Single-Cell Sequencing in Clinical Trials: Phase-I Studies

Single cell sequencing in clinical trials: how is the technology applied? Drugs take the shape of a t-SNE plot

Single Cell in Phase-I Clinical Trials  — In this blog post, we take a look at the first documented
Phase-I clinical trials that use single-cell RNA sequencing. What have the clinical researchers employed the technology for? What did it bring them?

“Methods to sequence the DNA and RNA of single cells are poised to transform many areas of biology and medicine.” Thus spoke Nature Methods when they awarded single-cell sequencing the title Method of the Year in 2013.

Ten years later, biology and medicine have indeed observably transformed. 2023 saw more than 5,000 publications documenting the technology’s utilization, in areas ranging from neuroscience to agriculture. It has been widely adopted to study disease, disentangle heterogeneous drug responses, find novel biomarkers, optimize cell and gene therapy development, preclinical drug testing, and much more.

What is also transformative in today’s single-cell landscape is that early adopters are pioneering single-cell sequencing in clinical trials. So far, at least five clinical trials have shared how the researchers integrated the technology and what they found. A deep dive into their setups tells much about the unique benefits of Single Cell.

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Phase-I clinical trials

Phase-I clinical trials have the primary objective of determining safety, maximum safe dose, and side effects. As a secondary objective, these studies can include an investigation of efficacy or mechanism. It is to the secondary objective that single-cell sequencing is showing powerful contributions.

In this section, we document five phase-1 clinical trials, covering a tumor vaccine, two antitumor CAR T-cell therapies, immunotherapy against rheumatoid arthritis, and peanut allergy.

Jump to a section:

  1. Finding proof of vaccine-induced tumor-infiltrating T cells
  2. Clinically validating antitumor activity in CAR-NKT therapy
  3. Exploring the impact of rheumatoid arthritis immunotherapy doses
  4. CAR-T cell mode of action in multiple myeloma
  5. Peanut oral immunotherapy mechanism of action in peanut allergy
  6. Takeaways


Finding proof of vaccine-induced tumor-infiltrating T cells

  • Indication: Advanced metastatic solid tumors
  • Therapy modality: Immunotherapy + neoantigen vaccine
  • scRNA-seq application: Study mechanism of action & find causal link for vaccine effect on disease outcome.
  • Single-cell analyses: Time-point analysis of T-cell clonal dynamics in response to treatment & gene expression analysis of immune cells in responsive patients.

A Phase-I clinical trial (Voon et al., 2022) employed single-cell sequencing to study increased antitumor effects of immunotherapy with vaccines. In it, patients with different types of advanced metastatic solid tumors got a treatment combination of checkpoint inhibitors and T-cell-inducing neoantigen vaccines. The researchers published the interim results in August 2022.

That team performed Single Cell Immune Profiling, a 5’ RNA sequencing technology that captures variable V(D)J regions of B or T-cell receptors along with full transcriptomes. The samples were patient-derived PBMCs from various time points during treatment. This setup can create a highly accurate picture of immune cell clonal dynamics during treatment and provide evidence for a therapy’s mechanism of action.

In the trial’s single-cell data, the team could identify distinct T-cell clonotypes whose clonal expansion was probably induced by the neoantigen vaccine. In addition, there was an overlap of T-cell clonotypes in PBMC and tumor cells. That indicates that the vaccine-induced T-cells can infiltrate tumors in vivo successfully — an important sign of therapy efficacy.

What's more, gene expression analysis in responsive patients showed a 12 to 98-fold increase in cytokine transcript expression after vaccination, across clonotypes. This further revealed the mechanism of the vaccine-activated immune response. Patients with such expression changes showed tumor stabilization and prolonged overall survival. The positive results led to a Phase-II/III clinical trial that compares this combination to other treatments, started in 2022, estimated to finish in 2027.


Clinically validating antitumor activity in CAR-NKT therapy

  • Indication: Pediatric neuroblastoma
  • Therapy modality: CAR natural killer T (CAR NKT)-cell therapy
  • scRNA-seq application: Study mechanism of action & identify therapeutic markers genes.
  • Single-cell analyses: Marker identification of in vivo antitumor activity & gene expression profiling of the CAR-NKT cell clusters significantly enriched in responders.

This Phase-I clinical trial studies (Heczey et al., 2022) the effects of a novel CAR natural killer T-cell (CAR-NKT) therapy platform on pediatric neuroblastoma patients. For its primary objective, the team aimed to determine the therapy’s maximum tolerated dose. For its secondary objective, they studied the mechanisms of anti-tumor activity, for which the clinical researchers employed single-cell sequencing.

Single-cell sequencing of the preinfusion products aimed to identify distinct CAR-NKT cell types and find specific markers for antitumor activity. This revealed CD62L as a marker in a CAR-NKT cell cluster enriched in responders.

In addition, the researchers used gene set enrichment analysis to identify a gene expression profile in this cluster that resembled memory-T-cell differentiation. This showed that in-patient CAR-NKT cell therapy efficacy was linked to the capacity to activate T-cell memory. Moreover, it suggested that efficacy may be improved by protocols that increase CAR NKT-cell memory differentiation, while CD62L may be a prognostic marker for response.


Exploring the impact of rheumatoid arthritis immunotherapy doses

  • Indication: Seropositive rheumatoid arthritis
  • Therapy modality: Immunotherapy
  • scRNA-seq application: Study immune response to different dosages
  • Single-cell analyses: Cell type identification & T-cell receptor clonotype analysis for each dosage & gene expression analysis per clone

This randomized Phase-I trial (Sonigra et al., 2022) set out to explore the impact of low, medium, and high doses of a novel nanoparticle-based immunotherapy drug called DEN-181. The drug combines native collagen-II with small molecule inhibitor calcitriol, which shows promise to reduce the severity of rheumatoid arthritis in patients by enticing a multimodal T-cell response.

The primary objective of the study was to assess the safety and efficacy of this drug in patients. The secondary objective was to study the immunological and clinical effects of each dose. With flow cytometry, the team observed that the expansion of specific T cells correlated with a good prognosis. With Single Cell Immune Profiling, the team identified a T-cell clonotype that underwent expansion after treatment in low, medium, and high doses. Its gene expression profile showed signs of exhaustion, which may underlie at least a part of the treatment’s effect.

Here, the single-cell data provided additional evidence that the expected T-cell response, important to therapy effectiveness, already occurred in low and medium doses.


CAR-T cell mode of action in multiple myeloma

  • Indication: Multiple myeloma in poor performance patients
  • Therapy modality: CAR-T cell therapy
  • scRNA-seq application: Study mode of action
  • Single-cell analyses: Comparing gene expression

Ever since it showcased great successes in treating B cell malignancies (Schuster et al., 2017), CAR-T cells have been a promising therapeutic modality against blood cancer types. This includes relapsed/refractory multiply myeloma. For a Phase I/II clinical trial that focuses on patients with poor performance (i.e., vulnerable patients often excluded from clinical trials), Du et al., 2022 showed a successful response to CAR-T cell therapy in these patients.

The team employed Single Cell Immune Profiling to elucidate the transcriptomic differences between responders and non-responders. Their slightly superficial investigation revealed 100+ differentially expressed genes, while gene set enrichment analysis highlighted a role for the mTORC1 signaling pathway in therapy response.


Peanut oral immunotherapy mechanism of action in peanut allergy

  • Indication: Peanut allergy
  • Therapy modality: Oral immunotherapy
  • scRNA-seq application: Study mechanism of action & identify markers
  • Single-cell analyses: Cell type identification & gene expression analysis

In a Phase-I/IIa study on peanut oral immunotherapy against peanut allergy in children, the researchers used scRNA-seq to study the Treg response during the first 24 weeks of treatment (Anvari et al., 2021). Single-cell analysis revealed alterations in gene expression for memory and naïve gamma-delta Tregs, which the authors claim can serve as an early marker of successful therapy.



As we study the Phase-I clinical studies that utilize single-cell sequencing, the following image arises. Single-cell sequencing is most frequent in trials for novel cell therapy drugs (CAR-T and CAR NKT-cell therapy) and novel immuno-oncology drugs (including cancer vaccines). In other words, therapy modalities in which heterogeneous (immune) cell populations play an important role.

What stands out is that the best of these studies leverage single cells' unique ability to study a heterogeneous immune response, disentangle clonal dynamics during treatment, and study how treatment affects gene expression in crucial immune cell populations.


This blog post leaves out a 2023 Phase-I observational study that combines traditional Chinese medicine Huayu-Qiangshen-Tongbi (HQT) formula with methotrexate to treat rheumatoid arthritis patients. The researchers carried out single-cell RNA sequencing to study gene expression changes after treatment. They document lipid metabolism changes correlated to plasma lipid concentrations and patient outcomes.

Find out more

Find out more information on single-cell sequencing application across the drug discovery pipeline, from biomarker identification to lead optimization, and from preclinical research to clinical studies.