Development and production of small molecule drugs bound to antibody drug conjugates (ADCs)

Abstract

The present invention relates to an antibody-drug conjugate (ADC) comprising an anti-Trop-2 antibody. Compositions comprising such an ADC, and methods for manufacturing and using the same are provided herein. The antitumor effect is superior to that of using adjuvants separately, the expression of various immune-related substances is increased, and the product can be used more stably in an individual. [Index] ADC, antibody-drug conjugate, cell lysate, CpG-ODN.

Description

Technology Field

[0001] The present invention relates to an antitumor composition comprising a lysate of tumor cells that have undergone photodynamic treatment and an immunoadjuvant selected from one or more of CpG-ODN. Background Technology

[0002] The mortality rate caused by malignant tumors, or cancer, has been rapidly increasing in Korea, becoming one of the top three diseases. It is known that approximately 50,000 to 60,000 new cancer patients are diagnosed annually, and the majority of them die as a result. Cancer treatment methods can be broadly classified into surgery, radiation therapy, and chemotherapy. Among these, while radiation therapy is useful for treating almost all cancer patients, its application has been limited due to problems such as the inevitable damage to normal tissues surrounding the tumor. The most important aspect of cancer treatment is to selectively treat only the tumor tissue while minimizing damage to normal tissues, but most existing treatment methods have significant weaknesses in this regard.

[0003] However, recently, photodynamic therapy (PDT) using lasers has been proposed as one of the clinically applicable methods to solve these problems and is known to be relatively effective.

[0004] Accordingly, while conducting research to solve the above-mentioned problems and develop more effective antitumor therapeutic agents, the inventors discovered that by using a composition comprising a photodynamically treated tumor cell lysate and an immunoadjuvant CpG-ODN—rather than a photodynamic treatment in which a photosensitizer and a light source are directly irradiated onto tumor tissue—such a composition not only exhibits a superior antitumor effect compared to the antitumor effects of conventional photodynamic treatment or immunoadjuvants individually, but also solves the above-mentioned problems, thereby completing the present invention. Prior art literature

[0005] US 2014 / 0271640 A1 (Katherine S. Bowdish) 2014,09,18.US 7,696,171 B1 (Michael Blaber) 2010,04,13 The problem to be solved

[0006] The present invention aims to provide an antitumor composition comprising a lysate of photodynamically treated tumor cells and an adjuvant selected from one or more of CpG-ODN. means of solving the problem

[0007] As described below in the specification, the amount of heat shock protein (HSP) 70 in the photodynamically treated tumor cell lysate increased compared to the tumor cell lysate not treated by photodynamic therapy (Fig. 5), and the tumor size decreased by 10 to 25%, exhibiting an antitumor effect (Fig. 6). When the photodynamically treated tumor cell lysate and the immunoadjuvant CpG-ODN were used simultaneously according to the present invention, the tumor size decreased by 28 to 31% compared to when each was used separately, resulting in a significantly increased antitumor effect (Fig. 7). Furthermore, the amount of released CTLs and the expression levels of various cytokines and immunoglobulins increased significantly (Figs. 8 to 12). From these results, it can be seen that the antitumor composition of the present invention exhibits a superior antitumor effect compared to using photodynamic therapy alone or an immunoadjuvant alone, and the antitumor composition of the present invention is also safe for the body (Fig. 13).

[0008] An antitumor composition comprising a photodynamically treated tumor cell lysate of the present invention and one or more adjuvants selected from CpG-ODN, ... can suppress and treat tumors by co-administering to the tumor tissue of a target subject or individual.

[0009] The above antitumor composition is typically administered parenterally (subcutaneously, intramuscularly, intravenously, subcutaneously, or peritoneally), orally, orally, orally, orally, orally, orally. The preferred method of administration is parenteral or transdermal administration.

[0010] The dosage of the above-mentioned antitumor composition may be determined by the method of administration, species, weight, or other standard factors, and such dosage can be easily and readily determined by a person skilled in the art for an effective amount and method of administration.

[0011] The present invention will be explained in more detail through the following examples. These examples are presented merely as illustrative examples to help the invention be better understood, and therefore, the scope of the invention should not be limited by these examples. Effects of the invention

[0012] An antitumor composition comprising a photodynamically treated tumor cell lysate and an immunoadjuvant selected from one or more of CpG-ODNs of the present invention can be effectively used as a tumor treatment agent because it exhibits a superior antitumor effect compared to using the photodynamically treated tumor cell lysate and the immunoadjuvant separately. Brief explanation of the drawing

[0013] Figure 1 is a figure showing the accumulation of the photosensitizer radachlorin in intracellular organelles, A, D, and G showing the distribution of radachlorin in small spot-like shapes around the nucleus, B, E, and H showing the distribution of molecular probes on mitochondria, lysosomes, and Golgi apparatus, respectively, and C, F, and I showing the distribution of radachlorin in the Golgi apparatus, mitochondria, and lysosomes, respectively. Figure 2 shows the growth inhibition effect of CaSki cell lines by photodynamic treatment according to the concentration of the photosensitizer radachlorin and the intensity of the light source. Figure 3 is a figure comparing the growth inhibitory effects of photodynamic treatment and the Adp53 gene when treated separately or in combination with CaSki cell lines. Figure 4 is a figure comparing the expression levels of p53 protein when photodynamic treatment and the Adp53 gene were treated separately or in combination. Figure 5 is a figure comparing the expression levels of p21 protein when photodynamic treatment and the Adp53 gene were treated separately or in combination. Specific details for implementing the invention

[0014] An antitumor composition comprising a photodynamically treated tumor cell lysate and one or more adjuvants selected from CpG-ODN, ... can suppress and treat tumors by co-administering to the tumor tissue of a target subject or individual.

[0015] The above antitumor composition is typically administered parenterally (subcutaneously, intramuscularly, intravenously, subcutaneously, or peritoneally), orally, orally, orally, orally, orally, orally. The preferred method of administration is parenteral or transdermal administration.

[0016] The dosage of the above-mentioned antitumor composition may be determined by the method of administration, species, weight, or other standard factors, and such dosage can be easily and readily determined by a person skilled in the art for an effective amount and method of administration.

[0017] The present invention will be explained in more detail through the following examples. These examples are presented merely as illustrative examples to help the invention be better understood, and therefore, the scope of the invention should not be limited by these examples.

[0018] Example 1: Materials and Analysis Method

[0019] 1-1. Cell Lines and Cell Culture

[0020] The cell line used for the implementation of the present invention is the TC-1 cell line, which was isolated from the lungs of C57BL / 6 mice and induced to cancer by introducing the HPV 16-E7 gene and the c-Ha-ras gene (Lin et al, 1996). The cell culture medium of the TC-1 cell line was prepared by adding 5% fetal bovine serum (FBS) (Gibco BRL), 0.22% sodium bicarbonate (Sigma-Aldrich, Spruce St, St. Louis), 400 mg / L of G418 (Sigma-Aldrich), and streptomycin / penicillin (Gibco BRL) to RPMI-1640 (Gibco BRL, Rockville, MD, USA). This TC-1 cell line was cultured at 37°C in a CO2 incubator.

[0021] 1-2. Photosensitizers and Light Sources

[0022] Radachlorin (RADA-PHARMA Co, Ltd., Moscow, Russia) was used as the photosensitizer, and a 662 nm laser device (Won-PDT D662, Won Technology, Daejeon, Korea) was used.

[0023] Example 2: Measurement of intracellular absorption of intracellular photosensitizers

[0024] 5 × 10⁶ TC-1 cell lines in a 6-well plate containing sterile cover glass 4After seeding into cells / well, the cells were treated with radachlorin at a concentration of 5 µg / ml and cultured for 12 hours. The medium containing radachlorin was removed, and the cells were washed twice with PBS buffer (Gibco BRL). Molecular probes that specifically bind to specific organelles within the cell (Invitrogen Co., Carlsbad, California) were added, and each was cultured separately. Mitochondria were stained with MitoTraker-Green FM (200 nM, 45 min, 37°C), lysosomes with LysoSensor-Green DND-189 (75 nM, 60 min, 37°C), and the Golgi apparatus and endoplasmic reticulum (ER) with NBD C6-sphingomyelin (200 nM, 60 min, 37°C). After staining, the cells were fixed in 1% paraformaldehyde (Sigma-Aldrich) solution for 15 minutes, washed twice with PBS buffer (Gibco BRL), and then prepared as specimens on slide glasses with mounting medium (Dako, Glostrup, Denmark). Fluorescent substances stained on radachlorin and intracellular organelles were confirmed using confocal microscopy (TCS SP2, Leica, Wetzlar, Germany).

[0025] The results are shown in Fig. 1, where radachlorin appeared as small spots around the nucleus (Fig. 1 A, D, G), and molecular probes specific to intracellular organelles indicated mitochondria, lysosomes, and the Golgi apparatus, respectively (Fig. 1 B, E, H). When radachlorin was treated together with organelle-specific molecular probes, it was found that the Golgi apparatus showed the highest uptake of radachlorin (Fig. 1 I), while uptake was also confirmed in mitochondria and lysosomes (Fig. 1 C, F).

[0026] Example 3: Observation of cell growth inhibitory effect after photodynamic treatment of cell lines

[0027] TC-1 cell lines were each placed in 3 × 10⁴ 96-well plates (SPL, Seoul, Korea). 3 After seeding into cells / well, they were cultured for 24 hours. After treating each of the three wells with rhadachlorin at concentrations of 1.25, 2.5, and 5 µg / ml for 12 hours, 6.25 J / Cm 2 The light source was irradiated. In addition, after treating each of the three wells with rhadachlorin at a concentration of 2.5 µg / ml for 12 hours, the three wells were irradiated with 6.25, 12.5, 25, and 50 J / Cm² 2 The light source was irradiated. 20 μl of MTT (5 mg / ml, Sigma-Aldrich) solution was added to each well and incubated at 37°C for 4 hours. After removing the supernatant, DMSO was added at a rate of 100 μl / well and shaken on a shaker for 1 minute. Subsequently, absorbance was measured at 570 nm using an ELISA reader (Spectra Max 340, Molecular Devices, Sunnyvale, CA). MTT assays were performed daily for 5 days.

[0028] 90 to 95% sodium chlorine (sodium chlorin e) 6The growth of TC-1 cells was inhibited depending on the concentration of radachlorin composed of ) and the intensity of the light source ( P <0.05, Figs. 2A and B) (--> In the data you sent, there are three structural forms of ladaclorine. What is the difference between these and the ladaclorine structures other than those in Fig. 1, and how are they used? 3. Ladachlorine is a photosensitizer containing a mixture of these types of chlorine.) After photodynamic treatment with ladaclorine at concentrations of 1.25, 2.5, and 5 µg / ml, the cell growth inhibitory effects on days 1, 2, 3, 4, and 5 were found to increase compared to the control group by 31.5%, 21.7%, 23.0%, 14.8%, and 22.6%, 47.1%, 28.1%, 42.9%, 30.2%, and 30%, respectively, and 80.8%, 98.5%, 98.7%, 80.8%, and 75.7% (Fig. 2A). After treatment with 2.5 µg / ml ladaclorine, 6.25, 12.5, 25, and 50 J / Cm 2 As a result of irradiating with light sources of different intensities, the cell growth inhibitory effect increased depending on the intensity of the light source ( P <0.05) (Fig. 2B). In the group treated with radachlorin or a light source alone, no cell growth inhibition was observed compared to the control group.

[0029] Example 4: Cell death irradiation by photodynamic treatment

[0030] 5 × 10⁴ TC-1 cell lines in a 100mm dish 5 After dispensing into cells / dish, incubate for 24 hours, then treat with 1.25, 2.5, and 5 µg / ml ladaclor, followed by 6.25 J / Cm 2 The cells were irradiated with a light source. After 24 hours, cells were collected after treatment with trypsin / EDTA (Gibco BRL), washed with PBS buffer (Gibco BRL), and then treated with 1 X binding buffer (10 mM Hepes / NaOH, pH 7.4 / 140 mM NaCl / 2.5 mM CaCl2) to reduce the cell count to 1 × 10⁶ 5Cells were prepared. 5 μl of Annexin V-FITC solution (BD, Flanklin Lakes, NJ) and 10 μl of PI solution (100 μg / ml, BD) were added to the prepared cells and mixed well, then reacted for 15 minutes at room temperature in the dark. Afterward, 400 μl of 1 X binding buffer was added, and the cells were analyzed using a flow cytometer (FACS Caliber, BD).

[0031] The rates of early apoptosis in the control group and the photodynamic treatment group were 0.35% and 2.35%, respectively, while the rates of late apoptosis and necrosis were 6.7% and 35.6%, respectively, which increased by 6.7 times and 5.3 times compared to the control group (Fig. 3).

[0032] Example 5: Increase in HSP 70 expression in photodynamically treated tumor cell lysates

[0033] Preparation of photodynamically treated tumor cell lysates

[0034] 1 × 10⁶ TC-1 cells in a 100 mm dish (SPL) 7 After dispensing into cells / dish, cultured for 24 hours. LD 99 To perform photodynamic treatment under these conditions, TC-1 cells were treated with 5 µg / ml of cradachlorin and cultured for 12 hours. After washing with FBS-free culture medium, 6.25 J / Cm² 2 The light source was irradiated. Cells and medium were collected within 48 hours after photodynamic treatment, centrifuged at 800g, and the supernatant was collected, stored at -70℃, and used. Cell lysates to be used as a control were frozen and thawed three times, then centrifuged at 800g, the supernatant was collected, and stored at -70℃ for use.

[0035] HSP70 ELISA and western blot

[0036] Protein quantification by the Bradford method was performed on photodynamically treated tumor cell lysates and cell lysates (control group) that underwent three cycles of freezing and thawing using the BioRad Protein-Assay kit (Bio-Rad Laboratories, Hercules, CA). The expression of HSP 70 was measured using 50 µg of protein and an ELISA kit (Stressgen Biotechnologies, San Diego, CA). Additionally, 25 µg of protein was transferred to a Hybond-ECL membrane (Amersham, Uppsala, Sweden) after electrophoresis on 8% SDS-PAGE. Western blot analysis was performed using pHSP70 and anti-actin (R&D Systems).

[0037] result

[0038] The amount of HSP 70 in the photodynamically treated tumor cell lysate was 0.37 ± 0.06 ng, which was 2.4 times higher than that of the tumor cell lysate prepared by repeated freezing and thawing (control group) (Fig. 4).

[0039] Example 6: Antitumor effect of photodynamically treated tumor cell lysate

[0040] After treating TC-1 cell lines with trypsin-EDTA (Gibco BRL) and collecting the cells, washing twice with sterile PBS buffer (Gibco BRL), then 5 × 10⁶ 6 The solution was prepared at cells / ml and injected 100 μl subcutaneously into the abdomen of 6-week-old C57BL / 6 mice. 10 to 12 mice were used per group, and the tumor size was measured using a caliper at intervals of 2 and 3 days for the long axis (A), short axis (B), and height. The tumor size was calculated as the mean diameter (A+B) / 2 according to the conventional calculation method.

[0041] On days 4, 8, and 13 following the injection of photodynamically treated tumor cell lysates, the tumor sizes of the control group were 10.8 ± 0.24 mm, 12.5 ± 0.25 mm, and 15.8 ± 0.71 mm, respectively; however, the groups injected with tumor cell lysates measured 8.8 ± 0.64 mm, 10.1 ± 0.87 mm, and 13.4 ± 1.14 mm, representing a decrease of 10.6%, 23.0%, and 25.0% compared to the respective control groups (P<0.05, Fig. 5). No tumor growth inhibitory effect was observed in the group injected with freezing-thawing cell lysates prepared by repeated freezing and thawing without photodynamic treatment.

Claims

Claim 1 An antitumor composition comprising photodynamically treated tumor cell lysate and an immunoadjuvant of CpG-ODN. Claim 2 In claim 1, the photodynamic treatment is selected from the group consisting of hematophorphyrin derivative (HpD), Photogem, tin etiopurpurin (SnET2), mono-1-aspartyl chlorin e6 (NPe6), benzoporphyrin derivative (BPD), meso-tetra-(hydroxyphenyl) chlorin (mTHPC), 5-amino levulinic acid (ALA), 5-ALA-induced protoporphyrin IX, radachlorin, ALPcS4 (aluminum tetrasulfophthalocyanine), and TPPS (meso-tetra (sulfonatophenyl) porphine). A composition formed by treating tumor cells with a photosensitizer. Claim 3 A composition according to paragraph 2, wherein the photosensitizer is radachlorin. Claim 4 A composition according to claim 1, wherein the photodynamic treatment involves treating tumor cells with a photosensitizer at a concentration of 3 to 7 μg / ml. Claim 5 In claim 1, the photodynamic treatment is 10 to 60 J / cm² 2 A composition that irradiates a light source onto tumor cells. Claim 6 A composition according to claim 1, wherein the photodynamically treated tumor cell lysate is obtained by centrifuging photodynamically treated tumor cells and taking the supernatant. Claim 7 In claim 1, the composition comprises administering a photodynamically treated tumor cell lysate and a CpG-ODN,... immunoadjuvant together or separately.

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