CN109678961A - A kind of construction method of the Chimeric antigen receptor based on BMCA nano antibody sequence and its application - Google Patents
A kind of construction method of the Chimeric antigen receptor based on BMCA nano antibody sequence and its application Download PDFInfo
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- CN109678961A CN109678961A CN201910036367.1A CN201910036367A CN109678961A CN 109678961 A CN109678961 A CN 109678961A CN 201910036367 A CN201910036367 A CN 201910036367A CN 109678961 A CN109678961 A CN 109678961A
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- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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Abstract
The present invention relates to cellular immunity technical fields, and disclose construction method and its application of a kind of Chimeric antigen receptor based on BMCA nano antibody sequence, step 1: BCMA single domain antibody library construction, step 2: the screening of BCMA specific nano antibody sequence, step 3: Chimeric antigen receptor T cell preparation；For main application in tumor therapeutic agent, the tumour is the relevant tumor disease of blood, the relevant tumor disease of the blood be mainly include each quasi-leukemia and malignant lymphoma.The construction method of the Chimeric antigen receptor based on BMCA nano antibody sequence and its application, the nano antibody expressed sequence prepared is shorter, limited slow virus carrier space is saved, the Chimeric antigen receptor T cell specificity based on the antibody construction is more preferable, and tumor-killing ability is stronger.
Description
Technical field
The present invention relates to cellular immunity technical field, specially a kind of chimeric antigen based on BMCA nano antibody sequence by
The construction method of body and its application.
Background technique
In recent years, swollen based on Chimeric antigen receptor (chimeric angiten receptor, CAR) modification T cell
Tumor adoptive immunotherapy makes substantial progress.T cell after modification not only has a killing ability of targets neoplastic cells, and can gram
It takes tumor by local immunosupress microenvironment and breaks the state of host immune tolerance.CAR has been sent out at present by continuously improving
Forth generation is opened up.First generation CAR is mainly made of the scFv of identification tumor associated antigen and ITAM (usually CD3 ζ), still
Due to only having CD3 ζ activation signals, T cell cannot sufficiently rise in value, and the time-to-live is short in vivo for the T cell activated.The second generation
Costimulatory molecules such as CD28, CD134, CD137 etc. are added in CAR on the basis of generation CAR, enhance t cell activation,
Proliferation is held time in vivo, to improve antitumor effect.Third generation CAR be re-introduced on the basis of two generation CAR one with
On costimulatory molecules.Forth generation CAR (TRUCKS, T cells redirected for universal cytokine
Killing) main by introducing IL-12, the cell factors such as IL-23, IL-27 call together inherent immunity cell-macrophage etc. to kill
Hurt the tumour cell of TAA feminine gender.
Chimeric antigen receptor T cell is the means by gene modification, by an artificial synthesized CAR molecule
(Chimeric Antigen Receptor) is expressed on T cell film, identifies T cell simultaneously in such a way that antigen-antibody combines
Killing tumor cell.CAR molecule includes extracellular antigen binding regions, hinge area, transmembrane region and signal segment intracellular.Compare often
The CAR antigen binding regions seen are made of the scFv that the variable region of the heavy chain and light chain of people or mouse forms.Compared with ScFV,
Nano antibody sequence VHH expression cassette from alpaca is shorter, and specificity is higher.We are obtained by immune alpaca by screening
The BCMA nano antibody sequence affinity obtained is higher, and the Chimeric antigen receptor based on the sequence construct shows excellent in vitro
Tumor-killing vigor.
Summary of the invention
(1) the technical issues of solving
In view of the deficiencies of the prior art, the present invention provides a kind of Chimeric antigen receptors based on BMCA nano antibody sequence
Construction method and its application, it is shorter to have a nano antibody expressed sequence prepared, save limited slow virus carrier space,
Chimeric antigen receptor T cell specificity based on the antibody construction is more preferable, and the stronger advantage of tumor-killing ability solves common
The scFv expressed sequence that is made of the variable region of the heavy chain and light chain of people or mouse of CAR antigen binding regions it is longer and tumour is killed
Hurt the low problem of ability.
(2) technical solution
In order to achieve the above object, the invention provides the following technical scheme: it is a kind of based on the embedding of BMCA nano antibody sequence
Close the construction method of antigen receptor, the specific steps are as follows:
Step 1: BCMA single domain antibody library construction
Alpaca is periodically immunized by the extracellular regions (1-54 amino acid) of Bacillus coli expression BCMA albumen, is immunized
Assessment immune response effect after being immunized, took peripheral blood to separate by lymphocyte separation medium and drenched by taking blood to sample period
Bar cell, extracts lymphocyte RNA and reverse transcription is at cDNA, is inserted into phage display technology after coming out VHH sequence amplification by primer
Show carrier, and the product for carrying single domain antibody genetic fragment is transferred to by competent E.coli by electrotransformation, to obtain
Single domain antibody non-immune libraries.
Step 2: BCMA specific nano antibody sequence screening
Using display technique of bacteriophage by single domain antibody molecular display in phage surface, and then filter out antigentic specificity
Single domain antibody, by BCMA albumen coating into 96 orifice plates, pass through Elisa test 3-5 wheel screening, the bacteriophage of BCMA specificity
Gradually it is enriched with.
Step 3: Chimeric antigen receptor T cell preparation
Exocytosis signal sequence, BCMA nano antibody sequence, hinge area, transmembrane region and signal segment intracellular are connected
Come, be cloned into slow virus carrier, with packaging plasmid cotransfection to 293T cell, purifying concentration slow virus postoperative infection T cell
Obtain the Chimeric antigen receptor T cell for being directed to BCMA.
Preferably, the process of the Bacillus coli expression BCMA albumen are as follows: e. coli bl21 is chosen, at 37 degrees Celsius,
Under IPTG induction, the extracellular regions (1-54 amino acid) of overexpression BCMA albumen, are then infused in LB liquid medium
Enter periodically being immunized in vivo for alpaca.
Preferably, described to take blood using detailed process are as follows: before blood sampling, iodine is used in the first cropping after alpaca neck finds vein accurately
Wine, alcohol carry out partly sterilised, then use the disposal vacuum heparin tube of volume 5ml in the venous blood collection 5.0ml of alpaca, then
Syringe needle, and gently rotation vacuum heparin tube are extracted rapidly, are mixed blood sufficiently with anti-coagulants, are immediately placed on the ice equipped with ice cube
In pot, and curling stone is taken back into laboratory in 20h.
Preferably, the Elisa tests detailed process are as follows: by single domain antibody and BCMA albumen in phage enzyme to substrate
Efficient catalytic effect is combined together.
Preferably, slow virus specific steps are concentrated in the purifying are as follows: a purifying: taking 6 Ultra-clear SW28 centrifugations
Pipe puts opening ultraviolet lamp in superclean bench and continues disinfection 30 minutes, each Ultra-clear after 70% ethanol disinfection
The pretreated vial supernatant of about 32ml is added in SW28 centrifuge tube, takes the pipette of a 10ml, draws 12ml 20%
Sucrose solution.Pipette is inserted into always to the bottom of centrifuge tube, sucrose solution is slowly got into 4ml, separately take one it is clean
Pipette, remaining 3 pipes are equally handled, the weight of each pipe is adjusted with PBS, makes the weight phase between corresponding centrifuge tube
Difference is no more than 0.1g, and all 6 centrifuge tubes are put into Beckman SW28 ultracentrifugation rotary head in order, outwell supernatant,
Tube bottom should have visible precipitating, and 100ml is added in every pipe, and the PBS of calcic and magnesium does not wash lower precipitating, by SW28 ultracentrifugation pipe
It is inserted into 50ml cone bottom centrifuge tube, closes the lid, softly being blown and beaten with 200 μ l pipettors is resuspended precipitating, will be in all pipes
Liquid focuses in a SW28 centrifuge tube, and the viral suspension after concentration is distributed into 50 every part of μ l, is stored in production tube, use is broken
- 80 DEG C are stored in after dry ice quick-frozen；Two concentration methods: 5X PEG8000+NaCl preparation weighs NaCl 8.766g；PEG800050g
It is dissolved in 200ml Milli-Q pure water；The damp and hot extinction 30min of 121 degrees Celsius of 30min；It is stored in 4 DEG C；It is filtered using 0.45 μm
Head filtering slow virus supernatant；5X PEG-8000+NaCl mother liquor 7.5ml is added in every filtered initial liquid of virus of 30ml；Often
20~30min mixing is primary, carries out 3-5 times altogether；4 degrees Celsius stand overnight；It inhales and abandons supernatant, stand pipe 1~2 minute, siphon away
Suitable slow virus lysate dissolution slow virus precipitating is added in residual liquid；Viral suspension after concentration is distributed into 50 every part of μ l,
It is stored in production tube, with being stored in -80 DEG C after broken dry ice quick-frozen.
A kind of application of the building of the Chimeric antigen receptor based on BMCA nano antibody sequence, main application are controlled in tumour
Treat in drug, the tumour is the relevant tumor disease of blood, the relevant tumor disease of the blood be mainly include all kinds of white
Blood disease and malignant lymphoma.
(3) beneficial effect
Compared with prior art, the present invention provides a kind of structures of Chimeric antigen receptor based on BMCA nano antibody sequence
Construction method and its application, have it is following the utility model has the advantages that
1, construction method and its application of the Chimeric antigen receptor based on BMCA nano antibody sequence, the nanometer prepared are somebody's turn to do
Antibody expression sequence is shorter, saves limited slow virus carrier space, and the Chimeric antigen receptor T cell based on the antibody construction is special
Anisotropic more preferable, tumor-killing ability is stronger.
Specific embodiment
Below in conjunction with real Shi Lizhong of the invention, technical solution in the embodiment of the present invention is carried out clearly and completely
Description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on this hair
Embodiment in bright, every other implementation obtained by those of ordinary skill in the art without making creative efforts
Example, shall fall within the protection scope of the present invention.
A kind of construction method of the Chimeric antigen receptor based on BMCA nano antibody sequence, the specific steps are as follows:
Step 1: BCMA single domain antibody library construction
Alpaca is periodically immunized by the extracellular regions (1-54 amino acid) of Bacillus coli expression BCMA albumen, is immunized
Assessment immune response effect after being immunized, took peripheral blood to separate by lymphocyte separation medium and drenched by taking blood to sample period
Bar cell, extracts lymphocyte RNA and reverse transcription is at cDNA, is inserted into phage display technology after coming out VHH sequence amplification by primer
Show carrier, and the product for carrying single domain antibody genetic fragment is transferred to by competent E.coli by electrotransformation, to obtain
Single domain antibody non-immune libraries.
Step 2: BCMA specific nano antibody sequence screening
Using display technique of bacteriophage by single domain antibody molecular display in phage surface, and then filter out antigentic specificity
Single domain antibody, by BCMA albumen coating into 96 orifice plates, pass through Elisa test 3-5 wheel screening, the bacteriophage of BCMA specificity
Gradually it is enriched with.
Step 3: Chimeric antigen receptor T cell preparation
Exocytosis signal sequence, BCMA nano antibody sequence, hinge area, transmembrane region and signal segment intracellular are connected
Come, be cloned into slow virus carrier, with packaging plasmid cotransfection to 293T cell, purifying concentration slow virus postoperative infection T cell
Obtain the Chimeric antigen receptor T cell for being directed to BCMA.
The process of Bacillus coli expression BCMA albumen are as follows: e. coli bl21 is chosen, under 37 degrees Celsius, IPTG induction,
The extracellular regions (1-54 amino acid) of overexpression BCMA albumen, are then injected into the internal of alpaca in LB liquid medium
Periodically it is immunized.
Take blood using detailed process are as follows: before blood sampling, the first cropping after alpaca neck finds vein accurately is carried out with the tincture of iodine, alcohol
Then then needle is extracted rapidly in the venous blood collection 5.0ml of alpaca with the disposal vacuum heparin tube of volume 5ml by partly sterilised
Head, and gently rotation vacuum heparin tube, mix blood sufficiently with anti-coagulants, are immediately placed in the curling stone equipped with ice cube, and
Curling stone is taken back into laboratory in 20h.
Elisa tests detailed process are as follows: makees single domain antibody and BCMA albumen in efficient catalytic of the phage enzyme to substrate
With being combined together.
Purifying concentration slow virus specific steps an are as follows: purifying: 6 Ultra-clear SW28 centrifuge tubes are taken, with 70% second
After alcohol disinfection, puts opening ultraviolet lamp in superclean bench and continue disinfection 30 minutes, each Ultra-clear SW28 centrifuge tube
The middle pretreated vial supernatant that about 32ml is added, takes the pipette of a 10ml, and the sucrose for drawing 12ml 20% is molten
Liquid.Pipette is inserted into always to the bottom of centrifuge tube, sucrose solution is slowly got into 4ml, separately takes a clean pipette,
Remaining 3 pipes are equally handled, the weight of each pipe is adjusted with PBS, the weight between corresponding centrifuge tube is differed and is no more than
All 6 centrifuge tubes are put into Beckman SW28 ultracentrifugation rotary head in order, outwell supernatant by 0.1g, tube bottom should
There is visible precipitating, 100ml is added in every pipe, and the PBS of calcic and magnesium does not wash lower precipitating, and SW28 ultracentrifugation pipe is inserted into
50ml is bored in the centrifuge tube of bottom, is closed the lid, and softly being blown and beaten with 200 μ l pipettors is resuspended precipitating, by the liquid collection in all pipes
In into a SW28 centrifuge tube, the viral suspension after concentration is distributed into 50 every part of μ l, is stored in production tube, with broken dry ice speed
- 80 DEG C are stored in after jelly；Two concentration methods: 5X PEG8000+NaCl preparation weighs NaCl 8.766g；PEG800050g is dissolved in
In 200ml Milli-Q pure water；The damp and hot extinction 30min of 121 degrees Celsius of 30min；It is stored in 4 DEG C；It is filtered using 0.45 μm of filter
Slow virus supernatant；5X PEG-8000+NaCl mother liquor 7.5ml is added in every filtered initial liquid of virus of 30ml；Every 20~
30min mixing is primary, carries out 3-5 times altogether；4 degrees Celsius stand overnight；It inhales and abandons supernatant, stand pipe 1~2 minute, siphon away remnants
Suitable slow virus lysate dissolution slow virus precipitating is added in liquid；Viral suspension after concentration is distributed into 50 every part of μ l, saves
In production tube, with being stored in -80 DEG C after broken dry ice quick-frozen.
A kind of application of the building of the Chimeric antigen receptor based on BMCA nano antibody sequence, main application are controlled in tumour
It treats in drug, tumour is the relevant tumor disease of blood, and the relevant tumor disease of blood is mainly including each quasi-leukemia and evil
Property lymthoma.
In conclusion being somebody's turn to do construction method and its application of the Chimeric antigen receptor based on BMCA nano antibody sequence, preparation
Nano antibody expressed sequence out is shorter, saves limited slow virus carrier space, based on the chimeric antigen of the antibody construction by
Body T cell specificity is more preferable, and tumor-killing ability is stronger.
It should be noted that term " includes " or any other variant thereof is intended to cover non-exclusive inclusion, thus
So that the process, method, article or equipment for including a series of elements not only includes those elements, but also including not clear
The other element listed, or further include for elements inherent to such a process, method, article, or device.Do not having more
In the case where more limitations, the element that is limited by sentence "including a ...", it is not excluded that including process, the side of the element
There is also other identical elements in method, article or equipment.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
A variety of variations, modification, replacement can be carried out to these embodiments without departing from the principles and spirit of the present invention by understanding
And modification, the scope of the present invention is defined by the appended.
Claims (6)
1. a kind of construction method of the Chimeric antigen receptor based on BMCA nano antibody sequence, it is characterised in that: specific steps are such as
Under:
Step 1: BCMA single domain antibody library construction
Alpaca is periodically immunized by the extracellular regions (1-54 amino acid) of Bacillus coli expression BCMA albumen, during being immunized
By taking blood to sample, assessment immune response effect after being immunized, takes peripheral blood thin by lymphocyte separation medium separation lymph
Born of the same parents, extract lymphocyte RNA and reverse transcription is at cDNA, and insertion phage display carries after being come out VHH sequence amplification by primer
Body, and the product for carrying single domain antibody genetic fragment is transferred to by competent E.coli by electrotransformation, to obtain single domain
Antibody mediated immunity library.
Step 2: BCMA specific nano antibody sequence screening
Using display technique of bacteriophage by single domain antibody molecular display in phage surface, and then filter out the list of antigentic specificity
Domain antibodies test 3-5 wheel screening by Elisa, the bacteriophage of BCMA specificity is gradually by BCMA albumen coating into 96 orifice plates
It is enriched with.
Step 3: Chimeric antigen receptor T cell preparation
Exocytosis signal sequence, BCMA nano antibody sequence, hinge area, transmembrane region and signal segment intracellular are together in series,
It is cloned into slow virus carrier, with packaging plasmid cotransfection to 293T cell, purifying concentration slow virus postoperative infection T cell can be obtained
Obtain the Chimeric antigen receptor T cell for BCMA.
2. a kind of construction method of Chimeric antigen receptor based on BMCA nano antibody sequence according to claim 1,
It is characterized in that: the process of the Bacillus coli expression BCMA albumen are as follows: choose e. coli bl21, at 37 degrees Celsius, IPTG is lured
It leads down, the extracellular regions (1-54 amino acid) of overexpression BCMA albumen, are then injected into alpaca in LB liquid medium
In vivo be periodically immunized.
3. a kind of construction method of Chimeric antigen receptor based on BMCA nano antibody sequence according to claim 1,
It is characterized in that: described to take blood using detailed process are as follows: before blood sampling, the first cropping after alpaca neck finds vein accurately, with the tincture of iodine, alcohol
Partly sterilised is carried out, is then then pulled out rapidly with the disposal vacuum heparin tube of volume 5ml in the venous blood collection 5.0ml of alpaca
Syringe needle out, and gently rotation vacuum heparin tube, mix blood sufficiently with anti-coagulants, are immediately placed in the curling stone equipped with ice cube,
And curling stone is taken back into laboratory in 20h.
4. a kind of construction method of Chimeric antigen receptor based on BMCA nano antibody sequence according to claim 1,
Be characterized in that: the Elisa tests detailed process are as follows: urging the efficient of substrate single domain antibody and BCMA albumen in phage enzyme
Change effect is combined together.
5. a kind of construction method of Chimeric antigen receptor based on BMCA nano antibody sequence according to claim 1,
Be characterized in that: slow virus specific steps are concentrated in the purifying are as follows: a purifying: taking 6 Ultra-clear SW28 centrifuge tubes, uses
After 70% ethanol disinfection, puts opening ultraviolet lamp in superclean bench and continue disinfection 30 minutes, each Ultra-clear SW28
The pretreated vial supernatant of about 32ml is added in centrifuge tube, takes the pipette of a 10ml, draws the sugarcane of 12ml 20%
Sugar juice.Pipette is inserted into always to the bottom of centrifuge tube, sucrose solution is slowly got into 4ml, separately takes a clean shifting
Liquid pipe equally handles remaining 3 pipes, the weight of each pipe is adjusted with PBS, differs the weight between corresponding centrifuge tube not
More than 0.1g, all 6 centrifuge tubes are put into Beckman SW28 ultracentrifugation rotary head in order, supernatant are outwelled, in tube bottom
There should be visible precipitating, 100ml is added in every pipe, and the PBS of calcic and magnesium does not wash lower precipitating, and SW28 ultracentrifugation pipe is inserted into
Into 50ml cone bottom centrifuge tube, close the lid, softly being blown and beaten with 200 μ l pipettors is resuspended precipitating, by the liquid in all pipes
It focuses in a SW28 centrifuge tube, the viral suspension after concentration is distributed into 50 every part of μ l, is stored in production tube, with broken dry ice
- 80 DEG C are stored in after quick-frozen；Two concentration methods: 5X PEG8000+NaCl preparation weighs NaCl 8.766g；PEG8000 50g dissolution
In 200ml Milli-Q pure water；The damp and hot extinction 30min of 121 degrees Celsius of 30min；It is stored in 4 DEG C；Use 0.45 μm of filter mistake
Filter slow virus supernatant；5X PEG-8000+NaCl mother liquor 7.5ml is added in every filtered initial liquid of virus of 30ml；Every 20~
30min mixing is primary, carries out 3-5 times altogether；4 degrees Celsius stand overnight；It inhales and abandons supernatant, stand pipe 1~2 minute, siphon away remnants
Suitable slow virus lysate dissolution slow virus precipitating is added in liquid；Viral suspension after concentration is distributed into 50 every part of μ l, saves
In production tube, with being stored in -80 DEG C after broken dry ice quick-frozen.
6. a kind of application of the building of the Chimeric antigen receptor based on BMCA nano antibody sequence, it is characterised in that: it is mainly answered
In tumor therapeutic agent, the tumour is the relevant tumor disease of blood, and the relevant tumor disease of the blood is main
Including each quasi-leukemia and malignant lymphoma.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111333729A (en) * | 2020-03-17 | 2020-06-26 | 深圳市南科生物工程有限公司 | Nano antibody for resisting B cell mature antigen and application |
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| WO2021043169A1 (en) * | 2019-09-02 | 2021-03-11 | 成都盛世君联生物技术有限公司 | Antibody that binds specifically to b-cell maturation antigen and use thereof |
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| CN111333729B (en) * | 2020-03-17 | 2022-12-06 | 深圳市南科生物工程有限公司 | Nanometer antibody for resisting B cell maturation antigen and application thereof |
| CN111909271A (en) * | 2020-08-12 | 2020-11-10 | 深圳市茵冠生物科技有限公司 | BCMA chimeric antigen receptor based on single domain antibody and application thereof |
| CN112028996A (en) * | 2020-10-30 | 2020-12-04 | 南京北恒生物科技有限公司 | Single domain antibodies targeting BCMA and uses thereof |
| CN112028996B (en) * | 2020-10-30 | 2021-01-22 | 南京北恒生物科技有限公司 | Single domain antibodies targeting BCMA and uses thereof |
| CN113087798A (en) * | 2020-12-31 | 2021-07-09 | 北京艺妙神州医药科技有限公司 | anti-BCMA antibody and application thereof |
| CN113087798B (en) * | 2020-12-31 | 2022-05-24 | 北京艺妙神州医药科技有限公司 | anti-BCMA antibody and application thereof |
| WO2022199590A1 (en) * | 2021-03-22 | 2022-09-29 | 浙江纳米抗体技术中心有限公司 | Nanobody targeting bcma and application thereof |
| CN117430709A (en) * | 2023-12-21 | 2024-01-23 | 康维众和(中山)生物药业有限公司 | Nanometer antibody and application thereof |
| CN117430709B (en) * | 2023-12-21 | 2024-03-26 | 康维众和(中山)生物药业有限公司 | Nanometer antibody and application thereof |
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