CN107245500B - A kind of leaching based on OCTS technology is leukaemia CAR-T therapy vector and its construction method and application - Google Patents
A kind of leaching based on OCTS technology is leukaemia CAR-T therapy vector and its construction method and application Download PDFInfo
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Abstract
本发明公开了一种基于OCTS技术的淋系白血病CAR‑T治疗载体，包括慢病毒骨架质粒、人EF1α启动子、OCTS嵌合受体结构域和IL6R单链抗体；OCTS嵌合受体结构域包括：CD8 leader嵌合受体信号肽、两组单链抗体：第一组选自以下四组单链抗体的任意一组：CD20单链抗体轻链VL、CD20单链抗体重链VH；CD22单链抗体轻链VL、CD22单链抗体重链VH；CD30单链抗体轻链VL、CD30单链抗体重链VH；CD123单链抗体轻链VL、CD123单链抗体重链VH；第二组为CD19单链抗体轻链VL以及CD19单链抗体重链VH；抗体内铰链Inner‑Linker、单链抗体间铰链Inter‑Linker、CD8 Hinge嵌合受体铰链、CD8 Transmembrane嵌合受体跨膜区、TCR嵌合受体T细胞激活域以及嵌合受体共刺激因子区域。此外，本发明还公开了该载体的构建方法及其在制备治疗淋系白血病的药物中的应用。
The invention discloses a lymphoid leukemia CAR-T treatment vector based on OCTS technology, comprising a lentiviral backbone plasmid, a human EF1α promoter, an OCTS chimeric receptor domain and an IL6R single-chain antibody; the OCTS chimeric receptor domain Including: CD8 leader chimeric receptor signal peptide, two groups of single-chain antibodies: the first group is selected from any one of the following four groups of single-chain antibodies: CD20 single-chain antibody light chain VL, CD20 single-chain antibody heavy chain VH; CD22 Single-chain antibody light chain VL, CD22 single-chain antibody heavy chain VH; CD30 single-chain antibody light chain VL, CD30 single-chain antibody heavy chain VH; CD123 single-chain antibody light chain VL, CD123 single-chain antibody heavy chain VH; second group It is CD19 single-chain antibody light chain VL and CD19 single-chain antibody heavy chain VH; Inner-Linker of intra-antibody hinge, Inter-Linker of single-chain antibody hinge, CD8 Hinge chimeric receptor hinge, CD8 Transmembrane chimeric receptor transmembrane region , TCR chimeric receptor T cell activation domain and chimeric receptor costimulator region. In addition, the invention also discloses a construction method of the vector and its application in preparing a drug for treating lymphocytic leukemia.
Description
Technical field
The invention belongs to field of medical biotechnology, and in particular to a kind of carrier more particularly to a kind of leaching based on OCTS technology
It is leukaemia CAR-T therapy vector.Moreover, it relates to construction method and the application of the carrier.
Background technique
The theoretical basis of immunotherapy of tumors is that immune system has identification tumor associated antigen, regulation body attack tumour
The ability of cell (cell dissolution of high degree of specificity).Generation nineteen fifty, Burnet and Thomas propose " immunosurveillance " reason
By, it is believed that the tumour cell for the mutation that often will appear in body can be identified by immune system and be removed, and be controlled for tumour immunity
Treatment established theoretical basis [Burnet FM.Immunological aspects of malignant disease.Lancet,
1967；1:1171-4].Then, various tumour immunotherapies include cytokine therapy, monoclonal antibody therapy, adoptive immunity
The sequential uses such as therapy, vaccine therapy are in clinic.
A kind of more advanced tumour immunotherapy in 2013 --- CAR-T therapy is used successfully to clinic, and is demonstrated by preceding institute not
Some clinical efficacies.CAR-T, full name are Chimeric Antigen Receptor T-Cell Immunotherapy, are fitted into
Antigen receptor T cell immunotherapy.The therapy is the means by transgenosis, by promoter, antigen recognizing district, costimulation because
The chimeric molecule that son, effect area etc. collectively constitute imports in T cell genome, to make T cell to the identification of target cell, letter
Number transduction, killing etc. functions combine together, realize specific killing [the Eleanor J.Cheadle, et to target cell
al.CAR T cells:driving the road from the laboratory to the clinic.
Immunological Reviews 2014.Vol.257:91–106].CAR-T therapy is clinically most leading Novartis
CLT019 recurs refractory Patients With Acute Lymphoblastic Leukemia, six months tumour Progression free survival rates using CLT019 treatment
Reach 67%, wherein longest response time reached more than 2 years.General headquarters are located at the Shanghai You Kadi biological medicine section of Chinese Shanghai
Skill Co., Ltd cooperates with hospital, and by the end of 2 months 2017, refractory Patients With Acute Lymphoblastic Leukemia 36 was recurred in treatment altogether
Example, wherein complete 24, alleviation ratio reaches 66.6%.This is that the subversiveness of anticancer research is broken through.CAR-T cell therapy can
Can be most possible one of the means for curing cancer, and by " Science " magazine be chosen as 2013 annual ten big technological breakthroughs it
It is first.
CAR-T is significant in efficacy in terms of the neoplastic hematologic disorder of the several types such as treatment B- lymphocytic leukemia at present, still
There is also some limitations, the previous Chimeric antigen receptor of mesh can only identify that a kind of antigenic targets, tumour cell are a complexity
Group, after the tumour cell containing corresponding antigens is removed, the tumour cell without corresponding antigens can be proliferated rapidly, and one section
Lead to tumor recurrence after time.So to make CAR-T identification that can identify two kinds of antigens simultaneously, just there are two scheme is optional: first is that
Two groups of Chimeric antigen receptor buildings are entered into a slow virus transgene carrier, disposably two groups of Chimeric antigen receptors are transduceed
Into primary T lymphocyte；Second is that being transduceed in two times with two slow virus transgene carriers, by two groups of Chimeric antigen receptors point
Primary T lymphocyte Zhuan Dao not entered.
The shortcomings that scheme one, is to occupy the valuable capacity of slow virus transgene carrier, is unfavorable for loading other Functional Units
Part；Transgene carrier packaging efficiency is low；Gene transduction efficiency is very low, is difficult transduction and enters in primary T lymphocyte.
The shortcomings that scheme two, is to need by transduceing twice, and the overall efficiency transduceed twice is lower, transduces cycle time
Long, primary cell is easy aging, proliferative capacity is caused to fail, and killing ability decline influences tumor clearance curative effect.
People B-lymphocyte antigen CD19, also known as CD19 are a kind of albumen encoded by people's CD19 gene
Matter.CD19 is mainly expressed in dendritic cells,follicular and B cell surface.CD19 is not expressed in hemopoietic stem cell surface, it exists
In the stage of development of Pro-B cell to Memory B cell, the surface thick liquid cell (Plasma cell) is disappeared to.CD19 is big
Most B cell malignant tumour surface expressions, including chronic lymphocytic leukemia (CLL), B acute lymphoblastic leukemia (B-
ALL), Diffuse Large B-Cell Lymphoma (DLBCL), follicular lymphoma (FL) lymphoma mantle cell (MCL).Therefore, CD19 is
Target spot [Scheuermann RH, the Racila E.CD19 antigen in of one good tumour cell immunization therapy
leukemia and lymphoma diagnosis and immunotherapy.Leukemia&Lymphoma.1995,18
(5-6): 385–97.]。
CD22 is that important work is mainly played in B cell signal transduction in I type transmembrane protein of mature bone-marrow-derived lymphocyte expression
With.A co-receptor of the CD22 as B-cell receptor (BCR), causes CD22 to be crosslinked with BCR by antigen, triggers CD22 phosphoric acid
Change, mainly make downstream signaling proteins dephosphorylation and inactivation, inhibits BCR signal transduction.CD22 is prevalent in normal B cells
In B cell malignant tumour.
CD20 has adjustment effect, and CD20 to the growth of bone-marrow-derived lymphocyte as a kind of ingredient of signal transduction compound
The specific expressed differential period in pre B cell to mature B cell, especially most of lymphoma cell surfaces
(Mohamed-Rachid Boulassel and Ahmed Galal. Immunotherapy for B-Cell Neoplasms
using T Cells expressing Chimeric Antigen Receptors.Sultan Qaboos University
Med J,August 2012,Vol.12,Iss.3,pp.273-285.)。
CD30 belongs to Tumor Necrosis Factor Receptors family, plays an important role in the apoptosis of lymphocyte and proliferation, almost
There is expression in all Hodgkin lymphomas and part non-Hodgkin lymphoma surface, is drenched at present in clinic as Huo Qijin
Important symbol (Carlos A.Ramos, the et al.Chimeric T-Cells of bar tumor and primary cutaneous type diagnosis
for Therapy of CD30+Hodgkin and Non-Hodgkin Lymphomas (HL&NHL).Biology of
Blood and MarrowTransplantation,2016,22(3):S145-S146.)。
CD123 is the alpha chain of human interleukin-13 receptor, in most of acute myeloid leukemia (AML) cell and very much
There is expression on hematopoietic cell surface, and CD123 is an extraordinary immunotherapeutic targets, because even seldom in CD123 expression
Cell in, its expression can increase at any time gradually increase, and acute myeloid leukemia is likely due in white blood
The candidate stem cell of sick early period can then play the role of clear marrow (Saar using CD123 as target spot by clone evolution
Gill,Carl H.June et al.Preclinical targeting of human acute myeloid leukemia
and myeloablation using chimeric antigen receptor-modified T
cells.Blood.2014；123(15):2343-2354.).
IL-6 receptor (IL-6R) is made of an IL6 receptor subunits and two gp130 signal transduction subunits, mainly
It is distributed in liver cell, neutrophil leucocyte, monokaryon, macrophage and lymphocytic cell surface；In addition there are also a kind of soluble recepters
(sIL-6R), this receptor deficiency cross-film ingredient and cytoplasmic components, in signal transduction, the sIL-6R of activation is in conjunction with film
Gp130 subunit combines, and plays it and acts on [RoseJS, SchellerJ, ElsonG, et al.Interleukin-
6biology is coordinated by membrane-bound and soluble receptors:role in in-
flammation and cancer.J Leukoc Biol,2006,80:227-236].Cell in CAR-T cell therapy because
Sub- storm (CRS) is just related to the overactivity of IL-6 signal path, blocks closing IL-6R, is conducive to block IL-6 signal logical
The excessive activation on road, to control CRS reaction.
Currently, there has been no overcome disadvantages mentioned above for CD19 and any one in CD22, CD20, CD30, CD123
The relevant report of the CAR-T treatment of the dual anti-original of kind antigen composition.
Summary of the invention
It is leukaemia CAR-T treatment that one of the technical problem to be solved in the present invention, which is to provide a kind of leaching based on OCTS technology,
Carrier.Firstly, it only needs once to transduce, transduction efficiency is high, does not influence the curative effect of CAR-T treatment；Secondly, it is not take up slowly
The valuable capacity of Viral Gene Transfer Vectors is conducive to load other function element.Third can effectively close IL6R, block IL6 letter
Number access prevents inflammatory factor storm (CRS) from upgrading.
The second technical problem to be solved by the present invention is to provide the construction method of the carrier.
The third technical problem to be solved by the present invention is to provide the application of the carrier.
In order to solve the above technical problems, the present invention adopts the following technical scheme:
In one aspect of the invention, providing a kind of leaching based on OCTS technology is leukaemia CAR-T therapy vector, including slow
Viral backbone plasmid, people EF1 α promoter, OCTS Chimerical receptor structural domain and IL6R single-chain antibody；
The slow virus skeleton plasmid includes: the AmpR containing ampicillin resistance gene for purpose bacterial strain massive amplification
Sequence, as shown in SEQ ID NO.1；For the prokaryotic replions pUC Ori sequence of plasmid replication, such as SEQ ID NO.2 institute
Show；For enhancing the Viral Replicon SV40Ori sequence of the duplication in eukaryocyte, as shown in SEQ ID NO.3；For slow
The slow virus of virus packaging packs cis element；ZsGreen1 green fluorescent protein, as shown in SEQ ID NO.11；IRES core
Sugared body binding sequence, as shown in SEQ ID NO.12；The enhanced marmot of eWPRE for enhancing the expression efficiency of transgenosis
Hepatitis B posttranscriptional regulatory element, as shown in SEQ ID NO.13；
The sequence of the people EF1 α promoter is as shown in SEQ ID NO.14；
The OCTS Chimerical receptor structural domain includes: the CD8 leader Chimerical receptor signal as shown in SEQ ID NO.15
Peptide, two groups of single-chain antibodies: first group is selected from any one group of following four groups of single-chain antibodies: as shown in SEQ ID NO.18
CD20 single-chain antibody light chain VL, the CD20 single-chain antibody heavy chain VH as shown in SEQ ID NO.19；As shown in SEQ ID NO.20
CD22 single-chain antibody light chain VL, the CD22 single-chain antibody heavy chain VH as shown in SEQ ID NO.21；Such as SEQ ID NO.22 institute
CD30 single-chain antibody light chain VL, the CD30 single-chain antibody heavy chain VH as shown in SEQ ID NO.23 shown；Such as SEQ ID NO.24
Shown in CD123 single-chain antibody light chain VL, the CD123 single-chain antibody heavy chain VH as shown in SEQ ID NO.25；Second group is such as
CD19 single-chain antibody light chain VL and the CD19 single-chain antibody heavy chain as shown in SEQ ID NO.17 shown in SEQ ID NO.16
VH；Between antibody inner hinge Inner-Linker, the single-chain antibody as shown in SEQ ID NO.27 as shown in SEQ ID NO.26
Hinge Inter-Linker, the CD8 Hinge Chimerical receptor hinge as shown in SEQ ID NO.28, as shown in SEQ ID NO.29
CD8 Transmembrane Chimerical receptor transmembrane region, the TCR Chimerical receptor t cell activation domain as shown in SEQ ID NO.32
And Chimerical receptor costimulating factor region；Chimerical receptor costimulating factor region be selected from 4-1BB, ICOS, CD27,
OX40、CD28、 MYD88、IL1R1、CD70、TNFRSF19L、TNFRSF27、TNFRSF1OD、TNFRSF13B、TNFRSF18、
The tumor necrosis factor superfamilies such as CD134 (tumor necrosis factor receptor superfamily, TNFRSF)
In the combination of any one or more.
The slow virus packaging cis element can use second generation slow virus carrier, can also use third generation slow virus
Carrier.The slow virus packaging cis element includes: the slow disease as shown in SEQ ID NO.5 using second generation slow virus carrier
Malicious 5terminal LTR, slow virus 3terminal Self-Inactivating LTR as shown in SEQ ID NO.6, such as
Gag cis element shown in SEQ ID NO.7, the RRE cis element as shown in SEQ ID NO.8, such as SEQ ID NO.9 institute
Env cis element, the cPPT cis element as shown in SEQ ID NO.10 shown.The slow virus packaging cis element uses
Third generation slow virus carrier includes: slow virus 5terminal LTR as shown in SEQ ID NO.5, such as SEQ ID NO.6 institute
3 terminal Self-Inactivating LTR of slow virus that shows, the Gag cis element as shown in SEQ ID NO.7, such as
RRE cis element shown in SEQ ID NO.8, the env cis element as shown in SEQ ID NO.9, such as SEQ ID NO.10 institute
The cPPT cis element shown, and the RSV promoter as shown in SEQ ID NO.4.Present invention preferably employs third generation slow virus
Carrier.
Preferably, two groups of single-chain antibodies use series connection mode or corner connection type；It is as shown in Figure 4:
When two groups of single-chain antibodies are CD20 single-chain antibody and CD19 single-chain antibody, the series connection mode specifically:
CD20 single-chain antibody light chain VL and CD19 single-chain antibody light chain VL uses hinge Inter-Linker connection between single-chain antibody,
CD20 single-chain antibody light chain VL is connect with CD20 single-chain antibody heavy chain VH using antibody inner hinge Inner-Linker, and CD19 is mono-
Chain antibody light chain VL is connect with CD19 single-chain antibody heavy chain VH using antibody inner hinge Inner-Linker, i.e. pOCTS2019s
(see Fig. 4 A, Fig. 4 C)；The corner connection type specifically: CD19 single-chain antibody light chain VL and CD19 single-chain antibody heavy chain VH
Using the Inner-Linker connection of antibody inner hinge, CD20 single-chain antibody light chain VL and CD19 single-chain antibody heavy chain VH are using single
Hinge Inter-Linker connection between chain antibody, CD20 single-chain antibody heavy chain VH and CD19 single-chain antibody light chain VL is using single-stranded
Hinge Inter-Linker connection between antibody, i.e. pOCTS2019t (see Fig. 4 B, Fig. 4 C)；
When two groups of single-chain antibodies are CD30 single-chain antibody and CD19 single-chain antibody, the series connection mode specifically:
CD30 single-chain antibody light chain VL and CD19 single-chain antibody light chain VL uses hinge Inter-Linker connection between single-chain antibody,
CD30 single-chain antibody light chain VL is connect with CD30 single-chain antibody heavy chain VH using antibody inner hinge Inner-Linker, and CD19 is mono-
Chain antibody light chain VL is connect with CD19 single-chain antibody heavy chain VH using antibody inner hinge Inner-Linker, i.e. pOCTS3019s
(see Fig. 4 A, Fig. 4 C)；The corner connection type specifically: CD19 single-chain antibody light chain VL and CD19 single-chain antibody heavy chain VH
Using the Inner-Linker connection of antibody inner hinge, CD30 single-chain antibody light chain VL and CD19 single-chain antibody heavy chain VH are using single
Hinge Inter-Linker connection between chain antibody, CD30 single-chain antibody heavy chain VH and CD19 single-chain antibody light chain VL is using single-stranded
Hinge Inter-Linker connection between antibody, i.e. pOCTS3019t (see Fig. 4 B, Fig. 4 C)；
When two groups of single-chain antibodies are CD22 single-chain antibody and CD19 single-chain antibody, the series connection mode specifically:
CD22 single-chain antibody light chain VL and CD19 single-chain antibody light chain VL uses hinge Inter-Linker connection between single-chain antibody,
CD22 single-chain antibody light chain VL is connect with CD22 single-chain antibody heavy chain VH using antibody inner hinge Inner-Linker, and CD19 is mono-
Chain antibody light chain VL is connect with CD19 single-chain antibody heavy chain VH using antibody inner hinge Inner-Linker, i.e. pOCTS2219s
(see Fig. 4 A, Fig. 4 C)；The corner connection type specifically: CD19 single-chain antibody light chain VL and CD19 single-chain antibody heavy chain VH
Using the Inner-Linker connection of antibody inner hinge, CD22 single-chain antibody light chain VL and CD19 single-chain antibody heavy chain VH are using single
Hinge Inter-Linker connection between chain antibody, CD22 single-chain antibody heavy chain VH and CD19 single-chain antibody light chain VL is using single-stranded
Hinge Inter-Linker connection between antibody, i.e. pOCTS2219t (see Fig. 4 B, Fig. 4 C)；
When two groups of single-chain antibodies are CD123 single-chain antibody and CD19 single-chain antibody, the series connection mode specifically:
CD123 single-chain antibody light chain VL and CD19 single-chain antibody light chain VL uses hinge Inter-Linker connection between single-chain antibody,
CD123 single-chain antibody light chain VL is connect with CD123 single-chain antibody heavy chain VH using antibody inner hinge Inner-Linker, CD19
Single-chain antibody light chain VL is connect with CD19 single-chain antibody heavy chain VH using antibody inner hinge Inner-Linker, i.e.,
POCTS12319s (see Fig. 4 A, Fig. 4 C)；The corner connection type specifically: CD19 single-chain antibody light chain VL and CD19 is single-stranded
Heavy chain of antibody VH uses antibody inner hinge Inner-Linker connection, CD123 single-chain antibody light chain VL and CD19 single-chain antibody weight
Chain VH uses hinge Inter-Linker connection between single-chain antibody, CD123 single-chain antibody heavy chain VH and CD19 single-chain antibody light chain
VL uses hinge Inter-Linker connection between single-chain antibody, i.e. pOCTS12319t (see Fig. 4 B, Fig. 4 C).
Preferably, the sequence of the IL6R single-chain antibody is as shown in SEQ ID NO.33.
Preferably, the enhanced marmot hepatitis B posttranscriptional regulatory element of the eWPRE has the enhancing of 6 nucleotide prominent
Become, specifically: g.396G > A, g.397C > T, g.398T > C, g.399G > A, g.400A > T, g.411A > T.
Preferably, entire OCTS expression of structural gene is started by the people EF1 α promoter, the CD8 leader is chimeric
Receptor signal peptide is located at the N-terminal of OCTS coded sequence, for guiding OCTS albumen to be positioned at cell membrane；Described two groups single-stranded anti-
Body is combined into double antigen recognizing districts, for identification corresponding target antigen；The CD8 Hinge Chimerical receptor hinge is used for scFv
It is anchored on the outside of cell membrane；The CD8 Transmembrane Chimerical receptor transmembrane region is for entire Chimerical receptor to be fixed on
On cell membrane；The CD28 Chimerical receptor costimulating factor is for stimulating T lymphocyte Activation In Vitro and interior tumor cell to kill
Wound effect；For promoting, T lymphocyte is proliferated the CD134 Chimerical receptor costimulating factor and cytokine secretion, enhancing tumour are exempted from
Epidemic disease is conducive to the long-term surviving of memory T cell；The TCR Chimerical receptor t cell activation domain is for activating downstream signaling pathway
Expression；The IL6R single-chain antibody is secreted into extracellularly, closes IL6R, is blocked IL6 signal path, is prevented inflammatory factor storm
Upgrading；When antigen recognition region is in conjunction with target antigen, signal is transferred into the cell by Chimerical receptor, to generate T cell
Proliferation, cytokine secretion increase, Anti-apoptotic proteins secretion increase, cell death delay, cracking target cell etc. are a series of
Biological effect.
Preferably, Chimerical receptor costimulating factor region uses the CD28 Chimerical receptor as shown in SEQ ID NO.30
Costimulating factor and the combination of the CD134 Chimerical receptor costimulating factor as shown in SEQ ID NO.31.
Preferably, CD19 single-chain antibody light chain VL, CD19 single-chain antibody heavy chain VH, CD20 single-chain antibody light chain VL,
CD20 single-chain antibody heavy chain VH, CD30 single-chain antibody light chain VL, CD30 single-chain antibody heavy chain VH, CD123 single-chain antibody light chain
VL, CD123 single-chain antibody heavy chain VH, IL6R single-chain antibody pass through humanization modified.
In the second aspect of the present invention, providing the above-mentioned leaching based on OCTS technology of one kind is leukaemia CAR-T therapy vector
Construction method, comprising the following steps:
(1) sequence of AmpR containing ampicillin resistance gene as shown in SEQ ID NO.1, such as SEQ ID NO.2 institute
The prokaryotic replions pUC Ori sequence shown, the Viral Replicon SV40Ori sequence as shown in SEQ ID NO.3, for slow disease
The slow virus of poison packaging packs cis element, the ZsGreen1 green fluorescent protein as shown in SEQ ID NO.11, such as SEQ ID
IRES ribosome binding sequence shown in NO.12, as the enhanced marmot hepatitis B of eWPRE shown in SEQ ID NO.13 turn
Controlling element is stored on slow virus skeleton plasmid after record；
(2) the people EF1 α promoter as shown in SEQ ID NO.14, the OCTS Chimerical receptor structural domain and such as SEQ
IL6R single-chain antibody shown in ID NO.33 is combined into OCTS Chimerical receptor design scheme, by digestion, connection, recombining reaction
It is cloned into slow virus skeleton plasmid, obtains the recombinant slow virus plasmid of third generation OCTS design；
(3) by obtained recombinant slow virus plasmid respectively with slow virus packaging plasmid pPac-GP, pPac-R and memebrane protein
Plasmid pEnv-G transfects HEK293T/17 cell jointly, after carrying out gene transcript expression in HEK293T/17 cell, is packaged into
Function recombined lentivirus vector can be discharged into cells and supernatant, collect the supernatant for the recombined lentivirus vector for including；
(4) obtained recombinant slow virus supernatant is purified using the column purification mode for filtering, adsorbing, eluting, respectively
Obtain recombined lentivirus vector.
Preferably, in step (4), the suction filtration step will control supernatant volume in 200ml~2000ml, control vacuum degree
In -0.5MPA~-0.9MPA, prevent due to plug-hole bring carrier loss；The pH value that the adsorption step will control solution exists
6~8, prevent the variation of PH from carrier being caused to inactivate；The elution step to control the ionic strength of eluent 0.5M~
1.0M prevents the variation of ionic strength from causing elution not exclusively or carrier inactivation.
In the third aspect of the present invention, application of the carrier in the drug that preparation treatment leaching is leukaemia is provided.
Compared with prior art, the invention has the following beneficial effects:
OCTS-CAR-T technology of the present invention is on the basis of current tradition CAR-T cell therapy, by right
The Optimizing Reconstruction of Chimeric antigen receptor (CAR) structure enables Chimeric antigen receptor to identify two kinds of antigens, expands significantly
The identification range of CAR-T cell, the removing for cancer colonies is more thorough, and curative effect is more longlasting；Avoid batch culture CAR-T thin
Born of the same parents greatly save cost；It avoids patient from repeatedly feeding back different targeting CAR-T cells, has saved the economic expenditure of patient, reduce
The probability of recurrence, improves life in patients indirectly.It only needs once to transduce, transduction efficiency is high, does not influence CAR-T treatment
Curative effect；It is not take up the valuable capacity of slow virus transgene carrier, is conducive to load other function element, transgene carrier packaging effect
Rate is high, and gene transduction efficiency is high.
The full name of OCTS is One CAR with Two ScFvs, passes through series connection OCTS (Series OCTS) or corner
The connection type of OCTS (Turn OCTS) by two sections of scFv and is integrated into a chimeric molecule (as shown in Figure 1), assigns T leaching
Bar cell HLA non-dependent mode identifies the ability of two kinds of tumour antigens, can identify more relative to traditional CAR-T cell
Extensive target, the further expansion removing range of tumour cell.It include that two tumour correlations are anti-in the basic engineering of OCTS
Former combined area (tumor-associated antigen, TAA) (is typically derived from monoclonal antibody antigen bond area
ScFv sections), an extracellular hinge area, a transmembrane region, Liang Ge intracellular signal transduction area and a response element area.The area scFv
Domain is crucial determinant for the safety of the specificity of OCTS, validity and genetic modification T cell itself.
As the clinical investigation phase that will enter of OCTS-CAR-T indicates that CAR-T cell therapy will enter for 2.0 epoch.
Carrier framework of the present invention can be applied in third generation slow virus carrier structure, also can be applied to
In two generation slow virus carrier structures.The difference of the second generation and third generation slow virus carrier in structure is as shown in Figure 2 B.The present invention
It is preferred that third generation slow virus carrier (as shown in Figure 2 A), 3 ' SIN LTR eliminate the region U3, eliminate slow virus carrier self
A possibility that duplication, substantially increases safety；CPPT and WPRE element is increased, transduction efficiency and transgenosis are improved
Expression efficiency；The lasting efficient transcription of core RNA when ensure that slow virus carrier packaging using RSV promoter；Using people itself
EF1 α promoter, enable CAR gene in human body long lasting for expression.
CD19 single-chain antibody light chain VL, CD19 single-chain antibody heavy chain VH, CD20 single-chain antibody light chain VL of the present invention,
CD20 single-chain antibody heavy chain VH, CD22 single-chain antibody light chain VL, CD22 single-chain antibody heavy chain VH, CD30 single-chain antibody light chain VL,
CD30 single-chain antibody heavy chain VH, CD123 single-chain antibody light chain VL, CD123 single-chain antibody heavy chain VH, IL6R single-chain antibody passes through
Cross it is humanization modified, can effectively reduce the anti-mouse of internal people resist (Human anti-mouse antibodies, HAMA) production
It is raw, extend half-life period and the function and effect of scFv, increase OCTS-CAR-T cell there are the times.
One kind of costimulating factor used in the present invention or several combination can increase the proliferation speed of cell after transduction
The characteristics such as rate, time-to-live, killing-efficiency, immunological memory.
After Workshop Production of the OCTS-CAR-T cell that the present invention uses by GMP rank, it can be used for human clinical trial.
Recombined lentivirus vector of the invention may be implemented on human T lymphocyte express CD19, CD20, CD22, CD30,
Double targeting Chimeric antigen receptors of the combinations such as CD123, guide and activated T lymphocytes to CD19, CD20, CD22, CD30,
The lethal effect of the positive cells such as CD123 clinically can be used for treating the treatment of bone-marrow-derived lymphocyte leukaemia, B lymthoma.
The present invention passes through the single of recombined lentivirus vector skeleton, OCTS structural domain, interleukin-6 receptor (IL6R)
Chain antibody (single-chain antibody) building forms recombined lentivirus vector, and the recombined lentivirus vector which obtains can
To realize the single-chain antibody for expressing IL-6R in human T lymphocyte, IL6R can be effectively closed, IL-6 signal path is blocked, faces
It can be used for alleviating cytokine release syndrome (Cytokine Release Syndrome, CRS) on bed, ensure cell therapy
The life security of patient in the process.
As it can be seen that OCTS-CAR-T cell of the present invention will be treated to tumour cell provides reliable guarantee.
Detailed description of the invention
Fig. 1 is the schematic diagram of OCTS Chimerical receptor of the present invention, contains series connection OCTS (Series OCTS) and turns
Angle OCTS (Turn OCTS) schematic diagram；
Fig. 2 slow virus carrier structural schematic diagram of the present invention；Wherein Fig. 2A is that the third generation that the present invention uses is sick slowly
Poisonous carrier structural schematic diagram, Fig. 2 B are the second generation and third generation slow virus carrier structure comparison schematic diagram；
Fig. 3 is the building flow chart that recombined lentivirus vector of the present invention is constructed in the embodiment of the present invention 1.Wherein,
(A) figure is the structural schematic diagram of slow virus skeleton plasmid pLenti-3G basic；(B) figure is the schematic diagram of 8 OCTS plasmids；
(C) figure is the structural schematic diagram of pPac-GP plasmid；(D) figure is the structural schematic diagram of pPac-R plasmid；(E) figure is pEnv-G packet
Fill the structural schematic diagram of plasmid；
Fig. 4 is the element orders schematic diagram of OCTS structure in the embodiment of the present invention 1, wherein A figure is series connection OCTS
The structural schematic diagram of (Series OCTS), B figure are the structural schematic diagrams of corner OCTS (Turn OCTS), and C figure is OCTS structure
Plasmid number (OCTS Symbol) list schematic diagram；
Fig. 5 be recombinant slow virus plasmid pOCTS12319s, pOCTS2219s in the embodiment of the present invention 1, pOCTS2019s,
The digestion prediction of pOCTS3019s, pOCTS12319t, pOCTS2219t, pOCTS2019t, pOCTS3019t and digestion agarose
Gel electrophoresis figure；Wherein Fig. 5 A is the digestion prediction schematic diagram of pOCTS12319s, and Fig. 5 B is the digestion agar of pOCTS12319s
Sugared gel electrophoresis figure；Fig. 5 C is the digestion prediction schematic diagram of pOCTS2219s, and Fig. 5 D is that the digestion agarose of pOCTS2219s is solidifying
Gel electrophoresis figure；Fig. 5 E is the digestion prediction schematic diagram of pOCTS2019s, and Fig. 5 F is the digestion Ago-Gel electricity of pOCTS2019s
Swimming figure；Fig. 5 G is the digestion prediction schematic diagram of pOCTS3019s, and Fig. 5 H is the digestion agarose gel electrophoresis of pOCTS3019s
Figure；Fig. 5 I is the digestion prediction schematic diagram of pOCTS12319t, and Fig. 5 J is the digestion agarose gel electrophoresis of pOCTS12319t
Figure；Fig. 5 K is the digestion prediction schematic diagram of pOCTS2219t, and Fig. 5 L is the digestion agarose gel electrophoresis figure of pOCTS2219t；
Fig. 5 M is the digestion prediction schematic diagram of pOCTS2019t, and Fig. 5 N is the digestion agarose gel electrophoresis figure of pOCTS2019t；Fig. 5 O
It is the digestion prediction schematic diagram of pOCTS3019t, Fig. 5 P is the digestion agarose gel electrophoresis figure of pOCTS3019t；In Fig. 5 A
Lane1 is 1kb DNA ladder Marker: band is from top to bottom successively are as follows: 10kb, 8kb, 6kb, 5kb, 4kb, 3.5kb,
3kb,2.5kb,2kb, 1.5kb,1kb,750bp,500bp,250bp；Lane2 in Fig. 5 A is the BamH I of pOCTS12319s
Digestion prediction: band is from top to bottom successively are as follows: 11384bp, 818bp；Lane1 in Fig. 5 B is 1kb DNA ladder
The electrophoresis result of Marker；Lane2 in Fig. 5 B is the BamH I restriction enzyme digestion and electrophoresis result of pOCTS12319s；In Fig. 5 C
Lane1 is 1kb DNA ladder Marker: band is from top to bottom successively are as follows: 10kb, 8kb, 6kb, 5kb, 4kb, 3.5kb,
3kb,2.5kb,2kb,1.5kb,1kb,750bp,500bp,250bp；Lane2 in Fig. 5 C is the Kpn I enzyme of pOCTS2219s
Cut prediction: band is from top to bottom successively are as follows: 7824bp, 4322bp；Lane1 in Fig. 5 D is 1kb DNA ladder Marker
Electrophoresis result；Lane2 in Fig. 5 D is the Kpn I restriction enzyme digestion and electrophoresis result of pOCTS2219s；Lane1 in Fig. 5 E is 1kb
DNA ladder Marker: band is from top to bottom successively are as follows: 10kb, 8kb, 6kb, 5kb, 4kb, 3.5kb, 3kb, 2.5kb,
2kb,1.5kb,1kb,750bp,500bp,250bp；Lane2 in Fig. 5 E is the ApaL I digestion prediction of pOCTS2019s: item
Band is from top to bottom successively are as follows: 4883bp, 3931bp, 1726bp, 1246bp, 497bp；Fig. 5 F lane1 is 1kb DNA
The electrophoresis result of ladder Marker；Fig. 5 F lane2 is the ApaL I restriction enzyme digestion and electrophoresis result of pOCTS2019s；In Fig. 5 G
Lane1 is 1kb DNA ladder Marker: band is from top to bottom successively are as follows: 10kb, 8kb, 6kb, 5kb, 4kb, 3.5kb,
3kb,2.5kb,2kb,1.5kb,1kb,750bp,500 bp,250bp；Lane2 in Fig. 5 G is the BamH I of pOCTS3019s
Digestion prediction: band is from top to bottom successively are as follows: 10268bp, 1197bp, 818bp；Lane1 in Fig. 5 H is 1kb DNA
The electrophoresis result of ladder Marker；Lane2 in Fig. 5 H is the BamH I restriction enzyme digestion and electrophoresis result of pOCTS3019s；In Fig. 5 I
Lane1 be 1kb DNA ladder Marker: band is from top to bottom successively are as follows: 10kb, 8kb, 6kb, 5kb, 4kb,
3.5kb,3kb,2.5kb,2kb,1.5kb,1kb,750bp,500bp,250bp；Lane2 in Fig. 5 I is pOCTS12319t
Sal I digestion prediction: band is from top to bottom successively are as follows: 6934bp, 5389bp；Lane1 in Fig. 5 J is 1kb DNA ladder
The electrophoresis result of Marker；Lane2 in Fig. 5 J is the Sal I restriction enzyme digestion and electrophoresis result of pOCTS12319t；Lane1 in Fig. 5 K
Be 1kb DNA ladder Marker: band is from top to bottom successively are as follows: 10kb, 8kb, 6kb, 5kb, 4kb, 3.5kb, 3kb,
2.5kb,2kb,1.5kb,1kb,750 bp,500bp,250bp；Lane2 in Fig. 5 K is the EcoR I digestion of pOCTS2219t
Prediction: band is from top to bottom successively are as follows: 10626bp, 1399bp, 311bp；Lane1 in Fig. 5 L is 1kb DNA ladder
The electrophoresis result of Marker；Lane2 in Fig. 5 L is the EcoR I restriction enzyme digestion and electrophoresis result of pOCTS2219t；Lane1 in Fig. 5 M
Be 1kb DNA ladder Marker: band is from top to bottom successively are as follows: 10kb, 8kb, 6kb, 5kb, 4kb, 3.5kb, 3kb,
2.5kb,2kb,1.5kb,1kb,750bp,500bp,250bp；Lane2 in Fig. 5 M is that the Pst I digestion of pOCTS2019t is pre-
Survey: band is from top to bottom successively are as follows: 9231bp, 2554bp, 617bp；Lane1 in Fig. 5 N is 1kb DNA ladder
The electrophoresis result of Marker；Lane2 in Fig. 5 N is the Pst I restriction enzyme digestion and electrophoresis result of pOCTS2019t；Lane1 in Fig. 5 O
Be 1kb DNA ladder Marker: band is from top to bottom successively are as follows: 10kb, 8kb, 6kb, 5kb, 4kb, 3.5kb, 3kb,
2.5kb,2kb,1.5kb,1kb,750 bp,500bp,250bp；Lane2 in Fig. 5 O is the Sac II digestion of pOCTS3019t
Prediction: band is from top to bottom successively are as follows: 11652bp, 887bp；Lane1 in Fig. 5 P is 1kb DNA ladder Marker
Electrophoresis result；Lane2 in Fig. 5 P is the Sac II restriction enzyme digestion and electrophoresis result of pOCTS3019t；
Fig. 6 is the titre testing result schematic diagram of recombined lentivirus vector in the embodiment of the present invention 1；
Fig. 7 is the step flow chart of OCTS-CAR-T cell construction described in the embodiment of the present invention 1, includes separation training
The stages such as feeding, activation, gene transfer, OCTS-CAR-T cellular identification；
Fig. 8 is the detection of mycoplasma result schematic diagram of OCTS-CAR-T cell in the embodiment of the present invention 2, wherein lane1 is
DL2000marker, counterband tape is from top to bottom successively from top to bottom are as follows: 2kb, 1kb, 750bp, 500bp, 250bp, 100bp；
Lane2 is positive control；Lane3 is negative control；Lane4 is PBS；Lane5 is lysate；Lane6 is OCTS12319s-
CAR-T cell；Lane7 is OCTS2219s-CAR-T cell；Lane8 is OCTS2019s-CAR-T cell；Lane9 is
OCTS3019s-CAR-T cell；Lane10 is OCTS12319t-CAR-T cell；Lane11 is that OCTS2219t-CAR-T is thin
Born of the same parents；Lane12 is OCTS2019t-CAR-T cell；Lane13 is OCTS3019t-CAR-T cell；
Fig. 9 is the transduction efficiency and immunophenotyping result of flow cytometer detection OCTS-CAR-T cell in the embodiment of the present invention 2
Schematic diagram；Wherein, Fig. 9 A indicates the transduction efficiency result of OCTS12319s-CAR-T cell；Fig. 9 B indicates OCTS12319s-
The immunophenotyping result of CAR-T cell；The transduction efficiency result of Fig. 9 C expression OCTS2219s-CAR-T cell；Fig. 9 D is indicated
The immunophenotyping result of OCTS2219s-CAR-T cell；The transduction efficiency result of Fig. 9 E expression OCTS2019s-CAR-T cell；
The immunophenotyping result of Fig. 9 F expression OCTS2019s-CAR-T cell；The transduction of Fig. 9 G expression OCTS3019s-CAR-T cell
Efficiencies；The immunophenotyping result of Fig. 9 H expression OCTS3019s-CAR-T cell；Fig. 9 I indicates OCTS12319t-CAR-T
The transduction efficiency result of cell；The immunophenotyping result of Fig. 9 J expression OCTS12319t-CAR-T cell；Fig. 9 K is indicated
The transduction efficiency result of OCTS2219t-CAR-T cell；The immunophenotyping result of Fig. 9 L expression OCTS2219t-CAR-T cell；
The transduction efficiency result of Fig. 9 M expression OCTS2019t-CAR-T cell；Fig. 9 N indicates immune point of OCTS2019t-CAR-T cell
Type result；The transduction efficiency result of Fig. 9 O expression OCTS3019t-CAR-T cell；Fig. 9 P indicates OCTS3019t-CAR-T cell
Immunophenotyping result；
Figure 10 is in the embodiment of the present invention 3 under the conditions of different effect target ratios, and OCTS-CAR-T cell is to target cell killing-efficiency
Histogram；Wherein, Figure 10 A indicates OCTS12319s-CAR-T cell and OCTS12319t-CAR-T cell to different target cells
Mortaility results；Figure 10 B indicates that OCTS2219s-CAR-T cell and OCTS2219t-CAR-T cell kill different target cells
Hurt result；Figure 10 C indicates OCTS2019s-CAR-T cell and OCTS2019t-CAR-T cell to the killing knot of different target cells
Fruit；Figure 10 D indicates OCTS3019s-CAR-T cell and OCTS3019t-CAR-T cell to the Mortaility results of different target cells.
Specific embodiment
The invention is further described combined with specific embodiments below.It should be understood that particular implementation described herein
It indicates by way of example, is not intended as limitation of the present invention.Without departing from the scope of the invention, this hair
Bright main feature can be used for various embodiments.Material
1, slow virus skeleton plasmid pLenti-3G basic, slow virus packaging plasmid pPac-GP, pPac-R and film egg
White matter grain pEnv-G, HEK293T/17 cell, homologous recombination enzyme, Oligo Annealing Buffer, mycoplasma test reagent
Box, endotoxin detection kit, CD19+K562、 CD20+K562、CD22+K562、CD30+K562、CD123+K562、CD19+
CD123+K562、CD19+CD20+K562、CD19+CD22+K562、 CD19+CD30+K562, K562 cell take wing (Shanghai) purchased from generation
Biological medicine Science and Technology Ltd.；The specific preparation method of slow virus skeleton plasmid pLenti-3G basic has been proposed in hair
It is bright entitled " a kind of based on the CAR-T transgene carrier and its construction method of replication defective recombinant slow virus and application ", specially
Application No. is in 201610008360.5 patent application specification for benefit；
2, people's fresh peripheral blood is provided by health donors；
3、OCTS12319s、OCTS2219s、OCTS2019s、OCTS3019s、OCTS12319t、OCTS2219t、
The combination of OCTS2019t, OCTS3019t DNA sequence dna designs (C referring to fig. 4) by Shanghai You Kadi company, and it is raw to give Shanghai JaRa
The synthesis of object Engineering Co., Ltd, and saved with oligonucleotides dry powder or plasmid form；
4, toolenzyme Cla I, Pst I, Sac II, Sal I, EcoR I, BamH I, ApaL I, Kpn I, T4DNA connection
Enzyme is purchased from NEB company；
5,0.22 μm of -0.8 μm of PES filter is purchased from millipore company；
6, D-PBS (-), 0.4% trypan blue, sieve, all types of Tissue Culture Dish, culture bag, culture plate are purchased from
Corning company；
7、Opti-MEM、Pen-Srep、Hepes、FBS、AIM-V、RPMI 1640、DMEM、lipofectamine 3000
Purchased from invitrogen company；
8, Biotinylated protein L is purchased from GeneScript company；
9, LDH detection kit is purchased from promega company；
10, Ficoll lymphocyte separation medium is purchased from GE company；
11,20% human serum albumin injection is purchased from Ztel's Belling company；
12, CryoPremium frozen stock solution, sorting buffer come from Shanghai You Kadi company；
13, rIL-2, rIL-7, rIL-15, rIL-21 are purchased from peprotech company；
14, CD3 monoclonal antibody, CD28 monoclonal antibody, CD3/CD28 magnetic bead CD4/CD8 magnetic bead is purchased from Germany
Miltenyi company；
15, refrigerated centrifuge (ThermoScientific company, the U.S.；
16, FACS flow cytometer is purchased from Thermo company；
17, fluorescence inverted microscope is purchased from Olympus company；
18, CD4-FITC, CD8-APC are purchased from BioLegend company；
19,0.9% physiological saline is purchased from Jin Mai company；
20, ProteinL Magnetic Beads is purchased from BioVision company；
21, PrimeSTAR, RetroNectin are purchased from Takara company；
22, phycoerythrin (PE)-conjugated streptavidin is purchased from BD Bioscience company；
23, plasmid extraction kit, Ago-Gel QIAquick Gel Extraction Kit are purchased from MN company；
24, competent cell TOP10 is purchased from tiangen company；
25、NaCl、KCl、Na2HPO4.12H2O、KH2PO4、Trypsin、EDTA、CaCl2, NaOH, PEG6000 be purchased from
Give birth to work in Shanghai；
26, DNeasy kit is purchased from Shanghai JaRa company；
27, SA-HRP is purchased from Shanghai Yi Sheng company；
28, primer: primer needed for designing amplification of DNA fragments and target site according to design of primers principle, the primer is by upper
The synthesis of marine growth company,
Specifically:
EF1 α-F:5 '-ATTCAAAATTTTATCGATGCTCCGGTGCCCGTCAGT-3 ' (SEQ ID NO.34)
EF1 α-R:5 '-TCACGACACCTGAAATGGAAGA-3 ' (SEQ ID NO.35)
OCTS-F:CATTTCAGGTGTCGTGAGGATCCGCCACCATGGCGCTGCCGGTGAC (SEQ ID NO.36)
OCTS-R:GGGGAGGGAGAGGGGCTTAGCGCGGCGGCAGCG (SEQ ID NO.37)
IRES-F:GCCCCTCTCCCTCCCCC (SEQ ID NO.38)
IRES-R:ATTATCATCGTGTTTTTCAAAGGAA (SEQ ID NO.39)
IL6Rscab-F:AAAACACGATGATAATGCCACCATGAACTCCTTCTCCACAAGCG (SEQ ID NO.40)
IL6Rscab-R:AATCCAGAGGTTGATTGTCGACGAATTCTCATTTGCCCGGGCTCA G (SEQ ID
NO.41)
WPRE-QPCR-F:5 '-CCTTTCCGGGACTTTCGCTTT-3 ' (SEQ ID NO.42)
WPRE-QPCR-R:5 '-GCAGAATCCAGGTGGCAACA-3 ' (SEQ ID NO.43)
Actin-QPCR-F:5 '-CATGTACGTTGCTATCCAGGC-3 ' (SEQ ID NO.44)
Actin-QPCR-R:5 '-CTCCTTAATGTCACGCACGAT-3 ' (SEQ ID NO.45)
29, in the present invention, SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ the ID NO.4, SEQ
ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID
NO.11, SEQ ID NO12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID
NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22, SEQ ID
NO.23, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO.28, SEQ
DNA fragmentation shown in ID NO.29, SEQ ID NO.30, SEQ ID NO.31, SEQ ID NO.32, SEQ ID NO.33 is by Shanghai
The JaRa bioengineering Co., Ltd sequent synthesis that people provides according to the present invention.
1 OCTS-CAR-T cell construction of embodiment
One, recombined lentivirus vector lvOCTS12319s, lvOCTS2219s, lvOCTS2019s, lvOCTS3019s,
The building of lvOCTS12319t, lvOCTS2219t, lvOCTS2019t, lvOCTS3019t, purifying, detection method.
Referring to Fig. 3, the construction method of recombined lentivirus vector of the present invention is as follows:
1, by people EF1 α promoter (SEQ ID NO.14), the OCTS structure [CD8 as shown in SEQ ID NO.15
Leader Chimerical receptor signal peptide, two groups of single-chain antibodies: first group is selected from any one group of following four groups of single-chain antibodies: such as SEQ
CD20 single-chain antibody light chain VL, the CD20 single-chain antibody heavy chain VH as shown in SEQ ID NO.19 shown in ID NO.18；Such as
CD22 single-chain antibody light chain VL, the CD22 single-chain antibody heavy chain VH as shown in SEQ ID NO.21 shown in SEQ ID NO.20；
CD30 single-chain antibody light chain VL, the CD30 single-chain antibody heavy chain as shown in SEQ ID NO.23 as shown in SEQ ID NO.22
VH；CD123 single-chain antibody light chain VL, the CD123 single-chain antibody as shown in SEQ ID NO.25 as shown in SEQ ID NO.24
Heavy chain VH；Second group for the CD19 single-chain antibody light chain VL as shown in SEQ ID NO.16 and as shown in SEQ ID NO.17
CD19 single-chain antibody heavy chain VH；Antibody inner hinge Inner-Linker, such as SEQ ID as shown in SEQ ID NO.26
Hinge Inter-Linker, the CD8 Hinge Chimerical receptor as shown in SEQ ID NO.28 between single-chain antibody shown in NO.27
Hinge, the CD8 Transmembrane Chimerical receptor transmembrane region as shown in SEQ ID NO.29, as shown in SEQ ID NO.30
CD28 Chimerical receptor costimulating factor, CD134 Chimerical receptor costimulating factor, such as SEQ ID as shown in SEQ ID NO.31
TCR Chimerical receptor t cell activation domain shown in NO.32.OCTS12319s,OCTS2219s,OCTS2019s,OCTS3019s,
OCTS12319t, OCTS2219t, OCTS2019t, OCTS3019t, structure are detailed in Fig. 4], IL6R single-chain antibody (SEQ ID
NO.33 it) is combined into OCTS Chimerical receptor design scheme, is cloned into slow virus skeleton plasmid by digestion, connection, recombining reaction
In pLenti-3G basic, respectively obtain recombinant slow virus plasmid pOCTS12319s, pOCTS2219s, pOCTS2019s,
POCTS3019s, pOCTS12319t, pOCTS2219t, pOCTS2019t, pOCTS3019t, element orders and number such as Fig. 4 C
It is shown.
(1) slow virus skeleton plasmid pLenti-3G basic is carried out using Cla I and EcoR I restriction enzyme double
Digestion, product pass through 1.5% agarose gel electrophoresis, confirm the segment V1 of 5823bp, and be tapped and recovered and be placed in
In Eppendorf pipe, corresponding segment (being shown in Table 1) is recycled with the Ago-Gel QIAquick Gel Extraction Kit of MN company, and measure product
Purity and concentration；
| 1, colloidal sol | Sol solutions are added in 200 μ l NTI/100mg gel ratios, 50 DEG C of water-baths are placed 5-10 minutes. |
| 2, in conjunction with DNA | 11000g is centrifuged 30 seconds, discards filtrate. |
| 3, film is washed | 700 μ l NT3,11000g is added centrifugation 30 seconds, discards filtrate. |
| 4, film is washed | It is primary to repeat third step |
| 5, it dries | 11000g is centrifuged 1 minute, and the collecting pipe renewed is placed at room temperature for 1 minute. |
| 6, eluted dna | 15-30 μ l NE is added, is placed at room temperature for 1 minute, 11000g is centrifuged 1 minute, collects filtrate. |
1 Ago-Gel recycling step of table
(2) with primer EF1 α-F and EF1 α-R with the people EF1 α promoter (SEQ ID NO.14) that synthesizes for template, use
System in table 2, PCR cycle condition are as follows: 98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 2min) * 35cycle, 72
℃10min.Product passes through 1.5% agarose gel electrophoresis, confirms the segment a of 1208bp, and be tapped and recovered and be placed in
In Eppendorf pipe, corresponding segment (being shown in Table 1) is recycled with the Ago-Gel QIAquick Gel Extraction Kit of MN company, and measure product
Purity and concentration；
| Reagent | Volume (μ l) |
| H2O | 32.5 |
| 5×Buffer(with Mg2+) | 10 |
| DNTP (each 2.5mM) | 4 |
| Primer1(+)(10μM) | 1 |
| Primer2 (-) (10 μM) | 1 |
| Template | 1 |
| PrimeSTAR | 0.5 |
2 50 μ l PCR reaction system of table
(3) with primer OCTS-F and OCTS-R using the OCTS12319s synthesized as template, using the system in table 2, PCR is followed
Ring condition are as follows: 98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 30sec) * 35cycle, 72 DEG C of 5min.Product passes through
1.5% agarose gel electrophoresis, confirms the segment b of 2363bp, and is tapped and recovered and is placed in Eppendorf pipe, with MN company
Ago-Gel QIAquick Gel Extraction Kit recycle corresponding segment (being shown in Table 1), and measure the purity and concentration of product；
(4) with primer OCTS-F and OCTS-R using the OCTS2219s synthesized as template, using the system in table 2, PCR is followed
Ring condition are as follows: 98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 30sec) * 35cycle, 72 DEG C of 5min.Product passes through
1.5% agarose gel electrophoresis, confirms the segment c of 2369bp, and is tapped and recovered and is placed in Eppendorf pipe, with MN company
Ago-Gel QIAquick Gel Extraction Kit recycle corresponding segment (being shown in Table 1), and measure the purity and concentration of product；
(5) with primer OCTS-F and OCTS-R using the OCTS2019s synthesized as template, using the system in table 2, PCR is followed
Ring condition are as follows: 98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 30sec) * 35cycle, 72 DEG C of 5min.Product passes through
1.5% agarose gel electrophoresis, confirms the segment d of 2381bp, and is tapped and recovered and is placed in Eppendorf pipe, with MN company
Ago-Gel QIAquick Gel Extraction Kit recycle corresponding segment (being shown in Table 1), and measure the purity and concentration of product；
(6) with primer OCTS-F and OCTS-R using the OCTS3019s synthesized as template, using the system in table 2, PCR is followed
Ring condition are as follows: 98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 30sec) * 35cycle, 72 DEG C of 5min.Product passes through
1.5% agarose gel electrophoresis, confirms the segment e of 2381bp, and is tapped and recovered and is placed in Eppendorf pipe, with MN company
Ago-Gel QIAquick Gel Extraction Kit recycle corresponding segment (being shown in Table 1), and measure the purity and concentration of product；
(7) with primer OCTS-F and OCTS-R using the OCTS12319t synthesized as template, using the system in table 2, PCR is followed
Ring condition are as follows: 98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 30sec) * 35cycle, 72 DEG C of 5min.Product passes through
1.5% agarose gel electrophoresis, confirms the segment f of 2390bp, and is tapped and recovered and is placed in Eppendorf pipe, with MN company
Ago-Gel QIAquick Gel Extraction Kit recycle corresponding segment (being shown in Table 1), and measure the purity and concentration of product；
(8) with primer OCTS-F and OCTS-R using the OCTS2219t synthesized as template, using the system in table 2, PCR is followed
Ring condition are as follows: 98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 30sec) * 35cycle, 72 DEG C of 5min.Product passes through
1.5% agarose gel electrophoresis, confirms the segment g of 2399bp, and is tapped and recovered and is placed in Eppendorf pipe, with MN company
Ago-Gel QIAquick Gel Extraction Kit recycle corresponding segment (being shown in Table 1), and measure the purity and concentration of product；
(9) with primer OCTS-F and OCTS-R using the OCTS2019t synthesized as template, using the system in table 2, PCR is followed
Ring condition are as follows: 98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 30sec) * 35cycle, 72 DEG C of 5min.Product passes through
1.5% agarose gel electrophoresis, confirms the segment h of 2411bp, and is tapped and recovered and is placed in Eppendorf pipe, with MN company
Ago-Gel QIAquick Gel Extraction Kit recycle corresponding segment (being shown in Table 1), and measure the purity and concentration of product；
(10) with primer OCTS-F and OCTS-R using the OCTS3019t synthesized as template, using the system in table 2, PCR is followed
Ring condition are as follows: 98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 30sec) * 35cycle, 72 DEG C of 5min.Product passes through
1.5% agarose gel electrophoresis, confirms the segment i of 2424bp, and is tapped and recovered and is placed in Eppendorf pipe, with MN company
Ago-Gel QIAquick Gel Extraction Kit recycle corresponding segment (being shown in Table 1), and measure the purity and concentration of product；
(11) with primer I RES-F and IRES-R with the IRES ribosome binding sequence (SEQ ID NO.12) that synthesizes for mould
Plate uses the system in table 2, PCR cycle condition are as follows: 98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 30sec) *
35cycle, 72 DEG C of 5min.Product passes through 1.5% agarose gel electrophoresis, confirms the segment j of 575bp, and be tapped and recovered and set
In in Eppendorf pipe, corresponding segment (being shown in Table 1) is recycled with the Ago-Gel QIAquick Gel Extraction Kit of MN company, and measure production
The purity and concentration of object；
(12) it is with the IL6R single-chain antibody (SEQ ID NO.33) synthesized with primer I L6Rscab-F and IL6Rscab-R
Template uses the system in table 2, PCR cycle condition are as follows: 98 DEG C of 3min, (98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 30sec) *
35cycle, 72 DEG C of 5min.Product passes through 1.5% agarose gel electrophoresis, confirms the segment k of 1569bp, and be tapped and recovered
It is placed in Eppendorf pipe, recycles corresponding segment (being shown in Table 1) with the Ago-Gel QIAquick Gel Extraction Kit of MN company, and measure
The purity and concentration of product；
(16) by recombinant slow virus Plasmid DNA fragment combination (being shown in Table 3) with 5 μ l total volumes and the ratio of molar ratio 1:1:1:1
Example is added in Eppendorf pipe, and 15 μ l of homologous recombination enzyme reaction solution is added, and is incubated for 30 minutes after mixing at 42 DEG C, is transferred to ice
Reaction solution is added in 50 μ l TOP10, gently rotates to mix content by upper placement 2-3 minutes, and 30 points are placed in ice
Pipe is put into pre-heating into 42 DEG C of thermostat water bath heat shock 90 seconds, quickly pipe is transferred in ice bath, keeps cell cold by clock
But 2-3 minutes, every pipe added 900 μ l LB culture solutions, and then pipe is transferred on 37 DEG C of shaking tables, and incubating 1 hour makes bacteria resuscitation,
It takes the transformed bacteria solution of 100 μ l to be coated on Amp LB agar plate, is inverted plate, 37 DEG C of cultures in constant incubator, 16 is small
When.
3 recombinant slow virus Plasmid DNA fragment combination of table
Picked clones progress bacterium colony PCR identification, the correct clone of identification as recombinant slow virus plasmid pOCTS12319s,
pOCTS2219s、 pOCTS2019s、pOCTS3019s、pOCTS12319t、pOCTS2219t、pOCTS2019t、
POCTS3019t carries out digestion identification (see Fig. 5) to correct clone, and send sequencing review result.
2, recombined lentivirus vector lvOCTS12319s, lvOCTS2219s, lvOCTS2019s, lvOCTS3019s,
The packaging of lvOCTS12319t, lvOCTS2219t, lvOCTS2019t, lvOCTS3019t.
(1) complete medium: taking out preheated fresh culture, and 10%FBS+5ml Pen-Srep is added, runs up and down
Mix；
(2) NaCl 8g, KCl 0.2, Na 1XPBS solution: are weighed2HPO4.12H2O 3.58g, KH2PO4 0.24g is placed in
In 1000ml beaker, the dissolution of 900ml Milli-Q grade ultrapure water is added, after the completion of dissolution, uses 1000ml graduated cylinder constant volume
To 1000ml, 121 DEG C of high-temperature heat sterilization 20min；
(3) 0.25%Trypsin solution: weighing Trypsin 2.5g, and EDTA 0.19729g is placed in 1000ml beaker,
900ml 1XPBS dissolution is added, after the completion of dissolution, is settled to 1000ml using 1000ml graduated cylinder, 0.22 μM of filtration sterilization is long
Phase use can be reserved for -20 DEG C of refrigerators；
(4) 0.5M CaCl2 solution: 36.75g CaCl is weighed2It is dissolved with 400ml Milli-Q grade ultrapure water；With
Total volume is settled to 500ml by Milli-Q grade ultrapure water, is mixed；0.22 μm of filtration sterilization, packing be saved in 50ml from
In heart pipe, every pipe 45ml or so, 4 DEG C of preservations.
(5) 4.09g NaCl, 0.269g Na 2XHBS solution: are weighed2HPO4,5.96g Hepes, with 400ml Milli-
The dissolution of Q grade ultrapure water；After calibrating pH instrument, the pH of HBS solution is transferred to 7.05 with 2M NaOH solution.Adjust every bottle of HBS
PH consumption 2M NaOH be 3ml or so；
(6) the HEK293T/17 cell frozen is taken out from liquid nitrogen container, is quickly transferred in 37 DEG C of water-baths, after 1~2min
It is transferred in super-clean bench, the liquid in cryopreservation tube is fully transferred to 10cm by sterile working2In culture dish, supply containing 10%FBS
DMEM to 8mL/10cm2Dish, rear micro- sem observation cell, the degree of cell confluency are greater than 80% and are passed on for 24 hours；
(7) selection cell state is good, free of contamination HEK293T/17 cell, and every 2-6 culture dish is one group, by cell
After pancreatin digestion, 4-12ml complete medium is drawn with electric pipettor, 2ml is added into each postdigestive culture dish, is avoided
Culture dish dries out；All cells are blown and beaten into single cell suspension using 1ml pipettor, are transferred in medium bottle；
(8) remaining cell in above-mentioned 2-6 culture dish is transferred in medium bottle, and rinsed again with culture medium once
Culture dish；
(9) culture medium bottle cap is covered tightly, turns upside down 10 times or so and mixes well cell suspension, cell is passed to 8-24
10cm2In culture dish, the cell density of every ware should about 4 × 106A/10ml complete medium or so.If cell density and
Expected difference is larger, then needs to count cell, then according to 4 × 106The amount of a/ware is inoculated with；
(10) every 6 culture dishes arrange piles up for one, pays attention to keeping the cooperation between ware up and down.By culture dish or so, front and back
It shakes for several times, spreads out cell sufficiently, be then placed in 5%CO2Incubator.Remaining cell does same processing；
(11) checking that institute's passage cell, cell confluency degree should be 70-80%, profile is full, and it is adherent good, it is trained in cell
It supports and is uniformly distributed in ware；
(12) liquid is changed for cell, culture medium is replaced with into fresh complete medium, every ware 9ml, and by the CO of incubator2It is dense
Degree setting value is increased to 8%；
(13) match DNA/CaCl according to N+0.52Solution.Every ware HEK293T/17 cell transfecting plasmid amount is according to following ratio
It uses: recombinant slow virus plasmid (20 μ g), pPac-GP (15 μ g), pPac-R (10 μ g), pEnv-G (7.5 μ g).Take one it is new
5ml centrifuge tube, be added 0.5M CaCl2:0.25ml, 20 μ g:pPac-GP of recombinant slow virus plasmid, 15 μ g:pPac-R, 10 μ
7.5 μ g of g:pEnv-G, supplement ultrapure water close the lid to 0.5ml, mix well；
(14) a 5ml centrifuge tube is separately taken, 0.5ml DNA/CaCl2 solution is added.Turbula shaker is opened, a hand is taken
The firmly upper end of 5ml centrifuge tube makes tube bottom contact oscillating end, and liquid is made to scatter on tube wall flowing, and another hand is by one 1mL
Liquid-transfering gun is drawn 2 × HBS of 0.5mL solution, is slowly added dropwise into centrifuge tube, coutroi velocity, being dripped off with half a minute is advisable.2×
It after HBS is added, after persistent oscillation 5 seconds, stops oscillation, can be directly added into the cell for needing to transfect；
(15) take a ware cell, the 1mL calcium in centrifuge tube turned into drop and is added, make as far as possible calcium turn reagent be distributed to it is whole
In a culture dish；
(16) it after calcium turns liquid addition, is marked in ware lid, culture dish is released to another 5%CO2In incubator.
Ensure that culture dish is horizontal positioned, every pile culture dish does not exceed 6.In 5%CO2(6-8h) is placed in incubator；
(17) by the CO of first incubator2Concentration set point adjusts back to 5%；
After (18) 24 hours, cell state is checked.Cell confluency degree should be 80-85% or so, in good condition.It will culture
Base siphons away, replacement 10ml fresh DMEM complete medium；
After (19) 48 hours, transfection efficiency is observed.Most cells are still adherent.It can be seen that thin more than 95%
Born of the same parents can have green fluorescence.By the same virus packaging supernatant collection to together, and continue addition 10mL into culture dish
Fresh culture；
After (20) 72 hours, the same vial supernatant is collected into the virus together, collected twice again to be placed on
Together, culture dish is abandoned；Contained in the supernatant collected at this time recombined lentivirus vector lvOCTS12319s,
lvOCTS2219s、lvOCTS2019s、lvOCTS3019s、 lvOCTS12319t、lvOCTS2219t、lvOCTS2019t、
lvOCTS3019t。
3, ion exchange chromatography recombined lentivirus vector；
(1) supernatant of collection is used into Thermo vacuum pump, is filtered through 0.22 μm -0.8 μm of PES filter, remove impurity elimination
Matter；
(2) 1.5M NaCl 250mM Tris-HCl (pH 6-8) is added into supernatant in the ratio of 1:1~1:10；
(3) 2 ion exchange columns are placed in series, with 4ml 1M NaOH, 4ml 1M NaCl, 5ml 0.15M NaCl
25mM Tris-HCl (pH 6-8) solution successively crosses column；
(4) solution obtained in step 2 is given to ion exchange column loading with the speed of 1-10ml/min by peristaltic pump；
(5) it after whole supernatants cross column, is cleaned using 10ml 0.15M NaCl 25mM Tris-HCl (pH 6-8) solution
One time；
(6) it is eluted according to applied sample amount using 1-5ml 1.5M NaCl 25mM Tris-HCl (pH 6-8), collection is washed
De- liquid；
(7) eluent is divided into 25 to 50 μ l mono- to manage, freezes -80 DEG C of refrigerators, carry out long-term preservation；
4, recombined lentivirus vector titer determination；
(1) 24 orifice plates is taken to be inoculated with 293T cell.Every hole cell is 5 × 104A, added culture volume is 500ul, different
Difference, cell confluency when carrying out virus infection are 40%-60% to the vitro growth rates of type；
(2) prepare 3 sterile EP tubes, the fresh complete medium (DMEM in high glucose+10% of 90ul is added in each pipe
FBS) after inoculating cell 24 hours, the cell in two holes is taken to be counted with blood counting chamber, the actual number of cell when determining infection
Mesh is denoted as N；
(3) it takes virus stock solution used 10ul to be determined to be added in first pipe, after mixing gently, 10ul is taken to be added to second
In a pipe, a to the last pipe is then successively operated；410ul complete medium (DMEM in high glucose+10% is added in every pipe
), FBS final volume 500ul；
(4) 20 hours after infection starts, culture supernatant is removed, 500 μ l complete medium (DMEM in high glucose+10% are changed to
FBS), 5%CO2Continue culture 48 hours；
After (5) 72 hours, luciferase expression situation is observed, under normal circumstances, fluorecyte number increases and phase with extension rate
It should reduce, and take pictures；
(6) 0.25% pancreas enzyme -EDTA solution digestion cell of 0.2ml is used, is placed 1 minute at 37 DEG C.It is purged with culture medium whole
A cell face, is collected by centrifugation cell.Illustrate extracting genomic DNA according to DNeasy kit.It is added in each sample cell
200 μ l eluents are washed lower DNA and are quantified；
(7) preparing target DNA detection qPCRmix general pipeline I, (QPCR primer sequence is SEQ ID NO.42---SEQ ID
NO.43):
N=number of reactions. is for example: overall reaction number is 40, by 2 × TaqMan of 1ml Universal
PCR Master Mix, 4 μ l forward primer, 4 μ l reverse primer, 4 μ l probe and 788 μ l H2O is mixed
With.It is placed on ice after concussion；
(8) preparing internal reference DNA detection qPCRmix pipe II, (QPCR primer sequence is SEQ ID NO.44---SEQ ID
NO.45):
2×TaqMan Master Mix 25μl×n
10×RNaseP primer/probe mix 2.5μl×n
H2O 17.5μl×n
N=number of reactions. is for example: overall reaction number is 40, by 2 × TaqMan of 1ml Universal
PCR Master Mix, 100 μ l 10 × RNaseP primer/probe mix and 700 μ l H2O is mixed.Ice is placed on after concussion
On；
(9) PCR system is completed in 96 hole PCR plates of pre-cooling to establish.45 μ l are respectively taken to be added to each row of A-D from general pipeline I
Hole in, from respectively taking 45 μ l to be added in the hole of each row of E-G in general pipeline II.
(10) 5 μ l plasmid standards and sample to be tested genomic DNA are taken to be added in A-D row respectively, each sample repeats 1
It is secondary.Separately stay 1 hole that the water of 5 μ l is added as no template control (no-template control).
(11) 5 μ l genome standard items and sample to be tested genomic DNA are taken to be added in E-G row respectively, each sample weight
It is 1 time multiple.Separately stay 1 hole that the water of 5 μ l is added as no template control (no-template control).
(12) used quantitative PCR apparatus is 7500 quantitative system of ABI PRISM.Cycling condition setting are as follows: 50 DEG C 2 minutes,
95 DEG C 10 minutes, followed by 95 DEG C 15 seconds, 60 DEG C of 40 of 1 minute circulations.
Data analysis: the slow virus carrier copy number integrated in the DNA sample measured is demarcated with genome number, is obtained
The viral copy number of every genome conformity.
Titre (integration units per ml, IU ml-1) calculation formula it is as follows:
IU ml-1=(C × N × D × 1000)/V
Wherein: C=is averaged the viral copy number of every genome conformity
The number (about 1 × 10 of cell when N=infects5)
The extension rate of D=viral vectors
The volume number for the dilution virus that V=is added
(13) recombined lentivirus vector lvOCTS12319s, lvOCTS2219s, lvOCTS2019s, lvOCTS3019s,
The titre results (as shown in Figure 6) of lvOCTS12319t, lvOCTS2219t, lvOCTS2019t, lvOCTS3019t；
Two, OCTS-CAR-T cell construction
Referring to Fig. 7, the construction method of OCTS-CAR-T cell of the present invention is as follows:
1, PBMC is separated.
(1) health donors fresh peripheral blood 50ml is extracted；
(2) blood taking bag spray is wiped twice of alcohol, and dried.
(3) haemocyte in bag is sucked out with 50ml syringe and is moved in new 50ml pipe.
(4) 400g, 20 DEG C of centrifugation 10min.
(5) upper plasma being moved on in new 50ml centrifuge tube, 56 DEG C, 30min inactivates blood plasma, restores to room temperature,
2000g is centrifuged 30min, takes supernatant stand-by into 50ml centrifuge tube.
(6) it is mended with D-PBS (-) to 50ml, tightens lid, be mixed by inversion.
(7) 2 new 50ml centrifuge tubes are taken, 15ml Ficoll lymphocyte separation medium is added in every pipe.
(8) haemocyte dilution 25ml is carefully added on every pipe Ficoll.800g, 20 DEG C of centrifugation 20min.
(9) liquid is divided into four layers in centrifuge tube, be respectively as follows: from top to bottom the plasma layer (recycling stand-by) of yellow, tunica albuginea layer,
The cell mixing layer of colorless and transparent Ficoll layer, reddish black.
(10) tunica albuginea layer is carefully drawn into new 50ml centrifuge tube, is added D-PBS (-) to 50ml, is mixed by inversion rear 500g,
20 DEG C of centrifugation 10min.
(11) 5% human serum albumin of 25ml is added and cell is resuspended, 400g, 20 DEG C of centrifugation 10min.
(12) supernatant is abandoned, 5% human serum albumin of 25ml is added, cell precipitation is resuspended, and cross 70um sieve, count.
(13) 1 part is taken to contain 1.25x108Cells is for activating；Remaining cell suspension 400g, 20 DEG C of centrifugation 10min add
CryoPremium simultaneously freezes.
2, CD4/CD8 positive T cell sorts.
(1) PBMC of acquisition is counted, with 80ul/107Sorting buffer is added in the ratio of cells, and cell precipitation is resuspended.
(2) again with 20ul/107CD4/CD8 magnetic bead is added in the ratio of cells, and piping and druming is put into 4 DEG C after mixing and is incubated for
15min。
(3) magnetic bead-cell mixture is taken out, with 2ml/107Sorting buffer is added in the ratio of cells, after being mixed by inversion,
250g, 4 DEG C of centrifugation 10min.
(4) with 500ul/108Sorting buffer is added in the ratio of cells, and cell precipitation is resuspended.
(5) on tweezers clamping LS splitter to magnetic frame.
(6) prepare 2 15ml centrifuge tubes simultaneously, mark respectively: CD4-/CD8- cell liquid (A pipe), CD4+/CD8+ cell
Liquid (B pipe).
(7) 3ml dissociating buffer rinse LS is used, and connects buffer with A pipe.
(8) cell-magnetic bead mixed liquor is added, 3ml buffer is added after dripping off and rinses pillar (when each no liquid remains again
New liquid is added), in total three times, collection obtains CD4/CD8- cell.
(9) LS splitter is separated with magnetic frame, connects cell suspension with B pipe, 5ml buffer is added, by and with pillar internal plug
It slightly firmly rinses, is collected as CD4+/CD8+ cell, sampling counts.
(10) 1x10 is pressed6/ml-4x106The cell density of/ml AIM-V culture medium resuspension cell precipitation, and addition 2 ×
105~1 × 106The U/L IFN-γ factor.
3, t cell activation.
(1) the previous day is mentioned by 1 × 103Ug/L~1 × 104Ug/L CD3 monoclonal antibody and 1 × 103Ug/L~1 ×
10424 orifice plates are added in ug/L CD28 monoclonal antibody, and sealed membrane sealing, 4 DEG C are coated with overnight.
(2) coated T75 bottles is taken out, coating buffer is outwelled, washed once with D-PBS (-), and the cell that sorting obtains is hanged
Liquid is inoculated into T75 bottles, is shaken up, and 37 DEG C, 5%CO are put into2It is cultivated in incubator.
4, CAR gene transfer and OCTS-CAR-T cell Fiber differentiation.
(1) the previous day coating 1 × 10 is proposed3Ug/L~1 × 104In in 24 orifice plates, sealed membrane seals ug/L RetroNectin
Mouthful, 4 DEG C are coated with overnight.
(2) into 24 orifice plates, according to every hole 5 × 105Cell concentration is separately added by the amount of MOI=5~20
lvOCTS12319s、lvOCTS2219s、 lvOCTS2019s、lvOCTS3019s、lvOCTS12319t、lvOCTS2219t、
LvOCTS2019t, lvOCTS3019t slow virus transgene carrier, while adding and containing 2 × 105~5 × 105U/L rIL-2,5 ×
103Ng/L~1 × 104Ng/L rIL-7,5 × 103Ng/L~1 × 104Ng/L rIL-15,5 × 103Ng/L~1 × 104ng/L
RIL-21 and 37 DEG C of AIM-V culture medium, 5%CO containing 10% autoserum2Continue to cultivate.
5, OCTS-CAR-T cell expansion ex vivo.
(1) every 2 days equivalent is added containing 2 × 105~5 × 105U/LrIL-2,5 × 103Ng/L~1 × 104Ng/LrIL-7,5
×103Ng/L~1 × 104Ng/L rIL-15,5 × 103Ng/L~1 × 104Ng/L rIL-21 and containing 10% autoserum
AIM-V culture medium, maintains pH value between 6.5~7.5, and cell density maintains 5 × 105~2 × 106Between/ml, 37
DEG C, 5%CO2Continue culture 10-14 days.
(2) the 7th days or so, the OCTS-CAR-T cell of culture was frozen for subsequent detection.
Embodiment 2
OCTS-CAR-T cell Pathogen test and detection of expression.
One, endotoxin detects；
(1), endotoxin working standard is 15EU/ branch；
(2), sensitivity of the limulus reagent λ=0.25EU/ml, 0.5ml/ pipe
(3), endotoxin standard dilutes: taking endotoxin standard one, is diluted to 4 λ and 2 λ in proportion with BET water respectively
Dissolution, sealed membrane sealing, concussion dissolution 15min；One step of every dilution should all mix 30s on eddy mixer when dilution；
(4), it is loaded: taking reagents several, every addition BET water 0.5ml dissolution, packing to several endotoxin-frees tries
Guan Zhong, every pipe 0.1ml.Wherein 2 are negative control pipe, and BET water 0.1ml is added；
2 are positive control pipe, and the endotoxin working standard solution 0.1ml of 2 λ concentration is added；
2 be Sample Positive control tube, be added 0.1ml containing 2 λ endotoxin standards sample solution (dilution 20 times to
The endotoxin standard solution 1ml=2ml of sample 1ml+4 λ contains 40 times of samples of dilution of 2 λ endotoxin standards).
Be added 0.1ml sample in sample cell, dilution ratio be shown in Table 4,37 ± 1 DEG C of water-baths (or incubator) heat preservation 60 ±
1min；
4 endotoxin dilution ratio of table and corresponding endotoxin content
(5), the endotoxin testing result (as shown in table 5) of OCTS-CAR-T cell, the endotoxin content of all cells are equal
Less than 2.5EU/ml, meet the standard for being less than 10EU/ml in the Pharmacopoeia of the People's Republic of China；
Table 5
Two, detection of mycoplasma；
(1) first three day is being tested, cell sample is cultivated with antibiotic-free culture medium；
(2) (cell number is greater than 1*10 to collection 1ml cell suspending liquid5), it is placed in 1.5ml centrifuge tube；
(3) 13000g is centrifuged 1min, collects precipitating, discards culture medium；
(4) 500ul PBS pipette tips pressure-vaccum or vortex oscillation is added, precipitating is resuspended.13000g is centrifuged 5min；
(5) step 4 is repeated once；
(6) 50 μ l Cell Lysis Buffer are added after mixing well, to be incubated in 55 DEG C of water-baths with pipette tips pressure-vaccum
20min；
(7) sample is placed in 95 DEG C and heats 5min；
(8) after 13000g is centrifuged 5min, take 5 μ l supernatants as template, 25 μ l PCR reaction systems are as follows: 6.5 μ l of ddH20,
1 μ l of Myco Mix, 2x Taq Plus Mix Master (Dye Plus) 12.5 μ l, 5 μ l of template；PCR cycle condition are as follows: 95
DEG C 30sec, (95 DEG C of 30sec, 56 DEG C of 30sec, 72 DEG C of 30sec) * 30cycle, 72 DEG C of 5min.
(9) detection of mycoplasma as the result is shown (as shown in Figure 8), is free of mycoplasma in OCTS-CAR-T cell.
Three, the detection of OCTS gene transduction efficiency and immunophenotyping detection；
(1) T cell after viral transduction is collected, cell is resuspended with D-PBS (-) solution containing 1~4% human serum albumin
And it is adjusted to 1 × 106/ml。
(2) D-PBS (-) solution 1ml containing 1~4% human serum albumin is added into centrifuge tube and mixes, 350g centrifugation
5min abandons supernatant.
(3) it is primary to repeat step 2.
(4) cell is resuspended with D-PBS (-) solution containing 1~4% human serum albumin of 0.2ml, and is added into centrifuge tube
1mg/ul protein L, 5ul CD4-FITC, the 5ul CD8-APC of 1ul is mixed, 4 DEG C of incubation 45min.
(5) D-PBS (-) solution of the 1ml containing 1~4% human serum albumin is added into centrifuge tube and mixes, 350g centrifugation
5min abandons supernatant.
(6) step 5 is repeated twice.
(7) cell is resuspended in D-PBS (-) solution with 0.2ml containing 1~4% human serum albumin, and is added into centrifuge tube
0.2ul PE-SA is mixed, and 37 DEG C are protected from light incubation 15min.
(8) D-PBS (-) solution of 1ml containing 1~4% human serum albumin is added into centrifuge tube to weigh and mix, 350g centrifugation
5min abandons supernatant.
(9) cell precipitation is resuspended with 1ml D-PBS (-) solution, 350g is centrifuged 5min, abandons supernatant.
(10) step 9 is repeated twice.
(11) cell precipitation is resuspended with 0.4ml D-PBS (-) solution, flow cytometer is detected.
(12) OCTS gene transduction efficiency and the testing result of immunophenotyping detection are as shown in figure 9, the OCTS-CAR- prepared
For most of efficiency of infection of T cell between 30%~40%, the ratio of CD4 positive cell and CD8 positive cell is located at 1:
Between 3~3: 1, follow-up function detection can be carried out.
The Function detection of 3 OCTS-CAR-T cell of embodiment.
One, target cell fragmentation effect is assessed.
(1) target cell [CD19 is cultivated respectively+K562、CD20+K562、CD22+K562、CD30+K562、CD123+K562、
CD19+CD123+K562、CD19+CD20+K562、CD19+CD22+K562、CD19+CD30+K562, K562 cell] and effector cell
[OCTS-CAR-T cell]；
(2) target cell 4x10 is collected5Cells and OCTS-CAR-T cell 2.8x106Cells, 800g, 6min are centrifuged, in abandoning
Clearly；
(3) target cell and effector cell are resuspended respectively with 1ml D-PBS (-) solution, supernatant is abandoned in 800g, 6min centrifugation；
(4) it is primary to repeat step 3；
(5) effector cell is resuspended with 700ul culture medium (+1~10%FBS of AIM-V culture medium), with 2ml culture medium (AIM-
+ 1~10% FBS of V culture medium) target cell is resuspended；
(6) experimental port that setting effect target ratio is 1: 1,5: 1,10: 1, effector cell respectively with single target cell and pair target cell
The grouping situation being incubated for altogether is as shown in table 6, and control group (K562 cell) is arranged, every group of 3 multiple holes；
Table 6
| Effector cell | Target cell 1 | Target cell 2 | Target cell 3 |
| OCTS12319s-CAR-T | CD123+K562 | CD19+K562 | CD19+CD123+K562 |
| OCTS2219s-CAR-T | CD22+K562 | CD19+K562 | CD19+CD22+K562 |
| OCTS2019s-CAR-T | CD20+K562 | CD19+K562 | CD19+CD20+K562 |
| OCTS3019s-CAR-T | CD30+K562 | CD19+K562 | CD19+CD30+K562 |
| OCTS12319t-CAR-T | CD123+K562 | CD19+K562 | CD19+CD123+K562 |
| OCTS2219t-CAR-T | CD22+K562 | CD19+K562 | CD19+CD22+K562 |
| OCTS2019t-CAR-T | CD20+K562 | CD19+K562 | CD19+CD20+K562 |
| OCTS3019t-CAR-T | CD30+K562 | CD19+K562 | CD19+CD30+K562 |
(7) centrifugation of 250g, 5min plate；
It (8) 37 DEG C, is cultivated 4 hours in 5%CO2 incubator；
(9) centrifugation of 250g, 5min plate；
(10) take the 50ul supernatant in each hole into new 96 orifice plate, and every hole is added 50ul substrate solution and (is protected from light behaviour
Make)；
(11) it is protected from light and is incubated for 25min；
(12) 50ul terminate liquid is added in every hole；
(13) microplate reader detects 490nm absorbance；
(14) 3 multiple holes are averaged；The light absorption value of all experimental ports, Target cell wells and effector cell hole is subtracted into training
Support the mean value of base background light absorption value；The light absorption value of target cell maximum value is subtracted to the mean value of volume correction control light absorption value.
(15) it brings the corrected value obtained in step 14 into following formula, it is thinner than generated to calculate each effect target
Cellular toxicity percentage.The results are shown in Figure 10, and OCTS-CAR-T, which has respective single target cell and double target cells, preferably to be killed
Hurt effect, the CAR-T cell of Turn OCTS structure is slightly above the CAR- of Series OCTS structure to the killing-efficiency of target cell
T cell；
Killing-efficiency=(experimental port-effector cell hole-Target cell wells)/(target cell largest hole-Target cell wells) X100%
(16) above-mentioned the experimental results showed that, pass through in traditional CAR structure antigen recognizing district transformation formed OCTS tie
Structure can significantly improve OCTS-CAR-T cell recognition and kill the range of target cell, therefore OCTS-CAR-T cell will be not
The CD19 positive/CD22 positive/CD20 positive/CD123 positive/bis- the positives of the CD30 positive/CD19, CD22/CD19, the CD20 come
The malignant tumours such as the bis- positives of double positives/CD19, CD123/CD19, CD30 bis- positives bone-marrow-derived lymphocyte leukaemia, B lymthoma
Huge effect is played in cell therapy.
Sequence table
<110>Shanghai You Kadi biological medicine Science and Technology Ltd.
<120>a kind of leaching based on OCTS technology is leukaemia CAR-T therapy vector and its construction method and application
<130> HJ17-13348
<160> 45
<170> PatentIn version 3.5
<210> 1
<211> 861
<212> DNA
<213>artificial sequence
<400> 1
atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 60
gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca 120
cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc 180
gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc 240
cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg 300
gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta 360
tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc 420
ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt 480
gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg 540
cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct 600
tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc 660
tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct 720
cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac 780
acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc 840
tcactgatta agcattggta a 861
<210> 2
<211> 674
<212> DNA
<213>artificial sequence
<400> 2
cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc 60
ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca 120
actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta 180
gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct 240
ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg 300
gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc 360
acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta 420
tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg 480
gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt 540
cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg 600
cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg 660
ccttttgctc acat 674
<210> 3
<211> 147
<212> DNA
<213>artificial sequence
<400> 3
atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt 60
tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga 120
ggcttttttg gaggcctaga cttttgc 147
<210> 4
<211> 228
<212> DNA
<213>artificial sequence
<400> 4
gtagtcttat gcaatactct tgtagtcttg caacatggta acgatgagtt agcaacatgc 60
cttacaagga gagaaaaagc accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg 120
tgccttatta ggaaggcaac agacgggtct gacatggatt ggacgaacca ctgaattgcc 180
gcattgcaga gatattgtat ttaagtgcct agctcgatac aataaacg 228
<210> 5
<211> 180
<212> DNA
<213>artificial sequence
<400> 5
ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac 60
tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt 120
gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca 180
<210> 6
<211> 234
<212> DNA
<213>artificial sequence
<400> 6
tgctagagat tttccacact gactaaaagg gtctgaggga tctctagtta ccagagtcac 60
acaacagacg ggcacacact acttgaagca ctcaaggcaa gctttattga ggcttaagca 120
gtgggttccc tagttagcca gagagctccc aggctcagat ctggtctaac cagagagacc 180
cagtacaagc aaaaagcaga tcttattttc gttgggagtg aattagccct tcca 234
<210> 7
<211> 353
<212> DNA
<213>artificial sequence
<400> 7
atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc 60
ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg 120
agctagaacg attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa 180
tactgggaca gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata 240
atacagtagc aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag 300
ctttagacaa gatagaggaa gagcaaaaca aaagtaagac caccgcacag caa 353
<210> 8
<211> 233
<212> DNA
<213>artificial sequence
<400> 8
aggagctttg ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcctcaat 60
gacgctgacg gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt 120
gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca 180
gctccaggca agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcc 233
<210> 9
<211> 489
<212> DNA
<213>artificial sequence
<400> 9
tggggatttg gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta 60
gttggagtaa taaatctctg gaacagattg gaatcacacg acctggatgg agtgggacag 120
agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca 180
agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt 240
taacataaca aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt 300
aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc 360
accattatcg tttcagaccc acctcccaac cccgagggga cccgacaggc ccgaaggaat 420
agaagaagaa ggtggagaga gagacagaga cagatccatt cgattagtga acggatctcg 480
acggttaac 489
<210> 10
<211> 119
<212> DNA
<213>artificial sequence
<400> 10
ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat 60
agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaatttta 119
<210> 11
<211> 696
<212> DNA
<213>artificial sequence
<400> 11
atggcccagt ccaagcacgg cctgaccaag gagatgacca tgaagtaccg catggagggc 60
tgcgtggacg gccacaagtt cgtgatcacc ggcgagggca tcggctaccc cttcaagggc 120
aagcaggcca tcaacctgtg cgtggtggag ggcggcccct tgcccttcgc cgaggacatc 180
ttgtccgccg ccttcatgta cggcaaccgc gtgttcaccg agtaccccca ggacatcgtc 240
gactacttca agaactcctg ccccgccggc tacacctggg accgctcctt cctgttcgag 300
gacggcgccg tgtgcatctg caacgccgac atcaccgtga gcgtggagga gaactgcatg 360
taccacgagt ccaagttcta cggcgtgaac ttccccgccg acggccccgt gatgaagaag 420
atgaccgaca actgggagcc ctcctgcgag aagatcatcc ccgtgcccaa gcagggcatc 480
ttgaagggcg acgtgagcat gtacctgctg ctgaaggacg gtggccgctt gcgctgccag 540
ttcgacaccg tgtacaaggc caagtccgtg ccccgcaaga tgcccgactg gcacttcatc 600
cagcacaagc tgacccgcga ggaccgcagc gacgccaaga accagaagtg gcacctgacc 660
gagcacgcca tcgcctccgg ctccgccttg ccctga 696
<210> 12
<211> 575
<212> DNA
<213>artificial sequence
<400> 12
gcccctctcc ctcccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt 60
gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc 120
ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag 180
gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct tcttgaagac 240
aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc 300
tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc 360
acgttgtgag ttggatagtt gtggaaagag tcaaatggct cacctcaagc gtattcaaca 420
aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt 480
gcacatgctt tacatgtgtt tagtcgaggt taaaaaacgt ctaggccccc cgaaccacgg 540
ggacgtggtt ttcctttgaa aaacacgatg ataat 575
<210> 13
<211> 592
<212> DNA
<213>artificial sequence
<400> 13
aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct 60
ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt 120
atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg 180
tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact 240
ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct 300
attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg 360
ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc 420
gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc 480
aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt 540
cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcc tg 592
<210> 14
<211> 1178
<212> DNA
<213>artificial sequence
<400> 14
gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg 60
gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg 120
atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag 180
tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg 240
tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta 300
cttccacctg gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg 360
agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc 420
ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt 480
tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc 540
aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg 600
cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag 660
cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg 720
gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag 780
ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga 840
cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt 900
cctcagccgt cgcttcatgt gactccactg agtaccgggc gccgtccagg cacctcgatt 960
agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg 1020
agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat 1080
tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag 1140
tggttcaaag tttttttctt ccatttcagg tgtcgtga 1178
<210> 15
<211> 63
<212> DNA
<213>artificial sequence
<400> 15
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccg 63
<210> 16
<211> 333
<212> DNA
<213>artificial sequence
<400> 16
gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc 60
atctcctgca aggccagcca aagtgttgat tatgatggtg atagttattt gaactggtac 120
caacagattc caggacagcc acccaaactc ctcatctatg atgcatccaa tctagtttct 180
gggatcccac ccaggtttag tggcagtggg tctgggacag acttcaccct caacatccat 240
cctgtggaga aggtggatgc tgcaacctat cactgccagc aaagtactga ggatccgtgg 300
acgttcggtg gaggcaccaa gctggaaatc aaa 333
<210> 17
<211> 375
<212> DNA
<213>artificial sequence
<400> 17
caggttcagc tgcagcagtc tggggctgag ctggtgaggc ctgggtcctc agtgaagatt 60
tcctgcaagg cttctggcta tgcattcagt agctactgga tgaactgggt gaagcagagg 120
cctggacagg gtcttgagtg gattggacag atttggcctg gagatggtga tactaactac 180
aatggaaagt tcaagggtaa agccactctg actgcagacg aatcctccag cacagcctac 240
atgcaactca gcagcctagc atctgaggac tctgcggtct atttctgtgc aagacgggag 300
actacgacgg taggccgtta ttactatgct atggactact ggggtcaagg aacctcagtc 360
accgtctcct cgagt 375
<210> 18
<211> 318
<212> DNA
<213>artificial sequence
<400> 18
gacattgtgc tgacccaatc tccagctatc ctgtctgcat ctccagggga gaaggtcaca 60
atgacttgca gggccagctc aagtgtaaat tacatggact ggtaccagaa gaagccagga 120
tcctccccca aaccctggat ttatgccaca tccaacctgg cttctggagt ccctgctcgc 180
ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagcagagt ggaggctgaa 240
gatgctgcca cttattactg ccagcagtgg agttttaatc cacccacgtt cggagggggg 300
accaagctgg aaataaaa 318
<210> 19
<211> 366
<212> DNA
<213>artificial sequence
<400> 19
gaggtgcagc tgcagcagtc tggggctgag ctggtgaagc ctggggcctc agtgaagatg 60
tcctgcaagg cttctggcta cacatttacc agttacaata tgcactgggt aaagcagaca 120
cctggacagg gcctggaatg gattggagct atttatccag gaaatggtga tacttcctac 180
aatcagaagt tcaaaggcaa ggccacattg actgcagaca aatcctccag cacagcctac 240
atgcagctca gcagcctgac atctgaggac tctgcggact attactgtgc aagatctaat 300
tattacggta gtagctactg gttcttcgat gtctggggcg cagggaccac ggtcaccgtc 360
tcctca 366
<210> 20
<211> 321
<212> DNA
<213>artificial sequence
<400> 20
gatattcaga tgacccagac caccagcagc ctgagcgcga gcctgggcga tcgcgtgacc 60
attagctgcc gcgcgagcca ggatattagc aactatctga actggtatca gcagaaaccg 120
gatggcaccg tgaaactgct gatttattat accagcattc tgcatagcgg cgtgccgagc 180
cgctttagcg gcagcggcag cggcaccgat tatagcctga ccattagcaa cctggaacag 240
gaagattttg cgacctattt ttgccagcag ggcaacaccc tgccgtggac ctttggcggc 300
ggcaccaaac tggaaattaa a 321
<210> 21
<211> 369
<212> DNA
<213>artificial sequence
<400> 21
gaagtgcagc tggtggaaag cggcggcggc ctggtgaaac cgggcggcag cctgaaactg 60
agctgcgcgg cgagcggctt tgcgtttagc atttatgata tgagctgggt gcgccagacc 120
ccggaaaaac gcctggaatg ggtggcgtat attagcagcg gcggcggcac cacctattat 180
ccggataccg tgaaaggccg ctttaccatt agccgcgata acgcgaaaaa caccctgtat 240
ctgcagatga gcagcctgaa aagcgaagat accgcgatgt attattgcgc gcgccatagc 300
ggctatggca gcagctatgg cgtgctgttt gcgtattggg gccagggcac cctggtgacc 360
gtgagcgcg 369
<210> 22
<211> 321
<212> DNA
<213>artificial sequence
<400> 22
gatattcaga tgacccagag cccgaccagc ctgagcgcga gcgtgggcga tcgcgtgacc 60
attacctgcc gcgcgagcca gggcattagc agctggctga cctggtatca gcagaaaccg 120
gaaaaagcgc cgaaaagcct gatttatgcg gcgagcagcc tgcagagcgg cgtgccgagc 180
cgctttagcg gcagcggcag cggcaccgat tttaccctga ccattagcag cctgcagccg 240
gaagattttg cgacctatta ttgccagcag tatgatagct atccgattac ctttggccag 300
ggcacccgcc tggaaattaa a 321
<210> 23
<211> 336
<212> DNA
<213>artificial sequence
<400> 23
caggtgcagc tgcagcagtg gggcgcgggc ctgctgaaac cgagcgaaac cctgagcctg 60
acctgcgcgg tgtatggcgg cagctttagc gcgtattatt ggagctggat tcgccagccg 120
ccgggcaaag gcctggaatg gattggcgat attaaccatg gcggcggcac caactataac 180
ccgagcctga aaagccgcgt gaccattagc gtggatacca gcaaaaacca gtttagcctg 240
aaactgaaca gcgtgaccgc ggcggatacc gcggtgtatt attgcgcgag cctgaccgcg 300
tattggggcc agggcagcct ggtgaccgtg agcagc 336
<210> 24
<211> 324
<212> DNA
<213>artificial sequence
<400> 24
gatattgtga tgacccagag cccgagcagc gtgagcgcga gcgtgggcga tcgcgtgacc 60
attacctgcc gcgcgagcca gaacgtggat agcgcggtgg cgtggtatca gcagaaaccg 120
ggcaaagcgc cgaaagcgct gatttatagc gcgagctatc gctatagcgg cgtgccgagc 180
cgctttagcg gccgcggcag cggcaccgat tttaccctga ccattagcag cctgcagccg 240
gaagattttg cgacctatta ttgccagcag tattatagca ccccgtggac ctttggccag 300
ggcaccaaag tggaaattaa acgc 324
<210> 25
<211> 381
<212> DNA
<213>artificial sequence
<400> 25
gaagtgaaac tggtggaaag cggcggcggc ctggtgcagc cgggccgcag cctgcgcctg 60
agctgcaccg cgagcggctt tacctttacc gattattata tgagctgggt gcgccaggcg 120
ccgggcaaag gcctggaatg ggtgggcctg attcgcagca aagcggatgg ctataccacc 180
gaatatagcg cgagcgtgaa aggccgcttt accattagcc gcgatgatag caaaagcatt 240
ctgtatctgc agatgaacag cctgaaaacc gaagataccg cggtgtatta ttgcgcgcgc 300
gatgcggcgt attatagcta ttatagcccg gaaggcgcga tggattattg gggccagggc 360
accctggtga ccgtgagcag c 381
<210> 26
<211> 45
<212> DNA
<213>artificial sequence
<400> 26
ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatct 45
<210> 27
<211> 57
<212> DNA
<213>artificial sequence
<400> 27
gcctccacca agggcccatc tgtcttcccc ctggccccca gctcctctgg ctccgga 57
<210> 28
<211> 141
<212> DNA
<213>artificial sequence
<400> 28
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgatatcta c 141
<210> 29
<211> 66
<212> DNA
<213>artificial sequence
<400> 29
atctgggcgc ccttggccgg gacttgtggg gtccttctcc tgtcactggt tatcaccctt 60
tactgc 66
<210> 30
<211> 123
<212> DNA
<213>artificial sequence
<400> 30
aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60
gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120
tcc 123
<210> 31
<211> 111
<212> DNA
<213>artificial sequence
<400> 31
cgccgcgatc agcgcctgcc gccggatgcg cataaaccgc cgggcggcgg cagctttcgc 60
accccgattc aggaagaaca ggcggatgcg catagcaccc tggcgaaaat t 111
<210> 32
<211> 336
<212> DNA
<213>artificial sequence
<400> 32
cgcgtgaaat ttagccgcag cgcggatgcg ccggcgtatc agcagggcca gaaccagctg 60
tataacgaac tgaacctggg ccgccgcgaa gaatatgatg tgctggataa acgccgcggc 120
cgcgatccgg aaatgggcgg caaaccgcgc cgcaaaaacc cgcaggaagg cctgtataac 180
gaactgcaga aagataaaat ggcggaagcg tatagcgaaa ttggcatgaa aggcgaacgc 240
cgccgcggca aaggccatga tggcctgtat cagggcctga gcaccgcgac caaagatacc 300
tatgatgcgc tgcatatgca ggcgctgccg ccgcgc 336
<210> 33
<211> 1506
<212> DNA
<213>artificial sequence
<400> 33
atgaactcct tctccacaag cgccttcggt ccagttgcct tctccctggg gctgctcctg 60
gtgttgcctg ctgccttccc tgccccagac atccagatga cccagagccc aagcagcctg 120
agcgccagcg tgggtgacag agtgaccatc acctgtagag ccagccagga catcagcagt 180
tacctgaatt ggtaccagca gaagccagga aaggctccaa agctgctgat ctactacacc 240
tccagactgc actctggtgt gccaagcaga ttcagcggta gcggtagcgg taccgacttc 300
accttcacca tcagcagcct ccagccagag gacatcgcta cctactactg ccaacagggt 360
aacacgcttc catacacgtt cggccaaggg accaaggtgg aaatcaaagg tggcggtggc 420
tcgggcggtg gtgggtcggg tggcggcgga tctcaggtcc aactgcagga gagcggtcca 480
ggtcttgtga gacctagcca gaccctgagc ctgacctgca ccgtgtctgg ctactcaatt 540
accagcgatc atgcctggag ctgggttcgc cagccacctg gacgaggtct tgagtggatt 600
ggatacatta gttatagtgg aatcacaacc tataatccat ctctcaaatc cagagtgaca 660
atgctgagag acaccagcaa gaaccagttc agcctgagac tcagcagcgt gacagccgcc 720
gacaccgcgg tttattattg tgcaagatcc ctagctcgga ctacggctat ggactactgg 780
ggtcaaggca gcctcgtcac agtctcctca gaaccgaaaa gctgcgataa aacccatacc 840
tgcccgccgt gcccggcgcc ggaactgctg ggcggcccga gcgtgtttct gtttccgccg 900
aaaccgaaag ataccctgat gattagccgc accccggaag tgacctgcgt ggtggtggat 960
gtgagccatg aagatccgga agtgaaattt aactggtatg tggatggcgt ggaagtgcat 1020
aacgcgaaaa ccaaaccgcg cgaagaacag tataacagca cctatcgcgt ggtgagcgtg 1080
ctgaccgtgc tgcatcagga ttggctgaac ggcaaagaat ataaatgcaa agtgagcaac 1140
aaagcgctgc cggcgccgat tgaaaaaacc attagcaaag cgaaaggcca gccgcgcgaa 1200
ccgcaggtgt ataccctgcc gccgagccgc gaagaaatga ccaaaaacca ggtgagcctg 1260
acctgcctgg tgaaaggctt ttatccgagc gatattgcgg tggaatggga aagcaacggc 1320
cagccggaaa acaactataa aaccaccccg ccggtgctgg atagcgatgg cagctttttt 1380
ctgtatagca aactgaccgt ggataaaagc cgctggcagc agggcaacgt gtttagctgc 1440
agcgtgatgc atgaagcgct gcataaccat tatacccaga aaagcctgag cctgagcccg 1500
ggcaaa 1506
<210> 34
<211> 36
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<223>primer
<400> 34
attcaaaatt ttatcgatgc tccggtgccc gtcagt 36
<210> 35
<211> 22
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<223>primer
<400> 35
tcacgacacc tgaaatggaa ga 22
<210> 36
<211> 46
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<223>primer
<400> 36
catttcaggt gtcgtgagga tccgccacca tggcgctgcc ggtgac 46
<210> 37
<211> 33
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<223>primer
<400> 37
ggggagggag aggggcttag cgcggcggca gcg 33
<210> 38
<211> 17
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<223>primer
<400> 38
gcccctctcc ctccccc 17
<210> 39
<211> 25
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<223>primer
<400> 39
attatcatcg tgtttttcaa aggaa 25
<210> 40
<211> 44
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<223>primer
<400> 40
aaaacacgat gataatgcca ccatgaactc cttctccaca agcg 44
<210> 41
<211> 46
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<223>primer
<400> 41
aatccagagg ttgattgtcg acgaattctc atttgcccgg gctcag 46
<210> 42
<211> 21
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<223>primer
<400> 42
cctttccggg actttcgctt t 21
<210> 43
<211> 20
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<223>primer
<400> 43
gcagaatcca ggtggcaaca 20
<210> 44
<211> 21
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<223>primer
<400> 44
catgtacgtt gctatccagg c 21
<210> 45
<211> 21
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<223>primer
<400> 45
ctccttaatg tcacgcacga t 21
Claims (10)
1. a kind of leaching based on OCTS technology is leukaemia CAR-T therapy vector, which is characterized in that including slow virus skeleton matter
Grain, people EF1 α promoter, OCTS Chimerical receptor structural domain and IL6R single-chain antibody；
The slow virus skeleton plasmid includes: the sequence of AmpR containing ampicillin resistance gene for purpose bacterial strain massive amplification
Column, as shown in SEQ ID NO.1；For the prokaryotic replions pUC Ori sequence of plasmid replication, as shown in SEQ ID NO.2；With
In the Viral Replicon SV40 Ori sequence of the duplication in enhancing eukaryocyte, as shown in SEQ ID NO.3；For slow virus packet
The slow virus of dress packs cis element；ZsGreen1 green fluorescent protein, as shown in SEQ ID NO.11；IRES ribosomes combines
Sequence, as shown in SEQ ID NO.12；The enhanced marmot hepatitis B of eWPRE for enhancing the expression efficiency of transgenosis turns
Controlling element after record, as shown in SEQ ID NO.13；
The sequence of the people EF1 α promoter is as shown in SEQ ID NO.14；
The OCTS Chimerical receptor structural domain include: the CD8 leader Chimerical receptor signal peptide as shown in SEQ ID NO.15,
Two groups of single-chain antibodies: first group is selected from any one group of following four groups of single-chain antibodies: as CD20 shown in SEQ ID NO.18 is mono-
Chain antibody light chain VL, the CD20 single-chain antibody heavy chain VH as shown in SEQ ID NO.19；The CD22 as shown in SEQ ID NO.20
Single-chain antibody light chain VL, the CD22 single-chain antibody heavy chain VH as shown in SEQ ID NO.21；As shown in SEQ ID NO.22
CD30 single-chain antibody light chain VL, the CD30 single-chain antibody heavy chain VH as shown in SEQ ID NO.23；As shown in SEQ ID NO.24
CD123 single-chain antibody light chain VL, the CD123 single-chain antibody heavy chain VH as shown in SEQ ID NO.25；Second group is such as SEQ
CD19 single-chain antibody light chain VL shown in the ID NO.16 and CD19 single-chain antibody heavy chain VH as shown in SEQ ID NO.17；Such as
Hinge between antibody inner hinge Inner-Linker, the single-chain antibody as shown in SEQ ID NO.27 shown in SEQ ID NO.26
Inter-Linker, the CD8 Hinge Chimerical receptor hinge as shown in SEQ ID NO.28, as shown in SEQ ID NO.29
CD8 Transmembrane Chimerical receptor transmembrane region, the Chimerical receptor t cell activation domain TCR as shown in SEQ ID NO.32 with
And Chimerical receptor costimulating factor region；Chimerical receptor costimulating factor region be selected from 4-1BB, ICOS, CD27, OX40,
CD28, MYD88, IL1R1, CD70, TNFRSF19L, TNFRSF27, TNFRSF1OD, TNFRSF13B, TNFRSF18, CD134 are swollen
The combination of any one or more in tumor necrosis factor superfamily.
2. carrier as described in claim 1, which is characterized in that the slow virus packaging cis element is sick slowly using the second generation
Poisonous carrier or third generation slow virus carrier；The second generation slow virus carrier includes: the slow virus 5 as shown in SEQ ID NO.5
Terminal LTR, 3 terminal Self-Inactivating LTR of slow virus, such as SEQ as shown in SEQ ID NO.6
Gag cis element shown in ID NO.7, the RRE cis element as shown in SEQ ID NO.8, as shown in SEQ ID NO.9
Env cis element, the cPPT cis element as shown in SEQ ID NO.10；The third generation slow virus carrier includes: such as SEQ
5 terminal LTR of slow virus, the 3 terminal Self- of slow virus as shown in SEQ ID NO.6 shown in ID NO.5
Inactivating LTR, the Gag cis element as shown in SEQ ID NO.7, the cis- member of RRE as shown in SEQ ID NO.8
Part, the env cis element as shown in SEQ ID NO.9, the cPPT cis element as shown in SEQ ID NO.10, and such as SEQ
RSV promoter shown in ID NO.4.
3. carrier as described in claim 1, which is characterized in that two groups of single-chain antibodies are using series connection mode or turn
Angle connection type；
When two groups of single-chain antibodies are CD20 single-chain antibody and CD19 single-chain antibody, the series connection mode specifically: CD20
Single-chain antibody light chain VL and CD19 single-chain antibody light chain VL uses hinge Inter-Linker connection between single-chain antibody, and CD20 is single-stranded
Antibody light chain VL is connect with CD20 single-chain antibody heavy chain VH using antibody inner hinge Inner-Linker, CD19 single-chain antibody light chain
VL is connect with CD19 single-chain antibody heavy chain VH using antibody inner hinge Inner-Linker；The corner connection type specifically:
CD19 single-chain antibody light chain VL is connect with CD19 single-chain antibody heavy chain VH using antibody inner hinge Inner-Linker, and CD20 is single-stranded
Antibody light chain VL and CD19 single-chain antibody heavy chain VH use hinge Inter-Linker connection between single-chain antibody, CD20 single-chain antibody
Heavy chain VH and CD19 single-chain antibody light chain VL use hinge Inter-Linker connection between single-chain antibody；
When two groups of single-chain antibodies are CD30 single-chain antibody and CD19 single-chain antibody, the series connection mode specifically: CD30
Single-chain antibody light chain VL and CD19 single-chain antibody light chain VL uses hinge Inter-Linker connection between single-chain antibody, and CD30 is single-stranded
Antibody light chain VL is connect with CD30 single-chain antibody heavy chain VH using antibody inner hinge Inner-Linker, CD19 single-chain antibody light chain
VL is connect with CD19 single-chain antibody heavy chain VH using antibody inner hinge Inner-Linker；The corner connection type specifically:
CD19 single-chain antibody light chain VL is connect with CD19 single-chain antibody heavy chain VH using antibody inner hinge Inner-Linker, and CD30 is single-stranded
Antibody light chain VL and CD19 single-chain antibody heavy chain VH use hinge Inter-Linker connection between single-chain antibody, CD30 single-chain antibody
Heavy chain VH and CD19 single-chain antibody light chain VL use hinge Inter-Linker connection between single-chain antibody；
When two groups of single-chain antibodies are CD22 single-chain antibody and CD19 single-chain antibody, the series connection mode specifically: CD22
Single-chain antibody light chain VL and CD19 single-chain antibody light chain VL uses hinge Inter-Linker connection between single-chain antibody, and CD22 is single-stranded
Antibody light chain VL is connect with CD22 single-chain antibody heavy chain VH using antibody inner hinge Inner-Linker, CD19 single-chain antibody light chain
VL is connect with CD19 single-chain antibody heavy chain VH using antibody inner hinge Inner-Linker；The corner connection type specifically:
CD19 single-chain antibody light chain VL is connect with CD19 single-chain antibody heavy chain VH using antibody inner hinge Inner-Linker, and CD22 is single-stranded
Antibody light chain VL and CD19 single-chain antibody heavy chain VH use hinge Inter-Linker connection between single-chain antibody, CD22 single-chain antibody
Heavy chain VH and CD19 single-chain antibody light chain VL use hinge Inter-Linker connection between single-chain antibody；
When two groups of single-chain antibodies are CD123 single-chain antibody and CD19 single-chain antibody, the series connection mode specifically:
CD123 single-chain antibody light chain VL and CD19 single-chain antibody light chain VL uses hinge Inter-Linker connection between single-chain antibody,
CD123 single-chain antibody light chain VL is connect with CD123 single-chain antibody heavy chain VH using antibody inner hinge Inner-Linker, and CD19 is mono-
Chain antibody light chain VL is connect with CD19 single-chain antibody heavy chain VH using antibody inner hinge Inner-Linker；The corner connection side
Formula specifically: CD19 single-chain antibody light chain VL and CD19 single-chain antibody heavy chain VH is connected using antibody inner hinge Inner-Linker
It connecing, CD123 single-chain antibody light chain VL and CD19 single-chain antibody heavy chain VH uses hinge Inter-Linker connection between single-chain antibody,
CD123 single-chain antibody heavy chain VH and CD19 single-chain antibody light chain VL uses hinge Inter-Linker connection between single-chain antibody.
4. carrier as described in claim 1, which is characterized in that the sequence of the IL6R single-chain antibody such as SEQ ID NO.33
It is shown.
5. carrier as described in claim 1, which is characterized in that start entire OCTS structure base by the people EF1 α promoter
Because of expression, the CD8 leader Chimerical receptor signal peptide is located at the N-terminal of OCTS coded sequence, for guiding OCTS albumen to position
In cell membrane；Two groups of single-chain antibodies are combined into double antigen recognizing districts, for identification corresponding target antigen；The CD8 Hinge
Chimerical receptor hinge is used to for scFv being anchored on the outside of cell membrane；The CD8 Transmembrane Chimerical receptor transmembrane region is used
It is fixed on cell membrane in by entire Chimerical receptor；The CD28 Chimerical receptor costimulating factor is for stimulating T lymphocyte body
Outer activation and interior tumor cell lethal effect；The CD134 Chimerical receptor costimulating factor is for promoting T lymphocyte to be proliferated
And cytokine secretion, enhance tumour immunity, is conducive to the long-term surviving of memory T cell；The TCR Chimerical receptor t cell activation domain
For activating the expression of downstream signaling pathway；The IL6R single-chain antibody is secreted into extracellularly, closes IL6R, blocks IL6 signal
Access prevents inflammatory factor storm from upgrading；When antigen recognition region is in conjunction with target antigen, signal is transferred to by Chimerical receptor
Into the cell, to generate T cell proliferation, cytokine secretion increases, Anti-apoptotic proteins secretion increases, cell death is prolonged
Late, a series of biological effects of target cell are cracked.
6. carrier as described in claim 1, which is characterized in that Chimerical receptor costimulating factor region uses such as SEQ
CD28 Chimerical receptor costimulating factor shown in ID NO.30 and the CD134 Chimerical receptor as shown in SEQ ID NO.31 pierce altogether
Swash combinations of factors.
7. carrier as described in claim 1, which is characterized in that CD19 single-chain antibody light chain VL, CD19 single-chain antibody weight
Chain VH, CD20 single-chain antibody light chain VL, CD20 single-chain antibody heavy chain VH, CD30 single-chain antibody light chain VL, CD30 single-chain antibody weight
Chain VH, CD123 single-chain antibody light chain VL, CD123 single-chain antibody heavy chain VH, IL6R single-chain antibody passes through humanization modified.
8. a kind of if the described in any item leaching based on OCTS technology of claim 1-7 are the structure of leukaemia CAR-T therapy vector
Construction method, which comprises the following steps: (1) ampicillin resistance gene will be contained as shown in SEQ ID NO.1
AmpR sequence, prokaryotic replions pUC Ori sequence, the virus replication as shown in SEQ ID NO.3 as shown in SEQ ID NO.2
Sub- SV40 Ori sequence, the slow virus packed for slow virus pack cis element, as shown in SEQ ID NO.11
ZsGreen1 green fluorescent protein, the IRES ribosome binding sequence as shown in SEQ ID NO.12, such as SEQ ID NO.13 institute
The enhanced marmot hepatitis B posttranscriptional regulatory element of the eWPRE shown is stored on slow virus skeleton plasmid；It (2) will be such as SEQ
People EF1 α promoter, the OCTS Chimerical receptor structural domain shown in ID NO.14 and the IL6R as shown in SEQ ID NO.33
Single-chain antibody is combined into OCTS Chimerical receptor design scheme, is cloned into slow virus skeleton plasmid by digestion, connection, recombining reaction
In, obtain the recombinant slow virus plasmid of third generation OCTS design；
(3) by obtained recombinant slow virus plasmid respectively with slow virus packaging plasmid pPac-GP, pPac-R and memebrane protein plasmid
PEnv-G transfects HEK293T/17 cell jointly, after carrying out gene transcript expression in HEK293T/17 cell, packs and successfully weighs
Group slow virus carrier can be discharged into cells and supernatant, collect the supernatant for the recombined lentivirus vector for including；
(4) obtained recombinant slow virus supernatant is purified using the column purification mode for filtering, adsorbing, eluting, is respectively obtained
Recombined lentivirus vector.
9. method according to claim 8, which is characterized in that in step (4), the suction filtration step will control supernatant volume and exist
200mL ~ 2000mL controls vacuum degree in -0.5MPA ~ -0.9MPA, prevents due to plug-hole bring carrier loss；The absorption
Step will control the pH value of solution 6 ~ 8, prevent the variation of pH from carrier being caused to inactivate；The elution step will control eluent
Ionic strength prevents the variation of ionic strength from causing elution not exclusively or carrier inactivation in 0.5M ~ 1.0M.
10. such as application of the described in any item carriers of claim 1-7 in the drug that preparation treatment leaching is leukaemia.
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| PCT/CN2017/110667 WO2018218876A1 (en) | 2017-05-27 | 2017-11-13 | Lymphoblastic leukaemia car-t therapeutic vector based on octs technology, and construction method and application thereof |
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| CN108148863B (en) * | 2016-12-05 | 2019-12-17 | 上海优卡迪生物医药科技有限公司 | A kind of CAR-T transgene vector for alleviating CRS that blocks IL6R and its construction method and application |
| CN107245500B (en) * | 2017-05-27 | 2019-05-17 | 上海优卡迪生物医药科技有限公司 | A kind of leaching based on OCTS technology is leukaemia CAR-T therapy vector and its construction method and application |
| CN107916269B (en) * | 2017-11-17 | 2020-12-11 | 山东兴瑞生物科技有限公司 | TCR gene targeting CD19, preparation method, plasmid with gene, kit and application |
| CN110129369B (en) * | 2018-02-09 | 2023-10-13 | 上海交通大学医学院附属上海儿童医学中心 | A chimeric antigen receptor genetic engineering vector, immune cells and their applications |
| CN110157677A (en) * | 2018-02-12 | 2019-08-23 | 深圳宾德生物技术有限公司 | A kind of targeting T lymphocyte and its preparation method and application |
| CN110157738B (en) * | 2018-02-13 | 2023-06-27 | 亘喜生物科技（上海）有限公司 | Engineered immune cells targeting CD19 and CD22 and their applications |
| CN110951689A (en) * | 2018-11-30 | 2020-04-03 | 北京美康基免生物科技有限公司 | CD19 and CD30 based double chimeric antigen receptor gene modified immune cell and application thereof |
| EP4182352A4 (en) * | 2020-07-16 | 2025-03-19 | Legend Biotech Ireland Limited | CD22-BINDING MOLECULES AND USES THEREOF |
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