Minimally modified off-the-shelf allogeneic CAR T cells

T cells modified with synthetic chimeric antigen receptors (CARs) have been enormously successful in treating hematological malignancies, and multiple products are now approved for use in patients with relapsed/refractory leukemia, lymphoma, and myeloma. However, on-demand manufacturing of an autologous engineered T cell product creates considerable financial, logistical, and availability challenges to the widespread implementation of the therapy. To complicate matters, patients with aggressive disease may require bridging therapy while the T cell product undergoes manufacturing and release testing, and the quality and function of the CAR T cells may be variable between patients following multiple courses of therapy. For these reasons, off-the-shelf strategies using alternative allogeneic cell sources for ex vivo engineering including healthy donor T cells, pluripotent stem cell-derived T cells1Michaels Y.S. Durland L.J. Zandstra P.W. Engineering T Cell Development for the Next Generation of Stem Cell-Derived Immunotherapies.GEN Biotechnol. 2023; 2: 106-119https://doi.org/10.1089/genbio.2023.0008Crossref PubMed Google Scholar, and natural killer (NK) cells2Marin D. Li Y. Basar R. Rafei H. Daher M. Dou J. Mohanty V. Dede M. Nieto Y. Uprety N. et al.Safety, efficacy and determinants of response of allogeneic CD19-specific CAR-NK cells in CD19+ B cell tumors: a phase 1/2 trial.Nat. Med. 2024; 30: 772-784https://doi.org/10.1038/s41591-023-02785-8Crossref PubMed Scopus (43) Google Scholar, and direct in vivo CAR T cell engineering3Short L. Holt R.A. Cullis P.R. Evgin L. Direct in vivo CAR T cell engineering.Trends Pharmacol. Sci. 2024; 45: 406-418https://doi.org/10.1016/j.tips.2024.03.004Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar are being explored preclinically and clinically. Using therapeutic allogeneic αβ T cells creates two challenges: (1) the native T cell receptor (TCR) can cause graft versus host disease (GVHD), and conversely, (2) alloreactive host T cells and NK cells can quickly reject the cells. To address the former, components of the TCR complex such as the TCR α constant (TRAC) gene have been disrupted by clustered regularly interspaced short palindromic repeats (CRISPR) or transcription activator-like effector nucleases (TALENs), or the locus repurposed to express the CAR.4van der Stegen S.J.C. Lindenbergh P.L. Petrovic R.M. Xie H. Diop M.P. Alexeeva V. Shi Y. Mansilla-Soto J. Hamieh M. Eyquem J. et al.Generation of T-cell-receptor-negative CD8αβ-positive CAR T cells from T-cell-derived induced pluripotent stem cells.Nat. Biomed. Eng. 2022; 6: 1284-1297https://doi.org/10.1038/s41551-022-00915-0Crossref PubMed Scopus (34) Google Scholar To address the latter problem of the mismatched human leukocyte antigen (HLA), deletion of the β2 microglobulin (B2M) and class II-transactivator (CIITA) genes creates stealth T cells that fail to express HLA class I and II respectively.5Jo S. Das S. Williams A. Chretien A.S. Pagliardini T. Le Roy A. Fernandez J.P. Le Clerre D. Jahangiri B. Chion-Sotinel I. et al.Endowing universal CAR T-cell with immune-evasive properties using TALEN-gene editing.Nat. Commun. 2022; 13: 3453https://doi.org/10.1038/s41467-022-30896-2Crossref PubMed Scopus (48) Google Scholar Since HLA-negative cells can be targeted by NK cells, overexpression of the minimally polymorphic HLA-E molecules that engage the NKG2A inhibitory receptor on NK cells can compensate for this problem.6Wang B. Iriguchi S. Waseda M. Ueda N. Ueda T. Xu H. Minagawa A. Ishikawa A. Yano H. Ishi T. et al.Generation of hypoimmunogenic T cells from genetically engineered allogeneic human induced pluripotent stem cells.Nat. Biomed. Eng. 2021; 5: 429-440https://doi.org/10.1038/s41551-021-00730-zCrossref PubMed Scopus (79) Google Scholar An alternative strategy involves targeting alloreactive host T cells by either deleting CD52 in the therapeutic T cells and concurrently treating with the anti-CD52 antibody alemtuzumab7Poirot L. Philip B. Schiffer-Mannioui C. Le Clerre D. Chion-Sotinel I. Derniame S. Potrel P. Bas C. Lemaire L. Galetto R. et al.Multiplex Genome-Edited T-cell Manufacturing Platform for “Off-the-Shelf” Adoptive T-cell Immunotherapies.Cancer Res. 2015; 75: 3853-3864https://doi.org/10.1158/0008-5472.Can-14-3321Crossref PubMed Google Scholar or by expressing an alloimmune defense receptor in therapeutic T cells that recognizes the activation marker 4-1BB.8Mo F. Watanabe N. McKenna M.K. Hicks M.J. Srinivasan M. Gomes-Silva D. Atilla E. Smith T. Ataca Atilla P. Ma R. et al.Engineered off-the-shelf therapeutic T cells resist host immune rejection.Nat. Biotechnol. 2021; 39: 56-63https://doi.org/10.1038/s41587-020-0601-5Crossref PubMed Scopus (76) Google Scholar These approaches can deplete host T cells globally or with some selectively (Figure 1 left). In a recent issue of Molecular Therapy Oncology, Quach et al. have astutely leveraged the biology of CD30 (also known as TNFRSF8), combined with the expansion of TCR antigen-specific T cells, to bypass the multiplexed genetic modifications required to prevent rejection and GVHD associated with allogeneic T cell therapies.9Quach D.H. Ganesh H.R. Briones Y.D. Nouraee N. Ma A. Hadidi Y.F. Sharma S. Rooney C.M. Rejection resistant CD30.CAR-modified Epstein-Barr virus-specific T cells as an off-the-shelf platform for CD30(+) lymphoma.Mol. Ther. Oncol. 2024; 32200814https://doi.org/10.1016/j.omton.2024.200814Abstract Full Text Full Text PDF PubMed Google Scholar Using Epstein-Barr virus (EBV)-specific T cells (EBVSTs) expanded in vitro with peptides representing latent and lytic genes (EBNA1, LMP1, LMP2, and BZLF1) simultaneously reduces the risk of GVHD and enables targeting of EBV-positive cells through the native TCR. CD30 is expressed not only on Reed-Sternberg cells in Hodgkin lymphoma and anaplastic large cell lymphomas but also subsets of activated T cells, B cells, and NK cells,10van der Weyden C.A. Pileri S.A. Feldman A.L. Whisstock J. Prince H.M. Understanding CD30 biology and therapeutic targeting: a historical perspective providing insight into future directions.Blood Cancer J. 2017; 7: e603https://doi.org/10.1038/bcj.2017.85Crossref PubMed Scopus (0) Google Scholar and they demonstrated multi-specificity toward EBV-positive and CD30-positive targets through the TCR and the CAR. Instead of genetically engineering hypoimmunogenic T cells, they exploited the expression of CD30 on activated T and NK cells. In mixed lymphocyte reactions (MLRs), they demonstrated that CD30 CAR EBVSTs inhibited the expansion of alloreactive T cells and thus resisted their killing. Given that CD30 is also upregulated on CAR T cells upon antigen recognition, it was surprising that the transduced cells did not undergo fratricide. However, the authors showed that the CAR binds to CD30 in cis, masking the epitope from surrounding CAR T cells (Figure 1 right). The effectiveness of the approach will be borne out in an ongoing phase 1 clinical trial where CD30 CAR EBVSTs are manufactured from healthy EBV-seropositive donors (NCT04288726). The study has already reported early positive outcomes, including a tolerable safety profile, and, of seven evaluable patients, two complete responses and three partial responses.11Quach D.H. Ramos C.A. Lulla P.D. Sharma S. Ganesh H.R. Hadidi Y.F. Thakkar S.G. Becerra-Dominguez L. Mehta B. Perconti S. et al.Safety and Efficacy of Off-the-Shelf CD30.CAR-Modified Epstein-Barr Virus-Specific T Cells in Patients with CD30-Positive Lymphoma.Blood. 2021; 138: 1763https://doi.org/10.1182/blood-2021-153421Crossref Google Scholar The efficacy of the CD30 virus-specific T cell strategy will likely be further improved by combination with vaccines12Tanaka M. Tashiro H. Omer B. Lapteva N. Ando J. Ngo M. Mehta B. Dotti G. Kinchington P.R. Leen A.M. et al.Vaccination targeting native receptors to enhance the function and proliferation of chimeric antigen receptor (CAR)-modified T cells.Clin. Cancer Res. 2017; 23: 3499-3509https://doi.org/10.1158/1078-0432.CCR-16-2138Crossref PubMed Scopus (70) Google Scholar or following viral reactivation.13Lapteva N. Gilbert M. Diaconu I. Rollins L.A. Al-Sabbagh M. Naik S. Krance R.A. Tripic T. Hiregange M. Raghavan D. et al.T-Cell Receptor Stimulation Enhances the Expansion and Function of CD19 Chimeric Antigen Receptor-Expressing T Cells.Clin. Cancer Res. 2019; 25: 7340-7350https://doi.org/10.1158/1078-0432.CCR-18-3199Crossref PubMed Scopus (35) Google Scholar Of note, the CD30 CAR EBVSTs were detected in the peripheral blood of patients, but there was no evidence of T cell expansion.11Quach D.H. Ramos C.A. Lulla P.D. Sharma S. Ganesh H.R. Hadidi Y.F. Thakkar S.G. Becerra-Dominguez L. Mehta B. Perconti S. et al.Safety and Efficacy of Off-the-Shelf CD30.CAR-Modified Epstein-Barr Virus-Specific T Cells in Patients with CD30-Positive Lymphoma.Blood. 2021; 138: 1763https://doi.org/10.1182/blood-2021-153421Crossref Google Scholar Although in vitro MLR cultures suggest that alloreactive T cells are eliminated, the lack of expansion of the CAR T cells in patients suggests that further insight is required into the levels and kinetics of CD30 expression in vivo across subsets of activated T cells and NK cells that contribute to rejection. When a CAR T cell expresses high levels of the target antigen, it can be subject to fratricide by other CAR-expressing T cells, for example as described with CD7.14Gomes-Silva D. Srinivasan M. Sharma S. Lee C.M. Wagner D.L. Davis T.H. Rouce R.H. Bao G. Brenner M.K. Mamonkin M. CD7-edited T cells expressing a CD7-specific CAR for the therapy of T-cell malignancies.Blood. 2017; 130: 285-296https://doi.org/10.1182/blood-2017-01-761320Crossref PubMed Scopus (307) Google Scholar What physical properties of a CAR allow for good cis binding and protection from fratricide? The location of the scFv epitope and the length and source of the hinge domain may determine whether the CAR molecule has enough flexibility to bind in the cis conformation. It would be interesting to test whether other CD30 CARs have similar functionality or whether it is specific to the construct used by Quach et al. Protection from fratricide may also be antigen dependent. Binding of the CAR to a surface-expressed antigen in cis could potentially activate signaling through both the CAR itself and the antigenic receptor. In this case, CD30 ligation could promote signal transduction through MAPK and NFKB, which could help the cell survive and proliferate.10van der Weyden C.A. Pileri S.A. Feldman A.L. Whisstock J. Prince H.M. Understanding CD30 biology and therapeutic targeting: a historical perspective providing insight into future directions.Blood Cancer J. 2017; 7: e603https://doi.org/10.1038/bcj.2017.85Crossref PubMed Scopus (0) Google Scholar Delineation of these properties may enable the design of other cis-protective CAR molecules. P.L. is supported by the BC Cancer Foundation and the Fondation Léon Fredericq. L.E. is supported by the Canadian Institute for Health Research, BC Cancer and the BC Cancer Foundation, Michael Smith Health Research BC, and the Terry Fox Research Institute. All authors declared no potential conflicts of interest.

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