Genome Editing in Engineered T Cells for Cancer Immunotherapy

Abstract

Advanced gene transfer technologies and profound immunological insights have enabled substantial increases in the efficacy of anticancer adoptive cellular therapy (ACT). In recent years, the U.S. Food and Drug Administration and European Medicines Agency have approved six engineered T cell therapeutic products, all chimeric antigen receptor-engineered T cells directed against B cell malignancies. Despite encouraging clinical results, engineered T cell therapy is still constrained by challenges, which could be addressed by genome editing. As RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats technology passes its 10-year anniversary, we review emerging applications of genome editing approaches designed to (1) overcome resistance to therapy, including cancer immune evasion mechanisms; (2) avoid unwanted immune reactions related to allogeneic T cell products; (3) increase fitness, expansion capacity, persistence, and potency of engineered T cells, while preserving their safety profile; and (4) improve the ability of therapeutic cells to resist immunosuppressive signals active in the tumor microenvironment. Overall, these innovative approaches should widen the safe and effective use of ACT to larger number of patients affected by cancer.

INTRODUCTION

Among therapeutic options available to increase the efficacy of anticancer T cells, genetic engineering can deliver intrinsic antineoplastic responses while simultaneously increasing therapeutic benefit through next-generation genome editing. The original concept, developed in the late 1980s, aimed to confer cancer cell recognition and killing by autologous or allogeneic T cells. Early pioneering approaches of adoptive T cell transfer relied on naturally occurring autologous tumor-infiltrating lymphocytes (TILs) obtained from resected solid tumors.

TILs include T cells capable of recognizing tumor-associated antigens, mainly those resulting from tumor-specific mutations or “neoantigens.” These T cells can be selected and expanded ex vivo, and reinfused into patients after lymphodepletion to create a favorable niche for their expansion.¹ TIL T cell therapy proved particularly effective against metastatic melanoma. Because TILs recognize the products of cancer mutations, identification of neoantigen-reactive T cells can now be facilitated by next-generation sequencing, easing their stimulation and selection before infusion into patients with solid tumors.^2
–4

In the allogeneic setting, cellular therapies against relapsed hematological malignancies first used donor lymphocyte infusions (DLIs) after allogeneic hematopoietic stem cell transplantation (allo-SCT), to exploit the alloreactive graft versus leukemia effect and to enhance post-transplant immune reconstitution. DLI has historically been useful against chronic myelogenous leukemia but has been less effective against other hematological malignancies.^5
–7

With the advent of cell engineering, antitumor T cells have been taken to the next level, eventually proving effective against difficult-to-treat malignancies and revolutionizing the field of cancer immunotherapy.⁸ By using viral vectors, T cells have been genetically engineered for enhanced persistence,⁹ improved safety,¹⁰ and, more recently, to express an artificial receptor that provides tumor-specific antigen recognition. Isolation and sequencing of T cell receptors (TCRs) specific for tumor-specific or tissue-restricted antigens enable the design and cloning of artificial TCRs.

Adoptive transfer of TCR-engineered T cells avoids the challenges associated with expanding TILs in vitro and is currently being applied to target multiple tumor types, including melanoma, synovial sarcoma, acute myeloid leukemia (AML), and myeloma.¹¹ To circumvent human leukocyte antigen (HLA) restriction, chimeric antigen receptor (CAR) molecules have been designed for both cellular and humoral immunity, by combining the antigen-recognition ability of antibodies with the effector functions of immune cells. Engagement of an antibody single chain fragment variable (ScFv) region redirects T cell antigen specificity to surface molecules expressed by the tumor.

The first generation of CAR T cells included only the CD3ζ chain as the intracellular signaling domain¹² and proved safe but with limited persistence.¹³ The use of CAR constructs based on ScFv linked to the signaling domain of the Fc receptor γ chain was also explored, resulting in suboptimal efficacy.¹⁴ Costimulation domains added to subsequent generations of CAR T cells improved T cell function, proliferation, and persistence, producing remarkable and long-lasting clinical responses against B cell malignancies.^15

–18

Since 2017, the U.S. Food and Drug Administration and European Medicines Agency have approved six CAR T cell products, including four against CD19 expressed in B cell lymphoma^19

–22 and acute lymphoblastic leukemia,²³ and two anti-B-cell maturation antigen CARs against multiple myeloma.^24,25 We are now witnessing a progressive shift toward approved adoptive cellular therapies (ACTs) performed in earlier lines of treatment. In addition, we are also seeing the emergence of therapies aimed at new targets with evidence of clinical efficacy not only for liquid but also for solid tumors.²⁶

Current research is focused on using advanced engineering approaches to increase efficacy and overcome mechanisms of resistance to engineered T cells. Although efficient transgene insertion has commonly been achieved using viral vectors, nonviral technologies are only recently emerging as sustainable alternatives.^27
–29 In this context, genome editing is progressively gaining momentum as the choice technology to precisely introduce artificial receptors and replace endogenous TCRs to facilitate applications in allogeneic settings. In addition, genome editing is being exploited to generate engineered T cells with increased potency, persistence, and resistance to inhibitory signals and fratricidal activity (Fig. 1).

Figure 1.

Genome editing applied to T cells for cancer immunotherapy: applications. TALENs, HE, ZFNs, and CRISPR/Cas9 platforms, including BE system can be used to obtain permanent gene disruption and/or to promote targeted integration of a selected transgene in a preselected genomic region. A summary of some of the genes edited to date in the context of adoptive T cell therapy is reported, together with the specific nuclease system used. ARID1A, AT-Rich Interaction Domain 1A; B2m, B2-microglobulin; BATF3, basic leucine zipper ATF-like transcription factor 3; BEs, base editors; CAR, chimeric antigen receptor; cBAF, canonical BAF complex; CBLC, Cbl Proto-Oncogene C; CIITA, Class II Major Histocompatibility Complex Transactivator; DGK, diacylglycerol kinase; DNMT3A, DNA methyltransferase 3 alpha; EGR1, early growth response protein 1; FAM49B, family with sequence similarity 49, member B; HE, homing endonucleases; ID3, inhibitor of DNA binding 3; IFPs, immunomodulatory fusion proteins; INO80, chromatin-remodeling ATPase INO80; RFX5, regulator factor X5; SOX4, SRY-box transcription factor 4; TALENs, transcription activator-like effector nucleases; TCR, T cell receptor; TET2, tet methylcytosine dioxygenase 2; UBASH3A, Ubiquitin Associated and SH3 Domain Containing A; ZFNs, zinc finger nucleases.

As we recently celebrated the 10-year anniversary of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology, we now critically review the results and emerging applications of T cell genome editing for cancer immunotherapy, including progress to date, clinical trials (Table 1) and challenges for the future.

Table 1.

Completed and active clinical trials with genome-edited T cells

Target Gene Editing	Gene Editing Platform	Indication	Transgene	Modality	Identifier	Sponsor	Refs.
CCR5	ZFNs	HIV infections	—	Adenoviral vector encoding ZFNs	NCT00842634	University of Pennsylvania	Tebas et al.¹⁹⁴
PD-1	CRISPR Cas9	Advanced esophageal cancer	—	n.a.	NCT03081715	Hangzhou Cancer Hospital	Jing et al.¹⁹⁵
PDCD1 TRAC TRBC	CRISPR Cas9	Multiple myeloma Melanoma Synovial sarcoma M/R cell liposarcoma	NY-ESO-1 TCR	Electroporation with CRISPR/Cas9 RNP and transduction with LV	NCT03399448	University of Pennsylvania	Stadtmauer et al.⁴³
PDCD1	CRISPR Cas9	Metastatic nonsmall cell lung cancer	—	Electroporation with CRISPR guides and Cas9 plasmids	NCT02793856	Sichuan University	Lu et al.¹¹⁹
PDCD1 TRAC	CRISPR Cas9	Solid tumors	Mesothelin CAR	Electroporation with sgRNAs and Cas9 protein and transduction with LV	NCT03545815	Chinese PLA General Hospital	Wang et al.¹¹⁰
TRAC CD52	TALEN	B cell acute lymphoblastic leukemia	CD19 CAR	Transduction with LV and electroporation with TALEN mRNA	NCT02808442, NCT02746952	Servier	Benjamin et al.⁷¹
TRAC	Arcus HE	Non-Hodgkin lymphoma	CD19 CAR	AAV and HE	NCT0366600	Precision	Jain et al.⁷⁶
CD52 TRAC	CRISPR Cas9	Acute lymphoblastic leukemia, non-Hodgkin lymphoma	CD19 and CD22 CARs	Transduction with LV and electroporation with CRISPR/Cas9 RNP	NCT04227015	He Huang, Zhejiang University	Hu et al.⁷²
TRAC CD52	TALEN	B cell acute lymphoblastic leukemia	CD22 CAR	Transduction with LV and electroporation with TALEN arm mRNA	NCT04150497	Cellectis	Jain et al.¹⁹⁶
TRAC B2M	CRISPR Cas9	R/R B cell malignancies	CD19 CAR	Site-specific CAR insertion at the TRAC locus using an AAV template	NCT04035434	CRISPR Therapeutics AG	McGuirk et al.⁴⁵
TRAC CD52	TALEN	R/R large B cell lymphoma	CD19 CAR	Transduction with LV and electroporation with TALEN arm mRNA	NCT04416984	Allogene Therapeutics	Locke et al.¹⁹⁷
CCR5	ZFNs	HIV infections	—	Electroporation with ZFN mRNAs	NCT02388594	University of Pennsylvania	Tebas et al.¹⁹⁸
CCR5	ZFNs	HIV infections	—	Adenoviral vector encoding ZFNs	NCT01044654	Sangamo Therapeutics	Zeidan et al.¹⁹⁹
CCR5	ZFNs	HIV infections	—	n.a.	NCT01252641	Sangamo Therapeutics	n.a.
CCR5	ZFNs	HIV infections	—	n.a.	NCT02225665	Sangamo Therapeutics	n.a.
PDCD1	CRISPR Cas9	B cell lymphoma	CD19 CAR	Electroporation with CRISPR guide and DNA template	NCT04213469	Bioray Laboratories	Zhang et al.¹⁸⁴
CD52 TRAC	CRISPR Cas9	B cell lymphoma	CD19 CAR	Transduction with LV incorporating guide RNA expression cassettes and electroporation with Cas9 mRNA	NCT04557436	GOSH	Ottaviano et al.⁴⁴
TRAC B2M	CRISPR Cas9	Refractory T or B cell malignancies	CD70 CAR	Site-specific CAR insertion at the TRAC locus using an AAV template	NCT04502446	CRISPR Therapeutics AG	Zain et al.²⁰⁰
TRAC B2M	CRISPR Cas9	R/R renal cell carcinoma	CD70 CAR	Site-specific CAR insertion at the TRAC locus using an AAV template	NCT04438083	CRISPR Therapeutics AG	Pal et al.²⁰¹
TRAC CD52	TALEN	R/R acute myeloid leukemia	CD123 CAR	Transduction with LV and electroporation with TALEN arm mRNA	NCT03190278	Cellectis	Sallman et al.¹⁸⁶
TRAC PDCD1	CRISPR Cas9	R/R B cell non-Hodgkin lymphoma	CD19 CAR	Cas9 and hybrid RNA–DNA guides	NCT04637763	Caribou Biosciences	Nastoupil et al.²⁰²
TRAC TRBC	CRISPR Cas9	Solid tumors	neoTCR	Electroporation with CRISPR/Cas9 RNP and DNA template	NCT03970382	PACT Pharma, Inc.	Foy et al.¹⁸⁵
CD7 CD52 TRBC	Base editing	T cell acute lymphoblastic leukemia	CD7 CAR	Electroporation with sgRNAs and BE3 mRNA and transduction with LV	NCT05397184	GOSH	Chiesa et al.⁸⁰
TRAC CD7	CRISPR Cas9	T cell acute lymphoblastic leukemia	CD7 CAR	Transduction with LV and CRISPR/Cas9	ChiCTR1900025311	Gracell	Wang et al.⁷⁸
TRAC B2M Regnase-1 TGF-βRII	CRISPR Cas9	R/R B cell malignancies	CD19 CAR	Site-specific CAR insertion at the TRAC locus using an AAV template	NCT05643742	CRISPR Therapeutics AG	Terrett et al.⁴⁶
TRAC B2M Regnase-1 TGF-βRII	CRISPR Cas9	R/R solid tumors	CD70 CAR	Site-specific CAR insertion at the TRAC locus using an AAV template	NCT05795595	CRISPR Therapeutics AG	Terrett et al.⁴⁶
TRAC B2M	CRISPR Cas12a	R/R multiple myeloma	BCMA CAR, B2M-HLA-E	Cas12a and hybrid RNA–DNA guides	NCT05722418	Caribou Biosciences	Berdeja et al.²⁰³
HPK1	CRISPR Cas9	CD19+ leukemia or lymphoma	CD19 CAR	Electroporation with CRISPR guide RNA and transduction with LV	NCT04037566	Xijing Hospital	Zhang et al.²⁰⁴
CCR5	ZFNs	HIV infections	CD4 CAR	n.a.	NCT03617198	University of Pennsylvania	n.a.
TRAC B2M	CRISPR Cas9	R/R multiple myeloma	BCMA CAR	Site-specific CAR insertion at the TRAC locus using an AAV template	NCT04244656	CRISPR Therapeutics AG	n.a.
TRAC B2M	TALEN	R/R multiple myeloma	CS1/SLAMF7 CAR	Electroporation with TALEN arm mRNA and transduction with LV	NCT04142619	Cellectis	n.a.
TRAC CD52	TALEN	B cell non-Hodgkin lymphoma	CD20 and CD22 CARs	Transduction with LV and electroporation with TALEN arm mRNA	NCT05607420	Cellectis	n.a.
TRAC TRBC	CRISPR Cas9	Acute myeloid leukemia	WT-1 TCR	Integration of WT-1–TCR into TRAC locus	NCT05066165	Intellia Therapeutics	n.a.
CD5	CRISPR Cas9	R/R CD5+ hematopoietic malignancies	CD5 CAR	n.a.	NCT04767308	Huazhong University of Science and Technology	n.a.
CISH	CRISPR Cas9	Metastatic gastrointestinal cancers	—	n.a.	NCT04426669	Intima Bioscience	n.a.
CISH	CRISPR Cas9	Metastatic nonsmall cell lung cancer	—	n.a.	NCT05566223	Intima Bioscience	n.a.
PDCD1	CRISPR Cas9	Advanced hepatocellular carcinoma	—	n.a.	NCT04417764	Central South University	n.a.
PDCD1	CRISPR Cas9	Advanced breast cancer	MUC1 CAR	CRISPR/Cas9 knockout and transduction with LV	NCT05812326	Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University	n.a.
TRAC	CRISPR Cas9	B cell non-Hodgkin's lymphoma	CD19 START	Knockin	NCT05631912	Chinese PLA General Hospital	n.a.
TGF-βRII	CRISPR Cas9	EGFR-positive solid tumors	EGFR CAR	n.a.	NCT04976218	Chinese PLA General Hospital	n.a.
CCR5	ZFNs	HIV infections	—	n.a.	NCT03666871	University of Cincinnati	n.a.

AAV, adeno-associated virus; B2M, B2-microglobulin; BCMA, B-cell maturation antigen; BEs, base editors; CAR, chimeric antigen receptor; CCR5, C–C chemokine receptor type 5; CISH, cytokine inducible SH2 containing protein; CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats; EGFR, epidermal growth factor receptor; GOSH, Great Ormond Street Hospital; HE, homing endonuclease; HLA-E, human leukocyte antigen E; HPK1, hematopoietic progenitor kinase 1; LV, lentiviral vector; M/R, myxoid and round; mRNA, messenger RNA; MUC1, mucin 1; n.a., not available; NY-ESO-1, New York esophageal squamous cell carcinoma 1 antigen; PD-1, programmed cell death protein 1; PDCD1, programmed cell death 1 gene; R/R, relapsed and refractory; RNP, ribonucleoprotein; sgRNA, single guide RNA; SLAMF7 (alias CS1), signaling lymphocytic activation molecule 7; START, synthetic TCR and antigen receptor; TALEN, transcription activator-like effector nucleases; TCR, T cell receptor; TGF-βRII, TGF-β receptor II; TRAC, TCRα constant chain; TRBC, TCRβ constant chain; WT-1, Wilms tumor antigen 1; ZFNs, zinc finger nucleases.

GENOME EDITING PLATFORMS APPLIED TO T CELLS

The T cell genome editing tools most widely tested in human studies can be broadly divided into those with protein-based DNA recognition moieties, fused to endonuclease domains, and RNA-guided nuclease enzymes that are designed to create highly specific double-strand DNA (dsDNA) breaks. Subsequent repair by nonhomologous end joining can efficiently disrupt coding sequences by creating gene knockout effects, such as insertions and deletions (indels). Alternatively, using a suitable DNA template for repair by homologous recombination enables an opportunity for site-specific gene insertions.

Replacing nuclease activity with alternative enzymes, including deaminases for nucleotide conversion, and reverse transcriptase for localized generation of repair template DNA, is providing an ever increasing toolbox for T cell engineering. These developments have evolved alongside techniques for delivery of molecular tools into T cells, including viral and nonviral platforms, as well as strategies for using autologous and allogeneic starting populations. The earliest reported experimental knockout studies in humans used zinc finger nucleases (ZFNs), which comprised a Fok1 DNA cleaving domain and dual DNA-binding finger-like modules for targeted DNA binding.

Autologous T cells from subjects with HIV infections were edited with adenoviral vector-delivered ZFN to disrupt the C–C chemokine receptor type 5 (CCR5) virus entry receptor.³⁰ ZFN enabled exploitation for the first time of homologous recombination for targeted transgene integration in primary T cells,³¹ and reprogramming of T cell specificity through TCR gene editing.³²

Subsequently, transcription activator-like effector nucleases (TALENs) were explored. TALEN are delivered as messenger RNA (mRNA) by electroporation and could incorporate longer programmable DNA-binding domains for high specificity to the target site. TALENs were used for highly efficient disruption of the TCRα constant chain (TRAC) gene to prevent graft versus host disease (GVHD), and knockout of CD52 to evade lymphodepletion by the CD52-binding monoclonal antibody (alemtuzumab), in the context of a universal allogeneic, CAR19-targeting CAR therapy.³³

Similar products were generated using homing endonucleases (meganucleases), integrating a CAR19 template into the TRAC locus that was delivered through adeno-associated virus (AAV).³⁴ Over the past decade, RNA-guided CRISPR/Cas9 technology^35

–38 has been widely translated from the laboratory^39

–42 to clinical T cell studies, that is, for programmed cell death protein 1 (PD1) disruption in recombinant TCR-⁴³ and CAR-redirected T cells.^44,45 The platforms rely on a carefully designed single guide RNA (sgRNA) sequence to direct Cas9 (or related enzymes) to specific DNA loci close to particular protospacer adjacent motif (PAM) sequences.

Improved electroporation systems, and the availability of highly purified recombinant Cas9 protein, sgRNAs and synthetic DNAs, have enabled nonviral guide-dependent CAR transgene insertions, at efficiencies suitable for clinical manufacture.²⁷ There are also hybrid delivery systems employing lentiviral delivery of a CAR transgene and guide RNAs for use in combination with Cas9 mRNA.⁴⁴ Targeted AAV-mediated delivery of CAR and nonpolymorphic HLA has been reported using CRISPR/Cas12a and chimeric RNA–DNA guides after multiplexed editing.⁴⁶

Thus there are now available numerous natural and synthetic Cas systems, with a variety of PAM dependencies and evermore stringent windows of operational activity. Although the CRISPR/Cas9 technology is bacterial in origin, CRISPR/Cas9-based ex vivo engineering strategies have not been particularly hampered by immunogenicity or toxic effects during T cell manufacture.

Additional advances have substituted Cas9 with deactivated derivatives that localize in a guide-dependent manner but operate in conjunction with deamination enzymes for highly targeted base conversion, including C > T and A > G editing within a specified distance from the PAM sequence. CRISPR-guided cytidine deamination base editors⁴⁷ have thus been used to introduce premature stop codons for knockout effects,⁴⁸ and targeted adenosine deamination⁴⁹ has been used to disrupt gene expression through modification of critical splice sites. These alternative platforms have the advantage of not causing dsDNA breaks and are ideal for multiplex editing where there is otherwise a risk of translocations and other chromosomal aberrations in T cells.^50
–52

Multiple base-editor variants have now been synthetically engineered, including highly efficient compact versions with narrow windows of activity.^53,54 A further alternative to nuclease-mediated DNA breakage and repair by homologous recombination has been prime editing, which incorporates an impaired Cas9 fused to a murine reverse transcriptase element and a prime editing guide RNA, enabling localized insertion, deletion, or base conversion without dsDNA breaks.⁵⁵

The platforms are still evolving; there are also untethered versions with split Cas and reverse transcriptase architecture.⁵⁶ For nonviral delivery of larger transgene cargos, or multicistronic cassettes, hybrid engineering platforms can support CRISPR-guided site-specific transposition.^57
–59 Finally, the transient expression of engineered transcriptional repressors, which bind at a target genetic locus, now enables mediation of repressive histone marks and DNA methylation for long-term targeted epigenome editing.⁶⁰

GENOME EDITING TO REDIRECT T CELL SPECIFICITY: TCR/CAR GENE EDITING

In general, T cells respond to a single antigenic epitope that is presented by an HLA molecule, by way of a specific TCR, which is a heterodimeric glycoprotein composed of an α and β chain associated with the CD3 complex. A critical step for effective ACT is the redirecting of T cells to recognize and kill tumor antigens. This aim can be achieved using tumor-specific TCRs isolated from patients or healthy donors, or with CARs.

One of the first applications of genome editing tools in cancer immunotherapy was TCR gene editing.³² By combining permanent genetic disruption of the endogenous TCR repertoire with transfer and expression of a tumor-specific TCR, gene editing first enabled redirection of T cell specificity and overcame some of the limitations encountered with conventional gene transfer of a tumor-specific TCR into mature lymphocytes.

TCR gene editing avoids the dilution of a tumor-specific TCR with endogenous TCRs and maximizes the expression level of the transgenic TCR in T cells, thus promoting the generation of high-avidity cellular products. Furthermore, this procedure eliminates the risks of unwanted antigen recognition through mispairing between the tumor-specific and endogenous TCR chains. Overall, TCR gene editing proved superior to TCR gene transfer in vivo and in vitro.³²

Although targeted transgene integration can be achieved in T cells with ZFNs,³¹ efficiency is low. Consequently, the first manufacturing protocols for TCR gene editing were based on sequential steps, including gene disruption and lentiviral-mediated TCR gene transfer.^32,61 The advent of highly efficient CRISPR/Cas9 editing has significantly increased the efficacy of gene disruption and of homology-directed repair, while also permitting simultaneous multiple gene editing. TCR-edited T cells proved safe in a pilot study of cancer patients.⁴³ Using CRISPR/Cas9, efficient orthotopic TCR gene replacement produced high levels of endogenously regulated TCR expression, similar to that observed with unmodified T cells, and with improved T cell function.^62
–64

TCR genetic disruption has been also applied to CAR T cell manufacture. In this setting, the TCR repertoire can be abrogated by disrupting either TCR α or β chain, and represents a major advance for generating allogeneic universal CAR T cells that are unlikely to mediate detrimental GVHD. Initially modeled with ZFNs,⁶⁵ TRAC-disrupted CAR T cells were then efficiently also obtained with TALENs. Qasim et al. were the first to report complete remissions in infants with B-cell acute lymphoblastic leukemia (B-ALL) infused with universal TALEN gene-edited CD19-redirected CAR T cells.³³

Similarly in the field of CAR T cells, the introduction of CRISPR/Cas9 allowed efficient targeted CAR gene integration. Eyquem et al. showed that the integration of a CD19-specific CAR into the TRAC locus of human peripheral blood T cells resulted in uniform CAR expression and enhanced T cell potency, possibly due to reduced tonic CAR signaling, effective internalization, and re-expression of the CAR after antigen encounter.⁶⁶

GENOME EDITING TO OVERCOME HLA BARRIERS

The majority of CAR T therapies to date have employed autologous T cells, but rely on complex harvest and manufacturing logistics, and poor-quality substrate cells from heavily pretreated patients. The use of donor-derived premanufactured T cells would address some of these limitations. A handful of studies using allogeneic T cells derived from HLA-matched hematopoietic stem cell donors showed feasibility.^67,68 A more ambitious option of generating banks of donor-derived T cells from nonmatched donors is now in development, but must address two major issues related to HLA barriers. First, the threshold of risk of GVHD from donor-derived cells is well characterized in the allo-SCT setting.^69,70

Second, host-mediated rejection of mismatched infused T cells mediated by host antibodies and cellular compartments, through recognition of HLA classes I and II molecules on incoming T cells. Both of these limitations can be addressed through genome editing approaches.

Genome editing to prevent alloreactivity

Disruption of either the TCR α or β chain, or the parts of the CD3 complex can be used to prevent cell surface assembly of a functional multimeric TCR. Residual T cells with TCRα or β expression can be further depleted using magnetic bead depletion systems, providing stringently enriched allogeneic T cell products that are unlikely to cause significant alloreactivity. Thresholds for how many TCRα or β T cells a product can carry without causing GVHD can be estimated from the mismatched SCT experience and is usually set at around 5 × 10⁴/kg,^69,70 although this number may be variable for T cells that have been manipulated and cultured to express CARs.

For removal of TCRα or β from allogeneic CAR T cells, a number of studies have reported the use of TALENs in therapies for B-ALL,^33,71,72 lymphomas,^73,74 and AML⁷⁵; homing endonucleases for B cell malignancies⁷⁶; CRISPR/Cas9 for B cell^45,77; and T cell cancers^78,79; and base editing for T-cell acute lymphoblastic leukemia (T-ALL).^80,81 In these studies, GVHD has generally been absent or limited in severity, or readily managed with immunosuppression.

Genome editing to address host-mediated rejection

Host-mediated immune rejection is a major barrier to allogeneic immune effector cells, even in immunosuppressed patients. Circulating anti-HLA antibodies can meditate rapid clearance and generally contraindicate administering the products, if directed against donor HLA. In addition, cellular responses mediated by host lymphocytes can reject mismatched cells within a period of days or weeks. Lymphodepletion can limit such responses and, compared with the autologous setting, may need to be augmented with, for example, the inclusion of alemtuzumab as serotherapy targeting CD52 to provide an advantage to CD52 knockedout CAR T cells for a period of ∼4 weeks.^33,71,77

Other strategies propose conferring an advantage to infused cells by reducing the reactivity of host CD3+ T cells with anti-CD3 monoclonal therapy (while infused TCR/CD3-disrupted cells remain unaffected)⁸² or disrupting deoxycytidine kinase in CAR T cells to confer fludarabine resistance.⁸³ Engineering approaches to express alloimmune defense receptors^84,85 have also been investigated for targeting host alloreactive cells. Clinical studies are underway to explore CD7-redirected CAR T cell-mediated lymphodepletion strategies for use in combination with CAR T cells targeting CD19 (CAR19).⁸⁶

Alternative approaches to directly remove HLA molecules on infused cells, targeting the conserved β2 microglobulin (B2m) domain of HLA class I molecules that interact with CD8 T cells, are also in early clinical phase testing.⁸⁷ For example, CRISPR/Cas9-edited universal site-specific CAR19 devoid of B2m has been used in patients with B cell non-Hodgkin lymphoma (B NHL), with 38% achieving remission without GVHD.⁴⁵ There had been concerns about triggering natural killer (NK)-mediated “missing-self” immunity against HLA class I negative cells, but it is unclear how relevant these effects may be, and mitigation strategies such as the inclusion of nonpolymorphic HLA-E are available, if required.⁸⁸

Also unknown is the extent to which additional removal of HLA class II, expressed on activated CD4 T cells, is required to avoid CD4 T cell-mediated responses. Class II molecules are highly polymorphic, without readily targeted conserved domains suitable for editing across multiple donors, but are dependent on the activity of critical transcription factors upon T cell activation.

CRISPR/Cas9 disruption of DNA-binding regulator factor X5 protein, part of a multifactor HLA control complex, has enabled the generation of universal anti-CD7 CAR T cell approaches that are now being studied.⁷² These immune-stealth approaches are at early experimental stages, mindful of the heightened potential risks associated with creating viral reservoirs or bypassing immunological surveillance in the event of transformation conferred by cells depleted of all antigen-presenting pathways.

GENOME EDITING TO INCREASE PERSISTENCE OF CAR T CELLS

Increasing persistence of autologous and allogeneic engineered T cells

CAR T cell persistence has corroborative efficacy in several clinical trials.^89,90 Persistence may be affected by both intrinsic factors (e.g., fitness, exhaustion, and memory formation) and extrinsic factors (e.g., immune rejection and homing to lymphoid organs). Therefore, various attempts have been undertaken to increase the persistence of CAR T cells by modifying the manufacturing process, CAR design, or genetic-engineering approaches. In particular, specific T cell subpopulations have been associated with improved immune memory formation and long-term remission in patients, making premanufacture selection of T cell populations critical for optimal cell products^91,92 and in particular for CAR T cells.⁹³

CAR construct intracellular domain selection (e.g., CD28, 4-1BB) has been shown to influence the fate of transferred cells, such that sophisticated approaches to CAR design or even combinations of different intracellular CAR domains may better sustain persistence.⁹⁴ In addition, a genome-wide T cell screen identified exhaustion-specific factors that can also negatively affect T cell persistence; the specific deletion of these genes led to an improvement in human T cell persistence and tumor control in vitro and in vitro.⁹⁵

Also being considered is the overexpression of specific genes that have been identified as beneficial for memory formation and persistence. For example, the basic leucine zipper ATF-like transcription factor 3 (BATF3) is induced shortly after CD8⁺ T cell activation and has long-lasting effects on contraction, cellular/mitochondrial fitness, and longevity, and thus impacts CD8 T cell memory. BATF3 overexpression further enhanced T cell persistence in vitro, making it a suitable target for CAR T cell optimization.⁹⁶

In contrast to the TCR replacement strategy mentioned above, Stenger et al. have shown that the endogenous TCR may contribute to the persistence of CAR T cells.⁹⁷ However, for the use of “off-the-shelf” allogeneic T cells, it is mandatory to address extrinsic factors, such as immune rejection by deletion of the endogenous TCR.⁹⁸ The introduction of an alloimmune defense receptor, such as the 4-1BB extracellular domain, HLA-E/HLA-G, or sialic acid-binding immunoglobulin-type lectin (Siglec)-7/9,^84,88,99 was able to prevent cytolytic reactions against CAR T cells by endogenous T or NK cells.

In addition, triple knockout of B2m, Class II Major Histocompatibility Complex Transactivator, and TRAC did not induce GVHD in xenograft mouse models, but improved the persistence of CD19 CAR T cells and maintained antitumor efficacy.¹⁰⁰ In some cases, CAR T cells do not reach the targeted tumor site. Therefore, specific homing markers (e.g., CCR4 or CXCR1/CXCR2) have been identified and modified in allogeneic CAR T cells to enhance extravasation to either target tissues or lymphoid organs.^101,102

Increasing persistence of inducible pluripotent stem cell-derived engineered T cells

As another option for an “off-the-shelf” solution, inducible pluripotent stem cells (iPSCs) are considered promising in terms of scalability and efficiency.¹⁰³ iPSCs can be generated from a single donor and differentiated into any immune cell type.^104,105 In addition, arbitrary gene modifications can be introduced during the manufacturing process to provide a hyperindividualized solution for patients. However, the production and maintenance of an iPSC culture are a highly complex and labor-intensive process. The iPSC production systems are mainly based on animal cell products, resulting in high variability and the risk of species cross-contamination.¹⁰⁶

Furthermore, CAR T cell persistence is positively influenced by a balanced ratio of CD4 and CD8 CAR T cells,¹⁰⁷ but the majority of iPSC-derived CAR T cells correspond to a CD8⁺ phenotype.¹⁰⁸ The factors responsible for a CD4-specific lineage have been identified, but these production systems still rely on murine feeder systems, which precludes clinical application.¹⁰⁵ Regardless of limitations of the manufacturing process, extensive studies have already aimed to improve the persistence of iPSC-derived CAR T cells.

Deletion of the diacylglycerol (DAG)α and the DAGζ molecules, and introduction of a membrane-bound interleukin (IL)-15/IL-15ra fusion protein, improved proliferation and persistence of iPSC-derived CAR T cells in vitro.¹⁰⁹ The induction of modifications before phenotypic lineage induction also has potential for hypoimmunogenic CAR T cells. Wang et al. demonstrated in an in vitro model that disruption of CD155 expression in HLA-2, HLA-I, and HLA-II null iPSCs enabled iPSC-derived CD20 CAR T cells to largely escape NKG2A⁺ DNAM-1⁺ NK cell-mediated cytolysis, as well as CD8 and CD4 T cell-mediated cytolysis, paving the way for off-the-shelf hypoimmunogenic cell products for CAR T cell therapy.¹¹⁰

PREVENTING T CELL DYSFUNCTION WITH GENOME EDITING APPROACHES

After antigen recognition, negative regulators of T cell activation are transiently upregulated, preventing T cells from becoming overly stimulated. However, chronic antigen exposure can lead to sustained overexpression of these negative regulators, leading to T cell exhaustion and/or dysfunction.¹¹¹ Significant effort has focused on preventing T cell dysfunction by inhibiting immunosuppressive signals in T cells.

As the PD1/programmed death ligand 1 (PD-L1) axis is considered key in driving T cell exhaustion, antibody-mediated blockade of the PD-1/PD-L1 axis has been combined with engineered T cells in preclinical studies and in clinical trials, with some signs of efficacy.^112,113 Antibody-mediated blockade of the PD-1/PD-L1 axis can control the timing and duration of inhibition, and allow the reinvigoration of neoantigen-specific tumor-infiltrating T cells. However, these therapies can be associated with loss of peripheral tolerance and severe toxicities.

An alternative is to use genome editing to ablate the gene encoding PD-1 (Pdcd1). This strategy has the advantage of specifically inhibiting the PD1–PD-L1 axis only in engineered tumor-specific T cells, perhaps mitigating toxicities related to autoreactivity. However, the consequences of long-term PD-1 elimination remain poorly understood, especially in the context of CAR T cells. Although some studies suggest that genetic absence of PD-1 can accelerate T cell exhaustion and impair T cell functionality,^114,115 others suggest the opposite.^41,116

PD-1 has also been described as a haplo-insufficient suppressor of T cell lymphomagenesis,¹¹⁷ although a recent study using CD19-CAR T cells in immunocompetent mice suggests that PD-1 knockout CAR T cells can differentiate, form memory, and persist in the presence of constant antigen exposure provided by continuous B cell renewal, with no evidence of malignant transformation.¹¹⁸

Early clinical trials with PD-1-disrupted T cells have shown feasibility and safety in the absence of unconstrained proliferation or persistence, but also in the absence of objective antitumor responses.^43,110,119 Missing consensus on whether PD-1 knockout is beneficial or detrimental may be due to the lack of clinically relevant preclinical models,¹²⁰ different levels of PD-L1 expression across treated patients, and different sensitivities of the engineered T cells to PD-1/PD-L1 axis signaling. In this regard, we and others have observed that CARs containing different ScFv and costimulatory domains can respond differently to inhibition of the PD-1/PD-L1 axis.¹²¹

An important observation from studies that knocked out PD-1 in tumor-specific T cells is that T cell exhaustion can occur in the absence of PD-1 expression. Expression levels of T-cell immunoglobulin and mucin-domain containing 3, lymphocyte activation gene 3, T-cell immunoreceptor with Ig and ITIM domains, and cytotoxic T-lymphocyte associated protein 4 (CTLA-4) are also hallmarks of T cell exhaustion, and inhibition of these markers is being actively investigated to promote antitumor responses. Antibody-mediated blocking or genetic ablation of any one or of a combination of these immune checkpoint can boost the therapeutic efficacy of tumor-directed T cells, although further studies are required to better understand their role in CAR T cell function.^122,123

Other pathways have also been targeted with genome editing to enhance CAR T cell function. For example, gene disruption of TGF-β receptor II (TGF-βRII) in CAR T cells was reported to reduce the induced Treg conversion, prevent the exhaustion of CAR T cells, and increase their antitumor activity.¹²⁴ In addition, gene disruption of Fas reduced activation-induced cell death in engineered T cells, and increased the antitumor effect of CAR T cells.⁴¹

In a different approach, elimination of the DAG kinase, to increase TCR signaling, rendered CAR T cells resistant to soluble immunosuppressive factors, preventing T cell dysfunction driven or exacerbated by the tumor microenvironment.¹²⁵ CD8⁺ T cells from humans' cancer express high levels of nuclear receptor transcription factors (NR4A) and display enrichment of NR4A-binding motifs in accessible chromatin regions. Triple gene disruption of NR4A transcription factors in CAR T cells promotes tumor regression and prolonged the survival of tumor-bearing mice.¹²⁶

Typically, these knockout strategies have been used to identify candidate genes, based on their previously described roles in inhibiting T cell activation or function. With the recent development of next-generation sequencing, high-throughput genetic perturbation technologies, and genetic screens, new candidate genes have been identified for roles driving T cell exhaustion. Examples include CD39, identified as a major driver of T cell exhaustion in both primary and metastatic colorectal tumor models testing HER-2-specific TCR-edited T cells for eliminating human epidermal growth factor receptor 2 (HER-2) expressing patient-derived organoids in vitro and in vitro. ¹²⁷

Another model of continuous antigen exposure, which is present in many solid tumor settings, used single-cell RNA sequencing to identify transcription factors associated with CAR T cell dysregulation associated with a CD8 T to NK-like T cell transition and identified SRY-box transcription factor 4 (SOX4) and inhibitor of DNA binding 3 (ID3) as key regulators of T cell exhaustion.¹²⁸ CRISPR/Cas9-mediated ID3 and SOX4 knockouts prevented or delayed T cell dysfunction and improved CAR T cell efficacy against solid tumors.

Signatures of T cell exhaustion and dysfunction include global chromatin remodeling and epigenetic changes, and genome-wide CRISPR screens have also identified genes involved in epigenetic regulation as candidates for therapeutic editing. Genome-editing disruption of regulators, such as DNA methyltransferase 3 alpha (DNMT3A) and AT-rich interaction domain 1A, has reversed exhausted epigenetic states in vitro and in animal models.^129,130 In addition, chronic CAR T stimulation can coordinate an early growth response protein 2 (EGR2)-dependent epigenomic resistance program in T cells, and knocking out the gene encoding the EGR2 transcriptional regulator renders CAR T cells impervious to dysfunction and improves memory differentiation.¹³¹

Finally, a recent study used an in vitro model of T cell exhaustion mediated by tonic signaling to identify the Mediator Complex Subunit 12 (MED12) and Cyclin C genes, which encode proteins in the kinase module of the multiprotein mediator complex, as regulators of CAR T cell effector functions. Indeed, MED12-deficient T cells showed enhanced potency in mediating antitumor effects.¹³² Although challenging in primary T cells and in vitro models, these types of CRISPR screenings can enable the unbiased discovery and functional characterization of gene targets and pathways with key roles in T cell function.¹³³

Whether these candidate genes or pathways can be targeted with small molecules, antibodies, or gene-editing approaches will require further preclinical studies. Any concomitant knockout of tumor-suppressor activity would have to be balanced against heightened risks of genotoxicity, even though the clinical experience with thousands of engineered and infused T cells indicates that these cells are particularly resistant to transformation.

GENOME EDITING TO BOOST THE POTENCY OF ADOPTIVELY TRANSFERRED CELLS

Genome-wide CRISPR/Cas9 editing offers further avenues to enhance T cell activation, proliferation, and survival, thereby overcoming the inhibitory tumor environment. In vitro screens have successfully identified potentially targetable genes involved in T cell function, including inhibitors of T cell proliferation (CBLB, CD5, FAM49B, RASA2, MAPK14),^134
–136 suppressors of the Jak/STAT signaling pathway (SOCS1, TCEB2, RNF, CUL5), and inhibitors of nuclear factor kappa B (NF-κB) signaling (UBASH3A, TNFAIBS, TNIP1).¹³³

Deletion of genes affecting T cell chromatin remodeling related to exhaustion (canonical BAF complex), metabolism (chromatin-remodeling ATPase INO80), and methylation-associated plasticity (DNMT3A) impact on cell fitness and antitumor activity,^95,130,137 with more efficiency compared with deleting individual genes specifically associated with activation/exhaustion such as PD1.⁴³ These approaches hold translational potential for human T cell products, but selection of the pathway/individual gene may be dependent on the transferred T cells' protein target and type of tumor targetted, highlighting the validity and rationale for in vitro screens.¹³⁷

In addition, many targeted pathways have been associated with tumorigenesis in different contexts, either by ablating negative regulation of oncogenes (e.g., CBLB, RASA2, FAM49B) or suppressing tumor-suppressor genes (e.g., MAPK14), underscoring the necessity of thorough safety screening and monitoring.

There is evidence that ablation or perturbation of inhibitory genes in substrate cells can enhance function in transferred cells, as in the unusual experience of tet methylcytosine dioxygenase 2 (TET2) gene inactivation by CAR construct integration in a patient with an additional hypomorphic TET2 allele.¹³⁸ Although TET2 is a tumor-suppressor gene and a master regulator of blood cell formation, its disruption alone has not been associated with overt oncogenesis.¹³⁹

Disrupting TET2 in a patient altered the differentiation state and proliferative capacity of CAR-expressing T cells, resulting in considerable and durable therapeutic effects without transformation during >4 years. However, further studies have indicated that biallelic TET2 ablation also enables antigen-independent CAR T cell clonal expansion, raising potential safety concerns associated with disruptions of genes linked to broad epigenetic changes.¹⁴⁰

In addition to gene ablation, the overexpression or knockingin of natural or synthetic genes holds promise for enhancing cell therapy. Although technically challenging, CRISPR activation screens are increasingly employed to identify genes that confer advantages to T cells through overexpression.¹⁴¹ As proof of principle, overexpressing the activator protein 1 (AP-1) factor c-Jun in CAR T cells driven by tonic-signaling demonstrated the ability to prevent exhaustion and enhance potency in solid tumor models.¹⁴² This strategy is now being implemented in patients.¹⁴³ Although c-Jun may not be implicated in mature T cell transformation, concerns regarding potential oncogenesis remain as c-Jun expression has been described in cancer settings.¹⁴⁴

To maintain T cell efficacy in the face of repeated antigen stimulation, T cell activation with associated proliferation and survival requires a costimulatory signal concurrent with TCR triggering.¹⁴⁵ Unlike CARs that already include a costimulatory domain, TCRs and other newer constructs, such as TCR fusion (T cell receptor fusion constructs [TRuC]) and HLA-independent T cell receptors, may require independent costimulatory receptor triggering for maximal efficiency.^146,147 Ligands for these coreceptors are seldom present on tumor cells, which further commonly upregulate inhibitory ligands that interfere with T cell activation.^148,149

To overcome both of these challenges, synthetic immunomodulatory fusion proteins (IFPs) that combine an inhibitory ectodomain with a costimulatory endodomain are being pursued, introduced either by gene knockins or lentiviral-mediated insertions. These constructs are geared to minimize/ablate the negative effects of suppressive ligands and transmit a positive cosignal to the binder (TCR/CAR/TRUC/Other).^150
–152 Such constructs fall into broad categories that include “Signal 2” CD28 family of receptors (CD28, ICOS, CTLA-4, and PD1), TNF superfamily (CD27, CD30, CD40, OX40, and 4-1BB), and fibronectin type III domains typically constituting “Signal 3” cytokine receptors (e.g., IL-2, IL-7, IL-15, and IL-21).

IFP choice may be target and tumor context dependent. For the IFPs to signal, they must first find their inhibitory receptors either on tumor cells or cells present in the tumor microenvironment. For example, not all negative regulators are consistently expressed across tumor types, emphasizing the need for careful selection of IFPs for a particular tumor type.¹⁵³ The type of intracelluar signal transmitted by the IFP also has to be considered. For example, upon cross-linking, the cytoplasmic CD28 domain binds to phosphaditylinositol 3-kinase that has been phosphorylated on tyrosine by lymphocyte-specific protein tyrosine kinase, enhancing NF-κB, nuclear factor of activated T cell and AP-1 signaling pathways that in turn mediate survival, proliferation, cytokine production, and differentiation.¹⁵⁴

4-1BB instead promotes the activity of NF-κB through the activation of the inhibitor of NF-κB (IkB) kinase complex, induces the antiapoptotic protein B cell lymphoma-extra large (Bcl-xL), proliferation, and promotes memory differentiation and mitochondrial biogenesis.¹⁵⁵ In CAR designs, CD28 signaling elicited faster larger magnitude changes in protein phosphorylation, which correlated with effector functions, compared with the slower memory-type activities of 4-1BB that produce more sustained antitumor activity against established tumors in vitro.¹⁵⁶

Another strategy being explored to boost transferred cell function is to express cytokine receptors' intracellular tails as a means for T cells to also secrete supportive cytokines either constitutively or upon synthetic cytokine triggering.^157,158 As most costimulation signals do not function in the absence of binding of the primary construct to the target, these approaches may offer modulable and safe translation strategies.

PREVENTING CAR T CELL FRATRICIDE USING GENOME EDITING APPROACHES

Treating T cell malignancies remains one of the key challenges for CAR T cell therapy. T cell neoplasms have a very poor prognosis, leading to high rates of relapse and mortality in both children and adults, which represents a significant unmet medical need. However, most T lineage antigens, which could potentially be targeted with CAR T cells, are similarly expressed in both normal and malignant T cells. Thus, the introduction of a T cell surface protein-redirected CAR into a normal T cell can result, as the product is being generated, in CAR-mediated killing—a phenomenon known as “fratricide.”

Fratricide of CAR T cells during the manufacturing process limits ex vivo T cell expansion, accelerates T cell differentiation and exhaustion due to constant CAR signaling, and results in impaired antitumor efficacy. Fratricide has been observed in CAR T cell product development for T cell and non-T cell malignancies targeting CD3, TCRβ, CD7, CD38, CD70, and NKG2D ligands. To avoid fratricide, one solution is to use genome editing to eliminate the targeted antigen from the surface of CAR T cells. Functional CAR T cells targeting CD3 or CD7 have been successfully generated by knocking out CD3 or CD7, respectively, using TALENs,¹⁵⁹ CRISPR/Cas9,¹⁶⁰ or base editors.^51,52

Collecting enough healthy autologous T cells from patients with T cell malignancies constitutes another limitation for the generation of CAR T cells. As a consequence, significant efforts are under way to develop allogeneic approaches to treat these patients. To generate off-the-shelf CAR T cells, genetic disruption of the CAR target is typically combined with the disruption of the TCR gene. In addition, the ablation of the CD52 gene can be incorporated into this multiple-target editing strategy to confer resistance to depletion by alemtuzumab.

The feasibility and potential of this approach have already been demonstrated in some clinical trials. Off-the-shelf anti-CD7 CAR T cells, which are resistant to fratricide through genome-editing¹⁶¹ or base-editing⁸¹ approaches, have induced deep and durable responses in a small number of selected patients with relapsed or refractory T-ALL.

Alternatives to mitigate fratricide during T cell manufacturing without genome editing are also being explored, especially in the context of autologous CAR T cells. This includes targeting T lineage antigens that are downregulated after CAR expression (such as CD5),¹⁶² blocking CAR signaling with pharmacological inhibitors of key signaling kinases,¹⁶³ or selecting a subpopulation of T cells for engineering that do not express the targeted antigen.¹⁶⁴ Finally, the expansion of naturally selected CD7 CAR T cells during manufacturing has also been proposed and tested in clinical trials, with impressive clinical results.¹⁶⁵

LONG-TERM SAFETY WITH GENOME-EDITED T CELL PRODUCTS: CONSIDERATIONS FROM CLINICAL TRIALS

Engineered T cells were first reported in a clinical study in 1990, using TILs transduced with a retroviral vector.⁹ This occurred almost concurrently with the first patient successfully treated with gene therapy using T cells transduced with the enzyme adenosine deaminase in children with severe combined immunodeficiency¹⁶⁶ and the first use of allogeneic T cells transduced with retroviral vectors encoding a suicide gene.¹⁰ There is now increasing long-term confidence that overt transformation risks from using gamma-retroviral and lentiviral vectors in T cells are low.^167,168

Gene addition into differentiated T cells appears safer than in stem cells,^167,169 perhaps because polyclonal T cells are well differentiated and lack expression of genes associated with stemness and embryonic development and perhaps their diversity prevents outgrowth of potentially malignant T cells.¹⁷⁰ In contrast, for hematopoietic stem cell modification, there have been concerns relating to gamma-retroviral-mediated genotoxicity due to insertional mutagenesis in patients with inherited genetic conditions,^171

–174 and although these have been largely mitigated by switching to sin-lentiviral configurations, safety issues continue to be carefully investigated as wider applications develop.¹⁷⁵

In T cells, clonal expansion due to integration near the Casitas B-lineage lymphoma oncogene and disruption of the TET2 tumor-suppressor gene has been reported in patients treated with CAR T cells.^138,176 Interestingly, TET2 gene disruption resulted in the promotion of CAR T cell expansion and antitumor activity, indicating that viral vectors are able to influence gene expression and host cell behavior. Although there was clonal dominance in these subjects, there was no evidence of malignant transformation.

However, transformation of engineered T cells has been reported in a clinical trial of Piggybac transposon-modified CAR T cells where 2 cases of T cell lymphoma arose in a study of 10 patients treated. The mechanism of transformation is still uncertain, but excessive transposase activity, insertional mutagenesis, high transgene copy number, and the manufacturing process may have played a role.¹⁷⁷ Other transposon systems are based on the synthetic Sleeping Beauty transposon and have been used for T cell gene therapy, and so far proven safe.^68,178

Compared with technologies that achieve transgene integration by random gene insertion, precise genomic targeting should reduce risks of insertional mutagenesis or other undesirable influences on regional genes. However, off-target DNA cleavage activity may lead to point mutations, indels, or other changes at unintended sites at low frequency, whereas chromosomal aberrations such as deletions, translocation, and chromosome loss could also occur. A recent report of recombination-induced karyotype changes in a patient treated with TALEN-edited CAR19 T cells highlighted the importance of investigating such events when they arise.¹⁷⁹

Moreover, as a consequence of the DNA damage response, there is a risk of selection of p53-inactive clones.¹⁸⁰ To understand whether unintended off-target events are clinically relevant, Cromer et al. performed ultradeep sequencing of >500 tumor suppressors and oncogenes in hematopoietic stem cells electroporated with high-fidelity Cas9 protein and guide RNA; no evidence of mutations in tumor suppressors or oncogenes was found.¹⁸¹ Conversely, large abnormalities were found in primary human T cells transfected with CRISPR/Cas9 and guide RNAs targeted to TCR and PD1.¹⁸²

The clinical relevance of Cas9-induced chromosomal loss was confirmed in preclinical CAR T cells, but reduced by clinical editing protocols that used a different order of operations in T cell manufacture.¹⁸³ Using this platform, early results from clinical trials of engineered TCR (NY-ESO-1 specific) T cells with TCR and PD-1 knockouts did not cause chromosome loss (NCT03399448). Moreover, the potential risk of immunogenicity did not appear to have affected the persistence of CRISPR/Cas9-edited T cells.⁴³

The safety of PD-1-edited T cells was also recently tested in a study for metastatic nonsmall lung cancer (NCT02793856), with an off-target event rate of 0.05%.¹¹⁹ High safety and a first demonstration of CRISPR/Cas9-edited T cell efficacy have also been shown with edited anti-CD19 CAR T cells specifically targeted to PD-1 locus in B NHL,¹⁸⁴ with universal anti-CD19 CAR T cells edited in the TRAC and CD52 loci of pediatric B-ALL patients,⁴⁴ and with TRAC and TCRβ constant chain-edited neoantigen-specific TCR (neoTCR) in patients with refractory solid cancers.¹⁸⁵ Safety has also been demonstrated for anti-CD123 allogeneic CAR T cells in patients with AML.¹⁸⁶ GVHD has been generally absent or limited in severity and readily managed with immunosuppression.

Evaluation of long-term safety of genome editing is essential to ensure its application in the clinic, and the risk/benefit balance must drive the design of new therapies. It is anticipated that reducing the time of Cas9 activity by ribonucleoprotein complexes,¹⁸⁷ high-fidelity nucleases,^188,189 usage of nickase (n) Cas9 variants,¹⁹⁰ base editors,⁴⁷ and prime editors⁵⁵ have the potential to increase gene editing specificity and safety.

CONCLUSION

Advancements in genome editing technology and the identification of major targets and pathways involved in intratumoral T cell dysfunction have enabled a wide range of next-generation T cell therapeutic products. This new therapeutic pillar holds immense potential for cancer patients. Although further research and careful consideration of target specificity and safety concerns are necessary to fully exploit these promising approaches, gene ablation, gene overexpression, and the use of IFPs all offer new avenues to enhance T cell function and overcome inhibitory tumor environments.

Additional cellular platforms, including engineered NK,¹¹⁹ Invariant natural killer-T cells,¹⁹¹ gamma delta T cells,¹⁹² and macrophages,¹⁹³ are also being investigated as part of an armamentarium of cancer immunotherapy using engineered cells. The proper clinical exploitation of engineered T cells will require efforts to increase sustainability and harmonization of manufacturing, use, and clinical monitoring. International coalitions and consortia, such as the GO-CART Coalition (https://thegocartcoalition.com) and the T2Evolve consortium (https://www.google.com/search?client=safari&rls=en&q=t2evolve&ie=UTF-8&oe=UTF-8), represent successful endeavors toward this outcome.

Footnotes

ACKNOWLEDGMENTS

We thank Susanna Cesarano, BSc, and Alessia Ugolini, BSc, for their support for ; and Dr. Deborah Banker for editing the article.

AUTHOR DISCLOSURE

C.B. is inventor on patent applications and has been granted patents related to T cell engineering that have been, in part, licensed to industry. C.B. has been member of Advisory Board and Consultant for Intellia Therapeutics, Novartis, GSK, Allogene, Kite/Gilead, Miltenyi, Kiadis, Evir, Janssen, Alia and received research support from Intellia Therapeutics.

A.G.C. is a scientific co-founder of Affini-T Therapeutics, is an inventor on patents related to TCRs, and has received funding from Affini-T, Amazon and Lonza.

S.G. is an inventor on patents related to CAR-T cell therapy, filed by the University of Pennsylvania and licensed to Novartis and Tmunity, and has received commercial research funding from Gilead.

C.F.M. is an inventor on patents related to CAR T and regulatory T cell therapy, filled by the Tettamanti Research Center (under license to Coimmune), and Yale University, San Raffaele Hospital and the Telethon Foundation.

M.H. is inventor on patent applications and has been granted patents related to CAR technology that have been, in part, licensed to industry. Co-founder and equity owner T-CURX GmbH, Würzburg, Germany. Funding: BMS. Speaker honoraria: Janssen, BMS, Novartis.

W.Q. is inventor in patents filed in relation to genome edited T cells; Funding- Servier, Cellectis, Miltenyi; Consultancy Wugen, Virocell, Skylark.

FUNDING INFORMATION

C.B. is funded by AIRC (Ig 18458 and Ig 24965), AIRC 5xMille, Rif. 22737, Italian Ministry of Research and University (PRIN 2015NZWsec; PRIN 2017WC8499), Italian Ministry of Health (Research project on CAR T cells for hematological malignancies and solid tumors and RF-2019-12370243).

A.G.C. has received funding from the U.S. NIH (P01CA18029-41). S.G. has received funding from the Spanish Ministry of Science and Innovation under a Ramon y Cajal grant (RYC2018-024442-I). C.M. is supported by the Swiss National Science Foundation (PR00P3_201621), the Comprehensive Cancer Center Zurich, the San Salvatore Foundation, and the Helmut Horten Foundation.

M.H.: BMS. Speaker honoraria: Janssen, BMS, Novartis. W.Q. is supported by National Institute of Health Research, MRC, Wellcome Trust.

References

Rosenberg

, Restifo

. Adoptive cell transfer as personalized immunotherapy for human cancer. Science, 2015; 348:62–68.

van den Berg

, Heemskerk

, van Rooij

, et al. Tumor infiltrating lymphocytes (TIL) therapy in metastatic melanoma: Boosting of neoantigen-specific T cell reactivity and long-term follow-up. J Immunother Cancer, 2020; 8(2):e000848.

Kim

, Vale

, Zacharakis

, et al. Adoptive cellular therapy with autologous tumor-infiltrating lymphocytes and T-cell receptor-engineered T cells targeting common p53 neoantigens in human solid tumors. Cancer Immunol Res, 2022; 10:932–946.

Chandran

, Ma

, Klatt

, et al. Immunogenicity and therapeutic targeting of a public neoantigen derived from mutated PIK3CA. Nat Med, 2022; 28:946–957.

Kolb

, Mittermüller

, Clemm

, et al. Donor leukocyte transfusions for treatment of recurrent chronic myelogenous leukemia in marrow transplant patients. Blood, 1990; 76:2462–2465.

Collins

Jr. , Shpilberg

, Drobyski

, et al. Donor leukocyte infusions in 140 patients with relapsed malignancy after allogeneic bone marrow transplantation. J Clin Oncol, 1997; 15:433–444.

Roddie

, Peggs

. Donor lymphocyte infusion following allogeneic hematopoietic stem cell transplantation. Expert Opin Biol Ther, 2011; 11:473–487.

Morgan

, Dudley

, Wunderlich

, et al. Cancer regression in patients after transfer of genetically engineered lymphocytes. Science, 2006; 314:126–129.

Rosenberg

, Aebersold

, Cornetta

, et al. Gene transfer into humans—immunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction. N Engl J Med, 1990; 323:570–578.

10.

Bonini

, Ferrari

, Verzeletti

, et al. HSV-TK gene transfer into donor lymphocytes for control of allogeneic graft-versus-leukemia. Science, 1997; 276:1719–1724.

11.

Manfredi

, Cianciotti

, Potenza

, et al. TCR redirected T cells for cancer treatment: Achievements, hurdles, and goals. Front Immunol, 2020; 11:1689.

12.

Gross

, Waks

, Eshhar

. Expression of immunoglobulin-T-cell receptor chimeric molecules as functional receptors with antibody-type specificity. Proc Natl Acad Sci U S A, 1989; 86:10024–10028.

13.

Park

, Digiusto

, Slovak

, et al. Adoptive transfer of chimeric antigen receptor re-directed cytolytic T lymphocyte clones in patients with neuroblastoma. Mol Ther, 2007; 15:825–833.

14.

Kershaw

, Westwood

, Parker

, et al. A phase I study on adoptive immunotherapy using gene-modified T cells for ovarian cancer. Clin Cancer Res, 2006; 12:6106–6115.

15.

Imai

, Mihara

, Andreansky

, et al. Chimeric receptors with 4-1BB signaling capacity provoke potent cytotoxicity against acute lymphoblastic leukemia. Leukemia, 2004; 18:676–684.

16.

Grupp

, Kalos

, Barrett

, et al. Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N Engl J Med, 2013; 368:1509–1518.

17.

Maude

, Frey

, Shaw

, et al. Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med, 2014; 371:1507–1517.

18.

Lee

, Kochenderfer

, Stetler-Stevenson

, et al. T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: A phase 1 dose-escalation trial. Lancet, 2015; 385:517–528.

19.

Neelapu

, Locke

, Bartlett

, et al. Axicabtagene ciloleucel CAR T-cell therapy in refractory large B-cell lymphoma. N Engl J Med, 2017; 377:2531–2544.

20.

Schuster

, Bishop

, Tam

, et al. Tisagenlecleucel in adult relapsed or refractory diffuse large B-cell lymphoma. N Engl J Med, 2019; 380:45–56.

21.

Abramson

, Palomba

, Gordon

, et al. Lisocabtagene maraleucel for patients with relapsed or refractory large B-cell lymphomas (TRANSCEND NHL 001): A multicentre seamless design study. Lancet, 2020; 396:839–852.

22.

Wang

, Munoz

, Goy

, et al. KTE-X19 CAR T-cell therapy in relapsed or refractory mantle-cell lymphoma. N Engl J Med, 2020; 382:1331–1342.

23.

Maude

. Tisagenlecleucel in pediatric patients with acute lymphoblastic leukemia. Clin Adv Hematol Oncol, 2018; 16:664–666.

24.

Berdeja

, Madduri

, Usmani

, et al. Ciltacabtagene autoleucel, a B-cell maturation antigen-directed chimeric antigen receptor T-cell therapy in patients with relapsed or refractory multiple myeloma (CARTITUDE-1): A phase 1b/2 open-label study. Lancet, 2021; 398:314–324.

25.

Munshi

, Anderson

Jr ., Shah

, et al. Idecabtagene vicleucel in relapsed and refractory multiple myeloma. N Engl J Med, 2021; 384:705–716.

26.

Labanieh

, Mackall

. CAR immune cells: Design principles, resistance and the next generation. Nature, 2023; 614:635–648.

27.

Moretti

, Ponzo

, Nicolette

, et al. The past, present, and future of non-viral CAR T cells. Front Immunol, 2022; 13:867013.

28.

Irving

, Lanitis

, Migliorini

, et al. Choosing the right tool for genetic engineering: Clinical lessons from chimeric antigen receptor-T cells. Hum Gene Ther, 2021; 32:1044–1058.

29.

Magnani

, Myburgh

, Brunn

, et al. Anti-CD117 CAR T cells incorporating a safety switch eradicate human acute myeloid leukemia and hematopoietic stem cells. Mol Ther Oncolytics, 2023; 30:56–71.

30.

Perez

, Wang

, Miller

, et al. Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. NatBiotechnol, 2008; 26:808–816.

31.

Lombardo

, Cesana

, Genovese

, et al. Site-specific integration and tailoring of cassette design for sustainable gene transfer. Nat Methods, 2011; 8:861–869.

32.

Provasi

, Genovese

, Lombardo

, et al. Editing T cell specificity towards leukemia by zinc finger nucleases and lentiviral gene transfer. Nat Med, 2012; 18:807–815.

33.

Qasim

, Zhan

, Samarasinghe

, et al. Molecular remission of infant B-ALL after infusion of universal TALEN gene-edited CAR T cells. Sci Transl Med, 2017; 9(374):eaaj2013.

34.

MacLeod

, Antony

, Martin

, et al. Integration of a CD19 CAR into the TCR alpha chain locus streamlines production of allogeneic gene-edited CAR T cells. Mol Ther, 2017; 25:949–961.

35.

Jinek

, Chylinski

, Fonfara

, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012; 337:816–821.

36.

Cong

, Ran

, Cox

, et al. Multiplex genome engineering using CRISPR/Cas systems. Science, 2013; 339:819–823.

37.

Cho

, Kim

, et al. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol, 2013; 31:230–232.

38.

Mali

, Yang

, Esvelt

, et al. RNA-guided human genome engineering via Cas9. Science, 2013; 339:823–826.

39.

Schumann

, Lin

, Boyer

, et al. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. Proc Natl Acad Sci U S A, 2015; 112:10437–10442.

40.

Ren

, Liu

, Fang

, et al. Multiplex genome editing to generate universal CAR T cells resistant to PD1 inhibition. Clin Cancer Res, 2017; 23(9):2255–2266.

41.

Ren

, Zhang

, Liu

, et al. A versatile system for rapid multiplex genome-edited CAR T cell generation. Oncotarget, 2017; 8(10):17002–17011.

42.

Georgiadis

, Preece

, Nickolay

, et al. Long terminal repeat CRISPR-CAR-coupled “universal” T cells mediate potent anti-leukemic effects. Mol Ther, 2018; 26:1215–1227.

43.

Stadtmauer

, Fraietta

, Davis

, et al. CRISPR-engineered T cells in patients with refractory cancer. Science, 2020; 367(6481):eaba7365.

44.

Ottaviano

, Georgiadis

, Gkazi

, et al. Phase 1 clinical trial of CRISPR-engineered CAR19 universal T cells for treatment of children with refractory B cell leukemia. Sci Transl Med, 2022; 14(668):eabq3010.

45.

McGuirk

, Bachier

, Bishop

, et al. A phase 1 dose escalation and cohort expansion study of the safety and efficacy of allogeneic CRISPR-Cas9–engineered T cells (CTX110) in patients (Pts) with relapsed or refractory (R/R) B-cell malignancies (CARBON). J Clin Oncol, 2021; 39:TPS7570.

46.

Terrett

, Kalaitzidis

, Dequeant

M-L

, et al. Abstract ND02: CTX112 and CTX131: Next-generation CRISPR/Cas9-engineered allogeneic (allo) CAR T cells incorporating novel edits that increase potency and efficacy in the treatment of lymphoid and solid tumors. Cancer Res, 2023; 83:ND02.

47.

Komor

, Kim

, Packer

, et al. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature, 2016; 533:420–424.

48.

Billon

. CRISPR-mediated base editing enables efficient disruption of eukaryotic genes through induction of stop codons. Mol Cell, 2017; 67(6):1068.e4–1079.e4.

49.

Gaudelli

. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature, 2017; 551(7681):464–471.

50.

Webber

, Lonetree

, Kluesner

, et al. Highly efficient multiplex human T cell engineering without double-strand breaks using Cas9 base editors. Nat Commun, 2019; 10:5222.

51.

Georgiadis

, Rasaiyaah

, Gkazi

, et al. Base-edited CAR T cells for combinational therapy against T cell malignancies. Leukemia, 2021; 35(12):3466–3481.

52.

Diorio

, Murray

, Naniong

, et al. Cytosine base editing enables quadruple-edited allogeneic CART cells for T-ALL. Blood, 2022; 140:619–629.

53.

Neugebauer

, Hsu

, Arbab

, et al. Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity. Nat Biotechnol, 2023; 41:673–685.

54.

Chen

, Zhang

, Xue

, et al. Engineering a precise adenine base editor with minimal bystander editing. Nat Chem Biol, 2023; 19:101–110.

55.

Anzalone

, Koblan

, Liu

. Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors. Nat Biotechnol, 2020; 38:824–844.

56.

Grunewald

, Miller

, Szalay

, et al. Engineered CRISPR prime editors with compact, untethered reverse transcriptases. Nat Biotechnol, 2023; 41:337–343.

57.

Klompe

, Vo

PLH

, Halpin-Healy

, et al. Transposon-encoded CRISPR–Cas systems direct RNA-guided DNA integration. Nature, 2019; 571:219–225.

58.

Lampe

, King

, Halpin-Healy

, et al. Targeted DNA integration in human cells without double-strand breaks using CRISPR-associated transposases. Nat Biotechnol, 2023; doi: 10.1038/s41587-023-01748-1.

59.

Tou

, Orr

, Kleinstiver

. Precise cut-and-paste DNA insertion using engineered type V-K CRISPR-associated transposases. Nat Biotechnol, 2023; 41(7):968–979.

60.

Amabile

, Migliara

, Capasso

, et al. Inheritable silencing of endogenous genes by hit-and-run targeted epigenetic editing. Cell, 2016; 167:219.e214–232.e214.

61.

Mastaglio

, Genovese

, Magnani

, et al. NY-ESO-1 TCR single edited stem and central memory T cells to treat multiple myeloma without graft-versus-host disease. Blood, 2017; 130:606–618.

62.

Schober

, Müller

, Gökmen

, et al. Orthotopic replacement of T-cell receptor α- and β-chains with preservation of near-physiological T-cell function. Nat Biomed Eng, 2019; 3:974–984.

63.

Roth

, Puig-Saus

, Yu

, et al. Reprogramming human T cell function and specificity with non-viral genome targeting. Nature, 2018; 559:405–409.

64.

Ruggiero

, Carnevale

, Prodeus

, et al. CRISPR-based gene disruption and integration of high-avidity, WT1-specific T cell receptors improve antitumor T cell function. Sci Transl Med, 2022; 14:eabg8027.

65.

Torikai

, Reik

, Liu

P-Q

, et al. A foundation for universal T-cell based immunotherapy: T cells engineered to express a CD19-specific chimeric-antigen-receptor and eliminate expression of endogenous TCR. Blood, 2012; 119:5697–5705.

66.

Eyquem

, Mansilla-Soto

, Giavridis

, et al. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection. Nature, 2017; 543:113–117.

67.

Kochenderfer

, Dudley

, Carpenter

, et al. Donor-derived CD19-targeted T cells cause regression of malignancy persisting after allogeneic hematopoietic stem cell transplantation. Blood, 2013; 122:4129–4139.

68.

Magnani

, Gaipa

, Lussana

, et al. Sleeping Beauty-engineered CAR T cells achieve antileukemic activity without severe toxicities. J Clin Invest, 2020; 130(11):6021–6033.

69.

Shah

, Elfeky

, Nademi

, et al. T-cell receptor alphabeta(+) and CD19(+) cell-depleted haploidentical and mismatched hematopoietic stem cell transplantation in primary immune deficiency. J Allergy Clin Immunol, 2018; 141:1417.e1411–1426.e1411.

70.

Aversa

, Tabilio

, Velardi

, et al. Treatment of high-risk acute leukemia with T-cell-depleted stem cells from related donors with one fully mismatched HLA haplotype. N Engl J Med, 1998; 339:1186–1193.

71.

Benjamin

, Graham

, Yallop

, et al. Genome-edited, donor-derived allogeneic anti-CD19 chimeric antigen receptor T cells in paediatric and adult B-cell acute lymphoblastic leukaemia: results of two phase 1 studies. Lancet, 2020; 396:1885–1894.

72.

, Zhou

, Zhang

, et al. CRISPR/Cas9-engineered universal CD19/CD22 dual-targeted CAR-T cell therapy for relapsed/refractory B-cell acute lymphoblastic leukemia. Clin Cancer Res, 2021; 27:2764–2772.

73.

Neelapu

, Nath

, Munoz

, et al. ALPHA Study: ALLO-501 produced deep and durable responses in patients with relapsed/refractory non-Hodgkin's lymphoma comparable to autologous CAR T. Blood, 2021; 138:3878.

74.

Jain

, Roboz

, Konopleva

, et al. Preliminary results from the flu/cy/alemtuzumab arm of the Phase I BALLI-01 trial of UCART22, an anti-CD22 allogeneic CAR-T cell product, in adult patients with relapsed or refractory (R/R) CD22+ B-cell acute lymphoblastic leukemia (B-ALL). Blood, 2021; 138:1746.

75.

Mailankody

, Liedtke

, Sidana

, et al. Universal updated Phase 1 data validates the feasibility of allogeneic anti-BCMA ALLO-715 therapy for relapsed/refractory multiple myeloma. Blood, 2021; 138:651.

76.

Jain

, Kantarjian

, Solomon

, et al. Preliminary safety and efficacy of PBCAR0191, an allogeneic ‘off-the-shelf’ CD19-directed CAR-T for patients with relapsed/refractory (R/R) CD19+ B-ALL. Blood, 2021; 138:650.

77.

Ottaviano

, Georgiadis

, Ghazi

, et al. CAR-coupled CRISPR-Cas9 engineered universal donor T cells: Phase 1 trial of TT52CAR19 in paediatric refractory B-cell leukemia. Sci Transl Med, 2022; 14(668):eabq3010.

78.

Wang

, Li

, Gao

, et al. Safety and efficacy results of GC027: The first-in-human, universal CAR-T cell therapy for adult relapsed/refractory T-cell acute lymphoblastic leukemia (r/r T-ALL). J Clin Oncol, 2020; 38:3013.

79.

Iyer

. The cobalt-lym study of CTX130: A phase 1 dose escalation study of CD70-targeted allogeneic CRISPR-CAS9–engineered CAR T cells in patients with relapsed/refractory (R/R) T-cell malignancies. EHA Library, 2022; 357126:S262.

80.

Chiesa

, Georgiadis

, Ottaviano

. Tvt CAR7: Phase 1 clinical trial of base-edited “universal” CAR7 T cells for paediatric relapsed/refractory T-ALL. Blood, 2022; 140(Suppl. 1):4579–4580.

81.

Chiesa

, Georgiadis

, Syed

, et al. Base-edited CAR7 T cells for relapsed T-cell acute lymphoblastic leukemia. N Engl J Med, 2023; 389(10):899–910.

82.

Kuhn

, Weiner

. Therapeutic anti-CD3 monoclonal antibodies: From bench to bedside. Immunotherapy, 2016; 8:889–906.

83.

Pires

, Martin

. 140 Allogeneic CAR T cells with deoxycytidine kinase knockdown demonstrate resistance to fludarabine. J ImmunoTher Cancer, 2021; 9:A149.

84.

, Watanabe

, McKenna

, et al. Engineered off-the-shelf therapeutic T cells resist host immune rejection. Nat Biotechnol, 2021; 39:56–63.

85.

Quach

, Becerra-Dominguez

, Rouce

, et al. A strategy to protect off-the-shelf cell therapy products using virus-specific T-cells engineered to eliminate alloreactive T-cells. J Transl Med, 2019; 17:240.

86.

, Yang

, Sun

, et al. Preclinical results of an allogeneic, universal CD19/CD7-targeting CAR-T cell therapy (GC502) for B cell malignancies. Blood, 2021; 138:1722.

87.

Kagoya

, Guo

, Yeung

, et al. Genetic ablation of HLA Class I, Class II, and the T-cell receptor enables allogeneic T cells to be used for adoptive T-cell therapy. Cancer Immunol Res, 2020; 8:926–936.

88.

Gornalusse

, Hirata

, Funk

, et al. HLA-E-expressing pluripotent stem cells escape allogeneic responses and lysis by NK cells. Nat Biotechnol, 2017; 35:765–772.

89.

, Sun

, Liang

, et al. Mechanisms of relapse after CD19 CAR T-cell therapy for acute lymphoblastic leukemia and its prevention and treatment strategies. Front Immunol, 2019; 10:2664.

90.

Hucks

, Rheingold

. The journey to CAR T cell therapy: The pediatric and young adult experience with relapsed or refractory B-ALL. Blood Cancer J, 2019; 9:10.

91.

Cieri

, Camisa

, Cocchiarella

, et al. IL-7 and IL-15 instruct the generation of human memory stem T cells from naive precursors. Blood, 2013; 121:573–584.

92.

Oliveira

, Ruggiero

, Stanghellini

, et al. Tracking genetically engineered lymphocytes long-term reveals the dynamics of T cell immunological memory. Sci Transl Med, 2015; 7:317ra198.

93.

Melenhorst

, Chen

, Wang

, et al. Decade-long leukaemia remissions with persistence of CD4+ CAR T cells. Nature, 2022; 602:503–509.

94.

Ying

, He

, Wang

, et al. Parallel comparison of 4-1BB or CD28 co-stimulated CD19-targeted CAR-T cells for B cell non-Hodgkin's Lymphoma. Mol Ther Oncolytics, 2019; 15:60–68.

95.

Belk

, Yao

, Ly

, et al. Genome-wide CRISPR screens of T cell exhaustion identify chromatin remodeling factors that limit T cell persistence. Cancer Cell, 2022; 40:768.e767–786.e767.

96.

Ataide

, Komander

, Knöpper

, et al. BATF3 programs CD8(+) T cell memory. Nat Immunol, 2020; 21:1397–1407.

97.

Stenger

, Stief

, Kaeuferle

, et al. Endogenous TCR promotes in vivo persistence of CD19-CAR-T cells compared to a CRISPR/Cas9-mediated TCR knockout CAR. Blood, 2020; 136:1407–1418.

98.

Champlin

, Passweg

, Zhang

, et al. T-cell depletion of bone marrow transplants for leukemia from donors other than HLA-identical siblings: Advantage of T-cell antibodies with narrow specificities. Blood, 2000; 95:3996–4003.

99.

Jandus

, Boligan

, Chijioke

, et al. Interactions between Siglec-7/9 receptors and ligands influence NK cell-dependent tumor immunosurveillance. J Clin Invest, 2014; 124:1810–1820.

100.

Liu

, Zhang

, Cheng

, et al. CRISPR-Cas9-mediated multiplex gene editing in CAR-T cells. Cell Res, 2017; 27:154–157.

101.

Di Stasi

, De Angelis

, Rooney

, et al. T lymphocytes coexpressing CCR4 and a chimeric antigen receptor targeting CD30 have improved homing and antitumor activity in a Hodgkin tumor model. Blood, 2009; 113:6392–6402.

102.

Jin

, Tao

, Karachi

, et al. CXCR1- or CXCR2-modified CAR T cells co-opt IL-8 for maximal antitumor efficacy in solid tumors. Nat Commun, 2019; 10:4016.

103.

Sadeqi Nezhad

, Abdollahpour-Alitappeh

, Rezaei

, et al. Induced pluripotent stem cells (iPSCs) provide a potentially unlimited T cell source for CAR-T cell development and off-the-shelf products. Pharm Res, 2021; 38:931–945.

104.

Goldenson

, Hor

, Kaufman

. iPSC-derived natural killer cell therapies—Expansion and targeting. Front Immunol, 2022; 13:841107.

105.

Netsrithong

, Wattanapanitch

. Advances in adoptive cell therapy using induced pluripotent stem cell-derived T cells. Front Immunol, 2021; 12:759558.

106.

Saha

, Jaenisch

. Technical challenges in using human induced pluripotent stem cells to model disease. Cell Stem Cell, 2009; 5:584–595.

107.

Turtle

, Hanafi

, Berger

, et al. CD19 CAR-T cells of defined CD4+:CD8+ composition in adult B cell ALL patients. J Clin Invest, 2016; 126:2123–2138.

108.

Iriguchi

, Yasui

, Kawai

, et al. A clinically applicable and scalable method to regenerate T-cells from iPSCs for off-the-shelf T-cell immunotherapy. Nat Commun, 2021; 12:430.

109.

Ueda

, Shiina

, Iriguchi

, et al. Optimization of the proliferation and persistency of CAR T cells derived from human induced pluripotent stem cells. Nat Biomed Eng, 2023; 7:24–37.

110.

Wang

, Li

, Feng

, et al. Phase I study of CAR-T cells with PD-1 and TCR disruption in mesothelin-positive solid tumors. Cell Mol Immunol, 2021; 18:2188–2198.

111.

McLane

, Abdel-Hakeem

, Wherry

. CD8 T cell exhaustion during chronic viral infection and cancer. Annu Rev Immunol, 2019; 37:457–495.

112.

Adusumilli

, Zauderer

, Rivière

, et al. A Phase I trial of regional mesothelin-targeted CAR T-cell therapy in patients with malignant pleural disease, in combination with the anti-PD-1 agent pembrolizumab. Cancer Discov, 2021; 11:2748–2763.

113.

Chong

, Melenhorst

, Lacey

, et al. PD-1 blockade modulates chimeric antigen receptor (CAR)-modified T cells: Refueling the CAR. Blood, 2017; 129:1039–1041.

114.

Wei

, Luo

, Wang

, et al. PD-1 silencing impairs the anti-tumor function of chimeric antigen receptor modified T cells by inhibiting proliferation activity. J ImmunoTher Cancer, 2019; 7:209.

115.

Odorizzi

, Pauken

, Paley

, et al. Genetic absence of PD-1 promotes accumulation of terminally differentiated exhausted CD8+ T cells. J Exp Med, 2015; 212:1125–1137.

116.

Rupp

, Schumann

, Roybal

, et al. CRISPR/Cas9-mediated PD-1 disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells. Sci Rep, 2017; 7:737.

117.

Wartewig

, Kurgyis

, Keppler

, et al. PD-1 is a haploinsufficient suppressor of T cell lymphomagenesis. Nature, 2017; 552:121–125.

118.

Dötsch

, Svec

, Schober

, et al. Long-term persistence and functionality of adoptively transferred antigen-specific T cells with genetically ablated PD-1 expression. Proc Natl Acad Sci USA, 2023; 120:e2200626120.

119.

, Xue

, Deng

, et al. Safety and feasibility of CRISPR-edited T cells in patients with refractory non-small-cell lung cancer. Nat Med, 2020; 26:732–740.

120.

Guedan

, Luu

, Ammar

, et al. Time 2EVOLVE: Predicting efficacy of engineered T-cells—How far is the bench from the bedside?. J ImmunoTher Cancer, 2022; 10:e003487.

121.

Cherkassky

, Morello

, Villena-Vargas

, et al. Human CAR T cells with cell-intrinsic PD-1 checkpoint blockade resist tumor-mediated inhibition. J Clin Invest, 2016; 126:3130–3144.

122.

Zou

, Lu

, Liu

, et al. Engineered triple inhibitory receptor resistance improves anti-tumor CAR-T cell performance via CD56. Nat Commun, 2019; 10:4109.

123.

Granier

, De Guillebon

, Blanc

, et al. Mechanisms of action and rationale for the use of checkpoint inhibitors in cancer. ESMO Open, 2017; 2:e000213.

124.

Tang

, Cheng

, Zhang

, et al. TGF-β inhibition via CRISPR promotes the long-term efficacy of CAR T cells against solid tumors. JCI Insight, 2020; 5(4):e133977.

125.

Jung

, Kim

, Yu

, et al. CRISPR/Cas9-mediated knockout of DGK improves antitumor activities of human T cells. Cancer Res, 2018; 78:4692–4703.

126.

Chen

, López-Moyado

, Seo

, et al. NR4A transcription factors limit CAR T cell function in solid tumours. Nature, 2019; 567:530–534.

127.

Potenza

, Balestrieri

, Spiga

, et al. Revealing and harnessing CD39 for the treatment of colorectal cancer and liver metastases by engineered T cells. Gut, 2023; 72(10):1887–1903; doi: 10.1136/gutjnl-2022-328042.

128.

Good

, Aznar

, Kuramitsu

, et al. An NK-like CAR T cell transition in CAR T cell dysfunction. Cell, 2021; 184:6081.e6026–6100.e6026.

129.

Kissick

, Ahmed

. New epigenetic regulators of T cell exhaustion. Cancer Cell, 2022; 40:708–710.

130.

Prinzing

, Zebley

, Petersen

, et al. Deleting DNMT3A in CAR T cells prevents exhaustion and enhances antitumor activity. Sci Transl Med, 2021; 13:eabh0272.

131.

Jung

, Bartoszek

, Rech

, et al. Type I interferon signaling via the EGR2 transcriptional regulator potentiates CAR T cell-intrinsic dysfunction. Cancer Discov, 2023; 13(7):1636–1655.

132.

Freitas

, Belk

, Sotillo

, et al. Enhanced T cell effector activity by targeting the Mediator kinase module. Science, 2022; 378:eabn5647.

133.

Shifrut

, Carnevale

, Tobin

, et al. Genome-wide CRISPR screens in primary human T cells reveal key regulators of immune function. Cell, 2018; 175:1958.e1915–1971.e1915.

134.

Carnevale

, Shifrut

, Kale

, et al. RASA2 ablation in T cells boosts antigen sensitivity and long-term function. Nature, 2022; 609:174–182.

135.

Gurusamy

, Henning

, Yamamoto

, et al. Multi-phenotype CRISPR-Cas9 screen identifies p38 kinase as a target for adoptive immunotherapies. Cancer Cell, 2020; 37:818.e819–833.e819.

136.

Shang

, Jiang

, Boettcher

, et al. Genome-wide CRISPR screen identifies FAM49B as a key regulator of actin dynamics and T cell activation. Proc Natl Acad Sci U S A, 2018; 115:E4051–E4060.

137.

Guo

, Huang

, Zhu

, et al. cBAF complex components and MYC cooperate early in CD8(+) T cell fate. Nature, 2022; 607:135–141.

138.

Fraietta

, Nobles

, Sammons

, et al. Disruption of TET2 promotes the therapeutic efficacy of CD19-targeted T cells. Nature, 2018; 558:307–312.

139.

Bowman

, Levine

. TET2 in normal and malignant hematopoiesis. Cold Spring Harb Perspect Med, 2017; 7(8):a026518.

140.

Jain

, Zhao

, Feucht

, et al. TET2 guards against unchecked BATF3-induced CAR T cell expansion. Nature, 2023; 615:315–322.

141.

Shi

, Doench

, Chi

. CRISPR screens for functional interrogation of immunity. Nat Rev Immunol, 2023; 23:363–380.

142.

Lynn

, Weber

, Sotillo

, et al. c-Jun overexpression in CAR T cells induces exhaustion resistance. Nature, 2019; 576:293–300.

143.

Lam

, Barragan

, Cheung

, et al. Preclinical development of LYL119, a ROR1-targeted CAR T-cell product candidate incorporating four novel T-cell reprogramming technologies to overcome barriers to effective cell therapy for solid tumors. In: ASGCT 2023, Los Angeles, May 16th–20th, 2023.

144.

Shaulian

. AP-1—The Jun proteins: Oncogenes or tumor suppressors in disguise?. Cell Signal, 2010; 22:894–899.

145.

Chen

, Flies

. Molecular mechanisms of T cell co-stimulation and co-inhibition. Nat Rev Immunol, 2013; 13:227–242.

146.

Baeuerle

, Ding

, Patel

, et al. Synthetic TRuC receptors engaging the complete T cell receptor for potent anti-tumor response. Nat Commun, 2019; 10:2087.

147.

Mansilla-Soto

, Eyquem

, Haubner

, et al. HLA-independent T cell receptors for targeting tumors with low antigen density. Nat Med, 2022; 28:345–352.

148.

Driessens

, Kline

, Gajewski

. Costimulatory and coinhibitory receptors in anti-tumor immunity. Immunol Rev, 2009; 229:126–144.

149.

Geiger

, Rubnitz

. New approaches for the immunotherapy of acute myeloid leukemia. Discov Med, 2015; 19:275–284.

150.

Oda

, Anderson

, Ravikumar

, et al. A Fas-4-1BB fusion protein converts a death to a pro-survival signal and enhances T cell therapy. J Exp Med, 2020; 217:(12):e20191166.

151.

Guo

, Kent

, Davila

. Chimeric non-antigen receptors in T cell-based cancer therapy. J Immunother Cancer, 2021; 9(8):e002628.

152.

Oda

, Daman

, Garcia

, et al. A CD200R-CD28 fusion protein appropriates an inhibitory signal to enhance T-cell function and therapy of murine leukemia. Blood, 2017; 130:2410–2419.

153.

Sprecher

, Bergman

, Meilick

, et al. Apoptosis, Fas and Fas-ligand expression in melanocytic tumors. J Cutan Pathol, 1999; 26:72–77.

154.

Esensten

, Helou

, Chopra

, et al. CD28 costimulation: From mechanism to therapy. Immunity, 2016; 44:973–988.

155.

Kawalekar

, O'Connor

, Fraietta

, et al. Distinct signaling of coreceptors regulates specific metabolism pathways and impacts memory development in CAR T cells. Immunity, 2016; 44:380–390.

156.

Salter

, Ivey

, Kennedy

, et al. Phosphoproteomic analysis of chimeric antigen receptor signaling reveals kinetic and quantitative differences that affect cell function. Sci Signal, 2018; 11(544):eaat6753.

157.

Saxton

, Glassman

, Garcia

. Emerging principles of cytokine pharmacology and therapeutics. Nat Rev Drug Discov, 2023; 22:21–37.

158.

Shum

, Omer

, Tashiro

, et al. Constitutive signaling from an engineered IL7 receptor promotes durable tumor elimination by tumor-redirected T cells. Cancer Discov, 2017; 7:1238–1247.

159.

Rasaiyaah

, Georgiadis

, Preece

, et al. TCRαβ/CD3 disruption enables CD3-specific antileukemic T cell immunotherapy. JCI Insight, 2018; 3(13):e99442.

160.

Gomes-Silva

, Srinivasan

, Sharma

, et al. CD7-edited T cells expressing a CD7-specific CAR for the therapy of T-cell malignancies. Blood, 2017; 130:285–296.

161.

, Wang

, Yuan

, et al. Eradication of T-ALL cells by CD7-targeted universal CAR-T cells and initial test of Ruxolitinib-based CRS Management. Clin Cancer Res, 2021; 27:1242–1246.

162.

Mamonkin

, Rouce

, Tashiro

, et al. A T-cell-directed chimeric antigen receptor for the selective treatment of T-cell malignancies. Blood, 2015; 126:983–992.

163.

Watanabe

, Mo

, Zheng

, et al. Feasibility and preclinical efficacy of CD7-unedited CD7 CAR T cells for T cell malignancies. Mol Ther, 2023; 31:24–34.

164.

Freiwan

, Zoine

, Crawford

, et al. Engineering naturally occurring CD7- T cells for the immunotherapy of hematological malignancies. Blood, 2022; 140:2684–2696.

165.

, Liu

, Yang

, et al. Naturally selected CD7 CAR-T therapy without genetic manipulations for T-ALL/LBL: First-in-human phase 1 clinical trial. Blood, 2022; 140:321–334.

166.

Blaese

, Culver

, Miller

, et al. T lymphocyte-directed gene therapy for ADA- SCID: Initial trial results after 4 years. Science, 1995; 270:475–480.

167.

Scholler

, Brady

, Binder-Scholl

, et al. Decade-long safety and function of retroviral-modified chimeric antigen receptor T cells. Sci Transl Med, 2012; 4:132ra153.

168.

Steffin

DHM

, Muhsen

, Hill

, et al. Long-term follow-up for the development of subsequent malignancies in patients treated with genetically modified IECs. Blood, 2022; 140:16–24.

169.

Bonini

, Grez

, Traversari

, et al. Safety of retroviral gene marking with a truncated NGF receptor. Nat Med, 2003; 9:367–369.

170.

Newrzela

, Al-Ghaili

, Heinrich

, et al. T-cell receptor diversity prevents T-cell lymphoma development. Leukemia, 2012; 26:2499–2507.

171.

Hacein-Bey-Abina

, Von Kalle

, Schmidt

, et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science, 2003; 302:415–419.

172.

Hacein-Bey-Abina

, Garrigue

, Wang

, et al. Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1. J Clin Invest, 2008; 118:3132–3142.

173.

Braun

, Boztug

, Paruzynski

, et al. Gene therapy for Wiskott-Aldrich syndrome—long-term efficacy and genotoxicity. Sci Transl Med, 2014; 6:227ra233.

174.

Ott

, Schmidt

, Schwarzwaelder

, et al. Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1. Nat Med, 2006; 12:401–409.

175.

Goyal

, Tisdale

, Schmidt

, et al. Acute myeloid leukemia case after gene therapy for sickle cell disease. N Engl J Med, 2022; 386:138–147.

176.

Shah

, Qin

, Yates

, et al. Clonal expansion of CAR T cells harboring lentivector integration in the CBL gene following anti-CD22 CAR T-cell therapy. Blood Adv, 2019; 3:2317–2322.

177.

Schambach

, Morgan

, Fehse

. Two cases of T cell lymphoma following Piggybac-mediated CAR T cell therapy. Mol Ther, 2021; 29:2631–2633.

178.

Kebriaei

, Singh

, Huls

, et al. Phase I trials using Sleeping Beauty to generate CD19-specific CAR T cells. J Clin Invest, 2016; 126:3363–3376.

179.

Sasu

, Opiteck

, Gopalakrishnan

, et al. Detection of chromosomal alteration after infusion of gene-edited allogeneic CAR T cells. Mol Ther, 2023; 31:676–685.

180.

Haapaniemi

, Botla

, Persson

, et al. CRISPR–Cas9 genome editing induces a p53-mediated DNA damage response. Nat Med, 2018; 24:927–930.

181.

Cromer

, Barsan

, Jaeger

, et al. Ultra-deep sequencing validates safety of CRISPR/Cas9 genome editing in human hematopoietic stem and progenitor cells. Nat Commun, 2022; 13:4724.

182.

Nahmad

, Lazzarotto

, Zelikson

, et al. In vivo engineered B cells secrete high titers of broadly neutralizing anti-HIV antibodies in mice. Nat Biotechnol, 2022; 40:1241–1249.

183.

Tsuchida

, Brandes

, Bueno

, et al. Mitigation of chromosome loss in clinical CRISPR-Cas9-engineered T cells. bioRxiv 2023:2023.2003.2022.533709.

184.

Zhang

, Hu

, Yang

, et al. Non-viral, specifically targeted CAR-T cells achieve high safety and efficacy in B-NHL. Nature, 2022; 609:369–374.

185.

Foy

, Jacoby

, Bota

, et al. Non-viral precision T cell receptor replacement for personalized cell therapy. Nature, 2023; 615:687–696.

186.

Sallman

, DeAngelo

, Pemmaraju

, et al. Ameli-01: A Phase I Trial of UCART123v1.2, an Anti-CD123 Allogeneic CAR-T Cell Product, in Adult Patients with Relapsed or Refractory (R/R) CD123+ Acute Myeloid Leukemia (AML). Blood, 2022; 140:2371–2373.

187.

Lin

, Staahl

, Alla

, et al. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife, 2014; 3:e04766.

188.

Kleinstiver

, Pattanayak

, Prew

, et al. High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects. Nature, 2016; 529:490–495.

189.

Vakulskas

, Dever

, Rettig

, et al. A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells. Nat Med, 2018; 24:1216–1224.

190.

Trevino

, Zhang

. Genome editing using Cas9 nickases. Methods Enzymol, 2014; 546:161–174.

191.

Delfanti

, Dellabona

, Casorati

, et al. Adoptive immunotherapy with engineered iNKT cells to target cancer cells and the suppressive microenvironment. Front Med, 2022; 9:897750.

192.

Sebestyen

, Prinz

, Déchanet-Merville

, et al. Translating gammadelta (γδ) T cells and their receptors into cancer cell therapies. Nat Rev Drug Discov, 2020; 19:169–184.

193.

Klichinsky

, Ruella

, Shestova

, et al. Human chimeric antigen receptor macrophages for cancer immunotherapy. Nat Biotechnol, 2020; 38:947–953.

194.

Tebas

, Stein

, Tang

, et al. Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. N Engl J Med, 2014; 370:901–910.

195.

Jing

, Zhang

, Ding

, et al. Safety and activity of programmed cell death-1 gene knockout engineered t cells in patients with previously treated advanced esophageal squamous cell carcinoma: An open-label, single-arm phase I study. J Clin Oncol, 2018; 36:3054.

196.

Jain

, Roboz

, Konopleva

, et al. Preliminary Results of Balli-01: A Phase I Study of UCART22 (allogeneic engineered T-cells expressing anti-CD22 Chimeric Antigen Receptor) in Adult Patients with Relapsed or Refractory (R/R) CD22+ B-Cell Acute Lymphoblastic Leukemia (B-ALL). Blood, 2020; 136:7–8.

197.

Locke

, Malik

, Tees

, et al. First-in-human data of ALLO-501A, an allogeneic chimeric antigen receptor (CAR) T-cell therapy and ALLO-647 in relapsed/refractory large B-cell lymphoma (R/R LBCL): ALPHA2 study. J Clin Oncol, 2021; 39:2529.

198.

Tebas

, Jadlowsky

, Shaw

, et al. CCR5-edited CD4+ T cells augment HIV-specific immunity to enable post-rebound control of HIV replication. J Clin Invest, 2021; 131(7):e144486.

199.

Zeidan

, Sharma

, Lee

, et al. Infusion of CCR5 Gene-Edited T Cells Allows Immune Reconstitution, HIV Reservoir Decay, and Long-Term Virological Control. bioRxiv 2021:2021.2002.2028.433290.

200.

Zain

, Iyer

, Sica

, et al. Ther-O-02 - The COBALT-LYM study of CTX130: A phase 1 dose escalation study of CD70-targeted allogeneic CRISPR-Cas9–engineered CAR T cells in patients with relapsed/refractory (R/R) T-cell malignancies. Eur J Cancer, 2022; 173:S21.

201.

Pal

, Tran

, Haanen

, et al. 558 CTX130 allogeneic CRISPR-Cas9–engineered chimeric antigen receptor (CAR) T cells in patients with advanced clear cell renal cell carcinoma: Results from the phase 1 COBALT-RCC study. J ImmunoTher Cancer, 2022; 10:A584.

202.

Nastoupil

, O'Brien

, Holmes

, et al. P1455: First-in-human trial of CB-010, a CRISPR-edited allogeneic anti-CD19 CAR-T cell therapy with a PD-1 knock out, in patients with relapsed or refractory B cell non-Hodgkin lymphoma (antler study). HemaSphere, 2022; 6:1337–1338.

203.

Berdeja

, Martin

, Rossi

, et al. A first-in-human phase 1, multicenter, open-label study of CB-011, a next-generation CRISPR-genome edited allogeneic anti-BCMA immune-cloaked CAR-T cell therapy, in patients with relapsed/refractory multiple myeloma (CAMMOUFLAGE trial). J Clin Oncol, 2023; 41:TPS8063.

204.

Zhang

, Si

, Li

, et al. Decreasing HPK1 expression in CD19 CAR-T cells: A novel strategy to overcome challenges of cell therapy for adult (r/r) B-ALL. J Clin Oncol, 2022; 40:7041.