WO2001065936A1

WO2001065936A1 - Method for immune switching

Info

Publication number: WO2001065936A1
Application number: PCT/US2001/007368
Authority: WO
Inventors: Kumarpal A. Shah
Original assignee: Shah Kumarpal A
Priority date: 2000-03-06
Filing date: 2001-03-02
Publication date: 2001-09-13
Also published as: AU2001245513A1

Abstract

A compound for modulating an immune response in medical application in the form of a hydrogel including an ultrapurified polymer, a complement modulator and a gelling agent. In a first embodiment the hydrogel has a pore size between 50 kDa and 100 kDa which generally functions as an immune response inhibitor. In a second embodiment, the hydrogel has a pore size greater than 200 kDa which generally functions as an immune response activator. The hydrogel may be applied directly, used to coat foreign materials, or used to encapsulate cellular or non-cellular material.

Description

Title of the Invention

METHOD FOR IMMUNE SWITCHING

Background of the Invention

1. Field of the Invention

This invention relates to the encapsulation of an immune modulator.

The pore size of the capsule acts as an immune switch. Encapsulation technology system and its targeted therapeutic applications are detailed.

2. The Prior Art:

Encapsulation technology is the packaging , carrier and delivery

system for cells and drugs. The technology immunoprotects cells and acts as

an extend-release-matrix for drugs. This approach is used to develop cure and treatment for many diseases. Several polymers have been used but

alginate is particularly favored for sensitive applications such as islet cell encapsulation. Alginates are commercially available. Use of these heterogeneous and impure commercial preparations have lead to bioincompatible, hemoincompatible and inflammatory reactions with fibrosis,

thrombosis, abscess formations, infections, immunorejections and graft failures. This led to bottleneck in technology and prevented it from advancement in larger animal models from 1970 to 1990s. Endotoxin content of commercial alginate preparations was 100,000 E.UJg or greater.

In recent years, purified alginate and other polymers have been

introduced commercially. Prokop A. and Wang T. G. have studied and

reviewed the subject in "Purification of Polymers Used for Fabrication of an Immunoisolation Barrier" published in 'Annals of NY academy of Medicine',

1997, Vol.831: 223-231. Several methods for cell encapsulations have been

described in recent years using purified alginate. U. S. Patent No. 5,459,054 to Skjak- Braek et al describes the method of encapsulating cells with

purified alginate. The material has been used successfully to

microencapsulate islet cells to cure type 1 diabetes. U. S. Patent 5,879,709 to Soon-Shiong et aland US. Patent No.5, 573, 934 to Hubell et al details the

method to further improve biocompatibility of alginate by additional coating

with PEG and its applications. US. Patent No. 5,874,099 to Dionne et al

describes applications of purified alginate material in cell transplant technology in general. Leblond F. A. et al evaluated biocompatibility, hemocompatibility and immune tolerance of alginate in "Studies on Smaller

(~ 315 micron ) Microcapsules: IV. Feasibility and Safety of Intrahepatic Implantation of Small Alginate Poly-L -Lysine Microcapsules". The findings

were published in "Cell Transplantation", Vol. 8, pp. 327-337, 1999. The removal of endotoxin appears to be the critical factor in above improvements of clinical results.

Endotoxins or Lipopolysaccharides(LPS) activate innate immune responses through CD 14 dependent and CD 14 independent pathways. CD 14

dependent pathway act through Human Toll-like Receptors(HTLRs), while CD 14 independent pathway act through complement mediated pathways.

Both pathways coordinate and translate mechanical and chemical signals

through dendritic cells to generate specific adaptive immune responses. Dendritic cells thus essentially act as a transducer. Further, translation of innate immune responses to adaptive immune responses also occur through breakdown products of C3 complement such as C3b and C3d. Recent

advances in endotoxin research suggest a human being is 1,000 times more

sensitive to the effects of endotoxin than small animal models. Fetal bovine

serum is frequently used in encapsulated technology for cells. It contains lipopolysaccharide binding protein and sensitizes the effects of endotoxins.

Bacterial and viral contaminants also act as endotoxin sensitizers. Above factors can also increase endotoxin sensitivity by 1,000 fold or more. The

combined effects in human beings therefore could be 1,000 to 1,000,000 fold increased sensitivity to endotoxin. This leads to activation of innate and adaptive immune responses and both causes or contributes to immune rejections, bioincompatibility and graft failure. Outside the capsule it leads

to fibrosis and choking of oxygen and nutritional supply, while inside the capsule death of cells occur by apoptotic and nonapoptotic mechanisms.

Complement inhibitors that affect complement pathways at various junctures such as at Cl, C3, C5a, C5b-C9 and Factor D have been used with success to control and inhibit endotoxin mediated effects. In experimental

models, both control of endotoxin mediated effects and improved survival have been demonstrated.

U. S. Patent No. 4,265,908 to Conrow et al describes the

complement inhibitory effects of sulfonic salts and its various formulations for therapeutic uses. Hydrogel formulation of sulfonic salts and its polymer

with agarose have been described by Iwata H. et al in "Strategy for

Developing Microbeads Applicable to Isletxenotransplantation into a

Spontaneous Diabetic NOD Mouse" in the J. of Biomedical Materials Research, 28(10), 1201-07, 1994. Sulfonic polymer, have been successfully

incorporated to improve biocompatibility in hollow fibers of polysulfone and

biosulfane used for hemodialysis. Sulfonic polymer component has additionally heparin-like properties. Labarre D. J. reviews this property in Ηeparin-like Polymer Surfaces: Control of Coagulation and Complement

Activation by Insoluble Functionalized Polymers" in The Int. J. of Artificial Organs 13:10, 651-657, 1990. Pascual M. et al details the "Specific

Interactions of Polystyrene Biomaterials with Factor D of Human Complement" in Biomaterials, 14, 9, 665-670, 1993. Kilpatrick J. M. et al details "Control of the Alternate Complement Pathway: Inhibition of Factor

D", Chapter 13, pp. 203-225, in controlling the complement system for novel

drug development, edited by Mazarakis H. and Swart S. J. published by IBC,

1997. Rustagi P. K. et al details "Development of Novel Broad Spectrum Serine Protease Inhibitors for use as Anticoagulants", Chapter 15, pp.307- 320, in "Anticoagulant, Antithrombotic and Thrombolytic Therapeutics II",

IBC series, 1998. US. Patent No. 5,660,825 to Sims P. et al describes the

composition of polypetide that has complement inhibitory property and its

therapeutic applications. U. S. Patent No. 5,679,345 to Sanfilippo et al

describes a method for preventing complement dependent rejection of organs or tissue transplants using antibodies that prevent the formation of C5b-C9, membrane attack complex.

As shown by these prior art examples, bioincompatibility,

hemoincompatibility and immune rejections are major problems in medicine and surgery including implantation and transplantation biology. Further, it

is obvious that the rate-limiting step in the clinical application of foreign materials is the discovery of chemicals and compounds that have inhibitory

effects on bioincompatible and immune reactions. LPS and complement mediated effects are best described in relation to microbial infections. It has been recently hypothesized that "the simple antigen migration-localization

principle should further our understanding of the events that occur with or

without therapeutic intervention in a variety of infectious, neoplastic, and

autoimmune diseases or after transplantation, and may offer improved rationales for prevention and treatment". (Starzl T.E. and Zinkernagel R.M.,

"Review Article: Antigen Localization and Migration in Immunity and

Tolerance", The New England Journal of Medicine, Vol. 339, No. 26, pp. 1905- 1913).

Summary of the Invention

It is an object of the present invention to improve biocompatibility, hemocompatibility and immune tolerance of polymers such as alginate by reducing its endotoxin content to a minimal level.

It is a further object of the present invention to combine ultrapure alginate with immune modulator for therapeutic purposes.

It is another object of the present invention to control the action and duration of immune modulators by combining ultrapurified alginate and sodium polystyrene sulfonate in a hydrogel formulation.

It is an additional object of the present invention to regulate the pore

size of the hydrogel to less than 100 kD to selectively permit inhibition of immune system for targeted therapeutic applications in implantations and transplantation biology.

It is a further object of the present invention to relax the pore size of

the hydrogel to more than 200 kD to permit selective activation of the

immune response for targeted therapeutic applications in certain infections, cancers and vaccine developments.

It is an additional object of the present invention to characterize

drug-device components of the encapsulation technology to develop encapsulation technology system and its targeted therapeutic applications.

It is a further object of the invention to develop an encapsulation technology system that obviates prior art problems and meet or exceed minimal safety criteria of endotoxin content specified by the FDA for drug-

devices.

I achieve the above objects of my invention by first developing an

Encapsulated Cell Device as detailed in my U.S. Patent No. 5,976,780.

Ultrapurified alginate and sulfonic polymer are combined and cellular material is processed to remove sensitizing factors. This strategy helps prevent the harmful effects of endotoxin.

When 2% alginate with 5% polystyrene sodium are reacted with Ca

100 mM/L, instant hydrogel formulation occurs. Both exchange Na and bind

with Ca. This leads to the encapsulation of polystyrene sulfonate in

ultrapurified alginate gel. The pore size of the gel obtained is 50-100 kD.

Polystyrene sulfonate is a 50 kD polymer but it does not leach out on repeated washing with water or saline and it forms strong, stable bonds with

alginate. Factor D, is a 25 kD protein. It readily permeates the gel and avidly binds with polystyrene sulfonate to provide complement inhibition. In the

prior art, agarose gel was combined with polystyrene sulfonate. The hydrogel

has pores of 10 kD. This would protect NOD mice from immune rejection simply because of its lower molecular cut off as detailed in my U. S. Patent

No. 5,976,780. Complement inhibition could not account for such effects because all complement proteins have molecular weight above 25 kD. Sodium polystyrene sulfonate thus was excluded to perform its complement

inhibitory effects. When combined with agarose, to prevent leaching,

additional coatings with polybrene and carboxy methylcellulose were required. In sensitive applications such as cell transplant, the procedure of agarose with polystyrene sulfonate requires repeated exposure to thermal trauma. When used intravenously, polystyrene sulfonate acts as a

suspension-solution and tends to precipitate out causing unreliable dosing,

has a tendency to clump and, due to more avid binding with Factor H, it may activate the alternate complement system. These problems have been

obviated when ultrapurified alginate is used in combination with polystyrene sulfonate.

Factor D circulates in the blood in the cone, of 1-2 meg/ ml but is a powerful complement activator. Thus, for example, cobra venom factor will not be able to convert C3 to C3c in the absence of Factor D. However, addition of even 1% of normal concentration of Factor D in -vitro is capable of

restoring C3 to C3c conversion. By contrast, bonded Factor D with sulfonic polymer in 100 times greater concentration fails to restore C3 conversion.

This indicates total loss of Factor D activity following its binding with sulfonic polymer. The pore size of 50-100 kD prevents binding of Factor H which is a complement inhibitor. Factor H, as a complement inhibitor, accentuates the degradation of C3 convertase, binds with breakdown

products of C3 convertase and prevents costimulation of B cells and work with Decay Accelerating Factor (DAF) and Factor I to rapidly inactivate C3

convertase. Its binding with polystyrene sulfonate and its inactivation of complement inhibitory property, is therefore not desirable. Selective binding

of Factor D and selective exclusion of Factor H may help potentiate complement inhibition in a 50-100 kD pore size gel. The strategy helps improve reliability of sulfonic polymer as a complement inhibitor. By preemptive blockage of the complement system, transducer function

of dendritic cells is not activated. This way, complement inhibitors also

act as an inhibitor of adaptive immune responses.

Encapsulation of sodium polystyrene sodium in 200 kD pore size alginate will have opposite effects viz. activation of complement system by more avid binding with Factor H. This in turn will lead to activation of

transducer function of dendritic cells. Thus, complement activation also

contributes to activation of adaptive immune responses. The relaxation of pore size can readily be obtained by using appropriate dilution of alginate

and CaCl concentration and by shortening the reaction time. The activation of immune response is beneficial in vaccine development as an adjuvant to speed immune responses. Thus, it can be used as military or civilian

preparedness against bioterrorism. Certain bacterial infections, viral

infections, parasitic infections and cancers evade immune system and thrive by binding with Factor H or by secreting Factor H-like substances.

Streptococcus pyogenes, Neisseria gonorrhoeae, HIV, Echincoccus granulosus and Yersinia entercolitica are such examples of infections that evade

immune responses by binding to Factor H. Cancers, such as of cervical, bladder and renal cells, evade immune response by secreting Factor H-like

substances. Use of Factor H antibodies has been shown to bring efficient

killing of resistant strains of N. Gonorrhea and HIV in in-vitro experiments. It is speculated that encapsulation of sodium polystyrene sulfonate in 200 kD

pore size polymer will allow direct binding of Factor H and deplete its

availability for pathogens and tumors. Thus, it will transiently activate the complement system. Concomitant administration of antibiotics, antivirals or antitumor agents will therefore be more effective in eradicating such

pathogens or tumors.

Sodium polystyrene sulfonate, when administered intravenously in

experimental animals, in doses of 10 mg/ kg or more, completely inhibits complement pathways. Further as Factor D inhibitor, it has broad spectrum inhibitory effects on serine protease of chymotrypsin family. This property

has protected death of mice from endotoxinemia. Ultrapurified alginate and

polystyrene sulfonate act synergistically to inhibit proinflammatory cytokines and complement system. By depriving survival stimuli, such as endotoxin,

C3b, C3d and cytokines, antiapoptotic proteins of the monocytes and Bel family are not activated. The strategy breaks the critical linkage, i.e.

breakdown products of C3 convertase and activation of monocyte/

dendrocytes to initiate adaptive immunity. This helps prevent activation of adaptive immunity as well. Dendritic cells are not activated and therefore do

not express second signal. This leads to arrest in T cell mediated adaptive responses. Lymphocytes and monocytes die " by neglect or passive death".

Cytokines and complement mediated signals are also needed to upregulate adhesion receptors and for cellular activation and aggregation. Failure to

provide such signal, leads to nonthrombotic endothelial surface and noninflammatory state. Different hydrogel formulations can readily be

prepared using commercially available devices for microencapsulation and hollow fibers for macroencapsulation. Such devices permit targeted

therapeutic applications in-vitro, in-vivo or ex-vivo. In-vivo applications could be both extravascular or intravascular. Brief Description of the Drawings

In the accompanying drawings to which reference is made in the

instant specification which is to be read in conjunction therewith, like

reference numerals are used to indicate the parts in the various views:

FIG. 1 shows endotoxin or LPS effects on afferent and efferent arm of immune responses.

FIG. 2 compares the endotoxin content of a purified alginate with

ultrapurified alginate.

FIG. 3 is the immune switch according to the invention.

Description of the Preferred Embodiment

As shown in FIG. 1, endotoxin causes activation and amplification of

effector or efferent arms of immune responses through its binding with LBP and complement. The items in the boxes 10, 12, 14, 16 and 18 show the activation of cellular pathways leading to activation of cytokines, costimuli

and adhesion receptors. The end result is bioincompatibility, hemoincompatibility and immune rejections. In a worst case scenario, septic shock and death may occur. Removal of endotoxin to its minimal level thus prevents the activation of items 10, 12, 14, 16 and 18. Blockage of cellular pathways 32, 34 ,36, and 38 occurs and contributes to biocompatibility, hemocompatibility and immune tolerance as shown 44.

Endotoxins contribute to activation of adaptive immunity through C3 breakdown products and monocyte/dendritic cell activation 10 and activation of dendritic cells leads to expression of costimulli, such as B7-1 and B7-2.

Costimulation of B 7-1 and B7-2 are the critical events that lead to migration

of dendritic cells to secondary lymphoid tissues and activate adaptive immunity. For the activation of CD 4+ T mediated humoral immune responses, CD + 8 T cell mediated immune responses and B cell mediated

antibody responses, at least two signals are needed. In the absence of second signal or B7 costimulli, T and B cells fail to cause immune responses and lead

to anergy. Prevention of dendritic cell maturation therefore is the critical

step to induce immune tolerance 42 to foreign materials including cell and

organ transplantations. This occurs by blockage 32 of dendritic cells and its maturation.

Majority of immunosuppressives such as cyclosporine, tacrolimus (FK

506), Azathioprine, Mycophenolatemofetil(CellCept), MuromonaCD3 (OKT3;Orthobiotech), Interleukin-2 receptor antagonist

(Basiliximab(Simulect, Novartis) and Daclizumab(Zenapax, Hoffmann-La- Roche) and antibodies to T cell or its costimulli used for induction protocol are targeted to inhibit T cell mediated effector or efferent immune responses

40 to cause immunosuppression after the expression of costimulli or after the activation of immune system or maturation of dendritic cell. Removal of

endotoxin, dampening of inflammatory cytokines and complement inhibition provide preemptive strategy to prevent activation of immune responses and

lead toward immune tolerance in cell and organ transplant. Presence of

memory clone of T and B cells may still activate the immune system. However, in the absence of costimuli the response tends to be milder. Hydrogel nature of the formulation allows one to combine any immuno-

suppressives to further strengthen immuno-suppressive effect 40 to cause immunosuppression. Complement inhibitor as a hydrogel formulation can be

used as a drug additionally to inhibit complement mediated events including

whole body inflammatory responses and septic shock. Hydrogel formulation permits encapsulation of any cell to further protect from immunorejections.

By enlarging pores of the hydrogel to 200 kD, the encapsulated drug

acts as an immune activator that has a range of applications in infections, cancers and vaccine developments. As shown in FIG. 3, by controlling the

pore size the immune switch function is regulated. Since any drug can be encapsulated, the immune switch function and its potency can be controlled for target specific applications. The micro hydrogel 60 according to the invention contains polystrene sulfonate 62 Factor D and Factor H inhibitor

64. Hydrogel 60 includes pores 50 which, on the one hand, may have a pore

size 66 having a nominal molecular weight cutoff between 50kd and lOOkd. Such pore size would exclude Factor H, as illustrated by arrow 68, and would allow selective entry of Factor D, as illustrated by arrow 70. This results in

immune inhibition. Alternatively, pores 50 may be of a pore size 72 having a

nominal molecular weight cutoff greater than 200kD. Pore size 72 would allow preferential entry of Factor H due to more avid and rapid binding of

Factor H as compared to Factor D, as illustrated by arrow 74. Arrow 76 indicates delayed entry of Factor D.

ENCAPSULATION TECHNOLOGY SYSTEM AND ITS TARGETED

THERAPEUTIC APPLICATIONS

Recently, FDA has defined encapsulation technology as 'Therapeutic Drug-Device'. Drug components may be cellular or noncellular and will vary

depending upon the targeted therapeutic application. The invention herein provides a device component which may be a capsule, syringe, microcapsule,

macrocapsule or ex -vivo instrument. It has the same variability as a drug component. A matrix component i.e. alginate, however, is the common component and its function is to provide biocompatibility,

hemocompatibility and immunetolerance. Addition of gelling agent, such as CaCl, imparts the immunoisolatory and extended-release functions to the

matrix. Thus standardization of the two common components, viz. matrix

and gelling agent, in the form of a kit permits the development of targeted therapeutic applications using different devices and cellular or noncellular

therapeutic agents. Encapsulation of an immune modulator broadens the range of targeted therapeutic applications in implantations, transplantations,

certain infections, cancers and vaccines. Since immune modulator is a sulfonic polymer, addition of sulfonic group improves basic biocompatibility,

hemocompatibility and immunetolerance of the encapsulated device. Use of

ultrapurified alginate of high 'G' monomer composition, removal of divalent

toxins and presence of sulfonic group contribute to increased mechanical strength and salt bridge stability over a wide pH range. In other words, encapsulation of an immune modulator such as sulfonic polymer results in

the development of an encapsulation technology system that overcomes prior

art problems and provides a wide range of targeted therapeutic applications. Described below are methods to formulate encapsulation devices of different shapes and sizes that allow targeted therapeutic applications.

1. Drug Formulation: Alginate is the most common polymer used for the encapsulating cellular and noncellular materials. Below, basic alginate

fomulation is described that meets or exceeds FDA acceptance criteria for minimal endotoxin content for encapsulated devices. Such formulation is standardized for its chemical properties as detailed in my U.S. Patent No.

5,976,780.

Step 1. Obtain UP alginate from sources as powder.

EX. UP MVG ( S. NO. Property Product Code: Ultrapurified 28023316).

Endotoxin content 700 E.UJg dry weight.

Step 2. Prepare 2% solution of defined quantity in 0.9% NaCl solution.

Endotoxin content in 2% 100 ml solution will be 1400 E.UJdl or 14 EU/ml.

Assuming 0.9% NaCl is endotoxin free.

Comment: Alginate powder dissolves poorly and tends to clump. Magnetic

shaker and vigorous shaking are required to adequately dissolve alginate powder. It may take up to 24 hours to completely dissolve alginate powder in the solution.

Step 3. Add appropriate preservative or antibiotics to prevent bacterial and fungal contamination and improve shelf life.

Step 4. Adjust pH to 7.4. Sterile filter solution through 0.45 micron filter. Step 5. Add dry or wetted PROSEP*-Rem Tox to above solution. Add 1 gm for every 10 ml solution.

Step 6: Gently mix the suspension for 3 hrs. The mixing can be carried out by placing the container on a roller-mixer. Do not use magnetic stirrer at this stage. This can cause break-up of glass beads.

Step 7. Remove the superpatent by allowing the PROSEP* Rem Tox to settle

and decant the solution. Alternately a glass sintered funnel can be used to separate the beads from the solution. For 2% 100 ml solution, endotoxin content will be reduced from 1400 EU/dl to 14 EU/dl.

Step 8. Supply alginate solution is 2% ready-to-use sterile solution in vials of 50 ml

size. Each vial will have endotoxin content of 7 E.U.

Above ready-to-use polymer solution should be further characterized for

mirobial growth and shelf life. Appropriate preservatives may be added. Prior to use, alginate solution is diluted to 1% for cellular or noncellular applications by

using 0.9% NaCl. Further pH adjustments are not necessary. For most cellular and noncellular applications, requirements of alginate are 5 ml to 100ml. 1% ml

alginate solution contains 7 E.U. of endotoxin. FDA requirement of endotoxin content limit is 5 E.U./kg i.e. 350 EU/70 kg or 20 EU/device. Above procedure meets

and exceeds such requirements. In addition to meeting FDA criteria of minimal endotoxin content, hyrogel formulations should be mechanically stable and should not expand or contract or crack or alter its pore size following implantation. Chemical composition of UP Alginate as defined in U.S. Patent 5,976,780 is free of

divalent toxins and rich in 'G' polymer content. Isotonic gel in 0.9% NaCl, will lead to mechanically stable hydrogel and will maintain its defined pore size. Addition of sulfonic group further increases salt bridge stability over wider pH range.

Above solution now can readily be used for encapsulating cellular as well as

noncellular material. Additionally, the solution can be used to develop target

specific therapeutic applications. Thus, for microencapsulation,

macroencapsulation or ex-vivo blood contacting applications, commercial devices such as INNOVA microencapsulator or A/G technology hollow fibers and cartridges can readily be used. Use of adaptable nozzle permit one to obtain preselected size

and provide protocol flexibility. Further sonification or fragmentation of

microcapsule lead to formation of ultramicrocapsules. Less than 5 micron size

particles can safely be administered due to its hemocompatibility. The mean size of capillaries is 8 micron and the mean size of RBC is 5 micron. Therefore, particles less than 5 micron will not lead to vascular obstruction. Encapsulation of sulfonic

polymer in 50-100 kDa pore size will allow prevention of ischemia-reperfusion

injuries. Porus nature of the gel will allow oxygen diffusion to readily take place

Claims

without resulting in hypoxic damage. This allows ultramicrocapsules to ferry blood and provide extend-release matrix for intravascular drugs such as antibiotics.EXAMPLES: TARGETED APPLICATIONS

1. Improvement in biocompatibility, hemocompatibility and immune

tolerance of foreign materials:

Hydrogel formation of ultrapurified alginate and polystyrene sulfonate form the basic template to improve biocompatibility, hemocompatibility and immune tolerance. A comparison of the endoxtoxin content of ultrapurified alginate 40 with

purified alginate 42 is illustrated in FIG. 2 This template can be used in abdomen, pelvic and spinal surgery to prevent postoperative adhesions. In burns it can serve

as dressing material. Further it can be used as biocot to improve biocompatibility of

foreign materials including other cell transplant procedures. Basic procedure in all these examples is to coat the surface with 2% ultrapurified alginate with or without polystrene sulfonate and developing 50-100 kD pore size hydrogel by spraying the

solution with 1-2% CaCl. In blood contacting surface prosthetic devices containing

ultrapurified alginate with polystrene sulfonate can be devised. CaCl in 1-2% added to form 50-100 kDa pore size. This will lead to improved hemocompatibility, biocompatibility and immune tolerance in a variety of blood contacting surfaces such as hemodialysis equipments, cardio-pulmonary bypass circuits, etc. In cell transplants, such as endocrine, neural, hepatic cells and somatic gene therapy, there are prior art problems that have defied appropriate solutions and have lead to

immune rejections and graft failures. Ultrapurified alginate with complement inhibitor can be used to prevent such occurrences with any foreign materials.

The strategy permits relaxation of pore control to 100 kD. This may be crucial for hepatic cell transplant for the egress of various proteins synthesized.

Further, in critical sites, cells can be directly mixed with alginate + complement inhibitor and can be co-injected with a gelling agent such as CaCl₂ in a dual lumen syringe. Such strategy is of value for ex. In neural transplant. The need for precise

pore control can be obviated due to immunoprivileged site, removal of endoxtoxin in the alginate and addition of complement inhibitor. The same approach is also

applicable to bring world-wide cure of diabetes by incorporating islet cells. In fact

any cellular or noncellular therapy can be achieved with this type of encapsulation technology system. FIG. 3 illustrates the components within such microhyrogel 60

contains one or more complement inhibitors — for example, sulfonic polymer like polystrene sulfonate 62 or any other Factor D inhibitor 64. The alginate hydrogel

60 has pore sizes 66 between 50 and 100 kilo-Daltons(kD) which excludes large molecules — for example Factor H 68 which has a molecular size of 150 kD.

2. Inhibition of innate responses: a. Septic shock.

Septic shock is a preterminal event with high mortality. It occurs

following gram-negative bacterial infections in the setting of major injuries, organ failure and extremely stressful situations in elderly subjects. Endotoxin causes antiapoptosis of hemopoetic cells while paraenchymal cells undergo apoptotic

changes leading to organ failures, hypotension and poor tissue perfusion lead to

complement mediated events. Widespread activation of serine proteases of chymotrypsin family occur. Multiple treatment approaches are required.

Hydration, resuscitation measures, use of antibiotics are common. Ultrapurified alginate with polystyrene sulfonate may be combined with therapeutic doses of antibiotic, antiapoptic substances such as Hepatocyte growth factor and may be

administered systematically to inhibit innate responses. Additionally, oral therapy of encapsulation of activated charcoal and "Prosep-RemTox" may be carried out to

remove endotoxins from G.I. tract. Similar measures may also be employed prophylactically prior to onset of septic shock to remove endoxtoxin by using a

prosthetic device containing encapsulated activated charcoal and "Prosep-RemTox".

b. Prevention of ischemia-rep erfusion injuries: Ischemia of small blood vessels and its reperfusion leads to activation of complement pathways and thrombosis. This can occur in hypovolummic conditions after surgery, following coronary angioplasty or bypass surgery; in transplant of organs at the time of organ

harvesting, rewarming phase or immediately after reperfusion phase of

transplanted organs. Hydrogel formulation of ultrapurified alginate with

complement inhibitor is expected to have protective effects in preventing thrombosis of vessels.

c. Xenotransplants: Endothelial surface of the transplanted organ is highly reactive. It binds with naturally circulating antibodies to xenoantigens.

Endothelial activation provides binding site for classical component such as Clq to initiate complement cascade. Upregulation of adhesion receptors, inflammatory

cytokines and thombotic responses may occur. Use of hydrogel formulation of

ultrapurified alginate and polstrene sulfonate will down regulate complement cascade and help prolong organ viability and its function.

3. Inhibition of adaptive immunity. a. In skin transplants: The wound or surface area where skin is to be

grafted is covered with ultrapurified alginate with 5% polystyrene sulfonate. This

is then sprayed with 2% CaCl2. This will lead to instant gelling. The skin is graft on top of this and sutured at the edges. Additionally, dressing is applied to prevent

slippage of the grant. The hydrogel act as immunoisolation devices and down regulate innate and adaptive immune responses. By preemptively blocking inflammatory cytokines, costimulli and inhibition of complements, dendritic cells will not be allowed to mature. This will lead to immune tolerance. Growth factors,

immunosuppressives or factors such as interleukin 10 that prevent dendritic cell maturation may be added as desired.

b. In organ transplants: It has been shown that 300 micron hydrogel containing

islets are well tolerated by endothelial linings of liver. It is biocompatible,

hemocompatible and does not lead to immunorejections. Porous nature of hydrogel allows oxygen diffusion to take place and prevent ischemic damage to the organ. A

critical difference between cell and organ transplant is that in cell transplants microcirculatory unit is disrupted, while in organ transplant this is preserved. In

both cell and organ transplant endothelial cells, as well as donor dendrocyte or

APC, causes MHC class 11 restricted immune reactions. However, in organ transplant, this results in endothelial blockage, ischemia and organ failure. In

transplanted organs, oxygen supply to cells is provided by microcirculatory unit in

accordance with Fick's law of gaseous diffusion, while this is not the case in cell transplant. It is reasoned, therefore, that endothelial gelling will not interfere with

oxygen supply and may help protect against ischemia-reperfusion injuries or organ

transplant rejections. This concept can be extended to transplanted liver, for example. A double lumen catheter can be placed in the portal vein. Ultrapurified alginate and polystyrene sulfonate can be injected through one lumen of the catheter. Other lumen is used for injection of CACI2 to form hydrogel in the endothehal capillaries and then followed with 2% CaCl. This will protect

endothehal lining and at the same time provide immunoisolation to Uver preventing dendritic ceU activation. This strategy is needed to avoid emboUc escape of geUed material. Like in skin transplant, hydrogel can be enriched with interleukin 10,

TGF-B, hepatocyte growth factor or immunosuppressives or can be combined with

bone marrow chimerism for better immunotolerance and graft acceptance. Site

specific deUvery of growth factors, drugs and ceUs could be done using this approach.

4. MisceUaneous appUcations. a. Isolation of ceUs from organs: Enzymes such as coUaginase can be

immobilized, for example, in pancreas isolation to selectively isolate islet ceUs and

avoid the need for cooUng of isolated ceUs. CoUaginase enzyme is the most costly component used in ceU isolation procedure. Currently there is no approach

described for the economical handling of this enzyme. During islet ceU isolation, coUaginase enzyme can be mixed with ultrapurified alginate with polystyrene

sulfonate and injected inside the pancreatic duct. CaCl2 can be co-injected simultaneously or sequentially to gel entrap coUaginase enzyme. In the presence of

Ca ions, coUaginase activity is increased during warming phase. Its leakage in surrounding bathfluid is reduced or prevented. Isolated islet ceUs need not be

cooled to inactivate enzymatic activity. Polystyrene sulfonate is a chymotrypsin inhibitor. It will protect islet ceUs from acinar proteolytic activity. It is expected

that this wiU lead to improved islet yield while reducing mechanical, chemical and thermal trauma to islet ceUs. CoUaginase enzyme can be inactivated by cooUng to

4*C. It can be regenerated by treating gel with EDTA or sodium citrate. It can be

sterilized and reused for most cost effective isolation of ceUs. Other

misceUaneous uses may involve preparations of dental and catilage molds which may help reduce inflammatory responses and scarring. SimUarly, the material can

be used to develop novel biomaterials such as sutures, tendons or hemodialysis fibers with improved biocompatibility and immune tolerance. In vitro, microhydrogel formulation can be used to prolong preservation of platelets, ceUs

and organs and its in-vitro culture.

b. Treatment of Goncoccal sepsis: A number of pathogens, such as streptococcal pyrogenes and Neisseria gonococci evade the immune system by

combining with Factor H. Factor H inactivates C3 convertase at microbial surface

and aUows ceUs to multiply. This phenomenon is responsible for virulence and

resistance to antibiotics resulting in severe sepsis and death of patients. Polystyrene sulfonate avidly binds to Factor H more than Factor D. Furthermore, such binding inactivates Factor H and activates the complement system. The hydrogel of polystyrene sulfonate of 200 kD pore size thus can readUy be prepared.

The size of hydrogel can also be reduced to 1 micron or less to encapsulate nonceUular material such as antibiotics. The use of such ultramicrohydrogel for

parenteral therapy of gonoccocal sepsis, wiU lead to competitive binding with Factor

H by polystyrene sulfonate and activate alternate complement pathways. Antibiotics that are also encapsulated then wiU provide bactericidal effects to exposed gonococci and cause improvement in chnical condition.

c. Treatment of HIV infection to prevent or halt progression to AIDS. GP 120/41 viral receptor binds to CD4+T ceUs. This binding is critical for i) gaining

entry into CD4 and dendritic ceUs, ii) multipUcation of viruses inside such ceUs, iii)

pathological destruction of CD4 and dendritic ceUs, and iv) destabiUzing immune responses. Rapid viremia, destruction of immune ceUs leads to AIDS. Both gp 120

and 41 evade activation of complement system by binding to Factor H and thus

obtain a foothold on CD4 and dendritic ceUs. Blockage of Factor H, for example, by Factor H antibodies have lead to efficient killing of HIV viruses by complement

activation in in-vitro experiments. Use of immunotoxins, such as cholera or diphtheria toxin is another approach pursued to prevent progression of HIV to

AIDS. By depleting Factor H with polystyrene sulfonate and providing antiviral medicine as encapsulated therapy, a complement system can be activated and at

the same time bring efficient killing of HIV viruses thus exposed. Ultrapurified alginate and sulfonic polymer can thus be combined to develop controUed release drugs that may help activate the complement system and cause efficient removal of pathogens, such as gonococci, streptococcal pyogenes and HIV

viruses. Activation of complement system provides the necessary costimuU and cytokines to activate adaptive immunity. Therefore, the same strategy can be used for developing vaccines against pathogens. In addition to bacterias and viruses, tumors such as renal ceU cancer, bladder cancer and cervical cancer also evade

complement system detection by secreting Factor H-Uke substances and may be

amenable for simUar therapy. Preparedness against bio-terrorism and military preparedness for overseas posts are of growing national importance for the U.S.A. as weU as several countries around the world. Encapsulation of CPG, ODN or

endoxtoxin with DNA vaccine against potential bio-threat agents such as Ebola,

anthrax, Usteria and tularemia in a 200kD pore size hydrogel, wiU help activate innate immune responses quickly and develop protective adaptive immunity faster

with lessening of side effects.

In conclusion, the invention accompUshes the object of providing a means for

immune switching in the form of a hydrogel containing an ultrapurified polymer

having an endotoxin content below 700E.UJg. The hydrogel may be applied in a

variety of forms or used to coat foreign materials or encapsulate ceUuar or nonceUular material. The hydrogel with pore size between 50 kDa and 100 kDa generally functions as a complement inhibitor whUe the embodiment having larger pores greater than 200 kDa generally functions as a complement activator.

It wiU be understood that certain features and subcombinations are of utility

and may be employed without reference to other features and subcombinations. This is contemplated by and is within the scope of my claims. It is further obvious that various changes may be made in detaUs within the scope of my claims without departing from the spirit of my invention. It is, therefore, to be understood that my

invention is not to be Umited to the specific detaUs deta ed shown and described.

Having thus described my invention, what I claim is:

The Claims

1. A compound for modulating an immune response in medical

appUcations comprising: a hydrogel including i) an ultrapurified, natural, biocompatible polymer, (ii) a

complement modulator, and (iii) a geUing agent; said hydrogel having a pore size which is selective to down regulate an

immune response and up regulate an immune response.

2. The compound of Claim 1, wherein said polymer has a

maximum endotoxin content of 700 E.U./g and is selected from the group consisting of alginate, carragenan, pectin and carboxy methyl ceUulose.

3. The compound of Claim 1, wherein said complement modulator is selected from the group consisting of inhibitor of Factor H and Factor D.

4. The compound of Claim 1, wherein said complement inhibitor comprises sulfonic polymer.

5. The compound of Claim 1, wherein said gelling agent is selected from the group consisting of a divalent cation compound and a polyvalent cation compound.

6. The compound of Claim 1, wherein said hydrogel comprises a

nonadhesive, nonfibrotic hydroscopic dressing.

7. The compound of Claim 1 further comprising ceUular material encapsulated by said hydrogel.

8. The compound of Claim 7, wherein said ceUular material is

selected from the group consisting of autologus ceUs, aUogenic ceUs and xenogenic

ceUs.

9. The compound of Claim 7, wherein said ceUular material is selected from the group consisting of islet ceUs, hepatic ceUs, neurocrine ceUs, endocrine ceUs, somatic ceUs, recombinant ceUs and in vitro cultured ceUs.

10. The compound of Claim 1, further comprising nonceUular

material encapsulated by said hyrogel.

11. The compound of Claim 10, wherein said nonceUular material is

a therapeutic substance selected from the group consisting of antibiotics, immunosuppressive agents, chemotherapeutic agents, enzymes, hormones,

cytokines, antibodies, growth factors, antiapoptotic factors, adsorbants, 1-125,

antiviral compounds, CPG ODN, vaccines and combinations thereof.

12. A method for modulating immune responses in medical appUcations comprising the steps of providing a mixture containing an ultrapurified, natural, biocompatible polymer and a complement modulator;

applying said mixture to a mammal; and converting said mixture to a hydrogel having pores with a nominal molecular weight cutoff between 50 kDa and 100 kDa by contacting said mixture with a geUing agent before, during or after said applying step.

13. The method of Claim 12 wherein said applying and converting steps comprise the steps of:

coating a foreign material with the hydrogel; contacting the mammal with the coated foreign material to improve the

biocompatibility, hemocompatibiUty and immune tolerance to the foreign material.

14. The method of Claim 12, wherein said hydrogel comprises a nonadhesive, nonfibrotic, hygroscopic dressing and said applying step comprises applying said mixture to a surgical site or wound area.

15. The method of Claim 12, wherein prior to said applying step said method comprises the step of encapsulating ceUular material with the

hydrogel.

16. The method of Claim 15, wherein said ceUular material is selected from the group consisting of autologus ceUs, aUogenic ceUs and xenogenic

ceUs.

17. The method of Claim 15, wherein said ceUular material is

selected from the group consisting of islet ceUs, hepatic ceUs, neurocrine ceUs, somatic ceUs, recombinant ceUs and in vitro cultured ceUs.

18. The method of Claim 12, wherein prior to said applying step

said method comprises the step of encapsulating nonceUular material with the

hydrogel.

19. The method of Claim 18, wherein said nonceUular material is selected from the group consisting of antibiotics, immunosuppressive agents,

chemotherapeutic agents, enzymes, hormones, cytokines, antibodies, growth factors, antiapoptotic factors, adsorbants, 1-125, antiviral compounds and combinations

thereof.

20. The method of Claim 12, wherein said applying and converting

steps comprise endothehal geUing of capiUaries of donor organs.

21. The method of Claim 12, wherein said applying and converting

steps comprise the steps of:

filling one lumen of a dual lumen catheter with the mixture;

fining the other lumen with the geUing agent; and injecting the contents of the dual lumen catheter into a vascular system for instant geUing in the endotheUum.

22. The method of Claim 12, wherein said applying and converting

steps comprise in vivo geUing of the mixture.

23. The method of Claim 22, wherein said in vivo geUing occurs in one of a donor organ, tumor or host organ.

24. The method of Claim 22, wherein said in vivo geUing occurs at

an immunoprivUeged site selected from the group consisting of the brain and testes.

25. The method of Claim 22, wherein said in vivo geUing occurs at an immunocompetent site selected from the group consisting of the Uver and the

pancreas.

26. The method of Claim 12, wherein said polymer is ultrapurified alginate having an endotoxin content of 700 EU/g or less.

27. The method of Claim 12, wherein said complement inhibitor is a Factor D inhibitor.

28. The method of Claim 12, wherein said complement inhibitor is a sulfonic polymer.

29. The method of Claim 12, wherein said geUing agent comprises a 1-2% CaC solution.

30. A compound for activating immune responses in medical appUcations comprising: a hydrogel including (i) an ultrapurified, natural, biocompatible polymer, (ii) a complement activator, and (iii) a geUing agent,

wherein said hydrogel has pores with a nominal molecular weight cutoff of 200 kDa or more.

31. The compound of Claim 30, wherein

said polymer comprises alginate,

said complement activator comprises a Factor H inhibitor, and said geUing agent comprises a CaC ; solution.

32. The compound of Claim 30, further comprising a material encapsulated by said hydrogel wherein said material is selected from the group consisting of:

CPG ODN,

CPG ODN and a ceUular vaccine,

CPG ODN and a nonceUular vaccine, an endotoxin,

an endotoxin and a ceUular vaccine, and

an endotoxin and a nonceUular vaccine.

33. The compound of Claim 32, wherein said ceUular vaccine is selected from the group consisting of Uve bacteria, five viruses,

Uve attenuated bacteria,

Uve attenuated viruses, killed bacteria, and kiUed viruses.

34. The compound of claim 32, wherein said nonceUular vaccine is

selected from the group consisting of protein and DNA material.

35. The compound of Claim 34, wherein said DNA material is selected from the group consisting of:

an HIV DNA vaccine,

a DNA vaccine against resistant forms of N. Gonnorhoeae,

an Ebola DNA vaccine, an anthrax DNA vaccine,

a Usteria DNA vaccine, and a tularemia DNA vaccine.

36. The compound of Claim 30, comprising a ready-to-use solution

which contains between 0.5% and 5% of said hydrogel.

37. The compound of Claim 36, wherein said ready-to-use solution contains a component selected from the group consisting of NaCl solution, Hank's solution, a tissue culture media and RPMI 1640.

38. The compound of Claim 30, further comprising an endotoxin

binding matrix, wherein said hydrogel contacts said matrix to remove endotoxins from said matrix.

39. The compound of claim 30, wherein said hydrogel is formed into microcapsules.

40. The compound of Claim 30, wherein said hydrogel is formed into ultramicroscopic capsules smaUer than 5 microns.