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WO1997038707A1 - Procede et dispositif servant a administrer des molecules provoquant l'apoptose - Google Patents

Procede et dispositif servant a administrer des molecules provoquant l'apoptose Download PDF

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Publication number
WO1997038707A1
WO1997038707A1 PCT/US1997/006409 US9706409W WO9738707A1 WO 1997038707 A1 WO1997038707 A1 WO 1997038707A1 US 9706409 W US9706409 W US 9706409W WO 9738707 A1 WO9738707 A1 WO 9738707A1
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Prior art keywords
apoptosis
fasl
cells
molecule
ofthe
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PCT/US1997/006409
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English (en)
Inventor
Adriano Fontana
Patrick Aebischer
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Cytotherapeutics, Inc.
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Priority to AU26740/97A priority Critical patent/AU2674097A/en
Publication of WO1997038707A1 publication Critical patent/WO1997038707A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0085—Brain, e.g. brain implants; Spinal cord
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/13—Tumour cells, irrespective of tissue of origin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0087—Galenical forms not covered by A61K9/02 - A61K9/7023
    • A61K9/0092—Hollow drug-filled fibres, tubes of the core-shell type, coated fibres, coated rods, microtubules or nanotubes

Definitions

  • This invention relates to a method and device for delivery of Fas ligand using encapsulated cells.
  • Fas/APO-1 CD95
  • FasL its ligand
  • the Fas receptor is typically expressed in mitotic tissues, but not in post-mitotic tissues, such as the mammalian central nervous system ("CNS").
  • CNS central nervous system
  • astrocytes, oligodendrocytes, and neurons do not express APO-1 (the Fas receptor) and are resistant to FasL mediated apoptosis.
  • APO-1 the Fas receptor
  • tumor cells including glioblastomas, express high levels of Fas receptor.
  • antigen-activated T cells and macrophages express high levels of Fas receptor.
  • the Fas receptor protein is also expressed in the thymus, liver, heart and ovary.
  • the Fas receptor is up-regulated by IFN- ⁇ and combinations of IFN- ⁇ and TNF- ⁇ in human B cells. Any lymphocyte transformed with human T cell leukemia, human immunodeficiency or Epstein Barr virus also expresses high levels of Fas receptor.
  • devices and methods are provided for delivery of FasL, or other suitable apoptosis-inducing molecule, to a suitable implant site in a patient, preferably directly to the CNS.
  • This invention provides novel methods and devices for delivery of a source of FasL, or other suitable apoptosis-inducing molecule.
  • one or more devices each containing between about 10 3 - 10 7 genetically modified cells surrounded by a permeable membrane, is implanted into a patient.
  • the encapsulated cells provide for release of FasL or other suitable apoptosis inducing molecule.
  • the device or devices should produce a level of FasL (or other suitable apoptosis-inducing molecule) sufficient to induce apoptosis ofthe target cells or tissue.
  • one or more devices are implanted into the CNS. Delivery to a localized area in the CNS is achieved, e.g., by implanting encapsulated cells into the brain parenchyma. Delivery over a more widespread area ofthe CNS is accomplished by implanting encapsulated cells into the brain ventricles or cerebrospinal fluid ("CSF') space.
  • CSF' cerebrospinal fluid
  • the device contains a permeable membrane that prevents immunologic rejection ofthe encapsulated cells and interposes a physical barrier between the cells and the patient. Moreover, each device may be retrieved from the patient.
  • FIG. 1 Supernatant of Neuro-2a FasL transfectants contains Fas/APO-1 specific cytotoxic activity.
  • Amplification ofthe full-length FasL transgene by RT-PCR yields a band of 875 bp in Neuro-2a cells transfected with the murine FasL gene (lane 2) but not in the Neuro-2a mock transfectants (lane 1).
  • PCR with the same primers was carried out using the BCMGS neo vector containing the FasL cDNA (lane 4) or the BCMGS neo plasmid without insert (lane 3) as a template. Size markers are indicated in base pairs (bp) on the left.
  • sFas sFas (500 ng/ml) (closed triangles) but not sTNF-R (open triangles) completely inhibited the cytotoxic effect ofthe FasL supernatant on Yac-1 cells.
  • Yac-1 cells were incubated with increasing concentrations ofthe anti-Fas antibody Jo2 (closed squares), Jo2 plus protein A (10 ⁇ g/ml) (closed triangles) or control hamster IgG (open squares) and viability was determined as described above.
  • FIG. 2 Detection of FasL in the supernatant of Neuro-2a FasL transfectants.
  • Concentrated supernatants obtained from Neuro-2a FasL transfectants (lane 1) or Neuro-2a mock transfectants (lane 2) were subjected to SDS-PAGE and subsequent Western blotting using anti-Fas/ APO- 1 ligand antibodies.
  • FIG. 3 The FasL induces apoptosis of Yac-1 cells in vivo.
  • Figure 4 Systemic but not local application of FasL-containing supernatant induced liver apoptosis. Groups of weight-matched mice were injected with:
  • Liver sections were prepared 16, 2, 2 and 4 h after injection, respectively. Sections were stained with haematoxylin and eosin, magnification is 298 x in all cases. Normal liver tissue is seen in (a) and (c), whereas the hepatocytes in (b) and (d) carry pyknotic and fragmented nuclei and are surrounded by red blood cells.
  • This invention relates to delivery of FasL or other suitable apoptosis- inducing molecule using encapsulated cells.
  • localized systemic delivery of FasL is contemplated.
  • This embodiment may provide a therapy for human tumors or cancers.
  • One benefit is the reduction or elimination of side effects resulting from systemic injections or other conventional administration routes. Such side effects include hepatoxicity.
  • FasL apoptosis-inducing molecule
  • this invention contemplates capsular delivery of an apoptosis-inducing molecule to the CNS, in a sufficient dosage to result in apoptosis ofthe target tumor or cancer, but not ofthe surrounding non-tumorigenic or non-cancerous tissue.
  • the dosage of FasL provided is sufficient to achieve destruction of activated T cells or macrophages.
  • Local application of FasL may be useful in the treatment of primary brain tumors, e.g., malignant glioma, and lymphoma ofthe CNS, as well as metastatic tumors provided they are Fas/APO-1 positive.
  • CSF cerebrospinal fluid
  • FasL is contemplated for the treatment of glioblastoma.
  • Glioma cells express Fas/APO-1 and are susceptible to anti-Fas/ APO- 1 -mediated apoptosis in vitro.
  • Fas/ APO- 1 expression is not detectable in normal brain tissue including neurons and glial cells, with the exception of endothelial cells.
  • FasL in vitro cultured neurons and oligodendrocytes treated with IFN-gamma and TNF- ⁇ resist FasL mediated killing.
  • intrathecal or intratumoral application of FasL may be a promising approach for the treatment of Fas/APO-1 expressing malignant tumor cells.
  • the devices and methods of this invention may also be used as part of al therapy regime that includes other anti-cancer or anti-tumor treatments, such as administration of chemotherapeutic agent(s).
  • the combined treatment of an apoptosis-inducing molecule with a chemotherapeutic agent(s) may provide an enhanced "killing" effect on the target tumor or cancer tissue.
  • This invention also contemplates the use of encapsulated cells that produce an apoptosis-inducing molecule (e.g. Fas-L) for the treatment of multiple sclerosis (MS), and other forms of immune-mediated demyelinating diseases.
  • apoptosis-inducing molecule e.g. Fas-L
  • MS multiple sclerosis
  • a FAS-ligand expressing cell line we can apply soluble FAS-ligand delivery to the CSF space for the treatment of autoimmune dysfunction (for example, multiple sclerosis ⁇
  • autoimmune dysfunction for example, multiple sclerosis ⁇
  • cytotoxic lymphocytes inciuding T-cells, neutrophils, macrophages, and B-cells are activated and attack the myelin sheaths of CNS axons.
  • Activated macrophages and lymphocytes express high levels of FAS/APO-1 receptor and are susceptible to FAS-ligand mediated apoptotic killing.
  • activated immune ceils traffic into the CNS to attack myelin sheaths, they will be induced to undergo apoptotic death in response to FAS-ligand expression.
  • CNS neurons, astrocytes and oligodendrocytes do not normally express the APO- 1 /FAS receptor, only the activated immune cells will be affected. This may be analogous to the environment created in the eye and testis where high levels of FAS-ligand are expressed and serve to immunosuppress these environments (Griffith et al., Science. 270, pp. 1 189-1192 (1995); Suda et al., Cfill, 75, pp. 1169-1 178 (1993)).
  • FAS and its receptor are members ofthe TNF gene family which includes TNF NGFR (low affinity), CD40, OX40, 4-13B, CD27 and CD30 (Suda et al., CeJl, 75, pp. 1 169- 1178 ( 1993); Rabizadeh and Bredesen, Dev Neurosci .. 16, pp. 207-211 (1994)). TNF and FAS share similar death domains. Interestingly, low affinity NGF receptor may be directly involved in mediating apoptotic actions but in the reverse fashion when the LNGF receptor is not occupied (Rabizadeh and Bredesen, Dev Neurosci .. 16, pp. 207-211 (1994)).
  • LNGF receptor may act directly to prevent apoptosis when occupied with NGF (Ibid.): Dobrowsky et al., Science, 265, pp. 1596-1599 (1994)) or in conjunction with other Trk receptors and BDNF, NT-3 or NT4/5 (Hantzopoulos et al., Neuron. 13, pp. 187-201 (1994)).
  • Drug can be delivered to the CNS directly, which will potentially reduce unwanted peripheral side effects including the toxicity expected for systemic delivery in human patients;
  • these cells should have advantages over pump delivery of drug stores, where drug is continuously degraded (or its potency drops) but is not continuously replenished.
  • FasL through cell therapy (i.e., via transplantation of encapsulated cells that produce or that have been genetically-engineered to release FasL) circumvents the majority of these problems.
  • FasL is approximately 40,000 molecular weight glycoprotein and member ofthe tumor necrosis factor family of proteins.
  • the murine gene is composed of five (5) exons and four (4) introns, with an open reading frame coding for 279 amino acids.
  • FasL contains a region of 22 hydrophobic amino acids in the middle ofthe molecule, suggesting that FasL is a Type II membrane protein.
  • the protein does not have a hydrophobic signal sequence.
  • FasL The genes encoding murine and human FasL are known.
  • the preferred FasL is the naturally-occurring murine or human ligand, such as described in EP 675 200, WO 95/32627, Cheng et al., Science, 263, pp. 1759-62 (1994), Suda et al., Cell, 75, pp. 1169-78 (1993) and Takahashi et al., Int. Immunolog y . 6, pp. 1567 (1994), each of which is incorporated herein by reference.
  • FasL The entire membrane form of FasL, as well as modified, truncated and mutein forms of FasL, as well as active fragments of FasL (i.e., those fragments of FasL having biological activity) are also known. All of these forms of FasL are contemplated by this invention. Each of these documents are specifically incorporated herein by reference.
  • FasL is the preferred ligand
  • any other suitable apoptosis- inducing molecule may also be delivered using the methods and devices of this invention.
  • lymphotoxin, CD40L and TNF- ⁇ may also useful in the methods and devices of this invention.
  • a gene of interest i.e., a gene that encodes a suitable biologically active molecule, e.g., FasL
  • a suitable biologically active molecule e.g., FasL
  • FasL a biologically active molecule
  • the expression vector containing the gene of interest may then be used to transfect the desired cell line.
  • Standard transfection techniques such as calcium phosphate co-precipitation, DEAE-dextran transfection or electroporation may be utilized.
  • Commercially available mammalian transfection kits may be purchased from e.g., Stratagene.
  • a wide variety of host/expression vector combinations may be used to express the gene encoding FasL, or other biologically-active molecule of interest.
  • Suitable promoters include, for example, the early and late promoters of S V40 or adenovirus and other known non-retroviral promoters capable of controlling gene expression.
  • Useful expression vectors may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences, such as various known derivatives of SV40 and known bacterial plasmids, e.g., pUC, pBlueScriptTM plasmids from E. coli including pBR322, pCRl , pMB9, pUC, pBlueScriptTM and their derivatives.
  • segments of chromosomal, non-chromosomal and synthetic DNA sequences such as various known derivatives of SV40 and known bacterial plasmids, e.g., pUC, pBlueScriptTM plasmids from E. coli including pBR322, pCRl , pMB9, pUC, pBlueScriptTM and their derivatives.
  • Expression vectors containing the geneticin (G418) or hygromycin drug selection genes are also useful. Expression vectors containing the zeocin drug selection gene are also contemplated. Examples of expression vectors that can be employed are the commercially available pRC/CMV, pRC/RSV, and pCDNAlNEO (InVitrogen).
  • the viral promoter regions directing the transcription ofthe drug selection and biologic genes of interest are replaced with one ofthe above promoter sequences that are not subject to the down regulation experienced by viral promoters within the CNS.
  • the GFAP promoter would be employed for the transfection of astrocytes and astrocyte cell lines, the TH promoter would be used in PC 12 cells, or the MBP promoter would be used in oligodendrocytes.
  • the pNUT expression vector is used. Baetge et al., PNAS. 83, pp. 5454-58 (1986).
  • the pNUT expression vector can be modified such that the DHFR coding sequence is replaced by the coding sequence for G418 or hygromycin drug resistance.
  • the SV40 promoter within the pNUT expression vector can also be replaced with any suitable constitutively expressed mammalian promoter, such as those discussed above.
  • the human FasL gene is expressed from the pNUT vector, which contains the cDNA ofthe mutant DHFR and the entire pUC18 sequence including the polylinker (Baetge et al., supra).
  • the DHFR transcription unit is driven by the SV40 promoter and fused at its 3' end with the hepatitis B virus gene polyadenylation signal (approximately 200 bp 3' untranslated region) to ensure efficient polyadenylation and maturation signals.
  • the FasL gene is expressed behind the mouse metallothionein I promoter and utilizes the human growth hormone polyadenylation signal sequence for 3' processing.
  • This invention also contemplates use of a "suicide" gene in the transformed cells.
  • the cell carries the HSV-TK (he ⁇ es simplex virus thymidine kinase) gene as a safety measure, permitting the host cell to be killed in vivo by treatment with ganciclovir.
  • HSV-TK simplex virus thymidine kinase
  • a "suicide” gene is known in the art. See, e.g., Anderson, published PCT application WO 93/10218, Hamre, published PCT application WO 93/02556.
  • the recipient's own immune system provides a first level of protection from adverse reactions to the implanted cells.
  • the presence ofthe TK gene (or other suicide gene) in the expression construct adds an additional level of safety to the recipient ofthe implanted cells.
  • the He ⁇ es Simplex Virus Thymidine kinase (HSV-TK) gene is inserted into the FasL vector construct.
  • This suicide gene was included to allow elimination ofthe transfected cells upon ganciclovir administration.
  • the 1800 bp fragment can be inserted into the Notl site ofthe pNUT vector.
  • a typical dosage of ganciclovir required to "kill" the cells in vivo is approximately 5 mg/kg.
  • One preferred cell chosen for the gene transfer technique are
  • Neuro-2A cells While Neuro-2A cells are one preferred cell, a wide variety of cells may be used. It is required that the engineered cell does not express Fas/APO-1 receptor to ensure lack of "self-killing" and to ensure a high level of expression of FasL. For example, mouse Neuro-2A cells, do not express the Fas/APO-1 receptor.
  • the cells contemplated include well known, publicly available immortalized (and conditionally immortalized) cell lines as well as dividing primary cell cultures.
  • suitable publicly available cell lines include, Chinese hamster ovary (CHO), mouse fibroblast (L-M), NIH Swiss mouse embryo (NTH/3 T3), African green monkey cell lines (including COS-1, COS-7, BSC-1, BSC-40, BMT-10 and Vero), rat adrenal pheochromocytoma (PC 12 and PC 12 A), AT3, rat glial tumor (C6), astrocytes and other fibroblast cell lines, ⁇ -TC cells, Hep-G2 cells, and myoblasts (including C2C12 cells).
  • Primary cells that may be used include, neurospheres, neural progenitor cells and neural stem cells derived from the CNS of mammals (Richards et al., PNAS 89, pp. 8591-8595 (1992); Ray et al., PNAS 90, pp. 3602-3606 (1993)), primary fibroblasts, Schwann cells, astrocytes, and oligodendrocytes and their precursors, and the like. To the extent that any ofthe foregoing cells may express the Fas/APO-1 receptor, the cells may be genetically modified to "knock out" expression of that receptor.
  • Cell lines offer several advantages including unlimited availability, the possibility of rapid screening in vitro for the presence of pathogens from which cell banks are established, and the suitability for stable gene transfer using non-viral- based recombinant DNA techniques.
  • Both allogeneic and xenogeneic cell 5 may be used.
  • Use of a cell line of xenogeneic origin provides an additional advantage since the transplanted cells are more likely to be rejected by the host immune system in the event of device breakage.
  • the cells are surrounded with a membrane which permits the diffusion of small molecules such as nutrients and trophic factors into and out of the polymer envelope, while excluding larger molecules ofthe immune system (antibodies, complement, etc.).
  • Increased expression can be achieved by increasing or amplifying the copy number ofthe transgene encoding FasL or other suitable biologically active molecule(s), using amplification methods well known in the art.
  • amplification methods include, e.g., DHFR amplification (see, e.g., Kaufman et al., United States patent 4,470,461) or glutamine synthetase ("GS”) amplification (see, e.g., United States patent 5, 122,464, and European published application EP 338,841).
  • the pNUT-FasL expression vector is transfected into Neuro-2A cells using a standard calcium phosphate transfection procedure and selected with increasing concentrations of methotrexate (1 to 200 ⁇ M) over 8 weeks to produce stable amplified cell lines. Following this selection, the engineered cells are maintained in vitro in 50-200 ⁇ M methotrexate.
  • FasL (or other apoptosis-inducing molecule) is continuously produced by the cells. While we prefer continuous production, we also contemplate regulated production ofthe desired apoptosis- inducing molecule. We also contemplate co-delivery of other molecules. For example, in the treatment of human tumors or cancers, co- delivery of FasL with one or more of lymphotoxin, TNF- ⁇ , and IFN- ⁇ , is contemplated.
  • FasL In the treatment of immune-mediated diseases, we contemplate co- delivery of FasL with neural growth factors, cytokines, hormones, and neurotransmitters (including peptide neurotransmitters).
  • co-delivery of IGF- 1, CNTF, GDNF, BDNF, NGF, NT-3, NT-4/5, NT-6, IFN- ⁇ , IFN- ⁇ , DL-4, IL-6, IL-10, CT-1, neuturin, persephin, TGF- ⁇ 's, EGF and FGF is contemplated. It will be appreciated that any combination ofthe foregoing molecules may be co- delivered with FasL.
  • Co-delivery can be accomplished in a number of ways.
  • Cells may be transfected with separate constructs containing the genes encoding the described molecules.
  • cells may be transfected with a single construct containing two or more genes. Multiple gene expression from a single transcript is preferred over that from multiple transcription units.
  • One approach for achieving expression of multiple genes from a single eukaryotic transcript takes advantage ofthe 5' non- translated region in encephalomyocarditis virus (EMCV) known as the internal ribosome entry site ("IRES").
  • EMCV encephalomyocarditis virus
  • IRES internal ribosome entry site
  • the IRES allows internal binding of ribosomes and translation of a downstream gene or genes. See, e.g., Macejak, Njtuie, 353, pp.
  • a patient may be implanted with a device device containing a first cell type (e.g., FasL-secreting Neuro-2A cells). If after time, the patient develops an immune response to that cell type, the device can be retrieved, or explanted, and a second device can be implanted containing a second cell type (e.g., FasL-secreting C2C12 cells). In this manner, continuous provision ofthe therapeutic molecule is possible, even if the patient develops an immune response to one ofthe encapsulated cell types.
  • a first cell type e.g., FasL-secreting Neuro-2A cells
  • Encapsulation hinders elements ofthe immune system from entering the device, thereby protecting the encapsulated cells from immune destruction.
  • This technology increases the diversity of cell types that can be employed in therapy.
  • the semipermeable nature ofthe device membrane also permits the molecule of interest to easily diffuse from the device into the surrounding host tissue. This technique prevents the inherent risk of tumor formation and allows the use of unmatched human or even animal tissue, without immunosuppression ofthe recipient.
  • the implant may be retrieved if necessary or desired. It is both undesirable and expensive to maintain a patient in an immunosuppressed state for a substantial period of time. Such retrievability may be essential in many clinical situations.
  • Tl Type 1
  • T2 Type 2
  • Tl/2 Type 1/2
  • Type 4 Type 4
  • a biocompatible device means that the device, upon implantation in a host mammal, does not elicit a detrimental host response sufficient to result in the rejection ofthe device or to render it inoperable, for example through degradation.
  • an immunoisolatory device means that the device upon implantation into a mammalian host minimizes the deleterious effects ofthe host's immune system on the cells within its core or with the outer surface ofthe device.
  • biocompatible devices are suitable for delivery of molecules according to this invention. Such devices will allow for the passage of metabolites, nutrients and therapeutic substances while minimizing the detrimental effects ofthe host immune system.
  • the device of this invention will be similar to those described in Aebischer et al., PCT publication WO 92/19195, inco ⁇ orated herein by reference.
  • Useful biocompatible devices comprise (a) a core which contains a cell or cells, either suspended in a liquid medium or immobilized within a biocompatible matrix (e.g., a hydrogel or extracellular matrix), and (b) a surrounding or peripheral region of permeable matrix or membrane (jacket) which does not contain isolated cells, which is biocompatible.
  • a biocompatible matrix e.g., a hydrogel or extracellular matrix
  • the jacket may be microporous or permselective.
  • the core ofthe polymer device is constructed to provide a suitable local environment for the continued viability and function ofthe cells isolated therein.
  • cells or cell lines are most advantageously isolated within a device having a liquid core.
  • cells can be isolated within a device whose core comprises a nutrient medium, optionally containing a liquid source of additional factors to sustain cell viability and function.
  • the inner wall ofthe device may be coated with one or more molecules that facilitate cell adhesion. See, e.g., PCT/US95/09281.
  • the core may be composed of a matrix formed by a hydrogel which stabilizes the position ofthe cells.
  • hydrogel herein refers to a three dimensional network of cross-linked hydrophilic polymers.
  • the network is in the form of a gel, substantially composed of water, preferably but not limited to gels being greater than 90% water.
  • compositions which form hydrogels fall into three classes.
  • the first class carries a net negative charge (e.g., alginate).
  • the second class carries a net positive charge (e.g., collagen and laminin).
  • Examples of commercially available extracellular matrix components include MatrigelTM and VitrogenTM. Fibroblasts generally survive well in a positively charged matrix and are thus suitably enclosed in extracellular-matrix type hydrogels.
  • the third class is net neutral in charge (e.g., highly crosslinked polyethylene oxide, or polyvinylalcohol).
  • any suitable matrix or spacer may be employed within the core, including agarose (including agarose derivatized with adhesion molecule fragments), precipitated chitosan, synthetic polymers and polymer blends, microcarriers and the like, depending upon the growth characteristics ofthe cells to be encapsulated.
  • the devices are immunoisolatory.
  • the surrounding or peripheral region ofthe device should confer protection ofthe cells from the immune system ofthe host in whom the device is implanted, by preventing harmful substances ofthe host's body from entering the core ofthe vehicle, and by providing a physical barrier sufficient to prevent detrimental immunological contact between the isolated cells and the host's immune system.
  • the thickness of this physical barrier can vary, but it will always be sufficiently thick to prevent direct contact between the cells and/or substances on either side ofthe barrier.
  • the thickness of this region generally ranges between 5 and 200 microns; thicknesses of 10 to 100 microns are preferred, and thickness of 20 to 75 microns are particularly preferred.
  • Types of immunological attack which can be prevented or minimized by the use ofthe instant vehicle include attack by macrophages, neutrophils, cellular immune responses (e.g. natural killer cells and antibody-dependent T cell-mediated cytolysis (ADCC), and humoral response (e.g., antibody-dependent, complement-mediated cytolysis).
  • immunoisolatory devices allow the implantation of xenogeneic cells or tissue, without a concomitant need to immunosuppress the recipient.
  • the exclusion of IgG from the core ofthe vehicle is not the touchstone of immunoisolation, because in most cases IgG alone is insufficient to produce cytolysis ofthe target cells or tissues.
  • Using immunoisolatory macrodevices it is possible to deliver needed high molecular weight products or to provide metabolic functions pertaining to high molecular weight substances, provided that critical substances necessary to the mediation of immunological attack are excluded from the immunoisolatory device.
  • MWCO Nominal molecular weight cutoff (MWCO) values up to 1000 kD are contemplated.
  • the MWCO is between 50-700 kD.
  • the MWCO is between 70-300 kD.
  • the device jacket can be formed from any suitable biocompatible permeable material, including, e.g, hydrogels. See, e.g., WO92/19195.
  • the device can be any configuration appropriate for maintaining biological activity and providing access for delivery ofthe product or function, including for example, cylindrical, rectangular, disk-shaped, patch-shaped, ovoid, stellate, or spherical. Moreover, the device can be coiled or wrapped into a mesh ⁇ like or nested structure. If the device is to be retrieved after it is implanted, configurations which tend to lead to migration ofthe devices from the site of implantation, such as spherical devices small enough to migrate in the patient, are not preferred. Certain shapes, such as rectangles, patches, disks, cylinders, and flat sheets offer greater structural integrity and are preferable where retrieval is desired. For local delivery of FasL, e.g., in the treatment of solid tumors or cancers, a disk shape may be preferred.
  • the device has a tether that aids in retrieval.
  • tethers are well known in the art.
  • macrodevices have a core of a preferable minimum volume of about 1 to lO ⁇ l and depending upon use are easily fabricated to have a volume in excess of 10O ⁇ l.
  • the fiber in a hollow fiber configuration, preferably has an inside diameter of less than 1500 microns, more preferably approximately 300-600 microns. If a semi-permeable membrane is used, the hydraulic permeability is preferably in the range of 1-100 mls/ ⁇ ii/M /mmHg, more preferably in the range of
  • the glucose mass transfer coefficient ofthe device is preferably greater than 10 cm/sec, more preferably greater .4 than 10 cm/sec.
  • both semi-permeable membranes and microporous membranes are contemplated for forming the device jacket.
  • Biocompatible, semi-permeable hollow fiber membranes, and methods of making them, are disclosed in United States patents 5,284,761, 5,158,881, and WO 95/0542, all herein inco ⁇ orated by reference.
  • the device is formed from a polyether sulfone membrane, such as those described in United States Patent Nos. 4,976,859 and 4,968,733 (describing both semi-permeable and microporous membranes), each herein inco ⁇ orated by reference. See also Aebischer et al., J. Biomed. Engin. ⁇ 1 13, p. 178 (1991).
  • any suitable method of sealing the devices may be used, including the employment of polymer adhesives and/or crimping, knotting and heat sealing. These sealing techniques are known in the art.
  • any suitable "dry” sealing method can also be used. In such methods, a substantially non-porous fitting is provided through which the cell-containing solution is introduced.
  • the device is sealed.
  • a mammalian host, recipient, patient, subject or individual preferably a primate, most preferably a human.
  • delivery of FasL is used as a potential treatment for glioblastoma.
  • Glioblastomas are the most common form of malignant primary brain tumor. For people 15-34 years of age, it is the third leading cause of death from cancer. Little progress has been made in the treatment of glioblastoma.
  • glioblastomas Conventional treatment consists of surgery to remove the bulk ofthe tumor followed by irradiation to ablate the remaining cancer or s cells. Other approaches include chemotherapy and conventional gene therapy. Prognosis is poor. Median survival for patients with diagnosed glioblastoma is approximately nine to twelve months after diagnosis, with a five-year survival rate at only 5%.
  • FasL encapsulated source of FasL (such as encapsulated FasL-producing cells), or other apoptosis-inducing molecule, to the site ofthe tumor in the patient's CNS. Delivery to the brain ventricles, brain parenchyma or CSF is contemplated. Device placement is preferably proximate to or in the tumor tissue.
  • the dosage ofthe apoptosis-inducing molecule can be varied by any suitable method known in the art. This includes changing the cellular production of FasL, achieved in any conventional manner, such as varying the copy number ofthe FasL gene in the transduced cell, or driving expression ofthe FasL using a higher or lower efficiency promoter, as desired. Further, the device volume and cell loading density can easily be varied, over at least three orders of magnitude. In addition, dosage may be controlled by implanting a fewer or greater number of devices. We prefer implanting between 1 and 10 devices per patient. EXAMPLES
  • FasL-containing supernatant mimicked the effect of intraperitoneally applied anti-Fas/ APO- 1 antibody, causing liver failure due to hemorrhage and hepatocyte apoptosis.
  • mice were purchased from WIGA (Hannover, Germany).
  • Balb/c (H-2 d ) and C57BL/6 (H-2 b ) mice were obtained from the breeding colony of the Institut fur Zuchthygiene, Tierspital Zurich, Switzerland. All experiments were carried out with 6-10-wk-old mice of either sex.
  • the murine neuroblastoma cell line Neuro-2a and the murine lymphoma cell line Yac-1 were obtained from the American Type Culture Collection (Rockville, MD). Neuro-2a cells were maintained in MEM supplemented with 10% heat-inactivated FCS, 2mM L-glutamine, non-essential amino acids (lx) and penicillin (100 U/ml)/streptomycin (100 mg/ml) (GIBCO, Parley, " Scotland) and Yac-1 cells in RPMI 1640 (Hyclone, Cramlington, UK) containing 10% FCS, L-glutamine and antibiotics as above. Reagents
  • Cycloheximide (CHX), acridine orange and protein A were obtained from Sigma Chemical Co. (St. Louis, MO).
  • Hamster-anti-mouse Fas mAb Jo2 was from PharMingen (San Diego, CA)
  • hamster IgG was purchased from Axell, Accurate Chemical & Scientific Co ⁇ oration (Westbury, NY).
  • Soluble human Fas/APO-1 was kindly provided by Dr. P. Barr (Richmond, CA), F(ab% fragments of anti-human APO-1 and control FH23 F(ab') 2 fragments were generated as described in Dhein et al., Mature, 373, p. 438 (1995).
  • F ⁇ sL and Fas/APO-1 transfectants were generated as described in Dhein et al., Mature, 373, p. 438 (1995).
  • the murine FasL cDNA was subcloned from pBS(-)KS (Stratagene, La Jolla, CA) into the Xhol and Notl sites ofthe BCMGS Neo expression plasmid (Karasuyama et al., J Exp Med 172, p. 969 (1990)).
  • the human Fas/APO-1 cDNA was cloned into the same expression vector.
  • Neuro-2a cells (5xl0 6 ) were transfected with 10 ⁇ g plasmid DNA by electroporation using a Biorad Gene Pulser (25 kV). Selection with G418 (500 ⁇ g/ml) (GIBCO) was started 48 h later and continued throughout the culture period.
  • RT-PCR Reverse transcription-polymerase chain reaction
  • the second primer bound to the vector sequences just 3'-prime to the FasL insert (5*- GTG GAT CCC CCG GGC TGC AG -3') (SEQ ID NO. 2).
  • the full-length FasL transgene but not the endogenous FasL gene was amplified from reverse transcribed RNA of Neuro-2a FasL transfectants.
  • Cytotoxicity and proliferation assays Yac-1 cells were seeded into 96-well microtiter F-plates (Falcon, Becton Dickinson, Oxnard, CA) at a density of 2 x IO 4 cells per well.
  • the cells were stimulated with serial dilutions of 100-fold concentrated 24 to 48 h supernatant from Neuro-2a FasL and mock transfectants or with Jo2 or control hamster IgG. After 18 h, cultures were pulsed with 1 ⁇ Ci ( 3 H)TdR (5 Ci/mmol; Amersham, Bucks. UK) per well for 6 h, harvested on filter paper strips using an automated sample harvester, and counted in a liquid ⁇ -scintillation counter. Survival of Neuro-2a human Fas/APO-1 and mock transfectants was determined by crystal violet staining after incubation for 16 h with the respective stimulant in the presence of cycloheximide ( 10 ⁇ g/ml) . SDS-PAGE and Western blot
  • Yac-1 cells enclosed in permeable devices were implanted into the peritoneum of mice.
  • Semi- permeable polyether sulfone hollow fiber membranes (outside diameter: 600 ⁇ m) with a molecular weight cut-off of 2 x 10 3 kD were loaded with 10 5 cells/ ⁇ l. See, e.g., Aebischer et al., J. Biomed. Enyin .. 113, p. 178 (1991).
  • Each membrane device contained 4-6 x 10 5 Yac-1 cells.
  • the mice were anesthetized by intramuscular injection of Innovar- Vet (0.16 mg sublimazine/kg and 8 mg inarisine/kg) and were placed in a sterile working unit for the surgical procedure.
  • the peritoneum was opened by a small incision, two to maximally four devices were placed in the peritoneal cavity and the skin closed with two wound clips. Eight hours later the mice were injected i.p. with concentrated FasL or control supernatant in a volume of 2 ml. Viability staining and detection of apoptosis After exposure to FasL supernatant in vitro or in vivo, Yac- 1 cells were removed from the devices and incubated with fluorescein diacetate (FDA) at a final concentration of 10 ⁇ g/ml for 5 min. Subsequently, viable and dead cells were counted by fluorescence microscopy and percentage viability was calculated.
  • FDA fluorescein diacetate
  • FasL For the production of recombinant FasL, Neuro-2a cells were transfected with the CDNA encoding murine FasL. Neuro-2a cells were chosen because they do not express Fas/ APO- 1 as determined by flow cytometry and are resistant to Fas/APO-1 mediated cytotoxicity (data shown below). Successful transfection was confirmed by reverse transcription polymerase chain reaction (RT- PCR). A band ofthe expected size was seen in FasL transfectants but not in Neuro-2a cells transfected with the empty vector (Fig. la).
  • Yac-1 cells were incubated with serial dilutions of supernatant for 24 h and proliferation assessed by ( 3 H) thymidine inco ⁇ oration during the last 6 h of the incubation period.
  • the FasL concentration which induced 50% cell killing of Yac-1 cells was defined as 1 unit/ml.
  • Yac-1 cells were also exposed to the antiFas/APO- 1 antibody Jo2 (see, e.g., Ogasawara et al., Nature. 364, p. 806 (1994)), which was clearly less effective: concentrations of up to 100 ⁇ g/ml caused only approximately 20% cell death or 30% cell death in the presence of protein A (Fig. lc).
  • Protein A promotes Fc-Fc interactions of antibodies and increases their ability to cross-link Fas/APO-1 receptors on the target cell surface. Receptor multimerization appears to be required for the generation ofthe apoptotic signal. Adso ⁇ tion of anti-Fas/ APO- 1 antibody to a solid phase support has also been described to enhance its cytotoxic activity but failed to augment killing of Yac-1 cells in our experiments (data not shown).
  • Fas/ APO- 1 positive and negative target cells were transfected with the human Fas/APO-1 cDNA or with the vector contro 1.
  • Mouse FasL has been shown to bind to human Fas/ APO- 1.
  • Stable transfectants should differ only in their expression level of human Fas/APO-1 and as a consequence in their susceptibility to Fas/APO-1 -mediated apoptosis.
  • supernatant of FasL transfectants efficiently killed Neuro-2a target cells expressing human Fas/APO-1 (Fig. Id), but did not affect viability ofthe mock transfectants (data not shown).
  • the specificity ofthe cytotoxic effect ofthe supernatant was further determined by adding soluble human Fas/APO-1 (sFas) or anti-Fas/ APO- 1 antibody F(ab') 2 fragments (see Dhein et al., NatUIfi, 373, p. 438 (1995)) to the FasL cytotoxicity assay. Both reagents would be expected to specifically block FasL- mediated cell death either by scavenging FasL from the supernatant or by competing with the binding of FasL to Fas/APO-1 on the target cells.
  • sFas completely blocked supernatant- mediated cytotoxicity on Yac-1 cells at a concentration of 500 ng/ml, while soluble TNF-receptor (sTNF-R) had no inhibitory effect.
  • F(ab') 2 anti-Fas/ APO- 1 reduced supematant-induced cell killing of Neuro-2a Fas/APO-l transfectants at a concentration of 10 ⁇ g/ml (Fig. Id). Inhibition by both reagents was dose-dependent (data not shown) and specific since no effect was seen after the addition of sTNF-R or FII23 control F(ab') 2 fragments (Fig. Id).
  • mice had complications from the surgical procedure or signs of irritation from the devices, reconfirming the biocompatibility ofthe membrane material. See, e.g., Christenson et al. Biomed Mater. Res.. 25. p. 1119 (1991).
  • encapsulated Yac-1 cells were exposed to FasL- containing supernatant or control supernatant for 16 h in vitro. Viability ofthe Yac-1 cells was assessed by FDA staining. As shown in Fig. 3a, FasL-containing supernatant reduced viability in a dose-dependent manner in vivo. The maximal dose of 2000 U resulted in 100% cell killing, while equally concentrated supernatant of mock transfectants did not impair viability. Thus, the FasL supernatant is also cytotoxic in vivo. Efficiency of FasL cell killing was dependent on the FasL concentration but not on the number of target cells, at least in the tested range of 5 x 10 s to 2 x 10* target cells (data not shown).
  • FasL supernatant also efficiently killed encapsulated Yac-1 cells in vitro (data not shown).
  • FasL supernatant was given also to Balb/c and C57BIJ6 mice. Likewise, i.p. application of FasL was well tolerated in these mice and liver sections were normal.
  • FasL compared to anti-Fas/APO-1 antibodies ii especially striking since a dose of FasL which efficiently kills 100% of the Yac- 1 cells in vivo has no systemic side effects when applied i.p., while a dose of anti- Fas/APO-1 antibodies which only kills about 20% of Yac-1 cells in vitro induces lethal liver toxicity in vivo.
  • FasL caused severe liver hemorrhage and hepatocyte apoptosis when injected i.v.
  • it induced changes analogous to those seen after i.p. administration of anti-Fas/APO-1 antibody (Fig. 4b and d).
  • Histology revealed altered liver tissue with dilated sinusoids, loss of sinusoidal endothelial cells and hemorrhages. Damaged hepatocytes of irregular size exhibited condensed and fragmented nuclei indicative of apoptosis.
  • i.v. injection of concentrated control supernatant did not induce liver damage (Fig. 4c). Normal liver tissue with regular trabecules and slight steatosis was observed.
  • FasL given i.p. acts locally on target cells without inducing hepatocyte apoptosis.
  • Example 2 In the above experiment, Yac-1 cells (which bear the Fas/APO-1 receptor) were encapsulated, and the killing effect of injected soluble FasL on those cells was demonstrated. In further experiments, the FasL-secreting cells are encapsulated and the effect ofthe secreted FasL on tumor tissue is demonstrated, both in vitro and in vivo. For these experiments, the following encapsulation procedure is suitable.
  • Hollow fibers are fabricated from a polyether sulfone (PES) with an outside diameter of 720 ⁇ m and a wall thickness of a 100 ⁇ m (AKZO-Nobel Wuppertal, Germany). These fibers are described in United States patents 4,976,859 and 4,968,733, herein inco ⁇ orated by reference. In some studies, including human studies, we contemplate using a polyether sulfone ("PES”) PES#5 membrane which lias a MWCO of about 280 kd. In other studies we contemplate using a PES#8 membrane which has a MWCO of about 90 kd.
  • the devices comprise:
  • the semipermeable PES membrane contemplated in the human studies has the following characteristics:
  • the components ofthe device are commercially available.
  • the LCM glue is available from Ablestik Laboratories (Newark, DE); Luxtrak Adhesives LCM23 and LCM24).
  • the tether material is available from Specialty Silicone Fabricators (Robles, CA). The tether dimensions are 0.79 mm OD x 0.43 mm ID x length 202 mm.
  • Fiber material is cut into 5 cm long segment and the distal end ofthe devices are sealed with a photopolymerized acrylic glue (LCM-25, ICI). Following sterilization with ethylene oxide and outgassing, the fiber segments are loaded with a suspension of between 10 3 to IO 7 cells via a Hamilton syringe and a 25 gauge needle through an attached injection port.
  • a collagen matrix e.g., ZyplastTM.
  • the proximal end ofthe device is sealed with the same acrylic glue.
  • the volume ofthe device is approximately 15-18 ⁇ l.
  • a silicone tether (Specialty Silicone Fabrication, Taunton, MA) (ID: 690 ⁇ m; OD: 1.25 ⁇ m) is placed over the proximal end of the fiber allowing easy manipulation and retrieval ofthe device.
  • a membrane-based implant containing between 10 3 to IO 7 cells producing FasL, as described above, is implanted into the tumor bed in the CNS and/or into the CSF fluid compartment.
  • Patient recruitment may be based on the following criteria: a) Entry criteria:
  • Implantation Patients receive a hollow fiber implant or a round implant (e.g., shaped as a disk) containing FasL-producing cells. Standard surgical procedure, such as that described in WO 94/15663, may be used to implant the devices of this invention. SEQUENCE LI ST ING
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO: 1: TCCGCCACCA TGCAGCAGCC 20
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO

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Abstract

Dispositif et procédé servant à administrer, sous forme de capsules, une molécule provoquant l'apoptose, à un malade, de préférence, dans le système nerveux central.
PCT/US1997/006409 1996-04-17 1997-04-17 Procede et dispositif servant a administrer des molecules provoquant l'apoptose WO1997038707A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000012066A1 (fr) * 1998-08-26 2000-03-09 Norsk Hydro Asa Capsules d'alginate utilisees pour le traitement des tumeurs cerebrales
WO2003031473A1 (fr) * 2001-09-27 2003-04-17 Leukocare Gmbh Module d'inactivation de leucocytes
WO2003092723A1 (fr) * 2002-05-03 2003-11-13 Nikolai Rainov Utilisation de fasl dans le traitement de maladies neurodegeneratives
US9567568B2 (en) 2005-05-04 2017-02-14 Enlivex Therapeutics Ltd. Method of preparing apoptotic monocytes

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WO1992019195A1 (fr) * 1991-04-25 1992-11-12 Brown University Research Foundation Vehicule implantable immuno-isolateur et biocompatible servant a apporter des produits therapeutiques selectionnes
WO1995005452A2 (fr) * 1993-08-12 1995-02-23 Cytotherapeutics, Inc. Compositions et procedes ameliores pour l'administration de molecuiles a activite biologique a l'aide de cellules modifiees genetiquement comprises dans des capsules biocompatibles immuno-isolatrices
WO1995016464A1 (fr) * 1993-12-14 1995-06-22 Johns Hopkins University School Of Medicine Liberation controlee de substances pharmaceutiquement actives pour l'immunotherapie

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WO1992019195A1 (fr) * 1991-04-25 1992-11-12 Brown University Research Foundation Vehicule implantable immuno-isolateur et biocompatible servant a apporter des produits therapeutiques selectionnes
WO1995005452A2 (fr) * 1993-08-12 1995-02-23 Cytotherapeutics, Inc. Compositions et procedes ameliores pour l'administration de molecuiles a activite biologique a l'aide de cellules modifiees genetiquement comprises dans des capsules biocompatibles immuno-isolatrices
WO1995016464A1 (fr) * 1993-12-14 1995-06-22 Johns Hopkins University School Of Medicine Liberation controlee de substances pharmaceutiquement actives pour l'immunotherapie

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Title
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NAGATA S ET AL: "THE FAS DEATH FACTOR", SCIENCE, vol. 267, 10 March 1995 (1995-03-10), pages 1449 - 1456, XP002026333 *
WELLER ET AL: "FAS/APO-1 GENE TRANSFER FOR HUMAN MALIGNANT GLIOMA", CANCER RESEARCH, vol. 55, 1 July 1995 (1995-07-01), pages 2936 - 2944, XP002036423 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000012066A1 (fr) * 1998-08-26 2000-03-09 Norsk Hydro Asa Capsules d'alginate utilisees pour le traitement des tumeurs cerebrales
JP2002523445A (ja) * 1998-08-26 2002-07-30 ノルスク・ヒドロ・アーエスアー 脳腫瘍治療用のアルギネートカプセル
AU760433B2 (en) * 1998-08-26 2003-05-15 Norsk Hydro Asa Alginate capsules for use in the treatment of brain tumour
US6926888B1 (en) 1998-08-26 2005-08-09 Fmc Biopolymer As Alginate capsules for use in the treatment of brain tumor
KR100699286B1 (ko) * 1998-08-26 2007-03-26 노르스크 히드로 아에스아 뇌 종양 치료용 알긴산염 캡슐
WO2003031473A1 (fr) * 2001-09-27 2003-04-17 Leukocare Gmbh Module d'inactivation de leucocytes
JP2005507908A (ja) * 2001-09-27 2005-03-24 レウコカーレ ゲーエムベーハー 白血球不活性化モジュール
CN100346822C (zh) * 2001-09-27 2007-11-07 白血球保健股份有限公司 白细胞失活元件
US7850969B2 (en) 2001-09-27 2010-12-14 Leukocare Gmbh Leukocyte inactivation module
WO2003092723A1 (fr) * 2002-05-03 2003-11-13 Nikolai Rainov Utilisation de fasl dans le traitement de maladies neurodegeneratives
US9567568B2 (en) 2005-05-04 2017-02-14 Enlivex Therapeutics Ltd. Method of preparing apoptotic monocytes

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