KR20040067687A - Neutralizing human monoclonal antibody specific to hepatitis A virus - Google Patents
Neutralizing human monoclonal antibody specific to hepatitis A virus Download PDFInfo
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- KR20040067687A KR20040067687A KR1020030004915A KR20030004915A KR20040067687A KR 20040067687 A KR20040067687 A KR 20040067687A KR 1020030004915 A KR1020030004915 A KR 1020030004915A KR 20030004915 A KR20030004915 A KR 20030004915A KR 20040067687 A KR20040067687 A KR 20040067687A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1009—Picornaviridae, e.g. hepatitis A virus
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
Abstract
Description
본 발명은 A형 간염 바이러스(Hepatitis A virus, 이하 "HAV"라 약칭함)에 특이적으로 결합하여 HAV를 중화시키는 인간 단일클론항체에 관한 것으로서, 보다 상세하게는 HAV에 특이적으로 결합하는 인간 단일클론항체의 경쇄 가변영역 및 중쇄 가변영역을 암호하는 유전자, 상기 유전자를 포함하는 발현벡터, 상기 발현벡터가 도입되어 인간 단일클론항체를 생산할 수 있는 형질전환체 및 상기 형질전환체를 배양하여 제조된 HAV를 중화시키는 인간 단일클론항체에 관한 것이다.The present invention relates to a human monoclonal antibody that specifically binds to Hepatitis A virus (abbreviated as "HAV") and neutralizes HAV, and more specifically, to a human that specifically binds to HAV. Gene encoding the light chain variable region and heavy chain variable region of the monoclonal antibody, the expression vector containing the gene, the expression vector is introduced to produce a transformant capable of producing a human monoclonal antibody and produced by culturing the transformant It relates to a human monoclonal antibody that neutralizes the HAV.
HAV는 A형 간염을 유발하는 바이러스로서, 특히 개발도상국 비롯한 세계 각지에서 인류의 건강을 위협하고 있다. 한국에서도 최근 들어 HAV 감염이 젊은층에서 새로이 증가함이 보고되어 문제로 대두되고 있다(Byun KS et al.,J. Gastroenterol. Hepatol.,2001, 16:519). A형 간염의 경우, 보통 자체 제한(self-limiting)이어서 발병 3-4주 후 회복되는 것이 보통이나, 노인환자의 경우 종종 극발성(fulminant) 간염으로 진행되어 사망에 이르기도 한다(Gust ID and Feinstone SM.,Hepatitis A., 1998, CRC Press, 145).HAV is a virus that causes hepatitis A, threatening human health, especially in developing countries and around the world. Recently, a new increase in HAV infection in young people has been reported as a problem (Byun KS et al., J. Gastroenterol. Hepatol., 2001, 16: 519). Hepatitis A is usually self-limiting and usually recovers three to four weeks after onset, but in older patients it often progresses to fulminant hepatitis and death (Gust ID and Feinstone SM., Hepatitis A. , 1998, CRC Press, 145).
HAV는 외피(envelope)가 없는 양성 가닥 RNA 바이러스로서 피코나비리디(Picornaviridae) 과로 분류된다. HAV의 게놈은 약 7,500 뉴클레오티드로 구성되어 있고 2,200개 아미노산 크기의 바이러스 폴리펩티드를 암호하고 있으며, 바이러스 단백질 효소에 의해 이 폴리펩티드가 각각의 구조 단백질(VP1, VP2, VP3, VP4)과 비구조 단백질로 가공된다. 각각의 구조 단백질(VP1, VP2, VP3, VP4)은 20면체의 HAV 캡시드(capsid)로 조립된다. 한편, HAV에는 대부분의 중화항체들이 인지하는 면역우성인(immunodominant) 에피토프(epitope)가 존재하고, 이 에피토프는 형태적(conformational) 에피토프로서 구조 단백질이 조립된 후에만 형성되는 것으로 알려져 있다. 따라서, HAV의 캡시드에 존재하는 중화 에피토프에 결합하는 단일클론항체는 HAV의 진단은 물론 감염의 예방에 유용할 수 있다.HAV is a positive stranded RNA virus with no envelope and is classified into the family Picornaviridae. The genome of HAV consists of approximately 7,500 nucleotides and encodes a viral polypeptide of 2,200 amino acids in size, which is processed by viral protein enzymes into individual structural proteins (VP1, VP2, VP3, VP4) and nonstructural proteins. do. Each structural protein (VP1, VP2, VP3, VP4) is assembled into icosahedron HAV capsids. On the other hand, there is an immunodominant epitope that is recognized by most neutralizing antibodies in HAV, and this epitope is known to be formed only after the structural protein is assembled as a conformational epitope. Thus, monoclonal antibodies that bind to neutralizing epitopes present in the capsid of HAV may be useful for the diagnosis of HAV as well as for the prevention of infection.
현재까지 HAV 감염을 예방하기 위해서는 인간 면역글로불린(human immune serum globulin)을 사용하고 있다(Pollock and Reid,Lancet,1969, 1:281). 상기 면역글로불린은 HAV에 노출된 2주 후에 투여하여도 HAV 감염을 예방시키는 것으로 보고되고 있다(Mosley 등,Am. J. Epidemiol., 1968, 87:539). 그러나, 현재 사용되고 있는 항체는 인간의 혈장으로부터 추출되기 때문에 특이성(specificity)이 낮고, 오염원에 노출될 수 있으며, 인간 혈액의 계속적인 공급이 필요하다는 단점이 있다.To date, human immune serum globulin has been used to prevent HAV infection (Pollock and Reid, Lancet, 1969, 1: 281). The immunoglobulin has been reported to prevent HAV infection even when administered two weeks after exposure to HAV (Mosley et al . , Am. J. Epidemiol. , 1968, 87: 539). However, currently used antibodies have a disadvantage of low specificity because they are extracted from human plasma, can be exposed to contaminants, and require continuous supply of human blood.
상기와 같은 문제점을 해결하기 위하여 HAV를 중화시킬 수 있는 단일클론항체의 필요성이 대두되었다. HAV에 대한 중화항체는 기존의 생쥐 하이브리도마 기술을 이용하여 만들 수 있으나, 생쥐항체에 대한 면역반응(Human anti-Mouse antibody; 이하 "HAMA"라 약칭함) 때문에 인체에 투여할 경우, 부작용이 생기거나 치료 효과가 감소된다. 이러한 HAMA 반응을 피하기 위해 생쥐항체의 항원 결합 부위를 제외한 나머지 부위를 인간항체의 것으로 치환하는 키메라 또는 인간화 항체기술이 개발되었고, 이들 항체는 실제로 환자에게 반복 투여할 경우 인체 면역반응이 많이 감소됨이 보고되었다(Brown et al.,Proc. Natl. Acad. Sci. USA, 1991, 88:2663). 그러나 이들 항체는 여전히 생쥐 유래의 아미노산 잔기들을 포함하고 있어서 면역반응을 일으킬 가능성을 가지고 있다. 따라서 인체에 투여할 목적으로는 완전히 인간 유래의 단일클론항체를 이용하는 것이 유리하다.In order to solve the above problems, the need for a monoclonal antibody that can neutralize HAV has emerged. Neutralizing antibodies against HAV can be made using conventional mouse hybridoma technology, but when administered to the human body due to a human anti-Mouse antibody (abbreviated as "HAMA"), Occurs or the therapeutic effect is reduced. To avoid this HAMA reaction, chimeric or humanized antibody technology has been developed that replaces the antigen-binding site of mouse antibodies with those of human antibodies, and these antibodies have been reported to significantly reduce the human immune response when administered repeatedly to patients. Brown et al., Proc. Natl. Acad. Sci. USA , 1991, 88: 2663. However, these antibodies still contain amino acid residues derived from mice and have the potential to trigger an immune response. Therefore, it is advantageous to use monoclonal antibodies completely derived from humans for the purpose of administration to the human body.
이에, 본 발명자들은 HAV에 감염된 후 회복된 환자의 혈액으로부터 HAV-프라임된(primed) B 세포를 분리한 후 인간-생쥐의 이형하이브리도마(heterohybridoma)와 융합시켜 HAV에 대한 항체를 생산하는 세포 클론들을 분리하였고, 이로부터 HAV 특이 항체의 가변영역 cDNA를 클로닝하고 이를 인간 항체 불변영역 유전자와 재조합시킨 후 동물세포에서 발현시켜 HAV에 특이적으로 결합하는 항체를 제조하였으며, 상기 항체가 HAV를 중화할 수 있음을 밝힘으로써 본 발명을 완성하였다.Therefore, the present inventors have isolated HAV-primed B cells from the blood of a patient recovered after HAV infection and then fused with heterohybridoma of human-mouse to produce antibodies against HAV. The clones were isolated, cloned the variable region cDNA of the HAV-specific antibody and recombined with the human antibody constant region gene and expressed in animal cells to prepare an antibody that specifically binds to HAV, and the antibody neutralizes HAV. The present invention was completed by revealing that it can be done.
본 발명의 목적은 A형 간염 바이러스의 감염을 효과적으로 예방하기 위하여, A형 간염 바이러스에 특이적으로 결합하여 중화시킬 수 있는 인간 단일클론항체를 제공하는 것이다.An object of the present invention is to provide a human monoclonal antibody that can specifically bind to and neutralize the hepatitis A virus in order to effectively prevent the infection of hepatitis A virus.
도 1은 이형하이브리도마 배양액으로부터 순수분리한, HAV 특이 인간 단일클론항체를 SDS-PAGE로 확인한 것이고, 1 shows the HAV-specific human monoclonal antibody isolated purely from heterologous hybridoma culture by SDS-PAGE,
레인 1 : Hb-HAV6 세포로부터 분리한 항체Lane 1: antibody isolated from Hb-HAV6 cells
레인 2 : Hb-HAV9 세포로부터 분리한 항체Lane 2: antibody isolated from Hb-HAV9 cells
도 2는 본 발명의 HAV 특이 항체 HAV6와 HAV9의 항원결합능을 ELISA로 분석한 결과를 보여주는 그래프이고, Figure 2 is a graph showing the results of the ELISA analysis of the antigen-binding ability of HAV-specific antibodies HAV6 and HAV9 of the present invention,
도 3은 HAV6와 HAV9 항체의 발현벡터를 나타낸 모식도이고, 3 is a schematic diagram showing expression vectors of HAV6 and HAV9 antibodies,
도 4는 HAV6와 HAV9 항체의 경쇄(A) 및 중쇄(B) 가변영역 아미노산 서열을 비교한 것이고, 4 is a comparison of the light chain (A) and heavy chain (B) variable region amino acid sequences of HAV6 and HAV9 antibodies,
도 5는 HAV6와 HAV9 항체가 생쥐 HAV 중화항체인 B5B3(A)과 7E7(B)의 HAV에 대한 결합을 저해함을 보여주는 그래프이고, 5 is a graph showing that HAV6 and HAV9 antibodies inhibit the binding of HA5 neutralizing antibodies of B5B3 (A) and 7E7 (B) to HAV.
도 6은 HAV6와 HAV9 항체가 HAV 감염을 중화시킬 수 있음을 보여주는 사진이다. 6 is a photograph showing that HAV6 and HAV9 antibodies can neutralize HAV infection.
상기 목적을 달성하기 위하여, 본 발명은 HAV에 결합하는 인간 항체의 경쇄 가변영역 및 중쇄 가변영역을 포함하는 인간 단일클론항체, 상기 인간 단일클론항체의 유전자를 포함하는 발현벡터 및 상기 발현벡터가 도입되어 인간 단일클론항체를 생산할 수 있는 형질전환체를 제공한다.In order to achieve the above object, the present invention provides a human monoclonal antibody comprising a light chain variable region and a heavy chain variable region of a human antibody that binds to HAV, an expression vector comprising the gene of the human monoclonal antibody and the expression vector is introduced To provide a transformant capable of producing human monoclonal antibodies.
또한, 본 발명은 상기 인간 단일클론항체를 유효성분으로 함유하는 HAV 중화용 약학적 조성물을 제공한다.The present invention also provides a pharmaceutical composition for neutralizing HAV containing the human monoclonal antibody as an active ingredient.
또한, 본 발명은 상기 인간 단일클론항체를 이용하여 HAV를 중화시키는 방법 및 HAV 감염을 예방 및 치료하는 방법을 제공한다.The present invention also provides a method for neutralizing HAV using the human monoclonal antibody and a method for preventing and treating HAV infection.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 A형 간염 바이러스에 결합하는 인간 항체의 경쇄 가변영역 및 중쇄 가변영역을 포함하는 인간 단일클론항체, 상기 인간 단일클론항체의 유전자를 포함하는 발현벡터 및 상기 발현벡터가 도입되어 인간 단일클론항체를 생산할 수 있는 형질전환체를 제공한다.The present invention provides a human monoclonal antibody comprising a light chain variable region and a heavy chain variable region of a human antibody that binds to hepatitis A virus, an expression vector comprising a gene of the human monoclonal antibody, and the expression vector are introduced into a human monoclonal antibody. Provided are transformants capable of producing antibodies.
본 발명의 인간 단일클론항체 가변영역은 HAV와 특이적으로 결합하는 경쇄 가변영역 및 중쇄 가변영역으로 구성되어 있다. 상기에서, 경쇄 가변영역은서열번호 5또는서열번호 6으로 기재되는 아미노산 서열로 이루어지는 것이 바람직하고, 중쇄 가변영역은서열번호 7또는서열번호 8로 기재되는 아미노산 서열로 이루어지는 것이 바람직하다. 구체적으로, 본 발명의 인간 단일클론항체는서열번호 5로 기재되는 경쇄 가변영역 및서열번호 7로 기재되는 중쇄 가변영역을 포함하거나, 또는서열번호 6으로 기재되는 경쇄 가변영역 및서열번호 8로 기재되는 중쇄 가변영역을 포함하는 것이 더욱 바람직하다.The human monoclonal antibody variable region of the present invention is composed of a light chain variable region and a heavy chain variable region that specifically bind to HAV. In the above, the light chain variable region preferably consists of an amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6 , and the heavy chain variable region preferably comprises an amino acid sequence represented by SEQ ID NO: 7 or SEQ ID NO: 8 . Specifically, the human monoclonal antibodies of the invention comprises a heavy chain variable region described in the light chain variable region, and SEQ ID NO: 7 is described in SEQ ID NO: 5, or SEQ ID NO: 6 light chain variable region and a sequence number written in 8 described in More preferably, the heavy chain variable region is included.
본 발명의 인간 단일클론항체 불변영역은 Cκ로 이루어진 경쇄 불변영역 및 Cγ1로 이루어진 중쇄 불변영역으로 구성되는 것이 바람직하나, 반드시 여기에 한정되는 것은 아니다. 인간 항체의 중쇄 불변영역에는 여러 다양한 이소타입(isotype)(예를 들면, Cγ1, Cγ2, Cγ3, Cγ4, Cα, Cδ, Cμ혹은 Cε 등)이 존재하고 중쇄 가변영역과 재조합되어 다양한 이소타입의 중쇄(예를 들면, IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgM, 혹은 IgE)가 만들어진다. 또한, 인간 항체의 경쇄 불변영역에는 Cκ혹은 Cλ의 두가지 이소타입이 존재하고 경쇄 가변영역과 재조합되어 카파(κ) 혹은 람다(λ) 경쇄가 만들어진다(E. S. Golub, Immunology: A synthesis, Sinauer Associates, Sunderland, Massachusetts, U.S.A., 1987). 따라서, 본 발명의 인간 단일클론항체는 동일한 경쇄 및 중쇄 가변영역에 상기와 같은 다양한 종류의 경쇄 및 중쇄 불변영역이 결합된 다양한 이소타입 형태의 항체로 제조될 수 있다.Human monoclonal antibody constant region of the present invention is preferably composed of a light chain constant region consisting of Cκ and a heavy chain constant region consisting of Cγ1, but is not necessarily limited thereto. In the heavy chain constant region of a human antibody, there are various isotypes (for example, Cγ1, Cγ2, Cγ3, Cγ4, Cα, Cδ, Cμ, or Cε, etc.) and recombine with the heavy chain variable region to make various isotype heavy chains. (Eg, IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgM, or IgE) are made. In addition, two isotypes, Cκ or Cλ, exist in the light chain constant region of a human antibody and are recombined with the light chain variable region to form a kappa (κ) or lambda (λ) light chain (ES Golub, Immunology: A synthesis, Sinauer Associates, Sunderland). , Massachusetts, USA, 1987). Therefore, the human monoclonal antibodies of the present invention can be prepared with various isotype antibodies in which the various light and heavy chain constant regions are combined with the same light and heavy chain variable regions.
본 발명의 발현벡터는 상기 인간 단일클론항체의 가변영역 및 불변영역을 코딩하는 유전자를 포함하는 것을 특징으로 한다.The expression vector of the present invention is characterized in that it comprises a gene encoding a variable region and a constant region of the human monoclonal antibody.
본 발명의 발현벡터는 HAV와 특이적으로 결합하는 가변영역을 코딩하는서열번호 1또는서열번호 2로 기재되는 염기서열을 포함하는 인간경쇄 가변영역 유전자 및서열번호 3또는서열번호 4로 기재되는 염기서열을 포함하는 인간중쇄 가변영역 유전자를 포함하는 것이 바람직하다.The expression vector of the present invention is a human light chain variable region gene comprising a nucleotide sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 encoding a variable region that specifically binds HAV and a base described in SEQ ID NO: 3 or SEQ ID NO: 4 It is preferred to include a human heavy chain variable region gene comprising a sequence.
상기에서, 본 발명의 발현벡터는서열번호 1로 기재되는 인간경쇄 가변영역 유전자 및서열번호 3으로 기재되는 인간중쇄 가변영역 유전자, 또는서열번호 2로 기재되는 인간경쇄 가변영역 유전자 및서열번호 4로 기재되는 인간중쇄 가변영역 유전자를 포함하는 것이 더욱 바람직하다.In the above, the expression vector of the present invention is a human light chain variable region gene described by SEQ ID NO: 1 and a human heavy chain variable region gene described by SEQ ID NO: 3 , or a human light chain variable region gene described by SEQ ID NO: 2 and SEQ ID NO: 4 More preferably, the human heavy chain variable region gene described is included.
본 발명자들은 HAV 감염을 중화할 수 있는 인간 항체를 제조하기 위하여, HAV 감염에서 회복된 환자의 혈액으로부터 HAV-프라임된 B 세포를 분리하여 EBV (Epstein-Barr virus)로 형질전환시킨 후, 인간과 생쥐의 이형하이브리도마와 융합시킨 하이브리도마를 제조하였다. 본 발명자들은 HAV에 대한 결합능이 있는 IgG를 생산하는 두 하이브리도마 클론들을 선별하여 각각 "Hb-HAV6" 또는 "Hb-HAV9"라 명명하였다.In order to prepare human antibodies capable of neutralizing HAV infection, the present inventors isolated HAV-primed B cells from blood of patients recovered from HAV infection, transformed with EBV (Epstein-Barr virus), Hybridomas fused with heterologous hybridomas of mice were prepared. We selected two hybridoma clones that produce IgG capable of binding to HAV and named them "Hb-HAV6" or "Hb-HAV9", respectively.
상기 하이브리도마의 경우, 항체 생산능이 불안정하여 3개월 정도 지나면 소멸되므로, 본 발명에서는 상기 단일클론항체를 안정하게 생산하는 동물세포주를 제공한다. 이를 위하여, 상기 Hb-HAV6와 Hb-HAV9 하이브리도마로부터 각각 HAV6와 HAV9 항체의 중쇄 및 경쇄 가변영역을 암호하는 cDNA를 클로닝한 후 인간 면역글로불린(immunoglobulin)의 Cκ 및 Cγ1의 불변영역 유전자를 포함하는 카셋트 벡터 pdCMV-dhfrC-AKA/HzK (대한민국 특허출원 제2001-47737호)에 클로닝하였고, 각각"pdCMV-dhfrC-HAV6" 및 "pdCMV-dhfrC-HAV9"라 명명하였다(도 3참조). 본 발명자들은 상기 pdCMV-dhfrC-HAV6 발현벡터를 2001년 8월 3일자로 생명공학연구원 유전자은행에 기탁하였고(수탁번호; KCTC 10027BP), 상기 pdCMV-dhfrC-HAV9 발현벡터를 2001년 8월 4일자로 생명공학연구원 유전자은행에 기탁하였다(수탁번호; KCTC 10029BP).In the case of the hybridoma, since the antibody production capacity is unstable and disappears after about three months, the present invention provides an animal cell line stably producing the monoclonal antibody. For this purpose, cloned cDNA encoding the heavy and light chain variable regions of HAV6 and HAV9 antibodies from the Hb-HAV6 and Hb-HAV9 hybridomas, respectively, and include the constant region genes of Cκ and Cγ1 of human immunoglobulins. Cloned into the cassette vector pdCMV-dhfrC-AKA / HzK (Korean Patent Application No. 2001-47737) and named "pdCMV-dhfrC-HAV6" and "pdCMV-dhfrC-HAV9", respectively (see FIG. 3 ). The present inventors deposited the pdCMV-dhfrC-HAV6 expression vector with the Biotechnology Research Institute Gene Bank on August 3, 2001 (accession number; KCTC 10027BP), and the pdCMV-dhfrC-HAV9 expression vector on August 4, 2001. Was deposited in the Genetic Bank of Korea Biotechnology Institute (Accession No .; KCTC 10029BP).
상기 발현벡터들의 경쇄 및 중쇄 가변영역의 염기서열을 분석한 결과, HAV6 경쇄의 가변영역은서열번호 5로 기재되는 아미노산 서열을 코딩하는서열번호 1로 기재되는 염기서열을 포함하고 있고, HAV6 중쇄의 가변영역은서열번호 7로 기재되는 아미노산 서열을 코딩하는서열번호 3으로 기재되는 염기서열을 포함하고 있음을 확인하였다. 또한, HAV9 경쇄의 가변영역은서열번호 6으로 기재되는 아미노산 서열을 코딩하는서열번호 2로 기재되는 염기서열을 포함하고 있고, HAV9 중쇄의 가변영역은서열번호 8로 기재되는 아미노산 서열을 코딩하는서열번호 4로 기재되는 염기서열을 포함하고 있음을 확인하였다. 상기 인간경쇄는 둘 다 가변영역 생식세포주 유전자 중 VK1 패밀리의 DPK9/012에서 유래한 것이고, 인간 중쇄의 경우는 서로 다른 생식세포주 유전자에서 유래한 것이다(도 4참조).As a result of analyzing the nucleotide sequences of the light and heavy chain variable regions of the expression vectors, the variable region of the HAV6 light chain contained the nucleotide sequence of SEQ ID NO: 1 encoding the amino acid sequence of SEQ ID NO: 5 , The variable region was confirmed to contain the nucleotide sequence set forth in SEQ ID NO: 3 encoding the amino acid sequence set forth in SEQ ID NO: 7 . In addition, the sequence that the variable region of HAV9 light chain comprising the nucleotide sequence represented by SEQ ID NO: 2 encoding the amino acid sequence described in SEQ ID NO: 6, and the variable region of the HAV9 heavy chain encodes the amino acid sequence represented by SEQ ID NO: 8 It was confirmed that the base sequence described in No. 4 was included. The human light chains are both derived from DPK9 / 012 of the VK1 family of variable region germline genes, and the human heavy chains are derived from different germline genes (see FIG. 4 ).
본 발명자들은 상기에서 제조한 발현 벡터 pdCMV-dhfr-HAV6 또는 pdCMV-dhfr-HAV9으로dhfr유전자가 결여된 CHO (Chinese Hamster Ovary) 세포를 형질전환시켜 항체를 안정하게 생산하는 세포주를 제조하고, 이를 각각 "HAV6-20" 및"HAV9-20"이라 명명하였다. 상기 세포 배양액으로부터 항체를 분리·정제한 후 HAV에 대한 항원결합능을 측정한 결과, 모두 HAV에 특이적으로 결합함을 알 수 있다(도 2참조).The present inventors transform the CHO (Chinese Hamster Ovary) cells lacking the dhfr gene with the expression vector pdCMV-dhfr-HAV6 or pdCMV-dhfr-HAV9 prepared above to produce cell lines stably producing antibodies, respectively. It was named "HAV6-20" and "HAV9-20". After the antibody was isolated and purified from the cell culture, the antigen binding ability to HAV was measured. As a result, all of them specifically bind to HAV (see FIG. 2 ).
본 발명자들은 상기 HAV6-20 세포주 및 HAV9-20 세포주를 2002년 12월 24일자로 각각 한국생명공학연구원 유전자은행에 기탁하였다(수탁번호; KCTC 10404BP, KCTC 10405BP).The present inventors deposited the HAV6-20 cell line and HAV9-20 cell line with the Korea Biotechnology Research Institute Gene Bank on December 24, 2002, respectively (Accession No .; KCTC 10404BP, KCTC 10405BP).
또한, 본 발명은 상기 인간 단일클론항체를 유효성분으로 함유하는 HAV 중화용 약학적 조성물을 제공한다.The present invention also provides a pharmaceutical composition for neutralizing HAV containing the human monoclonal antibody as an active ingredient.
본 발명의 HAV 중화용 약학적 조성물은 상기 인간 단일클론항체를 유효성분으로 함유한다. 본 발명의 약학적 조성물은 바람직하게는 비경구로 투여할 수 있으며, 비경구 투여는 피하주사, 정맥주사, 근육내 주사 또는 흉부내 주사 주입방식에 의한다. 비경구 투여용 제형으로 제제화하기 위해서는 상기 인간 단일클론항체를 안정제 또는 완충제와 함께 물에서 혼합하여 용액 또는 현탁액으로 제조하고 이를 앰플 또는 바이알의 단위 투여형으로 제제한다.The pharmaceutical composition for neutralizing HAV of the present invention contains the human monoclonal antibody as an active ingredient. The pharmaceutical composition of the present invention may preferably be administered parenterally, and parenteral administration may be by subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection. To formulate into a parenteral formulation, the human monoclonal antibody is mixed in water with a stabilizer or buffer to prepare a solution or suspension, which is formulated in unit dosage forms of ampoules or vials.
본 발명에 따른 유효성분인 인간 단일클론항체의 투여량은 체내에서 활성성분의 흡수도, 불활성화율 및 배설속도, 환자의 연령, 성별 및 상태, 치료할 질병의 중증 정도에 따라 적절히 선택될 수 있다.The dosage of the human monoclonal antibody, which is the active ingredient according to the present invention, may be appropriately selected depending on the absorption rate, inactivation rate and excretion rate of the active ingredient in the body, age, sex and condition of the patient, and the severity of the disease to be treated.
또한, 본 발명은 상기 인간 단일클론항체를 이용하여 HAV를 중화시키는 방법 및 HAV 감염을 예방 및 치료하는 방법을 제공한다.The present invention also provides a method for neutralizing HAV using the human monoclonal antibody and a method for preventing and treating HAV infection.
본 발명자들은 상기에서 얻은 HAV 특이 항체가 HAV 중화효능이 있는지 확인하기 위해, HAV에 대한 중화효능이 있는 생쥐 단일클론항체와 경쟁 결합 분석(competition binding assay)을 수행한 결과, HAV6 와 HAV9는 모두 중화항체 7E7와 B5B3의 HAV에 대한 결합을 저해함을 알 수 있었다(도 5참조). 상기 결과로부터, 본 발명의 인간항체 HAV6와 HAV9도 HAV 중화 에피토프에 결합함을 알 수 있다.The present inventors performed a competition binding assay with a mouse monoclonal antibody having a neutralizing effect on HAV to confirm whether the HAV-specific antibody obtained above had a neutralizing effect on HAV, and thus, both HAV6 and HAV9 were neutralized. It was found that the binding of antibodies 7E7 and B5B3 to HAV was inhibited (see FIG. 5 ). From the above results, it can be seen that the human antibodies HAV6 and HAV9 of the present invention also bind to HAV neutralizing epitopes.
본 발명자들은 상기에서 얻은 HAV 특이 항체가 실제로 HAV의 감염을 막을 수 있는지 RIFIA(Radio Immuno Foci Inhibition Assay) 방법(SM Lemon and LN Binn,J. Infect. Dis. 1983, 148:1033-1039)을 이용하여 확인한 결과, HAV의 감염이 완전히 저해됨을 알 수 있었다(도 6참조). 따라서 본 발명의 HAV에 대한 인간 단일클론항체는 HAV 감염에 대한 중화항체로 사용이 가능하고, 따라서 HAV의 감염 예방 및 치료에 유용하게 이용될 수 있다.The present inventors have used the RIFIA (Radio Immuno Foci Inhibition Assay) method (SM Lemon and LN Binn, J. Infect. Dis . 1983, 148: 1033-1039) to determine whether the HAV specific antibody obtained above can actually prevent HAV infection. As a result, it was found that the infection of HAV was completely inhibited (see FIG. 6 ). Therefore, the human monoclonal antibody against HAV of the present invention can be used as a neutralizing antibody against HAV infection, and thus can be usefully used for preventing and treating infection of HAV.
이하, 본 발명을 실시예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail by way of examples.
단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.
<실시예 1> HAV-프라임된(primed) 인간 B 림프구의 분리 및 EBV에 의한 형질전환Example 1 Isolation of HAV-primed Human B Lymphocytes and Transformation by EBV
본 발명자들은 HAV 감염을 중화할 수 있는 신규한 인간항체를 제조하기 위하여, 먼저 HAV-프라임된 인간 B 림프구를 분리한 후 EBV (Epstein-Barr virus)를 이용한 형질전환을 실시하였다. 구체적으로, HAV 감염 후 회복된 5명의 환자로부터 혈액을 취한 후, E-로세팅(rosetting) 방법(Kaplan ME and Clark C, J. Immunol. Methods, 1974, 5, 131-135)을 이용하여 T-림프구를 제거하고 B-림프구를 분리하였다. 상기 세포를 DMEM 배양액 5 ㎖에 부유시킨 다음, 미리 HAV (200 ng/㎖, Biogenesis 사)가 결합된 T-플라스크에 넣었고 1 시간동안 37℃, CO2배양기에 정치하였다. T-플라스크 바닥에 붙지 않은 림프구를 가볍게 흔들어 쏟아낸 후, 5 ㎖의 DMEM 배양액을 T-플라스크에 넣은 후 세게 흔들어서 바닥에 붙어있는 HAV에 특이적으로 결합하는 B 세포들을 부유시켰다.The present inventors first isolated HAV-primed human B lymphocytes and then transformed with EBV (Epstein-Barr virus) to prepare a novel human antibody that can neutralize HAV infection. Specifically, blood was taken from five patients recovered after HAV infection and then T-treated using E-rosetting method (Kaplan ME and Clark C, J. Immunol. Methods, 1974, 5, 131-135). Lymphocytes were removed and B-lymphocytes were separated. The cells were suspended in 5 ml of DMEM culture and then placed in a T-flask with HAV (200 ng / ml, Biogenesis) in advance and allowed to stand in a 37 ° C., CO 2 incubator for 1 hour. After gently shaking the lymphocytes that did not adhere to the bottom of the T-flask, 5 ml of DMEM culture was added to the T-flask, followed by vigorous shaking to float B cells that specifically bind to HAV attached to the bottom.
상기와 같은 방법으로 분리한 HAV-프라임된 B 세포를 변형시키기 위하여, 상기 세포를 침전시킨 후 EBV를 생산하는 마모셋(Marmoset) 세포주 B95-8 (ATCC, CRL-1612)을 배양하여 얻은 EBV 배양 상층액 5 ㎖에 부유시켰다. 상기 배양액을 T-플라스크에 넣어 37℃, CO2배양기에 2시간동안 정치시킨 다음, 새로운 배양액을 가하여 3주 동안 배양을 계속하였다.EBV culture obtained by culturing Marmoset cell line B95-8 (ATCC, CRL-1612) producing EBV after precipitation of the cells, in order to modify the HAV-primed B cells isolated by the above method. It was suspended in 5 ml of the supernatant. The culture solution was placed in a T-flask and allowed to stand for 2 hours in a CO 2 incubator at 37 ° C., followed by addition of fresh culture solution, and the culture was continued for 3 weeks.
상기와 같은 방법으로 EBV에 의해 변형된 B 세포를 세포융합에 필요한 양만큼 충분히 증식시켰으며, 배양 상층액을 회수하여 HAV에 대한 인간항체를 생산함을ELISA (Enzyme Linked Immunosorbent Assay)로 확인하였다. 이를 위하여, 먼저 ELISA 플레이트의 각 웰에 BSA (Bovine serum albumin)와 HAV 항원(Biogenesis 제품)을 4℃에서 하룻밤동안 반응시켜 결합시키고 4% 탈지유로 2시간 블록(block)시킨 후 PBST 용액(NaCl 8 g, KCl 0.2 g, Na2HPO41.15 g, KH2PO40.2 g, Tween20 0.5 ㎖/ℓ, pH 7.4)으로 4번 세척하였다. 상기 EBV로 변형된 인간 B 세포 배양액을 ELISA 플레이트의 각 웰에 첨가하여 37℃에서 2시간 동안 반응시킨 후 PBST 용액으로 세척하였다. 2차 항체인 항-인간 IgG (Fc specific)-HRP(horseradish peroxidase) (Sigma 사 제품)를 PBST를 사용하여 (1: 2000) 농도로 희석하여 상기 1차 항체와 결합시킨 후 ABTS(2',2'-Azino-Bis (3-Ethylbenz-thiazoline-6-Sulphonic Acid) diammonium salt, Sigma)와 H2O2로 발색하여 405 nm에서 흡광도를 측정하였다.In the same manner as described above, B cells modified by EBV were sufficiently grown by the amount necessary for cell fusion, and the culture supernatant was recovered to produce human antibodies against HAV by ELISA (Enzyme Linked Immunosorbent Assay). To this end, first, BSA (Bovine serum albumin) and HAV antigen (Biogenesis product) are reacted at 4 ° C. overnight at each well of an ELISA plate, and then blocked with 4% skim milk for 2 hours, followed by PBST solution (NaCl 8). g, KCl 0.2 g, Na 2 HPO 4 1.15 g, KH 2 PO 4 0.2 g, Tween20 0.5 mL / L, pH 7.4). The EBV-modified human B cell culture was added to each well of the ELISA plate, reacted at 37 ° C. for 2 hours, and then washed with PBST solution. Anti-human IgG (Fc specific) -HRP (horseradish peroxidase) (produced by Sigma), a secondary antibody, was diluted with PBST (1: 2000) to bind to the primary antibody, followed by ABTS (2 ', 2'-Azino-Bis (3-Ethylbenz-thiazoline-6-Sulphonic Acid) diammonium salt (Sigma) and H 2 O 2 and the absorbance was measured at 405 nm.
그 결과, 하기 표 1에서와 같이 상기 배양액에 존재하는 인간항체는 대조군인 BSA에는 결합하지 않고 HAV에 특이적으로 결합함을 확인하였다. 따라서, HAV에 특이적인 B 세포가 증식되었음을 확인하였다.As a result, it was confirmed that the human antibody present in the culture as shown in Table 1 specifically binds to HAV without binding to the control BSA. Therefore, it was confirmed that B cells specific for HAV were proliferated.
<실시예 2> 하이브리도마 제작Example 2 Preparation of Hybridoma
<2-1> HAT-민감성(sensitive) 인간 및 생쥐의 이형하이브리도마 제작<2-1> HAT-sensitive heterologous hybridoma production in humans and mice
Foung SKH 등의 방법(Foung SKH, Perkins S, Raubitschek A, Larrick J, Lizak G, Fishwild D, Engleman EG, and Grumet FC, J. Immunol. Methods, 1984, 70, 83-90)에 따라 HAT-민감성 인간 및 생쥐 이형하이브리도마를 제작하였다. 구체적으로, 인간 PBL (peripheral blood lymphocyte)로부터 분리한 HAT-저항성(resistant) B 세포(약 107세포)와 HAT-민감성 생쥐 마이엘로마(myeloma) Sp2/0-Ag14 세포 (약 107세포, ATCC No: CRL-1581)를 50% PEG 1500 (polyethylene glycol 1500, Sigma)을 이용하여 세포융합시킨 후, HAT 배지에 부유시켜 96-웰 플레이트에 분주하였다. 상기에서 생존한 인간-생쥐 이형하이브리도마의 HGPRT가 결여되게 하기 위해 8-아자구아닌(azaguanine) (2 ㎍/㎖)이 첨가된 배양액에서 상기 이형하이브리도마를 약 2 개월 동안 배양하였다.HAT-sensitive according to the methods of Foung SKH et al. (Foung SKH, Perkins S, Raubitschek A, Larrick J, Lizak G, Fishwild D, Engleman EG, and Grumet FC, J. Immunol. Methods, 1984, 70, 83-90) Human and mouse heterologous hybridomas were made. Specifically, HAT-resistant B cells (about 10 7 cells) isolated from human peripheral blood lymphocytes (PBL) and HAT-sensitive mouse myeloma Sp2 / 0-Ag14 cells (about 10 7 cells, ATCC No: CRL-1581) was cell fused using 50% PEG 1500 (polyethylene glycol 1500, Sigma) and then suspended in HAT medium and aliquoted into 96-well plates. In order to lack HGPRT of the human-mouse heterohybridoma surviving above, the heterohybridoma was incubated for about 2 months in a culture medium to which 8-azaguanine (2 µg / ml) was added.
생존한 세포들 중 일부를 HAT 배지에 배양한 결과 모든 세포들이 사멸하였다. 즉, 8-아자구아닌에는 내성이 있으나, HGPRT가 결여되어 HAT 배지에 민감한 인간-생쥐 이형하이브리도마 세포를 얻을 수 있었다. 또한, 상기 세포들은 사람 항체를 분비하지 않았다.Some of the surviving cells were cultured in HAT medium and all the cells were killed. That is, 8-azaguanine is resistant, but lacks HGPRT to obtain human-mouse heterohybridoma cells sensitive to HAT medium. In addition, the cells did not secrete human antibodies.
<2-2> HAV 특이 인간 및 인간과 생쥐의 이형하이브리도마 제작<2-2> HAV-Specific Human and Human and Mouse Heterologous Hybridomas
상기 실시예 <2-1>에서 제조한 HAT-민감성 인간-생쥐 이형하이브리도마 (107세포)를 상기 실시예 1에서 제조한 HAV-프라임된 인간 B 세포 (107세포)와 50% PEG를 이용하여 1 분간 세포융합을 시켰다. 이를 HAT와 1 μM 오우아바인(ouabain, Sigma)을 포함하는 배양액에 부유시킨 후, 96-웰 플레이트에 분주하여 배양하였다. 생존한 인간세포와 인간 및 생쥐세포의 이형하이브리도마 세포주들을 따로 분리한 후, 그들의 HAV에 대한 인간항체 발현 여부를 실시예 1에서와 동일하게 ELISA 방법으로 조사하였다.The HAT-sensitive human-mouse heterohybridoma (10 7 cells) prepared in Example <2-1> was prepared with HAV-primed human B cells (10 7 cells) prepared in Example 1 and 50% PEG. Cell fusion was carried out using for 1 minute. This was suspended in a culture solution containing HAT and 1 μM ouabain (Sigma), and then cultured by dispensing in a 96-well plate. After heterologous hybridoma cell lines of surviving human cells and human and mouse cells were separated separately, the expression of human antibodies against their HAV was examined in the same manner as in Example 1 by ELISA.
그 결과, HAV에 결합하는 IgG 항체를 분비하는 2개의 이형하이브리도마 세포주들(Hb-HAV6, Hb-HAV9)을 얻을 수 있었다. 이들 세포주들의 항체발현능력은 시간이 지남에 따라 점점 감소하여, 약 3 개월 후에는 항체 생산능이 완전히 소멸되었다.As a result, two heterologous hybridoma cell lines (Hb-HAV6 and Hb-HAV9) secreting IgG antibodies that bind HAV were obtained. Antibody-expressing ability of these cell lines gradually decreased over time, and after about three months, the ability to produce antibodies completely disappeared.
<실시예 3> HAV 특이 인간 단일클론항체의 정제Example 3 Purification of HAV Specific Human Monoclonal Antibodies
HAV 특이 인간 단일클론항체를 정제하기 위해, 상기 실시예 2에서 분리한 Hb-HAV6 및 Hb-HAV9 이형하이브리도마를 각각 무혈청 배지(PFHM-II, Gibco사 제품)에서 배양한 후 배양 상층액을 단백질 G-Sepharose 4B 컬럼(Pharmacia 사)을 통과시켰으며, 컬럼에 결합된 항체를 0.1 M 글라이신 용액(pH 2.7)으로 용출시킨 후 1.0 M Tris 용액(pH 9.0)으로 중화시키고 PBS 완충용액(pH 7.0)에서 투석하였다. 정제된 항체를 12% SDS-폴리아크릴아미드 겔 상에서 전기영동(SDS-PAGE)하였다.In order to purify HAV specific human monoclonal antibodies, the culture supernatants after culturing Hb-HAV6 and Hb-HAV9 heterologous hybridoma isolated in Example 2, respectively, in serum-free medium (PFHM-II, manufactured by Gibco) Was passed through a protein G-Sepharose 4B column (Pharmacia), the antibody bound to the column was eluted with 0.1 M glycine solution (pH 2.7), then neutralized with 1.0 M Tris solution (pH 9.0) and PBS buffer (pH). Dialysis). Purified antibodies were electrophoresed (SDS-PAGE) on 12% SDS-polyacrylamide gels.
그 결과, 상기 항체를 환원시켰을 때에 항체의 중쇄 및 경쇄의 분자량으로 알려진 55 kDa, 25 kDa의 분자량을 갖는 단백질 밴드를 관찰하였다(도 1). 상기 결과로부터, 인간 하이브리도마 세포주로부터 항체가 발현되어 정제되었음을 확인하였다.As a result, when the antibody was reduced, a protein band having a molecular weight of 55 kDa and 25 kDa known as the molecular weight of the heavy and light chains of the antibody was observed ( FIG. 1 ). From the above results, it was confirmed that the antibody was expressed and purified from the human hybridoma cell line.
<실시예 4> HAV 특이 인간항체의 항원결합능 측정Example 4 Determination of antigen binding ability of HAV specific human antibody
본 발명자들은 상기 실시예 3에서 정제한 항체들의 항원결합능을 ELISA에 의하여 측정하였다. 먼저, HAV 항원을 1:5로 코팅 버퍼(Na2CO31.59 g, NaHCO32.93 g/ℓ, pH 9.6)에 희석 후 면역플레이트(immunoplate)에 넣었고, 4℃에서 하룻밤동안 정치하여 플레이트에 결합시킨 후 2% BSA로 블록시키고 PBST로 3번 세척하였다. 상기에서 정제된 항체들과 CS13 항체(negative control)을 PBS를 사용하여 희석한 후 동일한 농도로 플레이트에 첨가하여 37℃에서 1 시간 동안 반응시켰고, 항원과 결합하지 않은 항체를 PBST로 세척하여 제거하였다. 2차 항체로 항-인간 IgG (Fc specific)-HRP를 PBST를 사용하여 1: 2000 농도로 희석하여 상기 1차 항체와 결합시키고 ABTS와 H2O2로 발색하여 405 nm에서 흡광도를 측정하였다. 그 결과, HAV6, HAV9 항체 모두 HAV에 특이적으로 결합함을 알 수 있었고, 항원결합능이 서로 차이가 있음을 확인하였다(도 2).The present inventors measured the antigen binding ability of the antibodies purified in Example 3 by ELISA. First, the HAV antigen was diluted 1: 5 in coating buffer (Na 2 CO 3 1.59 g, NaHCO 3 2.93 g / L, pH 9.6), placed in an immunoplate, and allowed to stand at 4 ° C. overnight to bind to the plate. After blocking with 2% BSA and washed three times with PBST. The purified antibodies and the CS13 antibody (negative control) were diluted using PBS, added to the plate at the same concentration, and reacted at 37 ° C. for 1 hour. Antibodies not bound to the antigen were washed out with PBST. . As a secondary antibody, anti-human IgG (Fc specific) -HRP was diluted to a concentration of 1: 2000 using PBST, bound to the primary antibody, developed with ABTS and H 2 O 2 , and absorbance was measured at 405 nm. As a result, it was found that both HAV6 and HAV9 antibodies specifically bind to HAV, and it was confirmed that antigen binding ability is different from each other ( FIG. 2 ).
<실시예 5> HAV 특이 인간항체 가변영역의 cDNA 클로닝 및 발현벡터 제조Example 5 cDNA Cloning and Expression Vector Preparation of HAV Specific Human Antibody Variable Regions
<5-1> HAV 특이 인간항체 가변영역의 cDNA 클로닝 및 발현벡터의 제조<5-1> cDNA Cloning and Expression Vector Preparation of HAV Specific Human Antibody Variable Regions
우선, 분리된 HAV 특이 인간 단일클론항체의 N-말단 아미노산 서열을 결정하기 위해, 55 kDa 중쇄(heavy chain)와 25 kDa 경쇄(light chain) 단백질 밴드를 PVDF 막으로 옮긴 다음, 각 단백질 밴드의 N-말단 아미노산 서열을 결정하였으며(Applied Biosystems Precise Sequencer), 그 결과를표 2에 나타내었다.First, to determine the N-terminal amino acid sequence of an isolated HAV specific human monoclonal antibody, 55 kDa heavy chain and 25 kDa light chain protein bands were transferred to the PVDF membrane, and then N of each protein band. The terminal amino acid sequence was determined (Applied Biosystems Precise Sequencer) and the results are shown in Table 2 .
상기표 2에서 알 수 있는 바와 같이, HAV6와 HAV9 항체의 경쇄 N-말단 아미노산 서열은 동일하였고, 중쇄 N-말단 아미노산 서열은 차이가 있었다. 또한, 상기 N-말단 아미노산 서열을 V-베이스(base)의 인간항체 점라인(germline) 서열 데이터베이스와 비교한 결과, HAV6 및 HAV9가 각각 인간 점라인 항체 유전자인 DP51, DP71의 N-말단 아미노산 서열과 가장 유사함을 알 수 있었고, 이에 근거하여 클론닝에 필요한 제한효소 인지서열을 포함하는 5' 프라이머를 제작하였다. 경쇄의 경우서열번호 10으로 기재되는 KCfor 프라이머를 제작하였고, HAV6 중쇄 및 HAV9 중쇄의 경우 각각서열번호 15로 기재되는 HCfor6 프라이머 및서열번호 16으로 기재되는 HCfor9 프라이머를 제작하였다.As can be seen in Table 2 , the light chain N-terminal amino acid sequences of HAV6 and HAV9 antibodies were identical, and the heavy chain N-terminal amino acid sequences were different. In addition, when comparing the N-terminal amino acid sequence with the human antibody germline sequence database of V-base, the N-terminal amino acid sequence of DP51 and DP71 where HAV6 and HAV9 are the human germline antibody genes, respectively It was found that the most similar to, based on this based on the 5 'primer containing a restriction enzyme recognition sequence required for cloning was prepared. For light chains, the KCfor primers described in SEQ ID NO: 10 were prepared, and for HAV6 heavy and HAV9 heavy chains, HCfor6 primers described in SEQ ID NO: 15 and HCfor9 primers described in SEQ ID NO: 16 were prepared.
상기 인간 항체의 경쇄 가변영역을 암호하는 cDNA 합성을 위해, Hb-HAV6 및 Hb-HAV9 이형하이브리도마로로부터 전체(total) RNA를 분리하여, 이를 주형으로 역전사효소 (Superscript II, Gibco BRL)와서열번호 9로 기재되는 프라이머 CK1d를 사용하여 인간경쇄 가변영역에 해당하는 cDNA를 선택적으로 합성하였다. 상기 인간경쇄 cDNA를 주형으로서열번호 10으로 기재되는 프라이머 KCfor와서열번호 11로 기재되는 프라이머 KCBsiWIback를 사용하여 PCR을 수행하여 인간경쇄 가변영역의 유전자를 합성하였다. 이때, 이들 PCR 반응조건은 95℃에서 5분간 예비변성 후, 95℃에서 50초, 55℃에서 50초, 72℃에서 1분으로TaqDNA 중합효소(polymerase)를 사용하여 30회 수행하였다.For cDNA synthesis encoding the light chain variable region of the human antibody, total RNA was isolated from Hb-HAV6 and Hb-HAV9 heterohybridoma, and the reverse transcriptase (Superscript II, Gibco BRL) and sequence were used as a template. The primer CK1d described as No. 9 was used to selectively synthesize cDNA corresponding to the human light chain variable region. The gene of the human light chain variable region was synthesized by PCR using the primer KCfor described in SEQ ID NO: 10 and the primer KCBsiWIback described in SEQ ID NO: 11 as a template of the human light chain cDNA. At this time, these PCR reaction conditions were performed 30 times using Taq DNA polymerase (preliminary modification at 95 ℃ for 5 minutes, 50 seconds at 95 ℃, 50 seconds at 55 ℃, 1 minute at 72 ℃).
경쇄 유전자의 신호 서열을 합성하기 위하여, pdCMV-dhfr-AKA/HzK (대한민국 특허출원 제2001-47737호)를 주형으로 하고서열번호 12로 기재되는 LHS42 프라이머와서열번호 13으로 기재되는 KCleaderBack 프라이머를 쌍으로 PCR을 수행하였다.To synthesize the signal sequence of the light chain gene, paired the LHS42 primer as shown in SEQ ID NO: 12 and the KCleaderBack primer as shown in SEQ ID NO: 13 with pdCMV-dhfr-AKA / HzK (Korean Patent Application No. 2001-47737) as a template. PCR was performed.
상기 신호서열과 인간 경쇄 가변영역을 연결하기 위해서열번호 12로 기재되는 LHS42프라이머와서열번호 11로 기재되는 KCBsiWIback 프라이머를 이용하여 재조합 PCR을 행하였다. 이 때, PCR 반응 조건은 95℃에서 5분간 예비변성 후, 94℃에서 50초, 55℃에서 50초, 72℃에서 1분으로TaqDNA 중합효소(polymerase)를 사용하여 30회 수행하였다. 상기 DNA 단편의 양끝을 제한효소 HindIII과 BsiWI으로 절단한 후 pdCMV-dhfrC-AKA/HzK의 HindIII-BsiWI 위치에 삽입하여 pdCMV-dhfrC-HAVKC6 및 pdCMV-dhfrC-HAVKC9를 제조하였다.In order to connect the signal sequence and the human light chain variable region, recombinant PCR was performed using the LHS42 primer described in SEQ ID NO: 12 and the KCBsiWIback primer described in SEQ ID NO: 11 . At this time, PCR reaction conditions were performed 30 times using Taq DNA polymerase (50 minutes at 94 ℃, 50 seconds at 55 ℃, 1 minute at 72 ℃ after 5 minutes pre-denatured at 95 ℃). Both ends of the DNA fragment were digested with restriction enzymes HindIII and BsiWI, and then inserted at the HindIII-BsiWI position of pdCMV-dhfrC-AKA / HzK to prepare pdCMV-dhfrC-HAVKC6 and pdCMV-dhfrC-HAVKC9.
한편, 상기 인간 항체의 중쇄 가변영역을 암호하는 cDNA 합성을 위해 Hb-HAV6 및 Hb-HAV9 이형하이브리도마로부터 전체 RNA를 분리하여, 이를 주형으로 역전사효소(Superscript II, Gibco BRL)와서열번호 14로 기재되는 프라이머 CG1d를 사용하여 인간중쇄 Fd 영역에 해당하는 cDNA를 선택적으로 합성하였다. 또한, 인간중쇄 Fd 영역의 cDNA를 주형으로서열번호 15로 기재되는 프라이머 HCfor6나서열번호 16으로 기재되는 프라이머 HCfor9와서열번호 17로 기재되는 프라이머 LHS11를 각기 쌍으로 사용하여 PCR을 수행하여 인간중쇄의 가변영역의 유전자를 합성하였다.On the other hand, for the synthesis of cDNA encoding the heavy chain variable region of the human antibody, the total RNA is isolated from Hb-HAV6 and Hb-HAV9 heterohybridoma, and as a template reverse transcriptase (Superscript II, Gibco BRL) and SEQ ID NO: 14 The cDNA corresponding to the human heavy chain Fd region was selectively synthesized using primer CG1d described as. In addition, PCR was carried out using a pair of primers HCfor6 described in SEQ ID NO: 15 , primers HCfor9 described in SEQ ID NO: 16 , and primers LHS11 described in SEQ ID NO: 17 , respectively, as pairs of cDNAs of the human heavy chain Fd region. The gene of the variable region was synthesized.
중쇄 유전자의 신호 서열을 합성하기 위하여, pdCMV-dhfr-AKA/HzK를 주형으로 하고서열번호 18로 기재되는 LHS39 프라이머와서열번호 19로 기재되는 HCleaderBack 프라이머를 쌍으로 PCR을 수행하였다.In order to synthesize the signal sequence of the heavy chain gene, PCR was performed in pairs with the pdCMV-dhfr-AKA / HzK as a template and the LHS39 primer as shown in SEQ ID NO: 18 and the HCleaderBack primer as shown in SEQ ID NO: 19 .
다시, 신호서열과 중쇄 가변영역을 연결하기 위해서열번호 18로 기재되는 LHS39 프라이머와서열번호 17로 기재되는 LHS11 프라이머를 이용하여 재조합 PCR을 행하였다. 이 때, PCR 반응 조건은 95℃에서 5분간 예비변성 후, 94℃에서 50초, 55℃에서 50초, 72℃에서 1분으로TaqDNA 중합효소를 사용하여 30회 수행하였다.Again, recombinant PCR was performed using the LHS39 primer shown in SEQ ID NO: 18 and the LHS11 primer shown in SEQ ID NO: 17 to connect the signal sequence and the heavy chain variable region. At this time, PCR reaction conditions were performed 30 times using Taq DNA polymerase for 5 minutes at 95 ℃, 50 seconds at 94 ℃, 50 seconds at 55 ℃, 1 minute at 72 ℃ after denaturation .
상기 DNA 단편의 양끝을 제한효소 EcoRI과 ApaI으로 절단한 후, 상기의 항 HAV 경쇄가 클로닝된 pdCMV-dhfrC-HAVKC6 또는 pdCMV-dhfrC-HAVKC9의 EcoRI-ApaI위치에 삽입하여 본 발명의 발현 플라스미드를 제조하였으며, 이들은 각각 "pdCMV-dhfrC-HAV6" 및 "pdCMV-dhfrC-HAV9"이라 명명하였다. 본 발명자들은 상기 pdCMV-dhfrC-HAV6 발현벡터를 2001년 8월 3일자로 생명공학연구원 유전자은행에 기탁하였고(수탁번호; KCTC 10027BP), 상기 pdCMV-dhfrC-HAV9 발현벡터를 2001년 8월 4일자로 생명공학연구원 유전자은행에 기탁하였다(수탁번호; KCTC 10029BP).After cutting both ends of the DNA fragments with restriction enzymes EcoRI and ApaI, the anti-HAV light chain was inserted into the EcoRI-ApaI position of pdCMV-dhfrC-HAVKC6 or pdCMV-dhfrC-HAVKC9 cloned to prepare an expression plasmid of the present invention. And they were named "pdCMV-dhfrC-HAV6" and "pdCMV-dhfrC-HAV9", respectively. The present inventors deposited the pdCMV-dhfrC-HAV6 expression vector with the Biotechnology Research Institute Gene Bank on August 3, 2001 (accession number; KCTC 10027BP), and the pdCMV-dhfrC-HAV9 expression vector on August 4, 2001. Was deposited in the Genetic Bank of Korea Biotechnology Institute (Accession No .; KCTC 10029BP).
<5-2> 염기서열 분석<5-2> Sequence Analysis
상기 클론들의 경쇄 및 중쇄의 가변영역에 대한 염기서열을 T7 시쿼네이즈(Sequenase) V2.0 DNA 시퀀싱 키트(Amersham)로 결정한 결과, HAV6 항체의 경쇄(HAV6KC) 및 중쇄(HAV6HC)는 각각서열번호 1및서열번호 3, HAV9 항체의 경쇄(HAV9KC) 및 중쇄(HAV9HC)는 각각서열번호 2및서열번호 4로 기재되는 염기서열을 갖고 있고, 상기 인간경쇄 및 인간중쇄의 아미노산 서열은 각각서열번호 5내지서열번호 8로 기재됨을 확인하였다.The base sequences for the variable regions of the light and heavy chains of the clones were determined by the T7 Sequence V2.0 DNA Sequencing Kit (Amersham). As a result, the light chain (HAV6KC) and the heavy chain (HAV6HC) of the HAV6 antibody were respectively SEQ ID NO: 1 And SEQ ID NO: 3 , the light chain (HAV9KC) and heavy chain (HAV9HC) of the HAV9 antibody have the nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 4 , respectively, and the amino acid sequences of the human light and human heavy chains are SEQ ID NOs: 5 to 7, respectively. It was confirmed that it is described by SEQ ID NO: 8 .
상기 경쇄의 경우, 선별된 클론 모두가 인간 경쇄 생식세포주 유전자 중에 VK1 패밀리의 DPK9/O12의 아미노산 서열과 매우 유사하여 인간경쇄가 DPK9/O12에서 유래한 것을 알 수 있었고, 반면에, 중쇄의 경우, 모두 서로 다른 인간 중쇄 생식세포주 유전자에서 유래하였음을 알 수 있었다(도 4). HAV6 및 HAV9의 중쇄 유전자는 각각 인간 점라인 항체 유전자인 DP51, DP71와 가장 유사하였다.In the light chain, all of the selected clones were very similar to the amino acid sequence of the DPK9 / O12 of the VK1 family in the human light chain germline gene, indicating that the human light chain was derived from DPK9 / O12, whereas in the heavy chain, All were derived from different human heavy chain germline genes ( FIG. 4 ). The heavy chain genes of HAV6 and HAV9 were most similar to the human pointline antibody genes DP51 and DP71, respectively.
<실시예 6> 항 HAV 항체의 대량생산Example 6 Mass Production of Anti-HAV Antibodies
우선, CHO 세포(ATCC CRL 9096)를 소 태아 혈청이 10% 첨가된 DMEM/F12 배양배지(GIBCO)에 접종하여 37℃, 5% CO2항온기에서 계대배양하였다. 상기 세포를 60 ㎜ 배양접시에 5x105세포로 접종하고 37℃에서 밤새 배양한 후 OPTI-MEM I (GIBCO) 용액으로 3회 세척하였다. 한편, 상기에서 제조한 항 HAV 항체 발현벡터 pdCMV-dhfrC-HAV6 및 pdCMV-dhfrC-HAV9를 각각 5 ㎍씩 취하여 OPTI-MEM I 500 ㎕로 희석하였고, 25 ㎕의 리포펙타민(Lipofectamine, GIBCO)도 동일하게 OPTI-MEM I 500 ㎕로 희석하였다. 상기 항체발현 벡터와 리포펙타민 희석액을 15 ㎖ 튜브에 혼합하여 DNA-리포펙타민 혼합물을 제조한 후 이를 15분 이상 실온에서 방치하였다. 상기 각각의 DNA-리포펙타민 혼합물에 2 ㎖의 OPTI-MEM I을 첨가하였고 이를 깨끗이 세척된 CHO 세포에 골고루 혼합한 후 37℃, 5% CO2항온기에서 6시간 방치 후, 20% 소태아혈청을 포함한 DMEM/F12를 3ml 첨가하여 다시 48시간 동안 배양하였다.First, CHO cells (ATCC CRL 9096) were seeded in DMEM / F12 culture medium (GIBCO) to which 10% fetal bovine serum was added and passaged at 37 ° C and 5% CO 2 incubator. The cells were seeded with 5 × 10 5 cells in a 60 mm dish and incubated overnight at 37 ° C. and washed three times with OPTI-MEM I (GIBCO) solution. Meanwhile, 5 μg of each of the anti-HAV antibody expression vectors pdCMV-dhfrC-HAV6 and pdCMV-dhfrC-HAV9 prepared above was diluted to 500 μl of OPTI-MEM I, and 25 μl of lipofectamine (Lipofectamine, GIBCO) was also obtained. Equally diluted with 500 μl OPTI-MEM I. The antibody expression vector and the lipofectamine dilution were mixed in a 15 ml tube to prepare a DNA-lipopeptamine mixture, which was left at room temperature for at least 15 minutes. 2 ml of OPTI-MEM I was added to each of the DNA-lipopeptamine mixtures, and the mixture was evenly mixed with cleanly washed CHO cells and left for 6 hours at 37 ° C. in a 5% CO 2 incubator. 3ml of DMEM / F12 was added thereto and incubated again for 48 hours.
형질전환된 세포주를 얻기 위해 상기 CHO 세포들을 트립신(Trypsin)으로 떼어낸 후, G418 (Gibco BRL, 550 ㎎/ℓ)을 포함하는 10% 투석된(dialyzed) 소태아혈청의 α-MEM 배지에서 약 2주간 배양하였다. 여기서 살아남은 형질전환된 클론들을 각각 배양하여 항체생산능을 확인한 후, 20 nM MTX를 포함하는 10% 투석된 소태아혈청의 α-MEM 배지에서 배양하여 항체유전자의 증폭을 유도하였다. 여기서 살아남은 클론들의 항체생산성을 확인 후, 가장 생산성이 높은 클론을 얻었으며, 각 발현벡터 pdCMV-dhfrC-HAV6, pdCMV-dhfrC-HAV9으로부터 제조된 세포주를 각각 "HAV6-20", "HAV9-20"으로 명명하였다.The CHO cells were detached with Trypsin to obtain a transformed cell line and then medicated in α-MEM medium of 10% dialyzed fetal bovine serum containing G418 (Gibco BRL, 550 mg / L). Incubated for 2 weeks. After culturing each of the surviving transformed clones to confirm the antibody production capacity, and incubated in α-MEM medium of 10% dialyzed fetal bovine serum containing 20 nM MTX to induce amplification of the antibody gene. After confirming the antibody productivity of the surviving clones, the most productive clones were obtained, and cell lines prepared from each of the expression vectors pdCMV-dhfrC-HAV6 and pdCMV-dhfrC-HAV9 were designated as "HAV6-20" and "HAV9-20", respectively. Named.
본 발명자들은 상기 HAV6-20 세포주 및 HAV9-20 세포주를 2002년 12월 일자로 각각 한국생명공학연구원 유전자은행에 기탁하였다(수탁번호; KCTC 10404BP, KCTC 10405BP).The present inventors deposited the HAV6-20 cell line and HAV9-20 cell line with the Korea Biotechnology Research Institute Gene Bank, respectively, as of December 2002 (Accession No .; KCTC 10404BP, KCTC 10405BP).
HAV 특이 인간 단일클론항체를 대량생산하기 위하여, 상기 HAV6-20 세포주와 HAV9-20 세포주를 무혈청배지(CHO-SFMII, Gibco BRL)에서 대량배양한 후, 실시예 3의 방법으로 배양상층액으로부터 Protein G 컬럼을 이용하여 항체를 정제하였다.In order to mass-produce HAV-specific human monoclonal antibodies, the HAV6-20 cell line and HAV9-20 cell line were cultured in serum-free medium (CHO-SFMII, Gibco BRL), and then cultured from the culture supernatant by the method of Example 3. Antibodies were purified using a Protein G column.
<실시예 7> 항 HAV 인간항체의 항원 결합 특성Example 7 Antigen Binding Properties of Anti-HAV Human Antibodies
대부분의 HAV 중화 생쥐 단일클론항체의 경우, HAV의 VP1과 VP3로 구성된 면역우성(immunodominant) 구조 에피토프 클러스터(conformational epitope cluster)를 인식하는 것으로 알려져 있다(L Ping and SM Lemon,J. Virol., 1992, 66, 2208-2216; JT Stapletonet al.,J. Virol., 1993, 67, 1080-1085). 본 발명에서 제조한 HAV6와 HAV9 항체가 생쥐 중화항체의 에피토프를 공유하는지 확인하기 위해, 생쥐중화항체 7E7 (Mediagnost) 및 B5B3 (Biogenesis)을 이용하여 항체 경쟁 분석(antibody competition assay)을 수행하였다(L Ping and SM Lemon,J. Virol., 1992, 66:2208-2216). 구체적으로, 생쥐중화항체 7E7 또는 B5B3을 다양한 농도의 HAV6 또는 HAV9 항체와 섞은 후, HAV가 코팅된 마이크로플레이트에 첨가하였다. 37℃에서 1시간 정치 후, PBST로 3회 세척하였다. HAV와 결합한 생쥐중화항체를 정량하기 위해 항-생쥐항체(Fc specific, Pierce)-HRP를 1/2000으로 희석하여 가한후, 37℃에서 1시간동안 정치하였다. 각 웰을 PBST로 3회 세척한 후, ABTS로 발색시켜 405 nm에서 흡광도를 측정하였다. 생쥐중화항체에 HAV6 또는 HAV9을 첨가하지 않은 웰의 흡광도를 생쥐중화항체의 100% 결합으로 간주했을 때, 상기에서 얻은 인간항체 HAV6 와 HAV9는 모두 0.4 ㎍/㎖ 농도에서 중화항체 7E7와 B5B3의 HAV에 대한 결합을 90% 이상 저해함을 알 수 있었다(도 5). 상기 결과로부터, 본 발명의 인간항체 HAV6와 HAV9도 HAV 중화 에피토프에 결합함을 확인하였다.Most HAV neutralizing mouse monoclonal antibodies are known to recognize immunodominant structural epitope clusters consisting of VP1 and VP3 of HAV (L Ping and SM Lemon, J. Virol. , 1992) . , 66, 2208-2216; JT Stapleton et al. , J. Virol. , 1993, 67, 1080-1085). In order to confirm that the HAV6 and HAV9 antibodies prepared in the present invention share the epitope of the neutralizing antibody of the mouse, an antibody competition assay was performed using the mouse neutralizing antibodies 7E7 (Mediagnost) and B5B3 (Biogenesis) (L). Ping and SM Lemon, J. Virol. , 1992, 66: 2208-2216). Specifically, mouse neutralizing antibodies 7E7 or B5B3 were mixed with various concentrations of HAV6 or HAV9 antibodies and then added to HAV coated microplates. After standing at 37 ° C. for 1 hour, the mixture was washed three times with PBST. Anti-mouse antibody (Fc specific, Pierce) -HRP was added to dilute to 1/2000 in order to quantify HAV-bound mouse neutralizing antibody, and then allowed to stand at 37 ° C for 1 hour. Each well was washed three times with PBST and then developed with ABTS to measure absorbance at 405 nm. When the absorbance of wells without adding HAV6 or HAV9 to the neutralizing antibody was considered to be 100% binding of the neutralizing antibody, the human antibodies HAV6 and HAV9 obtained above were both HAV6 and B5B3 at 0.4 μg / ml. It can be seen that inhibiting the binding to more than 90% ( Fig. 5 ). From the above results, it was confirmed that the human antibodies HAV6 and HAV9 of the present invention also bind to HAV neutralizing epitopes.
<실시예 8> 항 HAV 항체의 HAV 중화능 결정Example 8 Determination of HAV Neutralizing Capacity of Anti-HAV Antibody
상기에서 제조한 항 HAV 항체가 실제로 HAV의 감염을 막을 수 있는지 여부를 RIFIA(Radio Immuno Foci Inhibition Assay) 방법을 이용하여 확인하였다(SM Lemon and LN Binn,J. Infect. Dis., 1983, 148:1033-1039). 구체적으로, HAV 감염 가능한 BS-C-1 세포주(ATCC, CCL-26)를 더마녹스 라운드(Thermanox round) 25-mm 커버슬립(coverslip)이 부착된 6-웰 플레이트(Nunc사 제품)에서 10% 소태아혈청이 포함된 MEM 배지를 이용하여 90% 포화상태(confluency)로 배양하였다. HAV와 상기에서 정제한 항체(50 ㎍/㎖)를 섞어준 후 35℃에서 1시간동안 정치시켰고, BS-C-1 세포에 첨가하여 2시간 동안 35℃에서 HAV의 감염을 유도하였다. 여기에 1% 아가로스가 첨가된 2% 소태아혈청의 MEM 배지를 위에 덮은 후, 35℃, 5% CO2배양기에서 2주간 배양하였다. 커버슬립을 아세톤에 2분간 방치하여 고정한 후 잘 건조시켰고, 항 HAV 항체 7E7(Mediagnost, Ger)을 1:500으로 5% BSA PBS에 희석하여 커버슬립과4시간동안 반응시켰다. 커버슬립을 PBS로 3회 세척 후, 1:100으로 희석된125I-표지된 항-생쥐 IgG(Amersham)와 1시간 동안 반응시켰다. 다시 PBS로 5회 세척하여 잘 건조시킨 후, 자가방사기록법을 수행하여 HAV 감염으로 형성된 초점을 확인하였다.Whether the anti-HAV antibody prepared above can actually prevent the infection of HAV was confirmed using the Radio Immuno Foci Inhibition Assay (RIFIA) method (SM Lemon and LN Binn, J. Infect. Dis. , 1983, 148: 1033-1039). Specifically, HAV infectious BS-C-1 cell lines (ATCC, CCL-26) were 10% in a 6-well plate (Nunc) with a Thermanox round 25-mm coverslip. Cultured at 90% saturation (confluency) using MEM medium containing fetal bovine serum. HAV and the above-prepared antibody (50 μg / ml) were mixed and allowed to stand at 35 ° C. for 1 hour and added to BS-C-1 cells to induce HAV infection at 35 ° C. for 2 hours. Covered with MEM medium of 2% fetal bovine serum to which 1% agarose was added thereto, and incubated for 2 weeks in a 35 ° C., 5% CO 2 incubator. The coverslip was left in acetone for 2 minutes, fixed, and then dried well. Anti-HAV antibody 7E7 (Mediagnost, Ger) was diluted 1: 500 in 5% BSA PBS and reacted with the coverslip for 4 hours. The coverslips were washed three times with PBS and then reacted with 125 I-labeled anti-mouse IgG (Amersham) diluted 1: 100 for 1 hour. After washing 5 times with PBS again and dried well, autoradiography was performed to check the foci formed by HAV infection.
그 결과, 본 발명의 HAV6 또는 HAV9 항체에 의해 HAV의 감염이 저해됨을 알 수 있다(도 6). 상기 결과로부터, HAV6 및 HAV9 항체는 HAV 감염에 대한 중화항체로 사용 가능함을 알 수 있다.As a result, it can be seen that HAV infection is inhibited by the HAV6 or HAV9 antibodies of the present invention ( FIG. 6 ). From the above results, it can be seen that HAV6 and HAV9 antibodies can be used as neutralizing antibodies against HAV infection.
상기에서 살펴본 바와 같이, 본 발명의 HAV에 특이적으로 결합하는 인간 단일클론항체는 HAV에 대한 중화능을 나타내므로 HAV의 진단이나 HAV 감염의 예방 또는 치료에 유용하게 이용될 수 있다.As described above, since the human monoclonal antibody specifically binding to HAV of the present invention exhibits neutralizing ability against HAV, it can be usefully used for diagnosis of HAV or prevention or treatment of HAV infection.
<110> Korea Research Institute of Bioscience and Biotechnology <120> Neutralizing human monoclonal antibody specific to hepatitis A virus <130> 2p-03-32B <160> 22 <170> KopatentIn 1.71 <210> 1 <211> 318 <212> DNA <213> Artificial Sequence <220> <223> HAV6KC <400> 1 atccagatga cccagtctcc atcctccctg tctgcatctg taggagacag agtcaccatc 60 acttgccggg caagtcagag cattagcagc tatttaaatt ggtatcagca gaaaccaggg 120 aaagccccta agctcctgat ctatgctgca tccagtttgc aaagtggggt cccatcaagg 180 ttcagtggca gtggatctgg gacagatttc actctcacca tcagcagtct gcaacctgaa 240 gattttgcaa cttactactg tcaacagagt tacagtacca cgtggacgtt cggccaaggg 300 accaaggtgg aaatcaaa 318 <210> 2 <211> 318 <212> DNA <213> Artificial Sequence <220> <223> HAV9KC <400> 2 atccagatga cccagtctcc atcctcccta tctgcatctg taggagacag agtcaccatc 60 acttgccggg caagtcagag cattagcagc tatttaaatt ggtatcagca gaaaccaggg 120 caagccccta agctcttgat ctttgctgca tccagtttgc aaagtggggt cccatcaagg 180 ttcagtggca gtggatctgg gacagatttc actctcaccc tcagcagtct gcaacctgaa 240 gattttgcaa cttactactg tcaacagagt tacagtaccc cgtacacttt tggccagggg 300 accaaggtgg agatcaaa 318 <210> 3 <211> 366 <212> DNA <213> Artificial Sequence <220> <223> HAV6HC <400> 3 gtgcagctgc tcgagactgg gggaggcttg gtgcagcctg gggggtccct gagactctcc 60 tgtgcagcct ctggattcac ctttagaaac tatggcatga actgggtccg ccaggctcca 120 gggaaggggc tggagtgggt cgcagttatt tatggcggtg gtggtcgaac acactatgca 180 gactccgtgc aggggcggtt catcatctcc agagatgatt ccacgaacac gatgtatctg 240 caaatgaaca gcctgagagc cgaggacacg gccgtatatt actgtgcgaa agacagaatg 300 acttcaaaag tgtgggacta cggtatggac gtctggggcc aagggaccac ggtcaccgtc 360 tcctca 366 <210> 4 <211> 384 <212> DNA <213> Artificial Sequence <220> <223> HAV9HC <400> 4 gtgcagctgc aggagtgggg cccaggactg gtgaggcctt cggagaccct gtccctcacc 60 tgcactgtct ctggtggctc catcagtcct gactactgga gctggatccg tcagccccca 120 gggaaggaac tggagtggat tgggtatatc tattacagtg ggagcaccaa ctacaacccc 180 tccctcaaga gtcgagtcgc tatatcagtc gacacgtcca agaaccaatt ctccctgaaa 240 ctgagctctg tgaccgccgc agacacggcc gtgtattact gtgcgagatt ctcggattac 300 tatgttcggg attactatgg ttcgggaacc cagcgatacg gtttggacgt ctggggccaa 360 gggaccacgg tcacagtctc ctca 384 <210> 5 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> HAV6KC <400> 5 Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp 1 5 10 15 Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu 20 25 30 Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45 Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Thr Trp Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 6 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> HAV9KC <400> 6 Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp 1 5 10 15 Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu 20 25 30 Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Leu Ile Phe 35 40 45 Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Leu Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Tyr Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 7 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> HAV6HC <400> 7 Val Gln Leu Leu Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 1 5 10 15 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asn Tyr Gly 20 25 30 Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala 35 40 45 Val Ile Tyr Gly Gly Gly Gly Arg Thr His Tyr Ala Asp Ser Val Gln 50 55 60 Gly Arg Phe Ile Ile Ser Arg Asp Asp Ser Thr Asn Thr Met Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Asp Arg Met Thr Ser Lys Val Trp Asp Tyr Gly Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser 115 120 <210> 8 <211> 130 <212> PRT <213> Artificial Sequence <220> <223> HAV9HC <400> 8 Val Gln Leu Gln Glu Trp Gly Pro Gly Leu Val Arg Pro Ser Glu Thr 1 5 10 15 Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Pro Asp Tyr 20 25 30 Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Glu Leu Glu Trp Ile Gly 35 40 45 Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser 50 55 60 Arg Val Ala Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys 65 70 75 80 Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg 85 90 95 Phe Ser Asp Tyr Tyr Val Arg Asp Tyr Tyr Gly Ser Gly Thr Gln Arg 100 105 110 Tyr Gly Leu Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 Ala Ser 130 <210> 9 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> CK1d primer <400> 9 ccgtctagaa ttaacactct cccctgttga agctctttgt gacgggcgaa ctcag 55 <210> 10 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> KCfor primer <400> 10 ggtgttgaag gagacatcca gatgacccag tctcc 35 <210> 11 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> KCBSiWIback primer <400> 11 gccaccgtac gttt 14 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> LHS42 <400> 12 aaagcttcgg cacgagca 18 <210> 13 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> KCleaderback primer <400> 13 tccttcaaca ccag 14 <210> 14 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> CG1d primer <400> 14 tgtactagtt ttgtcacaag atttgg 26 <210> 15 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> HCfor6 primer <400> 15 acaggtgtcc actccgaggt gcagctgctc gagactgggg gagg 44 <210> 16 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> HCfor9 primer <400> 16 acaggtgtcc actcccaggt gcagctgcag gagtgggggc cagg 44 <210> 17 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> LHS11 primer <400> 17 cggttcgggg aagt 14 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> LHS39 <400> 18 gaattcactc taaccatgga a 21 <210> 19 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> HC leaderback primer <400> 19 gtggacacct atag 14 <210> 20 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> N-terminal amino acid sequence for HAV6 heavy chain <400> 20 Glu Val Gln Leu Leu Glu Thr Gly Gly Gly Leu 1 5 10 <210> 21 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> N-terminal amino acid sequence for HAV6 or HAV9 light chain <400> 21 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu 1 5 10 <210> 22 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> N-terminal amino acid sequence for HAV9 heavy chain <400> 22 Gln Val Gln Leu Gln Glu Trp Gly Pro Gly Leu 1 5 10<110> Korea Research Institute of Bioscience and Biotechnology <120> Neutralizing human monoclonal antibody specific to hepatitis A virus <130> 2p-03-32B <160> 22 <170> KopatentIn 1.71 <210> 1 <211> 318 <212> DNA <213> Artificial Sequence <220> <223> HAV6KC <400> 1 atccagatga cccagtctcc atcctccctg tctgcatctg taggagacag agtcaccatc 60 acttgccggg caagtcagag cattagcagc tatttaaatt ggtatcagca gaaaccaggg 120 aaagccccta agctcctgat ctatgctgca tccagtttgc aaagtggggt cccatcaagg 180 ttcagtggca gtggatctgg gacagatttc actctcacca tcagcagtct gcaacctgaa 240 gattttgcaa cttactactg tcaacagagt tacagtacca cgtggacgtt cggccaaggg 300 accaaggtgg aaatcaaa 318 <210> 2 <211> 318 <212> DNA <213> Artificial Sequence <220> <223> HAV9KC <400> 2 atccagatga cccagtctcc atcctcccta tctgcatctg taggagacag agtcaccatc 60 acttgccggg caagtcagag cattagcagc tatttaaatt ggtatcagca gaaaccaggg 120 caagccccta agctcttgat ctttgctgca tccagtttgc aaagtggggt cccatcaagg 180 ttcagtggca gtggatctgg gacagatttc actctcaccc tcagcagtct gcaacctgaa 240 gattttgcaa cttactactg tcaacagagt tacagtaccc cgtacacttt tggccagggg 300 accaaggtgg agatcaaa 318 <210> 3 <211> 366 <212> DNA <213> Artificial Sequence <220> <223> HAV6HC <400> 3 gtgcagctgc tcgagactgg gggaggcttg gtgcagcctg gggggtccct gagactctcc 60 tgtgcagcct ctggattcac ctttagaaac tatggcatga actgggtccg ccaggctcca 120 gggaaggggc tggagtgggt cgcagttatt tatggcggtg gtggtcgaac acactatgca 180 gactccgtgc aggggcggtt catcatctcc agagatgatt ccacgaacac gatgtatctg 240 caaatgaaca gcctgagagc cgaggacacg gccgtatatt actgtgcgaa agacagaatg 300 acttcaaaag tgtgggacta cggtatggac gtctggggcc aagggaccac ggtcaccgtc 360 tcctca 366 <210> 4 <211> 384 <212> DNA <213> Artificial Sequence <220> <223> HAV9HC <400> 4 gtgcagctgc aggagtgggg cccaggactg gtgaggcctt cggagaccct gtccctcacc 60 tgcactgtct ctggtggctc catcagtcct gactactgga gctggatccg tcagccccca 120 gggaaggaac tggagtggat tgggtatatc tattacagtg ggagcaccaa ctacaacccc 180 tccctcaaga gtcgagtcgc tatatcagtc gacacgtcca agaaccaatt ctccctgaaa 240 ctgagctctg tgaccgccgc agacacggcc gtgtattact gtgcgagatt ctcggattac 300 tatgttcggg attactatgg ttcgggaacc cagcgatacg gtttggacgt ctggggccaa 360 gggaccacgg tcacagtctc ctca 384 <210> 5 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> HAV6KC <400> 5 Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp 1 5 10 15 Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu 20 25 30 Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45 Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Thr Trp Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 6 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> HAV9KC <400> 6 Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp 1 5 10 15 Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu 20 25 30 Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Leu Ile Phe 35 40 45 Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Leu Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Tyr Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 7 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> HAV6HC <400> 7 Val Gln Leu Leu Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 1 5 10 15 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asn Tyr Gly 20 25 30 Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala 35 40 45 Val Ile Tyr Gly Gly Gly Gly Arg Thr His Tyr Ala Asp Ser Val Gln 50 55 60 Gly Arg Phe Ile Ile Ser Arg Asp Asp Ser Thr Asn Thr Met Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Asp Arg Met Thr Ser Lys Val Trp Asp Tyr Gly Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr Val Val Thr Ser Ser Ala Ser 115 120 <210> 8 <211> 130 <212> PRT <213> Artificial Sequence <220> <223> HAV9HC <400> 8 Val Gln Leu Gln Glu Trp Gly Pro Gly Leu Val Arg Pro Ser Glu Thr 1 5 10 15 Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Pro Asp Tyr 20 25 30 Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Glu Leu Glu Trp Ile Gly 35 40 45 Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser 50 55 60 Arg Val Ala Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys 65 70 75 80 Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg 85 90 95 Phe Ser Asp Tyr Tyr Val Arg Asp Tyr Tyr Gly Ser Gly Thr Gln Arg 100 105 110 Tyr Gly Leu Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 Ala ser 130 <210> 9 <211> 55 <212> DNA <213> Artificial Sequence <220> 223 CK1d primer <400> 9 ccgtctagaa ttaacactct cccctgttga agctctttgt gacgggcgaa ctcag 55 <210> 10 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> KC for primer <400> 10 ggtgttgaag gagacatcca gatgacccag tctcc 35 <210> 11 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> KCBSiWIback primer <400> 11 gccaccgtac gttt 14 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> LHS42 <400> 12 aaagcttcgg cacgagca 18 <210> 13 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> KCleaderback primer <400> 13 tccttcaaca ccag 14 <210> 14 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> CG1d primer <400> 14 tgtactagtt ttgtcacaag atttgg 26 <210> 15 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> HCfor6 primer <400> 15 acaggtgtcc actccgaggt gcagctgctc gagactgggg gagg 44 <210> 16 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> HCfor9 primer <400> 16 acaggtgtcc actcccaggt gcagctgcag gagtgggggc cagg 44 <210> 17 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> LHS11 primer <400> 17 cggttcgggg aagt 14 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> LHS39 <400> 18 gaattcactc taaccatgga a 21 <210> 19 <211> 14 <212> DNA <213> Artificial Sequence <220> HC leaderback primer <400> 19 gtggacacct atag 14 <210> 20 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> N-terminal amino acid sequence for HAV6 heavy chain <400> 20 Glu Val Gln Leu Leu Glu Thr Gly Gly Gly Leu 1 5 10 <210> 21 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> N-terminal amino acid sequence for HAV6 or HAV9 light chain <400> 21 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu 1 5 10 <210> 22 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> N-terminal amino acid sequence for HAV9 heavy chain <400> 22 Gln Val Gln Leu Gln Glu Trp Gly Pro Gly Leu 1 5 10
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| Application Number | Priority Date | Filing Date | Title |
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| KR10-2003-0004915A KR100476343B1 (en) | 2003-01-24 | 2003-01-24 | Neutralizing human monoclonal antibody specific to hepatitis A virus |
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| KR10-2003-0004915A KR100476343B1 (en) | 2003-01-24 | 2003-01-24 | Neutralizing human monoclonal antibody specific to hepatitis A virus |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016089181A1 (en) * | 2014-12-05 | 2016-06-09 | (주)셀트리온 | Adjuvant composition containing at least one influenza virus neutralizing and binding molecule and vaccine composition containing same |
| US10610587B2 (en) | 2014-12-05 | 2020-04-07 | Celltrion Inc. | Adjuvant composition containing at least one influenza virus neutralizing and binding molecule and vaccine composition containing same |
| CN113717284A (en) * | 2021-09-07 | 2021-11-30 | 江苏华创医药研发平台管理有限公司 | Hepatitis A virus specific nano antibody and application thereof |
| CN116514965A (en) * | 2023-06-15 | 2023-08-01 | 上海精翰生物科技有限公司 | Hepatitis A virus antibody and application thereof |
-
2003
- 2003-01-24 KR KR10-2003-0004915A patent/KR100476343B1/en not_active IP Right Cessation
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016089181A1 (en) * | 2014-12-05 | 2016-06-09 | (주)셀트리온 | Adjuvant composition containing at least one influenza virus neutralizing and binding molecule and vaccine composition containing same |
| US10610587B2 (en) | 2014-12-05 | 2020-04-07 | Celltrion Inc. | Adjuvant composition containing at least one influenza virus neutralizing and binding molecule and vaccine composition containing same |
| CN113717284A (en) * | 2021-09-07 | 2021-11-30 | 江苏华创医药研发平台管理有限公司 | Hepatitis A virus specific nano antibody and application thereof |
| CN116514965A (en) * | 2023-06-15 | 2023-08-01 | 上海精翰生物科技有限公司 | Hepatitis A virus antibody and application thereof |
| CN116514965B (en) * | 2023-06-15 | 2023-11-10 | 上海精翰生物科技有限公司 | Hepatitis A virus antibody and application thereof |
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| Publication number | Publication date |
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| KR100476343B1 (en) | 2005-03-16 |
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