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Following successful implementation of anti-PD1/PDL1 agents in the treatment of patients with multiple cancers, immune checkpoint inhibitors have been increasingly used to rescue exhausted T cells following antigen stress. Genetic engineering strategies are being explored and translated to clinical trials to prevent the exhaustion of adoptively transferred chimeric antigen receptor (CAR) T cells (e.g., PD1 or transforming growth factor β dominant-negative receptors or deletion of PD1 by use of CRISPR-Cas9 methodology).1Cherkassky L. Morello A. Villena-Vargas J. Feng Y. Dimitrov D.S. Jones D.R. Sadelain M. Adusumilli P.S. Human CAR T cells with cell-intrinsic PD-1 checkpoint blockade resist tumor-mediated inhibition.J. Clin. Invest. 2016; 126: 3130-3144https://doi.org/10.1172/JCI83092Crossref PubMed Scopus (772) Google Scholar,2Kloss C.C. Lee J. Zhang A. Chen F. Melenhorst J.J. Lacey S.F. Maus M.V. Fraietta J.A. Zhao Y. June C.H. Dominant-negative TGF-beta receptor enhances PSMA-targeted human CAR T cell proliferation and augments prostate cancer eradication.Mol. Ther. 2018; 26: 1855-1866https://doi.org/10.1016/j.ymthe.2018.05.003Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar In a recent issue of Molecular Therapy Oncology, a study by Veliz et al.3Veliz K. Shen F. Shestova O. Shestov M. Shestov A. Sleiman S. Hansen T. O'Connor R.S. Gill S. Deletion of CD38 enhances CD19 chimeric antigen receptor T cell function.Mol. Ther. Oncol. 2024; 32200819https://doi.org/10.1016/j.omton.2024.200819Abstract Full Text Full Text PDF PubMed Google Scholar provided insight into how CD38 deletion can improve tumor-specific CAR T cell response. Similar to CTLA4, PD1, TIM3, and LAG3, CD38 is an immune-metabolic checkpoint molecule that is upregulated shortly after T cell activation. CD38 plays an inhibitory role in activated T cells; the underlying mechanism is not yet fully understood. Structurally, CD38 is a multifunctional ectoenzyme with hydrolase and cyclase activity and functions as a nicotinamide adenine dinucleotide (NAD)-consuming enzyme that produces immunosuppressive adenosine by converting ATP to AMP.4Chini C.C.S. Peclat T.R. Warner G.M. Kashyap S. Espindola-Netto J.M. de Oliveira G.C. Gomez L.S. Hogan K.A. Tarrago M.G. Puranik A.S. et al.CD38 ecto-enzyme in immune cells is induced during aging and regulates NAD(+) and NMN levels.Nat. Metab. 2020; 2: 1284-1304https://doi.org/10.1038/s42255-020-00298-zCrossref PubMed Scopus (170) Google Scholar In a recent publication, Lio et al. reported that after deleting CD38 from CAR T cells, these CAR T cells showed enhanced efficacy against T cell acute lymphoblastic leukemia (T-ALL) cells, both in vitro and in vivo.5Liao C. Wang Y. Huang Y. Duan Y. Liang Y. Chen J. Jiang J. Shang K. Zhou C. Gu Y. et al.CD38-Specific CAR integrated into CD38 locus driven by different promoters causes distinct antitumor activities of T and NK cells.Adv. Sci. 2023; 10e2207394https://doi.org/10.1002/advs.202207394Crossref Scopus (5) Google Scholar Furthermore, chemical inhibition of CD38 in CAR T cells was associated with improved tumor control. The study by Veliz et al.3Veliz K. Shen F. Shestova O. Shestov M. Shestov A. Sleiman S. Hansen T. O'Connor R.S. Gill S. Deletion of CD38 enhances CD19 chimeric antigen receptor T cell function.Mol. Ther. Oncol. 2024; 32200819https://doi.org/10.1016/j.omton.2024.200819Abstract Full Text Full Text PDF PubMed Google Scholar focused on comparing second-generation anti-CD19-41BBz CAR T cells, in which either CD38 or PD1 have been knocked out using CRISPR-Cas9 technology. The deletion of CD38 confers the relative resistance of CAR T cells to exhaustion in vitro and improved tumor control in vivo. After the deletion of either CD38 or PD1, CAR T cells showed comparable proliferation and cytotoxicity against CD19-expressing NALM6 ALL cells in vitro, as demonstrated by CellTrace Violet dilution and luciferase assay. In addition, no difference was observed in the glycolytic metabolism of CD38-knockout (KO) CAR T cells, as demonstrated by Seahorse assay, which is in contradiction with previous reports of metabolic changes and increased oxidative phosphorylation found in CD38-KO T and natural killer (NK) cells.6Chatterjee S. Daenthanasanmak A. Chakraborty P. Wyatt M.W. Dhar P. Selvam S.P. Fu J. Zhang J. Nguyen H. Kang I. et al.CD38-NAD(+)axis regulates immunotherapeutic anti-tumor T cell response.Cell Metab. 2018; 27: 85-100.e8https://doi.org/10.1016/j.cmet.2017.10.006Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar An in vitro model of chronic antigen stimulation showed that the deletion of either CD38 or PD1 renders CAR T cells resistant to exhaustion, as the ability to eliminate tumor cells after repeated rounds of antigen stimulations was significantly greater than that of the control group. Nevertheless, compared to CD38 KO, PD1 KO appeared to be superior in conferring chronically stimulated CAR T cells the capacity to retain cytotoxicity and secrete tumor necrosis factor. An in vivo study that used NALM6 cells showed that both CD38-KO and PD1-KO CAR T cells significantly reduced tumor burden compared to the control group, but tumor eradication was not observed in either group. Although the groups treated with CD38-KO and PD1-KO CD19-41BBz CAR T cells had significantly prolonged survival compared to the control group, no tumor rechallenge was administered in the mouse model. We have previously shown that in vivo tumor rechallenge demonstrated the functional persistence of CAR T cells beyond CAR T cell exhaustion that was observed in an antigen stress test in vitro.1Cherkassky L. Morello A. Villena-Vargas J. Feng Y. Dimitrov D.S. Jones D.R. Sadelain M. Adusumilli P.S. Human CAR T cells with cell-intrinsic PD-1 checkpoint blockade resist tumor-mediated inhibition.J. Clin. Invest. 2016; 126: 3130-3144https://doi.org/10.1172/JCI83092Crossref PubMed Scopus (772) Google Scholar,7Quach H.T. Skovgard M.S. Villena-Vargas J. Bellis R.Y. Chintala N.K. Amador-Molina A. Bai Y. Banerjee S. Saini J. Xiong Y. et al.Tumor-targeted nonablative radiation promotes solid tumor CAR T-cell therapy efficacy.Cancer Immunol. Res. 2023; 11: 1314-1331https://doi.org/10.1158/2326-6066.CIR-22-0840Crossref PubMed Scopus (5) Google Scholar The demonstration of functional persistence in vivo with multiple cancer cell lines and models would have strengthened the study beyond the use of CD-19 CAR T cells. In contrast to the well-known mechanisms of action for PD1, which activates inhibitory pathways downstream of the T cell receptor, or CTLA4, which competitively binds the costimulatory molecules CD80 and CD86, the proposed effect of CD38 appears to be mediated by its intrinsic enzymatic activity. Specifically, Veliz et al. reported that CD38 has a cyclase activity that converts NAD to cADPR (cyclic ADP ribose) and a hydrolase activity that converts cADPR to ADPR, which is further metabolized to generate the immunosuppressive metabolite adenosine.3Veliz K. Shen F. Shestova O. Shestov M. Shestov A. Sleiman S. Hansen T. O'Connor R.S. Gill S. Deletion of CD38 enhances CD19 chimeric antigen receptor T cell function.Mol. Ther. Oncol. 2024; 32200819https://doi.org/10.1016/j.omton.2024.200819Abstract Full Text Full Text PDF PubMed Google Scholar By reintroducing two mutant versions of CD38, the authors have shown that increased NAD levels and decreased immunosuppressive adenosine levels induced by CD38 KO are beneficial for sustaining T cell metabolism and function and further improve CAR T cell resistance to exhaustion. Yet, proof-of-principle experiments that directly correlate high NAD and low adenosine levels to improved metabolic fitness and function of T cells are still needed to confirm this hypothesis. A KEGG pathway enrichment analysis revealed a higher enrichment in arginine biosynthesis as well as in valine, leucine, and isoleucine metabolism in CD38-expressing wild-type cells, as compared to KO cells. Future studies may provide insight into relevant pathways by which CD38 affects T cell metabolism. The deletion of CD38 that leads to exhaustion resistance and enhanced CAR function has been observed not only in CAR T cells but also in CAR NK cells.8Clara J.A. Levy E.R. Reger R. Barisic S. Chen L. Cherkasova E. Chakraborty M. Allan D.S.J. Childs R. High-affinity CD16 integration into a CRISPR/Cas9-edited CD38 locus augments CD38-directed antitumor activity of primary human natural killer cells.J. Immunother. Cancer. 2022; 10e003804https://doi.org/10.1136/jitc-2021-003804Crossref PubMed Scopus (16) Google Scholar Similar to this study, other investigators have observed that cytotoxicity and cytokine production were independent of CD38 and that superior antitumor control was observed with CD38-KO CAR T cells.6Chatterjee S. Daenthanasanmak A. Chakraborty P. Wyatt M.W. Dhar P. Selvam S.P. Fu J. Zhang J. Nguyen H. Kang I. et al.CD38-NAD(+)axis regulates immunotherapeutic anti-tumor T cell response.Cell Metab. 2018; 27: 85-100.e8https://doi.org/10.1016/j.cmet.2017.10.006Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar These observations suggested that internal changes related to T cell metabolism, rather than increased T cell proliferation or cytotoxicity, contributed to the results. Similar observations of improved CAR T cell function without increased proliferation or cytotoxicity by use of different costimulatory domains have been shown both in vitro and in vivo.9Xiong Y. Taleb M. Misawa K. Hou Z. Banerjee S. Amador-Molina A. Jones D.R. Chintala N.K. Adusumilli P.S. c-Kit signaling potentiates CAR T cell efficacy in solid tumors by CD28- and IL-2-independent co-stimulation.Nat. Cancer. 2023; 4: 1001-1015https://doi.org/10.1038/s43018-023-00573-4Crossref PubMed Scopus (5) Google Scholar Previously, CD38 deletion and its importance on tumor-specific CAR T cells was only addressed in regard to fratricide prevention due to CD38 expression on CD38-targeting CAR T cells,5Liao C. Wang Y. Huang Y. Duan Y. Liang Y. Chen J. Jiang J. Shang K. Zhou C. Gu Y. et al.CD38-Specific CAR integrated into CD38 locus driven by different promoters causes distinct antitumor activities of T and NK cells.Adv. Sci. 2023; 10e2207394https://doi.org/10.1002/advs.202207394Crossref Scopus (5) Google Scholar,10Karvouni M. Vidal-Manrique M. Susek K.H. Hussain A. Gilljam M. Zhang Y. Gray J.D. Lund J. Kaufmann G. Ljunggren H.G. et al.Challenges in αCD38-chimeric antigen receptor (CAR)-expressing natural killer (NK) cell-based immunotherapy in multiple myeloma: Harnessing the CD38dim phenotype of cytokine-stimulated NK cells as a strategy to prevent fratricide.Cytotherapy. 2023; 25: 763-772https://doi.org/10.1016/j.jcyt.2023.03.006Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar whereas the study by Veliz et al.3Veliz K. Shen F. Shestova O. Shestov M. Shestov A. Sleiman S. Hansen T. O'Connor R.S. Gill S. Deletion of CD38 enhances CD19 chimeric antigen receptor T cell function.Mol. Ther. Oncol. 2024; 32200819https://doi.org/10.1016/j.omton.2024.200819Abstract Full Text Full Text PDF PubMed Google Scholar was the first study to link how CD38 enzymatic activity could enhance CAR T cell function. The study by Veliz et al.3Veliz K. Shen F. Shestova O. Shestov M. Shestov A. Sleiman S. Hansen T. O'Connor R.S. Gill S. Deletion of CD38 enhances CD19 chimeric antigen receptor T cell function.Mol. Ther. Oncol. 2024; 32200819https://doi.org/10.1016/j.omton.2024.200819Abstract Full Text Full Text PDF PubMed Google Scholar and other published studies on CD38 deletion are significant, as the immunosuppressive role of CD38 in T cells is supported by the presence of CD38 in dysfunctional and exhausted tumor-specific murine and human CD8+ T cells.11Verma V. Shrimali R.K. Ahmad S. Dai W. Wang H. Lu S. Nandre R. Gaur P. Lopez J. Sade-Feldman M. et al.PD-1 blockade in subprimed CD8 cells induces dysfunctional PD-1(+)CD38(hi) cells and anti-PD-1 resistance.Nat. Immunol. 2019; 20: 1231-1243https://doi.org/10.1038/s41590-019-0441-yCrossref PubMed Scopus (209) Google Scholar In addition, interestingly, the association of CD38 expression in patients with resistance to checkpoint inhibitors has been recently shown.12Chen L. Diao L. Yang Y. Yi X. Rodriguez B.L. Li Y. Villalobos P.A. Cascone T. Liu X. Tan L. et al.CD38-mediated immunosuppression as a mechanism of tumor cell escape from PD-1/PD-L1 blockade.Cancer Discov. 2018; 8: 1156-1175https://doi.org/10.1158/2159-8290.CD-17-1033Crossref PubMed Scopus (320) Google Scholar A clinical study, which consisted of twelve patients with relapsed B cell non-Hodgkin lymphomas who received CART19 and pembrolizumab to block PD1/PDL1, reported CD38 expression clustered with other markers of T cell exhaustion, including PD1, in the CAR-expressing T cells of nonresponders.13Chong E.A. Alanio C. Svoboda J. Nasta S.D. Landsburg D.J. Lacey S.F. Ruella M. Bhattacharyya S. Wherry E.J. Schuster S.J. Pembrolizumab for B-cell lymphomas relapsing after or refractory to CD19-directed CAR T-cell therapy.Blood. 2022; 139: 1026-1038https://doi.org/10.1182/blood.2021012634Crossref PubMed Scopus (87) Google Scholar The study by Veliz et al.3Veliz K. Shen F. Shestova O. Shestov M. Shestov A. Sleiman S. Hansen T. O'Connor R.S. Gill S. Deletion of CD38 enhances CD19 chimeric antigen receptor T cell function.Mol. Ther. Oncol. 2024; 32200819https://doi.org/10.1016/j.omton.2024.200819Abstract Full Text Full Text PDF PubMed Google Scholar did not directly address combination therapy that involves both CD38 KO and PD1 blockade. However, the authors suggested that targeting CD38 enzymatic activity in combination with PD1 blockade could offer significant benefits in enhancing CAR T cell function. We believe that this research has welcomed future perspectives on the potential of metabolic reprogramming of CAR T cells, either as a synergistic strategy to increase PD1-blockade effectiveness or as an alternative strategy to address the therapeutic need of patients who do not respond to PD1 blockade. P.S.A.'s laboratory work is supported by grants from the National Institutes of Health (P30 CA008748, R01 CA236615-01, R01 CA235667, UG3 CA290241, and U01 CA214195), the US Department of Defense (CA180889, and CA200437), the Comedy vs. Cancer Award, the DallePezze Foundation, the Derfner Foundation, the Esophageal Cancer Education Fund, the Memorial Sloan Kettering Technology Development Fund, the Miner Fund for Mesothelioma Research, Mr. William H. Goodwin and Mrs. Alice Goodwin, the Commonwealth Foundation for Cancer Research, and the Experimental Therapeutics Center of Memorial Sloan Kettering Cancer Center. P.S.A.'s laboratory received research support from ATARA Biotherapeutics and receives support from Novocure. V.R. and U.G. wrote the first draft and revision of the manuscript. P.S.A. conceived and revised the commentary draft. P.S.A. declares research funding from ATARA Biotherapeutics and Novocure; is a scientific advisory board member and consultant for ATARA Biotherapeutics, Bio4T2, Carisma Therapeutics, Orion Pharma, and Outpace Bio; and has patents, royalties, and intellectual property on mesothelin-targeted CAR and other T cell therapies, which were licensed to ATARA Biotherapeutics, issued a patent method for the detection of cancer cells using virus, and pending patent applications on the PD1 dominant-negative receptor, a wireless pulse-oximetry device, and an ex vivo malignant pleural effusion culture system. Memorial Sloan Kettering Cancer Center licensed intellectual property related to mesothelin-targeted CARs and T cell therapies to ATARA Biotherapeutics and had associated financial interests.
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