Activation Of Immune Effectors Research Articles

Genetic Engineering of Antibody Molecules

Antibodies are proteins produced by the immune system to combat pathogens and have long been appreciated for their exquisite specificity. The development of the hybridoma technology made it possible to immortalize single B cells, resulting in the production of unlimited quantities of antibodies of a single, well-defined antigen-binding specificity, known as monoclonal antibodies. However, the initial hybridoma-derived monoclonal antibodies were murine and highly immunogenic in humans. Advances in genetic engineering and expression systems have been used to overcome problems of the immunogenicity of rodent-produced antibodies, and to improve the ability of the antibodies to trigger human immune effector activity. The development of chimeric, humanized, and totally human antibodies, as well as antibodies with novel structures and functional properties, has further expanded the potential use of monoclonal antibodies as targeted therapeutics. As a consequence, recombinant antibody-based therapies are now used to treat a variety of diverse conditions that include infectious diseases, inflammatory disorders, and cancer. Today, these therapies are one of the fastest growing classes of biopharmaceutical therapeutics. The different strategies for developing recombinant antibodies and their derivatives are summarized and compared in this review. Keywords: antibody/antigen; antibody-dependent cell-mediated cytotoxicity (ADCC); antibody-dependent cell-mediated phagocytosis (ADCP); bacteriophage; complement; constant region; Fab; Fc; Fv/scFv; hybridoma; isotype; variable region

Activation of HIF-1α and LL-37 by commensal bacteria inhibits Candida albicans colonization.

Candida albicans colonization is required for invasive disease1-3. Unlike humans, adult mice with mature intact gut microbiota are resistant to C. albicans gastrointestinal (GI) colonization2,4. But the factors that promote C. albicans colonization resistance are unknown. Here we demonstrate that commensal anaerobic bacteria – specifically Clostridial Firmicutes (Clusters IV and XIVa) and Bacteroidetes – are critical for maintaining C. albicans colonization resistance in mice. Using Bacteroides thetaiotamicron as a model organism, we find that HIF-1α, a transcription factor important for activating innate immune effectors, and the antimicrobial peptide LL37-CRAMP are key determinants of C. albicans colonization resistance. While antibiotic treatment enables C. albicans colonization, pharmacologic activation of colonic Hif1a induces CRAMP expression and results in a significant reduction of C. albicans GI colonization and a 50% decrease in mortality from invasive disease. In the setting of antibiotics, Hif1a and Cramp are required for B. thetaiotamicron-induced protection against CA colonization of the gut. Thus, C. albicans GI colonization modulation by activation of gut mucosal immune effectors may represent a novel therapeutic approach for preventing invasive fungal disease in humans.

Indian Major CarpCirrhinus mrigala(Hamilton, 1822) Inoculated with LiveAeromonas hydrophilaShows Dynamic Changes in Specific Immune-Cellular Activities

The Indian major carp, mrigal (Cirrhinus mrigala Ham.), sensitised with live Aeromonas hydrophila, were used to evaluate in vitro cellular immune-effector activities against A. hydrophila antigens. Fishes were maintained in the laboratory in four groups, namely, two control C1 (n = 10) and C2 (n = 10) and two test (treated) groups namely T1 (n = 10) and T2 (n = 10). Test fishes (T1 and T2) were inoculated (100 μl) intra-peritoneally (I/P) with live A. hydrophila at 2 × 107 cfu/ml for sensitisation. At 7th, 15th and 21st day post inoculation (DPI), fish (n = 2) from each tank were sampled, mononuclear cells from spleen were isolated and plated at 2 × 105 cells per well. The stimulation indices (SI) of leucocytes stimulated by crude somatic antigens (SAs) were observed to be much higher in the sensitised fish than in control of all DPI. However, stimulation was observed to be peak at 15 DPI. The in vitro percent cytotoxicity of sensitised leucocytes was observed to be 65.8% at 7 DPI, 38.1% at 15 DPI and 13.21% at 21 DPI. The results showed high specific effector activities by immune cells of Indian major carp mrigal in the initial 15 DPI. The study reveals for the first time the dynamic changes in specific immune-cellular activities of mrigal fish in initial infective phases, experimentally inoculated with live A. hydrophila.

Preclinical Mechanistic Studies Investigating Neutrophil and Lymphoid Cell Depletion By IMGN529, a CD37-Targeting Antibody-Drug Conjugate (ADC)

CD37 is a surface antigen widely expressed on malignant B cells in non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL). In normal tissues, CD37 expression is restricted to lymphoid tissues and blood cells, with high levels of expression on B lymphocytes and low levels on non-B lymphoid and myeloid cells. IMGN529 is a CD37-targeting ADC currently in a Phase I clinical study in adult patients with relapsed or refractory NHL (NCT01534715). This ADC uniquely combines the intrinsic pro-apoptotic and immune effector activities of its anti-CD37 antibody component with the potent cytotoxic mechanism provided by targeted delivery of its maytansinoid payload, DM1. In the Phase I study, IMGN529 has demonstrated early evidence of clinical activity. A reduction in lymphocyte counts was also observed in the majority of patients after dosing, consistent with the proposed mechanism of action of a CD37-targeted therapy. However, in the initial dose-escalation phase, some patients experienced transient, early-onset neutropenia.To investigate the potential mechanisms of this transient neutropenia observed in patients, different pre-clinical models were considered and utilized to recapitulate clinical findings. In vitro studies with peripheral blood cells from normal human donors demonstrated that incubation with IMGN529 for 1 hour or 24 hours resulted in significant B-cell depletion with no apparent neutrophil depletion detected, similar to observations after rituximab treatment. In contrast, alemtuzumab treatment in vitro resulted in both B-cell and neutrophil depletion. This is consistent with the high level of CD37 expression on target B cells and the relatively low CD37 expression level on other blood cells. Analysis of cytokine release by normal human donor peripheral blood cells incubated with IMGN529 revealed increased levels of IL-8, CCL2 (MCP-1) and CCL4 (MIP-1β), but not IL-6 or TNF, to a similar extent as rituximab but less pronounced than alemtuzumab. An anti-murine CD37 antibody was identified to enable in vivo studies in a murine model and characterize CD37 expression on murine blood cells. Similar to the expression profile of CD37 in human peripheral blood cells, CD37 expression on murine peripheral blood cells was highest in B cells, with much lower expression seen on T cells and granulocytes. In vivo activity of the anti-muCD37 antibody and the corresponding ADC, with the same SMCC-DM1 linker-payload combination as IMGN529, was evaluated to discern antibody and payload-mediated events in comparison to the classic cytotoxic cyclophosphamide (CPA). Treatment of C57/B6 mice with 1-10 mg/kg of anti-muCD37 antibody or anti-muCD37 ADC resulted in a significant decrease in absolute lymphocyte counts (ALC) lasting greater than 7 days and a transient decrease in absolute neutrophil counts (ANC) lasting 1-2 days. A non-targeted control SMCC-DM1 ADC had no effect on ALC or ANC counts, suggesting the decrease is a CD37-mediated effect. In contrast, treatment with CPA resulted in an ALC decrease with similar kinetics but a more pronounced ANC decline. No impact on bone marrow lymphocyte, myeloid or erythroid precursor cell counts was observed in response to the anti-muCD37 antibody or anti-muCD37 ADC, whereas CPA treatment caused reduced cellularity with a decrease in the percentage of mature myeloid precursors and neutrophils in bone marrow. Elevated levels of CCL2 and CCL4 chemokines were detected in mouse plasma after anti-muCD37 ADC treatment, which may contribute to a redistribution of circulating neutrophils into peripheral tissues. Studies are currently underway to assess neutrophil distribution in murine tissues post anti-muCD37 ADC treatment.Current preclinical studies provide no clear evidence for direct IMGN529-mediated depletion of normal human neutrophils in the context of B-cell depletion in vitro. In vivo studies with an anti-muCD37 ADC recapitulate transient peripheral lymphopenia and neutropenia with no impact on bone marrow precursors observed, indicative of a different mechanism than classic chemotherapy-induced bone marrow myelosuppression. These preliminary results suggest a role for chemokine-mediated neutrophil redistribution following CD37 engagement, which is the subject of further studies. DisclosuresDeckert:ImmunoGen, Inc.: Employment, Equity Ownership. Ponte:ImmunoGen, Inc.: Employment, Equity Ownership. Coccia:ImmunoGen, Inc.: Employment, Equity Ownership. Lanieri:ImmunoGen, Inc.: Employment, Equity Ownership. Chicklas:ImmunoGen, Inc.: Employment, Equity Ownership. Yi:ImmunoGen, Inc.: Employment, Equity Ownership. Watkins:ImmunoGen, Inc.: Employment, Equity Ownership. Ruiz-Soto:ImmunoGen, Inc.: Employment, Equity Ownership; sanofi: Employment. Romanelli:ImmunoGen, Inc.: Employment, Equity Ownership; sanofi: Employment. Lutz:ImmunoGen, Inc.: Employment, Equity Ownership.

A Phase I Study of IMGN529, an Antibody-Drug Conjugate (ADC) Targeting CD37, in Adult Patients with Relapsed or Refractory B-Cell Non-Hodgkin’s Lymphoma (NHL)

Abstract Background: While CD37 is widely expressed on malignant B cells in NHL and chronic lymphocytic leukemia (CLL), few therapies are in development exploiting this target. In normal tissues, high CD37 expression is restricted to blood cells and lymphoid tissues, making CD37 well suited to an ADC approach. IMGN529 is a CD37-targeting ADC consisting of a CD37-binding antibody with intrinsic pro-apoptotic and immune effector activities conjugated to the maytansinoid anti-mitotic, DM1. Its unique profile enables IMGN529 to potentially kill CD37-positive B cells via multiple mechanisms of action. In preclinical studies, IMGN529 exhibits targeted, potent activity against NHL cells via direct inhibition of cell survival and effector function by its antibody and tubulin-disruption by DM1. Methods: A Phase I study is being conducted to evaluate safety, pharmacokinetics, pharmacodynamics, exploratory biomarkers and preliminary evidence of activity of IMGN529 and to determine the maximum tolerated dose/recommended phase 2 dose of IMGN529 in adult patients (pts) with relapsed or refractory NHL. IMGN529 is given intravenously on Day (d) 1 of each 21d cycle (C). Efficacy is evaluated based on Cheson response criteria. CD37 is being evaluated by IHC in available tumor samples to assess expression of CD37 among different NHL subtypes. Results: To date, 31 pts have been enrolled (66% male), median age of 65 years: Diffuse large B-cell (DLBCL, n = 14), Follicular lymphoma (FL, n = 10), MCL (n = 5), MZL (n = 2). Dose escalation began at 0.1 mg/kg. Early onset, transient grade 3-4 neutropenia was reported in 6 patients receiving doses at or below 0.8 mg/kg, potentially attributed to cytokine release. Peri-infusional steroid administration was added to the study protocol, and the incidence and severity of this neutropenia was significantly reduced. Additional patients have been enrolled and increasingly higher dose levels evaluated. To date the highest dose evaluated is 1.0 mg/kg. Cytokine levels are currently being evaluated in trial patients to gain a better understanding of the mechanisms underlying the early onset neutropenia. At the 1.0 mg/kg dose cohort grade 3-4 neutropenia was reported (1 DLT of febrile neutropenia among 6 patients) around d10 of the cycle and granulocyte colony stimulating factor (G-CSF) was added as primary prophylaxis. The most common treatment-emergent adverse events (AEs) occurring in ≥ 20% of the 31 pts enrolled were neutropenia (30%), fever (27%), asthenia (20%) and fatigue (20%). A reduction in lymphocyte count seen early after dosing (d2) in the majority of pts suggests a CD37-mediated reduction in lymphocytes, consistent with the mechanism of action of a CD37-targeted therapy. Four objective responses have been reported in patients who had received multiple lines of prior therapy. At the 1.0 mg/kg dose level, two patients with DLBCL who were heavily pretreated and who relapsed following autologous transplant have achieved an objective response that is ongoing. One pt has achieved a PR and one patient has achieved a CR. As previously reported, at doses of 0.2 mg/kg and 0.4 mg/kg one pt with transformed FL, who progressed following an autologous transplant, and one pt with DLBCL achieved a PR. The maximum tolerated dose has not yet been achieved and dose escalation is ongoing with additional data expected. Conclusions: IMGN529, a CD37-targeting ADC, demonstrates clinical activity in patients with NHL and has the potential to be a novel therapeutic for B-cell lymphoproliferative malignancies. Disclosures Stathis: ImmunoGen, Inc: Travel assistance Other; Pfizer: Research Funding; Oncoethix SA: Research Funding. Flinn:ImmunoGen, Inc: Research Funding. Maddocks:Pharmacyclics, Seattle Genetics, MorphoSys: Advisory Board Other, Research Funding. Zucca:Roche, Mundipharma, Novartis, Jannsen, Celgene: Consultancy, Honoraria, Research Funding, travel assistance Other. Romanelli:ImmunoGen, Inc.: Employment, Equity Ownership; sanofi: Employment. Zildjian:ImmunoGen, Inc: Employment. Ruiz-Soto:ImmunoGen, Inc: Employment; Sanofi: Past employment within 1 year, Past employment within 1 year Other.

Comparison of Ibrutinib, Idelalisib and Olaptesed Pegol on the Immune Effector Function Mediated By Rituximab

Olaptesed pegol (NOX-A12), an SDF-1 binding Spiegelmer® was found to detach SDF-1 from the surface of BMSCs (Hoellenriegel et al., Blood 2013) and to mobilize CXCR4 expressing, malignant cells from protective niches in the bone marrow or other secondary lymphoid tissues, thereby sensitizing them to the action of standard therapy. In addition to malignant cells CXCR4 expressing immune cells, e.g. T cells, neutrophils and monocytes are effectively mobilized by olaptesed (Vater et al., Clin Pharmacol Ther 2013). Here we investigated the combination of olaptesed with an immunotherapeutic agent, in particular rituximab (RTX). Several mechanisms of action (MoA) of RTX have been described including antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). The aim was to analyze whether olaptesed influences the immune effector cell activation by RTX as has been shown for the Btk or PI3Kd inhibitors ibrutinib and idelalisib, respectively.ADCC activity was measured by the calcein release assay. Raji lymphoma cells were labeled with calcein, treated with 10 µg/mL RTX and co-cultured for 4 hours with various ratios of PBMCs as effector cells, which have been incubated with either 10 µM olaptesed, 1 µM ibrutinib or 1 µM idelalisib, respectively, overnight. In order to analyze the impact on phagocyte function, ADCP assays with neutrophils and monocytes were performed. Phagocytes were preincubated with either 10 µM olaptesed, 1 µM ibrutinib or 1 µM idelalisib, respectively, for 6 hours, and cocultured overnight with DiD-labeled primary B-cells or Raji cells which were opsonized with different concentrations of RTX. ADCP was quantified in a guava easyCyte flow cytometer by counting the CD15+ (or CD14+) and DiD+ population (gated on CD19-negative cells) and normalizing to total phagocyte counts. To analyze B-cell depletion, heparinized whole blood from healthy volunteers was diluted with HBSS (1:2.5) and incubated with either 10 µM olaptesed, 1 µM ibrutinib or 1µM idelalisib, respectively, for 6 hours followed by the addition of different RTX concentrations. After incubation for 16 hours B-cell depletion was evaluated by determining the CD19+ B-cell and CD3+ T-cell populations.We found that preincubation of PBMCs with 1 µM ibrutinib significantly decreased calcein release. 1 µM idelalisib led to a slight decrease of calcein release, while 10 µM olaptesed did not influence ADCC activity (Figure 1A). Preliminary results from ADCP assays point to a decreased phagocytic activity in terms of maximal ADCP for neutrophils after preincubation with ibrutinib or idelalisib (Figure 1B). Importantly, although olaptesed is being endocytosed by phagocytes, the uptake of the drug did not inhibit ADCP. In accordance, the whole blood assay showed that olaptesed had no negative impact on RTX-mediated B-cell depletion, while ibrutinib and idelalisib were found to decrease B-cell depletion regarding EC50 and maximal killing (Figure 1C). Taken together, we found that olaptesed retains the immune functional repertoire of RTX, whereas ibrutinib and idelalisib decrease the immune effector function. These small molecule kinase inhibitors possess low specificity and are inhibiting also other kinases which may be important for immune cell activation, such as ITK which is blocked by ibrutinib. The MoA of olaptesed is partially overlapping with kinase inhibitors with regard to lymphoid cell mobilization; however, olaptesed is highly selective and therefore does not impair immune-cell mediated contribution to monoclonal antibody efficacy. Instead, olaptesed may further enhance the contribution of immune effector cells through mobilization of T cells from the bone marrow which are described to be exhausted in the peripheral blood of hematological malignancies like CLL. Furthermore, neutrophils and monocytes which contribute to ADCP are effectively mobilized with olaptesed.Based on their complementary mechanisms of action involving direct and immune-mediated cell death (RTX) or long-term mobilization of not only the malignant cells but also effector immune cells (olaptesed), the combination has the potential for greater efficacy in treating B lymphoid malignancies without impairing immune effector activity. Olaptesed is currently being tested in a Phase 2a clinical trial in combination with RTX and bendamustine in patients with relapsed/refractory CLL (#NCT01486797). [Display omitted] DisclosuresZboralski:NOXXON Pharma AG: Employment. Vater:NOXXON Pharma AG: Employment. Kruschinski:NOXXON Pharma AG: Employment.

A low, non-toxic dose of paclitaxel can prevent dendritic cell-precursors from becoming tolerogenic dendritic cells with impaired functions.

Tumor infiltrating dendritic cells (TIDCs) are thought to be potent antigen-presenting cells able to activate tumor-specific cytotoxic T lymphocytes (CTLs) or tolerogenic DCs that suppress immune reaction against tumors to escape. We have recently reported that majority of these TIDCs were DEC-205(+) DCs having a cross-presenting ability of captured tumor antigens to CD8(+) T cells via class I MHC (MHC-I) molecules, nevertheless, when the TIDCs expressed down-modulated costimulatory molecules, such as CD80 and CD86, they will inhibit the priming and activation of immune effectors (Immunol. Cell Biol., 91: 545-555, 2013). Here, we show that DC-precursors (preDCs) but not the established DCs become tolerogenic DCs expressing down-regulated costimulatory molecules having low responsiveness to LPS or tumor cells, when exposed to soluble factors released from the encountered ovarian tumors in the early phase of their development. However, we found that we could reduce the secretion of those soluble factors with a low, nontoxic concentration of paclitaxel (PTX) and we could stop the preDCs to be tolerogenic DCs and maintain DC functions. These findings indicate that we could prevent the induction of tolerogenic DCs from preDCs by using low, non-toxic doses of anti-cancer drugs to establish DCs that effectively elicit tumor-specific CTLs.

Immunity to viruses

The analysis of the innate and adaptive immune response to viruses has provided fundamental insight into the functioning of the immune system. Early studies on the host response to virus infection were instrumental in establishing the concept of immunological tolerance (1). Similarly, the realization that T lymphocytes are ‘restricted’ in their recognition of antigens by gene products encoded within the major histocompatibility complex (MHC) locus came from the analysis of T-lymphocyte recognition of virus-infected cells (2). Likewise, important initial insights into antigen processing and presentation came from the analysis of viruses and virus-infected cells (3, 4). Until recently, it was convenient to view immunity to infectious agents like viruses as a separate branch of immunology distinct from immunity to tumors, self-molecules (antigens), or allergens. As demonstrated in many of the articles in this volume of Immunological Reviews, the mechanisms underlying the induction and regulation of the innate and adaptive immune response to viruses represent the same processes controlling immunity to tumor antigens, allergens, and self-constituents. Thus, the results summarized in these review articles and the implications of these findings are applicable not only to those of us who study immunity to viruses but also to the immunology community at large. In selecting the topics for review in this volume of Immunological Reviews, we were first and foremost limited by space. Consequently, many important contributors to the field of viral immunology (indeed several topics) are not represented in this volume. Our selection of topics and authors was biased towards emerging areas, such as the application of systems biology approaches to viral pathogenesis and immunity, the contribution of inflammatory and stress responses to the induction of Innate and adaptive immune responses, and the impact of the microbiome on immunity to virus infection. We also considered it relevant to include reviews focused on well defined areas where recent findings have resulted in potential paradigm shifts in our understanding of topics such as B-lymphocyte or T-lymphocyte responses to virus infection. This volume of Immunological Reviews is also somewhat weighted towards analyses of the immune response to respiratory viruses. With the recent episodic infections with the severe acute respiratory syndrome (SARS)-like coronavirus and the outbreak of human infections with the avian influenza A H7N9 virus, respiratory viruses such as these organisms are recognized as major human pathogens with the potential for pandemic spread. Therefore, this class of viruses is a focus of immunological research. The reviews in this volume can be grouped according to the following scheme: (i) molecules and cells regulating the induction of the innate and adaptive response, (ii) expression of immune effector activity, (iii) regulation of the antiviral immune response, and (iv) systems analysis of the host response to infection and vaccination (Fig. 1). Fig. 1 Immunity to viruses

RG7116, a Therapeutic Antibody That Binds the Inactive HER3 Receptor and Is Optimized for Immune Effector Activation

The EGF receptor (EGFR) HER3 is emerging as an attractive cancer therapeutic target due to its central position in the HER receptor signaling network. HER3 amplifies phosphoinositide 3-kinase (PI3K)-driven tumorigenesis and its upregulation in response to other anti-HER therapies has been implicated in resistance to them. Here, we report the development and characterization of RG7116, a novel anti-HER3 monoclonal antibody (mAb) designed to block HER3 activation, downregulate HER3, and mediate enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) via glycoengineering of the Fc moiety. Biochemical studies and X-ray crystallography revealed that RG7116 bound potently and selectively to domain 1 of human HER3. Heregulin binding was prevented by RG7116 at concentrations more than 1 nmol/L as was nearly complete inhibition of HER3 heterodimerization and phosphorylation, thereby preventing downstream AKT phosphorylation. In vivo RG7116 treatment inhibited xenograft tumor growth up to 90% relative to controls in a manner accompanied by downregulation of cell surface HER3. RG7116 efficacy was further enhanced in combination with anti-EGFR (RG7160) or anti-HER2 (pertuzumab) mAbs. Furthermore, the ADCC potency of RG7116 was enhanced compared with the nonglycoengineered parental antibody, both in vitro and in orthotopic tumor xenograft models, where an increased median survival was documented. ADCC degree achieved in vitro correlated with HER3 expression levels on tumor cells. In summary, the combination of strong signaling inhibition and enhanced ADCC capability rendered RG7116 a highly potent HER3-targeting agent suitable for clinical development.

TLR3 agonists and proinflammatory antitumor activities

Although tumor growth leads to inflammatory responses, the immune system develops tolerance to cancer. One way to break host tolerance to tumors is to activate key immune effector activities. Toward this end, various adjuvants are under investigation in an effort to harness the immune system to overcome tolerance to tumor-associated self-antigens. There is enthusiasm for the use of specific ligands for toll-like receptor 3 (TLR3) that play a key role in the innate immune system. TLR3 agonists serve as immune adjuvants because they potently induce innate immune responses by activating dendritic cell (DC) maturation and inflammatory cytokine secretion. These activities facilitate the bridge between the innate and adaptive immune systems promoting the expansion of cytotoxic T lymphocytes (CTL) that destroy cancer cells. TLR3 agonists either alone or in combination with tumor antigens have shown success in terms of enhancing immune responses and eliciting antitumor activity in preclinical models. However, TLR3 agonists can also impact regulatory cells that dampen immune responses. Thus, immune strategies that utilize TLR3 agonists should consider the relative induction of suppressive as well as beneficial antitumor immune activities. Herein, we summarize the TLR3 agonists that will hopefully come to clinical fruition.

Immune effector activities in challenged rohu, Labeo rohita after vaccinating with Aeromonas bacterin

The present study assessed the efficacy of formalin inactivated Aeromonas vaccines by determining the cellular immune-effector activities such as production of oxidative radical (ROS) and nitrite and lymphocyte proliferation in challenged rohu, Labeo rohita upon vaccination with Aeromonas hydrophila (AH), Aeromonas caviae (AC) and a cocktail of both (CKTL). The relative percentage survival was much higher in AH group (68.89) than in the AC (28.89) and CKTL (20.00) groups. The vaccinated L. rohita showed high immune-effector activities in the first 15days post-vaccination, which subsequently lowered at different post-challenge periods. The vaccination activated the synthesis of ROS in leucocytic cells of all vaccinated groups up on challenge with A. hydrophila-C36 strain as immediate protection against the bacteria and the activities of blood phagocytes progressively waned as the disease process progressed. The production of nitrite was higher in AH group when stimulated with OMP antigen than that of somatic antigen and LPS; while in AC group, both OMP and somatic antigen stimulation resulted in higher nitrite production. On the other hand, the cocktail group showed higher nitrite production upon somatic antigen stimulation followed by OMP and LPS. A high level of non-specific lymphocyte proliferation was observed on the 10th day post-vaccination and the 5th day post-challenge periods and an antigen specific proliferation when stimulated with somatic and OMP antigens on the 15th day post-challenge. The stimulation by specific somatic and OMP antigens resulted in higher HK leucocyte proliferation in A. hydrophila vaccinated group. The experiment revealed that the highest protection was generated in the AH group compared to the other two groups and this might be explained by the specific cellular immune response generated in this vaccinated group against the specific antigen as assessed at 15days post-challenge.

MMP1, MMP9, and COX2 Expressions in Promonocytes Are Induced by Breast Cancer Cells and Correlate with Collagen Degradation, Transformation-Like Morphological Changes in MCF-10A Acini, and Tumor Aggressiveness

Tumor-associated immune cells often lack immune effector activities, and instead they present protumoral functions. To understand how tumors promote this immunological switch, invasive and noninvasive breast cancer cell (BRC) lines were cocultured with a promonocytic cell line in a Matrigel-based 3D system. We hypothesized that if communication exists between tumor and immune cells, coculturing would result in augmented expression of genes associated with tumor malignancy. Upregulation of proteases MMP1 and MMP9 and inflammatory COX2 genes was found likely in response to soluble factors. Interestingly, changes were more apparent in promonocytes and correlated with the aggressiveness of the BRC line. Increased gene expression was confirmed by collagen degradation assays and immunocytochemistry of prostaglandin 2, a product of COX2 activity. Untransformed MCF-10A cells were then used as a sensor of soluble factors with transformation-like capabilities, finding that acini formed in the presence of supernatants of the highly aggressive BRC/promonocyte cocultures often exhibited total loss of the normal architecture. These data support that tumor cells can modify immune cell gene expression and tumor aggressiveness may importantly reside in this capacity. Modeling interactions in the tumor stroma will allow the identification of genes useful as cancer prognostic markers and therapy targets.

Dynamic changes in immune-effector activities of Indian major carp, catla (Catla catla) infected with Edwardsiella tarda

Dynamic changes in immunoreactivity (innate and specific) of catla (Catla catla) against the bacterial pathogen Edwardsiella tarda was evaluated in the present study. Healthy catla were injected intraperitoneally with the LD50 dose of 105.5CFU/ml to induce edwardsiellosis. Different innate and specific immune responses were measured for the injected and control group on 0th, 2nd, 4th, 7th, 10th, 14th, 20th and 27th day post-injection (p.i.). The clinical and gross signs were first visible at 1day p.i. and the infected fish showed typical signs of hemorrhagic septicemia. Study of innate immunological parameters viz. superoxide anion production and nitric oxide production showed initial decrease at 2nd and 4th day p.i. followed by increase at 7th and 10th day p.i., respectively. However, the lysozyme activity showed a different trend with no decrement at initial stage. Anti-Edwardsiella antibody production was measured by indirect ELISA that showed an increasing trend with the highest observed on 20th day p.i. Another specific immune parameter i.e. lymphoproliferation showed almost similar trend like antibody production with the highest response observed on 20th day p.i. The results suggest that the protective immunity of catla against E. tarda essentially depends on the co-operation of both innate and specific immune responses.

In this issue: Harnessing Immunity against Difficult Targets, through the CD40-CD40L Pathway

This issue highlights translational aspects related to the CD40-CD40L pathway, a veritable master-switch at the interface between the innate and adaptive immunity. From direct targeting of CD40 onto tumor cells to licensing professional antigen presenting cells (APC), and from in vivo activation of immune effectors to enabling adoptive cell immune interventions, this pathway already shows translational promise in oncology and infectious diseases. In light of the pivotal nature and versatility of the target, the authors discuss a diverse range of options to design and advance novel, safe and effective immune interventions or combinatorial therapies involving CD40 agonistic engagement.

Abstract 770: Lorvotuzumab mertansine (IMGN901) immune effector activity and its effect on human NK cells

Abstract Lorvotuzumab mertansine, or IMGN901, is a conjugate consisting of the cytotoxic maytansinoid, DM1, covalently linked to the monoclonal antibody, lorvotuzumab, which selectively targets CD56 (also called neural cell adhesion molecule or NCAM). This conjugate is currently being evaluated in clinical trials for the treatment of CD56+ solid tumors and multiple myeloma. It is able to inhibit tumor cell growth by disruption of microtubule dynamics through delivery of its cytotoxic DM1 payload; however, other potential cell killing mechanisms, such as the immune effector activity of its antibody component, have not been fully investigated. Herein, we report on the immune effector activity of the lorvotuzumab antibody and the lorvotuzumab mertansine conjugate and their effect on the function of CD56+ human primary NK cells in vitro. To investigate the effector activity of the antibody, lorvotuzumab was incubated with CD56+ tumor target cells in the presence of rabbit complement or purified human primary NK cells. The results show that this antibody fails to mediate complement dependent cytotoxicity (CDC) but induces antibody-dependent NK cell-mediated cytotoxity (ADCC) in dose-dependent manner. The complete conjugate showed ADCC activity comparable to lorvotuzumab alone, suggesting that DM1 conjugation did not alter the interaction between the Fc domain of the antibody and NK cell's Fc receptor. Since human primary NK cells express CD56, albeit at a low level, we tested whether lorvotuzumab mertansine or its antibody component could negatively impact the function of NK cells. The results demonstrate that incubation with this antibody or with this conjugate has no effect on rituximab-mediated ADCC activity of the NK cells. Furthermore, overnight exposure of human primary NK cells with lorvotuzumab mertansine did not change rituximab-mediated ADCC activity. In conclusion, the lorvotuzumab antibody has ADCC activity that is retained after DM1 conjugation. Furthermore, neither lorvotuzumab nor its DM1 conjugate, appear to affect the ability of NK cells (which are CD56+) to mediate ADCC through another antibody. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 770. doi:10.1158/1538-7445.AM2011-770

Variable Antibody-dependent Activation of Complement by Functionalized Phospholipid Nanoparticle Surfaces

A wide variety of nanomaterials are currently being developed for use in the detection and treatment of human diseases. However, there is no systematic way to measure and predict the action of such materials in biological contexts. Lipid-encapsulated nanoparticles (NPs) are a class of nanomaterials that includes the liposomes, the most widely used and clinically proven type of NPs. Liposomes can, however, activate the complement system, an important branch of innate immunity, resulting in undesirable consequences. Here, we describe the complement response to lipid-encapsulated NPs that are functionalized on the surface with various lipid-anchored gadolinium chelates. We developed a quantitative approach to examine the interaction of NPs with the complement system using in vitro assays and correlating these results with those obtained in an in vivo mouse model. Our results indicate that surface functionalization of NPs with certain chemical structures elicits swift complement activation that is initiated by a natural IgM antibody and propagated via the classical pathway. The intensity of the response is dependent on the chemical structures of the lipid-anchored chelates and not zeta potential effects alone. Moreover, the extent of complement activation may be tempered by complement inhibiting regulatory proteins that bind to the surface of NPs. These findings represent a step forward in the understanding of the interactions between nanomaterials and the host innate immune response and provide the basis for a systematic structure-activity relationship study to establish guidelines that are critical to the future development of biocompatible nanotherapeutics.

Molecular cross-talk between Helicobacter pylori and human gastric mucosa

Helicobacter pylori (H. pylori) has co-evolved with humans to be transmitted from person to person and to colonize the stomach persistently. A well-choreographed equilibrium between the bacterial effectors and host responses permits microbial persistence and health of the host, but confers a risk for serious diseases including gastric cancer. During its long coexistence with humans, H. pylori has developed complex strategies to limit the degree and extent of gastric mucosal damage and inflammation, as well as immune effector activity. The present editorial thus aims to introduce and comment on major advances in the rapidly developing area of H. pylori/human gastric mucosa interaction (and its pathological sequelae), which is the result of millennia of co-evolution of, and thus of reciprocal knowledge between, the pathogen and its human host.

Compartment-Specific Bioluminescence Imaging Platform for the Open-Ended Identification of Novel Immunomodulatory Agents and High-Throughput Evaluation of Anti-Tumor Immune Function

AbstractAbstract 451Given the heterogeneity of human cancers, to comprehensively interrogate the anti-tumor cytotoxicity of immune effector cells for the identification of novel treatments, high-throughput platforms are vital for screening the multitude of cell types and experimental conditions and drugs. Conventional assays for anti-tumor immune effector cell activity have limited high-throughput scalability (e.g. use of radioactivity for Cr release) or measure indirect markers (e.g. IFN-γ levels, LDH release). In addition, conventional cell viability assays (e.g. MTT, Alamar Blue, CellTiterGlo) are suboptimal because the background viability signal from immune cells confounds the selective readout of tumor cell viability. To address these limitations, we adapted the recently developed Compartment-Specific Bioluminescence Imaging (CS-BLI) technique (McMillin et al, Nature Med 2010) to study tumor-immune system interactions. We first applied CS-BLI to quantify the anti-tumor activity of IL-2 pre-stimulated PBMCs on luciferase (Luc)-expressing MM.1S multiple myeloma (MM) cells or KU812F leukemic cells. Immediate CS-BLI bioluminescence readout showed significant linear correlation with viable tumor cell number, both in the presence (r=0.9996, p<0.01) and absence (r=0.9994, p=0.01) of Luc-negative PBMCs. In 4hr co-cultures at increasing PBMC-to-tumor cell ratios were concordant with results obtained by Cr release assays under the same experimental conditions. Consistent with prior studies, CS-BLI confirmed that the anti-MM cytotoxicity of IL-2-stimulated (10 ng/mL, 24 hrs) healthy donor PBMCs was enhanced by PBMC pretreatment with lenalidomide (Len) or pomalidomide (Pom) (p<0.0001 for both compounds; 2-way ANOVA); or by independent pretreatment of both PBMCs and MM cells with either compound (p<0.0001 for both compounds). In contrast, Dex pretreatment of PBMCs attenuated their anti-MM activity (p=0.018). MM cytotoxicity was decreased by depletion of CD4+ (p<0.0001), CD8+ (p<0.0001), or CD56+ cells (p<0.0001), but the decrease was more pronounced after CD56+ cell depletion. Conversely, positively selected CD56+ cells had significantly higher anti-tumor activity than unselected PBMCs (p<0.0001). The tumor-selective nature of CS-BLI measurements allowed the evaluation the effect of stromal cells on the anti-tumor immune effector activity. Luc+ MM.1S target cells were co-cultured with IL-2-stimulated PBMCs from MM patients, both in the presence and absence of autologous bone marrow stromal cells (BMSCs). BMSCs from these matched donors significantly decrease the anti-tumor activity of IL-2-stimulated PBMCs over 4hrs. Len and Pom enhanced, while Dex decreased, the anti-tumor effect in the presence of stromal cells. We next utilized the CS-BLI platform to screen in an open-ended manner a library of compounds for immunostimulatory properties against MM cells. Compounds of an NCI drug library were pin-transferred (1μM final conc.) to plates with Luc+ MM.1S cells cultured alone or with IL-2-prestimulated PBMCs and tumor cell viability accessed. This screening allowed us to identify candidate immunomodulators, one of which, the depsipeptide Didemnin B, showed direct anti-tumor activity in the absence of PBMCs, but also enhanced the anti-MM cytotoxicity of PBMCs (or the NK cell line NKL). CS-BLI allows high-throughput screening of compounds not only on the basis of their direct cytotoxic activity in co-cultures, but also their ability to enhance anti-tumor immune responses. CS-BLI can be applied not only in oncology, but also other settings where agents with immuno-stimulatory (i.e. infection) or immunosuppressive (i.e. inflammation/autoimmunity and transplant immunology) properties are important. Our results therefore provide the framework to identify and validate novel pharmacologically or biologically active agents based on their ability to modulate the activity of immune effector cells. CS-BLI allows for rapid and specific evaluation of such agents in multiple conditions, including co-cultures with stromal cells. CS-BLI is a novel tool that can accelerate the identification and prioritization of novel immunomodulatory agents for clinical development Disclosures:McMillin:Axios Biosciences: Equity Ownership. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Mitsiades:Millennium: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Merck & Co.: Consultancy, Honoraria; Kosan Pharmaceuticals: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Centrocor: Consultancy, Honoraria; PharmaMar: Patents & Royalties; OSI Pharmaceuticals: Research Funding; Amgen Pharmaceuticals: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding.

Immunotherapy of Malignant Gliomas Using Autologous and Allogeneic Tissue Cells

Immunotherapy of brain tumors is rapidly emerging as a potential clinical option [1-3]. The quality and magnitude of immune responses evoked by the new generation anti-tumor vaccines is in general highly dependent on the source or choice of peptide antigens, and as well, a suitable immunopotentiator. Poorly immunogenic antigens, such as those present in tumor cell lysates, may not reliably provide stimulation like recombinant or DNA-encoded protein antigens might be expected to. In addition, the efficacy of the vaccine may depend on inherent counteracting measures of the tumor which dampen immune surveillance and immune effector activity triggered by immunization [4]. Our body has many means of limiting an immune response to our own (self) proteins. In particular, patients with gliomas exhibit a broad suppression of cell-mediated immunity [5-8]. Unfortunately, for most tumor vaccines the induction of local or systemic immune effector cells does not necessarily translate into objective clinical responses or increased survival [9]. Here we review immunotherapeutic approaches against gliomas and recent pre-clinical and clinical initiatives based on cellular or active immunization of the patient's immune system using autologous and allogeneic tissues or cultured cells. Available evidence shows that single modality cancer therapies likely remain suboptimal. Combination regimens targeting the immune system at multiple coordinated levels must be developed, and possibly combined with strategies to inhibit immune suppressive factors if significant clinical benefit is to be achieved.

Hypoxia-driven immunosuppression: A new reason to use thermal therapy in the treatment of cancer?

Hypoxia within the tumour microenvironment is correlated with poor treatment outcome after radiation and chemotherapy, and with decreased overall survival in cancer patients. Several molecular mechanisms by which hypoxia supports tumour growth and interferes with effective radiation and chemotherapies are now well established. However, several new lines of investigation are pointing to yet another ominous outcome of hypoxia in the tumour microenvironment: suppression of anti-tumour immune effector cells and enhancement of tumour escape from immune surveillance. This review summarises this important information, and highlights mechanistic data by which hypoxia incapacitates several different types of immune effector cells, enhances the activity of immunosuppressive cells and provides new avenues which help ‘blind’ immune cells to detect the presence of tumour cells. Finally, we discuss data which indicates that mild thermal therapy, through its physiologically regulated ability to alter vascular perfusion and oxygen tensions within the tumour microenvironment, as well as its ability to enhance the function of some of the same immune effector activities that are inhibited by hypoxia, could be used to rapidly and safely release the tight grip of hypoxia in the tumour microenvironment thereby reducing barriers to more effective immune-based therapies.