛋白可包括(1)利用重组DNA技术连接的不同来源的DNA分子组合表达产生的半合成或合成的多肽；(2)半合成或合成起源的多肽，该多肽不与其在自然状态下关联的部分相关联；(3)半合成或合成起源的多肽，该多肽与其在自然状态下连接的多肽以外的多肽连接；或(4)在自然状态下不存在的半合成或合成起源的多肽。例如，所述重组蛋白可以指本申请的间隔肽和本申请的重组灵芝免疫调节蛋白突变体(rLZ-8突变体)连接形成的多肽。
在本申请中，术语“间隔肽”通常是指起连接任何结构域功能的任何寡肽或多肽。在本申请中，间隔肽位于重组灵芝免疫调节蛋白(rLZ-8)N端。例如，间隔肽可以位于前导肽和重组灵芝免疫调节蛋白(rLZ-8)之间。例如，间隔肽可以是信号肽的一部分。例如，间隔肽可以有利于与其相连的蛋白质的分泌。
在本申请中，术语“灵芝免疫调节蛋白”也可称为LZ-8，LZ8，Ling Zhi-8或灵芝蛋白8，通常是指一种来源于灵芝的免疫调节蛋白。LZ-8还包含其等位基因变体、剪接变体、衍生变体、取代变体、缺失变体和/或***变体(包括N末端蛋氨酸的添加)、融合多肽和种间同源物。在本申请中，所述LZ-8可以指来源于Ganoderma lucidum的免疫调节蛋白。在本申请中，术语“重组灵芝免疫调节蛋白(rLZ-8)”通常是指使用重组方法得到的LZ-8。例如，所述LZ-8和rLZ-8的氨基酸序列可参见UniProtKB登录号P14945。在本申请中，所述LZ-8和rLZ-8可包含SEQ ID NO:10所示的氨基酸序列。
在本申请中，术语“突变体”通常是指因含有一个或多个差异而与参比序列不同的序列。该参比序列可以是重组灵芝免疫调节蛋白的氨基酸序列。该差异可以是氨基酸的缺失、***或优选取代。突变体可以具有与参比序列相同或不同功能作用。
在本申请中，术语“氨基酸突变”通常涵盖一个或多个氨基酸的取代、缺失、***和修饰。
在本申请中，术语“EA”通常是指谷氨酸(E)和丙氨酸(A)通过肽键连接而成的肽片段。
在本申请中，术语“分离的”通常是指大体上不含其天然存在的环境中通常伴随或与之相互作用的组分的生物材料(例如病毒、核酸或蛋白质)。
在本申请中，术语“分离的核酸分子”通常是指基因组、mRNA、cDNA或合成来源的DNA或RNA或其一定组合，其不与在自然界中发现的多核苷酸的全部或一部分缔合，或连接至其在自然界中不连接的多核苷酸。
在本申请中，术语“载体”通常是指能够在合适的宿主中自我复制的核酸分子，其将***的核酸分子转移到宿主细胞中和/或宿主细胞之间。所述载体可包括主要用于将DNA或RNA***细胞中的载体、主要用于复制DNA或RNA的载体，以及主要用于DNA或RNA的转录和/或翻译的表达的载体。所述载体还包括具有多种上述功能的载体。所述载体可以是当引入合适的宿主细胞时能够转录并翻译成多肽的多核苷酸。通常，通过培养包含所述载体的合适的宿主细胞，所述载体可以产生期望的表达产物。
在本申请中，术语“细胞”通常是指可以或已经含有包括本申请所述的核酸分子的质粒或载体，或者能够表达本申请所述的抗体或其抗原结合片段的个体细胞、细胞系或细胞培养物。所述细胞可以包括单个宿主细胞的子代。由于天然的、意外的或故意的突变，子代细胞与原始亲本细胞在形态上或在基因组上可能不一定完全相同，但能够表达本申请所述的抗体或其抗原结合片段即可。所述细胞可以通过使用本申请所述的载体体外转染细胞而得到。所 述细胞可以是原核细胞(例如大肠杆菌)，也可以是真核细胞(例如酵母细胞，例如COS细胞，中国仓鼠卵巢(CHO)细胞，HeLa细胞，HEK293细胞，COS-1细胞，NS0细胞或骨髓瘤细胞)。在某些情形中，所述细胞可以是哺乳动物细胞。例如，所述哺乳动物细胞可以是CHO-K1细胞。在本申请中，术语“重组细胞”通常是指在其中引入了重组表达载体的细胞。所述重组宿主细胞不仅包括某种特定的细胞，还包括这些细胞的后代。
在本申请中，术语“试剂盒”通常是指包含用于给予本申请的重组蛋白治疗具有EGFR表达和/或EGFR异常表达相关病症的组分的包装产品。试剂盒的组分可包含在分开的小瓶中(即具有分开部分的试剂盒)，或在单个小瓶内提供。试剂盒可包含试剂，诸如缓冲剂、蛋白稳定试剂、信号产生体系(例如，荧光信号生成体系)、抗体、对照蛋白、以及测试容器。试剂盒还可以包含实施所述方法的说明书。
在本申请中，术语“给药装置”包括：(i)输注模块，所述输注模块用于对受试者施用包括具有一活性成分的药物组合物；(ii)用于输注的药物组合物，所述药物组合物中含有一活性成分，所述活性成分选自下组：重组蛋白、核酸分子、细胞、载体或其组合；以及(iii)任选的药效监控模块。
在本申请中，术语“肿瘤”通常是指所有赘生性细胞生长和增殖(无论恶性还是良性)以及所有癌前和癌性细胞和组织。肿瘤可包括实体瘤和/或非实体瘤(例如，血液瘤、淋巴瘤)。
在本申请中，术语“药学上可接受的载体”通常包括药剂学可接受的载体、赋形剂或稳定剂，它们在所采用的剂量和浓度对暴露于其的细胞或哺乳动物是无毒的。通常，生理学可接受的载体是pH缓冲水溶液。生理学可接受载体的例子可包括缓冲剂，抗氧化剂，低分子量(少于约10个残基)多肽，蛋白质，亲水性聚合物，氨基酸，单糖，二糖和其它碳水化合物，螯合剂，糖醇，成盐反荷离子，诸如钠；和/或非离子表面活性剂。
在本申请中，术语“治疗”通常是指期望改变所治疗个体的天然病程，且可为实现防治或在临床病变过程中进行的临床介入。合乎需要的治疗效果包括但不限于防止疾病发生或复发性、减轻症状、减弱疾病的任何直接或间接病理学后果、防止转移、降低疾病进展速率、改善或缓解疾病状态以及缓和或改善预后。在一些情形中，本申请的重组载体可用来延迟疾病发展或减缓疾病进展。
在本申请中，术语“施用”通常是指向受试者(例如，患者)给予一定剂量的化合物(例如，抗癌治疗剂)或药物组合物(例如，包含抗癌治疗剂的药物组合物)的方法。施用可通过任何合适的方式进行，包括肠胃外、肺内和鼻内，以及(如果局部治疗需要)损伤内施用。 胃肠外输注包括例如肌肉内、静脉内、动脉内、腹膜内或皮下施用。
除了本文提到的特定蛋白质和核苷酸之外，本申请还可包括其功能性变体、衍生物、类似物、同源物及其片段。
术语“功能性变体”指与天然存在序列具有基本上同源的氨基酸序列或由基本上同源的核苷酸序列编码并能够具有天然存在序列的一种或多种活性的多肽。在本申请的上下文中，任何给定序列的变体是指其中残基的特定序列(无论是氨基酸或核苷酸残基)已经经过修饰而使得所述多肽或多核苷酸基本上保留至少一种内源功能的序列。可以通过天然存在的蛋白质和/或多核苷酸中存在的至少一个氨基酸残基和/或核苷酸残基的添加、缺失、取代、修饰、替换和/或变异来获得变体序列，只要保持原来的功能活性即可。
在本申请中，术语“衍生物”通常是指本申请的多肽或多核苷酸而言包括自/对序列的一个(或多个)氨基酸残基的任何取代、变异、修饰、替换、缺失和/或添加，只要所得的多肽或多核苷酸基本上保留其至少一种内源功能。在本申请中，术语“类似物”通常对多肽或多核苷酸而言，包括多肽或多核苷酸的任何模拟物，即拥有该模拟物模拟的多肽或多核苷酸的至少一种内源功能的化学化合物。通常，可以进行氨基酸取代，例如至少1个(例如，1、2、3、4、5、6、7、8、9、10或20个以上)氨基酸取代，只要经修饰的序列基本上保持需要的活性或能力。氨基酸取代可包括使用非天然存在的类似物。
用于本申请的蛋白质或多肽也可以具有氨基酸残基的缺失、***或取代，所述氨基酸残基产生沉默的变化并导致功能上等同的蛋白质。可以根据残基的极性、电荷、溶解性、疏水性、亲水性和/或两性性质的相似性进行有意的氨基酸取代，只要保留内源性功能即可。例如，带负电荷的氨基酸包括天冬氨酸和谷氨酸；带正电荷的氨基酸包括赖氨酸和精氨酸；并且含具有相似亲水性值的不带电极性头基的氨基酸包括天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸和酪氨酸。
在本申请中，术语“同源物”通常是指与野生型氨基酸序列和野生型核苷酸序列具有一定同源性的氨基酸序列或核苷酸序列。术语“同源性”可以等同于序列“同一性”。同源序列可以包括可以与主题序列是至少80％、85％、90％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％相同的氨基酸序列。通常，同源物将包含与主题氨基酸序列相同的活性位点等。同源性可以根据相似性(即具有相似化学性质/功能的氨基酸残基)来考虑，也可以在序列同源性方面表达同源性。在本申请中，提及的氨基酸序列或核苷酸序列的SEQ ID NO中的任一项具有百分比同源性的序列是指在所提及的SEQ ID NO的整个长度上具有所述百分比同源性的序列。
为了确定序列同源性，可进行序列比对，其可通过本领域技术人员了解的各种方式进行，例如，使用BLAST、BLAST-2、ALIGN、NEEDLE或Megalign(DNASTAR)软件等。本领域技术人员能够确定用于比对的适当参数，包括在所比较的全长序列中实现最优比对所需要的任何算法。
在本申请中，术语“和/或”应理解为意指可选项中的任一项或可选项的两项。
在本申请中，术语“包含”通常是指包括明确指定的特征，但不排除其他要素。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
发明详述
重组蛋白
一方面，本申请提供了一种重组蛋白，所述重组蛋白包含间隔肽和重组灵芝免疫调节蛋白(rLZ-8)的突变体，其中所述rLZ-8可以包含SEQ ID NO：10所示的氨基酸序列。在本申请中，所述rLZ-8可以包含与SEQ ID NO：10所示的氨基酸序列具有至少80％(例如，至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或更高)同源性的氨基酸序列。
一方面，本申请提供了一种重组蛋白，所述重组蛋白包含间隔肽和重组灵芝免疫调节蛋白突变体(rLZ-8突变体)，其中与SEQ ID NO：10所示的氨基酸序列相比，所述rLZ-8突变体的氨基酸序列可以包含至少一个氨基酸突变。
在本申请中，所述重组蛋白包含间隔肽和重组灵芝免疫调节蛋白突变体(rLZ-8突变体)，其中与SEQ ID NO：10所示的氨基酸序列相比，所述rLZ-8突变体的氨基酸序列可以包含一个氨基酸突变。
在本申请中，所述重组蛋白包含间隔肽和重组灵芝免疫调节蛋白突变体(rLZ-8突变体)，其中与SEQ ID NO：10所示的氨基酸序列相比，所述rLZ-8突变体的氨基酸序列可以包含两个氨基酸突变。
在本申请中，所述重组蛋白包含间隔肽和重组灵芝免疫调节蛋白突变体(rLZ-8突变体)，其中与SEQ ID NO：10所示的氨基酸序列相比，所述rLZ-8突变体的氨基酸序列可以包含一个氨基酸突变，所述一个氨基酸突变可以位于选自下组的氨基酸位置处：R9、L17、D20、D70、K46和K74。例如，所述一个氨基酸突变可以位于选自下组的氨基酸位置处：D70、L17、 K74和K46。
在本申请中，L17处的氨基酸突变可以为L17K。
在本申请中，D70处的氨基酸突变可以为D70K。
在本申请中，K46处的氨基酸突变可以为K46E。
在本申请中，K74处的氨基酸突变可以为K74E。
在本申请中，D20处的氨基酸突变可以为D20H。
在本申请中，R9处的氨基酸突变可以为R9A。
在本申请中，所述重组蛋白包含间隔肽和重组灵芝免疫调节蛋白突变体(rLZ-8突变体)，其中与SEQ ID NO：10所示的氨基酸序列相比，所述rLZ-8突变体的氨基酸序列可以包含一个氨基酸突变，且所述一个氨基酸突变可以选自下组：R9A、L17K、D20H、K46E、D70K和K74E。又例如，所述一个氨基酸突变可以选自下组：L17K、K46E、D70K和K74E。
在本申请中，所述重组蛋白包含间隔肽和重组灵芝免疫调节蛋白突变体(rLZ-8突变体)，其中与SEQ ID NO：10所示的氨基酸序列相比，所述rLZ-8突变体的氨基酸序列可以包含一个氨基酸突变，且所述一个氨基酸突变可以选自下组：L17K、D70K和K46E。
在本申请中，所述重组蛋白包含间隔肽和重组灵芝免疫调节蛋白突变体(rLZ-8突变体)，其中与SEQ ID NO：10所示的氨基酸序列相比，所述rLZ-8突变体的氨基酸序列可以包含两个氨基酸突变，且所述两个氨基酸突变可以选自下组：K46E和K74E。又例如，所述两个氨基酸突变可以选自下组：L17K和D70K。
例如，所述rLZ-8突变体可以包含SEQ ID NO：13和16-21中任一项所示的氨基酸序列。例如，所述rLZ-8突变体可以包含与SEQ ID NO：13和16-21中任一项所示的氨基酸序列具有至少80％(例如，至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或更高)同源性的氨基酸序列。
在本申请中，所述rLZ-8突变体可以包含SEQ ID NO：13所示的氨基酸序列。例如，所述rLZ-8突变体可以包含与SEQ ID NO：13所示的氨基酸序列具有至少80％(例如，至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或更高)同源性的氨基酸序列。在本申请中，所述rLZ-8突变体的氨基酸序列可以如SEQ ID NO：13所示。
在本申请中，所述重组蛋白可包含间隔肽和rLZ-8，且所述rLZ-8可包含SEQ ID NO:10所示的氨基酸序列，所述间隔肽可包含1-5个EA。例如，所述间隔肽可包含2个EA。
在本申请中，所述重组蛋白可包含间隔肽和rLZ-8突变体，且所述rLZ-8突变体可以包含SEQ ID NO：13和16-21中任一项所示的氨基酸序列，所述间隔肽可包含1-5个EA。例 如，所述间隔肽可包含2个EA。
在本申请中，所述重组蛋白可包含间隔肽和rLZ-8突变体，且所述rLZ-8突变体可包含SEQ ID NO：13所示的氨基酸序列，所述间隔肽可包含SEQ ID NO：8所示的氨基酸序列。例如，所述重组蛋白可包含SEQ ID NO:14所示的氨基酸序列。
在本申请中，所述重组蛋白可包含间隔肽和rLZ-8突变体，且所述rLZ-8突变体可包含SEQ ID NO：16所示的氨基酸序列，所述间隔肽可包含SEQ ID NO：8所示的氨基酸序列。例如，所述重组蛋白可包含SEQ ID NO:22所示的氨基酸序列。
在本申请中，所述重组蛋白可包含间隔肽和rLZ-8突变体，且所述rLZ-8突变体可包含SEQ ID NO：17所示的氨基酸序列，所述间隔肽可包含SEQ ID NO：8所示的氨基酸序列。例如，所述重组蛋白可包含SEQ ID NO:23所示的氨基酸序列。
在本申请中，所述重组蛋白可包含间隔肽和rLZ-8突变体，且所述rLZ-8突变体可包含SEQ ID NO：18所示的氨基酸序列，所述间隔肽可包含SEQ ID NO：8所示的氨基酸序列。例如，所述重组蛋白可包含SEQ ID NO:24所示的氨基酸序列。
在本申请中，所述重组蛋白可包含间隔肽和rLZ-8突变体，且所述rLZ-8突变体可包含SEQ ID NO：19所示的氨基酸序列，所述间隔肽可包含SEQ ID NO：8所示的氨基酸序列。例如，所述重组蛋白可包含SEQ ID NO:25所示的氨基酸序列。
在本申请中，所述重组蛋白可包含间隔肽和rLZ-8突变体，且所述rLZ-8突变体可包含SEQ ID NO：20所示的氨基酸序列，所述间隔肽可包含SEQ ID NO：8所示的氨基酸序列。例如，所述重组蛋白可包含SEQ ID NO:26所示的氨基酸序列。
在本申请中，所述重组蛋白可包含间隔肽和rLZ-8突变体，且所述rLZ-8突变体可包含SEQ ID NO：21所示的氨基酸序列，所述间隔肽可包含SEQ ID NO：8所示的氨基酸序列。例如，所述重组蛋白可包含SEQ ID NO:27所示的氨基酸序列。
在本申请中，所述重组蛋白可包含间隔肽和rLZ-8突变体，且所述rLZ-8突变体可包含SEQ ID NO：13所示的氨基酸序列，所述间隔肽可包含SEQ ID NO：9所示的氨基酸序列。例如，所述重组蛋白可包含SEQ ID NO:15所示的氨基酸序列。
在本申请中，所述重组蛋白可包含间隔肽和rLZ-8突变体，且所述rLZ-8突变体可包含SEQ ID NO：16所示的氨基酸序列，所述间隔肽可包含SEQ ID NO：9所示的氨基酸序列。例如，所述重组蛋白可包含SEQ ID NO:28所示的氨基酸序列。
在本申请中，所述重组蛋白可包含间隔肽和rLZ-8突变体，且所述rLZ-8突变体可包含SEQ ID NO：17所示的氨基酸序列，所述间隔肽可包含SEQ ID NO：9所示的氨基酸序列。 例如，所述重组蛋白可包含SEQ ID NO:29所示的氨基酸序列。
在本申请中，所述重组蛋白可包含间隔肽和rLZ-8突变体，且所述rLZ-8突变体可包含SEQ ID NO：18所示的氨基酸序列，所述间隔肽可包含SEQ ID NO：9所示的氨基酸序列。例如，所述重组蛋白可包含SEQ ID NO:30所示的氨基酸序列。
在本申请中，所述重组蛋白可包含间隔肽和rLZ-8突变体，且所述rLZ-8突变体可包含SEQ ID NO：19所示的氨基酸序列，所述间隔肽可包含SEQ ID NO：9所示的氨基酸序列。例如，所述重组蛋白可包含SEQ ID NO:31所示的氨基酸序列。
在本申请中，所述重组蛋白可包含间隔肽和rLZ-8突变体，且所述rLZ-8突变体可包含SEQ ID NO：20所示的氨基酸序列，所述间隔肽可包含SEQ ID NO：9所示的氨基酸序列。例如，所述重组蛋白可包含SEQ ID NO:32所示的氨基酸序列。
在本申请中，所述重组蛋白可包含间隔肽和rLZ-8突变体，且所述rLZ-8突变体可包含SEQ ID NO：21所示的氨基酸序列，所述间隔肽可包含SEQ ID NO：9所示的氨基酸序列。例如，所述重组蛋白可包含SEQ ID NO:33所示的氨基酸序列。
在本申请中，所述间隔肽可位于所述rLZ-8或其突变体的N端。在本申请中，所述重组蛋白从N端至C端，可依次包含所述间隔肽和所述rLZ-8突变体。
在本申请中，所述重组蛋白可包含间隔肽和rLZ-8，且所述rLZ-8可包含SEQ ID NO：10所示的氨基酸序列，所述间隔肽可包含SEQ ID NO：8所示的氨基酸序列。
在本申请中，所述重组蛋白可包含间隔肽和rLZ-8，且所述rLZ-8可包含SEQ ID NO：10所示的氨基酸序列，所述间隔肽可包含SEQ ID NO：9所示的氨基酸序列。
在本申请中，所述间隔肽可位于所述rLZ-8的N端。在本申请中，所述重组蛋白从N端至C端，可依次包含所述间隔肽和所述rLZ-8。
在本申请中，所述重组蛋白可包含SEQ ID NO：14、15以及22-33中任一项所示的氨基酸序列。在本申请中，所述重组蛋白可包含与SEQ ID NO：14、15以及22-33中任一项所示的氨基酸序列具有至少80％(例如，至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或更高)同源性的氨基酸序列。
例如，所述重组蛋白可包含SEQ ID NO：14所示的氨基酸序列。例如，所述重组蛋白可包含与SEQ ID NO：14所示的氨基酸序列具有至少80％(例如，至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或更高)同源性的氨基酸序列。
核酸、载体和细胞
另一方面，本申请提供了一种多肽，所述多肽包含所述的重组蛋白。在所述多肽中，所 述重组蛋白可以与其他蛋白质或多肽以共价或非共价的形式连接。
另一方面，本申请提供了一种或多种核酸分子，其可以编码本申请所述的重组蛋白和/或多肽。本申请所述的核酸分子可以为分离的。例如，其可以是通过以下方法产生或合成的：(i)在体外扩增的，例如通过聚合酶链式反应(PCR)扩增产生的，(ii)通过克隆重组产生的，(iii)纯化的，例如通过酶切和凝胶电泳分级分离，或者(iv)合成的，例如通过化学合成。在某些实施方式中，所述分离的核酸是通过重组DNA技术制备的核酸分子。
另一方面，本申请提供了一种载体，其可以包含本申请所述的核酸分子。此外，所述载体中还可包含其他基因，例如允许在适当的宿主细胞中和在适当的条件下选择该载体的标记基因。此外，所述载体还可包含允许编码区在适当宿主中正确表达的表达控制元件。这样的控制元件为本领域技术人员所熟知的，例如，可包括启动子、核糖体结合位点、增强子和调节基因转录或mRNA翻译的其他控制元件等。所述载体可以包括，例如质粒、粘粒、病毒、噬菌体或者在例如遗传工程中通常使用的其他载体。例如，所述载体为表达载体。例如，所述载体为适于真核细胞(例如，酵母细胞)表达的载体。
另一方面，本申请提供了一种细胞，其可以包含本申请所述的核酸分子或本申请所述的载体。在某些实施方式中，每种或每个细胞可包含一个或一种本申请所述的核酸分子或载体。在某些实施方式中，每种或每个细胞可包含多个(例如，2个或以上)或多种(例如，2种或以上)本申请所述的核酸分子或载体。例如，可将本申请所述的载体引入所述宿主细胞中，例如真核细胞，如来自植物的细胞、真菌或酵母细胞等。可通过本领域已知的方法将本申请所述的载体引入所述宿主细胞中，例如电穿孔、lipofectine转染、lipofectamin转染等。
另一方面，本申请提供了一种制备所述的重组蛋白的方法，所述方法包括在使得所述的重组蛋白表达的条件下，培养所述的细胞。例如，可通过使用适当的培养基、适当的温度和培养时间等。
药物组合物、试剂盒和给药装置
另一方面，本申请提供了一种药物组合物，所述药物组合物包含所述的重组蛋白、所述的分离的核酸分子、所述的载体、所述的多肽和/或所述的细胞，以及任选地药学上可接受的载体(carrier)。所述药学上可接受的载体在所采用的剂量和浓度下对接受者无毒性，并且可包括缓冲剂，抗氧化剂，防腐剂，低分子量(小于约10个残基)多肽，蛋白质，亲水性聚合物，氨基酸，碳水化合物，成盐反离子，金属络合物，和/或非离子表面活性剂。本申请中的药物组合物还可含有多于一种活性化合物，通常为不会不利地影响彼此的具有互补活性的那些活性化合物。此类药物的类型和有效量取决于例如制剂中存在的拮抗剂的量和类型，以及 受试者的临床参数。本申请所述的药物组合物可以包含预防和/或治疗有效量的所述重组蛋白和/或多肽。所述预防和/或治疗有效量是能够预防和/或治疗(至少部分治疗)患有或具有发展风险的受试者中的疾病或病症和/或其任何并发症而所需的剂量。
另一方面，本申请提供了一种试剂盒，所述试剂盒包含所述的重组蛋白、所述的分离的核酸分子、所述的载体、所述的细胞、所述的多肽和/或所述的药物组合物。其可在单一常用容器中包括本申请所述抗原结合蛋白，载体，核酸分子，细胞，免疫缀合物，和/或本申请所述的药物组合物，也可任选地与一种或多种治疗剂组合，任选地一起配制于试剂盒中。
另一方面，本申请提供了一种给药装置，所述给药装置所述的重组蛋白、所述的分离的核酸分子、所述的载体、所述的细胞、所述的多肽和/或所述的药物组合物。
治疗方法
另一方面，本申请提供了一种预防、缓解或***的方法，所述方法包括向有需要的受试者施用所述的重组蛋白、所述的分离的核酸分子、所述的载体、所述的细胞、所述的多肽和/或所述的药物组合物。
另一方面，本申请提供了所述的重组蛋白、所述的分离的核酸分子、所述的载体、所述的细胞、所述的多肽和/或所述的药物组合物在制备药物中的用途，所述药物用于预防、缓解或***。
另一方面，本申请提供了所述的重组蛋白、所述的分离的核酸分子、所述的载体、所述的细胞、所述的多肽和/或所述的药物组合物，其用于预防、缓解或***。
例如，所述肿瘤可以包括实体瘤核/或非实体瘤(例如，血液瘤)。
例如，所述肿瘤可以为具有EGFR表达的肿瘤。例如，所述肿瘤可以为与EGFR表达异常相关的肿瘤。例如，所述肿瘤可以为具有EGFR过表达的肿瘤。
例如，所述肿瘤可以包括与EGFR突变相关的肿瘤。例如，所述肿瘤可以包括与BRAF突变相关的肿瘤。例如，所述肿瘤可以包括与KRAS突变相关的肿瘤。例如，所述可以肿瘤包括与HER2突变相关的肿瘤。
例如，所述肿瘤可以选自下组中的一种或多种：结直肠癌、肺癌、肝癌、乳腺癌、胃癌、肾癌、膀胱癌、神经母细胞瘤、卵巢癌、上皮鳞癌和胰腺癌。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请发明的各个技术方案，而不用于限制本申请发明的范围。
实施例
实施例1 rLZ-8和rLZ-8突变体的表达载体构建
(1)分别以rLZ-8(氨基酸序列如SEQ ID NO:10所示)DNA和rLZ-8突变体(氨基酸序列如SEQ ID NO:13所示)DNA为模板，正向引物F和反向引物R进行PCR扩增rLZ-8和rLZ-8突变体基因片段。所获取的基因片段不包含EAEA的DNA序列，但在5’端和3’分别带有Xho I和Xba I酶切位点。
正向引物F和反向引物R的信息如下：
正向引物F:CCGCTCGAGAAAAGAATGTCTGATACTGCTTTGATCTTCA(SEQ ID NO:1)
反向引物R:GCTCTAGACTAGTTCCATTGAGCGATA(SEQ ID NO:2)
(2)将rLZ-8(氨基酸序列如SEQ ID NO:10所示)和rLZ-8突变体(氨基酸序列如SEQ ID NO:13所示)基因片段通过Xho I/Xba I双酶切并使用T4DNA连接酶连接到空表达载体中，获得重组表达载体。
实施例2 带有EAEA序列的rLZ-8和rLZ-8突变体的表达载体构建
(1)根据毕赤酵母密码子偏好性，综合考虑密码子适应指数和RNA二级结构分析等方面影响，对EAEA+rLZ-8(氨基酸序列如SEQ ID NO:11所示)和EAEA+rLZ-8突变体(氨基酸序列如SEQ ID NO:14所示)进行了密码子优化。为便于构建表达载体，分别在优化后的DNA序列5’-和3’-端加入Xho I和Xba I限制性酶切位点，DNA序列通过人工合成后装载于pUC57克隆载体，即pUC57-EAEA+rLZ-8和pUC57-EAEA+rLZ-8突变体。
(2)以EAEA+rLZ-8(氨基酸序列如SEQ ID NO:11所示)DNA为模板，正向引物F3和正向引物R3进行PCR扩增rLZ-8基因片段。所获取的基因片段包含EAEA的DNA序列，并在5’端和3’分别带有Xho I和Xba I酶切位点。
正向引物F3和正向引物R3的信息如下：
正向引物F3：CCGCTCGAGAAAAGAGAGGCTGAAGCT(SEQ ID NO:3)
反向引物R3：GCTCTAGATCACTAGTTCCATTG(SEQ ID NO:4)
(3)以EAEA+rLZ-8突变体(氨基酸序列如SEQ ID NO:14所示)DNA为模板，正向引物M13F和反向引物M13R进行PCR扩增rLZ-8突变体基因片段。所获取的基因片段包含EAEA的DNA序列，并在5’端和3’分别带有Xho I和Xba I酶切位点。
正向引物M13F和反向引物M13R的信息如下：
正向引物M13F:CCCAGTCACGACGTTGTAAAACG(SEQ ID NO:5)
反向引物M13R:AGCGGATAACAATTTCACACAGG(SEQ ID NO:6)
(4)将EAEA+rLZ-8和EAEA+rLZ-8突变体基因片段通过Xho I/Xba I双酶切并使用T4DNA连接酶连接到空表达载体中，获得重组表达载体。
以同样的方法，分别构建N端连接氨基酸序列“EAEA”的rLZ-8突变体1(氨基酸序列如SEQ ID NO:16所示)、rLZ-8突变体2(氨基酸序列如SEQ ID NO:17所示)、rLZ-8突变体3(氨基酸序列如SEQ ID NO:18所示)、rLZ-8突变体4(氨基酸序列如SEQ ID NO:19所示)、rLZ-8突变体5(氨基酸序列如SEQ ID NO:20所示)、rLZ-8突变体6(氨基酸序列如SEQ ID NO:21所示)的重组蛋白。
实施例3 带有EEAEAEAEPK序列的rLZ-8和rLZ-8突变体的表达载体构建
(1)分别以EEAEAEAEPK+rLZ-8DNA(氨基酸序列如SEQ ID NO:12所示)和EEAEAEAEPK+rLZ-8突变体(氨基酸序列如SEQ ID NO:15所示)DNA为模板，正向引物F2和反向引物R进行PCR扩增EEAEAEAEPK+rLZ-8和EEAEAEAEPK+rLZ-8突变体基因片段。所获取的基因片段包含EEAEAEAEPK的DNA序列，并且在5’端和3’分别带有Xho I和Xba I酶切位点。
正向引物F2和反向引物R信息如下：
正向引物F2:
Figure PCTCN2022110916-appb-000001
反向引物R:GCTCTAGACTAGTTCCATTGAGCGATA(SEQ ID NO:2)
(2)将EEAEAEAEPK+rLZ-8(氨基酸序列如SEQ ID NO:12所示)和EEAEAEAEPK+rLZ-8突变体(氨基酸序列如SEQ ID NO:15所示)基因片段通过Xho I/Xba I双酶切并使用T4DNA连接酶连接到空表达载体中，获得重组表达载体。
以同样的方法，分别构建N端连接氨基酸序列“EEAEAEAEPK”的rLZ-8突变体1(氨基酸序列如SEQ ID NO:16所示)、rLZ-8突变体2(氨基酸序列如SEQ ID NO:17所示)、rLZ-8突变体3(氨基酸序列如SEQ ID NO:18所示)、rLZ-8突变体4(氨基酸序列如SEQ ID NO:19所示)、rLZ-8突变体5(氨基酸序列如SEQ ID NO:20所示)、rLZ-8突变体6(氨基酸序列如SEQ ID NO:21所示)的重组蛋白。
实施例4 毕赤酵母表达菌株构建
(1)感受态细胞制备：
将毕赤酵母空菌株接种到YPD液体培养基中，28.5℃培养16-18h，直到OD 600＝1.3-1.5之间，进行感受态制备。菌液冰浴30min，在无菌超净台用中4个50mL离心管分装，每管40mL。1500g，4℃离心5min。弃上清，收集菌体，用预冷的灭菌双蒸水160mL充分重悬浮细胞，1500g，4℃离心5min。再次弃上清，收集菌体，用预冷的灭菌双蒸水80mL充分重悬浮细胞，1500g，4℃离心5min。收集菌体，用预冷的灭菌1M山梨醇溶液10mL充分重悬浮细胞，1500g，4℃离心5min。收集菌细胞沉淀，用预冷的灭菌1M山梨醇溶液500μL重悬待用。
(2)重组质粒线性化
将重组质粒用Bsp H I线性化便于转化进入酵母细胞。
(3)电转化
取80μL酵母感受态细胞到2mm电转杯中，加入5-10μg的线性化重组质粒，混匀冰上静置5min。电转化参数设置：V＝1.5kv，u＝25μF，R＝200Ω，放电时间在4.5-4.9ms之间。电转后立即加入1mL 1M Sorbitol，28.5℃度恒温孵1h。取50-200μL菌液涂含Zeocin抗性100μg/mL的YPDS固体培养基平板。28.5℃恒温孵育箱中孵育2-3天。
实施例5 高表达菌株筛选
挑取Zeocin阳性克隆菌落接种于10mL YPD液体培养基进行培养，分光光度计600nm检测OD值，直至OD值≈6。取40μl活化菌液接种于25mL新YPD液体培养基，在28.5℃，225rpm的条件下培养72h。取1mL菌液经13000g 4℃低温离心，取上清液SDS-PAGE电泳检测实施例1-3中构建的6种表达载体中目标蛋白的多个克隆的表达。
高表达酵母菌株的筛选结果如图1-6所示。对于rLZ-8(SEQ ID NO:10)，3号克隆具有较高表达量(图1)；对于rLZ-8突变体(SEQ ID NO:13)，10号克隆具有较高表达量(图2)；对于EAEA+rLZ-8(SEQ ID NO:11)，6号克隆具有较高表达量(图3)；对于EAEA+rLZ-8突变体(SEQ ID NO:14)，7号克隆具有较高表达量(图4)；对于EEAEAEAEPK+rLZ-8(SEQ ID NO:12)，7号克隆具有较高表达量(图5)；对于EEAEAEAEPK+rLZ-8突变体(SEQ ID NO:15)，2号克隆具有较高表达量(图6)。
实施例6 不同间隔序列对rLZ-8和rLZ-8突变体蛋白表达的影响
(1)
根据实施例5的方法比较实施例1-3中构建的多种表达载体中目标蛋白的表达，结果如图7所示。
EAEA对rLZ-8(SEQ ID NO:10)和rLZ-8突变体(SEQ ID NO:13)表达分泌均具有促进作用，并且对rLZ-8突变体(SEQ ID NO:13)的促进作用优于rLZ-8(SEQ ID NO:10)(图7的泳道1、2、4和5)。
EEAEAEAEPK对rLZ-8(SEQ ID NO:10)和rLZ-8突变体(SEQ ID NO:13)表达分泌均具有促进作用，并且EEAEAEAEPK对rLZ-8(SEQ ID NO:10)的促进作用优于rLZ-8突变体(SEQ ID NO:13)(图7的泳道1、3、4和6)。
EAEA和EEAEAEAEPK均具有提高蛋白质表达分泌的作用，且两者对不同蛋白质表达分泌的促进作用不同，其中，EAEA对rLZ-8突变体(SEQ ID NO:13)有最好的促进作用(图7的泳道1、2和3)，而EEAEAEAEPK对rLZ-8(SEQ ID NO:10)有最好的促进作用(图7的泳道4、5和6)。
(2)
根据实施例5的方法，比较实施例1-3中构建的9种表达载体中目标蛋白(基于rLZ-8突变体4～rLZ-8突变体6)的表达，结果如图25所示。
结果说明，EAEA具有提高蛋白质表达分泌的作用，并且对rLZ-8突变体4(SEQ ID NO:19)，rLZ-8突变体5(SEQ ID NO:20)和rLZ-8突变体6(SEQ ID NO:21)具有最好的促进作用。
实施例7 重组蛋白的结构鉴定
本实施例测定EAEA+rLZ-8(SEQ ID NO:11)、EAEA+rLZ-8突变体(SEQ ID NO:14)、EEAEAEAEPK+rLZ-8(SEQ ID NO:12)、EEAEAEAEPK+rLZ-8突变体(SEQ ID NO:15)、rLZ-8(SEQ ID NO:10)和rLZ-8突变体(SEQ ID NO:13)蛋白N端氨基酸序列。重组蛋白用胰蛋白酶水解处理后，经反向色谱将水解产生的肽段进行分离，并由质谱对各肽段氨基酸进行测定，以获得N端氨基酸序列。具体步骤如下：
(1)样品溶液制备
取重组蛋白250μg，加入200μl 10M尿素溶液，并用水补足250μl，混匀后置于恒温混匀仪65℃孵育30min。孵育结束后将样品冷却至室温，经SEC小柱将缓冲溶液置换成0.1M碳酸氢铵溶液，并用微量分光光度计测定最终蛋白质浓度。
(2)酶解
取50μg供试品蛋白，用0.1M碳酸氢铵溶液稀释至0.5μg/μl，加入0.5μg胰蛋白酶，混匀后于37℃孵育20h。孵育结束后，加入等体积0.1％甲酸溶液或置于4℃终止酶解反应；将上述溶液于4℃，12000rpm离心2min，取上清液上样分析检测。
(3)色谱条件及洗脱程序
3.1色谱条件
Figure PCTCN2022110916-appb-000002
3.2洗脱程序
Figure PCTCN2022110916-appb-000003
结果显示，上述重组蛋白的N端氨基酸序列与理论序列均一致。
实施例8 EAEA+rLZ-8突变体与EEAEAEAEPK+rLZ-8突变体对肿瘤细胞杀伤作用检测
采用CellTiterTM-Flour Cell Viability Assay Reagent试剂盒测定EAEA+rLZ-8突变体(SEQ ID NO:14)和EEAEAEAEPK+rLZ-8突变体(SEQ ID NO:15)在体外对肿瘤细胞(人类非小细胞肺癌细胞系A549细胞系)的杀伤作用。将重组蛋白用2％胎牛血清培养液进行稀释，制成浓度为100μg/mL、50μg/mL、25μg/mL、12.5μg/mL、6.25μg/mL、3.125μg/mL、1.5625μg/mL、0.78125μg/mL、0.390625μg/mL、0.1953125μg/mL、0.09765625μg/mL的供试品溶液共11个浓度。
取上述稀释液加入至含有A549细胞的96孔板中，100μL/孔，设每组浓度平行孔为3个，并做标记。设一组细胞对照组(即只含细胞)100μL/孔，平行孔为6个；放入CO 2浓度为5％，温度为37℃的CO 2培养箱中培养48h。
每孔加入100μL配制好的CellTiterTM-Flour Cell Viability Assay Reagent(1mlL Assay Buffer；1μl GF-AFC Substrate)，振板混匀，放置CO 2浓度为5％，温度为37℃的CO 2培养箱中孵育不少于30min，使用酶标仪在400EX/505EM波长下检测荧光值，并计算IC 50值。
结果如图24所示(其中组1和组2分别对应EAEA+rLZ-8突变体(SEQ ID NO:14)和EEAEAEAEPK+rLZ-8突变体(SEQ ID NO:15)的结果)，EAEA+rLZ-8突变体(SEQ ID NO:14)IC50为1.21μg/ml；EEAEAEAEPK+rLZ-8突变体(SEQ ID NO:15)IC50为1.91μg/ml。结果显示，EAEA+rLZ-8突变体(SEQ ID NO:14)和EEAEAEAEPK+rLZ-8突变体(SEQ ID NO:15)对肺癌细胞系A549均具有杀伤作用，其中EAEA+rLZ-8突变体(SEQ ID NO:14)杀伤效果优于EEAEAEAEPK+rLZ-8突变体(SEQ ID NO:15)。
实施例9 重组蛋白体外抑制肿瘤生长(PDXO模型)
实验前一天(day-1)，使用50％Matrigel以1:1的比例处理所需的类器官数量，以定义用于执行筛选的类器官(organoid)的正确尺寸。
第0天：类器官接种
a)将20μl 100x Dispase溶液加入6孔板(含有2ml有机体培养基)，从每个孔中收集类器官。b)将6孔板放回培养箱中，37℃培养30分钟。c)从6孔板中收集类器官，并通过预湿的100μm过滤器移入50ml塑料管中。d)一旦所有孔都通过100μm过滤器过滤后，通过预湿的20μm过滤器过滤流。e)倒转20μm过滤器并在新的50ml管中回收类器官。f)在相应的培养基中收集和重悬类器官。计算类器官以获得浓度。g)用培养基调整细胞浓度至合适浓度。h)添加Matrigel至终浓度为5％v/v，并将类器官悬浮在冰上。i)用Multidrop分配器将40μL细胞悬液加入384孔板中。接种密度如下表1所示，接种密度与类器官形态和类器官生长条件有关。j)在添加重组蛋白之前将筛选板放回培养箱中。在接种后2-4小时添加重组蛋白溶液处理类器官。实施例中使用的重组蛋白的间隔序列的氨基酸序列如SEQ ID NO:8所示，rLZ-8突变体的氨基酸序列如SEQ ID NO:13所示，重组蛋白的氨基酸序列如SEQ ID NO:14所示。
表1 类器官的接种密度
类器官系 接种密度
LI6669B B 500/孔
GA6833B 350/孔
CR3099B 400/孔
CR20035B 2000/孔
GA2434B 350/孔
LU5162B 350/孔
LI6677B 400/孔
CR5043B 400/孔
CR5082B 400/孔
PA20078B 400/孔
PA0787B 400/孔
LU11624B 400/孔
LU1235B 400/孔
BR9457B 3000/孔
第5天：在检测结束时读取检测板的发光CTG信号：通过Multidrop分配器每孔添加40μL CTG 3D，在板振荡器上混合内容物5分钟，然后在室温下孵育板30分钟黑暗的。在Envision读板机上读取发光信号。
数据分析：
数据将使用GraphPad Prism 5.0以图形方式显示。计算存活率的公式如下所示。
存活率(％)＝(Lum Test article-Lum Medium control)/(Lum None treated-Lum Medium control)×100％。培养基控制与阳性对照(星形孢菌素，5μM)相同，因为阳性对照(星形孢菌素，5μM)设置为杀死所有或几乎所有类器官。
(1)结直肠癌(colorectal Cancer)
表2 重组蛋白抑制结直肠癌生长
Figure PCTCN2022110916-appb-000004
结果如表2、图8至图11所示：SEQ ID NO:14所示重组蛋白对EGFR野生型(CR20035B，图8)、KRAS突变(CR5043B，图9)、BRAF突变(CR5082B，图10)和EGFR与KRAS 同时突变(CR3099B，图11)的结直肠癌细胞均具有抑制作用。
(2)肺癌
表3 重组蛋白抑制肺癌生长
Figure PCTCN2022110916-appb-000005
结果如表3、图12至图14所示：SEQ ID NO:14所示重组蛋白对EGFR野生型(LU1235B，图14)、KRAS突变(LU5162B，图12)和BRAF突变(LU11624B，图13)的肺癌细胞均具有抑制作用。
(3)肝癌
表4 重组蛋白抑制肝癌生长
Figure PCTCN2022110916-appb-000006
结果如表4、图15至图16所示：SEQ ID NO:14所示重组蛋白对EGFR野生型肝癌细胞(LI6677B，图15；LI6669B，图16)具有抑制作用。
(4)乳腺癌
表5 重组蛋白抑制乳腺癌生长
Figure PCTCN2022110916-appb-000007
结果如表5、图17至图18所示：SEQ ID NO:14所示重组蛋白对HER2突变的乳腺癌细胞(BR9457B，图17；BR9466B，图18)具有抑制作用。
(5)胃癌
表6 重组蛋白抑制胃癌生长
Figure PCTCN2022110916-appb-000008
结果如表6、图19至图20所示：SEQ ID NO:14所示重组蛋白对EGFR野生型(GA6833B，图19)和BRAF突变(GA2434B，图20)的胃癌细胞具有抑制作用。
(6)胰腺癌
表7 重组蛋白抑制胰腺癌生长
Figure PCTCN2022110916-appb-000009
结果如表7、图21至图22所示：SEQ ID NO:14所示重组蛋白对EGFR野生型(PA20078B，图21)和KRAS与BRAF同时突变(PA0787B，图22)的胰腺癌细胞均具有抑制作用。
实施例10 重组蛋白体内抑制肿瘤生长(PDX模型)
本实施例进行受试物SEQ ID NO:14所示重组蛋白在肝癌LI6669原位移植BALB/c雄性裸鼠模型中的药效学评价。
实验设计：
每组的动物数及详细的给药途径、剂量和方案见下表8。
表8 LI6669动物模型中的给药途径、剂量及方案
Figure PCTCN2022110916-appb-000010
其中，i.p.代表腹腔注射，i.v.代表尾静脉注射，p.o.代表口服灌胃。
本实验的主要观察指标为：
相对肿瘤增殖率，T/C(％)，即在某一时间点，治疗组和对照组相对肿瘤体积或瘤重的百分比值。计算公式为：
T/C％＝TRTV/CRTV×100％(TRTV：治疗组平均RTV；CRTV：对照组平均RTV；RTV＝Vt/V0，V0为分组时该动物的瘤体积，Vt为治疗后该动物的瘤体积)；
或T/C％＝TTW/CTW×100％(TTW：治疗组实验终结时平均瘤重；CTW：对照组实验终结时平均瘤重)。
相对肿瘤抑制率，TGI(％)，计算公式为：
TGI％＝(1-T/C)×100％(T和C分别为治疗组和对照组在某一特定时间点的相对肿瘤体积(RTV)或瘤重(TW))。
实验动物：
Figure PCTCN2022110916-appb-000011
实验动物饲养室的环境条件：
Figure PCTCN2022110916-appb-000012
实验方法：
LI6669是源于一名亚洲患者的肝癌灶建立的
Figure PCTCN2022110916-appb-000013
原位移植模型。该模型具有肿瘤一定的破溃倾向。
从
Figure PCTCN2022110916-appb-000014
肝癌原位移植模型LI6669荷瘤小鼠收取肿瘤组织，切成直径为2-3mm的瘤块接种于Balb/c裸小鼠肝左叶上部。
当荷瘤鼠平均肿瘤体积到达约50-150mm 3时，将小鼠基于体重按照“3.实验设计”表格3随机分组。分组当天设定为Day 0，给药开始于Day 1。分组当天定义为第0天。
基于小鼠体重使用StudyDirectorTM(版本号3.1.399.19，供应商Studylog System,Inc.,S.San Francisco,CA,USA)进行分组。
选择“Matched distribution”随机分组方法进行分组，该算法将所有选定动物的个体体重测量值与所有选定动物的平均值进行匹配。首先，选出那些平均值接近所有被选的动物平均值的配对动物，然后将它们分配到组中，使得该组均值与所有选定动物的平均值匹配(或尽可能接近)。每组测量结果的最终平均值将尽可能接近其他组的最终平均值。
实验观察和数据收集：
肿瘤细胞接种后，常规监测包括了肿瘤生长及治疗对动物正常行为的影响，具体内容有实验动物的活动性，摄食和饮水情况，体重增加或降低情况，眼睛、被毛及其它异常情况。试验过程中观察到的临床症状均记录在原始数据中。开始给药后，每周测量两次小鼠的体重。观察记录需要安乐死的小鼠和相应的生存期。
肿瘤体积将会在实验结束后，小鼠解剖时测量。肿瘤体积计算公式：肿瘤体积(mm 3)＝1/2×(a×b 2)(其中a表示长径，b表示短径)。实验中使用StudyDirectorTM(版本号3.1.399.19，供应商Studylog System,Inc.)软件收集数据，包括肿瘤的长短径的测量和动物体重的称量，原始数据由天平和游标卡尺测量后直接导入软件，数据的任何变动都将被记录在此软件中。给药、肿瘤测量及体重称量等全部过程都在生物安全柜或超净工作台中进行。
统计分析：
为比较不同处理组某天的肿瘤体积，首先我们使用Bartlett检验来验证所有组间的方差齐性假设。当Bartlett检验的p值不小于0.05时，单因素方差分析将被用于检验所有组均值是否相等。若单因素方差分析的p值小于0.05，我们将用Tukey HSD检验进行所有组之间两两比较，或用Dunnett’s t检验进行每个治疗组和对照组之间的两两比较。当Bartlett检验的p值小于0.05时，Kruskal Wallis检验将被用于检验所有组的中位数是否相等。若Kruskal Wallis检验的p值小于0.05，我们将用Conover检验进行所有组之间两两比较或每个治疗组和对照组之间的两两比较，并根据多重检验的组数进行相应的p值校正。
此外，基于探索性数据分析的目的，我们进行了任意时间点所有组之间的两两比较。因为这种比较只使用了特定时间点待比较两组的肿瘤体积数据，所以不需要进行多重检验校正。首先我们使用Bartlett检验来验证两组之间的方差齐性假设，当Bartlett检验的p值不小于0.05时，我们用Welch’s t检验来比较两组均值是否相等；当Bartlett检验的p值小于0.05时， 我们用Mann Whitney U检验来比较两组中位数是否相等。
所有的统计分析和图形绘制都在R语言环境中进行(3.3.1版)。非特别说明的情况下，所有检验均为双尾检验，p值小于0.05时被认为具有统计显著性。
实验结果：
结果如表9和图23所示，SEQ ID NO:14所示重组蛋白5mg/kg(i.v.Q3D)组中位生存期(大于28天)长于对照组(17天)，具有统计学意义(p<0.05)。且SEQ ID NO:14所示重组蛋白各组未表现出明显的小鼠毒性作用。说明本申请的重组蛋白能够有效延长肝癌小鼠的生存期。
表9 LI6669人肝癌原位移植肿瘤模型中治疗终点时各治疗组和对照组小鼠中位生存期和p-value
组别 中位生存期(天) P value b
组1，溶剂对照组 17 --
组2，索拉菲尼(Sorafenib)50mg/kg QD >28 0.275
组3，SEQ ID NO:14所示重组蛋白5mg/kg，QD >28 0.119
组4，SEQ ID NO:14所示重组蛋白2.5mg/kg，QD 17 0.066
组5，SEQ ID NO:14所示重组蛋白0.5mg/kg，QD >28 0.430
组6，SEQ ID NO:14所示重组蛋白5mg/kg，Q3D >28 0.032
组7，西妥昔单抗50mg/kg，Q4D 17 0.044
注：a.平均值±SEM；b.与第1组相比。
实施例11 重组蛋白的体外药效实验
本实验以Erbitux对A431细胞系的增殖抑制作用作为阳性对照，探究SEQ ID NO:14所示重组蛋白对多种癌细胞系的增殖抑制作用。本实验使用Promega Cell Titer-Glo试剂盒检测细胞活率。该试剂中含有重组萤光素酶及荧光素，萤光素酶催化底物发生氧化反应，并释放冷光信号。该氧化反应依赖活细胞裂解后释放的ATP，反应产生的冷光信号可间接反应活细胞的数量。
具体步骤如下：
1)收集靶细胞并计数，将细胞接种到96孔板中；
2)将96孔板放置在37度细胞培养箱中，并孵育约16～20个小时；
3)将预先配制的待测样品工作液和阳性参照工作液加入到相应的孔中；
4)于37度细胞培养箱中继续培养72个小时；
5)孵育结束后，加入Cell TiterGlo检测试剂工作液到相应的孔中，并在室温放置使信号稳定；
6)使用PHERAStar FSX进行读板检测。
结果如表10所示，SEQ ID NO:14所示重组蛋白浓度在0.1mg/mL的条件下，对5637、HCI-H292、Calu-1、ACHN、AsPC-1、OVCAR8、SK-N-AS、Bel-7402、SK-HEP-1、MDA-MB-468、A431、HEP G2、MDA-MB-453、HLE、A549和SNU-398均具有一定的生长抑制作用。
表10 SEQ ID NO:14所示重组蛋白对肿瘤细胞系的增殖抑制作用
Figure PCTCN2022110916-appb-000015
实施例12 重组蛋白的临床前安全评估试验
(1)人红细胞的体外溶血实验
本实施例研究SEQ ID NO:14所示重组蛋白在体外是否引起人红细胞溶解或凝聚。
采用体外试管法，观察了供试品(即SEQ ID NO:14所示重组蛋白)对人红细胞溶解及凝聚的影响，使用制剂浓度为2.5mg/mL。
采自一名健康人的红细胞用氯化钠注射液配制成2％(v/v)混悬液。将不同容量(2.5mL～2.9mL)的氯化钠注射液和不同容量(0.5mL～0.1mL)的供试品，分别加入到已有2.0mL2％人红细胞混悬液的玻璃试管中，同时将3.0mL氯化钠注射液和3.0mL灭菌注射用水加入到已有2.0mL 2％人红细胞混悬液的玻璃试管中分别作为阴性对照和阳性对照，每个试管的总体积为5.0mL，置于电热恒温箱中温育3小时，观察红细胞的溶解和凝聚情况。
结果发现，供试品管和阴性对照品氯化钠注射液管，可见红细胞沉于管底，上层溶液无色澄明，震摇后管底的红细胞均匀分散，无溶血和凝聚；阳性对照灭菌注射用水管溶液呈澄 明红色，未见分层，管底无细胞残留，阳性对照品灭菌注射用水管完全溶血。
由此可见，制剂浓度为2.5mg/mL的供试品在体外对人红细胞无溶血作用，不引起红细胞凝聚。
(2)对巴马小型猪的毒性试验
本实施例研究SEQ ID NO:14所示重组蛋白静脉输注巴马小型猪后的毒性反应和体内代谢情况，以及给药期结束后4周毒性反应恢复情况。
将巴马小型猪40只(20只/性别)，随机分为4组，每组5只/性别。第1组给予安慰剂作为辅料对照，第2、3、4组分别给予供试品(即SEQ ID NO:14所示重组蛋白)0.5mg/kg、1.5mg/kg、5.0mg/kg。静脉输注给药，每天给药1次，连续给药28天。给药容量为2mL/kg，输注速度为30mL/kg/h。前3只/性别/组动物于给药4周后(D29)实施安乐死，剩下每组每性别2只动物于末次给药结束恢复4周后(D57)实施安乐死。
试验期间对动物进行临床观察、体重、体温、心电图、眼科检查、血细胞计数、凝血功能、血液生化、尿液检查、T淋巴细胞亚群、抗体检测、毒代动力学、脏器重量、大体观察和组织病理学检查。
结果发现，以0.5mg/kg、1.5mg/kg、5mg/kg剂量重复静脉输注给予巴马小型猪，每天给药1次，连续给药28天，各组动物未见死亡或者濒死；未见严重临床毒副作用；各剂量组动物体重及体重增长量、眼科检查、尿液分析、T淋巴细胞亚群均未见毒理学意义的改变；供试品各剂量组D29或D57的脏器重量、脏器重量/脑重比值未见统计学意义的改变；给药期结束(D29)及恢复期结束(D57)，大体观察及显微镜检查均未见供试品相关的异常改变。
(3)对呼吸***功能影响的安全药理试验
本实施例研究SEQ ID NO:14所示重组蛋白静脉注射给药对大鼠呼吸***功能的影响。
将SD大鼠40只(20只/性别，SPF级)，随机分配为4组，每组10只(5只/性别)，分别给予辅料对照品和0.5、2、8mg/kg供试品(即SEQ ID NO:14所示重组蛋白)，单次静脉注射给药。给药容量为10mL/kg。于给药前一天，给药后10min、24h将动物放入描记箱内，检测动物呼吸功能指标。
结果发现，与辅料对照组相比，供试品组动物给药前一天、药后10min及24h潮气量、每分钟通气量及呼吸频率未见统计学差异(P>0.05)及变化趋势。
因此供试品在0.5、2、8mg/kg剂量下单次静脉注射给药对SD大鼠呼吸***功能无明显影响。
(4)对中枢神经***功能影响的安全药理试验
本实施例研究SEQ ID NO:14所示重组蛋白静脉注射给药对大鼠中枢神经***功能的影响。
将SD大鼠40只(20只/性别，SPF级)，随机分配为4组，每组10只(5只/性别)，分别给予辅料对照品和0.5、2、8mg/kg供试品(即SEQ ID NO:14所示重组蛋白)，单次静脉注射给药。给药容量为10mL/kg。盲法设计，给药信息对实验观察者保密，于给药前一天，给药后10min、24h由同一名实验观察者采用功能组合观察法(FOB)对动物进行观察并记录结果。功能组合观察法内容包括：笼内观察、手抓观察、开放环境观察、刺激性反应观察，对前肢爪抓力、后肢张开幅度、体温进行测定。将药后数据减去药前值所得的数据进行统计分析。
结果发现，与辅料对照组相比，供试品各组动物药后10min及24h笼内观察，手抓观察，开放环境观察，刺激性反应，前肢爪抓力、后肢张开幅度，体温均未见异常。
可见供试品在0.5、2、8mg/kg剂量下单次静脉注射给药对SD大鼠中枢神经***功能无明显影响。
(5)对心血管***功能影响的安全药理试验
本实施例研究SEQ ID NO:14所示重组蛋白静脉注射给药对清醒巴马小型猪心血管***功能的影响。
用8只巴马小型猪(4只/性别，普通级)，采用交叉设计，设0.5、3mg/kg剂量供试品(即SEQ ID NO:14所示重组蛋白)和一个辅料对照静脉输注给药(供试品0.5mg/kg和3mg/kg剂量分别约为药效剂量的8.4倍和50.6倍)，给药容量为2mL/kg，给药速度为30mL/kg/h。采集给药前至少2小时至药后约48小时动物心电、血压、体温各项指标的数据，在检测时间点：药前1.5h内，给药后0.25h(±5min)、0.5h(±5min)、1h(±10min)、1.5h(±10min)、2h(±10min)、3h(±15min)、4h(±20min)、6h(±30min)、8h(±45min)、12h(±45min)、24h(±1h)、48h(±1h)对心电、血压、体温的各指标数据进行统计学分析和评估。
结果发现，与辅料对照相比，3mg/kg供试品给药后0.5h-6h心率加快。2h-4h QTcB间期缩短。0.5h-6h T波电压升高。
与辅料对照相比，3mg/kg供试品给药后RR间期、PR间期及QT间期出现心率相关的改变。
供试品各剂量下动物其余心电指标(QRS时限、QRS电压、ST段电压、Tp-e间期、P波宽度)、血压指标(收缩压、舒张压、平均动脉压、脉压差)及体温与辅料对照相同时间点 各指标比较均无统计学差异(P>0.05)，未见改变趋势。
可见供试品在药效剂量范围内静脉输注给药对清醒巴马小型猪心血管功能及体温无明显影响。
综上可知，供试品在药效剂量范围内未发现潜在毒性靶器官，未发现不可逆毒性；对中枢神经***、呼吸***和心血管***功能无显著影响。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方式的范围内。

Claims (27)

  1. 重组蛋白，其包含间隔肽和重组灵芝免疫调节蛋白突变体(rLZ-8突变体)，其中与SEQ ID NO：10所示的氨基酸序列相比，所述rLZ-8突变体的氨基酸序列包含至少一个氨基酸突变。
  2. 根据权利要求1所述的重组蛋白，其中所述rLZ-8突变体包含SEQ ID NO：13和16-21中任一项所示的氨基酸序列。
  3. 根据权利要求1-2中任一项所述的重组蛋白，其中所述rLZ-8突变体包含SEQ ID NO：13所示的氨基酸序列。
  4. 根据权利要求1-3中任一项所述的重组蛋白，其中所述间隔肽包含1-5个EA。
  5. 根据权利要求1-4中任一项所述的重组蛋白，其中所述间隔肽包含2个EA。
  6. 根据权利要求1-5中任一项所述的重组蛋白，其中所述间隔肽包含SEQ ID NO：8-9中任一项所示的氨基酸序列。
  7. 根据权利要求1-6中任一项所述的重组蛋白，其中所述间隔肽包含SEQ ID NO：8所示的氨基酸序列。
  8. 根据权利要求1-7中任一项所述的重组蛋白，其中所述间隔肽位于所述rLZ-8突变体的N端。
  9. 根据权利要求1-8中任一项所述的重组蛋白，其包含SEQ ID NO：14、15、22-33中任一项所示的氨基酸序列。
  10. 根据权利要求1-9中任一项所述的重组蛋白，其包含SEQ ID NO：14所示的氨基酸序列。
  11. 分离的核酸分子，其编码权利要求1-10中任一项所述的重组蛋白。
  12. 载体，其包含权利要求11所述的分离的核酸分子。
  13. 细胞，其包含权利要求11所述的分离的核酸分子和/或权利要求12所述的载体。
  14. 根据权利要求13所述的细胞，其为真核细胞。
  15. 根据权利要求13-14中任一项所述的细胞，其为酵母细胞。
  16. 多肽，其包含权利要求1-10中任一项所述的重组蛋白。
  17. 制备权利要求1-10中任一项所述的重组蛋白的方法，其包括在使得权利要求1-10中任一项所述的重组蛋白表达的条件下，培养根据权利要求13-15中任一项所述的细胞。
  18. 药物组合物，其包含权利要求1-10中任一项所述的重组蛋白、权利要求11所述的分离的核酸分子、权利要求12所述的载体、权利要求13-15中任一项所述的细胞和/或权利要求16所述的多肽，以及任选地药学上可接受的载体。
  19. 试剂盒，其包含权利要求1-10中任一项所述的重组蛋白、权利要求11所述的分离的核 酸分子、权利要求12所述的载体、权利要求13-15中任一项所述的细胞、权利要求16所述的多肽和/或权利要求18所述的药物组合物。
  20. 给药装置，其包含权利要求1-10中任一项所述的重组蛋白、权利要求11所述的分离的核酸分子、权利要求12所述的载体、权利要求13-15中任一项所述的细胞、权利要求16所述的多肽和/或权利要求18所述的药物组合物。
  21. 预防、缓解或***的方法，其包括向有需要的受试者施用权利要求1-10中任一项所述的重组蛋白、权利要求11所述的分离的核酸分子、权利要求12所述的载体、权利要求13-15中任一项所述的细胞、权利要求16所述的多肽和/或权利要求18所述的药物组合物。
  22. 根据权利要求21所述的方法，其中所述肿瘤包括实体瘤。
  23. 根据权利要求21-22中任一项所述的方法，其中所述肿瘤包括具有EGFR表达和/或EGFR表达异常的肿瘤。
  24. 根据权利要求21所述的方法，其中所述肿瘤包括非实体瘤。
  25. 根据权利要求21-24中任一项所述的方法，其中所述肿瘤包括结直肠癌、肺癌、肝癌、乳腺癌、胃癌、肾癌、膀胱癌、神经母细胞瘤、卵巢癌、上皮鳞癌和/或胰腺癌。
  26. 权利要求1-10中任一项所述的重组蛋白、权利要求11所述的分离的核酸分子、权利要求12所述的载体、权利要求13-15中任一项所述的细胞、权利要求16所述的多肽和/或权利要求18所述的药物组合物在制备药物中的用途，所述药物用于预防、缓解或***。
  27. 权利要求1-10中任一项所述的重组蛋白、权利要求11所述的分离的核酸分子、权利要求12所述的载体、权利要求13-15中任一项所述的细胞、权利要求16所述的多肽和/或权利要求18所述的药物组合物，其用于预防、缓解或***。
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