WO2017041739A1 - 改造的白介素12及其在制备治疗肿瘤药物中的用途 - Google Patents

改造的白介素12及其在制备治疗肿瘤药物中的用途 Download PDF

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WO2017041739A1
WO2017041739A1 PCT/CN2016/098527 CN2016098527W WO2017041739A1 WO 2017041739 A1 WO2017041739 A1 WO 2017041739A1 CN 2016098527 W CN2016098527 W CN 2016098527W WO 2017041739 A1 WO2017041739 A1 WO 2017041739A1
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cancer
vector
engineered
nsil
amino acid
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PCT/CN2016/098527
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French (fr)
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王尧河
王鹏举
尼克·莱蒙
高冬玲
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北京锤特生物科技有限公司
郑州大学
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Priority to CN201680022405.2A priority Critical patent/CN108307642B/zh
Priority to EP16843677.2A priority patent/EP3348643B1/en
Priority to CN202311154075.0A priority patent/CN117736296A/zh
Priority to JP2018532498A priority patent/JP7340222B2/ja
Priority to US15/758,853 priority patent/US11345732B2/en
Publication of WO2017041739A1 publication Critical patent/WO2017041739A1/zh
Priority to JP2021215459A priority patent/JP7438552B2/ja
Priority to US17/700,991 priority patent/US20220213161A1/en

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Definitions

  • the invention relates to the field of genetic engineering biotechnology, in particular to an engineered interleukin 12 (hereinafter referred to as nsIL-12) and a gene thereof, a recombinant vector and the use thereof in preparing a medicament for treating tumor.
  • nsIL-12 engineered interleukin 12
  • Interleukin-12 is an immunoregulatory cytokine secreted mainly by antigen-presenting cells such as dendritic cells, monocytes, macrophages and B cells.
  • the heterodimer is composed of two subunits p35 and p40 through a disulfide bond.
  • the main function of IL-12 is to participate in the differentiation of T cells, activate T cells and natural killer cells (NK cells), stimulate T cells and NK cells to secrete interferon gamma, mediate the enhancement of NK cells and CD 8+ cytotoxic T cells. It stimulates the expression of IL-12 receptors IL-12R- ⁇ 1 and ⁇ 2, participates in innate immunity and secondary immunity, and is a multifunctional strong immunoregulatory factor.
  • IL-12 can cause the regression or reduction of human and mouse tumor cells, as well as the reduction of angiogenesis in tumors.
  • IL-12 Systemic administration of IL-12 causes potential lethal toxicity, resulting in it has not been used clinically.
  • many development strategies to reduce the toxicity of IL-12 are now the main research hotspots, and a series of methods for modifying IL-12 have been developed.
  • a system that relies on oral ligand drugs to control IL-12 expression which significantly reduces IL-12-induced toxicity, but does not efficiently transform tumor cells and lacks simultaneous induced inflammatory responses that limit the anti-tumor application of this method. .
  • IL-12 modified tumor-dissolving IL-12 gene was constructed by inserting the modified N-terminal signal peptide-depleted IL-12 into a new tumor-targeting lytic tumor vector, and its therapeutic effect and IL- 12 features. Based on this, the present invention provides an engineered interleukin 12 (nsIL-12) and a gene thereof, a recombinant vector, and use thereof in the preparation of a medicament for treating a tumor.
  • nsIL-12 engineered interleukin 12
  • a nucleotide sequence encoding an engineered interleukin 12 consisting of the nucleotide sequence set forth in SEQ NO: 1 is provided.
  • the nucleotide sequence encoding the engineered interleukin 12 may also be a nucleotide sequence derived from a human interleukin 12 nucleotide sequence but different from SEQ NO: 1, and the encoded protein exhibits an antitumor effect or may cause Tumors subside and have lower toxic side effects than existing IL-12.
  • an engineered interleukin 12 is provided which is encoded by the nucleotide sequence described above.
  • the engineered interleukin 12 consists of the amino acid sequence set forth in SEQ NO:2.
  • the engineered interleukin 12 may also be composed of an amino acid sequence derived from human IL-12 but different from SEQ NO: 2, and exhibits an anti-tumor effect or may cause tumor regression and is compared with existing IL-12. Has lower toxic side effects.
  • a recombinant vector comprising the nucleotide sequence encoding the engineered IL-12 is provided.
  • the recombinant vector can target tumor cells. More preferably, the recombinant vector can be obtained by constructing a vector selected from the group consisting of an adenovirus vector, a vaccinia virus vector, and a herpesvirus vector.
  • the recombinant vector is a tumor-injecting adenovirus vector Ad-TD-nsIL-12 (China Type Culture Collection, Wuhan, China. Wuhan University, deposit number CCTCC NO: V201520, date of deposit, May 21, 2015, human type 5 adenovirus mutant, also known as Ad-TD-nsIL12), It is a C subtype of adenovirus type 5 Ad5, but deletes three genes E1A-CR2, E1B19K and E3gp19K, and controls expression of the engineered IL-12 gene sequence using the E3gp19K promoter.
  • the engineered IL-12 or the nucleotide sequence encoding the engineered interleukin 12 or the use of the vector in the manufacture of a medicament for treating a tumor is provided.
  • the tumor is a solid tumor, a peritoneal disseminated tumor or a metastatic tumor.
  • the nucleotide sequence encoding the engineered interleukin 12 is the p35 subunit coding gene (ns-p35, nucleotide sequence as shown in SEQ NO: 3) of the engineered human IL-12 and the p40 of the engineered human IL-12.
  • the subunit coding gene (ns-p40, nucleotide sequence as shown in SEQ NO: 4) was obtained by ligation of the short sequence (GTTCCTGGAGTAGGGGTACCTGGGGTGGGC).
  • the tumor-infected adenovirus vector Ad-TD-nsIL-12 is a C subtype of adenovirus type 5 Ad5, but deletes three genes E1A-CR2, E1B19K and E3gp19K, and uses the E3gp19K promoter to control expression of the modified interleukin 12 gene sequence.
  • the recombinant adenoviral vector selectively replicates in tumor cells and expresses engineered human IL-12 upon entry into the tumor, and the engineered IL-12 is localized in the expression of the tumor cells; the expression is expressed as the tumor cells are lysed IL-12 is released and simultaneously releases tumor-associated antigens, and the released tumor-associated antigens synergize with IL-12 to produce a highly efficient and specific anti-tumor response, thereby further killing distant tumor cells that are not infected by the virus, including Transfer of tiny tumor cell foci. These tumors include solid tumors, metastases, and spread tumors that can be seen by the naked eye or visible under the microscope.
  • the recombinant adenoviral vector has a stronger tumor growth inhibitory effect on peritoneal disseminated tumors and in situ tumors, and has low toxicity.
  • a method for constructing a vector Ad-TD-nsIL-12 for targeting tumor-forming adenovirus of human tumor comprising the following steps:
  • nsIL-12 fusion gene cloning Total RNA extracted from cultured RPMI-8866 cells was reverse transcribed into cDNA, and primers containing SnaBI cleavage site and elastin sequence (GTTCCTGGAGTAGGGGTACCTGGGGTGGGC) were used (p35-F: GTTCCTGGAGTAGGGGTACCTGGGGTGGGCGCCAGAAACCTCCCCGTG, p35 -R:GCTACGTATTAGGAAGCATTCAGATA) PCR-cloned human IL-12 engineered p35 subunit (ns-p35, nucleotide sequence as shown in SEQ NO: 3) and engineered p40 subunit gene (primer: p40-F: CCTACGTAATGATATGGGAACTGAAGAAAG) , p40-R: GCCCACCCCAGGTACCCCTACTCCAGGAACACTGCAGGGCACAGATGC, ns-p40, nucleotide sequence shown in SEQ NO:
  • pSSE3gp19K-nsIL-12 vector was digested with EcoR V, and the phosphate group of the phosphatase was decapted, and then ligated with the nsIL-12 gene fragment to generate pSSE3gp19K-nsIL-12 vector, sequencing sequence ;
  • Ad-TD-nsIL-12-CHL pSSE3gp19K-nsIL-12 was linearized with Pme1, and a large fragment containing nsIL-12 was recovered and electroporated with pAd-TD vector (see patent ZL200910066130.4). Homologous recombination in BJ5183 competent cells, and the positive clone pAd-TD-nsIL-12-CHL was removed;
  • pAd-TD-nsIL-12 The positive vector pAd-TD-nsIL-12-CHL was digested with Swa1, the CHL gene was excised, the Swa1 and T4 ligase were inactivated, and the TOP10 competent cells were transformed. pAd-TD-nsIL-12 vector; and
  • Ad-TD-nsIL-12 viral vector The recombinant pAd-TD-nsIL-12 vector was linearized with Pac1 and transfected into 293 cells to produce the recombinant adenoviral vector Ad-TD. -nsIL-12.
  • the present invention provides an engineered IL-12 which is non-secretory. Since the engineered IL-12 provided by the present invention lacks an N-terminal signal peptide, it cannot be secreted outside of the cell to which it is expressed.
  • the IL-12 modified by the present invention includes any natural IL-12 and variants thereof, for example, by genetic engineering or protein engineering, provided that the engineered IL-12 is a non-secretory IL-12 containing no signal peptide. And retain anti-tumor activity. Based on the present disclosure, one skilled in the art can obtain various modified IL-12s of the present invention and verify their activity (for example, see Gene Therapy. 2015 Sep; 22(9): 696-706).
  • the present invention specifically provides an engineered IL-12 that is non-secretory.
  • the engineered IL-12 of the invention has the structure:
  • the non-secreted IL-12 described therein does not contain a secretion signal peptide.
  • neither p40 nor p35 in the above structure contains a signal peptide.
  • linkers those skilled in the art can design according to the prior art (for example, Adv Drug Deliv Rev. 2013 October 15; 65 (10): 1357 - 1369 and Front Immunol. 2015 Apr 7; 6: 136) or A suitable linker is selected among various linkers.
  • the linker has the sequence of (GTTCCTGGAGTAGGGGTACCTGGGGTGGGC).
  • Those skilled in the art will appreciate that one skilled in the art can design a linker having various other sequences based on the present disclosure (J Biomed Nanotechnol. 2015 Aug; 11(8): 1418-30).
  • p40 without a signal peptide and p40 without a signal peptide are separately expressed by the oncolytic virus, that is, p35 and p40 are allowed to bind intracellularly (for example, by a disulfide bond) and IL-12 which does not secrete a signal peptide is produced.
  • the situation is also feasible.
  • P35 and p40 include any of the native p35 and p40 and variants thereof, such as those obtained by genetic engineering or protein engineering, provided that the variant retains the activity of native p35 and p40.
  • p35 and p40 are native human p35 and human p40, as well as variants thereof. More preferably, the p35 has or consists of the sequence of SEQ NO: 5; p40 has or consists of the sequence of SEQ NO: 6.
  • Variants of the invention may comprise or consist of an amino acid sequence which has been substituted, deleted, inserted or added to one or several amino acids and retains the activity of substitution, deletion, insertion or addition prior to its natural amino acid sequence or parent sequence. .
  • p35 may comprise or consist of an amino acid sequence which has been substituted, deleted, inserted or added with one or several amino acids based on its natural amino acid sequence or parent sequence and which retains the activity of substitution, deletion, insertion or addition.
  • p35 may comprise or consist of an amino acid sequence which has been substituted, deleted, inserted or added with one or several amino acids at SEQ NO: 5 and which retains the activity of substitution, deletion, insertion or addition.
  • p40 may comprise or consist of an amino acid sequence which has been substituted, deleted, inserted or added one or more amino acids based on its natural amino acid sequence or the parent sequence and retains the activity of substitution, deletion, insertion or addition.
  • p40 may comprise or consist of an amino acid sequence which has been substituted, deleted, inserted or added to one or more amino acids on SEQ NO: 6 and which retains the activity of substitution, deletion, insertion or addition.
  • substitution, deletion, insertion or addition of one or several amino acids in the amino acid sequence according to the present invention means substitution, deletion, insertion or addition at any position in the sequence and in one or several amino acid sequences. 1 or a few amino acid residues (eg 2, 3, 4, 5, 6, 7, 8, or 9), and 2 or 2 of the substitutions, deletions, insertions, and additions The above can happen at the same time.
  • substitution, deletion, insertion or addition of one or several amino acids in the amino acid sequence according to the present invention can be carried out by using "Molecular Cloning 3" and “Current Protocols in Molecular Biology” (Modern Molecular Biology Operation) The site-directed mutagenesis method described in the regulations) is obtained.
  • deletion, insertion or pre-addition activity as described herein, it can mean that the activity of the protein or amino acid sequence after substitution, deletion, insertion or addition is more than 10%, 20, or 20 before the substitution, deletion, insertion or addition. % or more, 40% or more, 60% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% or more.
  • the number of amino acid residues deleted, substituted, inserted and/or added as described above is generally preferably a small number.
  • the variant has about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% of its parent or natural amino acid sequence.
  • amino acid sequence of the above or 99.9% or more identity has the activity of the parent or natural amino acid sequence.
  • the numerical values of the above homology are generally preferably large values.
  • Variants of the invention also include at least about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, or substantially the amino acid sequence or the parent sequence thereof, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, An amino acid sequence of 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more of the identity, and having the activity of the natural amino acid sequence or the parent sequence.
  • p35 further comprises at least about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more with its native amino acid sequence or parent sequence (eg, SEQ NO: 5) Above, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or Above, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or Above, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or The above amino acid sequence of 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more, and having the activity of the natural amino acid sequence
  • p40 further comprises at least about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more with its native amino acid sequence or parent sequence (eg, SEQ NO: 6). Above, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or Above, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or Above, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or Above, 99.5% Or an amino acid sequence of 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more of the identity, and having the activity of the natural amino acid sequence or parent
  • the invention specifically provides an engineered IL-12 which is non-secretory and which has or consists of the amino acid sequence set forth in SEQ NO: 2.
  • the non-secretory IL-12 of the invention may comprise or consist of an amino acid sequence which has been substituted, deleted, inserted or added with one or several amino acids based on SEQ NO: 2 and retains the activity of SEQ NO: 2. .
  • the non-secretory IL-12 of the invention may comprise at least about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more, and SEQ NO: 2, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, An amino acid sequence of 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more of identity, and having the activity of SEQ NO: 2.
  • the non-secretory type means that in the gene encoding the protein of interest, a fragment encoding a signal peptide is lacking, so that the protein cannot be transported to the secretory endoplasmic reticulum-Golgi-plasma membrane system, thereby failing to Secreted to the outside of the cell.
  • the invention also provides nucleotides encoding the engineered non-secretory IL-12 of the invention. Preferably it comprises or consists of the nucleotide sequence set forth in SEQ NO: 1 or its complement.
  • the nucleotide encoding the engineered non-secretory IL-12 of the invention comprises or consists of the sequence:
  • having non-secretory IL-12 activity means that the activity of a certain protein or amino acid sequence is 10% or more, 20% or more, 40% of the non-secretory IL-12 (SEQ NO. 2). Above, 60% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% or more.
  • nucleotide encoding the engineered non-secretory IL-12 of the invention comprises or consists of the following sequences:
  • the "stringent conditions” described herein may be any of low stringent conditions, medium stringent conditions, and high stringency conditions, preferably high stringency conditions.
  • the "low stringency conditions” may be 30 ° C, 5 x SSC, 5 x Denhardt's solution, 0.5% SDS, 52% formamide
  • “medium stringent conditions” may be 40 ° C, 5 x SSC, 5 x Denhardt's solution, 0.5% SDS, 52% formamide
  • “high stringency conditions” may be 50 ° C, 5 x SSC, 5 x Denhardt's solution, 0.5% SDS, 52% formamide.
  • the higher the temperature the more highly homologous polynucleotides can be obtained.
  • one skilled in the art can select a comprehensive result of a plurality of factors affecting the stringency of hybridization, probe concentration, probe length, ionic strength, time, salt concentration, etc. to achieve a corresponding stringency.
  • hybridizable polynucleotide may be a multi-core encoding the modified non-secretory IL-12 of the present invention when calculated by a system-determined default parameter by a homology search software such as FASTA or BLAST.
  • the glycosides have about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1 or more, 99.2 or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9 A polynucleotide of % or more identity.
  • BLAST For amino acid sequence, nucleotide sequence identity, Karlin and Altschul's algorithm rule BLAST can be used (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990; Proc. Natl. Acad. Sci. USA 90: 5873 , 1993) to determine. BLASTN, BLASTX, based on BLAST algorithm rules, has been developed (Altschul SF, et Al: J Mol Biol 215: 403, 1990).
  • the invention also provides recombinant vectors, such as plasmid vectors or viral vectors, comprising a nucleotide sequence of the invention or capable of expressing the engineered non-secretory IL-12 of the invention. Further, the present invention also provides a microorganism (e.g., a bacterium) or a cell, such as Escherichia coli and yeast, into which the recombinant vector of the present invention or the nucleotide of the present invention is introduced.
  • a microorganism e.g., a bacterium
  • a cell such as Escherichia coli and yeast
  • the cells into which the recombinant vector of the present invention is introduced are preferably human-derived cells, such as cells derived from various tissues or organs of humans, such as cells derived from human liver, kidney, pancreas, stomach, large intestine, small intestine, or stem cells. More preferably, it is a cancer cell or a stem cell.
  • the recombinant vector comprising the nucleotide sequence of the present invention is an adenovirus type 5 vector, a vaccinia virus vector, an adenovirus type 11 vector, a herpes virus, a reovirus, a measles virus, a Newcastle disease virus, a vesicular stomatitis Virus, poliovirus, etc.
  • the vector comprising the nucleotide sequence of the present invention is an oncolytic adenovirus vector Ad-TD-nsIL-12 (China Center for Type Culture Collection, Wuhan University, Wuhan, China, deposit number CCTCC NO: V201520, preservation) Date May 21, 2015, human type 5 adenovirus mutant).
  • the invention also provides a kit or medicament comprising the engineered non-secretory IL-12 of the invention, a nucleotide encoding the engineered non-secretory IL-12 of the invention, capable of expressing the modifications of the invention A vector that does not secrete IL-12, or a vector that comprises the nucleotide sequence of the present invention.
  • the kit or medicament is for treating or preventing a disease associated with IL-12.
  • the invention also provides an engineered non-secretory IL-12 of the invention, a nucleotide encoding the engineered non-secretory IL-12 of the invention, a vector capable of expressing the engineered non-secretory IL-12 of the invention, Or the use of a vector comprising a nucleotide sequence of the invention in the manufacture of a kit or medicament for the treatment of a disease associated with IL-12, such as cancer, in a subject.
  • the invention also provides a method of treating an IL-12-associated disease (eg, cancer) in a subject, comprising administering to the subject an effective amount of the engineered non-secretory IL-12 of the invention, a coding version A modified non-secretory IL-12 nucleotide of the invention, a vector capable of expressing the engineered non-secretory IL-12 of the invention, or a vector comprising the nucleotide sequence of the invention.
  • an IL-12-associated disease eg, cancer
  • the subject is a mammal, more preferably a human.
  • IL-12-associated disease preferably refers to a disease, such as cancer, that can be treated or prevented by the use of IL-12.
  • cancer may be a solid tumor, a peritoneal disseminated tumor, or a metastatic tumor.
  • the cancer treated using the present invention is preferably said pancreatic cancer, kidney cancer, head and neck tumor,
  • An engineered non-secretory IL-12 as an active ingredient of the present invention, a nucleotide encoding the engineered non-secretory IL-12 of the present invention, a vector capable of expressing the engineered non-secretory IL-12 of the present invention, or A vector comprising a nucleotide sequence of the invention can be used in conjunction with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier including excipients and auxiliaries which facilitate processing of the active ingredient into preparations.
  • formulations suitable for injection or infusion include aqueous and nonaqueous sterile injectable solutions and aqueous and nonaqueous sterile suspensions, which may optionally contain antioxidants, buffers, bacteriostatic agents, and A solute that is capable of compressing the preparation with the blood of the intended recipient, which may include suspending agents and thickening agents.
  • the formulation may be presented in unit or multi-dose containers, such as sealed ampoules, and may be stored in freeze-dried (lyophilized) conditions, requiring only the addition of a sterile liquid carrier, such as water for injection, for reconstitution prior to immediate use. .
  • the active ingredient of the present invention may optionally be combined with a solid excipient, and optionally the resulting mixture, and, if desired, a mixture of granules, after addition of suitable auxiliaries, to obtain the desired dosage form.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose or starch preparations, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, carboxy Sodium methylcellulose and/or polyvinylpyrrolidone (PVP).
  • a disintegrating agent such as cross-linked polyvinylpyrrolidone, agar or alginic acid or a salt thereof such as sodium alginate may be added as needed.
  • An effective amount of the active ingredient of the present invention may be any amount which treats cancer, or alleviates cancer symptoms or inhibits cancer cells, and may be a dosage unit equivalent to about 0.1 to 15 mg of the active ingredient, preferably 0.2 to 12 mg of the active ingredient. More preferably, the dosage unit comprises from about 2 to about 5 mg of the active ingredient. Most preferably, the dosage unit comprises from about 3-4 mg of active ingredient. Determination of an effective amount is within the abilities of those skilled in the art, especially in light of the disclosure provided herein.
  • the pharmaceutical product (drug, kit) of the present invention can be administered to a subject in any effective dose number.
  • the pharmaceutical product (drug, kit) of the invention may be administered in multiple doses, for example from about 2 to about 15 doses, more preferably from about 4 to 10 doses, and most preferably about 6 doses.
  • the pharmaceutical product (drug, kit) of the invention is administered to a subject, such as by injection, infusion or oral, at a frequency of about once a day, during administration.
  • the administration is by injection or infusion, most preferably intratumoral injection.
  • the pharmaceutical products (drugs, kits) of the invention may be formulated in any suitable manner for administration by any suitable route.
  • the dosage unit of the pharmaceutical product (drug, kit) of the present invention is administered to a subject based on routine administration.
  • the dosage unit can be administered more than once daily, once every two days, once a week, once a month, and the like.
  • the dosage unit can be administered on a two-week basis, ie twice a week, for example every three days.
  • the invention also provides a method of making a recombinant vector of the invention comprising inserting a nucleotide encoding an engineered non-secretory IL-12 of the invention into a vector to obtain an expressible non-secretory IL- a recombinant vector of 12; and transfecting the recombinant vector into a cell to produce a recombinant virus of the invention.
  • "Inclusion&quot is used synonymously with "includes", "includes" or "characterize" and is intended to be inclusive or open and does not exclude additional unreported elements or method steps.
  • Any expression of the term "comprising”, particularly when describing a method, use, or product of the invention, is understood to include consisting essentially of and consisting of the component or element or component or The products, methods, and uses of the steps.
  • the invention exemplarily described herein may suitably be practiced without any one or more of the elements, one or more limitations not specifically disclosed herein.
  • the instructions relating to the pharmaceutical product contained in the pharmaceutical product of the present invention may contain the following indications: indications (cancer, such as pancreatic cancer), administration dose (such as exemplified above), side effects that may occur, and the like.
  • the recombinant vector of the present invention can selectively replicate in tumor cells or cancer cells, and express engineered non-secretory IL-12 upon entry into tumors or cancer cells, engineered non-secretory IL -12 due to lack of signal peptide sequence is confined to tumor cells or cancer cells; as tumor cells or cancer cells lyse, IL-12 is released, and simultaneously releases tumor-associated antigen or cancer cell-associated antigen, released Tumor-associated antigen or cancer cell-associated antigen synergizes with IL-12 to produce a highly effective and specific anti-tumor or anti-cancer response, thereby further killing distant tumor cells or cancer cells that are not infected by the virus, including metastatic tumors.
  • a recombinant oncolytic virus can selectively replicate in tumor cells or cancer cells, and express engineered non-secretory IL-12 upon entry into tumors or cancer cells, engineered non-secretory IL -12 due to lack of signal peptide sequence is confined to tumor cells or cancer cells; as tumor cells or
  • a recombinant vector e.g., a recombinant oncolytic virus
  • a recombinant vector comprising an IL-12 nucleotide encoding no signal peptide
  • the existing IL-12 protein has short half-life and high level of expression, which may cause toxic side effects; in addition, it has stronger tumor growth inhibition effect and no significant toxicity; and contains IL-12 nucleotides encoding no signal peptide.
  • a recombinant vector eg, a recombinant oncolytic virus
  • the IL-12 expressed in the recombinant vector contains a signal peptide
  • the IL-12 expressing the signal peptide is secreted into the tumor cells or cancer cells in time, thereby causing the organism to suffer a larger dose.
  • the challenge of IL-12 causes greater toxic side effects on organisms.
  • the recombinant vector expresses IL-12 prematurely secreted into tumor cells or cancer cells
  • the recombinant vector for example, oncolytic virus
  • the organism An effective amount of IL-12 cannot be effectively recruited to synergize with the released tumor-associated antigen or cancer cell-associated antigen.
  • a recombinant vector eg, a recombinant oncolytic virus
  • a recombinant vector eg, a recombinant oncolytic virus
  • a recombinant vector comprising an IL-12 nucleotide encoding no signal peptide.
  • the modified IL-12 of the present invention is mainly distributed in local tumor tissues, which increases the specificity to tumor cells and reduces the systemic toxic and side effects of IL-12;
  • the IL-12 method modified by the tumor-targeting viral vector of the present invention can stably and continuously express the IL-12 protein, and make up for the short half-life and high level expression of the existing IL-12 protein. Insufficient toxic side effects;
  • the modified IL-12 of the present invention has a stronger tumor growth inhibiting effect on abdominal disseminated tumors and in situ tumors, and has no significant toxicity.
  • the oncolytic adenovirus Ad-TD-nsIL-12 of the invention can be used not only for intratumoral injection, but also for intraperitoneal and intrathoracic injection, which reduces the difficulty of administration, expands the range of treatable patients, and causes tumors to occur in different parts. Patients can achieve good results without significant side effects.
  • Figure 1 Map of pSS-CHL plasmid.
  • Figure 2 Map of the pVV-TK plasmid.
  • Figure 3 Map of pVV-TK-nsIL-12 plasmid.
  • Figure 4 Schematic diagram of adenovirus type 5 Ad-TD-nsIL-12, vaccinia virus vector VV-TK-nsIL-12 and type 11 adenovirus vector Ad11-Tel-nsIL-12.
  • Figure 5 IL-12 expression of Ad-TD-nsIL-12 in cells.
  • Figure 6 IL-12 expression of VV-TK-nsIL-12 in cells.
  • Figure 7 Anti-tumor effect of Ad-TD-nsIL-12 on different types of solid tumors.
  • FIG. 8 Antitumor effect of VV-TK-nsIL-12 on different types of solid tumors.
  • Figure 9 Expression of IL-12 in peripheral blood of tumor-bearing golden hamsters (also known as Syrian hamsters, Syrian hamster) after injection of different viral vectors.
  • Figure 10 Comparison of anti-tumor effects of Ad-TD-nsIL-12 with Ad-TD-LUC and Ad-TD-IL-12 in a golden hamster pancreatic cancer subcutaneous xenograft model.
  • Figure 11 Comparison of tumor clearance rates of Ad-TD-nsIL-12 and Ad-TD-LUC and Ad-TD-IL-12 in a golden hamster pancreatic cancer subcutaneous xenograft model.
  • Figure 12 Ad-TD-nsIL-12 with Ad-TD-LUC and Ad-TD-IL-12 in golden hamster pancreas Comparison of anti-tumor effects in an in situ model of adenocarcinoma.
  • Figure 13 Comparison of anti-tumor effects of Ad-TD-nsIL-12 with Ad-TD-LUC and Ad-TD-IL-12 in a peritoneal disseminated tumor model of golden hamster pancreatic cancer.
  • Figure 14 Comparison of hepatotoxicity of Ad-TD-nsIL-12 with Ad-TD-LUC and Ad-TD-IL-12 in a peritoneal disseminated tumor model of golden hamster pancreatic cancer.
  • Figure 15 Comparison of anti-tumor effects of Ad-TD-nsIL-12 in a golden hamster head and neck tumor subcutaneous xenograft model.
  • Figure 16 Comparison of anti-tumor effects of VV-TK-nsIL-12 with VV-TK-RFP and VV-TK-IL-12 in a peritoneal disseminated tumor model of golden hamster pancreatic cancer.
  • Figure 17 Comparison of anti-tumor effects of Ad11-Tel-nsIL-12 with Ad11-Tel-GFP and Ad11-Tel-IL-12 in a peritoneal disseminated tumor model of golden hamster pancreatic cancer.
  • Example 1 A method for constructing a vector Ad-TD-nsIL-12 of a tumor-inhibiting adenovirus type 5 for targeted treatment of a human tumor, comprising the following steps:
  • nsIL-12 fusion gene cloning Total RNA extracted from cultured RPMI-8866 cells was reverse transcribed into cDNA, and primers containing SnaBI cleavage site and elastin sequence (GTTCCTGGAGTAGGGGTACCTGGGGTGGGC) were used (p35-F: GTTCCTGGAGTAGGGGTACCTGGGGTGGGCGCCAGAAACCTCCCCGTG, p35 -R:GCTACGTATTAGGAAGCATTCAGATA) PCR-cloned human IL-12 engineered p35 subunit (ns-p35, nucleotide sequence as shown in SEQ NO: 3) and engineered p40 subunit gene (primer: p40-F: CCTACGTAATGATATGGGAACTGAAGAAAG) , p40-R: GCCCACCCCAGGTACCCCTACTCCAGGAACACTGCAGGGCACAGATGC, the nucleotide sequence of ns-p40 is shown in SEQ
  • the nsIL-12 full gene fragment was ligated into the cloning vector T vector and designated as T-nsIL-12; the T-nsIL-12 plasmid was digested with SnaBI to obtain the nsIL-12 gene fragment for use (see SEQ NO: 1);
  • pSS-CHL (constructed on pBR322 plasmid, see Figure 1) was digested with Sal1 and EcoRV at the multiple cloning site upstream of CHL, containing Sal1 and EcoRV cleavage sites at both ends Primer clone E1 6.7K gene stop codon upstream 1091 bp sequence, as the left arm and pSS-CHL linked to form pSSE3gp19K-L; SnaB1 and Xho1 digestion pSSE3gp19K-L, with both ends containing SnaB1 and Xho1 cleavage site The primer clone cloned the 1146 bp sequence downstream of the stop codon of the E3gp19K gene, and the two were ligated to form a pSSE3gp19K shuttle vector;
  • pSSE3gp19K-nsIL-12 vector was digested with EcoR V, and the phosphate group of the phosphatase was decapted, and then ligated with the nsIL-12 gene fragment to generate pSSE3gp19K-nsIL-12 vector, sequencing sequence ;
  • Ad-TD-nsIL-12-CHL pSSE3gp19K-nsIL-12 was linearized with Pme1, and a large fragment containing nsIL-12 was recovered and electroporated with pAd-TD vector (see patent)
  • pAd-TD-nsIL-12 The positive vector pAd-TD-nsIL-12-CHL was digested with Swa1, the CHL gene was excised, the Swa1 and T4 ligase were inactivated, and the TOP10 competent cells were transformed. pAd-TD-nsIL-12 vector.
  • Ad-TD-nsIL-12 viral vector The recombinant pAd-TD-nsIL-12 vector was linearized with Pac1 and transfected into 293 cells to produce the recombinant adenoviral vector Ad-TD. -nsIL-12.
  • Ad-TD-nsIL-12 human type 5 adenovirus mutant strain, China Type Culture Collection, Wuhan, China. Wuhan University, deposit number CCTCC NO: V201520, date of deposit May 21, 2015, also known as Ad-TD-nsIL12.
  • Example 2 A method for constructing a vector VV-TK-nsIL-12 for targeting tumor vaccinia virus of human tumor, comprising the following steps:
  • the nsIL-12 gene was cloned with a primer containing the Sal1 and Nhe1 cleavage sites, and digested with the corresponding enzymes, and the pVV-TK plasmid was digested with Sal1 and Nhe1 (see Figure 2, according to the method published by the inventors). Construction, Mol Ther Methods Clin Dev. 2015Sep 16; 2:15035.doi:10.1038/mtm.2015.35.eCollection 2015), the nsIL-12 gene and The pVV-TK was ligated to construct the pVV-TK-nsIL-12 vector (Fig. 3).
  • CV1 cells were cultured, and CV1 was inoculated into a 96-well plate.
  • CRISP-cas9 and gRNA plasmids were transfected, and vaccinia virus VVL15 was infected 24 hours later, and two hours later, pVV-TK was transfected. After 24 hours, it was observed under a fluorescence microscope, and a red fluorescent cell clone was picked, and it was confirmed that the VV-TK-nsIL-12 viral vector (Fig. 4) was successfully recombined.
  • the CV1 cells were again infected, and the red fluorescent clone was picked. After repeated screening for 5 times, the CV1 cells were infected to produce a virus. The genome was extracted for sequencing.
  • Example 3 A method for constructing the vector Ad11-Tel-nsIL-12 of a tumor-inhibiting tumor type 11 adenovirus targeting human tumor, comprising the following steps:
  • pSSE3-18.5K-nsIL-12 vector was digested with SnaB1, and the phosphate group of the phosphatase was decapted, and then ligated with the nsIL-12 gene fragment to generate pSSE3-18.5K- nsIL-12 vector, sequencing analysis sequence;
  • Ad11-Tel-nsIL-12 vector was linearized with Not1 and then transfected into 293 cells to produce the recombinant adenovirus vector Ad11-Tel-nsIL-12.
  • Example 4 IL-12 in tumor cells by Ad-TD-nsIL-12 and VV-TK-nsIL-12 expression
  • Figure 5 shows that Ad-TD-nsIL-12 expresses IL-12 in tumor cells
  • Figure 6 shows that VV-TK-nsIL-12 expresses IL-12 in tumor cells.
  • pancreatic cancer SUIT2 and Capan1 The cultured pancreatic cancer SUIT2 and Capan1, head and neck tumor EC9706, lung cancer A549 and H1299, esophageal cancer EC9706 and ZZB, ovarian cancer SKOV3, colorectal cancer SW620 and HCT116 and gastric cancer AGS were trypsinized and counted, and contained 2
  • a cell suspension of % FBS DMEM was prepared and seeded in the center of a 96-well plate, wherein B1 to G1 were cell-free medium and the other three sides were PBS.
  • VV-TK/Ad-TD-nsIL-12 VV-TK/Ad-TD-IL-12 (full length human IL-12) and Ad-TD-LUC (LUC, Luciferase) or VV-TK-RFP.
  • the initial concentration was 1 ⁇ 10 4 pfu/cell, and the virus solution was diluted 10 times to a total of nine gradients. The last column of cells was free of virus. After dilution, the gun was added to the center of the 96-well plate with a lance, and each column of 10 ⁇ l/well was the same virus gradient.
  • the virus-infected cells were returned to the 37 ° C incubator, and after 6 days, 20 ⁇ l of a mixture of MTS and PMS 20:1 (MTS: PMS) was added to the wells other than the PBS. After 1-4 h, the absorbance was measured at a wavelength of 490 nm using a microplate reader. The EC50 is calculated and the results are shown in Figures 7 and 8.
  • FIGS 7 and 8 show that Ad-TD-nsIL-12 and VV-TK-nsIL-12 have the ability to kill various solid tumor tumor cells.
  • Example 6 Comparison of changes in IL-12 expression of Ad-TD-nsIL-12 and Ad-TD-LUC and Ad-TD-IL-12 after intraperitoneal injection of tumor-bearing golden hamsters.
  • SHPC6 Golden hamster pancreatic cancer cells, supplied by WSM Wold, St. Louis University, USA was inoculated into the abdominal cavity of a 5-6 week old golden hamster (also known as Syrian hamster, Syrian hamster), and the animals were divided 4 days later. There are four groups of 9 animals each.
  • Ad-TD-LUC LEC from pGL3 vector luciferase, constructed according to the method of constructing Example 1 for Ad-TD-nsIL-12
  • Ad-TD-IL -12 full length human IL-12, constructed as described in Example 1 for the construction of Ad-TD-nsIL-12
  • intraperitoneal injection of Ad-TD-nsIL-12 once, after the injection, 1, 3, 5
  • the serum was collected for daily detection of changes in the expression of IL-12 in peripheral blood. The results are shown in Figure 9.
  • Figure 9 shows that the Ad-TD-IL-12 group expressed high levels of IL-12 on the first day after injection of the viral vector, while IL-12 expression decreased significantly on the third and fifth days, and the expression level changed very much.
  • IL-12 was expressed at a low level every day. From the first day to the fifth day, there was no significant difference in IL-12 expression level, suggesting that Ad-TD-nsIL-12 is Animals are not susceptible to toxic side effects due to the large production of IL-12.
  • Example 7 Anti-tumor application of tumor-targeted adenovirus Ad-TD-nsIL-12, and its comparison with Ad-TD-LUC and Ad-TD-IL-12.
  • HPD1-NR Golden hamster pancreatic cancer cells, donated by WSM Wold, St. Louis University, USA
  • the average tumor volume of each group of animals was about 330 mm 3 .
  • Figure 10 shows that Ad-TD-nsIL-12 and Ad-TD-IL-12 showed stronger antitumor effects than the control viral vectors Ad-TD-LUC and PBS;
  • Figure 11 shows Ad-TD-nsIL-12 and Ad
  • the tumor clearance rate of the TD-IL-12 treatment group was extremely high, up to 100%, while the tumor clearance rate of the Ad-TD-LUC group was only 42.8%.
  • Example 8 Therapeutic effect of Ad-TD-nsIL-12 on an in situ model of golden hamster pancreatic cancer, and its comparison with Ad-TD-LUC and Ad-TD-IL-12.
  • FIG 12 shows that Ad-TD-nsIL-12 has more than Ad-TD-LUC and Ad-TD-IL-12. Better therapeutic effect and significantly prolong the survival time of tumor animals.
  • the total death time of the animals in the Ad-TD-IL-12 group was significantly earlier than that in the PBS group and other groups, suggesting that full-length IL-12 is likely to cause animal death due to its toxicity.
  • Example 9 Therapeutic effects of Ad-TD-LUC, Ad-TD-IL-12 and Ad-TD-nsIL-12 on the peritoneal dissemination model of golden hamster pancreatic cancer.
  • SHPC6 cells were inoculated into the peritoneal cavity of 4 to 5 weeks old golden hamsters. After 4 days, the animals were divided into four groups of 10 animals each. Intraperitoneal injection was performed with 500 ⁇ l of PBS, 1 ⁇ 10 9 of PFU Ad-TD-LUC, Ad-TD-IL-12, or Ad-TD-nsIL-12, once every other day for 3 times. Animal survival time was observed and the results are shown in Figure 13.
  • Figure 13 shows that the animals in the Ad-TD-nsIL-12 treated group survived 100% during the observation period (200 days after the first treatment), while the Ad-TD-LUC group and the Ad-TD-IL-12 group animals.
  • the survival rates were 30% and 10%, respectively, and the deaths of the Ad-TD-IL-12 group occurred earlier than the PBS group and other groups, suggesting that full-length IL-12 is likely to cause animal death due to its toxicity.
  • Example 10 Comparison of hepatotoxicity of Ad-TD-nsIL-12 and Ad-TD-IL-12 after intraperitoneal injection in golden hamsters.
  • SHPC6 1 ⁇ 10 7 SHPC6 were inoculated into the abdominal cavity of 5-6 weeks old golden hamsters, and the animals were grouped 4 days later, 9 animals in each group, 500 ⁇ l PBS, 1 ⁇ 10 9 PFU Ad-TD-LUC, Ad, respectively.
  • -TD-nsIL-12 or 5 ⁇ 10 8 Ad-TD-IL-12 was injected intraperitoneally, and serum was collected on days 1, 3 and 5 after injection to detect alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkali.
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • alkali alkali.
  • the phosphatase (ALP) The results are shown in Figure 14.
  • Figure 14 shows that the increase in ALT, AST and ALP caused by Ad-TD-nsIL-12 is significantly lower than that of Ad-TD-IL-12, demonstrating that the hepatotoxicity of Ad-TD-nsIL-12 is significantly lower than that of Ad-TD-IL. -12.
  • Example 11 Comparison of anti-tumor effects of Ad-TD-nsIL-12 in a golden hamster head and neck tumor subcutaneous xenograft model.
  • HCPC1 golden hamster head and neck tumor cells
  • the average tumor volume of each group was about 330 mm 3 , respectively, using PBS and Ad-TD.
  • -LUC and Ad-TD-nsIL-12 were injected intratumorally.
  • the virus vector was injected 5 ⁇ 10 7 PFU each time, once a day for 6 times, and then the tumor growth curve and tumor clearance rate were observed. The results are shown in Fig. 15.
  • Figure 15 shows that Ad-TD-nsIL-12 showed a stronger antitumor effect than the control viral vectors Ad-TD-LUC and PBS.
  • Example 12 Comparison of anti-tumor effects of VV-TK-nsIL-12 and VV-TK-RFP and VV-TK-IL-12 in a model of peritoneal disseminated tumor of golden hamster pancreatic cancer
  • Figure 16 shows that VV-TK-nsIL-12 has a better therapeutic effect than VV-TK-RFP and VV-TK-IL-12, and can significantly prolong the survival time of tumor animals.
  • Example 13 Comparison of anti-tumor effects of Ad11-Tel-nsIL-12 and Ad11-Tel-GFP and Ad11-Tel-IL-12 in a peritoneal disseminated tumor model of golden hamster pancreatic cancer
  • 1 ⁇ 10 7 SHPC6 cells were inoculated into the peritoneal cavity of 4 to 5 weeks old golden hamsters. After 4 days, the animals were divided into four groups of 10 animals each. Respectively 500 ⁇ l PBS, 1 ⁇ 10 9 PFU Ad11-Tel-GFP ( green fluorescent protein gene inserted, constructed as Ad11-Tel-nsIL-12 construction method), Ad11-Tel-IL- 12 ( IL-12 inserted into the full-length human The gene was constructed according to the Ad11-Tel-nsIL-12 construction method, or Ad11-Tel-nsIL-12 was administered intraperitoneally, once every other day for 3 times. Animal survival time was observed and the results are shown in Figure 17.
  • Ad11-Tel-GFP green fluorescent protein gene inserted, constructed as Ad11-Tel-nsIL-12 construction method
  • Ad11-Tel-IL-12 IL-12 inserted into the full-length human The gene was constructed according to the Ad11-Tel-nsIL-12 construction
  • Figure 17 shows that Ad11-Tel-nsIL-12 has a better therapeutic effect than Ad11-Tel-GFP and Ad11-Tel-IL-12, and can significantly prolong the survival time of tumor animals.

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Abstract

提供了一种改造的白介素12(nsIL-12)及其基因、重组载体和在制备治疗肿瘤的药物中的用途。

Description

改造的白介素12及其在制备治疗肿瘤药物中的用途
本申请要求于2015年9月9日提交中国专利局、申请号为201510568718.5、发明名称为“改造的白介素12及其在制备治疗肿瘤的药物中的用途”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域：
本发明涉及基因工程生物技术领域，特别是涉及一种改造的白介素12(以下简称nsIL-12)及其基因、重组载体和在制备治疗肿瘤的药物中的用途。
背景技术：
白介素12全称为白细胞介素12(Interleukin-12，IL-12)，是一种主要由抗原递呈细胞如树突状细胞、单核细胞、巨噬细胞和B细胞分泌的免疫调节细胞因子，由p35和p40两个亚基通过二硫键连接组成异二聚体。IL-12的主要功能是参与T细胞的分化，激活T细胞和自然杀伤细胞(NK细胞)，刺激T细胞和NK细胞分泌干扰素γ，介导NK细胞和CD8+细胞毒T细胞的增强，刺激IL-12受体IL-12R-β1和β2的表达，参与先天免疫和继发免疫，是多功能的强免疫调节因子。
目前，已有许多实验室在用多种方式研究IL-12的抗肿瘤作用，如：利用含IL-12基因的质粒电击转化肿瘤细胞；利用生物降解微球体包裹含IL-12质粒转化肿瘤细胞；利用重组IL-12蛋白、抗体和IL-12嵌合体、免疫细胞表达IL-12、含IL-12的腺病毒/逆转录病毒载体等进行基因治疗；以及IL-12与化疗、放疗和免疫治疗结合进行肿瘤治疗等。上述已有研究表明这些IL-12能引起人和鼠肿瘤细胞的消退或减小，以及肿瘤内血管生成减少。但是，上述各种IL-12在体内的半衰期很短，需经持续注射才能够达到治疗效果，且所需IL-12剂量相对较大(1～10μg/天)，而高剂量的IL-12经常导致严重的全身性毒副作用。因此，急需解决IL-12在体 内作用半衰期短和毒副作用大的问题。
系统性给予IL-12引起潜在的致死毒性，导致其一直未用于临床应用。为了将IL-12进行临床应用，许多降低IL-12毒性的开发策略是现在主要的研究热点，一系列修饰IL-12的方法被开发。例如，依赖口服配体药物控制IL-12表达的系统，它能显著地降低IL-12引起的毒性，但不能有效地转化肿瘤细胞和缺乏同时诱导的炎症反应限制了这种方法的抗肿瘤应用。
发明内容：
为了应用IL-12的抗肿瘤潜能，本文通过重新构建新的IL-12，使其抗肿瘤能力保留或增强，但是毒性减弱或消除。本文通过将改造的去除N端信号肽的IL-12插入新的肿瘤靶向性溶肿瘤病毒载体，构建了表达非分泌性IL-12的溶肿瘤病毒，并在研究了其治疗效果和IL-12的功能。基于此，本发明提供了一种改造的白细胞介素12(nsIL-12)及其基因、重组载体和在制备治疗肿瘤的药物中的用途。
在本发明的第一个方面，提供一种编码改造的白介素12的核苷酸序列，其由SEQ NO：1所示的核苷酸序列组成。
所述编码改造的白介素12的核苷酸序列还可以是衍生自人白介素12核苷酸序列但不同于SEQ NO：1的核苷酸序列，且其编码的蛋白表现出抗肿瘤效果或者可引起肿瘤消退并且与现有IL-12相比具有更低的毒副作用。
在本发明的另一个方面，提供一种改造的白介素12，其由上述的核苷酸序列编码。
优选地，所述改造的白介素12由SEQ NO：2所示的氨基酸序列组成。
所述改造的白介素12还可以由衍生自人IL-12的氨基酸序列但不同于SEQ NO：2的氨基酸序列组成，且表现出抗肿瘤效果或者可引起肿瘤消退并且与现有IL-12相比具有更低的毒副作用。
在本发明的另一个方面，提供一种重组载体，其含有编码所述改造的IL-12的所述核苷酸序列。
优选地，所述重组载体可靶向肿瘤细胞。更优选地，所述重组载体可由选自腺病毒载体、痘苗病毒载体、疱疹病毒载体的载体构建获得。
在一个优选例中，所述重组载体为溶肿瘤腺病毒载体Ad-TD-nsIL-12 (中国典型培养物保藏中心，中国.武汉.武汉大学，保藏号CCTCC NO：V201520，保藏日期2015年5月21日，人5型腺病毒突变株，也称为Ad-TD-nsIL12)，其为C亚类的5型腺病毒Ad5，但删除了E1A-CR2、E1B19K和E3gp19K三个基因，并利用E3gp19K启动子控制表达所述改造的IL-12基因序列。
在本发明的另一个方面，提供所述的改造的IL-12或者所述编码改造的白介素12的核苷酸序列或者所述载体在制备治疗肿瘤的药物中的用途。
优选地，所述肿瘤为实体瘤、腹腔播散肿瘤或转移肿瘤。
编码改造的白介素12的核苷酸序列是将改造的人IL-12的p35亚基编码基因(ns-p35，核苷酸序列如SEQ NO：3所示)和改造的人IL-12的p40亚基编码基因(ns-p40，核苷酸序列如SEQ NO：4所示)通过短序列(GTTCCTGGAGTAGGGGTACCTGGGGTGGGC)连接获得的。
所述溶肿瘤腺病毒载体Ad-TD-nsIL-12为C亚类的5型腺病毒Ad5，但删除了E1A-CR2、E1B19K和E3gp19K三个基因，并利用E3gp19K启动子控制表达改造的白介素12基因序列。所述重组腺病毒载体能选择性地在肿瘤细胞中复制，并在进入肿瘤后表达改造的人IL-12，改造的IL-12局限在肿瘤细胞内表达；随着肿瘤细胞裂解，而表达的IL-12得以释放，并同时释放肿瘤相关抗原，释放的肿瘤相关抗原与IL-12协同作用，使机体产生高效、特异的抗肿瘤反应，从而进一步杀伤未被病毒感染的远处肿瘤细胞，包括转移的微小肿瘤细胞灶。这些肿瘤包括裸眼能看到或者显微镜下可见的实体瘤、转移瘤和扩散瘤。所述重组腺病毒载体对腹腔播散肿瘤和原位肿瘤具有更强的肿瘤生长抑制效果，且毒性低。
靶向性治疗人肿瘤的溶肿瘤腺病毒的载体Ad-TD-nsIL-12的构建方法，包括以下步骤：
(1)nsIL-12融合基因克隆：提取培养的RPMI-8866细胞的总RNA，逆转录为cDNA，用包含SnaBⅠ酶切位点和弹性蛋白序列(GTTCCTGGAGTAGGGGTACCTGGGGTGGGC)的引物(p35-F:GTTCCTGGAGTAGGGGTACCTGGGGTGGGCGCCAGAAACCTCCCCGTG,p35-R:GCTACGTATTAGGAAGCATTCAGATA)进行PCR克隆人IL-12的改造的p35亚基(ns-p35，核苷酸序列如SEQ NO：3所示)和改造的p40亚基基因(引物：p40-F：CCTACGTAATGATATGGGAACTGAAGAAAG，p40-R： GCCCACCCCAGGTACCCCTACTCCAGGAACACTGCAGGGCACAGATGC，ns-p40，核苷酸序列如SEQ NO：4所示)，将ns-p40亚基基因片段与ns-p35亚基基因片段通过PCR连接成为nsIL-12完整基因片段，将nsIL-12完整基因片段连接进克隆载体T载体，命名为T-nsIL-12；用SnaBⅠ酶切T-nsIL-12质粒，获得nsIL-12基因片段备用(SEQ NO：1)；
(2)pSSE3gp19K穿梭载体的构建：用Sal1和EcoRV酶切pSS-CHL(见图1)在CHL上游的多克隆位点，用两端含有Sal1和EcoRV酶切位点的引物克隆E3 6.7K基因终止密码子上游1091bp的序列，作为左臂与pSS-CHL连接，构成pSSE3gp19K-L；用SnaB1和Xho1酶切pSSE3gp19K-L,用两端含有SnaB1和Xho1酶切位点的引物克隆E3gp19K基因终止密码子下游1146bp的序列，二者相连构成pSSE3gp19K穿梭载体；
(3)pSSE3gp19K-nsIL-12载体的构建：将pSSE3gp19K载体用EcoR V酶切，且磷酸酶去末端的磷酸基团，之后与nsIL-12基因片段连接产生pSSE3gp19K-nsIL-12载体，测序分析序列；
(4)Ad-TD-nsIL-12-CHL的构建：将pSSE3gp19K-nsIL-12用Pme1线性化，回收含有nsIL-12的大片段，通过电转与pAd-TD载体(见专利ZL200910066130.4)在BJ5183感受态细胞内进行同源重组，挑去阳性克隆pAd-TD-nsIL-12-CHL；
(5)pAd-TD-nsIL-12的构建：将阳性载体pAd-TD-nsIL-12-CHL用Swa1酶切，切除CHL基因，灭活Swa1，T4连接酶后，转化TOP10感受态细胞，获得pAd-TD-nsIL-12载体；和
(6)Ad-TD-nsIL-12病毒载体的构建：将所述重组pAd-TD-nsIL-12载体用Pac1线性化后转染到293细胞中，生产所述的重组腺病毒载体Ad-TD-nsIL-12。
本发明提供了一种改造的IL-12，其是非分泌型。由于本发明所提供的改造的IL-12缺少N端信号肽，因此其不能被分泌至其被表达的细胞之外。本发明所改造的IL-12包括任何天然的IL-12及其变体，例如通过基因工程或者蛋白质工程等手段，前提是该改造的IL-12是不含信号肽的非分泌型IL-12，且保留抗肿瘤活性。基于本公开内容，本领域技术人员可以获得各种本发明所述的、改造的IL-12，并验证其活性(例如可以参见Gene Therapy.2015Sep；22(9):696-706).
因此，本发明具体提供了一种改造的IL-12，其是非分泌型的。
优选地，本发明的改造的IL-12具有如下结构：
p40-连接子-p35或p35-连接子-p40；
其中所述的非分泌型的IL-12不含有分泌信号肽。优选地，上述结构中的p40和p35均不含有信号肽。
对于“连接子”，本领域技术人员可以根据现有技术(例如Adv Drug Deliv Rev.2013October 15；65(10):1357–1369和Front Immunol.2015Apr 7；6:136)来设计或从已有各种连接子中选择出合适的连接子。优选地，连接子具有(GTTCCTGGAGTAGGGGTACCTGGGGTGGGC)的序列。本领域技术人员应当理解，基于本公开内容，本领域技术人员可以设计出具有各种其他序列的连接子(J Biomed Nanotechnol.2015Aug；11(8):1418-30)。
当然，通过溶瘤病毒分别表达没有信号肽的p35以及没有信号肽的p40，即让p35和p40在细胞内进行结合(例如通过二硫键)并生成没有分泌信号肽的IL-12，这种情形也是可行的。
p35和p40包括任何天然的p35和p40以及其变体，例如通过基因工程或者蛋白质工程等手段获得的变体，前提所述变体保留天然p35和p40的活性。优选地，p35和p40是天然的人p35和人p40，以及其变体。更优选地，所述p35具有或由SEQ NO：5的序列组成；p40具有或由SEQ NO：6的序列组成。
本发明的变体可包括或由在其天然的氨基酸序列或者母本序列的基础上经过取代、缺失、插入或添加一个或几个氨基酸且保留取代、缺失、插入或添加前活性的氨基酸序列组成。
示例性地，p35可包括或由在其天然的氨基酸序列或者母本序列的基础上经过取代、缺失、插入或添加一个或几个氨基酸且保留取代、缺失、插入或添加前活性的氨基酸序列组成。例如p35可包括或由在SEQ NO：5上经过取代、缺失、插入或添加一个或几个氨基酸且保留取代、缺失、插入或添加前活性的氨基酸序列组成。
示例性地，p40可包括或由在其天然的氨基酸序列或者母本序列的基础上经过取代、缺失、插入或添加一个或几个氨基酸且保留取代、缺失、插入或添加前活性的氨基酸序列组成。例如p40可包括或由在SEQ NO：6上经过取代、缺失、插入或添加一个或几个氨基酸且保留取代、缺失、插入或添加前活性的氨基酸序列组成。
本发明所述的在氨基酸序列中取代、缺失、插入或添加1个或几个的氨基酸，是指在序列中的任意且1个或几个氨基酸序列中的位置上取代、缺失、插入或添加1个或几个氨基酸残基(例如2个、3个、4个、5个、6个、7个、8个或9个)，并且取代、缺失、插入和添加中的2种或两种以上可同时发生。
本发明所述的在氨基酸序列中取代、缺失、插入或添加1个或几个的氨基酸可通过使用《Molecular Cloning 3》(分子克隆3)和《Current Protocols in Molecular Biology》(现代分子生物学操作规程)等记载的定点诱变法获得。
对于本发明所述的取代，优选在下列各组之内进行氨基酸残基的相互取代：
1：亮氨酸、缬氨酸、丙氨酸、蛋氨酸、丝氨酸、甘氨酸；
2：天冬氨酸、谷氨酸；
3：天冬酰胺、谷氨酰胺；
4：赖氨酸、精氨酸；
5：脯氨酸、羟基脯氨酸；
6：丝氨酸、苏氨酸；和
7：苯丙氨酸、酪氨酸。
对于本文中所述的保守取代、缺失、插入或添加前活性，可表示在取代、缺失、插入或添加后的蛋白质或氨基酸序列的活性是取代、缺失、插入或添加前的10％以上、20％以上、40％以上、60％以上、80％以上、90％以上、95％以上、96％以上、97％以上、98％以上、99％以上、或100％或更高。
上述缺失、取代、插入及/或添加的氨基酸残基的个数，一般优选小的数目。变体与其母本或天然的氨基酸序列有约60％或以上、约70％或以上、71％或以上、72％或以上、73％或以上、74％或以上、75％或以上、76％或以上、77％或以上、78％或以上、79％或以上、80％或以上、81％或以上、82％或以上、83％或以上、84％或以上、85％或以上、86％或以上、87％或以上、88％或以上、89％或以上、90％或以上、91％或以上、92％或以上、93％或以上、94％或以上、95％或以上、96％或以上、97％或以上、98％或以上、99％或以上、99.1％或以上、99.2％或以上、99.3％或以上、99.4％或以上、99.5％或以上、99.6％或以上、99.7％或以上、99.8％或 以上、或99.9％或以上的同一性的氨基酸序列、且具有其母本或天然氨基酸序列所具有的活性。上述同源性的数值一般优选大的数值。
本发明的变体还包括与其天然的氨基酸序列或者母本序列具有至少约60％或以上、约70％或以上、71％或以上、72％或以上、73％或以上、74％或以上、75％或以上、76％或以上、77％或以上、78％或以上、79％或以上、80％或以上、81％或以上、82％或以上、83％或以上、84％或以上、85％或以上、86％或以上、87％或以上、88％或以上、89％或以上、90％或以上、91％或以上、92％或以上、93％或以上、94％或以上、95％或以上、96％或以上、97％或以上、98％或以上、99％或以上、99.1％或以上、99.2％或以上、99.3％或以上、99.4％或以上、99.5％或以上、99.6％或以上、99.7％或以上、99.8％或以上、或99.9％或以上的同一性的氨基酸序列、且具有天然氨基酸序列或母本序列所具有的活性。上述同源性的数值一般优选大的数值。
示例性地，p35还包括与其天然的氨基酸序列或者母本序列(例如SEQ NO：5)具有至少约60％或以上、约70％或以上、71％或以上、72％或以上、73％或以上、74％或以上、75％或以上、76％或以上、77％或以上、78％或以上、79％或以上、80％或以上、81％或以上、82％或以上、83％或以上、84％或以上、85％或以上、86％或以上、87％或以上、88％或以上、89％或以上、90％或以上、91％或以上、92％或以上、93％或以上、94％或以上、95％或以上、96％或以上、97％或以上、98％或以上、99％或以上、99.1％或以上、99.2％或以上、99.3％或以上、99.4％或以上、99.5％或以上、99.6％或以上、99.7％或以上、99.8％或以上、或99.9％或以上的同一性的氨基酸序列、且具有天然氨基酸序列或母本序列所具有的活性。上述同源性的数值一般优选大的数值。
示例性地，p40还包括与其天然的氨基酸序列或者母本序列(例如SEQ NO：6)具有至少约60％或以上、约70％或以上、71％或以上、72％或以上、73％或以上、74％或以上、75％或以上、76％或以上、77％或以上、78％或以上、79％或以上、80％或以上、81％或以上、82％或以上、83％或以上、84％或以上、85％或以上、86％或以上、87％或以上、88％或以上、89％或以上、90％或以上、91％或以上、92％或以上、93％或以上、94％或以上、95％或以上、96％或以上、97％或以上、98％或以上、99％或以上、99.1％或以上、99.2％或以上、99.3％或以上、99.4％或以上、99.5％ 或以上、99.6％或以上、99.7％或以上、99.8％或以上、或99.9％或以上的同一性的氨基酸序列、且具有天然氨基酸序列或母本序列所具有的活性。上述同源性的数值一般优选大的数值。
优选地，本发明具体提供了一种改造的IL-12，其是非分泌型，其具有或者由SEQ NO：2所示的氨基酸序列组成。
示例性地，本发明改造的非分泌型IL-12可包括或由在SEQ NO：2的基础上经过取代、缺失、插入或添加一个或几个氨基酸且保留SEQ NO：2活性的氨基酸序列组成。
示例性地，本发明改造的非分泌型IL-12可包括与SEQ NO：2具有至少约60％或以上、约70％或以上、71％或以上、72％或以上、73％或以上、74％或以上、75％或以上、76％或以上、77％或以上、78％或以上、79％或以上、80％或以上、81％或以上、82％或以上、83％或以上、84％或以上、85％或以上、86％或以上、87％或以上、88％或以上、89％或以上、90％或以上、91％或以上、92％或以上、93％或以上、94％或以上、95％或以上、96％或以上、97％或以上、98％或以上、99％或以上、99.1％或以上、99.2％或以上、99.3％或以上、99.4％或以上、99.5％或以上、99.6％或以上、99.7％或以上、99.8％或以上、或99.9％或以上的同一性的氨基酸序列、且具有SEQ NO：2所具有的活性。上述同源性的数值一般优选大的数值。
在本发明中，非分泌型是指：在编码目的蛋白的基因中，缺乏编码信号肽的片段，故合成蛋白质后不能被运输到分泌相关的内质网-高尔基体-质膜系统，从而不能分泌至细胞外。
本发明还提供了编码本发明所述的改造的非分泌型IL-12的核苷酸。优选地其包括或者由SEQ NO：1所示的核苷酸序列或其互补序列组成。
优选地，编码本发明所述的改造的非分泌型IL-12的核苷酸包含或者由如下序列组成：
a)SEQ NO：1中所示的核苷酸序列；
b)在严谨条件下与SEQ NO：1的核苷酸序列进行杂交、且编码具有非分泌型IL-12活性的氨基酸序列的多核苷酸；或
c)上述a)或b)的互补序列。
本文中，具有非分泌型IL-12活性是指某一蛋白质或氨基酸序列的活性是非分泌型IL-12(SEQ NO.2)的10％以上、20％以上、40％ 以上、60％以上、80％以上、90％以上、95％以上、96％以上、97％以上、98％以上、99％以上、或100％或更高。
进一步优选地，编码本发明所述的改造的非分泌型IL-12的核苷酸包含或者由如下序列组成：
a)SEQ NO：1中所示的核苷酸序列；
b)在严谨条件下与SEQ NO：1的核苷酸序列进行杂交、且编码具有非分泌型IL-12活性的氨基酸序列的多核苷酸；或
c)上述a)或b)的互补序列。
本文所述的“严谨条件”，可以为低严谨条件、中严谨条件、高严谨条件中的任一种，优选为高严谨条件。示例性地，“低严谨条件”可为30℃、5×SSC、5×Denhardt液、0.5％SDS、52％甲酰胺的条件；“中严谨条件”可为40℃、5×SSC、5×Denhardt液、0.5％SDS、52％甲酰胺的条件；“高严谨条件”可为50℃、5×SSC、5×Denhardt液、0.5％SDS、52％甲酰胺的条件。本领域技术人员应当理解温度越高越能得到高同源性的多核苷酸。另外，本领域技术人员可以选择影响杂交的严谨度的温度、探针浓度、探针长度、离子强度、时间、盐浓度等多个因素形成的综合结果来实现相应的严谨度。
除此之外可杂交的多核苷酸还可以为，通过FASTA、BLAST等同源性检索软件用系统设定的默认参数进行计算时，与编码本发明的改造的非分泌型IL-12的多核苷酸具有约60％或以上、约70％或以上、71％或以上、72％或以上、73％或以上、74％或以上、75％或以上、76％或以上、77％或以上、78％或以上、79％或以上、80％或以上、81％或以上、82％或以上、83％或以上、84％或以上、85％或以上、86％或以上、87％或以上、88％或以上、89％或以上、90％或以上、91％或以上、92％或以上、93％或以上、94％或以上、95％或以上、96％或以上、97％或以上、98％或以上、99％或以上、99.1或以上、99.2或以上、99.3％或以上、99.4％或以上、99.5％或以上、99.6％或以上、99.7％或以上、99.8％或以上、或99.9％或以上同一性的多核苷酸。
氨基酸序列、核苷酸序列的同一性，可以使用Karlin及Altschul的算法规则BLAST(Proc.Natl.Acad.Sci.USA 87：2264-2268,1990；Proc.Natl.Acad.Sci.USA 90:5873,1993)来确定。基于BLAST算法规则的程序BLASTN、BLASTX已被开发(Altschul SF,et  al:J Mol Biol 215:403,1990)。使用BLASTN分析碱基序列时，如使参数为score＝100、wordlength＝12；此外使用BLASTX分析氨基酸序列时，如使参数为score＝50、wordlength＝3；使用BLAST和Gapped BLAST程序时，采用各程序的系统可设定默认参数值。
本发明还提供了包含本发明的核苷酸序列或者能表达本发明的改造的非分泌型IL-12的重组载体，例如质粒载体或者病毒载体。另外，本发明还提供了导入本发明所述的重组载体或本发明的核苷酸的微生物(例如细菌)或者细胞，例如大肠杆菌和酵母。导入了本发明所述的重组载体的细胞优选为来源于人的细胞，例如来源于人的各组织或器官的细胞，例如来源于人肝脏、肾脏、胰腺、胃、大肠、小肠的细胞或干细胞，更优选为癌细胞或干细胞。
优选地，包含本发明的核苷酸序列的重组载体为5型腺病毒载体、痘苗病毒载体、11型腺病毒载体、疱疹病毒、呼肠孤病毒、麻疹病毒、新城疫病毒、水泡性口炎病毒、脊髓灰质炎病毒等。
更优选地，包含本发明的核苷酸序列的载体为溶肿瘤腺病毒载体Ad-TD-nsIL-12(中国典型培养物保藏中心，中国.武汉.武汉大学，保藏号CCTCC NO：V201520，保藏日期2015年5月21日，人5型腺病毒突变株)。
本发明还提供了一种药盒或药物，其包括本发明的改造的非分泌型IL-12、编码本发明的改造的非分泌型IL-12的核苷酸、能表达本发明的改造的非分泌型IL-12的载体、或包含本发明的核苷酸序列的载体。优选地，所述药盒或药物用于治疗或者预防与IL-12相关的疾病。
本发明还提供了本发明的改造的非分泌型IL-12、编码本发明的改造的非分泌型IL-12的核苷酸、能表达本发明的改造的非分泌型IL-12的载体、或包含本发明的核苷酸序列的载体在制备用于治疗受试者中与IL-12相关的疾病(例如癌症)的药盒或药物中的用途。
本发明还提供了治疗受试者中与IL-12相关的疾病(例如癌症)的方法，其包括向所述受试者施用有效量的本发明的改造的非分泌型IL-12、编码本发明的改造的非分泌型IL-12的核苷酸、能表达本发明的改造的非分泌型IL-12的载体、或包含本发明的核苷酸序列的载体。
优选地，所述受试者为哺乳动物，更优选为人。
在本文中，“与IL-12相关的疾病”优选指能够通过使用IL-12治疗或预防的疾病，例如癌症。
虽然本发明以胰腺癌、头颈部肿瘤、肺癌、食管癌、卵巢癌、大肠癌、结肠癌和胃癌进行了举例说明，但术语“癌症”可为实体瘤、腹腔播散肿瘤或转移肿瘤，包括，但不限于原发性黑素瘤、转移性黑素瘤，腺癌、鳞状细胞癌、鳞腺细胞癌、胸腺瘤、肉瘤、非霍奇金淋巴瘤、霍奇金淋巴瘤、白血病、前列腺癌、卵巢癌、胰腺癌、结肠癌、多发性骨髓瘤、成神经细胞瘤、NPC、膀胱癌、子宫颈癌、肾癌、脑癌、骨癌、胃癌、食道癌、肠癌(例如直肠癌和十二指肠癌)、肝癌、胰腺癌、肺癌、骨癌、膀胱癌、卵巢癌、淋巴癌、血癌、乳腺癌、头颈癌、宫颈癌、子宫癌、黑色素瘤等各种癌症及相关癌症症状。优选地，使用本发明治疗的癌症为优选地，所述胰腺癌、肾癌、头颈部肿瘤、肺癌、食管癌、乳腺癌、卵巢癌、大肠癌或胃癌及其相关症状。
本发明的作为活性成分的改造的非分泌型IL-12、编码本发明的改造的非分泌型IL-12的核苷酸、能表达本发明的改造的非分泌型IL-12的载体、或包含本发明的核苷酸序列的载体可以连同药学上可接受的载体一起使用。除活性成分外，本发明的方法、用途和产品还可以包含合适的药学上可接受的载体，包括促进活性成分加工成制剂的赋形剂和助剂。
例如适于注射或输注的制剂包括水性和非水性无菌注射液和水性和非水性无菌混悬剂，所述无菌注射液可任选地包含抗氧化剂、缓冲剂、抑菌剂和能使制剂与目的接收者的血液等压的溶质，所述无菌混悬剂可包括悬浮剂和增稠剂。所述制剂可存在于单位剂量或多剂量容器中，例如密封的安瓿，并且可以保存在冻结干燥的(冻干)条件，在立即使用前仅需要加入无菌液体载体，例如注射用水进行重构。
本发明的活性成分任选地可与固体赋形剂相组合，且任选地磨碎所得到的混合物，并且需要时，在加入合适的助剂后，加工颗粒的混合物，以获得所需剂型。合适的赋形剂特别是填充剂例如糖，包括乳糖、蔗糖、甘露醇或山梨糖醇；纤维素或淀粉制剂、明胶、黄蓍胶、甲基纤维素、羟丙基甲基纤维素、羧甲基纤维素钠和/或聚乙烯吡咯烷酮(PVP)。需要时，可以加入崩解剂，例如交联聚乙烯吡咯烷酮、琼脂或海藻酸或其盐例如海藻酸钠。
本发明的活性成分的有效量可为治疗癌症、或缓解癌症症状或抑制癌细胞的任何量，其可以是相当于约0.1-15mg活性成分，优选0.2-12mg活性成分的剂量单位。更优选地，剂量单位包括约2-5mg的活性成分。最优选地，剂量单位包括约3-4mg的活性成分。有效量的测定在本领域技术人员的能力内，特别是根据本文提供的公开内容的启示下。
根据本发明，本发明的药学产品(药物、药盒)可以以任意有效剂量数施用给药受试者。优选地，本发明的药学产品(药物、药盒)可以以多次剂量给药，例如从约2至约15次剂量，更优选从约4-10次剂量，最优选约6次剂量。在特别优选的实施方案，在给药过程中，以隔天给药约一次的频率将本发明的药学产品(药物、药盒)给药至受试者，例如注射、输注或口服。在特别优选的实施方案，给药为通过注射或者输注，最优选为瘤内注射。
应当理解本发明的药学产品(药物、药盒)可以按用于通过任意适宜的途径给药的任意适宜的方式配制。
本发明的药学产品(药物、药盒)的剂量单位是基于常规进行给药受试者。例如，剂量单位可以给药多于每日一次、每两天一次、每周一次、每月一次等。剂量单位可以是以两次/周为基础给药，即每周两次，例如每三天一次。
本发明还提供了制备本发明的重组载体的方法，其包括将编码本发明所述的改造的非分泌型IL-12的核苷酸插入至载体中以获得可表达改造的非分泌型IL-12的重组载体；以及将重组载体转染至细胞中以生产本发明的重组病毒。
如本文所使用的，“包含”与“包括”、“含有”或“特征在于”同义，并且是包括在内的或开放性的，并且不排除另外的未陈述的元件或方法步骤。术语“包含”在本文中的任何表述，特别是在描述本发明的方法、用途或产品时，应理解为包括基本上由所述组分或元件或步骤组成和由所述组分或元件或步骤组成的那些产品、方法和用途。本文示例性描述的本发明适当地可以在不存在本文未具体公开的任何一种或多种元件、一种或多种限制的情况下进行实践。
本发明的药学产品中所包含的涉及该药学产品的说明书可以含有如下内容：适应症(癌症，例如胰腺癌)、施用剂量(例如上述所示例性说明的)以及可能产生的副作用等等。
本发明的重组载体(例如重组的溶瘤病毒)能选择性地在肿瘤细胞或癌细胞中复制，并在进入肿瘤或癌细胞后表达改造的非分泌型IL-12，改造的非分泌型IL-12由于缺少信号肽序列被局限在肿瘤细胞或癌细胞内表达；随着肿瘤细胞或癌细胞裂解，而表达的IL-12得以释放，并同时释放肿瘤相关抗原或癌细胞相关抗原，释放的肿瘤相关抗原或癌细胞相关抗原与IL-12协同作用，使机体产生高效、特异的抗肿瘤或抗癌反应，从而进一步杀伤未被病毒感染的远处肿瘤细胞或癌细胞，包括转移的微小肿瘤细胞灶或癌细胞灶。这些肿瘤或癌包括裸眼能看到或者显微镜下可见的实体瘤、转移瘤和扩散瘤。本发明的发明人出乎意料地发现，包含编码没有信号肽的IL-12核苷酸的重组载体(例如重组的溶瘤病毒)能够稳定、持续和低水平表达L-12蛋白，从而弥补了现有IL-12蛋白半衰期短、以及高水平表达易引起毒副作用的不足；另外，具有更强的肿瘤生长抑制效果，且无显著性毒性；而且包含编码没有信号肽的IL-12核苷酸的重组载体(例如重组的溶瘤病毒)对肿瘤或癌症(例如腹腔播散肿瘤和原位肿瘤)具有更强的肿瘤生长抑制效果，且毒性低。
相反，如果重组载体(例如溶瘤病毒)中所表达的IL-12含有信号肽，所表达含有信号肽的IL-12则被及时分泌至肿瘤细胞或癌细胞外，从而使得有机体遭受较大剂量的IL-12的挑战，进而对有机体造成较大的毒副作用。另外由于重组载体所表达IL-12过早地被分泌出肿瘤细胞或癌细胞，从而当重组载体(例如溶瘤病毒)裂解肿瘤细胞或癌细胞进而释放肿瘤相关抗原或癌细胞相关抗原时，有机体不能有效地募集有效量的IL-12与所释放的肿瘤相关抗原或癌细胞相关抗原协同作用。因此，相对于包含编码没有信号肽的IL-12核苷酸的重组载体(例如重组的溶瘤病毒)，包含编码具有信号肽的IL-12核苷酸的重组载体(例如重组的溶瘤病毒)具有强的毒副作用，且不能够使机体产生高效、特异的抗肿瘤或抗癌反应。
本文已采用的术语和表述用作描述性而不是限制性术语，并且在此种术语和表述的使用中不预期排除所示和所述特征或其部分的任何等价物，但应认识到各种修饰在请求保护的本发明的范围内是可能的。因此，应当理解尽管本发明已通过优选实施方案和任选特征具体公开，但本领域技术人员可以采用本文公开的概念的修饰和变化，并且此类修饰和变化被视为在如由附加权利要求定义的本发明的范围内。
为更清楚地说明本发明，现结合如下实施例进行详细说明，但这些实施例仅仅是对本发明的示例性描述，并不能解释为对本申请的限制。
本发明的有益效果是：
1.与现有IL-12相比，本发明的改造的IL-12主要分布在局部肿瘤组织，提高了对肿瘤细胞的特异性，降低了IL-12的全身毒副作用；
2.本发明所述的用肿瘤靶向性病毒载体表达改造的IL-12方法，能使IL-12蛋白稳定持续低水平表达，弥补了现有IL-12蛋白半衰期短、以及高水平表达易引起毒副作用的不足；
3.与现有IL-12相比，本发明的改造的IL-12对腹腔播散肿瘤和原位肿瘤具有更强的肿瘤生长抑制效果，且无显著性毒性。
本发明的溶肿瘤腺病毒Ad-TD-nsIL-12，不仅可用于肿瘤内注射，还可用于腹腔、胸腔内注射，降低了给药难度，扩大了可治疗患者范围，使不同部位发生肿瘤的患者均能获得良好的疗效，同时不会产生明显的副作用。
附图说明：
图1：pSS-CHL质粒图谱。
图2：pVV-TK质粒图谱。
图3：pVV-TK-nsIL-12质粒图谱。
图4：5型腺病毒载体Ad-TD-nsIL-12、痘苗病毒载体VV-TK-nsIL-12和11型腺病毒载体Ad11-Tel-nsIL-12的结构示意图。
图5：Ad-TD-nsIL-12在细胞中的IL-12表达。
图6：VV-TK-nsIL-12在细胞中的IL-12表达。
图7：Ad-TD-nsIL-12对不同类型实体瘤的抗肿瘤作用。
图8：VV-TK-nsIL-12对不同类型实体瘤的抗肿瘤作用。
图9：荷瘤金黄地鼠(又名叙利亚仓鼠，Syrian hamster)注射不同病毒载体后外周血中IL-12的表达情况。
图10：Ad-TD-nsIL-12与Ad-TD-LUC和Ad-TD-IL-12在金黄地鼠胰腺癌皮下移植瘤模型中抗肿瘤效果的比较。
图11：Ad-TD-nsIL-12与Ad-TD-LUC和Ad-TD-IL-12在金黄地鼠胰腺癌皮下移植瘤模型中肿瘤清除率的比较。
图12：Ad-TD-nsIL-12与Ad-TD-LUC和Ad-TD-IL-12在金黄地鼠胰 腺癌原位模型中抗肿瘤效果的比较。
图13：Ad-TD-nsIL-12与Ad-TD-LUC和Ad-TD-IL-12在金黄地鼠胰腺癌腹腔播散肿瘤模型中抗肿瘤效果的比较。
图14：Ad-TD-nsIL-12与Ad-TD-LUC和Ad-TD-IL-12在金黄地鼠胰腺癌腹腔播散肿瘤模型中肝毒性的比较。
图15：Ad-TD-nsIL-12在金黄地鼠头颈部肿瘤皮下移植瘤模型中抗肿瘤效果的比较。
图16：VV-TK-nsIL-12与VV-TK-RFP和VV-TK-IL-12在金黄地鼠胰腺癌腹腔播散肿瘤模型中抗肿瘤效果的比较。
图17：Ad11-Tel-nsIL-12与Ad11-Tel-GFP和Ad11-Tel-IL-12在金黄地鼠胰腺癌腹腔播散肿瘤模型中抗肿瘤效果的比较。
具体实施方式：
以下通过实例对本发明进一步说明，并不是限定本发明的保护范围，本领域的技术人员根据说明书可以对这些实施方案进行修改，只要不脱离本发明的精神和范围都可以得到类似或相同的结果，均在本发明的保护范围之内。
实施例1：靶向性治疗人肿瘤的溶肿瘤5型腺病毒的载体Ad-TD-nsIL-12的构建方法，包括以下步骤：
(1)nsIL-12融合基因克隆：提取培养的RPMI-8866细胞的总RNA，逆转录为cDNA，用包含SnaBⅠ酶切位点和弹性蛋白序列(GTTCCTGGAGTAGGGGTACCTGGGGTGGGC)的引物(p35-F:GTTCCTGGAGTAGGGGTACCTGGGGTGGGCGCCAGAAACCTCCCCGTG,p35-R:GCTACGTATTAGGAAGCATTCAGATA)进行PCR克隆人IL-12的改造的p35亚基(ns-p35，核苷酸序列如SEQ NO：3所示)和改造的p40亚基基因(引物：p40-F：CCTACGTAATGATATGGGAACTGAAGAAAG，p40-R：GCCCACCCCAGGTACCCCTACTCCAGGAACACTGCAGGGCACAGATGC，ns-p40的核苷酸序列如SEQ NO：5所示)，将ns-p40亚基基因片段与ns-p35亚基基因片段通过PCR连接成为nsIL-12完整基因片段，将nsIL-12完整基因片段连接进克隆载体T载体，命名为T-nsIL-12；用SnaBⅠ酶切T-nsIL-12质粒，获得nsIL-12基因片段备用(见SEQ NO：1)；
(2)pSSE3gp19K穿梭载体的构建：用Sal1和EcoRV酶切pSS-CHL(在pBR322质粒基础上构建，见图1)在CHL上游的多克隆位点，用两端含有Sal1和EcoRV酶切位点的引物克隆E3 6.7K基因终止密码子上游1091bp的序列，作为左臂与pSS-CHL连接，构成pSSE3gp19K-L；用SnaB1和Xho1酶切pSSE3gp19K-L,用两端含有SnaB1和Xho1酶切位点的引物克隆E3gp19K基因终止密码子下游1146bp的序列，二者相连构成pSSE3gp19K穿梭载体；
(3)pSSE3gp19K-nsIL-12载体的构建：将pSSE3gp19K载体用EcoR V酶切，且磷酸酶去末端的磷酸基团，之后与nsIL-12基因片段连接产生pSSE3gp19K-nsIL-12载体，测序分析序列；
(4)Ad-TD-nsIL-12-CHL的构建：将pSSE3gp19K-nsIL-12用Pme1线性化，回收含有nsIL-12的大片段，通过电转与pAd-TD载体(见专利
ZL200910066130.4)在BJ5183感受态细胞内进行同源重组，挑去阳性克隆pAd-TD-nsIL-12-CHL。
(5)pAd-TD-nsIL-12的构建：将阳性载体pAd-TD-nsIL-12-CHL用Swa1酶切，切除CHL基因，灭活Swa1，T4连接酶后，转化TOP10感受态细胞，获得pAd-TD-nsIL-12载体。
(6)Ad-TD-nsIL-12病毒载体的构建：将所述重组pAd-TD-nsIL-12载体用Pac1线性化后转染到293细胞中，生产所述的重组腺病毒载体Ad-TD-nsIL-12。
3.将所述重组Ad-TD-nsIL-12载体用Pac1线性化后转染到293细胞中，生产所述的重组腺病毒载体Ad-TD-nsIL-12(人5型腺病毒突变株，中国典型培养物保藏中心，中国.武汉.武汉大学，保藏号CCTCC NO：V201520，保藏日期2015年5月21日)，也可称为Ad-TD-nsIL12。
实施例2：靶向性治疗人肿瘤的溶肿瘤痘苗病毒的载体VV-TK-nsIL-12的构建方法，包括以下步骤：
1.构建穿梭载体pVV-TK-nsIL-12
如实施例1，用含有Sal1和Nhe1酶切位点的引物克隆nsIL-12基因，并用相应的酶切备用，用Sal1和Nhe1酶切pVV-TK质粒(见图2，按发明人发表的方法构建，Mol Ther Methods Clin Dev.2015Sep 16；2:15035.doi:10.1038/mtm.2015.35.eCollection 2015)，将nsIL-12基因和 pVV-TK连接，构建pVV-TK-nsIL-12载体(图3)。
2.VV-TK-nsIL-12病毒载体重组
培养CV1细胞，将CV1接种到96孔板中，当细胞长至90％融合度，转染CRISP-cas9和gRNA质粒，24小时后感染痘苗病毒VVL15，两小时后，转染pVV-TK。24小时后在荧光显微镜下观察，并挑取红色荧光细胞克隆，证明VV-TK-nsIL-12病毒载体(图4)重组成功。
3.VV-TK-nsIL-12病毒载体的筛选
将挑去的红色荧光克隆冻融一次后，再次感染CV1细胞，挑取红色荧光克隆，反复筛选5次后，感染CV1细胞生产病毒。提取基因组进行测序。
实施例3：靶向性治疗人肿瘤的溶肿瘤11型腺病毒的载体Ad11-Tel-nsIL-12的构建方法，包括以下步骤：
1.含有nsIL-12的穿梭载体pSSE3-18.5K-nsIL-12的构建
(1)pSSE3-18.5K的构建，用SnaB1和Xho1酶切pSS-CHL(在pBR322质粒基础上构建，见图1)的多克隆位点，用两端含有SnaB1和Xho1酶切位点的引物克隆E3 16.1K基因终止密码子上游535bp的序列，作为左臂与pSS-CHL连接，构成pSSE3-18.5K-L；用Sal1和EcoRV酶切pSSE3-18.5K-L,用两端含有Sal1和EcoRV酶切位点的引物克隆E3-18.5K基因终止密码子下游584bp的序列，二者相连构成pSSE3-18.5K穿梭载体；
(2)pSSE3-18.5K-nsIL-12载体的构建，将pSSE3-18.5K载体用SnaB1酶切，且磷酸酶去末端的磷酸基团，之后与nsIL-12基因片段连接产生pSSE3-18.5K-nsIL-12载体，测序分析序列；
(3)将pSSE3gp19K-nsIL-12用Pme1线性化，回收含有nsIL-12的大片段，通过电转与Ad11-Tel-GFP(构建方法见专利ZL20110143385.3)在BJ5183感受态细胞内进行同源重组，挑去阳性克隆Ad11-Tel-nsIL-12(见图4)。
2.将所述重组Ad11-Tel-nsIL-12载体用Not1线性化后转染到293细胞中，生产所述的重组腺病毒载体Ad11-Tel-nsIL-12。
实施例4：Ad-TD-nsIL-12和VV-TK-nsIL-12在肿瘤细胞中的IL-12 表达
将培养的人胰腺癌Suit2和Capan1、头颈部肿瘤EC9706、肺癌A549和H1299、食管癌EC9706和ZZB、卵巢癌SKOV3、大肠癌SW620和HCT116和胃癌AGS等细胞，胰酶消化，计数，2x105细胞/孔，铺6孔板，分别感染Ad-TD-nsIL-12和VV-TK-nsIL-12,分别收集上清及细胞的混合物，ELISA检测IL-12的表达，结果见图5和6。
图5显示在肿瘤细胞中Ad-TD-nsIL-12表达了IL-12，图6显示在肿瘤细胞中VV-TK-nsIL-12表达了IL-12。
实施例5：Ad-TD-nsIL-12对各种实体瘤的抗肿瘤作用
将培养的胰腺癌SUIT2和Capan1、头颈部肿瘤EC9706、肺癌A549和H1299、食管癌EC9706和ZZB、卵巢癌SKOV3、大肠癌SW620和HCT116和胃癌AGS等细胞，胰酶消化，计数，用含2％FBS的DMEM制成细胞悬液，接种于96孔板中央，其中B1至G1为无细胞的培养基，另外三边为PBS。
在14-18h之后，倍比稀释病毒VV-TK/Ad-TD-nsIL-12、VV-TK/Ad-TD-IL-12(全长人IL-12)和Ad-TD-LUC(LUC，萤光素酶)或VV-TK-RFP。以1x104pfu/细胞为起始浓度，10倍比稀释病毒溶液，共稀释九个梯度，最后一列细胞不加病毒。倍比稀释后，用排枪加入96孔板中央，10μl/孔每一列为相同的病毒梯度。
将感染病毒的细胞放回37℃培养箱，6天后，将除加PBS以外的孔中加入20μl MTS和PMS 20:1(MTS:PMS)混合液。1-4h后取出用酶标仪在波长490nm测量吸光度。计算EC50，结果见图7和8。
图7和8显示Ad-TD-nsIL-12和VV-TK-nsIL-12具有杀伤各种实体瘤肿瘤细胞的能力。
实施例6：Ad-TD-nsIL-12与Ad-TD-LUC和Ad-TD-IL-12在荷瘤金黄地鼠腹腔注射一次后的IL-12表达变化的比较。
将1×107的SHPC6(金黄地鼠胰腺癌细胞，由美国圣路易斯大学WSM Wold提供)接种到5～6周龄的金黄地鼠(又名叙利亚仓鼠，Syrian hamster)腹腔，4天后将动物分为四组，每组9只动物。分别用500μl PBS，1×109的PFU Ad-TD-LUC(LUC来自pGL3载体萤光素酶,按构建实施例1 中有关Ad-TD-nsIL-12的方法构建)，Ad-TD-IL-12(全长人IL-12，按实施例1中有关构建Ad-TD-nsIL-12的方法构建)，或Ad-TD-nsIL-12腹腔注射一次，在注射后第1、3、5天收集血清检测外周血中IL-12的表达量变化，结果见图9。
图9结果表明，Ad-TD-IL-12组动物在注射病毒载体后第一天，高水平表达IL-12，而第三天、第五天时IL-12表达明显下降，表达水平变化非常大；而Ad-TD-nsIL-12组动物，则每天低水平恒量表达IL-12，从第一天到第五天，IL-12表达水平并无明显差异，提示Ad-TD-nsIL-12在动物体内不易因IL-12大量产生而导致毒副作用。
实施例7：肿瘤靶向性腺病毒Ad-TD-nsIL-12的抗肿瘤应用，及其与Ad-TD-LUC和Ad-TD-IL-12的比较。
在5～6周龄的金黄地鼠背部右上侧接种1×106个HPD1-NR(金黄地鼠胰腺癌细胞，由美国圣路易斯大学WSM Wold赠送)，每组动物肿瘤平均体积约为330mm3时，分别用PBS、Ad-TD-LUC、Ad-TD-IL-12和Ad-TD-nsIL-12进行瘤内注射，病毒载体每次注射1×109PFU，每天注射一次，共6次，然后观察肿瘤生长曲线及肿瘤清除率，结果见图10和图11。
图10表明Ad-TD-nsIL-12和Ad-TD-IL-12比对照病毒载体Ad-TD-LUC和PBS显示出更强的抗肿瘤效果；图11表明Ad-TD-nsIL-12和Ad-TD-IL-12治疗组动物的肿瘤清除率极高，可达100％，而Ad-TD-LUC组动物的肿瘤清除率仅为42.8％。
实施例8：Ad-TD-nsIL-12对金黄地鼠胰腺癌原位模型的治疗作用，及其与Ad-TD-LUC和Ad-TD-IL-12的比较。
10％水合氯醛麻醉4～5周龄的金黄地鼠，打开金黄地鼠左侧腹腔，找到与脾脏连接的胰腺，将3×106个HapT1细胞(金黄地鼠胰腺癌细胞，由美国圣路易斯大学WSM Wold提供)接种到的胰腺实质。6天后，将成瘤动物分为四组，每组7只动物。分别用500μl PBS、1×109的PFU Ad-TD-LUC、Ad-TD-IL-12或Ad-TD-nsIL-12进行腹腔注射治疗，隔天注射一次，共6次。观察动物生存时间，结果见图12。
图12结果表明，Ad-TD-nsIL-12比Ad-TD-LUC和Ad-TD-IL-12具有 更好的治疗效果，且能显著延长肿瘤动物的存活时间。而Ad-TD-IL-12组的动物全部死亡时间显著早于PBS组及其他各组，提示全长IL-12因其毒性易导致动物死亡。
实施例9：Ad-TD-LUC、Ad-TD-IL-12和Ad-TD-nsIL-12对金黄地鼠胰腺癌腹腔播散模型的治疗作用。
将1×107个SHPC6细胞接种到4～5周龄的金黄地鼠的腹腔中。4天后，将动物分为四组，每组10只动物。分别用500μl PBS、1×109的PFU Ad-TD-LUC、Ad-TD-IL-12，或Ad-TD-nsIL-12进行腹腔注射治疗，隔天一次，共3次。观察动物生存时间，结果见图13。
图13结果表明，Ad-TD-nsIL-12治疗组的动物，在观察期内(第一次治疗200天内)100％存活，而Ad-TD-LUC组和Ad-TD-IL-12组动物的存活率分别为30％和10％，且Ad-TD-IL-12组动物死亡发生的时间早于PBS组及其他各组，提示全长IL-12因其毒性易导致动物死亡。
实施例10：Ad-TD-nsIL-12与Ad-TD-IL-12在金黄地鼠腹腔注射后的肝毒性比较。
将1×107个SHPC6接种到5-6周龄的金黄地鼠腹腔，4天后将动物分组，每组9只动物，分别用500μl PBS、1×109的PFU Ad-TD-LUC、Ad-TD-nsIL-12或5×108Ad-TD-IL-12腹腔注射一次，在注射后第1、3、5天收集血清，检测谷丙转氨酶(ALT)、谷草转氨酶(AST)和碱性磷酸酶(ALP)，结果见图14。
图14结果表明，Ad-TD-nsIL-12引起的ALT、AST和ALP增高显著低于Ad-TD-IL-12，证明Ad-TD-nsIL-12的肝毒性显著低于Ad-TD-IL-12。
实施例11：Ad-TD-nsIL-12在金黄地鼠头颈部肿瘤皮下移植瘤模型中抗肿瘤效果的比较。
在5～6周龄的金黄地鼠背部右上侧接种1×107个HCPC1(金黄地鼠头颈部肿瘤细胞),每组动物肿瘤平均体积约为330mm3时，分别用PBS、Ad-TD-LUC和Ad-TD-nsIL-12进行瘤内注射，病毒载体每次注射5×107PFU，每天注射一次，共6次，然后观察肿瘤生长曲线及肿瘤清除率，结果见图15。
图15结果表明，Ad-TD-nsIL-12比对照病毒载体Ad-TD-LUC和PBS显示出更强的抗肿瘤效果。
实施例12：VV-TK-nsIL-12与VV-TK-RFP和VV-TK-IL-12在金黄地鼠胰腺癌腹腔播散肿瘤模型中抗肿瘤效果的比较
将1×107个SHPC6细胞接种到4～5周龄的金黄地鼠的腹腔中。4天后，将动物分为四组，每组10只动物。分别用500μl PBS、4×107PFU VV-TK-RFP(插入红色荧光蛋白，按VV-TK-nsIL-12构建方法构建)、VV-TK-IL-12(插入全长人IL-12基因，按VV-TK-nsIL-12构建方法构建)或VV-TK-nsIL-12进行腹腔注射治疗，隔天一次，共3次。观察动物生存时间，结果见图16。
图16结果表明，VV-TK-nsIL-12比VV-TK-RFP和VV-TK-IL-12具有更好的治疗效果，且能显著延长肿瘤动物的存活时间。
实施例13：Ad11-Tel-nsIL-12与Ad11-Tel-GFP和Ad11-Tel-IL-12在金黄地鼠胰腺癌腹腔播散肿瘤模型中抗肿瘤效果的比较
将1×107个SHPC6细胞接种到4～5周龄的金黄地鼠的腹腔中。4天后，将动物分为四组，每组10只动物。分别用500μl PBS、1×109PFU Ad11-Tel-GFP(插入绿色荧光蛋白基因，按Ad11-Tel-nsIL-12构建方法构建)、Ad11-Tel-IL-12(插入全长人IL-12基因，按Ad11-Tel-nsIL-12构建方法构建)，或Ad11-Tel-nsIL-12进行腹腔注射治疗，隔天一次，共3次。观察动物生存时间，结果见图17。
图17结果表明，Ad11-Tel-nsIL-12比Ad11-Tel-GFP和Ad11-Tel-IL-12具有更好的治疗效果，且能显著延长肿瘤动物的存活时间。
最后应当说明的是，以上实施例仅用于说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围，其均应涵盖在本发明的权利要求范围内。

Claims (19)

  1. 一种改造的IL-12，其是非分泌型的。
  2. 根据权利要求1所述的改造的IL-12，其具有如下结构：
    p40-连接子-p35或p35-连接子-p40；
    其中所述的改造的IL-12不含有分泌信号肽，优选地，上述结构中的p40和p35均不含有分泌信号肽。
  3. 根据权利要求2所述的改造的IL-12，其中p35和p40是天然的p35和p40或其活性变体，优选地，p35和p40是天然的人p35和人p40或其活性变体；更优选地，所述p35具有或由SEQ NO：5的序列组成，p40具有或由SEQ NO：6的序列组成。
  4. 根据权利要求3所述的改造的IL-12，其中所述变体与其母本或天然的氨基酸序列有约60％或以上、约70％或以上、71％或以上、72％或以上、73％或以上、74％或以上、75％或以上、76％或以上、77％或以上、78％或以上、79％或以上、80％或以上、81％或以上、82％或以上、83％或以上、84％或以上、85％或以上、86％或以上、87％或以上、88％或以上、89％或以上、90％或以上、91％或以上、92％或以上、93％或以上、94％或以上、95％或以上、96％或以上、97％或以上、98％或以上、99％或以上、99.1％或以上、99.2％或以上、99.3％或以上、99.4％或以上、99.5％或以上、99.6％或以上、99.7％或以上、99.8％或以上、或99.9％或以上的同一性的氨基酸序列、且具有其母本或天然氨基酸序列所具有的活性。
  5. 根据权利要求3所述的改造的IL-12，其中所述变体包括或由在其天然的氨基酸序列或者母本序列的基础上经过取代、缺失、插入或添加一个或几个氨基酸且保留取代、缺失、插入或添加前活性的氨基酸序列组成。
  6. 根据权利要求1-5中任一项所述的改造的IL-12，其中所述的连接子具有或者由SEQ NO：7的氨基酸组成。
  7. 根据权利要求1-5中任一项所述的改造的IL-12，其具有或者由SEQ NO：2的氨基酸或其变体组成。
  8. 根据权利要求7所述的改造的IL-12，其中所述变体与SEQ NO：2的氨基酸序列有约60％或以上、约70％或以上、71％或以上、72％或以 上、73％或以上、74％或以上、75％或以上、76％或以上、77％或以上、78％或以上、79％或以上、80％或以上、81％或以上、82％或以上、83％或以上、84％或以上、85％或以上、86％或以上、87％或以上、88％或以上、89％或以上、90％或以上、91％或以上、92％或以上、93％或以上、94％或以上、95％或以上、96％或以上、97％或以上、98％或以上、99％或以上、99.1％或以上、99.2％或以上、99.3％或以上、99.4％或以上、99.5％或以上、99.6％或以上、99.7％或以上、99.8％或以上、或99.9％或以上的同一性的氨基酸序列、且具有其母本或天然氨基酸序列所具有的活性。
  9. 根据权利要求7所述的改造的IL-12，其中所述变体包括或由在SEQ NO：2的基础上经过取代、缺失、插入或添加一个或几个氨基酸且保留取代、缺失、插入或添加前活性的氨基酸序列组成。
  10. 编码权利要求1-9中任一项所述的改造的IL-12的核苷酸。
  11. 根据权利要求10所述的核苷酸，其包含或者由如下序列组成：
    a)SEQ NO：1中所示的核苷酸序列；
    b)在严谨条件下与SEQ NO：1的核苷酸序列进行杂交、且编码具有非分泌型IL-12活性的氨基酸序列的多核苷酸；或
    c)上述a)或b)的互补序列。
  12. 重组载体，其包含权利要求10或11核苷酸或者能表达权利要求1-9中任一项所述的改造的IL-12。
  13. 根据权利要求12所述的重组载体，其为质粒载体或者病毒载体。
  14. 根据权利要求12所述的重组载体，其为溶瘤病毒，优选为5型腺病毒载体、痘苗病毒载体或11型腺病毒载体，更优选为保藏号为CCTCC NO：V201520的病毒载体。
  15. 微生物或分离的细胞，其包含权利要求10或11核苷酸或者能表达权利要求1-9中任一项所述的改造的IL-12，其中所述分离的细胞优选为来源于人的细胞，例如来源于人肝脏、肾脏、胰腺、胃、大肠、小肠的细胞或干细胞，更优选为癌细胞或干细胞。
  16. 一种药物，其包括权利要求1-9中任一项所述的非分泌型IL-12、 权利要求10或11的核苷酸、或权利要求12-14中任一项所述的载体。
  17. 治疗与IL-12相关的疾病的方法，其包括向受试者施用权利要求12-14中任一项所述的载体，优选地，所述疾病为癌症，更优选为胰腺癌、头颈部肿瘤、肺癌、食管癌、卵巢癌、大肠癌、结肠癌和胃癌，最优选为腹腔播散型肿瘤。
  18. 权利要求1-9中任一项所述的非分泌型IL-12、权利要求10或11的核苷酸、或权利要求12-14中任一项所述的载体在制备用于治疗受试者中与IL-12相关的疾病的药物中的用途，优选地，所述疾病为癌症，更优选为胰腺癌、头颈部肿瘤、肺癌、食管癌、卵巢癌、大肠癌、结肠癌和胃癌，最优选为腹腔播散型肿瘤。
  19. 制备本重组载体的方法，其包括将权利要求10或11的核苷酸插入至载体中以获得可表达的非分泌型IL-12的重组载体。
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