CN101508970A - Immune cell modification method and cell modification device - Google Patents
Immune cell modification method and cell modification device Download PDFInfo
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Abstract
The present invention relates to a cell modification method for modifying human or mammal immune cells, especially effector cells outside the human or mammal body, wherein in a first step at least one encapsulated substance, preferably an active pharmaceutical ingredient, is introduced in and/or arranged on isolated human or mammal immune cells and wherein in a second step prior to or after the introduction of said at least one substance the cytotoxic effector function of the isolated immune cells is enhanced. The present invention also relates to a correspondingly modified human or mammal immune cell and to a cell modification device.
Description
Technical field
The present invention relates to immune cell modification method and cell modification device, it modifies the cellular component of human or animal's blood, especially the cell of body innate immune defence mechanism (for example T cell, NK cell, monocyte, scavenger cell or microglia), so that described cell is especially afterwards brought into play therapeutic action in the mammalian body in introducing human or animal body, for example anticancer disease (as the cancer of lung or pancreas or ovary, mammary gland or brain) or anti-other disease are perhaps brought into play diagnostic effect.The invention still further relates to immunocyte through corresponding modification.
Background technology
For a long time, people just understand the method for carrying out therapeutic treatment by activeconstituents.In these methods, often activeconstituents is delivered to human or animal's whole body.For example, but per os or use activeconstituents by injection; Described then activeconstituents makes in the whole machine body that himself is evenly distributed on human or animal body.As seen, the decisive shortcoming of methods of treatment is that zone not ill in human or animal's body also can be subjected to the influence of activeconstituents before, and only has the sub-fraction activeconstituents to play a role in target region.Therefore, to become divided dose be avoidable to corresponding high reactivity.
Summary of the invention
Main purpose of the present invention is to use the target vehicle of immunocyte as active substance or medicament (for example medicine, RNAi etc.) and/or diagnostic reagent.Independently deliver the target of diseased tissue (promptly to) in order to improve target, used again target (retargeting) strategy, bi-specific antibody for example, the genetic transformation cell relevant etc. with TCR.Because it uses as the vehicle of living, transfer place (for example tumour) of immunocyte guiding, and at first bring into play intrinsic cytotoxicity function.Release medicine or active substance are to strengthen then.Described release is by specific triggering at inner or outside physics that applies or physiologic factor (for example pH, temperature), perhaps discharge, for example discharge by causing particle/substance or shell to degrade to cause as the enzyme in tenuigenin or the lysosome/endosome or by pH in the time-dependent manner mode.Medicament or active substance have improved the natural action (intrinsic lysis/cytotoxicity feature) of immunocyte.
Herein with following chapters and sections in, described cellular component also abbreviates cell as.Described cell also can comprise in the human or animal body for example other the n cell of differentiation, perhaps undifferentiated cell, for example stem cell.Thereafter the therapeutic action as the intravital modified cell of medicine introducing Mammals can comprise for example controlled activeconstituents release or tissue regeneration or the like.
The therapeutic action of so modifying cell can comprise:
-release medicine or active substance, for example cytostatic medicament and/or angiogenesis inhibitor,
-discharge therapeutical agent based on antibody,
-release is used for the therapeutic DNA of transfection,
-discharging therapeutical agent based on RNAi, it is morbific gene of target and virus optionally; And/or
-discharging chemokine, for example cytokine or protein peptide or glycoprotein promptly promote immune other cells or cause the conditioning agent of apoptosis, for example TNF α;
Perhaps, with regard to the diagnosis purpose:
-make the cell loading contrast medium, ferromagnetic particle (for example being used for the NMR diagnosis) for example.
Treatment or diagnostic reagent also can use together, for example make up in immunocyte, and perhaps the immunocyte level that is loaded with therapeutical agent or diagnostic reagent by application assigns to carry out.
Therefore, task of the present invention is the such method and apparatus of exploitation, it allows to modify human or animal's cell, so that these cells are delivered to initiatively the expectation body part or the cell of target when introducing human or animal body again, and brings into play treatment or diagnostic effect there.By modifying animal or human's cell by this way, might for example resist disease with low dosage, perhaps strengthen or enhanced tissue, and do not influence zone not ill in the body in the target mode in the target mode, and/or the disease sites in the detection body, for example position of tumour and transfer.
Purpose of the present invention realizes by the method for claim 1, the modified immunocyte of claim 10 and the device of claim 13.Advantageous embodiment has been described in the dependent claims.Purposes of the present invention is described in claim 14 and 15.
The present invention provides immune cell modification method to the basic sides of the solution that the problems referred to above provided, wherein in first step, a kind of material (or more than a kind of material through sealing) (preferred active pharmaceutical ingredient) through sealing introduced in isolating human or animal's immunocyte and/or placed on it; And wherein in second step before or after introducing described at least a material, strengthen the function of cytotoxic effect device (cytotoxic effector) of described isolating immunocyte and/or target ability or the target that strengthens described isolating immunocyte respectively and seek ability through sealing.
The material of being sealed can be introduced in the cell; But, also this material can be placed, connects or adheres to the outside surface of cell.The latter can be for example by the electrostatic interaction technology, realize by (for example dual specific, monospecific or the tri-specific) antibody or the molecule (to allow that described material is placed or be fixed on cell surface) on press back institute encapsulating substance surface.
The implication that " strengthens the cytotoxic effect device function of described isolating immunocyte and/or strengthen the target ability of described isolating immunocyte " is understood in the present invention in due form: it strengthens according to the present invention immunocyte to be finished and discerns and/or look for every kind of cellular immunization methods of treatment (especially adoptive immunotherapy) of the ability of target cell (for example tumour cell) all think in the scope of corresponding statement in human or animal's body.In other words, in order to implement described second step (perhaps in order in the immunocyte of modifying according to the present invention, to realize corresponding modification), can use every kind of strategy of target again that is used for strengthening the ill target cell of human or animal's body identification.Especially, the implication that " strengthens cytotoxic effect device function " also should be understood as follows, promptly comprise modification to immunocyte, this is modified and makes that the cytotoxic effect device function that described carrier cell is located at target (for example tumour) can strengthen when these immunocytes (carrier cell) arrive described ill target place.Especially, can strengthen cytotoxic effect device function by discharge the material of being sealed at the target place.As hereinafter further describing ground, can be accomplished in several ways in second step enhancing: for example to described separating immune cell-targeting ability, the strategy of target again that the T cell that can use utilization to have Chimerical receptor carries out perhaps can be realized the combination of described immunocyte and bi-specific antibody etc.Wherein, maybe advantageously, modify this immunocyte, so that the cell of such modified/treated is enhanced in the cytotoxic effect device function of disease sites after being infused in the body again.
The immunocyte to be finished that can be used among the present invention can be any immunocyte type used in the adoptive immunotherapy, for example from body or homogeneous variant cell.For example, can modify the T cell, except patient self or donorcells, also can use clone, cytotoxic T cell clone for example is as TALL-104 cell or C CURE 709 cells.Can also modify the NK cell, NK clone for example is as NK-92.
Therefore, cell to be finished can be T cell (mono-clonal and a polyclone T cell), especially again the polyclone T cell of target (as mentioned above or by alternate manner target again), specific for tumour antigen T-lymphocyte, genetically modified T lymphocyte or cytotoxic T cell.
Described second step can be carried out before described first step, perhaps carried out after described first step.Can realize the cytotoxic effect device function of separating before from the immunocyte of human or animal body is strengthened by multitude of different ways, this be described hereinafter in more detail.
Basically, can be by means of known cellular segregation test kit (Stem CellTechnologies for example, 5070 West Seventh Avenue, Vancouver, BC, the RosetteSep of Canada
TMTest kit) separates specific immunocyte, thereby realize immune cell modification method of the present invention.Therapeutical agent or active substance or diagnostic substances are loaded on the isolating immunocyte of institute.This can be undertaken by hatching altogether; But, also can support described loading by electroporation.Yet as an alternative, immune cell modification method of the present invention also can use cell modification device of the present invention to implement, and described equipment also further describes hereinafter.
In order to use in applied immunology or oncology according to the present invention and/or to be used for diagnosing tumour, the method of the effector functions of enhancing immunity cell is conceived to strengthen or improve the disease-resistant effect of this immunocyte, described immunocyte is cytotoxic T cell for example, especially (preferred activatory) cytotoxic T cell, natural killer cell (NK cell) for example, for example monocyte or for example scavenger cell of cells of monocytic origin, for example microglia.Immunocyte through corresponding modification can be used in adoptive immunotherapy or the diagnosis.
As the immune part of human or animal, these effector cells can discern illing tissue or tumor tissues, and destroy these tissues under the help of its cytotoxic effect device function.These cells in virus or antitumor answering in non-Lymphoid tissue active migration and the natural ability of soaking into illing tissue determined these cells to be used for autonomous active material target or to be used for diagnosis.Tumor escape mechanism for example angery, often stop immunity system to destroy tumour itself to the inhibition of the apoptosis induction of immunocyte or effector functions.
In order to strengthen this effector functions and to stop tumor escape mechanism, use above-mentioned dual-step type method, wherein the material of extraly one or more temporarily being sealed (especially active pharmaceutical ingredient) is introduced in the immunocyte or is placed on the described immunocyte.Described material is sealed, so that described material can be in immunocyte or on immunocyte discharge immediately, but (after preferred 1 day to 25 days) discharge after a few hours at least.This arrives target tissue and damaged or destroy by institute's load material before not destroying tumour cell at immunocyte arrival target tissue and by the intrinsic cytotoxicity for immunocyte usually is essential.
Institute's encapsulating substance or institute's entrapped drug are of a size of about 10 nanometers to 1 micron.For example, sealing like this can be by means of liposome, the liposome of sealing (for example, use alginate such as Barium alginate to seal), multilamellar liposome, capsule plastid (vesosome), virion (virosome) or comprise porous material or realize by its microballoon of forming (for example by material is introduced wherein), described porous mass is for example poly(lactic acid) (polylactide) or polyglycolic acid-lactic acid (polyglycolic-lactic acid, PGLA) polymkeric substance, starch, phosphatic rock and/or compacting polymkeric substance (having or do not have outside dressing etc.) dextran, agarose, albumin, chitosan, silicon-dioxide or hollow silica particle, polymine (polyethylenimin), Polyalkylcyanoacrylanano, poly methyl methacrylate particle, the core-shell nano particle, branch-shape polymer and branch fluidized polymer, its core element is amine or sugar normally, has several identical shell binding site molecule points on the surface.Described shell changes between acid and the amine through being everlasting.
The above-mentioned corpusculum (liposome etc.) that is used to seal medicament/material also can be modified corpusculum: can be respectively under the help of antibody or antibody fragment or peptide (except the described material of load respectively or introduce the described material), modify corpusculum (liposome etc.), with guarantee to diseased cells (especially tumour cell) identification, with the combining and/or mix in this type of cell of this type of cell.In this case, corpusculum/the liposome of corresponding modification is loaded into immunocyte to be finished, when h substance/medicine, the film integrality of carrier cell is destroyed, liposome leaves cell, respectively can in conjunction with cancer cells or can mix in these cancer cells (for example, by means of acceptor etc.) thereafter.
In order to strengthen the efficient of mixing in the immunocyte (carrier cell), can use the part of the expressed acceptor in immunocyte surface to modify described sealing, for example so-called cell-penetrating peptides of described part (cellpenetrating peptide, CPP).CPP is short polycation sequence, and the peptide or proteinic the mixing of this sequence carried in its simplification.If these CPP and nano particle or liposome covalent attachment then strengthen mixing in cell.The example of these CPP is TAT and the penetrine peptide of HIV virus one by one.
Modifiedly especially be competent in the therapeutic DNA that will be used for transfection or based on the therapeutical agent of RNAi and mix carrier cell to improve the release in cancer cells, mix through sealing active substance or medicament.
Can carry out the efficient that load in carrier cell further strengthens this method by using the dual specific liposome; Especially, back one liposome both can load medicament and/or therapeutic DNA and/or based on therapeutical agent of RNAi or the like, can modify with antibody again, modify (at the tumour-specific surface marker, the tumor associated antigen (TAA) of internalization) especially rapidly and/or modify (be used for based on mixing of carrying out of acceptor etc.) with peptide with its derivative.Also might use the liposome that is mounted with described material/medicine, described liposome earlier discharges described material/medicine, and its sole purpose is to kill and wound carrier cell, discharges from cell in easier mode to allow above-mentioned liposome.Than the release of described liposome, the release of described material/medicine from above-mentioned liposome can realize time lag, so that medicine to be discharged is accurately located to discharge in illing tissue's (for example tumour).
At the immune cell modification method that is used for producing medicine or diagnostic reagent, immunocyte (for example precursor cell or the effector cell of end differentiation eventually) can for example extract certainly or separate from mammiferous peripheral blood or mammiferous tumor tissues.Preparation with separate these immunocytes so that after it can be used for material that load sealed, can multitude of different ways before or after the described material of load, strengthen second step of the effector functions of (isolating) immunocyte and/or target ability (preferably infusion after target site) again, look for ability of illing tissue or tumor tissues etc. to strengthen modified cell.
First kind of described second step of realization enhancing cytotoxic effect device function and/or target ability may be to introduce in the isolating immunocyte of institute from the immunocyte acceptor gene (for example TXi Baoshouti gene, tcr gene) of tumor associated antigen (TAA) specific immunity cell (especially TAA specific T-cells).Preferably, can carry out this introducing by transgenosis.It is known to a person of ordinary skill in the art (referring to for example Timothy M.Clay how carrying out corresponding transgenosis, " Efficient Transfer of a Tumor Antigen-ReactiveTCR to Human Peripheral Blood Lymphocytes Confers Anti-TumorReactivity " such as Mary C.Custer, The Journal of Immunology, 1999,163:507-513).
This have the following advantages (this also is applicable to possibility hereinafter described): compare with using the immunocyte of not modifying, can improve the ratio (for example tumor-killing rate) that kills and wounds illing tissue by at least a encapsulating substance of introducing/arrangement.
Strengthen cytotoxic effect device function and/or target ability in described second step second kind may be to make isolating immunocyte have genetically modified or chimeric immunocyte acceptor, wherein preferably, described isolating immunocyte is cytotoxic T cell (CTL) or natural killer cell (NK cell).Especially, described second step uses the transgenosis of gomphosis immunoglobulin (TXi Baoshouti) to carry out.This has obtained " immunocyte of design ", the cytotoxic T cell or the natural killer cell that for example have genetically modified or chimeric immunocyte acceptor, described acceptor is suitable for going up combining of predetermined flag thing with the surface and improving cancer identification by being incorporated into, thus activating immune cell and/or kill and wound cancer cells.In this case, can the described dual-step type method of following enforcement: at first activate these and have the immunocyte that strengthens cytotoxic effect device function, then respective substance or medicine are introduced in the cell, at last the cell of corresponding activation and load is introduced in the mammalian body.It is well known in the art how specifically to implement corresponding transgenosis, referring to for example Claudia
With Malcolm K.Brenner " Chimeric T-cell Receptors for the Targeting ofCancer Cells ", 2003, Acta Haematologica; 110:154-159.
The another kind that strengthens cytotoxic effect device function and/or target ability respectively or implement described second step may be: isolating immunocyte is combined with antibody and/or antibody fragment, wherein preferably use bi-specific antibody, three-specific antibody, double antibody (diabody), three antibody (triabody) and/or four antibody (tetrabody) or its fragment, discern the ability of illing tissue to improve described immunocyte.Wherein preferably, make separation from the described material/medicine of the polyclone T of mammalian organism cell load in first step, afterwards load antibodies, for example load dual specific or three-specific antibody, double antibody, three antibody and/or four antibody or its fragment.In an advantageous embodiment, the isolating T cell of activation exsomatized before first step.Before described step, can carry out the T cell amplification.Perhaps, also at least a material/medicine of having sealed of load of non-activated immunocyte.In the case, separately injection of antibody (especially bi-specific antibody or double antibody or other antibody) (preferably before the described cell of injection) is so that the activation of described cell is carried out in vivo.Activation is preferably carried out in tumor site in the body of these cells: for example, antibody can at first combine with tumour thereafter, and the cell of load can combine with tumour by means of the dual specific function of described antibody thereafter.At last, can again modified immunocyte be introduced in the mammalian body, to improve the cancer recognition rate and/or to make described immunocyte remain on activated state.
How implementing corresponding combination is well known to a person skilled in the art, referring to " Bispecific CD3 * CD19 diabody for T cell-mediated lysis ofmalignant human B cells " such as for example Sergey M.Kipriyanov, Int.J.Cancer (77), 1998, the 763-772 page or leaf.
At last, the 4th kind of method that strengthens second step of the cytotoxic effect device function of institute's separating immune cell and/or target ability is, specificity is selected predetermined immunocyte group from the immunocyte that the patient extracts, wherein known in described patient these immunocytes group move to illing tissue and/or tumor site efficiently: especially can select the T cell, for example T lymphocyte specific for tumour antigen or with target strategy bonded T cell or monocyte again (or derive from its scavenger cell) etc.
In all above-mentioned situations, at the described medicine/material of load and/or before strengthening cytotoxic effect device function and before introducing in the human or animal body subsequently again after modifying, selected/isolating immunocyte in case of necessity can increase.For with the situation of antibody (especially bi-specific antibody or double antibody) combination, might use polyclone T cell.Amplification is necessary in specific example only.After introducing and/or the material of arrangement, with antibody and the combination of T cell through sealing; Afterwards, the immunocyte modified like this of infusion again.Perhaps, at first load antibodies is carried out the load of described material/medicine then.
Perhaps, can followingly carry out the dual-step type method, wherein at first with in the introducing bodies such as bi-specific antibody, described bi-specific antibody is modified tumour etc. thereafter, afterwards the T cell is introduced in the body, and the T cell rests on tumor site by bi-specific antibody.
In all the method for the invention, use material/drug loading cell through sealing (that is: described material/medicine is introduced in the mammalian immune cell or will material place on the mammalian immune cell) can be by described cell and particle being hatched preferred 5 minutes to 2 hours (depending on selected sealing) together or being undertaken by transfection or electroporation through sealing.
After with modified immunocyte infusion again, described immunocyte can be at first moves in mammalian body and is not subjected to the influence of its load according to its natural function, and can be in illing tissue's place's accumulation.There, described cell can be brought into play its intrinsic lysis/cytotoxic effect, promptly kills and wounds cancer cells.Described then material/medicine release from seal (after preferred 3 to 14 days) causes initial apoptosis in carrier cell, promotes described material/medicine to be discharged into the illing tissue from carrier cell thus.Except cytostatics, other material for example angiogenesis inhibitor also can be used as through encapsulating substance and is written in the immunocyte.
When being used for diagnostic purpose, for example nano particle (being that mean diameter is about the particle of several nanometers to the approximate number micron) can be written in the immunocyte as contrast medium etc. as ferrite, be used for MRI etc.By these nano particles are introduced in the immunocyte, can position tumour or transfer.
Modified immunocyte of the present invention, immune cell modification method of the present invention and cell modification device of the present invention can be preferably used for treating cancer, for example be used for the treatment of lymphoma, colorectal carcinoma, lung cancer and/or carcinoma of the pancreas, and/or ovarian cancer and or the mammary cancer and/or the cancer of the brain etc.; Be used for the treatment of the cell of virus infection, be used for the treatment of chronic inflammatory diseases, promptly be used for the chronic inflammatory diseases treatment, and/or be used for the treatment of alzheimer's disease.
Modified cell of the present invention (and corresponding immune cell modification method and cell modification device) has the following advantages: compare with the modified cell of prior art, have the cancer therapy ability of raising, especially the tumor-killing effect of Ti Gaoing.
Especially, by making the described medicament/material of T cell loading and by in the T cell, discharging the latter, initial t cell proliferation, it causes film integrality to strengthen and drug release improves.
Description of drawings
Fig. 1 shows an example of cell modification device of the present invention.
Embodiment
Hereinafter, provide the example of cell modification device of the present invention, wherein can produce modified immunocyte of the present invention, perhaps wherein can carry out immune cell modification method of the present invention.
In Fig. 1, blood bank 1 (for example, the human bloodstream) shows that with synoptic diagram blood is from wherein offering cell separation equipment 4 by filter plant 3a.In cell separation equipment 4, with the cell of known manner selection expectation, cell promptly to be finished.Reference numeral 3 shows filtered fluid bank 3, and filtered fluid can be from wherein being added to filter plant 3a by valve 2.(for example be used for the equipment of impedance measurement) in known manner by means of counting equipment 5 determine in the separating device 4 the quantity of isolating isolated cell.In the first load equipment 6a, hatch institute's isolated cells and load expectation material/expectation activeconstituents: here, activeconstituents/material is encapsulated in the liposome, and described liposome adds by lock 7a from activeconstituents/liposome bank 8a.
Therefore, kind of carrier used herein is a liposome, and used liposome prescription can be following liposome:
(1) DPPC 30%, cholesterol 40%, DPPG 30%,
(2) DPPC 30%, cholesterol 40%, DODAC 30%,
(3) DOPE 70%, N-succinyl-DOPE 30%,
(4) DOPE 69.5%, N-succinyl-DOPE 30% and PEG 0.5%, or
(5) cholesterol 10 to 60mol%, Yelkin TTS 10 to 60mol%, 5 to 25% be selected from following electronegative carrier: phosphatidyl one glycerine, phosphatidyl two glycerine, phosphatidyl triglycerin or phosphatidyl four glycerine, phosphoric acid one glycerine cholesterol (cholesterol phosphomonoglycerol) or phosphoric acid oligomerization glycerine cholesterol (cholesterol phosphooligoglycerol).
Described carrier can also be a nm-class core-and-shell particles, and (poly (DL-lactide-co-glycolide) perhaps has the dextran copolymer grafted for example to gather (DL-lactide glycolide multipolymer).The nm-class core-and-shell particles that perhaps has Yelkin TTS lipid core and medicine (for example darubicin or Dx) and triblock copolymer polymer surfaces.Described carrier can also be polymkeric substance-medicine-conjugate, for example darubicin or Dx-PLGA oligomer conjugate.
Material to be encapsulated can be an anthracyclines, for example Dx, darubicin, daunorubicin or related compound, darubicin alcohol (idarubicinol) for example, material perhaps to be encapsulated can also be camptothecine and analogue, bleomycin, cis-platinum and/or angiogenesis inhibitor and/or angiostatin, angiostatin (angiostatin), Endostatin (endostatin), vitaxin, bevacizuma, recentin.
After the described material/activeconstituents of load, in second load step, in the second load equipment 6b, strengthen the cytotoxic effect device function of the separating immune cell that is loaded with material.This by make bi-specific antibody with the isolating cell surface that has been loaded with the immunocyte of material combine (based on corresponding epi-position and/or eptiope) carry out, this by means of the second bank 8b that comprises corresponding bi-specific antibody by second the lock 7b carry out.
In metering facility 6b, can measure the concentration of activeconstituents in the cell of twice load.At last by the Micropump 9 that places outlet 10 upstream sides, modified cell failed in the Huis' the blood flow by exporting 10.
The method of the invention can be combined with all adoptive immunotherapies and/or cellular immunization therapy.
Claims (15)
1, be used for outside people or mammalian body, modifying people or mammalian immune cell, especially effector cell's immune cell modification method,
Wherein in first step, at least a material, preferred active pharmaceutical ingredient through sealing introduced in isolating people or the mammalian immune cell and/or placed on it and
Wherein in second step before or after introducing described at least a material, strengthen the cytotoxic effect device function and/or the target ability of institute's separating immune cell, the target that preferably strengthens described isolating immunocyte is looked for ability.
2, according to the immune cell modification method of claim 1, it is characterized in that:
In order to strengthen cytotoxic effect device function and/or target ability, in described second step, in the genome of described isolating immunocyte, introduce from tumor associated antigen (TAA) the specific immunity cell especially at least a immunocyte acceptor gene of TAA specific T-cells, especially TXi Baoshouti gene (tcr gene), wherein preferably, described at least a immunocyte acceptor gene is introduced by transgenosis.
3, according to the immune cell modification method of each aforementioned claim, it is characterized in that:
In order to strengthen cytotoxic effect device function and/or target ability, in described second step, provide genetically modified or chimeric immunocyte acceptor to described isolating immunocyte, wherein preferably, described isolating immunocyte is cytotoxic T cell (CTL) or natural killer cell (NK cell)
Wherein preferably, described chimeric immunocyte acceptor is the gomphosis immunoglobulin TXi Baoshouti, and/or wherein preferably provides genetically modified or chimeric immunocyte acceptor by transgenosis to described isolating immunocyte.
4, according to the immune cell modification method of each aforementioned claim, it is characterized in that:
In order to strengthen cytotoxic effect device function and/or target ability, in described second step with described isolating immunocyte and at least a antibody and/or its at least a fragment combination, preferably with at least a bi-specific antibody, three-specific antibody, double antibody, three antibody and/or four antibody and/or its fragment combination, wherein preferably, described at least a antibody and/or its fragment are introduced in the described isolating immunocyte and/or are placed on it
And/or wherein preferably, described isolating immunocyte is mono-clonal T cell or polyclone T cell, preferred activatory polyclone T cell.
5, according to the immune cell modification method of each aforementioned claim, it is characterized in that:
In order to strengthen cytotoxic effect device function and/or target ability, the separating immune cell of selecting following particular type in described second step is as described isolating immunocyte: T cell, especially T lymphocyte specific for tumour antigen,
And/or
Wherein said isolating immunocyte is effector cell, T cell, cytotoxic T cell activatory cytotoxic T cell, TALL-104 cell, C CURE 709 cells and/or cytotoxic T cell, T lymphocyte specific for tumour antigen, NK cell NK-92 cell, monocyte and/or the scavenger cell scavenger cell of cells of monocytic origin especially especially especially of autogenous cell, homogeneous variant cell or precursor cell, eventually end differentiation.
6, according to the immune cell modification method of each aforementioned claim, it is characterized in that:
Described isolating immunocyte increases in introducing and/or before settling described at least a material through sealing.
7, according to the immune cell modification method of each aforementioned claim, it is characterized in that:
At least a described material through sealing is used for the treatment of, and it comprises cytostatic medicament and/or angiogenesis inhibitor and/or based on the therapeutical agent of antibody and/or therapeutic DNA and/or based on the therapeutical agent of RNAi,
And/or
At least a described material through sealing is used for diagnosis, comprises especially ferrite nanometer particle of metallicity nano particle, and promptly the tens of approximately nanometers of mean diameter are to the particle of approximate number micron.
8, according to the immune cell modification method of each aforementioned claim, it is characterized in that:
In described second step, hatch isolating immunocyte, preferably hatched about 5 minutes to about 2 hours, to introduce and/or to settle described at least a material through sealing, and/or with the introducing and/or the arrangement of the effector functions that is used to strengthen described isolating immunocyte
And/or
Wherein said at least a material through sealing is introduced described isolating immunocyte by electroporation and/or transfection and/or is settled thereon, and/or be used in wherein said second step to strengthen described isolating immunocyte effector functions introducing and/or settle and undertaken by electroporation and/or transfection
And/or
Wherein said at least a material is encapsulated, so that this material discharged after 25 days at about 1 day.
9, according to the immune cell modification method of each aforementioned claim, it is characterized in that:
Seal described at least a material by following material: liposome, liposome of preferably sealing and/or multilamellar liposome, the liposome that preferably uses alginate, especially Barium alginate to seal; The capsule plastid, virion and/or comprise the microballoon of porous material, preferred poly(lactic acid) or polyglycolic acid-lactic acid copolymer (PGLA), starch, phosphatic rock and/or compacting polymers dextran, agarose, albumin, chitosan, silicon-dioxide or hollow silica particle, polymine, Polyalkylcyanoacrylanano, poly methyl methacrylate particle, nm-class core-and-shell particles, branch-shape polymer and branch fluidized polymer, wherein preferred its core element is amine or sugar, have the branch-shape polymer and the branch fluidized polymer in several identical one-tenth key sites that are used for the shell molecule from the teeth outwards, wherein preferably described shell is through the conversion between acid and the amine of being everlasting.
10, modified people or mammalian immune cell, preferred effector cell,
It comprises and is introduced in people or the mammalian cell and/or at least a material through sealing placed on it, preferred active pharmaceutical ingredient, and
Show enhanced cell toxic effect device function and/or enhanced target ability, preferred enhanced target is looked for ability.
11, according to the modified immunocyte of claim 10, it is characterized in that:
Enhanced cell toxic effect device function and/or enhanced target ability are by with the realization of getting off in the described modified immunocyte: introduce at least a immunocyte acceptor gene from tumor associated antigen (TAA) specific immunity cell, especially TAA specific T-cells in the genome of this immunocyte to be finished, especially TXi Baoshouti gene (tcr gene)
And/or
Enhanced cell toxic effect device function and/or enhanced target ability are by with the realization of getting off in the wherein said modified immunocyte: for immunocyte to be finished provides genetically modified immunocyte acceptor or chimeric immunocyte acceptor,
And/or
Enhanced cell toxic effect device function and/or enhanced target ability are by with the realization of getting off in the wherein said modified immunocyte: immunocyte to be finished and at least a antibody and/or its at least a fragment is combined, preferably combined with at least a bi-specific antibody, three-specific antibody, double antibody, three antibody and/or four antibody and/or its fragment.
12, according to the modified immunocyte of claim 10 or 11, it is characterized in that:
Described immunocyte is effector cell, T cell, cytotoxic T cell activatory cytotoxic T cell, preferred TALL-104 cell or C CURE 709 cells and/or cytotoxic T cell, T lymphocyte specific for tumour antigen, NK cell NK-92 cell, monocyte and/or the scavenger cell scavenger cell of cells of monocytic origin especially especially especially of autogenous cell, homogeneous variant cell or precursor cell, eventually end differentiation.
13, be used for modifying outside people or mammalian body people or mammalian immune cell, especially effector cell's cell modification device, described cell modification device comprises:
Be used to separate described immunocyte at least a device (4) and
At least a device (6,7,8), it is suitable for introducing in the described isolating immunocyte and/or placed on it through the material of sealing at least a, also be suitable for strengthening the cytotoxic effect device function of described isolating immunocyte and/or strengthen its target ability, preferably strengthen its target and look for ability
Wherein said evaluation method selecting optimal equipment ground also comprise be used for introduce and/or settle described at least a material through sealing before and/or strengthening cytotoxic effect device function before fix the device (6) of described isolating immunocyte,
And/or
Wherein preferably, described cell modification device is suitable for implementing according to each immune cell modification method in the claim 1 to 9.
14, the purposes that is used for following purpose according to the cell modification device and/or the immune cell modification method of each aforementioned claim: be used to strengthen the effector functions of people or mammalian immune cell and/or be used for cancer therapy preferred treatment of colon cancer, lymphoma treating, lung cancer therapy, ovarian cancer treatment, breast cancer treatment, brain tumor and/or treatment of pancreatic cancer; Be used for the chronic inflammatory diseases treatment; Be used for treatment of alzheimer, and/or the active of applied immunology or oncology and/or material and/or autonomous target field, and/or be used for diagnosis, as diagnosing tumor, alzheimer's disease diagnosis.
15, be used to prepare the purposes of medicine according to each modified immunocyte in the claim 10 to 12, described medicine is the medicine that is used for cancer therapy, treatment of colon cancer, lymphoma treating, lung cancer therapy, treatment of pancreatic cancer, chronic inflammatory diseases treatment and/or treatment of alzheimer.
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| JP2008034313 | 2008-02-15 | ||
| JP2008034313 | 2008-02-15 | ||
| JP2008181172 | 2008-07-11 |
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Application publication date: 20090819 |