CN106397608A - CD20 specificity chimeric antigen receptor and application thereof - Google Patents
CD20 specificity chimeric antigen receptor and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/7051—T-cell receptor (TcR)-CD3 complex
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70517—CD8
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70521—CD28, CD152
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
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- C07—ORGANIC CHEMISTRY
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- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
- C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
Abstract
The invention discloses a CD20 specificity chimeric antigen receptor and application thereof. The chimeric antigen receptor is prepared from a single-chain antibody of mouse antihuman CD20, a CD8 hinge region of the mouse antihuman CD20, a transmembrane region of the mouse antihuman CD20, CD137 and an intracellular signal structure of CD3 zeta, wherein the CD137 and the intracellular signal structure of the CD3 zeta are in serial connection. The chimeric antigen receptor is used for modifying T lymphocytes, and the modified T lymphocytes are applied to medicine of B cell lymphoma and B cell lymphocytic leukemia.
Description
Technical field
The present invention relates to biological heredity field, especially a kind of nucleic acid of coding CD20 specific chimeric antigen receptor and table
Reach the T cell of Chimeric antigen receptor, can be used for the adoptive cellular treatment of B cell lymphoma, B cell Lymphocytic leukemia
(adoptive cell therapy, ACT) field.
Background technology
Chimeric antigen receptor (Chimeric antigen receptor, CAR) is the φt cell receptor of synthetic, by born of the same parents
Outer targeting bonding pad, and activation signal domain (hinge region, the transmembrane region, intracellular signal transduction area) composition of T cell.Extracellular
Targeting bonding pad is made up of single-chain antibody or part, has the function of specific binding target antigen.Hinge region often adopt CD8,
The hinge region of CD4 or IgG4 molecule.Transmembrane region is made up of lipophilic aminoacid sequence, often adopts the transmembrane region of CD8, CD28
Section.Intracellular signal transduction area is by CD3 ζ chain or Fc ε R I γ chain and costimulatory signal molecule CD28, CD137 (4-1BB), CD134
(OX40), CD27, ICOS, CD244 etc. are constituted.Containing only CD3 ζ chain or Fc ε R I γ chain is first generation CAR, and comprising 1 stimulates altogether
Signaling molecule is second filial generation CAR, and the CAR containing 2 and above costimulatory signal molecule is the third generation.The T cell that CAR modifies is led to
Cross extracellular targeting bonding pad identification TSA, then signal transmission will be identified to cell by intracellular signal transduction area
Interior, activation T cell simultaneously plays killing tumor cell effect.
CAR is capable of identify that cell surface albumen not through processing, is not limited by MHC；And the knot between CAR and part
The adhesion made a concerted effort between more normal φt cell receptor and peptide-MHC is bigger；The identification of CAR is not subject to costimulatory moleculeses coordinate expression yet
Limit.Therefore, CAR modification T cell is adopted and is treated the downward MHC molecule that can preferably overcome tumor cell or reduce stimulation altogether
The immune evasion mechanism such as factor expression.
The earliest period developed except B cell and late period, CD20 expression is including late period precursor B cells to memory B cell
The B cell surface in nearly all stage.And find that CD20 B cell lymphoma and 1/3 acute B lymphocyte more than 90% are white
Expression in disorders of blood (B-ALL).Therefore, CD20 is treatment B cell lymphoma and the leukemic targetedly tumor of bone-marrow-derived lymphocyte
Target antigen.
Content of the invention
The technical problem to be solved is to provide a kind of CD20 specific chimeric antigen receptor and its application, institute
State CAR to be specifically bound on target antigen, and play the cell toxic effect for B cell lymphoma, B cell Lymphocytic leukemia
Should.
In order to solve above-mentioned technical problem, the technical solution used in the present invention is：A kind of CD20 specific chimeric antigen is subject to
Body, this Chimeric antigen receptor by single-chain antibody scFv, CD8 hinge region of mouse anti human CD20 and transmembrane region, CD28 and/or
The intracellular signal structures in series of CD137T cell co-stimulatory signaling molecule and CD3 ζ is constituted；It is special that this Chimeric antigen receptor contains CD20
The single-chain antibody of the opposite sex, as shown in the SEQ ID NO.3 in sequence table, the amino of this Chimeric antigen receptor is last for its aminoacid sequence
End contain a signal peptide, its aminoacid sequence as shown in the SEQ ID NO.4 in table, the light chain of this Chimeric antigen receptor and weight
Between chain, by aminoacid sequence, the connection peptides as shown in the SEQ ID NO.6 in table are formed by connecting.
Preferably, described Chimeric antigen receptor is tied with the intracellular signal of CD8 α hinge region and transmembrane region and CD137 and CD3 ζ
The domain that structure domain is in series is T cell stimulus signal conducting structure, the SEQ ID in its aminoacid sequence such as sequence table
Shown in NO.5.
The aminoacid sequence of described Chimeric antigen receptor is as shown in sequence table SEQ ID NO.1.Described Chimeric antigen receptor
Nucleotide sequence as shown in sequence table SEQ ID NO.2.
Above-mentioned CD20 specific chimeric antigen receptor is in anti-B cell lymphoma, B cell Lymphocytic leukemia medicine
Application.
The invention has the beneficial effects as follows：The present invention is in the past patent《CD 20 antagonizing Chimeric antibody mutant gene and application》
(the patent No.：ZL 02158550.4) sequence in amplify weight chain variable district and the light chain variable district of antibody, and overlapped extension
Round pcr adds linker to form CD20 specificity scFv.And by the scFv fragment of structure be cloned into containing signal peptide and CD8 α-
In the Lentiviral of CD137-CD3 ζ, it is packaged into and carries the slow of CD20 scFv-CD8 α-CD137-CD3 ζ encoding gene
Viral vector.Using slow virus infection T cell, T cell is made to express this Chimeric antigen receptor.By flow cytometry, retting conditions
The cytokine of analysis experiment and ELISA detection T cell secretion is it was demonstrated that the T cell of this Chimeric antigen receptor modification is to expression
The lymphoma cell of CD20 has specific cytotoxicity.Chimeric antigen receptor CD20 scFv-CD8 α therefore of the present invention-
CD137-CD3 ζ can apply in B cell lymphoma, the treatment of B cell Lymphocytic leukemia.
Brief description
Fig. 1 is the light chain variable district (V of the CD20 monoclonal antibody of the present inventionL), weight chain variable district (VH) and both connect after
PCR amplification electrophoretogram (1 swimming lane of single chain antibody fragments：2kb nucleic acid molecular weight standard；2 swimming lanes：VL；3 swimming lanes：VH；4 swimming lanes：
CD20 scFv).
Fig. 2 is the Lentiviral CD20 scFv-CD8 α-CD137-CD3 ζ digestion with restriction enzyme of the present invention
Fragment electrophoretic qualification figure (1 swimming lane：15kb nucleic acid molecular weight labelling；2 swimming lanes：Slow with Cobra venom endonuclease Xba I and Not I double digestion
Coding CD20 scFv-CD8 α-CD137-CD3 ζ's obtained by viral expression plasmids CD20 scFv-CD8 α-CD137-CD3 ζ
DNA fragmentation (732bp) and carrier segments (7985bp)；3 swimming lanes：The CAR carrier of non-enzyme action).
Fig. 3 is that (wherein counterclockwise sequence is positive genetic fragment, clockwise for the Lentiviral schematic diagram of the present invention
For cdna reverse fragment).
Fig. 4 is that the CD20 scFv-CD8 α-CD137-CD3 ζ that the Flow cytometry present invention builds modifies in T cell
(A is the T cell of transfection empty carrier to the expression figure of CAR molecule；B transfects the T cell of CAR).
Fig. 5 is CD20 target antigen molecule in Flow cytometry B cell lymphoma cell line Raji and Namalwa cell
Expression figure (A be CD20 target antigen developed by molecule level fluidic cell result, B be CD20 specific fluorescence intensity (SFI)
Statistical result).
Fig. 6 is that the CD20 scFv-CD8 α-CD137-CD3 ζ of the present invention modifies the T cell Raji cell line positive with CD20
Namalwa cell line p+ with CD20 compares 1 by effect target:1、1:2、1:4、1:After 8 co-cultivations 48 hours, the B cell of residual is drenched
(CAR-T is the experimental group that CD20 scFv-CD8 α-CD137-CD3 ζ modifies T cell to bar oncocyte figure；VEC-T is that transfection is unloaded
The matched group of body T cell).
Fig. 7 is that the CD20 scFv-CD8 α-CD137-CD3 ζ of the present invention modifies T cell to Raji and Namalwa cell line
Compare 1 by effect target:(CAR-T is that CD20 scFv-CD8 α-CD137-CD3 ζ modifies T cell to the retting conditions detection figure of 1 lethal effect 4h
Experimental group；VEC-T is the matched group of the T cell of transfection empty carrier).
Fig. 8 is that the CD20 scFv-CD8 α-CD137-CD3 ζ of the present invention modifies T cell and Raji and Namalwa cell line
Compare 1 by effect target:After 1 co-cultures 48 hours, the cytokine IFN-γ of T cell release, IL-2, TNF-α, IL-6 level view (A:
FN-γ、B:IL-2、C:TNF-α、D:IL-6；CAR-T is the experiment that CD20 scFv-CD8 α-CD137-CD3 ζ modifies T cell
Group；VEC-T is the matched group of the T cell of transfection empty carrier).
Specific embodiment
With reference to the accompanying drawings and detailed description the present invention is described in further detail：
CD20 specific chimeric antigen receptor of the present invention, this Chimeric antigen receptor is by the single-chain antibody of mouse anti human CD20
(single chain variable fragment, scFv), CD8 hinge region and transmembrane region, CD28 and/or CD137T cell are altogether
The intracellular signal structures in series of stimulus signal molecule and CD3 ζ is constituted；It is specific single-stranded anti-that this Chimeric antigen receptor contains CD20
Body, as shown in the SEQ ID NO.3 in sequence table, the amino terminal of this Chimeric antigen receptor contains a letter to its aminoacid sequence
Number peptide, its aminoacid sequence as shown in the SEQ ID NO.4 in sequence table, between the light chain of this Chimeric antigen receptor and heavy chain by
Connection peptides as shown in the SEQ ID NO.6 in sequence table for the aminoacid sequence are formed by connecting.
Preferably, described Chimeric antigen receptor is tied with the intracellular signal of CD8 α hinge region and transmembrane region and CD137 and CD3 ζ
The domain that structure domain is in series is T cell stimulus signal conducting structure, the SEQ ID in its aminoacid sequence such as sequence table
Shown in NO.5.
The aminoacid sequence of described Chimeric antigen receptor is as shown in sequence table SEQ ID NO.1.Described Chimeric antigen receptor
Nucleotide sequence as shown in sequence table SEQ ID NO.2.
Above-mentioned CD20 specific chimeric antigen receptor is in anti-B cell lymphoma, B cell Lymphocytic leukemia medicine
Application.
The activation signal domain of the T cell in this Chimeric antigen receptor, except being CD8 α-CD137-CD3 ζ, also may be used
To be CD8 α-CD28-CD3 ζ and CD8 α-CD28-CD137-CD3 ζ.
The present invention is with the Fab sequence of the mouse hybridoma cell system of the secretion anti-CD-20 monoclonal antibody of the past preparation as base
Plinth, design synthesizes anti-CD20-scFv sequence.
Embodiment 1：The structure of Chimeric antigen receptor CD20 specific single-chain antibody
(1) PCR amplification CD20 monoclonal antibody VL、VHGenetic fragment：
P1：5’GCTAGCGACATCGAGCTCACTCAG3’
P2：5’AGAACCACCACCACCGGAGCCGCCGCCGCCAGAACCACCACCACCCCGTTTGATCTCCACCTTG
G3’
P3：5’GGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGT TCTCAGGTGAAGCTGCAGCAG
TC3’
P4：5’CCG GAATTCTGAGGAGACGGTGACCGTGA3’
Prepare VL、VHPCR reaction system (20 μ l) is as follows：2 × Taq PCR Master Mix (TianGen company):10μ
l；10μM P1/P2(VL):1μl；10μM P2/P4(VH):1μl；Fab plasmid:1μl；ddH2O:Complement to 20 μ l.Reaction
Condition：94 DEG C of denaturations 5 minutes；It is repeated below circulation 33 times：94 DEG C 30 seconds, 52 DEG C 30 seconds, 72 DEG C 1 minute；Finally, 72 DEG C are prolonged
Stretch 10 minutes.Agarose gel electrophoresiies are separated and recovered from VL、VHFragment.By the V after reclaimingL、VHFragment and pMD19-T
(simple) carrier (Takara company) is attached by T4 ligase (Takara company), and censorship is sequenced.Just retain sequencing
Really clone.Coupled reaction system and reaction condition are as follows：VLPCR primer/VHThe each 50ng of PCR primer, pMD19-T (simple)
Carrier 1 μ l, Solution I coupled reaction liquid 5 μ l；ddH2O complements to 10 μ l, and 16 DEG C connect overnight.Connection product is transformed into
In E.coli DH5 α competence antibacterial, after 37 DEG C of incubated overnight, picking single bacterium colony, after amplification culture, use plasmid extraction reagent
Box (Takara company) extract positive colony plasmid, through enzyme action and sequencing detection, by correct plasmid designations be respectively designated as L and
H.
(2) build VL-linker-VHDirection anti-CD20 scFv fragment
Prepare second step Overlap extension PCR reaction system (25 μ l):(TianGen is public for 2 × Taq PCR Master Mix
Department)；10 μM of P1 primers:1μl；10 μM of P4 primers:1μl；VL、VHFragment：Each 200ng；ddH2O：Complement to 20 μ l.Reaction bar
Part：94 DEG C of 5 minutes denaturations；It is repeated below circulation 28 times：94 DEG C 30 seconds, 58 DEG C 1 minute, 72 DEG C 1 minute 30 seconds；Then, 72
DEG C extend 10 minutes.Agarose gel electrophoresiies are separated and recovered from CD20 scFv fragment (as Fig. 1).By the scFv fragment after reclaiming
It is attached by T4 ligase (Takara company) with pMD19-T (simple) carrier (Takara company), coupled reaction body
System and reaction condition are as follows：CD20 scFv PCR primer 50ng, pMD19-T (simple) carrier 1 μ l, Solution I connect
Reactant liquor 5 μ l；ddH2O complements to 10 μ l, and 16 DEG C connect overnight.Connection product is transformed in E.coli DH5 α competence antibacterial,
After 37 DEG C of incubated overnight, picking single bacterium colony, after amplification culture, extract positive gram with plasmid extraction kit (Takara company)
Grand plasmid, through enzyme action and sequencing detection, correct plasmid designations is named as CD20 scFv.The albumen of the CD20 scFv obtaining
Sequence is SEQ ID NO.3, wherein VLAnd VHCatenation sequence be SEQ ID NO.6.
(3) build the carrier of CD20 scFv-CD8 α-CD137-CD3 ζ mesh：1. adopt Nhe I, EcoR I endonuclease
The plasmid containing CD8 α-CD137-CD3 ζ fragment that enzyme enzyme action early stage builds (comprises signal peptide sequence SEQ ID in plasmid
NO.4, membrane spaning domain and t cell activation domain SEQ ID NO.5), obtain CD8 α-CD137-CD3 ζ fragment.2. design two
End is respectively provided with the primer of NheI, EcoRI restriction enzyme site, expands CD20 scFv fragment.3. by CD20 scFv fragment and purpose
Carrier is attached, and the carrier of the CD20 scFv-CD8 α-CD137-CD3 ζ mesh of structure is carried out enzyme action mirror with NheI and EcoRI
Fixed (as Fig. 2 enzyme action result shows that positive colony contains purpose band, and sequencing identification is correct).The CD20 scFv-CD8 obtaining
α-CD137-CD3 ζ DNA sequence be SEQ ID NO.2, translated after protein sequence be SEQ ID NO.1,
Embodiment 2：Chimeric antigen receptor CD20 scFv-CD8 α-CD137-CD3 ζ slow viruss modify the preparation of T cell
(1) EndoFree Plasmid Maxi plasmid extraction test kit (QIAGEN company) is adopted to extract CD20 scFv-
CD8 α-CD137-CD3 ζ transferring plasmid and packaging plasmid psPAX2, PMD.2G.Three kinds of plasmids use 4：3：1 ratio Turbofect
Transfection reagent (Thermo company) is transfected (concrete grammar is shown in Turbofect transfection reagent description).Transfect latter 24 hours,
Collect viral supernatants respectively within 48 hours, in 4 DEG C, 3000rpm, it is centrifuged 10 minutes, after filtering through 0.45 μm of filter, adopt
50000g, 4 DEG C, after 3 hours ultracentrifugations concentrate 10 times, after proceed to -80 DEG C of preservations.
(2) preparation of T cell：Take fresh and healthy human peripheral 10ml, using RosetteSep T cell
Enrichment Cocktail (Stemcell company) and Ficoll-Paque PLUS (GE Healthcare company) extracts T
Cell (concrete steps are according to RosetteSep T cell enrichment Cocktail description).Press cell：Magnetic bead=1：
1 ratio adds AntiCD3 McAb/CD28 magnetic bead (Gibco company), the T cell that culture is before transfecting for 24 hours.
(3) slow virus infection T cell and infection after T cell culture：Take out viral supernatants from -80 DEG C, melt under room temperature
Change, by every 1 × 106T cell adds 100 μ l viral supernatants, adds Polybrene to final concentration of 8 μ g/ml.32 DEG C,
1800rpm, is centrifuged 1.5h, proceeds to 5%CO2, 37 DEG C of incubator cultures.
(4) Flow cytometry CAR modifies the expression of T cell film surface C AR：Using Alexa Fluor 647 labelling
Goat anti-mouse IgG (Fab ')2Antibody (Jackson ImmunoResearch company) dyes, after being incubated 30 minutes on ice,
The expression efficiency carrying out flow cytometry T cell surface C AR is 50-60% (see Fig. 4).
Embodiment 3：Chimeric antigen receptor CD20 scFv-CD8 α-CD137-CD3 ζ slow viruss are modified T cell and B cell are drenched
The lethal effect of bar oncocyte.
(1) CD20 expression in B cell lymphoma cell line：Raji, Namalwa cell line is purchased from U.S. ATCC.
After cultivating respectively, respectively draw 5 × 105Cell suspension, after PBS washes 2 times, (Biolegend is public for labelling APC anti-humen CD 20 monoclonal antibody
Department), with labelling APC-isotype as matched group, it is incubated 30 minutes on ice.With the various cell line of Flow cytometry
The level of CD20 expression.The percentage rate that Raji and Namalwa cell line expresses CD20 is respectively 100% and 0.21% (Fig. 5 A)；
Specific fluorescence intensity (specific fluorescence intensity, SFI) is respectively 33.4 and 0.744 (Fig. 5 B).
(2) pouring of Flow cytometry residual after the T cell that CAR modifies is co-cultured with Raji, Namalwa cell line
Bar oncocyte：Cell is pressed 1 × 105Cells/well inoculates 24 well culture plates, is separately added into 1 × 105(1:1)、5×104(1:2)、
2.5×104(1:4)、1.25×104(1:8), the T cell that CAR modifies, the T cell (VEC-T) of transfection empty carrier is set to compare
Group.By the cell marking APC anti-humen CD 20 monoclonal antibody (Biolegend company) after co-culturing, Flow cytometry residual cell.
Result is shown in CAR-T and Raji and compares 1 to imitate target:1、1:2、1:4 and 1:8 co-culture 48 hours, and in co-culture system, phenotype is
CD3-Raji cell respectively remaining 30%, 57%, 85% and 91%, co-culture 48 hours in VEC-T and Raji, there is CD3-
Raji cell be 86%, 95%, 102%, 99%；And CAR-T and Namalwa with imitate target than co-culture 48 little when, co-culture
In system, phenotype is CD3-Namalwa cell be respectively present 101%, 127%, 121% and 118%, VEC-T with
Namalwa co-cultures 48 hours, there is CD3-Namalwa cell be 136%, 141%, 123%, 111% (see Fig. 6).
(3) activation of the T cell that retting conditions experimental analysiss CAR modifies：
CAR-T and VEC-T cell is compared 1 with Raji and Namalwa cell according to effect target respectively:1 is co-cultured, and
Anti-CD107a antibody and monensin is added in co-culture system；Flow cytomery CD3 is applied after 4h+Cell surface
The expression of CD107a.In CAR-T and Raji co-culture system, CD3+Cell-stimulating percentage rate is 14%, VEC-T and Raji
In co-culture system, CD3+Cell-stimulating percentage rate is 0.4%, both have significant difference (P=0.007).CAR-T with
In Namalwa co-culture system, CD3+Cell-stimulating percentage rate is CD3 in 3%, VEC-T and Namalwa co-culture system+Carefully
Born of the same parents' activation percentage rate is 3%, and both do not have difference (see Fig. 7).
(4) cytokine IFN-γ in ELISA detection lymphoma cell line and CAR-T co-culture of cells supernatant, TNF-α,
The level of IL-2 and IL-6：
Respectively by Raji and Namalwa cell line according to 6.3 × 105Cells/well inoculates 24 orifice plates.By every hole 6.3 × 105
Cell is separately added into CAR-T, VEC-T cell, supplements culture fluid to 1.5mL, after co-culturing 12 hours in incubator.Using people
IFN-γ, TNF α, IL-2 and IL-6ELISA detection kit (R＆D company), are detected (concrete steps to co-culturing supernatant
See ELISA detection kit description).The Raji of expression CD20 is co-culturing IFN-γ, IL-2, TNF-α in supernatant with CAR-T
All significance is had to raise (P compared with VEC-T group with IL-6 cytokine levels<0.001), but thin in the Namalwa not expressing CD20
The IFN-γ co-culturing in supernatant of born of the same parents, IL-2, TNF-α and IL-6 cytokine levels do not have the rising of significance.(Fig. 8) tie
Fruit shows, the T cell that Chimeric antigen receptor CD20 scFv-CD8 α-CD137-CD3 ζ modifies is in the lymphoma cell of expression CD20
Under system stimulates, Th1 type cytokines can be secreted.
In sum, present disclosure is not limited in the above embodiments, and the knowledgeable people in same area can
Can propose other embodiments easily within the technological guidance's thought of the present invention, but this embodiment is included in this
Within the scope of bright.
Claims (5)
1. a kind of CD20 specific chimeric antigen receptor it is characterised in that this Chimeric antigen receptor by mouse anti human CD20 list
The intracellular letter of chain antibody scFv, CD8 hinge region and transmembrane region, CD28 and/or CD137T cell co-stimulatory signaling molecule and CD3 ζ
Number structures in series is constituted；This Chimeric antigen receptor contains the specific single-chain antibody of CD20, in its aminoacid sequence such as sequence table
SEQ ID NO.3 shown in, the amino terminal of this Chimeric antigen receptor contains a signal peptide, its aminoacid sequence such as sequence table
In SEQ ID NO.4 shown in, by aminoacid sequence such as the SEQ in sequence table between the light chain of this Chimeric antigen receptor and heavy chain
Connection peptides shown in ID NO.6 are formed by connecting.
2. CD20 specific chimeric antigen receptor according to claim 1 it is characterised in that described Chimeric antigen receptor with
The domain that the intracellular signal domain of CD8 α hinge region and transmembrane region and CD137 and CD3 ζ is in series stimulates for T cell
Signal transduction structure, its aminoacid sequence is as shown in the SEQ ID NO.5 in sequence table.
3. CD20 specific chimeric antigen receptor according to claim 2 is it is characterised in that described Chimeric antigen receptor
Aminoacid sequence is as shown in sequence table SEQ ID NO.1.
4. CD20 specific chimeric antigen receptor according to claim 3 is it is characterised in that described Chimeric antigen receptor
Nucleotide sequence is as shown in sequence table SEQ ID NO.2.
5. the CD20 specific chimeric antigen receptor as described in any one of claim 1-4 drenches in anti-B cell lymphoma, B cell
Application in bar chronic myeloid leukemia medicine.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107384963A (en) * | 2017-07-31 | 2017-11-24 | 山东兴瑞生物科技有限公司 | A kind of preparation method and applications of controllable type CD20 Chimeric antigen receptors modification T cell |
| CN108251442A (en) * | 2018-02-07 | 2018-07-06 | 中国医学科学院血液病医院（血液学研究所） | FLT3 Chimeric antigen receptors and its application |
| CN108285486A (en) * | 2018-01-15 | 2018-07-17 | 浙江阿思科力生物科技有限公司 | Using CD20 as the specific antibody of target spot, CAR-NK cells and its preparation and application |
| CN109306013A (en) * | 2017-07-26 | 2019-02-05 | 重庆精准生物技术有限公司 | The Chimeric antigen receptor and its application of anti-CD20 antigen |
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| CN107216395A (en) * | 2017-07-03 | 2017-09-29 | 上海科医联创生物科技有限公司 | A kind of TM4SF1 specific chimerics antigen receptor and its application |
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| CN109608548A (en) * | 2017-12-29 | 2019-04-12 | 郑州大学第附属医院 | Chimeric antigen receptor, Lentiviral and its application based on source of people CD20 antibody |
| CN108285486A (en) * | 2018-01-15 | 2018-07-17 | 浙江阿思科力生物科技有限公司 | Using CD20 as the specific antibody of target spot, CAR-NK cells and its preparation and application |
| CN108251442A (en) * | 2018-02-07 | 2018-07-06 | 中国医学科学院血液病医院（血液学研究所） | FLT3 Chimeric antigen receptors and its application |
| CN112673025A (en) * | 2018-06-20 | 2021-04-16 | 上海隆耀生物科技有限公司 | Chimeric antigen receptor containing third signal receptor and application thereof |
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