WO1992017130A1

WO1992017130A1 - Local immunosuppression by polymer release of defined combinations of immunosuppressants

Info

Publication number: WO1992017130A1
Application number: PCT/US1992/002413
Authority: WO
Inventors: Patrick Aebischer; Paul E. Lacy
Original assignee: Patrick Aebischer; Lacy Paul E
Priority date: 1991-03-29
Filing date: 1992-03-26
Publication date: 1992-10-15

Abstract

A method and device for local immunosuppression by polymer release of defined combinations of immunosuppressants, particularly useful for reducing rejection by a recipient of a transplanted graft.

Description

LOCAL IMMUNOSUPPRESSION BY POLYMER RELEASE OF DEFINED COMBINATIONS OF IMMUNOSUPPRESSANTS Description Government Support

Work described herein was supported in part by grants from the United States Public Health Service. Background of the Invention

Grafts between two genetically dissimilar individuals of the same species (allogeneic grafts) or between individuals of different species

(xenogeneic grafts) are not generally successful. An important reason for graft rejection is an immune response by the host to the cell-surface antigens of the graft and interaction of donor lynphoid cells in the graft with recipient lymphocytes.

Graft rejection is currently combatted using several approaches. A first approach can be referred to as "antigen non-specific immuno-suppression." This approach employs immuno-suppressive drugs (e.g., antimitotic agents, adrenal steroids and antilymphocyte sera) and other methods (e.g., low temperature culture of donor cells to destroy or alter antigen-presenting cells, UV irradiation) to specifically reduce T cell function.

Although non-specific immunosuppression offers promise for increasing graft survival, immuno-suppressed graft recipients are immunodeficient and, therefore, have lowered resistance to disease. In addition, antimitotic agents are primarily active against dividing cells and, as a result, have some functional specificity for any cells activated to divide by donor antigens. The use of these drugs is limited by the need to avoid damage to other dividing tissue (e.g., gut epithelium).

A second approach to combatting graft rejection can be called "antigen-specific immunosuppression." The goal of this approach is to induce immunological tolerance and can be accomplished, for example, by administering antibodies directed towards graft antigens (anti-allogenic antibodies) or by inducing anti-idiotypic antibodies to T cells which recognize the graft. Treatments involving antigen-specific immunization have thus far not found practical application in human organ transplantation.

A third approach is to destroy donor lymphoid cells in the grafts prior to transplantation. This has been accomplished, for example, by low

temperature culture of donor rat islets to destroy or alter antigen-presenting cells, and a single injection of mouse and rat antilymphocyte sera into diabetic mice, but this effect is systemic and therefore the graft recipient is immunodeficient. (Lacy, P.E. et al., Science, 209: 283 (1980)); and (Lacy, P.E. et al., Diabetes 30: 285 (1981)).

There is, therefore, a great need for a method of combatting graft rejection in an individual, particularly one which does not cause the recipient to become immunodeficient. Summary of the Invention

The invention described herein relates to a method of reducing rejection of a graft in a

recipient by local administration or co-release of two immunosuppressants: one which is donor specific and causes local destruction of donor lymphoid cells which initiate rejection by interaction with

recipient lymphocytes and one which is recipient specific and causes local destruction of recipient cells which would otherwise participate in the recipient's immune response to donor lymphoid cells. In particular, the present invention relates to prolonging survival of a graft by local

immunosuppression, which is accomplished by

introducing into the graft recipient a

donor-specific immunosuppressant and a

recipient-specific immunosuppressant at a site sufficiently close to the islets that co-release of the two types of immunosuppressants results in a localized immunosuppressive effect.

The present invention further relates to a device useful in the subject method of prolonging graft survival. The device of the present invention includes a biocompatible material and the two types of immunosuppressants. The device can be a

container or receptacle which has compartments or sections which hold the immunosuppressants

themselves or cells which produce an

immunosuppressant (e.g., cells which produce and release specific antibodies). Alternatively, the device can be made of a material which has

incorporated therein the immunosuppressants. In either case, the immunosuppressants pass out of the device into the graft recipient and result in delivery of an effective quantity of the two types of immunosuppressants (an amount sufficient to cause local immunosuppression).

In use, the device of the present invention can be introduced into the graft recipient at a location vicinal the graft or can be external the body and the immunosuppressants can be directed, or delivered to the region of the graft, such as by means of tubing which passes into the body. In those

instances in which the device is a material, such as a polymer matrix, within which the

immunosuppressants are incorporated, the device is implanted or otherwise introduced into the

recipient's body. In instances in which the device includes compartments or sections which hold the immunosuppressants or cells producing the

immunosuppressants, the device can be implanted into the recipient's body or remain outside the body, in which case the immunosuppressants are delivered to the desired location by means of tubing or otherwise introduced into the recipient's body.

The method and device of the present invention can be used to prolong survival of many types of grafts, such as islets of Langerhans, liver cells, spleen cells, neural cells and cells which provide a hormone or an enzyme needed by a recipient (e.g., parathyroid hormone, growth hormone). Detailed Description of the Invention

The present invention is based on the discovery that survival of a graft from a donor to a genetically different recipient can be prolonged in the recipient by the local co-release of two types of immunosuppressants: a donor-specific immuno-suppressant and a recipient-specific immuno-suppressant.

As described herein, a xenograft has been introduced into a recipient mammal, along with a source of xenograft-specific antilymphocyte sera, and a source of recipient- specific antilymphocyte sera, and shown to survive (i.e., not to be

rejected) for a longer period of time than was the case if only one of the immunosuppressant types was introduced with the xenograft or if the xenograft alone was introduced into the recipient.

Local immune suppression resulted in local destruction of donor lymphoid cells in the

transplanted cells and of recipient lymphocytes and prolonged graft survival time (compared with graft survival time if local destruction of only one type (recipient or donor) or neither type of cell

occurred). In particular, prolonged survival of an islet of Langerhans xenograft has been accomplished by local co-release in a recipient of a substance (donor antilymphocyte serum) which caused local destruction of donor lymphoid cells in the

transplanted islets and a substance (recipient antilymphocyte serum) which caused local destruction of recipient lymphocytes in the grafts.

The present invention is a method of reducing (partially or totally) rejection of a graft (e.g., a xenograft or an allograft) in a recipient and, thus, of prolonging survival time of the engrafted tissue or cells in the recipient, by local administration or co-release of a first substance, referred to as a donor-specific immunosuppressant, which causes local destruction of donor cells (e.g., lymphoid cells and macrophages) and a second substance, referred to as a recipient-specific immunosuppressant, which causes local destruction of recipient cells (e.g.,

lymphocytes) whose presence results in xenograft rejection. It is now possible to prolong survival (reduce rejection) of a graft introduced into an individual by local destruction of such donor and recipient cells.

As described in detail in the examples, rat islets of Langerhans have been transplanted into diabetic mice, alone or in combination with donor antilymphocyte sera, recipient antilymphocyte sera or both and the longevity of the transplanted cells has been assessed. As shown in the Table, the transplanted islets survived longest in recipients in which local immunosuppression was carried out, effected by local co-release of rat (donor-specific) antilymphocyte sera and mouse (recipient-specific) antilymphocyte sera. Islet survival time was

significantly shorter in control mice, mice in which there was local release of either rat antilymphocyte sera or mouse antilymphocyte sera, but not both, and mice in which there was co-release of rat

antilymphocyte sera and mouse antilymphocyte sera at a distance from the transplanted islets. Islet survival time did not differ significantly among these three groups.

The effectiveness of the present method of local immunosuppression was demonstrated by the fact that diabetic mice which received rat islet cells and local co-release of the two types of

immunosuppressants (rat antilymphocyte sera and mouse antilymphocyte sera) remained normoglycemic for extended periods of time.

As a result of the work described herein, local suppression of the immune response of a graft recipient can be accomplished and, as a result, survival time of the graft can be prolonged. That is, local immunosuppression (local destruction of donor lymphoid cells which initiate rejection by interaction with recipient lymphocytes and of recipient cells which destroy xenograft tissue) has been shown to be effective in prolonging survival of graft cells in a recipient.

The method of the present invention has

numerous possible applications. For example, islets of Langerhans from a non-human source (e.g., a pig, cow or dog) and two immunosuppressant substances (one xenograft-specific and one recipient-specific) can be introduced into a human recipient. The islets and the immunosuppressants (e.g.,

antilymphocyte sera) are introduced, as described in Example 2, in such a manner that they are

sufficiently close to one another that the islets produce insulin and the antilymphocyte sera are released close to the location of the islets

(sufficiently close that the antilymphocyte sera provide the desired immunosuppressive effect). The donor-specific immunosuppressant and the

recipient-specific immunosuppressant can be

introduced into a xenograft or allograft recipient in an appropriate device, from which it is released in the vicinity of the transplanted islets. As a result of the local co-release of the two

immunosuppressants, the islets are not rejected or destroyed by the recipient.

The two types of immunosuppressants released from the device can be a single immunosuppressant of each type or a mixture of each type (e.g., a single donor-specific immunosuppressant or a combination of donor-specific immunosuppressants). The

immunosuppressants themselves (e.g., antilymphocyte sera, monoclonal antibodies) can be contained in the device or a source of an immunosuppressant (e.g., cells producing the immunosuppressant) can be in the device.

Appropriate immunosuppressants for use in the subject invention include any immunosuppressant that does not act on the bone marrow of the recipient. Therefore appropriate immunosuppressants have a local, rather than a systemic effect. Such locally acting immunosuppressants include antilymphocyte sera, antibodies to lymphokines and lymphokine receptors and monoclonal antibodies to subtypes of lymphocytes. For example, hybridoma cells which produce a specific type of monoclonal antibody can be included in the device of the present invention and released locally. The device in which the graft-specific

immunosuppressant and the recipient-specific

immunosuppressant are introduced into the recipient can be made of any biocompatible material which allows passage or release of the immunosuppressants, either by slow passive release or by gradual

(pre-patterned) assisted release (e.g., through use of a pump or other means of assisting release into the recipient). The device can be a bioabsorbable material which slowly releases the

immunosuppressants as the device biodegrades. The device can also be a container or receptacle with compartments or sections which hold the

immunosuppressants. Alternatively, two or more separate devices can be used to deliver the two immunosuppressants. A container or receptacle device can be introduced into the graft recipient at a location sufficiently close to the transplanted tissue or cells to provide local co-release of the immunosuppressants. Alternatively, a container or receptacle device can be external the body and the two types of immunosuppressant co-released at the desired location through tubing. In either case, the device can be removed at an appropriate time, thus resulting in termination of delivery of the immunosuppressants.

The material used in the device can be a biocompatible material which can be impregnated or saturated with an immunosuppressant, which is released slowly after the device is introduced into a transplant recipient. The device can be of any configuration which allows passage of a sufficient quantity of the immunosuppressants to produce the desired effect. For example, the device can be a polymer rod, as described in the examples.

Alternatively, it can be a sphere, a flat sheet or patch or other configuration suitable for the location at which it is used and the quantity of immunosuppressants to be delivered.

Materials appropriate for the devices include any which can contain the immunosuppressants (either held within a compartment or section made of the material or incorporated within the material without adversely affecting them (e.g., by degrading or otherwise inactivating them)). As described (see Example 1), the device can be a biocompatible copolymer processed in such a manner that a matrix in which the immunosuppressants are present is produced.

In one embodiment, purified ethylene-vinyl acetate copolymer resin (EVAc) is combined with an appropriate solvent (e.g., methylene chloride) to produce an EVAc solution. An immunosuppressant (e.g., powdered antilymphocyte serum) is combined with the EVAc solution, which is treated (e.g., by rapid cooling) to produce a solid matrix and fix the immunosuppressant within. Strings or rods of the resulting immunosuppressant-containing solid matrix are then produced and used to deliver the

immunosuppressant. Co-release of the two

immunosuppressants occurs gradually (i.e., over time) and the two immunosuppressants may be released at approximately the same rate or at different rates. The quantity or concentration of each of the immunosuppressants released locally (vicinal the transplanted cells) must be sufficient to have the desired effect (reduction of the recipient's immune response to the transplanted tissue or cells, resulting in its prolonged survival) and will vary, depending on the type of transplanted tissue or cells and the type of immunosuppressants

administered. The quantity can be determined empirically, using known methods, such as

determination of quantities effective in an

appropriate animal model, as described herein. The rate of release of the immunosuppressants varies with the device and must be considered when

determining the concentration of immunosuppressants locally released.

The present invention will now be illustrated by the following examples, which are not intended to be limiting in any way.

EXAMPLE I Assessment of the Cytotoxicity of Donor-specific Immunosuppressant or Recipient-specific Immunosuppressant

Islets were isolated from male Vistar Furth rats by the collagenase technique (Lacy et al.,

Diabetes 16:35 (1967); Lacy et al., Diabetes, 21:87 (1972)) separated on a Ficoll gradient (Lindall etal., Endocrinology, 84:218 (1969)), and hand-picked and screened with the green light technique (Finkeet al., Diabetes 28 : 612 (1979)) for specific

identification of the islet. C57BL/B6 male mice were made diabetic by the intravenous injection of streptozotocin (160 mg/Kg). The donor islets were cultured overnight at 37ºC and 150 islets were used for transplantation beneath the renal capsule of diabetic mice (non-fasting plasma glucose levels >400 mg/dl). Nonfasting plasma glucose levels were monitored three times weekly and rejection was considered to have occurred when the glucose levels exceeded 200 mg/dl on two consecutive

determinations.

Purified ethylene-vinyl acetate copolymer (EVAc) resin (40% by weight vinyl acetate, Elvax 40x, DuPont, Inc., Wilmington, DE) was dissolved in methylene chloride to make a 10% (W/v) solution. Mouse (MALS) and rat (RALS) antilymphocytic sera (Accurate Chemical and Scientific Corp., Westbury, New York) were lyophilized and ground in a mortar to a fine powder. Either MALS or RALS fine powder was added to the EVAc solution to a final concentration of 20% (group 1, 2, 3 and 4a) or 30% (group 4b and 5). The solution was then agitated in a vortex mixer for 30 min. A solid matrix was obtained by rapidly cooling the ALS/EVAc solution in liquid nitrogen in order to fix the ALS particles in the polymer bulk. The methylene chloride was removed by lyophilization. Strings with a diameter of 0.5 mm were pressure extruded through a stainless steel nozzle at a temperature of 55ºC.

In vitro release of MALS and RALS from the polymer rods was determined by incubating 12 mm impregnated rods in 0.9% NaCl (100 μl) at room temperature and aliquots were removed at 3, 6 and 24 hrs for cytotoxicity assay using mouse or rat splenocytes and complement for the appropriate impregnated rods. Aliquots of saline extracts of the RALS rods revealed 70% killing at 1:2 dilution at 3 and 6 hrs with 90%, 70% and 55% killing at 1:2, 1:4 and 1:8 dilutions, respectively at 24 hrs.

Comparable cytotoxicity was found with MALS rods, with 60% killing at a dilution of 1:2 at 3 and 6 hrs and 55% killing at a dilution of 1:16 at 24 hrs.

The rate of protein release in vitro from 12 mm impregnated rods containing 20% lyophilized serum powder and placed in 0.9% NaCl at room temperature was also determined. The rods contained 580 μg protein per rod and the rate of release was 120 μg protein in the first 24 hrs with a constant

subsequent rate of release of 10 μg/day during a 2 week period of incubation.

Before transplanting the islets, a 12 mm segment of either a control or MALS or RALS rod was inserted under the kidney capsule and bent into a U shape. The islets were then inserted near the arch of the U. When two rods were used, 8 mm segment of MALS and RALS rods were inserted individually, the distal ends of the rod were pushed close together, and the islets were inserted near this juncture of the rods.

Histologic examination of the islet grafts was done either after rejection had occurred or when the kidney bearing the graft was removed to verify function of the grafts. The kidneys were fixed in Bouin's solution, processed for light microscopy, and stained with aldehyde fuchsin to identify beta cells and with hematoxylin eosin.

EXAMPLE 2 Assessment of the Effect of Donor- Specific Immunosuppressant and Recipient-Specific Immunosuppressant on Graft Survival

Control polymer rods not impregnated with antilymphocyte serum were placed in the kidney capsule, bent into a U shape and rat islets were implanted adjacent to the arch of the 12 mm rod in each of the transplants. The control rods had no effect on the survival of the grafts since the mean survival time (MST, Group 1, Table 1) was 14.7 +/- 2.5 days which is identical to the survival time of control rat islet xenografts (14.4 +/- 1.4 days) in previous studies. Lacy P.E. et al Transplantation 33: 588 (1982).

Individual polymer rods (12 mm) containing either MALS or RALS were placed under the kidney capsule and rat islets were inserted adjacent to them. The MST for the RALS polymer rods was 15.0 +/- 1.4 days and 15.2 +/- 1.6 days for the MALS rods (Group 2 and 3, Table 1). Neither the RALS nor the MALS polymer rods had any effect in prolonging survival since the survival times of these two groups was almost identical to the control group.

The effect of insertion of both a MALS rod and a RALS rod adjacent to the islet graft on survival of the xenografts was also assessed. An 8 mm segment of each type of rod was inserted into the kidney capsule and bent toward each other at one end and the islets were inserted near the apex of the two rods. A most interesting finding was that the use of the MALS and RALS rods at the transplant site produced a marked prolongation of xenograft survival (>44.5±10.9; Group 4a, Table), which was

significantly different from the control group

(P<0.005). One of the recipients was still

normoglycemic at 79 days after transplantation and removal of the graft produced hyperglycemia.

Histologic examination of the graft revealed intact islets and only a slight fibrotic reaction around the rods.

The same procedure was also carried out using rods containing a slightly higher (30%)

concentration of either MALS or RALS and again a marked prolongation of survival was obtained (MST >48.5±11.7 days; Group 4b, Table) which was

significantly different from the control group

(p<0.05).

One recipient in Group 4b was still

normoglycemic at 100 days and removal of the graft returned the animal to a diabetic state. Histologic examination revealed intact islets with a normal degree of beta granulation. Foci of lymphocytes were present adjacent to the graft, which is similar to findings in previous studies where foci of lymphocytes were present close to established rat islet xenografts but not invading them (Lacy, P.E.et al., Science, 209:283 (1980); Lacy, P.E. et al., Diabetes, 30:285 (1981)).

The tissue reaction to the rods was minimal, with only a tiny rim of collagen surrounding the rod adjacent to the kidney tissue. A few foreign body giant cells were present at the rim of the rod and an occasional giant cell was found in the

interstices of the rod. This indicated that the polymer rods were biocompatible in the mouse and did not elicit a chronic inflammatory reaction that might affect the islets.

EXAMPLE 3 Assessment of the Mechanism of Action of Co-release of Donor-specific Immunosuppressant and Recipient-specific Immunosuppressant

An assessment was also carried out to determine whether the MALS or RALS rods were preventing the rejection of the xenografts due to a local release of the antibodies or due to systemic

immunosuppression of the recipients. The RALS and MALS polymer rods were inserted under the capsule of the right kidney and the islets were transplanted under the capsule of the left kidney. The mean survival time in this group (Group 5, Table) was 22.9±3.1 days, which was slightly longer than the control group (14.7±2.5 days) but was not

significantly different from the control group

(p<0.1).

In contrast, the survival time of the two groups (Group 4a and 4b) in which the rods were inserted in the same kidney immediately adjacent to the islet groups was significantly longer than occurred when the rods and the islets were in

different kidneys (p<0.005 and p<0.05,

respectively). These findings clearly indicated that the local release of rat and mouse antilymphocyte sera from the rods was responsible for the marked prolongation of islet xenograft survival. The lack of effect of a MALS polymer rod alone or only a RALS rod on survival of the xenografts indicated that local destruction of both rat lymphoid cells in the islets and mouse lymphocytes of the recipients was needed to prolong survival. The site of immunodestruction or immunoalteration of the lymphoid cells may have occurred at the graft site and in the local lymph nodes draining the site of implantation. Presumably the released serum would be present in the lymph draining these areas.

In summary, these results demonstrate that marked prolongation of rat islet xenograft survival was obtained by the local release of rat and mouse antilymphocyte serum at the site of transplantation. In addition, they show that the polymer rods are remarkably biocompatible in the mouse. The rods can be coated with different layers of pure polymer which will impede the rate of release of the agent from the rods (Winn, S.R. et al., Exper. Neurol., 105:244 (1989)), thus, prolonging the in vivo

exposure. Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine

experimentaiton, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

TABLE 1

EFFECT OF RODS IMPREGNATED WITH MALS OR RALS ON ISLET XENOGRAFT SURVIVAL (RAT TO MOUSE) Rods Transplant Site Survival (days)

Group MALS RALS Rods Islets Individual Mean ± SEM

1 - - Left Left 11×3,14×2,27 14.7±2.5

Kidney Kidney

2 - + Left Left 13×4,17,21 15.0+1.4

Kidney Kidney

3 + - Left Left 8,15x2,17x2,19 15.211.6

Kidney Kidney

4(a) + + Left Left 13,34,57,74, >55.5110.9

Kidney Kidney 76,76*→

4(b) + + Left Left 14,21,28,31, >48.5111.7

Kidney Kidney 42,56,96,100*→

5 + + Right Right 12×2,16×2,23, 22.913.1

Kidney Kidney 26,31,33,37 * Nephrectomy for removal of islet xenograft

Claims

1. A method of prolonging survival of a graft in a recipient, comprising introducing into the recipient a donor-specific immunosuppressant and a recipient-specific immunosuppressant, wherein the donor-specific immunosuppressant and the recipient-specific immunosuppressant are co-released vicinal the xenograft or the allograft and result in local immunosuppression in the recipient.

2. A method of prolonging survival of a graft in a recipient, comprising introducing into the recipient a device comprising a biocompatible material containing therein a donor-specific immunosuppressant and a recipient- specific immunosuppressant, at a location vicinal the graft, and maintaining the device in the recipient, wherein the donor-specific

immunosuppressant and the recipient-specific immunosuppressant are co-released, thereby resulting in local immune suppression.

3. The method of Claims 2 wherein the graft is

transplanted islets of Langerhans.

4. The method of Claim 3 wherein the

donor-specific immunosuppressant is

donor-specific antilymphocyte sera and the recipient-specific immunosuppressant is

recipient-specific antilymphocyte sera.

5. A method of prolonging survival of a graft in a recipient, comprising the steps of:

a) providing a device comprising a

biocompatible polymer which is a solid matrix having incorporated therein a donor-specific immunosuppressant and a recipient-specific immunosuppressant;

b) introducing into the graft recipient the device of (a), at a location vicinal the graft; and

c) maintaining the device of (a) under

conditions appropriate for co-release of the donor-specific immunosuppressant and the recipient-specific immunosuppressant, whereby co-release of the donor-specific immunosuppressant and the recipient-specific immunosuppressant result in immunosuppression vicinal the graft in the individual.

6. The method of Claim 5 wherein the graft is

transplanted islets of Langerhans.

7. A device comprising a donor-specific

immunosuppressant, a recipient-specific

immunosuppressant, and a biocompatible

material, the biocompatible material containing the donor-specific immunosuppressant and the recipient-specific immunosuppressant and permitting passage therefrom of the

donor-specific immunosuppressant and the recipient-specific immunosuppressant.