KR20200132110A - An antibody specific for protein in cancer stem cell membrane and a use thereof - Google Patents

An antibody specific for protein in cancer stem cell membrane and a use thereof Download PDF

Info

Publication number
KR20200132110A
KR20200132110A KR1020190057050A KR20190057050A KR20200132110A KR 20200132110 A KR20200132110 A KR 20200132110A KR 1020190057050 A KR1020190057050 A KR 1020190057050A KR 20190057050 A KR20190057050 A KR 20190057050A KR 20200132110 A KR20200132110 A KR 20200132110A
Authority
KR
South Korea
Prior art keywords
cancer
seq
antibody
monoclonal antibody
ser
Prior art date
Application number
KR1020190057050A
Other languages
Korean (ko)
Other versions
KR102259298B1 (en
Inventor
김미경
김은희
이정현
정여진
Original Assignee
강원대학교산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 강원대학교산학협력단 filed Critical 강원대학교산학협력단
Priority to KR1020190057050A priority Critical patent/KR102259298B1/en
Priority to KR1020200127010A priority patent/KR102258015B1/en
Priority to KR1020200127007A priority patent/KR102258014B1/en
Publication of KR20200132110A publication Critical patent/KR20200132110A/en
Application granted granted Critical
Publication of KR102259298B1 publication Critical patent/KR102259298B1/en

Links

Images

Classifications

    • 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
    • C07K16/2881—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD71
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • 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
    • C07K16/2884—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD44
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70582—CD71
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70585—CD44

Abstract

The present invention relates to a monoclonal antibody specific for cell membrane protein of cancer stem cell selected from the group consisting of monoclonal antibodies of a), b) and c), or a functional fragment thereof, wherein the monoclonal antibody of the a) includes: a heavy chain comprising a CDR1 region described in SEQ ID NO: 1, a CDR2 region described in SEQ ID NO: 2, and a CDR3 region described in SEQ ID NO: 3; and a light chain comprising a CDR1 region described in SEQ ID NO: 4, a CDR2 region described in SEQ ID NO: 5, and a CDR3 region described in SEQ ID NO: 6, the monoclonal antibody of the b) includes: a heavy chain comprising a CDR1 region described in SEQ ID NO: 7, a CDR2 region described in SEQ ID NO: 8, and a CDR3 region described in SEQ ID NO: 9, and a light chain comprising a CDR1 region described in SEQ ID NO: 10, and a CDR2 region described in SEQ ID NO: 11, and a CDR3 region described in SEQ ID NO: 12, and the monoclonal antibody of the c) includes: a heavy chain comprising a CDR1 region described in SEQ ID NO: 13, a CDR2 region described in SEQ ID NO: 14, and a CDR3 region described in SEQ ID NO: 15; and a light chain comprising a CDR1 region described in SEQ ID NO: 16, a CDR2 region described in SEQ ID NO: 17, and a CDR3 region described in SEQ ID NO: 18.

Description

암 줄기세포 세포막 단백질에 특이적인 항체 및 그 응용{AN ANTIBODY SPECIFIC FOR PROTEIN IN CANCER STEM CELL MEMBRANE AND A USE THEREOF}Antibody specific to cancer stem cell membrane protein and its application {AN ANTIBODY SPECIFIC FOR PROTEIN IN CANCER STEM CELL MEMBRANE AND A USE THEREOF}

본 발명은 암 줄기세포 세포막 단백질에 특이적인 항체 및 그 응용에 관한 것으로, 더욱 상세하게는 암 줄기세포에 결합하는 항체 발굴과 암 줄기세포에 대한 druggable 타겟 단백질을 동시에 검출하는 방법에 의하여 얻어진 암 관련 항원 및 항원에 결합하는 신규 항체 기반 물질, 항원를 발현하는 다양한 암과 질환의 진단 및 치료제 개발에 대한 것이다.The present invention relates to antibodies specific to cancer stem cell membrane proteins and their applications, and more particularly, to cancer-related cancer obtained by a method of simultaneously detecting an antibody binding to cancer stem cells and a druggable target protein for cancer stem cells. The purpose of this study is to develop antigens and novel antibody-based substances that bind to antigens, and to diagnose and treat various cancers and diseases that express antigens.

현재 항암 치료에 있어 가장 큰 문제점은 암 치료 이후 암이 재발하거나 지속적인 항암제 투여로 인한 항암제 내성으로 인해 지속적인 치료가 불가능한 한계점이 있다. 특히 이러한 문제점을 극복해 줄 수 있는 암치료제 개발 및 치료법 개선을 위한 타겟이 극히 제한된 점이 가장 취약점이며 문제이다. 약물 내성과 재발에 관한 문제를 해결하기 위해 글로벌 제약사들이 면역반응 기반 치료제에 투자를 하고 있으나, 임상단계에 사용되어지고 있는 타겟의 수는 60개 미만으로 기존의 타겟에 집중적으로 치료 병행법 개발을 하고 있으며 현 문제점을 극복하는 데에는 한계가 있다. Currently, the biggest problem in anticancer treatment is that the cancer recurs after cancer treatment or continuous treatment is impossible due to resistance to anticancer drugs due to continuous administration of anticancer drugs. In particular, the most vulnerable and problem is that the target for developing cancer treatments and improving treatments that can overcome these problems is extremely limited. Global pharmaceutical companies are investing in immune response-based treatments to solve the problem of drug resistance and recurrence, but the number of targets being used in the clinical stage is less than 60. And there is a limit to overcoming the current problems.

따라서 신규 항암 치료제 개발과 현 암치료제의 항암내성 및 암의 재발을 극복할 수 있는 혁신적인 novel 또는 신규 타겟과 이에 대한 치료제가 절실히 필요한 실정이다.Therefore, there is an urgent need for new anticancer treatments and innovative novel or novel targets that can overcome the recurrence of cancer and the anticancer resistance of current cancer treatments and treatments for them.

그러나 표적 항체 치료제 개발의 문제점으로는 대부분 암세포에 과발현되는 단백질, 즉 선택된 타겟(pre-selected target)에 대한 재조합 단백질 제작 및 이에 대한 치료용 항체가 개발되고 있는 것이다. 이는 생체 내에서 인식되는 타겟 항원의 구조 및 암 조직환경(TME, tumor microenvironment)과 다를 수 있으며, 이를 반영하듯 임상시험 시 전임상보다 낮은 항암 효과가 보고될 때도 있다.However, a problem in the development of targeted antibody therapeutics is that most of the proteins overexpressed in cancer cells, that is, recombinant proteins for pre-selected targets, and therapeutic antibodies for them are being developed. This may differ from the structure of the target antigen recognized in vivo and the tumor microenvironment (TME), and as reflecting this, sometimes lower anticancer effects than preclinical tests are reported in clinical trials.

또한 기존 항암치료제는 질병 암세포에 과발현하는 단백질/타겟에 대한 치료제로서 암세포의 핵심인 암 줄기세포를 근본적으로 표적화하여 사멸시키지 못함으로 인해 암 줄기세포가 암의 재발 및 항암제 내성과 재발에 많은 기여를 하는 것으로 인식되고 있다. 따라서 암 줄기세포의 특이적 세포막 단백체 발굴과 이를 특이적으로 응용하는 신규 표적 치료 타겟 발굴 및 응용한 새로운 항암치료제 개발이 요구되고 있다.In addition, existing anticancer drugs are therapeutics for proteins/targets overexpressed in diseased cancer cells, and as they cannot target and kill cancer stem cells, the core of cancer cells, cancer stem cells contribute a lot to recurrence of cancer and anticancer drug resistance and recurrence. It is recognized as doing. Accordingly, there is a need to discover specific cell membrane proteins of cancer stem cells, discover new target therapeutic targets that specifically apply them, and develop new anticancer therapeutic agents that apply them.

[선행 특허 문헌][Prior patent literature]

대한민국 공개특허 10-2017-0104619Republic of Korea Patent Publication 10-2017-0104619

본 발명은 상기의 필요성에 의하여 안출된 것으로서 본 발명의 목적은 신규 항암제 타겟 항원 및 그 타겟 항원에 대한 phenotypic 구조특성을 반영한 항체를 제공하는 것이다.The present invention was conceived by the above necessity, and an object of the present invention is to provide a novel anticancer agent target antigen and an antibody reflecting the phenotypic structural characteristics of the target antigen.

상기 목적을 달성하기 위하여 본 발명은 In order to achieve the above object, the present invention

하기 a), b) 및 c)의 단클론 항체로 구성된 군으로부터 선택된 암 줄기세포및/또는 암세포의 세포막 단백질에 특이적인 단클론 항체, 또는 그 기능적 단편:A monoclonal antibody specific for a cell membrane protein of cancer stem cells and/or cancer cells selected from the group consisting of monoclonal antibodies of a), b) and c) below, or a functional fragment thereof:

a)서열번호 1로 기재되는 CDR1 영역, 서열번호 2로 기재되는 CDR2 영역 및 서열번호 3으로 기재되는 CDR3 영역을 포함하는 중쇄 및 a) a heavy chain comprising a CDR1 region shown in SEQ ID NO: 1, a CDR2 region shown in SEQ ID NO: 2, and a CDR3 region shown in SEQ ID NO: 3, and

서열번호 4로 기재되는 CDR1 영역, 서열번호 5로 기재되는 CDR2 영역 및 서열번호 6으로 기재되는 CDR3 영역을 포함하는 경쇄를 포함하는 단클론 항체;A monoclonal antibody comprising a light chain comprising a CDR1 region represented by SEQ ID NO: 4, a CDR2 region represented by SEQ ID NO: 5, and a CDR3 region represented by SEQ ID NO: 6;

b)서열번호 7로 기재되는 CDR1 영역, 서열번호 8로 기재되는 CDR2 영역 및 서열번호 9로 기재되는 CDR3 영역을 포함하는 중쇄 및 b) a heavy chain comprising a CDR1 region represented by SEQ ID NO: 7, a CDR2 region represented by SEQ ID NO: 8, and a CDR3 region represented by SEQ ID NO: 9, and

서열번호 10으로 기재되는 CDR1 영역, 서열번호 11로 기재되는 CDR2 영역 및 서열번호 12로 기재되는 CDR3 영역을 포함하는 경쇄를 포함하는 단클론 항체; 및 A monoclonal antibody comprising a light chain comprising a CDR1 region represented by SEQ ID NO: 10, a CDR2 region represented by SEQ ID NO: 11, and a CDR3 region represented by SEQ ID NO: 12; And

c) 서열번호 13으로 기재되는 CDR1 영역, 서열번호 14로 기재되는 CDR2 영역 및 서열번호 15로 기재되는 CDR3 영역을 포함하는 중쇄 및 c) a heavy chain comprising a CDR1 region represented by SEQ ID NO: 13, a CDR2 region represented by SEQ ID NO: 14, and a CDR3 region represented by SEQ ID NO: 15, and

서열번호 16으로 기재되는 CDR1 영역, 서열번호 17로 기재되는 CDR2 영역 및 서열번호 18로 기재되는 CDR3 영역을 포함하는 경쇄를 포함하는 단클론 항체를 제공한다.It provides a monoclonal antibody comprising a light chain comprising the CDR1 region of SEQ ID NO: 16, the CDR2 region of SEQ ID NO: 17, and the CDR3 region of SEQ ID NO: 18.

본 발명의 일 구현예에 있어서, 상기 암 줄기세포 및/또는 암세포의 세포막 단백질은 CD 44 또는 CD71인 것이 바람직하나 이에 한정되지 아니한다.In one embodiment of the present invention, the cell membrane protein of the cancer stem cell and/or cancer cell is preferably CD 44 or CD71, but is not limited thereto.

본 발명의 기능적 항체로는 IgG, IgA, IgD, IgE, IgM의 아이소타입(isotype) 및 subclass 등을 들 수 있다.Functional antibodies of the present invention include IgG, IgA, IgD, IgE, IgM isotypes and subclasses.

본 발명의 기능적 항체 단편은 경쇄, 중쇄, 가변 영역, Fab, Fab', F(ab')2, 단일가변분절(scFv), 디아바디(Diabody), 트리바디(Tribody), 이중쇄가변분절(dsFv), scFv-Fc(맥시바디, Maxibody), 싱글체인IgG (scIgG), 다중체 또는 CDR(Complementarity Determining Region)을 함유하는 펩타이드인 것이 바람직하나 이에 한정되지 아니한다.Functional antibody fragments of the present invention include light chain, heavy chain, variable region, Fab, Fab', F(ab')2, single variable segment (scFv), Diabody, Tribody, double chain variable segment ( dsFv), scFv-Fc (Maxibody), single chain IgG (scIgG), a peptide containing a multiplex or CDR (Complementarity Determining Region) is preferred, but is not limited thereto.

Fab는 IgG를 단백질 분해효소 파파인으로 처리하여 수득되는 단편중(H쇄의 224번째의 아미노산 잔기로 절단된다),H쇄의 N 말단측 약 절반과 L쇄 전체가 디설파이드 결합(S-S 결합)으로 결합된 분자량 약 5만의 항원결합 활성을 갖는 항체 단편이다.Fab is a fragment obtained by treating IgG with the protease papain (cut to the 224th amino acid residue of the H chain), about half of the N-terminal side of the H chain and the entire L chain are bound by a disulfide bond (SS bond). It is an antibody fragment having an antigen-binding activity of about 50,000 molecular weight.

본 발명의 Fab는 본 발명의 항체를 단백질 분해효소 파파인으로 처리하여 수득할 수 있다. 또는 당해 항체의 Fab를 암호화하는 DNA를 원핵생물용 발현 벡터 또는 진핵생물용 발현 벡터에 삽입하고, 당해 벡터를 원핵생물 또는 진핵생물로 도입함으로써 발현시켜 Fab를 제조할 수 있다.The Fab of the present invention can be obtained by treating the antibody of the present invention with the protease papain. Alternatively, the DNA encoding the Fab of the antibody is inserted into a prokaryotic expression vector or an expression vector for eukaryotes, and the vector is introduced into a prokaryote or eukaryote to express it, thereby producing a Fab.

F(ab')2 는 IgG를 단백질 분해효소 펩신으로 처리하여 수득되는 단편중(H쇄의 234번째의 아미노산 잔기로 절단된다), Fab가 힌지영역의 S-S 결합을 개재하여 결합된 것보다 약간 큰 분자량 약 10만의 항원 결합 활성을 갖는 항체 단편이다.F(ab')2 is a fragment obtained by treating IgG with the proteolytic enzyme pepsin (cut to the 234th amino acid residue of the H chain), where the Fab is slightly larger than the one bound through the SS bond of the hinge region. It is an antibody fragment having an antigen-binding activity of about 100,000 molecular weight.

본 발명의 F(ab')2 는 본 발명의 항체를 단백질 분해효소 펩신으로 처리하여 수득할 수 있다. 또는 하기의 Fab'를 티오에테르 결합 또는 S-S 결합시켜 작제할 수 있다.Fab'는 상기 F(ab')2 의 힌지영역의 S-S 결합을 절단한 분자량 약 5만의 항원 결합 활성을 갖는 항체 단편이다.F(ab')2 of the present invention can be obtained by treating the antibody of the present invention with the protease pepsin. Alternatively, the following Fab' can be constructed by thioether binding or S-S binding. Fab' is an antibody fragment having an antigen-binding activity of about 50,000 molecular weight by cleaving the S-S bond of the hinge region of F(ab')2.

싱글체인IgG(scIgG)는 싱글체인Fab(scFab)이 Fc에 연결된 폴리펩타이드로 항원 결합 활성을 갖는 1개의 폴리펩타이드로된 항체이다. 여기서 싱글체인Fab은 1개의 VH-CH1와 VL-CL을 30이상의 적당한 펩타이드 링커(P)를 사용하여 연결된 싱글체인Fab(scFab)으로 VH-CH1-P-VL-CL 내지 VL-CL-P-VH-CH1 폴리펩타이드로 항원 결합 활성을 갖는 항체 단편이다.Single chain IgG (scIgG) is a single chain Fab (scFab) is a polypeptide linked to Fc, and is an antibody composed of one polypeptide having antigen-binding activity. Here, the single chain Fab is a single chain Fab (scFab) that connects one VH-CH1 and VL-CL using 30 or more suitable peptide linkers (P), and is VH-CH1-P-VL-CL to VL-CL-P- It is an antibody fragment having antigen-binding activity as a VH-CH1 polypeptide.

scFv는 1개의 VH와 1개의 VL을 12잔기 이상의 적당한 펩타이드 링커(P)를 사용하여 연결한 VH-P-VL 내지는 VL-P-VH 폴리펩타이드로, 항원 결합 활성을 갖는 항체 단편이다.scFv is a VH-P-VL or VL-P-VH polypeptide in which one VH and one VL are linked using an appropriate peptide linker (P) of 12 or more residues, and is an antibody fragment having antigen-binding activity.

본 발명의 scFv는 본 발명의 항체의 VH 및 VL을 암호화하는 cDNA를 수득하고, scFv를 암호화하는 DNA를 작제하며 당해 DNA를 원핵생물용 발현 벡터 또는 진핵생물용 발현 벡터에 삽입하여 당해 발현 벡터를 원핵생물 또는 진핵생물로 도입함으로써 발현시켜 제조할 수 있다.The scFv of the present invention obtains cDNA encoding VH and VL of the antibody of the present invention, constructs a DNA encoding scFv, and inserts the DNA into an expression vector for prokaryote or an expression vector for eukaryotes to obtain the expression vector. It can be produced by expression by introduction into prokaryotes or eukaryotes.

Diabody는 항원 결합 특이성이 동일하거나 상이한 scFv가 이량체를 형성한 항체 단편이고, 동일한 항원에 대한 2가의 항원 결합 활성 또는 상이한 항원에 대한 2특이적인 항원 결합 활성을 갖는 항체 단편이다.Diabody is an antibody fragment in which scFvs having the same or different antigen-binding specificity form a dimer, and is an antibody fragment having a bivalent antigen-binding activity for the same antigen or a bispecific antigen-binding activity for different antigens.

본 발명의 Diabody는 예를 들면, 본 발명의 항체의 VH 및 VL을 암호화하는 cDNA를 수득하고, 3 내지 15 잔기의 폴리펩타이드 링커를 갖는 scFv를 암호화하는 DNA를 작제하며 당해 DNA를 원핵생물용 발현 벡터 또는 진핵생물용 발현 벡터에 삽입하여 당해 발현벡터를 원핵생물 또는 진핵생물로 도입함으로써 Diabody를 발현시켜 제조할 수 있다.Diabody of the present invention, for example, obtains cDNA encoding VH and VL of the antibody of the present invention, constructs a DNA encoding scFv having a polypeptide linker of 3 to 15 residues, and expresses the DNA for prokaryote By inserting into a vector or an expression vector for eukaryotes, the expression vector can be introduced into a prokaryote or eukaryote to express a Diabody.

또한, linker P 길이가 3-10 일 때는 tribody가 형성되어, tribody로 포함할 수 있다.In addition, when the linker P length is 3-10, a tribody is formed and may be included as a tribody.

dsFv는 VH 및 VL 중 각각 하나의 아미노산 잔기를 시스테인 잔기로 치환한 폴리펩타이드를 당해 시스테인 잔기 간의 S-S 결합을 개재하여 결합시킨 것을 말한다. 시스테인 잔기로 치환되는 아미노산 잔기는 Reiter 등에 의해 기재된 방법[참조: Protein Engineering, 7, 697(1994)]에 따라서 항체의 입체구조 예측에 근거하여 선택할 수 있다.dsFv refers to a polypeptide obtained by substituting a cysteine residue for one of the amino acid residues of VH and VL, which is bonded via an S-S bond between the cysteine residues. Amino acid residues substituted with cysteine residues can be selected based on prediction of the conformational structure of an antibody according to the method described by Reiter et al. (Protein Engineering, 7, 697 (1994)).

본 발명의 일 구현예에 있어서, 상기 a) 단클론 항체는 서열번호 19의 아미노산 서열로 이루어진 중쇄 가변영역 및 서열번호 20의 아미노산 서열로 이루어진 경쇄 가변영역을 포함하는 것이 바람직하고,In one embodiment of the present invention, the a) monoclonal antibody preferably comprises a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 19 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 20,

상기 b) 단클론 항체는 서열번호 21의 아미노산 서열로 이루어진 중쇄 가변영역 및 서열번호 22의 아미노산 서열로 이루어진 경쇄 가변영역을 포함하는 것이 바람직하며,The b) monoclonal antibody preferably comprises a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 21 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 22,

상기 c) 단클론 항체는 서열번호 23의 아미노산 서열로 이루어진 중쇄 가변영역 및 서열번호 24의 아미노산 서열로 이루어진 경쇄 가변영역을 포함하는 것이 바람직하나 이에 한정되지 아니한다.The c) monoclonal antibody preferably includes a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 23 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 24, but is not limited thereto.

또 본 발명은 상기 본 발명의 단클론 항체, 또는 그 기능적 단편을 유효성분으로 포함하는 암 진단용 조성물을 제공한다.In addition, the present invention provides a composition for diagnosis of cancer comprising the monoclonal antibody of the present invention or a functional fragment thereof as an active ingredient.

본 발명의 일 구현예에 있어서, 상기 암은 유방암, 폐암, 난소암, 신장암, 췌장암, 결장암, 직장결장암, 전립선암, 신장세포 암종, 투명세포 암종, 소세포 암종, 편평세포 암종, 위암, 임파종, 백혈병 및 다발성 골수종으로 구성된 군으로부터 선택된 암인 것이 바람직하나 이에 한정되지 아니한다.In one embodiment of the present invention, the cancer is breast cancer, lung cancer, ovarian cancer, kidney cancer, pancreatic cancer, colon cancer, colorectal cancer, prostate cancer, renal cell carcinoma, clear cell carcinoma, small cell carcinoma, squamous cell carcinoma, gastric cancer, lymphoma , It is preferably a cancer selected from the group consisting of leukemia and multiple myeloma, but is not limited thereto.

또한 본 발명은 상기 본 발명의 단클론 항체, 또는 그 기능적 단편을 유효성분으로 포함하는 암 치료용 조성물을 제공한다.In addition, the present invention provides a composition for treating cancer comprising the monoclonal antibody of the present invention or a functional fragment thereof as an active ingredient.

본 발명의 조성물에는 기타 약물 또는 다른 면역 보조제가 포함되어 추가적인 면역 자극 효과를 제공할 수 있다. 면역보강제의 종류들은 당 분야에 널리 공지되어 있다(Vaccine Design - The Subunit and Adjuvant Approach, 1995, Pharmaceutical Biotechnology, Volume 6, Eds. Powell, M.F., and Newman, M.J., Plenum Press, New York and London, ISBN 0-306-44867-X). 바람직하게는, 본 발명의 조성물에 포함되는 면역보강제는 알루미늄 염 또는 칼슘 염(예를 들어, 하이드록사이드 또는 포스페이트)을 포함한다.The composition of the present invention may contain other drugs or other immune adjuvants to provide additional immune stimulating effects. Types of adjuvants are well known in the art (Vaccine Design-The Subunit and Adjuvant Approach, 1995, Pharmaceutical Biotechnology, Volume 6, Eds. Powell, MF, and Newman, MJ, Plenum Press, New York and London, ISBN. 0-306-44867-X). Preferably, the adjuvant included in the composition of the present invention comprises an aluminum salt or a calcium salt (eg, hydroxide or phosphate).

바람직한 면역보강제의 구체적 예는 다음과 같다: 알루미늄염 또는 칼슘염(하이드록사이드 또는 포스페이트),수중유(oil-in-water) 에멀젼(WO 95/17210, EP 0 399 843) 또는 리포좀과 같은 미립성 캐리어(WO 96/33739),남아메리카 수목인 퀼라자 사포나리아 몰리나(Quillaja Saponaria Molina)로부터 유래된 면역학적으로 항원보강제 활성을 지닌 사포닌 분획(예를 들어, Quil A), 3 De-O-실화된 모노포스포릴 지질 A, 뮤라밀 디펩티드, 3DMPL(3-O-데아실화된 모노포스포릴 지질 A)을 포함하지만, 이에 한정되는 것은 아니다.Specific examples of preferred adjuvants are as follows: aluminum salts or calcium salts (hydroxides or phosphates), oil-in-water emulsions (WO 95/17210, EP 0 399 843) or fine particles such as liposomes. Sex carrier (WO 96/33739), a saponin fraction with immunologically adjuvant activity derived from the South American tree Quillaja Saponaria Molina (e.g., Quil A), 3 De-O- Silylated monophosphoryl lipid A, muramyl dipeptide, 3DMPL (3-O-deacylated monophosphoryl lipid A), but are not limited thereto.

본 발명의 약제학적 조성물에 포함되는 약제학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서,락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트,프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 본 발명의 약제학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제,보존제 등을 추가로 포함할 수 있다. 적합한 약제학적으로 허용되는 담체 및 제제는 Remington's Pharmaceutical Sciences(19th ed., 1995)에 상세히 기재되어 있다.Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. It does not become. The pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweetening agents, flavoring agents, emulsifying agents, suspending agents, preservatives, and the like in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

본 발명의 약제학적 조성물은 경구 또는 비경구로 투여할 수 있고, 바람직하게는 비경구 투여이고, 비경구 투여인 경우에는 정맥내 주입, 피하 주입, 근육 주입, 복강 주입, 경피 투여 등으로 투여할 수 있다.The pharmaceutical composition of the present invention can be administered orally or parenterally, preferably parenteral, and in the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc. have.

본 발명의 약제학적 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하게 처방될 수 있다. 한편,본 발명의 약제학적 조성물의 경구 투여량은 바람직하게는 1일 당 0.001-10,000 mg/kg(체중)이다.The appropriate dosage of the pharmaceutical composition of the present invention is prescribed in various ways depending on factors such as the formulation method, the mode of administration, the patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and response sensitivity. Can be. Meanwhile, the oral dosage of the pharmaceutical composition of the present invention is preferably 0.001-10,000 mg/kg (body weight) per day.

본 발명의 약제학적 조성물은 당해 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기내에 내입시켜 제조될 수 있다. 이때 제형은 오일 또는 수성 매질중의 용액, 현탁액 또는 유화액 형태이거나 엑스제, 분말제, 과립제, 정제 또는 캅셀제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다.The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person having ordinary knowledge in the art. Or it can be prepared by placing it in a multi-dose container. At this time, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or a stabilizer.

또한 본 발명은 상기 본 발명의 단클론 항체, 또는 그 기능적 단편에 특정 물질이 결합된 컨쥬게이트 또는 CAR-T를 제공한다.In addition, the present invention provides a conjugate or CAR-T in which a specific substance is bound to the monoclonal antibody of the present invention or a functional fragment thereof.

본 발명의 일 구현예에 있어서, 상기 컨쥬게이트는 항체-형광물질 결합체, 항체-독소, 항체-약물 결합체 또는 항체-항체 결합체인 것이 바람직하나 이에 한정되지 아니한다.In one embodiment of the present invention, the conjugate is preferably an antibody-fluorescent material conjugate, antibody-toxin, antibody-drug conjugate, or antibody-antibody conjugate, but is not limited thereto.

이하 본 발명을 설명한다.The present invention will be described below.

암 줄기 세포막 단백체의 phenotypic 구조특성을 반영한 암줄기세포막 표적 druggable 타겟 발굴 및 이에 대한 항체개발은 암치료제의 임상효능을 효과적으로 높이는 방법뿐만 아니라 신규 진단, 치료제 개발 및 치료법 개선의 방법이 될 수 있다.The discovery of druggable targets for cancer stem cell membranes reflecting the phenotypic structural characteristics of cancer stem cell membrane proteins and development of antibodies thereto can be a method of effectively enhancing the clinical efficacy of cancer treatments, as well as new diagnosis, treatments, and treatment improvements.

따라서 이에 본 발명은 현 암치료제 개발 및 치료법의 난관 점을 해결할 수 있는 혁신적인 druggable novel 타겟 또는 신규 타겟 발굴과 이 타겟에 대한 phenotypic 구조특성을 반영한 항암치료제 개발을 동시에 진행하는 것이며 특히 본 발명에 있어서 예시로는 질병세포주, 특히 암 줄기세포주를 응용함에 있다.Therefore, the present invention simultaneously proceeds with the discovery of an innovative druggable novel target or new target that can solve the difficulties of the current cancer treatment development and treatment and the development of an anticancer treatment that reflects the phenotypic structural characteristics of this target. The furnace is to apply disease cell lines, especially cancer stem cell lines.

암 줄기세포에 연관된 타겟 또는 암 줄기세포에 과발현된 phenotypic 구조특성의 타겟 단백질을 인지하는 항체와 타겟 단백질을 동시에 동정함으로써, 항암 치료, 항암 내성조절과 재발 억제 그리고 조기 암 진단 등에 기여함으로 암을 치유할 수 있을 것으로 예측됨에 따라 도 1과 같은 전략으로 연구를 진행하였다.By simultaneously identifying an antibody and a target protein that recognizes a target protein related to cancer stem cells or a target protein with phenotypic structural characteristics overexpressed in cancer stem cells, it helps to cure cancer by contributing to anti-cancer treatment, anti-cancer resistance control and recurrence inhibition, and early cancer diagnosis. As it was predicted to be possible, the study was conducted with the strategy shown in FIG. 1.

본 발명의 항체/항원 동시 동정 방법을 PhAbomics Technology (platform)으로 칭한다.The antibody/antigen simultaneous identification method of the present invention is referred to as PhAbomics Technology (platform).

본 발명 예시로 암 줄기 세포를 사용하였지만, 본 발명 기법으로 그 외 다른 특이적 질환 세포주(예, 관절염(arthritis), 다발성 경화증, 폐암, 췌장암 등)에 대한 세포 및 줄기세포 등을 사용하여도 질병 세포주에 대한 특이적 항원 및 항체 기반 물질을 확보하여 진단 및 치료제 개발을 할 수 있다.Although cancer stem cells were used as an example of the present invention, cells and stem cells for other specific disease cell lines (e.g., arthritis, multiple sclerosis, lung cancer, pancreatic cancer, etc.) are also used as the technique of the present invention. By securing specific antigens and antibody-based substances for cell lines, diagnosis and treatment can be developed.

본 발명의 기술로 동시 발굴된 항체는 타겟 단백질에 대한 검출, 암에 대한 진단 및 다양한 항암 치료제(예, 암줄기세포 및 유방암, 삼중음성유방암, 췌장암, 위암, 난소암 등등)로 개발에 응용할 수 있다.Antibodies discovered simultaneously with the technology of the present invention can be applied to detection of target proteins, diagnosis of cancer, and development as various anticancer therapeutics (e.g., cancer stem cells and breast cancer, triple negative breast cancer, pancreatic cancer, gastric cancer, ovarian cancer, etc.). .

도 1은 표현형 암 항원 및 항체 쌍의 동시 동정의 개략도,
도 2는 암줄기세포막에 특이적으로 결합하는 항체풀 검증. (A)라운드별 항체 풀의 암 줄기세포(CSC) 및 PBMC에 대한 binding histogram. control는 해당 세포에 마우스 항 M13 파지 항체와 항-마우스-형광 표지 항체가 결합한 historgram. (B)라운드별 항체 풀의 relative binding level
도 3은 암 줄기 세포주에 특이적인 선별 항체,
도 4는 (A)scFv PCR product(~770bp)을 agarose electrophoresis로 분석. (B)정제된 scFv-Fc를 reducing (R) and non-reducing(NR) SDS-PAGE로 분석,
도 5는 암 줄기 세포주에 대한 scFv-Fc 항체의 특이적 결합력. (A)유동세포분석법과 (B) 암 줄기 세포 표면에 특이적으로 결합하는 1G11항체에 대한 면역형광염색법,
도 6은 선별 항체의 타겟 항원 동정과정,
도 7은 (A)선별된 암 줄기세포 3C7 항체의 LC-MS/MS 분석 및 (B)3C7 항체의 항원 단백질 탐색 결과. 3C7 타겟 항원을 항원유전자를 knock out된 세포주와 상용항체를 이용하여 항체의 항원 검증함,
도 8은 (A)선별 항체 1E12의 LC-MS/MS 결과. (B) 타겟 항원에 대한 선별 항체 1E12와 2E1의 항원에 binding하는 것을 ELISA로 검증함,
도 9는 3C7 항체의 유방암 결합특이성,
도 10은 1E12 및 2E1 항체의 유방암 세포주에 대한 결합 특이성,
도 11은 1E12 항체의 위암, 췌장암, 난소암 세포주에 대한 결합 특이성,
도 12는 타겟 단백질 발현 level의 correlation 분석. (A)Proteomics database으로부터 알려진 각각 세포당 타겟 항원의 relative 발현양상 수준. (B)유동세포분석법을 통해 확인된 각각 세포당 타겟 항원 relative 발현양상 수준,
도 13은 1E12 항체와 3C7 항체의 in vitro 세포증식 저해 효과 (A)암 줄기세포와 parent 암세포에 대한 1E12 와 3C7항체의 세포증식저해 효능검증. (B)암 줄기 세포주에서 1E12 항체의 타겟항원의 리간드 유무에 따른 효능변화 평가. (C)4종의 유방암세포주 subtype과 1종의 위암세포주에 대한 1E12항체의 세포증식저해 효능검증,
도 14는 ADC 항체 2차 항체를 표지한 1E12, 2E1 항체의 효능평가. (A)ADC 2차 항체만 MDA-MB-453(CSC), MDA-MB-231(TNBC)에 각각 처리하였을 때, 세포에 대한 cytotoxic effect이 없음을 확인됨. (B)MDA-MB-453(CSC) 또는 (C)MDA-MB-231(TNBC)에 1E12항체와 Fab anti-Fc-MMAF(30nM)를 동시 처리하였을 때 1E12항체의 세포증식제어 효능평가. (D)MDA-MB-453(CSC)에 2E1항체와 IgG anti-Fc-MMAF(6.7nM)와 동시 처리하였을 때 2E1항체의 세포증식제어 효능평가, 및
도 15는 a) 3C7 항체, b) 1E12 항체 및 c) 2E1 항체의 아미노산 서열을 나타낸 그림으로 CDR region은 밑줄 친 청색으로 표시하였음.1 is a schematic diagram of simultaneous identification of phenotypic cancer antigen and antibody pairs,
2 is a verification of an antibody pool that specifically binds to cancer stem cell membranes. (A) Binding histogram for cancer stem cells (CSC) and PBMC of each round of antibody pools. Control is a historgram in which a mouse anti-M13 phage antibody and an anti-mouse-fluorescent-labeled antibody are bound to the cell. (B) Relative binding level of the antibody pool by round
3 is a selection antibody specific for a cancer stem cell line,
4 is an analysis of (A) scFv PCR product (~770bp) by agarose electrophoresis. (B) analyzing purified scFv-Fc by reducing (R) and non-reducing (NR) SDS-PAGE,
5 is a specific binding force of the scFv-Fc antibody to the cancer stem cell line. (A) flow cytometry and (B) immunofluorescence staining for 1G11 antibody that specifically binds to the surface of cancer stem cells,
6 is a process for identifying a target antigen of a selection antibody,
7 is (A) LC-MS/MS analysis of selected cancer stem cell 3C7 antibody and (B) antigen protein search result of 3C7 antibody. Antigen verification of 3C7 target antigen using a cell line knocked out of the antigen gene and a commercial antibody,
8 is (A) LC-MS/MS results of the selected antibody 1E12. (B) The binding to the antigens of the selection antibodies 1E12 and 2E1 against the target antigen was verified by ELISA,
9 is a breast cancer binding specificity of 3C7 antibody,
Figure 10 is the binding specificity of 1E12 and 2E1 antibodies to breast cancer cell lines,
Figure 11 is the binding specificity of the 1E12 antibody for gastric cancer, pancreatic cancer, and ovarian cancer cell lines,
12 is a correlation analysis of the target protein expression level. (A) Relative expression levels of target antigens per cell known from the Proteomics database. (B) the level of relative expression of the target antigen per cell, as determined by flow cytometry,
Figure 13 shows the in vitro cell proliferation inhibitory effect of 1E12 antibody and 3C7 antibody (A) Verification of cell proliferation inhibitory efficacy of 1E12 and 3C7 antibodies against cancer stem cells and parent cancer cells. (B) Evaluation of changes in efficacy according to the presence or absence of a ligand of the target antigen of the 1E12 antibody in a cancer stem cell line. (C) Verification of cell proliferation inhibitory efficacy of 1E12 antibody against 4 subtypes of breast cancer cell lines and 1 type of gastric cancer cell line,
Figure 14 is the efficacy evaluation of the 1E12, 2E1 antibodies labeled with the ADC antibody secondary antibody. (A) When only the ADC secondary antibody was treated with MDA-MB-453 (CSC) and MDA-MB-231 (TNBC), it was confirmed that there was no cytotoxic effect on cells. (B) MDA-MB-453 (CSC) or (C) MDA-MB-231 (TNBC) with 1E12 antibody and Fab anti-Fc-MMAF (30 nM) simultaneously treated with 1E12 antibody for cell proliferation control efficacy evaluation. (D) Evaluation of the cell proliferation control efficacy of 2E1 antibody when treated with 2E1 antibody and IgG anti-Fc-MMAF (6.7 nM) to MDA-MB-453 (CSC) simultaneously
15 is a diagram showing the amino acid sequences of a) 3C7 antibody, b) 1E12 antibody, and c) 2E1 antibody. CDR regions are indicated in underlined blue.

이하 비한정적인 실시예를 통하여 본 발명을 더욱 상세하게 설명한다. 단 하기 실시예는 본 발명을 예시하기 위한 의도로 기재한 것으로서 본 발명의 범위는 하기 실시예에 의하여 제한되는 것으로 해석되지 아니한다. Hereinafter, the present invention will be described in more detail through non-limiting examples. However, the following examples are described with the intention of illustrating the present invention, and the scope of the present invention is not to be construed as being limited by the following examples.

본 발명에서 사용된 모든 세포주는 별도의 명시가 없을 경우 공급자 지침에 따라 10% FBS, 1% penicillin/streptomycin이 포함된 배양배지를 사용하여 각각 37°C, 5% CO2에서 배양하였다. 유방암 줄기세포주 및 유방암 세포주는 10% FBS, 1% penicillin/streptomycin이 포함된 DMEM(Gibco), CHO-DG44 세포는 10% FBS, 1% penicillin/streptomycin, HT supplement(sodium hypoxanthine, thymidine 혼합물)가 포함된 DMEM/F12 HEPES(Gibco)으로 각각 37°C, 5% CO2 incubator에서 배양하였다. HEK293F 세포주는 37°C, 8% CO2 조건에서 단백질발현용 배지 freestyle 293F(GIBCO)을 사용하여 배양하였다.All cell lines used in the present invention were cultured at 37°C and 5% CO2 using a culture medium containing 10% FBS and 1% penicillin/streptomycin according to the supplier's instructions unless otherwise specified. Breast cancer stem cell lines and breast cancer cell lines contain DMEM (Gibco) containing 10% FBS, 1% penicillin/streptomycin, 10% FBS, 1% penicillin/streptomycin, and HT supplement (sodium hypoxanthine, thymidine mixture) for CHO-DG44 cells. DMEM/F12 HEPES (Gibco) was incubated at 37°C and 5% CO2 incubator, respectively. The HEK293F cell line was cultured using freestyle 293F (GIBCO) as a protein expression medium at 37°C and 8% CO2.

실시예Example 1:암1: Cancer 줄기세포에 대한 항체 및 항원 발굴 및 검증 Identification and verification of antibodies and antigens for stem cells

1)암 줄기세포주에 결합하는 항체 발굴 및 검증 1) Discovery and verification of antibodies that bind to cancer stem cell lines

암 줄기세포막 단백체의 표현형 구조(native phenotypic structure/microenvironment of the target protein)를 인식하는 항체를 발굴하고자, 기존의 잘 알려진(George Smith, Science 228, 1315-1317(1985); McCafferty et al., Nature 348, 552-554 (1990); Azzazy and Highsmith, Clin Biochem 35, 425-445 (2002); Smith and Petrenko, Phage Display, Chem Rev 97, 391-410 (1997); Daugherty, Curr Opin Struct Biol 17, 474-480 (2007)) 파지디스플레이 기법을 적용하여 암줄기 세포주에 바이오패닝을 진행하였다. To discover an antibody that recognizes the native phenotypic structure/microenvironment of the target protein of cancer stem cell membrane proteins, the existing well-known (George Smith, Science 228, 1315-1317 (1985); McCafferty et al., Nature) 348, 552-554 (1990); Azzazy and Highsmith, Clin Biochem 35, 425-445 (2002); Smith and Petrenko, Phage Display, Chem Rev 97, 391-410 (1997); Daugherty, Curr Opin Struct Biol 17, 474-480 (2007)) Biopanning was performed on cancer stem cell lines by applying a phage display technique.

암 줄기세포주에 결합하는 항체가 발굴되었는지 확인하기 위하여 각 라운드별로 파지항체 풀(pool)을 발현 후 암 줄기세포 및 PBMC세포에 결합력을 유동세포분석법으로 다음과 같이 진행하였다. In order to confirm whether an antibody binding to a cancer stem cell line was discovered, a pool of phage antibodies was expressed for each round, and the binding force to the cancer stem cells and PBMC cells was analyzed by flow cytometry as follows.

바이오패닝으로 확보한 라운드 1에서 라운드 4까지 파지 항체풀을 각각 발현시킨 후 1x106 세포와 1시간 동안 4°C incubation하여 결합시키고 버퍼(1%BSA, 0.03%NaN3, pH7.4 in PBS)로 3번 세척하였다. 마우스 항 M13 파지항체(Thermo scientific)와 Alexa Flour® 647 goat 항마우스 항체(Jackson Immunoresearch)를 각각 차례대로 넣어주어 세포에 특이적으로 결합한 파지항체를 형광 표지하였다.After expressing each of the phage antibody pools from round 1 to round 4 secured by biopanning, incubation with 1x10 6 cells for 1 hour at 4 °C to bind and buffer (1% BSA, 0.03% NaN3, pH 7.4 in PBS). Washed 3 times. Mouse anti-M13 phage antibody (Thermo scientific) and Alexa Flour® 647 goat anti-mouse antibody (Jackson Immunoresearch) were added in turn, respectively, and the phage antibody specifically bound to the cells was fluorescently labeled.

FACS calibur(BD Bioscience)와 소프트웨어 CellQuest Pro를 사용하여 항체에 표지된 형광 detection 및 data 분석을 진행하였다.FACS calibur (BD Bioscience) and software CellQuest Pro were used to detect and analyze the fluorescence labeled on the antibody.

상기 실시예의 결과 바이오패닝 라운드를 거듭 진행할수록 암 줄기세포주에 대한 결합력이 증가하지만, PBMC에 대한 결합력은 증가하지 않음을 유동세포분석법으로 확인할 수 있었다. 따라서 바이오패닝을 통해 암 줄기세포에 특이적으로 결합하는 항체 발굴 및 항체풀이 확보되었음을 확인할 수 있었다(도 2).As a result of the above example, it was confirmed by flow cytometry that the binding force to the cancer stem cell line increased as the biopanning round was repeated, but the binding force to PBMC did not increase. Therefore, it was confirmed that antibodies that specifically bind to cancer stem cells and an antibody pool were secured through biopanning (FIG. 2).

2) 암 줄기세포주에 결합하는 항체 선별2) Selection of antibodies that bind to cancer stem cell lines

암 줄기세포주에 특이적으로 결합하는 단일클론 항체를 선별하고자 항체 풀로부터 single colony를 분리하여 각 단일클론 항체의 암 줄기세포주에 대한 결합력을 위에서 설명한 유동세포분석법과 동일하게 진행하여 분석하였다.In order to select monoclonal antibodies that specifically bind to cancer stem cell lines, a single colony was isolated from the antibody pool, and the binding strength of each monoclonal antibody to cancer stem cell lines was analyzed in the same manner as in the flow cytometry method described above.

분석 진행한 단일클론 항체 중 정상세포에는 결합하지 않으면서 암 세포주에만 특이적으로 결합하는 단일클론 항체를 선별하였으며, 대표적인 항체의 분석결과를 도 3에 나타내었다. Among the analyzed monoclonal antibodies, monoclonal antibodies that specifically bind to cancer cell lines without binding to normal cells were selected, and the analysis results of representative antibodies are shown in FIG. 3.

3) 암 줄기세포에 대한 항체 발현 및 정제 3) Antibody expression and purification against cancer stem cells

상기 선별된 항체는 파지-scFv에서 whole IgG와 유사한 기능을 하는 scFv-Fc 항체 형태로 제작하기 위하여, 항체의 scFv를 PCR기법으로 증폭하고 동물세포 발현용 vector에 scFv-Fc 항체로 sub-cloning하였다. cloning 완료 후, HEK293F에 일시적으로 transfection하여 6일 동안 발현한 후 상층액을 확보하여 western blot으로 항체 발현 여부를 검증한 후 Protein A resin을 이용하여 affinity chromatography로 각각의 항체를 정제하고 농축하여 확보하였다.In order to produce the selected antibody in a form of scFv-Fc antibody that functions similar to whole IgG in phage-scFv, the scFv of the antibody was amplified by PCR method and sub-cloning with scFv-Fc antibody in an animal cell expression vector. . After cloning was completed, transiently transfected into HEK293F and expressed for 6 days, and then the supernatant was secured to verify antibody expression by western blot, and each antibody was purified and concentrated by affinity chromatography using Protein A resin. .

동물세포 발현 기반 parent vector에 sub-cloning하기 위해 각 항체의 증폭된 scFv fragment를 대략 770bp 크기에서 확인하였으며(예시 도 4A), affinity chromatography를 통해 정제된 항체를 reducing 또는 non-reducing 조건으로 각각 SDS-PAGE 분석한 결과 항체 시퀀스기반의 예측 항체크기와 유사한 크기에서 항체 band를 확인하였다(도 4B, reduced form: ~50kDa, non-reduced form: ~ 100kDa).In order to sub-cloning into the parent vector based on animal cell expression, the amplified scFv fragment of each antibody was confirmed at a size of approximately 770 bp (example Fig. 4A), and the purified antibody through affinity chromatography was reduced or non-reducing conditions, respectively, SDS- As a result of PAGE analysis, an antibody band was confirmed at a size similar to that of the predicted antibody size based on the antibody sequence (Fig. 4B, reduced form: ~50kDa, non-reduced form: ~100kDa).

따라서 파지항체로부터 scFv-Fc 항체를 확보 및 검증하였다. Therefore, the scFv-Fc antibody was obtained and verified from the phage antibody.

4) 암 줄기세포 항체의 특성분석 (FACS and IF)4) Characterization of cancer stem cell antibodies (FACS and IF)

scFv-Fc 항체의 암줄기세포주에 대한 결합력은 상기의 유동세포분석법으로 동일하게 진행하였다. 또한 암 줄기세포 표면에 항체의 localization을 다음과 같은 방법으로 면역 형광 염색하여 관찰하였다. The binding force of the scFv-Fc antibody to the cancer stem cell line was performed in the same manner by the above flow cytometry. In addition, the localization of the antibody on the surface of cancer stem cells was observed by immunofluorescence staining in the following manner.

Culture dish에 약 70% confluence로 배양한 암 줄기세포, 암세포, CHO-DG44에 각각 10μg/mL scFv-Fc 항체를 넣고 1시간 동안 ice 위에서 결합을 진행하였다. PBS로 두 번 세척하여 결합하지 않은 항체를 제거한 후, 4% paraformaldehyde를 넣어주어 상온에서 20분 동안 Fix과정을 진행하였다. Fix가 끝나면 PBS로 두 번 세척하고 난 후 바로 Alexa594 항-human IgG(Jackson Immunoresearch)로 scFv-Fc를 표지하였디. 두 번의 세척을 거친 후에, 300nM DAPI를 넣어주어 핵을 염색시키고 모든 반응이 끝나면 3번 세척한 후 형광현미경(Olympus 1x71)으로 세포를 관찰하고 image를 관찰하였다. 10 μg/mL scFv-Fc antibody was added to each of cancer stem cells, cancer cells, and CHO-DG44 cultured with about 70% confluence in a culture dish, and binding was performed on ice for 1 hour. After washing twice with PBS to remove unbound antibody, 4% paraformaldehyde was added and the Fix process was performed at room temperature for 20 minutes. After the fix was finished, it was washed twice with PBS and then immediately labeled scFv-Fc with Alexa594 anti-human IgG (Jackson Immunoresearch). After two washings, 300nM DAPI was added to stain the nucleus. After all reactions were completed, the cells were washed 3 times, and then the cells were observed with a fluorescence microscope (Olympus 1x71) and the image was observed.

scFv-Fc 형태 항체의 유동세포분석결과 검증한 모든 항체가 파지항체와 동일하게 암 줄기세포주에 강하게 결합하면서 상대적으로 암 세포주에서는 약한 결합력을 갖고, 정상세포(CHO)에서는 완전히 결합하지 않음을 확인하였다(도 5A). As a result of flow cytometric analysis of the scFv-Fc type antibody, it was confirmed that all the verified antibodies strongly bind to the cancer stem cell line in the same way as the phage antibody, but have relatively weak binding power in the cancer cell line, and not completely bind in the normal cells (CHO). (Figure 5A).

또한 면역 형광염색에서 선별된 항체는 암 줄기세포 표면에 강하게 결합하면서 암 세포주에는 극히 일부분에서만 형광이 관찰되며 또한 정상세포에서는 형광표지가 되지 않다. 암 줄기세포 표면에만 특이도로 결합하는 항체의 예시로 도 5B에 표시하였다. In addition, the antibody selected by immunofluorescence staining strongly binds to the surface of cancer stem cells, and fluorescence is observed only in a small part of cancer cell lines, and is not fluorescently labeled in normal cells. It is shown in Fig. 5B as an example of an antibody that specifically binds only to the surface of cancer stem cells.

따라서 유동세포분석법을 통해 분별된 암 줄기세포 및 암세포에 대한 항체 결합력 차이를 면역 형광염색을 통해 재검증하였으며, 선별된 암 줄기세포막 항체는 암세포 대비 암 줄기세포에 대한 특이도가 더 높은 것이 검증되었다.Therefore, the difference in antibody binding ability to cancer stem cells and cancer cells identified through flow cytometry was re-verified through immunofluorescence staining, and the selected cancer stem cell membrane antibody was verified to have higher specificity for cancer stem cells compared to cancer cells. .

5) 암 줄기세포 항체의 암 줄기세포막 타겟항원 동정 및 검증 (In situ IP 및 LC-MS/MS)5) Identification and verification of cancer stem cell membrane target antigen of cancer stem cell antibody ( In situ IP and LC-MS/MS)

암 줄기세포에 특이적으로 결합하는 선별 항체의 타겟항원 동정을 위하여 in-situ 면역침강 기법으로 암 줄기세포 항체가 인식하는 암세포막 항원을 다음과 같은 방법으로 pull-down하였다(도 5). In order to identify the target antigen of the selection antibody that specifically binds to cancer stem cells, the cancer cell membrane antigen recognized by the cancer stem cell antibody was pulled down by the following method by the in-situ immunoprecipitation technique (FIG. 5).

암 줄기세포 및 암세포에 culture plate에 배양한 후 각각의 세포주에 40μg의 IgG 또는 scFv-Fc 항체 단편을 넣고 1.5시간 동안 4°C에서 항체결합 반응을 진행하였다. 세포막 항원에 결합되지 않은 항체는 PBS로 3번 세척하여 제거한 후 lysis buffer(25mM HEPES, 150mM NaCl, 1% NP-40, 5mM MgCl2, protease inhibitor)를 사용하여 암줄기세포 및 암세포 lysate을 획득하였다. 세포 lysates은 5분 동안 4500rpm으로 spin down하여 상층액을 분리하였다. 분리한 상층액은 protein A resin으로 pull down을 진행한 후 50mM glycine을 사용하여 정제된 세포표면 단백체-항체 결합체를 proein A resin으로부터 분리하였다. 획득한 세포표면 단백체-항체 결합체 샘플은 western blot 및 SDS-PAGE로 분석하고 in-gel digestion한 후 LC-MS/MS peptide 분석을 통해 암줄기세포 항체가 인식하는 특이적 암줄기세포막 단백질을 식별하였다(도 6). After culturing cancer stem cells and cancer cells on a culture plate, 40 μg of IgG or scFv-Fc antibody fragment was added to each cell line, followed by antibody-binding reaction at 4°C for 1.5 hours. Antibody not bound to the cell membrane antigen was removed by washing 3 times with PBS, and then cancer stem cells and cancer cell lysate were obtained using lysis buffer (25mM HEPES, 150mM NaCl, 1% NP-40, 5mM MgCl2, protease inhibitor). Cell lysates were spin down at 4500 rpm for 5 minutes to separate the supernatant. The separated supernatant was pulled down with protein A resin, and then the purified cell surface protein-antibody conjugate was isolated from proein A resin using 50mM glycine. The obtained cell surface protein-antibody conjugate sample was analyzed by western blot and SDS-PAGE, in-gel digestion, and then LC-MS/MS peptide analysis to identify specific cancer stem cell membrane proteins recognized by cancer stem cell antibodies ( Fig. 6).

식별된 암줄기세포 타겟 단백질 validation을 위한 ELISA 분석방법은 다음과 같이 진행하였다. The ELISA analysis method for validation of the identified cancer stem cell target protein was carried out as follows.

96-well ELISA plate에 100nM의 재조합 단백질을 50mM NaHCO3, pH9 버퍼로 overnight coating한 후 PBS 버퍼로 washing 한 후, PBS/3% milk로 blocking 및 다시 PBS washing이 끝난 다음 선별된 항체 및 huIgG 항체를 각각 1μg/mL로 넣고 1.5시간 동안 상온에서 반응시켰다. PBS washing 진행 후 anti-huIgG-HRP 항체를 1/5000 희석하여 각 plate well에 넣고 상온에서 1시간 반응시켰다. PBS로 washing을 여섯 번 진행한 후 TMB substrate을 넣고 반응시킨 후, 0.25M sulphuric acid로 HRP 반응을 stop하여 450nm에서 signal을 detection하였다.After coating 100 nM of recombinant protein on a 96-well ELISA plate with 50 mM NaHCO3, pH9 buffer overnight, washing with PBS buffer, blocking with PBS/3% milk and washing with PBS again, the selected antibodies and huIgG antibodies were respectively It was put at 1 μg/mL and reacted at room temperature for 1.5 hours. After washing with PBS, the anti-huIgG-HRP antibody was diluted 1/5000, put into each plate well, and reacted at room temperature for 1 hour. After washing with PBS six times, TMB substrate was added to react, and the HRP reaction was stopped with 0.25M sulphuric acid to detect a signal at 450nm.

선별된 암줄기세포 항체와 pull-down된 암줄기세포막 타겟 항원을 LC-MS/MS로 분석하였다. 검출된 peptide의 mass/charge ratio 분석 및 peptide 서열을 PD 2.2, PEAKS, Sorcerer 등 단백질 탐색 tool을 사용하여 항체가 인식하는 암 줄기세포막 타겟 항원을 식별하였다. The selected cancer stem cell antibody and pull-down cancer stem cell membrane target antigen were analyzed by LC-MS/MS. The mass/charge ratio analysis of the detected peptide and the peptide sequence were identified using a protein search tool such as PD 2.2, PEAKS, and Sorcerer to identify the cancer stem cell membrane target antigen recognized by the antibody.

[예시 1] 선별된 3C7 항체의 경우 암줄기세포막 타겟 항원이 CD44인 것으로 LC-MS/MS 분석으로 식별되었고, CD44 특이적 상용항체와 3C7항체가 CD44이 발현하는 세포주 (CD44+), CD44 gene knockout 세포주 (CD44-)에 결합하는 것을 비교분석하여 3C7항체가 인식하는 암줄기세포막 타겟 단백질이 CD44임을 검증하였다(도 7).[Example 1] In the case of the selected 3C7 antibody, the cancer stem cell membrane target antigen was identified by LC-MS/MS analysis, and CD44-specific commercial antibody and 3C7 antibody were CD44-expressing cell lines (CD44+), and CD44 gene knockout. By comparative analysis of binding to the cell line (CD44-), it was verified that the cancer stem cell membrane target protein recognized by the 3C7 antibody was CD44 (FIG. 7).

[예시 2] 선별된 1E1항체를 항원동정실험을 진행하여 LC-MS/MS로 얻어진 peptide를 분석하여 항원 후보군으로 CD71이 식별되었다(도 8A). [Example 2] The selected 1E1 antibody was subjected to an antigen identification experiment, and the peptide obtained by LC-MS/MS was analyzed to identify CD71 as an antigen candidate group (Fig. 8A).

식별된 타겟 항원 CD71에 선별항체가 binding하는지 ELISA로 분석한 결과, 1E12항체와 2E1항체가 CD71 단백질(Pro1)에 특이적으로 강한 결합력을 갖는 것을 관찰하였다. 음성대조군으로 사용된 BSA나 CTLA-4 및 그 외 세포막 단백질 (Pro2~Pro6)에는 결합하지 않았다(도 8B).As a result of analysis by ELISA to see if the selection antibody binds to the identified target antigen CD71, it was observed that the 1E12 antibody and the 2E1 antibody specifically had strong binding power to the CD71 protein (Pro1). It did not bind to BSA or CTLA-4 and other cell membrane proteins (Pro2 to Pro6) used as a negative control (Fig. 8B).

암 줄기세포 항체가 특이적으로 결합하는 암 줄기세포막 단백질은 종양 성장, 암 전이, invadopodia 형성 및 림프구 활성화 등 종양과 밀접한 연관성이 있음이 알려진 단백질이었다(표 1). Cancer stem cell membrane proteins to which cancer stem cell antibodies specifically bind were known proteins that are closely related to tumors such as tumor growth, cancer metastasis, invadopodia formation, and lymphocyte activation (Table 1).

특이 CD44은 암 줄기세포 바이오마커로 응용되고 있으며 다중약물 내성을 촉진하는 것으로 보고되었다(참고문헌: Ravindranath et al., CD44 promotes multi-drug resistance by protecting P-glycoprotein from FBXO21-mediated ubiquitination, Oncotarget 22, 26308-26321(2015)). Specific CD44 has been applied as a cancer stem cell biomarker and has been reported to promote multi-drug resistance (Reference: Ravindranath et al., CD44 promotes multi-drug resistance by protecting P-glycoprotein from FBXO21-mediated ubiquitination, Oncotarget 22, 26308-26321 (2015)).

따라서 본 발명 기술법으로 암 줄기세포 및 그 외 특이적 질병세포를 대상으로 과발현 또는 특이적으로 발현하는 단백체 발굴 및 이에 대한 항체 발굴을 동시 진행을 하여 바이오마커 및 치료제 개발을 위한 타겟 발굴과 진단 및 치료용 항체 발굴 가능함을 증명하였다. Therefore, by using the present technology method to discover overexpressed or specifically expressed proteins and antibodies to cancer stem cells and other specific disease cells, the discovery and diagnosis and treatment of targets for biomarkers and therapeutics development It was proved that it was possible to find an antibody for use.

Figure pat00001
Figure pat00001

표 1은 암줄기세포 특이적 항체의 타겟 exclusive spectrum count 및 보고된 타겟 항원의 암 관련 functionTable 1 shows the target exclusive spectrum count of cancer stem cell-specific antibodies and cancer-related functions of the reported target antigens.

실시예Example 2: 발굴된 2: excavated 암줄기세포Cancer stem cells 항원 및 Antigen and 항체에 대한 응용 방법Application method for antibodies 예시 example

본 발명 방법으로 발굴되는 단백질이 유방암 및 그 외 암 질환에 대한 바이오마커 및 치료제 개발의 타겟으로 응용 가능함과 동시에 본 발명의 방법으로 발굴한 항체를 진단 및 치료제로 응용 가능함을 다음과 같은 방법으로 입증하였다. The following method proves that the protein discovered by the method of the present invention can be applied as a target for the development of biomarkers and therapeutic agents for breast cancer and other cancer diseases, and the antibody discovered by the method of the present invention can be applied as a diagnosis and treatment. I did.

1) 유방암, 위암, 췌장암, 난소암 세포주에 대한 선별항체의 결합1) Binding of selection antibodies against breast cancer, gastric cancer, pancreatic cancer, and ovarian cancer cell lines

선별항체 3C7, 1E12, 2E1 항체가 유방암 subtype, 위암, 췌장암, 난소암 등의 다양한 세포주에 결합하는지 유동세포분석법으로 분석하였으며, 앞서 설명한 scFv-Fc항체의 암 줄기세포 결합력 분석과 동일한 방법으로 진행하였다. 유방암 subtype 세포로는 삼중음성 유방암(TNBC; MDA-MB-468, MDA-MB-231, BT-20, MDA-MB-453) 및 ZR-75-1, BT-474, MCF-7, T47D, SKBR-3(표 2)을 사용하였고, 위암으로는 MKN45, NCI-87을, 췌장암 PANC-1, 난소암 SKOV-3 세포주(도 9~ 도 12)를 사용하였다. Selective antibodies 3C7, 1E12, 2E1 antibodies were analyzed by flow cytometry to determine whether they bind to various cell lines such as breast cancer subtype, gastric cancer, pancreatic cancer, ovarian cancer, etc., and proceeded in the same manner as the analysis of cancer stem cell binding ability of the scFv-Fc antibody described above. . Breast cancer subtype cells include triple negative breast cancer (TNBC; MDA-MB-468, MDA-MB-231, BT-20, MDA-MB-453) and ZR-75-1, BT-474, MCF-7, T47D, SKBR-3 (Table 2) was used, and as gastric cancer, MKN45, NCI-87, pancreatic cancer PANC-1, and ovarian cancer SKOV-3 cell lines (FIGS. 9 to 12) were used.

Figure pat00002
Figure pat00002

표 2는 유방암 세포주 subtypeTable 2 shows the breast cancer cell line subtype

[A]암 줄기세포주 바이오마커로 알려진 CD44를 타겟 항원으로 인지하는 3C7항체가 암 줄기세포주에 특이적으로 제일 높은 겹합력이 있음이 재검증되었다(도 9). 그리고 유방암 세포 중에서 BT-20에만 아주 약하게 결합하는 것이 관찰되었고 그 외 유방암 세포주에 모두 결합하지 않았다. [A] Cancer stem cell line It was re-verified that the 3C7 antibody, which recognizes CD44, known as a biomarker, as a target antigen has the highest binding ability specifically for cancer stem cell lines (FIG. 9). In addition, among breast cancer cells, it was observed that it binds only to BT-20 very weakly, and not all other breast cancer cell lines.

따라서 본 발명의 방법이 암 줄기세포주 특이적인 항원 및 항체 발굴 및 선별 가능함을 재검증 되었으며 본 발명의 방법으로 신규 치료용 표적 항원 및 이에 대한 항체를 동시 발굴하여 진단 및 치료법 개발이 가능함이 검증되었다. Therefore, it was re-verified that the method of the present invention can discover and select antigens and antibodies specific to cancer stem cell lines, and it was verified that the method of the present invention can simultaneously discover new therapeutic target antigens and antibodies thereto to diagnose and develop therapeutics.

또한, 흥미롭게도 본 연구로 인해 3C7 항체가 NCI-87에 약하게 결합됨이 관찰됨에 따라 CD4를 표적한 위암치료제 개발이 가능할 것으로 인지된다. In addition, interestingly, as it was observed that the 3C7 antibody weakly binds to NCI-87 due to this study, it is recognized that it is possible to develop a gastric cancer therapeutic agent targeting CD4.

CD44 배경CD44 background

3C7 항체가 타겟하는 CD44 항원은 세포막에 발현되는 단백질로서 세포외 기질 (ECM) 상에 존재하는 히알루론산 (hyaluronic acid,, HA), osteopontin (OPN), chondroitin, collagen, fibronectin, and serglycin/sulfated proteoglycan를 포함한 여러 리간드와 결합하여 활성화되며, 그 중 HA는 CD44 활성화를 위한 가장 특이적인 리간드로 보고된다(참고문헌: Chen Chen, Shujie Zhao, Anand Karnad and James W. Freeman Journal of Hematology & Oncology 2018;11:64).The CD44 antigen targeted by the 3C7 antibody is a protein expressed on the cell membrane and is present on the extracellular matrix (ECM), hyaluronic acid (HA), osteopontin (OPN), chondroitin, collagen, fibronectin, and serglycin/sulfated proteoglycan. Among them, HA is reported as the most specific ligand for CD44 activation (Reference: Chen Chen, Shujie Zhao, Anand Karnad and James W. Freeman Journal of Hematology & Oncology 2018; 11 :64).

CD44와 HA의 결합으로 인해 CD44의 세포 내 cytoplasmic tail과 어댑터 분자의 결합이 잘되도록 구조 변화를 일으키며 이러한 구조 변화를 통해 세포의 상호작용 및 Ras, MAPK, PI3K, Rho 및 NFκB 등 신호 전달 경로를 활성화하여 종양 질환 발달에 기여한다. 특히 위 신호 전달 경로는 세포 주기 및 증식의 활성화, 세포골격 재구성 및 세포 이동, 세포 생존 및 염증 반응 등의 신호 전달 경로 등에 관여하는 것으로 알려져 있으며, 또한 D44의 발현수준이 암 전이에 중요 인자인 EMT와 연관성 있음이 보고되었으며 이는 ECM을 통한 종양 침윤성 및 혈관신생 유도의 경향을 잠재적으로 증가시킬 수 있다(참고문헌: Cho SH, Park YS, Kim HJ, Kim CH, Lim SW, Huh JW, Lee JH, Kim HR. Int J Oncol. 2012;41(1):211-8).The combination of CD44 and HA causes structural changes to facilitate the binding of the cytoplasmic tail of CD44 to the adapter molecule, and through these structural changes, cell interactions and signaling pathways such as Ras, MAPK, PI3K, Rho and NFκB are activated. Thus contributes to the development of tumor diseases. In particular, the above signaling pathway is known to be involved in the activation of cell cycle and proliferation, cytoskeleton reconstruction and cell migration, cell survival, and signaling pathways such as inflammatory response, and the expression level of D44 is EMT, an important factor in cancer metastasis. Has been reported to be associated with, and this may potentially increase the tendency of tumor invasiveness and angiogenesis through ECM (References: Cho SH, Park YS, Kim HJ, Kim CH, Lim SW, Huh JW, Lee JH, Kim HR. Int J Oncol. 2012;41(1):211-8).

유방암 세포에서 CD44와 HA의 결합은 다중 약물 내성 유전자 P-glycoprotein 및 항-세포 사멸 유전자 Bcl의 발현 증가에 기여하며 그 결과 암세포의 증식과 생존을 촉진시키는 것으로 보고된다(참고문헌: Bourguignon LY, Xia W, Wong G. J Biol Chem. 2009;284(5):2657-71).The binding of CD44 and HA in breast cancer cells has been reported to contribute to increased expression of the multidrug resistance gene P-glycoprotein and the anti-apoptotic gene Bcl, and as a result promote the proliferation and survival of cancer cells (References: Bourguignon LY, Xia. W, Wong G. J Biol Chem. 2009;284(5):2657-71).

또한 CD44는 화학요법에 반응이 없는 암환자군 및 생존율이 낮은 암 환자군에서 과발현되어 있다(참고문헌:Gao Y, Feng Y, Shen JK, Lin M, Choy E, Cote GM, Harmon DC, Mankin HJ, Hornicek FJ, Duan Z. Sci Rep. 2015;5:11365). In addition, CD44 is overexpressed in cancer patients who do not respond to chemotherapy and cancer patients with low survival rates (Refs: Gao Y, Feng Y, Shen JK, Lin M, Choy E, Cote GM, Harmon DC, Mankin HJ, Hornicek FJ, Duan Z. Sci Rep. 2015;5:11365).

이는 CD44의 발현수준을 기반으로 암환자의 신규 치료 가이드라인 설정 및 환자 예후 측정을 할 수 있을 것으로 기대할 수 있다.This can be expected to be able to establish new treatment guidelines for cancer patients and measure patient prognosis based on the expression level of CD44.

종합적으로 CD44는 암 발달 과정에 많이 관여하며 그 중에서도 약물내성에 높은 연관성이 있음을 알 수 있다. 따라서 본 발명에서 개발된 항체 조성물이 CD44의 암세포 표면 발현을 인지하며 동시에 암세포 성장 및 전이억제에 기여하는 것으로 나타난다면 매우 유용한 진단 및 치료 과정이 실현될 것이다.Overall, it can be seen that CD44 is heavily involved in the process of cancer development, and has a high association with drug resistance among them. Therefore, if the antibody composition developed in the present invention recognizes the surface expression of CD44 on the surface of cancer cells and at the same time appears to contribute to the inhibition of cancer cell growth and metastasis, a very useful diagnostic and therapeutic process will be realized.

[B] CD71을 특이적으로 인식하는 1E12 항체와 2E1 항체는 유방암 줄기세포의 parent세포주 MDA-MB-453에도 특이적인 binding 특성을 보임을 확인하였다(도 10A). [B] It was confirmed that the 1E12 antibody and 2E1 antibody specifically recognizing CD71 showed specific binding properties to the parent cell line MDA-MB-453 of breast cancer stem cells (FIG. 10A).

또한 여러 유방암 세포주에 대한 CD71 발현률을 1E12 항체 binding을 FACS로 분석하였을 때, 대조군 항체보다 삼중음성 유방암을 포함한 그 외 모든 유방암세포주에 강하게 결합함을 확인되었으며 이는 CD71이 다양한 유방암 세포주에 발현됨에 치료제 개발 타겟으로도 적합함이 검증되었다(도 10).In addition, when the 1E12 antibody binding was analyzed by FACS for the CD71 expression rate of several breast cancer cell lines, it was confirmed that it binds more strongly to all other breast cancer cell lines, including triple negative breast cancer, than the control antibody. This is because CD71 is expressed in various breast cancer cell lines. It was verified that it is also suitable as a target (FIG. 10).

또한 1E12 항체는 위암 세포주(gastric cancer)와 췌장암 세포주(pancreatic cancer), 난소암 세포주(ovarian cancer)에도 특이적인 binding함을 확인하였다.In addition, it was confirmed that the 1E12 antibody specifically binds to gastric cancer, pancreatic cancer, and ovarian cancer.

따라서 CD71을 타겟하는 항체는 암 줄기세포주뿐만 아니라 다양한 암 종 세포에 강하게 결합하며 이를 제어하여 유방암 및 그 외 암질환 치료제에 사용할 수 있는 가능성이 있음을 확인되었다(도 11, 표 3). Therefore, it was confirmed that the antibody targeting CD71 strongly binds not only to cancer stem cell lines, but also to various carcinoma cells, and controls it to be used for the treatment of breast cancer and other cancer diseases (FIG. 11, Table 3).

Figure pat00003
Figure pat00003

표 3은 암 줄기세포 타겟항체의 여러 암종 세포주에 대한 특이적 결합 여부 Table 3 shows the specific binding of cancer stem cell target antibodies to various carcinoma cell lines

2) 유동세포분석법과 타겟 단백질 발현 correlation 분석을 통한 항원검증2) Antigen verification through flow cytometry and correlation analysis of target protein expression

본 발명에서 발굴된 세포막 타겟 단백질 protein X1B1의 발현수준(expression level)이 보고된 4종의 유방암 세포주 및 난소암 세포주를 대상으로 타겟 단백질 protein X1B1 발현수준을 유동세포분석법으로 비교분석하였다.The expression level of the target protein protein X1B1 was compared and analyzed by flow cytometry for 4 breast cancer cell lines and ovarian cancer cell lines in which the expression level of the cell membrane target protein X1B1 discovered in the present invention was reported.

ProteomicsDB(https ://www.ProteomicsDB.org)의 보고된 타겟 단백질 protein X1B1 발현비율과 실험으로 확보한 단백질 발현비율이 서로 일관 연관성(consistently correlation) 있음을 확인할 수 있었다(도 12). It was confirmed that the reported target protein protein X1B1 expression ratio in ProteomicsDB ( https://www.ProteomicsDB.org ) and the protein expression ratio obtained by the experiment were consistently correlated with each other (Fig. 12).

이와 같은 결과를 바탕으로 LC-MS/MS 분석을 통해 확보한 타겟 단백질과 항체의 결합 및 특이도를 재검증 할 수 있었다. Based on these results, the binding and specificity of the target protein and antibody secured through LC-MS/MS analysis could be re-verified.

3) 발굴된 항체의 세포 성장저해 효능평가 (In-vitro cell based assay)3) Cell growth inhibition efficacy evaluation of the discovered antibody (In-vitro cell based assay)

3x103 세포를 96-well plate 각 well에 분주하고 37°C, 5% CO2 조건에서 24시간 배양한 후 새로운 growth media로 교체한 다음 serial diluted된 1E12 항체 또는 대조군 항체를 구분하여 각 well에 첨가한 다음 약 4-6일간 세포를 37°C, 5% CO2조건에서 incubation한 다음 AlamarBlue (Invitrogen)를 처리하고 2, 4, 6시간 마다 Ex530, Em590에서 형광 값을 측정하였음(PerkinElmer Victor x4). Prism4(GraphPad)로 EC50 값을 분석하였다.Dispense 3x10 3 cells into each well of a 96-well plate, incubate at 37°C, 5% CO 2 for 24 hours, replace with new growth media, and add serially diluted 1E12 antibody or control antibody to each well. Then, the cells were incubated at 37°C and 5% CO 2 for about 4-6 days, and then treated with AlamarBlue (Invitrogen), and fluorescence values were measured in Ex530 and Em590 every 2, 4 and 6 hours (PerkinElmer Victor x4). . EC50 values were analyzed with Prism4 (GraphPad).

[예시]: 본 발명에서 발굴한 3C7 항체를 CD44 발현이 보고된 암 줄기세포주(CSC)에서 CD44를 발현하지 않는 대조군 세포주(parent 암세포) 대비 30% (p=0.0032, t-test) 이상의 세포성장 억제능력이 있음을 확인하였다(도 13A).[example]: The 3C7 antibody discovered in the present invention has the ability to inhibit cell growth by more than 30% (p=0.0032, t-test) compared to a control cell line (parent cancer cell) that does not express CD44 in a cancer stem cell line (CSC) in which CD44 expression is reported. Was confirmed (Fig. 13A).

CD44+ 또는 CD44-의 각각의 세포주에 항체를 처리하지 않은 세포의 성장률을 100%로 계산하였다. The growth rate of cells not treated with antibodies to each cell line of CD44+ or CD44- was calculated as 100%.

[예시]: 본 발명에서 발굴한 1E12 항체를 암 줄기세포주(CSC)에 처리하였을 때 세포 성장이 최대 32% 억제가 되었음을 확인할 수 있었다. 암 줄기세포주뿐만 아니라 parent 암세포(cancer cell)에서도 비슷한 세포성장 저해 효능을 나타내었다(도 13). [Example]: When the 1E12 antibody discovered in the present invention was treated with a cancer stem cell line (CSC), it was confirmed that cell growth was inhibited by up to 32%. A similar cell growth inhibitory effect was shown not only in cancer stem cell lines but also in parent cancer cells (FIG. 13 ).

대조군 항체 human IgG(hIgG)를 넣어준 CSC 및 cancer 세포에서는 효능이 없었다. 각 세포에 대한 EC50 값을 분석한 결과 암 줄기세포주에서 53.28 nM, parent암 세포주에서 18.33nM인 것으로 확인되었음(도 13A).There was no efficacy in CSC and cancer cells to which the control antibody human IgG (hIgG) was added. As a result of analyzing the EC50 value for each cell, it was found to be 53.28 nM in the cancer stem cell line and 18.33 nM in the parent cancer cell line (Fig. 13A).

1E12 항체가 타겟하는 CD71은 세포막에 발현되어 있는 수용체로서 iron이 결합된 transferrin(트렌스퍼린) ligand와 결합하여 세포성장에 기여하는 특성이 있다. 따라서 1E12항체가 transferrin ligand가 존재할 때에도 CD71과 결합하여 ligand와 CD71의 결합을 제어함과 이에 따른 세포 성장저해 효능 여부를 확인하였다. ligand를 첨가하였을 때도 암 줄기세포의 증식 저해효과를 유지하는 것으로 관찰되었다(도 13B). CD71, which is targeted by the 1E12 antibody, is a receptor expressed on cell membranes, and has a property of contributing to cell growth by binding to iron-bound transferrin (transferrin) ligand. Therefore, even in the presence of the transferrin ligand, the 1E12 antibody binds to CD71 and controls the binding of the ligand to CD71, and thus, it was confirmed whether the effect of inhibiting cell growth was confirmed. It was observed that even when the ligand was added, the effect of inhibiting proliferation of cancer stem cells was maintained (FIG. 13B).

또한 암 줄기세포 사멸에 있어 트렌스퍼린 ligand이 있을 시 ligand가 없을 때 보다도 더 높은 1E12 항체의 농도가 필요한 것으로 보아 1E1 항체가 transferrin ligand을 통해 매개된 암세포주 증식 활성화를 저해하는 것으로 확인되었다. In addition, it was confirmed that the 1E1 antibody inhibited the activation of cancer cell line proliferation mediated through the transferrin ligand, as a higher concentration of the 1E12 antibody was required when the transferrin ligand was present than in the absence of the ligand for cancer stem cell death.

또한 1E12 항체는 다양한 암 및 유방암 세포주 subtype에 강하게 binding하는 특성이 있음이 관찰되어 4종의 유방암 세포주 subtype과 1종의 위암 세포주의 세포증식저해 분석을 하였을 때에도 세포 증식저해 효능이 관찰되었다(도 13C). 따라서 본 발명에서 발굴된 CD71 단백질을 표적화하여 다양한 암치료제 개발을 할 수 있으며, 본 발명에서 개발한 1E12 항체가 삼중음성 유방암 및 그 외 항암치료제의 응용성을 검증되었다. 1E12항체의 각 세포주에 대한 효능 평가 결과를 표 4에 정리하였다. In addition, it was observed that the 1E12 antibody strongly binds to various cancer and breast cancer cell line subtypes, and thus cell proliferation inhibitory efficacy was observed even when cell proliferation inhibition analysis of 4 subtypes of breast cancer cell lines and 1 type of gastric cancer cell line was performed (Fig. 13C). ). Therefore, it is possible to develop various cancer treatment agents by targeting the CD71 protein discovered in the present invention, and the 1E12 antibody developed in the present invention has been verified for the applicability of triple negative breast cancer and other anticancer drugs. Table 4 summarizes the efficacy evaluation results for each cell line of the 1E12 antibody.

Figure pat00004
Figure pat00004

표 4는 1E12 항체의 각 세포주에 대한 효능Table 4 shows the efficacy of the 1E12 antibody for each cell line.

3) 항체-약물 결합체(Antibody-Drug Conjugate)의 세포 성장저해 효능평가 (In-vitro cell based assay)3) Cell growth inhibition efficacy evaluation of Antibody-Drug Conjugate (In-vitro cell based assay)

본 발명에서 발굴된 타겟에 대한 치료제 plateform으로는 naked 항체(whole IgG, 절편항체 등)외 Antibody-drug conjugate(ADC) 등 다양한 치료제 개발을 할 수도 있으므로, 본 발명에서 개발한 anti-CD71 항체, 1E12 항체와 2E1항체를 “ADC“ form으로 암세포주 증식저해를 점검하였다. In addition to the naked antibody (whole IgG, fragment antibody, etc.) as the therapeutic plateform for the target discovered in the present invention, various therapeutic agents such as Antibody-drug conjugate (ADC) can be developed, so the anti-CD71 antibody developed in the present invention, 1E12 Antibody and 2E1 antibody were tested for inhibition of proliferation of cancer cell lines in the form of "ADC".

ADC 2차 항체를 표지한 타겟 항체의 효능평가를 위해서는 항체의 세포 성장저해 효능평가와 동일 조건으로 세포에 항체를 처리하고 추가로 30nM Fab aHFc-MMAF 또는 6.6nM IgG aHFc-MMAF를 함께 처리 후 4일 incubation한 다음 AlamarBlue(Invitrogen)를 처리하고 2, 4, 6시간 마다 Ex530, Em590에서 형광 값을 측정하여(Victor x4, PerkinElmer ) EC50 값을 (Prism4, GraphPad)로 분석하였다.For the efficacy evaluation of the target antibody labeled with the ADC secondary antibody, the cells were treated with the antibody under the same conditions as for the cell growth inhibition efficacy evaluation of the antibody, and additionally treated with 30 nM Fab aHFc-MMAF or 6.6 nM IgG aHFc-MMAF. After day incubation, AlamarBlue (Invitrogen) was treated, and fluorescence values were measured in Ex530 and Em590 every 2, 4 and 6 hours (Victor x4, PerkinElmer), and EC50 values were analyzed with (Prism4, GraphPad).

암 줄기세포주에 anti-CD71항체(1E12 또는 2E1)와 MMAF이 결합된 2차 항체와 함께 처리하였을 때 유방암 줄기세포 증식저해 효능이 기존 naked 항체보다 증가되는 것을 1E12와 2E1항체에서 입증되었음(도 14B, 및 D). It was proved in the 1E12 and 2E1 antibodies that the anti-CD71 antibody (1E12 or 2E1) and the MMAF-conjugated secondary antibody were treated with the cancer stem cell line to increase the breast cancer stem cell proliferation inhibitory effect compared to the existing naked antibody (Fig. 14B. , And D).

또한 삼중 음성 유방암(TNBC) 세포주 MDA-MB-231에서도 CD71을 타겟하는 1E12 항체가 세포증식저해하는 효능도 있음을 검증하였다(도 14C). In addition, it was verified that the 1E12 antibody targeting CD71 also inhibits cell proliferation in the triple negative breast cancer (TNBC) cell line MDA-MB-231 (FIG. 14C).

반면에 본 실험에서 사용한 MMAF 2차 항체농도에서는 MMAF가 결합된 2차 항체만 처리하였을 때에는 세포증식저해가 없는 것으로 관찰되었다. On the other hand, at the concentration of the MMAF secondary antibody used in this experiment, it was observed that there was no inhibition of cell proliferation when only the secondary antibody bound with MMAF was treated.

따라서 본 발명의 기술로 동시 발굴된 항체는 타겟 단백질에 대한 다양한 항암 치료제(예, 암줄기세포 및 유방암, 삼중음성유방암, 췌장암, 위암, 난소암 등등)로 개발에 응용할 수 있음을 확인되었다.Therefore, it was confirmed that the antibodies simultaneously discovered with the technology of the present invention can be applied to development as various anticancer therapeutic agents for target proteins (e.g., cancer stem cells and breast cancer, triple negative breast cancer, pancreatic cancer, gastric cancer, ovarian cancer, etc.).

<110> KNU-Industry Cooperation Foundation <120> AN ANTIBODY SPECIFIC FOR PROTEIN IN CANCER STEM CELL MEMBRANE AND A USE THEREOF <130> P18-0052HS <160> 24 <170> KopatentIn 2.0 <210> 1 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> CDR1 OF VH <400> 1 Gly Asp Tyr Gly Met His 1 5 <210> 2 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 OF VH <400> 2 Gly Ile Ser Trp Asn Ser Asp Thr Ile Gly Leu Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 3 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> CDR3 OF VH <400> 3 Pro Ala Phe Ile Ser Gly Trp Tyr Pro Gly Phe Phe Asp Ile 1 5 10 <210> 4 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> CDR1 OF VL <400> 4 Ser Gly Thr Thr Arg Asp Val Gly Arg Tyr Asp Tyr Val Ser 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 OF VL <400> 5 Asp Val Ser Lys Arg Pro Ser 1 5 <210> 6 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDR3 OF VL <400> 6 Cys Ser Tyr Ala Gly Asn Ser Thr Tyr Val 1 5 10 <210> 7 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 OF VH <400> 7 Asp Tyr Tyr Met His 1 5 <210> 8 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 OF VH <400> 8 Gly Ile Ser Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Asn Phe Gln 1 5 10 15 Gly <210> 9 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> CDR3 OF VH <400> 9 Asp Gly Ala Gly Pro Glu Tyr Tyr Tyr Tyr Gly Leu Asp Val 1 5 10 <210> 10 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> CDR1 OF VL <400> 10 Thr Gly Thr Ser Ser Asp Ile Gly Gly Tyr Asn Tyr Val Ser 1 5 10 <210> 11 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 OF VL <400> 11 Glu Val Thr Lys Arg Pro Ser 1 5 <210> 12 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDR3 OF VL <400> 12 Ser Ser Tyr Ser Asp Ser Asp Asn Leu Leu 1 5 10 <210> 13 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 OF VH <400> 13 Ser Tyr Ala Ile Ser 1 5 <210> 14 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 OF VH <400> 14 Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 15 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR3 OF VH <400> 15 Glu Gln Asp Gly Gly Tyr Phe Asp Tyr 1 5 <210> 16 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR1 OF VL <400> 16 Gly Gly Asp Asn Ile Gly Arg Glu Ser Val His 1 5 10 <210> 17 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 OF VL <400> 17 Tyr Asp Ser Asp Arg Pro Ser 1 5 <210> 18 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR3 OF VL <400> 18 Gln Val Trp Asp Ser Ser Ser Asp His Val Val 1 5 10 <210> 19 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 19 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Asp Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Asp Thr Ile Gly Leu Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Pro Ala Phe Ile Ser Gly Trp Tyr Pro Gly Phe Phe Asp Ile 100 105 110 Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 20 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 20 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Ser Gly Thr Thr Arg Asp Val Gly Arg Tyr 20 25 30 Asp Tyr Val Ser Trp Tyr Arg Gln Tyr Pro Gly Glu Ala Pro Gln Leu 35 40 45 Leu Ile Tyr Asp Val Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Phe Asp Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Asp Asp Glu Ala Asp Tyr Tyr Cys Cys Ser Tyr Ala Gly Asn 85 90 95 Ser Thr Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 110 <210> 21 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 21 Gln Met Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Asp Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ser Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Asn Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Asn Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Gly Ala Gly Pro Glu Tyr Tyr Tyr Tyr Gly Leu Asp Val 100 105 110 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 22 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 22 Gln Ser Ala Leu Thr Gln Pro Pro Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Ile Gly Gly Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln His His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Val Ile Ser Glu Val Thr Lys Arg Pro Ser Gly Val Pro Gly Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Ser Asp Ser 85 90 95 Asp Asn Leu Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 23 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 23 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Glu Gln Asp Gly Gly Tyr Phe Asp Tyr Trp Ser Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 24 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 24 Gln Leu Val Leu Thr Gln Ser Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asp Asn Ile Gly Arg Glu Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Ala Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Tyr Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Trp Val Asp Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr His Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90 95 Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 <110> KNU-Industry Cooperation Foundation <120> ANTIBODY SPECIFIC FOR PROTEIN IN CANCER STEM CELL MEMBRANE AND A USE THEREOF <130> P18-0052HS <160> 24 <170> KopatentIn 2.0 <210> 1 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> CDR1 OF VH <400> 1 Gly Asp Tyr Gly Met His 1 5 <210> 2 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 OF VH <400> 2 Gly Ile Ser Trp Asn Ser Asp Thr Ile Gly Leu Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 3 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> CDR3 OF VH <400> 3 Pro Ala Phe Ile Ser Gly Trp Tyr Pro Gly Phe Phe Asp Ile 1 5 10 <210> 4 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> CDR1 OF VL <400> 4 Ser Gly Thr Thr Arg Asp Val Gly Arg Tyr Asp Tyr Val Ser 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 OF VL <400> 5 Asp Val Ser Lys Arg Pro Ser 1 5 <210> 6 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDR3 OF VL <400> 6 Cys Ser Tyr Ala Gly Asn Ser Thr Tyr Val 1 5 10 <210> 7 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 OF VH <400> 7 Asp Tyr Tyr Met His 1 5 <210> 8 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 OF VH <400> 8 Gly Ile Ser Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Asn Phe Gln 1 5 10 15 Gly <210> 9 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> CDR3 OF VH <400> 9 Asp Gly Ala Gly Pro Glu Tyr Tyr Tyr Tyr Gly Leu Asp Val 1 5 10 <210> 10 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> CDR1 OF VL <400> 10 Thr Gly Thr Ser Ser Asp Ile Gly Gly Tyr Asn Tyr Val Ser 1 5 10 <210> 11 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 OF VL <400> 11 Glu Val Thr Lys Arg Pro Ser 1 5 <210> 12 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDR3 OF VL <400> 12 Ser Ser Tyr Ser Asp Ser Asp Asn Leu Leu 1 5 10 <210> 13 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 OF VH <400> 13 Ser Tyr Ala Ile Ser 1 5 <210> 14 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 OF VH <400> 14 Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 15 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR3 OF VH <400> 15 Glu Gln Asp Gly Gly Tyr Phe Asp Tyr 1 5 <210> 16 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR1 OF VL <400> 16 Gly Gly Asp Asn Ile Gly Arg Glu Ser Val His 1 5 10 <210> 17 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 OF VL <400> 17 Tyr Asp Ser Asp Arg Pro Ser 1 5 <210> 18 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR3 OF VL <400> 18 Gln Val Trp Asp Ser Ser Ser Asp His Val Val 1 5 10 <210> 19 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 19 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Asp Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Asp Thr Ile Gly Leu Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Pro Ala Phe Ile Ser Gly Trp Tyr Pro Gly Phe Phe Asp Ile 100 105 110 Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 20 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 20 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Ser Gly Thr Thr Arg Asp Val Gly Arg Tyr 20 25 30 Asp Tyr Val Ser Trp Tyr Arg Gln Tyr Pro Gly Glu Ala Pro Gln Leu 35 40 45 Leu Ile Tyr Asp Val Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Phe Asp Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Asp Asp Glu Ala Asp Tyr Tyr Cys Cys Ser Tyr Ala Gly Asn 85 90 95 Ser Thr Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 110 <210> 21 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 21 Gln Met Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Asp Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ser Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Asn Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Asn Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Gly Ala Gly Pro Glu Tyr Tyr Tyr Tyr Gly Leu Asp Val 100 105 110 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 22 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 22 Gln Ser Ala Leu Thr Gln Pro Pro Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Ile Gly Gly Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln His His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Val Ile Ser Glu Val Thr Lys Arg Pro Ser Gly Val Pro Gly Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Ser Asp Ser 85 90 95 Asp Asn Leu Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 23 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 23 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Glu Gln Asp Gly Gly Tyr Phe Asp Tyr Trp Ser Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 24 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 24 Gln Leu Val Leu Thr Gln Ser Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asp Asn Ile Gly Arg Glu Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Ala Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Tyr Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Trp Val Asp Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr His Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90 95 Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105

Claims (12)

하기 a), b) 및 c)의 단클론 항체로 구성된 군으로부터 선택된 암 줄기세포및/또는 암세포의 세포막 단백질에 특이적인 단클론 항체, 또는 그 기능적 단편:
a)서열번호 1로 기재되는 CDR1 영역, 서열번호 2로 기재되는 CDR2 영역 및 서열번호 3으로 기재되는 CDR3 영역을 포함하는 중쇄 및
서열번호 4로 기재되는 CDR1 영역, 서열번호 5로 기재되는 CDR2 영역 및 서열번호 6으로 기재되는 CDR3 영역을 포함하는 경쇄를 포함하는 단클론 항체;
b)서열번호 7로 기재되는 CDR1 영역, 서열번호 8로 기재되는 CDR2 영역 및 서열번호 9로 기재되는 CDR3 영역을 포함하는 중쇄 및
서열번호 10으로 기재되는 CDR1 영역, 서열번호 11로 기재되는 CDR2 영역 및 서열번호 12로 기재되는 CDR3 영역을 포함하는 경쇄를 포함하는 단클론 항체; 및
c) 서열번호 13으로 기재되는 CDR1 영역, 서열번호 14로 기재되는 CDR2 영역 및 서열번호 15로 기재되는 CDR3 영역을 포함하는 중쇄 및
서열번호 16으로 기재되는 CDR1 영역, 서열번호 17로 기재되는 CDR2 영역 및 서열번호 18로 기재되는 CDR3 영역을 포함하는 경쇄를 포함하는 단클론 항체.A monoclonal antibody specific for a cell membrane protein of cancer stem cells and/or cancer cells selected from the group consisting of monoclonal antibodies of a), b) and c) below, or a functional fragment thereof:
a) a heavy chain comprising a CDR1 region shown in SEQ ID NO: 1, a CDR2 region shown in SEQ ID NO: 2, and a CDR3 region shown in SEQ ID NO: 3, and
A monoclonal antibody comprising a light chain comprising a CDR1 region represented by SEQ ID NO: 4, a CDR2 region represented by SEQ ID NO: 5, and a CDR3 region represented by SEQ ID NO: 6;
b) a heavy chain comprising a CDR1 region represented by SEQ ID NO: 7, a CDR2 region represented by SEQ ID NO: 8, and a CDR3 region represented by SEQ ID NO: 9, and
A monoclonal antibody comprising a light chain comprising a CDR1 region set forth in SEQ ID NO: 10, a CDR2 region set forth in SEQ ID NO: 11, and a CDR3 region set forth in SEQ ID NO: 12; And
c) a heavy chain comprising a CDR1 region represented by SEQ ID NO: 13, a CDR2 region represented by SEQ ID NO: 14, and a CDR3 region represented by SEQ ID NO: 15, and
A monoclonal antibody comprising a light chain comprising a CDR1 region set forth in SEQ ID NO: 16, a CDR2 region set forth in SEQ ID NO: 17, and a CDR3 region set forth in SEQ ID NO: 18. 제1항에 있어서, 상기 암 줄기세포 및/또는 암 세포의 세포막 단백질은 CD 44 또는 CD71인 것을 특징으로 하는 단클론 항체, 또는 그 기능적 단편.The monoclonal antibody or functional fragment thereof according to claim 1, wherein the cell membrane protein of the cancer stem cell and/or cancer cell is CD 44 or CD71. 제1항에 있어서, 상기 기능적 단편은 경쇄, 중쇄, 가변 영역, Fab, Fab', F(ab')2, 단일가변분절(scFv), 디아바디(Diabody), 트리바디(Tribody), 이중쇄가변분절(dsFv), 단일가변분절-C절편(scFv-Fc), 싱글체인IgG(scIgG), 다중체 또는 CDR(Complementarity Determining Region)을 함유하는 펩타이드인 것을 특징으로 하는 단클론 항체, 또는 그 기능적 단편.The method of claim 1, wherein the functional fragment is light chain, heavy chain, variable region, Fab, Fab', F(ab')2, single variable segment (scFv), Diabody, Tribody, double chain A monoclonal antibody, characterized in that it is a peptide containing a variable segment (dsFv), a single variable segment-C segment (scFv-Fc), a single chain IgG (scIgG), a multiplex or a CDR (Complementarity Determining Region), or a functional fragment thereof . 제1항에 있어서, 상기 a) 단클론 항체는 서열번호 19의 아미노산 서열로 이루어진 중쇄 가변영역 및 서열번호 20의 아미노산 서열로 이루어진 경쇄 가변영역을 포함하는 것을 특징으로 하는 단클론 항체, 또는 그 기능적 단편.The monoclonal antibody according to claim 1, wherein the a) monoclonal antibody comprises a heavy chain variable region consisting of an amino acid sequence of SEQ ID NO: 19 and a light chain variable region consisting of an amino acid sequence of SEQ ID NO: 20, or a functional fragment thereof. 제1항에 있어서, 상기 b) 단클론 항체는 서열번호 21의 아미노산 서열로 이루어진 중쇄 가변영역 및 서열번호 22의 아미노산 서열로 이루어진 경쇄 가변영역을 포함하는 것을 특징으로 하는 단클론 항체, 또는 그 기능적 단편.The monoclonal antibody of claim 1, wherein the b) monoclonal antibody comprises a heavy chain variable region consisting of an amino acid sequence of SEQ ID NO: 21 and a light chain variable region consisting of an amino acid sequence of SEQ ID NO: 22, or a functional fragment thereof. 제1항에 있어서, 상기 c) 단클론 항체는 서열번호 23의 아미노산 서열로 이루어진 중쇄 가변영역 및 서열번호 24의 아미노산 서열로 이루어진 경쇄 가변영역을 포함하는 것을 특징으로 하는 단클론 항체, 또는 그 기능적 단편.The monoclonal antibody according to claim 1, wherein the c) monoclonal antibody comprises a heavy chain variable region consisting of an amino acid sequence of SEQ ID NO: 23 and a light chain variable region consisting of an amino acid sequence of SEQ ID NO: 24, or a functional fragment thereof. 제1항 내지 제6항 중 어느 한 항의 단클론 항체, 또는 그 기능적 단편을 유효성분으로 포함하는 암 진단용 조성물.A composition for diagnosis of cancer comprising the monoclonal antibody of any one of claims 1 to 6 or a functional fragment thereof as an active ingredient. 제7항에 있어서, 상기 암은 유방암, 폐암, 난소암, 신장암, 췌장암, 결장암, 직장결장암, 전립선암, 신장세포 암종, 투명세포 암종, 소세포 암종, 편평세포 암종, 위암, 임파종, 백혈병 및 다발성 골수종으로 구성된 군으로부터 선택된 암인 것을 특징으로 하는 암 진단용 조성물.The method of claim 7, wherein the cancer is breast cancer, lung cancer, ovarian cancer, kidney cancer, pancreatic cancer, colon cancer, rectal colon cancer, prostate cancer, renal cell carcinoma, clear cell carcinoma, small cell carcinoma, squamous cell carcinoma, gastric cancer, lymphoma, leukemia and Cancer diagnosis composition, characterized in that the cancer selected from the group consisting of multiple myeloma. 제1항 내지 제6항 중 어느 한 항의 단클론 항체, 또는 그 기능적 단편을 유효성분으로 포함하는 암 치료용 조성물.A composition for treating cancer comprising the monoclonal antibody of any one of claims 1 to 6 or a functional fragment thereof as an active ingredient. 제9항에 있어서, 상기 암은 유방암, 폐암, 난소암, 신장암, 췌장암, 결장암, 직장결장암, 전립선암, 신장세포 암종, 투명세포 암종, 소세포 암종, 편평세포 암종, 위암, 임파종, 백혈병 및 다발성 골수종으로 구성된 군으로부터 선택된 암인 것을 특징으로 하는 암 치료용 조성물.The method of claim 9, wherein the cancer is breast cancer, lung cancer, ovarian cancer, kidney cancer, pancreatic cancer, colon cancer, rectal colon cancer, prostate cancer, renal cell carcinoma, clear cell carcinoma, small cell carcinoma, squamous cell carcinoma, gastric cancer, lymphoma, leukemia and Cancer treatment composition, characterized in that the cancer selected from the group consisting of multiple myeloma. 제1항 내지 제6항 중 어느 한 항의 단클론 항체, 또는 그 기능적 단편에 특정 물질 및/또는 유전자 재조합 물질이 결합된 컨쥬게이트.A conjugate in which a specific substance and/or a genetically recombinant substance is bound to the monoclonal antibody of any one of claims 1 to 6, or a functional fragment thereof. 제11항에 있어서, 상기 컨쥬게이트는 항체-형광물질 결합체, 항체-독소, 항체-약물 결합체 또는 항체-항체 결합체인 것을 특징으로 하는 컨쥬게이트.
The conjugate according to claim 11, wherein the conjugate is an antibody-fluorescent substance conjugate, antibody-toxin, antibody-drug conjugate, or antibody-antibody conjugate.
KR1020190057050A 2019-05-15 2019-05-15 An antibody specific for protein in cancer stem cell membrane and a use thereof KR102259298B1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
KR1020190057050A KR102259298B1 (en) 2019-05-15 2019-05-15 An antibody specific for protein in cancer stem cell membrane and a use thereof
KR1020200127010A KR102258015B1 (en) 2019-05-15 2020-09-29 An antibody specific for protein in cancer stem cell membrane and a use thereof
KR1020200127007A KR102258014B1 (en) 2019-05-15 2020-09-29 An antibody specific for protein in cancer stem cell membrane and a use thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
KR1020190057050A KR102259298B1 (en) 2019-05-15 2019-05-15 An antibody specific for protein in cancer stem cell membrane and a use thereof

Related Child Applications (2)

Application Number Title Priority Date Filing Date
KR1020200127007A Division KR102258014B1 (en) 2019-05-15 2020-09-29 An antibody specific for protein in cancer stem cell membrane and a use thereof
KR1020200127010A Division KR102258015B1 (en) 2019-05-15 2020-09-29 An antibody specific for protein in cancer stem cell membrane and a use thereof

Publications (2)

Publication Number Publication Date
KR20200132110A true KR20200132110A (en) 2020-11-25
KR102259298B1 KR102259298B1 (en) 2021-05-31

Family

ID=73645277

Family Applications (1)

Application Number Title Priority Date Filing Date
KR1020190057050A KR102259298B1 (en) 2019-05-15 2019-05-15 An antibody specific for protein in cancer stem cell membrane and a use thereof

Country Status (1)

Country Link
KR (1) KR102259298B1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022215679A1 (en) * 2021-04-05 2022-10-13 公立大学法人大阪 Antibody against pancreatic cancer stem cells

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20090094848A (en) * 2006-12-21 2009-09-08 메다렉스, 인코포레이티드 Cd44 antibodies

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20090094848A (en) * 2006-12-21 2009-09-08 메다렉스, 인코포레이티드 Cd44 antibodies

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022215679A1 (en) * 2021-04-05 2022-10-13 公立大学法人大阪 Antibody against pancreatic cancer stem cells

Also Published As

Publication number Publication date
KR102259298B1 (en) 2021-05-31

Similar Documents

Publication Publication Date Title
RU2746413C1 (en) Antibody specifically binding with muc1 and its application
CA3100260A1 (en) Axl-targeting antibody, antibody-drug conjugate, preparation method therefor, and use thereof
CN107001452B (en) anti-CK 8 antibodies for the treatment of cancer
KR20150032075A (en) Anticancer composition containing an anti-Ang2 antibody inducing binding to Tie2 receptor
KR20230060509A (en) Nectin-4 Antibodies and Uses Thereof
CA3012270A1 (en) Endosialin-binding antibody
CN114269389B (en) Antibody drug conjugates targeting claudin 18.2
CN113754775A (en) Bispecific antibody for resisting PD-L1 and HER2
IL266460A (en) Antibody binding to carbonic anhydrase and use thereof
TR201906781T4 (en) Anti-transglutaminase 2 antibodies.
KR102259298B1 (en) An antibody specific for protein in cancer stem cell membrane and a use thereof
JP2024020436A (en) HER2 targeting agent
KR101917098B1 (en) Antibody binding to Carbonic anhydrase and use thereof
US9499622B2 (en) Anti-EGFR/anti-HER2 bispecific antibodies with anti-EGFR DARPins
CN107108735B (en) Novel EGFRVIII antibodies and compositions comprising the same
KR102258015B1 (en) An antibody specific for protein in cancer stem cell membrane and a use thereof
KR102258014B1 (en) An antibody specific for protein in cancer stem cell membrane and a use thereof
KR102185755B1 (en) A method for characterizing cancer stem cell specific proteome and antibody in cell membrane and a use thereof
KR102312922B1 (en) Anti-Ang2 antibody inducing binding to Tie2 receptor
US10214591B1 (en) Monoclonal antibody to human line-1 ORF2 protein and method for early detection of transforming cells in pre-neoplastic tissues of a human subject
KR20180119676A (en) Grape calicics and TRA-related antibodies, production methods and uses thereof as anticancer therapies
AU2017219386A1 (en) Antibody against EGFRvIII and use thereof
KR20230042524A (en) Anti-HER2 / Anti-4-1BB Bispecific Antibodies and Uses Thereof
EP3980127A1 (en) Antigen-binding protein constructs and uses thereof
KR20160048405A (en) Antibody that reconizes the beta-1 integrin and use of the antibody

Legal Events

Date Code Title Description
A107 Divisional application of patent
E902 Notification of reason for refusal
E701 Decision to grant or registration of patent right
GRNT Written decision to grant