New indications and platforms for CAR‐T therapy in lymphomas beyond DLBCL

Abstract CD19‐directed chimeric antigen receptor T‐cell therapy (CAR‐T) represents a significant advancement for patients with relapsed/refractory large B‐cell lymphoma (LBCL). Long‐term follow‐up confirms durable remissions in nearly half of the patients, a population that was previously estimated to have a median survival of around 6 months with standard salvage therapy. This initial success of CAR‐T has led to significant expansion across other lymphoma histologies resulting in the recent regulatory approval of CAR‐T in mantle cell lymphoma and follicular lymphoma. Additionally, multiple novel platforms of CAR‐T therapy are under development to improve efficacy and limit toxicity such dual antigen targeting, allogeneic and natural killer CARs. In this review, we focus on the new indications of CAR‐T in lymphomas beyond LBCL as well as emerging platforms of CAR‐T therapy.

Significant progress has been made in MCL relapsing after front line CIT with the approval of multiple novel chemotherapy free treatments.
Most notable are the Bruton's tyrosine kinase inhibitor inhibitors (BTKi) [16][17][18]. Despite initial high responses with BTKi the disease remains incurable with median progression-free survival (PFS) of around 1 year [19]. Outcomes are particularly poor for patients relapsing after BTKi with a median overall survival (OS) of under 6 months, representing a strong clinical need for new treatments [20,21].
Liso-cel is another CD19-directed CAR-T with a CD3ζ signaling domain and a 4-1BB costimulatory domain [5]. During manufacturing of liso-cel, CD4+ and CD8+ T cells are separated from the leukapheresis product and thereafter individually activated, expanded, and administered as two separate sequential infusions of equal doses [5].
Preliminary results on the safety and efficacy of liso-cel in R/R MCL were reported at the American Society of Hematology (ASH) annual meeting, 2020 [23]. Forty patients underwent leukapheresis and lisocel was administered at dose level (DL) of 50 × 10 6 CAR T cells (n = 6) or 100 × 10 6 CAR T cells (n = 26) to 32 patients [23]. Median patient age was 67 years (range: 36-80) [23]. High-risk disease features such as blastoid morphology, high Ki67 index, TP53 mutation and complex karyotype were reported in 37.5%, 72%, 22%, and 34% of patients, respectively [23]. Twenty-eight (87.5%) had received prior BTKi and 11 (34%) were assessed to be refractory to BTKi [23]. BT was administered to 17 patients (53%) [23]. Twenty-seven (84%) had grade ≥ 3 AEs, most common being neutropenia followed by anemia and thrombocytopenia [23]. CRS was observed in 16 (50%) with grade ≥3 CRS in only one patient. NAEs were observed in 9 (28%); 3 patients experienced grade ≥3 NAE [23]. OR was observed in 27 patients (84%) with CR in 19 (59%) (  [36]. Grade ≥3 CRS and NAE were reported in 6% and 15% of patients with FL. A higher incidence of Grade ≥3 NAE was reported in patients MZL at 41% [36]. Outcomes were also reported for nine patients who received retreatment with axi-cel upon disease relapse [37]. These patients had disease relapse at 3-month post infusion after initially achieving a OR and maintained CD19 expression at relapse [37]. All patients showed evidence of OR to retreatment, and safety profile was not different from first infusion [37]. Updated outcomes for these patients and two additional patients with FL were recently reported and median DOR remains not reached at 11.4 months [38] (Table 1).
Tisa-cel has also shown efficacy and safety in R/R FL based on the planned interim analysis of ELARA trial [39]. Patients with grade 1 to 3a FL who had disease relapse within 6 months of second line or later CIT or had disease relapse post auto-HCT were included. Tisa-cel was infused at a dose of 0.6-6 × 10 8 CAR-T to 97  As noted, there are multiple treatment options available today for patients with R/R FL; however, short of an allo-HCT, none of the treatment options are curative [40]. Despite the increase in treatment options, long-term outcomes for patients decline sharply after second line of therapy with continued decrease in PFS and OS with each subsequent line [41,42]. Recently, a comparison of ZUMA-5 with SCHOLAR-5 was presented at the European Hematology Association Meeting [43]. SCHOLAR-5 is a retrospective external control cohort of R/R FL patients who had initiated 3rd or higher line of therapy after July, 2014. Eighty-six patients from ZUMA-5 and 85 from SCHOLAR-5 were included with median follow-up of 23.3 and 26.2 months, respectively; both cohorts were balanced through propensity scoring.
Baseline characteristics were similar between the two cohorts except performance status; ZUMA-5 had a higher number of patients with poor performance scores [43]. OR, CR, PFS, and OS favored ZUMA-5 over SCHOLAR-5. Similar trend was observed when patients who had received four or more lines of therapy were compared [43]. These data support the use of axi-cel in patients who have received at least two lines of prior systemic therapy, consistent with the current regulatory approval.

EXPERIMENTAL AUTOLOGOUS CAR-T PLATFORMS
CD-19-directed auto-CAR-T represents a significant milestone in the treatment of patients with R/R NHL. However, disease relapse remains a significant hurdle with long-term durable responses seen in only about 40-50% of patients [44]. Various mechanisms have been elucidated regarding failure of CAR-T including antigen loss, host immune dysregulation, and exhausted T-cell repertoire [1,45,46].
Here we discuss targets beyond CD19 and new auto-CAR-T platforms that are being investigated in lymphomas with promising early results. At a median follow-up of 533 days, 1-year PFS and OS was at 36%
CD30 targeting CAR-T holds promise for lymphomas beyond HL.
Early trials have shown safety and encouraging responses in patients with CD30 expressing R/R anaplastic large cell lymphomas [50,51] ( Table 2).

CD22-directed CAR-T
CD22 represents another target for CAR-T in patients with B-cell malignancies as it is expressed exclusively on malignant B cells [52].
The results of a Phase I dose escalation study of anti-CD22 CAR-T in R/R CD22+ B-cell malignancies were recently reported [53].

Dual antigen targeting in lymphoma
Traditional CARs are directed against a single tumor antigen (e.g., CD19) and their use has been associated with antigen negative (e.g., CD19-) relapses. CARs targeting more than one tumor antigen theoretically may have improved efficacy and/or lower probability of antigen negative disease at release. Investigators at the Medical College of Wisconsin conducted a first-in-human trial of bispecific anti-CD20, anti-CD19 CAR-T for adult patients with B-cell NHL or chronic lymphocytic leukemia (CLL) [54]. The study used on-site manufacturing using the CliniMACS Prodigy system. CAR-T cell dose ranged from 2.5 × 10 5 to 2.5 × 10 6 cells/kg. Grade ≥3 CRS occurred in one (5%) patient, and grade ≥3 NAEs occurred in three (14%) patients. Eighteen (82%) patients achieved an OR at day 28, including 14 (64%) CR. Notably, loss of the CD19 antigen was not seen in patients who relapsed [54] ( Table 2).
Early results from two Phase I trials with bispecific anti-CD22, anti-19 CAR-T in LBCL have also been encouraging [55,56]. Patients with CD19+ LBCL who had at least two lines of prior therapy received bispecific anti-CD19, anti-CD22 CAR-T (n = 21); no patient had prior receipt of CD19 CAR-T. CAR T-cell DL ranged from 1 × 10 6 /kg to 3 × 10 6 cells/kg [55]. Grade ≥3 CRS and NAE occurred in one patient each.
Interestingly, 29% (n = 4) patients relapsed with CD19 negative disease but retained expression of CD22 [55]. Sequential infusion of anti-CD22 and anti-CD19 CAR-T is another strategy for dual antigen targeting that has shown encouraging responses. Of note no patient had antigen negative disease relapse in this study (

Targeting T-cell antigens
T-cell lymphomas (TCL) represent a biologically heterogeneous group of lymphomas, typically having an aggressive disease presentation.
However, development of CAR-T in TCL in comparison to their B-cell counterparts is challenging due to antigen sharing between malignant T cells and CAR-T; this can lead to a higher risk of antigen masking, fratricide, and T-cell aplasia [58,59]. Targeting CD5 as it is a pan Tcell marker has been evaluated with modest results and other strategies are underway [60]. CD4 is uniformly expressed on most T-cell

LIMITATIONS OF AUTOLOGOUS CARS
Despite impressive activity in B-cell lymphomas and commercial availability, the auto-CAR-T construct suffers from several practical limitations (Table 3) gained [71]. Modifications in the manufacturing technology, for examples, decentralized model of CAR-T production [72] or use of off-theshelf CAR-T products may mitigate the costs compared to the current model.

PROMISE OF ALLOGENEIC CAR CONSTRUCTS
Allo-CARs (derived from healthy donors or stored cellular products) as a potential "off-the-shelf" treatment may circumvent some of limitations associated with auto-CARs (Table 3). If allo-CARs live up to their potential of being readily available cellular therapy products, they may obviate the need for bridging treatments and address manufacturing failure occasionally seen with autologous platforms. Whether donor pool, scaling, and manufacturing process would be efficient enough to meet demand remains to be seen. Theoretically allo products can have less variability in terms of T-cell composition and fitness, but available data to confirm this are not available. These products are also touted as cost friendly options, but this remains unknown at this point.

ALLOGENEIC CARS' POSSIBLE PITFALLS
Before the potential benefits of allo-CAR-T therapies are clinically realized, potential pitfalls associated with approach need close attention. The main barrier for universal CAR-T products is alloreactivity, which results from the donor-recipient human leukocyte antigen (HLA) disparity imparting a bidirectional risk, that is, to the cellular product (from the recipient immune system) and to the recipient in vivo by the host immune system [75][76][77][78][79]. Knocking out B2M reduces surface expression of HLA class I; however, these HLA-I negative universal T cells could still be rejected by recipient NK cells [79]. Employing an anti-NK-cell depleting antibody or engineering T cells with HLA-E expression are possible solutions to evade NK-mediated rejection [80,81].
In the other direction, the allo-CAR-T reactivity directed against the host can lead to the development of lethal graft-versus-host disease (GVHD) [82]. One strategy to reduce the risk of GVHD is the use of allo-virus-specific T cells (VST) CARs.  diseases [87]. The risk of alloimmunization is also a concern, where new CAR-specific antibody generation in the recipient may limit redosing of the allo-CAR-T [88]. Disrupting HLA expression alone may not be sufficient for CAR-T long-term persistence and efficacy. Further With genomic editing technologies, oncogenesis via oncogene activation or disruption of tumor suppressor genes is potential concerns [92]. It is critical to avoid insertional oncogenesis by using approaches such as optimized sgRNA design and Cas9 activity, prior off-target detection assays, and careful selection of target loci [93,94]

EXPERIMENTAL ALLOGENEIC CAR-T PLATFORMS
Several allo-cellular therapy platforms are being actively investigated in lymphoid malignancies (Table 4). Most allo-T-cell therapies have used αβ T cells with knocked out TCR to eliminate alloreactivity. Other platforms have restricted or invariant TCRs, like γδ T cells, VSTs, or natural killer (NK) cells.

Allogeneic NK-cell CARs
NK cells play a pivotal role in immune surveillance by targeting cancer or virally infected cells that down regulate HLA class I molecules [96]. Allo-NK cells have been used for adoptive immunotherapy for cancer patients with excellent safety profile [97], and now NK-cellderived CARs are being investigated in B-cell lymphomas and other malignancies. The group at MD Anderson Cancer Center pioneered the use of CD19-directed NK-CARs derived from umbilical cord blood (UCB) units [98]. The study used a retroviral vector carrying genes that encoded CD19-directed CAR, IL-15 to enhance the in vivo expansion and persistence of the transduced NK cells ("Armored CAR"), and an inducible caspase 9 to trigger apoptosis of the CAR-NK (as a safety switch). The results showed a promising safety and efficacy profile in 11 NHL and CLL patients with clinical responses observed in 73% of patients [98] (Table 4).
Other NK-cell source beyond UCB includes induced pluripotent stem cells (iPSCs) and NK-cell lines. FT516 is a CD19-specific NK-CAR in development against relapsed, refractory B-cell NHL, and is engineered from a clonal master iPSC line, with clustered regularly interspaced short palindromic repeats (CRISPR)-mediated insertion of the CAR at the TRAC locus ( Table 4). The key attributes include a proprietary CAR optimized for NK-cell biology that targets the antigen of interest, a novel high-affinity 158V, noncleavable CD16 (hnCD16) Fc receptor, which has been modified to prevent its downregulation and to enhance its binding to tumor-targeting antibodies, and an IL-15 receptor fusion (IL-15RF) that promotes enhanced NK-cell activity [99]. CAR-NK might represent a promising therapeutic option with all the benefits inherent to "off-the-shelf" therapies pending the clinical trial results.

Allogeneic alpha-beta CAR-T
Several allo-CAR-T clinical trials are employing conventional αβ T cells from healthy donors ( Table 4). The following gene-editing technologies have been used in generating allo-CAR-T.

Transcription activator-like effector nucleases
Transcription activator-like effector nucleases (TALEN) technology is arguably the first gene editing technology used in the generation of universal allo-CAR-T for lymphoma patients in clinical trials. TALENs are transcription factors (hybrid molecules) linked to an endonuclease that can be engineered to cut specific DNA sequences [100]. Knocking out the TRAC gene locus is an attractive approach to disrupt the expression αβ TCR, thereby limiting the GVHD initiating potential of allo-CAR-T. By simultaneously electroporating TALENs that disrupted TCR and CD52 expression in the T cells, in the preclinical model, this methodology produced allo-CAR-T that did not induce GVHD and were resistant to anti-CD52 monoclonal antibody used to eliminate host T cells [77]. The latter was employed as an immunosuppressive strategy to prevent recipient immune cell-mediated rejection of CAR-T. As shown in Table 4, Allogene 501 trial using this platform produced CR rates of 50% in patients DLBCL and FL with to date no reports of GVHD or frequent CRS or ICANS. These preliminary finds need confirmation with longer follow-up and a larger sample size [101,102].

Meganuclease-edited CARs
Meganucleases are a group of naturally occurring and highly specific restriction enzymes with gene-editing potential. Precision Bio-Sciences has developed a next-generation meganuclease platform called "ARCUS" that can produce nucleases with customized activity and specificity [103,104]. PBCAR0191 is an anti-CD19 allo-CAR-

Allogeneic gamma-delta CAR-T
Conventionally αβ T cells have been used for production of CAR-T, however, γδ T cells may offer unique advantages over αβ T cells [114].
Despite the small number of γδ T cells present in peripheral blood, these cells can be expanded ex vivo to produce clinically significant yield for therapeutic effect [115]. Preclinical data have demonstrated γδ T-cell expressing anti-CD19 CAR have potent cytotoxicity toward CD19+ leukemia cell lines in vitro and in vivo [116]. γδ T cells can also recognize pathogen (including viral) stressed and transformed target cells in an HLA-independent fashion and are activated in an allosetting without the concern of GVHD. γδ1 CAR T-cell product targeting CD20 is now entering clinical trials for treatment of B-cell malignancies (NCT04735471). The study is using selectively expanded γδ1 T cells from healthy donors that are engineered with a second-generation CAR construct (4-1BBz).

FUTURE DIRECTIONS
CAR-T therapy is a revolutionary treatment for patients with R/R B-cell lymphomas. Although the platform currently has multiple limitations as discussed, the future for CAR-T appears promising with multiple strategies underway to increase efficacy and limit toxicity. The approval of CAR-T in MCL and FL represents a significant advancement in the field, as these histologies have traditionally been considered incurable unlike LBCL. Whether CAR-T can lead to a cure in these lymphomas remains to be proven, pending long-term follow-up data. Combining novel targeted agents with CAR-T is another promising strategy. In preclinical MCL models, concurrent treatment with ibrutinib and CAR-T resulted in improved responses and decreased toxicity [117]. This combination is rational as ibrutinib blocks inducible T-cell kinase in addition to BTK and with resultant enhanced Th1-type cellular immunity [118]; ongoing TRANSCEND-004 clinical trial is evaluating this combination in patients with CLL (NCT03331198).
Expansion of CAR-T to additional lymphoma histologies such as HL and TCL is expected, pending the results of ongoing trials; primary and secondary central nervous system lymphoma (PCNSL/SCNSL) represent orphan diseases with particularly poor outcomes for patients with R/R disease. ZUMA-1 and JULIET excluded patients with CNSL; however, patients with SCNSL were allowed in TRANSCEND-NHL-001(5). Safety and efficacy were not significantly different from patients without CNS involvement; additional experience from the real-world setting is also consistent with that of TRANSCEND-NHL-001(5) [119]. The three currently approved CD-19-directed CAR-T in LBCL (i.e., axi-cel, tisa-cel, and liso-cel) are being actively investigated in patients with PCNSL and SCNSL (NCT04608487) (NCT04134117) (NCT03484702); results are currently awaited. A recently published report from an ongoing Phase I trial (NCT02153580) has shown encouraging responses with manageable toxicities in a small subset of patients (n = 5) with PCNSL receiving CD19-directed CAR-T [120].
Current experience with CAR-T therapy has clearly established that CAR-T in lymphoma is here to stay. However, with the various plat-forms of CAR-T therapy that are currently in development, there are multiple questions that emerge. Most importantly, whether a particular CAR design or cell type will be superior in terms of efficacy and safety.
Allo-CARs can potentially be the answer to the limitations currently experienced with auto-CARs; however, the platform is associated with notable risks such as that of bidirectional alloreactivity and insertional oncogenesis among others. Second, how can these various therapeutic cell products be sequenced to allow for best long-term outcomes in patients with lymphoma. Lastly, if the safety profile of a particular product would allow for widespread CAR-T expansion in the community, particularly outpatient (OP) administration, 10% and 18% of patients received CAR-T as an OP in TRANSCEND-NHL-001 and the ELARA trial, respectively. TRANSCEND-OUTREACH-007 (NCT03744676) is currently ongoing and is exploring the safety and efficacy of liso-cel in the outpatient setting.

AUTHOR CONTRIBUTIONS
MI and MH collected and analyzed the data, wrote the first draft, and approved the final version.