KR20230148991A - Method for producing complex to which tumor-specific peptides are attached and pharmaceutical composition for inhibiting tumor growth comprising complex prepared therefrom - Google Patents
Method for producing complex to which tumor-specific peptides are attached and pharmaceutical composition for inhibiting tumor growth comprising complex prepared therefrom Download PDFInfo
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- KR20230148991A KR20230148991A KR1020220048082A KR20220048082A KR20230148991A KR 20230148991 A KR20230148991 A KR 20230148991A KR 1020220048082 A KR1020220048082 A KR 1020220048082A KR 20220048082 A KR20220048082 A KR 20220048082A KR 20230148991 A KR20230148991 A KR 20230148991A
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- major histocompatibility
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Abstract
본 발명은 종양 특이적 펩타이드가 부착된 복합체의 제조방법 및 이로부터 제조된 복합체를 포함하는 종양 성장 억제용 약학 조성물에 관한 것이다. 본 발명의 종양 특이적 펩타이드가 부착된 복합체는 수지상세포의 성숙 및 활성화를 유도하며, 교차항원제시(cross-presentation)를 통해 CTL 반응을 효과적으로 유도함에 따라 종양 성장 억제 효과를 발휘한다. 상기 조성물은 종양 성장 억제용 약학 조성물로 사용될 수 있다.The present invention relates to a method for producing a complex to which a tumor-specific peptide is attached and a pharmaceutical composition for inhibiting tumor growth comprising the complex prepared therefrom. The complex to which the tumor-specific peptide of the present invention is attached induces the maturation and activation of dendritic cells and effectively induces a CTL response through cross-presentation, thereby exerting a tumor growth inhibition effect. The composition can be used as a pharmaceutical composition for inhibiting tumor growth.
Description
본 발명은 종양 특이적 펩타이드가 부착된 복합체의 제조방법 및 이로부터 제조된 복합체를 포함하는 종양 성장 억제용 약학 조성물에 관한 것이다.The present invention relates to a method for producing a complex to which a tumor-specific peptide is attached and a pharmaceutical composition for inhibiting tumor growth comprising the complex prepared therefrom.
수지상세포(dendritic cell, DC)는 항원 특이적 면역반응에서 중요한 역할을 담당하는 전문 항원제시세포(antigen presenting cell, APC)로, 휴지상태의 T 세포에 직접 작용하여 세포성 면역반응과 체액성 면역반응을 동시에 유도할 수 있는 세포로 알려져 있다. 일반적으로 내인성 항원은 전문 항원제시세포의 주조직적합항원-Ⅰ(major histocompatibility complex class Ⅰ, MHC-Ⅰ) 분자로 제시되어 CD8+ T 세포 활성화를 유도하고, 외인성 항원은 전문 항원제시세포에 내포작용(endocytosis) 또는 탐식작용(phagocytosis) 과정을 통해 MHC-Ⅱ 분자로 제시되어 CD4+ T 세포 활성화를 유도한다.Dendritic cells (DCs) are professional antigen presenting cells (APCs) that play an important role in antigen-specific immune responses. They act directly on resting T cells to promote cellular immune responses and humoral immunity. It is known as a cell that can induce reactions simultaneously. In general, endogenous antigens are presented as major histocompatibility complex class Ⅰ (MHC-Ⅰ) molecules on professional antigen-presenting cells to induce CD8+ T cell activation, and exogenous antigens are endocytosed on professional antigen-presenting cells ( It is presented as an MHC-Ⅱ molecule through the process of endocytosis or phagocytosis and induces CD4+ T cell activation.
활성화된 수지상세포는 교차항원제시(cross-presentation) 과정을 통해 외인성 항원에 의한 CD8+ 세포독성 T 림프구(cytotoxic T lymphocyte, CTL) 반응을 효과적으로 유도한다. 수지상세포는 생체 내에서 미성숙 상태로 분포하며, 병원체 유래 또는 다양한 사이토카인 자극에 의해 활성화 및 성숙과정을 거친다. 수지상세포는 성숙과정에서 항원 포획을 위한 수용체와 내포작용 능력이 감소되지만, MHC 분자, 공동자극분자(costimulatory molecule)의 발현 증가 등을 통해 항원제시능력이 증가된다.Activated dendritic cells effectively induce CD8+ cytotoxic T lymphocyte (CTL) responses to exogenous antigens through a cross-presentation process. Dendritic cells are distributed in an immature state in vivo and undergo activation and maturation processes derived from pathogens or stimulated by various cytokines. During the maturation process, dendritic cells have reduced receptors and endocytosis ability to capture antigens, but their antigen presentation ability increases through increased expression of MHC molecules and costimulatory molecules.
한편, 종양은 일반적으로 8~10개 아미노산 길이의 펩타이드 항원을 MHC-Ⅰ 분자를 통해 발현하며, 종양의 펩타이드 항원은 정상 조직에 비해 종양에서 과발현되는 종양 관련 항원과 정상 조직이 아닌 종양에서만 발현되는 종양 특이적 항원의 두 범주로 분류된다. 최근 MHC-Ⅰ 분자와 관련된 종양 펩타이드가 종양 특이적 CTL을 유도하는 점에 대해 주목을 받고 있으나, MHC-Ⅰ 분자에 결합하는 8~10개의 아미노산으로 구성된 짧은 펩타이드를 생체 내에 직접 투여할 경우에는 특이적 CTL 반응을 유도하는 활성(면역원성)이 낮은 문제가 있으며, 전문 항원제시세포 이외의 세포 표면에 발현되는 MHC-Ⅰ 분자에 무차별적으로 결합하는 문제가 있다.Meanwhile, tumors generally express peptide antigens of 8 to 10 amino acids in length through MHC-Ⅰ molecules, and tumor peptide antigens include tumor-related antigens that are overexpressed in tumors compared to normal tissues, and tumor-related antigens that are expressed only in tumors and not in normal tissues. Tumor-specific antigens are classified into two categories. Recently, tumor peptides related to MHC-Ⅰ molecules have been attracting attention for inducing tumor-specific CTL. However, when a short peptide consisting of 8 to 10 amino acids that binds to MHC-Ⅰ molecules is administered directly in vivo, There is a problem of low activity (immunogenicity) in inducing a CTL response, and there is a problem of indiscriminately binding to MHC-I molecules expressed on the surface of cells other than professional antigen-presenting cells.
이에 본 발명은 비전문 항원제시세포에서 발현되는 MHC-Ⅰ 분자에 대한 펩타이드의 결합, 면역원성, 수지상세포 활성 불량 등의 문제점을 해결하였으며, 생체 내에서 펩타이드 특이적 CTL을 효율적으로 유도하기 위한 펩타이드 기반 조성물을 개발하여, 이를 이용한 항암 면역치료용 약학 조성물을 제공하고자 한다.Accordingly, the present invention solves problems such as binding of peptides to MHC-Ⅰ molecules expressed in non-professional antigen-presenting cells, immunogenicity, and poor dendritic cell activity, and provides a peptide-based method for efficiently inducing peptide-specific CTL in vivo. We intend to develop a composition and provide a pharmaceutical composition for anticancer immunotherapy using the same.
본 발명은 상기와 같은 문제를 해결하기 위해, 카복실화 나노입자에 주조직적합항원-Ⅰ(MHC-Ⅰ)에 대한 항체와 주조직적합항원-Ⅰ 분자가 결합된 복합체를 제조하고, 종양 특이 펩타이드를 부착함에 따라, 약한 면역원성을 개선하고, 탐식세포를 타겟으로 하는 전달이 가능하며, 종양 특이적 CTL을 효과적으로 유도할 수 있는 종양 특이적 펩타이드가 부착된 복합체의 제조방법과 이로부터 제조된 종양 특이적 펩타이드가 부착된 복합체를 포함하는 종양 성장 억제용 약학 조성물을 제공하고자 한다.In order to solve the above problems, the present invention prepares a complex in which an antibody against major histocompatibility antigen-I (MHC-I) and a major histocompatibility antigen-I molecule are bound to carboxylated nanoparticles, and a tumor-specific peptide is prepared. A method of manufacturing a complex to which a tumor-specific peptide is attached, which improves weak immunogenicity, enables targeted delivery to phagocytic cells, and effectively induces tumor-specific CTL by attaching it, and tumors manufactured therefrom The present object is to provide a pharmaceutical composition for inhibiting tumor growth comprising a complex to which a specific peptide is attached.
상기한 과제를 해결하기 위하여, 본 발명은 PLGA(poly(lactic-co-glycolic acid)) 및 PEMA(poly(ethylene/maleic anhydride))를 혼합하여 에멀젼을 제조하는 단계 (a); 상기 단계 (a)의 에멀젼을 PVA(polyvinyl alcohol)와 PEMA 혼합용액에 혼합하여 카르복실화 나노입자를 제조하는 단계 (b); 및 상기 단계 (b)의 카르복실화 나노입자에 주조직적합항원-Ⅰ에 대한 항체와 종양 유래 막 단백질에 포함된 주조직적합항원-Ⅰ 분자를 결합하는 단계 (c); 및 상기 단계 (c) 후, 종양 특이적 펩타이드를 부착하는 단계 (d);를 포함하는 주조직적합항원-Ⅰ 분자가 결합된 복합체의 제조방법을 제공한다.In order to solve the above problem, the present invention includes the steps of preparing an emulsion by mixing PLGA (poly(lactic-co-glycolic acid)) and PEMA (poly(ethylene/maleic anhydride)); Step (b) of preparing carboxylated nanoparticles by mixing the emulsion of step (a) with a mixed solution of polyvinyl alcohol (PVA) and PEMA; and a step (c) of binding an antibody against major histocompatibility antigen-I and a major histocompatibility antigen-I molecule contained in a tumor-derived membrane protein to the carboxylated nanoparticles of step (b); And after step (c), a step (d) of attaching a tumor-specific peptide is provided.
본 발명의 일 실시예에 있어서, 상기 주조직적합항원-Ⅰ 분자가 결합된 복합체는, 바람직하게는 수지상세포의 탐식작용(phagocytosis)을 유도하는 것이 좋다.In one embodiment of the present invention, the complex to which the major histocompatibility antigen-I molecule is bound preferably induces phagocytosis of dendritic cells.
본 발명의 일 실시예에 있어서, 상기 주조직적합항원-Ⅰ 분자가 결합된 복합체는, 바람직하게는 수지상세포의 성숙 또는 활성화를 유도하는 것이 좋다.In one embodiment of the present invention, the complex to which the major histocompatibility antigen-I molecule is bound preferably induces maturation or activation of dendritic cells.
본 발명의 일 실시예에 있어서, 상기 주조직적합항원-Ⅰ 분자가 결합된 복합체는, 바람직하게는 수지상세포의 교차항원제시능을 유도하는 것이 좋다.In one embodiment of the present invention, the complex to which the major histocompatibility antigen-I molecule is bound preferably induces the cross-antigen presentation ability of dendritic cells.
본 발명의 일 실시예에 있어서, 상기 주조직적합항원-Ⅰ 분자가 결합된 복합체는, 바람직하게는 종양 특이적 CTL(cytotoxic T lymphocyte) 반응을 유도하는 것이 좋다.In one embodiment of the present invention, the complex to which the major histocompatibility antigen-I molecule is bound preferably induces a tumor-specific CTL (cytotoxic T lymphocyte) response.
또한, 본 발명은 상기 제조방법에 의해 제조된 주조직적합항원-Ⅰ 분자가 결합된 복합체를 유효성분으로 포함하는 종양 성장 억제용 약학 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for inhibiting tumor growth comprising as an active ingredient a complex bound to a major histocompatibility antigen-I molecule prepared by the above production method.
본 발명에 있어서, 상기 약학 조성물은, 바람직하게는 암 특이 CTL이 없거나 미약한 암환자를 대상으로 치료하는 것이 좋다.In the present invention, the pharmaceutical composition is preferably used to treat cancer patients with no or weak cancer-specific CTL.
또한, 본 발명은 상기 제조방법에 의해 제조된 주조직적합항원-Ⅰ 분자가 결합된 복합체를 유효성분으로 포함하는 면역 항암제를 제공한다.In addition, the present invention provides an anti-cancer immunological agent comprising as an active ingredient a complex bound to a major histocompatibility antigen-I molecule prepared by the above production method.
본 발명의 종양 특이적 펩타이드가 부착된 복합체는 수지상세포의 성숙 및 활성화를 유도하며, 교차항원제시(cross-presentation)를 통해 CTL 반응을 효과적으로 유도함에 따라 종양 성장 억제 효과를 가진다. 이를 통해 본 발명의 종양 특이적 펩타이드가 부착된 복합체는 종양 조직 주위의 국소 면역억제 환경을 회피하여 종양 특이 CTL의 생산을 유도할 수 있다.The complex to which the tumor-specific peptide of the present invention is attached induces the maturation and activation of dendritic cells and effectively induces a CTL response through cross-presentation, thereby suppressing tumor growth. Through this, the complex to which the tumor-specific peptide of the present invention is attached can avoid the local immunosuppressive environment around the tumor tissue and induce the production of tumor-specific CTL.
도 1은 본 발명의 일 실시예에 있어서, 본 발명의 종양 특이적 펩타이드가 부착된 복합체의 제조방법과 이로부터 제조된 종양 특이적 펩타이드가 부착된 복합체가 CTL 반응을 유도하는 과정을 나타낸 도이다.
도 2는 본 발명의 일 실시예에 있어서, 카르복실화 나노입자(bare NP)와 H-2Kb(Kb) 부착 나노입자(NP-Ab-Kb)의 평균 크기와 ζ-전위(도 2a) 및 카르복실화 나노입자의 주사전자현미경 분석 결과(도 2b)를 나타낸 도이다.
도 3은 본 발명의 일 실시예에 있어서, 카르복실화 나노입자(bare NP)와 Kb 부착 나노입자(NP-Ab-Kb)의 유세포 분석 결과를 나타낸 도이다.
도 4는 본 발명의 일 실시예에 있어서, Kb 부착 나노입자(NP-Ab-Kb)의 구조가 9일간 안정적으로 유지되는 것을 확인한 도이다.
도 5는 본 발명의 일 실시예에 있어서, 수지상세포의 Kb 부착 나노입자(NP-Ab-Kb)에 대한 탐식작용을 확인한 도이다.
도 6은 본 발명의 일 실시예에 있어서, Kb 부착 나노입자(NP-Ab-Kb)와 리소좀의 colocalization을 확인하기 위한 공초점현미경 분석 결과를 나타낸 도이다.
도 7은 본 발명의 일 실시예에 있어서, Kb 부착 나노입자(NP-Ab-Kb) 처리에 따른 미성숙 골수 유래 수지상세포의 성숙 및 활성화를 확인한 도이다.
도 8은 본 발명의 일 실시예에 있어서, Kb 부착 나노입자(NP-Ab-Kb)의 수지상세포에 대한 세포독성을 확인한 도이다.
도 9는 본 발명의 일 실시예에 있어서, 반응 시간에 따른 SIINFEKL 교환 반응 결과를 나타낸 도이다. 도 9a는 2시간 반응에 따른 SIINFEKL-Kb 복합체의 부착정도, 도 9b는 2시간 반응에 따른 MFI(mean fluorescence intensity) 결과, 도 9c는 18시간 반응에 따른 SIINFEKL-Kb 복합체의 부착정도, 도 9d는 18시간 반응에 따른 MFI 결과를 나타낸 도이다.
도 10은 본 발명의 일 실시예에 있어서, SIINFEKL-pulsed Kb 부착 나노입자 처리에 따른 수지상세포의 교차항원제시능을 확인한 도이다.
도 11은 본 발명의 일 실시예에 있어서, 마우스에서 SIINFEKL 특이적 CTL을 유도하기 위한 SIINFEKL-pulsed Kb 부착 나노입자의 최적 입자수를 확인한 도이다.
도 12는 본 발명의 일 실시예에 있어서, 혈액에서 SIINFEKL-pulsed Kb 부착 나노입자에 의한 SIINFEKL 특이적 CTL 유도 활성을 확인한 결과를 나타낸 도이다.
도 13은 본 발명의 일 실시예에 있어서, 표적 세포에서 SIINFEKL-pulsed Kb 부착 나노입자에 의한 SIINFEKL 특이적 CTL 유도 활성을 확인한 결과를 나타내 도이다.
도 14는 본 발명의 일 실시예에 있어서, 종양세포 접종 후 7일차에 혈액에서 SIINFEKL-pulsed Kb 부착 나노입자에 의한 SIINFEKL 특이적 CTL 유도 활성을 확인한 결과를 나타낸 도이다.
도 15는 본 발명의 일 실시예에 있어서, 종양세포 접종 후 21일차에 혈액에서 SIINFEKL-pulsed Kb 부착 나노입자에 의한 SIINFEKL 특이적 CTL 유도 활성을 확인한 결과를 나타낸 도이다.
도 16은 본 발명의 일 실시예에 있어서, 종양세포 접종 후 SIINFEKL-pulsed Kb 부착 나노입자에 의한 종양 성장 억제 효과를 확인한 도이다.Figure 1 is a diagram showing a method for producing a complex to which a tumor-specific peptide of the present invention is attached and the process by which the complex to which a tumor-specific peptide is attached, prepared thereby, induces a CTL response, in one embodiment of the present invention. .
Figure 2 shows the average size and ζ-potential of carboxylated nanoparticles (bare NP) and H-2K b (K b ) attached nanoparticles (NP-Ab-K b ) in one embodiment of the present invention. 2a) and the results of scanning electron microscopy analysis of carboxylated nanoparticles (FIG. 2b).
Figure 3 is a diagram showing the results of flow cytometry analysis of carboxylated nanoparticles (bare NP) and K b attached nanoparticles (NP-Ab-K b ) in one embodiment of the present invention.
Figure 4 is a diagram confirming that the structure of K b attached nanoparticles (NP-Ab-K b ) is maintained stably for 9 days in one embodiment of the present invention.
Figure 5 is a diagram confirming the phagocytosis of dendritic cells to K b- attached nanoparticles (NP-Ab-K b ) in one embodiment of the present invention.
Figure 6 is a diagram showing the results of confocal microscopy analysis to confirm the colocalization of K b attached nanoparticles (NP-Ab-K b ) and lysosomes in one embodiment of the present invention.
Figure 7 is a diagram confirming the maturation and activation of immature bone marrow-derived dendritic cells according to treatment with K b attached nanoparticles (NP-Ab-K b ) in one embodiment of the present invention.
Figure 8 is a diagram confirming the cytotoxicity of K b- attached nanoparticles (NP-Ab-K b ) to dendritic cells in one embodiment of the present invention.
Figure 9 is a diagram showing the results of SIINFEKL exchange reaction according to reaction time in one embodiment of the present invention. Figure 9a shows the degree of attachment of the SIINFEKL-K b complex according to the 2-hour reaction, Figure 9b shows the MFI (mean fluorescence intensity) result according to the 2-hour reaction, Figure 9c shows the degree of attachment of the SIINFEKL-K b complex according to the 18-hour reaction, Figure 9d is a diagram showing the MFI results according to the 18-hour reaction.
Figure 10 is a diagram confirming the cross-antigen presentation ability of dendritic cells according to treatment with SIINFEKL-pulsed K b attached nanoparticles in one embodiment of the present invention.
Figure 11 is a diagram confirming the optimal particle number of SIINFEKL-pulsed K b attached nanoparticles for inducing SIINFEKL-specific CTL in mice, according to an embodiment of the present invention.
Figure 12 is a diagram showing the results of confirming SIINFEKL-specific CTL inducing activity by SIINFEKL-pulsed K b attached nanoparticles in blood according to an embodiment of the present invention.
Figure 13 shows the results of confirming SIINFEKL-specific CTL inducing activity by SIINFEKL-pulsed K b attached nanoparticles in target cells according to an embodiment of the present invention.
Figure 14 is a diagram showing the results of confirming the SIINFEKL-specific CTL inducing activity by SIINFEKL-pulsed K b attached nanoparticles in the blood on the 7th day after tumor cell inoculation in one embodiment of the present invention.
Figure 15 is a diagram showing the results of confirming SIINFEKL-specific CTL inducing activity by SIINFEKL-pulsed K b attached nanoparticles in the blood on day 21 after tumor cell inoculation, in one embodiment of the present invention.
Figure 16 is a diagram confirming the tumor growth inhibition effect by SIINFEKL-pulsed K b attached nanoparticles after tumor cell inoculation in an embodiment of the present invention.
본 발명은 PLGA(poly(lactic-co-glycolic acid)) 및 PEMA(poly(ethylene/maleic anhydride))를 혼합하여 에멀젼을 제조하는 단계 (a); 상기 단계 (a)의 에멀젼을 PVA(polyvinyl alcohol)와 PEMA 혼합용액에 혼합하여 카르복실화 나노입자를 제조하는 단계 (b); 및 상기 단계 (b)의 카르복실화 나노입자에 주조직적합항원-Ⅰ에 대한 항체와 종양 유래 막 단백질에 포함된 주조직적합항원-Ⅰ 분자를 결합하는 단계 (c); 및 상기 단계 (c) 후, 종양 특이적 펩타이드를 부착하는 단계 (d);를 포함하는 주조직적합항원-Ⅰ 분자가 결합된 복합체의 제조방법을 제공한다.The present invention includes the step (a) of preparing an emulsion by mixing PLGA (poly(lactic-co-glycolic acid)) and PEMA (poly(ethylene/maleic anhydride)); Step (b) of preparing carboxylated nanoparticles by mixing the emulsion of step (a) with a mixed solution of polyvinyl alcohol (PVA) and PEMA; and a step (c) of binding an antibody against major histocompatibility antigen-I and a major histocompatibility antigen-I molecule contained in a tumor-derived membrane protein to the carboxylated nanoparticles of step (b); And after step (c), a step (d) of attaching a tumor-specific peptide is provided.
본 발명의 주조직적합항원-Ⅰ 분자가 결합된 복합체는 PLGA를 포함하는 생체적합성 나노입자(nano particle, NP)에 주조직적합항원-Ⅰ에 대한 항체(anti-MHC-Ⅰ) 및 주조직적합항원-Ⅰ 분자를 부착하고, 주조직적합항원-Ⅰ 분자의 기존의 펩타이드를 종양 특이적 펩타이드(tumor specific antigen peptide, TSA peptide)로 교체부착하여 제조한 것이다. 본 발명의 주조직적합항원-Ⅰ 분자가 결합된 복합체는 전문 항원제시세포에 의해 탐식될 수 있으며, 종양 특이적 펩타이드는 전문 항원제시세포의 주조직적합항원-Ⅰ 분자를 통해 교차제시되어 세포독성 T 림프구 반응을 유도한다. 본 발명은 종양 특이 CTL이 미약한 환자의 CTL 생산을 효과적으로 유도할 수 있다(도 1).The complex in which the major histocompatibility antigen-Ⅰ molecule of the present invention is bound to a biocompatible nanoparticle (NP) containing PLGA, an antibody (anti-MHC-Ⅰ) against the major histocompatibility antigen-Ⅰ, and major histocompatibility It was manufactured by attaching an antigen-I molecule and replacing the existing peptide of the major histocompatibility antigen-I molecule with a tumor specific antigen peptide (TSA peptide). The complex to which the major histocompatibility antigen-Ⅰ molecule of the present invention is bound can be phagocytosed by professional antigen-presenting cells, and the tumor-specific peptide is cross-presented through the major histocompatibility antigen-Ⅰ molecule of the professional antigen-presenting cell, causing cytotoxicity. Induces T lymphocyte response. The present invention can effectively induce CTL production in patients with weak tumor-specific CTL (Figure 1).
또한, 본 발명은 상기 제조방법에 의해 제조된 주조직적합항원-Ⅰ 분자가 결합된 복합체를 유효성분으로 포함하는 종양 성장 억제용 약학 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for inhibiting tumor growth comprising as an active ingredient a complex bound to a major histocompatibility antigen-I molecule prepared by the above production method.
또한, 본 발명은 상기 제조방법에 의해 제조된 주조직적합항원-Ⅰ 분자가 결합된 복합체를 유효성분으로 포함하는 면역 항암제를 제공한다.In addition, the present invention provides an anti-cancer immunological agent comprising as an active ingredient a complex bound to a major histocompatibility antigen-I molecule prepared by the above production method.
본 발명의 약학 조성물에는 유효성분 이외에 보조제(Adjuvant)를 추가로 포함할 수 있다. 상기 보조제는 당해 기술분야에 알려진 것이라면 어느 것이나 제한 없이 사용할 수 있다.The pharmaceutical composition of the present invention may further include adjuvants in addition to the active ingredients. Any auxiliary agent known in the art may be used without limitation.
본 발명에 따른 약학 조성물은 유효성분을 약학적으로 허용된 담체에 혼입시킨 형태로 제조될 수 있다. 여기서, 약학적으로 허용된 담체는 제약 분야에서 통상 사용되는 담체, 부형제 및 희석제를 포함한다. 본 발명의 약학 조성물에 이용할 수 있는 약학적으로 허용된 담체는 이들로 제한되는 것은 아니지만, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로스, 메틸 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다.The pharmaceutical composition according to the present invention can be prepared by incorporating the active ingredient into a pharmaceutically acceptable carrier. Here, pharmaceutically acceptable carriers include carriers, excipients, and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, Examples include calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
본 발명의 약학 조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀전, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 또는 멸균 주사용액의 형태로 제형화하여 사용될 수 있다.The pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injection solutions according to conventional methods. .
제제화할 경우에는 통상 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 그러한 고형 제제는 유효성분에 적어도 하나 이상의 부형제, 예를 들면 전분, 칼슘 카르보네이트, 수크로스, 락토오스, 젤라틴 등을 섞어 조제될 수 있다. 또한, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용될 수 있다. 경구투여를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데, 일반적으로 사용되는 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수용성용제, 현탁제, 유제, 동결건조 제제 및 좌제가 포함된다. 비수용성용제, 현탁제로는 프로필렌 글리콜, 폴리에틸렌 글리콜, 올리브유와 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(Witepsol), 트윈(TweenW) 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.When formulated, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations contain the active ingredient plus at least one excipient, such as starch, calcium carbonate, sucrose, lactose, and gelatin. It can be prepared by mixing etc. Additionally, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used diluents such as water and liquid paraffin, they contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives. You can. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, Witepsol, TweenW 61, cacao, laurel, glycerogelatin, etc. can be used.
본 발명에 따른 약학 조성물은 개체에 다양한 경로로 투여될 수 있다. 투여의 모든 방식이 예상될 수 있는데, 예를 들면 경구, 정맥, 근육, 피하, 복강내 주사에 의해 투여될 수 있다.The pharmaceutical composition according to the present invention can be administered to an individual through various routes. All modes of administration are contemplated, for example, by oral, intravenous, intramuscular, subcutaneous, or intraperitoneal injection.
상기 약학 조성물은 다양한 경구 또는 비경구 투여 형태로 제형화 될 수 있다. The pharmaceutical composition may be formulated into various oral or parenteral dosage forms.
경구 투여용 제형으로는 예를 들면 정제, 환제, 경질, 연질 캅셀제, 액제, 현탁제, 유화제, 시럽제, 과립제 등이 있는데, 이들 제형은 유효성분 이외에 희석제(예: 락토즈, 덱스트로즈, 수크로즈, 만니톨, 솔비톨, 셀룰로즈 및/또는 글리신), 활택제(예: 실리카, 탈크, 스테아르산 및 그의 마그네슘 또는 칼슘염 및/또는 폴리에틸렌 글리콜)를 추가로 포함할 수 있다. 또한, 상기 정제는 마그네슘 알루미늄 실리케이트, 전분 페이스트, 젤라틴, 트라가칸스, 메틸셀룰로즈, 나트륨 카복시메틸셀룰로즈 및/또는 폴리비닐피롤리딘과 같은 결합제를 함유할 수 있으며, 경우에 따라 전분, 한천, 알긴산 또는 그의 나트륨 염과 같은 붕해제 또는 비등 혼합물 및/또는 흡수제, 착색제, 향미제 및 감미제를 함유할 수 있다. 상기 제형은 통상적인 혼합, 과립화 또는 코팅 방법에 의해 제조될 수 있다.Dosage forms for oral administration include, for example, tablets, pills, hard and soft capsules, solutions, suspensions, emulsifiers, syrups, granules, etc. These dosage forms contain diluents (e.g. lactose, dextrose, water) in addition to the active ingredient. crose, mannitol, sorbitol, cellulose and/or glycine), lubricants (e.g. silica, talc, stearic acid and magnesium or calcium salts thereof and/or polyethylene glycol). Additionally, the tablets may contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine, and in some cases starch, agar, alginic acid. or disintegrants such as sodium salts thereof or effervescent mixtures and/or absorbents, colorants, flavoring and sweetening agents. The formulation can be prepared by conventional mixing, granulating or coating methods.
또한, 비경구 투여용 제형의 대표적인 것은 주사용 제제이며, 주사용 제제의 용매로서 물, 링거액, 등장성 생리식염수 또는 현탁액을 들 수 있다. 상기 주사용 제제의 멸균 고정 오일은 용매 또는 현탁 매질로서 사용할 수 있으며 모노-, 디-글리세라이드를 포함하여 어떠한 무자극성 고정오일도 이러한 목적으로 사용될 수 있다.In addition, representative formulations for parenteral administration are injectable formulations, and solvents for injectable formulations include water, Ringer's solution, isotonic saline solution, or suspension. The sterile fixed oil of the injectable preparation can be used as a solvent or suspending medium, and any non-irritating fixed oil, including mono- and di-glycerides, can be used for this purpose.
또한, 상기 주사용 제제는 올레산과 같은 지방산을 사용할 수 있다.Additionally, the injectable preparation may use fatty acids such as oleic acid.
본 발명에 있어서, 암컷 C57BL/6 마우스(8-12주령)는 KosaBio Inc.(Seongnam, Korea)에서 구입하였으며, 동물과 관련된 모든 실험 절차는 충북대학교 동물실험윤리위원회(CNBUA-1490-21-02)의 승인을 받았으며 지침 및 규정에 따라 수행되었다.In the present invention, female C57BL/6 mice (8-12 weeks old) were purchased from KosaBio Inc. (Seongnam, Korea), and all experimental procedures related to animals were in accordance with the Chungbuk National University Animal Experiment Ethics Committee (CNBUA-1490-21-02). ) was approved by and was performed in accordance with the guidelines and regulations.
이하, 실시예 및 실험예를 통하여 본 발명을 보다 자세히 설명한다. 다만, 하기 실시예 및 실험예는 본 발명에 대한 예시로 제시되는 것으로, 당업자에게 주지 저명한 기술 또는 구성에 대한 구체적인 설명이 본 발명의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우에는 그 상세한 설명을 생략할 수 있고, 이에 의해 본 발명이 제한되지는 않는다. 본 발명은 후술하는 특허청구범위의 기재 및 그로부터 해석되는 균등 범주 내에서 다양한 변형 및 응용이 가능하다.Hereinafter, the present invention will be described in more detail through examples and experimental examples. However, the following examples and experimental examples are provided as examples of the present invention, and if it is judged that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description is omitted. It can be done, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the description of the claims described below and the scope of equivalents interpreted therefrom.
<실시예 1> K<Example 1> K b b 부착 나노입자(주조직적합항원-Ⅰ 분자가 결합된 복합체)의 제조Preparation of attached nanoparticles (complex of major histocompatibility antigen-Ⅰ molecules)
1-1. 카르복실화 나노입자(bare NP)의 제조1-1. Preparation of carboxylated nanoparticles (bare NPs)
PLGA는 체내에서 젖산(lactic)과 글리콜산(glycolic acid)으로 가수분해되는 생체적합성 고분자, 생분해성 고분자로 의약품에서 담체로 사용된다. 일반적으로 PVA(polyvinyl alcohol)를 입자 안정제로 사용하지만, 본 발명에서는 카르복실화 나노입자를 제조하기 위해 PVA와 달리 나노입자 표면에 카르복실 잔기를 다수 노출시켜 항체(antibody, Ab)의 아민 잔기 사이의 카르보디이미드(carbodiimide) 커플링 반응으로 더 많은 Ab와의 공유결합을 가능하게 하는 PEMA를 사용하였다.PLGA is a biocompatible and biodegradable polymer that is hydrolyzed into lactic acid and glycolic acid in the body and is used as a carrier in pharmaceuticals. Generally, PVA (polyvinyl alcohol) is used as a particle stabilizer, but in the present invention, in order to produce carboxylated nanoparticles, unlike PVA, many carboxyl residues are exposed on the surface of the nanoparticles to form a residue between the amine residues of the antibody (Ab). PEMA was used, which enables covalent bonding with more Abs through a carbodiimide coupling reaction.
카르복실화 나노입자는 용매 증발 방법을 이용하여 제조하였다. 1 %(v/v) PEMA 수용액 8 ㎖와 에틸아세테이트에 용해된 5 %(v/v) PLGA 4 ㎖와 혼합하였다. 혼합물을 14,000 rpm에서 4분간 균질화하여 유중수(W/O) 에멀젼을 형성하였다. 나노입자를 고형화하기 위해 에멀젼을 0.2 %(v/v) PEMA 및 0.1 %(v/v) PVA와 혼합하고 18~24시간 교반하여 카르복실화 나노입자를 제조하였다. 카르복실화 나노입자를 4℃에서 20분간 3,500×g로 원심분리하고, 증류수로 2회 세척한 뒤, 10 ㎖의 멸균 증류수에 현탁하였다. 형광 표지된 나노입자는 에틸아세테이트에 FITC(Sigma-Aldrich)를 첨가하여 제조하였다.Carboxylated nanoparticles were prepared using a solvent evaporation method. 8 ml of 1% (v/v) PEMA aqueous solution and 4 ml of 5% (v/v) PLGA dissolved in ethyl acetate were mixed. The mixture was homogenized at 14,000 rpm for 4 minutes to form a water-in-oil (W/O) emulsion. To solidify the nanoparticles, the emulsion was mixed with 0.2% (v/v) PEMA and 0.1% (v/v) PVA and stirred for 18 to 24 hours to prepare carboxylated nanoparticles. The carboxylated nanoparticles were centrifuged at 3,500 × g for 20 minutes at 4°C, washed twice with distilled water, and then suspended in 10 ml of sterile distilled water. Fluorescently labeled nanoparticles were prepared by adding FITC (Sigma-Aldrich) to ethyl acetate.
카르복실화 나노입자의 평균 크기와 ζ-전위는 입도 분석기(ELS-Z, Otsuka, Japan)를 사용하여 측정한 결과, 평균 크기는 767.0±79.9 nm였으며, 이는 수지상세포에 의해 탐식될 수 있는 최적 입자 크기 범위에 포함되었다. 카르복실화 나노입자의 평균 다분산 지수는 0.258±0.021, ζ-전위는 -38.61±0.35 mV로 확인되었다(도 2a).The average size and ζ-potential of carboxylated nanoparticles were measured using a particle size analyzer (ELS-Z, Otsuka, Japan), and the average size was 767.0 ± 79.9 nm, which is optimal for phagocytosis by dendritic cells. included in the particle size range. The average polydispersity index of the carboxylated nanoparticles was 0.258 ± 0.021, and the ζ-potential was found to be -38.61 ± 0.35 mV (Figure 2a).
카르복실화 나노입자의 형태를 주사전자현미경(LEO-1530, Carl Zeiss, Germany)으로 분석한 결과, 도 2b에서 보듯이, 균일한 크기의 구형 입자가 관찰되었다.As a result of analyzing the shape of the carboxylated nanoparticles using a scanning electron microscope (LEO-1530, Carl Zeiss, Germany), spherical particles of uniform size were observed, as shown in Figure 2b.
1-2. H-2K1-2. H-2K bb 를 포함하는 막 단백질의 분리Isolation of membrane proteins containing
EG7.OVA 종양 1 g과 0.15 M NaCl, 0.05 M 인산나트륨 버퍼(pH 7.0), 1X protease inhibitor cocktail(Sigma-Aldrich)을 포함하는 추출 완충액(extraction buffer) 10 ㎖를 gentlMACS 분리기(Miltenyi Biotec, Bergisch Gladbach, Germany)를 이용하여 EG7.OVA 종양으로부터 H-2Kb(Kb)를 포함하는 막 단백질을 분리하였다. 세포 파편과 지질을 제거한 후, 상층액을 Ultracentrifuge(Beckman Coulter, Brea, CA, USA)를 사용하여 4℃에서 45분간 80,000×g에서 초원심분리하였다. Tissue Grinder(SHS-30E; SciLab, Seoul, Korea)를 사용하여 펠렛을 0.5 ㎖의 추출 완충액(extraction buffer)에서 균질화하였다. 균질물을 2% n-dodecyl β-D-maltoside 및 0.4% 3-[(3-cholamidopropyl)dimethyl ammonio]-1-propanesulfonate hydrate(Sigma-Aldrich)를 포함하는 가용화 완충액(solubilization buffer) 0.5 ㎖와 혼합하고 4℃에서 90분간 가용화하였다. 이후, 4℃에서 30분간 17,000 rpm으로 원심분리하여 Kb를 포함하는 막 단백질 상층액을 회수하였다. 분리된 막 단백질의 양은 마이크로비신코닌산 분석 키트(ThermoFisher Scientific, Rockford, IL, USA)를 사용하여 분석하였다.1 g of EG7.OVA tumor and 10 ml of extraction buffer containing 0.15 M NaCl, 0.05 M sodium phosphate buffer (pH 7.0), and 1X protease inhibitor cocktail (Sigma-Aldrich) were separated using a gentlMACS separator (Miltenyi Biotec, Bergisch Gladbach) , Germany) was used to isolate the membrane protein including H-2K b (K b ) from EG7.OVA tumor. After removing cell debris and lipids, the supernatant was ultracentrifuged at 80,000 × g for 45 minutes at 4°C using an Ultracentrifuge (Beckman Coulter, Brea, CA, USA). The pellet was homogenized in 0.5 ml of extraction buffer using a Tissue Grinder (SHS-30E; SciLab, Seoul, Korea). The homogenate was mixed with 0.5 ml of solubilization buffer containing 2% n-dodecyl β-D-maltoside and 0.4% 3-[(3-cholamidopropyl)dimethyl ammonio]-1-propanesulfonate hydrate (Sigma-Aldrich). and solubilized at 4°C for 90 minutes. Afterwards, the membrane protein supernatant containing K b was recovered by centrifugation at 17,000 rpm for 30 minutes at 4°C. The amount of isolated membrane proteins was analyzed using a microbicinchoninic acid assay kit (ThermoFisher Scientific, Rockford, IL, USA).
1-3. K1-3. K b b 부착 나노입자(주조직적합항원-Ⅰ 분자가 결합된 복합체)의 제조Preparation of attached nanoparticles (complex of major histocompatibility antigen-Ⅰ molecules)
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride(EDC; ThermoFisher Scientific)/N hydroxysuccinimide(NHS; Sigma-Aldrich) 커플링 방법을 사용하여 anti-Kb mAb(monoclonal antibody, 단일클론항체)를 카르복실화 나노입자에 공유결합하였다. anti-Kb mAb는 주조직적합항원-Ⅰ(MHC-Ⅰ)의 하나인 Kb에 대한 항체이다. anti-Kb mAb는 친수성 카르복실 측쇄와 Ab의 1차 아민 측쇄 사이의 카르보디이미드(carbodiimide) 커플링 반응을 사용하여 나노입자에 공유결합되었다.anti-K b mAb (monoclonal antibody) was synthesized using the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC; ThermoFisher Scientific)/N hydroxysuccinimide (NHS; Sigma-Aldrich) coupling method. It was covalently bound to the boxylated nanoparticles. anti-K b mAb is an antibody against K b , one of the major histocompatibility antigen-I (MHC-I). The anti-K b mAb was covalently attached to the nanoparticles using a carbodiimide coupling reaction between the hydrophilic carboxyl side chain and the primary amine side chain of the Ab.
10 mg의 카르복실화 나노입자(bare NP)를 1.2 ㎖의 50 mM 활성화 완충액(activation buffer, 4-morpholineethanesulfonic acid buffer, pH 6.0, Sigma-Aldrich)에 재현탁하고 300 mM EDC 4 ㎕ 및 300 mM NHS 8 ㎕로 24~26℃에서 1시간 처리하여 EDC-활성화 나노입자를 제조하였다. EDC-활성화 나노입자를 50 mM 활성화 완충액(activation buffer)으로 2회 세척한 뒤, 600 ㎍/㎖의 anti-Kb mAb(Clone Y-3; BioXcell, Lebanon, NH, USA)를 포함하는 1 ㎖의 50 mM 활성화 완충액(activation buffer)에서 18시간, 실온에서 재현탁하여 Ab-conjugated 나노입자(NP-Ab)를 제조하였다.10 mg of carboxylated nanoparticles (bare NPs) were resuspended in 1.2 ㎖ of 50 mM activation buffer (4-morpholineethanesulfonic acid buffer, pH 6.0, Sigma-Aldrich) and 4 ㎕ of 300 mM EDC and 300 mM NHS. EDC-activated nanoparticles were prepared by treating 8 μl at 24-26°C for 1 hour. EDC-activated nanoparticles were washed twice with 50 mM activation buffer and then added to 1 ml containing 600 ㎍/ml anti-K b mAb (Clone Y-3; BioXcell, Lebanon, NH, USA). Ab-conjugated nanoparticles (NP-Ab) were prepared by resuspending in 50 mM activation buffer for 18 hours at room temperature.
Ab-conjugated 나노입자(NP-Ab)를 30 ㎕ 에탄올아민으로 30분간 처리하여 유리 EDC 활성화 카르복실 잔기의 반응을 중지(quench)시키고 PBS로 2회 세척하였다. Ab-conjugated 나노입자(NP-Ab)를 Kb를 함유하고 있는 막 단백질과 함께 혼합한 뒤, 4℃에서 24시간 동안 반응시켜 Kb가 부착된 나노입자(NP-Ab-Kb, 주조직적합항원-Ⅰ 복합체)를 제조하였다. Kb가 부착된 나노입자(NP-Ab-Kb)를 PBS로 2회 세척하고 PBS 1 ㎖에 재현탁하여 하기 실험에서 사용하였다.Ab-conjugated nanoparticles (NP-Ab) were treated with 30 μl ethanolamine for 30 minutes to quench the reaction of free EDC-activated carboxyl residues and washed twice with PBS. Ab-conjugated nanoparticles (NP-Ab) were mixed with membrane proteins containing K b and reacted at 4°C for 24 hours to form nanoparticles with K b attached (NP-Ab-K b , main tissue). Suitable antigen-Ⅰ complex) was prepared. The nanoparticles (NP-Ab-K b ) to which K b was attached were washed twice with PBS, resuspended in 1 ml of PBS, and used in the following experiment.
<실험예 1> K<Experimental Example 1> K b b 부착 나노입자(주조직적합항원-Ⅰ 분자가 결합된 복합체)의 특성 분석Characterization of attached nanoparticles (complex of major histocompatibility antigen-Ⅰ molecules)
Kb 부착 나노입자(NP-Ab-Kb)는 anti-mIgG(BioLegend, San Diego, CA, USA) 및 anti-Kb mAb(BD Biosciences, San Jose, CA, USA)로 30분간 염색하고 유세포 분석기(FACS Canto II, BD Biosciences)를 사용하여 분석하였다. FlowJo software (TreeStar, Ashland, OR, USA)를 사용하여 유세포 분석 결과 데이터를 분석하였다. 나노입자의 입자수는 qNano Gold (IZON Science, Christchurch, New Zealand)를 사용하여 분석하였다.K b -attached nanoparticles (NP-Ab-K b ) were stained with anti-mIgG (BioLegend, San Diego, CA, USA) and anti-K b mAb (BD Biosciences, San Jose, CA, USA) for 30 min and analyzed by flow cytometry. Analysis was performed using an analyzer (FACS Canto II, BD Biosciences). Flow cytometry data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA). The particle number of nanoparticles was analyzed using qNano Gold (IZON Science, Christchurch, New Zealand).
유세포 분석 결과, Kb 부착 나노입자는 anti-Kb 98.2%, Kb 83.9%로 코팅된 것으로 확인되었다(도 3)다. Kb 부착 나노입자의 평균 크기는 767.0±79.9에서 90 3.1±32.2 nm로 증가하였으며(도 2a 및 표 1), ζ-전위는 -38.61±0.35에서 -18.68±3.64 mV로 감소하였다(도 2b 및 표 1). 이로부터 Ab와 Kb가 나노입자 표면에 고정되었음을 알 수 있다. As a result of flow cytometry, it was confirmed that the K b attached nanoparticles were coated with anti-K b 98.2% and K b 83.9% (Figure 3). The average size of K b attached nanoparticles increased from 767.0 ± 79.9 to 90 3.1 ± 32.2 nm (Figure 2a and Table 1), and the ζ-potential decreased from -38.61 ± 0.35 to -18.68 ± 3.64 mV (Figures 2b and Table 1). From this, it can be seen that Ab and K b were fixed to the surface of the nanoparticle.
또한, PBS에 현탁된 Kb 부착 나노입자(NP-Ab-Kb)는 평균 입자 크기, ζ-전위, Ab와 Kb의 부착에 대해, 4℃에서 최소 9일간 안정한 것으로 확인되었다(도 4). In addition, K b -attached nanoparticles (NP-Ab-K b ) suspended in PBS were confirmed to be stable at 4°C for at least 9 days in terms of average particle size, ζ-potential, and attachment of Ab and K b (Figure 4 ).
<실험예 2> 수지상세포의 K<Experimental Example 2> K of dendritic cells b b 부착 나노입자(주조직적합항원-Ⅰ 분자가 결합된 복합체) 탐식작용Phagocytosis of attached nanoparticles (complex of major histocompatibility antigen-Ⅰ molecules)
본 실험에서는 수지상세포(dendritic cell, DC)에 의한 Kb 부착 나노입자(NP-Ab-Kb) 탐식작용을 분석하기 위해 형광이 표지된 카르복실화 나노입자(NP[FITC])를 이용하여 Ab-conjugated 나노입자(NP[FITC]-Ab) 및 Kb 부착 나노입자(NP[FITC]-Ab-Kb)를 제조하였다.In this experiment, fluorescently labeled carboxylated nanoparticles (NP[FITC]) were used to analyze the phagocytosis of K b attached nanoparticles (NP-Ab-K b ) by dendritic cells (DC). Ab-conjugated nanoparticles (NP[FITC]-Ab) and K b -attached nanoparticles (NP[FITC]-Ab-K b ) were prepared.
2-1. 골수 유래 BMDC(Bone marrow dendritic cell) 제조2-1. Manufacturing bone marrow-derived BMDC (Bone marrow dendritic cells)
마우스 대퇴골에서 채취한 골수세포를 6 well 플레이트에서 5×106 cells/well로 분주하고, 40 ng/㎖ GM-CSF 및 20 ng/㎖ IL-4가 포함된 배양 배지에서 배양하였다. 3일차, 4일차에 플레이트를 부드럽게 흔들어 비부착 세포를 제거하고 배지를 교체하였다. 6일차에 미성숙 BMDC를 피펫으로 회수하여 실험에 사용하였다.Bone marrow cells collected from the mouse femur were distributed at 5×10 6 cells/well in a 6-well plate and cultured in culture medium containing 40 ng/ml GM-CSF and 20 ng/ml IL-4. On days 3 and 4, the plate was gently shaken to remove non-adherent cells and the medium was replaced. On day 6, immature BMDCs were recovered with a pipette and used in experiments.
2-2. 수지상세포(dendritic cell)의 탐식작용2-2. Phagocytosis of dendritic cells
FITC 표지된 나노입자는 6 well 플레이트에 2×109 particles/well로 분주하고, 마우스 유래 수지상세포의 세포주인 DC2.4 세포를 2×106 particles/well로 첨가하였다. 2시간 반응시킨 후, 따뜻한 PBS로 2회 세척하여 탐식되지 않은 나노입자를 제거하였다. 세포를 회수하고 PBS 중 1% 파라포름알데히드로 고정하고 유세포 분석기로 분석하였다. 공초점 분석을 위해, 상기 DC2.4 세포가 분주된 6 well 플레이트에 FITC 표지된 나노입자를 5×108 particles/well로 분주하고, 50 nM LysoTracker Red DND-99(Thermo Fisher Scientific)를 첨가하였다. 2시간 반응시킨 후, 따뜻한 PBS로 2회 세척하여 탐식되지 않은 나노입자를 제거하였다. 세포를 4% 파라포름알데히드로 고정하고 DAPI가 포함된 Antifade Mounting Medium (Vector Laboratories Inc., Burlingame, CA, USA)를 처리하여 공초점 현미경(K1-fluo; Nanoscope Systems Inc., Daejeon, Korea)을 사용하여 시각화하였다.FITC-labeled nanoparticles were distributed in a 6-well plate at 2×10 9 particles/well, and DC2.4 cells, a mouse-derived dendritic cell line, were added at 2×10 6 particles/well. After reacting for 2 hours, the unphagocytosed nanoparticles were removed by washing twice with warm PBS. Cells were recovered, fixed with 1% paraformaldehyde in PBS, and analyzed by flow cytometry. For confocal analysis, FITC-labeled nanoparticles were dispensed at 5×10 8 particles/well in the 6-well plate containing the DC2.4 cells, and 50 nM LysoTracker Red DND-99 (Thermo Fisher Scientific) was added. . After reacting for 2 hours, the unphagocytosed nanoparticles were removed by washing twice with warm PBS. Cells were fixed with 4% paraformaldehyde, treated with Antifade Mounting Medium (Vector Laboratories Inc., Burlingame, CA, USA) containing DAPI, and analyzed using a confocal microscope (K1-fluo; Nanoscope Systems Inc., Daejeon, Korea). It was visualized using.
도 5에서 보듯이, DC2.4 세포의 탐식작용은 카르복실화 나노입자(NP[FITC])에 비해 Ab-conjugated 나노입자(NP[FITC]-Ab) 및 Kb 부착 나노입자(NP[FITC]-Ab-Kb)의 경우에 더욱 효과적으로 나타났다. 또한, Ab-conjugated 나노입자(NP[FITC]-Ab)로 처리된 DC2.4 세포의 MFI(mean fluorescence intensity) 값은 대조군인 카르복실화 나노입자(NP[FITC])로 처리된 DC2.4 세포의 MFI 값보다 15.2배 높았으며, Kb 부착 나노입자(NP[FITC]-Ab-Kb)로 처리된 DC2.4 세포의 MFI 값은 대조군에 비해 11.6배 높게 나타났다. Ab-conjugated 나노입자 및 Kb 부착 나노입자에서 탐식작용이 증가하는 것은 Ab의 옵소닌화(opsonization) 효과 때문으로 추정된다. Ab-conjugated 나노입자는 Ab의 Fc 단편이 나노입자에 부착되어 항원 결합 부위가 외부로 노출되도록 제조되었다. 그러나 Ab 중 일부는 항원 결합 부위를 통해 접합되어 Fc 영역이 외부로 노출될 수 있다. 한편, Ab-conjugated 나노입자에 비해 Kb 부착 나노입자에서 탐식작용이 상대적으로 감소하는 것은 근처에 부착된 Kb에 의한 Fc의 입체 장애로 설명될 수 있다.As shown in Figure 5, the phagocytosis of DC2.4 cells was observed for Ab-conjugated nanoparticles (NP[FITC]-Ab) and K b- attached nanoparticles (NP[FITC]) compared to carboxylated nanoparticles (NP[FITC]). ]-Ab-K b ) was more effective. In addition, the mean fluorescence intensity (MFI) value of DC2.4 cells treated with Ab-conjugated nanoparticles (NP[FITC]-Ab) was similar to that of DC2.4 cells treated with carboxylated nanoparticles (NP[FITC]), which was the control. It was 15.2 times higher than the MFI value of cells, and the MFI value of DC2.4 cells treated with K b attached nanoparticles (NP[FITC]-Ab-K b ) was 11.6 times higher than that of the control group. The increased phagocytosis of Ab-conjugated nanoparticles and K b- attached nanoparticles is presumed to be due to the opsonization effect of Ab. Ab-conjugated nanoparticles were manufactured so that the Fc fragment of Ab was attached to the nanoparticle and the antigen binding site was exposed to the outside. However, some of the Abs may be conjugated through the antigen binding site, exposing the Fc region to the outside. Meanwhile, the relative decrease in phagocytosis in K b -attached nanoparticles compared to Ab-conjugated nanoparticles can be explained by steric hindrance of Fc caused by K b attached nearby.
공초점 현미경 분석 결과를 도 6에 나타내었다. 녹색 형광의 나노입자와 빨간색 형광의 리소좀을 오버레이하여 노란색 형광의 나노입자와 리소좀의 colocalization을 확인하였다.The results of confocal microscopy analysis are shown in Figure 6. By overlaying green fluorescent nanoparticles and red fluorescent lysosomes, colocalization of yellow fluorescent nanoparticles and lysosomes was confirmed.
2-3. 수지상세포(dendritic cell)의 성숙 및 활성화 유도2-3. Induces maturation and activation of dendritic cells
미성숙 BMDC(bone marrow derived dendritic cell)를 24 well 플레이트에 1×106 cells/well로 분주하고, 100 ng/㎖ LPS로 24시간 자극하거나 Kb 부착 나노입자(NP-Ab-Kb)를 1×109 cells/well로 24시간 처리하였다. 대조군으로 PBS 또는 카르복실화 나노입자(bare NP)를 사용하였다. BMDC를 회수하여 마우스 표면 마커(CD11c, H-2Kb, I-Ab, CD80 및 CD86) 및 이소타입 일치 대조군 mAb(BD Biosciences)로 염색한 뒤, 유세포 분석기를 이용하여 분석하였다. 또한, 상기 24시간 반응 후, 배양 상층액을 채취하고 ELISA kit(BD Biosciences)를 이용하여 IL-1β, IL-6, TNF-α의 생산량을 분석하였다.Immature BMDC (bone marrow derived dendritic cells) were distributed at 1×10 6 cells/well in a 24 well plate and stimulated with 100 ng/ml LPS for 24 hours or with K b attached nanoparticles (NP-Ab-K b ) for 1 Treated at × 109 cells/well for 24 hours. As a control, PBS or carboxylated nanoparticles (bare NPs) were used. BMDCs were recovered, stained with mouse surface markers (CD11c, H-2K b , IA b , CD80, and CD86) and isotype-matched control mAb (BD Biosciences), and then analyzed using flow cytometry. In addition, after the 24-hour reaction, the culture supernatant was collected and the production of IL-1β, IL-6, and TNF-α was analyzed using an ELISA kit (BD Biosciences).
도 7에서 보듯이, 수지상세포의 표현형 분석은 Kb 부착 나노입자(NP-Ab-Kb)로 처리된 BMDC가 대조군(PBS)에 비해 항원 특이적 T 세포 프라이밍과 관련된 CD80, CD86 및 MHC-Ⅱ(I-Ab) 분자를 높은 수준으로 발현하는 것을 확인하였다. 표현형 성숙 유도 활성은 카르복실화 나노입자(bare NP)에서 가장 약했으며, Kb 부착 나노입자(NP-Ab-Kb), Ab-conjugated 나노입자(NP-Ab), LPS 순서로 강한 활성이 나타났다(도 7a). Kb 부착 나노입자(NP-Ab-Kb)로 처리된 BMDC는 대조군(PBS) 미성숙 BMDC에 비해 염증성 사이토카인(IL-1β, IL-6, TNF-α)의 생산량이 증가하였으며, Ab-conjugated 나노입자(NP-Ab)로 처리된 BMDC에 비해 염증성 사이토카인(IL-1β, IL-6, TNF-α)의 생산량이 적었다. 이러한 결과는 상기 탐식작용을 비교한 실험과 일치하였다.As shown in Figure 7, phenotypic analysis of dendritic cells showed that BMDCs treated with K b attachment nanoparticles (NP-Ab-K b ) had CD80, CD86, and MHC- cells associated with antigen-specific T cell priming compared to the control group (PBS). It was confirmed that II(IA b ) molecule was expressed at a high level. The phenotypic maturation inducing activity was weakest for carboxylated nanoparticles (bare NP), with the strongest activity occurring in that order: K b attached nanoparticles (NP-Ab-K b ), Ab-conjugated nanoparticles (NP-Ab), and LPS. appeared (Figure 7a). BMDCs treated with K b- attached nanoparticles (NP-Ab-K b ) showed increased production of inflammatory cytokines (IL-1β, IL-6, TNF-α) compared to control (PBS) immature BMDCs, and Ab- Compared to BMDCs treated with conjugated nanoparticles (NP-Ab), the production of inflammatory cytokines (IL-1β, IL-6, and TNF-α) was lower. These results were consistent with the experiment comparing phagocytosis.
2-4. 수지상세포의 생존률2-4. Survival rate of dendritic cells
한편, Kb 부착 나노입자(NP-Ab-Kb)의 DC2.4 세포에 대한 세포독성을 확인하기 위해, 96 well 플레이트에 DC2.4 세포를 5×104 cells/well로 분주하고, 카르복실화 나노입자(bare NP) 또는 Kb 부착 나노입자(NP-Ab-Kb) 6.25×106, 1.25×107, 2.5×107, 5×107 particles/well로 처리하여 24시간 반응시킨 뒤, 37℃의 PBS로 2회 세척하였다. DC2.4 세포에 환원제인 2-mercaptoethanol이 함유되지 않은 배양배지(100 ㎕/well)를 첨가한 뒤, 10 ㎕/well의 WST-8 용액을 처리하여 4시간 배양하였다. 흡광도는 마이크로플레이트 리더를 사용하여 450 nm에서 측정하였다. 도 8a에서 보듯이, PLGA와 PEMA로 제조한 Kb 부착 나노입자(NP-Ab-Kb)에서는 세포독성이 나타나지 않았으며, 수지상세포의 증식을 유도하는 것을 확인하였다. 도 8b에서 보듯이, Meanwhile, to confirm the cytotoxicity of K b- attached nanoparticles (NP-Ab-K b ) to DC2.4 cells, DC2.4 cells were distributed at 5 × 10 4 cells/well in a 96 well plate, and Treated with boxylated nanoparticles (bare NP) or K b attached nanoparticles (NP-Ab-K b ) 6.25×10 6 , 1.25×10 7 , 2.5×10 7 , 5×10 7 particles/well and reacted for 24 hours After doing so, it was washed twice with PBS at 37°C. Culture medium (100 ㎕/well) without 2-mercaptoethanol, a reducing agent, was added to DC2.4 cells, and then treated with 10 ㎕/well of WST-8 solution and cultured for 4 hours. Absorbance was measured at 450 nm using a microplate reader. As shown in Figure 8a, the K b- attached nanoparticles (NP-Ab-K b ) prepared from PLGA and PEMA did not show cytotoxicity, and it was confirmed that they induced the proliferation of dendritic cells. As shown in Figure 8b,
또한, Kb 부착 나노입자(NP-Ab-Kb)의 BMDC에 대한 세포독성을 확인하기 위해, 96 well 플레이트에 BMDC를 5×105 cells/well로 분주하고, 카르복실화 나노입자(bare NP) 또는 Kb 부착 나노입자(NP-Ab-Kb) 6.25×107, 1.25×108, 2.5×108, 5×108 particles/well로 처리하여 24시간 반응시킨 뒤, 상기와 같이 450 nm에서 흡광도를 측정하였다. 도 8b에서 보듯이, Kb 부착 나노입자(NP-Ab-Kb)에서는 세포독성이 나타나지 않았으며, BMDC의 증식을 유도하는 것을 확인하였다.In addition, to confirm the cytotoxicity of K b- attached nanoparticles (NP-Ab-K b ) to BMDCs, BMDCs were distributed at 5 × 10 5 cells/well in a 96 well plate, and carboxylated nanoparticles (bare NP) or K b attached nanoparticles (NP-Ab-K b ) 6.25×10 7 , 1.25×10 8 , 2.5×10 8 , 5×10 8 particles/well and reacted for 24 hours as above. Absorbance was measured at 450 nm. As shown in Figure 8b, K b attached nanoparticles (NP-Ab-K b ) did not show cytotoxicity and were confirmed to induce proliferation of BMDCs.
<실험예 3> SIINFEKL 펩타이드 특이적 CTL 유도<Experimental Example 3> SIINFEKL peptide-specific CTL induction
3-1. SIINFEKL 펩타이드로 교환된 K3-1. K exchanged with SIINFEKL peptide b b 부착 나노입자의 제조Preparation of attached nanoparticles
Kb 부착 나노입자(NP-Ab-Kb)의 Kb 분자 내에 결합된 기존의 펩타이드 에피토프를 종양 특이적 펩타이드인 SIINFEKL(Peptron, Daejeon, Korea, 서열번호 1)로 교환하기 위해, Kb 부착 나노입자(NP-Ab-Kb) 5 mg을 0, 20, 100 μM SIINFEKL와 함께 37℃에서 2~18시간 배양하였다. 이후 PBS로 2회 세척하고, 0.5 ㎖ PBS에 재현탁하였다. Kb 부착 나노입자(NP-Ab-Kb)에서 SIINFEKL 펩타이드의 부착 정도는 SIINFEKL-Kb 복합체를 인식하는 mAb(Clone 25-D1.16)를 이용해 염색한 뒤, 유세포 분석기로 분석하였다. 도 9a 및 도 9b는 2시간 반응에 따른 결과, 도 9c 및 도 9d는 18시간 반응에 따른 결과이다. 도 9에서 보듯이, Kb 부착 나노입자(NP-Ab-Kb)에 SIINFEKL 펩타이드를 100 μM 농도로 18시간 처리한 경우 교환 반응이 최대로 일어나는 것을 확인하였다.In order to exchange the existing peptide epitope bound within the K b molecule of the K b attached nanoparticle (NP-Ab-K b ) with the tumor-specific peptide SIINFEKL (Peptron, Daejeon, Korea, SEQ ID NO. 1), K b was attached. 5 mg of nanoparticles (NP-Ab-K b ) were incubated with 0, 20, and 100 μM SIINFEKL at 37°C for 2 to 18 hours. Afterwards, it was washed twice with PBS and resuspended in 0.5 ml of PBS. The degree of attachment of the SIINFEKL peptide to the K b- attached nanoparticle (NP-Ab-K b ) was stained using a mAb (Clone 25-D1.16) that recognizes the SIINFEKL-K b complex, and then analyzed by flow cytometry. Figures 9a and 9b are the results of the reaction for 2 hours, and Figures 9c and 9d are the results of the reaction for 18 hours. As shown in Figure 9, it was confirmed that the maximum exchange reaction occurred when K b attached nanoparticles (NP-Ab-K b ) were treated with SIINFEKL peptide at a concentration of 100 μM for 18 hours.
3-2. MHC-Ⅰ 제한 교차항원제시능 분석3-2. MHC-Ⅰ restricted cross-antigen presentation analysis
활성화된 수지상세포는 교차항원제시 과정을 통해 외인성 항원에 의한 CD8+ 세포독성 T 림프구(cytotoxic T lymphocyte, CTL) 반응을 효과적으로 유도한다. 즉, 수지상세포는 SIINFEKL-Kb 복합체를 인식하고 이에 대한 반응으로 SIINFEKL-특이적 CD8+ T 세포를 자극하는 능력이 있다. Activated dendritic cells effectively induce CD8+ cytotoxic T lymphocyte (CTL) responses to exogenous antigens through the cross-antigen presentation process. In other words, dendritic cells have the ability to recognize SIINFEKL-K b complex and stimulate SIINFEKL-specific CD8+ T cells in response.
본 실험에서는 MHC-Ⅰ 제한 교차항원제시능 분석 방법(MHC-I-restricted cross-presentation assay)을 이용하여 SIINFEKL 펩타이드로 교환된 Kb 부착 나노입자(SIINFEKL-pulsed NP-Ab-Kb)로 처리된 수지상세포의 교차 프라이밍 용량(cross-priming capacity)을 분석하였다.In this experiment, the MHC-I-restricted cross-presentation assay was used to treat K b- attached nanoparticles (SIINFEKL-pulsed NP-Ab-K b ) exchanged with SIINFEKL peptide. The cross-priming capacity of dendritic cells was analyzed.
DC2.4 세포를 96 well 플레이트에 1×105 cells/well로 분주하고, SIINFEKL 펩타이드로 교환된 Kb 부착 나노입자(SIINFEKL-pulsed NP-Ab-Kb)와 2시간 배양한 뒤, 37℃의 PBS로 세척하고, 1% 파라포름알데히드로 고정한 다음 다시 PBS로 세척하였다. 이를 SIINFEKL-특이적 CD8+ T 세포 hybridoma CD8OVA 세포를 2×105 cells/well와 공동 배양하였다. 상층액을 채취하고 IL-2 생산량은 ELISA kit(BD Biosciences)로 측정하였다.DC2.4 cells were distributed at 1×10 5 cells/well in a 96 well plate, incubated with K b- attached nanoparticles exchanged with SIINFEKL peptide (SIINFEKL-pulsed NP-Ab-K b ) for 2 hours, and incubated at 37°C. Washed with PBS, fixed with 1% paraformaldehyde, and then washed again with PBS. This was co-cultured with SIINFEKL-specific CD8+ T cell hybridoma CD8OVA cells at 2×10 5 cells/well. The supernatant was collected, and IL-2 production was measured using an ELISA kit (BD Biosciences).
도 10에서 보듯이, SIINFEKL-pulsed Kb 부착 나노입자로 처리된 DC2.4 세포는 CD8OVA 세포를 강력하게 활성화하였으며, SIINFEKL-pulsed Kb 부착 나노입자 처리 농도 의존적으로 활성이 증가하였다. Kb 부착 나노입자로 처리된 DC2.4 세포는 CD8OVA 세포의 활성이 확인되었으나, SIINFEKL-pulsed Kb 부착 나노입자를 처리한 경우에 비해 현저하게 낮았다(도 10b). 한편, Kb 분자는 EG7.OVA 종양으로부터 분리된 것으로, 일부 Kb 분자를 통해 SIINFEKL 펩타이드가 발현되어 있다.As shown in Figure 10, DC2.4 cells treated with SIINFEKL-pulsed K b attached nanoparticles strongly activated CD8OVA cells, and the activity increased in a concentration-dependent manner upon treatment with SIINFEKL-pulsed K b attached nanoparticles. CD8OVA cell activity was confirmed in DC2.4 cells treated with K b attached nanoparticles, but it was significantly lower than when treated with SIINFEKL-pulsed K b attached nanoparticles (Figure 10b). Meanwhile, K b molecules were isolated from EG7.OVA tumors, and SIINFEKL peptide is expressed through some K b molecules.
3-3. SIINFEKL-pulsed K3-3. SIINFEKL-pulsed K b b 부착 나노입자의 SIINFEKL 특이적 CTL 유도SIINFEKL-specific CTL induction by attached nanoparticles
실험 당일 및 7일차에 C57BL/6 마우스에 PBS 또는 SIINFEKL-pulsed Kb 부착 나노입자 1×109 particles/mouse를 정맥 내 주사하였다. SIINFEKL 특이적 CTL을 유도하기 위한 SIINFEKL-pulsed Kb 부착 나노입자의 최적 입자수는 별도의 실험을 통해 확인하였다(도 11). 대조군으로 PBS 또는 카르복실화 나노입자(bare NP)를 사용하였다.On the day and 7th day of the experiment, C57BL/6 mice were intravenously injected with 1×10 9 particles/mouse with PBS or SIINFEKL-pulsed K b attached nanoparticles. The optimal particle number of SIINFEKL-pulsed K b attached nanoparticles to induce SIINFEKL-specific CTL was confirmed through a separate experiment (FIG. 11). As a control, PBS or carboxylated nanoparticles (bare NPs) were used.
실험 5일차 및 12일차에 안면 정맥에서 채혈한 말초 혈액은 T-Select H-2Kb OVA Tetramer(MBL, Tokyo, Japan)와 마우스 CD8α mAb(Clone KT15; BioLegend)로 염색하여 SIINFEKL 펩타이드 특이적 CD8+ T 세포를 상기 실험예 1과 같이 유세포 분석기(FACS Canto II, BD Biosciences)를 이용하여 분석하였다. 도 12에서 보듯이, SIINFEKL-pulsed Kb 부착 나노입자에 의해 면역이 유도된 경우 SIINFEKL 특이적 CTL 유도 활성이 강하게 나타났다. CD8+ T 세포 중 SIINFEKL 특이적 CTL의 비율은 20 μM SIINFEKL-pulsed Kb 부착 나노입자를 투여할 경우 1.8%, 100 μM SIINFEKL-pulsed Kb 부착 나노입자를 투여할 경우 2.5%로 증가하였다.Peripheral blood collected from the facial vein on days 5 and 12 of the experiment was stained with T-Select H-2K b OVA Tetramer (MBL, Tokyo, Japan) and mouse CD8α mAb (Clone KT15; BioLegend) to identify SIINFEKL peptide-specific CD8+ T. Cells were analyzed using a flow cytometer (FACS Canto II, BD Biosciences) as in Experimental Example 1 above. As shown in Figure 12, when immunity was induced by SIINFEKL-pulsed K b attached nanoparticles, SIINFEKL-specific CTL induction activity was strongly observed. The proportion of SIINFEKL-specific CTL among CD8+ T cells increased to 1.8% when 20 μM SIINFEKL-pulsed K b attached nanoparticles were administered and to 2.5% when 100 μM SIINFEKL-pulsed K b attached nanoparticles were administered.
실험 14일차에 SIINFEKL 특이적 CTL 활성에 대하여 in vivo CTL assay로 평가하였다. 표적 세포는 1 μM SIINFEKL 펩타이드로 펄스되고(pulsed) 25 μM CFSE로 표지된 동계 비장세포(syngeneic splenocytes)이며, SIINFEKL 펩타이드 펄스 없이 5 μM CFSE로 표지된 동계 비장세포를 대조군 표적 세포로 사용하였다. SIINFEKL-pulsed 표적 세포의 특이적 사멸은 각 마우스로부터 분리된 비장 세포(spleen cell) 및 림프절 세포(lymph node cell)를 유세포 분석을 통해 분석함으로써 결정되었다. 도 13에서 보듯이 SIINFEKL-pulsed 표적 세포의 특이적 사멸률은 20 μM SIINFEKL-pulsed Kb 부착 나노입자를 투여했을 때 29.7%였고, 100 μM SIINFEKL-pulsed Kb 부착 나노입자를 투여했을 때 62.7%로 증가하였다.On the 14th day of the experiment, SIINFEKL-specific CTL activity was evaluated by in vivo CTL assay. Target cells were syngeneic splenocytes pulsed with 1 μM SIINFEKL peptide and labeled with 25 μM CFSE, and syngeneic splenocytes labeled with 5 μM CFSE without SIINFEKL peptide pulse were used as control target cells. Specific killing of SIINFEKL-pulsed target cells was determined by analyzing spleen cells and lymph node cells isolated from each mouse using flow cytometry. As shown in Figure 13, the specific death rate of SIINFEKL-pulsed target cells was 29.7% when 20 μM SIINFEKL-pulsed K b attached nanoparticles were administered and 62.7% when 100 μM SIINFEKL-pulsed K b attached nanoparticles were administered. increased to.
<실험예 4> SIINFEKL-pulsed K<Experimental Example 4> SIINFEKL-pulsed K b b 부착 나노입자의 항종양 활성Antitumor activity of attached nanoparticles
C57BL/6 마우스의 오른쪽 옆구리에 EG7.OVA 세포를 3×105 cells/mouse로 피하 접종하였다. 접종 후 2일, 9일, 16일에 SIINFEKL-pulsed Kb 부착 나노입자를 1×109 cells/mouse로 정맥 투여하였다. 대조군으로 PBS 또는 카르복실화 나노입자(bare NP)를 정맥 투여하였다.EG7.OVA cells were inoculated subcutaneously at 3×10 5 cells/mouse on the right flank of C57BL/6 mice. On days 2, 9, and 16 after vaccination, SIINFEKL-pulsed K b attached nanoparticles were administered intravenously at 1×10 9 cells/mouse. As a control, PBS or carboxylated nanoparticles (bare NPs) were administered intravenously.
4-1. 혈액에서 SIINFEKL 특이적 CTL 분석4-1. SIINFEKL-specific CTL analysis in blood
실험 7일차(post-priming 5일 후) 및 21일차(post-boosting 5일 후)에 안면 정맥에서 채혈한 말초 혈액은 T-Select H-2Kb OVA Tetramer(MBL, Tokyo, Japan)와 마우스 CD8α mAb(Clone KT15; BioLegend)를 이용하여 SIINFEKL 특이적 CTL을 분석하였다. Peripheral blood collected from the facial vein on the 7th day (5 days after post-priming) and 21st day (5 days after post-boosting) of the experiment was incubated with T-Select H-2K b OVA Tetramer (MBL, Tokyo, Japan) and mouse CD8α. SIINFEKL-specific CTL were analyzed using mAb (Clone KT15; BioLegend).
도 14에 7일차, 도 15에 21일차 실험 결과를 나타내었다. SIINFEKL-pulsed Kb 부착 나노입자에 의한 면역화는 SIINFEKL 펩타이드 특이적 CTL을 효과적으로 유도하였다. 100 μM SIINFEKL-pulsed Kb 부착 나노입자를 투여한 마우스에서 CD8+ T 세포 중 SIINFEKL 특이적 CTL의 상대 빈도는 7일차에 1.8%, 21일차에 3.4%로 확인되었다. Figure 14 shows the experiment results on the 7th day, and Figure 15 shows the experimental results on the 21st day. Immunization with SIINFEKL-pulsed K b attached nanoparticles effectively induced SIINFEKL peptide-specific CTL. In mice administered 100 μM SIINFEKL-pulsed K b attached nanoparticles, the relative frequency of SIINFEKL-specific CTL among CD8+ T cells was found to be 1.8% on day 7 and 3.4% on day 21.
4-2. 종양 부피 확인4-2. Determination of tumor volume
C57BL/6 마우스의 오른쪽 옆구리에 EG7.OVA 세포를 3×105 cells/mouse로 피하 접종하였다. 접종 후 2일, 9일, 16일에 SIINFEKL-pulsed Kb 부착 나노입자를 1×109 cells/mouse로 정맥 투여하였다. 대조군으로 PBS 또는 카르복실화 나노입자(bare NP)를 정맥 투여하였다. 캘리퍼스(calipers)를 사용하여 2일마다 종양의 부피를 측정하였다. 부피(mm3)는 0.52 × 긴 직경(mm) × (짧은 직경(mm2)으로 계산하였다.EG7.OVA cells were inoculated subcutaneously at 3×10 5 cells/mouse on the right flank of C57BL/6 mice. On days 2, 9, and 16 after vaccination, SIINFEKL-pulsed K b attached nanoparticles were administered intravenously at 1×10 9 cells/mouse. As a control, PBS or carboxylated nanoparticles (bare NPs) were administered intravenously. Tumor volume was measured every 2 days using calipers. The volume (mm 3 ) was calculated as 0.52 × long diameter (mm) × (short diameter (mm 2 )).
도 16에서 보듯이, SIINFEKL-pulsed Kb 부착 나노입자를 정맥 투여한 경우에는 종양의 성장이 억제되었으며, 100 μM의 SIINFEKL-pulsed Kb 부착 나노입자를 정맥 투여한 경우에는 종양의 성장이 거의 억제되었다.As shown in Figure 16, when SIINFEKL-pulsed K b attached nanoparticles were administered intravenously, tumor growth was inhibited, and when 100 μM of SIINFEKL-pulsed K b attached nanoparticles were administered intravenously, tumor growth was almost inhibited. It has been done.
이상에서 살펴본 바와 같이, 본 발명의 구체적인 실시예를 상세하게 설명되었으나, 본 발명의 사상을 이해하는 당업자는 동일한 사상의 범위 내에서 다른 구성요소를 추가, 변경, 삭제 등을 통하여, 퇴보적인 다른 발명이나 본 발명 사상의 범위 내에 포함되는 다른 실시예를 용이하게 제안할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다. 본 발명의 범위는 상술한 상세한 설명보다는 후술하는 특허청구의 범위에 의하여 나타내어지며, 특허청구의 범위의 의미 및 범위 그리고 그 균등 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.As discussed above, specific embodiments of the present invention have been described in detail, but those skilled in the art who understand the spirit of the present invention may add, change, delete, etc. other components within the scope of the same spirit, thereby creating other regressive inventions. However, other embodiments that are included within the scope of the present invention can be easily proposed. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive. The scope of the present invention is indicated by the scope of the claims described below rather than the detailed description above, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts are included in the scope of the present invention. It should be interpreted as
<110> Chungbuk National University Industry-Academic Cooperation Foundation <120> Method for producing complex to which tumor-specific peptides are attached and pharmaceutical composition for inhibiting tumor growth comprising complex prepared therefrom <130> PN2202-130 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 8 <212> PRT <213> Gallus gallus <400> 1 Ser Ile Ile Asn Phe Glu Lys Leu 1 5 <110> Chungbuk National University Industry-Academic Cooperation Foundation <120> Method for producing complex to which tumor-specific peptides are attached and pharmaceutical composition for inhibiting tumor growth comprising complex prepared there from <130>PN2202-130 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 8 <212> PRT <213> Gallus gallus <400> 1 Ser Ile Ile Asn Phe Glu Lys Leu 1 5
Claims (8)
상기 단계 (a)의 에멀젼을 PVA(polyvinyl alcohol)와 PEMA 혼합용액에 혼합하여 카르복실화 나노입자를 제조하는 단계 (b);
상기 단계 (b)의 카르복실화 나노입자에 주조직적합항원-Ⅰ에 대한 항체와 종양 유래 막 단백질에 포함된 주조직적합항원-Ⅰ 분자를 결합하는 단계 (c); 및
상기 단계 (c) 후, 종양 특이적 펩타이드를 부착하는 단계 (d);를 포함하는 주조직적합항원-Ⅰ 분자가 결합된 복합체의 제조방법.
Step (a) of preparing an emulsion by mixing PLGA (poly(lactic-co-glycolic acid)) and PEMA (poly(ethylene/maleic anhydride));
Step (b) of preparing carboxylated nanoparticles by mixing the emulsion of step (a) with a mixed solution of polyvinyl alcohol (PVA) and PEMA;
Step (c) combining an antibody against major histocompatibility antigen-I and a major histocompatibility antigen-I molecule contained in a tumor-derived membrane protein to the carboxylated nanoparticles of step (b); and
A method of producing a complex to which a major histocompatibility antigen-I molecule is bound, comprising the step (d) of attaching a tumor-specific peptide after step (c).
상기 주조직적합항원-Ⅰ 분자가 결합된 복합체는,
수지상세포의 탐식작용(phagocytosis)을 유도하는 것인, 주조직적합항원-Ⅰ 분자가 결합된 복합체의 제조방법.
According to paragraph 1,
The complex to which the major histocompatibility antigen-I molecule is bound is,
A method of producing a complex containing major histocompatibility antigen-I molecules, which induces phagocytosis of dendritic cells.
상기 주조직적합항원-Ⅰ 분자가 결합된 복합체는,
수지상세포의 성숙 또는 활성화를 유도하는 것인, 주조직적합항원-Ⅰ 분자가 결합된 복합체의 제조방법.
According to paragraph 1,
The complex to which the major histocompatibility antigen-I molecule is bound is,
A method for producing a complex to which a major histocompatibility antigen-I molecule is bound, which induces maturation or activation of dendritic cells.
상기 주조직적합항원-Ⅰ 복합체는,
수지상세포의 교차항원제시능을 유도하는 것인, 주조직적합항원-Ⅰ 분자가 결합된 복합체의 제조방법.
According to paragraph 1,
The major histocompatibility antigen-I complex,
A method for producing a complex combining major histocompatibility antigen-I molecules, which induces the cross-antigen presentation ability of dendritic cells.
상기 주조직적합항원-Ⅰ 분자가 결합된 복합체는,
종양 특이적 CTL(cytotoxic T lymphocyte) 반응을 유도하는 것인, 주조직적합항원-Ⅰ 분자가 결합된 복합체의 제조방법.
According to paragraph 1,
The complex to which the major histocompatibility antigen-I molecule is bound is,
A method of producing a complex bound to a major histocompatibility antigen-I molecule, which induces a tumor-specific CTL (cytotoxic T lymphocyte) response.
A pharmaceutical composition for inhibiting tumor growth, comprising as an active ingredient a complex of major histocompatibility antigen-I molecules prepared by the method of claim 1.
상기 약학 조성물은,
암 특이 CTL이 없거나 미약한 암환자를 대상으로 치료하는 것인, 종양 성장 억제용 약학 조성물.
According to clause 6,
The pharmaceutical composition is,
A pharmaceutical composition for inhibiting tumor growth, which is used to treat cancer patients with no or weak cancer-specific CTL.
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