T-cell Expansion Research Articles

T-cell exhaustion induced by continuous bispecific molecule exposure is ameliorated by treatment-free intervals

AbstractT-cell–recruiting bispecific molecule therapy has yielded promising results in patients with hematologic malignancies; however, resistance and subsequent relapse remains a major challenge. T-cell exhaustion induced by persistent antigen stimulation or tonic receptor signaling has been reported to compromise outcomes of T-cell–based immunotherapies. The impact of continuous exposure to bispecifics on T-cell function, however, remains poorly understood. In relapsed/refractory B-cell precursor acute lymphoblastic leukemia patients, 28-day continuous infusion with the CD19xCD3 bispecific molecule blinatumomab led to declining T-cell function. In an in vitro model system, mimicking 28-day continuous infusion with the half-life–extended CD19xCD3 bispecific AMG 562, we identified hallmark features of exhaustion arising over time. Continuous AMG 562 exposure induced progressive loss of T-cell function (day 7 vs day 28 mean specific lysis: 88.4% vs 8.6%; n = 6; P = .0003). Treatment-free intervals (TFIs), achieved by AMG 562 withdrawal, were identified as a powerful strategy for counteracting exhaustion. TFIs induced strong functional reinvigoration of T cells (continuous vs TFI-specific lysis on day 14: 34.9% vs 93.4%; n = 6; P < .0001) and transcriptional reprogramming. Furthermore, use of a TFI led to improved T-cell expansion and tumor control in vivo. Our data demonstrate the relevance of T-cell exhaustion in bispecific antibody therapy and highlight that T cells can be functionally and transcriptionally rejuvenated with TFIs. In view of the growing number of bispecific molecules being evaluated in clinical trials, our findings emphasize the need to consider and evaluate TFIs in application schedules to improve clinical outcomes.

High-fat diet blunts T-cell responsiveness in Nile tilapia

The reduced stress resistance and increased disease risk associated with high-fat diet (HFD) in animals have attracted increasing attention. However, the effects of HFD on adaptive immunity in early vertebrates, especially non-tetrapods, remain unknown. In this study, using Nile tilapia (Oreochromis niloticus) as a model, we investigated the effects of HFD on the primordial T-cell response in fish. Tilapia fed with an HFD for 8 weeks showed impaired lymphocyte homeostasis in the spleen, as indicated by the decreased number of both T and B lymphocytes and increased transcription of proinflammatory cytokines interferon-γ and interleukin-6. Moreover, lymphocytes isolated from HFD-fed fish or cultured in lipid-supplemented medium exhibited diminished T-cell activation in response to CD3ε monoclonal antibody stimulation. Moreover, HFD-fed tilapia infected by Aeromonas hydrophila showed decreased T-cell expansion, increased T-cell apoptosis, reduced granzyme B expression, and impaired infection elimination. Additionally, HFD attenuated adenosine 5’-monophosphate (AMP)-activated protein kinase (AMPK) activity in tilapia lymphocytes, which in turn upregulated fatty acid synthesis but downregulated fatty acid β-oxidation. Altogether, our results suggest that HFD impairs lymphocyte homeostasis and T cell-mediated adaptive immune response in tilapia, which may be associated with the abnormal lipid metabolism in lymphocytes. These findings thus provide a novel perspective for understanding the impact of HFD on the adaptive immune response of early vertebrates.

Levamisole Suppresses CD4+ T-Cell Proliferation and Antigen-Presenting Cell Activation in Aplastic Anemia by Regulating the JAK/STAT and TLR Signaling Pathways.

Aplastic anemia (AA) is a life-threatening disease primarily caused by a metabolic disorder and an altered immune response in the bone marrow (BM) microenvironment, where cytotoxic immune cells attack resident cells and lead to hematopoietic failure. We previously reported an efficient strategy by applying cyclosporin (CSA) combined with levamisole (CSA+LMS-based regimen) in the treatment of AA, but the immunoregulatory mechanism of LMS was still unclear. Here, the therapeutic effects of LMS were examined in vivo using the BM failure murine model. Meanwhile, the proportion and related function of T cells were measured by flow cytometry in vivo and in vitro. The involved signaling pathways were screened by RNA-seq and virtual binding analysis, which were further verified by interference experiments using the specific antagonists on the targeting cells by RT-PCR in vitro. In this study, the CSA+LMS-based regimen showed a superior immune-suppressive response and higher recession rate than standard CSA therapy in the clinical retrospective study. LMS improved pancytopenia and extended the survival in an immune-mediated BM failure murine model by suppressing effector T cells and promoting regulatory T-cell expansion, which were also confirmed by in vitro experiments. By screening of binding targets, we found that JAK1/2 and TLR7 showed the highest docking score as LMS targeting molecules. In terms of the underlying molecular mechanisms, LMS could inhibit JAK/STAT and TLR7 signaling activity and downstream involved molecules. In summary, LMS treatment could inhibit T-cell activation and downregulate related molecules by the JAK/STAT and TLR signaling pathways, supporting the valuable clinical utility of LMS in the treatment of AA.

Chimeric antigen receptor T-cell therapy in adults: management of toxicities and implications for critical care

Chimeric antigen receptor (CAR) T-cell therapy is potentially life-changing in patients with haematological malignancies refractory to multiple lines of chemoimmunotherapy and stem cell transplantation. This adoptive cell immunotherapy involves harvesting and engineering patients' own T lymphocytes to express specific antitumour CARs, thereby directing them to target tumour cells. The CAR also has an intracellular, co-stimulatory domain causing in vivo CAR T-cell expansion and persistence. Chimeric antigen receptor T-cells destroy cancer cells but may also induce life-threatening, reversible toxicities that may require support from the critical care team.

γ9δ2 T-Cell Expansion and Phenotypic Profile Are Reflected in the CDR3δ Repertoire of Healthy Adults

γ9δ2T cells fill a distinct niche in human immunity due to the unique physiology of the phosphoantigen-reactive γ9δ2TCR. Here, we highlight reproducible TCRδ complementarity-determining region 3 (CDR3δ) repertoire patterns associated with γ9δ2T cell proliferation and phenotype, thus providing evidence for the role of the CDR3δ in modulating in vivo T-cell responses. Features that determine γ9δ2TCR binding affinity and reactivity to the phosphoantigen-induced ligand in vitro appear to similarly underpin in vivo clonotypic expansion and differentiation. Likewise, we identify a CDR3δ bias in the γ9δ2T cell natural killer receptor (NKR) landscape. While expression of the inhibitory receptor CD94/NKG2A is skewed toward cells bearing putative high-affinity TCRs, the activating receptor NKG2D is expressed independently of the phosphoantigen-sensing determinants, suggesting a higher net NKR activating signal in T cells with TCRs of low affinity. This study establishes consistent repertoire–phenotype associations and justifies stratification for the T-cell phenotype in future research on γ9δ2TCR repertoire dynamics.

Oligoclonal T-cell expansion in a patient with bone marrow failure after CD19 CAR-T therapy for Richter-transformed DLBCL

The advent of chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment landscape of refractory B-cell malignancies. Real-world evidence has highlighted the high incidence of hematological toxicity, including prolonged and profound cytopenia. Here, we present a case of bone marrow (BM) failure in a 57-year old man with DLBCL-type Richter transformation receiving tisagenlecleucel in a standard-of-care setting. His clinical course was notable for underlying BM infiltration, severe cytokine release syndrome, and multiple infectious complications in the setting of prolonged, profound neutropenia. Cytokine profiling revealed a signature consistent with acquired aplastic anemia - including downregulation of CD40-L and EGF. Flow cytometric analyses demonstrated migration of CAR T-cells to the BM, as well as peripheral blood expansion of both CAR and non-CAR bearing CD8+CD57+ T-cells, an immunophenotype linked to oligoclonality in aplastic anemia. From single-cell level transcriptomics, this cytotoxic T-cell population was characterized by T-cell receptor Vβ oligoclonal expansion and post-CAR clonal drift. Gene expression profiling revealed upregulation of exhaustion markers on (CAR) T-cells and decreased expression of genes involved in STAT signalling and inflammatory response. In conclusion, this case highlights the complex nature of CAR-T-related hematological toxicity and introduces oligoclonal (CAR) T-cell expansion as a potential contributing pathophysiologic mechanism.

Individuals With Higher CD4/CD8 Ratio Exhibit Increased Risk of Acute Respiratory Distress Syndrome and In-Hospital Mortality During Acute SARS-CoV-2 Infection.

BackgroundCD4/CD8 ratio has been used as a quantitative prognostic risk factor in patients with viral infections. This study aims to assess the association between in-hospital mortality and at admission CD4/CD8 ratio among individuals with acute SARS-CoV-2 infection.MethodsThis is a longitudinal cohort study with data of all consecutive patients admitted to the COVID-19 unit at Hospital del Mar, Barcelona, Spain for ≥48 h between March to May 2020. The CD4+ CD8+ T-cell subset differentiation was assessed by flow cytometry at admission as well as a complete blood test. Patients were classified according to CD4/CD8 ratio tertiles. The primary outcome was in-hospital mortality and the secondary outcome was acute respiratory distress (ARDS).ResultsA total of 338 patients were included in the cohort. A high CD4/CD8 ratio (third tertile) was associated with a higher in-hospital mortality [adjusted Cox model hazard ratio (HR) 4.68 (95%CI 1.56–14.04, p = 0.006), reference: second tertile HR 1]. Similarly, a high CD4/CD8 ratio (third tertile) was associated with a higher incidence of ARDS [adjusted logistic regression model OR 1.97 (95%CI 1.11–3.55, p = 0.022) reference: second tertile HR 1]. There was a trend of higher in-hospital mortality and incidence of ARDS in patients within the first tertile of CD4/CD8 ratio compared with the second one, but the difference was not significant. No associations were found with total lymphocyte count or inflammatory parameters, including D-dimer.ConclusionCD4/CD8 ratio is a prognostic factor for the severity of COVID-19, reflecting the negative impact on prognosis of those individuals whose immune response has abnormal CD8+ T-cell expansion during the early response to the infection.

P800: BLOCKADE OF COMMON GAMMA CHAIN CYTOKINE SIGNALING WITH REGN7257, AN INTERLEUKIN 2 RECEPTOR GAMMA (IL2RG) MONOCLONAL ANTIBODY, PROTECTED MICE FROM GRAFT-VERSUS-HOST DISEASE AND IMMUNE APLASTIC ANEMIA

Background: Pathogenic T-cell responses in T cell-mediated diseases can be driven by cytokines of the common gamma chain (γc) cytokine family (IL2, IL4, IL7, IL9, IL15, and IL21). γc cytokines signal through their corresponding receptors, expressed primarily on immune cells (including T cells), that share a common coreceptor, interleukin 2 receptor subunit gamma (IL2RG) that is required for signaling. Aims: To understand the roles of γc cytokines in driving graft-versus-host disease (GVHD) and bone marrow failure disorders such as immune aplastic anemia (AA), we generated REGN7257, a fully human IL2RG monoclonal antibody that inhibits γc cytokine-induced signaling, and we tested its ability to suppress pathogenic T-cell responses in mouse models of T cell-mediated disease. Methods: We evaluated the efficacy of REGN7257 in a xenogeneic mouse model of GVHD, where immunodeficient NOD-scid-IL2RGnull mice were engrafted with human peripheral blood mononuclear cells (huPBMC). Mice were treated with REGN7257 either prophylactically or therapeutically, and monitored for weight loss and survival, peripheral human T-cell engraftment as well as serum pro-inflammatory cytokine levels over time. In a separate experiment, mice were sacrificed at day 49 post-huPBMC injection (after 4 weeks of antibody treatment) for tissue analysis (liver, lung, skin and bone marrow) of immune cell infiltration (T cells and macrophages), inflammation and/or fibrosis. Results: In a xenogeneic model of GVHD, both prophylactic and therapeutic γc cytokine signaling blockade with REGN7257 effectively protected mice from weight loss and resulted in improved survival, by reducing T-cell expansion in blood and production of pro-inflammatory cytokines in serum. Consistent with the classic pathology of GVHD, lungs, liver and skin of control mice were highly infiltrated by T cells, while γc cytokine signaling blockade strongly reduced T-cell infiltration, with reduced tissue levels of pro-inflammatory cytokines. Importantly, therapeutic γc cytokine signaling blockade in established GVHD (i.e. when mice already showed weight loss) provided similar benefit. Blockade of γc cytokine signaling also led to a reduction in the severity of chronic GVHD, with decreased macrophage infiltration in liver and associated hepatic fibrosis. In this xenogeneic model of GVHD, hemoglobin levels and platelet numbers in blood were both reduced, indicating anemia and thrombocytopenia, respectively, which are two complications associated with aplastic anemia. In addition to peripheral pancytopenia, recipient mice that were engrafted with huPBMC also showed severe marrow aplasia. Importantly, this phenotype of aplastic anemia was prevented by blockade of γc cytokine signaling. Summary/Conclusion: Blockade of γc cytokine signaling with REGN7257 protected against T cell-mediated pathology in a xenogeneic GVHD mouse model that uniquely presents hallmarks of both acute and chronic GVHD, with T-cell expansion/infiltration into tissues and liver fibrosis, as well as hallmarks of immune aplastic anemia, with bone marrow aplasia and peripheral cytopenia. These data provide evidence of γc cytokines as key drivers of T cell-mediated responses, offering a potentially novel strategy for the management of T cell-mediated diseases, such as GVHD and immune AA.

S257: A PHASE 1, FIRST-IN-HUMAN, DOSE-ESCALATION CLINICAL TRIAL OF MEMORY-ENRICHED CD30-CAR T-CELL THERAPY FOR THE TREATMENT OF RELAPSED OR REFRACTORY HODGKIN LYMPHOMA AND CD30+ T-CELL LYMPHOMA

Background: Up to 30% of Hodgkin lymphoma (HL) patients are refractory or relapse (R/R) after first treatment and their prognosis is poor. We have developed a refined CD30-CAR-T (HSP-CAR30) targeting a proximal epitope within the CD30 molecule to overcome soluble CD30 and generated products enriched in memory T-cells to ensure efficient engraftment, persistence and enhancement of antitumor efficacy (Alvarez-Fernandez et al, 2021). Here, we report the results of our Phase 1 study evaluating HSP-CAR30 for the treatment of R/R HL and CD30+ T-cell non-Hodgkin lymphoma (T-NHL) (NCT04653649). Aims: Primary endpoints were to assess safety of HSP-CAR30 and to establish maximum tolerated dose (MTD) recommended for the following Phase 2. Secondary objectives include best response rates after infusion. Methods: We conducted a phase 1 dose-escalation study in 11 patients with R/R HL or CD30+ T-NHL. HL patients were R/R to treatments including chemotherapy, brentuximab and anti-PD-1 antibodies, while T-NHL patients were R/R to at least 2 chemotherapy treatments. T-cells were transduced with a lentivirus encoding a second-generation 4-1BB costimulated CAR, containing a scFv directed against an epitope from the proximal non-cleavable part of CD30 protein. Three cell-dose levels were evaluated: DL1 (3x106/kg), DL2 (5x106/kg) and DL3 (10x106/kg) CAR30+ T-cells. Results: From February 2021 to December 2021, 11 patients (9 HL and 2 CD30+ T-NHL) were enrolled and underwent leukapheresis. Of these, 10 patients received HSP-CAR30: 3 patients at DL1, 3 at DL2 and 4 at DL3. Demographic characteristics and baseline disease features are summarized in the Table. Median age was 49.9 years (range 21–65). Median number of prior lines of treatment was 4.6 (range 3–7). One patient did not received treatment due to lack of T-cell expansion. All patients received LD before infusion, fludarabine/bendamustine in HL patients (n=8) and fludarabine/cyclophosphamide in T-NHL (n=2). Mean HSP-CAR30 expression was 94.79±3,38% (±SD). Memory T-cell subset comprised 93.07±4,8% (±SD) in CD4+ and 91.64±4,9% (±SD) in CD8+. Mean time to HSP-CAR30 cell peak level across all doses was 29 days (range 6–98). CAR+ T-cells were detectable by flow cytometry up to 11 months after infusion. HSP-CAR30 infusion was well tolerated; there were no dose limiting toxicities (DLTs). Relevant adverse events are shown in the Table. Grade 1 cytokine release syndrome (CRS) was observed in 6 (60%) patients. No patient developed neurotoxicity. Self-limited skin rash was seen in 4 (40%) patients. One patient with history of cytomegalovirus (CMV) infections had CMV pneumonia. Another patient developed pulmonary tuberculosis. At data cutoff (February 21st, 2021), the median follow-up was 204 days (60–351). Best objective response was 100%, including 5 (50%) patients with complete response (CR), all with HL (DL1=1; DL2=3; DL3= 1). Three patients have died of progressive disease (2 T-NHL and 1 HL). There were no non-relapse mortality events. Median PFS and median overall survival (OS) was not reached. Six-month PFS for HL patients was 75%. Image:Summary/Conclusion: This is the first European academic CART clinical trial evaluating a T-cell memory-enriched CART 30. Our Phase 1 study provides evidence for feasibility and safety of HSP-CAR30. Additionally, HSP-CAR30 has shown promising efficacy in heavily treated HL patients that is being explored in a phase 2 trial already started.

Lenalidomide bypasses CD28 co-stimulation to reinstate PD-1 immunotherapy by activating Notch signaling

Programmed cell death protein 1 (PD-1) checkpoint blockade therapy requires the CD28 co-stimulatory receptor for CD8+ Tcell expansion and cytotoxicity. However, CD28 expression is frequently lost in exhausted Tcells and during immune senescence, limiting the clinical benefits of PD-1 immunotherapy in individuals with cancer. Here, using a cereblon knockin mouse model that regains invivo Tcell response to lenalidomide, an immunomodulatory imide drug, we show that lenalidomide reinstates the anti-tumor activity of CD28-deficient CD8+ Tcells after PD-1 blockade. Lenalidomide redirects the CRL4Crbn ubiquitin ligase to degrade Ikzf1 and Ikzf3 in Tcells and unleashes paracrine interleukin-2 (IL-2) and intracellular Notch signaling, which collectively bypass the CD28 requirement for activation of intratumoral CD8+ Tcells and inhibition of tumor growth by PD-1 blockade. Our results suggest that PD-1 immunotherapy can benefit from a lenalidomide combination when treating solid tumors infiltrated with abundant CD28- Tcells.

Membrane-Anchored and Tumor-Targeted IL12 (attIL12)-PBMC Therapy for Osteosarcoma.

Chimeric antigen receptor (CAR) T-cell therapy has shown great promise for treating hematologic malignancies but requires a long duration of T-cell expansion, is associated with severe toxicity, and has limited efficacy for treating solid tumors. We designed experiments to address those challenges. We generated a cell membrane-anchored and tumor-targeted IL12 (attIL12) to arm peripheral blood mononuclear cells (PBMC) instead of T cells to omit the expansion phase for required CAR T cells. This IL12-based attIL12-PBMC therapy showed significant antitumor efficacy in both heterogeneous osteosarcoma patient-derived xenograft tumors and metastatic osteosarcoma tumors with no observable toxic effects. Mechanistically, attIL12-PBMC treatment resulted in tumor-restricted antitumor cytokine release and accumulation of attIL12-PBMCs in tumors. It also induced terminal differentiation of osteosarcoma cells into bone-like cells to impede tumor growth. In summary, attIL12-PBMC therapy is safe and effective against osteosarcoma. Our goal is to move this treatment into a clinical trial. Owing to the convenience of the attIL12-PBMC production process, we believe it will be feasible.

Abstract 2726: Host myeloid response drives anti-PD-L1 resistance in murine models of triple negative breast cancer

Abstract Immune checkpoint inhibitors (ICI) have improved patient overall and progression-free survival in some cancer types with limited success in breast cancer. Clinical trials in triple negative breast cancer (TNBC) patients, who harbor extensive tumor-infiltrating lymphocytes within tumor stroma, have demonstrated increased progression-free survival (IMpassion130) and pathologic complete response (KEYNOTE-522). Thus, combinations of ICI and chemotherapy have been FDA-approved for metastatic TNBC. However, the therapeutic benefit of ICI alone is poorly characterized. We sought to model ICI response in vivo to ascertain the immune repertoire responsible for ICI efficacy in breast cancer and identify the therapeutic benefit of ICI alone or in combination with approved chemotherapeutics. We used an immunocompetent EMT6 orthotopic mammary tumor model to investigate the efficacy of single-agent ICI (anti-PD-L1) or in combination with standard-of-care chemotherapy (paclitaxel or doxorubicin). Analysis of the primary tumor immune landscape was performed by flow cytometry and single-cell RNA sequencing. Peripheral blood from mice was serially sampled by bulk and T-cell receptor (TCR) sequencing to identify systemic genomic alterations and T-cell expansion, respectively. Single-agent ICI robustly suppressed primary tumor growth (p =0.0046) and extended survival (p&amp;lt;0.0001) beyond the control group. While chemotherapy demonstrated moderate therapeutic efficacy, it did not enhance ICI benefit. Transcriptomic and phenotypic profiling of the tumor microenvironment (TME) revealed increased T cells, dendritic cells, and NK cells in the combination groups versus chemotherapy alone, but this did not translate into improved benefit. Interestingly, despite using a genetically identical tumor model and murine host, ICI induced heterogeneous responses, ranging from complete response to intrinsic resistance. The longitudinal analysis of peripheral blood from heterogeneously responding mice uncovered enriched myeloid signatures and clonal T cell expansion corresponding to ICI resistance and response, respectively. In conclusion, we identify a heterogeneously ICI-responsive in vivo model that emulates TNBC patient response to combinatorial ICI approaches. We report the efficacy of single-agent ICI in upregulating cytotoxic immune cell infiltration and expansion within the primary tumor, thereby diminishing tumor growth and enhancing survival. We describe host-specific signatures, specifically myeloid cells, that correlate with differential responses to immunotherapy, which models heterogeneous patient response to ICI. Ongoing characterization of matched peripheral blood and TME samples may identify systemic biomarkers and tumor antigen-specific T cell clones to accurately predict ICI response in patients and uncover mechanisms for sensitizing tumors refractory to ICI. Citation Format: Ann Hanna, Xiaopeng Sun, Paula I. Gonzalez-Ericsson, Violeta Sanchez, Justin M. Balko. Host myeloid response drives anti-PD-L1 resistance in murine models of triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2726.

Abstract CT016: MEDI5752, a novel PD-1/CTLA-4 bispecific checkpoint inhibitor for advanced solid tumors: First-in-human study

Abstract Background: MEDI5752 is a monovalent bispecific antibody targeting PD-1 and CTLA-4. The bispecific format is engineered to preferentially inhibit these two checkpoints on activated T cells in tumors, where co-expression is particularly abundant. This may maximize tumor-specific response and reduce the peripheral toxicity that is common when targeting these checkpoints independently. Here we present dose-escalation data from a phase I, open-label, multicenter study in advanced solid tumors (NCT03530397). Methods: Eligible patients (pts) were ≥18 years old (ECOG PS 0-1) with tumors not amenable to standard therapy. Pts were treated at 10 dose levels from 2.25-2500 mg IV Q3W until progression or unacceptable toxicity. Primary objectives were safety and identification of maximum tolerated dose (MTD). Secondary objectives included preliminary antitumor activity by RECIST v1.1, PK and immunogenicity. Exploratory objectives included evaluation of pharmacodynamic (PD) biomarkers. Results: At data cutoff (Sept 30, 2021), 86 pts (median age, 60.5 years; male, 75.6%; Asian, 57.0%) were enrolled. The most common tumor types were RCC (22.1%), NSCLC (16.3%), and head and neck cancer (8.1%); 95.2% had received prior systemic therapy and 90.7% were immunotherapy-naïve. MEDI5752 showed dose-dependent PK and sustained peripheral PD-1 receptor occupancy (&amp;gt;90%) at doses &amp;gt;225 mg. Dose-dependent increases in peripheral T cell proliferation (Ki67+) and activation (ICOS+) plateaued at doses ≥500 mg and demonstrated CTLA-4 specific blockade in the range of tremelimumab 6-10 mg/kg. At doses ≥500 mg, MEDI5752 significantly expanded new and existing T-cell clones, likely broadening the tumor antigen-driven T-cell response. Across all doses, objective responses were seen in 19.8% of pts (n=17, 1 complete and 16 partial [PR]). Median duration of response was 17.5 months. Molecular response (defined as ≥50% reduction in ctDNA) occurred in 36.5%. Doses ≥1500 mg (n=53) were poorly tolerated with Grade (G) 3/4 treatment-related AEs (TRAEs) in 50.9%; discontinuations due to TRAEs in 45.3%, and death in 1 (at 2000 mg). However, doses &amp;lt;1500mg (n=33) had improved tolerability with G 3/4 TRAEs in 18.2% and discontinuations due to TRAEs in 9.1%. An MTD was not reached by protocol-defined DLT criteria. Immune-related AEs were also less common at doses &amp;lt;1500 mg than at ≥1500 mg (G 3/4 in 18.2% vs 49.1%, respectively). Conclusions: MEDI5752 showed encouraging antitumor activity with durable clinical benefit. Doses &amp;lt;1500 mg were better tolerated than doses ≥1500 mg. Treatment led to robust dual checkpoint blockade, evidence of T-cell activation and expansion of new and existing T-cell clones at pharmacodynamic levels that exceeded what is clinically feasible with dosing of conventional anti-PD-1 plus anti-CTLA-4 antibodies. MEDI5752 is now being explored at doses &amp;lt;1500 mg in multiple expansion cohorts. Citation Format: Ben Tran, Mark Voskoboynik, Sang-We Kim, Charlotte Lemech, Enric Carcereny, Sun Young Rha, Myung-Ju Ahn, Enriqueta Felip, Ki H. Lee, Eduardo C. Alvarez, James Chih-Hsin Yang, Paolo A. Ascierto, Mariano P. Pulla, Dan Freeman, Xuyang Song, Shelby D. Gainer, Patrick Mitchell, Ikbel Achour, Deepa S. Subramaniam, Seock-Ah Im. MEDI5752, a novel PD-1/CTLA-4 bispecific checkpoint inhibitor for advanced solid tumors: First-in-human study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT016.

Abstract 2847: Simultaneous TCR and transcriptomic sequencing of single Tcells defines biological subtypes in pancreatic cancer for adoptive Tcell therapy

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly fatal tumor-type with very few effective treatment strategies. Attempts to improve outcomes using immune checkpoint blockade therapy have also failed, likely because the overall Tcell infiltration in this tumor-type is low. Despite this, the presence of CD3+CD8+ tumor-infiltrating lymphocytes (TIL) in PDAC is associated with improved survival outcomes, suggesting that other immune-based strategies could be more successful. Here, we examine ex vivo tumor-infiltrating Tcell expansion for adoptive Tcell therapy (ACT) as a potential strategy for treating PDAC. The focus of this study is to understand the transcriptional states of Tcells in the pancreas and PDACs and how they change with ex vivo expansion and re-infusion into patients as treatment strategy. We have performed both single cell transcriptome and TCR sequencing (scRNA-TCRseq) on 54,579 Tcells from 8 human PDAC samples and the ex vivo grown TIL from a subset of 6 patients and found 13 purported substates. Through TCR tracking of the Tcell clonotypes, we find that the expansion protocol is able to expand Tcells found in many different Tcell states in the primary tumor. Furthermore, we compared our thirteen tumor-infiltrating substates with 41,935 Tcells from two other independent single cell studies across 71 samples, and confirmed our substates to be present across all datasets. Citation Format: Aislyn Schalck, Donastas Sakellariou-Thompson, Marie-Andrée Forget, Emi Sei, Tara Hughes, Shanshan Bai, Min Hu, Tapsi Kumar, Mark Hurd, Matthew Katz, Chine-Wei Tzeng, Shubham Pant, Milind Javle, Anirban Maitra, Cara Haymaker, Michael Kim, Nicholas Navin, Chantale Bernatchez. Simultaneous TCR and transcriptomic sequencing of single Tcells defines biological subtypes in pancreatic cancer for adoptive Tcell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2847.

Abstract LB090: Targeting GCN2 kinase-driven stress response inactivation to restore immunity in AML Cancers

Abstract Introduction/Purpose: The most common leukemia in adults is Acute myeloid leukemia (AML) and the 5-year overall survival (OS) in all AML patients is approximately 24%. The highest of AML deaths in the US alone is among older patients at ages ≥ 65 emphasizing the need for better therapeutics. The older patients have more limited curative options available due to their inability to tolerate aggressive chemotherapy. Identifying ways to boost immunotherapy responses could change the paradigm of AML, a disease still difficult to treat despite several approved targeted immunotherapeutic. The development of effective small molecules would be significant since small molecules not only target immunosuppressive mechanisms like mAbs but can stimulate intracellular pathways where antibodies are unable to permeate. In addition, small molecules can provide optimal pharmacokinetics and pharmacodynamics for oral administration, amenable clinical dosing, and can induce relatively acute antitumor efficacy to avoid systemic immunogenicity, thus providing a superior therapeutic potential. The highly proliferative nature of tumor cells, along with infiltration of myeloid cells into the AML leads to depletion of nutrients such as functional/natural amino acids. A key meditator of this nutrient stress pathway, the cytoplasmic GCN2 (EIF2AK4) kinase, switches on reduction of amino acids, and this activity results in T-cell inactivation, T-cell death, regulatory T-cell expansion, and the potentiation of myeloid-derived suppressor cells (MDSCs). We have discovered a series of novel small molecule immunotherapeutic agents (HCI-1046) that reversibly bind to GCN2 kinase, competitively block the ATP site, and elicit responses in AML patient samples and cell lines. Experimental Procedures: GCN2 cell-free kinase binding, EIF2α family selectivity, pGCN2 inhibition assays were performed and confirmed its on-target efficacy and potency of lead inhibitor HCI-1046. Additional experiments were conducted including CellTiterGlo, IncuCyte Live Cell Imaging, FACS Flow Cytometry, Cytotoxicity, Western Blot, ELISpot, and ELISA assays. Results and Summary: GCN2 cell-free kinase binding, kinome selectivity, pGCN2, pEIF2α, ATF4 inhibition data confirmed on-target activities of our lead GCN2 inhibitor HCI-1046. HCI-1046 demonstrated potent activity, with an IC50 of 36 nM in inhibiting GCN2 kinase, and exhibited cellular efficacy with an IC50 of 0.1 to 0.5 μM range. Our preliminary results support the hypothesis that the inhibition of GCN2 reinstates anti-tumor immunity and blocks tumor progression in the AML cancer model in vivo. Our in vivo PK studies on HCI-1046 in rodent species showed excellent PK properties with 55% oral bioavailability, low clearance, and &amp;gt;5.0-hour half-life. Thus, HCI-1046 is nominated as a pre-clinical agent. Additional data regarding the evaluation of the effects of HCI-1046 on the MDSC-suppressive function on T-cells using ELISpot, ELISA assays in AML patient samples, and other mouse model efficacy studies results will be discussed. Citation Format: Zhaoliang Li, Kyle Medley, Dongqing Yan, Kimberly Coffman, Tony Pomicter, David Lum, David J. Bearss, Hariprasad Vankayalapati. Targeting GCN2 kinase-driven stress response inactivation to restore immunity in AML Cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB090.

Abstract 3603: Reverse fate mapping of CD19-targeted CAR T cells in patients with large B-cell lymphoma

Abstract Autologous T cells genetically engineered to express a chimeric antigen receptor targeting CD19 (CD19-CAR) have achieved high complete response rates in patients with hematologic malignancies, but &amp;gt;50% of patients progress following therapy. Here, we sought to understand key T-cell intrinsic factors impacting efficacy, namely CAR T-cell expansion, persistence, and homing to the tumor. Using an approach called reverse fate mapping, we followed individual T-cell clones at the single-cell level from pre-manufacture apheresis to the infusion product, tumor-involved lymph node, and blood at peak and late expansion in 12 adult patients with relapsed or refractory large B-cell lymphoma treated with axicabtagene ciloleucel, an FDA-approved CD19-CAR T-cell immunotherapy. The resulting CAR T-cell atlas comprises matched transcriptome (scRNA-seq) and surface protein expression (CITE-seq) for 322,028 cells from 44 samples, with 119,397 unique T-cell receptor (TCR) clonotypes identified. This atlas enabled us to ask questions like: “What were the phenotypes of the most successful CAR T-cell clones at the time of infusion or pre-manufacture apheresis?” We found that T-cell clonotypes with juvenile features at apheresis, including IL7R expression, were the most successful at expansion to higher frequencies in the infusion product, while clones with effector gene expression programs, such as those encoding perforin and granzymes, contracted between apheresis and product. Conversely, it was GZMK-expressing T cells in pre-manufacture apheresis that were dominant in the tumor early following CAR T-cell infusion. Further, T-cell clonotypes with active effector programs at infusion dominated at peak expansion. Finally, we defined active expression modules and pathways in the infusion product for CAR T-cell clones that homed to the tumor or became dominant at late expansion. These analyses pinpoint the molecular mechanisms that could be modulated to rationally steer CAR T-cell differentiation trajectories at the genetic or pharmacological level. This work was supported in part by the Parker Institute for Cancer Immunotherapy, California Institute for Regenerative Medicine, Kite Pharma, and Stanford Cancer Institute. Citation Format: Zinaida Good, Mark P. Hamilton, Jay Y. Spiegel, Sreevidya Kurra, Moksha Desai, Snehit Prabhu, Eric Yang, Michael G. Ozawa, Paul J. Hanson, Fang Wu, Matthew J. Frank, John H. Baird, Lori Muffly, Gursharan K. Claire, Juliana Craig, Katherine A. Kong, Dhananjay Wagh, John Coller, Sylvia K. Plevritis, Bita Sahaf, David B. Miklos, Crystal L. Mackall. Reverse fate mapping of CD19-targeted CAR T cells in patients with large B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3603.

Genetic tracing reveals transcription factor Foxp3-dependent and Foxp3-independent functionality of peripherally induced Treg cells

Regulatory T (Treg) cells expressing the transcription factor Foxp3 are an essential suppressive Tcell lineage of dual origin: Foxp3 induction in thymocytes and mature CD4+ Tcells gives rise to thymic (tTreg) and peripheral (pTreg) Treg cells, respectively. While tTreg cells suppress autoimmunity, pTreg cells enforce tolerance to food and commensal microbiota. However, the role of Foxp3 in pTreg cells and the mechanisms supporting their differentiation remain poorly understood. Here, we used genetic tracing to identify microbiota-induced pTreg cells and found that many of their distinguishing features were Foxp3 independent. Lineage-committed, microbiota-dependent pTreg-like cells persisted in the colon in the absence of Foxp3. While Foxp3 was critical for the suppression of a Th17 cell program, colitis, and mastocytosis, pTreg cells suppressed colonic effector Tcell expansion in a Foxp3-independent manner. Thus, Foxp3 and the tolerogenic signals that precede and promote its expression independently confer distinct facets of pTreg functionality.

Adoptively Transferred in vitro-Generated Myeloid-Derived Suppressor Cells Improve T-Cell Function and Antigen-Specific Immunity after Traumatic Lung Injury

Immune reactions after trauma are characterized by immediate activation of innate immunity and simultaneously downregulation of adaptive immunity leading to a misbalanced immunohomeostasis and immunosuppression of the injured host. Therefore, the susceptibility to secondary infections is strongly increased after trauma. Immune responses are regulated by a network of immune cells influencing each other and at the same time modifying their functions dependent on the inflammatory environment. Although myeloid-derived suppressor cells (MDSCs) are initially described as T-cell suppressors, their immunomodulatory capacity after trauma is mostly undefined. Therefore, in vitro-generated MDSCs were adoptively transferred into mice after blunt chest trauma (TxT). A single MDSC treatment-induced splenic T-cell expansion decreased apoptosis sensitivity and improved proliferation in the absence of T-cell exhaustion until 2 weeks after trauma. MDSC treatment had a long-lasting effect on the genomic landscape of CD4+ T cells by upregulating primarily Th2-associated genes. Remarkably, immune-activating functions of MDSCs supported the ability of TxT mice to respond to post-traumatic secondary antigen challenge. Secondary insults were mimicked by immunizing MDSC-treated TxT mice with ovalbumin (OVA), followed by OVA restimulation in vitro. MDSC treatment significantly increased the frequency of OVA-specific T cells, enhanced their Th1/Th2 cytokine expression, and induced upregulation of cytolytic molecules finally improving OVA-specific cytotoxicity. Overall, we could show that therapeutic MDSC treatment after TxT improves post-traumatic T-cell functions, which might enable the traumatic host to counterbalance trauma-induced immunoparalysis.

Changes to culture pH and dissolved oxygen can enhance chimeric antigen receptor T-cell generation and differentiation.

Chimeric antigen receptor (CAR) T-cell therapy is growing clinically and commercially as a powerful new approach to treat cancer. Understanding how key culture conditions such as pH and dissolved oxygen (DO) affect CAR T-cell generation and function is important in developing better CAR-T manufacturing processes and CAR T-cell therapies for patients. We used the automated mini-bioreactor (AMBR) 15 platform to assess how differences in pH and DO affect CAR T-cell transduction, proliferation, and differentiation. We found that higher pH can significantly improve CAR T-cell transduction and proliferation, and also biases CAR T-cells away from an effector memory and toward a more central memory phenotype. Both high and low DO negatively affect CAR T-cell generation, with both hypoxic and hyperoxic conditions reducing T-cell transduction into CAR T-cells. Collectively, this data underscores how pH and DO can significantly affect CAR T-cell expansion and differentiation, and provides insight into the optimal culture conditions to enhance CAR T-cell yield and phenotype in clinical and commercial processes.

Three-Year Follow-Up of KTE-X19 in Patients With Relapsed/Refractory Mantle Cell Lymphoma, Including High-Risk Subgroups, in the ZUMA-2 Study.

Brexucabtagene autoleucel (KTE-X19) autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy is approved for the treatment of relapsed/refractory mantle cell lymphoma (MCL). Outcomes after a 3-year follow-up in the pivotal ZUMA-2 study of KTE-X19 in relapsed/refractory MCL are reported, including for subgroups by prior therapy (bendamustine and type of Bruton tyrosine kinase inhibitor [BTKi]) or high-risk characteristics. Patients with relapsed/refractory MCL (one to five prior therapies, including prior BTKi exposure) received a single infusion of KTE-X19 (2 × 106 CAR T cells/kg). After a median follow-up of 35.6 months, the objective response rate among all 68 treated patients was 91% (95% CI, 81.8 to 96.7) with 68% complete responses (95% CI, 55.2 to 78.5); medians for duration of response, progression-free survival, and overall survival were 28.2 months (95% CI, 13.5 to 47.1), 25.8 months (95% CI, 9.6 to 47.6), and 46.6 months (95% CI, 24.9 to not estimable), respectively. Post hoc analyses showed that objective response rates and ongoing response rates were consistent among prespecified subgroups by prior BTKi exposure or high-risk characteristics. In an exploratory analysis, patients with prior bendamustine benefited from KTE-X19, but showed a trend toward attenuated T-cell functionality, with more impact of bendamustine given within 6 versus 12 months of leukapheresis. Late-onset toxicities were infrequent; only 3% of treatment-emergent adverse events of interest in ZUMA-2 occurred during this longer follow-up period. Translational assessments revealed associations with long-term benefits of KTE-X19 including high-peak CAR T-cell expansion in responders and the predictive value of minimal residual disease for relapse. These data, representing the longest follow-up of CAR T-cell therapy in patients with MCL to date, suggest that KTE-X19 induced durable long-term responses with manageable safety in patients with relapsed/refractory MCL and may also benefit those with high-risk characteristics.