Abstract
The remarkable clinical activity of chimeric antigen receptor (CAR) therapies in B cell and plasma cell malignancies has validated the use of this therapeutic class for liquid cancers, but resistance and limited access remain as barriers to broader application. Here we review the immunobiology and design principles of current prototype CARs and present emerging platforms that are anticipated to drive future clinical advances. The field is witnessing a rapid expansion of next-generation CAR immune cell technologies designed to enhance efficacy, safety and access. Substantial progress has been made in augmenting immune cell fitness, activating endogenous immunity, arming cells to resist suppression via the tumour microenvironment and developing approaches to modulate antigen density thresholds. Increasingly sophisticated multispecific, logic-gated and regulatable CARs display the potential to overcome resistance and increase safety. Early signs of progress with stealth, virus-free and in vivo gene delivery platforms provide potential paths for reduced costs and increased access of cell therapies in the future. The continuing clinical success of CAR T cells in liquid cancers is driving the development of increasingly sophisticated immune cell therapies that are poised to translate to treatments for solid cancers and non-malignant diseases in the coming years.
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Main
CARs are synthetic modular proteins that redirect immune cell reactivity toward a target of interest. This versatile platform has demonstrated substantial clinical effects in the treatment of B cell and plasma cell malignancies, and the potential to expand its application is driving rapid technological developments and large investments from academia and the biopharmaceutical industry. Six CAR T cell products have been approved by the US Food and Drug Administration (FDA) for 12 indications, including large B cell lymphoma1,2,3,4 (LBCL), B cell acute lymphoblastic leukaemia5,6,7 (B-ALL), mantle cell lymphoma8 and follicular lymphoma9. In pivotal trials, CD19-CAR therapy outperformed standard of care (SOC) as second line therapy for LBCL10,11, and was highly effective as a first line therapy12, paving the way for its application in earlier-stage disease. The generalizability of the CAR platform beyond CD19 targeting is now established, with two BCMA-CAR T cell therapies (BCMA is also known as TNF receptor superfamily member 17) having been approved by the FDA for treatment of multiple myeloma13,14, and high response rates with CD22-CARs in B-ALL15,16 and LBCL17, CD30-CARs in Hodgkin lymphoma18, CD7-CARs in T cell acute lymphoblastic leukaemia19,20,21,22 (T-ALL), CD20-CARs in LBCL23, and GPRC5D-CARs in multiple myeloma24 (Table 1). Standardized toxicity grading and management has resulted in low treatment-related mortality with current commercial CAR T cells1,2,3,4,5,6,7,8,9,10,11.
Despite this progress, many challenges remain. Fewer than 50% of patients treated with commercial CAR T cells for B cell malignancies experience durable disease control1,2,3,4,5,6,7. CAR T cells have shown signs of activity in solid tumours25,26,27,28,29, but high rates of consistent durable responses have not been demonstrated (Table 1). Autologous cell manufacturing is labour-intensive and expensive and commercial scaling is not yet adequate to meet clinical needs. This Perspective synthesizes current understanding of the immunobiology of CAR T cells, emphasizing resistance mechanisms in cancer, design principles and emerging approaches to enhance efficacy. We focus primarily on developing CAR T cells for cancer treatment, but many of the principles are relevant to other immune cell therapies for cancer and to nascent efforts to develop cell therapies for non-malignant diseases. Owing to space constraints, we focus primarily on the most recent literature and on the emerging efforts to enhance efficacy, and refer the reader to recent authoritative reports for additional information on CAR-related toxicities30 and clinical outcomes31.
CAR T immunobiology and mechanisms of resistance
Sustained broad-based advances by many groups focused on developing immune cell therapies for cancer have been essential for the success of CAR T therapy (Fig. 1). CARs were invented by Eshhar and colleagues with the goal of harnessing the expansion, killing and persistence of natural T cells while overcoming major histocompatibility complex (MHC) restriction of the T cell receptor (TCR), to enable broader therapeutic applicability32,33. After iterative optimization by many groups34,35, a receptor incorporating a scFv as the antigen-binding domain, a hinge/transmembrane domain, TCRζ and a CD28 or 4-1BB costimulatory endodomain emerged as the CAR prototype (Fig. 2). This architecture is utilized by five out of the six FDA-approved agents, with the sixth incorporating the same architecture with two nanobody heavy chains (Vhh) as the antigen-binding domains14. Antigen engagement of the prototype 1.5–2.2 kilobase (kb) receptor largely replicates antigen specific activation and killing mediated by the TCR–CD3 complex in natural T cells; however, significant distinctions exist between the biology of CAR T cells and natural T cells that provide opportunities and challenges for application of these therapeutic agents, as discussed below.
Prototype CARs comprise a target-binding domain such as a scFv, a hinge or spacer domain that projects the binder away from the cell surface and provides conformational flexibility, a transmembrane domain that anchors the receptor in the cell surface, and costimulatory and CD3ζ signalling domains that provide activation signals. Small modifications in CAR structure can have profound effects on CAR T cell function, including modulation of the antigen density threshold for activation, persistence, potency, tonic signalling, CAR expression level and the propensity for dimerization237,242,243,244,245,246,247,248,249,249. CDR, complementarity-determining region; FW, framework region.
Resistance due to antigen modulation
A major distinction between CAR and TCR signalling is that CARs require higher antigen density for full T cell activation36,37,38. Despite the higher affinity of single-chain variable fragments (scFvs) compared with TCRs and the generally higher density of CAR expression compared with TCR–CD3 complexes36, TCRs induce full activation in response to less than 100 peptides per antigen presenting cell39,40, whereas CARs require more than 1,000 target molecules per target cell15,41,42,43,44,45. The basis for the difference includes diminished proximal kinase recruitment by CARs38,44,46,47, a less developed immune synapse46, reduced engagement of co-receptors and greater induction of negative downstream regulators36—in part related to tonic signalling, in which CAR aggregation, often driven by the scFv, induces antigen-independent activation48. Modifications to the design of CAR prototypes can tune the antigen density threshold to some extent, with features that modulate signal strength, scFv affinity, CAR expression density, hinge/transmembrane architecture and synapse spacing, having significant effects44,49,50,51,52 (Fig. 2). Because greater signal strength lowers the antigen density threshold, features that enhance T cell fitness independently of CAR design also reduce the CAR antigen density threshold44,53. These insights are foundational for developing safe and effective CAR T cell therapies, since toxicity and efficacy are intimately related to the expression characteristics of the targeted antigen. CARs targeting molecules that are absent from vital tissues, such as B cell lineage antigens, should be engineered for activation at low antigen density to diminish the risk of low antigen recurrence, whereas CARs targeting molecules that are highly expressed in cancer but with low expression in vital tissues should be engineered with higher antigen density thresholds to exploit a therapeutic window on the basis of differential antigen density49.
Antigen modulation is a major cause of CAR T cell resistance in B cell malignancies, and is likely to pose an even greater challenge in solid tumours, where most targetable antigens show significant heterogeneity54,55,56. In B-ALL in children and young adults, approximately 50% of relapses are associated with CD19 loss5,57,58, and in LBCL, approximately 30% of relapses are CD19-negative and an additional 30% express CD19 at levels below the antigen density threshold for commercial CARs45,59. The role of antigen modulation in resistance to BCMA-CAR T therapy in multiple myeloma is less well defined. Baseline BCMA expression levels are heterogeneous among patients and have not been associated with clinical responses60,61,62. BCMA loss—associated with genetic mutation and deletion—is a rare cause of resistance13,63,64 (less than 5% of cases), however, antigen modulation is observed following BCMA-CAR treatment61,62. Antigen density can be modulated through a variety of mechanisms, including genetic mutation63,65,66, alternative RNA splicing65, cell lineage switching67, epigenetic and/or posttranscriptional mechanisms15, trogocytosis50, hyperglycosylation68 and antigen shedding69. Downregulation of some targets is amenable to therapeutic intervention with small molecules, such as CD22 upregulation by bryostatin70, CD70 upregulation by azacitidine71 and BCMA upregulation by γ-secretase inhibitors, which inhibit antigen shedding69,70.
Resistance due to inadequate T cell function
A second major cause of CAR T cell resistance is related to inadequate T cell potency, persistence, functional persistence and/or dysfunction, and is typically associated with disease recurrence in the absence of antigen modulation. Dysfunction often results from T cell exhaustion, characterized by global transcriptional and epigenetic reprogramming that converges on terminal differentiation53,72. T cells in the apheresis and/or manufactured CAR T cell product sometimes manifest exhaustion73,74, and high tumour burdens induce exhaustion following adoptive transfer. CAR-intrinsic factors also contribute to exhaustion, with the costimulatory domain having a major role. CD28-costimulated CARs manifest more rapid and greater expansion, secrete more inflammatory cytokines, and show limited persistence owing to T cell exhaustion when compared with 4-1BB and first-generation CARs, which in some cases, may persist for years5,58,75,76,77. The pro-exhaustion effect of the CD28 costimulatory domain is magnified in CARs with tonic signalling48,53. The effect of tonic signalling depends on the magnitude of the signal and is context-dependent, with some CARs demonstrating enhanced function and persistence in the presence of tonic signalling78. Tuning down the signalling strength of CD28-based CARs through mutations in CD3ζ or CD28 domains can attenuate their propensity for exhaustion and improve persistence79,80, as can mutations that interfere with downregulation and ubiquitination of 4-1BB-costimulated CARs81. Of interest, a recent long-term follow-up study demonstrated that long-lived CARs were CD4-positive, raising the prospect that this subset may be less susceptible to exhaustion and thereby exhibit greater persistence76,82.
The clinical effect of shorter persistence of CD28- versus 4-1BB-costimulated CARs varies by disease. In LBCL, tumour eradication occurs rapidly, and CD28 and 4-1BB costimulated CAR T cells demonstrate similar efficacies1,3,4,83. By contrast, in B-ALL, persistence of CAR T cells beyond 6 months is associated with increased rates of relapse—thus, CD28 costimulated CAR T cells are less effective unless patients receive a post-CAR bone marrow transplant to consolidate remission57,84. In multiple myeloma, functional persistence of anti-BCMA-CAR T cells is associated with a longer duration of response13. It remains unclear whether CD28 or 4-1BB costimulation is preferred for solid tumours, where both strong signalling strength and persistence are desirable. Prototype CARs incorporating both CD28 and 4-1BB costimulatory domains have not demonstrated superiority, leading investigators to integrate novel85,86 or synthetic costimulatory domains87 with the goal of endowing maximal signalling power alongside durable persistence. Pooled CAR screening has been used to identify optimal CAR signalling domains and designs and elucidate CAR design principles87,88,89. Genome-wide CRISPR screens have identified the CD2–CD58 axis as a mediator of T cell potency90 and IFNγR signalling has been demonstrated to be required for productive CAR T cell adhesion and cytotoxicity in solid but not liquid tumours91. CAR T cell potency is also limited by immunosuppressive molecules (TGFβ, IL-10, IL-6 and checkpoint molecules) in the tumour microenvironment (TME), and work is underway to combine CAR T cell therapies with immunomodulators designed to activate immunity within the TME and/or to arm immune cells to resist specific immunosuppressive mediators (Box 1).
Impaired trafficking and locoregional delivery
Impaired trafficking to the tumour site may also limit CAR T cell efficacy, especially in solid tumours. In preclinical models of central nervous system tumours, intratumoral or intracerebroventricular (ICV) administration has improved therapeutic benefit, with an approximate tenfold lower regional dose being required to achieve the same efficacy as intravenous administration92,93,94. Several clinical trials have demonstrated the safety of locoregional delivery of CAR T cells into the central nervous system27,28,95 and in a patient with glioblastoma multiforme, ICV delivery of IL-13Rα2 CAR T cells induced a complete response, whereas intratumoral administration was not effective27. In a study of patients with diffuse midline gliomas, ICV delivery of GD2-CAR T cells induced antitumour effects and clinical responses, and repeated dosing was associated with sustained benefit, raising the prospect that delivery to the central nervous system may abrogate immune sensitization, which has probably limited the effectiveness of multidose intravenous CAR T cell regimens28,96,97. In patients with lung cancer involving the pleura, regional delivery of mesothelin-CAR T cells in combination with PD-1 blockade mediated stable disease and metabolic responses98. Cell-intrinsic strategies to improve T cell homing to and persistence in the TME, such as secretion of IL-7 and CCL1999, are also being explored.
The next generation of CAR T cells
The various next-generation platforms being used to overcome tumour resistance mechanisms, augment immune cell fitness, improve specificity, tune CAR signalling, enhance safety, and increase antigen sensitivity are discussed in this section.
Platforms to diminish antigen escape
Bispecific CAR targeting may be achieved by administration of a mixed cell product, bicistronic expression of two receptors, two scFvs incorporated into a single receptor100, or co-transduction of multiple CARs, with each approach presenting opportunities and challenges. Co-infusion is financially, labour- and cell-intensive and co-infusion and co-transduction generate heterogeneous products, risking the emergence of a subpopulation that dominates the pool of cells after infusion101,102. Bicistronic vectors may result in reduced protein expression, and in one clinical trial, a bicistronic construct demonstrated limited persistence103. Several trials with bispecific receptors targeting CD19 plus CD20 or CD22 have been reported45,104,105, and in one, the receptor mediated diminished potency toward CD22 and tumour cell variants exhibiting low or no surface expression of CD19 emerged45. Cilta-cel, a BCMA-CAR recently approved by the FDA, incorporates two tandem Vhh binders in one receptor, which binds two different epitopes on BCMA. Clinical results with cilta-cel demonstrate a sCR of 83% and 55% PFS at 27 months, the highest reported so far using CARs for multiple myeloma14,106 (Table 1). In summary, clinical data with multispecific CARs is nascent but demonstrates safety and promise for improved efficacy by diminishing antigen escape.
Novel receptors designed to lower the antigen density threshold are also being developed. Katsarou and colleagues have expressed a chimeric costimulatory receptor (CCR), which lacks a CD3ζ domain, in trans with a prototype CAR, and reported that CCR engagement activated the prototype CAR at very low antigen density, preventing antigen low escape in preclinical models107. Induction of antitumour responses toward non-CAR T cell antigens—as reported following CAR T cell therapy in a patient with rhabdomyosarcoma—could diminish resistance due to antigen modulation54. Several approaches are under development to augment innate and adaptive immunity (Box 1), including CAR-mediated delivery of the immunostimulatory RNA RN7SL1108, coexpression of ligands or cytokines that reshape the TME such as IL-12109,110, IL-18111, CD40L112 or Flt3L113, engineering CAR T cells to secrete bispecific T cell engagers (BiTEs), taking advantage of CAR T cell accumulation within the tumour site and avoiding systemic toxicity of the BiTE114, or using non-traditional immune cells that may mediate more potent endogenous antitumour activity.
Enhancing T cell potency
Extensive work is underway to enhance immune cell fitness (Fig. 3 and Box 1). Significant effort is focused on epigenetic modulation, in part on the basis of an exceptional responder in a clinical trial of CD19-CAR for CLL—in which lentiviral integration disrupted the TET2 gene, a mediator of DNA methylation, resulting in substantial clonal T cell proliferation and a sustained antitumour response115. Similarly, knockout of the DNMT3A gene enhances the antitumour activity of CAR T cells in preclinical models116. Overexpression of transcription factors to prevent exhaustion has also shown promise, including overexpression of the AP-1 factor JUN, which enhances T cell expansion and persistence, diminishes terminal differentiation and lowers the antigen density threshold, presumably owing to increased signal strength53. Similarly, overexpression of BATF transcription factors has been reported to enhance T cell potency117. Manufacturing strategies are being developed to optimize CAR T cell phenotype towards stem-like and central memory subsets, including shorter culture duration118, inhibition of PI3K–mTOR–AKT119, BTK120 or tyrosine kinase121 signalling, and culture in memory-promoting cytokines122.
Investigators have leveraged numerous bioengineering strategies to develop advanced CAR platforms to improve the safety and efficacy of immune cell therapies. Multispecific (bivalent) CARs, which operate as an OR gate, may be able to overcome obstacles related to tumour heterogeneity and antigen loss, whereas combinatorial antigen sensing systems such as SynNotch or LINK CAR increase the specificity of CAR T cells by requiring two antigens for activity. Safety switches such as drug-regulated and adapter CARs could mitigate CAR-induced toxicities and enhance efficacy by tuning signalling. Knockout of TRAC, B2M and CIITA genes ablates TCR, MHC class I and MHC class II expression, respectively, and human leukocyte antigen (HLA)-E and CD47 overexpression shield stealth CAR T cells from natural killer cell- and macrophage-mediated rejection. Deletion of NR4A and TET2 genes or ectopic overexpression of c-Jun results in transcriptional rewiring that renders T cells exhaustion resistant. By fusing a scFv domain to TCR subunit, TRuC and HIT receptors redirect the specificity of the endogenous receptor and exhibit increased antigen sensitivity compared with CARs, while secreting lower levels of cytokine. Integration of a cytokine gene in the CAR vector provides a growth and survival signal to improve the persistence of T cells. Costim, costimulatory domain; TF, transcription factor; TM, transmembrane domain.
CRISPR-mediated gene editing was first applied clinically in the setting of adoptive T cell therapy, in which PD-1 was deleted from cells engineered to express NY-ESO-1, a cancer-specific TCR transgene123. The engineered cells did not demonstrate enhanced persistence or potency, but the study demonstrated the feasibility and safety of the approach, and accelerated efforts to apply gene editing technologies to enhance immune cell therapies. Several genes have been identified as candidates for editing to enhance T cell fitness72,124,125,126,127,128,129,130,131,132,133 (Box 1), and CRISPR-mediated disruption of T cell markers such as CD7 and CD5 has enabled CAR T cell therapies for T cell malignancies, while avoiding CAR T cell lysis134,135 (termed ‘fratricide’). We anticipate increasing clinical trial activity incorporating gene-edited immune cells into adoptive immune cell therapy platforms to enhance their potency, expand the landscape of targetable antigens and avoid immune sensitization.
To enhance persistence, some investigators have sought to integrate cytokine signals into the CAR receptor or express cytokines in trans136,137, including a clinical trial in which CAR-expressing natural killer cells transgenically expressing IL-15 demonstrated prolonged persistence138. Immune rejection may also limit CAR T cell persistence as anti-CAR immune responses—often targeting mouse, humanized or fully human scFvs—can be measured in many patients29,97,139,140. Consistently, clinical experience demonstrates the limited utility of second and subsequent intravenous CAR T cell doses, which can be improved using enhanced lymphodepleting regimens96. These findings raise the prospect that stealth platforms—which are currently being developed to enable off-the-shelf allogeneic products (discussed in ‘Platforms to enhance access and efficacy’)—could enhance CAR T cell efficacy by enhancing persistence or enabling multiple CAR T dosing regimens.
Diverse efforts are underway to address the suppressive TME (Box 1), including genetic ablation or expression of dominant-negative TBGβ141,142, PD-1143,144 or Fas receptors145, and engineering CAR T cells to secrete checkpoint-blocking scFvs146. Some investigators have engineered switch receptors, fusion proteins that convert a suppressive signal within the TME to an activating signal in the CAR T cells147,148. Whether tonic activating signals induced by such receptors result in long-term CAR T cell enhancement or predispose them to exhaustion and terminal differentiation remains to be determined. Biomaterials-based approaches for enhancing the expansion and persistence are also being explored149.
CAR tuning and regulatable platforms
Substantial efforts are underway to enhance safety and potency by tuning or dampening CAR signalling to diminish toxicity and exhaustion. This concept was first proposed by Eyquem and colleagues, who used CRISPR to knock-in CAR receptors into the TRAC locus and observed improved potency and diminished exhaustion due to antigen-induced CAR downregulation mediated by endogenous TRAC regulatory elements150. Weber and colleagues extended this principle using synthetic biology or small molecules to transiently cease CAR signalling, which enhanced CAR T cell potency when used during manufacturing and improved antitumour effects when applied in vivo after adoptive transfer121.
Kill switches such as iCasp9151, HSV tyrosine kinase152 (HSV-tk) and epitope tags153 enable the depletion of engineered cells in the event of severe toxicity, and a transgene-free safety switch that renders T cells auxotrophic for uridine has been developed154. Regulatable platforms can serve as reversible safety switches and also tune CAR signalling, thereby enhancing T cell potency by providing rest periods that prevent T cell exhaustion121. Numerous regulatable platforms have been developed using drug-sensitive promoters155, induced dimerization156,157, disruption of split CARs158, drug-dependent activation of binders159, proteolysis-targeting chimeras160 (PROTACs), chemically-dependent degron domains121,157,161 and drug-regulated CAR proteolysis162,163. These systems represent significant advances in synthetic biology, but remain challenged by leaky activity in the OFF state that risks toxicity, diminished CAR expression or activity in the ON state and the use immunosuppressive drugs as regulators121,155,156,157,158,159,160,161. Labanieh et al. recently developed a protease-regulated grazoprevir-induced ‘drug ON’ platform, signal neutralization by an inhibitable protease (SNIP), which shows no leaky activity and full functional capacity162 (Fig. 3). Similar to synNotch164, SNIP demonstrates superior antitumour efficacy compared with conventional CAR T cells owing to reduced exhaustion, and in an on-target off-tumour ROR1 toxicity model, decreased grazoprevir dosing tuned SNIP CARs to open a therapeutic window in which healthy tissue was spared but ROR1-expressing tumour cells were killed162. Similarly, Hernandez-Lopez et al. iterated the synNotch platform to target very highly expressed tumour antigens while avoiding lower levels of the antigens on normal tissues165. Thus, regulatable CARs show promise for enhancing efficacy and diminishing toxicity.
Enhancing specificity through Boolean logic
B cell and plasma cell malignancies are especially suited to CAR T cell therapy owing to the high, homogenous expression of lineage antigens that are co-expressed predominantly on B cells and plasma cells, the depletion of which is tolerable. However, a recent case report showed the development of parkinsonism in a patient after BCMA-CAR T cell therapy, with postmortem analysis revealing expression of BCMA on subsets of neurons and astrocytes in the patient’s basal ganglia166. In another study, single-cell RNA-sequencing analysis showed the expression of CD19 on brain mural cells, raising the prospect that on-target killing may be responsible for neurotoxicity after CD19-CAR T cell therapy. These results highlight the challenge of identifying targets that are not expressed on vital tissue.
So far, the paucity of tumour-specific surface targets on solid tumours has limited the application of the CAR prototype to solid tumours, with unacceptable off-tumour, on-target toxicity having been observed in trials of CARs targeting CAIX167 and CEACAM5168. However, several clinical trials of CAR T cells and other potent antibody-directed therapies have demonstrated good safety profiles in solid tumours (Table 1). The high CAR antigen density threshold is likely to explain the safe targeting of some antigens with known expression on vital tissues—such as GD2, which is expressed at low levels on neural tissues169,170. A recent trial demonstrated promising clinical activity of claudin-18.2-CARs was associated with significant but non-dose-limiting toxicity, potentially explained by antigen expression restricted to differentiated epithelial cells buried in gastric mucosa that may be less accessible to CAR T cells29. Identifying additional molecules with sufficient differential expression levels for safe targeting, such as oncofetal cell-surface targets is essential for expanding the reach of CAR T cells beyond B cell and plasma cell malignancies. However, the safety of specific targets will need to be continually reassessed as potency and persistence enhancements are deployed, as in recent studies with a PSMA-targeted CAR integrating a dominant-negative TGFβ receptor that was associated with lethal toxicity171,172.
Next-generation receptors incorporating logic gates could allow better discrimination between tumour and healthy tissue through combinatorial antigen sensing, and expand the repertoire of potential antigens (Fig. 3). Roybal et al. developed synNotch, an IF–THEN circuit incorporating a synthetic notch receptor against antigen A, which upon engagement, triggers the transcription of a conventional CAR against antigen B173,174. The synNotch system has not been tested clinically, but in preclinical models it prevented on-target, off-tumour toxicity when tumours and susceptible vital tissues are not colocalized175. Tousley et al. developed an AND gate platform called LINK, which utilizes the proximal TCR signalling molecules LAT and SLP76, each fused to a membrane-bound scFv specific for a unique antigen176. Engagement of both antigens colocalizes LAT and SLP76, leading to T cell activation. In an on-target, off-tumour ROR1 toxicity model, LINK CAR T cells cured mice of tumours expressing both antigens without ROR1-mediated toxicity, whereas mice treated with synNotch T cells succumbed to toxicity175. Other approaches for combinatorial antigen targeting that are under development include SUPRA177 and co-LOCKR178, which redirect CAR T cell specificity through protein switches. Although combinatorial antigen sensing could expand the landscape of targetable tumour antigens, the increased risk of tumour escape owing to loss of either antigen is a potential concern. An alternative approach to enhance specificity is to use an AND NOT gate, in which a prototype activating CAR is expressed in trans with an inhibitory CAR (iCAR) targeting an antigen that is expressed on healthy tissue but not on tumour tissue179,180,181. Limited engineering with NOT gates has been undertaken so far and these applications have not been tested clinically.
TCR-like CARs
With the goal of targeting antigens that are expressed at low levels, HLA-independent TCRs (HIT) are designed with the variable domain of the endogenous TCR being altered to target scFvs by gene editing the endogenous TRAC locus182. When CD80 and 4-1BBL are provided in trans, CD19-directed HITs display superior antigen sensitivity compared with prototype CD19-CARs (Fig. 3). Synthetic TCR and antigen receptors (STARs) have a similar design but are not knocked in to the TRAC locus; thus, the endogenous TCR specificity is retained183. Other approaches for redirecting TCR specificity include the antibody–TCR (AbTCR) platform184, which replaces the variable domains of TCRγδ with a Fab fragment and TCR fusion constructs185 (TRuC), which fuse an scFv to a CD3 subunit. A recent comparison of TCR-like chimeric receptors showed that STAR and HIT receptors reproduce TCR antigen sensitivity, whereas TruCs do not186. One potential drawback of CAR T cells compared with native T cells is the inability to target intracellular antigens, since most aberrant proteins that drive cancer are intracellular. Yarmarkovich et al. overcame this by developing a prototype CAR with specificity for peptides presented by MHC187 (pMHC). Using scFv binders specific for a PHOX2B peptide–MHC overexpressed in neuroblastoma, they targeted pMHCs across several HLA allotypes. This strategy could greatly expand the landscape of CAR targets, including key oncogenic drivers.
Platforms to enhance access and efficacy
Diverse approaches are under exploration to increase access of cell therapies, diminish the high manufacturing costs, create stealth immune cells resistant to rejection, and leverage the unique properties of alternative immune cells.
Distributed manufacturing and allogeneic products
Engineering advances have yielded automated closed-system manufacturing, which is providing opportunities for point-of-care manufacturing to diminish the costs, delays and logistical challenges associated with the centralized manufacturing models that are the industry standard. A recent multicentre trial demonstrated the safety and efficacy of cells manufactured at the point of care188. Defining the regulatory requirements for point-of-care manufacturing is an area of significant current interest, especially for therapies targeting rare indications, such as paediatric cancers189.
Allogeneic CAR T cells manufactured from healthy ‘super donors’ could improve potency by avoiding preexisting T cell dysfunction and decrease the cost and logistical challenges of manufacturing, thereby enhancing access. However, allogeneic T cell therapies must overcome the risk of GVHD mediated by the TCR and rejection of the transferred cells by the host immune system. Gene editing of the endogenous TCR eliminates the risk of GVHD190, but endowing stealth properties to avoid immune rejection remains a significant challenge, since CD8+, CD4+, natural killer and macrophage cells can reject allogeneic cells and each are regulated by distinct axes, necessitating multiple enhancements (Box 1). Knockout of β2-microglobulin can eliminate HLA class I surface expression, but paradoxically increases the risk of rejection by natural killer cells. Additional strategies for inducing allogeneic tolerance include knockout of the CIITA gene to ablate MHC class II expression191, and overexpression of HLA-E192 and CD47193 to ameliorate natural killer cell- and macrophage-mediated cell rejection.
Many allogeneic approaches use CRISPR–Cas9, and the risks of CRISPR-based mutagenic events could be magnified when producing hundreds or thousands of allogeneic products with a singular manufacturing process. Alternative platforms, such as base editing or prime editing may emerge as preferred alternatives to nuclease-based genome editing since they probably involve lower risk owing to an absence of double strand DNA breaks194. CRISPR–Cas systems targeting RNA could also provide opportunities for multiplexed gene knockdowns with greater specificity and efficiency compared with RNA-mediated interference. Although allogeneic donor-derived cells containing multiple gene edits could provide significant advantages, these technologies are nascent and their toxicity profiles remain unknown. Some groups have attempted to prevent immune rejection by augmenting immune suppression of the host using conventional chemotherapy or immunosuppressive antibodies for which the targets are edited from the CAR T cells190. Early response rates with this approach are promising, but long-term safety and efficacy have not been demonstrated and concerns remain regarding infectious risks associated with intensive immune-depleting regimens190.
Alternative immune cells
Several non-T immune cells, including natural killer cells, invariant natural killer T (iNKT) cells, γδ T cells and macrophages exhibit innate antitumour activity and do not induce GVHD, raising the prospect that they could provide an off-the-shelf source of cells with reduced toxicity, enhanced tumour trafficking and/or target antigen-negative variants through innate tumour recognition. However, allogeneic innate immune cells remain susceptible to rejection, raising concerns regarding the durability of their effects if they are not engineered for stealth. Cord blood-derived allogeneic natural killer cells incorporating ectopically expressed IL-15 have shown promise in a phase I trial for NHL and CLL138. iNKT-CAR cells mediated activity in mouse models, in part by cross-priming host CD8 cells towards tumour antigens195, and their safety and feasibility in a phase I trial for neuroblastoma have been demonstrated196. γδ T cells engineered to express a CD20-CAR have also shown impressive activity in early studies197. Expressing CARs in macrophages requires substantial adaptations of vectors and signalling domains, but antitumour effects associated with augmented phagocytosis, modification of the TME and recruitment of T cells198,199 have been demonstrated in preclinical models198, with CD3ζ-based CARs demonstrating equivalent phagocytic activity as Fcγ-based CARs Efforts are also underway to create induced pluripotent stem (iPS) cell-derived CAR T cells200, natural killer cells201 and macrophages202. The differentiation of iPS cells to natural killer cells has been particularly successful, and clinical testing of these off-the-shelf therapies is currently in progress203, whereas iPS cell differentiation to fully functional T cells has been more challenging204. Given their nearly inexhaustible expansion potential, iPS cell-derived products could enable mass production of a homogenous cell product integrating numerous enhancements to endow stealth properties, safety switches and potency, and the long-term safety and efficacy results of these emerging platforms are thus eagerly anticipated.
Next-generation gene delivery
Viral vector-based gene delivery has been the gold standard in the field, but vector production and qualification is costly and time consuming. Viral-free platforms for gene delivery are under development, with CRISPR-based gene delivery in human T cells demonstrating proof of principle, although DNA templates are toxic to T cells and the efficiency of this approach remains lower than with viral vectors205. Clinical feasibility has been demonstrated with a CD19-CAR site-specifically delivered to the PD-1 locus inducing a high CR rate in NHL, although the manufacturing process did not meet dose requirements for a relatively high proportion of patients206. Modifications to DNA templates and small-molecule inhibitor cocktails are improving knock-in efficiencies and cell yields207. Transposon-based gene delivery has also been used, although malignant transformation of CAR-engineered T cells was reported in two patients associated with high-copy-number integration using a Piggybac transposon platform208. In vivo gene delivery is another emerging approach that could improve accessibility and diminish cost. In this approach, DNA or RNA is delivered systemically using viral vectors209 or nanoparticles210 that preferentially target and transduce immune populations in vivo. Immunogenicity could prohibit repeat administration of viral vectors owing to the induction of neutralizing antibodies. Stable expression of a CD19-CAR has been demonstrated using lipid nanoparticles targeting CD3 in mice210 and T cell-targeted lipid nanoparticles incorporating optimized RNA diminished cardiac fibrosis in a mouse model211.
CAR therapy for non-malignant diseases
The CAR T platform has been optimized for cancer treatment, but the design principles and expansive synthetic biology toolbox used for CAR T cells are providing opportunities to extend this therapeutic approach to non-malignant diseases, including autoimmunity, senescence, fibrosis and infectious diseases. In preclinical studies, CD19-CAR T cells have demonstrated beneficial effects in systemic lupus erythematosus212, and a case study reported sustained activity of CD19-CAR therapy in a patient with refractory lupus nephritis213 . Chimeric autoantibody receptors (CAARs) are prototype CARs that incorporate a scFv targeting the idiotype of an autoreactive B cell clone or use autoantigens as the recognition domain. In preclinical studies, CAARs mediated therapeutic effects against pemphigus vulgaris, and clinical testing is underway. Adoptive transfer of T regulatory (Treg) cells, which mediate suppression rather than cytotoxicity, is an alternative approach for treating autoimmunity. Non-engineered Treg cells have demonstrated activity in mouse models of GVHD, allograft transplantation, type 1 diabetes, systemic lupus erythematosus and multiple sclerosis, and early clinical data demonstrate feasibility of manufacturing and a good safety profile214. Compared with non-engineered cells, Treg cells expressing a CAR targeting antigens expressed on the diseased tissues show enhanced specificity and potency215,216. Recent data have demonstrated that inadvertent expansion of CAR Treg cells limits the efficacy of commercial CAR T cells, providing proof-of-concept for the utility of CAR-engineered Treg cells217,218. Approaches are underway to engineer FOXP3 expression to enforce lineage stability and incorporate safety switches to diminish risk214. Recent promising preclinical data were generated in haemophilic mice, in which Treg cells expressing a factor VIII-targeted CAR and FOXP3 prevented the development of neutralizing anti-factor VIII antibodies219. Senolytic CAR T cells targeting urokinase-type plasminogen activator receptor have been demonstrated to target senescent cells in vitro and restore tissue homeostasis in models of liver fibrosis220. CARs targeting fibroblast activation protein (FAP) have improved cardiac function in a mouse model of cardiac fibrosis221 and in vivo generation of FAP-CARs using CD5-directed lipid nanoparticles loaded with mRNA also demonstrated benefit211. In this model, the non-integrating nature of mRNA ensured that CAR expression was transient, thereby mitigating the risk of toxicity associated with widespread elimination of activated fibroblasts.
Outlook
Adoptive immune cell therapy is established as a transformative therapeutic modality. The past decade has witnessed significant progress in understanding the biology of prototype CAR T cells, identifying antigen modulation and T cell dysfunction as major resistance mechanisms and highlighting the logistical challenges of delivering cell therapies to all patients who could benefit. Modifications to prototype CARs can augment their potency, but increasingly investigators are designing next-generation platforms to create advanced cellular therapies that incorporate a diverse array of enhancements. The fields of immunology, synthetic biology, genetic engineering and cell manufacturing are synergizing to create smarter, safer and more accessible cellular therapies that are poised for increased efficacy and access, diminished risk and cost, and broader utility, for the treatment of cancer as well as non-malignant diseases.
Change history
20 June 2023
A Correction to this paper has been published: https://doi.org/10.1038/s41586-023-06088-3
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Acknowledgements
C.L.M. and L.L are members of the Parker Institute for Cancer Immunotherapy, which supports the Stanford University Cancer Immunotherapy Program. This work was supported by the St Baldrick’s Foundation Empowering Pediatric Immunotherapies for Childhood Cancer (EPICC) Team and NCI 5P30CA124435 (C.L.M.). L.L. was supported by a Siebel Scholars Fellowship, Stanford Graduate Fellowship, National Science Foundation Graduate Research Fellowship (DGE-1656518), and Discovery Innovation Award philanthropically supported through the Biomedical Innovation Initiative.
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L.L. and C.L.M. are inventors on several patents related to CAR T cell therapies. C.L.M. is a cofounder of Lyell Immunopharma, CARGO Therapeutics and Link Cell Therapies, which are developing CAR-based therapies, and consults for Lyell, Syncopation, Link, Apricity, Nektar, Immatics, Ensoma, Mammoth, Glaxo Smith Kline and Bristol Myers Squibb. L.L. is a cofounder of, consults for, and holds equity in CARGO Therapeutics. L.L. is a consultant for and holds equity in Lyell Immunopharma.
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Labanieh, L., Mackall, C.L. CAR immune cells: design principles, resistance and the next generation. Nature 614, 635–648 (2023). https://doi.org/10.1038/s41586-023-05707-3
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DOI: https://doi.org/10.1038/s41586-023-05707-3
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