AU710637B2 - Method for synthesizing microcapsules of predetermined permeability - Google Patents

Description

WO 97/11685 PCT/US96/15649 METHOD FOR SYNTHESIZING MICROCAPSULES OF PREDETERMINED PERMEABILITY This application claims the benefit of U.S. Provisional Application No. 60/018,524, filed May 28, 1996, and of U.S.

Provisional Application No. 60/004,375, filed September 27, 1995, the contents of both of which are hereby incorporated by reference into the present application.

Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of this application, preceding the claims.

Background of the Invention There is a critical need for better insulin replacement therapy to circumvent the complications of insulindependent diabetes mellitus (IDDM). It would therefore be useful to develop techniques for transplantation of microencapsulated, xenogeneic islets to provide a durable, physiological source of insulin to diabetic patients. It has previously been shown that microcapsules are biocompatible and that xenogeneic islet grafts contained in microcapsules functioned indefinitely in the peritoneal cavity of mice with streptozotocin-induced (SZN) diabetes.

Thus, microcapsules may be intact and stable in vivo and factors that may be required for long-term survival and function of the xenogeneic islets are accessible. The microcapsules serve as a mechanical barrier that prevents cell-to-cell contact between recipient lymphocytes and donor islets. The mechanical barrier primarily prevents host sensitization rather than protecting the graft from WO 97/11685 PCT/US96/15649 -2immune destruction, because encapsulated islets are very rapidly destroyed by recipients that are presensitized to the islet donor cell antigens. Similarly, encapsulated xenogeneic islets were rejected (in two weeks) by NOD mice, which is possibly due to presensitization of NODs to islet antigens. Xenografts undergoing rejection in NOD mice were surrounded by large numbers of activated macrophages and immunoglobulins, with IL-la, TNFa, both documented by immunocytochemistry, and IL-4 messenger RNA detected by RT-PCR.

We have been able to improve the microencapsulation process to permit long-term survival of concordant, rat islet xenografts, even in NOD mice.

Insulin-Dependent Diabetes Mellitus The last several years have witnessed a remarkable increase in or knowledge of the effects of therapies for insulindependent diabetes mellitus (IDDM). The Diabetes Control and Complications Trial (DCCT) found that intensive insulin therapy delayed the onset and slowed progression of retinopathy, nephropathy, and neuropathy in patients with IDDM Unfortunately, intensive insulin therapy is not appropriate for many IDDM patients; and even with careful monitoring, DCCT patients had increased episodes of severe hypoglycemia Ironically, results of the DCCT support the rationale for pancreas and islet transplantation.

Since the inception of islet transplant experiments, it has been the hope that such grafts might supply insulin more homeostatically than exogenous insulin can, and that 'nearnormal' modulation of carbohydrate metabolism might prevent the secondary complications of IDDM Clinical pancreas allografts have improved outcomes with the advent of combination immunosuppression; and near normal of glucose homeostasis follows most pancreatic allo- and auto-grafts However, the first-year mortality of a human WO 97/11685 PCT/US96/15649 3 pancreatic allograft remains high immunosuppression is required, and only limited numbers of clinical wholeorgan pancreatic transplants are being done worldwide The Rationale for Microencapsulated Islet Xenografts Islet transplantation is an attractive therapy for patients with IDDM, since problems related to the exocrine pancreas may be avoided. However, allografts of donor human islets have not been successful long-term and availability and yield of human islets are limited. Therapeutic islet transplants for large number of patients almost certainly will require donor islets harvested from animals (xenografts) The optimal source of xenogeneic islets for clinical use remains controversial. Islets have been isolated from subhuman primates and xenografted into immunosuppressed, diabetic rodents, with short-term reversal of diabetes However, there are significant ethical issues surrounding use of primates, Other promising sources are porcine, bovine, canine, and rabbit islets, which function remarkably well, maintaining normoglycemia) in diabetic rodents until transplant rejection occurs (7-11).

Long-term human, bovine and porcine islet xenograft survival has been documented in nude mice and rats, suggesting that sufficient islet-specific growth factors are present in xenogeneic recipients (2,12-17). For sociologic/ethical reasons, canine islets are not clinically appropriate. Porcine islets are both difficult to isolate (intact) and to maintain in vitro; nevertheless, they are extremely promising for eventual clinical application (18-21). Isolation of bovine islets is technically easier (than porcine islets), and calf islets are glucose-responsive Recently, large scale rabbit WO 97/11685 PCTIUS96/15649 4 islets isolation has been developed (23) (see Preliminary Studies). Rabbit pancreas is an attractive source of islets. Rabbit, like porcine insulin, differs from human insulin at only one amino acid, and rabbit islets are glucose responsive (22,24). In addition, most humans do not possess natural anti-rabbit antibodies, which might improve the possibility of preventing xenograft rejection It is currently feasible to consider isolation of 1,000,000 donor islets/per human diabetic recipient from either calves, pigs or rabbits, utilizing multiple donors.

The most significant obstacle to islet xenotransplantation on human IDDM is the lack of an effective immunosuppressive regiment to prevent cross-species graft rejection (2,26- 28) Recently, it has been reported that human islets will survive long-term in SZN-diabetic mice treated either with anti-CD4 antibody (16) or CTLA4Ig (a high affinity fusion protein which blocks CD28-B7 interactions) or by exposure of donor islets to purified high affinity anti-HLA (ab) 2 However, with the exception of these studies, indefinite survival of islet xenografts has rarely been achieved, except with the aid of porous, mechanical barriers. Both intra- and extra-vascular devices are under development. However, potential clinical complications, such as bleeding, coagulation, and bioincompatibility mitigate against their current use in diabetic patients (30,31). For example, acrylic-copolymer hollow fibers placed subcutaneously maintained viability of human islet allografts for two weeks (50 islets per 1.5 cm fiber) (65,000 M.W. permeability) However, to implant 500,000 islet would require >150 meters of these hollow fibers, which is not clinically feasible.

One of the most promising islet envelopment methods is the polyamino acid-alginate microcapsule. A large number of

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WO 97/11685 PCT/US96/15649 5 recent studies have shown that intraperitoneal xenografts of encapsulated rat, dog, pig or human islets into streptozotocin-diabetic mice or rats promptly normalized blood glucose for 10-100' days (7,19,33-39). Long-term normalization of hyperglycemia by microencapsulated canine islet allografts, porcine islet xenografts, and one human islet allograft has been reported (21,40-42). The mechanisms by which microcapsules protect islet xenografts from host destruction are not fully understood. However, it has been suggested that prohibition of cell-cell contact with host immunocytes is important (30,35). The marked prolongation of widely unrelated encapsulated islet xenografts in rodents with induced diabetes has prompted studies in animals with spontaneous diabetes.

The Spontaneously Diabetic NOD Mouse As A Model Of Human

IDDM

Nonobese diabetic (NOD) mice develop diabetes spontaneously, beginning at approximately twelve weeks of age. NOD mice are the most appropriate model for studying the feasibility of islet xenotransplants because their disease resembles human IDDM in several ways. Macrophage, dendritic cell and lymphocytic infiltration of islets can be detected as early as four weeks of age and precedes overt hyperglycemia (43-46). NOD diabetes is T lymphocytedependent (43-45); and it is associated with (MHC) Class II genes (47-50). Cytotoxic T cells and antibodies specific for beta cells or for insulin have been identified, characterized and cloned from NOD mice (44,45,51-55) Loss of tolerance to islet antigens in NODs correlates with appearance of Thl immune responses to glutamic acid decarboxylase, a factor which has been reported to be a primary auto-antigen in human IDDM (55,56,57). The disease can be induced in non-diabetic, syngeneic mice by transfer of both CD8' and CD4' T cells or T-cell clones from diabetic

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WO 97/11685 PCT/US96/15649 6 NODs (44,52,55,58); and inhibition of NOD macrophages or CD4' T lymphocytes or treatment with anti-Class II monoclonal antibodies prevents or delays diabetes onset in NOD mice (59,50). Defects in NOD macrophages, complement and NK cell function have been reported (61).

It has been suggested that helper T-cells function to activate CD8 cells, which damage beta cells by direct cytotoxic attack. However, some recent studies have suggested that beta cell killing may be indirect, from a nonspecific inflammatory response which initially involves CD4' cells, but also includes infiltrating macrophages, which release cytokines and oxygen free-radicals (particularly nitric oxide), known beta cell toxins (62- Because of similarities to IDDM, NOD mice are the best model in which to study islet xenografts.

Recently, the Scid mutation has been back-crossed onto the NOD background, resulting in immuno-deficient NOD-Scid mice (66-69). These mice homologous for the Scid mutation, which results in an inability to rearrange T-cell receptor and immunoglobulin genes (66,67). The consequence is an absence of T and B-lymphocytes. These mice do not develop diabetes spontaneously; but they may be rendered diabetic with multiple low-dose streptozotocin (MLD-SZN) regimens, making them an optimal model for adoptive transfer experiments (67-69). NOD-Scids express NOD MHC genes and other genes that are relevant for development of the disease. They mount robust macrophage and limited NK-cell responses, but are functionally T- and B-lymphocyte deficient (69).

Islet Xenografts into Diabetic NOD Mice Unlike mice with SZN-induced diabetes, diabetic NOD mice rapidly reject unencapsulated islet xenografts, allografts WO 97/11685 PCT/US96/15649 7 and isografts (7,8,10,19,33,56,70,71). Conventional immunosuppressive regimens have little effect on this reaction (10,71-73). Treatment of NOD recipients with monoclonal antibodies directed against CD4' helper T lymphocytes or FK506 prolongs islet graft function (from to 25 days)(7,8,10,73); but long-term islet graft survival in NODs has not been reported.

Several laboratories have reported that intraperitoneal microencapsulated islets (allo- and xeno-geneic) function significantly longer than non-encapsulated controls, but eventually are destroyed also by recipients with spontaneous (autoimmune) diabetes (NOD mice or BB rats) (7,9,19,33,35,70,74-78). Rejection is accompanied by an intense cellular reaction, composed primarily of macrophages and lymphocytes, which entraps islet-containing microcapsules and recurrence of hyperglycemia within 21 days, in both NOD and BB recipients (7,19,74,76,77). The mechanism of encapsulated islet rejection by animals with spontaneous diabetes remains incompletely understood, but the fact that it rarely occurs in mice with induced (SZN) diabetes suggests that anti-islet autoimmunity may be involved in islet graft destruction.

Mechanisms of NOD Destruction of Encapsulated Islet Xenografts: Macrophages, T-Cells, and Cytokines It has been suggested by several investigators that microcapsules, like other bioartificial membrane devices promote survival of xenogeneic and allogeneic islets by: preventing or minimizing release of donor antigen(s), thereby reducing host sensitization, and/or preventing or reducing host effector mechanisms T-cell contact, anti-graft antibody binding, cytokine release).

Most studies of rejection of islets in microcapsules and WO 97/11685 PCT/US96/15649 8 other membrane devices have focused on effector mechanisms.

For example, Halle (35) and Darquy and Reach (79) reported that microcapsules protected donor islets from host immunoglobulins, specifically human anti-islet antibodies and complement effects, in vitro. Although complement components, are too large (>>150,000 Kd) to enter conventional poly-l-lysine microcapsules, it is possible that antibodies combine with shed donor antigens forming complexes which bind to FcR of macrophages in vivo (in the peritoneal cavity) which could initiate cytokine release causing encapsulated islet destruction Complement could facilitate binding of complexes to macrophages via the C3b receptor or by the release of chemotactic peptides that could increase the number of macrophages.

Involvement of NOD T-lymphocytes in rejection of encapsulated islets has been proposed by Iwata, et al.

who found significant prolongation of encapsulated hamster-to-NOD mouse encapsulated islet xenografts when NOD recipients were treated with deoxyspergualin (DSG), a Tcell inhibitory immunosuppressant (81) This data is consistent with prior finding of several laboratories, that treatment of NODs with monoclonal antibodies directed against CD4' helper T cells or FK-506 prolonged function of both encapsulated and nonencapsulated rat-to-NOD islet xenograft (7,8,10,73) and these finding are similar to observations of Auchincloss Pierson (82) and Gill that CD4* T cells play a dominate role in xenoreactivity.

A prominence of macrophages/monocytes in peri-microcapsular infiltrates of encapsulated islet allografts and xenografts in NOD mice and BB rats has been reported (7,33,36,74,76- 78,84). Cytokines known to be products of macrophages, including IL-1 and TNF (62,77,85,86), may be involved WO 97/11685 PCTIUS96/15649 9 destruction of encapsulated islets. Both IL-1 and TNF have been reported to reduce insulin secretion and cause progressive damage of islet cells in vitro (58,62-64,85- 87). Cytokine-mediated injury might occur directly or indirectly, by activation of an intraperitoneal inflammatory response (30,77). Recently, it has been reported by Dr. J. Corbett (IPITA conf. 6/95), that there are as many as ten macrophages within each islet. IL-1 induces nitric oxide synthase (NOS) (63-65), with resultant generation of nitric oxide which causes injury to mitochondria and to DNA in beta cells (63-65).

Furthermore, this pathway of islet damage is worsened by TNF (88,89). Theoretically, macrophages from within donor islets and host peritoneal cavity or within the down islets could be involved in cytokine-mediated damage to encapsulated islets.

Studies of cytokine messenger RNA profiles in hamster-torat liver and pig-to-mouse islet xenografts have found selective increases in Th2 cytokines (IL-4, IL-5, and no change from normal in IL-2 (11,90). These are distinctly different from those of O'Connell, et al.

(91,92), who reported IL-2 messenger RNA in biopsies of allograft rejections of nonencapsulated islets. Increased Th2 activity relative to Thl (93-95) activity is distinct from the known NOD 'Thl' anti-islet immune response (56,57,96). The Th2 response is characteristic of evoked antibody responses to foreign antigens and suggests that humoral reactions to encapsulated xenografts may be of critical importance. Furthermore, strategies designed to abrogate 'Th2' responses may significantly prolong encapsulated islet xenograft survival. The 'Th2' helper Tcell cytokine mRNA profile is characteristic of antibody responses to foreign antigens.

WO 97/11685 PCT/US96/15649 10 Costimulatory Molecules, APC's and Islet Xenograft Destruction by NOD Mice Involvement of APCs in immune responses to islet xenografts is suggested by recent studies of Lenschow, et al. (12), who found that blockade of the co-stimulatory molecule, B7 with the soluble fusion protein, CTLA4Ig, prolonged humanto-mouse islet xenografts in SZN-diabetic mice. Several studies, in vitro and in vivo, have shown that foreign molecules which interact with the T cell receptor (peptides, specific antibodies, mitogens) fail on their own to stimulate naive T cells to proliferate (95,97), and may induce antigen-specific anergy. At least one additional (costimulatory) signal is required, and it is delivered by APCs. In mice, one such costimulatory pathway involves the interaction of the T-cell surface antigen, CD28 with either one of two ligand, B7-1 and B7-2, on the APCs (95,97-102).

Once this full interaction of T-cells and APCs occurs, however, subsequent re-exposure of T-cells to peptide, mitogen, etc. will result in proliferation in the absence of costimulation. Recent studies have further illuminated helper T-cell-APC interactions, with recognition of the importance of binding of the APC-CD40 antigen to its ligand, GP39, on helper Tcells (103,104). A monoclonal hamster anti-murine GP39 antibody (MR1) blocks helper T-cell interactions with APCs, macrophages, effector T-cells and B-lymphocytes (103,104).

Dr. A. Rossini has reported recently (IPITA conf. 6/95) that MR1 plus B7 negative donor spleen cells day 7 allows long-term survival of both allo- and xeno-geneic islets in SZN-diabetic mice.

The Immunogenicity of Encapsulated Islets and Mechanisms of Graft of Destruction Empty microcapsules have been reported to elicit no WO 97/11685 PCT/US96/15649 11 cellular responses (33,35,36). On the other hand, others have found reactions to empty capsules, (30,76,77,105,106).

Impurities in reagents such as contamination with endotoxin or high concentrations of mannuronate most likely contribute to bioincompatibility (107). It is apparent that some formulations of poly-1-lysine microcapsules are biocompatible and some are not. Until standardized reagents are available, immunologic studies are microencapsulated islets can only be interpreted when investigators include empty microcapsule controls which document their biocompatibility.

Recently, de Vos, et al. (108) reported incomplete encapsulation or actual protrusion of islets through microcapsule membranes in some microcapsules, and suggested this biomechanical imperfection is one factor in microcapsule destruction. Similar observations have been made by Chang (109), who found incorporation of islets and hepatocytes within the walls of poly-l-lysine alginate microcapsules. Several other investigators have published photomicrographs of encapsulated islets showing obvious entrapment of islets in capsules, walls, but did not comment on this problem (35,110,111). Incomplete encapsulation would be anticipated to result in premature capsule fracture and exposure of donor islets to host cells; but there are no reports analyzing this as a source of donor antigen exposure, sensitization and host.

Relatively few studies have focused on the role of donor islet antigen(s) released from microcapsules in initiating host immune responses. Ricker, et al. (33) reported similar, intense cellular reactions by NOD mice to rat insulinoma, hepatoma and pheochromocytoma cell lines in microcapsules and concluded that the NOD immune reaction was not islet-specific. Horcher, et al. (36) reported

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WO 97/11685 PCT/US96/15649 12 week survival of 6/7 encapsulated Lewis rat islet isografts, compared to failure of 8/10 encapsulated Wistarto-Lewis islet allografts within 56 days. Isograft biopsies showed viable islets, intact capsules and no pericapsular immune reaction while biopsies of failed allografts revealed pericapsular cellular responses and nonviable islets. This is the only report in the literature with encapsulated islet isograft controls.

Although the Lewis rat model is not one with autoimmune diabetes, the results are significant, and suggest that donor antigen(s) are the stimulus for subsequent host responses.

Improving Microcapsule Design Xenotransplantation of microencapsulated islets is, potentially, an ideal treatment for diabetes in that insulin is delivered homeostatically, no immunosuppression is needed and the islet source is plentiful. Microcapsules prevent direct contact between donor islets and host lymphocytes, macrophages, platelets and xenoreactive antibodies. Rejection occurs via cytokines released from host cells sensitized by soluble xenoantigens that traverse the capsule.

Systematically altering capsule porosity would provide a useful tool to characterize the size range of those immunological constituents which sensitize the host. For purposes of this invention, the effect on capsule diffusion properties of PLL molecular weight changes, (ii) concentration changes, as well as, (iii) polyamino acid substitutes were analyzed.

WO 97/11685 PCT/US96/15649 13 Summary of the Invention The subject invention provides an assay for determining a poly-l-lysine concentration suitable for synthesizing microcapsules having a predetermined permeability, which assay comprises encapsulating a material which produces or is capable of producing detectable particles of a known molecular weight in a series of microcapsules, each microcapsule of said series being synthesized using a different poly-l-lysine concentration; initiating production of the detectable particles in each microcapsule if the material encapsulated in step is not already producing such particles; detecting detectable particles diffusing from each microcapsule in said series, thereby determining the permeability of each microcapsule in said series; ascertaining the presence or absence of a microcapsule in the series having the predetermined permeability, and; repeating steps through if a microcapsule having the predetermined permeability is absent, but varying the poly-l-lysine concentration used to synthesize each microcapsule in the series in step The subject invention further provides a method of synthesizing microcapsules having a predetermined permeability which comprises determining a suitable poly-1lysine concentration according to the aforementioned assay, and synthesizing microcapsules using the determined poly-llysine concentration.

Brief Description of the Drawings Figure 1: Encapsulated Lewis rat islet, day #150 after xenografting to unmodified diabetic NOD H&E. (x250). The microcapsule is a "double-wall" microcapsule.

Figure 2: Survival of islet xenograft, "double-wall"

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WO 97/11685 PCT/US96/15649 14 microcapsule.

Figure 3: Comparison of survival of rabbit islets encapsulated in microcapsules with a permeability of up to 70,000 Kd to survival of rabbit islets in microcapsules having a permeability of 100,000 Kd.

Detailed Description of the Invention This invention provides an assay for determining a poly-llysine concentration suitable for synthesizing microcapsules having a predetermined permeability, which assay comprises encapsulating a material which produces or is capable of producing detectable particles of a known molecular weight in a series of microcapsules, each microcapsule of said series being synthesized using a different poly-l-lysine concentration; initiating production of the detectable particles in each microcapsule if the material encapsulated in step is not already producing such particles; detecting detectable particles diffusing from each microcapsule in said series, thereby determining the permeability of each microcapsule in said series; ascertaining the presence or absence of a microcapsule in the series having the predetermined permeability, and; repeating steps through if a microcapsule having the predetermined permeability is absent, but varying the poly-l-lysine concentration used to synthesize each microcapsule in the series in step The subject invention also provides a method of synthesizing microcapsules having a predetermined permeability utilizing the aforementioned assay for determining a suitable poly-l-lysine concentration. This method of synthesizing microcapsules comprises determining a suitable poly-l-lysine concentration according to the WO 97/11685 PCT/US96/15649 15 invented assay, and synthesizing microcapsules using the poly-l-lysine concentration so determined.

Encapsulation of material in a microcapsule using poly-llysine is well-known in the art. Briefly, material is suspended in from 1.85% 2.0% sodium alginate in saline and droplets containing the material in alginate are produced by extrusion through a 22 gauge air-jet needle.

Droplets of alginate containing the material flow from a height of approximately 2 cm into a beaker containing 1.1% calcium chloride in saline. The negatively charged alginate droplets bind calcium and form a calcium alginate gel. The calcium alginate gel is temporary in that it may be solubilized under suitable conditions. Gelled droplets are decanted and subsequently incubated in calcium chloride for 10 minutes.

Thereafter, sequential washes with more dilute calcium chloride solutions are followed by a final wash in saline, after which .5 mg/ml poly-l-lysine is added and a subsequent incubation of 6 minutes allows for the positively charged poly-l-lysine to displace calcium ions and bind negatively charged alginate, producing a polyelectrolyte membrane. Initial studies utilized poly-llysine of 57,000 molecular weight. More recent studies have utilized smaller molecular weight poly-l-lysine (18- 25,000 molecular weight). Following the poly-l-lysine incubation, microcapsules are washed in CHES and a final 0.185% 0.2% solution of sodium alginate is added, forming a thin outer coating of sodium alginate. Finally capsulates are incubated in 55 mM sodium citrate, which solubalizes any calcium alginate which has not reacted with poly-l-lysine. This method produces what has been termed a "single-wall" capsule.

WO 97/11685 PCT/US96/15649 16 In an attempt to enhance the strength of the capsule wall, what has been termed the "double-wall" technique has been devised and published in detail (112, 114, 115).

Basically, after completion of the "single-wall" method, but prior to the sodium citrate incubation, additional poly-l-lysine is added and a second incubation of about 6 minutes is followed by a subsequent wash in CHES followed by an additional reincubation in dilute 0.185% 0.2% sodium alginate, followed by a final sodium citrate incubation.

Certain variations on the above-described encapsulation process are known in the art, and any such variation may be used in the subject invention.

As used herein, the term "microcapsule" means any polyamino acid spherical capsule. As explained above, microcapsules and their methods of manufacture are well known in the art and include, but are not limited, single layered, double layered, or multilayered polyamino acid spheres, as well as polyamino acid spheres comprising a layer or more than one layer of alginate.

In the subject assay, the concentration of the poly-llysine is varied in the encapsulation process so as to produce a series of microcapsules, each synthesized from a different poly-l-lysine concentration.

For purposes of the subject assay, detectable particles of known molecular weight, as well as material producing or capable of producing them, are well known to those of ordinary skill in the art. For example, the material for the subject assay may be a composition comprising particles of a known molecular weight, which particles are capable of fluorescing. Alternatively, the particles may be WO 97/11685 PCT/US96/15649 17 detectable in that a change in opacity of a medium into which the particles are diffusing may be detected with, for example, a spectrophotometer.

Examples of particles of known molecular weight which are detectable on the basis of their ability to fluoresce include fluoresceinated polymers, such as fluoresceinated dextrans, which may be obtained commercially or synthesized in a variety of different molecular weights.

In another example, the material is a composition comprising red blood cells, and the detectable particles in step are produced from the red blood cells after lysing the red blood cells so as to release hemoglobin from the red blood cells. In this example, the detectable substance of known molecular weight is the released hemoglobin, the diffusion of which from the microcapsules in the series may be detected with a spectrophotometer.

For purposes of the subject invention, "predetermined permeability" is any desired permeability. A predetermined permeability may be desired based on such factors as the size of cells or tissue to be contained within microcapsules, the size of any substances needed to permeate the microcapsules in order to sustain cells or tissue contained therein, or the size of a biologically active substance secreted by cells contained within microcapsules, diffusion of which from the microcapsules is desired. In one embodiment, the predetermined permeability is impermeable to lymphocytes. In another embodiment, the predetermined permeability is impermeable to lymphocytes and immunoglobulins. Impermeability to immunoglobulins and/or lymphocytes prevents contact between the immunoglobulins and/or lymphocytes of a host and viable cells contained within such microcapsules, and thereby I I WO 97/11685 PCT/US96/15649 18 prevents destruction of the contained cells which would result from such contact.

The subject assay may be used to determine a poly-l-lysine concentration capable of synthesizing microcapsules having the maximum permeability for preventing diffusion of a specific substance. Thus, an ideally permeable microcapsule for excluding or permitting diffusion of any specific substance may be synthesized according to the subject method. In one embodiment, the specific substance is viable cells or tissue chosen for encapsulation within microcapsules. The viable cells or tissue may be chosen for encapsulation within microcapsules for purposes of transplantation into a host.

Accordingly, the subject invention also provides microcapsules and compositions comprising microcapsules, which microcapsules have a predetermined permeability. The predetermined permeability may be a desired pore size for excluding certain substances, for example immunoglobulin, from the microcapsules. Furthermore the predetermined permability of the microcapsules may be a desired pore size for preventing certains substances, such as an antigen produced by a cells or tissue encapsulated within the microcapsules, from diffusing from the microcapsules. The predetermined permeability may also be the minimum pore size for permitting diffusion of a biological substance from the microcapsules, and/or the minimum pore size for permitting cellular nutrients, such as albumin or oxygen, to diffuse into the microcapsules.

In one embodiment, a composition of microcapsules having a predetermined permeability is a pharmaceutical composition comprising a pharmaceutically acceptable carrier. In a further embodiment, the pharmaceutical composition WO 97/11685 PCT/US96/15649 19 comprising microcapsules having a predetermined permeability according to the subject invention is a pharmaceutical composition suitable for transplantation into a host, for example by injection into the host.

For purposes of the subject invention, the viable cells or the tissue may be derived from any source for viable cells.

In one embodiment, the viable cells or tissue are derived from a xenogeneic donor, i.e. a donor which is a different species from the host into which the viable cells or tissue are transplanted. In another embodiment, the viable cells or the tissue comprising the viable cells are derived from an allogeneic donor, i.e. a donor which is of the same species as the host into which the viable cells or tissue are transplanted. In a further embodiment, the viable cells or the tissue comprising the viable cells are derived from the host into which they are transplanted, i.e. they are, inter alia, obtained from the host, encapsulated, and transplanted back into the host. Viable cells obtained from a host may, for example, be genetically engineered after they are obtained and before they are transplanted back into the host.

The host may be any host into which transplantation of viable cells is desired. Examples of hosts include, but are not limited to, humans and domesticated animals. If the host is a human, the viable cells, or tissue containing them, are in one embodiment derived from a mammal, for example a human.

As used herein, a domesticated animal is any animal subjected to human intervention. Domesticated animals include, for example, farm animals which are raised by humans and which are used as a resource for products for human consumption. Such products include, but are not WO 97/11685 PCT/US96/15649 20 limited to, meat, milk, and leather. Examples of domesticated animals include, but are not limited to, cows, pigs, sheep, horses, and chickens. Domesticated animals useful in applications of the subject invention may be adults, infants, or domesticated animals at any other developmental stage. Moreover, if the host is a domesticated animal, the viable cells may comprise cells which secrete a hormone which promotes growth in the domesticated animal. Such hormones are well known to those of ordinary skill, including hormones such as growth hormone and insulin. The viable cells secreting such a hormone are in one embodiment genetically engineered to secrete the hormone. That is they have been genetically engineered to contain the gene encoding the hormone and are capable of expressing the gene.

In this invention, the viable cells or tissue in one embodiment comprise cells which secrete a biologically active substance. The term "biologically active substance" as used herein means any substance which is capable of eliciting a physiological response in a subject. The biologically active substance may illicit a response in a host into which the microcapsules containing the cells or tissue producing the substance are transplanted. Cells which secrete biologically active substances are well known in the art, and any such cells may be used in the subject invention. Examples of cells which secrete a biologically active substance include, but are not limited to, endocrine cells, such as insulin-producing cells, hepatocytes, parathyroid cells, and pituitary cells; and neuroectodermal cells, such as adrenal cells and lymphocytes.

Furthermore, microcapsules synthesized according to the subject invention may contain cells which are genetically engineered to secrete a biologically active substance. For WO 97/11685 PCT/US96/15649 21 example, the cells may be genetically engineered to secrete a biologically active substance useful for treating a host into which they are transplanted. Thus, microcapsules synthesized according to the subject invention provide an advantageous drug delivery system for treatment of subjects afflicted with conditions including, but not limited to, cancer and HIV infection. If the subject is afflicted with cancer, the microcapsules may contain viable cells which, for example, may be genetically engineered to secrete Interleukin-2, a cytokine, or a lymphokine. If the subject is infected with HIV, the microcapsules may contain viable cells genetically engineered to secrete a substance which stimulates lymphocyte production in the subject, such as a T cell growth factor or the HIV T cell receptor.

In another embodiment, the subject assay is used to determine a poly-l-lysine concentration capable of synthesizing microcapsules having the maximum permeability for preventing diffusion of an antigen or antigens produced by cells or tissue chosen for encapsulation within the microcapsules.

As explained above, this invention also provides a method of synthesizing microcapsules having a predetermined permeability by determining a suitable poly-l-lysine concentration for synthesizing a microcapsule having the predetermined permeability according to the aforementioned assay, and synthesizing microcapsules using the determined poly-1-lysine concentration.

This invention will be better understood from the "Experimental Details" section which follows. However, one skilled in the art will readily appreciate that the specific methods and results discussed therein are not intended to limit, and rather merely illustrate, the WO 97/11685 PCT/US96/15649 22 invention as described more fully in the claims which follow thereafter.

Experimental Details Improvements in Microcapsule Design An improved formulation of poly-l-lysine-alginate microencapsulation which allows nearly indefinite survival of rat islets in spontaneously diabetic NOD mice is the "double-wall" microcapsule (Figures 1 and This doublewall microcapsule is more durable than conventional microcapsules, with fewer capsule wall defects, has a measured membrane permeability of approximately 100,000 Kd, and excludes IgG (unlike conventional design capsules, which allowed passage of IgG and 148,000 Kd fluoresceinated dextran) (9,19,20,112). These data support the relevance of encapsulated islet xenografts for eventual application in humans with IDDM.

Poly-L-Lysine (PLL) Concentration Alters Permeability of PLL-Alginate Microcapsules Methods: Five drops of packed Lewis rat red blood cells (RBC) were mixed with 8.5 mL of 2.0% sodium alginate (Kelrone LV,Lot #91502A Kelco, San Diego, Ca) and gently mixed for ten minutes. This mixture was extruded through an air jet needle into 10mL of 1.1% CaC12. The resulting capsules were then treated according to the protocol displayed in Tables 1 and 2.

Table 1: Encapsulation Protocol Step Incubation Solution Incubation Time Washes 1 1.1% CaC1 2 10 minutes 0.58% CaC12: 0.28% CaCI 2 saline 2 Polyamino Acids (Table 2) 6 minutes CHES. saline 3 0.2% Alginate 4 minutes saline 4 repeat step 2 6 minutes CHES. saline repeat step 3 4 minutes saline 6 56mM Na Citrate 10 minutes Three in saline washes WO 97/11685 PCT/US96/15649 24 Table 2: Polyamino Acid Variables for Step 2 of the Encapsulation Poly Amino Acid Molecular Weight (Kd) Concentration w/v) Poly-l-lysine 29.5 0.0500% Poly-l-lysine 29.5 0.1250% Poly-l-lysine 29.5 0.1375% Poly-l-lysine 29.5 0.1438% Poly-l-lysine 29.5 0.1500% Poly-l-lysine 29.5 0.2000% Poly-l-lysine 134 0.0500% Poly-l-lysine 537 0.0500% Poly-l-lysine 102 0.0500% Poly-l-lysine 4 0.0500% Poly-l-histidine 226 0.0500% Poly-l-ornithine 152 0.0500% The capsules were transferred to a petri dish and all extracapsular liquid was removed with the help of a cell strainer. Exactly 1.0 mL of RBC containing capsules were placed in a gas sterilized cuvette containing 3.0 mL of either TBAC buffer (to lyse RBC's) or saline (control).

Hemoglobin efflux was measured spectrophotometrically at 480 nm as a function of time.

It was postulated that microencapsulated islet xenograft survival would be influenced by microcapsule permeability. We found that microcapsule permeability may be altered by increasing or decreasing the SUBSTITUTE SHEET (RULE 26) WO 97/11685 PCT/US96/15649 25 concentration of PLL (poly-1lysine) in the microcapsule formula. As described above, red blood cells were encapsulated in alginate via an air jet system and then incubated with various polyamino acids including PLL. The RBCs were then lysed and hemoglobin (MW 64,500) efflux was measured spectrophotometrically at 480nm as a function of time alongside a concurrent control.

Permeability coefficient was calculated according to the following formula: (2.303 Cf* Vt S) (Ci At), where Ci and Cf are the initial and final hemoglobin concentrations, Vt and At are the total volumes and areas of capsules respectively, and S slope of In (Ct-Cf) (Ci-Ct)(115). Others have suggested that substitution of different amino acids for PLL, or changing PLL molecular weight would alter microcapsule permeability. On the contrary, we found that PLL substitutions (poly-lornithine, alanine, aspartate and histidine) did not result in viable capsules and thus PLL molecular weight alterations did not effect permeability. PLL concentration was the most critical factor in altering capsule diffusion.

These observations are supported by the recent findings of other investigators (115), however these investigators disclose other conflicting results, such as temperature influence during microcapsule synthesis. They do not extend the concept of altering permeability to in vivo uses of microcapsules. We noted a thirteen fold decrease in hemoglobin efflux occurring in capsules that had a fourfold increase in PLL (see Table 3).

I

WO 97/11685 PCT/US96/15649 26 Table 3. Increasing PLL Concentration Reduces Microcapsule Permeability to Hemoglobin PLL 0.050 0.125 0.137 0.144 0.150 0.200 Concentration w/v) Permeability 50 56 52 30 6.7 3.8 constant (E-06cm/sec) In experiments, encapsulated rabbit islet survival in spontaneously diabetic NODs was prolonged using microcapsules with permeability <70,000 Kd vs. 100,000 Kd controls (see Figure At sacrifice, day number 125, we found intact microcapsules with viable rabbit islets present within them and no NOD cellular reaction present.

This is the longest survival of encapsulated discordant islet xenografts of which we are aware.

Discussion: We present here data that confirms that poly-l-lysine concentration directly affects microcapsule permeability.

We did not find a similar permeability trend for poly-llysine molecular weight substitutions as has been reported elsewhere. This may be because the permeability changes 27 are not large enough to affect hemoglobin transport, but may still affect passage of smaller solutes. The polyamino acid substitutions for lysine with histidine and ornithine did not form viable capsules probably because they did not form ionic bonds with the calcium alginate gel.

Our viable capsule data was acquired using a bioassay that has many advantages over other techniques that have been utilized in the past to determine microcapsule permeability. The first advantage is that the assay does not commence until the investigator lyses the red blood cells. Therefore at time zero there is a negligible external concentration of hemoglobin. The second advantage is that the only equipment needed is a spectrophotometer.

Thirdly, no radioactivity is utilized. The final advantage is that the molecular weight of hemoglobin is perfectly suited for configuring the ideal microcapsule for use as a bioartificial pancreas. With pores that exclude hemoglobin, one can feel assured that immunoglobulin, 20 complement and direct cell contact have eliminated.

Further in vivo studies can determine whether microcapsules that just barely exclude hemoglobin prevent host sensitization from donor xenoantigens and host rejection 'via low molecular weiaht islet toxins. Further in-vitro 25 studies can, also, use this technique to quickly determine :new permeability trends.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

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Claims (9)

1. An assay for determining a poly-l-lysine concentration suitable for synthesizing microcapsules having a predetermined permeability, which assay comprises encapsulating a material which produces or is capable of producing detectable particles of a known molecular weight in a series of microcapsules, each microcapsule of said series being synthesized using a different poly-l-lysine concentration; initiating production of the detectable particles in each microcapsule if the material encapsulated in step is not already producing such particles; detecting detectable particles diffusing from each microcapsule in said series, thereby determining the permeability of each microcapsule in said series; ascertaining the presence or absence of a microcapsule in the series having the predetermined permeability, and; repeating steps through if a microcapsule having the predetermined permeability is absent, but varying the poly-1-lysine concentration used to synthesize each microcapsule WO 97/11685 PCT/US96/15649 48 in the series in step (a)

2. An assay according to claim 1, wherein the material is a composition comprising particles of a known molecular weight which are capable of fluorescing.

3. An assay according to claim 2, wherein the composition comprises fluoresceinated dextran of a known molecular weight.

4. An assay according to claim i, wherein the material is a composition comprising red blood cells, and wherein initiating production of the detectable particles in step comprises lysing the red blood cells so as to release hemoglobin from the red blood cells.

An assay according to claim 4, wherein hemoglobin diffusing form each microcapsule is detected using a spectrophotometer.

6. An assay according to claim i, wherein the predetermined permeability is the maximum permeability capable of preventing diffusion of a specific substance.

7. An assay according to claim 6, wherein the specific substance is immunoglobulin.

8. An assay according to claim 6, wherein the specific substance is cells or tissue chosen for encapsulation within the microcapsule having the predetermined permeability. WO 97/11685 PCT/US96/15649 49

9. An assay according to claim 6, wherein the specific substance is an antigen produced by cells or tissue chosen for encapsulation within the microcapsule having the predetermined permeability. A method of synthesizing microcapsules having a predetermined permeability which comprises determining a suitable poly-l-lysine concentration according to the assay of claim 1, and synthesizing microcapsules using the determined poly-l-lysine concentration.