JNCI J Natl Cancer Inst (2022) 114(7): djac088 https://doi.org/10.1093/jnci/djac088 First published online April 19, 2022 Commentary Biological and Molecular Factors Predicting Response to Adoptive Cell Therapies in Cancer Gerardo Ferrer, PhD  ,1,2,3,‡ Damiana Alvarez-Errico, PhD,1,3,‡ Manel Esteller, MD, PhD 1,3,4,5, * Downloaded from https://academic.oup.com/jnci/article/114/7/930/6570594 by guest on 26 May 2023 1 Cancer and Leukemia Epigenetics and Biology Program (PEBCL), Josep Carreras Leukaemia Research Institute (IJC), Badalona, Catalonia, Spain; 2Karches Center for Oncology Research, Institute of Molecular Medicine, Feinstein Institutes for Medical Research, Northwell Health, Manhasset, NY, USA; 3Centro de Investigacio n Biome dica en Red Ca ncer (CIBERONC), Madrid, Madrid, Spain; 4Institucio  Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Catalonia, Spain; and 5 Physiological Sciences Department, School of Medicine and Health Sciences, University of Barcelona (UB), Barcelona, Catalonia, Spain COMMENTARY ‡ Authors contributed equally to this work. *Correspondence to: Manel Esteller, MD, PhD, Josep Carreras Leukaemia Research Institute (IJC), Carretera de Can Ruti, Camı de les Escoles s/n, 08916 Badalona, Barcelona, Catalonia, Spain (e-mail: mesteller@carrerasresearch.org). Abstract Adoptive cell therapy (ACT) constitutes a major breakthrough in cancer management that has expanded in the past years due to impressive results showing durable and even curative responses for some patients with hematological malignancies. ACT leverages antigen specificity and cytotoxic mechanisms of the immune system, particularly relying on the patient’s T lymphocytes to target and eliminate malignant cells. This personalized therapeutic approach exemplifies the success of the joint effort of basic, translational, and clinical researchers that has turned the patient’s immune system into a great ally in the search for a cancer cure. ACTs are constantly improving to reach a maximum beneficial clinical response. Despite being very promising therapeutic options for certain types of cancers, mainly melanoma and hematological malignancies, these individualized treatments still present several shortcomings, including elevated costs, technical challenges, management of adverse side effects, and a limited population of responder patients. Thus, it is crucial to discover and develop reliable and robust biomarkers to specifically and sensitively pinpoint the patients that will benefit the most from ACT as well as those at higher risk of developing potentially serious toxicities. Although unique readouts of infused cell therapy success have not yet been identified, certain characteristics from the adoptive cells, the tumor, and/or the tumor microenvironment have been recognized to predict patients’ outcome on ACT. Here, we comment on the importance of biomarkers to predict ACT chances of success to maximize efficacy of treatments and increase patients’ survival. Immunotherapy has revolutionized cancer management in the (2,3). In this respect, reliable predictors of response or resistance past years, improving patients’ survival rates and reducing the as well as treatment monitoring markers remain a crucial need risk of recurrence for many cancer types. Immunotherapy to stratify patients and maximize clinical success. Predictive approaches, including the use of immunomodulatory agents to markers include biological markers as well as molecular, (epi)- enhance anticancer responses, started with the use of high- genomic, and transcriptomic signatures that allow rational dose synthetic IL-2 (aldesleukin) for the treatment of melanoma therapeutic decision making and enhance the progress of preci- and renal carcinoma in the early 1990s (1). To date, immuno- sion medicine. Multiomics technologies are becoming a major therapy has expanded to different kinds of treatments, includ- player in the search of novel biomarkers of response to immu- ing the use of monoclonal antibodies, immune checkpoint notherapy (2). A good example of molecular signatures that pre- inhibitors, vaccines against tumoral antigens, and cell-based dict the chances of success to immunotherapy is the presence therapies, all of which aim to leverage and boost the patient’s of the DNA methylation signature known as EPIMMUNE in met- immune response to fight malignant cells and prevent cancer astatic non-small cell lung cancer patients. EPIMMUNE associ- progression. However, despite impressive clinical results, many ates with good response and improved progression-free patients still cannot fully benefit from immunotherapies due to survival on treatment with immune checkpoint inhibitors (4). a lack of response to treatment, relapse, and/or development of These DNA methylation profiles that are able to predict clinical resistance, or life-threatening therapy-associated toxicities response to immunotherapy have also been extended to Received: November 23, 2021; Revised: February 8, 2022; Accepted: April 12, 2022 © The Author(s) 2022. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com 930 G. Ferrer et al. | 931 melanoma patients (5). In addition, recent development of so- inflammation and prevent effective antitumor responses (16). phisticated computational methods allows the inference of T- Nonetheless, certain T cells that infiltrate tumors (TILs) and rec- cell infiltration into tumor samples from whole exome sequenc- ognize cancer-specific antigens can effectively kill the malig- ing data, which can predict the response to immune checkpoint nant cells and reduce cancer growth. Exploiting the cytotoxic inhibitors independently of tumor mutational burden (6). potential of these tumor infiltrating memory-like CD8þT cells Finally, the advancement of single-cell technologies also holds by isolating them, expanding them ex vivo in the presence of great potential to refine the search for predictors of treatment growing factors like IL-2, and adoptively transferring them back response, personalize patients’ management, and increase the into the patient to prevent cancer progression constitutes a responders to nonresponders’ ratio (7-9). promising therapeutic option for solid tumors such as mela- noma and some epithelial cancers (14). TIL-based therapies were pioneered by the Rosenberg team at the Surgery Branch in T Cells in Cancer Therapy the National Cancer Institute, who first demonstrated that tu- mor-bearing mouse TILs cultured ex vivo showed in vivo cyto- T cells are central players of vertebrates’ adaptive immune sys- lytic activity against tumor cells after reinjection (14) and later tem and mediate immune defense against viral infections and confirmed those findings in cancer patients (17). A major deter- cancer (10). T cells are equipped with a specific receptor that Downloaded from https://academic.oup.com/jnci/article/114/7/930/6570594 by guest on 26 May 2023 minant of TIL therapy success is the presence of cells that rec- recognizes foreign peptides in the context of HLA molecules on ognize neoantigens expressed by tumor cells (18,19). In this the surface of antigen presenting cells and activates effector regard, a meta-analysis of several cancer cohorts’ datasets sup- programs to defend against infection and abnormal cells. The ports that tumor-specific CD8þ TILs are represented not only by T-cell receptor (TCR) is a heterodimeric complex composed of 2 the exhausted CD8þ T cells and their precursors (expressing COMMENTARY protein chains (usually a and b chains) that have an extracellu- thymocyte selection-associated HMG BOX (TOX), and TOX and lar domain that binds to the peptide-HLA complex. The intracel- transcription factor 7 (TCF7), respectively) but also by effector lular signaling module of the TCR is composed of the c, d, e, and memory-like T cells with high cytotoxic capacity that could be f subunits from 2 CD3 chains that transduce activating signals isolated and used in TIL-based therapeutic protocols (20). on engagement of the TCR, orchestrating specific transcrip- tional programs (11,12). The antitumor T-cell immunity results from the combined action of CD4þ helper T cells and the CD8þ T-Cell Engineered Receptor cytotoxic cells, where specific transcriptional programs shape different stages of cell differentiation into distinct functional Despite CD8þ T cells having great cytotoxic capacity, the sup- subsets that can be categorized as naı̈ve T cell, stem cell mem- pressive TME often dampens such response, constituting a ory T cell, central memory T cell, memory effector T cell, and ef- mechanism for tumor immune escape (16,21). An important fector T cell (13). breakthrough in cancer immunotherapy was the application of Adoptive cell therapy (ACT) accounts for different immuno- genetic modification of normal peripheral lymphocytes from therapy modalities based on the redirection of cytotoxic T cells cancer patients to express TCR that recognizes specific antigen- from the patient towards an efficient antitumor response. expressing tumor cells with high affinity. This method over- Therapies can be divided into those with no genetic manipula- comes the need to identify and isolate antitumor effector T cells tion of the T lymphocytes present in the tumor as tumor infil- specifically recognizing tumor-associated antigens (TAAs) from trating leucocytes (TILs) (14), and those that include the genetic each patient. In the case of solid tumors, TCRs specific for the manipulation and reprogramming of patients’ T cells to potenti- melanoma antigens melanoma antigen recognized by T cells 1 ate and direct killing activity towards the tumor. In the case of or MART-1 and glycoprotein 100 (gp100) were retrovirally trans- the latter, these are T cells expressing chimeric antigen receptor duced into melanoma patients’ peripheral blood lymphocytes, (CAR-T cells) and engineered TCRs that specifically recognize successfully promoting cancer regression (22). tumor antigens (15). In all ACT protocols, T cells are harvested from the patient (either from the tumor or from circulation), ex- panded ex vivo, and reinjected in the patient (Figure 1). Such T- Chimeric Antigen Receptor T Cells lymphocyte–based treatments hold curative potential and have CAR-T cell therapy has shown great efficacy in the treatment of provided promising clinical efficacy results for certain cancers; hematological cancers, with improved clinical responses since as such, this highly evolving field with increasing therapies is the development of first-generation CAR-T cells to the therapies being evaluated and approved at a high pace, either as stand- approved so far, which are the second-generation CAR-Ts alone therapies or in combination with other drugs (2). In this (23,24). In addition, many combinations are being investigated regard, Supplementary Table 1 (available online) summarizes for the successful treatment of solid tumors, which has been the different neoplasms that have been treated with ACT, elusive to date (25). To produce CARs, a synthetic fusion protein whereas Table 1 indicates the US Food and Drug Administration consisting of 2 different functional modules connected by a (FDA)-approved therapies. In the following subsections, we will transmembrane domain is generated and transduced via a viral discuss the most common ACT approaches: TILs, TCRs, and vector (most frequently retrovirus or lentivirus) into autologous CAR-T cells. or allogeneic T cells that, on expansion, are transfused into the patient to engraft in vivo and target malignant cells (26). First- generation CARs consist of a single-chain fragment variable re- Tumor Infiltrating Lymphocytes gion of antibodies extracellular domain that recognizes tumoral A hallmark of cancer is the presence of a complex immunosup- antigens and is connected by a linker to a transmembrane do- pressive tumor microenvironment (TME) characterized by cells main with a cytoplasmic signaling module from the CD3f chain. and factors that promote immune evasion by malignant cells. In the case of second-generation CAR-Ts, the CAR contains an Tumors recruit T regulatory cells, tumor-associated macro- additional costimulatory domain (from CD28 or 4-1BB) that phages, and myeloid derived suppressor cells that dampen transduces the activating signal to the T cells. To date, 932 | JNCI J Natl Cancer Inst, 2022, Vol. 114, No. 7 Downloaded from https://academic.oup.com/jnci/article/114/7/930/6570594 by guest on 26 May 2023 COMMENTARY Figure 1. Adoptive cell therapy generation strategies. Tumor infiltrating leucocytes (TILs) are extracted from the tumor tissue, selected, and expanded in vitro. On ex- pansion, TILs are reintroduced in the patient to redirect the antitumor response. T cells from the patient’s circulation are selected and transduced with either an engi- neered T-cell receptor (TCR) or a chimeric antigen receptor (CAR). Cells are then expanded in vitro and reinfused into the patient. autologous CAR-T cell therapy targeting CD19, and B-cell produced and infused in many patients. As an example, to treat maturation antigen have been approved by the FDA for 6 types B-cell malignancies, healthy donors’ T cells have been engi- of relapsed or refractory hematological cancers —diffuse large neered, using the Sleeping Beauty system to express an anti- B-cell lymphoma, follicular lymphoma, mantle cell CD19 CAR, at the time that endogenous TCR has been geneti- lymphoma, multiple myeloma, and B-cell acute lymphoblastic cally silenced to abolish MHC-dependent TCR activation, thus leukemia (ALL)—in pediatric and young adult patients up to age preventing graft-vs-host-disease (36). 25 years (23). As recently as October 2021, another anti-CD19 In addition to T cells, other immune effector cells (IECs) are (CAR) T-cell therapy (brexucabtagene autoleucel) named KTE- being engineered as alternative strategies to be used in adoptive X19 was approved by the FDA for the treatment of relapsed or cell therapy. Natural killer (NK) cells are well-suited with a cyto- refractory B-cell precursor ALL in adults, indicating a long-term toxic machinery to fight against tumoral cells that downregu- beneficial outcome for these patients (27). late HLA and are amenable of in vitro expansion and allogenic use. NK cells also show less potential of serious side effects, which makes them an excellent candidate for treatment design. Therapy Limitations of ACT A phase 1 and 2 clinical trial using manufactured allogenic anti- Despite the clinical benefit obtained from the different ACT CD19 CAR-NK cells in chronic lymphocytic leukemia (CLL) and approaches described above, some of them show short-lived non-Hodgkin’s lymphoma (NHL) patients revealed no associa- remissions and develop resistance to treatment due to several tion to cytokine release syndrome (CRS) or severe toxicities on factors, highlighting the importance of patients’ stratification infusion and clinical responses in 70% of patients, which could according to their risk of resistance for the rational use of these potentially make this strategy optimal for those patients at risk expensive and time-consuming therapies. Patients relapsing on of serious adverse reactions to CAR-T cell therapy (37). CAR-T cell infusion has been reported to be due to either early Other IECs being leveraged for adoptive cell immunotherapy poor CAR-T cell persistence leading to antigen-positive malig- are cells from the myeloid compartment in particular macro- nant cell expansion (28-30) or late resistance due to antigen re- phages, which are promising candidates for the treatment of duction or loss (31-33). solid tumors, given their tumor-mediated recruitment. Another major obstacle precluding the generalized use of Adenovirus transduction of an anti-HER2 CAR into human mac- immunotherapy, and particularly of ACT, in cancer patients is rophages and subsequent in vitro expansion yielded proinflam- the appearance of treatment-related severe adverse effects that matory M1-type macrophages that, on intravenous injection in are potentially life-threatening (34,35). mice, were recruited to the tumor in a xenograft model of ovar- In line with this, efforts are being made to generate less ian cancer showing in vivo antitumor activity that reduced tu- costly, allogenic universal off-the-shelf CAR-T cells that can be mor burden (38). Of potential interest in the clinic, induced G. Ferrer et al. | 933 Table 1. ACTs FDA-approved therapiesa Type of therapy Name Trademark Indications Year of first FDA approval TILs ITIL-168 N/A Adult melanoma stages IIB-IV Orphan drug designation 2021 TCR Tebentafusp-tebn Kimmtrak Adult unresectable or uveal metastatic 2022 melanoma CAR-T Tisagenlecleucel Kymriah Pediatric and young adults ALL 2017 CAR-T Axicabtagene ciloleucel YESCARTA r/r LBCL 2017 DLBCL CAR-T Brexucabtagene autoleucel TECARTUS Adult r MCL 2020 Adult r/r B-cell precursor ALL CAR-T Idecabtagene vicleucel ABECMA Multiple myeloma 2021 CAR-T Lisocabtagene maraleucel BREYANZI r/r LBCL 2021 DLBCL Downloaded from https://academic.oup.com/jnci/article/114/7/930/6570594 by guest on 26 May 2023 a ACT ¼ adoptive cell therapy; ALL ¼ acute lymphoblastic leukemia; CAR-T ¼ T cells expressing chimeric antigen receptor; r/r ¼ relapse or refractory; LBCL ¼ large B-cell lymphoma; DLBCL ¼ diffuse large B-cell lymphoma; FDA ¼ Food and Drug Administration; MCL ¼ mantle cell lymphoma; N/A ¼ not applicable; TCR ¼ T-cell receptor; TIL ¼ tumor infiltrating leucocyte. COMMENTARY pluripotent stem cells (iPSCs)-derived macrophages expressing success. In addition, a better performance in most solid tumors, CARs have shown promising in vitro and in vivo antitumor ac- in which an immunosuppressive TME dampens effective anti- tivity in preclinical studies but still need further investigation tumor immunity, remains an important challenge for this type (39). of immunotherapy. It is therefore paramount to find robust and There is currently great interest in both academia and indus- reliable biomarkers to establish not only which patients are try in the development of new strategies and uses, such as the more likely to benefit from ACT, but also which ones are at risk evaluation of CAR cells in addition to the CD19 antigen. of developing serious adverse effects to rationalize the use of Moreover, as learned from viral infections, the evaluation of the these costly custom-made therapeutic strategies. optimal ratio of monotypic to polytypic effector cells is optimal There are no clear factors dictating whether a particular in- for therapeutic success in each type of tumor (40). In fused cell therapy is going to be successful. Most of the data Supplementary Table 1 (available online), we show the targets reported correspond to TILs and, in the last few years, to CAR- presently under investigation with more than 500 clinical trials, Ts, but little has been reported on engineered TCRs, although opening this technology beyond cancer treatment and T cells common features may be shared by all T-cell therapies. Due to (38,41-48). Those targets represent neoantigens, TAAs, and the limited studies and heterogeneity of cellular products, it is other targetable antigens such as viral particles. Neoantigens difficult to identify a universal predictive set of biomarkers. The are tumor-specific antigens induced by a mutation or genetic al- main areas explored for the identification of biomarkers corre- teration; thus, tumors with higher tumoral load also present a spond to adoptive transferred cells’ intrinsic factors, the tumor- higher number of neoantigens. Due to HLA restrictions, only specific characteristics, and the microenvironment (Figure 2). A some of those might be immunogenic and can favor the expan- summary of biomarkers of ACT response, persistence, and tox- sion of TILs and therefore can be applied to vaccines, TCRs, and icity is shown in Table 2, and its value for TILs and CAR-T cells CARs, but their patient specificity limits broader application. is discussed in the following subheadings. Unlike neoantigens, TAAs are not restricted to tumor tissue be- cause they are also present in other normal cells, but their ex- pression in an “expendable” population like CD19 in B cells, or Biomarkers of TILs Therapies their higher expression in the tumor compared with normal cells, poise them as good targets, especially because they can be Unlike other types of cancer therapies, ACT is generated from a applied to a broader number of patients, providing attainable pool of heterogeneous T-cell subtypes at diverse ratios and management of toxicities. As mentioned, the target choice will stages of differentiation. Although the phenotype(s) and ratios have a great implication in therapy design because it will deter- responsible for clinical success are not completely clear, reports mine different technology and conditionings (49-51). To over- suggest that the presence of a stem-like fraction, a bias towards come current limitations, several new strategies have been CD8þ T cells with the company of CD4þ T lymphocytes, and the developed, including optimal expansion procedures, the use of presence of functional markers are indicators of more effective bispecific molecules, modifications in the recognition fragment, antitumor response in vivo. logistic strategies, modifications in the signaling section, knock- From the early 1990s, it was noted that shortened T-cell cul- in or knock-out of several molecules including endogenous ture periods were associated with a better response to TILs TCR, and combination with checkpoint inhibitors, immunother- (58). Shorter culture times were found to correlate with telo- apy, and other therapies (45,52-57). mere length provided that these regions got shorter in every cycle of cell division and have downstream influences on cel- lular function and antitumoral activity (59,60). TIL products that are rich in naı̈ve T cells, memory T cells, and stem cell Biomarkers in ACT memory T cells exhibit stronger antitumor responses and ACTs are still very costly and require substantial cellular manip- long-term persistence in vivo, with higher capacity for self-re- ulation, so it is essential to differentiate patients to identify po- newal and expansion (9). A higher proportion of CD8þ T cells tential responders and maximize the chances of therapeutic within the TILs population has been shown to correlate with a 934 | JNCI J Natl Cancer Inst, 2022, Vol. 114, No. 7 Downloaded from https://academic.oup.com/jnci/article/114/7/930/6570594 by guest on 26 May 2023 COMMENTARY Figure 2. Adoptive cell therapy outcome biomarkers. Several characteristics of adoptive transfer (AT) cells, the tumor, and tumor microenvironment depicted in the fig- ure have been linked with patient outcomes. These include adoptive transferred cell maturation, the presence of both CD8þ and CD4þ, and the level of exhaustion and genomic, epigenetic, and transcriptomic markers. In the tumor, the specificity, amount, and dependence of the antigen are very important to induce a strong dura- ble response, as are the tumor and the microenvironment suppressive strength. TN ¼ naı̈ve T cells; TSCM ¼ stem memory T cells; TCM ¼ central memory T cells; TEM ¼ effector memory T cells; TEF ¼ terminal effector T cells; T-regs ¼ regulatory T cells; MDSCs ¼ myeloid derived suppressor cells. better response to treatment (58), probably due to higher Biomarkers of CAR-T Therapies in vitro killing activity shown by those cells. CD4þ T cells, known as helper T cells, promote higher CD8þ T-cell activity Given their common origin, a number of biomarkers are shared and are now recognized as crucial for improving the response by CAR-T cells and TILs. For instance, the therapeutic efficacy of CD8þ cytotoxic TILs (61,62). Moreover, single-cell gene ex- and in vivo persistence of CAR-T cells statistically significantly pression of long-lasting TILs allowed identification of a profile correlate with their differentiation stage. CAR-T cell products enriched in surface markers and transcription factors (IL7R, that are rich in stem-like cells mediate more efficacious antitu- ITGB1, KLF2, and ZNF683) (7). mor responses and improve patients’ outcomes (65,66). In addition to TIL composition, there are biomarkers related However, infusion of CD8þ anti-CD19 CAR-T cells alone is suffi- to the tumor and its microenvironment that can also determine cient for long-term B-cell eradication in lymphoma patients the chances of therapeutic success. Currently, TIL therapy is (67). CD4þ T cells are also shown to be crucial in CAR-T cells only applied to immunogenic tumors, where the higher the ca- (68-70). CD4þ CAR-T cell dynamics showed initial slower tumor pacity of achieving antigenic-mediated activation, the higher killing and a reduced granzyme B secretion but presented better chance of tumor killing by the T cells. In TIL and TCR cell thera- persistence following antigen exposure compared with CD8þ pies, one of the main predictors of response remains the specif- counterparts (68-70). Most current approaches use CAR-T for- icity of the TCR against tumor antigens, especially against neo- mulations that include a heterogeneous random composition of epitopes. Sequence variation such as mutations, frameshifting CD4þ and CD8þ cells. A defined composition of CD4 to CD8 insertions or deletions, and tumor-specific alternative splicing CAR-T cells in a 1:1 ratio augments antitumor activity in vivo have been found to correlate with a successful immunotherapy and supports the synergism between both subsets (65,71). T-cell response (63,64). TILs are effective for patients with melanoma, biology complexity is worth considering; CD4þ T cells can be who usually present numerous neo-epitopes, whereas in further subdivided into Th subsets, and the expansion of Th1 tumors with low average mutational load, an optimization of cells expressing T-bet and Th9 cells during CAR-T cells TILs cultures to enrich T cells that recognize neo-epitopes is re- manufacturing has a positive impact on the antitumor response quired to potentiate tumor killing (63). (72,73). G. Ferrer et al. | 935 Table 2. TILs and CAR-T cells biomarkersa ACT Origin Biomarker Output References TILs TILs T cells stemness and memory Response, persistence (9, 58-60) CD8þ and CD4þ T cells Response, persistence (58, 61, 62) Surface markers and transcrip- Response, persistence (7) tomic phenotype Tumor and TME Abundance of neoantigens Response, persistence (63, 64) Suppressive TME Response, persistence (63) CAR-Ts CAR-Ts T cells stemness and memory Response, persistence (65, 66) phenotype CD8þ and CD4þ T cells Response, persistence (65, 67-73) CAR design Response, persistence, toxicity (23, 43, 88, 94, 95, 116) Cytokine production Response, persistence, toxicity (88, 93-102, 106, 108-110, 117) Tumor infiltration Response, persistence (88, 99, 101) Downloaded from https://academic.oup.com/jnci/article/114/7/930/6570594 by guest on 26 May 2023 Epigenetics (signatures) Response (79) Surface markers and transcrip- Response, persistence (52, 77, 78) tomic phenotype CAR integration site Response, persistence (30, 80) CAR methylation Response, persistence (81) COMMENTARY Tumor and TME Tumor load Response, persistence (82, 83) Antigen escape Response, persistence (32, 84) Suppressive TME Response, persistence (52, 87-91) Immunological clearance Response, persistence (102) Inflammatory cytokines Response, persistence, toxicity (93, 111-113, 117) a CAR-T ¼ T cells expressing chimeric antigen receptor; TIL ¼ tumor infiltrating leucocyte; TME ¼ tumor microenvironment. During in vitro expansion and after therapeutic infusion, re- follow-up study in adult ALL patients receiving CD19 CAR ther- peated antigen stimulation can induce CAR-T cells to enter an apy showed that high tumor burden correlates with relapse (82). exhaustion state (74), which is characterized by poor self-re- An article describing opposite risk factors for CD19-negative vs newal, expansions, and effector function; sustained expression CD19-positive relapse on anti-CD19 CAR-T treatment of pediat- of inhibitory receptors (eg, PD1, TIM-3 and LAG3); and epigenetic ric relapsed or refractory BCP-ALL has identified high tumor and gene expression alterations (75,76). Higher levels of the ex- burden as the major factor that correlates to increased risk of haustion markers PD1, TIM-3, and LAG3 at the moment of aphe- CD19 negative relapses in the presence of increased CAR-T cell resis, preinfusion, and postinfusion have been reported in persistence. On the other hand, low tumor burden was found to patients whose CAR-T cells persist for shorter periods of time be associated with decreased persistence and risk of CD19- and fail to respond to treatment (52,77,78). positive relapses (83). In addition, downregulation or loss of the At the molecular level, in addition to cell surface markers, targeted antigen is associated with reduced or abrogated re- some studies have identified indicators of response associated sponse to treatment. This evasion mechanism occurs via 2 dis- with CAR-T cell gene expression, CAR integration site, and its tinct pathways: antigen escape (84), where there is a lack of epigenetic regulation. Recently, our group described a DNA surface expression of the antigen, or lineage switch (80), where methylation signature in CAR-T cells that we named EPICART the patient relapses with a phenotypically different malignancy (79). EPICART, like a previously described gene expression signa- (32). To avoid immune elimination, cancer cells can present 1 or ture (77), associates good outcome with a signature enriched in several immune evasion mechanisms. Tumor cells can express naı̈ve-like or early memory cell populations, thus reinforcing inhibitory ligands such as PD-L1 and release immune antiin- the importance of the presence of these subsets at preinfusion flammatory cytokines, including arginase, transforming growth to determine overall response. CAR integrates in the genome, factor beta (TGF-b), indoleamine 2,3-dioxygenase (IDO), and IL- thereby marking cell lineages and modifying the cellular ge- 4, as well as orchestrate and sustain an inhibitory TME contain- nome by insertional mutagenesis. Interestingly, it has been ing T regulatory cells and myeloid derived suppressor cells shown that vector integration into the TET2 locus and subse- (16,85,86). All these immunomodulatory mechanisms diminish quent clonal expansion is associated with clinical success (30). CAR-T efficacy by reducing their proliferation, cytotoxicity, and Consequently, a wider study demonstrated that therapy res- persistence (52,87-91). ponders presented integration sites enriched in genes coding for cell-signaling and chromatin modifiers, suggesting that in- sertional mutagenesis into these pathways promoted therapeu- Persistence of CAR-T Therapy tic T-cell proliferation and preinfusion products able to support a better outcome (80). Finally, retroviral vectors encoding foreign Persistence remains an important impediment for the develop- genetic material have been shown to be prone to progressive ac- ment of effective CAR-T therapies in cancer and clearly predicts quisition of DNA methylation, leading to subsequent epigenetic the effectiveness of the treatment because long-lasting CAR-T silencing of the CAR, which abrogates CAR-T cell function (81). cells aim for the complete eradication and avoid tumor relapse. Regarding predictors of CAR-T therapies related to the tumor However, in some cases, transient rather than prolonged persis- characteristics and its microenvironment, an earlier long-term tence is desirable. Such is the case for the use of in vivo– 936 | JNCI J Natl Cancer Inst, 2022, Vol. 114, No. 7 generated CAR-T cells to eliminate fibrosis heart injuries that blood-brain barrier permeability have been suggested to play peak at 24 hours, post delivering modified mRNA in T cell–tar- key roles in the unfolding of ICANS, but recent results from a geted lipid nanoparticles and rapidly dropping over the follow- single-cell study identified mural cells that surround the endo- ing days, where transient presence of CAR-Ts is required to thelium and are critical to the integrity of the blood-brain bar- avoid affecting the normal injury-healing process (43). Several rier, express CD19, and present an off-target for CAR19 factors affect the persistence of CAR-T cells in patients, includ- strategies (116). The incidence of ICANS was associated with a ing activation-induced cell death, peripheral tolerance, periph- high pretreatment disease burden, a rate of CAR-T cell expan- eral ignorance, and immunological clearance (92). Some of sion, higher levels of proinflammatory cytokines, and endothe- these issues are being addressed by engineering better CARs, lial markers that can be used as predictors and for personalized but as mentioned above, the complex composition of the prod- monitoring (93,117). ucts being used is especially important, and T-cell stemness and T-cell exhaustion are key markers of persistence (65,66,69,93). On infusion, the lack of increase in inflammatory Conclusions and Perspectives markers such as interferon gamma (IFN-c), IL-6, and IL-10 and The refinement of related protocols to IECs in cancer manage- the absence of tumor infiltration or the elimination of the CAR- ment has opened up an unprecedented opportunity to reach Downloaded from https://academic.oup.com/jnci/article/114/7/930/6570594 by guest on 26 May 2023 T cells by the immune system have been identified to be poor therapeutic success through tailoring of the patients’ immune markers of persistence and response (88,94-102). cell response to therapies that fight the tumor. Such highly per- sonalized therapies are costly and labor intensive and are not exempt from generating potentially life-threatening adverse COMMENTARY CAR-T Adverse Side Effects and Toxicity side effects. This risk underscores the need for reliable and ro- bust predictive and prospective biomarkers of response that in- Toxicities or adverse side effects associated with CAR-T cell form of individual characteristics, enabling rational decision therapies can be severe and even life threatening if not man- making in therapeutic options to expand the responder popula- aged properly, and are less common and milder in TIL and TCR tion and increase survival. In addition, further research is regimes. For this reason, there has been a great interest in un- needed to discover novel biomarkers in specific T-cell products derstanding the mechanisms underlying these adverse effects to improve efficacy and safety through the identification of and identifying predictors and markers for their early detection patients who are greater risk of developing severe toxicities re- in CAR-T cell treatment. The main described toxicities include lated to treatment. CRS, immune effector cell associated neurotoxicity syndrome Off-the-shelf modalities have started to emerge, to elimi- (ICANS) and hematologic toxicities and infections, with CRS and nate the waiting period for patients to receive the treatment, ICANS being the most frequent. CRS is a major complication of normalize therapeutic agents, reduce costs, and allow these CAR-T cell therapy and is characterized by systemic inflamma- therapeutic options to reach a broader spectrum of individuals. tion (34). The symptoms of CRS vary depending on the severity Solid tumors also represent an unmet need because they, un- and range from mild to serious illness, the latter presenting like in hematological malignancies, do not express an ideal high fever, hypotension, shock, disseminated intravascular co- uniform target for adoptive cell therapy, increasing the risk of agulation (usually in the absence of bleeding), and even multi- on-target off-tumor toxicity (118). Finding predictive markers ple organ dysfunction (103-105). CRS generally develops in that inform of the risk of an unwanted immunological re- response to the engagement of the CARs by its specific antigens, sponse in each case remains a major challenge. In addition, tu- which subsequently stimulate other immune and nonimmune mor heterogeneity, mainly due to genomic instability and the cells (106,107). The IL-6, IL-10, and IFN-ccytokines are the stron- accumulation of mutations, in solid tumors makes it difficult gest contributors to CRS development, and IL-6 is a core cyto- to select the best TAA to target in each patient (119). In that re- kine in CRS pathophysiology (106,108-110). Cytokine responses spect, the target selection for the rational design of immuno- generated by CAR-T and bystander macrophage activation can therapeutic products deserves further investigation to lead to Macrophage Activation Syndrome that contributes to pinpoint targets that may warrant clinical success as well as therapy-derived immunotoxicity (111). A murine xenogeneic approaches to surpass the immunosuppressive TME. Clinical model of ALL with human 1928z CAR-T cell infusion was used breakthroughs in the biomarkers arena will likely come from to interrogate the role of different cell types in the production of currently used multiomics technologies and the analytical inflammatory cytokines such as IL6 and IL1b that drive acute computational tools that are constantly being advanced. Such CRS identified macrophages as the main contributors to the biomarkers will also contribute to the optimization of the pro- pathogenesis of CRS (112). In this line, INF-c produced on CAR-T duction process to generate affordable and scalable therapies cell activation has been shown to activate macrophages trigger- that reduce the risk of relapse and the appearance of resis- ing a CRS-like cytokine release, and IFN blockade abolished tance, coming closer to the ultimate goal of achieving curative macrophage-mediated cytokine production, diminishing cyto- responses. kine-derived toxicities, increasing CAR-T persistence, and im- proving overall antitumor response (111). Fever and high monocyte chemoattractant protein 1 (MCP-1) levels within Funding 36 hours after the infusion of CAR-T cells are considered to be predictors of severe CRS and ICANS with the best sensitivity GF is recipient of Marie Skłodowska-Curie Action individual and specificity (113). fellowship (H2020-MSCA-IF-2019 – 896403). We thank CERCA ICANS is another primary adverse event during CAR-T cell Programme/Generalitat de Catalunya for institutional sup- therapy and is characterized by encephalopathy, aphasia, delir- port. Work at ME laboratory is supported by the Health ium, seizures, and tremors (114,115). The mechanism underly- Department PERIS—project no. SLT/002/16/00374 and ing the development of ICANS is not fully understood. The AGAUR—project no. 2017SGR1080 of the Catalan massive release of inflammatory cytokines and changes in Government (Generalitat de Catalunya); Ministerio de G. Ferrer et al. | 937  n (MCI), Agencia Estatal de Investigacio Ciencia e Innovacio n 15. Hinrichs CS, Rosenberg SA. Exploiting the curative potential of adoptive T- cell therapy for cancer. Immunol Rev. 2014;257(1):56–71. doi: (AEI) and European Regional Development Fund (ERDF) proj- 10.1111/imr.12132. ect no. RTI2018-094049-B-I00; the Cellex Foundation; and “la 16. Labani-Motlagh A, Ashja-Mahdavi M, Loskog A. The tumor microenviron- Caixa” Banking Foundation (LCF/PR/GN18/51140001). ment: a milieu hindering and obstructing antitumor immune responses. Front Immunol. 2020;11:940.doi:10.3389/FIMMU.2020.00940/BIBTEX. 17. Muul LM, Spiess PJ, Director EP, Rosenberg SA. Identification of specific cyto- lytic immune responses against autologous tumor in humans bearing ma- Notes lignant melanoma. J Immunol. 1987;138(3):989–995. 18. Robbins PF, Lu YC, El-Gamil M, et al. Mining exomic sequencing data to Role of the funder: The funder had no role in the design, inter- identify mutated antigens recognized by adoptively transferred tumor-reac- pretation, writing or submission of the manuscript. tive T cells. Nat Med. 2013;19(6):747–752. doi:10.1038/nm.3161. 19. Tran E, Turcotte S, Gros A, et al. Cancer immunotherapy based on muta- Disclosures: ME is a consultant of Ferrer International and tion-specific CD4þ T cells in a patient with epithelial cancer. Science. 2014; 344(6184):641–645. doi:10.1126/science.1251102. Quimatryx. The other authors declare that they have no conflict 20. Andreatta M, Corria-Osorio J, Müller S, Cubas R, Coukos G, Carmona SJ. of interest. Interpretation of T cell states from single-cell transcriptomics data using reference atlases. Nat Commun. 2021;12(1):1–19. doi: Author contributions: Conceptualization: All authors. 10.1038/s41467-021-23324-4. 21. Silk JD, Abbott RJM, Adams KJ, et al. Engineering cancer antigen-specific T Downloaded from https://academic.oup.com/jnci/article/114/7/930/6570594 by guest on 26 May 2023 Supervision: ME. Writing—original draft: All authors. Writing— cells to overcome the immunosuppressive effects of TGF-b. J Immunol. 2022; review and editing: All authors. 208(1):169–180. doi:10.4049/JIMMUNOL.2001357/-/DCSUPPLEMENTAL. 22. Morgan RA, Dudley ME, Wunderlich JR, et al. Cancer regression in patients Acknowledgements: Figures have been created using Biorender after transfer of genetically engineered lymphocytes. Science. 2006; (https://biorender.com/). 314(5796):126–129. doi:10.1126/science.1129003. COMMENTARY 23. Irving M, Lanitis E, Migliorini D, Ivics Z, Guedan S. Choosing the right tool for genetic engineering: clinical lessons from chimeric antigen receptor-T cells. Hum Gene Ther. 2021;32:19–20. doi:10.1089/hum.2021.173. Data Availability 24. Sadelain M, Brentjens R, Rivière I. The promise and potential pitfalls of chi- meric antigen receptors. Curr Opin Immunol. 2009;21(2):215–223. doi: The data underlying this article are available in the article. 10.1016/j.coi.2009.02.009. 25. Lanitis E, Coukos G, Irving M. All systems go: converging synthetic biology and combinatorial treatment for CAR-T cell therapy. Curr Opin Biotechnol. References 2020;65:75–87. doi:10.1016/j.copbio.2020.01.009. 26. Zhang H, Zhao P, Huang H. Engineering better chimeric antigen receptor T 1. Buter J, Sleijfer DT, van der Graaf WT, de Vries EG, Willemse PH, Mulder NH. cells. Exp Hematol Oncol. 2020;9(1):1–13. doi:10.1186/S40164-020-00190-2. A progress report on the outpatient treatment of patients with advanced re- 27. Shah BD, Ghobadi A, Oluwole OO, et al. KTE-X19 for relapsed or refractory nal cell carcinoma using subcutaneous recombinant interleukin-2. Semin adult B-cell acute lymphoblastic leukaemia: phase 2 results of the single- Oncol. 1993;20(6, suppl 9):16–21.  arm, open-label, multicentre ZUMA-3 study. Lancet. 2021;398(10299): 2. Villanueva L, Alvarez-Errico D, Esteller M. The contribution of epigenetics to 491–502. Doi:10.1016/S0140-6736(21)01222-8. cancer immunotherapy. Trends Immunol. 2020;41(8):676–691. doi: 28. Maude SL, Laetsch TW, Buechner J, et al. Tisagenlecleucel in children and 10.1016/j.it.2020.06.002. young adults with B-cell lymphoblastic leukemia. N Engl J Med. 2018;378(5): 3. Lebrec H, Maier CC, Maki K, Ponce R, Shenton J, Green S. Nonclinical safety 439–448. doi:10.1056/NEJMoa1709866. assessment of engineered T cell therapies. Regul Toxicol Pharmacol. 2021;127: 29. Lee DW, Kochenderfer JN, Stetler-Stevenson M, et al. T cells expressing 105064.doi:10.1016/J.YRTPH.2021.105064. CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in chil- 4. Duruisseaux M, Martınez-Card us A, Calleja-Cervantes ME, et al. Epigenetic dren and young adults: a phase 1 dose-escalation trial. Lancet. 2015; prediction of response to anti-PD-1 treatment in non-small-cell lung cancer: 385(9967):517–528. doi:10.1016/S0140-6736(14)61403-3. a multicentre, retrospective analysis. Lancet Respir Med. 2018;6(10):771–781. 30. Fraietta JA, Nobles CL, Sammons MA, et al. Disruption of TET2 promotes the doi:10.1016/S2213-2600(18)30284-4. therapeutic efficacy of CD19-targeted T cells. Nature 2018;558(7709):307–312. 5. Jung H, Kim HS, Kim JY, et al. DNA methylation loss promotes immune eva- doi:10.1038/s41586-018-0178-z. sion of tumours with high mutation and copy number load. Nat Commun. 31. Fry TJ, Shah NN, Orentas RJ, et al. CD22-targeted CAR T cells induce remis- 2019;10(1):4278.doi:10.1038/s41467-019-12159-9. sion in B-ALL that is naive or resistant to CD19-targeted CAR immunother- 6. Bentham R, Litchfield K, Watkins TBK, et al.; TRACERx Consortium. Using apy. Nat Med. 2018;24(1):20–28. doi:10.1038/nm.4441. DNA sequencing data to quantify T cell fraction and therapy response. 32. Gardner R, Wu D, Cherian S, et al. Acquisition of a CD19-negative myeloid Nature. 2021;597(7877):555–560. doi:10.1038/S41586-021-03894-5. phenotype allows immune escape of MLL-rearranged B-ALL from CD19 7. Lu YC, Jia L, Zheng Z, Tran E, Robbins PF, Rosenberg SA. Single-cell tran- CAR-T-cell therapy. Blood. 2016;127(20):2406–2410. doi: scriptome analysis reveals gene signatures associated with T-cell persis- 10.1182/blood-2015-08-665547. tence following adoptive cell therapy. Cancer Immunol Res. 2019;7(11): 33. Samur MK, Fulciniti M, Aktas Samur A, et al. Biallelic loss of BCMA as a resis- 1824–1836. doi:10.1158/2326-6066.CIR-19-0299. tance mechanism to CAR T cell therapy in a patient with multiple myeloma. 8. Campillo-Marcos I, Alvarez-Errico D, Alandes RA, Mereu E, Esteller M. Nat Commun. 2021;12(1):868.doi:10.1038/s41467-021-21177-5. Single-cell technologies and analyses in hematopoiesis and hematological 34. Lee DW, Gardner R, Porter DL, et al. Current concepts in the diagnosis and malignancies c. Exp Hematol. 2021;98:1–13. doi: management of cytokine release syndrome. Blood. 2014;124(2):188–195. doi: 10.1016/j.exphem.2021.05.001. 10.1182/blood-2014-05-552729. 9. Krishna S, Lowery FJ, Copeland AR, et al. Stem-like CD8 T cells mediate re- 35. Schuster SJ, Svoboda J, Chong EA, et al. Chimeric antigen receptor T cells in sponse of adoptive cell immunotherapy against human cancer. Science. refractory B-cell lymphomas. N Engl J Med. 2017;377(26):2545–2554. doi: 2020;370(6522):1328–1334. doi:10.1126/science.abb9847. 10.1056/nejmoa1708566. 10. Germain RN, Stefanova  I. The dynamics of T cell receptor signaling: com- 36. Torikai H, Reik A, Liu P-Q, et al. A foundation for universal T-cell based im- plex orchestration and the key roles of tempo and cooperation. Annu Rev munotherapy: T cells engineered to express a CD19-specific chimeric-anti- Immunol. 1999;17(1):467–522. doi:10.1146/annurev.immunol.17.1.467. gen-receptor and eliminate expression of endogenous TCR. Blood. 2012; 11. Meuer SC, Acuto O, Hussey RE, et al. Evidence for the T3-associated 90K het- 119(24):5697–5705. doi:10.1182/blood-2012-01-405365. erodimer as the T-cell antigen receptor. Nature. 1983;303(5920):808–810. doi: 37. Liu E, Marin D, Banerjee P, et al. Use of CAR-transduced natural killer cells in 10.1038/303808a0. CD19-positive lymphoid tumors. N Engl J Med. 2020;382(6):545–553. doi: 12. Wucherpfennig KW, Gagnon E, Call MJ, Huseby ES, Call ME. Structural biol- 10.1056/nejmoa1910607. ogy of the T-cell receptor: insights into receptor assembly, ligand recogni- 38. Klichinsky M, Ruella M, Shestova O, et al. Human chimeric antigen receptor tion, and initiation of signaling. Cold Spring Harb Perspect Biol. 2010;2(4): macrophages for cancer immunotherapy. Nat Biotechnol. 2020;38(8):947–953. a005140. doi:10.1101/cshperspect.a005140. doi:10.1038/s41587-020-0462-y. 13. Busch DH, Fra € ßle SP, Sommermeyer D, Buchholz VR, Riddell SR. Role of 39. Zhang L, Tian L, Dai X, et al. Pluripotent stem cell-derived CAR-macrophage memory T cell subsets for adoptive immunotherapy. Semin Immunol. 2016; cells with antigen-dependent anti-cancer cell functions. J Hematol Oncol. 28(1):28–34. doi:10.1016/j.smim.2016.02.001. 2020;13(1):153.doi:10.1186/s13045-020-00983-2. 14. Rosenberg SA, Spiess P, Lafreniere R. A new approach to the adoptive immu- 40. Chen H, Ndhlovu ZM, Liu D, et al. TCR clonotypes modulate the protective notherapy of cancer with tumor-infiltrating lymphocytes. Science. 1986; effect of HLA class I molecules in HIV-1 infection. Nat Immunol. 2012;13(7): 233(4770):1318–1321. doi:10.1126/science.3489291. 691–700. doi:10.1038/NI.2342. 938 | JNCI J Natl Cancer Inst, 2022, Vol. 114, No. 7 41. Albinger N, Hartmann J, Ullrich E. Current status and perspective of CAR-T 65. Turtle CJ, Hanafi LA, Berger C, et al. CD19 CAR-T cells of defined CD4þ:CD8þ and CAR-NK cell therapy trials in Germany. Gene Ther. 2021;28(9):513–527. composition in adult B cell ALL patients. J Clin Invest. 2016;126(6):2123–2138. doi:10.1038/s41434-021-00246-w. doi:10.1172/JCI85309. 42. Wrona E, Borowiec M, Potemski P. CAR-NK cells in the treatment of solid 66. Sabatino M, Hu J, Sommariva M, et al. Generation of clinical-grade CD19- tumors. IJMS. 2021;22(11):5899. doi:10.3390/IJMS22115899. specific CAR-modified CD81 memory stem cells for the treatment of human 43. Rurik JG, Tomba  cz I, Yadegari A, et al. CAR T cells produced in vivo to treat B-cell malignancies. Blood. 2016;128(4):519–528. doi: cardiac injury. Science. 2022;375(6576):91–96. doi:10.1126/SCIENCE.ABM0594. 10.1182/blood-2015-11-683847. 44. Larrea C. D, Staehr M, Lopez AV, et al. Defining an optimal dual-targeted 67. Wang X, Naranjo A, Brown C, et al. Phenotypic and functional attributes of CAR T-cell therapy approach simultaneously targeting BCMA and GPRC5D lentivirus-modified CD19-specific human CD8þ central memory T cells to prevent BCMA escape–driven relapse in multiple myeloma. Blood Cancer manufactured at clinical scale. J Immunother. 2012;35(9):689–701. doi: Discov. 2020;1(2):146–154. doi:10.1158/2643-3230.BCD-20-0020. 10.1097/CJI.0B013E318270DEC7. 45. Chmielewski M, Abken H. TRUCKS, the fourth-generation CAR T cells: cur- 68. Agarwal S, Hanauer JDS, Frank AM, Riechert V, Thalheimer FB, Buchholz CJ. rent developments and clinical translation. Adv Cell Gene Ther. 2020;3(3): In vivo generation of CAR T cells selectively in human CD4þ lymphocytes. e84.doi:10.1002/ACG2.84. Mol Ther. 2020;28(8):1783–1794. doi:10.1016/j.ymthe.2020.05.005. 46. Zhao L, Cao YJ. Engineered T cell therapy for cancer in the clinic. Front 69. Wang D, Aguilar B, Starr R, et al. Glioblastoma-targeted CD4þ CAR T cells Immunol. 2019;10:2250. doi:10.3389/fimmu.2019.02250. mediate superior antitumor activity. JCI Insight. 2018;3(10):e99048. doi: 47. Zhao Q, Jiang Y, Xiang S, et al. Engineered TCR-T cell immunotherapy in an- 10.1172/jci.insight.99048. ticancer precision medicine: pros and cons. Front Immunol. 2021;12: 70. Melenhorst JJ, Chen GM, Wang M, et al. Decade-long leukaemia remissions 658753.doi:10.3389/FIMMU.2021.658753/BIBTEX. with persistence of CD4þ CAR T cells. Nature. 2022;602(7897):503–509. doi: 48. Wang S, Sun J, Chen K, et al. Perspectives of tumor-infiltrating lymphocyte 10.1038/S41586-021-04390-6. Downloaded from https://academic.oup.com/jnci/article/114/7/930/6570594 by guest on 26 May 2023 treatment in solid tumors. BMC Med. 2021;19(1):1–7. doi: 71. Sommermeyer D, Hudecek M, Kosasih P, et al. Chimeric antigen receptor- 10.1186/S12916-021-02006-4/FIGURES/4. modified T cells derived from defined CD8þ and CD4þ subsets confer supe- 49. Peng M, Mo Y, Wang Y, et al. Neoantigen vaccine: an emerging tumor immu- rior antitumor reactivity in vivo. Leukemia. 2016;30(2):492–500. doi: notherapy. Mol Cancer. 2019;18(1):1–14. doi:10.1186/S12943-019-1055-6. 10.1038/LEU.2015.247. 50. Haen SP, Löffler MW, Rammensee HG, Brossart P. Towards new horizons: 72. Nelson AS, Khan I, Kaplan MH. T helper differentiation of CAR T cells aug- COMMENTARY characterization, classification and implications of the tumour antigenic ments function and target cell killing. J Immunol. 2020;204(suppl 1):246.6. repertoire. Nat Rev Clin Oncol. 2020;17(10):595–610. doi:10.1038/ 73. Chmielewski M, Abken H. CAR T cells releasing IL-18 convert to T-Bethigh S41571-020-0387-X. FoxO1low effectors that exhibit augmented activity against advanced solid 51. Zhu G, Zhang Q, Zhang J, Liu F. Targeting tumor-associated antigen: a prom- tumors. Cell Rep. 2017;21(11):3205–3219. doi:10.1016/J.CELREP.2017.11.063. ising CAR-T therapeutic strategy for glioblastoma treatment. Front 74. Long A, Haso W, Shern J, et al. 4-1BB costimulation ameliorates T cell ex- Pharmacol. 2021;12:661606.doi:10.3389/FPHAR.2021.661606/BIBTEX. haustion induced by tonic signaling of chimeric antigen receptors. Nat Med. 52. Cherkassky L, Morello A, Villena-Vargas J, et al. Human CAR T cells with 2015;21(6):581–590. doi:10.1038/NM.3838. cell-intrinsic PD-1 checkpoint blockade resist tumor-mediated inhibition. J 75. Wherry E, Kurachi M. Molecular and cellular insights into T cell exhaustion. Clin Invest. 2016;126(8):3130–3144. doi:10.1172/JCI83092. Nat Rev Immunol. 2015;15(8):486–499. doi:10.1038/NRI3862. 53. Turtle CJ, Berger C, Sommermeyer D, et al. Anti-CD19 chimeric antigen re- 76. Pauken K, Wherry E. Overcoming T cell exhaustion in infection and cancer. ceptor-modified T cell therapy for B cell non-hodgkin lymphoma and Trends Immunol. 2015;36(4):265–276. doi:10.1016/J.IT.2015.02.008. chronic lymphocytic leukemia: fludarabine and cyclophosphamide lym- 77. Fraietta J, Lacey S, Orlando E, et al. Determinants of response and resistance phodepletion improves in vivo expansion and persistence of CAR-T cells to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lympho- and clinical outcomes. Blood. 2015;126(23):184–184. doi:10.1182/ cytic leukemia. Nat Med. 2018;24(5):563–571. doi:10.1038/S41591-018-0010-1. BLOOD.V126.23.184.184. 78. Finney OC, Brakke H, Rawlings-Rhea S, et al. CD19 CAR T cell product and 54. Grada Z, Hegde M, Byrd T, et al. TanCAR: a novel bispecific chimeric antigen disease attributes predict leukemia remission durability. J Clin Invest. 2019; receptor for cancer immunotherapy. Mol Ther Nucleic Acids. 2013;2(7):e105. 129(5):2123–2132. doi:10.1172/JCI125423 doi:10.1038/MTNA.2013.32. 79. Garcia-Prieto CA, Villanueva L, Bueno-Costa A, et al. Epigenetic profiling 55. Pollack IF, Jakacki RI, Butterfield LH, et al. Antigen-specific immune and response to CD19 chimeric antigen receptor T-cell therapy in B-cell ma- responses and clinical outcome after vaccination with glioma-associated lignancies. JNCI J Natl Cancer Inst. 2021. doi:10.1093/jnci/djab194. antigen peptides and polyinosinic-polycytidylic acid stabilized by lysine 80. Nobles CL, Sherrill-Mix S, Everett JK, et al. CD19-targeting CAR T cell immu- and carboxymethylcellulose in children with newly diagnosed malignant notherapy outcomes correlate with genomic modification by vector integra- brainstem and nonbrainstem gliomas. J Clin Oncol. 2014;32(19):2050–2058. tion. J Clin Invest. 2020;130(2):673–685. doi:10.1172/JCI130144. doi:10.1200/J Clin Oncol.2013.54.0526. 81. Nowicki TS, Farrell C, Morselli M, et al. Epigenetic suppression of transgenic 56. Sukumaran S, Watanabe N, Bajgain P, et al. Enhancing the potency and spe- t-cell receptor expression via gamma-retroviral vector methylation in adop- cificity of engineered T cells for cancer treatment. Cancer Discov. 2018;8(8): tive cell transfer therapy. Cancer Discov. 2020;10(11):1645–1653. doi: 972–987. doi:10.1158/2159-8290.CD-17-1298. 10.1158/2159-8290.CD-20-0300. 57. Muliaditan T, Halim L, Whilding LM, et al. Synergistic T cell signaling by 82. Park JH, Rivière I, Gonen M, et al. Long-term follow-up of CD19 CAR therapy 41BB and CD28 is optimally achieved by membrane proximal positioning in acute lymphoblastic leukemia. N Engl J Med. 2018;378(5):449–459. doi: within parallel chimeric antigen receptors. Cell Rep Med. 2021;2(12): 10.1056/NEJMoa1709919. 100457.doi:10.1016/J.XCRM.2021.100457. 83. Shen Q, Zhang Q, Shi Y, et al. Tet2 promotes pathogen infection-induced 58. Schwartzentruber DJ, Hom SS, Dadmarz R, et al. In vitro predictors of thera- myelopoiesis through mRNA oxidation. Nature. 2018;554(7690):123–127. doi: peutic response in melanoma patients receiving tumor-infiltrating lympho- 10.1038/nature25434. cytes and interleukin-2. J Clin Oncol. 1994;12(7):1475–1483. doi:10.1200/J Clin 84. Zhang Z, Chen X, Tian Y, et al. Point mutation in CD19 facilitates immune Oncol.1994.12.7.1475. escape of B cell lymphoma from CAR-T cell therapy. J Immunother Cancer. 59. Tran KQ, Zhou J, Durflinger KH, et al. Minimally cultured tumor-infiltrating 2020;8(2):e001150. doi:10.1136/jitc-2020-001150. lymphocytes display optimal characteristics for adoptive cell therapy. J 85. Gajewski T, Meng Y, Blank C, et al. Immune resistance orchestrated by the Immunother. 2008;31(8):742–751. doi:10.1097/CJI.0b013e31818403d5. tumor microenvironment. Immunol Rev. 2006;213(1):131–145. doi: 60. Zhou J, Shen X, Huang J, Hodes RJ, Rosenberg SA, Robbins PF. Telomere 10.1111/J.1600-065X.2006.00442.X. length of transferred lymphocytes correlates with in vivo persistence and 86. Rosenberg S, Restifo N. Adoptive cell transfer as personalized immunother- tumor regression in melanoma patients receiving cell transfer therapy. J apy for human cancer. Science. 2015;348(6230):62–68. doi: Immunol. 2005;175(10):7046–7052. doi:10.4049/jimmunol.175.10.7046. 10.1126/SCIENCE.AAA4967. 61. Antony PA, Piccirillo CA, Akpinarli A, et al. CD8 þ T cell immunity against a 87. Boulch M, Cazaux M, Loe-Mie Y, et al. A cross-talk between CAR T cell sub- tumor/self-antigen is augmented by CD4 þ T helper cells and hindered by sets and the tumor microenvironment is essential for sustained cytotoxic naturally occurring T regulatory cells. J Immunol. 2005;174(5):2591–2601. doi: activity. Sci Immunol. 2021;6(57):eabd4344. doi: 10.4049/jimmunol.174.5.2591 10.1126/SCIIMMUNOL.ABD4344. 62. Dudley ME, Wunderlich JR, Shelton TE, Even J, Rosenberg SA. Generation of 88. Gulati P, Ruhl J, Kannan A, et al. Aberrant Lck signal via CD28 costimulation tumor-infiltrating lymphocyte cultures for use in adoptive transfer therapy augments antigen-specific functionality and tumor control by redirected T for melanoma patients. J Immunother. 2003;26(4):332–342. doi: cells with PD-1 blockade in humanized mice. Clin Cancer Res. 2018;24(16): 10.1097/00002371-200307000-00005. 3981–3993. doi:10.1158/1078-0432.CCR-17-1788. 63. Bobisse S, Genolet R, Roberti A, et al. Sensitive and frequent identification of 89. Long AH, Highfill SL, Cui Y, et al. Reduction of MDSCs with all-trans retinoic high avidity neo-epitope specific CD8 þ T cells in immunotherapy-naive acid improves CAR therapy efficacy for sarcomas. Cancer Immunol Res. 2016; ovarian cancer. Nat Commun. 2018;9(1):1–10. doi:10.1038/ 4(10):869–880. doi:10.1158/2326-6066.CIR-15-0230. s41467-018-03301-0. 90. Ninomiya S, Narala N, Huye L, et al. Tumor indoleamine 2,3-dioxygenase 64. Wood MA, Weeder BR, David JK, Nellore A, Thompson RF. Burden of tumor (IDO) inhibits CD19-CAR T cells and is downregulated by lymphodepleting mutations, neoepitopes, and other variants are weak predictors of cancer drugs. Blood. 2015;125(25):3905–3916. doi:10.1182/BLOOD-2015-01-621474. immunotherapy response and overall survival. Genome Med. 2020;12(1):1–16. 91. Lee JC, Hayman E, Pegram HJ, et al. In vivo inhibition of human CD19-tar- doi:10.1186/S13073-020-00729-2. geted effector T cells by natural T regulatory cells in a xenotransplant G. Ferrer et al. | 939 murine model of B cell malignancy. Cancer Res. 2011;71(8):2871–2881. doi: 105. Maude SL, Frey N, Shaw PA, et al. Chimeric antigen receptor T cells for sus- 10.1158/0008-5472.CAN-10-0552. tained remissions in leukemia. N Engl J Med. 2014;371(16):1507–1517. doi: 92. Jafarzadeh L, Masoumi E, Fallah-Mehrjardi K, Mirzaei HR, Hadjati J. 10.1056/NEJMOA1407222. Prolonged persistence of chimeric antigen receptor (CAR) T cell in adoptive 106. Cifaldi L, Prencipe G, Caiello I, et al. Inhibition of natural killer cell cytotoxicity cancer immunotherapy: challenges and ways forward. Front Immunol. 2020; by interleukin-6: Implications for the pathogenesis of macrophage activation 11(702):702. doi:10.3389/FIMMU.2020.00702/BIBTEX. syndrome. Arthritis Rheumatol. 2015;67(11):3037–3046. doi:10.1002/ART.39295. 93. Turtle CJ, Hanafi LA, Berger C, et al. Immunotherapy of non-Hodgkin’s lym- 107. Obstfeld AE, Frey NV, Mansfield K, et al. Cytokine release syndrome associ- phoma with a defined ratio of CD8þ and CD4þ CD19-specific chimeric anti- ated with chimeric-antigen receptor T-cell therapy: clinicopathological gen receptor-modified T cells. Sci Transl Med. 2016;8(355):355ra116. doi: insights. Blood. 2017;130(23):2569–2572. doi:10.1182/BLOOD-2017-08-802413. 10.1126/scitranslmed.aaf8621. 108. Fitzgerald JC, Weiss SL, Maude SL, et al. Cytokine release syndrome after 94. Hombach A, Hombach AA, Abken H. Adoptive immunotherapy with geneti- chimeric antigen receptor T cell therapy for acute lymphoblastic leukemia. cally engineered T cells: modification of the IgG1 Fc ‘spacer’ domain in the Crit Care Med. 2017;45(2):e124–e131. doi:10.1097/CCM.0000000000002053. extracellular moiety of chimeric antigen receptors avoids ‘off-target’ activa- 109. Norelli M, Camisa B, Barbiera G, et al. Monocyte-derived IL-1 and IL-6 are dif- tion and unintended initiation of an innate immune response. Gene Ther. ferentially required for cytokine-release syndrome and neurotoxicity due to 2010;17(10):1206–1213. doi:10.1038/GT.2010.91. CAR T cells. Nat Med. 2018;24(6):739–748. doi:10.1038/S41591-018-0036-4. 95. Zhao Z, Condomines M, van der Stegen SJC, et al. Structural design of engi- 110. Tanaka T, Narazaki M, Kishimoto T. Immunotherapeutic implications of IL- neered costimulation determines tumor rejection kinetics and persistence 6 blockade for cytokine storm. Immunotherapy. 2016;8(8):959–970. doi: of CAR T cells. Cancer Cell. 2015;28(4):415–428. doi:10.1016/ 10.2217/IMT-2016-0020. J.CCELL.2015.09.004. 111. Bailey SR, Vatsa S, Larson RC, et al. Blockade or deletion of IFNg reduces 96. Lamure S, Van Laethem F, De Verbizier D, et al. Clinical and product features macrophage activation without compromising CAR-T function in hemato- Downloaded from https://academic.oup.com/jnci/article/114/7/930/6570594 by guest on 26 May 2023 associated with outcome of dlbcl patients to cd19-targeted car t-cell ther- logic malignancies. Blood Cancer Discov. 2022;3(2):136–153. doi: apy. Cancers (Basel). 2021;13(17):4279.doi:10.3390/CANCERS13174279/S1. 10.1158/2643-3230.BCD-21-0181. 97. Cappell KM, Sherry RM, Yang JC, et al. Long-term follow-up of anti-CD19 chi- 112. Giavridis T, van der Stegen SJC, Eyquem J, Hamieh M, Piersigilli A, Sadelain meric antigen receptor T-cell therapy. J Clin Oncol. 2020;38(32):3805–3815. M. CAR T cell–induced cytokine release syndrome is mediated by macro- doi:10.1200/J Clin Oncol.20.01467. phages and abated by IL-1 blockade. Nat Med. 2018;24(6):731–738. doi: COMMENTARY 98. Locke FL, Rossi JM, Neelapu SS, et al. Tumor burden, inflammation, and 10.1038/s41591-018-0041-7. product attributes determine outcomes of axicabtagene ciloleucel in large 113. Hay KA, Hanafi LA, Li D, et al. Kinetics and biomarkers of severe cytokine re- B-cell lymphoma. Blood Adv. 2020;4(19):4898–4911. doi:10.1182/ lease syndrome after CD19 chimeric antigen receptor–modified T-cell ther- BLOODADVANCES.2020002394. apy. Blood. 2017;130(21):2295–2306. doi:10.1182/blood-2017-06-793141. 99. Skovgard MS, Hocine HR, Saini JK, et al. Imaging CAR T-cell kinetics in solid 114. Santomasso B, Park J, Salloum D, et al. Clinical and biological correlates of tumors: translational implications. Mol Ther Oncolytics. 2021;22:355–367. doi: neurotoxicity associated with CAR T-cell therapy in patients with B-cell 10.1016/J.OMTO.2021.06.006/ATTACHMENT/854C5851-129F-4C45-B540-AB1 acute lymphoblastic leukemia. Cancer Discov. 2018;8(8):958–971. doi: BDD503238/MMC1.PDF. 10.1158/2159-8290.CD-17-1319. 100. Jensen MC, Popplewell L, Cooper LJ, et al. Antitransgene rejection 115. Hong R, Hu Y, Huang H. Biomarkers for chimeric antigen receptor T cell responses contribute to attenuated persistence of adoptively transferred therapy in acute lymphoblastic leukemia: prospects for personalized man- CD20/CD19-specific chimeric antigen receptor redirected T cells in agement and prognostic prediction. Front Immunol. 2021;12:627764. doi: humans. Biol Blood Marrow Transplant. 2010;16(9):1245–1256. doi: 10.3389/fimmu.2021.627764 10.1016/J.BBMT.2010.03.014. 116. Parker K, Migliorini D, Perkey E, et al. Single-cell analyses identify brain mu- 101. Caruana I, Savoldo B, Hoyos V, et al. Heparanase promotes tumor infiltra- ral cells expressing CD19 as potential off-tumor targets for CAR-T immuno- tion and antitumor activity of CAR-redirected T lymphocytes. Nat Med. 2015; therapies. Cell. 2020;183(1):126–142.e17. doi:10.1016/J.CELL.2020.08.022. 21(5):524–529. doi:10.1038/NM.3833. 117. Arcangeli S, Falcone L, Camisa B, et al. Next-generation manufacturing pro- 102. Lamers CHJ, Willemsen R, Van Elzakker P, et al. Immune responses to trans- tocols enriching TSCM CAR T cells can overcome disease-specific T cell gene and retroviral vector in patients treated with ex vivo-engineered T defects in cancer patients. Front Immunol. 2020;11:1217.doi: cells. Blood. 2011;117(1):72–82. doi:10.1182/BLOOD-2010-07-294520. 10.3389/FIMMU.2020.01217. 103. Yan ~ ez L, Sa nchez-Escamilla M, Perales MA. CAR T cell toxicity: current man- 118. Guha P, Heatherton KR, O’Connell KP, Alexander IS, Katz SC. Assessing the agement and future directions. HemaSphere. 2019;3(2):e186. doi: future of solid tumor immunotherapy. Biomed. 2022;10(3):655.doi: 10.1097/HS9.0000000000000186 10.3390/BIOMEDICINES10030655. 104. Maude SL, Barrett D, Teachey DT, Grupp SA. Managing cytokine release syn- 119. Patel AP, Tirosh I, Trombetta JJ, et al. Single-cell RNA-seq highlights intratu- drome associated with novel T cell-engaging therapies. Cancer J. 2014;20(2): moral heterogeneity in primary glioblastoma. Science. 2014;344(6190): 119–122. doi:10.1097/PPO.0000000000000035. 1396–1401. doi:10.1126/SCIENCE.1254257.