IndelTrace 2.0 | In Vivo Changes After Allogeneic CAR-T Infusion and Donor-derived Cell-free DNA Testing

How to observe the expansion, contraction and persistence of allogeneic CAR-T cells after infusion? In addition to measuring cell numbers and CAR transgene levels in peripheral blood, DNA release signals associated with cell damage and death provide a complementary perspective for understanding in vivo changes in treated cells.

AlloDx has introduced IndelTrace 2.0, which uses genetic differences between donor and recipient to identify donor-derived DNA. Serial testing of plasma donor-derived cell-free DNA (dd-cfDNA) provides molecular-level information for research on the in vivo dynamics of allogeneic cell therapy.

Technical upgrade: supporting low-abundance donor signal detection

Building on AlloDx's first-generation probe hybridisation capture dd-cfDNA quantification platform, IndelTrace 2.0 uses multiplex PCR targeted amplification with insertion-deletion polymorphism (Indel) markers to identify, quantify and serially monitor donor-derived DNA.

Identification based on genetic differences between donors and recipients eliminates the need to use specific CAR sequences as detection targets, providing a technical path for monitoring research on different allogeneic cell products. The specific application still needs to be verified based on the genotype of the donor and recipient, product composition and sample conditions.

Lower background interference: observing trace signals

In low-abundance donor DNA testing, lower background interference helps separate true signal from assay noise. The organ transplantation testing examples published in AlloDx's patent application “Methods for detecting organ transplant rejection risk and molecular markers for testing” report the following:

Table 1 Methodological metrics from the organ transplantation testing examples in patent application CN118406771A. Between the two mean background values, the Indel method is approximately 78% lower.

These data provide a methodological basis for the identification of low-abundance donor DNA. Combining continuous sampling with a pre-infusion baseline facilitates observation of changes in the donor-derived DNA signal over time, providing information for studying the DNA release process following cell infusion.

The above indicators come from the organ transplantation examples in the patent disclosures. They are limited by their samples, input volumes, and experimental conditions. They do not directly represent the verified performance of IndelTrace 2.0 in the allogeneic CAR-T scenario; the background reduction ratio is not equivalent to the detection sensitivity improvement ratio.

Three Assays, Complementary Observations

Allogeneic CAR-T studies have used flow cytometry, CAR-ddPCR and donor genetic marker testing for multidimensional monitoring, and the analytical performance of plasma donor-derived cfDNA testing has also been studied. The methods examine different targets, so combining them supports a more complete interpretation of in vivo dynamics.

Table 2 Targets and information boundaries of the three complementary assays.

dd-cfDNA cannot distinguish between apoptosis and necrosis alone, nor can it be directly converted into cell death rate or viable cell survival time. Donor-derived DNA is also not equivalent to CAR-positive cell-derived DNA; additional evaluation of signal attribution is required when other cells from the same donor or in a multi-donor background are present. The detection ratios, denominators and input amounts of the three methods are different, so it is not appropriate to directly compare sensitivities based on percentages.

Collaborate on Monitoring Research for Allogeneic Cell Therapies

AlloDx welcomes collaboration with cell therapy companies, clinical research teams and academic institutions to combine IndelTrace 2.0 with flow cytometry, CAR-ddPCR and other methods, build continuous observation protocols around expansion, contraction and persistence, and explore how donor-derived DNA signals relate to in vivo changes in the therapeutic cells. Technical approaches and collaboration options are described in theallogeneic cell transplantation monitoring topic page。

AlloDx IndelTrace 2.0 | Identify trace donor signals and study dynamic changes in the body.

References

1. Moreno et al. Expansion, Persistence and Pharmacodynamic Profile of ADI-001, a First-in-Class Allogeneic CD20-targeted CAR Gamma Delta T Cell Therapy, in Patients with Relapsed/Refractory Aggressive B-cell Non-Hodgkin's Lymphoma. ASH 2023. In this study, donor genetic marker testing used cellular DNA, not plasma dd-cfDNA.
2. Wong L et al. Analytical performance validation of the AlloCell cfDNA assay. Conference abstract. Transplantation and Cellular Therapy. 2024;30(Suppl):S229, abstract 299. DOI: 10.1016/j.jtct.2023.12.299.

Disclaimer: This content is provided for medical and scientific information exchange only and does not constitute medical advice. Testing services should be performed and interpreted by qualified professionals at accredited institutions, and results must be interpreted together with clinical findings, donor and recipient genotypes and sample conditions; they cannot be used for clinical diagnosis alone. The methodological figures cited here come from organ transplantation examples in a published patent application and do not represent performance validated in the allogeneic CAR-T setting.