CN109836496A - It is a kind of to target the single-chain antibody of CD317, Chimeric antigen receptor T cell and its preparation method and application - Google Patents

Abstract

The present invention provides a kind of single-chain antibody for targeting CD317, the single-chain antibody of the targeting CD317 includes the amino acid sequence as shown in SEQ ID NO:1, and the single-chain antibody of the targeting CD317 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 CD317.The Chimeric antigen receptor of the targeting CD317 can target CD317 in specific manner, promote T cell in the amplification of patient's body, efficiently and specifically killing tumor cell, and hardly cause to damage to normal cell.The Chimeric antigen receptor T cell of Humanized single chain antibody targeting CD317 obtained can enduringly maintain its self-renewal capacity and tumor-killing power simultaneously.The present invention also provides a kind of preparation method and application of Chimeric antigen receptor T cell for targeting CD317.

Description

A kind of single-chain antibody, Chimeric antigen receptor T cell and its preparation side targeting CD317 Method 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 CD317 Cell 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.CD317 is Lipid Rafts phase Albumen is closed, main expression is on a variety of solid tumor cells such as liver cancer, colon cancer, and the almost seldom expression in ordinary cells. The expression of CD317 is often that cell shows the response of gamma- interferon access, therefore has substantial connection with tumour.

CAR-T (Chimeric antigen receptor T cell) technology is a kind of novel cell therapy, it is will be by the T of CA R transformation Cell is fed back to human body, activates self immune system, kills to tumour cell, it is considered to be most effective at present pernicious swollen One of therapeutic modality of tumor.But the application of current CAR-T technology is also limited to blood tumor, does not have also to a variety of solid tumors such as liver cancer Progress.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 CD317, and including the targeting CD317's The Chimeric antigen receptor T cell of single-chain antibody.The Chimeric antigen receptor of the targeting CD317 can target CD317 in specific manner, Promote T cell in the amplification of patient's body, efficiently and specifically killing tumor cell, and hardly normal cell is caused Damage.The Chimeric antigen receptor T cell of Humanized single chain antibody targeting CD317 obtained can enduringly maintain its self simultaneously Updating ability and tumor-killing power.The present invention also provides a kind of preparation methods of Chimeric antigen receptor T cell for targeting CD317 And application.

In a first aspect, the present invention provides a kind of single-chain antibody for targeting CD317, the single-chain antibody of the targeting CD317 Including the amino acid sequence as shown in SEQ ID NO:1, the single-chain antibody of the targeting CD317 is Humanized single chain antibody.

Optionally, the encoding gene of the single-chain antibody of the targeting CD317 includes the nucleotide as shown in SEQ ID NO:2 Sequence.

Optionally, the single-chain antibody encoding gene of the targeting CD317 should consider degeneracy base, i.e., such as SEQ ID NO: The encoding gene of amino acid sequence shown in 1 includes the nucleotide sequence as shown in SEQ ID NO:2, and protection scope should also be protected 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.

With CD317 albumen specificity can occur for the single-chain antibody for the targeting CD317 that first aspect present invention provides In conjunction with having a fine targeting to the solid tumor cell of expression CD317, and hardly target normal cell.

Second aspect, the present invention provides a kind of Chimeric antigen receptor T cells for targeting CD317, including targeting CD317's Chimeric antigen receptor CAR-CD317, the CAR-CD317 include the sequentially connected targeting CD317 from aminoterminal to c-terminus Single-chain antibody, extracellular hinge area, transmembrane region and intracellular signal area amino acid sequence, wherein the targeting CD317's is single-stranded anti- Body includes 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 CD317 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.

The extracellular hinge area described in the present invention is used to promote on the single-chain antibody and tumour of the targeting CD317 CD317 is combined.

Optionally, the extracellular hinge area include CD8 α hinge area, CD28 hinge area, CD4 hinge area, CD5 hinge area, One of CD134 hinge area, 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。

The transmembrane region is used to fix the Chimeric antigen receptor CAR-CD317 of the targeting CD317 in the present invention.

Optionally, the transmembrane region includes one of CD3 transmembrane region, CD4 transmembrane region, CD8 transmembrane region, CD28 transmembrane region Or a variety of combination.

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 signaling zone, CD27 signal One of area, OX40 signaling zone, CD28 signaling zone, IL1R1 signaling zone, CD70 signaling zone, TNFRS F19L signaling zone are more The combination of kind.

Optionally, the intracellular signal area is 4-1BB signaling zone and CD3 ζ signaling zone.

Optionally, the amino acid sequence of the 4-1BB signaling zone includes the amino acid sequence as shown in SEQ ID NO:10.

Optionally, the encoding gene of the 4-1BB signaling zone includes the nucleotide sequence as shown in SEQ ID NO:11.

Optionally, the encoding gene of the 4-1BB 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-CD317 includes the amino acid sequence as shown in SEQ ID NO:3.

Optionally, the encoding gene of the CAR-CD317 includes the nucleotide sequence as shown in SEQ ID NO:4.

Optionally, the encoding gene of the CAR-CD317 should consider degeneracy base, i.e., as shown in SEQ ID NO:3 The encoding gene of amino 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。

The Chimeric antigen receptor T cell for the targeting CD317 that second aspect of the present invention provides, including targeting CD317's Chimeric antigen receptor CAR-CD317, this receptor for T cell targeted expression CD317 in specific manner tumour cell, After CAR-CD317 is in conjunction with CD317, the intracellular signal area of the T cell is activated, promote T cell patient's body amplification, And efficient and specific killing tumor cell, and normal cell is hardly caused to damage.In addition, the list of targeting CD317 Chain antibody is Humanized single chain antibody, this makes the Chimeric antigen receptor T cell of the targeting CD317 be avoided causing human organism's Immune response maintains ability (including vigor 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-CD317 of the Chimeric antigen receptor T cell of the targeting CD317.

Optionally, the encoding gene of the CAR-CD317 includes the nucleotide sequence as shown in SEQ ID NO:4.

Preferably, the encoding gene of the CAR-CD317 includes the nucleotide sequence as shown in SEQ ID NO:5.

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-CD317 encoding gene segment entered can be inserted into host genome by genetic recombination, obtain the chimeric antigen of targeting CD317 Recipient T cells, to make it continue, the Chimeric antigen receptor CAR-CD317 of steadily expression targeting CD317.

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 or 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 CD317, comprising:

(1) encoding gene of the Chimeric antigen receptor CAR-CD317 of targeting CD317 is provided, including suitable from 5 ' ends to 3 ' ends The encoding gene of the signal peptide of secondary connection, the encoding gene of single-chain antibody for targeting CD317, extracellular hinge area encoding gene, The encoding gene of transmembrane region, intracellular signal area encoding gene；The single-chain antibody of the targeting CD317 is anti-for Humanized single chain The encoding gene of body, the single-chain antibody of the targeting CD317 includes the nucleotide sequence as shown in SEQ ID NO:2；

(2) gene order of the CAR-CD317 is inserted into pWPXLD carrier, obtains pWP XLD-CAR-CD317 Recombinant plasmid；

(3) it by the pWPXLD-CAR-CD317 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, obtains the Chimeric antigen receptor T of targeting CD317 Cell.

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 CD317 are connected, the encoding gene of the single-chain antibody of the targeting CD317 3 ' ends of sequence are connected with 5 ' ends of the coding gene sequence of the extracellular hinge area, the encoding gene sequence of the extracellular hinge area 3 ' ends of column are connected with the 5 ' of the coding gene sequence of transmembrane region ends, the 3 ' of the coding gene sequence of the transmembrane region hold and 5 ' ends of the coding gene sequence in the intracellular signal area are connected.

The signal peptide is used to that the Chimeric antigen receptor CAR-CD317 to be instructed to express to cell surface in the present invention, The signal peptide is 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-CD317 includes the nucleotide sequence as shown in SEQ ID NO:5.

The coding gene sequence of the CAR-CD317 be inserted into pWPXLD carrier BamH I and I restriction enzyme site of EcoR it Between, and be located at after the extension factor 1 α (EF1 α) of pWPXLD carrier, using EF1 α as promoter.The gene of the CAR-CD317 When sequence is inserted into pWPXLD carrier, initiation codon (such as ATG) can be also added in 5 ' ends of the gene order of the CAR-CD317 It is connected with BamH1 restriction enzyme site in pWPXLD carrier, 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: into peripheral blood mononuclear cells by certain CD3/CD28 immunomagnetic beads are added in ratio, 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 CD317 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 CD317, 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.Suitable for entities such as the diagnosing and treatings etc., such as liver cancer, colon cancer, cervical carcinoma of expressing the tumour of CD317 Tumor.

The application specifically: provide a kind of kit, the kit includes targeting CD317 described in first aspect Single-chain antibody, the targeting Chimeric antigen receptor T cell of CD317 as described in second aspect, the recombination as described in the third aspect One of viral vectors, 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-CD317 recombinant plasmid provided in an embodiment of the present invention.

Fig. 2 is the positive rate of the Chimeric antigen receptor T cell of targeting CD317 provided in an embodiment of the present invention；In Fig. 2 (a) For negative control group, (b) is the experiment of the Chimeric antigen receptor T cell of targeting CD317 provided in an embodiment of the present invention in Fig. 2 Group.

Fig. 3 is the tumor cell in vitro killing of the Chimeric antigen receptor T cell of targeting CD317 provided in an embodiment of the present invention Effect picture.

Fig. 4 is the effect of the treatment mice with tumor of the Chimeric antigen receptor T cell of targeting CD317 provided in an embodiment of the present invention Fruit figure.

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 CD317, comprising the following steps:

(1) gene order of the Chimeric antigen receptor CAR-CD317 of preparation targeting CD317

Prepare respectively signal peptide, target the single-chain antibody of CD317, CD8 α hinge area, CD8 transmembrane region, 4-1BB 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 CD317 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 volume of the 4-1BB signaling zone Code gene order 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, target single-chain antibody, the CD8 α hinge area, CD8 transmembrane region, 4- of CD317 1BB signaling zone is successively connected together from 5 ' ends to 3 ' ends with the coding gene sequence of CD3 ζ signaling zone, obtains targeting CD317's The encoding gene of Chimeric antigen receptor CAR-CD317, the coding gene sequence of the CAR-CD317 as shown in SEQ ID NO:5, Wherein the single-chain antibody of the targeting CD317 is Humanized single chain antibody.

(2) pWPXLd-CAR-CD317 recombinant plasmid is constructed

The coding gene sequence of CAR-CD317 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-CD317 is inserted into pWPXLD When carrier, 5 ' ends of the coding gene sequence of the CAR-CD317 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-CD317 recombination as shown in Figure 1 Plasmid.

(3) recombinant slow virus constructs

PWPXLd-CAR-CD317 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 CD317 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 CD317, and It is stored in and feeds back in dedicated cells frozen storing liquid, used so that patient feeds back.

Effect example

In order to assess the Chimeric antigen receptor T cell effect for targeting CD317 of above method preparation described in the invention, Carry out following effect example.

Effect example one assesses the positive rate of the Chimeric antigen receptor T cell of targeting CD317 prepared by the present invention

It will be by the Chimeric antigen receptor T cell (experimental group) of the method for the present invention preparation targeting CD317 and without the T of preparation Lymphocyte (negative control group), using its positive rate of flow cytomery, as a result as shown in Fig. 2, wherein (a) is in Fig. 2 Negative control group, i.e., without the T cell of preparation, (b) is experimental group in Fig. 2, and targeting CD317's as produced by the present invention is chimeric Antigen receptor T cell.(a) can be obtained compared with (b) in Fig. 2, the Chimeric antigen receptor of targeting CD317 prepared by the present invention The positive rate of T cell is 25.3%.

The tumor cell in vitro of effect example two, the Chimeric antigen receptor T cell of assessment targeting CD317 kills situation

By the Chimeric antigen receptor T cell (being abbreviated as CAR-T-CD317) by targeting CD317 made from the method for the present invention It is compared with the Vitro Tumor fragmentation effect of the T lymphocyte (negative control group) without preparation, specifically: in vitro will effect Answer cell (CAR-T-CD317 or the T lymphocyte without preparation) and target cell (HeLa cell) by quantity ratio be 1:10,1:3, 1:1,3:1 and 10:1 ratio, at 37 DEG C, 5%CO₂Under co-cultured, after incubation 15-18 hours, collect cell, into The dyeing of row streaming, detects cell killing situation, as a result as shown in Figure 3.As can be seen from Figure 3, by method of the present invention The tumor-killing power of the CAR-T-CD317 cell of preparation is 20% or more, even up to 56%, significantly larger than negative control Group.The result of Fig. 3 illustrates that there is the Chimeric antigen receptor T cell of the targeting CD317 through the method for the present invention preparation strong tumour to kill Hurt ability.

Effect example three, the mouse interior tumor cell killing feelings of the Chimeric antigen receptor T cell of assessment targeting CD317 Condition

Will by the method for the present invention preparation targeting CD317 Chimeric antigen receptor T cell (CAR-T-CD317), without The T lymphocyte (negative control group) and physiological saline (blank control group) of preparation, in mouse tumor model, to every small Tail vein injection 1 × 10⁶A HeLa cell (n=9), obtains the survivorship curve of mouse, as a result as shown in Figure 4.It can be with from Fig. 4 Find out that the Chimeric antigen receptor T cell of the targeting CD317 by this method preparation makes mouse at culture 50 days or more, survives Rate is also higher than 55% or more, considerably beyond negative control group and blank control group.Fig. 4's the result shows that, prepared by this method Targeting CD317 Chimeric antigen receptor T cell can preferably protect mice against because of tumour caused by it is dead.

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

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<213>artificial sequence (Artificial Sequence)

<400> 3

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Tyr Met Ala Trp Val Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu

35 40 45

Gly Leu Ile Arg Asn Lys Gly Asn Gly Tyr Thr Thr Glu His Ser Ala

50 55 60

Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gln Ser Ile

65 70 75 80

Leu Tyr Leu Gln Met Asn Thr Leu Arg Pro Glu Asp Ser Ala Thr Tyr

85 90 95

Tyr Cys Ala Arg Asp Tyr Arg Ser Met Asp Tyr Trp Gly Ala Gly Thr

100 105 110

Ile Val Thr Val Asn Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Asp Ile Val Leu Thr Gln Ser Thr Pro Ser Leu Ala

130 135 140

Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser

145 150 155 160

Val Asp Ser Tyr Gly Asn Ser Phe Met His Trp Phe Gln Gln Lys Pro

165 170 175

Gly Gln Pro Pro Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser

180 185 190

Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr

195 200 205

Leu Thr Ile Asn Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys

210 215 220

Gln Gln Ser Asn Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu

225 230 235 240

Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro

245 250 255

Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro

260 265 270

Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

275 280 285

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

290 295 300

Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu

305 310 315 320

Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu

325 330 335

Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys

340 345 350

Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys

355 360 365

Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu

370 375 380

Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly

385 390 395 400

Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu

405 410 415

Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly

420 425 430

Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser

435 440 445

Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro

450 455 460

Pro Arg

465

<210> 4

<211> 1398

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

gaggtgaagc tggtggagtc tggaggaggc ttggtacagc ctgggggttc tctgagactc 60

tcctgtgcaa cttctgggtt cacgttctct gattactaca tggcctgggt ccgccagcct 120

ccaggaaagg cacttgagtg gttgggttta attagaaaca aaggtaatgg ttacacaaca 180

gagcacagtg catctgtgag gggtcggttc accatctcca gagataattc ccaaagcatc 240

ctctatcttc aaatgaacac cctgagacct gaggacagtg ccacttatta ctgtgcaaga 300

gattaccggt ctatggacta ctggggcgca ggaactatag tcacagtcaa tggtggcggt 360

ggctcgggcg gtggtgggtc gggtggcggc ggatctgaca ttgtgctcac acaatctaca 420

ccttctttgg ctgtgtctct agggcagagg gccaccatat cctgcagagc cagtgaaagt 480

gttgatagtt atggcaacag ttttatgcac tggttccagc agaaaccagg acagccaccc 540

aaactcctca tctatcgtgc atccaaccta gaatctggga tccctgccag gttcagtggc 600

agtgggtcta ggacagactt caccctcacc attaatcctg tggaggctga tgatgttgca 660

acctattact gtcagcaaag taatgaggat ccgtacacgt tcggaggggg gaccaagctg 720

gaaataaaaa ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 780

cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 840

agggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 900

gtccttctcc tgtcactggt tatcaccctt tactgcaaac ggggcagaaa gaaactcctg 960

tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt 1020

agctgccgat ttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 1080

agcgcagacg cccccgcgta caagcagggc cagaaccagc tctataacga gctcaatcta 1140

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1200

ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1260

atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1320

gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1380

caggccctgc cccctcgc 1398

<210> 5

<211> 1458

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

gccctgcctg tgacagccct gctgctgcct ctggctctgc tgctgcatgc cgctagaccc 60

gaggtgaagc tggtggagtc tggaggaggc ttggtacagc ctgggggttc tctgagactc 120

tcctgtgcaa cttctgggtt cacgttctct gattactaca tggcctgggt ccgccagcct 180

ccaggaaagg cacttgagtg gttgggttta attagaaaca aaggtaatgg ttacacaaca 240

gagcacagtg catctgtgag gggtcggttc accatctcca gagataattc ccaaagcatc 300

ctctatcttc aaatgaacac cctgagacct gaggacagtg ccacttatta ctgtgcaaga 360

gattaccggt ctatggacta ctggggcgca ggaactatag tcacagtcaa tggtggcggt 420

ggctcgggcg gtggtgggtc gggtggcggc ggatctgaca ttgtgctcac acaatctaca 480

ccttctttgg ctgtgtctct agggcagagg gccaccatat cctgcagagc cagtgaaagt 540

gttgatagtt atggcaacag ttttatgcac tggttccagc agaaaccagg acagccaccc 600

aaactcctca tctatcgtgc atccaaccta gaatctggga tccctgccag gttcagtggc 660

agtgggtcta ggacagactt caccctcacc attaatcctg tggaggctga tgatgttgca 720

acctattact gtcagcaaag taatgaggat ccgtacacgt tcggaggggg gaccaagctg 780

gaaataaaaa ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 840

cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 900

agggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 960

gtccttctcc tgtcactggt tatcaccctt tactgcaaac ggggcagaaa gaaactcctg 1020

tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt 1080

agctgccgat ttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 1140

agcgcagacg cccccgcgta caagcagggc cagaaccagc tctataacga gctcaatcta 1200

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1260

ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1320

atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1380

gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1440

caggccctgc cccctcgc 1458

<210> 6

<211> 46

<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 Ile

35 40 45

<210> 7

<211> 138

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgatatc 138

<210> 8

<211> 23

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 8

Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser

1 5 10 15

Leu Val Ile Thr Leu Tyr Cys

20

<210> 9

<211> 69

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

tacatctggg cgcccttggc cgggacttgt ggggtccttc tcctgtcact ggttatcacc 60

ctttactgc 69

<210> 10

<211> 42

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 10

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 11

<211> 126

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 11

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<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

agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgc 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 CD317, which is characterized in that the single-chain antibody of the targeting CD317 includes such as SEQ ID The single-chain antibody of amino acid sequence shown in NO:1, the targeting CD317 is Humanized single chain antibody.

2. the single-chain antibody of targeting CD317 as described in claim 1, which is characterized in that the single-chain antibody of the targeting CD317 Encoding gene include the nucleotide sequence as shown in SEQ ID NO:2.

3. a kind of Chimeric antigen receptor T cell for targeting CD317, which is characterized in that the Chimeric antigen receptor including targeting CD317 CAR-CD317, the CAR-CD317 include the single-chain antibody, extracellular of the sequentially connected targeting CD317 from aminoterminal to c-terminus The amino acid sequence of hinge area, transmembrane region and intracellular signal area, wherein the single-chain antibody of the targeting CD317 includes such as SEQ ID Amino acid sequence shown in NO:1.

4. the Chimeric antigen receptor T cell of targeting CD317 as claimed in claim 3, which is characterized in that the CAR-CD317 Amino acid sequence include the amino acid sequence as shown in SEQ ID NO:3.

5. a kind of recombinant viral vector, which is characterized in that the recombinant viral vector includes such as any one of claim 3 or 4 institute The coding gene sequence of the CAR-CD317 of the Chimeric antigen receptor T cell of the targeting CD317 stated.

6. recombinant viral vector as claimed in claim 5, which is characterized in that the coding gene sequence packet of the CAR-CD317 Include the nucleotide sequence as shown in SEQ ID NO:4.

7. a kind of host cell, which is characterized in that the host cell includes such as the described in any item recombinations of claim 5 or 6 Viral vectors.

8. a kind of preparation method for the Chimeric antigen receptor T cell for targeting CD317 characterized by comprising

(1) encoding gene of the Chimeric antigen receptor CAR-CD317 of targeting CD317 is provided, including is sequentially connected from 5 ' ends to 3 ' ends The encoding gene of the signal peptide connect, the encoding gene of single-chain antibody for targeting CD317, the encoding gene of CD8 α hinge area, CD8 across The encoding gene of the encoding gene in film area, the encoding gene of 4-1BB signaling zone and CD3 ζ signaling zone；The list of the targeting CD317 Chain antibody is Humanized single chain antibody, and the encoding gene of the single-chain antibody of the targeting CD317 includes as shown in SEQ ID NO:2 Nucleotide sequence；

(2) gene order of the CAR-CD317 is inserted into pWPXLD carrier, obtains pWPXLD-CAR-CD317 recombination matter Grain；

(3) by the pWPXLD-CAR-CD317 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 CD317.

9. the preparation method of the Chimeric antigen receptor T cell of targeting CD317 as claimed in claim 8, which is characterized in that described Envelope plasmid is PMD2G, and the packaging plasmid is psPAX2, and the host cell is HEK293T cell.

10. a kind of target the single-chain antibody of CD317, such as any one of claim 3-4 institute as claim 1-2 is described in any item It is stating or as made from the described in any item preparation methods of claim 8-9 target CD317 Chimeric antigen receptor T cell or As the described in any item recombinant viral vectors of claim 5-6 or host cell as claimed in claim 7 are preparing prevention, examining Application in disconnected and treatment malignant tumor medicine.