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. Author manuscript; available in PMC: 2022 Sep 1.
Published in final edited form as: Ann Surg. 2021 Sep 1;274(3):473–480. doi: 10.1097/SLA.0000000000004996

Anti-C5 Antibody Tesidolumab Reduces Early Antibody Mediated Rejection and Prolongs Survival in Renal Xenotransplantation.

Andrew B Adams 1, Brendan P Lovasik 2, David A Faber 2, Christopher Burlak 1, Cynthia Breeden 2, Jose L Estrada 3, Luz M Reyes 3, Rodrigo M Vianna 2, Matthew F Tector 4, Alfred J Tector 3
PMCID: PMC8915445  NIHMSID: NIHMS1713888  PMID: 34238812

Abstract

Objective:

Pig-to-primate renal xenotransplantation is plagued by early antibody mediated graft loss which precludes clinical application of renal xenotransplantation. We evaluated whether temporary complement inhibition with anti-C5 antibody Tesidolumab could minimize the impact of early antibody mediated rejection in rhesus monkeys receiving pig kidneys receiving costimulatory blockade-based immunosuppression.

Methods:

Double (Gal and Sda) and triple xenoantigen (Gal, Sda, and SLA I) pigs were created using CRISPR/Cas. Kidneys from DKO and TKO pigs were transplanted into rhesus monkeys that had the least reactive crossmatches. Recipients received anti-C5 antibody weekly for 70 days, and T cell depletion, anti-CD154, mycophenolic acid, and steroids as baseline immunosuppression (n = 7). Control recipients did not receive anti-C5 therapy (n = 10).

Results:

Temporary anti-C5 therapy reduced early graft loss secondary to antibody mediated rejection and improved graft survival (p <0.01, figure). Deleting class I MHC (SLA I) in donor pigs did not ameliorate early antibody mediated rejection (table). Anti-C5 therapy did not allow for the use of tacrolimus instead of anti-CD154 (table), prolonging survival to a maximum of 62 days.

Mini-Abstract

Temporary complement inhibition with anti-C5 antibody Tesidolumab minimizes early IgM mediated antibody mediated rejection in a GGTA1/β4GalNt2 knockout pig-to-rhesus kidney xenotransplant model. Once early antibody mediated rejection is avoided, prolonged survival more than 300 days is achieved in most recipients.

Introduction

Renal xenotransplantation could enable all patients with ESRD to receive renal transplants, but antibody mediated rejection has precluded clinical implementation(1–3). The development of triple xenoantigen knockout pigs (GGTA1/β4GalNT2/CMAH) has created a situation where a significant number of waitlisted patients have a negative crossmatch, and would be expected to be able to receive a renal xenograft without experiencing early AMR(4–6). Additionally, prolonged survival has been achieved in the pig-to-NHP renal xenograft model with a number of recipients surviving more than 1-year(7, 8).

The next steps in moving xenotransplantation into the clinic will include developing immunosuppressive protocols using medications that are clinically approvable and can prolong survival using the simplest genetically modified pig kidney donor possible. There are two important considerations in the pig-to-NHP preclinical model. First the insertion of multiple transgenes brings significant questions from a regulatory standpoint, and more importantly the transgenes have not appreciably altered long term graft survival or the pathophysiology of graft failure(9). The survival of renal xenografts has been more dependent upon the pre-transplant crossmatch(2). The second consideration is that there is no suitable NHP for use in a preclinical kidney transplant model that has a negative crossmatch to a genetically engineered pig so that all xenografts performed in this model are in fact CDC crossmatch positive(5, 7, 10). The net result of the positive crossmatch is that many renal xenografts fail early (first 100 days posttransplant), succumbing to AMR regardless of whether the pig kidney has none or as many as 6 human transgenes(7, 11).

Anti-C5 antibodies have been used to prevent early AMR in a mouse model of ABO incompatible model of heart allotransplantation. In this model, if early AMR was avoided the cardiac allografts were able to survive in recipients treated with a clinically available immunosuppressive regimen of tacrolimus, mycophenolic acid, and steroids(12). If temporary anti-C5 therapy can prevent early AMR of renal xenografts in a preclinical model that would be significant since eculizumab is readily available and there is clinical experience with this drug in renal allotransplantation(13). Since eculizumab does not bind to NHP C5, we tested anti-C5 antibody tesidolumab for its ability to prevent early AMR in the pig-to-Rhesus kidney xenotransplant model(14). Once it was clear that anti-C5 antibodies could prevent early AMR using our anti-CD154 based immunosuppressive regimen that is not clinically available because of thromboembolic complications in human trials, we evaluated FDA approved tacrolimus for its ability to provide consistently prolonged renal xenograft survival under the cover of tesidolumab.

Materials and Methods

Animals

Animal work was performed under Institutional Animal Care and Use Committee (IUCAC)-approved protocols. CRISPR/Cas9 was used to generate GGTA1, β4GalNt2, and SLA I KO cells as previously described(7, 15, 16). SCNT was performed using the GGTA1/β4GalNT2, and GGTA1/β4GalNt2/SLA I KO cells to create cloned piglets as previously described.

Pig-to-Rhesus Renal Transplantation

GGTA1/β4GalNt2 KO, or GGTA1/β4GalNT2/SLA I KO pigs had bilateral nephrectomies on the day of transplantation, and kidneys were cold stored in UW solution until implantation. Rhesus macaques also had bilateral nephrectomies followed by life-sustaining renal transplantation using either a GGTA1/β4GalNt2 KO, or GGTA1/β4GalNT2/SLA I KO pig kidney. Operations were performed in accordance with Institutional Animal Care and Use Committee regulations. Postoperatively graft function was monitored with daily urine output assessment and weekly serum chemistries. Quantitative cytomegalovirus (CMV) was measured using a PCR-based assay. Ultrasound guided biopsies were performed postoperatively.

Treatment Regimens

Recipients received T-cell depletion using anti-CD4, B-cell depletion with anti CD20, and costimulation blockade with anti-CD154, and daily mycophenolic acid and steroids. T-Cell depletion began 3 days prior to transplantation with a 1-time dose of anti-CD4 50 mg/kg IV (clone CD4R1; (clone CD4R1; NIH Nonhuman Primate Resource, Boston, MA). B-cell depletion began 2 days preoperatively with a one-time dose of anti-CD20 mg/kg IV (Rituximab, Genentech, San Francisco, CA). Recipients receiving anti-C5 (Tesidolumab, Novartis, Basel Switzerland) had a 30 mg/kg loading dose on the day of transplant, followed by weekly 10mg/kg IV for seven weeks, at which point anti-C5 was discontinued. Anti-CD-154 (5c8; NIH Nonhuman Primate Reagent Resource, Boston, MA) was given on days 0, 7, 14, and then biweekly at 20 mg/kg IV. Recipients also received daily MMF and steroids (figure 1).

FIGURE 1.

FIGURE 1.

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Flow cytometric crossmatch and immunosuppression protocol employed in transplants. (A) IgG flow cytometric MFI and demonstrated for 14 rhesus macaque recipients. (●) represents an individual pre-transplant serum sample tested against DKO cells (GGTA1/B4GalNT2 KO) who later received a DKO porcine kidney. (☐) represents an individual pre-transplant serum sample tested against TKO cells (GGTA1/B4GalNT2/SLA-1 KO) who later received a TKO porcine kidney. All recipients were selected for low pretransplant IgG antibody binding, but none achieved negative crossmatch. (B) Immunosuppression regimen used in all of the recipients.

To evaluate whether tacrolimus-based immunosuppression could prolong survival in tesidolumab treated recipients, a third group of 4 recipients received the same regimen except that anti-CD154 (5c8) was replaced with tacrolimus given daily intramuscularly with trough levels between 8–12 ng/mL tested weekly (figure 4). Prophylactic enrofloxacin, fluconazole, and ganciclovir were started on the day of transplant in all recipients. Fluconazole and enrofloxacin were discontinued when flow cytometry demonstrated sustained T-cell reconstitution. Ganciclovir was continued until CMV titers were negative on 2 consecutive measurements. Weekly subcutaneous Epogen injections were started for hemoglobin less than 9.5 gm/dL, and discontinued when hemoglobin reached 12 g/dL.

FIGURE 4.

FIGURE 4.

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(A) Immunosuppression regimen including Tacrolimus and Tesidolumab without CD154 monoclonal antibody therapy. (B) Kaplan-Meier curve analysis reveals that a tacrolimus based immunosuppression regimen fails to extend xenograft survival beyond 62 days despite the presence of C5 complement inhibition by tesidolumab. (C) Biopsy taken from explanted kidney at day 62 shows parenchymal hemorrhage and thrombotic microangiopathy, prominent IgG, IgM, C4d, and minimal C5b-9. (D) Creatinine and potassium levels rose above normal values days to weeks before rejection. Hemoglobin values remained relatively normal.

Renal biopsies and histology and immunofluorescence confocal microscopy

Protocol renal biopsies were performed on postoperative days 14 and 35, as well as at times of suspected rejection. Biopsies were performed with ultrasound guidance with a 20-guage Carefusion Achieve Soft Tissue Automatic Biopsy Needle (BD Biosciences, Franklin Lakes, NJ). Biopsies were analyzed using standard hematoxylin and eosin (H&E) staining. Biopsies and nephrectomy specimens were also evaluated for immunopathology using immunofluorescence confocal microscopy. Frozen tissue sections were fixed using 4% paraformaldehyde for 10 minutes. Fixed sections were blocked with 1% IgG-free BSA in PBS. The antibodies used to probe tissue sections were (Primary antibodies: anti-C4d (Lifespan Bioscience # LS-C140137–100) and anti-C5b-9 (Santa Cruz Biotechnology #SC-58935); secondary antibody: anti-human IgG # 309–486-003, anti-human IgM #309–606-043 both conjugated to alexafluor 488, and anti-mouse IgG conjugated to alexafluor 647 #115–605-003 (Jackson Immunoresearch). Following antibody staining, the sections were incubated with 40,6-diamidino-2 phenylindole (DAPI, Invitrogen) as a nuclear stain. Tissues were then mounted in Prolong Gold Anti-fade (Invitrogen) before analysis. Confocal microscopy was performed using an Olympus FV1000 (Olympus America Inc., Center Valley, PA).

RESULTS

Tesidolumab eliminates early graft loss from IgM-mediated AMR in GGTA1/β4GalNt2 KO +/− SLA I KO pig kidney xenografts in Rhesus monkeys.

Nine of 10 renal xenografts in the control arm were rejected within 100 days, with most being lost in the first 10 days posttransplant (figure 2A). Two of 7 tesidolumab treated recipients rejected their graft in the first 100 days with one animal being censored for weight loss (p<0.019) (figure 2A). Early AMR in the first 14 days post-transplant was characterized by severe anemia and thrombocytopenia, and was seen in all early graft losses including the tesidolumab treated graft that was rejected on posttransplant day 8. The other 6 tesidolumab treated recipients showed minimal or no signs of early AMR when compared to control recipients despite the presence of significant antibody binding in both groups. C4d binding was similar in both groups, but there was significantly less C5b-9 binding in the renal xenografts of tesidolumab treated recipients (figure 2B).

FIGURE 2.

FIGURE 2.

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Analysis of grafts surviving to 557 days. (A) Kaplan-Meier curve analysis of recipients who received genetically engineered porcine kidneys with or without Tesidolumab included in the immunosuppression regimen. (B) H&E and confocal microscope analysis of rejected xenografts. Confocal microscopy showing deposition of IgM, and C4d in the control and tesidolumab treated recipients. Tesidoluman treated recipients had minimal histological injury while control kindeys had parenchymal hemorrhage and thrombotic microangiopathy. Deposition of C5b-9 was reduced on xenografts in tesidolumab treated recipients.

Deletion of SLA I had no impact on early AMR as 3 of 4 recipients rejected their kidney within the first 11 days posttransplant, and all 4 recipients rejected their renal xenografts within 100 days (table 1). There appeared to be no difference in kidneys with SLA I deleted when transplanted into recipients with tesidolumab as the grafts survived 205 and 414 days with one recipient being censored out for weight loss at 43 days with a normal creatinine and favorable explant pathology.

Table 1.

Donor Kidney Genetics Number of Transplants Anti-C5 Baseline Immunosuppression Survival (Days)
GGTA1/B4GalNT2 KO 6 No Anti-CD154 5a, 6a, 6a, 35a, 100a, 435
GGTA1/B4GalNT2/SLA-l KO 4 No Anti-CD154 6a, 7a, 11a‘ 95a
GGTA1/B4GalNT2 KO 4 Yes Anti-CD154 8a, 70a, 547b, 557b
GGTA1/B4GalNT2/SI_A-l KO 3 Yes Anti-CD154 43c, 205, 414b
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a

Early AMR;

b

Experimental end-point reached;

c

Censored for weight loss

Tesidolumab protected renal xenografts from early antibody-mediated rejection.

All kidneys in the non-C5 group exhibited signs of early AMR on H&E, while the tesidolumab treated kidneys did not show signs of early AMR. Kidneys in both groups showed significant IgM binding and C4d deposition. C5b-9 was much more pronounced in the kidneys in the control group, while there was minimal early C5b-9 staining in the tesidolumab treated kidneys (figure 2B). Kidneys rejected during the first 14 days (n=6 of 10 in control group vs. n=1 out of 7 in tesidolumab group were evaluated as well as the single kidney the tesidolumab group rejected at 8 days. Platelet counts remained above 2 X103/μL throughout, and urine protein was not elevated until late in the course when grafts began to fail secondary to transplant glomerulopathy (data not shown)

Renal function is preserved until late in the post-transplant course in recipients of renal xenografts.

Renal function was preserved until late in the course in any renal xenograft that was not rejected in the first postoperative week (figure 2). Serum creatinine and potassium were well controlled throughout, unless there was late graft failure from rejection (figure 3a and b respectively). Hemoglobin remained above 9.5 g/dL in recipients surviving long-term (figure 3c).

FIGURE 3.

FIGURE 3.

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(A) Creatinine is shown to be well controlled until acutely rising at time of graft failure. (B) Serum potassium was well controlled throughout, except during late graft failure due to rejection (C) Early graft failure recipients encounter anemia as shown by decreased hemoglobin levels. Tesidolumab treated recipients-maintained hemoglobin levels at or above normal levels more frequently than control animals for the duration of graft survival.

Tesidolumab improves survival in Rhesus recipients of GGTA1/β4GalNt2KO pig kidneys immunosuppressed with tacrolimus, but not in a clinically meaningful way.

Since tesidolumab was able to diminish early AMR in our preclinical model, we next evaluated whether we could substitute anti-CD154 5c8 antibody which is not a clinically approved immunosuppressive agent with tacrolimus, which is the industry standard for kidney transplantation. Tesidolumab was able to eliminate graft loss in the first 10 days due to AMR, but all 4 transplants failed secondary to IgG related AMR between 16–62 days (figure 4B). Biopsies taken on day 14 were clean showing no signs of early AMR, or T cell infiltration (data not shown). Graft failure in all 4 transplants was secondary to AMR and grafts showed obliterative lesions in the glomerular capillary lumens, a paucity of cellular infiltrate, and significant signs of thrombotic microangiopathy (figure 4C).

Discussion

Renal xenotransplantation using genetically modified pig kidneys could allow all appropriate patients with ESRD to receive a life-saving kidney transplant. Clinical application of this form of therapy has been limited by the early AMR that occurs as the result of preformed DSA that patients have against glycan xenoantigens on the surface of pig cells. Pigs with three xenoantigens deleted (α-gal, Neu5Gc, and Sda) have been created and have a negative crossmatch to 30% of waitlisted patients for IgM and IgG, and 70% of patients have a negative IgG crossmatch. The negative crossmatch in patients suggests that a large fraction of the waitlist could be transplanted using these kidneys without the expectation of early AMR. Additionally, those who have a negative IgG crossmatch, but a positive IgM crossmatch could very well be transplanted using plasmapheresis or other immunomodulating approaches to eliminate the IgM specific DSA(17–19). The next step toward clinical application is the demonstration that consistent prolonged survival in a preclinical model of renal xenotransplantation can be achieved using immunosuppression that both clinicians and regulatory authorities are comfortable with from both efficacy and human toxicity profile standpoints.

A significant challenge to moving ahead with renal xenotransplantation is that there is no donor pig that has been created that has a negative crossmatch with either Rhesus monkeys or baboons, the two most readily available recipients for preclinical testing(5, 11). In fact, all rhesus monkeys we have tested have a CDC positive crossmatch to all pigs we have tested(7). The positive crossmatch is also seen in all baboons tested, and CDC assays while improved using GGTA1/β4GalNt2 KO rather than GGTA1/β4GalNt2/CMAH KO PBMCs, were still positive in all cases(10). We have shown that recipients with the most favorable crossmatches can survive long-term receiving GGTA1 KO/CD55Tg pig kidneys and being immunosuppressed with T cell depletion and anti-CD154 antibodies and mycophenolic acid(2, 8). Our initial results with the GGTA1/β4GalNt2 KO pig kidney transplants showed that it is possible to achieve prolonged survival but only if the renal xenograft survived the early IgM mediated AMR that occurred in the first week posttransplant(7).

Our previous data with the GGTA1 KO/CD55Tg and the GGTA1/β4GalNt2 KO kidney xenografts suggested that the pre-transplant crossmatch was very important to the outcome of graft survival. While all of these transplants were CDC crossmatch positive with the majority of the DSA being IgM, those recipients with less DSA had better function and performed longer than those recipients with higher pre-transplant DSA levels(2). While expression of human complement regulatory transgene CD55 seemed to help overcome the initial IgM mediated injury, others have shown that human transgenes are not effective for improving long term survival(9). Cooper recently showed that GGTA1/β4GalNt2/CMAH KO pig kidneys with six human transgenes transplanted into baboons failed to prolong transplant survival, with 2 of 3 grafts going down within the first three days posttransplant. The early failure of these kidneys in Cooper’s series was likely the result of the worsening of the crossmatch that occurs in NHPs with the CMAH deletion(11).

Studies from the hamster-to-rat xenograft model showed that temporary inhibition of the anti-proliferative response with antiproliferative reagents could lead to prolonged xenograft survival in heterotopic cardiac and orthotopic hepatic xenografts(20, 21). Later, the results were extended to show that marginal zone T cell independent IgM producing B cells could induce rapid production of xenoreactive IgM(22, 23). This mechanism was dependent upon NK cell help and was partially suppressed by anti-CD154(24). Park used an ABO incompatible mouse model to show that the use of temporary anti-C5 (4 weeks) improved cardiac allograft survival at 56 days from 15% to 75% compared to recipients who were placed on standard immunosuppression with tacrolimus, mycophenolic acid and steroids, but did not receive anti-C5 therapy. Humanized anti-C5 Eculizumab is clinically available and has been used for the treatment of AMR in renal allotransplantation, but this antibody binds to AA residues ARG885 and TRP917 which makes eculizumab specific for human C5(14). Since eculizumab is not active in Rhesus monkeys we used the anti-C5 antibody tesidolumab, a humanized anti-C5 antibody whose activity is cross-reactive with NHPs, and has demonstrated a favorable toxicity profile in humans during phase II clinical trials in patients with uveitis(25, 26). Our data shows that anti-C5 therapy can eliminate early AMR in renal xenotransplantation so that consistent long-term survival can be achieved. Tesidolumab could be used in a clinical trial since it has demonstrated a favorable toxicity profile in human phase II clinical trials. Alternatively, anti-C5 eculizumab could be substituted in a clinical trial on the basis of its nearly identical mechanism of action and FDA approval. Having circumvented the initial obstacle in the preclinical model using an acceptable clinical intervention with anti-C5, our next task was to evaluate whether prolonged renal xenograft survival could be achieved using a clinically acceptable baseline immunosuppressive reagent instead of the anti-CD154 (5c8) that is not a clinically acceptable drug based on thromboembolic complications encountered in clinical trials in renal allotransplantation. Tacrolimus based immunosuppression is the industry standard for renal allotransplantation, and was able to prolong survival to at least 56 days in the Park’s. ABO incompatible mouse cardiac allograft model(12). We tested tesidolumab for its ability to prolong survival of GGTA1/β4GalNt2 KO pig kidneys and the results were clear. While tesidolumab delayed the onset of AMR, it did not provide enough protection to allow tacrolimus to prolong survival in the rhesus monkey to the point where an argument could be made that clinical success was likely. While the clinical situation will likely be performed with a recipient that has a negative crossmatch, and tacrolimus works well to prevent T cell mediated rejection in the situation where no preformed DSA is present, we are currently unable to definitively show that tacrolimus will work in renal xenotransplantation with the currently available donor pigs. Our results push preclinical development into two possible directions: (1) identifying new xenoantigens in the rhesus monkey and creating new donor pigs with the offending xenoantigen deleted on the GGTA1/β4GalNt2 KO background. This path while very important and worthwhile, will be dependent upon the ability to identify a xenoantigen and produce a knockout pig. The second option (2) is to test CD40/CD40L costimulatory blockade reagents that have been tested in phase II clinical trials with favorable toxicity profiles. We are in the process of evaluating a number of the CD40/CD40L costimulatory that are in phase II and III trials. We will evaluate these reagents continuing to use tesidolumab to prevent early AMR, T cell depletion, mycophenolic acid and steroids. The results in these experiments are very important for several reasons pertaining to the translation of renal xenotransplantation to clinical practice. First, we can say that an immunosuppressive regimen consisting entirely of drugs with tested and favorable toxicity profiles in humans can prolong survival consistently in preclinical models. There is robust data from the cardiac xenotransplant literature that showing that anti-CD40 can effectively prolong graft survival in the preclinical model, so that blockade of the CD40/CD40L pathway can be blocked either with anti-CD154 or anti-CD40 to achieve prolonged survival(27, 28). The result is that we may have more options to choose from regarding selection of baseline immunosuppression for our initial clinical trials(29).

Anti-class I HLA antibodies are a serious barrier to transplantation for many patients on the transplant waitlist. Some anti-HLA class I antibodies cross-react with SLA I and pose a barrier to xenotransplantation(6, 30). If the donor graft had no SLA I, then these antibodies would not bind to the pig kidney, and the anti-class I HLA barrier to xenotransplantation could be circumvented. We evaluated the impact of deletion of SLA I on the immunological response to renal xenografts. Our results indicate that SLA deletion does not confer an immunological advantage to survival. Recipients who had graft loss in the first two weeks showed signs of classic AMR, and a relative absence of cellular infiltrate. Our earlier data with GGTA1 KO/CD55Tg pig kidneys transplanted into rhesus showed that CD8 T cells were less critical to rejection, and that CD4 T cell depletion was required for long term graft survival(8). We did not note any difference in CD8 T cell infiltration at any time point in recipients of GGTA1/β4GalNt2/SLA I KO kidneys when compared to recipients receiving GGTA1/β4GalNt2 KO kidneys (data not shown). The other potential important consideration for the use of SLA I KO pig kidneys is that absence of class I MHC is associated with increased susceptibility to NK cell damage. In renal allotransplantation, missing self in conjunction with DSA led to decreased graft survival and in endothelial cell and NK cell coculture, there was increased graft endothelial damage(31). There is also significant in vitro and small animal data suggesting that NK cells could play a role in xenograft rejection based upon species differences in class I MHC, but there has been little confirmation of this in a preclinical pig-to-non-human primate model(24, 32). Our results suggest that absence of SLA I did not impact renal xenograft survival, and confocal analysis of GGTA1/β4GalNt2/SLA I KO kidneys did not show an increase in infiltration of NK cells at any time point (days 14, 35, and explant) when compared to GGTA1/β4GalNt2 KO SLA I replete kidneys.

In conclusion, our studies show that temporary complement inhibition with anti-C5 antibody tesidolumab can prevent early IgM mediated rejection and prolonged survival of renal xenografts in a preclinical model using a CD154 based costimulation blockade immunosuppressive regimen. Tesidolumab was not able to prolong survival using tacrolimus-based immunosuppression.

Acknowledgments

This work was funded by grants from the National Institutes of Health (1RO1AI26322) and ORIP/OD P51OD011132. We thank Novartis for providing Tesidolumab.

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