CN110157675A - A kind of targeting T lymphocyte and its preparation method and application - Google Patents

A kind of targeting T lymphocyte and its preparation method and application Download PDF

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Publication number
CN110157675A
CN110157675A CN201810148269.2A CN201810148269A CN110157675A CN 110157675 A CN110157675 A CN 110157675A CN 201810148269 A CN201810148269 A CN 201810148269A CN 110157675 A CN110157675 A CN 110157675A
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car
bcma
targeting
gly
encoding gene
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CN110157675B (en
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张宏玲
龙丽梅
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Shenzhen Benta Biological Technology Co Ltd
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Shenzhen Benta Biological Technology Co Ltd
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    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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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
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    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
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Abstract

The present invention provides a kind of targeting T lymphocytes, including targeting the Chimeric antigen receptor CAR-CD19 of CD19 and/or targeting the Chimeric antigen receptor CAR-BCMA of BCMA, it is wide to the targets identification of tumour cell and strong, there is target spot escape in avoidable tumour cell, efficiently and specifically killing tumor cell, it is lethal to expand its wide spectrum.The present invention also provides the preparation method and application of the targeting T lymphocyte.

Description

A kind of targeting T lymphocyte and its preparation method and application
Technical field
The present invention relates to field of medical biotechnology, in particular to a kind of targeting T lymphocyte and its preparation method and application.
Background technique
Leukaemia is a kind of malignant disease of hemopoietic system, higher in the death rate of each age group malignant tumour in China. CAR-T (Chimeric antigen receptor T cell) technology is a kind of novel immune cell therapy, it is will to return by the T cell of CAR transformation Human body is transported to, self immune system is activated, tumour cell is killed, to achieve the purpose that remove malignant cell.
Although CAR-T technology achieves significant curative effect in the treatment of blood tumor etc., in complicated tumour micro-loop In border (such as kinds of tumors antigen occurs simultaneously), the identification of tumour cell, killing ability are substantially reduced, it is limited and faces Bed application.For example, for lymphocytic leukemia, the tumour cell of Most patients all expresses CD19, but there is also not The case where expressing CD19 (i.e. CD19 feminine gender is escaped).Therefore, it is necessary to which it is thin to provide T lymph stronger to malignant tumour targeting Born of the same parents.
Summary of the invention
In consideration of it, the present invention provides a kind of targeting T lymphocytes and its preparation method and application.The T lymphocyte It can be targeted to two tumor targets of B cell malignant tumour (such as lymphocytic leukemia), knowledge high to the targeting of tumour cell Other property is relatively wide and strong, and target spot escape occurs in avoidable tumour cell, and the wide spectrum for expanding the T cell is lethal.
In a first aspect, the present invention provides a kind of targeting T lymphocyte, the Chimeric antigen receptor including targeting CD19 The CAR-CD19 and/or Chimeric antigen receptor CAR-BCMA for targeting BCMA, wherein the CAR-CD19 includes from aminoterminal to carboxylic The sequentially connected targeting single-chain antibody of CD19 of cardinal extremity, extracellular hinge area, transmembrane region and intracellular signal area amino acid sequence, institute State single-chain antibody, the extracellular hinge area, transmembrane region that CAR-BCMA includes the sequentially connected targeting BCMA from aminoterminal to c-terminus With the amino acid sequence in intracellular signal area；Wherein, the amino acid sequence of the single-chain antibody of the targeting CD19 includes such as SEQ ID The amino acid sequence of amino acid sequence shown in NO:1, the single-chain antibody of the targeting BCMA includes as shown in SEQ ID NO:2 Amino acid sequence.
Preferably, the targeting T lymphocyte can be double target spot chimeric antigens with CAR-CD19 and CAR-BCMA Recipient T cells (that is, double target spot Chimeric antigen receptor T cells of targeting CD19 and BCMA), or with the embedding of CAR-CD19 Close the mixing of antigen receptor T cell and the Chimeric antigen receptor T cell with CAR-BCMA, can also for the CAR-CD19 and Double target spot Chimeric antigen receptor T cells, the Chimeric antigen receptor T cell with the CAR-CD19 of the CAR-BCMA, and The mixing of Chimeric antigen receptor T cell with the CAR-BCMA.
At this point, the targeting T lymphocyte, can both identify that surface expression had the tumour cell of CD19 antigen protein, It can also identify that surface expression has the tumour cell of BCMA antigen protein, certainly to having a CD19 antigen protein and BCMA antigen simultaneously The identity of the tumour cell of albumen is also preferable, and tumour cell can effectively be avoided the generation of CD19 immunologic escape occur.
Wherein, it when the targeting T lymphocyte is double target spot CAR-T with CAR-CD19 and CAR-BCMA, is fitted into The distributing position of antigen receptor CAR-CD19 and CAR-BCMA are not construed as limiting, can be and be alternately distributed (such as ABAB ..., AABABB ...) or it is sequentially distributed (such as AAAA ... BBB ...), but covalent linkage is had no between the two, they are corresponding at this time Also covalent linkage is had no between CD19 single-chain antibody and BCMA single-chain antibody, them can be made to keep preferable recognition capability in this way.
Above-mentioned " being sequentially connected with from aminoterminal to c-terminus " specifically: the single-chain antibody of the targeting CD19 or the targeting The c-terminus of the amino acid sequence of the single-chain antibody of BCMA is connected with the aminoterminal of the amino acid sequence of the extracellular hinge area, institute The c-terminus for stating the amino acid sequence of extracellular hinge area is connected with the aminoterminal of the amino acid sequence of the transmembrane region, the cross-film The c-terminus of the amino acid sequence in area is connected with the aminoterminal of the amino acid sequence in the intracellular signal area.
The above-mentioned CD19 referred to is B cell system marker in hematological system, passes through the knot with the B-cell receptor in B cell It closes and participates in the processes such as B cell development, Cellular Signaling Transduction Mediated.CD19 be B system malignant tumour (such as lymphocytic leukemia) compared with Common target spot, but also have in many malignant B cells and do not express CD19.And BCMA is that Tumor Necrosis Factor Receptors TNF surpasses house The member of race is a kind of more important B cell biomarker, does not express in most normal cells and organ, extensively It is general to be present in Huppert's disease (MM) cell and hematological system B system's malignant tumour surface.Therefore, BCMA is selected to be used as to CD19 The target for the B system malignant cell that feminine gender escape or CD19 antigen are lost, is avoided that CD19 escape occurs in tumour cell.
Optionally, the encoding gene of the single-chain antibody of the targeting CD19 includes the nucleotide as shown in SEQ ID NO:3 The encoding gene of sequence, the single-chain antibody of the targeting BCMA includes the nucleotide sequence as shown in SEQ ID NO:4.The present invention The single-chain antibody of the targeting CD19 remains with the affine activity to CD19 antigen, can efficient identification surface expression have CD19 anti- Former tumour cell.Similarly, the single-chain antibody of the targeting BCMA also remains with the affine activity to BCMA antigen, Neng Gougao Effect identification surface expression has the tumour cell of BCMA antigen.
Optionally, the encoding gene of the amino acid sequence of the single-chain antibody of the targeting CD19 should consider degeneracy base, I.e. the encoding gene of the amino acid sequence as shown in SEQ ID NO:1 includes the nucleotide sequence as shown in SEQ ID NO:3, is protected Shield range should also protect the nucleotide sequence for having base degeneracy matter with SEQ ID NO:3, these nucleotide sequences are corresponding Amino acid sequence remain as SEQ ID NO:1.The encoding gene of the amino acid sequence of the single-chain antibody of the targeting BCMA is same Sample should consider degeneracy base.
In the present invention, the extracellular hinge area in the CAR-CD19 is used to promote the single-chain antibody of the targeting CD19 and swells CD19 on oncocyte is combined；Similarly, the extracellular hinge area in the CAR-BCMA is used to promote the list of the targeting BCMA Chain antibody is in conjunction with the BCMA on tumour cell.
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.
Still optionally further, 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:9.
Optionally, the encoding gene of the CD8 α hinge area includes the nucleotide sequence as shown in SEQ ID NO:10.
Optionally, the encoding gene of the CD8 α hinge area should consider degeneracy base, i.e., as shown in SEQ ID NO:9 The encoding gene of amino acid sequence include the nucleotide sequence such as SEQ ID NO:10 shown in, protection scope should also protect and SEQ ID NO:10 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ ID NO:9。
In the present invention, the transmembrane region in the CAR-CD19 is used to fix the Chimeric antigen receptor CAR- of the targeting CD19 CD19；Similarly, the transmembrane region in the CAR-BCMA is used to fix the Chimeric antigen receptor CAR-BCMA of the targeting BCMA.
Optionally, the transmembrane region includes one of CD3 transmembrane region, CD4 transmembrane region, CD8 transmembrane region, CD28 transmembrane region Or a variety of combination.
Still optionally further, 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:11.
Optionally, the encoding gene of the CD8 transmembrane region includes the nucleotide sequence as shown in SEQ ID NO:12.
Optionally, the encoding gene of the CD8 transmembrane region should consider degeneracy base, i.e., as shown in SEQ ID NO:11 The encoding gene of amino acid sequence include the nucleotide sequence such as SEQ ID NO:12 shown in, protection scope should also protect and SEQ ID NO:12 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ ID NO:11。
In the present invention, the intracellular signal area for providing the signal of T cell activation, 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, TNFRSF19L signaling zone are a variety of Combination.
In an embodiment of the present invention, the intracellular signal Qu Weicong aminoterminal to the sequentially connected 4-1BB of c-terminus Signaling zone and CD3 ζ signaling zone.Correspondingly, the encoding gene in the intracellular signal area includes sequentially connected from 5 ' ends to 3 ' ends The encoding gene of 4-1BB signaling zone and the encoding gene of CD3 ζ signaling zone.
In another embodiment of the present invention, the intracellular signal area can also be to be sequentially connected with from aminoterminal to c-terminus CD27 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:13.
Optionally, the encoding gene of the 4-1BB signaling zone includes the nucleotide sequence as shown in SEQ ID NO:14.
Optionally, the encoding gene of the 4-1BB signaling zone should consider degeneracy base, i.e., as shown in SEQ ID NO:13 Amino acid sequence encoding gene include the nucleotide sequence such as SEQ ID NO:14 shown in, protection scope should also protect and SEQ ID NO:14 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ ID NO:13。
Optionally, the amino acid sequence of the CD3 ζ signaling zone includes the amino acid sequence as shown in SEQ ID NO:15.
Optionally, the encoding gene of the CD3 ζ signaling zone includes the nucleotide sequence as shown in SEQ ID NO:16.
Optionally, the encoding gene of the CD3 ζ signaling zone should consider degeneracy base, i.e., as shown in SEQ ID NO:15 Amino acid sequence encoding gene include the nucleotide sequence such as SEQ ID NO:16 shown in, protection scope should also protect and SEQ ID NO:16 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ ID NO:15。
It should be noted that in the present invention, extracellular hinge area, transmembrane region and intracellular signal area in the CAR-CD19 Amino acid sequence, with extracellular hinge area corresponding in the CAR-BCMA, the corresponding amino acid sequence of transmembrane region and intracellular signal area Column may be the same or different.
In an embodiment of the present invention, the amino acid sequence of the CAR-CD19 includes as shown in SEQ ID NO:5 Amino acid sequence.
Optionally, the encoding gene of the CAR-CD19 includes the nucleotide sequence as shown in SEQ ID NO:17.
Optionally, the encoding gene of the CAR-CD19 should consider degeneracy base, the i.e. ammonia as shown in SEQ ID NO:5 The encoding gene of base acid sequence includes the nucleotide sequence as shown in SEQ ID NO:17, and protection scope should also protect and SEQ ID NO:17 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ ID NO:5。
In an embodiment of the present invention, the amino acid sequence of the CAR-BCMA includes as shown in SEQ ID NO:6 Amino acid sequence.
Optionally, the encoding gene of the CAR-BCMA includes the nucleotide sequence as shown in SEQ ID NO:18.
Optionally, the encoding gene of the CAR-BCMA should consider degeneracy base, the i.e. ammonia as shown in SEQ ID NO:6 The encoding gene of base acid sequence includes the nucleotide sequence as shown in SEQ ID NO:18, and protection scope should also protect and SEQ ID NO:18 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 targeting T lymphocyte that first aspect present invention provides, the Chimeric antigen receptor CAR- including targeting CD19 The CD19 and/or Chimeric antigen receptor CAR-BCMA for targeting BCMA, can identify two tumor targets, enhance to tumour cell Identification, avoids tumour cell from escaping.In conjunction with CAR-CD19 and/or CAR-BCMA antigen protein corresponding on tumour cell Afterwards, the intracellular signal area of the targeting T lymphocyte is activated, and promotes T cell in the amplification of patient's body, and efficiently and special Anisotropic ground killing tumor cell, especially expresses the tumour cell of CD19 and/or BCMA, expands the wide spectrum killing of the T cell Property.Additionally due to the single-chain antibody of the CD19 and BCMA is Humanized single chain antibody, this makes the targeting T cell be avoided drawing The immune response for playing human organism maintains ability, such as activity and lethality in body with lasting.
Targeting T lymphocyte of the present invention with efficient identification and can kill expression and have the tumour of CD19 and/or BCMA Cell, especially malignant B, such as lymphocytic leukemia cell.
Second aspect, the present invention provides a kind of recombinant viral vectors, including targeting T lymph as described in relation to the first aspect The encoding gene of CAR-CD19 described in cell and/or CAR-BCMA.
Optionally, the encoding gene of CAR-CD19 and the encoding gene of CAR-BCMA are contained when the recombinant viral vector When, on the recombinant viral vector between the encoding gene of the CAR-CD19 and the encoding gene of the CAR-BCMA, also Including a special sequence, the special sequence is for making the encoding gene of the CAR-CD19 encoding gene and the CAR-BCMA exist Available two independent PROTEIN C AR-CD19 and CAR-BCMA after transcription and translation.Turn using such recombinant viral vector After contaminating CD3 positive t lymphocytes, resulting targeting T lymphocyte is chimeric for double target spots with CAR-CD19 and CAR-BCMA Antigen receptor T cell.Still optionally further, the special sequence can be RBS sequence, IRES sequence, T2A sequence or other eggs White enzyme sequence etc..
Optionally, the recombinant viral vector has the encoding gene of the CAR-CD19, or has the CAR- The encoding gene of BCMA.It is preferred that the recombinant viral vector using CAR-CD19 encoding gene and the weight with CAR-BCMA encoding gene Separately or simultaneously co-infection T lymphocyte, the targeting T that can be obtained as described in the first aspect of the invention drench group viral vectors Bar cell, and efficiency of infection is higher, gained targeting T lymphocyte can more fully hereinafter express CAR-CD19 and/or CAR-BCMA.
Optionally, the encoding gene of the CAR-CD19 includes the nucleotide sequence as shown in SEQ ID NO:17.
Preferably, the encoding gene of the CAR-CD19 includes the nucleotide sequence as shown in SEQ ID NO:19.Such as SEQ Nucleotide sequence shown in ID NO:19 is compared with the nucleotide sequence as shown in SEQ ID NO:17, more companies described below Connect the encoding gene of peptide.The encoding gene of the signal peptide can be expressed with Chimeric antigen receptor CAR-CD19 described in guide To cell surface.
Optionally, the encoding gene of the CAR-BCMA includes the nucleotide sequence as shown in SEQ ID NO:18.
Preferably, the encoding gene of the CAR-BCMA includes the nucleotide sequence as shown in SEQ ID NO:20.Such as SEQ Nucleotide sequence shown in ID NO:20 is compared with the nucleotide sequence as shown in SEQ ID NO:18, more companies described below Connect the encoding gene of peptide.The encoding gene of the signal peptide can be reached with Chimeric antigen receptor CAR-BCMA described in guide Cell surface.
Optionally, the viral vectors in the recombinant viral vector includes slow virus carrier, adenovirus vector or reverse transcription Viral vectors.Still optionally further, the viral vectors is slow virus carrier.In the preparation method that fourth aspect present invention provides The step of (1)-(3) show the preparation process of the recombinant viral vector.
The recombinant viral vector that second aspect of the present invention provides, efficiency of infection and transcriptional efficiency with higher, The encoding gene segment of CAR-CD19 and/or CAR-BCMA therein can be inserted into host genome by genetic recombination, obtain Targeting T lymphocyte is stated, continues it, play consistently targeting, killing effect.
The third aspect, the present invention provides a kind of host cell, the host cell includes the weight as described in second aspect Group slow virus carrier.
The host cell that third aspect present invention provides is used to assemble the recombinant viral vector as described in second aspect, Make it have infectivity.
Optionally, the host cell includes but is not limited to HEK293T cell, 293 cells, 293T cell, 293FT thin Born of the same parents, SW480 cell, u87MG cell, HOS cell, COS1 cell and COS7 cell.
Fourth aspect, the present invention provides a kind of preparation methods of targeting T lymphocyte, comprising:
(1) encoding gene of the Chimeric antigen receptor CAR-CD19 of targeting CD19 and the inosculating antibody of targeting BCMA are provided respectively The encoding gene of original receptor CAR-BCMA；
The encoding gene of the CAR-CD19 includes encoding gene, the targeting that signal peptide is sequentially connected with from 5 ' ends to 3 ' ends The encoding gene of the single-chain antibody of CD19, the encoding gene of extracellular hinge area, the encoding gene of transmembrane region and intracellular signal area Encoding gene；The encoding gene of the CAR-BCMA includes encoding gene, the targeting that signal peptide is sequentially connected with from 5 ' ends to 3 ' ends The encoding gene of the single-chain antibody of BCMA, the encoding gene of extracellular hinge area, the encoding gene of transmembrane region and intracellular signal area Encoding gene；
Wherein, the encoding gene of the single-chain antibody of the targeting CD19 includes the amino acid sequence as shown in SEQ ID NO:1 The encoding gene of the corresponding nucleotide sequence of column, the single-chain antibody of the targeting BCMA includes as shown in SEQ ID NO:2 Nucleotide sequence corresponding to amino acid sequence；
(2) encoding gene of the encoding gene of the CAR-CD19 and the CAR-BCMA pWPXLD is inserted respectively into carry In body, pWPXLD-CAR-CD19 recombinant plasmid and pWPXLD-CAR-BCMA recombinant plasmid are obtained；
(3) the pWPXLD-CAR-CD19 recombinant plasmid and the pWPXLD-CAR-BCMA recombinant plasmid are carried out respectively Packaging, obtains the first recombinant slow virus with CAR-CD19 encoding gene and the second recombinant lentiviral with CAR-BCMA encoding gene Virus；
(4) by first recombinant slow virus and second recombinant slow virus, separately or simultaneously co-transfection CD3 is positive Property T lymphocyte, through separation obtain targeting T lymphocyte.
It is above-mentioned " being sequentially connected with from 5 ' ends to 3 ' ends " specifically: the signal peptide by taking the encoding gene of CAR-CD19 as an example Coding gene sequence 3 ' end with it is described targeting CD19 single-chain antibody encoding gene 5 ' hold be connected, the targeting CD19 The 3 ' ends of encoding gene of single-chain antibody hold and be connected with the 5 ' of the extracellular hinge area encoding gene, the extracellular hinge area 3 ' ends of encoding gene are connected with 5 ' ends of the encoding gene of the transmembrane region, 3 ' ends of the encoding gene of the transmembrane region and institute 5 ' the ends for stating the encoding gene in intracellular signal area are connected.
In the present invention, the signal peptide is for instructing the Chimeric antigen receptor CAR-CD19 or CAR-BCMA to express to thin Cellular surface, the signal peptide are cut in protein translation maturation by signal peptidase.And believe in the encoding gene of CAR-CD19 The amino acid sequence of number peptide and signal peptide in the encoding gene of the CAR-BCMA may be the same or different, and be also possible to The amino acid sequence of their signal peptide is identical, and nucleotide sequence is different.
Optionally, the amino acid sequence of the signal peptide includes the amino acid sequence as shown in SEQ ID NO:21.
Optionally, the encoding gene of the signal peptide includes the nucleotide sequence as shown in SEQ ID NO:22.
Optionally, the encoding gene of the signal peptide includes the nucleotide sequence as shown in SEQ ID NO:23.
Optionally, the encoding gene of the signal peptide should consider degeneracy base, the i.e. ammonia as shown in SEQ ID NO:21 The encoding gene of base acid sequence includes such as SEQ ID NO:22 or the nucleotide sequence as shown in SEQ ID NO:23, protection scope The nucleotide sequence that there is base degeneracy matter with SEQ ID NO:22 or SEQ ID NO:23, these nucleotide should also be protected The corresponding amino acid sequence of sequence remains as SEQ ID NO:21.
It, can for the extracellular hinge area, transmembrane region, the specific choice in intracellular signal area and corresponding coding gene sequence Referring to described in first aspect present invention, which is not described herein again.
Optionally, the encoding gene of the CAR-CD19 includes as amino acid sequence shown in SEQ ID NO:7 is corresponding Nucleotide sequence.
Optionally, the encoding gene of the CAR-CD19 includes the nucleotide sequence as shown in SEQ ID NO:19.Certainly, The encoding gene of the CAR-CD19 may also comprise the nucleotide for having base degeneracy matter with sequence shown in SEQ ID NO:19 Sequence.
Optionally, the encoding gene of the CAR-BCMA includes as amino acid sequence shown in SEQ ID NO:8 is corresponding Nucleotide sequence.
Optionally, the encoding gene of the CAR-BCMA includes the nucleotide sequence as shown in SEQ ID NO:20.Certainly, The encoding gene of the CAR-BCMA may also comprise the nucleotide for having base degeneracy matter with sequence shown in SEQ ID NO:20 Sequence.
By taking CAR-CD19 as an example, compared with SEQ ID NO:19 nucleotide sequence shown in the SEQ ID NO:17, Duo Liaolian Connect the encoding gene of peptide, but when Chimeric antigen receptor CAR-CD19 expression is to T cell surface, signal peptide is in protein translation maturation It is cut in the process by signal peptidase.Therefore, in amino acid sequence (the SEQ ID of the Chimeric antigen receptor CAR-CD19 translated into NO:5 in) and not with the amino acid sequence as shown in SEQ ID NO:21.The case where CAR-BCMA, is similar therewith.
By taking CAR-CD19 as an example, the coding gene sequence of the CAR-CD19 is inserted into I He of BamH in pWPXLD carrier Between I restriction enzyme site of EcoR, and it is located at after the extension factor 1 α (EF1 α) of pWPXLD carrier, using EF1 α as promoter.It is described When the coding gene sequence of CAR-CD19 is inserted into pWPXLD carrier, 5 ' ends of the gene order of the CAR-CD19 can be also added Initiation codon (such as ATG) is connected with BamH1 restriction enzyme site in pWPXLD carrier, 3 ' end can also be added terminator codon with EcoR1 restriction enzyme site is connected in pWPXLD carrier.The case where CAR-BCMA, is same.
Optionally, described " respectively to the pWPXLD-CAR-CD19 recombinant plasmid and the pWPXLD- in step (3) CAR-BCMA recombinant plasmid is packed, and obtains the first recombinant slow virus with CAR-CD19 encoding gene and with CAR-BCMA Second recombinant slow virus of encoding gene ", comprising:
By the pWPXLD-CAR-CD19 recombinant plasmid and envelope plasmid and packaging plasmid cotransfection host cell, obtain First recombinant slow virus；By the pWPXLD-CAR-BCMA recombinant plasmid and envelope plasmid and packaging plasmid cotransfection place Chief cell obtains second recombinant slow virus.
Using method preparation and reorganization plasmid of the present invention and packaging virus, wherein the pWPXLD-CAR-CD19 weight On group plasmid and pWPXLD-CAR-CD22 recombinant plasmid, the encoding gene of the encoding gene of CAR-CD19 and the CAR-BCMA It has passed through codon optimization, and molecular weight is suitable for, the packaging efficiency of recombinant slow virus is high, while the disease as made from host cell The concentration of poison is higher.Correspondingly, using the first recombinant slow virus and the second recombinant slow virus come co-transfection CD3 positive T lymph When cell, the dosage of both recombinant slow virus is lower, can reduce experimental cost.
In an embodiment of the present invention, the envelope plasmid is PMD2G, and the packaging plasmid is psPAX2, 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 ?.The source of people peripheral blood mononuclear cells is from autologous vein blood, autologous bone marrow, Cord blood and placental blood etc..Further Optionally, the fresh peripheral blood or marrow acquired after cancer patient's operation one month, after chemicotherapy one month.
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.
Wherein, in step (4), first recombinant slow virus and second recombinant slow virus separately or simultaneously are joined Close transfection CD3 positive t lymphocytes, comprising:
After first transfecting CD3 positive t lymphocytes using first recombinant slow virus, then using the second recombinant lentiviral disease Poison is transfected；Or after first using second recombinant slow virus transfection CD3 positive t lymphocytes, then use first weight Group slow virus is transfected；Or CD3 sun is simultaneously transfected using first recombinant slow virus and second recombinant slow virus Property T lymphocyte.Here " co-transfection " refers to carries out for same group of cell.
Further, in step (4), the virus titer of used first recombinant slow virus and the first recombinant slow virus it Than being 1:(0.5-2).
In the present invention, the targeting T lymphocyte being prepared includes with the CAR-CD19 and the CAR- Double target spot Chimeric antigen receptor T cells of BCMA, the Chimeric antigen receptor T cell with the CAR-CD19 and with the CAR- At least one of Chimeric antigen receptor T cell of BCMA.Optionally, double targets with the CAR-CD19 and CAR-BCMA Point Chimeric antigen receptor T cell, or for the Chimeric antigen receptor T cell with the CAR-CD19 and with the CAR-BCMA's The mixing of Chimeric antigen receptor T cell, or for the Chimeric antigen receptor T cell with the CAR-CD19 and with the CAR- The mixing of the one or two and double target spot Chimeric antigen receptor T cells of the Chimeric antigen receptor T cell of BCMA.
Preferably, the targeting T lymphocyte is chimeric for double target spots with the CAR-CD19 and CAR-BCMA Antigen receptor T cell, or be the Chimeric antigen receptor T cell with the CAR-CD19 and the inosculating antibody with the CAR-BCMA The mixing of original receptor T cell, or it is thin for double target spot Chimeric antigen receptor T with the CAR-CD19 and CAR-BCMA Born of the same parents, the Chimeric antigen receptor T cell with the CAR-CD19, and the Chimeric antigen receptor T cell with the CAR-BCMA this Three kinds of mixing.
At this point, the surface tool of targeting T lymphocyte there are two it is independent, not covalently bound Chimeric antigen receptor ( Be there are two independent single-chain antibody), do not influence they to the identification of respective target, combine, can simultaneously, efficiently identify it is swollen CD19 and BCMA target on oncocyte.The targeting T lymphocyte swells to one or two kinds of in expression CD19 and BCMA Oncocyte can identify and kill, and target spot escape occurs in avoidable tumour cell, improve the range and intensity of its targets identification, And killing broad spectrum activity, also there is stronger tumor-killing ability under complicated tumor microenvironment.
In another embodiment of the present invention, when required targeting T lymphocyte is the inosculating antibody with the CAR-CD19 When the mixing of original receptor T cell and Chimeric antigen receptor T cell with the CAR-BCMA, it can also make in the following ways : using above-mentioned first recombinant slow virus transfect CD3 positive t lymphocytes, obtain the chimeric antigen with the CAR-CD19 by Body T cell；CD3 positive t lymphocytes are transfected using above-mentioned second recombinant slow virus, obtain the inosculating antibody with the CAR-BCMA Original receptor T cell；Then both Chimeric antigen receptor T cells are mixed.It can also be used for examining for B system malignant tumour in this way Disconnected and/treatment.
In the preparation method for the targeting T lymphocyte that fourth aspect present invention provides, base is encoded using band CAR-CD19 First recombinant slow virus of cause and the second recombinant slow virus with CAR-BCMA encoding gene either separately or simultaneously co-transfection CD3 positive t lymphocytes may make Chimeric antigen receptor CAR-CD19 and/or CAR- in targeting T lymphocyte obtained The expression efficiency of BCMA is higher, with the identification of preferable tumour, killing ability.
5th aspect, the present invention provides a kind of targeting T lymphocytes as described in the first aspect of the invention, such as this hair Recombinant viral vector described in bright second aspect, host cell as described in the third aspect of the present invention or such as fourth aspect present invention Application of the targeting T lymphocyte made from the preparation method in the drug that preparation diagnoses and/or treats malignant tumour.
Particularly, relevant swollen suitable for expressing the malignant tumour of CD19 and/or BCMA, especially malignant B The diagnosing and treating of tumor, such as bone-marrow-derived lymphocyte leukaemia, B cell lymphoma etc..
The application can be with specifically: provides a kind of kit, the kit includes target as described in relation to the first aspect Tropism T lymphocyte or the targeting T lymphocyte transfected using the recombinant viral vector as described in second aspect are adopted The targeting T lymphocyte obtained by the preparation method as described in fourth aspect, recombination as described in respect of the second aspect of the invention One of viral vectors, host cell as described in the fourth aspect of the present invention 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-CD19 recombinant plasmid provided in an embodiment of the present invention.
Fig. 2 is the plasmid map of pWPXLd-CAR-BCMA recombinant plasmid provided in an embodiment of the present invention.
Fig. 3 is the tumor cell in vitro fragmentation effect figure of targeting T lymphocyte provided in an embodiment of the present invention.
Fig. 4 is the effect picture that targeting T lymphocyte provided in an embodiment of the present invention treats mice with tumor.
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.
A kind of preparation method of the targeting T lymphocyte of embodiment one, comprising the following steps:
(1) gene order of the Chimeric antigen receptor CAR-CD19 of preparation targeting CD19
Prepare respectively signal peptide, target the single-chain antibody of CD19, CD8 α hinge area, CD8 transmembrane region, 4-1BB signaling zone and The encoding gene of CD3 ζ signaling zone, in the CAR-CD19, the encoding gene of signal peptide is as shown in SEQ ID NO:22, targeting The encoding gene of the single-chain antibody of CD19 is as shown in SEQ ID NO:3, the encoding gene of CD8 α hinge area such as SEQ ID NO:10 Shown, the encoding gene of the CD8 transmembrane region is as shown in SEQ ID NO:12, the encoding gene such as SEQ of the 4-1BB signaling zone Shown in ID NO:14, the encoding gene of the CD3 ζ signaling zone is as shown in SEQ ID NO:16.
By the method for PCR by above-mentioned signal peptide, target single-chain antibody, the CD8 α hinge area, CD8 transmembrane region, 4- of CD19 1BB signaling zone is successively connected together from 5 ' ends to 3 ' ends with the encoding gene of CD3 ζ signaling zone, obtains the coding of CAR-CD19 Gene, the encoding gene of the CAR-CD19 is as shown in SEQ ID NO:19.
(2) gene order of the Chimeric antigen receptor CAR-BCMA of preparation targeting BCMA
Prepare respectively signal peptide, target the single-chain antibody of BCMA, CD8 α hinge area, CD8 transmembrane region, 4-1BB signaling zone and The encoding gene of CD3 ζ signaling zone, the encoding gene of signal peptide used in the CAR-BCMA is as shown in SEQ ID NO:23, target To BCMA single-chain antibody encoding gene as shown in SEQ ID NO:4, the encoding gene of CD8 α hinge area such as SEQ ID NO: Shown in 10, the encoding gene of CD8 transmembrane region is as shown in SEQ ID NO:12, the encoding gene such as SEQ of the 4-1BB signaling zone Shown in ID NO:14, the encoding gene of the CD3 ζ signaling zone is as shown in SEQ ID NO:16.
By the method for PCR by above-mentioned signal peptide, target single-chain antibody, the CD8 α hinge area, CD8 transmembrane region, 4- of BCMA 1BB signaling zone is successively connected together from 5 ' ends to 3 ' ends with the encoding gene of CD3 ζ signaling zone, obtains the coding of CAR-BCMA Gene, the encoding gene of the CAR-BCMA is as shown in SEQ ID NO:20.
(3) pWPXLd-CAR-CD19 recombinant plasmid and pWPXLd-CAR-BCMA recombinant plasmid are constructed
The encoding gene of CAR-CD19 is inserted between I restriction enzyme site of BamH I and EcoR of pWPXLD carrier, and After pWPXLD carrier EF1 α, using EF1 α as promoter.When the encoding gene of the CAR-CD19 is inserted into pWPXLD carrier, institute I restriction enzyme site of BamH in initiation codon (such as ATG) and pWPXLD carrier can be added in the 5 ' ends for stating the encoding gene of CAR-CD19 It is connected, 3 ' ends can be added terminator codon (such as TAA) and be connected with I restriction enzyme site of EcoR in pWPXLD carrier.Then it is transferred to large intestine Bacillus competent cell DH5 α carries out positive colony PCR identification and sequencing identification.By PCR product detected through gel electrophoresis and survey Sequence identification meets target fragment size and sequence, successfully constructs pWPXLd-CAR-CD19 recombinant plasmid as shown in Figure 1.
The encoding gene of CAR-BCMA is inserted between I restriction enzyme site of BamH I and EcoR of pWPXLD carrier, and After pWPXLD carrier EF1 α, using EF1 α as promoter.Wherein carried when the encoding gene of the CAR-BCMA is inserted into pWPXLD When body, I enzyme of BamH in initiation codon (such as ATG) and pWPXLD carrier can be added in 5 ' ends of the encoding gene of the CAR-BCMA Enzyme site is connected, and 3 ' ends can be added terminator codon (such as TAA) and be connected with I restriction enzyme site of EcoR in pWPXLD carrier.Then turn Enter competent escherichia coli cell DH5 α, carries out positive colony PCR identification and sequencing identification.It is examined by PCR product gel electrophoresis It surveys and sequencing identification meets target fragment size and sequence, successfully construct pWPXLd-CAR-BCMA recombination matter as shown in Figure 2 Grain.
(4) recombinant slow virus constructs
PWPXLd-CAR-BCMA 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 merge with the viral supernatants of 48h harvest and are added together It in ultracentrifugation pipe, is put into Beckman ultracentrifuge one by one, setting parameter of noncentricity is 25000rpm, and centrifugation time is 2h, centrifuging temperature are controlled at 4 DEG C；It after centrifugation, discards supernatant, removal as far as possible remains in the liquid on tube wall, and virus is added Liquid is saved, gently piping and druming is resuspended repeatedly；Through after completely dissolution, high speed centrifugation 10000rpm takes supernatant fluorescence method after being centrifuged 5min Titre is measured, by virus according to 100 μ L, 2 × 108TU/mL packing, is stored in -80 DEG C of ultra low temperature freezers, obtains band CAR-CD19 The first recombinant slow virus.
PWPXLd-CAR-BCMA 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 merge with the viral supernatants of 48h harvest and are added together It in ultracentrifugation pipe, is put into Beckman ultracentrifuge one by one, setting parameter of noncentricity is 25000rpm, and centrifugation time is 2h, centrifuging temperature are controlled at 4 DEG C；It after centrifugation, discards supernatant, removal as far as possible remains in the liquid on tube wall, and virus is added Liquid is saved, gently piping and druming is resuspended repeatedly；Through after completely dissolution, high speed centrifugation 10000rpm takes supernatant fluorescence method after being centrifuged 5min Titre is measured, by virus according to 100 μ L, 2 × 108TU/mL packing, is stored in -80 DEG C of ultra low temperature freezers, obtains band CAR-BCMA The second recombinant slow virus.
(5) preparation of targeting T lymphocyte
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；PBS is washed It washs, after removing immunomagnetic beads, 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, while being added and CD3 positive cell The first recombinant slow virus with CAR-CD19 and the second recombinant slow virus with CAR-BCMA of the corresponding virus titer of number carry out Co-incubation, wherein the ratio between the first recombinant slow virus and the dosage (titre) of the second recombinant slow virus are 1:1.
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, and obtained targeting T lymphocyte, i.e. CAR-CD19 Dan Yang by the 9-11 days Property T lymphocyte and the mono- positive t lymphocytes of CAR-BCMA, the bis- positive t lymphocytes cells of CAR-19/CAR-BCMA, and protect In the presence of in the dedicated cells frozen storing liquid of feedback.
Effect example
Effect example one: the tumor cell in vitro of assessment targeting T lymphocyte of the present invention kills situation
It will be thin by targeting T lymphocyte (experimental group) made from the embodiment of the present invention one and the T lymph without preparation Born of the same parents' (negative control group), the T cell (independent group of CD19CAR-T) with individual CAR-CD19 and have individual CAR- The T cell (independent group of BCMA CAR-T) of BCMA is compared, and by above-mentioned four groups of effector cells, (Raji is thin with target cell in vitro Born of the same parents or Nalm-6 cell) in quantity than the ratio for 1:10,1:3,1:1,3:1 and 10:1, in 37 DEG C, 5%CO2Under carry out total training It supports, after incubation 15-18 hours, collects cell, carry out streaming dyeing, cell killing situation is detected, as a result such as Fig. 3 institute Show.As can be seen from Figure 3, the tumor-killing power of the targeting T lymphocyte by method of the present invention preparation is 60% More than, even up to 99%, significantly larger than other control groups, this illustrates the targeting T lymphocyte prepared through the method for the present invention Tumor-killing ability with super strength.
Effect example two assesses targeting T lymphocyte of the present invention to mouse interior tumor cell killing situation
The targeting T lymphocyte (experimental group) and the T lymph without preparation that prepare by the embodiment of the present invention one is thin Born of the same parents' (negative control group), the T cell (independent group of CD19 CAR-T) with individual CAR-CD19 and have individual CAR- The T cell (independent group of BCMACAR-T) of BCMA gives every mouse tail vein injection 1 in mouse lymphocyte Leukemia Model ×106A cell (n=9), obtains the survivorship curve of mouse, as a result as shown in Figure 4.It is prepared by the present invention as can be seen from Figure 4 Targeting T lymphocyte can enable mouse survival rate to stablize 80% or so in injection Mice Body after 30 days, considerably beyond Negative control group and independent group of two above.Fig. 4's the result shows that the targeting T lymphocyte of offer can preferably protect it is small Mouse is from because dead caused by lymphocytic leukemia.
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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275 280 285
Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser
290 295 300
Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr
305 310 315 320
Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu
325 330 335
Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu
340 345 350
Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln
355 360 365
Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu
370 375 380
Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly
385 390 395 400
Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
405 410 415
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
420 425 430
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
435 440 445
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
450 455 460
Arg
465
<210> 6
<211> 471
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 6
Asp Ile Val Leu Thr Gln Ser Pro Pro Ser Leu Ala Met Ser Leu Gly
1 5 10 15
Lys Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Thr Ile Leu
20 25 30
Gly Ser His Leu Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Thr Leu Leu Ile Gln Leu Ala Ser Asn Val Gln Thr Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp
65 70 75 80
Pro Val Glu Glu Asp Asp Val Ala Val Tyr Tyr Cys Leu Gln Ser Arg
85 90 95
Thr Ile Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly
100 105 110
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
115 120 125
Gly Gly Ser Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys
130 135 140
Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe
145 150 155 160
Thr Asp Tyr Ser Ile Asn Trp Val Lys Arg Ala Pro Gly Lys Gly Leu
165 170 175
Lys Trp Met Gly Trp Ile Asn Thr Glu Thr Arg Glu Pro Ala Tyr Ala
180 185 190
Tyr Asp Phe Arg Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser
195 200 205
Thr Ala Thr Leu Gln Ile Asn Asn Leu Lys Tyr Glu Asp Thr Ala Thr
210 215 220
Tyr Phe Cys Ala Leu Ala Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln
225 230 235 240
Gly Thr Ser Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro
245 250 255
Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro
260 265 270
Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu
275 280 285
Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys
290 295 300
Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly
305 310 315 320
Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val
325 330 335
Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu
340 345 350
Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp
355 360 365
Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn
370 375 380
Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg
385 390 395 400
Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly
405 410 415
Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu
420 425 430
Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu
435 440 445
Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His
450 455 460
Met Gln Ala Leu Pro Pro Arg
465 470
<210> 7
<211> 485
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 7
Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His
1 5 10 15
Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser
20 25 30
Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp
35 40 45
Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val
50 55 60
Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser
65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser
85 90 95
Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn
100 105 110
Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly
115 120 125
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val
130 135 140
Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu
145 150 155 160
Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val
165 170 175
Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val
180 185 190
Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg
195 200 205
Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met
210 215 220
Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His
225 230 235 240
Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr
245 250 255
Ser Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr
260 265 270
Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala
275 280 285
Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe
290 295 300
Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val
305 310 315 320
Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys
325 330 335
Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr
340 345 350
Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu
355 360 365
Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro
370 375 380
Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly
385 390 395 400
Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro
405 410 415
Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr
420 425 430
Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly
435 440 445
Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln
450 455 460
Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln
465 470 475 480
Ala Leu Pro Pro Arg
485
<210> 8
<211> 491
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 8
Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His
1 5 10 15
Ala Ala Arg Pro Asp Ile Val Leu Thr Gln Ser Pro Pro Ser Leu Ala
20 25 30
Met Ser Leu Gly Lys Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser
35 40 45
Val Thr Ile Leu Gly Ser His Leu Ile His Trp Tyr Gln Gln Lys Pro
50 55 60
Gly Gln Pro Pro Thr Leu Leu Ile Gln Leu Ala Ser Asn Val Gln Thr
65 70 75 80
Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr
85 90 95
Leu Thr Ile Asp Pro Val Glu Glu Asp Asp Val Ala Val Tyr Tyr Cys
100 105 110
Leu Gln Ser Arg Thr Ile Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu
115 120 125
Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
130 135 140
Gly Ser Gly Gly Gly Gly Ser Gln Ile Gln Leu Val Gln Ser Gly Pro
145 150 155 160
Glu Leu Lys Lys Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser
165 170 175
Gly Tyr Thr Phe Thr Asp Tyr Ser Ile Asn Trp Val Lys Arg Ala Pro
180 185 190
Gly Lys Gly Leu Lys Trp Met Gly Trp Ile Asn Thr Glu Thr Arg Glu
195 200 205
Pro Ala Tyr Ala Tyr Asp Phe Arg Gly Arg Phe Ala Phe Ser Leu Glu
210 215 220
Thr Ser Ala Ser Thr Ala Thr Leu Gln Ile Asn Asn Leu Lys Tyr Glu
225 230 235 240
Asp Thr Ala Thr Tyr Phe Cys Ala Leu Ala Tyr Ser Tyr Ala Met Asp
245 250 255
Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Thr Thr Thr Pro
260 265 270
Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu
275 280 285
Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His
290 295 300
Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu
305 310 315 320
Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr
325 330 335
Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe
340 345 350
Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg
355 360 365
Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser
370 375 380
Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr
385 390 395 400
Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys
405 410 415
Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn
420 425 430
Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu
435 440 445
Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly
450 455 460
His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr
465 470 475 480
Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
485 490
<210> 9
<211> 45
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 9
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> 10
<211> 135
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgat 135
<210> 11
<211> 24
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 11
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> 12
<211> 72
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60
accctttact gc 72
<210> 13
<211> 42
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 13
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> 14
<211> 126
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60
actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120
gaactg 126
<210> 15
<211> 112
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 15
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> 16
<211> 336
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
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> 17
<211> 1395
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60
atcagttgca gggcaagtca ggacattagt aaatatttaa attggtatca gcagaaacca 120
gatggaactg ttaaactcct gatctaccat acatcaagat tacactcagg agtcccatca 180
aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240
gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 300
gggaccaagc tggagatcac aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc 360
ggatctgagg tgaaactgca ggagtcagga cctggcctgg tggcgccctc acagagcctg 420
tccgtcacat gcactgtctc aggggtctca ttacccgact atggtgtaag ctggattcgc 480
cagcctccac gaaagggtct ggagtggctg ggagtaatat ggggtagtga aaccacatac 540
tataattcag ctctcaaatc cagactgacc atcatcaagg acaactccaa gagccaagtt 600
ttcttaaaaa tgaacagtct gcaaactgat gacacagcca tttactactg tgccaaacat 660
tattactacg gtggtagcta tgctatggac tactggggcc aaggaacctc agtcaccgtc 720
tcctcaacca cgacgccagc gccgcgacca ccaacaccgg cgcccaccat cgcgtcgcag 780
cccctgtccc tgcgcccaga ggcgtgccgg ccagcggcgg ggggcgcagt gcacacgagg 840
gggctggact tcgcctgtga tatctacatc tgggcgccct tggccgggac ttgtggggtc 900
cttctcctgt cactggttat caccctttac tgcaaacggg gcagaaagaa actcctgtat 960
atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc 1020
tgccgatttc cagaagaaga agaaggagga tgtgaactga gagtgaagtt cagcaggagc 1080
gcagacgccc ccgcgtacaa gcagggccag aaccagctct ataacgagct caatctagga 1140
cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga 1200
aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1260
gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1320
ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 1380
gccctgcccc ctcgc 1395
<210> 18
<211> 1413
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
gacatcgtgc tgacccagag ccctcctagc ctggccatga gcctgggcaa gagggccacc 60
atcagctgca gggccagcga aagcgtgacc atcctgggca gccacctgat ccactggtac 120
cagcagaagc ctggccagcc ccctaccctg ctgatccagc tggccagcaa cgtgcagaca 180
ggcgtgcctg ccaggtttag cggcagcggc agcaggaccg acttcaccct gaccatcgac 240
cctgtggagg aggacgacgt ggccgtgtac tactgcctgc agagcaggac catccctagg 300
accttcggcg gcggcaccaa gctggagatt aagggaggcg gaggatctgg cggcggagga 360
agtggcggag ggggatctgg gggaggcgga agccagatcc agctggtgca gagcggccct 420
gagctgaaga agcccggcga gaccgtgaag atcagctgca aggccagcgg ctacaccttc 480
accgactaca gcatcaactg ggtgaagagg gcccctggca agggcctgaa gtggatgggc 540
tggatcaaca ccgagaccag ggagcccgcc tacgcctacg acttcagggg caggttcgcc 600
ttcagcctgg agacaagcgc cagcaccgcc accctgcaga tcaacaacct gaagtacgag 660
gacaccgcca catacttctg cgccctggcc tacagctacg ccatggacta ctggggccag 720
ggcacatccg tgaccgtgag cagcaccacg acgccagcgc cgcgaccacc aacaccggcg 780
cccaccatcg cgtcgcagcc cctgtccctg cgcccagagg cgtgccggcc agcggcgggg 840
ggcgcagtgc acacgagggg gctggacttc gcctgtgata tctacatctg ggcgcccttg 900
gccgggactt gtggggtcct tctcctgtca ctggttatca ccctttactg caaacggggc 960
agaaagaaac tcctgtatat attcaaacaa ccatttatga gaccagtaca aactactcaa 1020
gaggaagatg gctgtagctg ccgatttcca gaagaagaag aaggaggatg tgaactgaga 1080
gtgaagttca gcaggagcgc agacgccccc gcgtacaagc agggccagaa ccagctctat 1140
aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg 1200
gaccctgaga tggggggaaa gccgagaagg aagaaccctc aggaaggcct gtacaatgaa 1260
ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 1320
aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 1380
gacgcccttc acatgcaggc cctgccccct cgc 1413
<210> 19
<211> 1455
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
gccttaccag tgaccgcctt gctcctgccg ctggccttgc tgctccacgc cgccaggccg 60
gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 120
atcagttgca gggcaagtca ggacattagt aaatatttaa attggtatca gcagaaacca 180
gatggaactg ttaaactcct gatctaccat acatcaagat tacactcagg agtcccatca 240
aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 300
gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 360
gggaccaagc tggagatcac aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc 420
ggatctgagg tgaaactgca ggagtcagga cctggcctgg tggcgccctc acagagcctg 480
tccgtcacat gcactgtctc aggggtctca ttacccgact atggtgtaag ctggattcgc 540
cagcctccac gaaagggtct ggagtggctg ggagtaatat ggggtagtga aaccacatac 600
tataattcag ctctcaaatc cagactgacc atcatcaagg acaactccaa gagccaagtt 660
ttcttaaaaa tgaacagtct gcaaactgat gacacagcca tttactactg tgccaaacat 720
tattactacg gtggtagcta tgctatggac tactggggcc aaggaacctc agtcaccgtc 780
tcctcaacca cgacgccagc gccgcgacca ccaacaccgg cgcccaccat cgcgtcgcag 840
cccctgtccc tgcgcccaga ggcgtgccgg ccagcggcgg ggggcgcagt gcacacgagg 900
gggctggact tcgcctgtga tatctacatc tgggcgccct tggccgggac ttgtggggtc 960
cttctcctgt cactggttat caccctttac tgcaaacggg gcagaaagaa actcctgtat 1020
atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc 1080
tgccgatttc cagaagaaga agaaggagga tgtgaactga gagtgaagtt cagcaggagc 1140
gcagacgccc ccgcgtacaa gcagggccag aaccagctct ataacgagct caatctagga 1200
cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga 1260
aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1320
gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1380
ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 1440
gccctgcccc ctcgc 1455
<210> 20
<211> 1473
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
gccctgcctg tgacagccct gctgctgcct ctggctctgc tgctgcatgc cgctagaccc 60
gacatcgtgc tgacccagag ccctcctagc ctggccatga gcctgggcaa gagggccacc 120
atcagctgca gggccagcga aagcgtgacc atcctgggca gccacctgat ccactggtac 180
cagcagaagc ctggccagcc ccctaccctg ctgatccagc tggccagcaa cgtgcagaca 240
ggcgtgcctg ccaggtttag cggcagcggc agcaggaccg acttcaccct gaccatcgac 300
cctgtggagg aggacgacgt ggccgtgtac tactgcctgc agagcaggac catccctagg 360
accttcggcg gcggcaccaa gctggagatt aagggaggcg gaggatctgg cggcggagga 420
agtggcggag ggggatctgg gggaggcgga agccagatcc agctggtgca gagcggccct 480
gagctgaaga agcccggcga gaccgtgaag atcagctgca aggccagcgg ctacaccttc 540
accgactaca gcatcaactg ggtgaagagg gcccctggca agggcctgaa gtggatgggc 600
tggatcaaca ccgagaccag ggagcccgcc tacgcctacg acttcagggg caggttcgcc 660
ttcagcctgg agacaagcgc cagcaccgcc accctgcaga tcaacaacct gaagtacgag 720
gacaccgcca catacttctg cgccctggcc tacagctacg ccatggacta ctggggccag 780
ggcacatccg tgaccgtgag cagcaccacg acgccagcgc cgcgaccacc aacaccggcg 840
cccaccatcg cgtcgcagcc cctgtccctg cgcccagagg cgtgccggcc agcggcgggg 900
ggcgcagtgc acacgagggg gctggacttc gcctgtgata tctacatctg ggcgcccttg 960
gccgggactt gtggggtcct tctcctgtca ctggttatca ccctttactg caaacggggc 1020
agaaagaaac tcctgtatat attcaaacaa ccatttatga gaccagtaca aactactcaa 1080
gaggaagatg gctgtagctg ccgatttcca gaagaagaag aaggaggatg tgaactgaga 1140
gtgaagttca gcaggagcgc agacgccccc gcgtacaagc agggccagaa ccagctctat 1200
aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg 1260
gaccctgaga tggggggaaa gccgagaagg aagaaccctc aggaaggcct gtacaatgaa 1320
ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 1380
aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 1440
gacgcccttc acatgcaggc cctgccccct cgc 1473
<210> 21
<211> 20
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 21
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> 22
<211> 60
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
gccttaccag tgaccgcctt gctcctgccg ctggccttgc tgctccacgc cgccaggccg 60
<210> 23
<211> 60
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 23
gccctgcctg tgacagccct gctgctgcct ctggctctgc tgctgcatgc cgctagaccc 60

Claims (10)

1. a kind of targeting T lymphocyte, which is characterized in that including target CD19 Chimeric antigen receptor CAR-CD19 and/or Target the Chimeric antigen receptor CAR-BCMA of BCMA, wherein the CAR-CD19 includes being sequentially connected with from aminoterminal to c-terminus The targeting single-chain antibody of CD19, extracellular hinge area, transmembrane region and intracellular signal area amino acid sequence, the CAR-BCMA packet Include single-chain antibody, extracellular hinge area, transmembrane region and the intracellular signal area of the sequentially connected targeting BCMA from aminoterminal to c-terminus Amino acid sequence；
Wherein, the amino acid sequence of the single-chain antibody of the targeting CD19 includes the amino acid sequence as shown in SEQ ID NO:1, The amino acid sequence of the single-chain antibody of the targeting BCMA includes the amino acid sequence as shown in SEQ ID NO:2.
2. targeting T lymphocyte as described in claim 1, which is characterized in that the volume of the single-chain antibody of the targeting CD19 Code gene includes the nucleotide sequence as shown in SEQ ID NO:3, and the encoding gene of the single-chain antibody of the targeting BCMA includes The nucleotide sequence as shown in SEQ ID NO:4.
3. targeting T lymphocyte as described in claim 1, which is characterized in that the amino acid sequence packet of the CAR-CD19 The amino acid sequence as shown in SEQ ID NO:5 is included, the amino acid sequence of the CAR-BCMA includes as shown in SEQ ID NO:6 Amino acid sequence.
4. a kind of recombinant viral vector, which is characterized in that thin including targeting T lymph as described in any one of claims 1-3 The encoding gene of CAR-CD19 described in born of the same parents and/or CAR-BCMA.
5. a kind of host cell, which is characterized in that the host cell includes recombinant viral vector as claimed in claim 4.
6. a kind of preparation method of targeting T lymphocyte characterized by comprising
(1) respectively provide targeting CD19 Chimeric antigen receptor CAR-CD19 encoding gene and targeting BCMA chimeric antigen by The encoding gene of body CAR-BCMA；
The encoding gene of the CAR-CD19 includes encoding gene, the targeting CD19 that signal peptide is sequentially connected with from 5 ' ends to 3 ' ends The encoding gene of single-chain antibody, the encoding gene of extracellular hinge area, the encoding gene of transmembrane region and the coding base in intracellular signal area Cause；The encoding gene of the CAR-BCMA includes encoding gene, the targeting BCMA that signal peptide is sequentially connected with from 5 ' ends to 3 ' ends The encoding gene of single-chain antibody, the encoding gene of extracellular hinge area, the encoding gene of transmembrane region and the coding base in intracellular signal area Cause；
Wherein, the encoding gene of the single-chain antibody of the targeting CD19 includes the amino acid sequence institute as shown in SEQ ID NO:1 The encoding gene of corresponding nucleotide sequence, the single-chain antibody of the targeting BCMA includes the amino as shown in SEQ ID NO:2 Nucleotide sequence corresponding to acid sequence；
(2) encoding gene of the encoding gene of the CAR-CD19 and the CAR-BCMA is inserted respectively into pWPXLD carrier In, obtain pWPXLD-CAR-CD19 recombinant plasmid and pWPXLD-CAR-BCMA recombinant plasmid；
(3) the pWPXLD-CAR-CD19 recombinant plasmid and the pWPXLD-CAR-BCMA recombinant plasmid are wrapped respectively Dress obtains the first recombinant slow virus with CAR-CD19 encoding gene and the disease of the second recombinant lentiviral with CAR-BCMA encoding gene Poison；
(4) by first recombinant slow virus and second recombinant slow virus, separately or simultaneously co-transfection CD3 positive T drenches Bar cell obtains targeting T lymphocyte through separation.
7. the preparation method of targeting T lymphocyte as claimed in claim 6, which is characterized in that the volume of the CAR-CD19 Code gene includes nucleotide sequence corresponding to the amino acid sequence as shown in SEQ ID NO:7, the coding of the CAR-BCMA Gene includes nucleotide sequence corresponding to the amino acid sequence as shown in SEQ ID NO:8.
8. the preparation method of targeting T lymphocyte as claimed in claim 6, which is characterized in that in step (4), used The first recombinant slow virus and the ratio between the virus titer of the second recombinant slow virus be 1:(0.5-2).
9. the preparation method of targeting T lymphocyte as claimed in claim 6, which is characterized in that the targeting T lymph is thin Born of the same parents are double target spot Chimeric antigen receptor T cells with the CAR-CD19 and CAR-BCMA, or for the CAR- The mixing of the Chimeric antigen receptor T cell of CD19 and the Chimeric antigen receptor T cell with the CAR-BCMA, or for described in band Double target spot Chimeric antigen receptor T cells of the CAR-CD19 and CAR-BCMA, the Chimeric antigen receptor T with the CAR-CD19 Cell, and the mixing of the Chimeric antigen receptor T cell with the CAR-BCMA.
10. recombinant viral vector as claimed in claim 4, host cell as claimed in claim 5, such as claim 1-3 Targeting T lymph made from described in any item targeting T lymphocytes or preparation method as described in claim 6-9 is thin Application of the born of the same parents in the drug that preparation diagnoses and/or treats malignant tumour.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110623977A (en) * 2018-06-25 2019-12-31 深圳宾德生物技术有限公司 Application of chimeric antigen receptor T cell targeting BCMA (brain cell activating receptor) in autoimmune diseases
CN110623979A (en) * 2018-06-25 2019-12-31 深圳宾德生物技术有限公司 Application of chimeric antigen receptor T cell targeting CD19 in autoimmune diseases
CN116444669A (en) * 2023-04-04 2023-07-18 上海科棋药业科技有限公司 Humanized antibodies targeting BCMA CAR-T cells

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013154760A1 (en) * 2012-04-11 2013-10-17 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Chimeric antigen receptors targeting b-cell maturation antigen
CN105837693A (en) * 2016-05-30 2016-08-10 李斯文 BCMA-based (B cell maturation antigen-based) chimeric antigen receptor and preparation method and application thereof
WO2016168773A2 (en) * 2015-04-15 2016-10-20 The California Institute For Biomedical Research Optimized pne-based chimeric receptor t cell switches and uses thereof
US20160362472A1 (en) * 2015-04-08 2016-12-15 Hans Bitter Cd20 therapies, cd22 therapies, and combination therapies with a cd19 chimeric antigen receptor (car)- expressing cell
WO2017075537A1 (en) * 2015-10-30 2017-05-04 Aleta Biotherapeutics Inc. Compositions and methods for treatment of cancer
CN107207598A (en) * 2014-12-12 2017-09-26 蓝鸟生物公司 Bcma chimeric antigen receptors
CN110157679A (en) * 2018-02-12 2019-08-23 深圳宾德生物技术有限公司 A kind of targeting T lymphocyte and its preparation method and application

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013154760A1 (en) * 2012-04-11 2013-10-17 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Chimeric antigen receptors targeting b-cell maturation antigen
CN107207598A (en) * 2014-12-12 2017-09-26 蓝鸟生物公司 Bcma chimeric antigen receptors
US20160362472A1 (en) * 2015-04-08 2016-12-15 Hans Bitter Cd20 therapies, cd22 therapies, and combination therapies with a cd19 chimeric antigen receptor (car)- expressing cell
WO2016168773A2 (en) * 2015-04-15 2016-10-20 The California Institute For Biomedical Research Optimized pne-based chimeric receptor t cell switches and uses thereof
WO2017075537A1 (en) * 2015-10-30 2017-05-04 Aleta Biotherapeutics Inc. Compositions and methods for treatment of cancer
CN105837693A (en) * 2016-05-30 2016-08-10 李斯文 BCMA-based (B cell maturation antigen-based) chimeric antigen receptor and preparation method and application thereof
CN110157679A (en) * 2018-02-12 2019-08-23 深圳宾德生物技术有限公司 A kind of targeting T lymphocyte and its preparation method and application

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LINGZHI YAN 等: ""Combined Infusion of CD19 and Bcma-Specific Chimeric Antigen Receptor T Cells for RRMM: Initial Safety and Efficacy Report from a Clinical Pilot Study"", 《BLOOD》 *
刘雪莹: ""CAR-T细胞增强抗多发性骨髓瘤的实验研究"", 《中国优秀硕士学位论文全文数据库(电子期刊) 医药卫生科技辑》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110623977A (en) * 2018-06-25 2019-12-31 深圳宾德生物技术有限公司 Application of chimeric antigen receptor T cell targeting BCMA (brain cell activating receptor) in autoimmune diseases
CN110623979A (en) * 2018-06-25 2019-12-31 深圳宾德生物技术有限公司 Application of chimeric antigen receptor T cell targeting CD19 in autoimmune diseases
CN116444669A (en) * 2023-04-04 2023-07-18 上海科棋药业科技有限公司 Humanized antibodies targeting BCMA CAR-T cells
CN116444669B (en) * 2023-04-04 2024-02-13 科弈(浙江)药业科技有限公司 Humanized antibodies targeting BCMA CAR-T cells

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