KR20230119856A - A Composition for Preventing or Treating Cancer Comprising Novel Bitargeting Fusion-Antibody as an Active Ingredient - Google Patents
A Composition for Preventing or Treating Cancer Comprising Novel Bitargeting Fusion-Antibody as an Active Ingredient Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
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- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/525—Tumour necrosis factor [TNF]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/72—Increased effector function due to an Fc-modification
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
Abstract
본 발명은 항체의 Fc 영역에 TNFα 돌연변이체가 결합된 새로운 형태의 항-CD20 항체; 및 이를 포함하는 암의 예방 또는 치료용 약학적 조성물에 관한 것이다. 본 발명은 TNF 수용체에는 결합하나 신호는 발생시키지 않는 TNFα 돌연변이체를 항-CD20 항체에 결합시킴으로써, CD20과의 결합을 통한 고유한 세포 사멸 기전과 함께 TNFR1에 의한 세포사멸 기전을 동시에 유도하여 항체의 암세포에 대한 사멸률을 증가시킬 뿐 아니라, 정상 TNFα에 대한 경쟁적 억제제로 작용하여 과도한 염증반응을 제어하는 효율적인 치료용 항체로 유용하게 이용될 수 있다.The present invention provides a new type of anti-CD20 antibody in which a TNFα mutant is bound to the Fc region of the antibody; And it relates to a pharmaceutical composition for preventing or treating cancer comprising the same. The present invention binds a TNFα mutant that binds to the TNF receptor but does not generate a signal to an anti-CD20 antibody, thereby simultaneously inducing an apoptosis mechanism by TNFR1 as well as a unique cell death mechanism through binding to CD20, thereby reducing the effect of the antibody. In addition to increasing the death rate of cancer cells, it can be usefully used as an efficient therapeutic antibody that controls excessive inflammatory responses by acting as a competitive inhibitor for normal TNFα.
Description
본 발명은 암 세포 사멸률을 크게 향상시켜 보다 효율적으로 B세포 림프종을 예방 또는 치료하는 동시에 TNF 신호에 대한 억제력을 추가로 제공하여 과도한 염증반응을 효율적으로 제어하는 새로운 조합의 융합항체를 제공하고자 한다.The present invention is intended to provide a new combination of fusion antibodies that effectively control excessive inflammatory responses by providing additional inhibitory power to TNF signals while more efficiently preventing or treating B-cell lymphoma by greatly improving cancer cell death rate. .
단클론 항체(monoclonal antibodies : mAbs)는 지난 몇 년간 치료제 분야에서 가장 빠르게 성장하는 분야였고, 암부터 자가면역질환까지 다양한 병증에 대하여 치료방법으로서 승인이 되었다. 그 중 가장 연구가 많이 된 항체의 타겟 중 하나는 B 세포의 마커인 CD20으로, 1997년 리툭시맙(Rituximab)이 비-호지킨성 림프종에 대해서 사용 허가가 내려진 이래로 많은 차세대 항-CD20 단클론 항체가 다양한 전략을 통해 개발되고 있다.Monoclonal antibodies (mAbs) have been the fastest-growing segment of therapeutics in the past few years, and have been approved as treatments for conditions ranging from cancer to autoimmune diseases. One of the most studied antibody targets is CD20, a B cell marker. is being developed through various strategies.
항-CD20 단클론항체(Anti-CD20 mAb)는 원형질막 내부에 있는 CD20을 지질뗏목(lipid rafts)속으로 재분배(redistribute)하는 능력을 가진다. 기능적인 면을 볼 때, 이러한 재분배 능력은 BCR 신호전달에서 있어서 CD20의 중요한 기능으로 예상된다. 그러나 이러한 재분배 능력은 항-CD20 항체 자체에 대해서도 중요한 의미를 가지는데, 이는 이러한 재분배 능력의 유무가 항-CD20 항체를 분류하는 유용한 기준이 되기 때문이다. 리툭시맙(Rituximab) 및 오파투무맙(Ofatumumab)과 같이 CD20에 부착하여 지질뗏목 속으로의 구역화(compartmentalization)을 일으키는 단클론 항체들은 1형(type I)으로 구분되는 반면에, CD20에 결합은 하지만 재분배는 일으키지 않는 오비누투주맙(Obinutuzumab)과 같은 항체는 2형(type II)로 구분된다.Anti-CD20 monoclonal antibody (Anti-CD20 mAb) has the ability to redistribute CD20 inside the plasma membrane into lipid rafts. From a functional point of view, this redistribution capacity is expected to be an important function of CD20 in BCR signaling. However, this redistribution ability is also important for the anti-CD20 antibody itself, because the presence or absence of this redistribution ability is a useful criterion for classifying the anti-CD20 antibody. Monoclonal antibodies that attach to CD20 and cause compartmentalization into lipid rafts, such as Rituximab and Ofatumumab, are classified as type I, whereas they bind to CD20 but Antibodies such as Obinutuzumab, which do not cause redistribution, are classified as type II.
이러한 CD20 및 결합한 단클론항체의 지질뗏목 속으로의 재분배는 분류에 대한 기준이 되어줄 뿐만 아니라, 기능적인 차이점도 제공한다. 1형 항체의 경우 항체의 Fc 영역이 더욱 뭉쳐지기 때문에 보체 의존성 세포독성(Complement-dependent cytotoxicity : CDC)를 잘 일으키는 반면, 2형 항체의 경우 1형만큼 강한 CDC는 일으키지 못한다. 그러나, 2형 항체의 경우 더욱 강한 비-아포토시스 경로의 직접세포사멸(non-apoptotic Direct Cell Death)과, 항체 의존성 세포독성(Antibody-Dependent Cellular Cytotoxicity : ADCC)을 일으키는 기작을 통하여 항암제로 작용한다.This redistribution of CD20 and bound monoclonal antibodies into lipid rafts not only serves as a criterion for classification, but also provides functional differences. In the case of type 1 antibody, because the Fc region of the antibody is more aggregated, complement-dependent cytotoxicity (CDC) is well caused, whereas in the case of type 2 antibody, CDC is not as strong as type 1. However, type 2 antibodies act as anticancer agents through mechanisms that cause non-apoptotic direct cell death and antibody-dependent cellular cytotoxicity (ADCC).
1형 항체의 경우 상기와 같은 잘 뭉쳐지는 성질로 인하여 세포 내부로 더욱 잘 들어가게(Internalization) 되며, 결과적으로 라이소좀 분해를 통하여 세포 표면의 CD20 발현을 줄이게 된다. 현재까지 정확하게 밝혀지지는 않았으나, 이러한 기작은 1형 항-CD20 항체의 저항성 발생에 대한 주요한 기작으로 생각된다.In the case of the type 1 antibody, due to the well-aggregated property as described above, it enters into the cell better (Internalization), and as a result, CD20 expression on the cell surface is reduced through lysosomal degradation. Although it has not been precisely identified to date, this mechanism is considered to be a major mechanism for the development of resistance to type 1 anti-CD20 antibodies.
반면 2형 항-CD20 항체의 경우, 많은 항암제의 주요 기전인 아포토시스(apoptosis)와 무관한 경로로 직접세포사멸을 일으키기 때문에, 아포토시스에 저항성을 가지는 B세포림프종(B lymphoma)에 대한 대안인 동시에, 1형 항-CD20 항체와 같이 세포 내부로 들어가지 않아서 항체에 대한 저항성 발생 가능성도 낮은 편이다. 또한, CDC가 주요기전인 1형 항-CD20 항체와 비교하여 자연살해세포(NK cell)과 같은 면역효과세포(Immune Effector Cell)을 이용한다는 점에서 세포 사멸률이 높은 장점을 가진다.On the other hand, in the case of type 2 anti-CD20 antibody, since it causes direct cell death in a pathway unrelated to apoptosis, which is the main mechanism of many anticancer drugs, it is an alternative to B-cell lymphoma that is resistant to apoptosis. Like the type 1 anti-CD20 antibody, it does not enter cells, so the possibility of developing resistance to the antibody is also low. In addition, CDC has a high cell death rate in that it uses immune effector cells such as natural killer cells (NK cells) compared to type 1 anti-CD20 antibody, which is the main mechanism.
최근, 이중항체(Bispecific antibodies)를 이용하여 더욱 강한 암세포사멸능력을 가지는 새로운 구조의 항체에 대한 연구가 활발하다. 이중항체란, 두 개의 서로 다른 항원을 인식하는 항체로, 일반적인 항체에서는 두 개의 항원 인식부위가 같은 항원을 인식하는 반면, 서로 다른 항원을 인식하게끔 디자인한 구조의 항체이다. 즉, 자연계에는 존재하지 않는 인공적인 항체로써 1961년 이중항체에 대한 개념이 도출된 이래로, 기술의 한계로 구현이 되지 못하다가 최근 10년 간 이중항체를 제작할 수 있는 관련 기술들이 발달하고 두 개의 단백질을 물리적으로 가깝게 위치시켰을 때 기대할 수 있는 시너지 효과들이 입증되면서 많은 제약사들이 관심을 보이고 있는 상황이다.Recently, studies on antibodies with a new structure that have a stronger ability to kill cancer cells using bispecific antibodies have been actively conducted. A bispecific antibody is an antibody that recognizes two different antigens. In a general antibody, two antigen recognition sites recognize the same antigen, but the antibody has a structure designed to recognize different antigens. In other words, since the concept of double antibody was derived in 1961 as an artificial antibody that does not exist in nature, it has not been implemented due to limitations in technology. As the synergistic effects that can be expected when placed physically close are proven, many pharmaceutical companies are showing interest.
이에, 본 발명자들은 기존 2형 항-CD20 항체와 TNFα 돌연변이체를 융합한 새로운 구조의 항체를 제작함으로써, CD20 및 TNFR1과 동시에 결합할 수 있게 하여 CD20에 대한 결합력이 같음에도 불구하고 더욱 증가된 직접세포사멸(Direct Cell Death : DCD)을 일으키는 동시에 TNF 수용체에 대한 경쟁적 억제제로 작용함으로써 자가 면역 질환 치료제로도 사용될 수 있는 항체 및 이를 이용하여 암 및 자가 면역 질환에 대한 다각도의 치료전략을 제공하고자 하였다.Accordingly, the present inventors prepared an antibody with a new structure by fusing the existing type 2 anti-CD20 antibody and the TNFα mutant, thereby enabling simultaneous binding to CD20 and TNFR1, resulting in a more increased direct Antibodies that can be used as a treatment for autoimmune diseases by causing direct cell death (DCD) and acting as competitive inhibitors for TNF receptors and using them to provide multifaceted treatment strategies for cancer and autoimmune diseases .
본 명세서 전체에 걸쳐 다수의 논문 및 특허문헌이 참조되고 그 인용이 표시되어 있다. 인용된 논문 및 특허문헌의 개시 내용은 그 전체로서 본 명세서에 참조로 삽입되어 본 발명이 속하는 기술 분야의 수준 및 본 발명의 내용이 보다 명확하게 설명된다.A number of papers and patent documents are referenced throughout this specification and their citations are indicated. The contents of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly describe the level of the technical field to which the present invention belongs and the contents of the present invention.
본 발명자들은 혈액암 중 가장 높은 유병률을 보이면서 고령의 환자가 많고 재발율이 높은 난치성 종양에 속하는 림프종에 대한 효율적인 치료 조성물을 개발하기 위하여 예의 연구 노력하였다. 그 결과, 항-CD20 항체에 TNFα(Tumor Necrosis Factor α)를 접합시키는 경우 암세포 사멸률이 크게 증가하고, 특히 TNFR1(Tumor Necrosis Factor Receptor 1)을 발현하는 B 림프종(B lymphoma)에 대해 현저한 효과를 보일 뿐 아니라, 염증인자인 TNF의 경쟁적 억제제로 작용하여 과도한 염증반응에 대한 효율적인 제어가 가능한 새로운 형태의 치료용 항체로 기능할 수 있음을 발견함으로써, 본 발명을 완성하게 되었다.The present inventors have intensively researched to develop an effective therapeutic composition for lymphoma, which has the highest prevalence among hematological cancers and belongs to intractable tumors with many elderly patients and high recurrence rates. As a result, when anti-CD20 antibody is conjugated with TNFα (Tumor Necrosis Factor α), the cancer cell death rate is greatly increased, and in particular, it has a remarkable effect on B lymphoma expressing TNFR1 (Tumor Necrosis Factor Receptor 1). The present invention was completed by discovering that it can function as a new type of therapeutic antibody capable of efficiently controlling an excessive inflammatory response by acting as a competitive inhibitor of TNF, an inflammatory factor, as well as being visible.
따라서 본 발명의 목적은 항체의 Fc 영역에 TNFα 돌연변이체가 결합된 새로운 형태의 항-CD20 항체를 제공하는 데 있다.Accordingly, an object of the present invention is to provide a new type of anti-CD20 antibody in which a TNFα mutant is bound to the Fc region of the antibody.
본 발명의 다른 목적은 항-CD20 항체 및 TNFα를 포함하는 암의 예방 또는 치료용 약학적 조성물을 제공하는 데 있다.Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer comprising an anti-CD20 antibody and TNFα.
본 발명의 또 다른 목적은 중 상기 새로운 형태의 항-CD20 항체를 유효성분으로 포함하는 암의 예방 또는 치료용 약학적 조성물을 제공하는 데 있다.Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer comprising the novel anti-CD20 antibody as an active ingredient.
본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다.Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.
본 발명의 일 양태에 따르면, 본 발명은 Fc 영역에 TNFα 돌연변이체가 결합된 항-CD20 항체를 제공한다.According to one aspect of the present invention, the present invention provides an anti-CD20 antibody in which a TNFα mutant is bound to an Fc region.
본 발명자들은 혈액암 중 가장 높은 유병률을 보이면서 고령의 환자가 많고 재발율이 높은 난치성 종양에 속하는 림프종에 대한 효율적인 치료 조성물을 개발하기 위하여 예의 연구 노력하였다. 그 결과, 항-CD20 항체에 TNFα(Tumor Necrosis Factor α)를 접합시키는 경우 암세포 사멸률이 크게 증가하고, 특히 TNFR1(Tumor Necrosis Factor Receptor 1)을 발현하는 B 림프종(B lymphoma)에 대해 현저한 효과를 보일 뿐 아니라, 염증인자인 TNF의 경쟁적 억제제로 작용하여 과도한 염증반응에 대한 효율적인 제어가 가능한 새로운 형태의 치료용 항체로 기능할 수 있음을 발견하였다.The present inventors have intensively researched to develop an effective therapeutic composition for lymphoma, which has the highest prevalence among hematological cancers and belongs to intractable tumors with many elderly patients and high recurrence rates. As a result, when anti-CD20 antibody is conjugated with TNFα (Tumor Necrosis Factor α), the cancer cell death rate is greatly increased, and in particular, it has a remarkable effect on B lymphoma expressing TNFR1 (Tumor Necrosis Factor Receptor 1). In addition, it was found that it can function as a new type of therapeutic antibody capable of efficiently controlling excessive inflammatory responses by acting as a competitive inhibitor of TNF, an inflammatory factor.
본 명세서에서 용어“항체(antibody)”는 CD20에 대한 항체로서, 이의 특정 에피토프를 특이적으로 인식하여 이에 결합하며, 완전한 항체 형태뿐만 아니라 항체 분자의 항원 결합 단편(항체 단편)을 포함하는 일부 절편일 수도 있다.As used herein, the term “antibody” refers to an antibody against CD20, which specifically recognizes and binds to a specific epitope thereof, and includes not only a complete antibody form but also a partial fragment including an antigen-binding fragment (antibody fragment) of an antibody molecule. It could be.
완전한 항체는 2개의 전체 길이의 경쇄 및 2개의 전체 길이의 중쇄를 가지는 구조이며 각각의 경쇄는 중쇄와 다이설파이드 결합으로 연결되어 있다. 중쇄 불변영역은 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 불변영역은 카파(κ) 및 람다(λ) 타입을 가진다.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 disulfide bonds. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and subclasses include gamma 1 (γ1), gamma 2 (γ2), and gamma 3 (γ3). ), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The constant region of the light chain has kappa (κ) and lambda (λ) types.
본 명세서에서 용어“항원 결합 단편(antigen binding fragment)”은 면역글로불린 전체 구조 중 항원이 결합할 수 있는 폴리펩티드의 일부를 의미하며, 예를 들어 F(ab')2, Fab', Fab, Fv 및 scFv를 포함하나, 이에 제한되는 것은 아니다. As used herein, the term “antigen binding fragment” refers to a part of a polypeptide capable of binding to an antigen in the overall immunoglobulin structure, and includes, for example, F(ab')2, Fab', Fab, Fv and scFvs, but are not limited thereto.
본 명세서에서, 용어 “CD20”은 일반적으로 영장류(예를 들어, 인간); 설치류(예를 들어, 쥐 또는 실험용 쥐) 등을 포함하는 척추동물에서 유래한 자연적인 CD20 혹은 이의 유전적 변이체를 포함하며, 더 나아가 전체 길이의 가공되지 않은 CD20는 물론; 스플라이싱 과정에서의 변이체(Splice Variants), 대립유전자 변이체(Allelic Variants)등의 세포 내에서의 가공 과정을 거친 모든 형태의 CD20가 포함된다.As used herein, the term “CD20” generally refers to primates (eg, humans); including native CD20 or genetic variants thereof derived from vertebrates, including rodents (eg, rats or mice) and the like, as well as full-length unprocessed CD20; All forms of CD20 that have undergone intracellular processing, such as splice variants and allelic variants in the splicing process, are included.
본 명세서에서 용어“중쇄”는 항원에 특이성을 부여하기 위한 충분한 가변영역 서열을 갖는 아미노산 서열을 포함하는 가변영역 도메인 VH 및 3 개의 불변영역 도메인 CH1, CH2 및 CH3을 포함하는 전체길이 중쇄 및 이의 단편을 모두 의미한다. 또한, 본 명세서 용어“경쇄”는 항원에 특이성을 부여하기 위한 충분한 가변영역 서열을 갖는 아미노산 서열을 포함하는 가변영역 도메인 VL 및 불변영역 도메인 CL을 포함하는 전체길이 경쇄 및 이의 단편을 모두 의미한다.As used herein, the term "heavy chain" refers to a full-length variable region domain V H comprising an amino acid sequence having sufficient variable region sequence to impart specificity to an antigen and three constant region domains C H1 , C H2 and C H3 . Both heavy chains and fragments thereof are meant. In addition, the term "light chain" herein refers to both a full-length light chain and fragments thereof 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. do.
본 명세서에서 용어“Fc 영역”은 면역글로불린(immunoglobulin)의 중쇄의 불변부위로써, 항체의 Fc 부분의 단량체(monomeric form)뿐만 아니라 이량체(dimeric form)까지 포함하는 의미이다. 바람직하게는, 단일쇄 Fc 융합 형태이다.In the present specification, the term “Fc region” is a constant region of the heavy chain of immunoglobulin, and includes not only the monomeric form of the Fc region of an antibody but also the dimer form. Preferably, it is a single-chain Fc fusion form.
본 명세서에서, 용어 “TNFα”는 일반적으로 종양괴사인자 알파라고 불리는 아디포카인 또는 사이토카인을 의미하며, TNF 도메인을 갖는 다양한 막 단백질로 구성된 TNF 수퍼패밀리(TNF superfamily)의 구성원이다. 본 명세서에서 TNFα에는 폴리텝타이드; TNF 수용체(TNFR)에 특이적으로 결합하는 능력이 유지된 일부 절편; 인간 TNFα와 그 변이체, 동형단백질, 종간 동족체(species homolog) 또는 적어도 하나의 에피토프(epitope)가 공통된 유사체(analog)를 포함하고; 설치류, 쥐, 토끼, 비인간 영장류, 돼지, 소 등의 다른 포유동물의 TNFα까지 모두 포함된다.As used herein, the term “TNFα” refers to an adipokine or cytokine commonly called tumor necrosis factor alpha, and is a member of the TNF superfamily composed of various membrane proteins having a TNF domain. In the present specification, TNFα includes polypeptide; Some fragments retain the ability to specifically bind to the TNF receptor (TNFR); human TNFα and its variants, isoforms, species homologs or analogs in which at least one epitope is in common; TNFα of other mammals such as rodents, rats, rabbits, non-human primates, pigs, and cattle are all included.
본 명세서에서 용어“돌연변이체”는 야생형의 유전자나 그 산물과 비교하여 서열이나 기능적인 특징에 변화가 생긴 유전자 또는 유전자의 산물을 의미한다.As used herein, the term "mutant" refers to a gene or gene product that has a change in sequence or functional characteristics compared to a wild-type gene or its product.
본 발명의 구체적인 구현예에 따르면, 상기 TNFα 돌연변이체는 수용성(soluble)이다.According to a specific embodiment of the present invention, the TNFα mutant is water-soluble.
본 명세서에서 용어 “수용성”은 임의의 생리학적 pH 범위에서 1g을 초과하는 전해질 물질이 100ml의 물에 녹는 정도의 용해도를 의미하며, 더욱 구체적으로는 임의의 생리학적 pH 범위에서 3g을 초과하는 전해질 물질이 100ml의 물에 녹는 정도의 용해도를 의미한다.copy In the specification, the term “water solubility” refers to the solubility of an electrolyte material exceeding 1 g in a certain physiological pH range in 100 ml of water, and more specifically, an electrolyte material exceeding 3 g in a certain physiological pH range. This means the degree of solubility that dissolves in 100 ml of water.
본 발명의 구체적인 구현예에 따르면, 상기 TNFα 돌연변이체는 A160S, V161T, S162T, Y163H, Q164N 및 T165Q로 구성된 군으로부터 선택되는 하나 이상의 아미노산 치환을 포함한다.According to a specific embodiment of the present invention, the TNFα mutant contains one or more amino acid substitutions selected from the group consisting of A160S, V161T, S162T, Y163H, Q164N and T165Q.
상기 돌연변이의 치환 위치를 특정하는 잔기 번호는 서열번호 7의 아미노산 서열을 기준으로 한다. The residue number specifying the substitution position of the mutation is based on the amino acid sequence of SEQ ID NO: 7.
본 발명의 구체적인 구현예에 따르면, 상기 TNFα 돌연변이체가 항-CD20 항체의 Fc 영역의 C-말단에 결합한다.According to a specific embodiment of the present invention, the TNFα mutant binds to the C-terminus of the Fc region of the anti-CD20 antibody.
본 명세서에서“항체에 결합”되었다는 기재는 특정 펩타이드가 항체의 아미노산 서열 중 특정 영역에 삽입되거나 또는 공유 또는 비공유적으로 결합되는 것을 의미하나, 여기에 국한되지 않고, 상기 펩타이드가 항체와 분리되지 않는 상태를 유지하게 하는 모든 수단을 포함한다.In the present specification, the description of "binding to an antibody" means that a specific peptide is inserted into a specific region of the amino acid sequence of the antibody or bound covalently or non-covalently, but is not limited thereto, and the peptide is not separated from the antibody. It includes all means to maintain the state.
본 발명의 구체적인 구현예에 따르면, 상기 항-CD20 항체는 단백질 링커(Protein linker)를 추가적으로 포함한다.According to a specific embodiment of the present invention, the anti-CD20 antibody additionally includes a protein linker.
본 명세서에서 용어 “단백질 링커”란 아미노산으로 구성된 연결 서열(linker sequence)를 의미하며, 이는 2개의 다른 단백질을 연결하면서도 전체 단백질 활성에 영향을 주지 않는 길이의 단백질 또는 핵산으로 이루어진 연결체를 의미한다.As used herein, the term "protein linker" refers to a linker sequence composed of amino acids, which means a linkage composed of proteins or nucleic acids of a length that does not affect the overall protein activity while linking two different proteins. .
본 명세서에서 단백질 링커는 4 - 8개의 아미노산으로 이루어진 길이를 가지는 링커이고; 보다 구체적으로는 4 - 7개의 아미노산으로 이루어진 길이를 가지며, 보다 더 구체적으로는 4 - 6개의 아미노산으로 이루어진 길이를 가지고, 가장 구체적으로는 5개의 아미노산으로 이루어진 길이를 가진다. The protein linker herein is a linker having a length of 4 to 8 amino acids; More specifically, it has a length of 4 to 7 amino acids, more specifically, it has a length of 4 to 6 amino acids, and most specifically, it has a length of 5 amino acids.
구체적으로는 상기 아미노산은 글리신(Glycine : G) 및 세린(Serine : S) 중 어느 하나이고; 더 구체적으로는 상기 단백질 링커는 GSGSG 링커(GSGSG linker, 서열번호 24)이다.Specifically, the amino acid is any one of glycine (G) and serine (S); More specifically, the protein linker is a GSGSG linker (SEQ ID NO: 24).
또한, 상기 단백질 링커는 항체에 2개의 TNFα를 연결할 때 2개가 사용될 수 있고, 여기서 2개의 링커는 서로 다른 서열을 가질 수 있다.In addition, two protein linkers may be used when linking two TNFα to an antibody, wherein the two linkers may have different sequences.
다만, 본 발명에서 항체와 TNFα 돌연변이체를 연결하기 위한 수단은 상기에서 정의한 단백질 링커에 국한되지 않고, 본 발명의 새로운 형태의 항체의 기능에 영향을 주지 않는 한, 당업계에서 통용되는 모든 연결 수단을 포함한다.However, the means for linking the antibody and the TNFα mutant in the present invention is not limited to the protein linker defined above, and any linking means commonly used in the art, as long as it does not affect the function of the new type of antibody of the present invention includes
본 발명의 구체적인 구현예에 따르면, 상기 단백질 링커는 항-CD20 항체의 Fc 영역에 결합하며, 수용성 TNFα 돌연변이체는 상기 단백질 링커를 통하여 항-CD20 항체와 연결된다.According to a specific embodiment of the present invention, the protein linker binds to the Fc region of the anti-CD20 antibody, and the soluble TNFα mutant is linked to the anti-CD20 antibody through the protein linker.
본 명세서에서, 용어 “항-CD20 항체”는 항원 CD-20과 특이적으로 결합할 수 있는 항원 결합 부위를 가지는 항체를 뜻한다.As used herein, the term "anti-CD20 antibody" refers to an antibody having an antigen-binding site capable of specifically binding to the antigen CD-20.
구체적으로는, 서열번호 1(RSSKSLLHSNGITYLY)의 LCDR1 영역, 서열번호 2(QMSNLVS)의 LCDR2 영역, 서열번호 3(AQNLELPYT)의 LCDR3 영역, 서열번호 4(GYAFSYS)의 HCDR1 영역, 서열번호 5(FPGDGD)의 HCDR2 영역 및 서열번호 6(NVFDGYWLVY)의 HCDR3 영역 중 어느 하나 이상의 CDR 서열을 가지는 것을 의미하며, 보다 구체적으로는 상기 경쇄 및 중쇄 가변영역의 CDR 서열을 모두 가지는 항체를 의미하고;Specifically, the LCDR1 region of SEQ ID NO: 1 (RSSKSLLHSNGITYLY), the LCDR2 region of SEQ ID NO: 2 (QMSNLVS), the LCDR3 region of SEQ ID NO: 3 (AQNLELPYT), the HCDR1 region of SEQ ID NO: 4 (GYAFSYS), SEQ ID NO: 5 (FPGDGD) means having one or more CDR sequences of the HCDR2 region and the HCDR3 region of SEQ ID NO: 6 (NVFDGYWLVY), and more specifically means an antibody having both the CDR sequences of the light chain and heavy chain variable regions;
가장 구체적으로는 상기 항-CD20 항체는 오비누투주맙(Obinutuzumab)이다.Most specifically, the anti-CD20 antibody is Obinutuzumab.
본 명세서에서 용어, "특이적으로 결합(specifically binding)" 은 "특이적으로 인식(specifically recognizing)"과 동일한 의미로서, 항원과 항체(또는 이의 단편)가 면역학적 반응을 통해 특이적으로 상호작용하는 것을 의미한다.As used herein, the term "specifically binding" has the same meaning as "specifically recognizing", and specifically interacts with an antigen and an antibody (or a fragment thereof) through an immunological reaction. means to do
본 명세서에서, 용어“CDR(complementarity determining region)”은 면역글로블린 중쇄 및 경쇄의 고가변 영역(hypervariable region)의 아미노산 서열을 의미한다(Kabat et al. Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987)). 중쇄(CDRH1, CDRH2 및 CDRH3) 및 경쇄(CDRL1, CDRL2 및 CDRL3)에는 각각 3개의 CDR이 포함되어 있으며, 이들 CDR은 항체가 항원 또는 에피토프에 결합하는 데 있어서 주요한 접촉 잔기를 제공한다.As used herein, the term "complementarity determining region (CDR)" refers to the amino acid sequence of the hypervariable region of immunoglobulin heavy and light chains (Kabat et al . Sequences of Proteins of Immunological Interest , 4th Ed., US Department of Health and Human Services, National Institutes of Health (1987)). The heavy chain (CDRH1, CDRH2, and CDRH3) and light chain (CDRL1, CDRL2, and CDRL3) each contain three CDRs, which provide key contact residues for antibody binding to an antigen or epitope.
본 발명의 항체 또는 항체 단편의 범위에는 CDR 영역에 보존적 아미노산 치환을 갖는 변이체가 포함된다. 또한, 본 발명의 항체 또는 항체 단편은, 인산화된 PLCγ2를 특이적으로 인식할 수 있는 범위 내에서 첨부한 서열목록에 기재된 아미노산 서열의 변이체를 포함할 수 있다. 예를 들면, 항체의 결합 친화도 및/또는 기타 생물학적 특성을 보다 더 개선시키기 위하여 항체의 아미노산 서열에 추가적인 변화를 줄 수 있다. 이러한 변형은, 예를 들어 항체의 아미노산 서열 잔기의 결실, 삽입 및/또는 치환을 포함한다. 이러한 아미노산 변이는 아미노산 곁사슬 치환체의 상대적 유사성, 예컨대, 소수성, 친수성, 전하, 크기 등에 기초하여 이루어진다. 아미노산 곁사슬 치환체의 크기, 모양 및 종류에 대한 분석에 의하여, 아르기닌, 라이신과 히스티딘은 모두 양전하를 띤 잔기이고; 알라닌, 글라이신과 세린은 유사한 크기를 갖으며; 페닐알라닌, 트립토판과 타이로신은 유사한 모양을 갖는다는 것을 알 수 있다. 따라서, 이러한 고려 사항에 기초하여, 아르기닌, 라이신과 히스티딘; 알라닌, 글라이신과 세린; 그리고 페닐알라닌, 트립토판과 타이로신은 생물학적으로 기능 균등물이라 할 수 있다.The scope of the antibody or antibody fragment of the present invention includes variants with conservative amino acid substitutions in the CDR regions. In addition, the antibody or antibody fragment of the present invention may include a variant of the amino acid sequence described in the accompanying sequence listing within the scope of specifically recognizing phosphorylated PLCγ2. For example, additional changes may be made to the amino acid sequence of the antibody to further improve its binding affinity and/or other biological properties. Such modifications include, for example, deletions, insertions and/or substitutions of residues in the amino acid sequence of the antibody. Such amino acid variations are made based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape and type of amino acid side chain substituents revealed that arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have similar shapes. Accordingly, based on these considerations, arginine, lysine and histidine; alanine, glycine and serine; And phenylalanine, tryptophan and tyrosine are biologically functional equivalents.
분자의 활성을 전체적으로 변경시키지 않는 단백질에서의 아미노산 교환은 당해 분야에 공지되어 있다(H. Neurath, R.L.Hill, The Proteins, Academic Press, New York, 1979). 가장 통상적으로 일어나는 교환은 아미노산 잔기 Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu 및 Asp/Gly 간의 교환이다.Amino acid exchanges in proteins that do not entirely alter the activity of the molecule are known in the art (H. Neurath, R.L. Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thr/Phe, Ala/ Exchange between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly.
상술한 생물학적 균등 활성을 갖는 변이를 고려한다면, 본 발명의 항체 또는 이를 코딩하는 핵산 분자는 서열목록에 기재된 서열과 실질적인 동일성(substantial identity)을 나타내는 서열도 포함하는 것으로 해석된다. 상기의 실질적인 동일성은, 상기한 본 발명의 서열과 임의의 다른 서열을 최대한 대응되도록 얼라인 하고, 당업계에서 통상적으로 이용되는 알고리즘을 이용하여 얼라인 된 서열을 분석한 경우에, 최소 61%의 상동성, 일특정예에 따르면 70%의 상동성, 다른 특정예에 따르면 80%의 상동성, 또 다른 특정예에 따르면 90%의 상동성을 나타내는 서열을 의미한다. 서열비교를 위한 얼라인먼트 방법은 당업계에 공지되어 있다. 얼라인먼트에 대한 다양한 방법 및 알고리즘은 Smith and Waterman, Adv. Appl. Math. (1981) 2:482 Needleman and Wunsch, J. Mol. Bio. (1970) 48:443; Pearson and Lipman, Methods in Mol. Biol. (1988) 24: 307-31; Higgins and Sharp, Gene (1988) 73:237-44; Higgins and Sharp, CABIOS (1989) 5:151-3; Corpet et al. Nuc. Acids Res. (1988) 16:10881-90; Huang et al. Comp. Appl. BioSci. (1992) 8:155-65 및 Pearson et al. Meth. Mol. Biol. (1994) 24:307-31에 개시되어 있다. NCBI Basic Local Alignment Search Tool(BLAST)(Altschul et al. J. Mol. Biol. (1990) 215:403-10)은 NBCI 등에서 접근 가능하며, 인터넷 상에서 blastp, blasm, blastx, tblastn 및 tblastx와 같은 서열 분석 프로그램과 연동되어 이용할 수 있다. BLAST는 www.ncbi.nlm.nih.gov/BLAST/에서 접속 가능하다. 이 프로그램을 이용한 서열 상동성 비교 방법은 www.ncbi.nlm.nih.gov/BLAST/blast_help.html에서 확인할 수 있다.Considering the mutations having the above-described biologically equivalent activity, the antibodies of the present invention or the nucleic acid molecules encoding them are construed to include sequences showing substantial identity with the sequences listed in the Sequence Listing. The above substantial identity is at least 61% when the sequence of the present invention and any other sequence described above are aligned so as to correspond as much as possible and the aligned sequence is analyzed using an algorithm commonly used in the art. Homology, according to one specific example, refers to a sequence exhibiting 70% homology, according to another specific example, 80% homology, and according to another specific example, 90% homology. Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are described in Smith and Waterman, Adv. Appl. Math . (1981) 2:482 Needleman and Wunsch, J. Mol. Bio . (1970) 48:443; Pearson and Lipman, Methods in Mol. Biol . (1988) 24: 307-31; Higgins and Sharp, Gene (1988) 73:237-44; Higgins and Sharp, CABIOS (1989) 5:151-3; Corpet et al . Nuc. Acids Res . (1988) 16:10881-90; Huang et al . Comp. Appl. BioSci . (1992) 8:155-65 and Pearson et al . Meth. Mol. Biol . (1994) 24:307-31. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al . J. Mol. Biol . (1990) 215:403-10) is accessible from NBCI and the like, and sequences such as blastp, blasm, blastx, tblastn and tblastx are found on the Internet. It can be used in conjunction with an analysis program. BLAST is accessible at www.ncbi.nlm.nih.gov/BLAST/. Sequence homology comparison methods using this program can be found at www.ncbi.nlm.nih.gov/BLAST/blast_help.html.
본 발명의 항체는 단일클론 항체, 인간 항체, 인간화 항체, 키메라 항체, 단쇄 Fvs(scFV), 단쇄항체, Fab 단편, F(ab')단편, 다이설파이드-결합 Fvs(sdFV) 및 항-이디오타입(항-Id) 항체, 그리고 상기 항체들의 에피토프-결합 단편 등을 포함하나, 이에 한정되는 것은 아니다.Antibodies of the present invention include monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain Fvs (scFV), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFV) and anti-idio type (anti-Id) antibodies, and epitope-binding fragments of the antibodies, and the like, but are not limited thereto.
본 발명의 또 다른 양태에 따르면, 본 발명은 전술한 본 발명의 항체를 유효성분으로 포함하는 암의 예방 또는 치료용 약학적 조성물을 제공한다.According to another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the above-described antibody of the present invention as an active ingredient.
본 명세서에서 용어“예방”은 질환 또는 질병을 보유하고 있다고 진단된 적은 없으나, 이러한 질환 또는 질병에 걸릴 가능성이 있는 대상체에서 질환 또는 질병의 발생을 억제하는 것을 의미한다. As used herein, the term “prevention” refers to suppressing the occurrence of a disease or disease in a subject who has not been diagnosed with the disease or disease, but is likely to suffer from the disease or disease.
본 명세서에서 용어“치료”는 (a) 질환, 질병 또는 증상의 발전의 억제; (b) 질환, 질병 또는 증상의 경감; 또는 (c) 질환, 질병 또는 증상을 제거하는 것을 의미한다. 본 발명의 조성물을 대상체에 투여하면 조성물에 포함된 본 발명의 항체의 에피토프 부분은 암세포의 CD20 항원에 특이적으로 결합하고, 본 발명 항체의 TNFα돌연변이체 부분은 TNFR1에 동시에 결합함으로써, 항체 결합만으로 암세포의 사멸을 일으키는 DCD(Direct Cell Death)반응과 NK 세포를 통한 ADCC(Antibody Dependent Cellular Cytotoxicity)을 통해 암세포를 선별적으로 제거하는 역할을 하는 동시에 TNFα 돌연변이체가 TNFα의 경쟁적 억제제로 작용하여 과도한 염증반응을 방지하는 역할도 수행한다. 따라서, 본 발명의 조성물은 그 자체로 이들 질환 치료의 조성물이 될 수도 있고, 혹은 다른 약리성분과 함께 투여되어 상기 질환에 대한 치료 보조제로 적용될 수도 있다. 이에, 본 명세서에서 용어“치료”또는“치료제”는“치료 보조”또는“치료 보조제”의 의미를 포함한다.As used herein, the term “treatment” refers to (a) inhibition of the development of a disease, condition or condition; (b) alleviation of the disease, condition or symptom; or (c) eliminating the disease, disorder or condition. When the composition of the present invention is administered to a subject, the epitope portion of the antibody of the present invention included in the composition specifically binds to the CD20 antigen of cancer cells, and the TNFα mutant portion of the antibody of the present invention binds to TNFR1 at the same time, resulting in only antibody binding. It plays a role in selectively removing cancer cells through DCD (Direct Cell Death) reaction that causes cancer cell death and ADCC (Antibody Dependent Cellular Cytotoxicity) through NK cells, and at the same time, TNFα mutant acts as a competitive inhibitor of TNFα, resulting in an excessive inflammatory response. It also plays a role in preventing Therefore, the composition of the present invention may be a composition for treating these diseases by itself, or may be administered together with other pharmacological ingredients to be applied as a treatment adjuvant for the above diseases. Accordingly, the term "treatment" or "therapeutic agent" in the present specification includes the meaning of "therapeutic aid" or "therapeutic aid".
본 발명의 약학적 조성물은 약학적으로 허용 가능한 담체를 포함할 수 있다. 상기 약학적으로 허용되는 담체는 경구 투여 시에는 결합제, 활탁제, 붕해제, 부형제, 가용화제, 분산제, 안정화제, 현탁화제, 색소, 향료 등이 사용될 수 있고, 주사제의 경우에는 완충제, 보존제, 무통화제, 가용화제, 등장제, 안정화제 등이 혼합되어 사용될 수 있으며, 국소투여용의 경우에는 기제, 부형제, 윤활제, 보존제 등이 사용될 수 있다. 본 발명의 약학 조성물의 제형은 상술한 바와 같은 약학적으로 허용되는 담체와 혼합하여 다양하게 제조될 수 있다. 예를 들어, 경구 투여시에는 정제, 트로키, 캡슐, 엘릭서(Elixir), 서스펜션, 시럽, 웨이퍼 등의 형태로 제조할 수 있으며, 주사제의 경우에는 단위 투약 앰플 또는 다수회 투약 형태로 제조할 수 있다. 기타, 용액, 현탁액, 정제, 캡슐, 서방형 제제 등으로 제형화할 수 있다.The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a colorant, a flavoring agent, etc. for oral administration, and in the case of an injection, a buffer, a preservative, Pain relievers, solubilizers, isotonic agents, stabilizers, etc. may be mixed and used, and in the case of topical administration, bases, excipients, lubricants, preservatives, etc. may be used. Formulations of the pharmaceutical composition of the present invention may be variously prepared by mixing with the pharmaceutically acceptable carrier as described above. For example, for oral administration, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, it can be prepared in unit dosage ampoules or multiple dosage forms. there is. In addition, it may be formulated into solutions, suspensions, tablets, capsules, sustained-release preparations, and the like.
한편, 제제화에 적합한 담체, 부형제 및 희석제의 예로는, 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말디톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로즈, 폴리비닐피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 또는 광물유 등이 사용될 수 있다. 또한, 충진제, 항 응집제, 윤활제, 습윤제, 향료, 유화제, 방부제 등을 추가로 포함할 수 있다.On the other hand, examples of carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil and the like can be used. In addition, fillers, anti-agglomerating agents, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, and the like may be further included.
본 발명의 상기 약학 조성물의 투여 경로는 이들로 한정되는 것은 아니지만 구강, 정맥내, 근육내, 동맥내, 골수내, 경막내, 심장내, 경피, 피하, 복강내, 비강내, 장관, 국소, 설하 또는 직장이 포함된다. The route of administration of the pharmaceutical composition of the present invention is not limited thereto, but is not limited to oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, This includes sublingual or rectal.
본 발명의 상기 약학 조성물은 사용된 특정 화합물의 활성, 연령, 체중, 일반적인 건강, 성별, 정식, 투여 시간, 투여 경로, 배출율, 약물 배합 및 예방 또는 치료될 특정 질환의 중증을 포함한 여러 요인에 따라 다양하게 변할 수 있고, 상기 약학 조성물의 투여량은 환자의 상태, 체중, 질병의 정도, 약무 형태, 투여 경로 및 기간에 따라 다르지만 당업자에 의해 적절하게 선택될 수 있고, 1일 0.0001 내지 50mg/kg 또는 0.001 내지 50mg/kg으로 투여할 수 있다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다. 본 발명에 따른 의약 조성물은 환제, 당의정, 캡슐, 액제, 겔, 시럽, 슬러리, 현탁제로 제형화될 수 있다.The pharmaceutical composition of the present invention depends on various factors including the activity of the specific compound used, age, body weight, general health, sex, diet, administration time, route of administration, excretion rate, drug combination and severity of the specific disease to be prevented or treated. It can vary widely, and the dosage of the pharmaceutical composition varies depending on the patient's condition, body weight, degree of disease, drug type, administration route and period, but can be appropriately selected by those skilled in the art, and is 0.0001 to 50 mg/kg per day. Alternatively, it may be administered at 0.001 to 50 mg/kg. Administration may be administered once a day, or may be administered in several divided doses. The dosage is not intended to limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated into a pill, dragee, capsule, liquid, gel, syrup, slurry, or suspension.
본 발명의 또 다른 양태에 따르면, 본 발명은 항-CD20 항체 및 TNFα 돌연변이체를 포함하는 암의 예방 또는 치료용 약학적 조성물을 제공한다.According to another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising an anti-CD20 antibody and a TNFα mutant.
본 발명에서 암은 구체적으로 혈액암을 의미하고, 더욱 구체적으로는 림프종(Lymphoma)을 의미하며, 가장 구체적으로는 상기 암은 B 세포 림프종(B lymphoma)이다.In the present invention, cancer specifically means blood cancer, more specifically means lymphoma, and most specifically, the cancer is B cell lymphoma.
본 발명의 특징 및 이점을 요약하면 다음과 같다:The features and advantages of the present invention are summarized as follows:
(a) 본 발명은 항체의 Fc 영역에 TNFα 돌연변이체가 결합된 새로운 형태의 항-CD20 항체; 및 이를 포함하는 암의 예방 또는 치료용 약학적 조성물을 제공한다.(a) The present invention provides a new type of anti-CD20 antibody in which a TNFα mutant is bound to the Fc region of the antibody; And it provides a pharmaceutical composition for preventing or treating cancer comprising the same.
(b) 본 발명은 TNF 수용체에는 결합하나 신호는 발생시키지 않는 TNFα 돌연변이체를 항-CD20 항체에 결합시킴으로써, CD20과의 결합을 통한 고유한 세포 사멸 기전과 함께 TNFR1에 의한 세포사멸 기전을 동시에 유도하여 항체의 암세포에 대한 사멸률을 증가시킬 뿐 아니라, 정상 TNFα에 대한 경쟁적 억제제로 작용하여 과도한 염증반응을 제어하는 효율적인 치료용 항체로 유용하게 이용될 수 있다.(b) The present invention binds a TNFα mutant that binds to the TNF receptor but does not generate a signal to an anti-CD20 antibody, thereby simultaneously inducing apoptosis mechanism by TNFR1 along with a unique cell death mechanism through binding to CD20 Thus, it can be usefully used as an efficient therapeutic antibody that not only increases the death rate of cancer cells, but also controls excessive inflammatory responses by acting as a competitive inhibitor for normal TNFα.
도 1a은 Raji 및 Ramos B 림프종 세포주에서 TNFR1 발현을 확인하기 위한 항-TNFR1 항체를 사용한 면역블롯팅 결과를 도시한 그림이다.
도 1b는 Ramos 세포주에서 막 TNFR1 발현의 유세포 분석 히스토그램으로써, 마우스 항-TNFR1 항체 및 FITC 접합된 염소 항-마우스 IgG 항체를 사용하였다.
도 1c는 오비누투주맙 처리 후 Ramos 세포에서 CD20 및 TNFR1의 면역형광 결과를 나타낸 그림으로, 왼쪽은 세포를 고정한 후 오비누투주맙 및 마우스 항-TNFR1 항체로 처리한 결과이고, 오른쪽은 세포에 오비누투주맙으로 10분간 전처리한 후 고정하고 마우스 항-TNFR1 항체로 처리한 결과이다. 오비누투주맙은 FITC-접합 항-인간 IgG 이차 항체(녹색)으로 표지되었고, TNFR1은 Alexa680-접합 항-마우스 IgG 이차 항체(붉은색)으로 표지되었으며 병합된 이미지는 공동국소화(노란색)을 나타낸다. 스케일 바는 5μm이다.
도2a는 항-CD20 / 항-TNFR1 의 이중특이성을 가지는 항체 디자인을 도시한 그림이다.
도2b는 Obi x TNFα WT 융합 항체를 도시한 그림으로써, 하단의 푸른색 타원형 도형 2개는 수용성 TNFα WT를 나타내고, 항체와 TNFα WT를 잇는 선은 GSGSG링커를 나타낸다.
도2c는 Obi x TNF 돌연변이체 융합 항체를 도시한 그림으로써, 주황색 타원형 도형 2개는 수용성 TNFα 돌연변이체를, 중간의 선은 GSGSG 링커를 나타내며, 붉은색 박스는 돌연변이가 일어난 부분의 아미노산 변이를 표현한 그림이다.
도3a는 환원 및 비환원 조건에서 SDS-PAGE를 사용한 각 항체의 무결성을 검증한 결과를 도시한 그림이다. 총 항체는 NanoDrop™ Lite Spectrophotometer로 정량화되었고, 샘플은 10% SDS-PAGE 젤로 분리되었고, 쿠마시 블루로 염색되었다.
도3b는 항체의 CD20에 대한 결합 친화도를 보여주는 유세포 분석 히스토그램을 나타낸 그림이다.
도3c는 항체의 TNFR1에 대한 결합 친화도를 보여주는 유세포 분석 히스토그램을 나타낸 그림이다.
도4a는 항체-유도 TNFα 신호전달 활성화 어세이의 과정을 개략적으로 나타낸 그림이다. HEK293 리포터 세포는 NF-κB 프로모터 의존적 GFP 유전자를 안정적으로 발현하고 있으며, NF-κB 신호전달이 TNFα 또는 항체에 의해 유도될 때, GFP는 세포질에서 발현되고 형광이 발생하게 되고, 이를 통하여 TNFα 신호전달의 강도가 GFP 형광에 의해 가시화 될 수 있다.
도4b는 TNFα 및 항체에 의해 유도된 HEK293 리포터 세포에서 GFP 형광을 나타내는 대표이미지로써, TNFα가 양성 조건으로 사용되었다.
도4c는 GFP 형광 강도를 정규화한 뒤 이를 각 항체별로 농도에 따라 도시한 그래프이다.
도5a는 항체-매개 TNFα 신호 전달 차단 어세이의 과정을 개략적으로 나타낸 그림이다. NF-κB 프로모터 의존적 GFP 유전자를 안정적으로 발현하는 HEK293 리포터 세포에 항체가 결합하여 신호가 차단되면 형광이 나타나지 않게 된다.
도5b는 항체에 의해 차단된 HEK293 리포터 세포에서 GFP 형광을 보여주는 대표 이미지로써, 엔브렐이 양성 조건으로 사용되었다.
도5c는 GFP 형광 강도를 정규화한 뒤 이를 각 항체별로 도시한 그래프이다.
도6a는 Raji 및 Ramos B 림프종 세포주에서 TNFR1 발현의 면역블롯팅 결과를 나타낸 그림이다. GAPDH가 단백질 로딩량의 대조군으로 사용되었다.
도6b는 Ramos 세포 결합 친화도 분석의 유세포 분석 결과를 항체 용량((0, 0.7. 2.1. 7. 21. 70. 210nM)을 따라 나타낸 것이다.
도6c는 Ramos 세포를 7 nM 항체로 처리한 경우의 항체 의존성 DCD의 결과를 각 항체별로 정량화한 그림이다.
도6d는 Ramos 세포를 이용한 ADCC의 결과를 각 항체별로 정량화한 그림이다.
도7a는 빈 벡터(EV) 및 TNFR1을 안정적으로 과발현하는 Raji 세포에서 TNFR1 발현을 관측하기 위해서 항-TNFR1 항체를 사용한 면역블롯팅의 결과를 도시한 그림이다. CD20을 사용하여 TNFR1 과발현에 의한 CD20 발현의 변화가 없음을 확인하였고, GAPDH는 단백질 로딩량이 대조군으로 사용되었다.
도7b는 EV 및 TNFR1을 안정적으로 과발현하는 Raji 세포에서 막 TNFR1 발현의 유세포 분석 히스토그램으로, 마우스 항-TNFR1 항체 및 FITC 접합된 염소 항-마우스 IgG 항체를 사용하였다.
도7c는 EV 및 TNFR을 안정적으로 과발현하는 Raji 세포를 사용한 항체 의존성 DCD의 결과를 각 항체별로 정량화한 그림이다.
도7d는 Ramos 세포를 이용한 ADCC의 결과를 각 항체별로 정량화한 그림이다.Figure 1a is a picture showing the results of immunoblotting using an anti-TNFR1 antibody to confirm TNFR1 expression in Raji and Ramos B lymphoma cell lines.
1B is a flow cytometric histogram of membrane TNFR1 expression in Ramos cell line using a mouse anti-TNFR1 antibody and a FITC conjugated goat anti-mouse IgG antibody.
Figure 1c is a picture showing the immunofluorescence results of CD20 and TNFR1 in Ramos cells after treatment with obinutuzumab. This is the result of pre-treatment with obinutuzumab for 10 minutes, fixation, and treatment with mouse anti-TNFR1 antibody. Obinutuzumab was labeled with FITC-conjugated anti-human IgG secondary antibody (green), TNFR1 was labeled with Alexa680-conjugated anti-mouse IgG secondary antibody (red) and merged images show colocalization (yellow) . Scale bar is 5 μm.
Figure 2a is a picture showing the antibody design with anti-CD20 / anti-TNFR1 bispecific.
Figure 2b is a picture showing the Obi x TNFα WT fusion antibody, two blue oval figures at the bottom represent soluble TNFα WT, and the line connecting the antibody and TNFα WT represents the GSGSG linker.
Figure 2c is a picture showing the Obi x TNF mutant fusion antibody, where two orange oval figures represent a soluble TNFα mutant, the middle line represents a GSGSG linker, and the red box represents the amino acid mutation of the mutated part. It is a picture.
Figure 3a is a picture showing the results of verifying the integrity of each antibody using SDS-PAGE under reducing and non-reducing conditions. Total antibody was quantified with a NanoDrop™ Lite Spectrophotometer, and samples were separated on a 10% SDS-PAGE gel and stained with Coomassie blue.
Figure 3b is a diagram showing a flow cytometry analysis histogram showing the binding affinity of the antibody to CD20.
Figure 3c is a diagram showing a flow cytometry analysis histogram showing the binding affinity of the antibody to TNFR1.
Figure 4a is a diagram schematically showing the process of antibody-induced TNFα signaling activation assay. HEK293 reporter cells stably express the NF-κB promoter-dependent GFP gene, and when NF-κB signaling is induced by TNFα or an antibody, GFP is expressed in the cytoplasm and emits fluorescence, through which TNFα signaling The intensity of can be visualized by GFP fluorescence.
Figure 4b is a representative image showing GFP fluorescence in HEK293 reporter cells induced by TNFα and antibodies, and TNFα was used as a positive condition.
Figure 4c is a graph showing the normalized GFP fluorescence intensity according to the concentration for each antibody.
Figure 5a is a diagram schematically showing the process of antibody-mediated TNFα signaling blocking assay. When the antibody binds to HEK293 reporter cells stably expressing the NF-κB promoter-dependent GFP gene and the signal is blocked, fluorescence is not displayed.
Figure 5b is a representative image showing GFP fluorescence in HEK293 reporter cells blocked by antibody, Enbrel was used as a positive condition.
5C is a graph showing the normalized GFP fluorescence intensity for each antibody.
Figure 6a is a picture showing the immunoblotting results of TNFR1 expression in Raji and Ramos B lymphoma cell lines. GAPDH was used as a control for protein loading.
Figure 6b shows the results of flow cytometry analysis of Ramos cell binding affinity according to the antibody dose ((0, 0.7. 2.1. 7. 21. 70. 210 nM).
Figure 6c is a picture quantifying the results of antibody-dependent DCD for each antibody when Ramos cells were treated with 7 nM antibody.
Figure 6d is a picture quantifying the results of ADCC using Ramos cells for each antibody.
Figure 7a is a picture showing the results of immunoblotting using an anti-TNFR1 antibody to observe TNFR1 expression in Raji cells stably overexpressing empty vector (EV) and TNFR1. Using CD20, it was confirmed that there was no change in CD20 expression due to TNFR1 overexpression, and the protein loading amount of GAPDH was used as a control.
7B is a flow cytometry histogram of membrane TNFR1 expression in Raji cells stably overexpressing EV and TNFR1 using a mouse anti-TNFR1 antibody and a FITC conjugated goat anti-mouse IgG antibody.
Figure 7c is a picture quantifying the results of antibody-dependent DCD for each antibody using Raji cells stably overexpressing EV and TNFR.
Figure 7d is a picture quantifying the results of ADCC using Ramos cells for each antibody.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .
실시예Example
실험방법 및 분석방법Experiment method and analysis method
세포주, 시약 및 용액Cell lines, reagents and solutions
Human Ramos, HEK293T 및 HeLa 세포주는 한국세포주은행에서 구입하였다. Raji 세포주는 김성환 교수(충남 국립 대학교, 대전)에게 제공받았다. Ramos와 Raji 세포들은 37℃, 5% CO₂의 10% FCS 및 1% 페니실린/스트렙토마이신이 포함된 RPMI1640 배지에서 배양 및 유지되었다. HEK293T 및 HeLA 세포들은 37℃, 5% CO₂의 10% FCS 및 1% 페니실린/스트렙토마이신이 포함된 고-포도당 DMEM 배지에서 배양 및 유지되었다. Human Ramos, HEK293T and HeLa cell lines were purchased from Korea Cell Line Bank. The Raji cell line was provided by Professor Seonghwan Kim (Chungnam National University, Daejeon). Ramos and Raji cells were cultured and maintained in RPMI1640 medium supplemented with 10% FCS and 1% penicillin/streptomycin at 37°C and 5% CO2. HEK293T and HeLA cells were cultured and maintained in high-glucose DMEM medium containing 10% FCS and 1% penicillin/streptomycin at 37°C and 5% CO2.
세포를 배양하기 위하여, DMEM 배양 배지(Dulbecco’s modified Eagle medium, 11995-065); RPMI 1640 (Roswell Park Memorial Institute medium, 11875-093); 소태아혈청 (FBS) (26140-079); 페니실린-스트렙토마이신 (15140-122, Gibco, Life technologiesTM,Carlsbad,CA,USA); 및 Trypsin-EDTA 0.05% solution (25300-062, Gibco, LifetechnologiesTM, Carlsbad, CA, USA)이 사용되었다.To culture the cells, DMEM culture medium (Dulbecco's modified Eagle medium, 11995-065); RPMI 1640 (Roswell Park Memorial Institute medium, 11875-093); fetal bovine serum (FBS) (26140-079); penicillin-streptomycin (15140-122, Gibco, Life technologies™, Carlsbad, CA, USA); and Trypsin-EDTA 0.05% solution (25300-062, Gibco, LifetechnologiesTM, Carlsbad, CA, USA) were used.
면역블롯팅을 수행하기 위하여, NaCl (S7653), Triton X-100 (T8787), Glycerol (G5516, Sigma Aldrich); EDTA (15694, Usb, USB Corporation, Cleveland, OH, USA); Tris Ultrapure (T1501, Duchefa, Haarlem, The Netherlands), Complete proteinase inhibitors (Roche Applied Science, Mannheim, Germany), HCl (084-05425), NaOH (196-05375, Wako, Osaka, Japan); BCA Protein Assay Kit (23227, PierceTM, ThermoScientific, Rockford, IL, USA), 5xsamplebuffer, pre-made 10%SDS-PAGEgels(KG7040)(KOMABiotech,Seoul,Korea)이 사용되었다.To perform immunoblotting, NaCl (S7653), Triton X-100 (T8787), Glycerol (G5516, Sigma Aldrich); EDTA (15694, Usb, USB Corporation, Cleveland, OH, USA); Tris Ultrapure (T1501, Duchefa, Haarlem, The Netherlands), Complete proteinase inhibitors (Roche Applied Science, Mannheim, Germany), HCl (084-05425), NaOH (196-05375, Wako, Osaka, Japan); BCA Protein Assay Kit (23227, PierceTM, ThermoScientific, Rockford, IL, USA), 5xsamplebuffer, pre-made 10% SDS-PAGEgels (KG7040) (KOMABiotech, Seoul, Korea) were used.
면역세포화학법을 수행하기 위하여 4% Paraformaldehyde (19943), BSA (10857, Affymetrix, Cleveland, Ohio, USA), Phosphate-buffered saline (PBS) (P5493, Sigma Aldrich) 이 사용되었다.For immunocytochemistry, 4% Paraformaldehyde (19943), BSA (10857, Affymetrix, Cleveland, Ohio, USA), and Phosphate-buffered saline (PBS) (P5493, Sigma Aldrich) were used.
항체antibody
사용된 항체들로는, 마우스 항-TNFR1 항체(sc-8436, Santa Cruz Biotechnology), 마우스 항-CD20 항체 (sc-393894, Santa Cruz Biotechnology), 항-GAPDH 항체 (sc-47724, Santa Cruz Biotechnology), 겨자무과산화효소(horseradish peroxidase, HRP) 접합 염소 항-마우스 항체(115-035-003, Jackson Laboratories, PA, USA), FITC 접합 염소 항-인간 IgG 항체 (109-095-003, Jackson Laboratories), Alexa Fluor-647 접합 염소 항-마우스 IgG 항체 (115-605-006, Jackson Laboratories), Alexa Fluor-680 접합 염소 항-마우스 IgG 항체 (115-625-146, Jackson Laboratories), 리툭시맙 (Mabthera, Roche, Basel, Switzerland)을 사용하였다.Antibodies used include mouse anti-TNFR1 antibody (sc-8436, Santa Cruz Biotechnology), mouse anti-CD20 antibody (sc-393894, Santa Cruz Biotechnology), anti-GAPDH antibody (sc-47724, Santa Cruz Biotechnology), mustard Horseradish peroxidase (HRP) conjugated goat anti-mouse antibody (115-035-003, Jackson Laboratories, PA, USA), FITC conjugated goat anti-human IgG antibody (109-095-003, Jackson Laboratories), Alexa Fluor-647 conjugated goat anti-mouse IgG antibody (115-605-006, Jackson Laboratories), Alexa Fluor-680 conjugated goat anti-mouse IgG antibody (115-625-146, Jackson Laboratories), Rituximab (Mabthera, Roche , Basel, Switzerland) was used.
오비누투주맙, 항-TNFR1 항체 (ATROSAB), 항-CD20 / TNFR1 이중특이성 항체, Obi x TNFα 야생형(WT) 및 돌연변이체(mutant) 융합 항체들이 있고, 본 발명자의 연구실에서 생산되었다.Obinutuzumab, anti-TNFR1 antibody (ATROSAB), anti-CD20 / TNFR1 bispecific antibody, Obi x TNFα wild type (WT) and mutant fusion antibodies, were produced in our laboratory.
면역블롯팅법 (Immunoblotting)Immunoblotting
세포는 차가운 PBS 용액으로 3회 세척되었고, 4℃에서 20분 동안 용해완충액(150 mM NaCl, 5 mM Na-EDTA, 10% glycerol, 20 mM Tris-HCl (pH 8.0), 0.5% triton X-100 및 proteinase inhibitor (Complete, Roche Applied Science))에서 용해되었다. 세포 분해물은 4 ℃에서 10분 동안 17,000 x g의 원심분리로 처리되었다. 원심분리 후 부유물만을 옮긴 다음, Bradford 정량법을 이용하여 총 단백질의 양을 측정하였다. 세포 분해물 샘플은 5x 샘플 완충액을 추가하여 95 ℃에서 5분동안 가열한 후, 10% 또는 SDS-PAGE 겔로 분해(resolve)한 후 니트로셀룰로오스 막으로 옮겼다. 니트로셀룰로오스 막으로 전기적으로 전달된 단백질 이외의 부분은 5% 무지방 우류를 포함하는 TBS-T로 실온에서 1시간 동안 차단되었다. 일차 항체, 마우스 항-TNFR1 항체 (sc-8436, Santa Cruz Biotechnology), 마우스 항-CD20 항체 (sc-393894, Santa Cruz Biotechnology), 2% BSA를 포함하는 TBS-T에서 희석된 항-GAPDH 항체 (sc-47724, Santa Cruz Biotechnology)를 추가하였고 4℃에서 밤새 배양되었다. Cells were washed three times with cold PBS solution and lysed in lysis buffer (150 mM NaCl, 5 mM Na-EDTA, 10% glycerol, 20 mM Tris-HCl (pH 8.0), 0.5% triton X-100 at 4°C for 20 minutes). and proteinase inhibitor (Complete, Roche Applied Science)). Cell lysates were subjected to centrifugation at 17,000 x g for 10 min at 4 °C. After centrifugation, only the suspension was transferred, and then the amount of total protein was measured using the Bradford quantification method. The cell lysate sample was heated at 95° C. for 5 minutes by adding 5x sample buffer, resolved with a 10% or SDS-PAGE gel, and then transferred to a nitrocellulose membrane. The portion other than proteins electrically transferred to the nitrocellulose membrane was blocked with TBS-T containing 5% fat-free broth for 1 hour at room temperature. Primary antibody, mouse anti-TNFR1 antibody (sc-8436, Santa Cruz Biotechnology), mouse anti-CD20 antibody (sc-393894, Santa Cruz Biotechnology), anti-GAPDH antibody diluted in TBS-T with 2% BSA ( sc-47724, Santa Cruz Biotechnology) was added and incubated overnight at 4°C.
그 후 막을 세척한 후 실온에서 1시간 동안 HRP-접합 염소 항-마우스 항체(115-035-003, JACKSON Lab, PA, USA)와 함께 배양하였다. Thereafter, the membrane was washed and incubated with an HRP-conjugated goat anti-mouse antibody (115-035-003, JACKSON Lab, PA, USA) for 1 hour at room temperature.
세척 후, 단백질 밴드는 Super Signal West Femto Luminol/enhancer solution (TC263618, Thermoscientific)로 시각화되었고, Chemi-luminescent Image Analyzer (LAS 4000 mini, Fujifilm)를 이용하여 디지털 방식으로 캡처하였다.After washing, protein bands were visualized with Super Signal West Femto Luminol/enhancer solution (TC263618, Thermoscientific) and digitally captured using a Chemi-luminescent Image Analyzer (LAS 4000 mini, Fujifilm).
막 단백질의 측정 Measurement of membrane proteins
Ramos 및 Raji 세포주는 100μl의 PBS에 도포되었고, 일차 항체, 마우스 항-TNFR1 항체(sc-8436, Santa Cruz Biotechnology) 및 인간 IgG 항체(I4506-10MG, Sigma)가 70 nM 투여되었으며, 4℃에서 30분 간 배양하였다.Ramos and Raji cell lines were plated in 100 μl of PBS, and 70 nM of primary antibody, mouse anti-TNFR1 antibody (sc-8436, Santa Cruz Biotechnology) and human IgG antibody (I4506-10MG, Sigma) were administered and incubated at 4°C for 30 days. Incubated for minutes.
이 후 차가운 PBS로 한 번 세척한 후, PBS에 1:500으로 희석한 후, FITC-접합 염소 항-인간 IgG Fc-특이적 이차 항체(109-095-003, Jackson Laboratories)를 추가하고 30분 동안 배양하였다. 세척 후, a total of 10,000 cells were counted by 유세포 분석 (FACS, BD Biosciences, Franklin Lakes, NJ, USA)으로 총 10,000개의 세포를 계수하였고, FlowJo 소프트웨어로 분석하였다.After washing once with cold PBS, after diluting 1:500 in PBS, FITC-conjugated goat anti-human IgG Fc-specific secondary antibody (109-095-003, Jackson Laboratories) was added and incubated for 30 minutes cultured for a while. After washing, a total of 10,000 cells were counted by flow cytometry (FACS, BD Biosciences, Franklin Lakes, NJ, USA) and analyzed with FlowJo software.
면역세포화학법 (Immunocytochemistry)Immunocytochemistry
Ramos 세포에 오비누투주맙을 처리하기 전이나 후에, 실온에서 10분 동안 PBS 중 4%의 파라포름알데히드를 이용하여 고정한 다음, PBS로 3회 세척하였다. 세포들은 실온에서 30분 동안 차단 용액(blocking solution : 5% horse serum, 1% BSA, 0.1% gelatin, 0.001% sodium azide in PBS pH7.4)과 함께 배양되었다.Before or after treatment with obinutuzumab, Ramos cells were fixed with 4% paraformaldehyde in PBS for 10 minutes at room temperature, and then washed with PBS three times. Cells were incubated with blocking solution (5% horse serum, 1% BSA, 0.1% gelatin, 0.001% sodium azide in PBS pH7.4) for 30 minutes at room temperature.
PBS로 2회 세척한 후, 세포들은 실온에서 1시간 동안 마우스 항-TNFR1 항체(sc-8436, Santa Cruz Biotechnology)와 함께 1% BSA에서 배양되었고, PBS로 3회 세척한 후, 실온 및 암실 조건에서 30분 동안 FITC-접합 염소 항-인간 IgG Fc-특이적 이차 항체 및 Alexa680-접합 염소 항-마우스 Fc 특이적 이차 항체와 함께 배양되었다.After washing twice with PBS, the cells were incubated in 1% BSA with mouse anti-TNFR1 antibody (sc-8436, Santa Cruz Biotechnology) for 1 hour at room temperature, washed three times with PBS, and then at room temperature and in the dark. FITC-conjugated goat anti-human IgG Fc-specific secondary antibody and Alexa680-conjugated goat anti-mouse Fc specific secondary antibody for 30 minutes.
세포들은 3회 세척되었고, 형광 마운팅 배지(fluorescent mounting medium, S3023, Dako, Glostrup, Denmark)에 마운팅한 후, 공초점 현미경(LSM 780 controlled with Zen software; Carl Zeiss, Jena, Germany)으로 시각화하였다.Cells were washed three times, mounted in fluorescent mounting medium (S3023, Dako, Glostrup, Denmark) and visualized with a confocal microscope (LSM 780 controlled with Zen software; Carl Zeiss, Jena, Germany).
플라스미드를 이용한 항체 복제 (Cloning of Antibodies Plasmid)Cloning of Antibodies Plasmid
항-TNFR1 항체 (ATROSAB)의 서열에 대해서는 #US8859739B2 특허를 참고하였다. 중쇄와 경쇄의 가변 영역은 Genscript의 DNA 합성을 통해 수행하였다. 경쇄는 pLVX 바이러스 벡터에 Xba1에 의하여 삽입되었고, 중쇄의 가변영역은 이미 중쇄 불변영역을 포함하는 pLVX 바이러스 벡터에 Xba1 및 Nhe1에 의하여 삽입되었다. 항-CD20 / TNFR1 이중특이성 항체 제작을 위하여, Q39K, K62E, H172A, F174G, T366S, L368A, Y407V를 포함하는 항-TNFR1 항체 중쇄 및 D1R, Q38D, L135Y, S176W를 포함하는 경쇄가 Genscript에서 합성되었다.For the sequence of the anti-TNFR1 antibody (ATROSAB), reference was made to patent #US8859739B2. The variable regions of the heavy and light chains were prepared through Genscript's DNA synthesis. The light chain was inserted into the pLVX viral vector by Xba1, and the heavy chain variable region was already inserted into the pLVX viral vector containing the heavy chain constant region by Xba1 and Nhe1. For anti-CD20 / TNFR1 bispecific antibody construction, anti-TNFR1 antibody heavy chains including Q39K, K62E, H172A, F174G, T366S, L368A, and Y407V and light chains including D1R, Q38D, L135Y, and S176W were synthesized in Genscript. .
오비누투주맙 부분에서는, 중쇄(Q39Y, T366W) 및 경쇄(G38R)의 돌연변이를 국부 돌연변이화(site-directed mutagenesis)를 통하여 생성하였다. 돌연변이화를 위한 프라이머는 PrimerX 프로그램을 이용하여 결정하였다.In the obinutuzumab portion, mutations in the heavy chain (Q39Y, T366W) and light chain (G38R) were generated through site-directed mutagenesis. Primers for mutagenesis were determined using the PrimerX program.
중쇄의 Q39Y 돌연변이는 프라이머 TTGTCCAGGGGCGTACCGCACCCA(서열번호 8) 및AACTGGGTGCGGTACGCCCCTGGA(서열번호 9)가 이용되었고, 중쇄의 T366W 돌연변이는, 프라이머 GAAGCCTTTGACCAGGCACCACAG(서열번호 10) 및 CAAGAACCAGGTCAGCCTGTGGTG(서열번호 11)가 이용되었으며, 경쇄의 G38R 돌연변이는, 프라이머 CTGCCCTGGCTTTCTCAGGTACCA(서열번호 12) 및 GTATTGGTACCTGAGAAAGCC AGG(서열번호 13)가 이용되었다. DNA 서열은 염기서열 분석을 통해 검증되었다.For the Q39Y mutation of the heavy chain, primers TTGTCCAGGGGCGTACCGCACCCA (SEQ ID NO: 8) and AACTGGGTGCGGTACGCCCCTGGA (SEQ ID NO: 9) were used, and for the T366W mutation of the heavy chain, primers GAAGCCTTTGACCAGGCACCACAG (SEQ ID NO: 10) and CAAGAACCAGGTCAGCCTGTGGTG (SEQ ID NO: 11) were used, and G38R of the light chain For mutation, primers CTGCCCTGGCTTTCTCAGGTACCA (SEQ ID NO: 12) and GTATTGGTACCTGAGAAAGCC AGG (SEQ ID NO: 13) were used. The DNA sequence was verified through sequencing.
Obi x TNF WT을 만들기 위하여, 2개의 삽입물(오비누투주맙 HC, GSGSG 링커 + 수용성 TNFα (aa77-233))이 BamH1을 통하여 연결되었고, Xba1 및 Not1을 통하여 pLVX-CIP 벡터 안으로 삽입되었다. GSGSG linker + sTNFα 는 연장 PCR을 통하여 제작하였다. 프라이머 GGATCCGGCAGCGGCGTCAGATCATCTTCTCGAAC(서열번호 14), GT TCGAGAAGATGATCTGACGCCGCTGCCGGATCCCG(서열번호 15), CGGGATCCGGCAGCGGCGGATCTGGTAGCGGCGTCAGATCATCTTCTCGAACC(서열번호 16), GGTTCGAGAAGATGATCTGACGCCGCTACCAGATCCGCCGCTGCCGGATCCCG(서열번호 17), CGGGATCCGGCAGCGGCGGATCTGGTAGCGGCGGGAGCGGGTCAGGCGTCAGATCATCTTCTCGAACC(서열번호 18), GGTTCGAGAAGATGATCTGACGCCTGACCCGCTCCCGCCGCTACCAGATCCGCCGCTGCCGGATCCCG(서열번호 19)가 사용되었다.To make Obi x TNF WT, two inserts (obinutuzumab HC, GSGSG linker + soluble TNFα (aa77-233)) were ligated via BamH1 and inserted via Xba1 and Not1 into the pLVX-CIP vector. GSGSG linker + sTNFα was prepared through extension PCR. Primers GGATCCGGCAGCGGCGTCAGATCATCTTCTCGAAC (SEQ ID NO: 14), GT TCGAGAAGATGATCTGACGCCGCTGCCGGATCCCG (SEQ ID NO: 15), CGGGATCCGGCAGCGGCGGATCTGGTAGC GGCGTCAGATCATCTTCTCGAACC (SEQ ID NO: 16), GGTTCGAGAAGATGATCTGACGCCGCTACCAGATCCGCCGCTGCCG GATCCCG (SEQ ID NO: 17), CGGGATCCGGCAGCGGCGGATCTGGTAGCGGCGGGAGCGGGTCAGGCGTCAGATCATCTTCTCGAACC (SEQ ID NO: 18), GGTTCGAGAAGATGATCTGACGCCTGACCCGCTCCCGCCGCTACCAGATCCGCCGCTGCCGGATCCCG (SEQ ID NO: 19) were used.
Obi x TNF 돌연변이체를 생산하기 위하여, sTNFα 돌연변이체 (A160S, V161T, S162T, Y163H, Q164N, T165Q)가 Obi x TNF WT 주형에서 국부 돌연변이화를 통하여 돌연변이를 발생시켰다. 프라이머는 AGCCGCATCAGCACCACCCACAACCAGAAGGTCAAC(서열번호 20) 및 GTTGACCTTCTGGTTGTGGGTGGTGCTGATGCGGCT(서열번호 21)가 사용되었다.To generate Obi x TNF mutants, sTNFα mutants (A160S, V161T, S162T, Y163H, Q164N, T165Q) were mutated through local mutagenesis in the Obi x TNF WT template. As primers, AGCCGCATCAGCACCACCCACAACCAGAAGGTCAAC (SEQ ID NO: 20) and GTTGACCTTCTGGTTGTGGGTGGTGCTGATGCGGCT (SEQ ID NO: 21) were used.
렌티바이러스 생산Lentivirus production
모든 단백질과 항체는 렌티바이러스 감염을 통하여 안정적으로 발현되었다. 렌티바이러스는 HEK293T 세포를 렌티 바이러스 발현 카세트와 패키징 플라스미드(pMD2.5G (12259, Addgene) 및 psPAX2 (12260, Addgene))를 공동감염시킴으로써 생산되었고, 폴리에틸렌이민(PEI, 23966-1, Polysciences, PA, USA)이 형질전환 시약으로 이용되었다. 형질감염 24시간 후 바이러스 상층액을 수집하였고, 1,000 xg에서 5분간 원심분리하고 0.45μm 필터로 여과하였다.All proteins and antibodies were stably expressed through lentiviral infection. Lentiviruses were produced by co-infecting HEK293T cells with lentiviral expression cassettes and packaging plasmids (pMD2.5G (12259, Addgene) and psPAX2 (12260, Addgene)) and polyethylenimine (PEI, 23966-1, Polysciences, PA; USA) was used as a transfection reagent. Viral supernatants were collected 24 hours after transfection, centrifuged at 1,000 xg for 5 minutes and filtered through a 0.45 μm filter.
복제, 렌티바이러스 생산 및 안정 세포주 Cloning, lentivirus production and stable cell lines
pCNS 벡터의 인간 MS4A1 및 TNFRSF1A cDNA는 한국 유전자 은행(Korean gen bank)에서 구입하였다. MS4A1 및 TNFRSF1A cDNA 전장은 Xba1를 사용하여 pLVX 렌티바이러스 벡터에 클로닝되었다. TNFRSF1A Δ CD(세포질 도메인 결여(aa233-455))는 Nhe1 및 Not1를 사용하여 pLVX 렌티바이러스 벡터에 클로닝되었다. 프라이머 CCCGCTAGCATGGGCCTCTCCACC(서열번호 22) 및GGGCGGCCGCTTAGTAGAGCTTGGACTTCCACC(서열번호 23)가 사용되었다. 렌티바이러스는 전술한 방법에 의하여 생산되었다. CD20 또는 TNFR1ΔCD을 안정적으로 과발현하는 HeLa 세포 및 빈 벡터(empty vector) 및 TNFR1을 안정적으로 과발현하는 Raij 세포를 생성하기 위하여, 다음과 같은 방법이 사용되었다:Human MS4A1 and TNFRSF1A cDNAs of the pCNS vector were purchased from Korean gen bank. The MS4A1 and TNFRSF1A cDNA full lengths were cloned into the pLVX lentiviral vector using Xba1. TNFRSF1A Δ CD (lacking the cytoplasmic domain (aa233-455)) was cloned into the pLVX lentiviral vector using Nhe1 and Not1. Primers CCCGCTAGCATGGGCCTCTCCACC (SEQ ID NO: 22) and GGGCGGCCGCTTAGTAGAGCTTGGACTTCCACC (SEQ ID NO: 23) were used. Lentivirus was produced by the method described above. To generate HeLa cells stably overexpressing CD20 or TNFR1ΔCD and Raij cells stably overexpressing TNFR1 with an empty vector, the following method was used:
HeLa 및 Raji 세포는 10μg/ml 폴리브렌(TR-1003-G, Sigma Aldrich)이 포함된 12웰 플레이트에 플레이팅되었고, 바이러스 함유 배지와 함께 24시간 동안 배양하였으며, 바이러스를 제거한 후 세포들은 1㎍/ml의 퓨로마이신(ant-pr-1, Invivogen)을 함유하는 신선한 배지로 48시간 동안 교체하였다. 단백질 발현은 유세포분석 및 면역블롯팅으로 확인하였다.HeLa and Raji cells were plated in 12-well plates containing 10 μg/ml polybrene (TR-1003-G, Sigma Aldrich) and cultured for 24 hours with virus-containing medium. After removing the virus, cells were plated with 1 μg /ml of puromycin (ant-pr-1, Invivogen) was replaced for 48 hours with fresh medium. Protein expression was confirmed by flow cytometry and immunoblotting.
항체 생산 CHO 세포(CHO cells)의 생성Generation of antibody-producing CHO cells
CHO-K1 세포를 12 웰 플레이트에 웰 당 5 x 104 세포의 비율로 플레이팅하였고, 24시간 동안 배양하였다.CHO-K1 cells were plated in a 12-well plate at a ratio of 5 x 10 4 cells per well and cultured for 24 hours.
오비누투주맙을 생성하기 위하여 항-TNFR1 항체; Obi x TNF 야생형 및 돌연변이체 융합 항체; 및 세포를, 항체 HC; LC를 포함하는 바이러스와 3:2의 비율로 함께 배양하였고, 이러한 배양은 10 μg/ml 폴리브렌 (TR-1003-G, Sigma Aldrich)과 함께 24시간 동안 이루어졌다. 바이러스를 제거한 후에, 10 ㎍/ml 퓨로마이신(ant-pr-1, Invivogen), 20 ㎍/ml 블라스티시딘 S(ant-bl-05, Invivogen)를 함유하는 신선한 배지에서 72시간 동안 배양하여 세포들을 선별하였다.anti-TNFR1 antibody to generate obinutuzumab; Obi x TNF wild type and mutant fusion antibodies; and cells, antibody HC; It was cultured with LC-containing virus at a ratio of 3:2, and this incubation was performed for 24 hours with 10 μg/ml polybrene (TR-1003-G, Sigma Aldrich). After removing the virus, it was cultured for 72 hours in a fresh medium containing 10 μg/ml puromycin (ant-pr-1, Invivogen) and 20 μg/ml blasticidin S (ant-bl-05, Invivogen). Cells were selected.
항-CD20 / TNFR1 이중특이성 항체를 생성하기 위하여, 세포들은 오비누투주맙 HC 돌연변이체, 오비누투주맙 LC 돌연변이체, 항-TNFR1 HC 돌연변이체, 항-TNFR1 LC 돌연변이체 벡터를 각각 3:2:3:2의 비율로 포함하는 바이러스와 배양하였고, 이러한 배양은 10 μg/ml 폴리브렌 (TR-1003-G, Sigma Aldrich)과 함께 24시간 동안 이루어졌다. 바이러스를 제한 후에, 10 ㎍/ml 퓨로마이신(ant-pr-1, Invivogen), 20 ㎍/ml 블라스티시딘 S(ant-bl-05, Invivogen), 200 μg/ml 하이그로마이신 B (H0192,Duchefa Biochemie), 400 μg/ml G418 (ant-gn-1, Invivogen)을 포함하는 신선한 배지에서 72동안 배양하여 세포들을 선별하였다. 선별 후에, 나머지 세포를 증식시켜 충분히 안정한 세포를 확보하였다. To generate the anti-CD20/TNFR1 bispecific antibody, cells were transfected with obinutuzumab HC mutant, obinutuzumab LC mutant, anti-TNFR1 HC mutant, and anti-TNFR1 LC mutant vectors at a ratio of 3:2, respectively. :3:2 ratio of the containing virus, and this incubation was done with 10 μg/ml polybrene (TR-1003-G, Sigma Aldrich) for 24 hours. After virus restriction, 10 μg/ml puromycin (ant-pr-1, Invivogen), 20 μg/ml blasticidin S (ant-bl-05, Invivogen), 200 μg/ml hygromycin B (H0192 , Duchefa Biochemie) and 400 μg/ml G418 (ant-gn-1, Invivogen) were cultured for 72 days in a fresh medium to select cells. After selection, the remaining cells were propagated to ensure sufficiently stable cells.
항체의 생산과 정제Antibody production and purification
10% FBS 및 1% 페니실린/스트렙토마이신을 함유하는 RPMI에서 80% 컨플루언스까지 성장한 항체생성세포를 PBS로 2회 세척하고; 8 nM L-글루타민 (25030081, Gibco, Life technologiesTM) 및 1 mM 부티르산나트륨 (LS 033-01, WELGENE, Daegu, Korea)을 포함하는 CD CHO 배지(10743029, Gibco, Life technologiesTM,Carlsbad,CA,USA)로 교체하였다. 항체를 포함하는 조건 배지는 5% CO2 / 95% 공기에서 30℃에서 2주 동안 추가 배양하여 수득하였다. 항체는 Pierce 단백질 A 아가로스(20333, Thermofisher)를 사용하여 친화성 크로마토그래피를 통해 정제되었다. 항체를 함유하는 배지를 단백질 A 아가로스 비드와 함께 4℃에서 24시간 동안 배양하였다. 원심분리로 상층액을 제거하고 세척 완충액(0.1M NaPO4, 0.15M NaCl, pH 7.4)으로 3회 세척하였다. 항체는 용출 완충액(0.2 M glycine, pH 3)으로 6개의 분획으로 용출하였다. 용출된 항체의 투석은 4℃에서 6시간 동안 2L PBS에서 총 2회 수행되었고 Amicon® Ultra-15 Centrifugal Filter Units(UFC900324, Merck, 3K)을 이용하여 농축하였다. 총 항체는 NanoDrop™ Lite Spectrophotometer로 정량하였고, 정량 후에 1μg 항체를 5x 램나이 샘플 완충액과 함께 첨가하고 Pre-cast 10% SDS-PAGE 젤로 분리한 뒤 쿠마시블루로 염색하였다.Antibody-producing cells grown to 80% confluence in RPMI containing 10% FBS and 1% penicillin/streptomycin were washed twice with PBS; CD CHO medium (10743029, Gibco, Life technologies™, Carlsbad, CA, USA) containing 8 nM L-glutamine (25030081, Gibco, Life technologies™) and 1 mM sodium butyrate (LS 033-01, WELGENE, Daegu, Korea) was replaced with A conditioned medium containing the antibody was obtained by further incubation for 2 weeks at 30° C. in 5% CO 2 /95% air. Antibodies were purified via affinity chromatography using Pierce protein A agarose (20333, Thermofisher). Medium containing the antibody was incubated with Protein A agarose beads at 4°C for 24 hours. The supernatant was removed by centrifugation and washed three times with washing buffer (0.1M NaPO4, 0.15M NaCl, pH 7.4). The antibody was eluted in 6 fractions with an elution buffer (0.2 M glycine, pH 3). Dialysis of the eluted antibody was performed twice in 2L PBS for 6 hours at 4°C and concentrated using Amicon® Ultra-15 Centrifugal Filter Units (UFC900324, Merck, 3K). Total antibody was quantified with a NanoDrop™ Lite Spectrophotometer, and after quantification, 1 μg of antibody was added with 5x Lamnai sample buffer, separated on a pre-cast 10% SDS-PAGE gel, and then stained with Coomassie blue.
항체 결합 친화도 어세이(Antibodies Binding affinity assay)Antibodies Binding affinity assay
Ramos 및 HeLa-CD20, TNFR1 ΔCD 세포를 PBS 100μl에 재현탁하고, 항체의 처리량을 늘리거나, 70nM으로 처리하고 4℃에서 30분 동안 배양하였다. PBS로 2회 헹군 후, 세포를 1:500 희석된 FITC-접합 항-인간 Ig Fc-특이적 이차 항체(109-095-003, Jackson Laboratories)로 처리하고 4℃에서 30분 동안 배양하였다. PBS로 두 번 세척한 후, 총 10,000개의 세포를 세포를 유세포분석기(FACS, BD Biosciences, Franklin Lakes, NJ, USA)로 계수하고 FlowJo 소프트웨어로 분석하였다.Ramos and HeLa-CD20, TNFR1 ΔCD cells were resuspended in 100 μl of PBS, treated with increasing antibody throughput or 70 nM and incubated at 4° C. for 30 minutes. After rinsing twice with PBS, cells were treated with a 1:500 dilution of FITC-conjugated anti-human Ig Fc-specific secondary antibody (109-095-003, Jackson Laboratories) and incubated at 4° C. for 30 minutes. After washing twice with PBS, a total of 10,000 cells were counted by flow cytometry (FACS, BD Biosciences, Franklin Lakes, NJ, USA) and analyzed with FlowJo software.
TNFR1 신호 리포터 어세이(TNFR1 signaling reporter assay)TNFR1 signaling reporter assay
NF-κB 프로모터 의존적 GFP 리포터 HEK293 세포는 이진우 교수(연세대학교 약학대학)로부터 제공받았다. 안정한 HEK 293 세포를 1회 세척한 후, 48웰 플레이트에 웰당 1 x 105개 세포 비율로 도포한 후, 24시간 동안 배양하였다. 항체-유도 NF-κB 신호전달 활성화 분석에서, 세포들은 지시된 용량(0, 0.1, 0.3, 1, 3, 10 nM)의 항체와 함께 24시간 동안 배양되었다. 항체 의존적 NF-κB 신호전달 차단 분석에서 세포는 분석 30분 전에 지시된 용량의 항체(70nM)로 전처리 되었고, 그 다음 24시간 동안 37℃의 CO2 인큐베이터에서 20ng/ml TNFα와 함께 배양되었다. 그 후 세포를 PBS로 1회 세척한 후 Versene 용액(15040066, Gibco Life TechnologiesTM)에 흡착시켰다. 그 후 세포를 96웰 불투명 플레이트(opaque plate)에 플레이팅하고 원심분리한 뒤 PBS로 2회 세척하였다. 웰당 GFP의 형광 강도는 488 nm/520 nm의 여기/방출 파장을 마이크로플레이트 판독기(Flexstation 3, Molecular Devices)통하여 관측하여 측정하였다. 정규화를 위해 세포를 Triton X 2%로 용해하고 PI 염색으로 염색하였다. 플레이트를 진탕 플레이트(shake plate) 상에서 배양하였고, 518 nm/620 nm의 여기/방출을 관측하는 마이크로플레이트 판독기(Flexstation 3, Molecular Devices)를 사용하여 웰당 PI의 형광 강도를 측정하였다.NF-κB promoter-dependent GFP reporter HEK293 cells were provided by Professor Jinwoo Lee (College of Pharmacy, Yonsei University). After washing the stable HEK 293 cells once, they were spread on a 48-well plate at a rate of 1×10 5 cells per well and cultured for 24 hours. In the antibody-induced NF-κB signaling activation assay, cells were incubated for 24 h with the indicated doses (0, 0.1, 0.3, 1, 3, 10 nM) of antibody. In the antibody-dependent NF-κB signaling blockade assay, cells were pre-treated with the indicated dose of antibody (70 nM) 30 min before assay, then incubated with 20 ng/ml TNFα in a CO 2 incubator at 37 °C for 24 h. Then, the cells were washed once with PBS and adsorbed to Versene solution (15040066, Gibco Life TechnologiesTM). Thereafter, the cells were plated in a 96-well opaque plate, centrifuged, and washed twice with PBS. The fluorescence intensity of GFP per well was measured by observing excitation/emission wavelengths of 488 nm/520 nm through a microplate reader (Flexstation 3, Molecular Devices). For normalization, cells were lysed with Triton X 2% and stained with PI stain. The plate was incubated on a shake plate and the fluorescence intensity of PI per well was measured using a microplate reader (Flexstation 3, Molecular Devices) observing excitation/emission of 518 nm/620 nm.
항체-유도 직접 세포사 어세이(Antibody induced direct cell death assay)Antibody induced direct cell death assay
직접세포사멸(DCD)를 측정하기 위하여, 1 x 105 세포/웰 비율로 세포를 100μl의 RPMI 전체 배지에 재현탁한 다음 지시된 용량(7, 21 및 70nM)의 항체와 함께 6시간 동안 배양하였다. 그 다음, 세포를 37℃에서 20분 동안 PI(Propidium iodide)로 염색하였다. PI의 형광을 판독값으로 사용하고 FACS Verse(FACS, BD Biosciences, Franklin Lakes, NJ, USA) 및 Flow Jo 소프트웨어를 이용하여 세포 용해 %(10,000개의 계산된 총 세포 중 형광 손실 세포 수의 %)를 계산하였다.To measure direct apoptosis (DCD), cells were resuspended in 100 μl of RPMI complete medium at a ratio of 1 x 10 5 cells/well and incubated for 6 hours with the indicated doses (7, 21 and 70 nM) of antibodies. . Then, the cells were stained with PI (Propidium iodide) for 20 minutes at 37°C. The fluorescence of PI was used as a readout and % cell lysis (% of cells losing fluorescence out of 10,000 counted total cells) was calculated using FACS Verse (FACS, BD Biosciences, Franklin Lakes, NJ, USA) and Flow Jo software. Calculated.
말초 혈액 단핵세포 분리 (PBMC isolation)Peripheral blood mononuclear cell isolation (PBMC isolation)
건강한 기증자의 혈액을 PBS와 1:1의 비율로 혼합하고, 이를 피콜(ficoll, HISTOPAQUE-1077, Sigma, 10771)이 미리 놓여진 튜브에 층을 쌓으면서 올렸다. 백혈구와 적혈구를 25℃에서 400 x g, 30분간 원심분리로 분리한 후, 백혈구만 채취하여 새 튜브로 옮겼다. PBS로 세척한 후, 상온에서 300 xg로 10분간 원심분리하여 상층액을 제거하였다. 이러한 세척과정을 두 번 반복하여 혈소판을 완전히 제거하였다. 그 다음, PBMC를 계수하고 사용하기 위해서 RPMI 전체 배지에 재현탁하였다.Blood from a healthy donor was mixed with PBS at a ratio of 1:1, and the blood was layered in a tube pre-loaded with ficoll (HISTOPAQUE-1077, Sigma, 10771). After separating white blood cells and red blood cells by centrifugation at 25° C. at 400 x g for 30 minutes, only white blood cells were collected and transferred to a new tube. After washing with PBS, the supernatant was removed by centrifugation at room temperature at 300 xg for 10 minutes. This washing process was repeated twice to completely remove platelets. PBMCs were then resuspended in RPMI complete medium for enumeration and use.
항체 의존성 세포독성 어세이 (Antibody dependent cellular cytotoxicity assay)Antibody dependent cellular cytotoxicity assay
항체 의존성 세포독성 어세이를 위하여 Ramos 및 Raji 세포를 PBS로 세척하고 0.25μM 칼세인-AM으로 염색하였다. 세포를 30분 동안 배양하고, PBS로 2회 세척하였다. 정제된 말초 혈액 단핵 세포(PBMC, 이펙터: 표적= 5:1)를 첨가하고, 지시된 용량(7, 70 nM)의 항체로 처리한 다음, 4시간 동안 CO2에서 37℃로 배양하였다. 60,000개의 계수된 총 PBMC 세포 중 살아있는 세포의 %는 FACS Verse(FACS, BD Biosciences, Franklin Lakes, NJ, USA) 및 FlowJo 소프트웨어에 의해 계산되었다. 정규화는 ADCC %= 100 - (살아 있는 세포의 % / 표적 세포 집단의 %) *100를 통하여 이루어졌다.For antibody-dependent cytotoxicity assays, Ramos and Raji cells were washed with PBS and stained with 0.25 μM calcein-AM. Cells were incubated for 30 minutes and washed twice with PBS. Purified peripheral blood mononuclear cells (PBMC, effector: target = 5:1) were added, treated with the indicated doses (7, 70 nM) of antibody, and then incubated at 37° C. in CO 2 for 4 hours. The percentage of viable cells out of 60,000 counted total PBMC cells was calculated by FACS Verse (FACS, BD Biosciences, Franklin Lakes, NJ, USA) and FlowJo software. Normalization was done via ADCC %= 100 - (% of live cells/% of target cell population) *100.
통계 분석statistical analysis
데이터는 평균±평균의 표준오차(표준편차)로 표시되었다. 스튜던트 t-검정으로 통계 분석을 수행한 후, 적절하게 GraphPad Prism 소프트웨어 패키지(버전 9.0)를 사용하여 Tukey 다중 비교를 수행하였다. P<0.05 인 경우는 통계적으로 유의한 것으로 간주하였다.Data are expressed as mean ± standard error of the mean (standard deviation). Statistical analysis was performed with Student's t-test followed by Tukey multiple comparisons using the GraphPad Prism software package (version 9.0) as appropriate. A case of P<0.05 was considered statistically significant.
실험결과Experiment result
오비누투주맙 처리에 의한 CD20과 TNFR1의 공동국소화(colocalization)를 확인하였다.Colocalization of CD20 and TNFR1 by obinutuzumab treatment was confirmed.
TNFR1은 B 세포 림프종에서 오비누투주맙에 의해 유도된 DCD에 관여하는 것으로 알려져 있다. 발명자들은 면역블롯팅을 통해 Ramos 및 Raji B 림프종 세포에서 TNFR1 발현을 확인하였다(도 1a). 그 결과, TNFR1은 Raji 세포가 아닌 Ramos에서 발현됨을 확인하였고, 이에 따라 발명자들은 Ramos 세포를 이용하여 TNFR1의 막 발현을 분석하였다(도 1b). 그 결과, Ramos 세포의 막에서 TNFR1도 발현되는 것을 확인하였다.TNFR1 is known to be involved in DCD induced by obinutuzumab in B-cell lymphoma. The inventors confirmed TNFR1 expression in Ramos and Raji B lymphoma cells through immunoblotting (FIG. 1a). As a result, it was confirmed that TNFR1 was expressed in Ramos, not Raji cells, and accordingly, the inventors analyzed the membrane expression of TNFR1 using Ramos cells (FIG. 1b). As a result, it was confirmed that TNFR1 was also expressed in the membrane of Ramos cells.
그 다음, 발명자들은 세포 고정 전·후에 오비누투주맙 처리에 의한 CD20 및 TNFR1 위치를 비교하였다(도 1c). 고정 후에 세포를 오비누투주맙으로 처리한 경우, CD20과 TNFR1은 덜 공동 국소화(co-localization)되었다. 이에 비해 세포를 오비누투주맙으로 처리하고 10분 동안 배양한 후 고정하였을 때는 세포는 더욱 공동 국소화되었다. 이러한 결과는 오비누투주맙이 CD20에 결합할 때 B 세포 림프종 막에 변화가 있음을 의미하고, 실험을 통하여 CD20과 TNFR1이 가깝게 위치하게 됨을 확인하였다. 해당 결과를 바탕으로, 본 발명자들은 항-CD20/TNFR1 이중특이성 및 융합항체 개발을 결정하였다.Then, the inventors compared CD20 and TNFR1 locations by obinutuzumab treatment before and after cell fixation (FIG. 1c). When cells were treated with obinutuzumab after fixation, CD20 and TNFR1 were less co-localized. In comparison, cells were more colocalized when cells were treated with obinutuzumab, incubated for 10 minutes and then fixed. These results indicate that there is a change in the B cell lymphoma membrane when obinutuzumab binds to CD20, and it was confirmed through the experiment that CD20 and TNFR1 are located close to each other. Based on these results, the present inventors decided to develop anti-CD20/TNFR1 bispecific and fusion antibodies.
항-CD20 / TNFR1 이중특이성 항체 및 융합 항체을 생산하였다.Anti-CD20 / TNFR1 bispecific antibodies and fusion antibodies were produced.
본 발명의 발명자들은, 항CD20/TNFR1 이중특이성 항체, 오비누투주맙 x soluble TNFα 야생형 융합항체(Obi x TNF WT) 및 오비누투주맙 x soluble TNFα 돌연변이체 융합항체(Obi x TNF Mutant)를 개발하였다(도 2a, 2b, 2c). 이중특이성 항체는 항체 이종이량체화(heterodimerization)가 될 Fc 영역에 ‘knobs into hole’ 돌연변이를 포함시켜 제작되었다. 오비누투주맙 Fc영역의 Knobs 돌연변이는 T366W를 포함하였고, 항-TNFR1 Fc의 holes 돌연변이는 T366S, L368A, Y407V를 포함하였다. 또한, 항체 Fab 영역은 각 항체의 중쇄 및 경쇄를 정확하게 쌍으로 연결하는 '직교 Fab 인터페이스' 돌연변이를 포함하였다(도 2a). 오비누투주맙 중쇄 Fab는 Q39Y를, 경쇄는 G38R을 포함하였고; 항-TNFR1 항체 중쇄 Fab는 Q39K, K62E, H172A, F174G을, 경쇄는 D1R, Q38D, L135Y, S176W를 포함하였다. 수용성 TNFα 야생형 및 TNFR1을 활성화시키는 기능을 상실한 TNFα 돌연변이체는 오비누투주맙 중쇄의 C-말단에 GSGSG 링커를 통하여 접합되었다(도 2b, 도 2c). The inventors of the present invention developed an anti-CD20/TNFR1 bispecific antibody, obinutuzumab x soluble TNFα wild type fusion antibody (Obi x TNF WT) and obinutuzumab x soluble TNFα mutant fusion antibody (Obi x TNF Mutant) (Fig. 2a, 2b, 2c). Bispecific antibodies were constructed by including 'knobs into hole' mutations in the Fc region that will result in antibody heterodimerization. Knobs mutations in the Fc region of obinutuzumab included T366W, and holes mutations in the anti-TNFR1 Fc included T366S, L368A, and Y407V. In addition, the antibody Fab region contained an 'orthogonal Fab interface' mutation that correctly paired the heavy and light chains of each antibody (Fig. 2a). The obinutuzumab heavy chain Fab contained Q39Y and the light chain contained G38R; The anti-TNFR1 antibody heavy chain Fab included Q39K, K62E, H172A, F174G, and the light chain included D1R, Q38D, L135Y, S176W. Soluble TNFα wild-type and TNFα mutants that lost their ability to activate TNFR1 were conjugated to the C-terminus of the heavy chain of obinutuzumab via a GSGSG linker (FIGS. 2b, 2c).
본 발명자들은 SDS-PAGE을 이용하여 상기 항체들이 의도대로 제작되었는지 검증하였다. 비환원 조건에서 이중특이성 항체는, 오비누투주맙과 항-TNFR1 항체의 중간 정도의 크기를 가지는 것으로 결과가 나왔다(도 3a). 환원조건에서 Obi x TNF WT 및 Obi x TNF Mutant 중쇄는 오비누투주맙보다 거의 15 kDa 더 높게 위치하였다. 두 표적에 대한 결합을 정확하게 검증하기 위하여 발명자들은 CD20 또는 TNFR1ΔCD가 과발현된 HeLa 안정 세포주를 사용하여 이러한 항체들의 친화도를 테스트하였다. 모든 항체는 오비누투주맙만큼 HeLa-CD20 안정 세포에 결합했지만, 이중특이성 항체는 CD20에 결합하는 Fab 영역이 하나만 남았음으로 인해 감소된 친화성을 보였다(도 3b). 이중특이성, Obi x TNF WT 및 Obi x TNF Mutant는 HeLa-TNFR1 ΔCD 안정 세포에 결합했지만, Obi x TNF WT만이 항-TNFR1 항체에 대해 동등한 친화성을 나타냈다(도 3c). 이러한 결과를 통하여, 항체가 제대로 생산되고 결합력이 유지되고 있음을 확인할 수 있었다.The present inventors verified whether the antibodies were produced as intended using SDS-PAGE. In non-reducing conditions, the bispecific antibody was found to have an intermediate size between obinutuzumab and anti-TNFR1 antibody (FIG. 3a). Under reducing conditions, the heavy chains of Obi x TNF WT and Obi x TNF Mutant were approximately 15 kDa higher than those of obinutuzumab. To accurately verify binding to the two targets, the inventors tested the affinity of these antibodies using HeLa stable cell lines overexpressing CD20 or TNFR1ΔCD. All antibodies bound HeLa-CD20 stable cells as well as obinutuzumab, but the bispecific antibody showed reduced affinity due to leaving only one Fab region that binds to CD20 (FIG. 3B). The bispecific, Obi x TNF WT and Obi x TNF Mutant bound to HeLa-TNFR1 ΔCD stable cells, but only Obi x TNF WT showed equivalent affinity to the anti-TNFR1 antibody (FIG. 3c). Through these results, it was confirmed that the antibody was properly produced and the binding force was maintained.
항-CD20 / TNFR1 이중특이성 항체(bispecific antibody)와 융합 항체(fusion antibody)의 NF-κB 연관 TNFα 신호 전달 활성화도의 비교하였다.Anti-CD20 / TNFR1 bispecific antibody and fusion antibody were compared for NF-κB-related TNFα signal transduction activation.
본 발명자들은 항-CD20/TNFR1 이중특이성 및 융합 항체의 NF-κB 매개 TNFα 신호전달 활성화 능력을 비교하기 위하여 NF-κB 프로모터 의존적 GFP 형광 HEK293 리포터 세포 시스템을 사용하였다. TNFα 신호전달의 강도는 NF-κB 프로모터에 반응하는 GFP 형광에 의해 시각화되었다(도 4a). TNFα가 양성조건으로 사용되었고, 다른 항체들과 결과를 비교하였다. 대표 이미지에서 TNFα는 Obi x TNF WT보다 GFP를 더 많이 형광을 나타내지만, 세포 수로 정규화하면 Obi x TNF WT가 TNFα보다 더 많이 형광을 활성화시킴을 알 수 있었다. 동일한 몰 농도에서 Obi x TNF WT는 2개의 TNFα를 가지기 때문에, 본 발명자들은 2배 양의 TNFα가 세포의 생존력에 영향을 미칠 수 있다고 추정하였다. Obi x TNF Mutant는 3 nM 이하에서 TNFα 신호전달을 활성화할 수 없었지만, 10 nM에서는 약간 활성화하였다. 다른 항체의 경우에는 10nM까지 TNFα 신호전달을 전혀 활성화하지 않았다(도 4b,도 4c). We used the NF-κB promoter dependent GFP fluorescent HEK293 reporter cell system to compare the ability of anti-CD20/TNFR1 bispecific and fusion antibodies to activate NF-κB mediated TNFα signaling. The intensity of TNFα signaling was visualized by GFP fluorescence in response to the NF-κB promoter (FIG. 4a). TNFα was used as a positive condition, and the results were compared with other antibodies. In the representative images, TNFα fluoresced more GFP than Obi x TNF WT, but when normalized to cell count, Obi x TNF WT showed more fluorescence activation than TNFα. Since Obi x TNF WT has two TNFα at the same molar concentration, we assumed that a double amount of TNFα could affect cell viability. Obi x TNF Mutant was unable to activate TNFα signaling at 3 nM or less, but slightly at 10 nM. Other antibodies did not activate TNFα signaling at all up to 10 nM (Fig. 4b, Fig. 4c).
TNFα에 의한 NF-κB 신호전달은 염증 반응 등과 관련된 많은 부작용이 있는 것으로 알려져 있기 때문에, 본 발명자들은 항체들이 TNFα에 의한 이 NF-κB 신호전달을 차단할 수 있는지도 확인하였다(도 5a). 이를 확인하기 위하여 항체로 전처리한 후, TNFα를 HEK 리포터 시스템에 추가하였다. 엔브렐(Enbrel)이 TNFα 신호를 차단하는 양성 조건에 사용되었으며, 엔브렐이 신호전달을 완전히 차단하는 것을 확인할 수 있었다. 발명자들은 항-TNFR1이 엔브렐만큼 TNFα 신호 전달을 차단할 것으로 예상했지만 엔브렐의 차단도의 절반만 차단함을 확인할 수 있었고, 이러한 결과는 해당 시스템이 TNFR2에 의하여 영향을 받을 수 있음을 시사함을 알 수 있었다.Since NF-κB signaling by TNFα is known to have many side effects related to inflammatory responses, the present inventors also confirmed that antibodies could block this NF-κB signaling by TNFα (FIG. 5a). To confirm this, after pretreatment with an antibody, TNFα was added to the HEK reporter system. Enbrel was used for positive conditions to block TNFα signaling, and it was confirmed that Enbrel completely blocked signal transduction. The inventors expected that anti-TNFR1 would block TNFα signaling as much as Enbrel, but it was confirmed that it blocked only half of Enbrel's blockage, suggesting that the system may be affected by TNFR2. there was.
다른 항체를 항-TNFR1과 비교한 경우, 이중특이성 항체는 항-TNFR1보다 덜 TNFα 신호전달을 차단했지만, Obi-TNF 돌연변이체는 항-TNFR1 항체와 비슷한 차단도를 보여주었다(도 5b, 5c). 그 외의 항체들은 TNFα 신호전달을 차단할 수 없는 것으로 나타났다. 이와 같은 결과를 종합하여, 본 발명자들은 이중특이항체와 Obi x TNF Mutant는 TNFα 신호전달을 거의 유도하지 않으면서 과도한 TNFα 효과를 차단할 수 있어, 부작용이 거의 없는 것을 확인할 수 있었다.When comparing other antibodies to anti-TNFR1, the bispecific antibody blocked TNFα signaling to a lesser extent than anti-TNFR1, but the Obi-TNF mutant showed comparable blocking to the anti-TNFR1 antibody (FIGS. 5B, 5C). . Other antibodies have been shown to be unable to block TNFα signaling. Taking these results together, the present inventors confirmed that the bispecific antibody and the Obi x TNF Mutant can block excessive TNFα effects while hardly inducing TNFα signaling, and thus have few side effects.
Obi x TNF 돌연변이체 융합 항체가 오비누투주맙에 비하여 TNFR1 의존 기작에서 높은 DCD 및 ADCC를 보였다.The Obi x TNF mutant fusion antibody showed higher DCD and ADCC in a TNFR1 dependent mechanism compared to obinutuzumab.
본 발명자들은 항체들이 CD20 및 TNFR1을 발현하는 B-세포 비호지킨성 림프종(B-cell non-Hodgkin Lymphoma, BNHL)에서 실제로 효과적인지 보여주기 위해, 내인성 TNFR1을 발현하는 Ramos 세포주에서 이중특이성 항체와 융합 항체의 결합 친화도, DCD 및 ADCC를 비교하였다(도 6a). 결합친화도는 항체 농도를 계속 증가시키면서 분석한 세포 결합 어세이(cell binding assay)에 의하여 측정되었다(도 6b). 이전 데이터와 일관되게, 리툭시맙은 오비누투주맙보다 거의 두 배의 친화도로 결합하였고, Obi x TNF WT 및 Mutant는 오비누투주맙과 거의 동일하게 결합하였으며, 이중특이항체는 오비누투주맙보다 낮은 결합력을 보였다. To show that the antibodies are indeed effective in B-cell non-Hodgkin Lymphoma (BNHL) expressing CD20 and TNFR1, we fused them with a bispecific antibody in a Ramos cell line expressing endogenous TNFR1. The binding affinities of the antibodies, DCD and ADCC were compared (FIG. 6a). The binding affinity was measured by a cell binding assay analyzed while continuously increasing the antibody concentration (FIG. 6b). Consistent with previous data, Rituximab binds with nearly twice the affinity of obinutuzumab, Obi x TNF WT and Mutant bind nearly equally to obinutuzumab, and the bispecific antibody binds to obinutuzumab almost equally. showed lower bonding strength.
TNFR1에 비해 CD20의 발현이 너무 높아서 TNFR1에 의한 결합 차이를 비교하기 어려웠으나, 항-TNFR1 항체와 hIgG 사이의 차이는 명확하게 관찰할 수 있었고, 이는 TNFR1이 친화성에 영향을 미친다는 것을 시사함을 알 수 있었다.Although the expression of CD20 compared to TNFR1 was too high, it was difficult to compare the difference in binding by TNFR1, but the difference between the anti-TNFR1 antibody and hIgG was clearly observable, suggesting that TNFR1 affects the affinity. Could know.
이러한 친화성 분석 결과를 바탕으로, 항체 의존성 DCD를 비교하였다. 그 결과, 이중특이성 항체는 오비누투주맙보다 DCD를 덜 유도함을 보였다(도 6c). 이는 이중특이성 항체의 결합 친화도가 오비누투주맙보다 낮았기 때문인 것으로 사료된다. 반면, Obi x TNF WT 및 Obi x TNF Mutant는 오비누투주맙에 비해 더 많이 세포 사멸을 유도하였다. 다만, DCD에 있어서는 융합 항체 사이에 유의한 차이가 관찰되지는 않았다. 이를 통하여 CD20과 TNFR1 단백질이 동시에 포획될 때 DCD가 효율적으로 발생한다는 것을 증명할 수 있었다. Based on the results of this affinity analysis, antibody-dependent DCD was compared. As a result, it was shown that the bispecific antibody induced less DCD than obinutuzumab (FIG. 6c). It is believed that this is because the binding affinity of the bispecific antibody was lower than that of obinutuzumab. On the other hand, Obi x TNF WT and Obi x TNF Mutant induced more apoptosis than obinutuzumab. However, no significant difference was observed between the fusion antibodies in DCD. Through this, it was proved that DCD occurs efficiently when CD20 and TNFR1 proteins are simultaneously captured.
또한, 본 발명자들은 이전 실험의 결과로써 항체에 의한 DCD 증가가 ADCC를 향상시킬 수 있다는 점을 확인했었고, 이를 통하여 융합 항체에 의해 증가된 DCD가 ADCC를 향상시킬 수 있다고 예측한 바 있었다. 이에, 본 발명자들은 이펙터와 표적 세포를 5:1의 비율로 하여 ADCC 어세이를 수행하였다(도 6d). 그 결과 Obi x TNF Mutant는 오비누투주맙보다 거의 2배의 ADCC를 유도함을 관찰할 수 있었고, Obi x TNF Mutant 융합 항체가 TNFR1 의존 기작에서 오비누투주맙에 비해 더 높은 DCD 및 ADCC를 유도한다는 결과를 얻을 수 있었다.In addition, the present inventors have confirmed that an increase in DCD by an antibody can improve ADCC as a result of a previous experiment, and through this, it was predicted that an increase in DCD by a fusion antibody can improve ADCC. Thus, the present inventors performed the ADCC assay using the effector and target cells at a ratio of 5:1 (FIG. 6d). As a result, it was observed that Obi x TNF Mutant induced ADCC almost twice that of obinutuzumab, and that the Obi x TNF Mutant fusion antibody induced higher DCD and ADCC compared to obinutuzumab in a TNFR1-dependent mechanism. I was able to get results.
증가된 DCD와 ADCC는 TNFR1과 관련이 있다.Increased DCD and ADCC are related to TNFR1.
증가된 DCD 및 ADCC가 TNFR1에 의해 유발되는지 확인하기 위해, 본 발명자들은 TNFR1을 안정적으로 과발현하는 Raji 세포로 DCD 및 ADCC를 테스트하였다. 이를 위하여 렌티바이러스를 사용하여 빈 벡터(EV)와 TNFR1을 안정적으로 발현하는 Raji 세포를 생성하였고, 면역블로팅을 통하여 생성된 Raji세포가 EV 및 TNFR1을 과발현함을 확인하였다(도 7a). 이를 통하여 TNFR1을 과발현하는 Raji 세포에서는 많은 양의 TNFR1이 발현되고, EV에서는 거의 발현되지 않는 것을 알 수 있었고, TNFR1이 과발현되어도 CD20의 발현에는 변화가 없다는 것 또한 확인할 수 있었다. 그리고 이 Raji 안정 세포주에서 막 TNFR1을 유세포 분석법으로 분석하였다(도 7b). 이를 통해 막 TNFR1은 EV에 비해 TNFR1를 과발현하는 Raji 세포에서 약간 증가할 뿐이여서, 막 TNFR1을 너무 많이 발현하는 세포는 TNFR1을 안정적으로 과발현하는 Raji 세포를 생성하는 과정에서 죽은 것으로 보임을 확인하였다. To confirm that increased DCD and ADCC are caused by TNFR1, we tested DCD and ADCC with Raji cells stably overexpressing TNFR1. To this end, an empty vector (EV) and Raji cells stably expressing TNFR1 were generated using lentivirus, and it was confirmed through immunoblotting that the generated Raji cells overexpressed EV and TNFR1 (FIG. 7a). Through this, it was found that a large amount of TNFR1 was expressed in Raji cells overexpressing TNFR1, but little was expressed in EVs, and it was also confirmed that there was no change in CD20 expression even when TNFR1 was overexpressed. And membrane TNFR1 was analyzed by flow cytometry in this Raji stable cell line (FIG. 7b). Through this, it was confirmed that membrane TNFR1 was only slightly increased in Raji cells overexpressing TNFR1 compared to EVs, and thus cells that overexpress membrane TNFR1 seemed to die in the process of generating Raji cells stably overexpressing TNFR1.
본 발명자들은, 상기와 같은 안정적인 세포주에서 DCD를 일으켰다(도 7c). 낮은 농도에서 Obi x TNF WT 및 돌연변이체 융합 항체는 TNFR1 Raji 세포에서만 오비누투주맙에 비해 더 높은 DCD를 유도하였다. 그리고 고농도에서 융합 항체와 오비누투주맙은 TNFR1 과발현 Raji 세포에서 DCD를 유사하게 유도했지만, EV와 비교해서는 더 높은 DCD를 유도하였다.The present inventors induced DCD in the above stable cell line (FIG. 7c). At low concentrations, Obi x TNF WT and mutant fusion antibodies induced higher DCD compared to obinutuzumab only in TNFR1 Raji cells. And at high concentrations, the fusion antibody and obinutuzumab induced similar DCD in TNFR1-overexpressing Raji cells, but higher DCD compared to EV.
그 다음, 본 발명자들은 상기와 같은 안정적인 세포주에서 ADCC 분석을 수행하였다(도 7d). Ramos 세포에서의 이전 결과(도 6d)와 비슷하게, Obi x TNF Mutant는 오비누투주맙에 비해 더 높은 ADCC를 유의하게 유도함을 확인할 수 있었다. 이러한 결과로부터, 본 발명자들은 CD20 및 TNFR1 단백질이 동시에 포획되고 DCD 및 ADCC가 TNFR1에 의해 강화될 때 DCD가 효율적으로 발생함을 알 수 있었다.Next, the present inventors performed ADCC analysis on the above stable cell line (FIG. 7d). Similar to the previous results in Ramos cells (FIG. 6d), it was confirmed that Obi x TNF Mutant induced significantly higher ADCC compared to obinutuzumab. From these results, the present inventors found that DCD occurs efficiently when CD20 and TNFR1 proteins are simultaneously captured and DCD and ADCC are enhanced by TNFR1.
이상으로 본 발명의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.Having described specific parts of the present invention in detail above, it is clear that these specific techniques are merely preferred embodiments for those skilled in the art, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.
<110> Yonsei University Industry Academic Cooperation Foundation <120> A Composition for Preventing or Treating Cancer Comprising Novel Bitargeting Fusion-Antibody as an Active Ingredient <130> PDPB214666 <160> 24 <170> KoPatentIn 3.0 <210> 1 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> anti-CD20 antibody LCDR1 <400> 1 Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr 1 5 10 15 <210> 2 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> anti-CD20 antibody LCDR2 <400> 2 Gln Met Ser Asn Leu Val Ser 1 5 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> anti-CD20 antibody LCDR3 <400> 3 Ala Gln Asn Leu Glu Leu Pro Tyr Thr 1 5 <210> 4 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> anti-CD20 antibody HCDR1 <400> 4 Gly Tyr Ala Phe Ser Tyr Ser 1 5 <210> 5 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> anti-CD20 antibody HCDR2 <400> 5 Phe Pro Gly Asp Gly Asp 1 5 <210> 6 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> anti-CD20 antibody HCDR3 <400> 6 Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr 1 5 10 <210> 7 <211> 157 <212> PRT <213> Artificial Sequence <220> <223> TNF alpha Mutant aa77-233 <400> 7 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ser Thr Thr His Asn Gln Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> OBI heavy chain Q39Y site-directed mutagenesis forward primer <400> 8 ttgtccaggg gcgtaccgca ccca 24 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> OBI heavy chain Q39Y site-directed mutagenesis reverse primer <400> 9 aactgggtgc ggtacgcccc tgga 24 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> OBI heavy chain T366W site-directed mutagenesis forward primer <400> 10 gaagcctttg accaggcacc acag 24 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> OBI heavy chain T366W site-directed mutagenesis reverse primer <400> 11 caagaaccag gtcagcctgt ggtg 24 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> OBI light chain G38R site-directed mutagenesis forward primer <400> 12 ctgccctggc tttctcaggt acca 24 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> OBI light chain G38R site-directed mutagenesis reverse primer <400> 13 gtattggtac ctgagaaagc cagg 24 <210> 14 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> GSGSG linker + sTNF alpha extension PCR forward primer 1 <400> 14 cgggatccgg cagcggcgtc agatcatctt ctcgaac 37 <210> 15 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> GSGSG linker + sTNF alpha extension PCR reverse primer 1 <400> 15 gttcgagaag atgatctgac gccgctgccg gatcccg 37 <210> 16 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> GSGSG linker + sTNF alpha extension PCR forward primer 2 <400> 16 cgggatccgg cagcggcgga tctggtagcg gcgtcagatc atcttctcga acc 53 <210> 17 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> GSGSG linker + sTNF alpha extension PCR reverse primer 2 <400> 17 ggttcgagaa gatgatctga cgccgctacc agatccgccg ctgccggatc ccg 53 <210> 18 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> GSGSG linker + sTNF alpha extension PCR forward primer 3 <400> 18 cgggatccgg cagcggcgga tctggtagcg gcgggagcgg gtcaggcgtc agatcatctt 60 ctcgaacc 68 <210> 19 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> GSGSG linker + sTNF alpha extension PCR reverse primer 3 <400> 19 ggttcgagaa gatgatctga cgcctgaccc gctcccgccg ctaccagatc cgccgctgcc 60 ggatcccg 68 <210> 20 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> sTNF alpha mutant site-directed mutagenesis forward primer <400> 20 agccgcatca gcaccaccca caaccagaag gtcaac 36 <210> 21 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> sTNF alpha mutant site-directed mutagenesis reverse primer <400> 21 gttgaccttc tggttgtggg tggtgctgat gcggct 36 <210> 22 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> TNFRSF1A(lack cytoplasmic domain (aa233-455)) pLVX lentiviral vector cloning forward primer <400> 22 cccgctagca tgggcctctc cacc 24 <210> 23 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> TNFRSF1A(lack cytoplasmic domain (aa233-455)) pLVX lentiviral vector cloning reverse primer <400> 23 gggcggccgc ttagtagagc ttggacttcc acc 33 <210> 24 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> GSGSG linker protein <400> 24 Gly Ser Gly Ser Gly 1 5 <110> Yonsei University Industry Academic Cooperation Foundation <120> A Composition for Preventing or Treating Cancer Comprising Novel Bitargeting Fusion-Antibody as an Active Ingredient <130> PDPB214666 <160> 24 <170> KoPatentIn 3.0 <210> 1 <211> 16 <212> PRT <213> artificial sequence <220> <223> anti-CD20 antibody LCDR1 <400> 1 Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr 1 5 10 15 <210> 2 <211> 7 <212> PRT <213> artificial sequence <220> <223> anti-CD20 antibody LCDR2 <400> 2 Gln Met Ser Asn Leu Val Ser 1 5 <210> 3 <211> 9 <212> PRT <213> artificial sequence <220> <223> anti-CD20 antibody LCDR3 <400> 3 Ala Gln Asn Leu Glu Leu Pro Tyr Thr 1 5 <210> 4 <211> 7 <212> PRT <213> artificial sequence <220> <223> anti-CD20 antibody HCDR1 <400> 4 Gly Tyr Ala Phe Ser Tyr Ser 1 5 <210> 5 <211> 6 <212> PRT <213> artificial sequence <220> <223> anti-CD20 antibody HCDR2 <400> 5 Phe Pro Gly Asp Gly Asp 1 5 <210> 6 <211> 10 <212> PRT <213> artificial sequence <220> <223> anti-CD20 antibody HCDR3 <400> 6 Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr 1 5 10 <210> 7 <211> 157 <212> PRT <213> artificial sequence <220> <223> TNF alpha Mutant aa77-233 <400> 7 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ser Thr Thr His Asn Gln Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155 <210> 8 <211> 24 <212> DNA <213> artificial sequence <220> <223> OBI heavy chain Q39Y site-directed mutagenesis forward primer <400> 8 ttgtccaggg gcgtaccgca ccca 24 <210> 9 <211> 24 <212> DNA <213> artificial sequence <220> <223> OBI heavy chain Q39Y site-directed mutagenesis reverse primer <400> 9 aactgggtgc ggtacgcccc tgga 24 <210> 10 <211> 24 <212> DNA <213> artificial sequence <220> <223> OBI heavy chain T366W site-directed mutagenesis forward primer <400> 10 gaagcctttg accaggcacc acag 24 <210> 11 <211> 24 <212> DNA <213> artificial sequence <220> <223> OBI heavy chain T366W site-directed mutagenesis reverse primer <400> 11 caagaaccag gtcagcctgt ggtg 24 <210> 12 <211> 24 <212> DNA <213> artificial sequence <220> <223> OBI light chain G38R site-directed mutagenesis forward primer <400> 12 ctgccctggc tttctcaggt acca 24 <210> 13 <211> 24 <212> DNA <213> artificial sequence <220> <223> OBI light chain G38R site-directed mutagenesis reverse primer <400> 13 gtattggtac ctgagaaagc cagg 24 <210> 14 <211> 37 <212> DNA <213> artificial sequence <220> <223> GSGSG linker + sTNF alpha extension PCR forward primer 1 <400> 14 cgggatccgg cagcggcgtc agatcatctt ctcgaac 37 <210> 15 <211> 37 <212> DNA <213> artificial sequence <220> <223> GSGSG linker + sTNF alpha extension PCR reverse primer 1 <400> 15 gttcgagaag atgatctgac gccgctgccg gatcccg 37 <210> 16 <211> 53 <212> DNA <213> artificial sequence <220> <223> GSGSG linker + sTNF alpha extension PCR forward primer 2 <400> 16 cgggatccgg cagcggcgga tctggtagcg gcgtcagatc atcttctcga acc 53 <210> 17 <211> 53 <212> DNA <213> artificial sequence <220> <223> GSGSG linker + sTNF alpha extension PCR reverse primer 2 <400> 17 ggttcgagaa gatgatctga cgccgctacc agatccgccg ctgccggatc ccg 53 <210> 18 <211> 68 <212> DNA <213> artificial sequence <220> <223> GSGSG linker + sTNF alpha extension PCR forward primer 3 <400> 18 cgggatccgg cagcggcgga tctggtagcg gcgggagcgg gtcaggcgtc agatcatctt 60 ctcgaacc 68 <210> 19 <211> 68 <212> DNA <213> artificial sequence <220> <223> GSGSG linker + sTNF alpha extension PCR reverse primer 3 <400> 19 ggttcgagaa gatgatctga cgcctgaccc gctcccgccg ctaccagatc cgccgctgcc 60 ggatcccg 68 <210> 20 <211> 36 <212> DNA <213> artificial sequence <220> <223> sTNF alpha mutant site-directed mutagenesis forward primer <400> 20 agccgcatca gcaccaccca caaccagaag gtcaac 36 <210> 21 <211> 36 <212> DNA <213> artificial sequence <220> <223> sTNF alpha mutant site-directed mutagenesis reverse primer <400> 21 gttgaccttc tggttgtggg tggtgctgat gcggct 36 <210> 22 <211> 24 <212> DNA <213> artificial sequence <220> <223> TNFRSF1A (lack cytoplasmic domain (aa233-455)) pLVX lentiviral vector cloning forward primers <400> 22 cccgctagca tgggcctctc cacc 24 <210> 23 <211> 33 <212> DNA <213> artificial sequence <220> <223> TNFRSF1A (lack cytoplasmic domain (aa233-455)) pLVX lentiviral vector cloning reverse primer <400> 23 gggcggccgc ttagtagagc ttggacttcc acc 33 <210> 24 <211> 5 <212> PRT <213> artificial sequence <220> <223> GSGSG linker protein <400> 24 Gly Ser Gly Ser Gly 1 5
Claims (11)
An anti-CD20 antibody with a TNFα mutant bound to the Fc region.
The anti-CD20 antibody according to claim 1, wherein the TNFα mutant is soluble.
3. The anti-CD20 antibody of claim 2, wherein the TNFα mutant comprises one or more amino acid substitutions selected from the group consisting of A160S, V161T, S162T, Y163H, Q164N and T165Q.
The anti-CD20 antibody according to claim 1, wherein the TNFα mutant binds to the C-terminus of the Fc region of the anti-CD20 antibody.
The anti-CD20 antibody according to claim 1, wherein the anti-CD20 antibody further comprises a protein linker.
6. The anti-CD20 antibody according to claim 5, wherein the protein linker is a GSGSG linker.
The anti-CD20 antibody according to claim 5, wherein the protein linker binds to the Fc region of the anti-CD20 antibody, and the soluble TNFα mutant is linked to the anti-CD20 antibody through the protein linker.
The anti-CD20 antibody according to claim 1, wherein the anti-CD20 antibody is Obinutuzumab.
A pharmaceutical composition for preventing or treating cancer comprising the antibody of any one of claims 1 to 7 as an active ingredient.
A pharmaceutical composition for preventing or treating cancer comprising an anti-CD20 antibody and a TNFα mutant as active ingredients.
The pharmaceutical composition for preventing or treating cancer according to claim 9 or 10, wherein the cancer is B cell lymphoma.
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