CN1163512C - Humanized neutralizing generatically engineering Fab antibody of hepatitis A virus - Google Patents

Abstract

The present invention relates to a Fab antibody of humanized anti-hepatitis a virus neutralizing genetic engineering, which comprises an antibody gene of a Fab fragment, gene products and application thereof. The present invention is characterized in that the recombination antibody is a functional neutralizing antibody of a specific binding hepatitis a virus, wherein the functional neutralizing antibody is determined by a hypervariable region (CDRs) specificity gene order existing in a variable region of a light chain and a heavy chain of the antibody and obtains effective expression in a prokaryotic cell. The probable application of the Fab antibody lies in that utilizing the obtained neutrality Fab antibody gene or IgG full antibody gene can produce the antibody in a prokaryotic cell, a yeast cell, an insect cell and a eukaryotic cell and any expression system. The Fab antibody can be used for clinically preventing or treating hepatitis a.

Description

Human source anti-hepatitis A virus neutrality engineered Fab antibody

Preparation and the application, the especially specificity that the present invention relates to prevent and treat personnel selection source gene engineering monoclonal antibody are at the proteic neutrality gene engineering monoclonal antibody of hepatitis A virus (HAV) VP1.

Since B lymphocyte hybridoma cell-fusion techniques in 1975 comes out, monoclonal antibody is used for fundamental research as a class, laboratory diagnosis, the novel product of clinical treatment and prevention, its applying value and DEVELOPMENT PROSPECT have obtained general affirming. and the researchdevelopment of molecular biology and molecular immunology has caused the generation and the development of genetic engineering antibody.Reorganization by the antibody molecule gene level can obtain diversified specific murine source and human antibody, and making has had breakthrough and more and more demonstrated its significance and practice prospect studies on Monoclonal Antibody.The phage antibody gene pool technology rise of rising the beginning of the nineties at the end of the eighties and the development in whole genetic engineering antibody technical study field make the development research of source, people from world today or genetic engineering antibody obtain remarkable progress and step into substantive applied research and development phase by the fundamental research stage.At present in the biological products that drugs approved by FDA is gone on the market or awaited the reply, various forms of monoclonal antibodies and genetic engineering antibody account for certain proportion, in 67 kinds of biological products with the approval listing, treatment and prevention have 9 kinds with monoclonal antibody and genetic engineering antibody at present.The medium antibody that awaiting the reply has 35 kinds.Wherein in four kinds of humanized genetic engineering antibodies of approved listing and generation tremendous economic and social benefit, a kind of antiviral gene engineered antibody that is is arranged wherein, its commodity are called " Synagis ^TM"; be humanization preventing respiratory combination of syndromes poison (RSV) genetic engineering antibody; and that the anti-RSV virus gene engineering antibody in people source that derives from phage antibody library screening has gone through to enter the II phase is clinical, another kind of antiviral antibody anti-HBs antibody is also among examining.

Up to now, most of virus diseases do not have the specific treatment medicine, the biological products that are used for clinical treatment and some virus disease of prevention are still based on blood product, as the haematogenous gamma-globulin that uses clinically for many years, be used for viral hepatitis that hepatitis A or hepatitis B virus cause and measles etc., not only non-specific foreign protein is many in these blood products, specific antibody content is very low, and maximum problem is the cause of disease pollution problem that blood source goods potential fails to detect, and considers and should abandon from long-term interest.Therefore become a general orientation of domestic and international research with human source gene engineering product Blood substitute goods, and progressively led to success.Still the precedent of not having any approval with pure mouse resource monoclonal antibody prevention and treatment virus disease in the world, domestic have a clinical treatment of filing an application to be used for anti-encephalitis and hemorrhagic fever, mouse source antibody human is a heterologous protein with maximum disadvantage, causes easily that in human body inherited immunity repels and antibody was lost efficacy and cause immunological disease.Therefore, specificity antivirus people source neutrality antibody is one of the most promising biological products of virus disease prevention and treatment.The research of people's source antivirus genetic engineering antibody is except that successful anti-rsv antibodies, present system by phage surface, gene engineering antibody library technology and molecular biology method unite utilization, made substantial progress people's source antivirus antibody of having succeeded in developing at present of the research in this field has the antibody of anti-following virus: respiratory syncytial virus, HIV (human immunodeficiency virus) (HIV), hepatitis B virus, hepatitis C virus, hsv (HSV), Hantaan virus, B19 virus, CMV virus, the anti-hepatitis A virus that VZV etc. and this institute that does not deliver have as yet succeeded in developing, rabies virus antibodies etc.Anti-hepatitis A antibody preparation is succeeded in developing, and will be domestic and international initiative, might obtain a kind new medicine certificate.

The objective of the invention is by genetic engineering means and phage display technique in conjunction with utilization, directly from the human immunoglobulin gene storehouse, filter out the gene engineering monoclonal antibody of the anti-hepatitis A virus (HAV) of neutrality, obtain its antibody gene, the specific antibody medicine that provides feasibility in the future possible clinical antiviral prevention and treatment.

The human source anti-hepatitis A virus neutrality engineered Fab antibody of the present invention's statement comprises:

(1) is the reorganization IgG Fab antibody that in prokaryotic cell prokaryocyte, obtains to stablize effective expression in a kind of people source, forms called after HAFab16 by heavy chain Fd and light chain Lambda chain.

(2) its antibody protein function is by complementary region (the Complementarity-Dertemining Regions of decision family that is present in antibody gene light chain and variable region of heavy chain, what CDRs) the specificity nucleotide sequence determined among CDR1, CDR2 and the CDR3, its amino acid sequence corresponding has constituted the specific antigens calmodulin binding domain CaM of antibody.

(3) specific light chain and heavy chain gene derive from the rich long-pending screening expression of the specificity of human source anti-hepatitis A antiviral antibody gene pool, and corresponding three the CDR region sequences of light chain and heavy chain are the distinctive brand-new sequence of this antibody.

(4) specific recognition hepatitis A virus (HAV) VP1 albumen, and have the neutralization activity that anti-hepatitis A virus (HAV) infects.

(5) utilize the neutrality Fab antibody gene of above-mentioned acquisition, can in prokaryotic cell prokaryocyte, yeast cell, eukaryotic cell and recombinant virus system, express other genes that contain this antibody gene after this antibody gene or the reconstruction based on this, obtain hepatitis A virus (HAV) infected and have the active antibody product of neutralization.

Traditional hybridoma cell technology of utilizing obtains quite difficulty of human monoclonal antibody, and the clone of setting up is unstable usually, and gene is easily lost.The human source anti-hepatitis A virus neutrality engineered Fab antibody of the present invention's statement, it is the expression that on the basis that obtains antibody gene, obtains gene product, this antibody gene can be along with plasmid DNA duplicating and stable duplicating in bacterium, and can reconstruct antibody gene arbitrarily, thereby obtain a kind of clinical treatment antibody preparation of feasibility according to different needs.

Following preferential embodiment elaborates to the present invention, but does not mean that restriction content of the present invention

In these embodiments, be explanation the present invention, the employing phage expression vector be pComb3 (Barbas C.III etal, Proc.Natl.Acas.Sci USA 1991,89:10164-10168).Used main bacterial strain is commercial prod XLI-Blu (U.S. Strategene company).Used phage is commercial prod VCSM13 (an American I nvitrogene company).Hepatitis A virus (HAV) be this institute hepatitis chamber from the isolating imperial first strain of China patient, existing preserve by the hepatitis chamber, can openly provide to the research institution or the unit of correlative technology field.

Embodiment 1-4 is the screening preparation method of human source anti-hepatitis A virus neutrality engineered Fab antibody HAFab16; Embodiment 5 is the gene expression characteristics of human source anti-hepatitis A virus neutrality engineered Fab antibody HAFab16; Example 6-9 is albumen and the functional character of human source anti-hepatitis A virus neutrality engineered Fab antibody HAFab16.

Example 1: the pcr amplification of humanized IgG Fab fragment gene: with isolated lymphocytes in the lymphocyte separation medium hepatitis A patient decubation anticoagulation, with Tril-Zon (U.S. Gibco, BRL) extract total cell RNA, with the Olig-dT primer RNA that extracts is passed through reversed transcriptive enzyme (U.S. Gibco, BRL) reverse transcription becomes cDNA, with a group-specific IgGFabGamma chain, Kampa chain and Lamda strand primer, people's endogenous light chain and heavy chain Fab gene are carried out pcr amplification.The PCR condition is: 94 ℃ 1 minute, 54 ℃ 1 minute, 72 ℃ 2 minutes, 35 circulations (PE480), above-mentioned PCR product reclaims through sepharose respectively, (U.S. Gibco BRL) behind the purifying, obtains Kamba, Lamda and Fd chain PCR product about 650-700bp through DNA purification column Spin-X.

The foundation of example 2 phage antibody gene pools: different primer synthetic Kamba and Lamda chain PCR product are mixed, different primer synthetic Fd chains are mixed, be cloned into phage vector pComb3 with SacI/XbaI and XhoI/SpeI respectively, to be cloned into light, the pComb3 carrier DNA of heavy chain gene connects product behind ethanol sedimentation, hang with the 10ul aquae destillata, the 200ul electricity that it is good that prepared beforehand is gone in the electricity transduction changes bacterium XLI-Blu, (electric commentaries on classics condition is: the Bio-Red electroporation, 0.2cm electric revolving cup, 2.5K volt), add the 10mlSOC nutrient solution after electricity changes, 37 ℃ 1 hour, adding 10ml has the SB nutrient solution (3) of penbritin and tsiklomitsin, 37 ℃ 1 hour, add the aforementioned SB of 80-100ml, 37 ℃ added helper phage VCSM13 1 * 10 after 2 hours ¹²PFU/ml adds kantlex (70ug/ml) after 1 hour, 37 ℃ of shaking table overnight incubation.With 4%PEG8000 and 3%NaCl precipitation phage supernatant, through 9000rpm, 20 minutes, 4 ℃ centrifugal after, with the resuspended precipitation of 2ml 0.02M PBS PH7.4, set up phage antibody library, packing is stored in-20 ℃ of refrigerator-freezers.

Example 3: be used for the antigenic preparation of hepatitis A virus (HAV) of antibody library enrichment screening: hepatitis A virus (HAV) is results after cultivating 21 days on the Frhk4 cell, basic document (the Hughes that presses, J.V., L.W.Stanton, J.E.Tomassini, et alNeutralizing monoclonal antibodies to hepatitis A virus:partial localizationof a neutralizing antigenic site.J.Virol, 52:465-473.1984) method is by sucrose bed course ultracentrifugation purifying hepatitis A virus (HAV) antigen, the hav antigen behind the purifying can be directly used in bag by elisa plate.

Example 4: enrichment screening: the hepatitis A virus (HAV) bag that adopts purifying has been carried out 4 to the anti-hepatitis A virus (HAV) antibody library of the people of above-mentioned foundation and has taken turns the enrichment screening by elisa plate.We take turns after the screening at random the clone of picking and have carried out restriction enzyme and cut identification and analysis every.After cutting with the Xbal/Xhol enzyme, the clone that band phage gene III and people's monoclonal antibody section weight chain gene insert should cut out the band of about 2.2Kb and the carrier band of 3.0Kb.With the increase of screening wheel number, have the also increase gradually of clone that double-stranded gene inserts, taking turns to last one has 90% clone to show the insertion of heavy chain and light chain gene approximately.

The nucleic acid sequence analysis of the variable region gene of example 5 human IgG Fab antibody HAFab16: carry out nucleic acid sequence analysis with QiagenMiniprep Kit (U.S. Qiagen) preparation plasmid DNA.Order-checking is automatic sequencing.At least 3 clones are used to determine same identical sequence.The sequence that obtains all uses DNA Strider (MS) sequence analysis software to carry out analyzing and processing, and compares the IgG sequence in the gene pool on the Internet network.Confirmer's source anti-hepatitis A virus neutrality engineered Fab antibody HAFab16 gene is made up of human IgG γ chain Fd and λ chain, its gene expression characteristics is made of the specificity nucleotide sequence and the amino acid in 6 CDR districts in VH and the VL structural domain, be VH-CDR1, VH-CDR2, VH-CDR3 and VL-CDR1, VL-CDR2 and VL-CDR3.Sequence data such as accompanying drawing 5 are the nucleotide sequence and the aminoacid sequence of the variable region gene of antibody HAFab16.

Example 6 will extract plasmid DNA after will having the positive colony amplification that HAFa16 antibody weight chain gene inserts according to a conventional method, with the gIII in SpeI and the NpeI excision carrier, become the FdgIII fusion rotein and be independent expressed proteins, connect the back and transform the XL1-Blu bacterium, the single bacterium colony of picking from the ammonia benzyl plate of overnight growth, inoculation SB or TB inoculum are when bacterium grows to OD=0.2-0.3, add 1mM IPTG, at 30 ℃ of abduction delivering 10-12 hours.Results bacterium, centrifugal back add 10 times of PBS (0.02M PH7.4) that concentrate amount of original fluid hangs, multigelation 3 times, and its supernatant is the Fab antibody of expression behind the high speed centrifugation.

Example 7: isolated lymphocytes from hepatitis A patient decubation blood, with the round pcr humanized IgG Fab antibody gene that increased, set up the phage antibody gene pool, present technology screening to the anti-hepatitis A virus (HAV) Fab antibody genes of 12 strains with phage surface, as Fig. 1, be that human source anti-hepatitis A virus gene engineering Fab antibody combines with purifying hepatitis A virus (HAV) antigen ELISA and combines with the competition of the mouse-anti hepatitis A virus (HAV) Mabs of HRP mark.Show simultaneously that from Fig. 1 the anti-hepatitis A virus (HAV) Fab antibody of 12 strains that filter out comprises that HAFab16 all can compete the neutrality mouse-anti hepatitis A monoclonal antibody Mab37 of HRP mark well.And this strain Mab has been determined and the proteic neutralizing antibody K3-4C8 of mouse-anti hepatitis A VP1 and the K2-4F2 perfect competition of generally acknowledging in the world.

Example 8: selecting two strain Fab monoclonal antibodies 9 and Fab16 further studies from the above-mentioned Fab antibody that obtains, as Fig. 2, is Fab antibody raw product and the unlabelled international standard strain monoclonal antibody K3-4C8 and the K2-4F2 competitive ELISA of escherichia coli expression.Fab16 energy and the combination of the anti-VP1 albumen of above-mentioned two strains monoclonal antibody.Illustrate that it may be at hepatitis A virus (HAV) VP1 albumen.Fig. 3 shows the good competition of monoclonal antibody 9 and Fab16 and hepatitis A human serum.Show that this two strain antibody is at the main paratope of hepatitis A virus (HAV).

Example 9: cell neutralization test: whether have extracorporeal neutralizing activity for understanding the anti-hepatitis A virus (HAV) Fab antibody of people that is obtained, bacterium cracking supernatant after the above-mentioned 8 strain positive colonies expression has been carried out external neutralization experiment, the result show all HAFab16 antibody according to have external in and the activity of hepatitis A virus (HAV) (be ELISA detects hepatitis A virus (HAV) negative), negative control then can not in and hepatitis A virus (HAV) (it is positive promptly to detect hepatitis A virus (HAV)), as Fig. 4, be the neutralization activity of the anti-HAV Fab antibody of reorganization to hepatitis A virus (HAV).

Claims (5)

1, human source anti-hepatitis A virus neutrality genetic engineering antibody is characterized in that:

(1) is a kind of human source anti-hepatitis A viral glycoprotein VP1 neutrality gene engineering monoclonal antibody Fab antibody that in prokaryotic cell prokaryocyte, obtains to stablize the gene recombination of effective expression, forms by Fd and Lambda chain;

(2) its antibody protein function is by specificity nucleotide sequence decision among the complementary region CDR1 of decision family, the CDR2 that are present in antibody gene light chain and variable region of heavy chain and the CDR3, and its amino acid sequence corresponding has constituted the specific antigens calmodulin binding domain CaM of antibody;

(3) specific light chain and heavy chain gene derive from the rich long-pending screening expression of the specificity of human source anti-hepatitis A antiviral antibody gene pool, its corresponding three CDR region sequences are the distinctive brand-new sequence of this antibody: its heavy chain CDR1 region amino acid sequence is DYGLS, the CDR2 district is YINRNGYRTGYADSVKG, and the CDR3 district is RYNYGVQYYFDY; Its light chain CDR1 region amino acid sequence is SGTSSNIGNNYVS, and the CDR2 district is DNNKRPS, and the CDR3 district is CGTWDSSLSGVV;

(4) specific recognition hepatitis A virus (HAV) VP1 albumen, and have the neutralization activity that anti-hepatitis A virus (HAV) infects.

2, according to claim 1 human source anti-hepatitis A virus neutrality engineered Fab antibody, it is characterized in that: the gene of the described specificity Fab antibody protein of encoding is for expressing the human source anti-hepatitis A virus neutrality antibody gene of proteic neutralizing antibody of anti-hepatitis A virus (HAV) VP1 or polypeptide product.

3, according to claim 1 human source anti-hepatitis A virus neutrality engineered Fab antibody, it is characterized in that: described antibody also combines with it with antigen in having on the identification hepatitis A virus (HAV) VP1 albumen, thus the function that the blocking-up hepatitis A virus (HAV) infects.

4, be used for preventing or treating the purposes of the medicine of the hepatitis A that causes by hepatitis A virus (HAV) in preparation as the arbitrary described human source anti-hepatitis A virus neutrality engineered Fab antibody of claim 1-3.

5, coding is as the purposes of the gene of the arbitrary described human source anti-hepatitis A virus neutrality engineered Fab antibody of claim 1-3, it is characterized in that: can in prokaryotic cell prokaryocyte, yeast cell, eukaryotic cell and recombinant virus system, express this antibody gene, obtain in having and the antibody product of hepatitis A virus (HAV) infection.

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