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WO2024152030A1 - Épreuve de compatibilité pour xénogreffe porcine - Google Patents

Épreuve de compatibilité pour xénogreffe porcine Download PDF

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Publication number
WO2024152030A1
WO2024152030A1 PCT/US2024/011527 US2024011527W WO2024152030A1 WO 2024152030 A1 WO2024152030 A1 WO 2024152030A1 US 2024011527 W US2024011527 W US 2024011527W WO 2024152030 A1 WO2024152030 A1 WO 2024152030A1
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cells
xenotransplantation
kidney
porcine
human
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Jayme Elizabeth LOCKE
Vera HAUPTFELD-DOLEJSEK
Paige PORRETT
Julie Aline HOUP
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UAB Research Foundation
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UAB Research Foundation
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    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56977—HLA or MHC typing
    • 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

  • kidney transplantation For most of the more than 700,000 Americans living with kidney failure, kidney transplantation — the gold standard treatment — remains elusive (Wolfe RA, et al. N Engl J Med. 1999 341 :1725-1730), despite efforts to increase the donor pool (Goldberg DS, et al. N Engl J Med. 2017 376:2394-2395; Montgomery RA, et al. N Engl J Med. 2011 365:318-326; Starzl TE, et al. Transplantation. 1964 2:752-776).
  • the domestic pig is a promising source of kidney xenografts.
  • a method for crossmatching a human subject for porcine transplantation involves assaying a serum sample from the subject for a swine leukocyte antigen (SLA) haplotype, wherein an SLA haplotype corresponding to a positive control is an indication that the subject is not a crossmatch for the xenotransplantation, and wherein an SLA haplotype corresponding to a negative control is an indication that the subject is a crossmatch for the xenotransplantation.
  • SLA swine leukocyte antigen
  • the subject has a positive crossmatch for the xenotransplantation, further comprising transplanting an organ (e.g. kidney, lung, liver, heart, or pancreas) from a donor pig to the subject.
  • an organ e.g. kidney, lung, liver, heart, or pancreas
  • the subject is not a crossmatch for the xenotransplantation, further comprising treating the subject with plasmapheresis prior to transplanting an organ from a donor pig to the subject.
  • assaying the serum sample involves contacting porcine cells with the serum sample, assaying for antibodies bound to the cells, and comparing antibody binding to a positive control and negative control.
  • the pig cells are peripheral blood mononuclear cells (PBMCs).
  • the method involves assaying for antibodies bound to porcine lymphocytes.
  • assaying the serum sample involves assaying the subject for a human leukocyte antigen (HLA) haplotype and comparing the HLA haplotype to a control haplotype based on HLA antibodies that cross-react with SLA antigens in a positive control.
  • HLA human leukocyte antigen
  • FIG. 1 shows a study timeline and event summary for Example 1.
  • FIG. 2 shows detection of swine leucocyte antigen and decedent flow crossmatch results.
  • PBMCs from a 10GE pig isolated and provided fresh or frozen by Revivicor Inc.
  • Porcine PBMCs were also incubated with negative and positive control sera that were identified from screening of sera banked in the histocompatibility laboratory at the University of Alabama at Birmingham.
  • FITC labeled secondary antibody (goat) was used to detect antibodies in the serum that were bound to the porcine lymphocytes. Histograms are shown for all cells or for lymphocytes gated based on FSC and SSC characteristics.
  • Prospective crossmatches were performed using previously frozen porcine PBMCs.
  • Retrospective crossmatches were performed using freshly isolated porcine PBMCs.
  • FIGs. 3A to 3D show reperfusion of porcine renal xenotransplants in the human decedent. Intraoperative photographs demonstrate viable kidney transplants bilaterally.
  • FIG. 3A shows reperfusion of the right kidney as shown over the course of approximately 1 min.
  • Panel A(i) shows appearance of the right kidney immediately prior to reperfusion after completion of the vascular anastomosis.
  • Vascular clamps are present in the operative field.
  • Panel A(ii) shows appearance of the right kidney immediately after removal of vascular clamps. Note darker pink color of the kidney and the appearance of blood on the kidney surface under surgeon's hand.
  • Panel A(iii) shows appearance of the right kidney 5-10 s after removal of clamps. Reperfusion is progressing from superior to inferior pole.
  • FIG. 3B shows sequential urine output after reperfusion of the right kidney is shown.
  • Right kidney is depicted by black arrowheads.
  • Panels B(i) and B(ii) showcase urine output prior to ureteral anastomosis. Right ureter is being held in the surgeon's hand alongside collection cup. Note increased volume of urine in the cup between Panels B(i) and B(ii).
  • Panel B(iii) shows urine output from the right kidney after anastomosis to the decedent bladder. Total volume in the collecting Foley bag is shown.
  • FIG. 3C shows comparable kinetics of reperfusion and absence of hyperacute rejection for the left porcine renal xenograft.
  • FIG. 3D show reperfusion biopsy results of the left kidney. There was no difference in gross appearance of the kidneys at the time of biopsy.
  • FIGs. 4A to 4C show annotated anesthesia report of intraoperative hemodynamic monitoring. Results demonstrate stability of the decedent during bilateral native nephrectomies and transplantation of bilateral kidney xenografts. Phenylephrine and dopamine dosing are shown as continuous infusions while ephedrine was administered as 10 mg boluses.
  • FIG. 4A shows anesthetic record from 10:30 to 14:00. During this time frame, the decedent underwent native nephrectomies and the xenografts were prepared on the backbench.
  • FIG. 4B shows anesthetic record from 14:00 to 17:30. Anastomosis and reperfusion of the xenografts is performed. Specific timing of xenograft reperfusions are shown.
  • FIG. 4C shows anesthetic record from 17:30 to completion of surgery. Ureteral anastomoses were performed during this time frame.
  • FIG. 5 shows longitudinal assessment of the porcine renal xenografts. Photographs from post-operative days 1 and 3 (POD 1 , POD 3) were taken intraoperatively while the kidneys were in vivo. Minor blood accumulation underneath the right kidney capsule on POD 1 occurred after biopsy was taken. Yellow tinge of left kidney on POD 3 likely reflects bilirubin staining given hyperbilirubinemia in the decedent.
  • FIGs. 6A and 6B show porcine renal xenotransplant function in the human decedent.
  • FIG. 6A shows cumulative posttransplant urine output from transplantation to study end from right and left xenografts.
  • FIG. 6B shows BUN and creatinine in the decedent's serum. Results prior to POD 0 reflect function of decedent's native kidneys prior to native nephrectomies.
  • FIG. 7 shows serial histologic examination of the porcine kidney xenografts. All biopsies represent core biopsies obtained ex vivo (panels A, B, G, and H) or in vivo (panels C, D, E and F). Sections are stained with PASH and are 10X, except for (panels C and D,40X) and (panel F, silver stain). C4d negative throughout. Panels A and B shows mild to moderate acute tubular injury from cold ischemia. Normal appearance of the capillary network, the mesangium, and the podocytes. Panels C and D shows glomerulus with multiple fibrin thrombi (circle).
  • FIG. 8 shows immunofluorescence, staining (left xenograft).
  • Core biopsies, of the left renal xenograft were, obtained, fixed in formalin and paraffin, embedded, and then submitted for, immunofluorescence microscopy to, Arkana Laboratories (Little Rock, AR)., Tissues were stained as indicated, following protease digestion.
  • POD 1 samples had 2 glomeruli present, for evaluation. No glomerular or, extraglomerular staining was noted., Kappa and lambda light chains stained, equally throughout the tubules and, interstitium. On POD 3, two intact, glomeruli were evaluated.
  • FIG. 9 shows immunofluorescence staining (right xenograft).
  • FIG. 10 shows longitudinal analysis of porcine endogenous retrovirus transmission and microchimerism in the decedent.
  • No PERV or microchimerism (pig-specific RPL4) was detected by RT-PCR using mRNA from different time intervals posttransplant.
  • Pig(+) is a PERVC-positive pig control.
  • GAPDH is an endogenous control showing presence of mRNA in all samples. Water is shown as a negative control.
  • FIG. 11 shows kidney function over time after 10GE pig-to-human xenotransplantion.
  • FIG. 12. shows kidney histopathology after 10GE pig-to-human xenotransplatation.
  • FIGs 13A to 13D show Xenotransplant recipient hormone plasma concentrations over time, Renin-Angiotensin-Aldosterone System (RAAS). Shaded areas represent normal human ranges for each hormone.
  • FIG. 13A shows renin (pg/ml), normal ⁇ 45.7 pg/ml. Plasma renin activity was ⁇ 0.6 ng/ml/hr at all time points.
  • FIG. 13B shows angiotensinogen (pg/ml), 71 pg/ml is the upper limit of normal.
  • FIG. 13C shows angiotensin II (pg/ml), normal range 3-30 pg/ml.
  • FIG. 13D shows aldosterone (pg/ml), normal range 31-354 pg/ml.
  • FIG. 14 shows parathyroid hormone (PTH) levels and ionized calcium levels in decedent following xenotransplantation.
  • PTH parathyroid hormone
  • FIGs. 15A to 15D show renal clearance physiology.
  • FIG. 15A shows inulin decay curve, concentration at timed intervals after a 10 g bolus injection.
  • FIG. 15B shows pig kidney clearance grouped by method of measurement.
  • FIG. 15C shows serum creatinine trend.
  • FIG. 15D shows tacrolimus pharmacokinetics.
  • FIGs. 16A to 16D show water and sodium balance.
  • FIG. 16A shows decedent’s daily urine output after xenotransplantation (liters). Intraoperative furosemide 100mg and mannitol 25g were administered intravenously right before reperfusion.
  • FIG. 16B shows serum sodium.
  • FIG. 16C shows water clearance after xenotransplantation (liters).
  • FIG. 16D shows urine osmolarity mOsm /kg H 2 O).
  • FIG. 17 shows aquaporin (AQP) expression in the 10 GE xenokidney.
  • Panel A shows AQP1 in the apical side of the proximal tubule.
  • Panel B shows AQP4 in the basolateral membrane of the principal cells of the collecting duct. Arrows indicate principal cells positive for AQP4.
  • Panel C shows AQP2 in the apical membrane of the principal cells, and
  • Panel D shows AQP2 phosphorylation S256, a known activated form of AQP2, is also expressed in the principal cells.
  • Panel E shows immunofluorescent labeling of principal cells with AQP2 488 and V ATPase positive staining of intercalated cells.
  • Panel F shows cortex, and Panel G shows medulla: representative trichrome stained sections with proximal tubules (PT) and collecting ducts lined by pale staining principal cells and rare darkly stained intercalated cells. Asterisks (*) denote intercalated cells. Scale bar represents 50 micrometers.
  • FIGs. 18A to 18C show crossmatch for pig-to-human xenotransplantation.
  • FIG. 18A shows crossmatch schema where the Decedent 1 crossmatch can also be found.
  • Flow cytometry with decedent sera and porcine donor lymphocytes were performed. For all tubes, lymphocytes and serum were incubated with fluorescein isothiocyanate conjugated goat antihuman IgG F(ab)’2. Pooled sera from human males blood type AB was used as a negative control. Human serum containing IgG known to react with porcine lymphocytes was used as a positive control.
  • FIG. 18B shows negative crossmatch for Decedent 2, with appropriate controls.
  • FIG. 18C shows negative crossmatch for Decedent 3, with appropriate controls.
  • FIG. 19 shows decedents’ tacrolimus levels following xenotransplantation. Note only Decedent 3 reached therapeutic range (8-12 ng/mL, dashed lines).
  • FIG. 20 shows eculizumab level of Decedent 3 after xenotransplantation. Note the subtherapeutic levels at post-operative day 5, when MAC staining became evident. >50 mcg/mL is the published therapeutic threshold for atypical hemolytic uremic syndrome (aHUS, dashed line), Mayo Clinic Laboratories.
  • Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of chemistry, biology, and the like, which are within the skill of the art.
  • pig refers to any pig known to the art, including a wild pig, a domestic pig, mini pigs, a Sus scrofa pig, a Sus scrofa domesticus pig, as well as inbred pigs.
  • the pig can be selected from the group consisting of, for example, Landrace, Hampshire, Duroc, Chinese Meishan, Chester White, Berkshire Goettingen, Landrace/York/Chester White, Yucatan, Barna Xiang Zhu, Wuzhishan, Xi Shuang Banna, and Pietrain pigs.
  • Porcine organs, tissue or cells are organs, tissue or cells from a pig.
  • the term “subject” refers to any individual who is the target of administration or treatment.
  • the subject can be a vertebrate, for example, a mammal.
  • the subject can be a human or veterinary patient.
  • patient refers to a subject under the treatment of a clinician, e.g., physician.
  • sample from a subject refers to a tissue, organ, cell (including a cell maintained in culture), cell lysate (or lysate fraction), biomolecule derived from a cell or cellular material (e.g. a polypeptide or nucleic acid), or body fluid from a subject.
  • body fluids include blood, urine, plasma, serum, tears, lymph, bile, cerebrospinal fluid, interstitial fluid, aqueous or vitreous humor, colostrum, sputum, amniotic fluid, saliva, anal and vaginal secretions, perspiration, semen, transudate, exudate, and synovial fluid.
  • an animal e.g., a transgenic porcine animal
  • the animal is an ungulate and more particularly, a porcine animal or pig.
  • organs are derived from a transgenic pig.
  • a transgenic pig for xenotransplantation is described in US2018/0249688, which is incorporated by reference in its entirety for the teaching of these genetically engineered pigs and uses of their organs for xenotransplantation.
  • Crossmatching is described in US2018/0249688, which is incorporated by reference in its entirety for the teaching of these genetically engineered pigs and uses of their organs for xenotransplantation.
  • a subject is crossmatched according to the disclosed methods by assaying a serum sample for immunoreactive antibodies.
  • a subject is crossmatched according to the disclosed methods by assaying a serum sample for an HLA haplotype indicative of HLA antibodies that cross-react with SI_A antigens in a positive control.
  • HI_A class I genes include HLA-A*, HLA-B*, HI_A-Cw* haplotype combinations.
  • the HI_A class II genes include HLA DRBI*, DRB3*, DRB4*, DRB5*, DQAI*, and DQBI* haplotype combinations.
  • biological products for xenotransplantation are derived from source animals produced and maintained according to methods known in the art.
  • biological products include, but are not limited to, liver, kidney, skin, lung, heart, pancreas, intestine, nerve and other organs, cells and/or tissues.
  • Harvesting of such biological products occurs in a single, continuous, and self- contained, segregated manufacturing event that begins with the sacrifice of the source animal through completion of the production of the final product.
  • the animal is euthanized via captive bolt euthanasia, may be moved, if necessary, in a sterile, non-porous bag, to an operating room where the procedure to harvest biological product from the source animal will occur. All members of the operating team should be in full sterile surgical gear, e.g., dressed in sterile dress to maintain designated pathogen free conditions prior to receiving the source animal and in some instanced be double-gloved to minimize contamination, and surgical areas and tools are sterilized.
  • the source animal is removed from the bag and container in an aseptic fashion.
  • the source animal is scrubbed by operating staff, e.g., for at least 1-10 minutes with antiseptic, e.g., Chlorhexidine, brushes over the entire area of the animal where the operation will occur, periodically pouring Chlorhexidine over the area to ensure coverage.
  • Surgical area(s) of the animal are scrubbed with opened Betadine brushes and sterile water rinse over the entire area of the animal where the operation will occur for, e.g., 1-10 minutes.
  • operators will be dressed in sterile dress in accordance with program and other standards to maintain designated pathogen free conditions. All organs, cells or tissue from the source animal that will be used for xenotransplantation is harvested within 15 hours of the animal being sacrificed.
  • Biological products can also include, but are not limited to, those disclosed herein (e.g., in the specific examples), as well as any and all other tissues, organs, and/or purified or substantially pure cells and cell lines harvested from the source animals.
  • tissues that are utilized for xenotransplantation as described herein include, but are not limited to, areolar, blood, adenoid, bone, brown adipose, cancellous, cartaginous, cartilage, cavernous, chondroid, chromaffin, connective tissue, dartoic, elastic, epithelial, Epithelium, fatty, fibrohyaline, fibrous, Gamgee, Gelatinous, Granulation, gut-associated lymphoid, Haller's vascular, hard hemopoietic, indifferent, interstitial, investing, islet, lymphatic, lymphoid, mesenchymal, mesonephric, mucous connective, multilocular adipose, muscle, mye
  • organs that are utilized for xenotransplantation as described herein include, but are not limited to, skin, kidneys, liver, brain, adrenal glands, anus, bladder, blood, blood vessels, bones, cartilage, cornea, ears, esophagus, eye, glands, gums, hair, heart, hypothalamus, intestines, large intestine, ligaments, lips, lungs, lymph, lymph nodes and lymph vessels, mammary glands, mouth, nails, nose, ovaries, oviducts, pancreas, penis, pharynx, pituitary, pylorus, rectum, salivary glands, seminal vesicles, skeletal muscles, skin, small intestine, smooth muscles, spinal cord, spleen, stomach, suprarenal capsule, teeth, tendons, testes, thymus gland, thyroid gland, tongue, tonsils, trachea, ureters, urethra, uterus, and vagina.
  • purified or substantially pure cells and cell lines that are utilized for xenotransplantation as describe herein include, but are not limited to, blood cells, blood precursor cells, cardiac muscle cells, chondrocytes, cumulus cells, endothelial cells, epidermal cells, epithelial cells, fibroblast cells, granulosa cells, hematopoietic cells, Islets of Langerhans cells, keratinocytes, lymphocytes (B and T), macrophages, melanocytes, monocytes, mononuclear cells, neural cells, other muscle cells, pancreatic alpha-1 cells, pancreatic alpha-2 cells, pancreatic beta cells, pancreatic insulin secreting cells, adipocytes, epithelial cells, aortic endothelial cells, aortic smooth muscle cells, astrocytes, basophils, bone cells, bone precursor cells, cardiac myocytes, chondrocytes, eosinophils, ery
  • Embodiment 1 A method for crossmatching a human subject for porcine transplantation, comprising assaying a serum sample from the subject for antibodies reactive with swine leukocyte antigen (SLA), wherein reactivity corresponding to a positive control (positive SLA haplotype) is an indication that the subject is not a viable candidate (negative crossmatch) for the xenotransplantation, and wherein reactivity corresponding to a negative control (negative SLA haplotype) is an indication that the subject is a candidate (positive crossmatch) for the xenotransplantation.
  • SLA swine leukocyte antigen
  • Embodiment 2 The method of embodiment 2, wherein the subject is a positive crossmatch for the xenotransplantation, further comprising transplanting an organ from a donor pig to the subject.
  • Embodiment 3 The method of embodiment 2, wherein the subject has a negative crossmatch for the xenotransplantation, further comprising treating the subject with plasmapheresis prior to transplanting an organ from a donor pig to the subject.
  • Embodiment 4 The method of any one of embodiments 1 to 3, wherein the organ is a kidney, lung, liver, heart, or pancreas.
  • Embodiment 5 The method of embodiment 1 , wherein assaying the serum sample comprises contacting porcine cells with the serum sample, assaying for antibodies bound to the cells, and comparing antibody binding to a positive control and negative control.
  • Embodiment 6 The method of embodiment 5, wherein the pig cells are peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • Embodiment 7 The method of embodiment 5 or 6, wherein the method comprises assaying for antibodies bound to porcine lymphocytes.
  • Embodiment 8 The method of embodiment 1 , wherein assaying the serum sample comprises assaying the subject for a human leukocyte antigen (HLA) haplotype and comparing the HLA haplotype to a control haplotype based on HLA antibodies that cross-react with SLA antigens in a positive control.
  • HLA human leukocyte antigen
  • Example 1 First clinical-grade porcine kidney xenotransplant using a human decedent model
  • Decedent inclusion and exclusion criteria Eligible human decedents included adults (>18 years), declared brain-dead, referred for organ donation but ruled out for donation of heart, lung, liver, pancreas, and/or intestine, whose next-of-kin authorized research and transport to the recovery center, and had a negative prospective crossmatch with the donor pig.
  • Source animals Porcine renal xenografts were procured from genetically engineered (GE) pigs provided by Revivicor, Inc.
  • the GE pigs harbor ten genetic modifications (10-GE pigs), including targeted insertion of two human complement inhibitor genes (hDAF, hCD46), two human anticoagulant genes (hTBM, hEPCR), and two immunomodulatory genes (hCD47, hHO1), as well as deletion (knockout) of 3 pig carbohydrate antigens and the pig growth hormone receptor gene.
  • 10-GE pigs do not express red blood cell antigens and are therefore universal donors with respect to blood type.
  • KO of genes encoding [31 ,4-N- acetylgalactosyltransferase (p4GalNT2, the enzyme responsible for synthesis of SDa), CMP-N- acetylneuraminic acid hydroxylase (CMAH, the enzyme responsible for synthesis of Neu5Gc) and growth hormone receptor (GHR) were assessed by Next-Gen DNA sequencing (MiSeq, Illumina) for the presence of large or frameshifting indels.
  • Phenotypes of GGTA1 KO, B4GALNT2KO, and CMAHKO were confirmed by flow cytometry of PBMC stained with IB4 lectin, DBA lectin and anti-Neu5Gc respectively, to reveal the absence of xenogeneic carbohydrate residues catalyzed by the knocked-out gene product.
  • GHRKO phenotype was determined by demonstrating reduced serum IGF-1 levels and body weight. Expression of individual transgenes was confirmed in kidney biopsies of the donor pig after transplantation by western blot and immunohistochemistry.
  • 10-GE pigs Housing and maintenance of 10-GE pigs:
  • the 10-GE pigs are housed in facilities on the UAB XPC and are free of specified infectious agents (e.g., porcine CMV and porcine endogenous retrovirus C) which is assured by rigorous documentation, maintenance of well- defined routine testing, and rigorous standard operating procedures and practices for herd husbandry and veterinary care.
  • Donor source 10-GE pigs are tested every three months for porcine viruses, including porcine endogenous retrovirus C (see Table 4).
  • cDNA synthesis 50-100 ng of DNAse treated mRNA was reverse transcribed using an oligo-dT primer and the GoScript Reverse Transcription System (Promega).
  • PCR reaction 1 pl of cDNA template or water and 0.2 pM of each primer were added to 1x EmeraldAmp GT PCR Master Mix (Takara Bio) and amplified for 35 cycles of denaturation (98°C/1 min), annealing (60°C/30 s), and extension (72°C/45 s).
  • the RT-PCR products were analyzed on a 1.5% agarose gel containing ethidium bromide and visualized (FluorChem R imager, ProteinSimple).
  • Kidney procurement After induction of general anesthesia, the 10-GE pig donor kidneys were procured en bloc in a standard operating room at the UAB XPC using an aseptic technique.
  • Immunosuppression consisted of daily methylprednisolone taper, anti-thymocyte globulin for a total of 6 mg/kg, and anti-CD20. Maintenance immunosuppression included mycophenolate mofetil, tacrolimus, and prednisone.
  • Biopsies were formalin fixed and sectioned for staining including PASH, immunohistochemistry, hematoxylin & eosin, silver, and immunofluorescence in standardized methods. Formalin fixation was performed in order to reduce potential infectious risk.
  • kidney xenotransplantation was designed using a human brain-dead decedent model that included a pretransplant phase (19 h), a transplant phase (4 h), and a posttransplant phase (74 h) ( Figure 1).
  • the primary goal of the study was to address core safety questions within the limits of the decedent model that would inform the development of an IRB-approved clinical trial (Table 1).
  • a secondary goal was to test our xenotransplantation program infrastructure by executing all the steps required to perform kidney xenotransplantation in living humans.
  • a 13-month-old, 350 lb, male 10-GE donor pig was identified at the UAB Xenotransplantation Procurement Campus (XPC).
  • the donor animal had normal renal function (BUN 19, creatinine 1.3, assessed ⁇ 60 days prior to donation) and was negative for porcine endogenous retrovirus C and other pathogens (Table 4).
  • Prospective flow crossmatch between the decedent and 10-GE pig was negative ( Figure 2).
  • the decedent was brought to an operating suite, and anuria was established by performing bilateral native nephrectomies. Simultaneously, surgical procurement of the porcine kidneys occurred in an operating suite at the XPC. Of note, a surgical injury to the left porcine renal vein during procurement was repaired intraoperatively after clamping of the left renal vein for approximately 20 min. The kidneys were packaged in sterile fashion and transported on ice from the XPC to the DRC. Backtable preparation of the porcine kidneys occurred in standard fashion. Anatomy of the porcine kidneys largely recapitulated human renal anatomy. Preimplantation biopsies demonstrated normal histology of the 10-GE pig kidneys that appeared similar to normal human kidney.
  • the 10-GE pig kidneys were transplanted sequentially into the decedent using conventional heterotopic allotransplantation technique.
  • the kidneys were transplanted into the bilateral iliac fossae, thereby replicating the retroperitoneal location used in most kidney transplant centers.
  • Warm ischemia time was 28 and 29 min for the right and left xenografts, respectively; cold ischemia time was 4h and 5h 37 min for the right and left xenografts, respectively.
  • CNI calcineurin inhibitor
  • the decedent was maintained in the operating room for the remainder of the study. He received intensive nursing care, monitoring, and laboratory investigations as required for maintenance of cardiovascular perfusion in the setting of brain death. Over the ensuing three days of the study, the decedent developed progressive multisystem organ failure with evidence of shock liver, pancytopenia, and disseminated intravascular coagulation. Acidemia was significant, and maintenance of a normal pH and serum bicarbonate level required continuous administration of sodium bicarbonate (i.e., sodium bicarbonate 150 mEq + Dextrose 5% in Water @ 50 ml/h daily). He received continuous infusion heparin, blood transfusions, and additional high dose methylprednisolone to counter the effects of brain death physiology.
  • sodium bicarbonate i.e., sodium bicarbonate 150 mEq + Dextrose 5% in Water @ 50 ml/h daily.
  • kidney xenografts were well-perfused with the maintenance of turgor and Doppler signals throughout the parenchyma at all time points ( Figure 5). Two hours after the surgical exploration on day 3, the decedent developed exsanguinating hemorrhage due to his severe coagulopathy. The study was thus terminated at 77 h and 32 min after reperfusion and 8 days post-declaration of brain death.
  • the right kidney made 700 cc of urine within the first 24 h, with scant urine production from the left ( Figure 6).
  • Urine output from each kidney was monitored separately as the right xenograft ureter was anastomosed to the decedent's bladder while the left xenograft ureter was exteriorized as a urostomy.
  • Urinalysis obtained from the right kidney on postoperative day 1 (POD 1) revealed a normal specific gravity and the presence of RBCs, mild proteinuria and mild glucosuria (Table 6). Serum creatinine did not decrease over the course of the study ( Figure 6), and neither kidney excreted significant creatinine into the urine (Table 7, results shown for right kidney). However, normal serum electrolytes were maintained, likely due in part to exogenous administration of sodium bicarbonate.
  • Decedent was typed for HLA loci used in deceased donor allocation (A, B, C, DRB1 , DRB3/4/5, DQA1 , DQB1 , DPA1 , DPB1) and tested for presence of HLA- specific antibodies (Single Antigen Bead assay). See Table 8 for results.
  • a number of safety goals of this study revolved around the consequences of connecting the circulation of a human with a porcine kidney.
  • the blood pressures of both a pig and a non-human primate are significantly less than a human, and we tested the assumption that a porcine kidney could withstand the non-trivial increase in human blood pressure.
  • xenograft vascular integrity was maintained at human mean arterial pressures. Equally important was the relative hemodynamic stability of the decedent upon reperfusion, indicating that washout of inflammatory mediators from the xenograft during reperfusion did not provoke cardiovascular collapse.
  • TMA is not mediated by complement or antibody in the xenografts and is instead the result of some other unknown mechanism or molecular incompatibility. Nevertheless, the observed TMA may still yet be the result of complement-mediated cytotoxicity (Poppelaars F, et al. Mol Immunol. 2017 84:77-83), as the alternative complement pathway does not require antibody or C4 to trigger the formation of the membrane attack complex (MAC; C5-9) (Yamamoto T, et al. Transplantation. 2019 103(10):2090-2104).
  • MAC membrane attack complex
  • the 10-GE xenograft has been engineered to contain complement inhibitor genes (decay accelerating factor, DAF; membrane cofactor protein, MCP/CD46) to address some of the histologic findings associated with acute humoral xenograft rejection, 30 these proteins merely slow MAC formation and do not necessarily prevent it (Poppelaars F, et al. Mol Immunol. 2017 84:77-83). Additional genetic and/or pharmacologic interventions which prevent complement-mediated cytotoxicity may thus be necessary to improve graft survival and function. Of note, an anti-C5 antibody is available (Hillmen P, et al. N Engl J Med.
  • porcine kidney procurement was administered for the purposes of porcine kidney procurement, and porcine donors were humanely euthanized thereafter.
  • porcine kidneys were flushed with University of Wisconsin solution, sterile packed, cold-stored on ice, labeled, and transported via ground to the transplant center. Results
  • Goal tacrolimus levels (8-10 ng/dL) were reached postoperative day (POD) 2 and maintained through study completion.
  • Xenografts were transplanted en bloc with pig vasculature anastomosed to the decedent’s right-side common iliac artery and distal inferior vena cava and pig ureters anastomosed to the decedent’s bladder.
  • the xenografts made urine, producing more than 37 L in the first 24 hours.
  • serum creatinine was 3.9 mg/dL after cessation of dialysis and bilateral native nephrectomy. After xenotranplant, serum creatinine decreased to 1 .9 mg/dL within the first 24 hours, normalized to 1 .1 mg/dL at 48 hours, remained within normal limits through study duration, and was 0.9 mg/dL on POD 7 at study completion.
  • Pigs with 10 genetic edits including 4 gene knockouts (GTKO, CMAH, B4GALNT2, GHR) and 6 human transgenes (CD46, CD55, CD47, THBD, PROCR, HMOX1), were maintained in a pathogen-free facility and negative for porcine endogenous retrovirus C, porcine cytomegalovirus, and other zoonoses (Table 9). Porcine donors received general anesthesia for procurement and were humanely euthanized thereafter. 10 gene-edited porcine kidneys were flushed with the University of Wisconsin solution, sterile packaged, cold-stored on ice, and transported via ground to UAB for xenotransplantation. The study was approved by IACUC (No.22015).
  • Tissue compatibility was assessed using flow crossmatch as previously described (Porrett PM, et al. Am J Transplant. 2022 22(4): 1037-1053). Bilateral native nephrectomies were performed through a midline incision. Both porcine donor kidneys were transplanted en bloc to the decedent’s right common iliac artery and distal inferior vena cava. Porcine donor kidney ureters were anastomosed to the decedent’s bladder. Xenotransplantation was performed in an operating theatre meeting Joint Commission on Accreditation of Healthcare Organizations standards.
  • a complement inhibitor (anti-C5, eculizumab) was administered intravenously 24 h prior to (1200 mg) and 24 h after (900 mg) xenotransplantation.
  • Induction immunosuppression included intravenous methylprednisolone (500 mg), anti-thymocyte globulin (1.5 mg/kg), and anti-CD20, rituximab, (375 mg/m 2 ).
  • Intravenous methylprednisolone was tapered over four days for immunosuppression. Additional steroid dosing was administered throughout the experiment to manage brain death.
  • calcineurin inhibitor tacrolimus, goal level 8-12 ng/dL
  • mycophenolate mofetil 1000 mg twice daily
  • prednisone 30 mg once daily
  • tacrolimus pharmacokinetic study was performed. Specifically, tacrolimus levels (ng/mL) were drawn at time of administration on post-operative day 4 and then 2, 4, 6, 8, 10, and 12 hours post-administration; an area under the curve (AUC) was then calculated.
  • RAAS Renin-Angiotensin-Aldosterone-System
  • Serum PTH levels ionized calcium, phosphorus, creatinine, total 25-OH Vitamin D, and 1 ,25 DI-OH Vitamin D, along with random urine concentrations of phosphorus and creatinine were measured daily. The fractional excretion of phosphorus was calculated.
  • Kidney clearance was assessed by measuring 24-hour flow, serum inulin clearance, creatinine clearance, and cystatin-C based glomerular filtration rate (GFR) estimation. Blood samples for sodium, chloride, creatinine, cystatin-C, and random urine samples for sodium, chloride, and creatinine occurred daily. Following a validation study of GFR measurement in NHPs, inulin clearance was measured by analyzing the decay curve after bolus dosing (Hansen-Estruch C, et al. Xenotransplantation. 2023 30(2):e12795) on post-operative day 5 after serum creatinine nadir and stabilization. Serum Inulin was measured by ELISA (BioPAL, #FIT-0416). The area under the inulin serum concentration versus time curve was calculated. Inulin total body clearance after the bolus dose was calculated using the formula:
  • Electrolyte free water clearance was calculated as a measure of kidney water handling. Electrolyte free water clearance (liters) was calculated using the formula:
  • AQP Aquaporins
  • Primary antibodies were AQP1 (Proteintech, 1/2500 dilution), AQP2 (Santa Cruz Biotechnology, clone E2 1/1000), AQP2-phosphorylated S256 (Abeam, 1/1000), and AQP4 (Abeam, 1/2000).
  • Primary antibodies were diluted in 2.5% normal horse serum (Vector Labs) and on the tissues for 24 h at 4°C.
  • the anti-V-ATPase was 1/100 (Santa Cruz Biotechnology, sc-55544) diluted in 2.5% normal horse serum placed on the tissue for 1 h and then detected with 1/1000 goat-anti-mouse 595. This was followed by incubation with 488-directely tagged AQP2 (clone E2, 1/100). Nuclei were visualized with DAPI. Negative controls lacking primary antibodies were included. Images were taken with an Olympus Bx53 microscope and DP28 digital camera. Urine pH was measured using serial dipstick urinalyses.
  • Plasma angiotensinogen levels ranged from 92 pg/mL on post-operative day 1 to 58.7 pg/mL on post-operative day 7, comparable to healthy humans (Figure 13B) (Katsurada A, et al. Am J Physiol Renal Physiol. 2007 293(3): F956-60). Plasma angiotensin II increased from 0.6 pg/mL on post-operative day 4 to 10.6 pg/mL on post-operative day 7 (Figure 13C). Plasma aldosterone levels were low, ranging from 65 pg/mL on post-operative day 1 to 44.2 pg/mL on post-operative day 7 ( Figure 13D).
  • Serum potassium concentrations remained between 3.1 to 4.6 mEq/L throughout the study duration with a reduction in intravenous potassium supplementation from 160-200 mEq/day on post-operative days 1-4 to 40mEq/day on day 6 (Table 10).
  • Serum magnesium levels were maintained near 1.9 mg/dL with minimal infusion support (Table 10).
  • Serum vasopressin concentrations averaged 6.4 pg/mL, and copeptin was 0.26 pg/L on post-operative day 5.
  • PTH level was elevated prior to xenotransplantation and rose to 1 ,015 pg/mL on post-operative day 1 with a corresponding ionized calcium level ⁇ 1.0 mmol/L.
  • intravenous supplementation Table 11
  • ionized calcium increased to >1 mmol/L on post-operative day 2 and remained stable through study duration.
  • PTH decreased to 455.9 pg/mL on postoperative day 2 and remained between 232.1 and 386.1 pg/mL through study duration (Figure 14).
  • the decedent was found to be vitamin D deficient prior to xenotransplantation with total 25- OH Vitamin D level of 7 ng/mL.
  • Serum phosphate levels remained between 4.2-7.4 mg/dL postxenotransplantation; however, urinary phosphate excretion remained normal with a fractional excretion of phosphate averaging 29% in the last three days of the study.
  • Glomerular filtration rate increased in the first 5 days after xenotransplantation, to a peak of 240.7 mL/min by 24 h urine creatinine clearance on post-op day 4 and 231.6 mL/min by inulin clearance on post-op day 5 ( Figure 15A & 15B).
  • GFR Glomerular filtration rate
  • the GFR returned to 150 mL/min by 24 h urine creatinine clearance on post-op day 7 ( Figure 15B).
  • Urine output was 37 L in the first 24 hours of xenotransplantation (Figure 16A). Serum sodium (Na) levels rose sharply, peaking at 167 mEq/L on post-operative day 2 ( Figure 16B). As per standard brain death management protocol at UAB, the decedent was on a low dose continuous vasopressin infusion to replace pituitary function. In response to rising serum Na levels, replacement fluid was switched to 1 normal saline and a total of 2 doses of DDAVP were administered intravenously (2 mcg and 1 mcg on post-operative days 2 and 3, respectively) with gradual decline of serum Na levels to the normal range on post-operative day 3.
  • Serum osmolality levels ranged 285-312 mOsm/kg and were often above 300 mOsm/kg, representing high blood glucose levels.
  • the presence of glucosuria (urinalyses detected glucose at 1+ to 2+ on all 7 post-operative days), kept urine osmolality high despite net water loss ( Figures 16C and 16D).
  • Urinary water loss peaked on post-operative day 3 at 9.5 L per day and stabilized post-operative days 5-7 between 3-4.5L per day (Figure 16C).
  • Urine osmolality was 230 mOsm/kg on post-operative day 1 and peaked at 429 mOsm/kg by day 6 (Figure 16D).
  • Protein levels in the urine were initially nephrotic-range at 8.9 grams of total protein and 3.5 grams of albumin on post-operative day 1. By post-operative day 6, 24-hour total protein had reduced to 3.24 grams with 0.95 grams of albumin.
  • porcine kidney xenograft For the first-time, we have established the ability of a 10-gene edited porcine kidney xenograft to maintain physiologic homeostasis in a human.
  • the porcine xenograft cleared both endogenous and exogenous substrates, including the most common maintenance immunosuppressant used in transplantation, provided sufficient RAAS activity to maintain normal hemodynamics and avoid hyperkalemia, sufficiently concentrated urine to make daily enteral water intake feasible, secreted acid, and demonstrated appropriate hormonal response to hypocalcemia.
  • Understanding the physiologic underpinnings of pig-to-human kidney xenotransplantation is critical to ensuring the safety and feasibility of porcine kidney xenografts as a treatment option for persons with end-stage kidney failure.
  • porcine kidney xenografts can maintain normal serum creatinine but have provided few details regarding clearance of endogenous and exogenous substrates. Renal clearance as a metric is pivotal to understanding the ability of a kidney graft (xeno or allo) to provide immediate and long-term life sustaining kidney function.
  • the pig kidney has a reduced ability to concentrate urine and retain water compared to human kidneys given data from the pig-to-NHP xenotransplant model where urine osmolality levels remained less than 400 mOsm/kg despite intermittent hypotension.
  • the likelihood of impaired urinary concentrating ability of porcine kidney xenografts due to speciesspecific differences between human arginine vasopressin and pig lysine vasopressin is a substantial knowledge gap in our understanding of pig kidney physiology and could have significant consequences for human xenograft recipients. Prior to the present study, the ability of pig kidneys to concentrate urine had never been tested in a human recipient.
  • AQP2 is vasopressin-responsive and vasopressin results in increased trafficking of AQP2 to the apical membrane to drive water reabsorption. This is mediated through the phosphorylation of Serine 256 in the c-terminus of AQP2, and AQP2-S526 was detected in the apical membrane of the principal cells of the pig kidney. Thus, the localization of these water channels was normal and consistent with the water reabsorption reported. In the brain-dead model, a vasopressin infusion is required to replace reduced hypothalamic-pituitary function. Low levels of copeptin ( ⁇ 1 pg/L) on post-operative day 5 confirmed little endogenous vasopressin release.
  • the undetectable PRA confirmed little ability of the pig renin to cleave human angiotensinogen, although angiotensin II and aldosterone were detected.
  • the ability to maintain blood pressure without use of any inotropes in the absence of native human kidney renin production combined with measured levels of angiotensin II and aldosterone after porcine kidney xenotransplantation supports residual RAAS activity.
  • most kidney transplants in living persons do not involve bilateral native nephrectomies, and as such, in the setting of phase I clinical trials of porcine kidney xenotransplantation in living persons RAAS activity will be maintained and hypoaldosteronism and hypotension will be avoided. Renin and aldosterone levels are persevered in patients on hemodialysis for at least 27 months.
  • the PTH axis principally defends the body’s active form of circulating calcium in the blood (e.g., ionized calcium). Since the body’s primary stores of calcium are in the form of hydroxyapatite located in bone, PTH has secondary effects on bone mineralization and interacts with vitamin D and phosphate balance. In kidney failure, a combination of vitamin D deficiency and reduced urinary phosphate excretion result in pathologic hyperparathyroidism, which is primarily treated by administering activated vitamin D analogs (e.g., calcitriol). Kidney allotransplantation typically restores PTH levels to normal.
  • activated vitamin D analogs e.g., calcitriol
  • Example 4 C5 inhibition with eculizumab prevents thrombotic microangiopathy in a case series of pig-to-human kidney xenotransplantation
  • Sex as a biologic variable was considered in our study design and as such a specific biologic sex was not excluded from enrollment. However, our study represents a case series, and as such, by random chance no decedents were female.
  • Immunosuppression included induction therapy with methylprednisolone, antithymocyte globulin (6 mg/kg total), and anti-CD20 (rituximab).
  • Anti-thymocyte globulin (rabbit) was given in four separate doses (1 .5 mg/kg), the first in the operating room and subsequent doses on post-operative days 1 , 2, and 3.
  • Rituximab was dosed at 375 mg/kg/m2 and given 12 hours before xenotransplantation.
  • Maintenance therapy included tacrolimus, mycophenolate mofetil, and prednisone.
  • Decedent 3 native kidney showed tubular atrophy and severe arteriosclerosis, consistent with the decedent’s known chronic kidney disease, without evidence of TMA, though MAC deposition was present (Table 13).
  • Xenograft biopsies on PODO, POD1 , and POD3 had no MAC deposition, though MAC deposition was observed on POD5 and POD7 (Table 13), in the setting of subtherapeutic eculizumab (Fig. 20, Table 13).
  • TMA was not observed during the 7-day study period.
  • Tacrolimus was 12 ng/mL on POD1 , then ranged from 8.5-11 .3 ng/mL before peaking at 19.7 ng/mL on POD7 (Fig. 19).
  • C5 inhibition may be beneficial in preventing TMA in pig-to-human xenotransplantation.
  • Complement activation in the setting of brain death is common and was observed in Decedents 2 and 3 with the native kidneys staining positively for MAC.
  • Decedent 1 native kidneys had no evidence of complement activation, yet after xenotransplantation MAC deposition and TMA progressed rapidly, suggesting an immune response to the xenograft rather than brain death physiology.

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Abstract

La divulgation concerne une méthode d'épreuve de compatibilité d'un sujet pour une greffe porcine qui implique le dosage d'un échantillon de sérum du sujet pour analyser un haplotype d'antigène leucocytaire porcin (SLA), la correspondance d'un haplotype SLA avec un contrôle positif étant une indication du fait que le sujet n'est pas compatible pour la xénogreffe, et la correspondance d'un haplotype SLA avec un contrôle négatif étant une indication du fait que le sujet est compatible pour la xénogreffe.
PCT/US2024/011527 2023-01-13 2024-01-13 Épreuve de compatibilité pour xénogreffe porcine Ceased WO2024152030A1 (fr)

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