A COVID-19 peptide vaccine for the induction of SARS-CoV-2 T cell immunity.

T cell immunity is central for the control of viral infections. CoVac-1 is a peptide-based vaccine candidate, composed of SARS-CoV-2 T cell epitopes derived from various viral proteins1,2, combined with the Toll-like receptor 1/2 agonist XS15 emulsified in Montanide ISA51 VG, aiming to induce profound SARS-CoV-2 T cell immunity to combat COVID-19. Here we conducted a phase I open-label trial, recruiting 36 participants aged 18-80 years, who received a single subcutaneous CoVac-1 vaccination. The primary end point was safety analysed until day 56. Immunogenicity in terms of CoVac-1-induced T cell response was analysed as the main secondary end point until day 28 and in the follow-up until month 3. No serious adverse events and no grade 4 adverse events were observed. Expected local granuloma formation was observed in all study participants, whereas systemic reactogenicity was absent or mild. SARS-CoV-2-specific T cell responses targeting multiple vaccine peptides were induced in all study participants, mediated by multifunctional T helper 1 CD4+ and CD8+ T cells. CoVac-1-induced IFNγ T cell responses persisted in the follow-up analyses and surpassed those detected after SARS-CoV-2 infection as well as after vaccination with approved vaccines. Furthermore, vaccine-induced T cell responses were unaffected by current SARS-CoV-2 variants of concern. Together, CoVac-1 showed a favourable safety profile and induced broad, potent and variant of concern-independent T cell responses, supporting the presently ongoing evaluation in a phase II trial for patients with B cell or antibody deficiency.

Absence of NKG2D ligands defines leukaemia stem cells and mediates their immune evasion.

Patients with acute myeloid leukaemia (AML) often achieve remission after therapy, but subsequently die of relapse1 that is driven by chemotherapy-resistant leukaemic stem cells (LSCs)2,3. LSCs are defined by their capacity to initiate leukaemia in immunocompromised mice4. However, this precludes analyses of their interaction with lymphocytes as components of anti-tumour immunity5, which LSCs must escape to induce cancer. Here we demonstrate that stemness and immune evasion are closely intertwined in AML. Using xenografts of human AML as well as syngeneic mouse models of leukaemia, we show that ligands of the danger detector NKG2D-a critical mediator of anti-tumour immunity by cytotoxic lymphocytes, such as NK cells6-9-are generally expressed on bulk AML cells but not on LSCs. AML cells with LSC properties can be isolated by their lack of expression of NKG2D ligands (NKG2DLs) in both CD34-expressing and non-CD34-expressing cases of AML. AML cells that express NKG2DLs are cleared by NK cells, whereas NKG2DL-negative leukaemic cells isolated from the same individual escape cell killing by NK cells. These NKG2DL-negative AML cells show an immature morphology, display molecular and functional stemness characteristics, and can initiate serially re-transplantable leukaemia and survive chemotherapy in patient-derived xenotransplant models. Mechanistically, poly-ADP-ribose polymerase 1 (PARP1) represses expression of NKG2DLs. Genetic or pharmacologic inhibition of PARP1 induces NKG2DLs on the LSC surface but not on healthy or pre-leukaemic cells. Treatment with PARP1 inhibitors, followed by transfer of polyclonal NK cells, suppresses leukaemogenesis in patient-derived xenotransplant models. In summary, our data link the LSC concept to immune escape and provide a strong rationale for targeting therapy-resistant LSCs by PARP1 inhibition, which renders them amenable to control by NK cells in vivo.

Publisher Correction: Absence of NKG2D ligands defines leukaemia stem cells and mediates their immune evasion.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Identification of CD105 (endoglin) as novel risk marker in CLL.

Several genetic and clinical markers are established as prognostic factors in chronic lymphocytic leukemia (CLL). However, additional markers are needed for risk stratification. Flow cytometric analysis is a mainstay of CLL diagnostics, thus identification of novel prognostic surface markers can improve risk assessment without increasing burden for patients and physicians. Furthermore, surface molecules preferentially expressed in high-risk cases could serve as therapeutic targets for immunotherapy. CD105 (endoglin) is a TGF-beta coreceptor and activates endothelial cells in healthy tissues and cancer. In addition, it is expressed on healthy hematopoietic precursors as well as lymphoid and myeloid leukemias. In acute myeloid leukemia (AML), a CD105 antibody is successfully applied in clinical studies. In CLL, mRNA expression of the CD105 gene ENG reportedly correlates with other risk factors but failed to show significant correlation with overall survival. However, CD105 protein expression in CLL has never been studied. We here analyzed CD105 surface expression on CLL cells from 71 patients by flow cytometry and report for the first time that substantial levels of CD105 are detectable on CLL cells in 70.4% of patients. Using receiver operating characteristics, we established a cutoff of 5.99% positive cells to distinguish between low and high CD105 levels, the latter correlating with decreased time to first treatment and overall survival. High CD105 expression further correlates with CD38 expression. Our study identified membrane expression of CD105 as a potential risk marker and therapeutic target in high-risk CLL. However, multivariant analyses of large cohorts should be performed in confirmatory studies.

C-Cbl regulates c-MPL receptor trafficking and its internalization.

Thrombocyte formation from megakaryocyte and their progenitor cells is tightly regulated by thrombopoietin (TPO) and its receptor c-MPL, thereby maintaining physiological functionality and numbers of circulating platelets. In patients, dysfunction of this regulation could cause thrombocytopenia or myeloproliferative syndromes. Since regulation of this pathway is still not completely understood, we investigated the role of the ubiquitin ligase c-Cbl which was previously shown to negatively regulated c-MPL signalling. We developed a new conditional mouse model using c-Cblfl/fl Pf4Cre mice and demonstrated that platelet-specific knockout of c-Cbl led to severe microthrombocytosis and impaired uptake of TPO and c-MPL receptor internalization. Furthermore, we characterized a constitutive STAT5 activation c-Cbl KO platelets. This study identified c-Cbl as a potential player in causing megakaryocytic and thrombocytic disorders.

Long-term efficacy of the peptide-based COVID-19 T cell activator CoVac-1 in healthy adults.

OBJECTIVES: T cell immunity is key for the control of viral infections including SARS-CoV-2, in particular with regard to immune memory and protection against arising genetic variants. METHODS: We recently evaluated a peptide-based SARS-CoV-2 T cell activator termed CoVac-1 in a first-in-human trial in healthy adults. Here, we report on long-term safety and efficacy data of CoVac-1 until month 12. RESULTS: CoVac-1 is well tolerated without long-term immune-related side effects and induces long-lasting anti-viral T cell responses in 100% of study participants, with potent expandability of clusters of differentiation (CD4+) and CD8+ T cells targeting multiple different CoVac-1 T cell epitopes. T cell responses were associated with stronger injection site reaction. Beyond induction of T cell immunity, 89% of subjects developed CoVac-1-specific immunoglobulin G antibodies which associated with the intensity of the T cell response, indicating that CoVac-1-specific CD4+ T cells support the induction of B-cell responses. Vaccination with approved COVID-19 vaccines boosted CoVac-1-specific T cell responses. Overall, a low SARS-CoV-2 infection rate (8.3%) was observed. CONCLUSION: Together, a single application of CoVac-1 elicits long-lived and broad SARS-CoV-2-specific T cell immunity, which further supports the current evaluation of our T cell activator in patients with congenital or acquired B-cell defects.

Phase I/II trial of a peptide-based COVID-19 T-cell activator in patients with B-cell deficiency.

T-cell immunity is central for control of COVID-19, particularly in patients incapable of mounting antibody responses. CoVac-1 is a peptide-based T-cell activator composed of SARS-CoV-2 epitopes with documented favorable safety profile and efficacy in terms of SARS-CoV-2-specific T-cell response. We here report a Phase I/II open-label trial (NCT04954469) in 54 patients with congenital or acquired B-cell deficiency receiving one subcutaneous CoVac-1 dose. Immunogenicity in terms of CoVac-1-induced T-cell responses and safety are the primary and secondary endpoints, respectively. No serious or grade 4 CoVac-1-related adverse events have been observed. Expected local granuloma formation has been observed in 94% of study subjects, whereas systemic reactogenicity has been mild or absent. SARS-CoV-2-specific T-cell responses have been induced in 86% of patients and are directed to multiple CoVac-1 peptides, not affected by any current Omicron variants and mediated by multifunctional T-helper 1 CD4+ T cells. CoVac-1-induced T-cell responses have exceeded those directed to the spike protein after mRNA-based vaccination of B-cell deficient patients and immunocompetent COVID-19 convalescents with and without seroconversion. Overall, our data show that CoVac-1 induces broad and potent T-cell responses in patients with B-cell/antibody deficiency with a favorable safety profile, which warrants advancement to pivotal Phase III safety and efficacy evaluation. ClinicalTrials.gov identifier NCT04954469.

BCR::ABL1 tyrosine kinase inhibitors hamper the therapeutic efficacy of blinatumomab in vitro.

PURPOSE: Acute B-lymphoblastic leukemia (B-ALL) is a malignant disease characterized by accumulation of clonal immature lymphocytes in the bone marrow and peripheral blood. The approval of BCR::ABL1 tyrosine kinase inhibitors (TKI) such as imatinib, dasatinib, nilotinib and ponatinib marked a milestone in targeted therapy only for a subset of patients carrying the translocation t(9;22)(q34;q11). Immunotherapy with the bispecific antibody (bsAb) blinatumomab targeting CD19xCD3 revolutionized treatment of all B-ALL cases. The combination of both TKI and bsAb, so-called "dual targeting", is currently under clinical investigation, although TKI might influence T cell effects. METHODS: We here investigated the combination of different TKI and blinatumomab in BCR::ABL1+ and BCR::ABL1- B-ALL cell lines and primary samples regarding T cell proliferation, differentiation, cytokine release and killing of tumor cells. RESULTS: In vitro analysis revealed profound reduction of T cell proliferation, differentiation, cytokine release and killing of tumor cells upon application of BCR::ABL1 TKI with blinatumomab. Inhibition was more pronounced with dasatinib and ponatinib compared to nilotinib and imatinib. T cell signalling after CD3 stimulation was impaired by TKI mirrored by inhibition of LCK phosphorylation. This known off-target effect might influence the efficacy of bsAb therapy when combined with BCR::ABL1 TKI. CONCLUSION: In conclusion, we propose that nilotinib and imatinib might also be suitable substances for combination with blinatumomab and suggest evaluation in clinical trials.

Receptor Activator of NF-κB (RANK) Confers Resistance to Chemotherapy in AML and Associates with Dismal Disease Course.

Although treatment options of acute myeloid leukemia (AML) have improved over the recent years, prognosis remains poor. Better understanding of the molecular mechanisms influencing and predicting treatment efficacy may improve disease control and outcome. Here we studied the expression, prognostic relevance and functional role of the tumor necrosis factor receptor (TNFR) family member Receptor Activator of Nuclear Factor (NF)-κB (RANK) in AML. We conducted an experimental ex vivo study using leukemic cells of 54 AML patients. Substantial surface expression of RANK was detected on primary AML cells in 35% of the analyzed patients. We further found that RANK signaling induced the release of cytokines acting as growth and survival factors for the leukemic cells and mediated resistance of AML cells to treatment with doxorubicin and cytarabine, the most commonly used cytostatic compounds in AML treatment. In line, RANK expression correlated with a dismal disease course as revealed by reduced overall survival. Together, our results show that RANK plays a yet unrecognized role in AML pathophysiology and resistance to treatment, and identify RANK as "functional" prognostic marker in AML. Therapeutic modulation of RANK holds promise to improve treatment response in AML patients.

Immunoprofiling of 4-1BB Expression Predicts Outcome in Chronic Lymphocytic Leukemia (CLL).

Recent success of novel therapies has improved treatment of chronic lymphocytic leukemia (CLL) patients, but most of them still require several treatment regimes. To improve treatment choice, prognostic markers suitable for prediction of disease outcome are required. Several molecular/genetic markers have been established, but accessibility for the entirety of all patients is limited. We here evaluated the relevance of GITR/4-1BB as well as their ligands for the prognosis of CLL patients. Surface expression of GITR/GITRL and 4-1BB/4-1BBL was correlated with established prognostic markers. Next, we separated our patient population according to GITR/GITRL and 4-1BB/4-1BBL expression in groups with high/low expression levels and performed Kaplan-Meier analyses. Interestingly, no correlation was observed with the defined prognostic markers. Whereas no significant difference between high and low expression of GITR, GITRL and 4-1BBL was observed, high 4-1BB levels on leukemic cells were associated with significantly shorter survival. Thereby we identify 4-1BB as prognostic marker for CLL.

Platelet-Expressed TNFRSF13B (TACI) Predicts Breast Cancer Progression.

Although treatment options in breast cancer have been improved significantly, predictive biomarkers for disease progression and metastasis are still lacking. Recent studies indicate that several TNF Receptor Superfamily members are involved in breast cancer cell proliferation and survival. Interestingly, TNFRSF13B (TACI) mRNA level were of prognostic relevance in breast cancer patients. In this study we provide evidence for TACI expression on platelets of breast cancer patients. The level of platelet-expressed TACI (pTACI) was significantly increased on platelets derived from breast cancer patients compared to healthy controls. Upon platelet activation, pTACI was downregulated on the platelet surface of healthy donors and breast cancer patients. Of note, inhibition of matrix metalloprotease (MMP) prevented downregulation of pTACI ex vivo, indicating that proteolytic cleavage of pTACI is responsible for reduction of pTACI level. Stimulation of pTACI via BAFF, BAFF 60-mer or APRIL did not influence platelet activation and function. Remarkably, pTACI was particularly regulated during tumor progression in our breast cancer cohort. TACI expression levels on platelets were correlated with clinical parameters including tumor stage, occurrence of metastasis and tumor cell proliferation (Ki67). In conclusion, our data emphasize the potential use of platelets as a liquid biomarker in breast cancer.

Neutralization of B-Cell Activating Factor (BAFF) by Belimumab Reinforces Small Molecule Inhibitor Treatment in Chronic Lymphocytic Leukemia.

The introduction of idelalisib, ibrutinib and venetoclax for treatment of chronic lymphocytic leukemia (CLL) has greatly improved long term survival of patients. However, many patients do not achieve complete remission and suffer from development of resistance upon treatment with these small molecule inhibitors. Here we report that the TNF family member B-cell activating factor (BAFF) mediates resistance of CLL cells to idelalisib, ibrutinib and venetoclax by sustaining survival and preventing apoptosis of the malignant B cells as revealed by analysis of cellular ATP levels and mitochondrial membrane integrity as well as caspase activation, respectively. As BAFF also plays a prominent role in autoimmune diseases, the BAFF-neutralizing antibody belimumab was developed and approved for treatment of systemic lupus erythematosus (SLE). When we employed belimumab in the context of CLL treatment with idelalisib, ibrutinib and venetoclax, BAFF neutralization was found to significantly increase the sensitivity of the leukemic cells to all three small molecule inhibitors. Notably, BAFF neutralization proved to be beneficial independently of clinical stage according to Binet and Rai or IgVH mutational status. Our results identify drug repurposing of belimumab for neutralization of BAFF to complement small molecule inhibitor treatment as a promising therapeutic approach in CLL that is presently undergoing clinical evaluation.

Genetic Loss of LCK Kinase Leads to Acceleration of Chronic Lymphocytic Leukemia.

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.

CD105 (Endoglin) as negative prognostic factor in AML.

While several genetic and morphological markers are established and serve to guide therapy of acute myeloid leukaemia (AML), there is still profound need to identify additional markers to better stratify patients. CD105 (Endoglin) is a type I transmembrane protein reported to induce activation and proliferation of endothelial cells. In addition, CD105 is expressed in haematological malignancies and the vessels of solid tumours. Here, CD105 associates with unfavourable disease course, but so far no data are available on the prognostic relevance of CD105 in haematological malignancies. We here generated a novel CD105 antibody for analysis of expression and prognostic relevance of CD105 in a cohort of 62 AML patients. Flow cytometric analysis revealed substantial expression in the various AML FAB types, with FAB M3 type displaying significantly lower surface levels. Next we established a cut-off specific fluorescence level of 5.22 using receiver-operating characteristics, which allowed to group patients in cases with CD105lo and CD105hi surface expression and revealed that high CD105 expression correlated significantly with poor overall and progression free survival. In conclusion, we here identify CD105 expression as a novel prognostic marker in AML, which may serve to optimize follow up and treatment decisions for AML patients.