RU2727059C1 - Method of suppressing growth of tumours with genetically modified version of trail cytokine - Google Patents

Abstract

FIELD: biotechnology; medicine.SUBSTANCE: invention refers to biotechnology and medicine, namely to oncology, and can be used for suppressing tumour growth in patients suffering colorectal cancer. Disclosed is a method for inhibiting the growth of colorectal cancer tumours in vivo by exposing a genetically modified version of the extracellular domain of TRAIL DR5-B recombinant protein, having an amino acid sequence as presented in SEQ ID NO. 1, on tumour-bearing animals, wherein DR5-B selectively binds to DR5 death receptor and causes death of tumour cells by apoptosis.EFFECT: tumour growth inhibition reaches 41-46 % and is stably maintained at a relatively high level during 15to 30day after the beginning of the exposure.1 cl, 5 dwg, 3 tbl, 3 ex

Description

The field to which the invention relates

The invention relates to medicine, pharmacy, namely to oncology, for suppressing the growth of colorectal cancer using a recombinant protein preparation of a modified version of the TRAIL DR5-B cytokine, the target of which is the TRAIL death receptor located on the cell surface (designated in the description as DR5). When DR5-B binds to the DR5 receptor, it causes apoptotic death of tumor cells without damaging normal cells.

State of the art

The cytokine TRAIL (TNF related apoptosis inducing ligand) induces apoptosis in transformed cell lines without affecting normal cells. TRAIL is a polypeptide of 281 amino acid residues and is a type II transmembrane protein, the C-terminus of which is located on the extracellular surface. It has been established that the extracellular domain of the TRAIL ligand can be cleaved by metalloproteinases and present in small amounts in a soluble form in human blood. Like other members of the tumor necrosis factor (TNF) family, the TRAIL protein forms a homotrimeric molecule that binds to the corresponding death receptors, initiating a signaling cascade. The ability of the TRAIL ligand to induce apoptosis mainly in tumor cells, with little effect on normal cells, makes it a potentially valuable candidate for use in the treatment of malignant neoplasms. The possible range of application of antitumor therapy using TRAIL death receptor agonists is very wide, since TRAIL death receptors are expressed in almost all types of tumors.

Currently, at least five TRAIL receptors have been identified, of which two, namely DR4 (death receptor 4, TRAIL-R1) and DR5 (death receptor 5, TRAIL-R2), have the ability to transduce an apoptosis signal, and three other receptors ( TRAIL-R3, TRAIL-R4 and soluble OPG) block TRAIL-mediated apoptosis, which is why they are called "trap receptors".

Despite the fact that the recombinant drug TRAIL (drug name Dulanermin) has shown high antitumor activity in a number of experimental studies, its therapeutic effect in clinical trials was limited to partial responses or disease stabilization. Thus, there is a need for the development of specific DR5 agonists that will induce tumor cell death and suppress tumor growth for the treatment of related diseases.

To date, several DR5-specific mutant TRAIL variants have been obtained in foreign laboratories. A known method of obtaining a mutant variant of TRAIL DR5-8 with six substitutions of amino acid residues, which binds to the death receptor DR5, showing no affinity for the death receptor DR4 (Kelley FR et al. Receptor-selective mutants of Apo2L / TRAIL reveal a greater contribution of DR5 than DR4 to apoptosis signaling J. Biol. Chem., 2005, Vol. 280, pp. 2205-2212). Another mutant variant of TRAIL with two substitutions of amino acid residues (D269H / E195R) with a reduced affinity for the DR4 receptor is known (Van der Sloot AM et al. Designed tumor necrosis factor-related apoptosis-inducing ligand variants initiating apoptosis exclusively via the DR5 receptor. Proc. Natl. Acad. Sci. USA, 2006, V. 103, pp. 8634-8639). The antitumor activity of this variant, tested on the A2780 human ovarian cancer xenograft model in immunodeficient mice, practically did not differ from the wild-type TRAIL preparation. Nevertheless, the preparation of the TRAIL D269H / E195R mutant variant together with cisplatin more effectively inhibited the growth of human tumor xenografts in animals. (Duiker E. W. et al. Enhanced antitumor efficacy of a DR5 specific TRAIL variant over recombinant human TRAIL in a bioluminescent ovarian cancer xenograft model. Clin. Cancer Res. 2009, Vol. 15, pp. 2048-2057).

A disadvantage of the known TRAIL mutant variants is their high affinity for the TRAIL DcR1 and DcR2 trap receptors, which not only do not conduct the apoptosis signal, but also inhibit the cytotoxic activity of the cytokine. Such mutant variants can more effectively inhibit the growth of only those tumors in which the expression of decoy receptors is absent. However, it is known that decoy receptors are expressed in most types of tumor cells. Therefore, to increase the antitumor activity of TRAIL, it is necessary to obtain such mutant variants of TRAIL, which selectively target only the death receptors DR4 or DR5. A known method of obtaining mutant variants of TRAIL with reduced affinity for receptor-traps (O'Leary L. et al. Decoy receptors block TRAIL sensitivity at a supracellular level: the role of stromal cells in controlling tumor TRAIL sensitivity. Oncogene. 2016. Vol. 35 , pp. 1261-1270), which are more effective in killing tumor cells than wild-type TRAIL. The in vivo antitumor activity of these mutants has not been investigated.

There is also known a method for the suppression of colon cancer using synthetic polyvalent peptides that do not have homology with TRAIL b binding to the DR5 receptor (V. PAVET et al. "Multivalent DR5 Peptides Activate the TRAIL Death Pathway and Exert Tumoricidal Activity" Cancer Research, 2010, v. 70, no. 3, p. 1 101-1110, DOI: 10.1158 / 0008-5472). However, the cytotoxic activity of these peptides was inferior to TRAIL in vitro. In addition, in subsequent publications (J. Beyrath et al. Synthetic ligands of death receptor 5 display a cell-selective agonistic effect at different oligomerization levels. Oncotarget. 2016 v. 4, no. 7, p. 64942-64956. Doi: 10.18632 / oncotarget. 10508, Chekkat N et al. Relationship between the agonist activity of synthetic ligands of TRAIL-R2 and their cell surface binding modes. Oncotarget. 2018 v. 17, no. 9, p. 15566-15578. Doi: 10.18632 / oncotarget.24526), the authors who obtained these peptides showed that while TRAIL is capable of effectively killing Jurkat or HCT116 tumor cells, polyvalent peptides do not affect the viability of these cells, since they form a weak complex with the DR5 receptor on the surface of these cells.

The technical problem solved by the present invention is the development of a method of antitumor action based on TRAIL-mediated death of tumor cells, more effective than using known death receptor agonists.

The technical result of the claimed invention is a new method for suppressing the growth of colorectal cancer tumors using a genetically modified version of the TRAIL DR5-B cytokine with increased antitumor activity.

To solve this technical problem and achieve the claimed technical result, it is proposed to use a genetically modified receptor-specific variant of the extracellular domain of the recombinant DR5-B protein containing 6 substitutions of amino acid residues (SEQ ID NO 1).

Previously, the authors obtained a unique variant of TRAIL DR5-B with substitutions of amino acid residues Y189N / R191K / Q193R / H264R / I266L / D269H in the TRAIL protein, which selectively binds to only one of the five TRAIL receptors, the DR5 death receptor (Gasparyan M.E. and etc. A method of obtaining mutant human TRAIL protein (RF Patent No. 2405038 dated November 27, 2010). Studies on tumor cell lines of various origins have shown that the mutant DR5-B variant induces apoptosis much more efficiently compared to wild-type TRAIL, both when used alone and in combination with chemotherapy drugs (Gasparyan M.E. et al. Method for inducing tumor cell death , RF patent No. 2620165, dated 23.05.2017).

The in vivo antitumor efficacy of DR5-B has been demonstrated in the HT116 human subcutaneous colon cancer (RTK) xenograft model in BALB / c nu / nu mice. Highly purified recombinant protein DR5-B was obtained from the E. coli SHuffle B strain as described earlier (Yagolovich A.V. et al. A new effective method for producing the recombinant antitumor cytokine TRAIL and its receptor of the selective DR5-B variant. Biochemistry, 2019, vol. 84 , issue 6, pp. 808-818).

Two modes of exposure have been investigated:

regimen I - DR5-B or control substance was injected 10 times, in the form of two consecutive cycles of daily intravenous injections, 5 injections in each cycle, with an interval of 2 days, total duration of exposure 12 days;

regimen II - DR5-B or the control substance was administered 7 times, twice a week, with an interval of 2 or 3 days, for a total duration of 12 days.

It was shown that after repeated intravenous administration of DR5-B in mode I, at a single dose of 10 mg / kg (total dose of 100 mg / kg), statistically significant inhibition of tumor growth is observed in mice with HCT116 xenograft, compared with the control group of tumor-bearing animals , received in the same mode of injection of saline. The value of inhibition of tumor growth (TPO) is 41-48%. The reproducibility of the antitumor effect was established in three independent experiments. According to the generalized results of independent experiments, repeated intravenous administration of DR5-B at a single dose of 10 mg / kg in regimen I leads to a statistically significant increase in the life expectancy of animals.

Prolongation of exposure by using DR5-B in mode II (single dose 10 mg / kg; total dose 70 mg / kg) leads to a deeper inhibition of the growth of HCT116 xenograft, the TPO value reaches 50-70%. However, no significant increase in the duration of the therapeutic effect is observed. In general, regimens I and II, which differ in the duration of the intervals between drug injections and the duration of exposure, are characterized by a comparable level of the resulting antitumor effect.

It was found that the antitumor effect of exposure depends on the dose of the DR5-B preparation. The optimal single dose for intravenous administration of DR5-B in the human RTK xenograft model HCT116 in BALB / c nu / nu mice is 10 mg / kg.

It has been shown that DR5-B, when administered systemically for 10 days at a single dose of 10 mg / kg, more effectively inhibits the growth of HCT116 xenograft than its unmodified analogue, wild-type TRAIL.

In a comparative study, it was shown that human RTK xenografts obtained using different cell lines differ in sensitivity to the action of DR5-B. Thus, when DR5-B is used at the optimal dose in mode I, xenograft HCT116 is the most sensitive to its action, while xenograft Saco-2 is less sensitive. Xenograft HT-29 can serve as a model of a tumor that is not sensitive to the action of DR5-B.

The invention is illustrated by the following figures:

Figure 1. The effect of DR5-B on the growth of human RTK xenografts HCT116 in BALB / c nu / nu mice after repeated intravenous administration in single doses of 2 mg / kg, 10 mg / kg and 25 mg / kg. Mode I - 10 times introduction; two cycles of daily injections, the duration of each cycle is 5 days; the interval between cycles is 2 days; the total duration of exposure is 12 days. Regimen II - 7-fold administration, twice a week, with an interval between injections of 2 or 3 days; the total duration of exposure is 22 days. Each experimental group consists of 5 animals. Data are presented as arithmetic mean and standard error of the mean.

Figure 2. Lifespan of BALB / c nu / nu mice with HCT116 human RTK xenografts after repeated intravenous administration of DR5-B at single doses of 2 mg / kg, 10 mg / kg and 25 mg / kg. Mode I - 10 times introduction; two cycles of daily injections, the duration of each cycle is 5 days; the interval between cycles is 2 days; the total duration of exposure is 12 days. Regimen II - 7-fold administration, twice a week, with an interval between injections of 2 or 3 days; the total duration of exposure is 22 days.

Figure 3. Dynamics of tumor growth in xenografts of human RTK HCT116 in BALB / c nu / nu mice with multiple intravenous administration of DR5-B at a single dose of 10 mg / kg in regimen I (10-fold administration; two cycles of daily injections, the duration of each cycle - 5 days; interval between cycles - 1 day). Results of three independent experiments. Each experimental group consists of 5-6 animals. Data are presented as arithmetic mean and standard error of the mean.

Figure 4. A - Cytotoxic activity of TRAIL and DR5-B on HCT116 cells after 24 hours of incubation, determined using the MTT test; B - Growth dynamics of human RTK xenografts HCT116 in BALB / c nu / nu mice with multiple intravenous administration of TRAIL and DR5-B in single doses of 10 mg / kg in mode I (10-fold administration; two cycles of daily injections, duration of each cycle - 5 days; interval between cycles - 2 days); C - Inhibition of tumor growth (TPO,%) in the groups of mice treated with TRAIL and DR5-B, relative to the control group of animals that were injected with saline. In in vivo experiments, each group consists of 5 animals. Data are presented as arithmetic mean and standard error of the mean.

Figure 5. A - Cytotoxic activity of TRAIL and DR5-B on human RTK tumor cell lines HCT116, Caco-2 and HT-29. The cells were incubated with ligands at the indicated concentrations for 24 hours, cell survival was determined using the MTT test. B - Dynamics of tumor growth in xenografts of human RTK HCT116, Caco-2 and HT-29 in BALB / c nu / nu mice with multiple intravenous administration of DR5-B at a single dose of 10 mg / kg in regimen I (10-fold administration; two cycles of daily injections, the duration of each cycle is 5 days; the interval between cycles is 2 days). Data are presented as arithmetic mean and standard error of the mean.

Implementation of the invention

The implementation of the claimed invention is illustrated by the following examples 1-3.

Example 1.

Influence of DR5-B on the growth of HCT116 human colon cancer xenografts in BALB / c nu / nu mice after repeated intravenous administration

To obtain a xenograft, cultured human RTK cells of the HCT116 line were used. Cells were cultured in plastic bottles with a surface area of 75 cm ² (Costar, USA) in DMEM medium with L-glutamine (PanEco, Russia) with 10% fetal bovine serum (FBS; PanEco, Russia) under standard culturing conditions (in a humidified atmosphere at a temperature of 37 ° C and 5% atmospheric CO ₂ ).

The cells of passage 8 were used for grafting animals. Immediately before inoculation with animals, the cells were removed from the vials with a Versene solution (PanEko, Russia), washed in a medium without ETS, and transferred into sterile tubes. The cells were sedimented in a CM-6M Elmi centrifuge at 1000 rpm for 10 min. The counting of the number of viable cells was performed in a Goryaev chamber, with the addition of 0.4% trypan blue solution to the cell suspension in a 1: 1 volumetric ratio. Based on the counting results, a cell suspension was prepared with a concentration of 8 × 10 ⁷ cells per ml. The cells were inoculated to animals in Matrigel (BD Matrigel ™ Basement Membrane Matrix, BD Biosciences). For the introduction of animals, 50 μl of the cell suspension (4 × 10 ⁶ cells) was transferred into a cooled test tube containing 50 μl of Matrigel, and after mixing, the cell material (100 μl) was injected subcutaneously on the right side of the animal.

The exposure began on the 6th day of tumor xenograft growth. DR5-B and a control substance were injected intravenously into the mice in the tail vein. The volume of administration of the drug to animals was calculated on the basis of the average body weight of animals in the group, in accordance with the dose of the drug.

We used two modes of exposure:

- mode I: administration is carried out daily for 10 days (with a break of 2 days), in single doses of 2 mg / kg, 10 mg / kg and 25 mg / kg. Control animals in the same regimen are injected with 0.9% sodium chloride solution (saline) in a volume equivalent to the volume of administration of the highest dose. The total duration of exposure is 12 days.

- regimen II: the introduction is carried out twice a week, at intervals of 2 or 3 days, in single doses of 2 mg / kg, 10 mg / kg and 25 mg / kg. Control animals in the same regimen are injected with physiological solution in a volume equivalent to the volume of administration of the highest dose. The total duration of exposure is 22 days.

During the observation period, the general condition of the mice was assessed (daily), the volume of subcutaneous formations (three times a week), the weight of the animals (once a week) were recorded. Tumor size was measured three times a week. The size of the tumor was determined by percutaneous measurement of tumor formations using a caliper in three mutually perpendicular projections. Tumor volume was calculated using the formula: V = d ₁ × d ₂ × d ₃ mm ³ , where d ₁ , d ₂ and d ₃ are three mutually perpendicular diameters of the tumor. According to the measurement results, the average tumor volume in each experimental group and the amount of tumor growth inhibition (TPO) were calculated:

TPO = [(Vk-Vop) / Vk] × 100%, where

Vop - the average tumor volume in animals in the experimental group; Vk is the average tumor volume in animals in the control group.

After intravenous administration of DR5-B to animals at a single dose of 10 mg / kg in regimen I (total dose of 100 mg / kg), statistically significant inhibition of HCT116 xenograft growth was observed. The value of TPO in the period from 8 to 20 days after the start of exposure, in comparison with the group of animals that received in the same mode of injection of saline, averaged 41-48% (Fig. 1, Mode I). With a decrease in a single dose of the drug to 2 mg / kg (total dose 20 mg / kg), the severity of the effect in regimen I significantly decreased, and an increase in a single dose to 25 mg / kg (total dose 250 mg / kg) did not have an inhibitory effect on growth tumors in animals.

With intravenous administration of DR5-B at a single dose of 10 mg / kg in regimen II (total dose 70 mg / kg), a tendency towards deeper inhibition of tumor growth was observed. The value of TPO in the period from 6 to 15 days after the start of exposure, in comparison with the group of animals that received injections of saline in the same mode, reached 50-70%. However, an increase in the total duration of exposure, compared with mode I, did not lead to a significant prolongation of the antitumor effect (Fig. 1, Mode II). When using this mode of exposure, a dependence of the antitumor effect on the dose of DR5-B was also observed.

Repeated intravenous administration of DR5-B preparation was satisfactorily tolerated by the animals. There were no reactions to the drug administration.

Thus, on the HCT116 human RTK xenograft model in immunodeficient mice, the optimal single dose of DR5-B when administered intravenously is 10 mg / kg. Regimes I and II, differing in the duration of the intervals between the injections of DR5-B and the duration of exposure, are characterized by a comparable level of the resulting antitumor effect.

The lifespan of BALB / c nu / nu mice with HCT116 xenografts in the experimental and control groups was traced to the endpoint of observation, which was determined by the state of the tumor-bearing animals. The conditions for immediate euthanasia were: weight reduction of the animal by more than 20%; an increase in the transverse size of the tumor up to 18 mm; the appearance of severe necrosis or ulceration on the surface of the tumor. The observation results show that the lifespan of the mice treated with intravenous injections of DR5-B in regimen I exceeds the lifespan of animals in the control group (Fig. 2). According to the generalized results of three independent experiments, the average lifespan of mice in the groups receiving DR5-B was 45.2 ± 9.6 days from the time of inoculation of tumor cells, in the control group of animals - 34.5 ± 7.3 days (p = 0.0016, Student's t-test).

The antitumor activity of DR5-B when administered intravenously in regimen I at a single dose of 10 mg / kg on the HCT116 xenograft model was confirmed in three independent experiments performed at different times (Fig. 3).

Thus, systemic repeated administration of DR5-B at a single dose of 10 mg / kg (total dose 100 mg / kg) reproducibly leads to a moderately pronounced, but statistically significant inhibition of the growth of subcutaneous human RTK xenograft HCT116 in immunodeficient mice. The TPO value varies in the range of 42-49% and is kept at the achieved level for at least two weeks after the end of the treatment of animals.

Example 2.

Comparative analysis of the effect of TRAIL and DR5-B on human PTK cells HCT116 in vitro and the growth of HCT116 xenografts in vivo in BALB / c nu / nu mice.

After removing the culture from cryostorage and switching to the mode of increasing the cell mass, the HCT116 cell line shows stable growth: the time for doubling the cell mass is about 24 hours, the frequency of reseeding is 2 times a week, the number of cells in a vial with a surface area of 75 cm ² upon reaching a confluent monolayer - from 11 × 10 ⁶ to 13 × 10 ⁶ . To study the biological activity of the preparations, the HCT116 colorectal carcinoma cell line was cultivated in a DMEM nutrient medium containing 10% (v / v) fetal bovine serum at 37 ° C in an atmosphere of 5% CO ₂ . Cells are transferred to sterile 96-well plates (1 x 10 ⁵ cells per well for HCT116 and incubated with TRAIL (SEQ ID NO 2) and DR5-B (SEQ ID NO 1) preparations for 24 hours. MTT is added to determine cell viability (3- [4,5-dimethylthiazole-2] -2,5-diphenyl tetrazolium bromide) at a final concentration of 0.5 mg / ml and incubated for 3 hours at 37 ° C. The plates are centrifuged at 3000 rpm (" ELMI ", Latvia) for 5 min, the supernatant is removed, dimethyl sulfoxide (DMSO) is added to each well, and the optical density of the well contents is measured using an iMark plate spectrophotometer (Bio-Rad, USA) at a wavelength of 450 nm with background subtraction at 655 nm The level of culture growth inhibition (IR) is calculated by the formula:

IR (%) = [(ODk-ODo) / ODk] * 100%, where ODo and ODk are the average optical density of the formazan solution in the test and control wells, respectively.

To calculate the IC ₅₀ value (the concentration of the test substance at which 50% inhibition of cell culture growth in vitro is observed), a growth curve of the dependence of inhibition of cell culture proliferation on the drug concentration is constructed.

As a result of the studies, it was found that the introduction of DR5-B into the culture medium at concentrations from 1 ng / ml to 500 ng / ml leads to a biologically significant inhibition of the growth of the HCT116 culture. The maximum inhibition of proliferation for DR5-B is 79%, and for TRAIL 72% (Fig. 4). Under these conditions, the IC ₅₀ for DR5-B is 0.886 ± 0.033 ng / ml, while for TRAIL it is 23.004 ± 0.3 ng / ml, which is 28.75 times higher than the IC ₅₀ calculated for DR5-B. Thus, DR5-B more effectively leads to the death of the HCT116 cell as compared to TRAIL.

Comparative analysis of the antitumor activity of TRAIL and DR5-B was carried out on the HCT116 xenograft model in BALB / c nu / nu mice. The procedure for preparing the model and evaluating the results are described in example 1. The test substances were administered to animals at a dose of 10 mg / kg for 10 days, every day with a break of 2 days after the fifth injection. It was shown that, compared to TRAIL, DR5-B more effectively inhibits the growth of HCT116 xenograft (Fig. 4). In the group of tumor-bearing mice treated with DR5-B, the maximum TPO value varied in the range of 41-46%, and at the end of observation (day 33 from the start of exposure) it was 33%. In the group of mice that received TRAIL at the same dose and in the same regimen, the maximum TPO value did not exceed 25%, and at the end of the observation period was 8%.

Thus, the totality of data obtained in vitro and in vivo indicates that the antitumor activity of the TRAIL cytokine significantly increases with the introduction of amino acid substitutions Y189N / R191K / Q193R / H264R / I266L / D269H, which increase the selectivity of the protein to the DR5 receptor.

Example 3

Comparative study of the sensitivity of human RTK cells HCT116, HT-29 and Caco-2 in vitro and model tumors HCT116, HT-29 and Caco-2 in vivo to the action of DR5-B.

Human RTK cells HCT116, HT-29, and Caco-2 are cultured in plastic flasks with a surface area of 75 cm ² (Costar, USA) in DMEM medium with L-glutamine (PanEko, Russia) supplemented with 10% fetal calf serum (FBS; PanEco , Russia) under standard cultivation conditions (in a humidified atmosphere at a temperature of 37 ° C and 5% atmospheric CO ₂ ).

The cytotoxic activity of the test substances is determined using the MTT test as described in Example 2.

The results of in vitro testing show that HCT116 cells are sensitive to both TRAIL and DR5-B, however, the effective concentrations of DR5-B are 30 times less than the effective concentrations of TRAIL (Fig. 5A). Cells of the Caco-2 line, as well as HT-29 practically do not die under the influence of TRAIL, while DR5-B at concentrations above 100 ng / ml inhibits the growth of Caco-2 cells by 60%, HT-29 cells - by 27% (Fig. . 5A).

The study of the antitumor activity of DR5-B in vivo was carried out using models of HCT116, HT-29 and Caco-2 xenografts in BALB / c nu / nu mice. The preparation of model HCT116 is described in Example 1. To obtain a Caco-2 xenograft, tumor cells were inoculated subcutaneously in mice in Matrigel, in an amount of 5 × 10 ⁶ cells per mouse, according to the same procedure. HT-29 cells (3 × 10 ⁶ cells per mouse) were inoculated subcutaneously, on the right side, in 100 μl of DMEM medium without fetal calf serum.

On the HCT116 and HT-29 models, the effect was started on the 6th day of tumor growth, on the Caco-2 model, on the 14th day of tumor growth. DR5-B and a control substance were injected intravenously into the mice in the tail vein. The introduction was carried out in mode I (see Example 1) in two cycles of 5 days each, with a break of 2 days (for HCT116 and HT-29) or 1 day (for Saco-2). A single dose of DR5-B was 10 mg / kg. Control animals in the same regimen were injected with 0.9% sodium chloride solution (saline) in a volume equivalent to the volume of DR5-B administration.

Tumors HCT-116 and HT29 were measured twice a week, tumors Caco-2 - once a week. The size of the tumors was determined as described in Example 1. During the observation time, the general condition of the mice was assessed (daily), the volume of subcutaneous formations was recorded (twice a week for HCT116 and HT-29; once every 7-10 days for Caco-2) , weight of animals (once a week).

The results of evaluating the dynamics of growth of HCT116, HT-29 and Caco-2 tumor xenografts in mice in the experimental groups receiving a course of treatment with DR5-B and in animals in the control groups that were injected with saline in the same regimen are shown in Figure 5B. The duration of observation of animals with HCT116 and NT-29 xenografts from the beginning of exposure was 30 days (after the completion of the exposure course - 18 days). Observation of animals with Saco-2 xenografts from the beginning of exposure was 68 days (after completion of the course of exposure - 57 days).

On the HCT116 xenograft model, the inhibition of tumor growth with repeated systemic administration of DR5-B reached 35-37% and was stably kept at a relatively high level in the period from 15 to 30 days after the start of exposure (Table 1). In the group of mice with Caco-2 xenograft treated with DR5-B, compared with the control group of animals, there was a weak tendency to inhibition of tumor growth (Fig. 5B). The value of TPO did not exceed 30%, and this tendency in this model was manifested after the completion of the course of exposure and was maintained until the end of observation of the animals (Table 2).

On the NT-29 xenograft model, a slight and short-term delay in tumor growth was observed 8-10 days after the start of exposure (TPO 20-30%), however, in general, the dynamics of tumor growth in the experimental and control groups of mice differed insignificantly (Table 3).

Thus, among the studied variants of human RTK models in immunodeficient mice, xenograft HCT116 is the most sensitive to the action of DR5-B in the applied mode of exposure, and xenograft Saco-2 is less sensitive. Xenograft HT-29 can serve as a model of a tumor resistant to DR5-B action.

SEQ ID NO 1.

SEQ ID NO 2.

Claims (1)

A method for inhibiting the growth of colorectal cancer tumors in vivo by exposure to a genetically modified variant of the extracellular domain of the recombinant TRAIL DR5-B protein having the amino acid sequence as set forth in SEQ ID NO. 1, wherein DR5-B selectively binds to the death receptor DR5 and causes the death of tumor cells by apoptosis.

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