CN109957021A - 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 PDF

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CN109957021A
CN109957021A CN201711422919.XA CN201711422919A CN109957021A CN 109957021 A CN109957021 A CN 109957021A CN 201711422919 A CN201711422919 A CN 201711422919A CN 109957021 A CN109957021 A CN 109957021A
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targeting
cell
chain antibody
antigen receptor
chimeric antigen
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张宏玲
龙丽梅
钟春颖
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Shenzhen Benta Biological Technology Co Ltd
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Shenzhen Benta Biological Technology Co Ltd
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
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    • A61K35/14—Blood; Artificial blood
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
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    • C07—ORGANIC CHEMISTRY
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    • C07K2319/00—Fusion polypeptide
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    • C12N2740/00—Reverse transcribing RNA viruses
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

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 T cell amplification, 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

It is a kind of to target the single-chain antibody of CD38, Chimeric antigen receptor T cell and preparation method thereof And application
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 the single-chain antibodies that one kind can target CD38, and including the targeting CD38 Single-chain antibody Chimeric antigen receptor T cell.The Chimeric antigen receptor of the targeting CD38 can target CD38 in specific manner, T cell is promoted to expand in patient's body, efficiently and the tumour cell of CD38 is expressed in specifically killing, while with Humanized single chain The Chimeric antigen receptor T cell of the targeting CD38 of antibody can enduringly maintain its self-renewing vigor and lethality.The present invention Additionally provide a kind of preparation method and application of Chimeric antigen receptor T cell for targeting 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 targeting C D38's Chimeric antigen receptor CAR-CD38, the CAR-CD38 include the single-stranded of the sequentially connected targeting CD38 from aminoterminal to c-terminus Antibody, extracellular hinge area, transmembrane region and intracellular signal area amino acid sequence, wherein it is described targeting CD38 single-chain antibody include The amino acid sequence as 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 N O:6 Amino acid sequence encoding gene include the nucleotide sequence such as SEQ ID NO:7 shown in, 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 believed to the sequentially connected CD28 signaling zone of c-terminus and C D3 ζ Number area.Correspondingly, the encoding gene in the intracellular signal area includes the volume from 5 ' ends to 3 ' the sequentially connected CD28 signaling zones in end The encoding gene of code gene and CD3 ζ signaling zone.
Optionally, the amino acid sequence of the CD28 signaling zone includes the amino acid sequence as shown in SEQ ID NO:10. The CD28 signaling zone is as costimulatory signal.
Optionally, the encoding gene of the CD28 signaling zone includes the nucleotide sequence as shown in SEQ ID NO:11.
Optionally, the encoding gene of the CD28 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 CD28 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 CD28 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) encoding gene of the CAR-CD38 is inserted into pWPXLD carrier, obtains pWPX LD-CAR-CD38 weight Group 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 B amH1 restriction enzyme site is connected in pWPXLD carrier, and EcoR1 digestion in terminator codon and pWPXLD carrier can be also added in 3 ' ends Site 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, CD28 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 CD28 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, CD28 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-C D38 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 p WPXLd-CAR-CD38 recombination as shown in Figure 1 Plasmid.
(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> 500
<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
Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu
325 330 335
Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser
340 345 350
Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly
355 360 365
Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala
370 375 380
Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala
385 390 395 400
Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg
405 410 415
Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu
420 425 430
Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn
435 440 445
Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met
450 455 460
Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly
465 470 475 480
Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala
485 490 495
Leu Pro Pro Arg
500
<210> 4
<211> 1500
<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
ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg 1020
gcctttatta ttttctgggt gaggagtaag aggagcaggc tcctgcacag tgactacatg 1080
aacatgactc cccgccgccc cgggcccacc cgcaagcatt accagcccta tgccccacca 1140
cgcgacttcg cagcctatcg ctcccgcgtg aaattcagcc gcagcgcaga tgctccagcc 1200
tacaagcagg ggcagaacca gctctacaac gaactcaatc ttggtcggag agaggagtac 1260
gacgtgctgg acaagcggag aggacgggac ccagaaatgg gcgggaagcc gcgcagaaag 1320
aatccccaag agggcctgta caacgagctc caaaaggata agatggcaga agcctatagc 1380
gagattggta tgaaagggga acgcagaaga ggcaaaggcc acgacggact gtaccaggga 1440
ctcagcaccg ccaccaagga cacctatgac gctcttcaca tgcaggccct gccgcctcgg 1500
<210> 5
<211> 1560
<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
ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg 1080
gcctttatta ttttctgggt gaggagtaag aggagcaggc tcctgcacag tgactacatg 1140
aacatgactc cccgccgccc cgggcccacc cgcaagcatt accagcccta tgccccacca 1200
cgcgacttcg cagcctatcg ctcccgcgtg aaattcagcc gcagcgcaga tgctccagcc 1260
tacaagcagg ggcagaacca gctctacaac gaactcaatc ttggtcggag agaggagtac 1320
gacgtgctgg acaagcggag aggacgggac ccagaaatgg gcgggaagcc gcgcagaaag 1380
aatccccaag agggcctgta caacgagctc caaaaggata agatggcaga agcctatagc 1440
gagattggta tgaaagggga acgcagaaga ggcaaaggcc acgacggact gtaccaggga 1500
ctcagcaccg ccaccaagga cacctatgac gctcttcaca tgcaggccct gccgcctcgg 1560
<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> 68
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 10
Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu
1 5 10 15
Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser
20 25 30
Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly
35 40 45
Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala
50 55 60
Ala Tyr Arg Ser
65
<210> 11
<211> 204
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg 60
gcctttatta ttttctgggt gaggagtaag aggagcaggc tcctgcacag tgactacatg 120
aacatgactc cccgccgccc cgggcccacc cgcaagcatt accagcccta tgccccacca 180
cgcgacttcg cagcctatcg ctcc 204
<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 CD28 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 the encoding gene of single-chain antibody of CD38, 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 CD38 is Humanized single chain antibody, the target Encoding gene to the single-chain antibody of CD38 includes the nucleotide sequence as shown in SEQ ID NO:2；
(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.
CN201711422919.XA 2017-12-25 2017-12-25 It is a kind of to target the single-chain antibody of CD38, Chimeric antigen receptor T cell and its preparation method and application Withdrawn CN109957021A (en)

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