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.

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.

Mouse strain specificity of the IgE response to the major allergens of Phleum pratense.

BACKGROUND: IgE immune responses against major allergens from Phleum pratense in low- and high-responder mouse strains were compared and the influence of alum was assessed, in order to evaluate the effect of the genetic background and adjuvants on IgE reactivity in a mouse model for P. pratense allergy. METHODS: Different mouse strains and F1 offspring were sensitized with P. pratense pollen extract. Serum IgE levels, the induction of specific IgE antibodies and immediate cutaneous hypersensitivity reactions were monitored by ELISA, Western blot and a skin test, respectively. RESULTS: All mouse strains investigated mounted an IgE response and exhibited a positive skin test to pollen extract. Differences were seen in the level of total serum IgE and in specific IgE reactivity to different major allergens of P. pratense. Notable differences were seen in IgE reactivity and immediate hypersensitivity against Phl p 1, which were only observed in SJL/j mice. The foremost influence of alum was on total IgE production levels. CONCLUSIONS: Alum is not necessary as an adjuvant to elicit IgE reactivity against the clinically relevant allergens of P. pratense, since even low-responder mouse strains mounted a hypersensitivity reaction after sensitization without the adjuvant using otherwise identical sensitization strategies. Moreover, when analyzing the allergenicity of a compound, the hypersensitivity response of different mouse strains should be considered, as implicated by the differential results obtained for IgE reactivity against Phl p 1. Lastly, a genetic component may be involved in IgE reactivity to Phl p 1.

Quantitative assessment of immediate cutaneous hypersensitivity in a mouse model exhibiting an IgE response to Timothy grass allergens.

BACKGROUND: A variety of allergic reactions can be induced in mice, as measured by the induction of specific IgE. Functional read-out parameters include skin reactions and airway constriction. The aim of this study was to establish an improved quantitative assessment of the immediate cutaneous hypersensitivity reaction in a mouse model. MATERIAL/METHODS: Timothy grass pollen was used to sensitize BALB/c mice, which were monitored for hypersensitivity reactions by Western blot analysis of the IgE response, determination of specific immunoglobulin subclasses by ELISA, and a skin test. The immediate cutaneous hypersensitivity reaction (ICHR) was assessed by pretreating the animals with an intravenous injection of Evans blue before anesthesia and provoking a reaction by intradermal application of the allergen. Ensuing blue coloring at the injection site could be seen as an indication of mast cell degranulation and the areas of 'wheal and flare' reaction were documented for digital imaging and quantitative analysis. RESULTS: Analysis using an antigen-specific ELISA showed that sensitization with pollen extract induced the production of specific IgE, IgG1, IgG2a and IgG2b antibodies. Western blot analyses demonstrated the production of specific IgE antibodies analogous to the IgE reactivity pattern found in allergic patients. The production of IgE was concurrent with a positive ICHR to these allergens. Quantitative analysis demonstrated that the area of ICHR increased in size in correlation to the dose of allergen applied. CONCLUSIONS: This model and the mode of skin reactivity analysis may serve as a tool to evaluate new hyposensitization and therapeutic strategies for pollen allergy.