Adoptive Therapy Research Articles

Azacitidine, Venetoclax and Allogeneic NK Cells in Newly Diagnosed Acute Myeloid Leukemia (ADVENT-AML): An Investigator-Initiated Multicenter Phase Ib Trial

BACKGROUND AND SIGNIFICANCE : Older/unfit patients with acute myeloid leukemia (AML) have limited treatment options. Outcomes in adverse-risk subgroups of AML are modest with CR/CRi rates of 45% to 60% and median overall survival (OS) of 5 to 12 months with current standard frontline therapy of hypomethylating agent with venetoclax (HMA-VEN).While allogeneic stem cell transplantation (allo-SCT) can significantly improve OS in AML, only 10 to 20% of older pts are able to undergo allo-SCT (Pratz et al. Blood 2019, Maiti et al. Blood 2022) Natural killer (NK) cells bridge the innate and adaptive immune system and play a major role in graft-vs-leukemia (GVL) effect responsible for the clinical benefit noted with allo-SCT. NK cell-based adoptive cellular therapies have shown good safety profiles and promising activity in AML with responses noted in 25% to 88% of patients. Furthermore, NK cells are less likely to induce graft-versus-host disease (GVHD), severe cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS). We hypothesize that the combination of azacitidine, venetoclax and allogeneic NK cells will be safe and improve cure rates in AML compared to either Aza/Ven or NK cells alone. Improved clinical activity may be mediated through multiple synergistic mechanisms including reduction of disease burden by Aza/Ven, upregulation of epigenetically silenced NKG2D ligands, dual activation of extrinsic and intrinsic apoptotic cascades, mitochondrial priming of leukemia cells by venetoclax, and bypassing significant morbidity and mortality associated allo-SCT. Significance: This will be the first trial to evaluate 1) cellular therapy in the frontline setting in AML; 2) the synergy of venetoclax with adoptive NK cell therapy; and 3) Aza/Ven as a lymphodepletion strategy. The results will have significant impact on the development of cellular therapies for hematological and solid tumors. STUDY DESIGN AND METHODS: This investigator-initiated, multicenter, open-label, phase Ib study will evaluate the combination of azacitidine, venetoclax and ex vivo-expanded, off-the-shelf cryopreserved allogeneic NK cells in patients with AML (NCT05834244). Eligibility criteria: The dose escalation phase will enroll adults with relapsed or refractory AML or MDS/AML, and the dose expansion phase will enroll older/unfit pts with newly diagnosed adverse-risk AML. Eligible Pts will be ≥ 75 years, or ≥ 18 years with at least one protocol-defined comorbidity. Patients with favorable risk AML, poor end-organ function, needing immunosuppression, uncontrolled infection or CNS leukemia will be excluded. Trial design: The dose escalation phase will enroll a maximum of 12 patients at 2 dose levels to establish a recommended phase 2 dose (RP2D) using a BOIN design. The dose expansion will enroll up to 20 patients at the RP2D ( Fig. 1). Study treatment: Patients will receive Aza/Ven as standard. Two dose levels of NK cells including 0.5 x10 9 and 1 x10 9 flat dose on day 8 and 15 of the first 4 cycles will be evaluated. Subsequently, pts will continue Aza/Ven indefinitely. Objectives: The primary objective is to determine the safety of this novel triplet combination in terms of dose-limiting toxicities including acute GVHD, CRS or ICANS. Secondary objectives include overall response rate, CR/CRi by 4 cycles, rate of negative measurable residual disease, OS and relapse-free survival. Exploratory objectives include additional response and survival endpoints, the persistence of NK cells using SNP-NGS chimerism, kinetics of allorejection, remodeling of host immune microenvironment and impact on AML stem/progenitor cell hierarchies using single cell CyTOF to understand response/resistance mechanisms. Status: Trial approved by the FDA and institutional IRB. Activation planned for 2H 2023. Funding: Gateway for Cancer Research, MDACC PRIME Award, Chimeric Therapeutics, NCI Cancer Center Support Grant.

Tumor immune evasion: insights from CRISPR screens and future directions.

Despite the clinical success of cancer immunotherapies including immune checkpoint blockade and adoptive cellular therapies across a variety of cancer types, many patients do not respond or ultimately relapse; however, the molecular underpinnings of this are not fully understood. Thus, a system-level understating of the routes to tumor immune evasion is required to inform the design of the next generation of immunotherapy approaches. CRISPR screening approaches have proved extremely powerful in identifying genes that promote tumor immune evasion or sensitize tumor cells to destruction by the immune system. These large-scale efforts have brought to light decades worth of fundamental immunology and have uncovered the key immune-evasion pathways subverted in cancers in an acquired manner in patients receiving immune-modulatory therapies. The comprehensive discovery of the main pathways involved in immune evasion has spurred the development and application of novel immune therapies to target this process. Although successful, conventional CRISPR screening approaches are hampered by a number of limitations, which obfuscate a complete understanding of the precise molecular regulation of immune evasion in cancer. Here, we provide a perspective on screening approaches to interrogate tumor-lymphocyte interactions and their limitations, and discuss further development of technologies to improve such approaches and discovery capability.

Research progress on the immune microenvironment and immunotherapy in gastric cancer.

The tumor microenvironment, particularly the immune microenvironment, plays an indispensable role in the malignant progression and metastasis of gastric cancer (GC). As our understanding of the GC microenvironment continues to evolve, we are gaining deeper insights into the biological mechanisms at the single-cell level. This, in turn, has offered fresh perspectives on GC therapy. Encouragingly, there are various monotherapy and combination therapies in use, such as immune checkpoint inhibitors, adoptive cell transfer therapy, chimeric antigen receptor T cell therapy, antibody-drug conjugates, and cancer vaccines. In this paper, we review the current research progress regarding the GC microenvironment and summarize promising immunotherapy research and targeted therapies.

Intragraft regulatory T cells in the modern era: what can high-dimensional methods tell us about pathways to allograft acceptance?

Replacement of diseased organs with transplanted healthy donor ones remains the best and often only treatment option for end-stage organ disease. Immunosuppressants have decreased the incidence of acute rejection, but long-term survival remains limited. The broad action of current immunosuppressive drugs results in global immune impairment, increasing the risk of cancer and infections. Hence, achievement of allograft tolerance, in which graft function is maintained in the absence of global immunosuppression, has long been the aim of transplant clinicians and scientists. Regulatory T cells (Treg) are a specialized subset of immune cells that control a diverse array of immune responses, can prevent allograft rejection in animals, and have recently been explored in early phase clinical trials as an adoptive cellular therapy in transplant recipients. It has been established that allograft residency by Tregs can promote graft acceptance, but whether intragraft Treg functional diversification and spatial organization contribute to this process is largely unknown. In this review, we will explore what is known regarding the properties of intragraft Tregs during allograft acceptance and rejection. We will summarize recent advances in understanding Treg tissue residency through spatial, transcriptomic and high-dimensional cytometric methods in both animal and human studies. Our discussion will explore properties of intragraft Tregs in mediating operational tolerance to commonly transplanted solid organs. Finally, given recent developments in Treg cellular therapy, we will review emerging knowledge of whether and how these adoptively transferred cells enter allografts in humans. An understanding of the properties of intragraft Tregs will help lay the foundation for future therapies that will promote immune tolerance.

Overexpression of AMPKγ2 increases AMPK signaling to augment human T cell metabolism and function

Cellular therapies are currently employed to treat a variety of disease processes. For T cell-based therapies, success often relies on the metabolic fitness of the T cell product, where cells with enhanced metabolic capacity demonstrate improved invivo efficacy. AMP-activated protein kinase (AMPK) is a cellular energy sensor which combines environmental signals with cellular energy status to enforce efficient and flexible metabolic programming. We hypothesized that increasing AMPK activity in human T cells would augment their oxidative capacity, creating an ideal product for adoptive cellular therapies. Lentiviral transduction of the regulatory AMPKγ2 subunit stably enhanced intrinsic AMPK signaling and promoted mitochondrial respiration with increased basal oxygen consumption rates, higher maximal oxygen consumption rate, and augmented spare respiratory capacity. These changes were accompanied by increased proliferation and inflammatory cytokine production, particularly within restricted glucose environments. Introduction of AMPKγ2 into bulk CD4 T cells decreased RNA expression of canonical Th2 genes, including the cytokines interleukin (IL)-4 and IL-5, while introduction of AMPKγ2 into individual Th subsets universally favored proinflammatory cytokine production and a downregulation of IL-4 production in Th2 cells. When AMPKγ2 was overexpressed in regulatory T cells, both invitro proliferation and suppressive capacity increased. Together, these data suggest that augmenting intrinsic AMPK signaling via overexpression of AMPKγ2 can improve the expansion and functional potential of human T cells for use in a variety of adoptive cellular therapies.

CAR-T-Cell Therapy in Multiple Myeloma: B-Cell Maturation Antigen (BCMA) and Beyond.

Significant progress has been achieved in the realm of therapeutic interventions for multiple myeloma (MM), leading to transformative shifts in its clinical management. While conventional modalities such as surgery, radiotherapy, and chemotherapy have improved the clinical outcomes, the overarching challenge of effecting a comprehensive cure for patients afflicted with relapsed and refractory MM (RRMM) endures. Notably, adoptive cellular therapy, especially chimeric antigen receptor T-cell (CAR-T) therapy, has exhibited efficacy in patients with refractory or resistant B-cell malignancies and is now also being tested in patients with MM. Within this context, the B-cell maturation antigen (BCMA) has emerged as a promising candidate for CAR-T-cell antigen targeting in MM. Alternative targets include SLAMF7, CD38, CD19, the signaling lymphocyte activation molecule CS1, NKG2D, and CD138. Numerous clinical studies have demonstrated the clinical efficacy of these CAR-T-cell therapies, although longitudinal follow-up reveals some degree of antigenic escape. The widespread implementation of CAR-T-cell therapy is encumbered by several barriers, including antigenic evasion, uneven intratumoral infiltration in solid cancers, cytokine release syndrome, neurotoxicity, logistical implementation, and financial burden. This article provides an overview of CAR-T-cell therapy in MM and the utilization of BCMA as the target antigen, as well as an overview of other potential target moieties.

The Emerging Role of Induced Pluripotent Stem Cells as Adoptive Cellular Immunotherapeutics.

Adoptive cell therapy (ACT) has transformed the treatment landscape for cancer and infectious disease through the investigational use of chimeric antigen receptor T-cells (CAR-Ts), tumour-infiltrating lymphocytes (TILs) and viral-specific T-cells (VSTs). Whilst these represent breakthrough treatments, there are subsets of patients who fail to respond to autologous ACT products. This is frequently due to impaired patient T-cell function or "fitness" as a consequence of prior treatments and age, and can be exacerbated by complex manufacturing protocols. Further, the manufacture of autologous, patient-specific products is time-consuming, expensive and non-standardised. Induced pluripotent stem cells (iPSCs) as an allogeneic alternative to patient-specific products can potentially overcome the issues outlined above. iPSC technology provides an unlimited source of rejuvenated iPSC-derived T-cells (T-iPSCs) or natural killer (NK) cells (NK-iPSCs), and in the context of the growing field of allogeneic ACT, iPSCs have enormous potential as a platform for generating off-the-shelf, standardised, "fit" therapeutics for patients. In this review, we evaluate current and future applications of iPSC technology in the CAR-T/NK, TIL and VST space. We discuss current and next-generation iPSC manufacturing protocols, and report on current iPSC-based adoptive therapy clinical trials to elucidate the potential of this technology as the future of ACT.

TMIC-61. INVESTIGATING REPROGRAMING OF THE GBM’S TUMOR MICROENVIRONMENT AS A RESULT OF CO-TRANSFER OF HEMATOPOIETIC STEM CELLS (HSCS) WITH ADOPTIVE CELLULAR THERAPY

Abstract Glioblastoma (GBM) is an incredibly aggressive and prevalent primary CNS tumor with dismal survival outcomes. GBM’s intra-tumor heterogeneity and the powerful regulatory mechanisms within the tumor microenvironment (TME) that hamper immune activation have proved to be formidable challenges to the development of effective therapies. Fortunately, immunotherapy has emerged as a potentially powerful approach to achieve of immune activation within the TME through adoptive immunotherapy and checkpoint inhibition Our previous work has demonstrated that the survival benefit of adoptive cellular therapy (ACT) is significantly enhanced by concomitant transfer of bone marrow derived hematopoietic stem cells (HSC) with tumor-reactive T cells in preclinical models. Using a novel organotypic brain slice culture (BSC) model study using glioma-bearing C57BL/6J mice, we interrogate tumor microenvironment and directly demonstrate that the presence of HSC-derived cells in the microenvironment increases T cell migration to tumor, and increased tumor cell death. In addition, we can investigate reprogramming of the TME as a result of treatment with this ACT+HSC platform. These studies are unique in that through this model system, we are capable of studying the cell-cell interactions occurring within the tumor microenvironment TME longitudinally while accounting for the complexity and the spatial orientation of the components within the TME that promote immunosuppression and tumor escape.

CTIM-21. T-CELL RECEPTOR REPERTOIRE ANALYSIS IN PBMCS FROM PEDIATRIC PATIENTS WITH RECURRENT MEDULLOBLASTOMA DURING ADOPTIVE CELLULAR THERAPY

Abstract BACKGROUND Adoptive cellular therapy (ACT), which involves transferring tumor-specific lymphocytes, has shown promising clinical efficacy across various cancer types. However, accurately tracking these transferred cells has been challenging. To overcome this limitation, we utilized T-cell receptor (TCR) sequencing to investigate the TCR repertoire in peripheral blood mononuclear cells (PBMCs). Specifically, we examined PBMCs stimulated ex vivo using autologous dendritic cells (DCs) loaded with total tumor RNA, which we applied for infusion in pediatric patients. We also collected blood samples prior to and following immunotherapy to compare the TCR repertoire dynamics. METHODS We isolated total RNA from patient PBMCs obtained before the initiation of adoptive cellular therapy (ACT) and continued collection on a weekly basis for one month, followed by monthly collections after immunotherapy treatment. To generate cDNA, we used RACE technology with the addition of a common adapter at the 5' end. The raw sequencing data obtained was then preprocessed using MiXCR software. Subsequent TCR repertoire analysis was performed using the Immunarch R-package and VDJtools software. RESULTS& CONCLUSIONS The analysis of T-cell receptor (TCR) diversity across ex vivo extended T-cell samples showed a range of 593 to 34,000 unique CDR3 clones among 28 patients with recurrent medulloblastoma. The observed range suggests that different patients exhibit distinct levels of TCR diversity. Our results revealed significant instability in the TCR repertoire within the peripheral blood during the initial 28 days after ACT for all patients. Subsequently, the regeneration was mainly driven by de novo thymic production of naïve T-cells. TCR sequencing of PBMCs stimulated with autologous DCs loaded with total tumor RNA allowed us to gain a deeper understanding of the TCR repertoire in the context of immunotherapy. This approach enabled us to track and analyze the tumor-specific T-cells in the repertoire, providing valuable insights into their behavior and response to the treatment.

EXTH-11. ADOPTIVE CELLULAR THERAPY OVERCOMES GLIOMA IMMUNE ESCAPE BY DISRUPTING MDSC PROLIFERATION AND RECRUITMENT

Abstract INTRODUCTION Our group has previously shown that adoptive cellular therapy (ACT) significantly extends survival in models of medulloblastoma, brain stem glioma, and glioblastoma. Further, ACT significantly decreases myeloid-derived suppressor cells (MDSC) in the tumor microenvironment (TME), suggesting that ACT may ameliorate glioma-mediated immunosuppression. We hypothesize that ACT-mediated reduction of MDSCs is driven by cell proliferation and recruitment mechanisms, but these mechanisms have not been defined. In this study, we quantified the proportions and cell cycle states of MDSCs in the TME and peripheral lymphoid tissues as well as chemokines in serum to determine how ACT affects MDSC recruitment. METHODS ACT was performed in C57BL/6J mice bearing a syngeneic high-grade glioma derived from KR158B-luciferase cell line. The treatment platform included total body irradiation, hematopoietic stem cell rescue, total tumor ribonucleic acid (ttRNA) electroporated dendritic cells, and tumor-reactive T cells. Following ACT completion, we evaluated the proportions and cell cycle states of MDSCs using flow cytometry. Bromodeoxyuridine (BRDU) and 7-aminoactinomycin (7-AAD) defined cell cycle phases. Serum chemokines were measured using a membrane-based sandwich immunoassay. RESULTS We replicated our previous findings that ACT reduces the level of MDSCs in the TME. Additionally, ACT significantly decreased the proportion of MDSCs in S-phase in the TME. Conversely, levels of MDSCs in peripheral lymphoid tissue were significantly increased in mice treated with ACT, with a significant increase of MDSCs in S-phase. Analysis of serum chemokines revealed significant decreases in multiple chemokines implicated in MDSC recruitment such as CXCL13 and CXCL16. CONCLUSION These findings suggest that ACT decreases proliferation and chemotactic recruitment of MDSCs to the TME, which may also explain MDSC accumulation peripheral lymphoid tissue in ACT-treated mice. These immune mechanisms likely contribute to ACT’s efficacy against brain cancer, preventing tumor immune escape, and offer new therapeutic targets for optimizing cellular immunotherapies.

IMMU-33. PRECISION ANTIGEN-DIRECTED IMMUNOTHERAPY TREATMENT OF BRAIN TUMORS INCREASES TUMOR-INFILTRATING EFFECTOR LYMPHOCYTE RESPONSES

Abstract BACKGROUND Despite considerable advancements in the successful use of immunotherapy in treating a variety of solid tumors, applications in treating brain tumors have lagged considerably. This is due, at least in part, to the lack of well-characterized tumor antigens expressed within brain tumors that can mediate tumor rejection; the limited mutational burden within these cancers that limits the abundance of targetable neoantigens; and the immunologically “cold” tumor microenvironment that hampers the generation of sustained and productive immunologic responses. Our OBJECTIVE is to develop a precision immunotherapy approach for brain tumor patients utilizing prospectively identified tumor-specific antigens using cancer immunogenomics and selective amplification of patients’ tumor-specific antigens for generating tumor antigen-specific lymphocyte therapy. METHODS We identified neoantigens and tumor-associated antigens that were uniquely expressed in preclinical glioblastoma and medulloblastoma tumors using our cancer immunogenomics pipeline called Open Reading Frame Antigen Network (ORAN). A tumor antigen-specific RNA library was created for each tumor model and tumor-bearing animals were treated with ex vivo expanded tumor-specific lymphocytes and tumor antigen-specific RNA-loaded dendritic cell (tsRNA-DC) vaccines. In a different approach, animals were treated with a combination of tsRNA-DC vaccines and anti-PD-1 checkpoint-blockade therapy. Tumor progression and survival outcomes were determined. The immune cell response was evaluated using flow cytometry, TCR sequencing, and single-cell RNAseq. RESULTS Our results demonstrate the effectiveness of tsRNA-DC vaccines in eliciting robust anti-tumor immune responses in combination with adoptive cellular therapy and checkpoint blockade therapy. Additionally, our findings substantiate a robust increase in tumor-infiltrating lymphocytes characterized by enhanced effector function both intratumorally and systemically after tumor antigen-specific RNA-directed immunotherapies. CONCLUSION This powerful approach to generating immunotherapy recognizing a plurality of tumor antigens uniquely addresses the challenge of dealing with tumor antigenic heterogeneity and confronting the reality of patient-to-patient variation in antigen expression in the development of antigen targeting strategies.

TSC2 S1365A mutation potently regulates CD8+T cell function and differentiation improving adoptive cellular cancer therapy.

MTORC1 integrates signaling from the immune microenvironment to regulate T cell activation, differentiation, and function. TSC2 in the tuberous sclerosis complex tightly regulates mTORC1 activation. CD8+ T cells lacking TSC2 have constitutively enhanced mTORC1 activity and generate robust effector T cells; however sustained mTORC1 activation prevents generation of long-lived memory CD8+ T cells. Here we show manipulating TSC2 at Ser1365 potently regulates activated but not basal mTORC1 signaling in CD8+ T cells. Unlike non-stimulated TSC2 knockout cells, CD8+ T cells expressing a phospho-silencing mutant TSC2-S1365A (SA) retain normal basal mTORC1 activity. PKC and T-cell Receptor (TCR) stimulation induces TSC2 S1365 phosphorylation and preventing this with the SA mutation markedly increases mTORC1 activation and T-cell effector function. Consequently, SA CD8+ T cells display greater effector responses while retaining their capacity to become long-lived memory T cells. SA CD8+ T cells also display enhanced effector function under hypoxic and acidic conditions. In murine and human solid-tumor models, CD8+ SA T cells used as adoptive cell therapy display greater anti-tumor immunity than WT CD8+ T cells. These findings reveal an upstream mechanism to regulate mTORC1 activity in T cells. The TSC2-SA mutation enhances both T cell effector function and long-term persistence/memory formation, supporting an approach to engineer better CAR-T cells for treating cancer.

Coordinated Immune Cell Networks in the Bone Marrow Microenvironment Define the Graft Versus Leukemia Response with Adoptive Cellular Therapy

Donor lymphocyte infusion (DLI) is an established therapy for relapsed acute myeloid leukemia (AML) after hematopoietic stem cell transplant (HSCT), but response rates are poor (~20%). Interactions between leukemia and immune cells within the leukemia microenvironment may determine responsiveness to adoptive cellular immunotherapies. We hypothesized that systematic characterization of the leukemic marrow microenvironment over treatment course with DLI would define leukemia-immune cell interactions critical to response and hence provide mechanistic understanding of the graft-versus-leukemia (GvL) effect. In particular, we anticipated that use of multi-modal single-cell sequencing across patients and time points before and after therapy accounting for temporal dependencies would facilitate disentangling of the complex microenvironment of leukemic marrow. To infer cell-cell interactions in a temporally resolved fashion from single cell RNA-sequencing (scRNA-seq) data, we developed DIISCO, a Bayesian model using a Gaussian process regression network. In total, we profiled 30 bone marrow aspirates (14 pre- and 16 post-DLI) from 9 patients (5 responder [R], 4 nonresponder [NR]) with post-HSCT relapsed AML treated with DLI by scRNA-seq, single cell TCR sequencing (scTCR-seq) and surface protein characterization (scCITE-seq). We obtained 51,371 high-quality transcriptomes from the marrow samples along with 43,918 cells from the DLI products of these patients (4 R, 3 NR) for a total of 95,289 cells. Cells were clustered with Phenograph resulting in 57 clusters, including 2 CD4+ T cell, 6 CD8+ T cell (C0, C5, C21, C25, C26, C40), 3 NK, 10 B cell, 3 AML leukemia, and several myelo-erythroid clusters. DIISCO revealed a cascading response post-DLI that centered around CD8 C0, CD8 C5, AML, and a B cell cluster in R, not observed in NR. Furthermore, C0 appeared to expand after DLI only in R. We observed a strong negative interaction from CD8 C0 to AML after DLI, suggesting an anti-leukemia immune response, supporting the notion that immune cell populations formed a coordinated interactome associated with effective GvL. Evaluation of inferred interacting pairs using DIISCO and with the tool CellPhoneDB pointed toward an activation circuit in R between CD226 on CD8 C0 and PVR/NECTIN2 on AML. Conversely, inhibitory interactions between the exhaustion marker TIGIT on CD8 C0 and AML were observed in NR. Because DIISCO pinpointed CD8 C0 as a central hub for response, we characterized the identity of this cluster and its post-DLI dynamics. scTCR-seq revealed that this cluster, but not other T cell clusters, underwent marked clonal expansion after DLI ( Figure 1A). scRNA-seq revealed high expression of activation/effector genes ( GZMB, IFNG, CX3CR1, PRF1) compared to other T cells. The clonally expanded cells of C0 also highly expressed ZNF683, a transcription factor associated with T cell cytotoxicity. scCITE-seq demonstrated expression of CD45RA and CD57 but not CD28, consistent with an effector memory re-expressing RA (TEMRA) phenotype. We validated post-DLI expansion of CD8 TEMRA cells (CD3+, CD8+, CD62L-, CD45RA+) in AML R by multi-parameter flow cytometry in an independent cohort of 53 AML patients (29 R, 24 NR) with post-HSCT relapse treated with DLI at our institution (fold change 2.56 pre to post for R, p<0.001; 1.3 pre to post for NR, p=0.474). Moreover, to examine whether CD8+ TEMRA cells were specifically activated after encounter with leukemia, we devised an in vitromodified mixed lymphocyte reaction. For 6 AML R, post-DLI donor-derived CD8+ TEMRA cells that were cultured with pre-DLI recipient leukemia cells consistently demonstrated greater CD137 (p=0.004) and IFN-γ secretion compared to co-culture of the leukemia cells with other T cell subsets (p=0.015) ( Figure 1B). CD8+ TEMRA cells cultured in the absence of leukemia cells did not show elevated expression of either CD137 or IFN-γ, indicating antigen-specific functional activation of CD8+ TEMRAs but not other cell types after encounter with autologous leukemia. Our results reveal clonal expansion and tumor-specific activation of CD8+ TEMRA cells, supporting an antigen specific functional role of these cells in mediating GvL in AML DLI responders. Further evaluation of this CD8 TEMRA population with the ability to recognize leukemia cells may translate to strategies to optimize adoptive cellular therapy or predict responsiveness to DLI.

DAP10 Co-Stimulation Imparts Memory-like Features to CD5 Targeting Cord Blood Derived CAR-NK Cells

Relapsed/refractory T-cell malignancies have a particularly poor prognosis and novel therapies are direly needed. CD5 is a great candidate for adoptive cellular therapy to target T-cell malignancies since it is ubiquitously expressed on T cells with restricted expression on other hematopoietic cells. NK cells are an attractive platform for CAR engineering to target CD5 since, unlike T cells, they do not express CD5 on their surface, which eliminates the risk of fratricide. Another advantage of NK cells for CAR engineering is their safety profile; in contrast to T cells, they do not cause cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS) and they are not associated with graft-versus-host disease (GVHD) in the allogeneic setting, opening the potential for a completely off-the-shelf cellular product to be used at point of care. Therefore, we sought to develop CD5 targeting CAR-NK cells for the treatment of T-cell malignancies. Multiple studies have demonstrated that the choice of co-stimulatory domain in CAR-T cells influences their function, persistence and metabolic profile. In the field of CAR-NK cells, the first constructs tested in the clinic have incorporated CD28, a T cell specific co-stimulatory domain, borrowing from the design of CAR-T cells. There is a sparsity of data regarding the impact of co-stimulatory domains on the proliferation, transcriptomic and proteomic profile, polyfunctionality, metabolism and fitness of CAR-NK cells. In this study, we aimed to do so, by comprehensively studying the impact of co-stimulatory domains including some that are more relevant for NK cell biology, namely DNAX-activating Protein 10 (DAP10), a major adaptor protein and the exclusive signaling intermediate of NKG2D in human NK cells, DNAX-activating Protein 12 (DAP12), an important adaptor molecule that associates with multiple activating receptors (e.g. NKG2C, NKp44, activating KIRs) and NKG2D, one of the most potent NK cytotoxicity receptors which is essential for anti-tumor immunity. Our results show that CD5 CAR-NK cells with DAP10 co-stimulatory domain show enhanced cytotoxicity against CD5+ T-cell leukemia targets even after multiple tumor rechallenges in an Incucyte live-cell imaging assay. They also show augmented polyfunctionality compared to CD5 CAR-NK cells with other co-stimulatory domains in an Isoplexis single-cell secretome assay. Moreover, DAP10 co-stimulation endowed CD5 CAR-NK cells with enhanced metabolic fitness as evidenced by increased oxidative phosphorylation compared to other co-stimulatory molecules. At the epigenetic level, CD5 CAR-NK cells with DAP10 co-stimulation interrogated by sc-ATACseq show enrichment in AP-1 complex and BATF transcription factors related to memory formation and exhaustion resistance. This translates to better in vivo performance as CD5 CAR-NK cells with DAP10 co-stimulatory domain significantly improve tumor control and survival in an NSG mouse model of CD5+ T-cell leukemia (CCRF-CEM) and show evidence of recall response following tumor rechallenge. In conclusion, our data show that DAP10 co-stimulation induces epigenetic reprogramming of CD5 CAR-NK cells leading to enhanced cellular fitness and memory formation ensuing better anti-tumor potential. Based on these preclinical data, a Phase I/II clinical trial evaluating the safety and efficacy of CD5 CAR-NK cells for the treatment of CD5+ malignancies is in preparation.

Time of Origin and Environmental Drivers of Leukemia Immune Escape after Transplantation

CONCLUSIONS One of the key questions regarding cancer therapies is whether treatment-resistant subclones pre-exist therapy or originate at a later time-point from residual cancer cells that were kept under control until that time. Here, we addressed this issue in HLA loss leukemia post-transplantation relapses, representing one of the best characterized mechanisms of selective immune escape from the adoptive immune therapy of allogeneic hematopoietic cell transplantation (allo-HCT) ( Vago, NEJM, 2009). Serial purified acute myeloid leukemia (AML) samples from 11 different patients, collected before allo-HCT and at the time of the post-transplant HLA loss relapse, were subjected to high-depth (average 100x) whole genome sequencing (WGS). Patient and donor germline controls were obtained through in vitro expansion of T cells collected from patients before and after allo-HCT, respectively, and sequenced at an average 30x depth. Starting from the notion that mutations can be employed as molecular clocks to date the age of cells ( Alexandrov, Nature Genetics, 2015) and that mutational signatures recapitulate their exposure to mutagens ( Alexandrov, Nature, 2020), we combined WGS with Poisson-based modelling to perform subclonal deconvolution ( Caravagna, Bioinformatics, 2020), and derive the timing of the HLA loss rearrangement, intersecting results with the patient clinical history. The AML cell line OCI-AML3 was employed for validation experiments, assessing in vitro the ability of selected compounds to induce DNA damage (quantifying ϒH2Ax through immunofluorescence and flow cytometry) and specific mutational signatures (by the WGS of single cell clones, performed at an average depth of sequencing of 30x). By WGS data of longitudinal AML samples we successfully reconstructed the clonal dynamics leading to 12 HLA loss relapses, and in each subclone deconvolved mutational signatures. By applying Poisson-based timing models to homozygous (i.e. occurred before the copy neutral loss of hetherozygosity - cn-LOH - rearrangement) and heterozygous (i.e. occurred after the event) mutations in the rearranged region, in 10/12 HLA loss relapses we confidently dated the event to a post-transplant time-point (Fig. 1A). In all HLA loss subclones we could detect a C>A mutational signature that was absent in pre-transplant samples (Fig. 1B): combining literature data ( Kanter, Cell Stem Cell, 2021) and in in vitro experiments on the OCI-AML3 cell line, we reconducted this signature to the antiviral drug ganciclovir (GCV). By in vitro experiments, we showed that GCV induces DNA damage and homologous recombination repair in AML, an effect that could be possibly potentiated by the concomitant exposure to the immunosuppressant mycophenolate ( Fang, Genome Medicine, 2022). Concordantly, by retrospective analysis of 126 AML relapses after a family haploidentical transplantation occurred at our Institute between August 2002 and December 2020, we observed that the incidence of HLA loss was 41/95 (43.1%) in patients exposed to GCV and 5/31 (16.1%) in unexposed ones (OR: 3.94, 95%CI 1.39 to 11.16, p=0.009). Our data indicate that in the majority of patients HLA loss variants originate after transplant, and that the genotoxicity of ganciclovir is a potent facilitator of the phenomenon. Defining the time of origin of therapy resistance has fundamental clinical consequences, implying that immune escape clones can only be targeted by post-transplant interventions, and warranting the development of new tools to anticipate the rapid detection of relapse.

Results of phase 2 randomized multi-center study to evaluate the safety and efficacy of infusion of memory T cells as adoptive therapy in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pneumonia and/or lymphopenia (RELEASE NCT04578210)

Background aimsThere are currently no effective anti-viral treatments for coronavirus disease 2019 (COVID-19)–hospitalized patients with hypoxemia. Lymphopenia is a biomarker of disease severity usually present in patients who are hospitalized. Approaches to increasing lymphocytes exerting an anti-viral effect must be considered to treat these patients. Following our phase 1 study, we performed a phase 2 randomized multicenter clinical trial in which we evaluated the efficacy of the infusion of allogeneic off-the-shelf CD45RA− memory T cells containing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cells from convalescent donors plus the standard of care (SoC) versus just the SoC treatment. MethodsEighty-four patients were enrolled in three Spanish centers. The patients were randomized into the infusion of 1 × 106/kg CD45RA− memory T cells or the SoC. We selected four unvaccinated donors based on the expression of interferon gamma SARS-CoV-2–specific response within the CD45RA− memory T cells and the most frequent human leukocyte antigen typing in the Spanish population. ResultsWe analyzed data from 81 patients. The primary outcome for recovery, defined as the proportion of participants in each group with normalization of fever, oxygen saturation sustained for at least 24 hours and lymphopenia recovery through day 14 or at discharge, was met for the experimental arm. We also observed faster lymphocyte recovery in the experimental group. We did not observe any treatment-related adverse events. ConclusionsAdoptive cell therapy with off-the-shelf CD45RA− memory T cells containing SAR-CoV-2–specific T cells is safe, effective and accelerates lymphocyte recovery of patients with COVID-19 pneumonia and/or lymphopenia. Trial registrationNCT04578210

CXCR5 + CD8 + T Cell-mediated Suppression of Humoral Alloimmunity and AMR in Mice Is Optimized With mTOR and Impaired With Calcineurin Inhibition.

Adoptive cellular therapy (ACT) with antibody-suppressor CXCR5 + CD8 + T cells (CD8 + T Ab-supp ) inhibits alloantibody production, antibody-mediated rejection (AMR), and prolongs graft survival in multiple transplant mouse models. However, it is not known how conventional immunosuppressive agents impact the efficacy of CD8 + T Ab-supp ACT. We investigated the efficacy of CD8 + T Ab-supp cell ACT when combined with calcineurin inhibitor (CNi) or mammalian target of rapamycin inhibitor (mTORi) in a murine model of kidney transplant. ACT-mediated decrease in germinal center B cells, posttransplant alloantibody titer, and amelioration of AMR in high alloantibody-producing CCR5 knockout kidney transplant recipients were impaired when ACT was combined with CNi and enhanced when combined with mTORi. CNi (but not mTORi) reduced ACT-mediated in vivo cytotoxicity of IgG + B cells and was associated with increased quantity of germinal center B cells. Neither CNi nor mTORi treatment impacted the expression of cytotoxic effector molecules (FasL, Lamp1, perforin, granzyme B) by CD8 + T Ab-supp after ACT. Concurrent treatment with CNi (but not mTORi) reduced in vivo proliferation of CD8 + T Ab-supp after ACT. The increase in quantity of splenic CD44 + CXCR5 + CD8 + T cells that occurs after ACT was reduced by concurrent treatment with CNi but not by concurrent treatment with mTORi (dose-dependent). Impaired efficacy of ACT by CNi is attributed to reduced persistence and/or expansion of CD8 + T Ab-supp cells after ACT. In contrast, concurrent immunosuppression with mTORi preserves CD8 + T Ab-supp cells quantity, in vivo proliferation, and in vivo cytotoxic effector function after ACT and enhances suppression of humoral alloimmunity and AMR.

Microphysiological model reveals the promise of memory-like natural killer cell immunotherapy for HIV± cancer

Numerous studies are exploring the use of cell adoptive therapies to treat hematological malignancies as well as solid tumors. However, there are numerous factors that dampen the immune response, including viruses like human immunodeficiency virus. In this study, we leverage human-derived microphysiological models to reverse-engineer the HIV-immune system interaction and evaluate the potential of memory-like natural killer cells for HIV+ head and neck cancer, one of the most common tumors in patients living with human immunodeficiency virus. Here, we evaluate multiple aspects of the memory-like natural killer cell response in human-derived bioengineered environments, including immune cell extravasation, tumor penetration, tumor killing, T cell dependence, virus suppression, and compatibility with retroviral medication. Overall, these results suggest that memory-like natural killer cells are capable of operating without T cell assistance and could simultaneously destroy head and neck cancer cells as well as reduce viral latency.

CD45 limits early Natural Killer cell development.

The clinical development of Natural Killer (NK) cell-mediated immunotherapy marks a milestone in the development of new cancer therapies and has gained traction due to the intrinsic ability of the NK cell to target and kill tumor cells. To fully harness the tumor killing ability of NK cells, we need to improve NK cell persistence and to overcome suppression of NK cell activation in the tumor microenvironment. The trans-membrane, protein tyrosine phosphatase CD45, regulates NK cell homeostasis, with the genetic loss of CD45 in mice resulting in increased numbers of mature NK cells. This suggests that CD45-deficient NK cells might display enhanced persistence following adoptive transfer. However, we demonstrate here that adoptive transfer of CD45-deficiency did not enhance NK cell persistence in mice, and instead, the homeostatic disturbance of NK cells in CD45-deficient mice stemmed from a developmental defect in the progenitor population. The enhanced maturation within the CD45-deficient NK cell compartment was intrinsic to the NK cell lineage, and independent of the developmental defect. CD45 is not a conventional immune checkpoint candidate, as systemic loss is detrimental to T and B cell development, compromising the adaptive immune system. Nonetheless, this study suggests that inhibition of CD45 in progenitor or stem cell populations may improve the yield of in vitro generated NK cells for adoptive therapy.

Effector and cytolytic function of natural killer cells in anticancer immunity.

Adaptive immune cells play an important role in mounting antigen-specific antitumor immunity. The contribution of innate immune cells such as monocytes, macrophages, natural killer (NK) cells, dendritic cells, and gamma-delta T cells is well studied in cancer immunology. NK cells are innate lymphoid cells that show effector and regulatory function in a contact-dependent and contact-independent manner. The cytotoxic function of NK cells plays an important role in killing the infected and transformed host cells and controlling infection and tumor growth. However, several studies have also ascribed the role of NK cells in inducing pathophysiology in autoimmune diseases, promoting immune tolerance in the uterus, and antitumor function in the tumor microenvironment. We discuss the fundamentals of NK cell biology, its distribution in different organs, cellular and molecular interactions, and its cytotoxic and noncytotoxic functions in cancer biology. We also highlight the use of NK cell-based adoptive cellular therapy in cancer.