Endogenous T-cell Research Articles

Intratumoral T-cell composition predicts epcoritamab-based treatment efficacy in B-cell non-Hodgkin lymphomas.

Leveraging endogenous tumor-resident T-cells for immunotherapy using bispecific antibodies (BsAb) targeting CD20 and CD3 has emerged as a promising therapeutic strategy for patients with B-cell non-Hodgkin lymphomas. However, features associated with treatment response or resistance are unknown. To this end, we analyzed data from patients treated with epcoritamab-containing regimens in the EPCORE NHL-2 trial (NCT04663347). We observed downregulation of CD20 expression on B-cells following treatment initiation both in progressing patients and in patients achieving durable complete responses (CR), suggesting that CD20 downregulation does not universally predict resistance to BsAb-based therapy. Single-cell immune profiling of tumor biopsies obtained following one cycle of therapy revealed substantial clonal expansion of cytotoxic CD4+ and CD8+ T-cells in patients achieving CR, and an expansion of follicular helper and regulatory CD4+ T-cells in patients whose disease progressed. These results identify distinct tumor-resident T-cell profiles associated with response or resistance to BsAb therapy.

IMMU-06. CAR DESIGN SHAPES THE BRAIN TUMOR IMMUNE MICROENVIRONMENT: IMPACT ON ACTIVITY AND PERSISTENCE OF ANTI-GLIOMA ADOPTIVE T CELL IMMUNOTHERAPY

Abstract BACKGROUND B7-H3 CAR T-cells effectively eradicate brain tumors in xenograft models and have proven safe in clinical trials, yet no objective responses were observed in patients. The limited efficacy is partly attributed to the immunosuppressive tumor microenvironment (TME), a characteristic not fully recapitulated in investigations using xenograft models. In this study, we set to optimize B7-H3 CAR structure and examine the effects of CAR design on the brain TME using an immunocompetent glioma model. METHODS We generated a panel of fully murine B7-H3 CARs with variations in transmembrane, co-stimulatory, and activation domains. This enabled us to comprehensively investigate their effector functions within the intact immune system. High-dimensional flow cytometry and single-cell RNA sequencing were then used to investigate changes in the brain TME following CAR T-cell therapy. RESULTS While five out of six B7-H3 CARs with single co-stimulatory domains exhibited potent functionality in vitro, optimizing co-stimulation and signaling failed to enhance their anti-glioma efficacy in vivo. To further enhance therapeutic effectiveness and persistence, we incorporated 4-1BB and CD28 co-stimulation through transgenic expression of 4-1BBL on CD28-based CAR T-cells. Although this design significantly improved anti-glioma efficacy in vitro, it conferred no additional benefits in vivo. Analysis of the TME revealed that CAR T-cell therapy influenced the immune composition of the brain TME. Successful anti-tumor responses were dictated by the recruitment, activation, and spatial localization of unique inflammatory ‘immune hubs’ containing specific subsets of macrophages and endogenous T-cells. Interestingly, depletion of brain macrophages using CSF1R inhibitor markedly inhibited CAR T-cell anti-tumor activity. CONCLUSION Our study highlights the critical role of CAR structural design and its modulation of the TME in mediating the efficacy of CAR T-cell therapy in brain tumors. Yet, more specific targeting and/or reprogramming of the TME is warranted for the successful design of effective adoptive immunotherapies for high-grade glioma.

Knocking Out CD70 Rescues CD70-Specific NanoCAR T Cells from Antigen-Induced Exhaustion.

CD70 is an attractive target for chimeric antigen receptor (CAR) T-cell therapy for the treatment of both solid and liquid malignancies. However, the functionality of CD70-specific CAR T cells is modest. We optimized a CD70-specific VHH-based CAR (nanoCAR). We evaluated the nanoCARs in clinically relevant models in vitro, using co-cultures of CD70-specific nanoCAR T cells with malignant rhabdoid tumor organoids, and in vivo, using a diffuse large B-cell lymphoma patient-derived xenograft (PDX) model. Although the nanoCAR T cells were highly efficient in organoid co-cultures, they showed only modest efficacy in the PDX model. We determined that fratricide was not causing this loss in efficacy but rather CD70 interaction in cis with the nanoCAR-induced exhaustion. Knocking out CD70 in nanoCAR T cells using CRISPR/Cas9 resulted in dramatically enhanced functionality in the diffuse large B-cell lymphoma PDX model. Through single-cell transcriptomics, we obtained evidence that CD70 knockout CD70-specific nanoCAR T cells were protected from antigen-induced exhaustion. In addition, we demonstrated that wild-type CD70-specific nanoCAR T cells already exhibited signs of exhaustion shortly after production. Their gene signature strongly overlapped with gene signatures of exhausted CAR T cells. Conversely, the gene signature of knockout CD70-specific nanoCAR T cells overlapped with the gene signature of CAR T-cell infusion products leading to complete responses in chronic lymphatic leukemia patients. Our data show that CARs targeting endogenous T-cell antigens negatively affect CAR T-cell functionality by inducing an exhausted state, which can be overcome by knocking out the specific target.

Individualized neoantigen therapy mRNA-4157 (V940) plus pembrolizumab in resected melanoma: 3-year update from the mRNA-4157-P201 (KEYNOTE-942) trial.

LBA9512 Background: mRNA-4157 is a novel, mRNA-based individualized neoantigen therapy designed to increase endogenous antitumor T-cell responses by targeting unique patient (pt) tumor mutations. In the primary analysis of the Ph 2 mRNA-4157-P201 (KEYNOTE-942) trial (median planned follow-up, 23 mo), pts with completely resected high-risk stage IIIB–IV cutaneous melanoma receiving mRNA-4157 + pembrolizumab (pembro; combo) had prolonged recurrence-free survival (RFS) and distant metastasis-free survival (DMFS) vs pembro alone (Weber JS, et al. Lancet. 2024). Methods: Pts were assigned 2:1 to mRNA-4157 (1 mg IM, max 9 doses) + pembro (200 mg IV, max 18 cycles) or pembro alone. The primary endpoint was investigator-assessed RFS; secondary endpoints were DMFS and safety. This planned supportive analysis was triggered when the last randomized pt had ≥2 y follow-up. Translational subgroup analyses were also reported. HLA genotypes were analyzed by exome sequencing of DNA from PBMC. RFS and DMFS were not formally tested; nominal 2-sided p-values are descriptive. Results: With an additional year follow-up (data cutoff, 03 Nov 2023; median [range], 34.9 [25.1–51.0] mo) after primary analysis, minimal new events occurred. RFS benefit in the combo vs pembro arm was maintained with 49% risk reduction in recurrence and/or death (HR [95% CI], 0.510 [0.288–0.906]; 2-sided nominal p-value 0.019). The 2.5-yr RFS rate of combo treatment (tx) vs pembro alone was 74.8% vs 55.6%. Combo tx also produced clinically meaningful, sustained improvement in DMFS vs pembro alone (HR [95% CI], 0.384 [0.172–0.858], 2-sided nominal p-value 0.0154). OS favored combo vs pembro alone; 2.5-y OS rate was 96.0% vs 90.2% (HR [95% CI], 0.425 [0.114–1.584]). RFS benefit of combo vs pembro was maintained in TMB high (HR [95% CI], 0.564 [0.253–1.258]), TMB non-high (0.571 [0.245–1.331]), PD-L1 positive (0.471 [0.226–0.979]), PD-L1 negative (0.147 [0.034–0.630]), and ctDNA negative (0.207 [0.091–0.470]) subgroups; ctDNA positive HR was not estimable. No significant associations between individual HLA alleles and RFS were observed in either tx arm. Maximal heterozygosity at HLA class I genotype loci (A, B, C) improved RFS vs homozygosity for ≥1 locus in the pembro arm (HR [95% CI], 0.425 [0.179–1.01]) but not combo arm (1.252 [0.498–3.146]). mRNA-4157 was well tolerated and combo tx had a safety profile consistent with previous analysis with no potentiation of immune-related AEs. Conclusions: The current analysis with ∼3 y median follow-up showed durable and meaningful long-term RFS and DMFS benefit with mRNA-4157 + pembro vs pembro alone. A trend for improved OS with combo tx was also observed. HLA and translational subgroup results suggest mRNA-4157 + pembro may benefit a broader pt population vs pembro alone. Clinical trial information: NCT03897881 .

Expression of VISTA regulated via IFN-γ governs endogenous T-cell function and exhibits correlation with the efficacy of CD19 CAR-T cell treated B-malignant mice

BackgroundDespite continuous improvements in the new target and construction of chimeric antigen receptor (CAR)-T, relapse remains a significant challenge following CAR-T therapy. Tumor microenvironment (TME) strongly correlates with the efficacy...

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.

Abstract 3607: Case study: Efficacy and persistence of autologous T cell therapy targeting SLC45A2 in uveal melanoma

Abstract Background: Metastatic uveal melanoma (UM) is an aggressive cancer, with a 1-year overall survival of < 50%. While the bispecific T-cell engager tabentafusp is approved for HLA-A2+ patients with gp100+ UM, there are limited treatment options for patients who do not express HLA-A2. We present a strategy and proof-of-concept for designing and manufacturing a tumor-selective endogenous T-cell therapy (ETC) for a patient with metastatic UM. Methods: PBMC were collected by apheresis in 2013 upon first diagnosis with uveal melanoma; 8 years later, following progression, cryopreserved PBMC were used to generate autologous antigen-specific T cells for adoptive therapy. The validated tumor antigen SLC45A2 was found to be overexpressed in the patient’s metastatic tumor based on RNA expression profiling. Peripheral blood derived SLC45A2-specific HLA-A2402-restricted central memory CD8+ T cells were generated via a process of in vitro priming, IL-21 treatment, tetramer-guided sorting and cell expansion. Clinical response was assessed via longitudinal CT imaging and ctDNA-based MRD monitoring. Persistence of transferred T cells was measured via serial flow cytometry and RNA-sequencing of PBMCs, along with TCR and RNA sequencing of tumor tissue collected post infusion. Results: A 39 year old male received SLC45A2-specific ETC therapy and checkpoint inhibitor treatment for advanced metastatic UM (GNAQ (Q209P), BAP1 (E398*, LOF). Bulk RNAseq data from baseline biopsies were queried for known tumor-associated antigens, and SLC45A2 was expressed at high levels. The patient experienced widespread disease stabilization with minimal toxicity. SLC45A2-specific CD8+ T cells persisted in circulation up to 181 days after the first infusion and 43 days after the second infusion, ranging from 6.7% - 1.5% of total circulating CD3 T cells. T cell clones from the infusion product included the dominating TCR subclone in tumor tissue collected 6 weeks post infusion, comprising 33% of the TCRá and 26% of TCRâ population. SLC45A2-specific T cells were identified in the tumor infiltrating lymphocytes in a separate lesion collected 10 months after the first infusion. A duodenum lesion progressed during treatment, and RNAseq demonstrated reduced expression of genes associated with HLA presentation and SLC45A2 and reduced TIL infiltration, suggesting tissue-specific immune editing of the tumor cell population. Conclusions: 1. Tumor-reactive antigen-specific T cells can be isolated and expanded for adoptive therapy using PBMCs cryopreserved for > 8 years. 2. Following infusion, peripheral blood and tumor profiling support the persistence of transferred T cells in a patient with metastatic uveal melanoma. 3. Disease stabilization in aggressive metastatic UM was achieved with minimal toxicity. Overall, our study provides evidence and rationale for utilizing autologous antigen-specific T cells for treating rare cancers. Citation Format: Shailbala Singh, Esther Ryu, Katie M. Campbell, Amber Smith, Suzanne Phillips, Luisa Soto, Ivy Lai, Gregory Lizee, Benjamin Vincent, Marshall Thompson, William Hoos, Sapna Patel, Cassian Yee. Case study: Efficacy and persistence of autologous T cell therapy targeting SLC45A2 in uveal melanoma [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 3607.

Targeting Epstein-Barr virus in multiple sclerosis: when and how?

Epidemiological evidence implicates Epstein-Barr virus (EBV) as the cause of multiple sclerosis (MS). However, its biological role in the pathogenesis of MS is uncertain. The article provides an overview of the role of EBV in the pathogenesis of MS and makes a case for targeting EBV as a treatment strategy for MS. EBV potentially triggers autoimmunity via molecular mimicry or immune dysregulation. Another hypothesis, supported by immunological and virological data, indicates that active EBV infection via latent-lytic infection cycling within the central nervous system or periphery drives MS disease activity. This supports testing small molecule anti-EBV agents targeting both latent and lytic infection, central nervous system-penetrant B-cell therapies and EBV-targeted immunotherapies in MS. Immunotherapies may include EBV-specific cytotoxic or chimeric antigen receptors T-cells, therapeutic EBV vaccines and immune reconstitution therapies to boost endogenous EBV-targeted cytotoxic T-cell responses. EBV is the probable cause of MS and is likely to be driving MS disease activity via latent-lytic infection cycling. There is evidence that all licensed MS disease-modifying therapies target EBV, and there is a compelling case for testing other anti-EBV strategies as potential treatments for MS.

DNAJB1-PRKACA fusion neoantigens elicit rare endogenous T cell responses that potentiate cell therapy for fibrolamellar carcinoma

Fibrolamellar carcinoma (FLC) is a liver tumor with a high mortality burden and few treatment options. A promising therapeutic vulnerability in FLC is its driver mutation, a conserved DNAJB1-PRKACA gene fusion that could be an ideal target neoantigen for immunotherapy. In this study, we aim to define endogenous CD8 Tcell responses to this fusion in FLC patients and evaluate fusion-specific Tcell receptors (TCRs) for use in cellular immunotherapies. We observe that fusion-specific CD8 Tcells are rare and that FLC patient TCR repertoires lack large clusters of related TCR sequences characteristic of potent antigen-specific responses, potentially explaining why endogenous immune responses are insufficient to clear FLC tumors. Nevertheless, we define two functional fusion-specific TCRs, one of which has strong anti-tumor activity invivo. Together, our results provide insights into the fragmented nature of neoantigen-specific repertoires in humans and indicate routes for clinical development of successful immunotherapies for FLC.

Enhanced tumor control activities of anti-mPD-L1 antibody and antigen-presenting cell-like natural killer cell in an allograft model

BackgroundDespite the utilization of immune checkpoint inhibitors (ICIs) in treating numerous types of cancers being approved, their efficacy in tumor control in the clinic is not satisfactory. Since adoptive cell therapy (ACT) can alter the tumor microenvironment, we hypothesized that ACT potentially synergized with ICI in tumor control and examined this hypothesis via a murine allograft model.MethodsFemale C57BL/6 mice were stimulated with interleukin 15 and granulocyte monocyte-colony stimulating factor, followed by collecting their bone marrow cells for murine NKDC cultivation. Then, female C57BL/6 mice, inoculated with lymphoma cancer cell line E.G7-OVA, were administrated with murine NKDC cells, murine anti-program cell death ligand-1 antibody (α-mPD-L1), or both for 28 days. After 28 days of treatment, mice were sacrificed whose inoculated tumors, spleen, sentinel lymph nodes, and peripheral blood were collected to measure tumor size, lymphocyte infiltration, and change of immune cell profile.ResultsCombined treatment of NKDCs with α-mPD-L1 exhibited significantly stronger tumor control efficacy than treatment of NKDCs or α-mPD-L1 alone. NKDCs/α-mPD-L1 combination increased migration of dendritic cells, CD4, CD8 T cells, and activated CD8 T cells to the tumor-bedding site, and promoted endogenous tumor-specific cytotoxic T-cell response.ConclusionThe current study confirmed our hypothesis that combining NKDC ACT with ICI therapy can potentiate tumor control efficacy by manipulating the tumor microenvironment. This study provided a novel circumstance on tumor immunotherapy.

Lymph Node-Targeted Vaccine Boosting of TCR T-cell Therapy Enhances Antitumor Function and Eradicates Solid Tumors.

T-cell receptor (TCR)-modified T-cell therapies have shown promise against solid tumors, but overall therapeutic benefits have been modest due in part to suboptimal T-cell persistence and activation in vivo, alongside potential tumor antigen escape. In this study, we demonstrate an approach to enhance the in vivo persistence and function of TCR T cells through combination with Amphiphile (AMP) vaccination including cognate TCR T peptides. AMP modification improves lymph node targeting of conjugated tumor immunogens and adjuvants, thereby coordinating a robust T cell-activating endogenous immune response. AMP vaccine combination with TCR T-cell therapy led to complete eradication and durable responses against established murine solid tumors refractory to TCR T-cell monotherapy. Enhanced antitumor efficacy was correlated with simultaneous in vivo invigoration of adoptively transferred TCR T cells and in situ expansion of the endogenous antitumor T-cell repertoire. Long-term protection against tumor recurrence in AMP-vaccinated mice was associated with antigen spreading to additional tumor-associated antigens not targeted by vaccination. AMP vaccination further correlated with pro-inflammatory lymph node transcriptional reprogramming and increased antigen presenting-cell maturation, resulting in TCR T-cell expansion and functional enhancement in lymph nodes and solid tumor parenchyma without lymphodepletion. In vitro evaluation of AMP peptides with matched human TCR T cells targeting NY-ESO-1, mutant KRAS, and HPV16 E7 illustrated the clinical potential of AMP vaccination to enhance human TCR T-cell proliferation, activation, and antitumor activity. Taken together, these studies provide rationale and evidence to support clinical evaluation of combining AMP vaccination with TCR T-cell therapies to augment antitumor activity.

Combined treatment with allogeneic Epstein–Barr- and human polyomavirus 1 specific T-cells in progressive multifocal leukoencephalopathy and EBV infection: a case report

Opportunistic viral infections in individuals with severe immunodeficiency can lead to fatal conditions such as progressive multifocal leukoencephalopathy (PML), for which treatment options are limited. These infections pose significant risks, especially when co-infections with other viruses occur. We describe a combined therapy approach using directly isolated allogeneic Human Polyomavirus 1 (also known as BKV) and Epstein–Barr virus (EBV) specific cytotoxic T-cells for the treatment of PML in conjunction with identified EBV in the cerebrospinal fluid (CSF) of a male patient infected with human immunodeficiency virus (HIV). A 53-year-old HIV-positive male, recently diagnosed with PML, presented with rapidly worsening symptoms, including ataxia, tetraparesis, dysarthria, and dysphagia, leading to respiratory failure. The patient developed PML even after commencing highly active antiretroviral therapy (HAART) 3 months prior. Brain magnetic resonance imaging (MRI) revealed multifocal demyelination lesions involving the posterior fossa and right thalamus suggestive of PML. In addition to the detection of human polyomavirus 2 (also known as JCV), analysis of CSF showed positive results for EBV deoxyribonucleic acid (DNA). His neurological condition markedly deteriorated over the following 2 months. Based on MRI, there was no evidence of Immune Reconstitution Inflammatory Syndrome contributing to this decline. The patient did not have endogenous virus-specific T-cells. We initiated an allogeneic, partially human leukocyte antigen-matched transfer of EBV and utilizing the cross-reactivity between BKV and JCV–BKV specific T-cells. This intervention led to notable neurological improvement and partial resolution of the MRI lesions within 6 weeks. Our case of a patient with acquired immune deficiency syndrome demonstrates that PML and concurrent EBV co-infection can still occur despite undergoing HAART treatment. This innovative experimental therapy, involving a combination of virus-specific T-cells, was demonstrated to be an effective treatment option in this patient.

The 4-1BBζ costimulatory domain in chimeric antigen receptors enhances CD8+ T-cell functionality following T-cell receptor stimulation

BackgroundChimeric antigen receptor (CAR) T-cells have revolutionized the treatment of CD19- and B-cell maturation antigen-positive haematological malignancies. However, the effect of a CAR construct on the function of T-cells stimulated via their endogenous T-cell receptors (TCRs) has yet to be comprehensively investigated.MethodsExperiments were performed to systematically assess TCR signalling and function in CAR T-cells using anti-mesothelin human CAR T-cells as a model system. CAR T-cells expressing the CD28 or 4-1BB costimulatory endodomains were manufactured and compared to both untransduced T-cells and CAR T-cells with a non-functional endodomain. These cell products were treated with staphylococcal enterotoxin B to stimulate the TCR, and in vitro functional assays were performed by flow cytometry.ResultsIncreased proliferation, CD69 expression and IFNγ production were identified in CD8+ 4-1BBζ CAR T-cells compared to control untransduced CD8+ T-cells. These functional differences were associated with higher levels of phosphorylated ZAP70 after stimulation. In addition, these functional differences were associated with a differing immunophenotype, with a more than two-fold increase in central memory cells in CD8+ 4-1BBζ CAR T-cell products.ConclusionOur data indicate that the 4-1BBζ CAR enhances CD8+ TCR-mediated function. This could be beneficial if the TCR targets epitopes on malignant tissues or infectious agents, but detrimental if the TCR targets autoantigens.

Prediction Modeling of CAR-T Cell Therapy for Diffuse Large B-Cell Lymphoma Using Artificial Neural Networks on Tumor Vascular Phenotype

Chimeric antigen receptor T (CAR-T) cell therapy targeting CD19 only results in complete remission in approximately 30-40% of relapsed/refractory disease patients with diffuse large B-cell lymphoma (DLBCL). Failure may result from lack of CAR-T entry into the tumor. Endogenous T cell homing is initiated by binding to post-capillary venular endothelial cells (ECs). Naïve and central memory T cells use L-selectin binding to peripheral node addressin (PNad) on high endothelial venules (HEVs) of the lymph node while circulating effector memory T cells, lacking L-selectin, use other molecules to bind cytokine-induced endothelial proteins E-selectin, ICAM-1 and VCAM-1 on flat venules of inflamed tissues. Both of these venular EC types are present in inflamed lymph nodes. We hypothesized that the expression patterns of adhesion molecules on venular ECs in pre-treatment tumor biopsies could predict CAR-T efficacy. To test this, we applied cyclic immunofluorescence to 18 archived pre-treatment DLBCL patient FFPE biopsies (8 responders and 10 non-responders, defined by relapse at 12 months). We detected venular ECs using a combination of anti-CD31 and UEA lectin on larger diameter vessels and assessed expression for PNad, E-selectin, ICAM-1, VCAM-1. We detected endogenous CD8+ T cells and evaluated their expression of L-selectin, alpha 4 integrin and PSGL-1. See table 1. DAPI nuclear staining was included in each cycle. Images of each cycle were combined in ImageJ and imported into QuPath for analysis. Using Warpy, affine transformations were applied to DAPI staining to create overlays between imaging cycles. After alignment, representative images of several samples were randomly chosen for image training using a combination of Artificial Neural Networks (ANNs) and classifiers to perform identification and mask tissue regions of vessels and T-cells within the tissue. Positive and negative cells for each marker of interest (EC PNad, ICAM-1, E-Selectin, VCAM-1, and T cell alpha 4 integrin, L-Selectin, and PSGL-1) were used to create classification algorithms to determine marker expression for ECs and T-cells within the tissue. Results were analyzed by unpaired t-test and one-way ANOVA conducted in Graph-Pad Prism 9 software. Preliminary results from quantitative analysis of pre-treatment biopsies show a significant increase in CD31+/PNAD- venular ECs compared to non-responders that were double positive for ICAM1 and VCAM-1 (p=0.0273). This analysis also revealed a significant increase in CD31+/PNAD+ HEV ECs from responders that were triple positive for E-Selectin, ICAM-1, and VCAM-1 (p=0.0242). See figure 1. Our initial semi-quantitative analysis showed a trend toward elevated numbers of tissue-infiltrating CD3 + CD8 + T cells in the pre-treatment biopsies of responders. T cells expressing PSGL-1, a glycoprotein that may carry carbohydrate moieties recognized by E-Selectin and linked to T-cell exhaustion, was increased in the non-responder cohort. These data suggest an increased extent and activation state of intra-tumoral venular ECs and increased endogenous T-cell infiltrates, which may predict the outcome of CAR-T therapy. Analysis of T-cells in the pre-treatment biopsies leveraging machine learning algorithms is in progress and both complete quantitative findings and prognostic value of this approach will be presented. While our findings require further validation thru additional biopsy analysis, they provide emerging evidence for a rationale involving vascular readiness in DLBCL responder differences to CART therapy.

MP0533, a CD3-Engaging Darpin Targeting CD33, CD123, and CD70 in Patients with Relapsed/Refractory AML or MDS/AML: Preliminary Results of a Phase 1/2a Study

BACKGROUND: The development of targeted immunotherapy for acute myeloid leukemia (AML) is challenging due to the clonal heterogeneity of the disease and the lack of single AML-specific target antigens. MP0533 is a multi-specific, half-life extended CD3-engaging DARPin (Designed Ankyrin Repeat Protein). It simultaneously targets CD33, CD123, and CD70, thereby enabling avidity-driven T cell-mediated killing of leukemic stem cells (LSCs) and malignant blast cells known to co-express these targets. MP0533's affinity to each target is tuned to preferentially kill malignant cells which co-express at least two of these three antigens, while preserving a therapeutic window towards healthy cells. MP0533 was shown to induce a tumor-specific endogenous T-cell response in ex vivo samples from AML patients and led to T-cell-mediated eradication of tumors in AML xenograft mouse models, with limited cytokine release or other serious adverse reactions (Bianchi et al., Blood 2022;140[Suppl 1]:2251-2). Herein, we present early observations of the first three MP0533 dose-escalation regimens (DR) of the ongoing first-in-human, multicenter, single-arm, open-label, phase 1/2a study. METHODS: The objectives of this ongoing trial are to evaluate the safety/tolerability, pharmacokinetics (PK), and pharmacodynamics, as well as preliminary antileukemic activity of MP0533 monotherapy in patients with relapsed/refractory (R/R) AML or myelodysplastic syndrome (MDS)/AML (NCT05673057). Patients with an Eastern Cooperative Oncology Group (ECOG) performance status of 0 to 2, and a life expectancy of ≥12 weeks are eligible. Up to 45 patients will be enrolled in the dose-escalation part of the trial. Patients receive MP0533 once per week starting at day 15 after a stepwise dose increase on days 1, 5, and 8, and until disease progression or unacceptable toxicity. One cycle corresponds to 28 days. DR escalation of MP0533 is guided by two Bayesian logistic regression models estimating the joint probability of cytokine release syndrome (CRS) and non-CRS dose-limiting toxicities (DLTs). Bone marrow examinations are performed at weeks 4, 8, and 12. Responses are determined using 2022 European LeukemiaNet (ELN) criteria with an assessment of hematologic improvement. Treatment-emergent adverse events (TEAEs) are assessed according to National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) v5.0. RESULTS: As of data cut-off (20 July 2023), 5 patients received ≥5 infusions of MP0533 across 3 DRs preceded by step-up dosing (1 patient each at DR 1 and DR 2, respectively, and 3 patients at DR 3), including 2 women and 3 men. The median age at enrollment was 79 years (range 68-81). All patients had adverse risk mutations. Patients received a median number of 1 prior antileukemic therapy line (range 1-2) and on average 2 cycles of MP0533 treatment (range 1-3). To date, one patient with a TP53 mutation achieved a morphologic leukemia-free state after 8 weeks and complete remission with incomplete hematologic recovery (CRi) after 12 weeks of treatment with DR 3. No DLTs have been observed in any DR so far. TEAEs considered to be related to MP0533 were Grade 2 infusion-related reaction (2 patients), Grade 1 CRS (2 patients), and Grade 1 diarrhea and fatigue (1 patient). No Grade ≥3 drug-related TEAEs were reported. The reported TEAEs (Figure) reflected the patients' disease course. Preliminary PK results up to DR 3 confirmed that all patients were exposed to MP0533 and exposure levels were broadly aligned with PK/PD model predictions of MP0533 in serum. CONCLUSION: The results of this ongoing phase 1/2a study indicated an acceptable safety profile of MP0533 monotherapy in 5 patients up to DR 3 with weekly infusions. Preliminary response data are encouraging, with one response observed in 1 of 2 patients evaluable for response in DR 3 to date.

Recent advances in cellular immunotherapy for lymphoid malignancies.

Cellular immunotherapy with chimeric antigen receptor (CAR) T-cells has revolutionized the treatment of lymphoid malignancies. This review addresses the need for CAR expression in our endogenous T-cells to kill tumor cells with a focus on the basic principles of T-cell receptor recognition of major histocompatibility complex-peptide complexes. We review the factors associated with CAR T-cell outcomes and recent efforts to employ CAR T-cells in earlier lines of therapy. We also discuss the value of bispecific T-cell engagers as off-the-shelf products with better toxicity profiles. Finally, natural killer cells are discussed as an important cellular immunotherapy platform with the potential to broaden immunotherapeutic applications beyond lymphoid malignancies.

IMMU-04. LOW-INTENSITY PULSED ULTRASOUND ENHANCES CAR T CELL THERAPY BY INDUCING TWO-WAY IMMUNE CELL TRAFFICKING – T CELL INTO TUMOR AND MYELOID CELL INTO PERIPHERY IN A SYNGENEIC MOUSE GLIOBLASTOMA MODEL

Abstract Chimeric Antigen Receptor (CAR) T-cell therapy has been successful against hematological malignancies. However, it has not established efficacy in glioblastoma (GBM) patients. Significant challenges currently limiting the effectiveness of CAR T-cell therapy for patients with GBM include poor CAR-T cell trafficking to GBM due to the blood-brain barrier, and the presence of highly immunosuppressive and pro-tumoral tumor-associated macrophages (TAMs) in the tumor microenvironment. Furthermore, the heterogeneous expression of target antigens in GBM can lead to antigen escape and therapeutic resistance. Low-intensity pulsed ultrasound with microbubbles (LIPU/MB) has been shown to enhance CAR T-cell trafficking and improve survival in immunocompromised xenograft mice GBM models. In this study, we investigated whether multiple rounds of LIPU/MB can improve not only CAR T-cell but also endogenous T-cell infiltration into the tumors and induce myeloid cell trafficking into lymph nodes (LNs), thus enhancing CAR T-cell therapy against GBM in the immunocompetent syngeneic mice model. We recently developed a novel immunocompetent transgenic (Tg) mouse that expresses an anti-EGFRvIII CAR on T-cells and enables us to investigate the interaction between CAR T-cells and the endogenous immune environment. C57BL6/J mice bearing intracerebral EGFRvIII+ GBM received an intravenous administration of anti-EGFRvIII CAR T-cells derived from Tg-mice. The mice were stratified to receive single-LIPU (1 min before CAR T-cells administration) or triple-LIPU/MB (1 min before CAR T-cells administration, then two and four days later). Triple-LIPU/MB significantly suppressed tumor growth and improved survival and was associated with increased CAR T-cells and endogenous T-cells infiltration into the tumors compared with single-LIPU/MB or without LIPU. Furthermore, draining cervical LNs showed increased macrophages trafficking after triple-LIPU/MB. Moreover, TAMs isolated from triple-LIPU/MB mice brains showed higher levels of MHC-II expression. These results suggest that LIPU/MB may promote the tumor antigen presentation to endogenous T-cells and enhance the efficacy of CAR T-cell therapy for GBM.

ATA3431: Allogeneic CD19/CD20 Bispecific CAR EBV T Cells for the Treatment of B-Cell Malignancies

BACKGROUND: Chimeric antigen receptor (CAR) therapy targeted against CD19 has yielded remarkable clinical responses in the treatment of relapsed/refractory B-cell malignancies, but about a third of patients ultimately relapse due to the loss or downregulation of CD19. Furthermore, technical and logistical challenges associated with autologous CAR T therapy limits access for a majority of eligible patients. To help address these challenges, we have developed an allogeneic bispecific tandem CAR directed against both CD19 and CD20 (ATA3431) built on our Epstein-Barr virus (EBV) T-cell platform. EBV T cells maintain expression of endogenous T-cell receptors that have inherently low alloreactivity due to their recognition of defined viral antigens. CAR EBV T cells have demonstrated a favorable safety profile with no associated evidence for graft-versus-host disease or high-grade cytokine release syndrome (Curran KJ, 2020). The bispecific tandem CAR design allows either CD19 or CD20 target antigen recognition to activate the CAR EBV T cell, with the goal of extending durability of response and limiting antigen escape. In clinical trials, autologous bispecific CAR T cells were shown to be efficacious and safe (Larson 2023, Shah 2020). Additionally, a 1XX mutated CD3ζ signaling domain and our optimized CAR EBV T-cell platform preserve a central memory phenotype, robust expansion, functional persistence, and potent antitumor activity of ATA3431. METHODS: We generated CD20/CD19 bispecific CAR EBV T cells containing a modified CD3ζ signaling domain 1XX using our EBV T-cell platform. As a benchmark comparator, we generated autologous CD20/CD19 bispecific CAR T cells using a 12-day process derived from OKT3/CD28-activated T cells. The final products were characterized via immunophenotyping and evaluated for in vitro and in vivo response to antigen expressing targets. RESULTS: CD20/CD19 bispecific CAR EBV T cells generated from multiple healthy donors demonstrated stable CAR expression, a high proportion of T-cell memory populations, and consistent HLA-independent killing of CD19 + and/or CD20 + tumor cells over repeated in vitrochallenges. ATA3431 mediated highly potent antitumor activity in an established disseminated tumor model of Burkitt's lymphoma and correlated with long-term in vivo persistence of CAR T cells without additional cytokine support. Moreover, ATA3431-treated animals showed superior tumor growth inhibition compared to animals treated with autologous CD20/CD19 bispecific benchmark CAR T cells. No treatment-related toxicities were observed in animals treated with ATA3431. CONCLUSIONS: Preclinical evaluations of ATA3431 show B-cell tumor growth inhibition without any treatment-related toxicities and thereby support advancing ATA3431 for clinical evaluation. Curran KJ, Sauter CS, Kernan CS, et al. Durable remission following “off-the-shelf” chimeric antigen receptor (CAR) T-cells in patients with relapse/refractory (R/R) B-cell malignancies. 2020 Transplantation & Cellular Therapy Meetings. Orlando. Larson SM, Walthers CM, Ji B, Ghafouri SN, et al. CD19/CD20 bispecific chimeric antigen receptor (CAR) in naive/memory T cells for the treatment of relapsed or refractory non-Hodgkin lymphoma. Cancer Discov. 2023 Mar 1;13(3):580-597. doi: 10.1158/2159-8290.CD-22-0964. PMID: 36416874; PMCID: PMC9992104. Shah NN, Johnson BD, Schneider D, et al. Bispecific anti-CD20, anti-CD19 CAR T cells for relapsed B cell malignancies: a phase 1 dose escalation and expansion trial. Nat Med. 2020 Oct;26(10):1569-1575. doi: 10.1038/s41591-020-1081-3. Epub 2020 Oct 5. PMID: 33020647.

Dual-locus, dual-HDR editing permits efficient generation of antigen-specific regulatory T cells with robust suppressive activity

Adoptive regulatory T (Treg) cell therapy is predicted to modulate immune tolerance in autoimmune diseases, including type 1 diabetes (T1D). However, the requirement for antigen (ag) specificity to optimally orchestrate tissue-specific, Treg cell-mediated tolerance limits effective clinical application. To address this challenge, we present a single-step, combinatorial gene editing strategy utilizing dual-locus, dual-homology-directed repair (HDR) to generate and specifically expand ag-specific engineered Treg (EngTreg) cells derived from donor CD4+ Tcells. Concurrent delivery of CRISPR nucleases and recombinant (r)AAV homology donor templates targeting FOXP3 and TRAC was used to achieve three parallel goals: enforced, stable expression of FOXP3; replacement of the endogenous Tcell receptor (TCR) with an islet-specific TCR; and selective enrichment of dual-edited cells. Each HDR donor template contained an alternative component of a heterodimeric chemically inducible signaling complex (CISC), designed to activate interleukin-2 (IL-2) signaling in response to rapamycin, promoting expansion of only dual-edited EngTreg cells. Using this approach, we generated purified, islet-specific EngTreg cells that mediated robust direct and bystander suppression of effector T (Teff) cells recognizing the same or a different islet antigen peptide, respectively. This platform is broadly adaptable for use with alternative TCRs or other targeting moieties for application in tissue-specific autoimmune or inflammatory diseases.

T-cell redirecting therapies for B-cell non-Hodgkin lymphoma: recent progress and future directions.

Several key advances in the treatment of B-cell non-Hodgkin lymphoma (B-NHL) over the past two decades have strategically exploited B-cell lineage markers suitable for targeting by immunotherapies. First, the addition of the anti-CD20 monoclonal antibody (mAb) rituximab to a range of standard therapies conferred remarkable outcomes improvements in diverse settings, perhaps most prominently an overall survival advantage in newly diagnosed diffuse large B-cell lymphoma (DLBCL). Subsequently, multiple chimeric antigen receptor (CAR) T-cell therapies targeting CD19 have revolutionized the treatment of relapsed/refractory (rel/ref) DLBCL and are active in other B-NHL subtypes as well. Most recently, the longstanding aspiration to exploit patients' endogenous T-cells to combat lymphoma has been achieved via T-cell redirecting therapies such as bispecific antibodies (BsAbs) that incorporate dual targeting of a T-cell antigen such as CD3 plus a B-cell antigen such as CD19 or CD20 expressed by the tumor. These novel agents have demonstrated impressive activity as monotherapies in patients with heavily pre-treated, rel/ref B-NHL of a variety of subtypes. Now, myriad clinical trials are exploring combinations of T-cell redirectors with targeted therapies, antibody-drug conjugates, conventional chemotherapy, and even new immunotherapies. Here, we highlight key landmarks in the development of T-cell redirecting therapies for the treatment of B-NHL, emerging evidence and lessons from recent clinical trials, and exciting new directions in this arena.