WO1999012584A1 - Methods for inhibiting rejection of transplanted tissues by bone grafting - Google Patents
Methods for inhibiting rejection of transplanted tissues by bone grafting Download PDFInfo
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- WO1999012584A1 WO1999012584A1 PCT/US1998/018866 US9818866W WO9912584A1 WO 1999012584 A1 WO1999012584 A1 WO 1999012584A1 US 9818866 W US9818866 W US 9818866W WO 9912584 A1 WO9912584 A1 WO 9912584A1
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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
- A61L27/38—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 containing added animal cells
- A61L27/3886—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 containing added animal cells comprising two or more cell types
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
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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
- A61L27/3604—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 characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
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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
- A61L27/3604—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 characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
- A61L27/3608—Bone, e.g. demineralised bone matrix [DBM], bone powder
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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
- A61L27/3641—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 characterised by the site of application in the body
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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
- A61L27/38—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 containing added animal cells
- A61L27/3804—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 containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3821—Bone-forming cells, e.g. osteoblasts, osteocytes, osteoprogenitor cells
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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
- A61L27/38—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 containing added animal cells
- A61L27/3804—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 containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
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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
- A61L27/38—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 containing added animal cells
- A61L27/3839—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 containing added animal cells characterised by the site of application in the body
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
Definitions
- the discovery herein involves transplantation of a donor bone graft with the bone marrow cells intact, preferably under the cover of effective immunosuppression, to allow establishment ofthe bone graft, but without the requirement for myeloablation in the recipient.
- This has the advantages of: (1) avoiding the morbidity of myeloablation; and (2) creating "space” in the recipient.
- This "space” could include important "facilitator cells” without the requirement for laborious isolation techniques as well as a microenvironment of structural and soluble factors which may be important for stem cell maturation. This may be particularly true for xenotransplantation, wherein cytokines and/or growth factors produced by stromal cells from the donor species may have a significant advantage over the recipient's growth factors.
- human GM-CSF has low activity in promoting development of mouse bone marrow colony development.
- the invention involves the discovery that grafting fragments of bone promotes long-term survival of skin grafts ofthe same donor type as the bone graft, without requiring irradiation ofthe host.
- the promotion of long-term graft survival using fragments of whole bone is superior to that obtained using bone marrow.
- transplantation model herein indicates that grafts of whole bone, which include stromal cells, are more effective than bone marrow for inducing long-term immune tolerance.
- This discovery provides methods that are new and more effective strategies to induce immunological tolerance and to suppress graft rejection.
- the present invention provides a method for inhibiting rejection of a transplanted tissue without myeloablating a recipient ofthe transplanted tissue.
- This method comprises grafting bone or fragments thereof into the recipient at a site that vascularizes the bone graft. Grafting can occur before or after transplantation ofthe tissue.
- the bone comprises stromal cells and marrow cells. The presence of bone so grafted results in immunological tolerance ofthe transplanted tissue by the recipient, thereby inhibiting rejection ofthe transplanted tissue.
- the bone graft comprises fragments of marrow-containing bone. In another embodiment, the bone comprises a vascularized segment of marrow-containing bone.
- rejection ofthe transplanted tissue is further inhibited by administration of an immunosuppressive agent.
- an immunosuppressive agent include, but are not limited to, soluble CTLA4 such as CTLA4 Ig, soluble CD28 such as CD281, soluble B7, such as B71g, soluble gp39, soluble CD40, anti-gp39 antibodies, anti-CD40 antibodies, anti-CD28 antibodies, anti-B7 antibodies, anti-CTLA4 antibodies, cyclosporin, azathioprine, methotrexate, cyclophosphamide, lymphocyte immune globulin, anti-CD3 antibodies, Rho (D) immune globulin, adrenocorticosteroids, sulfasalzine, FK-506, methoxsalen, mycophenolate mofetil (Cellcept), horse anti-human thymocyte globulin (ATGAM), humanized anti-TAC (HAT), basiliximab (Simulect),
- the immunosuppressive agent comprises a first soluble molecule, which prevents an endogenous molecule on a cell selected from the group consisting of gp39 and CD40 from binding its endogenous ligand.
- the immunosuppressive agent comprises a second soluble molecule which prevents an endogenous molecule on a cell selected from the group consisting of CTLA4. CD28, and B7 from binding its endogenous ligand.
- the immunosuppressive agent comprises a combination ofthe first soluble ligand and the second soluble ligand. The prevention of these molecules from binding their endogenous ligands blocks two independent signal pathways. The blockage of these two independent signal pathways inhibits the immune responses so that transplant rejection is further inhibited.
- multiple immunosuppressive agents can be used in the method ofthe invention, e.g., a combination of CTLA4Ig, anti-gp39 antibodies and cyclosporin.
- Figure 1 is a cumulative survival plot showing survival of secondary BALB/c skin grafts in C3H/He mice pre-treated with CTLA41g/MRI only (open triangles), CTLA41g/MRl and BALB/c bone grafts (closed circles), CTLA41g/MRI and BALB/c bone marrow (closed triangles), and CTLA41g/MRI and C57 bone grafts (open squares).
- Figure 2 is a line graph showing percent survival of secondary C57 skin grafts at 50 days after an initial BALB/c heart transplant.
- Animals in Group I open squares, received no further treatment
- Group 2 closed circles, received infusion of bone marrow
- Group 3 closed triangles, received BALB/c bone graft
- closed triangles follow same trajectory as all four open squares; closed circles follow trajectory of last two open squares In contrast, the animals in Group 4 (closed squares, received C57 bone graft) accepted secondary C57 skin grafts with all surviving >80 days.
- Figure 3 is a line graph showing percent survival of secondary skin grafts in mice pre- treated with CTLA41g/MRI only (closed squares), CTLA41g/MRI and C57 bone graft (closed circles, closed triangles), C57 bone graft only (closed diamonds), or no pre- treatment (open squares).
- CTLA41g/MRI CTLA41g/MRI
- C57 bone graft closed circles, closed triangles
- C57 bone graft only closed diamonds
- no pre- treatment open squares.
- One group (closed triangles) received a BALB/c skin graft; all others received a C57 skin graft.
- Figure 4 are graphs of two-color flow cytometry showing bone graft transplantation results in stable multi-lineage hematopoietic chimerism.
- Figures 5a-e are graphs and photographs showing transplanted bone grafts populate the recipient's thymus and influence negative selection.
- Figures 6a-d are graphs and photographs showing costimulation blockade and bone graft transplantation induce long-term donor-specific unresponsiveness in allogeneic hosts.
- Figure 7 includes line graphs showing that bone graft recipients treated with combination costimulatory molecule blockade accept donor specific skin grafts placed 12 hours later.
- a "bone” or “bone graft” means sufficient bone tissue to include stromal cells and marrow cells.
- the bone comprises fragments of bone.
- the bone grafts ofthe invention include portions of intact bones from, e.g., animals such as mice or primates. Typically, the bones are femur bones. Other bones may also be used.
- the graft may consist of a portion ofthe interior ofthe bone containing bone fragments or spicules and bone marrow as well as stromal cells.
- the graft may be removed or scooped, by an instrument such as a curette, from the bone marrow cavity after slicing an end ofthe bone.
- the amount of material for use in the graft is such that it is sufficient to induce at least 0.1% hematopoietic chimerism in the subject host. Optimization ofthe amount of material for the bone graft may be determined empirically.
- transplanted tissue includes autografts, isografts, allografts, and xenografts.
- transplanted tissue include, but are not limited to, solid organ grafts such as heart, liver, pancreas or kidney, skin grafts, bone marrow, pancreatic islet cells, cell suspensions, and genetically modified cells.
- MR1 monoclonal antibodies directed against gp39
- CD40 ligand any antibody molecule, fragment thereof, or recombinant binding protein that recognizes and binds gp39 (gp39 is also known in the literature as the CD40 ligand).
- monoclonal antibodies directed against CD40 includes any antibody molecule, fragment thereof, or recombinant binding protein that recognizes and binds CD40.
- administering means oral administration, administration as a suppository, topical contact, intravenous, intraperitoneal, intramuscular or subcutaneous administration, or the implantation of a slow-release device such as a miniosmotic pump, to the subject.
- pharmaceutically acceptable carrier includes any material which, when combined with the antibody, retains the antibody's binding specificity and is non-reactive with the subject's immune system. Examples include, but are not limited to, any ofthe standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil/water emulsion, and various types of wetting agents. Other carriers may also include sterile solutions, tablets, including coated tablets and capsules.
- such carriers typically contain excipients, such as starch, milk, sugar, certain types of clay, gelatin, stearic acid or salts, thereof, magnesium or calcium stearate, talc, vegetable fats or oils, gums, glycols, or other known excipients.
- excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid or salts, thereof, magnesium or calcium stearate, talc, vegetable fats or oils, gums, glycols, or other known excipients.
- Such carriers may also include flavor and color additives or other ingredients.
- Compositions comprising such carriers are formulated by well-known conventional methods.
- B7 includes B7-1 (also called CD80).
- B7-2 also called CD86
- B7-3 also called B7 family, e.g., a combination of B7-1, B7-2, and/or B7-3.
- a soluble ligand which recognizes and binds B7 antigen includes CTLA4- Ig, CD28-Ig or other soluble forms of CTLA4 and CD28, including recombinant CTLA4 and CD28, and includes any antibody molecule, ⁇ agment thereof or recombinant binding protein that recognizes and binds a B7 antigen.
- monoclonal antibodies directed against CTLA4 or CD28 includes any antibody molecule, fragment thereof, or recombinant binding protein that recognizes and binds CTLA4 or CD28.
- Applicants' discovery is related to a method for inhibiting rejection of a transplanted tissue in a subject.
- the method can be performed without myeloablating the subject.
- This method comprises grafting bone or fragments thereof into the subject. Grafting can occur before or after transplantation ofthe tissue.
- the bone comprises stromal cells and marrow cells. The presence of bone so grafted results in immunological tolerance ofthe transplanted tissue by the recipient, thereby inhibiting rejection ofthe transplanted tissue.
- the bone comprises an intact microenvironment.
- the microenvironment includes structural and soluble factors which contribute to stem cell maturation.
- the bone comprises fragments of bone containing marrow and stromal cells. Examples of sources of bone include, but are not limited to, vertebral bodies, rib, sternum or femur.
- the bone is taken from a donor antigenically matched with the transplant tissue.
- the bone is syngeneic with the transplant tissue.
- the bone may be grafted into the subject before, concurrently with, or after the tissue transplant.
- inhibition of rejection ofthe tissue transplant can be augmented by the administration of an immunosuppressive agent.
- an immunosuppressive agent is administered prior to, or concurrently with, the grafting of bone to inhibit rejection ofthe bone graft.
- the bone may be grafted into any site in the recipient that is capable of vascularizing the bone graft.
- the bone is grafted into a site having a rich blood supply.
- sites suitable for grafting include, but are not limited to omenturn or renal capsule.
- Vascularization and acceptance ofthe bone graft can be further enhanced by anastomosing one or more blood vessels ofthe graft with one or more blood vessels of the subject.
- the transplanted tissue can be an autograft, isograft, allograft, or xenograft.
- transplanted tissue include but are not limited to, solid organ grafts such as heart, pancreas, liver or kidney, skin grafts, bone marrow, pancreatic islet cells, cell suspensions, and genetically modified cells.
- rejection ofthe transplanted tissue is further inhibited by administration of an immunosuppressive agent.
- an immunosuppressive agent examples include, but are not limited to, soluble CTLA4 such as CTLA4Ig, soluble CD28 such as CD2Ig, soluble B7, such as BB7Ig, soluble gp39, soluble CD40, anti-gp39 antibodies, anti-CD40 antibodies, anti-CD28 antibodies, anti-B7 antibodies, anti-CTLA4 antibodies, cyclosporin, azathioprine, methotrexate, cyclophosphamide, lymphocyte immune globulin, anti-CD3 antibodies, Rho (D) immune globulin, adrenocorticosteroids, sulfasalzine, FK-506.
- soluble CTLA4 such as CTLA4Ig
- soluble CD28 such as CD2Ig
- soluble B7 such as BB7Ig
- soluble gp39 such as soluble gp39
- soluble CD40 anti-gp39 antibodies
- anti-CD40 antibodies anti-CD40 antibodies
- methoxsalen mycophenolate mofetil (Cellcept)
- mycophenolate mofetil Cellcept
- horse anti-human thymocyte globulin AGAM
- humanized anti-TAC HAT
- basiliximab Simulect
- rabbit anti-human thymocyte globulin TTYMOGLOBULIN
- sirolimus and thalidomide.
- the immunosuppressive agent comprises a first soluble molecule, such as a soluble ligand, which prevents an endogenous molecule on a cell selected from the group consisting of gp39 and CD40 from binding its endogenous ligand.
- the immunosuppressive agent comprises a second soluble molecule, such as a soluble ligand, which prevents an endogenous molecule on a cell selected from the group consisting of CTLA4. CD28, and B7 from binding its endogenous ligand.
- the immunosuppressive agent comprises a combination ofthe first soluble ligand and the second soluble ligand. The prevention of these molecules from binding their endogenous ligands blocks two independent signal pathways.
- multiple immunosuppressive agents can be used in the method ofthe invention, e.g., a combination of CTLA4Ig, anti-gp39 antibodies and cyclosporin.
- the first soluble molecule which prevents an endogenous molecule on a cell selected from the group consisting of gp39 and CD40 from binding its endogenous ligand, can be a soluble ligand which recognizes and binds a gp39 antigen on gp39-positive cells or CD40 antigen on CD40-positive cells.
- the soluble ligand can be a monoclonal antibody directed against gp39 or against CD40.
- the second soluble molecule which prevents an endogenous molecule on a cell selected from the group consisting of CTLA4, CD28 and B7 from binding its endogenous ligand, can be a soluble ligand which recognizes and binds a CD28 antigen on CD28-positive cells or a B7 antigen on B7 positive cells.
- the soluble ligand can be CTLA4Ig, CD28Ig, B7Ig or other soluble form of CTLA4, CD28 or B7, including recombinant CTLA4, CD28 or B7.
- the soluble ligand can be a monoclonal antibody or fragment thereof directed against a B7 antigen, CTLA4 or CD28.
- Immunosuppressive agents may be administered concurrently with the tissue transplant, before the tissue transplant, after the tissue transplant, concurrently with the bone graft, before the bone graft, or after the bone graft.
- Immunosuppressive agents may be administered by oral means, transdermal means, intravenous means, intramuscular means, intraperitoneal, or by subcutaneous administration.
- the most effective mode of administration and dosage regimen for the molecules ofthe present invention depends upon the location ofthe tissue or disease being treated, the severity and course ofthe medical disorder, the subject's health and response to treatment and the judgment ofthe treating physician. Accordingly, the dosages ofthe molecules should be titrated to the individual subject.
- the amount of bone graft required to achieve inhibition of transplant rejection may be determined by routine experimentation and optimized empirically.
- the interrelationship of dosages for animals of various sizes and species and humans based on mg/m 2 of surface area is described by Freireich, E. J., et al. Cancer Chemother., Rep. 50 (4): 219-244 (1966). Adjustments in the dosage regimen may be made to optimize suppression ofthe immune and inflammatory response resulting in graft rejection, e.g., doses may be divided and administered on a daily basis or the dose reduced proportionally depending upon the situation (e.g., several divided doses may be administered daily or proportionally reduced depending on the specific therapeutic situation). It would be clear that the dose ofthe immunosuppressive agent required to achieve an appropriate clinical outcome may be further reduced with schedule optimization.
- compositions useful in inhibiting graft rejection comprise an effective amount of fragments of bone and an acceptable carrier.
- the composition further comprises an immunosuppressive agent.
- the experiments described herein show that transplant tolerance can be achieved with the grafting of bone and without the use of myeloablation (e.g., irradiation) ofthe recipient.
- the experiment also demonstrates the generation of stable multi-lineage hematopoietic chimerism resulting from bone graft transplantation.
- the methods ofthe invention offer the advantages of avoiding the morbidity associated with myeloablation and avoiding laborious isolation techniques to obtain facilitator cells.
- the bone grafts ofthe invention provide a supportive microenvironment of structural and soluble factors which may be important for stem cell maturation.
- This example shows that the concomitant placement of a donor bone graft at the time of heart transplantation induces a long-term state of specific transplantation tolerance towards the donor antigens.
- mice Male C3H/He (H2 k), BALB/c (H2 d), and C57BL/6 (H2 b) mice (Jackson Laboratory, Bar Harbor, ME) were used at 8-12 weeks of age. Recipients were all C3H mice.
- Full thickness ear skin was grafted onto the posterior lateral thoracic wall of recipient mice. The grafts were then followed by daily visual inspection. Rejection was defined as the complete loss of visible epidermal graft tissue.
- BALB/c skin grafts were used for Part A ofthe experiment. For Part B, C57 skin grafts were used.
- the bones (one femur and one tibia) were sectioned into small pieces and were engrafted under the recipient renal capsules.
- the bone grafts consisted of fragments of whole bone, which includes cortex and marrow.
- the bone marrow in turn contained both stromal and hematopoietic elements.
- Bone marrow cells were flushed from the femurs and tibias of donor mice using a 27- gauge needle. The cells were prepared for injection by passage through a 70 um cell strainer (Becton Dickinson) and by red blood cell lysis with Tris-buffered ammonium chloride. Bone marrow cells (2x10 7 cells/dose) in sterile phosphate-buffered saline were administered intravenously to cardiac allograft recipients at the completion ofthe transplant procedure.
- Treatment Protocols 3CH He heart transplant or heart/bone transplant recipients were treated intravenously with 500 ⁇ g each of human CTLA4-Ig and MRI (hamster anti -mouse gp39 monoclonal antibody) at the time of transplantation of a BALB/c heart and intraperitoneally on days 2, 4 and 6 after transplantation.
- Group I received no further treatment.
- Group 2 received an infusion of 2x 1 07 BALB/c bone marrow cells.
- Group 3 received a BALB/c bone graft placed under the right kidney capsule while Group 4 received a C57 bone graft at the time of heart transplantation.
- Part A BALB/c skin grafts
- MST median survival time
- the MST in this group was >104 days, furthermore the long-term surviving skin grafts in these animals were large, supple, and healthy in appearance in contrast to the single long-term survivor in Group 2.
- This example shows that a bone graft can induce donor-specific tolerance to a subsequent skin graft.
- mice Male C3H/He (H2 k), BALB/c (H2 d), and C57BL/6 (H2 b) mice (Jackson Laboratory, Bar Harbor, ME) were used at 8-12 weeks of age.
- the bones (one femur and one tibia) were sectioned into small pieces and were engrafted unilaterally under the recipient renal capsule.
- Bone marrow cells were flushed from the femurs and tibias of donor mice using a 27- gauge needle. The cells were prepared for injection by passage through a 70-um cell strainer (Becton Dickinson) and by red blood cell lysis with Tris-buffered ammonium chloride. Bone marrow cells (2x10 7 cells/dose) in sterile phosphate-buffered saline were administered intravenously.
- mice were divided into five experimental groups. Group I received no pretreatment. Group 2 was treated intraperitoneally with 500 ⁇ g each of human CTLA4 Ig and MRI (hamster anti-mouse gp39 monoclonal antibody) on days 1, 2. 4, and 6.
- CTLA4 Ig human CTLA4 Ig
- MRI hamster anti-mouse gp39 monoclonal antibody
- Group 3 received a C57 bone graft under the right kidney capsule.
- Group 4 received a C57 bone graft under the right kidney capsule and was treated intraperitoneally with 500 ⁇ g each of human CTLA4-Ig and MRI (hamster anti-mouse gp39 monoclonal antibody) on days 1, 2, 4, and 6.
- Group 5 also received a C57 bone graft under the right kidney capsule and was treated intraperitoneally with 500 ⁇ g each of human CTLA4-Ig and MRI on days 0, 2, 4, and 6. Fifty days later the mice in groups 1-4 received a C57 skin graft and no additional treatment. The mice in group 5 received a Balb/c skin graft and no additional treatment.
- This example demonstrates the induction of hematopoietic chimerism using bone graft transplantation
- Femurs were harvested from donor mice and fragments of one femur were transplanted under the kidney capsule of recipient mice.
- Flow cytometric analysis Analysis of peripheral blood and spleen were carried out using fluorochrome-conjugated antibodies (anti-Ly5.1, anti-Ly5.2, anti-CD4, anti-CD 1 lc, anti- H2K d , anti-I-A d , anti-V ⁇ l 1, Pharmingen).
- Immunohistochemistry Cryosections (7 ⁇ m) were prepared and stained with biotinylated anti-I-A d (Pharmingen) followed by ABC complex (Vector). Peroxidase activity was visualized using DAB substrate (Pierce).
- Cytospin slides of bone graft cultures were stained with biotinylated primary antibodies (anti-H2K b , anti-H2K k , anti-I-A b , anti-I-A k ) followed by streptavidin-FITC or using FITC conjugated mAbs (Pharmingen).
- FIG. 4 shows bone graft transplantation results in stable multi-lineage hematopoietic chimerism.
- Peripheral blood WBC (Ly5+) were analyzed for the % of donor-derived (Ly5b+) cells within the granulocyte, monocyte and lymphocyte compartments using two color flow cytometry at 2, 10, and 30 weeks after bone graft transplantation. Results shown are from a single representative mouse of eight from two separate experiments.
- the viability ofthe bone graft was established by propagating donor derived cells from the bone grafts ex vivo in media containing recombinant granulocyte- macrophage colony stimulating factor (GM-CSF).
- GM-CSF granulocyte- macrophage colony stimulating factor
- the bone graft for transplantation consisted of crushed mouse femur containing bone fragments, bone marrow and stromal cells.
- hematopoietic progenitors from a transplanted bone graft were tested to determine whether they could populate the recipient thymus and influence negative selection of T cells.
- B6SCID mice were transplanted with bone grafts from B6 (H-2 b ) or B6CF1 (H-2 bxd , B6C) nude donor mice. Nude mice fail to develop T cells because of a thymic epithelial defect. In contrast, bone marrow cells from SCID mice fail to generate T cells because they lack a functional DNA-dependant kinase gene that is essential for T cell receptor arrangement. After 12 weeks the peripheral blood CD4+ T cells which developed in these mice were examined by flow cytometry. As expected, SCID mice that did not receive bone grafts failed to develop T cells, even after 20 weeks. In contrast, recipients of B6 of B6CF1 nude bone grafts slowly developed T cells.
- bone grafts from B6 mice were transplanted to C3H recipients which were treated with anti-CD40L antibody and CTLA4-Ig for six days. After 12 weeks, the ability ofthe experimental and control mice were assessed to respond to donor and third party alloantigen in vitro and in vivo.
- mice T cells from naive C3H mice generated strong proliferative responses when challenged with B6 or Balb/c dendritic cells. Similarly, mice that received costimulation blockade alone or a B6 bone graft and no costimulation blockade responded vigorously to both B6 and Balb/c stimulators. In contrast, 4 of 5 mice that received a B6 bone graft and costimulation blockade 12 weeks earlier displayed significantly diminished responses to B6 DC, but retained normal reactivity to Balb/c dendritic cells (Fig. 6a).
- Bone grafts from recipient mice were harvested and cultured in media containing
- Bone grafts from recipients that were not treated with costimulatory molecule blockade contains no viable hematopoietic progenitors and could not be propagated in culture demonstrating that costimulation blockade protects the hematopoietic potential of bone grafts transplanted to allogeneic hosts ( Figure 6b, bone marrow culture from a normal B6 mouse propagated in media containing GM-CSF (150 U/ml, left); nonviable B6 bone graft culture from an untreated C3H recipient (center); B6 bone graft culture from a C3H recipient treated with costimulation blockade 12 weeks after transplantation (right)).
- Figure 6b bone marrow culture from a normal B6 mouse propagated in media containing GM-CSF (150 U/ml, left); nonviable B6 bone graft culture from an untreated C3H recipient (center); B6 bone graft culture from a C3H recipient treated with costimulation blockade 12 weeks after transplantation (
- costimulation blockade was used with bone graft transplantation to determine the effects on donor-specific skin graft acceptance.
- Groups of C3H mice received either no treatment (Figure 6d, filled diamond); a B6 bone graft alone ( Figure 6d, open triangle); costimulation blockade and no bone graft ( Figure 6d, open circle); or costimulation blockade graft and a B6 bone graft ( Figure 6d, filled square). Twelve weeks later recipients were challenged with third-party (Balb/C, Figure 6d, left) or donor- specific (B6, Figure 6d, right) skin grafts. In a similar experiment 80% of mice receiving costimulation blockade and a donor bone graft accepted donor skin grafts for >100 days.
- mice were challenged 12 weeks after this induction protocol with donor (B6) or third-party (Balb/c) skin grafts (Fig. 7).
- C3H mice that received a B6 bone graft in the absence of costimulatory molecule blockade promptly rejected both donor-specific and third party skin grafts.
- mice that had been treated with costimulatory molecule blockade alone also promptly rejected B6 and Balb/c skin grafts, indicating no residual drug effect 12 weeks after administration.
- mice that received a B6 bone graft and costimulatory molecule blockade accepted donor-specific B6 skin grafts, while promptly rejecting third party Balb/c grafts.
- transplantation ofthe intact bone marrow microenvironment may provide distinct advantages in xenotransplantation, where the species selectivity of critical hematopoietic growth factors appears to be an important nonimmune barrier to long-term engraftment of xenogeneic HSCs (Nikolic, B. et al., Transplantation 65:1216-1224 (1998); Gritsch, H. A. and Sykes, M. Xenotransplantation 3:312-320 (1996); Warrens, A. N. et al., Transplantation 66:252-259 (1998)).
- Bone graft transplantation may also have potential to restore missing cell populations or immunologic functions in hereditary immunodeficiency syndromes (e.g. Bruton's agammaglobulinemia) or to treat hematopoietic diseases characterized by failure ofthe bone marrow microenvironment such as myelofibrosis without the need for myeloreductive conditioning.
- hereditary immunodeficiency syndromes e.g. Bruton's agammaglobulinemia
- myelofibrosis without the need for myeloreductive conditioning.
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AU95677/98A AU739277B2 (en) | 1997-09-10 | 1998-09-10 | Methods for inhibiting rejection of transplanted tissues by bone grafting |
EP98949333A EP1015043A1 (en) | 1997-09-10 | 1998-09-10 | Methods for inhibiting rejection of transplanted tissues by bone grafting |
CA002303008A CA2303008A1 (en) | 1997-09-10 | 1998-09-10 | Methods for inhibiting rejection of transplanted tissues by bone grafting |
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US8938741B2 (en) | 2004-07-20 | 2015-01-20 | Endress + Hauser Gmbh + Co. Kg | Electronic device and method for performing multiple processes with the electronic device |
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WO1994001534A1 (en) * | 1992-07-10 | 1994-01-20 | The University Of Pittsburgh | Hematopoietic facilitatory cells and their uses |
US5405390A (en) * | 1989-11-09 | 1995-04-11 | Osteotech, Inc. | Osteogenic composition and implant containing same |
US5580781A (en) * | 1986-04-18 | 1996-12-03 | Advanced Tissue Sciences, Inc. | Three-dimensional tumor cell and tissue culture system |
WO1996038543A1 (en) * | 1995-04-20 | 1996-12-05 | Diacrin, Incorporated | Modified cells and methods for inhibiting xenograft rejection |
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- 1998-09-10 AU AU95677/98A patent/AU739277B2/en not_active Ceased
- 1998-09-10 EP EP98949333A patent/EP1015043A1/en not_active Withdrawn
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US5580781A (en) * | 1986-04-18 | 1996-12-03 | Advanced Tissue Sciences, Inc. | Three-dimensional tumor cell and tissue culture system |
US5405390A (en) * | 1989-11-09 | 1995-04-11 | Osteotech, Inc. | Osteogenic composition and implant containing same |
WO1994001534A1 (en) * | 1992-07-10 | 1994-01-20 | The University Of Pittsburgh | Hematopoietic facilitatory cells and their uses |
WO1996038543A1 (en) * | 1995-04-20 | 1996-12-05 | Diacrin, Incorporated | Modified cells and methods for inhibiting xenograft rejection |
Non-Patent Citations (1)
Title |
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HISHA H ET AL: "Successful bone marrow transplantation by bone grafts in chimeric-resistant combination", EXPERIMENTAL HEMATOLOGY, vol. 23, no. 4, April 1995 (1995-04-01), pages 347 - 352, XP002091494 * |
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US8938741B2 (en) | 2004-07-20 | 2015-01-20 | Endress + Hauser Gmbh + Co. Kg | Electronic device and method for performing multiple processes with the electronic device |
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