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Novel cellular immunotherapies for hematological malignancies: recent updates from the 2021 ASH annual meeting

Cellular immunotherapy, including the chimeric antigen receptor T (CAR-T) cell therapy and CAR- natural killer (CAR-NK) cell therapy, has undergone extensive clinical investigation and development in recent years. CAR-T cell therapy is now emerging as a powerful cancer therapy with enormous potential, demonstrating impressive anti-tumor activity in the treatment of hematological malignancies. At the 2021 ASH annual meeting, numerous breakthroughs were reported concerning acute lymphocytic leukemia (ALL), lymphoma, acute myeloid leukemia (AML), and multiple myeloma (MM). Universal CAR-T cell and CAR-NK cell therapy, as well as induced pluripotent stem cell (iPSC)-derived immunotherapy, offer great “off-the-shelf” benefits. Major development and updates of cellular immunotherapy for hematological malignancies reported at the 2021 ASH annual meeting are summarized in this review.


Background
Chimeric antigen receptor T (CAR-T) cell therapy is a novel immunotherapy that directs genetically engineered T cells with specific target to antigen-expressing tumor cells [1]. With years of preclinical and clinical development, CAR-T cell therapy is now emerging as a desirable treatment strategy with a tolerable toxicity profile for many hematological malignancies [2]. So far, FDA have approved four CD19-targeted CAR-T cell products for refractory B cell malignancies, and two B cell maturation antigen (BCMA)-targeted CAR-T cell products for multiple myeloma (MM), which are provided for patients who relapse at least after four prior lines of treatment [3,4].
However, we are still confronted with challenges including the lack of more suitable tumor-specific targets, short duration of CAR-T cells or CAR-natural killer (CAR-NK) cells, high production cost, and high relapse rate. Excitingly, researchers are making advances in new target design, functional enhancement, precise regulation, invention of universal CAR-T cell, and transformation of different chassis cells. This review summarized the latest updates of novel cellular immunotherapies for hematological malignancies from the 2021 ASH annual meeting. used in many countries. Early real-world data for Tisacel from the Center for International Blood and Marrow Transplant Research (CIMBTR) registry reported high efficacy with tolerable adverse events (AEs) [5]. A non-interventional prospective study from the CIBMTR registry enrolled 451 patients aged ≤ 25y with R/R B-ALL who received commercial Tisa-cel since August 30, 2017 [6]. After a median follow-up of 21.5 months, the best overall response (BOR) of complete remission (CR) was 87.3% (95% confidence interval [CI], 83.1-90.7), of which 98.7% (148/150) were at negative minimal residual disease (MRD) status. Any-grade cytokine releasing syndrome (CRS) was observed in 58.0% (232/400) of patients, with grade ≥ 3 in 17.8% (71/400). Immune effector cell-associated neurotoxicity syndrome (ICANS) was observed in 27.3% (109/400) of patients, with grade ≥ 3 in 10.0% (40/400). Unfortunately, 20.5% (82/400) of patients died due to recurrence or progression of primary disease. Although early real-world evidence found lower cell dose and viability of CAR-T cells in commercial Tisa-cel product than those in previous pivotal trials, there was no apparent association with manufacturing parameters and clinical outcomes [5]. In addition, current prospective study identified greater disease burden and heavier pretreatment in young adult patients (age 18-25) than in pediatric patients (age < 18), and the older patients were more inclined to experience any-grade CRS or neurotoxicity, whereas the efficacy profile was also generally similar across age groups [6]. These results further confirmed the favorable efficacy of Tisa-cel with low toxicity in the real-world setting.
At the 2021 ASH annual meeting, several studies focused on the risk factors and predictors of relapse after CAR-T treatment, especially for highrisk patients. The risk of lineage switch (LS) following CD19-directed therapies in children and young adults with R/R B-ALL has been primarily limited to case reports. Of 420 CAR-T treated patients, after a median follow-up of 30.1 months, 2.9% (12/420) of patients experienced LS [7]. KMT2A rearrangement was presented in 75% (9/12) of patients with LS compared to 7.1% (20/408) of non-LS patients (P < 0.001). TP53 mutation was an independent prognostic factor of efficacy following CD19 CAR-T cell therapy. Among 64 B-ALL patients with TP53 mutation/chromosome 17p deletion who received CD19 CAR-T cell therapy, the presence of complex cytogenetics and not bridging into allogeneic hematopoietic stem cell transplantation (allo-HSCT) were two adverse factors affecting the long-term efficacy [8].

Novel strategies of CAR-T cell therapy to improve efficacy and overcome relapse
Although CD19-targeted CAR-T cell therapy has shown encouraging CR rate, up to 45% of patients eventually relapse, especially in adult B-ALL population [9]. Early (< 6 months from infusion) loss of B cell aplasia (BCA) is associated with high relapse risk [10]. In a single center experience, the maintenance therapy after CD19 CAR-T treatment achieved similar rate of overall survival (OS) and event-free survival (EFS) while being better-tolerated and lower-cost compared to HSCT in pediatric patients with B-ALL [11].
For CD19-positive relapse, the replacement of murine-derived binding domains by human-origin ones in the CAR construct or CAR-T cell reinfusion are common treatment approaches [12]. In an oral report from University of Pennsylvania at the meeting, reinfusion of the same CAR-T product was investigated [13]. Ten patients with CD19-postive relapse reinfused the same CD19 CAR-T cell product, 50% (5/10) of patients achieved CR, and 2 of the subsequently experienced another CD19-positive relapse.
To overcome CD19-negative relapse, CAR-T cells with different targets to B-cell surface markers could be applied. Dual-target CARs against CD19 and another antigen, such as CD22 or CD20, have been extensively studied in clinical trials [14][15][16][17][18][19]. At the 2021 ASH annual meeting, Seattle Children's Hospital reported the safety and feasibility of SCRI-CAR19 × 22v2, a dual transduced patient-derived product with lentiviral vectors encoding for either a CD19-or CD22-specific 4-1BB CAR [20]. Among 10 of 11 patients obtained CR with negative MRD, grade 1 CRS presented in 45% (5/11) of patients. ICANS occurred in 5 patients, with all grade 1 but one single self-limited grade 3. However, they also found that the peak engraftment of CAR-T cells in vivo lied most frequently between day + 7 and + 14, and it was predominated by CD22 CAR-T cells, with minimal contribution of the dual and CD19 CAR-T cells. Optimizing transduction methods may be required for a more balanced product to maintain effective dual targeting. Co-administration of CD19 CAR-T cells and CD22 CAR-T cells in adult patients with R/R B-cell malignancies also exhibited high efficacy and low toxicity [21]. Of the 11 patients who received co-administration with two humanized autologous CAR-T cells targeting CD19 and CD22, 100% (11/11) achieved CR with negative MRD in a month after infusion. Of the 13 treated patients, all experienced grade 1 or 2 CRS, and only 2 patients had ICANS. Moreover, CD19/CD123 dual-target CAR-T cells and tri-specific  [30]. Results from the US Lymphoma CAR-T Cell Consortium are similar to the ZUMA-1 trial, despite that the former included patients who did not meet ZUMA-1 eligibility criteria based on comorbidities [31]. However, CAR-T cell therapy in R/R LBCL still faces challenges. In DESCAR-T, a French nationwide registry, which enrolled all patients post commercialized CAR-T cell therapy, 43.3% (238/550) of treated patients relapsed within a median follow-up period of 7.9 months [32]. Therefore, to improve CAR-T cell efficacy and achieve prolonged duration of response (DOR), the application of lenalidomide or programmed cell death protein 1 (PD-1) inhibitors is a promising maintenance therapy that improved the OS in patients with R/R DLBCL [33,34].
The prognosis for patients with early R/R LBCL after first-line therapy remains poor. ZUMA-7 (NCT03391466), an international, randomized, phase 3 trial, enrolled 359 patients with second-line R/R LBCL treated by Axi-cel versus standard of care (SOC) treatment (high-dose chemotherapy with autologous stem cell transplantation (ASCT)) [35]. Among randomized patients, the ORR and CR rate were significantly higher in Axi-cel group than SOC group (ORR: 83% vs. 50%, CR: 65% vs. 32%, P < 0.0001]. The toxicity of Axi-cel was manageable and consistent with third-line Axi-cel therapy. Patient-reported outcomes in ZUMA-7 also showed that Axi-cel treatment led to clinically meaningful improvement in quality of life compared with SOC [36]. In a pivotal, global, randomized, multicenter, phase 3 TRANSFORM study, Liso-cel as a second-line therapy also demonstrated significant improvement in the CR rate (66% vs. 39%, P < 0.0001) and progression-free survival (PFS) (median PFS 14.8 vs. 5.7 months, P = 0.0001) compared with SOC [37]. However, the randomized, global, phase 3 BELINDA study, comparing Tisa-cel and SOC in patients with second-line R/R LBCL, revealed no significant difference of EFS between two groups. After a median follow-up of 10 months, the median EFS was 3 months (Hazard ratio [HR] 1.07; 95% CI, 0.82-1.40; P = 0.69) in both groups, while the ORR was 46% vs. 43% [38]. The different results among these three clinical trials may be attributed to diverse study design elements; For instance, no bridging therapy was allowed in TRANSFORM and ZUMA-7, whereas BELINDA study enrolled more aggressive patients with permission of bridging therapy and potential delay of Tisa-cel application. Details from these clinical trials are summarized in Table 2. Therefore, larger phase 3 clinical studies with longer follow-up period are warranted to determine the role of CAR-T cell therapy as a second-line treatment of R/R LBCL.

Indolent non-Hodgkin lymphoma (iNHL)
Follicular lymphoma (FL) is an indolent disease with a continuous relapse pattern that typically requires multiple lines of therapy. At the meeting, primary analysis of the single-arm, multicenter, phase 2 ELARA trial enrolling 94 R/R FL patients demonstrated that, after a 17-month median follow-up, Tisa-cel produced high ORR (86.2%) and CR rate (69.1%) [43]. Durable response was also achieved in high-risk patients with progression of disease within 2 years (POD24) (CR 59%, 12-month PFS 60.8%). Safety was consistent with known Tisa-cel profile.
ZUMA-5 is a phase 2, multicenter, single-arm study that evaluated Axi-cel for treatment of R/R iNHL

Novel strategies of CAR-T cell therapy to improve efficacy and overcome relapse
Although CD19-targeted CAR-T cell therapy has represented a paradigm shift in R/R B-NHL, many patients subsequently experienced disease progression or relapse with poor prognosis. Antigen escape or lack of adequate antigen expression contributes to the failure of CAR-T cell therapy [46]. CD22 and CD20, the other two lineage specific markers in B cell development, are attractive targets [19]. At the meeting, a single-center, phase 1 doseescalation clinical trial (NCT04088890) revealed that CD22 CAR-T cell therapy mediated high durable remission rates in 21 patients with DLBCL who relapsed after prior CD19 CAR-T treatment. The ORR at day 28 was 86% (CR, n = 11; partial response (PR), n = 7), while the median PFS and OS were not reached after a mean follow-up of 7.3 months (range, 1.2-21.3) [47]. In another ongoing phase 1/2 clinical trial of CD20 CAR-T cell therapy for high-risk B-NHLs and chronic lymphocytic leukemia (CLL), the ORR was 94% (15/16) with CR rate of 62% (10/16), while no grade 3 or 4 CRS or ICANS were noted [48]. Targeting two antigens simultaneously may overcome antigen escape in B-cell malignancies. In a phase 1/2 single-center, prospective trial (NCT04186520), an IL7/ IL15-expanded bispecific lentiviral anti-CD20, anti-CD19 (LV20.19) CAR-T cell product was tested [49]. Among 22 patients with DLBCL, FL, and MCL, the ORR was 91% (20/22), and the CR rate was 55% (12/22). Only one patient experienced grade 3 CRS, and 3 patients had grade 3 ICANS.
R/R NHL patients with TP53 gene disruption confer inferior prognosis. A clinical trial enrolled 60 patients with TP53 alteration who received CAR19/22 T-cell cocktail therapy [50]. After a median follow-up of 16.7 months (range, 3.1-41.0), the median OS was not reached, and the median PFS was 14.8 months (95% CI, 5.1-not estimated (NE)) in the patients harboring TP53

Update of T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoma therapy from the 2021 ASH annual meeting
Compared with the significant progress of CAR-T cell therapy in B-ALL and B-NHL, broadening the success to treating T cell malignancies, including T-ALL and T cell lymphoma, is still intractable. Treating T-cell malignancies with T cell therapy presents a unique problem, since cytotoxic cell may share the same antigens with malignant cell, causing risks for the fratricide of cytotoxic cells and the contamination of malignant cell during manufacturing in the autologous setting [51,52]. To avoid this problem, shared antigens such as CD7 can be knocked out of the CAR-T cell to specifically target CD7 on the malignant cell. At the meeting, more than 10 reports from preclinical and clinical trials with novel targets like CD7, CD5, CD21 and CCR9 were published (Fig. 1A). Table 3 illustrates the outcomes of phase 1 clinical trials of CAR-T therapy for T-ALL and T cell lymphoma. In a phase 1 clinical trial of patient or donor-derived CD7-targeting CAR-T cell therapy for R/R T-ALL (NCT04572308), 17 patients were enrolled. After a median follow-up of 105 days (range, 32-206), 92.9% (13/14) of patients achieved CR, or CR with incomplete hematological recovery (CRi), and negative MRD [53]. In addition, 80% (4/5) of patients with extramedullary disease (EMD) also achieved extramedullary remission at a median of day 32 after infusion. Another phase 1 clinical trial of patient or donor-derived CD7-targeting CAR-T cell therapy for R/R T-cell lymphoblastic lymphoma (T-LBL) (NCT04916860) also showed a high initial efficacy and a good safety profile [54]. Five patients who had prior bone marrow blasts all achieved CRi with negative MRD. Of the 7 patients who had diffuse EMD, 5 achieved EMD CR. Preclinical data from an "off-the-shelf " allogeneic CD7-targeted CAR-T cell therapy WU-CART-007 also supported the well-tolerance and anti-tumor efficacy in vivo [55,56]. Co-culture experiments in vitro confirmed strong cytotoxicity against CD7-expressing cells including T-ALL cell, primary T cell, and NK cell. Phase 1/2 studies are ongoing to further verify its efficacy and safety.
However, CD7-targeted CAR-T cell therapy for T-cell malignancies is challenging since target antigens are expressed on normal T cells. This leads to two main problems: loss of essential normal T cells and self-kill 'fratricide' of CAR-T cells. Therefore, exploring new targets in R/R T cell malignancies is inevitable. In a phase 1 clinical trial (NCT03081910) from the meeting, autologous T cells expressing a CD5-specific CAR were designed for patients with refractory or relapsed T cell lymphoma [57]. Forty-four percent (4/9) of patients responded, with 3 proceeding further allo-HSCT. After a long-term follow-up, 2 patients remained alive with CR for 29 months and 24 months, respectively. CCR9 is a G protein coupled receptor (GPCR) for the natural ligand CCL25, and is expressed in gut intraepithelial γδ T cells, some plasmacytoid dendritic cells, and doublepositive thymocytes, but in less than 5% of normal circulating T and B cells. Potent anti-leukemic function of anti-CCR9 CAR-T cell has been proved both in vitro and in animal models, whose efficacy is not associated with loss of essential normal T cells or with CAR-T cell fratricide [58]. More clinical trials with long-term follow-up are needed to further evaluate the potential benefits and side effects of CAR-T cell therapy for T-cell malignancies.

Update of multiple myeloma therapy from the 2021 ASH annual meeting
MM is an incurable hematological malignancy of plasma cells [59,60]. Although many novel therapies are available for MM [61,62], the treatment of MM still remains elusive. CAR-T cell therapy represents a promising immunotherapeutic approach with remarkable potential in relapsed and refractory multiple myeloma (RRMM) [63,64]. Sequential CAR-T cell infusion followed by lenalidomide maintenance after ASCT sustained MRD negativity for more than 2 years in patients with newly diagnosed MM [65]. BCMA is currently the main target for CAR-T cells in MM, as it is predominantly expressed on differentiated malignant plasma cells [66]. In March 2021, FDA approved Idecabtagene Vicleucel (Ide-cel) as the first BCMA-directed CAR-T cellular immunotherapy for RRMM based on results from the pivotal KarMMa trial [67]. Recently, the FDA authorized another BCMA CAR-T product Ciltacabtagene Autoleucel (Cilta-cel) for patients with MM who relapsed at least after four prior lines of treatment.

Novel target of CAR-T cell therapy for RRMM
BCMA-targeted CAR-T cell therapy has shown promising results in RRMM, but relapse is common. Additional treatment options with novel therapeutic targets are warranted (Fig. 1A). In a phase 1 clinical trial of MCARH109, the first-in-class GPCR Class C Group 5 Member D (GPRC5D)-targeted CAR-T cell therapy, 12 patients with RRMM were treated [68]. The ORR was 83% (10/12), with 2 patients achieved stringent complete response (sCR). After a median follow-up of 13 weeks (range, 2.0-39.1), 75% (9/12) of patients achieved progression free without additional treatment. CRS occurred in 92% (11/12) of patients, with grade 3 in only one patient, and no ICANS events or dose limiting toxicities were reported. More importantly, 6 patients who relapsed after previous BCMA CAR-T cell therapy all responded to GPRC5D-targeted CAR-T cell therapy, including 2 patients who achieved sCR. Therefore, GPRC5D-targeted CAR-T cell therapy will be another potential treatment option for patients with RRMM, especially for those who relapsed after BCMA CAR-T cell therapy.

Updated clinical data for BCMA-targeted CAR-T cell therapy for RRMM
Long-term safety and efficacy data from diverse BCMAtargeted CAR-T cell products were updated at the 2021 ASH annual meeting, including Cilta-cel [69], CT053 [70], and others (

Modified BCMA-target CAR-T therapy for RRMM
Many approaches for optimization of BCMA-targeted CAR-T cell therapy are under exploration and development. Bb21217 has the same CAR molecule as that in bb2121 (Ide-cel), but with a PI3K inhibitor motif bb007 added to the CAR construct during ex vivo culture to enrich the memory-like T cells in drug product and to decrease the proportion of highly differentiated or senescent T cells. Updated results from the phase 1 CRB-402 study showed that among 72 patients with RRMM, the ORR was 69% (50/72) and sCR was achieved in 28% (20/72) of patients [75]. Of the 15 patients evaluable for MRD with ≥ CR, 93% (14/15) of patients were MRD-negative. Eighty-one percent (30/37) of patients and 60% (9/15) of patients maintained detectable CAR-T cells at 6 and 12 months, respectively. The study also revealed that patients with higher levels of proliferative, less differentiated, memory-like CAR-T cells at peak expansion were more likely to experience prolonged DOR. CT103A, a fully human BCMA-specific CAR-T product, showed excellent safety and promising efficacy in heavily pretreated RRMM patients [76]. The unique CAR structure containing fully human single-chain variable fragment (scFv) may bypass the potential host anti-CAR immunogenicity and retain antitumor activity. In a multicenter, single- CAR density may influence antitumoral efficacy of BCMA CAR-T cell, which correlates with clinical outcomes. The combination of gamma secretase inhibitor (GSI) showed increased BCMA surface density on tumor cells and decreased soluble BCMA levels in the peripheral blood in an immunodeficient mouse model, therefore enhancing the efficacy of BCMA CAR-T cells (Fig. 1B). In a phase 1 first-in-human trial of BCMA-targeted CAR-T cells in combination with a GSI (JSMD194) for 18 patients with RRMM, all patients completed the 5-day run-in with JSMD194 [78]. The ORR was 89% (16/18), with 8 patients achieving CR. BCMA binding capacity increased from a median of 610 to 9563 receptors per cell. Therefore, GSI co-administration can increase BCMA surface density on plasma cells, augment anti-tumor activity of CAR-T cell, and induce durable and rapid response.

Update of acute myeloid leukemia (AML) therapy from the 2021 ASH annual meeting
R/R AML patients have a dismal prognosis. Numerous tumor antigens, such as CD33, CD123, and CLL-1, have been explored as potential target antigens for treatment of AML in the past few years (Fig. 1A) [79][80][81]. However, due to the lack of ideal specific antigen targets and the risk of fatal "off-tumor, on-target" side effects, CAR-T cell therapy remains challenging in AML [82]. More than 20 preclinical and clinical studies of CAR-T cell therapies with different targets for AML patients were presented at the meeting. The most remarkable results are listed in Table 5. PRGN-3006 UltraCAR-T is a novel CAR-T product manufactured from autologous T cells within 48 hours, while using non-viral gene delivery system to simultaneously express CD33 CAR, membrane-bound IL-15 (mbIL-15), and suicide switch. In a phase 1/1b doseexpansion study of PRGN-3006 in adult patients with R/R AML, 15 heavily pre-treated patients received PRGN-3006 at different dose levels up to 1 × 10 6 cells/ kg [83]. The ORR for 6 patients at dose level (1-3 × 10 5 cells/kg) was 50%. Among them, 2 patients with post-HSCT relapse also responded. Clinical therapeutic efficacy and safety of CD38-targeted CAR-T cells in AML patients who relapsed after HSCT were also investigated (NCT04351022) and revealed [84]. Four weeks after infusion, 66.7% (4/6) of patients achieved CR or CRi. The median OS and leukemia-free survival (LFS) were 7.9 months and 6.4 months, respectively. Side effects were clinically manageable among all patients. These novel CAR-T products may expand treatment options for patients with R/R AML.
Additionally, preclinical exploration is also important for the advancement of precise regulation and enhanced function (Fig. 1B). Dual targeting with FLT3 CAR-T immunotherapy and FLT3 inhibitor (gilteritinib) are promising therapeutic strategies in FLT3-mutant AML and KMT2A-rearrangement ALL [85]. SC-DARIC33, a pharmacologically controlled CD33-targeted anti-AML CAR-T product regulated by low concentrations of rapamycin, have been established and evaluated in an upcoming phase 1 clinical trial [86].
CAR-NK cell therapy has some considerable advantages over CAR-T cells, including unique recognition mechanism, powerful cytotoxic effect, and "off-the-shelf " nature [87,88]. It is particularly attractive in AML since the inherent graft-versus-leukemia (GVL) reaction of NK cells can be effectively augmented by a CAR directed to an AML expressed antigen [89,90]. The sources of CAR-NK cells include human peripheral blood NK cells, umbilical cord blood NK cells, or even NK cell lines. CAR-NK products targeting CD70 [91], CD33 or FLT3 [92], and CLL-1 [93] with different engineering methods, such as CRISPR/Cas9 or logic-gated CAR gene circuits, exhibit potent anti-leukemia activity in vitro and are now under preclinical evaluation (Fig. 1B). However, challenges of low persistence, low transduction efficiency, and immunosuppressive milieu of tumor microenvironment still exist.

Update of the universal CAR from the 2021 ASH annual meeting
The universal "Off-the-shelf " allogeneic CAR-T cells and other immune effector cells, such as NK or gamma delta (γδ) T cells, can be premanufactured from healthy donors and may offer alternatives to autologous strategies. The main design strategy of universal CAR-T (UCAR-T) cells is to genetically modified allogenic CAR-T cells without immunogenicity, which has specific anti-tumor activity without graft-versus-host disease (GvHD) or host rejection (Fig. 1C).
The traditional strategy of "Off-the-shelf " CAR-T cell therapy is to develop a universal CAR-T product by disruption of the T-cell receptor alpha constant gene (TRAC) and CD52 gene to avoid GvHD. ALLO-501A is a genetically modified anti-CD19 allogenic CAR-T product that uses TALEN ® technology to disrupt TRAC and CD52. In the updated phase 1/2 ALPHA2 (NCT04416984) study, 13 patients with R/R LBCL were enrolled with an additional anti-CD52 monoclonal antibody for selective and transitory host lymphodepletion [94]. The ORR and CR rate were 66.7% (4/6), with 3 patients PR converting to CR after consolidation. No CRS, ICANS, GvHD, or no dose-limiting toxicities occurred.
Another strategy with CD19 CAR knocked into the TRAC locus by a TRAC-specific ARCUS nuclease is also employed, which disrupts the endogenous T-cell receptor. PBCAR0191 is an allogeneic "off-the-shelf " CAR-T product targeting CD19 with T cells derived from non-HLA-matched healthy donors. In a phase 1/2 clinical trial, 21 patients with R/R B-cell malignancies were treated, including 16 NHL patients and 5 B-ALL patients [95]. Six patients progressed after treatment with auto-CD19 CAR-T cell, and 8 patients progressed after auto or allogeneic HSCT. Among the 13 evaluable patients with NHL, 8 patients (62%) achieved CR/CRi, while 4 out of 5 (80%) B-ALL patients obtained CR. PBCAR0191 also demonstrated a manageable safety profile with mild CRS and no evidence of GvHD [95,96].

Update of pluripotent stem cell-derived cellular therapy from the 2021 ASH annual meeting
While allogeneic CAR-T or CAR-NK cell therapies are already demonstrating clinical promise, these strategies remain limited due to donor variability and batch to batch heterogeneity [97]. It is desirable to utilize a single renewable cell source to standardize the manufacture and quality of these novel immunotherapies. Development of CAR-engineered induced pluripotent stem cells (iPSCs) has the potential to achieve true consistency and unlimited scalability [98]. Moreover, a single iPSC clone with relatively the best gene-editing efficiency can be selected and isolated to serve as a source for gene-edited cell bank. Fate Therapeutics has developed FT819, a first-of-kind, allogeneic, off-the-shelf CAR-T product derived from iPSC line, which is precisely engineered to insert a novel anti-CD19 CAR into the TRAC locus, to achieve more regulated CAR expression and abrogate risk of GvHD [99]. FT819 is currently being used in a multi-center phase 1 study (NCT04629729) for treatment of R/R B-cell lymphoma, CLL, and precursor B-ALL [100].
CAR-engineered iPSC-NK cells are also under development. FT596 is an off-the-shelf iPSC-derived CD19 CAR-NK product for R/R B-cell lymphoma. FT596 incorporates three genetically encoded functional components: a CD19-targeted CAR; a novel highaffinity, non-cleavable CD16 Fc receptor that enhances tumor targeting and antibody-dependent cell cytotoxicity in combination with a therapeutic monoclonal antibody; and an IL-15/IL-15 receptor fusion that promotes cytokine-autonomous persistence [101,102]. Twenty patients were enrolled and exhibited none dose-limiting toxicity or severe adverse events. Of the 17 efficacy-evaluable patients, 9 achieved an objective response after the first FT596 treatment cycle. At a single-dose level of ≥ 90 million cells, 8 of 11 efficacy-evaluable patients achieved an objective response, including 7 patients with CR [103]. FT576, a similar iPSC-derived CAR-NK product targeting BCMA, also showed efficiency in preclinical studies (Fig. 1B) [102]. Table 6, Fig. 1C, and Fig. 1D highlight some preclinical and clinical trials of universal CAR-T, CAR-NK products, and iPSC-derived immunotherapy at the 2021 ASH annual meeting.

Mechanism research of CAR-T therapy from the 2021 ASH annual meeting
Although CAR-T cell therapy has shown high clinical efficacy in hematological malignancies, there is unpredictable variability in the duration and depth of response. The mechanisms behind these divergent outcomes are not well understood yet. Heterogeneity of patients at the level of both tumor genomics and tumor microenvironment (TME) likely contributes to this important knowledge gap.
Single-cell multi-omics can provide a better understanding of the dynamic and evolution of CAR-T cells in human body. At the 2021 ASH annual meeting, Zachary Jackson et al. employed single cell RNA sequencing (scRNA-seq) and protein surface marker profiling in serial CD19 CAR-T cell samples from patients with NHL [104]. They revealed the evolution of CAR-T cells toward a non-proliferative, highly-differentiated, exhausted state in patients with poor response at the transcriptional and translational levels. David T. Melnekoff et al. also performed a longitudinal high resolution single cell genomic and proteomic analysis for patients with MM treated by BCMA CAR-T cell therapy [105]. They found significant up-regulation of anti-apoptotic genes at baseline and at relapse in poor responders, suggesting a novel mechanism of tumor-mediated escape. Molecular fate mapping of long-term persisting CAR-T cells from two leukemia patients with CR over a decade revealed functional persistence of CAR-T cells as a key predictor for durable remission [106].
CRS is the most common severe toxicity associated with CAR-T cell therapy. Caroline Diorio et al. performed comprehensive secretome profiling to measure more than 1400 serum analytes in serial samples collected from patients treated by CD19 CAR-T cells [107]. Two novel pre-infusion biomarkers, MILR1 and FLT3, were identified to predict the development of CRS. FLT3/ FLT3 ligand may play a potential biological role in severe CRS.
Modulation of the gut microbiota by using antibiotics can enhance the efficacy of tumor-specific T cells. In a retrospective cohort of patients with B-ALL receiving oral vancomycin after CD19 CAR-T cell therapy, higher CAR-T cell expansion and serum inflammatory cytokines were observed [108]. In preclinical models, vancomycinmediated modulation of the gut microbiota achieved better anti-tumor effect via cross-priming and enhanced CAR-T cell expansion in tumor samples.

Conclusions and perspectives
With the development of cutting-edge technologies in life science, cellular immunotherapy has achieved major impact on the treatment of hematological malignancies during the past decade. To date, six commercial CAR-T cell products have been approved by FDA for the treatment of R/R B-cell malignancy and MM. At the 2021 ASH annual meeting, real-world data from different countries and regions fully proved the efficacy and safety of CAR-T cell therapy for patients with multi-line treatment failure. CAR-T cell therapy has even advanced into first-line or second-line therapy for some high-risk patients with invasive B-cell malignancies. CD7 CAR-T products for T cell malignancies revealed exciting clinical efficiency with manageable safety profile. Novel targets of CAR-T cell therapy, such as CD33 for AML, GPRC5D for MM, as well as dual-target CAR-T products can provide more potential choices for heavily pre-treated patients. Universal CAR products with diverse gene-editing strategies, including allogenic CAR-T and CAR-NK, hold promise in early clinical trials. iPSC-derived immunotherapy highlights a future direction of cellular immunotherapy: clone selection, powerful gene-editing, unlimited cell sources, and precise manipulation. In-vivo induced CAR-T cells by nanocarriers loaded with CAR genes or gene-editing tools can potentially overcome the current limitations. Basic research provides a comprehensive knowledge of the dynamics of CAR-T cells and the interactions in TME. However, the underlying mechanisms of anti-tumor activity and exhaustion of CAR-T cell, and tumor relapse after treatment are very complicated and remain obscure. The combination with other treatment strategies, such as small molecule inhibitors or HSCT, may improve clinical outcomes. Further studies are warranted to comprehensively understand the advantages, efficacy, long-term complications, and major diversity among CAR-engineered cells.