Scalable generation of functional human iPSC-derived CAR-macrophages that efficiently eradicate CD19-positive leukemia.

Background: Macrophages have recently become attractive therapeutics in cancer immunotherapy. The potential of macrophages to infiltrate and influence solid malignancies makes them promising targets for the chimeric antigen receptor (CAR) technology to redirect their stage of polarization, thus enhancing their anticancer capacities. Given the emerging interest for CAR-macrophages, generation of such cells so far mainly depends on peripheral blood monocytes, which are isolated from the respective donor prior to genetic manipulation.

Clinical Implications and Dynamics of Clonal Hematopoiesis in Anti-CD19 CAR T-cell Treated Patients.

Recent evidence revealed important interactions between clonal hematopoiesis (CH) and cellular therapies established for the treatment of hematologic malignancies. The impact of CH on safety, efficacy, and outcome of chimeric antigen receptor (CAR) T-cell therapy is currently under investigation. We analyzed 110 patients with relapsed/refractory B-cell non-Hodgkin lymphoma (n = 105) or acute lymphoblastic leukemia (ALL) (n = 5), treated with Axicabtagene-Ciloleucel (39%), Tisagenlecleucel (51%), or Brexucabtagene autoleucel (10%).

Improved Activity against Acute Myeloid Leukemia with Chimeric Antigen Receptor (CAR)-NK-92 Cells Designed to Target CD123.

Anti-cancer activity can be improved by engineering immune cells to express chimeric antigen receptors (CARs) that recognize tumor-associated antigens. Retroviral vector gene transfer strategies allow stable and durable transgene expression. Here, we used alpharetroviral vectors to modify NK-92 cells, a natural killer cell line, with a third-generation CAR designed to target the IL-3 receptor subunit alpha (CD123), which is strongly expressed on the surface of acute myeloid leukemia (AML) cells.

Posttransplantation MRD monitoring in patients with AML by next-generation sequencing using DTA and non-DTA mutations.

Next-generation sequencing (NGS)-based measurable residual disease (MRD) monitoring in patients with acute myeloid leukemia (AML) is widely applicable and prognostic prior to allogeneic hematopoietic cell transplantation (alloHCT). We evaluated the prognostic role of clonal hematopoiesis-associated DNMT3A, TET2, and ASXL1 (DTA) and non-DTA mutations for MRD monitoring post-alloHCT to refine MRD marker selection. Of 154 patients with AML, 138 (90%) had at least one mutation at diagnosis, which were retrospectively monitored by amplicon-based error-corrected NGS on day 90 and/or day 180 post-alloHCT.

Targeted Inhibition of the NUP98-NSD1 Fusion Oncogene in Acute Myeloid Leukemia.

NUP98-NSD1-positive acute myeloid leukemia (AML) is a poor prognostic subgroup that is frequently diagnosed in pediatric cytogenetically normal AML. NUP98-NSD1-positive AML often carries additional mutations in genes including and . The purpose of our study was to characterize the cooperative potential of the fusion and its associated Neuroblastoma rat sarcoma (NRAS) mutation.

Effective drug treatment identified by in vivo screening in a transplantable patient-derived xenograft model of chronic myelomonocytic leukemia.

To establish novel and effective treatment combinations for chronic myelomonocytic leukemia (CMML) preclinically, we hypothesized that supplementation of CMML cells with the human oncogene Meningioma 1 (MN1) promotes expansion and serial transplantability in mice, while maintaining the functional dependencies of these cells on their original genetic profile. Using lentiviral expression of MN1 for oncogenic supplementation and transplanting transduced primary mononuclear CMML cells into immunocompromised mice, we established three serially transplantable CMML-PDX models with disease-related gene mutations that recapitulate the disease in vivo. Ectopic MN1 expression was confirmed to enhance the proliferation of CMML cells, which otherwise did not engraft upon secondary transplantation.

In vivo efficacy of mutant IDH1 inhibitor HMS-101 and structural resolution of distinct binding site.

Mutations in isocitrate dehydrogenase 1 (IDH1) are found in 6% of AML patients. Mutant IDH produces R-2-hydroxyglutarate (R-2HG), which induces histone- and DNA-hypermethylation through the inhibition of epigenetic regulators, thus linking metabolism to tumorigenesis. Here we report the biochemical characterization, in vivo antileukemic effects, structural binding, and molecular mechanism of the inhibitor HMS-101, which inhibits the enzymatic activity of mutant IDH1 (IDH1mut).

Molecular retargeting of antibodies converts immune defense against oncolytic viruses into cancer immunotherapy.

Virus-neutralizing antibodies are a severe obstacle in oncolytic virotherapy. Here, we present a strategy to convert this unfavorable immune response into an anticancer immunotherapy via molecular retargeting. Application of a bifunctional adapter harboring a tumor-specific ligand and the adenovirus hexon domain DE1 for engaging antiadenoviral antibodies, attenuates tumor growth and prolongs survival in adenovirus-immunized mice.

Preclinical Assessment of Suitable Natural Killer Cell Sources for Chimeric Antigen Receptor Natural Killer-Based "Off-the-Shelf" Acute Myeloid Leukemia Immunotherapies.

The introduction of chimeric antigen receptors (CARs) to augment the anticancer activity of immune cells represents one of the major clinical advances in recent years. This work demonstrates that sorted CAR natural killer (NK) cells have improved antileukemia activity compared to control NK cells that lack a functional CAR. However, in terms of viability, effectiveness, risk of side effects, and clinical practicality and applicability, an important question is whether gene-modified NK cell lines represent better CAR effector cells than primary human donor CAR-NK (CAR-dNK) cells.

Optimized induction of mitochondrial apoptosis for chemotherapy-free treatment of BCR-ABL+acute lymphoblastic leukemia.

BCR-ABL+acute lymphoblastic leukemia (ALL) in adults has a poor prognosis with allogeneic stem cell transplantation (SCT) considered the best curative option for suitable patients. We here characterize the curative potential of BH3-mimetics differentially targeting mitochondrial BCL2-family members using a combination therapy approach with dexamethasone and tyrosine kinase inhibitors targeting BCR-ABL. In BCR-ABL + ALL BH3-mimetics act by redistribution of mitochondrial activator BIM, which is strongly required for cytotoxicity of the BCL2-specific BH3-mimetic ABT-199, tyrosine kinase inhibitors (TKIs) and dexamethasone.

Measurable residual disease monitoring by NGS before allogeneic hematopoietic cell transplantation in AML.

Molecular measurable residual disease (MRD) assessment is not established in approximately 60% of acute myeloid leukemia (AML) patients because of the lack of suitable markers for quantitative real-time polymerase chain reaction. To overcome this limitation, we established an error-corrected next-generation sequencing (NGS) MRD approach that can be applied to any somatic gene mutation. The clinical significance of this approach was evaluated in 116 AML patients undergoing allogeneic hematopoietic cell transplantation (alloHCT) in complete morphologic remission (CR).

An optimized lentiviral vector system for conditional RNAi and efficient cloning of microRNA embedded short hairpin RNA libraries.

RNA interference (RNAi) and CRISPR-Cas9-based screening systems have emerged as powerful and complementary tools to unravel genetic dependencies through systematic gain- and loss-of-function studies. In recent years, a series of technical advances helped to enhance the performance of virally delivered RNAi. For instance, the incorporation of short hairpin RNAs (shRNAs) into endogenous microRNA contexts (shRNAmiRs) allows the use of Tet-regulated promoters for synchronous onset of gene knockdown and precise interrogation of gene dosage effects.

Administration of Gemcitabine After Pancreatic Tumor Resection in Mice Induces an Antitumor Immune Response Mediated by Natural Killer Cells.

Background & Aims: Even after potentially curative R0 resection, patients with pancreatic ductal adenocarcinoma (PDAC) have a poor prognosis owing to high rates of local recurrence and metastasis to distant organs. However, we have no suitable transgenic animal models for surgical interventions.

Methods: To induce formation of pancreatic tumor foci, we electroporated oncogenic plasmids into pancreata of LSL-KrasG12D × p53fl/fl mice; mutant Kras was expressed in p53fl/fl mice using a sleeping beauty transposon.

Retargeted oncolytic viruses provoke tumor-directed T-cell responses.

Intratumoral application of oncolytic viruses effectively induce tumor-directed immune responses. However, their systemic application is typically insufficient to stimulate the required extent of tumor tissue inflammation to elicit antitumor immunity. We recently discovered evidence that this barrier can be overcome by effective molecular retargeting of viral infection.

Viral Infection of Tumors Overcomes Resistance to PD-1-immunotherapy by Broadening Neoantigenome-directed T-cell Responses.

There is evidence that viral oncolysis is synergistic with immune checkpoint inhibition in cancer therapy but the underlying mechanisms are unclear. Here, we investigated whether local viral infection of malignant tumors is capable of overcoming systemic resistance to PD-1-immunotherapy by modulating the spectrum of tumor-directed CD8 T-cells. To focus on neoantigen-specific CD8 T-cell responses, we performed transcriptomic sequencing of PD-1-resistant CMT64 lung adenocarcinoma cells followed by algorithm-based neoepitope prediction.

PolySia-Specific Retargeting of Oncolytic Viruses Triggers Tumor-Specific Immune Responses and Facilitates Therapy of Disseminated Lung Cancer.

Polysialic acid (polySia) is expressed on several malignant tumors of neuroendocrine origin, including small cell lung cancer. In this study, we investigated the therapeutic efficacy of tumor-directed T-cell responses, elicited by polySia-retargeted oncolytic adenovirus infection, in an orthotopic murine model of disseminated polySia-positive lung cancer. In several cell lines, we demonstrated highly polySia-selective retargeting of adenoviral infection using a bispecific adapter comprising the ectodomain of the coxsackievirus/adenovirus receptor and a polySia-recognizing single-chain antibody domain.

Meganuclease-mediated virus self-cleavage facilitates tumor-specific virus replication.

Meganucleases can specifically cleave long DNA sequence motifs, a feature that makes them an ideal tool for gene engineering in living cells. In a proof-of-concept study, we investigated the use of the meganuclease I-Sce I for targeted virus self-disruption to generate high-specific oncolytic viruses. For this purpose, we provided oncolytic adenoviruses with a molecular circuit that selectively responds to p53 activation by expression of I-Sce I subsequently leading to self-disruption of the viral DNA via heterologous I-Sce I recognition sites within the virus genome.

Adjuvant gemcitabine therapy improves survival in a locally induced, R0-resectable model of metastatic intrahepatic cholangiocarcinoma.

Unlabelled: Complete surgical tumor resection (R0) for treatment of intrahepatic cholangiocarcinoma (ICC) is potentially curative, but the prognosis remains dismal due to frequent tumor recurrence and metastasis after surgery. Adjuvant therapies may improve the outcome, but clinical studies for an adjuvant approach are difficult and time-consuming for rare tumor entities. Therefore, animal models reflecting the clinical situation are urgently needed to investigate novel adjuvant therapies.

Virus-induced tumor inflammation facilitates effective DC cancer immunotherapy in a Treg-dependent manner in mice.

Vaccination using DCs pulsed with tumor lysates or specific tumor-associated peptides has so far yielded limited clinical success for cancer treatment, due mainly to the low immunogenicity of tumor-associated antigens. In this study, we have identified intratumoral virus-induced inflammation as a precondition for effective antitumor DC vaccination in mice. Administration of a tumor-targeted DC vaccine during ongoing virus-induced tumor inflammation, a regimen referred to as oncolysis-assisted DC vaccination (ODC), elicited potent antitumoral CD8+ T cell responses.

Selectivity of oncolytic viral replication prevents antiviral immune response and toxicity, but does not improve antitumoral immunity.

Oncolytic infection elicits antitumoral immunity, but the impact of tumor-selective replication on the balance between antiviral and antitumoral immune responses has not yet been investigated. To address this question, we constructed the highly tumor-selective adenovirus Ad-p53T whose replication in target tumor cells is governed by aberrant telomerase activity and transcriptional p53 dysfunction. Telomerase-dependent or nonselective adenoviruses were constructed as isogenic controls.

Antitumoural immunity by virus-mediated immunogenic apoptosis inhibits metastatic growth of hepatocellular carcinoma.

Background And Aims: Viral infection of a dying cell dictates the immune response against intracellular antigens, suggesting that virotherapy may be an effective tool to induce immunogenic cell death during systemic cancer treatment. Since viruses and proteasome inhibitors both induce accumulation of misfolded proteins, endoplasmic reticulum (ER) stress and immune responses during treatment of hepatocellular carcinoma (HCC) with bortezomib and the tumour-specifically replicating virus hTert-Ad (human telomerase reverse transcriptase promoter-regulated adenovirus) were investigated.

Methods: Unfolded protein response (UPR) pathways and ER stress-mediated apoptosis were investigated by western blots, caspase-3 assays, 4',6-diamidino-2-phenylindole (DAPI) and Annexin V staining in HCC cells following hTert-Ad/bortezomib treatment.