Mesenchymal stromal cells with chimaeric antigen receptors for enhanced immunosuppression.

Allogeneic mesenchymal stromal cells (MSCs) are a safe treatment option for many disorders of the immune system. However, clinical trials using MSCs have shown inconsistent therapeutic efficacy, mostly owing to MSCs providing insufficient immunosuppression in target tissues. Here we show that antigen-specific immunosuppression can be enhanced by genetically modifying MSCs with chimaeric antigen receptors (CARs), as we show for E-cadherin-targeted CAR-MSCs for the treatment of graft-versus-host disease in mice. CAR-MSCs led to superior T-cell suppression and localization to E-cadherin+ colonic cells, ameliorating the animals' symptoms and survival rates. On antigen-specific stimulation, CAR-MSCs upregulated the expression of immunosuppressive genes and receptors for T-cell inhibition as well as the production of immunosuppressive cytokines while maintaining their stem cell phenotype and safety profile in the animal models. CAR-MSCs may represent a widely applicable therapeutic technology for enhancing immunosuppression.

CD19 occupancy with tafasitamab increases therapeutic index of CART19 cell therapy and diminishes severity of CRS.

ABSTRACT: In the development of various strategies of anti-CD19 immunotherapy for the treatment of B-cell malignancies, it remains unclear whether CD19 monoclonal antibody therapy impairs subsequent CD19-targeted chimeric antigen receptor T-cell (CART19) therapy. We evaluated the potential interference between the CD19-targeting monoclonal antibody tafasitamab and CART19 treatment in preclinical models. Concomitant treatment with tafasitamab and CART19 showed major CD19 binding competition, which led to CART19 functional impairment. However, when CD19+ cell lines were pretreated with tafasitamab overnight and the unbound antibody was subsequently removed from the culture, CART19 function was not affected. In preclinical in vivo models, tafasitamab pretreatment demonstrated reduced incidence and severity of cytokine release syndrome and exhibited superior antitumor effects and overall survival compared with CART19 alone. This was associated with transient CD19 occupancy with tafasitamab, which in turn resulted in the inhibition of CART19 overactivation, leading to diminished CAR T apoptosis and pyroptosis of tumor cells.

AXL Inhibition Improves the Antitumor Activity of Chimeric Antigen Receptor T Cells.

The receptor tyrosine kinase AXL is a member of the TYRO3, AXL, and proto-oncogene tyrosine-protein kinase MER family and plays pleiotropic roles in cancer progression. AXL is expressed in immunosuppressive cells, which contributes to decreased efficacy of immunotherapy. Therefore, we hypothesized that AXL inhibition could serve as a strategy to overcome resistance to chimeric antigen receptor T (CAR T)-cell therapy. To test this, we determined the impact of AXL inhibition on CD19-targeted CAR T (CART19)-cell functions. Our results demonstrate that T cells and CAR T cells express high levels of AXL. Specifically, higher levels of AXL on activated Th2 CAR T cells and M2-polarized macrophages were observed. AXL inhibition with small molecules or via genetic disruption in T cells demonstrated selective inhibition of Th2 CAR T cells, reduction of Th2 cytokines, reversal of CAR T-cell inhibition, and promotion of CAR T-cell effector functions. AXL inhibition is a novel strategy to enhance CAR T-cell functions through two independent, but complementary, mechanisms: targeting Th2 cells and reversing myeloid-induced CAR T-cell inhibition through selective targeting of M2-polarized macrophages.

Emerging translational strategies and challenges for enhancing regulatory T cell therapy for graft-versus-host disease.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative therapy for many types of cancer. Genetic disparities between donor and host can result in immune-mediated attack of host tissues, known as graft versus host disease (GVHD), a major cause of morbidity and mortality following HSCT. Regulatory CD4+ T cells (Tregs) are a rare cell type crucial for immune system homeostasis, limiting the activation and differentiation of effector T cells (Teff) that are self-reactive or stimulated by foreign antigen exposure. Adoptive cell therapy (ACT) with Treg has demonstrated, first in murine models and now in patients, that prophylactic Treg infusion can also suppress GVHD. While clinical trials have demonstrated Treg reduce severe GVHD occurrence, several impediments remain, including Treg variability and practical need for individualized Treg production for each patient. Additionally, there are challenges in the use of in vitro expansion techniques and in achieving in vivo Treg persistence in context of both immune suppressive drugs and in lymphoreplete patients being treated for GVHD. This review will focus on 3 main translational approaches taken to improve the efficacy of tTreg ACT in GVHD prophylaxis and development of treatment options, following HSCT: genetic modification, manipulating TCR and cytokine signaling, and Treg production protocols. In vitro expansion for Treg ACT presents a multitude of approaches for gene modification to improve efficacy, including: antigen specificity, tissue targeting, deletion of negative regulators/exhaustion markers, resistance to immunosuppressive drugs common in GVHD treatment. Such expansion is particularly important in patients without significant lymphopenia that can drive Treg expansion, enabling a favorable Treg:Teff ratio in vivo. Several potential therapeutics have also been identified that enhance tTreg stability or persistence/expansion following ACT that target specific pathways, including: DNA/histone methylation status, TCR/co-stimulation signaling, and IL-2/STAT5 signaling. Finally, this review will discuss improvements in Treg production related to tissue source, Treg subsets, therapeutic approaches to increase Treg suppression and stability during tTreg expansion, and potential for storing large numbers of Treg from a single production run to be used as an off-the-shelf infusion product capable of treating multiple recipients.

Murine CAR19 Tregs suppress acute graft-versus-host disease and maintain graft-versus-tumor responses.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) efficacy is complicated by graft-versus-host disease (GVHD), a leading cause of morbidity and mortality. Regulatory T cells (Tregs) have shown efficacy in preventing GVHD. However, high Treg doses are often required, necessitating substantial ex vivo or in vivo expansion that may diminish suppressor function. To enhance in vivo suppressor function, murine Tregs were transduced to express an anti-human CD19 chimeric antigen receptor (hCAR19) and infused into lethally irradiated, hCD19-transgenic recipients for allo-HSCT. Compared with recipients receiving control transduced Tregs, those receiving hCAR19 Tregs had a marked decrease in acute GVHD lethality. Recipient hCD19 B cells and murine hCD19 TBL12-luciferase (TBL12luc) lymphoma cells were both cleared by allogeneic hCAR19 Tregs, which was indicative of graft-versus-tumor (GVT) maintenance and potentiation. Mechanistically, hCAR19 Tregs killed syngeneic hCD19+ but not hCD19- murine TBL12luc cells in vitro in a perforin-dependent, granzyme B-independent manner. Importantly, cyclophosphamide-treated, hCD19-transgenic mice given hCAR19 cytotoxic T lymphocytes without allo-HSCT experienced rapid lethality due to systemic toxicity that has been associated with proinflammatory cytokine release; in contrast, hCAR19 Treg suppressor function enabled avoidance of this severe complication. In conclusion, hCAR19 Tregs are a potentially novel and effective strategy to suppress GVHD without loss of GVT responses.

GM-CSF disruption in CART cells modulates T cell activation and enhances CART cell anti-tumor activity.

Inhibitory myeloid cells and their cytokines play critical roles in limiting chimeric antigen receptor T (CART) cell therapy by contributing to the development of toxicities and resistance following infusion. We have previously shown that neutralization of granulocyte-macrophage colony-stimulating factor (GM-CSF) prevents these toxicities and enhances CART cell functions by inhibiting myeloid cell activation. In this report, we study the direct impact of GM-CSF disruption during the production of CD19-directed CART cells on their effector functions, independent of GM-CSF modulation of myeloid cells. In this study, we show that antigen-specific activation of GM-CSFKO CART19 cells consistently displayed reduced early activation, enhanced proliferation, and improved anti-tumor activity in a xenograft model for relapsed B cell malignancies. Activated CART19 cells significantly upregulate GM-CSF receptors. However, the interaction between GM-CSF and its upregulated receptors on CART cells was not the predominant mechanism of this activation phenotype. GM-CSFKO CART19 cell had reduced BH3 interacting-domain death agonist (Bid), suggesting an interaction between GM-CSF and intrinsic apoptosis pathways. In conclusion, our study demonstrates that CRISPR/Cas9-mediated GM-CSF knockout in CART cells directly ameliorates CART cell early activation and enhances anti-tumor activity in preclinical models.

Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography.

T cells genetically engineered to express chimeric antigen receptors (CAR) have shown unprecedented results in pivotal clinical trials for patients with B cell malignancies or multiple myeloma (MM). However, numerous obstacles limit the efficacy and prohibit the widespread use of CAR T cell therapies due to poor trafficking and infiltration into tumor sites as well as lack of persistence in vivo. Moreover, life-threatening toxicities, such as cytokine release syndrome or neurotoxicity, are major concerns. Efficient and sensitive imaging and tracking of CAR T cells enables the evaluation of T cell trafficking, expansion, and in vivo characterization and allows the development of strategies to overcome the current limitations of CAR T cell therapy. This paper describes the methodology for incorporating the sodium iodide symporter (NIS) in CAR T cells and for CAR T cell imaging using [18F]tetrafluoroborate-positron emission tomography ([18F]TFB-PET) in preclinical models. The methods described in this protocol can be applied to other CAR constructs and target genes in addition to the ones used for this study.

Targeting cancer-associated fibroblasts in the bone marrow prevents resistance to CART-cell therapy in multiple myeloma.

Pivotal clinical trials of B-cell maturation antigen-targeted chimeric antigen receptor T (CART)-cell therapy in patients with relapsed/refractory multiple myeloma (MM) resulted in remarkable initial responses, which led to a recent US Food and Drug Administration approval. Despite the success of this therapy, durable remissions continue to be low, and the predominant mechanism of resistance is loss of CART cells and inhibition by the tumor microenvironment (TME). MM is characterized by an immunosuppressive TME with an abundance of cancer-associated fibroblasts (CAFs). Using MM models, we studied the impact of CAFs on CART-cell efficacy and developed strategies to overcome CART-cell inhibition. We showed that CAFs inhibit CART-cell antitumor activity and promote MM progression. CAFs express molecules such as fibroblast activation protein and signaling lymphocyte activation molecule family-7, which are attractive immunotherapy targets. To overcome CAF-induced CART-cell inhibition, CART cells were generated targeting both MM cells and CAFs. This dual-targeting CART-cell strategy significantly improved the effector functions of CART cells. We show for the first time that dual targeting of both malignant plasma cells and the CAFs within the TME is a novel strategy to overcome resistance to CART-cell therapy in MM.

Emerging approaches to improve allogeneic hematopoietic cell transplantation outcomes for nonmalignant diseases.

Many congenital or acquired nonmalignant diseases (NMDs) of the hematopoietic system can be potentially cured by allogeneic hematopoietic cell transplantation (HCT) with varying types of donor grafts, degrees of HLA matching, and intensity of conditioning regimens. Unique features that distinguish the use of allogeneic HCT in this population include higher rates of graft failure, immune-mediated cytopenias, and the potential to achieve long-term disease-free survival in a mixed chimerism state. Additionally, in contrast to patients with hematologic malignancies, a priority is to completely avoid graft-versus-host disease in patients with NMD because there is no theoretical beneficial graft-versus-leukemia effect that can accompany graft-versus-host responses. In this review, we discuss the current approach to each of these clinical issues and how emerging novel therapeutics hold promise to advance transplant care for patients with NMDs.

A novel Iowa-Mayo validated composite risk assessment tool for allogeneic stem cell transplantation survival outcome prediction.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative option for many hematologic conditions and is associated with considerable morbidity and mortality. Therefore, prognostic tools are essential to navigate the complex patient, disease, donor, and transplant characteristics that differentially influence outcomes. We developed a novel, comprehensive composite prognostic tool. Using a lasso-penalized Cox regression model (n = 273), performance status, HCT-CI, refined disease-risk index (rDRI), donor and recipient CMV status, and donor age were identified as predictors of disease-free survival (DFS). The results for overall survival (OS) were similar except for recipient CMV status not being included in the model. Models were validated in an external dataset (n = 378) and resulted in a c-statistic of 0.61 and 0.62 for DFS and OS, respectively. Importantly, this tool incorporates donor age as a variable, which has an important role in HSCT outcomes. This needs to be further studied in prospective models. An easy-to-use and a web-based nomogram can be accessed here: https://allohsctsurvivalcalc.iowa.uiowa.edu/ .

Regulatory T Cell Therapy of Graft-versus-Host Disease: Advances and Challenges.

Graft-versus-host disease (GVHD) is the leading cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Immunomodulation using regulatory T cells (Tregs) offers an exciting option to prevent and/or treat GVHD as these cells naturally function to maintain immune homeostasis, can induce tolerance following HSCT, and have a tissue reparative function. Studies to date have established a clinical safety profile for polyclonal Tregs. Functional enhancement through genetic engineering offers the possibility of improved potency, specificity, and persistence. In this review, we provide the most up to date preclinical and clinical data on Treg cell therapy with a particular focus on GVHD. We discuss the different Treg subtypes and highlight the pharmacological and genetic approaches under investigation to enhance the application of Tregs in allo-HSCT. Lastly, we discuss the remaining challenges for optimal clinical translation and provide insights as to future directions of the field.

Ruxolitinib resistance or intolerance in steroid-refractory acute graft-versus-host disease - a real-world outcomes analysis.

Ruxolitinib for steroid-refractory acute graft-versus-host disease (SR-aGVHD) results in resistance or intolerance in 1/5 of patients. Outcomes of such patients are undefined. We identified these patients in a multicentre review and reported outcomes. Ruxolitinib-resistant aGVHD was identified in 48/307 patients. Among patients receiving additional therapy, the overall response rate to next therapy was 36%. Median survival was 21 days. Ruxolitinib intolerance led to treatment discontinuation in 16/307 patients. Ten intolerant patients received additional therapy with 50% experiencing continued improvement of aGVHD. Median survival was 50 days in these patients. These data serve as a baseline for future SR-aGVHD studies.

Development of a Clinically Relevant Reporter for Chimeric Antigen Receptor T-cell Expansion, Trafficking, and Toxicity.

Although chimeric antigen receptor T (CART)-cell therapy has been successful in treating certain hematologic malignancies, wider adoption of CART-cell therapy is limited because of minimal activity in solid tumors and development of life-threatening toxicities, including cytokine release syndrome (CRS). There is a lack of a robust, clinically relevant imaging platform to monitor in vivo expansion and trafficking to tumor sites. To address this, we utilized the sodium iodide symporter (NIS) as a platform to image and track CART cells. We engineered CD19-directed and B-cell maturation antigen (BCMA)-directed CART cells to express NIS (NIS+CART19 and NIS+BCMA-CART, respectively) and tested the sensitivity of 18F-TFB-PET to detect trafficking and expansion in systemic and localized tumor models and in a CART-cell toxicity model. NIS+CART19 and NIS+BCMA-CART cells were generated through dual transduction with two vectors and demonstrated exclusive 125I uptake in vitro. 18F-TFB-PET detected NIS+CART cells in vivo to a sensitivity level of 40,000 cells. 18F-TFB-PET confirmed NIS+BCMA-CART-cell trafficking to the tumor sites in localized and systemic tumor models. In a xenograft model for CART-cell toxicity, 18F-TFB-PET revealed significant systemic uptake, correlating with CART-cell in vivo expansion, cytokine production, and development of CRS-associated clinical symptoms. NIS provides a sensitive, clinically applicable platform for CART-cell imaging with PET scan. 18F-TFB-PET detected CART-cell trafficking to tumor sites and in vivo expansion, correlating with the development of clinical and laboratory markers of CRS. These studies demonstrate a noninvasive, clinically relevant method to assess CART-cell functions in vivo.

Multi-institutional study evaluating clinical outcome with allogeneic hematopoietic stem cell transplantation after blinatumomab in patients with B-cell acute lymphoblastic leukemia: real-world data.

Safety and efficacy of allogeneic hematopoietic stem cell transplantation (alloHCT) consolidation after blinatumomab is largely undetermined. To address this issue, we assembled multi-center data of relapsed refractory (RR) acute lymphocytic leukemia (ALL) patients who received alloHCT after blinatumomab. From December 2014 to May 2019, 223 patients who received blinatumomab for RR ALL outside clinical trials were identified. Among them, 106 (47%) patients transplanted post blinatumomab were evaluated for response and toxicity. Ninety-two (87%) patients received alloHCT after achieving CR, while remaining received subsequent salvage prior to undergoing alloHCT. Progression free survival (PFS) and overall survival (OS) at 2 years post alloHCT was 48% (95% CI: 36-59%) and 58% (95% CI: 45-69%), respectively. The cumulative incidence of GIII-IV aGVHD at 3 months was 9.9% (95% CI: 5.0-16.6%). Similarly, cumulative incidence of moderate to severe cGVHD at 2 years was 34.4% (95% CI: 23.7-45.3%). The overall survival at 2 years was not significantly different in patient who achieved CR with MRD negative (68.4% [95% CI: 28.5-89.1%]) compared to CR with MRD positive (63.4% [95% CI: 47.8-75.4%]) prior to alloHCT (p = 0.8). Our real-world analysis suggests that alloHCT is feasible and effective post blinatumomab in patients with RR ALL.

Resistance to CART cell therapy: lessons learned from the treatment of hematological malignancies.

Chimeric antigen receptor T (CART) cell immunotherapy has yielded significant clinical success in treating certain hematological malignancies. However, despite high initial response rates, most patients eventually relapse. Resistance to CART cell therapy can stem from tumor cell mutations, T cell defects, and tumor microenvironment (TME) immunosuppression. Tumor cells can downregulate target antigen expression to evade CART cell detection or mutate death receptor pathways to resist CART cell cytotoxicity. Patient T cells can be intrinsically defective, and CART cells often undergo exhaustion. The TME is abundant with immunosuppressive cells and factors which contribute to suboptimal CART cell activity. Collectively, issues originating in tumor cells, T cells, and the TME present significant hurdles to long-term remission after CART cell therapy. Various strategies to combat CART cell resistance have shown promise in preclinical studies and early clinical trials and are crucial to achieving durable responses.

Leukemic extracellular vesicles induce chimeric antigen receptor T cell dysfunction in chronic lymphocytic leukemia.

Chimeric antigen receptor (CAR) T cell therapy has yielded unprecedented outcomes in some patients with hematological malignancies; however, inhibition by the tumor microenvironment has prevented the broader success of CART cell therapy. We used chronic lymphocytic leukemia (CLL) as a model to investigate the interactions between the tumor microenvironment and CART cells. CLL is characterized by an immunosuppressive microenvironment, an abundance of systemic extracellular vesicles (EVs), and a relatively lower durable response rate to CART cell therapy. In this study, we characterized plasma EVs from untreated CLL patients and identified their leukemic cell origin. CLL-derived EVs were able to induce a state of CART cell dysfunction characterized by phenotypical, functional, and transcriptional changes of exhaustion. We demonstrate that, specifically, PD-L1+ CLL-derived EVs induce CART cell exhaustion. In conclusion, we identify an important mechanism of CART cell exhaustion induced by EVs from CLL patients.

Sequencing of novel agents in relapsed/refractory B-cell acute lymphoblastic leukemia: Blinatumomab and inotuzumab ozogamicin may have comparable efficacy as first or second novel agent therapy in relapsed/refractory acute lymphoblastic leukemia.

BACKGROUND: The availability of novel agents (NAs), including blinatumomab and inotuzumab ozogamicin (InO), has improved the outcomes of patients with relapsed/refractory (RR) B-cell acute lymphoblastic leukemia (ALL). Because of the relative effectiveness, it is often a challenge for clinicians to determine how to best sequence these NAs with respect to efficacy and toxicity. METHODS: In this multicenter, retrospective study of patients with RR ALL treated with blinatumomab, InO, or both, their efficacy as a first or second NA was compared. RESULTS: Among 276 patients, 221 and 55 received blinatumomab and InO, respectively, as a first NA therapy. The complete remission (CR)/complete remission with incomplete count recovery (CRi) rate was 65% and 67% for the blinatumomab and InO groups, respectively (P = .73). The rate of treatment discontinuation due to adverse events was 4% and 7% in the blinatumomab and InO groups, respectively. Ninety-two patients (43%) in the blinatumomab group and 13 patients (29%) in the InO group proceeded with allogeneic hematopoietic stem cell transplantation. The median overall survival (OS) was 15 and 11.6 months in the blinatumomab and InO groups, respectively. A subset analysis was performed for 61 patients who received both NAs (blinatumomab and then InO [n = 40] or InO and then blinatumomab [n = 21]). The CR/CRi rate was 58% for patients who received InO as the second NA and 52% for patients who received blinatumomab as the second NA. The median OS was 10.5 for patients who received InO as the second NA and 5.9 months for patients who received blinatumomab as the second NA (P = .09). CONCLUSIONS: Although the limited power of this study to detect a significant difference between subgroups is acknowledged, the data suggest that blinatumomab and InO may have comparable efficacy as a first or second NA therapy in RR ALL.