Use of poxvirus display to select antibodies specific for complex membrane antigens.

Antibody discovery against complex antigens is limited by the availability of a reproducible pure source of concentrated properly folded antigen. We have developed a technology to enable direct incorporation of membrane proteins such as GPCRs and into the membrane of poxvirus. The protein of interest is correctly folded and expressed in the cell-derived viral membrane and does not require any detergents or refolding before downstream use. The poxvirus is selective in which proteins are incorporated into the viral membrane, making the antigen poxvirus an antigenically cleaner target for in vitro panning. Antigen-expressing virus can be readily purified at scale and used for antibody selection using any in vitro display platform.

Generation and preclinical characterization of an antibody specific for SEMA4D.

Semaphorin 4D (SEMA4D or CD100) is a member of the semaphorin family of proteins and an important mediator of the movement and differentiation of multiple cell types, including those of the immune, vascular, and nervous systems. Blocking the binding of SEMA4D to its receptors can result in physiologic changes that may have implications in cancer, autoimmune, and neurological disease. To study the effects of blocking SEMA4D, we generated, in SEMA4D-deficient mice, a panel of SEMA4D-specific hybridomas that react with murine, primate, and human SEMA4D. Utilizing the complementarity-determining regions from one of these hybridomas (mAb 67-2), we generated VX15/2503, a humanized IgG4 monoclonal antibody that is currently in clinical development for the potential treatment of various malignancies and neurodegenerative disorders, including multiple sclerosis and Huntington's disease. This work describes the generation and characterization of VX15/2503, including in vitro functional testing, epitope mapping, and an in vivo demonstration of efficacy in an animal model of rheumatoid arthritis.

Immunomodulation of the tumor microenvironment by neutralization of Semaphorin 4D.

Semaphorin 4D is highly expressed at the invasive tumor margin and acts as a guidance molecule, restricting movement of tumoricidal immune cells into the tumor microenvironment. We recently showed that antibody neutralization of SEMA4D augmented activated monocyte and anticancer T-cell tumor penetration and that anti-SEMA4D antibody potentiated other immunomodulatory therapies in murine tumor models.

CXCL13 antibody for the treatment of autoimmune disorders.

BACKGROUND: Homeostatic B Cell-Attracting chemokine 1 (BCA-1) otherwise known as CXCL13 is constitutively expressed in secondary lymphoid organs by follicular dendritic cells (FDC) and macrophages. It is the only known ligand for the CXCR5 receptor, which is expressed on mature B cells, follicular helper T cells (Tfh), Th17 cells and regulatory T (Treg) cells. Aberrant expression of CXCL13 within ectopic germinal centers has been linked to the development of autoimmune disorders (e.g. Rheumatoid Arthritis, Multiple Sclerosis, Systemic Lupus Erythematosis). We, therefore, hypothesized that antibody-mediated disruption of the CXCL13 signaling pathway would interfere with the formation of ectopic lymphoid follicles in the target organs and inhibit autoimmune disease progression. This work describes pre-clinical development of human anti-CXCL13 antibody MAb 5261 and includes therapeutic efficacy data of its mouse counterpart in murine models of autoimmunity. RESULTS: We developed a human IgG1 monoclonal antibody, MAb 5261 that specifically binds to human, rodent and primate CXCL13 with an affinity of approximately 5 nM and is capable of neutralizing the activity of CXCL13 from these various species in in vitro functional assays. For in vivo studies we have engineered a chimeric antibody to contain the same human heavy and light chain variable genes along with mouse constant regions. Treatment with this antibody led to a reduction in the number of germinal centers in mice immunized with 4-Hydroxy-3-nitrophenylacetyl hapten conjugated to Keyhole Limpet Hemocyanin (NP-KLH) and, in adoptive transfer studies, interfered with the trafficking of B cells to the B cell areas of mouse spleen. Furthermore, this mouse anti-CXCL13 antibody demonstrated efficacy in a mouse model of Rheumatoid arthritis (Collagen-Induced Arthritis (CIA)) and Th17-mediated murine model of Multiple Sclerosis (passively-induced Experimental Autoimmune Encephalomyelitis (EAE)). CONCLUSIONS: We developed a novel therapeutic antibody targeting CXCL13-mediated signaling pathway for the treatment of autoimmune disorders.

Antibody Blockade of Semaphorin 4D Promotes Immune Infiltration into Tumor and Enhances Response to Other Immunomodulatory Therapies.

Semaphorin 4D (SEMA4D, CD100) and its receptor plexin-B1 (PLXNB1) are broadly expressed in murine and human tumors, and their expression has been shown to correlate with invasive disease in several human tumors. SEMA4D normally functions to regulate the motility and differentiation of multiple cell types, including those of the immune, vascular, and nervous systems. In the setting of cancer, SEMA4D-PLXNB1 interactions have been reported to affect vascular stabilization and transactivation of ERBB2, but effects on immune-cell trafficking in the tumor microenvironment (TME) have not been investigated. We describe a novel immunomodulatory function of SEMA4D, whereby strong expression of SEMA4D at the invasive margins of actively growing tumors influences the infiltration and distribution of leukocytes in the TME. Antibody neutralization of SEMA4D disrupts this gradient of expression, enhances recruitment of activated monocytes and lymphocytes into the tumor, and shifts the balance of cells and cytokines toward a proinflammatory and antitumor milieu within the TME. This orchestrated change in the tumor architecture was associated with durable tumor rejection in murine Colon26 and ERBB2(+) mammary carcinoma models. The immunomodulatory activity of anti-SEMA4D antibody can be enhanced by combination with other immunotherapies, including immune checkpoint inhibition and chemotherapy. Strikingly, the combination of anti-SEMA4D antibody with antibody to CTLA-4 acts synergistically to promote complete tumor rejection and survival. Inhibition of SEMA4D represents a novel mechanism and therapeutic strategy to promote functional immune infiltration into the TME and inhibit tumor progression.

SEMA4D compromises blood-brain barrier, activates microglia, and inhibits remyelination in neurodegenerative disease.

Multiple sclerosis (MS) is a chronic neuroinflammatory disease characterized by immune cell infiltration of CNS, blood-brain barrier (BBB) breakdown, localized myelin destruction, and progressive neuronal degeneration. There exists a significant need to identify novel therapeutic targets and strategies that effectively and safely disrupt and even reverse disease pathophysiology. Signaling cascades initiated by semaphorin 4D (SEMA4D) induce glial activation, neuronal process collapse, inhibit migration and differentiation of oligodendrocyte precursor cells (OPCs), and disrupt endothelial tight junctions forming the BBB. To target SEMA4D, we generated a monoclonal antibody that recognizes mouse, rat, monkey and human SEMA4D with high affinity and blocks interaction between SEMA4D and its cognate receptors. In vitro, anti-SEMA4D reverses the inhibitory effects of recombinant SEMA4D on OPC survival and differentiation. In vivo, anti-SEMA4D significantly attenuates experimental autoimmune encephalomyelitis in multiple rodent models by preserving BBB integrity and axonal myelination and can be shown to promote migration of OPC to the site of lesions and improve myelin status following chemically-induced demyelination. Our study underscores SEMA4D as a key factor in CNS disease and supports the further development of antibody-based inhibition of SEMA4D as a novel therapeutic strategy for MS and other neurologic diseases with evidence of demyelination and/or compromise to the neurovascular unit.

Brucellar uveitis: intraocular fluids and biopsy studies.

BACKGROUND: Ocular brucellosis is usually diagnosed by clinical criteria and serological tests. Little is known with regard to the ocular immunology of brucellosis and the use of intraocular diagnostic tests. We report retrospectively the laboratory findings of patients with ocular involvement associated with brucellosis. MATERIALS AND METHODS: Patients with uveitis with no evident etiologic diagnosis were evaluated at the Instituto de Medicina Tropical "Alexander von Humboldt" of the Universidad Peruana Cayetano Heredia and the Hospital Nacional Cayetano Heredia. Patients were tested for brucellosis, tuberculosis, syphilis, toxoplasmosis, toxocariasis, and human T-cell lymphotropic virus-1. Blood and intraocular fluid samples were examined. Patients with a diagnosis of brucellar uveitis were selected as cases and patients with a diagnosis of uveitis of other etiology were included as controls. The Goldmann-Witmer coefficient was determined. RESULTS: Twelve patients with clinical and laboratory findings suggestive of brucellar uveitis were considered as cases. Seven patients with uveitis of other etiology were selected as controls. Four (33.3%) patients with ocular brucellosis had negative ocular agglutinations and eight (66.7%) had positive agglutinations. No control cases had positive agglutinations for Brucella melitensis. The sensitivity of the test was 66.7% and the specificity 100%. Only one patient had a positive culture for B. melitensis in subretinal fluid. The Goldmann-Witmer coefficient was calculated in six cases of brucellosis uveitis and five uveitis controls. It was highly positive in three patients with ocular brucellosis. Tissue samples showed lymphoplasmacytic infiltrates. CONCLUSIONS: Intraocular serological tests could be used to support the diagnosis of ocular brucellosis.

Ability of online drug databases to assist in clinical decision-making with infectious disease therapies.

BACKGROUND: Infectious disease (ID) is a dynamic field with new guidelines being adopted at a rapid rate. Clinical decision support tools (CDSTs) have proven beneficial in selecting treatment options to improve outcomes. However, there is a dearth of information on the abilities of CDSTs, such as drug information databases. This study evaluated online drug information databases when answering infectious disease-specific queries. METHODS: Eight subscription drug information databases: American Hospital Formulary Service Drug Information (AHFS), Clinical Pharmacology (CP), Epocrates Online Premium (EOP), Facts & Comparisons 4.0 Online (FC), Lexi-Comp (LC), Lexi-Comp with AHFS (LC-AHFS), Micromedex (MM), and PEPID PDC (PPDC) and six freely accessible: DailyMed (DM), DIOne (DIO), Epocrates Online Free (EOF), Internet Drug Index (IDI), Johns Hopkins ABX Guide (JHAG), and Medscape Drug Reference (MDR) were evaluated for their scope (presence of an answer) and completeness (on a 3-point scale) in answering 147 infectious disease-specific questions. Questions were divided among five classifications: antibacterial, antiviral, antifungal, antiparasitic, and vaccination/immunization. Classifications were further divided into categories (e.g., dosage, administration, emerging resistance, synergy, and spectrum of activity). Databases were ranked based on scope and completeness scores. ANOVA and Chi-square were used to determine differences between individual databases and between subscription and free databases. RESULTS: Scope scores revealed three discrete tiers of database performance: Tier 1 (82-77%), Tier 2 (73-65%) and Tier 3 (56-41%) which were significantly different from each other (p < 0.05). The top tier performers: MM (82%), MDR (81%), LC-AHFS (81%), AHFS (78%), and CP (77%) answered significantly more questions compared to other databases (p < 0.05). Top databases for completeness were: MM (97%), DM (96%), IDI (95%), and MDR (95%). Subscription databases performed better than free databases in all categories (p = 0.03). Databases suffered from 37 erroneous answers for an overall error rate of 1.8%. CONCLUSION: Drug information databases used in ID practice as CDSTs can be valuable resources. MM, MDR, LC-AHFS, AHFS, and CP were shown to be superior in their scope and completeness of information, and MM, AHFS, and MDR provided no erroneous answers. There is room for improvement in all evaluated databases.

Performance of online drug information databases as clinical decision support tools in infectious disease medication management.

Infectious disease (ID) medication management is complex and clinical decision support tools (CDSTs) can provide valuable assistance. This study evaluated scope and completeness of ID drug information found in online databases by evaluating their ability to answer 147 question/answer pairs. Scope scores produced highest rankings (%) for: Micromedex (82.3), Lexi-Comp/American Hospital Formulary Service (81.0), and Medscape Drug Reference (81.0); lowest includes: Epocrates Online Premium (47.0), Johns Hopkins ABX Guide (45.6), and PEPID PDC (40.8).

Ocular manifestations associated with brucellosis: a 26-year experience in Peru.

BACKGROUND: Brucellosis has unusual clinical manifestations. Ocular involvement caused by brucellosis remains poorly recognized in areas in which brucellosis is endemic. METHODS: A prospective study was performed to evaluate patients attending the Instituto de Medicina Tropical "Alexander von Humboldt" and the Hospital Nacional Cayetano Heredia (Lima, Peru) from January 1980 through December 2005 who received a diagnosis of brucellosis with ocular involvement. Diagnosis was made on the basis of clinical findings as well as agglutinations and/or culture positive for Brucella melitensis. RESULTS: During a period of 26 years, 1551 patients with brucellosis were seen, including 52 patients with ocular brucellosis. We found that 7 (0.7%) of 965 patients with acute brucellosis and 45 (7.9%) of 570 patients with chronic brucellosis had ocular brucellosis (P<.001). In 16 patients with brucellosis, the disease stage was unclassified. The most frequent ocular presentation was uveitis, which was found in 43 (82.7%) of the 52 patients with ocular brucellosis. Posterior uveitis was the most frequent uveal syndrome (21 cases; 45.7%). Patients with panuveitis had the worst visual prognosis: 8 of 9 patients with panuveitis were legally blind, including 5 patients with no light perception. CONCLUSIONS: Brucellosis may involve the eye and can lead to serious complications. In patients with brucellosis, early ophthalmologic evaluation can lead to prompt treatment and might prevent blindness from severe ocular damage.

Transforming growth factor-beta and Wnt signals regulate chondrocyte differentiation through Twist1 in a stage-specific manner.

We investigated the molecular mechanisms underlying the transition between immature and mature chondrocytes downstream of TGF-beta and canonical Wnt signals. We used two developmentally distinct chondrocyte models isolated from the caudal portion of embryonic chick sternum or chick growth plates. Lower sternal chondrocytes exhibited immature phenotypic features, whereas growth plate-extracted cells displayed a hypertrophic phenotype. TGF-beta significantly induced beta-catenin in immature chondrocytes, whereas it repressed it in mature chondrocytes. TGF-beta further enhanced canonical Wnt-mediated transactivation of the Topflash reporter expression in lower sternal chondrocytes. However, it inhibited Topflash activity in a time-dependent manner in growth plate chondrocytes. Our immunoprecipitation experiments showed that TGF-beta induced Sma- and Mad-related protein 3 interaction with T-cell factor 4 in immature chondrocytes, whereas it inhibited this interaction in mature chondrocytes. Similar results were observed by chromatin immunoprecipitation showing that TGF-beta differentially shifts T-cell factor 4 occupancy on the Runx2 promoter in lower sternal chondrocytes vs. growth plate chondrocytes. To further determine the molecular switch between immature and hypertrophic chondrocytes, we assessed the expression and regulation of Twist1 and Runx2 in both cell models upon treatment with TGF-beta and Wnt3a. We show that Runx2 and Twist1 are differentially regulated during chondrocyte maturation. Furthermore, whereas TGF-beta induced Twist1 in mature chondrocytes, it inhibited Runx2 expression in these cells. Opposite effects were observed upon Wnt3a treatment, which predominates over TGF-beta effects on these cells. Finally, overexpression of chick Twist1 in mature chondrocytes dramatically inhibited their hypertrophy. Together, our findings show that Twist1 may be an important regulator of chondrocyte progression toward terminal maturation in response to TGF-beta and canonical Wnt signaling.

C35 (C17orf37) is a novel tumor biomarker abundantly expressed in breast cancer.

Identification of shared tumor-specific targets is useful in developing broadly applicable therapies. In a study designed to identify genes up-regulated in breast cancer, a cDNA clone corresponding to a novel gene C35 (C17orf37) was selected by representational difference analysis of tumor and normal human mammary cell lines. Abundant expression of C35 transcript in tumors was confirmed by Northern blot and real-time PCR. The C35 gene is located on chromosome 17q12, 505 nucleotides from the 3' end of the ERBB2 oncogene, the antigenic target for trastuzumab (Herceptin) therapy. The chromosomal arrangement of the genes encoding C35 and ERBB2 is tail to tail. An open reading frame encodes a 12-kDa protein of unknown function. Immunohistochemical analysis detected robust and frequent expression of C35 protein, including 32% of grade 1 and 66% of grades 2 and 3 infiltrating ductal carcinomas of the breast (in contrast to 20% overexpressing HER-2/neu), 38% of infiltrating lobular carcinoma (typically HER-2/neu negative), as well as tumors arising in other tissues. C35 was not detected in 38 different normal human tissues, except Leydig cells in the testes and trace levels in a small percentage of normal breast tissue samples. The distinct and favorable expression profile of C35 spanning early through late stages of disease, including high frequency of overexpression in various breast carcinoma, abundant expression in distant metastases, and either absence or low level expression in normal human tissues, warrants further investigation of the relevance of C35 as a biomarker and/or a target for development of broadly applicable cancer-specific therapies.

Runx2-mediated regulation of the zinc finger Osterix/Sp7 gene.

The zinc finger transcription factor Osterix (Osx) regulates bone formation and osteoblast differentiation in vitro and in vivo. We investigated the transcriptional mechanisms underlying the mouse Osx expression by isolating and characterizing its 5' upstream region. We performed 5' RACE on mRNA isolated from murine chondroprogenitor cells and determined a cap site of Osx approximately -99 nucleotides upstream of the initiation codon. Sequence analysis of this TATA-less promoter shows several putative response elements for Sox9, VDRE, Runx and Sp1. Transfection of the Osx promoter driving the luciferase reporter gene into C3H10T1/2 and ATDC5 cells shows a strong basal promoter activity between 565 bp and 2 kb. Deletion mutant analyses show that the most proximal 852 kb of the Osx promoter contains the highest activating domains, while strong repressive domains were identified between 1.8 and 2 kb. Over-expression experiments indicate that Runx2 significantly transactivates the Osx promoter by at least 2 fold indicating that Osx is downstream of Runx2 in mesenchymal cells. This up-regulation was abrogated when the Runx2 responsive element on the Osx promoter was mutated. Finally, we show that Runx2 specifically binds to this DNA element in the Osx promoter. Thus our results show for the first time Osx transcriptional regulation through the bone and cartilage related transcription factor Runx2.

Variational Monte Carlo study of pentaquark states.

Accurate numerical solution of the five-body Schrödinger equation is effected via variational Monte Carlo calculations. The spectrum is assumed to exhibit a narrow resonance with strangeness S = +1. A fully antisymmetrized and pair-correlated five-quark wave function is obtained for the assumed nonrelativistic Hamiltonian, which has spin, isospin, and color dependent pair interactions and many-body confining terms, which are fixed by the nonexotic spectra. Gauge field dynamics are modeled via flux-tube exchange factors. The energy determined for the ground states with J(pi) = (1/2)- ((1/2)+) is 2.22 (2.50) GeV. A lower energy negative parity state is consistent with recent lattice results. The short-range structure of the state is analyzed via its diquark content.