CN110283249A - A kind of people's ScFv single-chain antibody of anti-interleukin 6 - Google Patents
A kind of people's ScFv single-chain antibody of anti-interleukin 6 Download PDFInfo
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- CN110283249A CN110283249A CN201711212019.2A CN201711212019A CN110283249A CN 110283249 A CN110283249 A CN 110283249A CN 201711212019 A CN201711212019 A CN 201711212019A CN 110283249 A CN110283249 A CN 110283249A
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- antibody
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
- C07K16/244—Interleukins [IL]
- C07K16/248—IL-6
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
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- General Health & Medical Sciences (AREA)
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- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
The invention discloses a kind of protein of the single-chain antibody of anti-interleukin 6, the single-chain antibody has the antigen binding ability of high degree of specificity, the single chain antibody protein had both had the specific recognition effect of antibody, this identity can be blocked again by specific receptor simultaneously, and the molecular weight of the antibody fusion protein is small, it can be expressed in procaryotic cell expression system, greatly reduce the production cost of antibody drug.
Description
Technical field
The invention discloses a kind of anti-IL6 people ScFv single-chain antibodies, belong to immunology and molecular biology field.
Background technique
Inflammation especially chronic inflammation has huge damage to body, and inflammation part discharges large amount of cell factor for a long time
And inflammatory mediator, not only cause local organization organ lesion, so that patient is generated pain, and prolonged inflammation is easier to cause
Tumour occurs.In inflammation two kinds of relatively conventional cell factors be tumor necrosis factor α and interleukin-6, for this two
The antibody drug of a factor research and development has also listed, and such as treats the adalimumab of rheumatoid arthritis.
Interleukin-6 (Interleukelin-6, IL-6) be with the active cell factor of various biological, and
Key factor in the cytokine network of body complexity, the effect of its other adjustable cell factor, be inflamed, it is bad
When increasing extremely or due to tumor-cell antigen stimulation immunocyte secretion IL-6, IL-6 increases in serum, overexpression
IL-6 (Keto etc., 1997) often related to certain diseases.The extensive biological activity as possessed by IL-6 and potential
Potential applicability in clinical practice receives significant attention, from discovery so far domestic and foreign scholars to its structure, biological characteristics, mechanism of action and
Numerous studies have been done in clinical application etc..
Mononuclear macrophage is the first line of defence of immune system, and energy characterization antigen simultaneously provides information transmitting, thus
Immune response is induced, it includes tumor necrosis factor (TNFa), interleukins that a series of monokines are discharged during these
One 6 (IL-VI), interleukin 1 (IL-I) etc., also known as pro-inflammatory cytokines.
Antibody drug has been used as one of the therapeutic agent of inflammatory patients routine, it is contemplated that the year two thousand twenty, the sales volume of monoclonal antibody medicine
It is up to 200,000,000,000 dollars.Since first genetic engineering antibody Chimeric antibody in 1984 is born, novel gene
Engineered antibody continuously emerges, such as humanized antibody, monovalent small molecular antibody (Fab, single-chain antibody, single domain antibody, hypervariable region polypeptide
Deng), multivalence small molecular antibody (double-chain antibody, three chain antibodies, miniantibody), certain type-specifics it is (bispecific antibody, anti-
Originalization antibody, intrabody, catalytic antibody, immunoliposome) and antibody fusion protein (immunotoxin, immune adherence element) etc.
Deng.In addition, the phage antibody library technique for being used to prepare novel antibodies becomes after hybridoma technology in life science
Another breakthrough progress.
At present small molecular antibody exploitation patent medicine object mainly in two forms based on, one is antibody coupling compounds
(Antibody conjugate complex, ADC), another kind is bispecific antibody.Antibody-drug conjugates are a kind of novel
Targeted therapy, such as in antineoplaston, ADCs is connected to one by an antibody or an antibody fragment ScFv and effectively carries
The drug (usually toxin) of lotus forms an immune complex.Antibody makes this immune complex be integrated to specific tumour
On cell, this usual immune complex can be internalized by inside tumor cells, and then drug release to causing tumour thin into the cell
Cellular damage and death.Although antibody coupling compound has good tumour recognition capability and Antineoplastic effect, it was prepared
It needs to be coupled with other chemical group in journey, not only increases preparation cost, be also difficult to ensure the homogeneity of product, coupling
The antigen recognizing district of chemical group and antibody is often also easy to generate space steric effect, also has malicious secondary work to body sometimes
With the actual anti-tumor effect of these factors affect antibody coupling compounds.
Newest tumor biotherapy method CAR-T is presently considered to be most effective oncotherapy means.But this side
If method will appear a kind of side reaction cytokine storm very serious with improper, this symptom may also appear in outer
In a variety of diseases such as wound, infection.Cytokine storm (cytokine storm) causes body fluid after referring to organism infection microorganism
Middle cytokine profiles such as TNF-α, IL-1, IL-6, IL-12, IFN-α, IFN-β, IFN-γ, MCP-1 and IL-8 etc. are big rapidly
The phenomenon that amount generates, is the major reason for causing acute respiratory distress syndrome and multiple organ failure.If can be in cell factor
A large amount of raising initial stages block the biological function of these factors in time, so that it may mitigate the damage to body.
The anti-interleukin 6 single-chain antibody of the present invention identifies and blocks the biological function of IL6, to reach reduction inflammatory reaction
Effect.
Summary of the invention
The purpose of the present invention is develop a kind of single-chain antibody with anti-inflammatory response.Based on foregoing invention purpose, originally
Invention provides a kind of single-chain antibody of anti-IL-8 6, the amino in the area CDR1, CDR2 and CDR3 of the light chain of the antibody
Acid sequence is respectively as shown in SEQ ID NO.1,2 and 3, the amino acid sequence in the area CDR1, CDR2 and CDR3 of the heavy chain of the antibody
Column are respectively as shown in SEQ ID NO.5,6 and 7.
In a preferred technical solution, the light chain variable region of the antibody and the amino acid sequence point of heavy chain variable region
Not as shown in SEQ ID NO.4 and 8.
In one more preferably technical solution, the light chain variable region and heavy chain variable region of the antibody are more by connecting
Peptide is connected.
Finally, the present invention provides the applications in preparing anti-inflammatory drugs of above-mentioned bispecific antibody.
The present invention chooses, and there is the interleukin-6 IL6 of certainty inflammatory factor to start with, and utilize phage antibody library method
Screen the ScFv of the anti-IL6 of high-affinity.
In combining test, each IL6 albumen of single-chain antibody has the affinity of higher level, and single-chain antibody and target
The combination of albumen can be blocked by their corresponding ligands.It, should and since select is antibody small molecule segment to the present invention
The molecular weight of bispecific antibody is small, can express in procaryotic cell expression system, greatly reduce the production of antibody drug
Cost.
Detailed description of the invention
Fig. 1 .VH and VL PCR product qualification figure
Fig. 2 Vector map and recombinant vector qualification figure
The combination for the anti-IL6 ScFv and IL6 albumen that Fig. 3 is filtered out is tested
Fig. 4 .IL6R is to the protein bound blocking test of IL6 ScFv and IL6
Fig. 5 .TNFa-IL6 bispecific antibody polyacrylamide gel electrophoresis qualification figure
Specific embodiment
The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and
It is apparent.But examples are merely exemplary for these, does not constitute any restrictions to protection scope of the present invention.
The preparation of the anti-IL6 single-chain antibody of embodiment 1
The foundation in 1.1 high storage capacity natural antibody libraries.
The separation single karyolymph cell of human peripheral: 100 normal adults are randomly selected, every human peripheral is extracted
10ml.It is diluted with the RPMI-1640 culture solution 1: 1 containing 10% heparin, is added to the centrifuge tube (dilution equipped with lymphocyte separation medium
The volume ratio of venous blood and lymphocyte separation medium is 2: 1) in, 2,000 × g is centrifuged 17 minutes.Draw lymphocyte separation medium
Interface milky mononuclearcell layer, PBS buffer solution are washed twice.
The extraction of 1.2 cell total rnas
By every 5 × 106Trizol reagent is added in the ratio of cells/ml, and piping and druming cell is allowed to crack.5min is incubated at room temperature,
It moves into the EP pipe handled through DEPC, the chloroform of 1/5 volume is added, acutely vibrate 15sec, be incubated at room temperature 3min.4 DEG C, 10,
000 × g is centrifuged 15min, draws upper strata aqueous phase into a new centrifuge tube, the isopropanol of 1/2 volume, ice bath 10min is added.
4 DEG C, 12,000 × g is centrifuged 10min, abandons supernatant, and 75% ethyl alcohol of 1ml cleaning precipitating is added.4 DEG C, 7,500 × g is centrifuged 5min,
Supernatant is abandoned, drying at room temperature precipitating is dissolved in the water of no RNase or is deposited in dehydrated alcohol, -80 DEG C save backup.
1.3 reverse transcriptions synthesize the first chain of cDNA
The genomic DNA of remaining is first eliminated with the DNase I processing total serum IgE of no RNase.Take processed RNA sample 2
μ g and Oligo (dT) 15 (500 μ g/ml) 1 μ l adds DEPC water to 12 μ l, 70 DEG C, heats 10min.It is placed at once after taking-up
In ice bath, 5 × Buffer, 5 μ l is sequentially added；dNTP(10mmol/L)5μl；1 μ l, MMLV reverse transcriptase of RNase inhibitor, 1 μ
L adds DEPC water to 25 μ l, and 42 DEG C, heat preservation 60min carries out reverse transcription reaction, and 70 DEG C, 15min inactivates enzymatic activity.
1.4PCR expands VH and VL gene
Using reverse transcription synthesis the first chain of cDNA as template, using VH and VL base expressed by people's ScFv antibody library primer amplification
Cause.Reaction system is as follows:
PCR parameter: after 94 DEG C of denaturation 3min, then with 94 DEG C of 30sec；61℃ 30sec；72 DEG C of 1min carry out PCR,
Totally 30 circulations, last 72 DEG C of extensions 10min.5 μ l reaction products are taken to carry out 1% agarose gel electrophoresis point after reaction
Analysis.
PCR product recycling
(1) PCR product carries out 1.5% agarose gel electrophoresis, and target DNA fragment is cut from Ago-Gel, is put into
In 1.5ml centrifuge tube.
(2) 400 μ l sol solutions A are added, 70 DEG C dissolve 5 minutes, until gel all dissolves.
(3) 200 μ l sol solutions B are added, suck whole liquid in recovery column after being mixed.
(4) 12,000 × g are centrifuged 1 minute, abandon waste liquid.
(5) 500 μ l neutralizers are added, 12,000 × g is centrifuged 1 minute, abandons waste liquid.
(6) 700 μ l cleaning solutions are added, 12,000 × g is centrifuged 1 minute, abandons waste liquid.
It repeats step 6) 1 time.
(7) 12,000 × g are centrifuged 2 minutes, are abandoned waste liquid, recovery column are moved into new reception pipe, drying at room temperature 5 minutes.
(8) 30 μ l deionized waters are added, 12,000 × g is centrifuged 1 minute, and eluted dna segment is saved backup in -20 DEG C.
The splicing of 1.5 bridging PCR methods progress VH and VL
Using VH the and VL segment of preparation as template, the series connection of VH and VL segment is carried out.
PCR reaction system:
PCR reaction condition be 94 DEG C initial denaturation 5 minutes, then by following parameter carry out 30 circulation: 94 DEG C be denaturalized 30 seconds,
60 DEG C are annealed 45 seconds, and 72 DEG C extend 1 minute half, and last 72 DEG C extend 10 minutes.Glue recycling step is repeated, PCR product is dissolved in 30 μ
In l ddH2O.Fig. 1 is VH and VL PCR product qualification figure, wherein swimming lane 1VL, swimming lane 2VH, swimming lane 3VH+VL concatermer.
1.6 are connected on phage vector,
Sfi1 digestion carrier and PCR product
37 DEG C digestion 2 hours, with identical method recycle endonuclease bamhi.
Connection reaction
After above-mentioned reactant mixes well and is centrifuged it is made to be sunken to tube bottom, 4 DEG C of connections are overnight.Segment after digestion with
The connection of pComb3XSS carrier segments, is built into recombinant plasmid.Fig. 2 is recombinant vector electrophoretic identification.Wherein, 1 is recombinant piece
Section pComb3XSS carrier restriction enzyme mapping, 2 be molecular weight marker.
1.7 conversions and expression
By the carrier built (plasmid) conversion (electricity turns) to (specific Escherichia coli) in Escherichia coli, large intestine bar is expanded
Bacterium simultaneously adds helper phage, and the bacteriophage after collecting recombination is exactly phage antibody library.
The storage capacity for being detected hundred naive ScFv antibody libraries is 2*1010, fully meet the needs of antibody screening.
The screening of 1.8IL6 antibody
It is expanded in Escherichia coli from 10ul is taken out in antibody library, the antibody library after collecting amplification, IL6 albumen wraps respectively
Antibody library is added after elisa plate, non-specific bacteriophage is rinsed after incubation, digests specific bond bacteriophage, it will digestion after enrichment
Phage-infect Escherichia coli coated plate, picking monoclonal cenobium simultaneously induces in a small amount, and the monoclonal bacterium supernatant after a small amount of inductions is done
ELISA screening positive clone, by multiple authentication, choosing affinity and specificity, all high (affinity reaches 10-8-10-9KD)
Positive colony carries out antibody expression.Fig. 4 is the combination test of IL6 ScFv and the IL6 albumen filtered out.Wherein OD value is highest
Anti- IL6 ScFv 5 clones are present invention sequences to be protected.Fig. 5 is that IL6R blocks anti-IL6 ScFv to test in conjunction with IL6.
IL6R can effectively block the combination of single-chain antibody and corresponding albumen as seen from the figure, and as the increase barrier effect of concentration also increases
By force.Illustrate that the antibody sequence of our patent protections can effectively block IL6R and the combination of its ligand.
Plasmid in positive colony is extracted and is transformed into other expression bacterium, or antibody gene is connected to other carriers again
Be transformed into corresponding expression bacterium, screening optimum condition of the expression carries out a large amount of inducing expressions, finally select optimal purification process and
Buffer obtains antibody protein.Fig. 3 is the polyacrylamide gel electrophoresis qualification figure of the anti-IL6 ScFv of expression.Swimming lane 1 is anti-
IL6 ScFv, anti-IL6 ScFv molecular weight are 30KD.
Bacillus coli DH 5 alpha is the preservation of this room, genotype are as follows: supE44 Δ lacU169
HsdR17 recA1 end1 gyr96 thi-1 relA1, amplification and conversion for plasmid.
E. coli bl21 (DE3), genotype are hsdS gal (λ cIts857ind1 Sam7 nin5 lacUV5-T7),
Expression for recombinant protein.
PGEM-T Easy: for the clone of PCR product, it is purchased from Promega company.
Prokaryotic expression plasmid vector pET30, pET42, pGEX-4t-1 are that this room saves.
The purifying of embodiment 2.ScFv antibody
It chooses single colonie to be inoculated in 5ml LB culture medium, 37 DEG C of violent shaken cultivations are stayed overnight.
Above-mentioned bacterium solution is inoculated into the conical flask containing 400ml LB (antibiotic) in 1: 100 ratio, 37 DEG C
Violent shaken cultivation 2h.
Final concentration of 1mmol/L IPTG is added, in 37 DEG C of inducing expression 3-4h.
Culture solution is collected, 5,000rpm centrifugation 10min abandon supernatant.PBS washes bacterial sediment.
It is resuspended with PBS according to 5ml/g, to work 10sec/300W/30 times under ice bath, the parameter of every minor tick 15sec surpasses
Sound smudge cells.12,000rpm centrifugation 20min, collect supernatant.
Purifying recombinant proteins
Will containing there are six histidine mark fusion protein TNFa ScFv-GH-His6 and IL6 ScFv-GH-His6 with
0.45 μm of membrane filtration, prepared column.
After HisTrap kit affinity column is balanced with Binding Buffer 10ml, the sample to be purified having had been prepared for is added
Product.Adjusting flow velocity is about 8-10 drop/min.
Column is washed using Binding Buffer.
Pillar is eluted using 6ml Elution Buffer.It is in charge of collection eluent.Take a small amount of progress SDS-PAGE mirror
It is fixed, each Guan Yu -70 DEG C of preservations rich in destination protein.
Claims (6)
1. a kind of single-chain antibody of anti-IL-8 6, which is characterized in that CDR1, CDR2 and CDR3 of the light chain of the antibody
The amino acid sequence in area respectively as shown in SEQ ID NO.1,2 and 3, the area CDR1, CDR2 and CDR3 of the heavy chain of the antibody
Amino acid sequence is respectively as shown in SEQ ID NO.5,6 and 7.
2. antibody according to claim 1, spy is being, the light chain variable region of the antibody and the ammonia of heavy chain variable region
Base acid sequence is respectively as shown in SEQ ID NO.4 and 8.
3. antibody according to claim 2, spy is being that the light chain variable region and heavy chain variable region of the antibody pass through
Connecting peptides are connected.
4. antibody according to claim 3, spy is being, the amino acid sequence of the connecting peptides such as SEQ ID
Shown in NO.9.
5. one kind contains ScFv single chain antibody protein described in claim 1,2 and 4.
6. application of the ScFv single chain antibody protein according to claim 5 in prevention of inflammation reaction.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201711212019.2A CN110283249A (en) | 2018-03-19 | 2018-03-19 | A kind of people's ScFv single-chain antibody of anti-interleukin 6 |
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| CN201711212019.2A CN110283249A (en) | 2018-03-19 | 2018-03-19 | A kind of people's ScFv single-chain antibody of anti-interleukin 6 |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07188056A (en) * | 1993-07-21 | 1995-07-25 | Chuzo Kishimoto | Suppressor for bone resorption containing antibody against interleukin 6 receptor as active ingredient |
| JP2003026699A (en) * | 2001-07-13 | 2003-01-29 | Inter Cyto Nano Science Co Ltd | Il-6 signal transmission-antagonizing peptide |
| CN1678744A (en) * | 2002-08-30 | 2005-10-05 | 财团法人化学及血清疗法研究所 | Human antihuman interleukin-6 antibody and fragment of the antibody |
| CN103261222A (en) * | 2010-09-10 | 2013-08-21 | 埃洛齐纳公司 | Novel antibody derivatives |
-
2018
- 2018-03-19 CN CN201711212019.2A patent/CN110283249A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07188056A (en) * | 1993-07-21 | 1995-07-25 | Chuzo Kishimoto | Suppressor for bone resorption containing antibody against interleukin 6 receptor as active ingredient |
| JP2003026699A (en) * | 2001-07-13 | 2003-01-29 | Inter Cyto Nano Science Co Ltd | Il-6 signal transmission-antagonizing peptide |
| CN1678744A (en) * | 2002-08-30 | 2005-10-05 | 财团法人化学及血清疗法研究所 | Human antihuman interleukin-6 antibody and fragment of the antibody |
| CN103261222A (en) * | 2010-09-10 | 2013-08-21 | 埃洛齐纳公司 | Novel antibody derivatives |
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