Bone marrow-derived mesenchymal stromal cells obstruct AML-targeting CD8+ clonal effector and CAR T-cell function while promoting a senescence-associated phenotype.

Bone marrow mesenchymal stromal cells (MSCs) have been described as potent regulators of T-cell function, though whether they could impede the effectiveness of immunotherapy against acute myeloid leukemia (AML) is still under investigation. We examine whether they could interfere with the activity of leukemia-specific clonal cytotoxic T-lymphocytes (CTLs) and chimeric antigen receptor (CAR) T cells, as well as whether the immunomodulatory properties of MSCs could be associated with the induction of T-cell senescence. Co-cultures of leukemia-associated Wilm's tumor protein 1 (WT1) and tyrosine-protein kinase transmembrane receptor 1 (ROR1)-reactive CTLs and of CD123-redirected switchable CAR T cells were prepared in the presence of MSCs and assessed for cytotoxic potential, cytokine secretion, and expansion. T-cell senescence within functional memory sub-compartments was investigated for the senescence-associated phenotype CD28-CD57+ using unmodified peripheral blood mononuclear cells. We describe inhibition of expansion of AML-redirected switchable CAR T cells by MSCs via indoleamine 2,3-dioxygenase 1 (IDO-1) activity, as well as reduction of interferon gamma (IFNγ) and interleukin-2 (IL-2) release. In addition, MSCs interfered with the secretory potential of leukemia-associated WT1- and ROR1-targeting CTL clones, inhibiting the release of IFNγ, tumor necrosis factor alpha, and IL-2. Abrogated T cells were shown to retain their cytolytic activity. Moreover, we demonstrate induction of a CD28loCD27loCD57+KLRG1+ senescent T-cell phenotype by MSCs. In summary, we show that MSCs are potent modulators of anti-leukemic T cells, and targeting their modes of action would likely be beneficial in a combinatorial approach with AML-directed immunotherapy.

Targeting CD10 on B-Cell Leukemia Using the Universal CAR T-Cell Platform (UniCAR).

Chimeric antigen receptor (CAR)-expressing T-cells are without a doubt a breakthrough therapy for hematological malignancies. Despite their success, clinical experience has revealed several challenges, which include relapse after targeting single antigens such as CD19 in the case of B-cell acute lymphoblastic leukemia (B-ALL), and the occurrence of side effects that could be severe in some cases. Therefore, it became clear that improved safety approaches, and targeting multiple antigens, should be considered to further improve CAR T-cell therapy for B-ALL. In this paper, we address both issues by investigating the use of CD10 as a therapeutic target for B-ALL with our switchable UniCAR system. The UniCAR platform is a modular platform that depends on the presence of two elements to function. These include UniCAR T-cells and the target modules (TMs), which cross-link the T-cells to their respective targets on tumor cells. The TMs function as keys that control the switchability of UniCAR T-cells. Here, we demonstrate that UniCAR T-cells, armed with anti-CD10 TM, can efficiently kill B-ALL cell lines, as well as patient-derived B-ALL blasts, thereby highlighting the exciting possibility for using CD10 as an emerging therapeutic target for B-cell malignancies.

Development and Functional Characterization of a Versatile Radio-/Immunotheranostic Tool for Prostate Cancer Management.

Due to its overexpression on the surface of prostate cancer (PCa) cells, the prostate stem cell antigen (PSCA) is a potential target for PCa diagnosis and therapy. Here we describe the development and functional characterization of a novel IgG4-based anti-PSCA antibody (Ab) derivative (anti-PSCA IgG4-TM) that is conjugated with the chelator DOTAGA. The anti-PSCA IgG4-TM represents a multimodal immunotheranostic compound that can be used (i) as a target module (TM) for UniCAR T cell-based immunotherapy, (ii) for diagnostic positron emission tomography (PET) imaging, and (iii) targeted alpha therapy. Cross-linkage of UniCAR T cells and PSCA-positive tumor cells via the anti-PSCA IgG4-TM results in efficient tumor cell lysis both in vitro and in vivo. After radiolabeling with 64Cu2+, the anti-PSCA IgG4-TM was successfully applied for high contrast PET imaging. In a PCa mouse model, it showed specific accumulation in PSCA-expressing tumors, while no uptake in other organs was observed. Additionally, the DOTAGA-conjugated anti-PSCA IgG4-TM was radiolabeled with 225Ac3+ and applied for targeted alpha therapy. A single injection of the 225Ac-labeled anti-PSCA IgG4-TM was able to significantly control tumor growth in experimental mice. Overall, the novel anti-PSCA IgG4-TM represents an attractive first member of a novel group of radio-/immunotheranostics that allows diagnostic imaging, endoradiotherapy, and CAR T cell immunotherapy.

Targeting Acute Myeloid Leukemia Using the RevCAR Platform: A Programmable, Switchable and Combinatorial Strategy.

Clinical translation of novel immunotherapeutic strategies such as chimeric antigen receptor (CAR) T-cells in acute myeloid leukemia (AML) is still at an early stage. Major challenges include immune escape and disease relapse demanding for further improvements in CAR design. To overcome such hurdles, we have invented the switchable, flexible and programmable adaptor Reverse (Rev) CAR platform. This consists of T-cells engineered with RevCARs that are primarily inactive as they express an extracellular short peptide epitope incapable of recognizing surface antigens. RevCAR T-cells can be redirected to tumor antigens and controlled by bispecific antibodies cross-linking RevCAR T- and tumor cells resulting in tumor lysis. Remarkably, the RevCAR platform enables combinatorial tumor targeting following Boolean logic gates. We herein show for the first time the applicability of the RevCAR platform to target myeloid malignancies like AML. Applying in vitro and in vivo models, we have proven that AML cell lines as well as patient-derived AML blasts were efficiently killed by redirected RevCAR T-cells targeting CD33 and CD123 in a flexible manner. Furthermore, by targeting both antigens, a Boolean AND gate logic targeting could be achieved using the RevCAR platform. These accomplishments pave the way towards an improved and personalized immunotherapy for AML patients.

Strength of clinical evidence leading to approval of novel cancer medicines in Europe: A systematic review and data synthesis.

We aimed to evaluate the quality of clinical evidence that substantiated approval of cancer medicines by the European Medicines Agency (EMA) in the last decade. We performed a systematic review and data synthesis of EMA documents in agreement with PRISMA guidelines. We included the European Public Assessment Reports, Summaries of Product Characteristics, and published randomized controlled trials (RCTs) on anti-cancer drugs approved by EMA from 2010 to 2019, and excluded drugs not indicated for targeting solid or hematological tumors and non-innovative treatments. We synthesized frequencies of approvals differentiating between unblinded and blinded RCTs with and without overall survival (OS) as a predefined primary outcome measure. We assessed the frequency of post-approval RCTs for indications without at least one RCT at the time of approval. Of 199 approvals, 159 (80%) were supported by at least one RCT, 63 (32%) by at least one RCT having OS as the primary or co-primary endpoint, 74 (37%) by at least one blinded RCT, and 30 (15%) by at least one blinded RCT having OS as the primary or co-primary endpoint. Whereas 40 approvals (20%) were not supported by any RCT and, of those, 9 (22%) were followed by a post-approval RCT. While the majority of approvals of cancer medicines approved by EMA was supported by at least one RCT, we noted substantial methodological heterogeneity of the studies. Clinical trial registration: PROSPERO registration number CRD42020206669.

UniCAR T cell immunotherapy enables efficient elimination of radioresistant cancer cells.

Induction or selection of radioresistant cancer (stem) cells following standard radiotherapy is presumably one of the major causes for recurrence of metastatic disease. One possibility to prevent tumor relapse is the application of targeted immunotherapies including, e.g., chimeric antigen receptor (CAR) T cells. In light of long-term remissions, it is highly relevant to clarify whether radioresistant cancer cells are susceptible to CAR T cell-mediated killing. To answer this question, we evaluated the anti-tumor activity of the switchable universal chimeric antigen receptor (UniCAR) system against highly radioresistant head and neck squamous cell carcinoma cells both in vitro and in vivo. Following specific UniCAR T cell engagement via EGFR or CD98 target modules, T cell effector mechanisms were induced including secretion of pro-inflammatory cytokines, up-regulation of granzyme B and perforin, as well as T cell proliferation. CD98- or EGFR-redirected UniCAR T cells further possess the capability to efficiently lyse radioresistant tumor cells. Observed anti-tumor effects were comparable to those against the radiosensitive parental cell lines. Finally, redirected UniCAR T cells significantly inhibited the growth of radioresistant cancer cells in immunodeficient mice. Taken together, our obtained data underline that the UniCAR system is able to overcome radioresistance. Thus, it represents an attractive technology for the development of combined radioimmunotherapeutic approaches that might improve the outcome of patients with metastatic radioresistant tumor diseases.

Adaptor CAR Platforms-Next Generation of T Cell-Based Cancer Immunotherapy.

The success of conventional chimeric antigen receptor (CAR) therapy in the treatment of refractory hematologic malignancies has triggered the development of novel exciting experimental CAR technologies. Among them, adaptor CAR platforms have received much attention. They combine the flexibility and controllability of recombinant antibodies with the power of CARs. Due to their modular design, adaptor CAR systems propose answers to the central problems of conventional CAR therapy, such as safety and antigen escape. This review provides an overview on the different adaptor CAR platforms available, discusses the possibilities and challenges of adaptor CAR therapy, and summarizes the first clinical experiences.

Radioimmunotherapy in Combination with Reduced-Intensity Conditioning for Allogeneic Hematopoietic Cell Transplantation in Patients with Advanced Multiple Myeloma.

Radioimmunotherapy (RIT) has the potential to reduce the incidence of relapse after allogeneic hematopoietic cell transplantation (allo-HCT) in patients with advanced-stage multiple myeloma (MM). In this study, we evaluated the efficacy of RIT in combination with chemotherapy-based reduced-intensity conditioning (RIC). RIT was based on the coupling of an anti-CD66 antibody to the beta emitter 188-rhenium (188-re) for targeted bone marrow irradiation. Between 2012 and 2018, 30 patients with MM, most of them heavily pretreated with various therapies including proteasome inhibitors, immunomodulatory drugs, anti-CD38 antibodies, and autologous hematopoietic cell transplantation (auto-HCT), were treated with a RIT-RIC combination before allo-HCT. In addition to a fludarabine plus melphalan- or treosulfan-based RIC, a median dose of 18.1 Gy (interquartile range [IQR], 14.6 to 24.1 Gy) was applied to the bone marrow. After a median duration of follow-up for surviving patients of 2.1 years (IQR, 1.3 to 3.0 years), the 2-year progression-free survival and overall survival rates were 43% (95% confidence interval [CI], 26% to 73%) and 55% (95% CI, 38% to 79%), respectively. The 2-year nonrelapse mortality and cumulative incidence of progression were 17% (95% CI, 3% to 30%) and 46% (95% CI, 25% to 67%), respectively. Renal toxicity and mucositis were the most frequent extramedullary side effects. In conclusion, the addition of RIT to RIC was safe and feasible and resulted in promising outcomes compared with those previously reported for RIC-based allo-HCT without the addition of RIT in patients with relapsed/refractory MM. Nevertheless, despite the addition of RIT, relapse after allo-HCT remained a major determinant of therapeutic failure. Therefore, the development of novel RIT strategies (eg, dual targeting strategies or combinations with adapter chimeric antigen receptor T cell-based therapies) is needed.

Midostaurin abrogates CD33-directed UniCAR and CD33-CD3 bispecific antibody therapy in acute myeloid leukaemia.

Combinatory therapeutic approaches of different targeted therapies in acute myeloid leukaemia are currently under preclinical/early clinical investigation. To enhance anti-tumour effects, we combined the tyrosine kinase inhibitor (TKI) midostaurin and T-cell mediated immunotherapy directed against CD33. Clinically relevant concentrations of midostaurin abrogated T-cell mediated cytotoxicity both after activation with bispecific antibodies and chimeric antigen receptor T cells. This information is of relevance for clinicians exploring T-cell mediated immunotherapy in early clinical trials. Given the profound inhibition of T-cell functionality and anti-tumour activity, we recommend specific FLT3 TKIs for further clinical testing of combinatory approaches with T-cell based immunotherapy.

Long-term efficacy of reduced-intensity versus myeloablative conditioning before allogeneic haemopoietic cell transplantation in patients with acute myeloid leukaemia in first complete remission: retrospective follow-up of an open-label, randomised phase 3 trial.

BACKGROUND: The impact of the intensity of conditioning before allogeneic haemopoietic cell transplantation (HCT) has been studied in a randomised phase 3 trial comparing reduced-intensity conditioning with myeloablative conditioning in patients with acute myeloid leukaemia in first complete remission. Because of the short follow-up of the original trial, whether reduced-intensity conditioning increases the risk of late relapse compared with myeloablative conditioning remained unclear. To address this question, we present retrospective 10-year follow-up data of this trial and focus on late relapse. METHODS: The original randomised phase 3 trial included patients aged 18-60 years, with intermediate-risk or high-risk acute myeloid leukaemia, an adequate organ function, and an available HLA-matched sibling donor or an unrelated donor with at least nine out of ten HLA alleles matched. Patients were randomly assigned (1:1) to 120 mg/m2 fludarabine combined with four 2 Gy doses of total-body irradiation (reduced-intensity conditioning) or six 2 Gy doses of total-body irradiation and 120 mg/kg cyclophosphamide (myeloablative conditioning). The primary and secondary efficacy endpoints of this trial have been published previously. In this retrospective, long-term follow-up analysis, data were collected from medical reports from individual participating study centres, and from physician and patient interviews. Endpoints included in this analysis were cumulative relapse incidence, overall survival, disease-free survival, and non-relapse mortality in the original study population and in patients alive and relapse-free at 12 months after HCT (landmark analysis). 10-year time to event rates were calculated in the intention-to-treat population and were compared with the Gray test. The trial is registered with ClinicalTrials.gov, number NCT00150878. FINDINGS: In the original trial, 195 patients were randomly assigned to receive reduced-intensity conditioning (n=99) or myeloablative conditioning (n=96). For this retrospective analysis, data were collected with a nearly complete follow-up (completeness index 99%). Median follow-up time for surviving patients was 9·9 years (IQR 8·5-11·4), during which the cumulative incidence of relapse in the complete study population was identical in both groups (30% [95% CI 20-39] in the reduced-intensity conditioning group vs 30% [21-40] in the myeloablative conditioning group; Gray test p=0·99). Relapse occurred at a median of 5·0 months (IQR 3·0-8·8) in the reduced-intensity conditioning group versus 9·5 months (4·5-20·5) in the myeloablative conditioning group. 10-year disease-free survival was 55% (95% CI 45-66) in the reduced-intensity conditioning group and 43% (34-55) in the myeloablative conditioning group (hazard ratio [HR] 0·76 [0·51-1·14]; p=0·19). 10-year non-relapse mortality was 16% (95% CI 8-24) in the reduced-intensity conditioning group and 26% (17-36) in the myeloablative conditioning group (subdistribution HR 0·60 [95% CI 0·32-1·11]; Gray test p=0·10). The incidence of long-term toxicities associated with total-body irradiation was comparable; secondary malignancies occurred in six (6%) of 94 patients in the reduced-intensity conditioning group and five (6%) of 90 in the myeloablative conditioning group (p=1·00). INTERPRETATION: There is no evidence that reduced-intensity conditioning increases the risk of late relapse compared with myeloablative conditioning. Given that the reduced-intensity conditioning group in the original trial was associated with lower early morbidity and toxicity, reduced-intensity conditioning with moderately reduced total-body irradiation doses could be the preferred conditioning strategy for patients with acute myeloid leukaemia who are younger than 60 years and transplanted in first complete remission. FUNDING: None.

Differential effects of mixed lymphocyte reaction supernatant on human mesenchymal stromal cells.

The concept that mesenchymal stromal cells (MSCs), a component of the hematopoietic microenvironment, can be a target for alloreactive effector cells in the context of graft-vs-host disease has not been investigated in detail. Mixed lymphocyte reaction (MLR) supernatant was used to mimic the inflammatory milieu induced by an allogeneic immune response in vitro. In addition to phenotype and proliferation, we monitored MSC differentiation, gene expression, and support of CD34(+) hematopoietic stem and progenitor cells after priming with MLR supernatant. Priming of MSCs with MLR supernatant led to an 11-fold decrease in cobblestone area-forming cells in the 4-week coculture (p < 0.05) and a threefold decrease of colony-forming unit macrophage in the colony-forming cell assay (p < 0.05). MSC proliferation over 8 days was increased 2.5-fold (p < 0.05). Osteogenic differentiation was enhanced, while adipogenesis was concurrently suppressed. In addition, the surface expression of HLA-DR and intercellular adhesion molecule-1 was increased 20-fold (p = 0.06) and 45-fold (p < 0.05), respectively. This was associated with increased adhesion of hematopoietic stem and progenitor cells to MLR-treated MSCs. In summary, our data shed light on the dysfunction of the stromal environment during graft-vs-host disease, possibly aggravating cytopenia and leading to an enhanced immunogenicity of MSCs.