WO2021034650A1 - Procédés pour déterminer la convenance d'un tissu de thymus cultivé pour l'implantation chez l'homme et procédés d'utilisation associés - Google Patents

Procédés pour déterminer la convenance d'un tissu de thymus cultivé pour l'implantation chez l'homme et procédés d'utilisation associés Download PDF

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WO2021034650A1
WO2021034650A1 PCT/US2020/046341 US2020046341W WO2021034650A1 WO 2021034650 A1 WO2021034650 A1 WO 2021034650A1 US 2020046341 W US2020046341 W US 2020046341W WO 2021034650 A1 WO2021034650 A1 WO 2021034650A1
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thymus
thymus tissue
days
donor
tissue
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PCT/US2020/046341
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Mary Louise MARKERT
Laura P. Hale
Alex TRACY
Kristin MARKS
Karin PIHEL
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Duke University & Medical Center
Enzyvant Therapeutics, Inc.
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Priority to CN202080057967.7A priority Critical patent/CN114340644A/zh
Priority to KR1020227007787A priority patent/KR20220045011A/ko
Priority to JP2022510851A priority patent/JP2022545215A/ja
Priority to AU2020332304A priority patent/AU2020332304A1/en
Priority to US16/994,061 priority patent/US20200405771A1/en
Priority to MX2022002091A priority patent/MX2022002091A/es
Application filed by Duke University & Medical Center, Enzyvant Therapeutics, Inc. filed Critical Duke University & Medical Center
Priority to EP20764236.4A priority patent/EP4017963A1/fr
Priority to CA3150732A priority patent/CA3150732A1/fr
Priority to BR112022003045A priority patent/BR112022003045A2/pt
Publication of WO2021034650A1 publication Critical patent/WO2021034650A1/fr
Priority to TW110121780A priority patent/TW202218671A/zh
Priority to IL290603A priority patent/IL290603A/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/065—Thymocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/26—Lymph; Lymph nodes; Thymus; Spleen; Splenocytes; Thymocytes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/521—Chemokines
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G01N2333/5446—IL-16
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders
    • G01N2800/245—Transplantation related diseases, e.g. graft versus host disease

Definitions

  • Biomarkers useful for determining the viability and suitability of T cell-depleted cultured pediatric thymus tissue also known as allogeneic cultured postnatal thymus tissue- derived product (or, sometimes “CTT”), for implantation and T cell reconstitution in subjects having thymus disorders, including congenital athymia and other immune system dysfunction due to thymus disorders.
  • CTT allogeneic cultured postnatal thymus tissue- derived product
  • Organ transplantation requires the preparation and harvesting of a human solid organ from a donor and transplantation into the recipient.
  • the major problem in solid organ transplantation is that the recipient is not tolerant of the donor.
  • the recipient T cells will reject the organ and the recipient B cells will develop antibodies to the organ causing its eventual failure.
  • the holy grail of solid organ transplantation is development of tolerance to the transplanted human organ by the recipient. More than 36,000 organ transplants are estimated to be performed per year in the U.S., and many more in in Europe, and other major countries. It is further estimated that there are more than 120,000 patients on waiting lists in the U.S. for organ transplants. Demand for healthy organs significantly exceeds the supply of suitable organs. In 2018, approximately 10,000 donors were identified.
  • Transplant rejection is a substantial challenge in solid organ transplantation. Transplant rejection by both T cells and B cells can lead to significant complications in organ function or even to transplant failure.
  • the 5-year graft survival for example, for heart transplants is 77.7%, for kidney transplants is 78.6%, for liver transplants is 72.8%, and for lung transplants is 53.4%.
  • MHC major histocompatibility complex
  • MHC major histocompatibility complex
  • the use of immunosuppressive regimens to manage the immunological response underlying transplant rejection has improved. However, tolerance has not been achieved and the mean survival for many organs is only 10 years.
  • Organ injury occurs primarily as a result of ischemia and hypothermia, but may also be related to reperfusion of the organ ex vivo or during implantation.
  • thymus tissue viability prior to and during the implantation procedure for allogeneic cultured postnatal thymus tissue-derived product in subjects with thymus disorders, including congenital athymia and other immune system dysfunction due to thymus disorders is an important factor in the practice of the various aspects and embodiments of the present disclosure.
  • the thymus is necessary for development of T cells that can appropriately respond to foreign antigens and pathogens, while avoiding damaging self-reactivity.
  • the thymus is large in infancy due to its need to establish the initial T cell repertoire, but soon becomes homeostatic, followed by a slow process of involution that continues throughout adulthood.
  • Work over the last two to three decades has established that while the overall output of adult thymus is decreased, the organ remains critical for producing T cells with novel specificities that can protect against infectious disease or cancer and repopulate the repertoire following immune insults such as radiation, chemotherapy, and some infections such as human immunodeficiency virus (HIV) (Gruver et al. 2007; Palmer et al. 2018; Sun et al. 2016; Wickemeyer and Sekhsaria 2014).
  • HIV human immunodeficiency virus
  • Age-related thymus involution in humans is characterized by loss of developing thymocytes and decreased numbers of thymic epithelial cells, with replacement of thymus parenchyma by adipose tissue. Determining whether the mechanisms driving these changes are thymus-intrinsic versus thymus-extrinsic is difficult to address using animal models due to constant trafficking to and from the thymus, and such questions are generally not possible to address in live humans.
  • Organ cultures of thymus tissue derived from young donors are useful for evaluating these questions, since in vitro culture of human thymus slices results in depletion of thymocytes, while generally maintaining the viability and function of the thymic epithelial and stromal cells. This is demonstrated by the ability of these slices to grow out monolayers (Markert et al. 1997b) and to facilitate T cell reconstitution when implanted into congenitally athymic recipients (Davies et al. 2017; Davis et al. 1997; Markert et al. 2004; Markert et al. 1999; Markert et al. 2007; Markert et al. 2010; Markert et al.
  • Allogeneic cultured postnatal thymus tissue-derived product has been shown to be useful for the treatment of T cell immunodeficiency (primary immune deficiency) resulting from congenital athymia, for example in the treatment of complete DiGeorge Anomaly (cDGA) associated with 22ql 1.2 deletion and CHARGE (coloboma, heart defect, choanal atresia, growth or mental retardation, genital hypoplasia and ear anomalies or deafness) syndrome associated with mutations in the chd7 (chromodomain-helicase-DNA-binding protein 7) gene and in athymic patients with forkhead box protein N1 (FOXN1) deficiency.
  • Congenital athymia is a rare, fatal condition and currently has no drug treatment options utilizing regulatory approved drug products.
  • DiGeorge Syndrome is defined as a condition in which there are variable defects in the heart, thymus and parathyroid gland. Approximately 1% of infants with DiGeorge syndrome have athymia and hence cannot make naive T cells that mature and fight infection. These infants are said to have complete DiGeorge syndrome. Without intending to be inclusive, there are subgroups of children who meet the criteria of complete DiGeorge syndrome, 22ql 1.2 deletion syndrome, CHARGE, infants of diabetic mothers, and infants with no syndromic or genetic defects. Congenital athymia may also be associated with mutations in the TBX1 or TBX2 genes.
  • Allogeneic cultured postnatal thymus tissue-derived product is a tissue-engineered product that is prepared, cultured and stored for up to 21 days (for example, a culturing regimen of about 6 to about 21 days) to produce partially T cell-depleted thymus tissue slices and which is differentiated from native thymus by a conditioning process.
  • the conditioning regimen partially depletes the donor thymocytes from the cultured thymus tissue slices.
  • Based on in vitro data a culture period between 6 and 21 days preserves the epithelial network as assessed using cytokeratin antibodies.
  • the culturing is preferably done at 37°C in a 5% CO2 incubator.
  • the culturing process significantly modifies the biological characteristics of the donor thymus tissue and constituent cells contained therein in the following manner to optimize the effective therapeutic properties of the allogeneic cultured postnatal thymus tissue-derived product slices.
  • the culturing process assures that a defined composition of the cultured cells/tissue having the pre-requisite biological characteristics is obtained in a manner suitable for surgical implantation into a subject to enable reconstitution of the subject’s immune system.
  • the culturing process results in a loss of thymocytes and relative enrichment of TECs and other stromal cells in the donor thymus tissue slices.
  • the culturing process further results in depletion of thymocytes and maintenance of TECs to enable reconstitution of the recipient’s immune system and allows tolerance to develop in the recipient to ELLA antigens in the donor thymus.
  • the culturing process is designed to deplete many of the thymocytes from the donor thymus tissue and to preserve the functional architecture of the thymic stroma (thymic epithelial cells and fibroblasts).
  • thymic stroma thymic epithelial cells and fibroblasts.
  • Common lymphoid progenitors that develop from stem cells migrate to the thymus and enter the thymus as thymus settling progenitors.
  • Markert ML et ah, 2008, “Use of allograft biopsies to assess thymopoiesis after thymus transplantation,” J Immunol 180(9):6354-6364; Markert ML, et ah, 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), which are incorporated herein by reference.
  • a new digital histology assay was developed using scanned images of H&E slides from previous clinical lots and from experimental lots of allogeneic, cultured postnatal thymus-tissue derived product. These images were analyzed for development into a quantitative release assay. The digital histology assay is described more completely in PCT/US2019/040275.
  • chemoattractant cytokines chemokines
  • chemokines chemoattractant cytokines
  • thymocyte progenitors and their CD4-/CD8- double negative (DN) progeny interact with cortical thymic epithelial cells, differentiate into CD4+/CD8+ double positive (DP) thymocytes, and then are positively selected to differentiate into CD4+ or CD8+ single positive thymocytes (Lancaster 2018) that migrate to the thymic medulla. After self-reactive cells are deleted by negative selection, the resulting naive mature T cells are released into the periphery.
  • Achieving donor-specific immune tolerance remains the ultimate immunologic goal in transplantation.
  • Most of the current approaches focus on controlling peripheral mature 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 thymus.
  • depletion e.g. alemtuzumab, thymoglobulin, etc.
  • suppression e.g. calcineurin inhibitors, basiliximab, etc.
  • the present inventors have shown that tolerance to solid organ transplants may be achieved through the implantation of allogeneic cultured postnatal thymus tissue-derived product (referred to herein also as “CTT” or as “RVT-802”), in a thymectomized recipient, to shorten the time period of use of post-transplantation immunosuppressive agents to prevent rejection of the transplanted organ.
  • CCT allogeneic cultured postnatal thymus tissue-derived product
  • RVT-802 allogeneic cultured postnatal thymus tissue-derived product
  • Tolerance induction by surgical insertion of allogeneic cultured postnatal thymus tissue-derived product is similar to tolerance induction via donor dendritic cells (“DC”) 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.).
  • DC donor dendritic cells
  • this group successfully used HLA-Class II matched/Class I mismatched donor (thymus and kidney or heart) as thymus composite tissues (thymokidney and thymoheart) with 12 days of cyclosporine (“CsA”) for transplant tolerance induction. They claimed that non-vascularized thymus did not induce tolerance in their model. More precisely, however, non-vascularized thymus that was not cultured did not engraft long term. As they indicated, the failure of engraftment of the uncultured thymus may have been due to ischemic injury in addition to alloimmunity (Yamada K, et al. , 2000).
  • DiGeorge Syndrome is defined as a condition in which there are variable defects in the heart, thymus and parathyroid gland. Approximately 1% of infants with DiGeorge syndrome have athymia and hence no T cells to fight infection. These infants are said to have complete DiGeorge syndrome. There are 4 subgroups of children who meet the criteria of complete DiGeorge syndrome, 22qll.2 deletion syndrome, CHARGE, infants of diabetic mothers, and infants with no syndromic or genetic defects. In all four groups, the infants with athymia represent a very tiny group, possibly 1% of the total children carrying the diagnosis, such as the diagnosis of 22ql 1.2 deletion syndrome.
  • Thymopoiesis 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).
  • Thymus Gland and Education of Thymocytes (Kwun, Jean, Li, Jie, Rouse, Clay, Park, Jae Berm, Farris, Alton B., Kuchibhatla, Maragatha, Turek, Joseph W. Knechtle, Stuart J. Kirk, Allan D. and Markert, M Louise, Cultured thymus tissue implantation promotes donor-specific tolerance to allogeneic heart transplants , JCI Insight . 2020 Jun 4;5(1 l):el29983. doi:
  • the thymus gland normally is located on top of the heart.
  • the thymus provides an essential microenvironment for T cell development and is critical to 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).
  • the thymus educates hematopoietic stem cells migrating from the bone marrow to the thymus gland.
  • the progenitor stem cells colonize the thymus thereby forming thymocytes.
  • the thymocytes thereafter undergo a series of maturation steps. This is evidenced by the expression of a number of observable cell surface markers appearing on the thymocytes.
  • T cells are critical for the protection of the body from infections.
  • T cells that develop in a normally functioning thymus develop a diverse set of T cell receptors (generally proteins on the surface of the cell), which enable the mature T cell to fight a wide variety of infections.
  • T cell receptors generally proteins on the surface of the cell
  • the developing T cells are instructed by the thymus not to attack the body’s normal proteins, such as insulin or parathyroid hormone (which regulate glucose and calcium levels in the blood). These instructions are carried out under the influence of the autoimmune regulator gene ⁇ AIRE gene).
  • Thymocytes present in the thymus gland, are formed from bone marrow stem cells.
  • Thymocytes are taught by thymus epithelial cells (“TECs”) and dendritic cells (“DCs”), located within the thymus, to not attack recipient major histocompatibility complex (MHC) proteins (antigens) such as HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DQA1, HLA- DPB1, HLA-DPA1 antigens.
  • MHC major histocompatibility complex
  • the HLA antigens have 2 proteins that hold a self-peptide in a groove.
  • the self-peptide could be from a thyroid protein or an insulin peptide 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.
  • TCR T cell receptor
  • the TCR is expressed on the cell surface of the T cells.
  • Each T cell expresses many copies of its unique TCR. If the TCR binds too tightly to the self peptide: MHC on a dendritic cell, the dendritic cell provides a signal to make the T cell undergo apoptosis and die. This mechanism prevents the development of autoimmunity to self.
  • the TEC can also provide a signal to thymocytes that they are binding too tightly.
  • the DC can grab bits of membrane from the TEC and present the TEC self peptide: MHC to the developing thymocyte.
  • T cells that leave the thymus are not self-reactive.
  • the T cells that leave the thymus are very variable and can recognize infections but they do not attack proteins of the body.
  • the two major constituents of thymus are epithelium and thymocytes that are produced in the thymus in the following manner.
  • Cells derived from bone marrow stem cells CLPs
  • CLPs common lymphoid progenitors
  • the CLPs migrate to the thymus as early thymic progenitors.
  • the CLPs enter the thymus in response to signals (chemokines) produced by the thymus epithelium and endothelium.
  • the CLPs differentiate into thymocytes and proliferate.
  • 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.
  • TCR T-cell receptor
  • a vast diversity of T cells develop rendering the cells capable of responding to infections throughout the life of the recipient.
  • Mixed lymphocyte reactions show tolerance of the recipient T cells in children who are treated with cultured thy
  • Self-reactive recipient thymocytes are deleted prior to exit from the thymus. This occurs by interaction of recipient thymocytes and recipient DCs that migrate to the thymus. Apoptosis is induced in recipient thymocytes that bind too tightly to the DCs as a measure to protect the body from autoimmune disease. After completion of this process, the thymocytes exit the thymus.
  • the new circulating T cells i.e., recent thymus emigrants, express the markers CD31, CD45RA and CD62L. After a few weeks, the CD31 marker is no longer expressed.
  • the T cells expressing CD45RA and CD62L are called naive T cells. These recipient T cells proliferate normally in response to mitogens. They protect the recipient from infection without having autoreactivity to self.
  • T cell immunodeficiency primary immune deficiency
  • T cell immunodeficiency due to athymia is associated with congenital disorders which prevent the development of a functional thymus, such as complete DiGeorge Anomaly (cDGA) associated with 22ql 1.2 deletion and CHARGE (coloboma, rieart defect, choanal atresia, growth or mental retardation, genital hypoplasia and ear anomalies or deafness) syndrome associated with mutations in the chd7 (chromodomain-helicase-DNA-binding protein 7) gene and in athymic patients with forkhead box protein N1 (FOXN1) deficiency.
  • cDGA complete DiGeorge Anomaly
  • CHARGE colonboma, rieart defect, choanal atresia, growth or mental retardation, genital hypoplasia and ear anomalies or deafness
  • Athymia Other genetic defects causing athymia include TBX1, TBX2, PAX1 and semaphorine 3E (SEMA3E), and Bernstock, Joshua D, Totten, AH, and Atkinson, T. Prescott, "Recurrent microdeletions at chromosome 2pl 1.2,” Bernstock, Joshua D, Totten, AH, and Atkinson, T. Prescott, JACI 145:358-367. Congenital athymia is a rare fatal condition and currently has no drug treatment options utilizing regulatory approved drug products.
  • SEMA3E semaphorine 3E
  • Allogeneic cultured postnatal thymus tissue-derived product is a tissue-engineered product. Based on disclosures in this specification and Examples, CTT is expected to be useful for the development of tolerance in a recipient receiving a transplanted solid organ.
  • the surgical administration of allogeneic, cultured postnatal thymus tissue-derived product leads to a cascade of events resulting in the development of a functional immune system.
  • T cells are educated by donor TECs and recipient DCs.
  • Donor TECs in conjunction with recipient DCs enable tolerance to the implanted donor thymus tissue, which is implanted as cultured thymus tissue slices. This is the same tolerance induction as in a normal thymus.
  • the recipient TECs in conjunction with recipient DCs lead to tolerance to self as described in this specification.
  • CTT induces donor-specific tolerance in a rat heart transplantation model.
  • the experiments reported herein used comparable CTT implantation methods that have been used clinically in subjects with congenital athymia, such as subjects afflicted by cDGA.
  • cDGA infants have essentially no naive T cells prior to surgical placement of CTT.
  • the infants developed naive T cells approximately 6 months after the surgical procedure.
  • the present invention substantiates donor thymus co-transplantation with solid organs as a method of tolerance induction with regard to the transplanted solid organ in the recipient.
  • the patient groups that would most benefit from the procedure is adults with heart failure as well as infants needing heart transplants. Since postnatal thymic tissue is present and could be removed from deceased infants, and the recipient thymus is routinely removed from infants undergoing heart transplantation, no additional procedure aside from cultured thymus tissue implantation (CTT) would be needed to transfer this approach to the clinic. Similar transplants may also be performed in adults.
  • CTT cultured thymus tissue implantation
  • Allogeneic, cultured postnatal thymus-tissue derived product is prepared, cultured and stored for up to 21 days (for example, a culturing regimen of about 6 days to about 21 days), and, on the day of implantation, placed in individual sterile cups for transport to the operating room, as described in more detail herein.
  • the CTT (cultured thymus tissue) is aseptically processed and cultured under current Good Manufacturing Practices (“cGMP”), for example, cGMPs established by the U.S. Food & Drug Administration (“FDA”), to produce partially T cell-depleted thymus tissue slices.
  • CTT is differentiated from native thymus by a conditioning process described in detail below.
  • CTT effects the normal positive and negative selection process of developing T cells in the thymus after implantation, enabling the T cells to be tolerant to both the donor thymus and the donor solid organ transplant plus recipient tissues.
  • these T cells can recognize foreign antigens in the context of recipient major histocompatibility (MHC) proteins so as to fight infection.
  • MHC major histocompatibility
  • the route of administration is by surgical implantation of CTT, in the manner described below.
  • a single administration is typically 1,000 to 22,000 mm 2 of CTT surface area per recipient body surface area (“BSA”) in m 2 .
  • the surface area is the total of all the surface areas of all cultured tissue slices.
  • the individual CTT slices are implanted in a single administration surgical procedure.
  • Recipient CLPs of the bone marrow migrate to the thymus allograft, enter as early thymic progenitors and there develop into recipient T cells.
  • the donor thymus graft provides a microenvironment in which the recipient thymocytes develop a broad repertoire of TCRs capable of recognizing pathogens.
  • Recipient bone marrow CLPs migrate to the thymus allograft where they develop into recipient T cells. Negative selection by recipient DCs that have migrated to the donor thymus results in tolerance to the recipient MHC antigens. Immunohistochemical evidence of thymopoiesis is observed in biopsies of the implanted cultured thymus tissue taken within approximately 2-3 months of transplantation. The thymopoiesis reflects the ability of the T cells to defend against and control infection, and prevent autoimmune disease.
  • Naive T cells are detected in the circulation 5-12 months post-transplantation, resulting in the ability to defend against and control infection, and the prevention of autoimmune disease.
  • Implantation of cultured thymus tissue was first shown to be beneficial in treating primary immune deficiency resulting from congenital athymia associated with conditions such as complete DiGeorge anomaly (cDGA) or forkhead box protein N1 (FOXN1) deficiency. It was discovered that replacement of defective thymus tissue with normal thymus tissue after culture (e.g. CTT and RVT-802) may also obviate the lack of tolerance observed in recipients of transplanted solid organs.
  • CTT e.g, RVT-802
  • placement of CTT will reconstitute an immune system and induce tolerance to the donor organ if the subject is first thymectomized and immunosuppressed prior to the implantation of the CTT that expresses the MHC of the donor organ.
  • Complete DiGeorge Anomaly patients may have defects in three glands that develop in the neck in the young embryo, the heart, the thymus and the parathyroid gland. Normally the heart and thymus descend into the chest and the parathyroid gland regulates calcium levels, and remain in the neck.
  • Treatment of cDGA subjects with CTT led to the survival rates at two years of age of 75% compared to a survival rate of 6% in patients treated by other modalities (unpublished data). As noted above, almost all deaths are in the first year prior to development of naive T cells. (Markert, et al., 2010). Of note, the CTT implantation does not affect the problems of the heart and the parathyroid gland that must be managed separately.
  • An aspect of the present disclosure provides methods for the surgical placement of allogeneic cultured postnatal thymus tissue-derived product in a recipient to induce tolerance to a solid organ transplant in an immunologically normal recipient.
  • Such methods comprise, consist of, or consist essentially of, removal of the thymus gland in an immunocompetent recipient followed by depleting the recipient’s T cells with an induction immunosuppressive regimen, comprising one or more immunosuppressive agent, such as with one or more antibody and/or one or more calcineurin inhibitor.
  • the induction immunosuppressive regimen is administered in a therapeutically effective amount 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 a thymus gland from a deceased donor is obtained and the solid organ is transplanted into the recipient.
  • a maintenance immunosuppressive regimen is administered for a period of time to suppress transplant rejection.
  • the thymus gland from the deceased donor is subjected to a conditioning regimen for a period up to 21 days (for example, a conditioning regimen of about 6 days to about 21 days), to aseptically process the donor thymus tissue in a thymus organ medium to produce partially T-cell depleted donor thymus tissue slices thereby comprising the allogeneic cultured postnatal thymus tissue-derived product.
  • the partially T-cell depleted donor thymus tissue slices show 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 presence of intact nuclei.
  • the allogeneic cultured postnatal thymus tissue-derived product is then surgically placed in the recipient, typically in the quadriceps muscle of the thigh.
  • the allogeneic cultured postnatal thymus tissue-derived product enables the recipient to develop naive T cells after implantation. All new T cells that develop are genetically recipient and are tolerant to both the recipient and to the donor.
  • the dosage of thymus tissue slices is about 1,000 - 22,000 mm 2 of thymus tissue surface area / recipient body surface area in m 2 Following implantation, the allogeneic cultured postnatal thymus tissue-derived product induces thymopoiesis and tolerance in the subj ect.
  • the donor would be a deceased donor.
  • the thymus would be removed from the donor at the same time that the heart is removed.
  • a heart transplantation is performed immediately with induction immunosuppression to decrease T cell numbers and suppress the remaining recipient T cells preventing them from attacking the donor heart.
  • the donor thymus is processed to form human allogeneic cultured postnatal thymus tissue-derived product that can be used for implantation to induce tolerance after a period of at least about 6 days to about 21 days of conditioning.
  • approximately half of the allogeneic cultured postnatal thymus tissue-derived product can be cryopreserved after conditioning, so that if there was a problem with later rejection of the heart necessitating the administration of high doses of steroids or other immunosuppressive agents to treat the rejection, and whereby the very high doses of steroid damage the allogeneic cultured postnatal thymus tissue-derived product, the cryopreserved allogeneic cultured postnatal thymus tissue-derived product would be available to implant after the rejection episode was controlled.
  • the donor thymus tissue matches the HLA alleles in the donor organ that are not in the recipient. All new T cells that develop are genetically recipient and are tolerant to both the recipient and to the donor.
  • a method for promoting donor-specific tolerance to an allogeneic solid organ transplant obtained from a deceased donor, in a recipient in need of a solid organ transplant comprising the steps of:
  • the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; wherein the partially T-cell depleted donor thymus tissue slices show areas positive for cytokeratin (CK) (using antibody AE1/AE3) scattered throughout the tissue, the presence of at least one Hassall body, CK14 staining scattered throughout the tissue and presence of intact nuclei; and (g) implanting the allogeneic cultured
  • CK cytokeratin
  • a method of promoting donor-specific tolerance to an allogeneic heart transplant in a recipient in need of a deceased donor heart comprises the following steps:
  • a maintenance immunosuppressive regimen comprising one or more immunosuppressive agents selected from the group consisting of a calcineurin inhibitor, an inosine monophosphate dehydrogenase inhibitor and an anti-thymocyte globulin for a period of time sufficient to prevent or suppress transplant rejection of the heart;
  • the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; wherein the partially T-cell depleted donor thymus tissue slices show 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 presence of intact nuclei;
  • step (h) cryopreserving a portion of the allogeneic cultured postnatal thymus tissue-derived product to be used in the recipient in the event that there is an early rejection episode requiring high doses of steroids that would damage the portion of allogeneic cultured postnatal thymus tissue-derived product that was implanted in step (g).
  • a method of promoting donor-specific tolerance to an allogeneic heart transplant in a recipient in need of a deceased donor heart comprises the following steps:
  • a maintenance immunosuppressive regimen comprising one or more immunosuppressive agents selected from the group consisting of a calcineurin inhibitor, an inosine monophosphate dehydrogenase inhibitor and an anti-thymocyte globulin for a period of time sufficient to prevent or suppress transplant rejection of the heart; wherein, if the post-operative condition of the recipient is too unstable to allow weaning of the glucocorticoids and safely implanting the allogeneic cultured postnatal thymus tissue-derived product in the recipient, the allogeneic cultured postnatal thymus tissue-derived product is cryopreserved to be implanted at a later time when the recipient is stable, wherein the donor thymus tissue is subjected to a conditioning regimen for a period of from about 6 to about 21 days to produce allogeneic cultured postnatal thymus tissue-derived product; further wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in
  • a method for promoting donor-specific tolerance to an allogeneic solid organ transplant obtained from a living human donor, in a human recipient in need of a solid organ transplant comprising the steps of:
  • cryopreserved allogeneic cultured postnatal thymus tissue-derived product maintained in a cryopreserved allogeneic cultured postnatal thymus tissue-derived product bank; wherein the cryopreserved allogeneic cultured postnatal thymus tissue-derived product was processed from thymus tissue from a thymus donor expressing HLA alleles matched to HLA alleles in the recipient that are not present in the solid organ transplant; wherein the donor thymus tissue was subjected to a conditioning regimen for a period from about 6 days to about 21 days; further wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T- cell depleted thymus tissue slices, wherein the thymus tissue slices show areas positive for keratin AE1/AE3 scattered throughout the tissue, the presence of at least one Hassall body,
  • a fourth aspect of the present disclosure provides a method for promoting donor- specific tolerance to an allogeneic solid organ transplant obtained from a deceased human donor, in a human recipient in need of a solid organ transplant, the method comprising the steps of:
  • cryopreserved allogeneic cultured postnatal thymus tissue-derived product maintained in a cryopreserved allogeneic cultured postnatal thymus tissue-derived product bank; wherein the cryopreserved allogeneic cultured postnatal thymus tissue-derived product is processed from thymus tissue from a thymus donor expressing HLA alleles matched to HLA alleles in the recipient that are not present in the solid organ transplant; wherein the donor thymus tissue was subjected to a conditioning regimen for a period from about 6 days to about 21 days; further wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T- cell depleted thymus tissue slices, wherein the thymus tissue slices show areas positive for keratin AE1/AE3 scattered throughout the tissue, the presence of at least one Hassall body,
  • the allogeneic cultured postnatal thymus tissue-derived product wherein the thymus, on the day of harvest, demonstrates that >50% of areas are positive for keratin in a lacy staining pattern, that Hassall bodies are present, that CK14 stains in a lacy pattern, and that >90% of nuclei are intact.
  • an allogeneic cultured postnatal thymus tissue-derived product for implantation into a subject undergoing a solid organ transplant prepared by obtaining suitable thymus tissue from a donor wherein the donor thymus tissue is subjected to a conditioning regimen for a period up to 21 days (for example, a conditioning regimen of about 6 days to about 21 days); further wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; wherein the donor thymus tissue slices show, between days 5 and 9 post-harvest, 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 presence of intact nuclei; recovering the partially T- cell depleted donor thymus tissue slices as allogeneic cultured postnatal thymus tissue-derived product
  • the thymus on the day of harvest from the donor, demonstrates that >50% of areas are positive for keratin in a lacy staining pattern, that Hassall bodies are present, that CK14 stains in a lacy pattern, and that >90% of nuclei are intact.
  • the allogeneic cultured postnatal thymus tissue-derived product is cryopreserved.
  • cryopreserved allogeneic cultured postnatal thymus tissue- derived product is maintained in liquid nitrogen for future use.
  • cryopreserved allogeneic cultured postnatal thymus tissue- derived product is maintained in a cryopreserved tissue bank.
  • the allogeneic cultured postnatal thymus tissue-derived product is prepared from suitable thymus tissue from a donor comprising HLA alleles matched to HLA alleles in a proposed recipient that are not present in the solid organ transplant.
  • the HLA alleles are: HLA-A, HLA-B, HLA-C, HLA-DRBl, HLA- DQB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA- DPB1, and HLA-DPAl.
  • a cryopreserved allogeneic cultured postnatal thymus tissue-derived product prepared by method comprising the steps of: (a) obtaining suitable thymus tissue from a donor; (b) typing HLA alleles: HLA-A, HLA-B, HLA-C, HLA-DRB 1 , HLA-DQB 1, HLA-
  • the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T- cell depleted donor thymus tissue slices; further wherein the donor thymus tissue slices show, on days 6 to 21, 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 presence of intact nuclei upon completion of the conditioning regimen;
  • the thymus on the day of harvest, demonstrates that >50% of areas are positive for keratin in a lacy staining pattern, that Hassall bodies are present, that CK14 stains in a lacy pattern, and that >90% of nuclei are intact.
  • cryopreserved allogeneic cultured postnatal thymus tissue- derived product in liquid nitrogen is held for future use by the recipient.
  • a method of preparing the donor thymus for implanting into a recipient subject is provided.
  • Such methods comprise, consist of, or consist essentially of culturing the donor thymus for up to about 5 days, up to about 6 days, up to about 7 days, up to about 8 days, up to about 9 days, up to about 10 days, up to about 11 days, up to about 12 days, up to about 13 days, up to about 14 days, up to about 15 days, up to about 16 days, up to about 17 days, up to about 18 days, up to about 19 days, up to about 20 days, or up to about 21 days, and then surgically placing the cultured thymus tissue into the recipient, as further described herein.
  • a culture period between about 6 and about 21 days results in good function.
  • the tissue is typically cultured for about 6 to about 21 days, and then cryopreserved.
  • an allogeneic cultured postnatal thymus tissue-derived product for implantation into a subject undergoing a solid organ transplant manufactured by the method of subjecting thymus tissue from a suitable donor to a conditioning regimen for a period up to 21 days (for example, a conditioning regimen of about 6 days to about 21 days); wherein the conditioning regimen for the allogeneic cultured postnatal thymus tissue-derived product comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T-cell depleted thymus tissue slices, wherein the thymus tissue slices show 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 presence of intact nuclei.
  • CTT allogeneic cultured postnatal thymus tissue-derived product
  • the donor thymus on the day of harvest, demonstrates that >50% of areas are positive for keratin in a lacy staining pattern, that Hassall bodies are present, that CK14 stains in a lacy pattern, and that >90% of nuclei are intact.
  • the recipient’s thymus is obtained by surgery.
  • the recipient’s thymus is obtained by robotic surgery.
  • the recipient’s thymus is obtained by thorascopic surgery.
  • the solid organ is a portion of a whole organ.
  • the method of the first to fourth aspects further comprises the step of cryopreserving peripheral blood mononuclear cells from the deceased donor for future use in a mixed lymphocyte reaction to demonstrate cellular tolerance.
  • the mixed lymphocyte reaction to demonstrate cellular tolerance is performed using peripheral blood mononuclear cells from the recipient and cryopreserved peripheral blood mononuclear cells from the donor following the implantation of allogeneic cultured postnatal thymus tissue-derived product in accordance the implantation procedure of CTT in this specification.
  • the mixed lymphocyte reaction to demonstrate cellular tolerance is performed with peripheral blood mononuclear cells from the recipient and cryopreserved peripheral blood mononuclear cells from the donor about 6 to 12 months following the implantation of allogeneic cultured postnatal thymus tissue-derived product.
  • the mixed lymphocyte reaction to demonstrate cellular tolerance is performed 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 total T cells in the recipient.
  • the implanted allogeneic cultured postnatal thymus tissue-derived product induces thymopoiesis in the subject within 12 months following the implantation of allogeneic cultured postnatal thymus tissue-derived product.
  • the development of tolerance is determined by a mixed lymphocyte reaction performed with cryopreserved peripheral blood mononuclear cells from the deceased donor and T cells from the recipient.
  • humoral tolerance is determined by the development of humoral immunity and the absence of donor reactive antibodies.
  • the solid organ transplant is a heart transplant, a kidney transplant, a liver transplant, a lung transplant, a heart/lung transplant, a pancreas transplant, an intestine transplant, a stomach transplant, an abdominal wall transplant, a craniofacial transplant, a scalp transplant, a penile transplant, a uterus transplant, a unilateral or bilateral upper limb transplant, a unilateral vascularized composite allograft, or combination thereof.
  • the method further comprises evaluating the recipient for HLA-Class I and HLA-Class II panel reactive antibodies (“PRA”) score prior to transplanting the solid organ.
  • PRA panel reactive antibodies
  • the solid organ transplant is a heart transplant.
  • the solid organ transplant is a pediatric heart transplant.
  • the solid organ transplant is an adult heart transplant.
  • the method further comprises evaluating the recipient for HLA-Class I and HLA-Class II panel reactive antibodies (“PRA”) score prior to transplanting the solid organ.
  • PRA panel reactive antibodies
  • recipients with HLA antibodies are cross-matched with potential donors.
  • recipients with HLA antibodies are virtually cross-matched with UNET.
  • the method will further comprise the step of performing plasmapheresis in the operating room at the time of solid organ transplant in the recipient.
  • the method will further comprise the step of performing an actual prospective donor cross-match and performing plasmapheresis in the operating room at the time of solid organ transplant in the recipient. Typically, transplants are not performed under these circumstance because of poor success rates.
  • the method further comprises the step of evaluating recipients with HLA antibodies by cross-matching virtually with UNET.
  • the method further comprises performing plasmapheresis in the operating room at the time of solid organ transplant in the recipient if the HLA panel reactive antibodies have a score >20%.
  • the method further comprises performing an actual prospective donor cross-match and performing plasmapheresis in the operating room at the time of solid organ transplant in the recipient if the HLA panel reactive antibodies have a score >70%.
  • the solid organ is HLA- matched, in for instance, from a living related donor of the kidney, partial liver and partial intestine transplants to the recipient.
  • the solid organ is HLA- mismatched.
  • the solid organ is HLA matched.
  • the HLA match is determined by typing HLA alleles: HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-
  • the solid organ transplants are ABO compatible.
  • the solid organ is HLA- mismatched.
  • HLA-mismatched is determined by typing HLA-A, HLA-B, HLA-C, HLA-DRBl, HLA-DQB1, HLA-DQA1, HLA-DPBl, HLA-DPAl in the donor and the recipient.
  • the cultured thymus tissue slices are surgically implanted into the quadriceps thigh muscle of the subject.
  • the cultured thymus tissue slices are surgically implanted into the body of the subject in an area other than the quadriceps.
  • a portion of the allogeneic cultured postnatal thymus tissue-derived product is surgically implanted into the quadriceps thigh muscle of the recipient.
  • the conditioning regimen is for a period of about 6 days to about 21 days.
  • the conditioning period of the donor thymus tissue is about 5 days to about 21 days, or about 5 days, or about 6 days, or about 7 days, or about 8 days, or about 9 days, or about 10 days, or about 11 days, or about 12 days, or about 13 days, or about 14 days, or about 15 days, or about 16 days, or about 17 days, or about 18 days, or about 19 days, or about 20 days, or about 21 days.
  • the induction immunosuppressive regiment comprises an induction immunosuppressive agent selected from the group of glucocorticoid, anti-thymocyte globulin (rabbit), anti -thymocyte globulin (equine), and alemtuzimab.
  • an induction immunosuppressive agent selected from the group of glucocorticoid, anti-thymocyte globulin (rabbit), anti -thymocyte globulin (equine), and alemtuzimab.
  • the ATG is antithymocyte globulin (rabbit).
  • the induction immunosuppressive regimen comprises administration of a glucocorticoid.
  • the glucocorticoid comprises methylprednisolone.
  • the glucocorticoid is methylprednisolone sodium succinate.
  • methylprednisolone sodium succinate is administered intravenously at no greater than 4 mg/kg/day.
  • the induction immunosuppressive regimen comprises rabbit-derived anti-thymocyte globulin.
  • the rabbit-derived anti -thymocyte globulin is administered intravenously in a dose of about 1.5 mg/kg.
  • the anti -thymocyte globulin is administered daily for four days.
  • the ATG is equine derived ATG.
  • the induction immunosuppressive regimen comprises basiliximab.
  • the basiliximab is administered at a dose of 10 mg intravenously for recipients less than 35 kg in body weight.
  • the basiliximab is administered at a dose of 20 mg intravenously for recipients more than 35 kg in body weight.
  • the second immunosuppressive regimen comprises one or more immunosuppressive agent selected from the group consisting of a glucocorticoid, calcineurin inhibitor, an inosine monophosphate dehydrogenase inhibitor, azathioprine, and anti -thymocyte globulin (“ATG”).
  • immunosuppressive agent selected from the group consisting of a glucocorticoid, calcineurin inhibitor, an inosine monophosphate dehydrogenase inhibitor, azathioprine, and anti -thymocyte globulin (“ATG”).
  • the immunosuppressive agent of the maintenance immunosuppressive regiment is anti -thymocyte globulin (ATG).
  • ATG is administered intravenously at a dose of about 1.5 mg/kg for a period of 3-14 days starting with administration in the operating room.
  • the anti -thymocyte globulin is administered daily for 3-14 days at about 15 mg/kg/day by intravenous administration.
  • the first immunosuppressive regimen comprises alemtuzumab.
  • the alemtuzumab is administered at a dose of about 0.25 mg/kg for 4 days intravenously for recipients less than 35 kg in body weight. In another, embodiment the alemtuzumab is administered at a dose of about 3 to 20 mg for 4 days intravenously for recipients more than 35 kg in body weight.
  • the second immunosuppressive regimen comprises one or more immunosuppressive agent selected from the group consisting of a calcineurin inhibitor, and inosine monophosphate dehydrogenase inhibitor, or azathioprine.
  • the immunosuppressive agent of the maintenance immunosuppressive regimen is a calcineurin inhibitor.
  • the immunosuppressive agent of the maintenance immunosuppressive regimen is an inosine monophosphate dehydrogenase inhibitor.
  • the immunosuppressive regimen comprises an inosine monophosphate dehydrogenase inhibitor, for example, mycophenolate mofetil.
  • mycophenolate mofetil is administered intravenously in a dose of about 15 to about 25 mg/kg.
  • the mycophenolate mofetil is administered intravenously two to three times a day.
  • inosine monophosphate dehydrogenase inhibitor is mycophenolic acid.
  • the mycophenolic acid is administered at a dose of about 25 to about 50 mg/kg in 2 or 3 divided doses.
  • the mycophenolic acid is administered at a dose of about for children about 400 mg/m 2 /dose twice daily with a maximum dose 720 mg, or BSA 1.19 to 1.59 m 2 about 540 mg twice daily, or for BSA > 1.58m 2 about 720 mg twice daily.
  • the mycophenolate mofetil is administered for children at a dose of about 15 to about 25 mg/kg/dose twice a day or for adults about 1500 mg orally or intravenously twice daily and adjusted for a WBC of >3500.
  • the second immunosuppressive regimen may further comprise a glucocorticoid selected from the group consisting of methylprednisolone, prednisone and prednisolone. In an embodiment, the dose of glucocorticoid is kept below 4mg/kg/day.
  • the glucocorticoid is administered in a tapered dosage reduction, as described elsewhere in the present disclosure.
  • the calcineurin inhibitor is tacrolimus. In another embodiment, the calcineurin inhibitor is cyclosporine A.
  • the administration of the second immunosuppressant regimen is weaned after naive T cells reach 10% of total T cells.
  • the second immunosuppressant regimen is weaned after implantation of allogeneic cultured postnatal thymus tissue-derived product.
  • a potentially important biomarker for thymocyte content of cultured thymus slices is L- selectin.
  • Another important biomarker for thymic epithelial cell viability and function relies on the secretion of the chemokine CCL21 6Ckine.
  • Figs. 50 and 56 Numerous additional potential biomarkers are set forth in Figs. 50 and 56. Of particular interest are the biomarkers set forth on Fig. 50, namely L-selectin, CCL21, CXCL16, M-CSF, galectin-7, CCL11, IL-16 and CXCL12. Also, of particular interest, are biomarkers set forth on Fig. 56, particularly biomarkers having a P-value of less than 0.05.
  • biomarkers would include: L-selectin, CXCL16, M-CSF, CCL21/6Ckine, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF-AA, CXCL12/SDFF-la, CCL20 (MIP-3a), IL-2Ra, ICAM-3, LIGHT, IGFBP-1, BCMA, EGF R, uPAR (CD87), MIP-lb, PIGF, PF4, CCL11/Eotaxin, HVEM, IGFBP-6, IL-6R. IL-12p40, RANTES, MICA, GCP-2, OPN, ALCAM, NRG1-B1, CEACAM-1, IL-lb, DKK-1 and ANG-1.
  • Osteopontin encoded by the SPP1 gene
  • This cytokine is increased during thymic stress and increased levels are associated with thymic atrophy (decrease in thymocyte number), decrease thymocyte numbers is a desirable state for cultured thymus (Wang 2009; Gridley 2013).
  • OPN is required to make corticosteroids, which have been well-established to induce thymocyte apoptosis.
  • CCL11 This chemokine is made by medullary TE (Bunting 2011). It was initially named for its ability to attract eosinophils and we have shown that eosinophil infiltrates may be prominent in thymus tissues with active thymopoiesis (Flores 1999). However, eotaxin has also been shown to serve as a chemoattractant for both double-positive (DP) and single positive (SP) human thymocytes (Bunting 2011).
  • DP double-positive
  • SP single positive
  • Urokinase receptor also known as urokinase plasminogen activator receptor
  • Urokinase receptor is expressed in both soluble and membrane-bound forms based on alternative splicing. It aids in local degradation of extracellular matrix. It has been shown to be expressed in human thymus and interestingly, by migrating epidermal keratinocytes (EK) at the edge of a wound (Loughner 2016). This latter characteristic is most interesting since TE cells mirror EK in expression of many genes (Patel 1995). Progressive increases in secretion by cultured thymus slices may reflect the activation of TE and thus be a marker for TE outgrowth following implantation.
  • EK epidermal keratinocytes
  • CXCL12 (SDF-la): This chemokine had an expression pattern that was different than the other analytes, in that it was only detected during the final 1/3 of the culturing period. First detectable on days 13-15, it rose linearly (in the In plots) to a much higher level over the next week in culture. CXCL12 has been documented to be produced by subcapsular cortical and medullary TE (Bunting 2011; Hernandez -Lopez 2002; Zaitseva 2002), but also can be made by thymic fibroblasts and endothelial cells present within the thymus.
  • CXCL12 has been shown to recruit B cells and antigen-presenting cells (APC) to the thymus (Weiss 2003), which is expected to be important in generation of full thymic function. It is also involved in localization of thymocyte subsets within the thymus and it enhances thymocyte proliferation to IL-7 (Hernandez-Lopez 2002). Of note, antibodies that neutralize CXCL12 have been shown to decrease thymopoiesis in human thymus organ cultures in vitro and addition of CXCL12 increases thymopoiesis in these cultures (Hernandez-Lopez 2002).
  • Additional biomarkers that may reflect thymocyte presence include L-selectin. This molecule is expressed at high levels on developing and naive T cells. It is released from the cell surface when thymocytes are cultured. Usually rapidly re-expressed when shed by healthy cells in vivo (Fitzhugh 2008), progressively decreased levels of shedding likely reflects the progressive loss of thymocyte viability because they normally do not re-express this molecule on their surface during culture (A. Macintyre, unpublished data).
  • Another biomarker that may reflect thymocyte presence is IL-16. This cytokine was included since its pattern (as decribed below) corresponded with that hypothesized for thymocytes. This biomarker is known to be made by lymphocytes.
  • CCL20 MIP-3a
  • IGFBP-1 Another biomarker that may reflect thymocycte presence is IGFBP-1.
  • IGFBP2 - 6 are known to be expressed by thymic epithelium, which does not express IGFBP-1 (Gosteli-Peter 1994; Ketcha 1999).
  • thymocyte-derived biomarkers that showed initially high levels that then decreased with time, a hypothesized characteristic of thymocyte-derived biomarkers, which, based on the data in the Examples and the literature, correlate with the presence of viable thymocytes: L-selectin, IL-16, MIF. CCL20 (MIP-3a), and IGFBP-1. Decreases in these biomarkers over time is consistent with our more qualitative observations that T cells are depleted while the thymus tissue slices are in culture.
  • CCL21 has been shown to be expressed by thymic epithelial cells (Lkhagvasuren et al. 2013) and to be functionally important due to its chemotactic activity for thymocyte precursors (Liu et al. 2005), as well as for thymocyte migration within the thymus (Hu et al. 2015).
  • This chemotactic property for thymocytes may be a critical determinant for successful immune reconstitution of the recipients following implantation of cultured thymus tissue, as described herein.
  • Fig. 50 examples include CC25(TECK), osteopontin (OPN), uPAR (CD87), MIF, CCL20 (MIP-3a) and IGFBP-1. Study of additional analytes whose release may reflect the viability and/or activation of critical cell types in human thymus may lead to clinically and mechanistically important insights, particularly regarding responses to thymocyte loss.
  • CCL25 Although detectable levels of TECK were only present in a few samples of supernatant late in the culture period of 2 of the 3 thymus cultures examined, it is of interest since this chemokine has been shown to be chemotactic for thymocytes (Liu 2005). It is known to be expressed by thymic dendritic cells (DC) and by both FoxNl+ and FoxNl- TE cells (Bunting 2011). However, its activity does not appear to be critical for thymus development based on studies of mice in which CCR9, the sole receptor for this chemokine, was deleted (Wurbel 2001).
  • Biomarkers that reflect the presence and function of thymic epithelium also can provide critical mechanistic information.
  • Studies set forth in the Examples focused on CCL21, since the microarray screen indicated that this chemokine began to be secreted into the media at high levels soon after the start of culturing.
  • CCL21 was previously shown to be expressed by thymic epithelium and to be chemotactic for thymocytes and their precursors (Liu et al. 2005).
  • Our studies showed that expression of CCL21 could also be readily quantitated by enzyme immunoassay. Immunohistochemistry confirmed CCL21 expression by TECs in cultured as well as non-cultured thymus, with strongest expression in the medullary and subcapsular cortical thymic epithelium.
  • CCL21 immunoreactivity of thymus slices did not necessarily increase as CCL21 secretion increased during culture. This may reflect that the additional CCL21 produced is secreted rather than being retained in the cytoplasm where it can be detected via immunohistochemistry.
  • CCL21 is a transcriptionally regulated, high turnover molecule with a short half-life (Dudal et al. 2015), so the positive immunohistochemical reactivity observed represents cells that are actively producing this chemokine.
  • CCL21 as a secreted biomarker that reflects the viability and function of TECs is also important clinically with regards to thymus transplantation.
  • Most established methods that can assess the quality of tissue to be implanted e.g. flow cytometry, immunohistochemistry, gene expression analysis) destroy the samples during analysis. In addition to decreasing the amount of tissue available for eventual implantation, such results are subject to sampling error since the slice(s) tested is not part of the slices that are eventually implanted.
  • assay of pooled spent media for CCL21 can integrate across all slices in a lot derived from any given thymus donor, providing a non-destructive picture of overall lot quality.
  • chemokine CXCL12 differed from most other analytes in the screening, in that it became detectable in conditioned media relatively late during the culturing period (Fig. 50H). First detectable on days 13-15, it rose linearly (in the In plots) to a much higher level over the next week in culture.
  • CXCL12 has been documented to be produced by subcapsular cortical and medullary TECs, but may also be made by thymic fibroblasts and endothelial cells present within the thymus (Bunting et al. 2011; Hernandez-Lopez et al. 2002; Zaitseva et al. 2002).
  • CXCL12 recruits B cells and antigen-presenting cells to the thymus (Weiss et al. 2013), which is expected to be important in generation of full thymic function. CXCL12 is also involved in localization of thymocyte subsets within the thymus and it enhances thymocyte proliferation to IL-7 (Hernandez-Lopez et al. 2002). Of note, antibodies that neutralize CXCL12 have been shown to decrease thymopoiesis in human thymus organ cultures in vitro and addition of CXCL12 increases thymopoiesis in these cultures (Hernandez-Lopez et al. 2002).
  • CXCL16 and CCL11 are likely biomarkers for assessing the viability and function of cultured thymus, since they also increase as thymocytes are lost during thymus organ culture. Both CXCL16 and CCL11 have previously been shown to be made by TECs (Bunting et al. 2011).
  • CCL11 was originally named eotaxin for its ability to attract eosinophils. We previously showed that eosinophil infiltrates may be prominent adjacent to thymus tissues with active thymopoiesis (Flores et al. 1999), although CCL11 levels were not directly measured in those studies. However, CCL11 was subsequently also shown to serve as a chemoattractant for both double-positive and single-positive human thymocytes (Bunting et al. 2011). Evidence for a specific role of CXCL16 in thymopoiesis is less clear.
  • thymus tissues from younger donors continued to express more CD3 epsilon and CD1A mRNAs and less keratin8 (KRT8) and keratin 14 (KRT14) mRNAs than thymus from older adults.
  • KRT8 keratin8
  • KRT14 keratin 14
  • CCL21 and CXCL12 are markedly increased for tissues derived from donors > 18 years, a timeframe when TEC content and active thymopoiesis is decreased compared to younger donors.
  • CCL21 and CXCL12 both increase as numbers of thymocytes decrease in both in vitro in thymus organ cultures and in vivo during aging raises the possibility that induction of these chemokines is part of homeostatic mechanisms that attempt to counter the decreased thymocyte numbers through enhanced recruitment of T cell precursors.
  • Increased secretion of thymocyte-attracting chemokines by thymocyte-depleted cultured thymus slices would be expected to enhance their colonization and ability to result in immunoreconstitution, as is observed when such slices are implanted into athymic infant recipients (Markert et al. 2008).
  • abundant secretion of CCL21 and CXCL12 may provide less benefit during aging, if the availability of thymocyte precursors or other critical aspects of the thymic microenvironment are limiting.
  • T cell-depleted cultured pediatric thymus must differ substantially from aged adult thymus in other ways, since implantation of T cell-depleted cultured pediatric thymus into athymic recipients results in immune reconstitution and protection from infections, whereas aged adults with involuted thymus are more vulnerable to infections than younger adults with more robust thymus function.
  • Fig. 56 The results presented here in Fig. 56 provide a rich source of additional molecules and pathways as potential biomarkers to model some aspects of human thymus aging in vitro using cultured infant thymus. This is important, since infant thymus is typically more readily available for research given the need to remove a portion of thymus from most infants to properly expose the operative field for corrective cardiac surgery.
  • Infant thymus tissue is typically less readily available, since it is generally not necessary to remove thymus tissue to provide access for many types of cardiac surgery common in adults.
  • any adult thymus tissue removed is not typically made available for research since it appears less organoid and grossly resembles fat.
  • thymus tissue would potentially be cultured and used to co-transplant with a solid organ if tolerance is desired in adult solid organ recipients.
  • the biomarkers also are useful in determining the suitability, functionality and viability of allogeneic, cultured postnatal thymus tissue-derived product derived from adult donors.
  • Thymus production of the thymocyte chemoattractants CCL21, CXCL16, CXCL12 and CCL11 increase as thymocyte content decreases. This suggests that thymocyte loss may activate homeostatic mechanisms that attempt to counteract potential atrophy, although ultimately unsuccessfully in the setting of aging, future studies to more fully elucidate these mechanisms will be useful for understanding and potentially reversing mechanisms that drive age-related thymus involution and may help to enhance thymus-driven immune reconstitution at all ages.
  • a method of producing an allogeneic cultured postnatal thymus tissue-derived product suitable for implantation into a human comprising the steps of subjecting donor thymus to a conditioning regimen for a period from about 6 to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; further comprising detecting increasing levels of CCL21 in the thymus organ medium during the course of the conditioning regimen; and recovering the partially T-cell depleted donor thymus tissue slices as allogeneic cultured postnatal thymus tissue-derived product suitable for implantation.
  • the method further comprises the step of cry opreserving the allogeneic cultured postnatal thymus tissue-derived product in liquid nitrogen for future implantation.
  • the method further comprises detecting decreasing levels of L-selectin in the thymus organ medium during the course of the conditioning regimen.
  • the method comprises detecting one or more of CCL21, CXCL12, CXCL16 or CCL11 in the thymus organ medium during the course of the conditioning regimen.
  • the method comprises detecting increasing levels of one or more of CXCL12, CXCL16 or CCL11 in the thymus organ medium during the course of the conditioning regimen.
  • the method comprises detecting increasing levels of CXCL12 in the thymus organ medium during the course of the conditioning regimen.
  • the method comprises detecting increasing CXCL16 in the thymus organ medium during the course of the conditioning regimen.
  • the method comprises detecting increasing levels of CCL11 in the thymus organ medium during the course of the conditioning regimen.
  • the method comprises detecting one or more of M-CSF, galectin-7 or IL-16 in the thymus organ medium during the course of the conditioning regimen. [00185] In an embodiment of the aspects and embodiments of the present disclosure, the method comprises detecting decreasing levels of one or more of M-CSF, galectin-7 or IL-16 in the thymus organ medium during the course of the conditioning regimen.
  • the method comprises detecting decreasing levels of M-CSF in the thymus organ medium during the course of the conditioning regimen.
  • the method comprises detecting decreasing levels of galectin-7 in the thymus organ medium during the course of the conditioning regimen.
  • the method comprises detecting decreasing levels of IL-16 in the thymus organ medium during the course of the conditioning regimen.
  • the conditioning regimen is for a period of five days, or six days, or seven days, or eight days, or nine days, or 10 days, or 11 days, or 12 days, or 13 days, or 14 days, or 15 days, or 16 days, or 17 days , or 18 days or 19 days, or 20 days, or 21 days; or for a period of 5-6 days, or 5-7 days, or 5-8 days, or 5-9 days, or 5-10 days, or 6 to 7 days, or 6 to 8 days, or 6 to 9 days, or 6 to 10 days, or 6 to 11 days, or 6 to 12 days, or 6 to 21 days, or 7 to 21 days, or 8 to 21 days, or 9 to 21 days, or 10 to 21 days, or 11 to 21 days, or 12 to 21 days, 13 to 21 days or 14 to 21 days, or 15 to 21 days, or 16 to 21 days or 17 to 21 days or 18 to 21 days, or 19 to 21 days, or 20 to 21 days.
  • the levels of CCL21 approximate the levels in Fig. 50E, and/or the levels of L-selectin approximate the levels in Fig. 50A, and/or the levels of M-CSF approximate the levels in Fig. 50B, and/or the levels of galectin-7 approximate the levels in Fig. 50C, and/or the levels of IL-16 approximate the levels in Fig. 50D, and/or the levels of CXCL16 approximate the levels in Fig. 50F, and/or wherein the levels of CCL11 approximate the levels in Fig. 50G, and/or the levels of CXL21 approximate the levels in Fig. 50H.
  • the method further comprises the step of determining in the donor thymus tissue slices between days 6 and 21, preferably between days 6 and 9 of the conditioning regimen areas positive for keratin AE1/AE3 scattered throughout the donor thymus tissue slices the presence of at least one Hassall body, CK14 staining scattered throughout the donor thymus tissue slices and the presence of intact nuclei.
  • the method further comprises detecting the level of at least one marker in the thymus organ medium during the culturing regimen, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or detecting at least eight markers in the thymus organ medium during the conditioning regimen, selected from L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, and CCL11.
  • the method further comprises detecting the level of at least one marker in the thymus organ medium during the culturing regimen selected from L-selectin, CXCL16, M-CSF, CCL21/6Ckine, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-
  • a method for determining whether allogeneic cultured postnatal thymus tissue-derived product is suitable for implantation into a human comprising the steps of conditioning donor thymus tissue slices in a thymus organ medium for a period of about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in the thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; and detecting the level of at least one marker selected from L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, and CCL11 in the thymus organ medium.
  • the at least one marker is L-selectin, and wherein the levels of L-selectin in the thymus organ medium decrease over time, i.e., during the course of the course of the conditioning regimen.
  • the at least one marker is M-CSF, and wherein the levels of M-CSF in the thymus organ medium decrease during the course of the conditioning regimen.
  • the at least one marker is galectin-7, and wherein the levels of galectin-7 in the thymus organ medium decrease during the course of the conditioning regimen.
  • the at least one marker is IL-16, and wherein the levels of IL-16 in the thymus organ medium decrease during the course of the conditioning regimen.
  • the at least one marker is CCL21, and wherein the levels of CCL21 in the thymus organ medium increase during the course of the conditioning regimen.
  • the at least one marker is CXCL12, and wherein the levels of CXCL12 in the thymus organ medium increase during the course of the conditioning regimen.
  • the at least one marker is CXCL16, and wherein the levels of CXCL16 in the thymus organ medium increase during the course of the conditioning regimen.
  • the at least one marker is CCL11, and wherein the levels of CCL11 in the thymus organ medium increase during the course of the conditioning regimen.
  • a method for determining whether allogeneic cultured postnatal thymus tissue-derived product is suitable for implantation into a human comprising the steps of conditioning donor thymus tissue slices in a thymus organ medium for a period of about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in the thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; and detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen selected from L-selectin, CXCL16, M-CSF, CCL21/6Ckine, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF- AA, CXCL12/SDFF-la, MIP-3a,
  • the method further comprises the step of determining in the donor thymus tissue slices between days 6 and 21 of the conditioning regimen areas positive for keratin AE1/AE3 scattered throughout the donor thymus tissue slices the presence of at least one Hassall body, CK14 staining scattered throughout the donor thymus tissue slices and the presence of intact nuclei.
  • the conditioning regimen is for a period of five days, or six days, or seven days, or eight days, or nine days, or 10 days, or 11 days, or 12 days, or 13 days, or 14 days, or 15 days, or 16 days, or 17 days , or 18 days or 19 days, or 20 days, or 21 days; or for a period of 5-6 days, or 5-7 days, or 5-8 days, or 5-9 days, or 5-10 days, or 6 to 7 days, or 6 to 8 days, or 6 to 9 days, or 6 to 10 days, or 6 to 11 days, or 6 to 12 days, or 6 to 21 days, or 7 to 21 days, or 8 to 21 days, or 9 to 21 days, or 10 to 21 days, or 11 to 21 days, or 12 to 21 days, 13 to 21 days or 14 to 21 days, or 15 to 21 days, or 16 to 21 days or 17 to 21 days or 18 to 21 days, or 19 to 21 days, or 20 to 21 days.
  • the levels of CCL21 approximate the levels in Fig. 50E, and/or the levels of L-selectin approximate the levels in Fig. 50A, and/or the levels of M-CSF approximate the levels in Fig. 50B, and/or the levels of galectin-7 approximate the levels in Fig. 50C, and/or the levels of IL-16 approximate the levels in Fig. 50D, and/or the levels of CXCL16 approximate the levels in Fig. 50F, and/or wherein the levels of CCL11 approximate the levels in Fig. 50G, and/or the levels of CXL21 approximate the levels in Fig. 50H.
  • the method further comprises detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen, or the levels of at least two, at least three, at least four, at least five, at least six, at least seven, or detecting at least eight markers, selected from L- selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, and CCL11, and wherein the levels of L-selectin, M-CSF, galectin-7, IL-16 decrease in the thymus organ medium during the course of the conditioning regimen, and further wherein the levels of CCL21, CXCL12, CXCL16, and CCL11 increase in the thymus organ medium during the course of the conditioning regimen.
  • a method for determining whether allogeneic cultured postnatal thymus tissue-derived product is suitable for implantation into a human comprising the steps of conditioning donor thymus tissue slices in a thymus organ medium for a period of about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in the thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; and detecting the level of at least one marker selected from the markers in Fig. 56.
  • a method for determining whether allogeneic cultured postnatal thymus tissue-derived product is suitable for implantation into a human comprising the steps of conditioning donor thymus tissue slices in a thymus organ medium for a period of about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in the thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; and detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen selected from L-selectin, CXCL16, M-CSF, CCL21/6Ckine, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF-AA, CXCL12/SDFF-la,
  • a method of treating a thymic disorder comprising implanting into a subject having a thymic disorder allogeneic cultured postnatal thymus tissue-derived slices subjected to a conditioning regimen in a thymus organ medium for a period of about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in the thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; and detecting the level of at least one marker selected from L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, and CCL11.
  • a method of treating thymic disorders comprising implanting into a subject having a thymic disorder allogeneic cultured postnatal thymus tissue-derived slices subjected to a conditioning regimen in a thymus organ medium for a period of about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in the thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; and detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen is selected from L-selectin, CXCL16, M-CSF, CCL21/6Ckine, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF- AA, CXCL12/SDFF-la,
  • At least one marker is L-selectin, and wherein the levels of L-selectin in the thymus organ medium decrease during the course of the conditioning regimen.
  • At least one marker is M-CSF, and wherein the levels of M-CSF in the thymus organ medium decrease during the course of the conditioning regimen.
  • At least one marker is galectin-7, and wherein the levels of galectin-7 in the thymus organ medium decrease during the course of the conditioning regimen.
  • At least one marker is IL-16, and wherein the levels of IL-16 in the thymus organ medium decrease during the course of the conditioning regimen.
  • At least one marker is CCL21, and wherein the levels of CCL21 in the thymus organ medium increase during the course of the conditioning regimen.
  • at least one marker is CXCL12, and wherein the levels of CXCL12 in the thymus organ medium increase during the course of the conditioning regimen.
  • At least one marker is CXCL16, and wherein the levels of CXCL16 in the thymus organ medium increase during the course of the conditioning regimen.
  • At least one marker is CCL11, and wherein the levels of CCL11 in the thymus organ medium increase during the course of the conditioning regimen.
  • the method further comprises the step of determining in the donor thymus tissue slices during the conditioning regimen areas positive for keratin AE1/AE3 scattered throughout the donor thymus tissue slices the presence of at least one Hassall body, CK14 staining scattered throughout the donor thymus tissue slices and the presence of intact nuclei.
  • the thymic disorder is congenital athymia associated with complete DiGeorge syndrome, 22ql 1.2 deletion, CHARGE (coloboma, heart defect, choanal atresia, growth or mental retardation, genital hypoplasia and ear anomalies or deafness), mutations in the CHD7 (chromodomain-helicase- DNA-binding protein 7) gene or forkhead box protein N1 (FOXN1) deficiency.
  • CHD7 chromodomain-helicase- DNA-binding protein 7
  • FOXN1 forkhead box protein N1
  • the thymic disorder is thymic involution. In another embodiment, thymic disorder is congenital athymia associated with mutations in the TBX-1 or TBX-2 gene. [00223] In an embodiment of the aspects and embodiments of the present disclosure, the thymic disorder is related to paired box 1 (PAX1), semaphorine 3E (SEMA3E) and recurrent microdeletions at chromosome 2pl 1.2.
  • PAX1 paired box 1
  • SEMA3E semaphorine 3E
  • recurrent microdeletions at chromosome 2pl 1.2 recurrent microdeletions at chromosome 2pl 1.2.
  • the thymic disorder is associated with a thymoma.
  • the thymoma is either non- malignant or malignant.
  • the thymic disorder is associated with myasthenia gravis (MG), pure red cell aplasia and hypogammaglobulinemia.
  • MG myasthenia gravis
  • pure red cell aplasia pure red cell aplasia
  • hypogammaglobulinemia hypogammaglobulinemia
  • a method for providing immune- competence in a human subject comprising the steps of conditioning donor thymus tissue slices in a thymus organ medium for a period of about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in the thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen selected from the markers L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, or CCL11; wherein the level is decreased if the marker is L-selectin, M-CSF, galectin-7, or IL-1 or increased if the marker is CCL21, CXCL12, CXCL16, or CCL11; and implanting the partially T-cell depleted donor
  • a method for providing immune-competence in a human subject comprising the steps of conditioning donor thymus tissue slices in a thymus organ medium for a period of about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in the thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen is selected from the markers from L-selectin, CXCL16, M-CSF, CCL21/6Ckine, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF-AA, CXCL12/SDFF-la, MIP-3a, IL-2Ra, IC AM-3, LIGHT, I
  • CCL11/Eotaxin HVEM, IGFBP-6, IL-6R. IL-12p40, RANTES, MICA, GCP-2, OPN, ALCAM, NRG1-B1, CEACAM-1, IL-lb, DKK-1 and ANG-1; and implanting the partially T-cell depleted donor thymus tissue slices into the human subject.
  • a method for providing immune-competence in a human subject undergoing a solid organ transplant comprising the following steps of removing the thymus of the human subject; obtaining thymus tissue from a donor matching HLA-Class I and HLA-Class II alleles in the solid organ; slicing the donor thymus; conditioning the donor thymus tissue slices in a thymus organ medium for a period of about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in the thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen selected from the markers L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, or CCL11; wherein the level is decreased
  • Fig 38 has a primate model of this procedure that will provide data to support a human study for generating donor-specific tolerance.
  • the experiment in Fig 38 has 3 monkeys. One is the thymus and heart donor (information in the left hand columns). The second is the thymus and heart recipient (information in the middle column on the 2 nd page. This column has the STAGE of the experiment). The third is the control (information in the right hand columns on the 3 rd page).
  • the original spreadsheet had the procedures for all three animals one page wide by many pages long. Because the spread sheet was wider than the width allowed, each row of the spread sheet was divided into 3 pages. The table continues in groups of 3 panels for many weeks and several stages.
  • a method for providing immune-competence in a human subject undergoing a solid organ transplant comprising the following steps of removing the thymus of the human subject; obtaining thymus tissue from a donor matching HLA-Class I and II alleles in the solid organ; slicing the donor thymus; conditioning the donor thymus tissue slices in a thymus organ medium for a period of about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in the thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regiment is selected from the markers L-selectin, CXCL16, M-CSF, CCL21/6Ckine, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1,
  • a method for providing immune- competence in a human subject undergoing a solid organ transplant comprising the following steps of removing the thymus of the human subject; obtaining thymus tissue from a donor matching both HLA-Class I and HLA-Class II alleles in the solid organ; slicing the donor thymus; conditioning the donor thymus tissue slices in a thymus organ medium for a period of about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in the thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen selected from the markers L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL 12, CXCL 16, or CCL11; wherein the level is decreased if
  • a method for providing immune-competence in a human subject undergoing a solid organ transplant comprising the following steps of removing the thymus of the human subject; obtaining thymus tissue from a donor matching both HLA-Class I and HLA-Class II alleles in the solid organ; slicing the donor thymus; conditioning the donor thymus tissue slices in a thymus organ medium for a period of about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in the thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen is selected from the markers L-selectin, CXCL16, M-CSF, CCL21/6Ckine, galectin-7, MIF,
  • GDNF GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF-AA, CXCL 12/ SDFF - 1 a, MIP-3a, IL-2Ra, IC AM-3, LIGHT, IGFBP-1, BCMA, EGF R, uPAR, MIP- lb, PIGF, PF4, CCL11/Eotaxin, HVEM, IGFBP-6, IL-6R.
  • a method of promoting donor-specific tolerance to an allogeneic solid organ transplant obtained from a deceased donor, in a recipient in need of a solid organ transplant comprising the following steps:
  • the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in the thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen selected from the markers L-selectin, M-CSF, galectin-7, IL- 16, CCL21, CXCL12, CXCL16, or CCL11; wherein the level of the marker in the thymus organ medium is decreased if the marker is L-selectin, M-CSF, galectin-7, or IL-1 or increased
  • a method of promoting donor-specific tolerance to an allogeneic solid organ transplant obtained from a deceased donor, in a recipient in need of a solid organ transplant comprising the following steps:
  • the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in the thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen is selected from the markers L-selectin, CXCL16, M-CSF, CCL21/6Ckine, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF-AA, CXCL12/SDFF
  • a method for promoting donor-specific tolerance to an allogeneic solid organ transplant obtained from a living human donor, in a human recipient in need of a solid organ transplant comprising the steps of:
  • cryopreserved allogeneic cultured postnatal thymus tissue-derived product maintained in a cryopreserved allogeneic cultured postnatal thymus tissue-derived product bank; wherein the cryopreserved allogeneic cultured postnatal thymus tissue-derived product was processed from thymus tissue from a thymus donor expressing HLA alleles matched to HLA- Class I and HLA-Class II alleles in the recipient that are not present in the solid organ transplant; wherein the donor thymus tissue was subjected to a conditioning regimen for a period of about 6 days to about 21 days; further wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T-cell depleted thymus tissue slices; detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen selected from the markers L-selectin, M-CSF
  • permissive mismatches for HLA-DP can be allowed (Pidala J et al 2014 Blood 124:2596-2606).
  • nonpermissive mismatches for HLA-DPB1 can be allowed if there is sufficient numerical functional distance (Crivello P et al 2016 Blood 128:120-129).
  • a method for promoting donor-specific tolerance to an allogeneic solid organ transplant obtained from a living human donor, in a human recipient in need of a solid organ transplant comprising the steps of:
  • cryopreserved allogeneic cultured postnatal thymus tissue-derived product maintained in a cryopreserved allogeneic cultured postnatal thymus tissue-derived product bank; wherein the cryopreserved allogeneic cultured postnatal thymus tissue-derived product was processed from thymus tissue from a thymus donor expressing HLA alleles matched to HLA- Class I and HLA-Class II alleles in the recipient that are not present in the solid organ transplant; wherein the donor thymus tissue was subjected to a conditioning regimen for a period of about 6 days to about 21 days; further wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T-cell depleted thymus tissue slices; detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen is selected from the markers L-selectin, CXCL
  • step (h) implanting the thawed cryopreserved allogeneic cultured postnatal thymus tissue- derived product into the recipient, wherein the dosage of the cryopreserved allogeneic cultured postnatal thymus tissue-derived product is about 1,000 - 22,000 mm 2 of thymus tissue surface area / recipient body surface area in m 2 , and further wherein the implanted allogeneic cultured postnatal thymus tissue-derived product induces thymopoiesis and tolerance in the recipient [00238] In an embodiment of the aspects and embodiments of the present disclosure, about one-half of the thawed cryopreserved allogeneic cultured postnatal thymus tissue-derived product is transplanted into the recipient and the remainder is cryopreserved for future use. [00239] In an embodiment of the aspects and embodiments of the present disclosure, step (h) is performed about one month or more after the transplantation of the solid organ.
  • a method for promoting donor-specific tolerance to an allogeneic solid organ transplant obtained from a deceased human donor, in a human recipient in need of a solid organ transplant comprising the steps of:
  • cryopreserved allogeneic cultured postnatal thymus tissue-derived product maintained in a cryopreserved allogeneic cultured postnatal thymus tissue-derived product bank; wherein the cryopreserved allogeneic cultured postnatal thymus tissue-derived product is processed from thymus tissue from a thymus donor expressing HLA alleles matched to HLA alleles in the recipient that are not present in the solid organ transplant; wherein the donor thymus tissue was subjected to a conditioning regimen for a period of about 6 days to about 21 days; further wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T-cell depleted thymus tissue slices, detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen selected from the markers L-selectin, M- CSF, galectin-7, IL-16
  • a method for promoting donor-specific tolerance to an allogeneic solid organ transplant obtained from a deceased human donor, in a human recipient in need of a solid organ transplant comprising the steps of:
  • cryopreserved allogeneic cultured postnatal thymus tissue-derived product maintained in a cryopreserved allogeneic cultured postnatal thymus tissue-derived product bank; wherein the cryopreserved allogeneic cultured postnatal thymus tissue-derived product is processed from thymus tissue from a thymus donor expressing HLA alleles matched to HLA alleles in the recipient that are not present in the solid organ transplant; wherein the donor thymus tissue was subjected to a conditioning regimen for a period of about 6 days to about 21 days; further wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T-cell depleted thymus tissue slices, detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen selected from the markers L-selectin, CXCL16, M-CSF, CCL21
  • IC AM-3 LIGHT, IGFBP-1, BCMA, EGF R, uPAR, MIP-lb, PIGF, PF4, CCL11/Eotaxin, HVEM, IGFBP-6, IL-6R.
  • IL-12p40 RANTES, MICA, GCP-2, OPN, ALCAM, NRG1-B1, CEACAM-1, IL-lb, DKK-1 and ANG-1; wherein the level of the marker in the thymus organ medium is increased or decreased in accordance with the levels in Fig. 7;
  • cryopreserved allogeneic cultured postnatal thymus tissue-derived product implanting the thawed cryopreserved allogeneic cultured postnatal thymus tissue- derived product into the recipient, wherein the dosage of the cryopreserved allogeneic cultured postnatal thymus tissue-derived product is about 1,000 - 22,000 mm 2 of thymus tissue surface area / recipient body surface area in m 2 , and further wherein the implanted allogeneic cultured postnatal thymus tissue-derived product induces thymopoiesis and tolerance in the recipient.
  • a cryopreserved allogeneic cultured postnatal thymus tissue-derived product prepared by a method comprising the steps of:
  • HLA-A typing HLA alleles: HLA-A, HLA-B, HLA-C, HLA-DRB 1, HLA-DQB 1, HLA- DRB3, HLA-DRB4, HLA-DRB 5, HLA-DQA1, HLA-DPBl, HLA-DPAl;
  • conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T- cell depleted donor thymus tissue slices;
  • cryopreserved allogeneic cultured postnatal thymus tissue-derived product prepared by a method comprising the steps of:
  • HLA-A typing HLA alleles: HLA-A, HLA-B, HLA-C, HLA-DRB 1, HLA-DQB 1, HLA- DRB3, HLA-DRB4, HLA-DRB 5, HLA-DQA1, HLA- DPB1, HLA-DPAl;
  • conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T- cell depleted donor thymus tissue slices;
  • IL- 12p40 IL- 12p40, RANTES, MICA, GCP-2, OPN, ALCAM, NRG1-B1, CEACAM-1, IL-lb, DKK-1 and ANG-1; wherein the level of the marker in the thymus organ medium is increased or decreased in accordance with the levels in Fig. 7;
  • a method for promoting donor-specific tolerance to an allogeneic solid organ transplant obtained from a deceased human donor, in a human recipient in need of a solid organ transplant comprising the steps of:
  • cryopreserved allogeneic cultured postnatal thymus tissue-derived product maintained in a cryopreserved allogeneic cultured postnatal thymus tissue-derived product bank; wherein the cryopreserved allogeneic cultured postnatal thymus tissue-derived product is processed from thymus tissue from a thymus donor expressing HLA alleles matched to HLA alleles in the recipient that are not present in the solid organ transplant; wherein the donor thymus tissue was subjected to a conditioning regimen for a period of a about 6 days to about 21 days; further wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T-cell depleted thymus tissue slices, detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regiment selected from the markers L-selectin, M- CSF, galectin-7,
  • cryopreserved allogeneic cultured postnatal thymus tissue-derived product prepared by a method comprising the steps of:
  • HLA-A typing HLA alleles: HLA-A, HLA-B, HLA-C, HLA-DRB 1, HLA-DQB 1, HLA- DRB3, HLA-DRB4, HLA-DRB 5, HLA-DQA1, HLA- DPB1, HLA-DPAl;
  • conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T-cell depleted donor thymus tissue slices;
  • a method for promoting donor-specific tolerance to an allogeneic solid organ transplant obtained from a deceased human donor, in a human recipient in need of a solid organ transplant comprising the steps of:
  • cryopreserved allogeneic cultured postnatal thymus tissue-derived product maintained in a cryopreserved allogeneic cultured postnatal thymus tissue-derived product bank; wherein the cryopreserved allogeneic cultured postnatal thymus tissue-derived product is processed from thymus tissue from a thymus donor expressing HLA alleles matched to HLA alleles in the recipient that are not present in the solid organ transplant; wherein the donor thymus tissue was subjected to a conditioning regimen for a period of about 6 days to about 21 days; further wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T-cell depleted thymus tissue slices, detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen selected from the markers L-selectin, CXCL16, M-CSF, CCL21
  • IC AM-3 LIGHT, IGFBP-1, BCMA, EGF R, uPAR, MIP-lb, PIGF, PF4, CCL11/Eotaxin, HVEM, IGFBP-6, IL-6R.
  • IL-12p40 RANTES, MICA, GCP-2, OPN, ALCAM, NRG1-B1, CEACAM-1, IL-lb, DKK-1 and ANG-1; wherein the level of the marker in the thymus organ medium is increased or decreased in accordance with the levels for the marker in Fig. 56;
  • the solid organ transplant is a heart transplant, a kidney transplant, a liver transplant, a lung transplant, a heart/lung transplant, a pancreas transplant, an intestine transplant, a stomach transplant, an abdominal wall transplant, a craniofacial transplant, a scalp transplant, a penile transplant, a uterus transplant, a unilateral or bilateral upper limb transplant, a unilateral vascularized composite
  • the solid organ transplant is a heart transplant, or a pediatric heart transplant, or an adult heart transplant.
  • the conditioning regimen is for a period of five days, or six days, or seven days, or eight days, or nine days, or 10 days, or 11 days, or 12 days, or 13 days, or 14 days, or 15 days, or 16 days, or 17 days , or 18 days or 19 days, or 20 days, or 21 days; or for a period of 5-6 days, or 5-7 days, or 5-8 days, or 5-9 days, or 5-10 days, or 6 to 7 days, or 6 to 8 days, or 6 to 9 days, or 6 to 10 days, or 6 to 11 days, or 6 to 12 days, or 6 to 21 days, or 7 to 21 days, or 8 to 21 days, or 9 to 21 days, or 10 to 21 days, or 11 to 21 days, or 12 to 21 days, 13 to 21 days or 14 to 21 days, or 15 to 21 days, or 16 to 21 days or 17 to 21 days or 18 to 21 days, or 19 to 21 days, or 20 to 21 days. [00250] In an aspect of
  • the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T- cell depleted donor thymus tissue slices; wherein levels of L-selectin, and/or M-CSF and/or galectin-7 and/or IL-16 in the thymus organ medium decrease during the course of the conditioning regimen; further wherein levels of CCL21 and/or CXCL12 and/or CXCL16 and/or CCL11 in the thymus organ medium increase during the course of the conditioning regimen;
  • the levels of CCL21 in the thymus organ medium increase during the course of the conditioning regimen
  • the levels of L-selectin in the thymus organ medium decrease during the course of the conditioning regimen.
  • the levels of one or more of M-CSF, galectin-7, and IL-16 in the thymus organ medium decrease during the course of the conditioning regimen.
  • the levels of one or more of CCL21, CXCL12, CXCL16, and CCL11 in the thymus organ medium increase during the course of the conditioning regimen.
  • the method further comprises the step of determining in the donor thymus tissue slices during the conditioning regimen areas positive for keratin AE1/AE3 scattered throughout the donor thymus tissue slices the presence of at least one Hassall body, CK14 staining scattered throughout the donor thymus tissue slices and the presence of intact nuclei.
  • kits for performing the methods of any of the foregoing aspects and embodiments together with instructions for use in determining whether allogeneic cultured postnatal thymus tissue-derived product is suitable for implantation into a human.
  • the kit comprises at least one antibody that specifically binds marker L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, or CCL11.
  • the kit comprises one or more antibody that specifically finds a marker set forth in Fig. 56.
  • kits for determining whether cryopreserved allogeneic cultured postnatal thymus tissue-derived product cultured in accordance with any one of the foregoing aspects and embodiments is suitable for implantation into a human together with instructions for use
  • the kit comprises at least one antibody that specifically binds marker L-selectin, M-CSF, galectin-7,
  • Fig. 1 describes the manner in which allogeneic, cultured postnatal thymus tissue- derived product (e.g CTT, RVT-802) provides for immune reconstitution in congenital athymia following implantation.
  • allogeneic, cultured postnatal thymus tissue- derived product e.g CTT, RVT-802
  • FIG. 2 shows a schematic of the steps for reconstituting the immune system in a rat, as described elsewhere in Example 5, by removing the thymus in an immunologically normal Lewis rat, administering an antibody to kill the recipient rat’s T cells, implanting cultured neonatal thymus tissue from a donor rat into the recipient rat, administering an immunosuppressive agent for about 4 months and evaluating T cell development in the recipient rat.
  • all rats in the treatment group had over 10% naive T cells prior to stopping the cyclosporine.
  • FIG. 3 shows the development of naive T cells in two experimental recipient rats of Example 5 (rising lines on right) versus two controls rat not receiving a thymus tissue implant (thick lines at baseline).
  • Fig. 4 shows a schematic of the manufacturing process for harvesting a thymus from a donor, culturing thin slices of the donor thymus tissue made with a hand microtome for up to 21 days and implanting the cultured thymus tissue in the quadriceps muscle of the recipient.
  • FIG. 5A shows a schematic showing the slicing of thymus tissue for characterization testing, as discussed in section
  • Fig.5B is a figure showing slices of thymus tissue on cellulose filters on surgical sponges in a tissue culture dish as is used for culture of the thymus.
  • Figs. 6A-H depict histology testing of thymus tissue slices from a lot (MFG-056) of cultured thymus tissue on day 5, 9, 12 and 21 after harvest of the thymus from a donor.
  • Hematoxylin and eosin-stained slices (left panels) and their corresponding reactivity with a cocktail of the anti-cytokeratin antibodies AE1/AE3 (right panels; brown color denotes positive reactivity) are shown at day 5 (Fig. 6A, Fig. 6B), day 9 (Fig. 6C, Fig. 6D), day 12 (Fig. 6E, Fig. 6F), and day 21 (Fig 6G, Fig. 6H), respectively. Bars in the lower left of each panel represent 100 pm. Panels with H&E show progression depletion of T cells with time. Fig. 6E and Fig. 6F are predominantly epithelial cells.
  • Figs. 7A and 7B depict the histology of thymus tissue slices on day 0 of the time course in a scale of 5 mm (Fig. 9A) and 100 pm (Fig. 7B), respectively. This shows the thymus and thymocytes at low power (bar 5 mm) and high power (bar 100 um) on day 0. This is normal thymus. At this time the cortex and medulla both have large numbers of thymocytes with dark blue nuclei contributing to the overall dark blue appearance of the tissue. Photo by Laura P.
  • Figs. 8A and 8B are images from H&E stained slide that depict the histology of thymus tissue slices on day 5 of the time course in a scale of 5 mm (Fig. 8 A) and 100 pm (Fig. 8B), respectively. Progression of depletion of the thymocytes results in a more eosinophil (pink) appearance of the tissue. Photo by Laura P. Hale, MD, PhD, Department of Pathology, Duke University.
  • Figs. 9A and Fig. 9B depict H&E staining of the thymus tissue slices on day 12 of the time course in a scale of 5 mm (Fig. 9A) and 100 pm (Fig. 9B), respectively.
  • Fig. 9A depicts H&E staining of the thymus tissue slices on day 12 of the time course in a scale of 5 mm (Fig. 9A) and 100 pm (Fig. 9B), respectively.
  • Fig. 9B depict H&E staining of the thymus tissue slices on day 12 of the time course in a scale of 5 mm (Fig. 9A) and 100 pm (Fig. 9B), respectively.
  • Fig. 9B depict H&E staining of the thymus tissue slices on day 12 of the time course in a scale of 5 mm (Fig. 9A) and 100 pm (Fig. 9B), respectively.
  • Fig. 10A and Fig. 10B depict H&E staining of thymus tissue slices on day 21 of the time course in a scale of 5 mm (Fig. 10A) and 100 pm (Fig. 10B), respectively. Note the preservation of the overall architecture of the tissue including in Fig. 10B the subcapsular cortex, cortical region and medullary region containing numerous Hassall bodies. The small dark cells are mostly necrotic thymocytes that have not yet undergone karyolysis. Photo by Laura P. Hale, MD, PhD, Department of Pathology, Duke University.
  • Figs. 11 A-E depict representative thymus slices which were immuno-stained with a cocktail of anti-cytokeratin antibodies (AE1/AE3).
  • Fig. 11 A Day 0; Fig. 11B. Day 5; Fig. 11C. Day 9; Fig. 1 ID. Day 12; and Fig. 1 IE. Day 21.
  • the structure of the thymic epithelial network remains intact as the culture progresses. Bar represents 400 pm. Photo by Laura P. Hale, MD, PhD, Department of Pathology, Duke University.
  • Figs. 12A and 12B depict the histology of thymus tissue slides after exposure to forced degradation conditions of 10X PBS.
  • Fig. 12A depicts the cortex at day 9 after exposure to forced degradation conditions.
  • Fig. 12B depicts the cortex at day 21 after exposure to forced degradation conditions.
  • the smear of blue is DNA released from cells. The majority of cells show evidence of degradation although small foci of cells with intact nuclei can be identified.
  • Fig. 13 depicts H&E stained histology sections for clinical sample MLM247. This is Day 0 of culture. The bar is 200 um. This is a frozen section from day 0. Because this was frozen, the tissue looks different from paraffin embedded formalin fixed tissue on day 0. Photo by Laura P. Hale, MD, PhD, Department of Pathology, Duke University.
  • Fig. 14 Frozen section, H&E stained histology sections for clinical sample MLM219. This is a frozen section so the tissue looks different from paraffin embedded formalin fixed tissue that was cultured and presented above. Nevertheless, the important histologic characteristics of thymocyte depletion and robust viability of TEC are well represented. Photo by Laura P. Hale, MD, PhD, Department of Pathology, Duke University.
  • Fig. 15 is a photograph of freshly harvested thymus tissue.
  • Fig. 16 is a schematic describing the harvesting, culturing, implantation and biopsy of the implantation of CTT under the rat kidney capsule, as presented in Example 5.
  • FIGs. 17A-D present photographs of the harvesting of thymus tissue from 3-day old FI (LWxDA) rats that was cut into four pieces as described in Example 5 (Fig. 17A).
  • Fig. 17C is a photograph of CTT implanted under the kidney capsule of an LW rat.
  • Fig. 17D is a photograph of a thymus graft harvested at 6 months after implantation. Arrows indicate the CTT under the kidney capsule.
  • FIGs. 18A-D are photographs depicting the histologic appearance of fresh thymus tissue (top frames) and CTT (bottom frames) at lOOx magnification.
  • Fig. 18A shows a comparison of medullary differentiation in H&E stained fresh thymus tissue (top frame) and CTT cultured for 5 days (bottom frame), as described in Example 5.
  • Fig. 18B shows the typical lacey pattern observable in CTT cultured for 5 days (bottom frame) when stained for cytokeratin compared with fresh thymus tissue (top frame), as described in Example 5.
  • Fig 18C shows fresh thymus tissue (top frame) and CTT depleted of T cells (bottom frame) when stained for Ki-67.
  • Fig. 18A shows a comparison of medullary differentiation in H&E stained fresh thymus tissue (top frame) and CTT cultured for 5 days (bottom frame), as described in Example 5.
  • Fig. 18B shows the typical lacey pattern observable in CTT culture
  • FIG. 18D shows fresh thymus tissue stained for CD3 (top frame) and CTT thymus tissue cultured for 5 days and then stained for CD3 (bottom frame).
  • the brown stain noted in the CD3 stained CTT (Fig. 18D, bottom frame), likely represents some viable cells plus the detritus of dead T cells that have not washed out of the tissue.
  • Figs. 19A-D are photographs depicting the histologic appearance of fresh thymus tissue (top frames) and CTT (bottom frames) at 600x magnification.
  • Fig. 19A shows a comparison of medullary differentiation in H&E stained fresh thymus tissue (top frame) and CTT cultured for 5 days (bottom frame), as described in Example 5.
  • Fig. 19B shows the typical lacey pattern observable in CTT cultured for 5 days (bottom frame) when stained for cytokeratin compared with fresh thymus tissue (top frame), as described in Example 5.
  • Fig 19C shows fresh thymus tissue (top frame) and CTT depleted of T cells (bottom frame) when stained for Ki-67.
  • Fig. 19A shows a comparison of medullary differentiation in H&E stained fresh thymus tissue (top frame) and CTT cultured for 5 days (bottom frame), as described in Example 5.
  • Fig. 19B shows the typical lacey pattern observable in CTT cultured
  • FIG. 19D shows fresh thymus tissue stained for DC3 (top frame) and CTT thymus tissue cultured for 5 days and then stained for CD3 (bottom frame).
  • the brown stain noted in the CD3 stained CTT (Fig. 19D, bottom frame), likely represents some viable cells plus the detritus of dead T cells that have not washed out of the tissue.
  • Fig. 20 is a schematic of the experimental design of the experiment reported in Example 5. This figure appears in Kwun, J. et al., JCI Insight (2020) Jun 4;5(11).
  • Fig. 21 shows repopulating recipient-type T cells are seen in the lower right quadrant after CTT imallogernicplantation. This figure appears in Kwun, J. et al., JCI Insight (2020) Jun 4;5(11).
  • Fig. 22A and Fig. 22B show implanted thymus explanted at 8.5 month after implantation showing positive cytokeratin staining (Fig. 22A), as well as T cell staining similar to native thymus (Fig. 22B).
  • Original magnification x 400 This figure appears in Kwun, J. et al., JCI Insight (2020) Jun 4;5(11).
  • Fig. 23 shows plots of significantly increased numbers of circulating CD4 and CD8 T cells compared to control animals without implantation of CTT. It also shows significantly increased numbers of naive CD4 and naive CD8 T cells in the cultured thymus tissue implantation (CTT) group compared to the control group that did not receive CTT and significantly increased numbers of CD4 and CD8 recent thymus emigrants (RTE) in the cultured thymus tissue imsplantation (CTT) group compared to the control group that did not receive CTT.
  • CTT cultured thymus tissue implantation
  • RTE thymus emigrants
  • FIG. 24A shows immunohistologic analysis of implanted CTT explanted on day 180 showing normal thymus histology under the capsule of the kidney (right hand side of Figure 24A).
  • Fig. 24B shows the explanted graft on H&E. Strains for viable T cells (CD3), T cell proliferation (Ki67), and cytokeratin (detected by a rabbit polyclonal antibody) are shown. In the panel stained for cytokeratin, a lacy pattern is seen with Hassall body formation (arrow) on TECs. This figure appears in Kwun, J. et ak, JCI Insight (2020) Jun 4;5(11).
  • Fig. 25 shows survival percentages of LW rats after thymectomy and immunosuppression with DA heart transplants with CTT (solid triangles, blue line) and without CTT (upside down triangles, red lines) transplants (CTT).
  • CTT solid triangles, blue line
  • CTT upside down triangles, red lines
  • the LW rats with CTT are tolerant; the LW rats without CTT are immunodeficient and thus do not reject the DA heart.
  • FIGs. 26A and 26B are photographs of transplanted allografts (DA hearts) from animals implanted with (Fig. 26A) and without (Fig. 26B) CTT showing mononuclear cell infiltration with no signs of rejection by 2004 International Society for Heart &Lung Transplantation (ISHLT) depicted in Fig. 26C (the solid blue squares and solid red triangles). This figure appears in Kwun, J. et ak, JCI Insight (2020) Jun 4;5(11).
  • Fig. 27 is a plot of BN heart graft survival in the neck percentage animal survival vs. graft survival days in LW rats with CTT (that were immunocompetent and rejected the cervical allogeneic BN heart) and control LW animals without CTT (that were immunodeficient because of lack of thymus and could not reject the cervical BN heart) inserted vs. BN control (LW rat rejecting a cervical BN heart) and syngeneic controls (LW rats do not reject cervical LW hearts).
  • CTT that were immunocompetent and rejected the cervical allogeneic BN heart
  • control LW animals without CTT that were immunodeficient because of lack of thymus and could not reject the cervical BN heart
  • syngeneic controls LW rats do not reject cervical LW hearts.
  • Fig. 28 A and Fig. 28B are photographs of BN heart tissue with (Fig. 28 A) and without (Fig. 28B) CTT insertions at 11 and 46 days, respectively. These pictures are the basis of the data in Fig. 27 and Fig. 29.
  • the heart in Fig. 28 A is not rejected because of tolerance.
  • the heart in Fig. 28B is not rejected because of immunodeficiency from lack of thymus. This figure appears in Kwun, J. et al., JCI Insight (2020) Jun 4;5(11).
  • Fig. 29 shows rejection grading of the cervical BN hearts. Syngeneic LW hearts placed into LW rats (open circles) were not rejected. BN hearts placed into LW rats (filled circles) were rejected. BN hearts placed in LW rats who received CTT were rejected (filled squares). BN hearts placed in LW rats who did not receive CTT were weakly rejected (shaded triangles) in 2 rats and not rejected by the other three rats. These data show that the rats with CTT were able to strongly reject 3 rd party hearts even while they accepted DA hearts (Fig. 26C) as the CTT expressed DA. The rats without CTT were immunodeficient and didn’t reject either the DA (Fig. 26C) or the BN hearts. This figure appears in Kwun, J. et al., JCI Insight (2020) Jun 4;5(11).
  • Fig. 30A and Fig. 30B are photographs of BN hearts in which rats received or did not receive CTT insertions compared to LW and DA hearts, respectively.
  • Fig. 30A after immunosuppression was removed and the BN heart transplanted, the BN heart was quickly rejected and thus is very large because of all the inflammation.
  • the LW heart is normal sized for the heart pumping blood through the body.
  • the DA heart is small as it was placed in the abdomen and didn’t need to pump blood.
  • the rat is immunodeficient and cannot reject either the BN or DA heart after immunosuppression is removed. This figure appears in Kwun, J. et al., JCI Insight (2020) Jun 4;5(11).
  • Fig. 31 A and Fig. 3 IB are plots of rejection grading for explanted cervical BN hearts from rats with and without CTT insertions vs. BN controls and syngeneic control rats.
  • Fig. 31A depicts quantification of inflammatory cells in the primary abdominal DA cardiac allograft.
  • the syngeneic control shows that LW rats do not reject LW hearts.
  • the DA control shows that LW rats do reject DA hearts.
  • the CTT group does not reject the DA heart because of tolerance.
  • Fig. 3 IB depicts quantification of inflammatory cells in secondary cervical BN cardiac allografts.
  • the syngeneic control shows that LW rats do not reject LW hearts.
  • the BN control shows that LW rats do reject BN hearts.
  • the CTT group rejects the BN heart because it is immunocompetent.
  • the group without CTT doesn’t reject the BN heart because of immunodeficiency from lack of a thymus.
  • Fig. 31C shows DA and BN heart rats that were harvested from the LW recipients along with the native LW heart at the time of the cervical BN heart rejection.
  • the lower right panel shows the T cells (brown) in the BN heart leading to its rejection.
  • Fig. 3 ID shows T cell infiltration in the LW, DA, and BN hearts from control animals without insertion of CTT. There is no T cell infiltration because the animals are immunodeficient. This figure appears in Kwun, J. et al., JCI Insight (2020) Jun 4;5(11).
  • Fig. 32A to Fig. 32C Humoral tolerance after CTT.
  • Fig. 32A shows representative histogram plots for post-transplant donor-specific alloantibody (anti-DA and anti-BN antibodies) measured by T cell flow crossmatch.
  • the upper left panel of Fig. 32A (DA control) shows the development of anti-DA antibody (thick line) in a normal LW rat after receiving a heterotopic abdominal DA heart transplant.
  • the upper middle panel of Fig. 32A shows lack of anti DA antibody in the LW rats that received CTT; this indicates tolerance.
  • the upper right panel shows no response by the LW rats without CTT; this reflects the immunodeficiency of the rats after thymectomy and T cell depletion without receipt of a donor thymus.
  • the lower left panel of Fig. 32A shows normal anti BN antibody formed by a normal LW rat receiving a cervical BN heart.
  • the lower middle panel of Fig. 32A shows a normal response of the LW rats with CTT against BN after receiving a cervical BN heart transplant, showing immunocompetence and ability to reject 3 rd party.
  • the lower right panel of Fig. 32A_ shows that there is no response of the LW rats without CTT against BN after having received a cervical BN heart transplant, showing immune-incompetence and lack of ability to reject 3 rd party.
  • Fig. 32B shows levels of anti-DA antibody after primary DA heart transplantation.
  • the LW rats with CTT from an LWxDA thymus donor do not make antLDA antibody after a DA heart transplant because they are tolerant to DA.
  • the LW rats without CTT do not make anti-DA antibody after DA heart transplantation because they are immunodeficient.
  • Fig. 32C shows levels of anti-BN antibody after secondary cervical BN heart transplantation.
  • the LW rats with CTT from an LWxDA donor make antibodies against BN showing immunocompetence against 3 rd party.
  • the LW rats without CTT do not make antibodies against BN showing immunoincompetence. This figure appears in Kwun, J. et ak, JCI Insight (2020) Jun 4;5(11).
  • Figs. 33 A-J present photomicrographs of immunohistochemical assessment of fresh and cultured non-human primate (NHP) thymus tissue from an 8-month old NHP.
  • the top row is NHP thymus on the day of harvest and the bottom row is NHP thymus after culture for 12 days.
  • the tissue was stained with hematoxylin and eosin (Fig. 33A and Fig. 33F), CD3 (Fig. 33B and Fig. 33G), pan cytokeratin (CK) antibody AE1/AE3 (Fig. 33C and Fig. 33H), Ki-67 (Fig. 33D and Fig. 331), and CK14 (Fig. 33E and Fig. 33J). All pictures are at 20X magnification.
  • Figs. 34A-P present analysis of cryopreserved cultured thymus tissue from an 8-month- old non-human primate (NHP) after 12 days of culture.
  • the top row is cytokeratin at harvest (Fig. 34A), day 6 of culture (Fig. 34B), day 12 of culture (Fig. 34C), and after 12 days of culture followed by 35 days cryopreservation then thawing for the photo (Fig. 34D).
  • the cytokeratin (AE1/AE3) in Fig. 34D resembles the cytokeratin in Fig. 34C.
  • the second row shows CK14 staining with the same time points in Fig. 34E (harvest), Fig. 34F (day 6 of culture), Fig.
  • Fig. 34G day 12 of culture
  • Fig. 34H after 12 days of culture followed by 35 days of cryopreservation then thawing
  • the CK14 in Fig. 34H is very similar to that in panel Fig. 34G.
  • the third row shows CD3 staining in Fig. 341, Fig. 34J, Fig. 34K and Fig. 34L at the same time points, which has the expected loss of viable T cells through time.
  • Panel Fig. 34L is similar to panel Fig. 34K in having very few T cells.
  • the fourth row Fig. 34M, Fig. 34N, Fig. 340 and Fig. 34P shows Ki-67 staining of proliferating T cells at the same time points.
  • Fig. 34N Staining with Ki-67 is absent by day 6 (Fig. 34N), as the T cells have mainly died.
  • This figure shows the ability to cryopreserve non-human primate thymus similarly to how cultured thymus tissue will be cryopreserved for patients. All pictures are at 40X magnification.
  • Fig. 35 presents a schematic diagram of the experimental transplantation strategy using maximally MHC-mismatched CMV-free rhesus macaques.
  • Recipient animals (Y) undergo complete thymectomy.
  • Donor animals (X) donate both cultured thymic tissue and a heart placed in a heterotopic position into recipient Y (first Tx and second Tx).
  • Immunosuppressive drugs are then withdrawn and donor-specific tolerance is demonstrated by i) continued beating of the donor heart, ii) tolerance to the donor in nMLR with reactivity against third party and iii) rejection of skin transplant from 3 rd party donor animal Z (third Tx).
  • Fig. 36 shows graphs of results from flow cytometry experiments that describe the general gating strategy for identifying recent thymic emigrants (RTEs).
  • Nonhuman primate peripheral blood mononuclear cells are collected and analyzed using polychromatic flow cytometry.
  • the first step is identification of single cells in the first panel top row.
  • the “Singlets” are used to identify the lymphocytes (low SSC, side scatter, and high CD45) in the second panel in top row.
  • the CD3 T cells in the lymphocytes are identified in the 3rd panel, in top row.
  • the CD4 and CD8 cells are gated off the CD3 cells as shown in the 4th panel of the top row.
  • 1st panel, CD28 and CD95 are used to identify, using the CD4 gate, the CD4+ naive subset, the central memory subset and the effector memory subset.
  • CD31 is used to show the percentage of CD4+ naive cells that are RTEs.
  • the 3rd and fourth panels on the bottom row show the same approach to RTEs but for CD8 naive T cells. As can be seen, the RTEs are 99.6% and 95.2% of the CD4 and CD8 naive subsets.
  • Fig. 37 presents photomicrographic images and a graph of CCL21 assessment in cultured infant thymus.
  • CCL21 is produced at high levels by cultured infant thymus.
  • Immunohistochemical reactivity with CCL21 antibody (Ab) (brown staining) on day 16 of culture is shown in the left panels (upper left panel, 2X magnification; lower left panel, 20X magnification).
  • a corresponding time course measuring daily CCL21 secretion into culture media is shown on the right for 3 infant thymus cultures (R&D Systems Duo-Set ELISA).
  • the cultured thymus tissue can produce a functionally important biomolecule, CCL21, the chemokine responsible for attracting immature thymocyte precursors to the thymus.
  • Fig. 38 presents an outline of the experimental design of Example 8 directed to an assessment of successful engraftment of cultured thymic tissue followed by tolerance to matched heart and rejection of unmatched skin in a CMV-free NHP model.
  • the recipient is T cell depleted and started on immunosuppression with tacrolimus.
  • Unmatched cultured donor thymus tissue from an unrelated NHP is engrafted into the recipient at week 3 of Stage 3.
  • a biopsy is done of the thymus graft at week 10 of Stage 3 to evaluate for thymopoiesis.
  • naive T cells develop a few months later, the recipient should be tolerant to the donor.
  • the recipient is then given a heterotopic heart transplant from the thymus donor (Week 4 of Stage 4). Immunosuppression is weaned off. The beating of the heart is followed (demonstrating tolerance). And a mixed lymphocyte reaction at week 10 of Stage 4 in done to show tolerance to cryopreserved donor cells and rejection of third party cells.
  • Stage 5 is used if more time is needed for naive T cells to develop.
  • Stage 6 is used if tolerance didn’t develop. Recipient thymus would be transplanted into the recipient NHP to prove that the thymus tissue transplant procedure is working in the NHP.
  • the experiment has 3 monkeys. Please note that the original spreadsheet had the procedures for all three animals in a document one page wide by many pages long. Because the spread sheet was wider than the width allowed in this patent application, each row of the spread sheet was divided into 3 pages.
  • the first monkey is the thymus and heart donor; procedures on this monkey are in the left hand columns on the 1 st , 4 th , 7 th etc pages.
  • the second monkey is the thymus and heart recipient; information in the middle columns are the 2 nd , 5 th , 8 th etc pages).
  • the third monkey is the control; information in the right hand columns are on the 3 rd , 6 th , 9 th etc pages).
  • Fig. 39 presents an outline of the experimental design of Example 9 which is the same as that in Example 8 except that an additional immunosuppression medication is added, mycophenylate mofetil (MMF).
  • MMF mycophenylate mofetil
  • the drug MMF is used routinely in heart transplantation. This study will assess if there is any detrimental effect of MMF on the cultured thymus tissue transplant.
  • Figs. 40A-D presents photomicrographs of slices of fresh thymus, dO of culture, showing thymic architecture.
  • hematoxylin and eosin (H&E) staining shows well-defined cortical and lighter-staining medullary areas, as expected for normal pediatric thymus.
  • Fig. 40C shows immunohistochemistry with a cocktail of pan-cytokeratin antibodies (AE1/AE3) that together detect all types of epithelial cells demonstrates that thymic epithelial cells are present beneath the capsule and in a light lacy network in both cortex and medulla (brown staining shows positive antibody reaction). Arrows in Fig.
  • Fig. 40D shows Cytokeratin 14 (CK14) antibody staining (brown).
  • CK14 antibody reacts with thymic epithelial cells in the sub-capsular cortex and in the medulla, as well as with scattered thymic epithelial cells in the cortex.
  • the dotted line highlights an area of medulla that is surrounded by cortex.
  • SCC denotes sub-capsular cortex
  • Cor denotes cortex
  • M denotes medulla.
  • Scale bar in Fig. 40A represents 1 mm; scale bars in Figs. 40B-D represent
  • Figs. 41 A-D present photomicrographs showing examples of Hassall bodies in cultured thymic slices. The histologic appearance of Hassall bodies is shown on day 0 (Figs. 41A-B) and day 9 (Figs. 41C-D) of culture.
  • Fig. 41 A and Fig. 41C show hematoxylin and eosin (H&E) staining;
  • Fig. 4 IB and Fig. 4 ID show reactivity with pan-cytokeratin (AE1/AE3) antibodies (brown color indicates a positive reaction). Arrowheads in Figs.
  • Hassall bodies which appear less prominent on H&E-stained sections of cultured thymus due to depletion and necrosis of surrounding thymocytes. However, Hassall bodies can still be readily identified by careful examination or by using immunohistochemistry. Scale bar in Figs. 41 A-D represents 100 pm.
  • Figs. 42A-D presents photomicrographs showing the architecture of cultured thymus, day 7.
  • Hematoxylin and eosin (H&E) staining in Figs. 42A-B shows marked depletion of thymocytes, although some cortical areas (Cor) still contain large numbers of thymocytes with retained nuclei.
  • Pan-cytokeratin (AE1/AE3) in Fig. 42C and cytokeratin 14 (CK14) immunohistochemistry in Fig. 42D show condensation of the thymic epithelium in the subcapsular cortex (SCC) and in the medulla (M). Brown color in Figs. 42C-D indicates a positive reaction with antibody.
  • Scale bar represents 1 mm in Fig. 42A and 500 pm in Figs. 42B-D.
  • FIGs. 43 A-D present photomicrographs showing the architecture of cultured thymus, day 9.
  • Figs. 43A-B show hematoxylin and eosin (H&E) staining. Few if any live T cells or thymic epithelial cells are present in the pale-staining area in Fig. 43 A that is enclosed by the dotted line, which is almost completely necrotic (Necr). Most nuclei formerly present in this region have been degraded via karyolysis. Other areas where the nuclei from residual thymocytes have not been completely degraded continue to stain dark blue with hematoxylin. Arrow in Fig. 43B points to a Hassall body. Fig.
  • FIG. 43C shows pan-cytokeratin (AE1/AE3) immunoreactivity (brown);
  • Fig. 43D shows cytokeratin 14 (CK14) immunoreactivity (brown).
  • Scale bar represents 1 mm in Fig. 43 A and 500 pm in Figs. 43B-D.
  • Figs. 44A-D presents photomicrographs showing the architecture of cultured thymus, day 12.
  • Figs. 44A-B show hematoxylin and eosin (H&E) staining. At this time point, many thymocytes have either been lost from the tissue or have died and their nuclei have been dissolved, making the tissue more eosinophilic (pink). Some areas retain architecture characteristic of normal uncultured thymus with cortical-like areas (Cor) that stain more basophilic (blue) and medullary-like areas (M), although with greatly decreased thymocyte cellularity.
  • Cor cortical-like areas
  • M medullary-like areas
  • Fig. 44C shows pan-cytokeratin (AE1/AE3) immunoreactivity (brown); Fig. 44D shows cytokeratin 14 (CK14) immunoreactivity (brown).
  • SCC sub-capsular cortex
  • Figs. 45A-D presents photomicrographs showing the architecture of cultured thymus, day 20.
  • Figs. 45A-B shows hematoxylin and eosin (H&E) staining. At this time point, most thymocytes have either been lost from the tissue or have died and their nuclei have been dissolved, making the tissue more eosinophilic (pink). Large groups of residual thymocytes are rare, although scattered cells with nuclear characteristics of thymocytes are evident.
  • Fig. 45C shows pan-cytokeratin (AE1/AE3) immunoreactivity (brown).
  • cytokeratin 14 (CK14) immunohistochemistry (brown) highlights former medullary areas and the subcapsular cortex. Arrows point to representative Hassall bodies. Scale bar represents 1 mm in Fig. 45A and 500 pm in Figs. 45B-D.
  • Figs. 46A-B present photomicrographs showing examples of intact nuclei in thymus slices. Examples of intact thymic epithelial cell nuclei (arrows) are shown in the subcapsular cortex on day 9 (Fig. 46A) and in the medulla on day 21 (Fig. 46B). Hematoxylin and eosin stain; scale bar represents 50 pm.
  • Figs. 47A-E present photomicrographs showing a comparison of the thymic epithelial network of cultured thymus tissue at different time points.
  • Fig. 47A shows day 0, Fig. 47B shows day 5, Fig. 47C shows day 9, Fig. 47D shows day 12, and Fig. 47E shows day 21.
  • Fig. 47A shows day 0, Fig. 47B shows day 5, Fig. 47C shows day 9, Fig. 47D shows day 12, and Fig. 47E shows day 21.
  • the structure of the thymic epithelial network (brown) remains intact as the culture progresses.
  • Both cortical and medullary epithelium may condense as intervening thymocytes are depleted. Brown color indicates a positive reaction with a cocktail of anti-cytokeratin antibodies (AE1/AE3); hematoxylin counterstain. Scale bar represents 400 pm.
  • AE1/AE3 anti-cytokeratin antibodies
  • Figs. 48A-K presents photomicrographs showing examples of CD3 immunohistochemistry in thymus slices as a function of time in culture.
  • Figs. 48A-B shows that on day 0, essentially all immature T cells in the cortex and more mature cells in the medulla react strongly with CD3 antibody.
  • Higher magnification shows pale blue nuclei surrounded by a ring of brown immunoreactivity, consistent with membrane expression of CD3.
  • tissue still shows extensive reactivity with CD3 antibody on day 7.
  • Figs. 48C-E tissue still shows extensive reactivity with CD3 antibody on day 7.
  • Figs. 48A-K presents photomicrographs showing examples of CD3 immunohistochemistry in thymus slices as a function of time in culture.
  • Figs. 48A-B shows that on day 0, essentially all immature T cells in the cortex and more mature cells in the medulla react strongly with CD3 antibody.
  • Higher magnification shows pale blue nuclei surrounded by a ring of
  • Scale bar represents 500 pm in Fig. 48A, Fig. 48C, Fig. 48F, Fig. 48H, and Fig. 48J, and 50 pm in Fig. 48B, Fig. 48D, Fig. 48E, Fig. 48G, Fig. 481, and Fig. 48K.
  • Figs. 49A-K presents photomicrographs showing examples of Ki-67 immunohistochemistry in thymus slices as a function of time in culture. The slices shown are all from a single lot that is representative of multiple lots examined at similar time points.
  • Figs. 49A-B shows that on day 0, the nuclei of the majority of immature T cells in the cortex (Cor) react strongly with antibody specific for Ki-67.
  • Higher magnification shows strong positive reactivity with the nuclei of cortical thymocytes (brown), whereas only rare lymphocytes in the medulla (M) react with Ki-67 antibody.
  • Figs. 49A-K presents photomicrographs showing examples of Ki-67 immunohistochemistry in thymus slices as a function of time in culture. The slices shown are all from a single lot that is representative of multiple lots examined at similar time points.
  • Figs. 49A-B shows that on day 0, the nuclei of the majority of immature T cells in the cortex (Cor)
  • FIG. 49C-E show that by day 7, the nuclei of most thymocytes that remain in cortical areas are small with indistinct nuclear borders consistent with apoptosis, and they fail to react with Ki-67-specific antibody.
  • the cells that react with antibody (Fig. 49E, arrows) have larger nuclei, suggesting that they are thymic epithelial cells.
  • a similar lack of Ki-67 labeling of residual thymocyte nuclei is seen on days 9 (Figs. 49F- G), 12 (Figs. 49H-I) and 21 (Figs. 49J-K).
  • Scale bar represents 500 pm in Fig. 49A, Fig. 49C, Fig. 49E, Fig. 49G, and Fig. 491 and 50 pm in Fig. 49B, Fig. 49D, Fig.49F, Fig. 4950H, and Fig. 49J.
  • Figs. 50A to Fig. 50H shows plots of selected soluble molecules detected in conditioned media from human thymus organ cultures.
  • Fig. 50A is a plot of L-selectin in Ln/pg versus days in culture
  • Fig. 50B is a plot of M-CSF in Ln/pg versus days in culture
  • Fig. 50C is a plot of galectin-7 in Ln/pg versus days in culture
  • Fig. ID is a plot of IL-16 in Ln/pg versus days in culture
  • Fig. 50E is a plot of CCL21 in Ln/pg versus days in culture
  • Fig. 50F is a plot of CXCL16 in Ln/pg versus days in culture
  • Fig. 50G is a plot of CCL11 in Ln/pg versus days in culture
  • Fig. 50H is a plot of CXCL12 in Ln/pg versus days in culture.
  • Fig. 51 shows plots assessing thymocyte content in cultured slices of human thymus in pg/ml from days 1 to 21 of the culturing process.
  • Fig. 52 shows photos of the immunohistochemical assessment of viable thymocytes in cultured slices of human thymus tissue as a function of time.
  • Fig. 52A is a photo of the immunohistochemistry with anti-CD3 antibodies that identify cells as T lineage and with anti-Ki- 67 antibody that identifies proliferating cells, demonstrates the rapid loss of thymocyte viability early in culture.
  • Fig. 52B is a photo on day 0 depicting the plasma membranes of essentially all immature T cells in the cortex and the medulla appear strongly reactive with anti-CD3 in a membrane pattern.
  • Fig. 52A is a photo of the immunohistochemistry with anti-CD3 antibodies that identify cells as T lineage and with anti-Ki- 67 antibody that identifies proliferating cells, demonstrates the rapid loss of thymocyte viability early in culture.
  • Fig. 52B is a photo on day 0 depicting the plasma membranes of essentially all immature
  • FIG. 52C depicts immunohistochemistry using antibody specific for the Ki- 67 proliferation marker shows abundant reactivity with cortical thymocytes on day.
  • Fig. 52D shows histology of cultured thymus on day 9, using hematoxylin and eosin stain. The decreased basophila (blue color) is indicative of loss of donor thymocytes during culture;
  • Fig. 52E depicts after several days of culture, the majority of the brown color is due to the anucleate, but still immunoreactive, debris that remains after dead thymocytes undergo karyolysis/nuclear dissolution.
  • Fig. 52F depicts thymocyte death that occurs during organ culture results in Ki-67 immunoreactivity only with larger cells morphologically consistent with TE cells at later time points during culture.
  • Fig. 53 A shows a plot of CCL21 levels in pg/ml of conditioned media from cultured thymus tissue versus the number of days in culture.
  • Fig. 53B is an illustration of the slicing procedure for thymus tissue to be subjected to conditioning.
  • Fig. 53C is a plot of the secretion of CCL21by slice of thymus organ tissue cultures as a function of time.
  • Figs. 54A to Fig. 54D are photographs depicting the immunoreactivity in cultured and non-cultured thymus tissue.
  • Fig. 54A is a photograph of the medullary region, but also includes TECS scattered throughout the cortex at Day 0 of culturing;
  • Fig. 54B is as photograph of the medullary region, but also includes TECS scattered throughout the image at Day 16 of culturing;
  • Fig. 54C is a photograph of TECs in medullary regions as well as in scattered TECs in cortical areas on Day 0 of culturing.
  • Fig. 54D is a photograph of TECs in medullary regions as well as in scattered TECs in cortical areas on Day 16 of culturing.
  • Figs. 55A to Fig. 55F are plots of the expression of selected mRNAs in thymus tissue from across the lifespan.
  • the relative amounts of the target mRNAs present in FFPE sections of thymus tissue were quantitated using the QuantiGene assay (Thermo Fisher) in accordance with the manufacturer’s directions.
  • Data for each target mRNA is presented normalized to GAPDH (“Not adjusted”), then further normalized to the % area containing thymic epithelium (“Adjusted by TE”) or to the % area containing CD la-positive cortical thymocytes (“Adjusted by Cor”).
  • Fig. 55C and 55D are photos depicting mRNAs encoding cytokeratins 8 (KRT8) and 14 (KRT14) were decreased relative to GAPDH in donors ⁇ 18 years compared with older adults (Fig. 55C, D), respectively.
  • Fig. 55C and 55D are photos depicting mRNAs encoding
  • Fig. 55E is a photo depicting unadjusted CCL21 gene expression which was consistently low relative to GAPDH in thymus from donors ⁇ 18 years of age.
  • Fig. 55F is a photo depicting unadjusted CXCL21 gene expression which was consistently low relative to GAPDH in thymus from donors ⁇ 18 years of age.
  • Fig. 56 is a table of proteins present in spent media of thymic organ cultures, as determined by multiplex antibody arrays.
  • Figs. 57A to 57C are photographs of morphological measurements of thymus tissues. Areas included in each measurement were outlined using the “pen tool” provided by the ImageScope software (Aperio Technologies, Leica Biosystems imaging, Inc.) in accordance with the manufacturer’s instructions.
  • Fig. 57A shows the total area of thymus tissue on the H&E- stained slide outlined in green. The proportion of this area that contains lymphocytes is further outlined in aqua.
  • Fig. 57B is a photo showing areas containing thymic epithelium (“TE area”) outlined in yellow on the section reacted with AE1/AE3 cocktail to identify pan-cytokeratins.
  • TE area thymic epithelium
  • Fig. 57C is a photo showing Areas containing immature thymocytes (“cortical area”) outlined in red on the section reacted with CDla antibody.
  • brown color indicates a positive reaction with antibody.
  • Figs. 58A to 58C are photographs depicting the few viable thymocytes in thymus tissue slices cultured for 21 days.
  • Fig. 58A is a photo depicting thymus slices containing few intact thymocytes on day 21 of culture, as indicated by the marked lack of basophilia (blue color) in H&E sections.
  • Fig. 58B is a photo depicting cultured thymus tissue slices in which most of the strong brown immunoreaction seen with CD3 immunohistochemistry is associated with anucleate cellular debris, although dead thymocytes that exhibit nuclear and cytoplasmic staining characteristic of necrotic cells that have not yet undergone karyolysis (inset) are not uncommon.
  • Fig. 58C is a photo depicting Ki-67 immunoreactivity on day 21 is limited to cells with larger nuclei that are characteristic of thymic epithelial cells. Bar represents 300 pm in the main panels and 50 pm in the insets.
  • Figs. 59A to 59C are graphs showing characteristics of human thymus tissues used for gene expression analysis.
  • Fig. 59A depicts the age and sex distribution of the thymus tissues studied, with the lower black-filled circles designating females, upper open circles designating males, and the middle gray circle designating the one donor of unknown sex.
  • Fig. 59B plots the % area containing thymic epithelial cells as a function of age for this panel of thymus tissues.
  • Fig, 59C plots the % area with active thymopoiesis as defined by CD la-positive thymocytes as a function of age for this panel of thymus tissues.
  • Fig. 60 is a photograph of freshly harvested thymus tissue.
  • Figs. 61 A and 61B depict the histology of thymus tissue slides after exposure to forced degradation conditions of 10X PBS.
  • Fig. 61 A depicts the cortex at day 9 after exposure to forced degradation conditions.
  • Fig. 6 IB depicts the cortex at day 21 after exposure to forced degradation conditions.
  • Fig. 61 A the smear of blue is DNA released from cells. The majority of cells show evidence of degradation although small foci of cells with intact nuclei can be identified. Photo by Laura P. Hale, MD, PhD, Department of Pathology, Duke University. [00324] Fig.
  • Fig.62A shows a schematic showing the slicing of thymus tissue for characterization testing, as discussed in section [00520]
  • Fig.62B is a figure showing slices of thymus tissue on cellulose filters on surgical sponges in a tissue culture dish as is used for culture of the thymus.
  • Figs. 63 A to 63H depict histology testing of thymus tissue slices from a lot (MFG-056) of cultured thymus tissue on day 5, 9, 12 and 21 after harvest of the thymus from a donor.
  • Hematoxylin and eosin-stained slices (left panels) and their corresponding reactivity with a cocktail of the anti-cytokeratin antibodies AE1/AE3 (right panels; brown color denotes positive reactivity) are shown at day 5 (Fig. 63 A, Fig. 63B), day 9 (Fig. 63C, Fig. 63D), day 12 (Fig. 63E, Fig. 63F), and day 21 (Fig 63G, Fig. 63H), respectively. Bars in the lower left of each panel represent 100 pm. Panels with H&E show progression depletion of T cells with time. Fig. 14E and Fig. 63F are predominantly epithelial cells.
  • Figs. 64A and 64B depict the histology of thymus tissue slices on day 0 of the time course in a scale of 5 mm (Fig. 64A) and 100 pm (Fig. 64B), respectively. This shows the thymus and thymocytes at low power (bar 5 mm) and high power (bar 100 um) on day 0. This is normal thymus. At this time the cortex and medulla both have large numbers of thymocytes with dark blue nuclei contributing to the overall dark blue appearance of the tissue. Photo by Laura P. Hale, MD, PhD, Department of Pathology, Duke University.
  • Figs. 65A and 65B are images from H&E stained slide that depict the histology of thymus tissue slices on day 5 of the time course in a scale of 5 mm (Fig. 65 A) and 100 pm (Fig. 65B), respectively. Progression of depletion of the thymocytes results in a more eosinophilic (pink) appearance of the tissue. Photo by Laura P. Hale, MD, PhD, Department of Pathology, Duke University.
  • Figs. 66A and Fig. 66B depict H&E staining of the thymus tissue slices on day 12 of the time course in a scale of 5 mm (Fig. 66A) and 100 pm (Fig. 66B), respectively.
  • Fig. 66A depicts H&E staining of the thymus tissue slices on day 12 of the time course in a scale of 5 mm (Fig. 66A) and 100 pm (Fig. 66B), respectively.
  • Fig. 66A depict H&E staining of the thymus tissue slices on day 12 of the time course in a scale of 5 mm (Fig. 66A) and 100 pm (Fig. 66B), respectively.
  • Fig. 66B depicts H&E staining of the thymus tissue slices on day 12 of the time course in a scale of 5 mm (Fig. 66A) and 100 pm (Fig. 66B), respectively.
  • Fig. 66B depicts H&E staining of the
  • Fig. 67A and Fig. 67B depict H&E staining of thymus tissue slices on day 21 of the time course in a scale of 5 mm (Fig. 67A) and 100 pm (Fig. 67B), respectively. Note the preservation of the overall architecture of the tissue including in Fig. 67B the subcapsular cortex, cortical region and medullary region containing numerous Hassall bodies. The small dark cells are mostly necrotic thymocytes that have not yet undergone karyolysis. Photo by Laura P. Hale, MD, PhD, Department of Pathology, Duke University.
  • Figs. 68A-E depict representative thymus slices which were immuno-stained with a cocktail of anti-cytokeratin antibodies (AE1/AE3).
  • Fig. 68A Day 0; Fig. 68B. Day 5; Fig. 68C. Day 9; Fig. 68D. Day 12; and Fig. 68E. Day 21.
  • the structure of the thymic epithelial network remains intact as the culture progresses. Bar represents 400 pm. Photo by Laura P. Hale, MD, PhD, Department of Pathology, Duke University.
  • Fig. 69 presents photomicrographic images and a graph of CCL21 assessment in cultured infant thymus.
  • CCL21 is produced at high levels by cultured infant thymus.
  • Immunohistochemical reactivity with CCL21 antibody (Ab) (brown staining) on day 16 of culture is shown in the left panels (upper left panel, 2X magnification; lower left panel, 20X magnification).
  • a corresponding time course measuring daily CCL21 secretion into culture media is shown on the right for 3 infant thymus cultures (R&D Systems Duo-Set ELISA).
  • the cultured thymus tissue can produce a functionally important biomolecule, CCL21, the chemokine responsible for attracting immature thymocyte precursors to the thymus.
  • Figs. 70A to 70D presents photomicrographs of slices of fresh thymus, dO of culture, showing thymic architecture.
  • hematoxylin and eosin (H&E) staining shows well-defined cortical and lighter-staining medullary areas, as expected for normal pediatric thymus.
  • Fig. 70C shows immunohistochemistry with a cocktail of pan-cytokeratin antibodies (AE1/AE3) that together detect all types of epithelial cells demonstrates that thymic epithelial cells are present beneath the capsule and in a light lacy network in both cortex and medulla (brown staining shows positive antibody reaction). Arrows in Fig.
  • Fig. 70D shows Cytokeratin 14 (CK14) antibody staining (brown).
  • CK14 antibody reacts with thymic epithelial cells in the sub-capsular cortex and in the medulla, as well as with scattered thymic epithelial cells in the cortex.
  • the dotted line highlights an area of medulla that is surrounded by cortex.
  • SCC denotes sub-capsular cortex
  • Cor denotes cortex
  • M denotes medulla.
  • Scale bar in Fig. 70A represents 1 mm; scale bars in Figs. 70B-D represent 500 pm.
  • Figs. 71 A to 71D present photomicrographs showing examples of Hassall bodies in cultured thymic slices. The histologic appearance of Hassall bodies is shown on day 0 (Figs. 71A-B) and day 9 (Figs. 71C-D) of culture.
  • Fig. 71A and Fig. 71C show hematoxylin and eosin (H&E) staining;
  • Fig. 71B and Fig. 71D show reactivity with pan-cytokeratin (AE1/AE3) antibodies (brown color indicates a positive reaction). Arrowheads in Figs.
  • Hassall bodies which appear less prominent on H&E-stained sections of cultured thymus due to depletion and necrosis of surrounding thymocytes. However, Hassall bodies can still be readily identified by careful examination or by using immunohistochemistry. Scale bar in Figs. 71A-D represents 100 pm.
  • Figs. 72A to 72D presents photomicrographs showing the architecture of cultured thymus, day 7.
  • Hematoxylin and eosin (H&E) staining in Figs. 72A-B shows marked depletion of thymocytes, although some cortical areas (Cor) still contain large numbers of thymocytes with retained nuclei.
  • Pan-cytokeratin (AE1/AE3) in Fig. 72C and cytokeratin 14 (CK14) immunohistochemistry in Fig. 72D show condensation of the thymic epithelium in the subcapsular cortex (SCC) and in the medulla (M). Brown color in Figs. 72C-D indicates a positive reaction with antibody.
  • Scale bar represents 1 mm in Fig. 23 and 500 pm in Figs. 72B- D.
  • Figs. 73 A to 73D present photomicrographs showing the architecture of cultured thymus, day 9.
  • Figs. 73A-B show hematoxylin and eosin (H&E) staining. Few if any live T cells or thymic epithelial cells are present in the pale-staining area in Fig. 73 A that is enclosed by the dotted line, which is almost completely necrotic (Necr). Most nuclei formerly present in this region have been degraded via karyolysis. Other areas where the nuclei from residual thymocytes have not been completely degraded continue to stain dark blue with hematoxylin. Arrow in Fig.
  • Fig. 73B points to a Hassall body.
  • Fig. 73C shows pan-cytokeratin (AE1/AE3) immunoreactivity (brown);
  • Fig. 73D shows cytokeratin 14 (CK14) immunoreactivity (brown).
  • Scale bar represents 1 mm in Fig. 73 A and 500 pm in Figs. 73B-D.
  • Figs. 74A to 74D presents photomicrographs showing the architecture of cultured thymus, day 12.
  • Figs. 74A-B show hematoxylin and eosin (H&E) staining. At this time point, many thymocytes have either been lost from the tissue or have died and their nuclei have been dissolved, making the tissue more eosinophilic (pink). Some areas retain architecture characteristic of normal uncultured thymus with cortical-like areas (Cor) that stain more basophilic (blue) and medullary-like areas (M), although with greatly decreased thymocyte cellularity.
  • 74C shows pan-cytokeratin (AE1/AE3) immunoreactivity (brown); Fig. 74D shows cytokeratin 14 (CK14) immunoreactivity (brown).
  • SCC sub-capsular cortex
  • Figs. 75A-B shows hematoxylin and eosin (H&E) staining. At this time point, most thymocytes have either been lost from the tissue or have died and their nuclei have been dissolved, making the tissue more eosinophilic (pink). Large groups of residual thymocytes are rare, although scattered cells with nuclear characteristics of thymocytes are evident.
  • Fig. 75C shows pan-cytokeratin (AE1/AE3) immunoreactivity (brown).
  • cytokeratin 14 (CK14) immunohistochemistry (brown) highlights former medullary areas and the subcapsular cortex. Arrows point to representative Hassall bodies. Scale bar represents 1 mm in Fig. 75A and 500 pm in Figs. 75B-D.
  • Figs. 76A-B present photomicrographs showing examples of intact nuclei in thymus slices. Examples of intact thymic epithelial cell nuclei (arrows) are shown in the subcapsular cortex on day 9 (Fig. 76A) and in the medulla on day 21 (Fig. 76B). Hematoxylin and eosin stain; scale bar represents 50 pm.
  • Figs. 77A to 77E present photomicrographs showing a comparison of the thymic epithelial network of cultured thymus tissue at different time points.
  • Fig. 77A shows day 0, Fig. 77B shows day 5, Fig. 77C shows day 9, Fig. 77D shows day 12, and Fig. 77E shows day 21.
  • Fig. 77A shows day 0
  • Fig. 77B shows day 5
  • Fig. 77C shows day 9
  • Fig. 77D shows day 12
  • Fig. 77E shows day 21.
  • Both cortical and medullary epithelium may condense as intervening thymocytes are depleted. Brown color indicates a positive reaction with a cocktail of anti-cytokeratin antibodies (AE1/AE3); hematoxylin counterstain. Scale bar represents 400 pm.
  • Figs. 78A-K presents photomicrographs showing examples of CD3 immunohistochemistry in thymus slices as a function of time in culture.
  • Figs. 78A-B shows that on day 0, essentially all immature T cells in the cortex and more mature cells in the medulla react strongly with CD3 antibody.
  • Higher magnification shows pale blue nuclei surrounded by a ring of brown immunoreactivity, consistent with membrane expression of CD3.
  • tissue still shows extensive reactivity with CD3 antibody on day 7.
  • Figs. 78C-E tissue still shows extensive reactivity with CD3 antibody on day 7.
  • Fig. 78D-E shows that when viewed under higher magnification, the majority of the immunoreaction (brown) is associated with debris from dead thymocytes, as most brown foci lack evidence of nuclei (Fig. 78D). Small foci of cells that demonstrate intact nuclei and membrane staining (arrows) can still be identified in areas away from the debris (Fig. 78E). As cultures progress through day 9 (Figs. 78F-G), day 12 (Figs. 78H-I), and day 21 (Figs. 78J-K), reactivity with thymocyte cellular debris remains strong, making it difficult to reliably detect potentially intact cells amidst the debris. The slices shown are all from a single lot that is representative of multiple lots examined at these time points.
  • Scale bar represents 500 pm in Fig. 78A, Fig. 78C, Fig. 78F, Fig. 78H, and Fig. 78J, and 50 pm in Fig. 78B, Fig. 78D, Fig. 78E, Fig. 78G, Fig. 781, and Fig. 78K.
  • Figs. 79A-K presents photomicrographs showing examples of Ki-67 immunohistochemistry in thymus slices as a function of time in culture. The slices shown are all from a single lot that is representative of multiple lots examined at similar time points.
  • Figs. 79A-B shows that on day 0, the nuclei of the majority of immature T cells in the cortex (Cor) react strongly with antibody specific for Ki-67.
  • Higher magnification Fig. 79B
  • Figs. 79A-K presents photomicrographs showing examples of Ki-67 immunohistochemistry in thymus slices as a function of time in culture. The slices shown are all from a single lot that is representative of multiple lots examined at similar time points.
  • Figs. 79A-B shows that on day 0, the nuclei of the majority of
  • 79C-E show that by day 7, the nuclei of most thymocytes that remain in cortical areas are small with indistinct nuclear borders consistent with apoptosis, and they fail to react with Ki-67-specific antibody.
  • the cells that react with antibody (Fig. 79E, arrows) have larger nuclei, suggesting that they are thymic epithelial cells.
  • a similar lack of Ki-67 labeling of residual thymocyte nuclei is seen on days 9 (Figs. 79F- G), 12 (Figs. 79H-I) and 21 (Figs. 79J-K).
  • Scale bar represents 500 pm in Fig. 79A, Fig. 79C, Fig. 79E, Fig. 79G, and Fig. 791 and 50 pm in Fig. 79B, Fig. 79D, Fig. 79F, Fig. 79H, and Fig. 79J.
  • Fig 80 is a plot of uPAR detected in conditioned media from human thymus organ cultures.
  • Fig. 81 is a plot of OPN detected in conditioned media from human thymus organ cultures.
  • Fig. 82 is a plot of MIP3a detected in conditioned media from human thymus organ cultures.
  • Fig. 83 is a plot of IGFBP-1 detected in conditioned media from human thymus organ cultures.
  • Fig. 84 is a plot of MIF detected in conditioned media from human thymus organ cultures.
  • Fig. 85 is a scatterplot of CCL21 Concentration in Spent Media vs. Day.
  • Fig. 86 is a boxplot of CCL21 Concentration in Spent Media vs. Day.
  • Fig. 87 is a graph of Figure 4: CCL21 Levels (pg/mL) in Forced Degradation Study - LotMFG-053 and Lot-054.
  • Fig. 88 is a graph of CCL21 Levels (pg/mL) in Forced Degradation Study - Lot MFG- 066.
  • Fig. 89 is a scatterplot of CXCL16 Concentration in Spent Media vs. Day.
  • Fig. 90 is a boxplot of CXCL16 Concentration in Spent Media vs. Day.
  • Fig. 91 is a scatterplot of CXCL16 Levels (pg/mL) in Forced Degradation Study - Lot MFG-053 and Lot MFG-054.
  • Fig. 92 is a scatterplot of CXCL21 Levels in Forced Degradation Study - Lot MFG- 066.
  • Fig. 93 is a scatterplot of L-Selectin Concentration in Spent Media vs. Day.
  • Fig. 94 is a boxplot of L-Selectin Concentration in Spent Media vs. Day.
  • Fig. 95 is a scatterplot of L-Selectin Levels (pg/mL) in Forced Degradation Study - Lot MFG-053 and Lot MFG-054.
  • Fig. 96 is a scatterplot of L-Selectin Levels in Forced Degradation Study - Lot MFG- 066.
  • Fig. 97 is a scatterplot of uPAR Concentration in Spent Media vs. Day.
  • Fig. 98 is a boxplot of uPAR Concentration in Spent Media vs. Day.
  • Fig. 99 is a scatterplot of uPAR Levels (pg/mL) in Forced Degradation Study - Lot
  • Fig. 100 is a scatterplot of uPAR Levels in Forced Degradation Study - Lot MFG-066.
  • Fig. 101 is a scatterplot of CXCL16 vs. CCL21.
  • Fig. 102 is a quadratic regression model of CCL21 vs. uPAR.
  • Fig. 103 is a quadratic regression model of CXCL16 vs. uPAR.
  • Fig. 104 is a scatterplot of CCL11 Concentration in Spent Media vs. Day.
  • Fig. 105 is a boxplot of CCL11 Concentration in Spendt Media vs. Day.
  • Fig. 106 is a linear regression model of CCL11 Concentration in Spent Media vs. Day.
  • Fig. 107 is a plot of CCL11 Levels (pg/mL) in Forced Degradation Study - Lot MFG- 053 and LotMFG-054.
  • Fig. 108 is a lot of CCL11 Levels in Forced Degradation Study-Lot MFG-066.
  • Fig. 109 is a scatterplot of OPN Concentration in Spent Media vs. Day.
  • Fig. 110 is a boxplot of OPN Concentration in spent Media vs. Day.
  • Fig. Ill is a quadratic regression model of OPN Concentration in Spent Media vs. Day.
  • Fig. 112 is a plot of OPN Levels (pg/mL) in Forced Degradation Study-Lot MFG-053 and Lot MFC-054.
  • Fig. 113 is a plot of OPN Levels in Forced Degradation Study-Lot MFG-066.
  • Fig. 114 is a scatterplot of CXCL12 Concentration in Spent Media vs. Day.
  • Fig. 115 is a boxplot of CXCL12 Concentration in Spent Media vs. Day.
  • Fig. 116 is a fitted line plot of CXCL12 Concentration in Spent Media vs. Day.
  • Fig. 117 is a scatterplot of CXCL12 Levels (pg/mL) in Forced Degradation Study-Lot MFG-053 and Lot MFG-054.
  • Fig. 118 is a scatterplot of CXCL12 Levels in Forced Degradation Study — Lot MFG- 066.
  • Fig. 119 is a scatterplot of CCL20 Concentration in Spent Media vs. Day.
  • Fig. 120 is a boxplot of CCL20 Concentration in Spent Media vs. Day.
  • Fig. 121 is a cubic regression model of CCL20 Concentration in Spent media vs. Day.
  • Fig. 122 is a scatterplot of CCL20 Levels (pg/mL) in Forced Degradation Study-Lot MFG-053 and Lot MFG-054.
  • Fig. 123 is a scatterplot of CCL20 Levels in Forced Degradation Study-Lot MFG-066.
  • Fig. 124 is a scatterplot of IL-16 Concentration in Spent Media vs. Day.
  • Fig. 125 is a boxplot of IL-16 Concentration in Spent Media vs. Day.
  • Fig. 126 is a fitted line plot of IL-16 Concentration in Spent Media vs. Day.
  • Fig. 127 is a scatterplot of IL-16 Levels (pg/mL) in Forced Degradation Study - Lot MFG-053 and Lot MFG-054.
  • Fig. 128 is a scatterplot of IL-16 Levels in Forced Degradation Study-Lot MFG-066.
  • Fig. 129 is a scatterplot of IGFBP-1 Concentration in Spent Media vs. Day.
  • Fig. 130 is a boxplot of IGFBP-1 Concentration in Spent Media vs. Day.
  • Fig. 131 is a fitted line plot of IGFBP-1 Concentration in Spent Media vs. Day.
  • Fig. 132 is a scatterplot of IGFBP-1 Levels (pg/mL) in Forced Degradation Study-Lot MFG-053.
  • Fig. 133 is a scatterplot of OGFBP-1 Levels in Forced Degradation Study-Lot MFG- 066.
  • Fig. 134 is a scatterplot of MIF Concentration in Spent Media vs. Day.
  • Fig. 135 is a boxplot of MIF Concentration in Spent Media vs. Day.
  • Fig. 136 is a linear regression model of MIF Concentration in Spent Media vs. Day.
  • Fig. 137 is a scatterplot of MIF Levels (pg/mL) in Forced Degradation Study-Lot- MFG-053 and Lot MFG-054.
  • Fig. 138 is a scatterplot of MIF Levels in Forced Degradation Study-Lot MFG-066.
  • Fig. 139 is a scatterplot of CCL25 Concentration in Spent Media vs. Day.
  • Fig. 140 is a boxplot of CCL25 Concentration in Spent Media vs. Day.
  • Fig. 141 is a linear regression model plot of MIF Concentration in Spent Media vs. Day.
  • Fig. 142 is a scatterplot of CCL25 Levels (pg/mL) in Forced Degradation Study-Lot- 053 and Lot-054.
  • Fig. 143 is a scatterplot of CCL25 Levels in Forced Degradation Study-Lot MFG-066.
  • the term “about” is used herein to mean approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of +/- 10%. As used herein, the term about refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated.
  • the term about generally refers to a range of numerical values (e.g., +/-5-10% of the recited range) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result).
  • the terms modify all of the values or ranges provided in the list.
  • the term about may include numerical values that are rounded to the nearest significant figure.
  • animal includes, but is not limited to, humans and non human vertebrates such as wild, domestic, and farm animals.
  • the animal can also be referred to as a "subject.”
  • biomarkers refer to the substances listed in Fig. 56. It is to be further understood that references to “decreased” and “increased” levels with respect to the appearance of the biomarkers in the thymus organ medium refers to increased measurements or decreasaed measurements of the particular biomarker over the time course of the conditioning regimen. [00413] As used here, “biocompatible” refers to any material, which, when implanted in a mammal, does not provoke an adverse response in the mammal.
  • a "graft” refers to a tissue or organ that is implanted into an individual, typically to replace, correct or otherwise overcome a defect.
  • the tissue or organ may consist of cells that originate from the same individual; this graft is referred to herein by the following interchangeable terms: “autograft”, “autologous transplant”, “autologous implant” and “autologous graft”.
  • autograft a tissue or organ that is implanted into an individual, typically to replace, correct or otherwise overcome a defect.
  • autograft autograft
  • autologous transplant autologous implant
  • autologous graft autologous graft
  • a graft from a genetically different individual of the same species is referred to herein by the following interchangeable terms: “allograft”, “allogeneic transplant”, “allogeneic implant” and “allogeneic graft”.
  • a graft from an individual to his identical twin is referred to herein as an "isograft", a “syngeneic transplant", a “syngeneic implant” or a “syngeneic graft”.
  • a "xenograft”, “xenogeneic transplant” or “xenogeneic implant” refers to a graft from one individual to another of a different species.
  • HLA matched refers to a donor recipient pair in which none of the HLA antigens are mismatched between the donor and recipient.
  • HLA matching in the methods of the invention comprise: HLA alleles: HLA-A, HLA-B, HLA-C, HLA-DRBl, HLA- DQB1, HLA-DQA1, HLA-DPBl, and HLA-DPAl.
  • HLA mismatched refers to matching in a donor and recipient HLA antigens, typically with respect to HLA-A, HLA-B, HLA-C, HLA-DRBl, HLA-DQBl, HLA-DQAl, HLA-DPBl, and HLA-DPAl wherein an HLA mismatch between the donor and recipient occurs.
  • HLA-A HLA-B
  • HLA-C HLA-DRBl
  • HLA-DQBl HLA-DQBl
  • HLA-DQAl HLA-DPBl
  • HLA-DPAl HLA mismatched
  • HLA antigens correspond to “human leukocyte antigens,” which are protein molecules expressed on the surface of cells that confer a unique antigenic identity to these cells. They are also known as “major histocompatibility complex antigens.” Thus the MHC or HLA antigens are target molecules that are recognized by T-cells as being “self’ or “non-self.” If the HLA antigens are derived from the same source of hematopoietic stem cells as the immune effector cells they are considered “self.” If, the HLA antigens are derived from another source of hematopoietic reconstituting cells, they are considered “non-self.”
  • HLA-Class I and HLA-Class II Two main classes of HLA antigens are recognized: HLA-Class I and HLA-Class II.
  • HLA-Class I antigens A, B, and C in humans
  • HLA- Class II antigens DRB1, DPB1, DPA1, DQB1, and DQA1 in humans
  • DRB1, DPB1, DPA1, DQB1, and DQA1 are involved in reactions between lymphocytes and antigen presenting cells. Both classes of HLA antigens have been implicated as targets of rejection of transplanted organs.
  • HLA genes are clustered on human chromosome position 6p21. This cluster of genes encodes the six classical transplantation HLA genes. The segment of 6p21 also encodes genes encoding proteins having important roles in the regulation of the immune system and other fundamental molecular and cellular processes. The complete cluster measures roughly 3.6 Mb, with at least 224 gene loci.
  • haplotypes occur (the set of alleles present on a single chromosome). The haplotypes inherited from one parent tend to be inherited as a group. The set of alleles inherited from each parent forms a haplotype, in which some alleles tend to be associated together. HLA matching is used to identify the recipient’s haplotypes and help in identifying suitable matching donors. Certain haplotypes are more prevalent than others and they vary in frequency in different racial and ethnic groups.
  • the phrase "in need thereof means that the subject has been identified as having a need for the particular method or treatment. In some embodiments, the identification can be by any means of diagnosis. In any of the methods and treatments described herein, the subject can be in need thereof.
  • integer from X to Y means any integer that includes the endpoints.
  • integer from X to Y means 1, 2, 3, 4, or 5.
  • the term "mammal” means a rodent (i.e., a mouse, a rat, or a guinea pig), a monkey, a cat, a dog, a cow, a horse, a pig, or a human. In some embodiments, the mammal is a human.
  • the term "organ” refers to a solid vascularized organ that performs a specific function or group of functions within an organism.
  • organ includes, but is not limited to heart, lung, kidney, liver, pancreas, skin, uterus, bone, cartilage, small or large bowel, bladder, brain, breast, blood vessels, esophagus, fallopian tube, gallbladder, ovaries, pancreas, prostate, placenta, spinal cord, limb including upper and lower, spleen, stomach, testes, thymus, thyroid, trachea, ureter, urethra, uterus.
  • the terms “prevent”, “preventing” and “prevention” refer to the administration of therapy to an individual who may ultimately manifest at least one symptom of a disease, disorder, or condition, but who has not yet done so, to reduce the chance that the individual will develop the symptom of the disease, disorder, or condition over a given period of time. Such a reduction may be reflected, for example, in a delayed onset of the at least one symptom of the disease, disorder, or condition in the patient.
  • the terms "subject,” “individual” or “patient,” used interchangeably, means any animal, including mammals, such as mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, such as humans.
  • the phrase "therapeutically effective amount” means the amount of active compound or pharmaceutical agent that elicits the biological or medicinal response that is being sought in a tissue, system, animal, individual or human by a researcher, veterinarian, medical doctor or other clinician.
  • the therapeutic effect is dependent upon the disorder being treated or the biological effect desired.
  • the therapeutic effect can be a decrease in the severity of symptoms associated with the disorder and/or inhibition (partial or complete) of progression of the disorder, or improved treatment, healing, prevention or elimination of a disorder, or side-effects.
  • the amount needed to elicit the therapeutic response can be determined based on the age, health, size and sex of the subject. Optimal amounts can also be determined based on monitoring of the subject's response to treatment.
  • tissue refers to any type of tissue in human or animals, and includes, but is not limited to, vascular tissue, skin tissue, hepatic tissue, pancreatic tissue, neural tissue, urogenital tissue, gastrointestinal tissue, skeletal tissue including bone and cartilage, adipose tissue, connective tissue including tendons and ligaments, amniotic tissue, chorionic tissue, dura, pericardia, muscle tissue, glandular tissue, facial tissue, ophthalmic tissue.
  • tissue bank in the context of the present disclosure refers to long-term storage of cryopreserved allogeneic cultured postnatal thymus tissue-derived product stored under liquid nitrogen.
  • General guidance for establishment of a repository for allogeneic cultured postnatal thymus tissue-derived product may be drawn from 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/GuidanceComplianceRegulatoryInfor mation/Guidances/Tissue/UCM285223.pdf.
  • CGTP Current Good Tissue Practice
  • HCT/Ps Cellular and Tissue-Based Products
  • Tissue engineer(ing)(-ed) refers to the process of generating tissues ex vivo for use in tissue replacement or reconstruction. Tissue engineering is an example of “regenerative medicine” which encompasses approaches to the repair or replacement of tissues and organs by incorporation of cells, genes or other biological building blocks, along with bioengineered materials and technologies.
  • the term "transplant rejection” encompasses both acute and chronic transplant rejection.
  • Acute rejection is the rejection by the immune system of a tissue transplant recipient when the transplanted tissue is immunologically foreign. Acute rejection is characterized by infiltration of the transplant tissue by immune cells of the recipient, which carry out their effector function and destroy the transplant tissue.
  • acute rejection The onset of acute rejection is rapid and generally occurs in humans within a few weeks after transplant surgery. Generally, acute rejection can be inhibited or suppressed with immunosuppressive drugs such as rapamycin, cyclosporin A, anti- CD40L monoclonal antibody and the like.
  • immunosuppressive drugs such as rapamycin, cyclosporin A, anti- CD40L monoclonal antibody and the like.
  • the terms “treat,” “treated,” or “treating” mean both therapeutic treatment and prophylactic measures wherein the object is to slow down (lessen) an undesired physiological condition, disorder or disease, or obtain beneficial or desired clinical results.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of extent of condition, disorder or disease; stabilized (i.e., not worsening) state of condition, disorder or disease; delay in onset or slowing of condition, disorder or disease progression; amelioration of the condition, disorder or disease state or remission (whether partial or total), whether detectable or undetectable; an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient; or enhancement or improvement of condition, disorder or disease.
  • any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. Ranges are approximate and may vary by more than an integer. [00435] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Measured values are understood to be approximate, taking into account significant digits and the error associated with the measurement.
  • SI Systeme International de Unites
  • Donor thymus tissue may be may be discarded during post-natal heart operations and may be used for CTT with informed consent of the donor's family. The removal of some thymus may be necessary to reveal the operative site. Thus, a portion of the thymus may be removed during the heart operation in post-natal heart surgeries, due to the nature of the surgical procedure.
  • thymus tissue may be discarded during the surgical procedure.
  • surgeon places the thymus tissue that has been discarded into a sterile container.
  • Thymus tissue donors for thymus tissue transplantation in infants with complete DiGeorge syndrome have been infants under nine months of age.
  • the drug substance allogeneic cultured postnatal thymus tissue-derived product is manufactured by processing and culturing the discarded thymus tissue as described herein.
  • Consent for use of the thymus in cultured thymus tissue implantation may be obtained before or after the thymus is harvested. However, consent allowing blood to be obtained from the infant prior to undergoing bypass is necessary and is always obtained prior to the surgery. This blood sample is used for donor screening.
  • tissue type matching is not required in the surgical procedures described herein, but such tissue type matching can be performed in certain situations.
  • Tissue may be processed immediately or stored refrigerated overnight for next day processing. If the thymus tissue is to be stored overnight, the tissue is aseptically added to thymus organ medium (“TOM” medium described below) sufficient to completely cover the thymus tissue in the original container. The container with the thymus is placed in refrigerator until ready to process the next day.
  • TOM thymus organ medium
  • the conditioning regimen depletes the donor thymocytes from the cultured thymus tissue slices. Based on in vitro data (immunohistochemistry) a culture period between 12 and 21 days preserves the epithelial network as assessed using cytokeratin antibodies. The culturing is preferably done at 37°C in a 5% CO2 incubator. [00444] For successful culture, the thymus tissue is preferably sliced and put on Millipore® cellulose or equivalent filters and placed on surgical sponges in tissue culture dishes. The medium comprises the thymus organ medium (TOM) and is changed daily.
  • TOM thymus organ medium
  • the thymus on receipt is assessed by pathology.
  • the test for identity must show >50% of areas positive for keratin in lacy staining pattern.
  • the test for potency must show Hassall bodies; it must also show CK14 staining in lacy pattern.
  • the test for viability must show >90% intact nuclei observed in sections.
  • the lot release for the tissue is done on one day between day 5 and day 21 (inclusive) and is performed by pathology.
  • For identity areas on tissue between days 5 and 21 must be positive for keratin, AE1/AE3.
  • the cultured thymus tissue between days 5 and 21 must show cytokeratin CK14 staining scattered throughout, and there must be at least one Hassall body identified.
  • the cultured thymus tissue between days 5 and 21 must show intact nuclei.
  • the thymus tissue slices are conditioned for about 12 days and then cryopreserved. In another embodiment all of the thymus tissue slices are conditioned for about 12 days, then about half are implanted in the recipient and the remaining thymus tissue slices are cryopreserved for future use.
  • the thymus is cut into thin slices.
  • the slices are held in culture for 12-21 days.
  • This culturing process depletes viable donor T cells and ultimately enables the surgically implanted tissue slices to reconstitute the athymic subject’s immune system, albeit at a immunologically effective level, although most subjects will have T cell counts below the 10th percentile for age.
  • the culturing process as outlined below, significantly modifies the biological characteristics of the donor thymus tissue and constituent cells contained therein in the following manner to optimize the effective therapeutic properties of the CTT slices.
  • the culturing process assures that a defined composition of the cultured cells/tissue having the pre-requisite biological characteristics is obtained in a manner suitable for surgical implantation into a subject to enable reconstitution of the subject’s immune system.
  • the culturing process results in a loss of thymocytes and relative enrichment of thymic epithelial cells and other stromal cells in the donor thymus tissue slices.
  • the culturing process further results in depletion of thymocytes and maintenance of TECs to enable reconstitution of the recipient’s immune system and allows tolerance to develop in the recipient to HLA antigens in the donor thymus.
  • the manufacturing process is designed to deplete thymocytes from the donor thymus tissue and to preserve the functional architecture of the thymic stroma (thymic epithelial cells and fibroblasts).
  • processed donor thymus tissue is an engineered thymus tissue product capable of inducing tolerance to the thymus tissue types (HLA antigens) in a subject in need thereof following a surgical implantation procedure.
  • HLA antigens thymus tissue types
  • the thymus sections are placed on Millipore cellulose filters and surgical sponges inserted into medium-containing tissue culture dishes.
  • the culture medium in each tissue culture dish is replaced daily from the day of harvest from the donor to the day of implantation (day 12 to day 21).
  • the culturing of donor thymus tissue depletes thymocytes in such processed tissue which minimizes the risk of graft versus host disease (“GvHD”), which could be highly problematic in profoundly immunodeficient subjects following a thymectomy.
  • GvHD graft versus host disease
  • thymocytes and their remnants that lack nuclei are hypothesized to be important for the intended function of the tissue- engineered product, because they help to preserve the open pockets in the three-dimensional network of thymic epithelial cells that is necessary for the entry of recipient bone marrow stem cells post-treatment.
  • the importance of having “space” for the entering bone marrow stem cells is supported by experience with patient DIG003 in Markert, 1999 (See list of reference infra).
  • the patient described in the foregoing reference had been given a very large dose of steroids (40 mg/kg/day x 3 days of methylprednisolone) 35 days after CTT implantation, which led to apoptosis of the thymocytes and condensation of the epithelium. No naive T cells ever developed, and the patient succumbed to infection.
  • the inserted thymus was a mass of viable epithelium with no space between the epithelial cells for thymocytes to enter.
  • HLA typing is performed to see if the patient (recipient) and donor tissue share any HLA alleles.
  • Anti-HLA antibody testing is performed in the recipient to determine if the recipient has any antibodies against HLA antigens in the thymus. If the recipient has antibodies targeting the donor’s MHC, another thymus would be sought.
  • the donor and the mother of the donor are checked for infection per 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 processed aseptically under the Code of Federal Regulations (CFR) 1271 subpart D “Current Good Tissue Practice.”
  • the tissue is released from manufacturing and provided to the surgical team for implantation, the tissue is surgically implanted into the recipient, as described in this specification.
  • the culturing process of the harvested thymus tissue significantly modifies the biological characteristics of the donor tissue and constituent cells contained therein in the following manners: loss of donor thymocytes and enrichment of thymic epithelial cells and other stromal cells, and depletion of donor thymocytes modifies the physiologic functions of the tissue (e.g., secretion of cytokines and growth factors) as well as its structural properties.
  • Thymus tissue during the first few days of culturing appears red due to residual blood on and within the tissue. See, e.g., Fig. 15.
  • days 5 to 21 viable tissue is observed minus the blood contamination that was evident on day 1.
  • thymic epithelial cells On day 0 following harvesting of the discarded thymus tissue the tissue is densely populated with viable thymocytes embedded in a stroma that contains thymic epithelial cells and fibroblasts. AE1/AE3 and CK14 staining confirm the presence of cytokeratin (CK)-positive thymic epithelial cells, which are characteristic of normal thymus. The thymic epithelial cells form a lacy three-dimensional network with delicate processes that surround neighboring thymocytes.
  • CK cytokeratin
  • thymocytes are washed out of the tissue, especially over the first 3 days. This depletion can be identified histologically as early as day 2 by H&E stains that show decreased thymocyte density, particularly in medullary areas. The majority of the thymocytes that remain in the tissue show nuclear changes consistent with apoptosis and/or necrosis or demonstrate karyolysis (complete loss of nuclei). Many of these dead thymocytes and their cellular remnants remain present throughout the thymus tissue and are believed to prevent total collapse of the space between epithelial cells.
  • Some epithelial condensation can be seen in external areas, such as in the subcapsular cortex where loss of thymocytes has led the epithelial cell network to collapse.
  • These condensed subcapsular cortical epithelial cells can form linear arrays several cell layers thick, which may add to the mechanical strength of the slices.
  • Some medullary epithelium may also condense to form patches of contiguous epithelial cells.
  • Thymus Tissue Detailed Culturing of Thymus Tissue.
  • thymus tissue for infants with complete DiGeorge syndrome is obtained as discarded tissue from infants under the age of 9 months undergoing cardiac surgery, as described previously.
  • discarded thymus will be obtained from individuals up to 50 years of age. Use of the thymus tissue will depend on whether it meets criteria for use as set forth in this specification.
  • the thymus tissue is aseptically processed and cultured under cGMP conditions to produce partially T cell-depleted thymus tissue slices.
  • CTT cultured thymus tissue
  • the thymus tissue slicing process entails using sterile, single-use scissors and forceps to cut off a piece of thymus tissue.
  • the operator removes the capsule of the thymus with forceps and scissors and places the capsule in the lid of the plate for disposal later.
  • the piece of thymus tissue is placed in the single-use tissue sheer using forceps.
  • the top of the sheer e.g Stadie-Riggs hand microtome (Thomas Scientific, Swedesboro, NJ)
  • the operator runs the blade through the tissue piece to cut off a slice.
  • the slices are approximately 0.5 to 1 mm thick. Approximately 50-90% of the filter space is filled without overlap of the tissue slices.
  • the filter and thymus slices are transferred to a gelatin surgical sponge saturated with TOM in a tissue culture dish.
  • the TOM wicks up wetting the Mihipore filters, keeping the tissue moist.
  • Two filters are placed per sponge and 2 sponges are used per tissue culture dish.
  • the process of slicing pieces of thymus is repeated until the required number of slices has been prepared.
  • Culture dishes are labeled with an operation number, dish number, and ISBT barcode label. Completed dishes are placed in a humidified incubator at 37°C with 5%
  • the tissue engineered drug substance comprises thymus tissue slices after they have been placed in a culture dish in media and cultured for about 6 days to about 21 days, as described below.
  • the tissue-engineered drug product comprises the thymus tissue slices after transfer into a drug product container. No other processing is conducted to create the drug product from the drug substance; the only processing of drug substance to create drug product is transfer of slices into the leak proof container and corresponding media change.
  • thymus tissue is obtained from the operating room as discarded tissue from infants aged 9 months and under undergoing cardiac surgery.
  • the tissue is then placed in a sterile specimen cup with a screw-cap top by the surgical team and transported to a GMP facility under ambient conditions for processing.
  • the sterile specimen container in which the thymus is received is labeled, including a barcode, with the donor’s name and medical record number.
  • the donor screening group gives the thymus a unique identifier (thymuses are numbered consecutively) and a unique medical record number. For manufacturing, each tissue has an operation number, and a unique label.
  • the drug substance container closure system may be a cell culture dish with lid. One slice of thymus tissue is placed on a filter and two filters are placed on each gelatin sponge in thymus organ media in the dish. Four slices are placed in each culture dish and the dishes are stored in the incubator, with daily media changes, until ready for release.
  • the culture dishes can be obtained from Corning. The dishes may be sterile, non-pyrogenic Falcon ® 100 mm polystyrene cell culture dishes (product #353003). The dishes are cleaned 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.
  • the Surgifoam ® sponge may be manufactured by Ethicon and it meets the requirements for Absorbable Gelatin Sponge, USP.
  • a suitable sponge is a sterile, water-insoluble, malleable, porcine gelatin absorbable sponge that is intended for hemostatic use.
  • An illustrative example of the mixed cellulose esters filter is manufactured by Millipore (product # SMWP 02500).
  • the 25 mm hydrophilic membrane has a 5.0 pm pore size.
  • the filter is made of biologically inert mixtures of cellulose acetate and cellulose nitrate.
  • the filter is sterilized by ethylene oxide prior to use.
  • the thymus After release and acceptance of the donor thymus into the processing laboratory, the thymus is cut into thin slices, which are placed on sterile filter papers that are put on the surgical sponges in sterile culture dishes. If the tissue is not processed immediately, it is stored in thymus organ media (TOM), as described below, at 2 - 8 °C for up to 24 hours after harvest from the donor before processing is initiated.
  • TOM thymus organ media
  • processing occurs in an ISO 5 space of a biological safety cabinet (BSC) in an ISO 7 manufacturing clean room.
  • BSC biological safety cabinet
  • Only one lot of thymus tissue from a single thymus is processed in the BSC at any time.
  • the BSC is cleaned before use.
  • the thymus is tested for appearance by visual inspection and weighed.
  • the thymus is then placed in a 150-mm tissue culture dish in TOM.
  • the capsule of the thymus is removed with sterile, single-use forceps and scissors. Tissue pieces are taken for testing and as retained samples.
  • the incoming thymus tissue is tested for identity by histology. Donor eligibility is also confirmed. Processing continues prior to receipt of histologic results and all donor screening results.
  • Donor screening is required per 21 CFR 1271 to protect the safety of the thymus tissue implant recipient. This screening minimizes the risk of disease transmission from donor to recipient.
  • the medium is made with ingredients approved for use in humans which are unlikely to cause allergic reactions, whenever such reagents are available.
  • Fetal Bovine Serum must be manufactured using US material because of the concern of Creutzfeldt-Jakob Disease. Information on each lot must be sent to the FDA prior to use.
  • TOM [00491] In an embodiment, the following materials are used to prepare TOM:
  • HAMS F12 Gibco #11765-054 (or case 11765-062), 500 ml bottles or equivalent source.
  • HEPES Gibco #15630-080 or equivalent, 1M solution, 100 ml bottles. Final concentration 25 mM.
  • L-Glutamine Gibco #25030-081 or equivalent source, (stock 200 mM).
  • FBS that is HI may be used in the following manner:
  • FBS must be heat inactivated at 56°C for 30 min.
  • TOM may be prepared in the following manner.
  • sterilization of the TOM preparation may be performed in the following manner. Dispense 1 liter TOM into one liter flask. Measure 80 ml TOM in a disposable sterile cylinder. Pour the 80 ml TOM into a 150 ml Corning filter sterilization unit. Attach house vacuum to filter and filter sterilize per manufacturer’s directions. Remove the filter unit from the container and discard. Cap the collection bottle with the sterile cap (provided with the unit). Label with TOM Lot No. Test one aliquot for bacterial culture with anaerobes; fungal culture, other; and Mycoplasma culture. Test one aliquot for endotoxin. Store all TOM aliquots in the -20° C freezer upright.
  • TOM media may be released for use if: LAL result must equal to or less than 2 EU/ml for samples diluted 20 fold for testing or 1 EU/ml for samples diluted 10 fold for testing; all culture results must be negative for growth.
  • a BSC must be used for the filtering and dispensing the medium.
  • TOM is tested for sterility and endotoxin before release. TOM is not released for culturing a donor thymus until after the 14-day sterility testing acceptance criterion has been met. Once prepared, TOM is stored at -20°C until thawed, at which point it may be stored for use in the refrigerator for up to two weeks.
  • the 14-day sterility testing may, for example, be conducted using the BacT/ALERT culture system.
  • the BacT/ALERT BioMerieux, Durham, NC
  • the BacT/ALERT is a commercially available culture system that can be used to test samples using an automated microbial detection system.
  • FBS may be obtained from GIBCO brand, Life Technologies. The FBS is prepared by an aseptic, validated process. FBS meets USDA requirements for abattoir-sourced animals, traceability and country of origin.
  • All fetal blood is collected from fetuses derived from healthy dams that have passed pre- and post-mortem certified veterinary inspection. All FBS are traceable by date and location of collection. FBS collected and processed in the United States is from USDA approved and inspected slaughter establishments. The United States is recognized by the USDA as being free of foot and mouth disease and rinderpest. To qualify the supplier, FBS is tested for pH, osmolality, endotoxin, total protein and identity before use [00512] Completed dishes are placed in a humidified incubator at 37°C with 5% CO2. Each lot of thymus tissue is stored in a separate incubator. After the thymus slices have been placed in the incubator, particle sampling and personnel monitoring is conducted.
  • Thymus slices are cultured for up to 21 days (for example a conditioning regimen of about 6 days to about 21 days), and during culture the medium is changed daily. These thymus slices are considered the drug substance. During the culture period, many thymocytes are washed out of the thymus tissue slices or the thymocytes undergo apoptosis while preserving the thymic stroma. All manufacturing steps are conducted using sterile, disposable equipment and supplies. Media is aspirated by pipette from the culture dish and pooled into a sterile collection container for in-process testing.
  • Ten (10) mL of fresh thymus organ media is then gently dispensed to each culture dish in a rinsing manner over the tissue slices. After the media change is completed, samples are taken from the pooled media for sterility and histology, if needed. Particle sampling and personnel monitoring are completed and line clearance is performed.
  • the medium is changed daily.
  • the slices are cultured for up to 21 days (for example a conditioning regimen of about 6 days to about 21 days).
  • In-process testing is conducted to provide insight into the process and product quality and to help ensure the safety and quality of the final drug product.
  • Samples are collected for sterility in-process testing on day 1 and day 7. Samples are collected for mycoplasma in-process testing on day 7. Samples are collected for in-process histology testing between days 5 and 9. The dose is determined on the day prior to release. Gram stain, BacT, mycoplasma and endotoxin are tested on the day of release.
  • the Gram stain is a bacteriological laboratory technique used to differentiate bacterial species into two groups, Gram-positive and Gram-negative. Gram stain is tested on pooled spent culture medium from the culture dishes. The method uses a staining technique to determine the classification based on the physical properties of the cell wall. This method is used to make a preliminary morphologic identification or to establish whether there are significant numbers of bacteria in a clinical specimen. Staining is conducted either manually or using an automated stainer. It has been demonstrated that the two different staining methods showed no qualitative differences that would impact culture results.
  • Histology testing is performed prior to implantation, and includes in an embodiment at a minimum: (1) determination that areas positive for keratin AE1/AE3 are scattered throughout tissue; (2) at least 1 Hassall body is microscopically identified; (3) CK14 staining of the tissue slices is scattered throughout the thymus tissue; and (4) intact nuclei are microscopically observed.
  • histology testing is performed between about days 6 to about 21 days)The presence of Hassall bodies and intact nuclei as well as successful CK14 staining are indicative of normal healthy thymus tissue that has been cultured.
  • Culture time is an important process parameter. As noted, culturing is performed for up to 21 days.
  • testing of thymus samples in culture prior to implantation is conducted to confirm whether histology results generated in culture are representative of histology testing of historical specimens of cultured thymus tissue. Based on the observations made by the pathologist for the samples discussed in the Examples, the histologic appearance of the tissue slices at day 5 reflects what is observed at each of the later time points (day 9, 12 and 21) of culturing. In an embodiment testing of thymus tissue may be performed at various timer periods and intervals between days 5 and 21 of culturing.
  • the testing may be performed during the conditioning regimen which is for a period of five days, or six days, or seven days, or eight days, or nine days, or 10 days, or 11 days, or 12 days, or 13 days, or 14 days, or 15 days, or 16 days, or 17 days , or 18 days or 19 days, or 20 days, or 21 days; or for a period of 5-6 days, or 5-7 days, or 5-8 days, or 5-9 days, or 5-10 days, or 6 to 7 days, or 6 to 8 days, or 6 to 9 days, or 6 to 10 days, or 6 to 11 days, or 6 to 12 days, or 6 to 21 days, or 7 to 21 days, or 8 to 21 days, or 9 to 21 days, or 10 to 21 days, or 11 to 21 days, or 12 to 21 days, 13 to 21 days or 14 to 21 days, or 15 to 21 days, or 16 to 21 days or 17 to 21 days or 18 to 21 days, or 19 to 21 days, or 20 to 21 days.
  • the conditioning regimen may be anytime between about 6 days and about 21
  • Acceptance criteria for incoming thymus tissue product include the tests identified in
  • CK cytokeratin
  • EU endotoxin unit
  • USP United States Pharmacopeia.
  • a thymus tissue is processed prior to obtaining all donor screening results.
  • the acceptance criterion for weight is greater than or equal to 3 grams. This is the minimal thymus weight that is accepted to ensure sufficient material is available for proper dosing of the final product. The acceptance criterion is based on experience in processing thymus tissue.
  • the acceptance criterion for identity is that thymus tissue identity is confirmed by histology on day 1 and at the midpoint (days 5-9).
  • a barcode is used for tracking of the tissue throughout the processing and the barcode is confirmed at release to verify the correct identity of the product.
  • the histology method is a standard method used by hospitals for all tissue types, as is known by a person of skill in the art.
  • Samples of the product are fixed in 10% formalin and transported to the laboratory. Containers are labeled with a coded identifier instead of the patient’s name to protect patient privacy, along a medical record number. Upon arrival in pathology, the specimens are assigned a unique pathology accession number, and barcoded. The subsequent blocks, slides, and paperwork are all barcoded with this pathology accession number.
  • the formalin-fixed tissue is grossly examined, and a written gross description of the material is prepared that will become part of the final report.
  • the formalin-fixed tissue is then processed and embedded into a paraffin block by standard methodology on an automated processor. Sections are cut from the paraffin block and the following stains are performed by ASCP-certified histotechnologists:
  • the incoming thymus sample also serves as a control for tissue slices that have been in culture for about 6 days to about 21 days, when samples are tested as part of potency testing.
  • the incoming thymus sample appears as a typical thymus sample and then changes occur to the tissue slices while they are cultured and then tested after about 6 days to about 21 days in culture. After about 6 days to about 21 days in culture, the sample must show 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 presence of intact nuclei.
  • Slides are interpreted by a pathologist certified in Anatomic Pathology, with additional experience in the histologic evaluation of thymic tissue. The final report is issued by the pathologist and the report documents the results.
  • Acceptance criteria for cultured thymus tissue drug substance testing is identified in Table 4 below.
  • the cultured thymus tissue must be free of microorganisms. In the sterility test performed on day 1 and day 7, there should be no growth of microorganisms. Mycoplasma should be negative upon testing on day 7. The sterility test should be gram stain negative. [00542] Product sterility is maintained using appropriate controls including aseptic technique; employing a training program and verifying the qualification of operators; utilizing appropriate clean room qualification procedures; employing establish clean media fill procedures and utilizing ready-to-use sterilized apparatus or apparatus sterilized utilizing validated sterilization cycles. [00543] The containers of processed thymus tissue are visually examined for damage. Tissue slices normally exhibit a yellow to reddish brown appearance with varying thickness and shape. [00544] Thymus tissue identity is confirmed by histology on day 1 and at midpoint (days 5-9). [00545] A barcode is used for tracking of the tissue throughout the processing and the barcode is confirmed at release.
  • the dosage (area) is 1,000 - 22,000 mm 2 of thymus tissue / recipient body surface area in m2. Dose is controlled by the surface area of slices released to the operating room as appropriate for the patient’s body surface area.
  • the acceptable dose range is defined as 1,000 - 22,000 mm 2 of thymus tissue per recipient body surface area (BSA) in m 2 .
  • the area of the thymus tissue is determined by photograph using software analysis (PAX-it Image Analysis Software).
  • BSA is determined using the patient’s height in cm and weight in kg.
  • the DuBois and DuBois formula is used to calculate the BSA:
  • BSA 0.007184 x [height (cm)] 0725 x [weight (kg)] 0425 .
  • the cultured thymus tissue is tested for endotoxin.
  • the specification is ⁇ 5 EU/kg body weight/hr.
  • Endotoxin testing may be performed, for example, by using the Endosafe PTS system.
  • the cartridges used with the Endosafe PTS use a chromogenic kinetic Limulus Amebocyte Lysate (LAL) test.
  • LAL chromogenic kinetic Limulus Amebocyte Lysate
  • Each cartridge contains precise amounts of LAL reagent, chromogenic substrate and control standard endotoxin.
  • Test sample is pipetted into four sample reservoirs.
  • the instrument draws and mixes the sample with LAL reagent in two channels (sample channels) and with the LAL reagent and positive product control in the other two (spike channels).
  • the sample is incubated 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 for each batch of cartridges is constructed using the log of the reaction time versus the log of the endotoxin standard concentration.
  • the sample and spike values are calculated by interpolation off the standard curve using the reaction time. This testing meets the requirements of United States Pharmacopeia (USP).
  • Testing for mycoplasma may be performed in the following manner. A sample of the pooled media is removed from the plates on day 7 and tested before product release.
  • the drug product undergoes similar visual inspections and histology testing before use.
  • the slices are transferred into drug product containers for transport to the operating room. Once received in the operating room, the slices are inserted into the thigh muscle of the recipient patient.
  • the container should be intact with no visible damage and the thymus tissue slices should appear as yellow to reddish-brown slices of tissue with varying thickness and shape. The tissue slices are visually examined to confirm that these acceptance criteria are met.
  • Cryopreservation and thawing of allogeneic cultured post-natal thymus tissue derived product may be performed in the following manner.
  • a cryopreserved allogeneic cultured postnatal thymus tissue-derived product prepared by method comprising the steps of:
  • cryopreserved allogeneic cultured postnatal thymus tissue-derived product in liquid nitrogen in a cryopreserved allogeneic cultured postnatal thymus tissue-derived product bank.
  • the cryopreserved allogeneic cultured postnatal thymus tissue- derived product of claim 66 wherein the thymus, on the day of harvest, demonstrates that >50% of areas are positive for keratin in a lacy staining pattern, that Hassall bodies are present, that CK14 stains in a lacy pattern, and that >90% of nuclei are intact.
  • the donor thymus is sliced and divided into roughly two equal portions, putting each slice with its cellulose filter in a separate cryovial (Nunc tube).
  • the filter is folded in half to insert it into the tube.
  • About 1 to about 1.5 ml of freezing medium [sterile filtered 90% heat inactivated fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO)] at room temperature is added to cover the tissue.
  • the sterile cap of the cryovial is replaced on the tube.
  • the tubes are placed overnight in a -80°C freezer.
  • each tissue plus filter in a 5ml CryoELITE tissue Vial (Wheaton). Put 3 to 5 ml of freezing medium at room temperature to cover the tissue. Put in a Styrofoam box and put in -80° freezer overnight. The vials are then transferred to the vapor phase of a liquid nitrogen freezer. Alternatively a controlled rate freezer can be used to bring the temperature of the cryovials to liquid nitrogen temperature.
  • the tissue on the filters are transferred to the sterile field into a tissue culture dish with approximately 2 ml of sterile saline.
  • the scrub nurse removes the tissue from the filter paper by scrapping or pulling with forceps.
  • the scrub nurse places the tissue, in an amorphous pile, back on the filter paper.
  • the tissue culture dish with approximately 4 filters and the tissue is transferred to the operative site where the surgeon can easily access the tissue.
  • the tissue is placed in the quadriceps muscles similarly to the procedure with CTT (RVT-802).
  • the Cryo- CTT resembles the CTT in that it is partially T-cell depleted, the thymus tissue slices show areas positive for keratin AE1/AE3 scattered throughout the tissue, slices contain at least one Hassall body, CK14 staining is scattered throughout the tissue and presence of intact nuclei.
  • unmatched thymus tissue slices from the donor are cultured for about 6 days to about 21 days.
  • steroids are usually given at the induction of anesthesia.
  • the thymus of the recipient is surgically removed at the time of the solid organ transplant.
  • the thymectomy may be done prior to the day of transplantation or on that day.
  • the thymectomy method would be surgical, thorascopic or robotic.
  • the recipient is given more steroids prior to receiving equine anti -thymocyte globulin (e.g ., rabbit anti -thymocyte globulin) over 3 to 7 days to kill most of the residual T cells (and NK cells) in the recipient or alemtuzumab over 4 days to kill the T, B and NK cells.
  • equine anti -thymocyte globulin e.g ., rabbit anti -thymocyte globulin
  • an immunosuppressant such as cyclosporine or tacrolimus
  • mycophenylate is then started until T cells develop and show greater than 10% naive T cells. It may take 6 to 12 months for the naive T cells to increase to this number.
  • Cultured thymus tissue is processed for the thymus of the donor of the solid organ.
  • Half of the CTT can be implanted into the quadriceps muscle between about 6 days to about 21 days.
  • the other half of the thymus will be cryopreserved for future use of the recipient.
  • the immunosuppressive regimens will suppress any remaining T cells until naive T cells are released by the cultured thymus tissue slices implanted in the recipient and the recipient meets criteria for weaning off the maintenance immunosuppression regimen. (Over 10% naive T cells are needed to wean the immunosuppression.)
  • the thymus is identified and carefully dissected away from the pleural investment of the lung, starting with the inferior horns and extending to the superior horns.
  • Chest tube placement One chest tube is always inserted (into the mediastinum). If a single pleural space is entered during the operation, the chest tube is continued from the mediastinum into that pleural space. If both pleural spaces are entered, a second chest tube is used in a similar fashion from the mediastinum to the other pleural space.
  • Sternum closure In neonates or infants, 0-Ticron sutures are used to close the sternum. At about 1-2 year of age, #1 sternal wires are used. At about 2-5 years of age, #4 sternal wires are used.
  • Allogeneic cultured postnatal thymus tissue-derived product should be implanted in accordance with the following instructions. Implantation of thymus tissue into the thigh requires a healthy bed of muscle tissue.
  • tissue slices on the filter papers that are on surgical sponges in medium are removed from the tissue culture dishes and placed in 120 ml sterile cups with 20 ml medium, packaged to maintain sterility, and delivered to the operating room or packaged for shipment. Tissue slices are not removed from the individual containers until ready to be used. Verify the product expiration date and time.
  • the sterile field team member will use a pair of forceps to remove the individual tissue slice with its filter paper from the container and place it in a sterile tissue culture dish containing approximately 2 ml preservative-free saline on the sterile prep table.
  • Four tissue slices with the filter papers taken from four containers are placed in one sterile tissue culture dish that is on the sterile field in front of the sterile field team member.
  • the sterile field team member then peels the tissue slice away from the filter paper using two pairs of forceps, one of which holds the filter in place while the other pulls the tissue or scrapes the tissue into a pile.
  • the tissue removed from each filter paper is than put on that filter paper in a pile in the middle of the filter paper.
  • the sterile tissue culture dish is then transferred to the sterile field.
  • the next set of four allogeneic cultured postnatal thymus tissue-derived product containers will then be processed the same way while the surgeon is implanting the first 4 slices.
  • the surgeon finishes implanting the first four slices the next dish with 4 pieces of tissue is put in the surgical field and the initial tissue culture dish is returned to the sterile field in front of the sterile field team member for loading the 3 rd set of four tissue slices. Continue this cycle until all the desired tissue is implanted. All of the tissue slices are not transferred at the beginning to avoid contamination from the air in the operating room.
  • Step 3 Muscle spreading and implantation.
  • Step 5 Repeat Steps 3-4 for each allogeneic cultured postnatal thymus tissue-derived product tissue slice up to the maximum intended dose.
  • Step 6 Incision closing.
  • [00597] Use mild analgesics as needed. Monitor for signs of infection or dehiscence.
  • the donor is a living related donor as for lung, kidney, intestine, or partial liver, a portion of that solid organ donors’ thymus may be adequate for culture and implantation. The pathology criteria listed above for the day of harvest and until implantation would need to be met.
  • Cryopreserved-cultured thymus tissue may be available from a 3 rd party donor.
  • the 3 rd party donor must express all the recipient HLA alleles that are not expressed by the solid organ donor. This includes HLA- A, HLA-B, HLA-C, HLA-DRBl, HLA-DQBl, HLA- DQA1, HLA-DPBl, and HLA-DPAl.
  • mismatching in HLA-DP alleles is acceptable if the mismatch is “permissive.” In other alleles minor mismatching is allowed, e.g., HLA-A*01:02 into a recipient carrying HLA-A*01 :01, in other words, the second field (after the colon) can be different, the first field (before the colon) must be identical.
  • Eligibility of a subject is determined based on a number of criteria including: Poor 12 to 24 month prognosis without cardiac transplantation despite current maximum supportive therapies; congenital or acquired heart disease with failure to thrive as defined by UNOS criteria; symptoms of advanced heart failure in the setting of congenital or acquired heart disease refractory to medical therapy; abnormal hemodynamics or increasing pulmonary vascular resistance; inoperable structural heart disease; symptomatic arrhythmias not amenable to medication or device therapy or poor exercise tolerance.
  • a number of absolute and relative contraindications to heart transplant surgery are assessed, including, for example, reversible renal dysfunction unless the subject is a candidate for heart/kidney transplant; irreversible liver disease unless candidate for heart/liver transplant; irreversible pulmonary dysfunction, use of non-conventional mechanical ventilator support (i.e . high frequency ventilator, maximal settings of CMV) or fixed pulmonary hypertension (TPG>15) unless the subject is a candidate for a heart-lung transplant; Diabetes mellitus with microvascular disease; or active, uncontrolled seizure disorder.
  • non-conventional mechanical ventilator support i.e . high frequency ventilator, maximal settings of CMV
  • TPG>15 fixed pulmonary hypertension
  • Other contraindications may include other diseases limiting long term survival and rehabilitation following a heart transplant; substance abuse, morbid obesity, malignancies; active psychiatric disorder; and other reasons such as documented medical non-compliance.
  • Relative contraindications include active infections; cognitive dysfunction; inadequate vascular access and significant allosensitization.
  • Donor and recipient histocompatibility management is also considered. Patients are evaluated for panel reactive antibodies (“PRA”) for pre-formed HLA antibodies that the recipient may have formed in response to a “sensitizing event” such as prior transfusions or major surgeries using bypass/blood products or homograft materials.
  • PRA panel reactive antibodies
  • Patients with an excessively high HLA-Class I or Class-II PRA or those undergoing repeat transplant may be candidates for desensitization strategies. Specific strategies will be individualized to the potential recipient and may include use of plasmapheresis, IVIG, rituximab and/or bortezomib. Significant antibodies are those that remain present after a 1 : 16 dilution.
  • Pre-sensitized patients may require an actual prospective cross-match with potential donors. Patients with a history of HLA antibodies will undergo virtual cross-match in UNET at the time a transplant is being considered.
  • Pre-sensitized patients may require an actual prospective cross-match with potential donors via the following general guidelines. All patients with a history of HLA antibodies will undergo virtual cross-match in UNET at the time an offer is being considered.
  • Immunosuppression management is determined based on the solid organ to be transplanted.
  • pre-transplant/induction therapy typically comprises administration of :
  • Methylprednisolone 10 mg/kg IV on induction (max dose 500 mg).
  • Methylprednisolone 10 mg/kg IV on release of x-clamp max dose 500 mg.
  • ATG antithymocyte globulin 1.5 mg/kg IV on release of x-clamp.
  • pre-transplant induction immunosuppressive therapy for heart and CTT implant candidates may comprise:
  • Second dose 20 mg IV administered 4 days after transplantation; hold second dose if complications occur (including severe hypersensitivity reactions or graft loss.
  • antithymocyte globulin (ATG, rabbit derived, Thymoglobulin®), may be given according to the following dosing schedule for heart transplant subjects:
  • patients may receive a dose of methylprednisolone (SoluMedrol) lOmg/kg (max dose 500mg) IV to be administered by anesthesia on induction of anesthesia before basiliximab and then a second dose of lOmg/kg (max 500mg) on reperfusion (before ATG).
  • Post-operative Maintenance Immunosuppression Maintenance immunosuppression regimen [00638]
  • post-transplant immunosuppression comprises:
  • ATG Thimoglobulin® 1.5 mg/kg IV daily for 5-7 doses.
  • [00649] Can substitute cyclosporine if IV medication needed or intolerance to tacrolimus.
  • tacrolimus (FK506, Prograf®) may be administered.
  • Usual dosage forms include intravenous solutions of 0.5mg/ml, and oral capsules of 0.5mg, lmg, and 5mg per capsule.
  • a starting dose of -0.05 mg/kg/dose - is given every 12 hours (maximum 5mg/dose) when the recipient is PO/NG/SL, usually started at -24 hours post-operatively if renal function acceptable.
  • Tacrolimus trough levels are monitored daily until therapeutic dosing is achieved. Increase administration to every 8 hours, if necessary, to achieve therapeutic trough levels. If the drug is given sublingually, the capsule contents are sprinkled under tongue (may result in higher levels). Oral and sublingual doses are not. Generally it will be necessary to administer ⁇ 1 ⁇ 2 of the oral dose to be given sublingually.
  • Tacrolimus dosing may be administered based on serum whole blood levels measured by mass spectrometry method 10-14 hours (7-9 hours if dosed every 8 hours) following the last dose.
  • cyclosporine is administered to patients unable to tolerate tacrolimus.
  • the starting dose 2 mg/kg/dose by mouth every 12 hours. If unable to achieve therapeutic levels (especially infants and young children) then the dosing frequency is increased to every 8 hours.
  • IV dose PO dose.
  • the dose may be held when ANC ⁇ 500 or WBC ⁇ 1000; may need to decrease dose in setting of decreasing ANC ( ⁇ 1000) or WBC ( ⁇ 3000).
  • dosages of Cellcept 500mg Myfortic (mycophenolate) 360mg.
  • Mycophenolate (Myfortic® dosage forms 180mg tablet, 360mg tablet) may be administered in lieu of Mycophenolate mofetil, according to the following dosage parameters:
  • Begin delayed-release tablet 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.
  • azathioprine may be administered in the following manner: [00671] Begin 2-4 mg/kg/day, given once daily
  • Azathioprine causes bone marrow suppression and dose may need to be reduced based on WBC/ANC.
  • the IV dose equals the oral dose.
  • steroid may be administered, as methylprednisolone (Solu- Medrol®), prednisone or prednisolone.
  • Intravenous steroids are started intraoperatively (as induction immunosuppressive therapy), and the continued post-operatively at 5 mg/kg/dose (maximum 125 mg/dose) IV every 8 hours x 6 doses.
  • Oral steroids may begin thereafter as prednisone tablets or prednisolone suspension 3 mg/ml. Intravenous methylprednisolone is continued if the transplant recipient is unable to tolerate the immunosuppression treatment regimen orally. A switch over to oral therapy may be made the recipient can tolerate oral medications.
  • Exemplary dosing ranges of steroids are: [00676] 0-10 kg. start at 2mg/kg/day divided BID, wean every 2 days, holding at 6mg daily [00677] 0-30kg. start at 2mg/kg/day divided BID (max single dose of 30mg, see below), wean by 5mg/day every two days then hold at lOmg daily.
  • Other drugs in the second immunosuppression regimen include the following: [00681] Sirolimus (Rapamune®) (0.5mg, lmg, 2mg caps): Initiated after diagnosis of coronary allograft vasculopathy or as otherwise clinically indicated by the transplant cardiologist.
  • Therapeutic level goal is 4-8, accept lower tacrolimus level (same 4-8 range) if on both drugs.
  • Pravastatin used in teenager s/older children and those with CAV
  • Begin 0.2 mg/kg/day administered once daily can give 1 ⁇ 4, 1 ⁇ 2 or up to 1-2 tabs per day at night.
  • Valganciclovir (Valcyte®) — for CMV prevention in all CMV + recipient or donor.
  • Monitor for drugs which may increase or decrease tacrolimus and cyclosporine levels, as known to the person of ordinary skill in the art.
  • the subject is treated in the following manner in the event transplant rejection is noted.
  • Methylprednisolone (SoluMedrol) 15mg/kg/day IV x 3 days (maximum 1000 mg).
  • inotrope therapy thymoglobulin; and/or plasmapheresis.
  • transplant recipients receive belatacept as maintenance therapy as part of the second immunosuppressive regimen.
  • This protocol is typically employed when the recipient is Epstein Barr Virus (EBV) Ig+, exhibits no DSA (donor specific alloantibody); irrespective of absolute or calculated panel reactive antibodies (PRA), the transplant involves a negative cross-match and the recipient is ⁇ 70 years old with a BMI of ⁇ 35. Further considerations include the recipient’s ability to tolerate induction (if they would not otherwise receive thymoglobulin).
  • the recipient should have no history of idiopathic focal segmental glomerulosclerosis (FSGS) and no previous non-kidney solid organ transplant.
  • the protocol is for kidney transplants only.
  • the induction immunosuppressive regimen comprises administration of methylprednisolone 500 mg intravenously intra-operatively.
  • Alemtuzumab 30 mg is administered by intravenous infusion over a period of three hours (2 hours post administration of steroids).
  • Belatacept 10 mg/kg is administered (TBW) (rounded to the nearest 12.5 mg) after the prior drug administrations.
  • the maintenance immunosuppressive regimen comprises belatacept lOmg/kg TBW on POD 4 and end of week 2, 4, 8, and 12; then 5mg/kg every month (rounded to nearest 12.5mg).
  • Sirolimus is administered 2mg daily with first trough level taken after 2 weeks (goal 8-10 ng/ml). No steroid maintenance therapy is normally required.
  • the induction immunosuppressive regimen may comprise no induction immunosuppressive regimen, z.e., the induction immunosuppressive regimen is optional.
  • the maintenance immunosuppressive regimen may comprise mycophenolic acid 1000 mg administered every 12 hours and tacrolimus administered 0.1 mg/kg/day with a maximum of 5 mg every 12 hours.
  • the induction immunosuppressive agent may comprise basalixiimab 20 mg to start in the operating room and post-operative day (POD) 4.
  • the maintenance immunosuppressive regimen may comprise mycophenolic acid 1000 mg every 12 hours and tacrolimus 0.1 mg/kg/day with a maximum of 5 mg every 12 hours with tapering dosages of steroids.
  • the following parameters are considered.
  • the induction immunosuppressive regimen may comprise Thymoglobulin 1.5mg/kg IBW (rounded to nearest 25mg) x 4 doses started in the operating room.
  • the induction immunosuppressive regimen may comprise thymoglobulin 1.5mg/kg IBW (rounded to nearest 25mg) x 4 doses started in the operating room.
  • the second immunosuppressive regimen (maintenance therapy) may comprise tapering dosages of steroids, such as 500 mg in the OR, 240 mg POD 1, 125 mg POD 2, 125 mg POD 3, 90 mg POD 4, with mycophenolic acid 1000 mg every 12 hours starting POD 4 and tacrolimus 0.1 mg/kg/day with a maximum of 5 mg every 12 hours.
  • the induction immunosuppressive regimen may comprise thymoglobulin 1.5mg/kg IBW (rounded to nearest 25mg) x 4 doses started in the operating room.
  • the second immunosuppressive regiment may comprise a steroid taper to lowest of 5 mg daily, mycophenolic acid 1000 mg administered every 12 hours starting POD 4 and tacrolimus administered 0.8 mg/kg/day with a maximum of 4 mg every 12 hours.
  • the initial dose may be chosen based on an assessment of patient and transplant factors.
  • induction immunosuppressive regimens are normally administered. Exceptions may occasionally be made when the risks of induction therapy are thought to outweigh the benefits of such therapy (i.e., the induction immunosuppressive regimen is optional).
  • the regimen when an induction immunosuppressive regimen is administered, the regimen may comprise Simulect® (basiliximab): 20mg IV given in the OR after reperfusion (cross-clamp removal) and repeated on post-operative day 4.
  • Simulect® basic anesthesia
  • the calcineurin inhibitor (CNI) administered in the second immunosuppressive regiment should be initiated at 48 hours post-operatively, but may be delayed further depending upon the patient’s renal condition.
  • Steroids may be administered in the peri-operative period, typically methylprednisolone 500 mg intravenously at induction of general anesthesia and 500 mg IV before reperfusion (cross-clamp removal).
  • the maintenance immunosuppressive regimen may comprise steroids, for example, methylprednisolone 125 mg IV q8 hours x 3 doses (Start 8 hours after reperfusion) followed by prednisone: 0.5 mg/kg twice daily beginning post-operative day 2; decrease dose by 5 mg twice daily every 2 days to 10 mg twice daily (or 20mg daily) and maintain this dose to 30 days post- transplant.
  • the second immunosuppressive regimen may also comprise calcineurin inhibitors (CNI), for example, Tacrolimus/FK506/Prograf ⁇ (Preferred Agent): lmg by mouth every 12 hours. The dose is titrated to trough goal level of 10-15 ng/ml. Typical dose adjustments are made after 5 doses of the calcineurin inhibitor to establish steady state;. Daily levels of the CNI are monitored initially to evaluate for CNI toxicity.
  • CNI calcineurin inhibitors
  • cyclosporine/CyA/Neoral® is administered at a dosage of 100 mg (1.5 to 5 mg/kg) by mouth every 12 hours. The dose is typically titrated to a trough goal level of 33 ng/ml.
  • the maintenance immunosuppressive regimen may also comprise an antiproliferative agent such as mycophenolate mofetil (Cellcept®) 1,000 to 1,500 mg orally, with the dosage adjusted to maintain a WBC count > 3,000.
  • the antiproliferative agent may be mycophenolate (Myfortic®) 360 mg - 720 mg administered orally twice a day, with the dosage adjusted to maintain a WBC count > 3,000.
  • the antiproliferative agent is azathioprine (Imuran®) administered orally in a dosage of 2 mg/kg daily, with the dosage adjusted to maintain a WBC count > 3,000.
  • the maintenance immunosuppressive regimen may comprise sirolimus (Rapammune®) (TOR-I) administered orally at a dosage of 2 mg daily.
  • the dosage is typically titrated to maintain a trough of 4-12 pg/ml
  • the maintenance immunosuppressive regimen may comprise a tapering dosage of steroids, for example, Month 1, decrease dose to 15.0 mg PO daily; Month 2, decrease dose to 12.5 mg PO daily; Month 3, decrease dose to 10.0 mg PO daily; Month 4, decrease dose to 7.5 mg PO daily; Month 5, decrease dose to 5.0 mg PO daily; Month 6, decrease dose to 2.5 mg PO daily.
  • the steroid taper should be reevaluated following > ISHLT Grade 1R with evidence of myocyte necrosis. The taper may be resumed after improvement in histological rejection guidelines.
  • the maintenance immunosuppressive regimen may comprise the following additional/adjunctive agents: methotrexate (MTX) administered 2.5 to 5 mg twice weekly for cell-mediated rejection.
  • MTX should be considered for 3 or more consecutive biopsies with > grade 1R/2 or 2 consecutive biopsies with grade 2R/3 A.
  • a liver transplant with no renal dysfunction may be performed without any induction immunosuppressive regimen, i.e. such induction immunosuppressive regimen is optional.
  • the maintenance immunosuppressive regimen may comprise, administration of mycophenolate 1 g every 12 hours and tacrolimus 2-3 mg every 12 hours along with a tapering dosage of steroids.
  • a typical tapering dosage of steroids is: methylprednisolone 500 mg IV intra-operatively; methylprednisolone 250 mg IV 6 hours post-operatively once; methylprednisolone 180 mg IV once POD 1; methylprednisolone 90mg IV once POD 2; methylprednisolone 60mg IV once POD 3; methylprednisolone 30mg IV once POD 4; prednisone 20 mg PO daily POD 5-14; decrease by 2.5 mg every 2 weeks.
  • the tapering dosage of prednisolone may be administered as 20 mg daily for the first 2 weeks; 17.5 mg daily for weeks 2-4; 15 m daily for weeks 4-6, 12.5 mg daily for weeks 6-8; 10 mg daily for weeks 8-10; 7.5 mg daily for weeks 10-12; 5 mg daily for weeks 12-14; 2.5 mg daily for weeks 14-16. Stop at 16 weeks. In certain circumstances the prednisolone is weaned to 5 mg daily and held for one year. [00747]
  • a renal sparing liver transplant is performed where there is pre operative dysfunction requiring HD or CVVHD/F.
  • a renal sparing liver transplant is performed where there is post-operative renal dysfunction with serum creatinine levels of > 2 mg/dL on POD 0-1.
  • the induction immunosuppressive regimen comprises thymoglobulin 1.5 mg/kg (rounded to the nearest 25 mg) every 48 hours for 4 doses.
  • dosage reductions are made for pancytopenia or neutropenia. If WBC count is 2-3 or platelets are 30-50, give 1 ⁇ 2 dosage. If WBC is ⁇ 2 or platelets are ⁇ 30, hold the dosage.
  • the premedication when premedication is indicated, the premedication may be administered 30-60 minutes prior to ATG infusion, acetaminophen 650 mg VT or orally, diphenhydramine 25-50 mg; and methylprednisolone 40 mg IV (or may use taper described above).
  • the transplant is an intestine or multivisceral transplant such as a liver, intestine, pancreas transplant where the recipient is at high immunologic risk (PRA > 0, prior pregnancy or transplant of isolated intestine, or where there is a low immunologic risk but a high risk of infection
  • the induction immunosuppressive regimen may comprise premedication, as described above, and may also comprise thymoglobulin 1.5 mg/kg (rounded to the nearest 25 mg every 24 hours for a total of 6 mg/kg, and basiliximab 20 mg in POD 0 and 4.
  • the maintenance immunosuppressive regimen may comprise a tapering dosage of steroids as described above and mycophenolate 1 g every 12 hours and tacrolimus 1 mg SL every 12 hours (goal 12-16 mg/ml).
  • the tacrolimus is administered at a dosage to achieve target levels of 5-8 (liver) or 12-17 (intestine, liver-intestine) while patient is on mycophenolate mofetil (Cellcept).
  • the target may be altered if the patient is participating in a drug study trial, has rejection, renal compromise, or for advancing age.
  • mycophenolate mofetil MMF, Cellcept
  • the standard dosage for adults is 1,000 mg every 12 hours. Dosage reductions may be made in patients with pancytopenia or neutropenia in accordance with the following: WBC 2-3 or platelets 30-50: consider giving 1 ⁇ 2 dose; for WBC ⁇ 2 or platelets ⁇ 30; consider holding dose.
  • WBC 2-3 or platelets 30-50 consider giving 1 ⁇ 2 dose; for WBC ⁇ 2 or platelets ⁇ 30; consider holding dose.
  • the following treatment may be administered: methylprednisolone 500 mg IV daily for 3 doses. If liver function tests are not improving, an additional two doses may be given (total of 5 doses of 500 mg IV daily), with a repeat lover biopsy performed on the eve of POD 3 or the morning of POD 4.
  • immunosuppressive management in lung implants follows the following induction and maintenance immunosuppressive regimens.
  • Induction immunosuppressive regimens may follow an induction immunosuppressive regimen described previously.
  • Maintenance immunosuppressive regimens may comprise the following: [00756] Calcineurin Inhibitors: tacrolimus every 12 hours with dosing adjusted to maintain trough tacrolimus levels. See Table 5 below.
  • Azathioprine 2 mg/kg may be administered orally on a daily basis. Important to ensure normal TMPT enzyme levels prior to start and to follow LFTs/CBC. Dosage adjustments may be considered if leukopenia is observed.
  • Mycophenolate mofetil (Cellcept®) may be administered at a usual dosage of 1,000 mg twice daily. The usual dosage for heart and lung transplants is 1,500 mg daily administered orally. CBC should be followed and dosage adjustments should be considered if leukopenia is observed.
  • Sirolimus is generally contraindicated in the first 3 months following transplantation due to concerns for anastomoctic dehiscence. If administered, sirolimus is typically administered 1 mg daily by oral administration, which is adjusted based on trough levels. For example, administered as a 3 rd agent or for CNI sparing, a target trough is 4-8 ng/ml; and as a CNI alternative, the trough level is 10-15 ng/ml.
  • Example 1 Intra-thymus variability study.
  • Intra-thymus variability was studied to determine whether histology testing results from one part of a thymus could be considered representative of histology testing results in any other part of the same thymus. The results of this test were used to determine how many samples should be tested during both routine release testing and for process validation testing.
  • Histology acceptance criteria were established, as noted previously, including the assessment of: areas positive for keratin AE1/AE3 scattered throughout tissue on days 5-9; at least 1 Hassall body identified; CK14 staining scattered throughout tissue; and intact nuclei observed.
  • Example 2 Whole thymus time-course study.
  • thymuses were sliced and cultured per SOP. On the day of slicing, the first, middle and last slices were prepared for immunohistochemistry. The remainder of each thymus was sliced and cultured in 6-well plates. Each thymus was designated for one of the following time points: baseline (day 0), day 5, day 9, day 12 and day 21. See Fig. 7 for a day 0 thymus slice, Fig. 8 for a day 5 slice, Fig. 9 for a day 12 slice, and Fig. 10 for a day 21 slice. [00774] The total number of slices from each thymus ranged from 21 to 62 slices. Slices were cultured per the procedures outlined above with daily media changes.
  • thymus tissue slices were treated to generate tissue slices that were considered degraded or non-viable. Three thymuses were used for these experiments. Control samples were taken from each thymus. The treatment conditions presented in Table 7 were tested. Table 7: Forced Degradation Treatment Conditions
  • Heat shock was accomplished by placing the 10 cm culture dish containing the slices into a Ziploc bag, and placing into a 55°C water bath. The plate rested on a support and was not submerged. Freeze/thaw was accomplished by placing the 10 cm culture dish into a -20°C freezer for 4 hours followed by thawing at ambient.
  • Thymocytes are progressively lost as thymus tissue is cultured. However, dead cells may persist in cultured thymus long-term due to inability to recruit phagocytes to clear them.
  • the nuclei of cells undergoing apoptotic cell death initially condense and stain more darkly (blue) with hematoxylin dye. As these cells deplete their energy but are not phagocytosed, they lose their membrane integrity and become necrotic.
  • Karyolysis dissolution of nuclei in necrotic cells typically occurs within 2-3 days in vivo, but appears to occur more slowly during thymus culture.
  • eosinophilic (pink) expanses of necrotic cell debris where thymocyte nuclei have undergone karyolysis. Some dead thymocytes retain their nuclei, which have ragged edges and altered staining characteristics compared to those of viable cells.
  • TE three-dimensional thymic epithelial
  • the nuclei of viable TE cells are typically oval, larger than those of thymocytes, and have a sharply defined nuclear membrane outlined by the hematoxylin (blue) stain, as well as one or more nucleoli. These TE nuclei typically look “open”, meaning they do not stain darkly with hematoxylin. This fits with an interpretation that they are alive and metabolically active, since active chromatin (“euchromatin”) cannot bind the hematoxylin dye.
  • the presence of nucleoli, which are the sites of ribosome synthesis, in many TE cells further confirms that they are alive and metabolically active.
  • the typical histologic appearance of control sections from days 5, 12 and 21 is shown Figs. 8, 9 and 10, respectively.
  • the pathologist indicated that the appearance of the slices did not differ significantly from those of the control.
  • the pathologist noted that the heat treatment may have “fixed” the cells, by coagulating proteins that prevent further degradation. Heat treatment has been used as a fixative for tissues, including thymus.
  • Fig. 12A and 12B depict the histology of thymus tissue slides after exposure to forced degradation conditions.
  • Fig. 10 depicts the histologic appearance of control thymus tissue slices at day 21.
  • Example 5 The overall experimental design of Example 5 is depicted in Fig. 20. A schematic representation of surgical procedures and treatment schedule is presented. The figures and much of the text below come from a manuscript in preparation for submission for publication: Kwun, J. et al., JCI Insight . 2020 Jun 4;5(11).
  • the schematic presentation of the experimental design depicts naive T cell reconstitution, thymopoiesis, and donor-specific tolerance induced by the surgical insertion of CTT.
  • All Lewis (LW) rats were thymectomized and T cell depleted via anti-CD5 mAh prior to heart transplantation and surgical insertion of a CTT.
  • CTT from FI (LWxDA) rats and hearts from DA rats were transplanted into thymectomized LW recipients.
  • Cyclosporine (CsA) was given for four months after transplantation via osmotic pump.
  • the third-party BN heart was transplanted into the neck 2 to 3 months after CsA discontinuation.
  • Example 5 demonstrates that CTT implanted in an immunoincompetent rat model, as described below, can induce tolerance to a transplanted solid organ.
  • FI Lewis x Dark Agouti, LWxDA
  • a implant of CTT cultured as described below
  • vascularized mismatched DA heart transplants into Lewis rats.
  • recipients Prior to the implant of CTT, recipients were thymectomized and T cell depleted.
  • Cyclosporine was administered for 4 months starting on the day of heart transplantation. The control group did not receive a implant of CTT.
  • Lewis (RT-11) and BN (RT-ln) rats were purchased from Charles River.
  • DA (RT-lavl) rats were purchased from Envigo.
  • F1(LEW/DA; RT-ll/avl) were bred by protocol staff at the Duke Breeding Core Division of Laboratory Animal Resources facility.
  • Lewis recipients received thymectomies 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).
  • Thymuses from three day’s old neonatal FI (LEW/DA) rat pups were harvested sterilely, cut into four pieces along the longitude natural seam, and transferred onto sterile nitrocellulose filters (MF-Millipore, Millipore Sigma) in a tissue culture dish with TOM medium (Fig. 17B). Thymus tissue was cultured in a CO2 incubator with 5% CO2 at 37° C for the desired length of time (5 to 7 days). The medium was changed daily.
  • the thymus organ medium was composed of HAMS F12 (Life Technologies) at 86.5%; Hepes (Life Technologies) at 25mM; L-Glutamine Life Technologies) at 2mM; Fetal Bovine Serum (Life Technologies) at 10%; and Pen-strep (Life Technologies) at lx.
  • HAMS F12 Life Technologies
  • Hepes Life Technologies
  • L-Glutamine Life Technologies Life Technologies
  • Fetal Bovine Serum Life Technologies
  • Pen-strep Life Technologies
  • RT-l avl Full MHC mismatched DA (RT-l avl ) donor hearts were transplanted into thymectomized Lewis (RT-1 1 ) recipients. Abdominal heart transplantation was performed using a modified technique of the methods described by Schmid C, Binder J, Heemann U, & Tilney NL, 1994,” Successful heterotopic heart transplantation in rat,” Microsurgery 15(4):279-281.
  • the donor heart was transplanted into the abdominal cavity of the recipient after a short period of cold ischemia in Euro-Collins solution.
  • the donor pulmonary artery and aorta were anastomosed to the recipient inferior vena cava and descending aorta with an end-side fashion as the inflow and outflow vessels for circulation, using running 9/0 non-absorbable monofilament sutures.
  • Cyclosporine A (CsA) was given via the osmotic pump (Model 2ML4, Alzet).
  • the recipients also received cyclosporine (CsA), approximately 2.5 mg/kg/day after thymus transplantation using osmotic pumps.
  • CsA was discontinued 4 months after thymus transplantation when the test group had naive T cells over 10%.
  • the pump was loaded sterilely and surgically inserted subcutaneously to mid-dorsal area of recipients. The osmotic pump was replaced every month for 4 months.
  • RT-l n third-party heart transplantation to the DA heart bearing Lewis recipients
  • the cervical vascularized heart transplantation method described by Heron, et al. (Heron T, 1971, “A technique for accessory cervical heart transplantation in rabbits and rats,” Acta Pathol Microbiol ScandA 79(4):366-372) was used in modified fashion.
  • the 3rd party BN heart was transplanted in the neck.
  • the third-party heart was transplanted into the right side of cervical area via a longitudinal incision from submaxilla to the xiphoid.
  • the donor pulmonary artery and external jugular vein were anastomosed end to end and the aorta was anastomosed to the right common carotid artery by cuffing technique.
  • the grafts were monitored by daily palpation and later confirmed by laparotomy at the time of sacrifice. Animals were sacrificed on the day of rejection (cessation of beating) or a designated time point.
  • Peripheral blood was obtained from the cranial vena cava and stained with antibodies.
  • DSA conjugated anti-mouse IgG
  • FITC-conjugated pan-rat immunoglobulin antibody was added to the samples and incubated after washing.
  • the T cells were stained with APC-conjugated anti-CD3. Samples were analyzed on a LSR fortessa (Beckman Coulter).
  • TEC thymic epithelial cells
  • recipient-derived T cells not expressing DA MHC, appeared in the peripheral blood of thymus implant recipients (Fig. 21). After implantation of CTT, increasingly repopulating recipient-type T cells are seen in the lower right quadrant of Fig. 21 at days 26, 55,
  • Circulating T cell repopulation after T cell depletion and thymus and heart transplantation are depicted in Fig. 23. All animals showed dramatic reduction of circulating T cells after T cell depletion. Cardiac allograft recipients with a CTT insertion (blue/dashed line) showed gradual repopulation of circulating T cells. Animals without a CTT insertion also showed some degree of circulating T cells (red/dotted line). However, naive and recent thymic emigrants CD4 and CD8 T cells were significantly increased (p ⁇ 0.01) in animals with a CTT insertion while control animals showed no circulating naive nor RTE CD4 and CD8 T cell.
  • Implanted thymus explanted at 8.5 month after implantation showed positive cytokeratin staining (Fig. 22A) as well as T cell staining similar to native thymus (Fig. 22B).
  • Fig. 24 Engrafted cultured thymus tissues under the renal capsule on day 180 in a recipient of cardiac allograft recipients is depicted in Fig. 24.
  • Histology showed distinct structures separate from renal tissue (Original magnification, x20).
  • Engrafted cultured thymus tissue showed a normal thymus structure (H&E), viable T cells (CD3), T cell proliferation (Ki67), and Hassall body formation (Black arrow) with a lacy pattern (Cytokeratin) on epithelial cells, confirming the viability of thymus with thymopoiesis (Fig.
  • Fig. 25 shows LW rats with DA heart transplants and without any immunosuppressive treatment rejected the DA heart grafts within 10 days (the DA control, open squares).
  • RTE CD90 + CD45RC +
  • a Kaplan-Meier survival curve (Fig. 25) showed significantly prolonged graft survival from animals with or without CTT and syngeneic controls (LW heart into LW rat) as compared to LW rats with DA heart transplants without any immunosuppression (DA control).
  • Representative scanned images of explanted graft at day 180 from animals with and without CTT are shown in Figs. 26A and 26B. The images were adapted from whole slide scan.
  • Fig. 29 shows rejection of BN hearts by LW rats.
  • the syngeneic control (open circles, Fig. 29) shows lack of rejection of LW hearts by LW rats.
  • Kaplan-Meier survival curve (Fig. 27) showed significant differences in the graft survival.
  • Representative scanned images of explanted BN heart graft at the time of rejection or 46 days post-transplantation are shown in Figs. 28 A and 28B, respectively.
  • BN heart grafts from animals with CTT inserted showed severe mononuclear cell infiltration (Fig. 28A), while BN heart grafts from animals without CTT inserted showed no sign of rejection (Fig. 28B). Images were adapted from whole slide scan.
  • ISHLT grading Histological analysis (ISHLT grading) of explanted BN hearts from rats with CTT inserted (filled squares, Fig. 29) showed grade 3R rejection with significantly increased inflammatory cell infiltration compared to syngeneic controls or rats without CTT inserted (shaded triangles, Fig. 29).
  • DA control grafts in LW rats with no immunosuppression
  • Animals inserted with CTT showed massive inflammatory cell infiltration in the third- party cardiac allograft (BN heart) (Fig. 3 IB).
  • BN control grafts in LW rats with no immunosuppression
  • BN grafts from recipients with CTT showed significantly elevated inflammatory cell infiltration compared to those in BN grafts from animals without CTT.
  • Rats not inserted with CTT showed no infiltrates in the BN heart (Fig. 3 IB) shaded triangles) because of their immunoincompetence.
  • T cell infiltration was evaluated with immunohistochemistry and confirmed a selective T cell infiltration in the BN (right hand panels, Fig. 31C), but not DA (middle panels, Fig.31C) hearts of the animals inserted with CTT and a lack of T cell infiltration in both hearts of animals without CTT inserted (Fig. 3 ID).
  • Heart allografts from DA and BN rats were harvested with native heart at the time of BN heart rejection. Grossly, native heart and DA heart (POD 196) did not show dramatic increase of T cells, while BN heart (POD14) showed a massive amount of T cells in recipients with CTT inserted. Images were adapted from whole slide scan. Total 3-5 animals per group were analyzed; student’s t-test, *P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001,
  • Anti-donor antibody responses were evaluated to determine whether the allogeneic T cell unresponsiveness noted in thymectomized LW rats followed by LWxDA transplants and the surgical insertion of CTT, was associated with humoral tolerance against donor DA MHC.
  • Serially collected recipient serum samples were collected and flow cross-match with PBMCs from DA and BN rats was performed. Animals that received DA or BN heart transplants without immunosuppression developed antibody against their donors (DA or BN, respectively). Animals with a syngeneic cardiac allograft did not produce antibody against either DA or BN MHC, as reported in Fig. 32A (horizontal shaded peaks in the left hand column, on top for DA, and on the bottom for BN).
  • Fig. 32A Representative histogram plots for post-transplant donor-specific alloantibody (anti-DA and anti-BN antibodies) measured by T cell flow cross-match are shown in Fig. 32A.
  • Recipients with or without CTT did not generate any antibodies against DA antigen (the top row, middle and right hand columns in Fig. 32A), while animals with CTT were able to generate antibody against BN antigen (lower row, middle panel, Fig. 32A).
  • Serum samples from recipients of DA heart transplantation without immunosuppression and from LW recipients of BN heart transplantation without immunosuppression were used as positive controls (DA control and BN control) for anti-DA (top row, left panel, bold line or anti-BN antibody (bottom row, left panel, dashed line), respectively.
  • thymus co-transplantation resulted in specific tolerance to the allogeneic DA MHC expressed in the donor thymus, and thus long-term survival of the DA heart transplant via preventing development of both the donor-specific anti-DA T cell repertoire as well as preventing the donor (DA)-specific humoral response. Immunocompetence was demonstrated in these rats by the rapid rejection of third-party BN hearts as well as alloantibody response against BN donor cells.
  • Patient l is a child born with complete DiGeorge anomaly. He had no T cells at birth.
  • a major problem for Patient 1 was profound hypoparathyroidism leading to many hospitalizations for hypocalcemia.
  • Patient 1 was given both a cultured thymus tissue transplant (CTT) and a parental parathyroid gland transplant on the same day. There were three other patients given thymus plus parental parathyroid in a small clinical trial.
  • Patient 1 received the two transplants at 4 months of life. Although Patient 1 had no T cells, Patient 1 was given RATGAM for immunosuppression prior to transplantation per protocol. No other immunosuppression was given.
  • Patient 1 developed naive T cells and normal proliferative T cell responses to mitogens.
  • Patient 1 All four patients who received both thymus and parathyroid in the trial developed normal parathyroid hormone levels.
  • Patient 1 was the only subject who was able to come off calcium supplementation long term (10 years). Of the three other Patients, one died prior to one year from pulmonary problems, and the other two with complete DiGeorge anomaly had to return to calcium supplementation by approximately one year.
  • Patient 1 was the only subject who had a negative mixed lymphocyte reaction (MLR) against the parental parathyroid donor at all time points. The other three subjects had positive MLRs starting with their first assay.
  • MLR mixed lymphocyte reaction
  • thymus donor thymic epithelial cells also delete thymocytes that bound tightly to them (marked “*” in Table 9 below). This is the mechanism of tolerance toward the alleles marked “*” in Table 9 below.

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Abstract

L'invention concerne des méthodes et des compositions permettant de favoriser la tolérance spécifique d'un donneur et l'immunocompétence pour un receveur d'un greffon d'organe solide, par l'implantation d'un organe solide allogénique chez un receveur ayant besoin d'un greffon d'organe solide et comprenant en outre l'implantation chirurgicale d'un produit tissulaire de thymus post-natal mis en culture allogénique modifié par génie tissulaire chez le receveur d'un organe solide provenant d'un donneur. L'invention concerne également des procédés de production d'un produit dérivé de tissu de thymus post-natal cultivé allogénique approprié pour une implantation chez un être humain; des procédés de culture d'un produit dérivé de tissu de thymus post-natal cultivé allogénique approprié pour une implantation chez un être humain et des procédés d'utilisation d'un produit dérivé de tissu de thymus post-natal cultivé allogénique par implantation chez un sujet humain.
PCT/US2020/046341 2018-02-23 2020-08-14 Procédés pour déterminer la convenance d'un tissu de thymus cultivé pour l'implantation chez l'homme et procédés d'utilisation associés WO2021034650A1 (fr)

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