nature reviews clinical oncology https://doi.org/10.

1038/s41571-023-00729-2

Review article Check for updates

Advancing CAR T cell

therapy through the use of
multidimensional omics data
Jingwen Yang1,6, Yamei Chen , Ying Jing
1,6
, Michael R. Green
2,6 3,4
& Leng Han 1,5

Abstract Sections

Despite the notable success of chimeric antigen receptor (CAR) T cell Introduction

therapies in the treatment of certain haematological malignancies, Multidimensional profiling

challenges remain in optimizing CAR designs and cell products, technologies

improving response rates, extending the durability of remissions, Target discovery for CAR T cell
therapy
reducing toxicity and broadening the utility of this therapeutic
modality to other cancer types. Data from multidimensional omics Understanding and enhancing
CAR T cell efficacy and
analyses, including genomics, epigenomics, transcriptomics, T cell persistence
receptor-repertoire profiling, proteomics, metabolomics and/or Minimizing CAR T cell-related
microbiomics, provide unique opportunities to dissect the complex toxicities
and dynamic multifactorial phenotypes, processes and responses of Conclusions
CAR T cells as well as to discover novel tumour targets and pathways
of resistance. In this Review, we summarize the multidimensional
cellular and molecular profiling technologies that have been used
to advance our mechanistic understanding of CAR T cell therapies.
In addition, we discuss current applications and potential strategies
leveraging multi-omics data to identify optimal target antigens and
other molecular features that could be exploited to enhance the
antitumour activity and minimize the toxicity of CAR T cell therapy.
Indeed, fully utilizing multi-omics data will provide new insights into
the biology of CAR T cell therapy, further accelerate the development
of products with improved efficacy and safety profiles, and enable
clinicians to better predict and monitor patient responses.
1
Center for Epigenetics and Disease Prevention, Institute of Biosciences and Technology, Texas A&M University,
Houston, TX, USA. 2Department of Biochemistry and Molecular Biology, The University of Texas Health Science
Center at Houston McGovern Medical School, Houston, TX, USA. 3Department of Lymphoma and Myeloma,
The University of Texas MD Anderson Cancer Center, Houston, TX, USA. 4Department of Genomic Medicine,
The University of Texas MD Anderson Cancer Center, Houston, TX, USA. 5Department of Translational Medical
Sciences, College of Medicine, Texas A&M University, Houston, TX, USA. 6These authors contributed equally:
Jingwen Yang, Yamei Chen, Ying Jing. e-mail: MGreen5@mdanderson.org; leng.han@tamu.edu

Nature Reviews Clinical Oncology | Volume 20 | April 2023 | 211–228 211

Review article
Key points
•• Multidimensional omics data, encompassing genomics, epigenomics,
transcriptomics, T cell receptor-repertoire profiling, proteomics,
metabolomics and microbiomics, have been exploited to advance our
mechanistic understanding of chimeric antigen receptor (CAR) T cell
therapy.
•• Utilization of multi-omics data derived from both tumour and non-
malignant tissues at the bulk and/or single-cell levels is a powerful
approach to identifying optimal targets for highly efficacious and safe
CAR T cell therapy.
•• Integration of bulk and/or single-cell multidimensional omics
data has been applied to investigate key determinants of CAR T cell
persistence and antitumour efficacy, including T cell states and
phenotypes, tumour cell characteristics, the tumour microenvironment
and the microbiome.
•• Leveraging multi-omics data promises to elucidate the mechanisms
underlying CAR T cell-related toxicities, including cytokine-release
syndrome, immune effector cell-associated neurotoxicity syndrome
and on-target, off-tumour toxicities.
Introduction
Chimeric antigen receptors (CARs) are synthetic fusion receptors
designed to redirect T cells to recognize and eliminate cancer cells that
express cognate antigens1. To achieve this aim, CARs have typically been
constructed using antibody-based moieties targeting specific tumour-
associated antigens, which are linked via a hinge and transmembrane
domain to intracellular signalling motifs derived from both the T cell
receptor (TCR) CD3ζ chain and a co-stimulatory receptor (such as CD28
and/or 4-1BB)1. Transgenes encoding these constructs are introduced
into autologous (patient-derived) or allogeneic (healthy donor-
derived) T cells ex vivo, followed by selection and in vitro expansion
of CAR-expressing T cells before they are infused into the patient.
CAR T cell therapy has moved to the forefront of therapy for relapsed
and/or refractory B cell malignancies, owing to impressive response
rates and durability in patients with B cell acute lymphoblastic leukae-
mia (B-ALL)2, large B cell lymphoma (LBCL)3, mantle cell lymphoma4,
follicular lymphoma5 or multiple myeloma6. In addition, CAR T cells
have been demonstrated to induce clinical responses in patients with
various advanced-stage solid tumours, including glioblastoma7, pancre-
atic ductal adenocarcinoma8, sarcoma9, gastrointestinal cancers10 and
castration-resistant prostate cancer11, albeit less frequently and more
transiently than in patients with B cell malignancies. Nevertheless, the
clinical success of CAR T cell therapy faces several challenges, includ-
ing the paucity of robustly expressed tumour-specific antigens in
many malignancies12,13, CAR T cell exhaustion14,15, limited CAR T cell
persistence16,17 and potential life-threatening toxicities18,19.
Multi-omics data, encompassing genomics, epigenomics, tran-
scriptomics, proteomics and beyond, can provide deep insights into
the abundance and/or variation of biological molecules across multiple
dimensions in various tissues or cells20,21. The rapid development of
multidimensional profiling strategies and approaches for their integra-
tion has facilitated the use of multi-omics data to characterize the com-
plex molecular features of diverse cellular processes in broad research
Nature Reviews Clinical Oncology | Volume 20 | April 2023 | 211–228
fields22,23, including CAR T cell therapy24–26. CAR T cells are living cells
that can actively sense and respond to a wide variety of extrinsic and
intrinsic factors27. Therefore, identification of detrimental phenotype
heterogeneity and pathway regulation of pre-manufacturing autolo-
gous T cells through bulk and single-cell profiling28–30 might contribute
to the quality control of CAR T cell products or inform strategies for
CAR design or product manufacture that improve their functionality.
Moreover, the use of multi-omics strategies to monitor the temporally
dynamic expansion of functional and/or dysfunctional CAR T cell sub-
populations24,31, characterize CAR T cell transcriptomic32, epigenomic33
and metabolic34 dynamics, delineate tumour microenvironment (TME)
features that restrict CAR T cell infiltration and suppress CAR T cell
cytotoxicity35, determine the origins and functional consequences of
key cytokines36,37, and predict on-target, off-tumour toxicities in non-
malignant tissues38 could facilitate biomarker discovery and improve
our mechanistic understanding of CAR T cell therapy responses and
toxicities.
In this Review, we highlight the potential of multi-omics data
to advance CAR T cell therapy. We first provide an overview of the
multidimensional profiling technologies that have been utilized in
the investigation of CAR T cell therapy. Next, we discuss strategies
leveraging multi-omics data to discover single and combinatorial
CAR targets. We further discuss molecular features and other key fac-
tors that are associated with enhanced antitumour responses and/or
decreased toxicity of CAR T cell therapy as uncovered by multi-omics
profiling. Exploiting current and emerging multidimensional
omics profiling technologies will enable a comprehensive understand-
ing of the molecular biology of CAR T cell therapies, ultimately enabling
the clinical benefits of these treatments to be maximized.
Multidimensional profiling technologies
Data from genomics, epigenomics, transcriptomics, TCR-repertoire
profiling, proteomics, metabolomics and microbiomics studies have
been applied to address the remaining key challenges for CAR T cell
therapy. Leveraging such multi-omics data can provide novel insights
into tumour and non-malignant cell characteristics, cell state tran-
sitions, cell types, and cell–cell interactions in the TME that affect
treatment outcomes of CAR T cell therapy (Fig. 1 and Table 1).
Genomics
Comparisons of DNA sequencing (DNA-seq) data from tumour and non-
malignant tissue samples have identified numerous tumour-associated
somatic mutations, some of which might generate tumour-specific
neoantigens that are presented on the cell surface and could potentially
serve as novel targets for CARs or TCR-like constructs39,40. To date,
however, attempts to target cancer cells with neoantigen-directed CAR
T cells have been limited to a few preclinical and clinical studies41–43.
Alternatively, tumour-associated mutations and complex genomics
alterations might influence clinical responses to CAR T cell therapy44–47.
Genome-wide pooled CRISPR–Cas9 knockout library screening fol-
lowed by sequencing-based quantification of the frequencies of single
guide RNAs enables large-scale, high-throughput studies to assess the
influence of individual genes under different conditions and thereby
identify cellular functional dependencies48,49. Such functional genom-
ics profiling has resulted in the identification of key genes involved in
T cell activation50, persistence51, cytotoxicity52–54 and dysfunction55
as well as genetic alterations in tumour cells that influence resist-
ance to treatment56, highlighting regulators that could potentially be
exploited to enhance the efficacy of CAR T cell therapy. Meanwhile,
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Sample Multi-omics
CAR design
Genome
Me Me Ac Me Epigenome
Bulk
CAR T cell
Transcriptome Cell functional phenotype
TCR repertoire
Single cell
P P
Proteome CAR T cell
Tumour microenvironment
Spatial Metabolome
Microbiome
Cellular composition and
cell–cell interactions
Fig. 1 | Overview of applications of multi-omics data in CAR T cell therapy.
Several types of samples, including bulk tissue specimens, single-cell suspensions
or spatially preserved tissue slides (left panel), can be used for multidimensional
omics analyses, including genomics, epigenomics, transcriptomics, T cell
receptor (TCR)-repertoire, proteomics, metabolomics and/or microbiomics
profiling (middle left panel), across a multitude of applications in the field of
pooled CRISPR-based gain-of-function screening in combination with
single-cell RNA sequencing (scRNA-seq) has been used to measure the
abundance and assess the cellular states of T cells expressing different
knock-in constructs and highlighted a novel chimeric cytokine–
co-stimulatory receptor for CAR T cells that improves solid tumour
clearance57. In summary, functional genomics profiling is a powerful
method to elucidate mechanistic regulators of CAR T cell functions
that might ultimately be leveraged to refine CAR design or cellular
engineering.
Epigenomics
Epigenetic factors, including DNA methylation, histone modifications,
and chromatin accessibility and 3D architecture, enable the integration
of multiple cellular signals to dynamically regulate the gene expression
programme and, ultimately, the phenotype and function of T cells58,59.
Epigenetic reprogramming of CAR T cells through perturbation of
epigenetic modulators therefore has the potential to boost antitu-
mour efficacy60–64. Genome-wide epigenomics profiling techniques
have become the optimal approach for investigating epigenetic
regulatory landscapes65, including those of CAR T cells. For example,
DNA methylation array analyses can help to identify differentially
methylated sites associated with response to CAR T cell therapy33,66.
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Features Clinical applications
Identify targets
Recognition
Tumour cells
Enhance efficacy
Non-malignant cells
Cytotoxic
Memory Reduce toxicity
Exhausted
Cytokine storm and
cytokine-release
syndrome
Immunosuppression
On-target,
off-tumour
Neurotoxicities toxicities
chimeric antigen receptor (CAR) T cell therapy. The valuable multi-omics data
facilitates the characterization of diverse tumour and non-malignant cell features,
cell functional phenotypes (for example, of CAR T cells both prior to and after
infusion), and various properties of the tumour microenvironment (middle right
panel), which will enable advances in the safety and efficacy of CAR T cell therapy
through improvements in target identification and cell engineering (right panel).
Combination analyses with assay for transposase-accessible chro-
matin using sequencing (ATAC-seq) and RNA sequencing (RNA-seq)
can facilitate the identification of epigenomics regulatory modules
that control cellular programmes related to CAR T cell exhaustion and
cytotoxicity32,66–69, thus providing a multitude of insights into potential
epigenetic reprogramming strategies to improve efficacy.
Advances in single-cell epigenomics profiling70 have enabled eval-
uation of the epigenetic regulatory landscape within individual CAR
T cells71. Combining data derived using single-cell ATAC-seq with tran-
scriptomics and proteomics data has helped to identify the patterns
of epigenetic regulation associated with functional subpopulations of
CAR T cells28. Collectively, the emerging evidence from epigenomics
profiling studies demonstrates the importance of dynamic epigenetic
alterations within CAR T cells in determining the molecular features
associated with therapeutic efficacy.
Transcriptomics
RNA-seq has an essential role in understanding cellular states and has
enabled tremendous progress in the characterization of tumour hetero­
geneity and the TME72–74. In the field of CAR T cell therapy, RNA-seq is
most commonly applied for differentially expressed gene analysis
under various conditions to delineate the cell-state transitions that
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Table 1 | Representative multi-omics profiling approaches applied to study CAR T cell therapy

Omics approach Profiling technology Examples of insights obtained

Genomics DNA-seq Identification of genomics alterations as potential targets or as biomarkers for therapeutic outcome44–46
CRISPR screening Defining dependencies for the cytotoxic activity of CAR T cells and determinants of the sensitivity of
cancer cells to CAR T cell-mediated cytotoxicity52,53
Epigenomics DNA methylation arrays Definition of differentially methylated genomic loci related to CAR T cell functions and therapeutic
responses33,66
ATAC-seq Characterization of changes in accessible chromatin regions of genes that affect CAR T cell
functions32,68
ChIP-seq Identification of transcriptional reprogramming of the exhaustion-associated epigenome in exhausted
CAR T cells63
scATAC-seq Discovery of epigenetic differences correlated with distinct CAR T cell subpopulations28,71
Transcriptomics RNA-seq Identification of transcriptional changes related to CAR T cell functions32,76; discovery of novel targets
based on differential expression and RNA dysregulation in malignant versus non-malignant tissues80,82
scRNA-seq Characterization of CAR T cell functional states that are associated with particular cellular conditions,
CAR designs/cell products, clinical responses and toxicities87; discovery of subpopulations of non-
malignant and tumour cells that cause toxicities and disease relapse, respectively90,203; identification of
hostile factors in the tumour microenvironment that affect CAR T cell function and, therefore, hamper
solid tumour elimination91
TCR-repertoire profiling TCR-seq Kinetic profiling of different TCR clonotypes throughout the treatment process96,97
Proteomics Mass spectrometry Information on the surfaceome (expression landscape of cell-surface proteins) and immunopeptidome
(repertoire of MHC-presented peptide epitopes) as a resource for CAR target identification100,103,106;
phosphoproteomics characterization to evaluate CAR downstream signalling109
Mass cytometry Identification of immune markers for monitoring of CAR T cell activation, proliferation and exhaustion114
Metabolomics Mass spectrometry Discovery of metabolic reprogramming in CAR T cells, which could enhance antitumour efficacy34,124,125
Metabolic flux analysis Assessment of dynamic metabolic pathway activity related to different cell conditions129
Mass cytometry of metabolic Characterization of the metabolic adaptation of CAR T cell products after infusion242
proteins
Microbiomics 16S rRNA-seq and Identification of features of the gut microbiota correlated with response and toxicities141
metagenomics shotgun
sequencing
16S rRNA-seq, 16S ribosomal RNA sequencing; ATAC-seq, assay for transposase-accessible chromatin with high-throughput sequencing; CAR, chimeric antigen receptor; ChIP-seq, chromatin
immunoprecipitation sequencing; DNA-seq, DNA sequencing; RNA-seq, RNA sequencing; scATAC-seq, single-cell ATAC-seq; scRNA-seq, single-cell RNA-seq; TCR, T cell receptor; TCR-seq,
TCR sequencing.

are associated with efficacy15,32,75,76. Moreover, monitoring changes in
the transcription of genes encoding various cytokines, which are key
mediators of CAR T cell activity, is also crucial to improve efficacy and
reduce the risk of cytokine-release syndrome (CRS), immune effec-
tor cell-associated neurotoxicity syndrome (ICANS) and other toxici-
ties67,77–79. Application of total RNA-seq provides additional information,
for example, on the expression of non-coding RNAs that can regulate
immunomodulatory molecules and thereby affect CAR T cell cyto-
toxicity80. Furthermore, dysregulation of post-transcriptional RNA
processing can lead to variant transcripts, thus potentially generating
different protein isoforms that enable escape from CAR recognition81.
Therefore, increasing interest is being placed on exploiting the existing
and expanding tumour RNA-seq data to discover novel CAR targets
derived from the differential expression and dysregulation of RNAs82,83.
scRNA-seq, which generates data on the transcriptome of indi-
vidual cells, has become a well-established method since 2009 (ref. 84)
and is widely applied in cancer research85,86. Notably, scRNA-seq is an
emerging method of investigating features of CAR T cells in preclini-
cal and translational research. For example, scRNA-seq enables gene
expression-based cell state examination of CAR T cell infusion prod-
ucts, with data demonstrating that signatures of cytotoxicity, central
memory, exhaustion and other cellular programmes can be affected by
variations in CAR T cell conditions or designs52,76,87,88 and are associated
with clinical response and toxicity15. Beyond profiling of the CAR T cell
products, applying scRNA-seq to non-malignant tissues and tumours
can help to identify rare cell subpopulations in order to elucidate the
mechanisms underlying treatment-related toxicities15,89,90. Addition-
ally, scRNA-seq can provide rich gene expression data across various
cell types and is therefore an optimal unbiased approach for exploring
factors in the TME that might affect the antitumour activity of CAR
T cells91,92. Such insights could offer potential opportunities to over-
come immunosuppressive features of the TME and seek solutions for
the expansion of CAR T cell applications in solid tumours91,93.
TCR-repertoire profiling
The TCR is a heterodimeric cell-surface protein complex comprising
heterogeneous TCRα and TCRβ or TCRγ and TCRδ chains generated
through random genetic recombination of different variable, diversity,
junctional and constant gene segments in individual T cell clones; this
process results in a varied T cell population with a highly diverse TCR
repertoire to mediate antigen-specific adaptive immune responses94.
High-throughput technologies have been developed to sequence the

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TCR chains expressed in each T cell and thus to characterize the TCR
repertoire and clonotypic diversity of the T cell population. Performing
TCR sequencing in combination with other omics approaches, such as
transcriptomics profiling, enables specific TCR clonotypes to be linked
with varying T cell phenotypes95. This combined analysis can help to
characterize T cell dynamics through tracing of T cell clonality among
different subpopulations over time95, and such studies have demon-
strated the cytotoxic and proliferative features of highly expanded CAR
T cell clonotypes in patients with cancer96,97. Moreover, advances in
single-cell technologies have enabled simultaneous TCR sequencing and
scRNA-seq in individual cells, which assigns a specific TCR clonotype to
an individual T cell with a distinct transcriptional phenotype and could
thus enable TCR clonotypes to be used as a surrogate for the expansion
and persistence of T cell functional states throughout therapy15. With
the caveat that individual TCR clonotypes can encompass a mixture
of cell states along a functional continuum, these profiling methods
offer a route to further explore whether specific clonotypically defined
T cell states are associated with CAR T cell performance or toxicities.
Proteomics
Proteomics profiling enables the identification and quantification of
proteins and their post-translational modifications (such as phospho-
rylation and glycosylation) to characterize cellular pathway activity and
cell functions. For example, emerging proteomics profiling approaches
in the field of immuno-oncology have substantially improved our
understanding of the mechanisms of tumour immune evasion98. Nota-
bly, applying single-cell proteomics profiling in the context of cancer
immunotherapy has shown early success in uncovering the clinical
relevance of distinct immune cell phenotypes in the TME93.
Traditional proteomics profiling technologies, such as protein
microarrays that provide data on the expression of select proteins,
have been applied to quantify plasma cytokine levels in patients
with lymphoma or leukaemia following treatment with CAR T cells99.
Advances in mass spectrometry-based proteomics techniques have
enabled robust high-throughput profiling of the membrane proteome,
including the cell surfaceome100 and immunopeptidome101, as well as
signalling pathway dynamics reflected in the phosphoproteome102.
The tumour surfaceome has been a source of novel therapeutic targets
in various haematological and solid malignancies103–105. The immun-
opeptidome and surfaceome of non-malignant tissues might serve as
a reference database for the selection of tumour-specific targets and
thus avoid on-target, off-tumour toxicities100,106. Phosphoproteomics
can be utilized to characterize CAR downstream signalling activity
to evaluate the functional states associated with different CAR con-
structs107–109. Thus, proteomics profiling has deepened our knowledge
of various aspects of CAR T cell therapy, especially CAR target discovery
and CAR function110.
Flow cytometry is the gold standard approach for both membrane
and intracellular protein detection at the single-cell level for immune
cell classification111. Advances in full-spectrum flow cytometry, whereby
highly sensitive detector arrays are used to capture the full emission
spectrum of a diverse range of fluorescent molecules, have improved
throughput by increasing the number of cellular parameters that
can be evaluated in an experimental run, thus presenting a power-
ful approach for protein expression analysis in studies of immuno-
therapy112. Mass cytometry, or cytometry by time of flight (CyTOF),
combines flow cytometry and mass spectrometry techniques to enable
high-throughput, single-cell proteomics profiling (through simultane-
ous evaluation of >40 proteins in millions of cells during one run)113 for
Nature Reviews Clinical Oncology | Volume 20 | April 2023 | 211–228
high-dimensional cellular phenotyping. Mass cytometry analysis of
protein expression has been utilized for functional characterization
of CAR T cells in preclinical and clinical settings, facilitating rapid
monitoring of the dynamic features of these cells114–116.
Technologies enabling single-cell secretome analyses have also
been developed. For example, single-cell microchamber, proteomics
barcode and multiplex immunoassay chips have been used to quan-
tify the secretion of ~32 selected effector and immunostimulatory
molecules by pre-infusion CAR T cell products as a measure of their
‘polyfunctionality’, which is associated with clinical response and
toxicities117,118.
Metabolomics
Metabolomics profiling catalogues a suite of small molecules produced
during metabolic processes, providing a functional readout of various
cellular activities119. T cell function is regulated by intrinsic and extrinsic
metabolic factors, and robust intrinsic metabolic activity is necessary
for effective T cell cytotoxicity120. Disrupted metabolism in the TME,
for example, owing to hypoxia and/or decreased nutrient availability,
can lead to unmet metabolic demands in infiltrating T cells and thus
limit their antitumour activity16. Therefore, maintaining the metabolic
fitness of CAR T cells is important to improve their therapeutic efficacy,
and especially to enhance their function in the immunosuppressive
TME of solid tumours121–123.
Mass spectrometry-based metabolomics enables both targeted
metabolite quantification and comprehensive untargeted metabolite
profiling. Several studies have profiled metabolites and their interme-
diates generated through various biochemical processes of interest in
T cells. Targeted quantification of metabolites involved in glycolysis
and the tricarboxylic acid cycle has highlighted impaired intrinsic
metabolic processes that affect the proliferation and exhaustion of
tumour-infiltrating T cells, presenting opportunities to enhance CAR
T cell efficacy through metabolic reprogramming124,125. Metabolomics
data can also be leveraged for metabolic reprogramming of CAR
T cells to enhance their fitness and resistance to unfavourable extrinsic,
microenvironmental factors34,121,126.
Cell metabolism is variable and adaptable to environmental con-
straints. Fluxomics describes the production and consumption rates
of metabolites involved in intracellular metabolic networks, which
reflect the dynamic metabolic processes within the cell127. A combina-
tion of stable isotopes and mass spectrometry can be used to trace
and quantify dynamic metabolic fluxes, referred to as metabolic flux
analysis, thereby enabling fluxomics profiling of metabolic pathways
of interest such as glycolytic and mitochondrial respiration128. This
approach has advantages for the dynamic assessment of metabolic
processes in CAR T cells with differing CAR designs or under different
environmental conditions, with emerging fluxomics data emphasizing
the pivotal role of maintaining the metabolic fitness of CAR T cells for
effective treatment123,129,130.
Microbiomics
The microbiota is emerging as a key factor regulating cancer progres-
sion and therapeutic responses131,132. Several studies have demonstrated
correlations between the gut microbiome or dietary interventions and
clinical responses to immunotherapies, in particular, immune-check-
point inhibitors targeting CTLA4, PD-1 or PD-L1 (refs. 133–136). Moreover,
microbial intervention through faecal microbial transplantation of
favourable microbiota from healthy donors or responders can improve
the efficacy of immune-checkpoint inhibitors134,137.
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Two main methods have been developed for the profiling of
microbiota: 16S ribosomal RNA-seq (16S rRNA-seq)138 and metagen-
omics shotgun sequencing139,140. 16S rRNA-seq is more cost-effective
for large-scale analyses of microbial taxonomics composition but
does not provide information on viral or eukaryotic components138.
Metagenomics shotgun sequencing comprehensively covers all micro-
organisms present in the sample, increasing the potential to discover
novel species with potential roles in cancer, and provides data at gene-
level resolution for analyses of functional variations, although these
benefits come at the cost of computational challenges. Microbiom-
ics profiling of faecal samples from patients receiving CAR T cells
has revealed correlations between gut microbiota composition and
response to therapy141, suggesting promising opportunities to exploit
links with the microbiota to improve CAR T cell therapy. The current
studies have mainly focused on the effects of the gut microbiota, yet
the intratumour microbiota might also have an important influence
on the antitumour immune response132,142, highlighting an additional
dimension for future exploration.
Incorporation of spatial contexture into omics profiling
Advances in spatial molecular profiling methods, especially spatial
transcriptomics and proteomics, have enabled the large-scale and high-
resolution characterization of localized variations in gene and protein
expression using tissue sections143. Spatial transcriptomics is an emerg-
ing technology that enables high-plex or transcriptome-wide quantifi-
cation of RNAs at near-cellular144 or subcellular145 resolution that, when
integrated with non-spatially resolved single-cell transcriptomics, might
reinforce analyses of cell–cell interactions through the precise location
of cells within the tissue architecture146. Similar approaches are being
developed for spatial epigenomics profiling, such as spatial ATAC-seq
and spatial cleavage under targets and tagmentation (CUT&Tag-seq),
which has proven utility in characterizing epigenetic regulation in
tissues at high resolution147,148. Established technologies for spatial
proteomics, such as imaging mass cytometry, enable the simultaneous
quantification and localization of ~40 proteins on a tissue slide, with
preservation of the tissue architecture149, to deconstruct the cellular
composition of the TME and explore the cytokine milieu at subcellular
resolution across various cancer types150–153. Furthermore, to explore
the spatial organization of T cell metabolic programmes in the TME,
a study has utilized CyTOF to quantify the expression of proteins
involved in the metabolic regulome in an individual cell and coupled
CyTOF to multiplexed ion beam imaging by time of flight (MIBI-TOF) to
characterize the distribution and expression of these proteins in tissue154.
Spatial fluxomics has also been developed for dynamic metabolic profil-
ing for cancer research155. These approaches have not yet been applied
to CAR T cell research, but the incorporation of spatial contexture into
omics profiling holds great promise to increase our understanding of
how cell–cell interactions and cellular neighbourhoods influence crucial
cellular programmes related to response and toxicity.
Challenges in multi-omics data processing
Despite the broad applications and remarkable progress of multi-omics
profiling in the realm of CAR T cell therapy, notable challenges remain
with regard to data analysis and interpretation. First, multi-omics
technologies using bulk tissues have broad utility in large-scale data
generation, but the potential masking of positive signals and averag-
ing of signals in the context of mixed cell populations might reduce
the potential for novel discoveries and the reproducibility of results.
This concern could potentially be addressed using newly developed
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deconvolution algorithms, provided that the underlying reference
signatures accurately reflect the transcriptional profiles of each cell
type and pathological states within the relevant microenvironment156.
Moreover, the revolutionary applications of high-resolution single-cell
and/or spatial multi-omics profiling technologies have provided a
variety of rich data on CAR T cell therapy, although these bring a level
of computational complexity that requires the development and
optimization of innovative bioinformatic tools.
Second, appropriately adapting analytical methods to new appli-
cations is a challenging yet crucial step towards achieving robust and
reproducible results for biological interpretation; careful evaluation
and correction for batch effects caused by differences in sample-
processing protocols and data-generating platforms is required for
most types of omics data157. Additionally, for single-cell and spatial
omics data, sparsity of data and background noise are common
challenges that necessitate the use of appropriate computational
algorithms for signal detection143,158.
Third, the kinetics of molecular fluctuations in cells might require
further omics studies with a temporally resolved design159. Indeed,
several studies have demonstrated the importance of monitoring
multi-omics variations at different stages of CAR T cell therapy to better
profile treatment-related biological changes96,97,160.
Finally, with the accumulating volume of multi-omics data, opti-
mized computational approaches to integrate data from multiple
omics modalities are desperately needed. Pioneering studies have
demonstrated the ability of a multi-omics machine learning model
to predict treatment response161, suggesting the promise of artificial
intelligence algorithms for data integration in future studies; however,
challenges relating to the complicated analytical procedures and to the
biological translation of results remain to be addressed162–164.
Target discovery for CAR T cell therapy
Identifying CAR targets with high levels of tumour specificity and cover-
age is crucial to ensure the antitumour efficacy and reduce the toxicity
of CAR T cell therapy16,165,166. In general, the ideal CAR target should be
specifically and highly expressed on the surfaces of all tumour cells but
not on essential non-malignant cells. However, owing to shared antigen
expression between tumour cells and non-malignant cells as well as
highly heterogeneous expression among tumour cells, the identifica-
tion of suitable CAR T cell targets has been challenging, particularly
for solid tumours13. In this context, the utilization and integration of
multidimensional omics data is a promising approach to identifying
optimal CAR targets.
Discovery of single CAR targets
Transcriptomics and/or proteomics data from analyses of both malig-
nant and non-malignant tissues have been widely applied to discover
CAR T cell targets that are overexpressed in tumours (Fig. 2). In a study
using a novel RNA-seq-based pipeline, glypican 2 was identified as a tar-
get selectively expressed at high levels on the surface of neuroblastoma
cells without being appreciably detected in non-malignant tissues167.
An analysis of data from non-malignant tissues included in the Human
Protein Atlas and the Genotype-Tissue Expression (GTEx) project and
from tumour samples included in the Human Protein Atlas pathology
database and the database for Differentially Expressed Proteins in
Cancer, encompassing 78 different tissues, 124 cell types and 20 can-
cer types, revealed the expression landscape of >100 candidate CAR
targets38. In another study, genomics, transcriptomics and immun-
opeptidomics data were integrated to identify MHC-binding peptides
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Inactive
Active CAR T cell
CAR T cell
Genomics
Antigen Antigen
positive negative
Non-malignant
Tumour tissue tissue
Transcriptomics
Overexpressed antigen
Proteomics
Non-malignant
tissues
Tumour tissue
A B A B
"AND" logic gating
Fig. 2 | Applications of multi-omics data in CAR target identification.
Comparative analyses of genomics, transcriptomics and/or proteomics data
from tumour and non-malignant tissue samples can facilitate the discovery
of novel targets and optimization of targeting strategies for chimeric antigen
receptor (CAR) T cell therapies. The top panels illustrate CAR T cell targeting
derived from intracellular oncoproteins highly expressed in neuroblas-
tomas; a non-mutated peptide (QYNPIRTTF) encoded by PHOX2B and
presented by HLA-A*24:02 was selected as a lead target for the genera-
tion of highly specific peptide-centric CARs, which induced complete
tumour regression in xenograft models168. Thus, bulk transcriptomics
and/or proteomics profiling has provided large and comprehensive
public resources supporting genome-wide screening for CAR targets
across diverse tissue, cell and cancer types. However, averaging gene or
protein expression data derived from bulk tissues might mask signals
from rare cell populations, with potentially important implications for
the efficacy and toxicity of CAR T cell therapy.
Single-cell technologies, such as scRNA-seq, enable omics profil-
ing at unprecedented resolution for CAR target discovery, including
the identification of potential safety signals. For example, an analysis
of three independent large-scale scRNA-seq datasets derived from
human brain tissue samples identified a rare population of mural cells
expressing CD19, which might contribute to on-target, off-tumour
neurotoxicity from CD19-directed CAR T cell therapy89. Similarly, an
analysis of two extensive scRNA-seq datasets to predict the on-target,
off-tumour toxicity landscape of 591 different CAR targets across a wide
range of tissue and cell types identified many as ‘potentially risky genes’
(including EGFR)90; a data portal, CARTSC, was also made available as a
resource providing data on CAR target expression at the single-cell level.
Nature Reviews Clinical Oncology | Volume 20 | April 2023 | 211–228
Mutant Normal
epitope peptide
Single targets
(neoepitope) epitope
Mutant antigen Wild-type
(neoantigen) antigen
Cell-surface antigen MHC-presented antigen
Neoantigen
Combinatorial targets
A B A B
"AND-NOT" logic gating
approaches predicated on single targets, including antigens overexpressed
on tumour cells and tumour cell-specific neoantigens or MHC-presented
neoepitopes; the bottom panels depict strategies based on combinatorial CAR
targets using “AND” or “AND-NOT” logic gates.
Neoantigens are novel tumour-specific proteins, generated
mainly by somatic mutations in the tumour cells, which might consti-
tute ideal targets for immunotherapy39,40. To screen for neoantigens,
comparative whole-exome or whole-genome sequencing of matched
tumour and non-malignant samples is usually performed to iden-
tify tumour-specific non-synonymous somatic mutations, followed by
RNA-seq and mass spectrometry to confirm expression of the mutated
mRNAs and peptides169. Several neoantigens have already been adopted
as promising CAR targets for the treatment of haematological or solid
malignancies41,43. For example, EGFR variant III (EGFRvIII) is a neoanti-
gen specifically expressed in certain solid tumours, including a subset
of glioblastomas, leading to ongoing clinical trials of EGFRvIII-directed
CAR T cells (such as NCT03726515, NCT03283631 and NCT03638206).
To date, high-throughput multidimensional omics approaches have
enabled the identification of a large number of neoantigens169–172,
providing a rich source of tumour-specific targets that could be further
developed for CAR T cell therapy.
Identification of combinatorial CAR targets
Adequately discriminating between tumour and non-malignant cells
using a single CAR target has proved challenging owing not only
to on-target, off-tumour toxicities but also to antigen escape that
results in therapy resistance38,100,104,173. This issue can potentially be
217
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overcome by combining multiple CAR targets using Boolean “AND”
and/or “AND-NOT” logic gating to increase tumour-targeting speci-
ficity and thus reduce toxicity174–178 (Fig. 2). In brief, CAR T cells with
“AND” logic gates can be triggered only if both target antigens are simul-
taneously expressed on the tumour cells, whereas CAR T cells with
“AND-NOT” logic gates can be activated only in the presence of a target
antigen expressed on tumour cells and the absence of an antigen nor-
mally expressed on non-malignant cells. Multi-omics data integration
has demonstrated the potential to improve CAR T cell design through
the identification of combinatorial CAR targets based on expression
profiles38,100,104,173. For example, an integrated analysis of extensive tran-
scriptomics and proteomics datasets derived from non-malignant and
malignant tissues was performed to propose optimal combinatorial
CAR targets in acute myeloid leukaemia, with four possible “AND” pairs
fulfilling the stringent efficacy and safety criteria104. A comparative
transcriptomics analysis of 3,567 genes encoding surface proteins,
based on mRNA expression data derived from tumours included in The
Cancer Genome Atlas (TCGA) and non-malignant tissues included in
GTEx, identified 179 “AND” and 443 “AND-NOT” pairs of CAR targets100.
Other studies have similarly applied multi-omics data to discover dual
targets for “AND” or “AND-NOT” logic-gate CARs38,173. This strategy can
also be applied to identify more complicated combinations of CAR tar-
gets such as triple targets. For example, large-scale mRNA expression
data on 33 tumour types from TCGA and 34 non-malignant tissues from
GTEx have been leveraged in a comprehensive computational screen of
2,358 predicted surface genes to investigate potential combinations
of two or three target antigens for “AND” or “AND-NOT” logic-gated
CARs173. This study demonstrated the better discriminatory power of
combinatorial CAR targets compared with single targets173; data on
the performance of all predicted antigen pairs are available through the
Antigen Explorer portal. Although Boolean logic-gated CARs have not
yet been used within the clinical setting, other combinatorial antigen-
targeting strategies that mitigate the potential for antigen escape,
for example, those predicated on the expression of CLL1 and CD33 in
acute myeloid leukaemia179,180, CD19 and CD22 in B-ALL and LBCL181,
and BCMA and GPRC5D in multiple myeloma182, have shown promise in
enhancing the antitumour activity of CAR T cells in preclinical and/or
clinical studies.
Single-cell expression data also provide high resolution for pre-
dicting the efficacy and safety of CAR T cells with combinatorial targets.
For example, an analysis90 using scRNA-seq data has predicted potential
toxicity risk for a predefined list of CAR target pairs identified based on
bulk expression data38. Theoretically, full utilization of comprehensive
single-cell multi-omics data from both tumour and non-malignant tis-
sues would be required to nominate optimal target antigens for CAR
T cell therapies and thus limit the risk of toxicities. Several scRNA-seq
data resources provide a human non-malignant cell atlas encompass-
ing multiple organs and cell types183–188, and large-scale, pan-cancer,
single-cell atlases are in development such as those being mapped by
the Human Tumour Atlas Network189. However, single-cell technolo-
gies also have limitations, including the large fraction of zero values
of scRNA-seq measurements, which could reflect either transcript
drop-out or a true absence of gene expression. At present, the discovery
of optimal CAR T cell targets with both high efficacy and safety might
best and most feasibly be accomplished by first nominating promis-
ing targets that are highly and ubiquitously expressed in tumour cells
based on bulk omics data, then using single-cell omics data to predict
the risk of on-target, off-tumour toxicities for the nominated targets.
Notably, identification of CAR targets based on RNA-seq data is not
Nature Reviews Clinical Oncology | Volume 20 | April 2023 | 211–228
straightforward, requiring integration with data on the human sur-
faceome190 and validation of target expression through proteomics
techniques (such as flow cytometry). Furthermore, expression of the
target protein on the cell surface does not necessarily correlate with
clinical responses, for example, owing to highly heterogeneous antigen
expression in tumour cells191, warranting careful evaluation of CAR
T cell products with novel targets in rigorous preclinical studies and
early phase clinical trials.
Understanding and enhancing CAR T cell efficacy
and persistence
A comprehensive understanding of the biological factors that affect
the antitumour efficacy of CAR T cells is crucial to the development of
strategies to achieve robust and durable therapeutic responses and
maximize the clinical benefit. The feasibility of integrating bulk and/or
single-cell multidimensional omics data to study the key features
related to treatment efficacy has been clearly demonstrated. The main
determinants of the efficacy and persistence of CAR T cells can be clas-
sified into four broad categories: T cell states and phenotypes, tumour
cell characteristics, the TME and the microbiota (Fig. 3 and Table 2).
T cell states and phenotypes
Pre-manufacture T cell features. The heterogeneity of source T cells
has a crucial role in determining the subsequent in vivo persistence of
the autologous CAR product28,30,192 (Fig. 3a). For example, an integrative
bulk and single-cell profiling study of pre-manufacture T cell popula-
tions revealed that a higher proportion of naive and early memory
T cells, especially in the CD8+ compartment, is associated with longer
CAR T cell persistence and greater efficacy in patients with B cell malig-
nancies28. Moreover, TCF7 (TCF1) network activity in effector T cells
was found to correlate with long-term CAR T cell persistence, whereas
upregulation of genes associated with the type I interferon response,
such as RSAD2, IRF7, MX1, ISG15, OASL and IFIT3, was associated with
poor CAR T cell persistence.
CAR T cell features. The composition of the CAR T cell products
infused into patients is also highly heterogeneous, and specific CAR
T cell populations have been associated with favourable or unfavour-
able responses15,30,97,117,193,194 (Fig. 3a). For example, bulk transcriptomics
profiling of CAR T cells before infusion into 41 patients with advanced-
stage chronic lymphocytic leukaemia (CLL) revealed that patients with
a subsequent complete response (CR) had CAR T cells enriched for early
memory programmes and expression of IL-6–STAT3 pathway genes30.
By contrast, CAR T cells of patients who had only a partial response or
of non-responders were enriched for the expression of genes associ-
ated with T cell differentiation and exhaustion, aerobic glycolysis and
apoptosis30. Features of the CD8+ CAR T cell subset are crucial deter-
minants of therapeutic efficacy, especially at the initial stage of the
antitumour response. In a single-cell multidimensional analysis of CAR
T cell infusion products received by patients with relapsed and/or
refractory LBCL, enrichment for memory CD8+ CAR T cells was associ-
ated with a CR at 3 months, whereas CAR T cell products enriched for
exhausted CD8+ T cells and with low levels of TCR clonotypic diversity
were associated with a partial response or progressive disease15.
The composition of CAR T cells also affects therapeutic persistence
and/or long-term disease remission. In a study involving 12 patients with
B-ALL, combined scRNA-seq and cellular indexing of transcriptomes
and epitopes by sequencing (CITE-seq) of the CD19-directed CAR T cell
infusion products indicated that deficiency of T helper 2 cell function
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a T cell states and phenotypes b Tumour cell characteristics

Pre-manufacture T cells
Antigen dependent
Memory Naive
Gene
Heterogeneity expresssion
Target
TF gene Gene Gene Gene
mutation splicing
Promoter CD19

Composition (for
example, memory or
exhaustion states)
Epitope masking

Expression of Antigen loss

Gene CAR T cells
specific genes Gene

Epigenetic Antigen independent

signature
Mutations and/or
Fas copy-number variations
Metabolic
↑ OXPHOS
state
FADD FADD

CAR integration CAR Caspase 8 Caspase 8

site Genomic alteration
(for example, of
Caspase 8
TP53 or FAS)
Different CAR e Proteome Active
intracellular m
lo Epigenome Cell death/apoptosis-
signalling
bo

related pathway
Genome
ta

(for example, Fas expression)

Me

ire
Different rto
CAR T cell pe ns cript
om
ra e
re

products
TCR

c Tumour microenvironment d Microbiota

Increase CAR T cell infiltration

e
m
e
lo
m
bo

a
io

et
ob

M r
ic
M

Transduction of Inhibition of
specific gene specific protein Diversity and composition

Gut
Strengthen CAR T cell against immunosuppression microbiome
Myeloid
Treg cell
cell Butyrate Pentanoate

+
Immune-
checkpoint Combination Co-engineering Microbial metabolites
inhibitors Tumour microbiome

Fig. 3 | Multidimensional omics characterization of molecular features associated
with the efficacy of CAR T cell therapy. a, T cell states and phenotypes, including
various features of both pre-manufacture T cells and chimeric antigen receptor (CAR)
T cell products, can be evaluated across multiple dimensions using omics technologies.
b, Multidimensional omics data can also provide insights on tumour cell characteristics
associated with response or resistance to CAR T cell therapy, classified into antigen-
dependent and antigen-independent factors. c, Transcriptomics and proteomics
data can reveal crucial features of the hostile tumour microenvironment that can
be leveraged to enhance CAR T cell efficacy, for example, through interventions to
increase CAR T cell infiltration and/or resistance to immunosuppression. d, Micro­
biomics and metabolomics data can be used to identify potential associations between
CAR T cell efficacy and the gut microbiota and/or microbial metabolites. FADD,
Fas-as­so­ciated death domain protein; OXPHOS, oxidative phosphorylation;
TCR, T cell receptor; TF, transcription factor; Treg, regulatory T.

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Review article

Table 2 | Examples of cellular features associated with CAR T cell efficacy identified using multi-omics data

Characteristic CAR T cell Cancer types Data types Key findings Ref.
targets

Pre-manufacture T cells
Heterogeneity CD19 B-ALL and Hodgkin RNA-seq, scRNA- A higher proportion of naive and early memory T cells was 28

lymphoma in patients seq, CITE-seq and correlated with longer CAR T cell persistence and greater
scATAC-seq efficacy
CAR T cells
Composition CD19 LBCL in patients scRNA-seq, scTCR-seq Memory signature of CD8+ T cells in the infusion product was 15

and DNA-seq enriched in patients with a complete response; patients with a

partial response or progressive disease had CD8+ T cells enriched
for an exhaustion signature and with low TCR clonotypic diversity
CD19 CLL in patients Mass cytometry, CITE- CD8+ CAR T cells were prominent at the initial time point of 97

seq, TCR-seq, scRNA- therapeutic response; cytotoxic characteristics of CD4+ CAR

seq and scTCR-seq T cells were key determinants of long-term remission and
CAR T cell persistence
Expression of CD19 Melanoma, thymoma and ATAC-seq, RNA-seq and Knocking out the three NR4A family transcription factors in CAR 29

specific genes colon adenocarcinoma in scRNA-seq T cells enhanced their antitumour activity
mouse models
Epigenetic CD19 B-ALL and NHL in DNA methylation A specific DNA methylation signature (EPICART) was associated 33

signature patients microarray with favourable clinical outcomes

Metabolic state CD22, BCMA Leukaemia, myeloma and CRISPR gain-of-function CAR T cells overexpressing PRODH2 had enhanced metabolic 34

or HER2 breast cancer in mouse screen, RNA-seq and and immune function as well as antitumour activity
models mass cytometry
CAR integration CD19 CLL in patients RNA-seq, DNA-seq and Integration of CAR sequence into the genome directly disrupted 62

site ATAC-seq the expression of TET2, which led to an enhanced antitumour

response
Intracellular CD19 or B cell lymphoma and Mass spectrometry T cells expressing a CD28–CD3ζ CAR including an additional 109

signalling mesothelin pancreatic cancer in CD3ε signalling module had reduced cytokine release and
domain mouse models enhanced antitumour activity and persistence
Tumour cells
Antigen- CD19 B-ALL in patients DNA-seq and RNA-seq CD19 mutations or mis-splicing result in loss of the CAR target 81

dependent antigen from the cell surface and disease relapse

resistance
CD19 B-ALL in patients RNA-seq and ChIP-seq Lineage switching of B-ALL to a CD19-negative myeloid 205

phenotype resulted in disease relapse

Antigen- CD19 B-ALL in patients Genome-wide loss- Knocking out genes involved in pro-apoptotic death receptor 55

independent of-function screen, signalling in tumour cells reduced their susceptibility to CAR
resistance ATAC-seq, RNA-seq and T cell-mediated cytotoxicity
scRNA-seq
CD19 LBCL in patients DNA-seq and RNA-seq TP53 alterations (mutations and/or copy-number loss) in tumour 44

cells were associated with poor complete response rates and

unfavourable overall survival
Tumour microenvironment
To increase CAR EGFRvIII Glioblastoma in mouse RNA-seq Inhibition of PAK4 improved T cell infiltration and sensitized 35

T cell infiltration models glioblastomas to CAR T cell therapy

To strengthen CD19 and B-ALL and ovarian cancer Mass cytometry IL-18-secreting CAR T cells could activate endogenous immune 222

CAR T cells MUC16 in mouse models cells and modulate the tumour microenvironment, thereby
against immune promoting the antitumour immune response
suppression
Microbiota
Diversity and CD19 B-ALL and NHL in 16S rRNA-seq and Baseline α-diversity was lower in CAR T cell recipients than in 141

composition patients metagenomics shotgun individuals without cancer; the abundance of specific bacterial
sequencing taxa of Lachnospiraceae, Ruminococcaceae and Bacteroidaceae
was higher in patients who had a complete response
Microbial ROR1 Pancreatic cancer in Mass spectrometry The microbial metabolites pentanoate and butyrate could 223

metabolites mouse models enhance the antitumour activity of CAR T cells

16S rRNA-seq, 16S ribosomal RNA sequencing; ATAC-seq, assay for transposase-accessible chromatin with high-throughput sequencing; B-ALL, B cell acute lymphocytic leukaemia;
CAR, chimeric antigen receptor; ChIP-sequencing, chromatin immunoprecipitation sequencing; CITE-seq, cellular indexing of transcriptomes and epitopes by sequencing; CLL, chronic
lymphocytic leukaemia; DNA-seq, DNA sequencing; EGFRvIII, EGFR variant III; LBCL, large B cell lymphoma; NHL, non-Hodgkin lymphoma; RNA-seq, RNA sequencing; scATAC-seq, single-cell
ATAC-seq; scRNA-seq, single-cell RNA-seq; scTCR-seq, single-cell T cell receptor sequencing; TCR, T cell receptor; TCR-seq, TCR sequencing.

Nature Reviews Clinical Oncology | Volume 20 | April 2023 | 211–228 220

Review article
and enrichment for effector-related phenotypes is predictive of CD19+
disease relapse194. Furthermore, data suggest that certain CD4+ CAR
T cell states are a predominant requirement for long-term remission
following CD19-directed CAR T cell therapy. In a longitudinal study,
both bulk and single-cell multidimensional omics data were used to
characterize the functional and molecular features of CD19-directed
CAR T cells in two patients with CLL who had durable CRs lasting more
than a decade; persistent CD4+ CAR T cell clones with cytotoxic, prolif-
erating and metabolically active phenotypes were identified as a key
determinant of long-term CAR T persistence97.
CAR T cell-intrinsic features, including expression of specific
genes29,32,195,196 (such as key transcription factors), epigenetic signa-
tures33 and metabolic states34, are associated with therapeutic efficacy
(Fig. 3a). For example, single-cell transcriptomics and chromatin acces-
sibility profiling data implicate the transcription factors NR4A1, NR4A2
and NR4A3 in CAR T cell dysfunction29. Moreover, ATAC-seq of Nr4a
triple-knockout CAR T cells showed enrichment of accessible chro-
matin regions containing binding motifs for the transcription factors
NF-κB and AP1 (ref. 29). Data from a multidimensional transcriptomics,
epigenomics and single-cell proteomics study indicate that the tran-
scription factor BATF can prevent CAR T cell exhaustion and facilitates
the transition of phenotypes and transcriptional profiles towards an
effector-like state196. In an alternative approach to gene expression
analysis, a predictive DNA methylation signature termed EPICART has
been generated using DNA methylation array data from 114 patients
with B cell malignancies treated with CD19-directed CAR T cells33.
Pre-infusion CAR T cells positive for this signature were associated with
better clinical outcomes in both the initial discovery cohort (n = 77)
and an independent validation cohort (n = 35) and were enriched for
naive-like or early memory T cell phenotypes33.
The metabolic state of the CAR T cells might influence antitumour
activity and toxicity (Fig. 3a). A guide RNA-based CRISPR gain-of-
function screening platform has been developed and applied to primary
CD8+ T cells, resulting in the identification of upregulation of PRODH2,
which encodes hydroxyproline dehydrogenase (an enzyme involved
in proline metabolism), as a means to augment CAR T cell function34.
Transcriptomics and metabolomics profiling of PRODH2-knock-in
CAR T cells revealed enhanced metabolic and immunological activity,
and these cells had improved antitumour activity in multiple mouse
models34. Furthermore, CAR T cell metabolic activity might lead to
imbalances, such as hypophosphataemia, that contribute to ICANS197.
Integration of the CAR transgene into the genome can affect
key genes, thereby influencing the clinical outcome of CAR T cell
therapy62,198 (Fig. 3a). For example, insertion of a CAR sequence into
T cells from a patient with CLL disrupted the transcription of TET2
(encoding a methylcytosine dioxygenase involved in epigenetic regu-
lation) and resulted in an enhanced antitumour response62. Further
transcriptomics and epigenomics analyses revealed that the patient
had a pre-existing hypomorphic mutation in the second TET2 allele and
that TET2-disrupted CAR T cells had increased expression of effector
molecules, such as perforin and granzyme B, as well as a central mem-
ory state that prevented cells from becoming terminally exhausted.
In a high-throughput sequencing study of CAR vector-integration
sites in 40 patients with CLL or B-ALL, integration sites in patients
who responded to therapy were enriched in genes related to pathways
mediating T cell proliferation, such as phosphatidylinositol, cyclic
AMP or TCR signalling, or covalent chromatin modification, suggest-
ing that insertional mutagenesis in these genes might lead to better
clinical outcomes198.
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The intracellular signalling domain of the CAR construct can also
influence CAR T cell activity (Fig. 3a). In a simultaneous examination
of the dynamics of immunoreceptor tyrosine-based activation motif
(ITAM) phosphorylation within the CD3ε, CD3δ, CD3γ and CD3ζ signal-
ling subunits of the TCR using quantitative mass spectrometry, high
levels of mono-phosphorylation and a basic residue-rich sequence
of CD3ε were found to restrain TCR signalling109. Notably, addition of
the CD3ε-derived ITAM–basic residue-rich sequence region into a
CD19–CD28−CD3ζ CAR construct resulted in a CAR T cell product asso-
ciated with reduced levels of cytokine release, enhanced antitumour
responses and increased persistence in a mouse xenograft model of
B cell lymphoma. Indeed, different CAR T products have different
phenotypes and expansion characteristics. Axicabtagene ciloleucel
(axi-cel) and tisagenlecleucel, for example, differ not only in the source
of their intracellular co-stimulatory domains (CD28 versus 4-1BB) but
also in their hinge–transmembrane domains (CD28 versus CD8α)
and their integration vectors (gammaretrovirus versus lentivirus)199.
In a scRNA-seq analysis of the infusion products and 105 peripheral
blood mononuclear cell samples collected at different pre-treatment
and post-treatment time points from 19 patients receiving axi-cel and
13 receiving tisagenlecleucel, expansion of central memory CD8+ cells
was only associated with responses to tisagenlecleucel; responders to
axi-cel had more heterogeneous T cell populations, while enrichment
of CAR-expressing regulatory T (Treg) cells in the axi-cel infusion prod-
uct were associated with a lack of response24. Together, these findings
underscore the application of multi-omics data in characterizing the
differences between distinct CAR designs and cell products.
Tumour cell characteristics
Antigen-dependent resistance. Tumour cell characteristics, includ-
ing both antigen-dependent and antigen-independent mechanisms,
are crucial drivers of resistance to CAR T cells166,200,201 (Fig. 3b). Antigen
escape in tumours (that is, loss of the target epitope) is the leading
cause of antigen-dependent resistance to CAR T cells and has been
shown to occur via downregulation202, mutation160, alternative splic-
ing81,203,204, lineage switching205,206 or antigen masking207. A DNA-seq
and RNA-seq analysis found that all 12 evaluated patients relapsing
with CD19-negative B-ALL after CD19-directed CAR T cell therapy had
somatic mutations in CD19 exons 2–5, which are postulated to result
in truncation of CD19 and subsequent loss of expression on the cell
surface160. An RNA-seq study demonstrated alternative splicing of
CD19 with exon 2 skipping, leading to a loss of the extracellular CD19
epitope81. By contrast, scRNA-seq in a patient with relapsing B-ALL
revealed antigen loss of CD19 via mis-splicing that resulted in a non-
functional CD19 transcript retaining intron 2, and this subclone was
present before CAR T cell treatment203. A study using flow cytometry
reported switching of B-ALL to a CD19– myeloid lineage in four patients
after CD19-directed CAR T cell therapy205. Combined RNA-seq and
ChIP-seq data from mouse models provided further validation that
the myeloid lineage switch was triggered following CD19-targeted CAR
T cell infusion205. In addition, combined genomics and transcriptomics
sequencing has uncovered a novel but rare mechanism of epitope
masking related to resistance to CAR T cell therapy: the CAR gene was
transduced into a single leukaemic B cell by accident, and expression
of the CD19-directed CAR on the progeny of this leukaemic clone pre-
vented recognition by, and thus conferred resistance to, CAR T cells
via competitive binding to CD19 on leukaemic cells in cis207. These
results demonstrate the complexity of antigen escape mechanisms in
B-ALL. However, despite approximately 28% of LBCLs having reduced
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CD19 expression at relapse after CD19-directed CAR T cell therapy202,
mutation and alternative splicing are not observed and the mechanisms
remain to be defined; thus, different mechanisms of antigen escape are
likely to exist in distinct malignancies. The application of genomics,
transcriptomics and/or proteomics profiling will be important to elu-
cidate the mechanisms of antigen escape across diverse contexts and
to develop strategies to circumvent antigen-dependent resistance, for
example, through the design of dual-targeted CAR T cell products181,182.
Antigen-independent resistance. Antigen-independent resistance
resulting from the intrinsic insensitivity of tumour cells to the mecha-
nisms of CAR T cell-mediated cytotoxicity also contributes substan-
tially to treatment failure (Fig. 3b). Emerging evidence indicates that
dysfunctional death receptor signalling in tumour cells can lead to
resistance to CAR T cell cytotoxicity. In a genome-wide CRISPR–Cas9
knockout screen, disruption of genes involved in pro-apoptotic death
receptor signalling (including FADD, BID, CASP8 and TNFRSF10B) in
B-ALL cell lines conferred resistance to CD19-directed CAR T cells,
which was further reinforced by induction of CAR T cell dysfunction as a
result of antigen persistence55. Conversely, knocking out anti-apoptotic
genes (such as CFLAR, TRAF2 and BIRC2) in leukaemic cells increased
their susceptibility to CAR T cell-mediated cytotoxicity55. Similarly,
another CRISPR–Cas9 screen demonstrated that expression of the
death receptor Fas on tumour cells is essential for antigen-dependent
CAR T cell-mediated killing as well as for bystander T cell-mediated
killing of antigen-negative tumour cells208. Notably, DNA-seq and RNA-
seq data indicate that patients with LBCL harbouring FAS deletions45 or
with low FAS expression208 have inferior survival outcomes following
CD19-directed CAR T cell therapy.
Other genomics alterations within tumour cells have also been
correlated with poor outcomes following CAR T therapy. Integrated
targeted DNA-seq of pre-treatment tumour samples from 82 patients
with relapsed and/or refractory LBCL receiving CD19-directed CAR
T cell therapy demonstrated that TP53 aberrations (mutations and/or
copy-number losses) are associated with reduced overall survival but
not with progression-free survival44. Bulk transcriptomics data from
a separate cohort of 562 patients further suggested that TP53 altera-
tions in tumours impair processes such as interferon signalling, death
receptor signalling and CD8+ T cell infiltration, which are important
for CAR T cell-mediated cytotoxicity44. TP53 alterations are also linked
with increased genomics complexity in a variety of contexts, including
LBCL209, and high levels of genomics complexity have been associated
with poor outcomes in patients with LBCL receiving CD19-directed CAR
T cells45,46. Taken together, these studies emphasize that integration of
multidimensional omics data is crucial to reveal the tumour-intrinsic
mechanisms of resistance to CAR T cell therapy.
The TME
The TME typically contains diverse immunosuppressive cells, including
Treg cells, cancer-associated fibroblasts, tumour-associated macro­
phages and myeloid-derived suppressor cells, and cytokines (such
as TGFβ, IL-10, IL-4 and VEGF) that might limit CAR T cell infiltration,
proliferation and effector function, and ultimately lead to CAR T cell
exhaustion16,210–212. The challenges to CAR T cell therapy posed by
the immunosuppressive TME and the engineering strategies aiming
to overcome them have been extensively reviewed elsewhere166,213.
Interrogation of the TME in patients treated with CAR T cells has been
limited to targeted gene expression assays25,210 and has not yet
been comprehensively explored with omics approaches, although the
Nature Reviews Clinical Oncology | Volume 20 | April 2023 | 211–228
findings suggest that intratumoural myeloid cells and cytokine levels
are associated with response to CD19-directed CAR T cells. Here, we
focus on the application of multidimensional omics data to obtain an
in-depth understanding of the hostile TME in order to facilitate the
development of new approaches to enhance the intratumoural abun-
dance, persistence and activity of CAR T cells and thereby improve
their clinical efficacy (Fig. 3c).
Increasing CAR T cell infiltration. Omics data have been utilized to
identify key regulators that could be leveraged to increase CAR T cell
infiltration into the TME. In a mouse model of breast cancer, the stimu-
lator of interferon genes (STING) agonist DMXAA greatly enhanced
CAR T cell recruitment to and persistence in the TME, with scRNA-seq
revealing favourable shifts in the chemokine milieu and the balance
of immunostimulatory versus immunosuppressive myeloid cells214.
In a kinome-wide screening study, RNA-seq revealed that PAK4 has an
important role in regulating mesenchymal-like transcription activa-
tion in glioblastoma-derived endothelial cells; inhibition of this kinase
improved T cell infiltration and sensitized glioblastoma tumours to
EGFRvIII-directed CAR T cells in a mouse model35.
Strengthening CAR T cells against immunosuppression. Multi-
omics analyses also present a powerful approach for identifying key
immunosuppressive factors in the TME, thereby enabling the engi-
neering of CAR T cells capable of resisting such factors1. A single-cell
transcriptomics analysis of oesophageal squamous cell carcinomas
indicated that PD-L1 is upregulated on tolerogenic dendritic cells in the
TME215, and data from a pan-cancer scRNA-seq atlas of T cells suggested
that binding of PD-L1 to PD-1 (ref. 216) inhibits T cell cytotoxicity and
facilitates immune evasion, underscoring the known role of the PD-L1–
PD-1 axis as a key mediator of immunosuppression in the TME. There-
fore, the efficacy of CAR T cells against poorly responding tumours
might be enhanced by combining CAR T cells with anti-PD-(L)1 anti-
bodies217, engineering PD-1-blocking CAR T cells218 or CAR T cells that
express PD-1 dominant-negative receptors219, or genetically disrupting
PD-1 expression in CAR T cells220.
A pan-cancer analysis based on RNA-seq data from TCGA and GTEx
revealed that FASLG (encoding Fas ligand; FasL) is often overexpressed
in the TME221. Accordingly, CAR T cells co-engineered to express dom-
inant-negative variants of Fas (the receptor of FasL that is typically
highly expressed on autologous T cells used for adoptive cell therapy)
had increased persistence and antitumour efficacy in mouse models of
various solid and haematological malignancies221. In addition, a CyTOF
analysis demonstrated that IL-18-secreting CAR T cells can modulate
the TME and enhance the activity of endogenous immune cells (for
example, promoting endogenous CD8+ T cells with a central memory
phenotype, macrophages with an M1 phenotype, and dendritic cells
with a more mature and activated phenotype) in diverse mouse models,
thereby promoting antitumour immune responses222. Taken together,
these studies demonstrate that leveraging multidimensional omics
data can greatly help to overcome factors in the TME that limit the
effectiveness of CAR T cell therapy for solid tumours.
The microbiota
The microbiome is emerging as a crucial factor affecting the efficacy
of CAR T cell therapy (Fig. 3d). In a multicentre study involving a pro-
spective cohort of 48 patients with B cell lymphomas or leukaemias
receiving CD19-directed CAR T cell therapy, 16S rRNA-seq and metagen-
omics shotgun sequencing of the baseline faecal samples revealed a
222
Review article
considerably lower level of α-diversity and a substantially different com-
position of the gut microbiota compared with that of individuals with-
out cancer141. Furthermore, specific bacterial taxa of Lachnospiraceae,
Ruminococcaceae and Bacteroidaceae were in greater abundance in
patients who had a CR at day 100 than those who did not. Preclinical
data from mouse models of melanoma and pancreatic cancer indi-
cate that two microbial metabolites identified using metabolomics
data from the human commensal bacteria Megasphaera massiliensis,
namely pentanoate and butyrate, can increase the antitumour activity
of CAR T cells through metabolic and epigenetic reprogramming223.
Prior knowledge that not only the intestinal microbiota but also the
intratumour microbiota can have multiple effects on human cancers
along with the development of technologies that enable quantitative
analyses of the microbiome132,142 will facilitate further investigations
to characterize the influence of the microbiota on the efficacy of CAR
T cell therapy.
Minimizing CAR T cell-related toxicities
The toxicities of CAR T cell therapies, which can be serious or even
fatal18, remain a substantial challenge to widespread clinical applica-
tion224–226. In the process of killing antigen-expressing target cells, CAR
T cells induce a series of inflammatory responses via the secretion
of various cytokines and chemokines — a double-edged sword that
amplifies antitumour immune responses but can also cause severe
toxicities16. The most common CAR T cell-induced adverse events
include CRS, ICANS and prolonged cytopenias227. Several studies have
b ICANS
Blood vessel Mural cell
CAR
T cell
a CRS
CAR T cell Tumour cell
IL-1, Cytokines
IL-6
e
om
Prote
Monocytes/
myeloid cells
Fig. 4 | Summary of factors related to major categories of CAR T cell-induced
toxicities revealed by multi-omics data. a, Factors involved in chimeric
antigen receptor (CAR) T cell-associated cytokine-release syndrome (CRS) that
have been characterized through transcriptomics and proteomics. b, Factors
related to the risk and/or mechanism of immune effector cell-associated
Nature Reviews Clinical Oncology | Volume 20 | April 2023 | 211–228
demonstrated the potential of multi-omics profiling to elucidate the
mechanisms underlying these toxicities and thus inform potential
prophylactic and therapeutic strategies.
CRS
CRS results from robust cytokine production by CAR T cells follow-
ing engagement with target cells as well as by subsequently activated
endogenous immune cells, which creates a loop of immune activation
that can cause a ‘cytokine storm’226,228. Characterization of cytokine lev-
els and immune cell interactions in the context of CRS has been crucial
to understanding its aetiology (Fig. 4a). Transcriptomics approaches
have been used to determine the key immune cell types and/or cellular
interactions that mediate this excessive cytokine release. In a mouse
model of CAR T cell-induced CRS, bulk RNA-seq of myeloid cells iso-
lated by fluorescence-activated cell sorting revealed that increased
expression of IL-1 receptors and IL-6 by these cells, and particularly by
macrophages, is associated with greater severity of CRS37. Moreover,
a scRNA-seq analysis of CD45+ leukocytes from a humanized mouse
model of high-burden leukaemia implicated circulating human mono-
cytes as the primary source of IL-6 and IL-1 released following CAR
T cell infusion, with a time-course analysis indicating that production
of IL-1 preceded IL-6 during the development of CRS36. Furthermore, a
cytokine signature generated through proteomics profiling of serum
samples from 51 patients receiving CD19-directed CAR T cell therapy
accurately predicted the likelihood of severe CRS, potentially enabling
early intervention229.
Microbiome Monocyte-like
composition CAR T cells Density of Treg cells
c On-target, off-tumour
toxicities
Non-malignant
cell
Microbiome
nscriptome
Tra
Pr
ote
CAR T cell
ome
Tumour
cell
CAR T cell
therapy-
related
toxicities
neurotoxicity syndrome (ICANS) have been identified using transcriptomics and
microbiomics data. c, On-target, off-tumour effects of CAR T cell products can
also be characterized, and potentially predicted, through transcriptomics and
proteomics analyses. Treg, regulatory T.
223
Review article
ICANS
ICANS is another major toxicity of CD19-directed CAR T cell ther-
apy19,227,230. Targeted gene expression analysis has identified an asso-
ciation between a high pre-treatment intratumoural density of Treg cells
and reduced risk of ICANS in patients with LBCL25, and targeted single-
cell cytokine analysis has shown that higher frequencies of polyfunc-
tional IL-17A-producing T cells within infusion products are associated
with increased risk of high-grade ICANS. Moreover, scRNA-seq of CD19-
directed CAR T cell infusion products for patients with LBCL revealed
a small subset of cells with a monocyte-like transcriptional signature
within the infusion products of patients with high-grade ICANS15. Nota-
bly, these cells express high levels of IL-1β; this finding, together with
the independent aforementioned support for the pathophysiological
role of IL-1 signalling in CRS36,37 and ICANS15,36, has prompted clinical
investigation of an IL-1 receptor antagonist for prophylaxis of CRS and
ICANS (NCT04432506). In addition, scRNA-seq data from human brain
tissue suggest that CD19 expression on brain mural cells might also
be a target for CD19-directed CAR T cells89. Furthermore, 16S rRNA-
seq and metagenomics shotgun sequencing of stool samples from
45 patients with B cell malignancies receiving CD19-directed CAR T cells
demonstrated an association between ICANS and faecal microbiota
composition141. The mechanisms underlying ICANS are multifactorial
and remain poorly understood; however, a clearer understanding might
be obtained by leveraging multi-omics data (Fig. 4b).
On-target, off-tumour toxicities
The targets of most CAR T cell products currently used in the clinic
and/or being tested in clinical trials are expressed to some extent on
non-malignant cells; therefore, CAR T cells can cause damage to non-
malignant tissues and result in on-target, off-tumour toxicities27,38,227,
especially when targeting solid tumours18. Predicting the risk of on-
target, off-tumour toxicities and monitoring patient safety through
multi-omics profiling during treatment could potentially enable more
efficient management of such adverse effects38. By combining bulk
transcriptomics and proteomics data, a targetable landscape encom-
passing >100 single CAR targets and >100,000 logic-gated target pairs
with minimal predicted toxicity has been reported38. Similarly, the
study of two independent large-scale scRNA-seq datasets90 defined
the safety landscape of 591 CAR targets and 320,884 logic-gated “AND”
design target pairs at the single-cell level. Notably, by leveraging scRNA-
seq to dissect rare cell populations and/or types, several potentially
risky targets were identified that might result in toxicities affecting
several important organs, including EGFR, prostate stem cell antigen
and VEGFR2, which were not identified as a safety risk in bulk expres-
sion analyses90. In summary, multi-omics analyses could substantially
broaden molecular insights on toxicities and thus facilitate the devel-
opment of approaches to mitigate the adverse effects of CAR T cell
therapies (Fig. 4c).
Conclusions
CAR T cell therapy has transformed the management of B cell malig-
nancies, yet substantial numbers of patients do not have a clinical
response or have disease relapse owing to poor CAR T cell function
and/or resistance of tumour cells to such therapies. Moreover, the
applicability of CAR T cell therapies remains limited owing to a series of
challenges that include a lack of appropriate CAR targets, the potential
for disease relapse and treatment-associated adverse events. Solu-
tions to these challenges are under exploration and development.
The expanding multi-omics data repertoire could be utilized to obtain
Nature Reviews Clinical Oncology | Volume 20 | April 2023 | 211–228
new molecular insights into CAR T cell therapy. Multi-omics profiling
of tumour and non-malignant tissues provides a feasible strategy for
large-scale screening of CAR targets and logical designs. Meanwhile,
characterization of T cells, tumour cells, the TME and the microbiota
can decipher the molecular features and pathways that determine
therapeutic efficacy as well as the potential underlying mechanisms and
pivotal regulators of different categories of toxicities associated with
CAR T cell therapy. The current FDA-approved CAR T cell products are
manufactured from autologous T cells owing to the advantage of avoid-
ing graft versus host disease, yet such products have disadvantages
related to long manufacturing times and impaired functional fitness of
patient-derived T cells231. Notably, multi-omics studies of donor-derived
allogeneic CAR T cell products and their comparison with autologous
products using transcriptomics, proteomics and metabolomics data
have demonstrated the impaired metabolic fitness of autologous CAR
T cells as well as a higher level of activation in allogeneic CAR T cells,
indicating a greater antitumour potential87,130,232. These studies have
also advanced the mechanistic understanding of allogeneic products
and delineated a promising future for ‘off the shelf’ CAR T cells.
Advances in single-cell technologies have greatly facilitated explo-
ration of the remarkable diversity of cellular phenotypes and states
among both tumour and immune cells in the TME, expanding our col-
lective knowledge in the field of immuno-oncology93,233. The parallel
improvements in analytical algorithms to interpret single-cell multi-
omics data have supported important advances in single-cell tech-
nologies70,113,234–237. Further application of single-cell technologies to
study various aspects of CAR T cell therapy is likely to provide rich and
high-resolution information, offering clinically relevant insights into
treatment strategies to maximize the potential for durable remissions.
Furthermore, spatial profiling technologies enable in situ cell charac-
terization and mapping of spatially preserved tissue sections. Although
not yet utilized to study CAR T cell therapy, spatial transcriptomics
and proteomics have been used to investigate interactions between
tumour cells and infiltrating immune cells in the context of immune-
checkpoint inhibitor therapy in order to explore the clinical relevance
of cell localization238,239. These advances in single-cell and spatial multi-
omics technologies have demonstrated promise in dissecting the
cellular components and their intercellular connections, providing
opportunities to overcome obstacles to effective therapeutic targeting,
including immune evasion and immunosuppression of CAR T cells.
Leveraging valuable multidimensional resources through multi-
omics data integration is also of great relevance in elucidating the evo-
lution and transitions of cells during therapy. For example, combined
RNA-seq, CITE-seq and single-cell ATAC-seq data have been used to
track the molecular determinants of long-term CAR T cell persistence28.
Furthermore, the integration of omics data from multiple studies
enables a substantially larger sample size and might thus lead to novel
discoveries of important biological processes or features formerly
underappreciated in studies with relatively small sample sizes240. To this
end, further optimization of computational frameworks for omics
data integration is required to fully leverage multi-omics data in future
research in the realm of CAR T cell therapy and beyond164,241.
Published online: 31 January 2023
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Acknowledgements
The work of the authors is supported by the US NIH (grants R01HG011633 and R01CA262623
to L.H.). The content is solely the responsibility of the authors and does not necessarily
represent the official views of the NIH. M.R.G. is a Scholar of the Leukaemia and Lymphoma
Society. The authors regret that page limitations have prevented them from including
all the relevant studies in this Review. Draft figures for this manuscript were created with
BioRender.com.
Author contributions
All authors made a substantial contribution to all aspects of the preparation of this manuscript.
Competing interests
M.R.G. has stock ownership interest in KDAc Therapeutics, receives funding from Abbvie,
Allogene, Kite/Gilead and Sanofi, and has received honoraria from Tessa Therapeutics. J.Y.,
Y.C., Y.J. and L.H. declare no competing interests.
Additional information
Correspondence should be addressed to Michael R. Green or Leng Han.
Peer review information Nature Reviews Clinical Oncology thanks M. Maus, who co-reviewed
with N. Haradhvala; R. Fan, who co-reviewed with Z. Bai; and the other, anonymous, reviewer(s)
for their contribution to the peer review of this work.
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Related links
Antigen Explorer: https://antigen.princeton.edu/
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