CN106939048A - A kind of CD3 ε × CD19 bispecific nano antibodies and preparation method thereof - Google Patents
A kind of CD3 ε × CD19 bispecific nano antibodies and preparation method thereof Download PDFInfo
- Publication number
- CN106939048A CN106939048A CN201710185332.5A CN201710185332A CN106939048A CN 106939048 A CN106939048 A CN 106939048A CN 201710185332 A CN201710185332 A CN 201710185332A CN 106939048 A CN106939048 A CN 106939048A
- Authority
- CN
- China
- Prior art keywords
- nano antibody
- bispecific
- antibody
- cell
- vhh
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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
- 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
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
-
- 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®
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Life Sciences & Earth Sciences (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a kind of CD3 ε × CD19 bispecific nano antibodies.The bispecific nano antibody, it is characterised in that：The bispecific nano antibody includes 3 parts, a）A kind of anti-CD19 nano antibody, has specific binding capacity, the tumor cell surface antigen preferably is CD19, CD20, CD30 and CD133 for tumor cell surface antigen, it is highly preferred that being that the tumor cell surface antigen is CD19；b）A kind of AntiCD3 McAb ε nano antibodies, for immunocyte be selected from T cell, NKT cells or CIK cell, it is preferred that there is specific binding capacity to immune cell surface antigenic CD3；c）Small peptide is connected, two kinds of nano antibodies are connected.Present invention also offers a kind of method for preparing bispecific nano antibody and the detection method of bispecific nano antibody.Purposes of the bispecific nano antibody in medicine is prepared, described medicine is used to treat the specific expressed caused tumours of CD19, or specific killing expresses CD19 cell.
Description
Technical field
The present invention relates to immunological technique field, the preparation method of bispecific nano antibody a kind of is specifically related to and double
Specific nano antibody.
Background technology
Bispecific antibody is the artificial antibody containing two species-specific antigen binding sites, can be in target cell and function point
Bridge is erected between sub (cell), the immune response with guidance quality is excited, is one kind of genetic engineering antibody, as anti-
The focus of body engineering field, has broad application prospects in the immunization therapy of tumour.It is characterized in bispecific antibody energy
Enough in combination with the target molecule on tumor associated antigen and immune effector cell, immune effector cell is directly triggered to tumour cell
Specific killing.
Nano antibody is the variable region fragment for the heavy chain antibody for being present in the natural deletions light chain inside hunchbacked class peripheral blood.
Be be currently known can combine antigen minimal structure unit.Nano antibody has that immunogenicity is low, molecular weight is small, soluble
High advantage.The country is not directed to the clinical treatment data of the bispecific nano antibody medicine of oncotherapy also at present.
Simple introduction is done below for the immune effector cell antigen and tumor-cell antigen studied, and introduces the related back of the body
The development of scape technology.
1.CD3
In immunology, CD3 molecules are made up of four different subunits.It is γ, δ, ε, ζ respectively.It is thin that CD3 exists only in T
Cellular surface, is made up of 6 peptide chains, often combines closely to form the TCR-CD3 complexs containing 8 peptide chains with TCR., specific signal
Figure is shown in figure one.CD3 kytoplasms section (immunoreceptor tyrosine-based containing ITAM
Activation motif, ITAM), TCR is recognized and combined by MHC (major histo-compatibility complex)
The Antigenic Peptide that molecule is offered, causes the CD3 ITAM tyrosine residue of conserved sequence by the LCK in T cell
P56lck phosphorylations, then can raise LCK that other contain SH2 (Scr homology 2) domain (such as
ZAP-70).ITAM phosphorylation and and ZAP-70 combination be T cell activation signal transduction process early stage important biochemistry
One of reaction.Therefore, the function of CD3 molecules is the activation signals produced by transduction TCR identification antigens.CD3 ε or γ chain gene lack
T cell signal transduction defect can be caused by falling into.
2.CD19
CD19 is expressed on B systems cell (not including mature plasme cell) and dendritic cells,follicular, and CD19 is a kind of important
Signal transduction molecule, growth activation and the activation of bone-marrow-derived lymphocyte are adjusted, in regulation bone-marrow-derived lymphocyte antigen receptor or other tables
Played an important role in the signal threshold value of face acceptor, be break up with bone-marrow-derived lymphocyte, activation, propagation and relevant important of antibody generation
Membranous antigen, is the preferably mark for diagnosing bone-marrow-derived lymphocyte system's tumour and identification bone-marrow-derived lymphocyte.
3. bispecific antibody preparation method
Preparing bispecific antibody has three kinds of methods such as chemical coupling method, hybridoma method and gene engineering method.It is chemical even
Connection method can quickly, largely prepare bispecific antibody, but this bispecific antibody is easily eliminated when being used for internal, influences it to apply；
Bispecific antibody bioactivity prepared by hybridoma method is high, but to be taken when preparing by cumbersome steps such as fusion, screenings
Long, gained bispecific antibody is difficult to separate with the other monoclonal antibodies produced simultaneously；Bispecific antibody point prepared by gene engineering method
Son amount is small, is easy to a large amount of preparations, has broad application prospects.
The content of the invention
Term and abbreviation：
VHH：Weight chain variable district
RNA：Ribonucleotide
cDNA：Complementary DNA (cDNA)
PCR：Polymerase chain reaction
Nano BiTE:Bispecific nano antibody
Tumor cell:Tumour cell
T cell：T cell
PBMC：PMNC
The invention provides a kind of bispecific nano antibody, for conventional monoclonal antibody or monoclonal nano antibody
Shortcoming and artificial reconstructed polyclonal antibody the bispecific nano antibody researched and developed of shortcoming, described bispecific
Nano antibody has stability high, soluble good, the characteristics of antigen binding power is strong.The bispecific nano antibody is special by mediating
Specific T cell lethal effect, substantially increases killing effect of tumour.Specifically, the invention provides following technical scheme：
In one embodiment, a kind of bispecific nano antibody, it is characterised in that：The bispecific nano antibody
Comprising 3 parts, a) a kind of anti-CD19 nano antibody, has specific binding capacity, preferably for tumor cell surface antigen
The tumor cell surface antigen be CD19, CD20, CD30 and CD133, it is highly preferred that being that the tumor cell surface antigen is
CD19；B) a kind of AntiCD3 McAb ε nano antibodies, for immunocyte be selected from T cell, NKT cells or CIK cell, it is preferred that
There is specific binding capacity to immune cell surface antigenic CD3；C) small peptide is connected, two kinds of nano antibodies are connected.
In one embodiment, the bispecific nano antibody is derived from hunchbacked class body.
In one embodiment, the bispecific nano antibody is the antibody of anti-human source antigen.
In one embodiment, the connection short peptide sequence of described bispecific nano antibody is
GGGGSGGGGSGGGGS。
In one embodiment, the amino acid sequence of described anti-CD19 nano antibody is the sequence shown in sequence number 1
Row.
In one embodiment, the amino acid sequence of the nano antibody of described AntiCD3 McAb is the sequence shown in sequence number 2
Row.
In one embodiment, the preparation method of described bispecific nano antibody comprises the following steps：
1) process of the separation of alpaca PMNC (PBMC)
2) process of fluidic cell sorting antibody positive clone
3) positive cell RNA, process of the reverse transcription into cDNA are extracted
4) amplification procedure of anti-human CD3 ε and CD19 nano antibody sequence
5) the structure qualification process of CD3 ε VHH-CD19VHH bispecific antibody prokaryotic expression carriers
6) pet32a-CD3 ε VHH-CD19VHH prokaryotic expression and qualification process
In one embodiment, in the preparation method of described bispecific nano antibody, connection carrier used is
pet32a(+)。
In one embodiment, in the preparation method of described bispecific nano antibody, two nano antibodies are connected
It is overlapping PCR method to pick up the method come.
In one embodiment, described bispecific nano antibody or bispecific is prepared according to claim 7
The purposes of bispecific nano antibody prepared by the method for nano antibody in medicine is prepared, described medicine is used to treat CD19
Specific expressed caused tumour, or specific killing express CD19 cell.
The beneficial effect of technical scheme has：
1 the invention provides a kind of bispecific nano antibody and preparation method, anti-relative to existing bispecific
For body, its molecular weight is small, soluble good, high expressivity and high antigenic binding property.
2 present invention increase the method for flux screening to prepare bispecific nano antibody with reference to fluidic cell sorting technology,
Fabrication cycle is short, and antibody yield is high.
Brief description of the drawings
Fig. 1 .CD3 schematic arrangements.
Fig. 2 bispecific nano antibody mechanism of action schematic diagrames.
Fig. 3 first step CD3 ε fragments are obtained
1. negative control；2.CD3 ε first steps PCR is expanded；3.DL2000marker
Fig. 4 first step CD19 fragments are obtained
4. negative control；5,6.CD19 first step PCR are expanded；7.DL2000marker
Fig. 5 second step CD3 ε and CD19 fragment are obtained
7. blank control；8.DL2000Marker；9.CD3 ε second steps PCR is expanded；10.CD19 second steps PCR is expanded.
Your VHH of Fig. 6 .CD3 ε VHH Yu CD19 agarose gel electrophoresis
11.CD3 ε-VHH and CD19-VHH connection product 12.DL2000 DNA Marker13. negative controls
The structure of Fig. 7 .pET32a-CD3 ε VHH-CD19VHH recombinant vectors
The 14.DL2000DNAMarker 15.pET32a-CD3 ε VHH-CD19VHH negative control of pcr amplification product 16.
The acquisition of Fig. 8 .pET32a-CD3 ε VHH-CD19VHH recombinant proteins
17. the bacterium solution of molecular weight of albumen Marker 18.pET32a-CD3 ε VHH-CD19VHH induced expressions 19. is compareed
The combination tendency chart of Fig. 9 .CD3 3-3 yupsilon proteins and bispecific nano antibody
The combination tendency chart of Figure 10 .CD19 albumen and bispecific nano antibody
Embodiment
The process of the separation of the alpaca PMNC (PBMC) of embodiment 1.
Whole experiment process sterile working：
1. being immunized with CD3 ε and CD19 after alpaca, taken a blood sample using 15mL centrifuge tube, blood is taken back behind laboratory
Directly centrifuge tube is put into centrifuge and centrifuged, whole process room temperature.Centrifugal condition, blood are determined according to blood sample amount
Sample size is more, and centrifugal force is bigger, and centrifugation time is longer.
2. drawing upper serum, the μ L of serum 800 often pipe packing marks are stored in -80 DEG C of refrigerators standby.
3. the blood drawn after supernatant is drawn onto in 50mL centrifuge tube, original is cleaned with the PBS of 2-3mL sterilizing
15mL centrifuge tubes, are drawn onto 50mL centrifuge tubes.Add 5mL PBS dilutions.A sterile 50mL centrifuge tube separately is taken, is poured into
15mL Ficoll separating liquids, the blood diluted is slowly and steadily laid in Ficoll separating liquids, centrifugation.
4. now from top to bottom it is divided into four layers in centrifuge tube after centrifugation.First layer is plasma layer, and the second layer is that ring-type is milky white
Color mononuclearcell layer, third layer is separation liquid layer, and the 4th layer is red blood cell layer.
5. the plasma layer that upper strata is carefully drawn with suction nozzle is discarded, (because serum has been separated, plasma layer liquid is not
It is many), intermediate second layer ring-type milky mononuclearcell layer is collected into a 50mL centrifuge tube, adds PBS to arrive in centrifuge tube
45mL, is mixed, and is centrifuged 10 minutes.
6. quickly outwelling PBS in super-clean bench, centrifugation bottom of the tube PBMC is gently rung, PBS is added to 45mL, mixed
It is even, centrifuge 10 minutes.7. adding 2mL cells frozen storing liquid, the cells frozen storing liquid for taking 10 μ L to mix cell adds 10 μ L trypan blues and mixed
It is even to be counted under the microscope with cell counting count board.PBMC concentration is adjusted with cells frozen storing liquid, it is 2 × 10 to preserve concentration6/mL。
8. the PBMC for mixing up concentration is divided in cell cryopreservation tube, it is put in slow interior -80 DEG C of the box that freezes and stays overnight, be put within second day
Preserved in liquid nitrogen.
9. the acquisition of negative control
Purchase alpaca is simultaneously marked, blood 30mL is taken in immune foreneck arteries and veins, is put into preprepared added with anti-coagulants
In 50mL centrifuge tubes.(anti-coagulants consumption 20-50U/mL) separating peripheral blood mononuclear cells (PBMC), Liquid nitrogen storage is standby.
The process of the fluidic cell of embodiment 2. sorting antibody positive clone
Required raw material is CD19-Biotin storing solutions for the CD19 albumen of biotin labeling, and molecular weight is 56.3KDa,
Molecular weight is 56-66KDa after the display glycosylation of SDS-PAGE results, and every million cells add algae red egg in 100 μ L systems
In vain-Streptavidin is that PE-Streptavidin amount is 0.015 μ g, and negative control resists not mark any fluorescein coupling
Cell after body CD19 is immune, blank control is non-immune natural alpaca PBMC.
Embodiment 3. extracts positive cell RNA, process of the reverse transcription into cDNA
Reagent used in whole experiment process, pipette tips, the equal deoxyribonuclease of centrifuge tube.
1. obtaining 50000 positive cells after first time airflow classification, collection liquid is transferred to from collecting pipe after sorting
In 1.5mL centrifuge tube, 12000r/ minutes, centrifugation abandoned supernatant in 5 minutes.
2. adding 1mL TRIZOL liquid in the positive cell into step 1, blown and beaten back and forth with rifle makes cell fully split several times
Solution.Plus 200 violent concussion 30S after μ L chloroforms, 5 minutes are stood, 12000r/ minute, is centrifuged 10 minutes, layering, absorption upper liquid
Body phase adds isometric isopropanol into another 1.5mL pipes, mixes, and -20 DEG C precipitate 20 minutes.
3 are transferred to precipitating liquid in RNA extraction columns, stand 3 minutes, are washed twice with 700 μ L bufferW2, then with 60
μ L deoxyribonuclease water elutions, obtain positive cell RNA.
4 RNA reverse transcriptions are into cDNA
0.2mL PCR pipe is taken, the μ L of 81 μ L, RNase-free water of μ L, OligodT of template ribonucleic acid 1.45 is added, mixes
Even rear brief centrifugation, is put into PCR instrument, and 65 DEG C are incubated 5 minutes, sample then are taken out into ice bath 3 minutes, brief centrifugation adds 5
× Reaction buffer 4 μ L, RNase inhibitor (20U/ μ L) 0.55 μ L, 10mM dNTP 4 μ L, Revert-M-M μ
The μ L of LvRT 1, are put into PCR instrument, and 42 DEG C are reacted 60 minutes.After the completion of reaction, obtained cDNA is transferred to sterilized
In 1.5mLEP pipes, -80 DEG C save backup
The anti-human CD3 ε of embodiment 4. and CD19 nano antibody sequences amplification
The anti-human CD3 ε of alpaca and CD19 nano antibodies are obtained by two-step pcr amplification
1. first step PCR primer is VHH-FP-F (CAGGTGAAGGTCATCGARTC), VHH-FP-R
(GATGCTCTTGTGACTCAGGAATC).First step PCR amplifications are carried out by the reaction system in table 1, response procedures are pre-degeneration
94 DEG C, 4 minutes；94 DEG C of denaturation, 30s；53 DEG C of annealing, 30s；72 DEG C of extension, 40s；Extend 72 DEG C, 10 points after the completion of reaction cycle
Clock；4 DEG C, ∞, totally 30 circulation.
Table 1：First step PCR amplifications obtain that alpaca is single-stranded and double-chain antibody system
2. second step PCR sense primer is Flag-VHH-F (GATTATAAAGATGATGATGACAAGCGCAGAGACAG
TGACCAGAGT), anti-sense primer is VHH-07R (TTAATCCAGCTGACTCAGCC), VHH-08R
(TTAATTGTTCTCWCCCAGTC).Second step PCR amplifications are carried out by the reaction system in table 2.Response procedures are pre-degeneration 94
DEG C, 4 minutes；94 DEG C of denaturation, 30s；53 DEG C of annealing, 30s；72 DEG C of extension, 40s；Extend 72 DEG C, 10 points after the completion of reaction cycle
Clock；4 DEG C, ∞, totally 30 circulation.
The second step PCR of table 2 amplifications obtain alpaca single-chain antibody reaction system
The structure qualification process of embodiment 5.CD3 ε-VHH-CD19-VHH bispecific antibody prokaryotic expression carriers
1.CD3 ε-VHH and CD19-VHH connection
Using GGGGSGGGGSGGGGS as connection peptide, CD3 ε-VHH and CD19-VHH sequences are connected using Overlap PCR methods
Row.As shown in table 3, second step PCR primer carries out substep PCR, reaction to Overlap PCR primers sequence as template using in step 5
System such as table 4, shown in table 5, response procedures are pre-degeneration 94 DEG C, 4 minutes；94 DEG C of denaturation, 30s；58 DEG C of annealing, 30s；Extension 72
DEG C, 1 minute；Extend 72 DEG C, 10 minutes after the completion of reaction cycle；4 DEG C, ∞, totally 30 circulation.By CD3 ε-VHH and CD19-VHH
PCR primer is entered after row agarose gel electrophoresis and glue reclaim, and Overlap PCR, the reaction system such as institute of table 6 are carried out as template
Show, response procedures are pre-degeneration 94 DEG C, 4 minutes；94 DEG C of denaturation, 30s；58 DEG C of annealing, 30s；72 DEG C, 2 minutes of extension；Reaction is followed
Extend 72 DEG C, 10 minutes after the completion of ring；4 DEG C, ∞, totally 30 circulation.Product after Overlap PCR is subjected to Ago-Gel electricity
Swim and glue reclaim, preserve and carry out next step experiment.
The Overlap PCR primers title of table 3 and sequence
The PCR amplification system of the CD3 ε-VHH sequences of table 4
The PCR amplification system of the CD19-VHH sequences of table 5
The Overlap PCR of table 6 reaction system
2.CD3 ε-VHH and CD19-VHH connection product are attached with pet32a (+)
The above-mentioned Overlap PCR CD3 ε VHH-CD19VHH connection products obtained and pet32a (+) are used into restriction enzyme
Enzyme EcoR I and Sal I carries out double digestion, and digestion system is as shown in table 7, table 8.After glue reclaim, connected with T4DNA ligases
Pet32a (+) and CD3 ε VHH-CD19VHH, linked system is as shown in table 9.After connection, by connection product conversion such as BL21 (DE3)
In competent cell, choose bacterium, shake bacterium enter performing PCR identification, as a result as shown in the figure.300 μ L bacterium solutions are taken to send sequencing company to be sequenced,
As a result show, CD3 ε-VHH-CD19-VHH sequences are not undergone mutation, lacked, and insertion vector is in the right direction, shows H-CD3
The success of ε-VHH-CD19-VHH bispecific antibodies Prokaryotic expression vector construction.
The H-CD3 ε VHH-CD19VHH digestion systems of table 7
Table 8 pet32a (+) digestion system
The pet32a (+) of table 9 and CD3 ε VHH-CD19VHH linked system
3. the PCR identifications of recombinant prokaryotic expression vector
The pET32a-CD3 ε VHH-CD19VHH prokaryotic expression carriers of restructuring are utilized into T7
(TAATACGACTCACTATAGGG) enter performing PCR with T7ter (TGCTAGTTATTGCTCAGCGG) primer and expand, reaction system 20
μ L (are shown in Table 10), and PCR response procedures are 95 DEG C, 5 minutes；95 DEG C, 30s, 58 DEG C, 30s, 72 DEG C, 45s (30 circulations)；72 DEG C,
8 minutes；4 DEG C, ∞.
Product after PCR is expanded carries out DNA electrophoresis experiments, as a result as shown in the figure.Stripe size and expection are in the same size,
Sample after identification is sequenced.Sequencing result shows that the non-producer of aim sequence is mutated, missing, sequence direction of insertion
Correctly, pET32a-CD3 ε VHH-CD19VHH recombinant prokaryotic expression vectors are successfully constructed.
The PCR identification systems of the pet32a-CD3 ε VHH-CD19VHH prokaryotic expression carriers of table 10
Embodiment 6.pet32a-CD3 ε VHH-CD19VHH prokaryotic expression and identification
1.pet32a-CD3 ε VHH-CD19VHH prokaryotic expression
Take the pet32a-CD3 ε VHH-CD19VHH bacterium solutions 2mL successfully constructed in 200mL 2YT culture mediums, 37 DEG C are shaken
Bacterium is to bacterium solution OD600nmFor 0.55~0.65 when, add IPTG, final concentration of 0.5 μM, be put into 37 DEG C shake bacterium induction 3h.Take out few
Amount bacterium solution, is centrifuged and adds appropriate PBS, and piping and druming is mixed, 20 μ L of taking-up, addition 20 μ L2 × sample-loading buffer, boiling water bath 3 minutes,
SDS-PAGE experiment detection expression is carried out, as a result as schemed.
The Elisa identifications of 2.CD3 ε antigens and CD19 antigens
CD3 ε antigens and CD19 antigens are diluted to 2ng/ μ L concentration with coating buffer (PH9.6), and enzyme is added to by every μ L of hole 100
In target, 4 DEG C of refrigerator overnights are positioned over, ELISA experiments are carried out within second day, ELISA steps are as follows：
Step 1, by every μ L of hole 200 PBS-T add coating after ELISA Plate cleaned, cleaning 5 times, each 3min,
Cleaning fluid is got rid of, ensures that washing lotion is all blotted in clappers on paper handkerchief；
Step 2, the skimmed milk power that 5% is added by every μ L of hole 200 into ELISA Plate, room temperature closing 1h；
Step 3, cleaned by cleaning step in step 1, then, successively by 100 μ L blank control liquid, negative right
It is added to according to, CD3e antibody after purification in ELISA Plate, is incubated at room temperature 2h；
Step 4, cleaned by cleaning step in step 1, then, HRP marks are added to ELISA Plate by every μ L of hole 100
Tag antibody, is incubated at room temperature 1h；
Step 5 is cleaned by cleaning step in step 1, and 100 μ L TMB, avoid light place are then added to every hole
10min；
Step 6, add to every hole 50 μ L 2mol/L sulfuric acid solution, ELISA Plate is put in ELIASA by terminating reaction,
The reading under the conditions of 450nm.
As a result it is as follows：
As a result show：When with ELISA method detect CD3 ε-CD19 bispecific nano antibody combination CD3 ε and CD19 eggs
In vain, Percentage bound is higher, is positive findings.Illustrate that the antigen binding rule of antibody is high.Specific linear graph is shown in Fig. 9 and Figure 10.
Finally it should be noted that：Obviously, above-described embodiment is only intended to clearly illustrate the application example, is excellent
The embodiment of choosing.And the not restriction to embodiment.For those of ordinary skill in the field, in described above
On the basis of can also make other changes in different forms.There is no need and unable to give thoroughly all embodiments
Lift.And among the obvious change or variation thus amplified out are still in the protection domain of the application type.
SEQUENCE LISTING
<110>Kang Zhong（Beijing）Bio tech ltd
<120>A kind of CD3 ε × CD19 bispecific nano antibodies and preparation method thereof
<130> 2017
<160> 16
<170> PatentIn version 3.3
<210> 1
<211> 131
<212> PRT
<213> human
<400> 1
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp
1 5 10 15
Ser Val Thr Leu Ser Cys Thr Ala Ser Gly Arg Thr Phe Ser Ser Tyr
20 25 30
Ala Leu Ala Trp Phe Arg Gln Arg Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Leu Arg Trp Gly Gly Pro Thr Asn Tyr Ala Asp Ser Val Lys
50 55 60
Asp Arg Phe Thr Ile Ser Gly Asp Ser Ala Lys His Thr Met Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Gly
85 90 95
Tyr Asn Pro Gly Gly Trp Ala Val Pro Ser Gln Tyr Glu Tyr Asp Ser
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala His His Ser Glu
115 120 125
Asp Pro Ser
130
<210> 2
<211> 137
<212> PRT
<213> human
<400> 2
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Pro Ser Cys Thr Ala Ser Gly Phe Ser Leu Asp Arg Leu
20 25 30
Gln Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Ala Val
35 40 45
Leu Cys Leu Arg Gly Thr Asp Asp Asp Ala Thr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Leu Ile Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Tyr Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Glu Pro Gly Ala Ala Arg Asp Thr Ala Gln Arg Met Cys Leu
100 105 110
Asp Thr Asp Phe Thr Ala Trp Gly Arg Gly Ala Arg Val Thr Val Ser
115 120 125
Ser Glu Pro Lys Thr Pro Lys Pro Gln
130 135
<210> 3
<211> 396
<212> DNA
<213> HUMAN
<400> 3
caggtgcagc tggtggagtc tgggggaggg ttggtgcagg ctggggactc tgtgacactc 60
tcctgtacag cctctggacg caccttcagt agctatgcct tggcctggtt tcgccaacgt 120
ccaggaaagg agcgtgagtt cgtcgcaggt ttgaggtggg gtggtcccac aaactacgca 180
gactccgtga aggaccgatt caccatctcc ggagacagcg ccaagcacac aatgtatctg 240
caaatgaaca gcttgaaacc tgaggacacg gccgtttatt actgtggata taatccgggg 300
ggttgggcgg tgccctccca atatgagtat gactcgtggg gccaggggac ccaggtcacc 360
gtctcctcag cgcaccacag cgaagacccc agctaa 396
<210> 4
<211> 414
<212> DNA
<213> HUMAN
<400> 4
caggtgcagc tggtggagtc tgggggaggc ttggtgcaac ctggggggtc tctgagaccc 60
tcctgtacag ccagtggatt ctctttggat cgacttcaag taggctggtt ccgccaggcc 120
ccagggaagg aacgtgaggc ggtcttatgt cttcggggta ctgatgatga cgctacgtat 180
gcagactctg tgaagggccg attcaccatc tccagggacc tcattaagaa cacggtgtat 240
ctgcaaatga actacctgaa acctgacgac acagccgttt attattgtgc agcggaaccc 300
ggggcggcga gggatactgc tcagcgtatg tgcctcgaca ctgactttac cgcctggggc 360
cggggggccc gggtcaccgt ctcctcggaa cccaagacac caaaaccaca ataa 414
<210> 5
<211> 20
<212> DNA
<213>Artificial sequence
<400> 5
caggtgaagg tcatcgartc 20
<210> 6
<211> 23
<212> DNA
<213>Artificial sequence
<400> 6
gatgctcttg tgactcagga atc 23
<210> 7
<211> 45
<212> DNA
<213>Artificial sequence
<400> 7
gattataaag atgatgatga caagcgcaga gacagtgacc agagt 45
<210> 8
<211> 20
<212> DNA
<213>Artificial sequence
<400> 8
ttaatccagc tgactcagcc 20
<210> 9
<211> 20
<212> DNA
<213>Artificial sequence
<400> 9
ttaattgttc tcwcccagtc 20
<210> 10
<211> 15
<212> DNA
<213>Artificial sequence
<400> 10
ggggsggggs ggggs 15
<210> 11
<211> 33
<212> DNA
<213>Artificial sequence
<400> 11
ccggaattcg attataaaga tgatgatgac aag 33
<210> 12
<211> 62
<212> DNA
<213>Artificial sequence
<400> 12
ggagcctcca ccaccagaac ctcctccgcc tgacccaccg cctccattgt tctcacccag 60
tc 62
<210> 13
<211> 66
<212> DNA
<213>Artificial sequence
<400> 13
ggaggcggtg ggtcaggcgg aggaggttct ggtggtggag gctcccgcag agacagtgac 60
cagagt 66
<210> 14
<211> 29
<212> DNA
<213>Artificial sequence
<400> 14
cgcgtcgact taatccagct gactcagcc 29
<210> 15
<211> 20
<212> DNA
<213>Artificial sequence
<400> 15
taatacgact cactataggg 20
<210> 16
<211> 20
<212> DNA
<213>Artificial sequence
<400> 16
tgctagttat tgctcagcgg 20
Claims (10)
1. a kind of bispecific nano antibody, it is characterised in that：The bispecific nano antibody includes 3 parts, a）One kind is anti-
CD19 nano antibody, has specific binding capacity for tumor cell surface antigen, and the tumor cell surface preferably resists
Original is CD19, CD20, CD30 and CD133, it is highly preferred that being that the tumor cell surface antigen is CD19；b）A kind of AntiCD3 McAb ε receives
Meter Kang Ti, for immunocyte be selected from T cell, NKT cells or CIK cell, it is preferred that to immune cell surface antigenic
CD3 has specific binding capacity；c）Small peptide is connected, two kinds of nano antibodies are connected.
2. bispecific nano antibody according to claim 1, it is characterised in that：Described bispecific nano antibody takes
From in hunchbacked class body.
3. bispecific nano antibody according to claim 1, it is characterised in that：The bispecific nano antibody is people
The antibody of source antigen.
4. bispecific nano antibody according to claim 1, it is characterised in that：It is described connection small peptide sequence be
GGGGSGGGGSGGGGS。
5. bispecific nano antibody according to claim 1, it is characterised in that：Described anti-CD19 nano antibody
Amino acid sequence is the sequence shown in sequence number 1.
6. bispecific nano antibody according to claim 1, it is characterised in that：The nano antibody of described AntiCD3 McAb
Amino acid sequence is the sequence shown in sequence number 2.
7. prepare the method for the bispecific nano antibody any one of claim 1-6：Characterized in that, described
Method comprises the following steps：
Alpaca PMNC（PBMC）Separation process
The process of fluidic cell sorting antibody positive clone
Extract positive cell RNA, process of the reverse transcription into cDNA
The amplification procedure of anti-human CD3 ε and CD19 nano antibody sequence
The structure qualification process of CD3 ε-VHH-CD19-VHH bispecific antibody prokaryotic expression carriers
Pet32a-CD3 ε VHH-CD19VHH prokaryotic expression and qualification process
The preparation method of bispecific nano antibody according to claim 7, it is characterised in that：Described step 5）With 6）
Carrier is pet32a (+).
8. the preparation method of bispecific nano antibody according to claim 7, it is characterised in that：Anti-human CD3 ε and CD19
The connection method of nano antibody is the method for over-lap PCR.
9. the nano antibody according to any one in claim 1-6 or the preparation according to any one of claim 7-9
The purposes of nano antibody prepared by the method for bispecific nano antibody in medicine is prepared, described medicine is used to treat CD19
Specific expressed caused tumour, or specific killing express CD19 cell.
10. the nano antibody according to any one in claim 1-6 or the preparation according to any one of claim 8-9
The purposes of nano antibody prepared by the method for bispecific nano antibody in medicine is prepared, described medicine is used to treat CD19
Specific expressed caused tumour, or specific killing express CD19 cell.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710185332.5A CN106939048A (en) | 2017-03-25 | 2017-03-25 | A kind of CD3 ε × CD19 bispecific nano antibodies and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710185332.5A CN106939048A (en) | 2017-03-25 | 2017-03-25 | A kind of CD3 ε × CD19 bispecific nano antibodies and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106939048A true CN106939048A (en) | 2017-07-11 |
Family
ID=59464208
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710185332.5A Pending CN106939048A (en) | 2017-03-25 | 2017-03-25 | A kind of CD3 ε × CD19 bispecific nano antibodies and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106939048A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110218254A (en) * | 2018-03-02 | 2019-09-10 | 广西医科大学 | Nano antibody CD3/Nb25 of AntiCD3 McAb and the preparation method and application thereof |
| CN110218255A (en) * | 2018-03-02 | 2019-09-10 | 广西医科大学 | Nano antibody CD3/Nb47 of AntiCD3 McAb and the preparation method and application thereof |
| CN110396128A (en) * | 2019-03-21 | 2019-11-01 | 南京东极医药科技有限公司 | The preparation of anti-CD19 nano antibody |
| WO2023221618A1 (en) * | 2022-05-18 | 2023-11-23 | 上海百英生物科技股份有限公司 | Anti-cd3 nano-antibody or antigen-binding part thereof and preparation method therefor |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103951754A (en) * | 2014-03-26 | 2014-07-30 | 中国药科大学 | Anti-tumor bispecific miniaturized antibody with double functions of targeting therapy and detection |
| WO2015095412A1 (en) * | 2013-12-19 | 2015-06-25 | Zhong Wang | Bispecific antibody with two single-domain antigen-binding fragments |
| CN106084046A (en) * | 2016-06-17 | 2016-11-09 | 中山大学 | AntiCD3 McAb single domain antibody |
| CN106188305A (en) * | 2015-06-01 | 2016-12-07 | 中山大学 | There is the bivalent antibody of the single domain Fab being fused to conventional Fab fragment |
| CN106282214A (en) * | 2016-08-03 | 2017-01-04 | 康众（北京）生物科技有限公司 | A kind of method of quick acquisition nano antibody and application thereof |
-
2017
- 2017-03-25 CN CN201710185332.5A patent/CN106939048A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015095412A1 (en) * | 2013-12-19 | 2015-06-25 | Zhong Wang | Bispecific antibody with two single-domain antigen-binding fragments |
| CN103951754A (en) * | 2014-03-26 | 2014-07-30 | 中国药科大学 | Anti-tumor bispecific miniaturized antibody with double functions of targeting therapy and detection |
| CN106188305A (en) * | 2015-06-01 | 2016-12-07 | 中山大学 | There is the bivalent antibody of the single domain Fab being fused to conventional Fab fragment |
| CN106084046A (en) * | 2016-06-17 | 2016-11-09 | 中山大学 | AntiCD3 McAb single domain antibody |
| CN106282214A (en) * | 2016-08-03 | 2017-01-04 | 康众（北京）生物科技有限公司 | A kind of method of quick acquisition nano antibody and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| KATJA ELS CONRATH: ""Camel Single-domain Antibodies as Modular Building Units in", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
| 李崴: "抗CD3/抗CD19微型双功能抗体的", 《中国癌症杂志》 * |
| 杨雨琪: "二硫键稳定的抗CD3 /抗CD19 微型双功能抗体的表达及活性测定", 《药物生物技术》 * |
| 郭婷婷: "双特异性抗体药物的研究进展", 《中国新药杂志》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110218254A (en) * | 2018-03-02 | 2019-09-10 | 广西医科大学 | Nano antibody CD3/Nb25 of AntiCD3 McAb and the preparation method and application thereof |
| CN110218255A (en) * | 2018-03-02 | 2019-09-10 | 广西医科大学 | Nano antibody CD3/Nb47 of AntiCD3 McAb and the preparation method and application thereof |
| CN110218255B (en) * | 2018-03-02 | 2020-12-04 | 广西医科大学 | CD 3-resistant nano antibody CD3/Nb47 and preparation method and application thereof |
| CN110218254B (en) * | 2018-03-02 | 2020-12-04 | 广西医科大学 | CD 3-resistant nano antibody CD3/Nb25 and preparation method and application thereof |
| CN110396128A (en) * | 2019-03-21 | 2019-11-01 | 南京东极医药科技有限公司 | The preparation of anti-CD19 nano antibody |
| CN110396128B (en) * | 2019-03-21 | 2022-09-06 | 南京东极医药科技有限公司 | Preparation of anti-CD 19 nano antibody |
| WO2023221618A1 (en) * | 2022-05-18 | 2023-11-23 | 上海百英生物科技股份有限公司 | Anti-cd3 nano-antibody or antigen-binding part thereof and preparation method therefor |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR101491835B1 (en) | A hybridoma cell line 10G4 and a monoclonal antibody against the total of aflatoxin B1, B2, G1 and G2 | |
| CN106939048A (en) | A kind of CD3 ε × CD19 bispecific nano antibodies and preparation method thereof | |
| CN102245767B (en) | Immunoassay method for human CXCL1 protein | |
| CN109734813A (en) | A kind of Chimeric antigen receptor and its application | |
| CN109406771A (en) | Cystatin C latex-enhanced turbidimetry detection kit and its preparation application method | |
| CN113151186B (en) | Monoclonal antibody of anti-human CD271 and application | |
| CN113265003B (en) | anti-TdT protein monoclonal antibody, cell strain thereof, preparation method and application | |
| CN108486064A (en) | The monoclonal antibody and application of hybridoma cell strain Anti-CLasMcAb1 and its secretion | |
| CN111848779A (en) | Chicken complement receptor 2(ChCR2) monoclonal antibody and application thereof | |
| CN110818778B (en) | Antigen, monoclonal antibody, polyclonal antibody and method for preparing Listeria monocytogenes monoclonal antibody | |
| CN113061186A (en) | Monoclonal antibody of anti CA125 protein, cell strain, preparation method and application thereof | |
| CN108659125B (en) | Monoclonal antibody of anti-helicobacter pylori protein, cell strain, preparation method and application thereof | |
| RU2008152009A (en) | MONOCLONAL ANTIBODIES AGAINST ANNEXIN A3 FOR DETECTION OF PROSTATE CARCINOMA | |
| CN113583132A (en) | anti-PR protein monoclonal antibody and preparation method and application thereof | |
| CN106244559B (en) | A kind of hybridoma cell strain that secreting anti-human IL-37b monoclonal antibody, anti-human IL-37b monoclonal antibody and its application | |
| CN110272494A (en) | The application of TIM-3 antigen, nano antibody and its screening and identification method, nano antibody | |
| RU2420588C2 (en) | MURINE MONOCLONAL ANTIBODIES BOUND WITH ANTIGEN F1 OF Yersinia pestis, METHOD FOR PRODUCING THEREOF WITH USING YEAST, METHOD AND KIT FOR Yersinia pestis DETECTION | |
| CN110511277A (en) | A kind of anti-HSP90 monoclonal antibody and its application | |
| CN113234158B (en) | anti-TIM 3 protein monoclonal antibody, cell strain thereof, preparation method and application | |
| CN113845592B (en) | anti-CK 5/6 protein monoclonal antibody, cell strain thereof, preparation method and application | |
| CN109503711A (en) | A kind of dual-functional nanometer antibody, encoding gene and its application for blood clotting method detection PCV2 virus | |
| CN103848888B (en) | A kind of people's C1Q/TNF α GAP-associated protein GAP 2（hCTRP2）Antigenic Peptide and its antibody | |
| CN105646713A (en) | Monoclonal antibody and application thereof | |
| CN108623683A (en) | The monoclonal antibody and its cell strain, preparation method and application of anti-Pax-5 albumen | |
| CN103214573A (en) | Anti human LOC339524 protein monoclonal antibody and a hybridomas cell strain and the purpose thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20170711 |
|
| WD01 | Invention patent application deemed withdrawn after publication |