Biology subjects

Dotti, G.

Publications and source records attributed to Dotti, G..

10 recordsLinked to original sources

bioRxiv2026

IL-12 restores the sequential cytotoxic capacities of anti-GD2 CAR-T and CAR-iNKT cells against glioblastoma

Glioblastoma (GBM) is a highly aggressive brain tumor characterized by rapid progression and a poor prognosis. CAR-based cellular therapies are promising approaches, and CAR-T cells targeting GD2 have demonstrated transient efficacy. Identifying how tumors evade these treatments is essential for advancing therapy development. In this study, we investigated the mechanisms through which GBM cells evade GD2.chimeric antigen receptor (CAR)-T and CAR-invariant natural killer T (iNKT) in vitro and explored ways to overcome tumor escape. GD2-targeted CAR-T and CAR-iNKT cells were tested in a stepwise in vitro model that repeatedly exposed them to GD2+ cell lines. While CAR effector cells effectively killed GD2+ GBM cells in short-term assays, their anti-tumor efficacy declined after repeated antigen exposures. Tumor escape mechanisms included reduced CAR expression, impaired proliferation, reduced production of cytokine, granzyme, and perforin, tumor downregulation of GD2, trogocytosis, and upregulation of the HLA-E/NKG2A inhibitory compared to MICA-B/NKG2D activation pathways on tumor and immune cells. Increasing effector cell numbers or adding IL-15 +/- IL-7 partially improved CAR persistence but did not fully restore CAR effector functions. By contrast, IL-12 addition optimized tumor-killing capacity by increasing CAR effector cell proliferation, CAR surface expression, IFN-y production, and balancing HLA-E/NKG2A versus MICA-B/NKG2D pathways. In conclusion, GD2.CAR-T and GD2.CAR-iNKT cells effectively target GBM but are susceptible to repeated antigen exposure, which IL-12 could counteract. These findings encourage further development of armored IL-12 CAR-T or CAR-iNKT cells and further investigation of the roles of HLA-E and MICA-B pathways in immunotherapy against GBM.

immunology↗

bioRxiv2026

NG2-targeting macrophages inhibit 3D invasion of patient-derived glioblastoma spheroids

Engineering macrophages with chimeric antigen receptors is emerging as a promising cancer therapeutic. Chimeric antigen receptor-expressing macrophages (CAR-Ms) engineered to recognize tumor-specific antigens have been shown to inhibit tumor growth and activate adaptive immune responses, leading to robust tumor control in animal studies. Based on this work, clinical trials have been initiated. While the trials have shown promise, challenges remain. The dynamic interactions between CAR-Ms and cancer cells and the exact mechanisms driving anti-tumor effects remain poorly defined. Defining the dynamic interactions between CAR-Ms and cancer cells will provide critical insights for optimizing future CAR-M design and improving therapeutic efficacy. We sought to directly visualize CAR-M interactions with glioblastoma cells at high-resolution and in real-time using CAR-Ms engineered to recognize Neural-Glial Antigen 2 (NG2), an antigen expressed on glioblastoma cells. Using patient-derived glioblastoma cells, we formed glioblastoma spheroids and embedded them in a 3D matrix together with CAR-Ms. Using time-lapse microscopy, as expected, we found that NG2-targeting CAR-Ms engulfed glioblastoma cells. However, excitingly, we found that NG2-targeting CAR-Ms blocked >85% of glioblastoma cell invasion in 3D. This inhibition of glioblastoma invasion was not due to a significant change in CAR-M polarization states. Together, these data suggest that NG2-targeting CAR-Ms both engulf glioblastoma cells and block glioblastoma invasive behavior.

cancer biology↗

bioRxiv2026

Latent Regulatory Programs Generate Synthetic T Cell States with Enhanced Therapeutic Potential

Transcription factors (TFs) govern cell fate through coordinated gene-regulatory networks, yet the full potential of these networks to generate non-native, therapeutically advantageous cell states in vivo remains largely unexplored. We hypothesized that systematic gain-of-function (GOF) overexpression of TFs in CD8 T cells, central mediators of immune protection, could reveal latent, or "hidden," regulatory programs capable of generating synthetic T cell states with therapeutic utility. To test this, we developed single-cell GOF sequencing (scGOF-seq), a multiplexed platform for unbiased, in vivo mapping of GOF effects on T cell fate in immunocompetent mouse models of infection and cancer. scGOF-seq uncovered unexpected regulators of T cell differentiation and accumulation, including SOX2, OCT4, and GATA2, which are normally silenced during T cell differentiation. Notably, outside its native regulatory context, supraphysiologic cMyc GOF reprogrammed CD8 T cells into a synthetic stem-effector hybrid state, enabling >5,000-fold antigen-dependent expansion and antitumor activity, contrasting sharply with its native function in driving terminal differentiation. scGOF-seq further identified TF modules that cooperate with cMyc GOF to promote robust CD8 T cell responses in solid tumors. Together, these findings establish GOF perturbation as a powerful strategy for revealing latent immune regulatory programs and engineering synthetic immune states with therapeutic potential. One-Sentence SummaryIn vivo single-cell gain-of-function screening reveals latent transcriptional programs that can reprogram T cells into highly functional synthetic states.

immunology↗

bioRxiv2025

Obesity Impairs the Antitumor Activity of CAR-T Cells in Triple-Negative Breast Cancer

BackgroundWe have reported that chimeric antigen receptor (CAR) T cells targeting B7-H3 (B7-H3.CAR) are effective in a preclinical model of triple negative breast cancer (TNBC), and have initiated a Phase I study to assess safety and efficacy. However, heterogeneous antigen expression and immunosuppressive tumor microenvironments (TME) remain roadblocks for effective CAR-T cell therapy. In particular, obesity represents a negative prognostic factor in TNBC partly due to chronic inflammation and impaired adaptive immune responses. Hence, we sought to determine if obesity can affect the antitumor activity of B7-H3.CAR-T cells. MethodsWe used qPCR and western blotting to determine if cytokines associated with obesity affect B7-H3 expression in TNBC cell lines. Furthermore, we used shRNA to suppress B7-H3 expression in a syngeneic orthotopic E0771 tumor model and measured tumor growth in control and diet-induced obese (DIO) mice. Finally, we evaluated the antitumor effects of B7-H3.CAR-T cells in both control and DIO mice orthotopically engrafted with the E0771 tumor cell line. Immune profiling was conducted using flow cytometry. ResultsObesity-related inflammatory cytokines promote B7-H3 expression in human and murine TNBC cells in vitro and B7-H3 expression correlates with tumor aggressiveness in vivo. CAR-T cells obtained from control or DIO mice were equally cytotoxic in vitro but activated T cells and B7-H3.CAR-T cells obtained from DIO mice show transcriptomic changes (enriched Tox2, Prdm1, Batf) and impaired glycolytic capacity, respectively. Finally, we demonstrated that obesity impairs CAR-T cell antitumor effects and durability of response in vivo with a near complete loss of memory formation. ConclusionsHere we identified a correlation between B7-H3 expression, obesity, and rate of tumor growth in TNBC. Furthermore, we showed that obesity constrains both the ability of B7-H3.CAR-T cells to control tumor growth and to elicit durable immunological memory. Taken together, these data identify obesity as an underappreciated and potent modulator of CAR-T cell functionality. What is already knownB7-H3 protein is upregulated in many human malignancies including TNBC and is often associated with worsened outcomes. B7-H3.CAR-T cells show promise in preclinical models of TNBC and entered clinical translation. What this study addsThis study identifies a previously unknown correlation between obesity, B7-H3 expression, and rate of tumor growth in TNBC. Furthermore, our preclinical model of B7-H3.CAR-T cell therapy demonstrates that obesity negatively affects the antitumor activity of B7-H3.CAR-T cells in TNBC. How this study affects other research/practiceThis study highlights obesity as an understudied and critically important covariate for adoptive T-cell therapy and demonstrates important links between systemic metabolism and antigen expression. This work paves the way for future mechanistic and translational research into how obesity impacts CAR-T cell functionality.

cancer biology↗

bioRxiv2025

IL-18 metabolically reprograms CAR-expressing natural killer T cells and enhances their antitumor activity against neuroblastoma

Invariant natural killer T cells (NKTs) have intrinsic anti-tumor properties that make them promising candidates for chimeric antigen receptor (CAR)-based immunotherapies. Transgenic cytokine expression has been shown to enhance the potency of cellular immunotherapies, and we hypothesized that co-expressing IL-18 alone or with IL-15 would boost CAR-NKT therapeutic potential. To test this hypothesis, we generated retroviral constructs expressing IL-15 and/or IL-18 with the inducible caspase 9 (iC9) safety switch and co-transduced them with a GD2-specific CAR into human NKTs. Co-expression of IL-18 or IL-15/IL-18 increased GD2.CAR-NKT in vitro cytotoxicity, proliferation, and cytokine secretion compared to IL-15 alone. In a metastatic neuroblastoma model, GD2.CAR-NKTs expressing constructs with IL-18 controlled tumor growth better than cells expressing IL-15 only, but mice in the IL-15/IL-18 group developed severe toxicities not observed in the IL-18-only group. Mechanistically, we found that IL-18 drives a distinct transcriptional profile from IL-15 in CAR-NKTs marked by lower expression of exhaustion gene signatures and enrichment of metabolism-related processes. Finally, targeted metabolomics revealed that IL-18 induces broad metabolic reprogramming in CAR-NKTs including enhancement of oxidative phosphorylation, glycolysis, glutaminolysis and purine metabolism. These results support the use of IL-18 in developing the next generation of cytokine-armed CAR-NKT cancer immunotherapy.

immunology↗

bioRxiv2024

Exploring Breast Cancer-Related Biochemical Changes in Circulating Extracellular Vesicles Using Raman Spectroscopy

Extracellular vesicles (EVs) are a subgroup of the circulating particles, released by cells in both normal and diseased states, carrying active biomolecules. They have gained significant attention as potential cancer biomarkers, particularly in breast cancer (BC). Previous research showed variations in EVs content and quantity between BC patients and healthy controls (HC). However, studying EVs biochemical profile remains challenging due to their low abundance and complex composition. Additionally, EVs may interact with other plasma components, like lipoproteins (LPs), forming a so called "biomolecular corona" that further complicates their analysis. Here, Raman spectroscopy (RS) is proposed as a fast tool to obtain the biochemical profile of circulating EVs in the context of BC. RS was employed to differentiate various extracellular particles (EPs) in blood, including LPs and EVs. The study also evaluated RSs capability to quantify major classes of biomolecules and compared these results with those obtained by traditional biochemical assays. Finally, compositional differences in large EVs (lEVs) and small EVs (sEVs) were assessed between 30 HC and 34 BC patients. RS revealed the existence of distinct biochemical signatures associated with BC, highlighting increased levels of nucleic acids and lipids in the BC group.

biophysics↗

bioRxiv2024

CSPG4-targeting CAR-macrophages inhibit melanoma growth

Chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment of hematological malignancies but has been clinically less effective in solid tumors. Engineering macrophages with CARs has emerged as a promising approach to overcome some of the challenges faced by CAR-T cells due to the macrophages ability to easily infiltrate tumors, phagocytose their targets, and reprogram the immune response. We engineered CAR-macrophages (CAR-Ms) to target chondroitin sulfate proteoglycan 4 (CSPG4), an antigen expressed in melanoma, and several other solid tumors. CSPG4-targeting CAR-Ms exhibited specific phagocytosis of CSPG4-expressing melanoma cells. Combining CSPG4-targeting CAR-Ms with CD47 blocking antibodies synergistically enhanced CAR-M-mediated phagocytosis and effectively inhibited melanoma spheroid growth in 3D. Furthermore, CSPG4-targeting CAR-Ms inhibited melanoma tumor growth in mouse models. These results suggest that CSPG4-targeting CAR-M immunotherapy is a promising solid tumor immunotherapy approach for treating melanoma. STATEMENT OF SIGNIFICANCEWe engineered macrophages with CARs as an alternative approach for solid tumor treatment. CAR-macrophages (CAR-Ms) targeting CSPG4, an antigen expressed in melanoma and other solid tumors, phagocytosed melanoma cells and inhibited melanoma growth in vivo. Thus, CSPG4-targeting CAR-Ms may be a promising strategy to treat patients with CSPG4-expressing tumors.

cancer biology↗

bioRxiv2022

Inadvertent Transfer of Murine VL30 Retrotransposons to CAR-T Cells.

For more than a decade genetically engineered autologous T-cells have been successfully employed as immunotherapy drugs for patients with incurable blood cancers. The active component in some of these game-changing medicines are autologous T-cells that express viral vector-delivered chimeric antigen receptors (CARs), which specifically target proteins that are preferentially expressed on cancer cells. Some of these therapeutic CAR expressing T-cells (CAR-Ts) are engineered via transduction with {gamma}-retroviral vectors ({gamma}-RVVs) produced in a stable producer cell line that was derived from murine PG13 packaging cells (ATCC CRL-10686). Earlier studies reported on the co-packaging of murine virus-like 30S RNA (VL30) genomes with {gamma}-retroviral vectors generated in murine stable packaging cells. In an earlier study VL30 mRNA was found to enhance the metastatic potential of human melanoma cells. These findings raise biosafety concerns regarding the possibility that therapeutic CAR-Ts have been inadvertently contaminated with potentially oncogenic VL30 retrotransposons. In this study, we demonstrated the presence of infectious VL30 particles in PG13 cells conditioned media and observed the ability of these particles to deliver transcriptionally active VL30 genomes to human cells. Notably, VL30 genomes packaged by HIV-1-based vector particles transduced naive human cells in culture. Furthermore, we detected transfer and expression of VL30 genomes in clinical-grade CAR-Ts generated by transduction with PG13 cells-derived {gamma}-retroviral vectors. Our findings raise biosafety concerns regarding the use of murine packaging cell lines in ongoing clinical applications.

microbiology↗

bioRxiv2019

CD16-158-VALINE CHIMERIC RECEPTOR T CELLS OVERCOME THE RESISTANCE OF KRAS-MUTATED COLORECTAL CARCINOMA CELLS TO CETUXIMAB

KRAS mutation hinders the therapeutic efficacy of epidermal-growth-factor-receptor (EGFR) mAb (cetuximab and panitumumab)-based immunotherapy of EGFR+ cancers. Although, cetuximab controls KRAS-mutated cancer cell growth in vitro utilizing a NK cell-mediated antibody-dependent-cellular-cytotoxicity-(ADCC) mechanism, KRAS-mutated colorectal carcinoma (CRC) cells can still escape NK cell immunosurveillance. To overcome this limitation, we used cetuximab and panitumumab to redirect Fc{gamma} chimeric receptor (CR) T cells against KRAS-mutated HCT116 CRC cells. We compared 4 polymorphic Fc{gamma}-CR constructs including CD16158F-CR, CD16158V-CR, CD32131H-CR, and CD32131R-CR which were transduced into T cells utilizing retroviral transduction. Percentages of transduced T cells expressing CD32131H-CR (83.5{+/-}9.5) and CD32131R-CR (77.7.{+/-}13.2) were significantly higher than those expressing with CD16158F-CR (30.3{+/-}10.2) and CD16158V-CR (51.7{+/-}13.7) (p<0.003). CD32131R-CR T cells specifically bound soluble cetuximab and panitumumab. However, only CD16158V-CR T cells released significantly higher levels of interferon gamma (IFN{gamma}=1145.5 pg/ml {+/-}16.5 pg/ml, p<0.001) and tumor necrosis factor alpha (TNF=614 pg/ml {+/-} 21 pg/ml, p<0.001) than non-transduced T cells when incubated with KRAS-mutated HCT116 cells opsonized with cetuximab. Only CD16158V-CR T cells combined with cetuximab controlled the growth of HCT116 cells subcutaneously engrafted in CB17-SCID mice. These results suggest that CD16158V-CR T cells combined with cetuximab represent useful reagents to develop an effective immunotherapy of EGFR+KRAS-mutated cancer.

immunology↗

bioRxiv2019

ELIMINATION OF EGFR-OVEREXPRESSING CANCER CELLS BY CD32 CHIMERIC RECEPTOR T CELLS IN COMBINATION WITH CETUXIMAB OR PANITUMUMAB

Cetuximab and panitumumab bind the human epidermal growth factor receptor (EGFR). While the chimeric cetuximab (IgG1) triggers antibody-dependent-cellular-cytotoxicity (ADCC) of EGFR positive target cells, panitumumab (a human IgG2) does not. The inability of panitumumab to trigger ADCC reflects a poor binding affinity of human IgG2 Fc for the Fc{gamma}RIII (CD16) on NK cells. However, both human IgG1 and IgG2 bind the Fc{gamma}RII (CD32) to a similar extent. Here, we have compared the ability of T cells, engineered with a novel low-affinity CD32131R -chimeric receptor (CR), and those engineered with the low-affinity CD16158F-CR T cells in eliminating EGFR positive epithelial cancer cells (ECCs) in combination with cetuximab or panitumumab. Following T cell transduction, the percentage of CD32131R-CR T cells was (74{+/-}10) significantly higher than that of CD16158F-CR T cells (46{+/-}15). Only CD32131R-CR T cells bound panitumumab. CD32131R-CR T cells combined with the mAb 8.26 (anti-CD32) and CD16158F-CR T cells combined with the mAb 3g8 (anti-CD16) eliminated colorectal carcinoma (CRC), HCT116Fc{gamma}R+ cells, in a reverse ADCC assay in vitro. Cross-linking of CD32131R-CR on T cells by cetuximab or panitumumab and CD16158F-CR T cells by cetuximab induced elimination of triple negative breast cancer (TNBC) MDA-MB-468 cells, and secretion of IFN gamma (IFN{gamma}) and tumor necrosis factor alpha (TNF). Neither cetuximab nor panitumumab induced Fc{gamma}-CR T anti-tumor activity against KRAS-mutated HCT116, non-small-cell-lung-cancer, A549 and TNBC, MDA-MB-231 cells. ADCC of Fc{gamma}-CR T cells was significantly associated with the over-expression of EGFR on ECCs. In conclusion, CD32131R-CR T cells are efficiently redirected by cetuximab or panitumumab against BC cells overexpressing EGFR.\n\nArticle categoryTumor Immunology and Microenvironment\n\nNovelty and ImpactMonoclonal antibody-redirected Fc{gamma}-CR T cell immunotherapy represents a promising approach in the fight against cancer. Here, we expand the application of this methodology to TNBC overexpressing the EGFR utilizing a novel CD32A131R-CR in combination with anti-EGFR mAbs. Our study supports the use of CD32A131R-CR T cells combined with panitumumab or cetuximab for targeting TNBC cells overexpressing the EGFR. Our results may be utilized as a platform for the rational design of therapies targeting TNBC overexpressing EGFR.

immunology↗