CA1339092C - Method for the treatment of grafts prior to transplantation - Google Patents
Method for the treatment of grafts prior to transplantationInfo
- Publication number
- CA1339092C CA1339092C CA000613971A CA613971A CA1339092C CA 1339092 C CA1339092 C CA 1339092C CA 000613971 A CA000613971 A CA 000613971A CA 613971 A CA613971 A CA 613971A CA 1339092 C CA1339092 C CA 1339092C
- Authority
- CA
- Canada
- Prior art keywords
- graft
- tgf
- antibodies
- host
- beta
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
- A61L2300/414—Growth factors
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/426—Immunomodulating agents, i.e. cytokines, interleukins, interferons
Abstract
A method is provided for the treatment of grafts such as tissue or organs prior to transplantation into a suitable host. The grafts are incubated, coated, or perfused with TGF-.beta. for an effective time for the uptake of the agent into the grafts and induction of its biological effects.
The thus-treated graft is then transplanted into a compatible host, preferably a human host.
The thus-treated graft is then transplanted into a compatible host, preferably a human host.
Description
~ ~3~9092 METHOD FOR THE TREATMENT
OF GRAFTS PR~OR TO TRANSPLANTATION
Tbis invention relates to the treatment of grafts prior to I~ I ;fm into a host. More specifically, this invention is directed to incubating or perfusing a graft such as tissue or an organ with TGF-13 and ~ lg the treated graft into . suitable recipient, The success of a transplant of an allograft in a host depends on such factors as the antigens on the 11n ~ tissue that are recognized by the recipient as foreign and can evoke the rejection response, the cells in the recipient's immune system that mediate rejection, and the reactions that modify either the ,ulc~ .ld~iu-l of the foreign antigen or the cellular response.
The major l~ u~.u~ua~ibili~y complex (MHC) is the most important ;~
system encoding ~ la~ldl~dtiun antigens. The MHC is genetically complex because it includes many different loci, each encoding separate cell-surface antigens, and because the loci have extensive polylllùl,ul-islll. The loci of the MHC fall into one of two classes, Class I or Class 11, based on their tissue distribution, the structure of the expressed antigens, and their functions.
Class I antigens, present on all nucleated cells, serve as the primary targets for cytotoxic T
(CD8+) Iymphocytes. Class 11 antigens are not distributed in the tissue as widely and serve as primary targets for helper T (CD4+) Iymphocytes.
The polymorphic forms of the individual loci of human leukocyte antigen (HLA), the ~0 MHC in humans, have been recognized by antibodies and by various in vitro techniques that measure T-lymphocyte recognition. These responses, mediated by the recipient's recognition of polymorphism in the donor, correlate with the strong rejection reactions that take place i~
vivo.
Investigation into the cellular basis of graft rejection, using both in v~ro and in vivo studies, reveals that both CD4+ and CD8+ Iyl~.,uhouyLel participate in the rejection response.
Attempts to prolong the survival of allografts and xenografts after ~ A~ ,., both in experimental models and in medical practice, have centered mainly on the suppression of the immuneapparatusoftherecipient. Thistreatmenthasasitsaimpreventive;,,,,,,-,,,,, i,l,.c~si and/or treatment of graft rejection.
Examples of agents used for i~ ullu~u,ul)leS~ivll include cytotoxic drugs, antimetabolites, co-~i~u~t~.uids, and antilymphocytic serum. Nonspecific illllllllllUaUUplC~SiVC agents found particularly effective in preventive ;'~''''''''''''I'l~'C~S;Ull (IL~IIlliU,Ulil.~,, blUl~U-,ly,UIil.~., methylprednisolone, prednisone, and most recently, cyclosporin A) have significantly improved the clinical success of IIA~ The nephrotoxicity of cyclosporin A after renal transplantation has been reduced by cud~ lion of steroids such as prednisolone, or prednisolone in conjunction with azathioprine. In addition, kidneys have been grafted successfully using anti-lymphocyte globulin followed by cyclosporin A. Another protocol being evaluated is total Iymphoid irradiation of the recipient prior to IIAI.~I~IAI~IAI;I~II followed by minimal illllllUllU~U,U,UlC~iOI~ after L-~ ,uld--~l~iu--.
Treatment of rejection has involved use of steroids, ~-amino-6-aryl-5-substituted pylilllidil.~s, heterologous anti-lymphocyte globulin, and monoclonal antibodies to vsrloos ~ -2- 1~39~92 leukocyte populations, including OKT-3. See generatly J, PediatTics. 111: 1004-1007 (1987), and specifically U.S. Pat. No. 4,665,077.
The principal complication of immuno~u,uu-c~ drugs is infections. Additionally, systemic immunu~ u.c~iol~ is ~C~r-~ "~ i~d by undesirable toxic effects (e.g., nephrotoxicity when cyclosporin A is used after renal l~ ,uldllldliull) and reduction in the level of the h~,~uu,uoic~h: stem cells. Immlln~ ,.c,~i~., drugs may also lead to obesity, poor wound healing, steroid h~pe.~ly~c~ a~ steroid psychosis, leukopenia, ~a.lluillt.,~lillàl bleeding, Iymphoma, and hYU~ iUII.
In view of these crmp1ira~irn~ ItA~ " imml~nr~lr~gicts have sought methods for suppressing immune IC~UU~ e~S in an antigen-specific manner (so that only the response to the donor alloantigen would be lost). Such specific ;~ IC~iUII generally has been achieved by modifying either the âllLi~ iuily of the tissue to be grafted or the specific cells capâble of mediating rejection. In certain instances, whether immunity or tolerance will be imduced depends on the manner in which the antigen is presented to the immune system.
Pretreating the allograft tissues by growth in tissue culture before ~ has been found in two murine models systems to lead to permanent acceptance across MHC barriers.
Lafferty et al., T~ ; 138-149 (1976); Bowen et al., Lancet, _:585-586 (1979). Ithas been lly,uùlll~;L~d that such treatment results in the depletion of passenger Iymphoid cells and thus the absence of a stimulator cell population necessary for tissue in~ ullù~,.,.lh,ily.
2û Lafferty et al., Annu. Rev. Imml~nr,l L: 143 (1983). See also Lafferty et al., Science, L88: 259-261 (1975) (thyroid held in organ culture) and Gores et al., J. Immunol., ~: 1482-1485 (1986) and Faustman et al., Proc, Natl. ~ra.1 SCi. U,.~ A,,. 78: 5156-5159 (1981) (islet cells treated with murine anti-la antisera and ,..,..1,1 .. 1 before l~ IAI;fm)~ Also, thyroids taken from donor animals pretreated with 1~ i ' - y Lulu~ drugs and gamma radiation and cultured for ten 25 days in vit~o were not rejected by any normal allogeneic recipient. Gose and Bach, J.ExD.Med..
~; 1254-1259 (1979). All of these techniques involve depletion or removal of donor Iymphocyte cells.
In some models such as vascular and kidney grafts, there exists a correlation between Class II matching and prolonged allograft survival, a correlation not present in skin grafts.
3û Pescovitz et al., J.ExD.Med.. L6Q:1495-15û8 (1984); Conti et al., TransDlant. Prûc.~ L9:6s2-654 (1987). Therefore, donor-recipient HLA matching has been utilized. Additionally, blood transfusions prior to IIA~ have been found to be effective. Opelz et al., TransPlant.
_ 253 (1973); Persijn et al., TransDlAnl Proc.. 23: 396 (1979). The cr-~hinati~m of blood transfusion before ~ . donor-recipient HLA matching, and i~ r~iùll 35 therapy (cyclosporin A) after trar~rlaAtatir~n was found to improve significantly the rate of graft survival, and the effects were found to be additive. Opelz et al., Tran~nla~ Prûc.. 17:
2179 (1985).
The I~ UIàjIIaIiU~I response may also be modified by antibodies directed at immune receptors for MHC antigens. Bluestone et al., Immunol. Rev. 9Q:5-27 (1986). Further, graft 4û survival can be prolonged in the presence of antigraft antibodies, which lead to a hûst reaction thatinturnproducesspecificimm~ .c~ion. Lancasteretal.,Nature,315:336-337(1985).
~ --3--Tbe immune response of the host to MHC antigens may be modif;ed specifically by using bone marrow ~ ll as a preparative procedure for organ grafting. Thus, anti-T-cell monoclonal antibodies are used to deplete mature T cells from the donor marrow inoculum to allow bone marrow ll~ l without incurring graft-versus-host disease. Mueller-Ruchholtz et al., Tr~r~nl~nt Proc.. 8:537-541 (1976). In addition, elements of the host's Iymphoid cells that remain for bone marrow ~ 'd~ solve the problem of occurring when fully allogeneic transplants are used.
The survival time of skin grafts has been prolonged by a factor of two by treatment ~n vitro with cortisone, i' ' ~ , or urethane before , ' into a laboratory animal. The 10 amount of drug locally applied to the skin was smaller than the amount required to achieve a similar effect by injecting the drug ~y~ y. In an additional study, the donor skin was treated in vitro with ~ .,h;l.~/strepto~ rr ~o, or with RNA and DNA pl~ liu-~s of the recipient. Further, treatment of transplant tissues with a solution of glutaraldehyde prior to '' was found to reduce their antigenicity. See U.S. Pat. No. 4,120,649.
The transforming growth factor-(TGF-B) molecules identified thus far are each dimers containing two identical 112 residue polypeptide chains linked by disulfide bonds. The molecular mass of these dimers is about 25 kd. Biologically active TGF-B has been defined as a molecule capable of inducing anchorage independent growth of target cell lines or rat fibroblasts in in v~t~o cell culture, when added together with EGF or TGF-~ as a co-factor.
TGF-B is secreted by virtually all cell types in an inactive form. This latent form is first activated by proteolytic cleavage of mature TGF-B from its precursor (at the Arg-Ala bond in position 278). A non-covalent complex is formed from the association of the mature TGF-B with the precursor remainder. This complex is disrupted so as to activate the TGF-B either by exposure to transient acidification or by the action of exogenous proteases.
There are at least three forms of TGF-B currently identified, TGF-B1, TGF-Bz, and TGF-B3. Suitable methods are known for purifying this family of TGF-Bs from platelets or placenta, for producing it in ~, hin~nt cell culture, and for d~t~ & its activity. See, for example, R. Derynck et al., Nature, ~lh,701 (1985) European Pat.Pub.Nos. 128,849 published December 19, 1984, 105,014 published April 4, 1984, 200,341 published December 10, 1986, 169,016 published January 22, 1986; 268,561 published May 25, 1988; and 267,463 published May 18, 1988; U.S. Pat. No. 4,774,322; Seyedin et al, J. Biol. ('h~m 262: 1946-1949 (1987);
Cheifetzetal,~1,48:409-415(1987);Jakowlewetal.,MolecularEndocrin _:747-755(1988);
and Dijke et al., Proc. ~tl ~r~l S~i U.~ 85: 4715-4719 (1988).
TGF-B has been shown to have numerous regulatory actions on a wide variety of both normal and neoplastic cells. Recent studies indicate an important role for TGF-B in cells of the immune system (J. Kehrl et al., J. EXD. M~-~ 163:1037 [1986]; H-J. Ristow, Proc. ~ tl A~
Sci. U.S.A., 83:5531 [1986]; A. Rook et al., J. Immunol.. 136:3916[1986]) and in proliferation of connective and soft tissue for wound healing ~ (M. Sporn et al., Science, ~;1329 [1983]; R. Ignotz et al., J, Biol. t'h~m 261:4337 [1986]; J. Varga et al., B.B.Res.Comm 138:974 [1986]; A. Roberts et al., Proc, Natl. Acad. S~i U.S.A., 78:5339 [1981]; A. Roberts et al., E~L
Proc., 42:2621 [1983]; A. Roberts et al., Proc, ~ -tl A~rl S~ i U.S.A., ~:4167 [1986]; U.S. Pat.
_4_ ~33~092 No. 4,774,228 to Seyedin et al.), as well as epithel;a (T. Matsui et al., Proc, Nqtl Arq~l ,cri U.S.A., ~:2438 [19861; G. Shipley et al. Cancer i?PC 46:2068 [1986]). Moreover, TGF-B has been described as a suppressor of cytokine (e.g., IFN-, TNF-~) production, indicating its use asan;,,,.. ,,n,~ ,ca~l.tfortreatingi~fi,qmmqfr,rydisorders(Espeviketal~J~F~tn~Med~
571-576 ~1987]; European Pat. Pub. Nos. 269,408 published June 1, 1988 and 213,776 published March 11, 1987), and as a promoter of cachexia (Beutler and Cerami, New Fno J. ~IP~I ~;
379 [1987]). Further, TGF-B induces collagenase secretion in human fibrobl~t cultures (Chua et al., J, Biol, ~'hPm 260:5213-5216 [1983]); stimulates the release of p,.,~ and -~ li7qfirn of calcium (A. Tashjian et al., Proc. i~qtl Arq~l ~ri U.S.A., ~ 4535 [1985]); and inhibits endotheliai regeneration (R. Heimark et al., ~ç~, _~;1078 [1986]).
TGF-B is multifunctional, as it can either stimulate or inhibit cell proliferation, differentiation, and other critical processes in cell function (M. Sporn, Science, 233:532 [1986]).
The multifunctional activity of TGF-B is modulated by the influence of other growth factors present together with the TGF-B. TGF-B can function as either an inhibitor or an enhancer of anchorage-il~ growth, depending on the particular set of growth factors, e.g., EGF or TGF-o~, operant in the cell together with TGF-B (Roberts et al., Proc. N,qfl Arq~i Sci, U.S.A., 82:119 [1985]). TGF-B also can act in concert with EGF to cause proliferation and piling up of normal (but not rheumatoid) synovial cells (Brinkerhoff et al., Arthrifis Rh ~1370 [1983]).
Most recently, TGF-B has been found to suppress the expression of Class 1~
1 - hility antigens on human celis induced by human interferon- and to inhibit constitutive expression of the Class 11 antigen message in the cells (Czarniecki et al., L
140: 4217-42223 [1988]; Czarniecki et al. J. !nfqrferon RP~ Z: 699 [19871; Palladino et al., J. Cell. Birr~om Supp. I IA [Jan. 17-Feb. 5, 1987], UCLA Symposia on Molecular and Cellular Biology, Alan R. Liss, Inc., New York, abstract A016, p. 10; Chiu et al., Triennial Symposium: Biology of Growth Factors, University of Toronto, Ontario, Canada, [June 17-19, 1987]; Palladino et al., ImmunobioloqoY~ 175: 42 [19871).
For a general review of TGF-~3 and its actions, see Sporn et al., J. Ceil Biol.. 105: 1039-1045 (1987) and Sporn and Roberts, Nature, 332: 217-219 (1988).
There is a need in the art for a method to prolong graft survival in transplant operations and minimize the toxicity and other adverse effects arising from the use of large doses of imm11n. ~ 6~
Accordingly, an object of this invention is to provide for longer graft survival in the host.
Another object is to provide for a LIA~ II method wherein lower amounts of iluluuuoau~ c~ , agents, if any, need be q illlilli~ ca to the host to achieve successful results, thereby reducing the side effects associated with systemic administration of illllllu drugs.
These and other objects will become apparent to one of ordinary skill in the art.
These objects are acc~ by a method for l.~ grafts from donors into 40 hosts which comprises treating a graft with a , ~ l . comprising a ~ ul ;~ ~lly effective amount of TGF-B; and lldll~ldlllillg the graft into a compatible host.
~ 13.~9092 In another aspect, this invention provides a graft treated with a uullluua;l;Ol~ comprising TGF-B, alone or in combination with another immUnuauuplt~aa;~, agent.
In a further aspect, this invention provides a . ' comprising TGF-13 in a perfluolucl.~...h,~.l emulsion.
The method herein results in grafts that are il.. ,llOIOgi-,ally stable in the hosts, biologically functional, and capable of being stored prior to L-~
This invention enables the P~ghl ' of a bank of treated grafts that can be utilized for short-term or long-term storage. In addition, the graft is not rejected when Llalla,ulalltt:d into suitable hosts. The method is applicable to both allografts and xenografts, and the use of lû xenografts overcomes the difficulties encountered by the limited supply of tissue from humans.
The term ''graft" as used herein refers to biological material derived from a donor for L.~ - into a recipient. Grafts include such diverse material as, for example, isolated cells such as islet cells, tissue such as the amniotic membrane of a newborn, bone marrow, hematopoietic precursor cells, and organs such as skin, heart, liver, spleen, pancreas, thyroid lobe, lung, kidney, tubular organs (e.g., intestine, blood vessels, or esophagus), etc. The tubular organs can be used to replace damaged portions of esophagus, blood vessels, or bile duct. The skin grafts can be used not only for burns, but also as a dressing to damaged intestine or to close certain defects such as dia,u~ ,t,-rl~h, hernia. The graft is derived from any source, preferably n~msli~n including human, whether from cadavers or living donors.
2û The term "host" as used herein refers to any compatible transplant recipient. By ", , ' '-" is meant a host that will accept the donated graft. Preferably, the host is mornn~slisn and more prefe~ably human. If both the donor of the graft and the host are human, they are preferably matched for HLA class 11 antigens so as to imprûve 1,;~",..",I.~lil ility.
The term "donor" as used herein refers to the species, dead or alive, from which the graft is derived. Preferably, the donor is mgmrn ~ n Human donors are preferably volunteer blood-related donors that are normal on physical PYsmir~s~inn and of the same major ABO blood group, because crossing major blood group barriers possibly prejudices survival of the allograft.
It is, however, possible to transplant, for example, a kidney of a type O donor into an A, B or AB recipient.
The term "transplant" and variations thereof refers to the insertion of a graft into a host, whether the ~ is syngeneic (where the donor and recipient are genetically identical), allogeneic (where the donor and recipient are of different genetic origins but of the same species), or xenogeneic (where the donor and recipient are from different species). Thus, in a typical scenario, the host is human and the graft is an isograft, derived from a human of the same or different genetic origins. In another scenario, the graft is derived from a species different from that into which it is transplanted, including animals from phylogenically widely separated species, for example, a baboon heart transplanted into a human host.
The term "TGF-13" refers to the family of molecules described l~ illal)u~,. Reference to TGF-B herein will be understûûd to be a reference to any one of the three currently identified forms, TGF-131, TGF-132 and TGF-fs3, as well as other molecules related to this 13~9092 ~ --6--family of proteins, and those idèntified in the future, their alleles, and their p~ t~ d amino acid sequence variants, so long as they are effective in the method described herein. The TGF-13 is a,uulupli~t~1y from any source, preferably r~qmmqliAn, and most preferably human.
TGF-13 from animals other than humans, for exampie, porcine or bovine sources, can be used 5 for soaking grafts to treat humans. Likewise, if it is desirable to treat other mammalian species such as domestic, farm, sports, or pet animals, human TGF-13, ~ well ~ TGF-~3 from other species, is suitably employed.
The term "immunosuppressive agent" as used herein refers to substances that act to suppress or mask the immune system of the host into which the graft is being lla~ ,Jldilt~l. This 10 would include substances that suppress cytokine production, downregulate or suppress self-antigen expression, or mask the MHC antigens. Examples of such agents include 2-amino-6-aryl-5-substituted ,uylilllidi~c~ (see U.S. Pat. No. 4,665,077, supra), aLa~lliùp~ . (or ~,y. ~ ,h.,~ ",hl~, if there is an adverse reaction to aLall.iuulil.~ y,uli.~C, glutaraldehyde (which masks the MHC antigens, as described in U.S. Pat. No. 4,120,649, supra);
15 antiidiotypic antibodies for MHC antigens; cyclosporin A; one or more steroids, preferably uulliuo~t~,.uhla, and most preferably ~lu~,ucOlli~u~t.,.uida such ~ prednisone, " yl,ulcdl~iaulull~, and ,' -~ ~; anti-interferon- antibodies; anti-tumor necrosis factor-~ antibodies; anti-tumor necrosis factor-B antibodies; anti-interleukin-2 antibodies;
anti-cytokine receptor ântibodies such as anti-lL-2 receptor antibodies; h.,t~,.ulob.,..~ anti-Iymphocyte globulin; pan-T antibodies, preferably OKT-3 monoclonal antibodies; antibodies to CD4; streptokin~e; streptodornase; or RNA or DNA from the host.
The preferred agent for this purpose will depend on many factors, including the type of A~ being performed, as well as the patient's history, but a general overall preference is that the agent be selected from cyclosporin A, a glu~ocolli-,u~.~,.ui:l (most preferably prednisone or methylprednisolone), OKT-3 monoclonal antibody, aLall-iop- i,~, bromocryptine, heterologous anti-lymphocyte globulin, or a mixture thereof.
TGF-L~ is appropriately used in its activated form, i.e., the mature form is cleaved from its precursor using a suitable enzyme and the resultant complex is treated with acid, a protease such as pl~min or cathepsin D, alkali, or chaotropic agents, to activate the TGF-fs. See Keski-Oja et al., J. ~ l Bin~hPm SurJnl IIA: 60 (1987); Kryceve-Martinerie et al., Int. J. ~An~qr, ~;553-558 (1985); Lawrence et al., Biochem. Bio~hYs. l~qc C~-mmlln ~; 1026-1034 (1985);
Lawrence et al., J. Cell PhYci~l 121: 184-188 (1984).
The ~ , ' ~ to be used in the therapy herein will be dosed in a fashion consistent with good medical practice taking into account the nature of the l.A,~ -- and the disorder to be treated, the species of the host, the medical condition of the individual patient, the presence of any other drug in the, , and other factors known to placl ' For purposes herein, the "Ll.~,.du~...i~qlly effective amount" of TGF-~3 used to treat the graft is an amount that is effective to reduce the ~ """.n~ ;ly of the graft so that it will be compatible with the host and not be rejected. A generally effective amount for this purpose is in a range 40 of I pg/ml to I mg/ml, more preferâbly, 10 pg/ml to l~g/ml, and most preferably, 10 pg/ml _7 to ~00 ng/rnl, but the practitioner will know more precisely what amount is to be used taking into ~u.~idelaliù.l the factors described above.
The TGF-B is optionally formulated with one or more ,, c~a;~, agents to enbance the ,, ca~(u~l effect on the graft. The effective amount of such other agents 5 depends on the amount of TGF-B present in the formulation, the type of transplant, and other factors discussed above, but generally also ran8e from about 0.1 pg/ml to 0.5 mg/ml as necessary to achieve the appropriate endpoint, i.e., ionger graft survival. Typically, the TGF-B is formulated by mixing it at ambient t~ , e at the appropriate pH, and at the desired de8ree of purity, with phy~ acceptable carriers, i.e., carriers that are non-toxic to 10 recipients at the dosages and employed. The pH of the formulation depends mainly on the particular use, and ~ of TGF-B, but preferably ranges anywhere from about 3 to about 8. Preferably the TGF-B is maintained at an acidic pH (e.g., about 4 to about 6) to prevent its adhering to the containers in which it is placed, as occurs at pH values .~uuluAA~ lg ,ully~;ùlù25h.dl levels, and to ensure activation of the TGF-B molecule. Thus, 15 formulation in an acetate buffer at pH 5 is a suitable '- ~ " If the TGF-B adheres to the containers in which the grafts are being treated, an appropriate ingredient, such as albumin, is optionally added, in an amount that does not prevent the TGF-B from perfusing or adhering to the 8raft being treated.
If the TGF-B formulation is to be applied topically, for example, if it is to be painted 20 onto a skin graft prior to IIA ~ , it is preferable to use a viscous solution such as a gel rather than a non-viscous solution. This may be r , '' ' 1, for example, by mixing the solution of the TGF-B with a gelling agent, such as a poly ~ , preferably a water-soluble poly~4~,cl.d-i-1e, such as, e.g., hyaluronic acid, starches, and cellulose derivatives, e.g., methylcellulose, llyJIuA.~ yl cellulose, and cAAIlluAy ' yl cellulose. The most preferred 25 8ellin8 agent is methylcellulose- The pùlya~.cl.dlide is generally present in a gel formulation ' in the range of 1-90% by weight of the gel, more preferably 1-20%. Examples of other suitable poly~ .iJ~, for this purpose, and a J~t~.ll-i..AA1iull of the solubility of the poly-are found in EP 267,015, published May 11, 1988.
If the 8raft to be treated is to be stored for any period of time, the TGF-B is preferably 30 formulated in or added to a perfl.,ù-u-,h~ l emulsion (acting as a blood substitute) to enable higher of oxygen to reach the 8raft. Such emulsions comprise a perfluol, ~ ' such as perfluu-ud~ and/or perf~olo~-iy-u~ ylAA.ll;l~e emulsified with a surfactant in water. Examples of suitable surfactants include the poloxamer surfactants (which represent a series of molecules that are blocked copolymers of ethylene oxide and propylene 35 oxide), alone or in admixture with a ~ .hA,I;~.;d such as egg lecithin. The perfluu.u~ ..-h,41 is chosen to be the least toxic in humans. One example of such an emulsion, which is commercially available from Green Cros~, is Fluosol-DA 20%, which contains perfluù.ùd~,~.lill and perfluu-uLliylu~uylAAlrli~.~ emulsified with the poloxamer surfactant, Pluronic F-68. The perfluo- . ' ~ ' emulsions and their effects in mammals are described more fully in Bollands 40 et al., J. Pharm. Pharmacol.. ~2: 1021-1024 (1987).
~trade-mark -8- 13~9092 The TGF-~3 for use in therapeutic A~ if m is preferably sterile. Sterility is readily , ' ' ~' by sterile filtration through (0.2 micron) br:~nPc TGF-~3 ordinarily will be stored as an aqueous solution since it is highly stable to thermal and oxidative ,~ ;fll~
although Iyophilized fu~ ulltiol~s for IC~ 11 are acceptable.
In accordance with the method of this invention, the graft is contacted with the TGF-!3, , - The contact suitably involves incubating or perfusing the organ with theor applying the ~ i"" to one or more surfaces of the graft. The treatment generally takes place for at least one minute, and preferably from I minute to 72 hours, and more preferably from 2 minutes to 24 hours, depending on such factors as the ~ iVII of TGF-13 in the formulation, the graft to be treated, and the particular type of formulation.
Perfusion is ~ ' by any suitable procedure. For e~cample, an organ can be perfused via a device that provides a constant pressure of perfusion having a pressure regulator and overflow situated between a pump and the organ, as described by DD 213,134 published Sept.
5, 1984. Alternatively, the organ is placed in a hyperbaric chamber via a sealing door and perfusate is delivered to the chamber by a pump that draws the fluid from the reservoir while spent perfusate is returned to the reservoir by a valve, as described in EP 125,847 published Nov. 21, 1984.
For skin grafts, the formulation is suitably painted on the lower surface of the skin to be grafted so that there is a layer of the TGF-3 between the tissue of the host and the lower surface of the graft. Alternatively, the whole skin graft is submerged in the f n.n.pf.~ci-if~n After the graft is treated, it is suitably stored for prolonged periods of time or is used immediately in the transplant procedure. Storage life can be enhanced as described above by using a blood substitute in the formulation (e.g., perfl--olv~,h~ l emulsion), or by perfusing the graft with a formulation of the TGF-~3 containing chilled isotonic agent and Alll;. .I~.rl~lAIII
followed by glycerol to allow for freezing of removed organs with no destruction of the cells, as described in JP 60061501 published April 9, 1985. In addition, the organs can be preserved with different liquids that include the formulation while the organs are cooled to freezing Leluu~,.,JLulc~, to preserve the organ semi-permanently without cell necrocytosis, as described by U.S. Pat. Nos. 4,462,215 and 4,494,385.
Respecting cardiac transplants specifically, Parent et al., CrvQbinlf)f~v~ .~; 571-576 ( 1981 ) describes that cold coronary perfusion prior to Ll~ ll at 5~C increases protection of the homograft during the initial period of illl~,l..lll.liif l~. Any of these procedures, or others, are within the scope of v'lis invention if deemed necessary upon or after TGF-.'3 treatment of the graft.
Before ~ ,., the graft is preferably washed free of the TGF- ~3 f l ll ~ ~
by soaking it in a physiological saline solution or by other means appropriate for this purpose.
Also, prior to ~ , the host is optionally given one or more donor-specific blood transfusions to aid in graft survival. An alternative procedure is to subject the host to total Iymphoid irradiation prior to the Ll~lllaul~ dLivl~ operation. Any other pre-transplant procedures that would be beneficial to the particular transplant recipient can be performed as part of the method of this invention.
-? 9 133t~92 The tran~rlanf~-ion procedure itself will depend on the particular disorder being treated, the condition of the patient, etc. The medical pldc~iliullrv. will recognize the appropriate procedure to employ in any given case. The transplants are optionally monitored ~y~lcllld~;crllly during the Gritical postoperative period (the first three months) using any suitable procedure.
5 One such procedure is Iddiu--u~ ie intravenous angiography using 99Tcm~ .Lalc, as described by Thomsen et al., Acta Farlirl 29:138-140 (1988). In addition, the method herein is amenable to ~;"",II~ , multiple organ perfusion and l-~ A~ (Toledo-Pereyra and n7iP, Am. Sur~., ~ 161-164 (1980). After the ~ IUI;..A, ;"""""'.' 'I.~.C~;UII
therapy, using TG~-13 and/or other; ~ l C~rlllltS), is utilized as necessary to ensure 10 graft survival.
In some instances, it is desirable to modify the surface of the graft so as to provide positively or negatively charged groups, as by using a suitable amino acid or POlymer or by attaching a physiologically acceptable source of charged functional groups. For example, a negatively charged surface is appropriate for blood vessels to diminsh blood clotting. It also is 15 desirableincertainui--:u-ll,Ldllcc,torenderthesurfacel-y~ '~' orhydrophilicbycoupling, e.g., phenylalanine, serine or Iysine to the surface. An ;.~ p.c,s;vc agent particularly effective for these surface - "rirafinn~ is gluteraldehyde.
Ir~ u~ c~a;OIl therapy typically involves the ~Idlllil~iaLIr~Li~ll of an effective amount of an ,.,UIC~S;~., agent, including TGF-f5 itself. The innrn~n~ es~ agent 20 ~ will be formulated and dosed in a fashion consistent with good medical practice.
Factors for consideration in this context include the clinical condition of the individual patient, the cause of the transplant, the site of delivery of the agent, the method of arlmini~frûtinn, the scheduling of ~ and other factors known to pldulii- The "effective amount"
in this context, which is determined by such rr~ncirl~r~irln~, is the minimum amount necessary 25 to prevent an immune response that would result in rejection of the graft by the host. Such amount is preferably below the amount that is toxic to the host or renders the host significantly more susceptible to infections. The amount of illllllullual.~Julca~lvc agent required for the invention herein may be lower than that normally required for ll~ila,ul.llllcd grafts that have not been pre-treated, and depends on the individual circumstances surrounding the transplant and 30 the type of immlln..~.,l,l,,c~s;v~ agent employed.
As a general proposition, the total pl~ la~ 411y effective amount of the immunuauu~J-c~aivc agent, cyclosporin A, ddl--;n;~e-cd parenterally per dose will be in the range of about 0.1 to 20 mg/kg of patient body weight per day, with the typical range of cyclosporin A currently used in ~,, c~S;v., therapy being 5 to 15 mg/kg/day. For renal transplants 35 it is useful to administer massive doses at short periods of ~ lu~u~olli~u~t~.ùid; e.g., methylprednisolone in several-gram doses per day is given for 3 to 5 days followed by 60 to 100 mg prednisone without TGF-fs pre-treatment of the graft. With such pre-treatment, lower doses would be expected to be useful.
As noted above, however, these suggested amounts of immunuau,up.e,,,~lll are subject to 40 a great deal of therapeutic discretion. The key factor in selecting an appropriate dose and scheduling is the result obtained, i.e., graft survival. For example, relatively higher doses may . . .
-~o be needed initially for the treatment of hyperacute graft rejection, which can be attributed to antibody-mediated graft destruction, or at a later stage for the treatment of acute rejection, which is ~ t~ by a,sudden decline in graft function.
The~ c~;~agentis~ld~l~;ùi~i~.cdbyanysuitablemeans,includingparenteral, 5 and, if desired for local ;- ~ ca~ treatment~ I IIC~iu~ Parenteral infusions include ' , intravenous, intraarterial, ' 1, or ~ 1,, l --- ~,.~
- ' ' ' In addition, the ' ,IulrJa~ agent is suitably ~ ' cd by pulse infusion, particularly with declining doses of the; ~ ca~ , agent, or by continuous infusion.
The invention will be more fully understood by reference to the following examples.
They should not, however, be construed as limiting the scope of the invention.
EXAMPLE I
Mllrjn~ Skin ~r ft Mr~
TGF-B: The TGF-~ used herein is from any one of several sources: (I) porcine platelel-15 derived TGF-Bl at 96 percent purity purchased in a Iyophilized form and suspended in 4 mM
HCI and phosphate buffered saline (PBS~ solution prior to use, (2) TGF-131, prepared by recombinant means as described by EP 200,341, supra, (3) TGF-13~, prepared as described by US. Pat. No. 4,774,322, sllpra, or (4) TGF-B3, prepared by ., hir.-.nt means as described by EP 267,463, sl pra see Fig. 29 and a~ u~ul~a~y ~e text of EP 267,463). The TGF-B from any of 20 these sources is recovered and formulated at ~00 ng/ml at pHS in an aqueous acetate buffer.
The resultant solution is sterile filtered through a 0 2 micron polysulfone filter Grafts: Skin allografts (I x 2 cm square) are obtained from BALB/c (H-2d) mice Hosts: Ihe hosts used are C57BL/6 (H-2s) mice Procedure: The skin grafts are incubated in one of the above TGF-B solutions at room 25 ~ p.,llull~ in a sterile glass vial for I minute to 24 hours, then ~vashed two times with PBS
' solution to remove any free TGF-B, The thus-treated skin is applied to the mouse recipient, and covered with vaseline gauze, and then with plaster of Paris, which are removed after ten days The treated grafts are tightly bound to the recipient They are initially soft, but become ylulyc~i.~ly stiffer with minimal shrinkage in size, and remain free from infection The histology shows that the grafts are viable and ~ , and the general structure of the skin (epidermis, adnexa, and dermis) is preserved for at least three months. Visual ~ f~rrnin~finn can also be made (e.g, looking for white skin on a black mouse).
R~t Skin Gr~ft Ml~
An aqueous-based gel of ' ylu(,llulu~c (Methocel A4M from Dow) is prepared by mixing it in water at a .I~iOII of 209~ by weight to give a viscosity useful for a topical skin application The TGF-BI ~, ' '~ produced as described in Example I is mixed with the gel to provide a co..~,G..LI~t;o~ of i mgjml.
This TGF-B gel is applied to the lower (back) side of skin taken from Fl Lewis/Brown 40 Norwegian rats, After 24 hours, the skin is placed on the graft bed of the Lewis parent rat, covered with gauze, and then with plaster of Paris, and ,~ ly removed after ten days *trade-mark : .
133~092 Alternatively, the graft is held in place with metal clips applied using a mechanical clipping device. The results are comparable to those observed for the mice as described above.
,MIlrinP an-l RRr J~IPt CPII T ~ ~ ~' l~odel Islet cells are extracted from the pancreas of a BALB/c mouse, soaked in one of the TGF-B compositions described in Example I for 48 hours, and ~ cd into the subrenal capsule space of a ~G~uLuLu~ill-treated allogeneic mouse such as C~7BL/6 male mouse. See, e.g., Faustman et al., sup~a. Similarly, islet cells are extracted from the pancreas of a Fl Lewis/Brown Norwegian rat, incubated in one of the TGF-B~ , described in Example1 for 24-48 hours, and Ll , ' ~' into the subrenal capsule space of a ~LIc~ ~JLul~,;u-treated CS~BL/6 mouse. The results indicate that the islet cells are not rejected by the host after three months.
EXAMPLE IV
~vlllrin~ an-i RRt ~PRrt TrRn~nlRnt ~n~PI
A heart is excised from a sacrificed BALB/c mouse and from a sacrificed Fl Lewis/Brown Norwegian rat. The respective hearts are ~ - ' ~ 'y incubated at room UIC with any of the TGF-B solutions of Example 1. The in~ubations are carried out for 24 to 48 hours. The hearts are washed free of the TGF-B solution using two washes of PBS
and then ' '~"I~ L,.~ using standard heart transplant operations into the ear of a C3H/HeJ BALB/c mouse and into the aorta of a Fl Lewis/Brown Norwegian rat from which tbe hearts had been removed, the mouse heart being received by a different allogeneic strain of mouse and the rat heart being received by a different allogeneic strain of rat. The gtafts are not rejected three months after rrRn~rlRntRtion Alternatively, after extraction the heart is stored in a perfluo..,~ l emulsion such 25 as Fluosol-DA 20% (Green Cross, Japan) to which one of the TGF-B solutions is added prior to trRn~rlRntRtinn in an amount sufficient to provide at least I ng/ml of TGF-B in the final emulstion.
OF GRAFTS PR~OR TO TRANSPLANTATION
Tbis invention relates to the treatment of grafts prior to I~ I ;fm into a host. More specifically, this invention is directed to incubating or perfusing a graft such as tissue or an organ with TGF-13 and ~ lg the treated graft into . suitable recipient, The success of a transplant of an allograft in a host depends on such factors as the antigens on the 11n ~ tissue that are recognized by the recipient as foreign and can evoke the rejection response, the cells in the recipient's immune system that mediate rejection, and the reactions that modify either the ,ulc~ .ld~iu-l of the foreign antigen or the cellular response.
The major l~ u~.u~ua~ibili~y complex (MHC) is the most important ;~
system encoding ~ la~ldl~dtiun antigens. The MHC is genetically complex because it includes many different loci, each encoding separate cell-surface antigens, and because the loci have extensive polylllùl,ul-islll. The loci of the MHC fall into one of two classes, Class I or Class 11, based on their tissue distribution, the structure of the expressed antigens, and their functions.
Class I antigens, present on all nucleated cells, serve as the primary targets for cytotoxic T
(CD8+) Iymphocytes. Class 11 antigens are not distributed in the tissue as widely and serve as primary targets for helper T (CD4+) Iymphocytes.
The polymorphic forms of the individual loci of human leukocyte antigen (HLA), the ~0 MHC in humans, have been recognized by antibodies and by various in vitro techniques that measure T-lymphocyte recognition. These responses, mediated by the recipient's recognition of polymorphism in the donor, correlate with the strong rejection reactions that take place i~
vivo.
Investigation into the cellular basis of graft rejection, using both in v~ro and in vivo studies, reveals that both CD4+ and CD8+ Iyl~.,uhouyLel participate in the rejection response.
Attempts to prolong the survival of allografts and xenografts after ~ A~ ,., both in experimental models and in medical practice, have centered mainly on the suppression of the immuneapparatusoftherecipient. Thistreatmenthasasitsaimpreventive;,,,,,,-,,,,, i,l,.c~si and/or treatment of graft rejection.
Examples of agents used for i~ ullu~u,ul)leS~ivll include cytotoxic drugs, antimetabolites, co-~i~u~t~.uids, and antilymphocytic serum. Nonspecific illllllllllUaUUplC~SiVC agents found particularly effective in preventive ;'~''''''''''''I'l~'C~S;Ull (IL~IIlliU,Ulil.~,, blUl~U-,ly,UIil.~., methylprednisolone, prednisone, and most recently, cyclosporin A) have significantly improved the clinical success of IIA~ The nephrotoxicity of cyclosporin A after renal transplantation has been reduced by cud~ lion of steroids such as prednisolone, or prednisolone in conjunction with azathioprine. In addition, kidneys have been grafted successfully using anti-lymphocyte globulin followed by cyclosporin A. Another protocol being evaluated is total Iymphoid irradiation of the recipient prior to IIAI.~I~IAI~IAI;I~II followed by minimal illllllUllU~U,U,UlC~iOI~ after L-~ ,uld--~l~iu--.
Treatment of rejection has involved use of steroids, ~-amino-6-aryl-5-substituted pylilllidil.~s, heterologous anti-lymphocyte globulin, and monoclonal antibodies to vsrloos ~ -2- 1~39~92 leukocyte populations, including OKT-3. See generatly J, PediatTics. 111: 1004-1007 (1987), and specifically U.S. Pat. No. 4,665,077.
The principal complication of immuno~u,uu-c~ drugs is infections. Additionally, systemic immunu~ u.c~iol~ is ~C~r-~ "~ i~d by undesirable toxic effects (e.g., nephrotoxicity when cyclosporin A is used after renal l~ ,uldllldliull) and reduction in the level of the h~,~uu,uoic~h: stem cells. Immlln~ ,.c,~i~., drugs may also lead to obesity, poor wound healing, steroid h~pe.~ly~c~ a~ steroid psychosis, leukopenia, ~a.lluillt.,~lillàl bleeding, Iymphoma, and hYU~ iUII.
In view of these crmp1ira~irn~ ItA~ " imml~nr~lr~gicts have sought methods for suppressing immune IC~UU~ e~S in an antigen-specific manner (so that only the response to the donor alloantigen would be lost). Such specific ;~ IC~iUII generally has been achieved by modifying either the âllLi~ iuily of the tissue to be grafted or the specific cells capâble of mediating rejection. In certain instances, whether immunity or tolerance will be imduced depends on the manner in which the antigen is presented to the immune system.
Pretreating the allograft tissues by growth in tissue culture before ~ has been found in two murine models systems to lead to permanent acceptance across MHC barriers.
Lafferty et al., T~ ; 138-149 (1976); Bowen et al., Lancet, _:585-586 (1979). Ithas been lly,uùlll~;L~d that such treatment results in the depletion of passenger Iymphoid cells and thus the absence of a stimulator cell population necessary for tissue in~ ullù~,.,.lh,ily.
2û Lafferty et al., Annu. Rev. Imml~nr,l L: 143 (1983). See also Lafferty et al., Science, L88: 259-261 (1975) (thyroid held in organ culture) and Gores et al., J. Immunol., ~: 1482-1485 (1986) and Faustman et al., Proc, Natl. ~ra.1 SCi. U,.~ A,,. 78: 5156-5159 (1981) (islet cells treated with murine anti-la antisera and ,..,..1,1 .. 1 before l~ IAI;fm)~ Also, thyroids taken from donor animals pretreated with 1~ i ' - y Lulu~ drugs and gamma radiation and cultured for ten 25 days in vit~o were not rejected by any normal allogeneic recipient. Gose and Bach, J.ExD.Med..
~; 1254-1259 (1979). All of these techniques involve depletion or removal of donor Iymphocyte cells.
In some models such as vascular and kidney grafts, there exists a correlation between Class II matching and prolonged allograft survival, a correlation not present in skin grafts.
3û Pescovitz et al., J.ExD.Med.. L6Q:1495-15û8 (1984); Conti et al., TransDlant. Prûc.~ L9:6s2-654 (1987). Therefore, donor-recipient HLA matching has been utilized. Additionally, blood transfusions prior to IIA~ have been found to be effective. Opelz et al., TransPlant.
_ 253 (1973); Persijn et al., TransDlAnl Proc.. 23: 396 (1979). The cr-~hinati~m of blood transfusion before ~ . donor-recipient HLA matching, and i~ r~iùll 35 therapy (cyclosporin A) after trar~rlaAtatir~n was found to improve significantly the rate of graft survival, and the effects were found to be additive. Opelz et al., Tran~nla~ Prûc.. 17:
2179 (1985).
The I~ UIàjIIaIiU~I response may also be modified by antibodies directed at immune receptors for MHC antigens. Bluestone et al., Immunol. Rev. 9Q:5-27 (1986). Further, graft 4û survival can be prolonged in the presence of antigraft antibodies, which lead to a hûst reaction thatinturnproducesspecificimm~ .c~ion. Lancasteretal.,Nature,315:336-337(1985).
~ --3--Tbe immune response of the host to MHC antigens may be modif;ed specifically by using bone marrow ~ ll as a preparative procedure for organ grafting. Thus, anti-T-cell monoclonal antibodies are used to deplete mature T cells from the donor marrow inoculum to allow bone marrow ll~ l without incurring graft-versus-host disease. Mueller-Ruchholtz et al., Tr~r~nl~nt Proc.. 8:537-541 (1976). In addition, elements of the host's Iymphoid cells that remain for bone marrow ~ 'd~ solve the problem of occurring when fully allogeneic transplants are used.
The survival time of skin grafts has been prolonged by a factor of two by treatment ~n vitro with cortisone, i' ' ~ , or urethane before , ' into a laboratory animal. The 10 amount of drug locally applied to the skin was smaller than the amount required to achieve a similar effect by injecting the drug ~y~ y. In an additional study, the donor skin was treated in vitro with ~ .,h;l.~/strepto~ rr ~o, or with RNA and DNA pl~ liu-~s of the recipient. Further, treatment of transplant tissues with a solution of glutaraldehyde prior to '' was found to reduce their antigenicity. See U.S. Pat. No. 4,120,649.
The transforming growth factor-(TGF-B) molecules identified thus far are each dimers containing two identical 112 residue polypeptide chains linked by disulfide bonds. The molecular mass of these dimers is about 25 kd. Biologically active TGF-B has been defined as a molecule capable of inducing anchorage independent growth of target cell lines or rat fibroblasts in in v~t~o cell culture, when added together with EGF or TGF-~ as a co-factor.
TGF-B is secreted by virtually all cell types in an inactive form. This latent form is first activated by proteolytic cleavage of mature TGF-B from its precursor (at the Arg-Ala bond in position 278). A non-covalent complex is formed from the association of the mature TGF-B with the precursor remainder. This complex is disrupted so as to activate the TGF-B either by exposure to transient acidification or by the action of exogenous proteases.
There are at least three forms of TGF-B currently identified, TGF-B1, TGF-Bz, and TGF-B3. Suitable methods are known for purifying this family of TGF-Bs from platelets or placenta, for producing it in ~, hin~nt cell culture, and for d~t~ & its activity. See, for example, R. Derynck et al., Nature, ~lh,701 (1985) European Pat.Pub.Nos. 128,849 published December 19, 1984, 105,014 published April 4, 1984, 200,341 published December 10, 1986, 169,016 published January 22, 1986; 268,561 published May 25, 1988; and 267,463 published May 18, 1988; U.S. Pat. No. 4,774,322; Seyedin et al, J. Biol. ('h~m 262: 1946-1949 (1987);
Cheifetzetal,~1,48:409-415(1987);Jakowlewetal.,MolecularEndocrin _:747-755(1988);
and Dijke et al., Proc. ~tl ~r~l S~i U.~ 85: 4715-4719 (1988).
TGF-B has been shown to have numerous regulatory actions on a wide variety of both normal and neoplastic cells. Recent studies indicate an important role for TGF-B in cells of the immune system (J. Kehrl et al., J. EXD. M~-~ 163:1037 [1986]; H-J. Ristow, Proc. ~ tl A~
Sci. U.S.A., 83:5531 [1986]; A. Rook et al., J. Immunol.. 136:3916[1986]) and in proliferation of connective and soft tissue for wound healing ~ (M. Sporn et al., Science, ~;1329 [1983]; R. Ignotz et al., J, Biol. t'h~m 261:4337 [1986]; J. Varga et al., B.B.Res.Comm 138:974 [1986]; A. Roberts et al., Proc, Natl. Acad. S~i U.S.A., 78:5339 [1981]; A. Roberts et al., E~L
Proc., 42:2621 [1983]; A. Roberts et al., Proc, ~ -tl A~rl S~ i U.S.A., ~:4167 [1986]; U.S. Pat.
_4_ ~33~092 No. 4,774,228 to Seyedin et al.), as well as epithel;a (T. Matsui et al., Proc, Nqtl Arq~l ,cri U.S.A., ~:2438 [19861; G. Shipley et al. Cancer i?PC 46:2068 [1986]). Moreover, TGF-B has been described as a suppressor of cytokine (e.g., IFN-, TNF-~) production, indicating its use asan;,,,.. ,,n,~ ,ca~l.tfortreatingi~fi,qmmqfr,rydisorders(Espeviketal~J~F~tn~Med~
571-576 ~1987]; European Pat. Pub. Nos. 269,408 published June 1, 1988 and 213,776 published March 11, 1987), and as a promoter of cachexia (Beutler and Cerami, New Fno J. ~IP~I ~;
379 [1987]). Further, TGF-B induces collagenase secretion in human fibrobl~t cultures (Chua et al., J, Biol, ~'hPm 260:5213-5216 [1983]); stimulates the release of p,.,~ and -~ li7qfirn of calcium (A. Tashjian et al., Proc. i~qtl Arq~l ~ri U.S.A., ~ 4535 [1985]); and inhibits endotheliai regeneration (R. Heimark et al., ~ç~, _~;1078 [1986]).
TGF-B is multifunctional, as it can either stimulate or inhibit cell proliferation, differentiation, and other critical processes in cell function (M. Sporn, Science, 233:532 [1986]).
The multifunctional activity of TGF-B is modulated by the influence of other growth factors present together with the TGF-B. TGF-B can function as either an inhibitor or an enhancer of anchorage-il~ growth, depending on the particular set of growth factors, e.g., EGF or TGF-o~, operant in the cell together with TGF-B (Roberts et al., Proc. N,qfl Arq~i Sci, U.S.A., 82:119 [1985]). TGF-B also can act in concert with EGF to cause proliferation and piling up of normal (but not rheumatoid) synovial cells (Brinkerhoff et al., Arthrifis Rh ~1370 [1983]).
Most recently, TGF-B has been found to suppress the expression of Class 1~
1 - hility antigens on human celis induced by human interferon- and to inhibit constitutive expression of the Class 11 antigen message in the cells (Czarniecki et al., L
140: 4217-42223 [1988]; Czarniecki et al. J. !nfqrferon RP~ Z: 699 [19871; Palladino et al., J. Cell. Birr~om Supp. I IA [Jan. 17-Feb. 5, 1987], UCLA Symposia on Molecular and Cellular Biology, Alan R. Liss, Inc., New York, abstract A016, p. 10; Chiu et al., Triennial Symposium: Biology of Growth Factors, University of Toronto, Ontario, Canada, [June 17-19, 1987]; Palladino et al., ImmunobioloqoY~ 175: 42 [19871).
For a general review of TGF-~3 and its actions, see Sporn et al., J. Ceil Biol.. 105: 1039-1045 (1987) and Sporn and Roberts, Nature, 332: 217-219 (1988).
There is a need in the art for a method to prolong graft survival in transplant operations and minimize the toxicity and other adverse effects arising from the use of large doses of imm11n. ~ 6~
Accordingly, an object of this invention is to provide for longer graft survival in the host.
Another object is to provide for a LIA~ II method wherein lower amounts of iluluuuoau~ c~ , agents, if any, need be q illlilli~ ca to the host to achieve successful results, thereby reducing the side effects associated with systemic administration of illllllu drugs.
These and other objects will become apparent to one of ordinary skill in the art.
These objects are acc~ by a method for l.~ grafts from donors into 40 hosts which comprises treating a graft with a , ~ l . comprising a ~ ul ;~ ~lly effective amount of TGF-B; and lldll~ldlllillg the graft into a compatible host.
~ 13.~9092 In another aspect, this invention provides a graft treated with a uullluua;l;Ol~ comprising TGF-B, alone or in combination with another immUnuauuplt~aa;~, agent.
In a further aspect, this invention provides a . ' comprising TGF-13 in a perfluolucl.~...h,~.l emulsion.
The method herein results in grafts that are il.. ,llOIOgi-,ally stable in the hosts, biologically functional, and capable of being stored prior to L-~
This invention enables the P~ghl ' of a bank of treated grafts that can be utilized for short-term or long-term storage. In addition, the graft is not rejected when Llalla,ulalltt:d into suitable hosts. The method is applicable to both allografts and xenografts, and the use of lû xenografts overcomes the difficulties encountered by the limited supply of tissue from humans.
The term ''graft" as used herein refers to biological material derived from a donor for L.~ - into a recipient. Grafts include such diverse material as, for example, isolated cells such as islet cells, tissue such as the amniotic membrane of a newborn, bone marrow, hematopoietic precursor cells, and organs such as skin, heart, liver, spleen, pancreas, thyroid lobe, lung, kidney, tubular organs (e.g., intestine, blood vessels, or esophagus), etc. The tubular organs can be used to replace damaged portions of esophagus, blood vessels, or bile duct. The skin grafts can be used not only for burns, but also as a dressing to damaged intestine or to close certain defects such as dia,u~ ,t,-rl~h, hernia. The graft is derived from any source, preferably n~msli~n including human, whether from cadavers or living donors.
2û The term "host" as used herein refers to any compatible transplant recipient. By ", , ' '-" is meant a host that will accept the donated graft. Preferably, the host is mornn~slisn and more prefe~ably human. If both the donor of the graft and the host are human, they are preferably matched for HLA class 11 antigens so as to imprûve 1,;~",..",I.~lil ility.
The term "donor" as used herein refers to the species, dead or alive, from which the graft is derived. Preferably, the donor is mgmrn ~ n Human donors are preferably volunteer blood-related donors that are normal on physical PYsmir~s~inn and of the same major ABO blood group, because crossing major blood group barriers possibly prejudices survival of the allograft.
It is, however, possible to transplant, for example, a kidney of a type O donor into an A, B or AB recipient.
The term "transplant" and variations thereof refers to the insertion of a graft into a host, whether the ~ is syngeneic (where the donor and recipient are genetically identical), allogeneic (where the donor and recipient are of different genetic origins but of the same species), or xenogeneic (where the donor and recipient are from different species). Thus, in a typical scenario, the host is human and the graft is an isograft, derived from a human of the same or different genetic origins. In another scenario, the graft is derived from a species different from that into which it is transplanted, including animals from phylogenically widely separated species, for example, a baboon heart transplanted into a human host.
The term "TGF-13" refers to the family of molecules described l~ illal)u~,. Reference to TGF-B herein will be understûûd to be a reference to any one of the three currently identified forms, TGF-131, TGF-132 and TGF-fs3, as well as other molecules related to this 13~9092 ~ --6--family of proteins, and those idèntified in the future, their alleles, and their p~ t~ d amino acid sequence variants, so long as they are effective in the method described herein. The TGF-13 is a,uulupli~t~1y from any source, preferably r~qmmqliAn, and most preferably human.
TGF-13 from animals other than humans, for exampie, porcine or bovine sources, can be used 5 for soaking grafts to treat humans. Likewise, if it is desirable to treat other mammalian species such as domestic, farm, sports, or pet animals, human TGF-13, ~ well ~ TGF-~3 from other species, is suitably employed.
The term "immunosuppressive agent" as used herein refers to substances that act to suppress or mask the immune system of the host into which the graft is being lla~ ,Jldilt~l. This 10 would include substances that suppress cytokine production, downregulate or suppress self-antigen expression, or mask the MHC antigens. Examples of such agents include 2-amino-6-aryl-5-substituted ,uylilllidi~c~ (see U.S. Pat. No. 4,665,077, supra), aLa~lliùp~ . (or ~,y. ~ ,h.,~ ",hl~, if there is an adverse reaction to aLall.iuulil.~ y,uli.~C, glutaraldehyde (which masks the MHC antigens, as described in U.S. Pat. No. 4,120,649, supra);
15 antiidiotypic antibodies for MHC antigens; cyclosporin A; one or more steroids, preferably uulliuo~t~,.uhla, and most preferably ~lu~,ucOlli~u~t.,.uida such ~ prednisone, " yl,ulcdl~iaulull~, and ,' -~ ~; anti-interferon- antibodies; anti-tumor necrosis factor-~ antibodies; anti-tumor necrosis factor-B antibodies; anti-interleukin-2 antibodies;
anti-cytokine receptor ântibodies such as anti-lL-2 receptor antibodies; h.,t~,.ulob.,..~ anti-Iymphocyte globulin; pan-T antibodies, preferably OKT-3 monoclonal antibodies; antibodies to CD4; streptokin~e; streptodornase; or RNA or DNA from the host.
The preferred agent for this purpose will depend on many factors, including the type of A~ being performed, as well as the patient's history, but a general overall preference is that the agent be selected from cyclosporin A, a glu~ocolli-,u~.~,.ui:l (most preferably prednisone or methylprednisolone), OKT-3 monoclonal antibody, aLall-iop- i,~, bromocryptine, heterologous anti-lymphocyte globulin, or a mixture thereof.
TGF-L~ is appropriately used in its activated form, i.e., the mature form is cleaved from its precursor using a suitable enzyme and the resultant complex is treated with acid, a protease such as pl~min or cathepsin D, alkali, or chaotropic agents, to activate the TGF-fs. See Keski-Oja et al., J. ~ l Bin~hPm SurJnl IIA: 60 (1987); Kryceve-Martinerie et al., Int. J. ~An~qr, ~;553-558 (1985); Lawrence et al., Biochem. Bio~hYs. l~qc C~-mmlln ~; 1026-1034 (1985);
Lawrence et al., J. Cell PhYci~l 121: 184-188 (1984).
The ~ , ' ~ to be used in the therapy herein will be dosed in a fashion consistent with good medical practice taking into account the nature of the l.A,~ -- and the disorder to be treated, the species of the host, the medical condition of the individual patient, the presence of any other drug in the, , and other factors known to placl ' For purposes herein, the "Ll.~,.du~...i~qlly effective amount" of TGF-~3 used to treat the graft is an amount that is effective to reduce the ~ """.n~ ;ly of the graft so that it will be compatible with the host and not be rejected. A generally effective amount for this purpose is in a range 40 of I pg/ml to I mg/ml, more preferâbly, 10 pg/ml to l~g/ml, and most preferably, 10 pg/ml _7 to ~00 ng/rnl, but the practitioner will know more precisely what amount is to be used taking into ~u.~idelaliù.l the factors described above.
The TGF-B is optionally formulated with one or more ,, c~a;~, agents to enbance the ,, ca~(u~l effect on the graft. The effective amount of such other agents 5 depends on the amount of TGF-B present in the formulation, the type of transplant, and other factors discussed above, but generally also ran8e from about 0.1 pg/ml to 0.5 mg/ml as necessary to achieve the appropriate endpoint, i.e., ionger graft survival. Typically, the TGF-B is formulated by mixing it at ambient t~ , e at the appropriate pH, and at the desired de8ree of purity, with phy~ acceptable carriers, i.e., carriers that are non-toxic to 10 recipients at the dosages and employed. The pH of the formulation depends mainly on the particular use, and ~ of TGF-B, but preferably ranges anywhere from about 3 to about 8. Preferably the TGF-B is maintained at an acidic pH (e.g., about 4 to about 6) to prevent its adhering to the containers in which it is placed, as occurs at pH values .~uuluAA~ lg ,ully~;ùlù25h.dl levels, and to ensure activation of the TGF-B molecule. Thus, 15 formulation in an acetate buffer at pH 5 is a suitable '- ~ " If the TGF-B adheres to the containers in which the grafts are being treated, an appropriate ingredient, such as albumin, is optionally added, in an amount that does not prevent the TGF-B from perfusing or adhering to the 8raft being treated.
If the TGF-B formulation is to be applied topically, for example, if it is to be painted 20 onto a skin graft prior to IIA ~ , it is preferable to use a viscous solution such as a gel rather than a non-viscous solution. This may be r , '' ' 1, for example, by mixing the solution of the TGF-B with a gelling agent, such as a poly ~ , preferably a water-soluble poly~4~,cl.d-i-1e, such as, e.g., hyaluronic acid, starches, and cellulose derivatives, e.g., methylcellulose, llyJIuA.~ yl cellulose, and cAAIlluAy ' yl cellulose. The most preferred 25 8ellin8 agent is methylcellulose- The pùlya~.cl.dlide is generally present in a gel formulation ' in the range of 1-90% by weight of the gel, more preferably 1-20%. Examples of other suitable poly~ .iJ~, for this purpose, and a J~t~.ll-i..AA1iull of the solubility of the poly-are found in EP 267,015, published May 11, 1988.
If the 8raft to be treated is to be stored for any period of time, the TGF-B is preferably 30 formulated in or added to a perfl.,ù-u-,h~ l emulsion (acting as a blood substitute) to enable higher of oxygen to reach the 8raft. Such emulsions comprise a perfluol, ~ ' such as perfluu-ud~ and/or perf~olo~-iy-u~ ylAA.ll;l~e emulsified with a surfactant in water. Examples of suitable surfactants include the poloxamer surfactants (which represent a series of molecules that are blocked copolymers of ethylene oxide and propylene 35 oxide), alone or in admixture with a ~ .hA,I;~.;d such as egg lecithin. The perfluu.u~ ..-h,41 is chosen to be the least toxic in humans. One example of such an emulsion, which is commercially available from Green Cros~, is Fluosol-DA 20%, which contains perfluù.ùd~,~.lill and perfluu-uLliylu~uylAAlrli~.~ emulsified with the poloxamer surfactant, Pluronic F-68. The perfluo- . ' ~ ' emulsions and their effects in mammals are described more fully in Bollands 40 et al., J. Pharm. Pharmacol.. ~2: 1021-1024 (1987).
~trade-mark -8- 13~9092 The TGF-~3 for use in therapeutic A~ if m is preferably sterile. Sterility is readily , ' ' ~' by sterile filtration through (0.2 micron) br:~nPc TGF-~3 ordinarily will be stored as an aqueous solution since it is highly stable to thermal and oxidative ,~ ;fll~
although Iyophilized fu~ ulltiol~s for IC~ 11 are acceptable.
In accordance with the method of this invention, the graft is contacted with the TGF-!3, , - The contact suitably involves incubating or perfusing the organ with theor applying the ~ i"" to one or more surfaces of the graft. The treatment generally takes place for at least one minute, and preferably from I minute to 72 hours, and more preferably from 2 minutes to 24 hours, depending on such factors as the ~ iVII of TGF-13 in the formulation, the graft to be treated, and the particular type of formulation.
Perfusion is ~ ' by any suitable procedure. For e~cample, an organ can be perfused via a device that provides a constant pressure of perfusion having a pressure regulator and overflow situated between a pump and the organ, as described by DD 213,134 published Sept.
5, 1984. Alternatively, the organ is placed in a hyperbaric chamber via a sealing door and perfusate is delivered to the chamber by a pump that draws the fluid from the reservoir while spent perfusate is returned to the reservoir by a valve, as described in EP 125,847 published Nov. 21, 1984.
For skin grafts, the formulation is suitably painted on the lower surface of the skin to be grafted so that there is a layer of the TGF-3 between the tissue of the host and the lower surface of the graft. Alternatively, the whole skin graft is submerged in the f n.n.pf.~ci-if~n After the graft is treated, it is suitably stored for prolonged periods of time or is used immediately in the transplant procedure. Storage life can be enhanced as described above by using a blood substitute in the formulation (e.g., perfl--olv~,h~ l emulsion), or by perfusing the graft with a formulation of the TGF-~3 containing chilled isotonic agent and Alll;. .I~.rl~lAIII
followed by glycerol to allow for freezing of removed organs with no destruction of the cells, as described in JP 60061501 published April 9, 1985. In addition, the organs can be preserved with different liquids that include the formulation while the organs are cooled to freezing Leluu~,.,JLulc~, to preserve the organ semi-permanently without cell necrocytosis, as described by U.S. Pat. Nos. 4,462,215 and 4,494,385.
Respecting cardiac transplants specifically, Parent et al., CrvQbinlf)f~v~ .~; 571-576 ( 1981 ) describes that cold coronary perfusion prior to Ll~ ll at 5~C increases protection of the homograft during the initial period of illl~,l..lll.liif l~. Any of these procedures, or others, are within the scope of v'lis invention if deemed necessary upon or after TGF-.'3 treatment of the graft.
Before ~ ,., the graft is preferably washed free of the TGF- ~3 f l ll ~ ~
by soaking it in a physiological saline solution or by other means appropriate for this purpose.
Also, prior to ~ , the host is optionally given one or more donor-specific blood transfusions to aid in graft survival. An alternative procedure is to subject the host to total Iymphoid irradiation prior to the Ll~lllaul~ dLivl~ operation. Any other pre-transplant procedures that would be beneficial to the particular transplant recipient can be performed as part of the method of this invention.
-? 9 133t~92 The tran~rlanf~-ion procedure itself will depend on the particular disorder being treated, the condition of the patient, etc. The medical pldc~iliullrv. will recognize the appropriate procedure to employ in any given case. The transplants are optionally monitored ~y~lcllld~;crllly during the Gritical postoperative period (the first three months) using any suitable procedure.
5 One such procedure is Iddiu--u~ ie intravenous angiography using 99Tcm~ .Lalc, as described by Thomsen et al., Acta Farlirl 29:138-140 (1988). In addition, the method herein is amenable to ~;"",II~ , multiple organ perfusion and l-~ A~ (Toledo-Pereyra and n7iP, Am. Sur~., ~ 161-164 (1980). After the ~ IUI;..A, ;"""""'.' 'I.~.C~;UII
therapy, using TG~-13 and/or other; ~ l C~rlllltS), is utilized as necessary to ensure 10 graft survival.
In some instances, it is desirable to modify the surface of the graft so as to provide positively or negatively charged groups, as by using a suitable amino acid or POlymer or by attaching a physiologically acceptable source of charged functional groups. For example, a negatively charged surface is appropriate for blood vessels to diminsh blood clotting. It also is 15 desirableincertainui--:u-ll,Ldllcc,torenderthesurfacel-y~ '~' orhydrophilicbycoupling, e.g., phenylalanine, serine or Iysine to the surface. An ;.~ p.c,s;vc agent particularly effective for these surface - "rirafinn~ is gluteraldehyde.
Ir~ u~ c~a;OIl therapy typically involves the ~Idlllil~iaLIr~Li~ll of an effective amount of an ,.,UIC~S;~., agent, including TGF-f5 itself. The innrn~n~ es~ agent 20 ~ will be formulated and dosed in a fashion consistent with good medical practice.
Factors for consideration in this context include the clinical condition of the individual patient, the cause of the transplant, the site of delivery of the agent, the method of arlmini~frûtinn, the scheduling of ~ and other factors known to pldulii- The "effective amount"
in this context, which is determined by such rr~ncirl~r~irln~, is the minimum amount necessary 25 to prevent an immune response that would result in rejection of the graft by the host. Such amount is preferably below the amount that is toxic to the host or renders the host significantly more susceptible to infections. The amount of illllllullual.~Julca~lvc agent required for the invention herein may be lower than that normally required for ll~ila,ul.llllcd grafts that have not been pre-treated, and depends on the individual circumstances surrounding the transplant and 30 the type of immlln..~.,l,l,,c~s;v~ agent employed.
As a general proposition, the total pl~ la~ 411y effective amount of the immunuauu~J-c~aivc agent, cyclosporin A, ddl--;n;~e-cd parenterally per dose will be in the range of about 0.1 to 20 mg/kg of patient body weight per day, with the typical range of cyclosporin A currently used in ~,, c~S;v., therapy being 5 to 15 mg/kg/day. For renal transplants 35 it is useful to administer massive doses at short periods of ~ lu~u~olli~u~t~.ùid; e.g., methylprednisolone in several-gram doses per day is given for 3 to 5 days followed by 60 to 100 mg prednisone without TGF-fs pre-treatment of the graft. With such pre-treatment, lower doses would be expected to be useful.
As noted above, however, these suggested amounts of immunuau,up.e,,,~lll are subject to 40 a great deal of therapeutic discretion. The key factor in selecting an appropriate dose and scheduling is the result obtained, i.e., graft survival. For example, relatively higher doses may . . .
-~o be needed initially for the treatment of hyperacute graft rejection, which can be attributed to antibody-mediated graft destruction, or at a later stage for the treatment of acute rejection, which is ~ t~ by a,sudden decline in graft function.
The~ c~;~agentis~ld~l~;ùi~i~.cdbyanysuitablemeans,includingparenteral, 5 and, if desired for local ;- ~ ca~ treatment~ I IIC~iu~ Parenteral infusions include ' , intravenous, intraarterial, ' 1, or ~ 1,, l --- ~,.~
- ' ' ' In addition, the ' ,IulrJa~ agent is suitably ~ ' cd by pulse infusion, particularly with declining doses of the; ~ ca~ , agent, or by continuous infusion.
The invention will be more fully understood by reference to the following examples.
They should not, however, be construed as limiting the scope of the invention.
EXAMPLE I
Mllrjn~ Skin ~r ft Mr~
TGF-B: The TGF-~ used herein is from any one of several sources: (I) porcine platelel-15 derived TGF-Bl at 96 percent purity purchased in a Iyophilized form and suspended in 4 mM
HCI and phosphate buffered saline (PBS~ solution prior to use, (2) TGF-131, prepared by recombinant means as described by EP 200,341, supra, (3) TGF-13~, prepared as described by US. Pat. No. 4,774,322, sllpra, or (4) TGF-B3, prepared by ., hir.-.nt means as described by EP 267,463, sl pra see Fig. 29 and a~ u~ul~a~y ~e text of EP 267,463). The TGF-B from any of 20 these sources is recovered and formulated at ~00 ng/ml at pHS in an aqueous acetate buffer.
The resultant solution is sterile filtered through a 0 2 micron polysulfone filter Grafts: Skin allografts (I x 2 cm square) are obtained from BALB/c (H-2d) mice Hosts: Ihe hosts used are C57BL/6 (H-2s) mice Procedure: The skin grafts are incubated in one of the above TGF-B solutions at room 25 ~ p.,llull~ in a sterile glass vial for I minute to 24 hours, then ~vashed two times with PBS
' solution to remove any free TGF-B, The thus-treated skin is applied to the mouse recipient, and covered with vaseline gauze, and then with plaster of Paris, which are removed after ten days The treated grafts are tightly bound to the recipient They are initially soft, but become ylulyc~i.~ly stiffer with minimal shrinkage in size, and remain free from infection The histology shows that the grafts are viable and ~ , and the general structure of the skin (epidermis, adnexa, and dermis) is preserved for at least three months. Visual ~ f~rrnin~finn can also be made (e.g, looking for white skin on a black mouse).
R~t Skin Gr~ft Ml~
An aqueous-based gel of ' ylu(,llulu~c (Methocel A4M from Dow) is prepared by mixing it in water at a .I~iOII of 209~ by weight to give a viscosity useful for a topical skin application The TGF-BI ~, ' '~ produced as described in Example I is mixed with the gel to provide a co..~,G..LI~t;o~ of i mgjml.
This TGF-B gel is applied to the lower (back) side of skin taken from Fl Lewis/Brown 40 Norwegian rats, After 24 hours, the skin is placed on the graft bed of the Lewis parent rat, covered with gauze, and then with plaster of Paris, and ,~ ly removed after ten days *trade-mark : .
133~092 Alternatively, the graft is held in place with metal clips applied using a mechanical clipping device. The results are comparable to those observed for the mice as described above.
,MIlrinP an-l RRr J~IPt CPII T ~ ~ ~' l~odel Islet cells are extracted from the pancreas of a BALB/c mouse, soaked in one of the TGF-B compositions described in Example I for 48 hours, and ~ cd into the subrenal capsule space of a ~G~uLuLu~ill-treated allogeneic mouse such as C~7BL/6 male mouse. See, e.g., Faustman et al., sup~a. Similarly, islet cells are extracted from the pancreas of a Fl Lewis/Brown Norwegian rat, incubated in one of the TGF-B~ , described in Example1 for 24-48 hours, and Ll , ' ~' into the subrenal capsule space of a ~LIc~ ~JLul~,;u-treated CS~BL/6 mouse. The results indicate that the islet cells are not rejected by the host after three months.
EXAMPLE IV
~vlllrin~ an-i RRt ~PRrt TrRn~nlRnt ~n~PI
A heart is excised from a sacrificed BALB/c mouse and from a sacrificed Fl Lewis/Brown Norwegian rat. The respective hearts are ~ - ' ~ 'y incubated at room UIC with any of the TGF-B solutions of Example 1. The in~ubations are carried out for 24 to 48 hours. The hearts are washed free of the TGF-B solution using two washes of PBS
and then ' '~"I~ L,.~ using standard heart transplant operations into the ear of a C3H/HeJ BALB/c mouse and into the aorta of a Fl Lewis/Brown Norwegian rat from which tbe hearts had been removed, the mouse heart being received by a different allogeneic strain of mouse and the rat heart being received by a different allogeneic strain of rat. The gtafts are not rejected three months after rrRn~rlRntRtion Alternatively, after extraction the heart is stored in a perfluo..,~ l emulsion such 25 as Fluosol-DA 20% (Green Cross, Japan) to which one of the TGF-B solutions is added prior to trRn~rlRntRtinn in an amount sufficient to provide at least I ng/ml of TGF-B in the final emulstion.
Claims (37)
1. The use of a composition comprising a therapeutically effective amount of transforming growth factor-.beta. (TGF-.beta.) for treating a graft prior to transplanting the graft into a compatible host.
2. The use of claim 1 wherein the graft is tissue.
3. The use of claim 1 wherein the graft is skin.
4. The use of claim 3 wherein the composition is coated on the lower surface of the skin.
5. The use of claim 4 wherein the composition comprises a polysaccharide.
6. The use of claim 1 wherein the graft comprises isolated cells.
7. The use of claim 6 wherein the graft comprises islet cells.
8. The use of claim 1 wherein the graft is an organ.
9. The use of claim 8 wherein the organ is selected from group consisting of heart, liver, spleen, pancreas, thyroid lobe, lung, kidney, intestine, blood vessel, and esophagus.
10. The use of claim 1 wherein the host is a mammal.
11. The use of claim 10 wherein the mammal is a human and the graft is derived from a human doner.
12. The use of claim 1 wherein the TGF-.beta. is human TGF-.beta..
13. The use of claim 12 wherein the TGF-.beta. is TGF-.beta.1, TGF-.beta.2 or TGF-.beta.3.
14. The use of claim 1 wherein the graft is keated in vitro for at least about one minute.
15. The use of claim 1 wherein the graft is treated in vitro for from about one minute to about 72 hours.
16. The use of claim 1 wherein the composition further comprises an effective amount of an immunosuppressive agent.
17. The use of claim 16 wherein the immunosuppressive agent is a 2-amino-6-aryl-5-substituted pyrimidine, glutaraldehyde, azathioprine, bromocryptine, cyclophosphamide, antiidiotypic antibodies for major histocompatibility complex antigens, cyclosporin A, a steroid, anti-interferon-antibodies, anti-tumor necrosis factor-a antibodies, anti-interleukin-2 antibodies, anti-cytokine receptor antibodies, anti-tumor necrosis factor-.beta.antibodies, anti-Ia antibodies, heterologous anti-lymphocyte globulin, pan-T
antibodies, antibodies to CD4, streptokinase, streptodornase, or RNA or DNA
from the host.
antibodies, antibodies to CD4, streptokinase, streptodornase, or RNA or DNA
from the host.
18. The use of claim 17 wherein the immunosuppressive agent is cyclosporin A, a glucocorticosteroid, OKT-3 monoclonal antibody, azathioprine, bromocryptine, heterologous anti-lymphocyte globulin, or a mixture thereof.
19. The use of claim 1 wherein the composition is acidic.
20. The use of claim 19 wherein the pH of the composition is about 4 to about 6.
21. The use of claim 1 wherein the composition is a perfluorochemical emulsion of the agent.
22. The use of claim 1 further comprising, after the treatment of the graft and prior to the transplantation, the graft is washed free of the agent.
23. The use of claim 1 wherein the therapeutically effective amount is 1 pg/ml to 1 mg/ml.
24. The use of claim 1 wherein the host undergoes a donor-specific blood transfusion or total lymphoid irradiation before the transplantation.
25. The use of claim 11 wherein the donor of the graft and the host are matched for HLA class II antigens.
26. The use of claim 25 wherein the host undergoes a donor-specific blood transfusion or total lymphoid irridiation before the transplantation.
27. The use of claim 1 further comprising the use of an effective amount of an mmunosuppressive agent for treatment of the host after the transplantation.
28. The use of claim 26 further comprising the use of an effective amount of an immunosuppressive agent for treatment of the host after the transplantation.
29. The use of claim 27 wherein the immunosuppressive agent is TGF-.beta., a 2-amino-6-aryl-5-substituted pyrimidine, glutaraldehyde, azathioprine, bromocryptine, cyclophosphamide, antiidiotypic antibodies for major histocompatibility complex antigens, cyclosporin A, a steroid, anti-interferon-antibodies, anti-tumor necrosis factor-a antibodies, anti-interleukin-2 antibodies, anti-cytokine receptor antibodies, anti-tumor necrosis factor-.beta.antibodies, anti-Ia antibodies, heterologous anti-lymphocyte globulin, pan-T
antibodies, antibodies to CD4, streptokinase, streptodornase, or RNA or DNA
from the host.
antibodies, antibodies to CD4, streptokinase, streptodornase, or RNA or DNA
from the host.
30. The use of claim 29 wherein the immunosuppressive agent is TGF-.beta., cyclosporin A, a glucocorticosteroid, OKT-3 monoclonal antibody, azathioprine, bromocryptine, heterologous anti-lymphocyte globulin, or a mixture thereof.
31. The use of claim 27 wherein the immunosuppressive agent is used to continuously infuse the host.
32. The use of claim 27 wherein the immunosuppressive agent is used to pulse infuse the host.
33. The use of claim 32 wherein declining doses of the immunosuppressive agent are used.
34. The use of claim 1 wherein the graft is to be transplanted into a mammalian species different from that from which the original graft was derived.
35. The use of claim 34 wherein the graft is to be transplanted into a human and was derived from a mammalian species other than human.
36. The use of claim 1 further comprising, after the treatment and prior to transplantation, the treated graft is stored.
37. A composition comprising TGF-.beta. in a perfluorochemical emulsion.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US07/258,276 | 1988-10-14 | ||
| US07/258,276 US5135915A (en) | 1988-10-14 | 1988-10-14 | Method for the treatment of grafts prior to transplantation using TGF-.beta. |
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| CA1339092C true CA1339092C (en) | 1997-07-29 |
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| CA000613971A Expired - Lifetime CA1339092C (en) | 1988-10-14 | 1989-09-28 | Method for the treatment of grafts prior to transplantation |
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| US (1) | US5135915A (en) |
| EP (1) | EP0438526B1 (en) |
| JP (1) | JPH04501264A (en) |
| AT (1) | ATE111361T1 (en) |
| CA (1) | CA1339092C (en) |
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| CA1340994C (en) * | 1989-09-21 | 2000-05-16 | Rudolf Edgar Dr. Falk | Treatment of conditions and disease |
| US5824297A (en) * | 1990-06-25 | 1998-10-20 | Oncogene Science, Inc. | Tissue-derived tumor growth inhibitors, methods of preparation and uses thereof |
| US5283058A (en) | 1990-08-30 | 1994-02-01 | The General Hospital Corporation | Methods for inhibiting rejection of transplanted tissue |
| US5192312A (en) * | 1991-03-05 | 1993-03-09 | Colorado State University Research Foundation | Treated tissue for implantation and methods of treatment and use |
| US6399569B1 (en) | 1991-03-11 | 2002-06-04 | Curis, Inc. | Morphogen treatments for limiting proliferation of epithelial cells |
| US6090776A (en) * | 1991-03-11 | 2000-07-18 | Creative Bio Molecules, Inc. | Morphogen treatment of organ implants |
| US6077823A (en) * | 1991-03-11 | 2000-06-20 | Creative Biomolecules, Inc. | Method for reducing tissue damage associated with ischemia-reperfusion or hypoxia injury |
| US6194376B1 (en) | 1991-03-11 | 2001-02-27 | Creative Biomolecules, Inc. | Method for modulating inflammatory response comprising administering morphogen |
| GB9106678D0 (en) * | 1991-03-28 | 1991-05-15 | Ferguson Mark W J | Wound healing |
| US5620971A (en) * | 1991-05-09 | 1997-04-15 | Vertex Pharmaceuticals Incorporated | Biologically active acylated amino acid derivatives |
| US5723459A (en) * | 1991-05-09 | 1998-03-03 | Vertex Pharmaceuticals Incorporated | Biologically active acylated amino acid derivatives |
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1989
- 1989-09-28 CA CA000613971A patent/CA1339092C/en not_active Expired - Lifetime
- 1989-10-10 AT AT89912606T patent/ATE111361T1/en not_active IP Right Cessation
- 1989-10-10 EP EP89912606A patent/EP0438526B1/en not_active Expired - Lifetime
- 1989-10-10 JP JP1511589A patent/JPH04501264A/en active Pending
- 1989-10-10 WO PCT/US1989/004429 patent/WO1990003812A1/en active IP Right Grant
- 1989-10-10 DE DE68918285T patent/DE68918285T2/en not_active Expired - Lifetime
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| EP0438526A1 (en) | 1991-07-31 |
| US5135915A (en) | 1992-08-04 |
| DE68918285T2 (en) | 1995-03-02 |
| DE68918285D1 (en) | 1994-10-20 |
| EP0438526B1 (en) | 1994-09-14 |
| WO1990003812A1 (en) | 1990-04-19 |
| ATE111361T1 (en) | 1994-09-15 |
| JPH04501264A (en) | 1992-03-05 |
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