CN109957023A - It is a kind of to target the single-chain antibody of CD22, 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 CD22, Chimeric antigen receptor T cell and its preparation method and application Download PDFInfo
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Abstract
The present invention provides a kind of single-chain antibody for targeting CD22, the single-chain antibody of the targeting CD22 includes the amino acid sequence as shown in SEQ ID NO:1, and the single-chain antibody of the targeting CD22 is Humanized single chain antibody.The present invention also provides a kind of Chimeric antigen receptor T cells of single-chain antibody including the targeting CD22, the Chimeric antigen receptor of the targeting CD22 can target CD22 in specific manner, the tumour cell for not expressing CD19 can be especially targeted to, promote amplification of the T cell in patient's body, efficient and specific killing tumor cell.In addition, the Chimeric antigen receptor T cell of Humanized single chain antibody targeting CD22 obtained has the vigor and lethality for preferably maintaining cell.The present invention also provides a kind of preparation method and application of Chimeric antigen receptor T cell for targeting CD22.
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 CD22 is thin
Born of the same parents and its preparation method and application.
Background technique
CAR-T (Chimeric antigen receptor T cell) technology is a kind of novel cell therapy, it is that the T by CAR transformation is thin
Born of the same parents are fed back to human body, activate self immune system, kill to tumour cell, in acute leukemia and non-Hodgkin lymphoma
Treatment on have significant curative effect, it is considered to be one of the therapeutic modality of current most effective malignant tumour.
CAR-T technology is in CD19 positive blood tumor (such as B cell lymphoma, acute and chronic bone-marrow-derived lymphocyte leukaemia) at present
Achieve the effect to attract people's attention.But since similar tumour often expresses different immunophenotype, for example, work as tumour
Cell occur CD19 feminine gender escape, that is, there is the tumour cell for not expressing CD19, then CD19 target spot fail, and CART-CD19 without
Method plays antitumous effect again, therefore, it is necessary to which the CAR-T for providing other wide expression target spots of energy targeted malignant tumour is thin
Born of the same parents.In addition, CAR-T cell is the guarantee of CAR-T long-term efficacy in body maintenance ability (persistence), but actually
CAR-T a few days to a few weeks after feedback just completely disappear, and fail 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 CD22, and the list including the targeting CD22
The Chimeric antigen receptor T cell of chain antibody.The Chimeric antigen receptor of the targeting CD22 can target CD22 in specific manner, promote
T cell is expanded in patient's body, efficiently and specifically killing tumor cell, while the targeting CD22 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 CD22.
In a first aspect, the present invention provides a kind of single-chain antibody for targeting CD22, the single-chain antibody packet of the targeting CD22
The amino acid sequence as shown in SEQ ID NO:1 is included, the single-chain antibody of the targeting CD22 is Humanized single chain antibody.
Optionally, the encoding gene of the single-chain antibody of the targeting CD22 includes the nucleotide as shown in SEQ ID NO:2
Sequence.
Optionally, the single-chain antibody encoding gene of the targeting CD22 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 CD22 albumen for the single-chain antibody for the targeting CD22 that first aspect present invention provides
It closes, it is particularly possible to be targeted to the tumour cell for not expressing CD19.In addition, the single-chain antibody of the targeting CD22 is Humanized single chain
Antibody, 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 CD22, including the embedding of targeting CD22
Antigen receptor CAR-CD22 is closed, the CAR-CD22 includes the single-stranded anti-of the sequentially connected targeting CD22 from aminoterminal to c-terminus
Body, extracellular hinge area, transmembrane region and intracellular signal area amino acid sequence, wherein it is described targeting CD22 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 CD22
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 CD22 knot on the single-chain antibody and tumour of the targeting CD22
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-CD22 of the targeting CD22.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-CD22 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-CD22 includes the nucleotide sequence as shown in SEQ ID NO:4.
Optionally, the encoding gene of the CAR-CD22 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-CD22 includes the nucleotide sequence as shown in SEQ ID NO:5.
The Chimeric antigen receptor T cell for the targeting CD22 that second aspect of the present invention provides, including the embedding of targeting CD22
Antigen receptor CAR-CD22 is closed, CD22 can be targeted in specific manner, after CAR-CD22 is in conjunction with the CD22 on tumour cell, institute
The intracellular signal area for stating T cell is activated, and promotes T cell in the amplification of patient's body, and efficient and specific killing tumour
Cell especially expresses the malignant tumour (including B cell malignant tumour etc.) of CD22, does not express CD19's especially suitable for killing
Tumour cell, CART-CD19 can not play antitumous effect again when tumour cell CD19 feminine gender being avoided to escape.In addition, targeting
The single-chain antibody of CD22 is Humanized single chain antibody, this makes the Chimeric antigen receptor T cell of the targeting CD22 be avoided causing
The immune response of human organism maintains ability (including activity and lethality) 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-CD22 of the Chimeric antigen receptor T cell of the targeting CD22.
Optionally, the encoding gene of the CAR-CD22 includes the nucleotide sequence as shown in SEQ ID NO:4.
Preferably, the encoding gene of the CAR-CD22 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-CD22 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-CD22 encoding gene segment entered can by genetic recombination be inserted into host genome, obtain targeting CD22 chimeric antigen by
Body T cell continues it, the Chimeric antigen receptor CAR-CD22 of steadily expression targeting CD22.
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 CD22, comprising:
(1) encoding gene of the Chimeric antigen receptor CAR-CD22 of targeting CD22 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 CD22
The encoding gene in area and the encoding gene in intracellular signal area；The single-chain antibody of the targeting CD22 is Humanized single chain antibody, institute
The encoding gene for stating the single-chain antibody of targeting CD22 includes the nucleotide sequence as shown in SEQ ID NO:2；
(2) encoding gene of the CAR-CD22 is inserted into pWPXLD carrier, obtains pWPXLD-CAR-CD22 recombination
Plasmid；
(3) it by the pWPXLD-CAR-CD22 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 CD22 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 CD22 are connected, the encoding gene sequence of the single-chain antibody of the targeting CD22
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-CD22 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-CD22 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-CD22 translated into not
Band is just like amino acid sequence shown in SEQ ID NO:14.
The coding gene sequence of the CAR-CD22 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-CD22
When being inserted into pWPXLD carrier, the gene order of the CAR-CD22 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 CD22 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 CD22, 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 CD22.Especially (such as B cell lymphoma, B drench B cell malignant tumour
Bar chronic myeloid leukemia etc.) prevention, diagnosing and treating.
The application specifically: provide a kind of kit, the kit includes targeting CD22 described in first aspect
Single-chain antibody, as described in second aspect targeting CD22 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-CD22 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 CD22, comprising the following steps:
(1) gene order of the Chimeric antigen receptor CAR-CD22 of preparation targeting CD22
Prepare respectively signal peptide, target the single-chain antibody of CD22, 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 CD22
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 CD22, 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 CD22
The encoding gene of antigen receptor CAR-CD22, the coding gene sequence of the CAR-CD22 is as shown in SEQ ID NO:5, wherein institute
The single-chain antibody for stating targeting CD22 is Humanized single chain antibody.
(2) pWPXLd-CAR-CD22 recombinant plasmid is constructed
The coding gene sequence of CAR-CD22 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-CD22 is inserted into pWPXLD load
When body, 5 ' ends of the coding gene sequence of the CAR-CD22 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-CD22 recombination matter as shown in Figure 1
Grain.
(3) recombinant slow virus constructs
PWPXLd-CAR-CD22 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 CD22 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 CD22, 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 CD22, Chimeric antigen receptor T cell and its preparation method and application
<160> 15
<170> SIPOSequenceListing 1.0
<210> 1
<211> 234
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 1
Asp Ile Val Leu Thr Gln Thr Pro Leu Thr Leu Ser Leu Ala Ile Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Ala Ser
20 25 30
Asp Gly Lys Thr Tyr Leu Asn Trp Leu Phe Gln Arg Pro Gly Gln Ser
35 40 45
Pro Lys Arg Leu Ile Tyr Leu Val Ser Ala Leu Asp Ser Gly Val Pro
50 55 60
Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Leu Glu Ala Glu Asp Leu Gly Leu Tyr Tyr Cys Trp Gln Gly
85 90 95
Ser His Phe Pro Phe Thr Phe Gly Glu Gly Thr Gly Gly Gly Gly Ser
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Met Leu Leu Glu
115 120 125
Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr Val Lys Ile Ser Cys
130 135 140
Lys Thr Ser Gly Tyr Thr Phe Thr Asn Phe Gly Met Asn Trp Val Lys
145 150 155 160
Gln Ala Pro Gly Lys Gly Leu Lys Trp Met Gly Trp Ile Asn Thr Tyr
165 170 175
Thr Gly Glu Pro Thr Tyr Gly Asp Asp Phe Lys Gly Arg Ile Val Phe
180 185 190
Ser Leu Glu Ile Ser Ala Ser Thr Ala Tyr Leu Gln Ile Asn Asn Leu
195 200 205
Lys Asn Asp Asp Met Ala Thr Tyr Phe Cys Thr Arg Asn Trp Asp Ala
210 215 220
Trp Tyr Phe Asp Val Trp Gly Glu Gly Thr
225 230
<210> 2
<211> 702
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gatattgttc tcacccagac tccactcact ttgtcgcttg ccattggaca accagcctcc 60
atctcttgca agtcaagtca gagcctctta gctagtgatg gcaagacata tttgaattgg 120
ttgtttcaga ggccaggcca gtctccaaag cgcctcatct atctggtgtc tgcactggac 180
tctggagtcc ctgacaggtt cactggcagt ggatcaggga cagatttcac actgaaaatc 240
agcagactgg aggctgagga tttgggactt tattattgct ggcaaggttc acattttcca 300
ttcacgttcg gcgaggggac aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc 360
ggatctgagg tgatgcttct ggagtctgga cctgagctga agaagcccgg tgagacagtc 420
aagatctcct gcaagacttc tgggtatacc ttcacaaact ttggaatgaa ctgggtgaag 480
caggctccag gaaagggttt aaagtggatg ggctggataa acacctacac tggagagcca 540
acatatggtg atgacttcaa gggacggatt gtcttctctt tggaaatttc cgccagcact 600
gcctatctgc agatcaacaa cctcaaaaat gatgacatgg ccacatattt ctgtacaaga 660
aactgggacg cctggtactt cgatgtctgg ggcgaaggaa ct 702
<210> 3
<211> 461
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 3
Asp Ile Val Leu Thr Gln Thr Pro Leu Thr Leu Ser Leu Ala Ile Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Ala Ser
20 25 30
Asp Gly Lys Thr Tyr Leu Asn Trp Leu Phe Gln Arg Pro Gly Gln Ser
35 40 45
Pro Lys Arg Leu Ile Tyr Leu Val Ser Ala Leu Asp Ser Gly Val Pro
50 55 60
Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Leu Glu Ala Glu Asp Leu Gly Leu Tyr Tyr Cys Trp Gln Gly
85 90 95
Ser His Phe Pro Phe Thr Phe Gly Glu Gly Thr Gly Gly Gly Gly Ser
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Met Leu Leu Glu
115 120 125
Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr Val Lys Ile Ser Cys
130 135 140
Lys Thr Ser Gly Tyr Thr Phe Thr Asn Phe Gly Met Asn Trp Val Lys
145 150 155 160
Gln Ala Pro Gly Lys Gly Leu Lys Trp Met Gly Trp Ile Asn Thr Tyr
165 170 175
Thr Gly Glu Pro Thr Tyr Gly Asp Asp Phe Lys Gly Arg Ile Val Phe
180 185 190
Ser Leu Glu Ile Ser Ala Ser Thr Ala Tyr Leu Gln Ile Asn Asn Leu
195 200 205
Lys Asn Asp Asp Met Ala Thr Tyr Phe Cys Thr Arg Asn Trp Asp Ala
210 215 220
Trp Tyr Phe Asp Val Trp Gly Glu Gly Thr Thr Thr Thr Pro Ala Pro
225 230 235 240
Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu
245 250 255
Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg
260 265 270
Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly
275 280 285
Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg
290 295 300
Lys Tyr Arg Ser Asn Lys Gly Glu Ser Pro Val Glu Pro Ala Glu Pro
305 310 315 320
Cys Arg Tyr Ser Cys Pro Arg Glu Glu Glu Gly Ser Thr Ile Pro Ile
325 330 335
Gln Glu Asp Tyr Arg Lys Pro Glu Pro Ala Cys Ser Pro Arg Val Lys
340 345 350
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln
355 360 365
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
370 375 380
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
385 390 395 400
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
405 410 415
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
420 425 430
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
435 440 445
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
450 455 460
<210> 4
<211> 1383
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
gatattgttc tcacccagac tccactcact ttgtcgcttg ccattggaca accagcctcc 60
atctcttgca agtcaagtca gagcctctta gctagtgatg gcaagacata tttgaattgg 120
ttgtttcaga ggccaggcca gtctccaaag cgcctcatct atctggtgtc tgcactggac 180
tctggagtcc ctgacaggtt cactggcagt ggatcaggga cagatttcac actgaaaatc 240
agcagactgg aggctgagga tttgggactt tattattgct ggcaaggttc acattttcca 300
ttcacgttcg gcgaggggac aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc 360
ggatctgagg tgatgcttct ggagtctgga cctgagctga agaagcccgg tgagacagtc 420
aagatctcct gcaagacttc tgggtatacc ttcacaaact ttggaatgaa ctgggtgaag 480
caggctccag gaaagggttt aaagtggatg ggctggataa acacctacac tggagagcca 540
acatatggtg atgacttcaa gggacggatt gtcttctctt tggaaatttc cgccagcact 600
gcctatctgc agatcaacaa cctcaaaaat gatgacatgg ccacatattt ctgtacaaga 660
aactgggacg cctggtactt cgatgtctgg ggcgaaggaa ctaccacgac gccagcgccg 720
cgaccaccaa caccggcgcc caccatcgcg tcgcagcccc tgtccctgcg cccagaggcg 780
tgccggccag cggcgggggg cgcagtgcac acgagggggc tggacttcgc ctgtgatatc 840
tacatctggg cgcccttggc cgggacttgt ggggtccttc tcctgtcact ggttatcacc 900
ctttactgca ggaaatatag atcaaacaaa ggagaaagtc ctgtggagcc tgcagagcct 960
tgtcgttaca gctgccccag ggaggaggag ggcagcacca tccccatcca ggaggattac 1020
cgaaaaccgg agcctgcctg ctccccccgc gtgaaattca gccgcagcgc agatgctcca 1080
gcctacaagc aggggcagaa ccagctctac aacgaactca atcttggtcg gagagaggag 1140
tacgacgtgc tggacaagcg gagaggacgg gacccagaaa tgggcgggaa gccgcgcaga 1200
aagaatcccc aagagggcct gtacaacgag ctccaaaagg ataagatggc agaagcctat 1260
agcgagattg gtatgaaagg ggaacgcaga agaggcaaag gccacgacgg actgtaccag 1320
ggactcagca ccgccaccaa ggacacctat gacgctcttc acatgcaggc cctgccgcct 1380
cgg 1383
<210> 5
<211> 1443
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
gccctgcctg tgacagccct gctgctgcct ctggctctgc tgctgcatgc cgctagaccc 60
gatattgttc tcacccagac tccactcact ttgtcgcttg ccattggaca accagcctcc 120
atctcttgca agtcaagtca gagcctctta gctagtgatg gcaagacata tttgaattgg 180
ttgtttcaga ggccaggcca gtctccaaag cgcctcatct atctggtgtc tgcactggac 240
tctggagtcc ctgacaggtt cactggcagt ggatcaggga cagatttcac actgaaaatc 300
agcagactgg aggctgagga tttgggactt tattattgct ggcaaggttc acattttcca 360
ttcacgttcg gcgaggggac aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc 420
ggatctgagg tgatgcttct ggagtctgga cctgagctga agaagcccgg tgagacagtc 480
aagatctcct gcaagacttc tgggtatacc ttcacaaact ttggaatgaa ctgggtgaag 540
caggctccag gaaagggttt aaagtggatg ggctggataa acacctacac tggagagcca 600
acatatggtg atgacttcaa gggacggatt gtcttctctt tggaaatttc cgccagcact 660
gcctatctgc agatcaacaa cctcaaaaat gatgacatgg ccacatattt ctgtacaaga 720
aactgggacg cctggtactt cgatgtctgg ggcgaaggaa ctaccacgac gccagcgccg 780
cgaccaccaa caccggcgcc caccatcgcg tcgcagcccc tgtccctgcg cccagaggcg 840
tgccggccag cggcgggggg cgcagtgcac acgagggggc tggacttcgc ctgtgatatc 900
tacatctggg cgcccttggc cgggacttgt ggggtccttc tcctgtcact ggttatcacc 960
ctttactgca ggaaatatag atcaaacaaa ggagaaagtc ctgtggagcc tgcagagcct 1020
tgtcgttaca gctgccccag ggaggaggag ggcagcacca tccccatcca ggaggattac 1080
cgaaaaccgg agcctgcctg ctccccccgc gtgaaattca gccgcagcgc agatgctcca 1140
gcctacaagc aggggcagaa ccagctctac aacgaactca atcttggtcg gagagaggag 1200
tacgacgtgc tggacaagcg gagaggacgg gacccagaaa tgggcgggaa gccgcgcaga 1260
aagaatcccc aagagggcct gtacaacgag ctccaaaagg ataagatggc agaagcctat 1320
agcgagattg gtatgaaagg ggaacgcaga agaggcaaag gccacgacgg actgtaccag 1380
ggactcagca ccgccaccaa ggacacctat gacgctcttc acatgcaggc cctgccgcct 1440
cgg 1443
<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 CD22, which is characterized in that the single-chain antibody of the targeting CD22 includes such as SEQ ID
The single-chain antibody of amino acid sequence shown in NO:1, the targeting CD22 is Humanized single chain antibody.
2. the single-chain antibody of targeting CD22 as described in claim 1, which is characterized in that the single-chain antibody of the targeting CD22
Encoding gene includes the nucleotide sequence as shown in SEQ ID NO:2.
3. a kind of Chimeric antigen receptor T cell for targeting CD22, which is characterized in that the Chimeric antigen receptor including targeting CD22
CAR-CD22, the CAR-CD22 include the single-chain antibody of sequentially connected targeting CD22, 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 CD22 includes such as SEQ ID
Amino acid sequence shown in NO:1.
4. the Chimeric antigen receptor T cell of targeting CD22 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 CD22 as claimed in claim 4, which is characterized in that the CAR-CD22'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 CD22 as described in claim 3 or 4, which is characterized in that the CAR-CD22
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 CD22 Chimeric antigen receptor T cell CAR-CD22 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 CD22 characterized by comprising
(1) encoding gene of the Chimeric antigen receptor CAR-CD22 of targeting CD22 is provided, including is sequentially connected with from 5 ' ends to 3 ' ends
Signal peptide encoding gene, target the encoding gene of single-chain antibody of CD22, the encoding gene of extracellular hinge area, transmembrane region
The encoding gene of encoding gene and intracellular signal area；The single-chain antibody of the targeting CD22 is Humanized single chain antibody, the target
Encoding gene to the single-chain antibody of CD22 includes the nucleotide sequence as shown in SEQ ID NO:2；
(2) encoding gene of the CAR-CD22 is inserted into pWPXLD carrier, obtains pWPX LD-CAR-CD22 recombination matter
Grain；
(3) by the pWPXLD-CAR-CD22 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 CD22.
10. a kind of target the single-chain antibody of CD22, 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 CD22 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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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109957018A (en) * | 2017-12-25 | 2019-07-02 | 深圳宾德生物技术有限公司 | It is a kind of to target the single-chain antibody of CD22, Chimeric antigen receptor T cell and its preparation method and application |
| CN110157677A (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 |
| CN110623978A (en) * | 2018-06-25 | 2019-12-31 | 深圳宾德生物技术有限公司 | Application of chimeric antigen receptor T cell targeting CD22 in autoimmune diseases |
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| CN109957018A (en) * | 2017-12-25 | 2019-07-02 | 深圳宾德生物技术有限公司 | It is a kind of to target the single-chain antibody of CD22, Chimeric antigen receptor T cell and its preparation method and application |
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| US20040038339A1 (en) * | 2000-03-24 | 2004-02-26 | Peter Kufer | Multifunctional polypeptides comprising a binding site to an epitope of the nkg2d receptor complex |
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