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Targeting T cell malignancies using CAR-based immunotherapy: challenges and potential solutions

Targeting T cell malignancies using CAR-based immunotherapy: challenges and potential solutions Chimeric antigen receptor (CAR) T cell therapy has been successful in treating B cell malignancies in clinical trials; however, fewer studies have evaluated CAR T cell therapy for the treatment of T cell malignancies. There are many challenges in translating this therapy for T cell disease, including fratricide, T cell aplasia, and product contamination. To the best of our knowledge, no tumor-specific antigen has been identified with universal expression on cancerous T cells, hindering CAR T cell therapy for these malignancies. Numerous approaches have been assessed to address each of these challenges, such as (i) disrupting target antigen expression on CAR- modified T cells, (ii) targeting antigens with limited expression on T cells, and (iii) using third party donor cells that are either non-alloreactive or have been genome edited at the T cell receptor α constant (TRAC) locus. In this review, we discuss CAR approaches that have been explored both in preclinical and clinical studies targeting T cell antigens, as well as examine other potential strategies that can be used to successfully translate this therapy for T cell disease. Keywords: CAR, Immunotherapy, T-ALL, T cell lymphoma Introduction (PTCL) [7]. Mycosis fungoides (MF) and Sezary syn- T cell malignancies encompass a heterogeneous group drome (SS) represent the two most common subtypes of of diseases, each reflecting a clonal evolution of dysfunc- CTCL, accounting for the majority of cases [8]. PTCL tional T cells at various stages of development. T cell can be classified into several different subtypes, among acute lymphoblastic leukemia (T-ALL) accounts for 15% which include anaplastic large cell lymphoma (ALCL), and 25% of childhood and adult ALL cases respectively, angioimmunoblastic T cell lymphoma (AITL), extrano- and is the most common form of T cell cancer seen in dal natural killer (NK)-T cell lymphoma (ENKTL), children [1, 2]. T-lymphoblastic lymphoma (T-LLy) is a enteropathy-associated T cell lymphoma (EATL), hepa- non-Hodgkin lymphoma with similar biology to T-ALL. tosplenic T cell lymphoma (HSTCL), and PTCL-not Adult T cell leukemia/lymphoma (ATLL) is an ex- otherwise specified (PTCL-NOS) which is the most tremely aggressive form of blood cancer driven by the common of the group [9, 10]. human T cell lymphocytic virus type 1 (HTLV1) [3–5]. The overall prognosis for T cell malignancies varies Other rare forms of T cell leukemia include T cell large depending on the type of disease, but in general is much granular lymphocytic leukemia (T-LGL) and T- poorer when compared to B cell malignancies. While prolymphocytic leukemia (T-PLL) [6]. T cell lymphomas the survival in T-ALL/LLy has significantly improved are broadly divided into two categories, cutaneous T cell with the intensification of chemotherapy, there still re- lymphoma (CTCL) and peripheral T cell lymphoma main very limited options for patients with relapsed/re- fractory disease [11–13]. ATLL remains a very challenging disease to treat, with a median survival of * Correspondence: sraikar@emory.edu less than 12 months for the acute form of this disease Cell and Gene Therapy Program, Department of Pediatrics, Aflac Cancer and [3–5]. Advanced stage CTCL has a median overall sur- Blood Disorders Center, Children’s Healthcare of Atlanta and Emory University School of Medicine, Atlanta, GA, USA vival of 5 years [14, 15], whereas outcomes of PTCL vary Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Fleischer et al. Journal of Hematology & Oncology (2019) 12:141 Page 2 of 21 depending upon the subtype, with ENKTL, EATL, and not restricted by major histocompatibility complex HSTCL having the poorest prognosis [9, 10]. While im- (MHC) interactions. Therefore, CARs can recognize cell munotherapy has revolutionized the treatment landscape surface proteins that have not been processed and pre- of various cancers with the use of monoclonal anti- sented by antigen presenting cells (APCs). Importantly, bodies, checkpoint inhibitors, bispecific T cell engagers, the interactions between scFvs and ligands have much and chimeric antigen receptor (CAR) T cell therapy, higher affinity and avidity compared to that of TCR- only limited responses have been seen in T cell disease ligand interactions [25]. Furthermore, the immune syn- [15]. Some promising results have been seen with use of apse formed from the interaction between a CAR and its brentuximab vedotin, a CD30-directed immunotoxin, in ligand likely results in a much greater functional avidity CD30-positive PTCL and CTCL [16, 17] and the use of than is observed using a targeted antibody approach pembroluzimab, a programmed cell death receptor 1 with the same antibody (25). (PD-1) inhibitor, in the treatment of ENKTL [18]; how- CARs targeting the B cell antigen CD19 have been ever, these positive results have been limited to very studied extensively for the treatment of B cell malignan- specific subsets of T cell disease. One form of immuno- cies. In 2017, the FDA approved the first CAR T cell therapy that has not yet been successfully translated to therapy, Kymriah, a CD19-directed CAR therapy for the T cell malignancies is that of chimeric antigen receptor treatment of relapsed/refractory B cell acute lympho- (CAR)-based immunotherapy. CAR T cell therapy has blastic leukemia (B-ALL) and in 2018, Yescarta was ap- been extremely successful in relapsed/refractory B cell proved to treat relapsed diffuse large B cell lymphoma malignancies as evidenced by the recent Food and Drug (DLBCL). These therapies, including others in clinical Administration (FDA) approval of two CAR T cell thera- trials, have been widely successful in eliminating malig- peutics for this disease [19–23]. However, implementing nant cells and re-inducing remission in patients who this technology to treat T cell malignancies has been dif- were otherwise treatment-refractory [19–21, 26, 27]. Pa- ficult, primarily due to the lack of a tumor-specific sur- tients receiving CAR therapy undergo leukapheresis face antigen in cancerous T cells. In this review, we will resulting in the collection of T cells, which are subse- discuss the challenges involved in translating this novel quently modified using a lentiviral or retroviral vector to technology to T cell disease, review all the preclinical express the CAR. These cells are expanded ex vivo while and clinical progress made in adapting this therapy for the patient undergoes lymphodepletion, a process in- this challenging disease, and examine potential solutions volving chemotherapeutic agents. Finally, the CAR T for the future development of this innovative therapy. cells are re-infused into the patient [28]. Lymphodeple- tion prior to re-infusion of the autologous T cells has CAR T cell therapy been shown to augment both CAR T cell proliferation as Genetic engineering of primary T cells was first pre- well as persistence [29–31]. The administered dose of sented in the late 1980s [24]. Since then, chimeric anti- CAR T cells and the pre-existing tumor burden do not gen receptor T cells have emerged as a promising appear to be the sole determinants of the degree of T technique for the treatment of relapsed/refractory malig- cell expansion, engraftment, and overall response. Other nancies. CAR therapy brings together numerous fields factors may be involved, such as the density of cognate including immunology, tumor biology, genetic engineer- antigen expression on the cancer cells [32]. However, ing, synthetic biology, and pharmacology. CARs are the optimal degree of persistence of CAR T cells re- comprised of the intracellular signaling domain from the quired to prevent leukemic relapse has not been deter- natural T cell receptor (TCR), CD3ζ, linked to a single- mined [25, 33]. chain variable fragment (scFv) which serves as the anti- One of the mechanisms of relapse post-CD19 gen recognition domain. The scFv sequence is derived CAR T cell therapy is due to surface antigen escape with from a monoclonal antibody by combining the variable relapsed leukemia cells being CD19-negative. The mech- heavy (V ) and light (V ) domains using a small peptide anism may be due to the expansion of a small subset of H L linker. Commonly used CARs also include one or two CD19-negative cancer cells or alternatively, the cells may costimulatory domains, such as CD28, 4-1BB, ICOS, downregulate CD19 from the cell surface in order to and/or OX40. Although the kinetics have yet to be fully evade detection by CAR T cells, rendering them resist- elucidated, it is essential that CAR T cells have mecha- ant [19, 21, 34–37]. Additionally, it was recently shown nisms of trafficking to the tumor site where they can that a phenomenon referred to as trogocytosis is a recognize their cognate antigen. This results in CAR T mechanism of antigen escape whereby the antigen is cell activation and expansion, and ultimately cytolytic transferred to the CAR T cell [38]. It has also been activity against cells expressing the target antigen. CAR- shown that transduction of a single leukemic blast with based ligand recognition is advantageous compared to an anti-CD19 CAR that was re-infused into a B-ALL pa- TCR-based ligand recognition because CAR-targeting is tient, ultimately resulted in relapse and death of the Fleischer et al. Journal of Hematology & Oncology (2019) 12:141 Page 3 of 21 patient [39]. Transduction of the leukemic cell resulted subsequent CAR-modification of tumor cells. Addition- in masking of the target antigen through interactions be- ally, expression of the targeted antigen on CAR T cells tween the CAR and the cognate antigen on the same results in fratricide and limited expansion of the CAR T cell. Clonal expansion of this population resulted in re- cells. Furthermore, targeting of an antigen regularly sistance to CAR therapy. This report emphasized the im- expressed on normal T cells would result in T cell apla- portance of strict and perfect isolation of normal, sia, leading to profound immunosuppression, likely to be healthy T cells for modification with the CAR construct. associated with high rates of morbidity and mortality As we discuss below, this is particularly challenging in T (Fig. 1). cell leukemia patients who are more likely to have circu- Various approaches have been used to overcome these lating cancerous T cells, and therefore have a higher challenges, including CRISPR-Cas9 genome editing to probability of these cells being inadvertently isolated, remove the antigen from the CAR T cells [45–47], Tet- transduced, and re-infused. OFF expression system to limit fratricide during ex vivo Of note, there are severe toxicities that have been as- expansion [48], protein expression blocker (PEBL) to re- sociated with CAR therapy. Cytokine release syndrome tain the antigen in the ER/Golgi to prevent cell surface (CRS) is a systemic inflammatory response directly expression [49, 50], or using CAR-modified natural killer resulting from robust T cell activation following infu- cells instead of T cells [47, 51–54]. Additionally, to date, sion. IL-6 is one pro-inflammatory cytokine that is se- four targets have been investigated as targets for CAR T creted at high levels during CRS. During a particularly cell therapy for the treatment of T cell malignancies with severe CRS condition, tocilizumab, an IL-6R antagonist limited to no expression in the normal population of T monoclonal antibody, was used to rapidly and effectively cells, CD30, CD37, TRBC1, and CD1a [55–58]. Table 1 reverse the symptoms of a pediatric patient [27]. Toci- provides a summary of potential solutions to the three lizumab has since been FDA approved for treatment of main challenges seen in adapting CAR technology for T CAR T cell-induced life-threatening CRS [40]. Neuro- cell malignancies—fratricide, T cell aplasia, and product logical toxicities have been reported following CAR T contamination. A list of all current CAR-based clinical cell infusion as well; however, preventative approaches trials targeting T cell disease is presented in Table 2. remain elusive [36, 41–44]. Compared to CRS and Below, we review all preclinical and clinical CAR studies neurotoxicity, a much more manageable consequence of targeting T cell malignancies categorized according to CAR T cell therapy targeting B cell malignancies is the the target antigen of interest. resulting B cell aplasia. This is a potentially lifelong out- come due to memory cell formation against a B cell anti- CD5 gen; but currently is managed by periodic infusions of CD5 expression is limited to normal T cells and a small intravenous immunoglobulins. Unfortunately, this is an subpopulation of B cells, called B-1a cells [65–69]. CD5 extremely problematic outcome for T cell malignancies, acts as a negative regulator of TCR signaling and has a as persistent T cell aplasia would be life threatening. role in protecting against autoimmunity [70, 71]. CD5 is There are currently > 200 clinical trials using CAR T highly expressed on many T cell malignancies, particu- cells registered at clinicaltrials.gov being carried out in larly T-ALL and PTCLs, rendering it a good target for the USA. However, the majority of these trials are enrol- CAR T cell therapy [72–74]. Since CD5 expression on T ling patients with B cell malignancies. Advances are be- cells is approximately ten times that on B cells [75], a ing made to expand CAR T cell therapy to the treatment low-affinity, high-avidity CAR targeting CD5 may steer of other cancers, and to minimize toxicities associated clear of CD5-positive B cells while selectively killing T with treatment while reducing difficulty and cost of cells [76, 77]. Furthermore, CD8 tumor-infiltrating lym- production. phocytes (TILs) express lower levels of CD5 compared to that of peripheral blood T cells, and one study Translating CAR T cell therapy for treatment of T showed downregulation of CD5 improves the ability of cell malignancies T cells to lyse malignant cells [78]. CD5 was previously Harnessing and redirecting the cytotoxicity of T cells to targeted as a tumor antigen in clinical trials using malignant B cells has been established, but reprogram- immunotoxin-conjugated CD5 monoclonal antibodies, ming T cells to kill malignant T cells, while sparing nor- with responses seen in patients with cutaneous T cell mal T cells, is much more complex and challenging. lymphoma and T-ALL [79, 80]. This requires aberrant expression of an antigen on ma- A preclinical study showed that expression of a CD5- lignant T cells that is absent or expressed at very low CAR with a CD28 costimulatory domain resulted in sur- levels on normal T cells. CAR therapy requires isolation face downregulation of CD5 in CAR T cells. As a result, of healthy T cells from malignant T cells, a complicated fratricide was observed only transiently, allowing the procedure that can result in product contamination and CD5-CAR T cells to expand. These cells had significant Fleischer et al. Journal of Hematology & Oncology (2019) 12:141 Page 4 of 21 Product Fratricide Contamination Anti-tumor T-cell aplasia cytolytic activity Fig. 1 Potential outcomes of CAR T cell therapy in a patient with T cell disease. Upon re-infusion into a patient, CAR T cells recognize their cognate antigen, expanding upon this recognition, and initiating an attack. However, due to shared antigen expression on CAR T cells, normal T cells, and tumor cells, numerous outcomes can be observed. CAR T cells target tumor cells as intended, reducing tumor burden. However, without further engineering, the CAR-modified T cells are likely to express the targeted antigen as well, resulting in fratricide. CAR T cells would also target healthy T cells, resulting in unintended T cell aplasia. Lastly, CAR T cell therapy involves isolating normal T cells from malignant T cells for CAR-modification. A single malignant cell contaminating this population can result in masking of the antigen, leading to antigen-positive relapse. *Figure was created using BioRender Table 1 Strategies to overcome challenges in translating CAR therapy to treat T cell malignancies Challenge Strategy Reference Fratricide Targeting downregulated antigens (e.g., CD5) [59] Genome editing of target antigen [45–47] Targeting antigens with limited expression on T cells [55–58] (e.g., CD30, CD37, TRBC1, CD1a) Tet-OFF expression system [48] Protein expression blockers (PEBLs) [49] Using NK cells or NK-92 cells [47, 51–54, 60] T cell aplasia Targeting antigens with limited expression on T cells [55–58] (e.g., CD30, CD37, TRBC1, CD1a) mRNA electroporation Adeno-associated viral (AAV) vector delivery Using NK cells or NK-92 cells [47, 51–54, 60] Using γδ T cells Suicide genes and safety switches Bridge to allogeneic hematopoietic stem cell transplant (HSCT) Product contamination Allogeneic CAR T cells with TRAC locus editing [46, 61] Using NK cells or NK-92 cells [47, 51–54, 60] Using γδ T cells Fleischer et al. Journal of Hematology & Oncology (2019) 12:141 Page 5 of 21 Table 2 Clinical CAR trials targeting T cell malignancies T cell Clinical Trials Sponsor CAR costimulatory Additional Phase Status Ref antigen domain intervention CD5 NCT03081910 Baylor College of Medicine CD28 None Phase I Recruiting (MAGENTA) CD7 NCT04004637 PersonGen BioTherapeutics Phase I Recruiting NCT04033302 Shenzhen Geno-Immune Medical Phase Recruiting Institute I/II NCT03690011 Baylor College of Medicine CD28 CRISPR/Cas9 Phase I Not yet CD7-editing recruiting NCT02742727 PersonGen BioTherapeutics CD28 and 4-1BB NK-92 cells Phase Unknown I/II CD4 NCT03829540 Stony Brook University CD28 and 4-1BB Phase I Recruiting CD30 NCT01192464 Baylor College of Medicine EBV-specific CTL Phase I Active, not recruiting nd NCT03383965 Immune Cell Inc 2 generation Phase I Recruiting NCT02690545 UNC Lineberger Comprehensive Phase Recruiting [62] Cancer Center I/II NCT02259556 Chinese PLA General Hospital 4-1BB Phase Recruiting [63] I/II NCT02958410 Southwest Hospital, China Phase Recruiting I/II NCT03049449 NCI Phase I Recruiting NCT01316146 UNC Lineberger Comprehensive CD28 Phase I Active, not [55] Cancer Center recruiting NCT02917083 (RELY- Baylor College of Medicine CD28 Phase I Recruiting [64] 30) rd NCT04008394 Wuhan Union Hospital, China 3 generation Phase I Recruiting NCT03602157 UNC Lineberger Comprehensive CCR4 Phase I Recruiting Cancer Center overexpression NCT02663297 UNC Lineberger Comprehensive CD28 Phase I Recruiting Cancer Center TRBC1 NCT03590574 Autolus Limited RQR8 safety Phase Recruiting mechanism I/II in vitro cytotoxicity against two T-ALL cell lines and persistence of treated T cells in NOD scid IL2Rγ-chain primary T-ALL cells and delayed leukemia progression knockout (NSG) mice [81]. in two different CD5-positive T-ALL models [59]. Based Interestingly, use of 4-1BB as the costimulatory do- on these results, CD5-CAR T cells with a CD28 costi- main in a CD5-CAR resulted in a significant fratricidal mulatory domain are being tested in patients with effect [48]. It was shown that tumor necrosis factor relapsed or refractory T cell disease (MAGENTA trial, (TNF) receptor-associated factor (TRAF) signaling from NCT03081910). Our group used CRISPR-Cas9 to the 4-1BB endodomain upregulated the intercellular ad- knockout CD5 expression in primary T cells prior to hesion molecule 1 (ICAM1), which subsequently stabi- transduction with the CD5-CAR. We showed that gene lized the fratricidal immunological synapse between editing of CD5 in effector CAR T cells increased CAR CD5-CAR T cells containing the 4-1BB costimulatory surface expression and decreased self-activation [47]. domain. To limit and control the effects of fratricide, a The increased CAR surface expression is predicted to Tet-OFF expression system was used, which allowed for enhance CAR T cell anti-tumor efficacy. We also controlled transgene expression using the small mol- showed antagonism of vasoactive intestinal peptide ecule inhibitor, doxycycline. In the presence of doxycyc- (VIP) signaling in conjunction with inhibition of the line, CD5-41BB-CAR T cells expanded ex vivo without PI3Kδ pathway increased expansion of CD5-CAR- evidence of fratricide, while maintaining a more naïve modified T cells as well as their cytotoxicity against genotype. Doxycycline was removed from the culture CD5-specific tumor cell lines. This combination of com- prior to injecting the CD5-41BB-CAR T cells into mice, pounds was also demonstrated to prolong in vivo resulting in CD5-CAR expression and improved survival Fleischer et al. Journal of Hematology & Oncology (2019) 12:141 Page 6 of 21 outcomes in a T-ALL mouse model. Furthermore, there was generated using CRISPR-Cas9 genome editing to dis- was a survival advantage in mice treated with Tet-OFF rupt the CD7 and TCRα constant (TRAC) loci. This study CD5-41BB-CAR T cells compared to survival of mice demonstrated that NSG mice engrafted with primary T- treated with CD5-CD28-CAR T cells without the Tet- ALL blasts and treated with UCART7 donor cells exhib- OFF expression system [48]. ited tumor clearance from the peripheral blood, and, did Alternatively, we expressed the CD5-CAR in NK-92 not develop graft versus host disease (GvHD) or other se- cells, an interleukin-2 (IL-2) dependent natural killer cell vere side effects [46]. line, which are inherently CD5-negative. Our data demon- A new technique using protein expression blockers strates that CD5-CAR-modified NK-92 cells have in- (PEBLs) has been established as an alternative to gen- creased cytotoxicity against T cell leukemia cell lines ome editing. This strategy couples an scFv with a reten- compared to the cytotoxicity of naïve NK-92 cells [47, 51], tion peptide to maintain the protein of interest in the and there is a significant improvement in survival of T- ER/Golgi preventing cell surface expression of the anti- ALL xenograft mouse models compared to survival of gen. PEBL-CD7-CAR T cells exhibited superior cytotox- mice treated with naïve NK-92 cells [47]. This data con- icity against primary T-ALL cells in vitro compared to firms previously published data illustrating significantly non-PEBL CD7-CAR T cells. Using a patient-derived improved survival and enhanced tumor reduction in irra- xenograft (PDX) model of ETP-ALL, upon detection of diated T-ALL mouse models treated with CD5-CAR- leukemic cell expansion in peripheral blood, PEBL-CD7- modified NK-92 cells compared to that of mice treated CAR T cells were injected. PEBL-CD7-CAR T cell- with control NK-92 cells [53]. Recently, another group treated mice had a significant survival advantage over tested CD5-CAR-modified NK-92 cells, using a NK- control mice. However, CD7-positive relapse did occur specific costimulatory domain 2B4 in their CAR con- in all PEBL-CD7-CAR T cell-treated mice [49]. structs [82]. Interestingly, the CD5-2B4-CAR NK-92 cells Despite CD7 expression on NK-92MI cells (IL-2 produ- displayed superiority to CD5-41BB-CAR NK-92 cells, in cing NK-92 cells), they have been used for CD7-CAR ther- both in vitro and in vivo experiments [82]. apy demonstrating only a small percentage of cells are CD7-positive, and upon CD7-CAR expression, fewer than CD7 1% CD7-positive NK-92MI cells remain [60]. Two CD7- CD7 is a transmembrane glycoprotein with expression on CAR constructs, a monovalent and bivalent construct, were T cells and NK cells [83]. The majority of T-ALLs are generated using a humanized CD7 nanobody sequence that CD7-positive, despite some populations lacking expres- had been previously developed in the laboratory. Both CAR sion of other common markers, such as the TCR [74, 84]. constructs demonstrated enhanced CD7-specific cytotox- Additionally, early T cell precursor acute lymphoblastic icity against T-ALL cell lines and primary patient cells leukemia (ETP-ALL), a high-risk subset of T-ALL, highly ex vivo when expressed in NK-92MI cells. The bivalent express CD7 [84–86]. Two clinical trials have been initi- CD7-CAR-modified-NK-92MI cells exhibited slightly ated in China studying CD7-CAR-modified T cells for the greater cytotoxicity compared to that of the monovalent treatment of CD7-positive malignancies (NCT04033302 CAR-modified cells, and significantly inhibited disease pro- and NCT04004637). However, preclinical studies showed gression in a T-ALL PDX model when compared to naïve significantly reduced expansion of CD7-CAR T cells com- unmodified NK-MI cells. pared to control T cells, as a result of fratricide [45, 49]. Fratricide appears to be observed to a greater extent in CD4 CD7-CAR T cells compared to CD5-CAR T cells [45]. It Most cancers derived from lineage-differentiated T cells is hypothesized that this is due to a more incomplete in- are likely to be of CD4-positive origin, making CD4 a ternalization mechanism of CD7 from the cell surface fol- potential target for CAR therapy. A preclinical study was lowing ligation of the antigen with an anti-CD7 scFv. performed to consider the cytotoxicity of CD4-CAR- CRISPR-Cas9 editing of CD7 from the cell surface of T modified T cells against T-ALL tumors in NSG mice. cells prior to CAR expression demonstrated a superior This study also included the use of alemtuzumab to method of developing CD7-CAR T cells. These cells ex- clear the CAR T cells as a safety mechanism. NSG mice hibited limited fratricide, expanded in vitro, and showed were injected with luciferase-expressing Jurkat T cells no evidence of impaired cytotoxicity in vitro nor in vivo. and subsequently treated with naïve T cells or CD4- Investigations in a T-ALL mouse xenograft model re- CAR-modified T cells. CAR-treated mice displayed a vealed a statistically significant prolonged survival of CD7- survival advantage and an ~ 80% reduction in tumor edited CD7-CAR-treated mice compared to survival of burden compared to mice treated with naïve T cells. control mice [45]. Based on these results, a phase I clinical CD4-CAR-modified T cells were also injected into mice trial has been initiated testing CD7-CD28-CAR T cells in to evaluate the ability of alemtuzumab to effectively T-ALL patients (NCT03690011). Additionally, a UCART7 eliminate CAR-modified T cells. Alemtuzumab was Fleischer et al. Journal of Hematology & Oncology (2019) 12:141 Page 7 of 21 administered 24 h post-CAR T cell injection. A > 95% expression of CD30 can also be found on a subset of depletion of CD4-CAR-modified T cells was observed PTCLs, including ALCL [92–94, 96]. One study demon- within 6 h following injection signifying the use of alem- strated that CD30 expression is upregulated during tuzumab as a safety mechanism for CAR T cell therapy chemotherapy regimens in T-ALL patients. Of 34 T- [87]. Additionally, a phase I clinical trial to assess the ALL patients, approximately 38% had CD30-positive T- safety and feasibility of CD4-CAR T cell infusions in pa- ALL [96]. Therefore, some T-ALL patients who relapse tients with relapsed/refractory T cell lymphoma and T following chemotherapy may still respond to CD30- cell leukemia has been initiated (NCT03829540). directed CAR therapy. However, expression of CD4 on T cells can complicate Preclinical studies have previously demonstrated CD4-CAR T cell therapy as previously described. NK-92 CD30-CAR T cell capacity for lysing tumor cells [97, 98] cells are inherently CD4-negative, and therefore the use and numerous clinical investigations into CD30-CAR T of NK-92 cells as opposed to T cells reduces risk of frat- cell therapy have been launched with encouraging re- ricide and avoids the need for further modifications. sults. Eleven phase I/II trials treating patients with Additionally, it abrogates the risk of aplasia of CD4- CD30-positive malignancies are currently active positive cells that can occur with long-term engraftment (NCT01316146 [55], NCT01192464, NCT03049449, of CAR T cells. Anti-CD4-CAR NK-92 cells have shown NCT02690545 [62], NCT02958410, NCT02663297, in vitro success eliminating PTCL cell lines and both NCT03383965, NCT02917083 [64], NCT04008394, adult and pediatric primary cells. Using a xenograft NCT02259556 [63], and NCT03602157). To date, no model in NSG mice, CD4-CAR NK-92 cell-treated mice toxicities related to CAR T cell infusion nor impaired demonstrate significantly prolonged survival compared immunity against common viruses has been reported to control-modified NK-92 cell-treated mice [54]. from these trials. However, one trial reported that the in vivo CAR T cell expansion and persistence was re- CD37 duced following subsequent infusions compared to those CD37 is a member of the tetraspanin superfamily with ex- following initial doses [55]. The decreased persistence of pression limited to lymphoid tissues, particularly B cells the CAR T cells may have prevented the development of [88, 89]. CD37 expression in cancer cells is typically char- severe adverse events such as CRS and neurotoxicity that acteristic of B cell malignancies; however, its expression are commonly observed following CAR T cell infusion. can be found in some cases CTCL and PTCL [90, 91]. Of the two ALCL patients in this trial, one patient was Since CD37 is not expressed in T cells, there is no evi- non-responsive to the therapy, while the other entered dence of fratricide occurring in anti-CD37 CAR T cells. complete remission lasting 9 months [55]. Results from However, in the presence of CD37-positive PTCL cell another phase I trial in China for patients with relapsed/ lines, CD37-CAR T cells exhibit increased activation and refractory CD30-positive lymphomas (NCT02259556) degranulation as well as specific cytolytic activity in vitro corroborate the limited toxicity and anti-tumor activity [56]. The restricted expression of CD37 makes it a safer of CD30-CAR T cells [63]. target for CAR T cell therapy, given there would be no concern of T cell aplasia. Additionally, CD37 is not TRBC1 expressed in NK cells, providing an opportunity to utilize T cells express the αβ TCR; the β-chain can either be NK cells as effector cells in place of T cells. The versatility encoded by the T cell receptor beta constant 1 (TRBC1) of CD37-CARs to treat B cell and T cell lymphomas sug- gene or TRBC2 gene [99, 100]. Therefore, expression of gests that this may be an important target for further in- TRBC1 and TRBC2 is mutually exclusive. Additionally, vestigations. While CD37 is predominantly being CD4- and CD8-positive T cell populations express both examined for dual targeting for B cell malignancies, the subsets and CD8-positive T cell populations specific for target has potential for CAR therapy against T cell common viruses also contain both TRBC1 and TRBC2 malignancies. cells [58]. However, as malignant T cells develop from a single cell, the entire population of cancerous cells will CD30 be either TRBC1- or TRBC2-positive. Numerous T cell CD30, a member of the tumor necrosis factor receptor malignancy cell lines and primary samples have been an- (TNFR) superfamily, promotes T cell proliferation and alyzed by flow cytometry to validate the homogeneity of cytokine production following TCR stimulation, while β-chain expression in a malignant cell population [58]. also having an opposing role in promoting apoptosis Many cancer cells downregulate the αβ TCR; however, it [92]. Expression is limited to a subset of activated lym- is expressed on > 95% of PTCLs [101] and > 30% of T- phocytes found around the follicular regions of lymphoid ALLs [102]. tissues [93–95]. While CD30 is well known for its strong Anti-TRBC1 CAR T cells exhibited specific and effi- expression in virtually all classical Hodgkin lymphoma, cient cytotoxicity against the JKO T cell line transduced Fleischer et al. Journal of Hematology & Oncology (2019) 12:141 Page 8 of 21 with TRBC1, but not against non-transduced cells or remaining at 5:1 effector to target ratios. Furthermore, cells transduced with TRBC2, even in a mixed popula- CD3-CAR NK-92-treated T-ALL NSG mice exhibited tion. Furthermore, in primary samples from patients prolonged survival with ~ 87% reduced tumor burden with T cell malignancies, the anti-TRBC1 CAR T cells through day 23 [52]. preserved a significant fraction of healthy T cells (TRBC2 cells), thereby circumventing a limitation of CD1a CAR T cell therapy for the treatment of T cell malignan- CD1a is a lipid-presenting molecule whose expression is cies [58]. In an NSG mouse model using TRBC1- restricted to developing cortical thymocytes, skin Lang- positive Jurkat T cells to establish cancer, mice treated erhans cells, and some circulating myeloid dendritic cells with the anti-TRBC1 CAR T cells exhibited reduced [103, 104]. Neither T cells nor CD34 hematopoietic tumor burden and elongated survival. In additional pre- progenitors express CD1a, making it a fratricide- clinical studies, NSG mice were injected with both resistant target, while limiting the risk of on-target/off- TRBC1 and TRBC2 cancer cells, and then treated with tumor toxicity. Expression in T cell malignancies is only either naïve T cells or anti-TRBC1 CAR T cells. TRBC1- limited to cortical T-ALL, a major subset of T-ALL ac- positive Jurkat T cells could not be detected in mice counting for ~ 35–40% of all T-ALL cases [105, 106]. A treated with anti-TRBC1 CAR T cells; however, TRBC2- study showed that CD1a-CAR T cells expanded without positive cells were identified. This is in contrast to mice fratricide, and had long-term persistence in an in vivo treated with naïve T cells, whose bone marrow con- model [57]. Additionally, these cells demonstrated spe- firmed the presence of both TRBC1-positive and cific cytotoxic activity against CD1a-positive T-ALL cell TRBC2-positive cells [58]. Thus, targeting TRBC1- lines and primary blasts in vitro, and exhibited potent positive malignant cells offers a unique approach to anti-leukemic activity in a PDX model of cortical T- avoiding T cell aplasia, a consequence of many proposed ALL. Thus, while not applicable to all T cell malignan- CAR T cell therapies for the treatment of T cell cies, targeting CD1a with CAR T cells may be successful malignancies. in the specific subset of cortical T-ALL cases. CD3 “Off-the-shelf” CAR T cell therapy CD3 is a pan T cell marker comprised of four distinct One of the greatest challenges in utilizing autologous polypeptide chains, epsilon, gamma, delta, and zeta, CAR T cell therapy for the treatment of T cell malignan- which form pairs of dimers, transmitting T cell activa- cies is the separation of healthy T cells from malignant tion signals. As CD3 is exclusively expressed on T cells, T cells, in order to generate a CAR T cell product that is it has been a popular target in preclinical CAR T cell not contaminated with cancerous T cells. To date, there therapies for the treatment of T cell malignancies. As has been one reported case from the University of Penn- expected, due to fratricidal issues, manufacturing of sylvania of CD19-CAR modification of a single leukemic anti-CD3 CAR T cells does not yield a viable cellular B cell, resulting in CD19-positive relapse and ultimately product [61]. Various approaches using an anti-CD3 death of the patient [39]. This task of isolating healthy T CAR have been investigated including the use of tran- cells is even more difficult when a proportion of the pa- scription activator-like effector nuclease (TALEN) tient’s T cells are malignant, especially in cases of T cell mRNA to disrupt the TRAC locus and using NK-92 cells leukemia where there is a high likelihood of circulating in place of T cells as the effector cell type. Disruption of cancerous T cells. Thus, manufacturing of autologous the TRAC locus prevents assembly of the TCRαβ/CD3 CAR T cells for the treatment of T cell malignancies has complex, allowing for anti-CD3-CAR expression without a very high likelihood of resulting in CAR-modified compromising cellular proliferation and viability. Enrich- leukemic cells. This would likely result in relapse as ment of the CAR-positive, CD3-negative population was these cells would likely escape recognition by normal observed. In patient T-ALL samples, anti-CD3 CAR T CAR-T cells. cells demonstrated specific cytotoxicity against CD3- Additionally, there remain numerous challenges to positive cells. In a T-ALL NSG model, anti-CD3 CAR T using a patient’s own cells to manufacture CAR T cells. cells were shown to clear luciferase-expressing CD3- Patients with advanced disease undergoing CAR T cell positive Jurkat cells, but showed no effect in NSG mice therapy typically are heavily pre-treated, having previ- engrafted with CD3-negative Jurkat cells [61]. To cir- ously undergone numerous rounds of chemotherapy, cumvent the need for additional modifications, NK-92 which can result in low T cell counts and/or T cells that cells can also be used to express the anti-CD3-CAR, may not be healthy enough to expand well making it since they are CD3-negative cells. CD3-CAR NK-92 cells very difficult to manufacture an efficacious CAR T cell demonstrated efficient ex vivo lysis of PTCL primary product [107]. This issue is much more prevalent in samples, resulting in less than 0.5% lymphoma cells adult patients due to the decreasing proportion of naïve Fleischer et al. Journal of Hematology & Oncology (2019) 12:141 Page 9 of 21 T cells associated with aging [107–110]. Additionally, severe side effects [46]. TALENs, an alternative genome given that many of these patients have advanced disease, editing technique, have also been used to prevent ex- a patient may experience disease progression, co- pression of the TRAC locus in order to limit fratricide of morbidities, or even death in the time it takes to manu- anti-CD3-CAR T cells and prevent MHC-recognition of facture autologous CAR T cells. This is especially true in foreign host cells. Genome editing the TRAC locus pre- most relapsed T cell malignancies, which tend to be ag- vents stable assembly of the TCRαβ/CD3 complex. Dis- gressive and chemo-resistant in nature. Lastly, each ruption of the TRAC locus using TALEN mRNA prior starting autologous T cell product is different—variable to transduction with an anti-CD3-CAR lentiviral vector function, maturation, CD4/CD8 ratios, and phenotypic yielded CAR T cells that proliferated well and greatly re- ratios—and the heterogeneity of each individual product duced tumor burden in an NSG mouse model of human has led to unpredictable results and variable potency of leukemia [61]. the therapy. As described above, PEBLs have been recently devel- An alternative to autologous CAR T cell manufactur- oped to selectively prevent expression of individual pro- ing is the use of allogeneic T cells as the cell source. In teins. PEBLs have been shown to effectively retain CD3ε order to make this approach feasible, expression of the in the ER/Golgi to prevent MHC recognition of host endogenous αβTCR in allogeneic CAR T cells must be cells during allogeneic use of anti-CD19 CAR T cells blocked as it would likely result in GvHD, unless the [50]. Disruption of TCRαβ signaling had no effect on T donor is a human leukocyte antigen (HLA) match. This cell proliferation. There was no evidence of GvHD in an process involves leukapheresis from a healthy donor, NSG mouse model of leukemia treated with the PEBL- followed by isolation of the donor’s T cells. Following CD19-CAR T cells, whereas 60% of the mice treated transduction of the T cells with a CAR-encoding retro- with CAR T cells that were not expressing the CD3ε viral vector, subsequent genome editing of the TRAC PEBL developed GvHD. Furthermore, both PEBL and locus is required to prevent expression of the endogen- CAR can be expressed from the same vector using a 2A ous TCR. Cells that remain TCR-positive are then de- sequence, resulting in only one transduction of the cells pleted from the expanded CAR T cell product prior to [50]. While this study utilized PEBL in conjunction with cryopreservation. This creates an “off the shelf” cellular an anti-CD19-CAR, this system can potentially be ap- product that can be banked until it is needed for ther- plied with other CAR constructs to target T cell apy. This approach resulted in successful remission in antigens. two infant B-ALL cases treated with allogeneic CD19- CAR T cells modified at the TRAC and CD52 loci. The Alternative effector cell types allogeneic CAR T cells persisted until conditioning for While CAR-modified αβ T cells can have a memory stem cell transplant [111]. Another group utilized phenotype resulting in T cell aplasia, NK cells and shRNA to knock down β2-microglobulin in conjunction gamma delta (γδ) T cells will not. Utilizing these innate with a knock-in strategy to insert a CD19-CAR into the cells for CAR therapy is a viable alternative that groups TRAC locus. Knock down of β2-microglobulin reduces are exploring. One disadvantage to preventing memory the ability of class I HLA molecules to form heterodi- cell formation and using effector cells with limited per- mers on the cell surface. Reducing expression of both sistence is reduced tumor control. However, this limita- β2-microglobulin and TRAC resulted in decreased allo- tion can potentially be overcome by utilizing these cells geneic attack by CD8 T cells and NK cells [112]. This in multiple dosing regimens. Repeated dosing of short- strategy may be useful to reduce allo-recognition in pa- lived CAR-expressing cells can be used to induce remis- tients receiving CAR T cell therapy. Other groups have sion; thus, providing a bridge to an allogeneic exploited similar approaches in preclinical CAR T cell hematopoietic stem cell transplant (HSCT) if needed. investigations targeting CD7 and CD3, as previously de- Since these products would be utilized in an allogeneic scribed [46, 61]. setting, they can be cryopreserved and would be readily CRISPR-Cas9 genome editing has become a popular available when needed for use. technique to prevent gene expression or to correct gene expression. One study targeting CD7 generated “fratri- Natural killer cells and NK-92 cells cide resistant, allo-tolerant” CAR T cells using CRISPR- Ex vivo-expanded NK cells are short-lived, and do not Cas9 to disrupt both CD7 and the TRAC loci (UCAR persist for extended periods of time in vivo compared to T7). NSG mice engrafted with primary T-ALL blasts de- that of αβ T cells [113]. CAR-modified NK cells have a veloped GvHD when treated with wildtype donor T turnover time of 1–2 weeks; therefore, there is reduced cells; however, mice treated with UCART7 donor cells concern of aplasia of antigen-expressing cells [114]. Cur- were able to clear the tumor cells from the peripheral rently, there are two active clinical trials using anti- blood, and, furthermore, did not develop GvHD or other CD19-CAR-modified NK cells (NCT00995137 and Fleischer et al. Journal of Hematology & Oncology (2019) 12:141 Page 10 of 21 NCT01974479). Additionally, some studies use NK-92 from a NK cell lymphoma, they require irradiation prior cells, an IL-2-dependent NK-lymphoma-derived cell line. to infusion into a patient to prevent expansion, resulting NK-92 cells are often used as an alternative to primary in persistence for about 1 week in vivo and potentially NK cells due to their ease of expansion under current exhibiting reduced cytotoxicity. Alternatively, suicide good manufacturing process (cGMP) conditions [115] mechanisms can be engineered into the cells to elimin- and transfection with CAR mRNA [116]. CAR-modified ate the risk of NK-92-cell persistence in vivo and elimin- NK or NK-92 cell infusion can result in tumor cell clear- ate the need for irradiation, thereby resulting in greater ance without the risk of GvHD. Therefore, these cells cytotoxicity of the infused cells. typically only require one genetic modification. Add- NK cells exhibit their cytotoxic activity through numer- itionally, with the exception of CD7, NK cells do not ex- ous means, including expression of FasL or TRAIL, secre- press antigens targeted in T cell malignancies. tion of perforin and granzyme, as well as through Therefore, neither fratricide nor T cell aplasia are of pri- antibody-dependent cellular cytotoxicity (ADCC) mecha- mary concern. nisms [131, 135, 136]. A major limitation to the use of CAR-expressing NK-92 cells have been extensively CAR T cells is antigen escape; however, as NK cells can assessed in preclinical studies targeting various cancers such kill through other mechanisms, downregulation of the as B cell malignancies [117–119], multiple myeloma [120], cognate antigen on tumor cells may not halt anti-tumor acute myeloid leukemia (AML) [121], breast carcinoma activity. NK cells also express the natural killer group 2D [122, 123], neuroblastoma [124], and glioblastoma [125]. As (NKG2D) receptor, which recognizes cellular stress li- previously discussed, multiple groups have initiated preclin- gands such as MHC class I chain-related protein A/B ical studies using CAR-modified NK-92 cells for the treat- (MICA/B) and UL16 binding proteins (ULBPs) [137, 138], ment of T cell malignancies, targeting antigens such as CD5, resulting in cytotoxicity against exceedingly stressed cells. CD7, CD4, and CD3, demonstrating reduced tumor burden As NK cells do not recognize targets on healthy cells, they and an overall survival benefit in NSG mouse models of T have limited off-target toxicity [131]. Additionally, their cell leukemia [47, 52–54, 60]. The safety and efficacy of NK- serial killing capability allows each individual NK cell to 92 cells has been evaluated in clinical trials displaying a good kill, on average, four tumor cells [139]. However, NK cells safety profile with few mild to moderate adverse events are notoriously difficult to expand ex vivo, transduce with [126–128] (NCT00900809, NCT00990717). To date, five viral vectors, cryopreserve, and they have limited life span clinical trials have been initiated involving infusion of CAR- in vivo [128, 140]. While autologous NK cells can be ob- modified NK-92 cells targeting a variety of antigens, includ- tained by leukapheresis followed by selection of CD56- ing CD33 [129], human epidermal growth factor receptor 2 positive cells, allogeneic NK cells derived from a third (HER2), B cell maturation antigen (BCMA), CD19, and the party donor requires an additional step for depletion of T cell antigen, CD7 (NCT02944162, NCT03383978, alloreactive T cells from the donor product [141]. NCT03940833, NCT02892695, and NCT02742727). Purification and expansion of NK cells from peripheral Inherent NK-cell cytotoxicity is dependent on the bal- blood mononuclear cells (PBMCs) have been optimized in ance of activating and inhibitory killer-cell cGMP protocols to clinically relevant numbers [142–144]. immunoglobulin-like receptor (KIR) signals. Inhibitory This is a time-consuming process as only 10% of PBMCs and activating KIRs on NK cells form a balance, as there are NK cells [145]. However, recently developed methods are often signals from both inhibitory and activating re- are being used to enhance NK-cell expansion, such as ceptors. The inhibitory signals predominate, typically through K562-feeder cell expression of OX40 ligand through higher affinity for their ligands; however, strong [146]. As mentioned above, a limitation to CAR-NK ther- activating signals can override the inhibitory signals, li- apy is the extreme sensitivity of NK cells to cryopreserva- censing NK cells to kill. If donor inhibitory KIRs do not tion. They have demonstrated poor viability and recognize patient HLA, there is reduced inhibitory sig- diminished cytotoxicity after cryopreservation. While naling to counteract the activating signaling [130, 131]. cytotoxicity can be restored to normal levels after a few While NK-92 cells lack many of the inhibitory KIRs days in culture with exogenous IL-2, the low viability expressed on primary NK cells, they have a wide range post-cryopreservation remains a concern [141]. of activating receptors [132]. Similar to NK cells, NK-92 cells have the capability to produce perforin and Gamma delta T cells granzyme upon activation, as well as display cytotoxic While αβ T cells function as a part of the adaptive im- activity through upregulation of TNF-related apoptosis- mune system, γδ T cells play roles in both the innate and inducing ligand (TRAIL), Fas ligand (FasL), and TNFα the adaptive immune systems. γδ T cells and αβ Tcells [133]. Additionally, NK-92 cells have demonstrated evi- originate from two distinct T cell lineages [224]. γδ Tcells dence of serial killing, with each cell killing numerous are the only innate immune cells expressing a TCR [147]; target cells [134]. However, as NK-92 cells were derived however, their target recognition is independent of MHC Fleischer et al. Journal of Hematology & Oncology (2019) 12:141 Page 11 of 21 recognition [148, 149]. Lack of MHCI- and MHCII- functioning as immune-surveillance of epithelial tissues restriction make γδ T cells optimal candidates for allogen- by scanning for inflammatory threats [161, 162]. The γδ eic cell therapy. The peripheral blood subset of γδ Tcells TCR recognizes self-antigens that serve as endogenous known as Vγ9Vδ2 T cells represents the most commonly danger signals such as heat shock proteins, which are studied subset in this context. Studies by our group have upregulated in cells with increased metabolism, like can- demonstrated that similar transduction efficiencies can be cer cells. Expression of scavenger receptors like the achieved in Vγ9Vδ2 T cells grown under cGMP serum- NKG2D receptor enables γδ T cell activation through free conditions as are achieved in αβ T cells using lenti- the interactions with antigens of cellular stress such as viral vectors. Additional studies were performed revealing MICA/B and ULBPs [147, 163–166]. Additionally, γδ T peak low-density lipoprotein receptor (LDL-R) expression cells express chemokine receptors that can detect che- on days 6–8of γδ T cell expansion [150]. As LDL-R is the mokines secreted by cancer cells, likely facilitating their major receptor for VSV-G-pseudotyped lentiviral vectors, migration toward the tumor site [167]. γδ T cells also this data suggests that greater transduction efficiency can express FasL (CD95L) as a means of recognizing Fas ex- be achieved on these days using lentiviral vectors com- pression on tumor cells and initiating apoptosis [168]. pared to earlier or later in the expansion [151]. Another mechanism by which γδ T cells recognize tumor To date, numerous preclinical studies have evaluated cells is through stimulation by phosphoantigens, such as CAR-modified γδ T cells targeting neuroblastoma [152, isopentenyl pyrophosphate (IPP), which are recognized by 153], melanoma [154], B cell malignancies [153, 155], and the γδ TCR. While there are many subsets of γδ T cells, epithelial cell adhesion molecule (epCAM)-positive adeno- phosphoantigens specifically expand the Vγ9Vδ2subset. carcinomas [156]. GD2-CAR-modified γδ T cells expressing IPPisusedasasubstratein the mevalonate pathwaybyfar- the RQR8 suicide gene were shown to expand 2.5-fold upon nesyl pyrophosphate synthase (FPPS). Bisphosphonates antigen exposure [152]. Furthermore, both GD2-CAR- and overproduced in cancer cells block FPPS, resulting in a CD19-CAR-modified γδ T cells were demonstrated to se- buildup of IPP, which is subsequently recognized by cyto- crete pro-inflammatory cytokines in the presence of GD2- toxic Vγ9Vδ2 T cells [169–172]. Bisphosphonate stimula- or CD19-expressing tumor cells, respectively [153]. While tion of γδ T cells has been applied to in vitro expansion of these studies utilized viral vectors to express the CAR, elec- γδ T cells in conjunction with IL-2 in serum-free condi- troporation of a Sleeping Beauty transposon has also been tions [150]. A preclinical study involving nude mice receiv- showntoresultinCD19-CARexpressionin γδ T cells, ing repeated dosing of γδ T cells resulted in decreased resulting in anti-tumor cytotoxicity in both the in vitro and tumor growth model; however, tumor growth resumed in vivo settings [155]. Additionally, expression of a CAR tar- upon completion of the γδ Tcell infusions [173]. In phase I geting melanoma-associated chondroitin sulfate proteogly- clinical trials, adoptive transfer of γδ T cells to patients re- can (MCSP) was established in γδ T cells using mRNA ceiving ex vivo expanded γδ T cells with a combination of transfection. Despite comparable anti-tumor cytotoxicity, IL-2 and bisphosphonate stimulation demonstrated the lower cytokine secretion was observed in MCSP-CAR- safety of the infused product and suggested that the therapy modified γδ T cells compared to that from conventional could be efficacious in slowing the progression of the dis- CAR-modified αβ T cells [154]. Reduced pro-inflammatory ease. However, mixed results were seen in terms of efficacy, cytokine secretion is favorable due to anticipated reduced suggesting that genetic modification with CAR expression severity of CRS. Lastly, epCAM CAR-modified γδ Tcells is likely to be more beneficial compared to γδ Tcelltherapy demonstrated high levels of in vitro cytotoxicity of tumor alone [169, 174–177]. cell lines when γδ T cells were both fresh and cryopreserved While the autologous transfer of CAR-modified αβ T [156]. These studies pave the way for additional trials using cells targeting a T cell malignancy can be used as a CAR-modified γδ T cells targeting T cell malignancies. They bridge to transplant (although the risk remains that a demonstrate that engineering of γδ T cells is feasible and re- single CAR T cell will be left behind ultimately resulting sults in enhanced in vitro and in vivo cytotoxicity upon in the development of T cell aplasia), it cannot be a CAR expression. curative option unless a near perfect design of a suicide CAR-modified γδ T cells may be able to overcome the gene, switch mechanism, or another system has been im- obstacle of antigen escape seen in some treatment- plemented to reliably eliminate all CAR T cells upon resistant cases by relying on their innate ability to completion of the treatment. Therefore, effector cells recognize tumor cells through other means. Naïve γδ T with a limited lifespan such as γδ T cells, NK-92 cells, or cells have been shown to have anti-tumorigenic activity NK cells are likely to be more effective in targeting T against leukemia, neuroblastoma, and colon cancer cell cell disease. Other techniques such as mRNA electropor- lines as well as primary cancer cells in vitro [157–160]. ation or adeno-associated viral (AAV) vector delivery They are found in peripheral blood, spleen, and lymph can also be useful in preventing long-term CAR T cell nodes, in addition to almost all mucosal tissues, persistence, as described below. Fleischer et al. Journal of Hematology & Oncology (2019) 12:141 Page 12 of 21 Prevention of memory cell formation and T cell using mRNA electroporation to deliver the CAR into T aplasia cells demonstrated the safety and efficacy of this treatment While current CAR T cell therapies for the treatment of in four relapsed/refractory classical Hodgkin lymphoma B cell malignancies have been hugely successful in indu- patients [225]. CAR mRNA was detected 48 h post- cing and maintaining remission, these therapies have infusion; however, no mRNA could be detected by day 21. prevented the re-emergence of endogenous B cells in pa- While only transient responses were seen, no severe toxic- tients in whom the CAR T cells have persisted. The ities were observed using this approach. Utilizing this CAR T cells can have a memory phenotype that allows non-viral strategy in T cell disease can be particularly ad- them to remain dormant until restimulation with the vantageous, as it prevents the risk of long-term T cell cognate antigen, CD19, expressed on all endogenous B aplasia. While the transient efficacy precludes this ap- cells. While B cell aplasia is an undesirable side effect of proach from being used as a definitive treatment, it could these therapies, it has been managed by continued peri- potentially serve as an effective bridge to transplantation. odic intravenous immunoglobulin injections [36]. The long-term implications of persistent B cell aplasia remain Adeno-associated viral vector unknown. In contrast, treatment of T cell malignancies AAV is an alternative viral delivery method that can over- using CAR T cells targeting antigens expressed on the come some of the disadvantages of using integrating viral majority of normal T cells is predicted to result in T cell vectors as previously discussed. AAV is a single-stranded, aplasia. While B cell aplasia is tolerable, there is no such non-enveloped DNA virus with a cargo capacity of ap- treatment for T cell aplasia. Patients who develop T cell proximately 4.7 kilobases [188]. Upon deletion of the Rep aplasia will have profound immunosuppression and can protein, the viral transgene forms circular concatamers potentially succumb to deadly infections [36]. Therefore, that exist episomally in the nucleus of the cell. AAV ex- prevention of memory cell formation of CAR T cells and pression is therefore diluted upon each mitotic division, subsequent T cell aplasia remains an essential challenge resulting in a transient transgene expression limited to the to translating CAR T cell therapy for the treatment of T lifespan of the cell [189, 190]. Thus, AAV delivery can cell malignancies. While bridging a patient to an allogen- control the duration of CAR expression, which is a desired eic HSCT following CAR T cell therapy may eliminate quality to regulate cytokine production and mediate toxic- the risk of life-threatening T cell aplasia by clearing out ities [191–193]. In particular, transient CAR expression the CAR T cells, safer and less invasive alternatives must may prove to be advantageous in the setting of T cell ma- also be explored to downregulate CAR activity after lignancies, by preventing unintended T cell aplasia. tumor clearance. Efficient transduction of innate immune cells, such as NK cells and γδ T cells, by an AAV vector would be par- mRNA electroporation ticularly invaluable in targeting this group of diseases. There are numerous disadvantages to using retroviral vec- As previously discussed, both NK cells and γδ T cells tors for CAR T cell therapy, including risk of clonal dom- are excellent candidates as CAR effector cells against T inance [178, 179], high cost of production [180], cell antigens. A common challenge reported in using maximum cargo size [181, 182], and the inability to “turn these cell types is the low transduction efficiency using off” transgene expression and unpredictable integration integrating viral vectors, delaying progress in the devel- sites potentially resulting in insertional oncogenesis [183, opment of these therapies. AAV gene transfer of a CAR 184]. The indefinite period of CAR expression can result into innate immune cells would offer the opportunity to in severe on-target off-tumor toxicities, which is particu- develop an allogeneic off-the-shelf CAR therapeutic that larly challenging to manage in T cell disease. To overcome can control CAR expression, thereby mitigating CRS and these unintended side effects, groups are alternatively ex- other adverse events. Additionally, the lack of memory ploring delivery of CAR mRNA through electroporation cell formation against T cell antigens in these cell types as a safer method [185–187]. As with the use of effector will completely negate the risk of T cell aplasia. The cells with limited persistence in vivo, therapies with transi- AAV capsid directs the infectivity of different tissues, ent CAR expression require multiple infusions into the and therefore the appropriate capsid serotype must be patients. Use of mRNA electroporation of T cells for used to maximize transduction of the desired cell type CD19-CAR expression has been reported in a preclinical [194, 195]. AAV6 has been shown to result in higher model, demonstrating reduced tumor burden 1 day post- transduction of hematopoietic stem and progenitor cells treatment. This study illustrated prolonged survival of a than have other serotypes [196–198]. xenograft mouse model after a single injection of CAR mRNA T cells; however, as predicted, as the mRNA levels Suicide genes and safety switches decreased the tumor burden increased [185]. Published re- While the motivation behind the incorporation of sui- sults from the first non-viral CD19-CAR clinical trial cide genes and switches into CAR constructs was to Fleischer et al. Journal of Hematology & Oncology (2019) 12:141 Page 13 of 21 mediate the severe adverse events commonly reported provide control to this therapy and increase the safety following extensive expansion of CAR T cells, they can profile [214]. In addition to CD19-CARs, iCas9 has been also serve an alternative purpose. Using pharmacologic included in other CAR constructs including an anti- agents, the apoptotic pathway in CAR T cells can be ac- CD20-CAR, demonstrating enhanced tumor clearance tivated, triggering selective cell death of the effector in vivo and a 90% reduction in CAR T cells in the per- cells, without destroying bystander cells. Therefore, they ipheral blood of mice following activation of the iCas9 can be valuable in the setting of T cell malignancies as suicide gene, compared to CAR T cells detected in per- they can prevent T cell aplasia. There are three main ipheral blood of control mice [215]. Additionally, a classes of suicide gene technologies, classified by the GD2-CAR including the iCas9 gene is being assessed for mechanism of action of the incorporated gene. They (i) the treatment of neuroblastoma (NCT01822652), sar- convert non-toxic compounds to toxic drugs via meta- coma (NCT01953900), osteosarcoma, and melanoma bolic pathways [199–202], (ii) induce dimerization of in- (NCT02107963) in phase I clinical trials. ducible caspase-9 [203, 204], or (iii) mediate ADCC In terms of utilizing ADCC for CAR T cell clearance, using monoclonal antibodies [205–207]. Co-expression administration of alemtuzumab, an anti-CD52 antibody of the suicide gene with the CAR in a bicistronic vector commonly used in lymphodepleting regimens, has been would result in two populations of cells—those that ex- tested in several studies. Specifically, alemtuzumab has press both the CAR and the suicide gene, and those that been assessed for CD4-CAR T cell elimination following express neither. This strategy negates the risk of generat- tumor cell eradication in NSG mice to prevent T cell apla- ing a CAR-positive population without the safety trans- sia [87]. Within 6 h following alemtuzumab infusion, > gene; thus, enabling one to confidently eliminate the 95% of the CAR T cells had been depleted. This approach entire CAR-positive population and thereby, in the con- was also tested in two other preclinical CAR studies tar- text of targeting T cell antigens, controlling T cell geting AML, both showing excellent results [216, 217]. aplasia. Multiple groups have also evaluated the retroviral transfer The first reported suicide gene utilized the herpes sim- of human CD20 into T cells as a novel suicide gene mech- plex virus thymidine kinase (HSV-TK) as a method of anism for adoptive T cell therapy. Their data supports that GvHD abrogation in the context of an allogeneic HSCT. infusion of the anti-CD20 antibody, rituximab, an ap- Expression of HSV-TK in donor lymphocytes prior to proved antibody for in vivo therapeutic applications, re- their infusion into a HSCT patient allows for selective de- sults in efficient, specific elimination of CD20-positive T pletion of the donor lymphocytes in patients that devel- lymphocytes through ADCC [205, 206, 218]. Studies have oped signs of GvHD upon administration of ganciclovir also demonstrated that rituximab can eliminate CD20- [199–201]. Metabolism of ganciclovir by the thymidine positive cells in vivo through inducing complement- kinase of HSV-TK results in a toxic substance, ultimately dependent cytotoxicity, a rapid and efficient mode of cell killing the cell [208]. However, there are a couple of limi- death [219]. CD20 co-expression with a CD123-CAR tations to this system including the potential for immuno- demonstrated strong and rapid anti-leukemia activity in a genicity and the slow T cell depletion, which requires human AML mouse model. Upon the infusion of rituxi- about 3 days [209–211]. mab, CAR T cells were cleared and mice were successfully More recently, the safety mechanism gaining the most engrafted with human bone marrow cells, mimicking an attention has been the inclusion of an inducible caspase- allogeneic HSCT [217]. Thus, ADCC-based safety systems 9-based suicide gene (iCas9) into the CAR construct. potentially allow for rapid and efficient elimination of Pharmacologic activation of the iCas9 results in effective CAR T cells [211]. and rapid elimination of CAR T cells. iCas9 inclusion in An epitope-based marker/suicide gene system (RQR8) a CD19-CAR construct has been shown to regulate CAR was recently developed to both track the transduced cells T cells in a dose-dependent manner, allowing for either and selectively deplete them by combining epitopes from control over the CAR T cells to reduce toxicities, or CD34 and CD20 [220]. Use of Miltenyi Biotec’s clinically complete elimination of all CAR T cells to facilitate B approved CliniMACS CD34 system allows for selection of cell reconstitution [212, 213]. This is especially signifi- the CAR-modified T cells while the binding of rituximab cant in cases with severe adverse events, such as GvHD results in ADCC and selective elimination of the adop- or CRS. iCas9 has recently been included in CAR con- tively transferred T cells. Co-expression of RQR8 with an structs containing an IL-15 gene to introduce control anti-GD2 CAR demonstrated selection of CAR T cells over CAR T cell function. The IL-15 gene arms the T with > 95% purity and clearance of > 97% of the CAR- cells to produce IL-15, which, while increasing T cell positive population. This RQR8 system is currently being survival and enhancing specific cytotoxicity, can also re- tested in clinical trials for the treatment of T cell non- sult in unrestricted proliferation and increased toxicity. Hodgkin lymphoma targeting TRBC1 (NCT03590574). Inclusion of an iCas9 gene in these CAR constructs can Another polypeptide that has been designed to facilitate Fleischer et al. Journal of Hematology & Oncology (2019) 12:141 Page 14 of 21 the selection of CAR-positive T cells, tracking of the cells antigen on CAR-modified cells. The identification of in vivo and selective elimination as a safety mechanism, is tumor-specific antigens would greatly enhance CAR the truncated human epidermal growth factor receptor therapy targeting T cell malignancies by avoiding fratri- (huEGFRt). Manipulation of this protein was done to re- cide. To date, only a few antigens with limited expres- move intracellular signaling domains, leaving it with an in- sion on normal T cells have been assessed as CAR tact epitope for binding cetuximab, an anti-EGFR targets to treat T cell malignancies; these include CD30, monoclonal antibody. Modification of T cells with the CD37, and CD1a. However, given their expression on CAR and huEGFRt allows for selection using GMP biotin only small subsets of T cell cancers, a focus on these an- immunomagnetic beads and biotinylated cetuximab, and tigens is unlikely to have a wide-ranging impact on the tracking using flow cytometry or immunohistochemistry. overall translation of CAR therapy for patients with T Upon administration of cetuximab, CAR T cells become cell disease. In contrast, TRBC1 is expressed on a much the targets for ADCC, resulting in in vivo depletion of larger population of T cells and therefore it is likely to CAR T cells. Successful T cell engraftment and ADCC- be found on a comparatively higher percentage of T cell mediated CAR T cell elimination with cetuximab were malignancies. To the best of our knowledge, only one demonstrated in a murine model [221]. The huEGFRt sui- study has evaluated anti-TRBC1-CAR T cell therapy. cide mechanism is currently being assessed in a phase I The data suggests that TRBC1 is a very promising ecto clinical trial in an anti-MUC-16 CAR construct to treat marker for targeting T cell malignancies and the field ecto+ patients with recurrent MUC16 solid tumors would benefit from studies further developing this (NCT02498912) [222]. therapy. A novel alternative approach to suicide genes is the Among other target antigens, CD5 has emerged as a generation of “ON-switch” CARs [223]. In this strategy, promising candidate given its ability to rapidly downreg- the CAR is a split receptor consisting of two distinct ulate from the cell surface upon interaction with the polypeptides: the antigen recognition domain and the CD5-CAR. Therefore, only transient and limited fratri- intracellular signaling domain. In order to act as a func- cide is observed, allowing for successful expansion of tional receptor, the two peptides must first dimerize, CD5-CAR T cells. While targeting CD5 or other T cell achieved through activation by a dimerization-inducing antigens using gene-edited CAR T cells may overcome small molecule. However, antigen stimulation is still re- the issue of fratricide, the concern regarding T cell apla- quired to facilitate a response. The small molecule can sia has not been addressed. The potential for life- be titrated for optimal response, controlling the timing threatening T cell aplasia emphasizes the need for a and dosage of active CAR T cells. Thus, removal of the safety mechanism that is completely effective at elimin- small molecule can reversibly regulate CAR T cell activ- ating CAR T cells following tumor eradication. Safer al- ity. These ON-switch CAR T cells demonstrate specific ternatives other than bridging to an allogeneic HSCT cytotoxicity in vitro and in vivo only when exposed to must be explored to limit CAR T cell persistence. the small molecule. In a mouse xenograft model, mice Adjusting the effector cell type to NK cells, NK-92 cells, treated with ON-switch CAR T cells displayed a reduc- or γδ T cells can limit the risk of a memory cell immune tion in K562 cells engineered to express CD19, only in response against a T cell antigen. However, given that the presence of the small molecule, similar to mice NK-92 cells require irradiation prior to infusion in a pa- treated with conventional CD19-CAR T cells. However, tient, their therapeutic effect may be limited. mRNA no benefit was seen in the absence of the small mol- electroporation or AAV delivery systems, which result in ecule. Given the tight control over CAR expression using transient CAR expression, could be utilized, thereby this innovative approach, it has the potential to be allowing for restoration of normal T cell immunity once adapted for T cell malignancies. the CAR effect has diminished. Additionally, the use of iCas9 and ADCC-based suicide genes, as well as other Summary and conclusions CAR safety switches should be explored in the context CAR therapies targeting CD19 have resulted in unparal- of T cell malignancies. leled success. However, there are many challenges in However, these strategies do not address the pressing translating these therapies beyond the treatment of B issue of isolating normal healthy T cells from malignant cell malignancies. We have highlighted some of these T cells upon leukapheresis, prior to modification with a challenges as it pertains to targeting T cell disease. CAR construct. A perfect system needs to be in place to While numerous antigens have been identified for the prevent transduction of a leukemic blast, a phenomenon treatment of T cell malignancies, targeting of many of that has occurred in a B-ALL patient, resulting in relapse these antigens results in fratricide and T cell aplasia. and ultimately death. In order to eliminate any risk of Multiple gene editing approaches are being evaluated to this event, third party donor cells must be used. Disrup- prevent fratricide by reducing expression of the targeted tion of TCR expression through genome editing of the Fleischer et al. Journal of Hematology & Oncology (2019) 12:141 Page 15 of 21 TRAC locus is required to prevent GvHD, when using concerns. CD7 is an exception as it is expressed on NK allogeneic αβ T cells for CAR expression. However, NK cells and therefore fratricide could occur. While several cells and γδ T cells can both be used in an allogeneic groups have published studies with CAR NK-92 cells setting given their MHC-independent activation, and are targeting T cell malignancies, more effort needs to be thus unlikely to cause GvHD. Use of allogeneic CAR- put into using primary NK cells for targeting this dis- modified cells also addresses the challenges of high cost ease, especially given the limitations of NK-92 cells. and difficulty of production, since healthy donor cells Other, equally promising approaches, such as utilizing can be expanded more easily and cryopreserved as an γδ T cells as the cellular vehicle for CAR therapy repre- off-the-shelf therapy until they are required for use. sents an alternative, less studied approach. Similar to Additionally, allogeneic cell delivery allows for titratable NK cells, γδ T cells are non-alloreactive and are unlikely dosing as well as multiple infusions, if such is required. to form a memory response against a T cell antigen. γδ Many avenues are currently being explored to enhance T cells are likely to succumb to fratricide in certain cir- the safety and efficacy of CAR therapy. However, the cumstances; however, targeting an antigen such as CD5 majority of these strategies do not address all three main that results in only transient and limited fratricide may challenges to utilizing CAR therapy to treat T cell malig- be especially advantageous. Furthermore, γδ T cells ex- nancies. Of the approaches evaluated in this review, only hibit innate MHC-independent mechanisms of cytotox- those incorporating NK cells or NK-92 cells can poten- icity by which they can recognize tumor cells. Thus, tially overcome all of these primary challenges (Fig. 2). CAR therapy using γδ T cells represents an understud- NK cells (i) are non-alloreactive and can be obtained ied avenue with the potential of developing into a super- from healthy donors, eliminating risk of product con- ior cellular product. tamination; (ii) do not form memory responses, prevent- Many advances have been made toward translating ing T cell aplasia; and (iii) do not express the same CAR therapy for the treatment of T cell malignancies. antigen repertoire as T cells, avoiding fratricidal Both academia and industry are focused on the Fig. 2 Venn diagram representing challenges and solutions in targeting T cell antigens with CAR therapy. Each circle represents a hurdle associated with translation of CAR therapy to T cell disease—fratricide, T cell aplasia, and product contamination. As seen in the figure, only the use of NK cells or NK-92 cells as the CAR-effector cell can potentially address all three issues concurrently. However, using NK cells or NK-92 cells comes with its own limitations as previously described. All other approaches require multiple modifications to generate a translatable CAR product to target T cell disease. Potential alternative solutions such as use of γδ T cells as the CAR-effector cell, transient CAR expression with mRNA electroporation or AAV viral delivery, as well as incorporating suicide genes and safety switches, remain largely unexplored. A greater focus on implementing such strategies is required to enable successful translation of this therapy for T cell malignancies Fleischer et al. Journal of Hematology & Oncology (2019) 12:141 Page 16 of 21 identification of tumor-specific antigens to enhance the Author details Molecular and Systems Pharmacology Graduate Program, Graduate Division safety and efficacy of CAR T cell products as well as on of Biological and Biomedical Sciences, Laney Graduate School, Emory the development of superior cellular products. Unfortu- 2 University School of Medicine, Atlanta, GA, USA. Cell and Gene Therapy nately, due to vast variability in the design and execution Program, Department of Pediatrics, Aflac Cancer and Blood Disorders Center, Children’s Healthcare of Atlanta and Emory University School of Medicine, of preclinical studies, it is often difficult to compare the Atlanta, GA, USA. different strategies. However, the numerous preclinical and clinical studies currently underway provide opti- Received: 26 August 2019 Accepted: 9 October 2019 mism for successful translation of this therapy to treat this aggressive and challenging group of diseases. References Abbreviations 1. Belver L, Ferrando A. The genetics and mechanisms of T cell acute AAV: Adeno-associated virus; ADCC: Antibody-dependent cellular lymphoblastic leukaemia. Nat Rev Cancer. 2016;16(8):494–507. cytotoxicity; AITL: Angioimmunoblastic T cell lymphoma; ALCL: Anaplastic 2. Hunger SP, Mullighan CG. Acute Lymphoblastic leukemia in children. 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Targeting T cell malignancies using CAR-based immunotherapy: challenges and potential solutions

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Copyright © 2019 by The Author(s).
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Medicine & Public Health; Oncology; Hematology; Cancer Research
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10.1186/s13045-019-0801-y
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Abstract

Chimeric antigen receptor (CAR) T cell therapy has been successful in treating B cell malignancies in clinical trials; however, fewer studies have evaluated CAR T cell therapy for the treatment of T cell malignancies. There are many challenges in translating this therapy for T cell disease, including fratricide, T cell aplasia, and product contamination. To the best of our knowledge, no tumor-specific antigen has been identified with universal expression on cancerous T cells, hindering CAR T cell therapy for these malignancies. Numerous approaches have been assessed to address each of these challenges, such as (i) disrupting target antigen expression on CAR- modified T cells, (ii) targeting antigens with limited expression on T cells, and (iii) using third party donor cells that are either non-alloreactive or have been genome edited at the T cell receptor α constant (TRAC) locus. In this review, we discuss CAR approaches that have been explored both in preclinical and clinical studies targeting T cell antigens, as well as examine other potential strategies that can be used to successfully translate this therapy for T cell disease. Keywords: CAR, Immunotherapy, T-ALL, T cell lymphoma Introduction (PTCL) [7]. Mycosis fungoides (MF) and Sezary syn- T cell malignancies encompass a heterogeneous group drome (SS) represent the two most common subtypes of of diseases, each reflecting a clonal evolution of dysfunc- CTCL, accounting for the majority of cases [8]. PTCL tional T cells at various stages of development. T cell can be classified into several different subtypes, among acute lymphoblastic leukemia (T-ALL) accounts for 15% which include anaplastic large cell lymphoma (ALCL), and 25% of childhood and adult ALL cases respectively, angioimmunoblastic T cell lymphoma (AITL), extrano- and is the most common form of T cell cancer seen in dal natural killer (NK)-T cell lymphoma (ENKTL), children [1, 2]. T-lymphoblastic lymphoma (T-LLy) is a enteropathy-associated T cell lymphoma (EATL), hepa- non-Hodgkin lymphoma with similar biology to T-ALL. tosplenic T cell lymphoma (HSTCL), and PTCL-not Adult T cell leukemia/lymphoma (ATLL) is an ex- otherwise specified (PTCL-NOS) which is the most tremely aggressive form of blood cancer driven by the common of the group [9, 10]. human T cell lymphocytic virus type 1 (HTLV1) [3–5]. The overall prognosis for T cell malignancies varies Other rare forms of T cell leukemia include T cell large depending on the type of disease, but in general is much granular lymphocytic leukemia (T-LGL) and T- poorer when compared to B cell malignancies. While prolymphocytic leukemia (T-PLL) [6]. T cell lymphomas the survival in T-ALL/LLy has significantly improved are broadly divided into two categories, cutaneous T cell with the intensification of chemotherapy, there still re- lymphoma (CTCL) and peripheral T cell lymphoma main very limited options for patients with relapsed/re- fractory disease [11–13]. ATLL remains a very challenging disease to treat, with a median survival of * Correspondence: sraikar@emory.edu less than 12 months for the acute form of this disease Cell and Gene Therapy Program, Department of Pediatrics, Aflac Cancer and [3–5]. Advanced stage CTCL has a median overall sur- Blood Disorders Center, Children’s Healthcare of Atlanta and Emory University School of Medicine, Atlanta, GA, USA vival of 5 years [14, 15], whereas outcomes of PTCL vary Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Fleischer et al. Journal of Hematology & Oncology (2019) 12:141 Page 2 of 21 depending upon the subtype, with ENKTL, EATL, and not restricted by major histocompatibility complex HSTCL having the poorest prognosis [9, 10]. While im- (MHC) interactions. Therefore, CARs can recognize cell munotherapy has revolutionized the treatment landscape surface proteins that have not been processed and pre- of various cancers with the use of monoclonal anti- sented by antigen presenting cells (APCs). Importantly, bodies, checkpoint inhibitors, bispecific T cell engagers, the interactions between scFvs and ligands have much and chimeric antigen receptor (CAR) T cell therapy, higher affinity and avidity compared to that of TCR- only limited responses have been seen in T cell disease ligand interactions [25]. Furthermore, the immune syn- [15]. Some promising results have been seen with use of apse formed from the interaction between a CAR and its brentuximab vedotin, a CD30-directed immunotoxin, in ligand likely results in a much greater functional avidity CD30-positive PTCL and CTCL [16, 17] and the use of than is observed using a targeted antibody approach pembroluzimab, a programmed cell death receptor 1 with the same antibody (25). (PD-1) inhibitor, in the treatment of ENKTL [18]; how- CARs targeting the B cell antigen CD19 have been ever, these positive results have been limited to very studied extensively for the treatment of B cell malignan- specific subsets of T cell disease. One form of immuno- cies. In 2017, the FDA approved the first CAR T cell therapy that has not yet been successfully translated to therapy, Kymriah, a CD19-directed CAR therapy for the T cell malignancies is that of chimeric antigen receptor treatment of relapsed/refractory B cell acute lympho- (CAR)-based immunotherapy. CAR T cell therapy has blastic leukemia (B-ALL) and in 2018, Yescarta was ap- been extremely successful in relapsed/refractory B cell proved to treat relapsed diffuse large B cell lymphoma malignancies as evidenced by the recent Food and Drug (DLBCL). These therapies, including others in clinical Administration (FDA) approval of two CAR T cell thera- trials, have been widely successful in eliminating malig- peutics for this disease [19–23]. However, implementing nant cells and re-inducing remission in patients who this technology to treat T cell malignancies has been dif- were otherwise treatment-refractory [19–21, 26, 27]. Pa- ficult, primarily due to the lack of a tumor-specific sur- tients receiving CAR therapy undergo leukapheresis face antigen in cancerous T cells. In this review, we will resulting in the collection of T cells, which are subse- discuss the challenges involved in translating this novel quently modified using a lentiviral or retroviral vector to technology to T cell disease, review all the preclinical express the CAR. These cells are expanded ex vivo while and clinical progress made in adapting this therapy for the patient undergoes lymphodepletion, a process in- this challenging disease, and examine potential solutions volving chemotherapeutic agents. Finally, the CAR T for the future development of this innovative therapy. cells are re-infused into the patient [28]. Lymphodeple- tion prior to re-infusion of the autologous T cells has CAR T cell therapy been shown to augment both CAR T cell proliferation as Genetic engineering of primary T cells was first pre- well as persistence [29–31]. The administered dose of sented in the late 1980s [24]. Since then, chimeric anti- CAR T cells and the pre-existing tumor burden do not gen receptor T cells have emerged as a promising appear to be the sole determinants of the degree of T technique for the treatment of relapsed/refractory malig- cell expansion, engraftment, and overall response. Other nancies. CAR therapy brings together numerous fields factors may be involved, such as the density of cognate including immunology, tumor biology, genetic engineer- antigen expression on the cancer cells [32]. However, ing, synthetic biology, and pharmacology. CARs are the optimal degree of persistence of CAR T cells re- comprised of the intracellular signaling domain from the quired to prevent leukemic relapse has not been deter- natural T cell receptor (TCR), CD3ζ, linked to a single- mined [25, 33]. chain variable fragment (scFv) which serves as the anti- One of the mechanisms of relapse post-CD19 gen recognition domain. The scFv sequence is derived CAR T cell therapy is due to surface antigen escape with from a monoclonal antibody by combining the variable relapsed leukemia cells being CD19-negative. The mech- heavy (V ) and light (V ) domains using a small peptide anism may be due to the expansion of a small subset of H L linker. Commonly used CARs also include one or two CD19-negative cancer cells or alternatively, the cells may costimulatory domains, such as CD28, 4-1BB, ICOS, downregulate CD19 from the cell surface in order to and/or OX40. Although the kinetics have yet to be fully evade detection by CAR T cells, rendering them resist- elucidated, it is essential that CAR T cells have mecha- ant [19, 21, 34–37]. Additionally, it was recently shown nisms of trafficking to the tumor site where they can that a phenomenon referred to as trogocytosis is a recognize their cognate antigen. This results in CAR T mechanism of antigen escape whereby the antigen is cell activation and expansion, and ultimately cytolytic transferred to the CAR T cell [38]. It has also been activity against cells expressing the target antigen. CAR- shown that transduction of a single leukemic blast with based ligand recognition is advantageous compared to an anti-CD19 CAR that was re-infused into a B-ALL pa- TCR-based ligand recognition because CAR-targeting is tient, ultimately resulted in relapse and death of the Fleischer et al. Journal of Hematology & Oncology (2019) 12:141 Page 3 of 21 patient [39]. Transduction of the leukemic cell resulted subsequent CAR-modification of tumor cells. Addition- in masking of the target antigen through interactions be- ally, expression of the targeted antigen on CAR T cells tween the CAR and the cognate antigen on the same results in fratricide and limited expansion of the CAR T cell. Clonal expansion of this population resulted in re- cells. Furthermore, targeting of an antigen regularly sistance to CAR therapy. This report emphasized the im- expressed on normal T cells would result in T cell apla- portance of strict and perfect isolation of normal, sia, leading to profound immunosuppression, likely to be healthy T cells for modification with the CAR construct. associated with high rates of morbidity and mortality As we discuss below, this is particularly challenging in T (Fig. 1). cell leukemia patients who are more likely to have circu- Various approaches have been used to overcome these lating cancerous T cells, and therefore have a higher challenges, including CRISPR-Cas9 genome editing to probability of these cells being inadvertently isolated, remove the antigen from the CAR T cells [45–47], Tet- transduced, and re-infused. OFF expression system to limit fratricide during ex vivo Of note, there are severe toxicities that have been as- expansion [48], protein expression blocker (PEBL) to re- sociated with CAR therapy. Cytokine release syndrome tain the antigen in the ER/Golgi to prevent cell surface (CRS) is a systemic inflammatory response directly expression [49, 50], or using CAR-modified natural killer resulting from robust T cell activation following infu- cells instead of T cells [47, 51–54]. Additionally, to date, sion. IL-6 is one pro-inflammatory cytokine that is se- four targets have been investigated as targets for CAR T creted at high levels during CRS. During a particularly cell therapy for the treatment of T cell malignancies with severe CRS condition, tocilizumab, an IL-6R antagonist limited to no expression in the normal population of T monoclonal antibody, was used to rapidly and effectively cells, CD30, CD37, TRBC1, and CD1a [55–58]. Table 1 reverse the symptoms of a pediatric patient [27]. Toci- provides a summary of potential solutions to the three lizumab has since been FDA approved for treatment of main challenges seen in adapting CAR technology for T CAR T cell-induced life-threatening CRS [40]. Neuro- cell malignancies—fratricide, T cell aplasia, and product logical toxicities have been reported following CAR T contamination. A list of all current CAR-based clinical cell infusion as well; however, preventative approaches trials targeting T cell disease is presented in Table 2. remain elusive [36, 41–44]. Compared to CRS and Below, we review all preclinical and clinical CAR studies neurotoxicity, a much more manageable consequence of targeting T cell malignancies categorized according to CAR T cell therapy targeting B cell malignancies is the the target antigen of interest. resulting B cell aplasia. This is a potentially lifelong out- come due to memory cell formation against a B cell anti- CD5 gen; but currently is managed by periodic infusions of CD5 expression is limited to normal T cells and a small intravenous immunoglobulins. Unfortunately, this is an subpopulation of B cells, called B-1a cells [65–69]. CD5 extremely problematic outcome for T cell malignancies, acts as a negative regulator of TCR signaling and has a as persistent T cell aplasia would be life threatening. role in protecting against autoimmunity [70, 71]. CD5 is There are currently > 200 clinical trials using CAR T highly expressed on many T cell malignancies, particu- cells registered at clinicaltrials.gov being carried out in larly T-ALL and PTCLs, rendering it a good target for the USA. However, the majority of these trials are enrol- CAR T cell therapy [72–74]. Since CD5 expression on T ling patients with B cell malignancies. Advances are be- cells is approximately ten times that on B cells [75], a ing made to expand CAR T cell therapy to the treatment low-affinity, high-avidity CAR targeting CD5 may steer of other cancers, and to minimize toxicities associated clear of CD5-positive B cells while selectively killing T with treatment while reducing difficulty and cost of cells [76, 77]. Furthermore, CD8 tumor-infiltrating lym- production. phocytes (TILs) express lower levels of CD5 compared to that of peripheral blood T cells, and one study Translating CAR T cell therapy for treatment of T showed downregulation of CD5 improves the ability of cell malignancies T cells to lyse malignant cells [78]. CD5 was previously Harnessing and redirecting the cytotoxicity of T cells to targeted as a tumor antigen in clinical trials using malignant B cells has been established, but reprogram- immunotoxin-conjugated CD5 monoclonal antibodies, ming T cells to kill malignant T cells, while sparing nor- with responses seen in patients with cutaneous T cell mal T cells, is much more complex and challenging. lymphoma and T-ALL [79, 80]. This requires aberrant expression of an antigen on ma- A preclinical study showed that expression of a CD5- lignant T cells that is absent or expressed at very low CAR with a CD28 costimulatory domain resulted in sur- levels on normal T cells. CAR therapy requires isolation face downregulation of CD5 in CAR T cells. As a result, of healthy T cells from malignant T cells, a complicated fratricide was observed only transiently, allowing the procedure that can result in product contamination and CD5-CAR T cells to expand. These cells had significant Fleischer et al. Journal of Hematology & Oncology (2019) 12:141 Page 4 of 21 Product Fratricide Contamination Anti-tumor T-cell aplasia cytolytic activity Fig. 1 Potential outcomes of CAR T cell therapy in a patient with T cell disease. Upon re-infusion into a patient, CAR T cells recognize their cognate antigen, expanding upon this recognition, and initiating an attack. However, due to shared antigen expression on CAR T cells, normal T cells, and tumor cells, numerous outcomes can be observed. CAR T cells target tumor cells as intended, reducing tumor burden. However, without further engineering, the CAR-modified T cells are likely to express the targeted antigen as well, resulting in fratricide. CAR T cells would also target healthy T cells, resulting in unintended T cell aplasia. Lastly, CAR T cell therapy involves isolating normal T cells from malignant T cells for CAR-modification. A single malignant cell contaminating this population can result in masking of the antigen, leading to antigen-positive relapse. *Figure was created using BioRender Table 1 Strategies to overcome challenges in translating CAR therapy to treat T cell malignancies Challenge Strategy Reference Fratricide Targeting downregulated antigens (e.g., CD5) [59] Genome editing of target antigen [45–47] Targeting antigens with limited expression on T cells [55–58] (e.g., CD30, CD37, TRBC1, CD1a) Tet-OFF expression system [48] Protein expression blockers (PEBLs) [49] Using NK cells or NK-92 cells [47, 51–54, 60] T cell aplasia Targeting antigens with limited expression on T cells [55–58] (e.g., CD30, CD37, TRBC1, CD1a) mRNA electroporation Adeno-associated viral (AAV) vector delivery Using NK cells or NK-92 cells [47, 51–54, 60] Using γδ T cells Suicide genes and safety switches Bridge to allogeneic hematopoietic stem cell transplant (HSCT) Product contamination Allogeneic CAR T cells with TRAC locus editing [46, 61] Using NK cells or NK-92 cells [47, 51–54, 60] Using γδ T cells Fleischer et al. Journal of Hematology & Oncology (2019) 12:141 Page 5 of 21 Table 2 Clinical CAR trials targeting T cell malignancies T cell Clinical Trials Sponsor CAR costimulatory Additional Phase Status Ref antigen domain intervention CD5 NCT03081910 Baylor College of Medicine CD28 None Phase I Recruiting (MAGENTA) CD7 NCT04004637 PersonGen BioTherapeutics Phase I Recruiting NCT04033302 Shenzhen Geno-Immune Medical Phase Recruiting Institute I/II NCT03690011 Baylor College of Medicine CD28 CRISPR/Cas9 Phase I Not yet CD7-editing recruiting NCT02742727 PersonGen BioTherapeutics CD28 and 4-1BB NK-92 cells Phase Unknown I/II CD4 NCT03829540 Stony Brook University CD28 and 4-1BB Phase I Recruiting CD30 NCT01192464 Baylor College of Medicine EBV-specific CTL Phase I Active, not recruiting nd NCT03383965 Immune Cell Inc 2 generation Phase I Recruiting NCT02690545 UNC Lineberger Comprehensive Phase Recruiting [62] Cancer Center I/II NCT02259556 Chinese PLA General Hospital 4-1BB Phase Recruiting [63] I/II NCT02958410 Southwest Hospital, China Phase Recruiting I/II NCT03049449 NCI Phase I Recruiting NCT01316146 UNC Lineberger Comprehensive CD28 Phase I Active, not [55] Cancer Center recruiting NCT02917083 (RELY- Baylor College of Medicine CD28 Phase I Recruiting [64] 30) rd NCT04008394 Wuhan Union Hospital, China 3 generation Phase I Recruiting NCT03602157 UNC Lineberger Comprehensive CCR4 Phase I Recruiting Cancer Center overexpression NCT02663297 UNC Lineberger Comprehensive CD28 Phase I Recruiting Cancer Center TRBC1 NCT03590574 Autolus Limited RQR8 safety Phase Recruiting mechanism I/II in vitro cytotoxicity against two T-ALL cell lines and persistence of treated T cells in NOD scid IL2Rγ-chain primary T-ALL cells and delayed leukemia progression knockout (NSG) mice [81]. in two different CD5-positive T-ALL models [59]. Based Interestingly, use of 4-1BB as the costimulatory do- on these results, CD5-CAR T cells with a CD28 costi- main in a CD5-CAR resulted in a significant fratricidal mulatory domain are being tested in patients with effect [48]. It was shown that tumor necrosis factor relapsed or refractory T cell disease (MAGENTA trial, (TNF) receptor-associated factor (TRAF) signaling from NCT03081910). Our group used CRISPR-Cas9 to the 4-1BB endodomain upregulated the intercellular ad- knockout CD5 expression in primary T cells prior to hesion molecule 1 (ICAM1), which subsequently stabi- transduction with the CD5-CAR. We showed that gene lized the fratricidal immunological synapse between editing of CD5 in effector CAR T cells increased CAR CD5-CAR T cells containing the 4-1BB costimulatory surface expression and decreased self-activation [47]. domain. To limit and control the effects of fratricide, a The increased CAR surface expression is predicted to Tet-OFF expression system was used, which allowed for enhance CAR T cell anti-tumor efficacy. We also controlled transgene expression using the small mol- showed antagonism of vasoactive intestinal peptide ecule inhibitor, doxycycline. In the presence of doxycyc- (VIP) signaling in conjunction with inhibition of the line, CD5-41BB-CAR T cells expanded ex vivo without PI3Kδ pathway increased expansion of CD5-CAR- evidence of fratricide, while maintaining a more naïve modified T cells as well as their cytotoxicity against genotype. Doxycycline was removed from the culture CD5-specific tumor cell lines. This combination of com- prior to injecting the CD5-41BB-CAR T cells into mice, pounds was also demonstrated to prolong in vivo resulting in CD5-CAR expression and improved survival Fleischer et al. Journal of Hematology & Oncology (2019) 12:141 Page 6 of 21 outcomes in a T-ALL mouse model. Furthermore, there was generated using CRISPR-Cas9 genome editing to dis- was a survival advantage in mice treated with Tet-OFF rupt the CD7 and TCRα constant (TRAC) loci. This study CD5-41BB-CAR T cells compared to survival of mice demonstrated that NSG mice engrafted with primary T- treated with CD5-CD28-CAR T cells without the Tet- ALL blasts and treated with UCART7 donor cells exhib- OFF expression system [48]. ited tumor clearance from the peripheral blood, and, did Alternatively, we expressed the CD5-CAR in NK-92 not develop graft versus host disease (GvHD) or other se- cells, an interleukin-2 (IL-2) dependent natural killer cell vere side effects [46]. line, which are inherently CD5-negative. Our data demon- A new technique using protein expression blockers strates that CD5-CAR-modified NK-92 cells have in- (PEBLs) has been established as an alternative to gen- creased cytotoxicity against T cell leukemia cell lines ome editing. This strategy couples an scFv with a reten- compared to the cytotoxicity of naïve NK-92 cells [47, 51], tion peptide to maintain the protein of interest in the and there is a significant improvement in survival of T- ER/Golgi preventing cell surface expression of the anti- ALL xenograft mouse models compared to survival of gen. PEBL-CD7-CAR T cells exhibited superior cytotox- mice treated with naïve NK-92 cells [47]. This data con- icity against primary T-ALL cells in vitro compared to firms previously published data illustrating significantly non-PEBL CD7-CAR T cells. Using a patient-derived improved survival and enhanced tumor reduction in irra- xenograft (PDX) model of ETP-ALL, upon detection of diated T-ALL mouse models treated with CD5-CAR- leukemic cell expansion in peripheral blood, PEBL-CD7- modified NK-92 cells compared to that of mice treated CAR T cells were injected. PEBL-CD7-CAR T cell- with control NK-92 cells [53]. Recently, another group treated mice had a significant survival advantage over tested CD5-CAR-modified NK-92 cells, using a NK- control mice. However, CD7-positive relapse did occur specific costimulatory domain 2B4 in their CAR con- in all PEBL-CD7-CAR T cell-treated mice [49]. structs [82]. Interestingly, the CD5-2B4-CAR NK-92 cells Despite CD7 expression on NK-92MI cells (IL-2 produ- displayed superiority to CD5-41BB-CAR NK-92 cells, in cing NK-92 cells), they have been used for CD7-CAR ther- both in vitro and in vivo experiments [82]. apy demonstrating only a small percentage of cells are CD7-positive, and upon CD7-CAR expression, fewer than CD7 1% CD7-positive NK-92MI cells remain [60]. Two CD7- CD7 is a transmembrane glycoprotein with expression on CAR constructs, a monovalent and bivalent construct, were T cells and NK cells [83]. The majority of T-ALLs are generated using a humanized CD7 nanobody sequence that CD7-positive, despite some populations lacking expres- had been previously developed in the laboratory. Both CAR sion of other common markers, such as the TCR [74, 84]. constructs demonstrated enhanced CD7-specific cytotox- Additionally, early T cell precursor acute lymphoblastic icity against T-ALL cell lines and primary patient cells leukemia (ETP-ALL), a high-risk subset of T-ALL, highly ex vivo when expressed in NK-92MI cells. The bivalent express CD7 [84–86]. Two clinical trials have been initi- CD7-CAR-modified-NK-92MI cells exhibited slightly ated in China studying CD7-CAR-modified T cells for the greater cytotoxicity compared to that of the monovalent treatment of CD7-positive malignancies (NCT04033302 CAR-modified cells, and significantly inhibited disease pro- and NCT04004637). However, preclinical studies showed gression in a T-ALL PDX model when compared to naïve significantly reduced expansion of CD7-CAR T cells com- unmodified NK-MI cells. pared to control T cells, as a result of fratricide [45, 49]. Fratricide appears to be observed to a greater extent in CD4 CD7-CAR T cells compared to CD5-CAR T cells [45]. It Most cancers derived from lineage-differentiated T cells is hypothesized that this is due to a more incomplete in- are likely to be of CD4-positive origin, making CD4 a ternalization mechanism of CD7 from the cell surface fol- potential target for CAR therapy. A preclinical study was lowing ligation of the antigen with an anti-CD7 scFv. performed to consider the cytotoxicity of CD4-CAR- CRISPR-Cas9 editing of CD7 from the cell surface of T modified T cells against T-ALL tumors in NSG mice. cells prior to CAR expression demonstrated a superior This study also included the use of alemtuzumab to method of developing CD7-CAR T cells. These cells ex- clear the CAR T cells as a safety mechanism. NSG mice hibited limited fratricide, expanded in vitro, and showed were injected with luciferase-expressing Jurkat T cells no evidence of impaired cytotoxicity in vitro nor in vivo. and subsequently treated with naïve T cells or CD4- Investigations in a T-ALL mouse xenograft model re- CAR-modified T cells. CAR-treated mice displayed a vealed a statistically significant prolonged survival of CD7- survival advantage and an ~ 80% reduction in tumor edited CD7-CAR-treated mice compared to survival of burden compared to mice treated with naïve T cells. control mice [45]. Based on these results, a phase I clinical CD4-CAR-modified T cells were also injected into mice trial has been initiated testing CD7-CD28-CAR T cells in to evaluate the ability of alemtuzumab to effectively T-ALL patients (NCT03690011). Additionally, a UCART7 eliminate CAR-modified T cells. Alemtuzumab was Fleischer et al. Journal of Hematology & Oncology (2019) 12:141 Page 7 of 21 administered 24 h post-CAR T cell injection. A > 95% expression of CD30 can also be found on a subset of depletion of CD4-CAR-modified T cells was observed PTCLs, including ALCL [92–94, 96]. One study demon- within 6 h following injection signifying the use of alem- strated that CD30 expression is upregulated during tuzumab as a safety mechanism for CAR T cell therapy chemotherapy regimens in T-ALL patients. Of 34 T- [87]. Additionally, a phase I clinical trial to assess the ALL patients, approximately 38% had CD30-positive T- safety and feasibility of CD4-CAR T cell infusions in pa- ALL [96]. Therefore, some T-ALL patients who relapse tients with relapsed/refractory T cell lymphoma and T following chemotherapy may still respond to CD30- cell leukemia has been initiated (NCT03829540). directed CAR therapy. However, expression of CD4 on T cells can complicate Preclinical studies have previously demonstrated CD4-CAR T cell therapy as previously described. NK-92 CD30-CAR T cell capacity for lysing tumor cells [97, 98] cells are inherently CD4-negative, and therefore the use and numerous clinical investigations into CD30-CAR T of NK-92 cells as opposed to T cells reduces risk of frat- cell therapy have been launched with encouraging re- ricide and avoids the need for further modifications. sults. Eleven phase I/II trials treating patients with Additionally, it abrogates the risk of aplasia of CD4- CD30-positive malignancies are currently active positive cells that can occur with long-term engraftment (NCT01316146 [55], NCT01192464, NCT03049449, of CAR T cells. Anti-CD4-CAR NK-92 cells have shown NCT02690545 [62], NCT02958410, NCT02663297, in vitro success eliminating PTCL cell lines and both NCT03383965, NCT02917083 [64], NCT04008394, adult and pediatric primary cells. Using a xenograft NCT02259556 [63], and NCT03602157). To date, no model in NSG mice, CD4-CAR NK-92 cell-treated mice toxicities related to CAR T cell infusion nor impaired demonstrate significantly prolonged survival compared immunity against common viruses has been reported to control-modified NK-92 cell-treated mice [54]. from these trials. However, one trial reported that the in vivo CAR T cell expansion and persistence was re- CD37 duced following subsequent infusions compared to those CD37 is a member of the tetraspanin superfamily with ex- following initial doses [55]. The decreased persistence of pression limited to lymphoid tissues, particularly B cells the CAR T cells may have prevented the development of [88, 89]. CD37 expression in cancer cells is typically char- severe adverse events such as CRS and neurotoxicity that acteristic of B cell malignancies; however, its expression are commonly observed following CAR T cell infusion. can be found in some cases CTCL and PTCL [90, 91]. Of the two ALCL patients in this trial, one patient was Since CD37 is not expressed in T cells, there is no evi- non-responsive to the therapy, while the other entered dence of fratricide occurring in anti-CD37 CAR T cells. complete remission lasting 9 months [55]. Results from However, in the presence of CD37-positive PTCL cell another phase I trial in China for patients with relapsed/ lines, CD37-CAR T cells exhibit increased activation and refractory CD30-positive lymphomas (NCT02259556) degranulation as well as specific cytolytic activity in vitro corroborate the limited toxicity and anti-tumor activity [56]. The restricted expression of CD37 makes it a safer of CD30-CAR T cells [63]. target for CAR T cell therapy, given there would be no concern of T cell aplasia. Additionally, CD37 is not TRBC1 expressed in NK cells, providing an opportunity to utilize T cells express the αβ TCR; the β-chain can either be NK cells as effector cells in place of T cells. The versatility encoded by the T cell receptor beta constant 1 (TRBC1) of CD37-CARs to treat B cell and T cell lymphomas sug- gene or TRBC2 gene [99, 100]. Therefore, expression of gests that this may be an important target for further in- TRBC1 and TRBC2 is mutually exclusive. Additionally, vestigations. While CD37 is predominantly being CD4- and CD8-positive T cell populations express both examined for dual targeting for B cell malignancies, the subsets and CD8-positive T cell populations specific for target has potential for CAR therapy against T cell common viruses also contain both TRBC1 and TRBC2 malignancies. cells [58]. However, as malignant T cells develop from a single cell, the entire population of cancerous cells will CD30 be either TRBC1- or TRBC2-positive. Numerous T cell CD30, a member of the tumor necrosis factor receptor malignancy cell lines and primary samples have been an- (TNFR) superfamily, promotes T cell proliferation and alyzed by flow cytometry to validate the homogeneity of cytokine production following TCR stimulation, while β-chain expression in a malignant cell population [58]. also having an opposing role in promoting apoptosis Many cancer cells downregulate the αβ TCR; however, it [92]. Expression is limited to a subset of activated lym- is expressed on > 95% of PTCLs [101] and > 30% of T- phocytes found around the follicular regions of lymphoid ALLs [102]. tissues [93–95]. While CD30 is well known for its strong Anti-TRBC1 CAR T cells exhibited specific and effi- expression in virtually all classical Hodgkin lymphoma, cient cytotoxicity against the JKO T cell line transduced Fleischer et al. Journal of Hematology & Oncology (2019) 12:141 Page 8 of 21 with TRBC1, but not against non-transduced cells or remaining at 5:1 effector to target ratios. Furthermore, cells transduced with TRBC2, even in a mixed popula- CD3-CAR NK-92-treated T-ALL NSG mice exhibited tion. Furthermore, in primary samples from patients prolonged survival with ~ 87% reduced tumor burden with T cell malignancies, the anti-TRBC1 CAR T cells through day 23 [52]. preserved a significant fraction of healthy T cells (TRBC2 cells), thereby circumventing a limitation of CD1a CAR T cell therapy for the treatment of T cell malignan- CD1a is a lipid-presenting molecule whose expression is cies [58]. In an NSG mouse model using TRBC1- restricted to developing cortical thymocytes, skin Lang- positive Jurkat T cells to establish cancer, mice treated erhans cells, and some circulating myeloid dendritic cells with the anti-TRBC1 CAR T cells exhibited reduced [103, 104]. Neither T cells nor CD34 hematopoietic tumor burden and elongated survival. In additional pre- progenitors express CD1a, making it a fratricide- clinical studies, NSG mice were injected with both resistant target, while limiting the risk of on-target/off- TRBC1 and TRBC2 cancer cells, and then treated with tumor toxicity. Expression in T cell malignancies is only either naïve T cells or anti-TRBC1 CAR T cells. TRBC1- limited to cortical T-ALL, a major subset of T-ALL ac- positive Jurkat T cells could not be detected in mice counting for ~ 35–40% of all T-ALL cases [105, 106]. A treated with anti-TRBC1 CAR T cells; however, TRBC2- study showed that CD1a-CAR T cells expanded without positive cells were identified. This is in contrast to mice fratricide, and had long-term persistence in an in vivo treated with naïve T cells, whose bone marrow con- model [57]. Additionally, these cells demonstrated spe- firmed the presence of both TRBC1-positive and cific cytotoxic activity against CD1a-positive T-ALL cell TRBC2-positive cells [58]. Thus, targeting TRBC1- lines and primary blasts in vitro, and exhibited potent positive malignant cells offers a unique approach to anti-leukemic activity in a PDX model of cortical T- avoiding T cell aplasia, a consequence of many proposed ALL. Thus, while not applicable to all T cell malignan- CAR T cell therapies for the treatment of T cell cies, targeting CD1a with CAR T cells may be successful malignancies. in the specific subset of cortical T-ALL cases. CD3 “Off-the-shelf” CAR T cell therapy CD3 is a pan T cell marker comprised of four distinct One of the greatest challenges in utilizing autologous polypeptide chains, epsilon, gamma, delta, and zeta, CAR T cell therapy for the treatment of T cell malignan- which form pairs of dimers, transmitting T cell activa- cies is the separation of healthy T cells from malignant tion signals. As CD3 is exclusively expressed on T cells, T cells, in order to generate a CAR T cell product that is it has been a popular target in preclinical CAR T cell not contaminated with cancerous T cells. To date, there therapies for the treatment of T cell malignancies. As has been one reported case from the University of Penn- expected, due to fratricidal issues, manufacturing of sylvania of CD19-CAR modification of a single leukemic anti-CD3 CAR T cells does not yield a viable cellular B cell, resulting in CD19-positive relapse and ultimately product [61]. Various approaches using an anti-CD3 death of the patient [39]. This task of isolating healthy T CAR have been investigated including the use of tran- cells is even more difficult when a proportion of the pa- scription activator-like effector nuclease (TALEN) tient’s T cells are malignant, especially in cases of T cell mRNA to disrupt the TRAC locus and using NK-92 cells leukemia where there is a high likelihood of circulating in place of T cells as the effector cell type. Disruption of cancerous T cells. Thus, manufacturing of autologous the TRAC locus prevents assembly of the TCRαβ/CD3 CAR T cells for the treatment of T cell malignancies has complex, allowing for anti-CD3-CAR expression without a very high likelihood of resulting in CAR-modified compromising cellular proliferation and viability. Enrich- leukemic cells. This would likely result in relapse as ment of the CAR-positive, CD3-negative population was these cells would likely escape recognition by normal observed. In patient T-ALL samples, anti-CD3 CAR T CAR-T cells. cells demonstrated specific cytotoxicity against CD3- Additionally, there remain numerous challenges to positive cells. In a T-ALL NSG model, anti-CD3 CAR T using a patient’s own cells to manufacture CAR T cells. cells were shown to clear luciferase-expressing CD3- Patients with advanced disease undergoing CAR T cell positive Jurkat cells, but showed no effect in NSG mice therapy typically are heavily pre-treated, having previ- engrafted with CD3-negative Jurkat cells [61]. To cir- ously undergone numerous rounds of chemotherapy, cumvent the need for additional modifications, NK-92 which can result in low T cell counts and/or T cells that cells can also be used to express the anti-CD3-CAR, may not be healthy enough to expand well making it since they are CD3-negative cells. CD3-CAR NK-92 cells very difficult to manufacture an efficacious CAR T cell demonstrated efficient ex vivo lysis of PTCL primary product [107]. This issue is much more prevalent in samples, resulting in less than 0.5% lymphoma cells adult patients due to the decreasing proportion of naïve Fleischer et al. Journal of Hematology & Oncology (2019) 12:141 Page 9 of 21 T cells associated with aging [107–110]. Additionally, severe side effects [46]. TALENs, an alternative genome given that many of these patients have advanced disease, editing technique, have also been used to prevent ex- a patient may experience disease progression, co- pression of the TRAC locus in order to limit fratricide of morbidities, or even death in the time it takes to manu- anti-CD3-CAR T cells and prevent MHC-recognition of facture autologous CAR T cells. This is especially true in foreign host cells. Genome editing the TRAC locus pre- most relapsed T cell malignancies, which tend to be ag- vents stable assembly of the TCRαβ/CD3 complex. Dis- gressive and chemo-resistant in nature. Lastly, each ruption of the TRAC locus using TALEN mRNA prior starting autologous T cell product is different—variable to transduction with an anti-CD3-CAR lentiviral vector function, maturation, CD4/CD8 ratios, and phenotypic yielded CAR T cells that proliferated well and greatly re- ratios—and the heterogeneity of each individual product duced tumor burden in an NSG mouse model of human has led to unpredictable results and variable potency of leukemia [61]. the therapy. As described above, PEBLs have been recently devel- An alternative to autologous CAR T cell manufactur- oped to selectively prevent expression of individual pro- ing is the use of allogeneic T cells as the cell source. In teins. PEBLs have been shown to effectively retain CD3ε order to make this approach feasible, expression of the in the ER/Golgi to prevent MHC recognition of host endogenous αβTCR in allogeneic CAR T cells must be cells during allogeneic use of anti-CD19 CAR T cells blocked as it would likely result in GvHD, unless the [50]. Disruption of TCRαβ signaling had no effect on T donor is a human leukocyte antigen (HLA) match. This cell proliferation. There was no evidence of GvHD in an process involves leukapheresis from a healthy donor, NSG mouse model of leukemia treated with the PEBL- followed by isolation of the donor’s T cells. Following CD19-CAR T cells, whereas 60% of the mice treated transduction of the T cells with a CAR-encoding retro- with CAR T cells that were not expressing the CD3ε viral vector, subsequent genome editing of the TRAC PEBL developed GvHD. Furthermore, both PEBL and locus is required to prevent expression of the endogen- CAR can be expressed from the same vector using a 2A ous TCR. Cells that remain TCR-positive are then de- sequence, resulting in only one transduction of the cells pleted from the expanded CAR T cell product prior to [50]. While this study utilized PEBL in conjunction with cryopreservation. This creates an “off the shelf” cellular an anti-CD19-CAR, this system can potentially be ap- product that can be banked until it is needed for ther- plied with other CAR constructs to target T cell apy. This approach resulted in successful remission in antigens. two infant B-ALL cases treated with allogeneic CD19- CAR T cells modified at the TRAC and CD52 loci. The Alternative effector cell types allogeneic CAR T cells persisted until conditioning for While CAR-modified αβ T cells can have a memory stem cell transplant [111]. Another group utilized phenotype resulting in T cell aplasia, NK cells and shRNA to knock down β2-microglobulin in conjunction gamma delta (γδ) T cells will not. Utilizing these innate with a knock-in strategy to insert a CD19-CAR into the cells for CAR therapy is a viable alternative that groups TRAC locus. Knock down of β2-microglobulin reduces are exploring. One disadvantage to preventing memory the ability of class I HLA molecules to form heterodi- cell formation and using effector cells with limited per- mers on the cell surface. Reducing expression of both sistence is reduced tumor control. However, this limita- β2-microglobulin and TRAC resulted in decreased allo- tion can potentially be overcome by utilizing these cells geneic attack by CD8 T cells and NK cells [112]. This in multiple dosing regimens. Repeated dosing of short- strategy may be useful to reduce allo-recognition in pa- lived CAR-expressing cells can be used to induce remis- tients receiving CAR T cell therapy. Other groups have sion; thus, providing a bridge to an allogeneic exploited similar approaches in preclinical CAR T cell hematopoietic stem cell transplant (HSCT) if needed. investigations targeting CD7 and CD3, as previously de- Since these products would be utilized in an allogeneic scribed [46, 61]. setting, they can be cryopreserved and would be readily CRISPR-Cas9 genome editing has become a popular available when needed for use. technique to prevent gene expression or to correct gene expression. One study targeting CD7 generated “fratri- Natural killer cells and NK-92 cells cide resistant, allo-tolerant” CAR T cells using CRISPR- Ex vivo-expanded NK cells are short-lived, and do not Cas9 to disrupt both CD7 and the TRAC loci (UCAR persist for extended periods of time in vivo compared to T7). NSG mice engrafted with primary T-ALL blasts de- that of αβ T cells [113]. CAR-modified NK cells have a veloped GvHD when treated with wildtype donor T turnover time of 1–2 weeks; therefore, there is reduced cells; however, mice treated with UCART7 donor cells concern of aplasia of antigen-expressing cells [114]. Cur- were able to clear the tumor cells from the peripheral rently, there are two active clinical trials using anti- blood, and, furthermore, did not develop GvHD or other CD19-CAR-modified NK cells (NCT00995137 and Fleischer et al. Journal of Hematology & Oncology (2019) 12:141 Page 10 of 21 NCT01974479). Additionally, some studies use NK-92 from a NK cell lymphoma, they require irradiation prior cells, an IL-2-dependent NK-lymphoma-derived cell line. to infusion into a patient to prevent expansion, resulting NK-92 cells are often used as an alternative to primary in persistence for about 1 week in vivo and potentially NK cells due to their ease of expansion under current exhibiting reduced cytotoxicity. Alternatively, suicide good manufacturing process (cGMP) conditions [115] mechanisms can be engineered into the cells to elimin- and transfection with CAR mRNA [116]. CAR-modified ate the risk of NK-92-cell persistence in vivo and elimin- NK or NK-92 cell infusion can result in tumor cell clear- ate the need for irradiation, thereby resulting in greater ance without the risk of GvHD. Therefore, these cells cytotoxicity of the infused cells. typically only require one genetic modification. Add- NK cells exhibit their cytotoxic activity through numer- itionally, with the exception of CD7, NK cells do not ex- ous means, including expression of FasL or TRAIL, secre- press antigens targeted in T cell malignancies. tion of perforin and granzyme, as well as through Therefore, neither fratricide nor T cell aplasia are of pri- antibody-dependent cellular cytotoxicity (ADCC) mecha- mary concern. nisms [131, 135, 136]. A major limitation to the use of CAR-expressing NK-92 cells have been extensively CAR T cells is antigen escape; however, as NK cells can assessed in preclinical studies targeting various cancers such kill through other mechanisms, downregulation of the as B cell malignancies [117–119], multiple myeloma [120], cognate antigen on tumor cells may not halt anti-tumor acute myeloid leukemia (AML) [121], breast carcinoma activity. NK cells also express the natural killer group 2D [122, 123], neuroblastoma [124], and glioblastoma [125]. As (NKG2D) receptor, which recognizes cellular stress li- previously discussed, multiple groups have initiated preclin- gands such as MHC class I chain-related protein A/B ical studies using CAR-modified NK-92 cells for the treat- (MICA/B) and UL16 binding proteins (ULBPs) [137, 138], ment of T cell malignancies, targeting antigens such as CD5, resulting in cytotoxicity against exceedingly stressed cells. CD7, CD4, and CD3, demonstrating reduced tumor burden As NK cells do not recognize targets on healthy cells, they and an overall survival benefit in NSG mouse models of T have limited off-target toxicity [131]. Additionally, their cell leukemia [47, 52–54, 60]. The safety and efficacy of NK- serial killing capability allows each individual NK cell to 92 cells has been evaluated in clinical trials displaying a good kill, on average, four tumor cells [139]. However, NK cells safety profile with few mild to moderate adverse events are notoriously difficult to expand ex vivo, transduce with [126–128] (NCT00900809, NCT00990717). To date, five viral vectors, cryopreserve, and they have limited life span clinical trials have been initiated involving infusion of CAR- in vivo [128, 140]. While autologous NK cells can be ob- modified NK-92 cells targeting a variety of antigens, includ- tained by leukapheresis followed by selection of CD56- ing CD33 [129], human epidermal growth factor receptor 2 positive cells, allogeneic NK cells derived from a third (HER2), B cell maturation antigen (BCMA), CD19, and the party donor requires an additional step for depletion of T cell antigen, CD7 (NCT02944162, NCT03383978, alloreactive T cells from the donor product [141]. NCT03940833, NCT02892695, and NCT02742727). Purification and expansion of NK cells from peripheral Inherent NK-cell cytotoxicity is dependent on the bal- blood mononuclear cells (PBMCs) have been optimized in ance of activating and inhibitory killer-cell cGMP protocols to clinically relevant numbers [142–144]. immunoglobulin-like receptor (KIR) signals. Inhibitory This is a time-consuming process as only 10% of PBMCs and activating KIRs on NK cells form a balance, as there are NK cells [145]. However, recently developed methods are often signals from both inhibitory and activating re- are being used to enhance NK-cell expansion, such as ceptors. The inhibitory signals predominate, typically through K562-feeder cell expression of OX40 ligand through higher affinity for their ligands; however, strong [146]. As mentioned above, a limitation to CAR-NK ther- activating signals can override the inhibitory signals, li- apy is the extreme sensitivity of NK cells to cryopreserva- censing NK cells to kill. If donor inhibitory KIRs do not tion. They have demonstrated poor viability and recognize patient HLA, there is reduced inhibitory sig- diminished cytotoxicity after cryopreservation. While naling to counteract the activating signaling [130, 131]. cytotoxicity can be restored to normal levels after a few While NK-92 cells lack many of the inhibitory KIRs days in culture with exogenous IL-2, the low viability expressed on primary NK cells, they have a wide range post-cryopreservation remains a concern [141]. of activating receptors [132]. Similar to NK cells, NK-92 cells have the capability to produce perforin and Gamma delta T cells granzyme upon activation, as well as display cytotoxic While αβ T cells function as a part of the adaptive im- activity through upregulation of TNF-related apoptosis- mune system, γδ T cells play roles in both the innate and inducing ligand (TRAIL), Fas ligand (FasL), and TNFα the adaptive immune systems. &g