WO2019000327A1 - Procédé d'administration de protéine trail de façon à inhiber en continu la croissance de cellules tumorales - Google Patents

Procédé d'administration de protéine trail de façon à inhiber en continu la croissance de cellules tumorales Download PDF

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WO2019000327A1
WO2019000327A1 PCT/CN2017/090826 CN2017090826W WO2019000327A1 WO 2019000327 A1 WO2019000327 A1 WO 2019000327A1 CN 2017090826 W CN2017090826 W CN 2017090826W WO 2019000327 A1 WO2019000327 A1 WO 2019000327A1
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trail
tumor
days
mur5s4tr
once
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陈守春
闫娟
徐琦
胡海洋
黄先洲
魏利佳
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成都华创生物技术有限公司
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Priority to PCT/CN2017/090826 priority Critical patent/WO2019000327A1/fr
Priority to CN201780009434.XA priority patent/CN109069584A/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the invention relates to the field of medicine, in particular to a method for administering TRAIL-like protein to continuously inhibit tumor cell growth.
  • TRAIL is a member of the Tumor necrosis factor (TNF) superfamily.
  • TNF Tumor necrosis factor
  • the gene sequences were independently cloned in 1995 by Wiley et al. and 1996 by Pitti et al., which was named apoptotic 2 Body (Apo2ligand, Apo2L). Later studies confirmed that Apo2L and TRAIL are essentially the same protein, so it is customary to call it Apo2L/TRAIL.
  • the function of TRAIL is firstly used as a regulator of congenital or acquired immunity in organisms, and secondly as an anti-tumor effect in immunocytochemistry in an exogenous apoptotic pathway.
  • TRAIL a multi-source human tumor cell line, including in vitro or in vivo, including nodules, lung cancer, breast cancer, prostate cancer, pancreatic cancer, kidney cancer, central nervous system tumors, Thyroid cancer, lymphoma, leukemia, and multiple myeloma all have the effect of inducing apoptosis.
  • apoptosis balance In tumor patients, the disruption of apoptosis balance—that is, the attenuation of pro-apoptotic signals and the enhancement of anti-apoptotic signals are very common. Therefore, repairing the uncontrolled apoptosis balance of cells is an important tumor treatment method.
  • the deep understanding of anti-tumor drugs has led to the understanding that whether cytotoxic drugs, molecularly targeted drugs or monoclonal antibodies play a role in the activation of tumor cell apoptosis pathways, signals that induce tumor cell apoptosis Access pathways are the hub and central link in which these drugs work, and apoptosis avoidance is an important mechanism for tumor development and drug resistance.
  • TRAIL has been developed as an important potential anti-tumor drug.
  • the clinical trial of TRAIL has entered Phase II abroad and has completed Phase III in China.
  • a large number of in vitro and in vivo tests have confirmed that TRAIL has tumor-specific cytotoxicity, especially when it is linked to low-dose chemotherapy drugs. It shows obvious synergy and synergism when used.
  • studies have found that TRAIL tolerance caused by loss of apoptotic machinery in the body is clearly associated with rapid growth and metastasis of tumor cells.
  • TRAIL-sensitive tumor cells have similar integrity and function in all aspects and factors in their apoptotic signaling pathway, and each TRAIL-resistant tumor cell has defects in some links and factors in the apoptotic signaling pathway. And mutations, these defects and mutations make these drug-resistant tumor cell apoptosis threshold abnormally elevated, easier to escape from apoptosis, and thus continue to grow and proliferate.
  • TRAIL Apo2L/TRAIL
  • factors that restrict Apo2L/TRAIL to better clinical efficacy include the rshTRAIL protein itself, because the trimer is a stable active form of TRAIL, and the preparation and storage of TRAIL trimer is difficult and the structure is highly heterogeneous (Trimer has low stability), TRAIL The in vivo short-half-life has no good pharmacokinetic properties.
  • tumor cell apoptosis signal pathway is a very complicated and huge system. It contains many pro-apoptotic factors and a large number of inhibitors of apoptosis. The interaction of these two factors determines the final destination of tumor cells. The soundness and function of the apoptotic signaling pathway are essential for tumor cell apoptosis, but they are not sufficient conditions.
  • the diversity of tumor apoptotic signaling pathways is manifested in: (1) exogenous and endogenous apoptotic signaling pathways, including pro-apoptotic factors and anti-apoptotic factors.
  • Proapoptotic factors include Caspases, DRs, FADD, Smac, Bax, Bak, etc.
  • Anti-apoptotic factors include c-FLIP, XIAP, Bcl-2, Mcl-1 and the like.
  • the TRAIL receptor agonist binds to the TRAIL receptor and forms a death-inducing complex through the classical signaling pathway to initiate apoptosis.
  • TRAIL receptor agonists also bind to TRAIL receptors to form secondary complexes that are directly or indirectly induced by activation of different kinases including I ⁇ B/NF- ⁇ B, MAPKs, PKC, PI3K/Akt, Src, etc. through non-canonical signaling pathways. Non-apoptotic response. Activation of these signaling pathways is associated with tumor proliferation and metastasis.
  • TRAIL receptor agonists need to be regulated by some substances to play a role in enhancing the anti-tumor effect.
  • a variety of different types of drug, molecular or genetic interventions can enhance the sensitivity of TRAIL to tumor cells, including different types of chemotherapeutic drugs, natural products, and small molecule kinase inhibitors. They enhance TRAIL-induced tumor cell apoptosis by potentiating extracellular apoptotic signaling pathways or mitochondrial apoptosis signaling pathways or by inhibiting other cellular survival signaling pathways or several pathways.
  • exogenous apoptosis signaling pathways includes up-regulating DRs, promoting the migration and aggregation of DRs to lipid raft microdomains, promoting the recruitment of FADD and DISC to receptor-ligand complexes, enhancing the activity of Caspases, and inhibiting apoptosis.
  • the link of mitochondrial apoptosis signaling pathway includes enhancing mitochondrial membrane potential depolarization, promoting mitochondrial release of Cytc, Smac or ARTS, stimulating Bid cleavage into tBid, promoting Bax and Bad oligomerization, inhibiting apoptosis and antagonizing Bcl-2 and Bcl-xL. , Bcl-w, Mcl-1, Survivin and other factors. Inhibition of other cell survival signaling pathways includes ERK/PI3K/Akt, MEK, Jak-STAT3, MAPK, NF- ⁇ B, and the like.
  • Apo2L/TRAIL showed linear pharmacokinetic properties over the dose range studied, and the area under the curve (AUC) of serum drug concentration and the increase in maximum plasma concentration (Cmax) increased in proportion to the dose.
  • AUC area under the curve
  • Cmax maximum plasma concentration
  • Apo2L/TRAIL route of administration On a nude mouse xenograft model, it was demonstrated that Apo2L/TRAIL intravenous injection is superior to intraperitoneal injection in inhibiting tumor growth. On a variety of nude mouse xenograft models, short-term intravenous injection of Apo2L/TRAIL (1 hour/day or 3 hours/day) or continuous intravenous injection (24 hours/day) significantly inhibited tumor growth, but short-term veins The efficacy of the injection is superior to long-term continuous administration. The results show that maintaining a relatively high blood drug concentration for a short period of time is stronger than an anti-tumor effect at a sustained exposure to a lower drug concentration. A direct comparison of 1 hour/day with 3 hours/day intravenous administration showed that the 1 hour/day administration mode has at least the same activity as the 3 hour/day administration mode, so the clinical selection is 1 hour/day. Method of administration.
  • the final dosing regimen is once daily (1 hour/day), continuous intravenous injection for 5 days, repeated every three weeks, which is also Clinically, the course of treatment of multiple chemotherapy drugs is consistent.
  • the dosing regimen with interval, repeat, and full-course drug exposure is the key to the present invention to greatly enhance the sustained inhibition of tumor growth by TRAIL-like proteins.
  • an object of the present invention is to provide a method for continuously inhibiting the growth of tumor cells by TRAIL-like proteins, which can greatly enhance the growth of TRAIL-like proteins to continuously inhibit the growth of various tumor cells.
  • a method for continuously inhibiting the growth of tumor cells by a TRAIL-like protein which is an administration of interval, repetition and full-course drug exposure, that is, prolonging the administration interval, increasing the drug exposure time of the tumor cells in the whole course of treatment, so that the drug is The effect of the full course of treatment is not attenuated, thereby continuing to inhibit tumor growth.
  • the TRAIL-like protein comprises a native or recombinant Apo2L/TRAIL protein cell membrane 114-281aa outer segment, a TRAIL receptor selective mutant, a TRAIL-penetrating peptide-like mutant of TRAIL-Mu3, TRAIL-MuR5 and TRAIL -MuR6, one or more of TRAIL-MuR5S4TR and TRAIL-MuR6S4TR and other mutants of the TRAIL transmembrane peptide mutein, wherein the amino acid sequence of the other mutant has a similarity to the wild-type protein of more than 75%.
  • the tumor cell is a solid tumor or a bone marrow-derived tumor
  • the solid tumor includes lung cancer, colorectal cancer, breast cancer, pancreatic cancer, liver cancer, gastric cancer, ovarian cancer, renal cancer, brain tumor, osteochondroma, prostate
  • the bone marrow-derived tumors include one or more of leukemia, non-Hodgkin's lymphoma.
  • the continuous inhibition of tumor cell growth includes in vitro cell-level tumor suppressor action and animal anti-tumor effect in vivo; in vitro, TRAIL-like protein interacts with cells in an effective dose range, and observes 24-96 hours of drug on tumor cells. Inhibition rate; TRAIL-like protein is the peak of inhibition of tumor cell growth in 24-72 hours in different sensitive tumor cells. For highly sensitive cell lines or higher concentration, the peak time of tumor inhibition continues. Up to 96 hours; in vivo experiments, animal transplanted tumors with different interval administration methods showed obvious growth inhibition state within 21 days, and the relative tumor growth rate T/C was ⁇ 40%.
  • the tumor inhibition rate in the tumor cell nude mice xenograft model is increased by at least 20% compared with the daily medication for five days, and the duration of action is prolonged for more than 5 days in a course of 21 days.
  • the dosing schedule for interval, repeat, and full course drug exposure includes any of the following:
  • TRAIL-like protein is administered intravenously once every other day. From the 0th day of treatment, the administration time is 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 or 0, 2, 4 respectively. 6,6,10,12,14,16,18,20, every 21 days for a course of treatment;
  • TRAIL-like protein is injected intravenously three times a week, from the 0th day of treatment, the administration time is 0, 2, 4, 7, 9, 11, 14, 16, 18, respectively, every 21 days for a course of treatment;
  • TRAIL protein is administered intravenously every three days. From the 0th day of treatment, the administration time is 0, 3, 6, 9, 12, 15, 18, and every 21 days is a course of treatment;
  • TRAIL-like protein is administered intravenously every four days. From the 0th day of treatment, the administration time is 0, 4, 8, 12, 16, 20, and every 21 days is a course of treatment.
  • the administration interval is higher than the minimum effective concentration in vitro and in vivo, and the administration interval is prolonged.
  • Rational drug use is a long-lasting subject of clinical pharmacy for many years. For this reason, many pharmacists have done a lot of effective work for this purpose. After the diagnosis of the disease, the correct choice of the treatment drug and the reasonable determination of the interval of administration are the early rehabilitation of the patient. The key is.
  • the half-life of drug metabolism (t 1/2 ), also known as the biological half-life and biological half-life, refers to the time required for the plasma drug concentration to decrease by half from the maximum value, usually expressed as t 1/2 .
  • a long half-life of the drug means slow elimination in the body and long residence time. Therefore, pay attention to the half-life of the drug. It is of great value to adjust the dosing schedule for grasping the residence time of the drug in the body, the degree of accumulation, and especially the interval of administration of the repeated medication.
  • the half-life of the drug determines the interval of administration, which plays an important role in ensuring the safety of the drug for repeated use and avoiding the side effects of the drug caused by drug accumulation.
  • the half-life of the drug is often inconsistent with the duration of the drug action, and the half-life of the drug is not equivalent to the duration of the drug action.
  • the interval between medications is mainly based on the half-life of the drug (the rate of drug elimination), which is a safer consideration for medication.
  • determining the dosing interval based on the half-life of the drug has its range of adaptation.
  • Some drugs with appropriate intervals of dosing, which are substantially consistent with their drug t 1/2 values, are suitable for determining the dosing interval based on t 1/2 .
  • Metoclopramide gastric ampoules
  • ampicillin etc.
  • sulfamethoxazole gliclazide (damecon)
  • ketoconazole risulfen
  • amphotericin etc.
  • Ultra-slow elimination drugs t 1/2 >24h
  • piroxicam Inflammation
  • nitrocodime digoxin
  • chlorpropamide etc.
  • Ultra-rapid elimination drugs (t 1/2 ⁇ 1h) and rapid elimination drugs (t 1/2 1 ⁇ 4h) with a short half-life of the drug, because they are eliminated quickly in the body, to maintain the desired blood concentration,
  • t 1/2 to arrange the interval of administration, it is necessary to increase the number of administrations and to administer frequently, so that not only the patient is unacceptable, but also clinically.
  • penicillin is very fast in metabolism and excretion in the body, and its half-life is only 0.5h. Frequent administration of t 1/2 to maintain the desired blood concentration is clearly undesirable.
  • the penicillin dosing interval is much longer than its t 1/2 .
  • the drug dose of t 1/2 is shorter than that of t 1/2 , and the drug with high therapeutic index can be prolonged. interval.
  • the biological half-life of Apo2L/TRAIL in rodents is 3 to 5 minutes, and in non-human primates, the biological half-life is 23 to 31 minutes, which is an ultra-rapid elimination drug.
  • the rapid clearance of Apo2L/TRAIL is mainly done by the kidney, and its clearance is highly correlated with glomerular filtration rate.
  • the maximum tolerated dose of Apo2L/TRAIL in a single dose exceeds 1500 mg/kg, which is very safe and has a particularly high therapeutic index.
  • Apo2L/TRAIL is not suitable for determining the interval of drug use strictly according to its drug metabolism half-life, so it is feasible to use a minimum concentration of action above its in vitro and in vivo effects to extend its dosing interval.
  • PAE Post antibiotic effect
  • MIC minimum inhibitory concentration
  • the use of previous antibiotics is based on the pharmacokinetics of the bacteria and the pharmacokinetic parameters such as the effective plasma concentration and half-life clearance rate, and the dose and interval of administration are determined, while the effect of the drug on the growth and reproduction of bacteria is neglected.
  • the role of human immune mechanisms in killing bacteria It has been observed that after the action of bacteria and antibiotics, when the drug is removed, the bacteria can produce a variety of detectable changes: such as the activity of enzymes and non-enzymatic proteins, cell morphology and the like. Bacterial metabolism and growth inhibit bacterial receptor changes, changes in susceptibility to phagocytosis, and changes in sensitivity to antibiotic exposure, such changes are antibacterial effects of antibiotics.
  • Apo2L/TRAIL also has a clear anti-tumor effect on tumor cell inhibition.
  • Apo2L/TRAIL has different sensitivities to different tumor cells.
  • Apo2L/TRAIL is allowed to adhere to tumor cells for 5 minutes at a certain concentration range, and then Apo2L/TRAIL is eluted with medium. After incubating at 37 ° C for 24 to 72 hours, the inhibition of tumors in the eluted culture wells did not differ between 24 and 72 hours as compared with the normal control wells.
  • the binding of Apo2L/TRAIL to the death receptor on the tumor cell membrane is strictly concentration-dependent and affinity-dependent, and there is no correlation with the number of receptors on the initial tumor cells. This binding differs depending on the drug sensitivity of the tumor cells. Sensitive cells bind very rapidly (5 minutes), while resistant cells bind slowly (1 hour).
  • Apo2L/TRAIL When Apo2L/TRAIL binds to death receptors (DR4, DR5) on the cell membrane, it multimerizes the receptor, rapidly causing aggregation and redistribution of the ligand/receptor complex in the cell membrane lipid raft microdomain, followed by ligand/ The receptor complex recruits the receptor molecule Fas-associated death domain (FADD) and pre-Caspase-8; forms a death-inducing signaling complex (DISC). Subsequently, Caspase-8 activates the biological effects of apoptosis via a mitochondria-dependent pathway and a non-mitochondria-dependent pathway.
  • FADD Fas-associated death domain
  • DISC death-inducing signaling complex
  • the non-mitochondria-dependent pathway is mainly the initial stage of Caspases, such as Caspase 8, 9, 10 activation, causing the Caspases protease cascade, further chain hydrolysis to activate the downstream homologous enzyme effect stage Caspases, such as Caspase 6, 7, and finally activate Caspase-3 .
  • the mitochondria-dependent pathway alters mitochondrial transmembrane potential through various apoptosis-promoting factors, increases mitochondrial permeability, leads to release of macroapoptic c-cytochrome c (Cytochrome c, Cyt c) and Smac/DIABLO, and caspase activation, apoptosis
  • the protein bcl-2 family is an important regulator of this.
  • the two apoptotic signaling pathways eventually merge into Caspase-3, and Caspase-3 catalyzes the breakdown of many apoptosis-related targeting molecules.
  • the transduction and transmission process of the apoptotic signaling pathway is complex and lengthy, and the elapsed steps are time-consuming. This is the molecular explanation for the effect of Apo2L/TRAIL on the apoptosis and tumor inhibition of tumor cells.
  • TRAIL-like proteins In vitro, TRAIL-like proteins (TRAIL-Mu3 and TRAIL-MuR5S4TR) interacted with cells at a concentration (dose) to observe the inhibition rate of the drug on tumor cells for 24-96 hours. In different sensitive tumor cells, TRAIL-like proteins are at the peak of inhibition of tumor cell growth for 24 to 72 hours. For highly sensitive cell lines (or higher concentration), the peak time of tumor inhibition continues. 96 hours.
  • the tumor inhibition rate showed significant difference; TRAIL-Mu3 72 mg/kg three times a week for three weeks.
  • Group TRAIL-Mu3 93mg/kg once every three days, for three consecutive weeks, TRAIL-Mu3 108mg/kg once every four days, for three consecutive weeks, there was no difference in efficacy between the groups; MuR5S4TR 105mg/kg three times a week, continuous In the three-week group, MuR5S4TR135mg/kg once every three days, for three consecutive weeks, MuR5S4TR 158mg/kg once every four days, there was no difference in efficacy between the three groups.
  • the method for administering the TRAIL-like protein of the present invention for continuously inhibiting the growth of tumor cells is understood to be a use of a TRAIL-like protein for the preparation of a medicament for continuously inhibiting the growth of tumor cells. And if necessary, do equivalent understanding or modification.
  • the design of the invention is not limited to the half-life of drug metabolism, but fully studies the duration of the drug's in vitro and in vivo effects and the anti-tumor effect, and is used to guide the design of a reasonable dosing interval, and finally the administration method is the interval.
  • New dosing regimens for repeated, full-course drug exposures Compared with the methods reported in the previous literature, the protocol prolongs the dosing interval, increases the duration of drug action of tumor cells in the whole course of treatment, and thus has stronger inhibitory effect on tumor growth, and does not attenuate the effect during the whole course of treatment, and has continuous inhibition. The role of tumor growth.
  • the dosing regimen of interval, repeat, and full-course drug exposure is a superior dosing regimen that optimizes the administration of TRAIL-like proteins and greatly enhances the continued inhibition of TRAIL-like proteins by various tumor cell growth. While improving the curative effect and duration of action, reducing the patient's treatment pain, improving patient compliance, in line with clinical application, great operability, and convenient clinical promotion.
  • Figure 1 is a graph showing the effect of TRAIL-Mu3 on the growth inhibition effect of NCI-H460 cells over time.
  • TRAIL-Mu3 was co-cultured with lung cancer cell NCI-H460 at different concentrations (dose) for 24, 48, 72, 84 or 96 hours, respectively, and the tumor growth inhibition rate of different drug concentrations (dose) at each time point was determined.
  • dose concentration of drug concentrations
  • the results showed that when the drug concentration (dose) was 0.02 ⁇ 5ug/ml, the tumor growth inhibition rate was at the peak plateau of tumor inhibition effect between 24 and 72 hours (the lowest concentration inhibition rate was 80.33%), and the tumor growth inhibition rate was The attenuation was still not significant at 96 hours (the lowest concentration inhibition rate was 46.30%).
  • Figure 2 is a graph showing the effect of TRAIL-MuR5S4TR on the growth inhibition effect of NCI-H460 cells over time.
  • TRAIL-MuR5S4TR was co-cultured with lung cancer cell NCI-H460 at different concentrations (dose) for 24, 48, 72, 84 or 96 hours, and the tumor growth inhibition rate of different drug concentrations (dose) at each time point was determined.
  • dose concentration of drug concentrations
  • the results showed that when the drug concentration (dose) was 0.04 ⁇ 10ug/ml, the tumor growth inhibition rate was at the peak plateau of tumor inhibition effect between 24 and 72 hours (the lowest concentration inhibition rate was 88.93%), and the tumor growth inhibition rate was The attenuation was still not significant at 96 hours (the lowest concentration inhibition rate was 68.76%).
  • Figure 3 is a graph showing the effect of TRAIL-Mu3 on the growth inhibition effect of Calu-1 cells over time.
  • TRAIL-Mu3 was co-cultured with Calu-1 cells at different concentrations (dose) for 24, 48, 72, 84 or 96 hours, and the tumor growth inhibition rate of different drug concentrations (dose) at each time point was determined.
  • dose concentration of drug concentrations
  • the results showed that when the drug concentration (dose) was 33.3-100 ug/ml, the tumor growth inhibition rate was at the peak plateau of tumor inhibition effect between 24 and 72 hours (the lowest concentration inhibition rate was 69.76%), and the tumor growth inhibition rate was The attenuation was still not significant at 96 hours (the lowest concentration inhibition rate was 78.28%).
  • Figure 4 is a graph showing the effect of TRAIL-MuR5S4TR on the growth inhibition effect of Calu-1 cells over time.
  • TRAIL-MuR5S4TR was co-cultured with lung cancer cell Calu-1 at different concentrations (dose) for 24, 48, 72, 84 or 96 hours, and the tumor growth inhibition rate of different drug concentrations (dose) at each time point was determined.
  • dose concentration of drug concentrations
  • the results showed that when the drug concentration (dose) was 33.3-100 ug/ml, the tumor growth inhibition rate was at the peak plateau of tumor inhibition effect between 24 and 72 hours (the lowest concentration inhibition rate was 80.75%), and the tumor growth inhibition rate was The attenuation was still not significant at 96 hours (the lowest concentration inhibition rate was 90.89%).
  • Figure 5 is a graph showing the effect of TRAIL-Mu3 on the growth inhibition effect of NCI-H1299 cells over time.
  • TRAIL-Mu3 was co-cultured with lung cancer cell NCI-H1299 at different concentrations (dose) for 24, 48, 72, 84 or 96 hours, and the tumor growth inhibition rate of different drug concentrations (dose) at each time point was determined.
  • dose concentration of drug concentrations
  • the results showed that when the drug concentration (dose) was 5.55-50 ug/ml, the tumor growth inhibition rate was at the peak plateau of tumor inhibition effect between 24 and 72 hours (the lowest concentration inhibition rate was 87.56%), and the tumor growth inhibition rate was The attenuation was still not significant at 96 hours (the lowest concentration inhibition rate was 71.20%).
  • Figure 6 is a graph showing the effect of TRAIL-MuR5S4TR on the growth inhibition effect of NCI-H1299 cells over time.
  • TRAIL-MuR5S4TR was co-cultured with lung cancer cell NCI-H1299 at different concentrations (dose) for 24, 48, 72, 84 or 96 hours, and the tumor growth inhibition rate of different drug concentrations (dose) at each time point was determined.
  • dose concentration of drug concentrations
  • the results showed that when the drug concentration (dose) was between 5.55 and 50 ug/ml, the tumor growth inhibition rate was at the peak plateau of tumor inhibition effect between 24 and 72 hours (the lowest concentration inhibition rate was 88.88%), and the tumor growth inhibition rate was The attenuation was still not significant at 96 hours (the lowest concentration inhibition rate was 75.15%).
  • Figure 7 is a graph showing the effect of different doses of TRAIL on tumor volume in animals in a human lung cancer NCI-H460 xenograft model.
  • paclitaxel 25mg/kg had significant difference in the inhibition of human lung cancer NCI-H460 xenograft tumor in nude mice.
  • T/C the relative tumor growth rate
  • TRAIL-Mu3 in three different dose frequency groups significantly inhibited human lung cancer NCI-H460 xenograft tumors in nude mice.
  • TRAIL once a day, 5 days in a row, protocol 1: 5 times in total
  • the inhibition effect on human lung cancer NCI-H460 xenograft tumors in Day3 was significantly different, and the relative tumor growth rate T/C was 39.30%.
  • the relative tumor growth rate T/C was 54.99% (P ⁇ 0.001), and to Day 21, the relative tumor growth rate T/C was 56.31% (P ⁇ 0.01).
  • TRAIL once a day, continuous administration for 5 days, interval 2 days, continuous administration for 5 days, protocol 2: a total of 10 times
  • T/C relative tumor growth rate
  • TRAIL once a day, 5 days after continuous administration, 5 days after continuous administration for 5 days, 2 days after repeated interval for 5 days, protocol 3: 15 times in total
  • the inhibition effect of transplanted tumor was significantly different in Day3.
  • the relative tumor growth rate was 36.79% (P ⁇ 0.001).
  • the relative tumor growth rate T/C was 34.46% (P ⁇ 0.001).
  • the relative tumor growth rate T/C was 32.87% (P ⁇ 0.001).
  • Figure 8 is a graph showing the effect of different doses of TRAIL on tumor weight in animals in a human lung cancer NCI-H460 xenograft model.
  • Figure 9 is a graph showing the effect of different doses of TRAIL-Mu3 and TRAIL-MuR5S4TR on tumor volume in animals in a human lung cancer NCI-H460 xenograft model.
  • paclitaxel (20mg/kg) had significant difference in the inhibition of human lung cancer NCI-H460 xenograft tumor in nude mice, but the relative tumor growth rate T/C>40% during the experiment.
  • TRAIL-Mu3 in three different dose frequency groups significantly inhibited human lung cancer NCI-H460 xenograft tumors in nude mice.
  • TRAIL-Mu3 once a day, 5 days for a total of two weeks
  • the inhibition of human lung cancer NCI-H460 xenograft tumors in Day4 was significantly different, the relative tumor growth rate T / C was 43.97% ( P ⁇ 0.001), at Day11, the relative tumor growth rate T/C was the smallest, being 24.75% (P ⁇ 0.001).
  • the relative tumor growth rate T/C was 33.5% (P ⁇ 0.01).
  • the inhibitory effect of TRAIL-Mu3 (3 times a week for 3 weeks) on human lung cancer NCI-H460 xenograft tumors was significantly different in Day4, and the relative tumor growth rate T/C was 48.94% (P ⁇ 0.001).
  • the relative tumor growth rate T/C was the smallest, 16.61% (P ⁇ 0.001), and to Day 21, the relative tumor growth rate T/C was 16.75% (P ⁇ 0.001).
  • MuR5S4TR in three different dosing frequency groups significantly inhibited human lung cancer NCI-H460 xenograft tumors in nude mice.
  • MuR5S4TR once a day, 5 days for a total of two weeks
  • the inhibition of human lung cancer NCI-H460 xenograft tumors in Day4 was significantly different
  • the relative tumor growth rate T / C was 59.57% (P ⁇ 0.001)
  • the relative tumor growth rate T/C was 38.92% (P ⁇ 0.01).
  • MuR5S4TR once every 2 days, a total of 10 times
  • the inhibition effect on human lung cancer NCI-H460 xenograft tumors in Day4 was significantly different, the relative tumor growth rate T/C was 54.61% (P ⁇ 0.001), to Day21
  • the relative tumor growth rate T/C was the smallest, being 24.43% (P ⁇ 0.001).
  • MuR5S4TR (3 times a week for 3 weeks) group had significant difference in the inhibition of human lung cancer NCI-H460 xenograft tumor in Day4, and the relative tumor growth rate T/C was 56.03% (P ⁇ 0.001). At day 21, the relative tumor growth rate T/C was the smallest, being 24.43% (P ⁇ 0.001).
  • Figure 10 is a graph showing the effect of different doses of TRAIL-Mu3 and TRAIL-MuR5S4TR on tumor weight in animals in a human lung cancer NCI-H460 xenograft model.
  • the average tumor weight of each group was 0.911 g, 0.658 g, 0.366 g, 0.170, respectively. g, 0.170 g, 0.416 g, 0.249 and 0.237 g.
  • Figure 11 is a graph showing the effect of different doses of TRAIL-Mu3 and TRAIL-MuR5S4TR on tumor volume in animals in a human colon cancer HT-29 nude mouse xenograft model.
  • CPT-11 Compared with the vehicle group, CPT-11 had a certain inhibitory effect on human colon cancer cell line HT-29 xenograft tumor in nude mice, and the tumor inhibition rate showed significant difference. At Day3, there was a significant difference in T/C, which was 88.74. % (P ⁇ 0.05); at Day 21, T/C was the smallest, 29.14% (P ⁇ 0.001)
  • T/C T/C
  • T/C T/C
  • MuR5S4TR 105mg/kg was administered three times a week, which had a certain inhibitory effect on human colon cancer cell line HT-29 xenograft tumor in nude mice.
  • T/C T/C was the smallest at 42.86% (P ⁇ 0.01).
  • T/C was 53.26% (P ⁇ 0.05).
  • T/C was the smallest at 29.03% (P ⁇ 0.01).
  • T/C was 51.53% (P ⁇ 0.05).
  • T/C was the smallest at 28.87% (P ⁇ 0.01).
  • Figure 12 is a graph showing the effect of different doses of TRAIL-Mu3 and TRAIL-MuR5S4TR on tumor weight in animals in a human colon cancer HT-29 nude mouse xenograft model.
  • Figure 13 is a graph showing the effect of different doses of TRAIL-Mu3 and TRAIL-MuR5S4TR on tumor volume in animals in a human pancreatic cancer PANC-1 nude mouse xenograft model.
  • TRAIL-Mu3 72mg/kg three times a week for three consecutive weeks, the human pancreatic cancer cell PANC-1 nude mice xenograft tumors have a certain inhibitory effect, the tumor inhibition rate showed significant differences, in Day4, T / C appeared significant The difference was 73.94% (P ⁇ 0.01); at Day21, the T/C was the smallest, 22.34% (P ⁇ 0.001).
  • TRAIL-Mu3 93mg/kg once every three days, the three-week group had a certain inhibitory effect on human pancreatic cancer cell PANC-1 xenograft tumor in nude mice, and the tumor inhibition rate showed significant difference.
  • T/C appeared. The significant difference was 63.38% (P ⁇ 0.01); at Day21, the T/C was the smallest, 23.25% (P ⁇ 0.001).
  • TRAIL-Mu3 108mg/kg once every four days, the three-week group had a certain inhibitory effect on human pancreatic cancer cell PANC-1 xenograft tumor in nude mice, and the tumor inhibition rate showed significant difference.
  • T/C appeared. The significant difference was 61.27% (P ⁇ 0.01); at Day21, the T/C was the smallest, 23.13% (P ⁇ 0.001).
  • T/C showed significantness. The difference was 65.49% (P ⁇ 0.001); at Day 21, the T/C was the smallest, 24.82% (P ⁇ 0.001).
  • MuR5S4TR 158mg/kg once every four days for three consecutive weeks has a certain inhibitory effect on human pancreatic cancer cell PANC-1 xenograft tumor in nude mice, and the tumor inhibition rate is significantly different.
  • T/C There was a significant difference of 73.24% (P ⁇ 0.001); at Day 21, the T/C was the smallest, 27.02% (P ⁇ 0.001).
  • Figure 14 is a graph showing the effect of different doses of TRAIL-Mu3 and TRAIL-MuR5S4TR on tumor weight in animals in a human pancreatic cancer PANC-1 nude mouse xenograft model.
  • TRAIL-Mu3 and TRAIL-MuR5S4TR are provided by Chengdu Huachuang Biotechnology Co., Ltd., batch number: 20160822.
  • the cells were cultured with NCI-H460, Calu-1, and NCI-H1299.
  • the culture medium and culture conditions were as follows.
  • the cells were changed once every 2 to 3 days, and mixed with 0.25% trypsin and 0.02% EDTA (1:1).
  • the logarithmic growth phase cells were connected to a 96-well plate.
  • the pre-test protein sample was diluted to the concentration of the following table with the corresponding medium of the cells, and then diluted 10 times in a gradient, diluted by 3 times, and 10 concentration points were added, and the cells were added at 25 ⁇ l/well.
  • tumor cell growth inhibition rate % [(Ac-As) / (Ac-Ab)] ⁇ 100%
  • TRAIL-like proteins In vitro, TRAIL-like proteins (TRAIL-Mu3 and TRAIL-MuR5S4TR) interacted with cells at a concentration (dose) to observe the inhibition rate of the drug on tumor cells for 24-96 hours. In different sensitive tumor cells, TRAIL-like proteins grow on tumor cells at 24-72 hours. At the peak of inhibition, for highly sensitive cell lines (or higher concentration of action), the peak duration of tumor inhibition lasts for 96 hours.
  • Figures 1 to 6 The relationship between the inhibitory effects of TRAIL-Mu3 and TRAIL-MuR5S4TR on three lung cancer cells with different sensitivity levels with time is shown in Figures 1 to 6.
  • Intravenous injection at a dose of 60 mg/kg once daily for 5 days (Scheme 1: 5 times in total), 60 mg/kg intravenously once daily, 5 days after continuous injection, and 5 consecutive days after 2 days. (Scheme 2: a total of 10 times) or a dose of 60 mg / kg intravenously, once a day, 5 days after continuous injection, 5 consecutive days after 5 days, and again after 2 days, 5 consecutive days (Scheme 3: 15 times in total)
  • Three different dosing regimens were compared for the difference in antitumor activity of human lung cancer NCI-H460 xenograft models in nude mice at three different dosing times.
  • Each squirrel cage carries an identity card with information such as the experiment number, experimental group, experimenter's name, mouse breed, and gender.
  • the mouse is labeled with the ear tag method.
  • the randomized block method was used to ensure that the tumor volume and the mouse body weight were uniform among the groups.
  • the mean value of the tumor volume of each group and the mean tumor volume of all the experimental animals did not exceed ⁇ 10%.
  • the irradiated large mouse feed was purchased from Beijing Keao Xieli Feed Co., Ltd. Free to eat.
  • mice were given the shortest environmental adaptation period before the experiment.
  • test article TRAIL and paclitaxel information are as follows:
  • a 1 ml sterile syringe was purchased from Shanghai Kangdelai Enterprise Development Group Co., Ltd. (Shanghai, China).
  • Human lung cancer cell line NCI-H460 was purchased from the Institute of Cell Biology, Shanghai Institute of Chinese Academy of Sciences.
  • NCI-H460 was cultured in F12-K medium (GIBCO, USA) containing 10% fetal bovine serum FBS (GIBCO, USA). Incubate in a 37 ° C incubator containing 5% CO 2 .
  • Matrigel Matrigel was purchased from BD Corporation of the United States
  • a human lung cancer NCI-H460 subcutaneous xenograft model was established. Each animal was inoculated with 3 ⁇ 10 6 cells, the inoculation volume was 0.1 ml/animal, and the cell suspension contained 50% Matrigel.
  • NCI-H460 cells were cultured in RPMI-1640 containing 10% fetal bovine serum FBS. The cells were cultured in a 5% CO 2 incubator at 37 °C.
  • the subcutaneous transplantation model of tumor nude mice was established by cell inoculation method: tumor cells in logarithmic growth phase were collected, counted, resuspended in 1 ⁇ PBS, 1:1 added Matrigel, and the cell suspension concentration was adjusted to 3 ⁇ 10 7 /ml. Tumor cells were inoculated subcutaneously in the right side of nude mice with a 1 ml syringe (4 gauge needle), 3 ⁇ 10 6 /0.1 ml/mouse, and a total of 60 cells were inoculated.
  • the animals were randomly grouped according to the randomized block method, so that the tumor difference of each group was less than 10% of the mean, and 8 mice in each group were grouped as Day 0, and according to the average body weight. Start administration.
  • TRTV treatment group RTV
  • CRTV negative control group RTV
  • the change in body weight (%) of the tumor-bearing animals was calculated as follows: (weight at the time of measurement - body weight at the time of grouping) / body weight at the time of grouping ⁇ 100.
  • the tumor growth curve was plotted with the time point as the X-axis and the tumor volume as the Y-axis; the body weight change value (%) was plotted on the Y-axis with the time point as the X-axis.
  • the t-test was used for comparison between groups. P ⁇ 0.05 was considered as significant difference, and P ⁇ 0.01 was extremely significant difference.
  • the vehicle control group was administered for 21 days, and the average body weight of the animals was not affected by the vehicle.
  • the average body weight of the animals continued to increase throughout the test period. Compared to Day 0, the average body weight of Day 21 increased by 8.34% (or 2.12 grams).
  • paclitaxel 25mg/kg had significant difference in the inhibition of human lung cancer NCI-H460 xenograft tumor in nude mice.
  • T/C the relative tumor growth rate
  • TRAIL-Mu3 in three different dose frequency groups significantly inhibited human lung cancer NCI-H460 xenograft tumors in nude mice.
  • TRAIL once a day, 5 days in a row, protocol 1: 5 times in total
  • the inhibition effect on human lung cancer NCI-H460 xenograft tumors in Day3 was significantly different, and the relative tumor growth rate T/C was 39.30%.
  • the relative tumor growth rate T/C was 54.99% (P ⁇ 0.001), and to Day 21, the relative tumor growth rate T/C was 56.31% (P ⁇ 0.01).
  • TRAIL once a day, continuous administration for 5 days, interval 2 days, continuous administration for 5 days, protocol 2: a total of 10 times
  • the relative tumor growth rate T/C was 32.68% (P ⁇ 0.001).
  • the relative tumor growth rate T/C was 34.12% (P ⁇ 0.001).
  • the relative tumor growth rate T/C was 37.48. % (P ⁇ 0.001).
  • TRAIL once a day, 5 days after continuous administration, 5 days after continuous administration for 5 days, 2 days after repeated interval for 5 days, protocol 3: 15 times in total
  • the inhibition effect of transplanted tumor was significantly different in Day3.
  • the relative tumor growth rate was 36.79% (P ⁇ 0.001).
  • the relative tumor growth rate T/C was 34.46% (P ⁇ 0.001).
  • the relative tumor growth rate T/C was 32.87% (P ⁇ 0.001).
  • Saline group, paclitaxel group, TRAIL once a day, continuous administration for 5 days, once daily, continuous administration for 5 days, interval 2 days, continuous administration for 5 days or once daily, continuous administration for 5 days after interval
  • the average tumor weight of each group in the group was 1.532 g, 0.728 g, 0.845 g, 0.646 g and 0.602 g, respectively, after 5 days of continuous administration for 5 days, and again for 2 days after repeated administration for 5 days.
  • TRAIL once a day, continuous administration for 5 days, once a day, continuous administration for 5 days, interval 2 days, continuous administration for 5 days and once daily, continuous administration 5
  • the rats were administered again for 5 days at 2 days after the interval, and again after 5 days and again for 5 days (three different administration times).
  • the group had a certain inhibitory effect on human lung cancer NCI-H460 xenograft tumor in nude mice, including intravenous injection.
  • the efficacy of the Tian group was significantly better than that of the once-daily, once-administered group for 5 days. After the administration, the tumor inhibition rate showed significant differences. However, there was no significant difference in the antitumor effect between regimen 3 and regimen 2 (P>0.05). Experiments have shown that increasing the number of administrations (prolonged administration time) can significantly increase the anti-tumor rate of the drug on the xenograft model of nude mice, but the increase in the tumor inhibition rate is not obvious when the number of administrations is 15 and 10 times.
  • human lung cancer NCI-H460 nude mouse xenograft model was used to evaluate the difference in in vivo antitumor activity between TRAIL-Mu3 and TRAIL-MuR5S4TR at different dosing intervals.
  • Each squirrel cage carries an identity card with information such as the experiment number, experimental group, experimenter's name, mouse breed, and gender.
  • the mouse is labeled with the ear tag method.
  • the randomized block method was used to ensure that the tumor volume and the mouse body weight were uniform between the groups.
  • the mean value of the tumor volume of each group and the mean tumor volume of all the experimental animals did not exceed ⁇ 10%.
  • the irradiated large mouse feed was purchased from Beijing Keao Xieli Feed Co., Ltd. Free to eat.
  • mice were given the shortest environmental adaptation period before the experiment.
  • test substances TRAIL-Mu3 and TRAIL-MuR5S4TR, paclitaxel information are as follows:
  • a 1 ml sterile syringe was purchased from Shanghai Kangdelai Enterprise Development Group Co., Ltd. (Shanghai, China).
  • Human lung cancer cell line NCI-H460 was purchased from the Institute of Cell Biology, Shanghai Institute of Chinese Academy of Sciences.
  • NCI-H460 was cultured in F12-K medium (GIBCO, USA) containing 10% fetal bovine serum FBS (GIBCO, USA). Incubate in a 37 ° C incubator containing 5% CO 2 .
  • Matrigel Matrigel was purchased from BD Corporation of the United States
  • a human lung cancer NCI-H460 subcutaneous xenograft model was established. Each animal was inoculated with 3 ⁇ 10 6 cells, the inoculation volume was 0.1 ml/animal, and the cell suspension contained 50% Matrigel.
  • NCI-H460 cells were cultured in RPMI-1640 containing 10% fetal bovine serum FBS. The cells were cultured in a 5% CO 2 incubator at 37 °C.
  • the subcutaneous transplantation model of tumor nude mice was established by cell inoculation method: tumor cells in logarithmic growth phase were collected, counted, resuspended in 1 ⁇ PBS, 1:1 added Matrigel, and the cell suspension concentration was adjusted to 3 ⁇ 10 7 /ml. Tumor cells were inoculated subcutaneously in the right side of nude mice with a 1 ml syringe (4 gauge needle), 3 x 10 6 / 0.1 ml / mouse, and a total of 90 cells were inoculated.
  • the animals were randomly grouped according to the randomized block method, so that the tumor difference of each group was less than 10% of the mean, and 8 mice in each group were grouped as Day 0, and started according to the average body weight. Dosing.
  • TRTV treatment group RTV
  • CRTV negative control group RTV
  • the change in body weight (%) of the tumor-bearing animals was calculated as follows: (weight at the time of measurement - body weight at the time of grouping) / body weight at the time of grouping ⁇ 100.
  • the tumor growth curve was plotted with the time point as the X-axis and the tumor volume as the Y-axis; the body weight change value (%) was plotted on the Y-axis with the time point as the X-axis.
  • the t-test was used for comparison between groups. P ⁇ 0.05 was considered as significant difference, and P ⁇ 0.01 was extremely significant difference.
  • the vehicle control group was administered for 21 days, and the average body weight of the animals was not affected by the vehicle.
  • the average body weight of the animals continued to increase throughout the test period. Compared to Day 0, the average body weight of Day 21 increased by 9.27% (or 2.22 grams).
  • paclitaxel (20mg/kg) had significant difference in the inhibition of human lung cancer NCI-H460 xenograft tumor in nude mice, but the relative tumor growth rate T/C>40% during the experiment.
  • TRAIL-Mu3 in three different dose frequency groups significantly inhibited human lung cancer NCI-H460 xenograft tumors in nude mice.
  • TRAIL-Mu3 once a day, 5 days for a total of two weeks
  • the inhibition of human lung cancer NCI-H460 xenograft tumors in Day4 was significantly different, the relative tumor growth rate T / C was 43.97% ( P ⁇ 0.001), at Day11, the relative tumor growth rate T/C was the smallest, being 24.75% (P ⁇ 0.001).
  • the relative tumor growth rate T/C was 33.5% (P ⁇ 0.01).
  • the inhibitory effect of TRAIL-Mu3 (3 times a week for 3 weeks) on human lung cancer NCI-H460 xenograft tumors was significantly different in Day4, and the relative tumor growth rate T/C was 48.94% (P ⁇ 0.001).
  • the relative tumor growth rate T/C was the smallest, 16.61% (P ⁇ 0.001), and to Day 21, the relative tumor growth rate T/C was 16.75% (P ⁇ 0.001).
  • MuR5S4TR in three different dosing frequency groups significantly inhibited human lung cancer NCI-H460 xenograft tumors in nude mice.
  • MuR5S4TR once a day, 5 days for a total of two weeks
  • the inhibition of human lung cancer NCI-H460 xenograft tumors in Day4 was significantly different
  • the relative tumor growth rate T / C was 59.57% (P ⁇ 0.001)
  • the relative tumor growth rate T/C was 38.92% (P ⁇ 0.01).
  • MuR5S4TR once every 2 days, a total of 10 times
  • the inhibition effect on human lung cancer NCI-H460 xenograft tumors in Day4 was significantly different, the relative tumor growth rate T/C was 54.61% (P ⁇ 0.001), to Day21
  • the relative tumor growth rate T/C was the smallest, being 24.43% (P ⁇ 0.001).
  • MuR5S4TR (3 times a week for 3 weeks) group had significant difference in the inhibition of human lung cancer NCI-H460 xenograft tumor in Day4, and the relative tumor growth rate T/C was 56.03% (P ⁇ 0.001). At day 21, the relative tumor growth rate T/C was the smallest, being 24.43% (P ⁇ 0.001).
  • the average tumor weight of each group was 0.911 g, 0.658 g, 0.366 g, 0.170, respectively. g, 0.170 g, 0.416 g, 0.249 and 0.237 g.
  • the paclitaxel group, the TRAIL-Mu3 group and the MuR5S4TR group had certain inhibitory effects on human lung cancer NCI-H460 xenograft tumors in nude mice.
  • the tumor inhibition rate showed significant differences, among which TRAIL -Mu3 and MuR5S4TR two drugs administered every other day and three times a week, three weeks
  • the anti-tumor effect of the drug group was superior to that of daily administration for 5 consecutive days for a total of two weeks.
  • the anti-tumor effect of TRAIL-Mu3 and MuR5S4TR in the drug-administered group and the three-week-weekly three-week administration group was equivalent.
  • a human colon cancer cell line HT-29 nude mouse xenograft model was used to evaluate the in vivo antitumor activity of TRAIL-Mu3 and TRAIL-MuR5S4TR at different dosing intervals.
  • Each squirrel cage carries an identity card with information such as the experiment number, experimental group, experimenter's name, mouse breed, and gender.
  • the mouse is labeled with the ear tag method.
  • the randomized block method was used to ensure that the tumor volume and the mouse body weight were uniform among the groups.
  • the mean value of the tumor volume of each group and the mean value of the tumor volume of all the experimental animals did not exceed ⁇ 10%.
  • the irradiated large mouse feed was purchased from Beijing Keao Xieli Feed Co., Ltd. Free to eat.
  • mice were given the shortest environmental adaptation period before the experiment.
  • the human colon cancer cell line HT-29 was purchased from the Institute of Cell Biology, Shanghai Institute of Chinese Academy of Sciences.
  • Trypsin-EDTA (purchased from GIBCO, USA)
  • Trypan Blue Trypan Blue (purchased from GIBCO, USA)
  • Biosafety cabinet (model: AC2-6E1), purchased from ESCO;
  • CO 2 water-tight cell incubator (model: 3111), purchased from Thermo Scientific Forma;
  • a 1 ml sterile syringe was purchased from Shanghai Kangdelai Enterprise Development Group Co., Ltd. (Shanghai, China).
  • a subcutaneous xenograft model of human colon cancer cell line HT-29 was established, and each animal was inoculated with 3 ⁇ 10 6 cells in an inoculation volume of 0.1 ml/animal.
  • CPT-11 was dispensed as a stock solution (20 mg/ml), 0.25 ml per tube. Store at room temperature.
  • HT-29 cells were cultured in McCoy's 5a medium containing 10% fetal bovine serum FBS. Incubate in a 37 ° C incubator containing 5% CO 2 . After cell resuscitation, sufficient cells are collected for animal inoculation after cell expansion and passage.
  • Tumor cells in the logarithmic growth phase were collected, counted, resuspended in serum-free McCoy's 5a medium, and the cell suspension concentration was adjusted to 3 ⁇ 10 7 /mL.
  • Tumor cells were inoculated subcutaneously in the right side of nude mice with a 1 mL syringe (4 gauge needle), and 3 animals were inoculated with 3 ⁇ 10 6 /0.1 mL/mouse.
  • the animals were randomly grouped according to the randomized block method, so that the tumor difference of each group was less than 10% of the mean, 8 rats in each group, a total of 8 groups. Group the day diary as Day 0. Administration according to "4. Experimental design".
  • TRTV treatment group RTV
  • CRTV negative control group RTV
  • the change in body weight (%) of the tumor-bearing animals was calculated as follows: (weight at the time of measurement - body weight at the time of grouping) / body weight at the time of grouping ⁇ 100.
  • the tumor growth curve was plotted with the time point as the X-axis and the tumor volume as the Y-axis; the body weight change curve was plotted on the Y-axis with the time point as the X-axis and the average animal weight (g).
  • the t-test was used for comparison between groups. P ⁇ 0.05 was considered as significant difference, and P ⁇ 0.01 was extremely significant difference.
  • the average body weight of the vehicle in the vehicle group was not affected by the vehicle solvent.
  • the average weight of animals continues to increase. Compared to Day 0 on the first day of dosing, the average body weight of Day 21 increased by 7.01% (ie 1.64 g).
  • the animals were dosed once every four days for TRAIL-Mu3 108 mg/kg, and the drug regimen was tolerated for three consecutive weeks. During the entire experimental period, compared with Day0, the average body weight of this group increased by 7.85% compared with Day0 (ie, 1.82 g).
  • the animals were administered three times a week to MuR5S4TR 105 mg/kg for three consecutive weeks.
  • the average body weight of the animals increased by 6.76% (ie 1.60 g) at Day 21 compared to Day 0 over the entire experimental period. .
  • CPT-11 Compared with the vehicle group, CPT-11 had a certain inhibitory effect on human colon cancer cell line HT-29 xenograft tumor in nude mice, and the tumor inhibition rate showed significant difference. At Day3, there was a significant difference in T/C, which was 88.74. % (P ⁇ 0.05); at Day 21, T/C was the smallest, 29.14% (P ⁇ 0.001)
  • T/C T/C
  • T/C T/C
  • MuR5S4TR 105mg/kg was administered three times a week, which had a certain inhibitory effect on human colon cancer cell line HT-29 xenograft tumor in nude mice.
  • T/C T/C was the smallest at 42.86% (P ⁇ 0.01).
  • T/C was 53.26% (P ⁇ 0.05).
  • T/C was the smallest at 29.03% (P ⁇ 0.01).
  • T/C was 51.53% (P ⁇ 0.05).
  • T/C was the smallest at 28.87% (P ⁇ 0.01).
  • the CPT-11 group Compared with the vehicle group, the CPT-11 group, TRAIL-Mu3 72 mg/kg three times a week, TRAIL-Mu 393 mg/kg once every three days, TRAIL-Mu3 108 mg/kg once every four days.
  • the tumor suppressive effect of TRAIL-Mu3 and TRAIL-MuR5S4TR administered once every three days and once every four days was significantly superior to the three-weekly administration group, and once every three days and once every four days. There was no significant difference in tumor suppression effects in the primary group.
  • human pancreatic cancer cell PANC-1 nude mouse xenograft model was used to evaluate the in vivo antitumor activity of TRAIL-Mu3 and TRAIL-MuR5S4TR at different dosing intervals.
  • Each squirrel cage carries an identity card with information such as the experiment number, experimental group, experimenter's name, mouse breed, and gender.
  • the mouse is labeled with the ear tag method.
  • the randomized block method was used to ensure that the tumor volume and the mouse body weight were uniform among the groups.
  • the mean value of the tumor volume of each group and the mean tumor volume of all the experimental animals did not exceed ⁇ 10%.
  • the irradiated large mouse feed was purchased from Beijing Keao Xieli Feed Co., Ltd. Free to eat.
  • mice were given the shortest environmental adaptation period before the experiment.
  • the human pancreatic cancer cell line PANC-1 was purchased from the Institute of Cell Biology, Shanghai Institute of Chinese Academy of Sciences.
  • DMEM medium (GIBCO, USA)
  • Trypsin-EDTA (purchased from GIBCO USA)
  • Trypan Blue Trypan Blue (purchased from GIBCO USA)
  • Biosafety cabinet (model: AC2-6E1), purchased from ESCO;
  • CO 2 water-tight cell incubator (model: 3111), purchased from Thermo Scientific Forma;
  • a 1 ml sterile syringe was purchased from Shanghai Kangdelai Enterprise Development Group Co., Ltd. (Shanghai, China).
  • a human pancreatic cancer cell PANC-1 subcutaneous xenograft model was established, and each animal was inoculated with 5 x 106 cells in an inoculation volume of 0.1 ml/animal.
  • PANC-1 cells were cultured in DMEM medium containing 10% fetal bovine serum FBS. Incubate in a 37 ° C incubator containing 5% CO 2 . After cell resuscitation, sufficient cells are collected for animal inoculation after cell expansion and passage.
  • Tumor cells in the logarithmic growth phase were collected, counted, resuspended in serum-free DMEM medium, and the cell suspension concentration was adjusted to 5 ⁇ 10 7 /mL.
  • Tumor cells were inoculated subcutaneously in the right side of nude mice with a 1 mL syringe (4 gauge needle), 5 x 10 6 /0.1 mL/mouse, and a total of 90 animals were inoculated.
  • the animals were randomly grouped according to the randomized block method, so that the tumor difference of each group was less than 10% of the mean, 8 rats in each group, a total of 8 groups. Group the journal as Day0. Administration according to "4. Experimental design".
  • TRTV treatment group RTV
  • CRTV negative control group RTV
  • the change in body weight (%) of the tumor-bearing animals was calculated as follows: (weight at the time of measurement - body weight at the time of grouping) / body weight at the time of grouping ⁇ 100.
  • the tumor growth curve was plotted with the time point as the X-axis and the tumor volume as the Y-axis; the body weight change curve was plotted on the Y-axis with the time point as the X-axis and the average animal weight (g).
  • the t-test was used for comparison between groups. P ⁇ 0.05 was considered as significant difference, and P ⁇ 0.01 was extremely significant difference.
  • the average body weight of the vehicle in the vehicle group was not affected by the vehicle solvent.
  • the average weight of animals continues to increase. Compared with Day 0 on the first day of dosing, the average body weight of Day 21 increased by 8.42% (ie 1.93 g).
  • the body weight of the animals decreased significantly at the initial stage of administration. Compared with Day 0 on the first day of administration, the average body weight of the animals decreased by 16.92%. That is, 3.18 g), and at the same time Day 7, an animal died; with the end of the dosing period, the weight of the animals began to recover and rose. At Day 21, the average weight of the animals increased by 8.57% (1.93 g).
  • the animals were dosed once every four days for TRAIL-Mu3 108 mg/kg, and the drug regimen was tolerated for three consecutive weeks. During the entire experimental period, compared with Day0, the average body weight of this group increased by 5.63% compared with Day0 (ie, 1.26g).
  • the animals were dosed once every three days for MuR5S4TR 135 mg/kg for three consecutive weeks. During the entire experimental period, compared with Day0, the average body weight of this group increased by 5.09% (ie 1.12 g) compared with Day0. ).
  • TRAIL-Mu3 72mg/kg three times a week for three consecutive weeks, the human pancreatic cancer cell PANC-1 nude mice xenograft tumors have a certain inhibitory effect, the tumor inhibition rate showed significant differences, in Day4, T / C appeared significant The difference was 73.94% (P ⁇ 0.01); at Day21, the T/C was the smallest, 22.34% (P ⁇ 0.001).
  • TRAIL-Mu3 93mg/kg once every three days, the three-week group had a certain inhibitory effect on human pancreatic cancer cell PANC-1 xenograft tumor in nude mice, and the tumor inhibition rate showed significant difference.
  • T/C appeared. The significant difference was 63.38% (P ⁇ 0.01); at Day21, the T/C was the smallest, 23.25% (P ⁇ 0.001).
  • TRAIL-Mu3 108mg/kg once every four days, the three-week group had a certain inhibitory effect on human pancreatic cancer cell PANC-1 xenograft tumor in nude mice, and the tumor inhibition rate showed significant difference.
  • T/C appeared. The significant difference was 61.27% (P ⁇ 0.01); at Day21, the T/C was the smallest, 23.13% (P ⁇ 0.001).
  • T/C There was a significant difference of 65.49% (P ⁇ 0.001); at Day 21, the T/C was the smallest, 24.82% (P ⁇ 0.001).
  • the gemcitabine group TRAIL-Mu3 72 mg/kg three times a week for three consecutive weeks, TRAIL-Mu3 93 mg/kg once every three days, for three consecutive weeks, TRAIL-Mu3 108 mg/kg once every four days,
  • MuR5S4TR 105 mg/kg three times a week for three consecutive weeks MuR5S4TR135 mg/kg once every three days for three consecutive weeks
  • PANC -1 nude mice xenograft tumors have obvious inhibitory effects, and there is a significant difference in tumor inhibition rate after administration.

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  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
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  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne un procédé d'administration d'une protéine TRAIL (ligand inducteur d'apoptose lié au facteur de nécrose tumorale (TNF)) de manière à inhiber en continu la croissance de cellules tumorales, le procédé spécifique comprenant l'administration au moyen d'une exposition à un médicament de traitement intermittente, répétée et complète ; par le biais de la prolongation d'un intervalle d'administration et de l'augmentation du temps d'exposition au médicament pour des cellules tumorales pendant un traitement complet, l'action du médicament pendant un traitement complet n'est pas atténuée, ce qui permet d'inhiber en continu la croissance tumorale.
PCT/CN2017/090826 2017-06-29 2017-06-29 Procédé d'administration de protéine trail de façon à inhiber en continu la croissance de cellules tumorales WO2019000327A1 (fr)

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CN201780009434.XA CN109069584A (zh) 2017-06-29 2017-06-29 一种trail类蛋白持续抑制肿瘤细胞生长的给药方法

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CN105916975A (zh) * 2014-01-10 2016-08-31 学校法人帝京平成大学 重组专性厌氧革兰氏阳性菌
CN106170300A (zh) * 2014-04-08 2016-11-30 西雅图基因公司 Cd19‑抗体药物偶联物的优化给药

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GB0724532D0 (en) * 2007-12-17 2008-01-30 Nat Univ Ireland Trail variants for treating cancer
CN102370987B (zh) * 2010-08-26 2013-05-29 复旦大学 一种包载抗肿瘤药物组合物的注射用脂质体
US9746370B2 (en) * 2014-02-26 2017-08-29 Sensity Systems Inc. Method and apparatus for measuring illumination characteristics of a luminaire
WO2016138618A1 (fr) * 2015-03-02 2016-09-09 成都华创生物技术有限公司 Mutant mur5 de type peptidique pénétrant dans la membrane de trail, son procédé de préparation et son application
CN106164090B (zh) * 2015-03-02 2019-07-23 成都华创生物技术有限公司 TRAIL穿膜肽样突变体MuR6、制备方法及应用
WO2017066963A1 (fr) * 2015-10-22 2017-04-27 成都华创生物技术有限公司 Mutéine mur6s4tr de trail à double cible, son procédé de préparation et son utilisation
EP3348577B1 (fr) * 2015-10-22 2020-02-12 Chengdu Huachuang Biotechnology Co., Ltd Mutéine mur5s4tr de trail à double cible, son procédé de préparation et son utilisation

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CN101636412A (zh) * 2007-03-30 2010-01-27 霍夫曼-拉罗奇有限公司 标记的和非标记的单克隆抗体的组合物
WO2009140469A2 (fr) * 2008-05-14 2009-11-19 Genentech, Inc. Procédés d'utilisation d'apo2l/trail pour traiter le cancer
CN105916975A (zh) * 2014-01-10 2016-08-31 学校法人帝京平成大学 重组专性厌氧革兰氏阳性菌
CN106170300A (zh) * 2014-04-08 2016-11-30 西雅图基因公司 Cd19‑抗体药物偶联物的优化给药

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