Cultured thymic tissue transplantation to promote donor-specific tolerance for allogeneic solid organ transplantation

Abstract

To provide methods and compositions for promoting donor-specific tolerance and immunocompetence to a recipient of a solid organ transplant, by implanting an allogeneic solid organ in the recipient in need of the solid organ transplant.SOLUTION: Methods and compositions comprise surgical implantation of a tissue-engineered allogeneic cultured postnatal thymus tissue product in a recipient of a solid organ from a donor.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001]

[0001] Methods and compositions for promoting donor-specific tolerance to allogeneic solid organ grafts in recipients receiving allogeneic solid organ grafts from a donor. [Background technology]

[0002]

[0002] Organ transplantation requires the preparation and harvesting of human solid organs from a donor, as well as their transplantation into a recipient. The main problem with solid organ transplantation is that recipients are not tolerant to the donor. Recipient T cells reject the organ, and recipient B cells produce antibodies against the organ, ultimately leading to failure. The difficult-to-achieve ideal of solid organ transplantation is the development of tolerance by the recipient to the transplanted human organ. It is estimated that more than 36,000 organ transplants are performed annually in the United States, with even more in Europe and other major countries. It is further estimated that there are more than 120,000 patients on organ transplant waiting lists in the United States. The demand for healthy organs far exceeds the supply of suitable organs. In 2018, approximately 10,000 donors were identified. See https: / / optn.transplant.hrsa.gov.

[0003]

[0003] Transplant rejection is a substantial challenge in solid organ transplantation. Transplant rejection by both T cells and B cells can lead to serious complications in organ function or even transplant failure. Five-year graft survival rates are, for example, 77.7% for heart transplants, 78.6% for kidney transplants, 72.8% for liver transplants, and 53.4% ​​for lung transplants. Typically, this problem is addressed in part through donor-recipient compatibility for major histocompatibility complex (MHC) antigens and by avoiding recipients who have antibodies against the recipient tissue type. In addition, the use of immunosuppressive regimens to manage the immunological responses underlying transplant rejection has improved. However, tolerance has not been achieved, and the mean survival for many organs is only 10 years.

[0004]

[0004] Preserving organ viability before and during transplantation is the second important challenge. Organ removal, preservation, and transplantation can have a profound impact on the internal structure and function of the organ, and can have a significant impact on the extent to which the recovery of normal organ function is delayed or prevented after the transplant is completed.

[0005]

[0005] The effective preservation time for human solid organs varies depending on the organ, ranging from 24 to 36 hours for the kidneys, 12 to 18 hours for the pancreas, 8 to 12 hours for the liver, and 4 to 6 hours for the heart and lungs. See https: / / unos.org / transplantation / matching-organs.

[0006]

[0006] Organ damage occurs mainly as a result of ischemia and hypothermia, but may also be related to reperfusion of organs ex vivo or during transplantation.

[0007]

[0007] Techniques for organ preservation, including ex vivo perfusion, are known in the art and are useful in minimizing organ damage and promoting optimal graft survival and function.

[0008]

[0008] The main solid organs targeted for transplantation include the kidneys, liver, heart, and lungs. The success of transplantation of these solid organs has been achieved with varying degrees of success. The main variability lies in the techniques used to interfere with immune-mediated graft rejection. Experience has shown that there is no single immunosuppressant or technique that is effective in all environments involved in solid organ transplantation. The limiting factor is usually the toxicity associated with each individual immunosuppressant. The toxicity associated with a given immunosuppressant can often interfere with the normal function of the transplanted solid organ or other organs such as the kidney, which can lead to failure when calcineurin inhibitors are used to prevent rejection.

[0009]

[0009] The toxic drawbacks associated with known immunosuppressants commonly used to prevent graft rejection in solid organ transplantation highlight the need to find new methods to prevent graft rejection in solid organ transplantation.

[0010]

[0010] The ability to distinguish between self-antigens and non-self-antigens is central to the immune response. This distinction leads to self-tolerance. Autoimmunity occurs when self-tolerance is lost. There is an unmet need in transplantation procedures to induce tolerance to solid organ grafts. [Overview of the project]

[0011]

[0011] Achieving donor-specific immune tolerance remains the ultimate immunological goal in transplantation. Most current approaches focus on controlling peripherally matured donor-reactive T cells by depletion (e.g., alemtuzumab, thymoglobulin, etc.) or suppression (e.g., calcineurin inhibitors, basiliximab, etc.) without targeting the production of alloreactive T cells in the thymus. However, even with dramatic advances in immunosuppressants and new immunomodulatory regimens, graft tolerance has not yet been consistently achieved.

[0012]

[0012] The inventors have shown that tolerance to solid organ grafts can be achieved in thymectomy recipients through the transplantation of an allogeneically cultured postnatal thymocyte-derived product (hereinafter also referred to herein as "CTT" or "RVT-802"), which can reduce the time required for post-transplant immunosuppressant use to prevent rejection of the transplanted organ. Removal of the recipient's thymus and replacement with an allogeneically cultured postnatal thymocyte-derived product results in the reconstruction of the solid organ recipient's immune system and tolerance of the recipient's own and transplanted allogeneic solid organs.

[0013]

[0013] Tolerance induction by surgical insertion of allogeneically cultured post-vitro thymocyte-derived products is similar to tolerance induction via donor dendritic cells ("DCs") in hematopoietic stem cell transplantation (Sharabi Y & Sachs DH, 1989, "Mixed chimerism and permanent specific transplantation tolerance induced by a nonlethal preparative regimen," J Exp Med 169(2):493-502; Manilay JO, Pearson DA, Sergio JJ, Swenson KG, & Sykes M, 1998, "Intrathymic deletion of alloreactive T cells in mixed bone marrow chimeras prepared with a nonmyeloablative conditioning regimen," Transplantation 66(1):96-102).A series of studies from the Transplantation Biology Research Center (TBRC, Boston, MA) demonstrated the important role of the thymus in tolerance induction (Yamada K, et al., 1997, “Role of the thymus in transplantation tolerance in miniature swine. I. Requirement of the thymus for rapid and stable induction of tolerance to class I-mismatched renal allografts,” J Exp Med 186(4):497-506.), and tested thymic transplantation with tolerance induction in large animal models (Yamada K, et al., 2000, “Thymic transplantation in miniature swine. II. Induction of tolerance by transplantation of composite thymokidneys to thymectomized recipients,” J Immunol 164(6):3079-3086;5, Yamada K, et al., 2003, “Thymic transplantation in miniature swine: III. Induction of In their series of studies, this group successfully used class II compatible / class I incompatible donors (thymus and kidney or heart) as thymic composite tissue (thymokidney and thymocinic heart) with 12 days of cyclosporine ("CsA") for transplant tolerance induction. (Transplantation 76(3):530-536, Nobori S, et al., 2006, "Thymic rejuvenation and the induction of tolerance by adult thymic grafts," Proc Natl Acad Sci USA 103(50):19081-19086)They argued that non-angiogenic thymus did not induce tolerance in their model. More precisely, however, uncultured non-angiogenic thymus did not engraft over the long term. As they demonstrated, the failure of engraftment of uncultured thymus may be due to ischemic injury in addition to alloimmunity (Yamada K, et al., 2000). This sophisticated concept of generating angiogenic thymus before transplantation to induce tolerance would be difficult to translate clinically without using xenotransplantation. (This section cites Kwan J et al., Cultured Thymus Transplantation Promotes Donor-specific Tolerance to Allogeneic Heart Transplants, submitted to the 2019 publication.)

[0014]

[0014] The limitations of non-angiogenic thymus transplantation, as well as T cell depletion, can be overcome in culture systems. Experimental transplantation of allogeneically cultured postnatal thymus tissue-derived products (CTT) that retain TECs has been successfully applied to treat pediatric patients with congenital athymus (Markert ML, Devlin BH, McCarthy EA, 2010, “Thymus transplantation,” Clin Immunol., 135(2):236-46; Markert ML, et al., 2004, “Postnatal thymus transplantation with immunosuppression as treatment for DiGeorge syndrome,” Blood 104(8):2574-2581; Markert ML, et al., 1999, “Transplantation of thymus tissue in complete DiGeorge syndrome,” N Engl J Med 341(16):1180-1189 27).

[0015]

[0015] In this reference, DiGeorge syndrome is defined as a condition characterized by various defects in the heart, thymus, and parathyroid glands. Approximately 1% of infants with DiGeorge syndrome have athymia and therefore lack T cells to fight infection. These infants are said to have complete DiGeorge syndrome. There are four subgroups of children that meet the criteria for complete DiGeorge syndrome, 22q11.2 deletion syndrome, CHARGE, infants of diabetic mothers, and infants without the syndrome or genetic defect. In all four groups, infants with athymia represent a very small group, perhaps 1% of all children who have received a diagnosis such as 22q11.2 deletion syndrome.

[0016]

[0016] Thymic lymphocyte formation has been documented by allograft biopsies and the presence of recipient naive T cells in the periphery (Markert ML, 2010; Markert ML, et al., 2008, “Use of allograft biopsies to assess thymopoiesis after thymus transplantation,” J Immunol 180(9):6354-6364; Markert ML, et al., 2007, “Review of 54 patients with complete DiGeorge anomaly enrolled in protocols for thymus transplantation: outcome of 44 consecutive transplants,” Blood 109(10):4539-454728). A study of children treated with the clinical trial CTT demonstrated tolerance to donor MHC due to a mixed lymphocyte response (Chinn IK, Devlin BH, Li YJ, & Markert ML, 2008, “Long-term tolerance to allogeneic thymus transplants in complete DiGeorge anomaly,” Clin Immunol 126(3):277-281). In addition, infants with congenital athymus can control infections such as Epstein-Barr virus after CTT transplantation (Markert ML, 2014, Thymus Transplantation. Stiehm's Immune Deficiences, eds Sullivan KE & Stiehm ER (Academic Press), 1st Ed, pp 1059-1067; Isakovic K, Smith SB, & Waksman BH, 1965, “Immunologic Tolerance in Thymectomized, Irradiated Rats Grafted with Thymus from Tolerant Donors,” Science 148(3675):1333-1335).Based on these data in humans with congenital athymia, it was determined that allogeneic cultured postnatal thymic tissue-derived products that express the MHC of solid organ donors in recipients after surgical insertion produce functional T cells that protect the recipient from infection while developing tolerance to both self and donor. Thymus and thymocyte education. (This section cites Kwan J et al Cultured Thymus Transplantation Promotes Donor-specific Tolerance to Allogeneic Heart Transplants, submitted for publication in 2019.)

[0017]

[0017] The thymus is usually located above the heart. The thymus provides the microenvironment essential for T cell development and is indispensable for the establishment and maintenance of the adaptive immune system (Boehm T and Takahama Y, 2014, Thymic Development and Selection of T Lymphocytes. Heidelberg: Springer-Verlag). During postnatal development, the thymus educates hematopoietic stem cells that migrate from the bone marrow to the thymus. The precursor stem cells colonize the thymus, thereby forming thymocytes. Thymocytes then undergo a series of maturation steps. This is evidenced by the expression of many observable cell surface markers that appear on thymocytes.

[0018]

[0018] T cells are important for the body's protection from infection. T cells that develop in a normally functioning thymus develop a diverse set of T cell receptors (generally proteins on the surface of cells), which enables mature T cells to fight various infections. During this educational process, the developing T cells are instructed by the thymus not to attack normal proteins of the body, such as insulin or parathyroid hormone (which regulate blood glucose and calcium levels). These instructions are carried out under the influence of the autoimmune regulator gene (the "AIRE gene").

[0019]

[0019] In short, the education process takes place in a properly functioning thymus. Thymocytes in the thymus, formed from bone marrow stem cells, are taught by thymic epithelial cells (TECs) and dendritic cells (DCs) located within the thymus not to attack recipient major histocompatibility complex (MHC) proteins (antigens) such as HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DQA1, HLA-DPB1, and HLA-DPA1 antigens. HLA antigens have two proteins that hold a self-peptide in a groove. The self-peptide can be derived from thyroid proteins or insulin peptides, or almost any other protein expressed in the body. Thymocytes developing in the thymus form a T cell receptor (TCR) composed of two proteins that cross the membrane. The TCR is expressed on the cell surface of T cells. Each T cell expresses many copies of its own TCR. If the binding of TCRs to the self-peptide:MHC on dendritic cells is too strong, the dendritic cells provide a signal to induce apoptosis and kill the T cells. This mechanism prevents the development of autoimmunity against self. TECs can also provide a signal to thymocytes that their binding is too strong. Finally, DCs can acquire a portion of the membrane from TECs and present TEC self-peptide:MHC to developing thymocytes. If the binding to the thymocytes is too strong, the DCs send a signal to the thymocytes to undergo apoptosis and die. Through these mechanisms, T cells leaving the thymus are not self-reactive. T cells leaving the thymus are highly diverse and can recognize infection, but they do not attack proteins in the body.

[0020]

[0020] The two main components of the thymus are the epithelium and thymocytes that are produced in the thymus in the following manner. Cells derived from bone marrow stem cells that are lymphoid lineage common progenitor cells (“CLP”) migrate to the thymus. CLP enters the thymus in response to signals (chemokines) produced by thymic epithelium and endothelium. In the thymus, CLP differentiates and proliferates into thymocytes. Thymocytes develop a unique T cell receptor (“TCR”) that is expressed on the cell surface. Thymocytes also begin to express the T cell molecules CD3, CD4, and CD8. A very diverse repertoire of T cells is generated so that the cells can respond to infection throughout the recipient's lifetime. The mixed lymphocyte reaction demonstrates tolerance of recipient T cells in pediatric patients treated with cultured thymic tissue (RVT-802) to the thymic donor MHC.

[0021]

[0021] Autoreactive recipient thymocytes are deleted before leaving the thymus. This occurs through the interaction of recipient thymocytes and recipient DCs that migrate to the thymus. Apoptosis is induced in recipient thymocytes that bind too strongly to DCs as a means of protecting the body from autoimmune diseases. After completion of this process, thymocytes leave the thymus. Newly circulating T cells, i.e., “naïve” T cells, express the markers CD45RA and CD62L. Flow cytometry and spectral typing demonstrate the development of a diverse T cell repertoire. These recipient T cells have a diverse TCR repertoire and normally proliferate in response to mitogens. They protect the recipient from infection without having autoreactivity against self.

[0022] Product derived from allogeneic cultured neonatal thymic tissue

[0022] Allogeneic cultured postnatal thymus tissue-derived products have been shown to be useful in the treatment of T-cell immunodeficiency (primary immunodeficiency) resulting from congenital athymus. T-cell immunodeficiency due to athymus is associated with congenital disorders that prevent the development of a functional thymus, such as complete DiGeorge anomaly (cDGA) associated with 22q11.2 deletion and CHARGE syndrome (coloboma, cardiac defects, posterior nasal atresia, growth or intellectual disability, genital hypoplasia, and ear abnormalities or hearing loss) associated with mutations in the chd7 (chromodomain-helicase-DNA-binding protein 7) gene, and forkheadbox protein N1 (FOXN1) deficiency in athymic patients. Congenital athymus is a rare and fatal condition and currently has no drug treatment options using regulatory-approved formulations. If left untreated and without therapeutic reconstitution of the child's immune system, primary immunodeficiency due to congenital athymus is fatal, with almost all infants dying before the age of two, most often from severe infections.

[0023]

[0023] Allogeneically cultured post-thymus tissue-derived products are tissue manipulation products. Based on the disclosures herein and in the examples, CTT is expected to be useful in developing tolerance in recipients receiving transplanted solid organs.

[0024]

[0024] Surgical administration of an allogeneically cultured postnatal thymocyte-derived product (e.g., "RVT-802") in a patient with athymopathy results in a series of events that lead to the development of a functional immune system. Following the surgical placement of the allogeneically cultured postnatal thymocyte-derived product (e.g., RVT-802) in the recipient, T cells are educated by donor TECs and recipient DCs. Donor TECs associated with recipient DCs enable tolerance to the transplanted donor thymocyte-derived tissue, which is transplanted as a cultured thymocyte-derived tissue fragment. This is the same tolerance induction as with a normal thymus. Recipient TECs associated with recipient DCs result in tolerance to themselves, as described herein.

[0025]

[0025] This complex process has been clinically shown to result in over 70% survival in patients with congenital athymus who receive allogeneically cultured postnatal thymus tissue-derived products (e.g., RVT-802) due to the recipient's ability to fight infection (Markert ML, Devlin BH, Alexieff MJ, Li J, McCarthy EA, Gupton SE, et al., 2007, “Review of 54 patients with complete DiGeorge anomaly enrolled in protocols for thymus transplantation: outcome of 44 consecutive transplants,” Blood, 109(10):4539-47; Markert ML, Devlin BH, McCarthy EA. Thymus transplantation. 2010, Clin. Immunol., 135(2):236-46). Recipients can control viral infections, such as Epstein-Barr virus, that were lethal before CTT. (Chinn IK, Devlin BH, Li YJ, & Markert ML, 2008).

[0026] Overview of tolerance induction in solid organ grafts combined with CTT transplantation

[0026] In accordance with the description, figures, examples, and claims herein, the inventors have shown that CTT induces donor-specific tolerance in a rat heart transplant model. The experiments reported herein used equivalent CTT transplantation methods clinically used in subjects with congenital athymus, such as subjects with cDGA. Infants with cDGA essentially lack naive T cells prior to surgical placement of CTT. After surgical placement of CTT, infants developed naive T cells approximately 6 months after the surgical procedure. (Markert ML, et al., 2004, “Postnatal thymus transplantation with immunosuppression as treatment for DiGeorge syndrome,” Blood 104(8):2574-2581; Markert ML, Devlin BH, & McCarthy EA, 2010, “Thymus transplantation,” Clin Immunol 135(2):236-246). The tolerance induction study in the rat model was based on the results of a survey of allogeneic postnatal cultured thymic tissue-derived product (CTT) transplantation in athymic infants with complete DiGeorge anomaly from 1993 to 2017. Reported studies of surgical placement of CTT in infants with congenital athymic syndrome have shown favorable results. Published results showed an overall survival rate of 71% (61 / 86) (median 11.7 years, range 1.2–25 years) in this other lethal condition (Markert, ML, et al., 2010). Biopsies of transplanted cultured thymic tissue showed thymic lymphocyte neogenesis based on immunohistochemistry (Markert, ML, et al., 2008). Flow cytometry and spectral typing showed development of a diverse T cell repertoire. Mixed lymphocyte reactions indicate tolerance of recipient T cells to thymic donor antigen-presenting cells (Chinn IK, Devlin BH, Li YJ, & Markert ML, 2008).

[0027]

[0027] Importantly, recipients of CTTs can control viral infections, such as Epstein-Barr virus, that were lethal before the CTT (Markert, ML, 2014, Thymus Transplantation. Stiehm's Immune Deficiences, eds Sullivan KE & Stiehm ER (Academic Press), 1st Ed, pp 1059-1067). Based on these human data demonstrating tolerance to incompatible thymic MHC antigens, CTT transplantation in rat models was evaluated using the same clinically used methods for its ability to induce donor-specific tolerance in rat cardiac transplantation models. These studies showed that transplanting an incompatible heart with a donor CTT expressing cardiac donor MHC class I and class II antigens (with early T cell depletion by anti-CD5 and immunosuppression with cyclosporine) induced tolerance to donor cardiac antigens while preserving alloreactivity to other MHC antigens.

[0028]

[0028] The present invention demonstrates co-transplantation of a solid organ and donor thymus as a method for inducing tolerance of transplanted solid organs in recipients. The patient group that would benefit most from the procedure is infants requiring heart transplants. Since postnatal thymic tissue is present and can be removed from deceased infants, and recipient thymus is routinely removed from infants undergoing heart transplants, no additional procedures other than cultured thymic tissue transplantation (CTT) are required to translate this approach into clinical practice.

[0029] Overview of the preparation of products derived from allogeneically cultured post-thymus tissue

[0029] Allogeneic cultured postnatal thymus tissue-derived products are prepared, cultured, and stored for up to 21 days as described in detail herein, and on the day of transplantation, they are placed in individual sterile cups for transport to the operating room.

[0030]

[0030] CTT (cultured thymic tissue) is processed and cultured under sterile conditions, for example, current Good Manufacturing Practices ("cGMP") established by the U.S. Food and Drug Administration ("FDA"), in order to produce thymic tissue fragments in which T cells are partially depleted. CTT is distinguished from the innate thymus by a conditioning process described in detail below. CTT is affected by the normal positive and negative selection processes that generate T cells in the thymus after transplantation, allowing the T cells to be tolerant to both the donor thymus and the donor solid organ transplant and the recipient tissue. In addition, these T cells can recognize foreign antigens in the context of recipient major histocompatibility (MHC) proteins in order to fight infection.

[0031]

[0031] The route of administration is by surgical implantation of CTT in the manner described below. A single dose is typically 1 m 2 1,000 to 20,000 mm² per recipient body surface area ("BSA") 2 This is the CTT surface area. The surface area is the sum of the total surface areas of all cultured tissue pieces. Each CTT piece is implanted in a single-dose surgical procedure.

[0032]

[0032] Surgical transplantation of allogeneically cultured post-thymic tissue-derived products in patients with athymia results in a series of events that lead to the development of a functional immune system. (Markert ML, 2007; Markert ML, et al., 2010; Markert ML, Devlin BH, McCarthy EA. Chapter 84 Thymic reconstitution. 2013. In: Fleisher TA, Shearer WT, Schroeder HW, Frew AJ, Weyand CM, editors. Clinical Immunology (Fourth Edition). London; pp. 1032-8).

[0033]

[0033] Recipient CLP from the bone marrow migrate to the donor thymus graft. The donor thymus graft provides a microenvironment that allows recipient thymocytes to develop a broad repertoire of TCRs that can recognize pathogens.

[0034]

[0034] The migration of recipient DCs to the donor thymus depletes the autoreactive recipient thymocytes that attack the recipient's tissues after the new T cells leave the thymus and enter circulation. Genetically naive recipient T cells appear in circulation approximately 5–12 months after administration. These recipient T cells have a diverse TCR repertoire and normally proliferate in response to mitogens. They protect the recipient from infection without having autoreactivity against themselves.

[0035]

[0035] Recipient bone marrow CLPs migrate to the thymic allogeneic graft, where they develop into recipient T cells. Negative selection by recipient DCs that have migrated to the donor thymus results in tolerance to recipient MHC antigens. Immunohistochemical evidence of thymic lymphocyte neogenesis is observed in biopsies of transplanted cultured thymic tissue taken within approximately 2-3 months after transplantation. Thymic lymphocyte neogenesis reflects the ability of T cells to defend against and control infection and prevent autoimmune diseases.

[0036]

[0036] Naive T cells are detected in circulation 5 to 12 months after transplantation and possess the ability to defend against and control infections, as well as prevent autoimmune diseases.

[0037]

[0037] The transplantation of cultured thymic tissue was first shown to be beneficial in treating primary immunodeficiency resulting from congenital athymopathy associated with conditions such as complete DiGeorge anomaly (cDGA) or forkheadbox protein N1 (FOXN1) deficiency. The inventors herein have found that replacement of defective thymic tissue with cultured normal thymic tissue (e.g., CTT and RVT-802) can also prevent the lack of tolerance observed in recipients of transplanted solid organs.

[0038]

[0038] Nonclinical and clinical studies underlying the treatment of congenital athymia through the placement of cultured thymic tissue have led to the recognition that placement of CTT (e.g., RVT-802) in patients may allow for the development of tolerance to the transplanted solid organ. Specifically, if the subject undergoes initial thymectomy and immunosuppression before transplantation of a CTT expressing the MHC of the donor organ, placement of the CTT will reconstruct the immune system and induce tolerance to the donor organ.

[0039]

[0039] Measuring the expression and distribution of specific markers related to thymic cellular components establishes the phenotype ex vivo. The culture conditions described herein and in the examples support the observation of in vivo thymic lymphocyte neogenesis after placement of CTT in athymic subjects.

[0040]

[0040] Importantly, the development of naive T cells and the presence of extensive TCR variable regions after surgical placement of CTTs in athymic recipients provide clear evidence that thymic tissue culture can promote the development of a functional endogenous T cell population. In addition, the expression of major regulatory and structural genes was confirmed in the cultured thymic tissue. Circulating naive (CD45RA+CD62L+) T cells can be first detected 3–5 months after surgical insertion of CTTs. These observations have been confirmed in the treatment of patients with complete DiGeorge anomaly (Markert ML, 2010, Markert ML, 2013).

[0041]

[0041] Nonclinical data described in the literature on thymic tissue transplantation are consistent with the robust clinical efficacy of transplanting allogeneically cultured postnatal thymic tissue and support its use in humans. (Markert ML, Watson TJ, Kaplan I, Hale LP, Haynes BF, 1997, “The human thymic microenvironment during organ culture,” Clin Immunol Immunopathol. Jan;82(1):2 6-36, Hong R, Schulte-Wissermann H, Jarrett-Toth E, Horowitz SD, Manning DD, 1979, “Transplantation of cultured thymic fragments.II.Results in nude mice,”J Exp Med.,149(2):398-415.Li B,Li J,Hsieh CS,Hale LP,Li YJ,Devlin BH,Markert ML,2009,“Characterization of cultured thymus tissue used for transplantation with emphasis on promiscuous expression of thyroid tissue-specific genes,”Immunol Res.2009;44(1-3):71-83, Li B, Li J, Devlin BH, Markert ML, 2011, “Thymic microenvironment reconstitution after postnatal human thymus transplantation,” Clin Immunol., Sep; 140(3):244-59).

[0042]

[0042] Patients with complete DiGeorge anomaly have defects in three glands that develop in the cervix, heart, thymus, and parathyroid glands during early embryonic development. Normally, the heart and thymus descend to the chest, while the parathyroid glands, which regulate calcium levels, remain in the cervix. Treatment with CTT in cDGA patients resulted in a 75% survival rate at age 2 compared to a 6% survival rate (unpublished data) in untreated patients. In the literature, children generally die within 2 years without treatment (Markert, et al., 2010). Notably, CTT transplantation does not affect the heart and parathyroid problems that must be managed separately.

[0043]

[0043] A first aspect of the present disclosure provides a surgical arrangement of an allogeneically cultured postnatal thymus tissue-derived product in an immunologically normal recipient for inducing tolerance to solid organ transplantation in the recipient. Such a method comprises, or essentially comprises, removal of the thymus in an immunoresponsive recipient, followed by depletion of the recipient's T cells with an inducible immunosuppressive regimen comprising one or more antibodies and / or one or more immunosuppressants such as one or more calcineurin inhibitors. The inducible immunosuppressive regimen is administered in a therapeutically effective dose to deplete mature T cells in the subject and / or to suppress the recipient's T cells from rejecting the transplanted solid organ. A suitable solid human organ and thymus are obtained from a deceased donor, and the solid organ is transplanted into the recipient. A maintenance immunosuppressive regimen is administered for a period of time to suppress transplant rejection. Thymus from deceased donors is subjected to a conditioning regimen for up to 21 days, in which the donor thymus tissue is sterilely treated in thymic organ medium to produce partially T-cell depleted donor thymus tissue fragments, thereby containing allogeneically cultured post-vivo thymus tissue-derived products. Partially T-cell depleted donor thymus tissue fragments show areas positive for keratin AE1 / AE3 scattered throughout the tissue, the presence of at least one Hassall's body, scattered CK14 staining throughout the tissue, and the presence of intact nuclei. The allogeneically cultured post-vivo thymus tissue-derived products are then surgically placed in the recipient, typically in the quadriceps femoris muscle. The allogeneically cultured post-vivo thymus tissue-derived products allow the recipient to generate naive T cells after transplantation. All newly generated T cells are genetically recipient-derived and tolerant to both the recipient and the donor. The dosage of thymus tissue fragments is approximately 1,000–20,000 mm³. 2 Thymus tissue surface area / 1m 2 This is the recipient body surface area. After transplantation, allogeneically cultured post-thymic tissue-derived products induce thymic lymphocyte ogenesis and tolerance in the subjects.

[0044]

[0044] Taking heart transplantation as an example, the donor is a deceased donor. The thymus is removed from the donor at the same time as the heart is removed. Heart transplantation is performed immediately with induced immunosuppression to reduce the number of T cells and suppress the remaining recipient T cells to prevent attack on the donor heart. The donor thymus is processed to form a human allogeneic cultured postnatal thymus tissue-derived product that can be used for transplantation to induce tolerance after a conditioning period of at least 12 to about 21 days. As a precautionary measure, about half of the allogeneic cultured postnatal thymus tissue-derived product can be cryopreserved after conditioning, and if there are problems in late rejection of the heart requiring the administration of high doses of steroids or other immunosuppressants to treat rejection, thereby very high doses of steroids damaging the allogeneic cultured postnatal thymus tissue-derived product, the cryopreserved allogeneic cultured postnatal thymus tissue-derived product can be used for transplantation after the rejection episode has been controlled.

[0045]

[0045] Importantly, after transplantation of allogeneically cultured post-vibrational thymus tissue-derived products, immune tolerance is maintained even in the presence of infection. Since approximately one-third of CD8 T cells are alloreactive, with other approaches such as co-stimulus deactivation, viral infection can lead to loss of tolerance. When the immune system is activated to fight infection, alloreactive CD8 T cells begin to reject solid organ transplants. In contrast, when tolerance is induced using thymus tissue treated with allogeneically cultured post-vibrational thymus tissue-derived products, all potential alloreactive T cells against the donor are deleted through a process of negative selection.

[0046]

[0046] In one embodiment, the donor thymus tissue is matched to the HLA alleles in the donor organ that are not present in the recipient. All new T cells that develop are genetically recipient and tolerant to both the recipient and the donor.

[0047]

[0047] A second aspect of the present disclosure provides a method for promoting donor-specific tolerance to allogeneic solid organ grafts obtained from deceased donors in recipients requiring solid organ grafts, the method being: (a) Steps of removing the recipient's thymus, (b) The recipient is treated with an inducible immunosuppressive regimen comprising one or more immunosuppressants to deplete the recipient's T cells and / or to suppress the recipient's T cells from rejecting the transplanted solid organ, (c) The step of providing both suitable solid human organs and thymus from a deceased donor, (d) The step of transplanting a solid human organ into a recipient, (e) The step of treating the recipient with a maintenance immunosuppressive regimen, (f) A step of providing an allogeneically cultured postnatal thymus tissue-derived product, wherein the allogeneically cultured postnatal thymus tissue-derived product is obtained from suitable thymus tissue of a solid organ donor, the donor thymus tissue is subjected to a conditioning regimen for a period of up to 21 days to produce the allogeneically cultured postnatal thymus tissue-derived product, and further the conditioning regimen for the donor thymus tissue comprises sterile treatment of the donor thymus tissue in thymic organ medium to produce a partially T-cell depleted donor thymus tissue fragment, the partially T-cell depleted donor thymus tissue fragment exhibiting areas positive for cytokeratin (CK) (using antibodies AE1 / AE3) scattered throughout the tissue, the presence of at least one Hassall body, scattered CK14 staining throughout the tissue, and the presence of intact nuclei, (g) A step in which a product derived from allogeneically cultured post-vibrational thymus tissue is transplanted into the recipient 12 to 21 days after the conditioning regimen, wherein the dose of thymus tissue is approximately 1,000 to 20,000 mm 2 Thymus tissue surface area / 1m 2 The recipient's body surface area is the recipient's surface area, and further, the transplanted allogeneically cultured post-thymic tissue-derived product induces thymic lymphocyte neogenesis and tolerance in the recipient.

[0048]

[0048] In one embodiment, a method is provided for promoting donor-specific tolerance to allogeneic heart grafts in recipients requiring a deceased donor heart. The method involves the following steps: (a) The step of obtaining a suitable human heart from a deceased donor for transplantation, (b) A step of obtaining a heart for conditioning into an allogeneically cultured post-vibrant thymic tissue-derived product while simultaneously removing a deceased donor thymus, The donor thymus matches the HLA alleles in the donor transplant organ, and (c) A step of treating the recipient with an induction immunosuppression regimen comprising one or more immunosuppressants to deplete and / or suppress the recipient's T cells, wherein the one or more immunosuppressants comprises glucocorticoids administered during induction of anesthesia and after reperfusion, (d) The step of transplanting the heart into the recipient, (e) The recipient is treated for a sufficient time to prevent or suppress cardiac graft rejection with a maintenance immunosuppression regime comprising one or more immunosuppressants selected from the group consisting of calcineurin inhibitors, inosine monophosphate dehydrogenase inhibitors, and antithymocyte globulins. (f) A step of providing an allogeneically cultured postnatal thymus tissue-derived product on days 12-21, wherein the allogeneically cultured postnatal thymus tissue-derived product is obtained from donor thymus tissue, the donor thymus tissue is subjected to a conditioning regimen for a period of approximately 12-21 days to produce the allogeneically cultured postnatal thymus tissue-derived product, and further comprising the conditioning regimen for the donor thymus tissue, sterile treatment of the donor thymus tissue in thymic organ medium to produce a partially T-cell depleted donor thymus tissue fragment, the partially T-cell depleted donor thymus tissue fragment showing areas positive for keratin AE1 / AE3 scattered throughout the tissue, the presence of at least one Hassall body, CK14 staining scattered throughout the tissue, and the presence of intact nuclei, (g) A step of transplanting a portion of a product derived from allogeneically cultured post-vibrational thymus tissue into a recipient, wherein the dose of the thymus tissue fragment is approximately 1,000 to 20,000 mm 2 Thymus tissue surface area / 1m 2 The recipient's body surface area is the recipient's body surface area, and furthermore, the transplanted allogeneically cultured post-thymic tissue-derived product induces thymic lymphocyte neogenesis and tolerance in the recipient, in the step of (h) The step of cryopreserving a portion of the allogeneically cultured postnatal thymus tissue-derived product for use in the recipient in the event of an early rejection episode requiring a high dose of steroids that damages a portion of the allogeneically cultured postnatal thymus tissue-derived product transplanted in step (g).

[0049]

[0049] In one embodiment, a method is provided for promoting donor-specific tolerance to allogeneic heart grafts in recipients requiring a deceased donor heart. The method involves the following steps: (a) The step of obtaining a suitable solid human heart from a deceased donor for transplantation, (b) A step of obtaining a heart for conditioning into an allogeneic post-vibrational thymic tissue-derived product and simultaneously removing a deceased donor thymus, wherein the donor thymus is compatible with the HLA alleles in the donor transplant organ. (c) A step of treating the recipient with an induction immunosuppression regimen comprising one or more immunosuppressants to deplete and / or suppress the recipient's T cells, wherein the one or more immunosuppressants comprises glucocorticoids administered during induction of anesthesia and after reperfusion, (d) The step of surgically removing the recipient's heart and thymus, (e) The step of transplanting a donor human heart into a recipient, (f) A step of treating the recipient for a sufficient time to prevent or suppress cardiac graft rejection with a maintenance immunosuppression regime comprising one or more immunosuppressants selected from the group consisting of calcineurin inhibitors, inosine monophosphate dehydrogenase inhibitors, and antithymocyte globulins, If the recipient's postoperative condition is too unstable to allow for glucocorticoid withdrawal and the safe transplantation of products derived from allogeneic cultured postnatal thymic tissue in the recipient, the products derived from allogeneic cultured postnatal thymic tissue are cryopreserved for transplantation at a time after the recipient is stable, the donor thymic tissue is subjected to a conditioning regimen for a period of about 12 to about 21 days to produce products derived from allogeneic cultured postnatal thymic tissue, and further, the conditioning regimen for the donor thymic tissue includes aseptically treating the donor thymic tissue in a thymic organ medium to produce donor thymic tissue pieces that are partially depleted of T cells, and the donor thymic tissue pieces that are partially depleted of T cells show regions positive for keratin AE1 / AE3 scattered throughout the tissue, the presence of at least one Hassall's corpuscle, CK14 staining scattered throughout the tissue, and the presence of intact nuclei, steps, (h) A step of transplanting a portion of the product derived from allogeneic cultured postnatal thymic tissue into the recipient after the patient has stabilized, wherein the dosage of the thymic tissue piece is about 1,000 to 20,000 mm 2 of thymic tissue surface area / 1 m 2 of the recipient's body surface area, and further, the transplanted product derived from allogeneic cultured postnatal thymic tissue induces thymic lymphopoiesis and tolerance in the recipient, steps, (i) A step of cryopreserving a portion of the product derived from allogeneic cultured postnatal thymic tissue for use in the recipient if there is a rejection episode that requires a high dose of steroid to damage a portion of the product derived from allogeneic cultured postnatal thymic tissue transplanted in step (h), including.

[0050]

[0050] The third aspect of the present disclosure provides a method for promoting donor-specific tolerance to an allogeneic solid organ transplant obtained from a living human donor in a human recipient who requires a solid organ transplant, the method comprising, (a) A step of removing the recipient's thymus, (b) Treating the recipient with an induction immunosuppressive regimen comprising one or more immunosuppressive agents to deplete the recipient's T cells and / or suppress the recipient's T cells from rejecting the transplanted solid organ, steps (c) The step of providing appropriate solid organs from a living human donor, (d) The step of transplanting a solid organ into the recipient, (e) The step of treating the recipient with a maintenance immunosuppressive regimen, (f) Providing cryopreserved allogeneic postnatal thymocyte-derived products maintained in a cryopreserved allogeneic postnatal thymocyte-derived product bank, wherein the cryopreserved allogeneic postnatal thymocyte-derived products are processed from thymocyte tissue from a thymocyte donor expressing an HLA allele matched to an HLA allele in the recipient that is not present in the solid organ graft, the donor thymocyte tissue is subjected to a conditioning regimen for a period of up to 12 days, the conditioning regimen for the donor thymocyte further comprises sterile treatment of the donor thymocyte tissue in thymocyte medium to produce a partially T cell-depleted thymocyte tissue fragment, the thymocyte tissue fragment exhibiting, upon completion of the conditioning regimen, regions positive for keratin AE1 / AE3 scattered throughout the tissue, the presence of at least one Hassall body, CK14 staining scattered throughout the tissue, and the presence of intact nuclei, (g) A step of thawing a cryopreserved, allocultured postnatal thymus tissue-derived product, (h) A step of transplanting a thawed cryopreserved allogeneic cultured postnatal thymus tissue-derived product into a recipient, wherein the dose of the cryopreserved allogeneic cultured postnatal thymus tissue-derived product is approximately 1,000 to 20,000 ml 2 Thymus tissue surface area / 1m 2 The recipient's body surface area is the recipient's surface area, and further, the transplanted allogeneically cultured post-thymic tissue-derived product induces thymic lymphocyte neogenesis and tolerance in the recipient.

[0051]

[0051] A fourth aspect of the present disclosure provides a method for promoting donor-specific tolerance to allogeneic solid organ transplants obtained from deceased human donors in human recipients requiring solid organ transplants, the method being: (a) Steps of removing the recipient's thymus, (b) The recipient is treated with an inducible immunosuppressive regimen comprising one or more immunosuppressants to deplete the recipient's T cells and / or to suppress the recipient's T cells from rejecting the transplanted solid organ, (c) The step of providing a suitable solid organ from a living human donor, (d) The step of transplanting a solid organ into the recipient, (e) The step of treating the recipient with a maintenance immunosuppressive regimen, (f) Providing cryopreserved allogeneic postnatal thymocyte-derived products maintained in a cryopreserved allogeneic postnatal thymocyte-derived product bank, wherein the cryopreserved allogeneic postnatal thymocyte-derived products are processed from thymocyte tissue from a thymocyte donor expressing an HLA allele matched to an HLA allele in the recipient that is not present in the solid organ graft, the donor thymocyte tissue is subjected to a conditioning regimen for a period of up to 12 days, the conditioning regimen for the donor thymocyte further comprises sterile treatment of the donor thymocyte tissue in thymocyte medium to produce a partially T cell-depleted thymocyte tissue fragment, the thymocyte tissue fragment exhibiting, upon completion of the conditioning regimen, regions positive for keratin AE1 / AE3 scattered throughout the tissue, the presence of at least one Hassall body, CK14 staining scattered throughout the tissue, and the presence of intact nuclei, (g) A step of thawing a cryopreserved, allocultured postnatal thymus tissue-derived product, (h) A step of transplanting a thawed cryopreserved allogeneic cultured postnatal thymus tissue-derived product into a recipient, wherein the dose of the cryopreserved allogeneic cultured postnatal thymus tissue-derived product is approximately 1,000 to 20,000 ml 2 Thymus tissue surface area / 1m 2 The recipient's body surface area is the recipient's surface area, and further, the transplanted allogeneically cultured post-thymic tissue-derived product induces thymic lymphocyte neogenesis and tolerance in the recipient.

[0052]

[0052] In one embodiment of the second to fourth aspects of the present disclosure, the allocultured postnatal thymus tissue product of claim 63 shows that on the day of collection, more than 50% of the region is positive for keratin in a lace-like staining pattern, Hassar bodies are present, CK14 stains in a lace-like pattern, and more than 90% of the nuclei are intact.

[0053]

[0053] A fifth aspect of the present disclosure provides an allogeneically cultured postnatal thymus tissue product for transplantation to a subject receiving solid organ transplantation, which is prepared by obtaining suitable thymus tissue from a donor, wherein the donor thymus tissue is subjected to a conditioning regimen for a period of up to 21 days, and the conditioning regimen for the donor thymus tissue further comprises sterile treatment of the donor thymus tissue in thymic organ medium to produce a partially T cell depleted donor thymus tissue fragment, the donor thymus tissue fragment showing, 5 to 9 days after collection, areas positive for keratin AE1 / AE3 scattered throughout the tissue, the presence of at least one Hassall body, CK14 staining scattered throughout the tissue, and the presence of intact nuclei, and recovering the partially T cell depleted donor thymus tissue fragment as an allogeneically cultured postnatal thymus tissue product.

[0054]

[0054] In one embodiment, the thymus shows that on the day of collection, more than 50% of the region is positive for keratin with a lace-like staining pattern, Hassall bodies are present, CK14 stains with a lace-like pattern, and more than 90% of the nuclei are intact.

[0055]

[0055] In one embodiment of a fifth aspect of the present invention, the allocultured postnatal thymus tissue-derived product is cryopreserved.

[0056]

[0056] In one embodiment, the cryopreserved alloculture postnatal thymus tissue-derived product is maintained in liquid nitrogen for subsequent use.

[0057]

[0057] In another embodiment, cryopreserved allocultured postnatal thymus tissue-derived products are maintained in a cryopreserved tissue bank.

[0058]

[0058] In one embodiment, the allogeneically cultured postnatal thymus tissue-derived product is prepared from suitable thymus tissue from a donor containing HLA alleles that match the HLA alleles in the recipient candidate that are not present in solid organ transplants.

[0059]

[0059] In one embodiment, the HLA alleles are HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DPB1, and HLA-DPA1.

[0060]

[0060] A sixth aspect of this disclosure is (a) Steps to obtain suitable thymic tissue from a donor, (b) HLA alleles: A step of typing HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DPB1, HLA-DPA1, (c) A step of subjecting thymic tissue to a conditioning regimen for a period of up to 12 days, wherein the conditioning regimen for donor thymic tissue comprises sterile treatment of the donor thymic tissue in thymic organ medium to produce a donor thymic tissue fragment in which the T cells are partially depleted, and further, the donor thymic tissue fragment shows, on days 5–9, areas positive for keratin AE1 / AE3 scattered throughout the tissue, the presence of at least one Hassall body, CK14 staining scattered throughout the tissue, and the presence of intact nuclei upon completion of the conditioning regimen. (d) A step of collecting a donor thymus tissue fragment in which T cells are partially depleted as an allogeneic cultured post-vitro thymus tissue-derived product, (e) A step of cryopreserving the product derived from allogeneic cultured postnatal thymus tissue in liquid nitrogen, (f) A cryopreserved allocultured postnatal thymus tissue-derived product prepared by a method comprising the step of maintaining the cryopreserved allocultured postnatal thymus tissue-derived product in liquid nitrogen in a cryopreserved allocultured postnatal thymus tissue-derived product bank.

[0061]

[0061] In one embodiment, the thymus shows that on the day of collection, more than 50% of the region is positive for keratin with a lace-like staining pattern, Hassall bodies are present, CK14 stains with a lace-like pattern, and more than 90% of the nuclei are intact.

[0062]

[0062] In one embodiment, the allogeneically cultured postnatal thymus tissue-derived product, which has been cryopreserved in liquid nitrogen, is kept for subsequent use by the recipient.

[0063]

[0063] A seventh aspect of the present disclosure provides a method for preparing a donor thymus for transplantation into a recipient subject. Such a method, as further described herein, comprises or is essentially derived from culturing the donor thymus for up to about 5, up to about 6, up to about 7, up to about 8, up to about 9, up to about 10, up to about 11, up to about 12, up to about 13, up to about 14, up to about 15, up to about 16, up to about 17, up to about 18, up to about 19, up to about 20, or up to about 21 days, and then surgically placing the cultured thymic tissue into the recipient. A culture period of about 6 to about 12 days yields good function. For successful transplantation of cryopreserved thymic tissue, the tissue is typically cultured for about 6 to about 12 days and then cryopreserved.

[0064]

[0064] An eighth aspect of the present disclosure provides an allogeneically cultured postnatal thymus tissue-derived product (CTT, RVT-802) for transplantation into subjects receiving solid organ transplants, which is produced by subjecting thymus tissue from a suitable donor to a conditioning regimen for a period of up to 21 days, the conditioning regimen for the allogeneically cultured postnatal thymus tissue-derived product comprising sterile treatment of donor thymus tissue in thymic organ medium to produce a partially T-cell depleted donor thymus tissue fragment, the thymus tissue fragment exhibiting keratin AE1 / AE3-positive regions scattered throughout the tissue, the presence of at least one Hassall body, CK14 staining scattered throughout the tissue, and the presence of intact nuclei.

[0065]

[0065] In one embodiment of the eighth aspect, the donor thymus shows on the day of collection that more than 50% of the region is positive for keratin with a lace-like staining pattern, Hassall bodies are present, CK14 stains with a lace-like pattern, and more than 90% of the nuclei are intact.

[0066]

[0066] In one embodiment of the first to fourth aspects of the present disclosure, the recipient's thymus is obtained by surgery.

[0067]

[0067] In one embodiment of the first to fourth aspects of the present disclosure, the recipient's thymus is obtained by robotic surgery.

[0068]

[0068] In one embodiment of the first to fourth aspects of the present disclosure, the recipient's thymus is obtained by thoracoscopic surgery.

[0069]

[0069] In one embodiment of the first to fourth aspects of the present disclosure, the solid organ is a part of the whole organ. In one embodiment, the methods of the first to fourth embodiments further include the step of cryopreserving peripheral blood mononuclear cells from a deceased donor for future use in a mixed lymphocyte reaction to demonstrate cell tolerance.

[0070]

[0070] In one embodiment, a mixed lymphocyte response demonstrating cell tolerance is performed using peripheral blood mononuclear cells from the recipient and cryopreserved peripheral blood mononuclear cells from the donor, after transplantation of an allogeneically cultured postnatal thymus tissue-derived product according to the transplantation procedure of CTT as described herein.

[0071]

[0071] In one embodiment, a mixed lymphocyte response demonstrating cell tolerance is performed approximately 6 to 12 months after transplantation of the allogeneically cultured post-thymocyte tissue-derived product using peripheral blood mononuclear cells from the recipient and cryopreserved peripheral blood mononuclear cells from the donor.

[0072]

[0072] In one embodiment, a mixed lymphocyte response demonstrating cell tolerance is carried out with peripheral blood mononuclear cells from the recipient and cryopreserved peripheral blood mononuclear cells from the donor, after naive T cells constitute about 10% of the total T cells in the recipient. In one embodiment of the first to fourth aspects, the transplanted allogeneically cultured post-thymic tissue-derived product induces thymic lymphocyte regeneration in the subject within 12 months after transplantation of the allogeneically cultured post-thymic tissue-derived product.

[0073]

[0073] In one embodiment of the first to fourth aspects, the development of tolerance is determined by a mixed lymphocyte response performed by cryopreserved peripheral blood mononuclear cells from a deceased donor and naive T cells from the recipient.

[0074]

[0074] In one embodiment of the first to fourth aspects, humoral tolerance is determined by the development of humoral immunity and the absence of donor-reactive antibodies.

[0075]

[0075] In one embodiment of the first to fourth aspects of the present disclosure, solid organ transplants include heart transplants, kidney transplants, liver transplants, lung transplants, heart / lung transplants, pancreas transplants, intestine transplants, stomach transplants, abdominal wall transplants, craniofacial transplants, scalp transplants, penile transplants, uterine transplants, unilateral or bilateral upper limb transplants, unilateral neovascular composite allografts, or combinations thereof.

[0076]

[0076] In one embodiment of the first to fourth aspects of the present disclosure, the method further includes evaluating the recipient for an HLA class I or HLA class II panel-reactive antibody ("PRA") score before transplantation of a solid organ.

[0077]

[0077] In one embodiment, the solid organ transplant is a heart transplant.

[0078]

[0078] In one embodiment, the solid organ transplant is a pediatric heart transplant.

[0079]

[0079] In one embodiment, the solid organ transplant is an adult heart transplant.

[0080]

[0080] In one embodiment of the first to fourth aspects of the present disclosure, the method further includes evaluating the recipient for an HLA class I or HLA class II panel-reactive antibody ("PRA") score before transplantation of a solid organ.

[0081]

[0081] In one embodiment, a recipient having HLA antibodies is cross-matched with a potential donor.

[0082]

[0082] In one embodiment, the recipient having an HLA antibody is substantially cross-matched with the UNET.

[0083]

[0083] In a further embodiment, if a PRA score of more than 20% virtual cross-matching is recorded, the method further includes the step of performing plasma exchange in the operating room at the time of solid organ transplantation in the recipient.

[0084]

[0084] In further embodiments, if a PRA score of more than 70% virtual crossmatch is recorded, the method further includes the step of performing actual prospective donor crossmatching in the operating room at the time of solid organ transplantation in the recipient and performing plasma exchange. Typically, transplantation is not performed under these circumstances due to the low success rate.

[0085]

[0085] In one embodiment of the first to fourth aspects of the present disclosure, the method further includes the step of evaluating a recipient having an HLA antibody by substantially cross-matching with a UNET.

[0086]

[0086] In one embodiment of the first to fourth aspects of the present disclosure, the method further includes performing plasma exchange in the operating room during solid organ transplantation in the recipient if the HLA panel-reactive antibody has a score greater than 20%.

[0087]

[0087] In one embodiment of the first to fourth aspects of the present disclosure, the method further includes performing actual prospective donor crossmatching and plasma exchange in the operating room at the time of solid organ transplantation in the recipient if the HLA panel reactive antibody has a score greater than 70%.

[0088]

[0088] In one embodiment, the solid organ is HLA-matched for, for example, a kidney, a partial liver, and a partial intestine transplant, with respect to the living donor and recipient.

[0089]

[0089] In another embodiment, the solid organ is HLA incompatible.

[0090]

[0090] In one embodiment, the solid organ is HLA compatible. In another embodiment, HLA compatibility is determined by typing the HLA alleles HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DPB1, and HLA-DPA1 in the donor and recipient.

[0091]

[0091] In one embodiment, solid organ transplantation is ABO compatible.

[0092]

[0092] In another embodiment, the solid organ is HLA incompatible. In one embodiment, HLA incompatible is determined by typing HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DQA1, HLA-DPB1, and HLA-DPA1 in the donor and recipient.

[0093]

[0093] In one embodiment, a cultured thymic tissue fragment is surgically implanted into the quadriceps femoris muscle of the subject.

[0094]

[0094] In one embodiment, the cultured thymic tissue fragment is surgically implanted into the body of the subject in a region other than the quadriceps muscle.

[0095]

[0095] In one embodiment, a portion of the allogeneically cultured post-thymic tissue-derived product is surgically transplanted into the quadriceps femoris muscle of the recipient.

[0096]

[0096] In another embodiment, the remaining portion of the allogeneically cultured post-thymocyte tissue-derived product is cryopreserved in liquid nitrogen for future transplantation.

[0097]

[0097] In one embodiment, the conditioning regimen is for a period of about 12 to 21 days.

[0098]

[0098] In one embodiment, the conditioning period for the donor thymus tissue is approximately 5 to 21 days, or approximately 5 days, or approximately 6 days, or approximately 7 days, or approximately 8 days, or approximately 9 days, or approximately 10 days, or approximately 11 days, or approximately 12 days, or approximately 13 days, or approximately 14 days, or approximately 15 days, or approximately 16 days, or approximately 17 days, or approximately 18 days, or approximately 19 days, or approximately 20 days, or approximately 21 days.

[0099]

[0099] It will be understood by those skilled in the art that there are numerous potential induction and maintenance immunosuppression regimens known in the art, and that appropriate induction and maintenance immunosuppressants can be selected without undue burden by those skilled in the art. The following exemplary induction and maintenance immunosuppression regimens are examples of the methods of the first to fourth aspects of the present invention and support the claimed invention.

[0100]

[0100] In one embodiment of the first to fourth aspects of the present disclosure, the inducible immunosuppression regimen comprises an inducible immunosuppressant selected from the group consisting of glucocorticoids, antithymocyte globulin (rabbit), antithymocyte globulin (horse), and alemtuzimab.

[0101]

[0101] In another embodiment, ATG is antithymocyte globulin (rabbit).

[0102]

[0102] In one embodiment, the inducing immunosuppression regimen includes the administration of a glucocorticoid. In one embodiment, the glucocorticoid includes methylprednisolone. In another embodiment, the glucocorticoid is methylprednisolone sodium succinate. In a further embodiment, methylprednisolone sodium succinate is administered intravenously at a dose of 4 mg / kg / day or less.

[0103]

[0103] In one embodiment, the inducible immunosuppression regimen includes rabbit-derived anti-thymocyte globulin. In another embodiment, rabbit-derived anti-thymocyte globulin is administered intravenously at a dose of approximately 1.5 mg / kg. In a further embodiment, anti-thymocyte globulin is administered daily for 4 days. In another embodiment, ATG is horse-derived ATG.

[0104]

[0104] In one embodiment, the inducing immunosuppression regimen includes basiliximab. In another embodiment, basiliximab is administered intravenously at a dose of 10 mg to recipients weighing less than 35 kg. In yet another embodiment, basiliximab is administered intravenously at a dose of 20 mg to recipients weighing more than 35 kg.

[0105]

[0105] In one embodiment of the first to fourth aspects of the present disclosure, the second immunosuppressive regimen comprises one or more immunosuppressants selected from the group consisting of glucocorticoids, calcineurin inhibitors, inosine monophosphate dehydrogenase inhibitors, azathioprine, and antithymocyte globulin ("ATG").

[0106]

[0106] In one embodiment, the immunosuppressant of the maintenance immunosuppression regimen is antithymocyte globulin (ATG).

[0107]

[0107] In one embodiment, ATG is administered intravenously at a dose of approximately 1.5 mg / kg for a period of 3 to 14 days, starting with administration in the operating room.

[0108]

[0108] In one embodiment, antithymocyte globulin is administered intravenously at a dose of approximately 15 mg / kg / day daily for 3 to 14 days.

[0109]

[0109] In one embodiment, the first immunosuppressive regimen includes alemtuzumab.

[0110]

[0110] In one embodiment, alemtuzumab is administered intravenously to recipients weighing less than 35 kg at a dose of approximately 0.25 mg / kg for 4 days. In another embodiment, alemtuzumab is administered intravenously to recipients weighing more than 35 kg at a dose of approximately 3 to 20 mg for 4 days.

[0111]

[0111] In one embodiment, the second immunosuppressive regimen comprises one or more immunosuppressants selected from the group consisting of calcineurin inhibitors, inosine monophosphate dehydrogenase inhibitors, or azathioprine.

[0112]

[0112] In one embodiment, the immunosuppressant of the maintenance immunosuppression regimen is a calcineurin inhibitor. In one embodiment, the immunosuppressant in the maintenance immunosuppression regimen is an inosine monophosphate dehydrogenase inhibitor.

[0113]

[0113] In one embodiment, the immunosuppressive regimen includes an inosine monophosphate dehydrogenase inhibitor, such as mycophenolate mofetil. In one embodiment, mycophenolate mofetil is administered intravenously at a dose of about 15 to about 25 mg / kg. In one embodiment, mycophenolate mofetil is administered intravenously two to three times a day.

[0114]

[0114] In another embodiment, the inosine monophosphate dehydrogenase inhibitor is mycophenolic acid. In another embodiment, mycophenolic acid is administered in two or three divided doses at a dose of about 25 to about 50 mg / kg.

[0115]

[0115] In one embodiment, mycophenolic acid is administered to children at a dose of approximately 400 mg / m². 2 Dosage: Twice daily, maximum dose 720 mg, or BSA 1.19-1.59 mg 2 Approximately 540 mg twice daily, or BSA > 1.58 mg 2 It is administered in doses of approximately 720 mg twice a day.

[0116]

[0116] In one embodiment, mycophenolate mofetil is administered orally or intravenously to children at a dose of about 15 to about 25 mg / kg / dose twice daily, or to adults at about 1500 mg twice daily, and is adjusted for more than 3500 WBCs.

[0117]

[0117] In one embodiment of the first to fourth aspects of the present disclosure, the second immunosuppressive regimen may further comprise a glucocorticoid selected from the group consisting of methylprednisolone, prednisone, and prednisolone. In one embodiment, the dose of the glucocorticoid is maintained at less than 4 mg / kg / day.

[0118]

[0118] In one embodiment, the glucocorticoid is administered in a tapering dose, as described elsewhere in this disclosure.

[0119]

[0119] In another embodiment of the first to fourth aspects of this disclosure, the calcineurin inhibitor is tacrolimus. In another embodiment, the calcineurin inhibitor is cyclosporine A.

[0120]

[0120] In another embodiment of the first to fourth aspects of the present disclosure, the administration of the second immunosuppressive regimen is discontinued after naive T cells have reached 10% of the total T cells. In yet another embodiment, the second immunosuppressive regimen is discontinued after transplantation of allogeneically cultured postnatal thymus tissue-derived products. [Brief explanation of the drawing]

[0121]

[0121] For a more complete understanding of the principles disclosed herein and their advantages, refer to the following description in conjunction with the accompanying drawings.

[0122] [Figure 1] This paper outlines the mechanism of action of allogeneic cultured post-thymus tissue-derived products (e.g., CTT, RVT-802) after administration for immune reconstitution in congenital athymia.

[0123] [Figure 2] As described elsewhere in Example 1, the steps to reconstruct the immune system in rats are outlined by removing the thymus in immunologically normal Lewis rats, administering an antibody that kills T cells in recipient rats, transplanting cultured neothyroid tissue from donor rats into recipient rats, administering immunosuppressants for approximately 4 months, and evaluating T cell development in recipient rats. Notably, all rats in the treatment group had more than 10% naive T cells before discontinuation of cyclosporine.

[0124] [Figure 3] This shows the development of naive T cells in two rats from the experimental recipe in Example 1 (upward line on the right) versus two control rats that did not receive thymic tissue transplantation (thick baseline line).

[0125] [Figure 4] This outlines the manufacturing process for harvesting the thymus from a donor, culturing a thin section of the donor thymus tissue for up to 21 days, and transplanting the cultured thymus tissue into the recipient's quadriceps femoris muscle.

[0126] [Figure 5A] A schematic diagram of thymic tissue sectioning for characteristic analysis testing, as discussed in Section

[0520] , is shown. [Figure 5B] This is a diagram of a section of thymic tissue on a cellulose filter on a surgical sponge in a tissue culture dish used for culturing the thymus.

[0127] [Figure 6]Histological examination of thymic tissue sections from a lot (MFG-056) of cultured thymic tissue at 5, 9, 12, and 21 days after thymic harvesting from a donor is shown. Their corresponding reactivity with hematoxylin and eosin-stained sections (left panel) and a cocktail of anti-cytokeratin antibodies AE1 / AE3 (right panel, brown indicates positive reactivity) is shown for day 5 (Figure 6A, 6B), day 9 (Figure 6C, 6D), day 12 (Figure 6E, 6F), and day 21 (Figure 6G, 6H), respectively. The bar in the lower left of each panel represents 100 μm. The H&E panels show the progression of T cell depletion over time. Figures 6E and 6F are primarily epithelial cells. As thymic cells are depleted over time, condensation of the subcapsular cortex epithelium occurs. Similar condensation occurs in the medullary region of the thymus. Photograph by Laura P. Hale, MD, PhD, Department of Pathology, Duke University.

[0128] [Figure 7A] The histology of thymic tissue fragments at day 0 of time progression is depicted at scales of 5 mm (Figure 9A) and 100 μm (Figure 7B), respectively. This shows the thymus and thymocytes at low power (bar 5 mm) and high power (bar 100 μm) at day 0. This is a normal thymus. At this point, both the cortex and medulla have numerous thymocytes with dark blue nuclei, contributing to the overall dark blue appearance of the tissue. Photograph by Laura P. Hale, MD, PhD, Department of Pathology, Duke University. [Figure 7B] The histology of thymic tissue fragments at day 0 of time progression is depicted at scales of 5 mm (Figure 9A) and 100 μm (Figure 7B), respectively. This shows the thymus and thymocytes at low power (bar 5 mm) and high power (bar 100 μm) at day 0. This is a normal thymus. At this point, both the cortex and medulla have numerous thymocytes with dark blue nuclei, contributing to the overall dark blue appearance of the tissue. Photograph by Laura P. Hale, MD, PhD, Department of Pathology, Duke University.

[0129] [Figure 8A]These are H&E-stained slides depicting the histology of thymic tissue samples at 5 mm (Figure 8A) and 100 μm (Figure 8B) scales, respectively, at 5 mm and 100 μm (Figure 8B) time points. Progression of thymic cell depletion results in a more eosinophilic (pink) appearance of the tissue. Photograph by Laura P. Hale, MD, PhD, Department of Pathology, Duke University. [Figure 8B] These are H&E-stained slides depicting the histology of thymic tissue samples at 5 mm (Figure 8A) and 100 μm (Figure 8B) scales, respectively, at 5 mm and 100 μm (Figure 8B) time points. Progression of thymic cell depletion results in a more eosinophilic (pink) appearance of the tissue. Photograph by Laura P. Hale, MD, PhD, Department of Pathology, Duke University.

[0130] [Figure 9A] H&E staining depicts the histology of thymic tissue sections at 12 days of time progression on 5 mm (Figure 9A) and 100 μm (Figure 9B) scales, respectively. We confirm progressive depletion of thymocytes. Higher magnification reveals numerous eosinophilic cell bodies lacking nuclei, indicating the state of necrotic cells that have undergone nuclear lysis (nuclear dissolution). This degree of necrosis is expected at this point in culture. Photograph by Laura P. Hale, MD, PhD, Department of Pathology, Duke University. [Figure 9B] H&E staining depicts the histology of thymic tissue sections at 12 days of time progression on 5 mm (Figure 9A) and 100 μm (Figure 9B) scales, respectively. We confirm progressive depletion of thymocytes. Higher magnification reveals numerous eosinophilic cell bodies lacking nuclei, indicating the state of necrotic cells that have undergone nuclear lysis (nuclear dissolution). This degree of necrosis is expected at this point in culture. Photograph by Laura P. Hale, MD, PhD, Department of Pathology, Duke University.

[0131] [Figure 10A]H&E staining depicts the histology of thymic tissue sections at 21 days of time progression on 5 mm (Figure 10A) and 100 μm (Figure 10B) scales, respectively. Note the preservation of the overall structure of the tissue in Figure 10B, including the subcapsular cortex, cortical region, and medullary region containing numerous Hassall bodies. Small dark cells are mostly necrotic thymocytes that have not yet undergone nuclear lysis. Photograph by Laura P. Hale, MD, PhD, Department of Pathology, Duke University. [Figure 10B] H&E staining depicts the histology of thymic tissue sections at 21 days of time progression on 5 mm (Figure 10A) and 100 μm (Figure 10B) scales, respectively. Note the preservation of the overall structure of the tissue in Figure 10B, including the subcapsular cortex, cortical region, and medullary region containing numerous Hassall bodies. Small dark cells are mostly necrotic thymocytes that have not yet undergone nuclear lysis. Photograph by Laura P. Hale, MD, PhD, Department of Pathology, Duke University.

[0132] [Figure 11] Representative thymic sections immunostained with a cocktail of anti-cytokeratin antibodies (AE1 / AE3) are shown. Figure 11A: Day 0, Figure 11B: Day 5, Figure 11C: Day 9, Figure 11D: Day 12, and Figure 11E: Day 21. The structure of the thymic epithelial network remains intact as the culture progresses. Bars represent 400 μm. Photograph by Laura P. Hale, MD, PhD, Department of Pathology, Duke University.

[0133] [Figure 12A]Histology of thymic tissue slides after exposure to forced degradation conditions with 10x PBS is shown. Figure 12A shows the cortex 9 days after exposure to forced degradation conditions. Figure 12B shows the cortex 21 days after exposure to forced degradation conditions. In Figure 12A, the blue smear represents DNA released from cells. While most cells show evidence of degradation, small foci with intact nuclei can be identified. Photograph by Laura P. Hale, MD, PhD, Department of Pathology, Duke University. [Figure 12B] Histology of thymic tissue slides after exposure to forced degradation conditions with 10x PBS is shown. Figure 12A shows the cortex 9 days after exposure to forced degradation conditions. Figure 12B shows the cortex 21 days after exposure to forced degradation conditions. In Figure 12A, the blue smear represents DNA released from cells. While most cells show evidence of degradation, small foci with intact nuclei can be identified. Photograph by Laura P. Hale, MD, PhD, Department of Pathology, Duke University.

[0134] [Figure 13] H&E-stained histological sections of clinical sample MLM247 are shown. This is day 0 of culture. Bars are 200 μm. This is a frozen section from day 0. Because it was frozen, the tissue looks different from paraffin-embedded formalin-fixed tissue from day 0. Photograph by Laura P. Hale, MD, PhD, Department of Pathology, Duke University.

[0135] [Figure 14] Frozen sections of clinical specimen MLM219, H&E-stained histological sections. Because these are frozen sections, the tissue appears different from the cultured and presented paraffin-embedded formalin-fixed tissues above. Nevertheless, the important histological characteristics of thymocyte depletion and the robust viability of TECs are well represented. Photograph by Laura P. Hale, MD, PhD, Department of Pathology, Duke University.

[0136] [Figure 15]This is a photograph of newly collected thymic tissue.

[0137] [Figure 16] This outlines the collection, culture, transplantation, and biopsy of CTT from beneath the rat kidney capsule, as presented in Example 5.

[0138] [Figure 17] Figure 17A shows a photograph of thymic tissue collected from a 3-day-old F1 (LWxDA) rat, cut into four pieces, as described in Example 5. Figure 17B shows a photograph of thymic tissue cultured for 5-7 days in a 37°C CO2 incubator on a sterile mixed cellulose ester filter, as described in Example 5. Figure 17C is a photograph of the CTT transplanted sub-renal capsule of an LW rat. Figure 17D is a photograph of the thymic graft collected 6 months after transplantation. The arrow indicates the CTT sub-renal capsule.

[0139] [Figure 18] These are photographs showing the histological appearance of fresh thymic tissue (top frame) and CTT (bottom frame) at 100x magnification. Figure 18A shows a comparison of medullary differentiation in fresh thymic tissue (top frame) and CTT cultured for 5 days (bottom frame) stained with H&E, as described in Example 5. Figure 18B shows the typical lace-like pattern observable in CTT cultured for 5 days (bottom frame) when stained for cytokeratin, compared to fresh thymic tissue (top frame), as described in Example 5. Figure 18C shows fresh thymic tissue (top frame) and T-cell depleted CTT (bottom frame) when stained for Ki-67. Figure 18D shows fresh thymic tissue (top frame) stained for CD3 and CTT thymic tissue stained for CD3 after being cultured for 5 days (bottom frame). The brown staining described in the CD3-stained CTT (Figure 18D, bottom frame) may represent some living cells and detritus of dead T cells not washed away from the tissue.

[0140] [Figure 19]These are photographs showing the histological appearance of fresh thymic tissue (top frame) and CTT (bottom frame) at 600x magnification. Figure 19A shows a comparison of medullary differentiation in fresh thymic tissue (top frame) and CTT cultured for 5 days (bottom frame) stained with H&E, as described in Example 5. Figure 19B shows the typical lace-like pattern observable in CTT cultured for 5 days (bottom frame) when stained for cytokeratin, compared to fresh thymic tissue (top frame), as described in Example 5. Figure 19C shows fresh thymic tissue (top frame) and T-cell depleted CTT (bottom frame) when stained for Ki-67. Figure 19D shows fresh thymic tissue (top frame) stained for DC3 and CTT thymic tissue stained for CD3 after being cultured for 5 days (bottom frame). The brown staining described in the CD3-stained CTT (Figure 19D, bottom frame) may represent some living cells and detritus of dead T cells not washed away from the tissue.

[0141] [Figure 20] This is a schematic diagram of the experimental design for the experiment reported in Example 5. This figure is from the manuscript being prepared for publication: Kwun J, Li J, Rouse DC, Park J, Farris AB, Turek JW, Knechtle SJ, Kirk AD, Markert ML, Cultured Thymus Transplantation Promotes Donor-specific Tolerance to Allogeneic Heart Transplants.

[0142] [Figure 21] This figure shows that recipient-type T cells that regrow are observed in the lower right quadrant after CTT transplantation. This figure is from a manuscript being prepared for publication: Kwun J, Li J, Rouse DC, Park J, Farris AB, Turek JW, Knechtle SJ, Kirk AD, Markert ML, Cultured Thymus Transplantation Promotes Donor-specific Tolerance to Allogeneic Heart Transplants.

[0143] [Figure 22A] This figure shows a transplanted thymus explanted 8.5 months post-transplantation, exhibiting positive cytokeratin staining (Figure 22A) and T-cell staining similar to that of an innate thymus (Figure 22B). The image is 400x magnification of the original. This figure is from a manuscript in preparation for publication: Kwun J, Li J, Rouse DC, Park J, Farris AB, Turek JW, Knechtle SJ, Kirk AD, Markert ML, Cultured Thymus Transplantation Promotes Donor-specific Tolerance to Allogeneic Heart Transplants. [Figure 22B] This figure shows a transplanted thymus explanted 8.5 months post-transplantation, exhibiting positive cytokeratin staining (Figure 22A) and T-cell staining similar to that of an innate thymus (Figure 22B). The image is 400x magnification of the original. This figure is from a manuscript in preparation for publication: Kwun J, Li J, Rouse DC, Park J, Farris AB, Turek JW, Knechtle SJ, Kirk AD, Markert ML, Cultured Thymus Transplantation Promotes Donor-specific Tolerance to Allogeneic Heart Transplants.

[0144] [Figure 23]This plot shows the significantly increased number of circulating CD4 and CD8 T cells compared to control animals that did not receive CTT transplantation. This also shows the significantly increased number of naive CD4 and naive CD8 T cells in the cultured thymus tissue transplantation (CTTT) group compared to the control group that did not receive CTTT, as well as the significantly increased number of CD4 and CD8 novel thymic exports (RTEs) in the cultured thymus tissue transplantation (CTT) group compared to the control group that did not receive CTT. This figure is from the manuscript in preparation for submission for publication: Kwun J, Li J, Rouse DC, Park J, Farris AB, Turek JW, Knechtle SJ, Kirk AD, Markert ML, Cultured Thymus Transplantation Promotes Donor-specific Tolerance to Allogeneic Heart Transplants.

[0145] [Figure 24] The immunohistochemical analysis of the transplanted CTT explanted on day 180 shows normal thymic histology beneath the renal capsule (right side of Figure 24A). Figure 24B shows the graft explanted in H&E. Staining for viable T cells (CD3), T cell proliferation (Ki67), and cytokeratin (detected with rabbit polyclonal antibody) is shown. In the panel stained for cytokeratin, a lace-like pattern is observed in Hassall's bodies on the TEC (arrow). This figure is from the manuscript being prepared for submission for publication: Kwun J, Li J, Rouse DC, Park J, Farris AB, Turek JW, Knechtle SJ, Kirk AD, Markert ML, Cultured Thymus Transplantation Promotes Donor-specific Tolerance to Allogeneic Heart Transplants.

[0146] [Figure 25]This figure shows the survival percentage of immunosuppressed LW rats after DA heart transplantation with and without thymectomy and CTT transplantation (solid triangles, blue lines) and without CTT (inverted triangles, red lines). LW rats with CTTT were tolerant, while LW rats without CTTT were immunodeficient and therefore did not reject the DA heart. The control group showed complete rejection of DA heart transplantation in unmanipulated LW rats (white squares). LW control animals also did not reject the LW heart graft (white circles with horizontal lines) (n=9). This figure is from a manuscript in preparation for submission for publication: Kwun J, Li J, Rouse DC, Park J, Farris AB, Turek JW, Knechtle SJ, Kirk AD, Markert ML, Cultured Thymus Transplantation Promotes Donor-specific Tolerance to Allogeneic Heart Transplants.

[0147] [Figure 26A] These are photographs of allogeneic grafts (DA hearts) transplanted from animals that received CTT (26A) and animals that did not receive CTT (26B), showing mononuclear cell infiltration without signs of rejection as indicated by the 2004 International Society for Heart & Lung Transplantation (ISHLT), as shown in Figure 26C (solid blue square and solid red triangle). This figure is from a manuscript in preparation for submission for publication: Kwun J, Li J, Rouse DC, Park J, Farris AB, Turek JW, Knechtle SJ, Kirk AD, Markert ML, Cultured Thymus Transplantation Promotes Donor-specific Tolerance to Allogeneic Heart Transplants. [Figure 26B]These are photographs of allogeneic grafts (DA hearts) transplanted from animals that received CTT (26A) and animals that did not receive CTT (26B), showing mononuclear cell infiltration without signs of rejection as indicated by the 2004 International Society for Heart & Lung Transplantation (ISHLT), as shown in Figure 26C (solid blue square and solid red triangle). This figure is from a manuscript in preparation for submission for publication: Kwun J, Li J, Rouse DC, Park J, Farris AB, Turek JW, Knechtle SJ, Kirk AD, Markert ML, Cultured Thymus Transplantation Promotes Donor-specific Tolerance to Allogeneic Heart Transplants.

[0148] [Figure 27] This figure plots the percentage of BN heart graft survival in the neck between animal survival days and graft survival days in LW rats with CTT (immune and rejected cervical allogeneic BN hearts) and control LW animals without CTT insertion (immunodeficient due to thymic absence and unable to reject cervical BN hearts) versus BN controls (LW rats that reject cervical BN hearts) and syngeneic controls (LW rats that do not reject cervical LW hearts). This figure is from the manuscript in preparation for submission for publication: Kwun J, Li J, Rouse DC, Park J, Farris AB, Turek JW, Knechtle SJ, Kirk AD, Markert ML, Cultured Thymus Transplantation Promotes Donor-specific Tolerance to Allogeneic Heart Transplants.

[0149] [Figure 28A]These are photographs of BN cardiac tissue at days 11 and 46 with and without CTT insertion (Figure 28A and Figure 28B), respectively. These photographs form the basis for the data in Figures 27 and 29. The heart in 28A is not rejected due to tolerance. The heart in 28B is not rejected due to immunodeficiency from the absence of thymus. These figures are from a manuscript in preparation for submission for publication: Kwun J, Li J, Rouse DC, Park J, Farris AB, Turek JW, Knechtle SJ, Kirk AD, Markert ML, Cultured Thymus Transplantation Promotes Donor-specific Tolerance to Allogeneic Heart Transplants. [Figure 28B] These are photographs of BN cardiac tissue at days 11 and 46 with and without CTT insertion (Figure 28A and Figure 28B), respectively. These photographs form the basis for the data in Figures 27 and 29. The heart in 28A is not rejected due to tolerance. The heart in 28B is not rejected due to immunodeficiency from the absence of thymus. These figures are from a manuscript in preparation for submission for publication: Kwun J, Li J, Rouse DC, Park J, Farris AB, Turek JW, Knechtle SJ, Kirk AD, Markert ML, Cultured Thymus Transplantation Promotes Donor-specific Tolerance to Allogeneic Heart Transplants.

[0150] [Figure 29]This shows rejection grading of cervical BN hearts. Syngeneic LW hearts placed in LW rats (white circles) were not rejected. BN hearts placed in LW rats (black circles) were rejected. BN hearts placed in LW rats that had undergone CTT were rejected (black squares). BN hearts placed in LW rats that had not undergone CTT were weakly rejected in two rats (shaded triangles) and not rejected in the other three rats. These data indicate that rats with CTT were able to strongly reject third-party hearts even when they accepted DA hearts as if CTTT had expressed DA (Figure 26C). Rats without CTT were immunodeficient and did not reject either DA (Figure 26C) or BN hearts. This figure is from a manuscript being prepared for publication: Kwun J, Li J, Rouse DC, Park J, Farris AB, Turek JW, Knechtle SJ, Kirk AD, Markert ML, Cultured Thymus Transplantation Promotes Donor-Specific Tolerance to Allogeneic Heart Transplants.

[0151] [Figure 30A] These are photographs of BN hearts in rats that underwent or did not undergo CTT insertion, compared to LW and DA hearts, respectively. In Figure 30A, after immunosuppression was removed and the BN heart was transplanted, the BN heart was immediately rejected and is therefore very large due to all inflammation. The LW heart is the normal size for a heart that pumps blood throughout the body. The DA heart is small because it was located in the abdomen and did not need to pump blood. In Figure 30B, after immunosuppression was removed, the rats were immunodeficient and could not reject either the BN or DA heart. This figure is from a manuscript in preparation for submission for publication: Kwun J, Li J, Rouse DC, Park J, Farris AB, Turek JW, Knechtle SJ, Kirk AD, Markert ML, Cultured Thymus Transplantation Promotes Donor-specific Tolerance to Allogeneic Heart Transplants. [Figure 30B] These are photographs of BN hearts in rats that underwent or did not undergo CTT insertion, compared to LW and DA hearts, respectively. In Figure 30A, after immunosuppression was removed and the BN heart was transplanted, the BN heart was immediately rejected and is therefore very large due to all inflammation. The LW heart is the normal size for a heart that pumps blood throughout the body. The DA heart is small because it was located in the abdomen and did not need to pump blood. In Figure 30B, after immunosuppression was removed, the rats were immunodeficient and could not reject either the BN or DA heart. This figure is from a manuscript in preparation for submission for publication: Kwun J, Li J, Rouse DC, Park J, Farris AB, Turek JW, Knechtle SJ, Kirk AD, Markert ML, Cultured Thymus Transplantation Promotes Donor-specific Tolerance to Allogeneic Heart Transplants.

[0152] [Figure 31] This is a plot of rejection grading for explanted cervical BN hearts from rats versus BN control and syngeneic control rats with and without CTT insertion. Figure 31A shows quantification of inflammatory cells in primary abdominal DA heart allografts. Syngeneic controls indicate that LW rats do not reject the LW heart. DA controls indicate that LW rats reject the DA heart. The CTT group does not reject the DA heart due to tolerance. The group without CTT does not reject the DA heart due to immunodeficiency from the absence of the thymus. Figure 31B shows quantification of inflammatory cells in secondary cervical BN heart allografts. Syngeneic controls indicate that LW rats do not reject the LW heart. BN controls indicate that LW rats reject the BN heart. The CTT group rejects the BN heart due to immune capacity. The group without CTT does not reject the BN heart due to immunodeficiency from the absence of the thymus. [Figure 31C] This image shows DA and BN heart rats taken from LW recipients along with their congenital LW hearts during cervical BN heart rejection. The lower right panel shows T cells (brown) in the BN heart that caused the rejection. [Figure 31D] This figure shows T cell infiltration in LW, DA, and BN hearts from control animals without CTT insertion. Since the animals are immunodeficient, there is no T cell infiltration. This figure is from a manuscript in preparation for publication: Kwun J, Li J, Rouse DC, Park J, Farris AB, Turek JW, Knechtle SJ, Kirk AD, Markert ML, Cultured Thymus Transplantation Promotes Donor-specific Tolerance to Allogeneic Heart Transplants.

[0153] [Figure 32]This shows humoral tolerance after CTTT. Figure 32A shows representative histogram plots of post-transplant donor-specific alloantibodies (anti-DA and anti-BN antibodies) as measured by T-cell flow cross-matching. The upper left panel (DA control) shows the development of anti-DA antibodies (thick line) in normal LW rats after ectopic abdominal DA heart transplantation. The upper center panel shows the absence of anti-DA antibodies in LW rats that underwent CTTT, which indicates tolerance. The upper right panel shows the absence of response in LW rats without CTTT, which reflects immunodeficiency in rats after thymectomy and T-cell depletion without acceptance of the donor thymus. The lower left panel shows normal anti-BN antibodies formed in normal LW rats receiving cervical BN hearts. The lower center panel shows the normal response to BN in LW rats with CTTT after cervical BN heart transplantation, indicating immune capacity and the ability to reject a third party. The lower right panel shows no response in LW rats without CTTT to BN after cervical BN heart transplantation, indicating immunodeficiency and a lack of ability to reject third parties. Figure 32B shows the level of anti-DA antibodies after primary DA heart transplantation. LW rats with CTTT from an LWxDA thymic donor are tolerant to DA and therefore do not produce anti-DA antibodies after DA heart transplantation. LW rats without CTTT are immunodeficient and therefore do not produce anti-DA antibodies after DA heart transplantation. Figure 32C shows the level of anti-BN antibodies after secondary cervical BN heart transplantation. LW rats with CTTT from an LWxDA donor produce antibodies against BN, demonstrating immunity to third parties. LW rats without CTTT do not produce antibodies against BN, demonstrating immunodeficiency. This figure is from a manuscript being prepared for publication: Kwun J, Li J, Rouse DC, Park J, Farris AB, Turek JW, Knechtle SJ, Kirk AD, Markert ML, Cultured Thymus Transplantation Promotes Donor-Specific Tolerance to Allogeneic Heart Transplants.

[0154] [Figure 33]This is a cryopreservation of cultured non-human primate thymus tissue 12 days after culture. The first row shows cytokeratin at collection (A), 6 days of culture (B), 12 days of culture (C), and 12 days after culture, followed by 35 days of cryopreservation, and then thawing in the photograph (D). Cytokeratin (AE1 / AE3) in D is similar to cytokeratin in C. The second row shows CK14 staining at the same time point. CK14 in H is very similar to CK14 in panel G. The third row shows CD3 staining, which is expected to result in loss of viable T cells over time. Panel L is similar to panel K in that it has very few T cells. The fourth row shows Ki-67 staining of proliferating T cells. Ki-67 staining is absent by day 6 because the T cells are mostly dead. This figure demonstrates the ability to cryopreserve non-human primate thymus, similar to how cultured thymus tissue is cryopreserved for patients. [Modes for carrying out the invention]

[0122]

[0155] The titles, headings, and subheadings provided herein should not be construed as limiting the various aspects of this disclosure. Accordingly, the terms defined below are more fully defined by reference to the entire specification. All references cited herein are incorporated in their entirety by reference.

[0123]

[0156] Unless otherwise specified, scientific and technical terms used herein shall have meanings generally understood by those skilled in the art. Furthermore, unless specifically required by the context, singular terms shall include plural forms and plural terms shall include singular forms. In this application, the use of “or” shall mean “and / or” unless otherwise specified. In the context of multiple dependent claims, the use of “or” shall, only if alternative, refer back to the multiple preceding independent or dependent claims.

[0124]

[0157] Furthermore, it should be noted that, as used herein and in the appended claims, the use of the singular forms “a,” “an,” and “the,” as well as the singular form of any word, includes multiple referents unless explicitly and obviously limited to one referent. As used herein, the term “includes” and its grammatical variations are intended to be non-restrictive so as not to exclude other similar items that may be substituted for or added to the listed items.

[0125]

[0158] The present invention is best understood by referring to the following definitions:

[0126]

[0159] The term “approximately” is used herein to mean roughly, nearly, approximately, or around. When the term “approximately” is used with a numerical range, it modifies that range by extending the boundary above and below the numerical value being stated. Generally, the term “approximately” is used herein to modify numerical values ​​above and below the stated value by a variance of + / - 10%. Where used herein, the term “approximately” refers to numerical values, including, for example, integers, fractions, and percentages, whether explicitly indicated or not. The term “approximately” generally refers to a range of numerical values ​​(e.g., + / - 5 to 10% of the listed range) that a person skilled in the art would consider equivalent to the listed values ​​(e.g., having the same function or result). Where terms such as “at least” and “approximately” are used prior to a list of numerical values ​​or ranges, the terms modify all of the values ​​or ranges provided in the list. In some cases, the term “approximately” may include numerical values ​​rounded to the nearest significant digit.

[0127]

[0160] As used herein, the term “animal” includes, but is not limited to, humans, as well as non-human vertebrates such as wild, domesticated, and livestock animals. Animals may also be referred to as “subjects.”

[0128]

[0161] As used herein, “biocompatible” means any material that, when implanted in a mammal, does not cause an adverse response in the mammal.

[0129]

[0162] "Chronic transplant rejection" generally occurs in humans within a few months to a few years after engraftment, even when immunosuppression for acute rejection is successful. Fibrosis is a common factor in chronic rejection of all types of organ transplants.

[0130]

[0163] As used herein, “comprising” (and any form of comprising, such as “comprise,” “comprises,” and “comprised”), “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 comprehensive or open-ended and do not exclude additional unenumerated elements or method steps. In addition, it is understood that any term used in conjunction with “comprising” may also be used in conjunction with the terms “consisting of” or “consisting essentially of.”

[0131]

[0164] As used herein, “graft” typically refers to a tissue or organ that is transplanted into an individual to replace, correct, or otherwise overcome a defect. The tissue or organ may consist of cells derived from the same individual, and this graft is referred herein to by the following interchangeable terms: “autograft,” “autologous transplant,” “autologous implant,” and “autologous graft.” As used herein, a graft from a genetically different individual of the same species is referred herein to by the following interchangeable terms: “allograft,” “allogeneic transplant,” “allogeneic implant,” and “allogeneic graft.” A graft from one individual to its identical twin is referred to herein as an "isograft," "syngeneic transplant," "syngeneic implant," or "syngeneic graft." A "xenograft," "xenogeneic transplant," or "xenogeneic implant" refers to a graft from one individual to another individual of a different species.

[0132]

[0165] As used herein, the term "HLA compatible" refers to a donor-recipient pair in which neither the donor nor the recipient has a mismatch in any HLA antigens. In the methods of the present invention, HLA compatibility includes HLA alleles: HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DQA1, HLA-DPB1, and HLA-DPA1.

[0133]

[0166] As used herein, the term “HLA mismatch” refers to a match in the donor and recipient HLA antigens, typically with respect to HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DQA1, HLA-DPB1, and HLA-DPA1, where HLA mismatch occurs between the donor and recipient. In some cases, one haplotype is compatible and the other is incompatible. This situation is common with organs from living or deceased donors. HLA mismatch in donor-recipient pairs results in an increased risk of graft rejection compared to HLA-compatible pairs.

[0134]

[0167] As background to the aforementioned definition, HLA antigens correspond to "human leukocyte antigens," which are protein molecules expressed on the surface of cells that confer specific antigenic identity to these cells. They are also known as "major histocompatibility complex antigens." Therefore, MHC or HLA antigens are target molecules that are recognized by T cells as either "self" or "non-self." If HLA antigens originate from the same hematopoietic stem cell source as immune effector cells, they are considered "self." If HLA antigens originate from a different source of hematopoietic reconstituted cells, they are considered "non-self."

[0135]

[0168] Two major classes of HLA antigens are recognized: HLA class I and HLA class II. HLA class I antigens (A, B, and C in humans) enable each cell to be recognized as "self." HLA class II antigens (DRB1, DPB1, DPA1, DQB1, and DQA1 in humans) are involved in the reaction between lymphocytes and antigen-presenting cells. Both classes of HLA antigens are involved as targets for transplanted organ rejection.

[0136]

[0169] HLA genes are clustered at human chromosome location 6p21. This gene cluster encodes six classic transplantable HLA genes. The 6p21 segment also encodes genes that encode proteins that play important roles in regulating the immune system as well as other fundamental molecular and cellular processes. The complete cluster is approximately 3.6 Mb and contains at least 224 loci. As a result of clustering, specific "haplotypes" (sets of alleles present on a single chromosome) arise. Haplotypes inherited from one parent tend to be inherited as a group. The set of alleles inherited from each parent forms a haplotype, within which some alleles tend to be associated together. HLA matching is used to identify the recipient's haplotype and to identify a suitable matched donor. Certain haplotypes are more common than others, and their frequencies vary across different racial and ethnic groups.

[0137]

[0170] As used herein, the phrase “needs it” means that the subject has been identified as having a need for a particular method or treatment. In some embodiments, identification may be by any diagnostic means. In any of the methods and treatments described herein, the subject may require it.

[0138]

[0171] As used herein, the phrase “an integer from X to Y” means any integer including the endpoints. For example, the phrase “an integer from X to Y” means 1, 2, 3, 4, or 5.

[0139]

[0172] As used herein, the term “mammal” means rodents (i.e., mice, rats, or guinea pigs), monkeys, cats, dogs, cattle, horses, pigs, or humans. In some embodiments, the mammal is a human.

[0140]

[0173] As used herein, the term “organ” refers to a solid, angiogenic organ that performs a specific function or set of functions within an organism. The term “organ” includes, but is not limited to, the heart, lungs, kidneys, liver, pancreas, skin, uterus, bone, cartilage, small or large intestine, bladder, brain, breast, blood vessels, esophagus, fallopian tubes, gallbladder, ovaries, pancreas, prostate, placenta, spinal cord, limbs including upper and lower limbs, spleen, stomach, testes, thymus, thyroid, trachea, ureters, urethra, and uterus.

[0141]

[0174] As used herein, the terms “prevent,” “prevent,” and “prevention” refer to the implementation of a therapy for an individual who may eventually develop, but has not yet developed, at least one symptom of a disease, disorder, or condition, in order to reduce the likelihood that the individual will develop symptoms of the disease, disorder, or condition over a period of time. Such reduction may be reflected, for example, in the delay of the onset of at least one symptom of the disease, disorder, or condition in the patient.

[0142]

[0175] As used herein, the terms “subject,” “individual,” or “patient” are interchangeable and mean any animal, including mammals, such as mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses, or primates such as humans.

[0143]

[0176] As used herein, “therapeutic dose” means the amount of an active compound or pharmaceutical preparation that elicits a desired biological or medical response in a tissue, system, animal, individual, or human, as determined by a researcher, veterinarian, physician, or other clinician. The therapeutic effect depends on the disorder being treated or the desired biological effect. Such effects may include a reduction in the severity of symptoms associated with the disorder and / or inhibition (partial or complete) of the progression of the disorder, or improved treatment, cure, prevention, or elimination of the disorder or its side effects. The amount required to induce a therapeutic response may be determined based on the age, health status, size, and sex of the subject. The optimal amount may also be determined based on monitoring the subject's response to the treatment.

[0144]

[0177] As used herein, the term “tissue” refers to, but is not limited to, any type of tissue in humans or animals, including, vascular tissue, skin tissue, liver tissue, pancreatic tissue, nerve tissue, genitourinary tissue, gastrointestinal tissue, skeletal tissue including bone and cartilage, adipose tissue, connective tissue including tendons and ligaments, amniotic tissue, chorionic tissue, dura mater, pericardium, muscle tissue, glandular tissue, facial tissue, and ocular tissue.

[0145]

[0178] In the context of this disclosure, “tissue bank” refers to the long-term storage of cryopreserved, allogeneically cultured postnatal thymus tissue-derived products stored under liquid nitrogen. General guidance for establishing repositories of allogeneically cultured postnatal thymus tissue-derived products can be found in Guidance for Industry.Current Good Tissue Practice (CGTP) and Additional Requirements for Manufacturers of Human Cells, Tissues, and Cellular and Tissue-Based Products (HCT / Ps) available at https: / / www.fda.gov / downloads / BiologicsBloodVaccines / GuidanceComplianceRegulatoryInformation / Guidances / Tissue / UCM285223.pdf.

[0146]

[0179] "Tissue manipulation" refers to the process of generating tissue ex vivo for use in tissue replacement or reconstruction. Tissue manipulation is an example of "regenerative medicine," which encompasses approaches to repairing or replacing tissues and organs by incorporating cells, genes, or other biological building blocks, along with bioengineered materials and techniques.

[0147]

[0180] The term "transplant rejection" encompasses both acute and chronic rejection. "Acute rejection" is the rejection by the recipient's immune system when the transplanted tissue is immunologically heterogeneous. Acute rejection is characterized by infiltration of the transplanted tissue by recipient immune cells, which perform their effector functions and destroy the transplanted tissue. The onset of acute rejection is rapid and typically occurs in humans within a few weeks after transplant surgery. Generally, acute rejection can be inhibited or suppressed with immunosuppressants such as rapamycin, cyclosporine A, and anti-CD40L monoclonal antibodies.

[0148]

[0181] As used herein, the terms “to treat,” “treated,” or “to treat” mean both therapeutic and preventive measures whose purpose is to slow (reduce) an undesirable physiological condition, disorder, or disease, or to obtain a beneficial or desired clinical outcome. Beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms; a reduction in the severity of a condition, disorder, or disease; a stabilized (i.e., non-worsening) state of a condition, disorder, or disease; a delay or slowing of the onset of the progression of a condition, disorder, or disease; improvement or remission (whether partial or complete), whether detectable or undetectable; improvement of at least one measurable physical parameter, which may not be identifiable by the patient; or improvement or improvement of a condition, disorder, or disease.

[0149]

[0182] As described herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer values ​​within the listed range, and, where appropriate, fractions thereof (such as 1 / 10 and 1 / 100 of an integer), unless otherwise specified. Ranges are approximate and may vary more than integers.

[0150]

[0183] Units, prefixes, and symbols are shown in the format approved by the Systeme International de Unites (SI). Numerical ranges include the numerical values ​​that define the range. Measurements are understood to be approximate, taking into account significant figures and measurement-related errors.

[0151]

[0184] Furthermore, for clarification, it is understood that certain features described herein, which are described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, for brevity, various features described in the context of a single embodiment may also be provided separately or in any suitable partial combination.

[0152] Donor thymus tissue collection

[0185] Donor thymic tissue can be obtained during postnatal cardiac surgery with the informed consent of the donor's family. Removal of some thymic tissue may be necessary to reveal the surgical site. Therefore, due to the nature of the surgical procedure, a portion of the thymus may be removed during cardiac surgery in postnatal cardiac surgery.

[0153]

[0186] During postnatal cardiac surgery, a portion of the thymic tissue may be discarded during the surgical procedure. For all cardiac surgeries, regardless of whether the thymus has been screened for transplantation, the surgeon places the removed thymic tissue in a sterile container.

[0154]

[0187] Thymic tissue donors for thymic tissue transplantation in infants with complete DiGeorge syndrome were infants under 9 months of age. The active pharmaceutical ingredient alloculture postnatal thymic tissue-derived product is prepared by processing and culturing discarded thymic tissue as described herein.

[0155]

[0188] Consent for the use of thymus tissue in cultured thymic tissue transplantation can be obtained before or after the thymus is harvested. However, consent to allow blood to be taken from an infant before the bypass is required and is always obtained before the surgery. This blood sample is used for donor screening.

[0156]

[0189] Discarded thymic tissue is placed in a sterile container. Routine testing is performed on the donor and the donor's birth mother in accordance with FDA guidelines for tissue transplantation. Tissue type matching is not required for the surgical procedures described herein, although such matching may be performed in certain circumstances.

[0157]

[0190] The tissue may be processed immediately or refrigerated overnight for processing the following day. If the thymic tissue is to be stored overnight, it is sterilely added to a sufficient amount of thymic culture medium (TOM medium, as described below) in the original container to completely cover the thymic tissue. The container containing the thymus is placed in the refrigerator until ready for processing the following day.

[0158] Overview of thymic tissue conditioning

[0191] The conditioning regimen depletes donor thymocytes from cultured thymic tissue sections. Based on in vitro data (immunohistochemistry), the culture period of 12–21 days preserves the epithelial network, which is evaluated using cytokeratin antibodies. The culture is preferably carried out at 37°C in a 5% CO2 incubator.

[0159]

[0192] For successful culturing, thymic tissue is preferably sectioned and placed on Millipore® cellulose or an equivalent filter, then placed on a surgical sponge in a tissue culture dish. The culture medium consists of thymic organ medium (TOM) and is replaced daily.

[0160]

[0193] The recipient's thymus is evaluated pathologically. Identity testing must show more than 50% of the area positive for keratin in a lace-like staining pattern. Efficacy testing must show Hassall's bodies and CK14 staining in a lace-like pattern. Viability testing must show more than 90% intact nuclei observed in the section. Tissue lot release is performed pathologically on one day between day 5 and day 9 (including this day). For identity testing, the tissue area on days 5-9 must be positive for keratin and AE1 / AE3. For efficacy testing, cultured thymic tissue on days 5-9 must show scattered cytokeratin CK14 staining throughout, and at least one Hassall's body must be identified. For viability testing, cultured thymic tissue on days 5-9 must show intact nuclei.

[0161]

[0194] In one embodiment, thymic tissue sections are conditioned for approximately 12 days and then cryopreserved. In another embodiment, all of the thymic tissue sections are conditioned for approximately 12 days, then about half are transplanted into the recipient, and the remaining thymic tissue sections are cryopreserved for future use.

[0162]

[0195] Within 24 hours of collection, the thymus is sectioned into thin sections. The sections are kept in culture for 12–21 days. This culture process depletes viable donor T cells, as detailed below, ultimately allowing the surgically transplanted tissue section to reconstruct the immune system targeting athymia at potentially lower but immunologically more effective T cell levels.

[0163]

[0196] The culture process, as outlined below, significantly alters the biological properties of the donor thymus tissue and its constituent cells in order to optimize the effective therapeutic properties of the CTT sections.

[0164]

[0197] The culture process ensures that a defined composition of cultured cells / tissues possessing the required biological properties is obtained in a manner suitable for surgical transplantation into the target, enabling the reconstruction of the target's immune system.

[0165]

[0198] The culture process results in the loss of thymocytes and the relative enrichment of thymic epithelial cells and other stromal cells in donor thymic tissue sections.

[0166]

[0199] The culture process further leads to thymocyte depletion and maintenance of TEC, enabling the reconstruction of the recipient's immune system and allowing tolerance to develop in the recipient against HLA antigens in the donor thymus.

[0167]

[0200] Overall, the manufacturing process is designed to deplete thymocytes from donor thymic tissue while preserving the functional structure of the thymic stroma (thymic epithelial cells and fibroblasts).

[0168]

[0201] In one embodiment, the processed donor thymic tissue is an engineered thymic tissue product that can induce tolerance to a thymic tissue type (HLA antigen) in subjects requiring it after surgical transplantation.

[0169]

[0202] To maintain the viability of the sectioned thymic tissue, the thymic sections are placed on a Millipore cellulose filter, and a surgical sponge is inserted into a tissue culture dish containing culture medium. The culture medium in each tissue culture dish is changed daily from the day of donor collection to the day of transplantation (days 12-21).

[0170]

[0203] Culturing donor thymic tissue depletes thymocytes in such treated tissue, minimizing the risk of graft-versus-host disease ("GvHD"), which can be a significant problem in severely immunocompromised patients after thymectomy.

[0171]

[0204] During the first few days of culture, many thymocytes "overflow" from the tissue section into the culture medium and are discarded during medium changes. As the culture continues throughout the culture period, donor thymocytes continue to die, but their cellular residues are retained within the CTT section.

[0172]

[0205] Without being constrained by theory, it is hypothesized that the presence of these nucleus-deficient, unviable thymocytes and their remnants is important for the intended function of the tissue manipulation product, as it helps preserve open pockets in the three-dimensional network of thymic epithelial cells necessary for the invasion of recipient bone marrow stem cells after treatment. The importance of having “space” for invading bone marrow stem cells is supported by the experience of patient DIG003 in Markert, 1999 (see the references list below). The patient described in the aforementioned reference was given a very large dose of steroids (methylprednisolone 40 mg / kg / day x 3 days) 35 days after CTT transplantation, which caused apoptosis of thymocytes and epithelial condensation. Naive T cells did not develop, and the patient succumbed to infection. At autopsy, the inserted thymus was a mass of viable cuboidal epithelium lacking space between epithelial cells for thymocytes to enter.

[0173]

[0206] During the culture period, HLA typing is performed to confirm whether the patient (recipient) and donor tissues share any HLA alleles. Anti-HLA antibody testing is performed in the recipient to determine whether the recipient has any antibodies against any HLA antigens in the thymus. If the recipient has antibodies targeting the donor's MHC, another thymus is requested. The donor and the donor's mother are checked for infection in accordance with the FDA guidance document "Guidance for Industry. Eligibility Determination for Donors of Human Cells, Tissues and Cellular and Tissue-Based Products (HCT / Ps)" and more recent guidance documents. The tissue is sterilized in accordance with Code of Federal Regulations (CFR) 1271 subpart D, "Current Good Tissue Practice."

[0174]

[0207] Following a review of batch records and QC testing, the tissue is released from manufacturing and provided to the surgical team for transplantation, where it is surgically implanted in the recipient as previously described.

[0175]

[0208] Cultured thymic tissue is produced by a process that is described more fully below and in the examples described herein.

[0176]

[0209] In summary, the culture process of harvested thymic tissue significantly alters the biological properties of the donor tissue and its constituent cells in the following ways: loss of donor thymocytes and enrichment of thymic epithelial cells and other stromal cells, as well as depletion of donor thymocytes, which alters the physiological function of the tissue (e.g., secretion of cytokines and growth factors) and its structural properties.

[0177]

[0210] During the first few days of culture, many thymocytes "overflow" from the tissue section into the culture medium and are discarded during medium changes.

[0178]

[0211] The operations performed during the manufacturing process result in changes to the overall and histological appearance of the resulting cells contained in the final product compared to the source or starting material obtained from the donor.

[0179]

[0212] During the first few days of culture, thymic tissue appears red due to residual blood on and within the tissue. See, for example, Figure 15.

[0180]

[0213] Viable tissue is observed between days 5 and 9, and the blood contamination that was apparent on day 1 is ruled out.

[0181]

[0214] During the remaining days of culture, the tissue depth decreases as thymocytes are depleted. The decrease in thymocyte density in the tissue is documented by immunohistochemistry and is described in detail below.

[0182]

[0215] On day zero after collection of discarded thymic tissue, the tissue is densely packed with viable thymocytes embedded in the stroma containing thymic epithelial cells and fibroblasts. AE1 / AE3 and CK14 staining confirm the presence of cytokeratin (CK)-positive thymic epithelial cells, characteristic of a normal thymus. The thymic epithelial cells form a lace-like three-dimensional network through a delicate process of surrounding adjacent thymic cells.

[0183]

[0216] During the culture process, thymic sections are cultured as described below. Numerous thymocytes are washed away from the tissue, particularly over the first three days. This depletion can be histologically identified on day two by H&E staining, which shows a reduced thymocyte density, especially in the medullary region. The majority of thymocytes remaining in the tissue show nuclear changes consistent with apoptosis and / or necrosis, or nuclear lysis (complete loss of the nucleus). Many of these dead thymocytes and their cellular remnants remain throughout the thymic tissue, presumably preventing the complete breakdown of inter-epithelial cell spaces.

[0184]

[0217] Some epithelial condensation can be seen in outer regions such as the subcapsular cortex, where thymic cell loss has disrupted the epithelial cell network. These condensed subcapsular cortical epithelial cells form linear arrays of several cell layer thicknesses, which can increase the mechanical strength of the section. Some medullary epithelium can also condense to form patches of adjacent epithelial cells.

[0185]

[0218] As the culture progresses, thymocyte death continues, and necrotic thymocyte fragments are retained within the tissue. Further condensation of the medulla and subcapsular cortical epithelium is minimal around days 7–19 of the culture.

[0186]

[0219] Regions with epithelial structures similar to those of a normal thymus can still be observed in each thymus in the later stages of culture using AE1 / AE3 staining. For longer-cultured tissues, the epithelial structures of the cortex and medulla can still be easily identified, and Hassall's bodies, for example, remain in the medullary region. However, the degree of thymic cell depletion results in a substantially different overall histological appearance in H&E compared to that of a normal thymus at point 1 and beyond.

[0187] Detailed culture of thymic tissue

[0220] A common procedure for preparing allogeneic cultured thymic tissue-derived products is that thymic tissue from infants with complete DiGeorge syndrome is obtained as discarded tissue from infants under 9 months of age undergoing cardiac surgery, as previously described. For solid organ transplantation, discarded thymus may be obtained from individuals up to 50 years of age. The use of thymic tissue will depend on whether it meets the criteria for use described herein.

[0188]

[0221] Thymic tissue is sterile-treated and cultured under cGMP conditions to produce thymic tissue sections with partially depleted T cells.

[0189]

[0222] The preparation of cultured thymic tissue (CTT) generally consists of the following steps: receiving and processing of incoming thymic tissue, sectioning, culturing, medium change, dose calculation, packaging, and transport of the thymus to the operating room. In addition, incoming thymic tissue is tested for receptivity, and in-process and release tests are performed on thymic tissue sections.

[0190]

[0223] In one embodiment, the thymic tissue sectioning process involves using sterile, disposable scissors and forceps to cut off a piece of thymic tissue. The surgeon removes the thymic capsule with the forceps and scissors and places the capsule on the plate lid for later disposal.

[0191]

[0224] A piece of thymic tissue is placed on a disposable tissue slicer using forceps. The top of the slicer (e.g., Stadie-Riggs hand microtome (Thomas Scientific, Swedesboro, NJ)) is placed on the central part of the slicer and fixed in place. The surgeon cuts the section by passing the blade through the tissue piece. The sections are approximately 0.5–1 mm thick. After cutting each section, the top of the slicer is removed, and the tissue sections are transferred using forceps onto a pre-moistened sterile Millipore filter. The filter is pre-moistened with TOM. Approximately 50–90% of the filter space is filled with tissue sections without overlap.

[0192]

[0225] Typically, three in-process sections are separated from the tissue at the start of sectioning, all approximately 3x3 mm in size. One is sent to histology, and two are retained. Once the thymus is sectioned, the thymocytes are free to flow into the culture medium.

[0193]

[0226] In one embodiment, the filters and thymic sections are transferred to gelatin surgical sponges saturated with TOM in tissue culture dishes. Two filters are placed in each sponge, and two sponges are used per tissue culture dish. The thymic sectioning process is repeated until the required number of sections are prepared. The culture dishes are labeled with the surgical number, dish number, and ISBT barcode label. The completed dishes are placed in a humidified incubator containing 5% CO2 at 37°C.

[0194]

[0227] The tissue manipulation drug substance (API) comprises thymic tissue sections that have been placed in culture dishes in culture medium and cultured for 12–21 days, as described below. The tissue manipulation formulation comprises thymic tissue sections after transfer to a formulation container. No other processing is performed from the API to prepare the formulation; the only processing of the API for preparation of the formulation is the transfer of sections to a leak-proof container and the corresponding change of culture medium.

[0195]

[0228] The culture of thymic tissue sections is described in more detail in the following paragraphs.

[0196]

[0229] In one embodiment, thymic tissue is obtained from the operating room as waste tissue from an infant undergoing cardiac surgery at 9 months of age. The tissue is then placed by the surgical team into a sterile specimen cup with a screw-cap top and transported to a GMP facility under ambient conditions for processing. The sterile specimen container into which the thymus is received includes a barcode and is labeled with the donor's name and medical record number. A donor screening group assigns each thymus a unique identifier (thymuses are numbered sequentially) and a unique medical record number. For manufacturing, each tissue has a surgical number and a unique label. All identifiers are recorded on a "Confidential Thymic Donor Form," which is maintained separately from batch records and kept confidential.

[0197]

[0230] In one embodiment, the active pharmaceutical ingredient container sealing system may be a cell culture dish with a lid. One section of thymic tissue is placed on a filter, and two filters are placed on each gelatin sponge in the thymic organ medium in the dish. Four sections are placed in each culture dish, and the dishes are stored in an incubator, with the medium being changed daily until ready for release.

[0198]

[0231] In one embodiment, the culture dish can be obtained from Corning. The dish may be a sterile, non-pyrogenic Falcon® 100 mm polystyrene cell culture dish (product #353003). The dish is purified by vacuum gas plasma treatment and sterilized by gamma irradiation. The dimensions of the dish are 89.43 mm O.D. x 19.18 mm.

[0199]

[0232] In exemplary embodiments, Surgifoam® sponge may be manufactured by Ethicon, which meets the requirements of absorbent gelatin sponge, USP. A suitable sponge is a sterile, water-insoluble, malleable porcine gelatin absorbent sponge intended for hemostatic applications. An example of a mixed cellulose ester filter is manufactured by Millipore (product number #SMWP 02500). The 25 mm hydrophilic membrane has a pore size of 5.0 μm. It is made of a biologically inert mixture of cellulose acetate and cellulose nitrate. The filter is sterilized with ethylene oxide before use.

[0200]

[0233] After release and acceptance of the donor thymus to the processing laboratory, the thymus is cut into thin sections, which are placed on sterile filter paper on a surgical sponge in a sterile culture dish. If the tissue is not processed immediately, it is stored in thymic organ medium (TOM) at 2–8°C for up to 24 hours after collection from the donor before processing begins, as described below. TOM consists of Ham's F-12 medium, HEPES buffer, L-glutamine, and heat-inactivated fetal bovine serum (FBS).

[0201]

[0234] In one embodiment, processing is carried out in an ISO5 space within a biological safety cabinet (BSC) in an ISO7 manufacturing cleanroom. Only one lot of thymic tissue from a single thymus is processed in the BSC at any given time. The BSC is purified before use. The thymus is tested visually for appearance and weighed. The thymus is then placed in a 150 mm tissue culture dish in a TOM. The thymic capsule is removed with sterile, disposable forceps and scissors. The tissue sample is taken as a retained sample for testing. The incoming thymic tissue is tested for identity by histology. Donor eligibility is also confirmed. Processing is continued before receiving the histological results and all donor screening results.

[0202]

[0235] The acceptance criteria for donor screening is that all donor eligibility requirements must be met. Donor screening is required in accordance with 21 CFR 1271 to protect the safety of thymic tissue transplant recipients. This screening minimizes the risk of infection transmission from donor to recipient.

[0203]

[0236] Using sterile, disposable scissors and forceps, a tissue section is cut off. The tissue section is placed on a disposable tissue slicer using forceps. As previously described, the top of the slicer is placed on the central part of the slicer and secured in place. The surgeon cuts the section by passing the blade through the tissue section. The sections are approximately 0.5–1 mm thick. After cutting each section, the top of the slicer is removed, and the tissue sections are transferred using forceps onto a pre-moistened sterile Millipore® filter. The filter is pre-moistened with TOM. Approximately 50–90% of the filter space is filled with tissue sections without overlap. The filter and thymic sections are transferred to a gelatin surgical sponge saturated with TOM in a tissue culture dish.

[0204] Thymus organ medium (TOM)

[0237] The culture medium will be prepared with human-approved ingredients that are less likely to cause allergic reactions, whenever such reagents are available.

[0205]

[0238] In the event of any problems, all reagents must be tracked so that all components can be identified after transplantation.

[0206]

[0239] Fetal bovine serum (FBS) must be manufactured using US materials due to concerns about Creutzfeldt-Jakob disease. Information on each lot must be sent to the FDA before use.

[0207]

[0240] Normal adults possess natural antibodies against FBS (antibodies against galactose-alpha-1,3-galactose (Gal-α-1-3Gal)), which are tested as IgM allohemagglutinins. Immunodeficient children with DiGeorge syndrome do not have these antibodies. (Parker W, Yu PB, Holzknecht ZE, Lundberg K, Buckley RH, Platt JL., 1997, “Specificity and function of 'natural' antibodies in immunodeficient subjects: Clues to B cell lineage and development,” J Clin Immunol. 17:311-321).

[0208]

[0241] The culture medium must be tested for bacterial, fungal, and mycoplasma contamination before use.

[0209]

[0242] In one embodiment, the following materials are used to prepare the TOM:

[0210]

[0243] HAMS F12, Gibco #11765-054 (or case #11765-062), 500 ml bottle or equivalent source.

[0211]

[0244] HEPES, Gibco #15630-080 or equivalent, 1M solution, 100ml bottle. Final concentration 25mM.

[0212]

[0245] L-glutamine, Gibco#25030-081 or equivalent source (stock 200mM).

[0213]

[0246] Fetal bovine serum, Gibco, #16140 (thermally inactivated) or #10082-147 (thermally inactivated, certified).

[0214]

[0247] In one embodiment, the HI FBS may be used in the following manner.

[0215]

[0248] FBS must be thermally inactivated at 56°C for 30 minutes.

[0216]

[0249] To reduce the possibility of contamination of the culture medium, it must be divided into aliquots, and these aliquots should not be reused.

[0217]

[0250] The remaining FBS aliquots can be frozen (-20°C) in 25 ml aliquots for research use.

[0218]

[0251] In one embodiment, TOM can be prepared by the following method.

[0219]

[0252] Thaw the fetal bovine serum overnight in the refrigerator, or at 37°C, stirring frequently and gently.

[0220]

[0253] If non-thermally inactivated fetal bovine serum is used, it should be heat-inactivated at 56°C for 30 minutes.

[0221]

[0254] To prepare 4 liters at once, place all the culture medium components together in a 4-liter flask and stir with a stirring rod on a magnetic stirring plate at a moderate speed (without creating bubbles) for 3-5 minutes.

[0222]

[0255] Sterilize using a 0.2 micron filter unit.

[0223]

[0256] In one embodiment, the sterilization of the TOM preparation can be performed by the following method. Dispense 1 liter of TOM into a 1 liter flask. Measure 80 ml of TOM using a disposable sterile cylinder. Pour 80 ml of TOM into a 150 ml Corning filter sterilization unit. Attach a household vacuum according to the manufacturer's instructions and filter and filter sterilize. Remove the filter unit from the container and discard. Plug the collection bottle with a sterile cap (provided with the unit). Label with the TOM lot number. Test one aliquot for bacterial culture with anaerobic bacteria; fungal culture, etc.; and mycoplasma culture. Test one aliquot for endotoxin. Store all TOM aliquots upright in a -20°C freezer.

[0224]

[0257] The TOM medium can be released for use if the LAL result is 2 EU / ml or less for a sample diluted 20-fold for testing, or 1 EU / ml or less for a sample diluted 10-fold for testing, and if all culture results are negative for growth.

[0225]

[0258] A BSC must be used to filter and dispense the medium.

[0226]

[0259] TOM is tested for sterility and endotoxin before release. TOM is not released for culturing donor thymus until the acceptance criteria for the 14-day sterility test are met. Once prepared, TOM is stored at -20°C until thawed, at which point it can be stored for up to 2 weeks in the refrigerator for use.

[0227]

[0260] In one embodiment, the 14-day sterility test can be performed, for example, using a BacT / ALERT culture system. BacT / ALERT (BioMerieux, Durham, NC) is a commercially available culture system that can be used to test samples using an automated microbial detection system.

[0228]

[0261] All in-process cultures and drug substance cultures are incubated for 14 days or reported immediately if the product turns positive. For positive tests, the organism(s) are identified and their antibiotic susceptibilities are determined. Culture bottles containing medium for aerobic growth and bottles containing medium for anaerobic growth are inoculated with samples to be tested on day 1, day 7, and the day of release. All bottles are incubated at 35 - 37 °C for 14 days.

[0229]

[0262] FBS can be obtained from the GIBCO brand, Life Technologies. FBS is prepared by a sterile and validated process. FBS meets the USDA requirements regarding animals sourced from meat processing plants, traceability, and country of origin. All fetal blood is collected from fetuses of healthy mother animals that passed the pre- and post-slaughter certified veterinarian inspections. All FBS is traceable by the date and location of collection. FBS collected and processed in the United States is from slaughter facilities approved and inspected by the USDA. The United States is recognized by the USDA as free of foot-and-mouth disease and rinderpest. To qualify suppliers, FBS is tested for pH, osmotic pressure, endotoxin, total protein, and identity prior to use.

[0230]

[0263] The completed dishes are placed in a humidified incubator containing 5% CO2 at 37 °C. Each lot of thymus tissue is stored in a separate incubator. After the thymus sections are placed in the incubator, particle sampling and operator monitoring are performed.

[0231]

[0264] Thymic sections are cultured for up to 21 days, during which the culture medium is changed daily. These thymic sections are considered the active pharmaceutical ingredient. During the culture period, many thymocytes are washed away from the thymocyte sections, or thymocytes undergo apoptosis while preserving the thymic stroma. All manufacturing steps are performed using sterile, disposable equipment and consumables. The medium is pipetted from the culture dish and pooled in a sterile collection container for in-process testing. 10 mL of fresh thymic organ medium is then gently dispensed into each culture dish, rinsing over the tissue sections. After the medium change is complete, samples are obtained from the pooled medium for sterility and histology, if necessary. Particle sampling and operator monitoring are completed, and line clearance is performed.

[0232]

[0265] The culture medium is replaced daily.

[0233]

[0266] The sections are cultured for up to 21 days.

[0234]

[0267] In-process testing is conducted to provide insights into process and product quality, helping to ensure the safety and quality of the final formulation.

[0235] In-process testing

[0268] Samples are collected for sterile in-process testing from day 1 to day 7. Samples are collected for mycoplasma in-process testing on day 7. Samples are collected for in-process histological testing from day 5 to day 9. The dose is determined the day before release. Gram staining, BacT, mycoplasma, and endotoxins are tested on the day of release.

[0236]

[0269] Gram staining is a bacteriological experimental technique used to distinguish bacterial species into two groups: Gram-positive and Gram-negative bacteria. Gram staining is performed on used culture media pooled from culture dishes. The method uses staining techniques to determine classification based on the physical characteristics of the cell wall. This method is used to perform preliminary morphological identification or to establish whether a significant number of bacteria are present in a clinical specimen. Staining can be performed either manually or using an automated staining machine. Studies have shown that two different staining methods do not show qualitative differences that affect culture outcomes.

[0237]

[0270] Histological examination performed on days 5–9 includes (1) determination that areas positive for keratin AE1 / AE3 are scattered throughout the tissue on days 5–9, (2) identification of at least one Hassall body under a microscope, (3) scattered CK14 staining of tissue sections throughout the thymic tissue, and (4) observation of intact nuclei under a microscope.

[0238]

[0271] The presence of Hassar bodies and intact nuclei, as well as successful CK14 staining, indicates cultured normal, healthy thymic tissue.

[0239]

[0272] Culture time is a crucial process parameter. As mentioned, culture is carried out for a maximum of 21 days.

[0240]

[0273] Testing of thymic samples at days 5, 9, 12, and 21 of the culture is performed to confirm whether the histological results produced at days 5–9 of the culture represent the histological test results produced at days 12–21 of the culture. Based on observations made by a pathologist on the samples considered in the examples, the histological appearance of the tissue section at day 5 reflects what is observed at each of the subsequent time points (days 9, 12, and 21). This supports the decision to perform release testing at days 5–9.

[0241]

[0274] The histological examination of any one section supports the conclusion regarding the acceptability of the entire lot. The relevant characteristics of any one section from the thymus reflect the characteristics of the entire thymus and support the continued use of a single section of tissue for histological testing.

[0242]

[0275] As shown in Figures 12A and 12B, the forced degradation tests showed that the cultured thymus tissue products were not easily degraded and were most sensitive to freezing / thawing, similar to changes in osmotic pressure. The other conditions tested during forced degradation showed little or no effect on the cultured thymus tissue products. Control of Cultured Thymus Product Drug Substances

[0243]

[0276] The acceptance criteria for incoming thymus tissue products include the tests specified in Table 1 below.

Table 1

[0244]

[0277] Abbreviations: CK, cytokeratin; EU, endotoxin unit; USP, United States Pharmacopeia. Thymus tissue is processed before all donor screening results are obtained.

[0245]

[0278] Generally, the acceptance criterion for weight is 3 grams or more. This is the minimum thymus weight accepted to ensure that sufficient material is available for proper dosing of the final product. The acceptance criteria are based on the processing experience of thymus tissue.

[0246]

[0279] The acceptance criteria for in-process testing are specified in Table 2 below.

Table 2

[0247]

[0280] The acceptance criteria for cultured thymus tissue drug substance testing are specified in Table 3 below.

Table 3

[0248]

[0281] The acceptance criteria for identity are that thymic tissue identity is confirmed histologically on day 1 and at the midpoint (days 5-9). Barcodes are used to track the tissue during processing, and the barcodes are checked at release to verify the correct identity of the product.

[0249] Histology using immunochemistry

[0282] Histological methods are standard practices used by hospitals for all tissue types, as is known to those skilled in the art.

[0250]

[0283] Product samples are fixed in 10% formalin and transported to the laboratory. Containers are labeled with coded identifiers in place of patient names to protect patient privacy, in accordance with the medical record number. Upon arrival at pathology, specimens are assigned a unique pathology accession number and barcoded. All subsequent blocks, slides, and documents are barcoded with this pathology accession number.

[0251]

[0284] After the specimen is received in the laboratory, the formalin-fixed tissue is examined overall, and a written overall description of the material, which will be part of the final report, is prepared. The formalin-fixed tissue is processed using standard methodologies in an automated processing machine and embedded in a paraffin block. Sections are cut from the paraffin block, and the following staining is performed by an ASCP-certified histologist.

[0252]

[0285] Hematoxylin & Eosin.

[0253]

[0286] Cytokeratin AE1 / AE3 immunohistochemistry

[0254]

[0287] Cytokeratin-14 immunohistochemistry

[0255]

[0288] CD3 immunohistochemistry.

[0256]

[0289] Ki-67 immunohistochemistry.

[0257]

[0290] During the immunohistochemical testing described above, appropriate control slides are also tested and re-examined. All control slides and internal controls exhibit the expected immunoreaction pattern. The incoming thymus sample also serves as a control for tissue sections cultured for 5–9 days if the sample is tested as part of an efficacy test. The incoming thymus sample appears as a typical thymus sample and is then tested 5–9 days after changes occur in the tissue sections during culture. After 5–9 days in culture, the sample shows areas positive for keratin AE1 / AE3 scattered throughout the tissue, the presence of at least one Hassall body, CK14 staining scattered throughout the tissue, and the presence of intact nuclei.

[0258]

[0291] The slides will be interpreted by a certified pathologist in anatomical pathology with additional experience in the histological evaluation of thymic tissue. The final report will be issued by the pathologist, and the report will document the results.

[0259]

[0292] The acceptance criteria for the cultured thymic tissue drug substance assay are specified in Table 4 below. [Table 4]

[0260]

[0293] Cultured thymic tissue must be microorganism-free. Sterility tests performed on days 1 and 7 should show no microbial growth. Mycoplasma should be negative on day 7. Sterility tests should be Gram stain-negative.

[0261]

[0294] Product sterility is maintained using appropriate controls, including aseptic techniques, such as using training programs to verify the qualifications of surgeons; utilizing appropriate cleanroom certification procedures; using established clean medium filling procedures and utilizing sterilized equipment using ready-to-use sterilization devices or validated sterilization cycles.

[0262]

[0295] The processed thymic tissue containers are visually inspected for damage. Tissue sections typically exhibit a yellow to reddish-brown appearance with varying thickness and shape.

[0263]

[0296] Thymic tissue identity is confirmed by histology on day 1 and at the midpoint (days 5-9).

[0264]

[0297] Barcodes are used to track organizations during processing, and these barcodes are verified upon release.

[0265]

[0298] The dosage (area) is 1,000 to 20,000 mm² of thymic tissue. 2 This is the recipient's body surface area per 1 m². The dose is controlled by the surface area of ​​the section released into the operating room, which is appropriate for the patient's body surface area.

[0266]

[0299] The permissible dose range is 1m 2 1,000-20,000 mm² per recipient body surface area (BSA) 2 It is defined as the thymic tissue. The area of ​​the thymic tissue is determined by photographs using software analysis (PAX-it image analysis software). The BSA is determined using the patient's height in cm and weight in kg. DuBois and the DuBois formula are used to calculate the BSA.

[0267]

[0300] BSA = 0.007184 × [Height (cm)] 0.725 ×[Weight (kg)] 0.425 .

[0268]

[0301] Cultured thymic tissue is tested for endotoxins. The specification is ≤5 EU / kg body weight / hour.

[0269]

[0302] Endotoxin testing can be performed, for example, by using the Endosafe PTS system. The cartridges used within the Endosafe PTS utilize a chromogenic kinetic Limulus Amebocyte Lysate (LAL) test. Each cartridge contains precise amounts of LAL reagent, chromogenic substrate, and control standard endotoxin. The test sample is pipetted into four sample reservoirs. The instrument aspirates the sample and mixes it with the LAL reagent in two channels (sample channels) and with the LAL reagent and positive product control in the other two channels (spike channels). The sample is incubated and then combined with the chromogenic substrate. After mixing, the optical density of the wells is measured and compared to a standard curve archived in the instrument. The instrument measures the reaction time in each channel. The archived standard curve, specific to each batch of cartridges, is constructed using a log of reaction time against a log of endotoxin standard concentration. Sample and spike values ​​are calculated by interpolation from the standard curve using reaction time. This test meets the requirements of the United States Pharmacopeia (USP).

[0270]

[0303] Mycoplasma testing can be performed in the following manner: Samples from the pooled culture medium are removed from the plate on day 7 and tested before product release.

[0271]

[0304] In the case of a positive culture during manufacturing, the lot is discarded and not administered. In the case of a positive culture after administration of the clinical product, the patient's attending physician and the trial sponsor treat the patient appropriately. A positive culture requires identification of the species of contaminating organism and determination of its antibiotic susceptibility. The attending physician initiates antibiotic therapy for the thymus recipient, if indicated.

[0272]

[0305] The preparation undergoes similar visual and histological examinations before use.

[0273]

[0306] After the thymic tissue sections are cultured for up to 21 days, they are transferred to a preparation container for transport to the operating room. Upon arrival in the operating room, the sections are inserted into the thigh muscle of the recipient patient.

[0274]

[0307] The container should be intact with no visible damage, and the thymic tissue sections should appear as yellow to reddish-brown tissue sections with varying thickness and shape. The tissue sections are visually examined to confirm that these acceptance criteria are met.

[0275] Freezing and thawing of products derived from allogeneic cultured post-thymus tissue.

[0308] Cryopreservation of products derived from allogeneic cultured post-thymus tissue can be carried out by the following methods.

[0276]

[0309]

[0310] (a) Steps to obtain suitable thymic tissue from a donor,

[0311] (b) HLA alleles: A step of typing HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DPB1, HLA-DPA1,

[0312] (c) A step of subjecting thymic tissue to a conditioning regimen for a period of up to 12 days, wherein the conditioning regimen for donor thymic tissue comprises sterile treatment of the donor thymic tissue in thymic organ medium to produce a donor thymic tissue fragment in which T cells are partially depleted, and further, the donor thymic tissue fragment exhibits, on days 5–9, areas positive for keratin AE1 / AE3 scattered throughout the tissue, the presence of at least one Hassall body, CK14 staining scattered throughout the tissue, and the presence of intact nuclei upon completion of the conditioning regimen.

[0313] (d) A step of collecting a donor thymus tissue fragment in which T cells are partially depleted as an allogeneic cultured post-vitro thymus tissue-derived product,

[0314] (e) A step of cryopreserving the product derived from allogeneic cultured postnatal thymus tissue in liquid nitrogen,

[0315] (f) A cryopreserved allocultured postnatal thymus tissue-derived product prepared by a method comprising the step of maintaining the cryopreserved allocultured postnatal thymus tissue-derived product in liquid nitrogen in a cryopreserved allocultured postnatal thymus tissue-derived product bank. In one embodiment, the cryopreserved allocultured postnatal thymus tissue-derived product according to claim 66, wherein on the day of collection, more than 50% of the region is positive for keratin in a lace staining pattern, Hassall bodies are present, CK14 stains in a lace staining pattern, and more than 90% of the nuclei are intact.

[0277]

[0316] In one embodiment, the donor thymus is sectioned and roughly divided into two equal parts, and each section is placed in a separate cryovial (Nunc tube) with its cellulose filter. The filter is folded in half and inserted into the tube. To cover the tissue, approximately 1 to 1.5 ml of cryo-medium [sterile filtered 90% heat-inactivated fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO)] at room temperature is added. The sterile caps of the cryovials are replaced with tubes. All tubes are placed in a Biocision Cool Cell or equivalent container at room temperature. Any empty slots in the Cool Cell should be filled with tubes containing 1 ml of cryo-medium. The tubes are placed in a -80°C freezer overnight. Alternatively, each tissue and filter is placed in a 5 ml CryoELITE tissue vial (Wheaton). To cover the tissue, 3 to 5 ml of cryo-medium at room temperature is added. Place in a styrofoam box and place in a -80°C freezer overnight. The vial is then transferred to the gas phase of a liquid nitrogen freezer. Alternatively, the temperature of the cryovial can be brought up to liquid nitrogen temperature using a freezer with a controlled rate.

[0278]

[0317] To recover the tissue, remove the cryovial or CryoELITE tissue vial from the liquid nitrogen freezer. Rapidly thaw the thymic fragments in the vial with a swirling motion in a 37°C water bath. The tube is sprayed with 70% ethanol and then placed in a biological safety cabinet (BSC). The thymic tissue and filters are removed from either the Nunc cryovial or CryoELITE tissue vial using forceps. The tissue and filters are placed in 50 ml conical tubes containing 20 ml of 4°C TOM. Up to five filters can be placed in each 50 ml conical tube containing 20 ml of 4°C TOM. Immediately transfer the five filters, along with the tissue, into a new conical tube containing 20 ml of 4°C TOM medium and leave at 4°C for 15 minutes. Repeat the washing three times. Maintaining the tissue at 4°C, transfer each fragment to its own 120 ml Starplex container containing 5 ml of 4°C TOM. All containers are transported to the operating room in temperature-controlled containers with cooling packs inside. Starplex containers containing tissue are transported to the operating room. The tissue on the filter is transferred to a sterile field and then to a tissue culture dish containing approximately 2 ml of sterile saline. The operating room nurse removes the tissue from the filter paper by scraping or pulling with forceps. The operating room nurse returns the tissue to the filter paper in an irregular stack. The tissue culture dish, containing approximately four filters and tissues, is transferred to the surgical site so that the surgeon can easily access the tissue. The tissue is placed in the quadriceps femoris muscle, similar to the procedure in CTT (RVT-802). Cryo-CTT is similar to CTT in that it is partially T-cell depleted, the thymic tissue section shows areas positive for keratin AE1 / AE3 scattered throughout the tissue, the section contains at least one Hassall's body, CK14 staining is scattered throughout the tissue, and intact nuclei are present.

[0279] CTT transplantation

[0318] In one embodiment, incompatible thymic tissue sections from a donor are cultured for 12–21 days. On the day of solid organ transplantation, steroids are usually administered during induction of anesthesia. For heart or lung transplants, the recipient's thymus is surgically removed at the time of solid organ transplantation. For other organ transplants, thymectomy may be performed the day before or on the day of transplantation. The method of thymectomy may be surgical, thoracoscopic, or robotic. At the end of surgery after reperfusion, the recipient is given more steroids before receiving equine anti-thymocyte globulin (e.g., rabbit anti-thymocyte globulin) over 3–7 days to kill most of the remaining T cells (and NK cells) in the recipient, or alemtuzumab over 4 days to kill T, B, and NK cells. Administration of immunosuppressants (such as cyclosporine or tacrolimus) and mycophenylate is initiated until T cells develop and show more than 10% naive T cells. It can take 6–12 months for naive T cells to increase to this number. Cultured thymic tissue is processed for the thymus of a solid organ donor. Half of the CTT can be transplanted into the quadriceps muscle in 12–21 days. The other half of the thymus will be cryopreserved for subsequent use in the recipient. The immunosuppressive regimen will suppress any remaining T cells until naive T cells are released by the cultured thymic tissue section transplanted into the recipient and the recipient meets the criteria for weaning from the maintenance immunosuppressive regimen. (More than 10% naive T cells are needed to wean from immunosuppression.)

[0280] Thymectomy protocol

[0319] The patient is taken to the operating room and placed under general anesthesia via an endotracheal tube.

[0281]

[0320] The chest and abdomen are prepared in a sterile condition and draped.

[0282]

[0321] The patient undergoes a complete sternotomy through a skin incision of approximately 4 cm.

[0283]

[0322] Both pleural cavities are accessed to ensure complete resection.

[0284]

[0323] The phrenic nerves are visualized on both sides, and care is taken not to damage them.

[0285]

[0324] The thymus is identified and carefully dissected from the pleural capsule of the lung, starting at the inferior horn and extending to the superior horn.

[0286]

[0325] A complete thymectomy is performed.

[0287]

[0326] Hemostasis is achieved within the mediastinum.

[0288]

[0327] Thoracic tube placement. One thoracic tube is always inserted (into the mediastinum). If a single pleural cavity is accessed during surgery, the thoracic tube continues from the mediastinum into that pleural cavity. If both pleural cavities are accessed, a second thoracic tube is used in a similar manner, from the mediastinum to the other pleural cavity.

[0289]

[0328] Drain size. #15 Blake drains are used for infants up to 2 years old. #19 Blake drains are used for children 2 years and older.

[0290]

[0329] Sternal closure: In newborns or infants, 0-Ticron sutures are used to close the sternum. At approximately 1-2 years of age, #1 sternal wires are used. At approximately 2-5 years of age, #4 sternal wires are used.

[0291]

[0330] The fascia, subcutaneous tissue, and skin are closed with continuous absorbable sutures.

[0292]

[0331] The skin wound VAC is positioned on the sternum.

[0293]

[0332] The patient is extubated in the operating room.

[0294]

[0333] Sponge, instrument, and needle counts must be performed and accurate at the end of the case. Surgical transplantation of products derived from allogeneic cultured post-thymic tissue.

[0295]

[0334] Allogeneic cultured post-thymic tissue-derived products should be transplanted according to the following instructions. Transplantation of thymic tissue into the thigh requires a healthy layer of muscle tissue.

[0296] Preparation for transplantation

[0335] The planned maximum and minimum doses of transplanted allogeneic cultured post-thym tissue-derived product should be calculated for each individual patient. The intended recipient should be correctly identified before administration.

[0297]

[0336] Under sterile conditions in a laminar flow hood, tissue sections on filter paper on a surgical sponge in culture medium are removed from the tissue culture dish, placed in a 120 ml sterile cup containing 20 ml of medium, packaged to maintain sterility, and delivered to the operating room or packaged for shipment. Tissue sections are not removed from their individual containers until ready for use. Check the expiration date and time of the product.

[0298]

[0337] Allogeneic cultured post-thymocyte-derived products (tissue sections) are always handled using strict aseptic techniques. Each container is inspected for evidence of leakage or damage. If there is evidence of contamination, it is not used. In the sterile field, the containers of allogeneic cultured post-thymocyte-derived products are unpacked from the shipping box. The rack containing the polypropylene containers is removed from the outer bag. When ready, a team member, outside the sterile field but adjacent to the sterile preparation table, uncaps and removes each container one at a time. Each opened container is then held by a team member outside the sterile field, with their arm extended over the sterile field without touching it.

[0299]

[0338] A sterile field team member uses a forceps to remove individual tissue sections from their containers along with their filter paper and places them in a sterile tissue culture dish containing approximately 2 ml of preservative-free saline on a sterile preparation table. The four tissue sections, each containing filter paper, removed from the four containers are placed in a sterile tissue culture dish on the sterile field in front of the sterile field team member. Using sterile forceps, the sterile field team member then uses two pairs of forceps to peel the tissue sections from the filter paper, one holding the filter in place and the other pulling the tissue or stacking and discarding the tissue. The tissue removed from each filter paper is placed on top of the filter paper, with the center of the filter paper overlapping. The sterile tissue culture dish is then transferred to the sterile field. The next set of four allogeneic cultured post-thymocyte-derived product containers is then processed in the same manner while the surgeon is transplanting the first four sections. When the surgeon has finished transplanting the first four sections, the next dish containing four tissue sections is placed in the surgical field, and the first tissue culture dish is returned to the sterile field in front of the sterile field team member to load a third set of four tissue sections. This cycle continues until all desired tissues have been transplanted. To avoid contamination from the air in the operating room, not all tissue sections are transplanted at once.

[0300]

[0339] Surgical treatment

[0340] Step 1. Skin incision After induction of general anesthesia, a longitudinal skin incision (typically about 5 cm long) is made across one of the anterior thigh compartments. Note: The size of the incision and the use of one or both legs for the grafting procedure are determined by the patient's size, the planned amount of tissue to be grafted, and their muscle mass. If all or most of the tissue can be grafted to one leg, only one leg should be used.

[0301]

[0341] Step 2. Open the fascia to expose the anterior compartment muscles.

[0302]

[0342] Step 3. Muscle diffusion and transplantation

[0343] The muscle is separated along the natural groove of the quadriceps femoris muscle using a tonsil clamp or similar instrument. Individual thymic sections of allogeneically cultured post-vibrational thymic tissue-derived product should be transplanted without cutting the muscle tissue. The individual tissue sections are placed in “pockets” about 1 cm deep and spaced about 1 cm apart within the quadriceps femoris muscle along the natural groove. Depending on the patient's size, the surgeon may place about 6-7 sections in 6-7 pockets along each groove. Individual allogeneically cultured post-vibrational thymic tissue-derived product sections may be cut in half before transplantation depending on the mass of tissue on each filter. Thick sections of tissue that completely covered the filter paper should be cut in half for optimal angiogenesis of each tissue. The required amount of tissue in each anterior compartment is transplanted up to the planned maximum dose.

[0303]

[0344] Step 4. Muscle Closure

[0345] To prevent the muscle from opening again and the graft from protruding, the muscle is closed with a single suture over the site where the thymic tissue was transplanted. Before closing the incision, ensure that the transplanted tissue is completely covered by muscle tissue that does not contain any exposed thymic tissue.

[0304]

[0346] Step 5. Repeat steps 3-4 for each allogeneically cultured post-thymic tissue-derived product tissue section up to the intended maximum dose.

[0305]

[0347] Step 6. Closing the incision

[0348] Confirm hemostasis. Close the skin incision with two layers of absorbable sutures and apply a standard dressing such as a wound closure strip or skin adhesive. Leave the fascia open to allow room for the muscle compartment to expand. A sealing dressing may be used to prevent contamination.

[0306]

[0349] Postoperative surgical / medical management Use a mild pain reliever as needed. Monitor for signs of infection or separation.

[0307]

[0350] If the donor is a living-related donor of organs such as the lungs, kidneys, intestines, or a portion of the liver, a portion of the thymus from that solid organ donor may be suitable for culture and transplantation. The pathological criteria listed above must be met on the day of collection and on days 5-9.

[0308]

[0351] Cryopreserved cultured thymic tissue may be available from a third-party donor. However, the third-party donor must express all recipient HLA alleles not expressed by the solid organ donor. These include HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DQA1, HLA-DPB1, and HLA-DPA1. Incompatibility in the HLA-DP allele is accepted if the incompatibility is "acceptable". For other alleles, small incompatibility is accepted, for example, HLA-A*01:02 to a recipient possessing HLA-A*01:01; in other words, the second field (after the colon) may differ, but the first field (before the colon) must be identical.

[0309] Human heart transplant procedure

[0352] Eligibility is determined based on several criteria, including poor prognosis of 12–24 months without heart transplantation despite current maximum supportive care, congenital or acquired heart disease with growth retardation as defined by UNOS criteria, symptoms of progressive heart failure in congenital or acquired heart disease resistant to medical therapy, abnormal hemodynamics or increased pulmonary vascular resistance, inoperable structural heart disease, symptomatic arrhythmias refractory to drug or device therapy, or low exercise tolerance.

[0310]

[0353] For example, many absolute and relative contraindications to heart transplant surgery are evaluated, including reversible renal impairment unless the patient is a candidate for heart / kidney transplant; irreversible liver disease unless the patient is a candidate for heart / liver transplant; irreversible pulmonary impairment, non-conventional ventilator support (i.e., radiofrequency ventilator, maximum CMV setting), or fixed pulmonary hypertension (TPG > 15) unless the patient is a candidate for heart-lung transplant; diabetes with microvascular disease; or active, uncontrolled paroxysmal disorders.

[0311]

[0354] Other contraindications may include other diseases that limit long-term survival and rehabilitation after heart transplantation; substance abuse, morbid obesity, malignant tumors; active mental disorders; and other reasons such as documented medical non-compliance.

[0312]

[0355] Relative contraindications include active infection; cognitive impairment; inadequate vascular access; and significant allosensitization.

[0313]

[0356] Donor and recipient tissue compatibility management is also considered. Patients are evaluated for panel-reactive antibodies ("PRAs") of pre-formed HLA antibodies that the recipient may form in response to “sensitization events” such as previous injections or major surgeries using bypass / blood products or allogeneic graft materials.

[0314]

[0357] The assessment will include the recipient's prior sensitization history, such as previous blood transfusions (number and date), previous surgeries, previous pregnancies, immunization history, and IVIG administration (with date).

[0315]

[0358] Patients with excessively high HLA class I or class II PRA, or those undergoing repeated transplants, may be candidates for desensitization strategies. Specific strategies are individualized to the potential recipient and may include the use of plasmapheresis, IVIG, rituximab, and / or bortezomib. Key antibodies are those that remain present after a 1:16 dilution.

[0316]

[0359] Pre-sensitized patients may require actual prospective cross-matching with potential donors. Patients with a history of HLA antibodies will undergo virtual cross-matching in UNET at the time transplantation is being considered.

[0317]

[0360] Pre-sensitized patients may require actual prospective cross-matching with potential donors via the following general guidelines. All patients with a history of HLA antibodies will undergo virtual cross-matching in UNET at the time donation is being considered.

[0318]

[0361] cPRA < 20%: In the case of virtual cross-matching in UNET, no additional measures are needed; routine retroactive donor cross-matching and routine immunosuppression should be carried out.

[0319]

[0362] cPRA > 20%: In the case of virtual cross-matching in UNET, the recipient undergoes plasma exchange in the operating room ("OR") at the time of transplantation.

[0320]

[0363] cPRA > 70%: In the case of virtual crossmatching, if time permits, consider obtaining actual prospective donor crossmatching and perform plasma exchange in the OR. Consider the placement of the ferresis catheter in the OR to continue ferresis after surgery.

[0321]

[0364] The decision to continue antibody reduction intervention is based on donor cross-matching, DSA, and clinical course.

[0322]

[0365] For all pre-sensitized patients, blood will be sent for donor-specific antibodies within the first two weeks post-transplant and will be repeated as clinically indicated. Routine testing for DSA will be performed on all post-transplant patients at least every six months post-transplant, and as needed if there are any clinical concerns.

[0323]

[0366] Standard blood product infusion protocols will be followed both before and after transplantation.

[0324] Immunosuppression management

[0367] Immunosuppressive management is determined based on the solid organ being transplanted.

[0325] Pediatric heart transplant

[0368] All patients will receive induction therapy with basiliximab or anti-thymocyte globulin based on their clinical condition and risk factors. The type of induction therapy used will be determined before transplantation (first immunosuppressive regimen drug therapy).

[0326]

[0369] In one embodiment, pre-transplant / induction therapy typically includes the following administrations:

[0327]

[0370] Mycophenolate mofetil (CellCept®) 25 mg / kg IV before entering the operating room.

[0328]

[0371] During induction, administer methylprednisolone 10 mg / kg IV (maximum dose 500 mg).

[0329]

[0372] Methylprednisolone 10 mg / kg IV (maximum dose 500 mg) is administered upon release of the X-clamp.

[0330]

[0373] ATG (anti-thymocyte globulin) 1.5 mg / kg IV was administered upon release of the X-clamp.

[0331]

[0374] Alternatively, basiliximab (Simulect®), administered according to the recipient's body weight, can be used.

[0332]

[0375] <35kg, 10mg upon release of the X-clamp, followed by a second dose 4 days later.

[0333]

[0376] >35kg, 20mg upon release of X-clamp, second dose 4 days later.

[0334]

[0377] In one embodiment, pre-transplant-induced immunosuppressive therapy for cardiac and CTT transplant candidates may include:

[0335]

[0378] Basiliximab (Simulect®) - (if ATG cannot be administered).

[0336]

[0379] Administration:

[0380] <35kg: Initial dose: 10 mg IV administered under anesthesia before surgery during anesthesia induction (after stabilization and after methylprednisolone has been administered).

[0337]

[0381] Second dose: 10 mg IV administered 4 days after transplantation. If complications (including severe hypersensitivity reaction or graft loss) occur, wait for the second dose.

[0382] >35kg: Initial dose: 20mg IV administered via anesthesia before surgery during the induction of anesthesia when the patient is stable.

[0338]

[0383] Second dose: 20 mg IV administered 4 days after transplantation. If complications (including severe hypersensitivity reaction or graft loss) occur, wait for the second dose.

[0339]

[0384] Administration: Intravenous injection over 20-30 minutes. Half-life: Children 1-11 years: 9.5 days, Adolescents 12-16 years: 9.1 days, Adults: 7.2 days.

[0340]

[0385] In one embodiment, anti-thymocyte globulin (ATG, rabbit-derived, Thymoglobulin®) may be administered according to the following dosing schedule for cardiac transplant recipients:

[0386] Based on lymphocyte count, markers, and platelet count, administer 1.5 mg / kg / day for 3-7 days. The first dose is administered after organ reperfusion upon release of cross-clamp following the second dose of steroids (during surgery).

[0341]

[0387] This will be continued daily based on the above parameters and will be determined by the transplanted MD.

[0342]

[0388] Administration: The first dose should be administered slowly via intravenous infusion over 6 hours, and subsequent doses can be given over 4 hours, depending on the patient's tolerance.

[0343]

[0389] In one embodiment, in addition to routine preoperative / intraoperative methylprednisolone for CPB / pump cases, patients may receive a dose of methylprednisolone (SoluMedrol) 10 mg / kg (maximum dose 500 mg) IV administered anesthesia during induction of anesthesia prior to basiliximab, and a second dose of 10 mg / kg (maximum dose 500 mg) then during reperfusion (prior to ATG).

[0344] Postoperative maintenance immunosuppression (maintenance immunosuppression regimen)

[0390] In one embodiment, post-transplant immunosuppression includes:

[0391] For doses 5-7, administer 1.5 mg / kg IV of ATG (Thymoglobulin®) daily.

[0345]

[0392] Wait if WBC < 2,000 or platelet count < 50,000.

[0346]

[0393] For WBC counts of 2,000-3,000 or platelet counts of 50,000-75,000, use half the dose.

[0347]

[0394] Mycophenolate 15-25 mg / kg IV / PO every 12 hours.

[0348]

[0395] Adjust for GI intolerance or leukopenia / neutropenia.

[0349]

[0396] In cases of GI intolerance, azathioprine can be substituted.

[0350]

[0397] In cases of GI intolerance, mycophenolic acid (Myfortic®) can be substituted.

[0351]

[0398] Tacrolimus should be started 24-48 hours after TX, depending on renal function and oral intolerance.

[0352]

[0399] The starting dose is approximately 0.05 mg POq per 12 hours and should be adjusted based on the level.

[0353]

[0400] The first 6 months yield approximately 10-15, 6 months to 3 years yield 8-12, and >3 years yield 4-8.

[0354]

[0401] If an IV drug is required, or if the patient is intolerant to tacrolimus, cyclosporine can be substituted.

[0355]

[0402] Methylprednisolone 5 mg / kg IVq, 8 hours x 6 doses (maximum dose 125 mg).

[0356]

[0403] Next, 1 mg / kg / dose IV / POq 12 hours (maximum 30 mg / dose).

[0357]

[0404] Based on the biopsy results, withdrawal will be gradually completed over the next two to three months.

[0358]

[0405] In one embodiment, tacrolimus (FK506, Prograf®) may be administered. Typical dosage forms include an intravenous solution of 0.5 mg / ml, as well as oral capsules containing 0.5 mg, 1 mg, and 5 mg per capsule.

[0359]

[0406] If the recipient is PO / NG / SL, an initial dose of approximately 0.05 mg / kg / dose is administered every 12 hours (maximum 5 mg / dose), usually started about 24 hours post-surgery, provided renal function is acceptable.

[0360]

[0407] Tacrolimus trough levels are monitored daily until a therapeutic dose is achieved. If necessary, the dose is increased every 8 hours to achieve a therapeutic trough level. When the drug is administered sublingually, the capsule contents are sprinkled under the tongue (which may result in higher levels). This is not the case for oral and sublingual doses. Generally, it will be necessary to administer approximately half the oral dose when administered sublingually.

[0361]

[0408] Tacrolimus administration may be based on serum whole blood levels measured by mass spectrometry 10–14 hours after the last dose (7–9 hours if administered every 8 hours).

[0362]

[0409] Renal and hepatic function, drug side effects, and a history of infection and rejection are all considered in managing the patient's tacrolimus levels. If the patient is clinically well-adjusted and the level is within the desirable range of + / - 1, dose adjustment is not necessary. In these cases, the decision rests with the transplant surgeon.

[0363]

[0410] In one embodiment, cyclosporine is administered to patients who cannot tolerate tacrolimus. Starting dose: 2 mg / kg / dose orally every 12 hours. If therapeutic levels cannot be achieved (especially in infants and young children), the frequency of administration is increased to every 8 hours.

[0364]

[0411] Typical administration is based on the following serum whole blood levels measured by mass spectrometry 10–14 hours after the last dose (7–9 hours if administered every 8 hours):

[0365]

[0412] Renal and hepatic function, drug side effects, and a history of infection and rejection are all considered in managing the patient's cyclosporine levels. If the patient is clinically well-adjusted and the level is within the desirable range of + / - 10-20, dose adjustment is not necessary. In these cases, the decision rests with the transplanting institution. Every effort should be made to record the patient's target cyclosporine level on a chart. An IV dose is generally equivalent to one-third of an oral dose.

[0366]

[0413] In one embodiment, mycophenolate mofetil (Cellcept®) (200 mg / ml, 250 mg, or 500 mg tablets) may be administered:

[0414] Start with 30-50 mg / kg / day in two divided doses (maximum dose: 2 g / day for children, 3 g / day for adults).

[0367]

[0415] IV dose = PO dose.

[0368]

[0416] Drug monitoring is not required, but it can be done if there are concerns about toxicity.

[0369]

[0417] Mycophenolic acids can cause bone marrow suppression and neutropenia.

[0370]

[0418] The dose can be maintained if ANC < 500 or WBC < 1000, and may need to be reduced in situations where ANC < 1000 or WBC < 3000. In practice, a 500 mg dose of Cellcept is equivalent to 360 mg of Myfortic (mycophenolic acid).

[0371]

[0419] In one embodiment, mycophenolic acid (Myfortic® dosage forms 180 mg tablets, 360 mg tablets) may be administered in place of mycophenolate mofetil according to the following dosage parameters.

[0372]

[0420] Start with delayed-release tablets: 400 mg / m2 / dose twice daily, maximum dose: 720 mg or BSA 1.19-1.58 m2: 540 mg twice daily, BSA > 1.58 m2: 720 mg twice daily.

[0373]

[0421] IV or suspension is not available. If IV or suspension is required, the drug will be converted to Cellcept (Cellcept 500mg = Myfortic 360mg). Mycophenolate can cause the same myelosuppression as mycophenolate mofetil.

[0374]

[0422] In one embodiment, if mycophenolate mofetil cannot be tolerated, azathioprine may be administered in the following manner:

[0423] Start with 2-4 mg / kg / day, administered once daily.

[0424] Azathioprine can cause myelosuppression, and the dose may need to be reduced based on WBC / ANC. The IV dose is equal to the oral dose.

[0375]

[0425] In one embodiment, the steroid may be administered as methylprednisolone (Solu-Medrol®), prednisone, or prednisolone.

[0376]

[0426] Intravenous steroids are initiated during surgery (as induced immunosuppressive therapy) and continued post-surgery at 5 mg / kg / dose (maximum 125 mg / dose) IV every 8 hours for 6 doses.

[0377]

[0427] Oral steroids may then be initiated as prednisone tablets or prednisolone suspension at 3 mg / ml. Intravenous methylprednisolone is continued if the transplant recipient cannot tolerate the immunosuppressive therapy regimen orally. A switch to oral therapy may be made if the recipient can tolerate oral medications. An exemplary range of steroid dosage is as follows:

[0378]

[0428] Start with 2 mg / kg / day divided BID for 0-10 kg, discontinue every 2 days, and maintain at 6 mg / day.

[0429] For patients weighing 0-30 kg, start with 2 mg / kg / day divided BID (maximum single dose of 30 mg, see below), discontinue with 5 mg / day every two days, then maintain with 10 mg / day.

[0379]

[0430] >30kg, starting with 30mg BID x 4 doses, 25mg BID x 4 doses, 20mg BID x 4 doses, 15mg BID x 4 doses, 10mg BID x 4 doses, then 15mg per day, further discontinued by a transplant cardiologist.

[0431] Based on the biopsy results, steroid withdrawal will continue over the first month after transplantation. If rejection occurs, steroids may be restarted at the discretion of the transplant cardiologist and may be continued indefinitely based on further biopsy results and the patient's clinical condition.

[0380]

[0432] Other drugs in the second immunosuppressive regimen include:

[0433] Sirolimus (Rapamune®) (0.5 mg, 1 mg, 2 mg caps): To be initiated after a diagnosis of coronary artery allograft vascular complications, or otherwise as clinically indicated by a transplant cardiologist.

[0381]

[0434] Starting dose: 1 mg / m2 (maximum dose: 3 mg) once daily, or divided into two daily doses if it is difficult to achieve an appropriate level.

[0382]

[0435] The treatment level target is 4-8, and a lower tacrolimus level (same 4-8 range) is acceptable when taking both medications.

[0383]

[0436] Trough levels are drawn 23–25 hours after the final dose, or 11–13 hours after the final dose if administered every 12 hours.

[0384]

[0437] When initiating sirolimus, discontinue mycophenolate (Cellcept®, Myfortic®) and azathioprine.

[0385]

[0438] Start bactrim prevention: It can cause bothersome mouth ulcers and delayed wound healing.

[0386]

[0439] Pravastatin (Pravachol®) - Used in teenagers / older children and patients with CAV.

[0387]

[0440] Start with 0.2 mg / kg / day administered once daily (supplied in tablet form, which can be given at night as 1 / 4, 1 / 2, or up to 1-2 tablets per day).

[0388]

[0441] The dosage should be gradually increased according to changes in body weight (maximum dose 20 mg / day).

[0389]

[0442] LFTs and CK levels will be monitored every 8 weeks.

[0390]

[0443] If joint or muscle pain occurs, discontinue the medication.

[0391]

[0444] Ganciclovir IV is given for CMV prophylaxis if the recipient or donor is CMV IgG positive.

[0392]

[0445] Induction therapy: 5mg / kg IV q 12 hours x 7-14 days.

[0393]

[0446] Maintenance therapy: 5 mg / kg IV per day.

[0394]

[0447] Monitor WBC levels and kidney function.

[0395]

[0448] If tolerated, switch to oral valganciclovir.

[0396]

[0449] Valganciclovir (Valcyte®) - For CMV prevention in all CMV+ recipients or donors.

[0397]

[0450] 450 mg tablets or 50 mg / ml suspension.

[0398]

[0451] 4 months to 16 years: Total daily oral dose (mg) = [7 x BSA x Cr clearance].

[0399]

[0452] >16 years old: 900 mg orally per day.

[0400]

[0453] CMV PCR will be monitored at each visit.

[0401]

[0454] Trimethoprim-sulfamethoxazole (Bactrim®, Septra®):

[0455] Single-strength tablets 80mg / 400mg, double-strength tablets 160mg / 800mg, suspension 40mg / 200mg per 5ml.

[0402]

[0456] To prevent PCP / toxicosis, this should be started similarly when the patient begins taking oral food before discharge.

[0403]

[0457] 1 month to 12 years: 5-10 mg / kg / day TMP div BID 3 times / week daily.

[0404]

[0458] >12 years old: 80-160 mg TMP orally per day or 160 mg TMP orally three times a week.

[0405]

[0459] As is known to those skilled in the art, we monitor drugs that may increase or decrease tacrolimus and cyclosporine levels.

[0406]

[0460] We will also monitor drugs that have synergistic nephrotoxicity with tacrolimus and cyclosporine.

[0407] Acute transplant rejection treatment

[0461] In one embodiment, the subject is treated in the following manner when transplant rejection is confirmed.

[0408]

[0462] In one embodiment, if the transplant recipient is Grade 0, 1R with no hemodynamic deterioration: treatment is not necessary, and steroid withdrawal may be considered.

[0409]

[0463] If the patient exhibits grade 2R cell rejection without hemodynamic deterioration, follow the treatment regimen below:

[0464] Optimize maintenance immunosuppression.

[0410]

[0465] Methylprednisolone 15 mg / kg / day IV x 3 days (maximum 1000 mg) can be administered, either in divided doses every 12 hours or once every 24 hours.

[0411]

[0466] For refractory cellular rejection, biopsies are repeated every 1-2 weeks, and methylprednisolone and, optionally, Thymoglobulin® are administered repeatedly.

[0412]

[0467] If rejection is resolved, a biopsy will be repeated one month later (six weeks after rejection), and if the rejection is successfully treated, the previously defined biopsy protocol will be resumed.

[0413]

[0468] For refractory cell rejection, repeated use of methylprednisolone and consideration of thymoglobulin are being investigated.

[0414]

[0469] If the patient exhibits grade 2R cell rejection or higher-grade rejection without hemodynamic deterioration.

[0415]

[0470] Optimize immunosuppression at higher levels (10-15).

[0416]

[0471] Administer methylprednisolone 15 mg / kg / day IV for 3 days (maximum 1000 mg).

[0417]

[0472] Administer Thymoglobulin®.

[0418]

[0473] If there is evidence or concern of antibody-mediated rejection, plasma exchange will be performed.

[0419]

[0474] If the patient exhibits antibody-mediated rejection with or without hemodynamic deterioration:

[0420]

[0475] Administer methylprednisolone 15 mg / kg / day IV for 3 days (maximum 1000 mg).

[0421]

[0476] Plasma exchange is performed 5 times (daily or every other day).

[0422]

[0477] Administer IVIG 1-2 gm / kg IV monthly for 6 months.

[0423]

[0478] Evidence of graft dysfunction or hemodynamic deterioration justifies a biopsy (if the patient is stable) for cell-mediated rejection and antibody-mediated rejection, including C4d staining. While awaiting biopsy results, initiate methylprednisolone (SoluMedrol) and consider initiating thymoglobulin and plasmapheresis.

[0424]

[0479] Grade 3R biopsy or hemodynamic deterioration should be treated as a high-grade rejection episode.

[0425]

[0480] Methylprednisolone (SoluMedrol) 15 mg / kg / day IV x 3 days (maximum 1000 mg). Consider inotropic therapy, thymoglobulin, and / or plasmapheresis.

[0426]

[0481] Antibody-mediated rejection with or without hemodynamic deterioration. Diagnosis: Identification of C4d deposition in myocardial tissue in the presence of donor-specific antibodies in peripheral serum samples. Consider steroids and ATG ferrection for AMR.

[0427] Renal and pancreatic immunosuppression management

[0482] Exemplary induction and maintenance immunosuppression regimens for kidney and pancreas transplant procedures are described below. In one embodiment, the transplant recipient receives beratacept as maintenance therapy as part of a second immunosuppression regimen. This protocol is typically used when the recipient is Epstein-Barr virus (EBV) Ig+ and does not show DSA (donor-specific alloantibody), and regardless of absolute or calculated panel-reactive antibody (PRA), the transplant includes negative crossmatching, and the recipient is under 70 years of age with a BMI of ≤35. Further considerations include the recipient's ability to tolerate induction (if not receiving thymoglobulin by other means). The recipient should not have a history of idiopathic focal segmental glomerulosclerosis (FSGS) or a previous non-renal solid organ transplant. The protocol is for kidney transplants only.

[0428]

[0483] In one embodiment, the inducing immunosuppression regimen includes intravenous administration of methylprednisolone 500 mg during surgery. Alemtuzumab 30 mg is administered by intravenous infusion over 3 hours (2 hours after steroid administration). Beratacept 10 mg / kg is administered after pre-drug administration (TBW) (rounded to 12.5 mg).

[0429]

[0484] In one embodiment, the maintenance immunosuppression regimen includes beratacept 10 mg / kg TBW at POD4 and at the end of weeks 2, 4, 8, and 12, followed by 5 mg / kg (rounded to 12.5 mg) monthly. Sirolimus is administered at 2 mg daily, with the first trough level achieved after 2 weeks (target 8-10 ng / ml). Steroid maintenance therapy is usually not required.

[0430]

[0485] In one embodiment, in low-risk kidney transplants where the recipient is not a candidate for beratacept, the inductive immunosuppression regimen may not include an inductive immunosuppression regimen; that is, the inductive immunosuppression regimen is optional. The maintenance immunosuppression regimen may include 1000 mg of mycophenolic acid administered every 12 hours and 0.1 mg / kg / day of tacrolimus administered every 12 hours at a maximum dose of 5 mg.

[0431]

[0486] In one embodiment, if the recipient is high-risk, has ATG contraindications, is frail, is over 70 years of age, and has evidence of recent infection and recent cancer activity, the inducing immunosuppressant may include basaliximab 20 mg initiated in the operating room and on postoperative day 4 (POD). The maintenance immunosuppression regimen may include mycophenolic acid 1000 mg every 12 hours and tacrolimus 0.1 mg / kg / day, up to 5 mg every 12 hours, along with a tapering dose of steroids.

[0432]

[0487] In one embodiment involving high-risk kidney transplantation or kidney and pancreas transplantation, the following parameters are considered: past peak PRA > 30, past donor-specific antibodies (regardless of DSA, absolute or calculated PRA), second transplant with presumed immunological early graft loss, third or subsequent transplants, pediatric en bloc (adult) recipients, kidney / pancreas, pancreas alone, and high-risk or high-risk DGF biopsy. In such transplants, the inductive immunosuppressive regimen (induction therapy) may include four doses of thymoglobulin 1.5 mg / kg IBW (rounded to 25 mg) initiated in the operating room.

[0433]

[0488] In one embodiment, if the recipient has zero PRA, no autoimmune disease, and high BMI, the inducing immunosuppression regimen may include four doses of thymoglobulin 1.5 mg / kg IBW (rounded to 25 mg) initiated in the operating room. A second immunosuppression regimen (maintenance therapy) may include tapering doses of steroids, e.g., 500 mg at OR, 240 mg POD1, 125 mg POD2, 125 mg POD3, 90 mg POD4, 1000 mg of mycophenolate every 12 hours starting at POD4, and tacrolimus 0.1 mg / kg / day, up to a maximum of 5 mg every 12 hours.

[0434]

[0489] In one embodiment where a pancreas transplant is performed after a kidney transplant, the inducible immunosuppression regimen may include four doses of thymoglobulin at 1.5 mg / kg IBW (rounded to 25 mg) starting in the operating room. A second immunosuppression regimen may include steroids tapered down to a minimum of 5 mg per day, 1000 mg of mycophenolate administered every 12 hours starting at POD4, and tacrolimus administered at 0.8 mg / kg / day, every 12 hours, up to a maximum of 4 mg.

[0435]

[0490] For all immunosuppressive regimens, including mycophenolic acid administration, the initial dose may be selected based on an assessment of the patient and graft factors.

[0436] Immunosuppressive management of adult heart transplant recipients

[0491] In one embodiment, an inducible immunosuppressive regimen is typically administered. Exceptions may occasionally occur (i.e., the inducible immunosuppressive regimen is optional) if the risks of induction therapy are considered to outweigh the benefits of such therapy.

[0437]

[0492] In one embodiment, if an inductive immunosuppressive regimen is administered, the regimen may include Simulect® (basiliximab): 20 mg IV, which is given to the OR after reperfusion (cross-clamp removal) and repeated on postoperative day 4. In patients receiving inductive immunosuppressive therapy, the calcineurin inhibitor (CNI) administered in the second immunosuppressive regimen should be initiated 48 hours postoperatively, but may be further delayed depending on the patient's renal status. Steroids may be administered perioperatively, typically methylprednisolone 500 mg intravenously during induction of general anesthesia, and 500 mg IV before reperfusion (cross-clamp removal).

[0438]

[0493] In one embodiment, a maintenance immunosuppression regimen (postoperative maintenance immunosuppression therapy) may include a steroid, e.g., methylprednisolone 125 mg IV q8 hours × 3 doses (starting 8 hours after reperfusion), followed by prednisone: 0.5 mg / kg twice daily, starting on postoperative day 2, and decreasing the dose by 5 mg twice daily every two days up to 10 mg twice daily (or 20 mg daily), maintaining this dose until post-transplant day 30. A second immunosuppression regimen may also include a calcineurin inhibitor (CNI), e.g., tacrolimus / FK506 / Prograf® (preferred agent): 1 mg orally every 12 hours. The dose is titrated to a trough target level of 10–15 ng / ml. Typical dose adjustments are made after 5 doses of the calcineurin inhibitor to establish a steady state. To assess CNI toxicity, the daily level of CNI is first monitored.

[0439]

[0494] In an alternative embodiment, cyclosporine / CyA / Neoral® is administered orally at a dose of 100 mg (1.5–5 mg / kg) every 12 hours. The dose is typically titrated to a trough target level of 33 ng / ml. The maintenance immunosuppression regimen includes an antiproliferative agent such as mycophenolate mofetil (Cellcept®) 1,000–1,500 mg orally, and the dose may be adjusted to maintain a WBC count > 3,000. Alternatively, the antiproliferative agent is mycophenolate (Myfortic®) 360–720 mg orally twice daily, and the dose may be adjusted to maintain a WBC count > 3,000. In another embodiment, the antiproliferative agent is azathioprine (Imuran®) orally at a dose of 2 mg / kg daily, and the dose is adjusted to maintain a WBC count > 3,000.

[0440]

[0495] In one embodiment, the maintenance immunosuppression regimen may include sirolimus (Rapammune®) (TOR-I) administered orally at a dose of 2 mg per day. The dose is typically titrated to maintain a trough of 4–12 μg / ml.

[0441]

[0496] In one embodiment, the maintenance immunosuppression regimen may include a tapering dose of steroid, for example, reducing the dose to 15.0 mg PO / day in month 1, to 12.5 mg PO / day in month 2, to 10.0 mg PO / day in month 3, to 7.5 mg PO / day in month 4, to 5.0 mg PO / day in month 5, and to 2.5 mg PO / day in month 6. Steroid tapering should be re-evaluated according to ≥ISHLT grade 1R with evidence of myocyte necrosis. Tapering may be resumed after improvements in histological rejection guidelines.

[0442]

[0497] In one embodiment, the maintenance immunosuppression regimen may include the following add-on / adjunct: methotrexate (MTX) administered at 2.5–5 mg twice weekly for cell-mediated rejection. MTX should be considered for three or more consecutive biopsies >= Grade 1R / 2 or two consecutive biopsies >= Grade 2R / 3A.

[0443] Management of hepatic and intestinal immunosuppression in adults

[0498] In one embodiment, liver transplantation without renal insufficiency may be performed without any inductive immunosuppression regimen; i.e., such an inductive immunosuppression regimen is optional. A maintenance immunosuppression regimen may include administration of 1 g of mycophenolic acid and 2-3 mg of tacrolimus every 12 hours, along with tapering doses of steroids. Typical tapering doses of steroids are: methylprednisolone 500 mg IV during surgery, methylprednisolone 250 mg IV once 6 hours postoperatively, methylprednisolone 180 mg IV once POD1, methylprednisolone 90 mg IV once POD2, methylprednisolone 60 mg IV once POD3, methylprednisolone 30 mg IV once POD4, prednisone 20 mg PO daily POD5-14, decreasing by 2.5 mg every two weeks. In one embodiment, the tapering dose of prednisolone may be administered as follows: 20 mg per day for the first two weeks, 17.5 mg per day for weeks 2-4, 15 mg per day for weeks 4-6, 12.5 mg per day for weeks 6-8, 10 mg per day for weeks 8-10, 7.5 mg per day for weeks 10-12, 5 mg per day for weeks 12-14, and 2.5 mg per day for weeks 14-16. It is discontinued after 16 weeks. In certain circumstances, prednisolone is withdrawn at 5 mg per day and retained for one year.

[0444]

[0499] In one embodiment, kidney-sparing liver transplantation is performed when there is preoperative renal failure requiring HD or CVVHD / F. In another embodiment, kidney-sparing liver transplantation is performed when there is postoperative renal failure with a serum creatinine level >2 mg / dL in POD0-1.

[0445]

[0500] In one embodiment, the inducing immunosuppression regimen includes four doses of thymoglobulin at 1.5 mg / kg (rounded to 25 mg) every 48 hours. In one embodiment, dose reduction is performed for pancytopenia or neutropenia. If the WBC count is 2-3 or the platelet count is 30-50, administer half the dose. If the WBC count is <2 or the platelet count is <30, wait for administration.

[0446]

[0501] In one embodiment, if premedication is indicated, premedication may be administered 30 to 60 minutes before ATG infusion, consisting of acetaminophen 650 mg VT or orally, diphenhydramine 25 to 50 mg, and methylprednisolone 40 mg IV (or the above tapering may be used).

[0447]

[0502] In one embodiment, transplantation is intestinal or multi-organ transplantation, such as liver, intestine, or pancreas transplantation, if the recipient is at high immunological risk (PRA>0, pre-pregnancy or isolated intestine transplantation) or low immunological risk but high risk of infection, and the inductive immunosuppression regimen may include premedication as described above, and may also include thymoglobulin 1.5 mg / kg (rounded to 25 mg every 24 hours, total 6 mg / kg), and basiliximab 20 mg at POD0 and 4. The maintenance immunosuppression regimen may include the tapering doses of steroids as described above, as well as mycophenolic acid 1 g every 12 hours and tacrolimus 1 mg SL every 12 hours (target 12-16 mg / ml).

[0448]

[0503] In certain embodiments, tacrolimus is administered at a dose that achieves a target level of 5-8 (liver) or 12-17 (intestinal, liver-intestinal) while the patient is taking mycophenolate mofetil (Cellcept). The target may be modified if the patient is participating in a drug trial, has refusal, has worsening renal function, or is aging.

[0449]

[0504] In certain embodiments in which mycophenolate mofetil (MMF, Cellcept) is administered, the standard dose for adults is 1,000 mg every 12 hours. Dose reduction may be performed in patients with pancytopenia or neutropenia as follows: WBC 2-3 or platelet count 30-50: Consider giving half a dose; WBC < 2 or platelet count < 30: Consider waiting for administration.

[0450]

[0505] In certain embodiments, including the treatment of acute liver allograft rejection, the following treatment may be administered: methylprednisolone 500 mg IV three times daily. If liver function tests do not improve, two additional doses are given (a total of five doses of 500 mg IV daily), and repeated liver biopsies may be performed the night before POD3 or the morning of POD4.

[0451]

[0506] In certain embodiments, patients who have previously tested negative for CMV IgG and have been treated with SoluMedrol® or Thymoglobulin® should be retested for CMV IgG at the start of treatment.

[0452] Immunosuppressive management in lung transplantation

[0507] In certain embodiments, immunosuppressive management in lung grafts follows the following induction and maintenance immunosuppression regimens. The induction immunosuppression regimen may follow the induction immunosuppression regimens previously described. The maintenance immunosuppression regimen may include:

[0453]

[0508] Calcineurin inhibitors: Tacrolimus every 12 hours in doses adjusted to maintain traftacrolimus levels. See Table 5 below. [Table 5]

[0454]

[0509] Cyclosporine administered every 12 hours in a dosage adjusted to maintain trough CyA levels according to Table 6 below. [Table 6]

[0455]

[0510] The steroid dose is gradually reduced from 0 to 3 months post-transplant: 20 mg orally daily; from 3 to 6 months post-transplant: 15 mg orally daily; from 6 to 9 months post-transplant: 10 mg orally daily; and from 9 months post-transplant: 5 mg orally daily.

[0456]

[0511] Azathioprine 2 mg / kg can be administered orally daily. It is important to confirm normal TMPT enzyme levels before starting treatment and to follow the LFT / CBC guidelines. If leukopenia is observed, dosage adjustment may be considered.

[0457]

[0512] Mycophenolate mofetil (Cellcept®) can be administered twice daily at a standard dose of 1,000 mg. The standard dose for heart and lung transplants is 1,500 mg orally per day. If leukopenia is observed, dosage adjustments should be considered according to the CBC.

[0458]

[0513] Sirolimus is generally contraindicated during the first three months after transplantation due to concerns about anastomotic dehiscence. When administered, sirolimus is typically given orally at a dose of 1 mg per day, adjusted based on trough levels. For example, when administered as a third drug or for CNI sparing, the target trough level is 4–8 ng / ml, and as a CNI surrogate, the trough level is 10–15 ng / ml. [Examples]

[0459]

[0514] Example 1: Study of intrathymic variability

[0515] We studied intrathymic variability to determine whether histological test results from a portion of the thymus can be representative of histological test results from any other portion of the same thymus. The results of this test are used to determine the number of samples to be tested during both the standard release test and the process validation test.

[0460]

[0516] As previously mentioned, histological acceptance criteria were established, including areas positive for keratin AE1 / AE3 scattered throughout the tissue on days 5–9, at least one identified Hassall body, CK14 staining scattered throughout the tissue, and evaluation of observed intact nuclei.

[0461]

[0517] For this study, three thymuses were sectioned in a directional manner, and the position of each section within each thymus was tracked. The sections were cultured in a 6-well plate to enable tracking of each section. Sectioning was performed as shown in Figure 5A.

[0462]

[0518] For each thymus in the study, sections were dedicated to analysis at the following time points: baseline (day 0), day 5, day 9, day 12, and day 21.

[0463]

[0519] Five to eleven sections were cultured at each time point for each thymus. The sections were cultured according to the method described above, with the culture medium changed daily. The sections were submitted for H&E staining in the pathology laboratory and analyzed for identity, efficacy, and viability. All sections in this study met the release acceptance criteria for histological testing specified above, namely: areas positive for keratin AE1 / AE3 scattered throughout the tissue at days 5–9, at least one identified Hassar body, CK14 staining scattered throughout the tissue, and observed intact nuclei.

[0464]

[0520] In addition to evaluating whether each section met the release acceptance criteria, a more detailed re-examination of each H&E and AE1 / AE3 slide was performed by the pathologist. Several images of these slides for cultured thymic tissue lot MFG-056 are shown in Figures 6A-H. The following observations were confirmed.

[0465]

[0521] All sections derived from the same donor thymus met the acceptable criteria at each time point. In different sections, cortical and medullary regions were similar to each other. However, variations were observed in the relative proportions of cortex and medullary regions between sections.

[0466]

[0522] The differences observed between different sections derived from the same thymus as a function of culture time were primarily associated with the amount of necrosis of thymocytes (which increased with increasing culture time) and the number of surviving thymocytes (which decreased with increasing culture time).

[0467]

[0523] Based on these observations, any single section from the thymus was representative of the entire thymus. In addition, the histological appearance of the tissue section on day 5 reflects what is observed at each of the subsequent time points (days 9, 12, and 21).

[0468]

[0524] Example 2: A study of the entire thymus over time.

[0525] For this study, five thymuses were sectioned and cultured according to the SOP. On the day of sectioning, the first, intermediate, and last sections were prepared for immunohistochemistry. The remainder of each thymus was sectioned and cultured in a 6-well plate. Each thymus was designated to one of the following time points: baseline (day 0), day 5, day 9, day 12, and day 21. See Figure 7 for the thymus section at day 0, Figure 8 for the section at day 5, Figure 9 for the section at day 12, and Figure 10 for the section at day 21.

[0469]

[0526] The total number of sections from each thymus ranged from 21 to 62. Sections were cultured according to the procedure outlined above, with the culture medium changed daily. Sections were submitted for H&E staining in the pathology laboratory and analyzed for identity, validity, and viability. All sections in this study met the release acceptance criteria for histological testing, namely: areas positive for keratin AE1 / AE3 scattered throughout the tissue on days 5–9, at least one identified Hassall's body, CK14 staining scattered throughout the tissue, and observed intact nuclei.

[0470]

[0527] In addition to evaluating whether each section met the release acceptance criteria, a more detailed re-examination of each H&E and AE1 / AE3 slide was performed by the pathologist. The following observations were confirmed:

[0471]

[0528] All sections derived from the same donor thymus and tested at the same time point similarly met the acceptance criteria at that time in terms of relative cortical and medullary volume, residual thymocytes, and / or necrotic variation, as described above.

[0472]

[0529] The differences observed were relative size, shape, relative content of thymic cortex versus medulla, amount of necrosis, condensation of thymic epithelium, and the number of remaining thymic cells.

[0473]

[0530] In addition, lots from different donors tested at different time points were also qualitatively similar to each other. The differences observed were related to the amount of necrosis (which increased with increasing culture time) and the number of residual thymocytes (which decreased with increasing culture time).

[0474]

[0531] Histological examination of any single section yielded the same conclusion regarding the receptivity of the entire lot. Based on these observations, the relevant features of any single section from the thymus reflect the entire thymus. In addition, the histological appearance of the tissue section on day 5 reflects what is observed at each of the subsequent time points (days 9, 12, and 21), although further necrosis is observed at the later time points. Figure 11 is a good example showing similarity of the epithelial network as assessed by antibodies AE1 / AE3 from day 0 to day 21.

[0475]

[0532] Example 3: Study on forced degradation of thymic tissue

[0533] In this study, thymic tissue sections were processed to produce sections that were degraded or deemed inviolable. Three thymuses were used in these experiments. Control samples were taken from each thymus. The processing conditions shown in Table 7 were tested. [Table 7]

[0476]

[0534] Thermal shock was achieved by placing a 10 cm culture dish containing the sections in a Ziploc bag and placing it in a 55°C water bath. The plates were left standing on a support and not immersed. Freezing / thawing was achieved by placing the 10 cm culture dish in a -20°C freezer for 4 hours, followed by thawing at ambient temperature.

[0477]

[0535] Samples were tested for histology on days 5 and 9 in culture. Some samples were also tested on day 21. All sections in this study met the release acceptance criteria for histological testing, namely: areas positive for keratin AE1 / AE3 scattered throughout the tissue on days 5–9, at least one identified Hassall's body, CK14 staining scattered throughout the tissue, and observed intact nuclei.

[0478]

[0536] In addition to evaluating whether each section met the release acceptance criteria, a more detailed re-examination of each slide was performed by the pathologist. The following observations were confirmed:

[0479]

[0537] Samples frozen / thawed or exposed to 10x PBS showed most necrosis, but some cells still appeared intact and met the histological criteria for viability. See Figure 12 for an example of exposure to 10X PBS.

[0480]

[0538] Sections kept at room temperature, dehydrated, incubated with saline or 1% DMSO, or subjected to heat shock showed less histological change.

[0481]

[0539] The following observations were confirmed by pathologists regarding the control sample.

[0482]

[0540] Thymocytes are gradually lost as thymic tissue is cultured. However, dead cells can persist for a long time in cultured thymus because they cannot be removed by recruiting phagocytic cells. The nuclei of cells undergoing apoptotic cell death initially condense and stain more intensely (blue) with hematoxylin dye. These cells lose their membrane integrity and become necrotic because they are not phagocytosed but their energy is depleted. Nuclear lysis (lysis of the nucleus in necrotic cells) typically occurs within 2-3 days in vivo, but appears to occur more slowly during thymic culture. Therefore, it is not uncommon to see large eosinophilic (pink) enlargements of necrotic cell debris where thymocyte nuclei have undergone nuclear lysis. Some dead thymocytes retained their nuclei, which had jagged edges and altered staining properties compared to those of living cells.

[0483]

[0541] As thymocytes are depleted from the tissue, thymic epithelial cells become more visible. The three-dimensional thymic epithelial (TE) network is usually shown in a section through the light and lace arrangement of (seemingly) scattered TE cells, which are not evident in the section where connected epithelial cells and / or their connections are examined. As thymocytes are lost during culture, the three-dimensional network shrinks. This results in condensation of the remaining epithelium, with the subcapsular cortical epithelial layer becoming thicker and the medullary TE cells becoming more densely packed. The nuclei of viable TE cells are typically oval, larger than those of thymocytes, and have a nuclear membrane clearly defined by hematoxylin (blue) staining, and one or more nucleoli. These TE nuclei typically appear "open," meaning they do not stain intensely with hematoxylin. This fits the interpretation that they are alive and metabolically active because active chromatin ("euchromatin") cannot bind to the hematoxylin dye. The presence of nucleoli, the site of ribosome synthesis, in many TE cells further confirms that they are alive and metabolically active. Typical histological appearances of control sections from days 5, 12, and 21 are shown in Figures 8, 9, and 10, respectively.

[0484]

[0542] Under treatment conditions including room temperature, dehydration, 1% DMSO, and heat shock, pathologists showed that the appearance of the sections did not differ significantly from that of the control. For heat-shocked samples, pathologists confirmed that the heat treatment may have "fixed" the cells by coagulating proteins that prevent further degradation. Heat treatment is used as a fixative for tissues, including the thymus.

[0485]

[0543] Figures 12A and 12B show the histology of thymic tissue slides after exposure to forced decomposition conditions. Figure 10 shows the histological appearance of a control thymic tissue section on day 21.

[0486]

[0544] The general histological appearance of the forced-degraded tissue is similar at these points in time, but fewer residual thymocytes are observed on day 21 (Figure 12B). Representative Hassall bodies in the medullary region are indicated by arrows in Figure 12B. Representative viable-looking thymic epithelial cells are indicated by arrows in Figures 12A and 12B. The cortical region shown in Figure 12B consists almost entirely of necrotizing lymphocytes on day 21. The bar in the lower left represents 100 μm.

[0487]

[0545] Example 4: Thymic tissue batch analysis of active pharmaceutical ingredient

[0546] The batch analysis data for 10 lots of thymic tissue is shown in Table 8 below. [Table 8]

[0488]

[0547] important: (a) These lots were tested according to the specifications at the time of manufacture. The active pharmaceutical ingredient test results shown in this table also represent the final formulation test results. (b) The external specifications did not constitute evidence of tampering or damage to the container. (c) Histological assays were used for both identity and efficacy. The histological specifications (tested on days 5–9) were as follows: (d) Keratin-positive regions scattered throughout the tissue. i. At least one identified Hassall body ii. CK14 staining scattered throughout the tissue iii. Observed intact nuclei iv. Sterile and mycoplasma samples were collected on days 1, 7, and 14.

[0489]

[0548] The current control library was generated using data from 56 clinical thymuses, intrathymic variation, interthymic variation, and 8 thymuses used for time-course studies, as well as 3 thymuses that underwent forced degradation. Forced degradation samples in the library (negative controls) were those that had been degraded by freezing / thawing or exposure to 10x PBS. The complete dataset resulted in 14 distinct clusters. All forced degradation samples clustered together, while clinical or characterization samples did not cluster with forced degradation samples.

[0490]

[0549] Example 5.

[0550] The overall experimental design for Example 5 is shown in Figure 20. A schematic diagram of the surgical procedure and treatment schedule is presented. Many of the following figures and text are from a manuscript being prepared for submission for publication: Kwun J, Li J, Rouse DC, Park J, Farris AB, Turek JW, Knechtle SJ, Kirk AD, Markert ML, Cultured Thymus Transplantation Promotes Donor-specific Tolerance to Allogeneic Heart Transplants.

[0491]

[0551] The schematic diagram of the experimental design shows donor-specific tolerance induced by naive T cell rearrangement, thymic lymphocyte regeneration, and surgical insertion of a CTT. All Lewis (LW) rats were depleted of T cells via thymectomy and anti-CD5 mAb prior to cardiac transplantation and surgical insertion of a CTT. CTTs from F1 (LWxDA) rats and hearts from DA rats were transplanted into thymectomized LW recipients. Cyclosporine (CsA) was administered via an osmotic pump for 4 months post-transplant. A third-party BN heart was transplanted into the neck 2-3 months after discontinuation of CsA. Control rats underwent the same procedure, except they did not undergo surgical insertion of a CTT.

[0492]

[0552] Example 5 demonstrates that transplanted CTTs can induce tolerance to transplanted solid organs in an immunodeficient rat model, as described below. We transplanted haplomatched F1 (Lewis x Dark Agouti, LWxDA) CTTs (cultured as described below) along with angiogenically incompatible DA hearts into Lewis rats.

[0493]

[0553] Prior to the CTT transplant, the recipient underwent a thymectomy and T-cell depletion.

[0494]

[0554] Cyclosporine was administered for 4 months, starting on the day of heart transplantation. The control group did not receive CTT transplantation. Two months after discontinuation of immunosuppression, recipients who received CTT transplantation showed regrowth of naive CD4 (CD62L+CD45RC+) T cells in peripheral blood, while control rats showed none (Figure 23). Even after the generation of new thymic-transferred CD4 (CD90+CD45RC+) T cells, recipients who received CTT transplantation did not reject the DA cardiac allogeneic graft (Figure 23). Controls did not reject the DA graft due to a lack of functional T cells (Figure 23).

[0495]

[0555] To confirm donor-specific unresponsiveness, MHC-incompatible Brown Norway (BN) hearts were transplanted 180 days after initial incompatible DA heart transplantation. LW rats with F1(LWxDA) CTT transplants rapidly rejected third-party BN hearts (mean rejection time, 10 days, n=5) (Figure 27). Controls did not reject third-party hearts (n=5). CTT recipients were able to produce antibodies against third-party BN donors, rather than against DA thymic donors that exhibit humoral donor-specific tolerance (Figure 32A). Immunohistochemistry of transplanted CTT at autopsy showed functional thymic tissue (Figure 24). In summary, F1(LWxDA)CTTT administered to Lewis rats resulted in tolerance specific to allogeneic DA MHC expressed in the donor thymus, and long-term survival after DA heart transplantation was achieved after withdrawal of all immunosuppression.

[0496]

[0556] material and method Animal models

[0557] In this Example 5, the same CTT was collected and cultured from 3-day-old F1 (Lewis x Dark Agouti ray offspring (Figures 17A and 17B)) as described below, and then thymectomy was performed in a manner equivalent to the treatment of human infants with athymopathy cDGA Lewis (RT-1) as previously described. l The cells were transplanted into recipient rats. (Markert, ML, et al., 2008; Market, ML, et al., 2010).

[0497]

[0558] Lewis (RT-1l) and BN (RT-1n) rats were purchased from Charles River. DA (RT-1av1) rats were purchased from Envigo. F1 (LEW / DA, RT-1l / av1) rats were reared by the protocol staff of the Duke Breeding Core Division of Laboratory Animal Resources facility. Lewis recipients underwent thymectomy as described in Rendell VR, Giamberardino C, Li J, Markert ML, & Brennan TV, 2014, “Complete thymectomy in adult rats with non-invasive endotracheal intubation.” J Vis Exp(94).

[0498]

[0559] In short, the submandibular gland and sternohyoid muscle were separated with blunt forceps to expose the tissue covering the trachea. A 1-1.5 cm incision was made in the manubrium. Using a 7 cm ALMS-type retractor, both halves of the manubrium and sternohyoid muscle were retracted to expose the sternum. The thymus was grasped with blunt forceps and excised. The cut end of the sternum was closed with a single 3-4-0 silk suture. Two drops of 2.5 mg / ml bupivacaine were applied to the incision, and the outer layer of skin was closed with three or four 9 mm wound clips.

[0499]

[0560] All thymectomized rats were maintained on a diet containing Septra (PMI Nutrition International, LLC). To induce T-cell depletion in vivo, 1 mg of anti-CD5 mAb (OX19, BioXCell, NH) was administered intraperitoneally on days 0, 5, and 10 post-thymectomy, and cyclosporine pump suppression at 0.25 mg / kg / d was administered from day 0 (at the time of cardiac transplantation and CTTT) to 4 months. All rats were used and maintained in accordance with the guidelines and compliance of the Duke Institutional Animal Research Ethics Committee.

[0500] In vitro thymic culture and CTT

[0561] Thymuses were aseptically harvested from 3-day-old neonatal F1 (LEW / DA) rat pups, cut into four sections along the natural longitudinal seam, and transferred to a sterile nitrocellulose filter (MF-Millipore, Millipore Sigma) in a tissue culture dish containing TOM medium (Figure 17B). The thymic tissue was cultured at 37°C in a CO2 incubator with 5% CO2 for the desired length of time (5-7 days). The medium was changed daily. The thymic organ medium (TOM) consisted of 86.5% HAMS F12 (Life Technologies), 25 mM Hepes (Life Technologies), 2 mM L-gluc (Life Technologies), 10% fetal bovine serum (Life Technologies), and 1x Pen-strep (Life Technologies). On the day of transplantation, the thymic sections were rinsed with fresh medium and transplanted under the kidney capsule of Lewis rats with a single safe suture (10-0 monofilament). See Figure 17C. All operations were performed under sterile conditions in a biological safety cabinet.

[0501] Abdominal and cervical heart transplants

[0562] Completely MHC incompatible DA(RT-1 av1 ) Donor heart, thymectomy Lewis (RT-1 l The heart was transplanted into the recipient. Abdominal heart transplantation was performed using a modified technique of the method described in Schmid C, Binder J, Heemann U, & Tilney NL, 1994, "Successful heterotopic heart transplantation in rat," Microsurgery 15(4):279-281.

[0502]

[0563] In short, a donor heart was transplanted into the recipient's abdominal cavity after a short period of cold ischemia with Eurocollins solution. The donor pulmonary artery and aorta were anastomosed to the recipient's inferior vena cava and descending aorta in an end-to-end manner, using serial 9 / 0 non-absorbable monofilament sutures, as inflow and outflow vessels for circulation. Cyclosporine A (CsA) was administered via an osmotic pump (Model 2ML4, Alzet). The recipient also received approximately 2.5 mg / kg / day of cyclosporine (CsA) using an osmotic pump after thym transplantation. CsA was discontinued 4 months after thym transplantation if the test group had more than 10% naive T cells. The pump was aseptically loaded and surgically subcutaneously inserted into the recipient's back region. The osmotic pump was replaced monthly for 4 months. Complete MHC mismatch BN(RT-1) to Lewis recipients with DA heart n For third-party heart transplantation, we used a modified version of the cervical angiogenesis heart transplantation method described by Heron, et al. (Heron I., 1971, “A technique for accessory cervical heart transplantation in rabbits and rats,” Acta Pathol Microbiol Scand A 79(4):366-372).

[0503]

[0564] At 6-7 months, a third-party BN heart was transplanted into the neck. Briefly, the third-party heart was transplanted to the right side of the cervical region through a longitudinal incision from the mandible to the xiphoid process. The donor pulmonary artery and external jugular vein were anastomosed end-to-end, and the aorta was anastomosed to the right common carotid artery using a cuffing technique. The graft was monitored daily by palpation and then confirmed by laparotomy at euthanasia. Animals were euthanized on the day of rejection (cessation of heartbeat) or at a specified time.

[0504] Flow cytometry analysis and monitoring DSA

[0565] Peripheral blood was obtained from the cranial vena cava and stained with antibodies. To analyze naive and novel thymic extrusion, we used combinations of anti-rat CD3 APC (BD Biosciences), anti-rat CD4 APC-Cy7 (Biolegend), anti-rat CD8a V450 (BD), anti-rat CD45 PE-Cy7 (BD), anti-rat CD45RC-PE (BD), anti-rat CD62L FITC (BD), and anti-rat CD90 BV 510 (Biolegend). To evaluate the percentage of T, B, and NK cells, we used combinations of anti-rat TCR FITC (BD), anti-rat CD4 APC-Cy7 (Biolegend), anti-rat CD8a V450 (BD), anti-rat CD45 PE-Cy7 (BD), anti-rat CD45RA PE (Invitrogen), and anti-rat NKR-P1A-APC (Invitrogen). To distinguish between host and donor, we used a combination of anti-rat TCR APC (Biolegend), anti-rat CD45 PE-Cy7 (BD), and MHC Class I RT1Aa (Santa Cruz Biotechnology). We also used secondary goat anti-mouse IgG (Invitrogen) with unbound MHC Class I RT1Aa. Donor-specific alloantibodies (DSAs) were assessed by flow cross-matching from recipient serum samples collected sequentially from DA donors or BN third-party rats. FITC-conjugated pan-rat immunoglobulin antibody was added to the samples, washed, and incubated. T cells were stained with APC-conjugated anti-CD3. Samples were analyzed using LSR fortessa (Beckman Coulter).

[0505] autopsy

[0566] Thymic grafts and all hearts were evaluated at autopsy 8 months after CTTT, when the test group rejected the cervical BN heart. As predicted, recipient-derived T cells that did not express DA MHC appeared in the peripheral blood of thymic graft recipients (Figure 21).

[0506] Histology, immunohistochemistry (IHC), and morphological analysis

[0567] All cultured thymus and CTTT samples from the subcapsular region of the kidney were frozen using OCT (Optimalcutting Compound, Tissue Tek). Control thymic tissue was obtained from neonatal to 5-day-old rat pups. 4–5 mm sections were stained for CD3 (polyclonal, Dako), Ki-67 (clone: ​​SP6, Thermo), and CK (polyclonal, Invitrogen). IHC images were acquired using an Olympus Vanox AH-3 microscope on an Olympus DP-70 digital camera system. Explanted hearts underwent serial sectioning (5 μm) from the mid-ventricular level to the base. H&E staining was performed for routine examination and rejection grading. Graft-infiltrating T cells were evaluated with polyclonal anti-CD3 (Dako) staining. All graft slides were scanned with an Aperio ScanScope XT (Aperio Technologies, Inc., Vista, CA).

[0507] statistical analysis

[0568] The experimental results were analyzed using GraphPad Prism (GraphPad Software 7.0, San Diego, CA). Log-rank tests and Student's t-tests or Mann-Whitney U tests were used for other data regarding differences in graft survival. All data were expressed as mean ± SD. A p-value less than 0.05 was considered statistically significant.

[0508]

[0569] result

[0570] Histological analysis (Figures 18C and 18D (100x magnification) and 19C and 19D (600x magnification)) showed reduced Ki67+, CD3+ cells in the thymus after culture, similar to the changes observed after culture of thymic tissue used in patients (Markert ML, et al., 2008), “Use of allograft biopsies to assess thymopoiesis after thymus transplantation.” J Immunol 180(9):6354-6364). Similar to cultured human thymus, the thymic epithelial cell (TEC) network was preserved in rat cultured thymic tissue based on cytokeratin (CK) staining (Figure 18B (100x magnification) and Figure 19B (600x magnification)).

[0509]

[0571] As predicted, recipient-derived T cells that do not express DA MHC appeared in the peripheral blood of thym transplant recipients (Figure 21). Recipient-type T cells that gradually regrow after CTT transplantation are seen in the lower right quadrant of Figure 21 at days 26, 55, 97, and 253.

[0510] Immunohistochemical analysis of engrafted allogeneic thymus tissue

[0572] Figure 23 shows circulating T cell depletion and regrowth after thymic and cardiac transplantation. All animals showed a dramatic reduction in circulating T cells after T cell depletion. Cardiac allograft recipients with CTT insertion (blue / dashed line) showed gradual regrowth of circulating T cells. Animals without CTT insertion also showed some circulating T cells (red / dotted line). However, naive and novel thymic transplant CD4 and CD8 T cells were significantly increased in animals with CTT insertion (p<0.01), while control animals did not show circulating naive or RTE CD4 and CD8 T cells.

[0511]

[0573] Eight.5 months after transplantation, the explanted thymus showed positive cytokeratin staining (Figure 22A) and T-cell staining similar to that of the innate thymus (Figure 22B). Original magnification: 400x.

[0512]

[0574] Animals with CTT insertion showed significantly increased regrowth of naive (CD62L+CD45RC+) CD4 and CD8 T cells, as well as novel thymic transfer (RTE) T cells, in peripheral blood, while the control group without thymic transplantation showed low levels of circulating naive CD4 and CD8 T cells and no circulating RTE CD4 and CD8 T cells (Figure 23). Total circulating CD3 T cell counts did not differ significantly between groups before transplantation. As expected, LW recipients with CTT transplantation showed significantly increased numbers of circulating CD4 and CD8 T cells compared to control animals without CTT transplantation (Figure 23).

[0513]

[0575] Figure 24 shows cultured thymic tissue engrafted under the renal capsule at 180 days in recipients of cardiac allografts.

[0514]

[0576] Histologically, it showed a distinct structure separate from that of kidney tissue (original magnification, 20x). The engrafted cultured thymic tissue showed normal thymic structure (H&E), viable T cells (CD3), T cell proliferation (Ki67), and Hassar's bodies with a lace-like pattern (cytokeratin) on epithelial cells (black arrow), confirming thymic viability with thymic lymphocyte regeneration (Figure 24B). Original magnification, 200x. (Data are expressed as mean ± SD, n = 8-9 animals per group, Student's t-test, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, NS, not significant (p>0.05)).

[0515]

[0577] In addition, immunohistochemical analysis of the transplanted CTT explanted at day 180 showed normal thymic histology, viable T cells (CD3), T cell proliferation (Ki67), and a lace-like pattern of CK with Hassar's bodies forming on the TEC from the surgical insertion of the CTT (arrow) (Figure 24B). These observations confirm the viability and function (thymic lymphocyte regeneration) of the transplanted thymus in animals undergoing allogeneic heart transplantation.

[0516]

[0578] In summary, rats transplanted with CTT showed thymic lymphocyte regeneration accompanied by naive T cell development in cardiac allograft recipients.

[0517]

[0579] T cells responsive to DA donors were not expected to occur because T cells were generated in CTT-expressing DA and LW.

[0518]

[0580] We evaluated the evidence for rejection of DA hearts. Figure 25 shows that LW rats with DA heart transplants and no immunosuppressive treatment rejected the DA heart graft within 10 days (DA control, white square). However, RTE (CD90) + CD45RC + Even after T cell generation, LW recipients with surgically inserted CTTs did not reject DA cardiac allografts (n=8, black triangles) (no cessation of heartbeat). Unexpectedly, LW control animals without CTT insertions also did not reject DA cardiac grafts (n=9, inverted black triangles). Both groups showed good heartbeat quality throughout the entire study period (180 days). Since continuous graft heartbeat does not necessarily mean the absence of rejection, two recipient rats were euthanized 2 months after cessation of immunosuppression (i.e., before third-party BN cervical heart transplantation) to confirm the absence of rejection. Explanted cardiac allografts (DA hearts) from both animals showed minimal mononuclear cell infiltration (Figure 26A, with CTT insertion, and Figure 26B, without CTT insertion) and showed no signs of rejection according to the 2004 International Society for Heart & Lung Transplantation (ISHLT) grading Figure 26C.

[0519]

[0581] The Kaplan-Meier survival curve (Figure 25) showed significantly extended graft survival from animals with or without CTT and syngeneic controls (LW hearts to LW rats) compared to LW rats with DA heart transplants without any immunosuppression (DA controls). Representative scan images of explanted grafts at 180 days from animals with and without CTT are shown in Figures 26A and 26B. Images were adapted from the entire slide scan.

[0520]

[0582] ISHLT grading did not show any difference in rejection grading between cardiac allografts from recipients with and without CTT (n=3-4 per group) (Figure 26C). Mann-Whitney U test, *P<0.05, NS, not significant (p>0.05).

[0521]

[0583] Based on the rearrangement of naive T cells after CTTT, we conclude that animals with CTT lost their donor-responsive T cell repertoire, while animals without thymic transplantation did not fully rearrange those T cell populations (general low responsiveness).

[0522] Alloresponsiveness to third-party neovascular heart transplantation

[0584] To confirm that donor-specific unresponsiveness (tolerance) was achieved, in contrast to general low responsiveness, additional completely MHC-mismatched BN heart transplants were performed in both animal groups 6–7 months (180–210 days) after DA heart transplantation.

[0523]

[0585] LW rats with inserted CTTs (solid triangles; Figure 27) rapidly rejected third-party BN hearts (graft pulsation cessation) (n=5, median survival time (MST) = 10 ± 1.0 days). However, control LW animals without inserted CTTs did not reject third-party hearts, likely due to a lack of alloreactive T cells (n=6, MST ≤ 38.5 ± 8.9 days).

[0524]

[0586] Following the absence of rejection of third-party hearts, histological analysis confirmed that animals with inserted CTTs (inverted black triangles, Figure 27) showed increased mononuclear cell infiltration in the cardiac allograft (Figure 28A), while animals without inserted CTTs showed the original BN cardiac allograft (Figure 28B). Recipients with CTTs rapidly rejected the BN heart (MST = 10 ± 1.0 days) (black squares, Figure 29), while recipients without CTTs did not reject the third-party BN heart (dashed triangles, Figure 29). BN controls (black circles, Figure 29) show rejection of BN hearts by LW rats. Syngeneic controls (white circles, Figure 29) show the absence of rejection of LW hearts by LW rats. Kaplan-Meier survival curves (Figure 27) showed significant differences in graft survival. Representative scan images of explanted BN heart grafts at rejection or 46 days post-transplant are shown in Figures 28A and 28B, respectively. BN heart grafts from animals with CTT implantation showed severe mononuclear cell infiltration (Figure 28A), while BN heart grafts from animals without CTT implantation showed no signs of rejection (Figure 28B). Images were adapted from the entire slide scan.

[0525]

[0587] Histological analysis (ISHLT grading) of BN hearts explanted from rats with CTT implantation (black squares, Figure 29) showed grade 3R rejection with significantly increased inflammatory cell infiltration compared to syngeneic control or rats without CTT implantation (shaded triangles, Figure 29). ISHLT grading showed significantly higher rejection grading from BN hearts from animals with CTT compared to BN hearts from animals without CTT (n=3-5 per group). Mann-Whitney U test, *P<0.05, **P<0.01, NS, not significant (p>0.05).

[0526]

[0588] It is noteworthy that while the cervical BN heart was significantly enlarged in recipients with CTT implantation (Figure 30A), the abdominal DA heart was smaller than the congenital heart (Figure 30A). BN hearts from recipients without CTT implantation showed no increase in size compared to BN hearts from recipients with CTT (Figure 30B).

[0527] Selective T cell infiltration in a third-party heart, rather than in a DA heart that shares DA MHC with CTT.

[0589] Two conventional methods were used to define graft rejection in this rat heart transplant model: cardiac beat / arrest measurement and the ISHLT human grading system. The former is insensitive to low-grade rejection, and the latter is insensitive to high-grade rejection. As a result, inflammatory cell infiltration was measured in DA hearts from three rats at day 180 and in BN hearts at sacrificial age of 7-8 months from five rats.

[0528]

[0590] Rats treated with T-cell depletion, surgical insertion of a CTT, and CsA administered for 4 months did not show increased levels of inflammatory cell infiltration in the DA heart after T-cell regrowth (Figure 31A). DA control grafts (in immunosuppressed LW rats) showed significantly increased graft infiltration of immune cells in the DA heart compared to rats with or without a CTT. Animals with inserted CTTs showed massive inflammatory cell infiltration in third-party cardiac allografts (BN hearts) (Figure 31B). BN control grafts (in immunosuppressed LW rats) and BN grafts from recipients with CTTs showed significantly increased inflammatory cell infiltration compared to BN grafts from animals without CTTs. Rats without inserted CTTs did not show infiltration in the BN heart (shaded triangle in Figure 31B) due to their immunodeficiency.

[0529]

[0591] T cell infiltration was evaluated by immunohistochemistry, confirming selective T cell infiltration in BN (right panel, Figure 31C) hearts rather than DA (center panel, Figure 31C) hearts in animals with CTT insertions, and the absence of T cell infiltration in both hearts of animals without CTT insertions (Figure 31D). Cardiac allografts from DA and BN rats were collected in the original hearts at the time of BN heart rejection. Broadly speaking, the original and DA hearts (POD 196) did not show a dramatic increase in T cells, while the BN heart (POD 14) showed a large number of T cells in recipients with CTT insertions. Images were adapted from the entire slide scan. A total of 3–5 animals per group were analyzed, and Student's t-tests were performed: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, NS, not significant (p>0.05).

[0530]

[0592] These data confirm that, in the group that underwent CTT, T cell infiltration occurred only in third-party BN grafts and not in grafts that shared MHC(DA) with the transplanted thymus, likely due to a lack of T cell repertoire against (DA cardiac) donor antigens (due to negative selection).

[0531] Humoral response to donor antigens after thym transplantation

[0593] Anti-donor antibody responses were evaluated to determine whether allogeneic T cell unresponsiveness observed in thymic LW rats, followed by LWxDA transplantation and surgical insertion of CTTs, was associated with humoral tolerance to donor DA MHCs. Serially collected recipient serum samples were gathered and cross-matched with PBMCs from DA and BN rats. Animals that received DA or BN heart transplants without immunosuppression developed antibodies against their respective donors (DA or BN, respectively). Animals with syngeneic cardiac allografts did not produce antibodies against either DA or BN MHCs, as reported in Figure 32A (horizontally shaded peaks in the left column, DA at the top and BN at the bottom). Representative histogram plots of post-transplant donor-specific alloantibodies (anti-DA and anti-BN antibodies) as measured by T cell flow cross-matching are shown in Figure 32A. Recipients, with or without CTT, did not produce any antibodies against the DA antigen (top, middle, and right columns in Figure 32A), but animals with CTT were able to produce antibodies against the BN antigen (bottom, middle panel, Figure 32A). Serum samples from recipients of DA heart transplants without immunosuppression and from LW recipients of BN heart transplants without immunosuppression were used as positive controls (DA control and BN control) for anti-DA (top, left panel, bold line) or anti-BN antibodies (bottom, left panel, dashed line), respectively.

[0532]

[0594] Interestingly, similar to T cell hyporesponsiveness, anti-DA Ab was not detected in animals, regardless of the presence or absence of CTTT (Figure 32B). Anti-BN Ab was readily detected in animals with LWxDA with inserted CTT, but not in animals without inserted CTT (p<0.01) (Figure 32C). Animals without CTT were generally immunodeficient. Animals with CTT exhibited specific tolerance to DA.

[0533]

[0595] In summary, thymic co-transplantation resulted in long-term survival of DA heart transplants by preventing both the development of specific tolerance to allogeneic DA MHC expressed in the donor thymus, thus preventing the development of a donor-specific anti-DA T cell repertoire, and by preventing a donor (DA)-specific humoral response. Immune capacity was demonstrated in these rats by rapid rejection of third-party BN hearts and an alloantibody response against BN donor cells.

[0534]

[0596] Further support for the above treatments can be inferred from clinical experience with patients with DiGeorge's anomaly.

[0535]

[0597] Patient 1 was a child born with complete DiGeorge anomaly. He was T-cell-free at birth. The primary problem for Patient 1 was severe hypoparathyroidism, which led to numerous hospitalizations due to hypocalcemia. Patient 1 was given both a cultured thymic tissue transplant (CTT) and a parental parathyroid transplant on the same day. There were three other patients in the small clinical trial who received both thymus and parental parathyroids. Patient 1 received two transplants at 4 months of age. Although Patient 1 was T-cell-free, he was given RATGAM for immunosuppression before transplantation according to the protocol. No other immunosuppression was given. Patient 1 developed a normal proliferative T-cell response to naive T cells and mitogens. All four patients who received both thymus and parathyroids in the trial developed normal parathyroid hormone levels. Patient 1 was the only subject who was able to discontinue long-term (10 years) calcium supplementation. Of the three other patients, one died a year earlier from lung problems, and the other two, who had complete DiGeorge anomaly, had to return to calcium supplementation after about a year. Patient 1 was the only subject to have a negative mixed lymphocyte reaction (MLR) to the parent parathyroid donor at all time points. The other three subjects had a positive MLR at the start of their first assay.

[0536]

[0598] A possible explanation for Patient 1's ability to maintain parathyroid function is that Patient 1's parathyroid donor possessed an HLA-class II allele that matched either the recipient class II allele (light gray shading in the graph below) or the thymus donor class II allele (dark gray shading in the chart below).

[0537]

[0599] In patient 1, stem cells developed into thymocytes in the thymus.

[0538]

[0600] Dendritic cells from patient 1 migrate to the thymus and lack T cells that strongly bind to MHC on patient 1 DCs. Tolerance to the allele is indicated by the light gray shading. See Table 9.

[0539]

[0601] Thymic donor thymic epithelial cells also lack thymic cells that are strongly bound to them (dark gray shading). This is a mechanism of tolerance to the dark gray allele. [Table 9]

[0540]

[0602] Notably, the parathyroid donor was found to have one HLA-B and one HLA-C allele that did not match either the recipient or the thymus donor. We did not understand why the parathyroid was not rejected. However, after 10 years, the child was given a live measles / mumps / rubella vaccine. Parathyroid function was destroyed within two weeks, and patient 1 returned to calcium supplementation.

[0541]

[0603] We conclude that the live vaccine activated CD8 T cells in patient 1. One-third of the CD8 T cells exhibited intrinsic alloreactivity. These alloreactive CD8 T cells likely responded to incompatible HLA-B and HLA-C alleles in the parathyroid donor (large, bolded HLA-B and HLA-C alleles in the table).

[0542]

[0604] Data from this patient clearly demonstrate that matching for both Class I and Class II is necessary to induce long-term tolerance. In addition, the second-field mismatch (bold italics) in DRB1 and DPB1 between the parathyroid and thymus donors indicates that second-field mismatches are tolerable and can allow tolerance to form. Notably, these second-field mismatches did not lead to graft dysfunction; they were tolerable. This child is growing up healthy with good T-cell count and function, as well as normal immunoglobulin levels. However, Patient 1 continues calcium due to parental parathyroid rejection.

[0543]

[0605] The embodiments and advantages described herein are merely illustrative and should not be construed as limiting the invention. These teachings can be readily applied to other types of apparatus, experiments, and surgical procedures. Furthermore, the description of embodiments of the invention is intended to be illustrative and not to limit the scope of the claims. Many alternatives, modifications, and variations will be apparent to those skilled in the art.

[0544]

[0606] Although the present invention has been described in conjunction with its detailed description, it should be understood that the foregoing description is intended to be illustrative and does not limit the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0545]

[0607] The references discussed in this application are incorporated in their entirety by reference for their intended purpose and are evident in their context.

[0546]

[0608] All patents, patent applications, and publications cited herein are incorporated herein in their entirety by reference. The disclosures of these publications are incorporated herein by reference in their entirety.

[0547]

[0609] Any patents, patent applications, publications, and accession number disclosures cited herein are incorporated herein in their entirety by reference.

[0548]

[0610] While this disclosure has been made with reference to various embodiments, it will be apparent that other embodiments and variations thereof can be devised by those skilled in the art without departing from the true spirit and scope of this disclosure. The appended claims are intended to be construed as encompassing all such embodiments and equivalent variations.

[0549]

[0611] The foregoing specification is considered sufficient to enable those skilled in the art to carry out the embodiments. The foregoing description and examples detail specific embodiments and illustrate the best mode intended by the inventors. However, it will be understood that, no matter how much the foregoing appears in the text, the embodiments may be carried out in many ways and should be interpreted in accordance with the appended claims and their equivalents.

[0550]

[0612] References

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[0624] Li B,Li J,Hsieh CS,Hale LP,Li YJ,Devlin BH,Markert ML.2009”Characterization of cultured thymus tissue used for transplantation with emphasis on promiscuous expression of thyroid tissue-specific genes,”2009,Immunol Res.2009,44(1-3):71-83.

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[0631] Markert ML,et al.,2008),“Use of allograft biopsies to assess thymopoiesis after thymus transplantation.”J Immunol 180(9):6354-6364.

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[0634] Markert ML.2014.“Thymus Transplantation,”Stiehm’s Immune Deficiences,eds Sullivan KE&Stiehm ER(Academic Press),1st Ed,pp 1059-1067.

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[0580]

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[0581]

[0644] Abbreviation

[0645] AIRE: Autoimmune regulatory gene

[0646] Ab: Antibody

[0647] Ag: antigen

[0648] APC: Antigen presenting cell

[0649] ATG: Thymoglobulin

[0650] Bid: Twice a day

[0651] BMI: Body mass index

[0652] BSA:Body surface area

[0653] BSC: Biological Safety Cabinet

[0654] cDGA: Complete DiGeorge abnormality

[0655] CFR: Federal Regulations

[0656] CK: Cytokeratin

[0657] CNI: Calcineurin inhibitor

[0658] CTT: Allogeneic cultured post-thymic tissue-derived product

[0659] CVVHD / F: Continuous venous hemodialysis filtration

[0660] DC: Dendritic cell

[0661] DSA: Donor-specific antibody

[0662] DGF: Delayed Graft Function

[0663] EBV: Epstein-Barr virus

[0664] EU: Endotoxin Units

[0665] FBS: Fetal Bovine Serum

[0666] FDA: Food and Drug Administration

[0667] FSGS: Focal segmental glomerulosclerosis

[0668] H&E: Hematoxylin and Eosin

[0669] HD: Hemodialysis

[0670] HEPES: N-2-hydroxyethylpeperazine N'-2-ethane-sulfonic acid

[0671] HI:HI-heat inactivation

[0672] HIP: intraperitoneal

[0673] HIV: Human Immunodeficiency Virus

[0674] IBW: ideal weight

[0675] IDDM: Insulin-dependent diabetes mellitus

[0676] ISHLT: International Society for Heart and Lung Transplantation

[0677] ISO: International Organization for Standardization

[0678] LAL: Horseshoe crab slime cell lysate

[0679] mAb: Monoclonal antibody

[0680] MHC: major histocompatibility complex

[0681] MST: Mean survival time

[0682] PBMC: Peripheral blood mononuclear cells

[0683] PBS: Phosphate-buffered saline

[0684] POD: Postoperative Day 1

[0685] PRA: Panel-reactive antibodies

[0686] SL: sublingual

[0687] TBW: Total Body Weight

[0688] TC: Tissue culture

[0689] Tfh: T follicular helper

[0690] TOM: Thymic Organ Medium

[0691] USP: United States Pharmacopeia

Claims

1. A cryopreserved allogeneic cultured postnatal thymus tissue-derived product for use in a method to promote donor-specific tolerance to allogeneic solid organ grafts obtained from a donor in recipients requiring solid organ transplantation, wherein the method is (a) The step of removing the thymus of the recipient, (b) The steps of treating the recipient with an inducible immunosuppressive regimen comprising one or more immunosuppressants to deplete the recipient's T cells and / or to suppress the recipient's T cells from rejecting the transplanted solid organ, (c) The step of providing a suitable solid human organ from a donor, (d) The step of transplanting the solid human organ into the recipient, (e) The step of treating the recipient with a maintenance immunosuppressive regimen, (f) Providing cryopreserved allogeneic postnatal thymus tissue-derived products maintained in a cryopreserved allogeneic postnatal thymus tissue-derived product bank, wherein the cryopreserved allogeneic postnatal thymus tissue-derived products are processed from thymus tissue from a thymus donor expressing an HLA allele that matches the recipient's HLA allele which is not present in the solid organ graft, the donor thymus tissue is subjected to a conditioning regimen for a period of up to 21 days to produce the allogeneic postnatal thymus tissue-derived products, and the conditioning regimen for the donor thymus tissue Gregmen comprises the steps of sterile treatment of the donor thymic tissue in thymic organ medium to produce a partially T-cell depleted donor thymic tissue fragment, wherein the partially T-cell depleted donor thymic tissue fragment shows areas positive for cytokeratin (CK) staining, the presence of at least one Hassall body, and the presence of intact nuclei of thymic epithelial cells and other stromal cells on days 5 to 9 of the conditioning regimen, and the partially T-cell depleted donor thymic tissue fragment is collected as an allogeneically cultured postnatal thymic tissue-derived product. (g) A step of producing a cryopreserved (h) A step of transplanting the cryopreserved allogeneic cultured post-vivary thymic tissue-derived product into the recipient 12 to 21 days after the conditioning regimen, wherein the dose of the partially T-cell-depleted thymic tissue fragment is approximately 1,000 to 20,000 ml ２ Thymus tissue surface area / 1 m ２ The cryopreserved allogeneic cultured post-thymocyte-derived product comprises the steps of: the recipient's body surface area, the transplanted allogeneic cultured post-thymocyte-derived product inducing thymic lymphocyte neogenesis and tolerance in the recipient, wherein the solid organ transplant is a heart transplant.

2. The cryopreserved allocultured postnatal thymocyte-derived product according to claim 1, wherein the partially T-cell-depleted donor thymocyte tissue fragment shows a region positive for cytokeratin (CK14) staining on days 5 to 9 of the conditioning regimen.

3. The cryopreserved allogeneic cultured postnatal thymocyte-derived product according to claim 1, wherein the inducing immunosuppression regimen comprises an immunosuppressant selected from the group consisting of glucocorticoids, anti-thymocyte globulin (rabbit), anti-thymocyte globulin (horse), and alemtuzimab.

4. The cryopreserved allogeneic cultured post-thymocyte-derived product according to claim 1, wherein the maintenance immunosuppression regimen comprises the administration of glucocorticoids.

5. The cryopreserved alloculture post-thymic tissue-derived product according to claim 4, wherein the glucocorticoid comprises sodium methylprednisolone succinate.

6. The cryopreserved allogeneic cultured post-thymus tissue-derived product according to claim 1, wherein the maintenance immunosuppression regimen further comprises administering glucocorticoids and tacrolimus or cyclosporine, and mycophenolate mofetil, mycophenolate mofetil equivalent, or azathioprine.

7. The cryopreserved allogeneic cultured postnatal thymus tissue-derived product according to claim 6, wherein the heart transplant is a pediatric heart transplant.

8. The cryopreserved allogeneic cultured postnatal thymus tissue-derived product according to claim 1, wherein the heart transplant is an adult heart transplant in the method described above.

9. The cryopreserved allogeneic cultured postnatal thymus tissue-derived product according to claim 1, further comprising evaluating the recipient for an HLA class I or HLA class II panel-reactive antibody ("PRA") score before transplantation of the solid organ.

10. The cryopreserved alloculture post-thymocyte-derived product according to claim 1, wherein a portion of the cryopreserved alloculture post-thymocyte-derived product is surgically transplanted into the quadriceps femoris muscle of the recipient.

11. The cryopreserved allocultured postthymocyte-derived product according to claim 1, wherein the conditioning regimen is for a period of about 12 to about 21 days.

12. The cryopreserved alloculture post-thymocyte-derived product according to claim 11, wherein the conditioning period is approximately 21 days.

13. The cryopreserved allocultured post-thymocyte tissue-derived product according to claim 3, wherein the inducible immunosuppression regimen includes rabbit-derived anti-thymocyte globulin.

14. The cryopreserved allogeneically cultured postthymocyte-derived product according to claim 1, wherein the maintenance immunosuppression regime comprises one or more immunosuppressants selected from the group consisting of calcineurin inhibitors, inosine monophosphate dehydrogenase inhibitors, and azathioprine.

15. The cryopreserved alloculture postthymic tissue-derived product according to claim 14, wherein the immunosuppressant is a calcineurin inhibitor.

16. The cryopreserved allogeneic cultured postthymic tissue-derived product according to claim 14, wherein the immunosuppressant is an inosine monophosphate dehydrogenase inhibitor.

17. The cryopreserved allogeneic cultured postthymic tissue-derived product according to claim 16, wherein the inosine monophosphate dehydrogenase inhibitor is mycophenolate mofetil.

18. The cryopreserved allogeneically cultured post-thymocyte-derived product according to claim 1, wherein the maintenance immunosuppression regimen further comprises a glucocorticoid selected from the group consisting of methylprednisolone, prednisone, and prednisolone.

19. The cryopreserved alloculture postthymic tissue-derived product according to claim 14, wherein the calcineurin inhibitor is tacrolimus.

20. The cryopreserved alloculture postthymic tissue-derived product according to claim 14, wherein the calcineurin inhibitor is cyclosporine A.

21. The cryopreserved allogeneic cultured postnatal thymocyte-derived product according to claim 1, wherein the administration of one or more immunosuppressants of the maintenance immunosuppressant regimen is discontinued after naive T cells have reached 10% of the total T cells.

22. The cryopreserved allogeneic cultured postnatal thymus tissue-derived product according to claim 1, wherein the transplanted cryopreserved allogeneic cultured postnatal thymus tissue-derived product is biopsied 2 to 12 months after transplantation to evaluate for evidence of thymic lymphocyte regeneration by immunochemistry.

23. Obtaining suitable donor thymic tissue from a thymic donor, wherein the donor thymic tissue is subjected to a conditioning regimen for a period of up to 21 days, and the conditioning regimen for the donor thymic tissue comprises sterile treatment of the donor thymic tissue in thymic organ culture medium to produce a partially T-cell depleted donor thymic tissue fragment, wherein the partially T-cell depleted donor thymic tissue fragment shows positive regions for cytokeratin (CK) staining, at least one hassal A cryopreserved allocellular postnatal thymus tissue product is prepared by: obtaining the presence of corpuscles and intact nuclei of thymic epithelial cells and other stromal cells; recovering the partially T-cell-depleted donor thymus tissue fragment as an allocellular postnatal thymus tissue-derived product; cryopreserving the allocellular postnatal thymus tissue-derived product in liquid nitrogen to produce a cryopreserved allocellular postnatal thymus tissue-derived product; and maintaining the cryopreserved allocellular postnatal thymus tissue-derived product in a cryopreserved allocellular postnatal thymus tissue-derived product bank in liquid nitrogen, wherein the cryopreserved allocellular postnatal thymus tissue-derived product is prepared by: obtaining the presence of corpuscles and intact nuclei of thymic epithelial cells and other stromal cells; recovering the partially T-cell-depleted donor thymus tissue fragment as an allocellular culture postnatal thymus tissue-derived product; cryopreserved allocellular culture postnatal thymus tissue-derived product in liquid nitrogen; and The cryopreserved alloculture post-thymus tissue-derived product shows partial T cell depletion, scattered keratin AE1 / AE3-positive regions throughout the tissue, the presence of at least one Hassall body, scattered CK14 staining throughout the tissue, and the presence of intact nuclei. Cryopreserved allogeneic cultured post-thymic tissue-derived product for transplantation to recipients of heart transplants.

24. The cryopreserved allogeneic cultured postnatal thymocyte-derived product according to claim 23, wherein the donor thymocyte tissue fragment with partially depleted T cells exhibits a region positive for CK14 staining on days 5 to 9 of the conditioning regimen.

25. The cryopreserved allocultured postnatal thymocyte-derived product according to any one of claims 1 to 24, wherein the donor thymocyte tissue fragment with partially depleted T cells exhibits a region positive for CK staining in a lace-like staining pattern on days 5 to 9 of the conditioning regimen.

26. The cryopreserved allocultured postnatal thymocyte-derived product according to any one of claims 1 to 25, wherein the partially T-cell-depleted donor thymocyte tissue fragment shows a region positive for CK14 staining in a lace-like staining pattern on days 5 to 9 of the conditioning regimen.

27. The cryopreserved allocultured post-thymic tissue-derived product according to any one of claims 1 to 26, wherein the solid organ graft is obtained from a deceased donor.

28. The cryopreserved allocultured post-thymic tissue-derived product according to any one of claims 1 to 6 and 9 to 26, wherein the solid organ graft is obtained from a living donor.

29. The cryopreserved allogeneic cultured post-thymus tissue-derived product according to any one of claims 1 to 28, wherein the heart transplant is performed in combination with a kidney transplant, liver transplant, lung transplant, pancreas transplant, or small intestine or large intestine transplant.

30. The cryopreserved allogeneic cultured post-thymic tissue-derived product according to any one of claims 1 to 6, 9 to 26, and 28, wherein the heart transplant is performed in combination with a kidney transplant, a liver transplant, or a lung transplant.

31. A method for producing allogeneic cultured post-thymic tissue, wherein the method is (a) A step of culturing thymic tissue obtained from a donor in thymic organ medium for a period of 6 to 12 days, wherein the conditioning regimen for the donor thymic tissue comprises sterile treatment of the donor thymic tissue in thymic organ medium to produce a partially T-cell depleted donor thymic tissue fragment, further comprising the steps of: (a) a step of culturing thymic tissue obtained from a donor in thymic organ medium for a period of 6 to 12 days, wherein the partially T-cell depleted donor thymic tissue fragment shows, on days 5 to 9, areas positive for keratin AE1 / AE3 scattered throughout the tissue, the presence of at least one Hassall body, CK14 staining scattered throughout the tissue, and the presence of intact nuclei, and (b) The step of collecting the donor thymus tissue fragments in which T cells are partially depleted as allogeneic cultured post-vivous thymus tissue. Methods that include...

32. The method according to claim 31, further comprising the step of typing the HLA alleles of the donor.

33. The method according to claim 32, wherein the HLA alleles include HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DPB1, and HLA-DPA1.

34. (c) The step of cryopreserving the cultured thymic tissue in liquid nitrogen, thereby producing cryopreserved cultured thymic tissue, and (d) The step of maintaining the cryopreserved cultured thymic tissue in liquid nitrogen in a cryopreserved cultured thymic tissue bank for subsequent use. The method according to any one of claims 31 to 33, further comprising:

35. (e) the step of thawing the cryopreserved cultured thymic tissue. The method according to claim 34, further comprising:

36. The method according to any one of claims 31 to 35, wherein the thymic tissue from the donor shows, on the day of collection, that more than 50% of the region is positive for keratin in a lace-like staining pattern, that Hassall bodies are present, that CK14 stains in a lace-like pattern, and that more than 90% of the nuclei are intact.

37. The method according to any one of claims 31 to 36, wherein the culturing step includes replacing the thymic organ medium daily.

38. The method according to any one of claims 31 to 37, wherein the donor thymus tissue fragment with partially depleted T cells does not show Ki67 staining on day 5 or day 6.

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