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WO2007102690A1 - Vecteur comprenant la cassette d'adn optimisée par un codon pour produire le dodécamère recombinant sécréteur trail - Google Patents

Vecteur comprenant la cassette d'adn optimisée par un codon pour produire le dodécamère recombinant sécréteur trail Download PDF

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Publication number
WO2007102690A1
WO2007102690A1 PCT/KR2007/001099 KR2007001099W WO2007102690A1 WO 2007102690 A1 WO2007102690 A1 WO 2007102690A1 KR 2007001099 W KR2007001099 W KR 2007001099W WO 2007102690 A1 WO2007102690 A1 WO 2007102690A1
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Prior art keywords
trail
dodecamer
recombinant
codon
nucleotide sequence
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PCT/KR2007/001099
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English (en)
Inventor
Young Chul Sung
Je-In Youn
Sang Hoon Park
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Postech Foundation
Lee, Ok Hee
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Application filed by Postech Foundation, Lee, Ok Hee filed Critical Postech Foundation
Priority to KR1020087021860A priority Critical patent/KR101451852B1/ko
Publication of WO2007102690A1 publication Critical patent/WO2007102690A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154

Definitions

  • the present invention relates to a novel codon- optimized DNA cassette for producing TRAIL, in which a secretion signal sequence, a dodecamer-forming domain and a TRAIL nucleotide sequence are sequentially linked, and a vector carrying the codon-optimized DNA cassette. Also, the present invention relates to a recombinant adenovirus, capable of expressing a secretory recombinant dodecamer TRAIL protein, anchoring the vector thereat. Further, the present invention is concerned with an anticancer composition comprising the vector, the recombinant dodecamer TRAIL protein, or the recombinant adenovirus.
  • TNF tumor necrosis factor
  • TRAIL tumor necrosis factor
  • TRAIL was found to exert cytotoxicity on various cancer cell lines without harm on normal cells (Ashkenazi et al., J. Clin. Invest., 104: 155-162, 1999; Walczak et al., Nat. Med. ,5:157-163, 1999).
  • the inability of TRAIL to induce apoptosis in normal cells is attributed to the decoy receptors, designated DcRl and DcR2, which exist only on the surface of normal cells (Sheridan et al., Science, 277:81 8-821, 1997).
  • DcRl and DcR2 although able to associate with TRAIL, do not induce the TRAIL-mediated apoptosis because they have no cytoplasmic domains capable of transferring the extracellular signal into the cell. Thanks to the ability thereof to selectively induce apoptosis, TRAIL has aroused scientist's interest in the use thereof as an excellent mediator in cancer therapy.
  • rTRAIL for anticancer drug.
  • rTRAIL for anticancer drug.
  • rTRAIL for anticancer drug.
  • approaches based on gene therapy have been devised.
  • gene therapy technology is advantageous with respect to economy and safety.
  • gene therapy has advantage over therapy with proteins not only in that genes persist for longer periods of time in vivo than do proteins, but also in that genes can be delivered specifically to targets of interest, thereby eliminating the side effects attributable to systemic diffusion over the body.
  • TRAIL extracellular domain
  • aa 114-281 members of the TNF superfamily exist as a homotrimer in cell membranes and bind to the TNF receptors located in other cell membranes so as to transmit signals.
  • the soluble domain of the TNF superfamily expressed from a vector carrying a gene encoding the extracellular domain of the TNF superfamily member, forms a homotrimer but in an incomplete form, thus having poor bioefficacy.
  • CD40L a representative member of the TNF superfamily, was reported to be improved in stability and biological activity when it was dimerized using a recombinant DNA technique and antibody Fc regions (Fanslow et al., J.Immunol. 136: 4099, 1986), dimerized using the extracellular domain of CD8 (Hollenbaugh et al., EMBO J.
  • dodecamer-forming domains were suggested for forming higher-mer than trimers of TNF superfamily members. It is disclosed that dodecamers of TNF superfamily members can be formed using SPD (surfactant protein D), a dodecamer-forming domain, and show enhanced biological effects (U.S. Pat. Publication No. 2005-0158831). It was found that when an SPD nucleotide sequence was joined to a CD40L nucleotide sequence using a recombinant DNA technique, dodacameric CD40L was formed and induced greater biological effects than did trimeric CD40L (Haswell et al., Eur. J. Immunol. 31: 3094, 2001).
  • SPD surfactant protein D
  • Codon optimization known to increase gene expression levels, is achieved by replacing a codon of low expression frequency with one of high expression frequency in a host cell.
  • there may be many degenerate codons which code for the same amino acid residue and when a gene is expressed through the transcription and translation process in a host cell, the host cell may prefer one codon to the other degenerate codons for the expression of the same amino acid. Codon optimization means to substitute for such codons of high preference, thereby increasing efficiency in the expression of the proteins encoded by a gene of interest.
  • the codon optimization of a nucleotide sequence of an antigen is known to increase the intracellular expression level of the antigen, thus maximizing the antigenicity.
  • the secretion signal sequence useful in the present invention is a codon-optimized tPA (tissue plasminogen activator) signal sequence which is far superior to the wild-type in terms of ability to induce secretion.
  • the dodecamer-forming domain useful in the present invention has a structure in which four homotrimers are associated together, each being encoded by a codon-optimized SPD
  • surfactant protein D (surfactant protein D) nucleotide sequence which shows an improved function in comparison with the wild-type.
  • TRAIL protein of the present invention is the soluble form, that is, extracellular domain of the entire TRAIL molecule and is encoded by a nucleotide sequence which is codon- optimized such that the modified extracellular domain is far superior to the wild-type extracellular domain in terms of expression efficiency and apoptotic induction. Consequently, the codon-optimized recombinant adenovirus featuring the three constitutional elements has an improved ability to kill cancer cells, compared to conventional TRAIL recombinant adenoviruses.
  • TRAIL TNF related apoptosis inducing ligand
  • TRAIL TNF related apoptosis inducing ligand
  • TRAIL which comprises (i) a nucleotide sequence encoding a secretion signal sequence, (ii) a nucleotide sequence encoding a dodecamer-forming domain and (iii) a nucleotide sequence encoding TRAIL, all of the nucleotide sequences being codon-optimized so as to express these nucleotide sequences at a high level in cells.
  • FIG. 1 is a schematic view of a DNA cassette in which codon-optimized nucleotide sequences coding respectively for a secretion signal sequence (tPA) , a dodecamer-forming domain (SPD) and an extracellular domain of TRAIL are sequentially linked;
  • FIG. 2 is a view showing codon-optimized nucleotide sequences of an tPA secretion signal sequence, an SPD dodecamer-forming domain, and TRAIL (114-281) compared with the wild-type nucleotide sequences thereof;
  • FIG. 3 is a view showing Western blots of a dodecameric form of TRAIL expressed by the recombinant adenovirus (rAd/tdTRAIL C0 ) ;
  • FIG. 4 is a view showing the cytolytic activity of the adenovirus (rAd/tdTRAIL co ) , expressing a dodecameric form of TRAIL, on U-87MG, A375 and HeLa cell lines. Best Mode for Carrying Out the Invention '
  • the present invention pertains to a DNA cassette for producing secretory recombinant dodecamer TRAIL (TNF related apoptosis inducing ligand) , which carries (i) a nucleotide sequence encoding a secretion signal sequence, (ii) a nucleotide sequence encoding a dodecamer-forming domain and (iii) a nucleotide sequence encoding TRAIL, and is codon- optimized so as to express these nucleotide sequences in a high level in cells.
  • TRAIL TNF related apoptosis inducing ligand
  • TRAIL recombinant dodecamer TRAIL
  • the dodecameric TRAIL of the present invention shows higher activity of inducing selective apoptosis of cancer cells than does monomeric or trimeric TRAIL.
  • a nucleotide sequence coding for a dodecamer-forming domain is located upstream of the nucleotide sequence encoding the TRAIL moiety so as to produce TRAIL as a dodecamer.
  • nucleotide sequence As long as a nucleotide sequence, whether full length or a part thereof, encodes a TRAIL protein capable of binding to the TRAIL receptor DR4 or DR5, it can be used in the present invention.
  • a nucleotide sequence encoding the amino acid sequence ranging from a. a. 114 to a. a. 281 in the full length of TRAIL.
  • the nucleotide sequence encoding TRAIL can be synthesized on the basis of the data from GeneBank (Gene ID: 8743, Pitti. R. M. et al., J. Biol. Chem. 271: 12687, 1996) by those skilled in the art.
  • TRAIL a wild type of the nucleotide sequence encoding TRAIL
  • the term "codon-optimized nucleotide sequence" or “codon optimization”, as used herein, is intended to refer to the substitution of some of the codons coding for a protein of interest (e.g., the TRAIL protein of the present invention) with such codons as increase the expression level of the protein of interest in mammalian cells, especially human cells.
  • Various combinations of the codons to be substituted can be applied for achieving the increase in the expression level by those skilled in the art.
  • Asp: D cysteine (Cys: C), glutamine (GIn: Q), glutamate (GIu: E), glycine (GIy: G), histidine (His: H), isoleucine (lie: I), leucine (Leu: L), lysine (Lys: K), phenylalanine (Phe: F), proline (Pro: P), serine (Ser: S), threonine (Thr: T), valine (VaI: V) and tyrosine (Tyr: Y) is substituted with one that is recognized in a higher frequency in human cells.
  • a wild-type nucleotide sequence corresponding to an amino acid sequence ranging from aa 114 to aa 281 of TRAIL is subjected to codon optimization and the resultant codon-optimized nucleotide sequence is represented as SEQ ID NO: 3. Comparison between the wild-type nucleotide sequence and the codon-optimized nucleotide sequence thereof is made in FIG. 2.
  • the codon-optimized TRAIL according to the present invention induces greater selective apoptotic effects on cancer cells in comparison with the conventional therapeutic consisting of the wild-type extracellular domains of TRAIL.
  • TRAIL proteins are associated with one another so as to form a dodecamer, but not a trimer, in accordance with the present invention.
  • a dodecamer-forming domain a nucleotide sequence coding for the dodecamer-forming domain is structured to be located upstream of the nucleotide sequence coding for the TRAIL protein.
  • the dodecamer-forming domain is SPD (surfactant protein D) .
  • the dodecamer-forming domain useful in the present invention is characterized in that it is optimized with at least one codon showing high expression frequency in mammalian cells, especially in human cells.
  • At least one of the codons responsible for the amino acid residues of TRAIL is substituted with one that is recognized in a higher frequency in human cells.
  • a nucleotide sequence ranging from nt. 79 to nt. 314 of the wild-type gene of SPD is subjected to codon optimization and the resultant codon-optimized nucleotide sequence is represented as SEQ ID NO: 2. Comparison between the wild-type nucleotide sequence and the codon-optimized nucleotide sequence thereof is made in FIG. 2. The codon-optimized SPD according to the present invention is expressed at a higher level than is the wild- type.
  • a secretion signal sequence which serves to secrete the TRAIL protein outside cells. Located in cell membranes, wild-type TRAIL lacks its own secretion signal sequence. The extracellular domain of TRAIL is known to be extracellularly guided by a secretion signal sequence and induce apoptotic signaling (Korean Pat. Appl'n No. 2000- 0038441) .
  • a secretion signal sequence is used to secrete the dodecameric TRAIL of the present invention, thus increasing the anticancer effect thereof.
  • the secretion signal sequence is located upstream of the 5' -terminus of the gene encoding the dodecamer-forming domain.
  • Examples of the secretion signal sequence useful in the present invention include tPA, HSV, gDs, SEC2, and SEC (CV) with preference for tPA.
  • the tPA secretion signal sequence used in a specific embodiment of the present invention shows excellent capability of inducing secretion.
  • a tPA signal sequence is utilized in developing a tuberculosis DNA vaccine. When linked to a tuberculosis antigen, the tPA signal sequence was reported to increase the secretion of the tuberculosis antigen outside cell membranes, thereby- enhancing cell-mediated immunity as well as antigen- specific humoral immunity (Li et al., Infection and Immunity, 67: 4780, 1999).
  • the tPA signal secretion sequence can more efficiently secrete the TRAIL protein of the present invention, thus achieving a high anticancer effect, the object of present invention.
  • the secretion signal sequence of the present invention is characterized in that it is optimized with at least one of the codons which have a high expression frequency in mammalian cells, especially in human cells.
  • at least one of the codons responsible for the amino acid residues of the secretion signal sequence that is, for alanine (Ala: A), arginine (Arg: R), asparagine (Asn: N), aspartate (Asp: D), cysteine (Cys: C), glutamine (GIn: Q), glutamate (GIu: E), glycine (GIy: G), histidine (His: H), isoleucine (lie: I), leucine (Leu: L), lysine (Lys: K), phenylalanine (Phe: F), proline (Pro: P), serine (Ser: S), threonine (Thr: T), valine (VaI: V) and tyrosine (Tyr: Y) is substitute
  • a nucleotide sequence ranging from nt. 1 to nt. 23 of the wild-type gene of tPA is subjected to codon optimization and the resultant codon-optimized nucleotide sequence is represented as SEQ ID NO: 1. Comparison between the wild-type nucleotide sequence of tPA and the codon- optimized nucleotide sequence thereof is made in FIG. 2. The codon-optimized tPA according to the present invention is found to be superior to the wild-type tPA in the induction of secretion.
  • the present invention provides a DNA cassette containing a codon-optimized secretion signal sequence, a codon-optimized dodecamer-forming domain and a codon-optimized TRAIL.
  • the codon-optimized secretion signal sequence is encoded by the nucleotide sequence of SEQ ID NO: 1, the codon-optimized dodecamer-forming domain by the nucleotide sequence of SEQ ID NO: 2, and the codon- optimized TRAIL by the nucleotide sequence of SEQ ID NO: 3, the arrangements of which are shown in FIG. 1.
  • the present invention pertains to a recombinant expression vector carrying the DNA cassette.
  • the recombinant expression vector according to the present invention which describes a vector capable of expressing a protein of interest, that is, the secretory recombinant dodecamer TRAIL protein, in a suitable host cell, refers to a genetic construct that comprises essential regulatory elements to which a gene insert (e.g., the DNA cassette) is operably linked in such a manner as to be expressed in a host cell.
  • a gene insert e.g., the DNA cassette
  • operably linked it is meant that there is a functional linkage between a nucleotide expression control sequence and a nucleotide sequence coding for a target protein, in such a manner as to perform general functions .
  • the operable linkage to a recombinant vector may be prepared using a genetic recombinant technique that is well known in the art, and site-specific DNA cleavage and ligation may be carried out using enzymes that are generally known in the art.
  • a vector suitable for use in the present invention may include expression regulatory elements, such as a promoter, an operator, an initiation codon, a stop codon, a polyadenylation signal and an enhancer. Both the initiation codon and the stop codon are generally regarded as a part of the nucleotide sequence coding for the antigenic target protein and are necessary in order to be functional in an individual to whom a genetic construct has been administered, and must be in frame with the coding sequence .
  • Examples of the expression vector useful in the present invention include plasmids, cosmids, bacteriophages, and viral vectors, but are not limited thereto, with preference for viral vectors. Of them, adenoviral vectors are most preferable.
  • An adenoviral vector which is one of the most prevalent vectors for use in gene therapy, shows excellent efficiency in transfection into various cells.
  • the adenovirus used in the present invention is type 5 and is incapable of self-replication due to the deficiency of the ElA gene essential for replication.
  • codons of a wild-type nucleotide sequence encoding TRAIL are substituted with ones which have high expression efficiency in human cells, and the resulting codon-optimized nucleotide sequence is linked to tPA and a dodeacmer-forming domain (SPD) to construct a recombinant vector for expressing secretory recombinant dodecamer TRAIL.
  • SPD dodeacmer-forming domain
  • the adenovirus (rAd/tdTRAIL co ) of the present invention was measured to be 5 to 15 times higher in apoptotic activity against cancer cells than the conventional recombinant adenovirus (rAd/stTRAIL) , which comprises a well-known secretion signal sequence, a trimerization-inducing leucine zipper motif and human TRAIL TRAIL (114-281) . Therefore, the vector carrying the DNA cassettes in accordance with the present invention can be more effective in the treatment of cancer than can conventional ones .
  • the present invention pertains to adenovirus, capable of producing the recombinant dodecamer TRAIL protein, anchoring the vector therein.
  • the adenovirus can be prepared by introducing the recombinant vector into a packaging cell line, culturing the packaging cell line, and separating the assembled adenovirus.
  • the adenovirus may be inducible adenovirus which comprises an inducible promoter which is activated in response to the presence of a particular compound.
  • the present invention employs CMV-TetlO as an inducible promoter for the inducible adenovirus in which expression is induced by tetracycline.
  • a tetracycline-inducible promoter is one of the most widely used inducible systems in vivo and is economically advantageous with the production of no significant side effects.
  • the vector expressing the secretory recombinant dodecamer TRAIL protein (ii) the recombinant dodecamer TRAIL protein produced using the vector, or (iii) the adenovirus, capable of producing the recombinant dodecamer TRAIL protein, anchoring the vector, may be provided as an active ingredient in an anticancer composition.
  • the anticancer composition according to the present invention may comprise a pharmaceutically acceptable carrier, and can be prepared into various dosage forms including tablets, troches, capsules, elixirs, suspensions, syrups, wafers, injections, etc.
  • a pharmaceutically acceptable carrier any form of solvents, dispersing media, antibacterial or antifungal agents, isotonics, and absorption retardants may be used as a carrier. These media and materials for effective use in pharmaceutically active ingredients are well known in the art.
  • the composition can be formulated into a suitable pharmaceutical preparation.
  • the vector capable expressing the secretory recombinant dodecamer TRAIL protein or an inducible adenovirus anchoring this vector therein is provided in a therapeutic vaccine composition.
  • mammals including humans can be vaccinated.
  • a composition comprising the secretory recombinant dodecamer TRAIL protein may be administered via oral routes or non-oral routes, such as intramuscular, intravenous, peritoneal, subcutaneous, and intradermal routes.
  • Preferable administration is to use an injection selected from among an intravenous injection, a subcutaneous injection, an intradermal injection, an intramuscular injection, instillation, and an intratumoral injection.
  • the composition may be administered in a single dose or in multiple doses.
  • composition must be administered in a pharmaceutically effective amount.
  • the pharmaceutically effective amount is determined depending on the route and frequency of administration, the kind and severity of the cancer to be treated, the age, gender and condition of patient, and other factors well-known in the pharmaceutical art.
  • the pharmaceutically effective amount may be administered once or in multiple doses.
  • EXAMPLE 1 Composition of tPA-SPD-TRAIL (114-281) DNA (tdTRAIL 00 ) Cassette
  • a nucleotide construct was chemically synthesized in which the codon-optimized tPA secretion signal nucleotide sequence of SEQ ID NO: 1 and the codon-optimized SPD dodecamer-forming domain sequence of SEQ ID NO: 2 were linked to each other.
  • the nucleotide construct was designed to have the restriction site Kpnl at its 5' terminus and the restriction site Notl at its 3' terminus.
  • the resulting tPA-SPD signal sequence was inserted into the inducible adenovirus shuttle vector pShuttle-TetlO which had been digested with Kpnl and Notl, so as to construct a pShuttle- TetlO/tPA-SPD vector.
  • the inducible adenovirus shuttle vector pShuttle-TetlO comprised the inducible promoter CMV- TetlO.
  • a codon-optimized human TRAIL (114- 281) nucleotide sequence of SEQ ID NO: 3 was chemically synthesized.
  • the TRAIL nucleotide sequence was designed to have the restriction site Notl at its 5' terminus and the restriction site Xbal at its 3' terminus. Afterwards, the TRAIL nucleotide sequence was inserted into the pShuttle- TetlO/tPA-SPD vector which had been digested with Notl and Xbal to construct a recombinant vector pShuttle-TetlO/tPA- SPD-hTRAIL co .
  • EXAMPLE 2 Generation of Recombinant Adenovirus Capable of Expressing the TRAIL Cassette
  • pShuttle-TetlO/tPA- SPD-hTRAIL C0 was subjected into homologous recombination with replication-incompetent pAd/Easy in BJ5183 competent cells.
  • pAd/tPA-SPD-hTRAIL co (hereinafter referred to as "pAd/tdTRAIL C0 ") in which the TRAIL cassette was embedded was selected.
  • This vector was treated with Pad to detect DNA bands of 35+4.5kb. 10 days after the transformation of pAd/tdTRAIL C0 into 293 cell line, recombinant adenovirus rAd/tdTRAIL C0 which expressed the TRAIL cassette was obtained.
  • rAd/tdTRAIL C0 expressed the dodecamer TRAIL
  • monkey kidney Cos7 cell line and human embryonic kidney 293 cell line were infected with rAd/tdTRAIL co at 200 MOI and 10 MOI, respectively.
  • culture supernatants were sampled and used to conduct Western blotting analysis under native and non-reducing conditions.
  • rAd/tdTRAIL co expressed monomeric, trimeric and dodecameric TRAIL proteins.
  • rAd/tdTRAIL In order to examine the apoptotic effect of rAd/tdTRAIL C0 on cancer cells, human glioma U-87MG cell line, human melanoma A375 cell line, and human cervical carcinoma HeIa cell line infected with rAd/tdTRAIL C0 or a control, rAd/stTRAIL, at 10 MOI, 100 MOI and 100 MOI, respectively.
  • rAd/stTRAIL which is a recombinant adnovirus consisting of a secretion signal sequence, an isoleucine zipper trimerization motif and a human TRAIL (114-281) , is reported to induce high apoptotic effects on cancer cells
  • rAd/tdTRAIL C0 was found to have 5 ⁇ 15 fold higher apoptotic activity against cancer cells as compared to rAd/stTRAIL as assayed with
  • the inducible recombinant adenovirus of the present invention which comprises a DNA cassette consisting of a codon-optimized secretion signal sequence, a codon-optimized dodecamer forming domain (SPD) , and a codon-optimized TRAIL nucleotide sequence, can be used as a gene therapeutic exhibiting superior anticancer activity to conventional TRAIL adenoviruses.
  • SPD codon-optimized dodecamer forming domain
  • the adenovirus vector comprising the DNA cassette in accordance with the present invention can be applied for the treatment of cancer.

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Abstract

L'invention porte sur une nouvelle cassette d'ADN optimisée par un codon pour produire le dodécamère recombinant sécréteur TRAIL, cassette dans laquelle trois séquences nucléotidiques différentes codant une séquence de signal de sécrétion, un domaine formant le dodécamère et TRAIL sont liées de manière séquentielle, l'invention portant également sur un vecteur supportant ladite cassette. L'invention porte, en outre, sur une protéine TRAIL recombinante sécrétrice produite par le vecteur, et sur un adénovirus, dans lequel est ancré le vecteur, capable de produire la protéine TRAIL dodécamère recombinante sécrétrice. La protéine TRAIL ou l'adénovirus peuvent être obtenus dans des préparations anticancéreuses.
PCT/KR2007/001099 2006-03-06 2007-03-06 Vecteur comprenant la cassette d'adn optimisée par un codon pour produire le dodécamère recombinant sécréteur trail WO2007102690A1 (fr)

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US9527897B2 (en) 2007-07-10 2016-12-27 Apogenix Ag CD40L collectin fusion proteins and encoding nucleic acid molecules
US8383774B2 (en) 2007-07-10 2013-02-26 Apogenix Gmbh Collectin fusion proteins comprising TNF or trail
WO2009007120A3 (fr) * 2007-07-10 2009-03-12 Apogenix Gmbh Protéines de fusion collectines de la superfamille des tnf
US9212211B2 (en) 2007-07-10 2015-12-15 Apogenix Gmbh Trail collectin fusion proteins
US9289388B2 (en) 2008-12-10 2016-03-22 Paka Pulmonary Pharmaceuticals, Inc. Methods and compositions for delivery of medicaments to the lungs
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WO2010078966A1 (fr) * 2009-01-09 2010-07-15 Apogenix Gmbh Protéines de fusion formant des trimères
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CN104160027A (zh) * 2012-02-01 2014-11-19 浦项工科大学校产学协力团 同时表达十二聚体trail及hsv-tk自杀基因的载体及利用其的抗癌干细胞治疗剂
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JP2015506697A (ja) * 2012-02-01 2015-03-05 ポステック アカデミー‐インダストリー ファウンデーション １２量体ｔｒａｉｌ及びｈｓｖ−ｔｋ自殺遺伝子を同時に発現するベクター並びにそれを用いた抗癌幹細胞治療剤
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CN102775497A (zh) * 2012-07-13 2012-11-14 浙江大学 肿瘤坏死因子相关的凋亡诱导配体融合蛋白及其制备方法
CN102775497B (zh) * 2012-07-13 2014-07-23 浙江大学 肿瘤坏死因子相关的凋亡诱导配体融合蛋白及其制备方法
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JP2014218510A (ja) * 2014-08-11 2014-11-20 アポゲニクスゲゼルシャフト ミット ベシュレンクテルハフツングＡｐｏｇｅｎｉｘ ＧｍｂＨ 三量体形成融合タンパク質
US11007251B2 (en) 2015-12-17 2021-05-18 The Johns Hopkins University Ameliorating systemic sclerosis with death receptor agonists
US11084879B2 (en) 2016-04-07 2021-08-10 The Johns Hopkins University Compositions and methods for treating pancreatitis and pain with death receptor agonists

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