WO2019000327A1

WO2019000327A1 - Method for administering trail protein so as to continuously inhibit tumor cell growth

Info

Publication number: WO2019000327A1
Application number: PCT/CN2017/090826
Authority: WO
Inventors: 陈守春; 闫娟; 徐琦; 胡海洋; 黄先洲; 魏利佳
Original assignee: 成都华创生物技术有限公司
Priority date: 2017-06-29
Filing date: 2017-06-29
Publication date: 2019-01-03
Also published as: CN109069584A

Abstract

Provided is a method for administering a tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) protein so as to continuously inhibit tumor cell growth, the specific method comprising administration by means of intermittent, repeated and full-course treatment drug exposure; by means of prolonging an administration interval and increasing drug exposure time for tumor cells during full-course treatment, the action of the drug during full-course treatment does not attenuate, thereby continuously inhibiting tumor growth.

Description

Method for administering TRAIL-like protein to continuously inhibit tumor cell growth

Technical field

The invention relates to the field of medicine, in particular to a method for administering TRAIL-like protein to continuously inhibit tumor cell growth.

Background technique

TRAIL is a member of the Tumor necrosis factor (TNF) superfamily. 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. The greatest advantage of TRAIL is that it can selectively induce apoptosis of a variety of tumor cells with little toxicity to normal cells. Studies have shown that Apo2L/TRAIL is 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.

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.

In the 20 years since its discovery, 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. In contrast, 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.

Recent research advances have shown that relying solely on Apo2L/TRAIL to treat many different types of tumors is still not enough. Although agonistic monoclonal antibodies to recombinant human Apo2L/TRAIL or TRAIL receptor DR4/DR5 achieved encouraging results in Phase I clinical trials, they did not show clear clinical benefit in subsequent Phase II clinical studies. . Clinical studies suggest that Apo2L/TRAIL drugs exhibit limited tumor responses to a variety of tumors, with a moderately disappointingly modest overall, and some patients have no Lack of therapeutic benefit.

Numerous studies have shown that normal cells and about half (or even up to 60%) of passaged tumor cell lines are resistant to TRAIL. According to a review by Roberta Di Pietro and Giorgio Zauli, Apo2L/TRAIL is sensitive to 61 of the 92 primary or passaged tumor cells that have been studied, with a sensitivity rate of 66.3%, while the remaining 31 strains are resistant. 33.7%. A large number of research literatures focus on the resistance of TRAIL. In summary, the research focuses on the following aspects: (1) Malignant tumors are resistant, and (2) originated from the body. Resistance in primary tumor cells, (3) Congenital and acquired resistance, (4) multi-target and multi-path resistance mechanisms have been Resistance mechanisms to TRAIL, (5) Resistance and effective therapies are effective in evading drug resistance.

Almost all 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.

The 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.

A large number of studies have confirmed that Apo2L/TRAIL alone does not produce high inhibition and killing effects on many tumor cells. The reason is that the 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. (2) Pro-survival signaling pathways, including NFκB, PI3K, Akt, MAPK, and the like. (3) Heterogeneity of tumor cell apoptosis defects, tumor cell apoptosis defects can occur in different aspects of the apoptotic signaling pathway. The study found that TRAIL itself has a variety of biological functions. 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 IκB/NF-κB, 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. The role of 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. Activity of factors FLIP, XIAP, IAPs, etc. 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-κB, and the like.

Genentech Inc. first studied the pharmacokinetics of ApoL/TRAIL in a variety of animal models. The plasma half-life of a single intravenous injection of Apo2L/TRAIL in rodents is 3 to 5 minutes, compared to 23 to 31 minutes in primates. Intravenous injection of Apo2L/TRAIL (0, 10, 30 or 100 mg/kg) in cynomolgus monkeys for 1 hour, rapid drug clearance, multiple doses (maximum dose of 100 mg/kg) of plasma The half-life is 28 to 30 minutes. Multiple administration does not change the half-life of the drug and does not cause drug accumulation. 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. In vivo studies of chimpanzees (administered doses of 1, 5, 50 mg/kg) also met the above rules.

Selection of 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.

Determination of the dose-effect relationship of Apo2L/TRAIL. On a nude mouse xenograft model, the peak dose of Apo2L/TRAIL for short-term intravenous injection (1 hour/day or 3 hours/day) was 30-90 mg/kg, in addition to a 5-day short-term continuous vein. The effect of injection (once a day) was better than a short-term continuous intravenous injection (once a day) for 3 days at the same dose, while intravenous injection for 5 days was sufficient to inhibit tumor growth. Combined with Apo2L/TRAIL's shorter drug metabolism half-life (21-31 minutes), 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.

Despite this, the experimental data published by Genentech Inc showed that once-a-day (1 hour/day), continuous intravenous injection for 5 days, the preclinical efficacy of the dosing regimen repeated every three weeks was not ideal, which also led to Apo2L/TRAIL. One of the important reasons for the unsatisfactory clinical efficacy. Preclinical data show that for apo2L/TRAIL highly sensitive cells (such as colon cancer cells COLO205, non-small cell lung cancer cells NCI-H2122, etc.) nude mouse xenograft model, once daily (1 hour / day), continuous intravenous injection After 5 days of dosing, the tumors were significantly reduced on days 0-5 (7), and the tumors continued to grow after 10 days. For moderately sensitive cells (such as non-small cell lung cancer NCI-H460) nude mouse xenograft model, once daily (1 hour / day), continuous intravenous injection for 5 days, the tumor grows on days 0-5 Slow, and the tumor grew rapidly after 5 days, no difference from the negative control group. The above experimental results show that once a day (1 hour/day), a 5-day continuous dosing regimen does not persist (within one treatment cycle of 21 days) inhibiting tumor growth, and this dosing regimen is not the optimal dosing regimen for Apo2L/TRAIL.

There are at least four defects in the dosing regimen identified by Genentech Inc. (1) Different sensitivity levels of tumor cells respond differently to Apo2L/TRAIL, and the data source determined by the Apo2L/TRAIL dosing regimen is primarily based on its sensitivity to high sensitivity and The inhibitory effect of moderately sensitive tumor cells does not fully represent its actual effect on relatively resistant tumor cells. Therefore, the experimental basis for determining the dosing regimen is not comprehensive enough and its applicability is limited. (2) Even for highly sensitive and moderately sensitive tumor cells, intravenous administration of Apo2L/TRAIL, once daily for 5 consecutive days, does not consistently inhibit tumor cell growth. Apo2L/TRAIL inhibited tumor growth significantly during drug administration, and the inhibition of tumor growth after drug withdrawal disappeared rapidly. The second half of the course of treatment was actually without drug effect. (3) Genentech Inc did not explore the difference in the efficacy of the Apo2L/TRAIL administration time (intravenous, once daily, for 10 or 15 days) and intravenous, once daily, and 5 consecutive days. According to our data, the anti-tumor effect of intravenous, once daily, continuous 10 or 15 day dosing regimens is superior to intravenous, once daily, continuous 5 day dosing regimens. (4) Genentech Inc did not explore the difference in efficacy of Apo2L/TRAIL intravenous, interval, repetitive, multiple-dose and intravenous, once daily, continuous 5-day dosing regimens.

Summary of the invention

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.

Therefore, in view of the deficiencies of the prior art, 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.

The technical solution of the present invention is implemented as follows:

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.

Further, 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%.

Further, the tumor cell is a solid tumor or a bone marrow-derived tumor, and the solid tumor includes lung cancer, colorectal cancer, breast cancer, pancreatic cancer, liver cancer, gastric cancer, ovarian cancer, renal cancer, brain tumor, osteochondroma, prostate One or more of the cancers; the bone marrow-derived tumors include one or more of leukemia, non-Hodgkin's lymphoma.

Further, 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%.

Further, 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.

Further, the dosing schedule for interval, repeat, and full course drug exposure includes any of the following:

(1) 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;

(2) 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;

(3) 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;

(4) 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.

Further, since the maximum tolerated dose of a single administration of Apo2L/TRAIL drug exceeds 1500 mg/kg, 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. However, 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. Traditionally, 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. In fact, 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 . For example, intermediate-effect elimination drugs (t _1/2 = 4 ~ 8h), such as ciprofloxacin, norfloxacin, atropine. Metoclopramide (gastric ampoules), ampicillin, etc., can be administered 3 times or 4 times / d. Slow-acting elimination drugs (t _1/2 = 8 ~ 12h), such as sulfamethoxazole, gliclazide (damecon), ketoconazole (risulfen), amphotericin, etc., daily Dosing 2 times. Ultra-slow elimination drugs (t _1/2 >24h), such as piroxicam (Inflammation), nitrocodime, digoxin, chlorpropamide, etc., can be administered once a day. 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, According to 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. For example, 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. Clinically, the penicillin dosing interval is much longer than its t _1/2 . This is due to the large amount of drug administered, which achieves a blood drug concentration that greatly exceeds the minimum inhibitory concentration for most microorganisms. In other words, the safety of penicillin is good, the therapeutic index is high, and the number of administrations can be appropriately reduced. The larger the dose, the longer the interval of administration. For drugs with a particularly long t _1/2 , such as digoxigenin in the ultra-slow elimination drug (t _1/2 =200h), if the dose is administered every 9 days according to t _1/2 , the blood concentration fluctuates. Large, adverse reactions are large, clinically often take a small dose once a day, so that the fluctuation of blood concentration is small, the safety range is expanded. In short, t _1/2 reflects the speed of drug elimination from the body. For rapid elimination, 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.

More and more studies have revealed that the metabolic indicators of many drugs are not completely consistent with the duration of their biological effects, and some drugs have post-drug effects. For example, many antibiotics have a Post antibiotic effect (PAE). PAE refers to the effect of continuous inhibition of bacterial growth when the concentration of the drug drops below the minimum inhibitory concentration (MIC) after repeated contact with the antibiotic. In the past two decades, research on PAE has been extensive and in-depth at home and abroad, and PAE has been used as an important parameter for evaluating antibiotics and an important basis for designing clinical drug delivery programs to guide the treatment of infectious diseases. It has long been believed that antibacterial drugs must achieve and maintain effective blood levels in order to exert good antibacterial effects. Therefore, 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. We found that Apo2L/TRAIL has different sensitivities to different tumor cells. For highly sensitive 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. For relatively resistant tumor cells, when Apo2L/TRAIL is in a higher concentration range with tumors The cells were allowed to act for 1 hour, and then Apo2L/TRAIL was eluted with the medium and cultured at 37 ° C for 24 to 72 hours. The eluted culture wells were inhibited against tumors for 24 to 72 hours compared with the normal control wells. difference. The above experiments show that Apo2L/TRAIL can rapidly bind to the death receptor on the sensitive tumor cell membrane and cause the transduction of the apoptotic signaling pathway. Once the above process is initiated, the subsequent process is no longer dependent on the presence of extracellular free Apo2L/TRAIL. For drug-resistant tumor cells, the contact initiation time required for the above process is longer. When Apo2L/TRAIL was applied to tumor cells in a certain concentration range, the inhibition rate of the drug on tumor cells was observed for 24 to 96 hours. It was found that the higher the concentration of Apo2L/TRAIL in the multiple tumor cells with different sensitivity, The faster the time to continuously inhibit tumor cell proliferation. For higher concentrations, Apo2L/TRAIL inhibited tumor cells at 24 to 96 hours, and for lower concentrations, Apo2L/TRAIL inhibited tumor cells at 24 hours. It gradually decreased in ~72 hours, and the effects disappeared completely in 84 hours and 96 hours. The above experiments show that the effect of Apo2L/TRAIL on tumor cells is rapid and lasting. 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). 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. 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.

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.

In vivo experiments with different doses of animals showed that compared with the saline group, paclitaxel group, 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 a day, 5 days after continuous administration, 5 days after continuous administration for 5 days, and again after 2 days, 5 consecutive days of continuous administration for three different administration times. The group has human lung cancer NCI-H460 xenograft tumor in nude mice. A certain inhibitory effect, in which intravenous injection, once a day, continuous administration for 5 days, 2 days apart, continuous administration for 5 days and once daily, continuous administration for 5 days, followed by continuous administration for 5 days at intervals of 2 days, The effect of the continuous administration for 5 days after the interval of 2 days was significantly superior to that of the single daily administration, and the continuous administration for 5 days, and the tumor inhibition rate showed significant difference after administration. 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.

In vivo experiments with different dosing intervals showed that compared with the saline group, 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. There was a significant difference in the rate. Among them, the TRAIL-Mu3 and MuR5S4TR drugs were administered every other day and three times a week. The anti-tumor effect of the three-week administration group was superior to that of daily administration for 5 consecutive days for 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.

In vivo experiments with different dosing intervals showed that compared with the vehicle group, CPT-11 group, TRAIL-Mu372 mg/kg three times a week, TRAIL-Mu3 93 mg/kg once every three days, TRAIL-Mu3 108 mg/ Kg was administered once every four days, MuR5S4TR 105 mg/kg was administered three times a week, MuR5S4TR 135 mg/kg was administered once every three days, MuR5S4TR 158 mg/kg was administered once every four days to human colon cancer cells HT- 29 nude mice xenograft tumors have a certain inhibitory effect, after the administration, the tumor inhibition rate is significantly different; TRAIL-Mu3 and TRAIL-MuR5S4TR every three days The tumor suppressive effect of the once-administered group and the four-day-administered group was significantly superior to the three-weekly-administered group, and there was no significant difference in the tumor suppressor effect between the three-day dosing group and the four-day dosing group. .

In vivo experiments with different dosing intervals showed that compared with the vehicle group, the gemcitabine group, TRAIL-Mu372 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 for three consecutive weeks, MuR5S4TR 105 mg/kg three times a week for three consecutive weeks, MuR5S4TR 135 mg/kg once every three days for three consecutive weeks, MuR5S4TR 158 mg/kg once every four days, continuous The three-week group had significant inhibitory effect on human pancreatic cancer cell PANC-1 xenograft tumor in nude mice. After administration, 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 beneficial effects of the invention are:

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.

DRAWINGS

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. 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. 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. 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. 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. 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. 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.

Compared with the vehicle group, paclitaxel (25mg/kg) had significant difference in the inhibition of human lung cancer NCI-H460 xenograft tumor in nude mice. At Day10, the relative tumor growth rate T/C was 34.88%; to Day21, T/ C is 46.12%.

Compared with the vehicle group, in this experiment, 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%. (P < 0.001), at Day 10, 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 inhibition of human lung cancer NCI-H460 xenograft tumors in Day3 was significant. The difference was relative tumor growth rate T/C of 32.68% (P<0.001). At Day10, relative tumor The value-added rate T/C was 34.12% (P<0.001), and to Day21, 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), for human lung cancer NCI-H460 nude mice The inhibition effect of transplanted tumor was significantly different in Day3. The relative tumor growth rate was 36.79% (P<0.001). At Day10, the relative tumor growth rate T/C was 34.46% (P<0.001). To Day21, 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.

At the end of the Day 21 experiment, all animals were sacrificed after weighing and tumor volume, tumors were isolated from animals and weighed. 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.

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.

Compared with the vehicle group, 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 (once a day, 5 days for a total of two weeks) group, 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). To Day21, the relative tumor growth rate T/C was 33.5% (P<0.01).

The inhibitory effect of TRAIL-Mu3 (once every 2 days for 10 times) on human lung cancer NCI-H460 xenograft tumors was significantly different in Day4, and the relative tumor growth rate T/C was 51.77% (P< 0.001), at Day14, the relative tumor growth rate T/C was the smallest, 16.16% (P < 0.001), and to Day21, the relative tumor growth rate T/C was 17.63% (P < 0.001).

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). At Day 18, 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).

Similar to TRAIL-Mu3, 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) group, 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), to Day21, the relative tumor growth rate T/C was 38.92% (P<0.01).

MuR5S4TR (once every 2 days, a total of 10 times) group, 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.

At the end of the Day 21 experiment, all animals were sacrificed after weighing and tumor volume, tumors were isolated from animals and weighed. In the saline group, the paclitaxel group, the TRAIL-Mu3 (three different administration frequency) group, and the MuR5S4TR (three different administration frequencies) group, 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.

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)

TRAIL-Mu3 72 mg/kg administered three times a week had a certain inhibitory effect on human colon cancer cells HT-29 xenograft tumors in nude mice. At Day 7, there was a significant difference in T/C, which was 54.69% (P< 0.05); At Day 21, T/C was the smallest, being 36.43% (P < 0.01).

TRAIL-Mu3 93mg/kg once every three days, had a certain inhibitory effect on human colon cancer cell line HT-29 xenograft tumor in nude mice. At Day3, there was a significant difference in T/C, which was 81.84% ( P < 0.05); at Day 21, T/C was the smallest at 28.00% (P < 0.01).

TRAIL-Mu3 108mg/kg once every four days, had a certain inhibitory effect on human colon cancer cell line HT-29 xenograft tumor in nude mice. At Day3, there was a significant difference in T/C, which was 85.49% (P <0.05); at Day 21, T/C was the smallest, being 28.69% (P < 0.01).

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. At Day3, there was a significant difference in T/C, which was 88.26% (P<0.05). At Day 21, T/C was the smallest at 42.86% (P < 0.01).

MuR5S4TR 135mg/kg once every three days, had a certain inhibitory effect on human colon cancer cell line HT-29 xenograft tumor in nude mice. At Day7, there was a significant difference in T/C, which was 53.26% (P<0.05). At Day 21, T/C was the smallest at 29.03% (P < 0.01).

MuR5S4TR 158mg/kg once every four days, had a certain inhibitory effect on human colon cancer cell line HT-29 xenograft tumor in nude mice. At Day7, there was a significant difference in T/C, which was 51.53% (P<0.05). At Day 21, 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.

At the end of the Day 21 experiment, all animals were sacrificed after weighing and tumor volume, tumors were isolated from animals and weighed. The vehicle group, CPT-11 group, TRAIL-Mu3 72 mg/kg three times a week, TRAIL-Mu3 93 mg/kg once every three days, TRAIL-Mu3 108 mg/kg once every four days, MuR5S4TR 105 mg/kg three times a week, MuR5S4TR 135 mg/kg once every three days, MuR5S4TR 158 mg/kg every four days, the average tumor weight was 1.339 g, 0.811 g, 0.898 g, 0.796 g , 0.805 g, 0.936 g, 0.823 g, and 0.812 g.

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.

Compared with the vehicle group, gemcitabine 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. At Day 4, the T/C showed significant difference, which was 80.28% (P <0.05); at Day 21, T/C was the smallest, being 36.14% (P < 0.001).

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. At Day4, 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. At Day4, 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).

MuR5S4TR 105mg/kg three times a week for three weeks, 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. At Day4, 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 135mg/kg once every three days for three consecutive weeks, 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. At Day4, T/C appeared significantly. The difference was 70.42% (P < 0.001); at Day 21, the T/C was the smallest, 27.00% (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. At Day4, 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.

At the end of the Day 21 experiment, all animals were sacrificed after weighing and tumor volume, tumors were isolated from animals and weighed. Solvent group, gemcitabine group (n=7), 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-Mu3108 mg/kg every four days One-time, three-week group, MuR5S4TR 105mg/kg three times a week for three consecutive weeks, MuR5S4TR 135mg/kg once every three days, for three consecutive weeks, MuR5S4TR 158mg/kg once every four days, three consecutive weeks of tumor average The weights were 0.131 g, 0.075 g, 0.042 g, 0.044 g, 0.043 g, 0.047 g, 0.050 g and 0.052 g, respectively.

Detailed ways

The invention is further illustrated by the following examples, but the scope of protection of the invention is not limited thereto.

Example 1

Time-effect relationship between TRAIL-Mu3 and TRAIL-MuR5S4TR in inhibiting tumor cell growth in vitro

1. Research purposes

To investigate the anti-tumor effect of TRAIL-Mu3 and TRAIL-MuR5S4TR at different concentrations (dose) on human lung cancer cells NCI-H460, Calu-1 and NCI-H1299 in vitro and their relationship with time.

2. Experimental materials, reagents and equipment

2.1 preliminary experiment

2.1.1 Cell selection: Three cells with different degrees of sensitivity were selected for formal experiments using previous experimental data (see Table 1).

Table 1. In vitro antitumor effect of TRAIL-Mu3 and TRAIL-MuR5S4TR on seven lung cancer cell lines

Based on the data in the above table, the effect of TRAIL-Mu3 and TRAIL-MuR5S4TR was combined to select a sensitive strain: NCI-H460; one insensitive strain: NCI-H1299; and one moderately sensitive strain: calu-1.

2.1.2 In the 20170314-LJ1 experiment (pre-experimental data), it was concluded that all the three cells showed the same anti-tumor cell proliferation inhibition rate in the absence of the drug concentration before 72h. the trend of. Therefore, the drug action interval was adjusted to 24h, 48h, 72h, 84h, 96h, in order to further observe the relationship between the time and the drug dose.

2.1.3 In the 20170314-LJ1 experiment, when the cell growth was observed under the microscope, it was found that the negative cell density was too large at 72 h, resulting in cell death. Therefore, in this experiment, the inoculation density of the 96-well plates of the three cells was appropriately reduced, and adjusted to NCI-H460 (6×10 ³ ), NCI-H1299 (4×10 ³ ), and Calu-1 (6×10 ^{4 , respectively).} ).

2.2 Materials

TRAIL-Mu3 and TRAIL-MuR5S4TR are provided by Chengdu Huachuang Biotechnology Co., Ltd., batch number: 20160822.

2.3 reagent

2.4 Instrumentation

3. Experimental methods and steps

3.1 Method: See the respective reagent instructions

3.2 steps:

First, the experimental steps

Cell culture

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). In the experiment, the logarithmic growth phase cells were connected to a 96-well plate.

2. IC ₅₀ experiment

(1) Collect logarithmic growth phase cells, count, resuspend cells with complete medium, adjust cell density to appropriate density NCI-H460 (6×10 ³ ), Calu-1 (6×10 ⁴ ), NCI-H1299 ( 4×10 ³ ) Inoculate 96-well plates, and add 100 μl of cell suspension to each well. The cells were incubated for 24 hours at 37 ° C in a 100% relative humidity, 5% CO ₂ incubator.

(2) 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 μl/well.

(3) The cells were placed in a 37 ° C, 100% relative humidity, 5% CO ₂ incubator, and each cell was incubated for 24 h, 48 h, 72 h, 84 h, and 96 h, respectively, and the medium was aspirated, and 10% CCK-8 was added. The complete medium was incubated in a 37 ° C incubator.

(4) When the negative hole is detected at a wavelength of 450 nm of the microplate reader, the absorbance at a wavelength of 450 nm is measured on a microplate reader (Infinite F50), and the inhibition rate is calculated.

Second, data processing

The inhibition rate of the drug on tumor cell growth was calculated as follows: tumor cell growth inhibition rate % = [(Ac-As) / (Ac-Ab)] × 100%

As: OA/RLU of the sample (cell + CCK-8 + test compound)

Ac: negative control OA/RLU (cell + CCK-8)

Ab: positive control OA/RLU (medium + CCK-8)

4. Experimental results

4.1 Antitumor effect of TRAIL-Mu3 and TRAIL-MuR5S4TR on different time of action of NCI-H460 cells

Table 2. Tumor inhibition rate of TRAIL-Mu3 and TRAIL-MuR5S4TR on different time of NCI-H460 cells

Table 3. Tumor inhibition rate of TRAIL-Mu3 and TRAIL-MuR5S4TR on different time of Calu-1 cells

Table 4. Tumor inhibition rate of TRAIL-Mu3 and TRAIL-MuR5S4TR on different time of NCI-H1299 cells

5. Experimental conclusion

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. 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.

Example 2

Anti-tumor effect of different doses of TRAIL on human lung cancer NCI-H460 cells xenograft tumor in nude mice

1. Experimental purpose

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.

2. Experimental animals

2.1 Animal species

Mouse.

2.2 varieties

Balb/c nude mouse.

2.3 gender

female.

2.4 quantity

Inoculate 60 and select 40.

2.5. Age

4 to 6 weeks.

2.6. Weight

16 ~ 18g ± 20% body weight average.

2.7. Animal source (supplier)

Shanghai Xipuer-Beikai Experimental Animal Co., Ltd. (BK), license number SCXK (Shanghai) 2013-0016, animal certificate number: 2008001661519.

2.8. Laboratory Animal Management

2.8.1 Animal identification method

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.

2.8.2 Random grouping

When the tumor volume reached 100-200 mm3, 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%.

2.8.3 Operation Management Specifications

All experimental animals are operated and managed in strict accordance with the guidelines for the use and management of experimental animals in Shanghai.

2.8.4 Feeding conditions

Living conditions: 3 per cage

Temperature: 20 ° C ~ 26 ° C

Humidity: 40% to 70%

Illumination: 12 hours and nights alternate

2.8.5 feed

The irradiated large mouse feed was purchased from Beijing Keao Xieli Feed Co., Ltd. Free to eat.

2.8.6 Drinking water

City tap water, filtered and autoclaved for drinking.

2.8.7 litter

Corn cob, Shanghai Maosheng Derivative Technology Co., Ltd., used after autoclaving, changing the litter twice a week.

2.8.8 Adaptation period

The mice were given the shortest environmental adaptation period before the experiment.

3. Experimental materials

3.1 test drugs

The test article TRAIL and paclitaxel information are as follows:

3.2 Other chemical materials

3.2.1 sterile syringe

A 1 ml sterile syringe was purchased from Shanghai Kangdelai Enterprise Development Group Co., Ltd. (Shanghai, China).

3.2.2 cell line

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 .

3.2.3 Matrigel (BD Matrigel)

Matrigel Matrigel was purchased from BD Corporation of the United States

4. Experimental design

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.

The dose and dosing schedule of this independent trial design are as follows.

Different doses of TRAIL for administration of human lung cancer NCI-H460 xenograft tumor in nude mice

5. Experimental methods

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.

When the tumor volume reached 100-200 mm ³ , 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.

Animal body weight and tumor size were measured twice weekly during the experiment. Daily observations record clinical symptoms. All animal experiments are strictly in accordance with Shanghai Medicil Biomedical Co., Ltd. Animal Use and Management Practices. The calculation of tumor-related parameters refers to the Chinese SFDA "Guidelines for Non-Clinical Research Techniques of Cytotoxic Antitumor Drugs" ^[1] .

The evaluation index of antitumor activity is the relative tumor growth rate T/C (%), and the calculation formula is: T/C (%) = TRTV / CRTV * 100%. (TRTV: treatment group RTV; CRTV: negative control group RTV); relative tumor volume (RTV), the formula is: RTV = Vt / V0. Where V0 is the measured tumor volume at the time of sub-cage administration (ie Day0), and Vt is the tumor volume at each measurement.

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.

According to the Chinese SFDA "Guidelines for Non-Clinical Research Techniques of Cytotoxic Antitumor Drugs" (November 2006), T/C (%) ≤ 40% and statistically analyzed P < 0.05 is effective. A dose of the drug is considered to be severely toxic if the body weight of the mouse drops by more than 20% or the number of drug-related deaths exceeds 20%.

6. Data analysis

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.

7. Results

7.1 Effects of different test substances on the body weight of human lung cancer NCI-H460 nude mice

In the experiment, 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).

Animals showed significant toxicity to paclitaxel (25 mg/kg, IV), and one animal died after the second and third doses, respectively. On the 7th to 14th day of the whole test period, the weight loss was obvious, and the maximum weight loss reached 18.52%. After Day14, the body weight gradually increased. By the end of the experiment, the body weight recovered.

Animals were tolerant to TRAIL (Scheme 1: 5 times, Protocol 2: 10 times, Protocol 3: 15 times) for different dosing times, and the average body weight of the animals increased throughout the test period. Compared with Day 0, the average body weight of the animals in the three different dosing frequency groups on Day 21 increased by 7.68% (ie 1.97 g), 5.25% (ie 1.20 g) and 5.34% (1.18 g), respectively.

7.2 Effect of each test substance on tumor volume of human lung cancer NCI-H460 nude mice xenograft tumor

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 inhibition of human lung cancer NCI-H460 xenograft tumors in Day3 was significant. The relative tumor growth rate T/C was 32.68% (P<0.001). At Day10, the relative tumor growth rate T/C was 34.12% (P<0.001). To Day21, the relative tumor growth rate T/C was 37.48. % (P < 0.001).

The detailed results are shown in Table 5. The results of the experiment are shown in Table 5 and Figure 7.

7.3 Effect of each test substance on tumor weight of human lung cancer NCI-H460 nude mice xenograft tumor

At the end of the Day21 experiment, all animals were sacrificed after weighing the body weight and tumor volume. The tumors were separated from the animals and weighed. Tumor tissues were collected. Each tumor tissue was divided into two parts: one liquid nitrogen was quickly frozen and stored with -80 The refrigerator was stored for subsequent analysis; one was fixed with formaldehyde and embedded in paraffin for subsequent analysis. 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.

The detailed results are shown in Table 5 and Figure 8.

Table 5. Effect of different administration time of TRAIL on tumor size in human lung cancer NCI-H460 nude mouse xenograft model

*: P < 0.05; **: P < 0.01; ***: P < 0.001 compared with the vehicle group

8. Summary

In this experiment, similar to the saline group, paclitaxel, 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 or once a day, 5 days after continuous administration, continuous administration for 5 days at intervals of 2 days, and repeated administration for another 5 days after 2 days interval, three different administration times) had little effect on animal body weight, and the toxicity was small and safe; the weight of paclitaxel group animals It decreased slightly and was gradually stable during the experiment and it was safer. Compared with the saline group, paclitaxel group, 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. Once a day, continuous administration for 5 days, 2 days apart, continuous administration for 5 days and once daily, continuous administration for 5 days, continuous administration for 5 days at 2 days, and repeated administration for another 2 days. 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.

9. References

[1] "Guidelines for Non-Clinical Research Techniques of Cytotoxic Antitumor Drugs" November 2006

Example 3

Therapeutic effects of different doses of TRAIL-Mu3 and TRAIL-MuR5S4TR on human lung cancer NCI-H460 xenograft xenografts in nude mice

1. Experimental purpose

In this study, 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.

2. Experimental animals

2.1 Animal species

Mouse.

2.2 varieties

Balb/c nude mouse.

2.3 gender

female.

2.4 quantity

Inoculate 90 and select 64.

2.5. Age

4-6 weeks.

2.6. Weight

16 ~ 18g ± 20% body weight average.

2.7. Animal source (supplier)

2.8. Laboratory Animal Management

2.8.1 Animal identification method

2.8.2 Random grouping

When the tumor volume reached 100-200 mm ³ , 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%.

2.8.3 Operation Management Specifications

2.8.4 Feeding conditions

Living conditions: 3 per cage

Temperature: 20 ° C ~ 26 ° C

Humidity: 40% to 70%

Illumination: 12 hours and nights alternate

2.8.5 feed

2.8.6 Drinking water

City tap water, filtered and autoclaved for drinking.

2.8.7 litter

Corn cob, Shanghai Maosheng Derivative Technology Co., Ltd., used after autoclaving. Change the litter twice a week.

2.8.8 Adaptation period

3. Experimental materials

3.1 test drugs

The test substances TRAIL-Mu3 and TRAIL-MuR5S4TR, paclitaxel information are as follows:

3.2 Other chemical materials

3.2.1 sterile syringe

3.2.2 cell line

3.2.3 Matrigel (BD Matrigel)

Matrigel Matrigel was purchased from BD Corporation of the United States

4. Experimental design

The dose and dosing schedule of this independent trial design are as follows:

Anti-tumor effect of TRAIL-Mu3 and MuR5S4TR in human lung cancer NCI-H460 xenograft model

5. Experimental methods

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.

When the tumor volume reached 100-200 mm ³ , 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.

6. Data analysis

7. Results

In the experiment, 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).

Animals were tolerant to paclitaxel (20 mg/kg, IV) and had a slight decrease in body weight throughout the test period. At Day 21, the average body weight of the animals decreased by 2.32% (or 0.52 g).

Animals were given TRAIL-Mu3 at different dosing frequencies (once a day for 5 days for a total of two weeks; for two days for a total of 10 times; for three times a week for a total of 3 weeks; for a dose of 60 mg/kg, IV) During the entire test period, the average animal weight continued to increase. Compared with Day 0, the average body weight of the animals in the three different dosing frequency groups on Day 21 increased by 8.93% (ie, 2.06 g), 5.38% (ie, 1.23 g) and 7.72% (ie, 1.78 g).

Animals were given MuR5S4TR at different dosing frequencies (once a day for 5 days for a total of two weeks; for 2 days for a total of 10 times; for 3 times a week for 3 weeks; for 60 mg/kg, IV) for tolerance throughout the trial During the cycle, the average animal weight continues to increase. Compared with Day 0, the average body weight of the animals in the three different dosing frequency groups on Day 21 increased by 8.02% (ie 1.79 g), 6.80% (ie 1.56 g) and 6.68% (ie 1.52 g).

The inhibitory effect of TRAIL-Mu3 (once every 2 days for 10 times) on human lung cancer NCI-H460 xenograft tumors was significantly different in Day4, and the relative tumor growth rate T/C was 51.77% (P<0.001). At Day 14, the relative tumor growth rate T/C was the smallest, 16.16% (P < 0.001), and to Day 21, the relative tumor growth rate T/C was 17.63% (P < 0.001).

The detailed results are shown in Table 6. The results of the experiments are shown in Table 6 and Figure 9.

At the end of the Day21 experiment, all animals were sacrificed after weighing the body weight and tumor volume. The tumors were separated from the animals and weighed. Tumor tissues were collected. Each tumor tissue was divided into two parts: one liquid nitrogen was quickly frozen and stored with -80 The refrigerator was stored for subsequent analysis; one was fixed with formaldehyde and embedded in paraffin for subsequent analysis. In the saline group, the paclitaxel group, the TRAIL-Mu3 (three different administration frequency) group, and the MuR5S4TR (three different administration frequencies) group, 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 detailed results are shown in Table 6 and Figure 10.

Table 6. Effect of TRAIL-Mu3 and MuR5S4TR on tumor size in human lung cancer NCI-H460 xenograft xenograft model

*: P < 0.05; **: P < 0.01; ***: P < 0.001 compared with the vehicle group

8. Summary

In this experiment, similar to the saline group, paclitaxel, TRAIL-Mu3 group and MuR5S4TR group had almost no effect on animal body weight, and the toxicity was small and safe. The weight of paclitaxel group was slightly decreased, and it was stable and safe during the experiment.

Compared with the saline group, 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. After administration, 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.

9. References

Example 4

Therapeutic effects of different doses of TRAIL-Mu3 and TRAIL-MuR5S4TR on human colon cancer cell line HT-29 xenograft xenografts in nude mice

1. Experimental purpose

In this study, 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.

2. Experimental animals

2.1 Animal species

Mouse.

2.2 varieties

Balb/c nude mouse.

2.3 gender

female.

2.4 quantity

The experiment used 64.

2.5 age

4 to 6 weeks.

2.6 weight

16 ~ 18g ± 20% body weight average.

2.7 animal source (supplier)

Shanghai Xipuer-Beikai Experimental Animal Co., Ltd. (BK), license number SCXK (Shanghai) 2013-0016, animal certificate number: 2008001665079.

2.8 Laboratory Animal Management

2.8.1 Animal identification method

2.8.2 Random grouping

When the tumor volume reached an average of 200 mm3, 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%.

2.8.3 Operation Management Specifications

2.8.4 Feeding conditions

Living conditions: 3 per cage.

Temperature: 20 ° C ~ 26 ° C

Humidity: 40% to 70%

Illumination: 12 hours and nights alternate

2.8.5 feed

2.8.6 Drinking water

City tap water, filtered and autoclaved for drinking.

2.8.7 litter

2.8.8 Adaptation period

3. Experimental materials

3.1 test drugs

Test objects TRAIL-Mu3 and MuR5S4TR information

3.2 cell line

The human colon cancer cell line HT-29 was purchased from the Institute of Cell Biology, Shanghai Institute of Chinese Academy of Sciences.

3.3 reagent

McCoy's 5a Medium (GIBCO, USA)

Fetal bovine serum FBS (GIBCO, USA)

Trypsin-EDTA (purchased from GIBCO, USA)

Trypan Blue Trypan Blue (purchased from GIBCO, USA)

Saline was purchased from Huayu (Wuxi) Pharmaceutical Co., Ltd. (Jiangsu, China).

3.4 Instruments

Biosafety cabinet (model: AC2-6E1), purchased from ESCO;

CO ₂ water-tight cell incubator (model: 3111), purchased from Thermo Scientific Forma;

Inverted microscope (model: CKX41SF), purchased from Olympus;

Electric suction device (Model YX930D), purchased from Shanghai Medical Device Industry (Group) Co., Ltd.;

Low speed centrifuge (model LD5-2A), purchased from Beijing Rebold Centrifuge Co., Ltd.

3.5 other

4. Experimental design

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.

The dose and dosing schedule of this independent experiment design are as follows:

Anti-tumor effect of different doses of TRAIL-Mu3 and MuR5S4TR in human colon cancer cell line HT-29 nude mice xenograft model

5. Experimental methods

5.1 test drug preparation

5.1.1 CPT-11 2.5mg/ml 2ml

CPT-11 was dispensed as a stock solution (20 mg/ml), 0.25 ml per tube. Store at room temperature.

Take one of the above-packed CPT-11 stock solutions (20 mg/ml).

Add 1.75 ml of physiological saline and mix.

Now ready to use, store at 18 ~ 25 ° C before use.

5.1.2 TRAIL-Mu3 10.8mg/ml 2.4ml

Solvent: 0.9% saline: water for injection = 1:1

Take TRAIL-Mu3 stock solution (24mg/ml) 1.08ml,

Add 1.32 ml of the above solvent and mix.

Now ready to use, keep at 4 ° C before use, use within 4 hours.

5.1.3 TRAIL-Mu3 9.3mg/ml 2.4ml

Solvent: 0.9% saline: water for injection = 1:1

Take TRAIL-Mu3 stock solution (24mg/ml) 0.93ml,

Add 1.47 ml of the above solvent and mix.

Now ready to use, keep at 4 ° C before use, use within 4 hours.

5.1.4 TRAIL-Mu3 7.2mg/ml 2.4ml

Take TRAIL-Mu3 stock solution (24 mg/ml) 0.72 ml.

Add 1.68 ml of the above solvent and mix.

Now ready to use, keep at 4 ° C before use, use within 4 hours.

5.1.5 MuR5S4TR 15.8mg/ml 2.4ml

Solvent: 0.9% saline: water for injection = 1:1

Take MuR5S4TR stock solution (24mg/ml) 1.58ml.

0.82 ml of the above solvent was added and mixed.

Now ready to use, keep at 4 ° C before use, use within 4 hours.

5.1.6 MuR5S4TR 13.5mg/ml 2.4ml

Solvent: 0.9% saline: water for injection = 1:1

Take the MuR5S4TR stock solution (24 mg/ml) at 1.35 ml.

1.05 ml of the above solvent was added and mixed.

Now ready to use, keep at 4 ° C before use, use within 4 hours.

5.1.7 MuR5S4TR 10.5mg/ml 2.4ml

Take MuR5S4TR stock solution (24 mg/ml) 1.05 ml.

Add 1.35 ml of the above solvent and mix.

Now ready to use, keep at 4 ° C before use, use within 4 hours

5.2 Experimental methods

5.2.1 Cell culture

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.

5.2.2 Cell inoculation method to establish a subcutaneous xenograft model of tumor in nude mice

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.

5.2.3 Group administration

When the tumor volume reached about 200 mm3, 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".

5.2.4 Indicator detection

Animal weight and tumor size were measured twice a week during the experiment. Clinical symptoms were recorded daily. After Day21 measurement, all animals were sacrificed, tumors were removed, tumor weight was weighed and photographed.

All animal experiments are strictly in accordance with Shanghai Medicil Biomedical Co., Ltd. Animal Use and Management Practices. The calculation of tumor-related parameters refers to the Chinese CFDA "Guidelines for Non-Clinical Research Techniques of Cytotoxic Antitumor Drugs" ^[1] .

5.3 Calculation

Calculation of tumor related parameters:

Tumor volume (TV) is calculated as: TV = a × b2 / 2. Where a and b represent the length and width of the tumor measurement, respectively.

The evaluation index of antitumor activity is relative tumor growth rate T/C (%) and tumor inhibition rate (%), and the calculation formulas are: T/C (%) = TRTV / CRTV * 100%. (TRTV: treatment group RTV; CRTV: negative control group RTV); relative tumor volume (RTV), the formula is: RTV = Vt / V0. Where V0 is the tumor volume measured when the cage is administered (ie, Day 0), and Vt is the tumor volume at each measurement.

According to the Chinese CFDA "Guidelines for Non-Clinical Research Techniques of Cytotoxic Antitumor Drugs" (November 2006), T/C (%) ≤ 40% and statistically analyzed P < 0.05 is effective. A dose of the drug is considered to be severely toxic if the body weight of the mouse drops by more than 20% or the number of drug-related deaths exceeds 20%.

6. Data analysis

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.

7. Results

7.1 Effects of various test substances on the body weight of human colon cancer cell line HT-29 in nude mice

During the entire experimental period, 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).

Animals were tolerant to CPT-11 25 mg/kg, and the average body weight of this group increased by 9.23% (ie 2.17 g) over Day 21 compared to Day 0 on the first day of dosing.

Animals were given TRAIL-Mu3 72 mg/kg three times a week for three consecutive weeks. The average body weight of this group increased by 10.62% (ie 2.46) compared to Day0 over Day0. g).

Animals were dosed once every three days for TRAIL-Mu3 93 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 5.83% compared to Day0 (ie, 1.44 grams).

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. .

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 5.62% (ie 1.30 g) compared with Day0. ).

Animals were dosed once every four days for MuR5S4TR 158 mg/kg for three consecutive weeks. During the entire experimental period, compared with Day0, the average body weight of this group increased by 7.60% (ie 1.78 g) compared to Day0. ).

7.2 Effect of each test substance on tumor volume of human colon cancer cell line HT-29 in nude mice

The experimental results are shown in Table 7 and Figure 11.

7.3 Effect of each test substance on tumor weight of human colon cancer cell line HT-29 in nude mice

At the end of the Day 21 experiment, all animals were sacrificed after weighing and tumor volume, tumors were isolated from animals and weighed. The vehicle group, CPT-11 group, TRAIL-Mu3 72 mg/kg three times a week, TRAIL-Mu3 93 mg/kg once every three days, TRAIL-Mu3 108 mg/kg once every four days, MuR5S4TR 105 mg/kg three times a week, MuR5S4TR 135 mg/kg once every three days, MuR5S4TR 158 mg/kg every four days, the average tumor weight was 1.339 g, 0.811 g, 0.898 g, 0.796 g , 0.805 g, 0.936 g, 0.823 g, and 0.812 g. The experimental results are shown in Table 7 and Figure 12.

Table 7. Effect of TRAIL-Mu3 and MuR5S4TR on tumor size in human colon cancer cell line HT-29 nude mice xenograft model

8. Summary

In this experiment, similar to the vehicle group, CPT-11 group, TRAIL-Mu3 72 mg/kg three times a week, TRAIL-Mu3 93 mg/kg once every three days, TRAIL-Mu3 108 mg/kg per four In the once-daily dosing group, MuR5S4TR 105 mg/kg was administered three times a week, MuR5S4TR 135 mg/kg was administered once every three days, and MuR5S4TR 158 mg/kg was administered once every four days, with little effect on animal body weight.

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. MuR5S4TR 105mg/kg three times a week, MuR5S4TR 135mg/kg once every three days, MuR5S4TR 158mg/kg once every four days for human colon cancer cells HT-29 nude mice xenografts A certain inhibitory effect, after administration, the tumor inhibition rate showed significant differences. 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.

9. References

Example 5

Therapeutic effects of different doses of TRAIL-Mu3 and TRAIL-MuR5S4TR on human pancreatic cancer cell line PANC-1 xenograft tumor in nude mice

1. Experimental purpose

In this study, 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.

2. Experimental animals

2.1 Animal species

Mouse.

2.2 varieties

Balb/c nude mouse.

2.3 gender

female.

2.4 quantity

The experiment used 64.

2.5 age

4 to 6 weeks.

2.6 weight

16 ~ 18g ± 20% body weight average.

2.7 animal source (supplier)

2.8 Laboratory Animal Management

2.8.1 Animal identification method

2.8.2 Random grouping

When the tumor volume reached an average of 160 mm3, 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%.

2.8.3 Operation Management Specifications

2.8.4 Feeding conditions

Living conditions: 3 per cage.

Temperature: 20 ° C ~ 26 ° C

Humidity: 40% to 70%

Illumination: 12 hours and nights alternate

2.8.5 feed

2.8.6 Drinking water

City tap water, filtered and autoclaved for drinking.

2.8.7 litter

2.8.8 Adaptation period

3. Experimental materials

3.1 test drugs

Test objects TRAIL-Mu3 and MuR5S4TR information

3.2 cell line

The human pancreatic cancer cell line PANC-1 was purchased from the Institute of Cell Biology, Shanghai Institute of Chinese Academy of Sciences.

3.3 reagent

DMEM medium (GIBCO, USA)

Fetal bovine serum FBS (GIBCO, USA)

Trypsin-EDTA (purchased from GIBCO USA)

Trypan Blue Trypan Blue (purchased from GIBCO USA)

3.4 Instruments

Biosafety cabinet (model: AC2-6E1), purchased from ESCO;

Inverted microscope (model: CKX41SF), purchased from Olympus;

3.5 other

4. Experimental design

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.

Anti-tumor effect of TRAIL-Mu3 and MuR5S4TR in human pancreatic cancer cell line PANC-1 xenograft model

5. Experimental methods

5.1 test drug preparation

5.1.1 Gemcitabine 4mg/ml 2ml

- Gemcitabine stock solution (20mg/ml)

- Gemcitabine (200 mg) was divided into four tubes (each containing 50 mg of gemcitabine). Store in a 4 degree refrigerator.

- Take one of the above-packed gemcitabine (50 mg).

- Add 2.5 ml of physiological saline, mix, dissolve to clarify, and prepare a 20 mg/ml gemcitabine stock solution.

- Take 0.4 ml of gemcitabine stock solution (20 mg/ml).

- Add 1.6 ml of physiological saline and mix.

- Now available.

5.1.2 TRAIL-Mu3 10.8mg/ml 2.4ml

- Solvent: 0.9% saline: water for injection = 1:1

- Take TRAIL-Mu3 stock solution (24mg/ml) 1.08ml,

- Add 1.32 ml of the above solvent and mix.

- Now ready to use, keep at 4 ° C before use, use within 4 hours.

5.1.3 TRAIL-Mu3 9.3mg/ml 2.4ml

- Solvent: 0.9% saline: water for injection = 1:1

- Take TRAIL-Mu3 stock solution (24mg/ml) 0.93ml,

- Add 1.47 ml of the above solvent and mix.

- Now ready to use, keep at 4 ° C before use, use within 4 hours.

5.1.4 TRAIL-Mu3 7.2mg/ml 2.4ml

- Take TRAIL-Mu3 stock solution (24 mg/ml) 0.72 ml.

- Add 1.68 ml of the above solvent and mix.

- Now ready to use, keep at 4 ° C before use, use within 4 hours.

5.1.5 MuR5S4TR 15.8mg/ml 2.4ml

- Solvent: 0.9% saline: water for injection = 1:1

- Take a MuR5S4TR stock solution (24 mg/ml) of 1.58 ml.

- Add 0.82 ml of the above solvent and mix.

- Now ready to use, keep at 4 ° C before use, use within 4 hours.

5.1.6 MuR5S4TR 13.5mg/ml 2.4ml

- Solvent: 0.9% saline: water for injection = 1:1

- Take 1.55 ml of MuR5S4TR stock solution (24 mg/ml).

- Add 1.05 ml of the above solvent and mix.

- Now ready to use, keep at 4 ° C before use, use within 4 hours.

5.1.7 MuR5S4TR 10.5mg/ml 2.4ml

- Take 1.05 ml of MuR5S4TR stock solution (24 mg/ml).

- Add 1.35 ml of the above solvent and mix.

- Now ready to use, keep at 4 ° C before use, use within 4 hours

5.2 Experimental methods

5.2.1 Cell culture

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.

5.2.3 Group administration

After the tumor volume reached 160 mm3, 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".

5.2.4 Indicator detection

All animal experiments are strictly in accordance with Shanghai Medicil Biomedical Co., Ltd. Animal Use and Management Practices. The calculation of tumor-related parameters refers to the Chinese CFDA "Guidelines for Non-Clinical Research Techniques of Cytotoxic Antitumor Drugs" [1].

5.3 Calculation

Calculation of tumor related parameters:

The evaluation index of antitumor activity is relative tumor growth rate T/C (%) and tumor inhibition rate (%), and the calculation formulas are: T/C (%) = TRTV / CRTV * 100%. (TRTV: treatment group RTV; CRTV: negative control group RTV); relative tumor volume (RTV), the formula is: RTV = Vt / V0. Where V0 is the measured tumor volume at the time of sub-cage administration (ie Day0), and Vt is the tumor volume at each measurement.

6. Data analysis

7. Results

7.1 Effects of various test substances on the body weight of human pancreatic cancer cell line PANC-1 in nude mice

During the entire experimental period, 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).

In the group of gemcitabine (40 mg/kg, IV, Day 0, 2, 4), 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).

Animals were administered TRAIL-Mu3 72 mg/kg three times a week for three consecutive weeks. The average body weight of the animals increased by 6.09% (ie 1.36) at Day 21 compared to Day0 over the entire experimental period. g).

Animals were dosed once every three days for TRAIL-Mu3 93 mg/kg. 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 5.76% compared with Day0. 1.35g).

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).

Animals were administered three times a week to MuR5S4TR 105 mg/kg for three consecutive weeks. The average body weight of this group increased by 5.53% (ie 1.24 g) over Day 21 compared to Day0. .

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. ).

Animals were dosed once every four days for MuR5S4TR 158 mg/kg. 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 5.89% (ie 1.33 g) compared with Day0. ).

7.2 Effect of each test substance on tumor volume of human pancreatic cancer cell line PANC-1 in nude mice

MuR5S4TR 105mg/kg three times a week for three weeks, 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. At Day4, 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).

MuR5S4TR 158mg/kg once every four days for three consecutive weeks, 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. At Day4, T/C appeared significantly. The difference was 73.24% (P < 0.001); at Day 21, the T/C was the smallest, 27.02% (P < 0.001).

The experimental results are shown in Table 8 and Figure 13.

7.3 Effect of each test substance on tumor weight of human pancreatic cancer cell line PANC-1 in nude mice

At the end of the Day 21 experiment, all animals were sacrificed after weighing and tumor volume, tumors were isolated from animals and weighed. Solvent group, gemcitabine group (n=7), 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-Mu3108 mg/kg every four days One-time, three-week group, MuR5S4TR 105mg/kg three times a week for three consecutive weeks, MuR5S4TR 135mg/kg once every three days, for three consecutive weeks, MuR5S4TR 158mg/kg once every four days, three consecutive weeks of tumor average The weights were 0.131 g, 0.075 g, 0.042 g, 0.044 g, 0.043 g, 0.047 g, 0.050 g and 0.052 g, respectively. The experimental results are shown in Table 8 and Figure 14.

Table 8. Effect of TRAIL-Mu3 and MuR5S4TR on tumor size in human pancreatic cancer cell PANC-1 nude mouse xenograft model

8. Summary

In this experiment, similar to the vehicle 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 days, TRAIL-Mu3 108 mg/kg every four days One-time, three-week group, MuR5S4TR 105mg/kg three times a week for three consecutive weeks, MuR5S4TR135mg/kg once every three days, three consecutive weeks, MuR5S4TR 158mg/kg once every four days, three weeks in a row, animal weight Almost no effect.

In the gemcitabine group, an animal died on Day 7.

Compared with the vehicle group, 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, For three consecutive weeks, MuR5S4TR 105 mg/kg three times a week for three consecutive weeks, MuR5S4TR135 mg/kg once every three days for three consecutive weeks, MuR5S4TR 158 mg/kg once every four days for three consecutive weeks on human pancreatic cancer cells PANC -1 nude mice xenograft tumors have obvious inhibitory effects, and there is a significant difference in tumor inhibition rate after administration. TRAIL-Mu3 72mg/kg three times a week for three consecutive weeks, TRAIL-Mu3 93mg/kg once every three days, for three consecutive weeks, TRAIL-Mu3 108mg/kg once every four days, for three consecutive weeks between groups There was no difference in efficacy. MuR5S4TR 105mg/kg three times a week for three consecutive weeks, MuR5S4TR 135mg/kg once every three days, for three consecutive weeks, MuR5S4TR158mg/kg every four days, for three consecutive weeks, there was no difference in efficacy between the groups.

9. References

The detailed description of the preferred embodiments of the present invention is not intended to limit the scope of the present invention. Changes are intended to be included within the scope of the invention.

Claims

A method for continuously inhibiting tumor cell growth by a TRAIL-like protein, which is characterized in that it is administered by interval, repetition and full-course drug exposure, that is, prolonging the administration interval and increasing the drug exposure time of the tumor cells in the whole course of treatment The effect of the drug on the whole course of treatment is not attenuated, thereby continuously inhibiting tumor growth.
The method for administering a TRAIL-like protein according to claim 1, wherein the TRAIL-like protein comprises a native or recombinant Apo2L/TRAIL protein extracellular segment 114-281aa, a TRAIL receptor a selective mutant, one or more of a TRAIL transmembrane peptide-like mutant TRAIL-Mu3, TRAIL-MuR5 and TRAIL-MuR6, a TRAIL transmembrane peptide mutein TRAIL-MuR5S4TR and TRAIL-MuR6S4TR, and other mutants, wherein The amino acid sequence of the other mutant has a similarity to the wild type protein of 75% or more.
The method for administering a TRAIL-like protein according to claim 1, wherein the tumor cell is a solid tumor or a bone marrow-derived tumor, and the solid tumor comprises lung cancer, colorectal cancer, breast cancer, One or more of pancreatic cancer, liver cancer, gastric cancer, ovarian cancer, renal cancer, brain tumor, osteochondroma, prostate cancer; the bone marrow-derived tumor includes one or more of leukemia and non-Hodgkin's lymphoma Kind.
The method for continuously inhibiting tumor cell growth by the TRAIL-like protein according to claim 1, characterized in that the continuous inhibition of tumor cell growth comprises in vitro cell-level tumor suppressing action and animal anti-tumor effect in vivo; in vitro, TRAIL The protein acts on the cells in an effective dose range, and observes the inhibition rate of the drug on the tumor cells for 24-96 hours. In different sensitivity tumor cells, the TRAIL protein is at the peak of inhibition in tumor cell growth for 24-72 hours. For highly sensitive cell lines or higher concentration, the peak time of tumor inhibition lasts for 96 hours; in vivo experiments, animal transplanted tumors with different interval administration methods show obvious growth inhibition state within 21 days. The relative tumor growth rate T/C is ≤ 40%.
The method for continuously inhibiting tumor cell growth by the TRAIL-like protein according to claim 1, which is characterized in that the tumor inhibition rate in the tumor cell xenograft model is compared with the treatment of the drug once per day for five days. Increase by at least 20%, and the duration of action is extended by more than 5 days within 21 days of a course of treatment.
The method of administering a TRAIL-like protein according to claim 1 for continuously inhibiting tumor cell growth, characterized in that the administration schedule of the interval, repetition and full-course drug exposure comprises any one of the following:

(1) 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;

(2) 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;

(3) 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;

(4) 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 method of administering a TRAIL-like protein according to claim 1, which is capable of continuously inhibiting the growth of tumor cells, characterized in that the administration interval is prolonged by using a minimum concentration which is higher than the onset of action in vitro and in vivo.
The method for continuously inhibiting tumor cell growth by the TRAIL-like protein according to claim 2, wherein each of the TRAIL-Mu3 group and the MuR5S4TR group has a certain inhibitory effect on human lung cancer NCI-H460 xenograft tumor in nude mice. After the administration, there was a significant difference in the tumor inhibition rate. The anti-tumor effect of the TRAIL-Mu3 and MuR5S4TR drugs administered every other day and three times a week, the three-week administration group was superior to the daily administration for 5 consecutive days. A total of two weeks group; TRAIL-Mu3 and MuR5S4TR two drugs every other day and three times a week, three weeks of treatment group anti-tumor effect is equivalent.
The method for continuously inhibiting tumor cell growth by the TRAIL-like protein according to claim 2, wherein TRAIL-Mu3 is administered at a dose of 72 mg/kg three times a week, and TRAIL-Mu3 is administered at a dose of 93 mg/kg every three days. TRAIL-Mu3 108 mg/kg once every four days, MuR5S4TR 105 mg/kg three times a week, MuR5S4TR 135 mg/kg once every three days, MuR5S4TR 158 mg/kg once every four days Human colon cancer cell line HT-29 has a certain inhibitory effect on xenograft tumors in nude mice. After administration, the tumor inhibition rate is significantly different. TRAIL-Mu3 and TRAIL-MuR5S4TR are administered once every three days and every four groups. The tumor suppressive effect of the once-daily administration group was significantly superior to the three-week administration group, and there was no significant difference in the tumor suppression effect between the three-day administration group and the four-day administration group.
The method for continuously inhibiting tumor cell growth by the TRAIL-like protein according to claim 2, wherein TRAIL-Mu3 72 mg/kg is administered three times a week for three consecutive weeks, and TRAIL-Mu3 93 mg/kg is administered once every three days. For three consecutive weeks, TRAIL-Mu3 108 mg/kg once every four days for three consecutive weeks, MuR5S4TR 105 mg/kg three times a week for three consecutive weeks, MuR5S4TR 135 mg/kg once every three days for three consecutive weeks, MuR5S4TR 158 mg /kg every four days, for three consecutive weeks, the human pancreatic cancer cell PANC-1 nude mice xenograft tumors have obvious inhibitory effect, after the administration, the tumor inhibition rate showed significant difference; TRAIL-Mu3 72mg / Three times per 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, no difference in efficacy between groups for three consecutive weeks; MuR5S4TR 105 mg/kg three times a week for three consecutive weeks, MuR5S4TR 135 mg/kg once every three days, for three consecutive weeks, MuR5S4TR 158 mg/kg once every four days, no difference in efficacy between the groups for three consecutive weeks.