NK Cell-Mediated Eradication of Ovarian Cancer Cells with a Novel Chimeric Antigen Receptor Directed against CD44.

Ovarian cancer is the most common cause of gynecological cancer-related death in the developed world. Disease recurrence and chemoresistance are major causes of poor survival rates in ovarian cancer patients. Ovarian cancer stem cells (CSCs) were shown to represent a source of tumor recurrence owing to the high resistance to chemotherapy and enhanced tumorigenicity. Chimeric antigen receptor (CAR)-based adoptive immunotherapy represents a promising strategy to reduce the risk for recurrent disease. In this study, we developed a codon-optimized third-generation CAR to specifically target CD44, a marker widely expressed on ovarian cancer cells and associated with CSC-like properties and intraperitoneal tumor spread. We equipped NK-92 cells with the anti-CD44 CAR (CD44NK) and an anti-CD19 control CAR (CD19NK) using lentiviral SIN vectors. Compared to CD19NK and untransduced NK-92 cells, CD44NK showed potent and specific cytotoxic activity against CD44-positive ovarian cancer cell lines (SKOV3 and OVCAR3) and primary ovarian cancer cells harvested from ascites. In contrast, CD44NK had less cytotoxic activity against CD44-negative A2780 cells. Specific activation of engineered NK cells was also demonstrated by interferon-γ (IFNγ) secretion assays. Furthermore, CD44NK cells still demonstrated cytotoxic activity under cisplatin treatment. Most importantly, the simultaneous treatment with CD44NK and cisplatin showed higher anti-tumor activity than sequential treatment.

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. Alpharetroviral vectors also contained a transgene cassette to allow constitutive expression of human IL-15 for increased NK cell persistence in vivo. The anti-AML activity of CAR-NK-92 cells was tested via in vitro cytotoxicity assays with the CD123+ AML cell line KG-1a and in vivo in a patient-derived xenotransplantation CD123+ AML model. Unmodified NK-92 cells or NK-92 cells modified with a truncated version of the CAR that lacked the signaling domain served as controls. Alpharetroviral vector-modified NK-92 cells stably expressed the transgenes and secreted IL-15. Anti-CD123-CAR-NK-92 cells exhibited enhanced anti-AML activity in vitro and in vivo as compared to control NK-92 cells. Our data (1) shows the importance of IL-15 expression for in vivo persistence of NK-92 cells, (2) supports continued investigation of anti-CD123-CAR-NK cells to target AML, and (3) points towards potential strategies to further improve CAR-NK anti-AML activity.

Prognostic role of docetaxel-induced suppression of free testosterone serum levels in metastatic prostate cancer patients.

To date, only few data concerning the biologically active, free form of testosterone (FT) are available in metastatic prostate cancer (mPC) and the impact of FT on disease, therapy and outcome is largely unknown. We retrospectively studied the effect of docetaxel on FT and total testosterone (TT) serum levels in 67 mPC patients monitored between April 2008 and November 2020. FT and TT levels were measured before and weekly during therapy. The primary endpoint was overall survival (OS). Secondary endpoints were prostate-specific antigen response and radiographic response (PSAR, RR), progression-free survival (PFS), FT/TT levels and safety. Median FT and TT serum levels were completely suppressed to below the detection limit during docetaxel treatment (FT: from 0.32 to < 0.18 pg/mL and TT: from 0.12 to < 0.05 ng/mL, respectively). Multivariate Cox regression analyses identified requirement of non-narcotics, PSAR, complete FT suppression and FT nadir values < 0.18 pg/mL as independent parameters for PFS. Prior androgen-receptor targeted therapy (ART), soft tissue metastasis and complete FT suppression were independent prognostic factors for OS. FT was not predictive for treatment outcome in mPC patients with a history of ART.

Retroviral gene therapy in Germany with a view on previous experience and future perspectives.

Gene therapy can be used to restore cell function in monogenic disorders or to endow cells with new capabilities, such as improved killing of cancer cells, expression of suicide genes for controlled elimination of cell populations, or protection against chemotherapy or viral infection. While gene therapies were originally most often used to treat monogenic diseases and to improve hematopoietic stem cell transplantation outcome, the advent of genetically modified immune cell therapies, such as chimeric antigen receptor modified T cells, has contributed to the increased numbers of patients treated with gene and cell therapies. The advancement of gene therapy with integrating retroviral vectors continues to depend upon world-wide efforts. As the topic of this special issue is "Spotlight on Germany," the goal of this review is to provide an overview of contributions to this field made by German clinical and research institutions. Research groups in Germany made, and continue to make, important contributions to the development of gene therapy, including design of vectors and transduction protocols for improved cell modification, methods to assess gene therapy vector efficacy and safety (e.g., clonal imbalance, insertion sites), as well as in the design and conduction of clinical gene therapy trials.

Conditionally immortalised leukaemia initiating cells co-expressing Hoxa9/Meis1 demonstrate microenvironmental adaptation properties ex vivo while maintaining myelomonocytic memory.

Regulation of haematopoietic stem cell fate through conditional gene expression could improve understanding of healthy haematopoietic and leukaemia initiating cell (LIC) biology. We established conditionally immortalised myeloid progenitor cell lines co-expressing constitutive Hoxa9.EGFP and inducible Meis1.dTomato (H9M-ciMP) to study growth behaviour, immunophenotype and morphology under different cytokine/microenvironmental conditions ex vivo upon doxycycline (DOX) induction or removal. The vector design and drug-dependent selection approach identified new retroviral insertion (RVI) sites that potentially collaborate with Meis1/Hoxa9 and define H9M-ciMP fate. For most cell lines, myelomonocytic conditions supported reversible H9M-ciMP differentiation into neutrophils and macrophages with DOX-dependent modulation of Hoxa9/Meis1 and CD11b/Gr-1 expression. Here, up-regulation of Meis1/Hoxa9 promoted reconstitution of exponential expansion of immature H9M-ciMPs after DOX reapplication. Stem cell maintaining conditions supported selective H9M-ciMP exponential growth. H9M-ciMPs that had Ninj2 RVI and were cultured under myelomonocytic or stem cell maintaining conditions revealed the development of DOX-dependent acute myeloid leukaemia in a murine transplantation model. Transcriptional dysregulation of Ninj2 and distal genes surrounding RVI (Rad52, Kdm5a) was detected. All studied H9M-ciMPs demonstrated adaptation to T-lymphoid microenvironmental conditions while maintaining immature myelomonocytic features. Thus, the established system is relevant to leukaemia and stem cell biology.

Use of Cell and Genome Modification Technologies to Generate Improved "Off-the-Shelf" CAR T and CAR NK Cells.

The broad success of adoptive immunotherapy to treat human cancer has resulted in a paradigm shift in modern medicine. Modification of autologous and allogenic immune cells with chimeric antigen receptors (CAR) designed to target specific antigens on tumor cells has led to production of CAR T and CAR NK cell therapies, which are ever more commonly introduced into cancer patient treatment protocols. While allogenic T cells may offer advantages such as improved anti-tumor activity, they also carry the risk of adverse reactions like graft-versus-host disease. This risk can be mitigated by use of autologous immune cells, however, the time needed for T and/or NK cell isolation, modification and expansion may be too long for some patients. Thus, there is an urgent need for strategies to robustly produce "off-the-shelf" CAR T and CAR NK cells, which could be used as a bridging therapy between cancer diagnosis or relapse and allogeneic transplantation. Advances in genome modification technologies have accelerated the generation of designer cell therapy products, including development of "off-the-shelf" CAR T cells for cancer immunotherapy. The feasibility and safety of such approaches is currently tested in clinical trials. This review will describe cell sources for CAR-based therapies, provide background of current genome editing techniques and the applicability of these approaches for generation of universal "off-the-shelf" CAR T and NK cell therapeutics.

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. Comparison of the functional activities of sorted CAR-NK cells generated using the NK-92 cell line with those generated from primary human dNK cells demonstrated that CAR-NK-92 cells had stronger cytotoxic activity against leukemia cells compared to CAR-dNK cells. CAR-NK-92 and CAR-dNK cells had similar CD107a surface expression upon co-incubation with leukemia cells. However, CAR-NK-92 cells secreted higher granzyme A and interleukin-17A levels, while CAR-dNK cells secreted more tumor necrosis factor alpha, interferon gamma, and granulysin. In addition, CAR-NK-92 cells revealed a significantly higher potential for adverse side effects against nonmalignant cells. In short, this work shows the feasibility for further development of CAR-NK strategies to treat leukemia.

Engineering CAR-T Cells for Improved Function Against Solid Tumors.

Genetic engineering T cells to create clinically applied chimeric antigen receptor (CAR) T cells has led to improved patient outcomes for some forms of hematopoietic malignancies. While this has inspired the biomedical community to develop similar strategies to treat solid tumor patients, challenges such as the immunosuppressive character of the tumor microenvironment, CAR-T cell persistence and trafficking to the tumor seem to limit CAR-T cell efficacy in solid cancers. This review provides an overview of mechanisms that tumors exploit to evade eradication by CAR-T cells as well as emerging approaches that incorporate genetic engineering technologies to improve CAR-T cell activity against solid tumors.

Human Teratoma-Derived Hematopoiesis Is a Highly Polyclonal Process Supported by Human Umbilical Vein Endothelial Cells.

Hematopoietic stem cells (HSCs) ensure a life-long regeneration of the blood system and are therefore an important source for transplantation and gene therapy. The teratoma environment supports the complex development of functional HSCs from human pluripotent stem cells, which is difficult to recapitulate in culture. This model mimics various aspects of early hematopoiesis, but is restricted by the low spontaneous hematopoiesis rate. In this study, a feasible protocol for robust hematopoiesis has been elaborated. We achieved a significant increase of the teratoma-derived hematopoietic population when teratomas were generated in the NSGS mouse, which provides human cytokines, together with co-injection of human umbilical vein endothelial cells. Since little is known about hematopoiesis in teratomas, we addressed localization and clonality of the hematopoietic lineage. Our results indicate that early human hematopoiesis is closely reflected in teratoma formation, and thus highlight the value of this model.

Chimeric Antigen Receptor T Cells: Extending Translation from Liquid to Solid Tumors.

Successful translation of chimeric antigen receptor (CAR) T cells designed to target and eradicate CD19+ lymphomas has emboldened scientists and physicians worldwide to explore the possibility of applying CAR T-cell technology to other tumor entities, including solid tumors. Next-generation strategies such as fourth-generation CARs (CAR T cells redirected for universal cytokine killing, also known as TRUCKs) designed to deliver immunomodulatory cytokines to the tumor microenvironment, dual CAR designs to improve tumor control, inclusion of suicide genes as safety switches, and precision genome editing are currently being investigated. One major ongoing goal is to determine how best to generate CAR T cells that modulate the tumor microenvironment, overcome tumor survival mechanisms, and thus allow broader applicability as universal allogeneic T-cell therapeutics. Development of state-of-the-art and beyond viral vector systems to deliver designer CARs coupled with targeted genome editing is expected to generate more effective off-the-shelf CAR T cells with activity against a greater number of cancer types and importantly solid tumors.

Uncoupling the Oncogenic Engine.

Inhibition of oncogenic signaling and correction of aberrant metabolic processes may be key paradigms to eliminate cancer cells. The high incidence of activating RAS mutations and hyperactivated ERK1/2 signaling observed in many human tumors and the lack of effective targeted therapies to elicit long-term inhibition of the RAS-ERK1/2 signaling pathway add to the importance of discovering novel strategies to treat malignancies characterized by elevated RAS-ERK1/2 signaling. In this review, we describe connections between oncogenic signaling and cancer cell metabolism and how these links may be exploited for novel modern molecular medicine approaches. Cancer Res; 77(22); 6060-4. ©2017 AACR.

Whole gut microbiome composition of damselfish and cardinalfish before and after reef settlement.

The Pomacentridae (damselfish) and Apogonidae (cardinalfish) are among the most common fish families on coral reefs and in the aquarium trade. Members of both families undergo a pelagic larvae phase prior to settlement on the reef, where adults play key roles in benthic habitat structuring and trophic interactions. Fish-associated microbial communities (microbiomes) significantly influence fish health and ecology, yet little is known of how microbiomes change with life stage. We quantified the taxonomic (16S rRNA gene) composition of whole gut microbiomes from ten species of damselfish and two species of cardinalfish from Lizard Island, Australia, focusing specifically on comparisons between pelagic larvae prior to settlement on the reef versus post-settlement juvenile and adult individuals. On average, microbiome phylogenetic diversity increased from pre- to post-settlement, and was unrelated to the microbial composition in the surrounding water column. However, this trend varied among species, suggesting stochasticity in fish microbiome assembly. Pre-settlement fish were enriched with bacteria of the Endozoicomonaceae, Shewanellaceae, and Fusobacteriaceae, whereas settled fish harbored higher abundances of Vibrionaceae and Pasteurellaceae. Several individual operational taxonomic units, including ones related to Vibrio harveyi, Shewanella sp., and uncultured Endozoicomonas bacteria, were shared between both pre and post-settlement stages and may be of central importance in the intestinal niche across development. Richness of the core microbiome shared among pre-settlement fish was comparable to that of settled individuals, suggesting that changes in diversity with adulthood are due to the acquisition or loss of host-specific microbes. These results identify a key transition in microbiome structure across host life stage, suggesting changes in the functional contribution of microbiomes over development in two ecologically dominant reef fish families.

Efficient generation of gene-modified human natural killer cells via alpharetroviral vectors.

UNLABELLED: Natural killer (NK) cells play an important role in tumor immunotherapy with their unique capability of killing transformed cells without the need for prior sensitization and without major histocompatibility complex (MHC)/peptide restriction. However, tumor cells can escape NK cell cytotoxicity by various tumor immune escape mechanisms. To overcome these escape mechanisms, NK cells can be modified to express chimeric antigen receptors (CARs), enhancing their tumor-specific cytotoxicity. To determine the most efficacious method to modify human NK cells, we compared different retroviral vector systems, retroviral pseudotypes, and transduction protocols. Using optimized transduction conditions, the highest transduction levels (up to 60%) were achieved with alpharetroviral vectors. Alpharetroviral-modified primary human NK cells exhibited no alteration in receptor expression and had similar degranulation activity as untransduced NK cells, thus demonstrating that alpharetroviral modification did not negatively affect NK cell cytotoxicity. Transduction of NK cells with an alpharetroviral vector containing a CD19 CAR expression cassette selectively enhanced NK cell cytotoxicity towards CD19-expressing leukemia cells, achieving nearly complete elimination of leukemia cells after 48 h. Taken together, alpharetroviral vectors are promising tools for NK cell-mediated cancer immunotherapy applications. KEY MESSAGES: Efficient modification of human NK cells using alpharetroviral vectors. Anti-CD19-CAR-NK cells exhibited improved cytotoxicity towards CD19(+) leukemia cells. Alpharetroviral vectors are promising tools for immunotherapy applications using NK cells.

Cetuximab Reconstitutes Pro-Inflammatory Cytokine Secretions and Tumor-Infiltrating Capabilities of sMICA-Inhibited NK Cells in HNSCC Tumor Spheroids.

Immunosuppressive factors, such as soluble major histocompatibility complex class I chain-related peptide A (sMICA) and transforming growth factor beta 1 (TGF-ß1), are involved in tumor immune escape mechanisms (TIEMs) exhibited by head and neck squamous cell carcinomas (HNSCCs) and may represent opportunities for therapeutic intervention. In order to overcome TIEMs, we investigated the antibody-dependent cellular cytotoxicity (ADCC), cytokine release and retargeted tumor infiltration of sMICA-inhibited patient NK cells expressing Fcγ receptor IIIa (FcγRIIIa, CD16a) in the presence of cetuximab, an anti-epidermal growth factor receptor (HER1) monoclonal antibody (mAb). Compared to healthy controls, relapsed HNSCC patients (n = 5), not currently in treatment revealed decreased levels of circulating regulatory NK cell subsets in relation to increased cytotoxic NK cell subpopulations. Elevated sMICA and TGF-ß1 plasma levels correlated with diminished TNFα and IFN-γ release and decreased NKG2D (natural killer group 2 member D)-dependent killing of HNSCC cells by NK cells. Incubation of IL-2-activated patient NK cells with patient plasma containing elevated sMICA or sMICA analogs (shed MICA and recombinant MICA) significantly impaired NKG2D-mediated killing by down-regulation of NKG2D surface expression. Of note, CD16 surface expression levels, pro-apoptotic and activation markers, and viability of patient and healthy donor NK cell subpopulations were not affected by this treatment. Accordingly, cetuximab restored killing activity of sMICA-inhibited patient NK cells against cetuximab-coated primary HNSCC cells via ADCC in a dose-dependent manner. Rapid reconstitution of anti-tumor recognition and enhanced tumor infiltration of treated NK cells was monitored by 24 h co-incubation of HNSCC tumor spheroids with cetuximab (1 µg/ml) and was characterized by increased IFN-γ and TNFα secretion. This data show that the impaired NK cell-dependent tumor surveillance in relapsed HNSCC patients could be reversed by the re-establishment of ADCC-mediated effector cell activity, thus supporting NK cell-based immunotherapy in combination with antineoplastic monoclonal mAbs.

Lack of noncanonical RAS mutations in cytogenetically normal acute myeloid leukemia.

Transforming mutations in RAS genes are commonly found in human malignancies, including myeloid leukemias. To investigate the incidence, spectrum, and distribution of activating K- and N-RAS mutations in cytogenetically normal acute myeloid leukemia (CN-AML) patients, 204 CN-AML patients were screened. Activating K- and N-RAS mutations were detected in 3 of 204 (1.5 %) and 22 of 204 (10.8 %) CN-AML samples, respectively. RAS mutated patients presented with a lower percentage of bone marrow blasts (65 vs 80 %, P = 0.022). RAS mutations tended to occur with nucleophosmin-1 (NPM1) mutations (P = 0.079), and all three samples containing K-RAS mutations had concomitant NPM1 mutations. There was no significant overlap between K-RAS mutations and N-RAS, FLT3, CEBPA, IDH1/2, WT1 or MLL mutations. RAS mutation status did not impact relapse-free or overall survival of CN-AML patients. In contrast to reports of noncanonical RAS mutations in other cancers, including some leukemia subtypes, we only observed K- and N-RAS mutations in codons 12, 13, or 61 in CN-AML samples. Our findings suggest that while K-RAS mutations are infrequent in CN-AML, activating K-RAS mutations may cooperate with mutated NPM1 to induce leukemia.