PMID- 34616392 OWN - NLM STAT- MEDLINE DCOM- 20211028 LR - 20211028 IS - 1664-3224 (Electronic) IS - 1664-3224 (Linking) VI - 12 DP - 2021 TI - Precision Engineering of an Anti-HLA-A2 Chimeric Antigen Receptor in Regulatory T Cells for Transplant Immune Tolerance. PG - 686439 LID - 10.3389/fimmu.2021.686439 [doi] LID - 686439 AB - Infusion of regulatory T cells (Tregs) engineered with a chimeric antigen receptor (CAR) targeting donor-derived human leukocyte antigen (HLA) is a promising strategy to promote transplant tolerance. Here, we describe an anti-HLA-A2 CAR (A2-CAR) generated by grafting the complementarity-determining regions (CDRs) of a human monoclonal anti-HLA-A2 antibody into the framework regions of the Herceptin 4D5 single-chain variable fragment and fusing it with a CD28-zeta signaling domain. The CDR-grafted A2-CAR maintained the specificity of the original antibody. We then generated HLA-A2 mono-specific human CAR Tregs either by deleting the endogenous T-cell receptor (TCR) via CRISPR/Cas9 and introducing the A2-CAR using lentiviral transduction or by directly integrating the CAR construct into the TCR alpha constant locus using homology-directed repair. These A2-CAR(+)TCR(deficient) human Tregs maintained both Treg phenotype and function in vitro. Moreover, they selectively accumulated in HLA-A2-expressing islets transplanted from either HLA-A2 transgenic mice or deceased human donors. A2-CAR(+)TCR(deficient) Tregs did not impair the function of these HLA-A2(+) islets, whereas similarly engineered A2-CAR(+)TCR(deficient)CD4(+) conventional T cells rejected the islets in less than 2 weeks. A2-CAR(+)TCR(deficient) Tregs delayed graft-versus-host disease only in the presence of HLA-A2, expressed either by co-transferred peripheral blood mononuclear cells or by the recipient mice. Altogether, we demonstrate that genome-engineered mono-antigen-specific A2-CAR Tregs localize to HLA-A2-expressing grafts and exhibit antigen-dependent in vivo suppression, independent of TCR expression. These approaches may be applied towards developing precision Treg cell therapies for transplant tolerance. CI - Copyright (c) 2021 Muller, Ferreira, Ronin, Ho, Nguyen, Faleo, Zhou, Lee, Leung, Skartsis, Kaul, Mulder, Claas, Wells, Bluestone and Tang. FAU - Muller, Yannick D AU - Muller YD AD - Department of Surgery, University of California, San Francisco, San Francisco, CA, United States. AD - Diabetes Center, University of California, San Francisco, San Francisco, CA, United States. FAU - Ferreira, Leonardo M R AU - Ferreira LMR AD - Department of Surgery, University of California, San Francisco, San Francisco, CA, United States. AD - Diabetes Center, University of California, San Francisco, San Francisco, CA, United States. AD - Sean N. Parker Autoimmune Research Laboratory, University of California, San Francisco, San Francisco, CA, United States. FAU - Ronin, Emilie AU - Ronin E AD - Department of Surgery, University of California, San Francisco, San Francisco, CA, United States. AD - Diabetes Center, University of California, San Francisco, San Francisco, CA, United States. FAU - Ho, Patrick AU - Ho P AD - Department of Surgery, University of California, San Francisco, San Francisco, CA, United States. AD - Diabetes Center, University of California, San Francisco, San Francisco, CA, United States. AD - Sean N. Parker Autoimmune Research Laboratory, University of California, San Francisco, San Francisco, CA, United States. FAU - Nguyen, Vinh AU - Nguyen V AD - Department of Surgery, University of California, San Francisco, San Francisco, CA, United States. AD - Diabetes Center, University of California, San Francisco, San Francisco, CA, United States. FAU - Faleo, Gaetano AU - Faleo G AD - Department of Surgery, University of California, San Francisco, San Francisco, CA, United States. AD - Diabetes Center, University of California, San Francisco, San Francisco, CA, United States. FAU - Zhou, Yu AU - Zhou Y AD - Department of Anesthesia and Perioperative Care, University of California, San Francisco, Zuckerberg San Francisco General Hospital and Trauma Center, San Francisco, CA, United States. FAU - Lee, Karim AU - Lee K AD - Department of Surgery, University of California, San Francisco, San Francisco, CA, United States. FAU - Leung, Kevin K AU - Leung KK AD - Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA, United States. FAU - Skartsis, Nikolaos AU - Skartsis N AD - Department of Surgery, University of California, San Francisco, San Francisco, CA, United States. AD - Department of Medicine, University of California, San Francisco, San Francisco, CA, United States. FAU - Kaul, Anupurna M AU - Kaul AM AD - Department of Surgery, University of California, San Francisco, San Francisco, CA, United States. FAU - Mulder, Arend AU - Mulder A AD - Department of Immunohaematology and Blood Transfusion, Leiden University Medical Center, Leiden, Netherlands. FAU - Claas, Frans H J AU - Claas FHJ AD - Department of Immunohaematology and Blood Transfusion, Leiden University Medical Center, Leiden, Netherlands. FAU - Wells, James A AU - Wells JA AD - Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA, United States. FAU - Bluestone, Jeffrey A AU - Bluestone JA AD - Diabetes Center, University of California, San Francisco, San Francisco, CA, United States. AD - Sean N. Parker Autoimmune Research Laboratory, University of California, San Francisco, San Francisco, CA, United States. FAU - Tang, Qizhi AU - Tang Q AD - Department of Surgery, University of California, San Francisco, San Francisco, CA, United States. AD - Diabetes Center, University of California, San Francisco, San Francisco, CA, United States. LA - eng GR - UC4 DK116264/DK/NIDDK NIH HHS/United States GR - P30 DK063720/DK/NIDDK NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't DEP - 20210920 PL - Switzerland TA - Front Immunol JT - Frontiers in immunology JID - 101560960 RN - 0 (Antibodies) RN - 0 (HLA-A2 Antigen) RN - 0 (Receptors, Antigen, T-Cell) RN - 0 (Receptors, Chimeric Antigen) SB - IM MH - Animals MH - Antibodies/*metabolism MH - Cell Engineering MH - Female MH - Graft vs Host Disease/immunology/metabolism MH - HLA-A2 Antigen/*immunology MH - Humans MH - Immunotherapy, Adoptive MH - Male MH - Mice MH - Mice, Inbred NOD MH - Receptors, Antigen, T-Cell/metabolism MH - Receptors, Chimeric Antigen/genetics/*metabolism MH - T-Lymphocytes, Regulatory/immunology/metabolism/*transplantation MH - *Transplantation Tolerance PMC - PMC8488356 OTO - NOTNLM OT - HLA OT - Treg OT - chimeric antigen receptor OT - genome editing OT - humanized mouse model OT - immune tolerance OT - regulatory T cells OT - transplantation COIS- A provisional patent on A2-CAR Tregs has been submitted. QT is a co-founder and scientific advisor of Sonoma Biotherapeutics. JB is a co-founder and the Chief Executive Officer and President of Sonoma Biotherapeutics. JW is co-Founder of Soteria Biotherapeutics developing small molecule switchable biologics, on the SAB of Spotlight, and recipient of sponsored research from Bristol Myers Squibb. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. EDAT- 2021/10/08 06:00 MHDA- 2021/10/29 06:00 PMCR- 2021/01/01 CRDT- 2021/10/07 06:55 PHST- 2021/03/26 00:00 [received] PHST- 2021/08/26 00:00 [accepted] PHST- 2021/10/07 06:55 [entrez] PHST- 2021/10/08 06:00 [pubmed] PHST- 2021/10/29 06:00 [medline] PHST- 2021/01/01 00:00 [pmc-release] AID - 10.3389/fimmu.2021.686439 [doi] PST - epublish SO - Front Immunol. 2021 Sep 20;12:686439. doi: 10.3389/fimmu.2021.686439. eCollection 2021.