Biomarker Insights: Tracking Donor Cell Dynamics After Cell Infusion

After cell infusion, when do donor cells become damaged and die? How do relevant signals change over time? How are these changes related to cell persistence and treatment response?

These issues run through the development and clinical research of allogeneic cell therapies. Understanding the process of therapeutic cells in vivo requires the establishment of assays that can continuously observe donor-derived signals in addition to efficacy evaluation. AlloDx is based on donor-derived cell-free DNA (dd-cfDNA) detection technology and explores providing molecular-level observation basis for this research need.

Cell therapy research requires continuous in vivo observations

The evaluation of allogeneic cell therapy requires attention to changes at different stages after infusion. Early cell damage and death, subsequent changes in donor-derived signals, and persistence of treated cells each provide different levels of information. Continuous sampling can help researchers observe these processes and correlate test results with administration time, cell dose, and treatment response.

Internationally, cell kinetic monitoring has been introduced into allogeneic cell therapy research. In 2024, TC BioPharm announced a collaboration with CareDx to use AlloCell to evaluate the expansion and persistence of the allogeneic γδ T cell product TCB008 in the ACHIEVE clinical trial. [1] This practice reflects the R&D team’s need for quantitative monitoring after cell infusion, and also suggests that the detection plan should match the specific research question.

dd-cfDNA provides molecular readout of processes associated with damage and death

When donor cells undergo processes such as apoptosis and necrosis, DNA fragments can be released into the circulation. Taking advantage of the genetic differences between donors and recipients, it is possible to identify donor-derived cell-free DNA in plasma and observe changes in its ratio over time.

This idea has been supported by cell therapy research. A study published in 2023 monitored 10 kidney transplant recipients who received allogeneic mesenchymal stromal cells (MSC). An increase in MSC-derived cfDNA was observed 4 hours after infusion, and the signal was no longer detected in subsequent sampling. This study provides a clinical research basis for using donor-derived cfDNA to observe MSC death-related processes. [2]

Therefore, through pre-infusion baseline and post-infusion serial sampling, researchers can observe the emergence, rise, fall, and duration of detectable signals from donor-derived cfDNA signals. However, plasma readings are also affected by DNA release, clearance, and changes in recipient background. Quantification of dd-cfDNA alone cannot distinguish between apoptosis and necrosis, nor can it be directly converted to the number of dead cells or death rate.

To identify low-abundance signals, you first need to control background noise.

When donor-derived DNA accounts for a low proportion of the sample, how to distinguish the true signal from the detection background is an important analytical issue in dynamic monitoring. The lower the detection background, the better it is for identifying weak donor-derived signals. Especially in longitudinal follow-up, the detection performance in the low-abundance stage will affect the researcher's judgment of signal changes.

AlloDx's patent application "Organ Transplant Rejection Risk Detection Method and Molecular Markers for Detection" discloses a dd-cfDNA quantitative method based on Indel markers, multiplex PCR library construction and high-throughput sequencing, and combines genotype and background noise models to correct the results. [3]

According to the examples in the patent publication, the average blank background value of the Indel method is 0.08%, and the method using the same number of SNP markers is 0.36%. The former is approximately 78% lower; the limit of detection (LOD) reported in the example is 0.1%, and the limit of quantification (LOQ) is 0.12%. [3]

Reducing background interference and identifying low-abundance donor-derived DNA are the technical basis for AlloDx to conduct dynamic monitoring research.

The above values are from the organ transplantation detection examples of the patent and reflect the methodological performance under corresponding experimental conditions. When applying this method to specific cell therapy products, it is still necessary to verify the applicability based on sample type, DNA input amount, and donor composition, and establish corresponding detection and quantitative performance.

Connecting dynamic signals to persistence research

In specific studies, dd-cfDNA can be laid out around the observation requirements at different stages: early after infusion, focus on changes in injury and death-related signals; during continuous follow-up, observe signal decline or persistence; in repeated dosing studies, compare signal trajectories at different dosing cycles and analyze them together with other research indicators.

Persistence assessment also requires further answers as to whether the treated cells persist. The continued detection of donor-derived cfDNA in plasma does not mean that viable cells continue to survive; the absence of detection does not mean that the donor cells have completely disappeared. Therefore, studies of expansion and persistence should be combined with validated donor DNA testing of cell components, flow cytometry, or other evidence of suitability for the cell product.

At the same time, test design needs to consider donor composition. For example, if you receive third-party MSC treatment after hematopoietic stem cell transplantation, the sample may involve three genetic sources: the recipient, the hematopoietic donor, and the MSC donor. The ability to distinguish between different sources needs to be verified. Only by clarifying the detection objects and interpretation boundaries can longitudinal data truly serve the research questions.

AlloDx enables allogeneic cell therapy monitoring research

AlloDx has accumulated experience in dd-cfDNA detection methods and application research in the field of transplantation. For allogeneic cell therapy, the Indel detection method with low background noise provides a technical basis for exploring the dynamic changes of donor-derived signals.

AlloDx looks forward to cooperating with cell therapy R&D companies and clinical research teams to conduct analytical verification and longitudinal monitoring studies around specific cell products, explore the connection between changes in donor-derived cfDNA, cell persistence, and treatment response, and add molecular evidence to understand the in vivo process after cell infusion.

Source

[1] TC BioPharm. TCBP Partners with CareDx to Support ACHIEVE Clinical Trial Using AlloCell for Pharmacokinetic Monitoring of Allogeneic Cell Therapy. 2024-12-11. https://www.prnewswire.com/news-releases/tcbp-partners-with-caredx-to-support-achieve-clinical-trial-using-allocell-for-pharmacokinetic-monitoring-of-allogeneic-cell-therapy-302327782.html

[2] Dreyer GJ, et al. Cell-free DNA measurement of three genomes after allogeneic MSC therapy in kidney transplant recipients indicates early cell death of infused MSC. Front Immunol. 2023;14:1240347. https://pmc.ncbi.nlm.nih.gov/articles/PMC10652747/

[3] Suzhou AlloDx Technology Co., Ltd. Organ transplant rejection risk detection method and molecular markers for detection: Chinese invention patent application, publication number CN118406771A, publication date July 30, 2024. https://patents.google.com/patent/CN118406771A/zh