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Cysteine-rich knob domains found in the ultralong complementarity determining regions of a subset of bovine antibodies are capable of functioning autonomously as 3–6 kDa peptides. While they can be expressed recombinantly in cellular systems, in this paper we show that knob domains are also readily amenable to a chemical synthesis, with a co-crystal structure of a chemically synthesized knob domain in complex with an antigen showing structural equivalence to the biological product. For drug discovery, following the immunization of cattle, knob domain peptides can be synthesized directly from antibody sequence data, combining the power and diversity of the bovine immune repertoire with the ability to rapidly incorporate nonbiological modifications. We demonstrate that, through rational design with non-natural amino acids, a paratope diversity can be massively expanded, in this case improving the efficacy of an allosteric peptide. As a potential route to further improve stability, we also performed head-to-tail cyclizations, exploiting the proximity of the N and C termini to synthesize functional, fully cyclic antibody fragments. Lastly, we highlight the stability of knob domains in plasma and, through pharmacokinetic studies, use palmitoylation as a route to extend the plasma half-life of knob domains in vivo. This study presents an antibody-derived medicinal chemistry platform, with protocols for solid-phase synthesis of knob domains, together with the characterization of their molecular structures, in vitro pharmacology, and pharmacokinetics.

ABSTRACTThe propensity for some monoclonal antibodies (mAbs) to aggregate at physiological and manufacturing pH values can prevent their use as therapeutic molecules or delay time to market. Consequently, developability assessments are essential to select optimum candidates, or inform on mitigation strategies to avoid potential late-stage failures. These studies are typically performed in a range of buffer solutions because factors such as pH can dramatically alter the aggregation propensity of the test mAbs (up to 100-fold in extreme cases). A computational method capable of robustly predicting the aggregation propensity at the pH values of common storage buffers would have substantial value. Here, we describe a mAb aggregation prediction tool (MAPT) that builds on our previously published isotype-dependent, charge-based model of aggregation. We show that the addition of a homology model-derived hydrophobicity descriptor to our electrostatic aggregation model enabled the generation of a robust mAb developability indicator. To contextualize our aggregation scoring system, we analyzed 97 clinical-stage therapeutic mAbs. To further validate our approach, we focused on six mAbs (infliximab, tocilizumab, rituximab, CNTO607, MEDI1912 and MEDI1912_STT) which have been reported to cover a large range of aggregation propensities. The different aggregation propensities of the case study molecules at neutral and slightly acidic pH were correctly predicted, verifying the utility of our computational method.

We present a comprehensive analysis of all ring systems (both heterocyclic and nonheterocyclic) in clinical trial compounds and FDA-approved drugs. We show 67% of small molecules in clinical trials comprise only ring systems found in marketed drugs, which mirrors previously published findings for newly approved drugs. We also show there are approximately 450 000 unique ring systems derived from 2.24 billion molecules currently available in synthesized chemical space, and molecules in clinical trials utilize only 0.1% of this available pool. Moreover, there are fewer ring systems in drugs compared with those in clinical trials, but this is balanced by the drug ring systems being reused more often. Furthermore, systematic changes of up to two atoms on existing drug and clinical trial ring systems give a set of 3902 future clinical trial ring systems, which are predicted to cover approximately 50% of the novel ring systems entering clinical trials.