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Background Chimeric antigen receptor (CAR) T-cell therapy has demonstrated significant benefits in the treatment of relapsed/refractory multiple myeloma (RRMM). However, these outcomes can be compromised by severe complications, including cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome (ICANS) and immune effector cell-associated hematotoxicity (ICAHT), predisposing for life-threatening infections.Methods This retrospective observational study examined a total of 129 patients with RRMM who had received idecabtagene vicleucel (ide-cel) at two major myeloma centers in Germany and one center in the USA to assess the Endothelial Activation and Stress Index (EASIX) as a risk marker for an unfavorable clinical course and outcome after CAR T-cell therapy. EASIX is calculated by lactate dehydrogenase (U/L) × creatinine (mg/dL) / platelets (109 cells/L) and was determined before lymphodepletion (baseline) and at the day of CAR T-cell infusion (day 0). The analysis was extended to EASIX derivatives and the CAR-HEMATOTOX score.Results An elevated baseline EASIX (>median) was identified as a risk marker for severe late ICAHT, manifesting with an impaired hematopoietic reconstitution and pronounced cytopenias during the late post-CAR-T period. Patients with high EASIX levels (>upper quartile) were particularly at risk, as evidenced by an increased rate of an aplastic phenotype of neutrophil recovery, severe late-onset infections and ICANS. Finally, we found associations between baseline EASIX and an inferior progression-free and overall survival. Moreover, the EASIX at day 0 also demonstrated potential to serve as a risk marker for post-CAR-T complications and adverse outcomes.Conclusions In conclusion, EASIX aids in risk stratification at clinically relevant time points prior to CAR T-cell therapy with ide-cel. Increased EASIX levels might help clinicians to identify vulnerable patients to adapt peri-CAR-T management at an early stage.

Abstract Background While quadruplet induction therapies deepen responses in newly diagnosed multiple myeloma patients, their impact on peripheral blood stem cell (PBSC) collection remains incompletely understood. This analysis aims to evaluate the effects of prolonged lenalidomide induction and isatuximab- or elotuzumab-containing quadruplet induction therapies on PBSC mobilization and collection. Methods A total of 179 transplant-eligible patients with newly diagnosed MM treated at a single academic center were included. The patients were evaluated based on PBSC mobilization and collection parameters, including overall collection results, CD34+ cell levels in peripheral blood, leukapheresis (LP) delays, overall number of LP sessions, and the rate of rescue mobilization with plerixafor. The patients underwent four different induction regimens: Lenalidomide, bortezomib, and dexamethasone (RVd, six 21-day cycles, n = 44), isatuximab-RVd (six 21-day cycles, n = 35), RVd (four 21-day cycles, n = 51), or elotuzumab-RVd (four 21-day cycles, n = 49). Results The patients' characteristics were well balanced across the different groups. Collection failures, defined as the inability to collect three sufficient PBSC transplants, were rare (n = 3, 2%), with no occurrences in the isatuximab-RVd and elotuzumab-RVd groups. Intensified induction with six 21-day cycles of RVd did not negatively impact the overall number of collected PBSCs (9.7 × 106/kg bw versus 10.5 × 106/kg bw, p = 0.331) compared to four 21-day cycles of RVd. Plerixafor usage was more common after six cycles of RVd compared to four cycles (16% versus 8%). Addition of elotuzumab to RVd did not adversely affect overall PBSC collection (10.9 × 106/kg bw versus 10.5 × 106/kg bw, p = 0.915). Patients treated with isatuximab-RVd (six cycles) had lower numbers of collected stem cells compared to those receiving RVd (six cycles) induction (8.8 × 106/kg bw versus 9.7 × 106/kg bw, p = 0.801), without experiencing significant delays in LP or increased numbers of LP sessions in a multivariable logistic regression analysis. Plerixafor usage was more common after isatuximab plus RVd compared to RVd alone (34% versus 16%). Conclusions This study demonstrates that stem cell collection is feasible after prolonged induction with isatuximab-RVd without collection failures and might be further explored as induction therapy. Trial registration Patients were treated within the randomized phase III clinical trials GMMG-HD6 (NCT02495922, 24/06/2015) and GMMG-HD7 (NCT03617731, 24/07/2018). However, during stem cell mobilization and -collection, no study-specific therapeutic intervention was performed.

Abstract Despite therapeutic advances, mortality of Acute Myeloid Leukemia (AML) is still high. Currently, the determination of prognosis which guides treatment decisions mainly relies on genetic markers. Besides molecular mechanisms, the ability of malignant cells to evade immune surveillance influences the disease outcome and, among others, the expression of checkpoints modulators contributes to this. In AML, functional expression of the checkpoint molecule OX40 was reported, but the prognostic relevance of OX40 and its ligand OX40L axis has so far not been investigated. Here we described expression and prognostic relevance of the checkpoint modulators OX40 and OX40L, analyzed on primary AML cells obtained from 92 therapy naïve patients. Substantial expression of OX40 and OX40L on AML blasts was detected in 29% and 32% of the investigated subjects, respectively, without correlation between the expression of the receptor and its ligand. Whereas OX40L expression was not associated with different survival, patients with high expression levels of the receptor (OX40high) on AML blasts survived significantly shorter than OX40low patients (p = 0.009, HR 0.46, 95% CI 0.24–0.86), which identifies OX40 as novel prognostic marker and a potential therapeutic target in AML patients.

Abstract The prostate‐specific membrane antigen (PSMA) has been demonstrated in numerous studies to be expressed specifically on prostate carcinoma cells and on the neovasculature of several other cancer entities. However, the simultaneous expression of PSMA on both, tumor cells as well as tumor vessels remains unclear, even if such “dual” expression would constitute an important asset to facilitate sufficient influx of effector cells to a given tumor site. We report here on the generation of a PSMA antibody, termed 10B3, which exerts superior dual reactivity on sections of prostate carcinoma and squamous cell carcinoma of the lung. 10B3 was used for the construction of T‐cell recruiting bispecific PSMAxCD3 antibodies in Fab‐ and IgG‐based formats, designated Fabsc and IgGsc, respectively. In vitro, both molecules exhibited comparable activity. In contrast, only the larger IgGsc molecule induced complete and durable elimination of established tumors in humanized mice due to favorable pharmacokinetic properties. Upon treatment of three patients with metastasized prostate carcinoma with the IgGsc reagent, marked activation of T cells and rapid reduction of elevated PSA levels were observed.

Background In lymphoid malignancies, the introduction of chimeric antigen receptor T (CAR-T) cells and bispecific antibodies (bsAbs) has achieved remarkable clinical success. However, such immunotherapeutic strategies are not yet established for acute myeloid leukemia (AML), the most common form of acute leukemia in adults. Common targets in AML such as CD33, CD123, and CLEC12A are highly expressed on both AML blasts and on normal myeloid cells and hematopoietic stem cells (HSCs), thereby raising toxicity concerns. In B-cell acute lymphoblastic leukemia (B-ALL), bsAbs and CAR-T therapy targeting CD19 and CD22 have demonstrated clinical success, but resistance via antigen loss is common, motivating the development of agents focused on alternative targets. An attractive emerging target is FLT3, a proto-oncogene expressed in both AML and B-ALL, with low and limited expression on myeloid dendritic cells and HSCs.Methods We developed and characterized CLN-049, a T cell-activating bsAb targeting CD3 and FLT3, constructed as an IgG heavy chain/scFv fusion. CLN-049 binds the membrane proximal extracellular domain of the FLT3 protein tyrosine kinase, which facilitates the targeting of leukemic blasts regardless of FLT3 mutational status. CLN-049 was evaluated for preclinical safety and efficacy in vitro and in vivo.Results CLN-049 induced target-restricted activation of CD4+ and CD8+ T cells. AML cell lines expressing a broad range of surface levels of FLT3 were efficiently lysed on treatment with subnanomolar concentrations of CLN-049, whereas FLT3-expressing hematopoietic progenitor cells and dendritic cells were not sensitive to CLN-049 killing. Treatment with CLN-049 also induced lysis of AML and B-ALL patient blasts by autologous T cells at the low effector-to-target ratios typically observed in patients with overt disease. Lysis of leukemic cells was not affected by supraphysiological levels of soluble FLT3 or FLT3 ligand. In mouse xenograft models, CLN-049 was highly active against human leukemic cell lines and patient-derived AML and B-ALL blasts.Conclusions CLN-049 has a favorable efficacy and safety profile in preclinical models, warranting evaluation of its antileukemic activity in the clinic.

Introduction Prostate cancer is the second most common cancer in men worldwide. When the disease becomes resistant to androgen-deprivation therapy, treatment options are sparse. To address the high medical need in castration-resistant prostate cancer (CRPC), we generated a novel PSMAxCD3 bispecific antibody termed CC-1. CC-1 binds to prostate-specific membrane antigen that is expressed on prostate cancer cells and tumour vessels, thereby allowing a dual anticancer effect.Methods and analysis This first in human clinical study is a prospective and multicentre trial which enrols patients with metastatic CRPC after failure of established third-line therapy. CC-1 is applied after prophylactic interleukin-6 receptor blockade with tocilizumab (once 8 mg/kg body weight). Each patient receives at least one cycle of CC-1 over a time course of 7 days in an inpatient setting. If clinical benefit is observed, up to five additional cycles of CC-1 can be applied. The study is divided in two parts: (1) a dose escalation phase with intraindividual dose increase from 28 µg to the target dose of 1156 µg based on a modified fast titration design by Simon et al to determine safety, tolerability and the maximum tolerated dose (MTD) as primary endpoints and (2) a dose expansion phase with additional 14 patients on the MTD level of part (1) to identify first signs of efficacy. Secondary endpoints compromise overall safety, tumour response, survival and a translational research programme with, among others, the analysis of CC-1 half-life, the induced immune response, as well as the molecular profiling in liquid biopsies.Ethics and dissemination The PSMAxCD3 study was approved by the Ethics Committee of The University Hospital Tübingen (100/2019AMG1) and the Paul-Ehrlich-Institut (3684/02). Clinical trial results will be published in peer-reviewed journals.Trial registration numbers ClinicalTrials.gov Registry (NCT04104607) and ClinicalTrials.eu Registry (EudraCT2019-000238-20).

Most patients with chronic lymphocytic leukemia (CLL) exhibit an indolent disease course and unresponsive B cell receptors (BCRs) exemplified by an anergic phenotype of their leukemic cells. In up to 5% of patients, CLL transforms from an indolent subtype to an aggressive form of B cell lymphoma (Richter's syndrome), which is associated with worse disease outcome and severe downregulation of NFAT2. Here we show that ablation of the tyrosine kinase LCK, which has previously been characterized as a main NFAT2 target gene in CLL, leads to loss of the anergic phenotype, thereby restoring BCR signaling, which results in an acceleration of CLL. Our study identifies LCK as a main player in mediating BCR unresponsiveness and its role as a crucial regulator of anergy in CLL.

Bispecific antibodies (bsAb) and chimeric antigen receptor (CAR) T cells allow for antibody guided recruitment of T cells against tumors. Both are successfully used for treatment of CD19 expressing leukemias, but may cause cytokine release syndrome (CRS) as a major dose-limiting side effect. For CRS prevention, steroids are recommended prior to bsAb treatment, despite their well-known lymphotoxic activity. The IL-6 receptor antibody tocilizumab is established for treatment of CRS induced by CAR T cells, but was not considered for CRS prevention in bsAb therapy. We here compared the influence of dexamethasone and tocilizumab on bsAb-mediated T cell proliferation and tumor lysis in vitro and in vivo and found that dexamethasone profoundly inhibited T cell proliferation and antitumor activity as induced by two different bsAb, particularly at low effector:target ratios, whereas tocilizumab did not affect efficacy. When we applied tocilizumab early during treatment of three patients with a newly developed PSMAxCD3 bsAb, significant CRS attenuation despite high IL-6 serum levels was observed. Thus, early IL-6 blockade may reduce the undesired sequelae of CRS upon bsAb therapy without affecting therapeutic activity, allowing in turn for safe application of effective doses.