Circadian oscillations in circulating leukocyte subsets including immature hematopoietic cells have been appreciated; the origin and nature of these alterations remain elusive. Our analysis of wild-type C57BL/6 mice under constant darkness confirmed circadian fluctuations of circulating leukocytes and clonogenic cells in blood and spleen but not bone marrow. Clock gene deficient Bmal1-/- mice lacked this regulation. Cell cycle analyses in the different hematopoietic compartments excluded circadian changes in total cell numbers, rather favoring shifting hematopoietic cell redistribution as the underlying mechanism. Transplant chimeras demonstrate that circadian rhythms within the stroma mediate the oscillations independently of hematopoietic-intrinsic cues. We provide evidence of circadian CXCL12 regulation via clock genes in vitro and were able to confirm CXCL12 oscillation in bone marrow and blood in vivo. Our studies further implicate cortisol as the conveyor of circadian input to bone marrow stroma and mediator of the circadian leukocyte oscillation. In summary, we establish hematopoietic-extrinsic cues as causal for circadian redistribution of circulating mature/immature blood cells.
Alectinib is a standard initial treatment for patients with advanced anaplastic lymphoma kinase (ALK) rearranged non-small-cell lung cancer (NSCLC). The current study analyzed a prospective cohort of 24 consecutive alectinib-treated patients and controls in order to comprehensively characterize longitudinal erythrocyte changes under treatment with ALK inhibitors. Upon starting alectinib, all examined patients developed reticulocytosis and abnormal erythrocyte morphology with anisocytosis and a predominance of acanthocytes (64% of red blood cells on average, range 36−100%) in the peripheral blood smear within approximately 2 weeks. Changes were accompanied by a gradual reduction in Eosin-5-maleimide (EMA) binding, which became pathologic (<80% of cells) within 1−2 months in all cases, mimicking an abortive form of hereditary spherocytosis. The latter could be ruled out in 3/3 of analyzed cases by normal sequencing results for the ANK1, EPB42, SLC4A1, SPTA1, or SBTB genes. The direct Coombs test was also negative in 11/11 tested cases. Besides, anemia, increased LDH, and increased bilirubin were noted in a fraction of patients only, ranging between 42 and 68%. Furthermore, haptoglobin decreases were infrequent, occurring in approximately 1/3 of cases only, and mild, with an average value of 0.93 g/L within the normal range of 0.3−2 g/dL, suggesting that hemolysis occurred predominantly in the extravascular compartment, likely due to splenic trapping of the deformed erythrocytes. These changes showed no association with progression-free survival under alectinib or molecular features, i.e., ALK fusion variant or TP53 status of the disease, and resolved upon a switch to an alternative ALK inhibitor. Thus, alectinib induces mild, reversible erythrocyte changes in practically all treated patients, whose most sensitive signs are aberrant red cell morphology in the peripheral smear, a pathologic EMA test, and reactive reticulocytosis. Frank hemolytic anemia is rare, but mild subclinical hemolysis is very frequent and poses differential-diagnostic problems. Alectinib can be continued under the regular control of hemolysis parameters, but the risk of long-term complications, such as cholelithiasis due to increased serum bilirubin in most patients, remains unclear at present.
Abstract BACKGROUND Novel concepts in immunotherapy have evolved with the identification of potential (neo)epitopes and/or combinations with immune checkpoint inhibitors (ICI). In contrast to many other solid tumors, ICI have not improved outcome for patients with glioblastoma (GB) in phase 3 studies. However, antigen-specific T cells induced by vaccines or adoptively transferred as chimeric antigen receptor (CAR) T cells produced encouraging responses in early clinical trials and case series. From one of these trials, the Actively Personalized Vaccine Consortium (GAPVAC) European Clinical Trial 101, we have identified a human leukocyte antigen (HLA)-A2-restricted T cell receptor (TCR) targeting protein tyrosine phosphatase receptor type zeta 1 (PTPRZ1). PTPRZ1 is strongly overexpressed in malignant gliomas, especially GB, and analyses of intratumoral heterogeneity revealed that the level of PTPRZ1 overexpression is strongly associated with cancer stemness. The PTPRZ1 epitope is naturally presented on HLA-A2. MATERIAL AND METHODS INVENT4GB is a German, multicenter, phase 1, first-in-human, first-of-kind dose-escalation window-of-opportunity investigator-initiated trial in recurrent GB, eligible for re-resection, assessing safety and feasibility of therapy with intravenously (iv) and intraventricularly (icv) infused TCR-engineered T cells (TCR-T). FPI is scheduled for 2024. HLA-A*02:01 positive patients will receive one neoadjuvant iv followed by up to three adjuvant icv infusions of PTPRZ1-specific TCR-transgenic autologous T cell therapy (TCR-T-001) with cyclophosphamide- and fludarabine-based lymphodepletion prior to iv infusion. Patient autologous T cells will be engineered by non-integrating nano scaffold matrix attachment region (SMAR) TCR DNA vectors. Icv infusions will be performed via intraventricular catheter implanted during re-resection. Biomarker discovery includes magnetic resonance imaging (MRI) and longitudinal flow cytometry-based immune monitoring of blood and cerebrospinal fluid. Primary endpoints are feasibility and safety (determination of maximum tolerated dose/ MTD), key secondary endpoints are progression-free survival according to the Immunotherapy Response Assessment in Neurooncology (iRANO) criteria. RESULTS TCR-T-001 robustly recognizes target-expressing cells in vitro and is efficacious in preclinical tumor models. Preclinical data and the trial concept will be presented. CONCLUSION INVENT4GB is a first-of-kind TCR-engineered T cell therapy for GB patients.
Abstract T cell receptor-engineered T cells (TCR-T) could be advantageous in glioblastoma by allowing safe and ubiquitous targeting of the glioblastoma-derived peptidome. Protein tyrosine phosphatase receptor type Z1 (PTPRZ1), is a clinically targetable glioblastoma antigen associated with glioblastoma cell stemness. Here, we identify a therapeutic HLA-A*02-restricted PTPRZ1-reactive TCR retrieved from a vaccinated glioblastoma patient. Single-cell sequencing of primary brain tumors shows PTPRZ1 overexpression in malignant cells, especially in glioblastoma stem cells (GSCs) and astrocyte-like cells. The validated vaccine-induced TCR recognizes the endogenously processed antigen without off-target cross-reactivity. PTPRZ1-specific TCR-T (PTPRZ1-TCR-T) kill target cells antigen-specifically, and in murine experimental brain tumors, their combined intravenous and intracerebroventricular administration is efficacious. PTPRZ1-TCR-T maintain stem cell memory phenotype in vitro and in vivo and lyse all examined HLA-A*02 + primary glioblastoma cell lines with a preference for GSCs and astrocyte-like cells. In summary, we demonstrate the proof of principle to employ TCR-T to treat glioblastoma.
Abstract Response to CAR-T cell therapy in glioblastoma (GB) patients is limited, particularly because of the paucity of cell surface immunotherapeutic targets or low antigen sensitivity. Moreover, tonic CAR signaling leads to T cell dysfunction eventually resulting in non-durable responses. T cell receptor-engineered T (TCR-T) cell therapy circumvents the latter limitation by allowing safe and ubiquitous targeting of the GB-derived peptidome. Protein tyrosine phosphatase receptor type Z1 (PTPRZ1), a GB antigen associated with GB cell stemness, was previously targeted in multipeptide vaccination trials. Here, we identify a therapeutic HLA-A*02-restricted PTPRZ1-reactive TCR retrieved from a vaccinated GB patient. Single-cell sequencing of primary brain tumor samples showed PTPRZ1 overexpression exclusively in malignant cells and most pronounced in GB stem cells and astrocyte (AC)- or oligodendrocyte-progenitor (OPC)-like cells. The validated vaccine-induced TCR recognized the endogenously processed antigen without cross-reactivity to in silico predicted low-risk off-targets. PTPRZ1-specific TCR-engineered human primary T cells (PTPRZ1-TCR-T) killed target cells antigen-specifically. Intravenous adoptive cell transfer against experimental brain tumors alone was not efficacious, but the addition of intracerebroventricular administration of PTPRZ1-TCR-T resulted in potent tumor rejection. PTPRZ1-TCR-T maintained stem cell memory phenotype in vitro and in vivo. PTPRZ1-TCR-T lysed all examined HLA-A*02+ primary GB cell lines with a preference for slow-cycling GB stem cells (GSCs) and AC-like cells. To further develop PTPRZ1-TCR-T as off-the-shelf therapy for HLA-A*02 GB patients, a phase I first-in-human clinical trial INVENT4GB is in preparation.
Immunocompromised patients are at high risk to fail clearance of SARS-CoV-2. Prolonged COVID-19 constitutes a health risk and a management problem as cancer treatments often have to be disrupted. As SARS-CoV-2 evolves, new variants of concern have emerged that evade available monoclonal antibodies. Moreover, antiviral therapy promotes SARS-CoV-2 escape mutations, particularly in immunocompromised patients. These patients frequently suffer from prolonged infection. No successful treatment has been established for persistent COVID-19 infection. Here, we report on a series of 21 immunocompromised patients with COVID-19-most of them hematologic malignancies-treated with plasma obtained from recently convalescent or vaccinated donors or a combination thereof. Repeated dosing of SARS-CoV-2-antibody-containing plasma could clear SARS-CoV-2 infection in 16 out of 21 immunocompromised patients even if COVID-19-specific treatments failed to induce sustained viral clearance or to improve clinical course of SARS-CoV-2 infection. Ten patients were major responders defined as an increase delta(d)Ct of > = 5 after the first administration of convalescent and/or vaccinated plasma (C/VP). On average, SARS-CoV-2 PCR Ct values increased from a median value of 22.55 (IQR = 19.10-24.25) to a median value of 29.57 (IQR = 27.55-34.63; p = <.0001) in the major response subgroup. Furthermore, when treated a second time with C/VP, even 4 out of 5 of the initial nonresponders showed an increase in Ct-values from a median value of 23.13 (IQR = 17.75-28.05) to a median value of 32.79 (IQR = 31.75-33.75; p = .013). Our results suggest that C/VP could be a feasible treatment of COVID-19 infection in patients with hematologic malignancies who did not respond to antiviral treatment.
