KR20230092466A - composition comprising PD-L1-inhibiting prodrug for the prevention or treatment of cancer - Google Patents
composition comprising PD-L1-inhibiting prodrug for the prevention or treatment of cancer Download PDFInfo
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- KR20230092466A KR20230092466A KR1020210181877A KR20210181877A KR20230092466A KR 20230092466 A KR20230092466 A KR 20230092466A KR 1020210181877 A KR1020210181877 A KR 1020210181877A KR 20210181877 A KR20210181877 A KR 20210181877A KR 20230092466 A KR20230092466 A KR 20230092466A
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Abstract
본 발명은 암세포의 면역원성 세포사멸 및 T 세포에 대한 자가 인식 과정 억제를 유도하는 암 예방 또는 치료 약학 조성물에 관한 것으로서, EPR(Enhanced Permeability and Retection) 효과를 통해 종양에 효과적으로 축적할 수 있으며, 암 전달 효율을 높여 정상조직에 대한 부작용은 최소화하면서 암을 효과적으로 예방 또는 치료할 수 있다.The present invention relates to a pharmaceutical composition for preventing or treating cancer, which induces immunogenic apoptosis of cancer cells and suppresses self-recognition process for T cells, and can effectively accumulate in tumors through EPR (Enhanced Permeability and Retection) effect, and can effectively accumulate in cancer cells. By increasing the delivery efficiency, it is possible to effectively prevent or treat cancer while minimizing side effects on normal tissues.
Description
본 발명은 암세포의 면역원성 세포사멸 및 T 세포에 대한 자가 인식 과정 억제를 유도하는 암 예방 또는 치료 약학 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for preventing or treating cancer, which induces immunogenic apoptosis of cancer cells and suppresses self-recognition process for T cells.
면역 관문 차단(Immune checkpoint blockade) 및 화학요법 (Chemotherapy)의 병용 요법을 통해 많은 완치 사례가 보고되면서, 암 치료를 위한 새로운 패러다임으로 급부상하고 있다. 병용 요법은 암세포와 T 세포 사이의 상호 작용에 관여하는 면역관문치료제와 항암제가 사용되었고, 이들의 조합을 통해 매우 우수한 항암 면역 치료가 가능하다는 것이 여러 임상 시험을 통해 밝혀졌다. 상기 면역관문치료제로는 PD-L1(Programmed death-ligand 1), PD-1(Programmed death-receptor) 및 CTLA-4(Cytotoxic T lymphocyte associated protein 4)에 선택적으로 결합하는 단일클론항체(Monoclonoal antibody)가 주로 이용되었고, 항암제로는 암세포의 면역원성 세포사멸을 유도할 수 있는 Doxorubicin, Paclitaxel, Oxaliplatin이 이용되었다. As many cases of complete cure have been reported through combination therapy of immune checkpoint blockade and chemotherapy, it is rapidly emerging as a new paradigm for cancer treatment. In combination therapy, immune checkpoint therapy and anticancer drugs involved in the interaction between cancer cells and T cells were used, and it was revealed through several clinical trials that excellent anticancer immunotherapy is possible through these combinations. The immune checkpoint therapeutic agent is a monoclonal antibody that selectively binds to PD-L1 (Programmed death-ligand 1), PD-1 (Programmed death-receptor) and CTLA-4 (Cytotoxic T lymphocyte associated protein 4) was mainly used, and as anticancer agents, Doxorubicin, Paclitaxel, and Oxaliplatin, which can induce immunogenic apoptosis of cancer cells, were used.
그러나 상기 병용 요법은 치료효과가 우수한 만큼, 그 부작용 또한 매우 위험하다는 치명적 단점이 있다. 특히 단일클론항체의 면역원성으로 인해 대부분의 장기에 치명적이고 예상하기 힘든 면역 관련 부작용(Immune-related-adverse events)이 유도되며, 항암제 역시 암 선택성이 매우 낮아 정상세포에서도 독성을 나타내어 심각한 장기 손상을 나타내는 부작용이 있다. 또한, 서로 다른 약물의 병용 투여로 인하여 환자부담비용이 높고, 전신 면역 억제 효과 때문에 면역 치료의 효능도 현저히 저하된다는 문제가 있다.However, the combination therapy has a fatal disadvantage that its side effects are also very dangerous as much as the therapeutic effect is excellent. In particular, the immunogenicity of monoclonal antibodies induces fatal and unpredictable immune-related-adverse events in most organs, and anticancer drugs also have very low cancer selectivity and are toxic to normal cells, causing serious organ damage. There are side effects that are indicated. In addition, due to the combined administration of different drugs, there is a problem that the patient burden is high and the efficacy of immunotherapy is significantly reduced due to the systemic immunosuppressive effect.
또한 단일클론항체와 항암제는 모두 종양에 전달되는 효율이 매우 낮기 때문에, 두 약물을 동시에 효과적으로 종양에 전달하여 면역 치료 효능을 극대화할 수 있는 새로운 치료법의 개발이 절실히 요구되고 있는 실정이다.In addition, since the delivery efficiency of both monoclonal antibodies and anticancer drugs to tumors is very low, there is an urgent need for the development of new therapies capable of maximizing the efficacy of immunotherapy by simultaneously and effectively delivering both drugs to tumors.
본 발명은 상기와 같은 문제점을 해결하기 위하여 안출된 것으로, 본 발명의 목적은 암세포의 면역원성 세포사멸 및 T 세포에 대한 자가 인식 과정의 억제를 유도할 수 있는 암 예방 또는 치료용 약학 조성물을 제공하고자 하는 것이다.The present invention has been made to solve the above problems, and an object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer that can induce immunogenic apoptosis of cancer cells and suppression of self-recognition process for T cells. It's what you want to do.
본 발명은 상기 목적을 이루기 위하여, 서열번호 1로 표시되는 펩타이드의 일 말단에 항암제가 결합된 접합체를 포함하는 전구약물을 유효성분으로 함유하는 암의 예방 또는 치료용 약학 조성물을 제공한다.In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating cancer containing, as an active ingredient, a prodrug comprising a conjugate in which an anticancer agent is bound to one end of the peptide represented by SEQ ID NO: 1.
상기 접합체의 분자량은 1 내지 5 kDa일 수 있다.The molecular weight of the conjugate may be 1 to 5 kDa.
상기 펩타이드는 암세포에서 과발현되는 카텝신 B에 의해 절단 가능한 것일 수 있다.The peptide may be cleavable by cathepsin B, which is overexpressed in cancer cells.
상기 항암제는 상기 펩타이드에 직접 또는 링커를 통해 공유적으로 결합된 것일 수 있다.The anticancer agent may be covalently bound to the peptide directly or through a linker.
상기 전구약물은 용액 상에서 자기조립에 의해 구형의 나노입자를 형성하는 것일 수 있다.The prodrug may form spherical nanoparticles by self-assembly in a solution.
상기 나노입자의 평균 직경은 100 내지 200 nm일 수 있다.The average diameter of the nanoparticles may be 100 to 200 nm.
상기 나노입자는 용액 상에서 30 내지 40일 동안 응집 또는 구조적 변형없이 장기간 보관이 가능한 것일 수 있다.The nanoparticles may be stored for a long period of time without aggregation or structural transformation for 30 to 40 days in a solution.
상기 항암제는 독소루비신(doxorubicin), 사이클로포스파아마이드(cyclophosphamide), 메클로레타민(mecholrethamine), 우라무스틴(uramustine), 멜파란(melphalan), 클로라부실(chlorambucil), 이포스파미드(ifosfamide), 벤다무스틴(bendamustine), 카르무스틴(carmustine), 로무스틴(lomustine), 스트렙토조신(streptozocin), 부설판(busulfan), 다카바진(dacarbazine), 테모졸로마이드(temozolomide), 티오테파(thiotepa), 알트레타민(altretamine), 듀오카르마이신(duocarmycin), 시스플라틴(cisplatin), 카르보플라틴(carboplatin), 네다플라틴(nedaplatin), 옥사리플라틴(oxaliplatin), 사트라플라틴(satraplatin), 트리플라틴 테트라나이트레이트(triplatin tetranitrate), 5-플루오로우라실(5-fluorouracil), 6-머캅토퓨린(6-mercaptopurine), 카페시타빈(capecitabine), 클라드리빈(cladribine), 클로파라빈(clofarabine), 시스타르빈(cystarbine), 플록스유리딘(floxuridine), 플루다라빈(fludarabine), 겜시타빈(gemcitabine), 하이드록시우레아(hydroxyurea), 메토트렉세이트(methotrexate), 페메트렉세드(pemetrexed), 펜토스타틴(pentostatin), 티오구아닌(thioguanine), 캠토테신(camptothecin), 토포테칸(topotecan), 이리노테칸(irinotecan), 에토포사이드(etoposide), 테니포시드(teniposide), 미토산트론(mitoxantrone), 파클리탁셀(paclitaxel), 도세탁셀(docetaxel), 이자베필론(izabepilone), 빈블라스틴(vinblastine), 빈크리스틴(vincristine), 빈데신(vindesine), 비노렐빈(vinorelbine), 에스트라머스틴(estramustine), 메이탄신(maytansine), DM1 (mertansine, 메르탄신), DM4, 돌라스타틴(dolastatin), 아우리스타틴 E(auristatin E), 아우리스타틴 F(auristatin F), 모노메틸 아우리스타틴 E(monomethyl auristatin E), 모노메틸 아우리스타틴 F(monomethyl auristatin F) 및 이들의 유도체로 이루어진 군으로부터 선택되는 어느 하나 이상일 수 있다.The anticancer agent is doxorubicin, cyclophosphamide, mecholrethamine, uramustine, melphalan, chlorambucil, ifosfamide , bendamustine, carmustine, lomustine, streptozocin, busulfan, dacarbazine, temozolomide, thiotepa ), altretamine, duocarmycin, cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, triple Triplatin tetranitrate, 5-fluorouracil, 6-mercaptopurine, capecitabine, cladribine, clofarabine ), cystarbine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, pentostatin (pentostatin), thioguanine, camptothecin, topotecan, irinotecan, etoposide, teniposide, mitoxantrone, paclitaxel ), docetaxel, izabepilone, vinblastine, vincristine, vindesine, vinorelbine, estramustine, maytansine ), DM1 (mertansine), DM4, dolastatin, auristatin E, auristatin F, monomethyl auristatin E, monomethyl It may be any one or more selected from the group consisting of auristatin F (monomethyl auristatin F) and derivatives thereof.
상기 약학 조성물은 정맥 주사로 투여되는 것일 수 있다.The pharmaceutical composition may be administered intravenously.
상기 암은 위암, 폐암, 비소세포성 폐암, 유방암, 난소암, 간암, 기관지암, 비인두암, 후두암, 췌장암, 방광암, 결장암, 자궁경부암, 골암, 비소세포성 골암, 혈액암, 피부암, 두부 또는 경부 암, 자궁암, 직장암, 항문 부근암, 결장암, 나팔관암, 자궁내막암, 질암, 음문암, 호지킨병(Hodgkin's disease), 식도암, 소장암, 내분비선암, 갑상선암, 부갑상선암, 부신암, 연조직 육종, 요도암, 음경암, 전립선암, 만성 또는 급성 백혈병, 림프구 림프종, 신장 또는 수뇨관암, 신장세포 암종, 신장골반암종, 침샘암, 육종암, 가성점액종, 간모세포종, 고환암, 교모세포종, 구순암, 난소생식세포종양, 기저세포암, 다발성골수종, 담낭암, 맥락막흑색종, 바터팽대부암, 복막암, 설암, 소세포암, 소아림프종, 신경모세포종, 십이지장암, 요관암, 성상세포종, 수막종, 신우암, 외음부암, 흉선암, 중추신경계(central nervous system, CNS) 종양, 1차 중추신경계 림프종, 척수종양, 뇌간 신경교종 및 뇌하수체 선종으로 이루어진 군으로부터 선택되는 어느 하나 이상일 수 있다.The cancer is gastric cancer, lung cancer, non-small cell lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colon cancer, cervical cancer, bone cancer, non-small cell bone cancer, blood cancer, skin cancer, head or Cervical cancer, uterine cancer, rectal cancer, perianal cancer, colon cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue Sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney or ureteric cancer, renal cell carcinoma, renal pelvic carcinoma, salivary gland cancer, sarcoma cancer, pseudomyxoma, hepatoblastoma, testicular cancer, glioblastoma, labrum Cancer, ovarian germ cell tumor, basal cell carcinoma, multiple myeloma, gallbladder cancer, choroidal melanoma, ampulla of Vater cancer, peritoneal cancer, tongue cancer, small cell cancer, juvenile lymphoma, neuroblastoma, duodenal cancer, ureteral cancer, astrocytoma, meningioma, renal pelvic cancer, It may be any one or more selected from the group consisting of vulvar cancer, thymus cancer, central nervous system (CNS) tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma.
본 발명에 따른 약학 조성물은 EPR(Enhanced Permeability and Retection) 효과를 통해 종양에 효과적으로 축적할 수 있으며, 면역관문치료제와 항암제의 암 전달 효율을 높여 정상조직에 대한 부작용은 최소화하면서 암을 효과적으로 예방 또는 치료할 수 있다.The pharmaceutical composition according to the present invention can effectively accumulate in tumors through the EPR (Enhanced Permeability and Retection) effect, and can effectively prevent or treat cancer while minimizing side effects to normal tissues by increasing the cancer delivery efficiency of immune checkpoint therapeutics and anticancer drugs. can
도 1은 본 발명에 따른 전구약물(PD-NPs)의 작동원리를 개략적으로 나타낸 도면이다.
도 1a는 본 발명에 따른 전구약물(PD-NPs)의 화학구조 및 작동원리를 도시한 것이고, 도 1b는 본 발명에 따른 전구약물(PD-NPs)을 정맥을 통해 체내로 주사하였을 때, EPR(Enhanced Permeability and Retection) 효과를 통해 종양에 효과적으로 축적되고, 서열번호 1의 펩타이드를 통해 암세포 표면의 PD-L1에 결합하는 과정을 도시한 것이다.
도 1c는 본 발명에 따른 전구약물(PD-NPs)이 암세포 내에 존재하는 카텝신 B 효소에 의해 항암제를 방출하여 면역원성 세포사멸(ICD)을 유도하는 전반적인 과정을 도시한 것이다.
도 1d는 본 발명에 따른 전구약물(PD-NPs)에 의한 면역원성 세포사멸 및 면역 관문 차단에 의해 암 조직에 많은 면역 세포를 모집하여 항암 면역 치료 효과를 유도하는 전반적인 과정을 도시한 것이다.
도 2는 실시예 1에 따른 전구약물(PD-NPs)의 합성 과정을 개략적으로 도시한 것이다.
도 3은 실시예 1을 통해 합성된 전구약물(PD-NPs)에 대한 HPLC 결과 그래프이다.
도 4는 실시예 1을 통해 합성된 전구약물(PD-NPs)에 대한 MALDI-TOF 결과 그래프이다.
도 5는 용액 상에서 실시예 1로부터 제조된 전구약물(PD-NPs)을 동적광산란(DLS)(좌) 및 투과전자현미경(TEM)(우)으로 측정한 결과이다.
도 6은 용액 상에서 실시예 1로부터 제조된 전구약물(PD-NPs)과 비교예 1로부터 제조된 전구약물을 분산시킨 후, 시간별로 동적광산란(DLS)으로 측정하여 나타낸 그래프이다.
도 7은 실시예 1로부터 제조된 전구약물(PD-NPs)를 카텝신 B 효소 또는 Cathepsin B 억제 siRNA 함께 0시간, 6시간 또는 24시간 배양한 후, 이를 HPLC 크로마토그래피로 분석한 그래프이다.
도 8은 실시예 1로부터 제조된 전구약물(PD-NPs)를 다양한 효소(cathepsin E, cathepsin D, caspase-9, caspase-3)와 함께 24시간 배양한 후, HPLC 크로마토그래피로 분석한 결과이다.
도 9는 실시예 1로부터 제조된 전구약물(PD-NPs)을 처리한 유방암세포(4T1)와 PD-L1 봉쇄세포(aPD-L1 Ab pre-treated)의 공초점 레이저 스캐닝 현미경(CLSM) 결과이다.
도 10은 실시예 1의 전구약물(PD-NPs)을 처리한 유방암세포(4T1)에서의 리소좀(Lysosome)과의 공동화(Co-localization)를 공초점 레이저 스캐닝 현미경(CLSM)으로 분석한 결과이다.
도 11은 실시예 1의 전구약물(PD-NPs) 또는 독소루비신(DOX)을 처리한 암세포(4T1)와 정상세포(H9C2)의 공초점 레이저 스캐닝 현미경(CLSM) 결과이다.
도 12는 농도별 실시예 1의 전구약물(PD-NPs)을 처리한 암세포(4T1) 혹은 정상세포(H9C2)의 생존율(cell viability, %)을 나타낸 그래프이다.
도 13은 농도별 독소루비신(DOX)만을 처리한 암세포(4T1) 혹은 정상세포(H9C2)의 생존율(cell viability, %)을 나타낸 그래프이다.
도 14a는 실시예 1의 전구약물(PD-NPs), 독소루비신(DOX) 또는 생리식염수(con)로 처리한 암세포를 DAPI와 APC-conjugated CRT 항체로 염색하고 공초점 형광 현미경(Confocal fluorescence microscope)으로 촬영한 사진이다.
도 14b는 실시예 1의 전구약물(PD-NPs), 독소루비신(DOX) 또는 생리식염수(con)로 처리한 암세포로부터 방출되는 HMGB1 발현량 및 ATP 발현량을 측정하여 나타낸 그래프이다.
도 15는 실시예 1의 전구약물(PD-NPs), PD-L1 단일클론항체(aPD-L1 Ab), PD-L1 결합 펩타이드(aPD-L1 Pep)를 처리한 각 암세포(4T1)를 T 세포와 공배양시 배양액에서 관측되는 INF-γ의 농도를 ELISA로 측정한 결과를 나타낸 그래프이다.
도 16은 독소루비신, 실시예 1의 전구약물(PD-NPs) 또는 비교예 1의 전구약물을 정맥 투여한 암 동물모델(1군, 2군, 3군)에 대한 NIRF(noninvasive near-infrared fluorescence) 이미지이다.
도 17은 도 16의 NIRF 이미지에서 종양 조직에서의 형광을 정량적으로 분석하여 나타낸 그래프이다.
도 17은 도 16의 각 그룹의 형광 이미지의 종양 내 형광 세기를 정량적으로 분석한 그래프이다.
도 18은 1군 내지 6군에서 암의 부피(V; mm3) 시간별로 측정하여 나타낸 그래프이다.
도 19는 1군 내지 6군에서 시간별로 체중을 측정하여 나타낸 그래프이다.
도 20은 1군 내지 6군에서 시간별로 생존율을 측정하여 나타낸 그래프이다.
도 21은 1군 내지 6군으로부터 적출한 암 조직 내에서의 T 세포 비율을 유세포 분석한 결과이다.1 is a diagram schematically showing the operating principle of prodrugs (PD-NPs) according to the present invention.
Figure 1a shows the chemical structure and operating principle of the prodrug (PD-NPs) according to the present invention, and Figure 1b shows the EPR when the prodrug (PD-NPs) according to the present invention is injected into the body through a vein. It shows the process of effectively accumulating in tumors through the (Enhanced Permeability and Retection) effect and binding to PD-L1 on the surface of cancer cells through the peptide of SEQ ID NO: 1.
Figure 1c shows the overall process of inducing immunogenic cell death (ICD) by releasing an anticancer drug by the cathepsin B enzyme present in cancer cells by the prodrug (PD-NPs) according to the present invention.
Figure 1d shows the overall process of inducing anticancer immunotherapeutic effect by recruiting many immune cells to cancer tissue by immunogenic cell death and immune checkpoint blockade by prodrugs (PD-NPs) according to the present invention.
2 schematically illustrates the synthesis process of prodrugs (PD-NPs) according to Example 1.
3 is a graph of HPLC results for prodrugs (PD-NPs) synthesized in Example 1.
4 is a graph of MALDI-TOF results for prodrugs (PD-NPs) synthesized in Example 1.
5 is a result of measuring the prodrug (PD-NPs) prepared in Example 1 in solution by dynamic light scattering (DLS) (left) and transmission electron microscopy (TEM) (right).
6 is a graph showing the dispersion of the prodrug (PD-NPs) prepared in Example 1 and the prodrug prepared in Comparative Example 1 in a solution and then measured by dynamic light scattering (DLS) over time.
7 is a graph obtained by incubating the prodrugs (PD-NPs) prepared in Example 1 with cathepsin B enzyme or Cathepsin B inhibitory siRNA for 0 hour, 6 hours or 24 hours, and then analyzing them by HPLC chromatography.
8 is a result of analysis by HPLC chromatography after incubating the prodrug (PD-NPs) prepared in Example 1 with various enzymes (cathepsin E, cathepsin D, caspase-9, and caspase-3) for 24 hours. .
9 shows the results of confocal laser scanning microscopy (CLSM) of breast cancer cells (4T1) and PD-L1 blockade cells (aPD-L1 Ab pre-treated) treated with the prodrug (PD-NPs) prepared in Example 1. .
10 is a result of analyzing co-localization with lysosomes in breast cancer cells (4T1) treated with the prodrug (PD-NPs) of Example 1 by confocal laser scanning microscopy (CLSM). .
11 is a confocal laser scanning microscopy (CLSM) result of cancer cells (4T1) and normal cells (H9C2) treated with the prodrug (PD-NPs) or doxorubicin (DOX) of Example 1.
12 is a graph showing the cell viability (%) of cancer cells (4T1) or normal cells (H9C2) treated with the prodrug (PD-NPs) of Example 1 at each concentration.
13 is a graph showing cell viability (%) of cancer cells (4T1) or normal cells (H9C2) treated only with doxorubicin (DOX) by concentration.
14a shows cancer cells treated with the prodrug (PD-NPs), doxorubicin (DOX), or physiological saline (con) of Example 1 were stained with DAPI and APC-conjugated CRT antibodies and analyzed using a confocal fluorescence microscope. this is a picture taken
Figure 14b is a graph showing the measurement of HMGB1 expression level and ATP expression level released from cancer cells treated with the prodrug (PD-NPs), doxorubicin (DOX), or physiological saline (con) of Example 1.
15 is a T cell treatment of each cancer cell (4T1) treated with the prodrug (PD-NPs), PD-L1 monoclonal antibody (aPD-L1 Ab), and PD-L1 binding peptide (aPD-L1 Pep) of Example 1. It is a graph showing the results of measuring the concentration of INF-γ observed in the culture medium during co-culture with ELISA.
16 is noninvasive near-infrared fluorescence (NIRF) analysis of cancer animal models (
FIG. 17 is a graph showing quantitative analysis of fluorescence in tumor tissue in the NIRF image of FIG. 16 .
FIG. 17 is a graph quantitatively analyzing fluorescence intensity in tumors of fluorescence images of each group of FIG. 16 .
18 is a graph showing the measurement of cancer volume (V; mm 3 ) over time in
19 is a graph showing body weight measured by time in
20 is a graph showing survival rates measured over time in
21 shows the results of flow cytometric analysis of the T cell ratio in cancer tissues extracted from
이하, 본 발명을 상세하게 설명한다.Hereinafter, the present invention will be described in detail.
본 발명의 일 측면은 서열번호 1로 표시되는 펩타이드의 일 말단에 항암제가 결합된 접합체로 구성된 전구약물을 유효성분으로 포함하는 암의 예방 또는 치료용 약학 조성물에 관한 것이다.One aspect of the present invention relates to a pharmaceutical composition for preventing or treating cancer comprising, as an active ingredient, a prodrug consisting of a conjugate in which an anticancer agent is bound to one end of the peptide represented by SEQ ID NO: 1.
[서열번호 1][SEQ ID NO: 1]
Cys-Val-Arg-Ala-Arg-Thr-Arg-Phe-Arg-Arg-GlyCys-Val-Arg-Ala-Arg-Thr-Arg-Phe-Arg-Arg-Gly
상기 펩타이드는 서열번호 2로 표시되는 카텝신 B에 의해 절단 가능한 펩타이드와 서열번호 3으로 표시되는 PD-L1 결합 펩타이드로 구분될 수 있다. 상기 서열번호 2로 표시되는 카텝신 B에 의해 절단 가능한 펩타이드는 암세포에 과량 존재하는 카텝신 B 효소에 의해 절단되는 펩타이드로, FRR' 또는 FRRG의 위치가 절단되며, 이러한 절단위치는 항암제와 PD-L1 결합 펩타이드의 활성에 전혀 악영향을 미치지 않는다.The peptide can be divided into a peptide cleavable by cathepsin B represented by SEQ ID NO: 2 and a PD-L1 binding peptide represented by SEQ ID NO: 3. The peptide cleaved by cathepsin B represented by SEQ ID NO: 2 is a peptide cleaved by cathepsin B enzyme present in excess in cancer cells, and the position of FRR' or FRRG is cleaved, and this cleavage site is anticancer agent and PD- It does not adversely affect the activity of the L1 binding peptide at all.
상기 서열번호 3으로 표시되는 PD-L1 결합 펩타이드는 암세포의 표면에 과량 존재하는 면역관문 중 PD-L1와 결합하여, 면역관물을 차단함으로써, 암세포의 면역원성 세포사멸(ICD)을 효과적으로 유도할 수 있다.The PD-L1 binding peptide represented by SEQ ID NO: 3 binds to PD-L1 among the immune checkpoints present in excess on the surface of cancer cells and blocks the immune checkpoint, thereby effectively inducing immunogenic cell death (ICD) of cancer cells. there is.
[서열번호 2][SEQ ID NO: 2]
Phe-Arg-Arg-GlyPhe-Arg-Arg-Gly
[서열번호 3][SEQ ID NO: 3]
Cys-Val-Arg-Ala-Arg-Thr-ArgCys-Val-Arg-Ala-Arg-Thr-Arg
상기 펩타이드의 서열이 어느 하나라도 달라질 경우 전구약물 전체 구조가 달라져 안정성, 나노입자 형성 또는 항암효과 등에 부정적 영향을 야기하는 문제가 발생할 수 있으므로, 서열번호 1로 표시되는 펩타이드를 사용하는 것이 가장 바람직하다.If the sequence of any one of the peptides is changed, the overall structure of the prodrug may be changed, causing problems such as stability, nanoparticle formation, or anticancer effect. Therefore, it is most preferable to use the peptide represented by SEQ ID NO: 1 .
서열번호 1로 표시되는 펩타이드와 항암제가 결합하여 형성된 접합체는 양친매성(Amphiphilic) 구조를 갖는다. 상기 접합체는 하기 화학식 1로 표시되는 것일 수 있다. 상기 접합체의 분자량은 1 내지 5 kDa일 수 있다. The conjugate formed by combining the peptide represented by SEQ ID NO: 1 and the anticancer agent has an amphiphilic structure. The conjugate may be represented by
[화학식 1][Formula 1]
상기 서열번호 1로 표시되는 펩타이드의 C 말단에는 항암제가 직접 또는 링커를 통해 공유적으로 결합된 것일 수 있다. 바람직하게는 직접 결합되는 것일 수 있다.An anticancer agent may be covalently bound to the C-terminus of the peptide represented by SEQ ID NO: 1 directly or via a linker. Preferably, it may be directly bonded.
상기 접합체를 포함하는 전구약물은 수용액 상태에서 자체적으로 나노입자를 형성할 수 있다(도 1). 종래 전구약물은 저분자 구조를 가져, 종양 전달 효율이 낮다는 문제가 있었다. 본 발명의 전구약물은 복수의 접합체가 용액 상에서 추가적인 담체 및 전달체 없이 자기조립을 통해 나노입자를 형성하는데, 용액 상에서 안정적인 구조를 장기간 유지할 수 있고, 종양 전달 효율이 현저히 우수할 뿐만 아니라 암세포의 면역원성 세포사멸을 유도하는 병용 효과를 가지므로, 그 자체만으로 하나의 제형으로 사용할 수 있다.The prodrug containing the conjugate can itself form nanoparticles in an aqueous solution (FIG. 1). Conventional prodrugs have a low molecular structure and have a problem of low tumor delivery efficiency. The prodrug of the present invention forms nanoparticles through self-assembly of a plurality of conjugates in solution without additional carriers and carriers, and can maintain a stable structure in solution for a long period of time, has remarkably excellent tumor delivery efficiency, and has immunogenicity of cancer cells. Since it has a combined effect of inducing apoptosis, it can be used as one formulation by itself.
본 발명에서 용액은 전구약물을 나노입자 형태로 자기조립되도록 하여, 안정적으로 보관하거나, 농도를 조절하거나, 액상으로 되게하여 투여에 용이하도록 하는 용도로 사용될 수 있다. 상기 용액은 천연 또는 비천연의 용액일 수 있고, 상기 천연 용액은 혈액, 혈장과 같은 체액일 수 있다. 비천연 용액은 증류수 또는 완충액일 수 있으며,상기 완충액은 헤페스(HEPES), 붕산염, 포스페이트, 탄산염, 트리스(Tris), 바르비탈 및 인산완충액(Phosphate-buffered saline, PBS)으로 이루어진 군으로부터 선택되는 어느 하나 이상일 수 있다.In the present invention, the solution can be used for the purpose of allowing the prodrug to self-assemble in the form of nanoparticles, stably storing it, adjusting the concentration, or making it into a liquid phase for easy administration. The solution may be a natural or non-natural solution, and the natural solution may be a bodily fluid such as blood or plasma. The non-natural solution may be distilled water or a buffer, wherein the buffer is selected from the group consisting of HEPES, borate, phosphate, carbonate, Tris, barbital, and phosphate-buffered saline (PBS). can be more than one.
본 발명에 따른 전구약물은 용액 내에서 자기조립에 의해 구형의 나노입자를 형성하므로, 체액이나 혈액 내에서도 안정적인 나노입자를 형성할 수 있다. 상기 나노입자 구조는 생체 내에서 최소한의 부작용을 갖는 안정적인 구조이다. 본 발명에 따른 전구약물은 나노 구조에도 불구하고 암세포에 대해 특이적으로 활성화되며, 뛰어난 종양 전달 효율을 갖는 것을 특징으로 한다.Since the prodrug according to the present invention forms spherical nanoparticles by self-assembly in a solution, stable nanoparticles can be formed in body fluids or blood. The nanoparticle structure is a stable structure with minimal side effects in vivo. The prodrug according to the present invention is characterized in that it is specifically activated against cancer cells despite its nanostructure and has excellent tumor delivery efficiency.
상기 전구약물은 용액 상에서 자기조립에 의해 구형의 나노입자를 형성하는데, 상기 나노입자의 평균 직경은 50 nm 이상일 수 있고, 바람직하게는 50 내지 500 nm일 수 있고. 보다 바람직하게는 100 내지 200 nm일 수 있으며, 가장 바람직하게는 140 내지 160 nm일 수 있다. 만일 상기 자가조립 나노입자의 평균 직경이 하한치 미만일 경우, 종양세포 내 축적 효율 및 약물 동력학 특성이 발휘되지 않을 수 있으며, 상기 상한치를 초과하는 경우에는 암세포 축적효율이 떨어질 뿐만 아니라 세포 내 유동성이 감소될 수 있다.The prodrug forms spherical nanoparticles by self-assembly in solution, and the average diameter of the nanoparticles may be 50 nm or more, preferably 50 to 500 nm. More preferably, it may be 100 to 200 nm, and most preferably, it may be 140 to 160 nm. If the average diameter of the self-assembled nanoparticles is less than the lower limit, accumulation efficiency and pharmacokinetic properties in tumor cells may not be exhibited. can
상기 서열번호 1로 표시되는 펩타이드는 나노입자의 내부 또는 외부 표면에 위치하는 것일 수 있으나, 바람직하게는 외부에 존재하는 것일 수 있다. 특히 서열번호 3으로 표시되는 PD-L1 결합 펩타이드가 나노입자의 표면에 가장 많이 노출되어 있는 것일 수 있다. 이때, 항암제는 나노입자의 구조 내부에 위치하는 것일 수 있다. 따라서 본 발명에 따른 자가조립 나노입자는 종양세포가 아닌 세포에 대해서는 항암제가 활성화되지 않으므로, 전혀 독성을 나타내지 않는 장점을 갖는다.The peptide represented by SEQ ID NO: 1 may be located on the inner or outer surface of the nanoparticle, but may preferably be present on the outside. In particular, the PD-L1 binding peptide represented by SEQ ID NO: 3 may be the most exposed on the surface of the nanoparticle. In this case, the anticancer agent may be located inside the nanoparticle structure. Therefore, the self-assembling nanoparticles according to the present invention have the advantage of not exhibiting toxicity at all because anticancer agents are not activated against cells other than tumor cells.
본 발명의 전구약물은 생체 내에 투여되면 EPR(Enhanced Permeability and Retection) 효과를 통해 암세포에 효과적으로 축적되고, 암 세포 표면에 과량 존재하는 면역 관문인 PD-L1에 결합하여, 면역관물을 차단함과 동시에, 암세포에 선택적으로 진입할 수 있다. 본 발명의 전구약물은 안정적인 나노입자 형태이고, 상기 나노입자에 표면에는 복수개의 PD-L1 결합 펩타이드가 노출되어 있으므로, 복수개의 암세포 표면의 PD-L1과 결합하여 다가 가교 결합을 형성함으로써, 암세포의 리소좀으로 진입하여 PD-L1이 재사용되지 않고 완전히 분해되도록 한다. 리소좀으로 진입한 본 발명의 전구약물은 암세포에서 과발현되는 카텝신 B에 의해 분해 및 활성화되어 항암제가 세포 핵 내로 전달되어 유의적이고 현저한 항암 효과와 를 가질 수 있다. 앞서 설명한 본 발명에 따른 전구약물의 작용 매커니즘에 대한 이해를 돕기 위해, 전반적인 과정을 도 1에 도시하였으므로, 이를 참고할 수 있다.When administered in vivo, the prodrug of the present invention effectively accumulates in cancer cells through the EPR (Enhanced Permeability and Retection) effect, binds to PD-L1, an immune checkpoint present in excess on the surface of cancer cells, and blocks immune checkpoints. , can selectively enter cancer cells. The prodrug of the present invention is in the form of stable nanoparticles, and since a plurality of PD-L1 binding peptides are exposed on the surface of the nanoparticles, it binds to PD-L1 on the surface of a plurality of cancer cells to form multivalent cross-links, thereby preventing cancer cells. It enters the lysosome and allows PD-L1 to be completely degraded rather than reused. The prodrug of the present invention entering the lysosome is degraded and activated by cathepsin B, which is overexpressed in cancer cells, so that the anticancer drug is delivered into the cell nucleus and can have a significant and significant anticancer effect. In order to help understand the mechanism of action of the prodrug according to the present invention described above, since the overall process is shown in FIG. 1, reference may be made to this.
본 발명의 전구약물은, 상술한 매커니즘을 통해 암세포의 PD-L1 면역관문을 차단하여 암세포의 자가인식회피를 억제함으로써, 항암제에 의한 면역원성 세포사멸과 면역관문 억제를 통해 생체 내에서 종양에 대한 면역세포 활성화를 유도할 수 있으므로, 기존의 항암효과와 함께 항암 면역치료의 병용 효과를 얻을 수 있다.The prodrug of the present invention inhibits self-recognition evasion of cancer cells by blocking the PD-L1 immune checkpoint of cancer cells through the above-described mechanism, thereby inhibiting immunogenic cell death by anticancer drugs and suppressing immune checkpoints against tumors in vivo. Since it can induce immune cell activation, it is possible to obtain the combined effect of anticancer immunotherapy together with the existing anticancer effect.
암세포의 표면에 PD-L1은, 정상세포에 비해 10배 이상 많이 발현되는 것으로 확인되었다. 따라서 본 발명에 따른 전구약물은 암세포에 보다 효율적으로 결합하므로, 암세포에 대한 전달과 축적이 우수하다. 반면 상대적으로 PD-L1의 발현이 낮은 정상세포에 대해서는 세포 내 진입되는 효율이 암세포 대비 상대적으로 낮기 때문에, 정상세포에 대한 독성, 부작용을 최소화할 수 있다.It was confirmed that PD-L1 is expressed on the surface of cancer cells more than 10 times as much as normal cells. Therefore, since the prodrug according to the present invention binds to cancer cells more efficiently, delivery and accumulation to cancer cells are excellent. On the other hand, since the efficiency of cell entry into normal cells with relatively low expression of PD-L1 is relatively low compared to cancer cells, toxicity and side effects to normal cells can be minimized.
카텝신 B 효소는 암세포에서 특이적으로 과발현되는데, 암세포는 정상세포에 비해 20 내지 30배 더 많이 카텝신 B 효소가 발현되는 것을 확인하였다. 따라서 본 발명에 따른 전구약물은 정상세포 내에 진입하더라도 카텝신 B의 발현이 낮기 때문에, 안정적인 비활성 상태를 유지하여 세포독성을 거의 나타내지 않는다. 그러나 카텝신 B 효소가 과발현되는 암세포에서는 활성화되어 세포 핵 내에서 높은 독성을 나타내므로 현저히 우수한 항암 효과를 나타낼 수 있다.Cathepsin B enzyme is specifically overexpressed in cancer cells, and it was confirmed that cancer cells express 20 to 30 times more cathepsin B enzyme than normal cells. Therefore, even if the prodrug according to the present invention enters normal cells, since the expression of cathepsin B is low, it maintains a stable inactive state and exhibits little cytotoxicity. However, in cancer cells in which cathepsin B enzyme is overexpressed, it is activated and exhibits high toxicity in the cell nucleus, so it can exhibit remarkably excellent anticancer effects.
본 발명에 따른 전구약물은 암세포에서 과발현되는 PD-L1과 카텝신 B 효소를 타겟으로 함으로써, 여러 단계를 거쳐 암세포에 대해 특이적 효과를 유도하므로, 정상조직에 대해서는 10 μM에서도 거의 세포 독성을 유발하지 않도록 매우 안정적으로 작용한다는 장점을 갖는다.The prodrug according to the present invention induces a specific effect on cancer cells through several steps by targeting PD-L1 and cathepsin B enzymes, which are overexpressed in cancer cells, and thus induces almost cytotoxicity in normal tissues even at 10 μM It has the advantage that it works very stably so that it does not fall.
본 발명에 따른 전구약물은 제조과정이 단순하고, 합성조건 제어가 용이하고, 봉입하거나 부착하는 등의 별도의 제형화 과정이 요구되지 않으므로 대량생산 및 의약품의 품질 관리(QC)가 용이하다는 장점이 있다.The prodrug according to the present invention has the advantages of simple manufacturing process, easy control of synthesis conditions, and easy mass production and quality control (QC) of pharmaceuticals because it does not require a separate formulation process such as encapsulation or attachment. there is.
상기 항암제는 항암효과를 갖는 약물이라면 특별히 제한되지 않으나, 상기 서열번호 1의 펩타이드와 결합하여 양친매성을 형성하는 것이 바람직하므로 소수성을 띄는 항암제일 수 있다. 바람직하게 상기 항암제는 독소루비신(doxorubicin), 클로라부실(chlorambucil), 테모졸로마이드(temozolomide), 시스플라틴(cisplatin), 옥사리플라틴(oxaliplatin), 5-플루오로우라실(5-fluorouracil), 겜시타빈(gemcitabine), 메토트렉세이트(methotrexate), 페메트렉세드(pemetrexed), 캠토테신(camptothecin), 토포테칸(topotecan), 이리노테칸(irinotecan), 에토포사이드(etoposide), 미토산트론(mitoxantrone), 파클리탁셀(paclitaxel), 도세탁셀(docetaxel), 아우리스타틴 E(auristatin E), 아우리스타틴 F(auristatin F), 모노메틸 아우리스타틴 E(monomethyl auristatin E), 모노메틸 아우리스타틴 F(monomethyl auristatin F) 및 이들의 유도체로 이루어진 군으로부터 선택되는 어느 하나 이상일 수 있고, 바람직하게는 독소루비신일 수 있다.The anti-cancer agent is not particularly limited as long as it has an anti-cancer effect, but it may be a hydrophobic anti-cancer agent since it is preferably combined with the peptide of SEQ ID NO: 1 to form an amphiphilic property. Preferably, the anticancer agent is doxorubicin, chlorambucil, temozolomide, cisplatin, oxaliplatin, 5-fluorouracil, gemcitabine ( gemcitabine), methotrexate, pemetrexed, camptothecin, topotecan, irinotecan, etoposide, mitoxantrone, paclitaxel, Docetaxel, auristatin E, auristatin F, monomethyl auristatin E, monomethyl auristatin F and their derivatives It may be any one or more selected from the group consisting of, preferably doxorubicin.
본 발명에 따른 전구약물은 암의 미세환경 즉, 암에서 과발현되는 카텝신 B 효소에 의해 작동되기 때문에, 일반 암뿐만 아니라 내성암, 재발암 및 전이암을 포함하는 것일 수 있다. 상기 전이암이란, 암세포가 원발 장기를 떠나 다른 장기로 이동하여 증식되어 발생된 암을 의미한다. 바람직하게는 특정 암에서 암세포가 주위 장기로 침습하거나, 혈관이나 림프관을 따라 다른 장기로 전이된 것을 의미할 수 있으나, 특별히 이에 제한되는 것은 아니다.Since the prodrug according to the present invention works by the microenvironment of cancer, that is, cathepsin B enzyme overexpressed in cancer, it may include not only general cancer but also resistant cancer, recurrent cancer, and metastatic cancer. The metastasis cancer refers to a cancer caused by cancer cells leaving a primary organ and moving to another organ and proliferating. Preferably, it may mean that cancer cells in a specific cancer invade nearby organs or metastasize to other organs along blood vessels or lymphatic vessels, but are not particularly limited thereto.
본 발명에서 재발암이란, 최초 암 발생 후 치료로 인해 완치 판정을 받은 후, 다시 암이 발생한 경우를 의미한다. In the present invention, recurrent cancer refers to a case in which cancer reoccurs after the first cancer has been diagnosed as cured due to treatment.
본 발명에서 내성암이란, 방사선 치료법과 같은 암 치료요범 또는 암 치료용 약물, 특히 항암제 치료에 대하여 극히 낮은 감수성을 나타내어, 상기 치료법에 의해 증세가 호전, 완화, 경감 또는 치료 증상을 나타내지 않는 암을 의미한다. 상기 내성암은 특정한 치료법에 대하여 처음부터 내성을 갖는 것일 수 있고, 최초에는 내성을 나타내지 않았으나, 긴 시간의 치료로 인하여 암 세포 내의 유전자 변이 등에 의하여 동일한 치료에 대해 더 이상 감수성을 나타내지 않게 되어 발생할 수도 있다.In the present invention, resistant cancer refers to cancer that exhibits extremely low sensitivity to cancer treatment guidelines such as radiation therapy or drugs for cancer treatment, particularly anticancer drug treatment, and does not improve, alleviate, reduce, or show symptoms of treatment by the treatment. it means. The resistant cancer may have resistance to a specific treatment from the beginning, and may not show resistance at first, but may occur due to long-term treatment and no longer showing sensitivity to the same treatment due to genetic mutations in cancer cells. there is.
상기 암은 위암, 폐암, 비소세포성 폐암, 유방암, 난소암, 간암, 기관지암, 비인두암, 후두암, 췌장암, 방광암, 결장암, 자궁경부암, 골암, 비소세포성 골암, 혈액암, 피부암, 두부 또는 경부 암, 자궁암, 직장암, 항문 부근암, 결장암, 나팔관암, 자궁내막암, 질암, 음문암, 호지킨병(Hodgkin's disease), 식도암, 소장암, 내분비선암, 갑상선암, 부갑상선암, 부신암, 연조직 육종, 요도암, 음경암, 전립선암, 만성 또는 급성 백혈병, 림프구 림프종, 신장 또는 수뇨관암, 신장세포 암종, 신장골반암종, 침샘암, 육종암, 가성점액종, 간모세포종, 고환암, 교모세포종, 구순암, 난소생식세포종양, 기저세포암, 다발성골수종, 담낭암, 맥락막흑색종, 바터팽대부암, 복막암, 설암, 소세포암, 소아림프종, 신경모세포종, 십이지장암, 요관암, 성상세포종, 수막종, 신우암, 외음부암, 흉선암, 중추신경계(central nervous system, CNS) 종양, 1차 중추신경계 림프종, 척수종양, 뇌간 신경교종 및 뇌하수체 선종으로 이루어진 군으로부터 선택되는 어느 하나 이상일 수 있고, 하나의 암종만이 아닌 복수의 암종에 대해서도 현저한 효과를 나타낼 수 있다.The cancer is gastric cancer, lung cancer, non-small cell lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colon cancer, cervical cancer, bone cancer, non-small cell bone cancer, blood cancer, skin cancer, head or Cervical cancer, uterine cancer, rectal cancer, perianal cancer, colon cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue Sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney or ureteric cancer, renal cell carcinoma, renal pelvic carcinoma, salivary gland cancer, sarcoma cancer, pseudomyxoma, hepatoblastoma, testicular cancer, glioblastoma, labrum Cancer, ovarian germ cell tumor, basal cell carcinoma, multiple myeloma, gallbladder cancer, choroidal melanoma, ampulla of Vater cancer, peritoneal cancer, tongue cancer, small cell cancer, juvenile lymphoma, neuroblastoma, duodenal cancer, ureteral cancer, astrocytoma, meningioma, renal pelvic cancer, It may be any one or more selected from the group consisting of vulvar cancer, thymus cancer, central nervous system (CNS) tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma, and not only one carcinoma. Significant effects can also be exhibited against multiple types of carcinoma.
본 발명의 전구약물은 생체 내에서 자기조립을 통해 구형의 나노입자로 존재하므로, 구조적 안정성이 높고, 암에 대한 뛰어난 특이적 반응성을 가질 뿐만 아니라, 체내에서 반감기의 증가로 인해 체류시간이 일반 항암제 또는 PD-L1 항체/펩타이드를 단독으로 사용하였을 때보다 3배 이상 유의적으로 뛰어나다.Since the prodrug of the present invention exists as spherical nanoparticles through self-assembly in vivo, it has high structural stability, excellent specific reactivity to cancer, and its retention time is similar to that of a general anticancer drug due to its increased half-life in the body. Or, it is significantly superior to 3 times or more when using the PD-L1 antibody/peptide alone.
본 발명에 따른 전구약물은 암의 미세환경에 의해 활성화되고, 이외에는 안정한 전구약물형태로 존재하는 것으로, 이를 유효성분으로 함유하여 암 예방 또는 치료용 약학 조성물로 제공될 수 있다.The prodrug according to the present invention is activated by the microenvironment of cancer and exists in a stable prodrug form, and can be provided as a pharmaceutical composition for preventing or treating cancer by containing it as an active ingredient.
본 발명에서 예방은 본 발명의 약학 조성물의 투여로 질환의 발병을 억제 또는 지연시키는 모든 행위를 의미하며, 치료란 본 발명의 약학 조성물의 투여로 인해 이미 유발된 질환의 증세가 호전되거나 이롭게 되는 모든 행위를 의미한다.In the present invention, prevention means any activity that suppresses or delays the onset of a disease by administration of the pharmaceutical composition of the present invention, and treatment means any activity that improves or benefits the symptoms of a disease already caused by the administration of the pharmaceutical composition of the present invention. means action.
본 명세서에서 용어 '유효성분으로 함유하는'이란 본 발명의 전구약물이 암에 대해 치료 또는 예방 효능 또는 활성을 달성하는 데 충분한 양을 포함하는 것을 의미한다.As used herein, the term 'contained as an active ingredient' means that the prodrug of the present invention contains an amount sufficient to achieve therapeutic or preventive efficacy or activity against cancer.
상기 약학 조성물 내의 유효성분의 함량은 약학 조성물의 사용 목적, 제형의 형태 등에 따라서 적절하게 조절 가능하며, 예컨대, 약학 조성물의 전체 중량 기준으로 0.001 내지 99 중량%, 0.001 내지 90 중량%, 0.001 내지 50 중량%, 0.01 내지 50 중량%, 0.1 내지 50 중량%, 또는 1 내지 50 중량%일 수 있으나, 이에 제한되는 것은 아니다The content of the active ingredient in the pharmaceutical composition can be appropriately adjusted according to the purpose of use of the pharmaceutical composition, the form of the dosage form, etc., for example, 0.001 to 99% by weight, 0.001 to 90% by weight, 0.001 to 50% by weight based on the total weight of the pharmaceutical composition. wt%, 0.01 to 50 wt%, 0.1 to 50 wt%, or 1 to 50 wt%, but is not limited thereto.
본 발명의 약학 조성물은 사용되는 유효성분의 활성, 연령, 체중, 일반적인 건강, 성별, 정식, 투여시간, 투여경로, 배출율, 약물 배합 및 예방 또는 치료될 특정 질환의 중증을 포함한 여러 요인에 따라 다양하게 변할 수 있고, 상기 약학 조성물의 투여량은 환자의 상태, 체중, 질병의 정도, 약물 형태, 투여경로 및 기간에 따라 다르지만 당업자에 의해 적절하게 선택될 수 있고, 1일 0.0001 내지 20 mg/kg, 바람직하게는 0.001 내지 5 mg/kg, 보다 바람직하게는 0.01 내지 10 mg/kg으로 투여할 수 있다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다. 본 발명에 따른 약학적 조성물은 환제, 당의정, 캡슐, 액제, 겔, 시럽, 슬러리, 현탁제로 제형될 수 있다.The pharmaceutical composition of the present invention varies depending on various factors, including the activity of the active ingredient used, age, body weight, general health, sex, diet, administration time, route of administration, excretion rate, drug combination, and severity of a specific disease to be prevented or treated. The dosage of the pharmaceutical composition may vary depending on the patient's condition, weight, disease severity, drug form, administration route and period, but may be appropriately selected by those skilled in the art, and may be 0.0001 to 20 mg/kg per day. , preferably 0.001 to 5 mg/kg, more preferably 0.01 to 10 mg/kg. Administration may be administered once a day, or may be administered in several divided doses. The dosage is not intended to limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated as a pill, dragee, capsule, liquid, gel, syrup, slurry, or suspension.
한편, 본 발명의 약학 조성물의 투여농도가 0.0001 내지 20 mg/kg, 바람직하게는 0.001 내지 10 mg/kg, 가장 바람직하게는 0.01 내지 5 mg/kg일 때, 정상 조직에 대한 최소한의 부작용을 가지면서 암을 예방 또는 치료하는 탁월한 효과가 있었다. 구체적으로 환자 체중 kg당 1 내지 5 mg를 정맥주사로 투여하는 경우 1회 투여만으로 암 질환을 개선하는 탁월한 효과가 있다.On the other hand, when the dosage concentration of the pharmaceutical composition of the present invention is 0.0001 to 20 mg/kg, preferably 0.001 to 10 mg/kg, and most preferably 0.01 to 5 mg/kg, it has minimal side effects on normal tissues. It has an excellent effect in preventing or treating cancer. Specifically, when 1 to 5 mg per kg of body weight of a patient is administered intravenously, there is an excellent effect of improving cancer disease with only one administration.
본 발명의 약학 조성물은 개체에게 다양한 경로로 투여될 수 있다. 예를 들어, 경구 또는 비경구로 투여될 수 있고, 비경구의 경우에는 정맥내, 복강내, 근육내, 동맥내, 구강, 심장내, 골수내, 경막내, 경피, 장관, 피하, 설하 또는 국소 투여할 수 있으나, 이에 제한되지 않으나, 바람직하게는 비경 투여일 수 있고, 가장 바람직하게는 정맥내 투여일 수 있다. The pharmaceutical composition of the present invention can be administered to a subject by various routes. For example, it can be administered orally or parenterally, and in the case of parenteral administration, intravenous, intraperitoneal, intramuscular, intraarterial, buccal, intracardiac, intramedullary, intrathecal, transdermal, enteral, subcutaneous, sublingual or topical administration It may be, but is not limited thereto, preferably parenteral administration, most preferably intravenous administration.
본 발명의 약학 조성물은 개별 치료제로 투여되거나 다른 치료제와 병용하여 투여될 수 있다. 다른 치료제와 병용하여 투여되는 경우, 본 발명의 약학 조성물과 다른 치료제는 순차적 또는 동시에 투여될 수 있다. 상기 다른 치료제는 암 퇴행을 촉진하거나 또는 추가로 종양 성장을 방지하는 화합물, 단백질 등의 약물일 수 있으며, 또한 방사선 치료 등 약물 요법 이외의 기타 항암 요법을 모두 포함한다.The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents. When administered in combination with another therapeutic agent, the pharmaceutical composition of the present invention and the other therapeutic agent may be administered sequentially or simultaneously. The other therapeutic agent may be a drug such as a compound or protein that promotes cancer regression or additionally prevents tumor growth, and also includes all other anti-cancer therapies other than drug therapy such as radiation therapy.
본 발명의 약학 조성물은 당해 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. The pharmaceutical composition of the present invention is prepared in a unit dose form or in a multi-dose container by formulating using a pharmaceutically acceptable carrier according to a method that can be easily performed by those skilled in the art. It can be prepared by incorporating into
상기 약제학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸 히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 본 발명의 약학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. 적합한 약제학적으로 허용되는 담체 및 제제는 Remington's Pharmaceutical Sciences(19th ed., 1995)에 상세히 기재되어 있다.The pharmaceutically acceptable carrier is one commonly used in formulation, and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).
본 발명의 다른 측면은 상기 약학 조성물을 암을 갖는 개체에 투여하는 단계를 포함하는 암 예방 또는 치료 방법을 제공한다.Another aspect of the present invention provides a method for preventing or treating cancer comprising administering the pharmaceutical composition to a subject having cancer.
본 발명에 따른 암 예방 또는 치료 방법에서 각 용어는 특별히 언급하지 않는 한, 상기 약학 조성물에서 상기한 바와 동일한 의미를 갖는다.In the method for preventing or treating cancer according to the present invention, each term has the same meaning as described above in the pharmaceutical composition, unless otherwise specified.
본 발명에서 '개체'는 임의의 인간 또는 비인간 동물을 포함한다. 상기 '비인간 동물'은 척추동물, 예컨대 비인간 영장류, 양, 개, 및 설치류, 예컨대 마우스, 래트 및 기니피그일 수 있다. 상기 개체는 바람직하게 인간일 수 있다. 상기 '개체'는 본 발명에서 '대상체' 또는 '환자'와 상호교환적으로 사용할 수 있다.In the present invention, 'individual' includes any human or non-human animal. The 'non-human animals' may be vertebrates such as non-human primates, sheep, dogs, and rodents such as mice, rats and guinea pigs. The subject may preferably be a human. The 'individual' may be used interchangeably with 'subject' or 'patient' in the present invention.
상기 암 치료는 예를 들면 비치료 개체에 비해 종양 성장을 약 10% 이상, 약 20% 이상, 약 40% 이상, 약 60% 이상, 또는 약 80% 이상 억제하는 것일 수 있다.The cancer treatment may be, for example, inhibition of tumor growth by about 10% or more, about 20% or more, about 40% or more, about 60% or more, or about 80% or more compared to an untreated subject.
본 발명의 암의 예방 또는 치료하는 방법에 있어서, 상기 약학 조성물은 그 자체로 이미 우수한 암세포 특이성을 갖고 있기 때문에, 투여 경로는 목적 조직에 도달할 수 있는 한 어떠한 일반적인 경로를 통하여도 투여될 수 있다. 본 발명의 약학 조성물은 특별히 이에 제한되지 않으나, 목적하는 바에 따라, 복강내 투여, 정맥내 투여, 근육내 투여, 피하 투여, 피내 투여, 경구 투여, 비내 투여, 폐내 투여, 직장내 투여될 수 있고, 바람직하게는 비경구 투여일 수 있으며, 가장 바람직하게는 정맥내 투여일 수 있다.In the method for preventing or treating cancer of the present invention, since the pharmaceutical composition itself already has excellent cancer cell specificity, it can be administered through any general route as long as it can reach the target tissue. . The pharmaceutical composition of the present invention is not particularly limited thereto, but may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, intranasally, intrapulmonaryly, or intrarectally, as desired. , Preferably it may be parenteral administration, and most preferably it may be intravenous administration.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시하나, 하기 실시예는 본 발명을 예시하는 것일 뿐 본 발명의 범주 및 기술사상 범위 내에서 다양한 변경 및 수정이 가능함은 당업자에게 있어서 명백한 것이며, 이러한 변형 및 수정이 첨부된 특허청구범위에 속하는 것도 당연한 것이다.Hereinafter, preferred embodiments are presented to aid understanding of the present invention, but the following examples are merely illustrative of the present invention, and various changes and modifications are possible within the scope and spirit of the present invention. It is obvious to those skilled in the art, It goes without saying that these variations and modifications fall within the scope of the appended claims.
또한 이하에서 제시되는 실험 결과는 상기 실시예 및 비교예의 대표적인 실험 결과만을 기재한 것이며, 아래에서 명시적으로 제시하지 않은 본 발명의 여러 구현예의 각각의 효과는 해당 부분에서 구체적으로 기재하도록 한다.In addition, the experimental results presented below are only representative experimental results of the above examples and comparative examples, and each effect of various embodiments of the present invention that is not explicitly presented below will be described in detail in the corresponding section.
실시예 1. 전구약물(PD-NPs) 합성Example 1. Synthesis of prodrugs (PD-NPs)
서열번호 1로 표시되는 펩타이드(CVRARTRFRRG)는 펩트론(대전, 한국)에 의뢰하여 제조 및 구입한 것을 사용하였다. 서열번호 1로 표시되는 펩타이드(CVRARTRFRRG)(1.8 g, 1.96 mmol), 독소루비신(Doxorubicin)(0.9 g, 1.66 mmol), EDC(1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride)(250 mg, 1.31 mmol) 및 NHS(N-Hydroxysuccinimide)(200 mg, 1.74 mmol)을 무수 DMF(Dimethylformamide)(100 mL)에 용해시킨 뒤 상온에서 6시간 동안 반응 후 DIPEA(Diisopropylethylamine)(200 mg, 1.55 mmol)을 넣고 추가로 6시간 반응한 뒤 HPLC를 이용하여 정제하였다. The peptide represented by SEQ ID NO: 1 (CVRATRFRRG) was manufactured and purchased at the request of Peptron (Daejeon, Korea). Peptide represented by SEQ ID NO: 1 (CVRARTFRFRG) (1.8 g, 1.96 mmol), Doxorubicin (0.9 g, 1.66 mmol), EDC (1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride) (250 mg, 1.31 mmol) and NHS (N-Hydroxysuccinimide) (200 mg, 1.74 mmol) were dissolved in anhydrous DMF (Dimethylformamide) (100 mL), and after reacting at room temperature for 6 hours, DIPEA (Diisopropylethylamine) (200 mg, 1.55 mmol) was added. After adding and reacting for an additional 6 hours, it was purified using HPLC.
합성과정은 도 2에 나타내었고, HPLC 결과는 도 3에 나타내었다. 그 결과 수율 99% 이상으로 항PD-L1 펩타이드와 카텝신B에 절단되는 펩타이드 및 항암제로 구성된 전구약물(PD-NPs)을 제조하였다.The synthetic process is shown in Figure 2, and the HPLC results are shown in Figure 3. As a result, prodrugs (PD-NPs) composed of an anti-PD-L1 peptide, a peptide cleaved by cathepsin B, and an anticancer agent were prepared with a yield of more than 99%.
비교예 1. 서열번호 4의 전구약물 합성Comparative Example 1. Synthesis of prodrug of SEQ ID NO: 4
서열번호 4로 표시되는 펩타이드(NYSKPTDRQYHF)는 펩트론(대전, 한국)에 의뢰하여 제조 및 구입한 것을 사용하였다. 서열번호 4로 표시되는 펩타이드(NYSKPTDRQYHF)(2 g, 1.91 mmol), 독소루비신(0.9 g, 1.66 mmol), EDC(250 mg, 1.31 mmol) 및 NHS(200 mg, 1.74 mmol)를 무수 DMF(100 mL)에 용해시킨 뒤, 상온에서 6시간 동안 반응 후 DIPEA(200 mg, 1.55 mmol)을 넣고 추가로 6시간 반응한 뒤 HPLC를 이용하여 정제하였다.The peptide represented by SEQ ID NO: 4 (NYSKPTDRQYHF) was manufactured and purchased at the request of Peptron (Daejeon, Korea). The peptide represented by SEQ ID NO: 4 (NYSKPTDRQYHF) (2 g, 1.91 mmol), doxorubicin (0.9 g, 1.66 mmol), EDC (250 mg, 1.31 mmol) and NHS (200 mg, 1.74 mmol) were mixed with anhydrous DMF (100 mL). ), and after reacting at room temperature for 6 hours, DIPEA (200 mg, 1.55 mmol) was added thereto, followed by further reaction for 6 hours, followed by purification using HPLC.
실험예 1. 전구약물(PD-NPs)의 분자량Experimental Example 1. Molecular weight of prodrugs (PD-NPs)
실시예 1로부터 제조된 전구약물(PD-NPs)의 분자량을 MALDI(matrix-assisted laser desorption/ionization)(AB Sciex TOF/TOF 5800 System, USA)(with cyano-4-hydroycinnamic acid matrix)로 분석하였다.The molecular weight of the prodrug (PD-NPs) prepared in Example 1 was analyzed by matrix-assisted laser desorption/ionization (MALDI) (AB Sciex TOF/TOF 5800 System, USA) (with cyano-4-hydroycinnamic acid matrix) .
도 4는 실시예 1의 전구약물(PD-NPs)에 대한 MALDI-TOF 결과 그래프로, 이에 따르면 전구약물(PD-NPs)은 성공적으로 합성되었으며, 전구약물(PD-NPs)의 분자량은 1945.8717 m/z [M]인 것을 알 수 있다.Figure 4 is a MALDI-TOF result graph for the prodrug (PD-NPs) of Example 1. According to this, the prodrug (PD-NPs) was successfully synthesized, and the molecular weight of the prodrug (PD-NPs) was 1945.8717 m /z [M].
실험예 2. 전구약물(PD-NPs)의 나노입자 분석Experimental Example 2. Nanoparticle analysis of prodrugs (PD-NPs)
실시예 1로부터 제조된 전구약물(PD-NPs)이 수용액 상에서 자기조립하여 나노입자 형태를 형성 및 유지하는지 분석하고자 하였다. 이를 위해, 실시예 1로부터 제조된 전구약물(PD-NPs) 1 mg를 1 mL의 생리 식염수에 분산시킨 뒤 동적광산란(DLS)으로 측정하였다.It was analyzed whether the prodrugs (PD-NPs) prepared in Example 1 self-assemble in an aqueous solution to form and maintain nanoparticle shapes. To this end, 1 mg of the prodrug (PD-NPs) prepared in Example 1 was dispersed in 1 mL of physiological saline and measured by dynamic light scattering (DLS).
도 5는 용액 상에서 실시예 1로부터 제조된 전구약물(PD-NPs)을 동적광산란(DLS)(좌) 및 투과전자현미경(TEM)(우)으로 측정한 결과이다.5 is a result of measuring the prodrug (PD-NPs) prepared in Example 1 in solution by dynamic light scattering (DLS) (left) and transmission electron microscopy (TEM) (right).
도 5에 나타난 바와 같이, 실시예 1로부터 제조된 전구약물(PD-NPs)은 수용액 상에서 나노입자 형태로 자기조립되는 것을 확인하였다. 실시예 1로부터 제조된 전구약물(PD-NPs)이 생리식염수 내에서 응집되거나 뭉개지지 않고, 구형의 나노입자 형태로 분산되어 존재하는 것을 확인하였다.As shown in FIG. 5, it was confirmed that the prodrug (PD-NPs) prepared in Example 1 was self-assembled in the form of nanoparticles in an aqueous solution. It was confirmed that the prodrugs (PD-NPs) prepared in Example 1 were dispersed in the form of spherical nanoparticles without being aggregated or crushed in physiological saline.
또한, 실시예 1로부터 제조된 전구약물(PD-NPs)의 자기조립된 나노입자의 평균 직경은 100 내지 200 nm인 것을 확인하였고, 바람직하게는 약 150 nm인 것을 확인하였다.In addition, it was confirmed that the average diameter of the self-assembled nanoparticles of the prodrug (PD-NPs) prepared in Example 1 was 100 to 200 nm, preferably about 150 nm.
도 6은 용액 상에서 실시예 1로부터 제조된 전구약물(PD-NPs)과 비교예 1로부터 제조된 전구약물을 분산시킨 후, 시간별로 동적광산란(DLS)으로 측정하여 나타낸 그래프이다.6 is a graph showing the dispersion of the prodrug (PD-NPs) prepared in Example 1 and the prodrug prepared in Comparative Example 1 in a solution and then measured by dynamic light scattering (DLS) over time.
도 6에 나타난 바와 같이, 용액 상에서 전구약물을 장기보관하였을 때 안정성을 분석하고자 하였다. 실시예 1로부터 제조된 전구약물(PD-NPs)과 비교예 1로부터 제조된 전구약물을 생리식염수 내에 1 mg/mL의 농도로 각각 분산시킨 뒤, 상온에서 5일 동안 크기 변화를 비교 분석하였다. 실시예 1로부터 제조된 전구약물(PD-NPs)은 5일 동안 본래의 평균 입경인 약 150 nm 크기를 그대로 유지하는 것을 확인할 수 있다. 이에 반해 비교예 1로부터 제조된 전구약물은 3일부터 마이크로 사이즈로 응집되는 것을 확인할 수 있다.As shown in Figure 6, the stability was analyzed when the prodrug was stored for a long time in solution. The prodrug (PD-NPs) prepared in Example 1 and the prodrug prepared in Comparative Example 1 were dispersed in physiological saline at a concentration of 1 mg/mL, respectively, and size changes were compared and analyzed for 5 days at room temperature. It can be seen that the prodrug (PD-NPs) prepared in Example 1 maintains its original average particle size of about 150 nm for 5 days. On the other hand, it can be seen that the prodrug prepared in Comparative Example 1 is aggregated into micro sizes from the 3rd day.
즉 실시예 1로부터 제조된 전구약물(PD-NPs)은 용액 상에서 매우 안정적인 형태의 자기조립 나노입자를 형성하며, 상기 나노입자는 매우 긴 시간동안 응집이나 크기변형없이 안정적으로 유지되는 것을 알 수 있다. 실시예 1로부터 제조된 전구약물(PD-NPs)는 용액 상에서 최소 5일 이상, 최대 30~40일동안 안정적인 보관이 가능함을 확인하였다. 실시예 1로부터 제조된 전구약물(PD-NPs)은 생체 내/외에서 장기간 안정적으로 존재한다는 것을 알 수 있다.That is, the prodrug (PD-NPs) prepared in Example 1 forms self-assembled nanoparticles in a very stable form in solution, and it can be seen that the nanoparticles remain stable for a very long time without aggregation or size change. . It was confirmed that the prodrugs (PD-NPs) prepared from Example 1 could be stored stably for a minimum of 5 days and a maximum of 30 to 40 days in solution. It can be seen that the prodrugs (PD-NPs) prepared in Example 1 exist stably for a long period of time in vivo/ex vivo.
실험예 3. 전구약물(PD-NPs)의 카텝신 B에 대한 선택적 활성화Experimental Example 3. Selective activation of prodrugs (PD-NPs) for cathepsin B
실시예 1로부터 제조된 전구약물(PD-NPs)의 카텝신 B에 대한 선택적 활성화를 확인하고자 하였다. 실시예 1로부터 제조된 전구약물(PD-NPs)는 PD-L1 결합 펩타이드가 FRRG 펩타이드에 인접해있기 때문에, 이들의 결합에 의한 간섭에도 불구하고 카텝신 B에 의해 성공적으로 절단되는지를 확인하고자 하였다.The selective activation of the prodrugs (PD-NPs) prepared in Example 1 for cathepsin B was confirmed. Since the prodrugs (PD-NPs) prepared from Example 1 are adjacent to the FRRG peptide, the PD-L1 binding peptide was successfully cleaved by cathepsin B despite interference by their binding. .
우선, 실시예 1로부터 제조된 전구약물(PD-NPs)에 cathepsin B 효소반응완충액(MES buffer; 50 ㎍/ml)을 가하여 상온에서 0시간 또는 6시간동안 배양하였다. 반응이 종료된 후 HPLC 크로마토그래프로 분석하였다. 대조군으로 실시예 1로부터 제조된 전구약물(PD-NPs)에 cathepsin B 효소반응완충액(MES buffer; 50 ㎍/ml) 및 Cathepsin B 억제 siRNA를 함께 넣고, 상온에서 24시간동안 배양하였다. 이는'W/ Cat-B_inh'로 표시하였다. Cathepsin B 억제 siRNA는 Santa Cruz Biotechnology에서 구입한 것을 사용하였고, 세포에 70 μmol 농도로 처리하였다.First, cathepsin B enzyme reaction buffer (MES buffer; 50 μg/ml) was added to the prodrug (PD-NPs) prepared in Example 1, followed by incubation at room temperature for 0 hour or 6 hours. After the reaction was completed, it was analyzed by HPLC chromatograph. As a control, cathepsin B enzyme reaction buffer (MES buffer; 50 μg/ml) and Cathepsin B inhibitory siRNA were added to the prodrug (PD-NPs) prepared in Example 1, and incubated at room temperature for 24 hours. This was marked as 'W/Cat-B_inh'. Cathepsin B inhibitory siRNA was purchased from Santa Cruz Biotechnology, and cells were treated at a concentration of 70 µmol.
다음, 실시예 1로부터 제조된 전구약물(PD-NPs)에 다양한 효소(cathepsin E, cathepsin D, caspase-9, caspase-3)를 포함하는 효소반응완충액(MES buffer; 50 ㎍/ml)을 가하고, 상온에서 배양하였다. 반응이 종료된 후 HPLC 크로마토그래프로 분석하였다. Next, enzyme reaction buffer (MES buffer; 50 μg/ml) containing various enzymes (cathepsin E, cathepsin D, caspase-9, caspase-3) was added to the prodrug (PD-NPs) prepared in Example 1, , and incubated at room temperature. After the reaction was completed, it was analyzed by HPLC chromatograph.
도 7은 실시예 1로부터 제조된 전구약물(PD-NPs)를 카텝신 B 효소 또는 Cathepsin B 억제 siRNA 함께 0시간, 6시간 또는 24시간 배양한 후, 이를 HPLC 크로마토그래피로 분석한 그래프이다. 도 8은 실시예 1로부터 제조된 전구약물(PD-NPs)를 다양한 효소(cathepsin E, cathepsin D, caspase-9, caspase-3)와 함께 24시간 배양한 후, HPLC 크로마토그래피로 분석한 결과이다.7 is a graph obtained by incubating the prodrugs (PD-NPs) prepared in Example 1 with cathepsin B enzyme or Cathepsin B inhibitory siRNA for 0 hour, 6 hours or 24 hours, and then analyzing them by HPLC chromatography. 8 is a result of analysis by HPLC chromatography after incubating the prodrug (PD-NPs) prepared in Example 1 with various enzymes (cathepsin E, cathepsin D, caspase-9, and caspase-3) for 24 hours. .
도 7에 나타난 바와 같이, 실시예 1로부터 제조된 전구약물(PD-NPs)는 카텝신 B를 처리한 6시간 후, 카텝신B 절단 펩타이드(FRRG)가 효과적으로 절단되어 항암제(Doxorubicin)가 방출되기 시작한다는 것을 확인하였다.As shown in FIG. 7, the prodrug (PD-NPs) prepared in Example 1 was treated with cathepsin B for 6 hours, and the cathepsin B cleavage peptide (FRRG) was effectively cleaved to release the anticancer drug (Doxorubicin). confirmed to start.
도 8에 나타난 바와 같이, 실시예 1로부터 제조된 전구약물(PD-NPs)은 카텝신 B가 아닌 다른 효소(cathepsin E, cathepsin D, caspase-9, caspase-3)에 대해서는, 그 어떤 유효한 수치 변화도 관찰되지 않는 것을 확인한 바, 본 발명의 전구약물(PD-NPs)은 다른 효소의 존재 하에서는 비활성화된 형태, 즉 doxorubicin 분자를 방출하지 않는 상태를 유지한다는 것을 알 수 있다.As shown in FIG. 8, the prodrugs (PD-NPs) prepared in Example 1 have no effective values for enzymes other than cathepsin B (cathepsin E, cathepsin D, caspase-9, caspase-3). As confirmed that no change was observed, it can be seen that the prodrugs of the present invention (PD-NPs) maintain an inactivated form, that is, a state in which doxorubicin molecules are not released, in the presence of other enzymes.
실시예 1로부터 제조된 전구약물(PD-NPs)은 암 세포에서 과발현되는 카텝신 B에 대해서만 선택적으로 활성화되므로, 안정적이고 현저히 우수한 전구약물이라는 것을 알 수 있다.It can be seen that the prodrug (PD-NPs) prepared in Example 1 is a stable and significantly superior prodrug because it is selectively activated only against cathepsin B overexpressed in cancer cells.
실험예 4. 전구약물(PD-NPs)의 PD-L1 결합 및 분해능Experimental Example 4. PD-L1 Binding and Resolution of Prodrugs (PD-NPs)
전구약물(PD-NPs)의 PD-L1 결합 효능 및 분해능을 확인하기 위하여 유방암세포 4T1을 이용하여 in vitro 세포실험을 진행하였다. In order to confirm the PD-L1 binding efficacy and degradation ability of prodrugs (PD-NPs), in vitro cell experiments were conducted using breast cancer cells 4T1.
공초점 현미경용 셀 배양 플레이트(24웰)의 각 웰에 2 × 104개의 암세포(유방암세포 4T1)를 각각 분주하였다. 상기 플레이트 중에서 12웰은 분주된 암세포에 PD-L1 항체(antibody)를 처리하여, 암세포 표면의 PD-L1을 봉쇄한 PD-L1 봉쇄세포(aPD-L1 Ab pre-treated)를 제조하였다. 상기 암세포를 24 시간 배양하여 안정화시킨 후, 각 웰에 실시예 1로부터 제조된 전구약물(PD-NPs)을 2 μM 농도로 처리하고, 37 ℃ 인큐베이터에서 다양한 시간별(1, 6, 9 및 24시간)로 배양하였다. 배양이 완료된 후, 각 웰에 DPBS(Dulbecco's phosphate buffered Saline)를 처리하여 잔류 약물을 제거하고, 세포 고정액을 15 분간 처리한 뒤, DAPI(4′,6-diamidino-2-phenylindole) 용액을 10분간 처리하여 세포의 핵을 염색하였다. 공초점 레이저 스캐닝 현미경(CLSM)으로 독소루비신(DOX)의 형광을 관찰하였다. 또한 세포 내 리소좀과의 상관관계를 관찰하기 위해, Rab7a antibody(제품명:PA5-109239, Invitrogen)를 이용하여 세포 내의 리소좀을 염색한 뒤 공초점 레이저 스캐닝 현미경(CLSM)으로 형광을 관찰하였다.2 × 10 4 cancer cells (breast cancer cells 4T1) were dispensed into each well of a cell culture plate (24 well) for confocal microscopy. In 12 wells of the plate, PD-L1-blocked cells (aPD-L1 Ab pre-treated) were prepared by treating the dispensed cancer cells with PD-L1 antibody to block PD-L1 on the cancer cell surface. After stabilization by culturing the cancer cells for 24 hours, each well was treated with the prodrug (PD-NPs) prepared in Example 1 at a concentration of 2 μM, and incubated at 37 ° C. at various times (1, 6, 9 and 24 hours). ) was cultured. After incubation was complete, each well was treated with DPBS (Dulbecco's phosphate buffered saline) to remove residual drug, treated with cell fixative for 15 minutes, and then with DAPI (4',6-diamidino-2-phenylindole) solution for 10 minutes. The nuclei of the cells were stained by treatment. The fluorescence of doxorubicin (DOX) was observed with a confocal laser scanning microscope (CLSM). In addition, in order to observe the correlation with intracellular lysosomes, Rab7a antibody (product name: PA5-109239, Invitrogen) was used to stain intracellular lysosomes, and then fluorescence was observed with a confocal laser scanning microscope (CLSM).
도 9는 실시예 1로부터 제조된 전구약물(PD-NPs)을 처리한 유방암세포(4T1)와 PD-L1 봉쇄세포(aPD-L1 Ab pre-treated)의 공초점 레이저 스캐닝 현미경(CLSM) 결과이다.9 shows the results of confocal laser scanning microscopy (CLSM) of breast cancer cells (4T1) and PD-L1 blockade cells (aPD-L1 Ab pre-treated) treated with the prodrug (PD-NPs) prepared in Example 1. .
도 9에 나타난 바와 같이, 실시예 1로부터 제조된 전구약물(PD-NPs)은 유방암세포(4T1)에서 효율적으로 세포 내로 흡수되어, 세포 핵 내로 독소루비신(DOX)을 성공적으로 방출하고 있다는 것을 확인할 수 있다.As shown in FIG. 9, it can be confirmed that the prodrug (PD-NPs) prepared in Example 1 is efficiently absorbed into the cells of breast cancer cells (4T1) and successfully releases doxorubicin (DOX) into the cell nucleus. there is.
실시예 1로부터 제조된 전구약물(PD-NPs)은 PD-L1 봉쇄세포(aPD-L1 Ab pre-treated)에 대해서는 세포 내로 흡수되는 양이 현저히 낮다는 것을 확인할 수 있다.It can be seen that the amount of prodrug (PD-NPs) prepared in Example 1 is absorbed into the cells of PD-L1 blocked cells (aPD-L1 Ab pre-treated) is remarkably low.
통상적으로 암세포가 정상세포보다 PD-L1 발현량이 현저히 높다고 알려져 있고, 실제 유방암세포(4T1)와 쥐 심근세포(H9C2)에서 PD-L1의 발현량을 비교한 결과, 유방암세포(4T1)가 심근세포(H9C2)보다 PD-L1 발현량이 10.23 ± 1.66배 더 많은 것을 확인하였다. 즉, 실시예 1로부터 제조된 전구약물(PD-NPs)은 세포 표면에 존재하는 PD-L1과 결합하여, 선택적으로 세포 내로 진입한다는 것을 확인할 수 있으므로, 실시예 1로부터 제조된 전구약물(PD-NPs)은 PD-L1이 다량 존재하는 암세포에 특이적일 것임을 알 수 있다.It is generally known that cancer cells have a significantly higher expression level of PD-L1 than normal cells, and as a result of comparing the expression level of PD-L1 in breast cancer cells (4T1) and mouse cardiomyocytes (H9C2), breast cancer cells (4T1) are cardiomyocytes. It was confirmed that the expression level of PD-L1 was 10.23 ± 1.66 times higher than that of (H9C2). That is, since it can be confirmed that the prodrug (PD-NPs) prepared in Example 1 binds to PD-L1 present on the cell surface and selectively enters cells, the prodrug (PD-NPs) prepared in Example 1 NPs) will be specific to cancer cells in which PD-L1 is present in large amounts.
도 10은 실시예 1의 전구약물(PD-NPs)을 처리한 유방암세포(4T1)에서의 리소좀(Lysosome)과의 공동화(Co-localization)를 공초점 레이저 스캐닝 현미경(CLSM)으로 분석한 결과이다.10 is a result of analyzing co-localization with lysosomes in breast cancer cells (4T1) treated with the prodrug (PD-NPs) of Example 1 by confocal laser scanning microscopy (CLSM). .
도 10에 나타난 바와 같이, 실시예 1의 전구약물(PD-NPs)을 처리한 유방암세포(4T1)에서 적색 형광(독소루비신)과 녹색 형광(리소좀)의 공동화(Co-localization) 형광인 주황색 형광이 분명하게 관찰되는 것을 확인할 수 있다. 이를 통해 실시예 1의 전구약물(PD-NPs)이 생체 내에 투여되면, 암세포의 표면에 존재하는 PD-L1을 인지하여, 암세포 특이적으로 결합하게 되고, 암세포 내로 진입하여 리소좀에 도달하게 됨으로써 PD-L1 분해가 이루어지게 되고, 항암제는 세포의 핵내로 방출되어 항암 효과를 달성할 수 있게 된다.As shown in FIG. 10, orange fluorescence, which is the co-localization fluorescence of red fluorescence (doxorubicin) and green fluorescence (lysosome) in breast cancer cells (4T1) treated with the prodrug (PD-NPs) of Example 1, It can be seen that it is clearly observed. Through this, when the prodrug (PD-NPs) of Example 1 is administered in vivo, it recognizes PD-L1 present on the surface of cancer cells, binds specifically to cancer cells, enters cancer cells and reaches lysosomes, thereby reaching PD-L1. -L1 decomposition takes place, and the anticancer agent is released into the nucleus of the cell to achieve an anticancer effect.
즉 실시예 1의 전구약물(PD-NPs)은 용액 상에서 나노입자 형태로 존재하고, 상기 나노입자의 표면에는 PD-L1 결합 펩타이드(서열 1)가 위치하고 있으므로, 하나의 나노입자에 다수의 암세포 표면의 PD-L1과 결합될 수 있다. 이를 다가 가교 결합(Multivalent cross-linking)이라고 한다. 임상에서 사용되는 종래 PD-L1 단일클론항체나 단일 PD-L1 결합 펩타이드(CVRARTR)은 세포 표면의 PD-L1에 다가 가교 결합을 유도할 수 없으므로 리소좀이 아닌 재사용 엔도좀(Recycling endosome)에 진입하여, 세포 내의 대사 과정을 통해 PD-L1이 재사용(Recycling)되어 면역치료의 효능이 크게 낮아지게 되는 문제가 발생한다. 그러나 본원발명의 실시예 1의 전구약물(PD-NPs)은 PD-L1에 결합하여 암세포의 리소좀으로 진입하여 PD-L1 분해가 유도되기 때문에 PD-L1 재사용이 이루어지지 않으므로, 우수한 항암 면역치료효과를 얻을 수 있다. That is, the prodrug (PD-NPs) of Example 1 exists in the form of nanoparticles in solution, and since the PD-L1 binding peptide (SEQ ID NO: 1) is located on the surface of the nanoparticles, one nanoparticle can be used on the surface of multiple cancer cells. can be combined with PD-L1 of This is called multivalent cross-linking. Conventional PD-L1 monoclonal antibodies or single PD-L1 binding peptides (CVRARTR) used in clinical practice cannot induce multivalent cross-linking to PD-L1 on the cell surface, so they enter the recycling endosome, not lysosome, , PD-L1 is reused (recycling) through intracellular metabolic processes, causing a problem in that the efficacy of immunotherapy is greatly reduced. However, since the prodrug (PD-NPs) of Example 1 of the present invention binds to PD-L1 and enters the lysosome of cancer cells and induces PD-L1 degradation, PD-L1 reuse is not achieved, resulting in excellent anticancer immunotherapeutic effect. can be obtained.
단독 또는 PD-L1 펩타이드/단일클론항체와 항암제의 단순 병용 투여에서는 PD-L1의 세포 내 흡수과정을 통한 재사용을 억제하거나 방지할 수 없어, 원래의 면역치료효과보다 현저히 낮은 효능만을 얻을 수 있었다. 그러나 본원발명에서는 PD-L1 펩타이드를 카텝신B 절단 펩타이드와 항암제와 결합시킴으로써, 용액상에서 자기조립을 통해 구형의 나노입자를 형성하도록 하면서, 30일이상 안정성을 가지며, PD-L1의 세포내 흡수과정을 리소좀에서 이루어지게 함으로써 '재사용'과정을 방지하여 면역치료효과를 유의적으로 현저히 향상되도록 하였다.In single or simple combined administration of the PD-L1 peptide/monoclonal antibody and anticancer drug, it was not possible to inhibit or prevent the reuse of PD-L1 through the intracellular absorption process, and only significantly lower efficacy than the original immunotherapeutic effect could be obtained. However, in the present invention, by combining the PD-L1 peptide with the cathepsin B cleavage peptide and the anticancer drug, spherical nanoparticles are formed through self-assembly in the solution phase, while having stability for more than 30 days, and the process of PD-L1 absorption into cells By making the lysosome, the 'reuse' process was prevented, and the immunotherapeutic effect was significantly improved.
실험예 5. 전구약물(PD-NPs)의 암세포 특이적 항암 효능Experimental Example 5. Cancer cell-specific anticancer efficacy of prodrugs (PD-NPs)
전구약물(PD-NPs)의 암세포 특이적 항암 효능을 확인하기 위하여 유방암세포 4T1과 쥐 심근세포 H9C2를 이용하여 in vitro 세포실험을 진행하였다. In order to confirm the cancer cell-specific anticancer efficacy of prodrugs (PD-NPs), in vitro cell experiments were conducted using breast cancer cells 4T1 and mouse cardiomyocytes H9C2.
공초점 현미경용 셀 배양 플레이트(24웰)의 각 웰에 2 × 104개의 암세포(유방암세포 4T1)와 정상세포(쥐 심근세포 H9C2)를 분주하였다. 24 시간 안정화 후, 각각의 세포에 실시예 1의 전구약물(PD-NPs) 2 μM 또는 독소루비신(doxorubicin) 2 μM 농도로 처리하여 37 ℃ 인큐베이터에서 24 시간 동안 배양하였다. 배양이 완료된 후, 각 웰에 DPBS(Dulbecco's phosphate buffered Saline)를 처리하여 잔류 약물을 제거하고, 2 × 10 4 cancer cells (breast cancer cells 4T1) and normal cells (rat cardiomyocytes H9C2) were dispensed into each well of a cell culture plate (24 wells) for confocal microscopy. After stabilization for 24 hours, each cell was treated with 2 μM of the prodrug (PD-NPs) of Example 1 or 2 μM of doxorubicin and cultured in a 37° C. incubator for 24 hours. After the incubation is complete, each well is treated with DPBS (Dulbecco's phosphate buffered saline) to remove residual drug,
Dulbecco's phosphate buffered Saline(DPBS)로 잔류 약물을 제거하고, 세포 고정액을 15 분간 처리한 뒤, DAPI(4′,6-diamidino-2-phenylindole) 용액을 10분간 처리하여 세포의 핵을 염색하였다. 공초점 레이저 스캐닝 현미경(CLSM)으로 독소루비신(DOX)의 형광을 관찰하였다.Residual drug was removed with Dulbecco's phosphate buffered saline (DPBS), cell fixative was treated for 15 minutes, and then cell nuclei were stained with DAPI (4',6-diamidino-2-phenylindole) solution for 10 minutes. The fluorescence of doxorubicin (DOX) was observed with a confocal laser scanning microscope (CLSM).
도 11은 실시예 1의 전구약물(PD-NPs) 또는 독소루비신(DOX)을 처리한 암세포(4T1)와 정상세포(H9C2)의 공초점 레이저 스캐닝 현미경(CLSM) 결과이다.11 is a confocal laser scanning microscopy (CLSM) result of cancer cells (4T1) and normal cells (H9C2) treated with the prodrug (PD-NPs) or doxorubicin (DOX) of Example 1.
암세포는 정상세포보다 카텝신 B 발현량이 현저히 높다고 알려져 있고, 유방암세포(4T1)와 심근세포(H9C2)에서 카텝신 B의 발현량을 비교한 실제 실험에서도 암세포의 카텝신 B 발현량이 정상세포(H9C2)보다 32.48 ± 3.14배 더 많은 것을 확인하였다.It is known that cancer cells have a significantly higher expression level of cathepsin B than normal cells, and in an actual experiment comparing the expression level of cathepsin B in breast cancer cells (4T1) and cardiomyocytes (H9C2), the amount of cathepsin B expression in cancer cells was higher than normal cells (H9C2). ), 32.48 ± 3.14 times more than that.
도 11에 나타난 바와 같이, 실시예 1의 전구약물(PD-NPs)은 정상세포(H9C2)와 암세포(4T1) 모두에서 세포 내 흡수가 있음을 확인하였다. 다만 정상세포(H9C2)에서는 핵 내에 독소루비신(DOX)의 형광이 전혀 관찰되지 않고, 세포질에서만 독소루비신(DOX)의 형광이 관찰되었다. 반면 암세포(4T1)에서는 핵 내에서 다량의 독소루비신(DOX) 형광이 관찰되었다.As shown in FIG. 11, it was confirmed that the prodrug (PD-NPs) of Example 1 was absorbed into cells in both normal cells (H9C2) and cancer cells (4T1). However, in normal cells (H9C2), no fluorescence of doxorubicin (DOX) was observed in the nucleus, and fluorescence of doxorubicin (DOX) was observed only in the cytoplasm. On the other hand, in cancer cells (4T1), a large amount of doxorubicin (DOX) fluorescence was observed in the nucleus.
또한 독소루비신을 단독으로 사용하는 경우(Free DOX)에는 정상세포(H9C2)와 암세포(4T1) 모두에서 핵 내에 다량의 독소루비신(DOX) 형광이 관찰되었다.In addition, when doxorubicin was used alone (Free DOX), a large amount of doxorubicin (DOX) fluorescence was observed in the nucleus in both normal cells (H9C2) and cancer cells (4T1).
이를 통해, 실시예 1의 전구약물(PD-NPs)은 항암제를 암세포의 경우에만 핵 내로 진입하도록 함으로써 세포의 핵내에 존재하는 DNA와 결합하여 독성을 효과적으로 유발하도록 하고, 정상세포에는 세포 핵내로 항암제가 방출되지 않으므로 항암효과를 나타내지 않는 안정한 상태를 유지한다는 것을 확인할 수 있다.Through this, the prodrug (PD-NPs) of Example 1 allows the anticancer drug to enter the nucleus only in the case of cancer cells, thereby effectively inducing toxicity by binding to the DNA present in the cell nucleus, and in normal cells, the anticancer drug enters the cell nucleus. Since it is not released, it can be confirmed that it maintains a stable state that does not exhibit anticancer effect.
따라서 실시예 1의 전구약물(PD-NPs)은 항암제 단독과 달리 카텝신 B를 과발현하는 암세포에 대해서만 특이적으로 활성화된다는 것을 알 수 있다.Therefore, it can be seen that the prodrug (PD-NPs) of Example 1 is specifically activated only against cancer cells overexpressing cathepsin B, unlike the anticancer agent alone.
실험예 6. 전구약물(PD-NPs)의 암세포 특이적 세포 독성Experimental Example 6. Cancer cell-specific cytotoxicity of prodrugs (PD-NPs)
실시예 1의 전구약물(PD-NPs)의 처리가 암세포(4T1) 혹은 정상세포(H9C2)에서 세포소멸을 유도하는지 알아보기 위하여, CCK를 이용하여 세포소멸을 분석하였다.In order to determine whether treatment with the prodrug (PD-NPs) of Example 1 induces apoptosis in cancer cells (4T1) or normal cells (H9C2), apoptosis was analyzed using CCK.
암세포(4T1) 혹은 정상세포(H9C2)는 1% 페니실린/스트렙토마이신이 첨가된 DMEM 배지(GIBCO BRL;Rockville, MD)에 10% 혈청(FBS;GIBCO BRL)을 첨가하여 37 ℃에서 5% CO2 및 95% 공기의 습한 환경에서 배양하였다. Cancer cells (4T1) or normal cells (H9C2) were prepared by adding 10% serum (FBS; GIBCO BRL) to DMEM medium (GIBCO BRL; Rockville, MD) supplemented with 1% penicillin/streptomycin at 37 °C in 5% CO 2 and in a humid environment of 95% air.
96-well 셀 배양 플레이트의 well 당 2 × 104개의 암세포(4T1) 혹은 정상세포(H9C2)를 분주하였다. 24 시간 안정화 후, 각 웰에 실시예 1의 전구약물(PD-NPs) 또는 독소루비신(DOX)을 다양한 농도(0, 0.01, 0.1, 1 및 10 μM)로 처리하여 37 ℃ 인큐베이터에서 24 시간 동안 배양하였다. 그 다음, 각 웰에 CCK(Cell Counting Kit-8) 용액을 10 ㎍씩 처리하여 30분 배양하고, 각각의 96-웰 플레이트의 흡광도를 마이크로 플레이트 판독기(VERSAmaxTM, Molecular Devices Corp., Sunnyvale, CA)를 사용하여 450 nm에서 분석하였다. 2 × 10 4 cancer cells (4T1) or normal cells (H9C2) were dispensed per well of a 96-well cell culture plate. After stabilization for 24 hours, each well was treated with the prodrug (PD-NPs) or doxorubicin (DOX) of Example 1 at various concentrations (0, 0.01, 0.1, 1 and 10 μM) and cultured in a 37 ° C incubator for 24 hours. did Then, each well was treated with 10 μg of CCK (Cell Counting Kit-8) solution, incubated for 30 minutes, and the absorbance of each 96-well plate was measured using a microplate reader (VERSAmaxTM, Molecular Devices Corp., Sunnyvale, CA). was analyzed at 450 nm.
통계분석을 위해 Student's t test를 이용하였고, *P < 0.05, **P < 0.01, ***P < 0.001로 유효성을 표기하였다.Student's t test was used for statistical analysis, and efficacy was expressed as *P < 0.05, **P < 0.01, and ***P < 0.001.
도 12는 농도별 실시예 1의 전구약물(PD-NPs)을 처리한 암세포(4T1) 혹은 정상세포(H9C2)의 생존율(cell viability, %)을 나타낸 그래프이고, 도 13은 농도별 독소루비신(DOX)만을 처리한 암세포(4T1) 혹은 정상세포(H9C2)의 생존율(cell viability, %)을 나타낸 그래프이다.12 is a graph showing the cell viability (%) of cancer cells (4T1) or normal cells (H9C2) treated with the prodrug (PD-NPs) of Example 1 by concentration, and FIG. 13 is a graph showing doxorubicin (DOX) by concentration. ) is a graph showing the survival rate (cell viability, %) of cancer cells (4T1) or normal cells (H9C2) treated only.
도 12에 나타난 바와 같이, 실시예 1의 전구약물(PD-NPs)은 암세포(4T1)에서만 선택적 독성을 나타내며, 정상세포(H9C2)에 대해서는 전혀 독성을 나타내지 않는 것을 확인하였다. As shown in FIG. 12, it was confirmed that the prodrug (PD-NPs) of Example 1 showed selective toxicity only to cancer cells (4T1) and showed no toxicity to normal cells (H9C2).
실시예 1의 전구약물(PD-NPs)은 암세포(4T1)에서 IC50가 3.01 μM이였으나, H9C2 세포에서는 IC50가 > 200 μM인 것으로 확인되었다. 실시예 1의 전구약물(PD-NPs)은 암세포에 대해 선택적이고 특이적이며 치명적인 독성을 나타내나, 정상세포에 대해서는 현저히 낮은 독성을 갖는다는 것을 의미하며, 정상세포에 대한 독성과 암세포에 대한 독성 차이가 70배 이상 유의적인 효과 차이가 있다는 것을 확인할 수 있다. 특히 실시예 1의 전구약물(PD-NPs)은 0.1 내지 10 μM에서 암세포는 사멸시키나, 정상세포에는 전혀 영향을 미치지 않는 것을 확인할 수 있다.The prodrug of Example 1 (PD-NPs) had an IC 50 of 3.01 μM in cancer cells (4T1), but an IC 50 of > 200 μM in H9C2 cells. The prodrugs (PD-NPs) of Example 1 exhibit selective, specific, and lethal toxicity to cancer cells, but have remarkably low toxicity to normal cells. Toxicity to normal cells and toxicity to cancer cells It can be confirmed that there is a significant effect difference of more than 70 times. In particular, it can be seen that the prodrug (PD-NPs) of Example 1 kills cancer cells at 0.1 to 10 μM, but does not affect normal cells at all.
도 13에 나타난 바와 같이, 가장 널리 사용되는 항암제인 독소루비신(DOX)는 암세포(4T1)와 정상세포(H9C2) 모두에서 세포독성이 나타난다는 것을 확인할 수 있다. 특히 독소루비신은 0.01 μM의 농도에서도 암세포와 정상세포를 모두 사멸시키고 있다는 것을 확인할 수 있는 바, 적은 농도로도 치명적인 부작용을 야기할 수 있다는 것을 알 수 있다.As shown in FIG. 13, it can be confirmed that doxorubicin (DOX), the most widely used anticancer agent, exhibits cytotoxicity in both cancer cells (4T1) and normal cells (H9C2). In particular, it can be confirmed that doxorubicin kills both cancer cells and normal cells even at a concentration of 0.01 μM, and it can be seen that even a small concentration can cause fatal side effects.
종합하면, 실시예 1의 전구약물(PD-NPs)은 암세포에 선택적이고 특이적인 항암효과를 나타내고, 정상세포에 대해서는 고농도(10 μM 이상/초과)에서도 최소한의 독성만을 나타내므로, 최소한의 부작용을 갖는 안정한 암 예방 또는 치료용 조성물로 활용될 수 있음을 알 수 있다.In summary, the prodrug (PD-NPs) of Example 1 exhibits a selective and specific anticancer effect on cancer cells, and exhibits minimal toxicity even at high concentrations (10 μM or more/exceeding) on normal cells, resulting in minimal side effects. It can be seen that it can be used as a stable composition for preventing or treating cancer.
실험예 7. 전구약물(PD-NPs)의 면역원성 세포사멸 효능 분석Experimental Example 7. Analysis of immunogenic apoptosis efficacy of prodrugs (PD-NPs)
암세포의 면역원성 세포사멸(immunogenic cell death; ICD)은 일부 항암제 계열(anthracycline 계, 옥살리플라틴(oxaliplatin), 보르테조밉(bortezomib) 등)에 의해 촉진될 수 있다. 이러한 ICD 유발은 사멸한 dying/dead 암세포는 항원으로 작용하여 수지상 세포를 활성화하여 지속적이고 강력한 항암 효능을 유도한다. ICD가 유도된 암세포는 DAMPs(damage-associated molecular patterns)로 통칭하는 ICD 특이적인 표지자를 세포 표면에 발현하거나 세포 밖으로 방출한다. ICD 표지자로는 칼레티쿨린(calreticulin, CRT), ATP, HMGB1와 같은 DAMP가 널리 알려져 있다. 상기 DAMP는 DC 표면의 수용체(CD91, P2RX7/P2RY2, IFNAR, TLR)에 결합하여 DC를 활성화하여 항원 제시능을 강화하고 공동 자극 수용체 발현을 증가시켜 면역반응을 유도할 수 있게 된다.Immunogenic cell death (ICD) of cancer cells can be promoted by some anticancer drugs (anthracycline, oxaliplatin, bortezomib, etc.). This ICD induces dying/dead cancer cells to act as antigens and activate dendritic cells to induce sustained and strong anticancer efficacy. ICD-induced cancer cells express ICD-specific markers collectively referred to as DAMPs (damage-associated molecular patterns) on the cell surface or release them outside the cell. DAMPs such as calreticulin (CRT), ATP, and HMGB1 are widely known as ICD markers. The DAMP binds to receptors (CD91, P2RX7/P2RY2, IFNAR, TLR) on the DC surface, activates the DC, enhances antigen presenting ability, and increases co-stimulatory receptor expression, thereby inducing an immune response.
실시예 1의 전구약물(PD-NPs)의 처리가 암세포(4T1)에서 면역원성 세포사멸(ICD)을 유도하는지 알아보기 위하여, Calreticulin, HSP70, HMGB1, ATP 발현을 이용하여 분석하였다.In order to determine whether treatment with the prodrug (PD-NPs) of Example 1 induces immunogenic cell death (ICD) in cancer cells (4T1), calreticulin, HSP70, HMGB1, and ATP expression were analyzed.
유방암세포(4T1)는 1% 페니실린/스트렙토마이신이 첨가된 DMEM 배지(GIBCO BRL;Rockville, MD)에 10% 혈청(FBS;GIBCO BRL)을 첨가하여 37 ℃에서 5% CO2 및 95% 공기의 습한 환경에서 배양하였다. 96-well 셀 배양 플레이트의 well 당 2 × 104개의 암세포(4T1)를 분주하였다. 24 시간 안정화 후, 각 웰에 실시예 1의 전구약물(PD-NPs) 2 μM 또는 독소루비신(DOX)을 2 μM 또는 생리식염수(con)를 처리하여 37 ℃ 인큐베이터에서 24 시간 동안 배양하여, 실시예 1의 전구약물(PD-NPs), 독소루비신(DOX) 또는 생리식염수(con)로 처리한 암세포를 각각 제조하였다. 상기 각 세포에 대하여 칼레티쿨린(CRT), HMGB1, ATP 발현을 다음과 같이 분석하였다.Breast cancer cells (4T1) were prepared by adding 10% serum (FBS; GIBCO BRL) to DMEM medium (GIBCO BRL; Rockville, MD) supplemented with 1% penicillin/streptomycin at 37 °C in 5% CO 2 and 95% air. Cultivated in a humid environment. 2 × 10 4 cancer cells (4T1) were dispensed per well of a 96-well cell culture plate. After stabilization for 24 hours, each well was treated with 2 μM of the prodrug (PD-NPs) of Example 1 or 2 μM of doxorubicin (DOX) or physiological saline (con) and cultured in a 37 ° C. incubator for 24 hours, Cancer cells treated with the prodrug of 1 (PD-NPs), doxorubicin (DOX) or physiological saline (con) were prepared, respectively. The expression of calreticulin (CRT), HMGB1, and ATP in each of the cells was analyzed as follows.
칼레티쿨린(CRT) 측정은, 상기 각 세포를 DAPI와 APC-conjugated CRT 항체로 12 시간 동안 염색한 후, 공초점 형광 현미경(Confocal fluorescence microscope)로 관찰하였다.For calreticulin (CRT) measurement, each of the cells was stained with DAPI and APC-conjugated CRT antibody for 12 hours, and then observed under a confocal fluorescence microscope.
HSP70, HMGB1(High-mobility group box 1) 측정은, APC-conjugated CRT 항체로 세포를 염색하기 전, 각 세포로부터 배양액을 회수하고, 웨스턴 블롯을 통해 발현을 분석하였다. 구체적으로 실시예 1의 전구약물(PD-NPs), 독소루비신(DOX) 또는 생리식염수(con)로 처리한 암세포로부터 배양액을 회수한 후, 4 ℃에서 30 분 동안 RIPA 완충액(Cell Signaling Technology)을 처리하고, 용해물의 단백질 양을 Pierce BCA protein assay kit(Thermo Fisher Scientific)를 사용하여 제조사의 프로토콜대로 분석하였다. SDS-PAGE 후, 단백질을 PVDF 막(Bio-Rad)에 옮긴 다음, 막을 5% skim milk와 0.1% Tween-20를 포함하는 TBS 완충액(Tris-buffered saline; TBS-T)으로 처리하였다. 그리고 1차 항체와 함께 4 ℃에서 밤새 배양하였다: HSP70, HMGB1 항체를 사용하였다. 막을 TBS-T로 세척하고 2차 항체로 상온에서 1 시간동안 배양하였다. 막을 TBS-T로 세척하고, EZ-Western Lumi Pico 또는 Femto reagent(DoGen)로 처리하였다. 밴드 세기를 측정하기 위하여 Fusion-Solo software(Vilber)을 사용하여 제조사의 프로토콜대로 분석하였다.To measure HSP70 and HMGB1 (High-mobility group box 1), the culture medium was collected from each cell before staining the cells with APC-conjugated CRT antibody, and the expression was analyzed through Western blotting. Specifically, after recovering the culture medium from the cancer cells treated with the prodrug (PD-NPs), doxorubicin (DOX) or physiological saline (con) of Example 1, RIPA buffer (Cell Signaling Technology) was treated for 30 minutes at 4 ° C. and the amount of protein in the lysate was analyzed using the Pierce BCA protein assay kit (Thermo Fisher Scientific) according to the manufacturer's protocol. After SDS-PAGE, the proteins were transferred to a PVDF membrane (Bio-Rad), and then the membrane was treated with TBS buffer (Tris-buffered saline; TBS-T) containing 5% skim milk and 0.1% Tween-20. And incubated overnight at 4 °C with primary antibodies: HSP70, HMGB1 antibodies were used. The membrane was washed with TBS-T and incubated with the secondary antibody for 1 hour at room temperature. Membranes were washed with TBS-T and treated with EZ-Western Lumi Pico or Femto reagent (DoGen). In order to measure the band intensity, Fusion-Solo software (Vilber) was used and analyzed according to the manufacturer's protocol.
ATP(Adenosine triphosphate) 측정은, 실시예 1의 전구약물(PD-NPs), 독소루비신(DOX) 또는 생리식염수(con)로 처리한 암세포를 준비하고, 상용화된 ATP 분석 키트(Beyotime Biotechnology)를 사용하여 제조사의 프로토콜에 따라 분석을 수행하였다.ATP (Adenosine triphosphate) was measured by preparing cancer cells treated with the prodrug (PD-NPs), doxorubicin (DOX) or physiological saline (con) of Example 1, and using a commercially available ATP assay kit (Beyotime Biotechnology). The assay was performed according to the manufacturer's protocol.
실험의 통계분석을 위하여 일원 분산 분석(one-way ANOVA test)을 사용하여 그룹 간 평균수치 유의차를 확인하였으며, 0.05보다 낮은 p 값을 가질 경우에는 *, 0.01보다 낮은 p 값을 가질 경우에는 **, 0.001보다 낮은 p 값을 가질 경우에는 ***로, 유의적 차이가 없는 경우는 N.S로 표기하였다. 에러바는 S.D(표준편차)를 나타낸다.For the statistical analysis of the experiment, a one-way ANOVA test was used to confirm the significant difference in mean values between groups. In the case of a p value lower than 0.05, *, and in the case of a p value lower than 0.01, * *, *** when the p value was lower than 0.001, and N.S when there was no significant difference. Error bars represent SD (standard deviation).
도 14a는 실시예 1의 전구약물(PD-NPs), 독소루비신(DOX) 또는 생리식염수(con)로 처리한 암세포를 DAPI와 APC-conjugated CRT 항체로 염색하고 공초점 형광 현미경(Confocal fluorescence microscope)으로 촬영한 사진이다.14a shows cancer cells treated with the prodrug (PD-NPs), doxorubicin (DOX), or physiological saline (con) of Example 1 were stained with DAPI and APC-conjugated CRT antibodies and analyzed using a confocal fluorescence microscope. this is a picture taken
도 14a에 나타난 바와 같이, 실시예 1의 전구약물(PD-NPs)이 처리된 암세포의 표면에 다량의 칼레티쿨린(CRT)이 발현되었음을 확인할 수 있다. 독소루비신을 처리한 암세포(4T1)의 표면에서도 칼레티쿨린(CRT)이 다량 발현되었다. 생리식염수를 처리한 암세포(con)에 비해서는 유의적으로 현저하게 증가하였으나, DOX와 PD-NPs는 유의적 차이가 없었다.As shown in FIG. 14a, it can be confirmed that a large amount of calreticulin (CRT) was expressed on the surface of cancer cells treated with the prodrug (PD-NPs) of Example 1. A large amount of calreticulin (CRT) was also expressed on the surface of doxorubicin-treated cancer cells (4T1). It was significantly increased compared to cancer cells (con) treated with physiological saline, but there was no significant difference between DOX and PD-NPs.
도 14b는 실시예 1의 전구약물(PD-NPs), 독소루비신(DOX) 또는 생리식염수(con)로 처리한 암세포로부터 방출되는 HMGB1 발현량 및 ATP 발현량을 측정하여 나타낸 그래프이다.Figure 14b is a graph showing the measurement of HMGB1 expression level and ATP expression level released from cancer cells treated with the prodrug (PD-NPs), doxorubicin (DOX), or physiological saline (con) of Example 1.
도 14b에 나타난 바와 같이, 실시예 1의 전구약물(PD-NPs)이 처리된 암세포로부터 다량의 HMGB1과 ATP이 방출되는 것을 확인할 수 있다. 독소루비신을 처리한 암세포(4T1)에서도 HMGB1, ATP이 다량 발현되었다. 생리식염수를 처리한 암세포(con)에 비해서는 유의적으로 현저하게 증가하였으나, DOX와 PD-NPs는 유의적 차이가 없었다. 실시예 1의 전구약물(PD-NPs)은 종래 항암제와 유사한 정도의 면역원성 세포사멸(ICD)이 발생한다는 것을 확인할 수 있다. 그러나 본원발명의 전구약물(PD-NPs)는 종래 항암제와 달리 정상세포에 대해서는 독성을 나타내지 않는 안정한 물질이라는 장점을 갖는다.As shown in FIG. 14b, it can be confirmed that a large amount of HMGB1 and ATP are released from the cancer cells treated with the prodrug (PD-NPs) of Example 1. HMGB1 and ATP were also expressed in large amounts in doxorubicin-treated cancer cells (4T1). It was significantly increased compared to cancer cells (con) treated with physiological saline, but there was no significant difference between DOX and PD-NPs. It can be confirmed that the prodrug (PD-NPs) of Example 1 causes immunogenic apoptosis (ICD) similar to that of conventional anticancer drugs. However, unlike conventional anticancer drugs, the prodrugs (PD-NPs) of the present invention have the advantage of being stable substances that do not exhibit toxicity to normal cells.
실험예 8. 전구약물(PD-NPs)의 T 세포 활성 효능 분석Experimental Example 8. Analysis of T cell activation efficacy of prodrugs (PD-NPs)
실시예 1의 전구약물(PD-NPs)의 처리가 암세포(4T1) 표면의 PD-L1을 차단하여 T 세포의 암세포 인식을 유도하는지 알아보기 위하여, IFN-γ 발현을 ELISA으로 분석하였다.To investigate whether treatment with the prodrug (PD-NPs) of Example 1 induces T cell recognition of cancer cells by blocking PD-L1 on the surface of cancer cells (4T1), IFN-γ expression was analyzed by ELISA.
유방암세포(4T1)는 1% 페니실린/스트렙토마이신이 첨가된 DMEM 배지(GIBCO BRL;Rockville, MD)에 10% 혈청(FBS;GIBCO BRL)을 첨가하여 37 ℃에서 5% CO2 및 95% 공기의 습한 환경에서 배양하였다. 96-well 셀 배양 플레이트의 well 당 2 × 104개의 암세포(4T1)를 분주하였다. 24 시간 안정화 후, 각 웰에 실시예 1의 전구약물(PD-NPs) 2 μM 또는 PD-L1 단일클론항체(aPD-L1 Ab) 2 μM 또는 PD-L1 결합 펩타이드(CVRARTR(서열 3); aPD-L1 Pep) 2 μM을 처리하여 37 ℃ 인큐베이터에서 24 시간 동안 배양하였다. 배양이 완료된 후 T 세포와 24시간 공동배양하였다. 상기 공동배양액에서 IFN-γ를 ELISA법으로 분석하였다.Breast cancer cells (4T1) were prepared by adding 10% serum (FBS; GIBCO BRL) to DMEM medium (GIBCO BRL; Rockville, MD) supplemented with 1% penicillin/streptomycin at 37 °C in 5% CO 2 and 95% air. Cultivated in a humid environment. 2 × 10 4 cancer cells (4T1) were dispensed per well of a 96-well cell culture plate. After stabilization for 24 hours, 2 μM of the prodrugs of Example 1 (PD-NPs) or 2 μM of PD-L1 monoclonal antibody (aPD-L1 Ab) or PD-L1 binding peptide (CVRARTR (SEQ ID NO: 3); aPD -L1 Pep) 2 μM and incubated for 24 hours in a 37 ℃ incubator. After the culture was completed, it was co-cultured with T cells for 24 hours. IFN-γ was analyzed in the co-culture medium by ELISA method.
도 15는 실시예 1의 전구약물(PD-NPs), PD-L1 단일클론항체(aPD-L1 Ab), PD-L1 결합 펩타이드(aPD-L1 Pep)를 처리한 각 암세포(4T1)를 T 세포와 공배양시 배양액에서 관측되는 INF-γ의 농도를 ELISA로 측정한 결과를 나타낸 그래프이다. 이때 대조군(Co-culture)으로 아무것도 처리하지 않은 암세포(4T1)과 T 세포의 공동배양액을 사용하였다.15 is a T cell treatment of each cancer cell (4T1) treated with the prodrug (PD-NPs), PD-L1 monoclonal antibody (aPD-L1 Ab), and PD-L1 binding peptide (aPD-L1 Pep) of Example 1. It is a graph showing the results of measuring the concentration of INF-γ observed in the culture medium during co-culture with ELISA. At this time, a co-culture of untreated cancer cells (4T1) and T cells was used as a control (Co-culture).
도 15에 나타난 바와 같이, 실시예 1의 전구약물(PD-NPs), PD-L1 단일클론항체(aPD-L1 Ab) 및 PD-L1 결합 펩타이드(CVRARTR; aPD-L1 Pep)를 처리한 공동배양액은 대조군(Co-culture)보다 IFN-γ 발현양이 유의적으로 증가하는 것을 관찰할 수 있었다.As shown in FIG. 15, the co-culture solution treated with the prodrug (PD-NPs) of Example 1, the PD-L1 monoclonal antibody (aPD-L1 Ab), and the PD-L1 binding peptide (CVRARTR; aPD-L1 Pep) It was observed that the amount of IFN-γ expression was significantly increased compared to the control group (Co-culture).
즉, 실시예 1의 전구약물(PD-NPs)은, PD-L1 단일클론항체(aPD-L1 Ab) 및 PD-L1 결합 펩타이드(CVRARTR; aPD-L1 Pep)보다 복합한 구조일 뿐만 아니라, 용액 상에서는 자기조립 나노입자 형태임에도 불구하고, 암세포 표면의 PD-L1을 효율적으로 차단하여 T 세포의 암세포 인식을 증가시킬 수 있다는 것을 확인하였다. 실시예 1의 전구약물(PD-NPs)은 암세포 표면의 PD-L1을 차단하여 T 세포가 면역활성 사이토카인 분비능이 증가할 수 있도록 하는 뛰어난 특성을 가지고 있으므로, 추가적인 항암 면역 반응을 유도할 수 있다는 것을 확인하였다.That is, the prodrug (PD-NPs) of Example 1 is not only more complex than the PD-L1 monoclonal antibody (aPD-L1 Ab) and PD-L1 binding peptide (CVRARTR; aPD-L1 Pep), In the above, it was confirmed that despite being in the form of self-assembling nanoparticles, cancer cell recognition by T cells can be increased by efficiently blocking PD-L1 on the surface of cancer cells. The prodrugs (PD-NPs) of Example 1 have the excellent property of blocking PD-L1 on the surface of cancer cells so that T cells can increase the ability to secrete cytokines that activate the immune system. confirmed that
실시예 1의 전구약물(PD-NPs)의 항암 면역 반응 유도 효과는 PD-L1 단일클론항체(aPD-L1 Ab) 및 PD-L1 결합 펩타이드(CVRARTR; aPD-L1 Pep)와 유사한 수준이라는 것을 알 수 있다.It can be seen that the anticancer immune response inducing effect of the prodrugs (PD-NPs) of Example 1 is similar to that of the PD-L1 monoclonal antibody (aPD-L1 Ab) and the PD-L1 binding peptide (CVRARTR; aPD-L1 Pep). can
실험예 9. 전구약물(PD-NPs)의 생체 내 거동 평가Experimental Example 9. In vivo behavior evaluation of prodrugs (PD-NPs)
본 발명에서의 동물실험은 한국과학기술연구원(KIST)의 지침에 따라 수행되었으며, 기관윤리회(institutional committees)로부터 승인을 받았다. 동물모델은 Nara Bio INC(경기도, Korea)으로 구입한 BALB/c 마우스(5.5 주령, 20-25 g, 암컷)를 이용하여 수행하였다. 상기 마우스에 1 × 107 개의 암세포(4T1)를 왼쪽 허벅지에 접종(n=6)하여 암 동물모델을 제작하였다. 암 크기가 250-300 ㎣이 되면, 각 동물모델의 꼬리 정맥을 통해 실시예 1의 전구약물(PD-NPs; 독소루비신 3 mg/kg에 해당하는 몰용량), 비교예 1의 전구약물(독소루비신 3 mg/kg에 해당하는 몰용량) 또는 독소루비신(DOX)(3 mg/kg)을 주입하였다. 생체 내 전구약물(PD-NPs)의 종양 축적 효능을 평가하기 위하여 각각의 약물을 투여하고 24 시간이 지난 후, 생체 내 형광 분석 장비 IVIS Luminar III를 사용하여 NIRF(noninvasive near-infrared fluorescence) 이미징 데이터를 얻었다. 상기 분석을 통해 도출된 이미지의 종양 부위의 형광 세기를 Living image software를 통해 정량 분석하였다.Animal experiments in the present invention were performed according to the guidelines of the Korea Institute of Science and Technology (KIST) and approved by the institutional committees. Animal models were performed using BALB/c mice (5.5 weeks old, 20-25 g, female) purchased from Nara Bio INC (Gyeonggi-do, Korea). A cancer animal model was prepared by inoculating 1 × 10 7 cancer cells (4T1) into the left thigh of the mouse (n=6). When the size of the cancer reaches 250-300 ㎣, the prodrug of Example 1 (PD-NPs; molar dose corresponding to 3 mg/kg of doxorubicin) and the prodrug of Comparative Example 1 (
3개 군의 동물모델3 groups of animal models
1군(Dox) : 암 동물모델의 꼬리 정맥을 통해 독소루비신 3 mg/kg 투여하고, 24 시간 경과Group 1 (Dox):
2군(실시예 1) : 암 동물모델의 꼬리 정맥을 통해 실시예 1의 전구약물(PD-NPs; 독소루비신 3 mg/kg에 해당하는 몰용량) 투여하고, 24 시간 경과Group 2 (Example 1): The prodrug of Example 1 (PD-NPs; molar dose corresponding to 3 mg/kg of doxorubicin) was administered through the tail vein of a cancer animal model, and 24 hours elapsed
3군(비교예 1) : 암 동물모델의 꼬리 정맥을 통해 비교예 1의 전구약물(독소루비신 3 mg/kg에 해당하는 몰용량) 투여하고, 24 시간 경과Group 3 (Comparative Example 1): The prodrug of Comparative Example 1 (molar dose corresponding to 3 mg/kg of doxorubicin) was administered through the tail vein of a cancer animal model, and 24 hours elapsed
통계분석을 위해 Student's t test를 이용하였고, *P < 0.05, **P < 0.01, ***P < 0.001로 유효성을 표기하였다.Student's t test was used for statistical analysis, and efficacy was expressed as *P < 0.05, **P < 0.01, and ***P < 0.001.
도 16은 독소루비신, 실시예 1의 전구약물(PD-NPs) 또는 비교예 1의 전구약물을 정맥 투여한 암 동물모델(1군, 2군, 3군)에 대한 NIRF(noninvasive near-infrared fluorescence) 이미지이고, 도 17은 도 16의 NIRF 이미지에서 종양 조직에서의 형광을 정량적으로 분석하여 나타낸 그래프이다.16 is noninvasive near-infrared fluorescence (NIRF) analysis of cancer animal models (
도 16 및 17에 나타난 바와 같이, 생체 내에서 실시예 1의 전구약물(PD-NPs)은 비교예 1의 전구약물 및 독소루비신에 비하여 3배 및 4배 더 많은 양이 종양 조직에 축적되었다.As shown in FIGS. 16 and 17 , the prodrug of Example 1 (PD-NPs) accumulated in tumor tissue in vivo in 3- and 4-fold greater amounts than the prodrug of Comparative Example 1 and doxorubicin.
독소루비신과 같이 항암제를 단독으로 사용할 경우, 저분자 구조로 인해 생체 내에서 종양 축적 효율이 매우 낮은 것을 알 수 있다. 비교예 1의 전구약물은 나노입자의 안정성이 낮으므로, 매우 낮은 종양 축적 효율을 갖는다는 것을 확인할 수 있다.It can be seen that when an anticancer agent such as doxorubicin is used alone, the tumor accumulation efficiency in vivo is very low due to its low molecular structure. Since the prodrug of Comparative Example 1 has low nanoparticle stability, it can be confirmed that it has a very low tumor accumulation efficiency.
실험예 1의 전구약물(PD-NPs)은 용액 내에서 매우 안정적인 나노입자를 형성할 뿐만 아니라 다양한 기전을 통해 종양세포를 표적화할 수 있으므로, 실제 생체 내에서도 비교예 1의 전구약물과 독소루비신 보다 유의적으로 현저히 우수한 종양 축적 효율을 갖는다는 것을 알 수 있다. Since the prodrug (PD-NPs) of Experimental Example 1 not only forms very stable nanoparticles in solution but also can target tumor cells through various mechanisms, it is significantly more effective than the prodrug of Comparative Example 1 and doxorubicin even in vivo. It can be seen that it has a remarkably excellent tumor accumulation efficiency.
실험예 10. 전구약물(PD-NPs)의 항암효과와 독성 평가Experimental Example 10. Anticancer effect and toxicity evaluation of prodrugs (PD-NPs)
본 발명의 전구약물(PD-NPs)은 생체 내 투여시, 자체적으로 나노입자를 형성함으로써, 암 조직에 축적됨과 동시에 높은 카텝신 B 선택성으로 인해 정상 세포에 대한 안정성을 확보할 수 있다는 것을 세포 실험을 통해 확인하였다. 이에 본 발명의 전구약물(PD-NPs)이 생체 내에서도 항암 효과와 정상 세포에 대한 안정성(부작용)을 갖는지 확인하고자 하였다.Cell experiments showed that the prodrugs (PD-NPs) of the present invention, when administered in vivo, by forming nanoparticles on their own, accumulate in cancer tissues and secure stability against normal cells due to their high cathepsin B selectivity. confirmed through Therefore, we tried to confirm whether the prodrugs (PD-NPs) of the present invention have anticancer effects and stability (side effects) to normal cells in vivo.
구체적으로 본 발명에서의 동물실험은 한국과학기술연구원(KIST)의 지침에 따라 수행되었으며, 기관윤리회(institutional committees)로부터 승인을 받았다. 동물모델은 Nara Bio INC(경기도, Korea)으로 구입한 BALB/c 마우스(5.5 주령, 20-25 g, 암컷)를 이용하여 수행하였다. 상기 마우스에 1 X 107 개의 MDA-MB-231 세포를 왼쪽 허벅지에 접종(n=6)하여 암 동물모델을 제작하였고, 5 주 후 암의 크기가 50-100 ㎣ 일 때 실험을 실시하였다.Specifically, animal experiments in the present invention were performed according to the guidelines of the Korea Institute of Science and Technology (KIST) and approved by the institutional committees. Animal models were performed using BALB/c mice (5.5 weeks old, 20-25 g, female) purchased from Nara Bio INC (Gyeonggi-do, Korea). A cancer animal model was prepared by inoculating 1
암 동물모델의 암조직의 크기가 50-100 ㎣ 일 때, 각 동물모델의 꼬리 정맥을 통해 하기에 기술된 바와 같이 주입하였다(6개 군의 동물 모델). 주입한 직후(0 day)부터 각 군의 체중과 암 조직의 부피 변화를 2일마다 측정하였고, 동시에 생존율을 분석하였다. 암 조직의 부피(V; mm3)는 0.53 × 최대 지름 × (최소 지름)2 으로 계산하였다. When the size of the cancer tissue in the cancer animal model was 50-100
6개 군의 동물모델6 groups of animal models
1군(Saline) : 암 동물모델의 꼬리 정맥을 통해 생리식염수 200 μL 투여Group 1 (Saline): Administration of 200 μL of physiological saline through the tail vein of cancer animal models
2군(PD-L1 peptide) : 암 동물모델의 꼬리 정맥을 통해 서열번호 3으로 표시되는 PD-L1 결합 펩타이드(CVRARTR)를 3 mg/kg 투여Group 2 (PD-L1 peptide): 3 mg/kg of PD-L1 binding peptide (CVRARTR) represented by SEQ ID NO: 3 was administered through the tail vein of a cancer animal model
3군(DOX) : 암 동물모델의 꼬리 정맥을 통해 독소루비신을 3 mg/kg 투여Group 3 (DOX): Doxorubicin administered at 3 mg/kg through the tail vein of cancer animal models
4군(DOX+PD-L1 Ab) : 암 동물모델의 꼬리 정맥을 통해 독소루비신 3 mg/kg 투여하고. 복강을 통해 PD-L1 단일클론항체 10 mg/kg를 병용 투여Group 4 (DOX+PD-L1 Ab):
5군(비교예 1) : 암 동물모델의 꼬리 정맥을 통해 비교예 1의 전구약물(독소루비신 3 mg/kg에 해당하는 몰용량) 투여Group 5 (Comparative Example 1): Administration of the prodrug of Comparative Example 1 (molar dose corresponding to 3 mg/kg of doxorubicin) through the tail vein of a cancer animal model
6군(PD-NPs) : 암 동물모델의 꼬리 정맥을 통해 실시예 1의 전구약물(독소루비신 3 mg/kg에 해당하는 몰용량) 투여Group 6 (PD-NPs): Administration of the prodrug of Example 1 (molar dose corresponding to 3 mg/kg of doxorubicin) through the tail vein of a cancer animal model
도 18은 1군 내지 6군에서 암의 부피(V; mm3) 시간별로 측정하여 나타낸 그래프이고, 도 19는 1군 내지 6군에서 시간별로 체중을 측정하여 나타낸 그래프이며, 도 20은 1군 내지 6군에서 시간별로 생존율을 측정하여 나타낸 그래프이다.18 is a graph showing the measurement of cancer volume (V; mm 3 ) over time in
도 18에 나타난 바와 같이, 실시예 1의 전구약물(PD-NPs)을 처리한 6군에서는 암의 크기가 271.1 ± 71.5 mm3 이였고, 1군부터 5군까지는 순서대로 암의 크기가 각각 1655.9 ± 320 mm3, 1209 ± 120.3 mm3, 1028.7 ± 218.2 mm3, 641.5 ± 97.2 mm3 및 724 ± 77.6 mm3인 것으로 확인되었다. 실시예 1의 전구약물(PD-NPs)은 다른 모든 군보다 암 성장을 유의적으로 현저하게 억제한다는 것을 확인할 수 있다.As shown in FIG. 18, in
도 19에 나타난 바와 같이, 실시예 1의 전구약물(PD-NPs)을 처리한 6군에서는 유의적인 체중변화가 관찰되지 않으나, 독소루비신이 처리된 3군, 독소루비신과 PD-L1 항체가 병용처리된 4군 및 비교예 1의 전구약물이 처리된 5군은 체중이 현저하게 감소하는 것을 확인하였다. 즉 독소루비신은 심각한 전신독성을 야기하여 16일 내에 사망에 이르고, 비교예 1의 전구약물은 낮은 나노입자 안정성으로 인해 생체 내에서 비특이적 독성을 나타낸다는 것을 알 수 있다. 실시예 1의 전구약물(PD-NPs)은 전신 독성을 나타내지 않으면서 우수한 항암효과를 달성할 수 있음을 확인하였다.As shown in FIG. 19, no significant weight change was observed in
도 20에 나타난 바와 같이, 실시예 1의 전구약물(PD-NPs)을 처리한 6군에서는 사망 개체가 없이 30일 이상 생존하는 것을 확인하였다. 이에 반해 독소루비신이 처리된 3군, 독소루비신과 PD-L1 항체가 병용처리된 4군 및 비교예 1의 전구약물이 처리된 5군은 암의 성장이 아닌 약의 독성으로 인해 각각 24일, 22일 및 26일 안에 모두 사망에 도달하는 것을 확인하였다. 생리식염수를 처리한 1군(대조군; Saline) 및 PD-L1 결합 펩타이드를 처리한 2군의 경우 암의 성장으로 인해 각각 16일 및 18일째부터 사망이 발생하였고, 22일 내에 암으로 인해 모두 사망하는 것을 확인하였다.As shown in FIG. 20, in the
본 발명에 따른 전구약물(PD-NPs)은 생체 내에서 자기조립을 통해 나노입자를 형성하며, 상술한 구조적 특징으로 인해 정상조직에 대해 부작용을 전혀 야기하지 않으면서, 암 조직의 성장만을 효과적으로 억제하므로, 화학요법적으로 안정적인 항암 예방, 치료 효능을 제공할 수 있다는 것을 확인하였다.The prodrug (PD-NPs) according to the present invention forms nanoparticles through self-assembly in vivo, and due to the above-described structural characteristics, it effectively inhibits only the growth of cancer tissues without causing any side effects to normal tissues. , it was confirmed that chemotherapy can provide stable anti-cancer prevention and treatment efficacy.
실험예 11. 전구약물(PD-NPs)의 면역 치료 효능 평가Experimental Example 11. Immunotherapy efficacy evaluation of prodrugs (PD-NPs)
실제 생체 내에서 전구약물(PD-NPs)이 암세포의 면역원성 세포사멸을 유도하여 암 조직으로의 T 세포 모집을 촉진하는 항암 면역 치료 효능을 나타내는지 확인하고자 하였다.It was intended to confirm whether prodrugs (PD-NPs) show anticancer immunotherapeutic efficacy by inducing immunogenic apoptosis of cancer cells in vivo and promoting T cell recruitment to cancer tissues.
본 발명에서의 동물실험은 한국과학기술연구원(KIST)의 지침에 따라 수행되었으며, 기관윤리회(institutional committees)로부터 승인을 받았다. 동물모델은 Nara Bio INC(경기도, Korea)으로 구입한 BALB/c 마우스(5.5 주령, 20-25 g, 암컷)를 이용하여 수행하였다. 상기 마우스에 1 × 107 개의 MDA-MB-231 세포를 왼쪽 허벅지에 접종(n=6)하여 암 동물모델을 제작하였고, 5 주 후 암의 크기가 50-100 ㎣ 일 때 실험을 실시하였다.Animal experiments in the present invention were performed according to the guidelines of the Korea Institute of Science and Technology (KIST) and approved by the institutional committees. Animal models were performed using BALB/c mice (5.5 weeks old, 20-25 g, female) purchased from Nara Bio INC (Gyeonggi-do, Korea). A cancer animal model was prepared by inoculating 1 × 10 7 MDA-MB-231 cells into the left thigh of the mouse (n=6), and the experiment was conducted 5 weeks later when the size of the cancer was 50-100
암 동물모델의 암조직의 크기가 50-100 ㎣ 일 때, 각 동물모델의 꼬리 정맥을 통해 하기에 기술된 바와 같이 주입하였다(6개 군의 동물 모델). 주입하고 7일이 지난 후, 각 군의 마우스로부터 암 조직을 적출하여 조직 내에 있는 T 세포의 비율을 유세포 분석기를 통해 분석하였다. T 세포의 분석을 위해 암 조직을 암 해리 키트(Tumor dissociation kit, Miltenyi Biotec)를 사용하여 제조사의 프로토콜대로 단일세포(monocytes)를 분리하였다. 다음 비특이적 결합을 피하기 위해 5 분 동안 FcBlock과 함께 배양하고, T 세포 마커인 CD45, CD3 및 CD8 염색으로 염색하여 분석하였다.When the size of the cancer tissue in the cancer animal model was 50-100
6개 군의 동물모델6 groups of animal models
1군(Saline) : 암 동물모델의 꼬리 정맥을 통해 생리식염수 200 μL 투여Group 1 (Saline): Administration of 200 μL of physiological saline through the tail vein of cancer animal models
2군(PD-L1 peptide) : 암 동물모델의 꼬리 정맥을 통해 서열번호 3으로 표시되는 PD-L1 결합 펩타이드(CVRARTR)를 3 mg/kg 투여Group 2 (PD-L1 peptide): Administer 3 mg/kg of PD-L1 binding peptide (CVRARTR) represented by SEQ ID NO: 3 through the tail vein of a cancer animal model
3군(DOX) : 암 동물모델의 꼬리 정맥을 통해 독소루비신을 3 mg/kg 투여Group 3 (DOX): Doxorubicin administered at 3 mg/kg through the tail vein of cancer animal models
4군(DOX+PD-L1 Ab) : 암 동물모델의 꼬리 정맥을 통해 독소루비신 3 mg/kg 투여하고. 복강을 통해 PD-L1 단일클론항체 10 mg/kg를 병용 투여Group 4 (DOX+PD-L1 Ab):
5군(비교예 1) : 암 동물모델의 꼬리 정맥을 통해 비교예 1의 전구약물(독소루비신 3 mg/kg에 해당하는 몰용량) 투여Group 5 (Comparative Example 1): Administration of the prodrug of Comparative Example 1 (molar dose corresponding to 3 mg/kg of doxorubicin) through the tail vein of a cancer animal model
6군(PD-NPs) : 암 동물모델의 꼬리 정맥을 통해 실시예 1의 전구약물(독소루비신 3 mg/kg에 해당하는 몰용량) 투여Group 6 (PD-NPs): Administration of the prodrug of Example 1 (molar dose corresponding to 3 mg/kg of doxorubicin) through the tail vein of a cancer animal model
도 21은 1군 내지 6군으로부터 적출한 암 조직 내에서의 T 세포 비율을 유세포 분석한 결과로, 실시예 1의 전구약물(PD-NPs)을 처리한 6군에서 확보한 암 조직 내의 T 세포의 비율은 약 20% 이상으로 현저히 증가한 반면, 1군 내지 5군은 모두 10% 이내로 유의하게 적은 것을 확인하였다.21 is a result of flow cytometric analysis of the T cell ratio in cancer tissues extracted from
상기의 결과를 통해 본 발명의 전구약물(PD-NPs)은 생체 내에서도 암 조직 내에 강력한 면역원성 세포사멸을 유도하여 DAMPs를 발생시켜, T 세포의 암 조직 내로의 침윤 및 활성을 유도한다는 것을 확인하였다.Through the above results, it was confirmed that the prodrugs (PD-NPs) of the present invention induce strong immunogenic apoptosis in cancer tissues in vivo, generate DAMPs, and induce T cell infiltration and activity into cancer tissues. .
<110> KOREA INSTITUTE OF SCIENCE AND TECHNOLOGY
<120> composition comprising PD-L1-inhibiting prodrug for the
prevention or treatment of cancer
<130> HPC10405
<160> 3
<170> KoPatentIn 3.0
<210> 1
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> PD-L1 binding peptide
<400> 1
Cys Val Arg Ala Arg Thr Arg Phe Arg Arg Gly
1 5 10
<210> 2
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> cathepsin B-cleavable peptide
<400> 2
Phe Arg Arg Gly
1
<210> 3
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> anti-PD-L1 peptide
<400> 3
Cys Val Arg Ala Arg Thr Arg
1 5
<110> KOREA INSTITUTE OF SCIENCE AND TECHNOLOGY
<120> composition comprising PD-L1-inhibiting prodrug for the
prevention or treatment of cancer
<130> HPC10405
<160> 3
<170> KoPatentIn 3.0
<210> 1
<211> 11
<212> PRT
<213> artificial sequence
<220>
<223> PD-L1 binding peptide
<400> 1
Cys Val Arg Ala Arg Thr Arg Phe
Claims (10)
상기 접합체의 분자량은 1 내지 5 kDa인 것을 특징으로 하는 암의 예방 또는 치료용 약학 조성물.According to claim 1,
A pharmaceutical composition for the prevention or treatment of cancer, characterized in that the molecular weight of the conjugate is 1 to 5 kDa.
상기 펩타이드는 암세포에서 과발현되는 카텝신 B에 의해 절단 가능한 것을 특징으로 하는 암의 예방 또는 치료용 약학 조성물.According to claim 1,
The peptide is a pharmaceutical composition for preventing or treating cancer, characterized in that it can be cleaved by cathepsin B, which is overexpressed in cancer cells.
상기 항암제는 상기 펩타이드에 직접 또는 링커를 통해 공유적으로 결합된 것을 특징으로 하는 암의 예방 또는 치료용 약학 조성물.According to claim 1,
The anti-cancer agent is a pharmaceutical composition for preventing or treating cancer, characterized in that covalently bound to the peptide directly or via a linker.
상기 전구약물은 용액 상에서 자기조립에 의해 구형의 나노입자를 형성하는 것을 특징으로 하는 암의 예방 또는 치료용 약학 조성물.According to claim 1,
The prodrug is a pharmaceutical composition for preventing or treating cancer, characterized in that to form spherical nanoparticles by self-assembly in solution.
상기 나노입자의 평균 직경은 100 내지 200 nm인 것을 특징으로 하는 암의 예방 또는 치료용 약학 조성물.According to claim 5,
A pharmaceutical composition for preventing or treating cancer, characterized in that the nanoparticles have an average diameter of 100 to 200 nm.
상기 나노입자는 용액 상에서 30 내지 40일 동안 응집 또는 구조적 변형없이 장기간 보관이 가능한 것을 특징으로 하는 암의 예방 또는 치료용 약학 조성물.According to claim 5,
The nanoparticle is a pharmaceutical composition for preventing or treating cancer, characterized in that it can be stored for a long time without aggregation or structural transformation for 30 to 40 days in solution.
상기 항암제는 독소루비신(doxorubicin), 사이클로포스파아마이드(cyclophosphamide), 메클로레타민(mecholrethamine), 우라무스틴(uramustine), 멜파란(melphalan), 클로라부실(chlorambucil), 이포스파미드(ifosfamide), 벤다무스틴(bendamustine), 카르무스틴(carmustine), 로무스틴(lomustine), 스트렙토조신(streptozocin), 부설판(busulfan), 다카바진(dacarbazine), 테모졸로마이드(temozolomide), 티오테파(thiotepa), 알트레타민(altretamine), 듀오카르마이신(duocarmycin), 시스플라틴(cisplatin), 카르보플라틴(carboplatin), 네다플라틴(nedaplatin), 옥사리플라틴(oxaliplatin), 사트라플라틴(satraplatin), 트리플라틴 테트라나이트레이트(triplatin tetranitrate), 5-플루오로우라실(5-fluorouracil), 6-머캅토퓨린(6-mercaptopurine), 카페시타빈(capecitabine), 클라드리빈(cladribine), 클로파라빈(clofarabine), 시스타르빈(cystarbine), 플록스유리딘(floxuridine), 플루다라빈(fludarabine), 겜시타빈(gemcitabine), 하이드록시우레아(hydroxyurea), 메토트렉세이트(methotrexate), 페메트렉세드(pemetrexed), 펜토스타틴(pentostatin), 티오구아닌(thioguanine), 캠토테신(camptothecin), 토포테칸(topotecan), 이리노테칸(irinotecan), 에토포사이드(etoposide), 테니포시드(teniposide), 미토산트론(mitoxantrone), 파클리탁셀(paclitaxel), 도세탁셀(docetaxel), 이자베필론(izabepilone), 빈블라스틴(vinblastine), 빈크리스틴(vincristine), 빈데신(vindesine), 비노렐빈(vinorelbine), 에스트라머스틴(estramustine), 메이탄신(maytansine), DM1 (mertansine, 메르탄신), DM4, 돌라스타틴(dolastatin), 아우리스타틴 E(auristatin E), 아우리스타틴 F(auristatin F), 모노메틸 아우리스타틴 E(monomethyl auristatin E), 모노메틸 아우리스타틴 F(monomethyl auristatin F) 및 이들의 유도체로 이루어진 군으로부터 선택되는 어느 하나 이상인 것을 특징으로 하는 암의 예방 또는 치료용 약학 조성물.According to claim 1,
The anticancer agent is doxorubicin, cyclophosphamide, mecholrethamine, uramustine, melphalan, chlorambucil, ifosfamide , bendamustine, carmustine, lomustine, streptozocin, busulfan, dacarbazine, temozolomide, thiotepa ), altretamine, duocarmycin, cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, triple Triplatin tetranitrate, 5-fluorouracil, 6-mercaptopurine, capecitabine, cladribine, clofarabine ), cystarbine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, pentostatin (pentostatin), thioguanine, camptothecin, topotecan, irinotecan, etoposide, teniposide, mitoxantrone, paclitaxel ), docetaxel, izabepilone, vinblastine, vincristine, vindesine, vinorelbine, estramustine, maytansine ), DM1 (mertansine), DM4, dolastatin, auristatin E, auristatin F, monomethyl auristatin E, monomethyl A pharmaceutical composition for preventing or treating cancer, characterized in that at least one selected from the group consisting of auristatin F (monomethyl auristatin F) and derivatives thereof.
상기 약학 조성물은 정맥 주사로 투여되는 것을 특징으로 하는 암의 예방 또는 치료용 약학 조성물.According to claim 1,
The pharmaceutical composition is a pharmaceutical composition for the prevention or treatment of cancer, characterized in that administered by intravenous injection.
상기 암은 위암, 폐암, 비소세포성 폐암, 유방암, 난소암, 간암, 기관지암, 비인두암, 후두암, 췌장암, 방광암, 결장암, 자궁경부암, 골암, 비소세포성 골암, 혈액암, 피부암, 두부 또는 경부 암, 자궁암, 직장암, 항문 부근암, 결장암, 나팔관암, 자궁내막암, 질암, 음문암, 호지킨병(Hodgkin's disease), 식도암, 소장암, 내분비선암, 갑상선암, 부갑상선암, 부신암, 연조직 육종, 요도암, 음경암, 전립선암, 만성 또는 급성 백혈병, 림프구 림프종, 신장 또는 수뇨관암, 신장세포 암종, 신장골반암종, 침샘암, 육종암, 가성점액종, 간모세포종, 고환암, 교모세포종, 구순암, 난소생식세포종양, 기저세포암, 다발성골수종, 담낭암, 맥락막흑색종, 바터팽대부암, 복막암, 설암, 소세포암, 소아림프종, 신경모세포종, 십이지장암, 요관암, 성상세포종, 수막종, 신우암, 외음부암, 흉선암, 중추신경계(central nervous system, CNS) 종양, 1차 중추신경계 림프종, 척수종양, 뇌간 신경교종 및 뇌하수체 선종으로 이루어진 군으로부터 선택되는 어느 하나 이상인 것을 특징으로 하는 암의 예방 또는 치료용 약학 조성물.According to claim 1,
The cancer is gastric cancer, lung cancer, non-small cell lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colon cancer, cervical cancer, bone cancer, non-small cell bone cancer, blood cancer, skin cancer, head or Cervical cancer, uterine cancer, rectal cancer, perianal cancer, colon cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue Sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney or ureteric cancer, renal cell carcinoma, renal pelvic carcinoma, salivary gland cancer, sarcoma cancer, pseudomyxoma, hepatoblastoma, testicular cancer, glioblastoma, labrum Cancer, ovarian germ cell tumor, basal cell carcinoma, multiple myeloma, gallbladder cancer, choroidal melanoma, ampulla of Vater cancer, peritoneal cancer, tongue cancer, small cell cancer, juvenile lymphoma, neuroblastoma, duodenal cancer, ureteral cancer, astrocytoma, meningioma, renal pelvic cancer, Prevention or treatment of cancer, characterized in that it is at least one selected from the group consisting of vulvar cancer, thymus cancer, central nervous system (CNS) tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma. pharmaceutical composition for use.
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