CN109957016A - It is a kind of to target the single-chain antibody of CD38, Chimeric antigen receptor T cell and its preparation method and application - Google Patents
It is a kind of to target the single-chain antibody of CD38, Chimeric antigen receptor T cell and its preparation method and application Download PDFInfo
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- CN109957016A CN109957016A CN201711421576.5A CN201711421576A CN109957016A CN 109957016 A CN109957016 A CN 109957016A CN 201711421576 A CN201711421576 A CN 201711421576A CN 109957016 A CN109957016 A CN 109957016A
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Abstract
The present invention provides a kind of single-chain antibody for targeting CD38, the single-chain antibody of the targeting CD38 includes the amino acid sequence as shown in SEQ ID NO:1, and the single-chain antibody of the targeting CD38 is Humanized single chain antibody.The present invention also provides the Chimeric antigen receptor T cells for the single-chain antibody for including the targeting CD38, the Chimeric antigen receptor of the targeting CD38 can targeted expression CD38 in specific manner tumour cell, promote the amplification of T cell, efficient and specific killing tumor cell.In addition, the Chimeric antigen receptor T cell of Humanized single chain antibody targeting CD38 obtained has the vigor and lethality for preferably maintaining cell.The present invention also provides the preparation method and application of the Chimeric antigen receptor T cell of targeting CD38.
Description
Technical field
The present invention relates to field of medical biotechnology, in particular to a kind of single-chain antibody, Chimeric antigen receptor T for targeting CD38 is thin
Born of the same parents and its preparation method and application.
Background technique
Malignant tumour (cancer) has become the arch-criminal for threatening human life, and disease incidence is at ascendant trend.CD38 is a kind of list
II type transmembrane glycoprotein of chain, is mainly expressed in myeloma, lymthoma etc., expression quantity is higher in malignant plasma cell.CAR-T is (embedding
Close antigen receptor T cell) technology is a kind of novel cell therapy, it is will to be fed back to human body by the T cell of CAR transformation, is swashed
Self immune system living, kills tumour cell, it is considered to be one of the therapeutic modality of current most effective malignant tumour.
There are not the preparation and research of the Chimeric antigen receptor T cell of targeting CD38 also at present.In addition, CAR-T cell maintains energy in body
Power (per sistence) is the guarantee of CAR-T long-term efficacy, but actually CAR-T a few days to a few weeks after feedback just disappear completely
It loses, and fails to play the ability of long-acting lasting killing tumor cell.
Summary of the invention
In view of this, the present invention provides a kind of single-chain antibodies for targeting CD38, and the list including the targeting CD38
The Chimeric antigen receptor T cell of chain antibody.The Chimeric antigen receptor of the targeting CD38 can target CD38 in specific manner, promote
T cell is expanded in patient's body, efficiently and specifically killing tumor cell, while the targeting CD38 with Humanized single chain antibody
Chimeric antigen receptor T cell can enduringly maintain its self-renewing vigor and lethality.The present invention also provides a kind of targets
To the preparation method and application of the Chimeric antigen receptor T cell of CD38.
In a first aspect, the present invention provides a kind of single-chain antibody for targeting CD38, the single-chain antibody packet of the targeting CD38
The amino acid sequence as shown in SEQ ID NO:1 is included, the single-chain antibody of the targeting CD38 is Humanized single chain antibody.
Optionally, the encoding gene of the single-chain antibody of the targeting CD38 includes the nucleotide as shown in SEQ ID NO:2
Sequence.
Optionally, the single-chain antibody encoding gene of the targeting CD38 should consider degeneracy base, i.e., such as SEQ ID NO:1
Shown in the encoding gene of amino acid sequence include the nucleotide sequence as shown in SEQ ID NO:2, protection scope should also protect
Shield has the nucleotide sequence of base degeneracy matter with SEQ ID NO:2, and the corresponding amino acid sequence of these nucleotide sequences is still
It is so SEQ ID NO:1.
Specificity knot can occur with CD38 albumen for the single-chain antibody for the targeting CD38 that first aspect present invention provides
It closes, has fine targeting to the tumour cell of expression CD38.In addition, the single-chain antibody of the targeting CD38 is anti-for Humanized single chain
Body, can be to avoid the immune response for causing human organism.
Second aspect, the present invention provides a kind of Chimeric antigen receptor T cells for targeting CD38, including the embedding of targeting CD38
Antigen receptor CAR-CD38 is closed, the CAR-CD38 includes the single-stranded anti-of the sequentially connected targeting CD38 from aminoterminal to c-terminus
Body, extracellular hinge area, transmembrane region and intracellular signal area amino acid sequence, wherein it is described targeting CD38 single-chain antibody include such as
Amino acid sequence shown in SEQ ID NO:1.
Wherein, described " being sequentially connected with from aminoterminal to c-terminus " specifically: the ammonia of the single-chain antibody of the targeting CD38
The c-terminus of base acid sequence is connected with the aminoterminal of the amino acid sequence of the hinge area, the amino acid sequence of the extracellular hinge area
The c-terminus of column is connected with the aminoterminal of the amino acid sequence of the transmembrane region, the c-terminus of the amino acid sequence of the transmembrane region
It is connected with the aminoterminal of the amino acid sequence in the intracellular signal area.
In the present invention, the extracellular hinge area is used to promote the CD38 knot on the single-chain antibody and tumour of the targeting CD38
It closes.Optionally, the extracellular hinge area includes CD8 α hinge area, CD28 hinge area, CD4 hinge area, CD5 hinge area, CD134 hinge
One of sequence, CD137 hinge area, ICOS hinge area or a variety of combinations.
Further alternative, the extracellular hinge area is CD8 α hinge area.
Optionally, the amino acid sequence of the CD8 α hinge area includes the amino acid sequence as shown in SEQ ID NO:6.
Optionally, the encoding gene of the CD8 α hinge area includes the nucleotide sequence as shown in SEQ ID NO:7.
Optionally, the encoding gene of the CD8 α hinge area should consider degeneracy base, i.e., as shown in SEQ ID NO:6
The encoding gene of amino acid sequence includes the nucleotide sequence as shown in SEQ ID NO:7, and protection scope should also protect and SEQ
ID NO:7 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ
ID NO:6。
In the present invention, the transmembrane region is used to fix the Chimeric antigen receptor CAR-CD38 of the targeting CD38.Optionally,
The transmembrane region includes one of CD3 transmembrane region, CD4 transmembrane region, CD8 transmembrane region, CD28 transmembrane region or a variety of combinations.
Further alternative, the transmembrane region is CD8 transmembrane region.
Optionally, the amino acid sequence of the CD8 transmembrane region includes the amino acid sequence as shown in SEQ ID NO:8.
Optionally, the encoding gene of the CD8 transmembrane region includes the nucleotide sequence as shown in SEQ ID NO:9.
Optionally, the encoding gene of the CD8 transmembrane region should consider degeneracy base, i.e., as shown in SEQ ID NO:8
The encoding gene of amino acid sequence includes the nucleotide sequence as shown in SEQ ID NO:9, and protection scope should also protect and SEQ
ID NO:9 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ
ID NO:8。
The intracellular signal area is for providing the signal of T cell activation in the present invention, maintain T cell life span and
Activate T cell proliferation signal access.Optionally, the intracellular signal area includes 4-1BB signaling zone, CD3 ζ signaling zone, ICOS letter
Number area, CD27 signaling zone, OX40 signaling zone, CD28 signaling zone, IL1R1 signaling zone, CD70 signaling zone, TNFRSF19L signaling zone
One of or a variety of combinations.
Optionally, the intracellular signal Qu Weicong aminoterminal is to the sequentially connected CD27 signaling zone of c-terminus and CD3 ζ signal
Area.Correspondingly, the encoding gene in the intracellular signal area includes the coding from 5 ' ends to 3 ' the sequentially connected CD27 signaling zones in end
The encoding gene of gene and CD3 ζ signaling zone.
Optionally, the amino acid sequence of the CD27 signaling zone includes the amino acid sequence as shown in SEQ ID NO:10.
The CD27 signaling zone is as costimulatory signal.
Optionally, the encoding gene of the CD27 signaling zone includes the nucleotide sequence as shown in SEQ ID NO:11.
Optionally, the encoding gene of the CD27 signaling zone should consider degeneracy base, i.e., as shown in SEQ ID NO:10
Amino acid sequence encoding gene include the nucleotide sequence such as SEQ ID NO:11 shown in, protection scope should also protect and
SEQ ID NO:11 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as
SEQ ID NO:10。
Optionally, the amino acid sequence of the CD3 ζ signaling zone includes the amino acid sequence as shown in SEQ ID NO:12.
Optionally, the encoding gene of the CD3 ζ signaling zone includes the nucleotide sequence as shown in SEQ ID NO:13.
Optionally, the encoding gene of the CD3 ζ signaling zone should consider degeneracy base, i.e., as shown in SEQ ID NO:12
Amino acid sequence encoding gene include the nucleotide sequence such as SEQ ID NO:13 shown in, protection scope should also protect and
SEQ ID NO:13 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as
SEQ ID NO:12。
Optionally, the amino acid sequence of the CAR-CD38 includes the amino acid sequence as shown in SEQ ID NO:3.This
When, antigentic specificity signal of the CD3 ζ signaling zone as shown in the amino acid sequence of SEQ ID NO:12 as the T cell
(that is, first signal), costimulation of the CD27 signaling zone as the T cell as shown in the amino acid sequence of SEQ ID NO:10
Signal, under their collective effect, T cell is activated completely after identifying antigen.Particularly, such as SEQ ID NO:10 is selected
CD27 signaling zone as costimulatory signal, can preferably promote amplification ability, the killing vigor of subsequent T cell.
Optionally, the encoding gene of the CAR-CD38 includes the nucleotide sequence as shown in SEQ ID NO:4.
Optionally, the encoding gene of the CAR-CD38 should consider degeneracy base, the i.e. ammonia as shown in SEQ ID NO:3
The encoding gene of base acid sequence includes the nucleotide sequence as shown in SEQ ID NO:4, and protection scope should also protect and SEQ
ID NO:4 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ
ID NO:3。
Further, the encoding gene of the CAR-CD38 includes the nucleotide sequence as shown in SEQ ID NO:5.
The Chimeric antigen receptor T cell for the targeting CD38 that second aspect of the present invention provides, including the embedding of targeting CD38
Close antigen receptor CAR-CD38, this receptor for T cell targeted expression CD38 in specific manner tumour cell, in CAR-
After CD38 is in conjunction with CD38, the intracellular signal area of the T cell is activated, and promotes T cell in the amplification of patient's body, and efficiently
And the killing tumor cell of specificity, and normal cell is hardly caused to damage.In addition, the single-chain antibody of targeting CD38 is
Humanized single chain antibody, this makes the Chimeric antigen receptor T cell of the targeting CD38 be avoided the immune response for causing human organism,
Ability (including vigor and lethality) is maintained in body with lasting.
The third aspect, the present invention provides a kind of recombinant viral vector, the recombinant viral vector includes such as second aspect
The encoding gene of the CAR-CD38 of the Chimeric antigen receptor T cell of the targeting CD38.
Optionally, the encoding gene of the CAR-CD38 includes the nucleotide sequence as shown in SEQ ID NO:4.
Preferably, the encoding gene of the CAR-CD38 includes the nucleotide sequence as shown in SEQ ID NO:5.Such as SEQ
Nucleotide sequence shown in ID NO:5 is compared with the nucleotide sequence as shown in SEQ ID NO:4, more coding bases of link peptide
Cause.The encoding gene of the signal peptide can be expressed with Chimeric antigen receptor CAR-CD38 described in guide to cell surface.
Optionally, the viral vectors in the recombinant viral vector includes slow virus carrier, adenovirus vector or reverse transcription
Viral vectors.
Further alternative, the viral vectors is slow virus carrier.
The recombinant viral vector that third aspect present invention provides has very high efficiency of infection and transcriptional efficiency, inserts
The CAR-CD38 encoding gene segment entered can by genetic recombination be inserted into host genome, obtain targeting CD38 chimeric antigen by
Body T cell continues it, the Chimeric antigen receptor CAR-CD38 of steadily expression targeting CD38.
Fourth aspect, the present invention provides a kind of host cell, the host cell includes the weight as described in the third aspect
Group viral vectors.
The host cell is used to assemble the recombinant viral vector as described in the third aspect, makes it have infectivity.
Optionally, the host cell may include HEK293T cell, 293 cells, 293T cell, 293FT cell,
SW480 cell, u87MG cell, HOS cell, COS1 cell, COS7 cell etc., but not limited to this.
Further alternative, the host cell is HEK293T cell.
5th aspect, the present invention provides a kind of preparation methods of Chimeric antigen receptor T cell for targeting CD38, comprising:
(1) encoding gene of the Chimeric antigen receptor CAR-CD38 of targeting CD38 is provided, including sequentially from 5 ' ends to 3 ' ends
The encoding gene of the signal peptide of connection, the encoding gene of single-chain antibody, the encoding gene of extracellular hinge area, cross-film for targeting CD38
The encoding gene in area, intracellular signal area encoding gene;The single-chain antibody of the targeting CD38 is Humanized single chain antibody, described
The encoding gene for targeting the single-chain antibody of CD38 includes the nucleotide sequence as shown in SEQ ID NO:2;
(2) gene order of the CAR-CD38 is inserted into pWPXLD carrier, obtains pWPXLD-CAR-CD38 recombination
Plasmid;
(3) it by the pWPXLD-CAR-CD38 recombinant plasmid and envelope plasmid, packaging plasmid cotransfection host cell, obtains
To recombinant slow virus;
(4) recombinant slow virus is transfected into CD3 positive t lymphocytes, the Chimeric antigen receptor T for obtaining targeting CD38 is thin
Born of the same parents.
It is above-mentioned " from 5 ' end to 3 ' end be sequentially connected with " specifically: the coding gene sequence of the signal peptide 3 ' end with it is described
5 ' the ends for targeting the coding gene sequence of the single-chain antibody of CD38 are connected, the encoding gene sequence of the single-chain antibody of the targeting CD38
3 ' ends of column are connected with 5 ' ends of the coding gene sequence of the extracellular hinge area, the coding gene sequence of the extracellular hinge area
3 ' ends be connected with the 5 ' of the coding gene sequence of transmembrane region ends, the 3 ' of the coding gene sequence of the transmembrane region are held and institute
5 ' the ends for stating the coding gene sequence in intracellular signal area are connected.
The signal peptide is for instructing the Chimeric antigen receptor CAR-CD38 expression to cell surface, institute in the present invention
Signal peptide is stated to be cut in protein translation maturation by signal peptidase.
Optionally, the amino acid sequence of the signal peptide includes the amino acid sequence as shown in SEQ ID NO:14.
Optionally, the encoding gene of the signal peptide includes the nucleotide sequence as shown in SEQ ID NO:15.
Optionally, the coding gene sequence of the signal peptide should consider degeneracy base, i.e., as shown in SEQ ID NO:14
Amino acid sequence encoding gene include the nucleotide sequence such as SEQ ID NO:15 shown in, protection scope should also protect and
SEQ ID NO:15 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as
SEQ ID NO:14。
It, can for the extracellular hinge area, transmembrane region, the specific choice in intracellular signal area and corresponding coding gene sequence
Referring to the description of second aspect of the present invention part, which is not described herein again.
Optionally, the encoding gene of the CAR-CD38 includes the nucleotide sequence as shown in SEQ ID NO:5.Such as SEQ
Nucleotide sequence shown in ID NO:5 is compared with the nucleotide sequence as shown in SEQ ID NO:4, more volumes of the link peptide
Code gene, but when Chimeric antigen receptor CAR-DR5 expression is to cell surface, the signal peptide is in protein translation maturation mistake
It is cut in journey by signal peptidase.Therefore, in the amino acid sequence of the Chimeric antigen receptor CAR-CD38 translated into not
Band is just like amino acid sequence shown in SEQ ID NO:14.
The coding gene sequence of the CAR-CD38 is inserted into pWPXLD carrier between I restriction enzyme site of BamH I and EcoR,
And be located at after the extension factor 1 α (EF1 α) of pWPXLD carrier, using EF1 α as promoter.The gene order of the CAR-CD38
When being inserted into pWPXLD carrier, the gene order of the CAR-CD38 5 ' end can also be added initiation codon (such as ATG) with
BamH1 restriction enzyme site is connected in pWPXLD carrier, and EcoR1 digestion position in terminator codon and pWPXLD carrier can be also added in 3 ' ends
Point is connected.
Optionally, the envelope plasmid is PMD2G, and the packaging plasmid is psPAX2, and the host cell is HEK293T
Cell.
The envelope plasmid PMD2G encodes vesicular stomatitis virus glycoprotein capsid, the vesicular stomatitis virus sugar egg
White capsid can assist recombinant slow virus to adhere to cell membrane, and keep the infectivity of recombinant slow virus.
Recombinant slow virus of the present invention can further contain the envelope protein from other viruses.For example, as this
Kind protein, is preferably the virus enveloped protein for carrying out self-infection human cell.To this protein, there is no particular limitation, can example
The amphophilic virus hand epithelium albumen etc. for enumerating retrovirus, can be used for example from mouse leukemia virus (MuMLV)
4070A plants of envelope protein.Alternatively, it is also possible to use the envelope protein from MuMLV 10Al.In addition, as herpetoviridae
Albumen, it can be cited for example that, gB, gD, gH, gp85 albumen of herpes simplex virus, gp350, gp220 albumen of Epstein-Barr virus
Deng.As the albumen of Hepadna Virus section, the S protein etc. of hepatitis B virus can be included.The envelope protein can also be morbilli
It is formed after viral glycoprotein and other single chain antibody fusions.
The packaging of recombinant slow virus is generallyd use transient transfection or is packed using cell line.It may be used as wrapping when transient transfection
Human cell's strain that dress cell uses, for example including 293 cells, 293T cell, 293FT cell, 293LTV cell, 293EBNA
Cell and other clones separated from 293 cells;SW480 cell, u87MG cell, HOS cell, C8166 cell, MT-4 are thin
Born of the same parents, Molt-4 cell, HeLa cell, HT1080 cell, TE671 cell etc..It can also be using the cell strain from monkey, example
Such as, COS1 cell, COS7 cell, CV-1 cell, BMT10 cell etc..Moreover, the calcium phosphate and PEI transfection reagent that generally use,
It is also well used there are also some transfection reagents such as Lipofectamine2000, FuGENE and S93fectin.
The packaging of recombinant slow virus also uses some slow virus package cell lines, such as most common Env glycoprotein of use,
Stable cell lines caused by VSVG albumen or HIV-1gag-pol albumen.
For the sake of security, the slow virus carrier system of large-scale use is all the method using split gene group, i.e., will
The assignment of genes gene mapping of different miscellaneous functions is played in different plasmids.There are four pUC pUCs (encoding gag-pol gene, Rev base at present
Cause, VSVG gene, SIN metastatic gene be located at four different plasmids), three pUC pUCs (eliminate coding Rev gene
Plasmid, gag-pol gene uses the codon of the preferences in people's cell in gag-pol plasmid) and two pUC pUCs are (slowly
Auxiliary gene necessary to viral vectors is packed is located on the same plasmid, these auxiliary genes are single gene orders;Separately
One is then transgenosis plasmid).Also the slow virus packaging system for having more than four pUC pUCs is using.
Optionally, in step (4), the CD3 positive t lymphocytes are to separate to obtain from source of people peripheral blood mononuclear cells
?.
Optionally, the source of people peripheral blood mononuclear cells derives from autologous vein blood, autologous bone marrow, Cord blood and placenta
Blood etc..It is further alternative, from cancer patient perform the operation one month after, the fresh peripheral blood that acquires after chemicotherapy one month or
Marrow.
Specifically, the acquisition process of the CD3 positive t lymphocytes is as follows: first by peripheral blood mononuclear cells by certain
CD3/CD28 immunomagnetic beads are added in ratio, and after being incubated for a period of time, are put into magnet and are screened, it is coated to obtain immunomagnetic beads
CD3 positive t lymphocytes after removing magnetic bead, obtain CD3 positive t lymphocytes.
6th aspect, the present invention provides the single-chain antibodies of targeting CD38 as described in relation to the first aspect a kind of, such as second party
Described in face or the Chimeric antigen receptor T cell of targeting CD38, such as third party made from the preparation method as described in terms of the 5th
Recombinant viral vector described in face or the host cell as described in fourth aspect are in preparation prevention, diagnosing and treating malignant-tumor agent
Application in object.It is particularly suitable for the malignant tumour of expression CD38.For example, can be used for the malignant tumours such as myeloma, lymthoma
Prevention, diagnosing and treating etc..
The application specifically: provide a kind of kit, the kit includes targeting CD38 described in first aspect
Single-chain antibody, as described in second aspect targeting CD38 Chimeric antigen receptor T cell, as described in the third aspect recombination disease
One of poisonous carrier, host cell as described in fourth aspect are a variety of.
Advantages of the present invention will be illustrated partially in the following description, and a part is apparent according to specification
, or can implementation through the embodiment of the present invention and know.
Detailed description of the invention
Fig. 1 is the plasmid map of pWPXLd-CAR-CD38 recombinant plasmid provided in an embodiment of the present invention.
Specific embodiment
The following is a preferred embodiment of the present invention, it is noted that for those skilled in the art
For, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also considered as
Protection scope of the present invention.
Embodiment one
A kind of preparation method for the Chimeric antigen receptor T cell targeting CD38, comprising the following steps:
(1) gene order of the Chimeric antigen receptor CAR-CD38 of preparation targeting CD38
Prepare respectively signal peptide, target the single-chain antibody of CD38, CD8 α hinge area, CD8 transmembrane region, CD27 signaling zone and
The encoding gene of CD3 ζ signaling zone, the coding gene sequence of the signal peptide is as shown in SEQ ID NO:15, the targeting CD38
Single-chain antibody coding gene sequence as shown in SEQ ID NO:2, the coding gene sequence such as SEQ of the CD8 α hinge area
Shown in ID NO:7, the coding gene sequence of the CD8 transmembrane region is as shown in SEQ ID NO:9, the coding of the CD27 signaling zone
Gene is as shown in SEQ ID NO:11, and the encoding gene of the CD3 ζ signaling zone is as shown in SEQ ID NO:13.
By the method for PCR by above-mentioned signal peptide, the single-chain antibody for targeting CD38, CD8 α hinge area, CD8 transmembrane region,
CD27 signaling zone is successively connected together from 5 ' ends to 3 ' ends with the encoding gene of CD3 ζ signaling zone, obtains the chimeric of targeting CD38
The encoding gene of antigen receptor CAR-CD38, the coding gene sequence of the CAR-CD38 is as shown in SEQ ID NO:5, wherein institute
The single-chain antibody for stating targeting CD38 is Humanized single chain antibody.
(2) pWPXLd-CAR-CD38 recombinant plasmid is constructed
The coding gene sequence of CAR-CD38 is inserted between BamH1 the and EcoR1 restriction enzyme site of pWPXLD carrier, and
After pWPXLD carrier EF1 α, using EF1 α as promoter.The coding gene sequence of the CAR-CD38 is inserted into pWPXLD load
When body, 5 ' ends of the coding gene sequence of the CAR-CD38 are added in initiation codon (such as ATG) and pWPXLD carrier
BamH1 restriction enzyme site is connected, and 3 ' ends are also connected added with terminator codon with EcoR1 restriction enzyme site in pWPXLD carrier.Then
It is transferred to competent escherichia coli cell DH5 α, carries out positive colony PCR identification and sequencing identification.By PCR product gel electrophoresis
Detection and sequencing identification meet target fragment size and sequence, successfully construct pWPXLd-CAR-CD38 recombination matter as shown in Figure 1
Grain.
(3) recombinant slow virus constructs
PWPXLd-CAR-CD38 recombinant plasmid, packaging plasmid psPAX2, envelope plasmid pMD2G three cotransfection are entered into training
The HEK293T cell supported.48h harvest is protected in -80 DEG C of ultra low temperature freezers containing the supernatant of virus through 0.45 μm of membrane filtration
It deposits;Supernatant of the 72h aftercrop containing virus, 0.45 μm of membrane filtration add together after merging with the viral supernatants of 48h harvest
Enter in the centrifuge tube that exceeds the speed limit, be put into Beckman ultracentrifuge one by one, setting parameter of noncentricity is 25000rpm, centrifugation time
For 2h, centrifuging temperature is controlled at 4 DEG C;It after centrifugation, discards supernatant, removal as far as possible remains in the liquid on tube wall, and disease is added
Poison saves liquid, and gently piping and druming is resuspended repeatedly;Through after completely dissolution, high speed centrifugation 10000rpm takes supernatant fluorescence after being centrifuged 5min
Method measures titre, and virus is according to 100 μ l, and 2 × 108A/mL packing, is stored in -80 DEG C of ultra low temperature freezers, obtains recombinant lentiviral disease
Poison.
(4) preparation of the Chimeric antigen receptor T cell of CD38 is targeted
A) separation of PBMC (peripheral blood mononuclear cells)
PBMC is from autologous vein blood, autologous bone marrow, Cord blood and placental blood etc..Preferably derive from cancer patient's hand
The fresh peripheral blood or marrow acquired after art one month, after chemicotherapy one month.
Extract patient blood, sample presentation to blood separating chamber;Peripheral blood mononuclear cells is acquired, is taken after Ficoll centrifuge separation
Intermediate layer cell;After PBS is washed, PBMC is obtained.
B) immunomagnetic beads antigenspecific T lymphocyte
Above-mentioned PBMC is taken, the basal medium for being free of serum is added, is made into cell suspension;Ratio in magnetic bead and cell is
3:1, is added CD3/CD28 immunomagnetic beads, and room temperature incubates 1-2h;The cell for being incubated for magnetic bead is screened using magnet;To screening
Cell out removes magnetic bead, and PBS washing obtains CD3 positive t lymphocytes.
C) virus transfection method prepares antigenspecific T lymphocyte
The above-mentioned CD3 positive t lymphocytes obtained by magnetic activated cell seperation are taken, are added and CD3 positive cell number phase
The recombinant slow virus for the virus titer answered is cultivated.
The 3rd day of culture carries out cell count and changes liquid, and adjustment cell concentration is 1 × 106A/mL is inoculated with, culture;Training
Cell state is observed in feeding the 5th day, if cell density increases, diluting cells concentration is 1 × 106A/mL detects cell
Activity continues to cultivate.Amplification cultivation collected cell by the 9-11 days, obtained the Chimeric antigen receptor T cell of targeting CD38, and
It is stored in and feeds back in dedicated cells frozen storing liquid, used so that patient feeds back.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention
Protect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Sequence table
<110>Shenzhen Bin De Bioisystech Co., Ltd
<120>a kind of single-chain antibody for targeting CD38, Chimeric antigen receptor T cell and its preparation method and application
<160> 15
<170> SIPOSequenceListing 1.0
<210> 1
<211> 251
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 1
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
115 120 125
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser
130 135 140
Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Asn Ser Phe Ala
145 150 155 160
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser
165 170 175
Ala Ile Ser Gly Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val Lys
180 185 190
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
195 200 205
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala
210 215 220
Lys Asp Lys Ile Leu Trp Phe Gly Glu Pro Val Phe Asp Tyr Trp Gly
225 230 235 240
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser
245 250
<210> 2
<211> 753
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60
ctctcctgca gggccagtca gagtgttagc agctacttag cctggtacca acagaaacct 120
ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg catcccagcc 180
aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag cctagagcct 240
gaagattttg cagtttatta ctgtcagcag cgtagcaact ggcctccgac gttcggccaa 300
gggaccaagg tggaaatcaa aggaggcgga ggatctggcg gcggaggaag tggcggaggg 360
ggatctgggg gaggcggaag cgaggtgcag ctgttggagt ctgggggagg cttggtacag 420
cctggggggt ccctgagact ctcatgtgca gtctctggat tcacctttaa cagctttgcc 480
atgagctggg tccgccaggc tccagggaag gggctggagt gggtctcagc tattagtggt 540
agtggtggtg gcacatacta cgcagactcc gtgaagggcc ggttcaccat ctccagagac 600
aattccaaga acacgctgta tctgcaaatg aacagcctga gagccgagga cacggccgta 660
tatttctgtg cgaaagataa gattctctgg ttcggggagc ccgtctttga ctactggggc 720
cagggaaccc tggtcaccgt ctcctcagcc tcc 753
<210> 3
<211> 478
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 3
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
115 120 125
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser
130 135 140
Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Asn Ser Phe Ala
145 150 155 160
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser
165 170 175
Ala Ile Ser Gly Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val Lys
180 185 190
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
195 200 205
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala
210 215 220
Lys Asp Lys Ile Leu Trp Phe Gly Glu Pro Val Phe Asp Tyr Trp Gly
225 230 235 240
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Thr Thr Pro Ala
245 250 255
Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser
260 265 270
Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr
275 280 285
Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala
290 295 300
Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys
305 310 315 320
Arg Lys Tyr Arg Ser Asn Lys Gly Glu Ser Pro Val Glu Pro Ala Glu
325 330 335
Pro Cys Arg Tyr Ser Cys Pro Arg Glu Glu Glu Gly Ser Thr Ile Pro
340 345 350
Ile Gln Glu Asp Tyr Arg Lys Pro Glu Pro Ala Cys Ser Pro Arg Val
355 360 365
Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn
370 375 380
Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val
385 390 395 400
Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg
405 410 415
Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys
420 425 430
Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg
435 440 445
Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys
450 455 460
Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
465 470 475
<210> 4
<211> 1434
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60
ctctcctgca gggccagtca gagtgttagc agctacttag cctggtacca acagaaacct 120
ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg catcccagcc 180
aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag cctagagcct 240
gaagattttg cagtttatta ctgtcagcag cgtagcaact ggcctccgac gttcggccaa 300
gggaccaagg tggaaatcaa aggaggcgga ggatctggcg gcggaggaag tggcggaggg 360
ggatctgggg gaggcggaag cgaggtgcag ctgttggagt ctgggggagg cttggtacag 420
cctggggggt ccctgagact ctcatgtgca gtctctggat tcacctttaa cagctttgcc 480
atgagctggg tccgccaggc tccagggaag gggctggagt gggtctcagc tattagtggt 540
agtggtggtg gcacatacta cgcagactcc gtgaagggcc ggttcaccat ctccagagac 600
aattccaaga acacgctgta tctgcaaatg aacagcctga gagccgagga cacggccgta 660
tatttctgtg cgaaagataa gattctctgg ttcggggagc ccgtctttga ctactggggc 720
cagggaaccc tggtcaccgt ctcctcagcc tccaccacga cgccagcgcc gcgaccacca 780
acaccggcgc ccaccatcgc gtcgcagccc ctgtccctgc gcccagaggc gtgccggcca 840
gcggcggggg gcgcagtgca cacgaggggg ctggacttcg cctgtgatat ctacatctgg 900
gcgcccttgg ccgggacttg tggggtcctt ctcctgtcac tggttatcac cctttactgc 960
aggaaatata gatcaaacaa aggagaaagt cctgtggagc ctgcagagcc ttgtcgttac 1020
agctgcccca gggaggagga gggcagcacc atccccatcc aggaggatta ccgaaaaccg 1080
gagcctgcct gctccccccg cgtgaaattc agccgcagcg cagatgctcc agcctacaag 1140
caggggcaga accagctcta caacgaactc aatcttggtc ggagagagga gtacgacgtg 1200
ctggacaagc ggagaggacg ggacccagaa atgggcggga agccgcgcag aaagaatccc 1260
caagagggcc tgtacaacga gctccaaaag gataagatgg cagaagccta tagcgagatt 1320
ggtatgaaag gggaacgcag aagaggcaaa ggccacgacg gactgtacca gggactcagc 1380
accgccacca aggacaccta tgacgctctt cacatgcagg ccctgccgcc tcgg 1434
<210> 5
<211> 1494
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
gccctgcctg tgacagccct gctgctgcct ctggctctgc tgctgcatgc cgctagaccc 60
gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 120
ctctcctgca gggccagtca gagtgttagc agctacttag cctggtacca acagaaacct 180
ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg catcccagcc 240
aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag cctagagcct 300
gaagattttg cagtttatta ctgtcagcag cgtagcaact ggcctccgac gttcggccaa 360
gggaccaagg tggaaatcaa aggaggcgga ggatctggcg gcggaggaag tggcggaggg 420
ggatctgggg gaggcggaag cgaggtgcag ctgttggagt ctgggggagg cttggtacag 480
cctggggggt ccctgagact ctcatgtgca gtctctggat tcacctttaa cagctttgcc 540
atgagctggg tccgccaggc tccagggaag gggctggagt gggtctcagc tattagtggt 600
agtggtggtg gcacatacta cgcagactcc gtgaagggcc ggttcaccat ctccagagac 660
aattccaaga acacgctgta tctgcaaatg aacagcctga gagccgagga cacggccgta 720
tatttctgtg cgaaagataa gattctctgg ttcggggagc ccgtctttga ctactggggc 780
cagggaaccc tggtcaccgt ctcctcagcc tccaccacga cgccagcgcc gcgaccacca 840
acaccggcgc ccaccatcgc gtcgcagccc ctgtccctgc gcccagaggc gtgccggcca 900
gcggcggggg gcgcagtgca cacgaggggg ctggacttcg cctgtgatat ctacatctgg 960
gcgcccttgg ccgggacttg tggggtcctt ctcctgtcac tggttatcac cctttactgc 1020
aggaaatata gatcaaacaa aggagaaagt cctgtggagc ctgcagagcc ttgtcgttac 1080
agctgcccca gggaggagga gggcagcacc atccccatcc aggaggatta ccgaaaaccg 1140
gagcctgcct gctccccccg cgtgaaattc agccgcagcg cagatgctcc agcctacaag 1200
caggggcaga accagctcta caacgaactc aatcttggtc ggagagagga gtacgacgtg 1260
ctggacaagc ggagaggacg ggacccagaa atgggcggga agccgcgcag aaagaatccc 1320
caagagggcc tgtacaacga gctccaaaag gataagatgg cagaagccta tagcgagatt 1380
ggtatgaaag gggaacgcag aagaggcaaa ggccacgacg gactgtacca gggactcagc 1440
accgccacca aggacaccta tgacgctctt cacatgcagg ccctgccgcc tcgg 1494
<210> 6
<211> 45
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 6
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 10 15
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
20 25 30
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
35 40 45
<210> 7
<211> 135
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgat 135
<210> 8
<211> 24
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 8
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu
1 5 10 15
Ser Leu Val Ile Thr Leu Tyr Cys
20
<210> 9
<211> 72
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60
accctttact gc 72
<210> 10
<211> 46
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 10
Arg Lys Tyr Arg Ser Asn Lys Gly Glu Ser Pro Val Glu Pro Ala Glu
1 5 10 15
Pro Cys Arg Tyr Ser Cys Pro Arg Glu Glu Glu Gly Ser Thr Ile Pro
20 25 30
Ile Gln Glu Asp Tyr Arg Lys Pro Glu Pro Ala Cys Ser Pro
35 40 45
<210> 11
<211> 138
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
aggaaatata gatcaaacaa aggagaaagt cctgtggagc ctgcagagcc ttgtcgttac 60
agctgcccca gggaggagga gggcagcacc atccccatcc aggaggatta ccgaaaaccg 120
gagcctgcct gctccccc 138
<210> 12
<211> 112
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 12
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
50 55 60
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
65 70 75 80
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
85 90 95
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110
<210> 13
<211> 336
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
cgcgtgaaat tcagccgcag cgcagatgct ccagcctaca agcaggggca gaaccagctc 60
tacaacgaac tcaatcttgg tcggagagag gagtacgacg tgctggacaa gcggagagga 120
cgggacccag aaatgggcgg gaagccgcgc agaaagaatc cccaagaggg cctgtacaac 180
gagctccaaa aggataagat ggcagaagcc tatagcgaga ttggtatgaa aggggaacgc 240
agaagaggca aaggccacga cggactgtac cagggactca gcaccgccac caaggacacc 300
tatgacgctc ttcacatgca ggccctgccg cctcgg 336
<210> 14
<211> 20
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 14
Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His
1 5 10 15
Ala Ala Arg Pro
20
<210> 15
<211> 60
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
gccctgcctg tgacagccct gctgctgcct ctggctctgc tgctgcatgc cgctagaccc 60
Claims (10)
1. a kind of single-chain antibody for targeting CD38, which is characterized in that the single-chain antibody of the targeting CD38 includes such as SEQ ID
The single-chain antibody of amino acid sequence shown in NO:1, the targeting CD38 is Humanized single chain antibody.
2. the single-chain antibody of targeting CD38 as described in claim 1, which is characterized in that the single-chain antibody of the targeting CD38
Encoding gene includes the nucleotide sequence as shown in SEQ ID NO:2.
3. a kind of Chimeric antigen receptor T cell for targeting CD38, which is characterized in that the Chimeric antigen receptor including targeting CD38
CAR-CD38, the CAR-CD38 include the single-chain antibody of sequentially connected targeting CD38, extracellular hinge from aminoterminal to c-terminus
The amino acid sequence of sequence, transmembrane region and intracellular signal area, wherein the single-chain antibody of the targeting CD38 includes such as SEQ ID
Amino acid sequence shown in NO:1.
4. the Chimeric antigen receptor T cell of targeting CD38 as claimed in claim 3, which is characterized in that the extracellular hinge area
Including CD8 α hinge area, the transmembrane region includes CD8 transmembrane region, the intracellular signal area include from aminoterminal to c-terminus sequentially
The CD27 signaling zone and CD3 ζ signaling zone of connection.
5. the Chimeric antigen receptor T cell of targeting CD38 as claimed in claim 4, which is characterized in that the CAR-CD38's
Amino acid sequence includes the amino acid sequence as shown in SEQ ID NO:3.
6. the Chimeric antigen receptor T cell of targeting CD38 as described in claim 3 or 4, which is characterized in that the CAR-CD38
Encoding gene include the nucleotide sequence as shown in SEQ ID NO:4.
7. a kind of recombinant viral vector, which is characterized in that the recombinant viral vector includes as described in claim any one of 3-6
Targeting CD38 Chimeric antigen receptor T cell CAR-CD38 coding gene sequence.
8. a kind of host cell, which is characterized in that the host cell includes recombinant viral vector as claimed in claim 7.
9. a kind of preparation method for the Chimeric antigen receptor T cell for targeting CD38 characterized by comprising
(1) encoding gene of the Chimeric antigen receptor CAR-CD38 of targeting CD38 is provided, including is sequentially connected with from 5 ' ends to 3 ' ends
Signal peptide encoding gene, target CD38 the encoding gene of single-chain antibody, the encoding gene of CD8 α hinge area, CD8 cross-film
The encoding gene of the encoding gene in area, the encoding gene of CD27 signaling zone and CD3 ζ signaling zone;The targeting CD38's is single-stranded anti-
Body is Humanized single chain antibody, and the encoding gene of the single-chain antibody of the targeting CD38 includes the core as shown in SEQ ID NO:2
Nucleotide sequence;
(2) encoding gene of the CAR-CD38 is inserted into pWPXLD carrier, obtains pWPXLD-CAR-CD38 recombination matter
Grain;
(3) by the pWPXLD-CAR-CD38 recombinant plasmid and envelope plasmid, packaging plasmid cotransfection host cell, weight is obtained
Group slow virus;
(4) recombinant slow virus is transfected into CD3 positive t lymphocytes, obtains the Chimeric antigen receptor T cell of targeting CD38.
10. a kind of target the single-chain antibody of CD38, such as any one of claim 3-6 institute as claim 1-2 is described in any item
Chimeric antigen receptor T cell, such as claim 7 of preparation method stating or as claimed in claim 9 targeting CD38 obtained
The recombinant viral vector or host cell as claimed in claim 8 are in preparation prevention, diagnosing and treating malignant-tumor agent
Application in object.
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CN110157679A (en) * | 2018-02-12 | 2019-08-23 | 深圳宾德生物技术有限公司 | A kind of targeting T lymphocyte and its preparation method and application |
CN113801228A (en) * | 2020-06-17 | 2021-12-17 | 常州费洛斯药业科技有限公司 | CD 38-targeting single-chain antibody, fully human chimeric antigen receptor, preparation method and application |
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
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CN110157679A (en) * | 2018-02-12 | 2019-08-23 | 深圳宾德生物技术有限公司 | A kind of targeting T lymphocyte and its preparation method and application |
CN110157679B (en) * | 2018-02-12 | 2022-05-27 | 深圳宾德生物技术有限公司 | Targeting T lymphocyte and preparation method and application thereof |
CN113801228A (en) * | 2020-06-17 | 2021-12-17 | 常州费洛斯药业科技有限公司 | CD 38-targeting single-chain antibody, fully human chimeric antigen receptor, preparation method and application |
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