WO2024152030A1 - Crossmatching for porcine xenotransplantation - Google Patents

Abstract

Disclosed herein is a method for crossmatching a human subject for porcine transplantation that 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.

Description

CROSSMATCHING FOR PORCINE XENOTRANSPLANTATION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63/479,833, filed January 13, 2023, which is hereby incorporated herein by reference in its entirety. BACKGROUND OF THE INVENTION

[0002] 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. Proof-of-concept work has been performed in pig to non-human primate (NHP) models, in which NHPs were thought to best recapitulate human biology (Sykes M, et al. Sci Immunol. 2019 4(41):eaau6298). Critically, this work identified a major immune barrier to xenotransplantation — namely, the existence of carbohydrate antigens on vascular endothelium that are not expressed in Old World NHPs and humans. Genetic modifications to remove these antigens have improved the outcome of porcine xenotransplants in NHPs by avoiding hyperacute rejection (Kuwaki K, et al. Nat Med. 2005 11 :29-31 ; Phelps CJ, et al. Science. 2003 299:411-414; Yamada K, et al. Nat Med. 2005 11 :32-34). Additional modifications designed to mitigate complement-mediated cytotoxicity and thrombosis have further refined the model (Cooper DKC, et al. Xenotransplantation. 2019 26:e12516).

[0003] Despite the power of the NHP model, it is unlikely that all immunologic and functional hurdles will be overcome given the many biologic differences that exist between NHPs and humans. For example, NHP models are inadequate to prospectively test crossmatching assays for human use. Moreover, there are safety concerns surrounding transmission of porcine viruses (Niu D, et al. Science. 2017 357:1303-1307; Yang , et al. Science. 2015 350:1101-1104), and whether porcine genetic modifications are sufficient to avert hyperacute rejection in humans can only be determined through in vivo human studies. Ultimately, a human xenotransplantation experience will be required to develop the necessary knowledge to achieve excellent outcomes in humans. Given the inevitability of this bold step into human testing, the primary questions confronting the field are thus when and how to make this leap. In light of the extreme lethality of the organ shortage crisis and the successes achieved thus far in NHP models, one could argue that human testing is perhaps overdue. Nevertheless, caution is warranted, and some degree of efficacy must be expected. One-off experiments performed outside of a committed xenotransplantation program that do not address key knowledge gaps should be discouraged.

SUMMARY OF THE INVENTION

[0004] Disclosed herein is a method for crossmatching a human subject for porcine transplantation that 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.

[0005] In some embodiments, 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. In some embodiments, 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.

[0006] In some embodiments, 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.

[0007] In some embodiments, the pig cells are peripheral blood mononuclear cells (PBMCs). In some embodiments, the method involves assaying for antibodies bound to porcine lymphocytes. In some embodiments, 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.

[0008] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF FIGURES

[0001] FIG. 1 shows a study timeline and event summary for Example 1.

[0002] 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.) were incubated with pretransplant sera from the decedent. 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.

[0003] 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. Panel A(iv) shows appearance of the right kidney 1 min after removal of clamps. Entirety of kidney is now re-perfused. 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.

[0004] 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.

[0005] 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.

[0006] 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.

[0007] 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). There is diffuse glomerular capillary congestion with swollen endothelial cells and near complete obliteration of the peripheral capillary lumina. There is presence of fibrin thrombi and fragmented red blood cells consistent with thrombotic microangiopathy (TMA). There is evidence of progressive tubular injury with extensive acute tubular necrosis (ATN). No mesangiolysis is appreciated. Panels E and F shows glomerular congestion and acute tubular necrosis. Endothelial cells remain segmentally swollen with partially obliterated lumina and rare fibrin thrombi with improvement of glomerular injury. Panels G and H shows acute tubular injury persists. Glomeruli with segmental endothelial swelling. No fibrin thrombi.

[0008] 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. These, glomeruli revealed 1+ IgM staining of, the mesangium and segmental capillary, loop thought to be due to non-specific, entrapment. IgA, IgG, and kappa light, chain stains were negative in the glomeruli, without significant extraglomerular, staining. At termination on POD 3, core, biopsies were performed of the explanted, xenograft and analyzed. Six glomeruli, were present for evaluation, without, significant glomerular or extraglomerular, staining. [0009] FIG. 9 shows immunofluorescence staining (right xenograft). Core biopsies of the right 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. On POD 1 , no significant staining is observed in the glomeruli (n = 3). There is also no significant extraglomerular staining, and kappa and lambda staining is homogenous throughout the tubulointerstitium. Similar results were observed on biopsies from POD 3 (8 glomeruli evaluated) and the explant at termination on POD 3 (5 glomeruli evaluated).

[0010] 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.

[0011] FIG. 11 shows kidney function over time after 10GE pig-to-human xenotransplantion.

[0012] FIG. 12. shows kidney histopathology after 10GE pig-to-human xenotransplatation.

[0013] 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.

[0014] FIG. 14 shows parathyroid hormone (PTH) levels and ionized calcium levels in decedent following xenotransplantation.

[0015] 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.

[0016] 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 H₂O). [0017] 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.

[0018] 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.

[0019] FIG. 19 shows decedents’ tacrolimus levels following xenotransplantation. Note only Decedent 3 reached therapeutic range (8-12 ng/mL, dashed lines).

[0020] 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.

DETAILED DESCRIPTION

[0021] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0022] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0023] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0024] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

[0025] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0026] 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.

[0027] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

[0028] Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible.

[0029] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0030] The term “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. Without limitation, the pig can be selected from the group consisting of, for example, Landrace, Yorkshire, 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.

[0031] 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. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.

[0032] The term “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. Non-limiting examples of 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.

Porcine Organs

[0033] Disclosed herein are methods for crossmatching an animal (e.g., a transgenic porcine animal) for use as a source for organs, organ fragments, tissues or cells for use in xenotransplantation. In exemplary embodiments, the animal is an ungulate and more particularly, a porcine animal or pig.

[0034] In some embodiments, organs are derived from a transgenic pig. For example, a multi-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

[0035] In some embodiments, a subject is crossmatched according to the disclosed methods by assaying a serum sample for immunoreactive antibodies.

[0036] In some embodiments, 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.

Xenotransplantation

[0037] As described herein, biological products for xenotransplantation are derived from source animals produced and maintained according to methods known in the art. Such biological products include, but are not limited to, liver, kidney, skin, lung, heart, pancreas, intestine, nerve and other organs, cells and/or tissues.

[0038] 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. For surgery, 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.

[0039] 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. In some aspects, 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, myeloid, nasion soft, nephrogenic, nerve, nodal, osseous, osteogenic, osteoid, periapical, reticular, retiform, rubber, skeletal muscle, smooth muscle, and subcutaneous tissue. In some aspects, 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.

[0040] In some aspects, 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, erythrocytes, fibroblasts, glial cells, hepatocytes, keratinocytes, Kupffer cells, liver stellate cells, lymphocytes, microvascular endothelial cells, monocytes, neuronal stem cells, neurons, neutrophils, pancreatic islet cells, parathyroid cells, parotid cells, platelets, primordial stem cells., Schwann cells, smooth muscle cells, thyroid cells, tumor cells, umbilical vein endothelial cells, adrenal cells, antigen presenting cells, B cells, bladder cells, cervical cells, cone cells, egg cells, epithelial cells, germ cells, hair cells, heart cells, kidney cells, leydig cells, lutein cells, macrophages, memory cells, muscle cells, ovarian cells, pacemaker cells, peritubular cells, pituitary cells, plasma cells, prostate cells, red blood cells, retinal cells, rod cells, Sertoli cells, somatic cells, sperm cells, spleen cells, T cells, testicular cells, uterine cells, vaginal epithelial cells, white blood cells, ciliated cells, columnar epithelial cells, dopaminergic cells, dopaminergic cells, embryonic stem cells, endometrial cells, fibroblasts fetal fibroblasts., follicle cells, goblet cells, keratinized epithelial cells, lung cells, mammary cells, mucous cells, non-keratinized epithelial cells, osteoblasts, osteoclasts, osteocytes, and squamous epithelial cells. An organ is a group of related cells that combine together to perform one or more specific functions within the body.

Specific Embodiments

[0041] 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.

[0042] 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.

[0043] 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.

[0044] Embodiment 4. The method of any one of embodiments 1 to 3, wherein the organ is a kidney, lung, liver, heart, or pancreas.

[0045] 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.

[0046] Embodiment 6. The method of embodiment 5, wherein the pig cells are peripheral blood mononuclear cells (PBMCs).

[0047] Embodiment 7. The method of embodiment 5 or 6, wherein the method comprises assaying for antibodies bound to porcine lymphocytes.

[0048] 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.

[0049] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

EXAMPLES

Example 1: First clinical-grade porcine kidney xenotransplant using a human decedent model

[0050] As part of a stepwise approach into human xenotransplantation testing, there was a need to develop a human pre-clinical model that would address fundamental questions regarding the safety and feasibility of porcine xenotransplantation into humans. This approach is founded on the premise that such questions must be answered before clinical trials of efficacy can be responsibly undertaken. It was hypothesized that human brain death might provide the necessary model to examine safety and feasibility (Parent B, et al. J Med Ethics. 2020 46:199- 204). Although brain death pathophysiology may create a hostile environment for transplantation and limit assessment of kidney function (Wood KE, et al. N Engl J Med. 2004 351 :2730-2739), such a model would allow for a priori assessment of multiple risks, including hyperacute rejection through the use of a xenotransplant-specific prospective crossmatch, lifethreatening surgical complications, and viral transmission, thereby facilitating the development of the first phase I clinical trial in living humans. Disclosed in this Example are the results of the first clinical-grade xenotransplant experience in vivo using a human decedent model.

Materials and Methods

[0051] 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.

[0052] 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. Importantly, 10-GE pigs do not express red blood cell antigens and are therefore universal donors with respect to blood type.

[0053] Development of 10-GE pigs: Two multi-cistronic vectors were generated that contained 2 human genes (DAF and CD46) or 4 human genes (TBM, EPCR, CD47, HO1), where the genes were separated by 2A sequences to achieve co-expression once introduced in pig cells. Correct, single copy targeting of these transgene constructs to landing pads was confirmed by PCR, Southern blot, and digital drop PCR. Knockout (KO) of a-1 ,3- galactosyltransferase (GGTA1 , the enzyme responsible for synthesis of Gal) was confirmed by PCR for the presence of a disruptive insertion in exon 9. 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.

[0054] 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).

[0055] Histocompatibility testing: Serologic compatibility was assessed between the donor pig and human decedent (recipient) prior to transplant. 10-GE donor lymphocytes were targets in a flow cytometric crossmatch with pre-xenotransplant decedent serum. Negative control was pooled human male AB serum. Positive control serum was human serum containing IgG known to react with porcine cells. For all tubes, 400 000 cells and 40 pl serum were incubated with FITC-conjugated goat anti-human IgG F(ab)’2 (Jackson ImmunoResearch Laboratories). Acquisition and analysis of flow crossmatch results were performed on a Beckman Coulter Cytoflex Flow Cytometer. Median channel shift of decedent sample was compared to negative and positive control sera to determine positivity.

[0056] Viral and chimerism testing: RNA was isolated from human PBMCs and pig tissues, following standard protocols (Direct-zol RNA Miniprep, ZymoResearch). For cDNA synthesis, 50-100 ng of DNAse treated mRNA was reverse transcribed using an oligo-dT primer and the GoScript Reverse Transcription System (Promega). For the 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).

[0057] 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.

[0058] Decedent bilateral native nephrectomies’. In an operating room meeting The Joint Commission standards, bilateral native nephrectomies were performed using a standard open donor nephrectomy technique to establish anuria and to allow the kidneys to be used for allotransplantation.

[0059] Backbench preparation of the porcine kidney xenograft’. En bloc kidneys were separated, and pre-implantation biopsies were obtained. While grossly normal, the porcine kidneys and the accompanying vascular structures were soft on palpation with an extremely thin capsule and reduced gross structural integrity compared to human kidneys. In addition, the ureters were larger in diameter than typically observed in human kidneys. These observations underscored the need for meticulous handling and surgical technique.

[0060] Porcine kidney xenotransplantation’. Right and left 10-GE pig kidneys were transplanted separately using conventional heterotopic allotransplantation techniques. The right ureter was anastomosed to the decedent's bladder, and the left ureter was brought through the skin as an end urostomy. A post-reperfusion biopsy of the left porcine renal xenograft was obtained in vivo. Due to the delicate nature of the porcine tissues, a complementary biopsy of the right porcine xenograft at this time point was deferred.

[0061] Immunosuppression: Induction 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.

[0062] Histology: 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.

[0063] Data management: Data were input in real time in a secure REDCap database by study personnel. The decedent was given an alias to preserve anonymity during the course of the study. All study personnel were aware of and instructed on the need to maintain the strictest of confidence about this study. All study personnel have received requisite training in data confidentiality and human subjects research. Results

[0064] Study overview and outcome measures

[0065] To test the core principles of the pig-to-NHP model in humans without risk to a living human being, a safety and feasibility study of 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. Of note, efficacy measures were collected as tertiary outcomes as we did not expect the altered physiologic milieu of brain death to provide an optimal environment to support renal recovery. Nevertheless, the goal was to capitalize on the opportunity to collect functional data as allowed within the constraints of the model; decedent bilateral native nephrectomies were therefore performed prior to xenotransplantation to permit interpretation of serum creatinine and other parameters of renal function (Figure 1).

[0066] Pretransplant phase

[0067] After exhausting the solid organ transplant lists, next-of-kin was approached regarding decedent study enrollment and provided informed consent authorizing the participation of the 57-year-old brain-dead male (Table 2). At the time of enrollment, the decedent was 5 days post-declaration of brain death and had mild-to-moderate acute kidney injury (Table 3). The decedent was maintained on phenylephrine (1 mcg/kg/min), vasopressin (0.008 units/min), levothyroxine (10 mcg/h), and methylprednisolone with normal hemodynamics (BP: 178/92, HR: 61 , temp: 98.8) as per routine management of brain-dead individuals prior to organ donation. 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).

[0068] 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.

[0069] Transplant phase

[0070] The 10-GE pig kidneys were transplanted sequentially into the decedent using conventional heterotopic allotransplantation technique. Of note, 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. Although results from some NHP studies suggest that calcineurin inhibitor (CNI)- based immunosuppression regimens may not be as effective as CD40-based regimens (Yamamoto T, et al. Transplantation. 2019 103(10):2090-2104), the precise mechanisms underlying graft loss in these NHP experiments are unknown and may not apply to human immune populations. As CNIs are highly effective in the prevention of cellular rejection in humans and the backbone of virtually all immunosuppression regimens in contemporary allotransplantation, we selected a conventional immunosuppression regimen to mimic “real- world” conditions of xenotransplantation. Methylprednisolone and anti-thymocyte globulin were thus administered immediately prior to xenotransplantation, and tacrolimus-based maintenance immunosuppression was started and maintained throughout the remainder of the study with effective depletion of lymphocytes (Table 5).

[0071] Both kidneys reperfused promptly with excellent color and turgor as judged independently by four experienced kidney transplant surgeons (Figure 3). Pulses were confirmed in the renal arteries with direct visual inspection and manual palpation. Doppler signals were normal in both the kidney parenchyma and the renal arteries bilaterally. There was no significant bleeding of the anastomotic suture lines or disruption of the renal parenchyma despite perfusion of the kidney with a human mean arterial blood pressure. The decedent remained on relatively stable doses of phenylephrine and dopamine prior to and after reperfusion (Figure 4). The right kidney made urine within 23 min of reperfusion. Urine output from the left kidney was more sluggish. The kidneys were observed under direct vision for at least 60 min prior to commencement of the ureteral anastomoses. No hyperacute rejection was observed and both kidneys maintained good coIor and turgor throughout the remainder of the operation. Post-reperfusion biopsy of the left kidney demonstrated mild to moderate acute tubular injury and normal glomeruli. There was no evidence of endothelial injury, fibrin thrombi, or staining for IgG, IgM, or C4d (Figure 3). [0072] Posttransplant phase

[0073] 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. Despite the severity of his physiologic derangement, his hemodynamics were sufficiently maintained to permit longitudinal data collection and exploration of the abdomen on days 1 and 3 for biopsies and kidney visualization. Notably, the 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.

[0074] 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.

[0075] Histologic findings on post-operative day 1 were consistent with thrombotic microangiopathy, with diffuse glomerular capillary congestion, swollen endothelial cells, and near complete obliteration of the peripheral capillary lumina along with the presence of fibrin thrombi (Figure 7). On post-operative day 3 there was evidence of progressive tubular injury with extensive acute tubular necrosis, but additional features of TMA including mesangiolysis were not observed. C4d was negative at both time points (Figure 7), as well as IgM, IgG, IgA, C1q, and C3 (Figures 8 and 9). Wedge biopsies from study termination demonstrated no evidence of cortical necrosis or interstitial hemorrhage and glomerular capillary congestion was no longer diffuse. Post-termination analysis of renal tissue confirmed expression of the human transgenes within the porcine kidney parenchyma.

[0076] Decedent blood samples were tested daily for the presence of porcine endogenous retroviruses and remained negative (Figure 10). In addition, chimerism, as measured by the presence of expression of the gene for a porcine large ribosomal protein (pRPL4), was not observed at any time point (Figure 10).

[0077] Decedent (recipient) 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.

Discussion

[0078] Xenotransplantation is arguably the most pragmatic solution to the organ shortage crisis, but safety and efficacy concerns have limited advancement into humans. In preparation for a phase I clinical trial of porcine renal xenotransplantation at the University of Alabama at Birmingham, we asked what gaps in knowledge must be filled before such a clinical trial could be ethically offered to research subjects. The aim was therefore to develop a human preclinical model which would permit the in vivo evaluation of critical safety and feasibility tenets of the pig-to-NHP model without risk to a living human. The study was designed to test five central questions: (1) Is the current suite of porcine genetic modifications sufficient to avoid hyperacute rejection in humans? (2) Would prospective flow-based crossmatching correlate with graft survival free of hyperacute rejection? (3) Would life-threatening intraoperative complications occur during a renal porcine xenotransplant? (4) Would porcine cells and/or pathogens be detected in the blood of a human recipient? (5) Could porcine renal xenotransplantation be safely performed under the conditions necessary for a clinical trial? To this end, we designed and performed this experiment under clinical-grade conditions which included the transplantation of 10-GE porcine kidneys designed specifically for human transplantation into the conventional anatomic position using processes and facilities in compliance with multiple regulatory agencies.

[0079] Similar to NHPs, hyperacute rejection was not observed in this human decedent, providing critical evidence that knockout of the genes encoding enzymes that synthesize carbohydrate xenoantigens (i.e., GGTA1 , [34GALNT2, CMAH) is indeed sufficient to prevent hyperacute rejection from this mechanism in humans. Importantly, our study addressed a second potential mechanism of hyperacute rejection in humans, which is preformed antibody against either the major histocompatibility complex in pigs (swine leukocyte antigen; SLA) or other unknown minor antigens. Although humans are not expected to possess anti-SLA antibody due to prior sensitization events, pre-existing anti-HLA antibody may cross-react with SLA alleles (Diaz Varela I, et al. J Am Soc Nephrol. 2003 14(10):2677-2683; Taylor CJ, et al. Transplantation. 1998 65(12): 1634-1641 ; Martens GR, et al. Transplantation. 2017 101(4):e86- e92; Ladowski JM, et al. Transplantation. 2018 102(2):249-254), particularly the class II loci, given the sequence homology between pig and human DR, DP, and DQ antigens (Lunney JK, et al. Dev Comp Immunol. 2009 33(3):362-374; Smith DM, et al. Tissue Antigens. 2005 65(2):136-14; Smith DM, et al. Tissue Antigens. 2005 66(6):623-639). To this end, we developed and tested a novel flow crossmatch assay which prospectively predicted that hyperacute rejection would not occur. Although preliminary testing of this assay suggested that either fresh or frozen pig PBMCs could be used at the time of crossmatching with a potential recipient, these results were validated during this decedent experiment. Although fluorescence intensities varied between fresh and frozen porcine PBMCs, the results overall were internally consistent and easily interpretable given the use of appropriate positive and negative controls.

[0080] A number of safety goals of this study revolved around the consequences of connecting the circulation of a human with a porcine kidney. Notably, 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. Reassuringly, 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. Finally, there was no evidence of porcine endogenous retrovirus transmission or peripheral chimerism in the decedent based on assays developed in our research laboratories. Although these initial results are encouraging, conclusions are limited given the short duration of the experiment and the unknown sensitivity and specificity of these first-generation research assays, despite the ability of PGR assays to detect as few as 100 copies of PERV-C (Kaulitz D, et al. J Virol Methods. 2011 175(1):60-65). Refinement of research laboratory assays to approximate those used in clinical laboratories will be necessary to support future clinical trial efforts, because veterinary diagnostic laboratories with clinical-grade porcine viral testing capabilities may not accept human specimens, and hospital clinical laboratories may not be equipped to test for porcine pathogens. Investment in the development of clinical-grade laboratory assays which utilize next generation sequencing technologies will likely increase the sensitivity and specificity of these assays — especially for the PERV-A/C recombinant virus (Kono K, et al. Sci Rep. 2020 10(1):21935) — and provide the foundation for a robust microbiologic safety plan to support a phase I clinical trial. Such goals can be accomplished as evidenced by recent studies published out of New Zealand, where seven-year follow-up data of human subjects transplanted with encapsulated porcine islets indicate no transmission of zoonotic disease (Wynyard S, et al. Xenotransplantation. 2014 21 (4):309-323; Matsumoto S, et al. Xenotransplantation. 2020 27(6):e12631). These data recapitulate other findings in preclinical animal models demonstrating no in vivo transmission of PERV (Denner J. Retrovirology. 2018 15(1):28).

[0081] Although this study was not designed to optimize renal performance or immunologic outcomes, the decedent model afforded the opportunity to perform serial renal biopsies and assess renal function. Urine output was initially robust from the right kidney but significantly less from the left kidney. While the etiology of this mismatch in renal performance is unknown, the accrual of additional warm ischemic time on the left kidney during clamping in the donor may have played a role.

[0082] Despite the transplantation of both kidneys, serum creatinine did not decrease and neither kidney excreted significant creatinine into the urine. The etiology of this renal dysfunction is unclear and is likely multifactorial. Although serum creatinine and BUN were normal in the donor pig and are normal in the 10-GE herd at the UAB XPC (BUN: 30 ± 9.4; creatinine: 0.92 ± 0.24 [avg, SD]; n = 17 measurements in 10 animals over 60 days), genetic modifications of these animals clearly altered the overall structural integrity of the renal parenchyma. To the best of our knowledge, such differences in tissue integrity have not been reported in other genetically modified pigs, and it is not yet clear to what degree these structural changes might impact renal function or recovery. Alternatively, poor renal recovery may reflect the deleterious milieu of brain death characterized by complement activation29 and hemodynamic decompensation requiring vasoactive agents (Wood KE, et al. N Engl J Med. 2004 351 :2730-2739). Finally, renal dysfunction in the decedent may have also been impacted by microvascular injury of unclear etiology. Of note, xenograft histology demonstrated endothelial injury with diffuse TMA on post-operative day 1 , but there was no evidence of progression to cortical necrosis or interstitial hemorrhage by post-operative day 3, as might be expected if TMA was due to significant antibody-mediated damage. As the xenograft biopsies were negative for IgM, IgG, C4d, C1q, and C3, we must consider the possibility that the 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). Moreover, although 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. 2006 355:1233-1243) and utilized for the treatment of severe antibody-mediated rejection in human allotransplantation (Locke JE, et al. Am J Transplant. 2009 9:231-235; Orandi BJ, et al. Transplantation. 2014 98:857-863), and prevention of MAC formation with anti-C5 antibody was recently shown to improve xenograft survival in a NHP model (Adams AB, et al. Ann Surg. 2021 274:473-480). Collectively, these findings highlight the need for ongoing work in this area and suggest that both NHP and human studies may be necessary to understand the molecular basis and clinical implications of these biopsy findings.

[0083] In conclusion, critical safety and feasibility questions in xenotransplantation were addressed using a novel pre-clinical human model under significant regulatory oversight. These results add significantly to the prior knowledge generated in non-human primate models and suggest that many barriers to xenotransplantation in humans have indeed been surmounted. The decedent model has significant potential to propel not only the field of xenotransplantation forward but to answer a multitude of other scientific questions unique to the human condition.

Example 2: Normal Graft Function After Pig-to-Human Kidney Xenotransplantation

Methods

[0084] In this case series, persons aged 18 years or older declared brain dead whose families provided informed consent for study participation were eligible for study entry after all organ donation options for transplantation were exhausted. The decedent received cardiopulmonary support in critical care setting for the duration of the study. Porcine animals with 10 gene modifications including 4 gene knockdowns and knockouts (GTKO, CMAH, B4GALNT2, GHR) and 6 human transgene insertions (CD46, CD55, CD47, THBD< PROCR, HMOX1) as previously described (Porrett PM, et al. Am J Transplant 2022 22(4): 1037-1053), were maintained in pathogen-free facility. General anesthesia was administered for the purposes of porcine kidney procurement, and porcine donors were humanely euthanized thereafter. Ten-gen-edited 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

[0085] A male in his 50s who was declared brain dead and had acute kidney injury superimposed on a history of CKD (stage 2) and hypertension underwent bilateral native nephrectomy and cessation of dialysis followed by crossmatched-compatible xenotransplant with 10-gene-edited pig kidneys (UKidney). The decedent received a complement inhibitor (anti- C5; ecluzumab) 24 hours before xenotransplantation followed by standard induction therapy, including a solumedrol taper, antithymocyte globulin (6 mg/kg total), and rituximab. Maintenace immunosuppression included tracrolimus, mycophenolate mofetil, and prednisone. 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. Within 4 minutes of reperfusion, the xenografts made urine, producing more than 37 L in the first 24 hours. Urine concentrated over time, with concurrent decreases in urine volume to a medial of 14.1 L (IQR, 13.8-20 L) on PODs 1 to 3 and a median of 5.1 L (IQR, 5-6 L) on PODs 4 to 7. Before xenotransplant, 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. Creatinine clearance also improved (POD 0, 0 mL/min; POD7, 200 mL/min) (Figure 11). Xenografts were serially biopsied and showed normal histology by light microscopy without evidence of thrombotic microangiopathy (Figure 12). Example 3. Physiologic Homeostasis after Pig-to-Human Kidney Xenotransplantation

Methods

Study decedents

[0086] In this case series, as previously described, adult (>18 years) brain-dead persons (decedents) whose families provided informed consent for study participation were eligible for study entry after all organ donation options were exhausted (Porrett PM, et al. Am J Transplant. 2022 22(4): 1037-1053). The study was approved by the University of Alabama at Birmingham (UAB) IRB (No.300004648). The study followed the Appropriate Use and Reporting of Uncontrolled Case Series in the Medical Literature reporting guideline.

Porcine kidney donors.

[0087] 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).

Pig-to-human xenotransplantation

[0088] 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.

Immunosuppression

[0089] Based on results from previous studies in NHPs, 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/m²). Intravenous methylprednisolone was tapered over four days for immunosuppression. Additional steroid dosing was administered throughout the experiment to manage brain death. Maintenance immunosuppression included calcineurin inhibitor (tacrolimus, goal level 8-12 ng/dL), mycophenolate mofetil (1000 mg twice daily), and prednisone (30 mg once daily). Tacrolimus, mycophenolic acid, and complement levels were followed daily. In addition, a 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.

Assessment of Renin-Angiotensin-Aldosterone-System (RAAS)

[0090] Levels of renin, angiotensinogen, angiotensin II, and aldosterone were measured following the protocol used in prior xenotransplant experiments in NHPs (Hansen-Estruch C, et al. Am J Transplant. 2023 23(3): 353-365).

PTH signaling

[0091] 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.

Measures of kidney clearance (endogenous and exogenous).

[0092] 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:

Measures of kidney salt and water handling.

[0093] Higher blood glucose levels from glucocorticoid use in the treatment of brain death resulted in glucosuria. To account for the osmotic effect of urinary glucose, electrolyte free water clearance was calculated as a measure of kidney water handling. Electrolyte free water clearance (liters) was calculated using the formula:

[0094] Daily measured urine osmolality and dipstick urinalyses provided specific gravity and glucose levels. Anti-diuretic hormone levels were measured by send-out hospital laboratory intermittently, and serum copeptin was measured prior to the inulin clearance by an ELISA (Cloud Corp, #CEA365Hu).

Measures of collecting duct function.

[0095] Aquaporins (AQP) were detected using immunohistochemistry methods previously reported (Mendoza LD, et al. Am J Physiol Renal Physiol. 2019 317(3):F547-f559). 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. Primary antibody binding was detected using ready-to-use secondary antibodies (Vector Labs) and ImmPACT DAB Peroxidase Substrate (Vector Labs). Additional slides were used to immunolocalize AQP2 (principal cells) and vacuolar-type H+-ATPase B1/2 (V-ATPase, intercalated cells) using immunofluorescent techniques as previous reported (Mendoza LD, et al. Am J Physiol Renal Physiol. 2019 317(3):F547-f559). 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.

Measurement of proteinuria.

[0096] Serial 24-hour urine collections allowed for quantification of albumin and total protein in the urine. Results

Demographics.

[0097] A 53-year-old male with a history of hypertension, diabetes mellitus type 2, and acute kidney injury superimposed on chronic kidney disease stage 2 was enrolled. Tissue compatibility was confirmed by negative flow crossmatch. Continuous renal replacement therapy (CRRT) was stopped, and bilateral native nephrectomies were performed. Serum creatinine peaked at 3.9 mg/dL after cessation of dialysis and bilateral native nephrectomy. Pig- to-human kidney xenotransplantation was then performed, and physiologic performance was assessed over the 7-day study period. The porcine donor was a 14-month-old, 92.2 kg, male, 10 gene-edited pig with normal kidney function (Table 10).

RAAS, vasopressin, copeptin, and electrolyte levels.

[0098] Mean arterial pressure remained >60 mmHg throughout the study period (Supplementary Figure S4). Notably, the decedent was not hypotensive after xenotransplantation and required a nicardipine drip to treat hypertension. Plasma renin concentrations ranged 37.1-61.3 pg/mL post xenotransplantation (Figure 13A). However, plasma renin activity (PRA) was below the level of detection (less than 0.6 ng/mL/hr) throughout the study duration. 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-axis.

[0099] 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. With intravenous supplementation (Table 11), ionized calcium increased to >1 mmol/L on post-operative day 2 and remained stable through study duration. In parallel, 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.

Clearance.

[0100] Glomerular filtration rate (GFR) 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). In the afternoon of post-op day 5, decline in urine flow due to urinary obstruction from a kinked Foley catheter was discovered and corrected. The GFR returned to 150 mL/min by 24 h urine creatinine clearance on post-op day 7 (Figure 15B). The trend in GFR changes mirrored trends in serum creatinine levels (Figure 15B & 15C). Estimated GFR by cystatin C measurement remained lower than other methods for assessing GFR (Figure 15B). Pharmacokinetic studies demonstrated tacrolimus trough levels ranging from 8-10 ng/dL with an AUCc of 103.34 ng*h/mL (Figure 3D).

Salt and water handling.

[0101] 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). Hemodynamic support with intravenous fluid offset urinary losses. Intravenous fluid administration decreased from 16L to 7L per day throughout the study (Table 12).

Collecting duct function.

[0102] Aquaporins (AQP) were immunolocalized in the pig kidney. AQP1 was abundant in the apical membrane of the proximal tubules (Figure 17, panel A). The basolateral membrane of the principal cells of the collecting duct had detectable AQP4 (Figure 17, panel B), while the apical/subapical membrane expressed AQP2 (Figure 17, panel C). Moreover, phosphorylation of AQP2-S256, an indicator of active AQP2, was also present in the principal cells (Figure 17D). V-ATPase were localized on the apical surface of intercalated cells (Figure 17, panel E) and the urine pH remained low at 5 for the last 5 days of the experiment. Trichrome stained kidney sections of the cortex (Figure 17, panel F) and medulla (Figure 17, panel G) showed no obvious pathology.

Proteinuria

[0103] 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.

Discussion

[0104] 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.

[0105] Limited studies in the genetic edited pig-to-NHP models indicate 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. Previously, in the pre-clinical human Parsons Model, we have demonstrated life sustaining kidney function after 10 gene-edited porcine kidney xenotransplantation as measured by the clearance of creatinine in the absence of native kidneys or dialysis support. In the current report, we build on those findings with evidence of exogenous clearance of inulin, as well as pharmacokinetic study of tacrolimus AUG consistent with findings after human-to-human allotransplantation. Moreover, we demonstrated that the endogenous clearance marker, cystatin-C, was not a reliable marker of GFR estimation in pig- to-human kidney xenotransplantation. Our findings suggest that porcine kidney xenografts will perform renal clearance of exogenous and endogenous substrates similar to human kidney allografts. [0106] 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.⁹ 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. Consistent with observations from NHP models, we observed voluminous urine output initially after xenotransplantation with corresponding hypernatremia. Over the course of the 7-day study period, the urine output decreased and serum sodium normalized. We calculated urinary water losses between 3-4.5 L/day, and given the GFR, this means 99% of the filtered water was reabsorbed. Aquaporin expression in the proximal tubules and principal cells of the collecting duct was abundant and found in expected subcellular compartments; AQP1 was abundant in the proximal tubules, AQP2 was in the apical membrane and AQP4 in the basolateral membrane of the collecting duct principal cells. 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. With an intact pig-kidney response to human vasopressin, water losses from anatomical limitations in urine concentration, could be managed by thirst and water intake. High volume of urine output, up to 20L, is common in the first day after a living kidney transplant with intra-operative diuretic dosing. However, a urine output of 6L per day by post-operative day 6 is high. The observed downtrend in urine output throughout the study, along with apical staining for AQP2 in the collecting duct, suggest that water balance could be maintained without intravenous support with longer follow-up. However, further studies are needed.

[0107] In addition to vasopressin-responsive principal cells, the intercalated cells of the collecting duct also appeared functional. The presence of V-ATPase, an ATP-dependent proton pump, on the apical surface of intercalated cells coupled with persistently low urinary pH supports intact urinary acidification in the pig kidney xenograft. [0108] Data from in vitro and in vivo NHP models support reduced ability of pig renin to cleave human angiotensinogen. Similar findings were observed in the present study. Renin concentrations measured on post-operative days 3-7 were the result of pig kidney production. 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. However, 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.

[0109] 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. In pig-to-NHP xenotransplantation experiments, mild alterations in serum calcium and phosphate levels were observed months after xenotransplantation. In contrast, in our human decedent study of pig-to-human xenotransplantation, PTH levels followed ionized calcium levels appropriately. Importantly, while serum phosphate levels remained high in the recipient, fractional excretion of phosphorus averaged 29% in the last three days of the study, which is above the normal human fractional excretion of phosphate of 5-20%. Taken together, these data support preserved PTH signaling with appropriate urinary phosphorus excretion in the days following xenotransplantation.

[0110] Our 7 day study aimed at exploring the physiologic function of the pig kidney within a human. In the study, nephrotic-range proteinuria was observed. Peaks levels of proteinuria were in the first 24 hours at 8.9 grams/day and down trended to 3.2 grams/day by post-operative day 6. Proteinuria soon after xenotransplantation may indicate early antibody- mediated injury. While still being explored, little evidence for antibody mediated rejection was seen. Other possible explanations for proteinuria could be hyperfiltration related to differences in mean arterial blood pressure between the donor pig and recipient human or factors affecting glomerular permeability. [0111] In summary, we have demonstrated for the first-time porcine kidney xenograft physiology in a human. Importantly, these results can be leveraged to develop protocols for Phase I studies in living persons, with the ultimate goal of establishing pig-to-human kidney xenotransplantation as a feasible option to expand kidney transplantation amidst a human organ shortage.

Example 4: C5 inhibition with eculizumab prevents thrombotic microangiopathy in a case series of pig-to-human kidney xenotransplantation

Methods

Sex as a biologic variable

[0112] 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.

Data availability

[0113] De-identified data used to create the figures have been supplied to the journal as a spreadsheet file, which will be made publically available at the time of publication as the Supporting Data Values file. Additional data are available upon reasonable request to the corresponding author.

Decedent enrollment and porcine donors

[0114] The study was approved by the University of Alabama at Birmingham Institutional Review Board for Human Use (No. 300004648) and the Institutional Animal Care and Use Committee (No. 22015). Brain-dead adult decedents precluded from organ donation whose families provided written informed consent for participation were considered for enrollment. Decedents were maintained within a critical care setting on a ventilator. Porcine donors had 10 genetic edits (10GE) (four knockouts: GTKO, CMAH, B4GALNT2, GHR; six human transgene insertions: CD46, CD55 (hDAF, decay accelerating factor), CD47, THBD, PROCR, HMOX1 ; as previously described) (references 2,5 in manuscript). Two of the transgenes, CD46 and CD55, are human complement inhibitor genes which were inserted to mitigate the effect of complement on the xenografts, primarily via the classical pathway (see Supplemental Figure S4 for phenotypic expression).

Immunosuppression

[0115] 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.

Biopsy protocol

[0116] Native kidneys were sent for pathologic evaluation, and xenograft biopsies were performed pre-implantation and every other day until termination. Specimens were fixed, stained with periodic acid-Schiff hematoxylin (PASH) and immunohistochemistry (IHC) for membrane attack complex (MAC, C5b-9; Arkana Laboratories, Abeam, #ab66768). Pathologic evaluation was performed by a renal pathologist.

Results

[0117] Although studies of porcine kidney xenotransplantation in non-human primates (NHPs) and brain-dead humans have improved our understanding of anti-xenograft immune responses , the optimal immunosuppression regimen for living human recipients is unknown. Prior NHP studies suggest that complement plays an important role in immune-mediated injury of xenografts, but the benefits of pharmacologic complement inhibition in human xenograft recipients have yet to be established. Here, we report the histologic outcomes of a series of brain-dead human recipients of a porcine kidney xenotransplant using exclusively FDA- approved medications, with and without anti-C5 monoclonal antibody.

[0118] Three male decedents, aged 57, 65, and 53 years, respectively, underwent bilateral native nephrectomies followed by crossmatch-compatible (Fig. 18) xenotransplantation with 10 gene-edited (10GE) pig kidneys, that expressed two human transgenes responsible for classical complement cascade inhibition (CD46, DAF), with standard immunosuppression. There was no evidence of hyperacute rejection in any decedent. Decedents 2 and 3 received anti-C5 monoclonal antibody therapy (eculizumab) 24 hours prior to (1200 mg) and 24 hours after (900 mg) xenotransplantation.

[0119] Decedent 1 native kidney and 10GE porcine donor kidney biopsies were histologically normal without membrane attack complex (MAC, C5b-9) deposition on immunohistochemistry (IHC) at the time of transplant (Table 13). On post-operative day 1 (POD1), the xenografts demonstrated evidence of early thrombotic microangiopathy (TMA) along with rare MAC reactivity along capillary loops. By POD3, TMA and MAC deposition were diffuse (Table 13). The observed TMA was seen in the absence of therapeutic tacrolimus levels (<2.0 ng/mL and 3.2 ng/mL, respectively; Fig. 19).

[0120] Decedent 2 native kidney biopsies (Table 13) demonstrated no histologic evidence of TMA; however, MAC staining was seen along capillary loops, indicative of complement activation pre-xenotransplant. Xenograft biopsies prior to implantation, on POD1 , and POD3 demonstrated no histologic evidence of TMA or MAC reactivity (Table 13). Tacrolimus remained subtherapeutic (<2.0 ng/mL and 4.5 ng/mL, respectively; Fig. 19).

[0121] 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).

[0122] We note that tacrolimus levels remained subtherapeutic in Decedents 1 and 2, but Decedent 3 had tacrolimus levels within therapeutic range for much of the study without evidence of TMA and in the presence of normal organ function. Altogether, these results suggested that the observed TMA was not the result of exposure to the calcineurin inhibitor, further reinforcing the use of tacrolimus as a maintenance immunosuppressant in the shortterm. Importantly, the immunosuppression regimen utilized in Decedents 2 and 3 followed an FDA-approved, standard-of-care regimen that involves complement inhibition at C5 for kidney allotransplant recipients with atypical hemolytic uremic syndrome (aHUS). Similar to human allograft recipients with aHUS, eculizumab levels will likely require surveillance, as indicated in Decedent 3 where subtherapeutic levels of eculizumab correlated with resurgence of MAC deposition (Fig. 20).

[0123] While complement activation in the setting of both brain death and xenotransplantation is difficult to decipher, 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. However, 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. Prior work by our group has demonstrated development of TMA in the absence of IgM and IgG or C1 , C3c or C4d deposition, suggesting activation of the alternative complement cascade via the innate immune system. Given that both the classical and alternative complement cascades converge at C3, downstream complement inhibition at C5 may be necessary to control the innate human immune response to porcine xenografts. Optimal inhibition of the complement cascade in crossmatch-compatible xenotransplant recipients may require inhibition of the alternative complement cascade, especially given the observation of TMA in the absence of therapeutic levels of eculizumab despite expression of transgenes which inhibit the classical complement cascade (CD46 and DAF) in 10GE xenografts.

[0124] In summary, our case series supports utilization of complement inhibition at C5 to control the innate human immune response to porcine kidney xenografts. Because our study reports three cases with short-term follow-up, generalizability is limited; however, our findings suggest a beneficial role of anti-C5 monoclonal antibody in pig-to-human kidney xenotransplantation, as previously suggested in the pig-to-NHP model. Our case series further demonstrates the utility of the Parsons Model in understanding the human immune response to xenografts, as well as the short-term efficacy of an FDA-approved, standard-of-care immunosuppression regimen in the setting of 10GE pig-to-human kidney xenotransplantation. Additional studies will be needed to define the long-term utility of this regimen.

[0125] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0126] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

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.

2. The method of claim 2, wherein the subject is a positive crossmatch for the xenotransplantation, further comprising transplanting an organ from a donor pig to the subject.

3. The method of claim 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.

4. The method of claim 1 , wherein the organ is a kidney, lung, liver, heart, or pancreas.

5. The method of claim 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.

6. The method of claim 5, wherein the pig cells are peripheral blood mononuclear cells (PBMCs).

7. The method of claim 5, wherein the method comprises assaying for antibodies bound to porcine lymphocytes.

8. The method of claim 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.