CN105296431A - Tumor binding specific gamma delta TCR gene modified alpha beta T cell and cancer suppression application thereof - Google Patents
Tumor binding specific gamma delta TCR gene modified alpha beta T cell and cancer suppression application thereof Download PDFInfo
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Abstract
The invention relates to a tumor binding specific gamma delta TCR gene modified alpha beta T cell and cancer suppression application thereof. The alpha beta T cell expresses the tumor binding specific gamma delta TCR; the tumor binding specific gamma delta TCR comprises a gamma 4 chain and a delta 1 chain; the amino acid sequence of the delta 1 chain CDR3 is shown as SEQ ID NO: 1. Experiment results prove that the cell has cytotoxicity for a plurality of tumor cells, and a novel method and a novel strategy are provided for adoptive immunotherapy of tumors.
Description
Technical field
The present invention relates to genetic modification lymphocyte adoptive immunotherapy field.In particular to a kind of α β T cell and but cancer purposes thereof of tumour binding specificity gamma delta T CR genetic modification.
Background technology
The immunotherapy of tumour is placed high hopes by people, its principle is by utilizing modern biotechnology, the immune functional state of artificial adjustment tumour patient body, balance between the links of regulation and control antineoplastic immune, thus reach the object controlling tumour or reduce the relevant toxic side effect that treatment brings.In the immunotherapy approach of all current exploitations, the adoptive immunotherapy of cell, by infusion patient body after Activated in Vitro and increase autologous or alloimmune effector cell, is considered to most potential Immunotherapy Strategy.
In adoptive immunotherapy, conventional has tumor infiltrating lymphocyte (TIL), Tumor-infiltrating lymphocytes (LAK cell), natural killer cell (NK cell), natural killer T cells (NKT cell), cytokine induced kill cell (CIK cell), cytotoxic T lymphocyte (CTL cell) etc. for the natural cell fed back of adopting.But, specific for tumour antigen is strong, avidity is good immunocyte source difficulty, comparatively small amt, and these cells are through external long-time cultivation amplification, time length in its body, kill the demand that tumor activity fails to reach clinical application, restrict the development of adoptive immunotherapy to a certain extent.
The immunocyte of genetic modification utilizes gene recombination technology, transforms naturally occurring immunocyte according to demand, can solve the problem continuing to kill tumor activity in tumour-specific and body.The T cell that Chimeric antigen receptor (Chimericantigenreceptor, CAR) is modified just belongs to the T cell that a genoid is modified, and it makes a breakthrough in clinical trial treatment bone-marrow-derived lymphocyte knurl.Except CAR-T cell, also having the T cell that a large genoid is modified, is the T cell that φt cell receptor (TCR) is modified.Using gene engineering technique makes the TCR molecule of all T Expressions In Lymphocytes specific recognition tumours, greatly can improve antineoplastic ability of T cell.
Another kind of as T cell colony, gamma delta T cells has unique advantage in anti-tumor immunotherapy: 1. gamma delta T cells is mainly with MHC non-limiting way identification antigen, which overcomes the restrictive limitation of MHC of α β T cell antineoplastic immune; 2. antigen recognition spectrum is extensively, as peptide class, non-peptide class, alcohols etc., and the antigen that identifiable design α β T cell can not identify, therefore, functionally can be used as the important supplement of α β T cell immunosurveillance; 3. tumor-infiltrated gamma delta T lymphocytes expresses Fc acceptor, in the expression of TCR-CD3 complex body or/and in assembling during defectiveness, still can transmission of signal, and this is not available for α β T cell; 4. effector function is played by cytotoxicity and generation cytokine; 5. identify that some endogenous stress antigen, fast activating, it is wide to kill knurl spectrum, all has lethal effect to hematological system tumor and solid tumor.
But because in peripheral blood, the ratio of gamma delta T cells is very low, natural gamma delta T cells preparation is used for the amplification for the treatment of and needing to carry out in vitro the long period of adopting of clinical tumor, just can reach the dosage needed for treatment.And immunosuppression in patient's body after Advanced cancers patient or chemotherapy, cause gamma delta T cells quantity to reduce and immunocompetence decline; The use of murine antibody and bovine serum composition can cause the cell fed back to cause in body the problems such as immune response, causes being prepared in for the treatment of gamma delta T cells method still exists some problems demand to solve.Therefore, utilize genetic engineering technique, prepare sufficient amount have cell tumour atopic, the research of nothing/hypotoxic gamma delta T immune effector cell is imperative.
Gamma delta T cells acceptor (gamma delta T CR) comprises γ chain (TCR γ) and δ chain (TCR δ), its antigen recognition site is mainly present in the complementary determining region (CDR) of δ chain variable region (V δ chain), wherein encoded by germline gene V in CDR1 and CDR2 district, and CDR3 district is formed by the somatocyte restructuring of V (D) and J gene.Result of study in the past shows, gamma delta T CR and B-cell receptor (BCR) and antibody structurally highly similar.CDR3 staple diagram shows, the length variations of heavy chain of antibody (H) and TCR δ chain CDR3 is maximum, and comparatively light chain (L) and γ chain person significantly long.It is the same that the prompting of these architecture basics and heavy chain of antibody CDR3 sequence in antibody recognition Process of Antigen play crucial recognition reaction, and δ chain CDR3 district (CDR3 δ) also plays keying action in gamma delta T cells identification antigen.
Existing research carries out sequential analysis to the CDR3 δ of the tumor-infiltrated gamma delta T cells of patients with gastric cancer, has found the advantage CDR3 delta sequences that a different patient is common, and by its called after GTM sequence.The GTM Toplink of synthetic specific with kinds of tumors tissue, the part MICA protein binding of tumor cell line and gamma delta T cells identification, TCR γ 4/ δ 1 (GTM)-Fc fusion rotein also can be specific with kinds of tumors tissue, tumor cell line and MICA protein binding, J.RT3-T3.5 cell is modified with δ 1 chain and γ 4 chain that contain GTM sequence, its cytotoxic activity to kinds of tumor cells can be strengthened, these results demonstrate GTM from different levels and have tumour binding specificity (YanJiang, YangGuo, XueyanXi, LianxianCui, andWeiHe*, FlankingVandJsequencesofcomplementarydeterminingregion3o fTcellreceptor (TCR) δ 1 (CDR δ 1) determiningthestructureandfunctionofTCR γ 4 δ 1.TheJ.Biol.Chem, 2011, 286 (29): 25611 – 25619).
Summary of the invention
Based on the above problem, the object of the present invention is to provide a kind of α β T cell of tumour binding specificity gamma delta T CR genetic modification, the adoptive immunotherapy for tumour provides new methods and strategies.
Applicant's early stage, the CDR3 delta sequences to the tumor-infiltrated gamma delta T cells of patients with gastric cancer were analyzed, and found that the advantage CDR3 delta sequences of the gamma delta T IL of Different Individual cancer of the stomach are tending towards identical.Wherein, the advantage CDR3 delta sequences that the gamma delta T IL of three routine patients with gastric cancer is common, as previously mentioned, in this specification sheets, by this advantage CDR3 delta sequences called after GTM sequence, its aminoacid sequence is as shown in SEQIDNO:1.
It is higher that α β T cell accounts for peripheral blood lymphocyte ratio, easily obtains, and the amplification in vitro time is short, as through modes such as CD3 monoclonal antibody, the stimulations of PHA, CD3-CD28 magnetic bead, can obtain a large amount of α β T cell in the short period of time.But the activation of α β T cell needs offering of antigen presenting cell, man-to-man for specific antigens, have MHC restricted, and the tumor microenvironment of costimulatory signal shortage or inhibition all inhibit the abundant fast activating of α β T cell, becomes the approach of tumor immune escape.And gamma delta T cells by gamma delta T CR identify a class kinds of tumors express stress molecule, fast activating, start the cytotoxic activity to target cell, have the advantages that to kill knurl and compose extensively and be easier to fully activation, there is unique advantage in the candidate cell of the adoptive immunotherapy of tumour.
Based on this, one aspect of the present invention provides a kind of α β T cell of tumour binding specificity gamma delta T CR genetic modification, described α β T cell expressing tumor binding specificity gamma delta T CR, described tumour binding specificity gamma delta T CR comprises γ 4 chain and δ 1 chain, and the aminoacid sequence of described δ 1 chain CDR3 is as shown in SEQIDNO:1.
In this manual by α β T cell called after γ 4 δ 1 (the GTM)-α β T cell of tumour binding specificity gamma delta T CR genetic modification.By tumour binding specificity gamma delta T CR called after γ 4 δ 1 (GTM) TCR that this α β T cell is expressed.
Preferably, the aminoacid sequence of described γ 4 chain is as shown in SEQIDNO:2, and the aminoacid sequence of described δ 1 chain is as shown in SEQIDNO:3.
Alternatively, described α β T cell is that expression vector by expressing described tumour binding specificity gamma delta T CR infects or transfection gained.
Preferably, described expression vector is recombinant slow virus.
Preferably, the carrier that sets out of described recombinant slow virus is pCDH-CMV-MCS-EF1-copGFP, insert the gene order of the expression described tumour binding specificity gamma delta T CR of this carrier that sets out as shown in SEQIDNO:4, this gene is the gene fragment of gene fragment by the gene fragment of described γ 4 chain, CMV promoter, described δ 1 chain, is obtained by digestion with restriction enzyme and ligation series connection.
Alternatively, the α β T cell of described tumour binding specificity gamma delta T CR genetic modification is after tumor cell proteins stimulates, IL-2 and IFN-γ secretes increase.
Alternatively, the α β T cell of described tumour binding specificity gamma delta T CR genetic modification plays cytotoxicity by pore-forming protein/granzyme approach to tumour cell.
Alternatively, the α β T cell of described tumour binding specificity gamma delta T CR genetic modification is the cell without autoimmune response.
The α β T cell that the present invention provides above-mentioned tumour binding specificity gamma delta T CR genetic modification is on the other hand preparing the purposes in antitumor cell preparation, or in preparation containing the purposes in the antitumor cell preparation of IL-2.
Alternatively, described tumour is hepatocellular carcinoma, cancer of the stomach, marrow series leukemia.
The present invention studies discovery, after the α β T cell of above-mentioned tumour binding specificity gamma delta T CR genetic modification and tumour cell are hatched altogether, the secretion of itself IL-2 and IFN-γ increases greatly in vitro, simultaneously, to the cytotoxic activity also significance enhancing of kinds of tumor cells, the expression of granzyme B significantly increases, and shows that above-mentioned cell can be activated by tumour antigen, and has antitumor action.Meanwhile, above-mentioned cell feeds back and can not cause autoimmune response.In vivo study finds, by lotus human hepatocellular carcinoma transplanted tumor nude mice model, cell described in locally injected into tumor, the growth of tumour is obviously suppressed.
To sum up, described cell has antitumor action widely, as hepatocellular carcinoma, cancer of the stomach, marrow series leukemia etc.Further, there is not the intersection interworking situation of external source TCR γ δ and endogenous TCR α β, cytotoxic activity is not had to normal cell, there is no autoreactivity risk, have and obtain preferably result for the treatment of, can be applied to and prepare antitumor cell preparation.Adoptive immunotherapy for tumour provides new methods and strategies.
Accompanying drawing explanation
The structure of Figure 1A-Fig. 1 F: γ 4 δ 1 (GTM)-α β T cell
Figure 1A: CDR3 delta sequences are genetically engineered γ 4 δ 1 (GTM) TCR molecular schematic diagram of GTM.Figure 1B: comprise specific complete γ 4 δ 1 (GTM) the TCR recombined lentivirus vector pCDH-δ 1TCR-CMV-γ 4TCR structural representation with the CDR3 delta sequences GTM of tumour binding specificity.Fig. 1 C: total length γ 4 and the pcr amplification electrophorogram of δ 1 chain and CMV.M, molecular weight marker; Zuo Tu: total length γ 4 chain, object fragment 975bp; Middle figure: total length δ 1 chain, object fragment 864bp; Right figure: total length CMV promoter, object fragment 589bp.The pcr amplification electrophorogram of Fig. 1 D: δ 1TCR-CMV-γ 4TCR total length.M, molecular weight marker, object fragment bp.Fig. 1 E:Westernblotting detects the expression of γ 4 δ 1 (GTM) TCR.1 is the 293T cell protein extract of transfection pCDH-δ 1TCR-CMV-γ 4TCR recombinant plasmid; 2: be the cell protein extract (negative control) of transfection empty carrier.δ 1 chain molecular weight is 36KD.Fig. 1 F: the expression of α β T cell surface external source γ 4 δ 1 (GTM) TCR of slow virus infection.Left: Isotype control; In: empty vector control; Right: γ 4 δ 1 (GTM)-α β T cell.Two positive cell ratio is 35.9%.
The Vitro Tumor killing ability of Fig. 2 A-Fig. 2 F3: γ 4 δ 1 (GTM)-α β T cell detects
Fig. 2 A: γ 4 δ 1 (GTM)-Fc schematic arrangement.Fig. 2 B1-Fig. 2 B3: flow cytometer detection γ 4 δ 1 (GTM)-Fc molecule and HepG2 (Fig. 2 B1), BGC-803 (Fig. 2 B2) and K562 (Fig. 2 B3) cell in conjunction with situation.Contrast molecule is irrelevant hIgG-Fc.Fig. 2 C1-Fig. 2 C3: laser confocal microscope detect γ 4 δ 1 (GTM)-Fc molecule and HepG2 (Fig. 2 C1), BGC-803 (Fig. 2 C2) and K562 (Fig. 2 C3) cell in conjunction with situation.Contrast molecule is irrelevant hIgG-Fc.Fig. 2 D:LDH method detects γ 4 δ 1 (GTM)-α β T cell to the cytotoxic activity of tumour cell.γ 4 δ 1 (GTM)-α β T cell and empty vector control cell are effector cell, from tumour cell (HepG2, BGC-803 and K562) different effect targets to hatch than 37 DEG C 6 little time, calculate each group of killing-efficiency.*, p<0.05vs empty vector control; *, p<0.01vs empty vector control.The expression killing and wounding associated molecule after Fig. 2 E1-Fig. 2 E2: γ 4 δ 1 (GTM)-α β T cell contacts with tumour cell detects.The expression of the granzyme B (GranzymeB) (Fig. 2 E2) in the FasL (Fig. 2 E1) and born of the same parents contacting rear γ 4 δ 1 (GTM)-α β T cell and compared with control cells surface with tumour cell is have detected with Flow Cytometry.After Fig. 2 F1-Fig. 2 F3: γ 4 δ 1 (GTM)-α β T cell contacts with tumour cell, cytokine secretion profile detects.The cytokine that Flow Cytometry detects is IL-2 (Fig. 2 F1), IFN-γ (Fig. 2 F2) and IL-4 (Fig. 2 F3) successively.Left lateral is compared with control cells, and right lateral is γ 4 δ 1 (GTM)-α β T cell.
The id reaction Journal of Sex Research of Fig. 3 A1-Fig. 3 D4: γ 4 δ 1 (GTM)-α β T cell
Interworking of intersecting Fig. 3 A1-Fig. 3 A3: external source γ 4 and δ 1 chain and endogenous TCR α β detects.α β T cell is infected, with the expression of flow cytometer detection cells infected surface gamma δ TCR respectively with pCDH-δ 1TCR and pCDH-γ 4TCR.Left lateral: empty vector control, right lateral: experimental group, is followed successively by and infects pCDH-δ 1TCR (Fig. 3 A1), pCDH-γ 4TCR (Fig. 3 A2) and infect pCDH-δ 1TCR-CMV-γ 4TCR (positive control) (Fig. 3 A3).Fig. 3 B1-Fig. 3 B4: γ 4 δ 1 (GTM) – Fc molecule and Normocellular in conjunction with situation.Used normal cell comprises PBMCs (Fig. 3 B1), 1308.1.86 (Fig. 3 B2), ccc-HEL-1 (Fig. 3 B3) and 293T (Fig. 3 B4) cell.Contrast molecule is irrelevant hIgG-Fc.Fig. 3 C: detect γ 4 δ 1 (GTM)-α β T cell to Normocellular cytotoxic activity by LDH method.Ns: do not have significant difference.The expression of IFN-γ after Fig. 3 D1-Fig. 3 D4: γ 4 δ 1 (GTM)-α β T cell and normal cell effect.Upper behavior compared with control cells, lower behavior γ 4 δ 1 (GTM)-α β T cell.Used normal cell comprises PBMCs (Fig. 3 D1), 1308.1.86 (Fig. 3 D2), ccc-HEL-1 (Fig. 3 D3) and 293T (Fig. 3 D4) cell.
Fig. 4 A-Fig. 4 C: γ 4 δ 1 (GTM)-α β T cell Anticancer effect in vivo
After the inoculation of HepG2 cell, treat that subcutaneous tumor nodules grows to 100mm
3left and right, is divided into two experimental group, treatment group at random by nude mice: intratumor injection γ 4 δ 1 (GTM)-α β T cell; Empty carrier group: the cell that intratumor injection empty carrier infects.Animal only gives abdominal injection IL-25000U/ simultaneously, every treatment in three days once, carries out four times altogether.Measured tumor size every 3 days, calculate inhibition rate of tumor growth, until 30 days, put to death mouse, be separated Subcutaneous tumor.Fig. 4 A: the subcutaneous tumor volumes changing conditions of lotus human hepatocellular carcinoma nude mice.Arrow place is for giving γ 4 δ 1 (GTM)-α β T cell or compared with control cells treatment, * *, p<0.01.Fig. 4 B: give γ 4 δ 1 (GTM)-α β T cell treatment group nude mouse tumor inhibiting rate curve.Fig. 4 C: treat after 30 days, the photo of tumor bearing nude mice subcutaneous transplantation knurl size.
Embodiment
Materials and methods
Experiment material:
1, cell cultures
HepG2: human liver tumor cell is, carries out adherent culture with the DMEM substratum containing 10%FCS; BGC-803: human gastric adenocarcinoma system, carries out adherent culture with the DMEM substratum containing 10%FCS; K562, myelogenous leukemia cell lines; Suspension culture is carried out with the RPMI-1640 substratum containing 10%FCS; The human embryonic kidney epithelial cells system that 293T:SV40 transforms, carries out adherent culture, for the packaging of slow virus with the DMEM substratum containing 10% foetal calf serum; Above-mentioned clone, all purchased from Chinese Academy of Medical Sciences's cell centre, is preserved in this laboratory.PBMC cell: be separated from normal human peripheral blood by human lymphocyte parting liquid density gradient centrifugation and obtain, for sorting α β T cell.
2, animal
BALB/c nude mouse, 4 ~ 6 weeks ages of mouse, body weight 15 ~ 20g, female, be purchased from biological products assay institute animal center, raise in the laminar-flow rack under Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences's Experimental Animal Center no-special pathogen (specificpathogenfree, SPF) condition.
3, bacterial strain and plasmid vector
Bacillus coli DH 5 alpha, purchased from precious biotechnology company limited.Genotype is: supE44 Δ lacU169
hsdR17recA1end1gyr96thi-1relA1, for amplification and the conversion of plasmid.
PCDH-CMV-MCS-EF1-copGFP: for building the slow virus recombinant expression vector of foreign gene δ 1TCR (i.e. δ 1 chain) and γ 4TCR (i.e. γ 4 chain), this plasmid is this experiment preservation.PsPAX2 and PMD2.G: be respectively the packaging plasmid in slow virus packaging system and envelope plasmid, opens industry teach problem group by Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and presents.
PREP7-γ 4TCR plasmid and pREP9-δ 1 (GTM) TCR plasmid: γ 4TCR and δ 1TCR gene source, provided by this laboratory doctor Jiang Yan.Draw from document: YanJiang, YangGuo, XueyanXi, LianxianCui, andWeiHe*, FlankingVandJsequencesofcomplementarydeterminingregion3o fTcellreceptor (TCR) δ 1 (CDR δ 1) determiningthestructureandfunctionofTCR γ 4 δ 1.TheJ.Biol.Chem, 2011,286 (29): 25611 – 25619.
4, primer (see table 1)
γ 4 chain of table 1. for γ 4 δ 1 (GTM) TCR that increases and the primer of δ 1 chain gene total length
Above primer is synthesized by Beijing Qing Ke company.
Experimental technique:
1, the structure of recombined lentivirus vector
(1) PCR of γ 4 chain of TCR, δ 1 chain and CMV full length gene.
With pREP9 δ 1, pCDH-CMV-MCS-EF1-copGFP and pREP7 γ 4 plasmid for template, (aminoacid sequence is as SEQIDNO:2 for pcr amplification δ 1 chain; Gene order is as shown in SEQIDNO:6), (its aminoacid sequence is as shown in SEQIDNO:3 for cytomegalovirus promoter sequence (Cytomegaloviruspromoter, CMV) (its gene order is as shown in SEQIDNO:5) and γ 4 chain gene total length; Gene order is as shown in SEQIDNO:7).
(2) purifying of PCR primer.
(3) pCDH-CMV-MCS-EF1-copGFP carrier and PCR primer endonuclease reaction.
(4) digestion products purifying and ligation.
Enzyme cut after total length γ 4 chain be connected with the carrier that δ 1 chain gene fragment is cut with enzyme respectively, midfeather CMV sequence, is built into recombinant plasmid pCDH-δ 1TCR-CMV-γ 4TCR (inserting the δ 1TCR-CMV-γ 4TCR gene order of lentiviral vectors as shown in SEQIDNO:4).
(5) conversion of recombinant plasmid and positive-selecting.
(6) a small amount of preparation of plasmid DNA.
(7) enzyme of recombinant plasmid cuts qualification.
(8) sequential analysis of DNA fragmentation.
Enzyme is cut assay certificate contain target DNA insert recombinant plasmid carry out determined dna sequence (being completed by Nuo Sai biotech company).With DNAMAN software analysis sequencing result.
2, the corresponding target protein detection of expression of slow virus expression plasmid pCDH-δ 1TCR-CMV-γ 4TCR
(1) slow virus expression plasmid transfection, according to the description operation of InvitrogenLipofectamine2000, proceeds to 293T cell by recombinant vectors.
(2) expression of fluorescence microscope GFP.
(3) expression of Westernblotting detection of delta 1TCR albumen.
3, slow virus packaging
(1) a large amount of extraction purification of plasmid.
According to the explanation of a large amount of extraction purification test kit of Vigorous plasmid, a large amount of extraction recombinant vectors pCDH-δ 1TCR-CMV-γ 4TCR, empty carrier pCDH, and viral package carrier pMD2.G and psPAX2.
(2) slow virus packaging and purifying.
According to the explanation of InvitrogenLipofectamine2000, cotransfection recombinant vectors pCDH-δ 1TCR-CMV-γ 4TCR and viral package carrier respectively, and empty carrier pCDH and viral package carrier are in 293T cell, collect culture supernatant, purified virus particles.
4, slow virus infection α β T cell and γ 4 δ 1 (GTM) TCR expression qualification after infecting
(1) magnetic bead sorting of α β T cell.
According to the explanation of Tianjin Hao foreigner's lymphocyte separation medium, density gradient centrifugation, separating health human peripheral blood single nucleus cell.After counting, illustrate according to the positive sorting test kit of MiltenyiTCR α β, separating alpha β T cell.
(2) slow virus infection α β T cell.
α β T cell sorting obtained stimulates in the environment of activation at immobilization anti-cd 3 antibodies cultivates 24 hours; 25 μ l serum-free RPMI-1640 substratum and 4 μ l slow virus infection toughener Envirus mix gently and are placed on 4 DEG C and hatch 5 minutes, add concentrated lentiviral particle and are placed on 4 DEG C and hatch 15 minutes in mixed solution; Mixed solution and the cell suspension (2 × 10 containing 200IU/mlIL-2
6individual/ml) mixing, be inoculated into and wrap by 24 orifice plates of anti-CD3 antibody in advance, at 32 DEG C, centrifugal 120 minutes of culture plate 1200 × g, then cell is placed in 37 DEG C, the cultivation of 5%CO2 incubator.This course of infection repeats twice, changes the fresh complete RPMI-1640 substratum containing 200IU/mlIL-2 continue cultivation 8 ~ 10 days every 2 ~ 3 days.
(3) qualification of metainfective α β T cell.
The empty vector control group slow virus infection α β T cell corresponding to experimental group is after the 7th day, and collecting cell, detects the expression of its surface gamma δ TCR with Flow Cytometry respectively.
5, γ 4 δ 1 (GTM)-α β T cell extracorporeal anti-tumor functional study
(1) γ 4 δ 1 (GTM)-Fc molecule and tumour cell in conjunction with situation.
With flow cytometry and laser confocal scanning microscope detect γ 4 δ 1 (GTM)-Fc molecule and HepG2, BGC-803 and K562 cell in conjunction with situation.
(2) LDH release (Lactatedehydrogenase, LDH) detects γ 4 δ 1 (GTM)-α β T cell to the cytotoxic activity of tumour cell.
Prepare target cell HepG2 respectively, the α β T cell that BGC-803 and K562 and effector cell's empty carrier infect and γ 4 δ 1 (GTM)-α β T cell.Equalizing effect cell concn, adds 50 μ l effector cells by different effect target ratio (10:1,20:1,40:1); Establish target cell spontaneous release group, target cell maximum release group, the spontaneous release group of effector cell simultaneously and only add the blank group of substratum; Often organize and all establish 4 multiple holes; Flat board centrifugal 3 minutes in room temperature 400 × g, is then placed in 37 DEG C, 5%CO
2hatch 6 hours altogether.According to PromegacytoTox
non-radioactive cell toxicity detection test kit illustrates, the killing-efficiency that detection computations is respectively organized.
(3) γ 4 δ 1 (GTM)-α β T cell kills and wounds the expression detection of associated molecule after contacting with tumour cell.
Effector cell, target cell preparation and the mixing of effect target are as previously mentioned.With the change of GranzymeB in the FasL of Flow Cytometry detection effect ultimate effect cell surface and born of the same parents.
(4) γ 4 δ 1 (GTM)-α β T cell contact with tumour cell after cytokine secretion profile detect.
Prepare α β T cell and γ 4 δ 1 (the GTM)-α β T cell of target cell HepG2 and the infection of effector cell's empty carrier respectively.Equalizing effect cell concn, adds 100 μ l effector cells than 3:1 by effect target and adds Protein transport inhibitor B FA (1000X), establishing control group and experimental group simultaneously; By flat board in 400 × g centrifugal 3 minutes.And be placed on 37 DEG C, 5%CO
2hatch 8 hours altogether; Collecting cell, rupture of membranes is fixed, and detects effector cell's intracellular cytokine IL-2 with Flow Cytometry, the expression change of IFN-γ and IL-4.
6, γ 4 δ 1 (GTM)-α β T cell autoreactivity detects
(1) external source γ 4 and δ 1 chain and endogenous TCR α β intersect interworking and detect.
With pREP7-γ 4TCR plasmid and pREP9-δ 1 (GTM) TCR plasmid for template, PCR builds the recombined lentivirus vector pCDH-δ 1TCR and the pCDH-γ 4TCR that comprise γ 4 and δ 1 chain respectively.Packaging virus respectively, and infect α β T cell, the expression of virus infected cell surface gamma δ TCR is detected with Flow Cytometry.Said process as previously mentioned.
(2) γ 4 δ 1 (GTM)-α β T cell is to Normocellular reactivity research.
With Flow cytometry γ 4 δ 1 (GTM)-Fc and normal cell, comprise PBMCs, 1308.1.86, ccc-HEL-1 and 293T cell in conjunction with situation; Detect γ 4 δ 1 (GTM)-α β T cell to the cytotoxic activity of these cells by LDH method, and with Flow Cytometry detect γ 4 δ 1 (GTM)-α β T cell with these cytosiies after the expression of IFN-γ.Said process as previously mentioned.
7, γ 4 δ 1 (GTM)-α β T cell Anticancer effect in vivo research
Establish lotus human liver cancer cell HepG2 transplanted tumor nude mice model.By HepG2 cell with after 0.25% trysinization counting, wash 3 times with PBS, be suspended from PBS, be seeded in the right shoulder dorsal sc (1 × 10 of nude mice
6, 100 μ l volumes).Treat that tumor nodule grows to 100mm
3left and right, is divided into two groups at random by nude mice.Treatment group, intratumor injection γ 4 δ 1 (GTM)-α β T cell (1 × 10
6cell/only); Empty carrier group, the same number of α β T cell that intratumor injection empty carrier infects, every treatment in three days once, carries out four times altogether.Animal only gives abdominal injection IL-25000U/ simultaneously.Measured tumor size every 3 days, calculate inhibition rate of tumor growth, until 30 days, put to death mouse, be separated Subcutaneous tumor.Tumor size calculation formula is: tumor volume (mm
3)=a × b
2× 0.5.A: knurl body major diameter (mm), b: knurl body minor axis (mm).Tumor control rate calculation formula is: the gross tumor volume of (gross tumor volume of the gross tumor volume-experimental group of control group)/control group.
Embodiment 1: the structure of recombined lentivirus vector pCDH-δ 1TCR-CMV-γ 4TCR
Because GTM sequence has tumour binding specificity, the present invention puts it in complete δ 1 chain, substitute original CDR3 delta sequences, combine with complete γ 4 chain again, form complete γ 4 δ 1 (GTM) TCR molecule (Figure 1A), it is made to be expressed in α β T cell surface, the α β T lymphocyte that this γ 4 δ 1 (GTM) tcr gene comprising tumour binding specificity CDR3 delta sequences (GTM) is modified will have the similar tumour binding specificity of GTM, can be used for the adoptive immunotherapy of tumour.
In order to make γ 4 chain of external source and δ 1 chain expression level in α β T lymphocyte suitable, so that successful combination, form complete γ 4 δ 1 (GTM) TCR molecule, the gene fragment of complete γ 4 chain and δ 1 chain (CDR3 delta sequences are GTM) is connected on a lentiviral vectors by the present invention, because containing a CMV promoter on carrier, therefore in the middle of two gene fragments, insert a CMV promoter again, build recombined lentivirus vector pCDH-δ 1TCR-CMV-γ 4TCR (Figure 1B).
Respectively with pREP9 δ 1, pCDH-CMV-MCS-EF1-copGFP and pREP7 γ 4 plasmid for template, pcr amplification δ 1 chain, CMV and γ 4 chain gene fragment (Fig. 1 C), by specific digestion with restriction enzyme and ligation, three gene fragments are connected in turn, be inserted in lentiviral vectors pCDH-CMV-MCS-EF1-copGFP, obtain recombined lentivirus vector pCDH-δ 1TCR-CMV-γ 4TCR.Qualification display is cut, pCDH-δ 1TCR-CMV-γ 4TCR vector construction success (Fig. 1 D) through enzyme.
Embodiment 2: recombined lentivirus vector pCDH-δ 1TCR-CMV-γ 4TCR corresponding target protein γ 4 δ 1 (GTM) TCR developed by molecule detects
Recombined lentivirus vector pCDH-δ 1TCR-CMV-γ 4TCR embodiment 1 obtained proceeds to 293T cell, and fluorescence microscope GFP expresses, and collecting cell, cracking, detects the expression of δ 1TCR in full cell pyrolysis liquid with Westernblotting.As referring to figure 1e, with the monoclonal antibody of mouse-anti people δ 1 for primary antibodie, HRP enzyme mark sheep anti-mouse antibody resists as two result, and compared with empty vector control, δ 1TCR albumen obtains good expression in 293T cell.And then prove that γ 4 δ 1 (GTM) TCR molecule is at 293T cells.
Embodiment 3: γ 4 δ 1 (GTM) the TCR expression qualification of slow virus infection α β T cell and the rear α β T cell of infection
The recombined lentivirus vector pCDH-δ 1TCR-CMV-γ 4TCR obtained in embodiment 1 is packaged as recombinant slow virus.
Separating health human PBMC, sorting α β T cell, and stimulate at immobilization anti-cd 3 antibodies in the environment of activation, use above-mentioned recombinant slow virus to infect.Infect latter 7th day, collecting cell, detects the expression of its surface gamma 4 δ 1 (GTM) TCR with Flow Cytometry.As shown in fig. 1f, compared with infecting contrast with empty carrier, the α β T cell of 35.9% expresses γ 4 δ 1 (GTM) TCR simultaneously, namely successfully obtains the α β T cell of expressing tumor binding specificity gamma delta T CR genetic modification, i.e. γ 4 δ 1 (GTM)-α β T cell.
Embodiment 4: γ 4 δ 1 (GTM)-α β T cell extracorporeal biology Function Identification
1) γ 4 δ 1 (GTM)-Fc molecule and tumour cell in conjunction with situation
The prerequisite of γ 4 δ 1 (GTM)-α β T cell to tumor-killing is the combination of γ 4 δ 1 (GTM) TCR tumor associated antigen corresponding to tumor cell surface.Have expressed γ 4 δ 1 (GTM)-Fc molecule (Fig. 2 A), γ 4 δ 1 (GTM) TCR extracellular region be connected with antibody Fc molecule, and detect itself and tumour cell in conjunction with situation.The result of Flow Cytometry (Fig. 2 B1-Fig. 2 B3) and laser scanning co-focusing microscope (Fig. 2 C1-Fig. 2 C3) all shows, compared with Isotype control group (hIgG1-Fc), γ 4 δ 1 (GTM)-Fc and HepG2, BGC-803 and K562 tumour cell can specific combinations.
2) γ 4 δ 1 (GTM)-α β T cell is to the cytotoxic activity of tumour cell
With HepG2, BGC-803 and K562 cell for target cell, be effector cell with γ 4 δ 1 (the GTM)-α β T cell obtained in embodiment 3, detect γ 4 δ 1 (GTM)-α β T cell to the killing activity (representing with kill rate) of target cell by LDH method, result as shown in Figure 2 D.Compared with empty vector control cell, the killing-efficiency of γ 4 δ 1 (GTM)-α β T cell to these tumour cells all significantly improves.
3) γ 4 δ 1 (GTM)-α β T cell kills and wounds approach to tumour cell
Gamma delta T cells kills and wounds mainly through two approach to tumour cell: Fas-FasL approach and pore-forming protein-granzyme approach.Flow cytometry and HepG2 is adopted to hatch the expression of rear γ 4 δ 1 (GTM)-α β T cell FasL and GranzymeB.Result shows, after hatching with tumour cell, FasL expression level and the empty vector control cell of γ 4 δ 1 (GTM)-α β T cell do not have difference, and the expression level of GranzymeB (40.7%) is apparently higher than control group (25.7%) (Fig. 2 E1-Fig. 2 E2), show that γ 4 δ 1 (GTM)-α β T cell plays killing and wounding tumour by pore-forming protein-granzyme approach.
4) secretion capacity of γ 4 δ 1 (GTM)-α β T cell cytokine
Except directly playing except cytotoxic activity, the antineoplastic immune function of gamma delta T cells also comprises secrete cytokines.Have detected after contacting 8 hours with tumour cell HepG2 with Flow Cytometry, the secretion capacity of γ 4 δ 1 (GTM)-α β T cell cytokine, cytokine comprises sub-IL-2, IFN-γ and IL-4.Result is as shown in Fig. 2 F1-2F3, and the secretion of γ 4 δ 1 (GTM)-α β T cell IL-2, IFN-γ ability is significantly higher than empty vector control cell, and both secretion capacities of IL-4 do not have significant difference.
Embodiment 5: γ 4 δ 1 (GTM)-α β T cell autoreactivity detects
1) external source γ 4 and δ 1 chain and endogenous TCR α β intersect interworking and detect
Research shows, interworking can occur to intersect TCR α β endogenous in external source α chain and β chain and α β T cell, and the novel TCR that this intersection interworking is formed causes autoimmune response most probably.If external source γ 4 δ 1 (GTM) TCR acceptor also occurs similar intersection interworking phenomenon when expressing in α β T cell, just likely cause autoreactivity.Therefore, whether by external source δ 1 chain and γ 4 chain modified alpha β T cell respectively, detecting cell surface has γ δ (GTM) TCR to express.Result is as shown in Fig. 3 A1-Fig. 3 A3, the α β T cell that δ 1TCR modifies and the α β T cell that γ 4TCR chain is modified all can't detect the expression of gamma delta T CR, and the α β T cell that pCDH-δ 1TCR-CMV-γ 4TCR modifies is as positive control, the expression of gamma delta T CR can be detected, illustrate that external source δ 1 chain or γ 4 chain with α TCR chain or β TCR chain, interworking do not occur to intersect, form complete TCR molecule, be transported to surface of cell membrane, therefore γ 4 δ 1 (GTM)-α β T cell does not produce new TCR and the autoimmune response risk brought.
2) γ 4 δ 1 (GTM)-α β T cell is to Normocellular reactivity research
Except intersection interworking, if the gamma delta T CR of external source itself can be combined with normal cell, also can cause the autoreactivity of γ 4 δ 1 (GTM)-α β T cell.First use Flow cytometry γ 4 δ 1 (GTM)-Fc molecule and normal cell, comprise PBMCs, 1308.1.86, ccc-HEL-1 and 293T cell in conjunction with situation.Result is as shown in Fig. 3 B1-Fig. 3 B4, and γ 4 δ 1 (GTM)-Fc molecule is not combined with normal cell.Compared with empty vector control cell, γ 4 δ 1 (GTM)-α β T cell does not change (Fig. 3 C) Normocellular lethal effect, and after hatching altogether with normal cell, the ability of γ 4 δ 1 (GTM)-α β T cell secretion of gamma-IFN does not also significantly improve (Fig. 3 D1-Fig. 3 D4).
Embodiment 6: γ 4 δ 1 (GTM)-α β T cell Anticancer effect in vivo
Establish lotus human liver cancer cell HepG2 transplanted tumor nude mice model.Treat that tumor nodule grows to 100mm
3left and right, is divided into two groups at random by nude mice.Treatment group, intratumor injection γ 4 δ 1 (GTM)-α β T cell (1 × 10
6cell/only); Empty carrier group, the same number of α β T cell that intratumor injection empty carrier infects, every treatment in three days once, carries out four times altogether.Animal only gives abdominal injection IL-25000U/ simultaneously.Fig. 4 A shows the subcutaneous tumor volumes changing conditions of different group nude mice, and the tumour of γ 4 δ 1 (GTM)-α β T cell treatment group nude mice is significantly less than control group.Fig. 4 B is the inhibition rate of tumor growth curve calculated.From the 3rd day, the treatment of γ 4 δ 1 (GTM)-α β T cell just reached 40% to the inhibiting rate of tumor growth, and along with time lapse, rises to 70%, and maintains in this level within 30 day time always.Fig. 4 C is after 30 days, puts to death mouse, the size of the Subcutaneous tumor be separated to.Above result fully shows, and γ 4 δ 1 (GTM)-α β T cell obviously inhibits the growth of mouse tumor in vivo.
As can be seen from above, the α β T cell of tumour binding specificity gamma delta T CR genetic modification provided by the present invention has antitumor action widely, as hepatocellular carcinoma, cancer of the stomach, marrow series leukemia etc.Further, there is not the intersection interworking situation of external source TCR γ δ and endogenous TCR α β, cytotoxic activity is not had to normal cell, there is no autoreactivity risk, have and obtain preferably result for the treatment of, can be applied to and prepare antitumor cell preparation.Adoptive immunotherapy for tumour provides new methods and strategies.
Claims (10)
1. the α β T cell of a tumour binding specificity gamma delta T CR genetic modification, this α β T cell expressing tumor binding specificity gamma delta T CR, described tumour binding specificity gamma delta T CR comprises γ 4 chain and δ 1 chain, and the aminoacid sequence of described δ 1 chain CDR3 is as shown in SEQIDNO:1.
2. the α β T cell of tumour binding specificity gamma delta T CR genetic modification according to claim 1, is characterized in that, the aminoacid sequence of described γ 4 chain is as shown in SEQIDNO:2, and the aminoacid sequence of described δ 1 chain is as shown in SEQIDNO:3.
3. the α β T cell of tumour binding specificity gamma delta T CR genetic modification according to claim 1, is characterized in that, described α β T cell is that the expression vector by expressing described tumour binding specificity gamma delta T CR infects or transfection gained.
4. the α β T cell of tumour binding specificity gamma delta T CR genetic modification according to claim 3, it is characterized in that, described expression vector is recombinant slow virus.
5. the α β T cell of tumour binding specificity gamma delta T CR genetic modification according to claim 4, it is characterized in that, the carrier that sets out of described recombinant slow virus is pCDH-CMV-MCS-EF1-copGFP, inserts the gene order of the expression described tumour binding specificity gamma delta T CR of this carrier that sets out as shown in SEQIDNO:4.
6. the α β T cell of the tumour binding specificity gamma delta T CR genetic modification according to any one of claim 1-5, is characterized in that, described α β T cell is after tumor cell proteins stimulates, IL-2 and IFN-γ secretes increase.
7. the α β T cell of the tumour binding specificity gamma delta T CR genetic modification according to any one of claim 1-5, it is characterized in that, described α β T cell plays cytotoxicity by pore-forming protein/granzyme approach to tumour cell.
8. the α β T cell of the tumour binding specificity gamma delta T CR genetic modification according to any one of claim 1-5, is characterized in that, described α β T cell is the cell without autoimmune response.
9. the α β T cell of the tumour binding specificity gamma delta T CR genetic modification according to any one of claim 1-5 is preparing the purposes in antitumor cell preparation, or in preparation containing the purposes in the antitumor cell preparation of IL-2.
10. purposes according to claim 9, is characterized in that, described tumour is selected from hepatocellular carcinoma, cancer of the stomach, marrow series leukemia.
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