CN105949323A - EpCAM-specific chimeric antigen receptor and encoding gene and application thereof - Google Patents
EpCAM-specific chimeric antigen receptor and encoding gene 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
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- C07K16/2821—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against ICAM molecules, e.g. CD50, CD54, CD102
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Abstract
The invention discloses an EpCAM (Epithelial cell adhesion molecule)-specific chimeric antigen receptor. The EpCAM-specific chimeric antigen receptor is obtained by serially connecting a single-chain antibody of a human anti-EpCAM, CH2CH3 of a human antibody IgG1, a transmembrane region and an intracellular signal structure of CD28, an intracellular signal structure of CD137 and an intracellular signal structure of CD3[zeta]. The invention further discloses an amino acid sequence and a nucleic acid sequence of the chimeric antigen receptor. The invention further discloses application of the chimeric antigen receptor to preparation of medicines for treating EpCAM-related tumors, namely modification of T lymphocytes, and the modified lymphocytes can be applied to treatment of the EpCAM-related tumors.
Description
Technical field
The present invention relates to cellular immunization technical field, particularly relate to a kind of EpCAM specific chimeric antigen and be subject to
Body and encoding gene, application.
Background technology
EpCAM (Epithelial cell adhesion molecule) epithelial cell adhesion molecule belongs to adhesion molecule
Family, also referred to as 17-A, ESA, EGP40, Trop-1, KSA, CD326, TACSTDI, C017-1A,
GA733-2 etc..EpCAM is a kind of to be correlated with Ca2+ oscillations transduction I (tumor-associated calcium by tumor
Signaltransducerl, TACSTDI) the single pass transmembrane albumen of a molecular weight 30～40kDa of gene code,
Participate in regulation cell adhesion, mediated signal transduction, cell migration, breed and the function such as differentiation (Kurtz JE,
DufourP.Adecatumumab:an ant1-EpCAM monoclonal antibody,from the bench to
the bedside[J].Expert Opin BioITher,2010,10(6):951-958；Trzpis M,McLaughlin
PM,de LeijLM,et al.Epithelial cell adhesion molecule.More than a carcinoma
marker andadhesion molecule[J].Am J Pathol,2007,171(2):386-395)。
EpCAM expresses on people's part normal epithelium cell and most of malignant epithelial cell surface, the most generally
Think that the biological characteristics of tumor is played an important role by it.Under pathologic condition, EpCAM is almost expressed in all
Adenocarcinoma in, including Colon and rectum adenocarcinoma, adenocarcinoma of stomach, breast carcinoma, ovarian cancer, adenocarcinoma of lung, carcinoma of prostate,
Cancer of pancreas and hepatocarcinoma, retinoblastoma (Kurtz JE, Dufour P.Adecatumumab:an
ant1-EpCAM monoclonal antibody,from thebench to the bedside[j].Expert Opin
BiolTher,2010,10(6):951-958).Research finds that EpCAM is also the labelling of some tumor stem cells
Thing, the research of Kimura etc. finds that liver-cancer stem cell positive for EpCAM can induce the mice of immunodeficiency
There is tumor (Kimura O, Takahashi T, Ishii N, et al.Characterization oftheepithelial cell
adhesion molecule(EpCAM)+cell population in hepatocellularcarcinoma cell lines
[J] .Cancer Sci, 2010,101 (10): 2145-2155.).By positive for hepatocarcinoma EpCAM and negative sub-
Group's cell is expelled in the Mice Body of immunodeficiency respectively, and result display forms the cancerous cell number needed for tumor
EpCAM positive is far fewer than EpCAM negative patient, and the mice of injection EpCAM positive cancer cell is produced
Raw tumor is bigger than injection EpCAM negative patient, and the positive cancerous cell oncogenicity ratio of EpCAM is described
Cancerous cell negative for EpCAM is big.Subgroup clone's speed positive for EpCAM in hepatocellular carcinoma cells system is far away
More than the subgroup that EpCAM is negative.Research also finds, cancerous cell positive for EpCAM can be divided into
The cell that EpCAM is positive and negative, and the cancerous cell of EpCAM feminine gender can only be divided into EpCAM feminine gender
Cell, illustrate that cancerous cell positive for EpCAM has the potential of multinomial differentiation.
The expression of EpCAM is relevant to the prognosis of tumor, can be as neoplasm targeted therapy a target site.
The antibody of EpCAM obtains FDA approval treatment cancer ascites at present in Europe, but there is presently no EpCAM
CART in clinical practice.
Summary of the invention
The technical problem existed based on background technology, it is an object of the invention to provide a kind of EpCAM special
Sex-mosaicism antigen receptor.
The present invention also aims to provide the gene encoding above-mentioned Chimeric antigen receptor.
The present invention also aims to provide the application of above-mentioned Chimeric antigen receptor.
To achieve these goals, a kind of EpCAM specific chimeric antigen receptor that the present invention proposes, its
The single-chain antibody of born of the same parents' exoantigen land behaviour anti-EpCAM.
Preferably, the aminoacid sequence of the single-chain antibody of described people's anti-EpCAM is as shown in SEQ ID NO.1.
Preferably, there is a connection between heavy chain and the light chain molecule of the single-chain antibody of described embedding people's anti-EpCAM
Peptide, its aminoacid sequence is as shown in SEQ ID NO.2.
Preferably, the amino terminal of described Chimeric antigen receptor contains a signal peptide, and its aminoacid sequence is such as
Shown in SEQ ID NO.3.
Preferably, described Chimeric antigen receptor is by the single-chain antibody of people's anti-EpCAM, people's IgG antibody 1
The cross-film district of CH2CH3, CD28 and intracellular signal structure, the intracellular signal structure of CD137, CD3 ζ
Intracellular signal structures in series is constituted.
Preferably, the cross-film district of described CD28 and intracellular signal structure, CD137 intracellular signal structure, CD3 ζ
Intracellular signal structures in series constitute domain be T cell costimulatory signal conducting structure.
Preferably, the aminoacid sequence such as SEQ ID NO.4 institute of described T cell costimulatory signal conducting structure
Show.
Preferably, the aminoacid sequence of described Chimeric antigen receptor is as shown in SEQ ID NO.5.
The gene encoding above-mentioned EpCAM specific chimeric antigen receptor that the present invention also proposes, its nucleic acid sequence
Row are as shown in SEQ ID NO.6.
The above-mentioned EpCAM specific chimeric antigen receptor that the present invention also proposes is in preparation treatment EpCAM phase
Close the application in tumour medicine.
The heavy chain of the human-derived anti-human EpCAM antibody that the present invention delivered according to calendar year 2001 and the variable region sequence of light chain
Row (Raum, 2001Cancer Immunol Immunother.Stefanie 2002, Int.J.Cancer), pass through
Codon optimized, the ScFv sequence of gene chemical synthesis anti-EpCAM, and synthesis ScFv is cloned into
In pSFG-CH2CH3-CD28-CD137-CD3 ζ, i.e. construction cost is invented
EpCAM-scFv-CH2CH3-CD28-CD137-CD3 ζ Chimeric antigen receptor.
The present invention utilizes Chimeric antigen receptor and reversal of viral packaging plasmid PeqPam3 and RD114env to exist
293T cell is packaged into reversal of viral, utilizes this virus to infect T lymphocyte.Elisa is utilized to detect CAR-T
Cell is the secretion of IFN-γ under antigenic stimulus, and by the flow cytometer detection tumor cell to the EpCAM positive
Lethal effect.
The present invention announces the structure of the specific Chimeric antigen receptor of EpCAM, and Infection in Vitro T lymph is thin
The technology of born of the same parents.The killing tumor cells such as the T lymphocyte release IFN-γ after modification.EpCAM specificity
Chimeric antigen receptor to may be used for EpCAM related neoplasms (as female thin in adenocarcinoma, hepatocarcinoma, retina
Born of the same parents' tumor etc.) targeted therapy.
Accompanying drawing explanation
Fig. 1 is the structural representation of the EpCAM specific chimeric antigen receptor that the present invention proposes.
Fig. 2 is the electrophoretogram of the PCR primer of gained EpCAM-scFv of the present invention.
Fig. 3 is the electrophoretogram of gained EpCAM specific chimeric antigen receptor of the present invention.
Fig. 4 is the result figure of the expression of CAR molecule in flow cytometer detection EpCAM specific C AR-T cell.
Fig. 5 is the ratiometric result figure of flow cytometry tumor cell and T cell.
Fig. 6 is gained Chimeric antigen receptor EpCAM specific C AR-T cell of the present invention and lymphoma cell
MTT testing result figure.
Fig. 7 is that ELISA detects gained CAR-T cell of the present invention IFN-γ after being stimulated by EpCAM
Expression of results figure with IL2.
Detailed description of the invention
Below, by specific embodiment, technical scheme is described in detail.
The structure of embodiment 1:EpCAM specific chimeric antigen receptor reversal of viral carrier
A kind of EpCAM specific chimeric antigen receptor that the present invention proposes, by the strand of people's anti-EpCAM
Antibody, the cross-film district of CH2CH3, CD28 of people's IgG antibody 1 and intracellular signal structure, the born of the same parents of CD137
Interior signal structure, the intracellular signal structures in series of CD3 ζ are constituted, its aminoacid sequence such as SEQ ID NO.1
Shown in.
1, utilize the human-derived anti-human EpCAM monoclonal antibody sequences that calendar year 2001 delivers, select VH and VL
Sequence, for designing the scFv sequence in CAR carrier, use Jin Sirui biotech company
Codon is optimized by OptimumGeneTM gene design software further, divides at the sequence two ends optimized
Not Jia Shang NcoI, BAMHI restriction enzyme site, the sequence after optimization is complete by Nanjing Jin Sirui biotech company
Becoming gene chemical synthesis, the sequence after synthesis is cloned in pCDNA3 carrier, and plasmid is named
EpCAM-scFv-pCDNA3。
The EpCAM-scFv Sequence wherein inserted can carry out amplification via PCR and obtain, and then carries out
Detection, as in figure 2 it is shown, wherein band 1 is the PCR primer of EpCAM-scFv, about 750bp；Bar
It it is the reference band of 100bp with 2.
2, EpCAM-scFv-pCDNA3 plasmid restricted enzyme NcoI and BAMHI enzyme action, enzyme
Cut system as follows: 2 μ g EpCAM-scFv-pCDNA3,1 μ L NcoI, 1 μ L BamHI, 2 μ L 10 × enzyme action
Buffer, mends 20 μ L with water, in 37 DEG C of water-baths overnight, digestion products with 1% agarose gel electrophoresis,
Under ultraviolet, cut purpose band, use DNA gel to reclaim test kit (AXYGEN company) and reclaim mesh
Fragment；With same method NcoI and BAMHI enzyme action pSFG-CH2CH3-CD28-CD137-CD3 ζ
Carrier, with agarose gel electrophoresis, reclaims purpose fragment.EpCAM-ScFv after reclaiming carries with enzyme action
Body is connected by T4DNA ligase, and reaction system is as follows: 2 μ L EpCAM-ScFv, 2 μ L carrier enzyme action
Product, 1 μ L 10 × connection buffer, 1 μ L ligase, mend 10 μ L with water, in 16 DEG C of water-baths overnight.
Take connection product to join in DH5 α competence [with reference to E. coli competent in " molecular cloning " third edition
Preparation (CaCl2Method)], place incubated overnight in 37 DEG C of bacteriological incubators.The single bacterium colony of picking, expands training
Support, extract the plasmid of positive colony, through enzyme action (Fig. 3) and order-checking, by named for correct carrier
The EpCAM specificity that EpCAM-scFv-CH2CH3-CD28-CD137-CD3 ζ, the i.e. present invention propose is embedding
Close antigen receptor.
Being detected by gained EpCAM specific chimeric antigen receptor, its result is as it is shown on figure 3, wherein
Band 1 is the comparison band of 100bp, and band 2 is gained EpCAM specific chimeric antigen receptor.
The preparation of the T lymphocyte that embodiment 2:EpCAM specific chimeric antigen receptor is modified
1. the packaging containing EpCAM specific chimeric antigen receptor reversal of viral
Retrovirus is extracted with without the operating instruction of (sky root is biological) in the big extraction reagent kit of endotoxin plasmid
Package carrier pRD114 and EpCAM-scFv-CH2CH3-CD28-CD137-CD3 ζ retroviral vector
Cotransfection is in 293T cell, and after transfection, 48h collects cell conditioned medium, and 4000rpm is centrifuged 10min, removes
Cell in supernatant and cell debris.With the membrane filtration of 0.45 μm, subpackage is frozen, standby.
The preparation of 2.T lymphocyte
Take the fresh anticoagulation of 20mL healthy volunteer, with lymph separation liquid (GE company) discrete periphery
Blood monocyte (PBMC).Cell CD3 and CD28 flat board after separation stimulate 48h, use T lymph
Cell culture medium GT-T551 (TAKARA company) adds 3% self blood plasma and induces, and obtains T lymph
Cell.
The preparation of 3.CAR-T cell
It is coated non-tissue culturing plate 24 orifice plate, often with the RetroNectin (TAKARA company) of 50 μ g/mL
Hole adds 500 μ L, and 4 DEG C overnight.Every hole adds 500 μ L viral supernatants, hatches 30min for 37 DEG C.Remove
Viral supernatants, adds 500 μ L viral supernatants, hatches 30min for 37 DEG C, and except viral supernatants, every hole adds
1.5mL viral supernatants, adds the T lymphocyte after 0.5mL dilution, thus obtains
EpCAM-scFv-CH2CH3-CD28-CD137-CD3 ζ cell, i.e. EpCAM specific C AR-T is thin
Born of the same parents.
4. the expression of CAR molecule in flow cytometer detection EPCAM specific C AR-T cell
CAR-T cell is added the anti-Fc antibody (Jackson company) of 5 μ L FITC labellings, hatches for 37 DEG C
After 1h, carry out flow cytometer detection, testing result as shown in Figure 4: the most left figure be comparison T lymphocyte,
Right figure is the retroviral T lymphocyte infecting Chimeric antigen receptor, as shown in Figure 4: this method institute
Obtain the efficiency of CAR-T cell more than 90%.
Embodiment 3: Chimeric antigen receptor EpCAM specific C AR-T cell is to EpCAM related neoplasms
Lethal effect
The lethality detection of 1.CAR-T cells against tumor
The colon cancer cell HT-29 culture medium that EpCAM is positive is adjusted to 5 × 106/ mL, every hole 50 μ L,
By effector lymphocyte and target cell ratio for 4:1, add oncocyte 2.5 × 105Individual, after cell is the most adherent, receive
Collection T cell and CAR-T cell, adjusting cell concentration is 1.25 × 106/ mL, every hole 50 μ L, then distinguish
Carry out fluidic cell (cultivating 24h) and MTT detection (cultivating 4h).
First, collect cell and carry out the dyeing of CD3, carry out flow cytometry tumor cell (CD3-) and
T cell (CD3+) ratio, its result is as shown in Figure 5.As shown in Figure 5: gained inosculating antibody of the present invention
Original receptor EpCAM specific C AR-T cell can effectively kill colon cancer cell.
Secondly, same experiment is abandoned supernatant and is added the CCK8 of 100 μ L dilutions, hatches 4 hours, microplate reader
The light absorption value of detection OD450.As shown in Figure 6, EpCAM is expressed its testing result by CAR-T cell
The tumor cell that the killing rate of tumor is not expressed higher than EpCAM；Related neoplasms is killed by CAR-T cell to be made
With higher than T cell, higher than media alone.
2.ELISA detection CAR-T cell IFN-γ and IL2 after being stimulated by EPCAM are expressed
Colon cancer cell HT-29 culture medium is adjusted to 5 × 106/ mL, every hole 50 μ L, add colon cancer
Cell HT-29 2.5 × 105Individual, after cell is the most adherent, collect T and CAR-T cell, adjust cell
Concentration is 1 × 106/ mL, every hole 50 μ L, cultivate 24h.Collect supernatant, utilize ELISA kit to examine
Survey IFN-γ and the expression of IL2.
Its result is as it is shown in fig. 7, the T lymphocyte that EpCAM specific chimeric antigen receptor is modified is being received
Express and secretion of gamma-IFN and IL2 after stimulating to colon cancer cell HT-29 positive for EpCAM.
The above, the only present invention preferably detailed description of the invention, but protection scope of the present invention not office
Being limited to this, any those familiar with the art is in the technical scope that the invention discloses, according to this
The technical scheme of invention and inventive concept thereof in addition equivalent or change, all should contain the protection in the present invention
Within the scope of.
Claims (10)
1. an EpCAM specific chimeric antigen receptor, it is characterised in that described Chimeric antigen receptor
The single-chain antibody of born of the same parents' exoantigen land behaviour anti-EpCAM.
EpCAM specific chimeric antigen receptor the most according to claim 1, it is characterised in that described
The aminoacid sequence of the single-chain antibody of people's anti-EpCAM is as shown in SEQ ID NO.1.
EpCAM specific chimeric antigen receptor the most according to claim 1 or claim 2, it is characterised in that
Individual connection peptides is had, its aminoacid between heavy chain and the light chain molecule of the single-chain antibody of described embedding people's anti-EpCAM
Sequence is as shown in SEQ ID NO.2.
4., according to EpCAM specific chimeric antigen receptor described in any one of claim 1-3, its feature exists
In, the amino terminal of described Chimeric antigen receptor contains a signal peptide, its aminoacid sequence such as SEQ ID
Shown in NO.3.
5., according to EpCAM specific chimeric antigen receptor described in any one of claim 1-4, its feature exists
In, described Chimeric antigen receptor by the single-chain antibody of people's anti-EpCAM, the CH2CH3 of people's IgG antibody 1,
The cross-film district of CD28 and intracellular signal structure, the intracellular signal structure of CD137, the intracellular signal of CD3 ζ
Structures in series is constituted.
6., according to EpCAM specific chimeric antigen receptor described in any one of claim 1-5, its feature exists
In, the cross-film district of described CD28 and intracellular signal structure, CD137 intracellular signal structure, the born of the same parents of CD3 ζ
Interior signal structure domain in series is T cell costimulatory signal conducting structure.
EpCAM specific chimeric antigen receptor the most according to claim 6, it is characterised in that described
The aminoacid sequence of T cell costimulatory signal conducting structure is as shown in SEQ ID NO.4.
8., according to EpCAM specific chimeric antigen receptor described in any one of claim 1-7, its feature exists
In, the aminoacid sequence of described Chimeric antigen receptor is as shown in SEQ ID NO.5.
9. coding gene of EpCAM specific chimeric antigen receptor as described in any one of claim 1-8,
It is characterized in that, its nucleotide sequence is as shown in SEQ ID NO.6.
10. as described in any one of claim 1-8, EpCAM specific chimeric antigen receptor is treated in preparation
Application in EpCAM related neoplasms medicine.
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| US20210145881A1 (en) * | 2018-04-09 | 2021-05-20 | Mayo Foundation For Medical Education And Research | Methods and materials for treating graft-versus-host disease |
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| CN113527509B (en) * | 2020-04-17 | 2023-09-26 | 深圳华大生命科学研究院 | Use of complexes containing recombinant proteins and protective proteins for the treatment of squamous cell lung carcinoma |
| WO2023196953A3 (en) * | 2022-04-08 | 2023-11-09 | The Wistar Institute Of Anatomy And Biology | Combinations of bispecific t cell engagers and methods of use thereof |
| WO2023196954A3 (en) * | 2022-04-08 | 2023-11-30 | The Wistar Institute Of Anatomy And Biology | Bispecific binding molecules that target fshr and cd3 |
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