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Humoral Reactivity of Renal Transplant-Waitlisted Patients to Cells From GGTA1/CMAH/B4GalNT2, and SLA Class I Knockout Pigs

Martens, Gregory R. MD; Reyes, Luz M. PhD; Butler, James R. MD; Ladowski, Joseph M. MS; Estrada, Jose L. DVM, PhD; Sidner, Richard A. PhD; Eckhoff, Devin E. MD; Tector, Matt PhD; Tector, A. Joseph MD, PhD

doi: 10.1097/TP.0000000000001646
Original Basic Science-General

Background: Antipig antibodies are a barrier to clinical xenotransplantation. We evaluated antibody binding of waitlisted renal transplant patients to 3 glycan knockout (KO) pig cells and class I swine leukocyte antigens (SLA).

Methods: Peripheral blood mononuclear cells from SLA identical wild type (WT), α1, 3-galactosyltransferase (GGTA1) KO, GGTA1/ cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) KO, and GGTA1/ CMAH /b1,4 N-acetylgalactosaminyl transferase (B4GalNT2) KO pigs were screened for human antibody binding using flow cytometric crossmatch (FCXM). Sera from 820 patients were screened on GGTA1/CMAH/B4GalNT2 KO cells and a subset with elevated binding was evaluated further. FCXM was performed on SLA intact cells and GGTA1/SLA class I KO cells after depletion with WT pig RBCs to remove cell surface reactive antibodies, but leave SLA antibodies. Lastly, human and pig reactive antibodies were eluted and tested for cross-species binding and reactivity to single-antigen HLA beads.

Results: Sequential glycan KO modifications significantly reduce antibody binding of waitlisted patients. Sera exhibiting elevated binding without reduction after depletion with WT RBCs demonstrate reduced binding to SLA class I KO cells. Human IgG, eluted from human and pig peripheral blood mononuclear cells, interacted across species and bound single-antigen HLA beads in common epitope-restricted patterns.

Conclusions: Many waitlisted patients have minimal xenoreactive antibody binding to the triple KO pig, but some HLA antibodies in sensitized patients cross-react with class I SLA. SLA class I is a target for genome editing in xenotransplantation.

Martens et al report that most waitlisted patients have minimal antibody binding to triple glycan KO cells, but HLA antibodies from some sensitized patients can cross-react with class I SLA, thus suggesting another antigen target for genome editing in xenotransplantation.

1 Department of Surgery, University of Alabama at Birmingham, Birmingham, AL.

2 Department of Surgery, Indiana University, School of Medicine, Indianapolis, IN.

3 Transplant Surgery, University of Alabama at Birmingham, Birmingham, AL.

Received 9 July 2016. Revision received 15 December 2016.

Accepted 22 December 2016.

G.R.M. is supported by a research scholarship from the American College of Surgeons. A.J.T. is the founder of Xenobridge LLC, has United Therapeutics funding and applied for patents. R.A.S. receives consulting fees related to xenotransplantation.

This study was funded by Indiana University. Indiana University Transplant Institute, and United Therapeutics.

This article has been revised approved by all authors. G.R.M., R.A.S., D.E.E., M.T., A.J.T. drafted this article developed refined study concepts. L.M.R., J.L.E. performed SLA sequencing, gene manipulations, pig cloning genotyping. G.R.M., R.A.S., J.R.B., J.M.L. performed data collection data analysis.

Correspondence: A. Joseph Tector, ZRB 701 1720 2nd Ave South Birmingham, AL 35294. (jtector@uab.edu).

Supplemental digital content (SDC) is available for this article. Direct URL citations appear in the printed text, and links to the digital files are provided in the HTML text of this article on the journal’s Web site (www.transplantjournal.com).

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