Transplant tolerance induction with carbodiimide-treated tolerizing vaccine

Abstract

To provide transplant tolerance induction with a carbodiimide-treated tolerizing vaccine.SOLUTION: The present disclosure is related to compositions and systems for inducing immune tolerance for transplanted cells, organs or tissues in a transplant recipient. Also provided herein are methods of making and administering tolerizing vaccines / regimens or preparatory regimens. In some aspects, disclosed herein is a preparatory regimen for allotransplantation or xenotransplantation, including: (a) apoptotic donor leukocytes fixed in a crosslinking agent; (b) an mTOR inhibitor; (c) an anti-tumor necrosis factor agent or an anti-tumor necrosis factor receptor agent; (d) an anti-interleukin 6 agent or an anti-interleukin 6 receptor agent; and (e) an anti-CD40 agent or an anti-CD40 ligand agent; where the preparatory regimen is for administration to a recipient of a transplant cell, tissue or organ.SELECTED DRAWING: None

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 748,115, filed October 19, 2018, and U.S. Provisional Patent Application No. 62 / 902,091, filed September 18, 2019, each of which is incorporated by reference herein in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Grant U01AI102463 awarded by the National Institutes of Health (NIH). The government has certain rights in this invention. [Background technology]

[0003] Transplantation has become the most effective treatment option for patients with end-stage organ failure. Current immunosuppressive treatments are effective in preventing acute rejection of transplanted organs and cells; however, due to their significant morbidity and lack of effectiveness in preventing chronic rejection, there is a serious unmet medical need in the growing population of chronically immunosuppressed transplant recipients. Xenotransplantation of organs, tissues, and cells from suitable porcine donors overcomes the donor shortage associated with allogeneic transplantation; however, as with allogeneic transplantation, recipients will be at increased risk of developing infections, malignancies, diabetes, cardiovascular complications, nephrotoxicity, and other morbidities associated with chronic immunosuppression. Induction of immune tolerance to allografts and xenografts will extend the longevity of graft survival by overcoming the need for maintenance immunosuppression and eliminating graft loss due to chronic rejection, thereby significantly improving patient satisfaction, outcomes, and the cost-effectiveness of transplantation. Incorporation by Reference

[0004] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Unless otherwise indicated, all publications, patents, and patent applications mentioned in this application are incorporated herein by reference in their entirety. Summary of the Invention [Means for solving the problem]

[0005] Disclosed herein in some aspects are conditioning regimens for allogeneic or xenogeneic transplants, the conditioning regimen comprising: (a) apoptotic donor leukocytes fixed in a crosslinking agent; (b) an mTOR inhibitor; (c) an anti-tumor necrosis factor agent or an anti-tumor necrosis factor receptor agent; (d) an anti-interleukin-6 agent or an anti-interleukin-6 receptor agent; and (e) an anti-CD40 agent or an anti-CD40 ligand agent, wherein the conditioning regimen is for administration to a recipient of transplanted cells, tissues, or organs.

[0006] In some embodiments, the conditioning regimen further comprises instructions for administering (a), (b), (c), (d), and (e) to the recipient. In some embodiments, (a), (b), (c), (d), and (e) are administered to the recipient about 10 days before to about 30 days after allogeneic or xenogeneic transplantation. In some embodiments, at least one of (a), (b), (c), (d), and (e) is administered to the recipient at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days before allogeneic or xenogeneic transplantation. In some embodiments, at least one of (a), (b), (c), (d) and (e) is administered to the recipient at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 15, 17, 18, 19, 20, 21, 22, 23, 24 or 25 days before allograft or xenograft. In some embodiments, at least one of (a), (b), (c), (d) and (e) is administered to the recipient subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly, intracerebroventricularly, intranasally, intracranially, intracavity, intracerebrally, intrathecally or transdermally, or topically. In some embodiments, (a), (b), (c), (d) and (e) are administered intravenously. In some embodiments, the mTOR inhibitor is rapamycin. In some embodiments, the target trough level of the mTOR inhibitor in the recipient is about 5 to 12 ng per mL. In some embodiments, the anti-tumor necrosis factor agent is an anti-tumor necrosis factor antibody or antigen-binding fragment thereof. In some embodiments, the anti-tumor necrosis factor agent comprises a tumor necrosis factor-binding domain of a tumor necrosis factor receptor. In some embodiments, the anti-tumor necrosis factor agent is etanercept. In some embodiments, the anti-tumor necrosis factor agent or anti-tumor necrosis factor receptor agent is administered to the recipient at a dose of about 0.1 mg / kg to about 10 mg / kg. In some embodiments, the anti-tumor necrosis factor agent or anti-tumor necrosis factor receptor agent is administered to the recipient at a dose of about 0.5 mg / kg to about 1 mg / kg. In some embodiments, the anti-interleukin-6 receptor agent is an antagonistic anti-interleukin-6 receptor antibody or antigen-binding fragment thereof. In some embodiments, the anti-interleukin-6 receptor agent is tocilizumab.In some embodiments, the anti-interleukin 6 agent or anti-interleukin 6 receptor agent is administered to the recipient at a dose of about 1 mg / kg to about 100 mg / kg. In some embodiments, the anti-interleukin 6 agent or anti-interleukin 6 receptor agent is administered to the recipient at a dose of about 10 mg / kg. In some embodiments, the anti-CD40 agent or anti-CD40 ligand agent is an antagonistic anti-CD40 antibody or antigen-binding fragment thereof. In some embodiments, the anti-CD40 agent or anti-CD40 ligand agent is 2C10 or 2C10R4. In some embodiments, the anti-CD40 agent or anti-CD40 ligand agent is administered to the recipient at a dose of about 1 mg / kg to 100 mg / kg. In some embodiments, the anti-CD40 agent or anti-CD40 ligand agent is administered to the recipient at a dose of about 50 mg / kg. In some embodiments, administration of the conditioning regimen to the recipient provides long-term tolerance to the transplanted cells, tissue, or organ. In some embodiments, the apoptotic leukocytes are mammalian leukocytes. In some embodiments, the apoptotic leukocytes are porcine leukocytes. In some embodiments, the apoptotic leukocytes are human leukocytes. In some embodiments, the apoptotic leukocytes are from cadaveric, brain-dead, non-heart-beating, or living donors. In some embodiments, the apoptotic leukocytes are ex vivo expanded leukocytes. In some embodiments, the apoptotic leukocytes are isolated from the spleen or peripheral blood. In some embodiments, the apoptotic leukocytes comprise B lymphocytes. In some embodiments, the apoptotic leukocytes comprise cells differentiated ex vivo from stem cells or induced pluripotent stem cells. In some embodiments, the stem cells are derived from a donor of the transplant cells, tissue, or organ. In some embodiments, the transplanted cells, tissues, or organs are kidney, liver, heart, lung, pancreas, pancreatic islet cells, small intestine, bone marrow, hematopoietic stem cells, embryonic stem cell-derived pancreatic islet beta cells, induced pluripotent stem cell-derived pancreatic islet beta cells, embryonic stem cell-derived pancreatic islets, induced pluripotent stem cell-derived pancreatic islets, stem cell-derived cells, tissues, or organs, or combinations thereof. In some embodiments, the recipient and the donor of the transplanted cells, tissues, or organs are MHC class I incompatible.In some embodiments, the recipient and the donor of the transplanted cells, tissue, or organ are MHC class II mismatched. In some embodiments, the recipient and the donor of the transplanted cells, tissue, or organ are haploidentical. In some embodiments, the recipient and the donor of the transplanted cells, tissue, or organ are matched for at least one MHC class II allele. In some embodiments, the recipient and the donor of the transplanted cells, tissue, or organ are matched for at least one MHC class II allele, MHC class IB allele, MHC class II DR allele, MHC class II DQ allele, or MHC class II DP allele. In some embodiments, the recipient and the donor of the transplanted cells, tissue, or organ are matched for at least one MHC class II DR allele, which is an MHC class II DRB allele. In some embodiments, the apoptotic leukocyte and the recipient are matched for at least one MHC class IA allele, MHC class IB allele, MHC class II DR allele, MHC class II DQ allele, or MHC class II DP allele. In some embodiments, the apoptotic leukocytes and the recipient are matched for MHC class II DR alleles, which are MHC class II DRB alleles. In some embodiments, the apoptotic leukocytes and the recipient are completely mismatched. In some embodiments, the apoptotic leukocytes and the graft are matched for at least one MHC class IA allele, MHC class IB allele, MHC class II DR allele, MHC class II DQ allele, or MHC class II DP allele. In some embodiments, the apoptotic leukocytes and the graft are haploidentical. In some embodiments, the apoptotic leukocytes are derived from the transplant donor. In some embodiments, the apoptotic leukocytes are derived from the differentiation of stem cells from the donor of the transplanted cells, tissue, or organ. In some embodiments, the apoptotic leukocytes contain one or more peptides conjugated on their surface derived from the recipient's MHC class II molecule.In some embodiments, the one or more peptides derived from an MHC class II molecule include peptides derived from the DR β chain, the DQ β chain, or the DP β chain, or a combination thereof. In some embodiments, the one or more peptides derived from an MHC class II molecule include the entire α1 or α2 domain of a DR, DP, or DQ. In some embodiments, the one or more peptides derived from an MHC class II molecule include the entire β1 or β2 domain of a DR, DP, or DQ. In some embodiments, the MHC class II molecule is HLA-DRB1 of the recipient. * 03 or HLA-DRB1 * The one or more peptides derived from the MHC class II molecule are encoded by the 04 allele. In some embodiments, the one or more peptides derived from the MHC class II molecule comprise a sequence derived from a hypervariable region. In some embodiments, the one or more peptides derived from the MHC class II molecule are at least 10 amino acids in length. In some embodiments, the one or more peptides derived from the MHC class II molecule are about 10-30 amino acids in length. In some embodiments, the one or more peptides derived from the MHC class II molecule are synthetic or recombinant. In some embodiments, the apoptotic leukocytes are derived from the recipient or derived during differentiation of stem cells derived from the recipient, and the apoptotic leukocytes comprise one or more peptides derived from an MHC class I molecule conjugated to their surface. In some embodiments, the MHC class I molecule is expressed in the transplant donor. In some embodiments, the MHC class I molecule is HLA-A1, HLA-A3, HLA-B7, or HLAB8. In some embodiments, the MHC class I molecule is HLA-A * 02, 24, 01 or HLA-B *35, 44, 51. In some embodiments, one or more peptides are conjugated to the surface of apoptotic leukocytes by treatment with a crosslinking agent. In some embodiments, the crosslinking agent comprises a carbodiimide. In some embodiments, the carbodiimide comprises 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI), N,N'-diisopropylcarbodiimide (DIC), N,N'-dicyclohexylcarbodiimide (DCC), or a combination thereof. In some embodiments, the crosslinking agent comprises 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI). In some embodiments, the crosslinking agent does not comprise a carbodiimide. In some embodiments, the crosslinking agent comprises genipin, acrylaldehyde, diformyl, osmium tetroxide, diimide ester, mercuric chloride, zinc sulfate, zinc chloride, trinitrophenol (picric acid), potassium dichromate, ethanol, methanol, acetone, acetic acid, or a combination thereof. In some embodiments, the diimide ester comprises cyanuric chloride, diisocyanate, diethylpyrocarbonate (DEPC), maleimide, benzoquinone, or a combination thereof. In some embodiments, the apoptotic leukocytes are fixed for a predetermined period of time. In some embodiments, the predetermined period of time is at least about 10, 20, 30, 40, 50, 60, 75, 90, 120, 150, 180, 210, or 240 minutes. In some embodiments, the apoptotic leukocytes are further contacted with an amount of one or more immunomodulatory molecules. In some embodiments, the amount of one or more immunomodulatory molecules is sufficient to modify the function of antigen-presenting cells of the recipient. In some embodiments, the one or more immunomodulatory molecules comprise IFN-γ, NF-kB inhibitor, vitamin D3, siCD40, cobalt protoporphyrin, insulin B9-23, all or a portion of a cluster of differentiation protein, or a combination thereof. In some embodiments, the NF-kB inhibitor is curcumin, triptolide, Bay-117085, or a combination thereof. In some embodiments, (a), (b), (c), (d), and (e) are administered separately or simultaneously.

[0007] In some aspects, disclosed herein is a tolerization regimen for post-transplant stabilization of an allograft or xenograft recipient, comprising: a carbodiimide crosslinker-modulated apoptotic leukocytes, wherein the apoptotic leukocytes are expanded in the presence of one or more of IL-2, IL-4, IL-21, BAFF, and CD40L prior to contact with the crosslinker; an mTOR inhibitor; an anti-tumor necrosis factor agent or anti-tumor necrosis factor receptor agent; an anti-interleukin-6 agent or anti-interleukin-6 receptor agent; and an anti-CD40 agent or anti-CD40 ligand agent, wherein the tolerization regimen is for administration to the recipient of transplanted cells, tissues, or organs.

[0008] In some embodiments, the tolerization regimen of claim 74, wherein the apoptotic leukocytes are expanded by at least about 3-fold, 5-fold, 10-fold, 50-fold, 100-fold, 150-fold, 200-fold, or 250-fold relative to the starting population prior to contact with the crosslinking agent. In some embodiments, (a), (b), (c), (d), and (e) are administered separately or simultaneously.

[0009] In some aspects, disclosed herein are conditioning regimens for transplantation of a cell, tissue, or organ graft into a recipient, the conditioning regimen comprising apoptotic leukocytes fixed in a crosslinker, wherein the apoptotic leukocytes comprise, conjugated on their surface, one or more peptides derived from MHC class II molecules of the recipient or one or more peptides derived from MHC class I molecules of a donor of the cell, tissue, or organ graft, and the conditioning regimen is for administration to the recipient.

[0010] In some embodiments, the conditioning regimen further comprises administering one or more agents for short-term immunosuppression of the transplant recipient. In some embodiments, the one or more agents for short-term immunosuppression comprise an mTOR inhibitor, an anti-tumor necrosis factor agent or anti-tumor necrosis factor receptor agent, an anti-interleukin 6 agent or anti-interleukin 6 receptor agent, an anti-CD40 agent or anti-CD40 ligand agent, or a combination thereof. In some embodiments, the conditioning regimen further comprises instructions for administering the one or more agents for short-term immunosuppression to the transplant recipient. In some embodiments, the one or more peptides derived from an MHC class II molecule comprise a peptide derived from a DR β chain, a DQ β chain, or a DP β chain, or a combination thereof. In some embodiments, the one or more peptides derived from an MHC class II molecule comprise the entire β1 or β2 domain of the DR. In some embodiments, the MHC class II molecule comprises an HLA-DRB1 * 03 or HLA-DRB1 *The peptides are encoded by the 04 allele. In some embodiments, the one or more peptides derived from an MHC class II molecule comprise the entire α1 or α2 domain of DR, DP, or DQ. In some embodiments, the one or more peptides derived from an MHC class II molecule comprise the entire β1 or β2 domain of DQ. In some embodiments, the one or more peptides derived from an MHC class II molecule comprise a sequence derived from a hypervariable region. In some embodiments, the one or more peptides derived from an MHC class II molecule are at least 10 amino acids in length. In some embodiments, the one or more peptides derived from an MHC class II molecule are about 10-30 amino acids in length. In some embodiments, the one or more peptides derived from an MHC class II molecule are synthetic or recombinant. In some embodiments, the apoptotic leukocytes fixed in a crosslinker comprise one or more peptides derived from a recipient's MHC class II molecule, and the apoptotic leukocytes are MHC class II-matched with the donor and MHC class II-mismatched with the recipient. In some embodiments, the MHC class I molecule is expressed in the transplant donor. In some embodiments, the MHC class I molecule is HLA-A1, HLA-A3, HLA-B7, or HLA-B8. * 02, 24, 01 or HLA-B *35, 44, 51. In some embodiments, the apoptotic leukocytes fixed in the crosslinker contain one or more peptides derived from donor MHC class I molecules, and the apoptotic leukocytes are MHC class I-matched, MHC class II-matched, or both, with the recipient. In some embodiments, the one or more peptides are conjugated to the surface of the apoptotic leukocytes by treatment with a crosslinker. In some embodiments, the crosslinker comprises a carbodiimide. In some embodiments, the carbodiimide comprises 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI), N,N'-diisopropylcarbodiimide (DIC), N,N'-dicyclohexylcarbodiimide (DCC), or a combination thereof. In some embodiments, the crosslinker comprises 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI). In some embodiments, the crosslinker does not comprise a carbodiimide. In some embodiments, the crosslinking agent comprises genipin, acrylaldehyde, diformyl, osmium tetroxide, diimidoester, mercuric chloride, zinc sulfate, zinc chloride, trinitrophenol (picric acid), potassium dichromate, ethanol, methanol, acetone, acetic acid, or a combination thereof. In some embodiments, the diimidoester comprises cyanuric chloride, diisocyanate, diethylpyrocarbonate (DEPC), maleimide, benzoquinone, or a combination thereof. In some embodiments, the apoptotic leukocytes are fixed for a predetermined period of time. In some embodiments, the predetermined period of time is at least about 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 75 minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes, 210 minutes, or 240 minutes. In some embodiments, the apoptotic leukocytes are further contacted with an amount of one or more immunomodulatory molecules.

[0011] In some aspects, disclosed herein are methods of inducing tolerance to a cell, tissue, or organ transplant in a recipient, the method comprising administering to the recipient an effective amount of a composition comprising: (a) apoptotic leukocytes fixed in a crosslinking agent; (b) an mTOR inhibitor; (c) an anti-tumor necrosis factor agent or an anti-tumor necrosis factor receptor agent; (d) an anti-interleukin-6 agent or an anti-interleukin-6 receptor agent; and (e) an anti-CD40 agent or an anti-CD40 ligand agent.

[0012] In some embodiments, the apoptotic leukocytes and the recipient are matched with the recipient for at least one MHC class II allele. In some embodiments, the apoptotic leukocytes and the recipient are MHC-mismatched. In some embodiments, the apoptotic leukocytes comprise one or more peptides derived from MHC class II molecules of the recipient, wherein the one or more peptides are conjugated to the surface of the apoptotic leukocytes. In some embodiments, the apoptotic leukocytes comprise cells differentiated ex vivo from stem cells, wherein the stem cells are derived from a donor of a cell, tissue, or organ transplant. In some embodiments, the apoptotic leukocytes are MHC-matched with the recipient. In some embodiments, the apoptotic leukocytes comprise one or more peptides derived from MHC class I molecules of a donor of a cell, tissue, or organ transplant, wherein the one or more peptides are conjugated to the surface of the apoptotic leukocytes. In some embodiments, the apoptotic leukocytes comprise cells differentiated ex vivo from stem cells, wherein the stem cells are derived from a recipient of a cell, tissue, or organ transplant. In some embodiments, the method further comprises transplanting a cell, tissue, or organ graft. In some embodiments, the transplanting occurs at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more days after administration of the composition. In some embodiments, the method further comprises administering at least one booster dose of the composition. In some embodiments, the booster dose is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 17, 20, 21, or 24 days after transplantation. In some embodiments, tolerance is for a period of at least 1 month. In some embodiments, tolerance is for a period of at least 100 days. In some embodiments, tolerance is for a period of at least 1 year.

[0013] Disclosed herein in some aspects is a method of inducing tolerance to a cell, tissue, or organ transplant in a recipient, comprising administering to the recipient an effective amount of a composition comprising apoptotic leukocytes fixed in a cross-linking agent, wherein the apoptotic leukocytes comprise, conjugated on their surface, one or more peptides derived from MHC class II molecules of the recipient or one or more peptides derived from MHC class I molecules of a donor of the cell, tissue, or organ transplant.

[0014] In some embodiments, tolerance exists in the absence of immunosuppressive therapy 50 days after transplantation, hi some embodiments, tolerance exists in the absence of a booster regimen.

[0015] In some aspects, disclosed herein are methods for inhibiting an immune response to a cell, tissue, or organ transplant in a recipient, the method comprising administering to the recipient effective amounts of: (a) apoptotic leukocytes fixed in a crosslinking agent; (b) an mTOR inhibitor; (c) an anti-tumor necrosis factor agent or an anti-tumor necrosis factor receptor agent; (d) an anti-interleukin-6 agent or an anti-interleukin-6 receptor agent; and (e) an anti-CD40 agent or an anti-CD40 ligand agent.

[0016] Disclosed herein in some aspects is a method for inhibiting an immune response to a cell, tissue, or organ transplant in a recipient, comprising administering to the recipient an effective amount of a composition comprising apoptotic leukocytes fixed in a cross-linking agent, wherein the apoptotic leukocytes comprise, conjugated on their surface, one or more peptides derived from MHC class II molecules of the recipient or one or more peptides derived from MHC class I molecules of a donor of the cell, tissue, or organ transplant.

[0017] In some embodiments, the immune response comprises B cell activation, T cell proliferation, B cell proliferation, macrophage activation, cytokine production, or a combination thereof.

[0018] In some aspects, disclosed herein are methods of post-transplant immune tolerization of a subject, the method comprising administering to the subject (a) carbodiimide crosslinker-modulated apoptotic leukocytes, where the apoptotic leukocytes are expanded in the presence of one or more of IL-2, IL-4, IL-21, BAFF, and CD40L prior to contact with the crosslinker; (b) an mTOR inhibitor; (c) an anti-tumor necrosis factor agent or anti-tumor necrosis factor receptor agent; (d) an anti-interleukin-6 agent or anti-interleukin-6 receptor agent; and (e) an anti-CD40 agent or anti-CD40 ligand agent.

[0019] In some embodiments, the apoptotic leukocytes are isolated from peripheral blood. In some embodiments, the apoptotic leukocytes are enriched for B cells by negative selection. In some embodiments, the apoptotic leukocytes are expanded at least 200-fold relative to the starting population prior to modulation with a carbodiimide crosslinker.

[0020] In some aspects, disclosed herein are kits for transplantation of a cell, tissue, or organ graft in a recipient, the kit comprising: (a) a first container comprising a first composition comprising apoptotic leukocytes fixed in a crosslinking agent; (b) a second container comprising a second composition comprising an mTOR inhibitor; (c) a third container comprising a third composition comprising an anti-tumor necrosis factor agent or an anti-tumor necrosis factor receptor agent; (d) a fourth container comprising a fourth composition comprising an anti-interleukin-6 agent or an anti-interleukin-6 receptor agent; and (e) a fifth container comprising a fifth composition comprising an anti-CD40 agent or an anti-CD40 ligand agent.

[0021] In some aspects, disclosed herein is a kit for transplantation of a cell, tissue, or organ graft in a recipient, the kit comprising: a first container containing a first composition comprising apoptotic leukocytes immobilized in a cross-linking agent, wherein the apoptotic leukocytes comprise, conjugated on their surface, one or more peptides derived from MHC class II molecules of the recipient or one or more peptides derived from MHC class I molecules of a donor of the cell, tissue, or organ graft.

[0022] In some embodiments, the kit further comprises a cell, tissue, or organ graft.

[0023]

[0010] In some aspects, disclosed herein is a transplant kit comprising a conditioning regimen and a tolerization regimen, wherein the conditioning regimen comprises apoptotic leukocytes fixed in a crosslinking agent; an mTOR inhibitor; an anti-tumor necrosis factor agent or anti-tumor necrosis factor receptor agent; an anti-interleukin-6 agent or anti-interleukin-6 receptor agent; and an anti-CD40 agent or anti-CD40 ligand agent; and the tolerization regimen comprises apoptotic leukocytes modulated with a carbodiimide crosslinking agent, wherein the apoptotic leukocytes are expanded in the presence of one or more of IL-2, IL-4, IL-21, BAFF, and CD40L prior to contact with the crosslinking agent; an mTOR inhibitor; an anti-tumor necrosis factor agent or anti-tumor necrosis factor receptor agent; an anti-interleukin-6 agent or anti-interleukin-6 receptor agent; and an anti-CD40 agent or anti-CD40 ligand agent;

[0024] In some embodiments, the transplant kit further comprises a cell, tissue, or organ transplant. In some embodiments, the apoptotic leukocytes of the tolerization regimen are isolated from peripheral blood. In some embodiments, the apoptotic leukocytes of the tolerization regimen are enriched for B cells by negative selection. In some embodiments, the apoptotic leukocytes of the tolerization regimen are expanded at least 200-fold relative to the starting population before contact with the cross-linking agent.

[0025] In one aspect, provided herein are mammalian leukocytes for allografts, wherein said mammalian leukocytes are cadaveric mammalian leukocytes, and said cadaveric mammalian leukocytes are fixed in carbodiimide.

[0026] In another aspect, the present disclosure provides mammalian leukocytes for allografts, wherein the mammalian leukocytes are ex vivo expanded mammalian leukocytes, and the ex vivo expanded mammalian leukocytes are fixed in carbodiimide.

[0027] In some embodiments, the ex vivo expanded mammalian leukocytes are from a living donor. In some embodiments, the living donor is human. In some embodiments, the cadaveric mammalian leukocytes are from a population of ex vivo expanded leukocytes. In some embodiments, the cadaveric mammalian leukocytes are from a cardiac-beating donor. In some embodiments, the cadaveric mammalian leukocytes are from a brain-dead donor. In some embodiments, the mammalian leukocytes are human leukocytes. In some embodiments, the mammalian leukocytes are isolated from the spleen. In some embodiments, the mammalian leukocytes are B cells. In some embodiments, the mammalian leukocytes are fixed for a predetermined period of time. In some embodiments, the predetermined period of time is at least about 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 75 minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes, 210 minutes, or 240 minutes.

[0028] In some embodiments of the above aspects, the mammalian leukocytes are further contacted with an amount of one or more immunomodulatory molecules. In some embodiments, the amount is sufficient to trigger apoptotic death of the donor-reactive cells when subsequently exposed to 37°C for 4 hours. In some embodiments, the one or more immunomodulatory molecules comprise IFN-γ, an NF-kB inhibitor, vitamin D3, siCD40, cobalt protoporphyrin, insulin B9-23, α1-antitrypsin, all or a portion of a cluster of differentiation protein, a gp39 antagonist, or a combination thereof. In some embodiments, the NF-kB inhibitor is curcumin, triptolide, Bay-117085, or a combination thereof. In some embodiments, the carbodiimide comprises 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI), N,N'-diisopropylcarbodiimide (DIC), N,N'-dicyclohexylcarbodiimide (DCC), or a combination thereof. In some embodiments, the carbodiimide agent comprises 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI).

[0029] In one aspect, provided herein is a method of inducing tolerance to a cell, tissue, or organ transplant, comprising administering to a subject a composition comprising an amount of the mammalian leukocytes disclosed above. In some embodiments, the cell or tissue transplant is derived from a donor of the mammalian leukocytes. In some embodiments, the cell or tissue transplant is derived from a donor that is MHC-matched to the donor of the mammalian leukocytes. In some embodiments, the cell or tissue transplant is derived from a donor that is haploidentical ... In some embodiments, the cell or tissue graft is derived from a donor who shares the B allele, the MHC class II DR allele, or the MHC class II DQ allele. In some embodiments, the cell or tissue graft is kidney, liver, heart, lung, pancreas, pancreatic islet cells, small intestine, bone marrow, hematopoietic stem cells, embryonic or induced pluripotent stem cell-derived pancreatic islet beta cells, embryonic or induced pluripotent stem cell-derived pancreatic islets, or a combination thereof.

[0030] In some embodiments, the method further comprises transplanting the cell or tissue graft into the subject. In some embodiments, the transplanting occurs at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more days after administration of the composition. In some embodiments, the transplanting occurs at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more days before administration of the composition. In some embodiments, the method aspects disclosed above further comprise administering at least one booster dose of the composition. In some embodiments, the method further comprises administering 1, 2, 3, 4, or 5 booster doses of the composition. In some embodiments, the booster dose is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more days after the transplantation. In some embodiments, the booster dose is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more days prior to the transplanting. In some embodiments, the booster dose comprises a lower amount of the mammalian leukocytes.

[0031] In one aspect, provided herein is a conditioning regimen for transplantation comprising leukocytes fixed in a crosslinking agent, wherein the leukocytes are treated with an agent that increases expression of anti-inflammatory cytokines in the recipient. In some embodiments, the anti-inflammatory cytokines include TGF-β, IL-10, IL-13, or a combination thereof. In some embodiments, the conditioning regimen provides long-term tolerance of the recipient to transplanted cells, tissues, or organs derived from the leukocyte donor. In some embodiments, the long-term tolerance extends for a period of at least one month. In some embodiments, the long-term tolerance exists in the absence of a booster regimen.

[0032] In some embodiments, the white blood cells are mammalian white blood cells. In some embodiments, the white blood cells are porcine white blood cells. In some embodiments, the white blood cells are human white blood cells. In some embodiments, the white blood cells are cadaveric white blood cells. In some embodiments, the white blood cells are ex vivo expanded white blood cells. In some embodiments, the cadaveric white blood cells are from a non-heart-beating donor. In some embodiments, the cadaveric white blood cells are from a brain-dead donor. In other embodiments, the ex vivo expanded white blood cells are from a living donor.

[0033] In some embodiments, the leukocytes are isolated from the spleen. In some embodiments, the leukocytes are fixed for a predetermined period of time. In some embodiments, the predetermined period of time is at least about 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 75 minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes, 210 minutes, or 240 minutes. In some embodiments, the agent comprises alpha 1-antitrypsin. In some embodiments, the leukocytes are further contacted with an amount of one or more immunomodulatory molecules. In some embodiments, the amount is sufficient to trigger apoptotic death of donor reactive cells.

[0034] In some embodiments, the one or more immunomodulatory molecules comprise IFN-γ, an NF-kB inhibitor, vitamin D3, siCD40, cobalt protoporphyrin, insulin B9-23, all or a portion of a cluster of differentiation protein, or a combination thereof. In some embodiments, the NF-kB inhibitor is curcumin, triptolide, Bay-117085, or a combination thereof. In some embodiments, the leukocytes are derived from a population of leukocytes isolated from a donor and expanded ex vivo. In some embodiments, the crosslinker comprises carbodiimide, genipin, acrylaldehyde, diformyl, osmium tetroxide, diimidoester, mercuric chloride, zinc sulfate, zinc chloride, trinitrophenol (picric acid), potassium dichromate, ethanol, methanol, acetone, acetic acid, or a combination thereof.

[0035] In some embodiments, the conditioning regimen includes a diimide ester including cyanuric chloride, diisocyanate, diethylpyrocarbonate (DEPC), maleimide, benzoquinone, or a combination thereof. In some embodiments, the conditioning regimen includes a carbodiimide including 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI), N,N'-diisopropylcarbodiimide (DIC), N,N'-dicyclohexylcarbodiimide (DCC), or a combination thereof. In some embodiments, the carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI). In some embodiments, the white blood cells include B lymphocytes. In some embodiments, the B lymphocytes are derived from a living donor.

[0036] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the present disclosure are utilized, and the accompanying drawings, in which: [Brief explanation of the drawings]

[0037] [Figure 1]Figure 1 illustrates positive, tolerizing, and pretreatment vaccines and regimens (alternatively referred to as negative vaccines). In certain embodiments of the present disclosure, a tolerizing or pretreatment vaccine or regimen, or components derived therefrom, can be provided as part of a pretreatment regimen described herein.

[0038] [Figure 2] FIG. 2 is a schematic diagram of a protocol for tolerance induction in donor transplantation, where the donor may be a living donor or a cadaveric donor, e.g., a deceased donor.

[0039] [Figure 3] FIG. 3 shows an exemplary approach to prolonging graft survival in subjects infused with apoptotic donor leukocytes, such as splenocytes, for tolerization or conditioning vaccinations or regimens, optionally with the support of transient immunosuppression.

[0040] [Figure 4]4 shows an exemplary transplantation regimen for inhibiting or minimizing rejection of the transplant and graft in a recipient and / or prolonging its survival, optionally in the absence of chronic and systemic immunosuppression of the recipient. In certain embodiments, the regimen comprises one or more of the following three components: i) transplanted cells or tissue, such as genetically engineered pancreatic islets, deficient and / or reduced in expression of αGal, NLRC5, B2M, MHC class I, complement C3, and / or CXCL10, and optionally transgenic expression of HLA-G and / or HLA-E; ii) transplanted cells or tissue, such as genetically engineered pancreatic islets, deficient and / or reduced in expression of αGal, Neu5Gc, NLRC5, B2M, human PD-L1, human PD-L2, and / or Sda / CAD, and optionally transgenic expression of HLA- 1) genetically engineered donor apoptotic and non-apoptotic mononuclear cells (e.g., genetically engineered cells in conditioning regimens or tolerization vaccines) that lack and / or reduce transgenic expression of G and / or HLA-E, with or without human CD47 (e.g., genetically engineered cells in conditioning regimens or tolerization vaccines), including ECDI-fixed leukocytes such as ECDI-fixed splenocytes or ECDI-fixed B lymphocytes); and ii) genetically engineered donor apoptotic and non-apoptotic mononuclear cells (e.g., genetically engineered cells in conditioning regimens or tolerization vaccines) that lack and / or reduce transgenic expression of G and / or HLA-E, with or without human CD47 (e.g., genetically engineered cells in conditioning regimens or tolerization vaccines), including ECDI-fixed leukocytes such as ECDI-fixed splenocytes or ECDI-fixed B lymphocytes); and iii) genetically engineered donor apoptotic and non-apoptotic mononuclear cells (e.g., genetically engineered cells in conditioning regimens or tolerization vaccines) that lack and / or reduce transgenic expression of G and / or HLA-E, with or without human CD47 (e.g., genetically engineered cells in conditioning regimens or tolerization vaccines), including but not limited to, antagonistic anti-CD40 This may include administration of transient immunosuppression comprising one or more components of the conditioning regimens described herein, such as one or more of: a mAb antibody, an Fc-engineered anti-CD40L antibody, rapamycin, compstatin or a compstatin derivative (e.g., the compstatin derivative APL-2), an anti-IL-6 receptor mAb, a soluble TNF receptor, a B-cell targeting strategy (e.g., a biologic that depletes B cells, e.g., a biologic that targets CD20, CD19, or CD22, and / or a biologic that modulates B cells, e.g., a biologic that targets BLyS, BAFF, BAFF / APRIL, CD40, IgG4, ICOS, IL-21, B7RP1), and / or alpha 1-antitrypsin. Non-limiting examples of B-cell-targeting biologics include rituximab, an anti-CD20 antibody.

[0041] [Figure 5] Figure 5 shows an exemplary protocol for leukocyte expansion for the preparation of apoptotic donor leukocytes (ADL), such as splenic B cells derived from human cadaveric donors, for use in conditioning or tolerization regimens. Splenocytes are isolated and subjected to RBC lysis and B cell enrichment on a nylon wool column. A first fraction of the isolated splenocytes (e.g., approximately 80%) can be used to generate a dose of apoptotic donor leukocytes (ADL) for infusion at the initial time point relative to transplantation (e.g., day -7). The splenocytes can be subjected to ECDI treatment as disclosed herein, subjected to release testing, and infused into the recipient. A second fraction of the isolated splenocytes (e.g., approximately 20%) can be set aside and used to generate a second dose of ADL for infusion on day +1. The second fraction may be subjected to selection to enrich for B cells (e.g., positive or negative selection using Miltenyi CliniMACS) and expanded ex vivo (e.g., for more than 8 days in the presence of hIL-2, hIL-4, h-IL21, hBAFF, and h-CD40L multimers). The enriched B cells may be subjected to at least 15-fold expansion before ECDI treatment, release testing, and infusion into the transplant recipient (e.g., on day 1).

[0042] [Figure 6] Figure 6 shows an exemplary protocol for tolerance induction to a living donor kidney allograft. On day -22 or -21 (±2 days) for the planned kidney transplant, B cells are obtained from a blood draw or leukapheresis transfusion. Ex vivo expanded, ECDI fixed donor B cells are infused IV into the recipient on days -7 and +1 for the day 0 kidney transplant. Short-term immunosuppressive and anti-inflammatory therapy is administered to the transplant recipient as disclosed herein.

[0043] [Figure 7]Figure 7 shows an exemplary protocol for generating a patient dose of tolerized apoptotic donor B cells obtained from donor peripheral blood and expanded ex vivo for infusion on days -7 and +1 prior to transplant. The protocol can be initiated at least approximately 3-4 weeks prior to transplant. One or more low-volume leukapheresis transfusion procedures are performed on living donors (e.g., kidney donors) that have not been cytokine-stimulated. B cells are enriched (e.g., by positive or negative selection using the CliniMACS) to provide an input cell yield of approximately 0.5 x 10^9 B cells. The enriched B cells are expanded at least 200-fold ex vivo to provide the target cell dose (e.g., expanded over approximately 16 days in the presence of hIL-2, hIL-4, h-IL-21, hBAFF, and h-CD40L multimers). After expansion, the B cells are ECDI treated as disclosed herein, subjected to release testing, and infused into the recipient (eg, on day -7 and +1 relative to transplant).

[0044] [Figure 8] 8A shows the conditioning regimen used in the examples of the present disclosure: Cohort B received short-term immunosuppression, while Cohort C received short-term immunosuppression and apoptotic donor leukocytes.

[0045] Figure 8B provides Kaplan-Meir estimates of rejection-free islet allograft survival. Administration of the apoptotic leukocyte-containing conditioning and tolerization regimen described herein was associated with significantly improved survival. P=0.021, Mantel-Cox.

[0046] [Figure 9]Figures 9A-F show that administration of the apoptotic leukocyte-containing conditioning and tolerization regimen described herein facilitates stable tolerance of pancreatic islet allografts. Figure 9A shows that monkeys receiving the apoptotic leukocyte-containing conditioning and tolerization regimen described herein became euglycemic shortly after transplantation and remained so even after cessation of immunosuppression and exogenous insulin on day 21 post-transplant. Pre- and post-prandial blood glucose levels are shown as lines, and daily insulin is shown as bars. The graphs show restoration of euglycemia (5547 IE / kg with DNA) after intraportal transplantation of 7-day cultured islets and maintenance of euglycemia despite cessation of insulin and immunosuppression on day +21 post-transplant. Figure 9B shows that recipient glycated hemoglobin (HbA1c) levels became and remained normal after transplantation. Figure 9C shows continued weight gain after transplantation. Figure 9D shows that strongly positive post-transplant fasting and random serum C-peptide levels and their increase after stimulation throughout 1-year follow-up in Cohort C monkeys confirmed stable islet allograft function. Figure 9E shows that stable post-transplant blood glucose disappearance rates (Kg) after IV glucose challenge were observed in the recipients, comparable to pre-STZ rates. Figure 9F shows that C-peptide levels derived from matching tests showed a substantial increase of more than 1 ng / mL throughout the post-transplant time course.

[0047] [Figure 10] Figures 10A-10C show histopathological analysis of livers from animals that underwent the apoptotic leukocyte-containing conditioning and tolerization regimen described herein and exhibited stable euglycemia. Figure 10A shows that histopathological analysis revealed several intact islets and no or minimal peri-islet infiltration. Transplanted, intrahepatic islets showed strong positive staining for insulin (Figure 10B); the absence of insulin-positive islet beta cells in the native pancreas at necropsy (Figure 10C) indicated that post-transplant euglycemia reflected graft function and was not due to remission of STZ-induced diabetes.

[0048] [Figure 11] Figures 11A-F show that transplant recipients receiving the disclosed conditioning regimen who did not succumb one year after transplantation demonstrated continued islet allograft function for more than two years after cessation of immunosuppression. Figure 11A provides pre- and post-prandial blood glucose (solid and dashed lines, respectively) and daily insulin (bars). Figure 11B shows positive and stable C-peptide levels (fasting, random, and mixed meal stimulated) throughout follow-up. Figure 11C shows restoration of near-normal HbA1c levels throughout follow-up. Figure 11D shows continued weight gain after transplantation, indicating that post-transplant normoglycemia is not due to a malabsorptive state. Figure 11E shows blood glucose before and after an IV infusion of 0.5 g / kg glucose (IVGTT) and Kg levels before and after diabetes induction and after transplantation. Figure 11F shows the acute C-peptide response to IV glucose (0.5 g / kg).

[0049] [Figure 12] 12 shows that histopathological analysis at necropsy confirmed rejection-free islet allograft survival in monkeys that demonstrated continued islet allograft function for more than two years after cessation of immunosuppression. The monkeys received the conditioning regimen of the present disclosure.

[0050] [Figure 13]Figures 13A-F show that monkeys that did not receive the apoptotic leukocyte-containing conditioning and tolerization regimen described herein exhibited a gradual decline in graft function, particularly evident 4 months after transplantation. Figure 13A shows pre- and post-prandial blood glucose levels (solid and dashed lines, respectively) and daily insulin (bars). Postprandial instability was evident starting at day 133 post-transplant, along with an upward trend, suggesting allograft loss due to rejection. Figure 13B shows that C-peptide levels (fasting, random, and mixed meal-stimulated) became positive after transplantation, with basal levels remaining approximately 1 ng / mL until day 161 post-transplantation. Figure 13C shows the recovery of near-normal HbA1c levels, which then began to rise around day 140 with a continuing upward trend. Figure 13D shows continued weight gain after transplantation, indicating that euglycemia after transplantation is not due to a malabsorptive state. Figure 13E shows blood glucose levels before and after IV infusion of 0.5 g / kg glucose (IVGTT) and Kg levels before and after diabetes induction and post-transplantation. Figure 13F shows the acute C-peptide response to IV glucose (0.5 g / kg).

[0051] [Figure 14]Figures 14A-F show effector cell responses in cohorts B and C at various time points before and after transplantation. Figure 14A shows the percentage of CD4+ T effector memory (TEM) cells in peripheral blood lymphocytes (PBLs) measured before transplantation and longitudinally at 3, 6, and 12 months after transplantation in recipients from cohorts B (n=7) and C (n=5). Figure 14B shows the percentage of CD4+ TEM cells in PBLs, liver mononuclear cells (LMNCs), and lymph nodes (LNs) at termination in recipients from cohorts B (n=3-7) and C (n=2-5). Figure 14C shows the fold change in proliferation (compared to pretransplant levels; naive) of carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled CD4+ T cells in cohorts B and C in response to irradiated donor PBLs at the indicated intervals before and after transplantation in a 6-day mixed lymphocyte reaction (MLR). Figure 14D shows the percentage of circulating CD8+ TEM cells in PBLs from cohorts B (n = 7) and C (n = 5). Figure 14E shows the percentage of CD8+ TEM cells in PBLs, LMNCs, and LNs at termination in recipients from cohorts B (n = 3-7) and C (n = 3-5). Figure 14F shows the fold change in proliferation (compared to pretransplant levels; naive) of CFSE-labeled CD8+ T cells in cohorts B and C in response to irradiated donor PBLs at the indicated intervals before and after transplantation in a 6-day MLR.

[0052] [Figure 15] Figures 15A-D show the frequencies of immune cell subsets in cohorts B and C at various time points pre- and post-transplant. Percentages are shown for circulating follicular helper cells (Tfh, Figure 15A), PD-1+CD4+ T cells (Figure 15B), PD-1+CD8+ T cells (Figure 15C), and CD20+ B cells (Figure 15D).

[0053] [Figure 16]Figures 16A-D show the percentages of immunoregulatory cell subsets in cohorts B and C. Figure 16A shows the percentage of circulating natural suppressor (NS) cells. Figure 16B shows the percentage of circulating Treg cells. Figure 16C shows the percentage of circulating Breg cells. Figure 16D shows the percentage of Breg cells in peripheral blood, liver, and lymph nodes at the time of sacrifice.

[0054] [Figure 17] Figures 17A-E show that ADL as part of the tolerization and conditioning regimen induces MDSC expansion and tolerogenic APCs. Figure 17A shows the gating strategy for MDSC identification. Singlets were first gated to remove doublets and exclude dead cells. MDSCs were identified based on coexpression of CD33 and CD11b among gated CD14+ cells within the Lin-HLA-DR- population, and Lin was used to delineate CD3-CD20- cells. Representative FACS profiles from one Cohort B (top) and one Cohort C (bottom) monkey are shown. Figure 17B shows a significant increase in the percentage of circulating MDSCs in Cohort C monkeys compared to Cohort B monkeys. Figure 17C shows the fold change in MFI of circulating HLA-DR+CD11b+ dendritic cells. Figure 17D shows the fold change in MFI of circulating HLA-DR+CD14+ monocytes. Figure 17E shows the fold change in MFI of circulating HLA-DR+CD20+ B cells. DETAILED DESCRIPTION OF THE INVENTION

[0055] The following discussion of the present disclosure is presented for purposes of illustration and description. It is not intended to limit the present invention to the form or forms disclosed herein. While the description of the present disclosure includes one or more embodiments and descriptions of certain variations and modifications, other variations and modifications are within the scope of the present disclosure, for example, as would be within the skill and knowledge of a person skilled in the art after understanding the present disclosure. It is intended to acquire the right to include, to the extent permitted by law, alternative embodiments that include alternative, interchangeable, and / or equivalent structures, functions, ranges, or steps to the claimed structures, functions, ranges, or steps, regardless of whether such alternative, interchangeable, and / or equivalent structures, functions, ranges, or steps are disclosed herein, and without intending to make any patentable subject matter publicly available.

[0056] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0057] While various features of the present disclosure may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, while the present disclosure may be described herein for clarity in the context of separate embodiments, various aspects and embodiments may also be implemented in a single embodiment.

[0058] The practice of some embodiments disclosed herein will employ, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, within the skill of the art. See, e.g., Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular Biology (FM Ausubel, et al. eds.); the series Methods In Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. Freshney, ed. (2010)). definition

[0059] The following definitions supplement the definitions in the art, and are directed to the current application, and should not be attributed to any related or unrelated cases, for example, any publicly owned patent or application.Any method and material similar or equivalent to the methods and materials described herein can be used in the implementation of the present disclosure for testing, but preferred materials and methods are described herein.Therefore, the terminology used herein is only for the purpose of describing specific embodiments, and is not intended to be limiting.

[0060] In this application, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Furthermore, the use of the term "including," as well as other forms such as "include," "includes," and "included," is not limiting.

[0061] As used herein, the terms "and / or" and "any combination thereof," as well as their grammatical equivalents, can be used interchangeably. These terms can convey that any combination is specifically contemplated. For purposes of explanation only, the following phrases "A, B and / or C" or "A, B, C, or any combination thereof" can mean "individually A, individually B, individually C, A and B, B and C, A and C, and A, B and C."

[0062] The term "or" can be used conjunctively or disjunctively, unless the context specifically dictates its disjunctive use.

[0063] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which may depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more standard deviations, as practiced in the art. Alternatively, "about" can mean within 20%, 10%, 5%, or 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. When particular values ​​are described in this application and claims, unless otherwise stated, the term "about" should be assumed to mean within an acceptable error range for the particular value.

[0064] As used in the specification and claims, the words "comprising" (and any form of "comprising," such as "comprise" and "comprises"), "having" (and any form of "having," such as "have" and "has"), "including" (and any form of "including," such as "includes" and "include"), or "containing" (and any form of "containing," such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, the compositions of the invention can be used to achieve the methods of the invention.

[0065] References herein to "some embodiments," "one embodiment," "one embodiment," or "other embodiments" mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least some embodiments of the present invention, but may not necessarily be included in all embodiments of the present invention.

[0066] As used herein, the term "transplantation" and its grammatical equivalents encompass any procedure involving the transplantation, implantation, or infusion of cells, tissues, or organs from a donor to a recipient. Non-limiting exemplary types of transplants include autotransplants, autografts, allotransplants, allografts, syngeneic transplants, syngeneic grafts, xenotransplants, xenografts, as well as split grafts and domino transplants. In one embodiment, the term "split graft" encompasses any procedure involving the transplantation of cells, organs, tissues, or even specific proteins from a donor that are split between two or more recipients.

[0067] As used herein, the terms "autotransplantation," "autograft," "autograft," or grammatically equivalent terms encompass any procedure involving the transplantation of organs, tissues, cells, or even specific proteins or expression products from one part of the body into another part of the same subject. In one embodiment, the subject is a member of the superorder Laurasiatheria. In one embodiment, the subject is an ungulate, such as a pig, giraffe, camel, deer, or cow. In one embodiment, the subject is a human or non-human primate. Autologous tissue (also called autogenous, autogeneic, or autogenic tissue) transplanted by such a procedure is called an autograft or autograft.

[0068] As used herein, the terms "allograft," "allograft," "allograft," or their grammatical equivalents encompass any procedure involving the transplantation, implantation, or infusion of cells, tissues, or organs into a recipient, where the recipient and donor are of the same species. In one embodiment, the recipient and / or donor are members of the superorder Laurasiatheria. In one embodiment, the recipient and / or donor are ungulates, such as pigs, giraffes, camels, deer, or cattle. In one embodiment, the cells, tissues, or organs described herein are transplanted into a human or non-human primate. Allografts include, but are not limited to, vascularized allografts, partially vascularized allografts, non-vascularized allografts, allograft dressings, allograft bandages, and allograft structures. In some cases, the allograft is a syngeneic graft or isograft, in which an organ or tissue is transplanted from a donor to a genetically identical recipient (such as an identical twin).

[0069] As used herein, the terms "xenotransplantation," "xenograft," or "xenograft," or grammatical equivalents, encompass any procedure involving the transplantation, implantation, or infusion of cells, tissues, or organs into a recipient, where the recipient and donor are of different species. In one embodiment, the recipient and / or donor are members of the superorder Laurasiatheria. In one embodiment, the recipient and / or donor are ungulates, such as pigs, giraffes, camels, deer, or cattle. In one embodiment, the donor is a pig and the recipient is a human or non-human primate. In one embodiment, the cells, tissues, or organs described herein are transplanted into a human or non-human primate. Xenotransplants include, but are not limited to, vascularized xenografts, partially vascularized xenografts, non-vascularized xenografts, xenodressings, xenobandages, and nanostructures.

[0070] As used herein, the term "graft rejection" and its grammatical equivalents may refer to one or more processes in which an organ transplant recipient's immune response mounts a reaction against the transplanted material (e.g., cells, tissues and / or organs) sufficiently to impair or destroy the function of the transplanted material.

[0071] As used herein, the term "hyperacute rejection" and its grammatical equivalents may refer to rejection of transplanted material or tissue that occurs or begins within the first 24 hours after transplantation. For example, hyperacute rejection may include, but is not limited to, "acute humoral rejection" and "antibody-mediated rejection."

[0072] As used herein, the term "antibody" refers to whole antibodies, including IgG (including IgG1, IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2), IgD, IgE, or IgM, and IgY, including single-chain whole antibodies, and antigen-binding (Fab) fragments thereof. Antigen-binding antibody fragments include, but are not limited to, Fab, Fab', and F(ab'), Fd (consisting of VH and CH1), single-chain variable fragments (scFv), single-chain antibodies, disulfide-linked variable fragments (dsFv), and fragments containing either the VL or VH domain. Antibodies can be derived from any animal origin. Antigen-binding antibody fragments, including single-chain antibodies, can contain the variable region alone or in combination with all or part of the following: hinge region, CH1, CH2, and CH3 domains. Any combination of the variable region and hinge region, CH1, CH2, and CH3 domains is also included. The antibodies can be monoclonal, polyclonal, chimeric, humanized, and human monoclonal and polyclonal antibodies.

[0073] As used herein, the terms "improve," "enhance," "enhance," and grammatical equivalents may refer to any improvement recognized by one of ordinary skill in the art. For example, improving transplantation may mean reducing hyperacute rejection, which may encompass a decrease, relief, or attenuation of undesirable effects or symptoms.

[0074] As used herein, the terms "pancreatic islet," "pancreatic islet cell," "pancreatic islet equivalent," "pancreatic islet-like cell," "pancreatic islet," "native pancreatic islet cell," "non-native pancreatic islet cell," "pancreatic islet-like cell culture," and their grammatical equivalents refer to endocrine (e.g., hormone-producing) cells present in the pancreas of an organism, or cells that mimic one or more functions of cells present in the pancreas of an organism. For example, pancreatic islet cells can include different types of cells, including, but not limited to, pancreatic alpha cells, pancreatic beta cells, pancreatic delta cells, pancreatic F cells, and / or pancreatic epsilon cells. Pancreatic islet cells can also refer to a group of cells, cell clusters, and the like, including cells cultured in vitro. In some embodiments, pancreatic islet cells are extracted from an islet donor and implanted or transplanted into a predetermined site in an islet recipient for differentiation, expansion, and vascularization to form a therapeutic beta cell mass by the methods, systems, and / or reagents described herein. In one embodiment, the predetermined site is the kidney subcapsular space of the islet recipient. In some embodiments, pancreatic islet cells extracted from a donor are implanted or transplanted into a recipient with the support of transient immunosuppression. Islets of Langerhans are regions of the pancreas that contain endocrine (e.g., hormone-producing) cells (e.g., beta cells). In some embodiments, provided herein are neonatal islet clusters (NICCs) or neonatal porcine islets (NPIs) comprising pancreatic lineage cells (e.g., beta-like cells or cell populations containing beta-like cells) isolated from a donor by a method, system, and / or reagent described herein. In some embodiments, NICCs or NPIs are extracted from an islet donor and implanted or transplanted into a predetermined site in an islet recipient for differentiation, expansion, and vascularization to form a therapeutic beta cell mass by a method, system, and / or reagent described herein. In one embodiment, the predetermined site is the kidney subcapsular space of the islet recipient. In some embodiments, NICCs or NPIs extracted from a donor are implanted or transplanted into a recipient with the support of transient immunosuppression. In some embodiments, the pancreatic islet cells can be stem cell-derived pancreatic islet cells, induced pluripotent stem cell-derived pancreatic islet cells, transdifferentiated or replacement pancreatic islet cells."Donor" is meant to include any mammalian organism, human or non-human, that can serve as a source of donor tissue or cells for transplantation and / or to induce donor cell tolerance. Non-human mammals include, but are not limited to, ungulates such as ungulates (e.g., pigs, peccaries, hippopotamuses, camels, llamas, mouse deer (mouse deer), deer, giraffes, pronghorns, antelopes, capricorn-antelopes (including sheep, goats, etc.), or cattle) or perissodactyls (e.g., horses, tapirs, and rhinoceroses), non-human primates (e.g., monkeys or chimpanzees), canines (e.g., dogs), or cats. The non-human animal may be a member of the superorder Laurasiatheria. The donor may be a living donor or a cadaveric donor. In some cases, the donor is a living donor. In some cases, the donor is a cadaveric donor. The cadaveric donor may be, for example, a brain-dead, heart-beating donor (BDD). The cadaveric donor may be, for example, a non-heart-beating donor (NHBD). Whether the donor is a living donor or a cadaveric donor (e.g., BDD or NHBD), the donor may be from any animal, for example, a human or a non-human animal. The donor may be at any stage of development, including, but not limited to, fetal, perinatal, neonatal, pre-weaning, post-weaning, child, young adult, or adult. The donor of cells used in the preparation of a tolerization vaccine or conditioning regimen may be fully or partially MHC (major histocompatibility complex)-matched with the transplant donor (e.g., the donor of cells, tissue, or organs used for transplantation). In some cases, the partially matched donor is haploidentical with the transplant donor. In some cases, the partially matched donor shares one or more MHC alleles with the transplant donor. For example, a partially matched donor can share with the transplant donor one or more of MHC class IA alleles, MHC class IB alleles, MHC class IC alleles, MHC class II DR alleles, MHC class II DQ alleles, MHC class II DP alleles, or combinations thereof.In some cases, the partially matched donor may share with the transplant donor one or more of the following: MHC class IA alleles, MHC class IB alleles, MHC class II DR alleles, MHC class II DQ alleles, MHC class II DP alleles, or a combination thereof. The partially matched donor may share one DR allele with the transplant donor.

[0075] The "recipient" may be a human or non-human animal that can receive, is receiving, or has received a transplant graft, and the tolerization vaccines, conditioning regimens for transplantation, and / or other compositions provided herein. The recipient may also require a transplant graft, tolerization vaccines, conditioning regimens for transplantation, and / or other compositions provided herein. In some cases, the recipient may be a human or non-human animal that can receive, is receiving, or has received a transplant graft. In some cases, the recipient may be a human or non-human animal that can receive, is receiving, or has received the tolerization vaccines or conditioning regimens for transplantation described herein.

[0076] As used herein, the term "non-human animal" and its grammatical equivalents include all animal species other than humans, including non-human mammals, which may be native or genetically modified non-human animals. Non-human mammals include ungulates such as ungulates (e.g., pigs, peccaries, hippopotamuses, camels, llamas, mouse deer (mouse deer), deer, giraffes, pronghorns, antelopes, caprine-antelopes (including sheep, goats, etc.), or cattle) or perissodactyls (e.g., horses, tapirs, and rhinoceroses), non-human primates (e.g., monkeys or chimpanzees), canines (e.g., dogs), or cats. The non-human animal may be a member of the superorder Laurasiatheria. The superorder Laurasiatheria may include the group of mammals described in Waddell et al., Towards Resolving the Interordinal Relationships of Placental Mammals. Systematic Biology 48 (1): 1-5 (1999). Members of the superorder Laurasiatheria may include Eulipotyphla (hedgehogs, shrews, and moles), Perissodactyla (rhinoceroses, horses, and tapirs), Carnivora (carnivores), Cetartiodactyla (artiodactyla and cetaceans), Chiroptera (bats), and Pholidota (pangolins). Members of the superorder Laurasiatheria may be ungulates as described herein, such as perissodactyla or even-ungulates. The ungulate may be a pig. The member may be a member of Carnivora, such as a cat or a dog. In some cases, the member of the superorder Laurasiatheria may be a pig.

[0077] As used herein, the terms "porcine," "porcine animal," "pig," and "swine," as well as grammatical equivalents thereof, may refer to animals of the genus Sus within the Suidae family of ungulates. For example, the pig may be a wild pig, a domestic pig, a miniature pig, a Sus scrofa pig, a Sus scrofa domesticus pig, or an inbred pig.

[0078] The term "fetal animal" and its grammatical equivalents can refer to any unborn offspring of an animal. In some cases, pancreatic cells or tissues are isolated from 6-week-old embryonic pigs for transplantation. The term "perinatal animal" and its grammatical equivalents can refer to an animal just before or just after birth. For example, the perinatal period can begin at 20-28 weeks of gestation and end 1-4 weeks after birth. The term "neonatal animal" and its grammatical equivalents can refer to any newly born animal. For example, a neonatal animal can be an animal within one month of birth. The term "preweaned non-human animal" and its grammatical equivalents can refer to any animal before it has stopped breastfeeding. The term "baby animal" and its grammatical equivalents can refer to any animal before it has become a young adult animal. For example, a juvenile pig can refer to any pig that is 2 years old or younger.

[0079] As used herein, the terms "genetically modified," "genetically engineered," "transgenic," "genetically modified," and grammatical equivalents refer to having one or more alterations of a nucleic acid, e.g., a nucleic acid in the genome of an organism. For example, genetic modification can refer to the alteration, addition, and / or deletion of a gene. A genetically modified cell can also refer to a cell having an added, deleted, and / or altered gene. A genetically modified cell can be derived from a genetically modified non-human animal. A genetically modified cell derived from a genetically modified non-human animal can be a cell isolated from such a genetically modified non-human animal. A genetically modified cell derived from a genetically modified non-human animal can be a cell originating from such a genetically modified non-human animal. A genetically modified cell or animal can contain an added or integrated transgene or other foreign DNA, or an altered endogenous gene, including targeted, recombined, interrupted, deleted, disrupted, replaced, suppressed, enhanced, or otherwise altered, to mediate a genotypic or phenotypic effect in at least one cell of the animal, and typically in at least one germline cell of the animal.

[0080] As used herein, the term "transgene" and its grammatical equivalents can refer to a gene or genetic material that can be transferred into an organism. For example, a transgene can be a section or segment of DNA containing a gene that is introduced into an organism. The gene or genetic material can be from a different species. The gene or genetic material can be synthetic. When a transgene is transferred into an organism, the resulting organism can be referred to as a transgenic organism. A transgene can retain its ability to produce RNA or a polypeptide (e.g., a protein) in the transgenic organism. A transgene can comprise a polynucleotide encoding a protein or a fragment (e.g., a functional fragment) thereof. The transgene polynucleotide can be an exogenous polynucleotide. A fragment (e.g., a functional fragment) of a protein can comprise at least, or at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the amino acid sequence of the protein. A fragment of a protein can be a functional fragment of the protein. A functional fragment of a protein can retain some or all of the function of the protein. The exogenous polypeptide can encode an exogenous protein or a functional fragment thereof.

[0081] As used herein, the terms "exogenous nucleic acid sequence," "exogenous polynucleotide," and grammatical equivalents thereof, may refer to a gene or genetic material that originates outside of a cell or animal and is transferred into a cell or animal. An exogenous nucleic acid sequence may be synthetically produced. An exogenous nucleic acid sequence may be derived from a different species, or from a different member of the same species. An exogenous nucleic acid sequence may be another copy of an endogenous nucleic acid sequence.

[0082] The term "gene knockout" or "knockout" can refer to any genetic modification that reduces expression of the gene being "knocked out." Reduced expression can include no expression. The genetic modification can include a genome disruption.

[0083] As used herein, the term "disrupt" and its grammatical equivalents may refer to the process of modifying a gene, for example, by deletion, insertion, mutation, transposition, or any combination thereof. For example, a gene may be disrupted by knockout. Disruption of a gene may be to partially reduce or completely suppress the expression of the gene (for example, mRNA and / or protein expression). Disruption may also include inhibitory techniques such as shRNA, siRNA, microRNA, dominant negative, or any other means of inhibiting the function or expression of a gene or protein.

[0084] As used herein, the term "gene editing" and its grammatical equivalents can refer to genetic manipulation in which one or more nucleotides are inserted, replaced or removed from a genome.For example, gene editing can be performed using nucleases (for example, naturally occurring nucleases or artificially engineered nucleases).

[0085] As used herein, the term "guide RNA" and its grammatical equivalents may refer to an RNA that can be specific to a target DNA and can form a complex with a Cas protein. The RNA / Cas complex can help "guide" the Cas protein to the target DNA.

[0086] As used herein, the term "condition" and its grammatical equivalents may refer to a disease, an event, or a change in health status.

[0087] As used herein, the term " diabetes mellitus " and its grammatical equivalents can refer to the disease characterized by long-term high blood sugar level.For example, as used herein, the term " diabetes mellitus " and its grammatical equivalents can refer to all or any type of diabetes, including but not limited to type 1, type 2, type 3c (including cystic fibrosis-related, and surgical, and hemochromatosis-related pancreatic diabetes), gestational diabetes and monogenic diabetes (such as HNF1A-MODY, GCK-MODY 2), and other forms of mitochondrial diabetes.In some cases, diabetes can be a form of hereditary diabetes.

[0088] As used herein, the term "phenotype" and its grammatical equivalents can refer to the composite of observable characteristics or traits of an organism, such as its morphology, development, biological or physiological properties, phenology, behavior, and products of behavior. Depending on the context, the term "phenotype" can sometimes refer to the composite of observable characteristics or traits of a population.

[0089] Some numerical values ​​disclosed throughout are referred to as, for example, "X is at least, or at least about 100, or 200 [or any numerical value]." This numerical value includes the number itself and all of the following: i) X is at least 100; ii) X is at least 200; iii) X is at least about 100; and iv) X is at least about 200.

[0090] All these different combinations are contemplated by the numerical values ​​disclosed throughout. All disclosed numerical values, whether referring to administration of therapeutic agents or to days, months, years, body weight, dosages, etc., shall be construed in this manner unless specifically indicated to the contrary.

[0091] Ranges disclosed throughout are sometimes referred to, for example, as "X is administered on days 1-2 or 2-3, or on about days 1-2 or about days 2-3 [or any number range]." The ranges include the numbers themselves (e.g., the endpoints of the range), as well as all of the following: i) X is administered on days 1-2; ii) X is administered on days 2-3; iii) X is administered on about day 1-2; iv) X is administered on about day 2-3; v) X is administered on day 1 to about day 2; vi) X is administered on day 2 to about day 3; vii) X is administered on about day 1 to about day 2; and viii) X is administered on about the second to third day.

[0092] All these different combinations are contemplated by the ranges disclosed throughout. All disclosed ranges, whether referring to administration of therapeutic agents or to days, months, years, body weight, dosages, etc., shall be construed in this manner unless specifically indicated to the contrary. transplant immunomodulation

[0093] Described herein are compositions, systems, and methods for inducing graft tolerance. In particular, the present disclosure relates to administering a tolerizing vaccine or conditioning regimen before, during, or after administration of donor transplant cells, tissues, or organs. The tolerizing vaccine or conditioning regimen can induce tolerance to an allograft or xenograft in the graft recipient.

[0094] Organs, tissues, or cells can be differentiated from stem cells, grown de novo, or isolated from an animal (e.g., a human or non-human animal) and transplanted into a recipient in need of a transplant from the same species (allograft) or a different species (xenotransplant). The donor of the organ, tissue, or cell can be referred to herein as a transplant donor. The transplanted organ, tissue, or cell can be referred to herein as a transplant or graft.

[0095] The donor of the transplant or graft can be any animal, including humans and non-human animals. In some cases, the donor is a human. In some cases, the transplant donor is a non-human animal. The transplant donor (e.g., a non-human animal donor) can be genetically modified, for example, to reduce or eliminate the possibility that the transplant or graft can be recognized by the recipient's immune system, or to reduce the immune response by the recipient's immune system when recognizing the transplant or graft.

[0096] The donor of the transplant or graft can be a living donor or a deceased donor. In some cases, the transplant donor is a living donor. In some cases, the transplant donor is a deceased donor. The deceased donor can be a brain-dead, heart-beating donor (BDD). Alternatively, the deceased donor can be a non-heart-beating donor (NHBD). Regardless of whether the transplant donor is a living donor or a deceased donor (e.g., BDD or NHBD), the donor can be from any animal, for example, a human or a non-human animal.

[0097] The transplant or graft donor can be of any age or stage of development. For example, the transplant donor can be a fetal, perinatal, neonatal, pre-weaning, post-weaning, child, young adult, or adult human or animal.

[0098] The transplant or graft can be used to treat a disease or disorder in a recipient in need thereof. Suitable diseases that can be treated include any disease in which the recipient's organ, tissue, or cells are defective or damaged, and the recipient can be treated by transplanting an organ, tissue, or cell (e.g., kidney, heart, lung, liver, vein, skin, endocrine pancreas, pancreatic islet cells, or a combination thereof). In some cases, the transplant includes kidney, liver, heart, lung, pancreas, endocrine pancreas, pancreatic islet cells, small intestine, bone marrow, hematopoietic stem cells, cells derived from embryonic or induced pluripotent stem cells, such as pancreatic islet beta cells or hepatocytes, embryonic or induced pluripotent stem cell-derived pancreatic islets, embryonic or induced pluripotent stem cell-derived hepatocytes, or a combination thereof. Graft / transplant rejection

[0099] Transplant / graft rejection can involve recognition of donor-specific antigens, for example, donor-specific antigens presented to T cells by host antigen-presenting cells (indirect) or donor antigen-presenting cells (direct). T cell activation in response to donor-specific antigens can lead to a pro-inflammatory response and graft rejection.

[0100] Transplant / graft rejection can be prevented by methods that moderate immune responses, including the methods described herein.The tolerization vaccine or pretreatment regimen of the present disclosure can be used to prevent or delay transplant rejection, for example, by reducing pro-inflammatory immune responses to the transplant and / or enhancing tolerance-promoting immune responses.In some cases, the tolerization vaccine or pretreatment regimen of the present disclosure can avoid the need for long-term immunosuppression of the recipient.

[0101] Graft rejection (e.g., T cell-mediated graft rejection) can be prevented by chronic immunosuppression with one or more immunomodulatory molecules. However, immunosuppression is costly and associated with the risk of serious side effects. To avoid the need for chronic immunosuppression, multifaceted T cell-targeted rejection prevention has been developed, such as those described in International Publication No. WO 2016 / 094679, U.S. Patent Application Publication No. 20160165861, and International Publication No. WO 2017 / 218714, each of which is incorporated herein by reference in its entirety. In some embodiments, T cell-targeted rejection prevention utilizes (i) genetically modified grafts lacking functional expression of MHC class I, thereby preventing direct specific activation of CD8+ T cells and preventing the cytolytic effector function of these CD8+ T cells; (ii) B cell (and other APC)-mediated T cell priming and antagonistic anti-CD40 Prevent memory development of anti-donor T cells using induction immunotherapy including mAbs, B cell targeting agents (e.g., that biodeplete B cells, e.g., that biotarget CD20, CD19, or CD22, and / or that biomodulate B cells, e.g., that biotarget BAFF, BAFF / APRIL, CD40, IgG4, ICOS, IL-21, B7RP1), mTOR inhibitors, TNF-alpha inhibitors, IL-6 inhibitors, nitrogen mustard alkylating agents (e.g., cyclophosphamide), complement C3 or C5 inhibitors, or combinations thereof, and / or (iii) deplete anti-donor T cells with indirect specificity via pre- or post-transplant infusion of an apoptotic donor cell vaccine (i.e., tolerization vaccine). In some cases, the B cell targeting agent can be an anti-CD20 mAb, rituximab, and / or a B cell-depleting antibody.

[0102] In some embodiments, the methods described herein for preventing transplant rejection or extending the time to transplant rejection, with or without minimal use of immunosuppressants (e.g., one or more immunomodulatory molecules), include using a genetically modified animal as a cell, organ, or tissue donor. Cells, organs, and / or tissues from the modified donor animal, e.g., a non-human donor animal, can be harvested and used in allografts or xenografts. Alternatively, cells, organs, or tissues can be extracted from the animal and used to generate genetically modified cells, organs, or tissues. In some cases, primary cells can be extracted from the animal and used to generate genetically modified cells. In some cases, cells, organs, or tissues derived from the animal (e.g., cell lines) can be used to generate genetically modified cells, organs, or tissues.

[0103] Transplant rejection can also be reduced or eliminated by inducing tolerance to the transplant or graft using tolerizing vaccines or conditioning regimens. Tolerance induction

[0104] Provided herein are methods and regimens for inducing tolerance to cell, tissue, or organ transplants. As used herein, the term "tolerance" or "immune tolerance" refers to a state in which the immune system is unresponsive to a substance or tissue capable of eliciting an immune response. The compositions, conditioning regimens, or tolerizing regimens of the present disclosure are useful for achieving tolerance or partial tolerance to a transplant upon transplantation of the transplant. As used herein, "partial tolerance" refers to partial immune tolerance that results in a reduced or inhibited immune response. In some embodiments, provided herein are methods and regimens for inhibiting an immune response and / or preventing the onset or development of GVHD. As used herein, the term "immune response" includes T cell-mediated and / or B cell-mediated immune responses. Exemplary immune responses include T cell responses, e.g., cytokine production and cytotoxic activity. In addition, the term immune response includes immune responses indirectly mediated by T cell activation, e.g., antibody production (humoral response), and activation of cytokine-responsive cells, e.g., macrophages. The immune cells involved in immune response include lymphocytes such as B cells and T cells (CD4+, CD8+, Th1 and Th2 cells); antigen-presenting cells (e.g., professional antigen-presenting cells such as dendritic cells); natural killer cells; myeloid cells such as macrophages, eosinophils, mast cells, basophils and granulocytes. For example, immune response is involved in transplant rejection and the accompanying physiological consequences of such immune response, such as interstitial fibrosis, chronic graft arteriosclerosis or vasculitis.

[0105] Thus, in comparison to subjects who are not administered the apoptotic leukocytes and other compositions, or conditioning or tolerizing regimens, disclosed herein, subjects who are administered the apoptotic cells, such as leukocytes and other compositions, or conditioning or tolerizing regimens, disclosed herein, exhibit, for example, a) a reduced level of immune response against the graft (believed to be mediated at least in part by a B-cell mediated immune response, more particularly donor-specific antibodies), b) a delayed initiation or progression of an immune response against the graft, c) a reduced risk of initiation or progression of an immune response against the graft, or d) an inhibition of or a reduced risk of the onset of GVHD. As used herein, the term "preventing or reducing graft rejection" is intended to encompass preventing or inhibiting immune graft rejection and delaying the onset or progression of immune graft rejection. This term is also intended to encompass extending a patient's graft survival or reversing graft failure. Furthermore, this term is intended to encompass ameliorating symptoms of immune graft rejection, including ameliorating immunological complications associated with immune rejection, such as interstitial fibrosis, chronic graft arteriosclerosis, or vasculitis. Thus, "inducing tolerance" can refer to changes in immune cell levels (e.g., an increase in the number of tolerogenic APCs, an increase in the number of Tregs, an increase in the number of Tr1 cells, a decrease in CD4+, CD8+, and / or CD20+ cells), changes in the levels of immunoregulatory molecules (e.g., an increase in IL-10 and TGF-β), or a combination thereof. In some embodiments, modulating an immune response can be suppression of an immune response or immunosuppression. As used herein, the terms level, number, count, and concentration can be used interchangeably. As used herein, the term "immunomodulatory molecule" refers to any molecule that can cause proliferation or activation of cells of the immune system of a subject. Such molecules include, but are not limited to, prostaglandin E2 (PGE2), transforming growth factor-β (TGF-β), indoleamine 2,3-dioxygenase (IDO), nitric oxide, hepatocyte growth factor (HGF), interleukin 6 (IL-6), and interleukin 10 (IL-10).

[0106] Those skilled in the art will understand that both the cell culture system and the subject's immune system contain basal levels of immune cells and immunoregulatory molecules. The terms basal level and normal level can be used interchangeably. As used herein, the basal level of an immune cell type or immunoregulatory molecule refers to the average number of cell types or immunoregulatory molecules present in an individual population under a certain reference state (e.g., in a healthy individual before transplantation, before administration of apoptotic cells such as the leukocyte composition or conditioning regimen or tolerization regimen disclosed herein, or before administration of immunosuppressive therapy, or a combination thereof), or the basal level of an immune cell type or immunoregulatory molecule refers to the average level of cell types or immunoregulatory molecules present in a population of unactivated cells. Those skilled in the art can determine the level of specific immune cells in such a population of cells or in a biological sample. As used herein, the term "biological sample" has its general meaning in the art and refers to any sample that can be obtained from a subject (e.g., a transplant recipient) for in vitro evaluation. A preferred biological sample is a blood sample (e.g., a whole blood sample, a serum sample, or a plasma sample).

[0107] Methods for measuring immune cells are well known in the art, including, for example, methods based on identifying the expression of specific surface marker proteins by flow cytometry. The level of immune cells can be measured, for example, by measuring proliferation by 3H-thymidine incorporation, bromodeoxyuridine incorporation (BrdU), ATP emission, fluorescent dye reduction (carboxyfluorescein succinimidyl ester (CFSE)-like dye); cytokine measurement, for example, using a Multi-Analyte ELISArray kit, a bead-based multiplex assay; measuring surface antigen expression, for example, by flow cytometry; measuring cytotoxicity, for example, by Two-Label flow cytometry, calcein AM dye release, luciferase-transduced target, or annexin V. Methods for measuring T cell responses and B cell responses are well known in the art, and are described, for example, in Expert Rev. Vaccines 9(6), 595-600 (2010), mBio. 2015 Jul-Aug; 6(4).

[0108] A reference or basal level of a cell or molecule can be a specific amount (e.g., a specific concentration) or can encompass a range of amounts. Basal levels or ranges of immune cells and immunoregulatory molecules are known to those skilled in the art. For example, in healthy individuals, the normal level of CD4+ T cells present in human blood is 500-1500 cells / ml. Basal levels of cells can also be reported as a percentage of the total cell population.

[0109] The number and function of immune cells may be monitored by assays that detect immune cells by activities such as cytokine production, proliferation, or cytotoxicity. For example, lymphoproliferation assays, which assay the ability of T cells to proliferate in response to an antigen, can be used as an indicator of the presence of antigen-specific CD4+ helper T cells. Typically, purified T cell or PBMC samples are mixed with various dilutions of antigen, or with antigen in the presence of stimulator cells (irradiated autologous or HLA-matched antigen-presenting cells). After 72-120 hours, [H]thymidine is added, and DNA synthesis (as a measure of proliferation) is quantified using a gamma counter that measures the amount of radiolabeled thymidine incorporated into DNA. A stimulation index can be calculated by dividing the number of cpm for the sample by the number of counts per minute in the control. Proliferation assays can be used to compare T cell responses before and after treatment with the compositions of the present disclosure. Another example of an assay that can be used to detect the proliferation of immune cells (e.g., T cells, B cells) is the use of intracellular fluorescent dyes, such as 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) in a mixed lymphocyte reaction (CFSE-MLR) and the determination of proliferating cells using flow cytometry. Another example of an assay that can be used is the detection of secreted cytokines by ELISA and ELISPOT assays.

[0110] Cytokine secretion by immune cells in a subject (e.g., in response to a transplant) may be detected by measuring either bulk cytokine production (by ELISA) or enumerating individual cytokines produced by immune cells (by ELISPOT assay). Macrophage activation can be determined, for example, by measuring the levels of chemokines such as IL-8 / CXCL8, IP-10 / CXCL10, MIP-1alpha / CCL3, MIP-1beta / CCL4, and RANTES / CCL5, which are released as chemoattractants for neutrophils, immature dendritic cells, natural killer cells, and activated T cells. The levels of released pro-inflammatory cytokines, including IL-1beta / IL-1F2, IL-6, and TNF-alpha, or anti-inflammatory cytokines, including IL-10 and TGF-β, can also be measured by assays well known in the art.

[0111] Provided herein are compositions, methods, and systems for inducing immune tolerance to a transplant or graft. The compositions, methods, and systems disclosed herein can include a tolerizing vaccine or a conditioning regimen including a tolerizing vaccine.

[0112] The benefits of transplantation are generally reduced by the infectious, metabolic, and malignant burdens of chronic immunosuppression that result. Chronic graft rejection remains a challenge in achieving long-term transplant success; however, the use of long-term immunosuppression (e.g., one or more immunomodulatory molecules) can be associated with significant risks, such as infection and drug toxicity. The compositions and methods of the present disclosure can induce long-term tolerance to the graft while avoiding long-term immunosuppression. The induction of stable donor-specific immune tolerance can eliminate the need for maintenance immune suppression. The induction of graft tolerance can improve transplant outcomes.

[0113] Induction of antigen-specific and stable tolerance to autoimmunity and allergy can be established through the intravenous delivery of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI)-crosslinked antigens to the surface of syngeneic apoptotic cells, such as leukocytes or mesenchymal stromal cells. In allograft models, intravenous infusion of ECDI-treated ADLs (apoptotic donor leukocytes) on days -7 and +1 before and after transplantation can effectively and safely induce long-lasting tolerance to minor antigen-mismatched skin grafts, completely MHC-mismatched pancreatic islet allografts, and even cardiac allografts when combined with short-term mTOR inhibitors (e.g., rapamycin, sirolimus, everolimus). IV infusion of ECDI-fixed apoptotic donor splenocytes has been shown to induce donor-specific tolerance to grafts such as pancreatic islets and cardiac allografts. After IV infusion, donor ECDI-fixed splenocytes can undergo rapid apoptosis, and after uptake of apoptotic bodies, maturation is arrested and they can be rapidly internalized by splenic dendritic cells (DCs), which selectively upregulate negative but not positive costimulatory molecules. Upon encountering such recipient DCs, T cells with indirect allospecificity rapidly expand, followed by significant clonal contraction, and the remaining T cells sequester in the spleen without transport to the graft or graft-draining lymph nodes. Donor ECDI-fixed splenocytes that are not internalized by host phagocytes can weakly activate T cells with direct allospecificity, rendering them resistant to subsequent stimulation (anergy). ECDI-fixed splenocytes can also induce / expand Tregs and myeloid-derived suppressor cells (MDSCs). Thus, the mechanisms of graft protection in these models may involve deletion, anergy, and modulation of T cell direct and indirect specificities. This antigen-specific tolerance strategy can both prevent priming of naive T cells and effectively control pre-existing memory / effector CD4+ and CD8+ T cell responses.

[0114] Strong tolerance to allografts can be achieved using a negative vaccination strategy without the need for same-donor bone marrow or hematopoietic stem cell transplantation associated with intensive conditioning regimens. An exemplary protocol involves same-donor islet allograft transplantation on day 0, followed by administration of a first dose of ECDI-fixed ADL, e.g., on day -7, to preempt the intended recipient's quiescent immune system, followed by a second dose of ADL, e.g., on day +1. Tolerance can be achieved and maintained, e.g., for ≥1 year, in unsensitized recipients. ADL infusions, added to short-term immunosuppression, also significantly alter the host's alloantigen-specific effector and regulatory immunity.

[0115] In one exemplary embodiment, a tolerizing vaccine or conditioning regimen of the present disclosure can include administering apoptotic donor leukocytes to a recipient and short-term immunosuppression including one or more of: (i) an mTOR inhibitor, (ii) an anti-tumor necrosis factor agent or anti-tumor necrosis factor receptor agent, (iii) an anti-interleukin-6 agent or anti-interleukin-6 receptor agent, and (iv) an anti-CD40 agent or anti-CD40 ligand agent. The short-term administration of these agents along with the apoptotic donor leukocytes disclosed herein can promote long-term tolerance to the transplanted cells, organ, or tissue, despite the recipient not being administered long-term maintenance immunosuppression.

[0116] In one exemplary embodiment, short-term immunosuppression comprises administering an mTOR inhibitor, an anti-tumor necrosis factor agent, an anti-interleukin-6 receptor agent, and an anti-CD40 agent. Short-term administration of these agents, along with the apoptotic donor leukocytes disclosed herein, can promote long-term tolerance to the transplanted cells, organ, or tissue, despite the recipient not being administered long-term maintenance immunosuppression.

[0117] In one exemplary embodiment, short-term immunosuppression comprises administering an mTOR inhibitor, a soluble anti-tumor necrosis factor receptor, an anti-interleukin-6 receptor antibody, and an antagonistic anti-CD40 antibody. Short-term administration of these agents together with the apoptotic donor leukocytes disclosed herein can promote long-term tolerance to the transplanted cells, organ, or tissue, despite the recipient not being administered long-term maintenance immunosuppression. Ex vivo leukocyte expansion

[0118] To translate the disclosed tolerance induction strategies into the clinical setting for living donor allotransplantation (e.g., kidney or pancreatic islet allotransplantation), clinically applicable sources of leukocytes, such as donor leukocytes or stem cell-derived / differentiated leukocytes, can be identified as effective for inducing donor-specific tolerance to the proposed allograft, for example, in short-term immunosuppressed recipients. Harvesting the spleen of a living donor as a source of donor leukocytes can compromise the donor's ability to fight off infection; therefore, alternative sources of donor leukocytes may be preferable. For example, B lymphocytes can be harvested from the donor in one or more blood collection and / or apheresis procedures and expanded ex vivo as needed, or a separate cell donor can be identified that is appropriately matched or partially matched to the transplant donor.

[0119] In some cases, separate donors can be used for tolerogenic leukocytes and allografts. For example, splenocytes from a cadaveric donor that is fully or partially matched (e.g., haploidentical) with the intended living donor are a clinically viable source of tolerogenic leukocytes. Once a matched, partially matched, or haploidentical spleen becomes available, a tolerization protocol can begin with an infusion of splenocytes, e.g., on day −7, followed by a living donor transplant (e.g., kidney transplant), e.g., on day 0, and an infusion of ex vivo-expanded splenic B cells, e.g., on day +1. Tolerance induction, enabled by the pre- and post-transplant infusion of splenocytes and / or expanded splenic B cells prepared from a cadaveric donor that shares a single MHC class I and II haplotype with the living donor, can rely on linkage suppression. Regulatory cells with specificity for these self-antigens can be activated and expanded by infusion of ADLs with shared haplotypes, and can prolong their effect of suppressing rejection responses directed against antigens from incompatible living donors due to their co-presentation with the shared antigen in antigen-presenting T cells of the same host, a phenomenon called linkage suppression.

[0120] For use in human allograft recipients, for example, a vaccine can be prepared from the spleen of a cadaveric donor on day -7 and infused into the prospective islet transplant recipient on the same day. This is possible in the context of islet transplantation, since isolated islets can be maintained in culture for, for example, 7 days before transplantation. A vaccine on day +1 can be based on ex vivo expanded B cells derived from an aliquot of the cadaveric donor's spleen. For use in porcine-human xenotransplantation, either kidney, neonatal porcine islets, or combined kidney-islet xenografts, apoptotic donor leukocytes can be derived from the graft donor if available in sufficient numbers or from a clonal porcine donor, and can be expanded ex vivo if necessary.

[0121] Cells (e.g., leukocytes) used in the preparation of a tolerizing vaccine / regimen or conditioning regimen can be expanded ex vivo. In some cases, the leukocytes are expanded in vivo in the presence of one or more reagents for a predetermined period of time prior to use as a tolerizing vaccine. For example, the cells can be contacted with at least one cytokine for about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 160, 165, 170, 175, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 390, or 300 hours. In some cases, the cells may be contacted with at least one cytokine for about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 days or longer. In some cases, the cells may be contacted with at least one cytokine for about 1-2, 2-3, 3-4, 1-4, 1-3, or 2-4 weeks. In some cases, the cytokine is one or more interleukins. In some cases, the interleukin (IL) is at least one of IL-2, IL-4, IL-21, BAFF, multimeric CD40L, IL-10, IL-12, and IL-15. In some cases, the cells are contacted with IL-2, IL-4, IL-21, BAFF, and multimeric CD40L.

[0122] The leukocytes used in the tolerization vaccine / regimen or conditioning regimen can comprise at least, or at least about 10%, e.g., 25%, CD19-, CD20-, or CD21-positive MHC class II-positive B cells. For example, donor splenocytes can comprise at least, or at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80%, e.g., about, at least, or at least about 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, or 60-70% CD19-, CD20-, and / or CD21-positive MHC class II-positive B cells. In some cases, the splenic B cells or leukocytes used in a tolerizing vaccine / regimen or conditioning regimen can comprise at least, or at least about 60%, e.g., 90%, CD19, CD20, or CD21-positive MHC class II-positive B cells. For example, the splenic B cells or leukocytes used in a tolerizing vaccine / regimen or conditioning regimen can comprise about, at least, or at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, e.g., at least, or at least about 60-70%, 70-80%, 80-90%, or 90-95% CD19, CD20, or CD21-positive MHC class II-positive B cells. In some cases, the donor's splenocytes or leukocytes used in the tolerization vaccine / regimen or conditioning regimen can comprise 50% to 100% or about 50% to 100%, for example, 60% to 100% or about 60% to 100%, or 80% to 100% or about 80% to 100% CD19, CD20, or CD21-positive MHC class II-positive B cells. In some embodiments, the MHC class II is swine leukocyte antigen (SLA) class II. In some embodiments, the MHC class II is human leukocyte antigen (HLA) class II.

[0123] In some cases, the tolerization vaccine or conditioning regimen includes cells derived from a living donor. For example, the cells can be peripheral blood leukocytes (e.g., peripheral blood B cells). The peripheral blood leukocytes can be obtained by one or more blood draws, apheresis, or any other method. The peripheral blood leukocytes can be enriched for B cells. The peripheral blood leukocytes can be used directly or can be expanded ex vivo. The tolerization vaccine or conditioning regimen can include directly isolated leukocytes, ex vivo expanded leukocytes, or a combination thereof. In some cases, the initial dose of the tolerization vaccine can include directly isolated peripheral blood leukocytes (e.g., B cells), while subsequent doses can include ex vivo expanded peripheral blood leukocytes (e.g., B cells).

[0124] In some cases, the tolerization vaccine / regimen or conditioning regimen includes cells from a cadaveric donor. The cadaveric donor can be a brain-dead, heart-beating donor (BDD). Alternatively, the cadaveric donor can be a non-heart-beating donor (NHBD). The donor can be from any animal, for example, a human or a non-human animal. For example, the cells can be cadaveric white blood cells obtained from the spleen (e.g., splenocytes, splenic B cells), liver, peripheral blood (including peripheral blood B cells), lymph nodes, thymus, bone marrow, or any combination thereof. In some cases, the tolerization vaccine / regimen or conditioning regimen includes splenic white blood cells (e.g., splenic B cells), peripheral blood white blood cells (e.g., peripheral blood B cells), or a combination thereof. The white blood cells can be used directly or expanded ex vivo. In some cases, an initial dose of a tolerization vaccine may include directly isolated peripheral blood leukocytes (e.g., peripheral blood B cells), splenic leukocytes (e.g., splenic B cells), or a combination thereof, while subsequent doses may include ex vivo expanded peripheral blood leukocytes (e.g., peripheral blood B cells), splenic leukocytes (e.g., splenic B cells), or a combination thereof.

[0125] The cells for preparing the tolerization vaccine / regimens or conditioning regimens can be stem cells or differentiated cell types (e.g., white blood cells) generated from stem cells. The stem cells can be embryonic stem cells, adult stem cells, or induced stem cells. The stem cells can be totipotent stem cells, pluripotent stem cells, or multipotent stem cells. In some cases, the stem cells cannot develop into fully developed animals (e.g., human animals). In some cases, the stem cells are induced pluripotent stem cells (iPSCs). In some cases, the stem cells can be obtained by mobilizing stem cells from bone marrow to peripheral blood with a mobilizing agent, such as granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), mozobil, or a combination thereof.

[0126] The donor of cells used in the preparation of tolerization vaccines or conditioning regimens can be fully or partially MHC (major histocompatibility complex) matched with the transplant donor (e.g., the donor of cells, tissues, or organs used for transplantation). In some cases, the partially matched donor is haploidentical with the transplant donor. In some cases, the partially matched donor shares one or more MHC alleles with the transplant donor. For example, the partially matched donor can share one or more of the following with the transplant donor: MHC class IA allele, MHC class IB allele, MHC class IC allele, MHC class II DR allele, MHC class II DQ allele, MHC class II DP allele, or a combination thereof. In some cases, the one or more shared MHC alleles do not include MHC class IC. The partially matched donor can share one DR allele with the transplant donor.

[0127] In some cases, a partially matched donor of cells used in preparing a tolerizing vaccine or conditioning regimen may share at least one MHC class I allele (e.g., an MHC class IA allele, an MHC class IB allele, an MHC class IC allele) and at least one MHC class II allele (e.g., an MHC class II DR allele, an MHC class II DQ allele, an MHC class II DP allele) with the transplant donor. In some cases, the one or more shared MHC alleles do not include MHC class IC. In some cases, a partially matched donor of cells used in preparing a tolerizing vaccine or conditioning regimen may share at least one MHC class I allele (e.g., an MHC class IA allele, an MHC class IB allele, an MHC class IC allele) and at least one MHC class II allele (e.g., an MHC class II DR allele, an MHC class II DQ allele, an MHC class II DP allele) with the transplant donor, and at least one MHC class II allele (e.g., an MHC class II DR allele, an MHC class II DQ allele, an MHC class II DP allele) with the transplant recipient, wherein the MHC class II alleles shared with the transplant donor and the transplant recipient are the same MHC class II allele. In some cases, the one or more shared MHC alleles do not include MHC class IC.

[0128] In some cases, the donor of the cells may be partially or completely MHC-mismatched with the transplant donor or transplant recipient. For example, in some cases, peptides derived from MHC molecules that are compatible with the transplant donor and / or transplant recipient may be conjugated to the ex vivo expanded donor cells disclosed herein. Tolerizing vaccines / regimens

[0129] Traditionally, vaccines are biological preparations that provide active acquired immunity to a specific disease in a host. Traditional vaccines may contain a drug that resembles the disease-causing pathogen and may be made from an attenuated or killed form of the pathogen, its toxin, or one of its surface proteins. The drug stimulates the host's immune system to recognize the drug as a threat, destroy it, and further recognize and destroy any pathogens associated with the drug that may be encountered in the future. For example, subcutaneous injection of an inactivated virus with an adjuvant can result in temporary or permanent immunity against an active and / or pathogenic version of that virus. This may be referred to as a positive vaccine (Figure 1).

[0130] However, inactivated or apoptotic cells injected or infused intravenously can result in immune tolerance to cells with similar cellular markers or antigens (e.g., transplanted donor cells). This can be referred to as a tolerization vaccine (also referred to as a negative vaccine) (Figure 1). In some embodiments, inactivated or apoptotic cells can be injected or infused without an adjuvant or additional agent (e.g., one or more immunomodulatory molecules). In some embodiments, inactivated or apoptotic cells can be injected with an additional agent (e.g., one or more immunomodulatory molecules). In some cases, the additional agent enhances the tolerogenicity of the tolerization vaccine by inhibiting the activation and / or maturation of antigen-presenting cells. Source of cells for tolerizing vaccines / regimens or conditioning regimens

[0131] Cells for preparation of a tolerization vaccine or conditioning regimen can be obtained from any source, including animals, cell lines, and / or laboratory-generated cells. For example, the cells can be obtained from a human or non-human animal. In another example, the cells can be derived from a cell line (e.g., a human or non-human cell line). In some cases, the cells are human cells. In other cases, the cells are non-human cells. In some cases, the cells are non-human primate cells. In some cases, the cells are cells of a member of the superorder Laurasiatheria. In some cases, the cells are cells of an ungulate, such as a camelid or a pig. In some cases, the cells are porcine cells. In some cases, the cells are from the same species as the transplant donor. In some cases, the cells are from the same species as the transplant recipient. In some cases, the cells are from the same species as the transplant donor and the transplant recipient. In some cases, the cells are from a different species than the transplant donor. In some cases, the cells are from a different species than the transplant recipient. In some cases, the cells are from a different species than the transplant donor and the transplant recipient.

[0132] Cells for preparing a tolerization vaccine or conditioning regimen can be obtained from a living donor or a cadaveric donor. In some cases, the donor is a living donor. In some cases, the donor is a cadaveric donor. A cadaveric donor can be, for example, a brain-dead, heart-beating donor (BDD). A cadaveric donor can be, for example, a non-heart-beating donor (NHBD). Whether the donor is a living donor or a cadaveric donor (e.g., BDD or NHBD), the donor can be from any animal, for example, a human or a non-human animal. In some cases, cells for preparing a tolerization vaccine can be from the same donor as the graft or transplant. In some cases, cells for preparing a tolerization vaccine can be from a different donor than the graft or transplant.

[0133] The cells for preparing tolerization vaccines or conditioning regimens can be obtained from donor animals of any age or developmental stage.For example, the donor animals can be fetal, perinatal, neonatal, pre-weaning, post-weaning, child, young adult or adult animals.In some cases, the non-human animals can be past weaning age.For example, the donor animals can be at least or at least about 6 months old.In some cases, the donor animals can be at least or at least about 18 months old.In some cases, the cells for preparing tolerization vaccines or conditioning regimens can be obtained (e.g., can be differentiated) from stem cells (e.g., embryonic stem cells, induced pluripotent stem cells and / or mesenchymal stem cells).

[0134] The cell used to make tolerization vaccine or conditioning regimen can comprise one or more cells from tissue, organ or body fluid.For example, cell can be derived from tissue, organ or body fluid, including but not limited to brain, lung, liver, heart, spleen, pancreas, small intestine, large intestine, skeletal muscle, smooth muscle, skin, bone, adipose tissue, hair, thyroid gland, trachea, gallbladder, kidney, ureter, urinary bladder, aorta, vein, esophagus, diaphragm, stomach, rectum, adrenal gland, bronchus, ear, eye, retina, reproductive organ, hypothalamus, larynx, nose, tongue, spinal cord or ureter, uterus, ovary, testis, blood, cerebrospinal fluid, lymphatic fluid, or combination thereof.

[0135] The cells used to make the tolerization vaccine or conditioning regimen may include one or more types of cells. For example, the cells may include, but are not limited to, trichocytes, keratinocytes, gonadotrophs, corticotrophs, thyrotrophs, growth hormones, mammary glands, chromaffin cells, parafollicular cells, glomus cells, melanocytes, nevus cells, Merkel cells, odontoblasts, cementoblasts, keratocytes, retinal Müller cells, retinal pigment epithelial cells, neurons, Glia (e.g., oligodendrocytes, astrocytes), ependymal cells, pineal cells, lung cells (e.g., type I pneumocytes and type II pneumocytes), Clara cells, goblet cells, G cells, D cells, ECL cells, gastric chief cells, parietal cells, pit cells, K cells, D cells, I cells, goblet cells, Paneth cells, enterocytes, fold cells, hepatocytes, hepatic stellate cells (e.g., mesodermally derived Kupffer cells), gallbladder cells, acinar center cells, pancreatic stellate cells, pancreatic α cells, pancreatic β cells, pancreatic δ cells, pancreatic F cells ( e.g., PP cells), pancreatic ε cells, thyroid gland (e.g., follicular cells), parathyroid gland (e.g., parathyroid chief cells), eosinophilic cells, urothelial cells, osteoblasts, osteocytes, chondroblasts, chondrocytes, fibroblasts, fibrocytes, myoblasts, myocytes, muscle satellite cells, tenocytes, cardiomyocytes, lipoblasts, adipocytes, interstitial cells of Cajal, angioblasts, endothelial cells, mesangial cells (e.g., intraglomerular mesangial cells and extraglomerular mesangial cells), juxtamembrane fimbriae The cells may include spheroid cells, macula densa cells, interstitial cells, interstitial cells, telocytes, simple epithelial cells, podocytes, renal proximal tubule brush border cells, Sertoli cells, Leydig cells, granulosa cells, PEG cells, germ cells, sperm, eggs, lymphocytes, myeloid cells, endothelial progenitor cells, endothelial stem cells, hemangioblasts, mesoangioblasts, pericytes, mural cells, mesenchymal stromal cells, or splenocytes (e.g., T lymphocytes, B lymphocytes, dendritic cells, macrophages, leukocytes). In some cases, the cells used to produce tolerization vaccines or conditioning regimens include cell types that express MHC class II. In some cases, the cells used to produce tolerization vaccines or conditioning regimens include cell types that do not express MHC class II.

[0136] The tolerization vaccine or conditioning regimen can include white blood cells. White blood cells can include, for example, neutrophils, eosinophils, basophils, lymphocytes, monocytes, or a combination thereof. Lymphocytes can include, for example, B lymphocytes (B cells), T lymphocytes (T cells), natural killer (NK) cells, or a combination thereof.

[0137] The leukocytes in the tolerization vaccine or conditioning regimen can be obtained from any source, including, for example, donors, cell lines, or differentiated stem cells. The leukocytes obtained from the donor can include leukocytes obtained from the spleen (e.g., splenocytes, splenic B cells), liver, peripheral blood (including peripheral blood B cells), lymph nodes, thymus, bone marrow, or any other organ, tissue, or body fluid, or any combination thereof. In some cases, the tolerization vaccine or conditioning regimen includes splenic B cells, peripheral blood B cells, or a combination thereof. In some cases, the tolerization vaccine or conditioning regimen includes cells mobilized from bone marrow to peripheral blood by a mobilizing agent, such as granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), mozobil, or a combination thereof. The leukocytes in the tolerizing vaccine or conditioning regimen obtained from the donor can include primary cells, ex vivo expanded cells, or a combination thereof. Genetically modified cells for tolerizing vaccines or conditioning regimens

[0138] The donor of the cells used in the preparation of tolerization vaccine or conditioning regimen can be genetically modified.Alternatively, or in addition, the cells obtained from the donor can be genetically modified ex vivo.In some cases, cell line is genetically modified to produce cells for use in tolerization vaccine or conditioning regimen.Genetically modified donor and / or cells can be produced by any method known in the art, including the method described herein.Regardless of whether genetically modified cells are isolated from genetically modified animals, produced in culture, or a combination thereof, genetically modified cells can be the genetically modified cells of any animal species, including human and non-human animals.

[0139] The genetically modified cells used in the tolerization vaccine or pretreatment regimen can contain one or more genetic modifications that reduce or eliminate the expression or gene or gene product (e.g., protein). The genetic modification can be a modification to the gene whose expression is reduced or eliminated. Such a gene can be referred to as a disrupted gene. The genetic modification can also be directed to a region of the genome that is separate from the gene whose expression is reduced or eliminated (e.g., a modification to a promoter, enhancer, silencer, transcription factor, etc.). The genetically modified cells used in the tolerization vaccine or pretreatment regimen can contain, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more genes whose expression is reduced or eliminated by genetic modification.

[0140] Non-limiting examples of genes whose expression may be reduced or eliminated by genetic modification in cells used in tolerizing vaccines or conditioning regimens include, but are not limited to, alpha 1,3 galactosyltransferase (GGTA1); putative cytidine monophosphatase-N-acetylneuraminic acid hydroxylase-like protein (CMAH); beta 1,4 N-acetylgalactosaminyltransferase (B4GALNT2); components of the major histocompatibility complex (MHC) I-specific enhanceosome (e.g., NOD-like receptor family CARD domain-containing 5 (NLRC5)); transporters of MHC I-bound peptides (e.g., transporter associated with antigen processing 1 (TAP1)); complement component 3 (C3); C-X-C chemokine receptor 3 ligand (CXCL3); C-X-C motif chemokine ligand 10 (CXCL10) genes; MHC II transactivator (MHCIITA); MHC class I polypeptide-related sequence A (MICA) gene; MHC class I polypeptide-related sequence B (MICB) gene; natural killer (NK) group 2D ligand (NKG2DL); tumor necrosis factor receptor (TNF-R); porcine endogenous retrovirus (PERV); B2M, PD-1, PD-L1, or any combination thereof.

[0141] In some cases, the genetically modified cells used in tolerization vaccines can contain disruption in one or more genes, including GGTA1, CMAH, B4GALNT2, B2M, NLRC5, or any combination thereof.For example, the genetically modified cells used to produce tolerization vaccines or pretreatment regimens can only have disruption in GGTA1, or only have disruption in CMAH, or only have disruption in B4GALNT2, B2M, or NLRC5.The genetically modified cells used to produce tolerization vaccines or pretreatment regimens can also have disruption in GGTA1 and CMAH, or have disruption in GGTA1 and B4GALNT2, or have disruption in CMAH and B4GALNT2, or have disruption in NLRC5 and B2M.The genetically modified cells used to produce tolerization vaccines or pretreatment regimens can have disruption in GGTA1, CMAH, and B4GALNT2. Without wishing to be bound by any particular theory, such destruction can minimize or eliminate cell-mediated immunity, antibody-mediated immunity, antibody-dependent cell-mediated immunity, and / or cell-dependent antibody-mediated immunity against organ, tissue, cell, and cell line grafts (e.g., xenografts or allografts containing the same genetic modifications as the cells used in the tolerizing vaccine).

[0142] The genetically modified cells used in the tolerization vaccine or pretreatment regimen can contain, or can further contain, one or more genetic modifications that increase the expression of one or more genes or gene products.The increased expression can be from zero expression, for example, the increased expression can be the expression of a gene or gene product that is not normally expressed in cells without genetic modification.The increased expression can be compared to a threshold level, for example, the level that is normally expressed in cells without genetic modification.The genetic modification can include one or more exogenous polynucleotides that encode a polypeptide (e.g., an endogenous or exogenous polypeptide).

[0143] Non-limiting examples of exogenous polynucleotides include, but are not limited to, MHC Examples of inhibitory ligands include polynucleotides encoding one or more of: suppressor of I formation (e.g., infected cell protein 47 (ICP47)); regulators of complement activation (e.g., CD46, CD55, or CD59); inhibitory ligands for NK cells; B7 family members (e.g., programmed death ligands such as PD-L1 or PD-L2); serine protease inhibitors (e.g., Spi9); galectins; interleukins (e.g., IL-37); CD40:CD40L blocking agents (e.g., CD40 antagonist polypeptides, anti-CD40 ligand polypeptides); Fas ligand (FasL); any functional fragments thereof; or any combination thereof. In some embodiments, the inhibitory ligand for NK cells is a human leukocyte antigen (HLA), such as human leukocyte antigen E (HLA-E), human leukocyte antigen G (HLA-G), beta-2-microglobulin (B2M), or any combination thereof. In some embodiments, the HLA-G is HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, HLA-G7, or any combination thereof. In some cases, the galectin is galectin-1, galectin-2, galectin-3, galectin-4, galectin-5, galectin-6, galectin-7, galectin-8, galectin-9, galectin-10, galectin-11, galectin-12, galectin-13, galectin-14, or galectin-15. For example, the galectin can be galectin-9. Generation of cells for tolerizing vaccines or conditioning regimens

[0144] The tolerizing vaccine or conditioning regimen can include apoptotic cells, non-apoptotic cells, or a combination thereof. In some cases, the tolerizing vaccine or conditioning regimen includes non-apoptotic cells. In some cases, the tolerizing vaccine or conditioning regimen includes both apoptotic and non-apoptotic cells. In some cases, the tolerizing vaccine or conditioning regimen includes apoptotic cells.

[0145] The term "anergy" may refer to the absence of a normal immune response to a specific antigen (e.g., the absence of a pro-inflammatory response). The term "T cell anergy" may refer to a tolerance mechanism in which immune cells (e.g., T cells) are essentially functionally inactivated after antigen encounter but remain viable for an extended period in a hyporesponsive state. The present disclosure is not limited to any particular mechanism. Without being bound by theory, apoptotic cells may be captured by host antigen-presenting cells (e.g., in the spleen) and presented to host immune cells (e.g., T cells) in a non-immunogenic manner, resulting in the induction of anergy in the immune cells (e.g., T cells). For example, apoptotic cells in tolerization vaccines (e.g., ECDI-treated splenocytes, leukocytes, or mesenchymal stromal cells, which are fixed to the cell surface and can inactivate many surface molecules) are unable to stimulate the activation and / or proliferation of antigen-specific normal T cell clones. Without wishing to be bound by theory, apoptotic cells in tolerization vaccines are unable to provide costimulatory signals to T cells. As a result, apoptotic cells can induce a state of long-term unresponsiveness called anergy. In some embodiments, tolerance induced by injection of a tolerizing vaccine or conditioning regimen may depend on the synergistic effect between the intact programmed death 1 receptor-programmed death ligand 1 signaling pathway and CD4+CD25+Foxp3+ regulatory T cells.

[0146] Cells for tolerization vaccines or conditioning regimens can be induced to apoptosis in several different ways. For example, cells can be contacted with chemicals (e.g., fixatives or cross-linking agents, cell-damaging agents, or combinations thereof) to induce apoptosis in some or all cells. In another example, cells can be irradiated (e.g., with ultraviolet light, gamma rays, etc.) to induce apoptosis in some or all cells.

[0147] Cells for tolerization vaccines or conditioning regimens can be contacted with chemicals such as fixatives or crosslinkers. Contact can induce apoptosis in some or all cells. Suitable fixatives or crosslinkers include, but are not limited to, amine-amine crosslinkers, sulfhydryl-sulfhydryl crosslinkers, amine-sulfhydryl crosslinkers, in vivo crosslinkers, sulfhydryl-carbohydrate crosslinkers, photoreactive crosslinkers, chemoselective ligation crosslinkers, carboxyl-amine crosslinkers, carbodiimides, genipin, acrylaldehyde, diformyl, osmium tetroxide, diimide esters, mercuric chloride, zinc sulfate, zinc chloride, trinitrophenol (picric acid), potassium dichromate, ethanol, methanol, acetone, acetic acid, or combinations thereof.

[0148] Cells for tolerization vaccines or pretreatment regimens can be contacted with a carbodiimide or a carbodiimide derivative. Treatment with a carbodiimide can chemically crosslink free amine and carboxyl groups, effectively inducing apoptosis in cells, organs, and / or tissues. The contact can be for a predetermined period of time. The contact can cause apoptosis in some or all cells. The carbodiimide can include ethylcarbodiimide, ethylenecarbodiimide, N,N'-diisopropylcarbodiimide (DIC), N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDCI, EDC, ECDI, or EDAC), or a combination thereof. In some cases, the carbodiimide includes ethylcarbodiimide. In some cases, the carbodiimide includes ethylenecarbodiimide. In some cases, the carbodiimide includes N,N'-diisopropylcarbodiimide (DIC). In some cases, the carbodiimide comprises N,N'-dicyclohexylcarbodiimide (DCC). In some cases, the carbodiimide comprises 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDCI, EDC, ECDI, or EDAC). In some cases, the tolerization vaccine comprises cells treated with an EDCI derivative and / or a functionalized EDCI.

[0149] Cells for tolerization vaccines or pretreatment regimens can be contacted with a diimide ester. The contact can be for a predetermined period of time. The contact can cause apoptosis of some or all of the cells. The diimide ester can include cyanuric chloride, diisocyanate, diethylpyrocarbonate (DEPC) or diethyldicarbonate, maleimide, benzoquinone, or a combination thereof.

[0150] Cells for a tolerization vaccine or conditioning regimen can be contacted with an amine-amine crosslinker. The contact can be for a predetermined period of time. The contact can cause apoptosis of some or all of the cells. In some cases, the amine-amine crosslinker comprises disuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS3), tris-(succinimidyl)aminotriacetate (TSAT), BS(PEG)5, BS(PEG)9, dithiobis(succinimidyl propionate) (DSP), 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP), disuccinimidyl tartrate (DST), bis(2-(succinimidooxycarbonyloxy)ethyl)sulfone (BSOCOES), ethylene glycol bis(succinimidyl succinate) (EGS), sulfo-EGS, or any combination thereof. In some cases, the amine-amine crosslinker comprises an imidoester, such as dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl 3,3'-dithiobispropionimidate (DTBP), or any combination thereof. In some cases, the amine-amine crosslinker comprises a difluoro, such as 1,5-difluoro-2,4-dinitrobenzene (DFDNB).

[0151] Cells for tolerization vaccines or conditioning regimens can be contacted with a sulfhydryl-sulfhydryl crosslinker. The contact can be for a predetermined period of time. The contact can cause apoptosis of some or all of the cells. In some cases, the sulfhydryl-sulfhydryl crosslinker comprises a maleimide, such as bismaleimidoethane (BMOE), 1,4-bismaleimidobutane (BMB), bismaleimidohexane (BMH), tris(2-maleimidoethyl)amine (TMEA), BM(PEG)2 (such as 1,8-bismaleimido-diethylene glycol); BM(PEG)3 (such as 1,11-bismaleimido-triethylene glycol), dithiobismaleimidoethane (DTME), or any combination thereof.

[0152] Cells for tolerization vaccines or conditioning regimens can be contacted with an amine-sulfhydryl crosslinker. The contact can be for a predetermined period of time. The contact can cause apoptosis of some or all of the cells. In some cases, the amine-sulfhydryl crosslinker includes an NHS-haloacetyl crosslinker, an NHS-maleimide, an NHS-pyridyldithiol crosslinker, a 4-(N-maleimidomethyl)cyclohexane-1-carboxylate sulfosuccinimidyl (SMCC) crosslinker, or any combination thereof. The NHS-haloacetyl crosslinker can include succinimidyl iodoacetate (SIA), succinimidyl 3-(bromoacetamido)propionate (SBAP), succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), sulfo-SIAB, or a combination thereof. NHS-maleimide includes N-α-maleimidoacet-oxysuccinimide ester (AMAS), N-β-maleimidopropyl-oxysuccinimide ester (BMPS), N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS), sulfo-GMBS, m-maleimidobenzoyl-N-hydrosuccinimide ester (MBS), sulfo-MBS, SMCC, sulfo-SMCC, N-ε-maleimidocaproyl-oxysuccinimide ester (EMCS), The crosslinker may include sulfo-EMCS, succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), sulfo-SMPB, succinimidyl 6-((beta-maleimidopropionamido)hexanoate) (SMPH), sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate) (LC-SMCC), N-κ-maleimidoundecanoyl-oxysulfosuccinimide ester (sulfo-KMUS), or a combination thereof. The NHS-pyridyldithiol crosslinker may include succinimidyl 3-(2-pyridyldithio)propionate (SPDP), succinimidyl 6-(3(2-pyridyldithio)propionamido)hexanoate (LC-SPDP), sulfo-LC-SPDP, or 4-succinimidyloxycarbonyl-alpha-methyl-α(2-pyridyldithio)toluene (SMPT).

[0153] Cells for a tolerization vaccine or conditioning regimen can be contacted with a sulfhydryl-carbohydrate crosslinker. The contact can be for a predetermined period of time. The contact can result in apoptosis of some or all of the cells. In some cases, the sulfhydryl-carbohydrate crosslinker comprises (N-β-maleimidopropionic acid hydrazide (BMPH), N-ε-maleimidocaproic acid hydrazide (EMCH), 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), N-κ-maleimidoundecanoic acid hydrazide (KMUH), 3-(2-pyridyldithio)propionyl hydrazide (PDPH), or any combination thereof.

[0154] In some cases, the carboxyl-amine crosslinker is dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDCI, EDC or EDAC), N-hydroxysuccinimide (NHS), sulfo-NHS, or any combination thereof.

[0155] Cells for tolerization vaccines or pretreatment regimens can be contacted with a photoreactive crosslinker. The contact can be for a predetermined time. The contact can cause apoptosis of some or all of the cells. In some cases, the photoreactive crosslinker includes an NHS ester / aryl azide, an NHS ester / diazirine, or a combination thereof. The NHS ester / aryl azide can include N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOS), sulfo-SANPAH, or a combination thereof. The NHS ester / diazirine may include SDA (NHS-diazirine / succinimidyl 4,4'-adipentanoate), sulfo-SDA, LC-SDA (NHS-LC-diazirine / succinimidyl 6-(4,4'-adipentanamido)hexanoate), sulfo-LC-SDA, SDAD (NHS-SS-diazirine / succinimidyl 2-((4,4'-adipentanamido)ethyl)1,3'-dithiopropionate), sulfo-SDAD, or combinations thereof.

[0156] Cells for tolerization vaccines or conditioning regimens can be contacted with an in vivo crosslinker. The contact can be for a predetermined period of time. The contact can induce apoptosis of some or all of the cells. The in vivo crosslinker can include BS3, DTSSP, sulfo-EGS, DSG, DSP, DSS, EGS, sulfo-SDA, sulfo-LC-SDA, sulfo-SDAD, SDA, LC-SDA, SDAD, NHS-ester diazirine, or any combination thereof.

[0157] In some cases, cells for use in tolerization vaccines or conditioning regimens are treated with a cell-damaging agent or apoptosis-inducing agent. In some cases, the cell-damaging agent induces apoptosis in some or all of the contacted cells. Non-limiting exemplary cell-damaging agents include doxorubicin, staurosporine, etoposide, camptothecin, paclitaxel, vinblastine, or any combination thereof. Non-limiting exemplary apoptosis inducers include marinopyrrole A, maritoclax, (E)-3,4,5,4'-tetramethoxystilbene, 17-(allylamino)-17-demethoxygeldanamycin, 2,4,3',5'-tetramethoxystilbene, 2OHOA, 6,8-bis(benzylthio)-octanoic acid, AT101, apotolidin, FU 40A, ara-G hydrate, arylquin 1, BAD, BAM7, BAX activator molecule 7, BH3I-1, BID, BMS-906024, BV02, bendamustine, borrelidin, borrelidine, cyclopentanecarboxylic acid, NSC 216128, treponemycin, brassinin, brassinine, brefeldin A, ascotoxin, BFA, cyanine, decumbin, bufalin, CCF642, CCT007093, CD437, CHM-1 hydrate, 2-(2-fluorophenyl)-6,7-Methylenedioxy-2-4-quinolone hydrate, NSC 656158, CIL-102, CP-31398, dihydrochloride hydrate, camalexin, 3-(thiazol-2-yl)-1H-indole, camalexin, carboxyatractyloside, cepharanthine, cepharanthine, cinnabarinic acid, cirsiliol, combretastatin A4, costunolide, DBeQ, DIM-C-pPhtBu, DMXAA, DPBQ, enniatin A1, enniatin A, enniatin B1, enniatin B, elastin, eupatorin, FADD, fluticasone propionate, fosbretabulin disodium, GO-201 trifluoroacetate, gambogic acid, HA 14-1, HMBA, hexaminolevulinate (HAL), IMB5046, IMS2186, ikarugamycin, imiquimod, iniparib, kurarinone, LLP-3, lipocalin-2, lometrexol, MI-4F, ML 210, ML291, Molgin, Muristerone A, NA-17, NID-1, NPC26, NSC59984, Nap-FF, Neocarzinostatin, Nifetepimine, Nitidine Chloride, Nutlin-3, Nutlin-3a, PKF118-310, PRIMA-1, PRT4165, Pemetrexed, Penta-O-galloyl-β-D-glucose Hydrate, Fenoxodiol, Prodigiosin (PG), Psoralidin, Pterostilbene, Raltitrexed, Raptinal, Ridaifen-B, Rifabutin, Roslin 2, sp-Bromobenzyl Glutathione Cyclopentyl Examples of suitable anti-apoptotic agents include diesters, SJ-17255, SMBA1, STF-62247, suprafenacine, syrosingopine, talniflumate, taurolidine, temoporfin, temozolomide, tetrazanbigen, thaxtomin A, thiocolchicine, tirapazamine, UCD38B, UMI-77, undecylprodigiosin, VK3-OCH3, vaquinol-1, violacein, vosaroxin, zerumbone, gAcrp30, gAcrp30 / adipolean, or any combination thereof. Cells contacted with a cell-damaging agent or apoptosis-inducing agent may then be contacted with a fixative or crosslinking agent.

[0158] Cells for tolerization vaccines or conditioning regimens can be induced to undergo apoptosis by contacting the cells with a chemical (e.g., a fixative or crosslinking agent, a cell-damaging agent, or a combination thereof) for a predetermined time. In some embodiments, cells in tolerization vaccines or conditioning regimens are induced to undergo apoptosis by fixation with a crosslinking agent (e.g., ECDI) for a predetermined time. In some cases, the predetermined time is about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, or about 72 hours. In some cases, the predetermined time is less than 1 hour. In some cases, the predetermined time is at least about 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 75 minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes, 210 minutes, or 240 minutes. In some cases, the predetermined time is at most about 30 minutes, 40 minutes, 50 minutes, 60 minutes, 75 minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes, 210 minutes, or 240 minutes. In some cases, the predetermined time period may be from about 1 minute to about 240 minutes, 1 minute to about 10 minutes, 10 minutes to about 240 minutes, from about 10 minutes to about 180 minutes, from about 10 minutes to about 120 minutes, from about 10 minutes to about 90 minutes, from about 10 minutes to about 60 minutes, from about 10 minutes to about 30 minutes, from about 30 minutes to about 240 minutes, from about 30 minutes to about 180 minutes, from about 30 minutes to about 120 minutes, from about 30 minutes to about 90 minutes, from about 30 minutes to about 60 minutes, from about 50 minutes to about 240 minutes, from about 50 minutes to about 180 minutes, from about 50 minutes to about 120 minutes, from about 50 minutes to about 90 minutes, from about 50 minutes to about 60 minutes, from about 10 minutes to about 20 minutes, from about 20 minutes to about 30 minutes, from about 30 minutes to about 40 minutes, from about 40 minutes to about 50 minutes, from about 50 minutes to about 60 minutes, about 60 to about 70 minutes, about 70 to about 80 minutes, about 80 to about 90 minutes, about 90 to about 100 minutes, about 100 to about 110 minutes, about 110 to about 120 minutes, about 10 to about 30 minutes, about 30 to about 50 minutes, about 50 to about 70 minutes, about 70 to about 90 minutes, about 90 to about 110 minutes, about 110 to about 130 minutes, about 130 to about 150 minutes, about 150 to about 170 minutes, about 170 to about 190 minutes, about 190 to about 210 minutes, about 210 to about 240 minutes, up to about 30 minutes, about 30 to about 60 minutes, about 60 to about 90 minutes, about 90 to about 120 minutes, or about 120 to about 150 minutes.

[0159] The contacting can be at any temperature. In some cases, the contacting is performed on ice (e.g., 4°C). In other cases, the contacting is performed at room temperature. In some cases, the contacting is performed at a temperature of at least about 0°C, 2°C, 4°C, 8°C, 15°C, 20°C, 25, 30°C, 35°C, or 37°C. In some cases, the contacting is performed at a temperature of up to about 4°C, 8°C, 15°C, 20°C, 25, 30°C, 35°C, 37°C, or 40°C. In some cases, the contacting is carried out at a temperature of about 0°C to about 37°C, about 0°C to about 25°C, about 0°C to about 15°C, about 0°C to about 10°C, about 0°C to about 8°C, about 0°C to about 6°C, about 0°C to about 4°C, about 0°C to about 2°C, about 2°C to about 10°C, about 2°C to about 8°C, about 2°C to about 6°C, about 4°C to about 25°C, about 4°C to about 10°C, about 15°C to about 37°C, about 15°C to about 25°C, about 20°C to about 40°C, about 20°C to about 37°C, or about 20°C to about 30°C.

[0160] The cells in tolerization vaccine or pretreatment regimen may aggregate as a result of the method that causes apoptosis of some or all cells.For example, cells may aggregate after contact with chemicals such as fixatives or cross-linking agents.The predetermined time that cells are contacted with chemicals can be selected to minimize the amount of aggregation in tolerization vaccine or pretreatment regimen.In some cases, aggregates can be removed, for example, by washing and / or filtration.

[0161] In some cases, the tolerization vaccine or pretreatment regimen can contain 0.01 to 10 or about 0.01 to 10 aggregates per μl. For example, the tolerization vaccine or pretreatment regimen can contain 0.01 to 1, 0.1 to 1, 0.25 to 1, 0.5 to 1, 1 to 5, or 1 to 10 aggregates per μl, or about 0.01 to 1, 0.1 to 1, 0.25 to 1, 0.5 to 1, 1 to 5, or 1 to 10 aggregates per μl. The tolerization vaccine or pretreatment regimen can contain less than about 0.1, 0.5, 0.75, 1, 5, or 10 aggregates per μl.

[0162] In some cases, the tolerizing vaccine or conditioning regimen can contain fewer than 5 aggregates per μl. For example, the tolerizing vaccine or conditioning regimen can contain less than about 5, 4, 3, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, or 0.01 aggregates per μl.

[0163] In some cases, the tolerization vaccine or conditioning regimen contains 1 or fewer aggregates per μL. For example, the tolerization vaccine or conditioning regimen can contain about 0.01, about 0.05, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, or about 1.0 aggregates per μL.

[0164] The tolerization vaccine or conditioning regimen can include 0.01% to 10%, or about 0.01% to 10%, e.g., 0.01% to 2%, or about 0.01% to 2% necrotic cells. For example, the cells of the tolerization vaccine or conditioning regimen can include 0.01% to 10%, 0.01% to 7.5%, 0.01% to 5%, 0.01% to 2.5%, or 0.01% to 1%, or about 0.01% to 10%, 0.01% to 7.5%, 0.01% to 5%, 0.01% to 2.5%, or 0.01% to 1% necrotic cells. In some embodiments, the cells of the tolerizing vaccine or conditioning regimen of the present disclosure may contain up to about 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% necrotic cells. Molecules conjugated on the surface of apoptotic leukocytes

[0165] immunomodulatory molecules

[0166] Cells (e.g., leukocytes) for tolerization vaccines or pretreatment regimens can be treated with a fixative or crosslinking agent (e.g., a carbodiimide such as ECDI) in the presence of one or more immunomodulatory molecules. The one or more immunomodulatory molecules can include all or part of IFN-γ, NF-kB inhibitors, vitamin D3, siCD40, anti-CD40 antibodies, cobalt protoporphyrin, insulin B9-23, α1-antitrypsin, cluster of differentiation proteins, gp39 antagonists, α1-antitrypsin, CD47, PD-L1, PD-L2, CTLA-4, rapamycin, compstatin, abatacept, ipilimumab, or a combination thereof. The NF-kB inhibitor can include dehydroxymethylepoxyquinomycin (DHMEQ), curcumin, triptolide, Bay-117085, or a combination thereof. The cluster of differentiation protein may include CD4, CD46, CD47, CD55, CD59, or portions thereof, or combinations thereof.

[0167] In some embodiments, the one or more immunomodulatory molecules may include all or a portion of a calcineurin inhibitor (e.g., cyclosporine or tacrolimus), a costimulatory signal blocker, an IL-2 signaling inhibitor (e.g., daclizumab or basiliximab), a cell cycle blocker (e.g., mycophenolate mofetil (MMF) or azathioprine), a T cell recirculation inhibitor (e.g., FTY720 or another sphingosine 1-phosphate (S1P) receptor agonist), a nitrogen mustard alkylating agent (e.g., cyclophosphamide), a complement C3 or C5 inhibitor, or any combination thereof.

[0168] In some embodiments, the immunomodulatory molecule is a T cell receptor (TCR), CD3e, FK506-binding protein 12 (FKBP12), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1, e.g., pembrolizumab), programmed death-ligand 1 (PD-L1, e.g., MPDL3280A), CD40L (CD154), CD40, inducible co-stimulatory factor (ICOS), IL-2, TNF-α (e.g., infliximab). The target may be IL-6 or IL-6R (e.g., tocilizumab, Actemra, clazakizumab, ALD518, siltuximab, elcilimomab, sirumab, sarilumab, olokizumab), IL-7, CD2, CD20, CD52, alpha-4 integrin, mTOR (e.g., rapamycin or everolimus), DNA synthesis, molecules in the pro-inflammatory pathway (e.g., cytokines, alpha 1-antitrypsin, NFkB), or any combination thereof.

[0169] Cells (e.g., leukocytes) for tolerization vaccine or pretreatment regimen can be treated with a fixative or cross-linking agent (e.g., carbodiimide such as ECDI) in the presence of an agent that increases the expression of anti-inflammatory cytokines in the recipient. Anti-inflammatory cytokines can include, for example, TGF-β, IL-10, IL-13, or a combination thereof. In some cases, the agent that increases the expression of anti-inflammatory cytokines in the recipient includes α1-antitrypsin.

[0170] In some cases where ADLs are administered to a transplant recipient multiple times, the ADLs from all doses may all be conjugated with the same immunomodulatory molecule, agent that increases the expression of anti-inflammatory cytokines, and / or antigen or epitope. In some cases where ADLs are administered to a transplant recipient multiple times, the ADLs from one or more doses may be conjugated with a first set of immunomodulatory molecules, agents that increase the expression of anti-inflammatory cytokines, and / or antigens or epitopes, while the ADLs from other doses may be conjugated with a different set of immunomodulatory molecules, agents that increase the expression of anti-inflammatory cytokines, and / or antigens or epitopes. In some cases where ADLs are administered to a transplant recipient multiple times, the ADLs from one or more doses may be conjugated with a set of immunomodulatory molecules, agents that increase the expression of anti-inflammatory cytokines, and / or antigens or epitopes, while the ADLs from other doses may lack any conjugated immunomodulatory molecules, agents that increase the expression of anti-inflammatory cytokines, and / or antigens or epitopes.

[0171] Peptides, antigens and epitopes

[0172] In one aspect, the present disclosure provides conditioning regimens and / or tolerization vaccines and regimens comprising apoptotic cells, such as leukocytes or mesenchymal stromal cells, comprising one or more peptides derived from MHC class II molecules. In some embodiments, the apoptotic cells further comprise one or more peptides derived from MHC class I molecules.

[0173] Thus, the sequence of amino acid residues in a peptide can be substantially similar or functionally equivalent to a polypeptide sequence in an MHC molecule. Thus, a "peptide derived from an MHC class II molecule" refers to a peptide having a sequence "derived from a region in an MHC class II molecule" (e.g., a hypervariable region) that has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, 99.95% or 100% identity to a naturally occurring MHC amino acid sequence in that region. In some embodiments, a peptide derived from an MHC class II molecule can comprise a sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, 99.95% or 100% identical to a hypervariable region of the MHC class II molecule. Thus, a "peptide derived from an MHC class I molecule" refers to a peptide having a sequence "derived from a region in an MHC class I molecule" (e.g., a hypervariable region), which has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, 99.95% or 100% sequence identity to a naturally occurring MHC amino acid sequence in that region. In some embodiments, a peptide derived from an MHC class I molecule comprises a sequence derived from a hypervariable region of an MHC class I molecule.

[0174] As used herein, the "hypervariable region" of an MHC molecule refers to a region of the molecule in which polypeptides encoded by different alleles at the same locus have high sequence variability or polymorphism. Polymorphism is typically concentrated in the α1 and α2 domains of class I molecules and the α1 and β1 domains of class II molecules. The number of alleles and the degree of polymorphism within an allele may vary at different loci. For example, in HLA-DR molecules, all polymorphism is attributed to the β chain, while the α chain is relatively invariant. For HLA-DQ, both the α and β chains are polymorphic.

[0175] The peptides of the present disclosure may be isolated peptides. The terms "isolated" or "biologically pure" refer to material that is substantially or essentially free from components that normally accompany it as found in its native state. Thus, peptides of the present disclosure do not contain materials normally associated with their in situ environment, e.g., other surface proteins on antigen-presenting cells. Even when proteins are isolated to a homogeneous or predominant band, there will be trace amounts of contaminants, ranging from 5 to 10% of the native protein that copurifies with the desired protein. Isolated peptides of the present disclosure do not contain such endogenous copurified proteins.

[0176] The term "residue" refers to an amino acid or amino acid mimetic incorporated into an oligopeptide by an amide bond or amide bond mimetic. Peptides suitable for use in the present disclosure can be obtained in a variety of ways. Conveniently, they can be synthesized by conventional techniques using an automated synthesizer, such as a Beckman, Applied Biosystems, or other commercially available peptide synthesizer using well-known protocols. They can also be synthesized manually using techniques well-known in the art. See, for example, Stewart and Young, Solid Phase Peptide Synthesis, (Rockford, Ill., Pierce), 2d Ed. (1984), incorporated herein by reference.

[0177] Alternatively, or in addition, DNA sequences encoding specific MHC molecules can be cloned and expressed to provide peptides. Cells containing various MHC genes are readily available; for example, they may be obtained from the American Type Culture Collection ("Catalogue of Cell Lines and Hybridomas," 6th edition (1988) Rockville, Md., USA). Standard techniques can be used to screen cDNA libraries to identify sequences encoding the desired sequences (see Sambrook et al., Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989, incorporated herein by reference). Fusion proteins (composed of all or part of the amino acid sequences of two or more proteins) can be produced recombinantly. Additionally, unrelated proteins can be mutated to contain the appropriate sequences using in vitro mutagenesis techniques.

[0178] MHC glycoproteins from various natural sources are also conveniently isolated using standard protein purification techniques. Peptides can be purified by any of a variety of known techniques, including, for example, reverse-phase high performance liquid chromatography (HPLC), ion exchange or immunoaffinity chromatography, size separation, or electrophoresis (see generally Scopes, R., Protein Purification, Springer-Verlag, NY (1982), incorporated herein by reference).

[0179] It is understood that the peptides of the present disclosure can be modified to provide various desirable attributes, such as improved pharmacological properties, while increasing or at least retaining substantially all of the biological activity of the unmodified peptide. For example, the peptides can be modified by lengthening or shortening the amino acid sequence of the peptide. Substitutions with different amino acids or amino acid mimetics can also be made.

[0180] In some embodiments, the peptides conjugated on the surface of apoptotic leukocytes are derived from MHC molecules. The term "MHC molecule" refers to a molecule comprising a protein sequence of a major histocompatibility complex (MHC) glycoprotein. As used herein, the term "MHC" is understood to refer to the major histocompatibility complex, which is defined as the set of genetic loci that specify the glycoprotein antigens of the major histocompatibility complex, including human leukocyte antigens (HLA). As used herein, the term "HLA" is understood to refer to human leukocyte antigens, which are defined as the major histocompatibility antigens found in humans. As used herein, "HLA" refers to the human form of "MHC" and can therefore be used interchangeably. Examples of HLA proteins that can be utilized in accordance with the presently disclosed and claimed inventive concepts include, but are not limited to, the HLA class I α chain, the HLA class II α chain, and the HLA class II β chain. Specific examples of HLA class II α and / or β proteins that may be utilized in accordance with the presently disclosed and claimed inventive concepts include, but are not limited to, those encoded by the following loci: HLA-DRA; HLA-DRB1; HLA-DRB3, 4, 5; HLA-DQA; HLA-DQB; HLA-DPA; and HLA-DPB.

[0181] In some embodiments, the peptides may be derived from MHC class I molecules or variants of such peptides derived from MHC class I molecules. In some embodiments, the peptides may be derived from MHC class II molecules or variants of peptides derived from MHC class II molecules. MHC molecules are heterodimeric glycoproteins expressed in the cells of higher vertebrates and play a role in immune responses. In humans, these molecules are called human leukocyte antigens (HLA). MHC glycoproteins are divided into two groups, class I and class II, which are structurally and functionally distinct from each other. In general, the primary function of MHC molecules is to bind antigenic peptides and display them on the surface of cells. MHC-encoded glycoproteins have been extensively studied in both human and mouse systems, and their nucleic acid and protein sequences are well known in the art. Many histocompatibility proteins have been isolated and characterized. For a general review of the structure and function of MHC glycoproteins, see Fundamental Immunology, 3d Ed., WE Paul, ed., (Ravens Press NY 1993).

[0182] MHC class I molecules are expressed on almost all nucleated cells and are recognized by cytotoxic T lymphocytes, which then destroy antigen-bearing cells. In mice, class I molecules are encoded by the K, D, and Qa regions of the MHC. Class II molecules are encoded by the IA and IE subregions. Isolated antigens encoded by the mouse IA and IE subregions have been shown to consist of two noncovalently linked peptide chains: a 32-38 kd α chain and a 26-29 kd β chain. A third, invariant 31 kd peptide is noncovalently associated with these two peptides, but this is not polymorphic and does not appear to be a component of the antigen on the cell surface. The α and β chains of several allelic variants of the IA region have been cloned and sequenced.

[0183] Human class I proteins have also been studied (Bjorkman, PJ, et (HLA-A2, HLA-A1, HLA-A2, HLA-A2-A2) (HLA-A2, HLA-A2 ...

[0184] Structurally, MHC class I molecules are heterodimers composed of two non-covalently linked polypeptide chains: a larger "MHC class I heavy chain (α)" and a smaller "light" chain (β-2-microglobulin). The polymorphic, polygenic heavy chain (45 kDa) is encoded within the MHC on chromosome 6. Chromosome 6 contains three loci, HLA-A, HLA-B, and HLA-C, the first two of which contain multiple alleles encoding the MHC class I heavy chain alloantigens, HLA-A and HLA-B, respectively. MHC class I heavy chain HLA (α) (e.g., HLA-A, HLA-B, and HLA-C) are subdivided into three extracellular domains (designated α1, α2, and α3), one intracellular domain, and one transmembrane domain. The two outermost extracellular domains, α1 and α2, together form a groove that binds to antigenic peptides. Thus, interaction with TCR occurs in this region of the protein. The third extracellular domain of the molecule contains the recognition site for the CD8 protein in CTLs, and this interaction serves to stabilize the contact between the T cell and the APC.

[0185] The invariant light chain (12 kDa) encoded outside the MHC on chromosome 15 consists of a single extracellular polypeptide. The terms "MHC class I light chain," "β-2-microglobulin," and "β2m" may be used interchangeably herein. Association of class I heavy and light chains is necessary for the expression of class I molecules on the cell membrane. In this context, the α3 domain of the β2-microglobulin protein and heavy chain associate. Therefore, the MHC class I molecule disclosed herein may refer to an MHC class I heterodimer, an MHC class I heavy chain (e.g., HLA-A, HLA-B, or HLA-C), an MHC class I light chain, or portions thereof. In some embodiments, the peptide may be derived from an MHC class I heavy chain, e.g., HLA-A or HLA-B. In some embodiments, the entire MHC class I heavy chain may be used. In some embodiments, the MHC class I molecule may be a domain (α1, α2, or α3) of the MHC class I heavy chain. In some embodiments, the peptide may comprise a sequence derived from the α1, α2, or α3 region of an MHC class I heavy chain, or may comprise a sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, 99.95%, or 100% identical thereto. The α1 and α2 domains of the heavy chain contain hypervariable regions that form the antigen-binding site to which the peptide binds. In some embodiments, the peptide may be derived from the α1 or α2 domain of an MHC class I heavy chain. In some embodiments, the peptide derived from an MHC class I molecule may comprise a sequence derived from a hypervariable region of an MHC class I molecule. In some embodiments, one or more peptides derived from MHC class I are selected from the tables disclosed herein or are 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, 99.95% or 100% identical to a peptide selected from the tables disclosed herein.

[0186] Cells (e.g., white blood cells) for tolerization vaccines or conditioning regimens can be treated with a fixative or cross-linking agent (e.g., a carbodiimide such as ECDI) in the presence of one or more antigens and / or epitopes. The antigens and / or epitopes can include antigens and / or epitopes from the transplant donor, transplant recipient, a third party, or a combination thereof. In some cases, cells in tolerization vaccines or conditioning regimens are coupled to antigens and / or epitopes from the recipient. In some cases, cells in tolerization vaccines or conditioning regimens are coupled to antigens and / or epitopes from a third party. In some cases, cells in tolerization vaccines or conditioning regimens are coupled to antigens and / or epitopes from the transplant donor.

[0187] MHC class II molecules are primarily expressed on cells involved in the initiation and support of immune responses, such as T lymphocytes, B lymphocytes, and macrophages. MHC class II molecules are recognized by helper T lymphocytes, inducing their proliferation and the amplification of the immune response to the specific antigen peptide displayed. Engagement of the T cell receptor induces a series of molecular events characteristic of cell activation, including increased tyrosine phosphorylation, Ca influx, PI turnover, cytokine and cytokine receptor synthesis, and cell division (see Altman et al., (1990) Adv. Immunol. 48:227-360). For a general discussion of how T cells recognize antigens, see Gray, HM, et al., Scientific American pp 56-64, (November, 1989). The MHC class II glycoproteins HLA-DR, HLA-DQ, and HLA-DP (encoded by alleles at the HLA-DR, DP, and DQ loci) have domain structures containing antigen-binding sites similar to those of class I. MHC class II molecules are heterodimers consisting of two nearly homologous subunits: α and β chains, both of which are encoded in the MHC. Thus, in some embodiments, an MHC class II molecule refers to a heterodimer of an MHC class II α chain and an MHC class II β chain (e.g., HLA-DQ, HLA-DR, HLA-DP). In some embodiments, an MHC class II molecule can be a subunit of a heterodimer. In some embodiments, an MHC class II molecule can be an MHC class II α chain (e.g., HLA-DPA, HLA-DQA, or HLA-DRA), or an MHC class II β chain (e.g., HLA-DPB, HLA-DQB, or HLA-DRB), or a domain thereof. In some embodiments, the MHC class II molecule is HLA-DRB.

[0188] HLA-DRB is encoded by four loci in humans (HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB4), however, no more than three functional loci are present in a single individual and no more than two functional loci are present on a single chromosome. In some embodiments, the MHC class II molecule that is HLA-DRB is encoded by the HLA-DRB1, HLA-DRB3, HLA-DRB4, or HLA-DRB4 locus. In some embodiments, the MHC class II molecule is encoded by the HLA-DRB1, HLA-DRB3, HLA-DRB4, or HLA-DRB4 locus. * 03 or HLA-DRB1 *The HLA-DRB1 locus is widely distributed and encodes a large number of functionally variable gene products (HLA-DR1 to HLA-DR17). The HLA-DRB3 locus, which specifically encodes HLA-DR52, is moderately variable and variably associated with certain HLA-DRB1 types. The HLA-DRB4 locus encodes HLA-DR53. In some embodiments, the MHC class II molecule that is an HLA-DRB is selected from HLA-DR1, HLA-DR2, HLA-DR3, HLA-DR4, or HLA-DR5.

[0189] Each subunit in a class II molecule consists of globular domains designated α1, α2, β1, and β2. All but the α1 domain are stabilized by intrachain disulfide bonds, typical of molecules of the immunoglobulin superfamily. Each chain in a class II molecule contains two ectodomains: a 33 kDa α chain containing α1 and α2 ectodomains, and a 28 kDa β chain containing β1 and β2 ectodomains. The membrane-proximal α2 and β2 domains, like the membrane-proximal third extracellular domain of class I heavy chain molecules, possess sequence homology to the immunoglobulin folding domain structure. The membrane-distal domain of a class II molecule is composed of the α1 and β1 domains, which form the antigen-binding cleft for processed peptide antigens. Thus, MHC class II molecules can contain globular domains, such as α1, α2, β1, or β2. Peptides derived from MHC class II molecules can contain the entire subunit (α or β chain), or a majority of them. For example, the peptide can comprise approximately 90-100 residues of an extracellular domain from an MHC class II subunit (e.g., β1 and β2, or α1 and α2, of a class II molecule), or can comprise 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, 99.95%, or 100% sequence identity thereto. The α1 and β1 domains in the N-terminal portions of the α and β chains contain hypervariable regions that are thought to contain most of the antigen-binding site (see Brown et al., Nature 364:33-39 (1993)). Thus, a peptide derived from an MHC class II molecule can include a sequence derived from a hypervariable region of an MHC class II molecule (e.g., the α1 and β1 domains of the α and β chain subunits, respectively), or a sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, 99.95% or 100% identical thereto.

[0190] In some embodiments, the peptide is derived from a hypervariable region of the α or β chain of an MHC class II molecule associated with a harmful immune response. In this manner, the ability of antigen-presenting cells (APCs) to present a target antigen (e.g., an autoantigen or an allergen) is inhibited.

[0191] Conjugating recipient-type MHC class II molecules to apoptotic cells such as leukocytes or mesenchymal stromal cells can enhance the tolerance-inducing efficacy of conditioning regimens for allogeneic or xenogeneic transplants (e.g., transplants of pancreatic islets, kidneys, or other cells, tissues, or organs, such as cells, tissues, and organs derived from embryonic stem cells or induced pluripotent stem cells (iPS)) in mammalian recipients. For example, for partially or completely MHC class I and class II-mismatched donor-recipient pairs, conjugating recipient-type MHC class II molecules to ADLs can enhance the effectiveness of the ADL in inducing tolerance to the transplanted cells, tissues, or organs. Coupling one or more peptides derived from one (or more) of the transplant recipient's MHC class II molecules to the surface of the completely mismatched ADL can provide abundant recipient-type MHC class II peptides for presentation by the recipient's MHC class II molecules after uptake of the ADL (e.g., by recipient splenic marginal zone antigen-presenting cells or liver sinusoidal endothelial cells). Recognition of self-MHC class II can promote tolerance in the recipient, for example, via regulatory T cell subsets.

[0192] An additional example of a means by which tolerance can be induced is through the activation of thymus-derived CD4+ Tregs (tTregs). For example, in experimental transplantation studies in mice, the development of MHC class II peptide-specific tTregs and tolerance of MHC-II-matched allografts can depend on the thymus expression of donor MHC class II-derived peptides. tTregs are selected by recognizing their own MHC class II peptides presented by their own MHC class II molecules. A significant proportion of self-peptides bound to and presented by MHC class II complexes are derived from MHC class II itself. Thus, many circulating (t)Tregs have specificity for self-MHC class II peptides. When this complex is presented on the surface of activated effector T cells, it can serve as a potent activation signal for tTregs, leading to tTreg activation and thus to the promotion of immune tolerance.

[0193] One way that tTregs can be activated is through trogocytosis of MHC class II peptides presented by MHC class II molecules into activated T cells. Trogocytosis involves the exchange of whole MHC class II molecules presenting MHC class II peptides. Trogocytosis of MHC class II complexes with bound self-MHC class II (e.g., DRB) peptides into activated T cells can transform these T cells into potent activators of tTregs with specificity for the same self-MHC class II peptides. Therefore, when recipient-type MHC class II molecules presenting the recipient's MHC class II peptides are delivered to and presented by activated recipient T cells, this can serve as a potent activation signal for tTregs. While tTreg activation requires antigen specificity, their regulatory function does not. Thus, activated tTregs can directly downregulate anti-donor immunity, including donor-specific CD4+ and CD8+ T cells of direct and indirect specificity, and can also downregulate anti-donor immunity through the expansion of other immune cell subsets with regulatory capacity, including Tr1 cells.

[0194] Self-MHC class II peptides (e.g., DRB peptides) that bind to self-MHC class II can also contribute to the induction of tolerance via LAG-3 receptor signaling. MHC class II peptides (e.g., DRB peptides) that bind to the MHC class II complexes from which they are derived can stabilize the peptide:MHC class II conformation required for recognition by and signal transduction through the LAG-3 receptor on T cells. LAG-3 is a TCR co-receptor that can distinguish stable peptides from unstable peptide:MHC class II complexes. The specificity of peptides that bind to self-MHC class II can therefore regulate the specificity of immune responses via LAG-3. Thus,

[0195] The expression and function of LAG-3 are associated with tolerance induction. For example, coexpression of LAG3 and CD49b can be used to identify Tr1 cells, and blocking LAG3 on Tr1 cells abrogates Tr1-induced tolerance. LAG3 is also expressed transiently in activated tTregs and at significantly lower levels in activated Teff cells, suggesting that LAG3 may be a reliable marker for cells with regulatory capacity. In addition, LAG3 crosslinking of MHC class II in DCs tolerizes DCs. Given that self-MHC class II peptides stabilize self-MHC class II peptides, which present peptides in a conformation relevant to LAG-3 recognition, delivery of abundant amounts of self-MHC class II peptides (e.g., DRB peptides) may enhance the presence of stable self-peptide-MHC class II complexes in DCs, thereby contributing to tolerance (e.g., via expansion of Tr1 cells). This is supported by data showing that apoptotic donor leukocytes matched with one MHC class II DRB allele promote tolerance in allogeneic transplant recipients.

[0196] One or more peptides (e.g., DRα chain, DRβ chain, DQα chain, DQβ chain, DPα chain, or DPβ chain) derived from one (or more) transplant recipient's MHC class II molecules can be conjugated to the surface of ADL leukocytes of a tolerization vaccine or conditioning regimen. In some cases, the one or more peptides derived from the transplant recipient's MHC class II molecules include a DRβ chain, a DQβ chain, a DPβ chain, or a combination thereof. In some cases, the one or more peptides are derived from one or more transplant recipient's MHC class II DRB alleles. In some cases, the one or more peptides are derived from one or more transplant recipient's MHC class II DRA alleles. In some cases, the one or more peptides are derived from one or more transplant recipient's MHC class II DQA alleles. In some cases, the one or more peptides are derived from one or more transplant recipient's MHC class II DQB alleles. In some cases, the one or more peptides are derived from one or more MHC class II DPA alleles of the transplant recipient. In some cases, the one or more peptides are derived from one or more MHC class II DPB alleles of the transplant recipient.

[0197] The DRα chain may be functionally monomorphic, and therefore, in some embodiments, peptides derived from other MHC class II molecules are preferred for tolerance induction. In other embodiments, the effectiveness of a conditioning regimen for inducing tolerance can be enhanced by conjugating recipient-type MHC class II DRA molecules to apoptotic donor leukocytes (ADLs). The DRα chain may be functionally monomorphic, making it a favorable target for tolerance induction. In some cases, recipient-type MHC class II presenting peptides derived from monomorphic DRα chains can serve as an activation signal for a subset of tTreg cells selected in the thymus for their cognate specificity. Any cell, such as any easily expandable T cell derived from a universal cell, can be conjugated with the same chain, domain, or peptide derived from a monomorphic DRA antigen and processed to generate ADLs that can be used to promote tolerance to the graft. This method may be broadly applicable to a range of transplant scenarios in which the recipient is positive for the DRA antigen. For example, this method can be used to induce tolerance using universal donor cells.

[0198] In some cases, the ADL is completely MHC-mismatched with the transplant recipient. In some cases, the ADL is partially MHC-matched with the transplant recipient (e.g., shares one or more MHC class I or MHC class II antigens with the recipient). In some cases, the ADL is haploidentical with the transplant recipient. In some cases, the ADL is from the same donor as the graft. In some cases, the ADL is not from the same donor as the graft.

[0199] Peptides derived from recipient MHC class II molecules may comprise the entire MHC class II molecule. Peptides derived from recipient MHC class II molecules may comprise the entire α chain of DR, DQ, or DP. Peptides derived from recipient MHC class II molecules may comprise the entire β chain of DR, DQ, or DP. Peptides derived from recipient MHC class II molecules may comprise the entire α1 and / or α2 domain of DR, DQ, or DP. Peptides derived from recipient MHC class II molecules may comprise the entire β1 and / or β2 domain of DR, DQ, or DP. Peptides derived from recipient MHC class II molecules may comprise MHC-DR1, MHC-DR2, MHC-DR3, MHC-DR4, and / or MHC-DR5.

[0200] Peptides derived from recipient MHC class II molecules may include fragments of the α1 and / or α2 domains of DR, DQ, or DP. Peptides derived from recipient MHC class II molecules may include fragments of the β1 and / or β2 domains of DR, DQ, or DP. Peptides derived from recipient MHC class II molecules may include fragments of MHC-DR1, MHC-DR2, MHC-DR3, MHC-DR4, and / or MHC-DR5. Peptides derived from recipient MHC class II molecules may include sequences derived from hypervariable regions. Peptides derived from recipient MHC class II molecules may include in silico identified high, medium, or low affinity peptides (e.g., peptides of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids) derived from the hypervariable regions of DRB molecules. The peptides derived from the recipient MHC class II molecule can include a variable region spanning the peptide-binding region (e.g., a peptide of about 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 40 amino acids). The peptides derived from the recipient MHC class II molecule can include dimeric peptides with cathepsin S-cleavable linkers that have different affinities for the DRB-binding groove. In some cases, the peptides derived from the recipient MHC class II molecule do not include peptides derived from the alpha chains of the DR, DQ, or DP.

[0201] The peptide derived from the recipient MHC class II molecule may be synthetic or recombinant. In some cases, the peptide derived from the recipient MHC class II molecule may be about 10-30 amino acids in length. The peptide derived from the recipient MHC class II molecule may be at least 10-30 amino acids in length. In some embodiments, the peptide derived from the recipient MHC class II molecule may be at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 275, 200, or 250 amino acids in length. In some embodiments, peptides derived from recipient MHC class II molecules can be up to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 275, 200 or 250 amino acids in length. In some embodiments, peptides derived from recipient MHC class II molecules can be about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 275, 200 or 250 amino acids in length. In some embodiments, peptides derived from recipient MHC class II molecules can be about 5-250, 10-250, 20-250, 30-250, 50-250, 100-250, 5-100, 10-100, 20-100, 30-100, 50-100, 5-50, 10-50, 20-50, 30-50, 5-30, 10-30, 15-30, or 20-30 amino acids in length.

[0202] In some cases, peptides derived from recipient MHC class II molecules may have high affinity for binding to the peptide-binding groove of HLA DR3 and DR4 molecules that are the most prevalent MHC class II alleles in patients with type 1 diabetes. Non-limiting examples of MHC class II DR3 and DR4 peptides with high affinity for binding to MHC class II DR3 and DR4 molecules in humans are provided in Example 14 (Tables 2-6).

[0203] In some cases, the cells in the tolerizing vaccine or conditioning regimen are coupled to HLA-G, HLA-E, or a combination thereof.

[0204] In some embodiments, a "cocktail" of peptides can be conjugated to the surface of apoptotic leukocytes. In some embodiments, apoptotic cells such as leukocytes can contain one or more peptides derived from MHC class II molecules. In some embodiments, apoptotic cells such as leukocytes can contain, for example, at least 2, 3, 5, 7, 10, 15, 20, 30, 40, 50, 100, or more peptides derived from MHC class II molecules. In some embodiments, apoptotic cells such as leukocytes can contain one or more peptides derived from MHC class I molecules. In some embodiments, apoptotic cells such as leukocytes can contain, for example, at least 2, 3, 5, 7, 10, 15, 20, 30, 40, 50, 100, or more peptides derived from MHC class I molecules. A mixture of two or more peptides derived from MHC class II molecules has the advantage of inducing an increased immune tolerance response in the recipient. Increased tolerance can be due, for example, to a mechanism called linkage abrogation. The mechanism of linkage abrogation is known to those skilled in the art. For example, peptides containing sequences from the hypervariable regions of the α and β chains may be used in combination.In certain embodiments, the size of the protein or polypeptide (wild type or modified) is, but is not limited to, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 0, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 1 2, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 7 The peptides may comprise 00, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500, or more amino acids, including any range or value derivable therein, or derivations thereof. In certain embodiments, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, or more consecutive amino acids and fragments of an MHC molecule, including derivatives, can be used. It is contemplated that peptides can be mutated by truncation to be shorter than their corresponding wild-type forms, but can also be altered by fusing or conjugating to heterologous protein sequences having specific functions (e.g., for presentation as a protein complex, for enhanced immunogenicity, etc.).

[0205] The MHC class II molecules, MHC class I molecules, and MHC status of a donor or recipient can be determined, for example, by conventional methods of HLA typing or tissue typing known in the art. Non-limiting examples of methods that can be used include serological, cellular, and DNA typing. Serology is used to identify HLA proteins on the surface of cells. Complement-dependent cytotoxicity tests or microlymphocytotoxicity assays can be used for serological identification of MHC molecules. Peripheral blood lymphocytes (PBLs) express MHC class I antigens and are used for serological typing of HLA-A, HLA-B, and HLA-C. MHC class II typing is performed using B lymphocytes isolated from PBLs, as these cells express class II molecules. HLA typing is performed in multiwell plastic trays, with each well containing serum of known HLA specificity.

[0206] Lymphocytes are plated into wells, incubated, and complement (rabbit serum as a source) is added to mediate lysis of antibody-bound lymphocytes (see Terasaki Pi, Nature. 1964). Cellular assays such as mixed lymphocyte culture (MLC) measure differences in class II proteins between individuals. This can be achieved in several ways, all of which are known to those skilled in the art; for example, subtyping can be achieved by mixed lymphocyte response (MLR) typing and by drug-stimulated lymphocyte testing (PLT). Both methods are described in Weir and Blackwell, eds., Handbook of Experimental Immunology, incorporated herein by reference. This can also be achieved by analyzing DNA restriction fragment length polymorphisms (RFLPs) using DNA probes specific to the MHC loci being investigated. Methods for preparing probes for MHC loci are known to those skilled in the art. See, for example, Gregersen et al. (1986), Proc. Natl. Acad. Sci. USA 79:5966, which is incorporated herein by reference. High-resolution differentiation of MHC molecules in grafts and recipients can be achieved by DNA typing. Different HLA alleles defined by DNA typing can identify indistinguishable HLA proteins using serological typing. For example, DRB1 * Individuals with the 040101 allele have DRB1 * will have the same serotype (DR4) as individuals with the 0412 allele. * 040101 and DRB1 * 0412 is a broad specificity DR4 split. These splits are identified by DNA typing.

[0207] Identification of MHC molecules can be achieved by sequencing the genomic DNA of the locus or the cDNA relative to the mRNA encoded within the locus. The sequenced DNA includes the section encoding the hypervariable region of the MHC-encoded polypeptide. Techniques for amplifying the desired region to specifically identify the desired DNA with a probe are known in the art, including, for example, polymerase chain reaction (PCR) techniques.

[0208] Alternatively, polymorphic DNA sequences can be used as amplification primers, in which case only alleles containing sequences complementary to these primers will anneal to the primers and undergo amplification.This second strategy of DNA typing is called sequence-specific primer (SSP) method, and is described, for example, in Altaf et al. World J Transplant. 2017, Erlich HA et al. Immunity, Vol. 14, 347-356, April 2001, Dunckley H, Methods Mol Biol. 2012, US Patent Application Publication No. 20090069190A1, and US Patent Application Publication No. US20110117553A1.Those skilled in the art can determine the protein product once the gene sequence of an MHC molecule is determined by DNA typing method.In some embodiments, DNA sequences can be used for recombinant synthesis of peptides of MHC class II molecules and MHC class I molecules.

[0209] Alternatively, or in addition, cells can be treated with a fixative or cross-linking agent in the presence of biotin or streptavidin.In this case, one or more immunomodulatory molecules, agents that increase the expression of anti-inflammatory cytokines, and / or antigens or epitopes can be coupled with streptavidin or biotin, and can be contacted with cells after contact with a fixative or cross-linking agent.One or more immunomodulatory molecules, agents that increase the expression of anti-inflammatory cytokines, and / or antigens or epitopes can be bound to cells via streptavidin-biotin interaction.

[0210] An alternative strategy may involve coupling one or more of immunomodulatory molecules, agents that increase expression of anti-inflammatory cytokines in the recipient, antigens and / or epitopes of the transplant donor, antigens and / or epitopes of the transplant recipient, and / or third party antigens and / or epitopes to nanoparticles, such as polystyrene nanoparticles. Such nanoparticles can be administered in conjunction with or as part of a tolerizing vaccine or conditioning regimen.

[0211] As used herein, terms such as "conjugate" or "conjugated to" refer to molecular entities (e.g., a peptide of the present disclosure and apoptotic leukocytes) being linked together by a covalent or non-covalent bond. Conjugation may be achieved by direct coupling of two molecular entities, for example, by creating an ester or amide from a hydroxyl group, an amino group, and a carboxylic acid. Conjugation may also be achieved by indirect coupling of two molecular entities, for example, by the incorporation of a linking group such as polyethylene glycol. Conjugation may also be achieved by modifying the molecular entities with chemical groups that react with each other, for example, an alkyne-functionalized entity with an azide-functionalized entity, or by the reduction of thiol groups on each entity to form a disulfide bond. Conjugates such as ethylene carbodiimide (ECDI), hexamethylene diisocyanate, propylene glycol diglycidyl ether containing two epoxy residues, and epichlorohydrin can be used to immobilize peptides or proteins on the surface of apoptotic leukocytes. Reactive carboxyl groups on the surface of apoptotic leukocytes can be conjugated to free amines (e.g., from Lys residues) on peptides or proteins by reacting them with, for example, 1-ethyl-3-[3,9-dimethylaminopropyl]carbodiimide hydrochloride (EDC) or N-hydroxysuccinimide ester (NHS). Similarly, the same chemicals can be used to conjugate free amines on the surface of apoptotic leukocytes with free carboxyls (e.g., from the C-terminus or from Asp or GIu residues) on peptides or proteins. Alternatively, free amino groups on the surface of apoptotic leukocytes can be covalently linked to peptides and proteins, or peptide or protein fusion proteins, using sulfo-SIAB chemistry essentially as described in Arano et al. (1991) Chem. 2:71-6. A wide variety of means well known in the art can be used to conjugate peptides to the surface of apoptotic leukocytes.These methods include any standard chemistry that allows for conjugation of a sufficient number of peptides to a surface in an orientation that allows for tolerance induction without destroying or significantly limiting the biological activity of the peptides and the biological activity of apoptotic leukocytes. In some embodiments, the C-terminal region of the peptide is conjugated. In other embodiments, the N-terminus of the peptide can be conjugated to the surface of the apoptotic leukocyte. Short-term immunosuppression

[0212] The conditioning regimens or tolerizing vaccines of the present disclosure can include administering one or more immunosuppressants and / or anti-inflammatory agents to the transplant recipient, for example, administering one or more immunosuppressants and / or anti-inflammatory agents in addition to apoptotic cells (such as apoptotic donor leukocytes or mesenchymal stromal cells).

[0213] In some cases, the conditioning regimen or tolerizing vaccine of the present disclosure can include short-term immunosuppression of the transplant recipient. In some cases, the transplant recipient's tolerance to the transplanted cells, organ, or tissue can be maintained despite the recipient not being administered long-term or maintenance immunosuppression.

[0214] Short-term immunosuppression can include administering to the transplant recipient any of the immunosuppressive and / or anti-inflammatory agents disclosed herein.

[0215] In some embodiments, the tolerizing vaccine or conditioning regimen of the present disclosure can include administering apoptotic cells (e.g., apoptotic donor leukocytes or mesenchymal stromal cells) to the recipient, and short-term immunosuppression including an mTOR inhibitor (e.g., rapamycin).

[0216] In exemplary embodiments, the tolerizing vaccine or conditioning regimen of the present disclosure can include administering apoptotic donor leukocytes to a recipient and short-term immunosuppression including one or more of: (i) an mTOR inhibitor, (ii) an anti-tumor necrosis factor agent or anti-tumor necrosis factor receptor agent, (iii) an anti-interleukin-6 agent or anti-interleukin-6 receptor agent, and (iv) an anti-CD40 agent or anti-CD40 ligand agent. The short-term administration of these agents along with the apoptotic donor leukocytes disclosed herein can promote long-term tolerance to the transplanted cells, organ, or tissue, despite the recipient not being administered long-term maintenance immunosuppression.

[0217] In one exemplary embodiment, short-term immunosuppression comprises administering an mTOR inhibitor, an anti-tumor necrosis factor agent, an anti-interleukin-6 receptor agent, and an anti-CD40 agent. Short-term administration of these agents, along with the apoptotic donor leukocytes disclosed herein, can promote long-term tolerance to the transplanted cells, organ, or tissue, despite the recipient not being administered long-term maintenance immunosuppression.

[0218] In one exemplary embodiment, short-term immunosuppression comprises administering an mTOR inhibitor, a soluble anti-tumor necrosis factor receptor, an anti-interleukin-6 receptor antibody, and an antagonistic anti-CD40 antibody. Short-term administration of these agents together with the apoptotic donor leukocytes disclosed herein can promote long-term tolerance to the transplanted cells, organ, or tissue, despite the recipient not being administered long-term maintenance immunosuppression.

[0219] Non-limiting examples of mTOR inhibitors include rapamycin, sirolimus, and everolimus. In some cases, the mTOR inhibitor of the present disclosure is rapamycin.

[0220] The anti-tumor necrosis factor agent or anti-tumor necrosis factor receptor agent may comprise an anti-tumor necrosis factor antibody or its antigen-binding fragment, an antagonistic anti-tumor necrosis factor receptor antibody or its antigen-binding fragment, or a soluble tumor necrosis factor binding domain of a tumor necrosis factor receptor (e.g., etanercept). The tumor necrosis factor may be TNF-alpha. In some cases, the anti-tumor necrosis factor agent or anti-tumor necrosis factor receptor agent is a soluble tumor necrosis factor binding domain of a tumor necrosis factor receptor (e.g., etanercept). Non-limiting examples of anti-tumor necrosis factor agents and anti-tumor necrosis factor receptor agents include etanercept, infliximab, adalimumab, certolizumab pegol, and golimumab.

[0221] The anti-interleukin 6 agent or anti-interleukin 6 receptor agent can be an anti-interleukin 6 antibody, an antagonistic anti-interleukin 6 receptor antibody, or an antigen-binding fragment thereof.In some cases, the anti-interleukin 6 agent or anti-interleukin 6 receptor agent of the present disclosure is an antagonistic anti-interleukin 6 receptor antibody (e.g., tocilizumab).Non-limiting examples of anti-interleukin 6 agents or anti-interleukin 6 receptor agents include tocilizumab, Actemra, clazakizumab, ALD518, siltuximab, elcilimomab, sirumab, sarilumab and olokizumab.

[0222] The anti-CD40 agent or anti-CD40 ligand agent may be, for example, an antagonistic anti-CD40 antibody or an antagonistic anti-CD40 ligand antibody, or an antigen-binding fragment thereof. The anti-CD40 agent or anti-CD40 ligand agent may be an antagonistic anti-CD40 antibody or an antigen-binding fragment thereof. Non-limiting examples of antagonistic anti-CD40 antibodies include 2C10, 2C10R4, ASKP1240, 4D11, bleselumab, BI-655064, HCD122, CFZ533, ch5D12, CDP7657, and FFP104. In some cases, the anti-CD40 agent is ASKP1240. The anti-CD40 agent or anti-CD40 ligand agent may be an antagonistic anti-CD40 ligand antibody or an antigen-binding fragment thereof. Non-limiting examples of antagonistic anti-CD40 ligand antibodies include BG9588, ruplizumab, toralizumab, IDEC-131, dapirolizumab, letolizumab, BMS-986004, VIB4920, and MEDI4920.

[0223] In some embodiments, short-term immunosuppression comprises administering an mTOR inhibitor. In some embodiments, short-term immunosuppression comprises administering an anti-tumor necrosis factor agent or an anti-tumor necrosis factor receptor agent. In some embodiments, short-term immunosuppression comprises administering an anti-interleukin-6 agent or an anti-interleukin-6 receptor agent. In some embodiments, short-term immunosuppression comprises administering an anti-CD40 agent or an anti-CD40 ligand agent.

[0224] In some embodiments, short-term immunosuppression comprises administering (i) an mTOR inhibitor, and (ii) an anti-tumor necrosis factor agent or an anti-tumor necrosis factor receptor agent. In some embodiments, short-term immunosuppression comprises administering (i) an mTOR inhibitor, and (ii) an anti-interleukin-6 agent or an anti-interleukin-6 receptor agent. In some embodiments, short-term immunosuppression comprises administering (i) an mTOR inhibitor, and (ii) an anti-CD40 agent or an anti-CD40 ligand agent. In some embodiments, short-term immunosuppression comprises administering (i) an anti-tumor necrosis factor agent or an anti-tumor necrosis factor receptor agent, and (ii) an anti-interleukin-6 agent or an anti-interleukin-6 receptor agent. In some embodiments, short-term immunosuppression comprises administering (i) an anti-tumor necrosis factor agent or an anti-tumor necrosis factor receptor agent, and (ii) an anti-CD40 agent or an anti-CD40 ligand agent. In some embodiments, short-term immunosuppression comprises administering (i) an anti-interleukin-6 agent or an anti-interleukin-6 receptor agent, and (ii) an anti-CD40 agent or an anti-CD40 ligand agent.

[0225] In some embodiments, short-term immunosuppression comprises administering (i) an mTOR inhibitor, (ii) an anti-tumor necrosis factor agent or anti-tumor necrosis factor receptor agent, and (iii) an anti-interleukin-6 agent or anti-interleukin-6 receptor agent. In some embodiments, short-term immunosuppression comprises administering (i) an mTOR inhibitor, (ii) an anti-tumor necrosis factor agent or anti-tumor necrosis factor receptor agent, and (iii) an anti-CD40 agent or anti-CD40 ligand agent. In some embodiments, short-term immunosuppression comprises administering (i) an mTOR inhibitor, (ii) an anti-interleukin-6 agent or anti-interleukin-6 receptor agent, and (iii) an anti-CD40 agent or anti-CD40 ligand agent. In some embodiments, short-term immunosuppression comprises administering (i) an anti-tumor necrosis factor agent or anti-tumor necrosis factor receptor agent, (iii) an anti-interleukin-6 agent or anti-interleukin-6 receptor agent, and (iii) an anti-CD40 agent or anti-CD40 ligand agent.

[0226] Short-term immunosuppression can include administering one or more immunosuppressants and / or anti-inflammatory agents of the present disclosure for up to about 100 days after transplantation. Short-term immunosuppression can include administering one or more immunosuppressants and / or anti-inflammatory agents of the present disclosure for, for example, up to about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 25, 28, 29, 30, 31, 32, 33, 34, 35, 40, 42, 49, 50, 55, 60, 70, 80, 90, or 100 days after transplantation. In some cases, short-term immunosuppression can be terminated within 28 days after transplantation. In some cases, short-term immunosuppression can be terminated about 21 days after transplantation.

[0227] In some embodiments, short-term immunosuppression can be initiated before transplantation. For example, short-term immunosuppression can be initiated about or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 25, 28, 29, 30, 35, or 50 days before transplantation. In some cases, short-term immunosuppression can be initiated at most about 10 days before transplantation. In some cases, short-term immunosuppression can be initiated about 7 days before transplantation.

[0228] The duration of short-term immunosuppression (e.g., the length of time between administration of the first and last doses of immunosuppressant and / or anti-inflammatory agent) can be about or up to about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 25, 28, 29, 30, 31, 32, 33, 34, 35, 40, 42, 49, 50, 55, 60, 70, 80, 90, or 100 days. In some cases, the duration of short-term immunosuppression can be about or up to about 30 days. In some cases, the duration of short-term immunosuppression can be about 28 days.

[0229] In some cases, short-term immunosuppression involves administering the mTOR inhibitor up to about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 25, 28, 29, 30, 31, 32, 33, 34, 35, 40, 42, 49, 50, 55, 60, 70, 80, 90, or 100 days after transplant. In some cases, short-term immunosuppression involves administering the mTOR inhibitor up to about 21 days after transplant.

[0230] In some cases, short-term immunosuppression involves administering an mTOR inhibitor starting about or up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 25, 28, 29, 30, 35, or 50 days prior to transplant. In some cases, short-term immunosuppression involves administering an mTOR inhibitor starting about or up to about 7 days prior to transplant.

[0231] In some cases, short-term immunosuppression involves administering an mTOR inhibitor for a period of about or up to about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 25, 28, 29, 30, 31, 32, 33, 34, 35, 40, 42, 49, 50, 55, 60, 70, 80, 90, or 100 days. In some cases, short-term immunosuppression involves administering an mTOR inhibitor for a period of about or up to about 28 days.

[0232] The mTOR inhibitor can be administered for short-term suppression on about any one or more of, for example, days −14, −13, −12, −11, −10, −9, −8, −7, −6, −5, −4, −3, −2, −1, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35 relative to implantation on day 0. In some cases, the mTOR inhibitor can be administered for short-term suppression from about day −7 to day 21 relative to implantation on day 0.

[0233] In some cases, short-term immunosuppression involves administering the anti-tumor necrosis factor agent or anti-tumor necrosis factor receptor agent up to about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 25, 28, 29, 30, 31, 32, 33, 34, 35, 40, 42, 49, 50, 55, 60, 70, 80, 90, or 100 days after transplantation. In some embodiments, short-term immunosuppression involves administering the anti-tumor necrosis factor agent or anti-tumor necrosis factor receptor agent up to about 21 days after transplantation.

[0234] In some cases, short-term immunosuppression involves administering the anti-tumor necrosis factor agent or anti-tumor necrosis factor receptor agent starting about or up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 25, 28, 29, 30, 35, or 50 days prior to transplant. In some cases, short-term immunosuppression involves administering the anti-tumor necrosis factor agent or anti-tumor necrosis factor receptor agent starting about or up to about 7 days prior to transplant.

[0235] In some cases, short-term immunosuppression comprises administering an anti-tumor necrosis factor agent or anti-tumor necrosis factor receptor agent for a period of about or up to about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 25, 28, 29, 30, 31, 32, 33, 34, 35, 40, 42, 49, 50, 55, 60, 70, 80, 90, or 100 days. In some embodiments, short-term immunosuppression comprises administering an anti-tumor necrosis factor agent or anti-tumor necrosis factor receptor agent for a period of about or up to about 28 days.

[0236] The anti-tumor necrosis factor agent or anti-tumor necrosis factor receptor agent can be administered for short-term suppression on any one or more of about, for example, days −14, −13, −12, −11, −10, −9, −8, −7, −6, −5, −4, −3, −2, −1, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35 relative to transplantation on day 0. In some cases, the anti-tumor necrosis factor agent or anti-tumor necrosis factor receptor agent can be administered for short-term suppression on about days −7, 0, 3, 7, 10, 14, and 21 relative to transplantation on day 0.

[0237] In some cases, short-term immunosuppression involves administering an anti-interleukin-6 agent or anti-interleukin-6 receptor agent up to about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 25, 28, 29, 30, 31, 32, 33, 34, 35, 40, 42, 49, 50, 55, 60, 70, 80, 90, or 100 days after transplantation. In some cases, short-term immunosuppression involves administering an anti-interleukin-6 agent or anti-interleukin-6 receptor agent up to about 21 days after transplantation.

[0238] In some cases, short-term immunosuppression involves administering an anti-interleukin-6 agent or anti-interleukin-6 receptor agent starting about or up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 25, 28, 29, 30, 35, or 50 days prior to transplant. In some cases, short-term immunosuppression involves administering an anti-interleukin-6 agent or anti-interleukin-6 receptor agent starting about or up to about 7 days prior to transplant.

[0239] In some cases, short-term immunosuppression involves administering an anti-interleukin-6 agent or anti-interleukin-6 receptor agent for a period of about or up to about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 25, 28, 29, 30, 31, 32, 33, 34, 35, 40, 42, 49, 50, 55, 60, 70, 80, 90, or 100 days. In some cases, short-term immunosuppression involves administering an anti-interleukin-6 agent or anti-interleukin-6 receptor agent for a period of about or up to about 28 days.

[0240] The anti-interleukin-6 agent or anti-interleukin-6 receptor agent can be administered for short-term suppression on about any one or more of, for example, days −14, −13, −12, −11, −10, −9, −8, −7, −6, −5, −4, −3, −2, −1, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35 relative to transplantation on day 0. In some cases, the anti-interleukin-6 agent or anti-interleukin-6 receptor agent can be administered for short-term suppression on about days −7, 0, 7, 14, and 21 relative to transplantation on day 0.

[0241] In some cases, short-term immunosuppression involves administering an anti-CD40 agent or anti-CD40 ligand agent up to about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 25, 28, 29, 30, 31, 32, 33, 34, 35, 40, 42, 49, 50, 55, 60, 70, 80, 90, or 100 days after transplantation. In some cases, short-term immunosuppression involves administering an anti-CD40 agent or anti-CD40 ligand agent up to about 14 days after transplantation.

[0242] In some cases, short-term immunosuppression involves administering an anti-CD40 agent or anti-CD40 ligand agent starting about or up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 25, 28, 29, 30, 35, or 50 days prior to transplant. In some cases, short-term immunosuppression involves administering an anti-CD40 agent or anti-CD40 ligand agent starting about or up to about 8 days prior to transplant.

[0243] In some cases, short-term immunosuppression involves administering an anti-CD40 agent or anti-CD40 ligand agent for a period of about or up to about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 25, 28, 29, 30, 31, 32, 33, 34, 35, 40, 42, 49, 50, 55, 60, 70, 80, 90, or 100 days. In some cases, short-term immunosuppression involves administering an anti-CD40 agent or anti-CD40 ligand agent for a period of about or up to about 22 days.

[0244] The anti-CD40 or anti-CD40 ligand agent can be administered for short-term suppression on about any one or more of, for example, days −14, −13, −12, −11, −10, −9, −8, −7, −6, −5, −4, −3, −2, −1, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35 relative to transplantation on day 0. In some cases, the anti-CD40 or anti-CD40 ligand agent can be administered for short-term suppression on about days −8, −1, 7, and 14 relative to transplantation on day 0.

[0245] In some cases, short-term immunosuppression can include administering one or more immunosuppressants (e.g., immunomodulatory molecules). One or more immunosuppressants / immunosuppressive drugs can be used together or sequentially. Immunosuppressants include, but are not limited to, anti-CD40 or anti-CD40L (CD154) agents (e.g., anti-CD40 antibodies), B cell targeting agents (e.g., B cell depleting biologics, e.g., biologics targeting CD20, CD19, or CD22, and / or B cell modulating biologics, e.g., biologics targeting BAFF, BAFF / APRIL, CD40, IgG4, ICOS, IL-21, B7RP1), mTOR inhibitors, TNF-alpha inhibitors, IL-6 inhibitors, alpha 1-antitrypsin, nitrogen mustard alkylating agents (e.g., cyclophosphamide), complement C3 or C5 inhibitors, IFN-γ, NFκB inhibitors, vitamin D3, siCD40, cobalt protoporphyrin, insulin B9-23, cluster of differentiation proteins (e.g., CD46, CD55, or CD59), any combination thereof, or any fragment thereof. In some cases, the NFκB inhibitor is dehydroxymethylepoxyquinomycin (DHMEQ), curcumin, triptolide, Bay-117085, or a combination thereof. Non-limiting examples of B cell-targeting biologics include the anti-CD20 antibody rituximab. Any of these additional immunosuppressants can be administered to the subject before and / or after transplantation.

[0246] In some cases, immunosuppressants used for short-term immunosuppression include MMF (mycophenolate mofetil (Cellcept)), ATG (antithymocyte globulin), anti-CD154 (CD40L), alemtuzumab (Campus), CTLA4-Ig (abatacept / Orencia), belatacept (LEA29Y), daclizumab (Zenapax), basiliximab (Simulect), infliximab (Remicade), cyclosporine, deoxyspergualin, and cyclospergualin. The antibody may be any one or more of soluble complement receptor 1, cobra venom factor, compstatin, anti-C5 antibody (eculizumab / soliris), methylprednisolone, FTY720, everolimus, anti-CD154-Ab, leflunomide, anti-IL-2R-Ab, anti-CXCR3 antibody, anti-ICOS antibody, anti-OX40 antibody, and anti-CD122 antibody, human anti-CD154 monoclonal antibody, CD40 antagonist, and CD40L (CD154) antagonist.

[0247] In some cases, the immunosuppressant used for short-term immunosuppression can target the T cell receptor (TCR), CD3e, FK506-binding protein 12 (FKBP12), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), CD40L (CD154), CD40, inducible costimulation (ICOS), IL-2, TNF-α, IL-6, IL-7, CD2, CD20, CD52, alpha-4 integrin, mTOR (mechanistic target of rapamycin, everolimus, cerolimus), DNA synthesis, or any combination thereof.

[0248] In some cases, the immunosuppressant used for short-term immunosuppression can be an MHC / TCR interaction blocker, a non-selective depleting agent, a calcineurin inhibitor, a costimulatory signal blocker, a cytokine blocker, a lymphocyte depleting agent, a cell adhesion inhibitor, an IL-2 signaling inhibitor, a cell cycle blocker, or any combination thereof. For example, the MHC / TCR interaction blocker can be anti-abTCR mAb T10B9. For example, the non-selective depleting agent can be anti-CD3 mAb (OKT3) or anti-thymocyte globulin (ATG). For example, the calcineurin inhibitor can be cyclosporine or tacrolimus. For example, the costimulatory signal blocker can be anti-CTLA-4 mAb, abatacept, ipilimumab, anti-PD-1 (such as pembrolizumab), anti-PD-L1 (such as MPDL3280A), anti-CD154 mAb, anti-CD40 mAb, or anti-ICOS mAb. For example, the cytokine blocker can be an anti-CD25 mAb (such as daclizumab or basiliximab), an anti-TNF (etanercept, infliximab, adalimumab, certolizumab pegol, and golimumab), an anti-IL-6 / IL-6R mAb (such as clazakizumab, ALD518, siltuximab, elcilimomab, sirukumab, olokizumab, sarilumab, tocilizumab, and Actemra), or an anti-IL-7 mAb. For example, the lymphocyte depleting agent can be an anti-CD2 mAb, a fusion protein with IgG1 (such as alefacept), an anti-CD20 mAb (such as rituximab), or an anti-CD52 mAb (such as alemtuzumab). For example, the cell adhesion inhibitor can be an anti-very large antigen 4 (VLA4) (such as natalizumab). For example, the IL-2 signaling inhibitor can be sirolimus (rapamycin) or everolimus. For example, the cell cycle blocker can be mycophenolate mofetil (MMF) or azathioprine. Administration method

[0249] Provided herein is a method for inducing tolerance to a cell, tissue, or organ transplant. The transplant may be a xenograft or an allograft. The method may include administering a composition containing a tolerizing vaccine or a conditioning regimen to a subject. The administration may be by intravenous infusion. The administration of the tolerizing vaccine or conditioning regimen can result in long-term tolerance to the cell, tissue, or organ transplant in the transplant recipient.

[0250] The tolerizing vaccines / regimens or conditioning regimens disclosed herein can increase the survival of a graft (e.g., a xenograft or allograft) in a recipient by at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, or at least 10 years. The tolerizing vaccines or conditioning regimens disclosed herein can also reduce or eliminate the need for immunosuppression after transplantation.

[0251] The xenograft or allograft graft can be an organ, tissue, cell, or cell line, including organs, tissues, cells, or cells differentiated from stem cells. The graft and tolerization vaccine can be from different donors or from the same donor. The graft and tolerization vaccine can be from different species or from the same species. In some cases, the xenograft graft and tolerization vaccine can be from different donors that are substantially genetically identical.

[0252] One or more doses of a tolerization vaccine / regimen or conditioning regimen can be administered to a transplant recipient. One or more doses of a tolerization vaccine or conditioning regimen can be administered before and / or during and / or after the cells, tissues, or organs are transplanted. The day of cell, tissue, or organ transplantation can be referred to as day 0. Days preceding day 0 (the day the recipient receives graft cells, tissues, or organs) can be described as negative numbers. For example, a tolerization vaccine or conditioning regimen administered 7 days before the graft cells, tissues, or organs can be described as being administered on day -7. Similarly, days after the day the recipient receives graft cells, organs, or tissues can be described as being administered on day +7. For example, a tolerization vaccine or conditioning regimen administered 7 days after the graft cells, tissues, or organs can be described as being administered on day 7 or day +7.

[0253] In some cases, the dose of the tolerizing vaccine / regimen or conditioning regimen is administered within at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 days of transplantation of the graft cells, tissue, or organ. , 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 days prior to the administration of the vaccine.

[0254] In some cases, the dose of the tolerizing vaccine / regimen or conditioning regimen is administered at most about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 ​​days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 61 days, 62 days, 63 days, 64 days, 65 days, 66 days, 67 days, 68 days, 69 days, 70 days, 71 days, 72 days, 73 days, 74 days, 75 days, 76 days, 77 days, 78 days, 79 days, 80 days, 81 days, 82 days, 83 days, 84 days, 85 days, 86 days, 87 days, 88 days, 89 days, 90 days, 91 days, 92 days, 93 days, 94 days, 95 days, 96 days, 97 days, 98 days, 99 days, It is administered no earlier than the 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, 40th, 41st, 42nd, 43rd, 44th, 45th, 46th, 47th, 48th, 49th, 50th, 51st, 52nd, 53rd, 54th, 55th, 56th, 57th, 58th, 59th, or 60th day.

[0255] In some cases, the dose of the tolerizing vaccine or conditioning regimen is administered within about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 ​​days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 61 days, 62 days, 63 days, 64 days, 65 days, 66 days, 67 days, 68 days, 69 days, 70 days, 71 days, 72 days, 73 days, 74 days, 75 days, 76 days, 77 days, 78 days, 79 days, 80 days, 81 days, 82 days, 83 days, 84 days, 85 days, 86 days, 87 days, 88 days, 89 days, 90 days, 91 days, 92 days, 93 days, 94 days, 95 days, 96 days, 97 days, 98 days, 99 days, 100 days Administered 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 days prior.

[0256] In some cases, a dose of the tolerizing vaccine / regimen or conditioning regimen is administered on the same day that the recipient receives the graft cells, tissue, or organ (e.g., the dose is administered on day 0). The dose administered on day 0 can be administered simultaneously with the graft cells, tissue, or organ, or can be administered within 24 hours of the graft cells, tissue, or organ. For example, a dose of the tolerizing vaccine or conditioning regimen can be administered at -23, -22, -21, -20, -19, -18, -17, -16, -15, -14, -13, -12, -11, -10, -9, -8, -7, -6, -5, -4, -3, -2, -1, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours relative to the grafted cells, tissue or organ.

[0257] In some cases, the dose of the tolerizing vaccine / regimen or conditioning regimen is administered within at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 days of transplantation of the graft cells, tissue, or organ. , 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 days later.

[0258] In some cases, the dose of the tolerizing vaccine / regimen or conditioning regimen is administered at most about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 ​​days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 61 days, 62 days, 63 days, 64 days, 65 days, 66 days, 67 days, 68 days, 69 days, 70 days, 71 days, 72 days, 73 days, 74 days, 75 days, 76 days, 77 days, 78 days, 79 days, 80 days, 81 days, 82 days, 83 days, 84 days, 85 days, 86 days, 87 days, 88 days, 89 days, 90 days, 91 days, 92 days, 93 days, 94 days, 95 days, 96 days, 97 days, 98 days, 99 days, It will be administered on the 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, 40th, 41st, 42nd, 43rd, 44th, 45th, 46th, 47th, 48th, 49th, 50th, 51st, 52nd, 53rd, 54th, 55th, 56th, 57th, 58th, 59th or 60th day.

[0259] In some cases, the dose of the tolerizing vaccine or conditioning regimen is administered within about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 ​​days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 61 days, 62 days, 63 days, 64 days, 65 days, 66 days, 67 days, 68 days, 69 days, 70 days, 71 days, 72 days, 73 days, 74 days, 75 days, 76 days, 77 days, 78 days, 79 days, 80 days, 81 days, 82 days, 83 days, 84 days, 85 days, 86 days, 87 days, 88 days, 89 days, 90 days, 91 days, 92 days, 93 days, 94 days, 95 days, 96 days, 97 days, 98 days, 99 days, 100 days It is administered on the 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, 40th, 41st, 42nd, 43rd, 44th, 45th, 46th, 47th, 48th, 49th, 50th, 51st, 52nd, 53rd, 54th, 55th, 56th, 57th, 58th, 59th or 60th day.

[0260] The conditioning regimen can include multiple doses of tolerization vaccine before and / or during and / or after transplantation of graft cells, tissues or organs.The multiple doses can be said to include a first dose and one or more booster doses.The first dose can be before or at the same time as the transplantation of graft cells, tissues or organs.If a booster dose is administered, it will be after the first dose.Depending on when the first dose of tolerization vaccine is administered, one or more booster doses can be administered before and / or at the same time as and / or after the transplantation of graft cells, tissues or organs.

[0261] Subsequent (e.g., booster) doses of the tolerization vaccine / regimen can be administered at any time interval after the preceding dose (e.g., initial dose). For example, the subsequent dose can be administered about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 ​​days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 61 days, 62 days, 63 days, 64 days, 65 days, 66 days, 67 days, 68 days, 69 days, 70 days, 71 days, 72 days, 73 days, 74 days, 75 days, 76 days, 77 days, 78 days, 79 days, 80 days, 81 days, 82 days, 83 days, 84 days, 85 days, 86 days, 87 days, 88 days, 89 days, 90 days, 91 days, 92 days, 93 days, The conditioning regimen may be administered 3 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 ​​days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 90 days, 120 days, 150 days, or 180 days later. Depending on when the initial dose is administered, subsequent (e.g., booster) doses can be administered before and / or simultaneously with and / or after transplantation of graft cells, tissues, or organs. In some cases, the conditioning regimen includes at least one dose of a tolerization vaccine before transplantation. In some cases, the conditioning regimen includes at least two doses (e.g., an initial dose and a booster dose) of a tolerization vaccine before transplantation. In some cases, the conditioning regimen includes at least three doses of a tolerizing vaccine (e.g., a primary dose and two booster doses) prior to transplantation. In some cases, the conditioning regimen includes a primary dose of a tolerizing vaccine prior to transplantation and at least one dose of a booster vaccine concurrently with or following transplantation of the graft cells, tissue, or organ.

[0262] In some cases, two doses of the tolerization vaccine or conditioning regimen may be administered. The first dose may be administered, for example, on day -14, -10, -9, -8, -7, -6, -5, -4, -3, -2, -1, 0, 1, 2, 3, 4, 5, 6, or 7, based on day 0 of transplantation of the donor cells, organs, and / or tissues. The second dose may be administered, for example, on day -3, -2, -1, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28, based on day 0 of transplantation of the donor cells, organs, and / or tissues. In some embodiments, the first dose is administered on day -8, and the second dose is administered on day -1. In some embodiments, the first dose is administered on day −8 and the second dose is administered on day 0. In some embodiments, the first dose is administered on day −8 and the second dose is administered on day 1. In some embodiments, the first dose is administered on day −7 and the second dose is administered on day −1. In some embodiments, the first dose is administered on day −7 and the second dose is administered on day 0. In some embodiments, the first dose is administered on day −7 and the second dose is administered on day 1. In some embodiments, the first dose is administered on day −12 and the second dose is administered on day −4. In some embodiments, the first dose is administered on day −11, −12, −13, or −14, and the second dose is administered on day −3, −4, −5, or −6.

[0263] In some cases, the second dose of the tolerization vaccine / regimen (e.g., a booster vaccine) may be administered on day 100, 90, 80, 70, 60, 50, 40, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 relative to donor cell, organ, and / or tissue transplantation on day 0. For example, the second dose of the tolerization vaccine (e.g., a booster vaccine) may be administered one day after donor cell, organ, and / or tissue transplantation. In some cases, a second dose of the tolerization vaccine is administered concomitantly with the transplant donor cells, organs and / or tissues on day 0. In some cases, a second dose of the tolerization vaccine is not required.

[0264] In some cases, a third dose of the tolerization vaccine / regimen or conditioning regimen may be administered. The first dose may be administered, for example, on day -14, -10, -9, -8, -7, -6, -5, -4, -3, -2, -1, 0, 1, 2, 3, 4, 5, 6, or 7 relative to day 0 of transplantation of the donor cells, organs, and / or tissues. The second dose may be administered, for example, on day -3, -2, -1, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 relative to day 0 of transplantation of the donor cells, organs, and / or tissues. The third dose can be administered, for example, on day 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 180, 200, 250, 300, 350, or 365 relative to transplantation of the donor cells, organs, and / or tissues on day 0. In some embodiments, the first dose is administered on day −8, the second dose is administered on day 1, and the third dose is administered on day 7. In some embodiments, the first dose is administered on day −8, the second dose is administered on day 1, and the third dose is administered on day 14. In some embodiments, the first dose is administered on day −8, the second dose is administered on day 1, and the third dose is administered on day 21. In some embodiments, the first dose is administered on day −7, the second dose is administered on day 1, and the third dose is administered on day 7. In some embodiments, the first dose is administered on day −7, the second dose is administered on day 1, and the third dose is administered on day 14. In some embodiments, the first dose is administered on day −7, the second dose is administered on day 1, and the third dose is administered on day 21.

[0265] In some cases, the third dose of the tolerization vaccine / regimen (e.g., a booster vaccine) may be administered on day 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 relative to transplantation of the donor cells, organs, and / or tissue on day 0. For example, the tolerization vaccine may be administered on about 300-200, 200-100, 100-50, 50-40, 40-30, 30-20, 20-10, 10-5, or 7-1 days relative to transplantation of donor cells, organs, and / or tissues on day 0.

[0266] In some cases, a fourth dose of the tolerization vaccine / regimen (e.g., a booster vaccine) may be administered on day 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 relative to transplantation of the donor cells, organs, and / or tissue on day 0. For example, the tolerization vaccine may be administered on or about days 600-500, 500-400, 400-300, 300-200, 200-100, 100-50, 50-40, 40-30, 30-20, 20-10, 10-5, 7-1, relative to day 0 of transplantation of donor cells, organs and / or tissues.

[0267] In some cases, a fifth dose of a tolerization vaccine (e.g., a booster vaccine) may be administered on day 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 based on transplantation of the donor cells, organs, and / or tissues on day 0. For example, tolerization vaccines may be administered at doses of 1,000-900, 900-800, 800-700, 700-600, 600-500, 500-400, 400-300, 300-200, 200-100, 100-50, 50-40, 40-30, 30-20, 200-300, 200-400, 300-500, 400-600, 400-700, 400-800, 400-900, 400-900, 400-100, 500-100, 500-100, 500-20, 500-30, 500-20, 500-20, 500-30, 500-20, 500-20, 500-30, 500-20, 500-30, 500-4 ...30, 500-40, 500-30, 500-20, 500-20, 500-30, 500-30, 500-30, 500-30, 500-30, 500-30, 500-30, 500-30, 500- It may be administered on days 0-10, 10-5, 7-1, or on about 1,000-900, 900-800, 800-700, 700-600, 600-500, 500-400, 400-300, 300-200, 200-100, 100-50, 50-40, 40-30, 30-20, 20-10, 10-5, 7-1.

[0268] The administration of a tolerization vaccine / regimen or conditioning regimen can result in long-term tolerance to cell, tissue, or organ transplants in transplant recipients. In some cases, long-term tolerance is at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 13 months, at least 14 months, at least 15 months, at least 16 months, at least 17 months, at least 18 months, at least 19 months, at least 20 months, at least 21 months, at least 22 months, at least 23 months, or at least 24 months. In some cases, long-term tolerance is at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, or at least 10 years. In some cases, long-term tolerance is achieved without the use of a booster vaccine or booster regimen. In some cases, long-term tolerance is achieved with administration of one or more doses of a booster vaccine or booster regimen. In some cases, the one or more booster vaccine doses are administered on the day of transplant or at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 ​​days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 61 days, 62 days, 63 days, 64 days, 65 days, 66 days, 67 days, 68 days, 69 days, 70 days, 71 days, 72 days, 73 days, 74 days, 75 days, 76 days, 77 days, 78 days, 79 days, 80 days, 81 days, 82 days, 83 days, 84 days, 85 days, 86 days, 87 days, 88 days, 89 days, 90 days, 91 days, 92 days, 93 days, 94 It may be administered 7, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 230, or 240 days later.In certain cases, one or more (e.g., 3) doses of the conditioning regimen are administered prior to transplantation, and one or more booster vaccine doses are administered 1, 7, 14, 21, 90, or up to 180 days after transplantation.

[0269] The dose of the tolerization vaccine or conditioning regimen can vary based on the weight of the recipient of the tolerization vaccine. For example, the dose of the tolerization vaccine or conditioning regimen can be about 1 x 10 1 cells / kg, 1×10 2 cells / kg, 1×10 3 cells / kg, 1×10 4 cells / kg, 1×10 5 cells / kg, 1×10 6 cells / kg, 1×10 7 cells / kg, 1×10 8 cells / kg, 1×10 9 cells / kg, 1×10 10 cells / kg, 1×10 11 cells / kg, 1×10 12 In some cases, the tolerization vaccine or conditioning regimen dose may comprise about 1 x 10 cells / kg or more. 1 ~1×10 2 cells / kg, 1×10 2 ~1×10 3 cells / kg, 1×10 3 ~1×10 4 cells / kg, 1×10 4 ~1×10 5 cells / kg, 1×10 5 ~1×10 6 cells / kg, 1×10 6 ~1×10 7 cells / kg, 1×10 7 ~1×10 8 cells / kg, 1×10 8 ~1×10 9 cells / kg, 1×10 9 ~1×10 10 cells / kg, 1×10 10 ~1×10 11 cells / kg, 1×10 11 ~1×10 12For example, the dose of a tolerization vaccine or conditioning regimen for administration may comprise about 0.01 x 10 cells / kg. 9 cells / kg, 0.02×10 9 cells / kg, 0.03×10 9 cells / kg, 0.04×10 9 cells / kg, 0.05×10 9 cells / kg, 0.06×10 9 cells / kg, 0.07×10 9 cells / kg, 0.08×10 9 cells / kg, 0.09×10 9 cells / kg, 0.1×10 9 cells / kg, 0.2×10 9 cells / kg, 0.21×10 9 cells / kg, 0.22×10 9 cells / kg, 0.23×10 9 cells / kg, 0.24×10 9 cells / kg, 0.25×10 9 cells / kg, 0.26×10 9 cells / kg, 0.27×10 9 cells / kg, 0.28×10 9 cells / kg, 0.29×10 9 cells / kg, 0.3×10 9 cells / kg, 0.4×10 9 cells / kg, 0.5×10 9 cells / kg, 0.6×10 9 cells / kg, 0.7×10 9 cells / kg, 0.8×10 9 cells / kg, 0.9×10 9 cells / kg, 1.0×10 9 cells / kg, 1.5×10 9 cells / kg, 2.0×10 9 cells / kg, 2.5×10 9 cells / kg, 3.0×10 9 cells / kg, 3.5×10 9 cells / kg, 4.0×10 9 cells / kg, 4.5×10 9 cells / kg, 5.0×10 9 cells / kg, 5.5×10 9 cells / kg, 6.0×109 cells / kg, 6.5×10 9 cells / kg, 7.0×10 9 cells / kg, 7.5×10 9 cells / kg, 8.0×10 9 cells / kg, 8.5×10 9 cells / kg, 9.0×10 9 cells / kg, 9.5×10 9 cells / kg, 10.0×10 9 cells / kg, or 25.0 × 10 9 It can be cells / kg.

[0270] In some cases, the tolerizing vaccine or conditioning regimen dose is at least about 1 x 10 4 cells / kg, 5×10 4 cells / kg, 1×10 5 cells / kg, 5×10 5 cells / kg, 1×10 6 cells / kg, 5×10 6 cells / kg, 1×10 7 cells / kg, 5×10 7 cells / kg, 1×10 8 cells / kg, 2×10 8 cells / kg, 3×10 8 cells / kg, 4×10 8 cells / kg, 5×10 8 cells / kg, 6×10 8 cells / kg, 7×10 8 cells / kg, 8×10 8 cells / kg, 9×10 8 cells / kg, 1×10 9 cells / kg, 1×10 10 It may include doses of cells / kg or higher.

[0271] The methods herein provide for a dose of at least or at least about 0.25 x 10 per kg of recipient body weight. 9For example, administering at least about 1 x 10 cells (e.g., apoptotic donor leukocytes (ADL), e.g., ECDI-treated cells, e.g., ECDI-treated leukocytes, or apoptotic mesenchymal stromal cells) per kg of recipient body weight. 7 , 1×10 8 , 0.25×10 9 , 0.50×10 9 , 0.75×10 9 , 1.00×10 9 , 1.25×10 9 , 1.50×10 9 , 1.75×10 9 Or 2 x 10 9 In one embodiment, ECDI-treated cells can be administered, such as ECDI-treated cells, e.g., ECDI-treated white blood cells.

[0272] The cells may include leukocytes, such as splenocytes, peripheral blood mononuclear cells (PBMCs), stem cell-derived leukocytes, or a combination thereof. The splenocytes, PBMCs, stem cell-derived leukocytes, or a combination thereof may include B cells or B lymphocytes. The cells may include primary cells, ex vivo expanded cells, cells of a cell line, or a combination thereof. The cells may include mesenchymal stromal cells.

[0273] The cells of the tolerization vaccine / regimen or conditioning regimen for administering each dose may be suspended in a volume suitable for infusion. For example, the cells may be in a volume of about 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml, 0.5 ml, 0.6 ml, 0.7 ml, 0.8 ml, 0.9 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 11 ml, 12 ml, 13 ml, 14 ml, 15 ml, 16 ml, 17 ml, 18 ml, 19 ml, 20 ml, 21 ml, 22 ml, 23 ml, 24 ml, 25 ml, 26 ml, 27 ml, 28 ml, 29 ml, 30 ml, 31 ml, 32 ml, 33 ml, 34 ml, 35 ml, 36 ml, 37 ml, 38 ml, 39 ml, 40 ml, 41 ml, 42 ml, 43 ml, 44 ml, 45 ml, 46 ml, 47 ml, 48 ml, 49 ml, 50 ml, 51 ml, 52 ml, 53 ml, 54 ml, 55 ml, 56 ml, 57 ml, 58 ml, 59 ml, 60 ml, 61 ml, 62 ml, 63 ml, 64 ml, 65 ml, 66 ml, 67 ml, 68 ml, 69 ml, 70 ml, 71 ml, 72 ml, 73 ml, 74 ml, 75 ml, 76 ml, 77 ml, 78 ml, 79 ml, 80 ml, 81 ml, 82 ml, 83 ml, 84 ml, 8 The cells may be suspended in a volume of 7 ml, 28 ml, 29 ml, 30 ml, 31 ml, 32 ml, 33 ml, 34 ml, 35 ml, 36 ml, 37 ml, 38 ml, 39 ml, 40 ml, 41 ml, 42 ml, 43 ml, 44 ml, 45 ml, 46 ml, 47 ml, 48 ml, 49 ml, 50 ml, 60 ml, 70 ml, 80 ml, 90 ml, 100 ml, 200 ml, 300 ml, 400 ml, or 500 ml. For example, the cells of the tolerization vaccine or conditioning regimen for administering each dose may be suspended in a volume of about 0.1 ml to 1 ml, 1 ml to 10 ml, 10 ml to 50 ml, 50 ml to 100 ml, 100 ml to 200 ml, 200 ml to 300 ml, 300 ml to 400 ml, or 400 ml to 500 ml. For example, 75 x 10 of a tolerizing vaccine or conditioning regimen 6 The cells may be suspended in a volume of 0.5 ml.

[0274] The tolerization vaccine / regimen or conditioning regimen can be administered (e.g., by intravenous infusion) in a volume that varies depending on the recipient's body weight. For example, the tolerization vaccine or conditioning regimen can be administered intravenously in a volume of at least about 0.01 ml, 0.1 ml, 0.5 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 10 ml, 20 ml, 30 ml, 40 ml, or 50 ml per kg of recipient body weight, for example, at least about 0.01-0.1, 0.1-1, 1-2, 2-3, 3-4, 4-5, 1-5, 5-10, 10-20, 20-30, 30-40, or 40-50 ml per kg of recipient body weight. In some cases, the tolerization vaccine (e.g., containing ECDI-treated cells) is administered intravenously in a volume of about 7 ml per kg of recipient body weight.

[0275] A booster dose of a tolerizing vaccine or conditioning regimen can contain fewer cells than the initial dose of the tolerizing vaccine or conditioning regimen. For example, a booster dose or subsequent dose of a tolerizing vaccine or conditioning regimen can contain about 1%, 2%, 3%, 4%, 5%, 7.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, or 75% fewer or fewer cells than the initial or preceding dose of the tolerizing vaccine or conditioning regimen.

[0276] The cells of the tolerization vaccine or conditioning regimen can have a circulating half-life after administration to a subject. In some cases, the tolerization vaccine or conditioning regimen described herein can have a circulating half-life of at least or at least about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 18, 24, 36, 48, 60, or 72 hours. For example, the circulating half-life of the tolerizing vaccine or conditioning regimen cells can be 0.1 to 0.5, 0.5 to 1.0, 1.0 to 2.0, 1.0 to 3.0, 1.0 to 4.0, 1.0 to 5.0, 5 to 10, 10 to 15, 15 to 24, 24 to 36, 36 to 48, 48 to 60, or 60 to 72 hours, or about 0.1 to 0.5, 0.5 to 1.0, 1.0 to 2.0, 1.0 to 3.0, 1.0 to 4.0, 1.0 to 5.0, 5 to 10, 10 to 15, 15 to 24, 24 to 36, 36 to 48, 48 to 60, or 60 to 72 hours. In some cases, the tolerizing vaccines or conditioning regimens described herein can have a circulating half-life of at least or at least about 3 hours.

[0277] Cells in the tolerization vaccine or pretreatment regimen can be treated to improve their circulating half-life. Such treatments include coating the cells with a protein, such as CD47. Cells treated to improve their circulating half-life can be non-apoptotic cells. Cells treated to improve their circulating half-life can be apoptotic cells. Alternatively, cells in the tolerization vaccine or pretreatment regimen can be genetically modified (e.g., by inserting a transgene, such as CD47, into their genome) to improve their circulating half-life. Cells genetically modified to improve their circulating half-life can be non-apoptotic cells. Cells genetically modified to improve their circulating half-life can be apoptotic cells.

[0278] Tolerization vaccine or conditioning regimen can be advantageous in transplantation, for example, in xenotransplantation or allotransplantation, because it can make graft recipient tolerant and prevent or delay graft rejection.Tolerization or conditioning regimen can be provided to graft recipient without using immunosuppressive therapy (for example, one or more immunomodulatory molecules).However, in some cases, other immunosuppressive therapy can be used in combination with tolerization vaccine to prevent, reduce or delay transplant rejection.

[0279] The tolerizing vaccine or conditioning regimen can be administered with or without an adjuvant (e.g., one or more immunomodulatory molecules). In some cases, the adjuvant enhances the tolerogenicity of the tolerizing vaccine by inhibiting the activation and maturation of antigen-presenting cells.

[0280] In some embodiments, the immunomodulatory molecule can target T cell receptor (TCR), CD3e, FK506-binding protein 12 (FKBP12), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), programmed cell death ligand 1 (PD-L1), CD40L (CD154), CD40, inducible co-stimulatory signaling (ICOS), IL-2, TNF-α, IL-6, IL-7, CD2, CD20, CD52, alpha-4 integrin, mTOR, DNA synthesis, molecules in the pro-inflammatory pathway (e.g., cytokines), alpha 1-antitrypsin, NFkB, or any combination thereof. In some embodiments, the immunomodulatory molecule is an NFkB inhibitor (e.g., dehydroxymethylepoxyquinomycin (DHMEQ)). In some embodiments, the one or more immunomodulatory molecules can target B cells (e.g., a B cell-depleting biologic, e.g., a biologic targeting CD20, CD19, or CD22, and / or a B cell-modulating biologic, e.g., a biologic targeting BAFF, BAFF / APRIL, CD40, IgG4, ICOS, IL-21, B7RP1). In some embodiments, the B cell-targeting biologic can be an anti-CD20 mAb (e.g., rituximab) or other B cell-depleting antibody. In some embodiments, the immunomodulatory molecule can be an MHC / TCR interaction blocker, a non-selective depleting agent, a calcineurin inhibitor, a costimulatory signal blocker, a cytokine blocker, a B cell-modulating agent, a lymphodepleting agent, a cell adhesion inhibitor, an IL-2 signaling inhibitor, a cell cycle blocker, or any combination thereof. For example, the MHC / TCR interaction blocker can be the anti-abTCR mAb T10B9. For example, the non-selective depleting agent can be anti-CD3 mAb (OKT3) or anti-thymocyte globulin (ATG). For example, the calcineurin inhibitor can be cyclosporine or tacrolimus.For example, the costimulatory signal blocker can be an anti-CTLA-4 mAb, abatacept, ipilimumab, anti-PD-1 (e.g., pembrolizumab), anti-PD-L1 (e.g., MPDL3280A), anti-CD154 mAb, an Fc-engineered anti-CD40L antibody, an anti-CD40 mAb, or an anti-ICOS mAb. For example, the cytokine blocker can be an anti-CD25 mAb (e.g., daclizumab or basiliximab), an anti-TNF (etanercept, infliximab, adalimumab, certolizumab pegol, and golimumab), an anti-IL-6 / IL-6R mAb (e.g., tocilizumab, Actemra, clazakizumab, ALD518, siltuximab, elsilimomab, sirumab, sarilumab, olokizumab), or an anti-IL-7 mAb. For example, the lymphodepleting agent can be an anti-CD2 mAb, a fusion protein with IgG1 (e.g., alefacept), an anti-CD20 mAb (e.g., rituximab), or an anti-CD52 mAb (e.g., alemtuzumab). For example, the cell adhesion inhibitor can be an anti-very large antigen 4 (VLA4) (e.g., natalizumab). For example, the mTOR inhibitor can be sirolimus (rapamycin) or everolimus or any other mTOR inhibitor.For example, the cell cycle blocker can be mycophenolate mofetil (MMF) or azathioprine.In some embodiments, the immunomodulatory molecule can be a T cell recirculation inhibitor (e.g., FTY720 and other sphingosine 1 phosphate (S1P) receptor agonists).

[0281] The tolerization vaccine or pretreatment regimen can be administered with or without one or more immunomodulatory molecules that inhibit T cell activation. The immunomodulatory molecule that inhibits T cell activation can be an anti-CD40 or anti-CD40L (CD154) agent. The anti-CD40 or anti-CD40L agent can be an antibody, such as an antagonist antibody. The anti-CD40 or anti-CD40L antibody can be the Fab' anti-CD40L monoclonal antibody fragment CDP7657. The anti-CD40 or anti-CD40L antibody can be an FcR-engineered, Fc-silenced anti-CD40L monoclonal domain antibody, a Fab' anti-CD40L antibody, or an otherwise Fc-engineered anti-CD40L antibody. The anti-CD40 or anti-CD40L agent can be 2C10, 2C10R4, ASKP1240, 4D11, bleselumab, BI-655064, HCD122, CFZ533, ch5D12, FFP104, CDP7657, BG9588, ruplizumab, toralizumab, IDEC-131, dapirolizumab, letolizumab, BMS-986004, VIB4920, or MEDI4920. The tolerizing vaccine or conditioning regimen can be further administered in conjunction with one or more additional immunomodulatory molecules described herein, e.g., one or more of a B cell-targeting biologic (e.g., a B cell-depleting biologic, e.g., a CD20, CD19, or CD22-targeting biologic, and / or a B cell-modulating biologic, e.g., a BAFF, BAFF / APRIL, CD40, IgG4, ICOS, IL-21, B7RP1-targeting biologic), an mTOR inhibitor, a TNF-alpha inhibitor, an IL-6 inhibitor, alpha 1-antitrypsin, a nitrogen mustard alkylating agent (e.g., cyclophosphamide), a complement C3 or C5 inhibitor, IFN-γ, an NFκB inhibitor, vitamin D3, siCD40, cobalt protoporphyrin, insulin B9-23, a cluster of differentiation protein (e.g., CD46, CD55, or CD59), any combination thereof, or any fragment thereof. In some instances, the NFκB inhibitor is curcumin, triptolide, Bay-117085, or a combination thereof.Non-limiting examples of biologics that target B cell include rituximab and anti-CD20 antibody.In some cases, the immunomodulatory molecule for administration as part of conditioning regimen can be adapted to allograft or xenograft.For example, tolerization vaccine or conditioning regimen is administered together with B cell depleting antibody during xenograft.For example, tolerization vaccine or conditioning regimen does not need to be administered together with B cell depleting antibody during allograft.

[0282] The tolerizing vaccine / regimen or conditioning regimen may be administered with or without other agents, including but not limited to MMF (mycophenolate mofetil (Cellcept)), ATG (antithymocyte globulin), anti-CD154 (CD40L), alemtuzumab (Campath), B cell targeting agents (e.g., biologics that deplete B cells, e.g., biologics targeting CD20, CD19, or CD22, and / or biologics that modulate B cells, e.g., BAFF, BAFF / APRIL, biologics targeting CD40, IgG4, ICOS, IL-21, B7RP1), anti-IL-6R antibodies (sarilumab, tocilizumab, Actemra), anti-IL-6 antibodies (clazakizumab, ALD518, siltuximab, elcilimomab, sirukumab, olokizumab), CTLA4-Ig (Abatacept / Orencia), belatacept ( LEA29Y), sirolimus (Rapamune), tacrolimus (Prograf), daclizumab, basiliximab (Simulect), infliximab (Remicade), cyclosporine, deoxyspergualin, soluble complement 1, cobra venom factor, compstatin, anti-C5 antibody (eculizumab / Soliris), methylprednisolone, FTY720, everolimus, anti-CD154-Ab, leflunomide, anti-IL-2R-Ab, rapamycin, anti-CXCR3 antibody, anti-ICOS antibody, anti-OX40 antibody, and anti-CD122 antibody, human anti-CD154 monoclonal antibody, CD40 antagonist, and CD40L (CD154) antagonist. A non-limiting example of a biologic that targets B cells includes Rituximab, an anti-CD20 antibody.

[0283] The tolerization vaccine / regimen or conditioning regimen can be administered together with or in addition to one or more immunosuppressive agents / drugs. For example, one or more immunosuppressive agents / drugs can be used in induction therapy or maintenance therapy. The same or different drugs can be used during the induction and maintenance phases. For example, daclizumab (Zenapax) is used in induction therapy, and tacrolimus (Prograf) and an mTOR inhibitor (e.g., sirolimus, rapamycin, everolimus) are used in maintenance therapy. In another example, daclizumab (Zenapax) is used in induction therapy, and low-dose tacrolimus (Prograf) and a low-dose mTOR inhibitor (e.g., sirolimus, rapamycin, everolimus) are used in maintenance therapy. Immunosuppression can also be achieved using non-medication regimens, including, but not limited to, total body irradiation, thymic irradiation, and total and / or partial splenectomy. These techniques can also be used in combination with one or more immunosuppressive drugs or agents.

[0284] In some embodiments, the tolerizing vaccine or conditioning regimen can be administered with at least one anti-CD40 / CD40L agent disclosed herein. In some embodiments, the anti-CD40 / CD40L agent is part of a conditioning regimen described herein that is administered before transplantation. In some cases, the tolerizing vaccine or conditioning regimen is administered simultaneously with, before, or after at least one anti-CD40 / CD40L agent. In some embodiments, the tolerizing vaccine or conditioning regimen can also be administered with, before, or after one or more additional immunosuppressive agents, and / or a CD40:CD40L blocking agent in combination with an anti-CD40 agent (e.g., an antibody) and / or an anti-CD40L (CD154) agent. The additional immunosuppressive agent can be administered to the subject, for example, to enhance the tolerogenic effectiveness of the tolerizing vaccine in the subject. Additional immunosuppressive agents may include anti-CD40 or anti-CD40L (CD154) agents (e.g., anti-CD40 antibodies), B cell targeting agents (e.g., B cell depleting biologics, e.g., biologics targeting CD20, CD19, or CD22, and / or B cell modulating biologics, e.g., biologics targeting BAFF, BAFF / APRIL, CD40, IgG4, ICOS, IL-21, B7RP1), mTOR inhibitors, TNF-alpha inhibitors, IL-6 inhibitors, alpha 1-antitrypsin, nitrogen mustard alkylating agents (e.g., cyclophosphamide), complement C3 or C5 inhibitors, IFN-γ, NFκB inhibitors, vitamin D3, siCD40, cobalt protoporphyrin, insulin B9-23, cluster of differentiation proteins (e.g., CD46, CD55, or CD59), any combination thereof, or any fragment thereof. In some cases, the NFκB inhibitor is dehydroxymethylepoxyquinomycin (DHMEQ), curcumin, triptolide, Bay-117085, or a combination thereof. Non-limiting examples of biologics that target B cells include Rituximab, an anti-CD20 antibody. Any of these additional immunosuppressants can be administered to the subject before and / or after transplantation (Figure 4).

[0285] The additional immunosuppressant can be administered before, after, and / or during administration of the tolerization vaccine or conditioning regimen. In some cases, the additional immunosuppressant can be administered between day -100 and day 0 relative to administration of the tolerization vaccine or conditioning regimen, e.g., day -90, day -80, day -70, day -60, day -50, day -40, day -30, day -20, day -15, day -14, day -13, day -12, day -11, day -10, day -9, day -8, day -7, day -6, day -5, day -4, day -3, day -2, day -1, or day 0. In some cases, the additional immunosuppressant can be administered concomitantly with the tolerization vaccine or conditioning regimen. In some cases, the additional immunosuppressant may be administered at or about days -100 to -50, -50 to -40, -40 to -30, -30 to -20, -20 to -10, -10 to -5, -10 to -1, -7 to -1, -10 to 0, or -7 to 0 relative to administration of the tolerizing vaccine or conditioning regimen.

[0286] In some cases, the additional immunosuppressant may be administered between days 0 and 100 relative to administration of the tolerizing vaccine or conditioning regimen, e.g., on day 100, 90, 80, 70, 60, 50, 40, 30, 28, 21, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1. For example, the immunosuppressant may be administered on or about days 100-50, 50-40, 40-30, 30-20, 20-10, 10-5, 21-1, 14-1, 7-1, 21-0, 14-0, or 7-0 relative to administration of the tolerizing vaccine or conditioning regimen.

[0287] In some cases, the additional immunosuppressant may be administered between days 0 and 300 relative to administration of the tolerizing vaccine or conditioning regimen, e.g., on day 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1. For example, the immunosuppressant may be administered on or about days 300-200, 200-100, 100-50, 50-40, 40-30, 30-20, 20-10, 10-5, or 7-1 relative to administration of the tolerizing vaccine or conditioning regimen.

[0288] In some cases, the additional immunosuppressant may be administered between days 0 and 600 relative to administration of the tolerizing vaccine or conditioning regimen, e.g., on day 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1. For example, the immunosuppressant may be administered on or about days 600-500, 500-400, 400-300, 300-200, 200-100, 100-50, 50-40, 40-30, 30-20, 20-10, 10-5, or 7-1 relative to administration of the tolerizing vaccine or conditioning regimen.

[0289] In some cases, the additional immunosuppressant may be administered between days 0 and 1000 relative to administration of the tolerizing vaccine or conditioning regimen, e.g., on day 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1. For example, immunosuppressants may be administered on days 1000-900, 900-800, 800-700, 700-600, 600-500, 500-400, 400-300, 300-200, 200-100, 100-50, 50-40, 40-30, 30-20, 20-10, 30-40, 30-10, 30-20, 30-10, 30-10, 30-20, 30-10, 30-10, 30-20, 30-10, 30-10, 30-20, 30-10, 30-20, 30-10, 30-20, 30-10, 30-20, 30-10, 30-20, 30-10, 30-20, 30-20, 30-10, 30-20, 30-20, 30-20, 30-3 ... , 10 to 5, 7 to 1 days, or on about 1000 to 900, 900 to 800, 800 to 700, 700 to 600, 600 to 500 days, 500 to 400 days, 400 to 300, 300 to 200, 200 to 100, 100 to 50, 50 to 40, 40 to 30, 30 to 20, 20 to 10, 10 to 5, 7 to 1 days.

[0290] In some cases, additional immunosuppressants can be administered on the day that tolerization vaccine or conditioning regimen is administered.In other cases, additional immunosuppressants can be administered before and after the administration of tolerization vaccine or conditioning regimen.For example, cyclophosphamide can be administered on or about the third day after the administration of tolerization vaccine or conditioning regimen.

[0291] The tolerogenic efficacy modulator (e.g., cyclophosphamide) can be administered at a dose of 5-100 mg / kg / day or about 5-100 mg / kg / day. The unit "mg / kg / day" can refer to the number of milligrams of tolerogenic efficacy modulator administered per kg of subject body weight per day. In some cases, the tolerogenic efficacy modulator (e.g., cyclophosphamide) may be administered at a dose of 20 mg / kg / day to 100 mg / kg / day, 30 mg / kg / day to 90 mg / kg / day, 40 mg / kg / day to 80 mg / kg / day, 50 mg / kg / day to 70 mg / kg / day, 50 mg / kg / day to 60 mg / kg / day, or 40 mg / kg / day to 60 mg / kg / day, or about 20 mg / kg / day to 100 mg / kg / day, 30 mg / kg / day to 90 mg / kg / day, 40 mg / kg / day to 80 mg / kg / day, 50 mg / kg / day to 70 mg / kg / day, 50 mg / kg / day to 60 mg / kg / day, or 40 mg / kg / day to 60 mg / kg / day.

[0292] A tolerizing vaccine or conditioning regimen can reduce the dose and / or duration of immunosuppression required to prevent or delay cell, organ, and / or tissue rejection. A tolerizing vaccine or conditioning regimen can extend cell, organ, and / or tissue survival without the need for maintenance immunosuppression.

[0293] In some cases, transplant recipients may not require immunosuppression after administration of a tolerizing vaccine or conditioning regimen.

[0294] A tolerizing vaccine or conditioning regimen can reduce the required dose of immunosuppression by at least or at least about 5%. For example, a tolerizing vaccine or conditioning regimen can reduce the required dose of an immunosuppressant by at least or at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. For example, a tolerizing vaccine or conditioning regimen can reduce the required dose of an immunosuppressant by at least or at least about 5-10, 5-25, 25-50, 50-75, 75-85, 85-90, 90-95, or 95-100%. As used herein, the term "reducing" and its grammatical equivalents can refer to using less immunosuppression compared to the required dose of immunosuppression when one or more cells, organs, and / or tissues are transplanted into a recipient (e.g., a human or non-human animal). The term "reducing" can also refer to using a lower immunosuppressant drug or agent compared to the required dose of the immunosuppressant when one or more cells, organs and / or tissues are transplanted into a recipient (e.g., a human or non-human animal).

[0295] The recipient (e.g., a human or non-human animal) may require a reduced dose of immunosuppressant for at least or at least about 1, 5, 7, 10, 14, 20, 21, 28, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 900, or 1,000 days after transplantation, e.g., at least or at least about 1-5, 5-10, 10-20, 20-30, 30-60, 60-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1,000 days. The recipient (e.g., a human or non-human animal) may require a reduced dose of immunosuppressant for at least or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 months after transplant, e.g., at least or at least about 1-2, 2-3, 3-6, 6-9, 9-12, 12-18, 18-24, 24-30, 30-36 months after transplant. The recipient (e.g., a human or non-human animal) may require a reduced dose of immunosuppressant for at least or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 years after transplantation, e.g., at least or at least about 1-2, 2-3, 3-4, 4-5, 1-5, 5-10, 10-15, 15-20, 20-25, 25-30 years after transplantation. In some cases, the recipient (e.g., a human or non-human animal) may require a reduced dose of immunosuppressant for up to the recipient's lifetime.

[0296] The recipient (e.g., a human or non-human animal) may not require immunosuppression for at least or at least about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 900, or 1,000 days after transplantation, e.g., at least or at least about 1-5, 5-10, 10-20, 20-30, 30-60, 60-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1,000 days. The recipient (e.g., a human or non-human animal) may require a reduced dose of immunosuppressant for at least or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 months after transplant, e.g., at least or at least about 1-2, 2-3, 3-6, 6-9, 9-12, 12-18, 18-24, 24-30, 30-36 months after transplant. The recipient (e.g., a human or non-human animal) may require reduced doses of immunosuppressant for at least or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 years after transplantation, e.g., at least or at least about 1-2, 2-3, 3-4, 4-5, 1-5, 5-10, 10-15, 15-20, 20-25, 25-30 years. In some cases, the recipient (e.g., a human or non-human animal) may not require immunosuppression for up to the recipient's lifetime.

[0297] Non-limiting exemplary approaches for extending the survival of a transplanted graft (e.g., an allograft or xenograft) in a subject (e.g., a human or non-human primate) are shown in Figures 3 and 4. In some embodiments, the subject (graft recipient) receives an infusion (e.g., intravenous infusion) of apoptotic cells derived from the graft donor or another donor (e.g., a tolerizing vaccination using apoptotic donor leukocytes (ADL) under the aid of transient immunosuppression). In some cases, the same donor may provide both the allograft or xenograft (e.g., pancreatic islets, kidney) for transplantation and cells as a tolerizing vaccine (e.g., splenocytes, splenic B cells, peripheral blood leukocytes, peripheral blood B cells, apoptotic leukocytes, or a combination thereof).

[0298] The cells of the tolerizing vaccine or conditioning regimen may have the same genotype and / or phenotype as the cells, organ, and / or tissue used in the transplant. The tolerizing vaccine or conditioning regimen may differ in genotype and / or phenotype from the graft. The tolerizing vaccine or conditioning regimen used in the transplant recipient may include cells from the transplant graft donor. The tolerizing vaccine or conditioning regimen used in the transplant recipient may include cells that are genetically and / or phenotypically different from the transplant graft. In some cases, the tolerizing vaccine or conditioning regimen used in the transplant recipient may include cells from the transplant graft donor and cells that are genetically and / or phenotypically different from the transplant graft. The cells that are genetically and / or phenotypically different from the transplant graft may be from an animal of the same species as the transplant graft donor.

[0299] In some cases, the tolerization vaccine or pretreatment regimen containing genetically modified cells can reduce, inhibit, or eliminate immune response.For example, genetic modification can reduce immune cell effector function, reduce immune cell proliferation, or reduce the persistence and / or expression of cytolytic effector molecules, such as granzyme B and CD107 alpha, in immune cells.Immune cells can be monocytes and / or macrophages.Immune cells can also be T cells and / or B cells.In some cases, T cell-derived cytokines, such as IFN-γ, can activate macrophages by secreting IFN-γ.In some cases, T cell activation can be inhibited, which can also cause macrophages to be inhibited.

[0300] The tolerogenic potential of any of these tolerizing cellular vaccines can be further optimized by coupling one or more of the following to the surface of the cells: IFN-γ, NF-κB inhibitors (e.g., dehydroxymethylepoxyquinomycin (DHMEQ), curcumin, triptolide, Bay-117085), vitamin D3, cobalt protoporphyrin, insulin B9-23, or other immunomodulatory molecules disclosed herein that modify the function of host antigen-presenting cells and host lymphocytes.

[0301] Tolerizing vaccines or conditioning regimens may include apoptotic cells and may be complemented by donor cells engineered to display surface molecules, such as FasL, PD-L1, galectin-9, CD8 alpha, that induce apoptotic death of donor-reactive cells.

[0302] A tolerizing vaccine or conditioning regimen may contain from 0.001 to about 5.0, or from about 0.001 to about 5.0, for example, 0.001 to 1.0, or from about 0.001 to 1.0, endotoxin units per kg of body weight of the intended recipient. For example, a tolerizing vaccine or conditioning regimen may be administered in a dose of 0.01 to 5.0, 0.01 to 4.5, 0.01 to 4.0, 0.01 to 3.5, 0.01 to 3.0, 0.01 to 2.5, 0.01 to 2.0, 0.01 to 1.5, 0.01 to 1.0, 0.01 to 0.9, 0.01 to 0.8, 0.01 to 0.7, 0.01 to 0.6, 0.01 to 0.5, 0.01 to 0.4, 0.01 to 0.3, 0.01 to 0.2, or may contain 0.01 to 0.1, or about 0.01 to 5.0, 0.01 to 4.5, 0.01 to 4.0, 0.01 to 3.5, 0.01 to 3.0, 0.01 to 2.5, 0.01 to 2.0, 0.01 to 1.5, 0.01 to 1.0, 0.01 to 0.9, 0.01 to 0.8, 0.01 to 0.7, 0.01 to 0.6, 0.01 to 0.5, 0.01 to 0.4, 0.01 to 0.3, 0.01 to 0.2, or 0.01 to 0.1 endotoxin units.

[0303] The tolerizing vaccine or conditioning regimen can induce the release of 0.001 pg / ml to 10.0 pg / ml, or about 0.001 pg / ml to 10.0 pg / ml, e.g., 0.001 pg / ml to 1.0 pg / ml, or about 0.001 pg / ml to 1.0 pg / ml, of IL-1 beta when about 50,000 frozen and thawed human peripheral blood mononuclear cells are incubated with about 160,000 cells (e.g., porcine cells) of the tolerizing vaccine. For example, the tolerizing vaccine or conditioning regimen induces the release of 0.001 to 10.0, 0.001 to 5.0, 0.001 to 1.0, 0.001 to 0.8, 0.001 to 0.2, or 0.001 to 0.1 pg / ml of IL-1 beta when about 50,000 frozen and thawed human peripheral blood mononuclear cells are incubated with about 160,000 cells (e.g., human or porcine cells) of the tolerizing vaccine. The tolerizing vaccine or conditioning regimen can induce the release of 0.001 to 2.0 pg / ml, or about 0.001 to 2.0 pg / ml, e.g., 0.001 to 0.2 pg / ml, or about 0.001 pg / ml to 0.2 pg / ml, of IL-6 when about 50,000 frozen and thawed human peripheral blood mononuclear cells are incubated with about 160,000 cells (e.g., human cells or porcine cells) of the tolerizing vaccine. For example, a tolerizing vaccine or conditioning regimen can induce the release of 0.001 to 2.0, 0.001 to 1.0, 0.001 to 0.5, or 0.001 to 0.1 pg / ml, or about 0.001 to 2.0, 0.001 to 1.0, 0.001 to 0.5, or 0.001 to 0.1 pg / ml, of IL-6 when about 50,000 frozen and thawed human peripheral blood mononuclear cells are incubated with about 160,000 cells (e.g., human or porcine cells) of the tolerizing vaccine.

[0304] The tolerizing vaccine or conditioning regimen may comprise greater than or greater than about 60%, e.g., greater than or greater than about 85%, Annexin V-positive apoptotic cells after 4 hours or about 4 hours of post-release incubation at 37° C. For example, the tolerizing vaccine comprises greater than 40%, 50%, 60%, 70%, 80%, 90%, or 99% Annexin V-positive apoptotic cells after about 4 hours of post-release incubation at 37° C.

[0305] Effective dose

[0306] The compositions (e.g., apoptotic leukocytes) of the present disclosure are administered in an effective amount. An "effective amount" is that amount of a composition that, alone or together with further doses, produces the desired response. In the case of transplantation, the desired response is inhibition of transplant rejection or increased transplant survival. When treating certain diseases, such as chronic kidney disease, inflammatory disease, autoimmune disease, cancer, etc., the desired response is inhibition of disease progression. This may involve only slowing the progression of the disease temporarily, but more preferably involves permanently halting the progression of the disease. This can be monitored by conventional methods.

[0307] Such amounts will, of course, depend on the particular condition being treated, the severity of the disorder, the activity of the particular compound, the route of administration, the clearance rate of the composition, the duration of treatment, any drugs used in combination with or concomitantly with the composition, the age, weight, sex, diet, and general health of the subject, and similar factors well known in the medical arts and sciences. Various general considerations that go into determining a "therapeutically effective amount" are known to those skilled in the art, and are described, for example, in Gilman et al. et al, eds., Goodman And Gilman's: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press, 1990; and Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., 1990. These factors are well known to those skilled in the art, and can be addressed with no more than routine experimentation. The maximum dose of each component or combination thereof used, i.e., the highest safe dose according to sound medical judgment, is generally preferred. The compositions used in the above-mentioned methods are preferably sterile and contain an effective amount of active agent to produce the desired response in a unit of weight or volume suitable for administration to a patient.

[0308] The efficacy of the methods disclosed herein, including administering a composition (e.g., apoptotic leukocytes) disclosed herein, can be assessed by a variety of endpoints commonly used in assessing graft survival or prevention of graft rejection, including, but not limited to, a) reduced level of immune response against the graft (believed to be mediated at least in part by a B-cell mediated immune response, more particularly donor-specific antibodies) or reduced incidence of graft-versus-host disease (GVHD), b) delayed initiation or progression of immune response against the graft, c) reduced risk of initiation or progression of immune response against the graft, d) increased level of tolerogenic APCs, e) increased level of Tregs, f) increased level of Tr1 cells, g) increased level of anti-inflammatory cytokines, such as IL-10, TGFβ, or h) combinations thereof.

[0309] In another embodiment, the methods described herein can significantly increase the response rate in a population of human subjects undergoing transplantation, with or without additional immunosuppression, where the response rate is defined as the percentage of transplant recipients who respond to treatment. Induction of tolerance to autoimmune diseases

[0310] The compositions, methods, kits, and systems provided herein can be used to prevent and / or treat autoimmune disorders. The terms "autoimmune disorder," "autoimmune disease," "autoimmune condition," and their grammatical equivalents can be used interchangeably when used herein. In some cases, the tolerance vaccines provided herein can be crosslinked to autoantigen peptides, autoantigens, or other cellular carriers and used as tolerance therapy for autoimmune disorders. In some cases, the cellular carriers are apoptotic cellular carriers. In some cases, the cellular carriers are syngeneic apoptotic cellular carriers.

[0311] The tolerizing vaccines described herein can be used with cellular carriers (e.g., autoantigens, autoantigenic peptides, apoptotic cell carriers) to induce antigen-specific T cell tolerance for the treatment of autoimmune conditions. Without being bound by theory, the tolerizing vaccines, with or without a carrier, can be taken up, processed, and presented in a tolerogenic manner by host splenic antigen-presenting cells, thereby inducing regulatory T cells and secreting immunosuppressive cytokines (e.g., IL-4, IL-10, IL-13, TGF-β).

[0312] Non-limiting examples of autoimmune disorders include inflammation, antiphospholipid syndrome, systemic lupus erythematosus, rheumatoid arthritis, autoimmune vasculitis, celiac disease, autoimmune thyroiditis, post-transfusion immunization, maternal-fetal incompatibility, transfusion reactions, immune deficiencies such as IgA deficiency, common variable immunodeficiency, drug-induced lupus, diabetes mellitus, type I diabetes, type II diabetes, juvenile-onset diabetes, juvenile rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, immunodeficiencies, allergies, asthma, psoriasis, atopic dermatitis, allergic contact dermatitis, chronic skin diseases, amyotrophic lateral sclerosis, chemotherapy-induced injury, graft-versus-host disease, bone marrow transplantation. Rejection, ankylosing spondylitis, atopic eczema, pemphigus, Behçet's disease, chronic fatigue syndrome, fibromyalgia, chemotherapy damage, myasthenia gravis, glomerulonephritis, allergic retinitis, systemic sclerosis, subacute cutaneous lupus erythematosus, cutaneous lupus erythematosus, e.g., chilblain-like lupus erythematosus, Sjogren's syndrome, autoimmune nephritis, autoimmune vasculitis, autoimmune hepatitis, autoimmune carditis, autoimmune encephalitis, autoimmune-mediated blood disorders, lc-SSc (limited cutaneous scleroderma), dc-SSc (diffuse cutaneous scleroderma), autoimmune thyroiditis (AT), Graves' diseasedisease) (GD), myasthenia gravis, multiple sclerosis (MS), ankylosing spondylitis, transplant rejection, immunosenescence, rheumatic / autoimmune diseases, mixed connective tissue disease, spondyloarthropathies, psoriasis, psoriatic arthritis, myositis, scleroderma, dermatomyositis, autoimmune vasculitis, mixed connective tissue disease, idiopathic thrombocytopenic purpura, Crohn's disease, human adjuvant disease, osteoarthritis, juvenile chronic arthritis, spondyloarthropathies, idiopathic inflammatory myopathy, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, thyroiditis, immune-mediated kidney disease, central or peripheral nervous system disorders Demyelinating diseases of the nervous system, idiopathic demyelinating polyneuropathy, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, hepatobiliary diseases, infectious or autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, sclerosing cholangitis, inflammatory bowel disease (including Crohn's disease (CD) and ulcerative colitis (UC)), gluten-sensitive enteropathy, Whipple's disease, autoimmune or immune-mediated skin diseases, bullous skin diseases, erythema multiforme, allergic rhinitis, atopic dermatitis, food allergies, urticaria, immune disorders of the lungs, eosinophilic pneumonia, idiopathic pulmonary fibrosis, hypersensitivity pneumonitis, transplant-related diseases Disease, graft rejection or graft-versus-host disease, psoriatic arthritis, psoriasis, dermatitis, polymyositis / dermatomyositis, toxic epidermal necrolysis, systemic sclerosis and sclerosis, reactions associated with inflammatory bowel disease, Crohn's disease, ulcerative colitis, respiratory distress syndrome, adult respiratory distress syndrome (ARDS), meningitis, encephalitis, uveitis, colitis, glomerulonephritis, allergic conditions, eczema, asthma, conditions with T-cell infiltration and chronic inflammatory responses, atherosclerosis, autoimmune myocarditis, leukocyte adhesion deficiency, allergic encephalomyelitis, acute myocardial infarction mediated by cytokines and T lymphocytes Immune responses associated with acute and delayed hypersensitivity, tuberculosis, sarcoidosis, granulomatous diseases such as Wegener's granulomatosis, granulocytopenia, vasculitis (e.g., ANCA), aplastic anemia, Diamond-Blackfan anemia, immune-mediated hemolytic anemia such as autoimmune hemolytic anemia (AIHA), pernicious anemia, pure red cell aplasia (PRCA), factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases with leukocyte emigration, central nervous system (CNS) inflammatory disorders, multiple organ injury syndrome, myasthenia gravisgravis), antigen-antibody complex-mediated disease, antiglomerular basement membrane disease, antiphospholipid syndrome, allergic neuritis, Behçet's disease, Castleman syndrome, Goodpasture syndrome, Lambert-Eaton myasthenic syndrome, Raynaud's syndrome, Sjögren's syndrome syndrome), Stevens-Johnson syndrome, bullous pemphigoid, pemphigus, autoimmune polyendocrinopathy, Reiter's disease, stiff man syndrome, giant cell arteritis, immune complex nephritis, IgA nephropathy, IgM polyneuropathy or IgM-mediated neuropathy, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, autoimmune diseases of the testes and ovaries, such as autoimmune orchitis and oophoritis, primary hypothyroidism, autoimmune endocrine disorders, such as autoimmune thyroiditis, chronic thyroiditis (Hashimoto's thyroiditis), subacute thyroiditis, idiopathic thyroiditis These include hypogonadism, Addison's disease, Graves' disease, polyglandular autoimmune syndrome (or polyendocrinopathy syndrome), Sheehan's syndrome, autoimmune hepatitis, lymphocytic interstitial pneumonia (HIV), bronchiolitis obliterans (non-transplant cases) vs. non-steroidal anti-inflammatory drug (NSIP), Guillain-Barré syndrome, large-vessel vasculitis (e.g., polymyalgia rheumatica and giant cell (Takayasu) arteritis), medium-sized vasculitis (e.g., Kawasaki disease and polyarteritis nodosa), ankylosing spondylitis, Berger's disease (IgA nephropathy), rapidly progressive glomerulonephritis, primary biliary cirrhosis, celiac sprue (gluten enteropathy), cryoglobulinemia, and amyotrophic lateral sclerosis (ALS). In some cases, the autoimmune disease is systemic lupus erythematosus (SLE), rheumatoid arthritis, or celiac disease. Pharmaceutical Composition

[0313] The present invention provides a kit for administering to a subject and a composition comprising a tolerization vaccine or a conditioning regimen.In some embodiments, the tolerization vaccine or conditioning regimen or its components (e.g., apoptotic donor leukocytes, such as ECDI-fixed splenocytes, or mesenchymal stromal cells) are combined with a pharmaceutically acceptable carrier, diluent, or excipient.The excipient that can be used is saline.The excipient that can be used is phosphate-buffered saline (PBS).Therefore, these pharmaceutical compositions can be used to treat patients who need transplantation.

[0314] The composition of the present disclosure can comprise buffering agents, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose or dextran; mannitol; protein; polypeptide or amino acid, such as glycine; antioxidant; chelating agent, such as EDTA or glutathione; preservative; or combinations thereof.The composition of the present disclosure can be formulated for intravenous administration (for example, intravenous injection or infusion).The composition of the present disclosure can be sterile liquid preparation, such as isotonic aqueous solution, suspension, emulsion, dispersion or viscous composition.

[0315] The compositions of the present disclosure can be buffered to a selected pH, for example, by buffering the compositions of the present disclosure to approximately 5-9, 5-8, 5-7, 5-6, 6-9, 6-8, 6-7, 7-9, 7-8, 6.5-8.5, 6.5-8, 6.5-7.7, 6.5-7.6, 6.5-7.5, 6.5-7.4, 6.5-7.3, 6.5-7.2, 6.5-7, 7-7.7, 7-7.6, 7-7.5, It can be buffered to a pH of 7-7.4, 7-7.3, 7-7.2, 7-7.1, 7.2-7.6, 7.2-7.5, 7.2-7.4, 7.3-7.7, 7.3-7.6, 7.3-7.5, 7.34-7.45, 7.0-7.2, 7.2-7.4, 7.3-7.5, 7.4-7.6, or 7.6-7.8. In addition, the composition may include a pH buffer, for example, 0.1 mM to 100 mM phosphate pH 6.0 to 9.0, 0.1 mM to 100 mM HEPES pH 6.0 to 9.0, 0.1 mM to 100 mM bicarbonate pH 6.0 to 9.0, 0.1 mM to 100 mM citrate pH 6.0 to 9.0, 0.1 mM to 100 mM acetate pH 4.0 to 8.0, or any combination thereof.

[0316] The composition may include an electrolyte, for example, 5 mM to 400 mM NaCl, 0.5 mM to 50 mM KCl, 0.05 mM to 50 mM CaCl2, 0.05 mM to 50 mM MgCl2, 0.05 mM to 50 mM LiCl2, 0.05 mM to 50 mM MnCl2, or any combination thereof.

[0317] The composition may include an antioxidant, for example, 0.05 to 10 mM glutathione (reduced), 0.05 to 10 mM glutathione (oxidized), 0.001 to 10 mM β-mercaptoethanol, 0.001 to 10 mM dithiothreitol, 0.01 to 100 mM ascorbate, 0.001 to 10 mM tris(2-carboxyethyl)phosphine, or any combination thereof.

[0318] The composition may contain a stabilizer, for example, 0.01% to 10% human serum albumin, 0.01% to 10% bovine serum albumin, 0.1% to 99% human serum, 0.1% to 99% fetal bovine serum, 0.01% to 10% IgG, 0.1% to 10% immunoglobulin, 0.06% to 60% trehalose, or a molecular polymer such as 0.1% to 20% polyethylene glycol (MW 200 to 20,000,000), or any combination thereof.

[0319] Liquid or viscous compositions may contain a carrier, which may be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof.

[0320] Various additives which enhance the stability and sterility of the compositions can be added, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like.

[0321] The composition can be isotonic, i.e., it can have the same osmotic pressure as blood and tears.The desired isotonicity of the composition of the present invention can be achieved using sodium chloride or other pharmaceutically acceptable agents, such as dextrose, boric acid, sodium tartrate, propylene glycol, or other inorganic or organic solutes.Sodium chloride is particularly preferred for buffers containing sodium ions.

[0322] In some embodiments, the tolerizing vaccine or conditioning regimen, or components thereof, is administered with one or more adjuvants (eg, one or more immunomodulatory molecules).

[0323] In some embodiments, the tolerizing vaccine or conditioning regimen is administered in conjunction with an immunosuppressant (e.g., one or more immunomodulatory molecules). In some cases, the immunosuppressant inhibits T cell activation. The immunosuppressant that inhibits T cell activation can be an anti-CD40 agent or an anti-CD40L (CD154) agent. In some embodiments, the anti-CD40 agent can be an anti-CD40 antibody. The anti-CD40 antibody can be an antagonist antibody. The anti-CD40 antibody can be the Fab' anti-CD40L monoclonal antibody fragment CDP7657. The anti-CD40 antibody can be an FcR-engineered, Fc-silenced anti-CD40L monoclonal domain antibody. The anti-CD40 or anti-CD40L agent can be 2C10, 2C10R4, ASKP1240, 4D11, bleselumab, BI-655064, HCD122, CFZ533, ch5D12, FFP104, CDP7657, BG9588, ruplizumab, toralizumab, IDEC-131, dapirolizumab, letolizumab, BMS-986004, VIB4920, or MEDI4920.

[0324] In some cases, the tolerizing vaccine or conditioning regimen may include one or more additional immunosuppressive agents described herein, e.g., an anti-CD40 agent or an anti-CD40L (CD154) agent (e.g., an anti-CD40 antibody), a B cell targeting agent (e.g., a biologic that depletes B cells, e.g., a biologic that targets CD20, CD19, or CD22, and / or a biologic that modulates B cells, e.g., BAFF, BAFF / APRIL, CD40, IgG4, ICOS, IL-21, B7RP1, etc.). The B-cell targeting biologic may be administered with one or more of the following: a biologic targeting B cells (e.g., a biologic targeting B cells), an mTOR inhibitor, a TNF-alpha inhibitor, an IL-6 inhibitor, a nitrogen mustard alkylating agent (e.g., cyclophosphamide), a complement C3 or C5 inhibitor, IFN-γ, an NFκB inhibitor, α1-antitrypsin, vitamin D3, siCD40, cobalt protoporphyrin, insulin B9-23, a cluster of differentiation protein (CD46, CD55, or CD59), any combination thereof, or any fragment thereof. In some cases, the NFκB inhibitor is dehydroxymethylepoxyquinomycin (DHMEQ), curcumin, triptolide, Bay-117085, or a combination thereof. In some cases, the B-cell targeting biologic may be Rituximab or another anti-CD20 antibody.

[0325] In some cases, the immunosuppressant is MMF (mycophenolate mofetil (Cellcept)), ATG (antithymocyte globulin), anti-CD154 (CD40L), alemtuzumab (Campath), B cell targeting agents (e.g., biologics that deplete B cells, e.g., biologics that target CD20, CD19, or CD22, and / or biologics that modulate B cells, e.g., BAFF, BAFF / APRIL, biologics that target CD40, IgG4, ICOS, IL-21, B7RP1), anti-IL-6R antibodies (sarilumab, tocilizumab, Actemra), anti-IL-6 antibodies (clazakizumab, ALD518, siltuximab, elcilimomab, sirukumab, olokizumab), CTLA4-Ig (Abatacept / Orencia) , belatacept (LEA29Y), mTOR inhibitors (sirolimus (e.g., Rapamune), rapamycin, everolimus), tacrolimus (Prograf), daclizumab (Ze-napax), basiliximab (Simulect), infliximab (Remicade), cyclosporine, deoxyspergualin, soluble complement receptor 1, cobra venom factor, compstatin, anti-C5 antibody (eculizumab / Soliris), methylprednisolone, FTY720, everolimus, anti-CD154-Ab, leflunomide, anti-IL-2R-Ab, anti-CXCR3 antibody, anti-ICOS antibody, anti-OX40 antibody, and anti-CD122 antibody, human anti-CD154 monoclonal antibody, CD40 antagonist, and CD40L (CD154) antagonist. Non-limiting examples of biologics that target B cells include antagonistic anti-CD40 These include mAb antibodies, Fc-engineered anti-CD40L antibodies, rituximab, and anti-CD20 antibodies. One or more immunosuppressants / drugs can be used together or separately. One or more immunosuppressants / drugs can be used for induction therapy or maintenance therapy. The same or different drugs can be used during the induction and maintenance phases. For example, daclizumab (Zenapax) can be used for induction therapy, and tacrolimus (Prograf) and sirolimus (Rapamune), or everolimus, or any other mTOR inhibitor, can be used for maintenance therapy. In another example, daclizumab (Zenapax) can be used for induction therapy, and low-dose tacrolimus (Prograf) and low-dose sirolimus (Rapamune) can be used for maintenance therapy. Immunosuppression can also be achieved using non-drug regimens, including, but not limited to, total body irradiation, thymic irradiation, and total and / or partial splenectomy. These techniques can also be used in combination with one or more immunosuppressive drugs.

[0326] In some embodiments, the one or more immunomodulatory molecules can target T cell receptor (TCR), CD3e, FK506-binding protein 12 (FKBP12), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), programmed cell death-ligand 1 (PD-L1), CD40L (CD154), CD40, inducible costimulation (ICOS), IL-2, TNF-α, IL-6, IL-7, CD2, CD20, CD52, alpha-4 integrin, mTOR (mechanistic target of rapamycin, everolimus, cerolimus), DNA synthesis, or any combination thereof. In some embodiments, the one or more immunomodulatory molecules can target B cells (B cell-depleting biologics, e.g., biologics targeting CD20, CD19, or CD22, and / or B cell-modulating biologics, e.g., biologics targeting BAFF, BAFF / APRIL, CD40, IgG4, ICOS, IL-21, B7RP1). In some cases, the B cell targeting agent can be an anti-CD20 mAb (e.g., rituximab) or other B cell-depleting antibody. In some embodiments, the immunosuppressant can be an MHC / TCR interaction blocker, a non-selective depleting agent, a calcineurin inhibitor, a costimulatory signal blocker, a cytokine blocker, a lymphocyte depleting agent, a cell adhesion inhibitor, an IL-2 signaling inhibitor, a cell cycle blocker, or any combination thereof. For example, the MHC / TCR interaction blocker can be the anti-abTCR mAb T10B9. For example, the non-selective depleting agent can be anti-CD3 mAb (OKT3) or anti-thymocyte globulin (ATG). For example, the calcineurin inhibitor can be cyclosporine or tacrolimus. For example, the costimulatory signal blocker can be anti-CTLA-4 mAb, abatacept, ipilimumab, anti-PD-1 (e.g., pembrolizumab), anti-PD-L1 (e.g., MPDL3280A), anti-CD154 mAb, anti-CD40 mAb, or anti-ICOS mAb.For example, the cytokine blocker can be an anti-CD25 mAb (e.g., daclizumab or basiliximab), an anti-TNF (etanercept, infliximab, adalimumab, certolizumab pegol, and golimumab), an anti-IL-6 / IL-6R mAb (e.g., clazakizumab, ALD518, siltuximab, elcilimomab, sirukumab, olokizumab, sarilumab, tocilizumab, Actemra), or an anti-IL-7 mAb. For example, the lymphodepleting agent can be an anti-CD2 mAb, a fusion protein with IgG1 (e.g., alefacept), an anti-CD20 mAb (e.g., rituximab), or an anti-CD52 mAb (e.g., alemtuzumab). For example, the cell adhesion inhibitor can be an anti-very large antigen 4 (VLA4) (e.g., natalizumab). For example, the IL-2 signaling inhibitor can be sirolimus (rapamycin) or everolimus. For example, the cell cycle blocker can be mycophenolate mofetil (MMF) or azathioprine. kit

[0327] Another embodiment of the present disclosure provides an article of manufacture in solution or lyophilized form containing a composition comprising apoptotic leukocytes, an mTOR inhibitor, an anti-tumor necrosis factor agent or anti-tumor necrosis factor receptor agent, an anti-interleukin-6 agent or anti-interleukin-6 receptor agent, and an anti-CD40 agent or anti-CD40 ligand agent, or a kit comprising the article of manufacture. The kit of the present disclosure may be for use during transplantation of a graft in a recipient or for use to reduce or suppress the occurrence of GVHD in a recipient. In some embodiments, the kit is useful as a pre-transplant conditioning regimen. In some embodiments, the kit is useful as a post-transplant tolerization regimen. The kit may include instructions for diluting the composition or for its reconstitution and / or use. The article of manufacture includes one or more containers. Suitable containers include, for example, bottles, vials (e.g., dual-chamber vials), syringes (e.g., dual-chamber syringes), and test tubes. The containers may be formed from a variety of materials, such as glass or plastic. The container may hold the composition, e.g., in lyophilized or solution form, and a label on or associated with the container may indicate instructions for reconstitution and / or use. For example, the label may indicate that the lyophilized composition is to be reconstituted to an effective amount as described above. The label may further indicate that the composition is useful for or intended for subcutaneous or intravenous administration. The container holding the composition may be a multi-use vial that allows for repeated administration of the composition (e.g., 2-6 administrations). The article of manufacture may further include a container containing a suitable diluent (e.g., BWFI). Upon mixing the diluent and the composition, the final concentration in the composition may be, for example, an effective amount suitable for administration. The article of manufacture may further include other materials desirable from a commercial and user standpoint, such as other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0328] The kit may have, for example, a single container containing apoptotic leukocytes fixed in a crosslinker with or without other components (e.g., an mTOR inhibitor, an anti-tumor necrosis factor agent or anti-tumor necrosis factor receptor agent, an anti-interleukin-6 agent or anti-interleukin-6 receptor agent, and an anti-CD40 agent or anti-CD40 ligand agent), or may have separate containers for each component. In some embodiments, the kit may have, for example, a single container containing apoptotic leukocytes fixed in a crosslinker and comprising, conjugated on their surface, one or more peptides derived from a recipient's MHC class II molecule or one or more derived from a donor's MHC class I molecule, with or without other components (e.g., an mTOR inhibitor, an anti-tumor necrosis factor agent or anti-tumor necrosis factor receptor agent, an anti-interleukin-6 agent or anti-interleukin-6 receptor agent, and an anti-CD40 agent or anti-CD40 ligand agent), or may have separate containers for each component.

[0329] Prior to administration to a patient, the kit compositions may be pre-complexed, or each component may be in a separate, distinct container. The kit components may be provided in one or more liquid solutions, preferably aqueous solutions, more preferably sterile aqueous solutions. The kit components may be provided as solids, which can be converted to liquids by the addition of a suitable solvent, preferably provided in a separate, distinct container. In some embodiments, the kits of the present disclosure include the components disclosed herein packaged for use in combination with the co-administration of an additional component (e.g., an anti-inflammatory agent, an immunomodulatory agent, an anti-tumor agent, a natural product, a hormone or antagonist, an anti-angiogenic agent or inhibitor, an apoptosis inducer, or a chelator). The kit containers may be vials, test tubes, flasks, bottles, syringes, or any other means for enclosing a solid or liquid. In some embodiments, the kit will include a second vial or other container that allows for separate administration. The kit may also include a separate container for a pharmaceutically acceptable liquid. Preferably, the kit will include a device (eg, one or more needles, syringes, eyedroppers, pipettes, etc.) that allows for administration of the compositions of the present disclosure that are components of the kit.

[0330] In some embodiments, the kits disclosed herein further comprise a transplant. In some embodiments, the transplant is a kidney, liver, heart, lung, pancreas, pancreatic islet cells, small intestine, bone marrow, hematopoietic stem cells, embryonic or induced pluripotent stem cell-derived pancreatic islet beta cells, embryonic or induced pluripotent stem cell-derived pancreatic islets, embryonic or induced pluripotent stem cell-derived hepatocytes, or a combination thereof. In some embodiments, the transplant can be an autograft, an allograft, or a xenograft. How to make genetic modifications

[0331] Various techniques can be used to generate the above-mentioned genetically modified cells or non-human animals.A few examples of the genetically modified cells or animals are disclosed herein.It should be understood that the methods disclosed herein are merely examples and are not intended to be limiting in any respect.

[0332] The methods disclosed herein may utilize techniques that can be used to introduce DNA or RNA constructs into host cells, including, but not limited to, calcium phosphate / DNA co-precipitation, microinjection of DNA into the nucleus, electroporation, bacterial protoplast fusion with intact cells, transfection, lipofection, infection, particle bombardment, sperm-mediated gene transfer, or any other technique known to those of skill in the art.

[0333] Certain embodiments disclosed herein may utilize vectors. Any plasmids and vectors can be used as long as they are replicable and viable in the selected host. Vectors known in the art and commercially available (and their variants or derivatives) can be engineered to contain one or more recombination sites for use in the method. Vectors that can be used include, but are not limited to, eukaryotic expression vectors such as pFastBac, pFastBacHT, pFastBacDUAL, pSFV, and pTet-Splice (Invitrogen), pEUK-C1, pPUR, pMAM, pMAMneo, pBI101, pBI121, pDR2, pCMVEBNA, and pYACneo (Clontech), pSVK3, pSVL, pMSG, pCH110, and pKK232-8 (Pharmacia, Inc.), p3'SS, pXT1, pSG5, pPbac, pMbac, pMClneo, and pOG44 (Stratagene, Inc.), as well as pYES2, pAC360, pBlueBa-cHis A, B and C, including pVL1392, pBlueBac111, pCDM8, pcDNA1, pZeoSV, pcDNA3, pREP4, pCEP4 and pEBVHis (Invitrogen, Corp.), and variants or derivatives thereof.

[0334] These vectors can be used to express genes, e.g., transgenes, or portions of genes of interest. The portions of genes, or genes, can be inserted using known methods, such as restriction enzyme-based techniques. Gene disruption

[0335] Gene disruption can be performed by any of the following methods, for example, knockout, knockdown, RNA interference, dominant negative, etc. Gene disruption can be performed in non-human animals. Gene disruption can be performed in human cells (e.g., human splenocytes, peripheral blood leukocytes, and / or stem cell-derived cells, tissues, or organs). Detailed descriptions of methods are disclosed in the section on genetically modified non-human animals below.

[0336] In some embodiments, the graft donor and / or apoptotic cells have reduced expression of one or more genes expressing alpha 1,3 galactosyltransferase (GGTA1), putative cytidine monophosphatase-N-acetylneuraminic acid hydroxylase-like protein (CMAH), and β1,4 N-acetylgalactosaminyltransferase (B4GALNT2). In some embodiments, the graft donor and / or apoptotic cells have disruption of one or more genes expressing GGTA1, CMAH, and B4GALNT2. This can minimize or eliminate cell-mediated immunity and cell-dependent antibody-mediated immunity against organ, tissue, cell, and cell line grafts (e.g., xenografts or allografts) from a graft donor animal that is genetically identical to the apoptotic cell vaccine donor animal.

[0337] For example, the cells may have one or more genes that can be disrupted (e.g., expression reduced), including GGTA1, CMAH, B4GALNT2, NLRC5, B2M, PD-L1, and / or any combination thereof. For example, the cells may have only GGTA1 disrupted, or only CMHA disrupted, or only B4GALNT2 disrupted. The cells may have GGTA1 and CMAH disrupted, or GGTA1 and B4GALNT2 disrupted, or CMAH and B4GALNT2 disrupted. The cells may have GGTA1, CMAH, and B4GALNT2 disrupted. In some cases, the disrupted genes do not include GGTA1. The cells may express HLA-G (endogenously or exogenously) even if GGTA1 and / or CMAH are disrupted. The cells may have C3 disrupted.

[0338] In some embodiments, the graft donor animal and / or the apoptotic cells comprise additional genetic modifications. In some embodiments, the graft donor animal and / or the apoptotic cells comprise suppression or disruption of one or more genes encoding a component of the major histocompatibility complex (MHC) I-specific enhanceosome (e.g., NOD-like receptor family CARD domain-containing 5 (NLRC5)), a transporter of MHC-I-bound peptides (e.g., antigen processing 1 (TAP1)), complement component 3 (C3), C-X-C chemokine receptor 3 ligand (CXCL3), C-X-C motif chemokine ligand 10 (CXCL10) gene, MHC II transactivator (MHCIITA), MHC class I polypeptide-related sequence A (MICA) gene, MHC class I polypeptide-related sequence B (MICB) gene, natural killer (NK) group 2D ligand (NKG2DL), tumor necrosis factor receptor (TNF-R), porcine endogenous retrovirus (PERV), PD-1, PD-L1, or any combination thereof.

[0339] In some embodiments, the graft donor animal and / or the apoptotic cells comprise one or more exogenous polynucleotides encoding one or more exogenous polypeptides. In some embodiments, the exogenous polypeptides express one or more of: a suppressor of MHC I formation (e.g., infected cell protein 47 (ICP47)), a regulator of complement activation (e.g., CD46, CD55, or CD59), an inhibitory ligand of NK cells, a B7 family member (e.g., a programmed death ligand, e.g., PD-L1 or PD-L2), a serine protease inhibitor (e.g., Spi9), a galectin, an interleukin (e.g., IL-37), a CD40:CD40L blocking agent (e.g., a CD40 antagonist polypeptide, an anti-CD40 ligand polypeptide), ST6 beta-galactoside alpha-2,6-sialyltransferase 1 (ST6Gal1), Fas ligand (FasL), any functional fragment thereof, or any combination thereof. In some embodiments, the NK cell inhibitory ligand is a human leukocyte antigen (HLA), such as human leukocyte antigen E (HLA-E), human leukocyte antigen G (HLA-G), beta-2-microglobulin (B2M), or any combination thereof. In some embodiments, the HLA-G is HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, HLA-G7, or any combination thereof. In some cases, the galectin is galectin-1, galectin-2, galectin-3, galectin-4, galectin-5, galectin-6, galectin-7, galectin-8, galectin-9, galectin-10, galectin-11, galectin-12, galectin-13, galectin-14, or galectin-15. For example, the galectin can be galectin-9.

[0340] In some cases, disruption is not limited to these genes. Gene homologs of genes within the scope of the present application (e.g., any mammalian version of a gene) are also contemplated. For example, the disrupted gene may exhibit a certain identity and / or homology to a gene disclosed herein, such as NLRC5, TAP1, GGTA1, B4GALNT2, CMAH, CXCL10, MICA, MICB, C3, and / or MHCIITA. Thus, it is contemplated that genes exhibiting at least or at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% homology (at the nucleic acid or protein level) may be disrupted, for example, genes exhibiting at least or at least about 50%-60%, 60%-70%, 70%-80%, 80%-90%, or 90%-99% homology. It is also conceivable that genes that exhibit at least or at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 99%, or 100% identity (at the nucleic acid or protein level) can be disrupted, for example, genes that exhibit at least or at least about 50%-60%, 60%-70%, 70%-80%, 80%-90%, or 90%-99% identity. Some gene homologs are known in the art, but in some cases, homologs are unknown. However, homologous genes between mammals can be found by comparing nucleic acid (DNA or RNA) sequences or protein sequences using publicly available databases such as NCBI BLAST.

[0341] Gene suppression can also be carried out in many ways.For example, gene expression can be reduced by modifying the promoter of the gene to knock out and / or by administering interference RNA (knockdown).This can be carried out at the organism level or at the tissue, organ and / or cellular level.When knocking down one or more genes in cells, tissues and / or organs, RNA interference reagents, such as siRNA, shRNA or microRNA, can be administered to reduce one or more genes.For example, nucleic acid capable of expressing shRNA can be stably transfected into cells to knock down expression.In addition, nucleic acid capable of expressing shRNA can be inserted into the genome of cells or non-human animals, resulting in knocking down genes in cells or non-human animals.

[0342] Disruption methods can also include overexpressing dominant-negative proteins. This method can result in a general reduction in the function of functional wild-type genes. In addition, expressing dominant-negative genes can produce phenotypes similar to those of knockout and / or knockdown.

[0343] In some cases, a stop codon can be inserted or created (e.g., by nucleotide substitution) in one or more genes, resulting in a non-functional transcript or protein (sometimes called a knockout). F...

Claims

[Claim 1] The invention described in the specification.

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