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.

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.

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.

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.

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.

GM-CSF inhibition reduces cytokine release syndrome and neuroinflammation but enhances CAR-T cell function in xenografts.

Chimeric antigen receptor T (CAR-T) cell therapy is a new pillar in cancer therapeutics; however, its application is limited by the associated toxicities. These include cytokine release syndrome (CRS) and neurotoxicity. Although the IL-6R antagonist tocilizumab is approved for treatment of CRS, there is no approved treatment of neurotoxicity associated with CD19-targeted CAR-T (CART19) cell therapy. Recent data suggest that monocytes and macrophages contribute to the development of CRS and neurotoxicity after CAR-T cell therapy. Therefore, we investigated neutralizing granulocyte-macrophage colony-stimulating factor (GM-CSF) as a potential strategy to manage CART19 cell-associated toxicities. In this study, we show that GM-CSF neutralization with lenzilumab does not inhibit CART19 cell function in vitro or in vivo. Moreover, CART19 cell proliferation was enhanced and durable control of leukemic disease was maintained better in patient-derived xenografts after GM-CSF neutralization with lenzilumab. In a patient acute lymphoblastic leukemia xenograft model of CRS and neuroinflammation (NI), GM-CSF neutralization resulted in a reduction of myeloid and T cell infiltration in the central nervous system and a significant reduction in NI and prevention of CRS. Finally, we generated GM-CSF-deficient CART19 cells through CRISPR/Cas9 disruption of GM-CSF during CAR-T cell manufacturing. These GM-CSFk/o CAR-T cells maintained normal functions and had enhanced antitumor activity in vivo, as well as improved overall survival, compared with CART19 cells. Together, these studies illuminate a novel approach to abrogate NI and CRS through GM-CSF neutralization, which may potentially enhance CAR-T cell function. Phase 2 studies with lenzilumab in combination with CART19 cell therapy are planned.

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.

Utilization and Outcomes of Fertility Preservation Techniques in Women Undergoing Allogeneic Hematopoietic Cell Transplant.

Iatrogenic menopause with consequent infertility is a major complication in reproductive-age women undergoing hematopoietic cell transplantation (HCT). Recent guidelines recommend a discussion of the possibility of infertility and the options for fertility preservation as part of informed consent before initiation of any cancer-directed therapy, including HCT. Women age 15 to 49 years at the time of allogeneic HCT, between the years 2001 and 2017, were identified from the Mayo Clinic Rochester institutional HCT database. One hundred seventy-seven women were eligible, of whom 49 (28%) were excluded due to documented postmenopausal state or prior hysterectomy. The median age of the cohort was 31 years (range, 15 to 49 years) with median gravidity and parity being G1P1 (range, G0 to G8, P0 to P6). Fifty-four (42%) women were nulligravid at the time of HCT. Eighty-two percent underwent myeloablative conditioning (MAC), whereas 18% underwent reduced-intensity conditioning (RIC). Only 34 women (27%) had documented fertility counseling within 72 hours of diagnosis, and a total of 61 (48%) received fertility counseling prior to HCT. Thirty-eight women (30%) were referred to a reproductive endocrinologist, of whom 13 (10%) underwent assisted reproductive technologies (ART; nine oocyte cryopreservation, four embryo cryopreservation). Of these, nine procedures yielded successful cryopreserved tissue (two completed at outside institutions). The median time to completion of the seven successful ART procedures at Mayo Clinic was 13 days (range, 9 to 15 days). The remainder of women referred to reproductive endocrinology did not undergo ART due to disease severity (68%), financial barriers (20%), and/or low antral follicle count (12%). Ninety-three women (73%) received leuprolide for ovarian suppression prior to conditioning. Three (4%) of 75 women who underwent MAC and were alive >365 days after HCT had spontaneous menstrual recovery after HCT (median time, 14 months; range, 6 to 21 months), in comparison to 10 (50%) of 20 women who underwent RIC and were alive >365 days after HCT (P < .01) (median, 21.5 months; range, 5 to 83 months). In the latter cohort, there were two spontaneous pregnancies, occurring at 71 and 72 months after HCT, respectively. Oncofertility is an emerging field due to an increasing number of young cancer survivors. Herein, we document that even at a large tertiary HCT center, the rate of documented fertility counseling and reproductive endocrinology referrals was low and the rate of ART was even lower. Spontaneous menstrual recovery was rare but more likely in the setting of nonmalignant disease and RIC HCT. A concerted multidisciplinary effort is needed to understand parenthood goals and to explore the impact of HCT on decision making about fertility preservation and parenthood. These efforts could improve oncofertility referral, ART utilization, and reproductive outcomes.

Extracorporeal Photopheresis Improves Survival in Hematopoietic Cell Transplant Patients with Bronchiolitis Obliterans Syndrome without Significantly Impacting Measured Pulmonary Functions.

We carried out the first matched retrospective cohort study aimed at studying the safety and efficacy of extracorporeal photopheresis (ECP) for bronchiolitis obliterans syndrome (BOS) after allogeneic hematopoietic cell transplantation (HCT). Medical records of 1325 consecutive adult patients who underwent HCT between 2005 and 2015 were reviewed. Seventy-four patients (median age, 51 years) with a diagnosis of BOS were included in the study. After propensity-score matching for BOS severity, 26 patients who underwent ≥3 months of ECP were matched to 26 non-ECP-treated patients, who were assigned an index date corresponding to the ECP start date for their matched pairs. The rate of decline in FEV1 percentage predicted (FEV1PP) decreased after ECP initiation (and after index date in the non-ECP group), with no significant difference between the 2 groups (P = .33). On a multivariable analysis that included baseline transplant and pulmonary function test variables, matched related donor HCT (HR, .1; 95% CI, .03 to .5; P = .002), ECP (HR, .1; 95% CI, .01 to .3; P = .001), and slower rate of decline in FEV1PP before the ECP/index date (HR, .7; 95% CI, .6 to .8; P = .001) were associated with a better overall survival. At last follow-up, non-ECP-treated patients were more likely to be on >5 mg daily dose of prednisone (54% versus 23%; P = .04) and had a greater decline in their Karnofsky performance score (mean difference, -9.5 versus -1.6; P = .06) compared with ECP-treated-patients. In conclusion, compared with other BOS-directed therapies, ECP was found to improve survival in HCT patients with BOS, without significantly impacting measured pulmonary functions. These findings need prospective validation in a larger patient cohort.

Safety and Efficacy of Infliximab Therapy in the Setting of Steroid-Refractory Acute Graft-versus-Host Disease.

Acute graft-versus-host disease (aGVHD) is the leading cause of morbidity and mortality after allogenic hematopoietic cell transplantation (HCT). Corticosteroids are the first-line treatment; however, less than one-half of patients achieve durable remission. Studies suggest that TNF-α, a cytokine released from the bone marrow during conditioning, is involved in the pathogenesis of aGVHD. We retrospectively evaluated the outcome of anti-TNF-α therapy with infliximab in 35 patients with steroid refractory (SR) aGVHD. Infliximab was administered intravenously at 10 mg/kg for a median of 4 doses (range, 1 to 6) on a weekly basis. The overall response rates were 40% (17% complete response [CR], 23% partial response [PR]) at 4 weeks, 23% (9% CR, 14% PR) at 8 weeks, and 17% (all CR) at 12 weeks. Twenty-nine (83%) patients had infectious complications within 12 weeks of initiation of infliximab. These infections included 40 bacterial infections, 6 invasive fungal infections, and 5 viral reactivations. Twelve patients (34%) died secondary to infections. Overall survival at 12 weeks and 6 months from the start of infliximab therapy was 37% (13 of 35) and 17% (6 of 35), respectively; with most deaths secondary to complications from GVHD and infections. In conclusion; the use of infliximab therapy in patients with SR-aGVHD is associated with a modest poorly sustained response along with a heightened risk of severe infections. Future studies with more effective and less toxic therapies are needed for these patients.

Fludarabine-Busulfan Reduced-Intensity Conditioning in Comparison with Fludarabine-Melphalan Is Associated with Increased Relapse Risk In Spite of Pharmacokinetic Dosing.

Fludarabine with busulfan (FB) and fludarabine with melphalan (FM) are commonly used reduced-intensity conditioning (RIC) regimens. Pharmacokinetic dosing of busulfan (Bu) is frequently done for myeloablative conditioning, but evidence for its use is limited in RIC transplants. We compared transplant outcomes of FB versus FM using i.v. Bu targeted to the area under the curve (AUC). A total of 134 RIC transplants (47 FB and 87 FM) for acute myelogenous leukemia and myelodysplastic syndrome were identified, and median follow-up of the cohort was 40 months (range, 0 to 63.3). A significantly higher 2-year cumulative incidence of relapse (CIR) was associated with FB versus FM at 35.6% versus 17.3%, respectively (P = .0058). Furthermore, 2-year progression-free survival rates were higher for FM versus FB at 60.5% versus 48.7%, respectively (P = .04). However, 2-year rates of nonrelapse mortality (NRM) and overall survival (OS) were similar. The need for dose adjustment based on AUC did not alter relapse risk or NRM. Patients with Karnofsky performance status ≥ 90 who received FM had a 2-year OS rate of 74.8% versus 48.3% for FB (P = .03). FB use remained prognostic for relapse in multivariable analysis (hazard ratio, 2.75; 95% confidence interval, 1.28 to 5.89; P = .0097). In summary, in spite of AUC-directed dosing, FB compared with FM was associated with a significantly higher CIR.

ABO blood group incompatibility as an adverse risk factor for outcomes in patients with myelodysplastic syndromes and acute myeloid leukemia undergoing HLA-matched peripheral blood hematopoietic cell transplantation after reduced-intensity conditioning.

BACKGROUND: ABO incompatibility is not a contraindication to hematopoietic cell transplantation (HCT), but it has been associated with additional risks including delayed engraftment, pure red cell aplasia (PRCA), and higher transfusion needs. Data on these events and on patient survival after reduced-intensity conditioning (RIC) HCT are limited. STUDY DESIGN AND METHODS: A total of 127 consecutive patients, 86 with acute myeloid leukemia and 41 with myelodysplastic syndromes, who underwent HLA-matched peripheral blood RIC allogenic HCT between 2005 and 2014 were retrospectively analyzed. RESULTS: Eighty ABO-compatible, 26 major/bidirectional, and 21 minor-ABO-mismatch HCT were identified. Compared to the ABO-compatible group, major/bidirectional mismatches had increased red blood cell (RBC) transfusion requirement during the first 100 days (p = 0.009), delayed RBC and PLT engraftment (p = 0.0011 and p = 0.005, respectively), and higher incidence of grade II to IV acute graft-versus-host disease (aGVHD; p = 0.037). In multivariable analysis, major/bidirectional mismatches had significantly higher non-relapse mortality (NRM) and inferior disease-free survival (DFS) and overall survival (OS) compared with ABO-compatible patients (p = 0.01, p = 0.04, and p = 0.035, respectively). Minor ABO mismatch had no impact on survival (p = 0.99). Four (15%) of 26 major/bidirectional mismatches developed PRCA. There was a significant association between fludarabine plus busulfan conditioning and PRCA (p = 0.0046). CONCLUSION: Major/bidirectional ABO mismatch is associated with higher NRM and shortened DFS and OS in the setting of RIC HCT. Increased transfusion need, delayed RBC and platelet engraftment, PRCA, and increased severity of aGVHD are additional complications contributing to the morbidity.

Vancomycin-resistant Enterococcus colonization and bloodstream infection: prevalence, risk factors, and the impact on early outcomes after allogeneic hematopoietic cell transplantation in patients with acute myeloid leukemia.

BACKGROUND: Screening for vancomycin-resistant Enterococcus (VRE) is performed at many transplant centers, but data on the impact of VRE colonization and bloodstream infection (BSI) on hematopoietic cell transplantation (HCT) outcomes remain conflicting. METHODS: Consecutive adults with acute myeloid leukemia who underwent allogeneic HCT between 2004 and 2014 were retrospectively reviewed. Patients were screened by perirectal PCR swabs targeting vanA and vanB twice weekly while inpatient. RESULTS: Of a total of 203 patients (median age 54 years), 73 (36%) were VRE colonized prior to HCT, 23 (11%) became colonized within the first 100 days, and 107 (53%) remained non-colonized through day 100 post HCT. A landmark analysis on HCT day 0 revealed no significant difference in overall survival according to pre-transplant colonization status (P=.20). However, patients with subsequent VRE colonization within the first 100 days of HCT had a significantly worse survival on both univariable (P=.04) and multivariable (P=.03) analyses. During the first 30 days post HCT, 11 (5% of total and 11% of the VRE colonized) patients developed VRE BSI. Ten (91%) of these had screened positive for VRE colonization before the bacteremia. Age ≥60 years, HCT-comorbidity index ≥3, and VRE colonization were independent risk factors for VRE BSI on multivariable analysis (P=.04, .03, .003, respectively). Only 1 (9%) patient with VRE BSI died within the first 100 days post HCT. CONCLUSION: VRE colonization is a surrogate marker and not an independent predictor of worse outcomes post HCT. VRE BSI is associated with increased morbidity, but does not impact post-HCT survival.

Surrogates of Endothelial Injury Predict Survival After Post-transplant Cyclophosphamide.

Post-transplant cyclophosphamide (PT-Cy) is becoming the standard of care for preventing graft-versus-host disease (GVHD) following allogeneic hematopoietic stem cell transplant (alloHCT). Cyclophosphamide is associated with endothelial injury. We hypothesized that the endothelial activation and stress index (EASIX) score, being a marker of endothelial dysfunction, will predict non-relapse mortality (NRM) in alloHCT patients receiving PT-Cy for GVHD prophylaxis. We evaluate the prognostic ability of the hematopoietic cell transplantation-specific comorbidity index (HCT-CI) and EASIX scores, and report other factors influencing survival, in patients with hematologic malignancies undergoing alloHCT and receiving PT-Cy-based GVHD prophylaxis. Adult patients with hematologic malignancies who underwent alloHCT and received PT-Cy for GVHD prophylaxis at the three Mayo Clinic locations were included in this study. We retrospectively reviewed the Mayo Clinic database and the available electronic medical records to determine the patient, disease, and transplant characteristics. An HCT-CI score of ≥3 was considered high. The EASIX score was calculated from labs available between day -28 (of alloHCT) to the day of starting conditioning and analyzed on log2 transformed values. A log2-EASIX score ≥2.32 was considered high. The cumulative incidence of NRM was determined using competing risk analysis, with relapse considered as competing risk. Overall survival (OS) from transplant was determined using Kaplan-Meier and log-rank methods. Cox-proportional hazard method was used to evaluate factors impacting survival. A total of 199 patients were evaluated. Patients with a high log2-EASIX score had a significantly higher cumulative incidence of NRM at 1 year after alloHCT (34.5% versus 12.3%, P = .003). Competing risk analysis showed that a high log2-EASIX score (HR 2.92, 95% CI 1.38 to 6.17, P = .005) and pre-alloHCT hypertension (HR 2.15, 95% CI 1.06 to 4.36, P = .034) were independently predictive of 1 year-NRM. Accordingly, we combined the two factors to develop a composite risk model stratifying patients in low, intermediate, and high-risk groups: 111 (55.8%) patients were considered low-risk, 76 (38.2%) were intermediate and 12 (6%) were high-risk. Compared to patients in the low-risk group, the intermediate (HR 2.38, 95% CI 1.31 to 4.33, P = .005) and high-risk (HR 5.77, 95% CI 2.31 to 14.39, P < .001) groups were associated with a significantly inferior 1-year OS. Multiorgan failure (MOF) was among the common causes of NRM (14/32, 43.8%) particularly among patients with prior pulmonary comorbidities [7 (50%) patients]. Our study shows that EASIX score is predictive of survival after PT-Cy. The novel EASIX-HTN composite risk model may stratify patients prior to transplant. MOF is a common cause of NRM in patients receiving PT-Cy, particularly among patients with pulmonary comorbidities.

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.

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.

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.

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.