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.

A glycoengineered anti-CD19 antibody with potent antibody-dependent cellular cytotoxicity activity in vitro and lymphoma growth inhibition in vivo.

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.

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.

B-cell depletion in vitro and in vivo with an afucosylated anti-CD19 antibody.

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.

Prophylactic and therapeutic benefits of a monoclonal antibody against the fusion protein of human metapneumovirus in a mouse model.

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 50mg/kg of MAb 338 either 24h before or 48h 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 50mg/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.

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.

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.

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.

Combination of rituximab with blinatumomab (MT103/MEDI-538), a T cell-engaging CD19-/CD3-bispecific antibody, for highly efficient lysis of human B lymphoma cells.

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.

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.

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.

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.

Type I interferon: potential therapeutic target for psoriasis?

BACKGROUND: Psoriasis is an immune-mediated disease characterized by aberrant epidermal differentiation, surface scale formation, and marked cutaneous inflammation. To better understand the pathogenesis of this disease and identify potential mediators, we used whole genome array analysis to profile paired lesional and nonlesional psoriatic skin and skin from healthy donors. METHODOLOGY/PRINCIPAL FINDINGS: We observed robust overexpression of type I interferon (IFN)-inducible genes and genomic signatures that indicate T cell and dendritic cell infiltration in lesional skin. Up-regulation of mRNAs for IFN-alpha subtypes was observed in lesional skin compared with nonlesional skin. Enrichment of mature dendritic cells and 2 type I IFN-inducible proteins, STAT1 and ISG15, were observed in the majority of lesional skin biopsies. Concordant overexpression of IFN-gamma and TNF-alpha-inducible gene signatures occurred at the same disease sites. CONCLUSIONS/SIGNIFICANCE: Up-regulation of TNF-alpha and elevation of the TNF-alpha-inducible gene signature in lesional skin underscore the importance of this cytokine in psoriasis; these data describe a molecular basis for the therapeutic activity of anti-TNF-alpha agents. Furthermore, these findings implicate type I IFNs in the pathogenesis of psoriasis. Consistent and significant up-regulation of type I IFNs and their associated gene signatures in psoriatic skin suggest that type I IFNs may be potential therapeutic targets in psoriasis treatment.

Strictly target cell-dependent activation of T cells by bispecific single-chain antibody constructs of the BiTE class.

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.

Selective targeting and potent control of tumor growth using an EphA2/CD3-Bispecific single-chain antibody construct.

The EphA2 receptor tyrosine kinase is frequently overexpressed and functionally altered in malignant cells and thus provides opportunities for selective targeting of tumor cells. We describe here the development of a novel, bispecific single-chain antibody (bscAb) referred to as bscEphA2xCD3. This molecule simultaneously targets EphA2 on tumor cells and the T-cell receptor/CD3 complex on T cells and possesses structural and functional characteristics of the recently developed BiTE technology. An EphA2-specific single-chain antibody was selected for recognition of an epitope that is preferentially exposed on malignant cells based on the concept of epitope exclusion; this was fused to a CD3-specific single-chain antibody to generate bscEphA2xCD3. The resultant bscAb redirected unstimulated human T cells to lyse EphA2-expressing tumor cells both in vitro and in vivo. In separate experiments, efficient tumor cell lysis was achieved in vitro at drug concentrations