KR20230052017A - Human antibodies for binding to human Fc alpha receptor - Google Patents
Human antibodies for binding to human Fc alpha receptor Download PDFInfo
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- KR20230052017A KR20230052017A KR1020210135095A KR20210135095A KR20230052017A KR 20230052017 A KR20230052017 A KR 20230052017A KR 1020210135095 A KR1020210135095 A KR 1020210135095A KR 20210135095 A KR20210135095 A KR 20210135095A KR 20230052017 A KR20230052017 A KR 20230052017A
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Abstract
Description
본 발명은 인간 Fc알파 수용체와의 결합력이 증대된 항체 또는 이의 면역학적 활성을 가진 단편에 관한 것이다.The present invention relates to an antibody having increased binding ability to a human Fcalpha receptor or a fragment having immunological activity thereof.
항체는 체액성 및 세포성 면역계 사이의 연결고리를 제공하며, 항체의 Fab 영역이 항원을 인식하는 반면, Fc 부분은 모든 면역 적격 세포에 의해 차별적으로 발현되는 Fc 수용체 (FcR)에 결합한다. 다가 항원/항체 복합체에 의한 수용체의 가교로, 표적 세포의 탈과립화, 세포용해 또는 포식작용 및 사이토카인-암호화 유전자의 전사-활성화가 촉발된다 (Deo, Y.M. et al., Immunol. Today 18(3):127-135 (1997)). 항체는, 항체 Fc 영역 상의 Fc 수용체 결합 부위가 세포 상의 Fc 수용체 (FcR) 에 결합함으로써 Fc 영역을 통해 세포에 결합한다. 면역글로블린의 Fc 영역에 대한 수용체는 단핵구, 대식세포 및 다형핵 세포의 많은 방어적 기능을 촉발하는 데 중요하다. 이들 세포 상의 면역글로불린에 대한 수용체 (Fc 수용체 또는 FcR)들은 광범위하게 연구되었으며, 이들 수용체에 대한 모노클로날 항체를 발생시켜 이것이 치료적으로 유효하다는 것을 밝혔다. 항체가 세포 표면 상의 Fc 수용체에 결합하면 항체-코팅 입자의 포식 및 파괴, 면역 복합체의 제거, 살세포에 의한 항체-코팅 표적 세포의 용해 (항체-의존적 세포-매개 세포독성, 또는 ADCC 라 알려짐), 염증 매개체의 방출, 태반 이동 및 면역글로불린 생성의 제어를 포함하여 중요하고도 다양한 여러 생물학적 반응을 촉발한다. Antibodies provide a link between the humoral and cellular immune systems, and while the Fab region of an antibody recognizes an antigen, the Fc portion binds to an Fc receptor (FcR) that is differentially expressed by all immunocompetent cells. Cross-linking of receptors by multivalent antigen/antibody complexes triggers degranulation, cytolysis or phagocytosis of target cells and transcription-activation of cytokine-encoding genes (Deo, Y.M. et al., Immunol. Today 18(3 ):127-135 (1997)). An antibody binds to a cell through the Fc region by binding an Fc receptor binding site on the antibody Fc region to an Fc receptor (FcR) on the cell. Receptors for the Fc region of immunoglobulins are important in triggering many protective functions of monocytes, macrophages and polymorphonuclear cells. Receptors for immunoglobulins on these cells (Fc receptors or FcRs) have been extensively studied, and monoclonal antibodies against these receptors have been raised and shown to be therapeutically effective. Binding of antibodies to Fc receptors on the cell surface results in phagocytosis and destruction of antibody-coated particles, elimination of immune complexes, and lysis of antibody-coated target cells by killer cells (known as antibody-dependent cell-mediated cytotoxicity, or ADCC) It triggers a number of important and diverse biological responses, including release of inflammatory mediators, control of placental migration and immunoglobulin production.
한편, 다수의 연구 결과는 Fc-수용체-의존성 메카니즘이 종양에 대한 세포독성 항체의 작용에 실질적인 원인이 됨을 시사하며, 종양에 대한 최적 항체는 활성화 Fc 수용체에 우선적으로 결합한다는 것을 보여준다 (Clynes, R. A., et al., Nature Medicine 6(4):443-446 (2000); Kalergis, A. M.,and Ravetch, J. V., J. Exp. Med. 195 (12):1653-1659 (2002년 6월). 종종, 성공적인 항암 단일클론 항체는, ADCC 외에, 세포 신호전달 캐스케이드를 활성화시키거나 또는 성장 인자에의 접근을 차단하여 표적 세포 생존, 증식 또는 세포사를 조절하는 Fc-독립적인 직접적 신호전달 메카니즘을 유도한다. (Selenko, N., et al., J Clin. Immunol. 22 (3):124-130 (2002)). 예를 들어, CD20+ B 세포를 리툭시맙으로 처리하면 Mab-유도된 세포자가사멸(apoptosis)의 유도 및 보체 매개된 세포용해와 ADCC를 유도하는 것으로 확인된 바 있다. (Selenko, N., et al., J. Clin. Immunol. 22 (3):124-130 (2002)). 또한, 림프종 세포의 리툭시맙 유도된 세포자가사멸은 세포를 죽일 뿐 아니라 항원 제시 수지상 세포(DC)에 의한 림프종 세포 유도된 펩티드의 흡수 및 교차 제시를 촉진하며, DC의 성숙을 유도하고, 특이적 세포독성 T 림프구(CTL)를 생성한다.On the other hand, a number of studies suggest that the Fc-receptor-dependent mechanism is a substantial cause for the action of cytotoxic antibodies on tumors, and show that optimal antibodies against tumors preferentially bind to activated Fc receptors (Clynes, RA , et al., Nature Medicine 6(4):443-446 (2000), Kalergis, AM, and Ravetch, JV, J. Exp. Med. 195 (12):1653-1659 (June 2002). , Successful anticancer monoclonal antibodies, in addition to ADCC, induce Fc-independent direct signaling mechanisms that activate cell signaling cascades or block access to growth factors to regulate target cell survival, proliferation or cell death. (Selenko, N., et al., J Clin. Immunol. 22 (3):124-130 (2002)) For example, treatment of CD20 + B cells with rituximab results in Mab-induced apoptosis (Selenko, N., et al., J. Clin. Immunol. 22 (3):124-130 (2002)). In addition, rituximab-induced apoptosis of lymphoma cells not only kills the cells, but also promotes uptake and cross-presentation of lymphoma cell-derived peptides by antigen-presenting dendritic cells (DCs), and induces maturation of DCs; Generates specific cytotoxic T lymphocytes (CTLs).
IgG (감마 수용체), IgE (엡실론 수용체), IgA (알파 수용체) 및 IgM (뮤 수용체)를 포함하는 상이한 종류의 항체에 특이적인 몇 가지 Fc 수용체가 존재한다. 이 중, IgA 수용체 (Fcα수용체 또는 CD89)는 효과기(effector) 세포의 기능을 증진시킬 수 있다. 리간드가 Fcα수용체에 결합하면 백혈구 및 Fcα수용체-보유 세포주의 식세포작용(antibody-dependent cellular phagocytosis, ADCP)과 항체-매개성 세포독성작용(antibody dependent cellular cytotoxicity, ADCC)이 촉발된다. 또한, Fcα수용체는 효과기 세포 상의 IgG에 대한 수용체와 협력하여 표적 세포에 대한 식세포작용을 강화시킬 수 있다. 항체 Fc 영역에 의해 매개되는 효과기 기능(effector function)은 두 카테고리로 나눌 수 있다: (1) 항체가 항원에 결합한 후 작동하는 효과기 기능 (이러한 기능은, 예컨대 보체 연쇄증폭반응에의 관여 또는 Fc 수용체 (FcR)-보유 세포를 포함함); 및 (2) 항원 결합과는 별개로 작동하는 효과기 기능 (이러한 기능은, 예컨대 트랜스사이토시스(transcytosis)에 의해 세포 장벽을 가로질러 이동하는 능력 및 순환 중 지속성을 부여함). 예를 들어, 보체의 Cl 성분이 항체에 결합하면 보체 시스템이 활성화된다. 보체의 활성화는 세포 병원체의 옵소닌화 및 용해에 있어 중요하다. 보체의 활성화는 또한 염증 반응을 자극하며 자가면역 과민증에 관련될 수도 있다. 이와 같이, Fcα수용체 또는 CD89는 IgA의 Fc 부분과 결합하는 수용체이며 (Kerr, M. A. 1990, Biochem. J. 271: 285-296), 주로 다형핵 백혈구 (PMN), 단핵구, 대식세포, 호중구 및 호산구를 비롯한 세포독성 면역 효과기 세포상에서 항상 발현된다 (Morton, H. C., et al., 1996, Critical Reviews in Immunology 16: 423). 또한, Fcα수용체가 림프구 아개체군 상에서 발현 (Morton, H. C., et al., 1996, Critical Reviews in Immunology 16: 423)되고, 사구체 혈관사이 세포(mesangial cell) 상에서도 발현되는 것이 보고되었다 (Gomez-Guerrero, C., et al., 1996, J. Immunol. 156: 4369-4376). 인간 Fcα수용체의 α-쇄는 많이 글리코실화되어 있고, IgG 및 IgE에 대한 수용체도 포함하는 Ig 수퍼-유전자 군에 속하는 타입 I 막횡단 분자이다. 19번 염색체 상에 위치한 1개의 유전자는 FcαRI 알파 쇄의, 다양하게 스플라이싱 (splicing)된 다수의 이소타입들을 코딩한다 (55 내지 110 kDa; Morton, H. C., et al., 1996, Critical Reviews in Immunology 16: 423). 골수세포성 Fcα수용체는, Fcα수용체 신호 전달에 역할을 하는 FcR γ-쇄와 연관되어 있는 것으로 밝혀졌다 (Morton, H. C. et al. 1995, J. Biol. Chem. 270: 29781; Pfefferkorn, L. C., et al. 1995, J. Immunol. 153: 3228-3236; Saito, K. et al., 1995, J. Allergy Clin. Immunol. 96: 1152). Fcα수용체는 항원이 결합된 IgA1 및 IgA2, 및 모노머성 IgA1 및 IgA2에 모두 결합하며 (Mazangera, R. L. et al., 1990, Biochem. J. 272: 159-165), 이것은 FcγR 및 FcεRI이 각각 IgG 및 IgE로 포화되는 것과 같은 동일한 방법으로 생체내에서 상기 수용체가 모노머성 IgA 항원으로 포화되어 있는 것과 일치한다. 골수 효과기 세포 상의 Fcα수용체와, 중합성 IgA, IgA 면역 복합체, 또는 리간드 결합 도메인 내부 또는 외부의 에피토프에 대해 특이적인 mAb의 교차 결합은 탈과립화, 과산화물 방출, 염증성 사이토카인의 분비, 내포작용(endocytosis) 및 식세포작용을 자극한다 (Patty, C.,A. Herbelin, A. Lihuen, J. F. Bach, and R. C. Monteiro, 1995, Immunology 86: 1-5; Stewart, W. W., R. L. MazYegera, L. Shen, and M. A. Kerr, 1994, J. Leucocyte Biology 56: 481-487; Stewart, W. W., and M. A. Kerr, 1990, Immunology 71: 328-334; Shen, L., 1992, J. Leukocyte Biology 51: 373-378). Fcα수용체에 의해 촉발된 이러한 생리학적 반응은 점막 표면 상에서 제1선의 체액성 방어에 중요할 수 있다 (Morton, H. C., M. van Egmond, and J. G. J. van de Winkel, 1996, Critical Reviews in Immunology 16: 423).There are several Fc receptors specific for different classes of antibodies including IgG (gamma receptor), IgE (epsilon receptor), IgA (alpha receptor) and IgM (mu receptor). Among them, the IgA receptor (Fcα receptor or CD89) can enhance the function of effector cells. When ligands bind to Fcα receptors, phagocytosis (antibody-dependent cellular phagocytosis, ADCP) and antibody-dependent cellular cytotoxicity (ADCC) of leukocytes and Fcα receptor-bearing cell lines are triggered. Additionally, Fcα receptors can enhance phagocytosis on target cells in concert with receptors for IgG on effector cells. Effector functions mediated by antibody Fc regions can be divided into two categories: (1) effector functions that operate after binding of an antibody to an antigen (such functions include, for example, involvement in complement cascade amplification or Fc receptors). (including FcR)-retaining cells); and (2) effector functions that operate independently of antigen binding (these functions confer persistence in circulation and the ability to migrate across cell barriers, such as by transcytosis). For example, the complement system is activated when the Cl component of complement binds to an antibody. Activation of complement is important for the opsonization and lysis of cellular pathogens. Activation of complement also stimulates the inflammatory response and may be involved in autoimmune hypersensitivity. Thus, the Fcα receptor or CD89 is a receptor that binds to the Fc portion of IgA (Kerr, M. A. 1990, Biochem. J. 271: 285-296), mainly polymorphonuclear leukocytes (PMN), monocytes, macrophages, neutrophils and eosinophils. It is constitutively expressed on cytotoxic immune effector cells, including (Morton, H. C., et al., 1996, Critical Reviews in Immunology 16: 423). In addition, it has been reported that the Fcα receptor is expressed on the lymphocyte subpopulation (Morton, H. C., et al., 1996, Critical Reviews in Immunology 16: 423) and also expressed on mesangial cells (Gomez-Guerrero, C., et al., 1996, J. Immunol. 156: 4369-4376). The α-chain of the human Fcα receptor is a highly glycosylated, type I transmembrane molecule belonging to the Ig super-gene family, which also includes receptors for IgG and IgE. One gene, located on
그러나, 이와 같이 포유류에서 가장 높은 비율을 가지는 백혈구의 효과기 기능을 이용하는 IgA는 이합체 형태가 존재할뿐만 아니라, 복잡한 글리코실화, 테일피스(tailpiece) 등을 거쳐 IgG보다 생산성이 현저히 떨어지며, 체내에서 항체의 재활용(recycle)에 관여한다고 알려진 FcRn에 결합하지 못해 3주의 반감기를 가지는 IgG와 비교하여 짧은 1주의 반감기를 가진다는 매우 큰 단점이 있다.However, IgA, which uses the effector function of leukocytes, which has the highest ratio in mammals, not only has a dimer form, but also has significantly lower productivity than IgG through complicated glycosylation and tailpiece, and is recycled in the body. Compared to IgG, which has a half-life of 3 weeks because it does not bind to FcRn known to be involved in (recycle), it has a very short half-life of 1 week.
본 발명에서는 생산성이 떨어지고 짧은 반감기를 가지는 IgA 항체의 단점을 개선하기 위해, Fc 알파 수용체에 결합하는 IgG 항체 또는 이의 면역학적 활성을 가진 단편을 제공하는 것을 목적으로 한다.An object of the present invention is to provide an IgG antibody that binds to an Fc alpha receptor or a fragment having immunological activity thereof in order to improve the disadvantages of an IgA antibody having low productivity and a short half-life.
상기 목적을 달성하기 위해, 본 발명은 Fc 알파 수용체에 특이적인 항체 또는 이의 면역학적 활성을 가진 단편을 제공한다.In order to achieve the above object, the present invention provides an Fc alpha receptor-specific antibody or a fragment having immunological activity thereof.
또한, 본 발명은 상기 항체 또는 이의 면역학적 활성을 가진 단편을 코딩하는 단리된 핵산 분자, 이를 포함하는 벡터 및 상기 벡터로 형질전환된 숙주 세포를 제공한다.In addition, the present invention provides an isolated nucleic acid molecule encoding the antibody or immunologically active fragment thereof, a vector containing the same, and a host cell transformed with the vector.
아울러, 본 발명은 Fc 알파 수용체에 특이적인 항체 또는 이의 면역학적 활성을 가진 단편을 제조하는 방법을 제공한다.In addition, the present invention provides a method for preparing an antibody specific to an Fc alpha receptor or a fragment having immunological activity thereof.
본 발명에 따른 Fc 알파 수용체에 결합력을 가지는 인간 항체 또는 이의 면역학적 활성을 가진 단편들은, IgG 형태이므로 IgA 항체의 단점들을 극복하였으며, 효과기(effector) 세포들, 특히, 포유류에서 가장 높은 비율을 차지하는 호중구의 Fc 알파 수용체와 특이적으로 결합하여 효과기 기능 (ADCC 및 ADCP)을 극대화시키는 효과가 있으므로, 이를 효과기 기능이 향상된 항체 의약품으로 유용하게 활용할 수 있다.Since the human antibodies having binding ability to the Fc alpha receptor or immunologically active fragments thereof according to the present invention are in the form of IgG, they overcome the disadvantages of IgA antibodies and account for the highest proportion of effector cells, particularly in mammals. Since it specifically binds to the Fc alpha receptor of neutrophils and has the effect of maximizing effector functions (ADCC and ADCP), it can be usefully used as an antibody drug with improved effector functions.
도 1은 제작된 이합체 Fc 알파 수용체-ECD 영역 항원 단백질 및 사합체 Fc 알파 수용체-ECD 영역 항원 단백질의 벡터 및 이의 SDS-PAGE 분석 결과를 나타낸 도이다.
도 2는 유세포 분석기를 이용한 항체 탐색 모식도를 나타낸 도이다.
도 3은 Fc 알파 수용체-ECD에 높은 결합 친화도를 보이는 scFv 인간 항체 변이체 6종의 아미노산 서열 분석결과를 나타낸 도이다.
도 4는 6종의 scFv 항체 변이체의 Fc 알파 수용체 결합력 분석 결과이다.
도 5는 6종의 scFv 항체 변이체를 각각 포함하는 IgG (JS9, JS19, JS30, JS40, JS41 및 JS48)들의 벡터, 및 발현 및 정제 후 SDS-PAGE 젤 사진을 나타낸 도이다.
도 6은 6종의 scFv 항체 변이체를 각각 포함하는 IgG 항체 (JS9, JS30, JS40, JS41 및 JS48)들의 항원 결합력을 확인한 도이다.
도 7은 JS40의 VH 영역을 가지면서 JS48의 VL 영역을 가지는 JS40-1 항체 및 JS48의 VH 영역을 가지면서 JS40의 VL 영역을 가지는 JS48-1 항체를 제작하기 위한 동물세포 발현 벡터와 이를 동물세포에서 발현 및 정제한 후 SDS-PAGE로 확인한 도이다.
도 8은 JS40-1 항체 및 JS48-1 항체의 Fc 알파 수용체-ECD에 대한 결합력을 ELISA로 확인한 결과를 나타낸 도이다.1 is a diagram showing the prepared vectors of dimeric Fc alpha receptor-ECD region antigen protein and tetrameric Fc alpha receptor-ECD region antigen protein and the results of SDS-PAGE analysis thereof.
2 is a diagram showing a schematic diagram of antibody search using a flow cytometer.
Figure 3 is a diagram showing the amino acid sequence analysis results of six scFv human antibody variants showing high binding affinity to Fc alpha receptor-ECD.
Figure 4 is the Fc alpha receptor binding force analysis results of six scFv antibody variants.
5 is a diagram showing vectors of IgG (JS9, JS19, JS30, JS40, JS41 and JS48) each containing 6 scFv antibody variants, and SDS-PAGE gel pictures after expression and purification.
Figure 6 is a diagram confirming the antigen-binding ability of IgG antibodies (JS9, JS30, JS40, JS41 and JS48) each containing six scFv antibody variants.
Figure 7 shows an animal cell expression vector for preparing JS40-1 antibody having a JS40 VH region and a JS48 VL region and a JS48-1 antibody having a JS48 VH region and a JS40 VL region and an animal cell expression vector thereof It is a diagram confirmed by SDS-PAGE after expression and purification in .
8 is a diagram showing the results of confirming the binding ability of the JS40-1 antibody and the JS48-1 antibody to the Fc alpha receptor-ECD by ELISA.
이하, 첨부된 도면을 참조하여 본 발명의 구현예로 본 발명을 상세히 설명하기로 한다. 다만, 하기 구현예는 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 detail as an embodiment of the present invention with reference to the accompanying drawings. However, the following embodiments are presented as examples of the present invention, and if it is determined that detailed descriptions of well-known techniques or configurations may unnecessarily obscure the gist of the present invention, the detailed descriptions may be omitted. , the present invention is not limited thereby. Various modifications and applications of the present invention are possible within the scope of the claims described below and equivalents interpreted therefrom.
또한, 본 명세서에서 사용되는 용어(terminology)들은 본 발명의 바람직한 실시예를 적절히 표현하기 위해 사용된 용어들로서, 이는 사용자, 운용자의 의도 또는 본 발명이 속하는 분야의 관례 등에 따라 달라질 수 있다. 따라서, 본 용어들에 대한 정의는 본 명세서 전반에 걸친 내용을 토대로 내려져야 할 것이다. 명세서 전체에서, 어떤 부분이 어떤 구성요소를 "포함"한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성 요소를 더 포함할 수 있는 것을 의미한다.In addition, the terms used in this specification (terminology) are terms used to appropriately express preferred embodiments of the present invention, which may vary according to the intention of a user or operator or customs in the field to which the present invention belongs. Therefore, definitions of these terms will have to be made based on the content throughout this specification. Throughout the specification, when a certain component is said to "include", it means that it may further include other components without excluding other components unless otherwise stated.
본 명세서 전반을 통하여, 천연적으로 존재하는 아미노산에 대한 통상의 1문자 및 3문자 코드가 사용될 뿐만 아니라 Aib(α-아미노이소부티르산), Sar(N-methylglycine) 등과 같은 다른 아미노산에 대해 일반적으로 허용되는 3문자 코드가 사용된다. 또한 본 발명에서 약어로 언급된 아미노산은 하기와 같이 IUPAC-IUB 명명법에 따라 기재되었다:Throughout this specification, conventional one-letter and three-letter codes for naturally occurring amino acids are used, as well as generally accepted for other amino acids such as Aib (α-aminoisobutyric acid), Sar ( N -methylglycine), etc. A three-letter code is used. Amino acids also referred to by abbreviations in the present invention are described according to the IUPAC-IUB nomenclature as follows:
알라닌: A, 아르기닌: R, 아스파라긴: N, 아스파르트산: D, 시스테인: C, 글루탐산: E, 글루타민: Q, 글리신: G, 히스티딘: H, 이소류신: I, 류신: L, 리신: K, 메티오닌: M, 페닐알라닌: F, 프롤린: P, 세린: S, 트레오닌: T, 트립토판: W, 티로신: Y 및 발린: V. Alanine: A, Arginine: R, Asparagine: N, Aspartic acid: D, Cysteine: C, Glutamic acid: E, Glutamine: Q, Glycine: G, Histidine: H, Isoleucine: I, Leucine: L, Lysine: K, Methionine : M, phenylalanine: F, proline: P, serine: S, threonine: T, tryptophan: W, tyrosine: Y, and valine: V.
일 측면에서, 본 발명은 Fc 알파 수용체에 특이적인 항체 또는 이의 면역학적 활성을 가진 단편에 관한 것이다.In one aspect, the present invention relates to an Fc alpha receptor-specific antibody or immunologically active fragment thereof.
일 구현예에서, 본 발명의 Fc 알파 수용체에 특이적인 항체 또는 이의 면역학적 활성을 가진 단편은 (i) 서열번호 1의 아미노산 서열을 포함하는 CDRH(Complementarity determining regions Heavy chain)1, 서열번호 2의 아미노산 서열을 포함하는 CDRH2, 및 서열번호 3의 아미노산 서열을 포함하는 CDRH3를 포함하는 VH 도메인; 및/또는 (ⅱ) 서열번호 4의 아미노산 서열을 포함하는 CDRL1, 서열번호 6의 아미노산 서열을 포함하는 CDRL2, 및 서열번호 8의 아미노산 서열을 포함하는 CDRL3를 포함하는 VL 도메인을 포함하는 V 도메인을 포함할 수 있다.In one embodiment, the Fc alpha receptor-specific antibody or immunologically active fragment thereof of the present invention is (i) CDRH (Complementarity determining regions Heavy chain) 1 comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 a VH domain comprising CDRH2 comprising the amino acid sequence and CDRH3 comprising the amino acid sequence of SEQ ID NO: 3; and/or (ii) a V domain comprising a VL domain comprising CDRL1 comprising the amino acid sequence of SEQ ID NO: 4, CDRL2 comprising the amino acid sequence of SEQ ID NO: 6, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 8. can include
일 구현예에서, 본 발명의 Fc 알파 수용체에 특이적인 항체 또는 이의 면역학적 활성을 가진 단편은 (i) 서열번호 1의 아미노산 서열을 포함하는 CDRH1, 서열번호 2의 아미노산 서열을 포함하는 CDRH2, 및 서열번호 3의 아미노산 서열을 포함하는 CDRH3를 포함하는 VH 도메인; 및/또는 (ⅱ) 서열번호 5의 아미노산 서열을 포함하는 CDRL1, 서열번호 7의 아미노산 서열을 포함하는 CDRL2, 및 서열번호 9의 아미노산 서열을 포함하는 CDRL3를 포함하는 VL 도메인을 포함하는 V 도메인을 포함할 수 있다.In one embodiment, the antibody specific to the Fc alpha receptor of the present invention or a fragment having immunological activity thereof is (i) CDRH1 comprising the amino acid sequence of SEQ ID NO: 1, CDRH2 comprising the amino acid sequence of SEQ ID NO: 2, and a VH domain comprising CDRH3 comprising the amino acid sequence of SEQ ID NO: 3; and/or (ii) a V domain comprising a VL domain comprising CDRL1 comprising the amino acid sequence of SEQ ID NO: 5, CDRL2 comprising the amino acid sequence of SEQ ID NO: 7, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 9. can include
일 구현예에서, 본 발명의 Fc 알파 수용체에 특이적인 항체 또는 이의 면역학적 활성을 가진 단편은 (i) 서열번호 10의 아미노산 서열을 포함하는 FR1, 서열번호 12의 아미노산 서열을 포함하는 FR2, 서열번호 13의 아미노산 서열을 포함하는 FR3 및 서열번호 14의 아미노산 서열을 포함하는 FR4를 포함하는 VH 도메인; 및/또는 (ⅱ) 서열번호 16의 아미노산 서열을 포함하는 FR1, 서열번호 18의 아미노산 서열을 포함하는 FR2, 서열번호 20의 아미노산 서열을 포함하는 FR3 및 서열번호 22의 아미노산 서열을 포함하는 FR4를 포함하는 VL 도메인을 포함할 수 있다.In one embodiment, the antibody specific for the Fc alpha receptor of the present invention or a fragment having immunological activity thereof comprises (i) FR1 comprising the amino acid sequence of SEQ ID NO: 10, FR2 comprising the amino acid sequence of SEQ ID NO: 12, sequence a VH domain comprising FR3 comprising the amino acid sequence of SEQ ID NO: 13 and FR4 comprising the amino acid sequence of SEQ ID NO: 14; and/or (ii) FR1 comprising the amino acid sequence of SEQ ID NO: 16, FR2 comprising the amino acid sequence of SEQ ID NO: 18, FR3 comprising the amino acid sequence of SEQ ID NO: 20, and FR4 comprising the amino acid sequence of SEQ ID NO: 22 It may include a VL domain that includes.
일 구현예에서, 본 발명의 Fc 알파 수용체에 특이적인 항체 또는 이의 면역학적 활성을 가진 단편은 (i) 서열번호 11의 아미노산 서열을 포함하는 FR1, 서열번호 12의 아미노산 서열을 포함하는 FR2, 서열번호 13의 아미노산 서열을 포함하는 FR3 및 서열번호 15의 아미노산 서열을 포함하는 FR4를 포함하는 VH 도메인; 및/또는 (ⅱ) 서열번호 17의 아미노산 서열을 포함하는 FR1, 서열번호 19의 아미노산 서열을 포함하는 FR2, 서열번호 21의 아미노산 서열을 포함하는 FR3 및 서열번호 23의 아미노산 서열을 포함하는 FR4를 포함하는 VL 도메인을 포함할 수 있다.In one embodiment, the antibody specific for the Fc alpha receptor or a fragment having immunological activity thereof of the present invention comprises (i) FR1 comprising the amino acid sequence of SEQ ID NO: 11, FR2 comprising the amino acid sequence of SEQ ID NO: 12, sequence a VH domain comprising FR3 comprising the amino acid sequence of SEQ ID NO: 13 and FR4 comprising the amino acid sequence of SEQ ID NO: 15; and/or (ii) FR1 comprising the amino acid sequence of SEQ ID NO: 17, FR2 comprising the amino acid sequence of SEQ ID NO: 19, FR3 comprising the amino acid sequence of SEQ ID NO: 21, and FR4 comprising the amino acid sequence of SEQ ID NO: 23 It may include a VL domain that includes.
일 구현예에서, 본 발명의 Fc 알파 수용체에 특이적인 항체 또는 이의 면역학적 활성을 가진 단편은 (i) 서열번호 10의 아미노산 서열을 포함하는 FR1, 서열번호 1의 아미노산 서열을 포함하는 CDRH1, 서열번호 12의 아미노산 서열을 포함하는 FR2, 서열번호 2의 아미노산 서열을 포함하는 CDRH2, 서열번호 13의 아미노산 서열을 포함하는 FR3, 서열번호 3의 아미노산 서열을 포함하는 CDRH3 및 서열번호 14의 아미노산 서열을 포함하는 FR4를 포함하는 VH 도메인; 및/또는 (ⅱ) 서열번호 16의 아미노산 서열을 포함하는 FR1, 서열번호 4의 아미노산 서열을 포함하는 CDRL1, 서열번호 18의 아미노산 서열을 포함하는 FR2, 서열번호 6의 아미노산 서열을 포함하는 CDRL2, 서열번호 20의 아미노산 서열을 포함하는 FR3, 서열번호 8의 아미노산 서열을 포함하는 CDRL3 및 서열번호 22의 아미노산 서열을 포함하는 FR4를 포함하는 VL 도메인을 포함하는 V 도메인을 포함하는 scFv 변이체 JS40-1일 수 있다.In one embodiment, the Fc alpha receptor-specific antibody or fragment having immunological activity thereof of the present invention comprises (i) FR1 comprising the amino acid sequence of SEQ ID NO: 10, CDRH1 comprising the amino acid sequence of SEQ ID NO: 1, sequence FR2 comprising the amino acid sequence of SEQ ID NO: 12, CDRH2 comprising the amino acid sequence of SEQ ID NO: 2, FR3 comprising the amino acid sequence of SEQ ID NO: 13, CDRH3 comprising the amino acid sequence of SEQ ID NO: 3, and the amino acid sequence of SEQ ID NO: 14 VH domain comprising FR4 comprising; and/or (ii) FR1 comprising the amino acid sequence of SEQ ID NO: 16, CDRL1 comprising the amino acid sequence of SEQ ID NO: 4, FR2 comprising the amino acid sequence of SEQ ID NO: 18, CDRL2 comprising the amino acid sequence of SEQ ID NO: 6, scFv variant JS40-1 comprising a V domain comprising FR3 comprising the amino acid sequence of SEQ ID NO: 20, CDRL3 comprising the amino acid sequence of SEQ ID NO: 8, and VL domain comprising FR4 comprising the amino acid sequence of SEQ ID NO: 22 can be
일 구현예에서, 본 발명의 Fc 알파 수용체에 특이적인 항체 또는 이의 면역학적 활성을 가진 단편은In one embodiment, an antibody specific to the Fc alpha receptor of the present invention or a fragment having immunological activity thereof
(i) 서열번호 11의 아미노산 서열을 포함하는 FR1, 서열번호 1의 아미노산 서열을 포함하는 CDRH1, 서열번호 12의 아미노산 서열을 포함하는 FR2, 서열번호 2의 아미노산 서열을 포함하는 CDRH2, 서열번호 13의 아미노산 서열을 포함하는 FR3, 서열번호 3의 아미노산 서열을 포함하는 CDRH3 및 서열번호 15의 아미노산 서열을 포함하는 FR4를 포함하는 VH 도메인; 및/또는 (ⅱ) 서열번호 17의 아미노산 서열을 포함하는 FR1, 서열번호 5의 아미노산 서열을 포함하는 CDRL1, 서열번호 19의 아미노산 서열을 포함하는 FR2, 서열번호 7의 아미노산 서열을 포함하는 CDRL2, 서열번호 21의 아미노산 서열을 포함하는 FR3, 서열번호 9의 아미노산 서열을 포함하는 CDRL3 및 서열번호 23의 아미노산 서열을 포함하는 FR4를 포함하는 VL 도메인을 포함하는 V 도메인을 포함하는 scFv 변이체 JS48-1일 수 있다.(i) FR1 comprising the amino acid sequence of SEQ ID NO: 11, CDRH1 comprising the amino acid sequence of SEQ ID NO: 1, FR2 comprising the amino acid sequence of SEQ ID NO: 12, CDRH2 comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 13 A VH domain comprising FR3 comprising the amino acid sequence of CDRH3 comprising the amino acid sequence of SEQ ID NO: 3 and FR4 comprising the amino acid sequence of SEQ ID NO: 15; and/or (ii) FR1 comprising the amino acid sequence of SEQ ID NO: 17, CDRL1 comprising the amino acid sequence of SEQ ID NO: 5, FR2 comprising the amino acid sequence of SEQ ID NO: 19, CDRL2 comprising the amino acid sequence of SEQ ID NO: 7, scFv variant JS48-1 comprising a V domain comprising FR3 comprising the amino acid sequence of SEQ ID NO: 21, CDRL3 comprising the amino acid sequence of SEQ ID NO: 9, and VL domain comprising FR4 comprising the amino acid sequence of SEQ ID NO: 23 can be
일 구현예에서, 본 발명의 Fc 알파 수용체에 특이적인 항체 또는 이의 면역학적 활성을 가진 단편은 서열번호 24의 아미노산 서열을 포함하는 CL, 서열번호 25의 아미노산 서열을 포함하는 CH1, 서열번호 26의 아미노산 서열을 포함하는 CH2 또는 서열번호 27의 아미노산 서열을 포함하는 CH3 서열을 포함할 수 있다.In one embodiment, the antibody specific to the Fc alpha receptor of the present invention or a fragment having immunological activity thereof comprises CL comprising the amino acid sequence of SEQ ID NO: 24, CH1 comprising the amino acid sequence of SEQ ID NO: 25, and SEQ ID NO: 26. CH2 comprising the amino acid sequence or CH3 sequence comprising the amino acid sequence of SEQ ID NO: 27.
일 구현예에서, 본 발명의 Fc 알파 수용체에 특이적인 항체 또는 이의 면역학적 활성을 가진 단편은 Fc 알파 수용체의 세포외부영역(Extra Cellular Domain, ECD)에 특이적으로 결합할 수 있으며, Fc 알파 수용체의 세포외부영역은 서열번호 28의 아미노산 서열을 포함할 수 있다.In one embodiment, the Fc alpha receptor-specific antibody or immunologically active fragment thereof of the present invention may specifically bind to the extra cellular domain (ECD) of the Fc alpha receptor, and may bind to the Fc alpha receptor. The extracellular region of may include the amino acid sequence of SEQ ID NO: 28.
일 구현예에서, 본 발명의 면역학적 활성을 가진 단편은 Fab, Fd, Fab', dAb, F(ab'), F(ab')2, scFv(single chain fragment variable), Fv, 단일쇄 항체, Fv 이량체, 상보성 결정 영역 단편, 인간화 항체, 키메라 항체 및 디아바디(diabody)로 이루어진 군으로부터 선택되는 어느 하나일 수 있으며, scFv인 것이 더욱 바람직하다.In one embodiment, the fragment having immunological activity of the present invention is Fab, Fd, Fab', dAb, F(ab'), F(ab') 2 , scFv (single chain fragment variable), Fv, single chain antibody , Fv dimers, complementarity determining region fragments, humanized antibodies, chimeric antibodies, and may be any one selected from the group consisting of diabodies, more preferably scFv.
일 구현예에서, 본 발명의 Fc 알파 수용체에 특이적인 항체 또는 이의 면역학적 활성을 가진 단편은 효과기 작용을 증가시킬 수 있으며, 백혈구 및 CD89-보유 세포주의 식세포작용(antibody-dependent cellular phagocytosis, ADCP) 또는 항체-매개성 세포독성작용(antibody dependent cellular cytotoxicity, ADCC)을 증가시킬 수 있다.In one embodiment, the Fc alpha receptor-specific antibody or immunologically active fragment thereof of the present invention can increase effector action, and phagocytosis of leukocytes and CD89-bearing cell lines (antibody-dependent cellular phagocytosis, ADCP) or antibody-mediated cytotoxicity (antibody dependent cellular cytotoxicity, ADCC) may be increased.
본 발명에서 용어, "Fc 알파 수용체(Fcα수용체)"는 IgA 수용체 또는 CD89로도 혼용될 수 있다.In the present invention, the term "Fc alpha receptor (Fcα receptor)" may also be used interchangeably with an IgA receptor or CD89.
상기 항체는 전체(whole) 항체 형태일 뿐 아니라 항체 분자의 기능적인 단편을 포함한다. 전체 항체는 2개의 전체 길이의 경쇄(light chain) 및 2개의 전체 길이의 중쇄(heavy chain)를 가지는 구조이며 각각의 경쇄는 중쇄와 다이설파이드 결합(disulfide bond)으로 연결되어 있다. 항체 분자의 기능적인 단편이란 항원 결합 기능을 보유하고 있는 단편을 뜻하며, 항체 단편의 예는 (i) 경쇄의 가변영역(VL) 및 중쇄의 가변영역(VH)과 경쇄의 불변영역(CL) 및 중쇄의 첫번째 불변 영역(CH1)으로 이루어진 Fab 단편; (ii) VH 및 CH1 도메인으로 이루어진 Fd 단편; (iii) 단일 항체의 VL 및 VH 도메인으로 이루어진 Fv 단편; (iv) VH 도메인으로 이루어진 dAb 단편(Ward ES et al., Nature 341:544-546 (1989)]; (v) 분리된 CDR 영역; (vi) 2개의 연결된 Fab 단편을 포함하는 2가 단편인 F(ab')2 단편; (vii) VH 도메인 및 VL 도메인이 항원 결합 부위를 형성하도록 결합시키는 펩타이드 링커에 의해 결합된 단일쇄 Fv 분자(scFv); (viii) 이특이적인 단일쇄 Fv 이량체(PCT/US92/09965) 및 (ix) 유전자 융합에 의해 제작된 다가 또는 다특이적인 단편인 디아바디(diabody) WO94/13804) 등을 포함한다. The antibodies include whole antibody forms as well as functional fragments of antibody molecules. A full antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is connected to the heavy chain by a disulfide bond. A functional fragment of an antibody molecule refers to a fragment having an antigen-binding function, and examples of antibody fragments include (i) a light chain variable region (VL) and a heavy chain variable region (VH) and a light chain constant region (CL) and a Fab fragment consisting of the first constant region of the heavy chain (CH1); (ii) a Fd fragment consisting of the VH and CH1 domains; (iii) an Fv fragment consisting of the VL and VH domains of a single antibody; (iv) a dAb fragment consisting of a VH domain (Ward ES et al., Nature 341:544-546 (1989)]; (v) an isolated CDR region; (vi) a bivalent fragment comprising two linked Fab fragments. F(ab')2 fragments; (vii) single-chain Fv molecules (scFv) joined by a peptide linker that links the VH and VL domains to form an antigen-binding site; (viii) bispecific single-chain Fv dimers. (PCT/US92/09965) and (ix) a multivalent or multispecific fragment produced by gene fusion (diabody WO94/13804).
본 발명의 항체 또는 이의 면역학적 활성을 가진 단편은 동물 유래 항체, 키메릭 항체, 인간화 항체, 인간 항체, 및 이들의 면역학적 활성을 가진 단편으로 이루어진 군에서 선택된 것일 수 있다. 상기 항체는 재조합적 또는 합성적으로 생산된 것일 수 있다.The antibody or immunologically active fragment thereof of the present invention may be selected from the group consisting of animal-derived antibodies, chimeric antibodies, humanized antibodies, human antibodies, and immunologically active fragments thereof. The antibody may be produced recombinantly or synthetically.
원하는 항원을 피면역 동물에게 면역시켜 생산하는 동물 유래 항체는 일반적으로 치료 목적으로 인간에 투여시 면역거부반응이 일어날 수 있으며, 이러한 면역거부반응을 억제하고자 키메릭 항체(chimeric antibody)가 개발되었다. 키메릭 항체는 유전공학적 방법을 이용하여 항-아이소타입(anti-isotype) 반응의 원인이 되는 동물 유래 항체의 불변 영역을 인간 항체의 불변 영역으로 치환한 것이다. 키메릭 항체는 동물 유래 항체에 비하여 항-아이소타입 반응에 있어서 상당 부분 개선되었으나, 여전히 동물 유래 아미노산들이 가변 영역에 존재하고 있어 잠재적인 항-이디오타입(anti-idiotypic) 반응에 대한 부작용을 내포하고 있다. 이러한 부작용을 개선하고자 개발된 것이 인간화 항체(humanized antibody)이다. 이는 키메릭 항체의 가변 영역 중 항원의 결합에 중요한 역할을 하는 CDR(complementaritiy determining regions) 부위를 인간 항체 골격(framework)에 이식하여 제작된다.Animal-derived antibodies produced by immunizing an animal to be immunized with a desired antigen may generally cause immune rejection when administered to humans for therapeutic purposes, and chimeric antibodies have been developed to suppress such immune rejection. A chimeric antibody is one in which the constant region of an animal-derived antibody, which causes an anti-isotype reaction, is substituted with the constant region of a human antibody using a genetic engineering method. Compared to animal-derived antibodies, chimeric antibodies have improved significantly in anti-isotype response, but animal-derived amino acids are still present in the variable region, resulting in side effects for potential anti-idiotypic responses. are doing What was developed to improve these side effects is a humanized antibody. It is produced by grafting complementarity determining regions (CDRs), which play an important role in antigen binding, in the variable region of a chimeric antibody to a human antibody framework.
인간화 항체를 제작하기 위한 CDR 이식(grafting) 기술에 있어서 가장 중요한 것은 동물 유래 항체의 CDR 부위를 가장 잘 받아들일 수 있는 최적화된 인간 항체를 선정하는 것이며, 이를 위하여 항체 데이터베이스의 활용, 결정구조(crystal structure)의 분석, 분자모델링 기술 등이 활용된다. 그러나, 최적화된 인간 항체 골격에 동물 유래 항체의 CDR 부위를 이식할지라도 동물 유래 항체의 골격에 위치하면서 항원 결합에 영향을 미치는 아미노산이 존재하는 경우가 있기 때문에, 항원 결합력이 보존되지 못하는 경우가 상당수 존재하므로, 항원 결합력을 복원하기 위한 추가적인 항체 공학 기술의 적용은 필수적이라고 할 수 있다.The most important thing in the CDR grafting technology for producing humanized antibodies is to select an optimized human antibody that can best accept the CDR region of an animal-derived antibody. structure analysis, molecular modeling techniques, etc. are utilized. However, even when the CDR region of an animal-derived antibody is grafted onto an optimized human antibody backbone, there are cases in which amino acids that affect antigen binding are present while being located in the backbone of an animal-derived antibody, so antigen-binding ability is not preserved in many cases. Since it exists, it can be said that the application of additional antibody engineering techniques for restoring antigen binding ability is essential.
상기 항체 또는 이의 면역학적 활성을 가진 단편은 생체에서 분리된 (생체에 존재하지 않는) 것 또는 비자연적으로 생산(non-naturally occurring)된 것일 수 있으며, 예컨대, 합성적 또는 재조합적으로 생산된 것일 수 있다.The antibody or immunologically active fragment thereof may be isolated from a living body (not present in a living body) or non-naturally occurring, for example, synthetically or recombinantly produced. can
본 발명에서 "항체"라 함은, 면역계 내에서 항원의 자극에 의하여 만들어지는 물질을 의미하는 것으로서, 그 종류는 특별히 제한되지 않으며, 자연적 또는 비자연적(예컨대, 합성적 또는 재조합적)으로 얻어질 수 있다. 항체는 생체 외뿐 아니라 생체 내에서도 매우 안정하고 반감기가 길기 때문에 대량 발현 및 생산에 유리하다. 또한, 항체는 본질적으로 다이머(dimer) 구조를 가지므로 접착능(avidity)이 매우 높다. 완전한 항체는 2개의 전장(full length) 경쇄 및 2개의 전장 중쇄를 가지는 구조이며 각각의 경쇄는 중쇄와 이황화 결합으로 연결되어 있다. 항체의 불변 영역은 중쇄 불변 영역과 경쇄 불변 영역으로 나뉘어지며, 중쇄 불변 영역은 감마(γ), 뮤(μ), 알파(α), 델타(δ) 및 엡실론(ε) 타입을 가지고, 서브클래스로 감마1(γ1), 감마2(γ2), 감마3(γ3), 감마4(γ4), 알파1(α1) 및 알파2(α2)를 가진다. 경쇄의 불변 영역은 카파(κ) 및 람다(λ) 타입을 가진다.In the present invention, "antibody" refers to a substance produced by stimulation of an antigen in the immune system, and the type is not particularly limited, and may be obtained naturally or non-naturally (e.g., synthetically or recombinantly). can Antibodies are advantageous for mass expression and production because they are very stable in vitro as well as in vivo and have a long half-life. In addition, since antibodies essentially have a dimer structure, avidity is very high. A complete antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is linked to the heavy chain by a disulfide bond. The antibody constant region is divided into a heavy chain constant region and a light chain constant region, and the heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types, subclasses It has gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The constant region of the light chain is of the kappa (κ) and lambda (λ) type.
본 발명에서 용어, "중쇄(heavy chain)"는 항원에 특이성을 부여하기 위해 충분한 가변 영역 서열을 갖는 아미노산 서열을 포함하는 가변 영역 도메인 VH 및 3개의 불변 영역 도메인 CH1 , CH2 및 CH3 과 힌지(hinge)를 포함하는 전장 중쇄 및 이의 단편을 모두 포함하는 의미로 해석된다. 또한, 용어 "경쇄(light chain)"는 항원에 특이성을 부여하기 위한 충분한 가변 영역 서열을 갖는 아미노산 서열을 포함하는 가변 영역 도메인 VL 및 불변 영역 도메인 CL을 포함하는 전장 경쇄 및 이의 단편을 모두 포함하는 의미로 해석된다.As used herein, the term "heavy chain" refers to a variable region domain V H and three constant region domains C H1 , C H2 and C H3 comprising an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen. It is interpreted as meaning including both a full-length heavy chain and a fragment thereof including a hinge. In addition, the term "light chain" refers to both a full-length light chain comprising a variable region domain V L and a constant region domain CL comprising an amino acid sequence having sufficient variable region sequence to impart specificity to an antigen and fragments thereof. be interpreted in a sense that includes
본 발명에서 용어, "가변 영역(variable region) 또는 가변 부위 (variable domain)"는 항원과 특이적으로 결합하는 기능을 수행하면서 서열상의 많은 변이를 보이는 항체 분자의 부분을 의미하고, 가변 영역에는 상보성 결정 영역인 CDR1, CDR2 및 CDR3가 존재한다. 상기 CDR 사이에는 프레임 워크 영역(framework region, FR) 부분이 존재하여 CDR 고리를 지지해주는 역할을 한다. 상기 "상보성 결정 영역"은 항원의 인식에 관여하는 고리모양의 부위로서 이 부위의 서열이 변함에 따라 항체의 항원에 대한 특이성이 결정된다.As used herein, the term "variable region or variable domain" refers to a portion of an antibody molecule that exhibits many variations in sequence while performing a function of specifically binding to an antigen, and the variable region has complementarity There are the determining regions CDR1, CDR2 and CDR3. A framework region (FR) portion exists between the CDRs to support the CDR rings. The "complementarity determining region" is a ring-shaped region involved in antigen recognition, and as the sequence of this region changes, the specificity of the antibody to the antigen is determined.
본 발명에서 사용되는 용어 "scFv(single chain fragment variable)"는 유전자 재조합을 통해 항체의 가변영역만을 발현시켜 만든 단쇄항체를 말하며, 항체의 VH 영역과 VL 영역을 짧은 펩티드 사슬로 연결한 단일쇄 형태의 항체를 말한다. 상기 용어 "scFv"는 달리 명시되지 않거나, 문맥상 달리 이해되는 것이 아니라면, 항원 결합 단편을 비롯한 scFv 단편을 포함하고자 한다. 이는 통상의 기술자에게 자명한 것이다.As used herein, the term "scFv (single chain fragment variable)" refers to a single-chain antibody made by expressing only the variable region of an antibody through genetic recombination, and is a single-chain form in which the VH and VL regions of an antibody are connected by a short peptide chain. refers to the antibody of The term "scFv" is intended to include scFv fragments, including antigen-binding fragments, unless otherwise specified or otherwise understood from the context. This is obvious to a person skilled in the art.
본 발명에서 용어, "상보성결정영역(complementarity determining region, CDR)"은 면역글로불린의 중쇄 및 경쇄의 고가변 영역 (hypervariable region)의 아미노산 서열을 의미한다. 중쇄 및 경쇄는 각각 3개의 CDR을 포함할 수 있다 (CDRH1, CDRH2,CDRH3 및 CDRL1, CDRL2, CDRL3). 상기 CDR은 항체가 항원 또는 항원결정부위에 결합하는 데 있어서 주요한 접촉 잔기를 제공할 수 있다. In the present invention, the term "complementarity determining region (complementarity determining region, CDR)" refers to the amino acid sequence of the hypervariable region of the heavy chain and light chain of immunoglobulin. Heavy and light chains may each contain three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). The CDRs may provide key contact residues for antibody binding to antigens or epitopes.
본 발명에서, 용어, "특이적으로 결합" 또는 "특이적으로 인식"은 당업자에게 통상적으로 공지되어 있는 의미와 동일한 것으로서, 항원 및 항체가 특이적으로 상호작용하여 면역학적 반응을 하는 것을 의미한다.In the present invention, the term "specifically binding" or "specifically recognizing" has the same meaning commonly known to those skilled in the art, and means that an antigen and an antibody specifically interact to cause an immunological reaction. .
본 발명에서 용어, "항원결합단편"은 면역글로불린 전체 구조에 대한 그의 단편으로, 항원이 결합할 수 있는 부분을 포함하는 폴리펩타이드의 일부를 의미한다. 예를 들어, scFv, (scFv) 2 , scFv-Fc, Fab, Fab' 또는 F(ab') 2 일 수 있으나, 이에 한정되지 않는다. 상기 항원결합단편 중 Fab는 경쇄 및 중쇄의 가변 영역과 경쇄의 불변 영역 및 중쇄의 첫 번째 불변 영역(CH1)을 가지는 구조로 1개의 항원 결합 부위를 가진다. Fab'는 중쇄 CH1 도메인의 C-말단에 하나 이상의 시스테인 잔기를 포함하는 힌지 영역(hinge region)을 가진다는 점에서 Fab와 차이가 있다. F(ab') 2 항체는 Fab'의 힌지 영역의 시스테인 잔기가 디설파이드 결합을 이루면서 생성된다. Fv는 중쇄 가변 영역 및 경쇄 가변 부위만을 가지고 있는 최소의 항체조각으로 Fv 단편을 생성하는 재조합 기술은 당업계에 널리 공지되어 있다. 이중쇄 Fv(two-chain Fv)는 비공유 결합으로 중쇄 가변 부위와 경쇄 가변 부위가 연결되어 있고 단쇄 Fv(single-chain Fv)는 일반적으로 펩타이드 링커를 통하여 중쇄의 가변 영역과 단쇄의 가변 영역이 공유 결합으로 연결되거나 또는 C-말단에서 바로 연결되어 있어서 이중쇄 Fv와 같이 다이머와 같은 구조를 이룰 수 있다. 상기 링커는 1 내지 100개 또는 2 내지 50개의 임의의 아미노산으로 이루어진 펩타이드 링커일 수 있으며, 당업계에 적절한 서열이 알려져 있다. 상기 항원결합단편은 단백질 가수분해 효소를 이용해서 얻을 수 있고(예를 들어, 전체 항체를 파파인으로 제한 절단하면 Fab를 얻을 수 있고 펩신으로 절단하면 F(ab') 2 단편을 얻을 수 있다), 유전자 재조합 기술을 통하여 제작할 수 있다.As used herein, the term "antigen-binding fragment" is a fragment of the entire structure of immunoglobulin, and refers to a portion of a polypeptide including a portion capable of binding to an antigen. For example, it may be scFv, (scFv) 2 , scFv-Fc, Fab, Fab' or F(ab') 2 , but is not limited thereto. Among the antigen-binding fragments, Fab has a structure having variable regions of light and heavy chains, constant regions of light chains, and a first constant region (C H1 ) of heavy chains, and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain C H1 domain. An F(ab') 2 antibody is produced by forming a disulfide bond between cysteine residues in the hinge region of Fab'. Fv is a minimal antibody fragment having only a heavy chain variable region and a light chain variable region, and a recombination technique for generating an Fv fragment is widely known in the art. In two-chain Fv, the heavy chain variable region and the light chain variable region are connected by a non-covalent bond, and in single-chain Fv, the heavy chain variable region and the short chain variable region are generally shared through a peptide linker. They are linked by bonds or directly linked at the C-terminus, so that they can form a dimer-like structure like double-chain Fv. The linker may be a peptide linker consisting of 1 to 100 or 2 to 50 amino acids, and appropriate sequences are known in the art. The antigen-binding fragment can be obtained using a proteolytic enzyme (eg, Fab can be obtained by restriction digestion of whole antibody with papain, and F(ab') 2 fragment can be obtained by digestion with pepsin), It can be produced through genetic recombination technology.
본 발명에서 용어 "힌지 영역(hunge region)"은 항체의 중쇄에 포함되어 있는 영역으로서, CH1 및 CH2 영역 사이에 존재하며, 항체 내 항원 결합 부위의 유연성(flexibility)를 제공하는 기능을 하는 영역을 의미한다. 예컨대, 상기 힌지는 인간 항체로부터 유래한 것일 수 있으며, 구체적으로, IgA, IgE, 또는 IgG, 예컨대, IgG1, IgG2, IgG 3, 또는 IgG4로부터 유래한 것일 수 있다.In the present invention, the term "hinge region" is a region included in the heavy chain of an antibody, which is present between the C H1 and C H2 regions, and functions to provide flexibility of the antigen binding site in the antibody. means area. For example, the hinge may be derived from a human antibody, specifically, IgA, IgE, or IgG, such as IgG1, IgG2,
일 측면에서, 본 발명은 본 발명의 항체 또는 이의 면역학적 활성을 가진 단편을 코딩하는 단리된 핵산 분자, 이를 포함하는 벡터, 및 이로 형질전환된 숙주 세포에 관한 것이다.In one aspect, the invention relates to an isolated nucleic acid molecule encoding an antibody of the invention or an immunologically active fragment thereof, a vector comprising the same, and a host cell transformed therewith.
본 발명의 핵산 분자는 단리된 것이거나 재조합된 것일 수 있으며, 단일쇄 및 이중쇄 형태의 DNA 및 RNA뿐만 아니라 대응하는 상보성 서열이 포함된다. 단리된 핵산은 천연 생성 원천에서 단리된 핵산의 경우, 핵산이 단리된 개체의 게놈에 존재하는 주변 유전 서열로부터 분리된 핵산이다. 주형으로부터 효소적으로 또는 화학적으로 합성된 핵산, 예컨대 PCR 산물, cDNA 분자, 또는 올리고뉴클레오타이드의 경우, 이러한 절차로부터 생성된 핵산이 단리된 핵산분자로 이해될 수 있다. 단리된 핵산분자는 별도 단편의 형태 또는 더 큰 핵산 구축물의 성분으로서의 핵산 분자를 나타낸다. 핵산은 다른 핵산 서열과 기능적 관계로 배치될 때 작동가능하게 연결된다. 예를 들면, 전서열 또는 분비 리더(leader)의 DNA는 폴리펩타이드가 분비되기 전의 형태인 전단백질(preprotein)로서 발현되는 경우 폴리펩타이드의 DNA에 작동가능하게 연결되고, 프로모터 또는 인핸서는 폴리펩타이드 서열의 전사에 영향을 주는 경우 코딩 서열에 작동가능하게 연결되며, 또는 리보솜 결합 부위는 번역을 촉진하도록 배치될 때 코딩 서열에 작동가능하게 연결된다. 일반적으로 작동가능하게 연결된은 연결될 DNA 서열들이 인접하여 위치함을 의미하며, 분비 리더의 경우 인접하여 동일한 리딩 프레임 내에 존재하는 것을 의미한다. 그러나 인핸서는 인접하여 위치할 필요는 없다. 연결은 편리한 제한 효소 부위에서 라이게이션에 의해 달성된다. 이러한 부위가 존재하지 않는 경우, 합성 올리고뉴클레오타이드 어댑터 또는 링커를 통상적인 방법에 따라 사용한다.Nucleic acid molecules of the present invention may be isolated or recombinant, and include DNA and RNA in single-stranded and double-stranded form, as well as corresponding complementary sequences. An isolated nucleic acid is a nucleic acid that has been separated from surrounding genetic sequences present in the genome of the individual from which the nucleic acid was isolated, in the case of a nucleic acid isolated from a naturally occurring source. In the case of a nucleic acid synthesized enzymatically or chemically from a template, such as a PCR product, cDNA molecule, or oligonucleotide, the nucleic acid resulting from such a procedure can be understood as an isolated nucleic acid molecule. An isolated nucleic acid molecule refers to a nucleic acid molecule in the form of a separate fragment or as a component of a larger nucleic acid construct. Nucleic acids are operably linked when placed into a functional relationship with another nucleic acid sequence. For example, DNA of a full sequence or secretory leader is operably linked to DNA of a polypeptide when the polypeptide is expressed as a preprotein in its pre-secreted form, and a promoter or enhancer is the polypeptide sequence. is operably linked to a coding sequence when it affects transcription of, or when the ribosome binding site is positioned to facilitate translation. In general, operably linked means that the DNA sequences to be linked are contiguous, and in the case of a secretory leader, contiguous and in the same reading frame. However, enhancers need not be contiguous. Linkage is achieved by ligation at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used according to conventional methods.
본 발명의 항체 또는 이의 면역학적 활성을 가진 단편을 코딩하는 단리된 핵산 분자는 코돈의 축퇴성(degeneracy)으로 인하여 또는 상기 항체를 발현시키고자 하는 생물에서 선호되는 코돈을 고려하여, 코딩영역으로부터 발현되는 항체의 아미노산 서열을 변화시키지 않는 범위 내에서 코딩영역에 다양한 변형이 이루어질 수 있고, 코딩영역을 제외한 부분에서도 유전자의 발현에 영향을 미치지 않는 범위 내에서 다양한 변형 또는 수식이 이루어질 수 있으며, 그러한 변형 유전자 역시 본 발명의 범위에 포함됨을 당업자는 잘 이해할 수 있을 것이다. 즉, 본 발명의 핵산 분자는 이와 동등한 활성을 갖는 단백질을 코딩하는 한, 하나 이상의 핵산 염기가 치환, 결실, 삽입 또는 이들의 조합에 의해 변이될 수 있으며, 이들 또한 본 발명의 범위에 포함된다. 이러한 핵산 분자의 서열은 단쇄 또는 이중쇄일 수 있으며, DNA 분자 또는 RNA(mRNA)분자일 수 있다.The isolated nucleic acid molecule encoding the antibody of the present invention or a fragment having immunological activity thereof is expressed from the coding region due to codon degeneracy or considering codons preferred in organisms intended to express the antibody. Various modifications may be made to the coding region within a range that does not change the amino acid sequence of the antibody to be used, and various modifications or modifications may be made to a part other than the coding region within a range that does not affect gene expression, and such modifications Those skilled in the art will understand that genes are also included in the scope of the present invention. That is, as long as the nucleic acid molecule of the present invention encodes a protein having an activity equivalent thereto, one or more nucleic acid bases may be mutated by substitution, deletion, insertion, or a combination thereof, and these are also included in the scope of the present invention. The sequence of such a nucleic acid molecule may be single- or double-stranded, and may be a DNA molecule or an RNA (mRNA) molecule.
본 발명에 따른 본 발명의 항체 또는 이의 면역학적 활성을 가진 단편을 코딩하는 단리된 핵산 분자는 단백질 발현을 위해 발현벡터에 삽입될 수 있다. 발현벡터는, 통상 조절 또는 제어 (regulatory) 서열, 선별마커, 임의의 융합 파트너, 및/또는 추가적 요소와 작동가능하게 연결된, 즉, 기능적 관계에 놓인 단백질을 포함한다. 적절한 상태에서, 핵산으로 형질전환된 숙주세포, 바람직하게는, 본 발명의 항체 또는 이의 면역학적 활성을 가진 단편을 코딩하는 단리된 핵산 분자 함유 발현벡터를 배양하여 단백질 발현을 유도하는 방법에 의해 본 발명의 항체 또는 이의 면역학적 활성을 가진 단편이 생산될 수 있다. 포유류 세포, 박테리아, 곤충 세포, 및 효모를 포함하는 다양한 적절한 숙주세포가 사용될 수 있으나, 이에 제한하는 것은 아니다. 외인성 핵산을 숙주세포에 도입하는 방법은 당해 기술분야에 공지되어 있으며, 사용되는 숙주세포에 따라 달라질 것이다. 바람직하게는, 생산비가 저렴하여 산업적 이용가치가 높은 대장균을 숙주세포로 생산할 수 있다.An isolated nucleic acid molecule encoding an antibody of the present invention or an immunologically active fragment thereof according to the present invention may be inserted into an expression vector for protein expression. An expression vector usually contains a protein operably linked, i.e., in a functional relationship, with regulatory or regulatory sequences, selectable markers, optional fusion partners, and/or additional elements. A method for inducing protein expression by culturing a host cell transformed with a nucleic acid, preferably, an expression vector containing an isolated nucleic acid molecule encoding an antibody of the present invention or an immunologically active fragment thereof, under appropriate conditions. Antibodies of the invention or immunologically active fragments thereof can be produced. A variety of suitable host cells may be used including, but not limited to, mammalian cells, bacteria, insect cells, and yeast. Methods for introducing exogenous nucleic acids into host cells are known in the art and will vary depending on the host cell used. Preferably, it is possible to produce E. coli, which has high industrial value due to low production cost, as a host cell.
본 발명의 벡터는 플라스미드 벡터, 코즈미드 벡터, 박테리오 파아지 벡터 및 바이러스 벡터 등을 포함하나 이에 제한되지 않는다. 적합한 벡터는 프로모터, 오퍼레이터, 개시코돈, 종결코돈, 폴리아데닐화 시그널 및 인핸서 같은 발현 조절 엘리먼트 외에도 막 표적화 또는 분비를 위한 시그널 서열 또는 리더 서열을 포함하며 목적에 따라 다양하게 제조될 수 있다. 벡터의 프로모터는 구성적 또는 유도성일 수 있다. 상기 시그널 서열에는 숙주가 에쉐리키아속(Escherichia sp.)균인 경우에는 PhoA 시그널 서열, OmpA 시그널 서열 등이, 숙주가 바실러스속(Bacillus sp.)균인 경우에는 α-아밀라아제 시그널 서열, 서브틸리신 시그널 서열 등이, 숙주가 효모(yeast)인 경우에는 MFα 시그널 서열, SUC2 시그널 서열 등이, 숙주가 동물세포인 경우에는 인슐린 시그널 서열, α-인터페론 시그널 서열, 항체 분자 시그널 서열 등을 이용할 수 있으나, 이에 제한되지 않는다. 또한 벡터는 벡터를 함유하는 숙주 세포를 선택하기 위한 선택 마커를 포함할 수 있고, 복제 가능한 발현벡터인 경우 복제 기원을 포함한다.Vectors of the present invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors and viral vectors. Suitable vectors include expression control elements such as promoters, operators, initiation codons, stop codons, polyadenylation signals and enhancers, as well as signal sequences or leader sequences for membrane targeting or secretion, and may be prepared in various ways depending on the purpose. The vector's promoter may be constitutive or inducible. The signal sequence includes a PhoA signal sequence and an OmpA signal sequence when the host is Escherichia sp., and an α-amylase signal sequence and a subtilisin signal when the host is Bacillus sp. Sequences such as MFα signal sequence, SUC2 signal sequence, etc. can be used when the host is yeast, and insulin signal sequence, α-interferon signal sequence, antibody molecule signal sequence, etc. can be used when the host is an animal cell. Not limited to this. In addition, the vector may include a selectable marker for selecting a host cell containing the vector, and in the case of a replicable expression vector, an origin of replication.
본 발명에서 용어, "벡터"는 핵산 서열을 복제할 수 있는 세포로의 도입을 위해서 핵산 서열을 삽입할 수 있는 전달체를 의미한다. 핵산 서열은 외생 (exogenous) 또는 이종 (heterologous)일 수 있다. 벡터로서는 플라스미드, 코스미드 및 바이러스(예를 들면 박테리오파지)를 들 수 있으나, 이에 제한되지 않는다. 당업자는 표준적인 재조합 기술에 의해 벡터를 구축할 수 있다(Maniatis, et al., Molecular Cloning , A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1988; 및 Ausubel et al., In: Current Protocols in Molecular Biology , John, Wiley & Sons, Inc, NY, 1994 등).As used herein, the term "vector" refers to a delivery vehicle into which a nucleic acid sequence can be inserted for introduction into a cell capable of replicating the nucleic acid sequence. A nucleic acid sequence may be exogenous or heterologous. Vectors include, but are not limited to, plasmids, cosmids, and viruses (eg, bacteriophages). One skilled in the art can construct vectors by standard recombinant techniques (Maniatis, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1988; and Ausubel et al., In: Current Protocols in Molecular Biology, John, Wiley & Sons, Inc, NY, 1994, etc.).
일 구현예에서, 상기 벡터의 제작 시, 상기 항체를 생산하고자 하는 숙주세포의 종류에 따라 프로모터(promoter), 종결자(terminator), 인핸서(enhancer) 등과 같은 발현조절 서열, 막 표적화 또는 분비를 위한 서열 등을 적절히 선택하고 목적에 따라 다양하게 조합할 수 있다.In one embodiment, when constructing the vector, an expression control sequence such as a promoter, terminator, enhancer, etc., for membrane targeting or secretion, according to the type of host cell to produce the antibody. Sequences and the like can be selected appropriately and combined in various ways depending on the purpose.
본 발명에서, 용어 "발현 벡터"는 전사되는 유전자 산물 중 적어도 일부분을 코딩하는 핵산 서열을 포함한 벡터를 의미한다. 일부의 경우에는 그 후 RNA 분자가 단백질, 폴리펩타이드, 또는 펩타이드로 번역된다. 발현 벡터에는 다양한 조절서열을 포함할 수 있다. 전사 및 번역을 조절하는 조절서열과 함께 벡터 및 발현 벡터에는 또 다른 기능도 제공하는 핵산 서열도 포함될 수 있다.In the present invention, the term "expression vector" refers to a vector comprising a nucleic acid sequence encoding at least a portion of a gene product to be transcribed. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. Expression vectors may contain various regulatory sequences. Along with regulatory sequences that control transcription and translation, vectors and expression vectors may also contain nucleic acid sequences that serve other functions.
본 발명에서, 용어 "숙주세포"는 진핵생물 및 원핵생물을 포함하며, 상기 벡터를 복제할 수 있거나 벡터에 의해 코딩되는 유전자를 발현할 수 있는 임의의 형질 전환 가능한 생물을 의미한다. 숙주세포는 상기 벡터에 의해 형질감염(transfected) 또는 형질전환(transformed) 될 수 있으며, 이는 외생의 핵산분자가 숙주세포 내에 전달되거나 도입되는 과정을 의미한다.In the present invention, the term "host cell" includes eukaryotes and prokaryotes, and refers to any transformable organism capable of replicating the vector or expressing a gene encoded by the vector. The host cell may be transfected or transformed by the vector, which means a process in which an exogenous nucleic acid molecule is delivered or introduced into the host cell.
일 구현예에서, 상기 숙주 세포는 박테리아 또는 동물세포일 수 있으며, 동물 세포주는 CHO 세포, HEK 세포 또는 NSO 세포일 수 있고, 박테리아는 대장균일 수 있다.In one embodiment, the host cell may be a bacterial or animal cell, the animal cell line may be a CHO cell, a HEK cell or a NSO cell, and the bacteria may be Escherichia coli.
일 측면에서, 본 발명은 본 발명의 항체 또는 이의 면역학적 활성을 가진 단편을 코딩하는 단리된 핵산 분자를 포함하는 벡터로 형질전환된 숙주 세포를 배양하는 단계; 및 숙주 세포 배양물로부터 항체 또는 이의 면역학적 활성을 가진 단편을 회수하는 단계를 포함하는 Fc 알파 수용체에 특이적인 항체 또는 이의 면역학적 활성을 가진 단편을 제조하는 방법에 관한 것이다.In one aspect, the present invention provides a method comprising culturing a host cell transformed with a vector comprising an isolated nucleic acid molecule encoding an antibody of the present invention or an immunologically active fragment thereof; And it relates to a method for preparing an Fc alpha receptor-specific antibody or immunologically active fragment thereof, comprising the step of recovering the antibody or immunologically active fragment thereof from the host cell culture.
하기의 실시예를 통하여 본 발명을 보다 상세하게 설명한다. 그러나 하기 실시예는 본 발명의 내용을 구체화하기 위한 것일 뿐 이에 의해 본 발명이 한정되는 것은 아니다.The present invention will be described in more detail through the following examples. However, the following examples are only for specifying the content of the present invention, and the present invention is not limited thereto.
실시예 1.Example 1. Fc 알파 수용체를 이용한 항원 제작 Antigen production using Fc alpha receptor
Fc 알파 수용체와 결합하여 효과기 기능(effector function)을 갖는 IgG를 발굴하기 위하여 Fc 알파 수용체의 세포외부영역(Extra Cellular Domain, ECD)을 항원으로 준비하였다. 또한, 유세포 분리기를 이용하여 항체를 스크리닝하기에 적합한 항원을 제작하고자 GST(Glutathione-S-transferase) 태그를 융합(fusion)한 이합체(dimeric) 형태 및 스트렙타비딘(Streptavidin) 태그를 융합한 사합체(tetrameric) 형태의 항원을 생산할 동물 세포 발현용 벡터를 제작하였다 (도 1). 그 후, 2x106 cells/ml의 밀도로 하루 동안 배양한 Expi293F 세포에 PEI (Polyscience, 23966)을 이용하여 트랜스펙션하였다. 그 후 진탕배양기에서 37℃, 125 rpm 및 8 % CO2의 조건으로 7일간 배양한 후 원심 분리하여 상등액만 취하였다. 상등액을 25x PBS를 이용해 평형을 맞춘 뒤 바틀탑 필터를 이용해 0.2 μm 필터 (Merck Millipore)로 여과하였다. 여과된 배양액에 각각 GSH 레진(resin) 및 Ni-NTA 레진 1 ml을 넣어 주고 4 ℃에서 16 시간 교반한 뒤 컬럼을 통해 레진을 회수 한 후 5 ml PBS로 세척하고 pH 8.0의 50 mM Tris-HCl & 10 mM 환원된 글루타치온 (SIGMA) 4 ml와 250 mM 이미다졸로 용출하였다. Centrifugal filter units 3K (Merck Millipore)을 사용하여 PBS로 버퍼 교체한 후 SDS-PAGE를 통해 각각 정제된 2종 항원 단백질 (GST-ECD 및 스트렙타비딘-ECD)의 크기와 순도를 분석하였다 (도 1). In order to discover an IgG having an effector function by binding to the Fc alpha receptor, the extra cellular domain (ECD) of the Fc alpha receptor was prepared as an antigen. In addition, in order to produce an antigen suitable for antibody screening using flow cytometry, a dimeric form fused with a GST (Glutathione-S-transferase) tag and a tetramer fused with a streptavidin tag A vector for animal cell expression to produce a (tetrameric) antigen was constructed (FIG. 1). Then, Expi293F cells cultured for one day at a density of 2x10 6 cells/ml were transfected using PEI (Polyscience, 23966). Then, after culturing for 7 days under conditions of 37° C., 125 rpm and 8% CO 2 in a shaking incubator, only the supernatant was taken by centrifugation. The supernatant was equilibrated with 25x PBS and filtered through a 0.2 μm filter (Merck Millipore) using a bottle top filter. 1 ml of GSH resin and Ni-NTA resin were added to the filtered culture medium, stirred at 4 ° C for 16 hours, then the resin was recovered through a column, washed with 5 ml PBS, and washed with 50 mM Tris-HCl pH 8.0. & 4 ml of 10 mM reduced glutathione (SIGMA) and 250 mM imidazole. After replacing the buffer with PBS using centrifugal filter units 3K (Merck Millipore), the size and purity of the two purified antigen proteins (GST-ECD and streptavidin-ECD) were analyzed by SDS-PAGE (Fig. 1 ).
실시예 2.Example 2. 초고속 탐색 시스템을 이용한 인간 항체 스크리닝Human antibody screening using an ultrafast search system
유세포 분리기를 이용한 스크리닝에 이용하기 위해 상기 실시예에서 준비한 항원 단백질 중 스트렙타비딘이 융합된 항원 단백질에 Alexa488 형광분자를 표지화하였다. 이 후, 본 발명자들이 구축한 박테리아 디스플레이된 인간 scFv 항체 라이브러리를 이용하여 Fc 알파 수용체의 ECD 영역에 결합하는 인간 항체 스크리닝을 진행하였다. 구체적으로, 라이브러리 세포 1 ml을 2% 글루코스 및 40 ㎍/ml의 클로람페니콜(chloramphenicol)이 포함된 25 ml TB 배지에서 37℃에서 250 rpm으로 4시간 배양한 뒤, 배양된 세포를 40 ㎍/ml의 클로람페니콜이 포함된 100 ml의 TB 배지에 1:100 비율로 OD600=0.5까지 배양하고 20분간 25℃ 및 250 rpm에서 쿨링(cooling)한 후 1 mM IPTG를 첨가하여 25℃ 및 250 rpm로 5시간 동안 인덕션(Induction)하여 단백질이 과발현된 대장균을 OD600=8을 기준으로 수득(harvest)하였다. 세포들을 유세포 분리기를 이용한 스크리닝에 적합한 스페로플라스트(spheroplasts) 형태로 만들기 위해 세포를 1ml의 10Mm Tris-HCl (pH 8.0)을 사용하여 재부유하여 2번 세척하여 잔여 배지를 제거한 뒤, 1 ml의 STE [0.5 M sucrose, 10 Mm Tris-HCl 및 10 Mm EDTA (pH 8.0)] 용액에서 37℃로 30분간 로테이션하여 삼투압 충격(osmotic shock)을 주는 방법으로 세포 외막을 제거하였다. 그 후 1 ml의 Solution A 및 50 mg/ml lysozyme solution 20 ㎕를 혼합한 용액에서 37℃로 15분간 로테이션하여 펩티도글리칸(peptidoglycan) 층을 제거하였고 1 ml의 PBS로 세척한 뒤 300 ㎕를 700 ㎕의 PBS와 200 nM (monomer 기준)의 Fc 알파 수용체-ECD-스트렙타비딘-Alexa488 프로브를 넣고 상온에서 1시간 동안 로테이션하여 스페로플라스트에 형광 프로브를 표지하였다. 이 후 항원 결합력 증가로 높은 형광을 보이는 클론들을 유세포분리기를 이용하여 회수하였으며, 회수된 클론들에서 scFv 유전자들을 PCR방법으로 증폭하고, 배양, 발현유도, 세포외막 및 펩티도글리칸 층을 제거하는 스페로플라스팅(spheroplasting) 과정, 항원 표지 및 유세포 분석의 선택적인 게이팅(gating)을 통해 Fc 알파 수용체-ECD 영역에 높은 친화도를 보이는 스페로플라스트들을 선별하는 과정을 반복하였다. 유세포 분석기를 이용한 선별 과정 및 선별 순도(purity)를 높이기 위한 재선별 과정 후에, 농축 과정, PCR 증폭을 통한 선별된 scFv 변이체 유전자 확보 과정, 서브클로닝 과정, 및 트랜스포메이션 과정을 거치고, 원하는 클론들을 증폭(enrich)하기 위해 다음 라운드의 선별 과정을 수행하였다 (도 2). 이와 같은 반복되는 여러 회차(round)의 탐색 과정으로 Fc 알파 수용체-ECD 영역에 결합력이 우수한 scFv 변이체 클론들을 확보하였다.For screening using a flow cytometer, Alexa488 fluorescent molecules were labeled on the antigen proteins fused with streptavidin among the antigen proteins prepared in the above example. Thereafter, human antibody binding to the ECD region of the Fc alpha receptor was screened using the bacterially displayed human scFv antibody library constructed by the present inventors. Specifically, after incubating 1 ml of library cells in 25 ml TB medium containing 2% glucose and 40 μg/ml of chloramphenicol at 37° C. at 250 rpm for 4 hours, the cultured cells were cultured at 40 μg/ml Incubated in 100 ml of TB medium containing chloramphenicol at a ratio of 1:100 to OD600 = 0.5, cooled at 25 ° C and 250 rpm for 20 minutes, and then added with 1 mM IPTG at 25 ° C and 250 rpm for 5 hours. E. coli in which the protein was overexpressed by induction was harvested based on OD600=8. To make the cells into spheroplasts suitable for screening using a flow cytometer, the cells were resuspended in 1 ml of 10 Mm Tris-HCl (pH 8.0), washed twice to remove residual medium, and The cell outer membrane was removed by rotation in STE [0.5 M sucrose, 10 Mm Tris-HCl and 10 Mm EDTA (pH 8.0)] solution at 37° C. for 30 minutes to give osmotic shock. Thereafter, a mixture of 1 ml of Solution A and 20 μl of 50 mg/ml lysozyme solution was rotated at 37° C. for 15 minutes to remove the peptidoglycan layer, washed with 1 ml of PBS, and 300 μl of 700 μl of PBS and 200 nM (monomer basis) of the Fc alpha receptor-ECD-streptavidin-Alexa488 probe were added and rotated at room temperature for 1 hour to label the fluorescent probe on the spheroplasts. Afterwards, clones showing high fluorescence due to increased antigen-binding ability were recovered using flow cytometry, and scFv genes were amplified by PCR in the recovered clones, followed by culturing, inducing expression, and removing the outer membrane and peptidoglycan layer. The process of selecting spheroplasts showing high affinity to the Fc alpha receptor-ECD region through selective gating of spheroplasting, antigen labeling, and flow cytometry was repeated. After a selection process using a flow cytometer and a re-selection process to increase selection purity, a process of enrichment, a process of securing the selected scFv mutant gene through PCR amplification, a process of subcloning, and a process of transformation, amplification of desired clones The next round of screening was performed to enrich (Fig. 2). Through this repeated search process of several rounds, scFv mutant clones having excellent binding ability to the Fc alpha receptor-ECD region were secured.
실시예 3.Example 3. Fc 알파 수용체 결합 항체 변이체들의 유전자 서열 확인Gene sequence identification of Fc alpha receptor binding antibody variants
상기 실시예 2에서 확보한 scFv 변이체 클론들의 유전자 서열을 확인하기 위하여 Sanger sequencing으로 DNA 염기 서열을 분석하였으며, 분석을 통해 항체 서열을 갖는 scFv 항체 변이체 6종 JS9, JS30, JS41, JS19, JS40 및 JS48을 선별하고, 상기 6종의 scFv 변이체들의 VH 및 VL 아미노산 서열과 각 변이체들의 VH의 FR1, CDRH1, FR2, CDRH2, FR3, CDRH3 및 FR4의 아미노산 서열, 및 VL의 FR1, CDRL1, FR2, CDRL2, FR3, CDRL3 및 FR4의 아미노산 서열을 확인하였다 (도 3).DNA sequencing was analyzed by Sanger sequencing to confirm the genetic sequences of the scFv mutant clones obtained in Example 2, and through the analysis, six scFv antibody variants having antibody sequences JS9, JS30, JS41, JS19, JS40 and JS48 was selected, and the VH and VL amino acid sequences of the six scFv variants and the amino acid sequences of FR1, CDRH1, FR2, CDRH2, FR3, CDRH3 and FR4 of VH of each variant, and FR1, CDRL1, FR2, CDRL2 of VL, The amino acid sequences of FR3, CDRL3 and FR4 were confirmed (FIG. 3).
실시예 4.Example 4. Fc 알파 수용체 ECD 영역에 대한 결합력 분석 Analysis of binding affinity to the Fc alpha receptor ECD region
상기 실시예 3에서 선별한 6종 scFv 항체 변이체들의 Fc 알파 수용체 ECD 영역에 대한 결합력을 확인하기 위해 유세포 분석기를 이용한 결합력 분석을 진행하였다. 이를 위하여 상기 실시예에서 사용된 방식으로 각각의 변이체와 대조군으로 사용될 Fc (야생형)들을 스페로플라스트들로 제조하였다. 준비된 스페로플라스트들에 Fc 알파 수용체-ECD-스트렙타비딘-Alexa488 항원을 50 nM의 농도로 결합시키고 상온에서 1시간 동안 인큐베이션하였다. 그 후 비특이적 결합을 제거하기 위해 PBS로 두 차례 세척하고 유세포 분석기를 이용하여 Fc 알파 수용체 ECD 영역에 대한 결합력을 분석하였다. 그 결과, 6종의 scFv 변이체들이 대조군인 IgG_Fc 및 IgA_Fc에 비해 월등히 증가된 형광 신호를 나타냈으며 (도 4), 이를 통해 Fc 알파 수용체 ECD 영역에 결합하는 scFv 항체 변이체들을 검증하였다.In order to confirm the binding ability of the six scFv antibody variants selected in Example 3 to the Fc alpha receptor ECD region, avidity analysis was performed using a flow cytometer. To this end, each variant and Fc (wild type) to be used as a control were prepared as spheroplasts in the manner used in the above examples. Fc alpha receptor-ECD-streptavidin-Alexa488 antigen was bound to the prepared spheroplasts at a concentration of 50 nM and incubated for 1 hour at room temperature. Thereafter, the cells were washed twice with PBS to remove non-specific binding, and the binding ability to the Fc alpha receptor ECD region was analyzed using a flow cytometer. As a result, the six scFv variants exhibited significantly increased fluorescence signals compared to the control groups IgG_Fc and IgA_Fc (FIG. 4), through which scFv antibody variants binding to the Fc alpha receptor ECD region were verified.
실시예 5. scFv 변이체 포함 항체 IgG 생산 및 정제Example 5. Production and purification of antibody IgG containing scFv variants
상기 실시예 3에서 선별한 6종의 scFv 항체 변이체가 IgG 형태로도 Fc 알파 수용체와의 결합력을 가지는지를 확인하기 위하여 6종의 scFv의 중쇄(heavy chain) 및 경쇄(light chain) 발현 벡터를 각각 제작한 뒤, Freestyle 293 expression 배양액 (Gibco, 12338-018) 100 ml에 각 변이체들의 중쇄유전자와 경쇄유전자를 1:1의 비율로 먼저 섞고 PEI:변이체유전자=4:1의 비율로 섞어 상온에서 20 분간 두었다가 전날 2x106 cells/ml의 밀도로 계대배양해 놓은 Expi293F 세포에 트랜스펙션하였다. 그 후 진탕배양기에서 37 ℃, 125 rpm 및 8 % CO2의 조건으로 7일간 배양하고 원심 분리하여 상등액만 취하였다. 상등액을 25x PBS를 이용해 평형을 맞춘 뒤 바틀탑 필터를 이용해 0.2 μm 필터 (Merck Millipore)로 여과하였다. 여과된 배양액에 Protein A 레진 500 μl을 넣어 주고 4 ℃에서 16 시간 교반한 뒤 컬럼을 통해 레진을 회수 한 후 5 ml PBS로 세척하고 pH 2.7의 100 mM 글리신(glycine) 버퍼 3 ml로 용출하고 1M Tris-HCl pH 8.0을 이용하여 중화하였다. Centrifugal filter units 3K (Merck Millipore)을 사용하여 PBS로 버퍼 교체한 후 정제된 6종의 scFv 항체 변이체를 각각 포함하는 IgG (JS9, JS30, JS41, JS19, JS40 및 JS48)들의 크기와 순도를 SDS-PAGE를 통해 분석하였다 (도 5).In order to confirm whether the 6 scFv antibody variants selected in Example 3 have binding ability to the Fc alpha receptor even in the form of IgG, the heavy chain and light chain expression vectors of 6 scFv were prepared, respectively. After production, the heavy chain gene and light chain gene of each variant were first mixed in 100 ml of Freestyle 293 expression culture medium (Gibco, 12338-018) at a ratio of 1:1, and then mixed at a ratio of PEI: mutant gene = 4:1 and incubated at room temperature for 20 minutes. After being allowed to stand for 10 minutes, Expi293F cells subcultured the previous day at a density of 2x10 6 cells/ml were transfected. Thereafter, the culture was cultured for 7 days under conditions of 37 °C, 125 rpm and 8% CO 2 in a shaking incubator, and only the supernatant was taken by centrifugation. The supernatant was equilibrated with 25x PBS and filtered through a 0.2 μm filter (Merck Millipore) using a bottle top filter. Add 500 μl of Protein A resin to the filtered culture solution, stir at 4 ° C for 16 hours, recover the resin through the column, wash with 5 ml PBS, elute with 3 ml of 100 mM glycine buffer at pH 2.7, and Neutralized using Tris-HCl pH 8.0. The size and purity of IgGs (JS9, JS30, JS41, JS19, JS40 and JS48) containing each of the six scFv antibody variants purified after buffer exchange with PBS using centrifugal filter units 3K (Merck Millipore) were measured by SDS- Analyzed via PAGE (FIG. 5).
실시예 6. scFv 변이체 포함 항체들의 Fc 알파 수용체에 대한 결합력 분석Example 6. Analysis of binding ability of antibodies containing scFv variants to Fc alpha receptor
상기 실시예 5에서 제작한 scFv 변이체 포함 항체들 JS9, JS30, JS41, JS40 및 JS48의 Fc 알파 수용체에 대한 항체 결합력을 측정하여 대조군인 트라스트주맙과 비교하기 위하여 ELISA 실험을 진행하였다. 구체적으로, pH 9.6의 0.05 M Na2CO3 용액 내의 스트렙타비딘 태그를 융합한 사합체 형태의 Fc 알파 수용체 (항원) 4 μg/ml을 50 μl씩 Flat Bottom Polystyrene High Bind 96웰 마이크로플레이트 (costar)에 4℃에서 16 시간 동안 고정화한 후 100 μl의 4% 스킴밀크 (GenomicBase) (in 0.05% PBST pH 6.0/pH 7.4)로 상온에서 2 시간 동안 블로킹하였다. 0.05% PBST 180 μl로 4 회씩 세척한 뒤 1% 스킴밀크 (in 0.05% PBST)로 연속 희석한 항체 JSA-9, JSA-30, JSA-40, JSA-41 및 JSA-48를 50 μl씩 각 웰에 분주하여 상온에서 1 시간 동안 반응시켰다. 세척 과정 후 50 μl의 항-인간 IgG(H+L)-HRP 컨쥬게이트 (Jackson Immunoresearch)과 상온에서 1 시간 동안 각각 항체 반응을 진행하고 세척하였다. 그 후 1-Step Ultra TMB-ELISA Substrate Solution(Thermo Fisher Scientific)를 50 μl씩 첨가해 발색한 뒤 50 μl의 2 M H2SO4를 첨가하여 반응을 종료시킨 다음 Epoch Microplate Spectrophotometer (BioTek)을 이용해 각 항체의 항원인 Fc 알파 수용체에 대한 결합력을 분석하였다 (도 6).An ELISA experiment was performed to measure the antibody binding ability to the Fc alpha receptor of the antibodies JS9, JS30, JS41, JS40 and JS48 containing the scFv variants prepared in Example 5 and compare them with trastuzumab as a control group. Specifically, 50 μl of 4 μg/ml of the tetrameric Fc alpha receptor (antigen) fused with a streptavidin tag in a 0.05 M Na 2 CO 3 solution at pH 9.6 was added to a Flat Bottom Polystyrene High Bind 96-well microplate (costar ) at 4° C. for 16 hours, and then blocked with 100 μl of 4% skim milk (GenomicBase) (in 0.05% PBST pH 6.0/pH 7.4) at room temperature for 2 hours. After washing four times with 180 μl of 0.05% PBST, 50 μl each of antibodies JSA-9, JSA-30, JSA-40, JSA-41 and JSA-48 serially diluted with 1% skim milk (in 0.05% PBST) The wells were dispensed and reacted at room temperature for 1 hour. After the washing process, the antibody reaction was carried out with 50 μl of anti-human IgG (H+L)-HRP conjugate (Jackson Immunoresearch) at room temperature for 1 hour, respectively, and then washed. Then, 50 μl of 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) was added to develop color, and 50 μl of 2 MH 2 SO 4 was added to terminate the reaction. The binding ability of the antibody to the Fc alpha receptor, which is an antigen, was analyzed (FIG. 6).
실시예 7. JS40 및 JS48의 VH 영역과 VL 영역이 치환된 항체 생산 및 정제Example 7. Production and purification of antibodies in which the VH and VL regions of JS40 and JS48 are substituted
상기 실시예 6에서 발굴한 항체 6종 중, 단백질 상에서 Fc 알파 수용체와 가장 높은 결합력을 보인 두 항체인 JS40 및 JS48의 VH 영역은 서열이 매우 비슷하지만 VL은 서열이 전혀 다른 것을 확인하여, 모항체로 이용하였던 JS40 및 JS48의 VH 영역 및 VL 영역을 서로 치환하여 포함하는 IgG 형태의 항체를 제작하였다. 이를 위하여 JS40의 VH 영역을 가지면서 JS48의 VL 영역을 가지는 JS40-1 항체 및 JS48의 VH 영역을 가지면서 JS40의 VL 영역을 가지는 JS48-1 항체의 scFv의 중쇄(heavy chain) 및 경쇄(light chain) 발현 벡터를 각각 제작한 뒤, Freestyle 293 expression 배양액 (Gibco, 12338-018) 100 ml에 각 변이체들의 중쇄유전자와 경쇄유전자를 1:1의 비율로 먼저 섞고 PEI:변이체유전자=4:1의 비율로 섞어 상온에서 20 분간 두었다가 전날 2x106 cells/ml의 밀도로 계대배양해 놓은 Expi293F 세포에 트랜스펙션하였다. 그 후 진탕배양기에서 37 ℃, 125 rpm 및 8 % CO2의 조건으로 7일간 배양하고 원심 분리하여 상등액만 취하였다. 상등액을 25x PBS를 이용해 평형을 맞춘 뒤 바틀탑 필터를 이용해 0.2 μm 필터 (Merck Millipore)로 여과하였다. 여과된 배양액에 Protein A 레진 500 μl을 넣어 주고 4 ℃에서 16 시간 교반한 뒤 컬럼을 통해 레진을 회수 한 후 5 ml PBS로 세척하고 pH 2.7의 100 mM 글리신(glycine) 버퍼 3 ml로 용출하고 1M Tris-HCl pH 8.0을 이용하여 중화하였다. Centrifugal filter units 3K (Merck Millipore)을 사용하여 PBS로 버퍼 교체한 후 정제된 2종의 JS40-1 및 JS48-1를 각각 포함하는 IgG들의 크기와 순도를 SDS-PAGE를 통해 분석하였다 (도 7).Among the six antibodies discovered in Example 6, the VH regions of JS40 and JS48, the two antibodies that showed the highest binding affinity to the Fc alpha receptor on the protein, had very similar sequences, but the VL sequences were completely different, confirming that the parent antibody An antibody in the form of an IgG containing the VH and VL regions of JS40 and JS48, which were used as JS40, was prepared by replacing each other. To this end, heavy chains and light chains of the scFv of the JS40-1 antibody having the VH region of JS40 and the VL region of JS48 and the JS48-1 antibody having the VH region of JS48 and the VL region of JS40 ) After constructing each expression vector, first mix the heavy chain gene and light chain gene of each variant in 100 ml of Freestyle 293 expression culture medium (Gibco, 12338-018) at a ratio of 1: 1, PEI: variant gene = 4: 1 ratio After mixing at room temperature for 20 minutes, it was transfected into Expi293F cells subcultured the previous day at a density of 2x10 6 cells/ml. Thereafter, the culture was cultured for 7 days under conditions of 37 °C, 125 rpm and 8% CO 2 in a shaking incubator, and only the supernatant was taken by centrifugation. The supernatant was equilibrated with 25x PBS and filtered through a 0.2 μm filter (Merck Millipore) using a bottle top filter. Add 500 μl of Protein A resin to the filtered culture solution, stir at 4 ° C for 16 hours, recover the resin through the column, wash with 5 ml PBS, elute with 3 ml of 100 mM glycine buffer at pH 2.7, and Neutralized using Tris-HCl pH 8.0. After replacing the buffer with PBS using centrifugal filter units 3K (Merck Millipore), the size and purity of IgGs containing the two purified JS40-1 and JS48-1, respectively, were analyzed by SDS-PAGE (FIG. 7) .
실시예 8. JS40-1 및 JS48-1 항체의 Fc 알파 수용체에 대한Example 8. JS40-1 and JS48-1 antibodies to the Fc alpha receptor 결합력 분석binding force analysis
상기 실시예 7에서 제작한 항체 JS40-1 및 JS48-1의 단백질 상에서 Fc 알파 수용체에 대한 결합력을 확인하기 위해 ELISA 실험을 수행하였다. 구체적으로, pH 9.6의 0.05 M Na2CO3에 4 μg/ml로 스트렙타비딘 태그를 융합한 사합체 형태의 Fc 알파 수용체 (항원)를 50 μl씩 Flat Bottom Polystyrene High Bind 96웰 마이크로플레이트 (costar)에 4℃에서 16 시간 동안 고정화한 후 100 μl의 4% 스킴밀크 (GenomicBase) (in 0.05% PBST pH 6.0/pH 7.4)로 상온에서 2 시간 동안 블로킹하였다. 0.05% PBST 180 μl로 4 회씩 세척한 뒤 1% 스킴밀크 (in 0.05% PBST)로 연속 희석한 상기 실시예 7에서 제작된 2종의 항체들 (JS40-1 및 JS48-1)과 대조군으로서 기존의 항체 2종 (JS40 및 JS48)를 50 μl씩 각 웰에 분주하여 상온에서 1 시간 동안 반응시켰다. 세척 과정 후 50 μl의 항-인간 IgG(H+L)-HRP 컨쥬게이트 (Jackson Immunoresearch)과 상온에서 1 시간 동안 각각 항체 반응을 진행하고 세척하였다. 그 후 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific)를 50 μl씩 첨가해 발색한 뒤 50 μl의 2 M H2SO4를 첨가하여 반응을 종료시킨 다음 Epoch Microplate Spectrophotometer (BioTek)을 이용해 각 항체의 항원인 Fc 알파 수용체에 대한 결합력을 분석하였다 (도 8).An ELISA experiment was performed to confirm the binding ability to the Fc alpha receptor on the proteins of the antibodies JS40-1 and JS48-1 prepared in Example 7. Specifically, 50 μl of a tetrameric Fc alpha receptor (antigen) fused with a streptavidin tag at 4 μg/ml in 0.05 M Na 2 CO 3 at pH 9.6 was added to a Flat Bottom Polystyrene High Bind 96-well microplate (costar ) at 4° C. for 16 hours, and then blocked with 100 μl of 4% skim milk (GenomicBase) (in 0.05% PBST pH 6.0/pH 7.4) at room temperature for 2 hours. After washing four times with 180 μl of 0.05% PBST, the two antibodies (JS40-1 and JS48-1) prepared in Example 7 serially diluted with 1% skim milk (in 0.05% PBST) and the existing 50 μl of two types of antibodies (JS40 and JS48) were dispensed into each well and reacted at room temperature for 1 hour. After the washing process, the antibody reaction was carried out with 50 μl of anti-human IgG (H+L)-HRP conjugate (Jackson Immunoresearch) at room temperature for 1 hour, respectively, and then washed. Then, 50 μl of 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) was added to develop color, and 50 μl of 2 MH 2 SO 4 was added to terminate the reaction. The binding ability of the antibody to the Fc alpha receptor, which is an antigen, was analyzed (FIG. 8).
<110> Korea University Research and Business Foundation <120> Human antibodies for binding to human Fc alpha receptor <130> PN <160> 28 <170> KoPatentIn 3.0 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDRH1 (JS40-1, JS48-1) <400> 1 Gly Phe Thr Phe Ser Asn Phe Ala 1 5 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDRH2 (JS40-1, JS48-1) <400> 2 Ile Ser Asp Asp Gly Thr Ile Thr 1 5 <210> 3 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> CDRH3 (JS40-1, JS48-1) <400> 3 Val Lys Val Pro Ser Pro Ala Pro Met Gln Gly Pro Asp Tyr 1 5 10 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDRL1 (JS40-1) <400> 4 Gln Gly Ile Ser Asn Tyr Leu Ala Trp Phe 1 5 10 <210> 5 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDRL1 (JS48-1) <400> 5 Gln Ser Ile Ser Thr Tyr Leu Asn Trp Tyr 1 5 10 <210> 6 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDRL2 (JS40-1) <400> 6 Ala Ala Ser Ser Leu Gln Ser Gly Val Pro 1 5 10 <210> 7 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDRL2 (JS48-1) <400> 7 Gly Ala Ser Asn Leu Gln Ser Gly Val Ser 1 5 10 <210> 8 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDRL3 (JS40-1) <400> 8 Gln Gln Tyr His Ser Tyr Pro Leu Thr 1 5 <210> 9 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDRL3 (JS48-1) <400> 9 Gln Gln Gly Asn Ser Val Pro Leu Thr 1 5 <210> 10 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> VH-FR1 (JS40-1) <400> 10 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Ala Gly Ser 20 25 <210> 11 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> VH-FR1 (JS48-1) <400> 11 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ala Cys Ala Gly Ser 20 25 <210> 12 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VH-FR2 (JS40-1, JS48-1) <400> 12 Met Ala Trp Val Arg Leu Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 1 5 10 15 Gly <210> 13 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> VH-FR3 (JS40-1, JS48-1) <400> 13 Asp Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 1 5 10 15 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 14 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> VH-FR4 (JS40-1) <400> 14 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 15 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> VH-FR4 (JS48-1) <400> 15 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 1 5 10 <210> 16 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> VL-FR1 (JS40-1) <400> 16 Glu Thr Thr Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 20 25 <210> 17 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> VL-FR1 (JS48-1) <400> 17 Asp Val Val Met Thr Gln Ser Pro Ser Ala Met Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 20 25 <210> 18 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> VL-FR2 (JS40-1) <400> 18 Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile Tyr 1 5 10 <210> 19 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> VL-FR2 (JS48-1) <400> 19 Gln Gln Lys Pro Gly Lys Val Pro Lys Arg Leu Ile Tyr 1 5 10 <210> 20 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> VL-FR3 (JS40-1) <400> 20 Ser Gln Phe Ser Gly Arg Gly Pro Gly Thr Asp Phe Thr Leu Thr Ile 1 5 10 15 Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 20 25 <210> 21 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> VL-FR3 (JS48-1) <400> 21 Ser Arg Phe Arg Gly Arg Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile 1 5 10 15 Ser Cys Leu Gln Ser Glu Asp Phe Ala Thr Tyr Tyr Cys 20 25 <210> 22 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> VL-FR4 (JS40-1) <400> 22 Phe Gly Gly Gly Thr Lys Val Asp Ile Lys Arg 1 5 10 <210> 23 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> VL-FR4 (JS48-1) <400> 23 Phe Gly Gly Gly Thr Lys Leu Ser Val Leu Gly 1 5 10 <210> 24 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> CL <400> 24 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 1 5 10 15 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 20 25 30 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 35 40 45 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 50 55 60 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 65 70 75 80 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 85 90 95 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 25 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> CH1 <400> 25 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val <210> 26 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> CH2 <400> 26 Glu Pro Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 15 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 20 25 30 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 35 40 45 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 50 55 60 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 65 70 75 80 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 85 90 95 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 100 105 110 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 115 120 125 <210> 27 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> CH3 <400> 27 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 1 5 10 15 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 105 <210> 28 <211> 206 <212> PRT <213> Artificial Sequence <220> <223> FcaR-ECD <400> 28 Gln Glu Gly Asp Phe Pro Met Pro Phe Ile Ser Ala Lys Ser Ser Pro 1 5 10 15 Val Ile Pro Leu Asp Gly Ser Val Lys Ile Gln Cys Gln Ala Ile Arg 20 25 30 Glu Ala Tyr Leu Thr Gln Leu Met Ile Ile Lys Asn Ser Thr Tyr Arg 35 40 45 Glu Ile Gly Arg Arg Leu Lys Phe Trp Asn Glu Thr Asp Pro Glu Phe 50 55 60 Val Ile Asp His Met Asp Ala Asn Lys Ala Gly Arg Tyr Gln Cys Gln 65 70 75 80 Tyr Arg Ile Gly His Tyr Arg Phe Arg Tyr Ser Asp Thr Leu Glu Leu 85 90 95 Val Val Thr Gly Leu Tyr Gly Lys Pro Phe Leu Ser Ala Asp Arg Gly 100 105 110 Leu Val Leu Met Pro Gly Glu Asn Ile Ser Leu Thr Cys Ser Ser Ala 115 120 125 His Ile Pro Phe Asp Arg Phe Ser Leu Ala Lys Glu Gly Glu Leu Ser 130 135 140 Leu Pro Gln His Gln Ser Gly Glu His Pro Ala Asn Phe Ser Leu Gly 145 150 155 160 Pro Val Asp Leu Asn Val Ser Gly Ile Tyr Arg Cys Tyr Gly Trp Tyr 165 170 175 Asn Arg Ser Pro Tyr Leu Trp Ser Phe Pro Ser Asn Ala Leu Glu Leu 180 185 190 Val Val Thr Asp Ser Ile His Gln Asp Tyr Thr Thr Gln Asn 195 200 205 <110> Korea University Research and Business Foundation <120> Human antibodies for binding to human Fc alpha receptor <130> PN <160> 28 <170> KoPatentIn 3.0 <210> 1 <211> 8 <212> PRT <213> artificial sequence <220> <223> CDRH1 (JS40-1, JS48-1) <400> 1 Gly Phe Thr Phe Ser Asn Phe Ala 1 5 <210> 2 <211> 8 <212> PRT <213> artificial sequence <220> <223> CDRH2 (JS40-1, JS48-1) <400> 2 Ile Ser Asp Asp Gly Thr Ile Thr 1 5 <210> 3 <211> 14 <212> PRT <213> artificial sequence <220> <223> CDRH3 (JS40-1, JS48-1) <400> 3 Val Lys Val Pro Ser Pro Ala Pro Met Gln Gly Pro Asp Tyr 1 5 10 <210> 4 <211> 10 <212> PRT <213> artificial sequence <220> <223> CDRL1 (JS40-1) <400> 4 Gln Gly Ile Ser Asn Tyr Leu Ala Trp Phe 1 5 10 <210> 5 <211> 10 <212> PRT <213> artificial sequence <220> <223> CDRL1 (JS48-1) <400> 5 Gln Ser Ile Ser Thr Tyr Leu Asn Trp Tyr 1 5 10 <210> 6 <211> 10 <212> PRT <213> artificial sequence <220> <223> CDRL2 (JS40-1) <400> 6 Ala Ala Ser Ser Leu Gln Ser Gly Val Pro 1 5 10 <210> 7 <211> 10 <212> PRT <213> artificial sequence <220> <223> CDRL2 (JS48-1) <400> 7 Gly Ala Ser Asn Leu Gln Ser Gly Val Ser 1 5 10 <210> 8 <211> 9 <212> PRT <213> artificial sequence <220> <223> CDRL3 (JS40-1) <400> 8 Gln Gln Tyr His Ser Tyr Pro Leu Thr 1 5 <210> 9 <211> 9 <212> PRT <213> artificial sequence <220> <223> CDRL3 (JS48-1) <400> 9 Gln Gln Gly Asn Ser Val Pro Leu Thr 1 5 <210> 10 <211> 25 <212> PRT <213> artificial sequence <220> <223> VH-FR1 (JS40-1) <400> 10 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Ala Gly Ser 20 25 <210> 11 <211> 25 <212> PRT <213> artificial sequence <220> <223> VH-FR1 (JS48-1) <400> 11 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ala Cys Ala Gly Ser 20 25 <210> 12 <211> 17 <212> PRT <213> artificial sequence <220> <223> VH-FR2 (JS40-1, JS48-1) <400> 12 Met Ala Trp Val Arg Leu Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 1 5 10 15 Gly <210> 13 <211> 38 <212> PRT <213> artificial sequence <220> <223> VH-FR3 (JS40-1, JS48-1) <400> 13 Asp Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 1 5 10 15 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 14 <211> 11 <212> PRT <213> artificial sequence <220> <223> VH-FR4 (JS40-1) <400> 14 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 15 <211> 11 <212> PRT <213> artificial sequence <220> <223> VH-FR4 (JS48-1) <400> 15 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 1 5 10 <210> 16 <211> 26 <212> PRT <213> artificial sequence <220> <223> VL-FR1 (JS40-1) <400> 16 Glu Thr Thr Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 20 25 <210> 17 <211> 26 <212> PRT <213> artificial sequence <220> <223> VL-FR1 (JS48-1) <400> 17 Asp Val Val Met Thr Gln Ser Pro Ser Ala Met Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 20 25 <210> 18 <211> 13 <212> PRT <213> artificial sequence <220> <223> VL-FR2 (JS40-1) <400> 18 Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile Tyr 1 5 10 <210> 19 <211> 13 <212> PRT <213> artificial sequence <220> <223> VL-FR2 (JS48-1) <400> 19 Gln Gln Lys Pro Gly Lys Val Pro Lys Arg Leu Ile Tyr 1 5 10 <210> 20 <211> 29 <212> PRT <213> artificial sequence <220> <223> VL-FR3 (JS40-1) <400> 20 Ser Gln Phe Ser Gly Arg Gly Pro Gly Thr Asp Phe Thr Leu Thr Ile 1 5 10 15 Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 20 25 <210> 21 <211> 29 <212> PRT <213> artificial sequence <220> <223> VL-FR3 (JS48-1) <400> 21 Ser Arg Phe Arg Gly Arg Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile 1 5 10 15 Ser Cys Leu Gln Ser Glu Asp Phe Ala Thr Tyr Tyr Cys 20 25 <210> 22 <211> 11 <212> PRT <213> artificial sequence <220> <223> VL-FR4 (JS40-1) <400> 22 Phe Gly Gly Gly Thr Lys Val Asp Ile Lys Arg 1 5 10 <210> 23 <211> 11 <212> PRT <213> artificial sequence <220> <223> VL-FR4 (JS48-1) <400> 23 Phe Gly Gly Gly Thr Lys Leu Ser Val Leu Gly 1 5 10 <210> 24 <211> 106 <212> PRT <213> artificial sequence <220> <223> CL <400> 24 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 1 5 10 15 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 20 25 30 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 35 40 45 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 50 55 60 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 65 70 75 80 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 85 90 95 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 25 <211> 98 <212> PRT <213> artificial sequence <220> <223> CH1 <400> 25 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val <210> 26 <211> 126 <212> PRT <213> artificial sequence <220> <223> CH2 <400> 26 Glu Pro Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 15 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 20 25 30 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 35 40 45 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 50 55 60 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 65 70 75 80 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 85 90 95 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 100 105 110 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 115 120 125 <210> 27 <211> 107 <212> PRT <213> artificial sequence <220> <223> CH3 <400> 27 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 1 5 10 15 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 105 <210> 28 <211> 206 <212> PRT <213> artificial sequence <220> <223> FcaR-ECD <400> 28 Gln Glu Gly Asp Phe Pro Met Pro Phe Ile Ser Ala Lys Ser Ser Pro 1 5 10 15 Val Ile Pro Leu Asp Gly Ser Val Lys Ile Gln Cys Gln Ala Ile Arg 20 25 30 Glu Ala Tyr Leu Thr Gln Leu Met Ile Ile Lys Asn Ser Thr Tyr Arg 35 40 45 Glu Ile Gly Arg Arg Leu Lys Phe Trp Asn Glu Thr Asp Pro Glu Phe 50 55 60 Val Ile Asp His Met Asp Ala Asn Lys Ala Gly Arg Tyr Gln Cys Gln 65 70 75 80 Tyr Arg Ile Gly His Tyr Arg Phe Arg Tyr Ser Asp Thr Leu Glu Leu 85 90 95 Val Val Thr Gly Leu Tyr Gly Lys Pro Phe Leu Ser Ala Asp Arg Gly 100 105 110 Leu Val Leu Met Pro Gly Glu Asn Ile Ser Leu Thr Cys Ser Ser Ala 115 120 125 His Ile Pro Phe Asp Arg Phe Ser Leu Ala Lys Glu Gly Glu Leu Ser 130 135 140 Leu Pro Gln His Gln Ser Gly Glu His Pro Ala Asn Phe Ser Leu Gly 145 150 155 160 Pro Val Asp Leu Asn Val Ser Gly Ile Tyr Arg Cys Tyr Gly Trp Tyr 165 170 175 Asn Arg Ser Pro Tyr Leu Trp Ser Phe Pro Ser Asn Ala Leu Glu Leu 180 185 190 Val Val Thr Asp Ser Ile His Gln Asp Tyr Thr Thr Gln Asn 195 200 205
Claims (10)
(ⅱ) 서열번호 4의 아미노산 서열을 포함하는 CDRL1, 서열번호 6의 아미노산 서열을 포함하는 CDRL2, 및 서열번호 8의 아미노산 서열을 포함하는 CDRL3를 포함하는 VL 도메인을 포함하는 V 도메인을 포함하는, Fc 알파 수용체에 특이적인 항체 또는 이의 면역학적 활성을 가진 단편.The method of claim 1, wherein (i) CDRH (Complementarity determining regions Heavy chain)1 comprising the amino acid sequence of SEQ ID NO: 1, CDRH2 comprising the amino acid sequence of SEQ ID NO: 2, and CDRH3 comprising the amino acid sequence of SEQ ID NO: 3 VH domain containing; and/or
(ii) a V domain comprising a VL domain comprising CDRL1 comprising the amino acid sequence of SEQ ID NO: 4, CDRL2 comprising the amino acid sequence of SEQ ID NO: 6, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 8, Antibodies specific for Fc alpha receptors or immunologically active fragments thereof.
(ⅱ) 서열번호 16의 아미노산 서열을 포함하는 FR1, 서열번호 18의 아미노산 서열을 포함하는 FR2, 서열번호 20의 아미노산 서열을 포함하는 FR3 및 서열번호 22의 아미노산 서열을 포함하는 FR4를 포함하는 VL 도메인을 포함하는, Fc 알파 수용체에 특이적인 항체 또는 이의 면역학적 활성을 가진 단편.The method of claim 2, wherein (i) FR1 comprising the amino acid sequence of SEQ ID NO: 10, FR2 comprising the amino acid sequence of SEQ ID NO: 12, FR3 comprising the amino acid sequence of SEQ ID NO: 13, and comprising the amino acid sequence of SEQ ID NO: 14 A VH domain comprising FR4; and/or
(ii) VL comprising FR1 comprising the amino acid sequence of SEQ ID NO: 16, FR2 comprising the amino acid sequence of SEQ ID NO: 18, FR3 comprising the amino acid sequence of SEQ ID NO: 20, and FR4 comprising the amino acid sequence of SEQ ID NO: 22 An antibody or immunologically active fragment thereof that is specific for an Fc alpha receptor, comprising a domain.
(ⅱ) 서열번호 5의 아미노산 서열을 포함하는 CDRL1, 서열번호 7의 아미노산 서열을 포함하는 CDRL2, 및 서열번호 9의 아미노산 서열을 포함하는 CDRL3를 포함하는 VL 도메인을 포함하는 V 도메인을 포함하는, Fc 알파 수용체에 특이적인 항체 또는 이의 면역학적 활성을 가진 단편.According to claim 1, (i) a VH domain comprising CDRH1 comprising the amino acid sequence of SEQ ID NO: 1, CDRH2 comprising the amino acid sequence of SEQ ID NO: 2, and CDRH3 comprising the amino acid sequence of SEQ ID NO: 3; and/or
(ii) a V domain comprising a VL domain comprising CDRL1 comprising the amino acid sequence of SEQ ID NO: 5, CDRL2 comprising the amino acid sequence of SEQ ID NO: 7, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 9, Antibodies specific for Fc alpha receptors or immunologically active fragments thereof.
(ⅱ) 서열번호 17의 아미노산 서열을 포함하는 FR1, 서열번호 19의 아미노산 서열을 포함하는 FR2, 서열번호 21의 아미노산 서열을 포함하는 FR3 및 서열번호 23의 아미노산 서열을 포함하는 FR4를 포함하는 VL 도메인을 포함하는, Fc 알파 수용체에 특이적인 항체 또는 이의 면역학적 활성을 가진 단편.The method of claim 4, wherein (i) FR1 comprising the amino acid sequence of SEQ ID NO: 11, FR2 comprising the amino acid sequence of SEQ ID NO: 12, FR3 comprising the amino acid sequence of SEQ ID NO: 13, and comprising the amino acid sequence of SEQ ID NO: 15 A VH domain comprising FR4; and/or
(ii) VL comprising FR1 comprising the amino acid sequence of SEQ ID NO: 17, FR2 comprising the amino acid sequence of SEQ ID NO: 19, FR3 comprising the amino acid sequence of SEQ ID NO: 21, and FR4 comprising the amino acid sequence of SEQ ID NO: 23 An antibody or immunologically active fragment thereof that is specific for an Fc alpha receptor, comprising a domain.
b) 숙주 세포 배양물로부터 항체 또는 이의 면역학적 활성을 가진 단편을 회수하는 단계를 포함하는 Fc 알파 수용체에 특이적인 항체 또는 이의 면역학적 활성을 가진 단편을 제조하는 방법.a) culturing a transformed host cell with a vector comprising an isolated nucleic acid molecule encoding the antibody of claim 1 or an immunologically active fragment thereof; and
b) a method for preparing an antibody specific for an Fc alpha receptor or an immunologically active fragment thereof comprising the step of recovering the antibody or immunologically active fragment thereof from a host cell culture.
Priority Applications (5)
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KR1020210135095A KR20230052017A (en) | 2021-10-12 | 2021-10-12 | Human antibodies for binding to human Fc alpha receptor |
CN202180075236.XA CN116419928A (en) | 2020-11-06 | 2021-11-03 | Fc alpha receptor binding antibodies |
PCT/KR2021/015795 WO2022098084A1 (en) | 2020-11-06 | 2021-11-03 | Fc alpha receptor binding antibody |
EP21889549.8A EP4242233A1 (en) | 2020-11-06 | 2021-11-03 | Fc alpha receptor binding antibody |
US18/035,609 US20230399407A1 (en) | 2020-11-06 | 2021-11-03 | Fc alpha receptor binding antibody |
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