Promising Tools For Immunotherapy Research Articles

Abstract 4106: Non-autonomous enhancement of gPDL1 CAR-T annihilates TNBC development

Abstract Triple-negative breast cancer (TNBC) is the most aggressive and challenging breast cancer subtype, which does not respond to traditional endocrine and anti-HER2-targeted therapies. PD-L1 is highly enriched in TNBC and has been considered a therapeutic target. Despite the excellent anti-cancer activity, the atezolizumab-based chimeric antigen receptor (CAR) T cells showed a robust off-target effect. In addition, the treatment of solid tumors with CAR-T is limited by abnormal glycosylation in malignant tumors. Targeting glycosylated PD-L1 (gPD-L1) provided tissue specificity against TNBC, implying it can prevent antigen escape and off-target effect. In this study, we generated gPD-L1 CAR-T cells using lentiviral vectors expressing the scFv regions of the anti-gPD-L1 antibody. The gPD-L1 CAR-T cells exhibited antigen-specific activation, cytokine production, and cytolytic activity against TNBCs in vitro and in the xenograft tumors model. CyTOF and single-cell RNA sequencing (scRNA-seq) showed distinct IFNγ-positive cell types. Mechanistically, IFNγ crosstalked with EGFR signaling through Src activation and, in turn, triggered B3GNT3-mediated PD-L1 glycosylation. Inhibition of Src resulted in reduced gPD-L1 expression in TNBC. CRISPR/Cas9 knockout of B3GNT3 in TNBC cells impaired gPD-L1 CAR-T response. As a result of nonautonomous gPDL1 amplification in TNBCs, gPD-L1 CAR-T cells continued to annihilate TNBCs. Additionally, since gPD-L1 CAR-T cells provided higher specificity on TNBC, they had lower normal tissue toxicity. Overall, gPD-L1 CAR-T exhibits excellent anti-tumor activity against TNBCs, and it could be a promising immunotherapy tool to treat TNBCs in clinic. Furthermore, targeting glycosylation moiety on the tumor antigen is a novel approach to lessen CAR-T toxicity in patients. Citation Format: Chia-Wei Li, Shih-Han Wang, Yun-Ju Lai, Jyun Wang, Chun-Tse Kuo, Shih-Duo Hsu Hung, Shou-Hou Liu. Non-autonomous enhancement of gPDL1 CAR-T annihilates TNBC development. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4106.

Generation and Screening of Antigen-Specific Nanobodies from Mammalian Cells Expressing the BCR Repertoire Library Using Droplet-Based Microfluidics.

Nanobodies, also known as VHHs, originate from the serum of Camelidae. Nanobodies have considerable advantages over conventional antibodies, including smaller size, more modifiable, and deeper tissue penetration, making them promising tools for immunotherapy and antibody-drug development. A high-throughput nanobody screening platform is critical to the rapid development of nanobodies. To date, droplet-based microfluidic systems have exhibited improved performance compared to the traditional phage display technology in terms of time and throughput. In realistic situations, however, it is difficult to directly apply the technology to the screening of nanobodies. Requirements of plasma cell enrichment and high cell viability, as well as a lack of related commercial reagents, are leading causes for impeding the development of novel methods. We overcame these obstacles by constructing a eukaryotic display system that secretes nanobodies utilizing homologous recombination and eukaryotic transformation technologies, and the significant advantages are that it is independent of primary cell viability and it does not require plasma cell enrichment in advance. Next, a signal capture system of "SA-beads + Biotin-antigen + nanobody-6 × His + fluorescence-labeled anti-6 × His (secondary antibody)" was designed for precise localization of the eukaryotic-expressed nanobodies in a droplet. Based on this innovation, we screened 293T cells expressing anti-PD-L1 nanobodies with a high positive rate of targeted cells (up to 99.8%). Then, single-cell transcriptomic profiling uncovered the intercellular heterogeneity and BCR sequence of target cells at a single-cell level. The complete complementarity determining region (CDR3) structure was obtained, which was totally consistent with the BCR reference. This study expanded the linkage between microfluidic technology and nanobody applications and also showed potential to accelerate the rapid transformation of nanobodies in the large-scale market.

Comparison of the PU.1 transcriptional regulome and interactome in human and mouse inflammatory dendritic cells.

Dendritic cells (DCs) are key immune modulators and are able to mount immune responses or tolerance. DC differentiation and activation imply a plethora of molecular and cellular responses, including transcriptional changes. PU.1 is a highly expressed transcription factor in DCs and coordinates relevant aspects of DC biology. Due to their role as immune regulators, DCs pose as a promising immunotherapy tool. However, some of their functional features, such as survival, activation, or migration, are compromised due to the limitations to simulate in vitro the physiologic DC differentiation process. A better knowledge of transcriptional programs would allow the identification of potential targets for manipulation with the aim of obtaining "qualified" DCs for immunotherapy purposes. Most of the current knowledge regarding DC biology derives from studies using mouse models, which not always find a parallel in human. In the present study, we dissect the PU.1 transcriptional regulome and interactome in mouse and human DCs, in the steady state or LPS activated. The PU.1 transcriptional regulome was identified by performing PU.1 chromatin immunoprecipitation followed by high-throughput sequencing and pairing these data with RNAsequencing data. The PU.1 interactome was identified by performing PU.1 immunoprecipitation followed by mass spectrometry analysis. Our results portray PU.1 as a pivotal factor that plays an important role in the regulation of genes required for proper DC activation and function, and assures the repression of nonlineage genes. The interspecies differences between human and mouse DCs are surprisingly substantial, highlighting the need to study the biology of human DCs.

Oncolytic Adenovirus in Cancer Immunotherapy.

Simple SummaryOncolytic adenoviruses are engineered to selectively replicate in and destroy cancer tissue. Moreover, these viruses are promising tools to restore antitumor immune response in cancer patients due to their high immunogenicity and the ability to interfere with the immunosuppressive tumor microenvironment. Due to these characteristics, oncolytic adenoviruses can activate tumors for already existing, systemic immunotherapies. The goal of this review is to provide an introduction into the common concepts of oncolytic adenoviruses, and to present their current status in clinical development. We also want to report in detail on strategies to optimize the immunoactivating properties of these agents for future application in multistage cancer immunotherapies.Tumor-selective replicating “oncolytic” viruses are novel and promising tools for immunotherapy of cancer. However, despite their first success in clinical trials, previous experience suggests that currently used oncolytic virus monotherapies will not be effective enough to achieve complete tumor responses and long-term cure in a broad spectrum of cancers. Nevertheless, there are reasonable arguments that suggest advanced oncolytic viruses will play an essential role as enablers of multi-stage immunotherapies including established systemic immunotherapies. Oncolytic adenoviruses (oAds) display several features to meet this therapeutic need. oAds potently lyse infected tumor cells and induce a strong immunogenic cell death associated with tumor inflammation and induction of antitumor immune responses. Furthermore, established and versatile platforms of oAds exist, which are well suited for the incorporation of heterologous genes to optimally exploit and amplify the immunostimulatory effect of viral oncolysis. A considerable spectrum of functional genes has already been integrated in oAds to optimize particular aspects of immune stimulation including antigen presentation, T cell priming, engagement of additional effector functions, and interference with immunosuppression. These advanced concepts have the potential to play a promising future role as enablers of multi-stage immunotherapies involving adoptive cell transfer and systemic immunotherapies.

Self-Adjuvanting Cancer Vaccines from Conjugation-Ready Lipid A Analogues and Synthetic Long Peptides.

Self-adjuvanting vaccines, wherein an antigenic peptide is covalently bound to an immunostimulating agent, have been shown to be promising tools for immunotherapy. Synthetic Toll-like receptor (TLR) ligands are ideal adjuvants for covalent linking to peptides or proteins. We here introduce a conjugation-ready TLR4 ligand, CRX-527, a potent powerful lipid A analogue, in the generation of novel conjugate-vaccine modalities. Effective chemistry has been developed for the synthesis of the conjugation-ready ligand as well as the connection of it to the peptide antigen. Different linker systems and connection modes to a model peptide were explored, and in vitro evaluation of the conjugates showed them to be powerful immune-activating agents, significantly more effective than the separate components. Mounting the CRX-527 ligand at the N-terminus of the model peptide antigen delivered a vaccine modality that proved to be potent in activation of dendritic cells, in facilitating antigen presentation, and in initiating specific CD8+ T-cell-mediated killing of antigen-loaded target cells in vivo. Synthetic TLR4 ligands thus show great promise in potentiating the conjugate vaccine platform for application in cancer vaccination.

Induction of breast cancer stem cells by M1 macrophages through Lin-28B-let-7-HMGA2 axis.

Proinflammatory macrophage (M1) is now being suggested as a potential therapeutic strategy for cancer because of its tumoricidal capacity. However, few studies have been focused directly on the effects of M1 macrophages on cancer cells. Here, we found that M1 induced a subpopulation of CD44high/CD24-/low or ALDH1+ cells with CSC-like phenotypes from different types of breast cancer cells (BCCs) in a paracrine manner. Stat3/NF-κB pathways in BCCs were activated by proinflammatory cytokines, igniting Lin-28B-let-7-HMGA2 axis to induce CSC through epithelial-mesenchymal transition (EMT). Previously, we reported that Stat3-coordinated Lin-28B-let-7-HMGA2 axis initiated EMT in BCCs. Here, inhibition of Stat3/NF-κB pathways or Lin-28B-let-7-HMGA2 axis suppressed EMT/CSCs program. Notably, HMGA2 knockdown directly repressed M1-induced CSC formation and expression of Klf-4 and Nanog. Meanwhile, prolonged coculture with BCCs endowed M1 with M2 properties. M1 supernatant induced CSC from non-stem cancer cells, while M2 supernatant sustained a higher proportion of ALDH1+ cells. Our data suggest that macrophages might modulate CSC formation and maintenance by transferring between M1/M2 phenotype. Given that M1 are being considered as a promising immunotherapy tool, it is important to inhibit their CSC-inducing potential by targeting key molecules and pathways.

Reducing on-Target Off-Tumor Effects of CD38-Chimeric Antigen Receptors By Affinity Optimization

Chimeric Antigen Receptors (CARs) are engineered transmembrane proteins consisting of an antibody (ab)-derived antigen recognition domain linked to intracellular T cell signaling domains. Cytotoxic T cells endowed with tumor-reactive CARs are highly promising tools for immunotherapy of cancer. There are however only a few truly tumor specific molecules that can be targeted by CARs, a drawback for the broad application of CAR T cell therapy. Indeed, when we recently aimed at targeting CD38high multiple myeloma (MM) with T cells transduced with high affinity CD38CARs, we observed that they not only lysed the CD38high MM cells but also CD38+ normal hematopoietic cells, pointing towards potential safety issues of such tumor-associated, but not entirely tumor-specific CARs. Therefore, using CD38 as a model for antigen we now tested whether it would be possible to reduce the on target, off-tumor effects of such CARs by optimizing their target cell affinity. To this end, we generated a new panel of CD38 abs through the "light chain exchange" method, in which heavy chains of two high affinity CD38 abs were combined with 176 different germ line light chains. This approach revealed around 100 new abs, which displayed 10- >1000 fold lower affinity to CD38 as compared to the parental abs. After categorizing them in three classes based on CD38 binding affinity, we used 8 abs from each class to generate 24 different CD38-CAR constructs. Testing the cytotoxic activity of T cells transduced with these CD38-CARs against CD38++ MM cell lines, primary MM cells and CD38+ normal hematopoietic cells in vitro and in vivo demonstrated that CD38-CAR T cells with ca. 1000 fold lower affinity to CD38 could still effectively lyse CD38++ MM cells while there was little or no cytotoxicity against CD38+ healthy hematopoietic cells. The results of this study reveal that it is possible to reduce the on-target off-tumor effects of CARs by optimizing their affinity. Thus, tailored affinity of the ab binding domain may open up new roads for CAR therapy. Disclosuresvan de Donk:Janssen: Research Funding; Celgene: Research Funding; BMS: Research Funding; Amgen: Research Funding. Lokhorst:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genmab: Research Funding. Mutis:Celgene: Research Funding; Genmab: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Versatile polyion complex micelles for peptide and siRNA vectorization to engineer tolerogenic dendritic cells

Dendritic cells (DCs) are professional antigen-presenting cells that play a critical role in maintaining the balance between immunity and tolerance and, as such are a promising immunotherapy tool to induce immunity or to restore tolerance. The main challenge to harness the tolerogenic properties of DCs is to preserve their immature phenotype. We recently developed polyion complex micelles, formulated with double hydrophilic block copolymers of poly(methacrylic acid) and poly(ethylene oxide) blocks and able to entrap therapeutic molecules, which did not induce DC maturation. In the current study, the intrinsic destabilizing membrane properties of the polymers were used to optimize endosomal escape property of the micelles in order to propose various strategies to restore tolerance. On the first hand, we showed that high molecular weight (Mw) copolymer-based micelles were efficient to favor the release of the micelle-entrapped peptide into the endosomes, and thus to improve peptide presentation by immature (i) DCs. On the second hand, we put in evidence that low Mw copolymer-based micelles were able to favor the cytosolic release of micelle-entrapped small interfering RNAs, dampening the DCs immunogenicity. Therefore, we demonstrate the versatile use of polyionic complex micelles to preserve tolerogenic properties of DCs. Altogether, our results underscored the potential of such micelle-loaded iDCs as a therapeutic tool to restore tolerance in autoimmune diseases.

CD38 Chimeric Antigen Receptor Engineered T Cells As Therapeutic Tools for Multiple Myeloma

Chimeric Antigen Receptors (CARs) are engineered constructs consisting of an antibody-derived antigen recognition domain linked to intracellular T cell signaling domains. Cytotoxic T cells transduced to express tumor-reactive CARs are highly promising tools for immunotherapy of cancer. The CD38 molecule, with its high and homogenous expression on Multiple Myeloma (MM) tumor cells, is considered a suitable target for antibody therapy of MM. Prompted by this, we evaluated the feasibility and efficacy of targeting MM cells with CD38-CAR-transduced T cells (CD38-CAR T cells). To this end, we generated three different retroviral CAR constructs based on human CD38 antibodies as antigen recognition domain, CD3zeta and 41BB (CD137) as signaling domains and transduced them into PBMCs of a healthy donor. After in vitro selection and expansion, all CD38-CAR T cells, either unsorted or CD4/CD8 sorted, effectively lysed MM cell lines in a dose-, and CD38 expression-dependent manner, with a better efficacy for the CD8+ fraction. CD38-CAR T cells also effectively eradicated primary MM cells in the bone marrow mononuclear cells derived from MM patients, indicating their clinical relevancy. Although CD38-CAR T cells also displayed cytotoxic activity against the CD38+ fraction of mature monocytes and NK cells and to a lesser extent CD38+ B and T cells, they did not affect the outgrowth of CD34+ cells into various myeloid lineages. In addition,CD38-CAR T cell activity was effectively controllable by transducing them with a caspase 9-based inducible suicide gene. More interestingly, we discovered that the CD38-CAR T cells were themselves devoid of CD38 surface expression, indicating that CD38 was not essential for T cell expansion and function. Finally, in a novel in vivo xenotransplant model (UM9 cell line), in which myeloma cells were grown in a humanized bone marrow microenvironment, i.v. as well as intra tumor administration of CD38-CAR T cells established significant anti-tumor effects, proving that CD38-CAR endowed cytotoxic T lymphocytes, even with no CD38 expression, can efficiently migrate, infiltrate and eliminate human MM tumors growing in their natural niche. These results demonstrate the feasibility and potency of CAR mediated targeting of CD38+ MM cells. Optimization of CD38-CAR and suicide-gene control of CD38 CAR T cellsmay provide next steps towards safe clinical implementation of CD38-CAR T cell immune therapy. DisclosuresDrent:Genmab BV: Guest employee (unpaid) Other. Lammerts van Bueren:Genmab : Employment. Parren:Genmab: Employment, Equity Ownership. van de Donk:Genmab BV: Research Funding; J&J: Research Funding; Celgene: Research Funding. Martens:Genmab BV: Research Funding; J&J: Research Funding; Celgene: Research Funding. Lokhorst:Celgene: Research Funding; J&J: Research Funding; Genmab: Research Funding. Mutis:Celgene: Research Funding; J&J: Research Funding; Genmab BV: Research Funding.

Deprivation of human natural killer cells and antitumor immune response.

IntroductionCell-based immunotherapy has been given increased attention as a treatment for cancer. Human natural killer (NK) cells are resident lymphocyte populations. They exhibit potent antitumor activity without human leukocyte antigen matching and without prior antigen exposure. They also are a promising tool for immunotherapy of solid and hematologic cancers. However, most cancer patients do not have enough NK cells to induce an effective antitumor immune response. This demonstrates a need for a source of NK cells that can supplement the endogenous cell population.Material and methodsIn this study, we derived induced pluripotent stem cells (iPSCs) from peripheral blood T-lymphocytes using Sendai virus vectors.ResultsGenerated iPSCs exhibited monoclonal T cell receptors (TCR) rearrangement in their genome, a hallmark of mature terminally differentiated T cells. These iPSCs were differentiated into NK cells using a two-stage coculture system: iPSCs into hematopoietic CD34+ cells with feeder cells M210-B4 (ATCC, USA) and CD34+ cells into mature NK cells with AFT024 cells (ATCC, USA). Our results showed that iPSC-derived NK cells expressed CD56, CD16, NKp 44 and NKp 46, possessed high cytotoxic activity and produced high level of interferon-γ.ConclusionBased on our data, derivation of NK cells from induced pluripotent stem cells should be considered in the treatment of oncologic diseases. This would allow for the development of cell therapy for cancer using immunologically compatible NK cells derived from iPSCs. This may contribute to a more efficient treatment of oncologic diseases in addition to traditional cancer treatment.

Percentages of NKT cells in the tissues of patients with non-small cell lung cancer who underwent surgical treatment.

IntroductionNatural killer T (NKT) cells are involved in the antitumor response by direct cytotoxicity and indirectly through activation of effector cells. Recent studies have shown a relationship between the number and function of NKT cells and clinical outcomes. NKT cells seem to represent a promising tool for immunotherapy of cancer.The aim of the studyThe aim of the study was to evaluate the distribution of NKT cells in peripheral blood, lymph nodes and tumor tissue of non-small cell lung cancer (NSCLC) patients, as well as development of the most efficient set of cytokines stimulating differentiation of NKT cells.Material and methodsWe evaluated the percentage of iNKT+CD3+ cells in the tissues collected from patients with NSCLC. For the generation of NKT cells, we cultured cells isolated from the blood of 20 healthy donors and from the tissues of 4 NSCLC patients. Cells were stimulated with α-GalCer in combinations with cytokines.ResultsWe noted significant differences in the percentages of NKT cells in the patients’ tissues. The highest percentage of these cells was observed in the tumor tissue and the lowest in the lymph nodes. In vitro, in healthy donors all α-GalCer-cytokine combinations were effective in stimulation of NKT cells’ proliferation. NKT cells’ proliferation was the most efficiently stimulated by α-GalCer+IL-2+IL-7 and α-GalCer+IL-2+IFN-γ.ConclusionsOur results suggest that in the course of NSCLC, NKT cells migrate to the primary tumor and accumulate therein. All tested combinations of α-GalCer and cytokines were capable of generation of NKT cells in vitro.

A model of dendritic cell therapy for melanoma.

Dendritic cells are a promising immunotherapy tool for boosting an individual’s antigen-specific immune response to cancer. We develop a mathematical model using differential and delay-differential equations to describe the interactions between dendritic cells, effector-immune cells, and tumor cells. We account for the trafficking of immune cells between lymph, blood, and tumor compartments. Our model reflects experimental results both for dendritic cell trafficking and for immune suppression of tumor growth in mice. In addition, in silico experiments suggest more effective immunotherapy treatment protocols can be achieved by modifying dose location and schedule. A sensitivity analysis of the model reveals which patient-specific parameters have the greatest impact on treatment efficacy.

Endogenous HLA class II epitopes that are immunogenic in vivo show distinct behavior toward HLA-DM and its natural inhibitor HLA-DO

AbstractCD4+ T cells play a central role in adaptive immunity. The acknowledgment of their cytolytic effector function and the finding that endogenous antigens can enter the HLA class II processing pathway make CD4+ T cells promising tools for immunotherapy. Expression of HLA class II and endogenous antigen, however, does not always correlate with T-cell recognition. We therefore investigated processing and presentation of endogenous HLA class II epitopes that induced CD4+ T cells during in vivo immune responses. We demonstrate that the peptide editor HLA-DM allowed antigen presentation of some (DM-resistant antigens) but abolished surface expression of other natural HLA class II epitopes (DM-sensitive antigens). DM sensitivity was shown to be epitope specific, mediated via interaction between HLA-DM and the HLA-DR restriction molecule, and reversible by HLA-DO. Because of the restricted expression of HLA-DO, presentation of DM-sensitive antigens was limited to professional antigen-presenting cells, whereas DM-resistant epitopes were expressed on all HLA class II–expressing cells. In conclusion, our data provide novel insights into the presentation of endogenous HLA class II epitopes and identify intracellular antigen processing and presentation as a critical factor for CD4+ T-cell recognition. This opens perspectives to exploit selective processing capacities as a new approach for targeted immunotherapy.

Nicotinamide, a Form of Vitamin B3, Promotes Expansion of Natural Killer Cells That Display Increased In Vivo Survival and Cytotoxic Activity,

Abstract 4035NK cells are cytotoxic lymphocytes that have drawn considerable attention in recent years as a promising tool for immunotherapy in patients with various refractory hematological malignancies and metastatic solid tumors. Clinical results of experimental protocols have shown only a partial response attributed mainly to the relatively low number of NK cells infused and their short in vivo persistence.An important challenge, therefore, in advancing the clinical applicability of NK cells is to expand ex vivo NK cells that display increased functionality upon in vivo infusion. In efforts to induce NK cell expansion, different combinations of cytokines have been studied. However, most reports show a modest expansion and demonstrate a need for additional stimuli.Nicotinamide (NAM) is a form of Vitamin B3 and a potent inhibitor of enzymes that use NAD for their activity. Hence, NAM is directly involved in the control of redox sensitive enzymes, mitochondrial functions, cell metabolism, the production of energy, and cell motility.Here we show that NAM (2.5–5 mM) enhances expansion (60-80 fold) of functional NK cells in feeder-free cultures stimulated with IL-2 and IL-15 for two weeks. This effect was observed in cultures initiated with purified CD56+ (CD56 enriched/CD3 depleted) or with CD3 depleted, peripheral blood and cord blood cells.Immunophenotyping of the cultured NK cells has so far revealed that NAM substantially modulates three cell surface receptors. CD200R and programmed death receptor-1 (PD-1) expressed on NK cells interact with their ligands on tumor cells which leads to a suppression in NK cell anti-tumor activity and tumor immunoevasion. These two receptors are down-regulated by NAM. CD62L (L-selectin) defines an NK subset with increased self-renewal capacity and its expression was reported to be pivotal for NK cells trafficking to lymphoid organs and their homeostatic proliferation. Following expansion in culture with IL-2, CD62L is down-regulated, whilst NAM increased its expression with a dose-dependent effect.Using a CFSE-based cytotoxicity assay we have demonstrated that NK cells cultured with NAM display higher cytotoxic activity against K562, BL2, NK-resistant COLO 205 cell lines and primary leukemia cells. In a Transwell migration assay, NK cells cultured with NAM demonstrated increased migration towards the CXCR4 ligand SDF-1. To test in vivo homing and retention, irradiated (350 RAD) NOD/SCID mice were transplanted with a similar number of cells (15–20×106 /mouse) derived from two week cultures treated with or without NAM. Mice were infused with 50μg/mouse IL-2 and 5μg/mouse IL-15 every other day. To test homing, mice were sacrificed 24 hour post infusion. Number of human NK cells (CD45+CD56+) detected in the spleen and BM were significantly (p< 0.05) higher in the cohort of mice infused with NK cells cultured with NAM (7.9 and 1.39 respectively) compared to mice infused with NK cells cultured without NAM (4.13 and 0, respectively). In a different set of experiments, persistence of human NK cells was analyzed 4 and 12 days post infusion. Four days post infusion, the percentage of human NK cells in the spleen, BM, lung and liver were substantially higher in mice infused with NK cells cultured with NAM compared to mice infused with NK cells cultured without NAM (Fig 1). Even though 12 days post infusion, a decrease in the number of human NK cells was observed in comparison to day 4, still cell retention in the spleen, liver and lung was significantly greater in the cohort infused with NK cells cultured with NAM (13.45, 0.6, 9.21% Vs. 1.26, 0.12, 2.85%, (p<0.05), respectively). The calculated decrease in the number of human NK cells from day 4 to 12 was 50% less in the NAM cohort, suggesting enhanced in vivo survival of NK cells cultured with NAM. [Display omitted] In conclusion, expansion of NK cells with NAM was found to increase in vivo homing and survival and to augment tumor cytotoxic effect of NK cells. This suggests a potential for enhancing the clinical efficacy of adoptively transferred NK cells. Based on these intriguing findings we are developing a cell product for adoptive cell-mediated immune therapy. Disclosures:Frei:Gamida Cell: Employment. Persi:Gamida Cell: Employment. Lador:Gamida Cell: Employment, Equity Ownership. Peled:Gamida Cell: Consultancy. Nagler:Gamida Cell: Consultancy. Peled:Gamida Cell: Employment, Equity Ownership, Patents & Royalties.

Role of IgA and IgA Fc Receptors in Inflammation

Signals delivered by serum monomeric IgA (mIgA) are essential in controlling the immune system by preventing the development of autoimmunity and inflammation. However, IgA can also, when aggregated, be deleterious to the host, inducing inflammatory diseases. This Janus-like nature of IgA is mainly due to their heterogeneity in molecular forms and their interaction with IgA receptors. While serum mIgA are mainly involved in FcalphaRI-mediated inhibition of immune responses, macromolecular serum IgA or circulating IgA immune complexes are often deleterious to the host by inducing sustained activation through IgA receptors including FcalphaRI and transferrin receptor. FcalphaRI-mediated inhibitory function is able to suppress several inflammatory diseases in mice including asthma and glomerulonephritis. Intravenous mIgA (mIgAIV) and anti-FcalphaR monovalent antibodies represent thus promising tools for immunotherapy.

The Role of Immunostimulants in Immunotherapy and Immunoprophylaxis

ABSTRACTMedical practice faces serious problems due to the constantly increasing bacterial poly-resistance to antibiotics, and the side effects of the latter, often including allergic and immunosuppressive reactions. One way to resolve this problem is the application of immunomodulators that increase resistance to bacterial and viral infections by stimulating non-specific immunity mechanisms. In the course of more than 20 years a number of oral immunomodulators have been developed and investigated in the NCIPD, Sofia: Respivax, Urostim and Dentavax which are widely and successfully applied in clinical practice for immunotherapy and immunomodulation.Data about the effects of polybacterial immunomodulators demonstrate a pronounced stimulation of phagocytosis, synthesis of secretory IgA, surfactant, interferon and a number of Th1 type of cytokines. Numerous investigations, including double-blind studies, have proved their efficacy in the prevention and treatment of non-specific respiratory, uro-genital and periodontal infections as well as in the complex therapy of AIDS. Based on this, polybacterial immunostimulators seem a very promising tool for immunotherapy and immunoprevention, allowing the modulation of immune responses in a most beneficial way.

Increased chondroitin sulphate proteoglycan expression (B5 immunoreactivity) in metastases of uveal melanoma

BackgroundMetastatic uveal melanoma has a poor prognosis and limited therapeutic options. Proteoglycans are involved in tumor cell invasion and metastatic behavior. The mAbB5 stains a chondroitin sulphate proteoglycan (CSPG) on cutaneous melanoma cells. Here, we compare the B5-staining of CSPG in primaries and metastases of uveal melanoma. Material and methodsImmunohistopathological staining was performed in 15 cutaneous and 39 uveal melanoma samples. A score for intracellular and surface staining was established. B5 staining was compared in primaries and metastases of uveal melanoma using Student's t-test. ResultsEight of 11 (73%) uveal melanoma metastases were positive for B5-staining whereas only 5 of 28 (18%) primary uveal melanoma samples were B5-positive (P < 0.001). Nine of 15 cutaneous melanoma samples (60%) were B5-positive without significant difference between primary and metastatic lesions. Surface staining was found both on uveal melanoma metastases and cutaneous melanomas. ConclusionsCSPG was expressed significantly more often in metastases than in primaries of uveal melanoma. It potentially may be one factor associated with metastatic spread. Further studies are needed to determine its use as prognostic factor. The mAbB5 may also be a promising tool for immunotherapy due to its strong staining of CSPG on the surface of cutaneous and metastatic uveal melanoma cells.

Lipopolysaccharide preferentially induces 4-1BB ligand expression on human monocyte-derived dendritic cells

Dendritic cells (DCs) represent a promising tool for immunotherapy. A key feature in their action is to provide co-stimulatory signals for full activation of T cells. In view of recent studies demonstrating the critical role of 4-1BB co-stimulation in T cell response, it is of importance to optimize 4-1BB ligand (4-1BBL) expression on human monocyte-derived DCs (MDDCs), the DC source of many clinical studies. In this study, two types of MDDCs, generated in granulocyte-macrophage colony-stimulating factor and interleukin-4 (GM-CSF/IL-4-DCs) or in interferon-β and IL-3 (IFN-β/IL-3-DCs), were analyzed for 4-1BBL expression in response to several known DC activators. Immature MDDCs expressed 4-1BBLs at very low levels. Lipopolysaccharide (LPS) was the only activator that preferentially triggered 4-1BBL expression on either MDDCs, but 4-1BBL-positive cells were significantly more frequently observed on LPS-activated GM-CSF/IL-4-DCs (30.2±2.6% versus 14.3±1.2%). Combinations of multiple activating signals did not bring about enhanced 4-1BBL stimulatory capacity. In addition, plasmid DNA transfection and necrotic cell pulsing of GM-CSF/IL-4-DCs for antigen loading also resulted in 4-1BBL up-regulation. However, in all circumstances, the induced 4-1BBL levels were low in comparison with CD80 co-stimulatory molecule. Finally, by demonstrating LPS-matured GM-CSF/IL-4-DCs from sorted 4-1BBLhigh population augmented T cell expansion and survival, we propose that efforts are required to increase 4-1BBL levels on MDDCs achieved by current activation schemes.

The human fascin gene promoter is highly active in mature dendritic cells due to a stage-specific enhancer.

Dendritic cells (DC), regarded as the most efficient APCs of the immune system, are capable of activating naive T cells. Thus, DC are primary targets in immunotherapy. However, little is known about gene regulation in DC, and for efficient transcriptional targeting of human DC, a suitable promoter is still missing. Recently, we successfully used the promoter of the murine actin-bundling protein fascin to transcriptionally target DC by DNA vaccination in mice. In this study, we report on isolation of the human fascin promoter and characterization of its regulatory elements. The actively expressed gene was distinguished from a conserved inactive genomic locus and a continuous region of 14 kb covering the gene and 3 kb of 5'-flanking sequences was subcloned, sequenced, and analyzed for regulatory elements. Regulatory sequences were found solely in the 5'-flanking promoter region. The promoter exerted robust activity in DC and a fascin-positive neuronal cell line, but not in the fascin-negative cells tested. Notably, promoter activity in DC markedly increased with maturation of DC. By progressive 5' deletion, we identified a core promoter region, harboring a putative GC box, a composite cAMP responsive element/AP-1 binding site and a TATA box. By internal deletion, we demonstrated functional importance of either regulatory element. Furthermore, we identified a more distal stage-specific enhancer region also containing silencer elements. Taken together, the human fascin promoter allows for transcriptional targeting of mature DC and represents a promising tool for immunotherapy. To our knowledge, this study reports for the first time on promoter activity in human monocyte-derived DC.