CONTENTS
5.3 Finding the mode of action of
1. Introduction engineered T cells
1. 1 The three main techniques used in
single-cell transcriptomics
1.2 Immune profiling of T- and B-cell
receptors
1.3 Single-cell data analysis
2. Mechanism of disease
2.1. A novel hypothesis for athero-
sclerosis
2.2 Gaining insight into persistent
EBOOK cancer cells
2.3 Unraveling the tumor
Single-Cell Sequencing
microenvironment
2.4 Key takeaways
3. Biomarker discovery exhaustion
in Drug Development
3.1. Creating single-cell atlases for
biomarker discovery
3.2 Using tumor immune atlases for
precision oncology
3.3 Key takeaways
4. Target identification & verification
4.1. Exploiting single-cell atlases to
identify CAR T-cell antigen targets
4.2 Finding high-grade targets for
cancer therapy
4.3 Utilizing single-cell CRISPR screens
to unravel genetic components of
disease
4.4 Key takeaways
5. Mechanism of action 7.6 Key takeaways
5.1. Unraveling the mechanism of action
in existing diabetes drugs
5.2 Characterizing drug-affected cell
types in precancerous liver
Single Cell Discoveries
Single-Cell Sequencing in Drug Development | 1 2 | | 2
Single-Cell Sequencing in Drug Sequencing
5.4 Key takeaways
6. Lead identification & verification
6.1. Guiding CAR T-cell engineering
with single-cell technology
6.2 Optimizing AAV biodistribution
6.3 Key takeaways
7. Preclinical research
7.1. Unearthing causes of CAR T
neurotoxicity in mice
7.2 Enhancing Treg presistence after
adoptive transfer
7.3 Characerizing causes of T-cell
7.4 Mapping biodistribution of AAV-
basd gene therapy
7.5 Key takeaways
8. Clinical research
7.1. Phase I: Clinical validation of
antitumor activity in CAR NKT-cell
therapy
7.2 Phase I: Proof of vaccine-induced
tumor-infiltrating cells
7.3 Phase II: Clinical validation of
antitumor activity in CAR NKT-cell
therapy
7.4 Phase II: Proof of vaccine-induced
tumor-infiltrating cells
7.5 Phase III: Optimizing hemato-
poietic stem cell transplantation
9. Conclusion
10. About Single Cell Discoveries
Single-Cell
Developmentin Drug Development
1. Introduction With the goal of providing a valuable resource for pharmaceutical researchers,
this eBook focuses on single-cell sequencing applications across the drug
The start of the single-cell sequencing revolution is often placed in 2009 when Tang development pipeline. It outlines where single-cell sequencing can significantly
et al. published the transcriptome of a single mouse blastomere. Until then, acquiring contribute to various stages of drug development, including biomarker discovery,
the transcriptome of blastomeres was impossible because one organism would not target identification & validation, mechanism-of-action studies, lead identification
contain enough of this cell type, and each cell contained too little RNA for successful & optimization, preclinical development, and clinical research. For each phase of
sequencing. the drug development pipeline, this eBook highlights published examples of crucial
discoveries and decisions enabled by single-cell sequencing.
Single-cell sequencing overcame these two challenges with the ability to add unique
barcodes to RNA transcripts in each cell and the ability to sequence these transcripts First, we briefly review the basics relevant to the examples put forward in later
from the tiny amounts of material available from a single cell. With its invention, a chapters.
more accurate study of developmental trajectories of early life was made possible, a
precedent for the many advancements fueled by single-cell sequencing today. 1.1 The three main techniques used in single-cell sequencing
Single-cell sequencing technologies broadly fall into three main categories: plate-
based methods, microwell-based methods, and microfluidics-based methods.
Drug development pipeline Plate-based methods
Plate-based methods were the chronologically first scRNA-seq methods. The protocol
starts with FACS sorting of single cells into individual wells, which undergo library
preparation and sequencing. During library preparation, unique cell-specific barcodes
are added to mRNA molecules and unique molecular identifiers. After sequencing,
these barcodes facilitate mRNA counts per cell, creating a gene expression profile
for each individual cell in the studied sample. Commercially available plate-based
Mechanism Biomarker Target ID & Mechanism Lead ID & Preclinical Clinical options include SORT-seq, VASA-seq (which goes beyond mRNA sequencing to
of Disease Discovery Validation of Action Optimization Research Trials
include noncoding RNA subtypes), SMART-Seq, and Evercode Whole Transcriptome.

For drug developers, single-cell sequencing provides unprecedented accuracy in

identifying and characterizing cells from healthy and diseased tissue before and after
treatment. Early important discoveries included unraveling intratumor heterogeneity
in colorectal cancer and fueling the discovery of Alzheimer’s disease-associated
microglia in the brain.

As the technology matures, becoming more scalable and compatible with additional
sample types like FFPE, the use cases are expanding with increasing applications to
target identification, mechanism-of-action, and lead optimization. Recent years have
also seen translational appliances in preclinical development, and the first Phase I, II,
Figure 1 Plate-based single-cell sequencing method SORT-seq (left) and microfluidics-
and III trials utilizing single-cell sequencing.
based 10x Genomics (right) are both performed at Single Cell Discoveries.

Single-Cell Sequencing in Drug Development | 3 4 ||

4 Single-Cell Sequencing in Drug Development
 Microwell-based methods
Microwell-based methods use chips with micro- or sometimes even pico-sized wells.
The main difference is that cells float to the bottom of the wells by gentle gravity.
Hence, it is suitable for sensitive cells like granulocytes. Commercially available
methods include Hivesource and Gexscope.
Microfluidics-based methods
Microfluidics-based methods differentiate themselves from plate- and microwell-
based methods by using microfluidics technology to isolate cells not in wells but
(water) droplets. This system has allowed for high-throughput methods enabling
sequencing of thousands of cells per sample. In drug development and elsewhere,
it is the most widely used technology, although plate-based methods can achieve
higher sensitivity. The leading technology in this field is 10x Genomics.
1.2 Immune Profiling of T- and B-cell receptors
Immune profiling is a powerful tool in studies on the tumor microenvironment,
immunotherapy, autoimmunity, or infections.
1.3 Single-cell data analysis
Single-cell data analysis requires customization to the specific experimental setup
and to what a researcher wants to conclude from the data. The most used single-cell
data analyses for drug development are differential gene expression analysis, cell
clustering, cell type annotation, and trajectory interference. Here, we explain them
shortly.
Cell clustering and cell type annotation are methods that provide cell type
identification, i.e., identifying which types and subtypes are present in a sample.
Clustering is based on the gene expression profiles of single cells. To further investigate
cell clusters, differential gene expression analysis can be used to characterize how
cells are transcriptionally different from each other. Often, differential gene expression
analysis paves the way for biomarker discovery or the identification of therapeutic
targets.

T and B cell receptors (TCR and BCRs, resp.) contain variable V(D)J regions that
enable great diversity in antigen recognition. Yet, the technologies mentioned above
generally exclude these regions during sequencing for technical reasons. Hence,
studying the immune repertoire
requires additional V(D)J
sequencing (often called TCR-
seq or BCR-seq) at single-cell
resolution. Figure 4 Example cell clustering results (left) and single-cell
gene expression results from real data. Image adapted from
Most common is the 10x Genomics Wehrens et al. (2022)
Single Cell Immune Profiling
solution. This technology enables
combining the transcriptomic Trajectory inference is a method to use gene expression profiles to trace the
data with the immune repertoire. developmental lineages of cells across samples. Here, it is important to sequence
The result is the full-length enough relevant cells to be able to perform the analysis. Common applications
immunoglobin/BCR sequence, include developmental diseases, cancer progression, and regenerative medicine.
including isotypes and α/β TCRs Beyond these key analyses, there are numerous other bioinformatic tools that prove
(sequencing another TCR type, useful to drug developers. A recent review by Van de Sande et al. provides a good
gamma-delta (γδ) TCR is possible summary.
with a custom service provided
by Single Cell Discoveries).
Figure 3 Single Cell Immune Profiling is compatible
with 10x Genomics single-cell technology

Single-Cell Sequencing in Drug Development | 5 6 | Single-Cell Sequencing in Drug Development

 2. Mechanism of disease
A decade of single-cell sequencing has fueled mechanism-of-disease studies by
identifying specific cell types, states, programs, and contexts wherein diseases arise.
Often, diseases are the result of more complex, multicellular changes such as a
remodeling of cellular composition or a cell-state transitions, traits detectable at
single-cell resolution. In this chapter, we detail three examples that showcase single-
cell sequencing’s unique capabilities in propelling complex disease understanding in
atherosclerosis, cancer persistence after treatment, and the tumor microenvironment.
2.1 A novel hypothesis for atherosclerosis
Scientists from Sheffield University applied single-cell sequencing on endothelial
cells in the bends of the aorta to test a new hypothesis for atherosclerosis. Previous
work had identified the JAG1 gene as a potential driver of disease at specific aortal
locations. Working with Jag1 knockout mice, the team mimicked atherosclerotic
conditions and characterized the effects on the endothelial cells with SORT-seq.
The results showed that knocking out Jag1 resulted in a shift in cellular composition,
with a larger cell subpopulation showing transcriptional signs of pathology and a
smaller subpopulation showing a gene expression profile related to vascular repair.
The results suggest that atherosclerosis is caused
by an unbalance in endothelial cell composition,
Find a detailed case study of and JAG1 disruption could be one driver of this
using SORT-seq to uncover phenomenon. This study exemplifies how the
a new mechanism of disease granularity achieved by single-cell sequencing
for atherosclerosis here. and its capacity to generate full gene-expression
profiles of cell subpopulations can propel a novel
understanding of disease.
2.2 Gaining insight into persistent cancer cells
An area of special interest in oncology is the study of persistent cancer cells, so-called
because they persist after treatment. Using single-cell sequencing, researchers from
the Broad Institute discovered that while most persistent cancer cells are arrested
during treatment, a small subpopulation comprising only 8% of all cells re-enters the
cell cycle and can lead to recurrence.
Since bulk RNA sequencing generally cannot reach the resolution to accurately
characterize such small cell numbers, the researchers continued their study with
Single-Cell Sequencing in Drug Development
single-cell analyses such as single-cell trajectory inference. The results showed that
persister cells that re-enter the cell cycle originate from a specific cell lineage with
distinct transcriptional programs, which are associated with a metabolic shift to fatty
acid oxidation and decreased antioxidation.
These programs were detectable in the bulk transcriptomes of varying cancer types
treated with targeted therapies such as osimertinib and lapatinib, establishing the
idea that preventing the induction of such programs in these cells with adjuvant
therapies may delay or even prevent disease recurrence.
In line with this promising research, a consortium of pharma like AstraZeneca,
Merck, and Bayer and institutes, including Single Cell Discoveries, are working on
understanding these persistent cells with single-cell sequencing. More can be read
on the website of the PERSIST-SEQ consortium.
2.3 Unraveling the tumor microenvironment
There has recently been a strong focus in oncology on investigating the tumor
microenvironment for immunotherapy and cell therapy and several studies have
utilized single-cell sequencing to demystify the multiple immune cell types and
various immune cell states in this heterogenous tissue (such as here, here, and here).
For example, researchers of the Fuijan Medical University Union Hospital used Single
Cell Immune Profiling to investigate the tumor microenvironment in pancreatic cancer.
The results indentified distinct immune profiles within compartments of the tumor
microenvironment of the pancreatic
tumor that associated with shorter
and longer survival. One of the highly
expressed proteins associated with
worse survival was CD47.
With this information, the team turned
again to single-cell sequencing to
uncover the mechanism underlying
anti-CD47 effectiveness in mouse
models and demonstrated that it
induced a significant remodeling of
the intratumor macrophages and
T-cell clusters to a more activated Figure 5 Single-cell clustering results show a shift in
state. cellular composition of the tumor microenvironment
after anti-CD47 treatment. From: Pan, Y. et al.
| 7 8 | Single-Cell Sequencing in Drug Development

2.4 Key takeaways
Mechanism-of-disease studies have used single-cell sequencing to:
• Identify and characterize rare cell types related to disease (e.g., persistant
cancer cells)
• Investigate mechanisms of disease in crucial subpopulations (e.g. tumor
microenvironment compartments)
• Detect multicellular changes like tissue remodelling or cell-state transitions
underlying disease (e.g. atherosclerosis)
astrocyte subpopulation appearing at early disease stages and expanding with age.
Importantly, the team of scientists could find this subtype in human postmortem
brain astrocytes. The presence of cells within this subpopulation is a biomarker for
the development of Alzheimer’s disease.
3.2 Tumor immune atlases for precision oncology
In recent years, immune cells of the tumor microenvironment have received special
interest for their potential role as therapeutic targets and diagnostic markers. In
particular, cancer immunotherapies (e.g., checkpoint inhibitors) target tumor-
specific T cells and thus benefit from accurate T-cell characterization.

In fact, one of the most striking insights from single-cell sequencing has been
the extensive heterogeneity discovered in T-cell populations that were previously
assumed to be homogeneous based on a set of commonly assayed surface markers
3. Biomarker discovery or cytokines. In some cases, this has led to the discovery of new T-cell subsets, which
can serve as biomarkers for disease outcomes.
Recent years have seen a plethora of single-cell characterizations of healthy and
diseased tissues. This has led to the identification of many novel biomarkers,
implications of rare cell subtypes in complex pathologies, and more accurate detection
of therapeutically interesting genes connected to disease-related cell subtypes. In
this chapter, we delineate how single-cell sequencing has led to these achievements.

3.1 Single-cell atlases reveal cellular biomarkers of disease

Many research efforts of the past decade concentrated on creating complete single-
cell resolution tissue atlases. These tissues often included heterogeneous tissues
that benefited greatly from more granular cellular subtyping, such as the pancreas,
the brain, and tumor tissue.

In extension, comparing healthy versus diseased tissue has led to identifying novel
targets. An early example is the single-cell sequencing of the healthy and diseased
heart by scientists from Hubrecht Institute and UMC Utrecht. Leveraging the high
granularity of the technology, the team could identify disease-specific subpopulations
that were enriched with cytoskeleton-associated protein CKAP4. As a result, CKAP4
was defined as a biomarker for fibroblast activation, a trait leading to cardiac fibrosis.

Furthermore, cell-state changes have key roles in pathogenesis and can serve as
biomarkers for disease, but changes in the cell’s internal programs and shifts in
cellular composition are often confounded in bulk profiling. A seminal example
concerns a single-cell atlas of mouse hippocampi that mimic Alzheimer’s Disease. A
high-resolution view of the nonneuronal brain cells revealed an Alzheimer-associated
Figure 6 Extensive single-cell atlases require high-throughput sequencing
with top-shelf NGS solutions like the Illumina NovaSeq X Plus used by Single
Cell Discoveries.

Single-Cell Sequencing in Drug Development | 9 10 | Single-Cell Sequencing in Drug Development

In line with this, many studies have focused on sequencing the microenvironment
of various cancer types, an example of which is detailed in Chapter 2. Mechanism
of Disease. Findings include a disease-defining T-cell subset in the tumor
microenvironment of classic Hodgkin lymphoma and a memory T-cell subtype in
breast cancer associated with improved prognosis.
4. Target identification & validation
Single-cell sequencing serves as a powerful tool for target identification and
validation by providing detailed insights into gene expression patterns and functional
consequences at the single-cell level. In this chapter, we delve into three examples.

A compelling example is a study on epithelial ovarian cancer (EOC) recurrence led 4.1 Exploiting single-cell atlases to identify CAR T-cell antigen
by scientists at the City University of Hong Kong. Due to intratumor heterogeneity, targets
the nature of EOC recurrence remains unknown until recently. Applying single-cell
Single-cell atlases can catalyze target identification in a more general sense. As
sequencing to primary and relapse tumors, the team discovered seven distinct
mentioned in the previous chapter, single-cell atlases empower researchers to map
subpopulations in primary tumors, of which they could identify one as a relapse
the gene expression profiles of all cell types and subtypes in multiple tissues. These
initiator.
atlases provide opportunities for target selection with high-grade specificity and
unlikely off-target effects.
The relapse-initiating cancer cells expressed the cell adhesion protein CYR61.
Subsequent study of the tumor microenvironment revealed a crucial supportive role
Take the following example. Chimeric
for a fibroblast subpopulation expressing RGS5, a known arterial growth–promoting
antigen receptor T-cells (CAR T-cells) CAR T-cell therapy
protein. The combined CYR61/RGS5 expression score was shown to be a useful
have emerged as a powerful treatment
biomarker to predict EOC recurrence in the team’s dataset and simultaneously
against B cell lymphomas, although they
advanced the proteins as potential therapeutic targets.
have no proven efficacy against other
blood cancers such as acute myeloid
Interestingly, researchers have hypothesized that generic markers may exist that can
lymphoma (AML), partially due to a lack
guide cancer immuno- and CAR T-cell therapy development for more than one cancer
of safe targets. Safe target antigens for
type. A single-cell pan-cancer immune atlas has consequently been an important
CAR T-cell therapy must be present in
focus in single-cell research, and two large collaborations have since published their
tumor cells but absent in healthy cells
atlases (here and here). A good example of a discovered biomarker based on this
and T cells in order to minimize off-
data is the identification of the Gαs–PKA signaling pathway to mark CD8+ T cells for
target toxicity.
immunotherapy failure.

To find these targets, scientists from

3.3 Key takeaways Munich and Nurnberg leveraged single-
cell atlases of malignant and healthy
Biomarker discovery studies have used single-cell sequencing to: bone marrow and nine vital human
tissues. Using a computational screening
• Create comprehensive single-cell atlases of healthy and diseased tissue that approach, they identified two targets
can be interrogated for disease-specific biomarkers and prognostic markers selectively expressed in AML cells
(e.g., heart disease and cancer) but almost entirely absent in healthy
cells. Previously unrecognized by bulk
• Discover disease-specific cells or cell states whose presence serves as a
sequencing, the team developed CAR Figure 7 Overview of the steps in CAR T-cell therapy
marker for disease (e.g. Alzheimer’s disease)
T-cells for these novel targets. With development. From: National Cancer Institute.
validation experiments in cell lines and
mouse models, the CAR T-cells indeed

Single-Cell Sequencing in Drug Development | 11 12 | Single-Cell Sequencing in Drug Development

 showed strong and durable treatment response and minimal toxicities, highlighting
the efficacy of using single-cell data for target selection and drug development in
CAR T-cell development.
4.2 Identifying high-grade targets for cancer therapy
In contrast to other technologies, single-cell sequencing creates an unbiased
characterization of complex tissue.
An example is the single-cell analysis of human glioma, immune, and other stromal
cells performed by scientists of the Houston Methodist Research Institute. They
discovered extensive heterogeneity in infiltrating T cells and could identify S100A4
as a novel regulator of immune suppressive T and myeloid cells in glioblastoma.
In functional experiments, they demonstrated that deleting S100A4 in non-cancer
cells was sufficient to reprogram the immune landscape and significantly improved
survival, providing evidence for the involvement of this target gene.
Another example concerns neuroblastoma, a diverse
type of childhood cancer that arises from aberrant
Read our blog on identifying
differentiation of the neural crest during early
a pan-neuroblastoma cell in
pediatric patients. development. Akin to Wilms tumor, it is expected
that neuroblastoma exhibits a cellular hierarchy
adopted from tissue development in utero.
However, in a collaboration between institutes in the Netherlands and the UK and
applying 10x Genomics and SORT-seq, the team uncovered a single widespread
phenotype across neuroblastoma tumors across patients. The phenotype represents
the sympathoblast, a pluripotent cell important in fetal development. Subsequently,
focusing on the gene expression profile of this cell type, the team could identify
genes that are expressed in neuroblastoma and during fetal development but are
absent in healthy tissue after birth. This, the team notes, makes these genes favored
novel targets for drugs or gene-silencing tools due to likely low off-target toxicity.
4.3 ScCRISPR-seq unravels genetic components of disease
Single-cell CRISPR screens (scCRISPR-seq) combine gene editing with single-cell
sequencing and provide a powerful source of discovery in pharmaceutical research.
Genetic perturbations created by CRISPR/Cas9 technology serve to validate targets
or reveal related genetic components. These components (genes, promotors,
epigenetic alterations, et cetera) may serve as drug targets, infer how drugs operate,
or provide direction for optimization strategies. For example, when applied to patient-
Single-Cell Sequencing in Drug Development | 13
derived melanoma cells, a scCRISPR-seq approach revealed CD58 loss as a new
marker for immune evasion in this cancer type.
4.4 Target validation
After identifying potential drug targets, single-cell sequencing can be used to assess
gene expression profile changes after interference with drug targets using genetic
knockout or silencing studies. This helps confirm whether the target is consistently
dysregulated in cells relevant to the disease and whether its modulation could
be therapeutic. Moreover, the technique provides information about the target’s
expression dynamics, such as whether it is upregulated or downregulated during
disease progression. This information is critical for validating the target’s involvement
in the process of disease.
Integrating scRNA-seq data with clinical data, such as patient outcomes or treatment
responses, allows researchers to correlate the target’s expression profile with real-
world clinical scenarios. This correlation can provide additional evidence of the
target’s relevance and potential as a therapeutic target.
4.5 Key takeaways
Target identification and validation studies have used single-cell sequencing to:
• Interrogate heterogenous tissue for disease-driving target genes (e.g., glioma)
• Uncover overlooked targets with low off-target potential (e.g. neuroblastoma
or CAR T-cell antigens)
• Unravel genetic components of disease in combination with perturbation
screens like scCRISPR-seq (e.g., melanoma)
• Validate the effects of target interference on cellular composition or rare cell
subsets
5. Mechanism of action
The combination of single-cell sequencing’s ability to characterize all cell subtypes
in a tissue and unbiasedly capture their whole transcriptome has given researchers
a toolset highly suited for demystifying potent drugs’ mechanisms of action. In this
chapter, we highlight three examples.
14 | Single-Cell Sequencing in Drug Development
 5.1 Unraveling mechanism of actions in existing diabetes drugs As such, they implicated liver macrophages in initial liver cancer stages and found a
way to test nizatidine’s mechanism of action. In their case, SORT-seq proved suitable
The power of single-cell sequencing is rarely more apparent than when it unravels
due to its high sensitivity and compatibility with the relatively low cell input from
the mechanisms of action of existing drugs.
such precious tissue.

A team from Washington University, St. Louis, and Janssen R&D performed single
nucleus RNA sequencing on almost 1 million cells from mouse models for diabetic
kidney disease, exposed to combinations of three different treatments, two days and
two weeks after treatment. Analysis of the resulting atlas revealed that the different
medications affected strikingly different cell types and caused non-overlapping
transcriptional changes.
Focusing on a specific cell subtype of the proximal tube, they observed that one
effective drug class, SGLT2i, induced starvation mimicry, and hypoxia responses,
features related to diabetes reversal. Functional experiments showed that SGLT2i
reversed how diabetes affected a splicing regulator in these specific cells, implicating
alternative splicing as a driver for diabetic kidney disease and its rescue as SGLT2i’s
mechanism of action. The results moreover support the development of new
combination therapies since different effective drugs target different cell types in
the kidney.
5.3 Identifying successful TEG therapy cells
An alternative strategy for creating T-cell therapies is equipping αβ T cells (used in
CAR-T production) with oncolytic γδ T-cell receptors. These so-called Engineered
T cells (TEGs) recognize surface protein patterns on cancer cells and proceed to
kill them. The Rios group from the Princess Máxima Center for Pediatric Oncology
engineered a platform utilizing single-cell sequencing, live imaging, and organoid
technology to study the mode of action of TEGs.
Based on the single-cell data, the team could identify ‘super engager’ TEG clusters
that were most efficient at killing tumors. Exploring the single-cell gene expression
profiles of these TEGS further, they could uncover the gene signature associated with
super engagement that provides opportunities to engineer T cells with potent killing
abilities. Moreover, they could lay out the optimal sequence of T-cell combination

5.2 Characterizing drug-affected cell types in precancerous liver

For example, a team of scientists from INSERM Strasbourg developed an organoid
system expressing a poor prognosis gene expression signature for precancerous liver
disease. By performing a drug screen on this model and detecting the expression
signature with single-cell sequencing, the team could identify the drug nizatidine as a
potentially effective medicine and could verify that it operated by blocking histamine
receptors. Then, the team could leverage a single-cell atlas of the healthy liver to find
a specific liver cell prominently expressing these receptors: liver macrophages.

By treating liver-derived white blood cells with

nizatidine or an empty control and using SORT-
Click to read an in-depth
seq to measure the gene expression changes after
case study on the mechanism
of action of nizatidine in pre- treatment, the team discovered that nizatidine
cancerous liver models. indeed specifically affected liver macrophages.
Moreover, the nizatidine-treated macrophages
showed an immunoregulatory signature and a
decreased pro-fibrotic/tumorigenic signature. Then, the team could repeat these
signatures by knocking out the histamine receptor in a macrophage cell line. The
data indicate that nizatidine operates by halting macrophage-driven liver fibrosis, Figure 9 SORT-seq has a high sensitivity for genes with low transcript
which prevents precancerous liver disease and consequently liver cancer. counts and is compatible with relatively low cell input.

Single-Cell Sequencing in Drug Development | 15 16 | Single-Cell Sequencing in Drug Development

therapy by demonstrating that priming organoids with type I interferon boosted TEG-
mediated killing of resistant tumors, key information for future treatment design.
5.4 Key takeaways
Mechanism-of-action studies have used single-cell sequencing to:
• Accurately identify gene expression programs in specific cell subtypes affected
after drug exposure (e.g. precancerous liver cells or diabetic kidney disease)
• Characterize and study the mode of action in key constituents of successful
cell therapy (e.g. TEG therapy)
6. Lead identification & optimization
By accurately identifying responsive cell subtypes or heterogeneous responses,
single-cell sequencing aids in evaluating drug and off-target effects at the single-
cell level. In addition, researchers can examine how gene expression changes vary
with different concentrations of the drug or with different drug combinations, aiding
in optimal dosing strategies.
What is more, single-cell sequencing is increasingly playing a role in optimizing
complex pharmaceutical platforms such as cell and gene therapy. For example,
combining cell type identification with single-cell transgene detection reveals if gene
therapy leads are targeting the cell type of choice in biodistribution studies. In this
chapter, we highlight three of those studies.
6.1 CAR T-cell optimization
As mentioned in Chapter 4. Target identification & validation, CAR T-cell therapy
successes in B-ALL patients have yet to be translated to other cancers and solid
tumors. Optimization efforts to minimize T-cell exhaustion and maximize long-term
persistence after infusion are key to this effort, and rely on accurate measurement of
exhaustion and memory T-cell signatures. Single-cell sequencing has been proposed
as a powerful technology to guide the design and engineering of the next generation
of CAR T cells by accounting for cell heterogeneity, enabling robust comparisons
of transcriptomic signatures, and tracking cell lineages. Moreover, as CAR T-cell
production creates multiple subpopulations that act differently once infused back
into the patient, single-cell sequencing of these populations informs how to optimize
production conditions.
Single-Cell Sequencing in Drug Development | 17
For example, two research groups from the University of Cambridge analyzed over
50,000 single CAR T-cells to identify cells not responding to antigens. They identified
T-cell exhaustion in a proportion of the antigen-responsive groups with previously
unknown gene expression markers. These findings can be used to change the
manufacturing protocol to improve the antigen response and exhaustion-resistant
CAR T-cell therapies by genetic editing and filtering out redundant T cells by their
genetic markers.
6.2 AAV optimization
In vivo gene therapy platforms that utilize
adenovirus-associated viral vectors (AAVs) for
gene delivery have recently entered the market Want to know more about
for some rare diseases and are the most popular single-cell resolution biodis-
vehicle for gene therapies being developed tribution studies? Read our
[source]. Yet, shortcomings in target specificity application note here.
cause a relatively low therapeutic index, meaning
high dosages are needed and off-target risks
and immunogenicity rise. Many companies are engineering novel capsids to address
such issues, and crucial to those efforts is the ability to test AAV specificity (and
consequent efficiency) with single-cell resolution biodistribution.
Single-cell sequencing can be combined with AAV transgene detection to improve
AAV-based gene therapy delivery. While AAV transgene detection marks successfully
transduced cells, single-cell data can characterize which cell subtypes are targeted
and how they respond transcriptionally. When performed on AAV candidates, it can
detail efficacy, specificity, and safety indices to select and optimize leads. Single-cell
AAV detection protocols are unpublished but can be custom-developed by Single
Cell Discoveries.
6.3 Key takeaways
Lead identification and optimization studies have used single-cell sequencing to:
• Characterize effects on cell therapy from genetic editing, selection or
production conditions (e.g. CAR T-cell optimization)
• Identify lead by studying their biodistribution and on-target and off-target
transcriptional effects (e.g. AAV candidate identification)
• Test heterogeneous gene expression effects of different drug dosages and
combinations
18 | Single-Cell Sequencing in Drug Development
7. Preclinical research
In preclinical studies, using single-cell sequencing has the benefit of capturing drug
effect, side effects, and mechanism-of-action data within one experiment. Again,
cell and immunotherapy studies benefit greatly from the granularity of single-cell
sequencing, as evidenced by multiple publications. In this chapter, we detail four of
those publications.
and to their heterogeneous cellular composition immediately after transfer, traits
detectable with single-cell sequencing.
Hence, in 2020 the first study of adoptive Treg transfer in non-human primates to
employ single-cell sequencing was performed. A team of Harvard Medical School
scientists performed 10x Genomics to study the effects of a drug combination, low-
dose IL-2 + rapamycin, on Treg persistence after transfer.

Overview of related preclincal studies The results reveal that the drug combination greatly enhanced Treg persistence in
monkeys, while gene expression analysis revealed that the heterogeneity immediately
after transfer changed to a beneficial uniformity of resting Tregs after twenty days.
Study Modality Model Disease Single-cell sequencing application
The data not only provided evidence for the benefits of the drug combination but also
Parker et al. 2020 CAR T-cells Mice B-cell malignancies
Reveal CAR T-cell antigen target presence in proved the power of single-cell sequencing to inform adoptive Treg immunotherapy
non-targeted cells that cause neurotoxicity
precisely.
Adoptive Non-human Autoimmune disease Characterize Treg composition dynamics
Furlan et al. 2020
Treg transfer primates & transplantation after transfer in the context of different drugs

Tracy et al. 2022 Immunotherapy Mice Leukemia

Measure T-cell exhaustion dynamics in the
context of novel immunotherapeutic combinations 7.3 Characterizing causes of T-cell exhaustion
Hypertrophic Validate delivery of adenine base
Reichart et al. 2023 Gene therapy Mice cardiomyophathy editor transcripts for biodistribution study In B-cell acute lymphoblastic lymphoma (B-ALL), CD4+ T-cell exhaustion may be
driving relapse after chemotherapy but could be rescued by checkpoint inhibitors.
Hence, in a preclinical mouse study, scientists from the University of Minnesota tested
combining checkpoint inhibitor PD-L1 with the established B-ALL drug nilotinib
7.1 Unearthing causes of CAR T-cell toxicity
[source]. They employed Single Cell Immune Profiling in combination with specific
One type of CAR T-cell therapy targets the CD19 antigen, a protein located on CITE-seq antibodies against immune
the surface of B cells. These CAR T cells effectively track and kill malignant cells receptors to characterize the T-cell
in conditions like B-cell leukemia, lymphoma, and multiple myeloma. However, this repertoire before and after treatment.
treatment causes neurotoxicity in some patients.
The results showed improved long-
While CD19 was thought to be restricted to B cells, results of a study in mice show term survival in mice after therapy,
that a rare subset of mural cells located at the blood-brain barrier express low levels which correlated with decreased
of CD19, likely causing anti-CD19 CAR T-cell toxicity. This on-target side effect exhaustion signatures in CD4+ T cells.
cautions the use of intrathecal administration in the spine and informs strategies to Moreover, they identified a unique
develop therapies with improved safety profiles. subset of CD4+ T cells with cytotoxic
and helper-T-cell signatures and
could show that the clonal expansion
7.2 Enhancing Treg persistence after adoptive transfer of this subset may prevent relapse
by reducing T-cell exhaustion. As a
Suppressive immunotherapy with regulatory T-cells (Tregs) answers to a growing
result, they define exhausted CD4+ T
clinical need in autoimmune disease and transplantation, and the long-term
cells as a negative prognostic marker
persistence of Tregs is key to addressing those needs. Relatedly, choosing an
for immunotherapy in B-ALL, and
immunosuppressant to synergize with the suppressive function of Tregs is important
identify the reinvigoration of a CD4+
in optimizing adoptively transferred Treg efficacy. That persistence in suppressive
T cell subset as a powerful target for Figure 10 The Illumina NovaSeq X Plus
function is linked to Tregs acquiring a “resting”-like state sometime after transfer
supportive therapy.

Single-Cell Sequencing in Drug Development | 19 20 | Single-Cell Sequencing in Drug Development

 Overview of related clinical trials
7.4 Preclinical AAV-based gene therapy research
Study Phase Modality Disease Single-cell sequencing application
Introduced in Chapter 6. Lead identification & optimization AAV-based gene therapy
Study the antitumor activity and gene
studies can utilize single-cell sequencing to detail AAV candidates’ specificity, Heczey et al. 2023 I CAR NKT-cells Neuroblastoma
expression of CAR NKT-cells in responders
efficacy, and safety indices. A recent study conducted by geneticists from Harvard Palmer et al. 2022 I Cancer vaccines Metastatic Trace vaccine-induced tumor infiltration
solid tumors of T cells
Medical School demonstrated that single-cell sequencing also has a great benefit in
Tian et al. 2023 II Immunotherapy BRAF-V600E Measure the cooperative effect of two
the preclinical phase of AAV-based gene therapy studies. colorectal cancer immunotherapeutics on T cells

Cascone et al. 2023 II Immunotherapy Non-small cell Measure the cooperative effect of two
In this study, they conducted experiments using two genome editing platforms; lung cancer immunotherapeutics on T cells

an adenine base editor (ABE) and a CRISPR/Cas9 system.These platforms were Crees et al. 2023 III Stem cell transfer Multiple myeloma Characterize drug effects on stem cell
administered in mice to prevent hypertrophic cardiomyopathy. As the ABE requires composition and gene expression

dual delivery (i.e., using two AAVs in parallel), the research team tested whether
ABE transcripts were detectable in cardiomyocytes. Using single-nucleus RNA
sequencing, they could quantify the biodistribution rates per heart region and link 8.1 Phase I clinical trials
the gene editing effects to these numbers. As a result of this analysis, the team
Clinical validation of antitumor activity in CAR-NKT therapy
decided to increase the ABE dose, which boosted infection of the heart atria, and
test how some of the therapy’s effects on the atria improved. A Phase I clinical trial for CAR natural killer T-cells (CAR NKT) was conducted in
children with neuroblastoma [source]. The primary objective was to determine the
safety of the therapy and the maximum tolerated dose. The secondary objective was
7.5 Key takeaways
to study the anti-tumor activity, for which the clinical researchers employed single-
In preclinical studies, single-cell sequencing has been used to: cell sequencing.

• Identify cell populations that underlie off-target or on-target side effects of

treatments (e.g., CAR T-cell neurotoxicity)
• Cumulate evidence for cell therapy efficacy in longitudinal studies in model
organisms and inform treatment choices (e.g. Treg persistence or T-cell
exhaustion)
• Validate gene therapy biodistribution in model organisms (e.g. ABE delivery
in heart regions)
The researchers applied single-cell sequencing on the preinfusion products of all
patients to identify markers for antitumor activity. This revealed CD62L as a marker
in a CAR NKT-cell cluster enriched in responders to CAR NKT-cell treatment. In
addition, the researchers identified a gene expression profile in this cluster that
resembled memory-T-cell differentiation, which validated earlier in vitro work. In
summary, in-patient CAR NKT-cell therapy efficacy may effectively be improved by
protocols that increase memory differentiation.

Proof of vaccine-induced tumor-infiltrating T cells

8. Clinical research
The adoption of single-cell sequencing in clinical trials is on the rise due to its ability
to provide detailed insights. It offers additional evidence for emerging therapy
approaches, such as assessing the anti-tumor activity of immunotherapy at clinically
relevant doses, identifying cooperative interactions between different cancer drugs,
and confirming the effectiveness of stem cell therapies in patients. In this chapter, we
delve into five clinical trials where the application of single-cell sequencing played a
significant role in shaping the outcomes and findings.
Another Phase I clinical trial employed single-cell sequencing with the same goal of
studying anti-tumor activity. Patients with advanced metastatic solid tumors were
treated with a combination of checkpoint inhibitors and T-cell-inducing neoantigen
vaccines. The interim results were published in August 2022.
The team performed Single Cell Immune Profiling specifically, on patient-derived
PBMCs at various time points during treatment. As a result, they identified several
distinct T-cell clonotypes after treatment. An overlap of T-cell clonotypes in
PBMCs and tumors was detected, proving that the vaccine-induced T-cells are
able to infiltrate tumors. Additionally, the transcriptome data showed a 12 to 98-fold
increase in cytokine transcript expression across clonotypes following vaccination,

Single-Cell Sequencing in Drug Development | 21 22 | Single-Cell Sequencing in Drug Development

and patients with such responses showed tumor stabilization and prolonged overall
survival, again suggesting a causal effect. A Phase II/III clinical trial of this combination
treatment was started in 2022.
8.2 Phase II clinical trials
Cooperativity between BRAF inhibitors and immunotherapy
One of the first Phase II clinical trials to incorporate systematic single-cell sequencing
of paired pre- and on-treatment tumor biopsies from patients was started in 2018
and published in 2023. In this trial, the research team focused on unraveling the
mechanism underlying a novel combination therapy approach for targeting metastatic
colorectal cancer, which involved the use of BRAF/MEK inhibitors and checkpoint
inhibitors.
Overall survival rates with the combination therapy appeared three times higher in
this clinical trial than in trials with standalone BRAF/MEK inhibitors. Using single-
cell sequencing and Single Cell Immune Profiling to study this cooperative effect,
the team observed gene expression changes both before and during therapy.
These changes indicated the activation of genetic immune programs in tumor cells,
specifically types I and II interferon response, antigen-presenting genes, and T-cell
recruiting chemokines. The findings were directly linked to more complete inhibition
of tumor growth and notably improved progression-free survival.
Combining two immunotherapy drugs in operable lung cancer
Ipilimumab (IPI) and nivolumab (NIVO) are successful immune checkpoint inhibitors
that have different mechanisms, and their cooperativity is established in metastatic
non-small cell lung cancer (NSCLC) patients. The NEOSTAR trial, a Phase II study,
researched the prevention of recurrence in patients with non-metastatic, operable
NSCLC by augmenting the current first-line treatment of neoadjuvant chemotherapy
with NIVO by adding IPI to treatment. To measure the cooperative effect on both
immune and tumor cells, the research team used single-cell sequencing.
The major pathological response was one-fifth higher in patients with chemo + NIVO
+ IPI as compared to those receiving only chemo + NIVO, with acceptable levels of
toxicity. Utilizing 10x Genomics, they observed that subclusters of CD8+ T, CD4+
T, B, and NK cells were relatively increased in patients treated with chemo + NIVO
+ IPI, whereas Tregs were decreased. When comparing gene expression changes
between the two treatment arms, the team observed an overall enhanced antitumor
and reduced immunosuppressive effect from the immunotherapy combination,
providing additional evidence for the stronger anti-tumor effects of the chemo +
NIVO+ IPI treatment.
Single-Cell Sequencing in Drug Development | 23
8.3 Phase III clinical trials
Optimizing hematopoietic stem cell transplantation
Currently, various clinical trials are being conducted to find methods to optimize
autologous hematopoietic stem-cell transplantation (ASCT), for which the ability
to harvest enough stem cells from the patient is key. In addition, mobilizing stem
cells that exhibit transcriptional markers for beneficial traits like self-renewal and
persistence is becoming a more important qualifier. For this, single-cell sequencing
provides a suitable readout.
Currently, granulocyte colony-stimulating factor (G-CSF) is administered to patients
before donation to increase mobilization. In a Phase III trial, researchers from
Washington University studied the effect of combining novel inhibitor motixafortide
with G-CSF to mobilize more and more suitable stem cells for ASCT in multiple
myeloma patients.
The results showed that patients with G-CSF + motixafortide had mobilized almost
four times more hematopoietic stem cells, common myeloid progenitor cells, and
multipotent progenitors. Additionally, the single-cell data showed that motixafortide
alone in healthy donors helped mobilize transcriptionally unique subsets of
hematopoietic stem cells that show signs of enhanced self-renewal, regeneration,
and quiescence. These data suggest motixafortide may mobilize long-lived stem
cells that are advantageous for multiple myeloma patients.
8.4 Key takeaways
In Phase I-III clinical studies, single-cell sequencing has been used to:
• Gather additional evidence for therapy efficacy during clinical trials (e.g. CAR
NKT-cell therapy or cancer vaccines)
• Offer insights into the mechanism of action of therapy in patients to stockpile
proof for using or combining therapies (e.g., immunotherapy on metastatic
colorectal cancer or operable lung cancer)
• Characterize the effects and mechanisms of transplantations (e.g.,
autologogous hematopoeitic stem-cell transplantation)
24 | Single-Cell Sequencing in Drug Development
9. Conclusion
Reviewing a decade’s worth of single-cell studies across the drug development
pipeline demonstrates massive utility in drug discovery and clinical development.
Disease-related cell subpopulations that were undistinguishable with traditional
bulk sequencing o have consequently been identified, leading to a new or revised
understanding of human diseases and their potential treatments. Extensive single-
cell atlases and in-depth study of healthy and diseased tissues have revealed novel,
high-grade biomarkers, and drug targets.
Moreover, single-cell sequencing stands out as the most accurate method for
identifying which cell subtypes undergo gene expression changes after drug
exposure, unraveling drugs’ mechanisms of action, and informing lead identification
and optimization. Especially in promising therapeutic avenues such as CAR T-cell
therapy, gene therapy, immunotherapy, and stem cell therapy, single-cell sequencing
can provide essential proof for therapeutic efficacy in (pre)clinical settings.
Still, ongoing advances in single-cell technologies promise a future of even greater
possibilities. Spatial transcriptomics at cellular or subcellular resolution adds single-
Figure 11 Single Cell Discoveries’ purpose-bulit lab for single-cell
sequencing solutions.
Single-Cell Sequencing in Drug Development
cell transcriptomic data to microscopy and immunohistochemistry analysis. This
enables the simultaneous localization and morphological characterization of cell
subpopulations whose full gene expression profiles can be traced as they progress
through disease or are affected by therapy.
At the same time, the application of single-cell multiomics is gaining momentum,
which combines transcriptomics with technologies that map other traits at single-
cell resolution, such as their genome, epigenome, proteome or metabolic properties.
As a result, researchers can ascertain a multidimensional phenotypic picture of
single cells that distinguishes between cellular states and substates at even higher
resolution, already leading to revised theories on mechanism of disease and potential
new targets source (for examples, see here or here).
While single-cell sequencing has led to some remarkable discoveries described here,
the lion’s share is yet to come. How will you use single-cell sequencing to transform
the lives of patients?
10. About Single Cell Discoveries
At Single Cell Discoveries, we are focused on providing cutting-edge single-
cell sequencing services to biopharmaceutical companies, health systems, and
academic research centers globally. Located in our brand-new laboratory in Utrecht,
the Netherlands, our team of PhD scientists is dedicated to developing customized
solutions for your unique scientific questions, all while ensuring rapid turnaround
times and high quality.
Five years after spinning out from one of the world’s first single-cell core facility,
quality service, intelligent automation, bleeding-edge technology, and hard work are
still central to Single Cell Discoveries’ expanding operations. Each member of the
growing team is committed to advancing single-cell sequencing and empowering
clients with impactful single-cell discoveries with the ultimate goal of improving the
human condition.
For a deeper dive into our expertise, services, and projects, we invite you to explore
our website.
GO TO SCDISCOVERIES.COM
| 25 26 | Single-Cell Sequencing in Drug Development