Development of KoRV-pseudotyped lentiviral vectors for efficient gene transfer into freshly isolated immune cells.

Allogeneic cell therapies, such as those involving macrophages or Natural Killer (NK) cells, are of increasing interest for cancer immunotherapy. However, the current techniques for genetically modifying these cell types using lenti- or gamma-retroviral vectors present challenges, such as required cell pre-activation and inefficiency in transduction, which hinder the assessment of preclinical efficacy and clinical translation. In our study, we describe a novel lentiviral pseudotype based on the Koala Retrovirus (KoRV) envelope protein, which we identified based on homology to existing pseudotypes used in cell therapy. Unlike other pseudotyped viral vectors, this KoRV-based envelope demonstrates remarkable efficiency in transducing freshly isolated primary human NK cells directly from blood, as well as freshly obtained monocytes, which were differentiated to M1 macrophages as well as B cells from multiple donors, achieving up to 80% reporter gene expression within three days post-transduction. Importantly, KoRV-based transduction does not compromise the expression of crucial immune cell receptors, nor does it impair immune cell functionality, including NK cell viability, proliferation, cytotoxicity as well as phagocytosis of differentiated macrophages. Preserving immune cell functionality is pivotal for the success of cell-based therapeutics in treating various malignancies. By achieving high transduction rates of freshly isolated immune cells before expansion, our approach enables a streamlined and cost-effective automated production of off-the-shelf cell therapeutics, requiring fewer viral particles and less manufacturing steps. This breakthrough holds the potential to significantly reduce the time and resources required for producing e.g. NK cell therapeutics, expediting their availability to patients in need.

CD44v6 specific CAR-NK cells for targeted immunotherapy of head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a major challenge for current therapies. CAR-T cells have shown promising results in blood cancers, however, their effectiveness against solid tumors remains a hurdle. Recently, CD44v6-directed CAR-T cells demonstrated efficacy in controlling tumor growth in multiple myeloma and solid tumors such as HNSCC, lung and ovarian adenocarcinomas. Apart from CAR-T cells, CAR-NK cells offer a safe and allogenic alternative to autologous CAR-T cell therapy. In this paper, we investigated the capacity of CAR-NK cells redirected against CD44v6 to execute cytotoxicity against HNSCC. Anti-CD44v6 CAR-NK cells were generated from healthy donor peripheral blood-derived NK cells using gamma retroviral vectors (gRVs). The NK cell transduction was optimized by exploring virus envelope proteins derived from the baboon endogenous virus envelope (BaEV), feline leukemia virus (FeLV, termed RD114-TR) and gibbon ape leukemia virus (GaLV), respectively. BaEV pseudotyped gRVs induced the highest transduction rate compared to RD114-TR and GaLV envelopes as measured by EGFP and surface CAR expression of transduced NK cells. CAR-NK cells showed a two- to threefold increase in killing efficacy against various HNSCC cell lines compared to unmodified, cytokine-expanded primary NK cells. Anti-CD44v6 CAR-NK cells were effective in eliminating tumor cell lines with high and low CD44v6 expression levels. Overall, the improved cytotoxicity of CAR-NK cells holds promise for a therapeutic option for the treatment of HNSCC. However, further preclinical trials are necessary to test in vivo efficacy and safety, as well to optimize the treatment regimen of anti-CD44v6 CAR-NK cells against solid tumors.

Optimized peptide nanofibrils as efficient transduction enhancers for in vitro and ex vivo gene transfer.

Chimeric antigen receptor (CAR)-T cell therapy is a groundbreaking immunotherapy for cancer. However, the intricate and costly manufacturing process remains a hurdle. Improving the transduction rate is a potential avenue to cut down costs and boost therapeutic efficiency. Peptide nanofibrils (PNFs) serve as one such class of transduction enhancers. PNFs bind to negatively charged virions, facilitating their active engagement by cellular protrusions, which enhances virion attachment to cells, leading to increased cellular entry and gene transfer rates. While first-generation PNFs had issues with aggregate formation and potential immunogenicity, our study utilized in silico screening to identify short, endogenous, and non-immunogenic peptides capable of enhancing transduction. This led to the discovery of an 8-mer peptide, RM-8, which forms PNFs that effectively boost T cell transduction rates by various retroviral vectors. A subsequent structure-activity relationship (SAR) analysis refined RM-8, resulting in the D4 derivative. D4 peptide is stable and assembles into smaller PNFs, avoiding large aggregate formation, and demonstrates superior transduction rates in primary T and NK cells. In essence, D4 PNFs present an economical and straightforward nanotechnological tool, ideal for refining ex vivo gene transfer in CAR-T cell production and potentially other advanced therapeutic applications.

Efficient Redirection of NK Cells by Genetic Modification with Chemokine Receptors CCR4 and CCR2B.

Natural killer (NK) cells are a subset of lymphocytes that offer great potential for cancer immunotherapy due to their natural anti-tumor activity and the possibility to safely transplant cells from healthy donors to patients in a clinical setting. However, the efficacy of cell-based immunotherapies using both T and NK cells is often limited by a poor infiltration of immune cells into solid tumors. Importantly, regulatory immune cell subsets are frequently recruited to tumor sites. In this study, we overexpressed two chemokine receptors, CCR4 and CCR2B, that are naturally found on T regulatory cells and tumor-resident monocytes, respectively, on NK cells. Using the NK cell line NK-92 as well as primary NK cells from peripheral blood, we show that genetically engineered NK cells can be efficiently redirected using chemokine receptors from different immune cell lineages and migrate towards chemokines such as CCL22 or CCL2, without impairing the natural effector functions. This approach has the potential to enhance the therapeutic effect of immunotherapies in solid tumors by directing genetically engineered donor NK cells to tumor sites. As a future therapeutic option, the natural anti-tumor activity of NK cells at the tumor sites can be increased by co-expression of chemokine receptors with chimeric antigen receptors (CAR) or T cell receptors (TCR) on NK cells can be performed in the future.

Taking Lessons from CAR-T Cells and Going Beyond: Tailoring Design and Signaling for CAR-NK Cells in Cancer Therapy.

Cancer immunotherapies utilize the capabilities of the immune system to efficiently target malignant cells. In recent years, chimeric antigen receptor (CAR) equipped T cells showed promising results against B cell lymphomas. Autologous CAR-T cells require patient-specific manufacturing and thus extensive production facilities, resulting in high priced therapies. Along with potentially severe side effects, these are the major drawbacks of CAR-T cells therapies. Natural Killer (NK) cells pose an alternative for CAR equipped immune cells. Since NK cells can be safely transferred from healthy donors to cancer patients, they present a suitable platform for an allogeneic "off-the-shelf" immunotherapy. However, administration of activated NK cells in cancer therapy has until now shown poor anti-cancer responses, especially in solid tumors. Genetic modifications such as CARs promise to enhance recognition of tumor cells, thereby increasing anti-tumor effects and improving clinical efficacy. Although the cell biology of T and NK cells deviates in many aspects, the development of CAR-NK cells frequently follows within the footsteps of CAR-T cells, meaning that T cell technologies are simply adopted to NK cells. In this review, we underline the unique properties of NK cells and their potential in CAR therapies. First, we summarize the characteristics of NK cell biology with a focus on signaling, a fine-tuned interaction of activating and inhibitory receptors. We then discuss why tailored NK cell-specific CAR designs promise superior efficacy compared to designs developed for T cells. We summarize current findings and developments in the CAR-NK landscape: different CAR formats and modifications to optimize signaling, to target a broader pool of antigens or to increase in vivo persistence. Finally, we address challenges beyond NK cell engineering, including expansion and manufacturing, that need to be addressed to pave the way for CAR-NK therapies from the bench to the clinics.

Role of HLA-G in Viral Infections.

The human leukocyte antigen (HLA)-G is a non-classical HLA class I molecule, which has distinct features to classical HLA-A, -B, -C antigens, such as a low polymorphism, different splice variants, highly restricted, tightly regulated expression and immune modulatory properties. HLA-G expression in tumor cells and virus-infected cells, as well as the release of soluble HLA-G leads to escape from host immune surveillance. Increased knowledge of the link between HLA-G expression, viral infection and disease progression is urgently required, which highlights the possible use of HLA-G as novel diagnostic and prognostic biomarker for viral infections, but also as therapeutic target. Therefore, this review aims to summarize the expression, regulation, function and impact of HLA-G in the context of different viral infections including virus-associated cancers. The characterization of HLA-G-driven immune escape mechanisms involved in the interactions between host cells and viruses might result in the design of novel immunotherapeutic strategies targeting HLA-G and/or its interaction with its receptors on immune effector cells.

The germinal center reaction depends on RNA methylation and divergent functions of specific methyl readers.

Long-lasting immunity depends on the generation of protective antibodies through the germinal center (GC) reaction. N6-methyladenosine (m6A) modification of mRNAs by METTL3 activity modulates transcript lifetime primarily through the function of m6A readers; however, the physiological role of this molecular machinery in the GC remains unknown. Here, we show that m6A modifications by METTL3 are required for GC maintenance through the differential functions of m6A readers. Mettl3-deficient GC B cells exhibited reduced cell-cycle progression and decreased expression of proliferation- and oxidative phosphorylation-related genes. The m6A binder, IGF2BP3, was required for stabilization of Myc mRNA and expression of its target genes, whereas the m6A reader, YTHDF2, indirectly regulated the expression of the oxidative phosphorylation gene program. Our findings demonstrate how two independent gene networks that support critical GC functions are modulated by m6A through distinct mRNA binders.

Human Metapneumovirus Escapes NK Cell Recognition through the Downregulation of Stress-Induced Ligands for NKG2D.

The Pneumoviridae family includes human metapneumovirus (HMPV) and human orthopneumovirus, which is also known as a respiratory syncytial virus (HRSV). These are large enveloped, negative single-strand RNA viruses. HMPV and HRSV are the human members, which commonly infect children. HMPV, which was discovered in 2001, infects most children until the age of five, which causes an influenza-like illness. The interaction of this virus with immune cells is poorly understood. In this study, we show that HMPV evades natural killer (NK) cell attack by downregulating stress-induced ligands for the activating receptor NKG2D including: Major histocompatibility complex (MHC) class I polypeptide-related sequences A and B (MICA, MICB), UL16 binding proteins ULBP2, and ULBP3, but not ULBP1. Mechanistically, we show that the viral protein G is involved in the downregulation of ULBP2 and that the viral protein M2.2 is required for MICA and MICB downregulation. These findings emphasize the importance of NK cells, in general, and NKG2D, in particular, in controlling HMPV infection, which opens new avenues for treating HMPV.

Activation of Siglec-7 results in inhibition of in vitro and in vivo growth of human mast cell leukemia cells.

Advanced systemic mastocytosis is a rare and still untreatable disease. Blocking antibodies against inhibitory receptors, also known as "immune checkpoints", have revolutionized anti-cancer treatment. Inhibitory receptors are expressed not only on normal immune cells, including mast cells but also on neoplastic cells. Whether activation of inhibitory receptors through monoclonal antibodies can lead to tumor growth inhibition remains mostly unknown. Here we show that the inhibitory receptor Siglec-7 is expressed by primary neoplastic mast cells in patients with systemic mastocytosis and by mast cell leukemia cell lines. Activation of Siglec-7 by anti-Siglec-7 monoclonal antibody caused phosphorylation of Src homology region 2 domain-containing phosphatase-1 (SHP-1), reduced phosphorylation of KIT and induced growth inhibition in mast cell lines. In SCID-beige mice injected with either the human mast cell line HMC-1.1 and HMC-1.2 or with Siglec-7 transduced B cell lymphoma cells, anti-Siglec-7 monoclonal antibody reduced tumor growth by a mechanism involving Siglec-7 cytoplasmic domains in "preventive" and "treatment" settings. These data demonstrate that activation of Siglec-7 on mast cell lines can inhibit their growth in vitro and in vivo. This might pave the way to additional treatment strategies for mastocytosis.

NKG2D Ligands-Critical Targets for Cancer Immune Escape and Therapy.

DNA damage, oncogene activation and excessive proliferation, chromatin modulations or oxidative stress are all important hallmarks of cancer. Interestingly, all of these abnormalities also induce a cellular stress response. By upregulating "stress-induced ligands," damaged or transformed cells can be recognized by immune cells and cleared. The human genome encodes eight functional "stress-induced ligands": MICA, MICB, and ULBP1-6. All of them are recognized by a single receptor, NKG2D, which is expressed on natural killer (NK) cells, cytotoxic T cells and other T cell subsets. The NKG2D ligand/NKG2D-axis is well-recognized as an important mediator of anti-tumor activity; however, patient data about the role of NKG2D ligands in immune surveillance and escape appears conflicting. As these ligands are often actively transcribed, tumor cells are urged to manipulate the expression of these ligands on post-transcriptional or post-translational level. Although our knowledge on the regulation of NKG2D ligand expression remains fragmentary, research of the past years revealed multiple cellular mechanisms that are adopted by tumor cells to reduce the expression of "stress-induced ligands" and therefore escape immune recognition. Here, we review the post-transcriptional and post-translational mechanisms by which NKG2D ligands are modulated in cancer cells and their impact on patient prognosis.We discuss controversies and approaches to apply our understanding of the NKG2D ligand/NKG2D-axis for cancer therapy.

Human cytomegalovirus escapes immune recognition by NK cells through the downregulation of B7-H6 by the viral genes US18 and US20.

Human cytomegalovirus (HCMV) is a major human pathogen, causing serious diseases in immunocompromised populations and congenially infected neonates. One of the main immune cells acting against the virus are Natural Killer (NK) cells. Killing by NK cells is mediated by a small family of activating receptors such as NKp30 that interact with the cellular ligand B7-H6. The outcome of B7-H6-NKp30 interaction was, so far, mainly studied with regard to NK recognition and killing of tumors. Here, we demonstrated that the expression of B7-H6 is upregulated following HCMV infection and that HCMV uses two of its genes: US18 and US20, to interfere with B7-H6 surface expression, in a mechanism involving endosomal degradation, in order to evade NK cell recognition.

Disarming Cellular Alarm Systems-Manipulation of Stress-Induced NKG2D Ligands by Human Herpesviruses.

The coevolution of viruses and their hosts led to the repeated emergence of cellular alert signals and viral strategies to counteract them. The herpesvirus family of viruses displays the most sophisticated repertoire of immune escape mechanisms enabling infected cells to evade immune recognition and thereby maintain infection. The herpesvirus family consists of nine viruses that are capable of infecting humans: herpes simplex virus 1 and 2 (HSV-1, HSV-2), varicella zoster virus (VZV), Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), roseoloviruses (HHV-6A, HHV-6B, and HHV-7), and Kaposi's-sarcoma-associated herpesvirus (KSHV). Most of these viruses are highly prevalent and infect a vast majority of the human population worldwide. Notably, research over the past 15 years has revealed that cellular ligands for the activating receptor natural-killer group 2, member D (NKG2D)-which is primarily expressed on natural killer (NK) cells-are common targets suppressed during viral infection, i.e., their surface expression is reduced in virtually all lytic herpesvirus infections by diverse mechanisms. Here, we review the viral mechanisms by which all herpesviruses known to date to downmodulate the expression of the NKG2D ligands. Also, in light of recent findings, we speculate about the importance of the emergence of eight different NKG2D ligands in humans and further allelic diversification during host and virus coevolution.

Vigilin Regulates the Expression of the Stress-Induced Ligand MICB by Interacting with Its 5' Untranslated Region.

NK cells are part of the innate immune system, and are able to identify and kill hazardous cells. The discrimination between normal and hazardous cells is possible due to an array of inhibitory and activating receptors. NKG2D is one of the prominent activating receptors expressed by all human NK cells. This receptor binds stress-induced ligands, including human MICA, MICB, and UL16-binding proteins 1-6. The interaction between NKG2D and its ligands facilitates the elimination of cells under cellular stress, such as tumor transformation. However, the mechanisms regulating the expression of these ligands are still not well understood. Under normal conditions, the NKG2D ligands were shown to be posttranscriptionally regulated by cellular microRNAs and RNA-binding proteins (RBPs). Thus far, only the 3' untranslated regions (UTRs) of MICA, MICB, and UL16-binding protein 2 were shown to be regulated by RBPs and microRNAs, usually resulting in their downregulation. In this study we investigated whether MICB expression is controlled by RBPs through its 5'UTR. We used an RNA pull-down assay followed by mass spectrometry and identified vigilin, a ubiquitously expressed multifunctional RNA-binding protein. We demonstrated that vigilin binds and negatively regulates MICB expression through its 5'UTR. Additionally, vigilin downregulation in target cells led to a significant increase in NK cell activation against said target cells. Taken together, we have discovered a novel mode of MICB regulation.

Human Herpesvirus 6B Downregulates Expression of Activating Ligands during Lytic Infection To Escape Elimination by Natural Killer Cells.

The Herpesviridae family consists of eight viruses, most of which infect a majority of the human population. One of the less-studied members is human herpesvirus 6 (HHV-6) (Roseolovirus), which causes a mild, well-characterized childhood disease. Primary HHV-6 infection is followed by lifelong latency. Reactivation frequently occurs in immunocompromised patients, such as those suffering from HIV infection or cancer or following transplantation, and causes potentially life-threatening complications. In this study, we investigated the mechanisms that HHV-6 utilizes to remain undetected by natural killer (NK) cells, which are key participants in the innate immune response to infections. We revealed viral mechanisms which downregulate ligands for two powerful activating NK cell receptors: ULBP1, ULBP3, and MICB, which trigger NKG2D, and B7-H6, which activates NKp30. Accordingly, this downregulation impaired the ability of NK cells to recognize HHV-6-infected cells. Thus, we describe for the first time immune evasion mechanisms of HHV-6 that protect lytically infected cells from NK elimination. IMPORTANCE: Human herpesvirus 6 (HHV-6) latently infects a large portion of the human population and can reactivate in humans lacking a functional immune system, such as cancer or AIDS patients. Under these conditions, it can cause life-threatening diseases. To date, the actions and interplay of immune cells, and particularly cells of the innate immune system, during HHV-6 infection are poorly defined. In this study, we aimed to understand how cells undergoing lytic HHV-6 infection interact with natural killer (NK) cells, innate lymphocytes constituting the first line of defense against viral intruders. We show that HHV-6 suppresses the expression of surface proteins that alert the immune cells by triggering two major receptors on NK cells, NKG2D and NKp30. As a consequence, HHV-6 can replicate undetected by the innate immune system and potentially spread infection throughout the body. This study advances the understanding of HHV-6 biology and the measures it uses to successfully escape immune elimination.

The RNA binding protein IMP3 facilitates tumor immune escape by downregulating the stress-induced ligands ULPB2 and MICB.

Expression of the stress-induced ligands MICA, MICB and ULBP 1-6 are up-regulated as a cellular response to DNA damage, excessive proliferation or viral infection; thereby, they enable recognition and annihilation by immune cells that express the powerful activating receptor NKG2D. This receptor is present not exclusively, but primarily on NK cells. Knowledge about the regulatory mechanisms controlling ULBP expression is still vague. In this study, we report a direct interaction of the oncogenic RNA binding protein (RBP) IMP3 with ULBP2 mRNA, leading to ULBP2 transcript destabilization and reduced ULBP2 surface expression in several human cell lines. We also discovered that IMP3 indirectly targets MICB with a mechanism functionally distinct from that of ULBP2. Importantly, IMP3-mediated regulation of stress-ligands leads to impaired NK cell recognition of transformed cells. Our findings shed new light on the regulation of NKG2D ligands and on the mechanism of action of a powerful oncogenic RBP, IMP3.

Cytokine secretion and NK cell activity in human ADAM17 deficiency.

Genetic deficiencies provide insights into gene function in humans. Here we describe a patient with a very rare genetic deficiency of ADAM17. We show that the patient's PBMCs had impaired cytokine secretion in response to LPS stimulation, correlating with the clinical picture of severe bacteremia from which the patient suffered. ADAM17 was shown to cleave CD16, a major NK killer receptor. Functional analysis of patient's NK cells demonstrated that his NK cells express normal levels of activating receptors and maintain high surface levels of CD16 following mAb stimulation. Activation of individual NK cell receptors showed that the patient's NK cells are more potent when activated directly by CD16, albeit no difference was observed in Antibody Depedent Cytotoxicity (ADCC) assays. Our data suggest that ADAM17 inhibitors currently considered for clinical use to boost CD16 activity should be cautiously applied, as they might have severe side effects resulting from impaired cytokine secretion.

Characterization of a bispecific FLT3 X CD3 antibody in an improved, recombinant format for the treatment of leukemia.

FLT3 is a receptor-tyrosine-kinase that is expressed on leukemic cells of the myeloid and lymphoid lineage rather specifically. We here report on the construction and selection of bispecific FLT3 X CD3 antibodies in a new recombinant format, termed Fabsc, that resembles the normal antibody structure more closely than the well-established bispecific single chain (bssc)-format. Our preferred antibody, which emerged from an initial selection procedure utilizing different FLT3- and CD3-antibodies, contains the FLT3-antibody 4G8 and the CD3-antibody UCHT1. The 4G8 X UCHT1 Fabsc-antibody was found to be superior to a bssc-antibody with identical specificities with respect to (i) affinity to the target antigen FLT3, (ii) production yield by transfected cells, and (iii) the diminished formation of aggregates. T-cell activation in the presence and absence of cultured leukemic cells and killing of these cells was comparable for both molecules. In addition, the 4G8 X UCHT1 Fabsc-antibody was found to induce T-cell activation and efficient killing of leukemic blasts in primary peripheral blood mononuclear cell (PBMC) cultures of acute myeloid leukemia (AML) patients. In these experiments, the bispecific molecule was clearly superior to an Fc-optimized monospecific FLT3-antibody described previously, indicating that within PBMC of AML patients the recruitment of T cells is more effective than that of natural killer cells.

MiR-520d-5p directly targets TWIST1 and downregulates the metastamiR miR-10b.

MicroRNAs are key players in most biological processes. Some microRNAs are involved in the genesis of tumors and are therefore termed oncomiRs, while others, termed metastamiRs, play a significant role in the formation of cancer metastases. Previously, we identified ten different cellular microRNAs that downregulate the expression of MICB, a ligand of the activating NK receptor NKG2D. Interestingly, several of the ten MICB-targeting microRNAs, such as miR-10b, are involved in tumor formation and metastasis. In this work, we identify a complex interplay between these different microRNAs. Specifically, we demonstrate that three of the MICB-targeting microRNAs: miR-20a, miR-17-5p and miR-93, also target the same site in the 3'UTR of TWIST1, a transcription factor implicated in cancer metastasis. Additionally, we show that miR-520d-5p targets a different site in the 3'UTR of TWIST1. We next show that the miR-520d-5p-mediated decrease of TWIST1 expression results in reduced expression of one of its targets, miR-10b, and in the restoration of E-Cadherin expression, which in turn results in reduced cellular motility and invasiveness. Finally, we show that miR-520d-5p leads to reduced proliferation of tumor cells, and that high levels of miR-520d-5p correlate with higher survival rates of cancer patients.