Maturation Of Antigen-presenting Cells Research Articles

Synergistic effect of dual targeting vaccine adjuvant with aminated β-glucan and CpG-oligodeoxynucleotides for both humoral and cellular immune responses

Presently, clinically approved adjuvants (such as aluminum salts) fail to induce cellular immune responses, which is crucial to defend against intracellular pathogens (including HIV, malaria, tuberculosis and Ebola) and cancer. However, Freund’s complete adjuvant potently stimulates both humoral and cellular immune responses, accompanying by high toxicity and severe side reactions. Here in this work, a CpG-oligodeoxynucleotides (CpG-OND) crosslinked aminated β-glucan-Ovalbumin dual targeting nanoparticle (CpG-OND-AG-OVA) is prepared through a simple and mild ionic complexation method. The aminated β-glucan plays dual roles as antigen presenting cells (APCs) targeted carrier and immunopotentiator (targeting and activating dectin-1 on APCs). Meanwhile, CpG-OND also plays dual roles as ionic crosslinker and immunopotentiator (targeting and activating Toll-like receptor 9 in APCs). The adjuvant activity of the particles is evaluated through in vitro and in vivo experiments. The particles significantly enhance uptake and sustained proteolytic processing of antigens, and result in APCs maturation, inducing robust Th1 and Th2-type immune responses comparable to Freund’s adjuvant without obvious toxicity. The potent adjuvant activity of the nanoparticles may originate from dual targeting synergistic effects between aminated β-glucan and CpG-OND. Accordingly, the dual targeting nanoparticles may be a promising vaccine adjuvant for inducing robust humoral and cellular immune responses against infectious diseases and cancers. Statement of SignificanceAn ideal adjuvant for subunit vaccine should act as both a carrier to enhance the uptake, sustained processing and cytosolic delivery of antigens, and an immunopotentiator to stimulate antigen presenting cells (APCs) for activation of naive T cells. Additionally, it should be easy to obtain and safe with negligible toxicity. Unfortunately, both synthetic and natural polymers that have been developed into antigen delivery system cannot completely fulfill the requirements. In the present study, the authors design nanoparticles with aminated β-glucan and CpG-oligodeoxynucleotides (CpG-OND) through a simple and mild method. β-Glucan (a dectin-1 and TLR2 targeted PAMP) and CpG-OND (a TLR9 targeted PAMP) are readily accessible. Aminated β-glucan plays dual roles in the nanoparticle as APCs targeted carrier and immunopotentiator. Meanwhile, CpG-OND also plays dual roles as crosslinker and APCs targeted immunopotentiator. By making use of synergistic effect of the dual targeting vaccine adjuvant with aminated β-glucan and CpG-OND, the nanoparticles induce robust antigen specific immune responses comparable to Freund’s adjuvant without obvious toxicity.

Abstract 1760: The hexavalent CD40 agonist HERA-CD40L augments multi-level crosstalk between T cells and antigen-presenting cells

Abstract Introduction: HERA-CD40L is a novel hexavalent CD40 agonist engineered with the HERA-Technology developed by Apogenix. We have previously shown that the natural binding mode via the receptor/ligand binding domains and the high clustering capacity for the cognate receptor clearly distinguish HERA-CD40L from other, e.g. antibody-based, CD40-targeting compounds. Here, we report on the effects of HERA-CD40L on crosstalk between T cells and antigen presenting cells (APC) and the functional consequences in vitro. Materials & Methods: Biological activity of CD40 agonists was analyzed using co-cultures of primary T cells with B cells or monocytes/macrophages. All primary cells were isolated by negative selection using magnetic sorting from healthy donor buffy coats. Expression of CD markers upon CD40 ligation on B cells and monocytes was analyzed by flow cytometry (FC). Monitoring of T cell-induced killing of tumor cells primed in direct co-cultures with APC was done on a real-time cell analysis system (xCELLigence). For analysis of phagocytosis, we developed an FC-based assay employing primary monocytes/macrophages and Jurkat A3 cells. Results: Treatment of primary B cells and monocytes with HERA-CD40L induced expression of co-stimulatory molecules, like CD86, and promotes M1 maturation of naïve (M0) monocytes. In vitro, treatment of alternatively activated M2 macrophages with HERA-CD40L induced an M2 to M1 phenotype switch (re-programming) which concurs with CD16 downregulation and a dose-dependent decrease of phagocytic activity of re-programmed macrophages compared to M0 or M2 macrophages. Primary B cells and M1 macrophages enhanced the proliferation and cytotoxic activity of naïve T cells in direct co-cultures in the presence of HERA-CD40L. The activating effect on T cells required direct cell-cell contact with APC and was not observed in indirect co-cultures. Functionally, neutralization of either MHC-I or CD80/CD86 in direct co-cultures inhibited full activation of the T cells in vitro as shown by kill assays with various tumor cell lines. Conclusion: The hexavalent CD40 agonist HERA-CD40L produced by the Apogenix HERA-Technology is a potent immune-regulator acting on B cells and myeloid cells. HERA-CD40L promotes activation of B cells, maturation of APC and induces an M2 to M1 phenotype switch which inhibits tolerance-inducing phagocytic activity of the repolarized macrophages in vitro. In response to CD40 ligation on APC, an efficient anti-tumor response is conferred to primary T cells through cell-cell interactions via MHC-I and CD80/CD86. Citation Format: Christian Merz, Jaromir Sykora, Viola Marschall, David M. Richards, Meinolf Thiemann, Harald Fricke, Oliver Hill, Christian Gieffers. The hexavalent CD40 agonist HERA-CD40L augments multi-level crosstalk between T cells and antigen-presenting cells [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 1760.

Abstract 4936: Combination CD40 agonist and PD-1 antagonist antibody therapy enhances vaccine induced T cell responses in non-immunogenic cancers

Abstract A hallmark of many non-immunogenic cancers is the lack of tumor infiltrating lymphocytes (TIL) and/or failure to mount a robust anti-tumor T cell response via multiple mechanisms. The presence of T regulatory cells and myeloid derived suppressor cells (MDSCs) serve to dampen the immune response, and furthermore, tumor antigen-specific T cell tolerance limits the efficacy of therapeutic cancer vaccines. CD40 signaling is critical to the decision of whether cytotoxic T lymphocytes become primed or tolerized. Administration of monoclonal CD40 agonistic antibody (Ab) has been shown to promote CD8 activation in vivo, and likely alters the myeloid component of the tumor microenvironment. Our study asks the question of whether combining a T cell inducing vaccine and PD1 inhibition with CD40 agonistic Ab can induce T cell priming and TIL activation in non-immunogenic solid malignancies. We utilized mouse models of pancreatic ductal adenocarcinoma (PDAC) and breast cancer to assess the effects of drug combinations on intratumoral immune responses. Tumor-bearing mice were treated with a GM-CSF secreting vaccine (GVAX) + anti-PD1 Ab alone or in combination with CD40 Ab, or isotype control Ab, and monitored for survival. A separate cohort of mice were analyzed by immunohistochemistry and multi-color flow cytometry to assess T cell infiltration/activation and myeloid maturation. In a hemisplenectomy model of PDAC in which tumor cells were surgically implanted into wild-type recipients, mice treated with isotype control Abs succumbed to disease with extensive liver and peritoneal metastases at 35-70 days. GVAX + anti-PD1 Ab treatment displayed some efficacy, although 70% of mice eventually developed fatal liver metastases. In contrast, CD40 Ab was highly active, with 90% long-term survival afforded by a single administration of Ab, and mice treated with GVAX + anti-PD1 Ab + CD40 Ab had 100% survival. Similar trends in treatment efficacy were observed following subcutaneous tumor implantation of PDAC tumor cells in the lower limb. In an orthotopic model in which HER2/neu-expressing breast tumor cells were implanted into the mammary fat pad of syngeneic neu-N mice, we demonstrated delayed tumor progression and increased median survival in mice treated with GVAX + anti-PD1 Ab + CD40 Ab relative to either therapy alone. Further characterization of immune populations was carried out by high dimensional flow cytometric analysis utilizing PhenoGraph clustering and visualized by t-SNE. Changes were observed in monocytic and dendritic cell infiltration and maturation in the tumors of combination-treated mice. A significant decrease in granulocytic MDSCs was associated with response, as well as an increase in mature antigen presenting cells. In conclusion, GVAX, anti-PD1 and CD40 agonist Ab have potential synergy in modulating anti-tumor immunity in non-immunogenic cancers. Citation Format: Hayley S. Ma, Evanthia Roussos Torres, Bibhav Poudel, Tara Robinson, Brian Christmas, Kayla Cruz, Skylar Woolman, Christine Rafie, Blake Scott, Valerie Wall, Todd Armstrong, Elizabeth Jaffee. Combination CD40 agonist and PD-1 antagonist antibody therapy enhances vaccine induced T cell responses in non-immunogenic cancers [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 4936.

Abstract 1758: Intratumoral IL-12 mRNA promotes cytotoxic T cell-dependent anti-tumor immunity and enhances responses to PD-L1 blockade in pre-clinical models

Abstract As a central mediator of TH1 immune responses, interleukin 12 (IL-12) in the tumor microenvironment (TME) plays a key role in driving anti-tumor immunity. Recombinant IL-12 has been reported to promote CD8+ cytotoxic T cell (CTL) dependent anti-tumor immunity in a wide variety of preclinical models. However, systemic recombinant IL-12 was poorly tolerated in early clinical trials. We are therefore investigating whether intra-tumoral (IT) delivery of a messenger RNA encoding IL-12 formulated in lipid nanoparticles can drive local IL-12 expression in solid tumors to induce TME transformation and anti-tumor immunity. Here we report that IT IL-12 mRNA induces dose-dependent local IL-12 protein expression in mice bearing both subcutaneous syngeneic and patient-derived xenograft tumors. In multiple syngeneic tumor models (e.g. MC38, A20), mouse IL-12 mRNA (mIL-12 mRNA) treatment promoted dose-dependent tumor regression and significantly improved overall survival. Flow cytometry and luminex analysis revealed that anti-tumor activity of mIL-12 mRNA was accompanied by transformation of the TME characteristic of a TH1 immune response. IFNγ induction and natural killer cell activation was accompanied by antigen presenting cell maturation, increased CTL numbers and activation, and increased PD-L1 expression. Furthermore, rejection of established MC38 tumors in response to mIL-12 mRNA was CTL-dependent, and animals demonstrated immunity to subsequent re-challenge with MC38 tumor cells. Since mIL-12 mRNA enhanced PD-L1 expression in the TME, we next investigated whether PD-L1 blockade would enhance responses to mIL-12 mRNA. We found that the combination of a single dose of mIL-12 mRNA and PD-L1 blockade led to significantly more complete responses compared to mIL-12 mRNA monotherapy in the PD-L1 resistant MC38 model. Furthermore, A single dose of mIL-12 mRNA induced rejection of un-injected MC38 tumors in a dual flank model, and this was further enhanced by combination with PD-L1 blockade. These preclinical data demonstrate the potential for IT IL-12 mRNA to drive TME transformation into a proinflammatory state consistent with a TH1 immune response and to ignite CTL-dependent anti-tumor immunity, even in a PD-L1 resistant setting. Citation Format: Susannah L. Hewitt, Nadia Luheshi, Dyane Bailey, John Zielinski, Ameya Apte, Faith Musenge, Russell Karp, Grace Adjei, Andrew Leinster, Sushma Gurumurthy, Ankita Mishra, Kristen Arnold, Darren Potz, Robert W. Wilkinson, Ronald Herbst, Joshua P. Frederick. Intratumoral IL-12 mRNA promotes cytotoxic T cell-dependent anti-tumor immunity and enhances responses to PD-L1 blockade in pre-clinical models [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 1758.

Properdin: A multifaceted molecule involved in inflammation and diseases

Properdin, the widely known positive regulator of the alternative pathway (AP), has undergone significant investigation over the last decade to define its function in inflammation and disease, including its role in arthritis, asthma, and kidney and cardiovascular diseases. Properdin is a glycoprotein found in plasma that is mainly produced by leukocytes and can positively regulate AP activity by stabilizing C3 and C5 convertases and initiating the AP. Promotion of complement activity by properdin results in changes in the cellular microenvironment that contribute to innate and adaptive immune responses, including pro-inflammatory cytokine production, immune cell infiltration, antigen presenting cell maturation, and tissue damage. The use of properdin-deficient mouse models and neutralizing antibodies has contributed to the understanding of the mechanisms by which properdin contributes to promoting or preventing disease pathology. This review mainly focusses on the multifaceted roles of properdin in inflammation and diseases, and how understanding these roles is contributing to the development of new disease therapies.

Lymph Node-Targeting Nanovaccine through Antigen-CpG Self-Assembly Potentiates Cytotoxic T Cell Activation.

Therapeutic vaccines that arouse the cytotoxic T cell immune response to reject infected cells have been investigated extensively for treating disease. Due to the large amounts of resident antigen-presenting cells (APCs) and T cells in lymph nodes, great efforts have been made to explore the strategy of targeting lymph nodes directly with nanovaccines to activate T cells. However, these nanovaccines still have several problems, such as a low loading efficiency and compromised activity of antigens and adjuvants derived from traditional complicated preparation. There are also safety concerns about materials synthesized without FDA approval. Herein, we construct an assembled nanoparticle composed of an antigen (ovalbumin, OVA) and adjuvant (CpG) to ensure its safety and high loading efficiency. The activity of both components was well preserved due to the mild self-assembly process. The small size, narrow distribution, negative charge, and good stability of the nanoparticle endow these nanovaccines with superior capacity for lymph node targeting. Correspondingly, the accumulation at lymph nodes can be improved by 10-fold. Subsequently, due to the sufficient APC internalization and maturation in lymph nodes, ~60% of T cells are stimulated to proliferate and over 70% of target cells are specifically killed. Based on the effective and quick cellular immune response, the assembled nanoparticles exhibit great potential as therapeutic vaccines.

325 - Effect of fluoropolymer-based culture vessel surface on monocyte differentiation

Most cell therapy products require an ex vivo cell processing step in a polymeric culture device. The transition from open systems such as tissue culture polystyrene (TCPS) flasks to functionally closed systems such as cell culture bags often entails a change in materials in contact with cells. It is well established that cell-material interactions affect cell behavior and cell fate decisions. Here, we investigated the effect of different culture surface on monocyte-derived dendritic cells (Mo-DCs), a commercially relevant immunotherapy product. Monocytes were isolated from the peripheral blood of healthy donors and differentiated into Mo-DCs in fluoropolymer (FEP) bags and conventional TCPS vessels. Quartz crystal microbalance with dissipation monitoring (QCM-D) measurements indicated rapid adsorption of albumin, insulin and transferrin proteins from the medium onto fluoropolymer surfaces to reach equilibrium within the first 2 minutes in the medium. Consistent with these observations, contact angle and atomic force microscopy experiments showed a significant increase in fluoropolymer surface hydrophilicity and a decrease in rigidity at the surface following incubation in cell culture medium. After 7 days of differentiation and 2 days of maturation, Mo-DCs obtained both in FEP and TCPS cell culture vessels expressed the surface markers for mature antigen-presenting cells based on flow cytometry. Overall, although FEP is much more hydrophobic than TCPS, differences in surface properties decrease after protein adsorption, leading to similar performance for the production of Mo-DCs.

Curdlan sulfate-O-linked quaternized chitosan nanoparticles: potential adjuvants to improve the immunogenicity of exogenous antigens via intranasal vaccination.

IntroductionThe development of ideal vaccine adjuvants for intranasal vaccination can provide convenience for many vaccinations. As an ideal intranasal vaccine adjuvant, it should have the properties of assisting soluble antigens to pass the mucosal barrier and potentiating both systemic and mucosal immunity via nasal administration.MethodsBy using the advantages of polysaccharides, which can promote both T-helper 1 and 2 responses, curdlan sulfate (CS)–O-(2-hydroxyl)propyl-3-trimethyl ammonium chitosan chloride (O-HTCC) nanoparticles were prepared by interacting CS with O-HTCC, and the adjuvancy of the nanoparticles was investigated.ResultsThe results showed that the polysaccharide-based nanoparticles induced the proliferation and activation of antigen-presenting cells. High protein-loading efficiency was obtained by testing with the model antigen ovalbumin (Ova), and the Ova adsorbed onto the cationic CS/O-HTCC complexes was taken up easily by the epithelium. To evaluate the capacity of the Ova/CS/O-HTCC nanoparticles for immune enhancement in vivo, we collected and analyzed immunocytes, serum, and mucosal lavage fluid from intranasally vaccinated mice. The results showed that Ova/CS/O-HTCC nanoparticles induced activation and maturation of antigen-presenting cells and provoked the proliferation and differentiation of lymphocytes more significantly compared to the immunization of Ova mixed with aluminum hydroxide gel. Furthermore, CS/O-HTCC evoked a significantly higher level of Ova-specific antibodies.ConclusionTherefore, these results suggest that CS/O-HTCC nanoparticles are ideal vaccine adjuvants for soluble antigens used in intranasal or mucosal vaccination.

Induction of DNA Damage in Ovarian Cancer Induces Type I Interferon Signaling

ObjectiveEpithelial ovarian cancer patients initially respond to cytotoxic chemotherapy, however the majority develop recurrent disease that is resistant to current treatments and 5‐year survival is less than 50%. Immunotherapies have shown promise in some solid tumors, yet response rates in ovarian cancer are only 10–15%. We therefore sought to identify new approaches to increase ovarian cancer tumor immunogenicity with the goal of improving immunotherapy response rates. We previously found that the DNA damage repair protein DEK is overexpressed in ovarian cancer cell lines and primary tumors, and that decreasing DEK levels induces DNA damage and apoptotic cell death. Cytoplasmic DNA from damaged cells can activate the DNA‐sensing protein STING, resulting in induction of the type I interferon (IFN‐I) signaling pathway that facilitates antigen‐presenting cell (APC) maturation and cross‐priming of T cells. Therefore, our objective was to determine if decreasing DEK levels induces IFN‐I signaling to enhance ovarian cancer immune signaling.MethodsWe developed a new machine learning tool, Fast and Robust Deconvolution of Expression Profiles (FARDEEP) to characterize the landscape of tumor‐infiltrating lymphocytes (TIL) in 299 high grade serous ovarian cancers from the Cancer Genome Atlas (TCGA). We then performed RNA‐Seq analysis on OVCAR8 and OVCAR3 ovarian cancer cell lines with stable expression of shRNA targeting DEK (shDEK) or control shRNA. Gene expression patterns were analyzed by Gene Set Enrichment Analysis and confirmed by RT‐PCR. In addition, IFN‐I signature transcripts were further validated by RT‐PCR after ovarian cancer cell lines were infected with lentivirus expressing shDEK or control. ID8 cells from an immunocompetent ovarian cancer mouse model were analyzed by RT‐PCR for IFN‐I signatures after being transfected with siRNA targeting Dek or control without or with Sting expression plasmid.ResultsFARDEEP analysis demonstrated that elevated levels of IFN‐I signature genes including CXC chemokines CXCL9 and CXCL10 are correlated with TIL subsets essential for anti‐tumor immunity, indicating IFN‐I signaling is important in preventing ovarian cancer immune escape. RNA‐Seq in ovarian cancer cell lines revealed that decreasing DEK levels resulted in an upregulation of interferon response signals. Further evaluation of IFN‐I signaling demonstrated that decreasing DEK levels enhanced transcription of IFN‐I signature genes, including interferon beta 1 (IFNB1), CXCL9 and CXCL10. In addition, Dek‐deficiency in ID8 cells enhanced Sting‐mediated induction of Ifnb1, Cxcl9 and Cxcl10.ConclusionOur TIL deconvolution findings support the hypothesis that IFN‐I signaling is pivotal for the immunogenicity of ovarian cancer. Decreasing DEK expression induces DNA damage and resultant IFN‐I signaling. Further studies are underway with the ID8 ovarian cancer mouse model system. Our results suggest a potential approach to increase immune signaling in the tumor microenvironment and thus improve response rates to immunomodulatory therapies in ovarian cancer.Support or Funding InformationMichigan Ovarian Cancer Alliance Geri Fournier Ovarian Cancer Research AwardsThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

In vitro uptake of oligomannose-coated liposomes leads to differentiation of inflammatory monocytes into mature antigen-presenting cells that can activate T cells

Oligomannose-coated liposomes (OMLs), containing entrapped antigens, serve as effective antigen delivery vehicles and as a novel adjuvant to induce antigen-specific cellular immune responses. However, in vitro activation of antigen-presenting cells (APCs) by OMLs has not yet been demonstrated. In this paper, we found that OMLs can deliver the antigens and the stimulatory signals into inflammatory monocytes in vitro, leading to differentiation of the cells to mature APCs. When OMLs were co-cultured with peripheral blood mononuclear cells from C57BL/6 mice in the presence of mouse serum, OMLs were preferentially incorporated into both Ly6Chigh monocytes and Ly6Clow monocytes, which are referred to as murine inflammatory and resident monocytes, respectively. The expression of CD11c, CD80, CD86, CCR7, and MHC class II on the Ly6Chigh monocytes was significantly enhanced during the 24 h after OML uptake, whereas upregulation of these molecules on the Ly6Clow monocytes was limited. In addition, the antigenic peptide of OVA encased in OMLs was presented on MHC class I of only Ly6Chigh monocytes. Furthermore, OVA-encasing OML-ingesting monocytes can activate CD8+ T cells from OT-1 mice, suggesting that antigens encapsulated in OMLs were cross-presented in inflammatory monocytes. Adoptive transfer of the monocytes that engulf OVA-encasing OMLs led to induction of an antigen-specific Th1 immune response in mice. Taken together, mature APCs can be generated from inflammatory monocytes in peripheral blood by ex vivo treatment of the cells with OMLs without any additional stimuli.

Abstract OT1-01-04: A phase 2, open-label study of imprime PGG (Imprime), a novel beta glucan, with pembrolizumab (Pembro) in chemotherapy-resistant metastatic triple negative breast cancer (TNBC)

Abstract By blocking the interaction of PD-L1 with PD-1, immune checkpoint inhibitors (CPI) can unleash specific, anti-cancer killing function of activated cytotoxic T cells in patients (pts) for whom there is evidence of an ongoing anti-cancer immune response (PD-L1 expression and/or activated T cells within the tumor bed). Single agent CPI therapy has provided substantial clinical benefit to pts with multiple cancer types. Though effective, response rates are typically limited (˜15-30% of pts depending on tumor type) and therapy fails to benefit the majority of pts. For these pts there is often limited or no evidence of an ongoing T cell-based immune response. Agents that stimulate the anti-cancer immune response may be particularly promising in expanding the clinical responsiveness to CPI therapies. Imprime is a novel beta glucan derived from Saccharomyces acting mechanistically as a pathogen-associated molecular pattern (PAMP) or non-self danger signal, to awaken and activate the innate immune system. Imprime drives a cascade of immune activating events activating tumor-specific cytotoxic T cells. Imprime treatment elicits repolarization of the immunosuppressive microenvironment while activating the maturation of antigen presenting cells. Imprime has significantly enhanced the efficacy of CPI therapy in preclinical tumor models. In humans, Imprime-mediated innate immune activation requires the formation of an immune complex with naturally-occurring anti-beta glucan antibodies (ABA). Formation of this complex is dependent upon sufficient ABA levels. Imprime is now being studied in combination with pembro (KEYTRUDA®), a humanized mAb against PD-1 which has been previously studied in TNBC pts.This phase 2 study explores the treatment combination in pts with metastatic TNBC who progressed following at least one line of chemotherapy and pts with metastatic melanoma who progressed following CPI therapy with sufficient pre-treatment ABA levels (˜50% of screened patients). The study is a Simon 2-stage design. Specific to the TNBC tumor type, a sample size of 12 pts in Stage 1 is planned. Criteria to advance to Stage 2 are ≤4 GR 3/4 AEs and ≥2 objective responses in TNBC. An additional 30 TNBC pts may be enrolled in Stage 2. Main eligibility criteria are metastatic TNBC after chemotherapy in the metastatic setting and serum ABA ≥20 µg/mL. The primary endpoints are ORR and safety; secondary endpoints are TTR, CRR, DoR, PFS, and OS. Efficacy will be analyzed for ORR and CRR as point estimates with 95% CI and for PFS, OS, DoR and TTR as descriptive summaries. Safety parameters will be summarized. Exploratory endpoints include ORR and PFS based on irRECIST. This study aims to collect pre- and early on-treatment tumor (6 wks post-1st dose) biopsies and peripheral blood to assess the impact of the treatment combination on immune activating events in the periphery and at the tumor site. As of June 2017, 11 sites were open and 4 pts were in treatment. The trial is sponsored by Biothera Pharmaceuticals, Inc. in collaboration with Merck & Co. (ClinicalTrials.gov NCT02981303) For information, contact Richard D. Huhn, MD, Biothera Med Dir at rhuhn@biothera.com or 651-256-4657. Citation Format: O'Day SJ, Stopeck AT, Huhn RD, Gargano MA, Prathikanti R, Ma B, Mattson PM, Lowe JR, Bose N, Ertelt KE, Ottoson NC, Uhlik MT, Graff JR, Chisamore MJ. A phase 2, open-label study of imprime PGG (Imprime), a novel beta glucan, with pembrolizumab (Pembro) in chemotherapy-resistant metastatic triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr OT1-01-04.

Immune response to reovirus (REO) in phase I study with chemotherapy in patients with KRAS mutant metastatic colorectal cancer (mCRC).

217 Background: Viruses are well known immune sensitizing agents. The therapeutic efficacy of oncolytic reovirus in combination with chemotherapy is underway in a phase 1 study of mCRC. This study evaluates the nature of immune response by determining the distribution of antigen presenting cells (APCs) and activated T lymphocytes along with the cytokine expression pattern in peripheral circulation. Methods: REO was administered as a 60-minute intravenous infusion for 5 consecutive days every 28 days, at a tissue culture infective dose (TCID50) of 3x106. Serum was collected pre- and post- REO on days 1, pre REO on days 2-5, and days 8, 15, 22, and 29. Peripheral blood mononuclear cells (PBMC) were isolated and stained with fluorophore labelled antibodies against CD4, CD8, CD56, CD70, and CD123. Stained cells were fixed and evaluated by flow cytometry. The expression profile of 25 cytokines in plasma was assessed (post PBMC isolation) on an EMD Millipore multiplex Luminex platform. Results: Patients mount a robust immune response with dendritic cell maturation at 48 hrs (p < 0.01) followed by activation of cytotoxic T (CD8+) cells at Day 8 (p < 0.01). Cytokine assay indicated upregulation of Interleukin 1 beta (IL-1β; p = 0.004), Granulocyte-macrophage colony-stimulating factor (GM-CSF; p = 0.05), the chemokine Macrophage Inflammatory Proteins (MIP-1β; p = 0.05) at day 15. Furthermore, consistent upregulation of inflammatory cytokine IL-6 was seen from days 3 through 8 (p < 0.05), and decrease in IL-8 at 72 hrs (p = 0.03) was observed. Conclusions: REO induces strong immune response in patients with mCRC. APCs are stimulated within 48 hrs and activated (CD8+ CD70+) T cells within 168 hrs. Cytokine profiling indicates stimulation for maturation of APCs, chemotactic induction for macrophages and activation of T cells as highlighted by release of IL-1β, GM-CSF and MIP-1β respectively. Sustained increased expression of IL-6 (triggering lymphocyte maturation) and downregulation of IL-8 (pro-angiogenic cytokine) is also observed. REO thus functions bimodally as an oncolytic agent causing lysis of tumor cells, and facilitator of immune-mediated recognition and destruction of tumor cells. Clinical trial information: NCT01274624.

Tumor infiltrating lymphocyte recruitment in patients with early stage breast cancer after intramammary IRX-2 cytokine immunotherapy.

7 Background: The IRX-2 biologic is an injectable immunotherapy containing cytokines derived from stimulated lymphocytes. In preclinical models, IRX-2 activates T-cells, natural killer (NK) cells, macrophages, and dendritic cells, and facilitates maturation of antigen-presenting cells. Stromal tumor-infiltrating lymphocytes (sTILs) are associated with improved outcomes in early stage breast cancer (ESBC), and neoadjuvant IRX-2 increased TILs in a head/neck squamous cell carcinoma trial. We conducted a phase Ib trial to evaluate the feasibility/activity of IRX-2 in ESBC. Methods: Beginning 21 days prior to surgical resection, enrolled operable patients with stage I-III ESBC received the pre-operative IRX-2 regimen consisting of a single low dose of cyclophosphamide (300 mg/m2 to facilitate T-regulatory cell depletion), followed by 10 days of subcutaneous peri-areolar injections of IRX-2 into the affected breast (1 mL × 2 at tumor axis and at 90°). Endpoints were feasibility (primary), sTIL count (secondary), and immune monitoring by flow cytometry and immune transcriptome analysis (Nanostring PanCancer Immune Panel). Results: As of October 2017, 12 patients are enrolled and evaluable. All patients received all planned injections with no treatment-related surgical delays, complications, or grade III/IV toxicities. Treatment was associated with a mean 52% relative increase in sTILs (range –25% to +166%, p = 0.02). RNA expression profiling revealed the greatest increases in Th1 cell signature (mean +28.5%, range -5.3% to +283%), but also increases in CD8+, NK, and DC signatures. PDL1 RNA expression increased in 10/12 patients (mean +131.5%, range –83% to +1112%). In blood, increases in T-cell activation markers (ICOS, HLADR, and CD38) and T-reg depletion were observed at 1-month follow-up. Conclusions: Peri-lymphatic IRX-2 was well tolerated with preliminary evidence of sTIL recruitment, PDL1 upregulation, and peripheral lymphocyte activation. Study enrollment is ongoing, including patients for a pre-neoadjuvant-chemotherapy triple-negative ESBC cohort. Clinical trial information: NCT02950259.

Oxidation of factor VIII increases its immunogenicity in mice with severe hemophilia A

The development of antibodies against therapeutic factor VIII (FVIII) represents the major complication of replacement therapy in patients with severe hemophilia A. Amongst the environmental risk factors that influence the anti-FVIII immune response, the presence of active bleeding or hemarthrosis has been evoked. Endothelium damage is typically associated with the release of oxidative compounds. Here, we addressed whether oxidation contributes to FVIII immunogenicity. The control with N-acetyl cysteine of the oxidative status in FVIII-deficient mice, a model of severe hemophilia A, reduced the immune response to exogenous FVIII. Ex vivo exposure of therapeutic FVIII to HOCl induced a mild oxidation of the molecule as evidenced by the loss of free amines and resulted in increased FVIII immunogenicity in vivo when compared to native FVIII. The increased immunogenicity of oxidized FVIII was not reverted by treatment of mice with N-acetyl cysteine, and did not implicate an increased maturation of professional antigen-presenting cells. Our data document that oxidation influences the immunogenicity of therapeutic FVIII.

Generation and Characterization of Alloantigen-Specific Regulatory T Cells For Clinical Transplant Tolerance

Donor-specific CD4+CD127−CD25+FOXP3+ regulatory T cells (AgTregs) have the potential to induce clinical transplant tolerance; however, their expansion ex vivo remains challenging. We optimized a novel expansion protocol to stimulate donor-specific Tregs using soluble 4-trimer CD40 ligand (CD40L)-activated donor B cells that expressed mature antigen-presenting cell markers. This avoided the use of CD40L-expressing stimulator cells that might otherwise result in potential cellular contamination. Purified allogeneic “recipient” CD4+CD25+ Tregs were stimulated on days 0 and 7 with expanded “donor” B cells in the presence of IL-2, TGFβ and sirolimus (SRL). Tregs were further amplified by polyclonal stimulation with anti-CD3/CD28 beads on day 14 without SRL, and harvested on day 21, with extrapolated fold expansion into the thousands. The expanded AgTregs maintained expression of classical Treg markers including demethylation of the Treg-specific demethylated region (CNS2) and also displayed constricted TcR repertoire. We observed AgTregs more potently inhibited MLR than polyclonally expanded Tregs and generated new Tregs in autologous responder cells (a measure of infectious tolerance). Thus, an optimized and more clinically applicable protocol for the expansion of donor-specific Tregs has been developed.

Characterizing the effects of insertion of a 5.2 kb region of a VACV genome, which contains known immune evasion genes, on MVA immunogenicity

Modified Vaccinia virus Ankara (MVA) is an attenuated Vaccinia virus (VACV) that is a popular vaccine vector candidate against many different pathogens. Its replication-restricted nature makes it a safe vaccine. However, higher doses or multiple boosts of MVA are necessary to elicit an immune response similar to wild-type VACV. Multiple strategies have been used to create modified MVA viruses that remain safe, but have increased immunogenicity. For example, one common strategy is to delete MVA immunomodulatory proteins in hopes of increasing the host immune response. Here, we take the opposite approach and examine, for the first time, how re-introduction of a 5.2 kb region of VACV DNA (that codes for multiple immunomodulatory proteins) into MVA alters viral immunogenicity. Since antigen presenting cells (APCs) are critical connectors between the innate and adaptive immune system, we examined the effect of MVA/5.2 kb infection in these cells in vitro. MVA/5.2 kb infection decreased virus-induced apoptosis and virus-induced NF-κB activation. MVA.5.2 kb infection decreased TNF production. However, MVA/5.2 kb infection did not alter APC maturation or IL-6 and IL-8 production in vitro. We further explored MVA/5.2 kb immunogenicity in vivo. VACV-specific CD8+ T cells were decreased after in vivo infection with MVA/5.2 kb versus MVA, suggesting that the MVA/5.2 kb construct is less immunogenic than MVA. These results demonstrate the limitations of in vitro studies for predicting the effects of genetic manipulation of MVA on immunogenicity. Although MVA/5.2 kb did not enhance MVA’s immunogenicity, this study examined an unexplored strategy for optimizing MVA, and the insight gained from these results can help direct how to modify MVA in the future.

Cancer-Targeted Oncolytic Adenoviruses for Modulation of the Immune System.

Adenovirus is one of the most commonly used vectors for gene therapy and it is the first approved virus-derived drug for treatment of cancer. As an oncolytic agent, it can induce lysis of infected cells, but it can also engage the immune system, promoting activation and maturation of antigen- presenting cells (APCs). In essence, oncolysis combined with the associated immunostimulatory actions result in a "personalized in situ vaccine" for each patient. In order to take full advantage of these features, we should try to understand how adenovirus interacts with the immune system, what are the receptors involved in triggering subsequent signals and which kind of responses they elicit. Tackling these questions will give us further insight in how to manipulate adenovirus-mediated immune responses for enhancement of anti-tumor efficacy. In this review, we first highlight how oncolytic adenovirus interacts with the innate immune system and its receptors such as Toll-like receptors, nucleotide-binding and oligomerization domain (NOD)- like receptors and other immune sensors. Then we describe the effect of these interactions on the adaptive immune system and its cells, especially B and T lymphocytes. Finally, we summarize the most significant preclinical and clinical results in the field of gene therapy where researchers have engineered adenovirus to manipulate the host immune system by expressing cytokines and signalingmediators.

Comparative immunological study of penetrating and anterior lamellar keratoplasty in a murine model

In lamellar keratoplasty, the diseased part of a cornea is replaced while the healthy tissue remains lamellar keratoplasty has the advantage of better graft survival compared to penetrating keratoplasty (PK). We compared the immunological responses to PK and anterior lamellar keratoplasty (ALK) in a murine model. PK or ALK was performed using C57BL/6 donor grafts and BALB/c recipients, and graft opacity was assessed to evaluate graft rejection up to 8 weeks. We evaluated the immunological responses in both groups, which were not clinically considered as graft failure on postoperative day 21. PK mice showed reduced clinical graft survival compared to ALK mice. The mRNA expression of inflammatory mediators, such as IL-1β, IFN-γ, and granzyme B, in grafted corneas of PK mice, was significantly increased compared to the levels in ALK mice at postoperative day 21. PK led to a higher delayed-type hypersensitivity response and IFN-γ secretion in an in vitro T cell assay from draining lymph nodes (LNs), as compared to ALK. Furthermore, PK showed increased angiogenesis and lymphangiogenesis in grafted corneas compared to ALK and led to greater infiltration of CD3+ T cells into grafted corneas and increased frequencies of mature antigen presenting cells (APC; MHC-IIhighCD11c + cells) and IL-12 + dendritic cells (DCs) in the draining LNs of transplanted mice. In conclusion, PK results in increased graft rejection compared to ALK through relatively increased neovascularization and lymphangiogenesis, which can induce infiltration of pathologic T cells and mature APC migration into grafted corneas and draining LNs.

Dry eye syndrome: developments and lifitegrast in perspective.

Dry eye (DE) is a chronic ocular condition with high prevalence and morbidity. It has a complex pathophysiology and is multifactorial in nature. Chronic ocular surface inflammation has emerged as a key component of DE that is capable of perpetuating ocular surface damage and leading to symptoms of ocular pain, discomfort, and visual phenomena. It begins with stress to the ocular surface leading to the production of proinflammatory mediators that induce maturation of resident antigen-presenting cells which then migrate to the lymph nodes to activate CD4 T cells. The specific antigen(s) targeted by these pathogenic CD4+ T cells remains unknown. Two emerging theories include self-antigens by autoreactive CD4 T cells or harmless exogenous antigens in the setting of mucosal immunotolerance loss. These CD4 T cells migrate to the ocular surface causing additional inflammation and damage. Lifitegrast is the second topical anti-inflammatory agent to be approved by the US Food and Drug Administration for the treatment of DE and the first to show improvement in DE symptoms. Lifitegrast works by blocking the interaction between intercellular adhesion molecule-1 and lymphocyte functional associated antigen-1, which has been shown to be critical for the migration of antigen-presenting cells to the lymph nodes as well as CD4+ T cell activation and migration to the ocular surface. In four large multicenter, randomized controlled trials, lifitegrast has proven to be effective in controlling both the signs and symptoms of DE with minimal side effects. Further research should include comparative and combination studies with other anti-inflammatory therapies used for DE.

Immunomodulatory Role of Vitamin D: A Review.

Vitamin D is well known for its classical hormonal action related to the maintenance of mineral and skeletal homeostasis. However, the discovery that vitamin D receptor (VDR) is expressed in most non-skeletal tissues points to its broad role in the human organism. Current literature emphasizes a multidirectional role of vitamin D, with a special focus on its immunomodulatory properties. As VDR and the enzyme 1-α-hydroxylase are expressed in most immune cells, vitamin D modulates the phagocytic activity of macrophages and natural killer cells. In addition, it induces the microbicidal activity of phagocytes. In contrast, vitamin D suppresses differentiation and maturation of antigen-presenting dendritic cells and B lymphocytes, and it inhibits proliferation of Th1 and Th17 cells. In this review we aimed to describe the current scientific discoveries on the role of vitamin D as immunomodulator.