Bi-epitope SPR surfaces: a solution to develop robust immunoassays.

Surface plasmon resonance (SPR)-based immunoassays have numerous applications and require high affinity reagents for sensitive and reliable measurements. We describe a quick approach to turn low affinity antibodies into appropriate capture reagents. We used antibodies recognizing human ephrin type A receptor 2 (EphA2) and a ProteOn XPR36 as a model system. We generated so-called 'bi-epitope' sensor surfaces by immobilizing various pairs of anti-EphA2 antibodies using standard amine coupling. The apparent binding affinities to EphA2 and EphA2 detection sensitivities of the bi-epitope and 'single-epitope' surfaces were then compared. For all antibody pairs tested, bi-epitope surfaces exhibited an ∼ 10-100-fold improvement in apparent binding affinities when compared with single-epitope ones. When pairing 2 antibodies of low intrinsic binding affinities (∼ 10(-8) M) and fast dissociation rates (∼ 10(-2) s(-1)), the apparent binding affinity and dissociation rate of the bi-epitope surface was improved up to ∼ 10(-10) M and 10(-4) s(-1), respectively. This led to an ∼ 100-200-fold enhancement in EphA2 limit of detection in crude cell supernatants. Our results show that the use of antibody mixtures in SPR applications constitutes a powerful approach to develop sensitive immunoassays, as previously shown for non-SPR formats. As SPR-based assays have significantly expanded their reach in the last decade, such an approach promises to further accelerate their development

Enhancement of Immune Effector Functions by Modulating IgG's Intrinsic Affinity for Target Antigen.

Antibody-mediated immune effector functions play an essential role in the anti-tumor efficacy of many therapeutic mAbs. While much of the effort to improve effector potency has focused on augmenting the interaction between the antibody-Fc and activating Fc-receptors expressed on immune cells, the role of antibody binding interactions with the target antigen remains poorly understood. We show that antibody intrinsic affinity to the target antigen clearly influences the extent and efficiency of Fc-mediated effector mechanisms, and report the pivotal role of antibody binding valence on the ability to regulate effector functions. More particularly, we used an array of affinity modulated variants of three different mAbs, anti-CD4, anti-EGFR and anti-HER2 against a panel of target cell lines expressing disparate levels of the target antigen. We found that at saturating antibody concentrations, IgG variants with moderate intrinsic affinities, similar to those generated by the natural humoral immune response, promoted superior effector functions compared to higher affinity antibodies. We hypothesize that at saturating concentrations, effector function correlates most directly with the amount of Fc bound to the cell surface. Thus, high affinity antibodies exhibiting slow off-rates are more likely to interact bivalently with the target cell, occupying two antigen sites with a single Fc. In contrast, antibodies with faster off-rates are likely to dissociate each binding arm more rapidly, resulting in a higher likelihood of monovalent binding. Monovalent binding may in turn increase target cell opsonization and lead to improved recruitment of effector cells. This unpredicted relationship between target affinity and effector function potency suggests a careful examination of antibody design and engineering for the development of next-generation immunotherapeutics

A Novel Dual Expression Platform for High Throughput Functional Screening of Phage Libraries in Product like Format.

High throughput screenings of single chain Fv (scFv) antibody phage display libraries are currently done as soluble scFvs produced in E.coli. Due to endotoxin contaminations from bacterial cells these preparations cannot be reliably used in mammalian cell based assays. The monovalent nature and lack of Fc in soluble scFvs prevent functional assays that are dependent on target cross linking and/or Fc functions. A convenient approach is to convert scFvs into scFv.Fc fusion proteins and express them in mammalian cell lines for screening. This approach is low throughput and is only taken after primary screening of monovalent scFvs that are expressed in bacteria. There is no platform at present that combines the benefits of both bacterial and mammalian expression system for screening phage library output. We have, therefore, developed a novel dual expression vector, called pSplice, which can be used to express scFv.Fc fusion proteins both in E.coli and mammalian cell lines. The hallmark of the vector is an engineered intron which houses the bacterial promoter and signal peptide for expression and secretion of scFv.Fc in E.coli. When the vector is transfected into a mammalian cell line, the intron is efficiently spliced out resulting in a functional operon for expression and secretion of the scFv.Fc fusion protein into the culture medium. By applying basic knowledge of mammalian introns and splisosome, we designed this vector to enable screening of phage libraries in a product like format. Like IgG, the scFv.Fc fusion protein is bi-valent for the antigen and possesses Fc effector functions. Expression in E.coli maintains the speed of the bacterial expression platform and is used to triage clones based on binding and other assays that are not sensitive to endotoxin. Triaged clones are then expressed in a mammalian cell line without the need for any additional cloning steps. Conditioned media from the mammalian cell line containing the fusion proteins are then used for different types of cell based assays. Thus this system retains the speed of the current screening system for phage libraries and adds additional functionality to it

Compositional Principles In Lucija Garuta'S Oratorio "God, Thine Earth Is Aflame!". - Page 28

Annexin A1 (anxA1) is an immunomodulatory protein that has been proposed as a tumor vascular target for antitumor biologic agents, yet to date the vascular expression of anxA1 in specific tumor indications has not been systematically assessed. Attempts to evaluate vascular anxA1 expression by immunohistochemistry are complicated by a lack of available antibodies that are both specific for anxA1 and bind the N-terminal-truncated form of anxA1 that has previously been identified in tumor vasculature. To study the vascular expression pattern of anxA1 in non-small-cell lung carcinoma (NSCLC), we isolated an antibody capable of binding N-terminal-truncated anxA127-346 and employed it in immunohistochemical studies of human lung specimens. Lung tumor specimens evaluated with this antibody revealed vascular (endothelial) anxA1 expression in five of eight tumor samples studied, but no vascular anxA1 expression was observed in normal lung tissue. Tumor microarray analysis further demonstrated positive vascular staining for anxA1 in 30 of 80 NSCLC samples, and positive staining of neoplastic cells was observed in 54 of 80 samples. No correlation was observed between vascular and parenchymal anxA1 expression. Two rodent tumor models, B16-F10 and Py230, were determined to have upregulated anxA1 expression in the intratumoral vasculature. These data validate anxA1 as a potential vascular anti-tumor target in a subset of human lung tumors and identify rodent models which demonstrate anxA1 expression in tumor vasculature

Discovery and characterization of potent IL-21 neutralizing antibodies via a novel alternating antigen immunization and humanization strategy.

Interleukin-21 (IL-21), a member of the common cytokine receptor γ chain (γc) family, is secreted by CD4+ T cells and natural killer T cells and induces effector function through interactions with the IL-21 receptor (IL-21R)/γc complex expressed on both immune and non-immune cells. Numerous studies suggest that IL-21 plays a significant role in autoimmune disorders. Therapeutic intervention to disrupt the IL-21/IL-21R/γc interaction and inhibit subsequent downstream signal transduction could offer a treatment paradigm for these diseases. Potent neutralizing antibodies reported in the literature were generated after extensive immunizations with human IL-21 alone and in combination with various adjuvants. To circumvent the laborious method of antibody generation while targeting a conserved functional epitope, we designed a novel alternating-antigen immunization strategy utilizing both human and cynomolgus monkey (cyno) IL-21. Despite the high degree of homology between human and cyno IL-21, our alternating-immunization strategy elicited higher antibody titers and more potent neutralizing hybridomas in mice than did the immunization with human IL-21 antigen alone. The lead hybridoma clone was humanized by grafting the murine complementarity-determining regions onto human germline framework templates, using a unique rational design. The final humanized and engineered antibody, MEDI7169, encodes only one murine residue at the variable heavy/light-chain interface, retains the sub-picomolar affinity for IL-21, specifically inhibits IL-21/IL-21R-mediated signaling events and is currently under clinical development as a potential therapeutic agent for autoimmune diseases. This study provides experimental evidence of the immune system's potential to recognize and respond to shared epitopes of antigens from distinct species, and presents a generally applicable, novel method for the rapid generation of exceptional therapeutic antibodies using the hybridoma platform