Dextran-based T-cell expansion nanoparticles for manufacturing CAR T cells with augmented efficacy

Domain: chemistry
Published: 2026-01-20
Editorial outcome: major_revision

Abstract
Adoptive T cell therapy using chimeric antigen receptor (CAR) engineered T cells is currently being explored in multiple cancer types beyond leukemia/lymphoma. A key step in CAR-T cell manufacturing is the activation and expansion of T cells, which facilitates viral transduction, however, may hamper T cell fitness and reduce in vivo persistence. “T-Expand” is developed for T cell activation and expansion, comprising dextran-based nanoparticles conjugated with anti-CD3 and anti-CD28 antibodies. The nanoparticles trigger robust polyclonal expansion of human T cells with efficiency in the range of commercial microbeads (Dynabeads™). Engineered in the presence of T-Expand, CD19 CAR T cells display enhanced proliferative capacity, cytotoxicity and persistence in vitro, and furthermore, exhibit potent anti-lymphoma activity in mouse models, resulting in complete tumor clearance at one fourth of the CAR T cell dose. Importantly, T-Expand is biocompatible with no observed toxicity, circumventing removal steps after T cell expansion compared to DynabeadsTM. As a biocompatible T cell expansion platform, T-Expand simplifies the manufacturing process while enhancing T cell persistence and functionality, and thereby holds promise for increasing clinical efficacy of CAR T cell therapy.

Introduction
T cells genetically modified to express a chimeric antigen receptor (CAR) has shown tremendous potential against a variety of hematological malignancies1,2. Despite the clinical success, a substantial number of patients do not gain long-term treatment benefit, potentially owing to T cell intrinsic factors that lead to lack of functional capacity and in vivo persistence3,4. While the genetic modification of T cells requires potent T cell activation to enter a proliferative state susceptible to gene modification, the expansion phase imprints the functional characteristics on the resulting CAR T cells5.

Current conventional CAR-T cell expansion platforms are based on immobilized anti-CD3 and anti-CD28 antibodies on magnetic microbeads (DynabeadsTM) or on a polymer matrix (TransActTM)6,7. However, these artificial antigen-presenting cell (aAPC) platforms can lead to overstimulation of T cells causing cell exhaustion and ultimately result in lack of persistence after infusion8. Alternative micro-scale aAPC platforms such as lipid-coated mesoporous silica scaffolds promote T cell expansion with demonstrated anti-tumor activity in vivo9,10. Recently, aAPC platforms based on graphene oxide flakes and viscoelastic microgel have been reported11,12. These platforms could enhance expansion kinetics as compared to conventional platforms. However, graphene oxide based aAPCs possess a technical challenge when isolating expanded T cell products from scaffolds, which may prevent generation of therapeutic CAR T cell products9. Furthermore, the microgel-based aAPC platforms have difficulty reproducing and maintaining mechanical properties when used at large scale, thus posing challenges for scale-up manufacturing13.

To address these limitations, we designed a unique dextran-based nano-aAPC, called T-Expand, comprising dextran nanoparticles (NPs) decorated with anti-CD3 and anti-CD28 antibody on its surface. Dextran presents notable advantages, such as high density of modifiable functional groups, which enables conjugation of high amount of T cell stimulatory molecules. We hypothesize that enhancing the surface density of these stimulatory molecules could effectively compensate for the reduced physical contact area inherent to nanoscale aAPCs, thereby maintaining robust T-cell expansion. Meanwhile, we hypothesize that the avidity between T-Expand and T cells is not as strong as that between microbeads and T cells. Upon T-cell activation, T-Expand is either internalized by T cells via the TCR or detached from the T-cell surface, which may help prevent T-cell exhaustion by avoiding prolonged stimulation. In addition, T-Expand is biocompatible, thereby avoiding the extra separation step and therefore suitable for large-scale production.

We demonstrate that T-Expand offers an alternative approach to enhance the quality of CAR T-cell products and simplify the CAR T-cell manufacturing process with beneficial functionality. CAR T cells expanded with T-Expand exhibit significantly improved persistence and therapeutic efficacy in both in vitro and in vivo settings. Specifically, CD19 CAR T cells expanded using T-Expand display reduced exhaustion markers compared to those generated using DynabeadsTM. Additionally, T-Expand-produced CAR T cells are applicable for long-term tumor clearance, underscoring their potential clinical advantages over conventional aAPC platforms.

Results
Characterization of the T-Expand
Dextran was selected as the material for T-Expand due to its biodegradability, biocompatibility, abundant functional groups and ease of chemical modification14–16. Herein, we modified the dextran polymer with multiple copies of azido groups, which enabled flexible conjugation of antibodies in the aqueous phase via bio-orthogonal approaches such as azido-alkyne cycloaddition and Staudinger ligation (Fig. 1a). Firstly, the azido linker was activated using carbonyldiimidazole (CDI) (Supplementary Figs. 1– 4). Subsequently, the CDI-functionalized azido linker was reacted with the dextran to produce azido-modified dextran polymer (Supplementary Figs. 5, 6), which was then acetylated to form the acid-sensitive azido-dextran polymer (Supplementary Fig. 7). The resulting hydrophobic azido-dextran NPs (“naked” NPs) were prepared by double emulsion technology. Measurement by NP tracking analysis (NTA) system showed that the naked NPs had a homogenous size distribution of about 150 nm (Supplementary Fig. 8a, b).

The hydrophobicity of the azido linker plays an important role in determining the orientation of the azido groups during the water/oil/water emulsion formation17. To investigate the effect of the linker on the amount of azido groups on the surface, we selected two types of linkers, hydrophobic azido linker and amphiphilic azido linker with three PEG segments, to modify the dextran backbone before synthesizing the NPs. The distribution and orientation of the two linkers were investigated using a click-based colorimetric labeling method. Specifically, Cy5 molecules with dibenzocyclooctyne (DBCO) groups were clicked onto the naked NPs via click chemistry, and the overall density of azido group was determined by measuring the optical density (OD) value of Cy5 on the NPs. Higher OD indicates a higher number of azido functional groups on the surface of naked NPs, whereas lower absorption suggests that more azido groups are oriented inside the NPs (Fig. 1b). The amphipathic linker with PEG segments orients more at the water–organic solvent interface, producing a higher density of azido groups on the NP surface compared to NPs without PEG segments (Fig. 1c). This linker was applied for further use. The available azido groups on the surface of NPs for antibody conjugation was 24.6 nmol per mg dextran NP as determined by UV-Vis spectroscopy analysis (Supplementary Fig. 8c, d).

Next, DBCO modified anti-CD3 and anti-CD28 antibodies were confirmed by nanodrop measurement, which showed the distinct absorption peak of DBCO from 300–320 nm, indicating that the DBCO linker has been conjugated to the antibody (Supplementary Fig. 9a, b). Furthermore, the DBCO modified antibodies were conjugated to the surface of the naked NPs through click chemistry. After the conjugation, the zeta potential decreased from −8.5 ± 0.25 to −12.5 ± 0.87 mV which is similar to the surface charge of DynabeadsTM (Fig. 1d).

To evaluate whether the conjugated anti-CD3 and anti-CD28 antibodies were successfully linked and retained their function for T cell activation, the T-Expand was co-cultured with primary T cells and analyzed for the expression of activation markers (CD25 and CD69) via flow cytometry. T cells were activated following formation of T cell clusters visible by microscopy (Supplementary Fig. 9c), with subsequent expression percentage of CD25 and CD69 similar to those induced by Dynabeads™ (Fig. 1e). In contrast, the naked NPs did not activate T cells, demonstrating the role of the conjugated antibodies on T-Expand.

To verify whether the surface stoichiometric ratio of anti-CD3 to anti-CD28 on T-Expand precisely mirrored the input feed, anti-CD3 was labeled with Cy5-NHS and anti-CD28 with Alexa Fluor 488-NHS, and both antibodies were simultaneously functionalized with DBCO-Sulfo-NHS (Supplementary Fig. 10a, b). In every case, the measured surface stoichiometry matched the input ratio, confirming feed-ratio control over ligand composition (Source data).

Next, to determine the absolute amount of antibody loading without potential competition between fluorophore and DBCO labeling, we employed a single bifunctional tag-anti-CD28 was derivatized with Cy5-DBCO-NHS (Supplementary Fig. 10c, d). Applying the calibration curve to the post-reaction supernatants allowed us to calculate the antibody mass bound per mg of dextran nanoparticle. As shown in Fig. 1f, T-Expand achieved an average loading of 18.2 ± 3.1 µg (4.5 µg anti-CD3, 13.6 µg anti-CD28) antibody per mg of naked NP. Compared to current T cell expansion scaffolds, our T-Expand achieves the highest antibody coupling density among reported nanoscale T cell expanders, even surpassing some micron-scale scaffolds (Supplementary Table 1). Additionally, cryogenic transmission electron microscopy (cryo-TEM) visualization (Fig. 1g) revealed that T-Expand exhibited the size of 173.1 ± 37.27 nm (Fig. 1h), while the NTA and dynamic light scattering (DLS) revealed size distribution at 204 ± 45.8 nm (Supplementary Fig. 11a) and 197 ± 39.9 nm (Supplementary Fig. 11b), respectively. This is because the physical diameter of the NP (as determined by cryo-TEM imaging) is typically smaller than its hydrodynamic diameter18,19. Overall, these data demonstrated that the click chemistry-mediated antibody conjugation method effectively anchors antibodies onto the naked NPs.

Next, we explored the presence of serum on T-Expand performance since several T-cell expansion protocols requires serum supplementation. The proteomics analysis revealed that the click chemistry-mediated antibody conjugation method also limits the protein corona formation on the surface of T-Expand (Supplementary Figs. 12, 13) and show no functional compromise for T cell activation (Supplementary Fig. 13h, i). Biocompatibility experiments indicated that neither naked dextran NPs nor T-Expand affect cell viability when co-cultured with human Jurkat T cells (Supplementary Fig. 14).

We further assessed the stability of T-Expand under physiologically relevant conditions. First, T cells were expanded with either T-Expand (20 µL, 0.75 mg/mL) or Dynabeads™ (75 µL/mL, bead-to-cell ratio 3:1), and culture supernatants were harvested at various time points. These supernatants were diluted 1:1 and applied to fresh, resting primary T cells. After a 20-hour incubation, T-cell activation was evaluated by co-expression of CD69 and CD25 (Fig. 1i). The stimulatory potency of the T-Expand supernatants declined progressively from day 6 onward. To eliminate any cell-mediated contributions, we next incubated T-Expand and Dynabeads™ separately in cell-free medium. These cell-free supernatants were then directly used to stimulate primary T cells. Under these conditions, T-Expand lost significant activity by day 6 (Fig. 1j), whereas Dynabeads™ maintained relatively stable stimulatory capacity.

Additionally, T-Expand could be effectively removed by standard T-cell washing procedures due to the nanoscale of T-Expand, obviating the need for magnetic bead separation as required for Dynabeads™.

Transmission electron microscopy of T cells harvested at various time points post–T-Expand activation further confirmed the absence of residual T-Expand both on the cell surface and in the post-wash supernatant (Supplementary Fig. 15).

Together, these data indicate that the active components of T-Expand gradually degrade or become depleted over time. Importantly, residual T-Expand that has not been fully removed does not activate T cells and can be readily cleared by simple centrifugation and washing, thereby underscoring its applicability for CAR T cell manufacturing.

Investigation of the interaction of T-Expand with T cells
To study the interaction of T-Expand with T cells, we engineered Jurkat T cells to express a cathepsin L-mCherry fluorescent reporter. Cathepsin L is a lysosomal protein co-localizing with perforin and Granzyme B in T cell granules20. Using the Jurkat cathepsin L-mCherry reporter system, we investigated the ability of T-Expand to activate T cells by viewing the movement of cathepsin L-mCherry granules. Confocal imaging of engineered Jurkat cells in 3D and simulation shows the mCherry-Cathepsin L reporter has been successfully expressed in Jurkat cells (Fig. 2a). Colocalization imaging indicated that T-Expand effectively activated Jurkat cells, accompanied by the translocation of mCherry-Cathepsin L to the T-Expand contact site, ultimately resulting in colocalization. In contrast, the naked NPs did not directly interact with the Jurkat cells (Fig. 2b). Furthermore, we combined a 3D perspective simulation (Fig. 2c) and measured the distance between the T-Expand and the granules (Fig. 2d). The distance between T-Expand and the granules suggests colocalization, underscoring the specificity of T-Expand in activating Jurkat cells. The real time-3D imaging shows that the T-Expand forms a multicentric granules cluster that is different from the monocentric granule formed with Dynabeads™ (Fig. 2e, Supplementary Movie 1&2), indicating that T-Expand could activate Jurkat cells at multiple interfaces between T-Expand and Jurkat cells.

Manufacturing of CAR T cells with T-Expand results in cell products with high proliferative capacity, cytotoxicity and persistence
We investigated two parameters known to influence T cell expansion: the ratio of conjugated anti-CD3:anti-CD28 antibodies on T-Expand and the dose of T-Expand. First, we investigated how the stoichiometric ratio of anti-CD3:anti-CD28 influences T cell activation. Optimization experiments revealed that a Dynabeads™:T-cell ratio of 3:1 yielded the most efficient CAR-T cell production (Supplementary Fig. 16a–h). Consequently, this ratio was selected as the benchmark condition for comparison with T-Expand. To avoid potential interference caused by variations in the concentration of anti-CD3 and anti-CD28, we ensured that the surface density of these factors was saturated in all experiments. Based on the T cell activation assay, the different stoichiometric ratios of anti-CD3:anti-CD28 antibodies did not influence significantly on the percentage cells positive for the activation markers CD25 and CD69. For all stoichiometric ratios, the percentage of these activation markers increased with higher concentrations of T-Expand, reaching saturation at 3.75 μg of T-Expand (0.75 mg/mL) per 0.5 × 10^6 T cells (Fig. 3a). At lower concentrations of T-Expand, we observed that T-Expand (anti-CD3:anti-CD28 ratio 1:3) demonstrated the highest fold expansion by day 14 (Supplementary Fig. 17a–c).

Then we followed the viability and fold expansion of the T cells over a 10-day cultivation period and compared T-Expand with gold standard of Dynabeads™ with a beads to cell of 3:1, a commonly-recommended dose21–25, and TransAct™ as the standards. (Fig. 3b, c). T-Expand at the dose of 15 µg induced T cell expansion at a similar degree as Dynabeads™ by day 10 and superior to TransAct™ (Fig. 3c). The T-Expand-expanded T cells predominantly consisted of 35–50% effector memory T cells (TEM) and approximately 40% effector memory T cells re-expressing CD45RA (TEMRA), which mirrored the distribution observed in DynabeadsTM-expanded T cells (Supplementary Fig. 17d). Notably, this condition yielded higher CD27+ CD28+ expression (Fig. 3d) and lower levels of PD1+ and LAG3+ on T cells (Fig. 3e). Consequently, the dose at 15 µg T-Expand/million T cells was selected for future use.

Next, the efficacy of T-Expand for engineering T cells with a CAR was evaluated. Activation with T-Expand prior to viral transduction resulted in a T cell transduction efficacy of ~50% CAR expressing T cells when infected at a multiplicity of infection (MOI) of 3 (MOI-3), which was similar to levels obtained with Dynabeads™. In contrast, activation with TransAct™ resulted in 20% transduction efficiency (Fig. 3f) and an overall lower yield of T cells (Supplementary Fig. 18a) after 10 days of expansion (Fig. 3g). Phenotypic analysis revealed that T-Expand CAR T cells had a significantly higher percentage of CD27+ CD28+ CAR T cells (Fig. 3h–j) and higher percentage of TEM (Fig. 3k and Supplementary Fig. 18b) with an increased CD8+ CAR T cell population compared to DynabeadsTM and TransActTM (Supplementary Fig. 18c, d).

For further functional analysis, we compared T-Expand against Dynabeads™ expanded CAR T cells.

To determine the proliferative potential of anti-CD19 CAR T cells after expansion with T-Expand, expanded and cryopreserved CAR T cells were stained with CellTrace Violet and stimulated with irradiated CD19+ Jeko-1 cells to assess their expansion potential upon target recognition (Fig. 4a). On day 6, flow cytometry analysis showed that ~85% of T-Expand expanded CD19 CAR T cells had undergone up to four rounds of population doublings, in comparison to only 20% of Dynabeads™-expanded CAR T cells (Fig. 4b). To determine the cytotoxic potential of CAR T cells expanded with T-Expand, we performed an incucyte based cytotoxic assay with co-cultivation of CAR T cells and Jeko-1 cells at effector:target ratio (E:T) 5:1; 1:1; 1:5 or 1:10. (Fig. 4c). CAR T cells expanded by T-Expand exhibited potent cytotoxicity against CD19+ Jeko-1 cells. Compared to CAR T cells expanded with Dynabeads™, CAR T cells expanded with T-Expand showed significant tumor cell-killing efficacy across all E:T ratios. To further investigate the serial cytotoxicity and phenotypic effect on CAR T cells after serial exposure to target cells, we performed a re-challenge killing assay, by continuedly exposing remaining CAR T cells with CD19 target cells every 2-3 days. Based on the E:T ratio at 5:1, T-Expand-expanded CAR T cells exhibited significantly higher killing capacity even after three rounds of re-challenge (Fig. 4d, e, and Supplementary Fig 19).

To further characterize CAR T cells with enhanced cytotoxic capacity after repetitive stimulation, remaining cells from the third re-challenge were harvested and analyzed by flow cytometry (Supplementary Fig 19a). T cell expanded with Dynabeads™ primarily had a terminally differentiated phenotype whereas CAR T cells expanded with T-Expand were primarily effector memory (Fig. 4f, Supplementary Fig 19b). Additionally, the percentage of the CAR T cells expressing exhaustion markers such as PD1, LAG3, TIM3, and TIGIT were all significantly lower in T-Expand compared with Dynabead™ expanded CAR T cells (Fig. 4g, Supplementary Fig 19c). Next, we investigated the cytotoxicity of CAR T cells expanded using T-Expand under low E:T ratios. Similarly, CAR T cells expanded using T-Expand demonstrated significantly higher cytotoxicity, even after three rounds of re-challenge (Fig. 4h). Flow cytometry analysis revealed that under co-culture conditions with E:T ratios of 1:1 and 5:1, the T-Expand group exhibited T cell persistence rates of approximately 70% and 99%, respectively, whereas the Dynabeads™ group showed only 0.2% and 45%. (Fig. 4i, Supplementary Fig 19d). This suggests that the superior cytotoxic capacity of T-Expand CAR T cells is likely attributed to a combination of enhanced proliferative capacity, effector memory phenotype and lower exhaustive profile.

T-Expand expanded CAR T cells show superior in vivo tumor killing efficacy
Subsequently, we tested the in vivo anti tumor efficacy of the CD19 CAR T cells expanded with T-Expand in NXG mice injected with the lymphoma cell line Jeko-1 engineered to express luciferase (luc-mCherry+) for monitoring tumor burden using bioluminescence (BLI). Initially we tested a dose of 1 × 10^6 CAR T cells expanded with either T-Expand or Dynabeads™ (Fig. 5a). The tumor growth measured by BLI shows that, the treatment with T-Expand expanded CAR T cells lead to complete tumor elimination by day 7 without signs of tumor relapse at study termination on day 21. However, the Dynabead™ expanded CAR T cell treated group showed insufficient tumor clearance on day 7, further leading to the gradual increase in tumor burden until day 21. (Fig. 5b, c and Supplementary Fig. 20). At study termination, mice were euthanized and spleen and bone marrow were collected for detection of CAR T cells. The results show that the percentage of the CAR T cells detected both in bone marrow and spleen is similar between the T-Expand and the DynabeadsTM group (Fig. 5d). Interestingly, mice treated with T-Expand expanded CAR T cells had a significantly higher proportion of CD4+ T cells in the spleen (Fig. 5e) and higher levels of CD137 (4-1BB) in bone marrow compared to Dynabeads™ expanded CAR T cells (Fig. 5f). CD137 expression on T cells serves as a marker for antigen specific activation often correlated with enhanced anti-tumor efficacy. CD137 receptor interaction with 4-1BB ligand also plays a significant role in enhancing the survival and proliferation of these T cells26. Next, to further elucidate differences between CAR T cells expanded with T-Expand or Dynabeads™, mice were injected with 1 × 10^6 luc-mCherry+ Jeko-1 cells and treated on day 7 with 0.25 × 10^6, 0.5 × 10^6 or 1 × 10^6 CAR T cells expanded with either Dynabeads™ or T-Expand (Fig. 5g). On day 21, blood was collected for further analysis. A higher percentage of circulating T cells in mice receiving CAR T cells expanded with T-Expand (Supplementary Fig. 21a). The mice treated with T-Expand expanded CAR T cells at lowest doses (0.25 × 10^6 and 0.5 × 10^6) showed some tumor control and the high dose (1 × 10^6) completely eradicated the tumors (Fig. 5h). In contrary, Dynabeads™ expanded CAR T cells at all doses failed to show any significant tumor control with the majority of mice developing systemic hematological tumors until day 33 (Fig. 5i, Supplementary Fig. 21b).

RNA sequencing analysis of expanded CAR T cells
To investigate the transcriptomic profile of expanded CAR-T cells, we collected CAR T cells, generated from four donors, on Day 10 of expansion using either T-Expand or DynabeadsTM for bulk RNA sequencing from four healthy donors. The principal component analysis (PCA) revealed that the overall gene profiles of T-Expand expanded CAR T cells markedly differed from those of DynabeadsTM expanded CAR T cells (Supplementary Fig. 22a, b). T-Expand expanded CAR T cells exhibited high expression of genes associated with a putative transitional state between cytotoxic effector and differentiation phenotypes (Fig. 6a). These transitional phenotypes were identified by the co-expression of multiple cytotoxic genes, including LTB and GZMA27.

The analysis showed that the T-Expand expanded CAR T cells also showed upregulation of Xcl1 and Xcl2 genes that are involved in dendritic cell recruitment and activation. The stem-like T cells express high levels of Xcl1 that promotes the infiltration of migratory cDC1s that are associated with improving CD8 T cell anti-tumor activity through cross-priming28,29. Among the selected gene set (Fig. 6b), CAR T cells expanded with T-Expand showed increased expression of genes associated with anti-tumor cytokines and effector molecules (IFNg, GZMA), as well as T cell infiltration and activation (CXCL9, CXCL10, CCL5), compared with DynabeadsTM expanded CAR T cells30–32 (Supplementary Fig 22b).

To further elucidate the global transcriptomic landscape related to T-cell functionality in T-Expand expanded CD19 CAR T cells, only significantly expressed genes (p.adj < 0.001) were selected, and the top 10 gene sets were characterized via gene set enrichment analysis (GSEA) (Fig. 6c). The results revealed a significant enrichment of gene sets related to immune responses, inflammatory regulation, and signal transduction in T-Expand expanded CAR T cells. In contrast, DynabeadsTM expanded CAR T cells did not show notable enrichment of these gene sets, indicating substantial differences in gene expression regulation between these two expansion strategies.

Methods
Chemicals
Sodium azide (for synthesis), 3-Bromo-1-propanol (97%), dimethylformamide (ACS reagent, ≥99.8%), ethyl acetate (ACS reagent, ≥99.5%), magnesium sulfate (anhydrous, ReagentPlus®, ≥99.5%), Carbonyldiimidazole (CDI, ≥97.0% (T), for peptide synthesis), dextran (from Leuconostoc mesenteroides, average mol wt 9000–11,000), anhydrous dimethyl sulfoxide (DMSO), pyridinium p-toluenesulfonate (98%), 2-methoxypropene (97%), spermine (≥99.0% (GC)), Dimethyl sulfoxide-d6, poly(vinyl alcohol) (Mw 9000–10,000, 80% hydrolyzed) were purchased from Sigma-Aldrich.

Preparation of dextran NPs (naked NPs)
Acetalated azido modified Dextran (10 KDa, 20 mg) was dissolved in cold dichloromethane (DCM, 500 µL). The PBS (100 µL, from DTU life science) was added to undergo sonication for 20 s every 10 s on ice using a probe sonicator9 (Qsonica L.L.C, USA) flat tip, an output setting power of 5. Then the poly(vinyl alcohol) (PVA, Mw 9000–10,000 g/mol, 80% hydrolyzed) (1 mL, 3% w/w in PBS) was added into the primary W/O solution and further sonicated for an additional 20 s on ice using the same settings. The resulting W/O/W emulsion was immediately transferred to the PVA solution (5 mL, 0.3% w/w in PBS) and stirred for 2 h at room temperature for evaporating the DCM solvent. The NPs were obtained by centrifugation (10,600 × g, 10 min, Heraeus fresco 21, Thermo Fisher Scientific) and washed with PBS 3 times.

Formation of DBCO functionalized antibody and T-Expand
First, the anti-CD3 (clone OKT-3, BioXCell) and anti-CD28 (clone 9.3, BioXCell) antibodies were dissolved in PBS buffer (0.5 mg/ml, 200 µl), and the DBCO-Sulfo-NHS (CCT-A124, Vector Laboratories) was dissolved in DMSO to final concentration at 10 mM. Next, 3.32 µL of the above DBCO-Sulfo-NHS Ester was added to each antibody solution and incubated at room temperature for 30 min. Then, the reaction was quenched with 18 µl of Tris-buffer (1 M, pH 8, Thermo Fisher Scientific) for 15 min. Finally, any unbound DBCO-Sulfo-NHS Ester was removed using Zeba spin desalting columns (7 kDa or 40 kDa MWCO, Thermo Fisher Scientific) and final dissolved in 200 µl PBS.

Then, the above DBCO-functionalized antibodies (DBCO-anti-CD3 and DBCO-anti-CD28) were conjugated onto the surface of azido modified dextran NPs via click chemistry. The DBCO-anti-CD3 (50 µl, 0.38 mg/ml) and DBCO-anti-CD28 (150 µl, 0.38 mg/ml) was mixed with naked NPs (100 µl, 3 mg/ml) at final volume of 600 µl PBS according to the ratio of 1:3. Similarly, naked NPs were conjugated with anti-CD3 and anti-CD28 at volume ratios of 3:1 (150 µL:50 µL in PBS) and 1:1 (100 µL:100 µL in PBS) to generate T-Expand formulations with different surface antibody ratios. After the incubation in room temperature for 2 h, the T-Expand was obtained by centrifugation (10.6 × g, 10 min) and wished with PBS 2 times, followed stored in 4-degree fridge for further using.

Nanoparticle characterization
The number and size of the NPs was measured by Nanoparticle Tracking Analysis (NTA, NANOSIGHT, Malvern Instruments). The aliquot of NPs solution was diluted in MiliQ water pre-filtered by 20 nm filter at the same numbers used for expansion. The setup for NTA test was shown as follows (Camera Type: sCMOS Laser Type: Green; Camera Level: 10; Slider Shutter: 696; Slider Gain: 55; FPS 25.0; Number of Frames: 1498; Temperature: 23.9 °C; Viscosity: (Water) 0.910–0.911 cP; Syringe Pump Speed: 100).

Szie and ζ-Potential measurement of T-Expand
Hydrodynamic diameter of T-Expand were determined by Dynamic Light Scattering (DLS) (Malvern DLS Zetasizer, Malvern Panalytical) at 25 °C. T-Expand (10 µL, 0.75 mg/ml) were dispersed in MiliQ water (2 ml) with gently sonicated water bath for 1 min and loaded into disposable cuvettes. For each sample, three independent measurements were recorded and reported as mean ± SD (n = 3). Solvent viscosity and refractive index were set to those of water at the measurement temperature. Zeta potential of naked dextran NPs, T-Expand and DynabeadsTM was processed using Malvern DLS Zetasizer. Naked dextran NPs (10 µL, 0.75 mg/ml), T-Expand (10 µL, 0.75 mg/ml) and DynabeadsTM (2 µL) were dispersed in MiliQ water (2 ml) with gently sonicated water bath for 1 min. For each sample, three independent measurements were recorded and reported as mean ± SD (n = 3).

Cryo-Transmission electron microscopy
Three µL of the sample was applied on a hydrophilized lacey carbon 300 mesh copper grid (Ted Pella Inc., California, USA). The excess sample on the grid was blotted with filter paper at blotting time of 3 s, blotting force 0, temperature 4 °C, and 100% humidity (FEI Vitrobot IV, Eindhoven, The Netherlands), and was rapidly plunged into liquid-nitrogen cooled ethane (−180 °C). Sample observations were performed using a Tecnai G2 20 transmission electron microscope (FEI, Eindhoven, The Netherlands) at a voltage of 200 kV under a low-dose rate. Images were recorded with an FEI Eagle camera 4k × 4k at a nominal magnification of 29k ×.

Quantifying the Azido molar on the surface of Dextran NPs
The sulfo-Cyanine5 DBCO (1 mg, cat 133F0, Lumiprobe) was dissolved in 1 mL of DMSO. The concentration calibration curve was obtained by detecting the servals concentration (0.25 µg/ml, 0.5 µg/ml, 0.75 µg/ml, 1 µg/ml, 2.5 µg/ml) of sulfo-Cyanine5 DBCO. Next, 10 µl of dextran NPs were incubated with 1 µl of sulfo-Cyanine5 DBCO (1 mg/ml) overnight. The NPs were pelleted at the bottom of the centrifuge tube by centrifugation at 10600 g for 10 minutes. The supernatant of un-clicked sulfo-Cyanine5 DBCO was measured by plate reader (Synergy H1, Biotek). The amount of clicked sulfo-Cyanine5 DBCO was determined by calculating the concentration difference before and after coupling.

Fluorometric analysis of antibody conjugation
First, the anti-CD3 (clone OKT-3, BioXCell) and anti-CD28 (clone 9.3, BioXCell) antibodies were dissolved in PBS buffer (0.5 mg/ml, 200 µl), Next, DBCO-Sulfo-NHS (3.32 µl, 10 mm) and Cy5-NHS (8 µl, 1.5 mM in DMSO, Cytiva, PA15101) was added into anti-CD3 solution, DBCO-Sulfo-NHS (3.32 µl, 10 mm) and Alexa Fluor (AF) 488-NHS (8 µl, 1.5 mM in DMSO, Thermo Fisher Scientific) was added into anti-CD28 solution and further incubated for 3 h at room temperature. After incubation, the DBCO-Cy5-anti-CD3 and DBCO-AF488-anti-CD28 was purified by using Zeba spin columns (500 µl, 40 kDa MWCO) according to the manufacturer’s protocol. In order to remove as much unreacted linker (DBCO-Sulfo-NHS, Cy5-NHS and AF488-NHS) as possible, we purified the labeled antibody separately using two pre-equilibrated Zeba spin columns. For detailed procedures, see Figure Supplementary Fig. 10a.

To construct precise calibration curves relating antibody fluorescence intensity to concentration, we combined 150 µL of DBCO–Cy5–anti-CD3 (0.0283 mg/mL) with 50 µL of DBCO–AF488–anti-CD28 (0.0283 mg/mL) to prevent spectral crosstalk between fluorophores, and from this mixture established individual calibration curves for anti-CD3 and anti-CD28.

To obtain a more accurate estimate of the antibody density on the nanoparticle surface, a single bifunctional tag strategy was employed. Anti-CD28 was first reacted with Cy5-DBCO-NHS (3.32 µl, 10 mM in DMSO, Broadpharm), thereby avoiding competition between fluorophore-NHS and DBCO-Sulfo-NHS reagents Supplementary Fig. 10c. A standard calibration curve for Cy5-DBCO–anti-CD28 was generated by measuring fluorescence intensities of known concentrations in a microplate reader. Nanoparticle conjugation reactions were performed as described in Supplementary Fig. 10d, after which the supernatants were collected. Fluorescence measurements of these supernatants were converted to antibody mass using the calibration equation.

Primary T-cell isolation and activation
Peripheral blood mononuclear cells (PBMCs) were sourced from healthy donors from Rigshospitalet, Copenhagen as approved by the Danish ethics committee. PBMCs were isolated from whole blood by density centrifugation using SepMate tubes (StemCell) and frozen at −80 °C in fetal bovine serum (FBS) (S1810, BIOWEST) and 10% DMSO. Primary T cells from healthy donor PBMCs were isolated using the EasySep™ Human T cell enrichment kit supplied by STEMCELL technologies (as per supplier instructions). The purified T cells were resuspended in 0.5 × 10⁶ cells/ml in Roswell Park Memorial Institute (RPMI 1640) + 10% FBS + 1% penniciln streptomycin (Penstrep) (Thermo Fischer Scientific) and IL2 (20 IU/µl) (PeproTech®). For T cells activation, 100,000 purified T cells were plated in 100 µL culture medium in a 96-well cell culture plate, then specific concentration (0.75 mg/ml: 1 µl, 2 µl, 5 µl, 10 µl) of T-Expand (or) DynabeadsTM at a bead-to-cell ratio of 3:1, (or) TransActTM were added to each well and incubated in a humidified CO2 incubator at 37 °C for 20 h. Next, the activated T cells were harvested and washed with PBS buffer for further flow cytometry analysis. The T cell activation is measured by staining the T cell activation markers CD69 and CD25. The expression of CD69 and CD25 was evaluated as the percentage of fluorochrome-labelled cells within the total cell population, using an LSR Fortessa™ flow cytometer (BD Biosciences, USA).

For T cell expansion, purified T cells were seeded at a density of 1 × 10⁶ cells/mL in a 48-well culture plate (or others suitable tissue culture plates/flasks) supplemented with rhIL-2 (20 IU/mL). Subsequently, 20 or 40 µL of T-Expand (0.75 mg/mL), Dynabeads™ at a bead-to-cell ratio of 3:1, or TransAct™ was added. Cultures were maintained in a humidified CO2 incubator at 37 °C. The T cells were counted on day 3, 5, and 7, and their viability was reassessed using LUNA-FL™ Dual Fluorescence Cell Counter. The cells are resuspended in fresh media to maintain a concentration of 1 × 10^6 cells/mL, and rhIL-2 is replenished to a final concentration of 20 IU/mL to support continued growth and expansion. On Day 10, the culture contained DynabeadsTM was thoroughly resuspended to ensure uniform mixing of cells and beads which was then removed using a magnetic separator (DynaMag-15 magnetic rack, Thermo Fisher Scientific), while the T-Expand expanded CAR T cells, were washed with fresh cell medium and further transferred to a clean container. Finally, the cells are cryopreserved for downstream application.

Cell lines and culture
The human cell lines Jeko-1 (B cell lymphoma) and Jurkat obtained from American Type Culture Collection (ATCC) and cultured in RPMI 1640 supplemented with 10% FBS and 1% Penstrep. For cytotoxicity assay and in-vivo tumor models, these cell lines were transduced with mCherry fluorescent protein and luciferase genes using lentiviral vectors and were sorted on a FACS melody (BD Biosciences, USA) to establish fresh cultures.

Biocompatibility assay of T-Expand
Jurkat T cells were co-incubated with a broad range of either naked dextran NPs or T-Expand at different doses (0.75 µg, 1.875 µg, 3.75 µg, 7.5 µg, 15 µg and 75 µg) and seeded into 96-well plates at 5 × 10^5 cells per well in 200 µl RPMI 1640 supplemented with 10% FBS and 1% Penstrep. Cell viability was assessed with the NucleoCounter NC-200 (ChemoMetec A/S, Denmark) on days 1, 4, 7, and 10, following the manufacturer’s protocol.

Airyscan microscope imaging of T-Expand interacting with Jurkat cells
The engineered Jurkat cells were cultured with T-Expand or DynabeadsTM for 24 hours. Following incubation, centrifugation was conducted to remove the culture medium and resuspend the cells in FACS buffer (PBS + 1% BSA). Cell membrane was stained with anti-human CD45 and incubated on ice for 20 min. The cells were subsequently collected by centrifugation and 5 µl of the cell suspension was transferred to a glass slide (Thermo Fisher Scientific). The cell droplet was mounted with a coverslip and the edges sealed with nail polish. Confocal images were obtained by using Airyscan confocal microscope (LSM900 Airyscan 2, Carl Zeiss Microscopy GmbH). For real-time imaging, engineered Jurkat cells were cultured in an 8-well Glass µ-slide chamber (ibidi) at 37 °C and stimulated with T-Expand or DynabeadsTM for 24 h. 3D Z-stack imaging was conducted using an Airyscan laser confocal microscope, with subsequent analysis performed using Imaris software (v. 10.1.0).

CAR construct and Lentiviral production
A second-generation CAR were designed with an anti-CD19 (FMC63) CD8TM/hinge domain, 4-1bb, and CD3ζ. Expression of the CAR was linked with expression of a green fluorescent protein (GFP) reporter gene. The CAR was inserted into a third-generation lentiviral vector under the control of a human EF1α promoter. All plasmids were synthesized by GenScript, USA. Lentiviral particles were generated in HEK-293T cells by Lipofectamine transfection using packaging plasmids pRSV.REV (Addgene plasmid #12253), pMDLg/p.RRE (Addgene plasmid #12251), and pMD2.G (Addgene plasmid #12259), all gifted by Didier Trono47, along with a transfer plasmid, a modified version of pLenti-puro (Addgene plasmid # 39481) gifted by Ie-Ming Shih48, containing the CAR construct of interest. The transfected HEK-293T cells were incubated at 37 °C and 5% CO2 and the cell culture supernatant were collected at 24 and 48 h after transfection. Collected supernatants were up concentrated for lentiviral particles using Lenti-X™ Concentrator according to the maufacturer’s recommendations (Takara bio).

The titter of the produced lentivirus particles was assessed through a titration experiment by infecting SUP-T1 cells and subsequently analyzing GFP or surface expression of the receptor via tetramer staining and flow cytometry.

Engineering of Jurkat with mCherry-Cathpesin L
A cathepsin L-mCherry fragment was synthesized and cloned into transfer vector by GenScript, USA. Lentivirus was created following the same protocol as the previous setup, and Jurkats were transduced using an MOI of 5, followed by sorting of the cells based on mCherry expression.

Production of CAR T cells in vitro
On Day 0, the purified human T cells from healthy donors were diluted to 1 × 10⁶ cells/mL in RPMI 1640 media containing recombinant human IL-2 (rhIL-2) at a final concentration of 20 IU/mL, which was then cultured with T-Expand (0.75 mg/ml, 20 ul), TransActTM (10 µl) or DynabeadsTM (Gibco) based on a bead to cell ratio, typically 3:1 for 24 h. Then the CD19 CAR lentiviral particles were added to the culture at an MOI of 3 to ensure efficient transduction. Further the CAR T cells were expanded using the T cell expansion protocol.

In vitro T-cell phenotype analysis
The phenotype of T cells and CAR T cells was evaluated using flow cytometry. The transduction efficiency of CAR was determined by the co-expression of two markers: the intracellular fluorescent protein GFP encoded by the CAR construct and CD19 tetramer. Flow cytometry staining was performed on ice for 20 min in FACS buffer. The concentration of antibodies used for staining was determined according to the manufacturer’s instructions. Stained cells were analyzed using an LSR Fortessa flow cytometer, and data analysis was conducted using FlowJo v10 software. Cell lines were sorted on a FACSAria™ flow cytometer (BD Biosciences, USA) using a 100 µm nozzle. Amplitude was adjusted to optimize droplet break-off, and droplet calibration was performed before each sort using AccuDrop™ beads (BD, USA). Gates were set for each time point and sample independently based on fluorescence minus one (FMO) control. Cells were centrifuged at 500 × g for 5 min at 4 °C, and the supernatant was discarded. The pelleted cells were resuspended in 5 µL of 1 µM Dasatinib (LC Laboratories, D3307), antigen tetramers were added, and the cells were incubated for 15 min at 37 °C in the dark. The cells were washed once with FACS buffer, followed by staining with Near-IR28 (NiR) viability dye (Invitrogen™, L34976) and additional antibodies for surface staining for surface markers 30 min at 4 °C in the dark. Finally, the cells were washed twice in 200–300 µL of FACS buffer, and immediately analyzed on a LSRFortessa™ flow cytometer. Alternatively, the cells were fixed with 50 µL of 1% paraformaldehyde for 1–2 h (if required), washed twice with FACS buffer, and analyzed 2–24 h later.

Fluorochrome-conjugated antibodies including BB700-anti-human CD25 (IL-2R) (Clone M-A251), BV786-anti-human CD69 (Clone FN50), BV421-anti-human CD3 (Clone SP34-1), BV711-anti-human CD45RA (HI100), APC-anti-human CD197 (CCR7) (Clone 2-L1-A), BUV737-anti-human 279 (PD-1) (Clone EH12.1), BV650-anti-human CD223 (LAG-3) (Clone 11C3C65), BV786-anti-human CD39 (Clone TU66), BV605-anti-human TIGHT (Clone 741182), BV480-anti-human CD4 (Clone L200), PerCP-anti-human CD8 (Clone SK1), BV480-anti-human CD8 (Clone RPA-T8), BUV737-anti-human CD28 (Clone CD28.2), PE-anti-human CD137 (Clone 4B4-1), BV711-anti-CD19 (Clone SJ25C1), PE-Cy7-anti-CD4 (Clone RPA-T4), PE/Cy7-Streptavidin were purchased from BD Biosciences. PE-anti-human CD69 (Clone FN50), PE/Cy7-anti-human TIM-3 (Clone F38-2E2), PE-Cy7-anti-human CD57 (Clone QA17A04), BV605-anti-human CD27 (Clone O323) were purchased from Biolegend. The catalog numbers of all antibody and protein information was provided in Supplementary Table 3.

Proliferation assay
The T cells were resuspended 1 × 10⁶ cells/mL in PBS and 0,75 ul/ml of CellTrace violet was added to the T cells and incubated at 37 °C for 15 min. After incubation and co-culturing with irradiated Jeko-1 target cells at a 1:5 effector to target ratio for 6 days, flow cytometry was used to measure CellTrace Violet dilution, and the proliferation index was calculated using FlowJo software.

In vitro CAR T cell killing assays and re-challenge assay
The CD19 CAR T cells and the CD19 target cells, Jeko1 lymphoma cell line modified to express mCherry, was resuspended in RPMI 1640 supplemented with 10% FBS and 1% Penstrep and seeded at the indictaed E:T ratio. On Day 0, 1 × 10⁴ target cells and 1 × 10⁴ CAR T cells were co-cultured in a 96-well plate with 200 µL of T-cell culture medium. After a 48-h incubation, the supernatant was collected for ELISA test, followed by the addition of another 1 × 10⁴ target cells to each well. This process was repeated every 48 hours to simulate the repeated in vivo challenge faced by CAR T cells.

In vivo efficacy studies
For in vivo studies, 6-week-old female NXG Immunodeficient mice (NOD-Prkdcscid-IL2rgTm1/Rj) were acquired from Janvier Labs and housed at the Bio Facility, Department of Health Technology, Technical University of Denmark. The mice were housed separately from the immunocompetent mice. The mice were housed in groups of 4–5 mice per cage, and the feeding was replenished every 3 days or as required. All procedures were approved by the Danish National Animal Experiment Inspectorate and the institutional ethical board (Approval no. 2020-15-0201-00748).

For efficacy studies, 1 × 10⁶ Jeko-1 lymphoma cells expressing luciferase were injected intravenously into 7–10-week-old NXG mice. Tumors were allowed to engraft for 7 days before randomization and treatment start. On treatment day, indicated amount of untransduced, DynabeadsTM-expanded T cells or T-Expand expanded CD19 CAR T cells in 200 µl PBS were administered i.v. by tail vein injection. Tumor growth was monitored weekly with bioluminescence imaging (Optical Imaging unit, MILabs). Mice received 30 mg/mL of D-Luciferin intraperitoneally and 20 min later bioluminescence was measured. Measurements were analyzed using fixed-size regions of interest using MILabs optical imaging software. Mouse body weight and appearance were monitored throughout the experiment.

At study termination, spleen and bone marrow were harvested for further analysis by flow cytometry. Spleens and bone marrow were passed through 70 µm filters and washed in PBS to create a single-cell suspension. Splenocytes subsequently went through red blood cell lysis before being passed through a 70 µm filter again. Blood was harvested on day 21 from the saphenous vein. Red blood cells were lysed before washing with PBS. Blood, splenocytes and bone marrow cells were surface stained for Near-IR28 (NiR) viability dye and antibodies against surface markers, CD3, CD4, CD8, CD19, CD137, CD69 and CD19 CAR tetramer and run on LSRFortessa™ for flow cytometry analysis.

Differential gene expression analysis and GSEA
Differential gene expression analysis was used to identify differentially expressed genes in PBMCs of four donors expanded with either T-Expand or DynabeadsTM. Raw RNA-Seq reads were trimmed using TrimGalore and transcript abundances were quantified using Kallisto Quant49. The transcript abundances from Kallisto were imported into DESeq250 for differential expression analysis. Log2FoldChange > 2 and < −2 with an adjusted p value < 0.05 was used as threshold for over- and under-expressed genes for the analysis. The volcano plot was generated with ggplot2 and the heatmap was generated with Complex Heatmap1.

Gene set enrichment analysis (GSEA) was performed using clusterProfiler51,52 to identify significantly enriched Gene Ontology (GO) terms. As an input for the GSEA we used a ranked gene list of all significant genes (p.adj < 0.001) from the DESeq2 analysis, ordered by Log2FoldChange. This ranked gene list was then used as input for the gseGO function from the clusterProfiler package to identify significantly enriched Gene Ontology (GO) terms. To retain only highly significant GO terms, this analysis was performed using minGSSize > 30 (selecting only gene sets with more than 30 genes) and p value Cut off < 0.001.

Statistical analysis
Data are represented as the mean ± standard deviation. When necessary, a two-tailed Student’s t-test was used to identify statistically significant differences between the two groups, using GraphPad Prism 8 for computations. In instances of comparison among more than two groups, a one-way analysis of variance (ANOVA) was conducted, followed by Dunnet’s multiple comparisons test or Holm Šídák’s multiple comparisons test. Levels of statistical significance were denoted as follows: not significant, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Tao Zheng and colleagues presents a comprehensive and innovative study describing the development and evaluation of a novel dextran-based nanoparticle platform, termed T-Expand, for the ex vivo expansion of CAR T cells. According to the authors, this platform addresses key limitations of current artificial antigen-presenting cell (aAPC) systems such as Dynabeads™ and TransAct™, including overstimulation, poor scalability, and challenges in manufacturing. In the first part of the study, the authors design and characterize the T-Expand nanoparticles, which feature a high surface density of anti-CD3 and anti-CD28 antibodies conjugated via click chemistry to the modifiable side chains of dextran. They claim that the nanoscale size and biocompatibility of T-Expand eliminate the need for post-expansion removal, simplifying the manufacturing process and improving scalability. In the second part of the study, the functional characteristics of CAR T cells expanded using T-Expand are assessed. The results suggest that these cells possess enhanced phenotypic and functional attributes, including a greater proportion of CD27⁺ and CD28⁺ effector memory T cells, reduced expression of exhaustion markers, and improved anti-tumor efficacy both in vitro and in vivo. Furthermore, bulk RNA sequencing reveals upregulation of effector genes, including cytokines, and chemokine receptors implicated in T cell trafficking and immune modulation. The authors propose that these transcriptional changes correlate with the superior in vivo persistence and anti-tumor performance observed in xenograft lymphoma models, where T-Expand-expanded CAR T cells outperformed higher-dose Dynabeads™-expanded cells. This is a timely and overall well-executed study that introduces a promising new platform for CAR T-cell manufacturing. It represents a meaningful contribution to the field of adoptive cell therapy, with the potential to improve therapeutic efficacy and streamline production. However, before publication, the authors should address the following major and minor concerns:\n\nMajor concerns\n1. The authors report the optimization of anti-CD3 and anti-CD28 antibody ratios used for the functionalization of the T-Expand particles. However, no data are provided regarding the final amount of each antibody successfully conjugated to the particles. Since conjugation efficiency may differ from the input ratios, quantification (e.g., using ELISA or fluorophore-tagged antibodies) is important to clearly report the final antibody composition present on the T-Expand particles used in the experiments.\n2. While the authors emphasize the biocompatibility of the T-Expand nanoparticles as a key feature, no clear experimental data are provided to support this claim. The presented T cell proliferation data do not address the concentration-dependent effects of the particles on the overall cell population. In this context, I recommend including a biocompatibility assessment, such as cytotoxicity or cell proliferation assays using T-Expand particles at varying concentrations on T cells and/or other relevant cell lines, to confirm the system's safety profile.\n3. Did the authors use equivalent concentrations of Dynabeads™, TransAct™, and T-Expand particles, considering their differing physical and biochemical nature? Specifically, do these platforms contain comparable amounts of anti-CD3 and anti-CD28 antibodies? Given that T-Expand is a nanoparticle-based system, while Dynabeads™ and TransAct™ are microscale or polymer-based platforms, direct comparison may be confounded by differences in particle size, surface area, and antibody density. For example, was normalization performed based on the number of particles, the total antibody content, surface density of antibodies, or functional activity? The authors should clearly describe and justify the approach used to compare the different platforms and discuss whether the different concentrations used may lead to different results.\n4. The authors claim that the particles do not require post-expansion removal due to their nanoscale size and biocompatibility. However, this assumes the particles remain stable and do not aggregate, accumulate, or degrade over time. I strongly recommend assessing the stability and integrity of T-Expand particles over relevant time points under physiological experimental conditions.\n5. RNA-seq data indicate substantial upregulation of IFN-γ, pro-inflammatory cytokines (e.g., IL-6, IL-17), and other immune mediators in T-Expand-expanded CAR T cells. While these signatures may correlate with increased functionality, they also raise concerns about the potential for cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), two major clinical risks associated with CAR T-cell therapy. The authors should explicitly discuss these risks in the manuscript and consider whether the upregulated cytokine profiles observed for the T-Expand approach may pose translational safety concerns. In this context, it would be informative if the authors could report and discuss whether they observed any adverse reactions in their CAR T-cell experiments in mice.\n6. Additional discussion is needed regarding the differing functional outcomes observed between the Dynabeads™ and T-Expand platforms. This is particularly important, as the two systems yield distinct clinical-relevant T cell activities and gene expression profiles. How do the authors explain the observed divergence in activity? Is the unique nature of the T-Expand nanoparticles, such as their size, flexibility, or nanoscale interaction dynamics, sufficient to account for these changes? Could the chemical composition of the dextran polymer itself contribute to altered T cell activation or downstream signaling? Alternatively, might these differences arise from distinct physicochemical properties of the particles, such as surface charge, stiffness, or antibody orientation and density? The manuscript currently lacks a detailed analysis and discussion regarding this point and a thoughtful discussion of these mechanisms would greatly strengthen the manuscript and support the claims made about the advantages of the T-Expand system over conventional aAPC platforms.\n\nOther comments:\n1. In lines 142–143, the authors state that T-Expand particles exhibit high uniformity in cryo-EM images. However, no quantitative analysis is presented from the cryoEM micrographs. Instead, the authors rely on NTA data showing a mean particle size of 199.8 ± 70.7 nm. Figure 1h shows a secondary peak at ~120 nm, suggesting the presence of a distinct subpopulation. The authors should discuss this heterogeneity and clarify whether the smaller peak represents unbound particles. If possible, quantify and report the proportion of each population.\n2. Overall, the authors should provide more comprehensive descriptions of their experimental procedures, as the current level of detail is insufficient for reproducibility. For example, in lines 501–514, the section describing the formation of T-Expand particles for T cell activation and expansion lacks critical experimental information. This includes the concentrations of antibodies used, reaction volumes, buffer compositions, solvents, and technical details of the instrumentation employed. The authors are encouraged to thoroughly revise and complete all experimental sections. Specifically, the sources and references for the anti-CD3 and anti-CD28 antibodies should be clearly stated. Additionally, the origin of all cell lines used, along with their corresponding unique identifiers or commercial catalog numbers, should be provided in the methods to ensure traceability.", "reviewer_2": "This manuscript describes dextran nanoparticles to which T cell stimulating antibodies have been conjugated using click chemistry, and their use to promote T cell and CAR-T cell expansion, along with analysis of the function of the resultant cells. Overall, the authors provide a set of very nice studies characterizing the nanoparticles, and T cell expansion with these dextran-based nanoparticles. One major concern is the novelty of this work. A number of nanoparticle and microparticle artificial antigen presenting cells (aAPCs) have been developed and published, so the novelty of the concept is not high. The authors highlight the improvements, as versus Dynabeads, that they find when using these particles to expand T cells, particularly in relation to the functionality of the resultant cell populations. However, it is not clear the authors provide a fair comparison, as they only compare to a single Dynabead condition. The authors first optimize the dose of their nanoparticles to obtain optimal expansion and T cell phenotype, and then utilize that optimal dose for subsequent studies. The finding that stimulation dose is critical to expansion and function of the T cells is consistent with past literature using other aAPCs. So a different, optimized, dose of Dynabeads in these present studies may result in very similar, beneficial impact on CAR-T expansion, differentiation status, and function. Without a comparison of the optimized Dynabeads and optimized dextran nanoparticles, one cannot know if one provides a true advantage.\n\nTechnical question – the size distribution of the dextran nanoparticles have a much larger size after antibody conjugation than prior. How do the authors explain? Also, the size distribution post-antibody conjugation has two peaks; one approx. the same size as reported for the naked NPs, the other much larger – how do the authors account for this", "reviewer_3": "The manuscript by Zheng et al. presents a well-conceived and technically rigorous study that introduces “T-Expand,” a novel dextran-based nanoparticle platform for T cell activation and CAR T cell manufacturing. The authors demonstrate that T-Expand enables robust T cell expansion, enhanced CD19 CAR T cell cytotoxicity, and in vivo tumor clearance at reduced cell doses. The biocompatibility, high antibody loading capacity, and simplified downstream processing of T-Expand make it a promising alternative to traditional expansion systems such as Dynabeads™. The manuscript is well-structured, the experiments are thorough, and the conclusions are generally well-supported. However, there are several areas where additional data, clarifications, or contextualization would significantly strengthen the claims and translational relevance.\n\nMajor issues:\n1.The use of azido-dextran and DBCO click chemistry is well-established in immunomaterials literature (e.g., Broaders et al., PNAS, 2009; Adv Mater. 2017;29(7):1603239.). The manuscript should more clearly articulate what is unique about this specific implementation and cite foundational references to distinguish engineering novelty from repurposed techniques.\n2.In the transmission electron microscopy (TEM) image shown in Figure 1g, the T-Expand nanoparticle on the left appears to exhibit a double-layered structure or possibly contains a smaller particle within it. This morphology is not addressed in the text and may lead to confusion regarding particle uniformity. The authors could either clarify this observation in the figure legend or main text, or consider replacing the image with a more representative example that better reflects the described nanoparticle population.\n3. In Figure 5i, the bioluminescence images in row 2, columns 3 and 6 appear to be identical, despite representing different treatment groups. Please verify whether this is a case of inadvertent image duplication.\n\nMinor issues:\n1. The nanoparticle tracking analysis (NTA) data in Figure 1h suggests a potential bimodal distribution of particle size, with what appears to be two peaks. This raises the question of whether the T-Expand formulation consists of two distinct nanoparticle populations. The authors should elaborate on this in the Results section-either to explain the source of heterogeneity, or to clarify whether this is an artifact of measurement or a true feature of the formulation.\n2. The representative contour plot in Supplementary Figure 16a appears to display an unusually low number of events, which limits the interpretability of the flow cytometry data. The authors should clarify the number of events collected and justify whether this sample is truly representative. If possible, consider replacing it with a plot from a higher-quality acquisition to support the conclusions drawn."}, "author_response": "Point-by-Point Response to Reviewer's Comments (Zheng et al. formerly manuscript NCOMMS-25-30476)\nFor ease of reading, the reviewer’s comments are in regular typeface, our responses are highlighted in italics\nPoint by point reply to Reviewer #1 (Remarks to the Author):\nReviewer #1 (Remarks to the Author):\nThe manuscript by Tao Zheng and colleagues presents a comprehensive and innovative study describing the development and evaluation of a novel dextran-based nanoparticle platform, termed T-Expand, for the ex vivo expansion of CAR T cells. According to the authors, this platform addresses key limitations of current artificial antigen-presenting cell (aAPC) systems such as Dynabeads™ and TransAct™, including overstimulation, poor scalability, and challenges in manufacturing.\nIn the first part of the study, the authors design and characterize the T-Expand nanoparticles, which feature a high surface density of anti-CD3 and anti-CD28 antibodies conjugated via click chemistry to the modifiable side chains of dextran. They claim that the nanoscale size and biocompatibility of T-Expand eliminate the need for post-expansion removal, simplifying the manufacturing process and improving scalability.\nIn the second part of the study, the functional characteristics of CAR T cells expanded using T-Expand are assessed. The results suggest that these cells possess enhanced phenotypic and functional attributes, including a greater proportion of CD27⁺ and CD28⁺ effector memory T cells, reduced expression of exhaustion markers, and improved anti-tumor efficacy both in vitro and in vivo. Furthermore, bulk RNA sequencing reveals upregulation of effector genes, including cytokines, and chemokine receptors implicated in T cell trafficking and immune modulation. The authors propose that these transcriptional changes correlate with the superior in vivo persistence and anti-tumor performance observed in xenograft lymphoma models, where T-Expand-expanded CAR T cells outperformed higher-dose Dynabeads™-expanded cells.\nThis is a timely and overall well-executed study that introduces a promising new platform for CAR T-cell manufacturing. It represents a meaningful contribution to the field of adoptive cell therapy, with the potential to improve therapeutic efficacy and streamline production. However, before publication, the authors should address the following major and minor concerns:\nMajor concerns\n1. The authors report the optimization of anti-CD3 and anti-CD28 antibody ratios used for the functionalization of the T-Expand particles. However, no data are provided regarding the final amount of each antibody successfully conjugated to the particles. Since conjugation efficiency may differ from the input ratios, quantification (e.g., using ELISA or fluorophore-tagged antibodies) is important to clearly report the final antibody composition present on the T-Expand particles used in the experiments.\nResponse: In the revised manuscript, we have incorporated a two-step fluorometric analysis to investigate the amount of conjugated antibody density on the surface of NP, now detailed in the Results sections and Methods (Figure 1f in revised manuscript, Supplementary Fig. 10 in revised supplementary materials), with all raw data provided in the source data. Details of the added data, is described below:\nStep A – Stoichiometric verification.\nTo confirm that the surface ratio of anti-CD3 to anti-CD28 matches the input ratio, anti-CD3 was labeled with Cy5-NHS and anti-CD28 with Alexa Fluor 488-NHS; both antibodies were simultaneously functionalized with DBCO-Sulfo-NHS (Figure S1, a).\nA microplate reader was used to quantify the Anti-CD3 and Anti-CD28 fluorescence calibration curves (Figure S1, b). Then, DBCO-Cy5-anti-CD3 and DBCO-Alexa Fluor 488-anti-CD28 at different concentration ratios (3:1; 1:1; 1:3) were subjected to click coupling reaction with naked NP. After the click-coupling reaction, NPs were pelleted by centrifugation. Fluorescence intensities of the supernatants were quantified against calibration curves for each antibody, allowing us to back-calculate the amount of antibody bound. The bounded anti-CD3:anti-CD28 ratio on the particle surface matched the input ratio within experimental error (Supplementary Fig. 10a, b in revised supplementary materials and added below).\nStep B – Absolute loading determination.\nTo obtain a more accurate estimate of the amount of antibody on the nanoparticle surface, we first confirmed that the CD3:CD28 ratio on the particles matched the input ratio, because concurrent labeling of an antibody with both a fluorophore and DBCO-Sulfo-NHS can introduce competition between the two reagents and potentially underrepresent the total amount conjugated. We next adopted a single bifunctional tag. Specifically, anti-CD28 was derivatized with Cy5-DBCO-NHS, eliminating any competition and enabling direct fluorescence quantification. A calibration curve for Cy5-DBCO-anti-CD28 was generated with a microplate reader, and the resulting equation was applied to the supernatant measurements to calculate the mass of antibody bound per milligram of nanoparticles (Supplementary Fig. 10c, d in revised supplementary materials and added below). The final loading was 18.2 ± 3.1 µg of antibody per milligram of dextran nanoparticles (Figure 1f in revised manuscript and added below).\nTogether, these experiments demonstrate (i) that the final anti-CD3/anti-CD28 stoichiometry on T-Expand is controlled by the feed ratio, and (ii) that the platform attains an average antibody amount of ~18 µg mg⁻¹ NP. We believe these additions substantially strengthen the rigor and reproducibility of the study, and we are grateful for the reviewer’s constructive recommendation. The related description was provided in the result section, line 137 to 147 in revised manuscript.\nSupplementary Fig. 10. Quantification of conjugated antibody density on the surface of NP.\nThe antibody conjugation efficiency on the surface of naked NPs measured by Nanodrop. a, Quantification of the conjugated anti-CD3 and anti-CD28 antibodies ratio on the surface of dextran NPs using a fluorescence spectroscopy microplate reader. b, anti-CD3 calibration curve used for final quantification and anti-CD28 calibration curve used for final quantification. c, Quantification of the conjugated anti-CD28 antibodies on the surface of dextran NPs using a fluorescence spectroscopy microplate reader. d, anti-CD28 calibration curve used for final quantification.\nFigure 1f. The antibody conjugation efficiency on the surface of naked NPs measured by microplate reader.\n2. While the authors emphasize the biocompatibility of the T-Expand nanoparticles as a key feature, no clear experimental data are provided to support this claim. The presented T cell proliferation data do not address the concentration-dependent effects of the particles on the overall cell population. In this context, I recommend including a biocompatibility assessment, such as cytotoxicity or cell proliferation assays using T-Expand particles at varying concentrations on T cells and/or other relevant cell lines, to confirm the system's safety profile.\nResponse: To verify the biocompatibility of our platform, we have added a dedicated biocompatibility study to the revised manuscript. Jurkat T cells were co-incubated with a broad range of either naked dextran nanoparticles or T-Expand and seeded into 96-well plates at 5 × 105 cells per well. Cell viability was assessed with the NucleoCounter NC-200 on days 1, 4, 7, and 10, following the manufacturer’s protocol. Across all concentrations and time points, cell survival consistently exceeded 90%, demonstrating excellent biocompatibility for both naked NPs and T-Expand. The complete experimental protocol has been added as a new “Biocompatibility Assay of T-Expand” subsection in the Methods (line 696 to 701 in revised manuscript.) and related discussion has been inserted in the line of 162 to 164 in the revised manuscript. the quantitative data and statistical analyses are presented in the Results section (Supplementary Fig. 14 in revised supplementary materials and added below).\nSupplementary Fig. 14. Quantification of cell viability (determined by NucleoCounter NC-200 machine) at day 1, 4, 7 and 10 by co-culturing naked NPs and T-Expand with Jurkat T cells.\n3. Did the authors use equivalent concentrations of Dynabeads™, TransAct™, and T-Expand particles, considering their differing physical and biochemical nature? Specifically, do these platforms contain comparable amounts of anti-CD3 and anti-CD28 antibodies? Given that T-Expand is a nanoparticle-based system, while Dynabeads™ and TransAct™ are microscale or polymer-based platforms, direct comparison may be confounded by differences in particle size, surface area, and antibody density. For example, was normalization performed based on the number of particles, the total antibody content, surface density of antibodies, or functional activity? The authors should clearly describe and justify the approach used to compare the different platforms and discuss whether the different concentrations used may lead to different results.\nResponse: We thank the reviewer for highlighting the critical issue of dose normalization. Because the total antibody load, CD3:CD28 stoichiometry, and conjugation chemistry for Dynabeads™ and TransAct™ are proprietary, only limited information is publicly available, that Dynabeads™ display around 2.8 × 105 antibody molecules per bead with an anti-CD3:anti-CD28 molar ratio of ~3:1 [1], whereas TransAct™ is reported to present a 1:1 ratio but without a disclosed antibody amount [2]. These commercial microscale carriers therefore differ markedly from our nanoscale T-Expand particles in diameter, specific surface area, and ligand density, making it challenging to normalize on the basis of absolute antibody site number.\nWe therefore adopted a clinically oriented comparison, The T-Expand and DynabeadsTM were seeded with the same initial number of T cells; the dose of Dynabeads™ (beads to cell ratio = 3:1) were used exactly according to commercial (thermo fisher scientific) recommendation for clinical CAR T cell manufacture protocol [3] and adopted by many research group [4-8] while T-Expand was tested across a 0.75µg to 30 µg dose of range T-Expand to identify the optimal concentration. The unified read-out was the fold expansion achieved after 10 days. The dose–response data show that T-Expand at ≥15 µg attains a cell-expansion efficiency comparable to Dynabeads™ used at a 3:1 bead-to-cell ratio (Figure 3c in revised manuscript, Figure S16 in revised supplementary materials). This design ensures the comparator was tested at its recommended clinical baseline while allowing a fair search for T-Expand’s optimum.\n4. The authors claim that the particles do not require post-expansion removal due to their nanoscale size and biocompatibility. However, this assumes the particles remain stable and do not aggregate, accumulate, or degrade over time. I strongly recommend assessing the stability and integrity of T-Expand particles over relevant time points under physiological experimental conditions.\nResponse: Thank you for your attention to our statement that “no removal is required.” By this, we mean that T-Expand, due to their nanoscale dimensions, are effectively eliminated during standard T-cell washing procedures, obviating the magnetic‐separation step necessary for Dynabeads™. In support of this, We conducted an additional experiment in which T-Expand was incubated with T cells for varying durations, followed by centrifugal washing. TEM imaging of the washed T cells was then performed to assess whether T-Expand remained on the cell surface or in the suspension, thereby demonstrating that T-Expand can be effectively removed by standard washing procedures. The TEM imaging confirmed the absence of T-Expand on the T-cell surface or in the post-wash suspension (Figure S15 in revised supplementary materials and inserted below). To further substantiate this attribute and assess nanoparticle stability under physiologically relevant conditions, we have incorporated the following two experimental series.\nFirst, supernatants were collected at various time points following T-cell expansion with either T-Expand (15 µg/mL) or Dynabeads™ (75 µL/mL, 3:1 beads to cell ratio). These supernatants were then diluted 1:1 and applied to fresh, unactivated primary T cells, and activation was measured 20 hours later by CD69/CD25 co-expression. The stimulatory potency of the T-Expand supernatant declined progressively after day 6 (Figure 1i in revised manuscript and inserted below). To eliminate any cell-mediated contributions, we incubated T-Expand and Dynabeads™ in cell-free RPMI 1640 (10% FBS, 37 °C, 5% CO₂), collected the media on days 2, 6, and 10, and used them directly to stimulate primary T cells. Under these acellular conditions, T-Expand lost significant activity by day 6, whereas Dynabeads™ retained a relatively stable stimulatory effect (Figure 1j in revised manuscript and inserted below). These data indicate that the active moieties of T-Expand are gradually lost over time. This means, that in a clinical use scenario, even residual, inadequately washed T-Expand fails to activate T cells.\nThe experimental design, results, and corresponding discussion have been added to the revised manuscript to address the concerns related to stability, and need for removal of T-Expand. The related discussion has been added in the line of 165 to 179 in the revised manuscript.\nSupplementary Fig. 15. Representative transmission electron microscopy (TEM) images of primary T cell and activated at different time point (15 mins, 30 mins, 120 mins and 24 h). Scale bars: 5 um.\nFigure 1. i, the T cell activation profile in terms of % of T cells expressing CD69+CD25+ after 20 hrs of co-incubation with the supernatant obtained from the expansion cultures at different time point. j, The T-Expand (20µl) and DynabeadsTM (75µl) were incubated in Roswell Park Memorial Institute (RPMI) 10%FCS at 37°C and 5% CO culture conditions for 10 days and the 2 media was removed at day 2,6 and 10 timepoints of culture and was added to unactivated T cells at 1:1 dilution. Data in panels i, j represented as mean ± SD, of biological replicates with each dot indicating one healthy donor sample(n=1).\n5. RNA-seq data indicate substantial upregulation of IFN-γ, pro-inflammatory cytokines (e.g., IL-6, IL-17), and other immune mediators in T-Expand-expanded CAR T cells. While these signatures may correlate with increased functionality, they also raise concerns about the potential for cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), two major clinical risks associated with CAR T-cell therapy. The authors should explicitly discuss these risks in the manuscript and consider whether the upregulated cytokine profiles observed for the T-Expand approach may pose translational safety concerns. In this context, it would be informative if the authors could report and discuss whether they observed any adverse reactions in their CAR T-cell experiments in mice.\nResponse: We thank the reviewer for this important and clinically relevant comment. We agree that the upregulation of IFN-γ and pro-inflammatory cytokines, such as IL-6 and IL-17, observed in our RNA-seq analysis could, in principle, be associated with cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS) in the clinical setting. In the revised Discussion section, we now explicitly address these potential translational safety concerns. Related discussions are provided in line from 540 to 553 in revised manuscript and added below:\n“There is marked upregulation of pro-inflammatory cytokine transcripts (IFN-γ, IL-6, IL-17) in T-Expand–expanded CAR T cells relative to Dynabeads™. While such signatures are consistent with heightened effector programming, they overlap with pathways implicated in clinical cytokine release syndrome (CRS) and immune-effector cell–associated neurotoxicity syndrome (ICANS) [9]. Importantly, bulk transcriptomic elevation does not equate to systemic cytokine release, and in patients the surge in IL-6 during CRS is predominantly mediated by monocyte/macrophage activation rather than by T cells themselves [10, 11].\nIn our NSG mouse xenograft experiments, we did not observe any adverse reactions, including distress, weight loss, behavioral changes, during the treatment period, despite the potent antitumor activity of T-Expand–expanded CAR T cells. Considering that NSG mice are not an appropriate system to model CRS and ICANS, this should be evaluated in future studies using immune-competent and humanized animal models in which myeloid-driven cytokine biology is preserved”.\n6. Additional discussion is needed regarding the differing functional outcomes observed between the Dynabeads™ and T-Expand platforms. This is particularly important, as the two systems yield distinct clinical-relevant T cell activities and gene expression profiles. How do the authors explain the observed divergence in activity? Is the unique nature of the T-Expand nanoparticles, such as their size, flexibility, or nanoscale interaction dynamics, sufficient to account for these changes? Could the chemical composition of the dextran polymer itself contribute to altered T cell activation or downstream signaling? Alternatively, might these differences arise from distinct physicochemical properties of the particles, such as surface charge, stiffness, or antibody orientation and density? The manuscript currently lacks a detailed analysis and discussion regarding this point and a thoughtful discussion of these mechanisms would greatly strengthen the manuscript and support the claims made about the advantages of the T-Expand system over conventional aAPC platforms.\nResponse: Based on the activation assay, we confirm that neither naked dextran nanoparticles nor the azide-modified polymer backbone activate T cells or alter downstream signaling (Figure 1e in the revised manuscript and inserted below). We also show that T-Expand and Dynabeads™ have similar surface zeta potentials (Figure 1d in the revised manuscript and inserted below), and although Atomic Force Microscopy (AFM) imaging measurements reveal that T-Expand’s surface Young’s modulus (488 Kpa, Figure 1 and table 1 shown below) is much lower than Dynabeads™ (20–40 MPa), both platforms still effectively expand T cells, implying stiffness is not a decisive factor here. Although we lack orientation data for Dynabeads™ or TransAct™, the fact that T-Expand retains high stimulatory potency after serum incubation (Figure S12,13 in revised supplementary materials) indicates that antibody orientation is not limiting on our platform. Related discussions are provided in line from 489 to 506 in revised manuscript and added below:\n“Mechanistically, the divergent functional outcomes of T-Expand and Dynabeads™ appear to be driven by differences in stimulus intensity and kinetics. T-Expand presents a higher surface density of anti-CD3/anti-CD28, delivering more activation signals and enabling the rapid formation of multiple immunological synapses. This brief, high-density “burst” is sufficient to initiate T-cell activation and proliferation without prolonged high-strength engagement. By contrast, we observed that Dynabeads™ remain tightly connected to T-cell membranes throughout the expansion period, providing continuous strong signaling that while promoting early proliferation, may also predispose cells to activation-induced exhaustion. In addition, the size and biocompatibility of T-expand facilitates receptor-mediated internalization, which aligns with the natural T cell behavior upon TCR activation. We hypothesize that the internalization capacity contributes to the observed phenotype.\nConsistent with this model, our stimulation-stability assays show that, under cell-free conditions, T-Expand’s stimulatory activity declines by day 6, indicating natural dissipation/degradation that limits prolonged overstimulation, whereas Dynabeads™ maintain relatively constant potency. In line with this, CAR-T cells expanded with Dynabeads™ exhibit higher exhaustion-marker expression in a three-round in vitro tumor rechallenge assay, whereas those expanded with T-Expand retain a less-exhausted CD27⁺CD28⁺ phenotype and superior durability under repeated challenge”.\nFigure 1. d, Zeta potential of naked NPs, T-Expand and DynabeadsTM (n=3). e, Percent of T cells expressing the activation markers CD25 and CD69 after co-incubation with T-Expand (number of donors: n = 3).\nFigure 1. AFM topography image of the particles.\nTable 1. Nanomechanical properties measured by AFM\nSample Young Modulus\n(KPa)\nT-Expand 488\nDynabeadsTM 20000-40000 [1]\nOther comments:\n1. In lines 142–143, the authors state that T-Expand particles exhibit high uniformity in cryo-EM images. However, no quantitative analysis is presented from the cryoEM micrographs. Instead, the authors rely on NTA data showing a mean particle size of 199.8 ± 70.7 nm. Figure 1h shows a secondary peak at ~120 nm, suggesting the presence of a distinct subpopulation. The authors should discuss this heterogeneity and clarify whether the smaller peak represents unbound particles. If possible, quantify and report the proportion of each population.\nResponse: In accordance with the suggestion, we re-imaged T-Expand by cryo-TEM. The resulting mean diameters for T-Expand were 173.1 ± 37.27 nm as quantify by image J. These data, now presented as histograms with population percentages in figure 1h in revised manuscript and inserted below.\nFigure 1. h, The size distribution of T-Expand, as determined by Cryo-TEM and measured by software of image J with 3 independent figures.\nAdditionally, we do not attribute peak of 120 nm to unconjugated particles. Normally, the strain-promoted alkyne–azide cycloaddition (SPAAC) between azide-decorated NPs and DBCO-antibodies proceeds with high efficiency, and NanoDrop spectroscopy at 309 nm confirms successful incorporation of DBCO into the antibody preparation (Figure S9 in revised supplementary materials). Owing to the high efficiency of the strain-promoted alkyne–azide cycloaddition between azide-functionalized nanoparticles and DBCO-modified antibodies, the ~120 nm peak does not represent nanoparticle populations lacking antibody conjugation.\nFinally, we repeated the NTA measurements and investigated the previously observed subpopulation peak. We identified minor contaminants in the original NTA microfluidic chip. After thorough cleaning of the chip and re-analysis, the subpopulation peak was no longer detected (Figure S11a in revised supplementary materials and added below). Concurrently, we performed DLS-based particle sizing as a complementary control (Figure S11b in revised supplementary materials and inserted below), which yielded an average T-Expand particle diameter of approximately 200 nm and consistent with the NTA results. The size measured by NTA and DLS is large than Cryo-TEM imaging, this is because the physical diameter of the NP (as determined by TEM imaging) is typically smaller than its hydrodynamic diameter.\nSupplementary Fig. 11. a, The size distribution of T-Expand, measured by NTA. b, The size distribution of T-Expand, measured by DLS.\n2. Overall, the authors should provide more comprehensive descriptions of their experimental procedures, as the current level of detail is insufficient for reproducibility. For example, in lines 501–514, the section describing the formation of T-Expand particles for T cell activation and expansion lacks critical experimental information. This includes the concentrations of antibodies used, reaction volumes, buffer compositions, solvents, and technical details of the instrumentation employed. The authors are encouraged to thoroughly revise and complete all experimental sections. Specifically, the sources and references for the anti-CD3 and anti-CD28 antibodies should be clearly stated. Additionally, the origin of all cell lines used, along with their corresponding unique identifiers or commercial catalog numbers, should be provided in the methods to ensure traceability.\nResponse: We thank the reviewer for raising this point. We have revised the Methods section throughout to ensure that details are provided. Specifically, we have expanded the description of T-Expand particle preparation and T-cell activation to include all concentrations, reaction volumes, buffer compositions, solvents, and relevant instrument specifications. We have also provided the sources, catalog numbers, and unique identifiers for anti-CD3 and anti-CD28 antibodies, as well as the origin and catalog information for all cell lines used. These additions ensure full traceability and reproducibility of our experiments.\nPoint by point reply to Reviewer #2 (Remarks to the Author):\nThis manuscript describes dextran nanoparticles to which T cell stimulating antibodies have been conjugated using click chemistry, and their use to promote T cell and CAR-T cell expansion, along with analysis of the function of the resultant cells. Overall, the authors provide a set of very nice studies characterizing the nanoparticles, and T cell expansion with these dextran-based nanoparticles. One major concern is the novelty of this work. A number of nanoparticle and microparticle artificial antigen presenting cells (aAPCs) have been developed and published, so the novelty of the concept is not high. The authors highlight the improvements, as versus Dynabeads, that they find when using these particles to expand T cells, particularly in relation to the functionality of the resultant cell populations. However, it is not clear the authors provide a fair comparison, as they only compare to a single Dynabead condition. The authors first optimize the dose of their nanoparticles to obtain optimal expansion and T cell phenotype,and then utilize that optimal dose for subsequent studies. The finding that stimulation dose is critical to expansion and function of the T cells is consistent with past literature using other aAPCs. So a different, optimized, dose of Dynabeads in these present studies may result in very similar, beneficial impact on CAR-T expansion, differentiation status, and function. Without a comparison of the optimized Dynabeads and optimized dextran nanoparticles, one cannot know if one provides a true advantage.\nTechnical question – the size distribution of the dextran nanoparticles have a much larger size after antibody conjugation than prior. How do the authors explain? Also, the size distribution post-antibody conjugation has two peaks; one approx. the same size as reported for the naked NPs, the other much larger – how do the authors account for this\nResponse: We thank the reviewer for their positive comments related to our work. We have addressed each comment in a point-by-point manner.\nFirst of all, based on the novelty concerns, while various nanoparticle- and microparticle-based aAPC platforms have been reported, T-Expand offers three key innovations that substantially enhance its novelty and translational potential (shown below). Related discussions are provided in line from 455 to 465 in revised manuscript.\n1. High-Density Ligand Loading.\nIt is well established that optimal T-cell activation and proliferation depend on parameters such as particle size, shape, ligand density, and rigidity. Micron-scale particles typically excel at T-cell stimulation because their larger contact area promotes more immunological synapse formation and greater ligand–receptor pairing. However, to achieve comparable activation efficiency at the nanoscale, aAPCs must present very high ligand densities within their limited contact footprint. Existing polymer-based systems, such as PLGA and PEG-PDLLA nanoparticles, only introduce functional groups at chain termini, resulting in sparse surface click-handles after self-assembly. In contrast, our azide-functionalized dextran features dense azido groups along every side chain, dramatically increasing antibody loading per particle. This high ligand density enables powerful T-cell activation and expansion at the nanoscale. Moreover, we have carried out extensive functional studies of CD19 CAR-T cells expanded with T-Expand, demonstrating enhanced cytotoxicity, persistence, and phenotype both in vitro and in vivo.\n2. One-Step Bioorthogonal Conjugation.\nMost reported aAPC platforms ranging from inorganic beads to polymeric microparticles which require multiple surface modification steps and secondary linkers to attach stimulatory antibodies [1, 12, 13]. By contrast, azide-modified dextran nanoparticles react directly with DBCO-anti-CD3 and DBCO-anti-CD28 in a single strain-promoted alkyne–azide cycloaddition (“click”) step. This one-pot process eliminates the need for additional activation or coupling chemistry, greatly simplifying manufacturing and reducing batch variability.\nFigure 1. Reference of related aAPCs. a. alginate microgel based aAPCs. b. graphite micro-sheets based aAPCs. c. Silica microrod based aAPCs.\n3．Beyond T-Cell Stimulation: Impact of T-Expand on CAR T-Cell Expansion and Function\nIn addition to the general advances of nanoscale aAPCs, we would like to emphasize that our study goes beyond T-cell stimulation alone. Specifically, we extend the application of the T-Expand platform into the clinically critical context of CAR T-cell manufacturing. We provide an in-depth analysis of how T-Expand–expanded CAR T cells differ in phenotype, persistence, and anti-tumor efficacy. To our knowledge, very few reports of nanoscale aAPCs have systematically investigated their impact on CAR T-cell expansion and downstream therapeutic performance. This extension into CAR T-cell production and functional evaluation further distinguishes our work and underscores its translational relevance.\nWithout a comparison of the optimized Dynabeads and optimized dextran nanoparticles, one cannot know if one provides a true advantage.\nResponse: We thank the reviewer for highlighting the optimized dose comparison. Because the total antibody load, CD3:CD28 stoichiometry, and conjugation chemistry for Dynabeads™ and TransAct™ are proprietary, only limited information is publicly available. According to the reported work that Dynabeads™ display ≈2.8 × 105 antibody molecules per bead with an anti-CD3:anti-CD28 molar ratio of ~3:1 [1], whereas TransAct™ is reported to present a 1:1 ratio but without a disclosed antibody amount [2]. These commercial microscale carriers therefore differ markedly from our nanoscale T-Expand particles in diameter, specific surface area, and ligand density, making it challenging to normalize on the basis of absolute antibody site number.\nWe therefore adopted a clinically oriented comparison, The T-Expand and DynabeadsTM were seeded with the same initial number of T cells; the dose of Dynabeads™ (beads to cell ratio = 3:1) were used exactly according to commercial (thermo fisher scientific) recommendation for clinical CAR T cell manufacture protocol [3] and adopted by many research works [4-8] while T-Expand was tested across a 0.75 µg–30 µg dose of range T-Expand to identify the optimal concentration. The unified read-out was the fold expansion achieved after 10 days. The data of dose–response data show that T-Expand at ≥15 µg attains a cell-expansion efficiency comparable to Dynabeads™ used at a 3:1 bead-to-cell ratio (Figure 3c in revised manuscript, Figure S16 in revised supplementary materials). This design ensures the comparator was tested at its recommended clinical baseline while allowing a fair search for T-Expand’s optimum.\nTechnical question – the size distribution of the dextran nanoparticles have a much larger size after antibody conjugation than prior.\nResponse: We attribute the apparent increase in particle size after antibody conjugation to two main factors. First, the intrinsic size of the antibody and its hydration layer: an IgG is ~150 kDa with an approximate diameter of ~10 nm; once tethered to the nanoparticle surface, the antibody together with its surrounding hydration/ion shell substantially increases the measured hydrodynamic diameter, potentially by over than 10 nm. Secondly, as noted in the revised manuscript, our T-Expand display a high surface density of antibodies; their orientation and conformational extension can enlarge the effective diameter, forming a thicker “soft corona.” While beneficial for functionalization, high conjugation density can thus increase the hydrodynamic size.\nTo quantify this phenomenon more systematically, we added multimodal measurements. Cryo-TEM analysis of three randomly selected fields yielded a mean T-Expand diameter of 173.1 ± 37.27 nm (Figure 1 h in revised manuscript and added below). Repeat NTA and DLS measurements gave 204 ± 45.8 nm (Figure S11a in revised supplementary materials and inserted below) and 197 ± 39.9 nm (Figure S11b in revised supplementary materials and inserted below), respectively, approximately 24-31 nm larger than cryo-TEM which is consistent with the expected methodological difference that physical diameter (imaging) less than hydrodynamic diameter.\nRegarding the “two peaks” distribution observed after antibody conjugation, troubleshooting identified trace contaminants in an earlier NTA microfluidic cartridge; after cleaning and re-measurement, the anomalous subpopulation disappeared. All raw data and images have been included in the revised supplementary materials, source data and are clearly cross-referenced in the response for ease of review.\nFigure 1. h, The size distribution of T-Expand, as determined by Cryo-TEM and measured by software of image J with 3 independent figures.\nSupplementary Fig. 11. a, The size distribution of T-Expand, measured by NTA. b, The size distribution of T-Expand, measured by DLS.\nPoint by point reply to Reviewer #3 (Remarks to the Author):\nRemarks to the authors:\nThe manuscript by Zheng et al. presents a well-conceived and technically rigorous study that introduces “T-Expand,” a novel dextran-based nanoparticle platform for T cell activation and CAR T cell manufacturing. The authors demonstrate that T-Expand enables robust T cell expansion, enhanced CD19 CAR T cell cytotoxicity, and in vivo tumor clearance at reduced cell doses. The biocompatibility, high antibody loading capacity, and simplified downstream processing of T-Expand make it a promising alternative to traditional expansion systems such as Dynabeads™. The manuscript is well-structured, the experiments are thorough, and the conclusions are generally well-supported. However, there are several areas where additional data, clarifications, or contextualization would significantly strengthen the claims and translational relevance.\nResponse: We thank the reviewer for their generous assessment of our work. We are delighted that you find this study “well-conceived and technically rigorous” and that you recognize the superior performance of the T-Expand platform in T-cell proliferation, CAR T-cell cytotoxicity, and in vivo tumor clearance. We also appreciate your encouragement regarding its biocompatibility, high antibody-loading capacity, and streamlined downstream processing. In the revised manuscript, we will further supplement our data, clarify key details, and expand our Discussion to more fully articulate the advantages and translational potential of T-Expand relative to conventional expansion systems such as Dynabeads™. We have addressed each comment in a point-by-point manner.\nMajor issues:\n1.The use of azido-dextran and DBCO click chemistry is well-established in immunomaterials literature (e.g., Broaders et al., PNAS, 2009; Adv Mater. 2017;29(7):1603239.). The manuscript should more clearly articulate what is unique about this specific implementation and cite foundational references to distinguish engineering novelty from repurposed techniques.\nResponse: Yes, as the reviewer rightly noted, azide–DBCO click chemistry has been extensively applied in immune-materials. We have already cited the references mentioned by the reviewer in our original manuscript (reference14, 15 ,16 in the revised manuscript). In the revised version, we have added detailed text to underscore the unique features of our azide-modified dextran nanoplatform and have cited the pertinent literature to delineate its distinctions and innovations compared to existing technologies. Related discussions are provided in line from 455 to 465 in revised manuscript and added below:\n“Compared to existing artificial antigen-presenting nanoparticle carriers, we employed azide-modified dextran polymers to install a high density of azido groups along the side chains for downstream antibody conjugation. In contrast, current polymer platforms, such as poly(lactic-co-glycolic acid and poly(ethylene glycol)-block-poly(d,l-lactide), typically localize azido groups at a single terminus of the polymer chain, yielding a low surface functional-group density after nanoparticle assembly. Furthermore, our azide-functionalized dextran nano-platform requires no additional surface modification to undergo click chemistry with DBCO-anti-CD3 and DBCO-anti-CD28, thereby providing an efficient, convenient, and controllable antibody-conjugation system. Finally, these azide-modified dextran nanoparticles remain stable in PBS buffer without loss of azido-group reactivity, offering a ready-to-use antibody-clickable nanocarrier platform”.\n2.In the transmission electron microscopy (TEM) image shown in Figure 1g, the T-Expand nanoparticle on the left appears to exhibit a double-layered structure or possibly contains a smaller particle within it. This morphology is not addressed in the text and may lead to confusion regarding particle uniformity. The authors could either clarify this observation in the figure legend or main text, or consider replacing the image with a more representative example that better reflects the described nanoparticle population.\nResponse: Our T-Expand does not exhibit a bilayer architecture; the apparent bilayer‐like features likely arose from impurity particles introduced during sample preparation. To avoid any further confusion, we have replaced them with more representative micrographs (Figure 1 g in revised manuscript) and also provide below.\nFigure 1. g, Cryo-TEM visualizing of T-Expand, scale bar: 100 nm.\n3. In Figure 5i, the bioluminescence images in row 2, columns 3 and 6 appear to be identical, despite representing different treatment groups. Please verify whether this is a case of inadvertent image duplication.\nResponse: We thank the reviewer for carefully examining Figure 5i and for identifying this issue. This duplication was entirely our oversight during figure assembly. We have now corrected the error by replacing the duplicated panel with the appropriate image from the original dataset and have updated the figure accordingly (Figure 5 i in revised manuscript and added below). We sincerely appreciate the reviewer’s attention to detail in helping us identify and correct this mistake.\nFigure 5. i, Representative IVIS overlay images showing bioluminescent signals for each treatment group.\nMinor issues:\n1. The nanoparticle tracking analysis (NTA) data in Figure 1h suggests a potential bimodal distribution of particle size, with what appears to be two peaks. This raises the question of whether the T-Expand formulation consists of two distinct nanoparticle populations. The authors should elaborate on this in the Results section-either to explain the source of heterogeneity, or to clarify whether this is an artifact of measurement or a true feature of the formulation.\nResponse: In response to the reviewer’s concern regarding the nanoparticle size distribution, we conducted a thorough investigation. First, we confirmed that T-Expand does not comprise multiple particle populations, since it is formed exclusively via strain-promoted azide–alkyne cycloaddition between azide-functionalized dextran nanoparticles and antibodies. Click chemistry is an exceptionally efficient reaction. Therefore, it is unlikely that a fraction of nanoparticles remained unconjugated with antibodies, which could otherwise account for an additional peak population. Two peaks observed in the original NTA data were traced to contamination within the microfluidic sizing channel, which compromised the accuracy of those measurements.\nTo quantify this phenomenon more systematically, we added multimodal measurements. Cryo-TEM analysis of three randomly selected fields yielded a mean T-Expand diameter of 173.1 ± 37.27 nm (Figure 1 h in revised manuscript and inserted below). Repeat NTA and DLS measurements gave 204 ± 45.8 nm (Figure S11a in revised supplementary materials and added below) and 197 ± 39.9 nm (Figure S11b in revised supplementary and inserted below), respectively, approximately 24-31 nm larger than cryo-TEM which is consistent with the expected methodological difference that physical diameter (imaging) less than hydrodynamic diameter (solution measurements). Regarding the “two peaks” distribution observed after antibody conjugation, troubleshooting identified trace contaminants in an earlier NTA microfluidic cartridge; after cleaning and re-measurement, the anomalous subpopulation disappeared. All raw data and images have been included in the revised supplementary materials and also inserted below.\nFigure 1. h, The size distribution of T-Expand, as determined by Cryo-TEM and measured by software of image J with 3 independent figures.\nSupplementary Fig. 11. a, The size distribution of T-Expand, measured by NTA. b, The size distribution of T-Expand, measured by DLS.\n2. The representative contour plot in Supplementary Figure 16a appears to display an unusually low number of events, which limits the interpretability of the flow cytometry data. The authors should clarify the number of events collected and justify whether this sample is truly representative. If possible, consider replacing it with a plot from a higher-quality acquisition to support the conclusions drawn.\nResponse: We have introduced a new gating strategy of Figure S19a in revised supplementary and added below.\nSupplementary Fig. 19. T-expand expanded CAR T display antitumor activity in a xenograft model of B-cell lymphoma. (a) Gating strategy of mice spleen and bone marrow on using Flowjo.\nReference\n1. Liu, Z., Li, YR., Yang, Y. et al. Viscoelastic synthetic antigen-presenting cells for augmenting the potency of cancer therapies. Nat. Biomed. Eng 8, 1615–1633 (2024).\nhttps://doi.org/10.1038/s41551-024-01272-w.\n2. Casati, A., Varghaei-Nahvi, A., Feldman, S.A. et al. Clinical-scale selection and viral transduction of human naïve and central memory CD8+ T cells for adoptive cell therapy of cancer patients. Cancer Immunol Immunother 62, 1563–1573 (2013). https://doi.org/10.1007/s00262-013-1459-x\n3. https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0008945_SPC0021034_Dynabeads_CD3CD28CTS.pdf\n4. Feng, B., Bai, Z., Zhou, X. et al. The type 2 cytokine Fc–IL-4 revitalizes exhausted CD8+ T cells against cancer. Nature 634, 712–720 (2024). https://doi.org/10.1038/s41586-024-07962-4\n5. Kalbasi, A., Siurala, M., Su, L.L. et al. Potentiating adoptive cell therapy using synthetic IL-9 receptors. Nature 607, 360–365 (2022). https://doi.org/10.1038/s41586-022-04801-2\n6. Yamada-Hunter, S.A., Theruvath, J., McIntosh, B.J. et al. Engineered CD47 protects T cells for enhanced antitumour immunity. Nature 630, 457–465 (2024). https://doi.org/10.1038/s41586-024-07443-8\n7. Zhang, A.Q., Hostetler, A., Chen, L.E. et al. Universal redirection of CAR T cells against solid tumours via membrane-inserted ligands for the CAR. Nat. Biomed. Eng 7, 1113–1128 (2023). https://doi.org/10.1038/s41551-023-01048-8\n8. Zhang, D.K.Y., Adu-Berchie, K., Iyer, S. et al. Enhancing CAR-T cell functionality in a patient-specific manner. Nat Commun 14, 506 (2023). https://doi.org/10.1038/s41467-023-36126-7\n9. Jennifer N. Brudno, James N. Kochenderfer; Toxicities of chimeric antigen receptor T cells: recognition and management. Blood 2016; 127 (26): 3321–3330. doi: https://doi.org/10.1182/blood-2016-04-703751\n10. Norelli, M., Camisa, B., Barbiera, G. et al. Monocyte-derived IL-1 and IL-6 are differentially required for cytokine-release syndrome and neurotoxicity due to CAR T cells. Nat Med 24, 739–748 (2018). https://doi.org/10.1038/s41591-018-0036-4.\n11. Giavridis, T., van der Stegen, S.J.C., Eyquem, J. et al. CAR T cell–induced cytokine release syndrome is mediated by macrophages and abated by IL-1 blockade. Nat Med 24, 731–738 (2018). https://doi.org/10.1038/s41591-018-0041-7.\n12. Zhu, E., Yu, J., Li, YR. et al. Biomimetic cell stimulation with a graphene oxide antigen-presenting platform for developing T cell-based therapies. Nat. Nanotechnol. 19, 1914–1922 (2024). https://doi.org/10.1038/s41565-024-01781-4\n13. Zhang, D.K.Y., Brockman, J.M., Adu-Berchie, K. et al. Subcutaneous biodegradable scaffolds for restimulating the antitumour activity of pre-administered CAR-T cells. Nat. Biomed. Eng 9, 268–278 (2025). https://doi.org/10.1038/s41551-024-01216-4"}, {"round": 2, "reviewer_comments": {"reviewer_1": "After a thorough evaluation of the revised version of the manuscript “Dextran-based Nanoparticle Platform (T-Expand) for CAR T-Cell Expansion” by Zheng et al. (Ref. NCOMMS-25-30476A), I am fully convinced that the authors have satisfactorily addressed all my previous major concerns.\n\nI particularly appreciate the authors’ efforts in performing new, well-designed experiments that directly respond to the reviewers’ major critiques. The inclusion of quantitative stoichiometric analysis, detailed biocompatibility and stability assessments, and expanded methodological descriptions has considerably improved the quality of the manuscript.\n\nFurthermore, the revised discussion now provides a thoughtful and balanced interpretation of the translational relevance and safety considerations, including potential implications for cytokine release and functional divergence relative to existing aAPC platforms.\n\nOverall, the manuscript now presents a comprehensive, methodologically sound, and compelling body of work. The additional data and clarifications convincingly demonstrate the novelty and translational value of the reported T-Expand platform. I am satisfied that all major concerns have been fully resolved, and I believe the manuscript is now suitable for publication in its current form.", "reviewer_2": "The authors have provided a variety of new/altered data and analysis in response to the original reviews. Unfortunately, however, they did not respond adequately to the main technical concern from the original review - a comparison of the optimized Dynabeads and optimized dextran nanoparticles is required to establish if this new platform provides a true advantage. The experiment here is quite straightforward - repeat previous experitments with a range of Dynabead:cell ratios, similarly to how the studies were performed with their particles, but the authors did not perform.", "reviewer_3": "The authors have fully addressed my prior concerns with appropriate additional data, figure corrections, and mechanistic clarification. Therefore I support the publication of this work."}, "author_response": "Point-by-Point Response to Reviewer's Comments (Zheng et al. formerly manuscript NCOMMS-25-30476A)\nFor ease of reading, the reviewer’s comments are in regular typeface, our responses are highlighted in italics\nPoint by point reply to Reviewer #2 (Remarks to the Author):\nThe authors have provided a variety of new/altered data and analysis in response to the original reviews. Unfortunately, however, they did not respond adequately to the main technical concern from the original review - a comparison of the optimized Dynabeads and optimized dextran nanoparticles is required to establish if this new platform provides a true advantage. The experiment here is quite straightforward - repeat previous experitments with a range of Dynabead:cell ratios, similarly to how the studies were performed with their particles, but the authors did not perform.\nResponse：We thank the reviewer for emphasizing the importance of testing optimized Dynabeads™ conditions. To address this concern, we conducted additional experiments evaluating a range of Dynabeads™:T-cell ratios (0.3:1, 1:1, 3:1, and 5:1), alongside a control group without beads. We have confirmed that a 3:1 Dynabeads™:cell ratio represents the optimized condition for a fair comparison with T-Expand, based on the following factors:\n(1) Expansion efficiency:\nAll Dynabeads™-stimulated groups achieved measurable T-cell expansion by day 10 compared to the unstimulated control. Among the tested conditions, ratios of 3:1 and 5:1 yielded the highest cell numbers (Supplementary Fig. 16a, b in revised supplementary materials and added below), reaching levels consistent with those typically required for clinical-scale CAR T-cell manufacture.\n(2) CAR transduction and expansion:\nFollowing lentiviral transduction of CD19 CAR, both the transduction efficiency and expansion fold change were maximal at Dynabeads™:cell ratios of 3:1 and 5:1 (Supplementary Fig. 16 c, d in revised supplementary materials and added below). However, given that the 5:1 condition produced T cells with marginally higher exhaustion marker expression, we identified the 3:1 ratio as the optimal balance between proliferation and phenotypic preservation.\n(3) Phenotype analysis:\nFlow cytometry profiling revealed that increasing the Dynabeads™:cell ratio above 1:1 resulted, resulted in increased proportion of Terminally differentiated effector memory (TEMRA) T cells accompanied by a gradual decline in the effector-memory (TEM) subset (Supplementary Fig. 16e in revised supplementary materials and added below). Also, the higher bead-to-cell ratios slightly elevated the expression of exhaustion-associated markers (e.g., PD-1, LAG-3, TIGIT) (Supplementary Fig. 16f, g, h in revised supplementary materials and added below).\nCollectively, these results confirm that the standard 3:1 Dynabeads™:T-cell ratio represents the optimal condition for robust clinical T-cell activation and CAR T-cell expansion. This finding validates that the comparison in our study that between T-Expand and Dynabeads™ using this optimized (and provide recommended) dose.\nWe have added the following sentence in the results section: Optimization experiments revealed that a Dynabeads™:T-cell ratio of 3:1 yielded the most efficient CAR-T cell production (Supplementary Fig. 16a-h). Consequently, this ratio was selected as the benchmark condition for comparison with T-Expand.\nAnd included the new Supplementary Fig. 16, as shown below.\nSupplementary Fig. 16 T cells activated and transduced with CD19 CAR and expanded for 10 days using different DynabeadsTM to cell ratio. (a) Fold expansion of T cell from Day 0 to Day 10. (b) Total absolute cell numbers on Day 10 of expansion (c) Percentage of CAR T cells in total CD3+ T cell population analyzed using flowcytometry (d) Total number of CAR+ T cells on Day 10 (e) Phenotypic distribution of T-cells based on CCR7 and CD45RA expression, showing TN (CD45RA⁺CCR7⁺), TCM (CD45RA⁻CCR7⁺), TEM (CD45RA-CCR7-), and TEMRA (CD45RA+CCR7-) subsets. (f-h) T cells from Day 10 stained for exhaustion markers (f) LAG3 (g) TIGIT (h) PD1 and analyzed using flowcytometry."}, {"round": 3, "reviewer_comments": {"reviewer_2": "The authors have appropriately addressed the earlier concern with the inclusion of new data addressing the question."}, "author_response": ""}]

Decision letter