WO2002040675A1 - Nouvelle proteine mutante du facteur onconecrosant humain et leur procede de preparation et application - Google Patents

Nouvelle proteine mutante du facteur onconecrosant humain et leur procede de preparation et application Download PDF

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WO2002040675A1
WO2002040675A1 PCT/CN2000/000424 CN0000424W WO0240675A1 WO 2002040675 A1 WO2002040675 A1 WO 2002040675A1 CN 0000424 W CN0000424 W CN 0000424W WO 0240675 A1 WO0240675 A1 WO 0240675A1
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tumor necrosis
human tumor
necrosis factor
mutant protein
human
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PCT/CN2000/000424
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Chinese (zh)
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Changqing Chen
Hui Liu
Fang Lu
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Shanghai Research Center Of Biotechnology, Chinese Academy Of Sciences
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Publication of WO2002040675A1 publication Critical patent/WO2002040675A1/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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/525—Tumour necrosis factor [TNF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Novel human tumor necrosis factor mutant protein preparation method and application thereof
  • the invention relates to the fields of genetic engineering, protein engineering and disease treatment, and more particularly, to a new low-toxic human tumor necrosis factor mutant protein and a preparation method thereof, a coding sequence encoding the mutant protein, an expression vector containing the coding sequence, and transformation. Host cells, and pharmaceutical compositions containing the novel human tumor necrosis factor mutant protein.
  • Human tumor necrosis factor TNF ⁇ is mainly produced by macrophages and monocytes. It can be induced by BCG, endotoxin, etc .; some tumor cells, such as human premyeloid leukemia cell HL-60, are induced by phorbol ester. Macrophages are differentiated in the direction and can also produce TNF. In 1984, Pennica et al.
  • hTNF-a human tumor necrosis factor
  • hTNF- ⁇ has large toxic and side effects, which limits its clinical use (Old LJ., Tumor Necrosis Factor: Structure, Mechanism of Action, Role in Disease and Therapy, 1990, l- 30, Karger, Pasel.).
  • Van Ostade et al.'S study also found that the activity of the alanine at position 84 was severely reduced after replacement with other amino acids, and the activity was completely lost when it was changed to valine; and the amino acid change at position 86 also had a great impact on the activity.
  • the substitution of the C-terminal amino acid of hTNF-a has a greater effect on the viability, especially when the leucine at position 157 is changed to phenylalanine with a 5-fold higher activity than the wild type (Van Ostade X., 1991, supra).
  • the activity of arginine at position 2 can be doubled after being converted to lysine (high longevity, Mao Shenlan, Yu Yue, etc., a new high-efficiency, low-toxicity human tumor necrosis factor mutant in E. coli High-efficiency expression in Chinese, Journal of Biochemistry and Biophysics, 1996, 28 (1): 49-55) 0
  • the two receptors R55 and R75 of hTNF- mainly mediate different biological functions.
  • R55 mediates cytotoxic activity, while R75 mediates endotoxicity, (Louis A., Tartaglia LA, David V. et al ., Two TNF Receptors, Immun. Today, 1992, 13 (5): 151-153; Kamijo R., Takeda K., Nagumo lines by TNF mutants, Biochem. Biophys. Res. Commun., 1989, 160 (2) : 820-827.).
  • Van Ostade et al. Reported that the 29th and 32th positions were replaced. After binding to the other 8 amino acids, the binding to R55 and R75 decreased (Van Ostade X., Vandenabeele P., Everaerdt B.et al "Human TNF mutants with selective activity on the R55 receptor, Nature, 1993, 361 (2): 166-169. When the serine at position 86 becomes threonine, its binding to the R55 receptor is the same as that of the wild type, but its binding to R75 decreases. Many.
  • Aspartic acid at position 143 was changed to tyrosine, phenylalanine, and asparagine, respectively, and the affinity with R55 decreased more than the affinity with R75 (Van Ostade X., Vandenabeele P., Tavernier J. et al. Human tumor necrosis factor mutants with preferential binding to and activity on either the R55 or R75 rceptor, Eur J Biochem, 1994, 220 (3): 771 -779 .; Loetscher H., Stueber D., Banner D. et al "Human tumor necrosis factor alpha (TNF alpha) mutants with exclusive specificity for the 55-Kda or 75-Kda receptors, J Biol. C em., 1993, 268: 2635046357.).
  • the purpose of the present invention is to overcome the above disadvantages in the prior art, and provide a human tumor necrosis factor TNFa with low toxicity and / or high efficiency, thereby promoting its clinical application.
  • a low-toxicity and / or high-efficiency human tumor necrosis factor mutant protein which protein contains the Arg32Trp / Leul57Plie mutation.
  • the mutant protein may further contain the Arg2Lys / Asn30Ser mutation;
  • the mutein has the amino acid sequence shown in SEQ ID NO. 1 or 2.
  • a DNA sequence encoding the novel human tumor necrosis factor mutant protein is provided. .
  • a vector containing the above-mentioned DNA sequence and a corresponding transformed host cell are provided.
  • a method for producing the aforementioned human tumor necrosis factor mutant protein includes the steps of:
  • a DNA sequence encoding a human tumor necrosis factor TNFct mutant protein is operably linked to an expression control sequence to form a human tumor necrosis factor mutant egg.
  • a white expression vector wherein the human tumor necrosis factor mutant protein contains an Arg32Trp / Leul57Phe mutation
  • the mutant protein may further contain an Arg2Lys / Asn30Ser mutation; most preferably, the mutant protein has an amino acid sequence shown in SEQ ID N0.1 or 2;
  • step (a) Transforming the expression vector in step (a) into a human host cell
  • novel human tumor necrosis factor mutant protein and a pharmaceutical composition containing the novel human tumor necrosis factor mutant protein are provided.
  • the composition contains an effective amount of the human tumor necrosis factor mutation of the present invention.
  • Protein and pharmaceutically acceptable carriers and / or excipients are provided.
  • tumor necrosis factor TNFcc generally means human tumor necrosis factor TNFoc, but can also refer to tumor necrosis factor TNFoc that is then derived from mouse, pig, horse, or bovine.
  • tumor necrosis factor TNFa is human tumor necrosis Factor TNFa.
  • the tumor necrosis factor TNFa may be a TNFa with a natural wild-type sequence, or a derivative or recombinant TNFa with a mutated sequence (as opposed to a wild-type sequence), as long as the mutated sequence does not include the Arg32Trp / Leul57Phe mutation.
  • the amino acid sequence of natural wild-type human tumor necrosis factor TNFa is shown in SEQ ID No: 3.
  • Arginine (Arg) at position 32 is mutated to tryptophan (Trp), and leucine (Leu) at position 157 is mutated to (Phe).
  • Trp tryptophan
  • Leu leucine
  • Arg32Trp / Leul57Plie is equivalent to "32Trp / 157Phe”, that is, whether or not arginine at position 32 and / or leucine at position 157 is provided, as long as the mutation is tryptophan and benzene Alanine can be expressed as "Arg32Trp / Leul57Plie" or "32Trp / 157Phe".
  • the description of mutations 2 and 30 is analogous. '
  • the novel mutein of the present invention can be prepared by synthesizing primers based on the published sequence of human tumor necrosis factor and amplifying the coding sequence of human tumor necrosis factor by the PCR method. For example, according to the method described in Li Changben et al., Science in China (Series B), 1991, 6: 497, a natural hTNFa gene was constructed. Human TNFa coding sequences can also be artificially combined. In addition, base sequences can be substituted for coding sequences to facilitate high expression (e.g., for expression in E. coli, natural codons can be replaced with E. coli preferred codons encoding the same amino acid).
  • the DNA sequence of human tumor necrosis factor is used to genetically modify the mutant form indicated in the present invention.
  • the technique of genetic modification is known in the art, for example, see “Mutagenesis: a Practical Approach", MJ McPerson , Ed., (IRL Press, Oxford, UK. (1991), which include, for example, site-directed mutagenesis, cassette mutagenesis, and polymerase chain reaction (PCR) mutagenesis.
  • the DNA sequence encoding the novel mutant protein of the present invention after site-directed mutation was ligated into a suitable expression vector and then transferred into a suitable host cell. Finally, the transformed main cells are cultured, and the new mutant protein of the present invention is obtained through isolation and purification.
  • various vectors known in the art such as commercially available vectors can be selected.
  • a commercially available vector is selected, and then a nucleotide sequence encoding the novel mutant protein of the present invention is operably linked to an expression control sequence to form a protein expression vector.
  • operably linked refers to a condition where certain parts of a linear DNA sequence can affect the activity of other parts of the same linear DNA sequence. For example, if the signal peptide DNA is expressed as a precursor and is involved in the secretion of the polypeptide, then the signal peptide (secret leader) DNA is operably linked to the polypeptide DNA; if the promoter controls the transcription of the sequence, it is operably linked to the 3 ⁇ 4k Coding sequence; if the ribosome binding site is placed at a position that enables it to translate, it is operably linked to the coding sequence. In general, "operably linked to” means adjacent, while for secreted leader sequences it means adjacent in reading frame.
  • the term "host cell” includes prokaryotic cells and eukaryotic cells.
  • prokaryotic host cells include E. coli, Bacillus subtilis, and the like.
  • eukaryotic host cells include yeast cells, insect cells, and mammalian cells.
  • the host cell is a prokaryotic cell, more preferably E. coli.
  • the SGI protein imaging workstation has established a model for the binding of hTNF- ⁇ to its receptors (R55 and R75 receptors) and conducted extensive and in-depth research.
  • Prior studies of the present invention have shown that mutation of arginine at position 32 to tryptophan results in a significant decrease in the killing activity of human tumor necrosis factor TNF ⁇ (Van Ostade, X., 1991, supra).
  • the present inventors have unexpectedly discovered that, 'After mutating arginine at position 32 and leucine at position 157 of human tumor necrosis factor hTNF- ⁇ to tryptophan at position 32 and phenylalanine at position 157 (in other words, that is, The introduction of the Arg32Trp / Leul57Phe mutation) can significantly reduce the toxicity of human tumor necrosis factor TNF c [, and at the same time the killing activity of human tumor necrosis factor TNF ⁇ on human tumor cells does not decrease.
  • hTNF- ⁇ mainly binds to receptors in a trisomy, and each receptor binds in a groove between two TNF molecules.
  • the C-terminal 157-position becomes Phe, which stabilizes the TNF trisomy form due to its increased hydrophobicity, thereby increasing its biological activity.
  • asparagine at position 30 does not participate in binding to two receptors based on the spatial structure of the hTNF- ⁇ trisomy.
  • asparagine at position 30 can change to hTNF- ⁇
  • the trisomics are tighter, which increases the stability of the trisomics.
  • Asn30Ser mutation can be introduced again.
  • increasing the activity of hTNF- ⁇ at the N-terminal basic amino acid can increase its activity (the specific mechanism cannot be explained), for example, the activity can be doubled by changing arginine at position 2 to lysine.
  • the present inventors constructed a novel mutant protein of human tumor necrosis factor TNFa using the method of Over-Lap PCR. According to the measurement of the properties of the mutant proteins, they were found to be highly effective in killing human tumor cells and the LD50 of the acute toxicity test was much lower than that of the wild type.
  • novel mutein of the present invention is helpful for people to further understand the mechanism of action of TNF, and to modify and modify the TNF molecule by using genetic engineering methods to locate functional regions with unique activity.
  • novel mutant protein of the present invention improves the anti-tumor activity of TNF and reduces the toxic and side effects, it will undoubtedly provide a broad prospect for the anti-tumor clinical treatment of TNF.
  • mutant protein is named MTNF1 (abbreviated as M1), and its amino acid sequence As shown in SEQ ID NO: 1.
  • mutant protein having the Arg2Lys / Asn30Ser / Arg32Trp / Leul57Phe mutation, and the mutant protein is It is named MTNF2 (abbreviated as M2), and its amino acid sequence is shown in SEQ ID NO: 2.
  • the pharmaceutical composition of the present invention comprises an effective amount of one or several novel T Foc muteins of the present invention, and at least one pharmaceutically acceptable carrier, diluent or excipient.
  • the active ingredient is usually mixed with, or diluted with, an excipient, or enclosed in a carrier that can be in the form of a capsule or sachet.
  • an excipient functions as a diluent, it can be a solid, semi-solid, or liquid material as an excipient, carrier, or medium for the active ingredient. Therefore, the composition may be a tablet, a pill, a powder, a solution, a syrup, a sterile injection solution, or the like.
  • excipients examples include: lactose, glucose, sucrose, sorbitol, mannitol, starch, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, and the like.
  • the formulation may also include: wetting agents, emulsifiers, preservatives (such as methyl and propyl hydroxybenzoate), sweeteners, and the like.
  • the composition can be made into unit or multiple dosage forms. Each dosage form contains a predetermined amount of active substance calculated to produce the desired therapeutic effect, as well as suitable pharmaceutical excipients.
  • the pharmaceutical composition can be administered according to the disease to be treated and in a dose beneficial to the patient (depending on weight and other considerations), which can be determined by the medical staff.
  • Fig. 1 shows the construction of a novel human tumor necrosis factor mutant protein M1 and M2 expression plasmid of the present invention.
  • Figure 2 is a 1.5% agarose electrophoresis image of the Over-Lap PCR product. Among them, lane 1, mutant gene Ml; lane 2, mutant gene M2; lane 3, molecular weight marker (pGEM7Zf (+) DNA / HaeIII).
  • - Figure 3 is the EcoRI / BamHI digestion identification map of each mutant protein gene expression plasmid.
  • Lane 1 molecular weight marker (pGEM7Zf (+) DNA / Haeni);
  • lane 2 EcoRI + BamHI digestion result of the expression plasmid pSB-MTNFl;
  • lane 3 EcoRI + BamHI digestion result of the expression plasmid pSB-MTNF2;
  • lane 4 EcoRI + BamHI digestion results of the expression vector pSB-92.
  • Figure 4 shows the corresponding Western-Blot results after high expression of each mutant protein gene expression plasmid in E. coli.
  • Lane 1 the mutant gene M1 is induced to express for 4 hours;
  • lane 2 the mutant gene M2 is induced to express for 4 'hours;
  • lane 3 the wild-type gene hTNF- ⁇ is induced to express for 4 hours;
  • lane 5 wild-type Protein imprinting results of gene hTNF-ct;
  • lane 6 protein imprinting results of mutant gene M2; 'lane 7, protein imprinting results of mutant gene M1.
  • Figure 5 shows the SDS-PAGE results of each mutant protein after purification. Among them, lane 1, Ml; lane 2, M2; lane 3, hTNF-a; lane 4, protein molecular weight standard.
  • Figure 6 shows the full sequence of plasmid PSB-92.
  • Figure 7 is an amino acid sequence diagram of natural human TNFa.
  • the present invention is further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention.
  • the experimental methods in the following examples are not marked with specific conditions, usually according to conventional conditions such as Sambrook et al. Molecular cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer.
  • the expression vector pSB-92 see Figure 6 for the complete sequence.
  • Expression plasmid pSB-TR The expression vector pSB92 containing the human TNFa gene.
  • pSB-TK An expression vector pSB92 containing a mutated human TNFa gene, in which the arginine at position 2 is mutated to lysine (high longevity, Mao Shenlan, Yu Yue, etc.), a novel high-efficiency, low-toxic human tumor necrosis factor Efficient expression of mutein in E. coli, Journal of Biochemistry and Biophysics, 1996, 28 (1): 49-55) pUC-19 plasmid was purchased from Huamei Bioengineering Company.
  • E. coli YK537 (supE44hsdRhsdMrecAlpho48LeuB6thilacYrpsL20galK2ara-
  • the DNA sequencer was determined by the American ⁇ automatic tester. .
  • the human laryngeal cancer cell line HEp-2 was a gift from Professor Pedersen of Aarhus University in Denmark.
  • the human gastric cancer cell line MGC was Professor Liu Quanhai of the Shanghai Pharmaceutical Industry Research Institute of the State Medical Administration.
  • a gift from Professor Jiao Binghua of the Second Military Medical University, and a human liver cancer cell line HepG-2 was given by Professor Wen Yumei of Shanghai Medical University. '
  • Mouse anti-hTNF- ⁇ antiserum was purchased from the Immunology Laboratory of the Fourth Military Medical University. Method
  • trypsin L929 cells With 0.25% trypsin L929 cells in logarithmic growth phase, the cell concentration adjusted to 2-2.5 X 10 5 / ml, added to 96-well culture plates, lOOul per well were incubated for 18 hours, serially diluted TNF sample, Continue to incubate for 18 hours.
  • the dilution solution is Actinomycin D containing 2ug / ml (Actinomycin D is a product of Sigma), ELPMI1640 with 5% NCS. Each dilution is made into 3 replicate wells, and a blank control is set at the same time. Cell control, actinomycin] control, the sample titer is set to the corresponding dilution factor that causes 50% of the cells to survive.
  • the induced expression products were separated by SDS-PAGE, and then transferred to a nitrocellulose membrane.
  • the pure live curve of tumor cells was set with blank control, cell control, and D control. The killing titer of the sample to human tumor cells was determined as the corresponding dilution factor that caused 50% of cell survival.
  • mice Six Kunming mice were randomly selected from each group, male and female, weighing 18-22 g (provided by the Animal Room of Tianjin Pharmaceutical Research Institute, animal certificate: Jin Shidong Facility Standard No. 001 :), and intravenous injection of different doses
  • LD50 LD50 of each sample was obtained by the Bliss method.
  • Rhesus monkeys were randomly divided into two groups. Each group consisted of 4 males and half females. One group was injected with wild-type hTNFo intravenously and the other group was injected with hTNFa mutant protein M2. The dose was calculated based on the body surface area of the animal. The daily injection dose was 200 ⁇ 8 / ⁇ 2 , and the drug was administered continuously for 10 days to observe the death of the animal.
  • the hTNFa mutant protein M2 injection group weigh each time before and after the administration; before and after the end of the administration, the hind limbs are quiet: pulse blood, coagulate at room temperature and separate the serum, and perform the following with a clinical biochemical analyzer Measurement of indicators: total protein (TP :), albumin
  • ALB blood glucose
  • GLU blood glucose
  • T-BIL 'total bilirubin
  • Crea creatinine
  • T-CHO total cholesterol
  • BUN urea nitrogen
  • ALT alanine aminoconverting enzyme
  • AST sky door Aspartate aminoconvertase
  • ALP alkaline phosphatase
  • mice Take well-growing S180 and HAC ascites, and dilute 1: 4 with normal saline.
  • 0.2ml of each mouse is subcutaneously axillary, randomly divided into groups of 10 mice.
  • the next day after inoculation 7 consecutive days of injection. 10 days after inoculation
  • the animals were sacrificed by neck dissection, tumor masses were dissected, and the tumor weights of each dose group were compared. The results were determined according to the following formula: l00%
  • CP-3 ascites of healthy human bladder cancer were separately taken, diluted 1: 4 with normal saline, 0.2ml subcutaneously axillary in each nude mouse, randomly divided into groups, and 10 nude mice in each group were provided with a normal saline group, a CTX group,
  • M2 was set in three dose groups of 2.5, 0.25, and 0.025 mg / kg, respectively, and the dose was 0.5 ml / head.
  • the next day after the inoculation the injection was continued for 7 days.
  • the animals were sacrificed 10 days after inoculation, and tumor masses were dissected out to compare the tumor weight of each dose group.
  • Example 1 The expression vectors pSB-TR (containing the natural hTNF- ⁇ gene) and pSB-TK (containing the hTNF- DK2 gene) were constructed.
  • the coding sequence shown in SEQ ID NO: 4 was artificially synthesized based on the amino acid sequence of natural human TNFa, and then.
  • the human TNFa gene hTNF-TR was then cloned between the EcoRI and BamHI sites of the expression vector pSB_92 (see Figure 6 for the complete sequence) to obtain the expression plasmid pSB-TR, which contains the coding sequence SEQ ID NO: encoding natural human TNFa: 4 expression vector pSB92.
  • No. 1 a sequence of the vector pSB-92 (upstream primer)
  • 100ng of template was added to the 100ul reaction system, and 50pmol of each of the two primers were denatured at 95 ° C for 5 minutes to enter the cycle.
  • the temperature of the cycle was 94 ° C for 40 seconds; 52 ° C for 50 seconds and 72 for 1 minute. A total of 35 cycles , Then 72 ° C for 10 minutes.
  • a PCR product (amplified fragment I or II) of about .480bp was digested with EcoRI and BamHI, and inserted into the pSB-92 expression vector digested with the same enzyme to transform E. coli YK537.
  • the transformants containing the inserted fragments were screened to obtain the mutant genes pSB-MTNF1 and pSB-MTNF2 (Fig. 1).
  • Each expression plasmid was digested with EcoRI and BarnHI to cut out a 480 bp fragment ( Figure 3). This fragment was inserted into Puc-19 to extract double-stranded DNA, and sequenced on the automatic sequencer of ABI Corporation in the United States. The results were as expected (results not shown).
  • E. coli YK537 which respectively contained the expression plasmids pSB-MTNF1 and pSB-MTNF2
  • the expression plasmids pSB-MTNF1 and pSB-MTNF2 were isolated and transformed into E. coli BL21, respectively, to obtain transformed engineered bacteria.
  • the engineered bacteria obtained from E. coli BL21 transformed with the expression plasmid pSB-MTNF2 is named E. coli BL21 / pSB-T4;
  • the engineered bacteria obtained from E. coli BL21 transformed with the expression plasmid pSB-MTNF1 is named E. coli BL21 / pSB-T2.
  • Escherichia coli BL21 / pSB-T4 which expresses the mutant protein M2 of human tumor necrosis factor TNFa, was deposited at CCTCC (Wuhan, China) on March 3, 1999 under CCTCC M99004.
  • hTNF- ⁇ mutein expression plasmid in E. coli for expression and protein blotting method.
  • expressing pick a single colony in LB medium at 30. ° C overnight and incubate at 1:50 ratio.
  • X In YT 3 ⁇ ⁇ was cultured with vigorous shaking for 2 hours, and culture was continued for 4 hours after induction at 42 ° C, and the cells were collected.
  • the bacterial cells were sonicated and purified according to the following purification method: Take the sonicated centrifugal supernatant 40-60% of the ammonium sulfate component by DEAE-Sepharose FF, collect the TNF peak, and CM-Sepharose FF After further purification, the TNF peak was collected and precipitated by dialysis with ammonium sulfate precipitation, and the pure TNF product was obtained by Sephacryl-100 (Fig. 5). The purity was more than 95% by laser grayscale scanning.
  • the activity was measured using wild-type hTNF-a as a control.
  • the activities were: wild-type hTN a was 8. lxl0 6 U / mg, MutTNF-1 was 7.94xl0 2 U / mg, MutTNF- 2 The activity was 4. OxlO mg.
  • the results showed that the activity of the mutant protein M1 on mouse fibroblast tumor cell L929 decreased by 4 orders of magnitude, and the activity of the mutant protein M2 was almost completely lost, indicating that the 30-position Asn ⁇ Ser, 32-position Arg -Tyr modification severely reduced the killing activity of each mutant protein on L929 cells.
  • Human tumor cells laryngeal cancer Hep-2 cells, liver cancer HepG-2 cells, gastric cancer MGC cells, and ovarian cancer 3A0 cells were plated, and then serially diluted mutant protein samples (more than 95% purity) were added, and purified wild-type The sample was used as a control, and the killing activity of the sample on various human tumor cells was measured (Table 1). It is known from the table that although the activity of each mutant protein on mouse fibroblast cells L929 has been severely reduced, the killing of several human tumor cells is similar to that of the wild type.
  • the activity ratio of Ml to liver cancer HepG-2 cells The wild-type hTNF-a is 6 times higher, indicating that the results of hTNF-a on mouse tumor cells and human tumor cells are not parallel, that is, the biological activity on L929 cells does not represent killing human tumors. Cell results.
  • mice were injected with different doses of mutant proteins Ml and M2 at one time, and wild-type hTNF-a was used as a control.
  • the LD50 value of each mutant protein was determined by the Bliss method (Table 2).
  • the mutant protein M2 could not be restricted by the concentration of the sample.
  • the LD50 value was obtained, but according to the pre-experiment results, it can be known that the 30% lethal dose to mice is 107 mg / kg.
  • the results showed that the LD50 of the mutant protein M1 was 300 times lower than that of the wild type, and the 30% lethal dose of M2 to the mice was 700 times lower than that of the wild type LD50.
  • the gender death difference after the injection of MTNF-1 it remains to be seen 'For further research. ,
  • hTNFa mutant protein M2 on human bladder cancer CP-3 transplanted in nude mice are as follows: At a dose concentration of 0.025 mg / kg, the tumor inhibition rate of M2 is 45.50%, while that of wild-type hTNFa is only 15.50%. Tumor inhibition rate. And M2 also achieved high tumor inhibition rates of 52.10% and 64.10% at the injection doses of 0.25mg / kg and 2.5mg / kg (Table 8). hTNFa mutant protein M2 on nude mice
  • the TNFct mutant protein of the present invention can efficiently kill human tumor cells and the LD50 of the acute toxicity experiment is much lower than that of the wild type, but the biological activity of the mouse fibroblast cell L929 is almost lost.
  • the mutant protein Ml had an activity of 157 amino acids and decreased L929 activity by four orders of magnitude compared with the wild type, but its activity against human laryngeal cancer cells Hep-2 was consistent with that of the single 32-bit mutant protein, which was similar to that of the wild type. ;
  • Mutant protein M2 After increasing the N-terminal 2 and 30 mutations, the activity on L92Q decreased by five orders of magnitude compared with the wild type, but the killing activity on tumor cells was still maintained. The most unexpected is the LD50 of the mutant proteins M1 and M2. The results show that they have extremely low toxicity, of which M2 is even less toxic than Ml.
  • the in vivo curative effect test of the hTNPa mutant protein M2 of the present invention on mouse sarcoma S180 shows that at a concentration of 0.025 mg / kg, the tumor inhibition rate of M2 is about 23 percentage points higher than that of wild-type hTNFa, and at this concentration, The inhibitory rate of M2 on liver cancer HAC solid tumors is slightly lower than that of wild-type hTNFa. However, because M2 mice have very low toxicity in vivo, higher concentrations can be used.
  • the tumor inhibition rates of M2 on S180 reached 59.35% and 69.06%, respectively, while the tumor inhibition rates of M2 on HAC reached very high 62.41% and 75.94%, respectively.
  • the efficacy of hTNFa mutant protein M2 on human bladder cancer CP-3 transplanted in nude mice further demonstrated the antitumor effect of M2.
  • the tumor inhibition rate of M2 was 45.50%, while that of wild-type hTNFa was only 15.50%.
  • M2 also achieved high tumor inhibition rates of 52.10% and 64.10% at the injection doses of 0.25mg / kg and 2.5mg / kg.
  • mice fibroblast L929 was used as a cell model to test the biological activity of hTNFa.
  • Ml and M2 the activities of two hTNFa mutant proteins, Ml and M2, were measured, their results on L929 cells and their effects on human tumor cells were found.
  • the results are not parallel; in addition, the hTNFa mutein M2 tumor inhibition experiments in mice further prove that the mutein has a good effect on tumors of mouse origin. This shows that there is a certain limitation on the biological activity of hTNFcc measured by murine L929 cells alone '.

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Abstract

L'invention concerne un nouveau type de protéine mutante du facteur onconécrosant humain à faible toxicité, leur procédé de préparation, les séquences codant les protéines mutantes, les vecteurs d'expression, les cellules hôtes transformées contenant les séquences codantes et les compositions pharmaceutiques contenant les nouvelles protéines mutantes du facteur onconécrosant humain. Les nouveaux facteurs contiennent des mutants Arg32Trp/Leu157Phe ou des mutants Arg2Lys/Asn30Ser/Arg32Trp/Leu157Phe. Les protéines mutantes font preuve d'une toxicité considérablement inférieure aux souris mais d'un effet mortel égal ou accru sur les cellules tumorales humaines par rapport à un type de protéine sauvage. Les quatre protéines mutantes font preuve d'une toxicité relativement inférieure sur les macaques et d'un effet inhibiteur de cancer accru sur les souris tout en ayant un effet thérapeutique amélioré sur le carcinome humain de la vessie CP-3 transplantée dans des souris atrichiques.
PCT/CN2000/000424 2000-11-15 2000-11-15 Nouvelle proteine mutante du facteur onconecrosant humain et leur procede de preparation et application WO2002040675A1 (fr)

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AU2001213790A AU2001213790A1 (en) 2000-11-15 2000-11-15 Novel human tumor necrosis factor mutant proteins and their preparing methods and uses
PCT/CN2000/000424 WO2002040675A1 (fr) 2000-11-15 2000-11-15 Nouvelle proteine mutante du facteur onconecrosant humain et leur procede de preparation et application

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PCT/CN2000/000424 WO2002040675A1 (fr) 2000-11-15 2000-11-15 Nouvelle proteine mutante du facteur onconecrosant humain et leur procede de preparation et application

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CN114220492A (zh) * 2021-12-16 2022-03-22 江南大学 基于等温压缩系数扰动对酶进行再设计的方法、应用及通过此方法筛选出的突变体

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JPH01231891A (ja) * 1988-03-11 1989-09-18 Tanpaku Kogaku Kenkyusho:Kk 変異ヒト腫瘍壊死因子
JPH03297388A (ja) * 1990-04-17 1991-12-27 Ube Ind Ltd 新規なｔｎｆ変異体、その製造法及びそれを有効成分とする抗腫瘍剤
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104761631A (zh) * 2015-04-15 2015-07-08 中国药科大学 多位点引入对硝基苯丙氨酸的人肿瘤坏死因子-α
CN114220492A (zh) * 2021-12-16 2022-03-22 江南大学 基于等温压缩系数扰动对酶进行再设计的方法、应用及通过此方法筛选出的突变体
CN114220492B (zh) * 2021-12-16 2023-02-28 江南大学 基于等温压缩系数扰动对酶进行再设计的方法、应用及通过此方法筛选出的突变体

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