Recruitment Of NK Cells Research Articles

The impact of previous live births on peripheral and uterine natural killer cells in patients with recurrent miscarriage

BackgroundPeripheral and uterine natural killer cells (pNK and uNK cells) are key players in the establishment and maintenance of pregnancy and are disturbed in patients with recurrent miscarriage (RM). Different immunologic risk factors have been proposed between patients with primary RM (pRM, no previous live birth) and secondary RM (sRM, ≥ 1 previous live birth). However, so far, the study populations mainly consisted of small subgroups. Therefore, we aimed to analyse pNK and uNK cells in a large, well defined study population within a prospective study.MethodsIn total, n = 575 RM patients (n = 393 pRM, n = 182 sRM) were screened according to a standard protocol for established risk factors as well as pNK and uNK cells. Peripheral blood levels of CD45+CD3−CD56+CD16+ NK cells were determined by flow cytometry and uterine CD56+ NK cells by immunohistochemistry in mid-luteal non-pregnant RM patients. Exclusion of patients with ≥1 established risk factor revealed n = 248 idiopathic RM patients (iRM, n = 167 primary iRM (ipRM), n = 81 secondary iRM (isRM)).ResultsPatients with pRM and ipRM showed significant higher absolute numbers and percentages of pNK cells compared to sRM and isRM patients (pRM/ipRM vs sRM/isRM, mean ± SD /μl: 239.1 ± 118.7/244.9 ± 112.9 vs 205.1 ± 107.9/206.0 ± 105.6, p = 0.004/ p = 0.009; mean ± SD %: 12.4 ± 5.5/12.8 ± 5.4 vs 11.1 ± 4.6/11.1 ± 4.3, p = 0.001; p = 0.002). Only patients with isRM showed significantly higher uNK levels compared to patients with ipRM (mean ± SD /mm2 288.4 ± 239.3 vs 218.2 ± 184.5, p = 0.044).ConclusionsThe demonstrated differences in pNK and uNK cells in RM patients depending on previous live birth might indicate differences in NK cell recruitment and potentially different underlying immune disorders between pRM and sRM. As there is an overlap in the distribution of the NK cell results, further studies with focus on NK cell function are needed in order to clearly identify RM patients with distinct immune abnormalities. The clinical relevance of our findings should be interpreted cautiously until specificity and sensitivity are further evaluated.

Mass Production of Highly Active NK Cells for Cancer Immunotherapy in a GMP Conform Perfusion Bioreactor.

NK cells have emerged as promising candidates for cancer immunotherapy, especially due to their ability to fight circulating tumor cells thereby preventing metastases formation. Hence several studies have been performed to generate and expand highly cytotoxic NK cells ex vivo, e.g., by using specific cytokines to upregulate both their proliferation and surface expression of distinct activating receptors. Apart from an enhanced activity, application of NK cells as immunotherapeutic agent further requires sufficient cell numbers and a high purity. All these parameters depend on a variety of different factors including the starting material, additives like cytokines as well as the culture system. Here we analyzed PBMC-derived NK cells of five anonymized healthy donors expanded under specific conditions in an innovative perfusion bioreactor system with respect to their phenotype, IFNγ production, and cytotoxicity in vitro. Important features of the meander type bioreactors used here are a directed laminar flow of medium and control of relevant process parameters. Cells are cultivated under “steady state” conditions in perfusion mode. Our data demonstrate that expansion of CD3+ T cell depleted PBMCs in our standardized system generates massive amounts of highly pure (>85%) and potent anti-cancer active NK cells. These cells express a variety of important receptors driving NK cell recruitment, adhesion as well as activation. More specifically, they express the chemokine receptors CXCR3, CXCR4, and CCR7, the adhesion molecules L-selectin, LFA-1, and VLA-4, the activating receptors NKp30, NKp44, NKp46, NKG2D, DNAM1, and CD16 as well as the death ligands TRAIL and Fas-L. Moreover, the generated NK cells show a strong IFNγ expression upon cultivation with K562 tumor cells and demonstrate a high cytotoxicity toward leukemic as well as solid tumor cell lines in vitro. Altogether, these characteristics promise a high clinical potency of thus produced NK cells awaiting further evaluation.

A Bispecific Antibody-Based Approach for Targeting Mesothelin in Triple Negative Breast Cancer.

Triple negative breast cancers (TNBC) remain a major medical challenge due to poor prognosis and limited treatment options. Mesothelin is a glycosyl-phosphatidyl inositol-linked membrane protein with restricted normal expression and high level expression in a large proportion of TNBC, thus qualifying as an attractive target. Its overexpression in breast tumors has been recently correlated with a decreased disease-free survival and an increase of distant metastases. The objective of the study was to investigate the relevance of a bispecific antibody-based immunotherapy approach through mesothelin targeting and CD16 engagement using a Fab-like bispecific format (MesobsFab). Using two TNBC cell lines with different level of surface mesothelin and epithelial/mesenchymal phenotypes, we showed that, in vitro, MesobsFab promotes the recruitment and penetration of NK cells into tumor spheroids, induces potent dose-dependent cell-mediated cytotoxicity of mesothelin-positive tumor cells, cytokine secretion, and decreases cell invasiveness. MesobsFab was able to induce cytotoxicity in resting human peripheral blood mononuclear cells (PBMC), mainly through its NK cells-mediated antibody dependent cell cytotoxicity (ADCC) activity. In vivo, the anti-tumor effect of MesobsFab depends upon a threshold of MSLN density on target cells. Collectively our data support mesothelin as a relevant therapeutic target for the subset of TNBC that overexpresses mesothelin characterized by a low overall and disease-free survival as well as the potential of MesobsFab as antibody-based immunotherapeutics.

Abstract 4982: Anti-mCTLA-4 treatment results in early and late immune response effects in a murine model of colorectal carcinoma

Abstract To determine therapeutic mechanism of action (MOA), it is standard to analyze the tumor immune response at a single timepoint. Therefore, to examine the impact of checkpoint inhibition on early and late phases of the immune response, we dosed CT26 tumor-bearing mice with anti-mCTLA-4, or isotype control, and analyzed tumors at day 12 and day 18 post-implant. Tumor measurements at day 17 revealed a 77% growth inhibition in response to anti-mCTLA-4 treatment. To explore MOA, 11 tumor-infiltrating myeloid and lymphoid subsets were quantified and analyzed for various phenotypic and functional markers. We first examined immune cell recruitment in the control group tumors from day 12 to day 18. As expected, dynamic changes were observed in various subsets. Absolute numbers of CD8+ T cells, M1 and M2 tumor-associated macrophages (TAM) increased by 203%, 208%, and 100% respectively. In contrast, a 31% reduction in monocytic myeloid-derived suppressor cells (M-MDSC) was observed. The effects of anti-mCTLA-4 on tumor infiltration was then examined. Enhanced CD4+ helper, CD8+, and regulatory T cell recruitment was observed at both timepoints compared to the isotype control group. While NK and NKT cell recruitment was also enhanced, the increase in NK cells was only transient (130% at day 12). A similar transient increase was observed for M1 TAMs. In contrast, a 33% reduction in M2 TAMs occurred following treatment from day 12 to day 18, compared to controls. Finally, an increase in M-MDSC was observed in the anti-mCTLA-4 group at both timepoints (33% and 58% respectively). To evaluate effects of anti-mCTLA-4 on proliferation, we measured Ki-67 expression as a surrogate marker in CD8+ T cells. A 43% increase in Ki-67 expression was induced by anti-mCTLA-4 compared to controls at day 12. No difference was observed on day 18 suggesting anti-mCTLA-4 increased CD8+ T cell infiltration by triggering an early and transient enhancement in proliferation. Analysis of CD62L and CD44 in the control group demonstrated the pool of memory CD8+ T cells increased in the tumors over time. Anti-mCTLA-4 treatment delayed memory formation resulting in a larger pool of CD8+ T cells with an effector phenotype (CD62L-CD44+). To examine anti-tumor activity in CD8+ T cells, IFNγ production was measured after ex vivo stimulation. Anti-mCTLA-4 enhanced IFNγ responses on day 18 in CD8+ T cells and NKT cells (25% and 17% respectively), but similar responses were observed between groups on day 12. Taken together, these results suggest that checkpoint blockade inhibited tumor growth in part by enhancing early T cell proliferation and recruitment of NK and M1 TAMs. This was followed by a late enhancement of anti-tumor T cell and NKT responses, as well as reduced M2 TAM infiltration. These findings demonstrate that kinetic analysis can help gain insight into therapeutic MOA by revealing dynamic responses that can be missed by analysis at a single point. Citation Format: David W. Draper, Alden Wong, Stacey Roys, Scott Wise, Maryland Franklin. Anti-mCTLA-4 treatment results in early and late immune response effects in a murine model of colorectal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4982.

PARP-1 controls NK cell recruitment to the site of viral infection.

The activation and recruitment of NK cells to the site of viral infection are crucial for virus control. However, it remains largely unknown what controls the recruitment of the activated NK cells to the infection site. In a model of intraperitoneal infection with vaccinia virus (VV), we showed that poly(ADP-ribose) polymerase-1 (PARP-1), a sensor of DNA damage, is critical for NK cell recruitment to the site of infection and viral control in vivo. We further demonstrated that PARP-1 promotes the production of CCL2 and that the CCL2-CCR2 axis is essential for NK cell recruitment to the infection site. In addition, we demonstrated that peritoneal macrophages are the main producer of PARP-1-dependent CCL2 secretion. Mechanistically, PARP-1 functions as a regulator of NF-κB by promoting its nuclear translocation and binding to its response sequences in macrophages upon VV infection. Taken together, our results reveal a potentially previously unknown role for PARP-1-dependent CCL2 production in NK cell migration and viral control and may provide important insights into the design of effective NK cell-based therapies for viral infections and cancer.

NKT Cell-Driven Enhancement of Antitumor Immunity Induced by Clec9a-Targeted Tailorable Nanoemulsion.

Invariant natural killer T (iNKT) cells are a subset of lymphocytes with immune regulatory activity. Their ability to bridge the innate and adaptive immune systems has been studied using the glycolipid ligand α-galactosylceramide (αGC). To better harness the immune adjuvant properties of iNKT cells to enhance priming of antigen-specific CD8+ T cells, we encapsulated both αGC and antigen in a Clec9a-targeted nanoemulsion (TNE) to deliver these molecules to cross-presenting CD8+ dendritic cells (DC). We demonstrate that, even in the absence of exogenous glycolipid, iNKT cells supported the maturation of CD8α+ DCs to drive efficient cross-priming of antigen-specific CD8+ T cells upon delivery of Clec9a/OVA-TNE. The addition of αGC to the TNE (Clec9a/OVA/αGC) further enhanced activation of iNKT cells, NK cells, CD8α+ DCs, and polyfunctional CD8+ T cells. When tested therapeutically against HPVE7-expressing TC-1 tumors, long-term tumor suppression was achieved with a single administration of Clec9a/E7 peptide/αGC TNE. Antitumor activity was correlated with the recruitment of mature DCs, NK cells, and tumor-specific effector CD8+ T cells to the tumor-draining lymph node and tumor tissue. Thus, Clec9a-TNE codelivery of CD8+ T-cell epitopes with αGC induces alternative helper signals from activated iNKT cells, elicits innate (iNKT, NK) immunity, and enhances antitumor CD8+ T-cell responses for control of solid tumors.

Chemerin Suppresses Breast Cancer Growth by Recruiting Immune Effector Cells Into the Tumor Microenvironment.

Infiltration of immune cells into the tumor microenvironment (TME) can regulate growth and survival of neoplastic cells, impacting tumorigenesis and tumor progression. Correlations between the number of effector immune cells present in a tumor and clinical outcomes in many human tumors, including breast, have been widely described. Current immunotherapies utilizing checkpoint inhibitors or co-stimulatory molecule agonists aim to activate effector immune cells. However, tumors often lack adequate effector cell numbers within the TME, resulting in suboptimal responses to these agents. Chemerin (RARRES2) is a leukocyte chemoattractant widely expressed in many tissues and is known to recruit innate leukocytes. CMKLR1 is a chemotactic cellular receptor for chemerin and is expressed on subsets of dendritic cells, NK cells, and macrophages. We have previously shown that chemerin acts as a tumor suppressive cytokine in mouse melanoma models by recruiting innate immune defenses into the TME. Chemerin/RARRES2 is down-regulated in many tumors, including breast, compared to normal tissue counterparts. Here, using a syngeneic orthotopic EMT6 breast carcinoma model, we show that forced overexpression of chemerin by tumor cells results in significant recruitment of NK cells and T cells within the TME. While chemerin secretion by EMT6 cells did not alter their phenotypic behavior in vitro, it did significantly suppress tumor growth in vivo. To define the cellular effectors required for this anti-tumor phenotype, we depleted NK cells or CD8+ T cells and found that either cell type is required for chemerin-dependent suppression of EMT6 tumor growth. Finally, we show significantly reduced levels of RARRES2 mRNA in human breast cancer samples compared to matched normal tissues. Thus, for the first time we have shown that increasing chemerin expression within the breast carcinoma TME can suppress growth by recruitment of NK and T cells, thereby supporting this approach as a promising immunotherapeutic strategy.

CCR5-mediated recruitment of NK cells to the kidney is a critical step for host defense to systemic Candida albicans infection

Abstract CCR5 regulates the trafficking of various immune cells to the site of infection. Expression of CCR5 and its ligands were rapidly increased in the kidney after systemic Candida albicans infection and infected CCR5−/− mice showed increased mortality and morbidity, indicating that CCR5 contributes to an effective defense mechanism against systemic C. albicans infection. The susceptibility of CCR5−/− mice to C. ablicans infection was due to their impaired capacity of fungal clearance, which in turn resulted in exacerbated renal inflammation and damage. CCR5-mediated recruitment of NK cells to the kidney in response to C. albicans infection was necessary for the anti-microbial activity of neutrophils, the main fungicidal effector cells. Mechanistically, C. albicans rapidly induced expression of IL-23 and IL-15 by CD11b+CD11c+ dendritic cells (DCs) and Ly6C+ monocytes, respectively. IL-23 and IL-15 in turn augmented the fungicidal activity of neutrophils through GM-CSF production by NK cells. As GM-CSF potentiated production of IL-23 and IL-5 in response to C. albicans, a positive feedback loop formed between NK cells and DCs or Ly6C+ monocytes seemed to function as a central amplification point for host defense. Taken together, our results suggest that CCR5-mediated recruitment of NK cells to the site of fungal replication is a critical step that underlies innate resistance to systemic C. albicans infection.

Endometrial Tumor Microenvironment Alters Human NK Cell Recruitment, and Resident NK Cell Phenotype and Function.

Endometrial Cancer is the most common cancer in the female genital tract in developed countries, and with its increasing incidence due to risk factors such as aging and obesity tends to become a public health issue. However, its immune environment has been less characterized than in other tumors such as breast cancers. NK cells are cytotoxic innate lymphoid cells that are considered as a major anti-tumoral effector cell type which function is drastically altered in tumors which participates to tumor progression. Here we characterize tumor NK cells both phenotypically and functionally in the tumor microenvironment of endometrial cancer. For that, we gathered endometrial tumors, tumor adjacent healthy tissue, blood from matching patients and healthy donor blood to perform comparative analysis of NK cells. First we found that NK cells were impoverished in the tumor infiltrate. We then compared the phenotype of NK cells in the tumor and found that tumor resident CD103+ NK cells exhibited more co-inhibitory molecules such as Tigit, and TIM-3 compared to recruited CD103− NK cells and that the expression of these molecules increased with the severity of the disease. We showed that both chemokines (CXCL12, IP-10, and CCL27) and cytokines profiles (IL-1β and IL-6) were altered in the tumor microenvironment and might reduce NK cell function and recruitment to the tumor site. This led to hypothesize that the tumor microenvironment reduces resident NK cells cytotoxicity which we confirmed by measuring cytotoxic effector production and degranulation. Taken together, our results show that the tumor microenvironment reshapes NK cell phenotype and function to promote tumor progression.

Highlights of This Issue

AKT kinase, as a component of the PI3K/mTOR/AKT cascade, mediates chemotherapy resistance in ovarian cancer. Following preclinical evidence that AKT inhibition restores chemotherapy efficacy, Blagden and colleagues conducted a Phase IB combination study of the AKT inhibitor afuresertib given with carboplatin and paclitaxel chemotherapy in 29 patients with platinum-resistant ovarian cancer. A 32% response rate was observed, and the combination showed acceptable tolerability. These findings support a future randomized evaluation of this combination in treatment-resistant ovarian cancer.CNS involvement worsens the prognosis of patients with T-cell acute lymphoblastic leukemia (T-ALL), because most anticancer drugs cannot pass the blood brain barrier. The histone demethylase LSD1 is considered to be a therapeutic target of T-ALL, in which LSD1 is overexpressed and acts as a founder abnormality. T-ALL cells, however, are resistant to tranylcypromine-based conventional LSD1 inhibitors. Saito and colleagues developed a novel LSD1 inhibitor, S2157, via N-alkylation of tranylcypromine and demonstrated its ability to eradicate CNS leukemia in a murine T-ALL model. The brain-permeable LSD1 inhibitor S2157 would be highly beneficial for T-ALL patients with CNS involvement.JX-594 (Pexa-vec) is one of the most promising oncolytic virus platforms in clinical development as one of the few oncolytic viruses in phase III clinical trials. Chon and colleagues show that a murine version of JX-594 (JX) remodels the tumor microenvironment by facilitating the accumulation of T cells. As a result, poorly immunogenic tumors become sensitive to cancer immunotherapy. Of note, the triple-combination therapy of JX, αPD-1, and αCTLA-4 maximizes anticancer immunity and induces durable regression with improved overall survival. These findings demonstrate the potential of JX in combination with immune checkpoint inhibitors for improving anticancer immune responses.Tumor-infiltrating NK cell (TI-NK) numbers were an independent predictor of pathological complete response, a surrogate marker of patient outcome, to anti-HER2 antibody-based treatment in primary HER2 positive breast cancer. The findings of Muntasell and colleagues add strong support for a role of antibody-dependent cellular cytotoxicity for the antitumor effects of anti-HER2 antibodies and, hence, for future research strategies to overcome resistance directed at the recruitment of NK cells in NK “cold” tumors. Furthermore, combination of HLA class I expression and TI-NK cell numbers improved the ability to predict long-term clinical outcomes, suggesting new ways to tailor anti-HER2 therapeutic strategies.

Elucidating the Immune-Related Mechanisms by Which Probiotic Strain Lactobacillus casei BL23 Displays Anti-tumoral Properties.

We have recently described antitumor properties of Lactobacillus casei BL23 strain in both a mouse allograft model of human papilloma virus (HPV)-induced cancer and dimethylhydrazine-associated colorectal cancer. However, the mechanisms underlying these beneficial effects are still unknown. Interestingly, in vitro cellular models show that this bacterium is able to stimulate the production of high levels of IL-2. Because this cytokine has well-known antitumor properties, we decided to explore its role in the anti-cancer effects of BL23 using the HPV-induced cancer model. We found a negative correlation between IL-2 and tumor size confirming the necessity of IL-2 to protect from tumor development. Then, we blocked IL-2 synthesis using neutralizing monoclonal antibodies in mice that were challenged with lethal levels of tumor cells; this led to a significant reduction in the protective abilities of BL23. Next, we used a genetically modified strain of Lactococcus lactis to deliver exogenous IL-2 to the system, and in doing so, we were able to partially mimic the antitumor properties of BL23. Additionally, we showed the systemic role of T-cells in tumor protection through a negative correlation between tumor size and T-cells subpopulations and an increasement of BL23-specific local Foxp3 levels in tumor-bearing mice. Finally, we observed a negative correlation between tumor size and NK+ cells, but local recruitment of NK cells and cytotoxic activity appeared specific to BL23 treatment. Taken together, our data suggest that IL-2 signaling pathway plays an important role in the anti-tumoral effects of probiotic strain L. casei BL23. These results encourage further investigation in the use of probiotic strains for potential therapeutic applications to clinical practice, in particular for the treatment of colorectal cancer. Furthermore, our approach could be extended and applied to other potential beneficial microorganisms, such as gut microbiota, in order to better understand the crosstalk between microbes and the host.

Intravesical treatment of chemotherapeutic agents sensitizes bacillus Calmette‑Guerin by the modulation of the tumor immune environment.

Intravesical treatment with bacillus Calmette‑Guerin (BCG) is the most common treatment for preventing progression and recurrence of non‑muscle invasive bladder cancer. Our previous study using the N‑butyl‑N‑(4‑hydroxybutyl) nitrosamine (BBN)‑induced orthotopic bladder cancer model demonstrated that intravesical treatment with mitomycin C (MMC) and adriamycin (ADM) suppressed pro‑tumoral immunity, including the aggregation of tumor‑associated macrophages (TAMs) and regulatory T cells (Tregs) in the tumor microenvironment. Previous evidence supports the association of resistance to intravesical treatment of BCG with TAMs and Tregs. In the present study, we investigated the antitumoral efficacy of sequential intravesical treatments with chemotherapeutic agents and BCG in a BBN‑induced orthotopic bladder cancer model. Thirty‑six C57BL/6J mice bearing bladder cancer were randomly divided into six treatment groups as follows: control, BCG, MMC, ADM, MMC‑BCG and ADM‑BCG. Intravesical treatment was performed once a week for six weeks. One week after the completion of intravesical treatment, bladder and blood were harvested. MMC‑BCG and ADM‑BCG were more effective antitumor activities than BCG monotherapy. Bladders were subjected to immunohistochemical analysis and revealed that intravesical BCG treatment combined with MMC/ADM promoted the local recruitment of NK cells to the bladder as effectively as BCG monotherapy and reduced TAMs and Tregs in the bladder. Interleukin (IL)‑17 and granulocyte‑colony stimulating factor (G‑CSF) in serum were analyzed by enzyme‑linked immunosorbent assay and these levels were revealed to be elevated in mice treated with sequential treatments similar to levels following monotherapy with MMC and ADM. Our findings indicated that intravesical sequential treatment could suppress the resistance to BCG through the enhancement of antitumor immunity (induction of NK cells) and inhibition of pro‑tumoral immunity (reduction of TAMs and Tregs). Systemic changes in IL‑17 and G‑CSF may be involved in topical immunomodulation. Further studies including clinical trials may be required to establish an appropriate strategy based on the immunomodulation of the tumor microenvironment.

Targeting the Glyco-Antigen CD75s with the Tetravalent, Fc-Engineered Antibody ‘Ebu-141 Tetra’ Induces Potent Killing of B Cell Lymphoma and Plasma Cell Tumors

While monoclonal antibodies (MoAb) are already well established for the treatment of B cell-derived malignancies and usually show a good safety profile, not all patients benefit and relapses may be a problem. In order to identify novel surface structures suitable for antibody-based therapies and to improve killing mechanisms, ‘EBU-141 Tetra’ was developed.The parental MoAb EBU-141 is of mouse IgMk isotype, was generated in our laboratory and recognizes the glyco-antigen CD75s (previously CDw75), which are α-2,6-sialylated lactosamines on cell surface glycoproteins and glycolipids. CD75s was found on normal mature B cells and subsets of T cells, but is also expressed on certain lymphatic and solid tumors, e.g. mature B cell lymphoma, pancreatic and prostate cancer cells. EBU-141 specifically binds to CD75s on most B cell lymphoma, including Burkitt's lymphoma, FL, DLBCL, MCL, CLL, and interestingly plasma cell tumors. In addition, EBU-141 showed reactivity on a few cases of peripheral T-cell lymphoma, whereas classical Hodgkin lymphomas were consistently negative. Previously a chimeric IgG1k antibody, chEBU-141, was derived from EBU-141. Compared to the parental IgM antibody, chEBU-141 showed strongly reduced binding avidity, but was moderately effective in triggering antibody-dependent cell-mediated cytotoxicity (ADCC) of mature B cell lymphoma and malignant plasma cells via recruitment of NK cells. However, chEBU-141 lacked the potent complement-dependent cytotoxicity (CDC) observed with the parental EBU-141 antibody. The aim of this study was to generate a tetravalent binding, Fc-engineered chEBU-141 IgG1 antibody with enhanced binding avidity for CD75s and potent effector functions for antibody-based therapy of mature B cell lymphomas and multiple myeloma.Using the variable regions of EBU-141, the chimeric IgG1κ antibody with a protein-engineered Fc and tetravalent binding properties, named ‘EBU-141 Tetra’, was generated. This MoAb and relevant controls were produced by transient transfection of 293T cells and purified from cell culture supernatants by affinity chromatography. Direct anti-tumor effects and Fc-mediated modes of action were investigated in cell proliferation assays and chromium release experiments using lymphoma and myeloma cell lines. Peripheral blood mononuclear cells and serum of healthy donors were used as source of human effector cells and complement in the cytotoxicity experiments.The ‘EBU-141 Tetra’ showed improved binding to CD75s on cell surface of mature B cell lymphoma as well as myeloma plasma cells compared to the bivalent binding chEBU-141 IgG1. The higher avidity for CD75s resulted in markedly improved ADCC activity of the ‘EBU-141 Tetra’ against Daudi Burkitt's lymphoma and U266 plasma cells with EC50 values in the picomolar range and higher maximum lysis rates. In addition, the ‘EBU-141 Tetra’ regained CDC activity of the parental EBU-141 and demonstrated efficient killing of Burkitt's lymphoma and myeloma cell lines with human serum as complement source. Thus, recruitment of immune effector cells and activation of the complement system are the main modes of action of the novel, tetravalent, chimeric, Fc-engineered antibody ‘EBU-141 Tetra’ antibody.Our findings further demonstrate that highly potent IgG-like antibodies against glycan-structures can be generated from mouse IgM antibodies and may open a new therapeutic window for therapy of patients with mature B cell lymphomas and multiple myeloma. DisclosuresKlausz:Affimed: Research Funding. Otte:Affimed: Research Funding. Klapper:HTG Molecular Diagnostics, Inc.: Research Funding; Amgen: Honoraria, Research Funding; F.Hoffman-La Roche: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Regeneron: Honoraria, Research Funding. Peipp:Affimed: Research Funding. Gramatzki:Affimed: Research Funding.

Clinical and Biological Evaluation of the Novel CD30/CD16A Tetravalent Bispecific Antibody (AFM13) in Relapsed or Refractory CD30-Positive Lymphoma with Cutaneous Presentation: A Biomarker Phase Ib/IIa Study (NCT03192202)

Introduction: Natural Killer cells (NK cells) play an important role in tumor immune-surveillance. NK-mediated cell killing can occur through distinct mechanisms: (1) by activating the receptor NKG2D and natural cytotoxicity receptors (NCR), and (2) through the potent activating receptor CD16 (FcγRIII) which mediates antibody-dependent cell-mediated cytotoxicity (ADCC). Therapeutic strategies to harness the ability of NK cells to induce an ADCC response are emerging. AFM13 is a CD30/CD16A targeting high affinity bispecific tetravalent antibody that engages and activates NK cells. This study evaluates the ability of AFM13 to engage innate immunity through specific activation and recruitment of NK cells to tumors expressing CD30 and the impact of these effects on clinical outcome. In addition, it examines the immunologic changes in the tumor and peripheral blood as a function of the dose and method of administration of AFM13 over time.Methods: Subjects with relapsed or refractory CD30 expressing lymphoma with cutaneous involvement were recruited into 3 treatment cohorts: (1) 1.5 mg/kg IV weekly, (2) 7 mg/kg IV weekly and (3) 7 mg/kg continuous intravenous infusion (CIVI) over 5 days weekly. Each cohort consisted of 3 patients. Subjects received 8 weeks of therapy each cycle. Response assessment was performed on week 11 of each cycle by mSWAT, photography, PET imaging and peripheral blood flowcytometry. A second cycle was administered if there was no progression of disease. Subjects underwent mandatory tumor biopsies and peripheral blood immunologic studies during the first cycle of therapy as follows: pretreatment, day 5 post first dose, week 4 and week 8 of therapy. An optional biopsy was also collected at end of study. Tumor biopsies were analyzed and evaluated by a pathologist and IHC image analyzer to characterize immune cell subpopulations. Peripheral blood samples were analyzed by flowcytometry.Results: Nine subjects were accrued. Their age ranged from 37-79 years, 3 of 9 where white and 6 of 9 were men. The median number of prior therapies was 4 (1-11), 2 patients had progressed on Brentuximab vedotin, and 5 patients received total skin electron beam radiotherapy. The disease etiologies were as follows: 4 patients with transformed mycosis fungoides, 2 patients with systemic anaplastic large cell lymphoma-ALK negative, 2 patients with non- transformed mycosis fungoides and 1 patient with cutaneous anaplastic large cell lymphoma.Among the 8 subjects who were evaluable for response 4 of 8 (50%) achieved an objective response rate (ORR). In cohort 1 we observed 1 complete response (CR), 1 partial response (PR) and 1 stable disease (SD). In cohort 2, we observed 3 SD. In cohort 3, we observed 2 PRs and one subject awaiting disease assessment. Infusion related reaction (IRR) were observed in 4 of 9 subjects and managed conservatively. Cellulitis was noted in 3 subjects of which 2 developed bacteremia requiring intravenous antibiotics, and was not considered related to AFM13 treatment. There were no other treatment-emergent adverse events.In the peripheral blood, we observed a decrease in circulating NK cells during therapy (Weeks 1- 8) with post therapy recovery (by week 11) by following cells that were CD56+ CD3- , CD56+ CD16+ and NKp46+ by Flow Cytometry. We observed an increase over time in the expression of the activation marker CD69 on NK cells from responders compared to non-responders. Similarly, tumor biopsies in responders showed increased infiltration of CD56+ NK cells as opposed to non-responders. Flow quantitation of circulating CD4+ CD25+ T cells (Tregs) shows a decrease in responders vs. non-responders.Conclusions: AFM13 demonstrated a high ORR of 50 % among a population of heavily pretreated patients with a CD30 positive lymphoproliferative T-cell malignancy. AFM 13 exhibited activity post Brentuximab vedotin failure. In addition, biological changes in NK cells may predict response. This data are the first to demonstrate the therapeutic advantages of this bispecific, and the first to correlate changes in NK cells as a function of dose and schedule. AFM13 is able to engage the innate immune system through a specific activation and recruitment of circulating NK cells to tumors expressing CD30. This immunological tumor response may correlate with clinical response. We have begun to expand the patient cohorts, and pursue additional comprehensive correlative studies in this population. DisclosuresRodig:KITE: Research Funding; Bristol Myers Squibb: Research Funding; Affimed: Research Funding; Merck: Research Funding. O'Connor:Seattle Genetics: Research Funding; ADC Therapeutics: Research Funding; Celgene: Research Funding.

PO-378 Exercise-dependent regulation of immune cell function in a clinical cancer perspective

IntroductionExercise is used as supportive therapy for cancer patients, aiming to improve patients’ physical functioning, quality-of-life and cancer-related fatigue. Emerging evidence suggests that exercise may also have direct anti-oncogenic effects on disease progression. Yet, in order to pursue ‘exercise as cancer medicine’, detailed insight into the underlying mechanisms is warranted. In murine cancer models immune cell mobilisation leads to control of tumour growth through increased infiltration by immune cells in the tumour tissue in a manner dependent on release of adrenalin and IL-6. To look further into these mechanisms in humans, we studied the NK cell mobilisation -and characteristics in healthy subjects or cancer patients undergoing neo-adjuvant chemotherapy participating in an acute bout of exercise.Material and methodsBlood immune cell profiles were evaluated in 5 healthy subjects performing either 2 hours of interval-based cycling or 2 hours of cycling at constant Watt intensity, and in 4 patients with cancer of the gastro-esophageal junction (GEJ) performing an acute bout of interval-biking followed by resistance training. Blood was collected before training, after warm-up, during training, and after a resting period. The samples were analysed by flow cytometry staining for CD3, CD19, CD14, CD16, CD56, and HLA-DR for differentiation of T cells, B cells, NK cells, and monocytes. The abundance of Granzyme B and Ki67 reflected cytotoxic profile and proliferation status, respectively.Results and discussionsIn healthy subjects, we observed a rapid up to 4-fold mobilisation of NK cells to the circulation, as well as increased Granzyme B content, decreased Ki-67 expression, and increased cellular size in the mobilised NK cells. These findings were particularly evident with interval-based training. In the 4 GEJ cancer patients we found up to 8-fold increase in circulating NK-cells after 35 min of interval-based cycling, along with increases in Granzyme B abundance and decreased Ki67 expression.ConclusionAn acute bout of interval-based exercise mediates recruitment of cytotoxic NK cells to the circulation in both healthy young men and GEJ cancer patients undergoing neo-adjuvant chemotherapy. Furthermore, immune cell mobilisation in the cancer patients was comparable to (if not better than) the recruitment observed in healthy subjects.

Abstract 1740: Epigenetic reprogramming of the tumor microenvironment by entinostat increases tumor sensitivity to multivalent immunotherapy combinations with an IL-15 superagonist plus vaccine or immune checkpoint blockade

Abstract The clinical promise of cancer immunotherapy relies on the immune system recognizing and eliminating tumor cells identified as non-self. However, the tumor microenvironment (TME) can greatly impede a tumor targeted immune response via increasing immunosuppressive cells or hampering T and NK cell maturation, recruitment, and function through numerous pathways, including upregulation of immune checkpoints such as PD-L1. Hence, there is an unmet clinical need to develop effective therapeutic strategies that can reprogram the TME to restore tumor immune recognition and reverse immune evasion. We recently demonstrated that Entinostat, a class I histone deacetylase (HDAC) inhibitor, reverses tumor immune escape to T cell-mediated lysis. We hypothesize that the immune-mediated tumor elimination promoted by the IL-15/IL-15Rα superagonist ALT803 in combination with PD-L1 checkpoint blockade or a therapeutic adenoviral vaccine targeting CEA (Ad-CEA) will be augmented by the epigenetic reprograming of the TME induced by Entinostat. In preclinical studies, ALT803 has been shown to exhibit potent antitumor activity in multiple murine models of cancer through the expansion of NK and CD8+ T cells with high effector function. Here, we demonstrate that Entinostat increased extracellular expression of immune-relevant proteins on murine colon and breast carcinoma cells in vitro. Additionally, the frequency of T cells with an activated phenotype were increased in non-tumor bearing mice. In the MC38-CEA murine model of colon carcinoma, the addition of Entinostat augmented the antitumor activity promoted by ALT803 plus Ad-CEA resulting in increased survival. Furthermore, in the 4T1 murine model of triple-negative breast cancer, the combination of Entinostat with ALT803 and a monoclonal antibody targeting PD-L1 significantly reduced primary tumor weight relative to ALT803 plus anti-PD-L1 therapy and resulted in a significant reduction of the number of 4T1 tumor-forming cells in the lung. The immune mechanism associated with antitumor efficacy of these multivalent therapies was examined in both the periphery and TME. Overall, these studies provide a rationale for combining Entinostat with multivalent immunotherapy combinations, including cytokines, antibodies targeting PD-L1, and therapeutic cancer vaccines. Citation Format: Kristin C. Hicks, Karin M. Knudson, Frank R. Jones, Peter Ordentlich, Shahrooz Rabizadeh, Hing C. Wong, James W. Hodge, Jeffrey Schlom, Sofia R. Gameiro. Epigenetic reprogramming of the tumor microenvironment by entinostat increases tumor sensitivity to multivalent immunotherapy combinations with an IL-15 superagonist plus vaccine or immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1740.

Anti-inflammatory consequences of bile acid accumulation in virus-infected bile duct ligated mice.

Cholestatic patients exhibiting high bile acid serum levels were reported to be more susceptible to bacterial and viral infections. Animal studies in bile duct ligated (BDL) mice suggest that cholestasis leads to an aggravation of hepatic bacterial infections. We have investigated the impact of cholestasis on mouse cytomegalovirus (MCMV)-induced immune responses and viral replication. While MCMV did not aggravate BDL-induced liver damage, BDL markedly reduced MCMV-triggered chemokine expression and immune cell recruitment to the liver. MCMV-infected BDL mice showed diminished trafficking of Ly6C+/F4/80+ myeloid cells and NK1.1+ NK cells to the liver compared to MCMV infected control mice. Moreover, virus-driven expression of CCL7, CCL12, CXCL9 and CXCL10 was clearly impaired in BDL- compared to sham-operated mice. Furthermore, production of the anti-inflammatory cytokine IL-10 was massively augmented in infected BDL mice. In contrast, intra- and extrahepatic virus replication was unaltered in BDL-MCMV mice when compared to sham-MCMV mice. Cholestasis in the BDL model severely impaired pathogen-induced chemokine expression in the liver affecting CCR2- and CXCR3-dependent cell trafficking. Cholestasis resulted in reduced recruitment of inflammatory monocytes and NK cells to the liver.

Dual TLR2/9 Recognition of Herpes Simplex Virus Infection Is Required for Recruitment and Activation of Monocytes and NK Cells and Restriction of Viral Dissemination to the Central Nervous System.

The importance of TLR2 and TLR9 in the recognition of infection with herpes simplex virus (HSV) and HSV-caused diseases has been described, but some discrepancies remain concerning the benefits of these responses. Moreover, the impact of TLR2/9 on innate and adaptive immune responses within relevant mucosal tissues has not been elucidated using natural mucosal infection model of HSV. Here, we demonstrate that dual TLR2/9 recognition is essential to provide resistance against mucosal infection with HSV via an intravaginal route. Dual TLR2/9 ablation resulted in the highly enhanced mortality with exacerbated symptoms of encephalitis compared with TLR2 or TLR9 deficiency alone, coinciding with highly increased viral load in central nervous system tissues. TLR2 appeared to play a minor role in providing resistance against mucosal infection with HSV, since TLR2-ablated mice showed higher survival rate compared with TLR9-ablated mice. Also, the high mortality in dual TLR2/9-ablated mice was closely associated with the reduction in early monocyte and NK cell infiltration in the vaginal tract (VT), which was likely to correlate with low expression of cytokines and CCR2 ligands (CCL2 and CCL7). More interestingly, our data revealed that dual TLR2/9 recognition of HSV infection plays an important role in the functional maturation of TNF-α and iNOS-producing dendritic cells (Tip-DCs) from monocytes as well as NK cell activation in VT. TLR2/9-dependent maturation of Tip-DCs from monocytes appeared to specifically present cognate Ag, which effectively provided functional effector CD4+ and CD8+ T cells specific for HSV Ag in VT and its draining lymph nodes. TLR2/9 expressed in monocytes was likely to directly facilitate Tip-DC-like features after HSV infection. Also, dual TLR2/9 recognition of HSV infection directly activated NK cells without the aid of dendritic cells through activation of p38 MAPK pathway. Taken together, these results indicate that dual TLR2/9 recognition plays a critical role in providing resistance against mucosal infection with HSV, which may involve a direct regulation of Tip-DCs and NK cells in VT. Therefore, our data provide a more detailed understanding of TLR2/9 role in conferring antiviral immunity within relevant mucosal tissues after mucosal infection with HSV.

Dose intensification of TRAIL-inducing ONC201 inhibits metastasis and promotes intratumoral NK cell recruitment.

ONC201 is a first-in-class, orally active antitumor agent that upregulates cytotoxic TRAIL pathway signaling in cancer cells. ONC201 has demonstrated safety and preliminary efficacy in a first-in-human trial in which patients were dosed every 3 weeks. We hypothesized that dose intensification of ONC201 may impact antitumor efficacy. We discovered that ONC201 exerts dose- and schedule-dependent effects on tumor progression and cell death signaling in vivo. With dose intensification, we note a potent anti-metastasis effect and inhibition of cancer cell migration and invasion. Our preclinical results prompted a change in ONC201 dosing in all open clinical trials. We observed accumulation of activated NK+ and CD3+ cells within ONC201-treated tumors and that NK cell depletion inhibits ONC201 efficacy in vivo, including against TRAIL/ONC201-resistant Bax-/- tumors. Immunocompetent NCR1-GFP mice, in which NK cells express GFP, demonstrated GFP+ NK cell infiltration of syngeneic MC38 colorectal tumors. Activation of primary human NK cells and increased degranulation occurred in response to ONC201. Coculture experiments identified a role for TRAIL in human NK-mediated antitumor cytotoxicity. Preclinical results indicate the potential utility for ONC201 plus anti-PD-1 therapy. We observed an increase in activated TRAIL-secreting NK cells in the peripheral blood of patients after ONC201 treatment. The results offer what we believe to be a unique pathway of immune stimulation for cancer therapy.

Cancer vaccine based on a combination of an infection-enhanced adenoviral vector and pro-inflammatory allogeneic DCs leads to sustained antigen-specific immune responses in three melanoma models

ABSTRACTAutologous patient-derived dendritic cells (DCs) modified ex vivo to present tumor-associated antigens (TAAs) are frequently used as cancer vaccines. However, apart from the stringent logistics in producing DCs on a patient basis, accumulating evidence indicate that ex vivo engineered DCs are poor in migration and in fact do not directly present TAA epitopes to naïve T cells in vivo. Instead, it is proposed that bystander host DCs take up material from vaccine-DCs, migrate and subsequently initiate antitumor T-cell responses. We used mouse models to examine the possibility of using pro-inflammatory allogeneic DCs (alloDCs) to activate host DCs and enable them to promote antigen-specific T-cell immunity. We found that alloDCs were able to initiate host DC activation and migration to draining lymph node leading to T-cell activation. The pro-inflammatory milieu created by alloDCs also led to recruitment of NK cells and neutrophils at the site of injection. Vaccination with alloDCs combined with Ad5M(gp100), an infection-enhanced adenovirus encoding the human melanoma-associated antigen gp100 resulted in generation of CD8+ T cells with a T-cell receptor (TCR) specific for the gp10025-33 epitope (gp100-TCR+). Ad5M(gp100)-alloDC vaccination in combination with transfer of gp100-specific pmel-1 T cells resulted in prolonged survival of B16-F10 melanoma-bearing mice and altered the composition of the tumor microenvironment (TME). We hereby propose that alloDCs together with TAA- or neoepitope-encoding Ad5M can become an “off-the-shelf” cancer vaccine, which can reverse the TME-induced immunosuppression and induce host cellular anti-tumor immune responses in patients without the need of a time-consuming preparation step of autologous DCs.