The NK cell checkpoint NKG2A maintains expansion capacity of human NK cells

Human natural killer (NK) cells are cytotoxic effector cells that are increasingly harnessed in cancer immunotherapy. NKG2A/CD94 is an inhibitory receptor on NK cells that has established regulatory functions in the direct interaction with target cells when engaged with its ligand, the non-classical HLA class I molecule HLA-E. Here, we confirmed NKG2A as a checkpoint molecule in primary human NK cells and identified a novel role for NKG2A in maintaining NK cell expansion capacity by dampening both proliferative activity and excessive activation-induced cell death. Maintenance of NK cell expansion capacity might contribute to the preferential accumulation of human NKG2A$^{+}$ NK cells after hematopoietic cell transplantation and enrichment of functionally impaired NK cells in human cancers. Functional silencing of NKG2A for cancer immunotherapy is highly attractive but will need to consider that this might also lead to a reduced survival by driving activation-induced cell death in targeted NK cells

The NK cell checkpoint NKG2A maintains expansion capacity of human NK cells

Human natural killer (NK) cells are cytotoxic effector cells that are increasingly harnessed in cancer immunotherapy. NKG2A/CD94 is an inhibitory receptor on NK cells that has established regulatory functions in the direct interaction with target cells when engaged with its ligand, the non-classical HLA class I molecule HLA-E. Here, we confirmed NKG2A as a checkpoint molecule in primary human NK cells and identified a novel role for NKG2A in maintaining NK cell expansion capacity by dampening both proliferative activity and excessive activation-induced cell death. Maintenance of NK cell expansion capacity might contribute to the preferential accumulation of human NKG2A⁺ NK cells after hematopoietic cell transplantation and enrichment of functionally impaired NK cells in human cancers. Functional silencing of NKG2A for cancer immunotherapy is highly attractive but will need to consider that this might also lead to a reduced survival by driving activation-induced cell death in targeted NK cells

Exit pathways of therapeutic antibodies from the brain and retention strategies

Treating brain diseases requires therapeutics to pass the blood-brain barrier (BBB) which is nearly impermeable for large biologics such as antibodies. Several methods now facilitate crossing or circumventing the BBB for antibody therapeutics. Some of these exploit receptor-mediated transcytosis, others use direct delivery bypassing the BBB. However, successful delivery into the brain does not preclude exit back to the systemic circulation. Various mechanisms are implicated in the active and passive export of antibodies from the central nervous system. Here we review findings on active export via transcytosis of therapeutic antibodies - in particular, the role of the neonatal Fc receptor (FcRn) - and discuss a possible contribution of passive efflux pathways such as lymphatic and perivascular drainage. We point out open questions and how to address these experimentally. In addition, we suggest how emerging findings could aid the design of the next generation of therapeutic antibodies for neurologic diseases

Rationale for Combining Bispecific T Cell Activating Antibodies With Checkpoint Blockade for Cancer Therapy

T cells have been established as core effectors for cancer therapy;this has moved the focus of therapeutic endeavors to effectively enhance or restore T cell tumoricidal activity rather than directly target cancer cells. Both antibodies targeting the checkpoint inhibitory molecules programmed death receptor 1 (PD1), PD-ligand 1 (PD-L1) and cytotoxic lymphocyte activated antigen 4 (CTLA4), as well as bispecific antibodies targeting CD3 and CD19 are now part of the standard of care. In particular, antibodies to checkpoint molecules have gained broad approval in a number of solid tumor indications, such as melanoma or non-small cell lung cancer based on their unparalleled efficacy. In contrast, the efficacy of bispecific antibody-derivatives is much more limited and evidence is emerging that their activity is regulated through diverse checkpoint molecules. In either case, both types of compounds have their limitations and most patients will not benefit from them in the long run. A major aspect under investigation is the lack of baseline antigen-specific T cells in certain patient groups, which is thought to render responses to checkpoint inhibition less likely. On the other hand, bispecific antibodies are also restricted by induced T cell anergy. Based on these considerations, combination of bispecific antibody mediated on-target T cell activation and reversal of anergy bears high promise. Here, we will review current evidence for such combinatorial approaches, as well as ongoing clinical investigations in this area. We will also discuss potential evidence-driven future avenues for testing

Exit pathways of therapeutic antibodies from the brain and retention strategies

Treating brain diseases requires therapeutics to pass the blood-brain barrier (BBB) which is nearly impermeable for large biologics such as antibodies. Several methods now facilitate crossing or circumventing the BBB for antibody therapeutics. Some of these exploit receptor-mediated transcytosis, others use direct delivery bypassing the BBB. However, successful delivery into the brain does not preclude exit back to the systemic circulation. Various mechanisms are implicated in the active and passive export of antibodies from the central nervous system. Here we review findings on active export via transcytosis of therapeutic antibodies - in particular, the role of the neonatal Fc receptor (FcRn) - and discuss a possible contribution of passive efflux pathways such as lymphatic and perivascular drainage. We point out open questions and how to address these experimentally. In addition, we suggest how emerging findings could aid the design of the next generation of therapeutic antibodies for neurologic diseases.</p

Enabling comprehensive optogenetic studies of mouse hearts by simultaneous opto-electrical panoramic mapping and stimulation

During the last decade, cardiac optogenetics has turned into an essential tool for investigating cardiac function in general and for assessing functional interactions between different myocardial cell types in particular. To advance exploitation of the unique research opportunities offered by this method, we develop a panoramic opto-electrical measurement and stimulation (POEMS) system for mouse hearts. The core of the experimental platform is composed of 294 optical fibers and 64 electrodes that form a cup which embraces the entire ventricular surface of mouse hearts and enables straightforward 'drop&go' experimentation. The flexible assignment of fibers and electrodes to recording or stimulation tasks permits a precise tailoring of experiments to the specific requirements of individual optogenetic constructs thereby avoiding spectral congestion. Validation experiments with hearts from transgenic animals expressing the optogenetic voltage reporters ASAP1 and ArcLight-Q239 demonstrate concordance of simultaneously recorded panoramic optical and electrical activation maps. The feasibility of single fiber optical stimulation is proven with hearts expressing the optogenetic voltage actuator ReaChR. Adaptation of the POEMS system to larger hearts and incorporation of additional sensors can be achieved by redesigning the system-core accordingly

HDR-based CRISPR/Cas9-mediated Knockout of PD-L1 in C57BL/6 Mice

The immune-inhibitory molecule programmed cell death ligand 1 (PD-L1) has been shown to play a role in pathologies such as autoimmunity, infections, and cancer. The expression of PD-L1 not only on cancer cells but also on non-transformed host cells is known to be associated with cancer progression. Generation of PD-L1 deficiency in the murine system enables us to specifically study the role of PD-L1 in physiological processes and diseases. One of the most versatile and easy to use site-specific gene editing tools is the CRISPR/Cas9 system, which is based on an RNA-guided nuclease system. Similar to its predecessors, the Zinc finger nucleases or transcription activator-like effector nucleases (TALENs), CRISPR/Cas9 catalyzes double-strand DNA breaks, which can result in frameshift mutations due to random nucleotide insertions or deletions via non-homologous end joining (NHEJ). Furthermore, although less frequently, CRISPR/Cas9 can lead to insertion of defined sequences due to homology-directed repair (HDR) in the presence of a suitable template. Here, we describe a protocol for the knockout of PD-L1 in the murine C57BL/6 background using CRISPR/Cas9. Targeting of exon 3 coupled with the insertion of a HindIII restriction site leads to a premature stop codon and a loss-of-function phenotype. We describe the targeting strategy as well as founder screening, genotyping, and phenotyping. In comparison to NHEJ-based strategy, the presented approach results in a defined stop codon with comparable efficiency and timelines as NHEJ, generates convenient founder screening and genotyping options, and can be swiftly adapted to other targets

Neoadjuvant in situ vaccination with cowpea mosaic virus as a novel therapy against canine inflammatory mammary cancer

BackgroundInflammatory mammary cancer (IMC), the counterpart of human inflammatory breast cancer (IBC), is the deadliest form of canine mammary tumors. IMC patients lack specific therapy and have poor outcomes. This proof-of-principle preclinical study evaluated the efficacy, safety, and effect on survival of neoadjuvant intratumoral (in situ) empty cowpea mosaic virus (eCPMV) immunotherapy in companion dogs diagnosed with IMC.MethodsTen IMC-bearing dogs were enrolled in the study. Five dogs received medical therapy, and five received weekly neoadjuvant in situ eCPMV immunotherapy (0.2–0.4 mg per injection) and medical therapy after the second eCPMV injection. Efficacy was evaluated by reduction of tumor growth; safety by hematological and biochemistry changes in blood and plasma; and patient outcome by survival analysis. eCPMV-induced immune changes in blood cells were analyzed by flow cytometry; changes in the tumor microenvironment were evaluated by CD3 (T lymphocytes), CD20 (B lymphocytes), FoxP3 (Treg lymphocytes), myeloperoxidase (MPO; neutrophils), Ki-67 (proliferation index, PI; tumor cell proliferation), and Cleaved Caspase-3 (CC-3; apoptosis) immunohistochemistry.ResultsTwo neoadjuvant in situ eCPMV injections resulted in tumor shrinkage in all patients by day 14 without systemic adverse events. Although surgery for IMC is generally not an option, reduction in tumor size allowed surgery in two IMC patients. In peripheral blood, in situ eCPMV immunotherapy was associated with a significant decrease of Treg$^{+}$/CD8$^{+}$ ratio and changes in CD8$^{+}$Granzyme B$^{+}$ T cells, which behave as a lagging predictive biomarker. In the TME, higher neutrophilic infiltration and MPO expression, lower tumor Ki-67 PI, increase in CD3$^{+}$ lymphocytes, decrease in FoxP3$^{+}$/CD3$^{+}$ ratio (p<0.04 for all comparisons), and no changes in CC-3$^{+}$ immunostainings were observed in post-treatment tumor tissues when compared with pretreatment tumor samples. eCPMV-treated IMC patients had a statistically significant (p=0.033) improved overall survival than patients treated with medical therapy.ConclusionsNeoadjuvant in situ eCPMV immunotherapy demonstrated anti-tumor efficacy and improved survival in IMC patients without systemic adverse effects. eCPMV-induced changes in immune cells point to neutrophils as a driver of immune response. Neoadjuvant in situ eCPMV immunotherapy could be a groundbreaking immunotherapy for canine IMC and a potential future immunotherapy for human IBC patients

De novo expression of gastrokines in pancreatic precursor lesions impede the development of pancreatic cancer

Molecular events occurring in stepwise progression from pre-malignant lesions (pancreatic intraepithelial neoplasia; PanIN) to the development of pancreatic ductal adenocarcinoma (PDAC) are poorly understood. Thus, characterization of early PanIN lesions may reveal markers that can help in diagnosing PDAC at an early stage and allow understanding the pathology of the disease. We performed the molecular and histological assessment of patient-derived PanINs, tumor tissues and pancreas from mouse models with PDAC (KC mice that harbor K-RAS mutation in pancreatic tissue), where we noted marked upregulation of gastrokine (GKN) proteins. To further understand the role of gastrokine proteins in PDAC development, GKN-deficient KC mice were developed by intercrossing gastrokine-deficient mice with KC mice. Panc-02 (pancreatic cancer cells of mouse origin) were genetically modified to express GKN1 for further in vitro and in vivo analysis. Our results show that gastrokine proteins were absent in healthy pancreas and invasive cancer, while its expression was prominent in low-grade PanINs. We could detect these proteins in pancreatic juice and serum of KC mice. Furthermore, accelerated PanIN and tumor development were noted in gastrokine deficient KC mice. Loss of gastrokine 1 protein delayed apoptosis during carcinogenesis leading to the development of desmoplastic stroma while loss of gastrokine 2 increased the proliferation rate in precursor lesions. In summary, we identified gastrokine proteins in early pancreatic precursor lesions, where gastrokine proteins delay pancreatic carcinogenesis

Neoadjuvant Intratumoral Immunotherapy with Cowpea Mosaic Virus Induces Local and Systemic Antitumor Efficacy in Canine Mammary Cancer Patients

The lack of optimal models to evaluate novel agents is delaying the development of effective immunotherapies against human breast cancer (BC). In this prospective open label study, we applied neoadjuvant intratumoral immunotherapy with empty cowpea mosaic virus-like particles (eCPMV) to 11 companion dogs diagnosed with canine mammary cancer (CMC), a spontaneous tumor resembling human BC. We found that two neoadjuvant intratumoral eCPMV injections resulted in tumor reduction in injected tumors in all patients and in noninjected tumors located in the ipsilateral and contralateral mammary chains of injected dogs. Tumor reduction was independent of clinical stage, tumor size, histopathologic grade, and tumor molecular subtype. RNA-seq-based analysis of injected tumors indicated a decrease in DNA replication activity and an increase in activated dendritic cell infiltration in the tumor microenvironment. Immunohistochemistry analysis demonstrated significant intratumoral increases in neutrophils, T and B lymphocytes, and plasma cells. eCPMV intratumoral immunotherapy demonstrated antitumor efficacy without any adverse effects. This novel immunotherapy has the potential for improving outcomes for human BC patients