Peptide-HLA-based immunotherapeutics platforms for direct modulation of antigen-specific T cells.

Targeted pharmacologic activation of antigen-specific (AgS) T cells may bypass limitations inherent in current T cell-based cancer therapies. We describe two immunotherapeutics platforms for selective delivery of costimulatory ligands and peptide-HLA (pHLA) to AgS T cells. We engineered and deployed on these platforms an affinity-attenuated variant of interleukin-2, which selectively expands oligoclonal and polyfunctional AgS T cells in vitro and synergizes with CD80 signals for superior proliferation versus peptide stimulation.

CUE-101, a Novel E7-pHLA-IL2-Fc Fusion Protein, Enhances Tumor Antigen-Specific T-Cell Activation for the Treatment of HPV16-Driven Malignancies.

PURPOSE: To assess the potential for CUE-101, a novel therapeutic fusion protein, to selectively activate and expand HPV16 E711-20-specific CD8+ T cells as an off-the shelf therapy for the treatment of HPV16-driven tumors, including head and neck squamous cell carcinoma (HNSCC), cervical, and anal cancers. EXPERIMENTAL DESIGN: CUE-101 is an Fc fusion protein composed of a human leukocyte antigen (HLA) complex, an HPV16 E7 peptide epitope, reduced affinity human IL2 molecules, and an effector attenuated human IgG1 Fc domain. Human E7-specific T cells and human peripheral blood mononuclear cells (PBMC) were tested to demonstrate cellular activity and specificity of CUE-101, whereas in vivo activity of CUE-101 was assessed in HLA-A2 transgenic mice. Antitumor efficacy with a murine surrogate (mCUE-101) was tested in the TC-1 syngeneic tumor model. RESULTS: CUE-101 demonstrates selective binding, activation, and expansion of HPV16 E711-20-specific CD8+ T cells from PBMCs relative to nontarget cells. Intravenous administration of CUE-101 induced selective expansion of HPV16 E711-20-specific CD8+ T cells in HLA-A2 (AAD) transgenic mice, and anticancer efficacy and immunologic memory was demonstrated in TC-1 tumor-bearing mice treated with mCUE-101. Combination therapy with anti-PD-1 checkpoint blockade further enhanced the observed efficacy. CONCLUSIONS: Consistent with its design, CUE-101 demonstrates selective expansion of an HPV16 E711-20-specific population of cytotoxic CD8+ T cells, a favorable safety profile, and in vitro and in vivo evidence supporting its potential for clinical efficacy in an ongoing phase I trial (NCT03978689).

Monoclonal antibody therapeutics with up to five specificities: functional enhancement through fusion of target-specific peptides.

The recognition that few human diseases are thoroughly addressed by mono-specific, monoclonal antibodies (mAbs) continues to drive the development of antibody therapeutics with additional specificities and enhanced activity. Historically, efforts to engineer additional antigen recognition into molecules have relied predominantly on the reformatting of immunoglobulin domains. In this report we describe a series of fully functional mAbs to which additional specificities have been imparted through the recombinant fusion of relatively short polypeptides sequences. The sequences are selected for binding to a particular target from combinatorial libraries that express linear, disulfide-constrained, or domain-based structures. The potential for fusion of peptides to the N- and C- termini of both the heavy and light chains affords the bivalent expression of up to four different peptides. The resulting molecules, called zybodies, can gain up to four additional specificities, while retaining the original functionality and specificity of the scaffold antibody. We explore the use of two clinically significant oncology antibodies, trastuzumab and cetuximab, as zybody scaffolds and demonstrate functional enhancements in each case. The affect of fusion position on both peptide and scaffold function is explored, and penta-specific zybodies are demonstrated to simultaneously engage five targets (ErbB2, EGFR, IGF-1R, Ang2 and integrin αvß3). Bispecific, trastuzumab-based zybodies targeting ErbB2 and Ang2 are shown to exhibit superior efficacy to trastuzumab in an angiogenesis-dependent xenograft tumor model. A cetuximab-based bispecific zybody that targeting EGFR and ErbB3 simultaneously disrupted multiple intracellular signaling pathways; inhibited tumor cell proliferation; and showed efficacy superior to that of cetuximab in a xenograft tumor model.

Simultaneous targeting of TNF and Ang2 with a novel bispecific antibody enhances efficacy in an in vivo model of arthritis.

Despite the clinical success of anti-tumor necrosis factor (TNF) therapies in the treatment of inflammatory conditions such as rheumatoid arthritis, Crohn disease and psoriasis, full control of the diseases only occurs in a subset of patients and there is a need for new therapeutics with improved efficacy against broader patient populations. One possible approach is to combine biological therapeutics, but both the cost of the therapeutics and the potential for additional toxicities needs to be considered. In addition to the various mediators of immune and inflammatory pathways, angiogenesis is reported to contribute substantially to the overall pathogenesis of inflammatory diseases. The combination of an anti-angiogenic agent with anti-TNF into one molecule could be more efficacious without the risk of severe immunosuppression. To evaluate this approach with our Zybody technology, we generated bispecific antibodies that contain an Ang2 targeting peptide genetically fused to the anti-TNF antibody adalimumab (Humira®). The bispecific molecules retain the binding and functional characteristics of the anti-TNF antibody, but with additional activity that neutralizes Ang2. In a TNF transgenic mouse model of arthritis, the bispecific anti-TNF-Ang2 molecules showed a dose-dependent reduction in both clinical symptoms and histological scores that were significantly better than that achieved by adalimumab alone.

Targeting the junction of CɛmX and ɛ-migis for the specific depletion of mIgE-expressing B cells.

Monoclonal antibodies targeting the extracellular region of the human IgE heavy chain membrane-tethering domain have been proposed for treating allergies caused by hyperproliferative monoclonal expansion of IgE-producing B cells. Antibodies against this target are expected to deplete membrane IgE (mIgE) displaying B cells and leave B cells of other immunoglobulin isotypes intact. Because of alternative splicing, the mIgE heavy chain has two isoforms that differ in their membrane-proximal segment. In the long isoform, the CH4 domain is followed by a 67-amino acid-long extracellular portion. Out of these 67 amino acids, the first 52 amino acids following the CH4 domain constitute the CɛmX segment while the rest of the 15 amino acids immediately adjacent to the membrane constitute the ɛ-migis. In the short isoform the CɛmX segment is absent and the CH4 domain is followed only by the 15-amino acid-long ɛ-migis segment. Using antibodies derived from a phage display library, we investigated: (1) ɛ-migis and (2) the junction of CɛmX and ɛ-migis (CɛmX.migis), as potential therapeutic antibody targets. Our results indicate that antibodies obtained from our phage library that target ɛ-migis bind to a variety of human cells irrespective of mIgE expression, possibly due to homology between ɛ-migis and a region of phosphoinositide-binding protein (ARAP3). In contrast, antibodies specific for the CɛmX.migis junctional region, bound specifically to transfected and primary B cells expressing human mIgE and elicited antibody-dependent cellular cytotoxicity and reduction in IgE production. These antibodies did not bind secreted IgE or the mIgE isoform in which CɛmX is absent. These results suggest that CɛmX.migis junctional region is a promising antibody target and the human antibodies we describe warrant further evaluation.

Patients with systemic lupus erythematosus, myositis, rheumatoid arthritis and scleroderma share activation of a common type I interferon pathway.

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.

IL-9 governs allergen-induced mast cell numbers in the lung and chronic remodeling of the airways.

RATIONALE: IL-9 is a pleiotropic cytokine that has multiple effects on structural as well as numerous hematopoietic cells, which are central to the pathogenesis of asthma. OBJECTIVES: The contribution of IL-9 to asthma pathogenesis has thus far been unclear, due to conflicting reports in the literature. These earlier studies focused on the role of IL-9 in acute inflammatory models; here we have investigated the effects of IL-9 blockade during chronic allergic inflammation. METHODS: Mice were exposed to either prolonged ovalbumin or house dust mite allergen challenge to induce chronic inflammation and airway remodeling. MEASUREMENTS AND MAIN RESULTS: We found that IL-9 governs allergen-induced mast cell (MC) numbers in the lung and has pronounced effects on chronic allergic inflammation. Anti-IL-9 antibody-treated mice were protected from airway remodeling with a concomitant reduction in mature MC numbers and activation, in addition to decreased expression of the profibrotic mediators transforming growth factor-ß1, vascular endothelial growth factor, and fibroblast growth factor-2 in the lung. Airway remodeling was associated with impaired lung function in the peripheral airways and this was reversed by IL-9 neutralization. In human asthmatic lung tissue, we identified MCs as the main IL-9 receptor expressing population and found them to be sources of vascular endothelial growth factor and fibroblast growth factor-2. CONCLUSIONS: Our data suggest an important role for an IL-9-MC axis in the pathology associated with chronic asthma and demonstrate that an impact on this axis could lead to a reduction in chronic inflammation and improved lung function in patients with asthma.

Respiratory syncytial virus (RSV) RNA loads in peripheral blood correlates with disease severity in mice.

BACKGROUND: Respiratory Syncytial Virus (RSV) infection is usually restricted to the respiratory epithelium. Few studies have documented the presence of RSV in the systemic circulation, however there is no consistent information whether virus detection in the blood correlates with disease severity. METHODS: Balb/c mice were inoculated with live RSV, heat-inactivated RSV or medium. A subset of RSV-infected mice was treated with anti-RSV antibody 72 h post-inoculation. RSV RNA loads were measured by PCR in peripheral blood from day 1-21 post-inoculation and were correlated with upper and lower respiratory tract viral loads, the systemic cytokine response, lung inflammation and pulmonary function. Immunohistochemical staining was used to define the localization of RSV antigens in the respiratory tract and peripheral blood. RESULTS: RSV RNA loads were detected in peripheral blood from day 1 to 14 post-inoculation, peaked on day 5 and significantly correlated with nasal and lung RSV loads, airway obstruction, and blood CCL2 and CXCL1 expression. Treatment with anti-RSV antibody reduced blood RSV RNA loads and improved airway obstruction. Immunostaining identified RSV antigens in alveolar macrophages and peripheral blood monocytes. CONCLUSIONS: RSV RNA was detected in peripheral blood upon infection with live RSV, followed a time-course parallel to viral loads assessed in the respiratory tract and was significantly correlated with RSV-induced airway disease.

Safety profile, pharmacokinetics, and biologic activity of MEDI-563, an anti-IL-5 receptor alpha antibody, in a phase I study of subjects with mild asthma.

BACKGROUND: Increased eosinophil levels have been linked to airway inflammation and asthma exacerbations. IL-5 is responsible for eosinophil differentiation, proliferation, and activation; IL-5 receptors are expressed on eosinophils and their progenitors, and targeting such receptors induces eosinophil apoptosis. OBJECTIVE: To evaluate the safety profile, pharmacokinetics, and pharmacodynamics of MEDI-563, a humanized mAb targeting the IL-5 receptor alpha chain. METHODS: Single, escalating, intravenous doses (0.0003-3 mg/kg) of MEDI-563 were administered to subjects with mild atopic asthma (n = 44) over approximately 3 to 30 minutes in this open-label study. Pulmonary function, symptom scores, adverse events, MEDI-563 pharmacokinetics, and levels of C-reactive protein (CRP), IL-6, eosinophil cationic protein (ECP), and eosinophils were evaluated. RESULTS: Mean peripheral blood (PB) eosinophil levels decreased in a dose-dependent fashion (baseline +/- SD, 0.27 +/- 0.2 x 10(3)/microL; 24 hours postdose, 0.01 +/- 0.0 x 10(3)/microL); 94.0% of subjects receiving >or=0.03 mg/kg exhibited levels between 0.00 x 10(3)/microL and 0.01 x 10(3)/microL. Eosinopenia lasted at least 8 or 12 weeks with doses of 0.03 to 0.1 and 0.3 to 3 mg/kg, respectively. ECP levels were reduced from 21.4 +/- 17.2 microg/L (baseline) to 10.3 +/- 7.0 microg/L (24 hours postdose). The most frequently reported adverse events were reduced white blood cell counts (34.1%), nasopharyngitis (27.3%), and increased blood creatine phosphokinase (25.0%). Mean C-reactive protein levels increased approximately 5.5-fold at 24 hours postdose but returned to baseline by study end; mean IL-6 levels increased approximately 3.9-fold to 4.7-fold at 6 to 12 hours postdose, respectively. Pharmacokinetic activity was dose proportional at doses of 0.03 to 3 mg/kg. CONCLUSION: Single escalating doses of MEDI-563 had an acceptable safety profile and resulted in marked reduction of PB eosinophil counts within 24 hours after dosing.

MEDI-563, a humanized anti-IL-5 receptor alpha mAb with enhanced antibody-dependent cell-mediated cytotoxicity function.

BACKGROUND: Peripheral blood eosinophilia and lung mucosal eosinophil infiltration are hallmarks of bronchial asthma. IL-5 is a critical cytokine for eosinophil maturation, survival, and mobilization. Attempts to target eosinophils for the treatment of asthma by means of IL-5 neutralization have only resulted in partial removal of airway eosinophils, and this warrants the development of more effective interventions to further explore the role of eosinophils in the clinical expression of asthma. OBJECTIVE: We sought to develop a novel humanized anti-IL-5 receptor alpha (IL-5Ralpha) mAb with enhanced effector function (MEDI-563) that potently depletes circulating and tissue-resident eosinophils and basophils for the treatment of asthma. METHODS: We used surface plasmon resonance to determine the binding affinity of MEDI-563 to FcgammaRIIIa. Primary human eosinophils and basophils were used to demonstrate antibody-dependent cell-mediated cytotoxicity. The binding epitope of MEDI-563 on IL-5Ralpha was determined by using site-directed mutagenesis. The consequences of MEDI-563 administration on peripheral blood and bone marrow eosinophil depletion was investigated in nonhuman primates. RESULTS: MEDI-563 binds to an epitope on IL-5Ralpha that is in close proximity to the IL-5 binding site, and it inhibits IL-5-mediated cell proliferation. MEDI-563 potently induces antibody-dependent cell-mediated cytotoxicity of both eosinophils (half-maximal effective concentration = 0.9 pmol/L) and basophils (half-maximal effective concentration = 0.5 pmol/L) in vitro. In nonhuman primates MEDI-563 depletes blood eosinophils and eosinophil precursors in the bone marrow. CONCLUSIONS: MEDI-563 might provide a novel approach for the treatment of asthma through active antibody-dependent cell-mediated depletion of eosinophils and basophils rather than through passive removal of IL-5.

Cell type-specific recognition of human metapneumoviruses (HMPVs) by retinoic acid-inducible gene I (RIG-I) and TLR7 and viral interference of RIG-I ligand recognition by HMPV-B1 phosphoprotein.

Human metapneumoviruses (HMPVs) are recently identified Paramyxoviridae that contribute to respiratory tract infections in children. No effective treatments or vaccines are available. Successful defense against virus infection relies on early detection by germ line-encoded pattern recognition receptors and activation of cytokine and type I IFN genes. Recently, the RNA helicase retinoic acid-inducible gene I (RIG-I) has been shown to sense HMPV. In this study, we investigated the abilities of two prototype strains of HMPV (A1 [NL\1\00] and B1 [NL\1\99]) to activate RIG-I and induce type I IFNs. Despite the abilities of both HMPV-A1 and HMPV-B1 to infect and replicate in cell lines and primary cells, only the HMPV-A1 strain triggered RIG-I to induce IFNA/B gene transcription. The failure of the HMPV-B1 strain to elicit type I IFN production was dependent on the B1 phosphoprotein, which specifically prevented RIG-I-mediated sensing of HMPV viral 5' triphosphate RNA. In contrast to most cell types, plasmacytoid dendritic cells displayed a unique ability to sense both HMPV-A1 and HMPV-B1 and in this case sensing was via TLR7 rather than RIG-I. Collectively, these data reveal differential mechanisms of sensing for two closely related viruses, which operate in cell type-specific manners.

The design and characterization of oligospecific antibodies for simultaneous targeting of multiple disease mediators.

Monoclonal antibodies are traditionally used to block the function of a specific target in a given disease. However, some diseases are the consequence of multiple components or pathways and not the result of a single mediator; thus, blocking at a single point may not optimally control disease. Antibodies that simultaneously block the functions of two or more disease-associated targets are now being developed. Herein, we describe the design, expression, and characterization of several oligospecific antibody formats that are capable of binding simultaneously to two or three different antigens. These constructs were generated by genetically linking single-chain Fv fragments to the N-terminus of the antibody heavy and light chains and to the C-terminus of the antibody C(H)3 domain. The oligospecific antibodies were expressed in mammalian cells, purified to homogeneity, and characterized for binding to antigens, Fcgamma receptors, FcRn, and C1q. In addition, the oligospecific antibodies were assayed for effector function, protease susceptibility, thermal stability, and size distribution. We demonstrate that these oligospecific antibody formats maintain high expression level, thermostability, and protease resistance. The in vivo half-life, antibody-dependent cellular cytotoxicity function, and binding ability to Fcgamma receptors and C1q of the test oligospecific antibodies remain similar to the corresponding properties of their parental IgG antibodies. The excellent expression, biophysical stability, and potential manufacturing feasibility of these multispecific antibody formats suggest that they will provide a scaffold template for the construction of similar molecules to target multiple antigens in complex diseases.

Antibody-dependent cell-mediated cytotoxicity effector-enhanced EphA2 agonist monoclonal antibody demonstrates potent activity against human tumors.

EphA2 is a receptor tyrosine kinase that has been shown to be overexpressed in a variety of human tumor types. Previous studies demonstrated that agonist monoclonal antibodies targeting EphA2 induced the internalization and degradation of the receptor, thereby abolishing its oncogenic effects. In this study, the in vitro and in vivo antibody-dependent cell-mediated cytotoxicity (ADCC) activity of EphA2 effector-enhanced agonist monoclonal antibodies was evaluated. With tumor cell lines and healthy human peripheral blood monocytes, the EphA2 antibodies demonstrated approximately 80% tumor cell killing. In a dose-dependent manner, natural killer (NK) cells were required for the in vitro ADCC activity and became activated as demonstrated by the induction of cell surface expression of CD107a. To assess the role of NK cells on antitumor efficacy in vivo, the EphA2 antibodies were evaluated in xenograft models in severe compromised immunodeficient (SCID) mice (which have functional NK cells and monocytes) and SCID nonobese diabetic (NOD) mice (which largely lack functional NK cells and monocytes). Dosing of EphA2 antibody in the SCID murine tumor model resulted in a 6.2-fold reduction in tumor volume, whereas the SCID/nonobese diabetic model showed a 1.6-fold reduction over the isotype controls. Together, these results demonstrate that the anti-EphA2 monoclonal antibodies may function through at least two mechanisms of action: EphA2 receptor activation and ADCC-mediated activity. These novel EphA2 monoclonal antibodies provide additional means by which host effector mechanisms can be activated for selective destruction of EphA2-expressing tumor cells.

Neutralization of interferon-alpha/beta-inducible genes and downstream effect in a phase I trial of an anti-interferon-alpha monoclonal antibody in systemic lupus erythematosus.

OBJECTIVE: Type I interferons (IFNs) play an important role in the pathogenesis of systemic lupus erythematosus (SLE). This phase Ia trial was undertaken to evaluate the safety, pharmacokinetics, and immunogenicity of anti-IFNalpha monoclonal antibody (mAb) therapy in SLE. During the trial, we also examined whether overexpression of an IFNalpha/beta-inducible gene signature in whole blood could serve as a pharmacodynamic biomarker to evaluate IFNalpha neutralization and investigated downstream effects of neutralizing IFNalpha on BAFF and other key signaling pathways, i.e., granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-10 (IL-10), tumor necrosis factor alpha (TNFalpha), and IL-1beta, in SLE. METHODS: Affymetrix Human Genome U133 Plus 2.0 microarrays were used to profile whole blood and lesional skin of patients receiving standard therapy for mild to moderate SLE. Selected IFNalpha/beta-inducible proteins were analyzed by immunohistochemistry. RESULTS: With the study treatment, we observed anti-IFNalpha mAb-specific and dose-dependent inhibition of overexpression of IFNalpha/beta-inducible genes in whole blood and skin lesions from SLE patients, at both the transcript and the protein levels. In SLE patients with overexpression of messenger RNA for BAFF, TNFalpha, IL-10, IL-1beta, GM-CSF, and their respective inducible gene signatures in whole blood and/or skin lesions, we observed a general trend toward suppression of the expression of these genes and/or gene signatures upon treatment with anti-IFNalpha mAb. CONCLUSION: IFNalpha/beta-inducible gene signatures in whole blood are effective pharmacodynamic biomarkers to evaluate anti-IFNalpha mAb therapy in SLE. Anti-IFNalpha mAb can neutralize overexpression of IFNalpha/beta-inducible genes in whole blood and lesional skin from SLE patients and has profound effects on signaling pathways that may be downstream of IFNalpha in SLE.

Potent control of tumor growth by CEA/CD3-bispecific single-chain antibody constructs that are not competitively inhibited by soluble CEA.

Carcinoembryonic antigen (CEA, CD66e) is a well-characterized tumor-associated antigen that is frequently overexpressed in tumors. Phospholipases release CEA from tumor cells resulting in high circulating serum levels of soluble CEA (sCEA) that has been validated as marker for progression of colorectal, breast, and lung cancers. sCEA also acts as a competitive inhibitor for anticancer strategies targeting membrane-bound CEA. As a novel therapeutic approach for treatment of tumors expressing CEA on their cell surface, we constructed a series of bispecific single-chain antibodies (bscAb) combining various single-chain variable fragments recognizing human CEA with a deimmunized single-chain variable fragments recognizing human CD3. CEA/CD3-bscAbs redirected human T cells to lyse CEA-expressing tumor cells in vitro and in vivo. Efficient tumor cell lysis was achieved in vitro at bscAb concentrations from 1 pg/mL (19 fM) to 8.9 pg/mL with preactivated CD8 T cells, and 200 to 500 pg/mL with unstimulated peripheral blood mononuclear cell. The cytotoxic activity of a subset of CEA/CD3-bscAbs was not competitively inhibited by sCEA at concentrations that exceeded levels found in the serum of most cancer patients. Treatment with CEA/CD3-bscAbs prevented the growth of human colorectal cancer lines in a severe combined immunodeficiency mouse model modified to show human T cell killing of tumors. A murine surrogate CEA/CD3-bscAb capable of recruiting murine T cells for redirected tumor lysis in immunocompetent mice prevented the growth of lung tumors expressing human CEA. Together, our results reveal a unique opportunity for targeting cytotoxic T cells toward CEA-expressing tumors without being competitively inhibited by sCEA and establish CEA/CD3-bscAb as a promising and potent therapeutic approach.

Development of Potential Pharmacodynamic and Diagnostic Markers for Anti-IFN-α Monoclonal Antibody Trials in Systemic Lupus Erythematosus.

To identify potential pharmacodynamic biomarkers to guide dose selection in clinical trials using anti-interferon-alpha (IFN-α) monoclonal antibody (mAb) therapy for systemic lupus erythematosus (SLE), we used an Affymetrix human genome array platform and identified 110 IFN-α/ß-inducible transcripts significantly upregulated in whole blood (WB) of 41 SLE patients. The overexpression of these genes was confirmed prospectively in 54 additional SLE patients and allowed for the categorization of the SLE patients into groups of high, moderate, and weak overexpressers of IFN-α/ß-inducible genes. This approach could potentially allow for an accurate assessment of drug target neutralization in early trials of anti-IFN-α mAb therapy for SLE. Furthermore, ex vivo stimulation of healthy donor peripheral blood mononuclear cells with SLE patient serum and subsequent neutralization with anti-IFN-α mAb or anti-IFN-α receptor mAb showed that anti-IFN-α mAb has comparable effects of neutralizing the overexpression of type I IFN-inducible genes as that of anti-IFNAR mAb. These results suggest that IFN-α, and not other members of type I IFN family in SLE patients, is mainly responsible for the induction of type I IFN-inducible genes in WB of SLE patients. Taken together, these data strengthen the view of IFN-α as a therapeutic target for SLE.

Identification of antibody neutralization epitopes on the fusion protein of human metapneumovirus.

Human metapneumovirus (hMPV) is genetically related to respiratory syncytial virus (RSV); both cause respiratory tract illnesses ranging from a mild cough to bronchiolitis and pneumonia. The F protein-directed monoclonal antibody (mAb) palivizumab has been shown to prevent severe lower respiratory tract RSV infection in animals and humans. We have previously reported on a panel of mAbs against the hMPV F protein that neutralize hMPV in vitro and, in two cases, in vivo. Here we describe the generation of hMPV mAb-resistant mutants (MARMs) to these neutralizing antibodies. Sequencing the F proteins of the hMPV MARMs identified several neutralizing epitopes. Interestingly, some of the epitopes mapped on the hMPV F protein coincide with homologous regions mapped previously on the RSV F protein, including the site against which the broadly protective mAb palivizumab is directed. This suggests that these homologous regions play important, conserved functions in both viruses.

A human antibody-drug conjugate targeting EphA2 inhibits tumor growth in vivo.

The EphA2 receptor tyrosine kinase is selectively expressed on the surface of many different human tumors. We have previously shown that tumor cells can be targeted by EphA2 monoclonal antibodies and that these antibodies function, in part, by inducing EphA2 internalization and degradation. In this report, we describe the isolation and characterization of a fully human monoclonal antibody (1C1) that selectively binds both the human and rodent EphA2 receptor. After cell binding, the antibody induces rapid tyrosine phosphorylation, internalization, and degradation of the EphA2 receptor. Because monoclonal antibodies that selectively bind tumor cells and internalize provide a vehicle for targeted delivery of cytotoxics, 1C1 was conjugated to the microtubule inhibitor monomethylauristatin phenylalanine using a stable maleimidocaproyl linker. The anti-EphA2 antibody-drug conjugate [1C1-maleimidocaproyl-MMAF (mcMMAF)] stimulated the activation of caspase-3/caspase-7 and the death of EphA2-expressing cells with IC(50) values as low as 3 ng/mL. Similarly, the conjugate induced degradation of the EphA2 receptor and inhibited tumor growth in vivo. Administration of 1C1-mcMMAF at doses as low as 1 mg/kg once weekly resulted in significant growth inhibition of EphA2-expressing tumors without any observable adverse effects in mouse xenograft and rat syngeneic tumor models. Our data support the use of an antibody-drug conjugate approach to selectively target and inhibit the growth of EphA2-expressing tumors.

Macrophage impairment underlies airway occlusion in primary respiratory syncytial virus bronchiolitis.

Although respiratory syncytial virus (RSV) infection is the most important cause of bronchiolitis in infants, the pathogenesis of RSV disease is poorly described. We studied histopathologic changes in a panel of lung tissue specimens obtained from infants with fatal cases of primary RSV infection. In these tissues, airway occlusion with accumulations of infected, apoptotic cellular debris and serum protein was consistently observed. Similar observations were found after RSV infection in New Zealand black (NZB) mice, which have constitutive deficiencies in macrophage function, but not in BALB/c mice. A deficiency in the number of alveolar macrophages in NZB mice appears to be central to enhanced disease, because depletion of alveolar macrophages in BALB/c mice before RSV exposure resulted in airway occlusion. In mice with insufficient numbers of macrophages, RSV infection yielded an increased viral load and enhanced expression of type I interferon-associated genes at the height of disease. Together, our data suggest that innate, rather than adaptive, immune responses are critical determinants of the severity of RSV bronchiolitis.

YKL-40 is elevated in patients with chronic obstructive pulmonary disease and activates alveolar macrophages.

YKL-40 is a chitin-binding protein that is elevated in patients with various inflammatory conditions associated with ongoing remodeling. We investigated whether the levels of YKL-40 were up-regulated in the circulation and the airways of patients with chronic obstructive pulmonary disease (COPD), and whether it promoted the production of inflammatory mediators from macrophages. Serum, bronchoalveolar lavage (BAL), bronchial biopsies, lung tissue specimens, and alveolar macrophages from never-smokers (n = 15), smokers without COPD (n = 20), and smokers with COPD (n = 30) were assessed for YKL-40 levels and immunolocalization. In addition, YKL-40-induced mediator release from alveolar macrophages was examined. We found that smokers with COPD had elevated levels of YKL-40 in serum (p