Stem Cell Memory Research Articles

A Phase II Open-Label, Randomized Clinical Trial of Atezolizumab with or without Human Recombinant IL-7 (CYT107) in Advanced Urothelial Cancer.

Advanced urothelial cancer generally has high mortality despite modern anti-PD-1/L1 antibody-based combinations. Augmenting checkpoint inhibitor-mediated immune responses with lymphocyte growth factors may improve outcomes. We conducted a randomized phase II study (Cancer Immunotherapy Trials Network-14) in 47 patients to explore whether human recombinant IL-7 (CYT107) could be safely combined with PD-L1 inhibition to enhance responses. Patients with urothelial cancer after platinum chemotherapy were randomized to atezolizumab alone or with CYT107 weekly for four doses. The primary objective was clinical efficacy by the objective response rate (ORR). Secondary objectives included safety, toxicity, and other clinical outcomes. Correlative endpoints included peripheral immunophenotyping and quantification of cytokines. CYT107 plus atezolizumab was well-tolerated, without dose-limiting toxicities and lower grade 3 to 4 treatment-related adverse events compared with atezolizumab monotherapy. The ORR was 26.3% for the combination therapy versus 23.8% for atezolizumab alone (P = 0.428). The complete response rate was 10.5% for the combination therapy versus 4.8% for monotherapy. Three patients on combination therapy had responses >21 months versus one with monotherapy. CD4+ and CD8+ T-lymphocyte expansion occurred in patients with response to combination therapy, with the greatest effect in T memory stem cells. Patients who responded to treatment exhibited elevated baseline levels of CCL4 and reduced levels of VEGFA and TNF. Combining CYT107 with atezolizumab was safe and resulted in lymphocyte expansion, a doubling of the complete response rate, and durable responses exceeding 2 years. However, the ORR was similar to atezolizumab alone. Increased and sustained doses of CYT107 coupled with patient selection strategies should be further investigated.

Breast cancer stem cells origins and the memory stem cells: clinical significance of biomarkers and the active therapeutic approaches

Despite recent improvements in diagnosis and treatment, breast cancer (BC) remains one of the leading causes of cancer-related deaths among women. In this complex disease, breast cancer stem cells (BCSCs) are a small but significant subset of different cancer cells with the ability to proliferate and self-renew. According to an increasing amount of studies, BCSCs are essential for breast cancer metastasis, drug resistance, and recurrence. Due to its diverse nature, BC includes numerous subtypes, each of which displays unique BCSC types and concentrations that are connected to different therapy outcomes and outcomes. Despite significant advancements in the treatment of early-stage breast cancer, there are still few effective therapy approaches for metastatic BC. The development, progression, and dissemination of BC are largely attributed to cancer stem-like cells (CSCs), which are characterized by their exceptional adaptability and self-renewal ability. An overview of the development of BCSCs, their biomarkers, clinical significance, and the mechanisms behind their behavior is the goal of the current study. The active therapy strategies being employed to address BCSCs will also be examined.

Viscoelastic synthetic antigen-presenting cells for augmenting the potency of cancer therapies.

The use of synthetic antigen-presenting cells to activate and expand engineered T cells for the treatment of cancers typically results in therapies that are suboptimal in effectiveness and durability. Here we describe a high-throughput microfluidic system for the fabrication of synthetic cells mimicking the viscoelastic and T-cell-activation properties of antigen-presenting cells. Compared with rigid or elastic microspheres, the synthetic viscoelastic T-cell-activating cells (SynVACs) led to substantial enhancements in the expansion of human CD8+ T cells and to the suppression of the formation of regulatory T cells. Notably, activating and expanding chimaeric antigen receptor (CAR) T cells with SynVACs led to a CAR-transduction efficiency of approximately 90% and to substantial increases in T memory stem cells. The engineered CAR T cells eliminated tumour cells in a mouse model of human lymphoma, suppressed tumour growth in mice with human ovarian cancer xenografts, persisted for longer periods and reduced tumour-recurrence risk. Our findings underscore the crucial roles of viscoelasticity in T-cell engineering and highlight the utility of SynVACs in cancer therapy.

Manufacturing CD20/CD19-targeted iCasp9 regulatable CAR-TSCM cells using a Quantum pBac-based CAR-T engineering system.

CD19-targeted chimeric antigen receptor (CAR) T cell therapies have driven a paradigm shift in the treatment of relapsed/refractory B-cell malignancies. However, >50% of CD19-CAR-T-treated patients experience progressive disease mainly due to antigen escape and low persistence. Clinical prognosis is heavily influenced by CAR-T cell function and systemic cytokine toxicities. Furthermore, it remains a challenge to efficiently, cost-effectively, and consistently manufacture clinically relevant numbers of virally engineered CAR-T cells. Using a highly efficient piggyBac transposon-based vector, Quantum pBac™ (qPB), we developed a virus-free cell-engineering system for development and production of multiplex CAR-T therapies. Here, we demonstrate in vitro and in vivo that consistent, robust and functional CD20/CD19 dual-targeted CAR-T stem cell memory (CAR-TSCM) cells can be efficiently produced for clinical application using qPB™. In particular, we showed that qPB™-manufactured CAR-T cells from cancer patients expanded efficiently, rapidly eradicated tumors, and can be safely controlled via an iCasp9 suicide gene-inducing drug. Therefore, the simplicity of manufacturing multiplex CAR-T cells using the qPB™ system has the potential to improve efficacy and broaden the accessibility of CAR-T therapies.

Stem cell memory EBV-specific T cells control EBV tumor growth and persist in vivo.

Adoptive T cell therapy (ACT), the therapeutic transfer of defined T cell immunity to patients, offers great potential in the fight against different human diseases including difficult-to-treat viral infections, but persistence and longevity of the cells are areas of concern. Very-early-differentiated stem cell memory T cells (TSCMs) have superior self-renewal, engraftment, persistence, and anticancer efficacy, but their potential for antiviral ACT remains unknown. Here, we developed a clinically scalable protocol for expanding Epstein-Barr virus (EBV)-specific TSCM-enriched T cells with high proportions of CD4+ T cells and broad EBV antigen coverage. These cells showed tumor control in a xenograft model of EBV-induced lymphoma and were superior to previous ACT protocols in terms of tumor infiltration, in vivo proliferation, persistence, proportion of functional CD4+ T cells, and diversity of EBV antigen specificity. Thus, our protocol may pave the way for the next generation of potent unmodified antigen-specific cell therapies for EBV-associated diseases, including tumors, and other indications.

Detection and characterization of autoreactive memory stem T-cells in children with acute immune thrombocytopenia

Primary immune thrombocytopenia (ITP) is an acquired autoimmune disorder characterized by an isolated decrease in platelets below 100 × 109/l after the exclusion of other conditions associated with thrombocytopenia. We investigated the role of different memory T-cell subsets, including T stem cell memory (TSCM), in children diagnosed with primary ITP and its association with therapeutic duration. This case–control study included 39 pediatric patients with acute ITP admitted to the Children's Hospital at Assiut University. Using a FACSCanto flow cytometer, CD8 + and CD4 + T-lymphocytes were gated. Five different subsets were characterized in each of these cells according to CD45RO and CD45RA expression. Afterward, gating was performed based on CCR7, CD95, and CD27. Examination of the CD8 + T cells subpopulation showed that Central memory T (TCM) and CD8+ Naïve T (TN) cells were significantly lower in ITP patients than in healthy children (p < 0.0001) and (p = 0.01), respectively. In addition, CD8 + TEMRA was significantly higher in ITP children than in controls (p = 0.001). CD4 + TCM cells were significantly lower in the ITP patient group (p = 0.04). However, CD4 + TEM was significantly higher in patients than controls (p = 0.04). Our research found that ITP patients had an imbalance in the ratio of CD4+ to CD8+ T cells in the peripheral blood and that TCM cells may be involved in the pathogenetic mechanism of ITP. TCMs could help in prediction of patients with higher risk of developing ITP.

Abnormal epigenetic memory of mesenchymal stem and progenitor cells caused by fetal malnutrition induces hypertension and renal injury in adulthood.

Fetal malnutrition has been reported to induce hypertension and renal injury in adulthood. We hypothesized that this hypertension and renal injury would be associated with abnormal epigenetic memory of stem and progenitor cells contributing to organization in offspring due to fetal malnutrition. We measured blood pressure (BP) for 60 weeks in offspring of pregnant rats fed a normal protein diet (Control), low-protein diet (LP), and LP plus taurine (LPT) in the fetal period. We used western blot analysis to evaluate the expression of αSMA and renin in CD44-positive renal mesenchymal stem cells (MSCs) during differentiation by TGF-β1. We measured kidney label-retaining cells (LRCs) at 11 weeks of age and formation of endothelial progenitor cells (EPCs) at 60 weeks of age from the offspring with fetal malnutrition. Epigenetics of the renal MSCs at 14 weeks were investigated by ATAC-sequence and RNA-sequence analyses. BP was significantly higher in LP than that in Control and LPT after 45-60 weeks of age. Numbers of LRCs and EPC colonies were significantly lower in LP than in Control. Renal MSCs from LP already showed expression of h-caldesmon, αSMA, LXRα, and renin before their differentiation. Epigenetic analyses identified PAR2, Chac1, and Tspan6 genes in the abnormal differentiation of renal MSCs. These findings suggested that epigenetic abnormalities of stem and progenitor cell memory cause hypertension and renal injury that appear in adulthood of offspring with fetal malnutrition.

A culture method with berbamine, a plant alkaloid, enhances CAR-T cell efficacy through modulating cellular metabolism

Memory T cells demonstrate superior in vivo persistence and antitumor efficacy. However, methods for manufacturing less differentiated T cells are not yet well-established. Here, we show that producing chimeric antigen receptor (CAR)-T cells using berbamine (BBM), a natural compound found in the Chinese herbal medicine Berberis amurensis, enhances the antitumor efficacy of CAR-T cells. BBM is identified through cell-based screening of chemical compounds using induced pluripotent stem cell-derived T cells, leading to improved viability with a memory T cell phenotype. Transcriptomics and metabolomics using stem cell memory T cells reveal that BBM broadly enhances lipid metabolism. Furthermore, the addition of BBM downregulates the phosphorylation of p38 mitogen-activated protein kinase and enhanced mitochondrial respiration. CD19-CAR-T cells cultured with BBM also extend the survival of leukaemia mouse models due to their superior in vivo persistence. This technology offers a straightforward approach to enhancing the antitumor efficacy of CAR-T cells.

Impact of urolithin A supplementation, a mitophagy activator on mitochondrial health of immune cells (MitoIMMUNE): A randomized, double-blind, placebo-controlled trial in healthy adults.

e14562 Background: Mitochondrial dysfunction and stem cell exhaustion are hallmarks of aging, driving inflammation and limiting immune function. Likewise, cancer is characterized by repression of mitochondrial dynamics in tumor-infiltrating leukocytes that fosters terminal T cell exhaustion. Yet, interventions to reliably improve immune function are still lacking. Urolithin A (UA) is a postbiotic known to improve muscle function in aged adults upon oral intake. We have recently shown that UA induces mitophagy in human CD8+ cells to induce formation of T memory stem cells, while sensitizing murine cancer to immunotherapy. Methods: In this double-blind, prospective, placebo-controlled trial (NCT05735886) we sought to characterize for the first time the effects of UA on the human immune system. Fifty healthy participants aged 45-70 years were randomized (1:1) to an intervention of single-dose (1000mg) UA or placebo per day for a study duration of 28 days. Randomization was performed based on age, gender and BMI. Exclusion criteria were prior medical conditions, BMI &gt;35 kg/m2, smoking, and intake of prescription medication or other dietary supplements. The primary end points of the study were changes in peripheral CD3+ cells, as well as alterations in mitochondrial activity in select immune populations of the peripheral blood as assessed by flow cytometry. Results: Intake of UA was safe and well-tolerated in the intervention cohort of 25 participants. After the study period, total peripheral lymphocytes and circulating NK cells were expanded in the UA group. Performing a complete immunophenotyping via spectral flow cytometry, we found that UA elicits immune remodeling characterized by broad changes of immune surface markers and mitochondrial measurements. CD8+ cells of participants in the intervention arm displayed greater mitochondrial mass, while preferably taking on a naive phenotype and expressing more Ki67. In addition, circulating monocytes exhibited a less inflammatory signature. UA intake reduced plasma levels of several proinflammatory cytokines. Conclusions: Collectively, our study constitutes an unprecedented intervention-based assessment of immune aging that globally characterizes the effect of UA on the immune system, proposing validation in future confirmatory studies and potential combination with cancer immunotherapy. Clinical trial information: NCT05735886 .

Investigation of a potential protein biomarker signature that may predict clinical benefit of NT-I7 and pembrolizumab in patients with cold gastrointestinal tumors.

2563 Background: Microsatellite stable colorectal (MSS-CRC) and pancreatic cancer (PDAC) are immunologically cold tumors due to low immunogenicity and lack of genomic diversity. NT-I7, a long-acting IL-7, and pembrolizumab (pembro) show efficacy in these hard-to-treat indications. While a limited set of patients (pts) achieve objective response, frequency and duration of the disease control rate point to a larger subset obtaining clinical benefit. To identify novel predictive biomarkers, we analyzed baseline peripheral and biopsy samples from pts based on treatment duration. Methods: Open-label Phase 2a study in pts with relapsed/refractory checkpoint inhibitor-naïve MSS-CRC and PDAC; NT-I7 1200 µg/kg IM every 6 weeks (Q6W), pembro 200 mg IV Q3W. Subjects were grouped by treatment duration, measured in NT-I7 doses administered before treatment discontinuation for any cause: 1 dose was short (ST), 2-3 doses medium (MT) and ≥4 doses long (LT). Correlative studies included peripheral (proteomics, T cell receptor sequencing [TCRseq], single cell RNA sequencing [scRNAseq]), and biopsy (genomics, transcriptomics, TCRseq). Results: As of 02OCT2023, 53 evaluable pts completed or discontinued treatment; 5 are still on follow up. ST group included 21 pts, MT 22 and LT 10 (including all 5 partial responders). Tumor biopsies were confirmed MSS with low tumor mutational burden (TMB). LT pts had similar age (59.0 [53.0-71.5] vs 66.0 [47.3-73.5]; ST vs LT) and lower baseline tumor burden (81.0 mm vs 58.0 mm; ST vs LT, p = 0.022). Biopsy analysis showed LT pts had, at baseline, upregulated pathways related to immune activity despite confirmed cold tumor status. Baseline scRNAseq in peripheral blood showed that stem-like CD8 T cells (precursors of exhausted [TPEX] and stem-cell memory [TSCM]) had a differential activation pattern in LT pts; those pathways were enriched in memory effector subsets in ST pts. Preserved antigen-specific stemness may be needed for NT-I7 + pembro efficacy. Baseline concentrations of 3 proteins that can be produced by growing tumors were significantly increased in ST pts. Pts were classified based on elevated levels of these potential biomarkers: POSITIVE (≥2 biomarkers; 20 pts) and NEGATIVE (≤1 biomarker; 33 pts). Pts with NEGATIVE signature at baseline had significantly higher overall survival, regardless of ST, MT or LT status (13.2 vs 8.9 months, p = 0.030). Conclusions: Preserved tumor-specific TPEX activity may be required for NT-I7 + pembro activity based on its presence in LT pts, who remained on treatment the longest. According to this analysis, there are 3 potentially predictive protein biomarkers that may help identify a pt subset more likely to experience clinical benefits from the combination treatment of NT-I7 + pembro. Further verification of the predictive nature of this signature in independent cohorts is ongoing. Clinical trial information: NCT04332653 .

Optimization of T Cell Activation Protocols to Enrich Stem Cell Memory Phenotypes: Metabolic and Health Assays as Predictive Tools

Abstract The generation of T cells with a stem cell memory phenotype (T_SCM) holds significant promise for immunotherapy due to their longevity and potent antitumor activity. This study aimed to optimize T cell activation and expansion conditions to selectively enrich for the T_SCM population. Utilizing a suite of cell health and metabolism assays, we monitored T cell cultures to identify predictive markers of T_SCM enrichment. We observed that early activation steps correlated with a surge in ATP production and alterations in cell reducing potential, indicative of metabolic reprogramming. Subsequent activation phases were marked by an upregulation in both catabolic and anabolic pathways, as evidenced by the increased turnover of key metabolites including glucose, lactate, glutamine, glutamate, branched-chain amino acids (BCAA), and pyruvate. Notably, the rate of T cell activation influenced the composition of the resultant T cell populations, with the media composition further affecting these dynamics. Our findings suggest that metabolic assays, particularly those measuring changes in ATP levels and primary metabolite flux, can serve as effective early predictors of T_SCM enrichment. These insights may guide the development of more efficacious T cell-based immunotherapies by facilitating the targeted generation of T cells with a stem cell-like memory phenotype.

Transpiration of neurological phenomena in cancer: Long‐term depression (LTD) abrogates cancer stem cell memory and sensitizes it to metabolic therapy

AbstractTherapy resistance and recurrence are irrefutably ascribed to the unique attributes of the cancer stem cell (CSC) population. Therefore, discerning the underlying mechanism is imperative in arbitrating a strategic therapeutic approach. In this study, we have observed the coexistence of neuron‐like cell (NLC) subpopulations in different cancer cell lines. The NLC subset was further validated by transfection studies and was found to be positive for PSD‐95. As a proof of concept, we have also demonstrated the transpiration of neurological phenomena such as long‐term potentiation (LTP) and long‐term depression (LTD) in cancer. We have utilized PMA drug and IL‐13 cytokine to study LTD and LTP, respectively, and observed a correlation between the memory marker (GLUN2B) and stemness markers (CD95 and CD58). Moreover, the transcriptional protein pCREB, which plays a crucial role in memory formation, was found to influence CD44 protein levels. These findings suggest that the instigation of LTD can impair cellular memory and sensitize CSC to the metabolic inhibitor, 3Bromopyruvate. Furthermore, this study interfaces the neurobiology of cancer and nanomedicine, wherein we have used biomimetic nanocarriers for the active targeting of drugs and demonstrate their ability to augment therapeutic efficiency.

Metabolic priming of GD2 TRAC-CAR T cells during manufacturing promotes memory phenotypes while enhancing persistence

Manufacturing chimeric antigen receptor (CAR) Tcell therapies is complex, with limited understanding of how medium composition impacts Tcell phenotypes. CRISPR-Cas9 ribonucleoproteins can precisely insert a CAR sequence while disrupting the endogenous Tcell receptor alpha constant (TRAC) gene resulting in TRAC-CAR Tcells with an enriched stem cell memory Tcell population, a process that could be further optimized through modifications to the medium composition. In this study we generated anti-GD2 TRAC-CAR Tcells using "metabolic priming" (MP), where the cells were activated in glucose/glutamine-low medium and then expanded in glucose/glutamine-high medium. Tcell products were evaluated using spectral flow cytometry, metabolic assays, cytokine production, cytotoxicity assays invitro, and potency against human GD2+ xenograft neuroblastoma models invivo. Compared with standard TRAC-CAR Tcells, MP TRAC-CAR Tcells showed less glycolysis, higher CCR7/CD62L expression, more bound NAD(P)H activity, and reduced IFN-γ, IL-2, IP-10, IL-1β, IL-17, and TGF-β production at the end of manufacturing exvivo, with increased central memory CAR Tcells and better persistence observed invivo. MP with medium during CAR Tcell biomanufacturing can minimize glycolysis and enrich memory phenotypes exvivo, which could lead to better responses against solid tumors invivo.

Hepatitis B virus-specific human stem cell memory T cells differentiate into cytotoxic T cells and eradicate HBV-infected hepatocytes in mice.

Chronic infection with the hepatitis B virus (HBV) induces progressive hepatic impairment. Achieving complete eradication of the virus remains a formidable challenge. Cytotoxic T lymphocytes, specific to viral antigens, either exhibit a numerical deficiency or succumb to an exhausted state in individuals chronically afflicted with HBV. The comprehension of the genesis and dissemination of stem cell memory T cells (TSCMs) targeting HBV remains inadequately elucidated. We identified TSCMs in subjects with chronic HBV infection and scrutinized their efficacy in a murine model with human hepatocyte transplants, specifically the TK-NOG mice. TSCMs were discerned in all subjects under examination. Introduction of TSCMs into the HBV mouse model precipitated a severe necro-inflammatory response, resulting in the elimination of human hepatocytes. TSCMs may constitute a valuable tool in the pursuit of a remedial therapy for HBV infection.

Abstract CT070: Solid tumor patients require higher cyclophosphamide (cy) dose than multiple myeloma (MM) patients to achieve adequate lymphodepletion (LD) necessary to enable allogeneic CAR-T expansion

Abstract Introduction: Poseida is developing innovative allogeneic T stem cell memory rich CAR-T both for hematologic malignancies (HM) and solid tumors (ST) including P-BCMA-ALLO1 which targets BCMA for MM, and P-MUC1C-ALLO1 targeting MUC1-C for epithelial-derived ST. Optimal LD for allogeneic cell therapies remains to be established. Most research has focused on HM, where patients (pts) have likely undergone HSCT and are, therefore, LD experienced in contrast to ST pts. Consequently, ST pts may require higher doses of LD chemotherapy to achieve LD depth comparable to HM pts. This study sought to compare LD with higher doses of cy across our early phase 1 trials (NCT04960579/NCT05239143) which are enrolling ST and MM pts, respectively. Methods: P-BCMA-ALLO1 and P-MUC1C-ALLO1 are 3+3 phase 1 dose escalation studies enrolling heavily pre-treated pts. LD arm S used cy 300 mg/m2 + fludarabine (flu) 30 mg/m2 × 3 days, followed by CAR-T. Alternative arms tested higher cy doses: P1 (500 mg/m2) and P2 (1,000 mg/m2), both with flu. We assessed LD depth and cellular kinetics (CK) by measuring WBC, serum IL-15, and CAR-T transcripts (qPCR). Results: By Jan 4, 2024, 42 pts received P-BCMA-ALLO1 (22 in S, 13 in P1, 7 in P2), and 30 pts received P-MUC1C-ALLO1 (19 in S, 9 in P1, 2 in P2). ST pts presented with higher baseline WBC compared to MM pts with median counts of 5.7×103 vs. 4.4×103/µL. LD was less pronounced in ST pts with a median WBC nadir of 1.1×103/µL for both S and P1. In MM pts median WBC nadir was 0.6×103 for S vs. 0.2×103 for P1. For P-BCMA-ALLO1, CK at 2×106 cells/kg dose showed a mean Cmax of 130,811 copies (cp)/µg in P2 and 6,191 in P1 compared to 170 in S. Median WBC nadir was significantly (p=0.0002) lower for P2 (0.1×103/µL) compared to S (0.6×103), with corresponding AUC0-13d values of 3.5×103/µL for P2 vs. 10×103 for S (p=0.0013). Peak serum IL-15 in P2 was 66.1 pg/mL, significantly higher than 29.7 in S (p=0.0110). In P1, there was a trend towards improved LD with median WBC nadir for P1 of 0.3×103/µL and AUC0-13d of 6.4×103/µL.P1 LD for P-MUC1C-ALLO1 did not improve CK, with WBC nadir and AUC 0-13d values comparable to S. P-MUC1C-ALLO1 CK at 2×106 cells/kg showed a mean Cmax of 692 cp/µg DNA in S, 23 in P1, and 4,200 in P2. P-MUC1C-ALLO1 pts receiving P2 achieved the deepest LD (WBC nadir: 0.1×103/µL). Peak IL-15 in ST pts was increased and delayed to day 8 in P2, compared to day 0 - 1 in S or P1. Conclusion: Increasing cy dose to 500 or 1,000 mg/m2 improves CK for P-BCMA-ALLO1 pts due to enhanced LD depth. For P-MUC1C-ALLO1 pts, increasing cy to 500 does not improve LD depth or CK compared to 300. This difference suggests the need for higher LD doses in ST pts than MM pts to achieve optimal CK. This differential need for LD chemo for these two studies is unlikely due to cellular characteristics because of similar manufacturing technology with the only difference being the binder. Citation Format: Sabrina Haag, Jeff D. Eskew, Katherine McArthur, Joanne McCaigue, Sepideh Vaziri, Samuel DePrimo, Christopher E. Martin, Catherine Gregovics, Ann Murphy, Hamid Namini, Ellen Christie, Marcela Marinez-Prieto, Rajesh Belani, Stacey Cranert, Julia Coronella, Devon Shedlock. Solid tumor patients require higher cyclophosphamide (cy) dose than multiple myeloma (MM) patients to achieve adequate lymphodepletion (LD) necessary to enable allogeneic CAR-T expansion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT070.

Abstract 6424: A patient centric full spectrum flow cytometry assay for comprehensive phenotyping of CAR T cells in clinical trials

Abstract Purpose While there is a strong desire to fully understand next generation CAR-T product characteristics in clinical trials, limited amounts of blood specimens are available as the enrolled patients are very sick and lymphodepleted. To address this high unmet need, we developed a single tube 20-color full spectrum flow cytometry assay for an in-depth characterization of CAR-T cells that support fulfilment of five key clinical trial endpoints, namely, manufacturing success (fitness and memory state), pharmacokinetics, response or resistance (ICIs), efficacy (gross tumor burden) and safety (B cell regeneration) for use across a broad range of CAR-T therapies and clinical indications. Study Design: The single tube 20-parameter full spectrum flow cytometry (FC) assay was designed to include CD45, HLA-DR, CD3, CD4, CD8, CD56 as backbone markers, CD45RA, CCR7, CD95, CD25 and CD127 antibodies allowed for the identification of all major subsets of CD4 and CD8 T cells. CXCR3, CD38, PD1, LAG3, and TIM3 were used for identification of activation/exhaustion phenotypes, while CD20 and HLA-DR were useful for identification of B cells and monocytes respectively. Additionally, we left two parameters (PE and AF647) open for customized CAR-T detection reagents or other customized markers. This assay was developed using Cytek® Northern Lights™ and frozen PBMCs. Results This robust 20-color full spectrum FC assay allowed a thorough analysis of the major T cell populations cells. FMO (Fluorescence-Minus-One) control studies confirmed the specificity of the antibodies and of the panel in identifying Naïve, EMRA, CM, EM, Th17, T stem cell memory and regulatory T cells with minimum background on all parameters. This method showed to be highly reproducible and can be adapted for use with many CAR-T detection reagents. Conclusion In summary, we developed a robust 20-parameter multiparameter full spectrum flow cytometry assay focused on different populations of T cells in a single tube. The inclusion of drop ins for prospective CAR-T cell detection reagents allows for easy integration of future advances in CAR-T cell research. This universal panel shows great potential for usage across CAR-T clinical trials, as it serves as a valuable tool understand product fitness, PK, biomarker monitoring and disease burden simultaneously. Citation Format: Keegan Vaughan, Selvam Muthusamy, Coral Zhao, Austin Carbajal, Charlie Fu, Naveen Dakappagari, Zeni Alfonso. A patient centric full spectrum flow cytometry assay for comprehensive phenotyping of CAR T cells in clinical trials [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6424.

Abstract 17: Metabolic screening of T cells guides the enrichment of stem cell memory phenotype

Abstract The efficacy of T cell-mediated immunotherapy is improved by the proliferation of T cells with a stem cell memory phenotype (Tscm). These cells are capable of long-term persistence, self-renewal, multipotency, and robust engagement with cancer cells. This study focuses on refining T cell activation protocols to bolster the presence of Tscm within the therapeutic arsenal against cancer. Through a series of cell health and metabolic assays, we traced the activation-induced metabolic reprogramming that results from Tscm enrichment. Ex vivo activation was characterized by a rapid 2.5-3 fold increase in ATP production and a 2 fold shift in intracellular reducing potential, paralleling the metabolic demands of Tscm in the cancer microenvironment. Progression of activation intensified the metabolic flux through catabolic and anabolic pathways, with significant fluctuations in metabolites such as glucose, lactate, glutamine, glutamate, branched chain amino acids, and pyruvate. These metabolic alterations were accompanied by a 2 fold increase in the prevalence of Tscm cells, and demonstrated a dependency on media composition. Our findings reveal that metabolic profiling during T cell activation provides early and actionable insights into the enrichment of Tscm, and may serve as a strategic guideline for the enhancement of cancer immunotherapies. Citation Format: Anthony Lauer, Natasha Karassina, Kayla Sylvester, Jolanta Vidugiriene. Metabolic screening of T cells guides the enrichment of stem cell memory phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 17.

Abstract 2: T cells selected from lymph node acquisition for adoptive cell therapyin NSCLC

Abstract Background: A primary limitation of PD-1 inhibitors in non-small cell lung cancer (NSCLC) arises from their inability to act on 'cold' tumors without tumor-reactive T cells, necessitating alternative approaches. Adoptive cell therapy (ACT) using (CAR)-engineered cells, strives to enhance antitumor immunity but faces several challenges such as identifying safe antigens, tumor heterogeneity, antigen escape, cell trafficking, and T cell persistence. To address these issues, we explored a new source of T cells from the benign tumor draining lymph nodes (tdLNs) of NSCLC patients. Our initial results have shown that tdLNs is a reservoir for of tumor-relevant 'stem-like' T cells. We posit that employing these pluripotent T cells for ACT could achieve significant tumor rejection in NSCLC. Methods: Resected tumors, tdLN, non-draining (ndLN), and blood from NSCLC patients, as well as a syngeneic murine lung cancer model (344SQ), underwent analysis. T cells were profiled using flow cytometry, cytotoxicity and proliferation assays. TCR and single cell (sc)RNA sequencing assessed clonal expansion, diversity, and transcriptional profiles of tumor-relevant T cells. T cells were then transduced with an ICAM-1 targeting CAR, in vivo efficacy was evaluated in an A549 murine lung cancer model. Results: T cell subsets with stem-cell memory characteristics, as indicated by PD-1+, TCF1hi, CXCR5+, and CD8+ expression, which were not significantly found in the tumor or PB. These T cells exhibited progenitor-like transcriptional signatures, enhanced SELL and TCF-1 expression, fewer exhaustion markers, and superior in vitro proliferation compared to TILs from both a murine lung cancer model and patient-derived tissues. scRNA sequencing, coupled with TCR "tumor matching" (TM) techniques, exposed a rich clonal diversity of tumor-relevant clones within tdLNs, which showcased a broader transcriptional memory profile and distinct CD4+ and CD8+ phenotypes. Upon analyzing the top 100 expanded (n&amp;gt;3) TM clones, 47 featured the presence of tdLN-derived T cells, covering progenitor, stem cell-like, and central memory clusters. T cell subsets were then transduced with a CAR targeting ICAM-1—a cell surface protein frequently overexpressed in NSCLC tumors. Manufacturing protocol yielded high transduction efficiency and T cell expansion within two weeks in 6/6 patients, consistent with PB-derived CAR T cells and on par with the optimal dosing requirements of an ICAM-1 CAR Phase I trial (NCT04420754). tdLN-CAR T cells showed potent antitumor efficacy compared to the control in an aggressive NSCLC murine model (Median survival 103d vs 66d; respectively; p=0.006). Conclusions: This represents the first reported use of T cells from tdLN for genetically engineered ACT. The data indicate that modifying antigen-experienced, stem-like T cells from tdLN with CAR is a promising and efficient method in a NSCLC murine model, warranting further research. Citation Format: Tatiana Delgado Cruz, Yanping Yang, Rachel Honigsberg, Geoffrey Markowitz, Nasser K Altorki, Vivek Mittal, Moonsoo M. Jin, Jonathan Villena-Vargas. T cells selected from lymph node acquisition for adoptive cell therapyin NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2.

Harnessing Biomaterials to Amplify Immunity in Aged Mice through T Memory Stem Cells.

The durability of a protective immune response generated by a vaccine depends on its ability to induce long-term T cell immunity, which tends to decline in aging populations. The longest protection appears to arise from T memory stem cells (TMSCs) that confer high expandability and effector functions when challenged. Here we engineered artificial antigen presenting cells (aAPC) with optimized size, stiffness and activation signals to induce human and mouse CD8+ TMSCs in vitro. This platform was optimized as a vaccine booster of TMSCs (Vax-T) with prolonged release of small-molecule blockade of the glycogen synthase kinase-3β together with target antigens. By using SARS-CoV-2 antigen as a model, we show that a single injection of Vax-T induces durable antigen-specific CD8+ TMSCs in young and aged mice, and generates humoral responses at a level stronger than or similar to soluble vaccines. This Vax-T approach can boost long-term immunity to fight infectious diseases, cancer, and other diseases.

CXCR5+TIM-3-PD-1+ stem-like cytotoxic CD8+ T cells: elevated in chronic rhinosinusitis and associated with disease severity.

Chronic rhinosinusitis (CRS) is a chronic inflammatory disease with an autoimmune background. Altered expression levels of T cell immunoglobulin and mucin-domain containing-3 (TIM-3), C-X-C chemokine receptor type 5 (CXCR5), and programmed cell death protein 1 (PD-1) are implicated in the progression of inflammatory and autoimmune diseases. Moreover, CXCR5+TIM-3-PD-1+ stem-like cytotoxic T cells function as memory stem cells during chronic disease processes and retain cytotoxicity-related gene networks. To explore the expressions of CXCR5, TIM-3, and PD-1 on T cells and their correlation with clinical parameters in CRS. Flow cytometry was used to assess the expressions and co-expressions of CXCR5, TIM-3, and PD-1 on T cells in the tissues of the paranasal sinus and peripheral blood of patients with CRS as well as healthy controls. Immunofluorescence was used to assess the co-localization of TIM-3, CXCR5, and PD-1 with T cells. The disease severity of our patients with CRS was evaluated using the Lund-Mackay score. A complete blood count was also performed for the patients with CRS. Expression levels of CXCR5 and PD-1 on T cells were significantly increased in the nasal tissues of patients with CRS. Compared with those in healthy controls, patients with CRS had high percentages of CXCR5+TIM-3-PD-1+ CD8+ and CD4+ T cells in nasal tissues, while no significant difference was observed in peripheral blood levels. Patients with CRS had a higher density of nasal CXCR5+TIM-3-PD-1+ T cells than that in healthy controls. CXCR5+TIM-3-PD-1+ CD8+ T cell levels in the nasal polyps of patients with CRS were negatively correlated with the patients' Lund-Mackay scores. The levels of CXCR5+TIM-3-PD-1+ T cells in nasal tissues were also negatively associated with disease duration and positively associated with the chronic inflammatory state of CRS. The level of CXCR5+TIM-3-PD-1+ stem cell-like T cells, especially CXCR5+TIM-3-PD-1+ CD8+ T cells, is increased in CRS. Therefore, inducing CXCR5+TIM-3-PD-1+ T cell exhaustion may be an effective immunotherapy for CRS.