Cytokine Release In Whole Blood Research Articles

Differential cellular and humoral immune responses in immunocompromised individuals following multiple SARS-CoV-2 vaccinations.

The heterogeneity of the immunocompromised population means some individuals may exhibit variable, weak or reduced vaccine-induced immune responses, leaving them poorly protected from COVID-19 disease despite receiving multiple SARS-CoV-2 vaccinations. There is conflicting data on the immunogenicity elicited by multiple vaccinations in immunocompromised groups. The aim of this study was to measure both humoral and cellular vaccine-induced immunity in several immunocompromised cohorts and to compare them to immunocompetent controls. Cytokine release in peptide-stimulated whole blood, and neutralising antibody and baseline SARS-CoV-2 spike-specific IgG levels in plasma were measured in rheumatology patients (n=29), renal transplant recipients (n=46), people living with HIV (PLWH) (n=27) and immunocompetent participants (n=64) post third or fourth vaccination from just one blood sample. Cytokines were measured by ELISA and multiplex array. Neutralising antibody levels in plasma were determined by a 50% neutralising antibody titre assay and SARS-CoV-2 spike specific IgG levels were quantified by ELISA. In infection negative donors, IFN-γ, IL-2 and neutralising antibody levels were significantly reduced in rheumatology patients (p=0.0014, p=0.0415, p=0.0319, respectively) and renal transplant recipients (p<0.0001, p=0.0005, p<0.0001, respectively) compared to immunocompetent controls, with IgG antibody responses similarly affected. Conversely, cellular and humoral immune responses were not impaired in PLWH, or between individuals from all groups with previous SARS-CoV-2 infections. These results suggest that specific subgroups within immunocompromised cohorts could benefit from distinct, personalised immunisation or treatment strategies. Identification of vaccine non-responders could be critical to protect those most at risk.

Abstract 5100: Combination of κλ bispecific antibodies targeting innate (CEAxCD47, NILK-2401) and adaptive immunity (CEAxCD3, NILK-2301 and CEAxCD28, NILK-3301) for next generation immunotherapy of CEA-expressing cancers

Abstract Background: The CEAxCD3 bispecific antibody (bsAb) NILK-2301 couples CEA (CEACAM5) on cancer cells and CD3 on T-cells inducing T-cell activation (signal 1) and tumor cell killing (TDCC). T-cell activation can be boosted by CEA-targeted CD28-costimulation (NILK-3301; signal 2). NILK-2401, carrying a fully effective IgG1 Fc, induces antibody-dependent phagocytosis (ADCP) and antibody-dependent cytotoxicity (ADCC) of tumor cells by co-targeting CEA and the innate immune checkpoint CD47 (“don’t eat me” signal). We present here next generation immunotherapy to overcome limited single class activity in CEA-expressing solid cancers. Methods: BsAbs were generated using LCB’s fully human κλ body platform. TDCC, ADCP, and ADCC with human PBMC or monocyte-derived macrophages were assessed using CEA+ colorectal (n=3), lung (n=2), and gastric (n=2) cancer lines. Combination activity of NILK-2401 + NILK-2301 (± NILK-3301) was assessed by flow cytometry. In vivo activity was tested in xenograft NOG or NSG/human PMBC-, HIS-, and hSIRPα/hCD47/hCD3/hCD28 transgenic mice. Safety data include binding to other CEACAMs, cytokine release in whole blood, erythrophagocytosis, platelet activation, exclusion of superagonism (NILK-3301), as well as PK- and tolerability in cynomolgus monkeys and Tg32-mice. Results: NILK-2301 induced dose-dependent killing of all tested cell lines, which was also visualized by live cell imaging. Combination of NILK-2301 (1 nM) + NILK-3301 vs. NILK-2301 alone (10 nM) increased TDCC (3-8-fold), T-cell activation (CD25, CD69, HLA-DR), cytokine secretion (interferon-γ, granzyme B, perforin), and CD4+/CD8+ T-cell proliferation. NILK-2401 blocked CD47-SIRPα interaction and induced ADCP/ADCC-mediated elimination of all cell lines. NILK-2301 + NILK-2401 treatment increased maximum activity (Emax) and reduced necessary dose of the T-cell bsAb to reach Emax. E.g., Emax of 30% killing (NILK-2301 alone) was increased in combination with NILK-2401 at 0.1/1/10 µg/mL to 40%, 80%, and 80%. In vivo, NILK-2301 (10 mg/kg IV, BIW) decreased tumor progression. NILK-2301/-3301 combination induced tumor regression in 8/8 mice. NILK-2401 delayed tumor growth vs. mean of control in 100% (15/15) of mice and prevented establishment of detectable tumors (&amp;gt;50mm3) in 53% (8/15). Results of double and quadruple transgenic mice, including triple bsAb combinations, will be presented at the meeting. No relevant safety signals were detected. Conclusions: NILK-2301 and NILK-2401 are active as single agents. Addition of NILK-2401 or NILK-3301 to NILK-2301 significantly increases activity, already at 10 -100x lower CEAxCD3 doses. GMP drug substance has been produced for NILK-2301 and NILK-2401. Generation of the clonal cell line for NILK-3301 clinical material production is ongoing. Citation Format: Anja Seckinger, Lise Nouveau, Sara Majocchi, Valéry Moine, Vanessa Buatois, Bruno Daubeuf, Franck Gueneau, Ulla Ravn, Krzysztof Masternak, Yves Poitevin, Emeline Rousset, Giovanni Magistrelli, Pauline Malinge, Limin Shang, Nicolas Fischer, Klaus Strein, Walter Ferlin, Dirk Hose. Combination of κλ bispecific antibodies targeting innate (CEAxCD47, NILK-2401) and adaptive immunity (CEAxCD3, NILK-2301 and CEAxCD28, NILK-3301) for next generation immunotherapy of CEA-expressing cancers. [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 5100.

Longitudinal T Cell Responses against Ancestral, Delta, and Omicron SARS-CoV-2 Variants Determined by Rapid Cytokine Release Assay in Whole Blood.

T cell immunity to natural SARS-CoV-2 infection may be more robust and longer lived than Ab responses. Accurate assessment of T cell responses is critical for understanding the magnitude and longevity of immunity across patient cohorts, and against emerging variants. By establishing a simple, accurate, and rapid whole blood test, natural and vaccine-induced SARS-CoV-2 immunity was determined. Cytokine release in whole blood stimulated with peptides specific for SARS-CoV-2 was measured in donors with previous PCR-confirmed infection, suspected infection, or with no exposure history (n = 128), as well as in donors before and after vaccination (n = 32). Longitudinal assessment of T cell responses following initial vaccination and booster vaccination was also conducted (n = 50 and n = 62, respectively). Cytokines were measured by ELISA and multiplex array. IL-2 and IFN-γ were highly elevated in PCR-confirmed donors compared with history-negative controls, with median levels ∼33-fold and ∼48-fold higher, respectively. Receiver operating curves showed IL-2 as the superior biomarker (area under the curve = 0.9950). Following vaccination, all donors demonstrated a positive IL-2 response. Median IL-2 levels increased ∼32-fold from prevaccination to postvaccination in uninfected individuals. Longitudinal assessment revealed that T cell responses were stable up to 6 mo postvaccination. No significant differences in cytokine production were observed between stimulations with Wuhan, Delta, or Omicron peptides. This rapid, whole blood-based test can be used to make comparable longitudinal assessments of vaccine-induced T cell immunity across multiple cohorts and against variants of concern, thus aiding decisions on public health policies.

Preclinical anti-tumor activity of small-molecule oral PD-L1 checkpoint inhibitors.

e14558 Background: The PD-1/PD-L1 checkpoint axis is a validated target in oncology, and immunotherapy with antibody approaches have proven efficacy across various tumor types. Oral small-molecule inhibitors of PD-L1 may offer advantages over antibody approaches by enabling tunable on-target engagement and the potential for better tissue penetration and improved efficacy. Here we report the preclinical in vitro activity of small-molecule inhibitors of PD-L1 possessing a novel mechanism of action, favorable pharmacokinetics, and demonstrated in vivo efficacy in a mouse colon adenocarcinoma model. Methods: In vitro activity was assessed in a Jurkat T cell NFAT reporter assay and PD-L1 reduction was confirmed in CHO-K1 cells expressing human PD-L1 (CHO-K1-hPD-L1) and in peripheral blood mononuclear cells (PBMCs) from healthy donors. T cell activation assays were conducted with PBMCs from healthy donors. Non-specific cytokine release was evaluated in human whole blood. Pharmacokinetic (PK) evaluations were conducted in rodents and non-human primates (NHP). In vivo efficacy was evaluated in an MC38 tumor humanized PD-L1 and PD-1 mouse model. Results: Small-molecule inhibitors of PD-L1 were able to disrupt PD-1:PD-L1 and PD-L1:CD80 interactions through the reduction of PD-L1 expression on the cell surface via a novel internalization mechanism, resulting in PD-L1 degradation (EC50s ranged from 1.9 – 24 nM in CHO-K1-hPD-L1 and primary human myeloid cells). Lead PD-L1 inhibitors mediated potent activation of T cells in a NFAT reporter assay (EC50s of 13 and 18 nM) as well as dose responsive elevations in IL-2 production in human PBMCs stimulated with staphylococcal enterotoxin B. Compound treatment did not elicit non-specific cytokine release in human whole blood, supportive of favorable immune safety. PK profiles showed low systemic clearance in rodents and NHP. In an MC38 tumor model, once daily oral administration of compounds at 3, 10, and 30 mg/kg resulted in dose responsive tumor reduction that was associated with T cell activation and infiltration into tumors. Conclusions: Oral small-molecule PD-L1 inhibitors possessing a novel mechanism of action and the ability to mediate T cell activation in primary human immune cell types were identified. These compounds display in vivo anti-tumor efficacy comparable to anti-PD-L1 antibody and possess favorable preclinical profiles for further development, with the potential for all-oral treatment regimens for oncology.

Safety and immunogenicity of a QazCovid-in® inactivated whole-virion vaccine against COVID-19 in healthy adults: A single-centre, randomised, single-blind, placebo-controlled phase 1 and an open-label phase 2 clinical trials with a 6 months follow-up in Kazakhstan.

BackgroundA new inactivated whole-virion QazCovid-in® vaccine against COVID-19 was developed from SARS-CoV-2 isolated in Kazakhstan, inactivated by formaldehyde, and adjuvanted with aluminium hydroxide. Phase 1 and 2 clinical trials aimed at assessing the vaccine's safety, immunogenicity, and the duration of immunity induced by the QazCovid-in® vaccine after one or two immunisations.MethodsFrom 23.09.2020 to 19.03.2021 we performed a randomised, single-blind, placebo-controlled phase 1 clinical trial and from 18.10.2020 to 17.04.2021 an open-label phase 2 clinical trials of the QazCovid-in® vaccine with a 6 months follow-up at a single centre in Almaty, the Republic of Kazakhstan. Eligible healthy adults aged 18 years and older with no history of laboratory-confirmed SARS-CoV-2 infection were randomly assigned to the treatment groups using a computerised randomisation scheme generator. In the phase 1 clinical trial, two doses of the vaccine (5 μg each) or placebo (0·9% NaCl) were administered intramuscularly to 44 subjects aged 18–50 years, 21 days apart. In the phase 2 trial, 200 healthy participants were randomised into four equal-sized groups according to the age (18–49 or ≥50 years) and either single (day 1) or double (day 1 and 21) vaccination protocol. The primary outcomes were safety and tolerability. The secondary outcome was immunogenicity. The cellular response was measured by a whole-blood cytokine release assay (phase 1 only). The trials were registered with ClinicalTrials.gov NCT04530357.FindingsThe QazCovid-in® vaccine was safe and well-tolerated and induced predominantly mild adverse events; no serious or severe adverse events were recorded in both trials. In the phase 1 trial, the percentage of subjects with a fourfold increase of antibody titres (sero conversion) in MNA was 59% after one vaccine dose and amounted to 100% after two doses. Neutralizing antibody titres reached the geometric mean titre (GMT) of 100 after administration of two doses. A statistically significant increase in the levels of pro-inflammatory cytokines after vaccination indicated the Th1-biased response. On day 180, 40% of placebo-treated subjects demonstrated a statistically significant increase in the levels of antibodies measured by both ELISA and MNA, which suggests the infection with SARS-CoV-2. In the phase 2 trial, 100% of subjects aged 18–49 years seroconverted for SARS-CoV-2 on day 21 after the first dose, as indicated by MNA yielding the GMTs of 32 or 30 in the one- and two-dose groups, respectively. Amongst ≥50-year-old subjects, the number of sero conversions in the two- and one-dose groups on day 21 was 94% and 92% with the respective GMTs of 25 and 24. After the second dose, the sero conversion rate reached 100%; however, the GMT was significantly lower when compared with the corresponding value measured in subjects aged 18–49 years (83 vs 143). In both trials, specific antibodies were detected in MNA and ELISA on study day 180, but the titres dropped in comparison to day 42. The results of this study serve as the rationale for the phase 3 study.InterpretationThe QazCovid-in® vaccine is safe and well-tolerated and promotes pronounced humoral immunity which lasts for at least 6 months after double intramuscular immunisation.FundingThe work was funded by the Science Committee of the Ministry of Education and Science of the Republic of Kazakhstan within the framework of the Scientific and Technical Program "Development of a vaccine against coronavirus infection COVID-1900 . State registration number ?.0927.

HMBD-002 is a novel, neutralizing, anti-VISTA antibody exhibiting strong preclinical efficacy and safety, being developed as a monotherapy and in combination with pembrolizumab.

e14569 Background: Inhibition of immune checkpoints has been an effective strategy against several cancers, but efficacy gaps remain. VISTA is an immunoregulatory protein expressed on both hematopoietic and tumor cells and has been shown to induce an immunosuppressive environment by repressing T-cell activation and cytokine production. VISTA expression has been shown to be upregulated following treatment with immune checkpoint inhibitors. Hummingbird Bioscience is developing HMBD-002, an IgG4 antibody against VISTA with high binding specificity across species (human, cyno and rodent) and low pM affinity to the region of VISTA required for engagement with interaction partners, including VSIG3. As HMBD-002 is an IgG4 isotype, it uniquely blocks the inhibitory function of VISTA and releases immune suppression without depleting VISTA+ cells. Here we establish the preclinical efficacy and safety of HMBD-002 along with the rationale for development of HMBD-002 as a monotherapy and in combination with PD1 inhibitors in triple negative breast cancer (TNBC) and non-small cell lung cancer (NSCLC). Methods: The effect of HMBD-002 in an in vitro model of human immune activation was evaluated using an allogenic Mixed Lymphocyte Reaction (MLR) assay. Tumor growth inhibition studies were conducted in syngeneic murine cell-derived xenograft (CDX) models. The PK and tolerability of HMBD-002 was evaluated in rats and cynomolgus monkeys with a 21 or 28-day observation period. The expression profile of VISTA was examined in different cancers by IHC staining of FFPE TMAs of various solid cancers. Results: HMBD-002 induced a significant dose dependent increase in the levels of inflammatory cytokines, IFN-γ and TNF- α and suppressed the expansion of CD14+ monocytes. Treatment with HMBD-002 significantly inhibited tumor growth and improved survival in CDX models. HMBD-002 is well tolerated in rats and cynomolgus monkeys with acceptable serum half-life, no HMBD-002 related morbidity/mortality or clinical signs, and no HMBD-002 related changes were observed in body weight, food consumption, clinical chemistry, or hematology parameters. HMBD-002 did not induce cytokine release in human PBMCs or whole blood. TNBC and NSCLC patients were found to harbor the highest VISTA expression across cancer types tested, providing a rationale for exploring these indications in upcoming clinical studies. Conclusions: These studies provide the rationale for development of HMBD-002 as a monotherapy and in combination with pembrolizumab to treat patients with solid tumors expressing VISTA in the tumor microenvironment. A Phase 1 study will be initiated in 2021.

Subvisible Particles in Solutions of Remicade in Intravenous Saline Activate Immune System Pathways in In Vitro Human Cell Systems.

Among patients that receive Remicade® therapy, more than 20% have adverse infusion related reactions and approximately 50% have immunogenic responses.1-3 Upon characterization of initial Remicade®-IV solution we observed a high concentration of subvisible particles that could inadvertently be delivered to patients. This solution was processed through the IV infusion system, mimicking the typical clinical administration setup - either with or without an in-line filter connected to the IV line. The samples generated thereafter were tested using various in vitro assays for activation of the innate immune system via cytokine release in whole blood and in peripheral blood mononuclear cell (PBMC) cultures, and activation of the Toll like receptors (TLRs). Activation of the adaptive immune system was evaluated by monitoring upregulation of surface receptors on dendritic cells (DCs) and CD4+ T cell proliferation in response to IV solution of Remicade®. Our results indicate that subvisible particles in Remicade®-saline solution have a significant role in activation of the immune system but there are extrinsic factors potentially contributed by the in-line filters or other process parameters that also contribute to immune system activation.

TLR8 and complement C5 induce cytokine release and thrombin activation in human whole blood challenged with Gram-positive bacteria.

We recently showed that TLR8 is critical for the detection of Gram-positive bacteria by human monocytes. Here, we hypothesized that TLR8 and complement together regulate antibacterial responses in human blood. Anticoagulated blood was treated with selective inhibitors of TLR8 and/or complement C5, and then challenged with live Streptococcus agalactiae (Group B streptococcus, GBS), Staphylococcus aureus, or Escherichia coli. Cytokine production, plasma membrane permeability, bacterial survival, phagocytosis, and activation of coagulation was examined. GBS and S. aureus, but not E. coli, triggered TLR8-dependent production of IL-12p70, IL-1β, TNF, and IL-6 in fresh human whole blood. In purified polymorphonuclear neutrophils (PMN), GBS and S. aureus induced IL-8 release in part via TLR8, whereas PMN plasma membrane leakage and extracellular DNA levels increased independently of TLR8. TLR8 was more important than C5 for bacteria-induced production of IL-12p70, IL-1β, and TNF in blood, whereas IL-8 release was more C5 dependent. Both TLR8 and C5 induced IL-6 release and activation of prothrombin cleavage, and here their combined effects were additive. Blocking of C5 or C5aR1 attenuated phagocytosis and increased the extracellular growth of GBS in blood, whereas TLR8 inhibition neither reduced phagocytosis nor intracellular killing of GBS and S. aureus. In conclusion, TLR8 is more important than C5 for production of IL-12p70, IL-1β, and TNF upon GBS and S. aureus infection in blood, whereas C5 is central for IL-8 release and phagocytosis. Both TLR8 and C5 mediate IL-6 release and activation of coagulation during challenge with Gram-positive bacteria in blood.

Preclinical Evaluation of MCLA117, a CLEC12AxCD3 Bispecific Antibody Efficiently Targeting a Novel Leukemic Stem Cell Associated Antigen in AML

Although chemotherapy regimens induce initial remissions in most acute myeloid leukemia (AML) patients, their long term prognosis is very poor. Novel targeted therapies that effectively eradicate both AML blasts and their progenitors are needed.MCLA-117, a potent human CLEC12AxCD3 bispecific IgG antibody redirects patient's cytotoxic T cells to induce AML tumor lysis. CLEC12A is a myeloid differentiation antigen that is expressed on 90-95% of newly diagnosed and relapsed AML. Moreover, CLEC12A is selectively expressed on leukemic stem cells (LSCs) but not normal early hematopoietic progenitors, including hematopoietic stem cells (HSCs).MCLA-117 is a full length human bispecific IgG that incorporates CH2 region amino acid substitutions to abrogate Fcγ receptor and C1q interactions while retaining its binding to FcRn. MCLA-117 specifically binds to CLEC12A expressing myeloid cells and CD3 expressing T cells, as evaluated on healthy donor samples by flow cytometry. In line with the CLEC12A expression profile within the hematopoietic progenitor compartment, MCLA-117 did not bind the HSCs, nor the common myeloid progenitor cells. As determined by Surface Plasmon Resonance, MCLA-117 had an affinity of 3 nM for human CLEC12A and 177 nM for human CD3.The efficacy of MCLA-117 to induce T cell mediated lysis of CLEC12A+ tumor cells was evaluated in cytotoxicity assays. In co-cultures with resting healthy donor T cells and AML tumor cells MCLA-117 efficiently induced CLEC12A antigen dependent T cell activation, T cell proliferation and tumor target cell lysis. MCLA-117 induced tumor target lysis with an EC50 of 66±37 ng/mL. Upregulation of the activation CD69 marker on CD8 T cells was the most sensitive read-out for the activity of MCLA-117 (EC20 of 11±3 ng/mL). Therefore the MABEL (minimum anticipated biological effect level) concentration for MCLA-117 was defined as 10 ng/mL and is used to calculate a safe starting dose in a planned first in human study. MCLA-117 was able to activate and redirect AML patient-derived T cells to CLEC12APOS tumor cells as potently as that of healthy donor-derived T cells.The efficacy of MCLA-117 to induce lysis of AML blasts by autologous T cells in primary AML bone marrow (BM) samples with low T cell to AML blast ratios (E:T ratios of 1:7-1:80) was examined. AML patient BM samples taken at diagnosis were cultured in medium with low amounts of a cytokine cocktail to support in vitro survival of AML blasts. MCLA-117 induced up to 30-fold T cell expansion after 7-10 days. More importantly, MCLA-117 efficiently induced AML blast lysis (up to 88%) in 6/6 tested primary AML patient BM samples, even at very low effector to target ratios (see figure 1).The cytokine release potential was assessed by incubation of human whole blood and PBMC preparations with MCLA-117. Moderate to strong cytokine release in whole blood was observed (IFNγ, IL-6, IL-8, IL-10 and TNFα) after 24 hours at concentrations of 10,000 and 1,000 ng/mL MCLA-117. At concentration of 100 ng/mL and 10 ng/mL, moderate to low cytokine release and no cytokine release was observed, respectively. In PBMC preparations, moderate to strong IFNγ release was observed after 48 hours at MCLA-117 concentrations of 10,000, 1,000 and 100 ng/mL. At these concentrations, only low release of IL-2, IL-10 and TNFα and no release of IL-1β, IL-6 and IL-8 was observed.MCLA-117 may provide a novel treatment option for all subtypes of AML with highly specific targeting of myeloid blasts and progenitors and sparing the normal HSCs, as well as eradicating residual LSCs residing in the BM niche compartments. A first clinical study is planned to evaluate the safety and preliminary efficacy of MCLA-117 in adult AML patients. [Display omitted] DisclosuresVan Loo:Merus B.V.: Employment. Doornbos:Merus B.V.: Employment. Shamsili:Merus B.V.: Employment. Bakker:Merus B.V.: Employment.

G.P.132: Pre-clinical development of peptide-conjugated antisense oligonucleotides for myotonic dystrophy type 1 (DM1)

Myotonic dystrophy type 1 (DM1) is a multisystemic neuromuscular disorder caused by an expanded CTG repeat in the 3′ UTR of the DM protein kinase (DMPK) gene, resulting in formation of toxic RNA aggregates that interfere with RNA splicing and dysregulate normal cell function. RNA-modulation by antisense oligonucleotides (AONs) represents an interesting therapeutic approach for DM1 and is currently in pre-clinical development. We have previously obtained molecular proof-of-concept for a panel of AONs that target the CUG expansion and result in efficient knockdown of toxic RNA in DM1 patient myoblasts with different repeat expansion lengths and in cultured muscle cells derived from DM500 mice. To facilitate systemic AON delivery and uptake, we used a 7-amino acid linear muscle targeting peptide (PP08) and conjugated this to different CUG-targeting AONs. We have shown that these peptide-conjugated AONs are well tolerated and do not result in complement activation in human and monkey plasma or induce cytokine release in human whole blood in vitro safety assays. Subcutaneous administration of peptide-conjugated AONs in different DM1 mouse models (DM500, HSALR) resulted in enhanced tissue PK levels in several tissues of relevance for DM1 such as skeletal muscle and brain, indicating that the peptide-AON displayed efficient tissue uptake and was even able to pass the blood–brain-barrier. The higher tissue levels were accompanied by an increased knockdown of mutant RNA compared to non-conjugated AONs. Besides inducing a significant RNA knockdown, peptide-conjugated AONs resulted in a mild but statistically significant reduction of myotonia in gastrocnemius and tibialis muscles of HSALR mice, as determined by EMG analysis. These data demonstrate that peptide-AONs targeting the CUG-repeat have potential in a multisystemic therapeutic approach for DM1.

The Absence of Mrp4 Has No Effect on the Recruitment of Neutrophils and Eosinophils into the Lung after LPS, Cigarette Smoke or Allergen Challenge

The multidrug resistance protein 4 (Mrp4) is an ATP-binding cassette transporter that is capable of exporting the second messenger cAMP from cells, a process that might regulate cAMP-mediated anti-inflammatory processes. However, using LPS- or cigarette smoke (CS)-inflammation models, we found that neutrophil numbers in the bronchoalveolar lavage fluid (BALF) were similar in Mrp4−/− and Mrp4+/+ mice treated with LPS or CS. Similarly, neutrophil numbers were not reduced in the BALF of LPS-challenged wt mice after treatment with 10 or 30 mg/kg of the Mrp1/4 inhibitor MK571. The absence of Mrp4 also had no impact on the influx of eosinophils or IL-4 and IL-5 levels in the BALF after OVA airway challenge in mice sensitized with OVA/alum. LPS-induced cytokine release in whole blood ex vivo was also not affected by the absence of Mrp4. These data clearly suggest that Mrp4 deficiency alone is not sufficient to reduce inflammatory processes in vivo. We hypothesized that in combination with PDE4 inhibitors, used at suboptimal concentrations, the anti-inflammatory effect would be more pronounced. However, LPS-induced neutrophil recruitment into the lung was no different between Mrp4−/− and Mrp4+/+ mice treated with 3 mg/kg Roflumilast. Finally, the single and combined administration of 10 and 30 mg/kg MK571 and the specific breast cancer resistance protein (BCRP) inhibitor KO143 showed no reduction of LPS-induced TNFα release into the BALF compared to vehicle treated control animals. Similarly, LPS-induced TNFα release in murine whole blood of Mrp4+/+ or Mrp4−/− mice was not reduced by KO143 (1, 10 µM). Thus, BCRP seems not to be able to compensate for the absence or inhibition of Mrp4 in the used models. Taken together, our data suggest that Mrp4 is not essential for the recruitment of neutrophils into the lung after LPS or CS exposure or of eosinophils after allergen exposure.

Reassessment of Immune Correlates in Human Visceral Leishmaniasis as Defined by Cytokine Release in Whole Blood

Depressed cell-mediated immunity in human visceral leishmaniasis (VL) (also known as kala-azar), revealed as the inability of peripheral blood mononuclear cells (PBMCs) to respond to Leishmania antigen, remains a hallmark of and is thought to underlie the progressive nature of this disease. We recently reported the ability of a whole-blood, gamma interferon (IFN-γ) release assay to detect subclinical infections among healthy individuals living in an area where kala-azar is endemic (Bihar, India) and the surprising result that patients with active VL also secreted significant levels of antigen-specific IFN-γ in this assay. We were interested in ascertaining whether these findings would be true for a larger cohort of subjects and in employing the whole-blood assay to detect additional cytokines that might better correlate with the disease status of infected individuals. We evaluated IFN-γ, tumor necrosis factor alpha (TNF-α), and interleukin-10 (IL-10) release in 35 patients with active VL, 54 patients with VL who were cured, 27 patients with other diseases, 52 healthy controls who lived in regions where VL or kala-azar is not endemic (NEHCs [for nonendemic healthy controls]), and 147 healthy controls who lived in regions where kala-azar is endemic (EHCs [for endemic healthy controls]). The cellular responses of the EHCs were correlated with their serological antibody titers against Leishmania donovani and Phlebotomus argentipes saliva. The whole-blood cells from the majority of both active (80%) and cured (85%) VL patients, as well as 24% of EHCs with presumed subclinical infections, produced significantly elevated levels of IFN-γ. The findings do not support a severe Th1 response defect in kala-azar. Importantly, only the patients with active VL also produced IL-10, which in conjunction with IFN-γ better reflects the immune responses that distinguish individuals with active disease from cured or subclinically infected, immune individuals.

Abstract LB-236: M4-3-ML2, a novel glycoengineered humanized IgG1 antibody, targeting a membrane-proximal epitope of MCSP/CSPG4 exhibits potent ADCC induction in vitro and in vivo anti-tumoral efficacy in disseminated melanoma models

Abstract MCSP/CSPG4 is a large transmembrane proteoglycan identified in melanomas as HMW-MAA. In the mouse it is known as neurite growth factor 2 (NG2), a marker of pericyte recruitment. MCSP has been used as a target for clinical imaging of (uveal) melanomas by immunoscintigraphy. MCSP shows uniform and abundant expression in ca. 60-80% of melanoma, and was described in lobular breast carcinoma, glioblastoma, osteo- &amp; chondrosarcoma, and basal cell carcinoma. It is present at high levels on pericytes of tumor neovasculature, but down-regulated as vessels mature. Normal tissue expression is low and it is not detected on PBMCs. We have generated human/Cynomolgus cross-reactive antibodies against a membrane-proximal MCSP epitope by mouse immunization with a linear peptide derived from the membrane proximal D3 domain followed by boosting with melanoma cells. The mouse antibody LC007 was selected for humanization due to its potent induction of ADCC as a chimeric antibody, compared to antibodies to membrane distal epitopes of MCSP. LC007 as chimeric IgG1 and its humanized IgG1 derivative M4-3-ML2 are characterized by the following properties: i) Specific binding to the native epitope on MCSP+ melanoma cells, but no induction of internalization; ii) Specific IHC staining of MCSP+ cells in FFPET samples; iii) ca 10 nM monovalent affinity for hMCSP D3 domain. Moreover, glycoengineering of LC007 and M4-3-ML2 antibodies using GlycoMab technology resulted in increased binding affinity for hFcgRIIIa and enhanced ADCC potency and absolute killing of melanoma cell lines. As expected, neither up to 10 ug/mL wildtype, nor glycoengineered M4-3-ML2 induced relevant cytokine (IL-6, TNF-α, IFN-γ) release in human whole blood supporting that MCSP is not expressed there. Subsequently, we studied anti-tumoral efficacy of the chimeric antibody LC007 and the humanized antibody M4-3-ML2 in disseminated models of MV3 and MDA-MB435 melanoma after i.v. injection of tumor cells in hCD16 transgenic Scid mice, which express the functional human high affinity FcgRIIIa receptor on NK cells. Both, glycoengineered LC007 and M4-3-ML2 mediated efficacy in terms of enhanced median and overall survival in both disseminated xenograft models, and were superior to the respective non-glycoengineered antibodies. Taken together, our studies support MCSP/CSPG4 as an attractive target for antibody-based cancer immunotherapy. Further studies investigating the anti-angiogenic effect of MCSP antibodies via their action on pericytes/vascular smooth muscle cells are ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-236. doi:1538-7445.AM2012-LB-236

Abstract LB-158: MEDI4736: Delivering effective blockade of immunosupression to enhance tumour rejection: Monoclonal antibody discovery and preclinical development

Abstract Cancerous cells emerge within the body following accumulation of deleterious genetic mutations. These mutations alter the phenotype of a cancer cell marking it as distinct from the surrounding host; an immunological state termed “altered self”. These cells, like other non-self entities such as viruses and bacteria, are recognised by the immune system and marked for destruction, a process known as “immune surveillance”. B7-H1 expression by tumour cells is believed to aid tumours in evading detection and elimination by the immune system. B7-H1 functions in this respect via several alternative mechanisms including driving exhaustion and anergy of tumour infiltrating T lymphocytes, stimulating secretion of immune repressive cytokines into the tumour micro-environment, stimulating repressive regulatory T cell function and protecting B7-H1 expressing tumour cells from lysis by tumour cell specific cytotoxic T cells. Using hybridoma technology and high throughput screening MedImmune has identified a series of fully human antibodies specific for human B7-H1. Further characterisation of these antibodies led to the identification of a single high affinity antibody, MEDI 4736, with the ability to relieve B7-H1 mediated suppression of T cell activation in vitro and to enhance sub-optimal T cell activation in a mixed lymphocyte reaction. In vitro testing shows that MEDI 4736 does not trigger non-specific cytokine release in whole blood, and is only able to activate T cells in the context of an active T cell receptor signal. A surrogate anti-mouse B7-H1 antibody shows significant anti-tumour activity in a syngeneic model when dosed in combination with chemotherapy. Similarly MEDI 4736 is able to inhibit tumour growth in a novel in vivo xenograft model, via a mechanism that is dependent on the presence of tumour specific human T cells. 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 LB-158. doi:10.1158/1538-7445.AM2011-LB-158

S47 RAGE-mediated cytokine release from leukocytes: implications for systemic inflammation

IntroductionSystemic inflammatory response syndrome (SIRS) is the physiological and biochemical changes that result from an inflammation of the body, which commonly occurs in patients after cardiac surgery necessitating cardiopulmonary bypass...

LPS interactions with immobilized and soluble antimicrobial peptides

A promising approach in sepsis therapy is the use of peptides truncated from serum- and membrane-proteins with binding domains for LPS: antimicrobial peptides (AMPs). AMPs can be useful in combination with conventional antibiotics to increase killing and neutralize LPS. Although many AMPs show a high specificity towards bacterial membranes, they can also exhibit toxicity, i.e. non-specific membrane lysis, of mammalian cells such as erythrocytes and therefore, unsuitable as systemic drugs. A way to overcome this problem may be an extracorporeal therapy with immobilized peptides. This study will compare neutralization of LPS using different AMPs in solution and when immobilized on to solid phases. The peptides ability to neutralize LPS-induced cytokine release in whole blood will also be tested. The peptides are truncated derivates from the known AMPs LL-37, SC4, BPI, S3Δ and CEME. Two different methods were used to immobilize peptides, biomolecular interaction analysis, and Pierce SulfoLink Coupling Gel. To investigate LPS binding in solution the LAL test was used. After whole blood incubation with LPS and AMPs ELISA was used to measure TNFα, IL-1β and IL-6 production. The results suggest that immobilization of antimicrobial peptides does not inhibit their capacity to neutralize LPS, although there are differences between the peptides tested. Thus, peptides derived from LL-37 and CEME were more efficient both in LPS binding and neutralizing LPS-induced cytokine production.

2‐Acylaminopyridin‐4‐ylimidazoles as p38 MAP Kinase Inhibitors: Design, Synthesis, and Biological and Metabolic Evaluations

Targeting cytokines has become an important focus in the treatment of many inflammatory disorders. p38 MAP kinase (MAPK) is the key enzyme in regulating the biosynthesis and release of pro-inflammatory cytokines such as IL-1beta and TNFalpha. Inhibition of p38 MAPK results in decreased expression of these cytokines. Tri- and tetrasubstituted pyridinylimidazoles are potent inhibitors of p38 MAPK. Substitution on the pyridinyl moiety allows the design of inhibitors that show increased selectivity and activity by targeting the enzyme's hydrophobic region II. The objective of this study was to synthesize novel 1,2,4,5-tetrasubstituted imidazole derivates and to characterize them not only for their ability to inhibit p38 MAPK and modulate cytokine release in human whole blood, but also to evaluate their metabolic stability. Biological data and metabolic studies demonstrate that the introduction of a 2-acylamino function at C2 of the pyridine results in highly efficient and metabolically stable inhibitors relative to C2-alkylamino derivatives. A series of novel candidates was investigated for metabolic stability in human liver microsomes and in human whole blood. Additionally, metabolic S-oxidation was investigated, and possible metabolites were synthesized.

Effects of remifentanyl and fentanyl on LPS-induced cytokine release in human whole blood in vitro

Aim The present study sought insight into the effects of remifentanyl and fentanyl on LPS-induced release of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) and IL-10 in human whole blood. Methods Whole blood was incubated in the presence and absence of remifentanyl and fentanyl. Effects of remifentanyl and fentanyl on spontaneous and endotoxin (lipopolysaccharide; 100 ng ml(-1))-stimulated cytokine release were studied in whole blood from volunteers (n = 10) cultured for 6 h. Results IL-6, TNF-alpha and IL-10 concentrations in groups added with LPS were significantly higher than those in control group (P < 0.01). IL-6, TNF-alpha and IL-10 concentrations in activation groups treated with remifentanyl or fentanyl were significantly lower than those in LPS treated group (P < 0.05). There were no significant differences on IL-6,TNF-alpha and IL-10 concentrations in drug-alone groups compared with control group (P > 0.05). Conclusion Remifentanyl or fentanyl alone has no effects on IL-6, TNF-alpha and IL-10 production, but could attenuate LPS-induced IL-6,TNF-alpha and IL-10 production in human whole blood. Remifentanyl and fentanyl could inhibit the expressions of IL-6, TNF-alpha and IL-10 induced by LPS.

Effects of C‐reactive Protein and Homocysteine on Cytokine Production: Modulation by Pravastatin

ObjectiveC-reactive protein (CRP) and homocysteine are markers of cardiovascular risk that may have inflammatory effects. HMG coenzyme A reductase inhibitors (statins) have anti-inflammatory effects in vitro, but it is not clear if such responses in vivo are secondary to lipid lowering. We examined the hypothesis that CRP and homocysteine would stimulate cytokine release in human whole blood and that short-term treatment with a statin would inhibit it.MethodsThe time course of IL-6 and MCP-1 production was determined in whole blood incubated with saline, 1 µg/mL lipopolysaccaride (LPS), 50 and 100 µM/L DL-homocysteine, and 5 µg/mL human recombinant CRP for 24 hours at 37°C under 5% CO2 atmosphere. Cytokine responses were determined in blood drawn from 15 healthy volunteers before and after administration of pravastatin 40 mg daily for 2 days.ResultsBoth human recombinant CRP and LPS significantly increased the production of IL-6 and MCP-1 in whole blood samples more than 4-fold (P < 0.001) but homocysteine did not. Oral administration of pravastatin, 40mg daily for 2 days, decreased CRP-stimulated IL-6 production by approximately 20% (P = 0.02) 6 hours after incubation, but did not affect MCP-1 production (P = 0.69). Pravastatin treatment did not affect LPS-stimulated MCP-1 but increased IL-6 modestly.ConclusionsCRP stimulated the production of the proatherogenic mediators MCP-1 and IL-6 in human whole blood, but homocysteine did not. CRP-stimulated production of IL-6, but not MCP-1, was modestly attenuated by short-term treatment with pravastatin.

Response to Comment on “Not Lipoteichoic Acid but Lipoproteins Appear to Be the Dominant Immunobiologically Active Compounds in Staphylococcus aureus”

Von Aulock et al. demonstrated in their letter ([1][1]) that the lipoteichoic acids (LTAs) of Staphylococcus aureus Δ lgt mutant and its wild type (WT) are equipotent in stimulating cytokine release in human whole blood. However, we were not able to confirm their results in our experimental systems