Human cluster of differentiation (CD) antigen 19 is a B cell-specific surface antigen and an attractive target for therapeutic monoclonal antibody (mAb) approaches to treat malignancies of B cell origin. MEDI-551 is an affinity-optimized and afucosylated CD19 mAb with enhanced antibody-dependent cellular cytotoxicity (ADCC). The results from in vitro ADCC assays with Natural Killer cells as effector cells, demonstrate that MEDI-551 is effective at lower mAb doses than rituximab with multiple cell lines as well as primary chronic lymphocytic leukaemia and acute lymphoblastic leukaemia samples. Targeting CD19 with MEDI-551 was also effective in several severe combined immunodeficiency lymphoma models. Furthermore, the combination of MEDI-551 with rituximab resulted in prolonged suppression of tumour growth, demonstrating that therapeutic mAbs with overlapping effector function can be combined for greater tumour growth inhibition. Together, the data demonstrate that MEDI-551 has potent antitumour activity in preclinical models of B cell malignancies. The results also suggest that the combination of the ADCC-enhanced CD19 mAb with an anti-CD20 mAb could be a novel approach for the treatment of B cell lymphomas.

Objective To characterise activation of the type I interferon (IFN) pathway in patients with systemic lupus erythematosus (SLE), dermatomyositis (DM), polymyositis (PM), rheumatoid arthritis (RA) and systemic scleroderma (SSc) and to evaluate the potential to develop a molecular diagnostic tool from the peripheral blood that reflects this activation in disease-affected tissues.Methods Overexpressed transcripts were identified in the whole blood (WB) of 262 patients with SLE, 44 with DM, 33 with PM, 28 with SSc and 89 with RA and compared with 24 healthy subjects using Affymetrix microarrays. A five gene type I IFN signature was assessed in these subjects to identify subpopulations showing both activation and concordance of the type I IFN pathway in the peripheral blood and disease-affected tissues of each disease and to correlate activation of this pathway in the WB with clinical measurements.Results A common set of 36 type I IFN inducible transcripts were identified among the most overexpressed in the WB of all subjects. Significant activation of the type I IFN pathway in subgroups of each of the five diseases studied was observed. Baseline disease activity measurements correlated with a type I IFN gene signature in the WB of subjects with SLE, PM and SSc, as did various serum autoantibody levels in subjects with SLE and DM. This signature was also well correlated between disease-affected tissue and WB in subjects with SLE, DM, PM and SSc.Conclusions The results indicate that the type I IFN pathway is activated in patient subsets of five rheumatic diseases and suggest that these subsets may benefit from anti-IFN therapy.

The pan B-cell surface antigen CD19 is an attractive target for therapeutic monoclonal antibody (mAb) approaches. We have generated a new afucosylated anti-human (hu)CD19 mAb, MEDI-551, with increased affinity to human FcγRIIIA and mouse FcγRIV and enhanced antibody-dependent cellular cytotoxicity (ADCC). During in vitro ADCC assays with B-cell lines, MEDI-551 is effective at much lower mAb concentrations than the fucosylated parental mAb anti-CD19-2. Furthermore, the afucosylated CD19 mAb MEDI-551 depleted B cells from normal donor peripheral blood mononuclear cell samples in an autologous ADCC assay, as well as blood and tissue B cells in human CD19/CD20 double transgenic (Tg) mice at lower concentrations than that of the positive control mAb rituximab. In huCD19/CD20 Tg mice, both macrophage-mediated phagocytosis and complement-dependent cytotoxicity contribute to depletion with rituximab; MEDI-551 did not require complement for maximal B-cell depletion. Furthermore, extended B-cell depletion from the blood and spleen was achieved with MEDI-551, which is probably explained by bone marrow B-cell depletion in huCD19/CD20 Tg mice relative to the control mAb rituximab. In summary, MEDI-551 has potent B-cell-depleting activity in vitro and in vivo and may be a promising new approach for the treatment of B-cell malignancies and autoimmune diseases.

Human metapneumovirus (HMPV) is a paramyxovirus causing acute respiratory tract infections in humans. The effects of a monoclonal antibody (MAb 338, MedImmune, Inc.) directed against the HMPV fusion protein were assessed in vivo. Different groups of BALB/c mice received an intraperitoneal injection of 25 or 50 mg/kg of MAb 338 either 24 h before or 48 h after viral infection. Lung samples were collected on days 5 and 42 after infection for determination of viral titers and histopathological changes. Pulmonary functions were also evaluated by plethysmography. On day 5 post-infection, lung viral titers were significantly decreased in mice treated with 25 or 50 mg/kg before or after viral infection compared to HMPV-infected control mice. Similarly, HMPV copy numbers on day 42 were decreased for all prophylactic and therapeutic interventions. Histopathological changes were also less severe in all treated groups of mice on days 5 and 42 post-infection, correlating with decreased airways obstruction. Finally, on day 42, all treated groups had a significant decrease in airways hyperresponsiveness following treatment with MAb 338. Both prophylactic and, to a lesser extent, therapeutic administration of MAb 338 improved acute and late consequences of HMPV infection in a relevant mouse model.

MEDI-565 (MT111) is a bispecific single chain antibody of the BiTE (Bi-specific T-cell engager) class that transiently links carcinoembryonic antigen (CEA; CEACAM5; CD66e) on tumor cells and human CD3 on T cells. This results in the activation of T cells and the subsequent redirected lysis of target cells expressing CEA. MEDI-565 is being developed as a clinical candidate for the treatment of CEA-positive tumors. With the exception of the chimpanzee, MEDI-565 does not cross-react with CD3 orthologs. Therefore, as a potential tool for evaluating mechanistic safety in non-human primates, we constructed a biosimilar antibody, CyS111, which combines the CEA-specific component of MEDI-565 with one that binds cynomolgous CD3. This study characterizes the biological activity of CyS111 as it compares to MEDI-565 to determine its potential utility in assessing safety in a relevant species. CyS111 and MEDI-565 both demonstrated specific lysis of CEA-expressing target cells with similar half-maximal lysis (EC50) values and kinetics of lysis, despite moderate binding affinity differences for species-appropriate CD3 and CEA. However, CyS111 non-specifically activated T cells in the presence of CEA-negative target cells or in the absence of target cells entirely, a feature not shared by MEDI-565. In addition, CyS111 induced proliferation of both CD4+ and CD8+ T cells, whereas MEDI-565 induced CD8+ T cell proliferation with a slower kinetics, and only poorly induced proliferation of CD4+ T cells. Interestingly, aggregated forms of MEDI-565 displayed significantly enhanced potency of BiTE-mediated T cell activation and target cell lysis, whereas CyS111 aggregated forms did not. Taken together, these findings may have implications for the use of biosimilar surrogate molecules for evaluating mechanistic safety. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 3247.

Abstract Systemic sclerosis (SSc) is a complex autoimmune disease involving autoreactive B and T cells and excessive extracellular matrix deposition in target organs such as kidneys. We have developed a novel human collagen V (hCollV) induced autoimmune model which shares some common pathological and immunological features with Ssc. Mice immunized with hCollV have increased proteinuria, renal fibrosis, serum autoantibodies (anti- RNP, -SSA, -collagen I, II) (p<0.01), and renal IgG depositions by day 42. Levels of proinflammatory cytokines and chemokines associated with tissue fibrosis, such as IL-6 and MCP-1, were elevated in the sera of immunized mice when compared to controls, as were gene expression levels in the kidneys by qPCR analysis. Depletion of B cells by afucosylated anti-hCD19 antibody resulted in reduced proteinuria, tissue fibrosis, and IgG depositions in the kidneys of immunized mice, decreased cytokines and chemokines levels, and greatly declined serum autoantibodies. Therefore, our data indicated that the B cells play crucial roles in causing renal damage in the hCollV-induced systemic sclerosis model, suggesting a potential therapeutic target to benefit Ssc patients.

We have compared the cytotoxic activity of rituximab with that of blinatumomab (MT103/MEDI-538), a single-chain CD19-/CD3-bispecific antibody engaging human T cells. Blinatumomab consistently led to a higher degree of lysis of human lymphoma lines than rituximab, and was active at much lower concentration. The cytotoxicity mediated by blinatumomab and rituximab both caused a potent activation of pro-caspases 3 and 7 in target cells, a key event in induction of granzyme-mediated apoptotic cell death. Combination of rituximab with blinatumomab was found to greatly enhance the activity of rituximab, in particular at low effector-to-target cell ratios and at low antibody concentration.

Background: Human metapneumovirus (hMPV) is one of the most frequent causes of respiratory tract infections in children. Our objective was to assess the prophylactic benefit of a monoclonal antibody (mAb) against the hMPV fusion protein in a murine model.Methods: BALB/c mice received one intramuscular injection of either 5 or 10 mg/kg of mAb 338 (MedImmune, Inc.) and were infected intranasally 24 h later with 1x10(8) TCID50 (50% tissue culture infectious dose) of hMPV. On days 5 and 42 post-infection, lung samples were collected for determination of viral titres and for histopathological studies. Pulmonary function was characterized by plethysmography.Results: Mean lung viral titres were significantly lower in mice treated with 5 or 10 mg/kg of mAb 338 compared with infected controls on day 5 (283, 45.6 and 1.49x10(5) TCID50/g, respectively; P<0.05). Similarly, lung viral RNA copies were significantly reduced in treated mice on day 42 (292, 101 and 607 copies per 0.01 g of lungs for mice that received 5 mg/kg, 10 mg/kg or no mAb, respectively; P<0.05). Histopathological changes characterized by important alveolar and interstitial inflammation were less severe in treated mice on days 5 and 42 compared with control. Airways obstruction was also significantly reduced in both treated groups on days 5 and 42, but development of hyperresponsiveness following the acute phase of infection was only significantly reduced in 10 mg/kg treated mice.Conclusions: Prophylactic administration of mAb 338 attenuates acute and late consequences of hMPV disease in this mouse model.

BACKGROUND The evolutionarily conserved 18-glycosyl-hydrolase family contains true chitinases and chitinase-like proteins that lack enzymatic activity. Acidic mammalian chitinase has recently been associated with animal models of asthma. The related chitinase-like protein, YKL-40 (also called human cartilage glycoprotein 39 [HCgp-39] and chitinase 3-like 1), can be readily measured in the serum. However, its relationship to asthma has not been evaluated. METHODS We quantified serum YKL-40 levels in three cohorts of patients with asthma--one recruited from the patient population at Yale University, one from the University of Paris, and one from the University of Wisconsin--as well as in controls from the surrounding communities. In the Paris cohort, immunohistochemical analysis and morphometric quantitation were used to evaluate the locus of expression of YKL-40 in the lung. The clinical characteristics of the patients with high serum or lung YKL-40 levels were also evaluated. RESULTS Serum YKL-40 levels were significantly elevated in patients with asthma as compared with controls. In the Paris cohort, lung YKL-40 levels were elevated and were correlated with circulating YKL-40 levels (r=0.55, P<0.001) and with airway remodeling (measured as the thickness of the subepithelial basement membrane) (r=0.51, P=0.003). In all three cohorts, serum YKL-40 levels correlated positively with the severity of asthma and inversely with the forced expiratory volume in 1 second. Patients with elevated levels of YKL-40 had significantly more frequent rescue-inhaler use, greater oral corticosteroid use, and a greater rate of hospitalization than patients with lower levels. CONCLUSIONS YKL-40 is found in increased quantities in the serum and lungs in a subgroup of patients with asthma, in whom expression of chitinase in both compartments correlates with the severity of asthma. The recovery of YKL-40 from these patients indicates either a causative or a sentinel role for this molecule in asthma.

Bispecific antibodies have been extensively studied in vitro and in vivo for their use in redirected tumor cell lysis. A particular challenge of bispecific antibody constructs that recognize the invariant CD3 signaling complex is a controlled polyclonal activation of T cells that, ideally, is exquisitely dependent on the presence of target cells. Otherwise, overt production of inflammatory cytokines and secondary reactions may occur as side effects, as can be observed with constitutively T-cell activating monoclonal antibodies to CD3 or CD28, and with bispecific antibodies bearing Fc gamma portions. Here we analyzed 2 distinct bispecific single-chain antibody constructs of the BiTE class, called MT110 and MT103 (or MEDI-538), for conditional T-cell activation. In the presence of target-expressing cell lines, low picomolar concentrations of the BiTE molecules were sufficient to stimulate a high percentage of peripheral human T cells to express cytokines and surface activation markers, enter into cell cycle, and induce redirected lysis of target cells. However, in the absence of target cells, the 2 BiTE molecules even at high concentrations did not detectably activate T cells. Our data show that T cell activation by monomeric forms of MT110 and MT103 is highly conditional in that it is strictly dependent on the presence of cells expressing the proper target antigen. BiTE molecules therefore qualify for a highly controlled polyclonal T-cell therapy of cancer.

Multiple lines of evidence suggest that inhibition of Type I Interferons (IFNs), including IFN(, may provide a therapeutic benefit in treating a number of autoimmune diseases such as Systemic Lupus Erythematosus (SLE). Herein, we describe the generation and use of AvalonRx-HITS®, a proprietary genomic based high-throughput screen (HTS), followed by immunological assays in primary human cells to identify inhibitors of the IFN( pathway. In the HTS step, THP1 cells were stimulated with IFN( and 268 defined small molecule inhibitors targeting 41 different intracellular signaling pathways were screened for their ability to block a specific IFN(-evoked gene signature. Four classes of compounds emerged from the HTS: NF-(B, JAK/STAT, Ubiquitin/Proteasome and HDAC inhibitors. In secondary assays, these compounds blocked both IFN(-induced human monocyte activation and differentiation into dendritic cells, and IFNs-associated gene signature induced by SLE serum in human monocytes. In addition to their dose-dependent anti-inflammatory effects, inhibitors targeting NF- (B or JAK/STAT signaling did not modulate IFN( anti-viral effects in an in vitro HSV-1 replication assay. In summary, we have developed a novel and robust platform using genomic, immunological and viral assays that may lead to the identification of small molecule inhibitors for the treatment of IFN(-associated autoimmune disorders.

B61 The EphA2 receptor tyrosine kinase is selectively expressed on the surface of many different human tumors. We have previously demonstrated that the EphA2 on tumor cells can be targeted by monoclonal antibodies and that these antibodies function, in part, by inducing EphA2 internalization and degradation. Monoclonal antibodies that selectively bind and internalize into tumor cells provide a vehicle for targeted delivery of cytotoxics. Thus, antibody-drug conjugates (ADC) increase the therapeutic index of the attached cytotoxics by reducing systemic toxicity and enhancing tumor targeting and efficacy. Here we explore the relationship between the pharmacokinetics of the EphA2 ADC, the pharmacodynamics of EphA2 in the prostate tumor (PC3) and tumor efficacy. The EphA2 ADC has a serum half life of approximately 50 hours and a tumor half life of approximately 60 hours. The tumor exposures were approximately dose proportional while the serum exposures were not. Following treatment with the EphA2 ADC, the EphA2 receptor is degraded in a time and dose dependent manner. We used this observation as our pharmacodynamic marker. A single dose of the EphA2 ADC at the 1, 5 and 10 mg/kg dose levels resulted in rapid and prolonged degradation of the EphA2 receptor in a PC3 tumor xenograft model, which began one hour post dose and lasted for at least six days at the higher doses. The 10 mg/kg dose of the EphA2 ADC did not result enhanced degradation of EphA2, thus setting our dosing limits between 1 and 5 mg/kg. We show that once/week administration of the EphA2 ADC gave comparable efficacy to a twice/week dosing schedule in the PC3 tumor model. In conclusion we report that the use of pharmacokinetic and pharmacodynamic data can provide guidance in the planning of tumor efficacy studies using the EphA2 ADC.

Cette invention se rapporte a de nouvelles compositions immunogeniques (p. ex. des vaccins), a la production desdites compositions immunogeniques et a des procedes d'utilisation desdites compositions. Plus particulierement, cette invention concerne des molecules immunogeniques uniques comprenant un polypeptide HMGB1 (p. ex. un polypeptide HMGB1 B-box) et un antigene. Cette invention concerne encore plus particulierement de nouvelles proteines de fusion comprenant un polypeptide HMGB1 isole et un antigene, l'administration de ces proteines de fusion permettant de fournir les deux signaux necessaires a l'activation des lymphocytes T natifs.