Stem Cell-like Memory Research Articles

Transient-resting culture after activation enhances the generation of CD8+ stem cell-like memory T cells from peripheral blood mononuclear cells

Adoptive cell therapy (ACT) has revolutionized the treatment of patients with cancer. The success of ACT depends largely on transferred T cell status, particularly their less-differentiated state with stem cell-like properties, which enhances ACT effectiveness. Stem cell-like memory T (TSCM) cells exhibit continuous self-renewal and multilineage differentiation similar to pluripotent stem cells. TSCM cells are promising candidates for cancer immunotherapies, whereas maintenance of a more stem-cell-like state before transfer is challenging. Here, we established a highly efficient protocol for generating CD8+ TSCM cells from peripheral blood mononuclear cells (PBMCs). The process involved activating PBMCs using anti-CD3 monoclonal antibody and RetroNectin, followed by a transient-resting culture period (24 h) and subsequent long-term expansion in vitro with interlukien-2. We report that this transient-resting culture after activation preserves CD8+ T cells in a stem memory phenotype (CD95+ CD45RA+ CCR7+) compared to the conventional culture method. Further, this approach reduces the expression of T cell immunoglobulin mucin-3, an exhaustion marker, and increases the expression of T cell factor-1, a master regulator of stemness even after long-term culture compared to the conventional culture method. In conclusion, our study presents a simplified and cost-effective method for generating and expanding CD8+ TSCM cells ex vivo. This approach streamlines the optimization of cancer immunotherapy using ACT.

A novel mitochondrial pyruvate carrier inhibitor drives stem cell-like memory CAR T cell generation and enhances antitumor efficacy

Adoptive cell transfer with chimeric antigen receptor (CAR) expressing T cells can induce remarkable complete responses in cancer patients. Therapeutic success has been correlated with central and stem cell-like memory T cell subsets in the infusion product which are better able to drive efficient CAR T cell in vivo expansion and long-term persistence. We previously reported that inhibition of the mitochondrial pyruvate carrier (MPC) during mouse CAR T cell culture, induces a memory phenotype and enhances antitumor efficacy against melanoma. Here, we use a novel MPC inhibitor, MITO-66, which robustly induces a stem cell-like memory phenotype in CD19-CAR T cells generated from healthy donors and patients with relapsed/refractory B cell malignancies. MITO-66-conditioned CAR T cells were superior in controlling human pre-B cell acute lymphoblastic leukemia in mice. Following adoptive cell transfer, MITO-66-conditioned CAR T cells maintained a memory phenotype and protected cured mice against tumor rechallenge. Furthermore, in an in vivo B cell leukemia stress model, CD19-CAR T cells generated in the presence of MITO-66 largely outperformed clinical-stage AKT and PI-3Kδ inhibitors. Thus, we provide compelling preclinical evidence that MPC inhibition with MITO-66 during CAR T cell manufacturing dramatically enhances their antitumor efficacy, thereby paving the way to clinical translation.

Elucidating the Immune Response to SARS-CoV-2: Natural Infection versus Covaxin/Covishield Vaccination in a South Indian Population.

A natural infection or a vaccination can initially prime the immune system to form immunological memory. The immunity engendered by vaccination against COVID-19 versus natural infection with SARS-CoV-2 has not been well studied in the Indian population. In this study, we compared the immunity conferred by COVID-19 vaccines to naturally acquired immunity to SARS-CoV-2 in a South Indian population. We examined binding and neutralizing antibody (NAb) levels against the ancestral and variant lineages and assessed the ex vivo cellular parameters of memory T cells, memory B cells, and monocytes and finally measured the circulating cytokine response. COVID-19 vaccination stimulates heightened levels of IgG antibodies against the original strain of SARS-CoV-2, as well as increased binding to the spike protein and neutralizing antibody levels. This enhanced response extends to variant lineages such as B.1.617.2 (Delta, India), B.1.1.529 (Omicron, India), B.1.351 (Beta, South Africa), and B.1.1.7 (Alpha, UK). COVID-19 vaccination differs from SARS-CoV-2 infection by having increased frequencies of classical memory B cells, activated memory B and plasma cells, CD4/CD8 T cells of effector memory, effector cells, stem cell-like memory T cells, and classical and intermediate monocytes and diminished frequencies of CD4/CD8 T cells of central memory and non-classical monocytes in vaccinated individuals in comparison to those with natural infection. Thus, COVID-19 vaccination is characterized by enhanced humoral responses and robust activation of innate and memory T cell responses in comparison to natural infection in a South Indian population.

RhIL-7-hyFc, a long-acting interleukin-7, improves efficacy of CAR-T cell therapy in solid tumors

BackgroundChimeric antigen receptor T-cell (CAR-T) therapy has achieved remarkable remission in patients with B-cell malignancies. However, its efficacy in treating solid tumors remains limited. Here, we investigated a combination therapy...

Preservation of naive-phenotype CD4+ T cells after vaccination contributes to durable immunity.

Memory T cells are conventionally associated with durable recall responses. In our longitudinal analyses of CD4+ T cell responses to the yellow fever virus (YFV) vaccine by peptide-MHC tetramers, we unexpectedly found CD45RO-CCR7+ virus-specific CD4+ T cells that expanded shortly after vaccination and persisted months to years after immunization. Further phenotypic analyses revealed the presence of stem cell-like memory T cells within this subset. In addition, after vaccination T cells lacking known memory markers and functionally resembling genuine naive T cells were identified, referred to herein as marker-negative T (TMN) cells. Single-cell TCR sequencing detected expanded clonotypes within the TMN subset and identified TMN TCRs shared with memory and effector T cells. Longitudinal tracking of YFV-specific responses over subsequent years revealed superior stability of TMN cells, which correlated with the longevity of the overall tetramer+ population. These findings uncover additional complexity within the post-immune T cell compartment and implicate TMN cells in durable immune responses.

Genetically-modified, redirected T cells target hepatitis B surface antigen-positive hepatocytes and hepatocellular carcinoma lesions in a clinical setting.

Hepatitis B virus (HBV)-DNA integration in HBV-related hepatocellular carcinoma (HBV-HCC) can be targeted by HBV-specific T cells. SCG101 is an autologous, HBV-specific T-cell product expressing a T-cell receptor (TCR) after lentiviral transduction recognizing the envelope-derived peptide (S20-28) on HLA-A2. We here validated its safety and efficacy preclinically and applied it to an HBV-related HCC patient (NCT05339321). Good Manufacturing Practice-grade manufactured cells were assessed for off-target reactivity and functionality against hepatoma cells. Subsequently, a patient with advanced HBV-HCC (Child-Pugh class A, Barcelona Clinic Liver Cancer stage B, Eastern Cooperative Oncology Group performance status 0, hepatitis B e antigen-, serum hepatitis B surface antigen [HBsAg]+, HBsAg+ hepatocytes 10%) received 7.9×107 cells/kg after lymphodepletion. Safety, T-cell persistence, and antiviral and antitumor efficacy were evaluated. SCG101, produced at high numbers in a closed-bag system, showed HBV-specific functionality against HBV-HCC cells in vitro and in vivo. Clinically, treatment was well tolerated, and all adverse events, including transient hepatic damage, were reversible. On day 3, ALT levels increased to 1,404 U/L, and concurrently, serum HBsAg started decreasing by 3.84 log10 and remained <1 IU/mL for over six months. HBsAg-expressing hepatocytes in liver biopsies were undetectable after 73 days. The patient achieved a partial response according to modified RECIST with a >70% reduction in target lesion size. Transferred T cells expanded, developed a stem cell-like memory phenotype, and were still detectable after six months in the patient's blood. SCG101 T-cell therapy showed encouraging efficacy and safety in preclinical models and in a patient with primary HBV-HCC and concomitant chronic hepatitis B with the capability to eliminate HBsAg+ cells and achieve sustained tumor control after single dosing.

Increased levels of GM-CSF and CXCL10 and low CD8+ memory stem T Cell count are markers of immunosenescence and severe COVID-19 in older people

BackgroundAgeing leads to altered immune responses, resulting in higher susceptibility to certain infections in the elderly. Immune ageing is a heterogeneous process also associated with inflammaging, a low-grade chronic inflammation. Altered cytotoxic T cell responses and cytokine storm have previously been described in severe COVID-19 cases, however the parameters responsible for such immune response failures are not well known. The aim of our study was to characterize CD8+ T cells and cytokines associated with ageing, in a cohort of patients aged over 70 years stratified by COVID-19 severity.ResultsOne hundred and four patients were included in the study. We found that, in older people, COVID-19 severity was associated with (i) higher level of GM-CSF, CXCL10 (IP-10), VEGF, IL-1β, CCL2 (MCP-1) and the neutrophil to lymphocyte ratio (NLR), (ii) increased terminally differentiated CD8+T cells, and (ii) decreased early precursors CD8+ T stem cell-like memory cells (TSCM) and CD27+CD28+. The cytokines mentioned above were found at higher concentrations in the COVID-19+ older cohort compared to a younger cohort in which they were not associated with disease severity.ConclusionsOur results highlight the particular importance of the myeloid lineage in COVID-19 severity among older people. As GM-CSF and CXCL10 were not associated with COVID-19 severity in younger patients, they may represent disease severity specific markers of ageing and should be considered in older people care.

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.

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.

High CD62L expression predicts the generation of chimeric antigen receptor T cells with potent effector functions.

Efficient generation of chimeric antigen receptor (CAR) T cells is highly influenced by the quality of apheresed T cells. Healthy donor-derived T cells usually proliferate better than patients-derived T cells and are precious resources to generate off-the-shelf CAR-T cells. However, relatively little is known about the determinants that affect the efficient generation of CAR-T cells from healthy donor-derived peripheral blood mononuclear cells (PBMC) compared with those from the patients' own PBMC. We here examined the efficiency of CAR-T cell generation from multiple healthy donor samples and analyzed its association with the phenotypic features of the starting peripheral blood T cells. We found that CD62L expression levels within CD8+ T cells were significantly correlated with CAR-T cell expansion. Moreover, high CD62L expression within naïve T cells was associated with the efficient expansion of T cells with a stem cell-like memory phenotype, an indicator of high-quality infusion products. Intriguingly, genetic disruption of CD62L significantly impaired CAR-T cell proliferation and cytokine production upon antigen stimulation. Conversely, ectopic expression of a shedding-resistant CD62L mutant augmented CAR-T cell effector functions compared to unmodified CAR-T cells, resulting in improved antitumor activity in vivo. Collectively, we identified surface expression of CD62L as a concise indicator of potent T cell proliferation. CD62L expression is also associated with functional properties of CAR-T cells. These findings are potentially applicable to selecting optimal donors to massively generate CAR-T cell products.

Abstract 2465: A novel agonist of CD137 as a single agent shows cancer immunoprevention efficacy against various tumor types that is not dependent on the trained immunity

Abstract Introduction: We recently demonstrated that pretreatment of mice with SA-4-1BBL, a novel oligomeric form of CD137 agonist, as a single agent prevents the development of various tumor types in mice. The tumor immunoprevention efficacy is long-lasting (&amp;gt;14 weeks) and contingent upon CD4+ immune cells, NK cells, and IFN-γ, but not CD8+ T and B cells. Trained immunity mediated by previously activated myeloid cells has been shown to have efficacy against cancer. The objective of this study was twofold: i) assess the role of trained immunity and ii) elucidate the mechanistic underlying of cancer immunoprevention. Study Design: Wild-type C57BL/6 mice and various transgenic (CD4+ OT-II.Rag+/+, CD4+ OT-II.Rag−/−, CD8+ OT-I/Rag2−/- KO, and Rag1−/−) mice on C57BL/6 background were pretreated subcutaneously with SA-4-1BBL or an agonistic Ab that does not have immunoprevention efficacy (3H3) twice two weeks apart. Mice were challenged two weeks later with a cervical cancer cell line (TC-1) and monitored for tumor development. A group of mice was also treated with monophosphoryl lipid A (MPLA), an agonist of TLR4 involved in trained immunity, using the above scheme. Mice treated with saline served as the control throughout all studies. Deep immunophenotyping was conducted to assess immune correlates of cancer immunoprevention. Results: Pretreatment with SA-4-1BBL prevented tumor growth in wild-type mice (&amp;gt; 60%), whereas all MPLA treated mice developed tumor. SA-4-1BBL did not show tumor immunoprevention efficacy in mice lacking adaptive immune cells (Rag1−/- KO), OT-II cells on Rag−/- or Rag+/+ background, and OT-I on Rag2−/− background. Deep immunophenotyping revealed a significant increase in the absolute number of CD4+ T central memory and CD4+ T stem cell-like memory cells, NK cells, NKT cells, and various myeloid cells in mice treated with SA-4-1BBL as compared with those treated with the 3H3 Ab or saline treated controls. Conclusions: These results demonstrate that cancer immunoprevention efficacy of SA-4-1BBL requires a diverse T cell receptor repertoire as OT-II mice on Rag+/+ background, restricted to a TCRβ chain, but have all other myeloid and NK cells, do not show efficacy. Furthermore, treatment with MPLA involved in trained immunity has no efficacy. Altogether, these observations do not implicate trained immunity as a mechanism of cancer immunoprevention. Elucidation of the mechanistic basis of SA-4-1BBL immunoprevention may have significant clinical implications. Citation Format: Feyza Nur Arguc, Mohammad Tarique, Vahap Ulker, Esma S. Yolcu, Haval Shirwan. A novel agonist of CD137 as a single agent shows cancer immunoprevention efficacy against various tumor types that is not dependent on the trained immunity [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 2465.

The construction of modular universal chimeric antigen receptor T (MU-CAR-T) cells by covalent linkage of allogeneic T cells and various antibody fragments

BackgroundChimeric antigen receptor-T (CAR-T) cells therapy is one of the novel immunotherapeutic approaches with significant clinical success. However, their applications are limited because of long preparation time, high cost, and interpersonal variations. Although the manufacture of universal CAR-T (U-CAR-T) cells have significantly improved, they are still not a stable and unified cell bank.MethodsHere, we tried to further improve the convenience and flexibility of U-CAR-T cells by constructing novel modular universal CAR-T (MU-CAR-T) cells. For this purpose, we initially screened healthy donors and cultured their T cells to obtain a higher proportion of stem cell-like memory T (TSCM) cells, which exhibit robust self-renewal capacity, sustainability and cytotoxicity. To reduce the alloreactivity, the T cells were further edited by double knockout of the T cell receptor (TCR) and class I human leukocyte antigen (HLA-I) genes utilizing the CRISPR/Cas9 system. The well-growing and genetically stable universal cells carrying the CAR-moiety were then stored as a stable and unified cell bank. Subsequently, the SDcatcher/GVoptiTag system, which generate an isopeptide bond, was used to covalently connect the purified scFvs of antibody targeting different antigens to the recovered CAR-T cells.ResultsThe resulting CAR-T cells can perform different functions by specifically targeting various cells, such as the eradication of human immunodeficiency virus type 1 (HIV-1)-latenly-infected cells or elimination of T lymphoma cells, with similar efficiency as the traditional CAR-T cells did.ConclusionTaken together, our strategy allows the production of CAR-T cells more modularization, and makes the quality control and pharmaceutic manufacture of CAR-T cells more feasible.

Digital twins elucidate critical role of Tscm in clinical persistence of TCR-engineered cell therapy

Despite recent progress in adoptive T cell therapy for cancer, understanding and predicting the kinetics of infused T cells remains a challenge. Multiple factors can impact the distribution, expansion, and decay or persistence of infused T cells in patients. We have developed a novel quantitative systems pharmacology (QSP) model of TCR-transgenic T cell therapy in patients with solid tumors to describe the kinetics of endogenous T cells and multiple memory subsets of engineered T cells after infusion. These T cells undergo lymphodepletion, proliferation, trafficking, differentiation, and apoptosis in blood, lymph nodes, tumor site, and other peripheral tissues. Using the model, we generated patient-matched digital twins that recapitulate the circulating T cell kinetics reported from a clinical trial of TCR-engineered T cells targeting E7 in patients with metastatic HPV-associated epithelial cancers. Analyses of key parameters influencing cell kinetics and differences among digital twins identify stem cell-like memory T cells (Tscm) cells as an important determinant of both expansion and persistence and suggest that Tscm-related differences contribute significantly to the observed variability in cellular kinetics among patients. We simulated in silico clinical trials using digital twins and predict that Tscm enrichment in the infused product improves persistence of the engineered T cells and could enable administration of a lower dose. Finally, we verified the broader relevance of the QSP model, the digital twins, and findings on the importance of Tscm enrichment by predicting kinetics for two patients with pancreatic cancer treated with KRAS G12D targeting T cell therapy. This work offers insight into the key role of Tscm biology on T cell kinetics and provides a quantitative framework to evaluate cellular kinetics for future efforts in the development and clinical application of TCR-engineered T cell therapies.

IL-10-expressing CAR T cells resist dysfunction and mediate durable clearance of solid tumors and metastases.

The success of chimeric antigen receptor (CAR) T cell therapy in treating several hematopoietic malignancies has been difficult to replicate in solid tumors, in part because of T cell exhaustion and eventually dysfunction. To counter T cell dysfunction in the tumor microenvironment, we metabolically armored CAR T cells by engineering them to secrete interleukin-10 (IL-10). We show that IL-10 CAR T cells preserve intact mitochondrial structure and function in the tumor microenvironment and increase oxidative phosphorylation in a mitochondrial pyruvate carrier-dependent manner. IL-10 secretion promoted proliferation and effector function of CAR T cells, leading to complete regression of established solid tumors and metastatic cancers across several cancer types in syngeneic and xenograft mouse models, including colon cancer, breast cancer, melanoma and pancreatic cancer. IL-10 CAR T cells also induced stem cell-like memory responses in lymphoid organs that imparted durable protection against tumor rechallenge. Our results establish a generalizable approach to counter CAR T cell dysfunction through metabolic armoring, leading to solid tumor eradication and long-lasting immune protection.

Development of optimized cytotoxicity assays for assessing the antitumor potential of CAR-T cells

CAR-T cells are T cells expressing a chimeric antigen receptor (CAR) rendering them capable of killing tumor cells after recognition of a target antigen. CD19 CAR-T cells have revolutionized the treatment of hematological malignancies. Their function is typically assessed by cytotoxicity assays using human allogeneic cell lines expressing the target antigen CD19 such as Nalm-6. However, an alloreactive reaction is observed with these cells, leading to a CD19-independent killing. To address this issue, we developed a fluorescence microscopy-based potency assay using murine target cells to provide an optimized cytotoxicity assay with enhanced specificity towards CD19.Murine NIH/3T3 (3T3) fibroblast-derived cell line and EL4 T-cell lymphoma-derived cell line were used as targets (no xenoreactivity was observed after coculture with human T cells). 3T3 and EL4 cells were engineered to express eGFP (enhanced Green Fluorescent Protein) and CD19 or CD22 using retroviral vectors. CD19 CAR-T cells and non-transduced (NT) control T cells were produced from several donors. After 4 h or 24 h, alloreactive cytotoxicity against CD19+ Nalm-6-GFP cells and CD19− Jurkat-GFP cells was observed with NT or CAR-T cells. In the same conditions, CAR-T but not NT cells specifically killed CD19+ but not CD19− 3T3-GFP or EL4-GFP cells. Both microscope- and flow cytometry-based assays revealed as sensitive as impedance-based assay. Using flow cytometry, we could further determine that CAR-T cells had mostly a stem cell-like memory phenotype after contact with EL4 target cells.Therefore, CD19+ 3T3-GFP or EL4-GFP cells and fluorescence microscopy- or flow cytometry-based assays provide convenient, sensitive and specific tools to evaluate CAR-T cell function with no alloreactivity.

Safety and Feasibility of a 16-Week Progressive Exercise Intervention in Treatment Naïve Chronic Lymphocytic Leukaemia

A growing body of evidence from preclinical and human epidemiology studies of multiple cancer types indicate that physical activity can delay or avert the outgrowth of cancer, in a mechanistic process that may involve exercise-induced alterations to anti-cancer immunity. Many Chronic Lymphocytic leukaemia (CLL) patients present with asymptomatic, early-stage disease that is monitored until disease progression. Thus, exercise may be an effective way to manage disease burden and delay progression in treatment naïve CLL. The primary objective of this pilot study was to investigate the safety and feasibility of an exercise programme in people with treatment naïve CLL, and preliminarily explore the effects of exercise training on CLL counts, body composition, cardiorespiratory fitness, and immune cell phenotypes including T-cells. We approached N = 100 treatment naïve CLL patients (Binet stage A and B) (Figure 1). Trial uptake was 40%, thus n = 40 participants with treatment naïve CLL were screened. After assessing suitability for exercise (e.g., resting electrocardiogram and other safety tests), n = 11 participants were excluded - the majority of these, n = 9, were due to the presence of cardiac abnormalities. Consequently, n = 28 participants were randomised into a 16-week, home-based, supervised, personalised, progressive exercise intervention ( n = 14: mean ± SD: age = 62 ± 12 years) or 16-weeks of usual care, control group ( n = 14: mean ± SD: age = 61 ± 10 years). The overall retention rate was 86%, with 79% of the exercise group and 93% of the control group completing the trial. Adherence to the exercise intervention was 92 ± 8%. One serious adverse event was reported (hospitalisation for pneumonia) that was unrelated to the trial and one adverse event was reported (syncope following exercise) that was related to the trial. Together, this evidence indicates that exercise training is both safe and feasible in people with treatment naïve CLL who passed pre-trial screening. The exercise intervention elicited a 2% increase in DEXA-derived lean mass in the exercise group compared to a 0.4% decrease in the control group ( p = .01) (Table 1). DEXA-derived total body fat percentage decreased by 4% and 1% and fat mass decreased by 3% and 2% ( p &amp;lt; .05) respectively in the exercise and control groups but there was no significant difference between the groups ( p &amp;gt; 0.05). Resting systolic and diastolic blood pressure was lower at post-intervention in both groups ( p &amp;lt; .05); the exercise group reduced systolic and diastolic blood pressure by 5% and 2% respectively and the control group reduced by 6% and 7% respectively, but there was no significant difference between groups ( p&amp;gt;0.05) suggesting the observed changes could be the result of “white coat hypertension” pre-intervention. Additionally, no changes were observed for whole-body mass, BMI, bone mineral density, resting heart rate, or measures of cardiorespiratory fitness (all p&amp;gt;0.05). This trial provided a unique opportunity to investigate the effects of regular exercise on neoplastic activity in humans (i.e., CLL counts) without the confounding presence of anti-cancer therapy. Resting blood samples collected pre- and post-intervention were analysed by flow cytometry to enumerate CD5 +CD19 + CLL cells clonally restricted to kappa or lambda. No differences were observed for clonal CLL cells over time or between conditions ( p&amp;gt;0.05) (Table 1). We also analysed resting blood samples collected pre- and post-intervention by flow cytometry to enumerate T cell subsets. No statistically significant changes were observed between conditions pre-intervention to post-intervention for CD4 + or CD8 + T-cell subsets including, naïve (CD27 +CD45RA +), stem cell-like memory (CD27 +CD45RA +CD127 +CD95 +), central memory (CD27 +CD45RA -), effector memory (CD27 -CD45RA -), EMRAs (CD27 -CD45RA +) or exhausted T-cells (PD1 +, Tim3 +) or FoxP3 T-regulatory cells (CD4 +CD127 lowCD25 +FoxP3 +) (all p&amp;gt;0.05). Our results show that exercise is safe and feasible in people with treatment naïve CLL who passed pre-trial screening. In addition, exercise training increased lean mass. No changes were observed to CLL cells. The latter finding is unsurprising given the poorly immunogenic profile of CLL.

Adaptive Manufacturing of LV20.19 CAR T-Cells for Relapsed, Refractory Mantle Cell Lymphoma

Introduction: Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy characterized by the presence of t(11;14) and bright CD20 expression. Approved CD19 CAR T-cell therapy for relapsed/refractory (R/R) MCL is limited by both relapse and high rates of Grade 3+ cytokine release syndrome (CRS) and ICANS with 15% and 31% patients (pts) experiencing such toxicities, respectively. To improve outcomes we utilized dual targeted lentiviral anti-CD20/anti-CD19 (LV20.19) CAR T-cells with an adaptive manufacturing (MF) process to optimize the final CAR product as part of a Phase 1/2 clinical trial in R/R MCL. Methods: We conducted a Phase 1/2 single center, prospective trial (NCT04186520) evaluating LV20.19 CAR T-cells at a fixed dose of 2.5x10e6 cells/kg for pts with R/R B-cell non-Hodgkin Lymphoma. We report results of MCL pts from a fully enrolled Phase I safety run-in and Phase II efficacy cohort. The primary outcome for the Phase II cohort was day 90 complete response (CR) rate. The Phase II cohort utilized an adaptive 8/12 day MF process to enhance the percentages of T-SCM (stem cell-like memory) and T-CM (central memory) CAR T-cells in the final product by shortening culture time. CAR T-cells were harvested either on day 8 of MF or extended to a 12-day MF process depending on achieving target cell dose in culture. Fludarabine/cyclophosphamide lymphodepletion was started during MF to facilitate fresh infusion in eligible pts. Descriptive statistics were utilized for demographic data and the Kaplan Meier methodology for survival analysis. Results: In total 17 pts with R/R MCL received LV20.19 CAR T-cells (Phase 1=3 pts and Phase 2=14 patients). All pts achieved target cell dose. 13 pts received an 8-day product while 4 received a 12-day product. 14/17 pts received a fresh (non-cryopreserved) infusion. 8-day products were enriched for T-SCM/CM phenotype cells with significantly lower number of terminally differentiated effector memory (EMRA) T-cells in the CD4 compartment (p&amp;lt;0.05) than 12-day products. The median age was 63 (50-74 years) and 15 (88%) were cisgender male. The median number of lines of therapy was 4 (3-8). p53 deletions or mutation were present in 7 pts. Day 28 overall response rate (ORR) was 100%; CR rate=76% and partial response (PR) =24%. Initial ClonoSEQ MRD assay performed within the first 90 days of LV20.19 CAR T (n=13) was negative in 10 pts (77%). Among Phase II patients with day 90 results available (n=12) the ORR was 100%, CR rate=92% and PR rate=8% exceeding the Phase II efficacy threshold. Two pts have relapsed to date, +8 months and +2 years after infusion. There were two non-relapse mortality (NRM) events that occurred beyond day 28: gram negative rod sepsis &amp; COVID19 infection resulting in a 1-year NRM rate of 12%. The 1-year progression free survival (PFS) was 77%, 1-year duration of response (DOR) was 92%, and 1-year overall survival (OS) of 84%. Median PFS/DOR/OS have not been met with median follow-up of 14 months of surviving pts (Figure 1). In terms of safety, 94% (n=16) experienced CRS, all grade 1-2. 18% (n=3) had ICANS in the first 28-days, 2 pts with Grade 3 toxicity. There were two pts with late (&amp;gt;day 28) episodes of Grade 1 neurological toxicity. Both pts had immense expansion of lymphocytes in the CSF (total nucleated cell count of 386/µLand 1005/µL) with CAR+ T cells present. Both received only a single dose of intrathecal hydrocortisone (no systemic steroids) with resolution of neurotoxicity within 24 hours of administration. Conclusions: Bispecific LV20.19 CAR T-cells with adaptive MF process is feasible, safe, and efficacious for R/R MCL with ORR 100%, no Grade 3+ CRS and low rates of Grade 3+ ICANS (12%). Adaptive MF enriched the final product with higher percentages of T-SCM/T-CM CAR cells and allowed most pts to receive CAR-T cells within 8 days of apheresis. Dual targeting of CD20 and CD19 with CAR-T cells may improve outcomes in pts with relapsed, refractory MCL.

A Phase 1 Study of RJMty19: Anti-CD19 Humanized CAR-Engineered Allogeneic Double Negative T Cells in Adults with B-Cell Non-Hodgkin's Lymphoma

Introduction: Autologous CAR-T cell therapies has been shown to benefit patients with relapsed/refractory B-cell non-Hodgkin's lymphoma (B-NHL). However, the intrinsic characteristics of autologous CAR-T cell products such as preparation failure, high cost, long production cycle, and bridging therapy required for some patients make it less accessible for a wide range of patients. RJMty19 is a first-in-class humanized anti-CD19 CAR-engineered allogeneic double negative T cells (DNTs). RJMty19 exhibits the advantages of both adaptive and natural immunity and has a high proportion of stem cell-like memory T cells (Tscm). Furthermore, RJMty19 fulfills the requirements of an off-the-shelf cellular immunotherapy, including not causing graft-versus-host disease (GvHD), resistance to host-versus-graft (HvG) rejection, scalability, and storability (WYZE 2022). Preclinical studies had shown that CD19-CAR-DNT has a better safety profile compared to conventional CD19-CAR-T (Zhang 2022). Hence, the first-in-human clinical evaluation of RJMty19 is being undertaken in this Phase 1 study in patients with B-NHL. Methods: This is a first-in-class, open-label, single-dose, phase 1 study of CD19-CAR-DNT cells. The study is designed to evaluate five dose levels (DL) of RJMty19 in a 3+3 dose-escalation scheme: 1×10 6, 3×10 6, 9×10 6, 2×10 7 and 3×10 7 viable CAR + DNT cells per kilogram of body weight. Eligibility criteria included the presence of measurable lesions, at least 2 lines of prior therapy, ECOG score of 0 to 1. All patients received lymphodepleting chemotherapy with fludarabine and cyclophosphamide. The primary endpoint is dose-limiting toxicities, incidence of adverse events and clinically significant laboratory abnormalities. Secondary endpoints include evaluation of standard cellular PK parameters, PD, immunogenicity, objective response rates (ORR) and disease control rate (DCR) per Lugano 2014 Criteria. This phase 1 study is registered on clinicaltrials.gov (NCT05453669). Results ： As of July 31 2023, 15 patients with B-NHL were enrolled and 12 patients were evaluable (including one subject finished infusion for just 10 days). Of the 11 patients who had completed DLT evaluations at Day 28, five (45.5%) were male, and the average age was 59.7 years (range 45-74). At baseline, 73% of patients with IPI score ≥ 3 (range 0-4); the median volume of target lesion was 169,000 (4520-513,688) mm 3. The median number of prior therapies was three (range2-9). Furthermore, five patients had prior BTKi therapy and four patients had prior lenalidomide/venetoclax therapy before recruiting into the trial. No patients had been treated with autologous CD19-CART products. Following single RJMty19 infusion, there was no cases of GvHD, ICANS and SAEs. Only one patient developed a grade 2 CRS which was resolved within 24 hours. Except for fever, all of ≥grade 3 adverse events were hematologic disorders including leukopenia, lymphocytopenia, neutropenia, reduced platelet count and anemia (Table 1). Among patients who received ≥ 9×10 6 CAR-DNT cells/kg (DL ≥3; N=5), best DCR and ORR were 100% (5/5) and 40% (2/5) respectively at Day 28 with B-ultrasound imaging. One of patients in DL4 achieved 87% reduction of tumor lesions (SPD) at Day 31 and Day 61. CD19-CAR-DNT cell kinetics improved in a dose-dependent manner with peak cell expansion occurring between Day 5 and 7 at DL4 based on flow cytometry. Additionally, the mean of C max in DL4 is 384 CAR + DNT cells per microliter (μl) blood (N=3), and the highest peak value of 936 CAR + DNT cells/μl was detected in the recent enrolled patient.

Characteristics of premanufacture CD8+ T cells determine CAR-T efficacy in patients with diffuse large B-cell lymphoma

Although chimeric antigen receptor (CAR) T cells have become an important treatment option for patients with relapsed/refractory B-cell malignancies, more than 60% of patients with diffuse large B-cell lymphoma (DLBCL) treated with CAR-T cell therapies fail to achieve a durable response. To reveal changes in CAR-T cell therapy and identify response biomarkers, we conducted a retrospective analysis of pre-manufacture source T cells and CAR-T cell products and their association with outcome in 58 patients with r/rDLBCL who received tandem CD19/CD20 CAR-T cell therapy. We performed bulk RNA-Seq, single-cell RNA-Seq, and paired T cell receptor sequencing on CAR-T cell products and pre-manufacture T cells from DLBCL patients. We note that a CD8+ stem cell-like memory T cell population with a higher proportion and enhanced activating capacity of the CAR-T cell products was key to achieving durable clinical response. By analysing autologously-derived, pre-manufacture T cells, our data suggest that heterogeneity in the cellular and molecular features of pre-manufacture T cells contribute to the variation in efficacy after CAR-T cell therapy in DLBCL. The differences in anti-tumour efficacy of CAR-T cells among patients with different clinical outcomes appear to be due to the loss of CCR7 gene expression, coupled with increased expression of activation- and inhibitor-related genes in the CD8+ naïve-T cell populations among the apheresis T cells from patients with a poor molecular response. These findings significantly advance our understanding of the underlying molecular determinants of pre-manufacture T cell function.

Erucic acid improves the progress of pregnancy complicated with systemic lupus erythematosus by inhibiting the effector function of CD8+ Tcells.

Pathogenic CD8+ Tcells are pivotal contributors to the onset of systemic lupus erythematosus (SLE). Erucic acid (EA) has been proven to have anti-inflammatory activity. However, the capacity of EA to regulate pathogenic CD8+ Tcells in the context of pregnancy complicated with SLE (pSLE) remains unclear. In our investigation, we observed augmented CD8+ T cell effector function juxtaposed with diminished EA levels in pSLE patients relative to healthy pregnant controls. Significantly, plasma EA levels exhibited a negative correlation with the severity of pSLE-associated complications. In blood from patients with pSLE, EA inhibited the effector function of CD8+ Tcells, concurrently dampening the maintenance of stem cell-like memory CD8+ Tcells. Mechanistically, EA orchestrated the inhibition of CD8+ T cell effector function by impeding signal transducer and activator of transcription 3 phosphorylation and promoting ferroptosis. Moreover, EA supplementation in pregnant MRL/lpr mice manifested as the attenuation of uterine CD8+ T cell effector function, culminating in the mitigation of placental pathological damage. Our findings uncover the immune response modulatory effects of EA upon pathogenic CD8+ cells, thereby unveiling new perspectives for therapeutic strategies targeting pSLE patients.