CA2264205A1 - Use of flexible plastic tanks in gene therapy - Google Patents

Abstract

The present invention relates to the use of flexible plastic tanks for extracorporeal operations in gene therapy.

Description

CA 02264205 1999-02-10 or»Use of flexible plastic vessels in gene therapyDescriptionThe present invention concerns the use of flexibleplastic vessels, for example blood bags, in genetherapy.Blood bags are composed of flexible closed plasticvessels which contain at least one inlet or outletopening. They are sold in various sizes by differentcompanies e.g. Baxter, Biotrans, Cobe, Fresenius andMacop arm and are used in hospitals and blood banks forvarious areas of application and for withdrawing wholeblood or bone marrow as well as for leukapheresis. It isknown that blood cells can be stored, frozen andprocessed in the bags and that special instruments areavailable e.g. for washing and centrifugation.In the field of gene therapy there is a great need formethods which allow the provision of the respectiveproducts such as vectors and cells on a large scale in asimple manner and in a form that is safe and easy tohandle. Previously, reaction vessels and devices fromresearch laboratories have been used for genetherapeutic methods whose handling, however, causesconsiderable problems for clinical applications.WO 87/06119 concerns a system for collecting blood whichis suitable for preventing the transmission of pathogensin the blood. This system contains a blood bag forcollecting the blood and a second container for theregulatable introduction of neutralizing reagents forCA 02264205 l999-02- 10pathogens. There is no connection at all for theapplication of blood bags in gene therapy.WO 92/19285 concerns a method for disinfecting a bloodsample contained in a blood bag in order to destroybacteria and viruses in the sample. The disinfectant iscomposed of an anionic surface—active agent, at leastone non—anionic surface—active agent, a stabilizer, twosalts and two phosphates. There is no reference to theapplication of blood bags in gene therapy.EP-A-0 471 947 describes the culture of cells in bloodbags that are permeable to gases. The culture ofhybridoma cells is described. There is no reference tothe use of blood bags in gene therapy.EP—A—0 725 134 describes flexible bioreactors for theextracorporeal culture of cells of therapeutic interest.However, there is no reference to the use of blood bagsin treatment steps for cells in gene therapy.JP-3 007 575 concerns a device for culturing lymphocytescollected from cancer patients in a device whichcomprises a blood bag on a rotatable and pivoted base.There is no reference to an application in gene therapy.WO 96/00782 concerns the culture of immobilized stemcells in blood bags that are coated with a bindingmatrix for the cells. The stem cells are collected froma patient and returned again after culture. A geneticmanipulation of the stem cells in vitro can also takeamended pageCA 02264205 l999-02- 10_2a..place during the culture. However, the method disclosedin WO 96/00782 is limited to stem cells and cannot besimply adapted to other cell types.It is described that tumour-infiltrating lymphocytes(TILs) can be cultured in plastic bags. However,treatment of TILs in plastic bags which goes beyond mereculturing is not disclosed.Hence the object of the present invention was to providenew means and methods in gene therapy which enable asimpler and safer handling of cells and othersubstances.amended pageCA 02264205 l999-02- 10...3.-This object according to the invention is achieved bythe use of flexible plastic vessels to performextracorporeal processing steps in gene therapy. Thepresent application focuses on gene therapy usingsuspended cells and in particular T cells. The cellsgrowing in suspension can be stimulated, transduced,cultured and labelled in flexible plastic vessels.The processing steps in gene therapy that can be carriedout using flexible plastic vessels include for examplefilling them with vectors that are used to introducegenetic material into cells derived from a donor,contacting the vectors with the donor cells in order totransfect or transduce the cells, stimulation or/andculture of suspended cells, in particular of T cells andthe filling or/and elution of cell separation devices.The use of flexible plastic vessels according to theinvention which have at least one opening for suitableconnecting elements, e.g. connecting tubes, enablesextracorporeal treatment of cells derived from a donorto be partially or completely carried out in a closedsystem.The flexible plastic vessels which generally have avolume of 10 - 5000 ml and in particular of 10 - 2000 mlare preferably closed plastic bags which have at leastone opening e.g. an attachment for connecting tubes.These connecting tubes which are preferably composed ofsealable plastic material, enable bags to be connectedand separated using a suitable instrument, e.g. thesterile sealing apparatus from the Terumo Company, insuch a manner that a closed system is always maintained.As a result, biological substances such as cells,viruses etc. can be handled with a considerably reducedrisk of contamination so that even sterile work benchesCA 02264205 l999-02- 10do not have to be used.A first embodiment of the method according to theinvention concerns filling transduction vectors e.g.retroviral vectors as described for example by Kotani etal., (Human Gene Therapy 5 (1994), 19-28) and which areused in gene therapy studies (Anderson, Science 256(1992), 808-813). Retroviral vectors that contain thegenetic information for a modified version of the humanLNGFR (low affinity nerve growth factor receptor)(Malvilio et al., Blood 82 (1993), 1988-1997) areespecially preferred. At present these vectors arefilled into rigid propylene vessels with a screw cap andthe open product is dispensed in sterile work benches.In contrast when blood bags are used according to theinvention, a liquid containing the vectors, e.g. a cellculture supernatant, can be directly filled through afilter e.g. 0.22 pm or 0.45 pm into small bags with avolume of for example 20 - 50 ml via appropriatelyprepared sterile tube connections . After the filling,the transduction vectors are preferably lyophilizedor/and frozen. After sealing the bags it is possible tostore as before at —80°C. It was surprisingly found thatretroviral vectors can be stored for very long periodsin a frozen or lyophilized state in the plastic bagse.g. for more than one—and-awhalf years. The bagcontaining the vectors can then be connected in thehospital to another bag which contains the cells to betransduced. In this process all steps are carried out ina closed system which almost completely excludes a riskof contamination during harvesting and filling.A second embodiment of the method according to theinvention is the transduction of cells. For transductionthe suspended donor cells contained in a blood bag whichCA 02264205 l999-02- 10are preferably T cells or subpopulations thereof orperipheral blood lymphocytes obtained by leukapheresisor antigen-presenting cells such as tumour cells arecoupled with a bag containing the transduction vectorand both volumes are combined in this manner. Thetransduction then takes place in this combined volumei.e. the cells are genetically changed by the vector.The subsequent steps such as washing and centrifugingthe cells can be carried out with the aid ofcommercially available apparatus and preferably alsousing a bag system.When using flexible plastic vessels for transducing Tcells, the transduction rate can be improved bycentrifuging the plastic vessels. Surprisingly apronounced increase in the efficiency of thetransduction was found when the bags were not placed asusual in a swing—out rotor with the bottom facingdownwards but are arranged such that the cells candisperse during the centrifugation over one of thelargest available surfaces. For this purpose the bagscan for example be placed on one of their two sides. Itis also possible to pile several bags on top of oneanother in the rotor. In this case the bags can liedirectly on top of one another or be placed in a specialholder which is composed of parallel plates.A third embodiment of the method according to theinvention concerns the multiplication of suspendedcells, in particular the multiplication of T cells orantigen-presenting cells. This multiplication of T cellsoccurs after stimulation or/and transduction and can becarried out in gas-permeable bags that are known in theprior art for producing thrombocytes. The culture iscarried out in CO2 incubators optionally with the use ofCA 02264205 l999-02- 10shaking devices. In the cell multiplication the culturecan be extended as required by coupling bags filled withfresh medium. An aliquot can be withdrawn in a sterilemanner after connecting a small bag to the culture bagin order to take a sample for cell counting in a cellchamber or to analyse components of the medium. Afterthe desired cell count has been reached the cells canthen be centrifuged in bags, taken up in a small volumeand prepared for the transplantation.A fourth preferred embodiment of the invention is theuse of flexible plastic vessels for filling or/andeluting cell separation devices e.g. cell separationcolumns (de Wynter et al., Stem Cells 13 (1995), 524-532). In the separation the blood bags can be used tofill the system as well as to withdraw the separatedcells. In order to make a completely closed system ofthe whole process, the preparation of the cells e.g.washing, centrifuging, adding antibodies etc. is alsopreferably carried out within bags. A particularlypreferred cell separation device is the avidin—biotinimmunoadsorption column described by Auditore-Hargreaveset al. (Bioconjugate Chem. 5 (1994), 287-300).A fifth preferred embodiment of the present invention isthe culture or/and stimulation of suspended cells, inparticular of T cells e.g. in graft—versus-leukaemiamethods, for adoptive immunotherapy, for the preparationof vaccination cell lines or for the stimulation ofkiller T cells. Also in this case the use of blood bagsavoids an open handling of the cells and thus a risk ofcontamination.For use in graft-versus-leukaemia methods the T cellsCA 02264205 l999-02- 10that have been collected from a healthy donor do nothave to be stimulated separately. A transduction iscarried out to label the cells and preferably with aretroviral vector as described above.In adoptive immunotherapy the T cells are stimulated forexample by tumour cells which have been collected fromthe patient. The isolation and possible multiplicationof the tumour cells depends on the type of the tumour.For the stimulation the tumour cells are added directlyto the bag containing the T cells of the donor.Afterwards all subsequent steps such as transductionwith a vector, multiplying the T cells and cellseparation can be carried out in bags i.e. in a closedsystem.Suitable vaccination cell lines are genetically modifiedtumour cell lines e.g. derivatives of the RCC-26 cellline (Schendel et al., J. Immunol. 151 (1993), 4209-4220). These cell lines which are for example derivedfrom a human renal carcinoma can be administered topatients with tumours, e.g. with kidney tumours, for animmune response to metastasising kidney tumour cells. Inthis case the vaccination cell lines are multiplied inbags and can be subsequently directly administered tothe patients.T cells stimulated with inactivated human B cells canalso attack tumour cells. In this case the inactivatedcells can be filled into small blood bags and directlyconnected to blood bags containing the T cells of thepatient. The further multiplication of the T cells thenproceeds as described above. An advantage of thisprocedure compared to previous methods in which theCA 02264205 l999-02- 10stimulating cells were frozen in cryotubes after cultureand inactivation is that it is possible to work in acompletely closed system.Furthermore coated plastic bags can also be used in themethod according to the invention. This coating can forexample be achieved using tumour cells or parts oftumour cells e.g. cell membrane vesicles that are freeof cell nuclei. In this manner it is possible tostimulate T cells without the T cells coming intocontact with live tumour cells. In addition the surfacesof the plastic bags can be coated with other substancese.g. proteins in order to further improve the T cellstimulation. The plastic surface can for example becoated according to the techniques described in theEuropean Patent Application 96 108 288.0 i.e. byimmobilization of cells or vesicles which contain partsof the natural surface of a cell. The cell or thevesicle can be modified with a first partner of abiological binding pair and can be immobilized on thesolid phase by means of the second partner of thebinding pair. Examples of suitable binding pairs arehigh affinity binding pairs such as biotin/streptavidin,sugar/lectin, CD/anti-CD antibody, MHC molecules orhapten/anti-hapten antibody.Yet a further subject matter of the present invention isa method for adoptive immunotherapy comprising theextracorporeal steps: stimulation of T cells,transduction with a vector, multiplying the T cells andseparation in order to obtain the desired T cellswherein this method is characterized in that at leastone of the processing steps is carried out using aflexible plastic bag. Preferably all steps are carriedout in a closed system.CA 02264205 l999-02- 10Yet a further subject matter of the present invention isa plastic bag, in particular a blood bag with sealableinlet or/and outlet openings which contains atransduction vector preferably a retroviral vector in asuitable buffer. The transduction vector can be storedfor a surprisingly long period in a frozen orlyophilized state. Alternatively or in addition theplastic bags can also contain stimulating or/andantigen-presenting T cells. Finally the invention alsoconcerns plastic bags which contain T cells geneticallymodified with a transduction vector in a suitablebuffer.Figure 1 is a chart showing the use of plastic bags inex-vivo gene therapy. The T cells collected from thedonor are stimulated by treatment with stimulatingagents. This is followed by a transduction preferablywith viral vectors. After the subsequent culture, thecells are labelled with an antibody and separated bymeans of a separation column. This can in turn befollowed by a culture. Finally the T cells aretransplanted into a patient.Some of the individual processing steps can be omittedor some new steps can be added according to needs. Thusthe culture after separation is not always necessary.Instead culture steps can be carried out at other stagesor it may be necessary to store the cells in deep frozenbags.The figure shows in particular a flow diagram for agraft—versus-leukaemia method or for an adoptiveimmunotherapy. In both these applications the retroviralvectors that are filled into the bags are used.CA 02264205 l999-02- 10-10.-When culturing vaccination cell lines, only filling withthe cells is used and the cells are subsequentlydirectly injected into the patient e.g. via a cannula.When ex-vivo stimulated killer T cells are expanded theprocessing steps of leukapheresis, stimulation, cultureand transplantation are used.As can be seen in the figure the use of flexible plasticvessels enables all processing steps to be carried outin a closed system which considerably simplifies thehandling and almost completely excludes a risk ofcontamination.

Claims (21)

Claims

1. Use of flexible plastic vessels for filling transduction vectors in order to carry out extracorporeal processing steps in gene therapy.

2. Use as claimed in claim 1 for filling retroviral vectors.

3. Use as claimed in claim 1 or 2, wherein the transduction vectors are lyophilized or/and frozen after the filling.

4. Use of flexible plastic vessels for the extracorporeal transduction of suspended cells in gene therapy.

5. Use as claimed in claim 4 for the transduction of leukocytes or subpopulations thereof or of antigen-presenting cells.

6. Use as claimed in claim 4 or 5, wherein the transduction includes a centrifugation step.

7. Use as claimed in claim 6, wherein the plastic vessels are arranged in such a way that the cells can disperse over one of the largest available surfaces.

8. Use of flexible plastic vessels for the extracorporeal filling or/and elution of cell separation devices in gene therapy.

9. Use of flexible plastic vessels for the extracorporeal stimulation and optionally culture of suspended cells in gene therapy.

10. Use as claimed in claim 9 for the stimulation and optionally culture of T cells for graft-versus-leukaemia processes.

11. Use as claimed in claim 9 for the stimulation and optionally culture of T cells for adoptive immunotherapy.

12. Use as claimed in claim 9 for the stimulation and optionally culture of vaccination cells.

13. Use as claimed in claim 9 for the stimulation and optionally culture of killer T cells.

14. Use as claimed in one of the claims 9 - 13, wherein a stimulation is carried out in a coated plastic bag.

15. Use as claimed in one of the claims 1 to 14, wherein the flexible plastic vessels have a volume of 10 - 5000 ml.

16. Use as claimed in one of the claims 1 to 15, wherein the plastic vessels are closed bags which contain at least one attachment for sealable connections.

17. Method for adoptive immunotherapy or graft-versus-leukaemia methods, comprising the extracorporeal steps: stimulation of T cells, transduction with a vector, multiplication of the T cells and separation, wherein at least one of the processing steps: stimulation of T cells, transduction with a vector and separation, is carried out in a flexible plastic bag.

18. Method as claimed in claim 17, wherein all steps are carried out in a closed system.

19. Plastic bag, wherein it contains a transduction vector in a suitable buffer.

20. Plastic bag as claimed in claim 19, wherein it contains a retroviral vector.

21. Plastic bag as claimed in claim 19 or 20 in a frozen or lyophilized state.