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TUTORIAL OMICS Figure 2. Epitope map of SDIX GPCR CXCR4 mAbs. Highlighted extracellular loops represent regions of critical binding. Diversity was also demonstrated by epitope mapping with cells expressing mutant MMPs. Most currently known CXCR4 mAbs bind primarily to the second extracellular loop. CXCR4 mAbs from DNA-immunized animals, however, exhibited eight different binding patterns including previously unreported, novel specifcities (Figure 2). Similarly, most existing CD20 mAbs fall into two classes (rituximab-like and ofatumumab-like). The CD20 mAbs isolated from DNA-immunized mice exhibit nine known and novel specifcities. The intrinsically gentle antigen delivery process of DNA immunization may explain this surprisingly diverse epitope pattern as these novel epitopes may be destroyed with conventional protein-based immunizations. The performance of mAbs isolated from DNA-immunized animals was compared to existing benchmark therapeutic antibodies. In fow cytometry, SDIX mAbs exhibit high titer binding and unique patterns of reactivity. All of the SDIX ADORA2A mAbs react positively in fow cytometry, and multiple CXCR4 and CD20 mAbs exhibit greater binding than existing benchmark antibodies. Performance in functional assays including apoptosis and receptor modulation was also evaluated, and many of the mAbs functioned as well as, or better than, existing benchmark antibodies (Figure 3). There is a great need for high-performance fow cytometry, functional, therapeutic, biomarker, and companion diagnostic antibodies with performance specifcations exceedingly more rigorous than antibodies generated from peptide antigens for Western blots. This is especially true for MMP where target sites of antibody binding are composed of multiple discontinuous protein sequences that are dependent on the cell membrane for proper 3D structure. DNA immunization is key to eliciting antibodies that recognize this class of critical therapeutic and diagnostic targets and the technologies described here represent a signifcant step toward routine, low-cost development of mAbs to MMP. Think Outside the Plate Array Tape™ – The New Standard for High Throughput Applications The Nexar¨ Optimized for Real Time Nucleic Acid Processing supports high throughput liquid handling, amplification, and real time detection of isothermal and qPCR chemistries in an automated, inline instrument. Utilizing the Array TapeTM consumable, the system operates with high precision and accuracy, allowing the user to generate repeatable data with walk-away operation. Figure 3. Inhibition of SDF-1–induced cAMP modulation by SDIX CXCR4 mAbs compared to three benchmark mAbs and the small molecule drug AMD 3100 cell-based protocols. DNA immunization results in the production of pure target MMP in the membranes of cells within the immunized animal, and, importantly, without introducing other foreign contaminants that could distract the immune system. The newly expressed protein is recognized as foreign, and antibodies are produced. Because the protein is expressed in vivo by mammalian cells, there is no purifcation or attendant protein denaturation. This is particularly critical for MMPs since most are highly fragile and easily denature. Following immunization, hybridomas are derived by fusion technology and supernatants are screened by multiplexed, highthroughput fow cytometry using cells expressing the target MMP. To demonstrate the effcacy of this approach we undertook the development of mAbs to three MMPs (GPCRs CXCR4, ADORA2A, and CD20). An important aspect of the technology is the capacity to make signifcant numbers of different highperformance mAbs with diverse binding sites and potential effects. For all three targets, panels of mAbs were generated with low numbers of hybridoma fusions. Ninety-three CXCR4 mAbs were isolated from two hybridoma fusions, 15 ADORA2A mAbs were isolated from a single fusion, and 51 CD20 mAbs from three fusions. Antibody gene sequences from SDIX mAbs revealed highly diverse antibody responses to all three MMPs. Seventy-fve of the 93 CXCR4 mAbs were shown to have unique sequences, (i.e., derived from a unique VDJ recombination event), as well as 14 of the 15 ADORA2A mAbs and 34 of the 51 CD20 mAbs. In addition, the level of somatic hypermutation in these mAbs was shown to be comparable to a benchmark set of 29 therapeutic mAbs suggesting similar levels of affnity maturation. Nexar¨ Optimized for Real Time Nucleic Acid Processing Key features: Up to 50% savings on consumables. Up to 90% savings on reagents. Automated, inline system providing walk-away operation Miniaturized reaction volumes of 1,200 to 1,800 nL Offset labor costs by 60% or more. Higher throughput Flexibility to utilize isothermal DNA amplification or qPCR chemistries www.DouglasScientifc.com Contact us today! Endless Array Tape. Endless Possibilities. Genetic Engineering & Biotechnology News | GENengnews.com | August 2013 | 29