Cell-delivered CD3×CD19 T-cell engager by STAb-T19 cells mediates leukemia clearance with limited systemic exposure

CD19-targeted T cell-engaging bispecific antibodies (TCEs) have demonstrated efficacy in B-cell acute lymphoblastic leukemia (B-ALL) but require sustained systemic exposure, increasing the risk of dose-limiting toxicities. STAb-T19 cells are engineered T lymphocytes designed to constitutively secrete a CD19×CD3 TCE (19-TCE), combining cellular delivery with local antibody-mediated T-cell redirection. While prior preclinical studies established their potent antitumor activity, the systemic pharmacokinetics, biodistribution, and safety of TCE-secreting T cells remain unclear.

We performed a comprehensive preclinical evaluation of STAb-T19 cells in NSG xenograft models of B-ALL. Quantitative short- and long-term pharmacokinetics of circulating 19-TCE were determined using a validated functional bioassay. Longitudinal biodistribution and persistence of engineered T cells were assessed across blood and tissues, complemented by spatial histopathology, immunohistochemistry, and sex-disaggregated analyses.

Intravenous administration of STAb-T19 cells resulted in minimal early systemic exposure to 19-TCE, with circulating concentrations remaining low during the first 48 hours. In leukemia-bearing mice, sustained and controlled serum 19-TCE levels were detected over time, consistent with in vivo secretion by persisting engineered T cells. STAb-T19 cells exhibited limited biodistribution, predominantly in peripheral blood, spleen, and lung, with no detectable accumulation in the brain. Histopathological analysis revealed no treatment-related tissue damage. Despite limited systemic TCE exposure and the absence of extensive cellular expansion, complete leukemia clearance was achieved.

STAb-T19 cells exhibit a distinct immunopharmacological profile characterized by controlled systemic TCE exposure and antitumor activity in a preclinical setting. These findings provide initial insights into the pharmacology of TCE-secreting cellular therapies and support their clinical development in CD19⁺ hematological malignancies, while recognizing that clinical safety remains to be established.

Graphical abstract

Graphical abstract.

Introduction

Adoptive T-cell therapies and T cell-engaging bispecific antibodies (T-cell engagers, TCEs) have transformed the treatment landscape of B-cell malignancies.1 Systemically administered TCEs, including the CD19×CD3 format, induce potent antitumor activity by redirecting endogenous T cells toward malignant B cells.2 However, non-IgG-based TCEs have short plasma half-lives due to their small size, requiring continuous systemic infusion to maintain therapeutic activity.3 4 In contrast, IgG-based TCEs have been engineered with extended serum half-lives that allow intermittent dosing but may result in prolonged systemic exposure.5 Sustained high circulating concentrations of TCEs can increase the risk of off-tumor toxicity and dose-limiting adverse effects.6

The STAb-T platform represents an alternative approach that combines cellular delivery with antibody-mediated T-cell redirection by engineering T cells to secrete TCEs in vivo.7–10 Prior studies demonstrated that STAb-T19 cells mediate durable leukemia eradication, prevent CD19 downmodulation-mediated escape, and recruit bystander T cells, achieving superior antitumor activity compared with conventional CD19-targeting chimeric antigen receptor (CAR)-T therapies in preclinical models.7

Despite these advances, the in vivo immunopharmacology of TCE-secreting T cells remains poorly defined.11–14 In contrast to systemically administered TCEs, whose efficacy depends on sustained plasma exposure,3 4 STAb-T19-secreted TCEs are not IgG-based molecules and therefore are expected to exhibit distinct pharmacokinetic (PK) properties, including rapid systemic clearance despite continuous in vivo production. Moreover, unlike conventional CAR-T cell products, whose activity depends largely on in vivo expansion,15 16 STAb-T cells represent a novel therapeutic modality in which the TCE production is spatially distributed and dynamically regulated in vivo, and in which the recruitment of endogenous bystander T cells may reduce the dependence on extensive expansion of the engineered T-cell population. As a result, their systemic exposure, tissue distribution, and pharmacological behavior cannot be inferred from existing therapeutic paradigms.

Here, we provide a comprehensive preclinical characterization of STAb-T19 cells focused on quantitative PKs, tissue biodistribution, spatial histopathology, and sex-disaggregated analyses in leukemia-bearing mouse models. Overall, these studies establish the immunopharmacological framework required for clinical translation of TCE-secreting cellular therapies.

Methods

Generation of STAb-T19 cells

Peripheral blood (PB) samples from volunteer healthy donors were provided by Madrid Blood Transfusion Center. All donors provided written informed consent in accordance with the Declaration of Helsinki. PB mononuclear cells (PBMCs) were isolated by density-gradient centrifugation using Ficoll-Paque Plus. PBMCs were directly activated with anti-CD3/CD28 Dynabeads (Thermo Fisher Scientific) at a 3:1 bead-to-cell ratio and cultured in Roswell Park Memorial Institute 1640 supplemented with 10% fetal bovine serum and 10 ng/mL interleukin (IL)-7 and IL-15 (Miltenyi Biotec) at 37°C and 5% CO_₂. After 24 hours, activated T cells were left non-transduced (NT-T cells) or transduced with 19-TCE (STAb-T19 cells) encoding lentiviruses at a multiplicity of infection of 10. Beads were removed 72 hours post-transduction, and T cells were further expanded in the medium described above. A 6–10 days expansion period was carried out before conducting experiments. Transduction efficiency (percentage of 19-TCE⁺T cells), determined by intracellular 19-TCE expression, as well as phenotype and viability, were analyzed by flow cytometry.

Flow cytometry

Flow cytometry staining was performed using fluorochrome-conjugated antibodies against surface and intracellular markers (see online supplementary Methods for full details). For surface staining, samples were incubated for 30 min at 4°C in the dark, washed, and fixed with 2% paraformaldehyde. Intracellular staining was performed using a commercial kit following the manufacturer’s instructions. Data acquisition was carried out on BD flow cytometers and analyzed using FlowJo software.

Animal models

5–6 weeks old female and male NOD.Cγ-Prkdcscid-IL2rgtm1Wjl/SzJ mice (NSG; The Jackson Laboratory) were used in all experiments. Animals were housed under specific pathogen-free conditions. Sex was included as a biological variable in all analyses. For short-term PK studies, healthy tumor-free NSG mice were used. For long-term PK, biodistribution, efficacy, and toxicity studies, NSG mice bearing human CD19⁺ tumors (NALM6^Luc) were employed.

Short-term TCE pharmacokinetics

To assess early PKs of the TCE secreted by STAb-T19 cells, a total of 12 healthy NSG mice (6 females and 6 males) received a single intravenous infusion of 5×10⁶ STAb-T19 cells, of which 30% (1.5×10⁶) were 19-TCE⁺. At early time points after injection (6, 24, and 48 hours), two animals of each sex were euthanized, while one phosphate-buffered saline-treated mouse of each sex served as a negative control for each time point, and PB samples were collected. Functional 19-TCE concentrations in serum were estimated using a validated cell-based bioassay calibrated against blinatumomab.17 Briefly, an activation assay was conducted in which Jurkat and NALM6 cells were co-cultured for 24 hours in the presence or absence of the corresponding serum samples. Jurkat cell activation was subsequently quantified by measuring the expression of CD69 by flow cytometry. To generate a standard curve, co-cultures were performed with different concentrations of blinatumomab (50 ng/mL to 49 pg/mL). The quantification threshold was set at 49 pg/mL (online supplemental figure 1). Because the standard curve was generated using blinatumomab, the resulting values represent blinatumomab-equivalent functional concentrations rather than absolute 19-TCE concentrations.

Long-term TCE pharmacokinetics, antitumor efficacy, and cellular biodistribution

A total of 48 NSG mice (24 females and 24 males) were intravenously injected with 1×10⁶ NALM6^Luc cells. 2 days later, mice were randomized in a sex-balanced manner into two treatment groups, receiving a total of 5×10⁶ NT-T or 1.5×10⁶ 19-TCE⁺ T cells (corresponding to 5×10⁶ STAb-T19 cells, containing 30% 19-TCE⁺). As treatment was initiated at an early time point following tumor inoculation, prior to robust establishment of measurable bioluminescent signal, randomization was based on uniform tumor cell inoculation and balanced allocation across experimental groups. This early treatment schedule was selected in accordance with previously described early-intervention NALM6 xenograft protocols18 19 to ensure synchronized disease establishment and minimize inter-animal variability. Tumor growth was monitored over time using bioluminescence imaging (BLI).

For longitudinal assessment of long-term PKs, cellular biodistribution, and tissue localization, animals were sacrificed at defined time points post-T cell infusion (days 7, 14, 21, and 28). At each time point, three mice per sex and treatment group were euthanized and tissues (PB, bone marrow (BM), spleen, liver, lungs, and brain) were harvested and split for molecular and cellular analyses or fixed for immunohistochemistry (IHC). In addition, plasma samples were collected to evaluate long-term exposure to the secreted TCE as previously indicated for the short-term PK study.

Cellular biodistribution of human T cells and, more specifically, of 19-TCE⁺ T cells, was assessed by using complementary methods. PB and BM samples were processed immediately after collection and analyzed by flow cytometry, whereas the remaining tissues (spleen, lungs, liver and brain) were preserved in RNA later at −20°C and analyzed by reverse transcription quantitative polymerase chain reaction (RT-qPCR), using specific primers for human CD3 (hCD3 (F): 5’-CTACCCCAGAGGAAGCAAACC-3’; hCD3 (R): 5’-GACATCACATCCATCTCCATGC-3’), to detect human T cells, and for A3B1 transcripts, corresponding to the CD19-binding scFv used in the 19-TCE construct, (A3B1 (F): 5’-AACTTCGGCAACAGCTTCATG-3’; A3B1 (R): 5’-GCTTTCCAGGTTGCTGGCGAT-3’), to detect 19-TCE⁺ T cells, normalized to mouse GAPDH expression (mGAPDH (F): 5’-GCGAGACCCCACTAACATCA-3’; mGAPDH (R): 5’-GGCGGAGATGATGACCCTTT-3’). These data were generated using well-established, standardized procedures by the Genomics Unit at Madrid Science Park Foundation (Spain).

Tissue localization and toxicity assessment

All steps related to IHC, including tissue processing and staining, were performed by the CNIO Histopathology Unit. Briefly, formalin-fixed, paraffin-embedded tissue sections were stained with H&E for morphological evaluation to assess tissue integrity and presence of tissue damage. Histopathological findings were evaluated by an expert pathologist and semiquantitatively scored using a four-tier scale: 0 (absent), 1 (mild), 2 (moderate), and 3 (severe), based on tissue architecture alterations, neoplastic infiltration, and immune cell infiltration. A double immunostaining was performed using an anti-CD3 antibody (alkaline phosphatase, red) to identify T lymphocytes, together with nucleoli staining (3,3’-diaminobenzidine, brown) to distinguish tumor cells (CD3⁻/nucleoli⁺) from T cells (CD3⁺/nucleoli⁺). Digital image analysis was performed using QuPath software.

Statistical analysis

Data are presented as mean±SD unless otherwise stated. Statistical comparisons between two groups were performed using a two-tailed unpaired Student’s t-test. A p value<0.05 was considered statistically significant. No statistical testing was performed for short-term PK analyses due to limited sample size, and these data are presented descriptively.

Results

Systemic exposure and pharmacokinetics of secreted 19-TCE

To characterize early systemic exposure to the 19-TCE secreted by STAb-T19 cells, we performed short-term PK analyses in non-leukemia-bearing NSG mice following intravenous infusion of the STAb-T19 cell product (figure 1A–C), where panel A shows transduction efficacy, while panel B illustrates that the phenotypic profile is comparable between STAb-T19 and NT-T cells. Blinatumomab-equivalent functional 19-TCE concentrations were below the limit of quantification at 6 hours post-infusion and increased only modestly at 24 and 48 hours. Overall, similar exposure profiles were observed in male and female mice across all the analyzed time points (figure 1D). Importantly, these concentrations remained lower than ranges reported for systemically administered TCEs in clinical settings; however, direct quantitative comparisons are not appropriate due to cross-species and modality differences20 (figure 1D).

Figure 1.

Short-term PKs of 19-TCE in blood following i.v. administration of STAb-T19 cells. (A) Percentage of 19-TCE+cells on day 11 post-transduction. (B) Distribution of CD4+ and CD8+ T-cell subsets and proportions of effector, effector memory, central memory and naïve T cells, among NT-T or STAb-T19 cells. The percentage of 19-TCE+T cells was adjusted to 30% prior to administration. (C) Experimental timeline of NSG mice receiving i.v. STAb-T19 cells (n=6 per sex) or PBS (n=3 per sex) as control. (D) At the indicated time points, two STAb-T19-treated mice from each sex and one PBS-treated control mouse from each sex were euthanized, and blood was collected for serum isolation. Circulating blinatumomab-equivalent functional 19-TCE levels were quantified using a validated and calibrated cell-based calibrated bioassay (online supplemental figure 1). Data are presented as mean±SD; each dot represents an individual mouse. i.v., intravenous; NT-T, non-transduced T cells; PBS, phosphate-buffered saline; TCE, T-cell engager.

Anti-leukemic activity and histopathological assessment of STAb-T19 cells

Using a B-cell acute lymphoblastic leukemia (B-ALL)-bearing NSG xenograft model (figure 2A), we evaluated the antitumor activity and safety profile of STAb-T19 cells. Consistent with previous observations,7 STAb-T19 treatment was not associated with treatment-related body weight loss (figure 2B). All mice from both sex groups treated with STAb-T19 cells showed complete leukemia eradication, as demonstrated by longitudinal BLI (figure 2C,D), whereas mice receiving NT-T cells showed progressive tumor growth.

Figure 2.

In vivo antitumor activity and histopathological characterization of mouse tissues following adoptive transfer of NT-T or STAb-T19 cells. (A) Experimental timeline of the cell line-derived xenograft model. NSG mice (n=12/sex/group) were i.v. injected with NALM6Luc cells, followed by i.v. administration of 5×106 T cells, either NT-T or STAb-T19 cells. At the indicated time points, three female and three male mice per group were euthanized for blood and tissue analysis. (B) Longitudinal monitoring of body weight. (C) Quantification of total bioluminescent radiance to assess leukemia burden. Data are presented as mean±SD. Statistical significance was determined using an unpaired Student’s t-test. (D) Representative BLI of male and female mice demonstrating disease progression over the study period. (E) Representative images of double immunohistochemical staining for nucleoli (DAB, brown) and CD3 (AP, red) in tissue samples collected 7 and 14 days post-adoptive transfer, showing the distribution and relative abundance of human tumor cells and T cells across tissues. (F) High-magnification views of representative organ sections collected on day 14, highlighting the spatial localization of human tumor cells (yellow arrows or circles) and human T cells within the tissue microenvironment. Scale bars are indicated for each organ. (G) Quantitative immunohistochemical analysis of mouse tissues following adoptive transfer of human NT-T or STAb-T19 cells at the indicated time points. Human CD3⁺ T cells and tumor cells (identified as nucleoli⁺ CD3⁻) were quantified in tissue sections at the indicated time points. Data are shown as mean±SD; each dot represents an individual mouse. AP, alkaline phosphatase; BLI, bioluminescence imaging; DAB, 3,3’-diaminobenzidine; i.v., intravenous; NT-T, non-transduced T cells; TCE, T-cell engager.

Histopathological evaluation of spleen, liver, lung, and brain sections by H&E staining revealed no treatment-related macroscopic or histological abnormalities in STAb-T19-treated mice compared with NT-T controls (online supplemental figure 2, online supplemental table 1). No abnormalities or immune cell infiltrates were observed in the brain. Spleen, liver, and lung showed preserved architecture with only mild immune cell infiltration in both groups (online supplemental figure 2, online supplemental table 1). Spatial immunohistochemical analyses further supported these findings. Double staining for nucleoli (human cells) and CD3 (human T cells) demonstrated malignant cell infiltration exclusively in NT-T-treated animals, whereas STAb-T19-treated mice showed no detectable tumor cells (figure 2E–G). Mild immune cell infiltration in peripheral organs was consistent with expected trafficking of human T cells in this xenogeneic setting.

Biodistribution, persistence, and long-term PK in leukemia-bearing mice

To further characterize the in vivo behavior of the infused T cells in the NALM6 leukemia-bearing mice described above, we evaluated the biodistribution and persistence of NT-T and STAb-T19 cells across tissues (figure 3A,B). Quantitative analyses revealed overall low frequencies of human T cells in most organs at early time points (days 7–14). Cells were predominantly detected in PB, spleen, and lungs, whereas no accumulation was observed in the brain. Direct comparison between NT-T-treated and STAb-T19-treated groups demonstrated comparable tissue distribution patterns (figure 3A). From day 21 onward, a marked increase in total human T cells was detected in PB, BM, spleen, and lungs (figure 3A,B), most likely reflecting the onset of xenogeneic graft-versus-host disease (xGvHD), a known limitation of this model.

Figure 3.

Biodistribution, sex-specific dynamics, and long-term PKs of STAb-T19 cells and 19-TCE in vivo. (A) Comparative biodistribution of human T cells (CD3⁺ cells) in the NT-T-treated and STAb-T19-treated mice (figure 2) across multiple organs at the indicated time points. (B) Total T-cell infiltration levels across organs displayed side by side for direct comparison in the STAb-T19-treated group. (C) Sex-disaggregated distribution of 19-TCE⁺ STAb-T19 cells in male and female STAb-T19-treated mice. (D) Pooled quantification of 19-TCE⁺ cell infiltration across organs. Results are shown as mean±SD of six biological replicates. (E, F) Percentage of 19-TCE⁺ cells in PB of treated males and female mice, respectively. (G, H) Corresponding serum blinatumomab-equivalent functional concentrations of 19-TCE (pg/mL), measured by cell-based functional bioassay at the indicated time points. Each dot represents an individual mouse. BM, bone marrow; NT-T, non-transduced T cells; PB, peripheral blood; PKs, pharmacokinetics; TCE, T-cell engager.

A comparative analysis of male and female STAb-T19-treated mice was performed to assess potential sex-related differences in cellular biodistribution and 19-TCE levels in the long-term (figure 3C). Slightly higher frequencies of engineered T cells were observed in PB and BM of female mice, whereas similar levels were observed across other organs (figure 3C). Overall, pooled analysis of 19-TCE-secreting STAb-T19 cells (19-TCE⁺ T cells) revealed detectable transcript levels primarily in PB and lung tissue; however, their abundance remained below 6% in all assessed tissues (figure 3D). Analysis of 19-TCE⁺ T-cell frequencies within the total human STAb-T19 cell compartment further supported persistence of engineered cells throughout the study period (online supplemental figure 3).

To assess systemic 19-TCE exposure under conditions that more closely resemble therapeutic activity, we performed long-term PK analyses in this B-ALL-bearing NSG xenograft model. This setting enables evaluation of circulating 19-TCE levels in the context of antigen engagement, leukemia interaction, and T-cell expansion. Following infusion of STAb-T19 cells, blinatumomab-equivalent functional 19-TCE concentrations remained consistently detectable in serum over an extended period, supporting sustained in vivo production by persisting engineered T cells (figure 3E–H). Notably, the progressive increase in 19-TCE levels over time (figure 3G,H) is likely attributable, at least in part, to the expansion of human T cells associated with the onset of xGvHD. Therefore, late 19-TCE measurements should be interpreted in the context of model-associated human T-cell expansion and distinguished from earlier pharmacological observations obtained before the onset of overt xGvHD. Long-term PK analyses further showed comparable circulating 19-TCE concentrations in male and female mice (figure 3G,H), with no significant differences in systemic exposure.

These data indicate that STAb-T19 administration is associated with limited systemic exposure to circulating 19-TCE while retaining potent antitumor activity in this preclinical model.

Discussion

This study delivers the first in vivo immunopharmacological characterization of STAb-T19 cells, describing the systemic exposure, tissue biodistribution, and safety profile of a TCE-secreting T-cell therapy. While earlier studies showed the potent antitumor efficacy and relapse-preventing capacity of this approach in vivo, here we address a key barrier to clinical translation by examining its PK and tissue-distribution properties in a disease-relevant model of B-ALL.

Complete leukemia eradication was achieved despite relatively limited circulating 19-TCE levels in this model. However, local TCE concentrations and exposure-response relationships were not directly measured, and therefore these observations should be interpreted cautiously. These observations suggest that potent antitumor activity can be achieved despite limited systemic exposure in this preclinical model.3 4 This pharmacological profile is consistent with the hypothesis that STAb-T19 cells may provide sustained TCE production at sites of tumor involvement. However, because local TCE concentrations and exposure-response relationships were not directly measured, this proposed mechanism remains to be experimentally validated.

This behavior defines a distinct profile that differs not only from the systemically administered TCEs but also from conventional CAR-T cell therapies, whose efficacy largely depends on extensive in vivo expansion.21–24

The biodistribution profile of STAb-T19 cells further supports a controlled pharmacological behavior, with engineered T cells predominantly detected in PB, spleen, and lung, and no evidence of accumulation in the brain. Analysis of 19-TCE⁺ cell frequencies within the human T-cell compartment further supported persistence of engineered cells during the analyzed period. Although intravenously administered STAb-T19 cells transiently accumulated in the lungs shortly after infusion, the secreted 19-TCE is a soluble protein that is expected to freely diffuse within the vascular compartment and reach systemic circulation. Overall, these findings are consistent with expected trafficking patterns in this model and suggest that constitutive TCE secretion does not result in a clear altered tissue homing. At later time points, an increased number of human T cells were detected across multiple tissues, likely reflecting xGvHD-associated expansion inherent to the NSG model and contributing to the observed increase in circulating 19-TCE levels. Accordingly, these late PK measurements should be interpreted in the context of model-associated T-cell expansion rather than as a direct measure of the therapy pharmacological behavior. Importantly, in vitro analyses performed in the presence or absence of CD19⁺ target cells at different effector:target ratios reveal no substantial changes in 19-TCE transcript expression, supporting relatively stable EF1α-driven transgene expression under the conditions evaluated (online supplemental figure 4).

Histopathological and immunohistochemical analysis provided complementary descriptive evidence supporting the biodistribution and pharmacological findings. No clear structural abnormalities or treatment-related tissue damage were detected in any of the analyzed organs, including the brain. Although no treatment-related histopathological abnormalities were observed, the NSG model does not recapitulate clinically relevant immune-mediated toxicities such as cytokine release syndrome or immune effector cell-associated neurotoxicity syndrome. Therefore, these findings support a favorable preclinical pharmacological profile but should not be interpreted as evidence of improved clinical safety. The absence of detectable tumor cells in STAb-T19-treated mice, together with preservation of normal tissue architecture, is consistent with the robust antitumor activity observed in this setting. Sex-disaggregated analyses did not reveal clear differences in systemic exposure, biodistribution, or efficacy; however, these comparisons should be interpreted cautiously given the exploratory nature of some analyses.

This study presents certain limitations. The NSG xenograft model lacks a fully competent immune system and is susceptible to xGvHD, which likely contributes to late T-cell expansion and may influence long-term PK measurements. Furthermore, the model does not recapitulate human cytokine release dynamics or neurotoxicity mechanisms observed clinically with T cell-redirecting immunotherapies. While alternative models such as major histocompatibility complex class I/II-deficient mice may reduce xGvHD-related effects,25 the NSG model was selected for consistency with prior studies and its widespread use in preclinical evaluation of T-cell therapies. Besides, this study does not include direct benchmarking against CAR-T cells or recombinant TCEs evaluated in parallel within the same experimental setting. Functional comparison with conventional CD19 CAR-T cells was previously reported for the STAb-T19 platform,7 although dedicated head-to-head studies incorporating both efficacy and pharmacokinetic analyses were not performed here. In particular, direct experimental comparison with blinatumomab in murine models is especially challenging because clinically relevant drug exposure requires continuous intravenous infusion, a dosing regimen that is technically difficult to replicate in NSG xenograft models. As such, conclusions regarding comparative efficacy or pharmacological advantages should be interpreted with caution. Finally, treatment was initiated at an early time point following tumor inoculation, corresponding to a low tumor burden setting. This early intervention design is commonly used in aggressive NALM6 xenograft models to ensure synchronized disease establishment and reduce inter-animal variability during evaluation of T cell-based therapies. Nevertheless, this approach does not fully recapitulate established or advanced leukemia and therefore, future studies evaluating efficacy in more established disease models will be required to further define therapeutic potential. In addition, although the present study provides an initial immunopharmacological characterization of STAb-T19 cells, future studies including larger animal cohorts will be required to validate these findings and strengthen the statistical robustness of the pharmacological analyses.

Collectively, these data describe the immunopharmacological profile of STAb-T19 cells, characterized by limited systemic TCE exposure, restricted tissue biodistribution without brain accumulation, absence of detectable tissue damage and potent anti-leukemic activity despite limited circulating TCE concentrations. Rather than representing a variation of existing CAR-T or systemically infused TCE approaches, these results provide initial insights into the behavior of TCE-secreting cellular therapies and support their continued preclinical and clinical investigation.

Conclusion

STAb-T19 cells represent a T cell-based approach capable of mediating antitumor activity under conditions of limited systemic TCE exposure and restricted biodistribution in a preclinical model. This study extends the initial proof-of-concept description of STAb-T19 cells by providing an in vivo pharmacological characterization of TCE-secreting cellular therapies. Although focused on STAb-T19 cells, this study contributes to the broader understanding of TCE delivery by engineered T cells and may support the further development of this class of therapeutic strategies.

Data availability statement

Data are available upon reasonable request. All data relevant to the study are included in the article or uploaded as supplementary information. Materials can be shared on an MTA basis.

Ethics statements

Patient consent for publication

Not applicable.

Ethics approval

Human samples were obtained after written informed consent from the donors, and all studies were performed according to the principles expressed in the Declaration of Helsinki and approved by the Instituto de Salud Carlos III Research Ethics Committee (CEI PI 15_2024). The in vivo studies were conducted at the Spanish National Cancer Research Centre (CNIO) in compliance with institutional animal care and ethical guidelines, following approval by the CNIO Institutional Animal Care and Use Committee (IACUC) and the Research Ethics and Animal Welfare Committee (CEyBA) of the Instituto de Salud Carlos III (ISCIII), and authorization by the Consejería de Medio Ambiente, Agricultura e Interior of the Comunidad de Madrid under protocol PROEX 272.2/23.