Antibody drug conjugates (ADCs) are a therapeutic class offering promise for cancer therapy. The attachment of cytotoxic drugs to antibodies can result in an effective therapy with better safety potential than nontargeted cytotoxics. To understand the role of conjugation site, we developed an enzymatic method for site-specific antibody drug conjugation using microbial transglutaminase. This allowed us to attach diverse compounds at multiple positions and investigate how the site influences stability, toxicity, and efficacy. We show that the conjugation site has significant impact on ADC stability and pharmacokinetics in a species-dependent manner. These differences can be directly attributed to the position of the linkage rather than the chemical instability, as was observed with a maleimide linkage. With this method, it is possible to produce homogeneous ADCs and tune their properties to maximize the therapeutic window.

Antibody repertoire diversity, potentially as high as 10 11 unique molecules in a single individual, confounds characterization by conventional sequence analyses. In this study, we present a general method for assessing human antibody sequence diversity displayed on phage using massively parallel pyrosequencing, a novel application of Kabat column-labeled profile Hidden Markov Models, and translated complementarity determining region (CDR) capture-recapture analysis. Pyrosequencing of domain amplicon and RCA PCR products generated 1.5 ؋ 10 6 reads, including more than 1.9 ؋ 10 5 high quality, full-length sequences of antibody variable fragment (Fv) variable domains. Novel methods for germline and CDR classification and fine characterization of sequence diversity in the 6 CDRs are presented. Diverse germline contributions to the repertoire with random heavy and light chain pairing are observed. All germline families were found to be represented in 1.7 ؋ 10 4 sequences obtained from repeated panning of the library. While the most variable CDR (CDR-H3) presents significant length and sequence variability, we find a substantial contribution to total diversity from somatically mutated germline encoded CDRs 1 and 2. Using a capture-recapture method, the total diversity of the antibody library obtained from a human donor Immunoglobulin M (IgM) pool was determined to be at least 3.5 ؋ 10 10 . The results provide insights into the role of IgM diversification, display library construction, and productive germline usages in antibody libraries and the humoral repertoire.HMM ͉ phage display ͉ pyrosequencing ͉ CDRs

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Summary B cells produce a diverse antibody repertoire by undergoing gene rearrangements. Pathogen exposure induces the clonal expansion of B cells expressing antibodies that can bind the infectious agent. To assess human B cell responses to trivalent seasonal influenza and monovalent pandemic H1N1 vaccination, we sequenced gene rearrangements encoding the immunoglobulin heavy chain, a major determinant of epitope recognition. The magnitude of B cell clonal expansions correlates with an individual’s secreted antibody response to the vaccine and the expanded clones are enriched for those expressing influenza-specific mAbs. Additionally, B cell responses to pandemic influenza H1N1 vaccination and infection in different people show a prominent family of convergent antibody heavy chain gene rearrangements specific to influenza antigens. These results indicate that microbes can induce specific signatures of immunoglobulin gene rearrangements and that pathogen exposure can potentially be assessed from B cell repertoires.

A diverse antibody repertoire is essential for an effective adaptive immune response to novel molecular surfaces. Although past studies have observed common patterns of V-segment use, as well as variation in V-segment use between individuals, the relative contributions to variance from genetics, disease, age, and environment have remained unclear. Using high-throughput sequence analysis of monozygotic twins, we show that variation in naive V H and D H segment use is strongly determined by an individual's germ-line genetic background. The inherited segment-use profiles are resilient to differential environmental exposure, disease processes, and chronic lymphocyte depletion therapy. Signatures of the inherited profiles were observed in class switched germ-line use of each individual. However, despite heritable segment use, the rearranged complementarity-determining region-H3 repertoires remained highly specific to the individual. As it has been previously demonstrated that certain V-segments exhibit biased representation in autoimmunity, lymphoma, and viral infection, we anticipate our findings may provide a unique mechanism for stratifying individual risk profiles in specific diseases.heritable variation | next generation sequencing | V-gene S pecific biases in the antibody repertoire have been found in many diseases, from viral infections to cancers to autoimmune disorders (1-15). Although it is possible that heritable variation in the composition of the antibody repertoire could alter inherent risk to specific diseases, the diversity of the antibody repertoire has hindered direct characterization of heritable influences.Early twin studies provided some evidence of genetic variation affecting reactive titers from the antibody repertoire. Multiple studies observed both total Ig and antigen-specific titers to be more correlated in monozygotic twins than dizygotic twins or unrelated individuals (16)(17)(18). In some cases of monozygotic twins discordant for autoimmune diseases, the healthy twin often shared high autoantibody reactive titers with their affected twin (16,19,20).Early sequencing studies were able to identify some systematic biases in the antibody repertoire with limited sampling depth. The first sequencing studies to characterize V(D)J diversification mechanisms identified the gene segment recombination process, but also implied a repertoire too diverse to exhaustively interrogate by traditional sequencing technologies (21). Complete characterization of V-segment loci established ∼50 V H , 40 V κ , and 30 V λ segments in an individual, with a number of allelic variants for the majority of segments (22)(23)(24). Evaluation of use across individuals revealed biased V-gene representation that preceded selection (25)(26)(27). Quantitative PCR of V-gene families showed family use largely stable over time, with fluctuations in use correlated to antigen-specific responses (28). In the T-cell receptor (TCR) repertoire, TCRB-V use was more highly correlated in healthy monozygotic twins than unrelated individua...

Allostery is a phenomenon that couples effector ligand binding at an allosteric site to a structural and/or dynamic change at a distant regulated site. To study an allosteric transition, we vary the size of the allosteric site and its interactions to construct a series of energy landscapes with pronounced minima corresponding to both the effector bound and unbound crystal structures. We use molecular dynamics to sample these landscapes. The degree of perturbation by the effector, modeled by the size of the allosteric site, provides an order parameter for allostery that allows us to determine how microscopic motions give rise to commonly discussed macroscopic mechanisms: (i) induced fit, (ii) population shift, and (iii) entropy driven. These mechanisms involve decreasing structural differences between the effector bound and unbound populations. A metric (ligand-induced cooperativity) can measure how cooperatively a given regulated site responds to effector binding and therefore what kind of allosteric mechanism is involved. We apply the model to three proteins with experimentally characterized transitions: (i) calmodulin-GFP Ca(2+) sensor protein, (ii) maltose binding protein, and (iii) CSL transcription factor. Remarkably, the model is able to reproduce allosteric motion and predict coupling in a manner consistent with experiment.

Background: An antagonistic anti-PCSK9 antibody exhibits target-mediated clearance, resulting in a dose-dependent PK. Results: Engineering of an antibody with pH-sensitive binding to PCSK9 decreases target-mediated clearance, resulting in increased PK and efficacy in vivo. Conclusion: pH-sensitive anti-PCSK9 antibodies are excellent candidates for therapeutic development. Significance: pH-sensitive antibodies may enable less frequent or lower dosing of antibodies hampered by target-mediated clearance and high antigen load.