CN106084047A - AntiCD3 McAb single domain antibody - Google Patents
AntiCD3 McAb single domain antibody Download PDFInfo
- Publication number
- CN106084047A CN106084047A CN201610435670.5A CN201610435670A CN106084047A CN 106084047 A CN106084047 A CN 106084047A CN 201610435670 A CN201610435670 A CN 201610435670A CN 106084047 A CN106084047 A CN 106084047A
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- Prior art keywords
- antibody
- cell
- single domain
- domain antibody
- anticd3 mcab
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Classifications
-
- 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®
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 comprise the expression vector of this nucleotide coding sequence and host cell.
Background technology
After monoclonal antibody technique in 1975 comes out, the antibody of antigenic specificity has changed the many aspects of biology and medical science.Except the research tool as importance, the availability of monoclonal antibody is the development diagnosis of human diseases and new therapy opens road.But, monoclonal antibody has technical restriction and shortcoming in range of application, such as based on mammalian expression systems costly.
In the past few decades, a series of new technique and method are for improving the performance of conventional antibodies, especially in oncotherapy.Such as, add poisonous compound to existing antibody, be linked to monoclonal antibody including radiosiotope.But, even up to today, the Clinical practice monoclonal anti scale of construction is the most little.Another method improving tumorcidal efficiency is to use the antibody of bispecific, immunocyte is guided to tumor cell, thus kills tumor cell.And it is most commonly used that T cell is guided to tumor cell by use anti-cd 3 antibodies.It is by binding the surface antigen of CD3 and tumor cell (TAA) simultaneously, and this bi-specific antibody can trigger the oncolysis of T cell mediation.CD3(cluster differentiation 3) φt cell receptor contributes to activating cytotoxic T cell.The protein of a kind of complexity that it is made up of four different chains.In mammal, comprise CD3 γ chain, CD3 δ chain and two CD3 ε chains.
A lot of bi-specific antibodys use single-chain antibody.Single-chain antibody is the complete minimum Fab obtained from traditional IgG molecule.Unfortunately, the scFvs yield of bacterial expression is the lowest.Camel and alpaca comprise the antibody of a kind of unique types, this kind of antibody deficiency light chain.Because lacking CH1 than conventional antibody, these so-called heavy chain antibodies have relatively low molecular weight.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 that minimum available intact antigen combines 15 kDa fragments, is referred to as nano antibody.Nano antibody, compared with Fab, Fv or single-chain antibody, 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 the more selection of design function albumen offer.
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.In one embodiment, this nucleotide sequence such as SEQ
Shown in 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 invention still further relates to 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 the mensuration of such as 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 total serum IgE of the lymphocyte that second time is drawn blood；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 purificationHH
The agarose gel electrophoresis figure of PCR primer.M swimming lane, DNA marker Marker III；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, uses 8 V to the purification that primer obtains from the PBMC of third time blood drawingHH PCR primer.
Fig. 5. the insertion rate of sdAb phage display library.Use M13R (-48) and M13F
(-47) primer expands 96 library clones randomly selected by PCR.Have ~ clone of 1100 bp DNA bands has VHH insert.
Detailed description of the invention
The present invention now will explain, it should be noted that these experimental examples and accompanying drawing should not be construed as limitation of the present invention in conjunction with following experiment and accompanying drawing further.
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 immune and immune response test, the structure of sdAb phage display library, phage display elutriation and FASEBA screening and FACS checking.
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)
PrimeScriptTM 1st
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 extract, 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 is by immunity five times.Peripheral blood sample is gathered respectively before immunity and after the 4th and the 5th immunity.Gradient separations method isolates lymphocyte.Cell adds RNAlater and is stored in-80 ° of C.The centrifugal blood adding anticoagulant obtains serum, and is stored in-80 ° of C.
3.1.2
Immune response is tested
Blood serum sample is used to be evaluated for fixing immunogenic immune response by ELISA.Serum before have rated immunity and after the 4th and the 5th immunity.Antigen (CD3-His and CD34-Fc) is diluted in being coated in buffer of 4 μ g/ml respectively.Titer plate is coated overnight by the antigen using dilution, 4 ° of C.Subsequently, wash titer plate 3 times with lavation buffer solution, close 2 hours then at 37 ° of C Block buffer.Titer plate is washed 4 times again with lavation buffer solution.The serum of a series of dilutions is added on plate, hatches 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 anti-camel IgG [HRP], hatch 1 hour at 37 ° of C.After washing, reactant adds tmb substrate and reacts 10 minutes, be subsequently added 1 M HCl and terminate 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.Total serum IgE is carried out qualitative and quantitative by gel electrophoresis and OD260/280 method.
3.2.2
RT-PCR and VHH expands
According to PrimeScriptTM 1st
The handbook of Strand cDNA synthetic agent box uses oligo (dT) 20 primer to be cDNA by total serum IgE reverse transcription.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 digests this PCR primer and passes through gel-purified.The fragment of gel-purified is inserted in the phagemid vector (Fig. 1) that GenScript is own.Build experimental displaying storehouse to connect and conversion condition to optimize.The connection and the conversion condition that optimize are used to develop actual displaying storehouse.Sub-fraction converts cell and is diluted and lines on 2 × YT flat board with 100 μ g/ml ampicillin.Bacterium colony is calculated library size.Random picking positive colony also checks order, to assess the quality in library.Remaining converts cell and is scribed on 15 cm 2 × YT flat boards with 100 μ g/ml ampicillin and 2% glucose.Lawn is scraped off from flat board.Fraction cell is used for Library plasmid and 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.Containing 7.4 × 108The big sdAb phage display library of individual clone (CFU) is used for this screening.Library is grown on logarithmic (log) phase, reclaims with M13KO7 helper phage, and expands overnight at 30 ° of C on agitator in 2 × YT culture plate with 100 μ g/ml ampicillin and 50 μ g/ml kanamycin.Make phages with PEG/NaCl, and be resuspended in PBS, be stored in-80 ° of C.For carrying out phage elutriation, microwell plate (Pierce, Prod# 15100) being coated CD3-His, this is by realizing them in 4 ° of C overnight incubation with 100 μ g/ml in PBS (pH 7.0).Meanwhile, phage particle is at room temperature hatched 1 hour to block non-specific binding with the PBS Block buffer containing 2% defatted milk powder.Rinsing after 3 times with PBS, phage particle is being added in micropore, and on the oscillator in incubated at room 1 hour.After hatching, by with PBST(containing the PBS of 0.05% Tween-20) rinsing aperture washes away the unconjugated and phage of non-specific binding for 2 times followed in turn by PBS rinsing 6 times.In conjunction with phage be used to immediately infect exponential phases at 37 ° of CE. coliTG1 cell (OD600 is about 0.5) 1 hour.After each panning rounds, infected cell and 10% glycerol are mixed, is subsequently stored in-80 ° of C.When carrying out the elutriation of next circulation, by the infection of 10 mlE.
coliTG1 cell liquid storage adds in the culture medium that 30 ml contain 200 μ g/ml ampicillin and 2% glucose, and grows to logarithmic (log) phase.Culture reclaims with M13KO7 helper phage, amplification precipitating phage, screens 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 that CD3-His specificity is cloned by Phage-ELISA.Use 2 μ g/ml CD3-His to be coated 96 hole flat boards (in 4 ° of C overnight incubation in being coated buffer), close with the PBS containing 2% defatted milk powder.Every hole adds the phage supernatants from overnight cell culture of about 50 μ l, and hatches 2 hours at 4 ° of C, washs four times with PBST subsequently.Unconjugated sdAb phage detects by hatching 1 hour at 37 ° of C with the anti-M13 monoclonal antibody of HRP labelling.With PBST washing hole four times again, each hole adds substrate solution.Absorption value is measured at 450 nm.
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 the ELISA screening sdAb with separation specific recognition CD3-His albumen, and select the expression culture medium of the affine classification for Octet.Octet RED96 instrument (ForteBio) carries out dissociation rate screening (Off-rate screening).All tests are all carried out at 30 ° of C.SdAb-SASA albumen in expression culture medium is caught into and is coated the biosensor of BSA and hatches together in solution (1x PBS, pH 7.4,0.05% Tween-20) with CD3 albumen.The CD3 of one of which concentration (100 nM) is used for dissociation rate classification.Baseline and dissociation steps are only at buffer (1x
PBS, pH 7.4,0.05% Tween-20) in carry out.Fortebio data processing software is used to measure binding kinetics with dynamics data analytical model by 1:1 Langmuir binding pattern.The conjugate that interacts with high-affinity with CD3 screened go out and carry out DNA sequencing.
3.4.2
FACS verifies
Combine Jurkat cell to filter out and do not combine the sdAb of KPCN cell, culture supernatants is carried out flow cytometry.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.After washing with PBS, 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.Use FACSCalibur (BD
Bioscience, San Jose, CA) and the sdAb combination of Flowjo software analysis cell.
4. result
4.1 immune response tests
Fig. 2 shows serum titer experimental result.After immunity, the potency ratio preimmune serum of first time and second time test sera is 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 different primers that the product obtained is mixed together and carries out gel-purified (Fig. 4).Obtain altogether the about 24 pure V of gHH
PCR primer.General PCR primer is used for library construction.
4.2.3
Library extends
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).Checked order altogether 72 clones, and 64 clones are positioned at frame.Neither one has identical aminoacid sequence (data are not shown), shows that 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 |
| 1<sup>st</sup> | 2.0×10<sup>11</sup> | 3.0×10<sup>5</sup> | ~ 5% |
| 2<sup>nd</sup> | 3.5×10<sup>10</sup> | 9.7×10<sup>7</sup> | ~ 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 screen guide's antibody by ELISA in FASEBA carrier.32 clones are obtained by FASEBA ELISA screening.These 32 clones carry out DNA sequencing, and carry out the combination test with CD3, carry out dissociation rate analysis by Octet RED96 subsequently.In conjunction with the result (4.4.2) of FACS, FortteBio data analysis software is utilized to select the independent clone of low part of dissociating in curve.Table 2 shows the dynamics data of selected sdAb clone.
The dynamics data of table 2. sdAb clone
| Sample ID | Serial number | k<sub>off</sub>(1/s) |
| A30325 | SEQ ID NO. 1 | <1.0E-07 |
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 control 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 | A30325 | NC-sdAb | TG1 |
| Jurkat cell | 443.03 | 115.03 | 132.03 |
| KPCN cell | 96.6 | 51 | 54.5 |
*: the culture supernatants of NC-sdAb (the non-correlation sdAb of same form) is arranged to negative control, and the culture supernatants of TG1 is arranged to ground control.
SEQUENCE
LISTING
<110>
Zhongshan University
<120>
AntiCD3 McAb single domain antibody
<130>
16504CN
<160>
2
<170>
PatentIn version 3.5
<210>
1
<211>
122
<212>
PRT
<213>
Artificial sequence
<400>
1
Gln Val Gln
Leu Val Glu Ser 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 Ser Ser Arg
20
25
30
Ala Val Gly
Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Gln Phe Val
35
40
45
Ala Ala Ile
Asp Ser Gly Gly Gly Glu Thr Gly Tyr Ala Asp Ser Val
50
55
60
Lys Gly Arg
Phe Ala Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65
70
75
80
Leu Gln Met
Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85
90
95
Ala Val Ala
Asp Leu Leu Val Thr Trp Pro Arg Ala Tyr Lys Tyr Trp
100
105
110
Gly Gln Gly
Thr Gln Val Thr Val Ser Ser
115
120
<210>
2
<211>
366
<212>
DNA
<213>
Artificial sequence
<400>
2
caggtacagc
tggtggagtc tgggggggga ttggtgcagg ctgggggctc
tctgagactc 60
tcctgtgcag
cctctggacg caccttcagt agccgtgccg tgggctggtt
ccgccaggct 120
ccagggaagg
agcgtcagtt tgtagcggct attgattcag gtggtggtga gacaggctat
180
gcagactccg
tgaagggccg attcgccatc tcccgagaca acgccaagaa
cacggtgtat 240
ctgcaaatga
acagcctgaa acctgaggac acggccgttt attactgtgc
agtcgcggac 300
cttttggtaa
catggcccag agcgtataag tactggggcc aggggaccca
ggtcaccgtc 360
tcctca
366
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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|---|---|---|---|
| CN201610435670.5A CN106084047A (en) | 2016-06-17 | 2016-06-17 | AntiCD3 McAb single domain antibody |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610435670.5A CN106084047A (en) | 2016-06-17 | 2016-06-17 | AntiCD3 McAb single domain antibody |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106084047A true CN106084047A (en) | 2016-11-09 |
Family
ID=57235551
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
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| CN201610435670.5A Pending CN106084047A (en) | 2016-06-17 | 2016-06-17 | AntiCD3 McAb single domain antibody |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021116699A1 (en) | 2019-12-11 | 2021-06-17 | Precision Immunotherapeutics Limited | Reversibly inhibited binding molecules |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1388136A (en) * | 2001-05-24 | 2003-01-01 | 中国科学院遗传研究所 | Double-specificity antibody resisting human ovary cancer and human CD3 |
| 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 CN201610435670.5A patent/CN106084047A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1388136A (en) * | 2001-05-24 | 2003-01-01 | 中国科学院遗传研究所 | Double-specificity antibody resisting human ovary cancer and human CD3 |
| CN104487587A (en) * | 2012-04-20 | 2015-04-01 | 新兴产品开发西雅图有限公司 | Cd3 binding polypeptides |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021116699A1 (en) | 2019-12-11 | 2021-06-17 | Precision Immunotherapeutics Limited | Reversibly inhibited binding molecules |
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