CN106928348A

CN106928348A - A kind of full human monoclonal antibody of regulation and control human body inherent immunity and its preparation method and application

Info

Publication number: CN106928348A
Application number: CN201710128502.6A
Authority: CN
Inventors: 张凤民; 翟爱霞; 宋武琦; 考文萍; 吴静; 郭彩玲; 卜桐
Original assignee: Harbin Medical University
Current assignee: Harbin Engineering University; Harbin Medical University
Priority date: 2017-03-06
Filing date: 2017-03-06
Publication date: 2017-07-07
Anticipated expiration: 2037-03-06
Also published as: CN106928348B

Abstract

The invention discloses a kind of full human monoclonal antibody of regulation and control human body inherent immunity and its preparation method and application.The invention also discloses the complete genome and amino acid sequence of encoding said antibody.The present invention filters out the monoclonal antibody of regulation and control human body inherent immunity from human antibody hybridoma storehouse, and cells and supernatant is collected by large-scale culture, is purified by affinity column and obtained.The antibody cDNA sequence is obtained by antagonist gene sequencing, and is cloned into carrier for expression of eukaryon, by human body cell expression system large-scale culture, the antibody is obtained by affinity column purifying.Research shows, the full human monoclonal antibody that the present invention is obtained can regulate and control human body inherent immunity, activation I type IFN signal systems, and virus multiplication can be suppressed, therefore full human monoclonal antibody of the invention can be used for the prevention and treatment of infectious diseases, tumour and disease of immune system.

Description

It is a kind of regulate and control human body inherent immunity full human monoclonal antibody and preparation method thereof and Using

Technical field

The present invention relates to a kind of full human monoclonal antibody and its preparation method and application, more particularly to a kind of regulation and control human body Full human monoclonal antibody of inherent immunity and preparation method thereof and the purposes in human body inherent immunity is regulated and controled, the invention belongs to Pharmaceutical technology field.

Background technology

Monoclonal antibody (monoclonal antibody) is after most important class biology skill after vaccine, recombinant protein Art product, it is high with target site affinity with clear and definite action site, it has been successfully applied to treatment tumour, LADA disease Disease, infectious diseases and graft-rejection etc..Monoclonal antibody technology experiences the development of 40 years, experiences mouse (murine), is fitted together to Body (chimeric), humanization (humanize) and full people source (full human) four-stage (Fig. 1).Mouse monoclonal antibody is by mouse Hybridoma is secreted, and immunogenicity is very strong, can induce and produces HAMA, and toxic and side effect is obvious；Chimera monoclonal antibody is The Fab section of mouse monoclonal antibody is retained, Fc sections is humanized；Humanized antibody refers to constant region (the i.e. CH and CL of antibody Area) as coded by human antibody gene；Full Human monoclonal antibody refers to all parts by coded by human antibody gene.Quan Renyuan is mono- Clonal antibody adverse reaction is small, long half time, good effect, is the direction of future studies development.Full human monoclonal antibody targeting Property it is strong, specificity is high, toxic and side effect is low, has obtained pushing in the diagnosis of research, disease in disease mechanisms and therapy field Extensively.

Full human monoclonal antibody Tanibirumab carries out intravitreal injection, with anti-vascular endothelial growth factor The effect of (vascular endothelial growth factor receptor) 2, can partly suppress rat model laser Induction CNV.P-cadherin (P-cadherin) mediate tumor cell sticks, breeds and attacks, full people The anti-P-cadherin antibody PF-03732010 in source is by disturbing P-cadherin signal pathways to suppress the growth of primary tumo(u)r and turn Move.

Inherent immunity is the first line of defence that body resists pathogenic microorganism, the I type interferon induced after virus infection (interferon, IFN) is the important mediated factor of human body antiviral natural immune response.Virus, bacterium or other IFN are induced After agent with cell membrane by acting on, promote the IFN genes disinhibition of I types and be activated, start to transcribe to form IFN-α and IFN-β MRNA, and the precursor of I types IFN is further translated on cytoribosome, I type IFN precursors are transported on cell membrane, precursor The final I types IFN for being cut into maturation.The classical path of I type IFN signal transductions --- JAK/STAT (janus kinase/ Signal transducers and activators of transcription) path：IFN-α and IFN-β are corresponding to its Coupled receptors after, cause the phosphorylation of I type IFN receptor tyrosine residues, promote the intracellular JAK tyrosine coupled with acceptor Kinases is activated, and the activation of JAK further promotes STAT phosphorylations, forms homologous or heterodimer.The dimerization of activation Body is combined composition tripolymer with associated proteins, is further diverted into nucleus, causes a series of antiviral proteins The expression of (antiviral protein, AVP) gene, so as to play the various BAs of I types IFN.

Full human monoclonal antibody is the Main way of therapeutic antibodies development at present, and the present invention passes through people source hybridoma skill Art obtains human antibody clone bank, and for the preparation screening of antibody provides good technology platform, the antibody for screening is Spontaneous antibody in human body, it is to avoid the artificial processing factors that humanizing process introduces, more meet in human body natural gives birth to Reason process, is with a wide range of applications.

The content of the invention

It is an object of the invention to provide it is a kind of can be used to regulate and control human body inherent immunity, activation I type IFN signal systems it is complete Human monoclonal antibody and preparation method thereof.

In order to achieve the above object, present invention employs following technological means：

(1) screening obtains one plant I types IFN can be promoted to express elevated people's hybridoma from human antibody storehouse Strain.

(2) expand, purify the full human monoclonal antibody, evaluate security of the antibody to normal liver cell.

(3) biological function identification is carried out with tumour cell effect to the monoclonal antibody for obtaining and is compared, as a result shown The full human monoclonal antibody can promote I type IFN mRNA to express to be increased, and suppresses hepatitis type B virus (hepatitis B Virus, HBV) HBeAg expression, with HepG2.215 cells (derive from HepG2 cells, the inside has transfected two sections of complete HBV Full genome) can combine.

(4) antibody gene sequencing is carried out to the hybridoma for producing the antibody；

(5) monoclonal antibody heavy sequence and sequence of light chain described in (4) are cloned into carrier for expression of eukaryon, transfecting host Cell, the monoclonal antibody of acquisition carries out affinity again and other biological function compares.

On the basis of the studies above, present invention screening obtains one plant of full people source Dan Ke that can regulate and control human body inherent immunity Grand antibody, is made up of light chain and heavy chain, and the amino acid sequence of the light chain variable district is the heavy chain shown in SEQ ID NO.6 The amino acid sequence of variable region is shown in SEQ ID NO.8.

In the present invention, it is preferred to, the amino acid sequence such as SEQ ID NO of the light chain:Shown in 3, the ammonia of the heavy chain Base acid sequence such as SEQ ID NO:Shown in 4.

Those skilled in the art will know that the conservative amino acid in antibody is replaced can't substantially be influenceed to resist The affinity and structure of body, the especially conservative are replaced to be occurred in constant region.Therefore, if the change of amino acid occurs The BA of antibody is had no effect on after constant region and change, then the antibody also should be within protection scope of the present invention.

Further, the invention allows for the single heavy chain comprising the full human monoclonal antibody and/or single light The single domain antibody of chain, chimeric antibody, antibody fusion protein antibody, antibody/antibody fragment-factor fusion protein or antibody/antibody Fragment-chemical coupling thing.And

The antigen-binding fragment of described full human monoclonal antibody, wherein, the antigen-binding fragment may be selected from Fab, Fab'、F(ab')₂, scFv, Fv, dsFv, double antibody, Fd and Fd' fragments.

Further, the invention allows for a kind of DNA molecular of separation, it is the antibody or described described in coding The nucleotide sequence of antigen-binding fragment, it is preferred that described nucleotide sequence includes SEQ ID NO:5 and/or SEQ ID NO:Nucleotide sequence shown in 6, it is furthermore preferred that described nucleotide sequence includes SEQ ID NO:1 and/or SEQ ID NO:2 Shown nucleotide sequence.

Containing described nucleotide sequence or its combination recombinant dna expression vector and contain described nucleotide sequence Or the host cell of described recombinant dna expression vector is also within protection scope of the present invention, it is preferred that described host is thin Born of the same parents are selected from prokaryotic, yeast or mammalian cell, it is furthermore preferred that the host cell is thin selected from HEK293F cells, CHO Born of the same parents or NSO cells.

The HEK293F cells are human embryo kidney (HEK) 293F cells (human embryonic kidney 293F cell)；CHO Cell is Chinese hamster ovary cell (chinese hamster ovary cell)；NSO cells are mouse NSO thymomas (mouse thymoma NSO cells)。

Further, the invention allows for described antibody, described antigen-binding fragment, described nucleotides sequence Row, described carrier or described host cell are preparing regulation and control human body inherent immunity, activate the medicine of I type IFN signal systems In purposes, the medicine is for preventing and treating infectious diseases, tumour and disease of immune system.

Wherein, it is preferred that the infectious diseases includes but is not limited to inhibition of HIV infection, A type, B-mode and hepatitis C Viral infection, herpesvirus infection, influenza infection, Coxsackie virus infection, respiratory syncytial virus infection, human papilloma Viral infection, ebv infection, enterovirns type 71 infection；The tumour include but is not limited to glioma, Hodgkin lymphoma, Follicularis lymph cancer, melanoma, acute or chronic leukaemia, lung cancer, breast cancer, liver cancer, stomach cancer, cancer of pancreas, kidney, prostatitis Gland cancer, carcinoma of urinary bladder, oophoroma, cutaneum carcinoma, squamous cell carcinoma, osteocarcinoma, head and neck cancer, colorectal cancer, solid tumor；The immune system Disease includes being not limited to systemic loupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, myasthenia gravis, multiple sclerosis Disease, autoimmune hemolytic anemia, oneself immunity hepatitis, chorionitis, PAN, Wegener granulomatosis.

Further, the invention allows for the side of the full human monoclonal antibody for preparing regulation and control human body inherent immunity Method, comprises the following steps：

(1) expression vector is provided, the expression vector contains the DNA molecular of encoding antibody or antigen-binding fragment, and The expression regulation sequence being connected operable with the DNA molecular；

(2) host cell is converted with the expression vector；

(3) under conditions of the expression antibody is adapted to, host cell of the culture containing the expression vector；

(4) monoclonal antibody described in obtaining is isolated and purified；

Wherein, it is preferred that described expression vector is the carrier for expression of eukaryon containing antibody-secreting signal peptide sequence PcDNA3.4, described host cell is HEK293F cells.

The monoclonal antibody that the present invention is provided can be by regulating and controlling human body inherent immunity, and activation I type IFN signal systems come pre- Anti- and treatment disease, the monoclonal antibody that the present invention is provided is full people source, the Dan Ke with other animal derived (such as mouses) Grand antibody is compared, and because immunogenicity greatly reduces caused by species difference, and specific good, affinity is high, if for clinic Side effect will be substantially reduced.To be had a good application prospect in terms of prevention and treatment of diseases.

Brief description of the drawings

Fig. 1 is the four-stage of monoclonal antibody technique development；

Fig. 2 is full human monoclonal antibody to human hepatoma cell line HepG2's cell (human liver Hepatocellular cells) in I types IFN expression influence；

Each group compares with control group mock, * * P<0.01, * * * P<0.001；

Fig. 3 is the influence that full human monoclonal antibody is expressed HepG2.215 cell I types IFN；

Each group compares with control group mock, * * * P<0.001；

Fig. 4 is full human monoclonal antibody to Human normal hepatocyte LO2 cells (human normal liver cells) The influence of propagation；

Fig. 5 is the protein electrophoresis figure of full human monoclonal antibody identification；

A：SDS-PAGE electrophoresis；B：Western blot are tested；

Fig. 6 is pcDNA3.4 plasmid maps；

Fig. 7 is the influence that human monoclonal antibodies are expressed HBeAg；

3B5 groups compare with control group mock, * * * P<0.001；

Fig. 8 is the combination situation of human monoclonal antibodies and HepG2.215 cells.

Specific embodiment

The detailed implementation of the present invention is referring to embodiment, experimental technique and reagent described in embodiment, if without special Illustrate to be normal experiment method and reagent.Following examples are merely to illustrate and explain the present invention, rather than by any way The limitation present invention.

The screening of embodiment 1, the monoclonal antibody of regulation and control human body inherent immunity

Full human monoclonal antibody storehouse is set up using people source B cell clone technology.HepG2 cells are inoculated with 96 orifice plates, will be complete Antibody in human monoclonal antibody storehouse is separately added into the HepG2 cells of culture, and after effect 48h, TRIzol reagents are collected carefully Born of the same parents, extract RNA, carry out RT-PCR and obtain cDNA.Draw using Genrunner and with reference to foreign literature design Real-time PCR Thing, the SYBR kits produced using TaKaRa companies, Roche quantitative PCR apparatus are expanded.Analysis method：According to period Cp Value, using 2^-△△CpMethod analyzes mRNA expressions.What result was represented is the expression of experimental group genes of interest relative to control group Change multiple.

Computing formula is：△ △ Cp=(Cp target gene-Cp house-keeping genes)_{Experimental group}- (Cp target gene-Cp house keeper's bases Cause)_{Control group}

The expression of detection I type IFN mRNA, so as to analyze the influence that antibody is expressed HepG2 cell I types IFN.

Obtaining one plant by the final screening from human antibody storehouse of the method can promote I types IFN to express elevated people Hybridoma cell strain, is named as 3B5, and the full human monoclonal antibody can promote the generation (Fig. 2) of I type IFN mRNA. HepG2.215 cells obtain similar result, and the full human monoclonal antibody can promote the table of IFN-α and IFN-β mRNA Up to (Fig. 3).

The security of embodiment 2, the full human monoclonal antibody 3B5 of evaluation to LO2 cells

The influence that the full human monoclonal antibody 3B5 of MTT experiment research breeds to LO2 cells.Monoclonal antibody action is in LO2 After cell 24h, 48h, 72h, MTT solution (5mg/mL PBS are prepared, pH=7.4) 20 μ L are added per hole.Continue to be incubated 4h, terminate Culture, careful suction abandons culture supernatant in hole,.Add 150 μ L DMSO per hole, vibrate 10min, crystal is fully melted.Selection 490nm wavelength, determines each hole absorbance value on enzyme linked immunological monitor, records result, and with the time as abscissa, light absorption value is Ordinate draws cell growth curve.As shown in figure 4, Fig. 4 results show, the antibody is acted on the unrestraint of LO2 cells result, is said The bright full human monoclonal antibody concentration is on the propagation of normal liver cell without influence.

The monoclonal antibody of embodiment 3, purifying regulation and control human body inherent immunity

The hybridoma cell strain 3B5 of the regulation and control human body inherent immunity positive that embodiment 1 is obtained is enlarged culture, collects Serum-free cell culture supernatant, the expression of protein electrophoresis detection antibody, such as Fig. 5.Purified by protein A affinity columns, taken off Salt and concentration, vacuumize it is lyophilized after, obtain the monoclonal antibody of purifying.

Embodiment 4, antibody dna construction of recombinant plasmid and expression

The hybridoma 3B5 of the regulation and control human body inherent immunity positive that embodiment 1 is obtained carries out antibody full genome sequencing, Obtain the gene order of antibody light chain and heavy chain：The nucleotides sequence of the light chain (1G3-L1) is classified as SEQ ID NO.1, amino acid As shown in SEQ ID NO.3, the nucleotides sequence of the heavy chain (1G3-H1) is classified as SEQ ID NO.2 to sequence, and amino acid sequence is such as Shown in SEQ ID NO.4.

Wherein, light chain variable district nucleotide acid sequence is SEQ ID NO.5, and orresponding amino acid sequence is SEQ ID NO.6. Weight chain variable district nucleotide acid sequence is SEQ ID NO.7, and orresponding amino acid sequence is SEQ ID NO.8.

Above-mentioned light chain and heavy chain gene are cloned into the carrier for expression of eukaryon equipped with antibody-secreting signal peptide sequence respectively In pcDNA3.4 (Fig. 6).HEK293F cells are transiently transfected, intact antibody is carried out, the expression of protein electrophoresis detection antibody is such as schemed 5.Purified using protein A affinity columns and obtain antibody protein.

Regulating and controlling effect of the embodiment 5, human monoclonal antibodies to virus multiplication

Full human monoclonal antibody 3B5 acts on HepG2.215 cell 48h, and ELISA method detection antibody is to HBeAg's Influence, by antigen used with coating diluted to debita spissitudo, 5% calf serum puts 37 DEG C of closing 40min, washs hydroful Hole is washed 3 times, every all over 3min.Detected sample is added, 37 DEG C are placed in, 60min is washed 3 times, every time with washing hydroful hole 3min.Add enzyme labelled antibody, 37 DEG C of 1h.Substrate solution is added, 37 DEG C of avoid light place 3-5min are put, terminate liquid colour developing is added, used 490nm wavelength is detected.Result display human monoclonal antibodies substantially suppress the expression (Fig. 7) of HBeAg.

The intracellular Binding experiment of embodiment 6, the monoclonal antibody of regulation and control human body inherent immunity

With HepG2.215 cells as substrate, with normal human serum as negative control (NC), analyze full people's resource monoclonal and resist The fluorescence distribution of body 3B5.(cover glass is positioned over the culture of 6 holes to cultured cells on the cover glass pre-processed through 4% poly-D-lysine In plate) culture, rinsed with the 0.1M PBS of precooling, 4% paraformaldehyde fixes 30min, lowlenthal serum closing 30min (37 ℃)；Primary antibody is incubated, and 4 DEG C overnight, and negative control replaces primary antibody with 0.1M PBS；0.1M PBS are rinsed 3 times；It is anti-that FITC- is marked Human IgG, hatches 37 DEG C of 1h；PBS is rinsed 3 times, fluorescence microscopy Microscopic observation.Result is as shown in figure 8, illustrate that full people's resource monoclonal resists Body 3B5 and HepG2.215 cells can be combined.

For the ordinary skill in the art, specific embodiment is that the present invention is exemplarily described, The obvious present invention is implemented and is not subject to the restrictions described above, as long as take method of the present invention design entering with technical scheme Capable various unsubstantialities are improved, or it is not improved design of the invention and technical scheme are directly applied into other occasions, Within protection scope of the present invention.

Sequence table

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Claims

1. a kind of full human monoclonal antibody of regulation and control human body inherent immunity, is made up of, it is characterised in that described light chain and heavy chain The amino acid sequence of light chain variable district is that shown in SEQ ID NO.6, the amino acid sequence of the weight chain variable district is SEQ ID Shown in NO.8.

2. the full human monoclonal antibody of human body inherent immunity is regulated and controled as claimed in claim 1, it is characterised in that the light chain Amino acid sequence such as SEQ ID NO:Shown in 3, the amino acid sequence such as SEQ ID NO of the heavy chain:Shown in 4.

3. comprising the single domain antibody of the single heavy chain of full human monoclonal antibody and/or single light chain described in claim 1 or 2, Chimeric antibody, antibody fusion protein antibody, antibody/antibody fragment-factor fusion protein or antibody/antibody fragment-chemical coupling Thing.

4. the antigen-binding fragment of the full human monoclonal antibody described in claim 1 or 2, it is characterised in that the antigen binding Fragment is selected from Fab, Fab', F (ab')₂, scFv, Fv, dsFv, double antibody, Fd and Fd' fragments.

5. a kind of DNA molecular of separation, it is characterised in that the DNA molecular be encode it is anti-described in claim any one of 1-3 The nucleotide sequence of the antigen-binding fragment described in body or claim 4, it is preferred that described nucleotide sequence includes SEQ ID NO:5 and/or SEQ ID NO:Nucleotide sequence shown in 6, it is furthermore preferred that described nucleotide sequence includes SEQ ID NO:1 And/or SEQ ID NO:Nucleotide sequence shown in 2.

6. the nucleotide sequence described in claim 5 or its recombinant dna expression vector for combining are contained.

7. the host containing the recombinant dna expression vector described in the nucleotide sequence or claim 6 described in claim 5 is thin Born of the same parents, it is preferred that described host cell is selected from prokaryotic, yeast or mammalian cell, it is furthermore preferred that the host cell Selected from HEK293F cells, Chinese hamster ovary celI or NSO cells.

8. described in the antibody described in any one of claim 1-3, the antigen-binding fragment described in claim 4, claim 5 The host cell described in carrier or claim 7 described in nucleotide sequence, claim 6 is inherently exempted from preparation regulation and control human body Epidemic disease, activate I type IFN signal systems medicine in purposes, the medicine be for prevent and treat infectious diseases, tumour and Disease of immune system.

9. purposes as claimed in claim 8, it is characterised in that the infectious diseases include but is not limited to inhibition of HIV infection, A type, B-mode and infection with hepatitis C virus, herpesvirus infection, influenza infection, Coxsackie virus infection, respiratory tract are closed Cellular virus infection, human papilloma virus infection, ebv infection, enterovirns type 71 infection；The tumour is included but is not limited to Glioma, Hodgkin lymphoma, follicularis lymph cancer, melanoma, acute or chronic leukaemia, lung cancer, breast cancer, liver cancer, Stomach cancer, cancer of pancreas, kidney, prostate cancer, carcinoma of urinary bladder, oophoroma, cutaneum carcinoma, squamous cell carcinoma, osteocarcinoma, head and neck cancer, Colon and rectum Cancer, solid tumor；The disease of immune system include be not limited to systemic loupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, Myasthenia gravis, multiple sclerosis, autoimmune hemolytic anemia, oneself immunity hepatitis, chorionitis, nodositas are moved more Arteries and veins is scorching, Wegener granulomatosis.

10. the method for preparing the full human monoclonal antibody of regulation and control human body inherent immunity, it is characterised in that comprise the following steps：

(1) expression vector is provided, the expression vector contains the antibody or claim 4 of coding claim any one of 1-3 The DNA molecular of antigen-binding fragment, and the expression regulation sequence being connected operable with the DNA molecular；

(2) host cell is converted with the expression vector；

(4) monoclonal antibody described in obtaining is isolated and purified；

Wherein, it is preferred that described expression vector is the carrier for expression of eukaryon pcDNA3.4 containing antibody-secreting signal peptide sequence, Described host cell is HEK293F cells.