The availability of stable human antibody reagents would be of considerable advantage for research, diagnostic, and therapeutic applications. Unfortunately, antibody variable heavy and light domains (V H and V L ) that mediate the interaction with antigen have the propensity to aggregate. Increasing their aggregation resistance in a general manner has proven to be a difficult and persistent problem, due to the high level of sequence diversity observed in human variable domains and the requirement to maintain antigen binding. Here we outline such an approach. By using phage display we identified specific positions that clustered in the antigen binding site (28, 30-33, 35 in V H and 24, 49-53, 56 in V L ). Introduction of aspartate or glutamate at these positions endowed superior biophysical properties (non-aggregating, well-expressed, and heat-refoldable) onto domains derived from common human germline families (V H 3 and V κ 1). The effects of the mutations were highly positional and independent of sequence diversity at other positions. Moreover, crystal structures of mutant V H and V L domains revealed a surprising degree of structural conservation, indicating compatibility with V H ∕V L pairing and antigen binding. This allowed the retrofitting of existing binders, as highlighted by the development of robust high affinity antibody fragments derived from the breast cancer therapeutic Herceptin. Our results provide a general strategy for the generation of human antibody variable domains with increased aggregation resistance.biotechnology | monoclonal antibodies | protein aggregation | protein engineering | antibody therapeutics P rotein aggregation represents a key bottleneck in the generation of antibody-based reagents and hinders the development and production of human therapeutics (1). It is generally believed that the aggregation propensity of larger antibody reagents (such as immunoglobulin G and Fab) is mostly determined by their variable domain components (V H and V L ), although there is currently little understanding of the mechanisms involved (1, 2). Indeed, significant differences of aggregation rates have been reported for antibodies that differ exclusively in their variable domains (1, 2). Aggregation propensity is even more pronounced for smaller antibody reagents, which lack the interdomain stabilization of their larger counterparts (3, 4). This is a major problem in biotechnology due to an increasing trend toward smaller antibody formats for imaging and tumor targeting applications (4). Common formats include human single chain fragments (scFv) and human single domain antibodies, both of which frequently display poor biophysical properties (1, 3).The aggregation propensity of human variable domains is in marked contrast to the variable heavy domains of camels and llamas, which are generally nonaggregating and soluble ("V HH domains") (5-7). More favorable properties have also been described for "camelized" and other engineered human V H model domains (8-12) but not for human V L domains. Their p...

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DNA-based individual identification and RNA-based tissue identification represent two commonly-used tools in forensic investigation, aiming to identify crime scene sample donors and helping to provide links between DNA-identified sample donors and criminal acts. Currently however, both analyses are typically performed separately. In this proof-of-principle study, we developed an approach for the simultaneous analysis of forensic STRs, amelogenin, and forensic mRNAs based on parallel targeted DNA/RNA sequencing using the Ion Torrent Personal Genome Machine(®) (PGM™) System coupled with the AmpliSeq™ targeted amplification. We demonstrated that 9 autosomal STRs commonly used for individual identification (CSF1PO, D16S539, D3S1358, D5S818, D7S820, D8S1179, TH01, TPOX, and vWA), the AMELX/AMELY system widely applied for sex identification, and 12 mRNA markers previously established for forensic tissue identification (ALAS2 and SPTB for peripheral blood, MMP10 and MMP11 for menstrual blood, HTN3 and STATH for saliva, PRM1 and TGM4 for semen, CYP2B7P1 and MUC4 for vaginal secretion, CCL27 and LCE1C for skin) together with two candidate reference mRNA markers (HPRT1 and SDHA) can all be successfully combined. Unambiguous mRNA-based tissue identification was achieved in all samples from all forensically relevant tissues tested, and STR sequencing analysis of the tissue sample donors was 100% concordant with conventional STR profiling using a commercial kit. Successful STR analysis was obtained from 1ng of genomic DNA and mRNA analysis from 10ng total RNA; however, sensitivity limits were not investigated in this proof-of-principle study and are expected to be much lower. Since dried materials with noticeable RNA degradation and small DNA/RNA amplicons with high-coverage sequencing were used, the achieved correct individual and tissue identification demonstrates the suitability of this approach for analyzing degraded materials in future forensic applications. Overall, our study demonstrates the feasibility of simultaneously obtaining multilocus STR, amelogenin, and multilocus mRNA information for combined individual and tissue identification from a small sample of degraded biological material. Moreover, our study marks the first step towards combining many DNA/RNA markers for various forensic purposes to increase the effectiveness of molecular forensic analysis and to allow more forensically relevant information to be obtained from limited forensic material.

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