CN106117361A - AntiCD3 McAb single domain antibody - Google Patents
AntiCD3 McAb single domain antibody Download PDFInfo
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- CN106117361A CN106117361A CN201610435687.0A CN201610435687A CN106117361A CN 106117361 A CN106117361 A CN 106117361A CN 201610435687 A CN201610435687 A CN 201610435687A CN 106117361 A CN106117361 A CN 106117361A
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- antibody
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- single domain
- domain antibody
- anticd3 mcab
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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
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
-
- 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
- C07K2317/626—Diabody or triabody
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- General Health & Medical Sciences (AREA)
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- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Life Sciences & Earth Sciences (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention provides a kind of AntiCD3 McAb single domain antibody, and its aminoacid sequence is as shown in SEQ ID NO. 1.The present invention further provides the coding nucleotide sequence of this AntiCD3 McAb single domain antibody, the expression vector comprising this nucleotide sequence and host cell.AntiCD3 McAb single domain antibody antagonism people's CD3 ε N end section that the present invention provides, these antibody may be used for the mensuration of such as CD3, and other need the functional protein of CD3, such as bi-specific antibody.
Description
Technical field
The present invention relates to AntiCD3 McAb single domain antibody (sdAb) and aminoacid, nucleotide coding sequence.The invention still further relates to bag
Expression vector containing this nucleotide coding sequence and host cell.
Background technology
After monoclonal antibody technique in 1975 comes out, the antibody of antigenic specificity has changed biology and medical science
Many aspects.Except the research tool as importance, the availability of monoclonal antibody is that the diagnosis of development human diseases is with new
Therapy opens road.But, monoclonal antibody has technical restriction and shortcoming in range of application, such as, move based on suckling
Thing expression system is costly.
In the past few decades, a series of new technique and method are for improving the performance of conventional antibodies, especially swollen
In tumor treatment.Such as, add poisonous compound to existing antibody, be linked to monoclonal antibody including radiosiotope.So
And, even up to today, the Clinical practice monoclonal anti scale of construction is the most little.Another method improving tumorcidal efficiency is to make
With the antibody of bispecific, immunocyte is guided to tumor cell, thus kills tumor cell.And it is most commonly used that use is anti-
T cell is guided to tumor cell by CD3 antibody.It is by binding the surface antigen of CD3 and tumor cell (TAA) simultaneously, this
Bi-specific antibody can trigger the oncolysis of T cell mediation.CD3(cluster differentiation 3) φt cell receptor helps
In activating cytotoxic T cell.The protein of a kind of complexity that it is made up of four different chains.In mammal, bag
Containing CD3 γ chain, CD3 δ chain and two CD3 ε chains.
A lot of bi-specific antibodys use single-chain antibody.Single-chain antibody be from traditional IgG molecule obtain complete
Little Fab.Unfortunately, the scFvs yield of bacterial expression is the lowest.Camel and alpaca comprise a kind of unique types
Antibody, this kind of antibody deficiency light chain.Because lacking CH1 than conventional antibody, these so-called heavy chain antibodies have relatively low molecule
Amount.The heavy chain of this kind of immunoglobulin variable is called for short VHH, to be different from the variable region of heavy chain of classics.Therefore, individual domain VHH is
Minimum available intact antigen combines 15 kDa fragments, is referred to as nano antibody.Nano antibody and Fab, Fv or single-chain antibody phase
Ratio, has the advantage of some uniquenesses, as more stable, be easier at bacterial expression.
In prior art, anti-cd 3 antibodies form is single and can not meet actual demand, it is therefore necessary to develop more
Anti-cd 3 antibodies, thus for producing and design function albumen provides more and selects.
Summary of the invention
One aspect of the present invention relates to a kind of AntiCD3 McAb single domain antibody, and its aminoacid sequence is as shown in SEQ ID NO. 1.
Another aspect of the present invention relates to the nucleotide sequence of the AntiCD3 McAb single domain antibody of a kind of code book invention.A reality
Executing in mode, this nucleotide sequence is as shown in SEQ ID NO. 2.
Another aspect of the present invention relates to a kind of expression vector, and it comprises the nucleotide sequence of the present invention.The present invention also relates to
And comprise the host cell of this expression vector.In one embodiment, this host cell is escherichia coli.
The invention still further relates to a kind of bi-specific antibody, it comprises arbitrary CD3 single domain antibody of the present invention.
AntiCD3 McAb single domain antibody antagonism people's CD3 ε N-end section that the present invention provides, these antibody may be used for such as
The mensuration of CD3, and other need the functional protein of CD3, such as bi-specific antibody.
Accompanying drawing explanation
Fig. 1 is the skeleton drawing of sdAb phage display library.
Fig. 2 shows serum titer experimental result.
Fig. 3. the agarose gel electrophoresis figure of the total serum IgE of separation of lymphocytes.M swimming lane, DNA marker Marker III；
1-3 swimming lane, is isolatable from the total serum IgE of the lymphocyte drawn blood for the first time；4-7 swimming lane, is isolatable from the lymph that second time is drawn blood
The total serum IgE of cell；8-10 swimming lane, is isolatable from the total serum IgE of the lymphocyte that third time is drawn blood.
Fig. 4. the V of purificationHThe agarose gel electrophoresis figure of H PCR primer.M swimming lane, DNA marker Marker III；The
1-8 swimming lane, uses 8 V to the purification that primer obtains from the PBMC of blood drawing for the first timeHH PCR primer；9-16 swimming lane, uses 8
V to the purification that primer obtains from the PBMC of second time blood drawingHH PCR primer；17-24 swimming lane, use 8 to primer from the 3rd
The V of the purification that the PBMC of secondary blood drawing obtainsHH PCR primer.
Fig. 5. the insertion rate of sdAb phage display library.M13R (-48) and M13F (-47) primer is used to be expanded by PCR
Increase 96 library clones randomly selected.Have ~ clone of 1100 bp DNA bands has VHH insert.
Detailed description of the invention
The present invention now will explain in conjunction with following experiment and accompanying drawing further, it should be noted that these experimental examples and accompanying drawing
Should not be construed as limitation of the present invention.
1 strategy
The present invention have developed the single domain antibody of AntiCD3 McAb by display technique of bacteriophage, and has carried out following steps: animal is exempted from
Epidemic disease and immune response test, the structure of sdAb phage display library, phage display elutriation and FASEBA screening and FACS are tested
Card.
2 materials
Antigen protein: CD3-His albumen (MP-5)
Cell antigen system: Jurkat target cell, KPCN compared with control cells
TRIzol® Reagent (Ambion, Cat. No. : 15596-026)
PrimeScriptTM1st Strand cDNA synthetic agent box (Takara, Cat. No.: 6110A)
SfiI enzyme (NEB, Cat. No.: R0123S)
Host Strains:E.coli SS320
Ampicillin, 100 mg/ml
Isopropyl-β-D-thiogalactoside (IPTG), 1 M
PBS: 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.4
ELISA titer plate (Corning, Cat. No.: 9018)
It is coated buffer: 0.05 M NaHCO3, pH 9.6
Block buffer (PBST): PBS, pH 7.4, add 5% defatted milk powder
Lavation buffer solution: PBS, pH 7.4, add 0.05% Tween 20
M13KO7 helper phage (NEB, Cat. No.: N0315S)
HRP/ anti-M13 monoclonal antibody (GE Healthcare, Cat. No.: 27-9421-01)
Goat-anti camel IgG [HRP] (Novus Biological, Cat. No.: NB7242)
Rabbit anti-camel IgG [HRP] (GenScript)
FACSCalibur (BD Bioscience, San Jose, CA)
Flowjo: Flowjo 7.6.1 Min software
2 × YT:16 g tryptone, 10 g yeast extracts, 5 g NaCl are dissolved in 1 L ddH2O.
OctetRED96 (ForteBio)
Super Streptavidin (SSA) Dip and ReadTM Biosensors (ForteBio)
10 mM glycine-HCl, pH 2.0
3 methods
3.1 animal immunes and immune response test
3.1.1 animal immune
CD3-His immunogen is mixed with adjuvant or PBS and is expelled to yamma (llama).In whole Project Process, animal
By immunity five times.Peripheral blood sample is gathered respectively before immunity and after the 4th and the 5th immunity.Gradient separations method is divided
Separate out lymphocyte.Cell adds RNAlater and is stored in-80 ° of C.The centrifugal blood adding anticoagulant obtains serum,
And it is stored in-80 ° of C.
3.1.2 immune response test
Blood serum sample is used to be evaluated for fixing immunogenic immune response by ELISA.Before have rated immunity and the 4th
With the serum after the 5th immunity.Antigen (CD3-His and CD34-Fc) is diluted in being coated in buffer of 4 μ g/ml respectively.Make
With the antigen of dilution, titer plate is coated overnight, 4 ° of C.Subsequently, titer plate is washed 3 times with lavation buffer solution, then at 37 ° of C
Close 2 hours with Block buffer.Titer plate is washed 4 times again with lavation buffer solution.The serum of a series of dilutions is added plate
On, hatch 2 hours at 37 ° of C.Titer plate is washed 4 times subsequently with lavation buffer solution.Add goat-anti camel IgG [HRP] or rabbit resists
Camel IgG [HRP], hatches 1 hour at 37 ° of C.After washing, reactant adds tmb substrate and reacts 10 minutes, be subsequently added 1 M
HCl terminates reaction.MK3 spectrometer measures every hole absorption value at 450 nm.
The structure of 3.2 sdAb phage display libraries
3.2.1 RNA extracts
From the lymphocyte (see 3.1.1) separated, total serum IgE is extracted according to TRIzol reagent handbook.Gel electrophoresis and
Total serum IgE is carried out qualitative and quantitative by OD260/280 method.
3.2.2 RT-PCR and VHH expands
According to PrimeScriptTMThe handbook of 1st Strand cDNA synthetic agent box uses oligo (dT) 20 primer by total
RNA reverse transcription is cDNA.Design four justice and two antisense specific degenerate primers expand VHH fragment, introduces twoSfiI restriction site.V is expanded according to GenScript S.O.P. (SOP)HH fragment.
3.2.3 show storehouse extension
Use different primers that amplification is obtained VHH PCR primer.UseSfiI digest this PCR primer and by gel pure
Change.The fragment of gel-purified is inserted in the phagemid vector (Fig. 1) that GenScript is own.Build experimental show storehouse with
Optimize and connect and conversion condition.The connection and the conversion condition that optimize are used to develop actual displaying storehouse.Sub-fraction converts cell
It is diluted and lines on 2 × YT flat board with 100 μ g/ml ampicillin.Bacterium colony is calculated library big
Little.Random picking positive colony also checks order, to assess the quality in library.Remaining converts cell and is scribed in having 100 μ g/ml
On 15 cm 2 × YT flat boards of ampicillin and 2% glucose.Lawn is scraped off from flat board.Fraction cell is used for
Library plasmid separates.Remaining adds glycerol, is stored in-80 ° of C standby.
3.3 phage library elutriations
3.3.1 biopanning
The standardization program using GenScript exploitation carries out the elutriation of CD3-His albumen to constructed sdAb phage library.Contain
Have 7.4 × 108The big sdAb phage display library of individual clone (CFU) is used for this screening.Library is grown on logarithmic (log) phase, uses
M13KO7 helper phage is reclaimed, and trains at the 2 × YT with 100 μ g/ml ampicillin and 50 μ g/ml kanamycin
Support in plate and expand overnight at 30 ° of C on agitator.Make phages with PEG/NaCl, and be resuspended in PBS, be stored in-
80°C.For carrying out phage elutriation, microwell plate (Pierce, Prod# 15100) being coated CD3-His, this is by by them
Realize in 4 ° of C overnight incubation in PBS (pH 7.0) with 100 μ g/ml.Meanwhile, phage particle is taken off with containing 2%
The PBS Block buffer of fat milk powder at room temperature hatches 1 hour to block non-specific binding.After rinsing 3 times with PBS, will
Phage particle adds in micropore, and on the oscillator in incubated at room 1 hour.After hatching, by containing 0.05% with PBST(
The PBS of Tween-20) rinsing aperture 6 times washes away biting of unconjugated and non-specific binding for 2 times followed in turn by PBS rinsing
Thalline.In conjunction with phage be used to immediately infect exponential phases at 37 ° of CE. coli(OD600 is about TG1 cell
0.5) 1 hour.After each panning rounds, infected cell and 10% glycerol are mixed, is subsequently stored in-80 ° of C.Carry out
During the elutriation that the next one circulates, by the infection of 10 mlE. coliTG1 cell liquid storage adds 30 ml and contains 200 μ g/ml
In the culture medium of ampicillin and 2% glucose, and grow to logarithmic (log) phase.Culture reclaims with M13KO7 helper phage, expands
Increase and precipitating phage, screen for next round.Conventional phage display elutriation repeated as described above.
3.3.2 Phage-ELISA
Single output phage clone is grown in 96 deep well plate and screens to confirm CD3-His by Phage-ELISA
Specificity is cloned.2 μ g/ml CD3-His are used to be coated 96 hole flat boards (in 4 ° of C overnight incubation in being coated buffer), to contain
The PBS having 2% defatted milk powder closes.Every hole adds the phage supernatants from overnight cell culture of about 50 μ l, and at 4 °
C is hatched 2 hours, washs four times with PBST subsequently.Unconjugated sdAb phage by the anti-M13 monoclonal antibody with HRP labelling at 37 °
C is hatched 1 hour and is detected.With PBST washing hole four times again, each hole adds substrate solution.Measure at 450 nm and absorb
Value.
3.4 FASEBA screening and FACS verify
3.4.1 FASEBA screening and affine classification
Amplification exports the DNA of the coding sdAb fragment of phage and is inserted intopTo filter out guide's antibody in FASEBA carrier.
Single FASEBA library clone abduction delivering is inoculated in 96 deep well plate.Carry out ELISA screening to know to separate specificity
The sdAb of other CD3-His albumen, and select the expression culture medium of the affine classification for Octet.At Octet RED96 instrument
(ForteBio) dissociation rate screening (Off-rate screening) is carried out on.All tests are all carried out at 30 ° of C.Express training
SdAb-SASA albumen in foster base is caught into and is coated the biosensor of BSA and with CD3 albumen at solution (1x PBS, pH
7.4,0.05% Tween-20) in hatch together.The CD3 of one of which concentration (100 nM) is used for dissociation rate classification.Base
Line and dissociation steps are only carried out in buffer (1x PBS, pH 7.4,0.05% Tween-20).Use Fortebio data
Process software and measure binding kinetics with dynamics data analytical model by 1:1 Langmuir binding pattern.With CD3 with
High-affinity interact conjugate screened go out and carry out DNA sequencing.
3.4.2 FACS checking
Combine Jurkat cell to filter out and do not combine the sdAb of KPCN cell, culture supernatants is carried out fluidic cell
Analyze.Cell is collected by being centrifuged when Jurkat cell and KPCN cell grow to 70-80% fusion rate.Every hole inoculation about 4 ×
105Individual cell, washs 2 times with PBS.In cell, add the culture supernatants of 200 l and at room temperature hatch 1 hour.With
After PBS washing, in cell, add the sdAb that antibody combines with detection.After 30 minutes hatch, with PBS washed cell twice, and
Cell is resuspended in PBS.FACSCalibur (BD Bioscience, San Jose, CA) and Flowjo software is used to divide
The sdAb of analysis cell combines.
4. result
4.1 immune response tests
Fig. 2 shows serum titer experimental result.First time and the potency ratio preimmune serum of second time test sera after immunity
Much higher.For the first time and significant difference is not observed between the titer of second time test sera after immunity.
4.2 sdAb phage display libraries build
4.2.1 Total RNAs extraction
Isolating about 449 g total serum IgE (Fig. 3) lymphocyte from all acquisition, the total serum IgE of about half is used to build high-quality
Library.
4.2.2 RT-PCR and VHH expands
SOP according to GenScript uses 8 primer (x2 antisense primer of 4 sense primers) is carried out RT-PCR.Use difference
The product obtained is mixed together and carries out gel-purified (Fig. 4) by primer.Obtain altogether the about 24 pure V of gHH PCR primer.
General PCR primer is used for library construction.
4.2.3 library extension
At least 7.4 × 107Individual transformant can obtain from one group of electricity converts, and converts so having carried out 10 times abreast, to obtain
Final library.Library size about ~ 7.4 × 108For.Screening according to bacterium colony, insertion rate is 97.92% (94/96) (Fig. 5).One
Checked order 72 clones altogether, and 64 clones are positioned at frame.Neither one has identical aminoacid sequence (data are not shown), table
Bright sdAb library has the multiformity of height.
4.3 phage library elutriations
Having carried out two-wheeled elutriation and Phage-ELISA, table 1 lists correlative detail.
Table 1. elutriation and the details of Phage-ELISA experiment
| Round | Input (pfu) | Output (pfu) | Phage-ELISA positive rate |
| 1st | 2.0×1011 | 3.0×105 | ~ 5% |
| 2nd | 3.5×1010 | 9.7×107 | ~ 51% |
4.4 FASEBA screenings and FACS verify
4.4.1 FASEBA screening and affine classification
The DNA of the coding sdAb fragment of output phage is taken turns in amplification second, and insertspTo pass through ELISA in FASEBA carrier
Screening guide's antibody.32 clones are obtained by FASEBA ELISA screening.These 32 clones carry out DNA sequencing, and carry out
Combination with CD3 is tested, and carries out dissociation rate analysis by Octet RED96 subsequently.In conjunction with the result (4.4.2) of FACS, profit
The independent clone of low part of dissociating in curve is selected by FortteBio data analysis software.Table 2 shows selected sdAb
The dynamics data of clone.
The dynamics data of table 2. sdAb clone
| Sample ID | Serial number | koff(1/s) |
| A30322 | SEQ ID NO. 1 | 1.43E-05 |
4.4.2 FACS checking
Being verified that by FACS the Jurkat cell of positive culture (from 4.4.1) supernatant combines, KPCN is used as negative right
Photo cell system.In conjunction with the result (4.4.1) of affine classification, it is listed in table 3. in conjunction with MFI
Table 3. culture supernatants and Jurkat cell and the combination MFI of KPCN cell *
| Sample | A30322 | NC-sdAb | TG1 |
| Jurkat cell | 521.03 | 115.03 | 132.03 |
| KPCN cell | 116.6 | 51 | 54.5 |
*: the culture supernatants of NC-sdAb (the non-correlation sdAb of same form) is arranged to negative control, the training of TG1
Support thing supernatant and be arranged to ground control.
SEQUENCE LISTING
<110>Zhongshan University
<120>AntiCD3 McAb single domain antibody
<130> 16503CN
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 117
<212> PRT
<213>artificial sequence
<400> 1
Glu Val Gln Leu Val Glu Cys Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Asp Thr Met
20 25 30
Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ala
35 40 45
Val Arg Trp Ser Ser Gly Asn Thr Tyr Tyr Gly Asn Thr Val Lys Gly
50 55 60
Arg Phe Thr Ile Ser Arg Asp Thr Ala Thr Asn Thr Val Tyr Leu Glu
65 70 75 80
Met Asn Ser Leu Lys His Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala
85 90 95
Arg Leu Ala Gly Arg Gly Ala Ala Asp His Trp Gly Gln Gly Thr Gln
100 105 110
Val Thr Val Ser Ser
115
<210> 2
<211> 351
<212> DNA
<213>artificial sequence
<400> 2
gaggtgcagc tggtggagtg tgggggagga ttggtgcagg ctgggggctc cctgagactc 60
tcctgtgcag cctctggacg caccttcgat accatgggct ggttccgcca ggctccaggg 120
aaggagcgtg agtttgtagc agcggttaga tggagtagtg gtaatacata ctatggaaac 180
accgtgaagg gccgattcac catctccaga gacactgcca cgaacacggt gtatctggaa 240
atgaacagtc tgaaacatga ggacacggcc gtttattact gtgcagcccg attggctggt 300
aggggcgcgg ctgaccactg gggccagggg acccaggtca ccgtctcctc a 351
Claims (7)
1. an AntiCD3 McAb single domain antibody, its aminoacid sequence is as shown in SEQ ID NO. 1.
2. the nucleotide sequence of the AntiCD3 McAb single domain antibody that a kind encodes described in claim 1.
Nucleotide sequence the most according to claim 2, wherein said nucleotide sequence is as shown in SEQ ID NO. 2.
4. an expression vector, it comprises the nucleotide sequence described in Claims 2 or 3.
5. the host cell of the expression vector comprised described in claim 4.
Host cell the most according to claim 5, wherein said host cell is escherichia coli.
7. a bi-specific antibody, it comprises the aminoacid sequence of the AntiCD3 McAb single domain antibody described in claim 1.
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| Application Number | Priority Date | Filing Date | Title |
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| Publication Number | Publication Date |
|---|---|
| CN106117361A true CN106117361A (en) | 2016-11-16 |
Family
ID=57470793
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| Application Number | Title | Priority Date | Filing Date |
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| CN201610435687.0A Pending CN106117361A (en) | 2016-06-17 | 2016-06-17 | AntiCD3 McAb single domain antibody |
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| Country | Link |
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| CN (1) | CN106117361A (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104487587A (en) * | 2012-04-20 | 2015-04-01 | 新兴产品开发西雅图有限公司 | Cd3 binding polypeptides |
-
2016
- 2016-06-17 CN CN201610435687.0A patent/CN106117361A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104487587A (en) * | 2012-04-20 | 2015-04-01 | 新兴产品开发西雅图有限公司 | Cd3 binding polypeptides |
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