CD27⁻CD39⁻ CAR T Cell Count Predicts Response at Low Doses, Study Finds
核心洞察
Researchers identified the absolute number of CD27 (搜索)-negative, CD39 (搜索)-negative CAR T cells as a biomarker of therapeutic response in patients receiving low CAR T cell doses.
In the phase I/II HD-CAR-1 trial, 28 patients with relapsed or refractory B-cell malignancies (搜索) received third-generation CD19 (搜索)-directed CAR T cells across a wide dose range.
Low-dose responders showed enrichment of non-exhausted effector and effector memory-like CAR T cells, while non-responders had naive or dysfunctional CD39 (搜索)-positive populations.
A distinct population of CAR T cells may allow clinicians to predict whether an under-filled infusion product will still drive a therapeutic response, according to a study published in Nature Communications. Researchers from the Berlin Institute of Health (搜索) identified the absolute number of CD27 (搜索)-negative, CD39 (搜索)-negative CAR T cells in an infusion product as a potential biomarker of response at low doses — a finding with direct implications for manufacturing decisions when products fall short of their target cell count.
CAR T manufacturing does not always yield the intended dose. The researchers cite failure rates of 1–13 percent due to insufficient cell numbers, which can leave patients unable to receive treatment or force clinicians to consider administering an out-of-specification product.
Dose Escalation in HD-CAR-1
The discovery analysis drew on CAR T products and corresponding leukapheresis starting material from 28 patients enrolled in the phase I/II HD-CAR-1 trial (NCT03676504), a dose-escalation study evaluating third-generation CD19 (搜索)-directed CAR T cells in relapsed and/or refractory B-cell malignancies (搜索). Patients received doses ranging from 1 × 10⁶ to 200 × 10⁶ CAR T cells per square meter of body surface area.
Patients were categorized into three groups: high-dose responders (n = 8), low-dose non-responders (n = 12), and low-dose responders (n = 6) — a subset who achieved robust clinical responses despite receiving low cell doses. Response was not significantly associated with sex, age, previous allogeneic stem cell transplantation, or disease indication. Within the low-dose group, responses appeared durable, with a trend toward longer overall survival and progression-free survival among responders compared with non-responders.
Notably, low-dose responders and low-dose non-responders had comparable numbers of CAR-positive cells in their infusion products and similar in vivo CAR T cell expansion, as reflected by similar peak levels within the first 28 days after infusion. High-dose responders, by contrast, showed significantly higher frequencies of CAR-positive cells in the infusion product and higher absolute CAR T cell numbers in vivo.
A Phenotype Defined by Effector States
Using full-spectrum flow cytometry with a 36-marker panel, the team profiled 10,129,369 high-quality cells across the CAR T products. CD4 and CD8 CAR T cells from high-dose responders were enriched in naive and central memory-like states, consistent with previous reports. Low-dose responders displayed a distinct phenotype, highly enriched in non-exhausted effector-memory and effector-like states.
Within patients responding to therapy, the administered dose was negatively correlated with the relative abundance of effector memory-like CD8 CAR T cells, while naive-like and central memory-like populations correlated positively with dose. Comparing low-dose responders with low-dose non-responders produced closely overlapping results: non-responders were predominantly enriched in naive-like states or showed features of exhaustion and dysfunction, marked by elevated co-expression of CD39 (搜索) and PD1.
Absolute Numbers Outperform Relative Frequencies
The researchers hypothesized that in patients receiving few cells, the absolute number of subsets with immediate therapeutic efficacy might matter more than relative frequencies. Although patients receiving high doses had significantly higher numbers of most CAR T subpopulations, the absolute numbers of effector-like subsets were comparable between low-dose and high-dose responders.
Quantification of absolute numbers of CD4 and CD8 CAR T cell subsets generally outperformed relative frequency analyses in predicting response, both at low dose levels and across all dose levels. The combined absence of CD27 (搜索) and CD39 (搜索) expression proved a robust biomarker combination for identifying effector-like states without phenotypic exhaustion, outperforming previously suggested predictor combinations.
"The greater the number of these specific CAR T cells, the more effective the therapy was, even when the overall number of cells was relatively low," said senior author Michael Schmitt. "Our findings suggest that the absolute number of these highly functional cells could serve as a biomarker for predicting the success of low-dose CAR T-cell therapy."
Lactate dehydrogenase levels, a proxy for tumor burden, showed a more modest association with response and were largely independent of the biomarker combination. LDH was associated with overall survival but not progression-free survival, whereas the CD27 (搜索)⁻CD39 (搜索)⁻ biomarker combination was associated with PFS but not OS. In a composite stratification, patients with low LDH and high biomarker levels had the most favorable OS, while those with high LDH and low biomarker levels had the poorest outcomes.
Functional Characterization In Vitro
Co-culture experiments with sorted CD27 (搜索)⁻CD39 (搜索)⁻ CAR T cells from six healthy donors and Nalm6 leukemia cells at a 1:1 effector-to-target ratio across two sequential 72-hour rounds showed that the CD27⁻CD39⁻ subset maintained a stable terminal effector phenotype with minimal plasticity. CD27⁺CD39⁺ control cells generated a broader spectrum of phenotypes, including CD27⁻CD39⁻ progeny, and expanded robustly during the first co-culture, more than doubling in number. Both subsets mediated potent leukemia clearance compared with non-transduced controls, but when normalized for CAR T expansion and leukemia growth, CD27⁻CD39⁻ cells displayed more efficient killing in the first round, converging in the second round as both populations shifted toward a predominantly CD27⁻CD39⁻ effector phenotype.
Pre-Manufacturing Immune Landscape
Deep phenotyping of pre-manufacturing peripheral blood mononuclear cells from all 28 patients, capturing 14,266,855 high-quality cells, showed that low-dose responders had blood significantly enriched in CD8 T cells and in effector and effector memory states. Low-dose non-responders were highly enriched in myeloid populations, including classical and non-classical monocytes, conventional and plasmacytoid dendritic cells, and eosinophils/basophils. No major differences in cellular composition were observed between disease subtypes.
Single-cell proteo-genomics on pre-manufacturing blood, yielding 112,442 high-quality cells, revealed a marked pro-inflammatory, M1-like state in myeloid populations of low-dose responders, with upregulated TNF signaling via NF-κB and antigen presentation machinery, plus elevated MHC molecule expression on monocytes and dendritic cells. Myeloid cells from non-responders showed reduced antigen presentation and diminished pro-inflammatory signaling, and were enriched in non-classical monocytes expressing complement regulators CD55 (搜索), clusterin, and CR1. CD55 expression correlated with M2-like polarization pathways including STAT3 and IL-10 signaling, and inversely with M1-like features.
Reversible Myeloid Influence on Product Quality
Analyses linking pre-manufacturing myeloid traits to post-manufacturing T cell states found that high antigen presentation capacity and M1-polarization signatures in monocytes correlated positively with production of functional CAR T cells and inversely with dysfunctional products. Signatures of TGF-β signaling and IL-4 or IL-10 production were linked to dysfunctional CD39 (搜索)⁺ CAR T cells.
In co-culture experiments, PBMCs exposed to M2-polarizing cytokines (IL-4, IL-10, IL-13, and TGF-β) before being used directly as manufacturing starting material generated significantly more CD39 (搜索)⁺ CAR T cells with a dysfunctional phenotype and showed a trend toward reduced targeted cell killing. When cytokines were washed out before manufacturing, CD39⁺ frequencies remained comparable to untreated controls, and cytotoxic killing capacity was restored — indicating the effect is largely transient and can be prevented by removing the myeloid-like cytokine milieu prior to CAR T generation.
Validation Across Products and Targets
The biomarker was tested in an independent cohort of 42 patients spanning B-cell non-Hodgkin lymphoma (n = 25) and multiple myeloma (搜索) (n = 17), second-generation CAR constructs with either CD28 or 4-1BB co-stimulatory domains, and anti-CD19 (搜索) or anti-BCMA (搜索) targets. Patients received four commercial products: Breyanzi, Yescarta, Kymriah, and Carvykti. Because of regulatory constraints, biomarker profiling of commercial products was performed on the earliest available post-infusion peripheral blood sample (day 1–10) as a proxy for the infused product; such samples were available for 29 patients.
In B-NHL, the CD27 (搜索)⁻CD39 (搜索)⁻ combination stratified responders from non-responders when quantified as absolute CAR T cell numbers, with similar stratification using relative frequencies given the comparable dose range in that setting. In multiple myeloma (搜索), all but one patient responded to anti-BCMA (搜索) therapy; the single non-responder ranked among the lowest in both absolute counts and relative frequencies of CD27⁻CD39⁻ CAR T cells, though the authors note that more non-responders will need to be investigated for confirmation. The biomarker outperformed previously described biomarkers for CD19 (搜索)-directed therapies in both sensitivity and specificity when using absolute numbers. Myeloid-associated markers were also validated in leukapheresis samples: CD55 (搜索) was significantly elevated on non-classical monocytes in non-responders, while CD69 was enriched on conventional dendritic cells and classical monocytes in responders.
The authors conclude that the CD27 (搜索)⁻CD39 (搜索)⁻ biomarker combination is reproducibly associated with clinical response across different diseases, CAR targets, and CAR constructs, suggesting it captures a core biological property of highly potent and therapeutically effective CAR T cells.