The teachings of both earlier-filed patent applications are incorporated herein by reference.

Field of the Invention The invention relates generally to the suppression of unwanted immune responses, particularly of counter-adaptive T-lymphocyte mediated immune responses.

The invention relates in particular to the prevention, treatment, suppression or reversal of immune system driven rejection of an implant or implanted device, such as an immunoisolation device containing foreign cells or tissue, in the body of a recipient.

Background of the Invention The term"immunoisolation"refers to the protection of transplanted living cells or tissue from the immune system of a recipient by enclosure in a semipermeable membrane or barrier. Historically, naturally occurring and synthetic polymers have been used to protect transplanted cells and tissues from the recipient's cellular and humoral immune system responses. Several types of immunoisolation systems or devices have been studied, including devices which are anastomosed to the vascular system such as arteriovenous shunts, as well as diffusion chambers, and micro-or macro-encapsulation devices including microcapsules. In general, such encapsulation systems can serve to block the access of antibodies and complement to the tissue within the device, or to block contact with mononuclear cells : however, there remains the possibility that antigens released from the capsule (such as secreted cellular products, shed cell surface components, or cellular debris) could stimulate a recipient immune response. Such an immune response in turn can lead to an allergic response, or immune complex disease. Cell-mediated immunity also can be induced by released antigens, leading to production of proinflammatory cytokines. which are low molecular weight molecules, and therefore able to pass through the semi-permeable membrane.

The device itself may also elicit an inflammatory response, such as a foreign body response, including the production of antibodies, proinflammatory mediators such as cytokines and nitric oxide, which ultimately can lead to the deposition of a relatively impermeable fibrotic shell around the device. Additionally, encapsulation technologies suffer from the risk of breakage or weakening of membrane areas, which impair the effectiveness of the capsule as an immunobarrier.

Thus, any interaction between implanted devices, such as capsules, and the recipient's immune system depends in part upon the device's composition, contents, and integrity. In addition, when the devices contain live cells or tissue, the recipient's response may depend upon the nature of cellular products which escape the device, such as by crossing a capsule membrane. These products may be antigenic and stimulate a recipient immune reaction.

Immunoisolation has potential in the treatment of many chronic conditions. For example, some cells and tissues which have already been used in the study of immunoisolation include: adrenal chromaffin cells for neurodegenerative disease and chronic pain; genetically engineered cells designed to produce clotting factor IX for hemophilia, growth hormone for the treatment of dwarfism, erythropoeitin for anemia, and nerve growth factors or neurotrophic factors for treatment of neurodegenerative conditions; islet cells for producing insulin for diabetics; parathyroid cells for hypocalcemia; and, hepatocytes for the correction of metabolic disorders.

Of the foregoing, immunoisolation of islet cell transplants for the treatment of insulin dependent diabetes mellitus (IDDM) has been most extensively studied. Until recently, the only success achieved in microencapsulated islet grafts without the need for immunosuppression was in rodent to rodent transplants. Other studies have required concurrent treatment with immunosuppressive agents, thus leading to various undesirable side effects. Thus, despite the advances in the field, the technology has yet to be perfected.

Studies in islet immunoisolation methods, and particularly islet microencapsulation, while leading to encouraging observations of successful transplantation in selected models, have also uncovered a plethora of barriers to further success. Besides immunogenicity of the substances used for the engineering of the

immunoisolation devices and the inflammatory and fibrotic responses to the geometry of such devices, problems also exist associated with tissue procurement or cell sourcing, tissue density constraints, diffusion of substances across membrane polymers, blood and nutrient supply and the structural integrity of the devices themselves. Each of the foregoing concerns applies similarly to the immunoisolation of other cell or tissue types.

There is accordingly a need for improved or more effective immunosuppressive or immunomodulatory treatments for implant recipients, including humans. In particular, there is a need for treatments that do not require pan-T cell immunosuppression, i. e., treatments that do not leave the recipient vulnerable to malignancies or opportunistic infection. More pointedly, there is a need for treatments, including devices, that have lesser toxicity than currently available therapeutic agents.

Similarly, there is a need for treatments that promote lasting functional integration of an immunoisolation device, i. e., integration that persists beyond termination of the course of treatment.

Summarv of the Invention It is an object of this invention to provide an immunomodulatory agent that mitigates counter-adaptive T cell responses without the need for pan-T cell immunosuppression. Another object is to provide an immunomodulatory agent that promotes functional integration of a therapeutic implant or device, particularly an immunoisolation device having viable cells or tissue disposed therein, in a recipient, particularly a human. Another object is to provide an immunomodulatory agent that inhibits immunological rejection of an implanted device, particularly of an immunoisolation or encapsulation device, such as a micro-or macro-capsule. A further object is to provide an immunomodulatory agent that interrupts delivery of a costimulatory signal to activated T cells of the recipient. A particular object is to provide a CD40: CD154 binding interruptor, such as a CD154 blocking agent, for use in therapy, particularly for use in therapy to mitigate, delay or reverse immunological rejection of an implanted device. A more general object of the invention is to improve the availability of immunoisolation technology, by providing immunomodulatory compositions that allow functional integration of immunoisolation devices into a

recipient. A further general object is to prevent, mitigate, attenuate or treat a condition in which the recipient is in need of a product secreted by cells or tissue in an immunoisolation device (e. g., dopamine producing cells needed by an individual with parkinsonism or another neurodegenerative disorder), or in which the recipient is in need of an enzymatic process catalyzed by cells or tissue in the immunoisolation device (e. g., detoxification processes catalyzed by hepatocytes, which are needed by individuals with liver failure).

The present invention rests on the discovery that use of a CD40: CD154 binding interruptor, such as a CD 154 blocking agent, whether used alone or in combination with another therapeutic agent, such as an immunomodulatory agent, attenuates, suppresses, prevents, delays or reverses counter-adaptive immune system rejection of an implanted device in a recipient, without the need for pan-suppression of the recipient's immune system.

The invention accordingly provides methods and compositions for immunomodulatory therapy for recipients of implanted devices, such as immunoisolation devices, drug delivery devices, and prosthetic devices. A first method inhibits rejection of an implanted device by a recipient thereof. A second method prolongs survival of cells or tissue contained within an implanted device. A third method reverses rejection of the implanted device. A fourth method preserves function of cells or tissue contained within the implanted device. A fifth method restores function of an impaired device, particularly of impaired cells or tissue within the device. All of the foregoing methods involve treating the recipient with a CD40: CD154 binding interruptor, by which is meant any agent that interrupts the binding of CD40 Ligand (i. e., CD40L, also known as CD154 or the 5c8 antigen, and sometimes referred to in the art as gp39) to its counter or cognate receptor (e. g., CD40).

Preferably, the binding interruptor is a CD154 (CD40L) blocking agent, by which is meant any agent that binds to CD154 and prevents or interferes with its binding to counter receptors (e. g., CD40). An exemplary CD154 blocking agent is a monoclonal antibody (MAb), particularly one having the antigen-specific binding characteristics of the 5c8 MAb disclosed in U. S. Patent the teachings of which are incorporated herein by reference.

The foregoing methods can be practiced with all types of implanted devices, such as drug delivery devices or prosthetic devices; however, the invention is particularly suitable for use with immunoisolation devices which contain viable cells or tissue. Practice of the present invention advantageously attenuates a foreign body response, a humoral immune response, or a cellular immune response, to the implanted device. The invention accordingly is useful wherever the recipient is in need of a product is secreted by cells or tissue that are suitable for containment in an immunoisolation device. Similarly, the invention is useful wherever the recipient is in need of an enzymatic process that is carried out by cells or tissue suitable for containment in an immunoisolation device. By way of example and without limitation, the cells or tissue can be allogeneic, xenogeneic, autologous, postmitotic, mitotically competent, differentiated, partially differentiated, derived from an adult donor, of fetal origin, cultured, immortalized, or genetically modified. The device can be any type of immunoisolation device, including without limitation an arteriovenous shunt, a diffusion chamber, or a micro-or macro-capsule made of any conventional material or combination of materials, that is sufficient to provide a physical barrier to cell/cell contact between tissues of the recipient's body and the cells or tissue contained within the device. The barrier, together with any other structural members of the device to which the barrier may be joined, defines an isolation chamber in which the cells or tissue are disposed. Preferably, the barrier is a semipermeable matrix or membrane which selectively excludes recipient cells and unwanted macromolecules (e. g., complement factor Clq, immunoglobulins) from the interior of the device (i. e., the isolation chamber), yet allows low molecular weight nutrients, metabolites, and dissolved gasses such as oxygen, to pass through freely. The device is implanted at any site within the recipient's body that is appropriate to delivery of the needed product, or enzymatic process, to the recipient. For example, the device may be implanted subcutaneously, intramuscularly, intravascularly, intraperitoneally, intrahepatically, retroperitoneally, within a bone marrow cavity, within a joint capsule, intraocularly, intracranially, intraspinally, or the like. The recipient can be any mammal, but preferably is a primate, most preferably a human.

In accordance with the foregoing, the invention provides a method of implanting cells or tissue into a recipient. The method involves providing an

immunoisolation device in which a semipermeable barrier defines an isolation chamber in which the cells or tissue are disposed. The device is implanted into the recipient by any conventional technique, such as by surgery, and a CD40: CD154 binding interruptor is administered to the recipient either concurrently, prior to the implantation, or subsequent to the implantation, in an ammount sufficient to attenuate an immune response (whether a foreign body response, humoral response, or cellular response) to the implanted device.

The invention furthermore provides a composition for the implantation of cells or tissue into a recipient. The present composition comprises an immunoisolation device in which a semipermeable barrier defines an isolation chamber (either by completely surrounding the chamber, or by sealing an aperture within or between one or more structural members of the device) and separates the chamber contents physically from tissue surrounding the device once implanted into the body of a recipient. The present composition further provides cells or tissue disposed within the isolation chamber. The composition still further comprises a CD40: CD154 binding interruptor, which can be (without limitation) dispersed in a physiologically acceptable carrier in which the immunoisolation device is immersed; or, adsorbed on the surface of the device; or, produced by cells or tissue within the isolation chamber. In another embodiment, the composition is for the delivery of a drug or other therapeutic agent to the recipient, and the composition comprises a reservoir or matrix having the drug/therapeutic agent formulated therein in the isolation chamber in lieu of the above- mentioned cells or tissue. In still another embodiment, the device is a prosthetic device (including, without limitation, a suture, staple, stent, catheter, pin, bolt, screw, lead line, artificial tooth, bone or joint replacement device, or artificial valve) having adsorbed thereon or formulated therein a CD40: CD154 binding interruptor as defined herein.

Any of the foregoing compositions are suitable for use in therapy, including without limitation, for the specific medical uses disclosed herein.

The foregoing and other objects, features and advantages of the present invention, as well as the invention itself, will be more fully understood from the following description of preferred embodiments.

Detailed Description of the Invention T cell activation, and immunological processes dependent thereon, requires both T cell receptor (TCR) mediated signals and simultaneously delivered costimulatory signals. An important costimulatory signal is delivered by the ligation of CD40 on an antigen-presenting cell, such as a B cell, by CD40L (CD 154) on a T cell. Human CD40 is a 50 kD cell surface protein expressed on mature B cells, as well as on macrophages and activated endothelial cells. CD40 belongs to a class of receptors involved in programmed cell death, including Fas/CD95 and the tumor necrosis factor (TNF) alpha receptor. Human CD154 (CD40L) is a 32 kD type II membrane glycoprotein with homology to TNF alpha that is transiently expressed, primarily on activated T cells. CD40: CD154 binding has been shown to be required for all T cell- dependent antibody responses. In particular, CD40: CD154 binding provides anti- apoptotic and/or lymphokine stimulatory signals.

The importance of CD40: CD154 binding in promoting T cell dependent biological responses was more fully appreciated when it was discovered that X-linked hyper-IgM syndrome (X-HIGM) in humans is the phenotype resulting from genetic lack of functional CD154. Affected individuals have normal or high IgM levels, but fail to produce IgG, IgA or IgE antibodies, and suffer from recurrent, sometimes severe, bacterial and parasitic infections, as well as an increased incidence of lymphomas and abdominal cancers. A similar phenotype is observed in non-human animals rendered nullizygous for the gene encoding CD154 (knockout animals). B cells of CD154 nullizygotes can produce IgM in the absence of CD40L: CD154 binding, but are unable to undergo isotype switching, or to survive normally after affinity maturation. Histologically, lymph node germinal centers fail to develop properly, and memory B cells are absent or poorly developed. Functionally, these defects contribute to a severe reduction or absence of a secondary (mature) antibody response. Defects in cellular immunity are also observed, manifested by an increased incidence of bacterial and parasitic infections. Many of these cell-mediated defects are reversible by administration of IL-12 or IFN-gamma. These observations substantiate the view that normal CD40: CD154 binding promotes the development of Type I T- helper cell immunological responses.

A number of preclinical studies have established that agents capable of interrupting CD40: CD154 binding have promise as immunomodulating agents. In particular, studies involving small-animal organ or tissue transplantation models have shown that CD40: CD154 interruptors promote survival of allogeneic grafts. In selected models, transient administration of agents interfering with T cell costimulation has resulted in the induction of indefinite graft acceptance. Interruption of CD40: CD154 binding in particular has yielded promising results, since it appears that engagement of this counter-receptor pair precedes other costimulatory signals in chronology and hierarchy (Shinde et al. (1996), J. Immunol. 157: 2764-2768; Yang et al. (1996), Science 273: 1862-1864; Grewal et al. (1996), Science 273: 1864-1867; Roy et al.

(1995), Eur. J. Immunol. 25: 596-603; Han et al. (1995), J. Inmunol. 155: 556-567; Ranheim et al. (1993), J. Exp. Med. 177: 925-935; Lederman et al. (1992), J. Immunol.

149: 3817-3826). Blockade of CD40: CD154 binding has resulted in prolongation of cardiac (Larsen et al. ( 1996), Transplantation 61: 4-9; Larsen et al. (1996), Nature 381: 434438), cutaneous (Larsen et al. (1996), Nature 381: 434-438; Markees et al.

(1997), Transplantation 64: 329-335) and islet allografts (Parker et al. (1995), Proc.

Natl. Acad. Sci. USA 92: 9560-9564; Rossini et al., Cell Transplant 5: 49-52) in rodents, and of allogeneic kidneys in primates (Kirk et al. (1997), Proc. Natl. Acad. Sci. USA 194: 8789-8794). It has also been demonstrated to delay onset of autoimmune diabetes in non-obese diabetic (NOD) mice (Balasa et al. (1997), J. Immunol. 159: 4620-4627).

Lastly, it has been reported that interference with CD40: CD154 binding prevents the production of inflammatory cytokines (Dechanet et al. (1997), J. Immunol. 159: 5640- 5647; Kiener et al. (1995), J. Immunol. 155: 4917-4925.

CD40: CD154 blockade technology thus may provide potentially powerful therapeutic avenues for prevention of immunological complications, including rejection and failure, which currently impede the further development and wider use of implanted devices. However, immunological studies in rodent model systems have have not correlated consistently with the outcome of testing or therapy of large animals, including humans. Similarly, immunological studies in induced-disease model systems have not correlated consistently with the outcome of testing in individuals having chronic or ongoing disease states.

Disclosed herein are presently-preferred, exemplary model systems for assessing the effects of a preferred CD 154 blocking agent, a humanized MAb having the antigen-specific binding properties of MAb 5c8 (Lederman et al. (1992), J. Exp.

Med. 175: 1091-1101), in a variety of preclinical models of implant treatment of selected disease states or impaired conditions. Studies using these model systems are expected to yield information of predictive value in developing implant therapies for the corresponding disease states or impairments (such as organ failures) in humans.

The following discussion accordingly illustrates and exemplifies the variety of contexts and circumstances in which the invention can be practiced.

Implantable Devices All types of implantable devices can be used in practicing the present invention, including, without limitation, immunoisolation devices, drug delivery devices, and prosthetic devices. However, the invention is particularly suitable for use with immunoisolation devices which contain viable cells or tissue. For present purposes, an "immunoisolation device"is a container or vessel fabricated of a biocompatible material or combination of materials, in which a semipermeable barrier, such as a membrane, defines an isolation chamber in the interior of the device, in which viable cells or tissue can be disposed. The contents of the isolation chamber are separated, and therefore protected, from the environment external to the device by the semipermeable barrier. In some embodiments, the semipermeable barrier completely surrounds or encapsulates the isolation chamber; however, in other embodiments, the semipermeable barrier seals an aperture within or between one or more structural members of the device. In each case, the barrier separates the chamber contents physically from the external environment, in particular from body tissue (s) of the recipient which surround the device after its implantation into the body of a recipient.

Preferably, the barrier is a semipermeable matrix or membrane which selectively excludes recipient immune effector cells (e. g., leukocytes) and unwanted macromolecules (e. g., complement factor C lq, immunoglobulins) from the interior of the device (i. e., the isolation chamber), yet allows low molecular weight nutrients, metabolites, and dissolved gasses such as oxygen, to pass through freely. Thus, preferred barriers (whether membranes or matrices) have a pore size (also referred to as

a cutoff size) allowing diffusion of molecules having molecular weights of no more than kD. This pore size is small enough to prevent contact of all recipient cells and most soluble products of the recipient's immune system with the contents of the isolation chamber. However, small molecules, such as cytokines, and the low molecular weight reactive metabolites of oxygen and nitrogen produced by macrophages and certain other cells, can still diffuse through such semipermeable membranes. Recent reports, however, suggest that even a partial barrier to antibodies and complement factors is sufficient to allow successful engraftment and survival of cells contained by an implant. Conversely, other reports indicate the presence of circulating antibodies against immunoisolated tissues 2 to 6 weeks after implantation.

Thus, there is no clear consensus as to the degree of protection from the recipient's immune system necessary for survival and functional integration of cells within an immunoisolation device when disposed in the body of the recipient. Accordingly, some embodiments of the present invention include devices in which the pore size of the semipermeable barrier is larger than 50,000 to 100,000 kD. For example, the invention encompasses devices in which the pore size range is 100,000 to 150,000 kD (large enough to allow diffusion of some immunoglobulins, such as IgG, but small enough to exclude Clq).

The present invention can be practiced with any size or shape of immunoisolation device, including without limitation an arteriovenous shunt, a diffusion chamber, or a micro-or macro-capsule, made of any conventional material or combination of materials. Thus, the invention can be practiced with devices sufficient to enclose an entire organ, a section of an organ, a sample or biopsy of tissue, a cluster or colony of cells, or individually isolated cells. The configuration and size of the immunoisolation device will vary depending on the site of implantation in the recipient's body, the recipient's body weight, age, sex and health status, and in particular on the number or amount of cells/tissue required to fulfill the recipient's need for a secreted product or an enzymatic process supplied by the cells/tissue within the immunoisolation device. In any case, an effective amount of cells or tissue is implanted, by which is meant an amount sufficient to attenuate (detectably mitigate or diminish) the recipient's need for the secreted product or the enzymatic process.

Optimally, the amount is sufficient to restore the recipient to substantially normal

homeostasis-that is, to free the recipient from dependence on conventional drugs or other therapy for the disease or condition being treated.

Specific immunoisolation devices useful in the present invention include any porous membrane, envelope, or capsule suitable for encapsulating living tissue.

Numerous types of microcapsules are known to those of skill in the art : thus, the choice of a particular encapsulation material can be made in light of the desired application.

Alginate polylysine microcapsules are well known in the art, but are known to weaken over time. Other microcapsules comprise alginate chemically cross-linked with barium; however, barium is toxic to most cells. Preferred microcapsules comprise biocompatible polymers, free of immunogenic substances, formed into a structurally sound device or capsule which can be tolerated by the recipient. (See, e. g., Methods in Cell Transplantation (Ricordi, ed.), R. G. Landes Co., Austin, TX (1995), especially Ch. G15, pp. 587-615; and, Jaink, et al.,"Long Term Preservation of Islets of Langerhans in Hydrophilic Macrobeads", Transplantation 1996 Feb. 27; 61 (4), both of which are incorporated herein by reference.) Synthetic polymers such as PEG (Polyethylene glycol), available from Neocrin Company, Irvine, CA, are also useful.

Exemplary alginate microcapsules can be either single walled or double walled, as described in U. S. Patent No. (July 13,1993), or uncoated, as described in U. S. Patent No. 5,651,980 (July 29,1997) the specifications of both of which are hereby incorporated by reference. Additional devices which can be used in the context of the present invention include, without limitation, the biological artificial liver described in U. S. Patent No. 5,270,192 (December 14,1993); the bioartificial endocrine device of U. S. Patent No. 5,614,205 (March 25,1997); the vascularizing bioartificial pancreas of U. S. Patent No. 5,674,289 (October 7,1997); the close vascularizing implant of U. S. Patent No. (September 1,1998) or, the permselective bioartificial organ of U. S. Patent No. 5,837,234 (November 17,1998).

The present immunoisolation device is implanted at any site within the recipient's body that is appropriate to delivery of the needed product, or enzymatic process, to the recipient. Thus, the device can be implanted at a site sufficient to deliver the product or process systemically, for example to the recipient's bloodstream, or locally, for example to a body compartment or a wound site. Suitable sites for implantation for systemic delivery include, without limitation: subcutaneous,

intramuscular, intravascular (including intraarterial, intravenous, and arteriovenous), intraperitoneal and intrahepatic sites. Suitable sites for implantation for local delivery include: subcutaneous, intramuscular, subgingival, retroperitoneal, intraosteal (within a bone marrow cavity), subcapsular (within a joint capsule), intraocularly, intracranially, or intraspinally.

In other embodiments of the invention, the implantable device is for the delivery of a drug or other therapeutic agent not of cellular origin to the recipient. In such embodiments, the device includes a reservoir or matrix having a drug or other therapeutic agent disposed or formulated therein in lieu of the isolation chamber containing cells or tissue. In still other embodiments, the device is a prosthetic device, such as a suture, staple, stent, catheter, pin, bolt, screw, lead line, artificial tooth, bone or joint replacement device, or artificial valve.

Any of the foregoing immunoisolation or other devices can, as desired, be immersed prior to implantation in a physiologically acceptable carrier (such as a preservative solution, a cell culture medium, or a balanced salts solution) in which a CD40: CD154 binding interruptor is dispersed or otherwise formulated. Alternatively, the CD40: CD154 binding interruptor can be adsorbed on the surface of the device, or otherwise incorporated or fabricated into the device material. Still further, the CD40: CD154 binding interruptor can be a product that is secreted by cells or tissue that are either within the isolation chamber or otherwise associated with the device (for example, adhered to the device surface). In such cases, the device may deliver products made by two or more distinct cell or tissue types, that is, the device may include cells which produce the CD40: CD154 binding interruptor (facilitating acceptance and engraftment), and in addition also include the isolated cells or tissue which produce a product, or carry out an enzymatic process, which is needed by the recipient.

Implant Recipients The invention can be used for treatment or prophylaxis of any mammalian recipient in need of cells or tissue suitable for implantation in an immunoisolation device. Recipients accordingly are afflicted with, or at risk of, any defect or deficiency which can be overcome by products or processes which can be supplied by living cells or tissue. For example, the recipient can be afflicted with diabetes, which can be

treated using insulin produced by isolated islets or islet cells as described in prior related International S. N. PCT/US98/12892, the teachings of which are incorporated herein by reference. Alternatively, the recipient can be afflicted with chronic pain or a neurodegenerative disease, such as epilepsy, amyotrophic lateral sclerosis, parkinsonism, Huntington's disease, Alzheimer's disease, motor neuron disease, bipolar disease, or muscular dystrophy, which can be treated with dopamine, epinephrine, acetylcholine, tryptophan, serotonin, or another neurotransmitter supplied by neural or neuroendocrine cells, such as adrenal chromaffin cells. Similarly, the recipient can be afflicted with an endocrine disorder, such as hypocalcemia, which can be treated with a hormone supplied by endocrine cells, such as parathyroid hormone produced by parathyroid gland cells. The recipient alternatively can be afflicted with infertility, which can be treated with reproductive hormone (s) supplied by reproductive endocrine tissue, such as uterine tissue. Other recipients can be afflicted with a metabolic disorder or failure, such as liver failure, which can be treated using immunoisolated hepatocytes to supply enzymatic processes, such as detoxification reactions, to the recipient. Finally, the recipient can be in need of any factor which can be supplied by genetically engineered cells, particularly cells which supply a factor genetically missing or defective in the recipient. Exemplary factors include, without limitation, clotting factors, such as factor IX, for treatment of hemophiliac recipients; and, growth hormone for treatment of recipients with dwarfism. Still further, the recipient can be in need of a genetically engineered factor to treat an acquired condition (e. g., erythropoeitin for a recipient with anemia), or to assist in healing a wound (e. g., nerve growth factor or neurotrophic factor for treatment of a recipient with nerve tissue trauma, such as spinal cord injury).

Preferably, the recipient is a primate, more preferably a higher primate, most preferably a human. In other embodiments, the recipient may be another type of mammal, particularly a mammal of commercial importance, or a companion animal or other animal of value, such as a member of an endangered species. Thus, recipient hosts also include, but are not limited to, sheep, horses, cattle, goats, pigs, dogs, cats, rabbits, guinea pigs, hamsters, gerbils, rats and mice.

Cells or Tissue for Immunoisolation

The invention can be practiced for immunoisolation of any type of cells or tissue which secrete a product needed by the recipient, or supply an enzymatic process needed by the recipient. The invention is particularly suitable for use in any context wherein the cells or tissue are not histocompatible (MHC-compatible) with the recipient, or would not be acceptable to the recipient's immune system, or otherwise would not be compatible with the recipient's body. Thus, in addition to autologous or syngeneic cells or tissue, the invention can be used with allogeneic or even xenogeneic cells or tissue. Differentiated cells or tissue can be derived, by conventional means, from a volunteer or other living donor, or from a cadaveric donor. If derived from a cadaver, preferably the cells or tissue have been exposed to cold ischemic conditions for no more than about eight hours, more preferably no more than about two hours.

Preferably, the donor is as histocompatible as practicable with the recipient. Thus, where the recipient is a human, the cells or tissue should preferably be autologous or allogeneic to the recipient. However, the cells or tissue can be sourced from a heterologous species, such as a non-human primate (e. g., a chimpanzee or a baboon), or another relatively compatible mammal (e. g., a pig).

The cells or tissue for use in the present invention can be sourced from an adult donor, a neonatal or juvenile donor, or can be of fetal origin, depending on the desired cellular properties, the availability of particular tissue sources, and other parameters.

Thus, fibroblasts, vascular lining cells, hepatocytes, islet cells, epithelial cells, or other differentiated cell types can be obtained from an adult. In contexts where the needed cellular product is produced by a partially differentiated or progenitor cell type, the cells or tissue can be obtained from a neonatal or fetal donor (examples would be neural cells, myoblasts, hepatoblasts, and the like). Cells for immunoisolation can be postmitotic or mitotically competent cells; in the latter case, the cells are preferably conditionally mitotic. For example, the cells divide until a specific stimulus is applied, such as contact dependent growth inhibition, whereupon they enter a postmitotic state.

Myoblasts are a preferred example of conditionally mitotic cells for immunoisolation.

Furthermore, cells for immunoisolation can be derived from biopsied or resected, extracted or excised tissue sourced directly from a donor. Many techniques are known for deriving a desired cell or tissue type from a donor organ, for example for preparing donor islets or islet cell suspensions from whole pancreata (see, e. g., Ricordi

et al. (1988), 37 Diabetes 413-420; Tzakis et al. (1990), 336 Lancet 402-405; Linetsky et al. (1997), 46 Diabetes 1120-1123). Analogous techniques are available for deriving desired cells from other organs comprising a mixture of tissues. Alternatively, the cells for immunoisolation can be cells grown in culture. Such cultured cells can be a population of primary cells, or can be immortalized cells (e. g., a cell line). Although not preferred, cultured cells useful in the invention can be tumorigenic cells. The invention makes the use of tumorigenic cells possible because the cells are constrained by the immunoisolation device. However, where tumorigenic cells are used, vigilant care must be exercised to ensure structural integrity of the device.

The invention still further encompasses the use of genetically modified cells, such as genetically engineered cells which have been (or are derived from ancestor cells which have been) transformed or transfected by conventional means with nucleic acid which encodes a desired secreted product, or encodes one or more enzymes for carrying out a desired enzymatic process. Such cells can be engineered to express the encoded product or enzyme either constitutively or inducibly (e. g., under control of a stimulus-responsive promoter or enhancer). Virtually any of the above-discussed types of cells can be genetically modified for use in this invention. For example, peripheral blood leukocytes, bone marrow, fibroblasts, epithelial cells, hepatocytes or muscle satellite cells can be obtained from the recipient, transfected ex vivo with nucleic acid encoding the desired product, and re-implanted in an immunoisolation device according to the present invention. Alternatively, fetal cells, or cultured neonatal cells such as human umbilical cord vascular endothelial cells (HUVEC) cells, foreskin fibroblasts or myoblasts can be transfected ex vivo and immunoisolated. Still further, dedifferentiated or immortalized cells from a cell line (e. g., a hepatic cell line, or a fetal myoblastic cell line) can be transfected ex vivo and immunoisolated. In a presently preferred example, myoblast cell lines are transfected with a vector encoding the secreted product of interest, along with a selectable marker such as the herpes simplex thymidine kinase gene, which allows for selective removal of dividing cells (see, e. g., Deglon et al. (1996), 7 Hum. Gene Ther. 2135-2146). In yet further embodiments, cells for immunoisolation can be derived from a transgenic mammal that has been engineered to include foreign nucleic acid encoding the desired product or enzyme in some or all of its body tissues.

The invention is suitable for use with numerous, diverse cell or tissue types.

For example, the cells or tissue for isolation can be insulin-producing tissue, such as islets, islet beta cells, or engineered cells which express recombinant insulin. Indeed, the cells or tissue can be any naturally-sourced cell or tissue which produces any endocrine hormone, neuroendocrine hormone, neurotransmitter, neurotrophic factor, wound healing factor, angiogenic factor, morphogen, cytokine, lymphokine, chemokine, differentiation promoting factor, survival factor, immunomodulator, histocompatibility factor, blood clotting factor, antithrombotic factor, or other biological signalling molecule or structural protein. Specific examples of the foregoing include, without limitation: insulin, growth hormone, estrogen, progesterone, testosterone, parathyroid hormone, dopamine, epinephrine, acetylcholine, tryptophan, serotonin, substance P, nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), fibroblast growth factor (FGF), transforming growth factor beta (TGF-beta) (or any TGF-beta superfamily member), any colony stimulating factor (CSF), erythropoeitin, any interleukin, tumor necrosis factor (TNF) (or any TNF superfamily member).

Exemplary tissue and cell types include, without limitation, those sourced from solid organs, such as brain, spinal cord, liver, heart, kidney, lung, uterus, pituitary, thyroid gland, parathyroid gland, adrenal gland, etc.; those sourced from tissue samples, organ sections, or biopsies, such as pancreatic islets, adrenal chromaffin cells, sertoli cells, blood vessels, nerve fibers or ganglia, skin plugs or sections (full-or partial-thickness), fat, bone, or cartilage; or those sourced from cell suspensions, isolates, or aspirates, such as peripheral blood leukocytes, or bone marrow cells (including sorted cells, such as stem cells or colony forming cells). In addition to the foregoing, engineered cells useful in the present invention include cells which secrete recombinant versions of the above-mentioned naturally occurring products, or engineered versions thereof (truncations, fusion proteins, sequence mutants, and the like). Still further, cells or tissue useful in the present invention include those which naturally carry out needed enzymatic processes needed by the recipient, or which have been engineered to do so.

Hepatic tissue or hepatocytes are nonlimiting examples of such cells.

Exemplary CD40: CD154 Binding Interruntor Therapeutic compounds useful for practice of the invention include any compound that blocks the interaction of cell surface CD40 (e. g., on B cells) with

CD40L (CD154) expressed, e. g., on the surface of activated T cells. CD40: CD154 binding interruptor compounds, such as CD154 blocking agents, that are specifically contemplated include polyclonal antibodies and monoclonal antibodies (MAbs), as well as antibody derivatives such as chimeric molecules, humanized molecules, molecules with reduced effector functions, bispecific molecules, and conjugates of antibodies. In a preferred embodiment, the antibody has the antigen-specific binding characteristics of MAb Sc8, as described in U. S. Patent 5,474,771, the disclosure of which is hereby incorporated by reference. In a currently highly preferred embodiment, the antibody is a humanized 5c8. Other known antibodies against CD 154 include antibodies ImxM90, ImxM91 and ImxM92 (obtained from Immune), an anti-CD40L mAb commercially available from Ancell (clone 24-31, catalog # 353-020, Bayport, MN), and an anti- CD40L mAb commercially available from Genzyme (Cambridge, MA, catalog # 80- 3703-01). Also commercially available is an anti-CD40L mAb from PharMingen (San Diego, catalog #33580D). Numerous additional anti-CD40L antibodies have been produced and characterized (see, e. g., WO 96/23071 of Bristol-Myers Squibb, the specification of which is hereby incorporated by reference).

The invention also includes use of CD154 blocking agents of other types, such <BR> <BR> <BR> <BR> as complete Fab fragments, F (ab) 2 compounds, VH regions, Fv regions, single chain antibodies (see, e. g., WO 96/23071), polypeptides. fusion constructs of polypeptides, fusions of CD40 (such as CD40Ig, as in Hollenbaugh et al., J. Immunol. Meth. 188: 1-7, 1995, which is hereby incorporated by reference), and small molecule compounds such as small semi-peptidic compounds or non-peptide compounds, all capable of blocking or interrupting CD40: CD154 binding. Procedures for designing, screening and optimizing small molecules are provided in PCT/US96/10664, filed June 21,1996, the specification of which is hereby incorporated by reference.

Various forms of antibodies also can be produced using standard recombinant DNA techniques (Winter and Milstein, Nature 349: 293-99,1991). For example, "chimeric"antibodies may be constructed, in which the antigen binding domain from an animal antibody is linked to a human constant domain (an antibody derived initially from a nonhuman mammal in which recombinant DNA technology has been used to replace all or part of the hinge and constant regions of the heavy chain and/or the constant region of the light chain, with corresponding regions from a human

immunoglobin light chain or heavy chain) (see, e. g., Cabilly et al., United States Pat.

No. 4,816,567; Morrison et al.. Proc. Natl. Acad. Sci. 81: 6851-55,1984). Chimeric antibodies reduce the immunogenic responses elicited by animal antibodies when used for human therapy or prophylaxis.

In addition, recombinant"humanized"antibodies can be synthesized.

Humanized antibodies are antibodies initially derived from a nonhuman mammal in which recombinant DNA technology has been used to substitute some or all of the amino acids not required for antigen binding with amino acids from corresponding regions of a human immunoglobin light or heavy chain. That is, they are chimeras comprising mostly human immunoglobulin sequences into which the regions responsible for specific antigen-binding have been inserted (see, e. g., PCT patent application WO 94/04679). Animals are immunized with the desired antigen, the corresponding antibodies are isolated and the portion of the variable region sequences responsible for specific antigen binding are removed. The animal-derived antigen binding regions are then cloned into the appropriate position of the human antibody genes in which the antigen binding regions have been deleted. Humanized antibodies minimize the use of heterologous (inter-species) sequences in antibodies for use in human therapies, and are less likely to elicit unwanted immune responses. Primatized antibodies can be produced similarly.

Another embodiment of the invention includes the use of human antibodies, which can be produced in nonhuman animals, such as transgenic animals harboring one or more human immunoglobulin transgenes. Such animals may be used as a source for splenocytes for producing hybridomas, as described in U. S. 5,569,825.

Antibody fragments and univalent antibodies also can be used in practice of this invention. Univalent antibodies comprise a heavy chain/light chain dimer bound to the Fc (or stem) region of a second heavy chain."Fab region"refers to those portions of the chains which are roughly equivalent, or analogous, to the sequences which comprise the Y branch portions of the heavy chain and to the light chain in its entirety, and which collectively (in aggregates) have been shown to exhibit antibody activity.

A Fab protein includes aggregates of one heavy and one light chain (commonly known as Fab'), as well as tetramers which correspond to the two branch segments of the antibody Y, (commonly known as F (ab) 2), whether any of the above are covalently or

non-covalently aggregated, so long as the aggregation is capable of selectively reacting with a particular antigen or antigen family.

In addition, standard recombinant DNA techniques can be used to alter the binding affinities of recombinant antibodies with their antigens by altering amino acid residues in the vicinity of the antigen binding sites. The antigen binding affinity of a humanized antibody may be increased by mutagenesis based on molecular modeling (Queen et al., Proc. Natl. Acad. Sci. 86: 10029-33,1989; PCT patent application WO 94/04679). It may be desirable to increase or to decrease the affinity of the antibodies for CD40L, depending on the targeted tissue type or the particular treatment schedule envisioned. This may be done utilizing phage display technology (see, e. g., Winter et al., Ann. Rev. Immunol. 12: 433-455,1994; and Schier et al., J. Mol. Biol. 255: 28-43, 1996, which are hereby incorporated by reference). For example, it may be advantageous to treat a patient with constant levels of antibodies with reduced affinity for CD40L for semi-prophylactic treatments. Likewise, antibodies with increased affinity for CD40L may be advantageous for short-term treatments.

Routes of Administration The CD40: CD154 binding interruptors, including CD 154 blocking agents, used in the invention can be administered in any manner which is medically acceptable.

Depending on the specific circumstances, local or systemic administration may be desirable. Preferably, the agent is administered via a parenteral route such as by an intravenous, intraarterial, subcutaneous, intramuscular, intraorbital, intraventricular, intraperitoneal, subcapsular, intracranial, intraspinal, or intranasal injection, infusion or inhalation. The agent also can be administered by implantation of an infusion pump, a biocompatible or bioerodable sustained release implant or other drug delivery device (e. g., capsules, liposomes), into the recipient, either before or after implantation of the immunoisolation device. If desired, the implant for supplying the interruptor is disposed at or near the site of implantation of the immunoisolation device, so as to deliver the interruptor to the local environment of the immunoisolation device.

Alternatively, certain compounds of the invention, or formulations thereof, may be appropriate for oral or enteral administration. Still other compounds of the invention will be suitable for topical administration.

In further embodiments, the CD40: CD154 binding interruptor is provided indirectly to the recipient, by administration of a vector or other expressible genetic material encoding the interruptor. The genetic material is internalized and expressed in cells or tissue of the recipient, thereby producing the interruptor in situ. For example, a suitable nucleic acid construct would comprise sequence encoding one or more of the MAb 5c8 immunoglobulin (Ig) chains as disclosed in U. S. Pat. Other suitable constructs would comprise sequences encoding chimeric or humanized versions of the MAb 5c8 Ig chains or antigen-binding fragments thereof. Still other suitable constructs would comprise sequences encoding part or all of other CD 154- specific MAbs. The construct is delivered systemically or locally, e. g., to a site vicinal to the site of device implantation.

Alternatively, the vector or other genetic material encoding the interruptor is internalized within a suitable population of isolated cells to produce interuptor- producing host cells. These host cells then are implanted or infused into the recipient, either locally or systemically, to provide in situ production of the CD40: CD154 binding interruptor. Appropriate host cells include cultured cells, such as immortalized cells, as well as cells obtained from the recipient (e. g., autologous peripheral blood or lymph node cells, such as natural killer (NK) cells, or fibroblasts or muscle satellite cells).

Preferably, the host cells are implanted at or near the site of device implantation, and optionally may be attached to a support matrix or enclosed within a macroporous device (which allows the interruptor to diffuse freely).

Formulation In general, the CD40: CD154 binding interruptor compounds used in practice of the invention are suspended, dissolved or dispersed in a pharmaceutically acceptable carrier or excipient. The resulting therapeutic composition does not adversely affect the recipient's homeostasis, particularly electrolyte balance. Thus, an exemplary carrier comprises normal physiologic saline (0.15M NaCI, pH 7.0 to 7.4). Other acceptable carriers are well known in the art and are described, for example, in Remington's Pharmaceutical Sciences, Gennaro, ed., Mack Publishing Co., 1990.

Acceptable carriers can include biocompatible, inert or bioabsorbable salts, buffering

agents, oligo-or polysaccharides, polymers, viscosity-improving agents, preservatives, and the like.

Any CD40: CD154 binding interruptor, such as a CD 154 blocking agent, that is used in practice of the invention is formulated to deliver a pharmaceutically-effective or therapeutically-effective amount or dose, which is an amount sufficient to produce a detectable, preferably medically beneficial effect on the recipient. Medically beneficial effects would include preventing, delaying or attenuating deterioration of, or detectably improving, the recipient's medical condition. As an example, renal function and health of a kidney allograft or xenograft can be monitored by routinely measuring the concentrations of blood urea nitrogen or creatinine, or the volume or solute contents of urine, or the rate of clearance of relevant solutes from the blood into the urine.

Similarly, glucoregulatory function and health of insulin-producing allograft or xenograft can be monitored by routinely measuring the concentrations of blood or urine glucose, glucose metabolites, or insulin, or measuring insulin response to glucose challenge, e. g., in a conventional glucose tolerance test. Thus, an effective amount of a therapeutic compound of the invention, such as a CD154 blocking agent, is any amount which detectably decreases the recipient's dependence on exogenous drug or other therapy. An optimal effective amount is one which substantially frees the recipient of dependence on exogenous drug. More specifically, an effective amount is one which induces partial or substantially complete engraftment (acceptance and function) of the implanted immunoisolation device.

Dosages and Frequencv of Treatment The amount of and frequency of dosing for any particular compound to be used in practice of the invention is within the skills and clinical judgement of ordinary practitioners of the tissue transplant arts, such as transplant surgeons. The general dosage and administration regime is established by preclinical and clinical trials, which involve extensive but routine studies to determine effective, e. g., optimal, administration parameters for the desired compound. Even after such recommendations are made, the practitioner will often vary these dosages for different recipient hosts based on a variety of considerations, such as the recipient's age, medical status, weight, sex, and concurrent treatment with other pharmaceuticals. Determining

an effective dosage and administration regime for each CD40: CD154 binding interruptor used to inhibit graft rejection is a routine matter for those of skill in the pharmaceutical and medical arts. The dosage amount and timecourse of should be sufficient to produce a clinically beneficial change in one or more indicia of the recipient's health status. Numerous exemplary timecourse and dosage regimes will be apparent to skilled practitioners. In one exemplary embodiment, the invention involves administration of a CD40: CD154 binding interruptor (exemplified by a humanized MAb Sc8, hu5c8) in an acceptance-inducing regime, followed if deemed prudent by an acceptance-maintaining regime, as described in prior PCT/US98/12892.

To exemplify dosing considerations for an anti-CD40L compound, the following examples of administration strategies are given for an anti-CD40L MAb.

The dosing amounts could easily be adjusted for other types of anti-CD40L compounds. In general, single dosages of between about 0.05 and about 50 mg/kg patient body weight are contemplated, with dosages most frequently in the 1-20 mg/kg range. For acute treatment, such as before or at the time of implantation, or in response to any evidence that implant rejection is beginning, an effective dose of antibodies ranges from about 1 mg/kg body weight to about 20 mg/kg body weight, administered daily for a period of about 1 to 5 days, preferably by bolus intravenous administration.

The same dosage and dosing schedule may be used in the load phase of a load- maintenance regimen, with the maintenance phase involving intravenous or intramuscular administration of antibodies in a range of about 0.1 mg/kg body weight to about 20 mg/kg body weight, for a treatment period of anywhere from weekly to 3 month intervals. Chronic treatment may also be carried out by a maintenance regimen, in which antibodies are administered by intravenous or intramuscular route, in a range of about 0.1 mg/kg body weight to about 20 mg/kg body weight, with interdose intervals ranging from about 1 week to about 3 months. In addition, chronic treatment may be effected by an intermittent bolus intravenous regimen, in which between about 1.0 mg/kg body weight and about 100 mg/kg body weight of antibodies are administered, with the interval between successive treatments being from 1 to 6 months. For all except the intermittent bolus regimen, administration may also be by oral, pulmonary, nasal or subcutaneous routes.

If desired, the effectiveness of the antibodies can be increased by administration serially or in combination with conventional anti-rejection therapeutic agents or drugs such as, for example, corticosteroids or immunosuppressants. Alternatively, the antibodies may be conjugated to a conventional agent. This advantageously permits the administration of the conventional agent in an amount less than the conventional dosage, for example, less than about 50% of the conventional dosage, when the agent is administered as monotherapy. Accordingly, the occurrence of many side effects associated with that agent should be avoided.

Combination therapies according to this invention for treatment of implant rejection include the use of anti-CD40L antibodies together with agents targeted at B cells, such as anti-CD19, anti-CD28 or anti-CD20 antibody (unconjugated or radiolabeled), IL-14 antagonists, LJP394 (LaJolla Pharmaceuticals receptor blocker), IR-1116 (Takeda small molecule) and anti-Ig idiotype monoclonal antibodies.

Alternatively, the combinations may include T cell/B cell targeted agents, such as CTLA4Ig, IL-2 antagonists, IL-4 antagonists, IL-6 antagonists, receptor antagonists, anti-CD80/CD86 monoclonal antibodies, TNF, LFA1/ICAM antagonists, VLA4/VCAM antagonists, brequinar and IL-2 toxin conjugates (e. g., DAB), prednisone, anti-CD3 MAb (OKT3), mycophenolate mofetil (MMF), cyclophosphamide, and other immunosuppressants such as calcineurin signal blockers, including without limitation, tacrolimus (FK506). Combinations may also include T cell targeted agents, such as CD4 antagonists, CD2 antagonists and IL-12.

For maintenance of implant integration, or in a period following suppression of an acute episode of implant rejection, a maintenance dose of anti-CD40L antibodies, alone or in combination with a conventional anti-rejection agent is administered, if necessary. Subsequently, the dosage or the frequency of administration, or both, may be reduced. Where no sign of implant rejection is evident, treatment might cease, with vigilant monitoring for signs of rejection. In other instances, as determlned by the ordinarily skilled practitioner, occasional treatment might be administered, for example at intervals of four weeks or more. Recipients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.

Pre-Clinical Model Svstems for Evaluatinz CD40: CD154 Interruotor Treatment Ré rimes The following published, well characterized exemplary model systems can be routinely adapted for testing the efficacy of a CD40: CD154 interrupting compound (e. g., an anti-CD40L compound or a CD154 blocking agent, such as a MAb having the specificity of MAb 5c8) for use in the present invention for inhibiting rejection of immunoisolated cells or tissue: The progressive motoneuropathy (pmn) mouse system described in Sagot et al.

(1995), 7 Eur. J. Neurosci. 1313-1322 for testing neurodegenerative diseases such as amyotrophic lateral myopathy; The lesion-induced rotational asymmetry system (hemiparkinsonism) in rat described in Aebischer et al. (1991), 560 Brain Res. 43-49; Date et al. (1996), 7 Neuroreport. 1813-1818; Date et al. (1996), 84 J. Neurosure. 1006-1012; Date et al.

(1997), 147 Exp. Neurol. 10-17; Lindner et al. (1998), 7 Cell. Transplant 165-174 for Parkinson's disease; The unilateral transection system in cynomolgus monkeys described in Emerich et al. (1994), 349 J. Comp. Neurol. 148-164 for Alzheimer's disease; The poultry and dwarf mouse systems described, respectively, in Vasilatos- Younken (1987), 66 Poult. Sci. 899-903 and Chang (1997), 831 Ann. N. Y. Acad. Sci.

461-473 for dwarfism; The galactosamine induction model system in mice described in Wong et al.

(1988), 16 Biomater. Artif. Cells Artif. Organs 731-739 for liver failure, along with the Gunn rat model system described in Bruni et al. (1989), 17 Biomater. Artif. Cells Artif.

Organs 403-411 for hyperbilirubinemia; The uremia model system in rats described in Chang (1998), 22 Artif. Organs 958-965 for renal failure; and, The Gus/Gus mouse model system described in Chang (1997), 831 Ann. N. Y.

Acad. Sci. 461-473 for lysosomal storage disease.

Equivalents The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative of, rather than limiting on, the invention disclosed herein. Scope of the invention thus is indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are intended to be embraced therein.