Chimeric antigen receptor (CAR) T cell therapies have developed over the past decade to become an increasingly important treatment for cancers, especially relapsed/refractory lymphoid malignancies. While promising, currently approved CAR T cell therapy products have limitations, including cost, consistency, and speed of manufacturing, toxicities including cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), and prolonged cytopenias. Moreover, in the common indication for relapsed and refractory large B cell lymphoma (LBCL), the majority of patients experience progression of disease (POD) following CAR T cell therapy. Those working to develop next-generation CAR T cell products are seeking to address these deficiencies, with a notable such effort by Zhang et al. recently published in Nature.1 Zhang et al. manufactured CAR T cells using a new non-viral, gene-specific integration approach using CRISPR-Cas9 introduced via electroporation. They subsequently knocked out several genes of interest by inserting a 4-1BB and CD3ζ CAR into the target locus, most notably PD1. These PD1-19bbz cells outperformed a CAR T cell product similar to current FDA-approved therapies that utilize lentiviral CAR transduction in an in vitro assay and were then tested in a small (8 patient) phase I clinical trial, demonstrating promising efficacy. These promising data were made more impressive by the fact that relatively small cell doses were used, specifically 560,000 cells/kg, which is compatible with low doses used in FDA-approved products (shown in Table 1). This is likely in part due to the high percentage of memory CAR T cells generated. This work is potentially transformational by offering a new CRISPR-Cas9-based CAR T cell manufacturing process that can edit out an undesirable target gene while enabling greater T cell persistence and efficacy. The phase I clinical trial only included 8 patients, and longer-term follow up will be needed to fully validate the findings.
Table 1.
Current FDA-approved chimeric antigen receptor (CAR) T cell products + PD1-19bbz
| CAR T product name (trade name) | Approved indications | Registration trials (year, indication) and key efficacy outcomes | Registration trial: Toxicity outcomes | CAR T properties |
|---|---|---|---|---|
| Axicabtagene ciloleucel (YESCARTA) | R/R PMBCL; R/R HGBL; R/R DLBCL (+transformed); R/R FL (3L+) | ZUMA-1 (2017, R/R PMBCL, HGBL, and DLBCL, FL grade 3B): 108 treated patients; ORR 83%, CRR 58%, mDOR 11.1 months, mPFS 5.9 months, mOS NR at 2 year follow-up; similar real-world data ZUMA-5 (2021, R/R FL, MZL): 104 treated patients eligible for analysis (84 FL, 20 MZL); ORR 92%, CRR 74%, mDOR NR (11.1 month MZL), mPFS NR (12 month MZL), mOS NR with 17.5 month median follow-up ZUMA-7 (2022, R/R DLBCL in 2nd line compared with autologous BMT): 170 patients treated with Axi-cel ORR 83%, CRR 65%. mPFS 14.7 mos, mOS NR, mEFS 8.3 mos with 24.9 mos median follow-up |
ZUMA-1: all grade ≥3 AEs 48%; 18% cytopenias >3 months, CRS 11%, NEs 32%; TRM ∼2%. ZUMA-5: all grade ≥3 AEs 50%; cytopenias 70%, infection 18%, CRS 7%, NEs 19%; TRM 3% |
- CD19 targeted- lentiviral transduction - CD28 costim- 17 day median manufacturing time - cell dose: 2–200 million cells/kg |
| Brexucabtagene autoleucel (TECARTUS) | R/R MCL; R/R B-ALL (adults) | ZUMA-2 (2020, MCL): 68 treated patients; ORR 91%, CRR 68%, mDOR 28.2 months, mPFS 25.8 months, mOS 46.6 months with 35.6 month median follow-up ZUMA-3 (2021, B-ALL): 45 treated patients; ORR 75%, CRR 69%, mDOR 14.5 months, mPFS 7.3 months, mOS 12.1 months with 22.1 month median follow-up |
ZUMA-2: grade ≥3 cytopenias 94%; infection 32%, CRS 15%, NEs 31%; TRM ∼3% ZUMA-3: grade ≥3 CRS 31%, NEs 38%; TRM ∼4% |
- CD19 targeted - lentiviral transduction - CD28 costim- 15–16 day median manufacturing time - cell dose: 2–200 million cells/kg |
| Lisocabtagene maraleucel (BREYANZI) | R/R DLBCL (+transformed); R/R HGBL; R/R PBMCL; R/R FL grade 3B | TRANSCEND (2021, R/R DLBCL, HGBL, PBMCL, FL grade 3B): 269 treated patients, ORR 73%, CRR 53%, mOS 18.8 months TRANSFORM: 92 treated patients, mEFS 10.1 months at 6.2 month median follow-up |
TRANSCEND: grade ≥3 neutropenia 60%, anemia 37%, thrombocytopenia 27%, CRS 2%, NEs 10%; TRM <1% TRANSFORM: similar to TRANSCEND |
- CD19 targeted - lentiviral transduction - 4-1BB costim - 26 day median manufacturing time - cell dose: 50–110 million cells |
| Tisagenlecleucel (KYMRIAH) | R/R DLBCL; R/R FL (3L+); R/R B-ALL age ≤25 years | JULIET (2018, R/R DLBCL): 93 treated patients; ORR 52%, 40% CRR, 12 months PFS 65% (79% with prior CR) ELARA (2022, R/R FL): 97 treated patients; ORR 86.2%, CRR 69.1%, median follow-up of 16.59 months CCTL019B2202 (2017, R/R ALL): CRR 63%, median follow-up of 4.8 months |
JULIET: grade ≥3 CRS 22%, NEs 12%, cytopenias >28 days 32%, infections 20%, febrile neutropenia 14%; 0% TRM but 3 deaths from PD within 30 days. ELARA: 48.5% CRS (0% grade ≥3), NEs 37.1% (3% grade ≥3); no TRM CCTL019B2202: CRS 79%, NEs 65% |
- CD19 targeted - lentiviral transduction - 4-1BB costim - 54 day median manufacturing time - cell dose: 600,000 to 600 million cells |
| Idecabtagene vicleucel (ABECMA) | R/R MM (5L + after prior immunomodulatory agent, proteasome inhibitor, and an anti-CD38 mAB) | KarMMa (2021, R/R MM): 128 treated patients; ORR 73%, CRR 33%, MRD-negative rate 26%, mDOR 10.7 months, mPFS 8.8 months, mOS 19.4 months with median follow-up of 13.3 months | KarMMa: grade ≥3 neutropenia 89%, anemia 60%, thrombocytopenia 52%, infection 22%, CRS 4% (84% overall), 3% NEs (18%); ∼3% TRM | - BCMA targeted - lentiviral transduction - 4-1BB costim - 15 day median manufacturing time - cell dose: 300–460 million cells |
| Ciltacabtagene autoleucel (CARVYKTI) | R/R MM (5L + after prior immunomodulatory agent, proteasome inhibitor, and an anti-CD38 mAB) | CARTITUDE-1 (2022, R/R MM): 97 treated patients; ORR 97%, CRR 67%, mDOR NR, mPFS N with 12 months PFS 77% and OS 89% with median follow-up of 12.4 months | CARTITUDE-1: grade ≥3 neutropenia 95%, anemia 68%, thrombocytopenia 60%, CRS 4% (95% overall), NEs 9% (21% overall); ∼6% TRM | - BCMA targeted - lentiviral transduction - 4-1BB costim - 29 day median manufacturing time - cell dose: 500,000 to 1 million cells/kg |
| PD1-19bbz (Zhang et al.1) | N/A | “Non-viral, specifically targeted CAR T cells achieve high safety and efficacy in B-NHL” (2022, aggressive B-NHL); 8 patients treated, ORR 100%, CRR 87.5% (62.5% durable) with median follow-up of 12 months | Grade <3 CRS in 50% (none grade ≥3), no NEs, transient/low-grade cytopenias with chemotherapy | - CD19 + PD1 knockout targeted - CRISPR-Cas9 electroporation (non-viral) - cell dose: 560,000 to 2.35 million/kg |
R/R, relapsed/refractory; diffuse large B cell lymphoma, DLBCL; B-ALL, B cell acute lymphoblastic leukemia; PMBCL, primary mediastinal large B cell lymphoma; HGBL, high-grade B cell lymphomas; FL, follicular lymphoma; #L+, indicated line of therapy or later; MCL, mantle cell lymphoma; MM, multiple myeloma; MZL, marginal zone lymphoma; ORR, overall response rate; CRR, complete response rate; mDOR, median duration of remission; mPFS, median progression-free survival; mOS, median overall survival; AE, adverse event; CRS, cytokine release syndrome; NEs, neurologic events; TRM, treatment-related mortality; MRD, minimal residual disease.
As of this writing, there are five FDA-approved CAR T cell products for relapsed/refractory B cell acute lymphoblastic leukemia (B-ALL), diffuse LBCL (DLBCL), grade 3B and relapsed/refractory follicular lymphoma (FL), mantle cell lymphoma (MCL), primary mediastinal BCL (PMBCL), other high-grade lymphomas, and multiple myeloma (see Table 1). All five products currently utilize a manufacturing approach where a lentiviral or γ-retroviral genome is edited to contain a CAR gene product, which contain several key domains crucial for CAR T cell function.
The FDA-approved CAR T cell products utilize autologous T cells for manufacturing and must be collected via leukapheresis, washed, undergo T cell enrichment and activation (often using anti-CD3 and anti-CD28 ligands), be transduced with the viral vector containing CAR-encoding sequences, and then quality controlled before being sent to treating institutions. While this process has produced effective cellular therapies that have revolutionized care for thousands of patients, this process has several important drawbacks that must be overcome. First, the manufacturing process takes time, typically from 15 to 30 days, during which time patients can experience progressive disease and may require “bridging” treatments after leukapheresis. In the pivotal studies’ designs, there has been ubiquitous lack of accounting for subjects that did not make it to planned CAR T cell therapy in the “modified” intention-to-treat designs. Also, viral transduction can introduce off-target genomic alterations in target T cells that, along with variation in leukapheresis parameters and quality control techniques, may adversely impact product quality and thereby contribute to inferior outcomes for some patients.
While most patients treated with currently approved CAR T cell therapies achieve at least a partial remission (Table 1), the majority of patients will experience POD, often with an abysmal prognosis.2 There are multiple mechanisms by which CAR T cell failure occurs. While tumoral antigenic loss/escape can occur in up to a third of patients due to negative selective pressure exerted by effector cells, the majority are thought to result from “T cell exhaustion,” often characterized by a general shift in T cell phenotype to terminally differentiated CD8+ effector cells that lose stem-like characteristics including the ability to persist and retain memory.3,4 Variability in manufacturing, pre-leukapheresis treatment effects (e.g., bendamustine lymphodepletion), tumor microenvironmental factors, and many other factors all contribute to T cell exhaustion. Combating T cell exhaustion is an active area of research, with some ongoing investigation of PD1/PDL1 checkpoint inhibition, BTK inhibition, and others, with encouraging, but overall modest, results to date.5
The novel CAR T cell manufacturing approach by Dr. Zhang et al. has the potential to improve upon current cellular therapies in several important ways. First, utilizing non-viral electroporation and CRISPR-Cas9-based gene editing for CAR insertion appears to produce a highly precise genetic alteration in transduced CAR T cells that could allow for faster manufacturing. Streamlining the manufacturing process is critical for allowing patients to receive these life-saving treatments while also limiting the need for bridging therapies that may adversely impact patients’ conditions prior to treatment. Second, while viral transduction has been shown to produce effective cellular therapies, emerging CRISPR-Cas9-based methods appear to be more precise, less likely to introduce random insertions into important T cell coding regions, and easier to target specific genes of interest (such as PD1). Third, by suppressing PD1 expression through integration of the CAR gene product into T cells, Zhang et al. have demonstrated an important strategy to circumvent possible T cell exhaustion and appear to have found another way to produce CAR T cells with a greater proportion of memory cells, which are increasingly recognized to have greater persistence in vivo as well as greater clinical activity. By infusing CAR T cells optimized for expansion and persistence, lower cell doses may be used. This may advantageously mitigate toxicities, such as CRS, ICANS, and cytopenias, that are common with our currently approved CAR T products. Ensuring that cellular therapy products are fit and resistant to exhaustion is an important strategy for next-generation CAR T cell production.
The phase I clinical trial in Zhang et al.’s study was small, with only 8 patients, and their findings need to be validated in a larger prospective clinical trial. Disabling PD1 expression may present some risks for checkpoint inhibitor-related toxicities, and a larger patient cohort would do much to not only confirm efficacy but also safety in this respect. Despite this limitation, which is expected in a phase I cellular therapy study, the approach outlined elegantly serves as an important milestone in the burgeoning field of cellular therapies and is worthy of further investigation and validation.
References
- 1.Zhang J., Hu Y., Yang J., Li W., Zhang M., Wang Q., Zhang L., Wei G., Tian Y., Zhao K., et al. Non-viral, specifically targeted CAR-T cells achieve high safety and efficacy in B-NHL. Nature. 2022;609:369–374. doi: 10.1038/s41586-022-05140-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Di Blasi R., Le Gouill S., Bachy E., Cartron G., Beauvais D., Le Bras F., Gros F.X., Choquet S., Bories P., Feugier P., et al. Outcome of relapsed/refractory aggressive B-cell lymphoma patients relapsing after anti-CD19 CAR T-cells and enrolled in the descar-T French national registry. Blood. 2021;138:885. [Google Scholar]
- 3.Deng Q., Han G., Puebla-Osorio N., Ma M.C.J., Strati P., Chasen B., Dai E., Dang M., Jain N., Yang H., et al. Characteristics of anti-CD19 CAR T cell infusion products associated with efficacy and toxicity in patients with large B cell lymphomas. Nat. Med. 2020;26:1878–1887. doi: 10.1038/s41591-020-1061-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Van der Leun A.M., Thommen D.S., Schumacher T.N. CD8+ T cell states in human cancer: insights from single-cell analysis. Nat. Rev. Cancer. 2020;20:218–232. doi: 10.1038/s41568-019-0235-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Mohty R., Gauthier J. Current combinatorial CAR T cell strategies with Bruton tyrosine kinase inhibitors and immune checkpoint inhibitors. Bone Marrow Transpl. 2021;56:2630–2636. doi: 10.1038/s41409-021-01420-9. [DOI] [PubMed] [Google Scholar]