CN108840930A - Anti- CD19 monoclonal antibody and the preparation method and application thereof - Google Patents
Anti- CD19 monoclonal antibody and the preparation method and application thereof Download PDFInfo
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Abstract
The present invention provides a kind of anti-CD19 monoclonal antibody, VHThe complementary determining region CDR of chain has the amino acid sequence of CDR selected from the group below：SEQ ID NO：Heavy chain CDR1 shown in 3, SEQ ID NO：Heavy chain CDR2 shown in 4, SEQ ID NO：Heavy chain CDR3 shown in 5；Its VLThe complementary determining region CDR of chain has the amino acid sequence of CDR selected from the group below：SEQ ID NO：Light chain CDR1 shown in 6, SEQ ID NO：Light chain CDR2 shown in 7, SEQ ID NO：Light chain CDR3 shown in 8.The present invention also provides the preparation method and applications of the monoclonal antibody.The monoclonal antibody can specific recognition cell surface people's CD19 albumen；With 1 × 10-7M or lower KD in conjunction with people CD19；Have many advantages, such as that immunogenicity is low, does not cause HAMA to react.
Description
Technical field
The present invention relates to biological immune technical field more particularly to anti-CD19 monoclonal antibody and preparation method thereof with answer
With.
Background technique
CD19 is the transmembrane protein on B cell surface.It is activated with B cell, signal transduction and growth regulating are closely related, is
It is dual anti-to constitute B cell when B cell antigen receptor (BCR) identifies antigen for a kind of functional receptor molecule on bone-marrow-derived lymphocyte surface
Former binding model participates in Ca in B cell2+Transhipment, adjust the activation and proliferation of B cell.CD19 is wide as important marker
General leukaemia, the diagnosis of lymthoma and disease of immune system and the Index for diagnosis of being applied to is simultaneously using CD19 as immunization therapy
An important target spot, CD19 is important the target spot of B cell malignant tumour.CD19 is almost expressed in all of B cell development
In period and various B cell tumours, it is however important that CD19 is not expressed in pluripotent hematopoietic stem cells.These are special
Point is so that CD19 becomes the target spot of good B cell tumour.Novel antibodies drug is ground in terms for the treatment of leukaemia and lymthoma
Issue includes non-binding antibody, drug conjugated antibodies, bispecific antibody and Chimeric antigen receptor.
Having shown that CD19 is combined both enhances or inhibits B cell to activate and be proliferated, this depends on the amount of the crosslinking occurred
(Tedder, ibid).CD19 is expressed on 90% or more B cell lymphoma, and predicts it in vitro and in vivo and influence leaching
The growth (Ghetie, ibid) of bar tumor.The anti-CD 19 antibodies generated are mouse antibody.Use mouse Antybody therapy human experimenter
One the disadvantage is that patient application after occur people's anti-mouse (HAMA) response.Human body has immune response to source of mouse antibody, this
It is also the major obstacle that source of mouse antibody is applied to clinical treatment.It can be transformed in gene level by genetic engineering antibody technology anti-
Body reduces the immunogenicity of source of mouse antibody, improves antibody specificity, stability and affinity.Humanization is carried out to source of mouse antibody
Transformation, can retain antibody variable region and merge with constant area of human antibody, improve affinity of antibody；Or engineered antibody structure, building
Only retain the scFv of the antibody variable region or Fab containing antibody variable region and portion constant area, the absorption of antibody in vivo can be improved
Percentage and half-life period.Single-chain antibody (single-chain antibody fragment, scFv) is the one of monoclonal antibody
Kind form forms single-stranded variable region, also referred to as single-chain antibody by the way that the variable region of antibody is connected by hinge area.
ScFv remains the minimum unit of the antigen binding capacity of antibody.Since its structure is smaller, more conducively genetic engineering is operated, so
It is widely used in the building, screening and application of human antibody.Such as CAR-T technology just uses the antibody of scFv structure as chimeric
The region of Receptor recognition antigen.Therefore, it is necessary to more effectively treat and/or prevent the therapeutic of the improvement of the disease of CD19 mediation
Anti-CD 19 antibodies.
Summary of the invention
It is an object of the invention to overcome the defect of the prior art, a kind of anti-CD19 monoclonal antibody and its preparation are provided
Method and application can have Hela cell, the K562 cell of the CD19 positive with people's CD19 albumen of specific recognition cell surface
There is significant lethal effect；The antibody is with 1 × 10-7M or lower KD in conjunction with people CD19；It is swollen with Raji and DaudiB cell
Oncocyte combines；Have many advantages, such as that immunogenicity is low, do not cause HAMA to react, there is better stability and safety.
The invention is realized in this way：
In the first aspect of the present invention, a kind of anti-CD19 monoclonal antibody V is providedHChain, its complementary determining region CDR tool
There is the amino acid sequence of CDR selected from the group below：
SEQ ID NO：Heavy chain CDR1 shown in 3, SEQ ID NO：Heavy chain CDR2 shown in 4, SEQ ID NO：Shown in 5
Heavy chain CDR3.
It is highly preferred that the heavy chain variable region includes and is selected from SEQ ID NO：1 amino acid sequence at least 80% is homologous
Amino acid sequence；
Preferably, the anti-CD19 monoclonal antibody VHChain, it has such as SEQ ID NO：Amino acid sequence shown in 1
Column.
In the second aspect of the present invention, a kind of anti-CD19 monoclonal antibody V is providedLChain, its complementary determining region CDR
Amino acid sequence with CDR selected from the group below：
SEQ ID NO：Light chain CDR1 shown in 6, SEQ ID NO：Light chain CDR2 shown in 7, SEQ ID NO：Shown in 8
Light chain CDR3.
Preferably, the light chain variable region includes and is selected from SEQ ID NO：2 amino acid sequence at least 80% is homologous
Amino acid sequence；
It is highly preferred that the anti-CD19 monoclonal antibody VLChain, it has such as SEQ ID NO：Amino acid sequence shown in 2
Column.
In the third aspect of the present invention, a kind of anti-CD19 monoclonal antibody, V are providedHChain and VLChain be respectively provided with as
SEQ ID NO：1 and SEQ ID NO：Amino acid sequence shown in 2.
In other embodiments, VHAnd/or VLAmino acid sequence can with above-mentioned sequence 85%, 90%, 95%,
96%, 97%, 98% or 99% are homologous.With the V with above-mentioned sequenceHAnd VLThe homologous VH of area's height (i.e. 80% or higher) and
The antibody in the area VL can obtain as follows：Mutagenesis (such as direct mutagenesis or PCR mediate mutagenesis) coding SEQ ID NO：1,2,3,4
Nucleic acid molecules, then with functions described herein testing inspection encode the reservation for being changed antibody function.
The anti-CD19 monoclonal antibody is with 1 × 10-7M or lower KD in conjunction with people CD19；With Raji and DaudiB cell
Tumour cell combines.
In the fourth aspect of the present invention, a kind of nucleic acid molecules are provided, it is anti-that it encodes any anti-CD19 monoclonal of power
Body.
The nucleic acid can reside in intact cell, in cell lysate, or with partial purification or substantially pure form is deposited
?.When passing through standard technique including the processing of alkali/SDS, the aobvious band of CsCl, column chromatography, agarose gel electrophoresis and this field
Well known other methods and other cell components or other pollutants.Nucleic acid of the invention can be such as DNA or RNA, and
It can contain or can be free of intron sequences.In a preferred embodiment, which is cDNA nucleic acid molecules.
Nucleic acid of the invention can use standard molecular biological technique acquisition.For hybridoma (for example, by it is as follows into
One step description carrying human immunoglobulin gene transgenic mice preparation hybridoma) expression antibody, encode by hybridizing
The antibody light chain of tumor preparation and the cDNA of heavy chain can be obtained with standard PCR amplification or cDNA clone technology.For from immune ball
The antibody (such as using display technique of bacteriophage) obtained in protein gene library can recycle from library and encode the antibody
One or more nucleic acid.Available standards PCR amplification obtains：Go out the segment of CD19 single-chain antibody using primer pair amplifies；It is double
Digestion, with T4DNA ligase, in same double digestion vector plasmid, (carrier is anti-in multiple cloning sites downstream amalgamation and expression human IgG1
The Fc segment of body is connected and is converted in host strain, and picked clones are identified positive colony by PCR and confirmed by sequencing, is obtained
Eukaryon expression plasmid.
In the present invention, it is converted into scFv gene using by variable region gene, once obtain coding VHAnd VLThe DNA piece of segment
Section, can be by these DNA fragmentations of standard recombinant dna technology further operating, such as convert overall length for variable region gene and resist
Body chain gene, Fab fragment gene or scFv gene.
In these operations, V is encodedLOr VHDNA fragmentation and coding another protein such as antibody constant region or flexibility
Another DNA fragmentation of connector effectively connects.Term " effectively connection " as used herein means two DNA fragmentation connections
Together, so that the amino acid sequence holding of the two DNA fragmentations coding meets reading frame.
By the way that V will be encodedHDNA and encoding heavy chain constant (CH1、CH2And CH3) another DNA molecular effectively connect
It connects, it can be by isolated coding VHThe DNA in area is converted into total length heavy chain gene.The sequence of people's weight chain constant area gene is in this field
In it is known, the DNA fragmentation including these regions can be obtained by standard PCR amplification.Heavy chain constant region can be IgG1,
IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably IgG1 or IgG4 constant region.For Fab
Fragment heavy chain gene encodes VHDNA can be with only encoding heavy chain CH1Another DNA molecular of constant region effectively connects.
By the way that V will be encodedLDNA and coding constant region of light chain CLAnother DNA molecular effectively connect, can will point
From coding VLThe DNA in area is converted into full-length light chains gene (and Fab light chain gene).The sequence of people's light chain constant region gene exists
DNA fragmentation known in the art including these regions can be obtained by standard PCR amplification.In preferred embodiments,
Constant region of light chain can be κ or λ constant region.
In order to generate scFv gene, V will be encodedHAnd VLDNA fragmentation and coding flexible connection body for example encode amino acid sequence
Another segment of column (Gly4-Ser) 3 effectively connects, so that VHAnd VLSequence can be expressed as continuous single chain protein matter,
Its VLAnd VHArea is connected by the flexible connection body.
In the fifth aspect of the invention, expression vector is provided, it includes to encode any anti-CD19 monoclonal antibody
Amino acid sequence, the nucleic acid can be expressed in protokaryon or eukaryotic host cell.The carrier can be conventional load
Body.Preferably, the present invention is using the third generation from slow virus carrier system is inactivated, and there are three plasmids to encode albumen altogether for the system
Gag/Pol, the packaging plasmid psPAX2 for encoding Rev albumen；It encodes the envelope plasmid pMD2.G of vesicular stomatitis virus-G protein and is based on empty carrier
The extracellular regions encoding human CD19 of pCDH-CMV-MCS-EF1-GFP-T2A-Puro (being purchased from Addgene company) and trans-membrane region
Recombinant plasmid pCDH-CMV-huCD19-EF1-GFP-T2A-Puro.It is provided according to Genbank accession number NM_001178098
People's CD19 sequence, using based on PCR put up a bridge method for synthesizing gene, synthesis include signal peptide, the extracellular regions people CD19, cross-film
Region, intracellular region, the extracellular identification region sequence for the CAR gene that the extracellular domain includes are the anti-CD19 monoclonal
The amino acid sequence of antibody.
In the sixth aspect of the present invention, host cell is provided, is that the protokaryon comprising the carrier or eucaryon host are thin
Born of the same parents.When the building of people's CD19 stable expression cell line, by the successful slow virus carrier pCDH-CMV-huCD19- of above-mentioned building
EF1-GFP-T2A-Puro transfects 293T cell and packs slow virus；Packaged recombinant slow virus can be used for infecting leukaemia cell
Hela or K562 etc..Flow cytometry experiments show that the antibody can be with people's CD19 albumen of specific recognition cell surface.The host
Cell can be the leukaemia cell Hela or K562 etc. infected with the packaged slow virus, be also possible to of the invention
The T lymphocyte infected with the packaged slow virus that eight aspects are mentioned.
In the seventh aspect of the present invention, the preparation method of anti-CD19 monoclonal antibody is provided, including in the host
The anti-CD19 monoclonal antibody is expressed in cell, and the CD19 antibody is separated from the host cell.The anti-CD19 of the present invention
Monoclonal antibody has preferable lethal effect to Hela cell, the K562 cell of the CD19 positive.
In the eighth aspect of the present invention, a kind of CAR-T cell is provided, it includes at least one extracellular ligand binding arrangements
Domain, transmembrane domain and at least one intracellular signal transduction structural domain, wherein the born of the same parents for the CAR gene that the extracellular domain includes
Outer identification region sequence is the amino acid sequence of the anti-CD19 monoclonal antibody.CAR-T technology just uses the anti-of scFv structure
Body constructs the T cell of the chimeric antigen carrier containing above-mentioned CDR segment as the region of Chimerical receptor identification antigen.By above-mentioned packet
The slow-virus infection T lymphocyte of the gene containing scFv installed is to get CAR-T cell of the invention.With traditional immunization therapy phase
Better targeting and lethal is shown in test than, CAR-T cell.
In the ninth aspect of the present invention, it is anti-swollen in preparation to provide the anti-CD19 monoclonal antibody and CAR-T cell
Purposes in tumor medicine.
The present invention is used as target thin using Human cervical cancer cell lines Hela with the Hela cell (CD19-Hela) for having transfected CD19
Born of the same parents, effector cell is the CAR-T cell of aforementioned preparation, when CAR-T cell killing tumour cell, T cell first can with carry it is swollen
The tumour cell of tumor antigen carries out identification verification, then forms immunological synapse, and then discharges killer factor and complete the immune of T cell
Monitoring and killing ability.CAR-T cell of the invention, which has Hela cell, the K562 cell of the CD19 positive, more significantly to kill
Hurt effect.
The invention has the advantages that：
1, anti-CD19 monoclonal antibody (scFv-3B5-Fc) provided by the invention its be to the apparent affinity of CD19
475pM can have Hela cell, the K562 cell of the CD19 positive aobvious with people's CD19 albumen of specific recognition cell surface
The lethal effect of work；
2, CAR-T cell provided by the invention, compared with traditional immunization therapy, CAR-T cell is shown in test
Better targeting and lethal, has more significant lethal effect to Hela cell, the K562 cell of the CD19 positive；
3, CAR-T cell of the invention has many advantages, such as that immunogenicity is low, does not cause HAMA to react, and has and preferably stablizes
Property and safety.
Detailed description of the invention
Fig. 1 is Isotype control IgG, the fluorescence peak of anti-CD19 monoclonal antibody (scFv-3B5-Fc) of the invention is in CD19
It expresses to compare on positive Raji cell and has significant difference；
Fig. 2 is that the bacteriophage for the expression scfv for proving that screening obtains can combine the native antigen of CD19, preliminary purification
Bacteriophage carries out dyeing and flow analysis chart to Raji cell；
Fig. 3 antibody-Fc fusion protein scFv-3B5-Fc, polyacrylamide gel electrophoresis qualification result；
Fig. 4 is the killing CD19-Hela cell of the CAR-T cell-specific of Scfv-3B5 amalgamation and expression of the invention；
Fig. 5 is the extracorporeal anti-tumor cytotoxicity of the CAR-T cell of Scfv-3B5 amalgamation and expression of the invention；
Fig. 6 is that anti-CD19 monoclonal antibody (scFv-3B5-Fc) of the invention obtains in concentration gradient ELISA experiment
Binding curve is exemplary；
Fig. 7 is that the plasmid construct of CAR-T cell provided by the invention forms figure.
Specific embodiment
Embodiment 1 combines the preparation of the specific single-chain antibody (scFv) of people CD19
One, the screening of the anti human CD 19 specific antibody based on phage display
1, the full source of people list of phage display to reach this purpose, is inoculated in 400ml 2 × YT/ ampicillin medium
The glycerol stock (self-built library) in the natural library of chain antibody, makes cell density reach OD600=0.1, under the conditions of 37 DEG C and 200rpm
Shaken cultivation is until cell density reaches OD600=0.5.With 1012(purchase is certainly for the M13KO7 helper phage of Pfu
ThermoFisher company) infection, it is cultivated 30 minutes under the conditions of 30 DEG C and 50rpm.It is added after 50mg/L kanamycins 37
DEG C and 200rpm under the conditions of after shaken cultivation 30 minutes, by centrifugation (15 minutes, 1600 × g, 4 DEG C) precipitation and separation, be resuspended in
2 × YT/ of 400ml ampicillin/kanamycins culture medium, shaken cultivation 16 hours under the conditions of 37 DEG C and 200rpm.Finally
It by centrifugation (20 minutes, 5000 × g, 4 DEG C) precipitation and separation and abandons, after 0.45 μm of specification membrane filtration of supernatant, is added 1/4
Volume 20% (w/v) PEG8000,2.5M NaCl solution is simultaneously incubated for 1 hour precipitating phage particles in ice bath.Then centrifugation
(20 minutes, 8000 × g, 4 DEG C), abandon supernatant, by phages be resuspended in 25ml pre-cooling PBS solution (137mM NaCl,
2.7mM KCl, 8mM Na2HPO4, 2mM KH2PO4) in, it is centrifuged (5 minutes, 20000 × g, 4 DEG C).1/4 body is added to supernatant
20% (w/v) PEG8000,2.5M NaCl solution of product, and the precipitating phage particles again of ice bath 30 minutes.(30 points of centrifugation
Clock, 20000 × g, 4 DEG C), phages are resuspended in 2ml pre-cooling PBS again, is kept for 30 minutes on ice and is centrifuged (30
Minute, 17000 × g, 4 DEG C).Supernatant is with the PBS solution containing 4% (w/v) BSA with 1:1 mixing, is placed on impeller,
30 minutes are kept the temperature at room temperature, is then directly used for screening.
2, it using above-mentioned phage antibody library, (is purchased from for people's Fc-CD19 recombinant protein of biotin labeling
Acrobiosystems company) 3 wheel directed screenings are implemented, screening scheme is as follows：
3,2 hours are incubated at room temperature by the recombined human CD19 antigen of phage antibody library and biotin labeling, then with
The marked by streptavidin closed through 2%BSA solutionMagnetic bead (being purchased from ThermoFisher company) exists
It is incubated for 30 minutes at room temperature.Then wash magnetic bead with PBST (contain 0.1% Tween-20) buffer, removing non-specific binding or
The weaker bacteriophage of binding ability.The strong bacteriophage of binding ability, then with glycine-HCI buffer (pH 2.2) from magnetic bead
It elutes, after being neutralized with Tris neutralizer (pH 9.1), for infecting the Escherichia coli ER2738 for being in logarithmic growth phase, and
It is used for next round screening.In 3 wheel screenings, the dosage of magnetic bead is respectively 50 μ l, 20 μ l and 10 μ l, the people of biotin labeling
Fc-CD19 antigen concentration is respectively 100nM, 10nM and 1nM, and the washing times of PBST are respectively 10 times, 15 times and 20 times.
Two, people CD19 specifically binds the identification of antibody
1, it is screened from third round and selects monoclonal in resulting clone at random, and (enzyme linked immunological is inhaled with single Phage-ELISA
Attached experiment) analyze the binding ability of itself and people Fc-CD19.For this purpose, each 300 μ l 2 × YT/ ammonia benzyl of single colonie inoculation is green
Mycin culture medium (contain 2% glucose) is in 96 hole depth well culture plates, and shaken cultivation 16 hours under 37 DEG C and 250rpm.With 20
μ l culture is inoculated into 500 μ 2 × YT/ of l ampicillin mediums (containing 0.1% glucose), shakes under 37 DEG C and 250rpm
Swing culture 1.5 hours.Prepare helper phage liquid solution, take 75 μ l M13KO7 (titre be 3 × 1012Pfu/ml) it is mixed into 15ml
In 2 × YT culture medium, 50 holes μ l/ are added in culture plate.In 37 DEG C and 150rpm CMC model 30 minutes, then it is added and is ready to
50 hole μ l/ of kanamycins solution (taking the 50mg/ml kanamycins of 180 μ l, be added to 2 × YT of 15ml culture medium), at 37 DEG C
With shaken cultivation 16 hours under 250rpm.Last centrifugation cell (30 minutes, 5000 × g, 4 DEG C), supernatant is transferred to newly
96 hole depth well culture plates.
2, as shown in Fig. 2, in order to primarily determine whether the bacteriophage for expressing scfv that screening obtains can be in conjunction with CD19's
Native antigen, we are dyed with the bacteriophage of preliminary purification to Raji cell and flow cytometer showed.The analysis of Fig. 2 the result shows that
The bacteriophage for screening obtained expression scfv can be in conjunction with the native antigen of CD19.(A) figure is the expression that screening obtains in Fig. 2
The bacteriophage of scfv, figure (B) are negative control group.
3, to carry out single Phage-ELISA, on 96 hole MediSorp elisa plates (being purchased from ThermoFisher) respectively
Using the hole 100ng/ people Fc-CD19 recombinant antigen and negative control protein Fc (100 hole μ l/), stayed overnight in 4 DEG C of coatings.Each
It is closed with the PBST solution containing 2%BSA in hole.Hole then is cleaned three times with PBST and is clapped net.Then wheat flour on 100 holes μ l/ is added
On every kind of standby phage solution to plate in each hole.After 37 DEG C keep the temperature 2 hours, washed three times with PBST.In order to detect combination
Bacteriophage, by anti-M13 antibody superoxide dismutase conjugate (being purchased from three hawk of Wuhan) with 1:5000 are diluted in PBST, take
100 μ l are added in each hole.37 DEG C heat preservation 1 hour after with PBST rinsing three times, then with PBS rinsing three times.Finally draw 50 μ
L tmb substrate is added in hole, and is developed the color 10 minutes at room temperature, and the 2MH of every 50 μ l of hole is then added2SO4Color development stopping is anti-
It answers.Extinction value is measured in 450nm with enzyme-linked immunosorbent assay instrument (Bio-Rad).It is determined as positive hole according to extinction value, extraction is bitten
The sequence verification of thallus plasmid progress scfv sequence.
4, in conjunction with sequencing analysis, single-chain antibody 3B5 (SEQ ID NO is observed:9), people CD19 is tied in ELISA experiment
It is extremely strong to close signal, to Fc recombinant protein without combination.
5, monoclonal antibody 3B5 (monoclonal antibody i.e. of the invention) comprising：(a) comprising such as SEQIDNO：Shown in 5
The heavy chain CDR1 of amino acid sequence；(b) comprising such as SEQIDNO：The heavy chain CDR2 of amino acid sequence shown in 6；(c) comprising such as
SEQIDNO：The heavy chain CDR3 of amino acid sequence shown in 7；(d) comprising such as SEQIDNO：The light chain of amino acid sequence shown in 8
CDR1；(e) comprising such as SEQIDNO：The light chain CDR2 of amino acid sequence shown in 9；(f) comprising such as SEQIDNO：Shown in 10
The light chain CDR3 of amino acid sequence, wherein the antibody specificity in conjunction with people CD19.
The expression and purifying of the single-chain antibody of the anti-CD19 of embodiment 2
1, the expression of people CD19 single-chain antibody
The recombination to construct of carrier for expression of eukaryon is carried out to selected single-chain antibody, transfection HEK293F induction recombinant expression is simultaneously
Purifying.Use primer pair 3B5-F (SEQ ID NO:And 3B5-R (SEQ ID NO 10):11) institute's clone from embodiment 1
ScFv-3B5 segment is amplified in pCantab-3B5；By NheI/BamHI (being purchased from Takara company) double digestion, with T4DNA
Ligase is in equally with Nhe I/BamH I double digestion vector plasmid pCMV-V5-Fc, (carrier merges in multiple cloning sites downstream
Express the Fc segment of human IgG1's antibody, hereinafter referred to as V5-Fc) it connects and converts in host strain TOP10, picked clones pass through
PCR identifies positive colony and by sequencing confirmation, obtains scFv-3B5-Fc eukaryon expression plasmid.
2, the purifying of people CD19 single-chain antibody
Above-mentioned expression plasmid is transfected into well-grown HEK-293F cell respectively, 37 DEG C, 5%CO2,125rpm shaking table connects
Continuous culture 7 days, 4000rpm are centrifuged 10min, and removal precipitating collects supernatant, and with 0.45 μm of membrane filtration, the sample that will be handled well
Product carry out affinity purification, the final antibody-Fc fusion protein scFv- for obtaining purifying with ProteinA (being purchased from GE company) affinity column
3B5-Fc, polyacrylamide gel electrophoresis qualification result are as shown in Figure 3.Fig. 3 shows the antibody-Fc fusion protein scFv- of purifying
3B5-Fc is constructed successfully.
Embodiment 3, anti human CD 19 single-chain antibody antigen-binding activity analysis
1, combination activity of the elisa assay recombinant antibodies to people's CD19 antigen
It is tested by concentration gradient ELISA, measures the combination activity of the antibody confrontation protoplast CD19 screened.Mesh thus
, antigen people Fc-CD19 is diluted with 0.1M NaHCO3 (pH 9.6) coating buffer, every hole is coated with 100ng, 50 holes μ l/, 4 DEG C of coatings
Overnight, and with the PBST confining liquid containing 2%BSA and 0.01% (v/v) Tween-20 it closes at room temperature 2 hours.Then it uses
PBST rinses plate three times and removes clean.Then, to each orifice plate be added 100 μ l containing a series of concentration (initial concentration 10nM,
3 times of gradient dilutions are carried out, until being diluted to 1:729) the PBST solution of each antibody protein, the measurement of each sample use parallel
The analysis of three holes.After 37 DEG C keep the temperature 2 hours, three times with PBST rinsing, it is then added 1:20000 diluted band horseradish peroxidases
Goat anti-human antibody (being purchased from three hawk of Wuhan) 100 holes μ l/ of label, 37 DEG C are reacted 1 hour.In order to detect, hole three is rinsed with PBST
It is secondary, it then three times with PBS rinsing, is eventually adding TMB and shows 15 minutes, reacted with the 2M H2SO4 color development stopping of every 50 μ l of hole,
Extinction value is measured in 450nm with enzyme-linked immunosorbent assay instrument (Bio-Rad).With the obtained photon absorbing intensity of GraphPad software evaluation
Value, the bond strength of calculating antibody.For this purpose, the extinction value measured in each case maps to corresponding antibody concentration, and
Curve obtained is fitted with following nonlinear regression.
Wherein identifying combination/dissociation equilibrium between fixed antigen and antibody protein is：
The concentration of x=antibody protein；
The concentration (being measured indirectly by the light absorption value after chromogenic reaction) of y=antigen/antibody complex；
The total concentration of a=immobilized antigen；
B=dissociation constant (KD).
Binding curve exemplary representation such as Fig. 6 institute that antibody ScFv-3B5-Fc is obtained in concentration gradient ELISA experiment
Show, wherein the apparent affinity of ScFv-3B5-Fc is 475pM.
2, combination activity of the facs analysis recombinant antibodies to Raji cell surface CD19
It is tested by facs analysis, the antibodies on tumor cell system Raji cell surface CD19 antigen that detection screening obtains
In conjunction with activity.
4 flow cyctometry of embodiment analyzes the combination of each individual's CD19 stable expression cell line and anti-CD19 single-chain antibody
Situation
One, the building of people CD19 stable expression cell line
1, the building of plasmid vector
(1) carrier system that the present embodiment uses belongs to the third generation from slow virus carrier system is inactivated, which shares three
A plasmid is the packaging plasmid psPAX2 for encoding Protein G ag/Pol, encoding Rev albumen；Encode the envelope plasmid of vesicular stomatitis virus-G protein
The pMD2.G and encoding human CD19 for being based on empty carrier pCDH-CMV-MCS-EF1-GFP-T2A-Puro (being purchased from Addgene company)
The recombinant plasmid pCDH-CMV-huCD19-EF1-GFP-T2A-Puro of extracellular regions and trans-membrane region.
(2) the people's CD19 sequence provided according to Genbank accession number NM_001178098, the base put up a bridge using based on PCR
Because of synthetic method, synthesis includes signal peptide, the extracellular regions people CD19, trans-membrane region, intracellular region, passes through primer pair huCD19F
(SEQ ID NO：12)/R(SEQ ID NO：13) PCR amplification is carried out, amplification condition is initial denaturation：94 DEG C, 4min；Denaturation：94
DEG C, 30s；Annealing：58 DEG C, 30s；Extend：68 DEG C, 80s；30 circulations.The segment theory size of acquisition is 1716bp, and amplification produces
Object confirms in the same size with theory through agarose electrophoresis.Wherein Xba I and BamH I enzyme are introduced in the upstream and downstream of open reading frame
Enzyme site.The target gene of above-mentioned acquisition is connected into the pCDH-CMV-MCS- of same double digestion by Xba I and BamH I double digestion
In EF1-GFP-T2A-Puro carrier, successful slow virus carrier pCDH-CMV-huCD19-EF1-GFP-T2A-Puro is constructed,
Slow virus packaging is carried out after Xba I and BamH I digestion identification and sequencing are correct.
2, plasmid transfection 293T cell packs slow virus
(1) with 6 × 106Density inoculated and cultured to the 6th~10 generation 293T cell (ATCC：CRL-11268) in 10cm
In culture dish, 37 DEG C, 5%CO2Overnight incubation prepares for transfecting.Culture medium is containing 10% without bacteriophage fetal calf serum (Hangzhou
Chinese holly) DMEM (ThermoFisher company), next day, about 2 hours replacement culture solutions are serum-free DMEM before transfection.
The step of transfection, is as follows：
By 5 μ g target gene plasmid pCDH-CMV-huCD19-EF1-GFP-T2A-Puro, respectively with 7.5 μ g packaging plasmids
PSPAX2 and 2.5 μ g envelope plasmid pMD2.G dissolves in 500 μ L Mill Q water, mixes；
62 μ L 2.5M CaCl2 (Sigma company) are added dropwise, are mixed with 1200rpm/min vortex,
500 2 × HBS of μ L (280mM NaCl, 10mM KCl, 1.5mM Na2HPO4,12mM grape is finally added dropwise
Sugar, 50mM Hepes (Sigma company), pH7.05,0.22 μM of filtration sterilization), 1200rpm/min oscillation mixes 10s,
It is added dropwise in culture dish, gently shakes up immediately, 37 DEG C, 5%CO2, after cultivating 4~6h, be changed to containing 10% tire
The DMEM of cow's serum.
(2) after transfecting 48h or 72h, it is centrifuged off cell fragment, then with 0.45 μm of filter (Millipore company)
Virus is collected by filtration.
3, recombinant slow virus infects leukaemia cell Hela or K562
After the concentrated titration of the virus liquid of above-mentioned collection, the Hela cell or K562 that infection is laid in 6 orifice plates respectively are thin
Born of the same parents.Infection collected cell after three days, takes part mixing clone, is examined using flow cytometry and the fluorescent labeled antibody for targeting CD19
The expression of cell surface CD19 is surveyed, the method and step of flow cytometry are the same as embodiment 5.After remaining cell expansion culture
A part is frozen, another part passes in 6 orifice plates, and the Puromycin that 2ug/ml is added (is bought from Sigma) antibiotic, sieve
1-2 weeks time is selected, microscopically observation freezes part cell at this time and be used for until whole cells express GFP into the visual field
Subsequent Flow cytometry and killing experiments.
Two, flow cyctometry is analyzed
1, the single-chain antibody for the anti human CD 19 that embodiment 2 obtains is analyzed by flow cytometer (CytoFLEX, Beckman)
The binding ability of the people CD19 of scFv-3B5-Fc and cell surface.
The specific method is as follows：
(1) cell Raji cell, K562-CD19 and the K562 of logarithmic growth phase are inoculated into T25 Tissue Culture Flask respectively
In, inoculating cell density is about 5x105A/ml, 37 DEG C of incubators are incubated overnight.
(2) cell is collected by centrifugation in the culture solution in weak vibrations culture dish, 200g × 5min.With 2~3 × 106/ mL's is dense
Degree is resuspended in 1% phosphate buffer (NBS PBS) containing calf serum, and streaming dedicated pipe is added by the amount of 100ul/ pipe
In.
(3) 200g × 5min is centrifuged, and abandons supernatant.
(4) two experimental groups are separately added into test antibodies scFv-3B5-Fc, FMC63 positive antibody, separately set a control group
For the PBS blank control that antibody is not added.The final concentration of each antibody is 20 μ g/ml, and 100ul is added in every pipe.Ice bath, 45 minutes.
(5) 2ml 1%NBS PBS is added in every pipe, is centrifuged with 200g × 5min, totally two times.
(6) supernatant is abandoned, is added 1:100 diluted goat anti-human antibody-FITC (three hawk of Wuhan biology), 100ul is added in every pipe.
Ice bath, 45 minutes.
(7) 2ml 1%NBS PBS is added in every pipe, is centrifuged with 200g × 5min, totally two times.
(8) supernatant is abandoned, is resuspended in 300ul 1%NBS PBS, flow cytomery.
(9) data are analyzed using Flow cytometry data analysis software CytoExpert2.0.
2, as shown in Figure 1, flow cytometric analysis results show that the fluorescence peak of Isotype control IgG, scFv-3B5-Fc exist
Compared to there is significant difference on the positive Raji cell of CD19 expression, without obvious poor on the negative K562 cell of people CD19 expression
Not, show that scFv-3B5-Fc can express positive Raji cell with specific recognition people CD19, but express feminine gender with people CD19
K562 cell does not combine.Therefore, antibody scFv -3B5-Fc can be with people's CD19 albumen of specific recognition cell surface.Because
Figure is the same before and after the streaming figure of K562, and no difference in figure so do not show.
Embodiment 5 prepares CAR-T cell
One, the building of the slow virus plasmid of Chimeric antigen receptor albumen
1, slow virus plasmid vector system used in the present embodiment belongs to four pUC pUC of third generation slow virus, the system
It is altogether the envelope plasmid pCMV-VSV-G (being purchased from addgene) for encoding vesicular stomatitis virus-G protein, the packet of coding Rev albumen there are four plasmid
Fill plasmid pRSV-Rev (being purchased from addgene), the pMDLg/pRRE (being purchased from addgene) of coding Gal and Pol and based on empty carrier
The recombinant expression carrier of the coding purpose CAR gene of pLVX-EF1 (our company's building), the system can be effectively reduced to be formed again
The risk of property lentiviral particle processed.
2, the gene promoter of the CAR expression slow virus carrier of all buildings is all made of the patent having disclosed
- 1 α of elongation factor (elongation factor-1 α, EF-1 α) on carrier in 201310164725.X.Construct specific method
It is as follows：
(1) full length amino acid sequence of CAR gene is designed
Usual one typical CAR structure should include epicyte protein encoding transport signals peptide, tumour antigen combined area, hinge
Area, transmembrane region and T cell costimulatory signal.Specific to the present embodiment, as shown in fig. 7, we are turned using the cell membrane of the CD8 of people
Signal peptide is transported, combined area of the scfv-3B5 as tumour antigen CD19 is connected, hereafter reconnects the extracellular nearly film of the CD8 of people
The transmembrane region of the area Hinge and CD8, cytoplasmic domains part connects the intracellular domain of 4-1BB and the intracellular domain of CD3 ζ is made
For the costimulation structural domain of T cell.In order to facilitate the detection of CAR, we are behind CAR structure by being connected to from montage peptide
GFP coexpression.Entire fusion and its corresponding amino acid sequence are as follows：
(2) nucleic acid sequence optimizes
In order to enhance the translation of expression and increase protein of the CAR gene in T cell, we use website http://
The source of people codon optimization algorithm of www.jcat.de/ optimizes the codon of CAR gene, and the sequence after optimization is as follows：
(3) complete sequence synthesis and sequence verification
For the Codon sequences of optimization, DNA synthesis supplier (Wuhan Jin Kairui) is transferred to carry out segment synthesis and total order
Column splicing, sequence verification after splicing is completed, by EcoR I/BamH I restriction enzymes double zyme cutting after verifying is errorless, even
Enter in the pLVX-EF1 carrier of same double digestion, to construct the slow virus carrier pLVX-EF1- for expressing each Chimeric antigen receptor
3B5-CAR,pLVX-EF1-FMC63-CAR.Successful carrier is constructed through EcoR I/BamH I digestion identification and sequencing just
After really, it can be used for slow virus packaging.As previously mentioned, correlation CAR gene is a peptide chain through transcription and translation, in GM-CSF signal
Under the guidance of peptide, anti-CD19 Chimeric antigen receptor be will be located on the cell membrane of T cell.The present embodiment uses similar strategy,
Construct the CAR fusion being made of scfv-3B5 segment and scfv-FMC63 segment component.
Two, plasmid transfection 293T packs slow virus
1, with 6 × 106Density inoculated and cultured to the 6th~10 generation HEK-293T cell (ATCC：CRL-11268) in
In 10cm culture dish, 37 DEG C, 5%CO2Overnight incubation prepares for transfecting.Culture medium is (equal containing 10% fetal calf serum and DMEM
Purchased from Hyclone company).
2, the step of transfecting is as follows：
A liquid is prepared：By each target gene plasmid pLVX-EF1-3B5-CAR of 5.2 μ g, respectively with 6.2 μ g packaging plasmids
PMDLg RRE and pRSV-REV and 2.4 μ g envelope plasmid pCMV-VSV-G, dissolves in the serum-free DMEM culture solution of 800 μ L
In, it mixes.
B liquid is prepared：60 μ g PEI (1 μ g/ μ l of polyethyleneimine is purchased from Polysciences company) are dissolved in 800 μ L
Serum-free DMEM culture solution in, mix gently, be incubated at room temperature 5min.
The formation of transfection composite：A liquid is added in B liquid and is gently mixed, vortex mixed or is mixed gently immediately after addition,
It is incubated for 20min at room temperature.
Transfection composite 1.6ml is added dropwise in HEK-293T cell, after 4-5h hours, is given with the DMEM of 2%FBS training base
The 293T cell of transfection changes liquid.
3, after transfection is incubated for 72h, virus is collected by filtration using 0.45 μm of filter (being purchased from Millipore company), then
Using Beckman Optima L-100XP ultracentrifuge 28000rpm, 4 DEG C are centrifuged 2 hours, abandon centrifugation supernatant, centrifugation gained
The AIM-V culture solution (being purchased from Life company) for precipitating 1/10~1/50 stoste volume is resuspended, and is dispensed and is frozen with 100 μ L/ pipes
- 80 DEG C are stored in, to titration of virus or infection T lymphocyte.
Three, recombinant slow virus infection T cell prepares CAR-T cell
1, human peripheral blood single nucleus cell is obtained (in Wuhan City's blood by density-gradient centrifugation method by healthy human peripheral blood
The heart provides), facs analysis confirms CD3 positive rate, is then 1 according to CD3 positive T cell and magnetic bead ratio:2 are added while being coated with
The recombinant human il-2 of the magnetic bead (ThermoFisher company) and final concentration 300U/mL that have AntiCD3 McAb and CD28 antibody is (purchased from Shanghai
Magnificent neoformation Hitek Ltd) stimulation culture is for 24 hours.Then T cell is infected with above-mentioned recombinant slow virus for 5 with MOI.Infection
Cell afterwards every other day uses 5 × 105The density of/mL is passed on, while final concentration is added in lymphocyte culture medium
The recombinant human il-2 of 300U/mL.
2, the T cell infected is expressed when cultivating the 5th day by FCM analysis Chimeric antigen receptor.Firstly, infection
People's Fc-CD19 protein 37 DEG C of CAR-T cell and biotin labeling afterwards is incubated for 30min, and D-PBS is washed 2 times, and PE- is then added
The Streptavidin of label is in 37 DEG C of incubation 30min.Flow cytomery positive cell ratio after D-PBS washes 3 times.With not
The T lymphocyte of infection and the T cell of the Streptavidin for only having added PE- to mark dyeing are as negative control, identification expression
The virus infection T cell and its positive rate of Chimeric antigen receptor are horizontal.It can also confirm expression GFP's by facs analysis simultaneously
The positive rate of T cell, the Positive Level of the T cell expression CAR gene of control Fc-CD19 recombinant antigen detection, can be confirmed and take
CAR structure successfully can be shown to the positive rate on the cell membrane of T cell by the T cell of the slow-virus infection with CAR gene
The result shows that the T cell of expression Chimeric antigen receptor receptor can be obtained by the method for slow-virus infection.
3, T cell is after infection is packaged with the virus of Chimeric antigen receptor respectively, with cell density for 5 × 105/ ml is every other day
Passage is counted and is added IL-2 (final concentration of 300U/ml) to the cell culture fluid of passage, and there are about 50~200 within the 11st day for culture
Amplification again shows that the CAR-T cell for expressing Chimeric antigen receptor is able to carry out a certain number of amplifications in vitro, is subsequent body
Outer fragmentation test and in vivo studies provide guarantee.
6 in vitro toxicity effect experiment of embodiment
One, in the cell culture system for being suitable in vitro, target cell or embodiment 5 of the mixing with specific tumor antigen
The CAR-T cell of preparation, i.e. T cell or CAR-T cell, which can be observed, implements identification (aggregation) to target cell after a period of time
With killing (reduction of tumour cell quantity) behavior.Such as this experiment mixing is overexpressed the Hela cell (people in embodiment 4 of CD19
Obtained in the building of CD19 stable expression cell line) and common amplification cultivation T cell or FMC63CAR-T (positive control),
3B5CAR-T.This experiment of cell cell used is Human cervical cancer cell lines Hela and has transfected the Hela cell (CD19- of CD19
Hela it) is used as target cell, effector cell is the CAR-T cell of aforementioned preparation, and effect target is than being optionally respectively 2.5,12.5:1 He
25:1, target cell numbers are 10000/hole, according to different effect targets than adding an appropriate number of effector cell.All cells are trained
It supports in 5%CO2, in 37 DEG C of cell incubator.
Two, microscope imaging analyzes its in vitro toxicity effect
1, method：When experiment starts, the CD19-Hela cell in 10000/hole is first inoculated with into 6 orifice plates, after cultivating 12h
According to effect target than adding an appropriate number of T cell or CAR-T cell, continue in 5%CO2, after cultivating 4h in 37 DEG C of incubators, take
Culture plate observes imaging under inverted microscope out.
2, result is as shown in figure 4,3B5-CAR-T has significant lethal effect to the Hela cell of the CD19 positive.
Three, real-time n cell analytical technology analyzes its in vitro toxicity effect experiment
1, principle：When CAR-T cell killing tumour cell, T cell first can with carry tumour antigen tumour cell into
Row identification verification, then forms immunological synapse, and then discharges immunosurveillance and killing ability that killer factor completes T cell, because
This this process is a lasting process.The method of traditional detection T cell killing tumor cell often takes a time
Point carries out end point determination, and often error is larger for the result obtained, can not really reflect T cell killing tumor cell
Complete procedure.In order to break through this kind of detection means technical bottleneck limitation, real-time n cell analytical technology (Real
Time Cellular Analysis, RTCA) electrical impedance based on cell adherent rear generation in the culture plate with microelectrode
The detection of value, may be implemented it is real-time, unmarked, continue dynamically to monitor small molecule compound, antibody drug, T cell etc. and cause
Cytotoxic effect.Real-time cell based on this electrical impedance kills analytical technology, and researcher can obtain very highly sensitive
Quantized data facilitates the specific mechanism of action for studying and disclosing antitumoral compounds or cell, while also can significantly reduce
The cost of experiment and the accuracy for improving result.RTCA technology continues dynamically to monitor the adherent of target cell, stretching, extension and breeding
Process, the growth conditions of target cell are indicated using cell index Cell Index, or can be equal to the quantity of target cell.
2, method：When whole experiment process is in addition to addition effector cell, it should ensure that culture plate and detecting instrument are in and stablize
Culture environment in, avoid Cell Index data from biggish fluctuation occur.In order to obtain time dependent cytological effect feature
The culture medium of 90ul is added in curve in the culture hole of the E-plate in 16 holes, and the thin of 100ul is being added after measuring background baseline
Born of the same parents' suspension is placed in CO after mixing2On monitor station in incubator, and continue to obtain the impedance value of reacting cells growth conditions
Cell Index.After 18h after the complete adherent growth of target cell for a period of time afterwards isometric effector cell's suspension is added, then again
It is put on the monitor station in incubator, continues the impedance value Cell Index data for collecting reacting cells growth conditions.
3, result：Terminate entire experiment after 48h, the data for exporting acquisition are as shown in Figure 5.In figure, SKOV-3-CD19 is real
Apply the stable cell lines for stablizing expression CD19 in example.Cell Index index is straight by the impedance value of measurement culture plate in Fig. 5
That sees shows the quantity of the tumour cell grown on culture plate.The effector cell of addition is suspension cell, therefore to culture plate
Impedance value have no significant effect.It is that Cell Index gradually rises that experiment initial stage, effector cell was not added, and cell is prompted to exist
Growth is proliferated under suitable condition of culture, the decline of Cell Index shows the target cell of adherent growth after addition effector cell
The quantity of CD19-SKOV3 is reduced, i.e., apoptosis is occurred by the killing of effector cell etc..Show 3B5- provided by the invention
CAR-T has significant lethal effect to the Hela cell of the CD19 positive.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.Core
Acid sequence is as follows：
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Claims (10)
1. a kind of anti-CD19 monoclonal antibody, which is characterized in that its VHThe complementary determining region CDR of chain is with CDR's selected from the group below
Amino acid sequence：SEQ ID NO：Heavy chain CDR1 shown in 3, SEQ ID NO：Heavy chain CDR2 shown in 4, SEQ ID NO：5 institutes
The heavy chain CDR3 shown；
Its VLThe complementary determining region CDR of chain has the amino acid sequence of CDR selected from the group below：SEQ ID NO：Light chain shown in 6
CDR1, SEQ ID NO：Light chain CDR2 shown in 7, SEQ ID NO：Light chain CDR3 shown in 8.
2. a kind of anti-CD19 monoclonal antibody, which is characterized in that its VHThe amino acid sequence of chain such as SEQ ID NO：Shown in 1 or with
It has the amino acid sequence of at least 80% homology；Its VLThe amino acid sequence SEQ ID NO of chain：2 or with its have extremely
Shown in the amino acid sequence of few 80% homology.
3. the anti-CD19 monoclonal antibody as described in claim 1-2 is any, which is characterized in that its for human antibody, humanization or
Chimeric antibody.
4. a kind of nucleic acid molecules, which is characterized in that it encodes any anti-CD19 monoclonal antibody of claim 1-3.
5. a kind of expression vector comprising nucleic acid described in claim 4, which is characterized in that it can be in protokaryon or eucaryon host
The nucleic acid is expressed in cell.
6. a kind of host cell, which is characterized in that it is protokaryon or eucaryon host comprising the expression vector described in claim 5
Cell.
7. a kind of preparation method of anti-CD19 monoclonal antibody, which is characterized in that be included in host cell as claimed in claim 6
Middle expression anti-CD19 monoclonal antibody, and the CD19 monoclonal antibody is separated from the host cell.
8. a kind of CAR-T cell, which is characterized in that it includes at least one extracellular ligand binding domains, transmembrane domain and
At least one intracellular signal transduction structural domain, wherein the extracellular identification region sequence for the CAR gene that the extracellular domain includes is
The amino acid sequence of any anti-CD19 monoclonal antibody of claim 1-3.
9. a kind of preparation method of CAR-T cell as claimed in claim 8, includes the following steps：
S1, the separation of T lymphocyte, activation and amplification：Amplification will be cultivated after isolated CD3+T lymphocyte activator；
S2, the recombinant slow virus for carrying CAR infect the step S1 treated T lymphocyte, and the CAR-T cell is made,
Wherein, the extracellular identification region sequence of the CAR gene carried in the recombinant slow virus for carrying CAR is that claim 1-3 is any
The amino acid sequence of the anti-CD19 monoclonal antibody.
10. prepared by any anti-CD19 monoclonal antibody of claim 1-3 and CAR-T cell according to any one of claims 8
Purposes in anti-tumor drug.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110331134A (en) * | 2019-07-19 | 2019-10-15 | 上海交通大学医学院附属瑞金医院 | A kind of universal cell therapy product and its preparation method and application for expressing antigen recognition region |
| CN114286683A (en) * | 2019-07-17 | 2022-04-05 | 新加坡国立大学 | Functional conjugates synthesized and secreted by immune cells |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105017422A (en) * | 2014-04-30 | 2015-11-04 | 山东百因制药技术有限公司 | Anti-CD3/anti-CD19 dual-specific antibody and application thereof |
| CN105837689A (en) * | 2015-01-13 | 2016-08-10 | 博生吉医药科技(苏州)有限公司 | Anti-CD19 monoclonal antibody and preparation method thereof |
| CN107188964A (en) * | 2016-12-15 | 2017-09-22 | 上海科医联创生物科技有限公司 | The anti-CD19 monoclonal antibodies of Quan Renyuan and its production method |
| CN107557388A (en) * | 2017-07-26 | 2018-01-09 | 生研医药科技（武汉）有限公司 | A kind of slow virus carrier prepared for CAR T and its construction method and application |
-
2018
- 2018-06-29 CN CN201810698095.7A patent/CN108840930B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105017422A (en) * | 2014-04-30 | 2015-11-04 | 山东百因制药技术有限公司 | Anti-CD3/anti-CD19 dual-specific antibody and application thereof |
| CN105837689A (en) * | 2015-01-13 | 2016-08-10 | 博生吉医药科技(苏州)有限公司 | Anti-CD19 monoclonal antibody and preparation method thereof |
| CN107188964A (en) * | 2016-12-15 | 2017-09-22 | 上海科医联创生物科技有限公司 | The anti-CD19 monoclonal antibodies of Quan Renyuan and its production method |
| CN107557388A (en) * | 2017-07-26 | 2018-01-09 | 生研医药科技（武汉）有限公司 | A kind of slow virus carrier prepared for CAR T and its construction method and application |
Non-Patent Citations (4)
| Title |
|---|
| SOMMERMEYER D 等: "Fully human CD19-specific chimeric antigen receptors for T-cell therapy", 《LEUKEMIA》 * |
| 孟兆丽: "人源单链抗体噬菌体展示文库的构建及抗LOX-1单链抗体的筛选", 《CNKI》 * |
| 王晓良: "《应用分子药理学》", 30 September 2015, 中国协和医科大学出版社 * |
| 陈森 等: "抗人CD19单链抗体基因的构建、表达及功能测定", 《生物工程学报》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114286683A (en) * | 2019-07-17 | 2022-04-05 | 新加坡国立大学 | Functional conjugates synthesized and secreted by immune cells |
| EP3999082A4 (en) * | 2019-07-17 | 2022-11-30 | National University of Singapore | Functional binders synthesized and secreted by immune cells |
| CN110331134A (en) * | 2019-07-19 | 2019-10-15 | 上海交通大学医学院附属瑞金医院 | A kind of universal cell therapy product and its preparation method and application for expressing antigen recognition region |
| WO2021012857A1 (en) * | 2019-07-19 | 2021-01-28 | 上海交通大学医学院附属瑞金医院 | Universal cell therapy product for expressed antigen recognition region, and preparation method therefor and application thereof |
| CN110331134B (en) * | 2019-07-19 | 2023-09-08 | 上海交通大学医学院附属瑞金医院 | Universal cell therapy product expressing antigen recognition region and preparation method and application thereof |
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