WO2000035427A1

WO2000035427A1 - Methods for reducing the immunogenicity of proteins, tissues, or organs

Info

Publication number: WO2000035427A1
Application number: PCT/US1999/029452
Authority: WO
Inventors: Dale P. Devore
Original assignee: Collagenesis, Inc.
Priority date: 1998-12-15
Filing date: 1999-12-13
Publication date: 2000-06-22

Abstract

Disclosed herein is a method for generating a substantially non-immunogenic protein suitable for implanting into an animal. The method involves exposing the protein to an acylating agent under conditions which allow acylation of the protein to occur.

Description

METHODS FOR REDUCING THE IMMIJNOGENICITY OF PROTEINS- TISSUES. OR ORGANS Background of the Invention In general, this invention relates to methods for reducing the immunogenicity of proteins, tissues, processed tissues, tissue constructs, or whole organs, for example, as a means to facilitate their implantation into an animal recipient.

Surgical implants find a number of uses in the medical field, including burn treatment, skin grafting, dermal contouring, soft tissue reconstruction, and bone, ligament, and tendon repair. Ideally, such an implant is composed of a material that is immunologically inert and retains its implant properties for the life of the patient. Biocompatible materials such as silicone or collagen are available. However, in a number of animal models and clinical studies, collagen, for example, in various forms including commercially available forms designed for surgical implantation, has been found to be somewhat immunogenic.

In one particular example, using a rabbit model, it was found that animals exposed to cross-linked bovine type I collagen implants mounted both a humoral and cell-mediated response to the implant material (Meade et al., Biomaterials, 11(3): 176-180 (1990)). In this study, while cross-linked collagen was found to be less antigenic, extensive crosslinking did not entirely eliminate immunogenicity. In another example, also using an animal model, it was observed that, when bovine type II collagen was introduced intradermally into rats, almost 50% of the rats developed chronic arthritis (Morgan et al., Ann. Rheum. Dis., 39(3):285-290(1980)). In human patients as well, collagen has been found to sometimes elicit immunogenic responses. For example, in a survey of 103 human patients, it was noted that all patients that had received bovine or human collagen implants generated antibodies to the implants (Sellem et al., J. Dermatol. Surg. Oncol, 13(11): 1199-1202 (1987)). And, in a number of other studies that tested commercial preparations of collagen-based biomaterial, researchers found that different implants provoked inflammatory responses and elevated levels of anti-implant antibodies in 3% to 90% of patients tested (J. Am. Acad. Dermatol., 10(4):647-651 (1984); J. Biomed. Mater. Res., 20(1): 109-120 (1986); and Plast. Reconstr. Surg., 79(4):581-594(1987)). While many of these immune responses were considered mild, the long term effects of immunogenic implants in aging patients was not determined.

In addition, the immunogenicity of implant materials, such as collagen, may increase with repeated exposure. For example, some implant procedures involve multiple treatments to either correct a defect or maintain a desired result, and certain patients, after multiple exposures to an implant, go on to develop immune responses (DeLustro et al, Plast. Reconstr. Surg., 79(4): 581- 594 (1987)). Moreover, patients are sometimes exposed to more than one implant material, and there exists evidence that implant materials can sometimes act in synergy to provoke an immune response. In one particular study, it was found that collagen or silicone implants alone provoked only a minimal immune response; however, when co-introduced into an animal, these materials were notably immunogenic (Takayama et al., J. Laryngol. OtoL, 106(8):704-708 (1992)). In addition, human subjects with silicone implants often generate antibodies to collagen at a fairly high frequency (35%), and a subset of these patients go on to develop autoimmune diseases (J. Autoimmun., 6(3):367-377 (1993)). Moreover, some percentage of humans are unusually sensitive to collagen antigens, even though those collagen antigens may be their own. Human patients with Goodpasture's syndrome produce antibodies against the alpha 3 chain of their type IN collagen and go on to develop a sudden multisystem disease (Turner et al., J. Clin Invest., 89(2):592-601 (1992); Hellmark et al., Kidney Int., 46(3):823-829 (1994)). Another debilitating variation of Goodpasture's syndrome, termed Alport's syndrome, develops in 15%) of patients (Fleming et al, Transplantation, 46(6):857-859 (1988)). Patients with this X-linked recessive disease produce collagen autoantibodies against an immunogenic epitope of the alpha 5 chain of type IN collagen and suffer from sensorineural deafness, nephritis, and ocular malformations (Weber et al., Clin. Investig., 70(9):809-815 (1992)).

Finally, in addition to the immunogenicity of implant materials or endogenous collagens, there also exists similar immunogenicity problems with tissues and organs for transplantation into animal recipients. In 1996, over 53,000 patients were waiting for an organ donor (Organ Procurement and Transplantation Network, 1997 Annual Report, U.S. Dept. Of Health and Human Services), and a shortage of suitable tissues and organs from closely matched donors (typically, identical twins or siblings) forced the use of less well-matched allografts and xenografts. The ability to reduce or eliminate the immunogenicity of such tissues or organs would greatly improve transplant success and save lives.

Summary of Invention The present invention provides a method for generating a substantially non-immunogenic protein suitable for implanting into an animal recipient. This method involves acylating the protein by exposing the protein to an acylating agent under conditions that preferably allow for at least one free amine group (and more preferably, 90% of the amine groups) to be acylated. In other preferred embodiments, the acylating agents derivatize a deprotonated free amine group of the protein with an anionic group to reduce immunogenicity. This derivatizing step preferably reduces the pKa of the protein. Any acylating agent may be employed to carry out the methods of the invention, but preferred acylating agents include anhydrides (for example, succinic anhydride), acid chlorides, and sulfonyl chlorides.

In particular applications of the invention, the protein rendered non- immunogenic by acylation is collagen (for example, type I or type II collagen) or a protein associated with a tissue, processed tissue, tissue construct, or whole organ (for example, skin, cartilage, ligaments, tendons, muscle, nervous tissue, islet cells, corneal tissue, heart valves, blood vessels, bone marrow, peripheral blood, heart, lung, liver, kidney, adrenal gland, pancreas, or intestine). Preferably, the acylated protein, tissue, processed tissue, tissue construct, or whole organ is suitable for implanting into a mammal, and, more preferably, into a human. In addition, the mammal preferably exhibits no substantial production of antibodies to the acylated protein.

The invention also provides a method for reducing an immunogenic response to an implanted collagen, preferably, a heterologous collagen, by exposing the collagen to an acylating agent which causes acylation of the collagen to occur. This method is useful for reducing an immunogenic response such as arthritis.

In addition, in preferred embodiments of both of the above aspects, the invention provides a method for generating a non-immunogenic protein, for example, collagen that is derived from a primate, cow, pig, sheep, or, preferably, a human. As used herein, by "protein" is meant a compound composed of two or more linked amino acids, synthesized in vitro or in vivo, and existing as a monomer or multimer. This term includes proteins of the extracellular matrix (for example, collagen types I-XIX or a combination thereof, elastin, fibronectin, and proteoglycans), structural proteins (for example, cytoskeletal proteins), enzymatic proteins, cell surface proteins (for example, MHC class I proteins, MHC class II proteins, LFA proteins, integrins, and ICAM proteins), and ligands (for example, peptide hormones and cytokines).

By "immunogenic" is meant the capacity to stimulate an immune response (for example, a humoral response or cell-mediated response) in an animal.

By "acylated protein" is meant a protein in which at least one free amine group has been modified by exposure to an acylating agent.

By "acylating agent" is meant an agent that transfers an acyl group to another nucleophile. Examples of acylating agents include anhydrides, acid chlorides, and sulfonyl chlorides.

By "heterologous" is meant a protein that is foreign in origin to the recipient animal.

By "collagen" is meant any of the collagens (for example, type I-XIX or a mixture, variant, or fragment thereof) derived from a multicellular organism (see, e.g., Prockop et al., Annu. Rev. Biochem., 64:403-434 (1995)). The term includes collagen in any form, whether produced naturally, recombinantly, or treated mechanically, chemically, or enzymatically.

By "biomaterial" is meant any material that includes a protein, cell, tissue, processed tissue, tissue construct, or organ. A processed tissue may include addition manipulation beyond the method of the invention (e.g., to facilitate, harvesting, implanting, tolerance, or surgical success). Similarly, a tissue construct may involve further manipulation of the tissue such that a particular structural feature is obtained (e.g., skin being grown or shaped for a particular graft application or endothelial cells being grown or shaped into tubes for use as blood vessels). By "implant" is meant any biomaterial which is introduced into an animal and which has been derived from a source other than that animal recipient.

As described herein, one advantage of the present technique is that the immunogenicity of proteins may be reduced by acylating lysine and arginine residues. Since most proteins have lysine and arginine residues, the method of the invention is suitable for reducing the immunogenicity of virtually any protein, or any protein associated with a tissue or organ. In addition, this technique is relatively inexpensive and facilitates the reduction of immunogenicity with minimal processing of the protein, tissue, or organ. Other features and advantages of the invention will be apparent from the following detailed description and from the claims.

Detailed Description of the Invention The invention described herein features methods for reducing the immunogenicity of a protein, tissue, processed tissue, tissue construct, or organ such that it is suitable for implanting into an animal recipient. In a preferred approach, immunogenicity is reduced by acylation with succinic anhydride. However, any acylating agent may be used for this purpose, and, in particular examples, any anhydride, acid chloride, or sulfonyl chloride may be utilized. All of these acylating agents work by reacting with, and derivatizing, the deprotonated free amines of a protein. In many cases, the free amine group is derivatized with a chemical moiety that provides an anionic group, thereby reducing the pKa of the protein. For certain proteins, such as collagen, this also renders the protein more soluble at physiological pH.

Amine-reactive acylating agents useful in the invention include, without limitation, carboxylic acid anhydrides (e.g., glutaric anhydride, benzoic anhydride, and 1,2,4,5-benzene tertracarboxylic acid dianhydride), carboxylic acid esters (e.g., monophenyl terephthalate, ethyl benzoate, and alpha- naphthoic acid ethyl ether), carboxylic acid halides (e.g., succinic acid chloride), sulfonic acids (e.g., 1,3,-benzene-disulfonic acid, aniline-2-sulfonic acid, and 3-nitrobenzene-sulfonic acid), 2-formylbenzene-sulfonic acid halides (e.g., 4,4' biphenyl-disulfonyl chloride and benzene sulfonyl chloride), and mixtures thereof.

In general, the acylating agent may be an aliphatic or aromatic, mono-, di-, or higher functional carboxylic acid anhydride, ester or halide; or sulfonic acid or halide, such as a lower alkanoic, lower alkane-dioic, or higher functional lower alkane carboxylic, or aryl mono-, di-, or higher functional carboxylic (e.g., benzoic or naphthoic), acid anhydride, ester or halide, or lower alkyl, or aryl (e.g., phenyl or naphthyl), mono-, di-, or higher functional sulfonic acid or halide, to provide the corresponding acyl (carbonyl or sulfonyl) moiety on the amine group, for example, lower alkanoyl, aroyl (e.g., phenoyl or naphthoyl), alkyl sulfonyl, aryl (e.g., phenyl or naphthyl), sulfonyl, or substituted amino (ami do or sulfonamido).

The acylating agent may be added directly to a reaction mixture as a solid material (e.g., a powder) or dissolved in a suitable organic solvent such as acetone, N,N-dimethylformamide (DMF), ethanol, or methyl pyrrolidone. The total quantity of acylating agent depends on the extent of modification required or desired. In the case of collagen, the quantity of acylating agent required should generally satisfy the weight ratio of acylating agent to wet tissue of approximately 0.005-0.5:1, and preferably 0.05-0.1 : 1.

Using any of the above reagents, the protein acylation reaction (i.e., the amine modifying reaction) generally proceeds within a pH range of 7 to 11, although it is preferably carried out at a mildly basic pH (for example, pH 8-10, and, more preferably 8.5-9) to increase the reaction speed and reduce the processing time. For acylating the free amine groups on proteins associated with cells, tissues, or organs, an acylation buffer at physiological pH is preferably utilized. In addition to the above-mentioned methods, the acylation methods provided in any of DeVore et al. and Kelman et al. (U.S. Pat. Nos. 5,412,135; 5,104,957; 5,201,764; and 5,480,427) may also be utilized to reduce protein immunogenicity, and these patents are incorporated herein by reference.

There now follow detailed examples describing preferred techniques and experimental results. These examples are provided for the purpose of illustrating this invention, and should not be construed as limiting.

EXAMPLE 1 Methods for Reducing the Immunogenicity of a Protein As described above, the present invention provides a technique for reducing the immunogenicity of an implant through protein acylation. The acylation occurs via free amine groups found on the protein's lysine and arginine residues. To demonstrate the effectiveness of this technique, an immunogenic protein sample

(type I bovine collagen) was rendered non-immunogenic by protein acylation as follows.

A type I bovine collagen sample was prepared and acylated using standard procedures (as similarly described in U.S. Pat. Nos. 5,104,957; 5,492,135; and 5,631,243). Briefly, collagen containing bovine dermis was diced with a scalpel and, in 5g aliquots in 20 ml volumes of deio ized water, throughly macerated with a dispersion tool. Each aliquot was poured into a 50 ml centrifuge tube, diluted with 10 ml of deionized water, shaken vigorously, and centrifuged at 4000 rpm for 10 minutes at 4°C.

The tubes were decanted, and the material was consolidated and resuspended in a 350 ml volume of 0.5M NaOH in a 500 ml container and rotated for one hour at 4°C. The sample was then divided into 50 ml tubes, centrifuged as above, rinsed twice with 50 mM sodium phosphate buffer, and combined in a 500 ml container with 350 ml of a 50 mM sodium phosphate buffer. This solution was titrated with 0.5M phosphoric acid to pH 2.5, rocked for three hours at 4°C, and then titrated with 0.5M NaOH to pH 7.1. The sample was again divided, centrifuged as above, and washed three times with a 4 mM phosphate buffer. The resultant material was homogenized in 5.0 g aliquots in 20 ml of 20 mM disodium phosphate buffer (pH 9.0) using the "beaker in a beaker" method to disperse the bovine collagen matrix. This material was then derivatized with three additions (1.85 ml) of 0.08% glutaric anhydride solution (the pH being adjusted to 9.0 between additions). The material was centrifuged and treated again with a glutaric anhydride solution ( 1 ml) and retrieved. The material was then diluted with 4 mM phosphate buffer (pH 7J), filtered (100 μm pore size), centrifuged (at 10,000 rpm for 10 minutes in SS-34 rotor), and adjusted to a concentration of about 30 mg/ml.

EXAMPLE 2

Demonstration of Acylated Collagen Having Reduced Immunogenicity as Measured by Reduced Lymphocyte Response To demonstrate reduced immunogenicity by collagen treatment, we immunized a number of test animals with treated and untreated collagen and analyzed whether the lymphocytes of these animals recognized the treated collagen as immunogenic. A standard rat lymphocyte assay as previously described in Sayegh et al. (Proc. Natl. Acad. Sci, U.S.A. 89(16):7762-7766 (1992)) and Nella et al. (American Society of Transplant Physicians, Annual Meeting (May 1997)) was used for measuring the cellular immune response of an animal immunized with either treated or untreated bovine collagen. This rodent model is favored for testing the immunogenic potential of materials to be used in human subjects because the MHC of these animals is fully characterized, and the animals are known to be fully allogeneic.

In this immunogenicity test, immunologically naive rats were immunized with a collagen sample (at approximately 30 mg/ml in Freund's adjuvant) derivatized with glutaric anhydride as described in Example 1. An untreated collagen control (i.e., Zyderm I™, a non-derivatized, commercially available collagen) was diluted to 20 mg/ml in PBS, and administered to other test animals in parallel. Seven to ten days after immunization, animals were sacrificed and lymphocytes were purified from the lymph nodes. These lymphocytes were then tested in vitro for their ability to proliferate when incubated with the same collagen sample used to immunize the test animal. Lymphocyte proliferation was assayed by measuring the amount of ³H- thymidine incorporated by the cells after co-incubation with either treated or untreated collagen. Parallel experiments were performed with adjuvant (complete Freund's adjuvant) in order to set a baseline against which the immunogenic potential of the various collagen products were measured.

Results obtained showed that non-derivatized bovine collagen induced a lymphocyte response nearly two-fold higher than that of the Con A control. By contrast, derivatized bovine collagen induced a lymphocyte response only one- half that of the Con A control (Table 1). From these results, we concluded that a collagen implant derivatized according to the method of the invention failed to provoke any significant cellular immune response in the recipient animals tested.

Table 1

If desired, to extend these results, test animals may be immunized with treated and untreated collagen to determine if the implants cause induce antibody formation. Further, using established methods, such animals may also be monitored for the occurrence of any delayed type hypersensitivity response (DTH) at the immunization site.

In addition to the foregoing animal tests for implant immunogenicity, similar human tests can be performed using techniques known in the art. For example, peripheral venous blood may be drawn from human subjects and tested in vitro for the presence of any T cells responsive to an implant. Ideally, such studies are performed either in parallel or in addition to other appropriate tests (e.g., skin testing).

Briefly, to carry out this assay, 10-20 mis of heparinized venous blood is obtained from the patient, and peripheral blood mononuclear cells are separated by density gradient centrifugation (for example, by Ficoll-Hypaque™ density gradient centrifugation), washed twice in RPMI media, counted, and resuspended in an appropriate cell culture medium (RPMI 1640 (Biowhittaker, Walkersville, MD), 10% normal human serum (Normalceraplus, NABI, Miami, FL), lOmM Hepes buffer, 100 μg/ml penicillin, and streptomycin)). These cells are then co-cultured ( 37°C, 5% C0₂, and 100% humidity) with various test immunogens for variable periods in 96 well microtiter plates (round bottom 96 well plates from Costar™ Corp., Cambridge, MA). Lymphocyte proliferation is assayed by measuring the amount of ³H-thymidine incorporated by the cells (added for the final 18 hours of culture) using a β scintillation counter (Beta Plate, Wallac, Gaithersburg, MD) as previously described in Vella et al. (Transplantation, 64 (6): 795-800 (1997)). Preferably, blood samples from a patient are drawn prior to the introduction of an implant and at various time points thereafter so the clinician can assess how well the patient tolerates the implant over time. In addition, if the patient has several different implants, this assay can assist the clinician in determining which implants are tolerated and which are immunogenic.

EXAMPLE 3 In Vivo Demonstration of Acylated Collagen Having Reduced Immunogenicity

To further demonstrate that acylation can reduced the immunogenicity of a protein, treated and untreated protein samples were used to immunize a number of mice (DBA/IJ strain). Mice immunized with an acylated collagen failed to develop antibodies to the immunogen. In contrast, the untreated collagen controls provoked a notable humoral response in animals tested in parallel. These experiments were carried out as follows.

Type I collagen samples were prepared from bovine hide using the following procedure as similarly described in U.S. Pat. Nos. 5,354,336, 5,631,243, and 5,476,515. Briefly, bovine hide was cut into pieces approximately l x l cm and washed in 0.2M NaCl for 1 hour with stirring. These pieces were then rinsed in 20 volumes of deionized water and subsequently added to 10 volumes of 0.5M acetic acid at 4°C. After 72 hours, the resultant viscous mass was filtered through cheesecloth to remove any insoluble material. The filtrate was then diluted with an equal volume of 0.5M acetic acid (1 : 1) and digested with pepsin (Sigma Chemical Co.; 1 :60,000), added to a concentration of 2% (w/w), for 48 hours at 4°C. Any undigested hide was removed by centrifugation at 20,000 x g, and the remaining digested collagen was precipitated using dialysis against 0.01M disodium phosphate. Next, the collagen precipitate was redissolved in 0.5M acetic acid and then reprecipitated by the addition of NaCl to 0.8M. The resultant pellet was redissolved in 0.5M acetic acid, and the pH was adjusted to 9.5 by the careful addition of ION and IN NaOH at 2-4°C. This mixture was held at the above pH for 3 hours, and the denatured pepsin was removed by centrifugation at 20,000 x g at 4°C. The remaining solution was then carefully adjusted to pH 7.2, and solid NaCl was added to 2.5M. The precipitated collagen was recovered by centrifugation and redissolved in 0.5M acetic acid. Solid NaCl was once again added to 0.8M to re-precipitate the collagen. The collagen- containing pellet was then recovered by centrifugation and dissolved in 0JM acetic acid to an approximate concentration of 3 mg/ml collagen concentration. Lastly, the collagen-containing solution was dialyzed against three changes of 0JM acetic acid and filtered through a 0.45μ membrane. This collagen sample was used as the untreated collagen control sample (i.e., non-derivatized by acylation).

A treated collagen sample was made by derivatizing the above sample by acylation using succinic anhydride (Aldrich Chemical Co.). In particular, 500 mis of soluble collagen at a concentration of about 3 mg/ml was adjusted to pH 9.0 using ION and IN NaOH. Then, solid succinic anhydride was added to a concentration of 10% (w/w) collagen solids. This mixture was allowed to react at pH 9.0 for 30 minutes, and the reaction was stopped by raising the pH to 12.0 using IN NaOH. After 5 minutes, the solution was filtered through a 0.45μ filter, and the pH was adjusted to 4.3 using 6N and IN HCl. The mixture was stirred for 10 minutes as the derivatized collagen precipitated. The treated collagen precipitate was then dissolved in 0.05M sodium phosphate buffer at pH 12 to a concentration of 5 mg/ml. This treated collagen solution was stored at 2-8°C and designated as lot 108.

To quantitate the immunogenicity of the above acylated collagen implants (i.e., lot 108) as compared to untreated controls, anti-collagen antibody levels were assayed in a mouse model. DBA/IJ mice were immunized by intradermal injection with either 100 or 500 μg of a treated or untreated type I bovine collagen emulsified in 50% complete Freund's adjuvant. Mice serving as negative controls received a control buffer of 0.1 N acetic acid alone.

Blood samples from mice with implants and control mice were drawn at several time points and analyzed by solid phase radioimmunoassay (Wint et al., Analytical Biochemistry •, 104: 175-181, 1980). The results are shown in Tables 2 and 3, and the radioimmunoassay was performed as follows. First, 96 multi-well flat bottom plates (Linbro^®) were coated in duplicate or triplicate with collagen solutions at a concentration of 1 mg/ml dialyzed in a 0.1 M phosphate buffer at pH 1.2 -7.4, and incubated at 37°C for 1 hour. The plates were then decanted and washed with PBS. The multi-well plates were then incubated with a 10% BSA solution for 1 hour at room temperature, decanted, and washed three times or more with PBS (until visible protein bubbles disappeared). Next, 25 μl of antisera, from either a test animal or control animal, was added to each well and allowed to incubate for 1 hour at room temperature. Wells were then washed three times with PBS and exposed to 25-50 μl of ¹² I-labeled protein A (at least 50,000 CPM/well). Wells were then washed three times with PBS to remove non-specific counts. Samples were harvested with 1 N NaOH and assayed for the presence of anti-collagen antibodies in a gamma counter. Unless otherwise described, the numbers presented indicate counts per minute (CPM) emitted by ¹²⁵I-labeled protein A, revealing the amount of anti-collagen antibodies bound to a particular collagen substrate (Tables 2 and 3).

Tables 2 and 3 represent the results of these radioimmunoassays. Each table indicates the presence of antibodies capable of binding a particular collagen substrate. The substrates tested in Tables 2 and 3 are treated (lot 108) and untreated type I bovine collagen.

The dramatic result observed was that, regardless of the collagen substrate used to measure antibody levels, anti-collagen antibody levels in mice immunized with untreated bovine collagen were much higher than levels measured in mice immunized with acylated collagen (e.g., bovine collagen (lot 108)) (see Tables

2 and 3, compare striped boxes). In fact, anti-collagen antibody levels in mice immunized with acylated bovine collagen (lot 108) were similar to levels measured in mice immunized with only a control buffer. For example, the antibody titers measured for animals immunized with treated collagen (lot 108) were 90% of the antibody titers measured for the acetic acid negative control.

Another indication that acylated collagen was significantly less antigenic than untreated collagen is shown in Table 2, where acylated bovine collagen (lot 108) showed a 2-6 fold lower cross reactivity with various other anti- collagen antibodies than did untreated material (compare, for example, shaded boxes of Tables 2 and 3). In addition, we discovered that antibody titers measured for animals immunized with treated collagen (lot 108) were only 46% of the antibody titers measured for animals immunized with untreated collagen, representing a 54% reduction in the antibody titers for the derivatized collagen. By contrast, untreated collagen induced antibody titers that were 196% higher than the acetic acid controls. Similarly, when antibodies that bind an untreated collagen substrate were measured, we discovered that treatment (acylation) resulted in a reduction of antibody titers by 82% compared to untreated collagen (Table 3). Further, treated collagen (lot 108) induced titers that were only 82% of the acetic acid control where as untreated collagen induced titers that were 427% higher than the acetic acid control (Table 3).

These results demonstrated that the acylation of an immunogenic protein can dramatically reduce the immunogenicity of the protein in an animal recipient.

Table 2

Antibodv Titers to Modified Bovine Collagen (lot 108) in Mice Immunized with Various Collaαens

Table 3 Antibody Titers to Bovine Collagen in Mice Immunized with Various Coilaqens

EXAMPLE 4 Methods for Reducing the Immunogenicity of a Tissue or Organ for Implanting

Into a Mammal To reduce the immunogenicity of tissues, processed tissues, tissue constructs, or organs, such biomaterials are obtained and prepared by standard medical techniques. These biomaterials are then treated by the acylation techniques described above.

Modification of the acylation conditions designed to reduce or prevent cell lysis are preferably employed, including acylation reactions carried out at physiological pH. Biomaterials that have been acylated may be tested for reduced immunogenicity using any standard in vitro immunoassay technique. In addition, a reduction in the immunogenicity of a biomaterial may also be tested in an animal model, with appropriate modifications, for example, as described herein.

Once a tissue has been demonstrated to be non-immunogenic, the biomaterial can be introduced into the recipient animal or patient using appropriate surgical techniques. Unused non-immunogenic biomaterial may be tissue-banked for future applications. One particular biomaterial amenable to this method is skin which can be grown in large sheets in vitro using donor cells. This skin tissue is then removed from culture and processed using the methods of the invention. Essentially, the sheets of skin are dipped in a compatible buffer (for example, at physiological pH) in which an acylation reaction can proceed without compromising the integrity of the tissue. Cartilage, ligaments, tendons, corneal tissue, heart valves, and blood vessels may also be harvested and acylated in this manner. In addition, bone marrow or peripheral blood may be treated using a similar method. Bone marrow or peripheral blood may be harvested according to conventional methods, centrifuged, and resuspended in a buffer that maintains the integrity of the blood cells while allowing acylation of immunogenic cell surface proteins. The reaction is allowed to proceed for a period of time appropriate for reducing immunogenicity. The reaction is then stopped, the cells resuspended in an appropriate physiological buffer, and the resuspended cells introduced into an animal recipient. This method is particularly advantageous for treating peripheral blood for use in transfusions when blood typing is unavailable or not practical. The blood group antigens may be rendered non-immunogenic using the acylation reaction of the invention. Accordingly, the resultant blood product can then be administered to any patient without concern for the patient's blood type.

Organ transplantation may also be facilitated by the methods of the invention. Techniques for manipulating organs ex vivo for a limited time (24- 48 hours), for example, using hypothermic conditions and maintenance buffers, have been established. These conditions can be adapted to include an acylation step to reduce the immunogenicity of the organ. Organs which may be treated by this technique include heart, lung, liver, kidney, adrenal gland, pancreas, and intestine.

To carry out such a technique, procedures for the rapid surgical dissection of the transplant organ are employed to ensure that the organ is not damaged during preparation (see, for example, Starzl et al., Surg. Gynecol. Obstet., 165:343-348 (1987)). Next, the organ is perfused with a preservation solution designed to maintain high oncotic pressure and the maintenance of a near-intracellular ionic concentration. Perfusion conditions can be modified to bathe the organ with an acylating reagent. Acylation conditions may be modified depending on the degree of donor to recipient mismatch, with a higher degree of mismatch warranting more extended acylation. Following acylation, the organ is reperfused with preservation solutions to ready the organ for implanting into a patient. The remaining steps of the transplant operation are carried out according to conventional transplant protocols.

This general method represents an improvement to conventional transplant procedures. First, the method reduces graft rejection mediated by humoral or cellular immune responses. In addition, implanting a tissue or organ of reduced immunogenicity requires the use of fewer immunosuppressive drugs to insure graft survival. These drugs, while essential for conventional transplantation success involving the use of allografts or xenografts, carry the dangerous side effects of liver toxicity and the risk of opportunistic infections. These side effects can be lessened or eliminated using the methods of the invention.

Other Embodiments In general, the methods described herein are used for the treatment of human patients, but may also be used to treat any other mammal, for example, any pet or livestock. In addition, the methods of the invention may also be used to reduce the immunogenicity of any immunogenic protein or compound having free amine groups capable of being acylated, for example, any implant composed of, or coated with, a compound or protein having free amine groups. In particular, this method may be used to reduce the immunogenicity of other immunogenic proteins including, without limitation, MHC class I proteins, MHC class II proteins, integrins, LFA proteins, and ICAM proteins. To reduce immunogenicity, acylation reactions would be carried out on these proteins as described herein.

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.

Other embodiments are within the claims.

What is claimed is:

Claims

1. A method for generating a substantially non-immunogenic protein suitable for implanting into an animal, said method comprising exposing said protein to an acylating agent under conditions which allow acylation of said protein to occur.

2. The method of claim 1 , wherein at least one free amine group of said protein is acylated.

3. The method of claim 1, wherein at least 90% of the free amine groups of said protein are acylated.

4. The method of claim 1 , wherein said acylating agent reacts with a deprotonated free amine group on said protein.

5. The method of claim 4, wherein said amine group is derivatized with an anionic group.

6. The method of claim 5, wherein the pKa of said protein is reduced.

7. The method of claim 1, wherein said acylating agent is selected from the group consisting of anhydrides, acid chlorides, and sulfonyl chlorides.

8. The method of claim 7, wherein said acylating agent is succinic anhydride.

9. The method of claim 1, wherein said protein is a collagen.

10. The method of claim 9, wherein said collagen is Type I or Type II collagen.

11. The method of claim 1 , wherein said protein is associated with a tissue, processed tissue, or tissue construct.

12. The method of claim 11, wherein said tissue is selected from the group consisting of skin, cartilage, ligaments, tendons, muscle, nervous tissue, islet cells, corneal tissue, heart valves, blood vessels, bone marrow, and peripheral blood.

13. The method of claim 1, wherein said protein is associated with a whole organ.

14. The method of claim 13, wherein said whole organ is selected from the group consisting of heart, lung, liver, kidney, adrenal gland, pancreas, and intestine.

15. The method of claim 1, wherein said animal is a mammal.

16. The method of claim 15, wherein said mammal is a human.

17. The method of claim 15, wherein said mammal exhibits no substantial production of antibodies to said acylated protein.

18. A method for reducing an immunogenic response to an implanted collagen, said method comprising exposing said collagen to an acylating agent under conditions which allow acylation of said collagen to occur.

19. The method of claim 18, wherein said protein is heterologous collagen.

20. The method of claim 18, wherein said immunogenic response is arthritis.

21. The method of claim 1 or 18, wherein said protein is derived from a primate, cow, pig, or sheep.

22. The method of claim 1 or 18, wherein said protein is derived from a human.