Neutrophils in cancer.

Neutrophils play an important role in cancer. This does not only relate to the well-established prognostic value of the presence of neutrophils, either in the blood or in tumor tissue, in the context of cancer progression or for the monitoring of therapy, but also to their active role in the progression of cancer. In the current review, we describe what is known in general about the role of neutrophils in cancer. What is emerging is a complex, rather heterogeneous picture with both pro- and anti-tumorigenic roles, which apparently differs with cancer type and disease stage. Furthermore, we will discuss the well-known role of neutrophils as myeloid-derived suppressor cells (MDSC), and also on the role of neutrophils as important effector cells during antibody therapy in cancer. It is clear that neutrophils contribute substantially to cancer progression in multiple ways, and this includes both direct effects on the cancer cells and indirect effect on the tumor microenvironment. While in many cases neutrophils have been shown to promote tumor progression, for instance by acting as MDSC, there are also protective effects, particularly when antibody immunotherapy is performed. A better understanding of the role of neutrophils is likely to provide opportunities for immunomodulation and for improving the treatment of cancer patients.

Dexamethasone promotes granulocyte mobilization by prolonging the half-life of granulocyte-colony-stimulating factor in healthy donors for granulocyte transfusions.

BACKGROUND: Granulocyte transfusion (GTX) is a potential approach to correcting neutropenia and relieving the increased risk of infection in patients who are refractory to antibiotics. To mobilize enough granulocytes for transfusion, healthy donors are premedicated with granulocyte-colony-stimulating factor (G-CSF) and dexamethasone. Granulocytes have a short circulatory half-life. Consequently, patients need to receive GTX every other day to keep circulating granulocyte counts at an acceptable level. We investigated whether plasma from premedicated donors was capable of prolonging neutrophil survival and, if so, which factor could be held responsible. STUDY DESIGN AND METHODS: The effects of plasma from G-CSF/dexamethasone-treated donors on neutrophil survival were assessed by annexin-V, CD16. and CXCR4 staining and nuclear morphology. We isolated an albumin-bound protein using α-chymotrypsin and albumin-depletion and further characterized it using protein analysis. The effects of dexamethasone and G-CSF were assessed using mifepristone and G-CSF-neutralizing antibody. G-CSF plasma concentrations were determined by Western blot and Luminex analyses. RESULTS: G-CSF/dexamethasone plasma contained a survival-promoting factor for at least 2 days. This factor was recognized as an albumin-associated protein and was identified as G-CSF itself, which was surprising considering its reported half-life of only 4.5 hours. Compared with coadministration of dexamethasone, administration of G-CSF alone to the same GTX donors led to a faster decline in circulating G-CSF levels, whereas dexamethasone itself did not induce any G-CSF, demonstrating a role for dexamethasone in increasing G-CSF half-life. CONCLUSION: Dexamethasone increases granulocyte yield upon coadministration with G-CSF by extending G-CSF half-life. This observation might also be exploited in the coadministration of dexamethasone with other recombinant proteins to modulate their half-life.

The identification and developmental requirements of colonic CD169⁺ macrophages.

CD169-positive macrophages in the marginal zone of the spleen and subcapsular sinus of lymph nodes play an important role as gatekeepers, strategically located to capture pathogens. Here we identified a population of CD169-positive macrophages in the colon and investigated which factors influenced their development. Murine colonic CD115+ F4/80(lo) CD11c(lo) macrophages expressing CD169 were present in the lamina propria, mainly surrounding the crypts. In spite of the high levels of bacterial flora in the colon and the importance of Toll-like receptor signalling in mucosal homeostasis, the presence of CD169+ macrophages was not affected in mice that were deficient in MyD88-mediated Toll-like receptor signalling and in mice in which the bacterial flora was eradicated. Whereas the development of splenic CD169+ macrophages was dependent on lymphotoxin α, colonic CD169+ macrophages were present in normal numbers in lymphotoxin α-deficient mice. In contrast, reduced numbers of CD169+ macrophages were found in the colon of mice deficient in vitamin A, whereas CD169+ macrophages in the spleen were unaffected. In conclusion, we identified a new macrophage subset in the lamina propria of the colon characterized by the expression of CD169. Its differentiation, unlike CD169+ macrophages in lymphoid organs, is independent of lymphotoxin α signalling, but requires vitamin A.

Cytotoxicity of the CD3×CD20 bispecific antibody epcoritamab in CLL is increased by concurrent BTK or BCL-2 targeting.

Chronic lymphocytic leukemia (CLL) is an immunosuppressive disease characterized by increased infectious morbidity and inferior antitumor activity of immunotherapies. Targeted therapy with Bruton's tyrosine kinase inhibitors (BTKis) or the Bcl-2 inhibitor venetoclax has profoundly improved treatment outcomes in CLL. To overcome or prevent drug resistance and extend the duration of response after a time-limited therapy, combination regimens are tested. Anti-CD20 antibodies that recruit cell- and complement-mediated effector functions are commonly used. Epcoritamab (GEN3013), an anti-CD3×CD20 bispecific antibody that recruits T-cell effector functions, has demonstrated potent clinical activity in patients with relapsed CD20+ B-cell non-Hodgkin lymphoma. Development of CLL therapy is ongoing. To characterize epcoritamab-mediated cytotoxicity against primary CLL cells, peripheral blood mononuclear cells from treatment-naive and BTKi-treated patients, including patients progressing on therapy, were cultured with epcoritamab alone or in combination with venetoclax. Ongoing treatment with BTKi and high effector-to-target ratios were associated with superior in vitro cytotoxicity. Cytotoxic activity was independent of CD20 expression on CLL cells and observed in samples from patients whose condition progressed while receiving BTKi. Epcoritamab induced significant T-cell expansion, activation, and differentiation into Th1 and effector memory cells in all patient samples. In patient-derived xenografts, epcoritamab reduced the blood and spleen disease burden compared with that in mice receiving a nontargeting control. In vitro, the combination of venetoclax with epcoritamab induced superior killing of CLL cells than either agent alone. These data support the investigation of epcoritamab in combination with BTKis or venetoclax to consolidate responses and target emergent drug-resistant subclones.

Preclinical anti-tumour activity of HexaBody-CD38, a next-generation CD38 antibody with superior complement-dependent cytotoxic activity.

BACKGROUND: HexaBody®-CD38 (GEN3014) is a hexamerization-enhanced human IgG1 that binds CD38 with high affinity. The E430G mutation in its Fc domain facilitates the natural process of antibody hexamer formation upon binding to the cell surface, resulting in increased binding of C1q and potentiated complement-dependent cytotoxicity (CDC). METHODS: Co-crystallization studies were performed to identify the binding interface of HexaBody-CD38 and CD38. HexaBody-CD38-induced CDC, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), trogocytosis, and apoptosis were assessed using flow cytometry assays using tumour cell lines, and MM patient samples (CDC). CD38 enzymatic activity was measured using fluorescence spectroscopy. Anti-tumour activity of HexaBody-CD38 was assessed in patient-derived xenograft mouse models in vivo. FINDINGS: HexaBody-CD38 binds a unique epitope on CD38 and induced potent CDC in multiple myeloma (MM), acute myeloid leukaemia (AML), and B-cell non-Hodgkin lymphoma (B-NHL) cells. Anti-tumour activity was confirmed in patient-derived xenograft models in vivo. Sensitivity to HexaBody-CD38 correlated with CD38 expression level and was inversely correlated with expression of complement regulatory proteins. Compared to daratumumab, HexaBody-CD38 showed enhanced CDC in cell lines with lower levels of CD38 expression, without increasing lysis of healthy leukocytes. More effective CDC was also confirmed in primary MM cells. Furthermore, HexaBody-CD38 efficiently induced ADCC, ADCP, trogocytosis, and apoptosis after Fc-crosslinking. Moreover, HexaBody-CD38 strongly inhibited CD38 cyclase activity, which is hypothesized to relieve immune suppression in the tumour microenvironment. INTERPRETATION: Based on these preclinical studies, a clinical trial was initiated to assess the clinical safety of HexaBody-CD38 in patients with MM. FUNDING: Genmab.

Semimechanistic Physiologically-Based Pharmacokinetic/Pharmacodynamic Model Informing Epcoritamab Dose Selection for Patients With B-Cell Lymphomas.

Epcoritamab is a CD3xCD20 bispecific antibody (bsAb) that induces T-cell-mediated cytotoxicity against CD20-positive B cells. Target engagement and crosslinking of CD3 and CD20 (trimer formation) leads to activation and expansion of T cells and killing of malignant B cells. The primary objective of the dose-escalation part of the phase I/II trial of epcoritamab was to determine the maximum tolerated dose, recommended phase II dose (RP2D), or both. For bsAbs, high target saturation can negatively affect trimer formation. The unique properties and mechanisms of action of bsAbs require novel pharmacokinetic (PK) modeling methods to predict clinical activity and inform RP2D selection. Traditional PK/pharmacodynamic (PD) modeling approaches are inappropriate because they may not adequately predict exposure-response relationships. We developed a semimechanistic, physiologically-based PK/PD model to quantitatively describe biodistribution, trimer formation, and tumor response using preclinical, clinical PK, biomarker, tumor, and response data from the dose-escalation part of the phase I/II trial. Clinical trial simulations were performed to predict trimer formation and tumor response in patients with diffuse large B-cell lymphoma (DLBCL) or follicular lymphoma (FL). Model-predicted trimer formation plateaued at doses of 48 to 96 mg. Simulation results suggest that the 48-mg dose may achieve optimal response rates in DLBCL and FL. Exposure-safety analyses showed a flat relationship between epcoritamab exposure and risk of cytokine release syndrome in the dose range evaluated. This novel PK/PD modeling approach guided selection of 48 mg as the RP2D and provides a framework that may be applied to other CD3 bsAbs.

c-Jun activating binding protein 1 binds to the IgA receptor and modulates protein levels of FcalphaRI and FcRgamma-chain.

The receptor for IgA, FcalphaRI or CD89, is expressed on myeloid cells and can trigger phagocytosis, tumor cell lysis, and release of inflammatory mediators. These functions critically depend on the associated FcR gamma-chain; however, some biological functions, like receptor internalization, are solely mediated by FcalphaRI alpha-chain. Little is known as to how FcalphaRI regulates these processes and the FcalphaRI intracellular domain does not contain recognized signalling motifs. We searched for associating proteins and identified c-Jun activating binding protein 1 (JAB1) as a binding partner specifically for FcalphaRI. We found increased FcalphaRI surface expression after ectopic expression of JAB1 as well as diminished protein levels of total FcR gamma-chain levels after JAB1 knock-down. These data functionally link JAB1 with controlling protein expression levels of FcalphaRI-FcR gamma-chain protein complex.

Epcoritamab induces potent anti-tumor activity against malignant B-cells from patients with DLBCL, FL and MCL, irrespective of prior CD20 monoclonal antibody treatment.

Epcoritamab (DuoBody-CD3xCD20, GEN3013) is a novel bispecific IgG1 antibody redirecting T-cells toward CD20+ tumor cells. Here, we assessed the preclinical efficacy of epcoritamab against primary tumor cells present in the lymph node biopsies from newly diagnosed (ND) and relapsed/refractory (RR) B-NHL patients. In the presence of T-cells from a healthy donor, epcoritamab demonstrated potent activity against primary tumor cells, irrespective of prior treatments, including CD20 mAbs. Median lysis of 65, 74, and 84% were achieved in diffuse large B-cell lymphoma (n = 16), follicular lymphoma (n = 15), and mantle cell lymphoma (n = 8), respectively. Furthermore, in this allogeneic setting, we discovered that the capacity of B-cell tumors to activate T-cells was heterogeneous and showed an inverse association with their surface expression levels of the immune checkpoint molecule Herpesvirus Entry Mediator (HVEM). In the autologous setting, when lymph node (LN)-residing T-cells were the only source of effector cells, the epcoritamab-dependent cytotoxicity strongly correlated with local effector cell-to-target cell ratios. Further analyses revealed that LN-residing-derived or peripheral blood-derived T-cells of B-NHL patients, as well as heathy donor T-cells equally mediated epcoritamab-dependent cytotoxicity. These results show the promise of epcoritamab for treatment of newly-diagnosed or relapsed/refractory B-NHL patients, including those who became refractory to previous CD20-directed therapies.

Different MDSC Activity of G-CSF/Dexamethasone Mobilized Neutrophils: Benefits to the Patient?

Human neutrophils exert a well-known role as efficient effector cells to kill pathogenic micro-organisms. Apart from their role in innate immunity, neutrophils also have the capacity to suppress T cell-mediated immune responses as so-called granulocyte-myeloid-derived suppressor cells (g-MDSCs), impacting the clinical outcome of various disease settings such as cancer. Patients undergoing chemotherapy because of an underlying malignancy can develop prolonged bone marrow suppression and are prone to serious infections because of severe neutropenia. Concentrates of granulocytes for transfusion (GTX) constitute a therapeutic tool and rescue treatment to fight off these serious bacterial and fungal infections when antimicrobial therapy is ineffective. GTX neutrophils are mobilized by overnight G-CSF and/or Dexamethasone stimulation of healthy donors. Although the phenotype of these mobilized neutrophils differs from the circulating neutrophils under normal conditions, their anti-microbial function is still intact. In contrast to the unaltered antimicrobial effector functions, G-CSF/Dexamethasone-mobilized neutrophils were found to lack suppression of the T cell proliferation, whereas G-CSF-mobilized or Dexamethasone-mobilized neutrophils could still suppress the T cell proliferation upon cell activation equally well as control neutrophils. Although the mechanism of how G-CSF/Dex mobilization may silence the g-MDSC activity of neutrophils without downregulating the antimicrobial activity is presently unclear, their combined use in patients in the treatment of underlying malignancies may be beneficial-irrespective of the number of circulating neutrophils. These findings also indicate that MDSC activity does not fully overlap with the antimicrobial activity of human neutrophils and offers the opportunity to elucidate the feature(s) unique to their T-cell suppressive activity.

DuoBody-CD3xCD20 induces potent T-cell-mediated killing of malignant B cells in preclinical models and provides opportunities for subcutaneous dosing.

BACKGROUND: DuoBody®-CD3xCD20 (GEN3013) is a full-length human IgG1 bispecific antibody (bsAb) recognizing CD3 and CD20, generated by controlled Fab-arm exchange. Its Fc domain was silenced by introduction of mutations L234F L235E D265A. METHODS: T-cell activation and T-cell-mediated cytotoxicity were measured by flow cytometry following co-culture with tumour cells. Anti-tumour activity of DuoBody-CD3xCD20 was assessed in humanized mouse models in vivo. Non-clinical safety studies were performed in cynomolgus monkeys. FINDINGS: DuoBody-CD3xCD20 induced highly potent T-cell activation and T-cell-mediated cytotoxicity towards malignant B cells in vitro. Comparison of DuoBody-CD3xCD20 to CD3 bsAb targeting alternative B-cell antigens, or to CD3xCD20 bsAb generated using alternative CD20 Ab, emphasized its exceptional potency. In vitro comparison with other CD3xCD20 bsAb in clinical development showed that DuoBody-CD3xCD20 was significantly more potent than three other bsAb with single CD3 and CD20 binding regions and equally potent as a bsAb with a single CD3 and two CD20 binding regions. DuoBody-CD3xCD20 showed promising anti-tumour activity in vivo, also in the presence of excess levels of a CD20 Ab that competes for binding. In cynomolgus monkeys, DuoBody-CD3xCD20 demonstrated profound and long-lasting B-cell depletion from peripheral blood and lymphoid organs, which was comparable after subcutaneous and intravenous administration. Peak plasma levels of DuoBody-CD3xCD20 were lower and delayed after subcutaneous administration, which was associated with a reduction in plasma cytokine levels compared to intravenous administration, while bioavailability was comparable. INTERPRETATION: Based on these preclinical studies, a clinical trial was initiated to assess the clinical safety of subcutaneous DuoBody-CD3xCD20 in patients with B-cell malignancies. FUNDING: Genmab.

Dynamic Transcriptome-Proteome Correlation Networks Reveal Human Myeloid Differentiation and Neutrophil-Specific Programming.

Human neutrophilic granulocytes form the largest pool of innate immune cells for host defense against bacterial and fungal pathogens. The dynamic changes that accompany the metamorphosis from a proliferating myeloid progenitor cell in the bone marrow into a mature non-dividing polymorphonuclear blood cell have remained poorly defined. Using mass spectrometry-based quantitative proteomics combined with transcriptomic data, we report on the dynamic changes of five developmental stages in the bone marrow and blood. Integration of transcriptomes and proteome unveils highly dynamic and differential interactions between RNA and protein kinetics during human neutrophil development, which can be linked to functional maturation of typical end-stage blood neutrophil killing activities.

Neutrophils as Suppressors of T Cell Proliferation: Does Age Matter?

Whereas, neutrophils have long been considered to mainly function as efficient innate immunity killers of micro-organisms at infected sites, they are now recognized to also be involved in modulation of adaptive immune responses. Immature and mature neutrophils were reported to have the capacity to suppress T cell-mediated immune responses as so-called granulocyte-myeloid-derived suppressor cells (g-MDSCs), and thereby affect the clinical outcome of cancer patients and impact the chronicity of microbial infections or rejection reactions in organ transplantation settings. These MDSCs were at first considered to be immature myeloid cells that left the bone marrow due to disease-specific signals. Current studies show that also mature neutrophils can exert suppressive activity. In this study we investigated in a robust T cell suppression assay whether immature CD11b+ myeloid cells were capable of MDSC activity comparable to mature fully differentiated neutrophils. We compared circulating neutrophils with myeloid cell fractions from the bone marrow at different differentiation stages. Our results indicate that functional MDSC activity is only becoming detectable at the final stage of differentiation, depending on the procedure of cell isolation. The MDSC activity obtained during neutrophil maturation correlated with the induction of the well-known highly mobile and toxic effector functions of the circulating neutrophil. Although immature neutrophils have been suggested to be increased in the circulation of cancer patients, we show here that immature neutrophils are not efficient in suppressing T cells. This suggests that the presence of immature neutrophils in the bloodstream of cancer patients represent a mere association or may function as a source of mature neutrophils in the tumor environment but not a direct cause of enhanced MDSC activity in cancer.

Activated neutrophils exert myeloid-derived suppressor cell activity damaging T cells beyond repair.

Myeloid-derived suppressor cells (MDSCs) have the capacity to suppress T-cell-mediated immune responses and impact the clinical outcome of cancer, infections, and transplantation settings. Although MDSCs were initially described as bone marrow-derived immature myeloid cells (either monocytic or granulocytic MDSCs), mature neutrophils have been shown to exert MDSC activity toward T cells in ways that remain unclear. In this study, we demonstrated that human neutrophils from both healthy donors and cancer patients do not exert MDSC activity unless they are activated. By using neutrophils with genetically well-defined defects, we found that reactive oxygen species (ROS) and granule-derived constituents are required for MDSC activity after direct CD11b-dependent interactions between neutrophils and T cells. In addition to these cellular interactions, neutrophils are engaged in the uptake of pieces of T-cell membrane, a process called trogocytosis. Together, these interactions led to changes in T-cell morphology, mitochondrial dysfunction, and adenosine triphosphate depletion, as indicated by electron microscopy, mass spectrometry, and metabolic parameters. Our studies characterize the different steps by which activated mature neutrophils induce functional T-cell nonresponsiveness and irreparable cell damage.

Dynamics of Transcription Regulation in Human Bone Marrow Myeloid Differentiation to Mature Blood Neutrophils.

Neutrophils are short-lived blood cells that play a critical role in host defense against infections. To better comprehend neutrophil functions and their regulation, we provide a complete epigenetic overview, assessing important functional features of their differentiation stages from bone marrow-residing progenitors to mature circulating cells. Integration of chromatin modifications, methylation, and transcriptome dynamics reveals an enforced regulation of differentiation, for cellular functions such as release of proteases, respiratory burst, cell cycle regulation, and apoptosis. We observe an early establishment of the cytotoxic capability, while the signaling components that activate these antimicrobial mechanisms are transcribed at later stages, outside the bone marrow, thus preventing toxic effects in the bone marrow niche. Altogether, these data reveal how the developmental dynamics of the chromatin landscape orchestrate the daily production of a large number of neutrophils required for innate host defense and provide a comprehensive overview of differentiating human neutrophils.

Intestinal epithelial cell transported TLR2 ligand stimulates Ly6C⁺ monocyte differentiation in a G-CSF dependent manner.

Microflora-induced TLR signaling in the intestinal epithelium is essential for a proper intestinal barrier function. Because of the close interactions of this epithelial layer with underlying mononuclear phagocytes, we hypothesized that epithelial TLR signaling may affect the differentiation of myeloid cell populations. In in vitro cultures we observed that colonic epithelial monolayers actively transported TLR2 ligands towards their basolateral side. The transported TLR2 ligands strongly stimulated the development of Ly6C(+) monocytes, while dendritic cell differentiation was inhibited. The TLR2 effect on monocyte and dendritic cell differentiation was mediated by the production of G-CSF. Mice lacking TLR signaling and mice that were treated with antibiotics showed decreased numbers of Ly6C(+) monocytes in bone marrow and spleen, which points to a role for microbial derived TLR-ligands in the homeostasis of Ly6C(+) monocytes. In conclusion, our results indicate that TLR ligands that are transported by intestinal epithelial cells stimulate Ly6C(+) monocyte development and suggest that this process may be involved in the maintenance of systemic Ly6C(+) monocyte numbers.

Nod2 improves barrier function of intestinal epithelial cells via enhancement of TLR responses.

Intestinal epithelial cells (IECs) form a physical barrier between the internal milieu and the intestinal microflora via the expression of tight junctions. TLR-mediated recognition of intestinal microflora by IECs is important for tight junction preservation, production of chemokines, and cell survival. Disturbance of the IEC barrier function results in bacterial invasion and contributes to the development of inflammatory bowel disease. We observed that muramyl dipeptide (MDP), a breakdown product of bacterial peptidoglycan, strongly enhances subsequent Toll-like receptor (TLR) responses in murine colonic epithelium cell lines. Prior exposure to MDP significantly increased the production of chemokines and cytokines and improved the barrier function induced by different TLR2 and TLR4 ligands. shRNA knock-down studies showed that MDP recognition by Nod2 mediated the enhancement of TLR responses. Our studies indicate that Nod2 stimulation by MDP significantly enhances TLR-mediated IEC barrier function and chemokine production. Failure of this protective mechanism may contribute to the increased risk of Crohn's disease in individuals with a loss-of-function mutation in NOD2.

Penetration of the blood-brain barrier by Staphylococcus aureus: contribution of membrane-anchored lipoteichoic acid.

Staphylococcus aureus is one of the most prevalent organisms responsible for nosocomial infections, and cases of community-acquired S. aureus infection have continued to increase despite widespread preventative measures. Pathologies attributed to S. aureus infection are diverse; ranging from dermal lesions to bacteremia, abscesses, and endocarditis. Reported cases of S. aureus-associated meningitis and brain abscesses have also increased in recent years, however, the precise mechanism whereby S. aureus leave the bloodstream and gain access to the central nervous system (CNS) are not known. Here we demonstrate for the first time that S. aureus efficiently adheres to and invades human brain microvascular endothelial cells (hBMEC), the single-cell layer which constitutes the blood-brain barrier (BBB). The addition of cytochalasin D, an actin microfilament aggregation inhibitor, strongly reduced bacterial invasion, suggesting an active hBMEC process is required for efficient staphylococcal uptake. Furthermore, mice injected with S. aureus exhibited significant levels of brain bacterial counts and histopathologic evidence of meningeal inflammation and brain abscess formation, indicating that S. aureus was able to breech the BBB in an experimental model of hematogenous meningitis. We found that a YpfP-deficient mutant, defective in lipoteichoic acid (LTA) membrane anchoring, exhibited a decreased ability to invade hBMEC and correlated to a reduced risk for the development of meningitis in vivo. Our results demonstrate that LTA-mediated penetration of the BBB may be a primary step in the pathogenesis of staphylococcal CNS disease.