PURPOSE. To design and select the next generation of ocular therapeutics, we performed a comprehensive ocular and systemic pharmacokinetic (PK) analysis of a variety of antibodies and antibody fragments, including a novel-designed bispecific antibody. METHODS.Molecules were administrated via intravitreal (IVT) or intravenous (IV) injections in rabbits, and antibody concentrations in each tissue were determined by ELISA. A novel mathematical model was developed to quantitate the structure-PK relationship RESULTS. After IVT injection, differences in vitreal half-life observed across all molecules ranged between 3.2 and 5.2 days. Modification or elimination of the fragment crystallizable (Fc) region reduced serum half-life from 9 days for the IgG to 5 days for the neonatal Fc receptor (FcRn) null mAb, to 3.1 to 3.4 days for the other formats. The F(ab') 2 was the optimal format for ocular therapeutics with comparable vitreal half-life to full-length antibodies, but with minimized systemic exposure. Concomitantly, the consistency among mathematical model predictions and observed data validated the model for future PK predictions. In addition, we showed a novel design to develop bispecific antibodies, here with activity targeting multiple angiogenesis pathways.CONCLUSIONS. We demonstrated that protein molecular weight and Fc region do not play a critical role in ocular PK, as they do systemically. Moreover, the mathematical model supports the selection of the ''ideal therapeutic'' by predicting ocular and systemic PK of any antibody format for any dose regimen. These findings have important implications for the design and selection of ocular therapeutics according to treatment needs, such as maximizing ocular halflife and minimizing systemic exposure.

A primary barrier to the success of T cell–recruiting bispecific antibodies in the treatment of solid tumors is the lack of tumor-specific targets, resulting in on-target off-tumor adverse effects from T cell autoreactivity to target-expressing organs. To overcome this, we developed an anti-HER2/CD3 T cell–dependent bispecific (TDB) antibody that selectively targets HER2-overexpressing tumor cells with high potency, while sparing cells that express low amounts of HER2 found in normal human tissues. Selectivity is based on the avidity of two low-affinity anti-HER2 Fab arms to high target density on HER2-overexpressing cells. The increased selectivity to HER2-overexpressing cells is expected to mitigate the risk of adverse effects and increase the therapeutic index. Results included in this manuscript not only support the clinical development of anti-HER2/CD3 1Fab–immunoglobulin G TDB but also introduce a potentially widely applicable strategy for other T cell–directed therapies. The potential of this discovery has broad applications to further enable consideration of solid tumor targets that were previously limited by on-target, but off-tumor, autoimmunity.

Summary The essential Mycobacterium tuberculosis Ser/Thr protein kinase (STPK), PknB, plays a key role in regulating growth and division, but the structural basis of activation has not been defined. Here we provide biochemical and structural evidence that dimerization through the kinase-domain (KD) N-lobe activates PknB by an allosteric mechanism. Promoting KD pairing using a small-molecule dimerizer stimulates the unphosphorylated kinase, and substitutions that disrupt N-lobe pairing decrease phosphorylation activity in vitro and in vivo. Multiple crystal structures of two monomeric PknB KD mutants in complex with nucleotide reveal diverse inactive conformations that contain large active-site distortions that propagate >30 Å from the mutation site. These results define flexible, inactive structures of a monomeric bacterial receptor KD and show how “back-to-back” N-lobe dimerization stabilizes the active KD conformation. This general mechanism of bacterial receptor STPK activation affords insights into the regulation of homologous eukaryotic kinases that form structurally similar dimers.

Many bacterial species express ‘eukaryotic-like’ Ser/Thr or Tyr protein kinases and phosphatases that are candidate mediators of developmental changes and host/pathogen interactions. The biological functions of these systems are largely unknown. Recent genetic, biochemical and structural studies have begun to establish a framework for understanding the systems for Ser/Thr and Tyr protein phosphorylation in Mycobacterium tuberculosis (Mtb). Ser/Thr protein kinases (STPKs) appear to regulate diverse processes including cell division and molecular transport. Proposed protein substrates of the STPKs include putative regulatory proteins, as well as six proteins containing Forkhead-associated domains. Structures of domains of receptor STPKs and all three Mtb Ser/Thr or Tyr phosphatases afford an initial description of the principal modules that mediate bacterial STPK signaling. These studies revealed that universal mechanisms of regulation and substrate recognition govern the functions of prokaryotic and eukaryotic STPKs. Several structures also support novel mechanisms of regulation, including dimerization of STPKs, metal-ion binding to PstP and substrate mimicry in PtpB.

To define how extracellular signals activate bacterial receptor Ser/Thr protein kinases, we characterized the regulatory functions of a weak dimer interface identified in the Mycobacterium tuberculosis PknB and PknE receptor kinases. Sequence comparisons revealed that the analogous interface is conserved in PknD orthologs from diverse bacterial species. To analyze the roles of dimerization, we constructed M. tuberculosis PknD kinase domain (KD) fusion proteins that formed dimers upon addition of rapamycin. Dimerization of unphosphorylated M. tuberculosis PknD KD fusions stimulated phosphorylation activity. Mutations in the dimer interface reduced this activation, limited autophosphorylation, and altered substrate specificity. In contrast, an inactive catalytic site mutant retained the ability to stimulate the wild-type KD by dimerization. These results support the idea that dimer formation allosterically activates unphosphorylated PknD. The phosphorylated PknD KD was fully active even in the absence of dimerization, suggesting that phosphorylation provides an additional regulatory mechanism. The conservation of analogous dimers in diverse prokaryotic and eukaryotic Ser/Thr protein kinases implies that this mechanism of protein kinase regulation is ancient and broadly distributed.Tight regulation of Ser/Thr protein kinases (STPKs) 2 is essential for signaling in all three kingdoms of life. In keeping with the importance of phospho-signaling pathways, STPKs are subject to multiple mechanisms of allosteric regulation (1). Diverse regulatory surfaces of kinase domains (KDs) can bind additional proteins (as in the cyclin-dependent kinases and cAMP-dependent protein kinase) or other domains of the kinase itself (such as the Src homology 2 and linker domains of Src). These interactions often affect the assembly of the catalytic site by positioning a conserved, substrate-binding element called the C helix. In addition, phosphorylation of a conserved motif called the activation loop often relieves a steric blockade of the active site and promotes assembly of a substrate-binding platform (1). Although many regulatory interactions have been characterized in eukaryotic protein kinases, the mechanisms by which environmental signals regulate prokaryotic STPKs have yet to be elucidated.Prokaryotic STPKs occur in numerous pathogens and organisms with complex developmental pathways (2). Like their eukaryotic homologs, the bacterial STPKs adopt a characteristic two-domain fold with the ATP-binding site located between the N-and C-terminal lobes (3-5). Unexpectedly, the catalytic domain of the receptor STPK PknB from Mycobacterium tuberculosis formed an unstable, backto-back dimer through an interface in the N-terminal lobe (Fig. 1A) (3-5). Residues in the PknB dimer interface were found to be strictly conserved in orthologs from dozens of bacterial genera, supporting the proposal that KD dimerization plays an important functional role (5). Because the dimer interface included contacts near the C terminus of the C helix, it was ...

IgA antibodies have broad potential as a novel therapeutic platform based on their superior receptor-mediated cytotoxic activity, potent neutralization of pathogens, and ability to transcytose across mucosal barriers via polymeric immunoglobulin receptor (pIgR)-mediated transport, compared to traditional IgG-based drugs. However, the transition of IgA into clinical development has been challenged by complex expression and characterization, as well as rapid serum clearance that is thought to be mediated by glycan receptor scavenging of recombinantly produced IgA monomer bearing incompletely sialylated N-linked glycans. Here, we present a comprehensive biochemical, biophysical, and structural characterization of recombinantly produced monomeric, dimeric and polymeric human IgA. We further explore two strategies to overcome the rapid serum clearance of polymeric IgA: removal of all N-linked glycosylation sites creating an aglycosylated polymeric IgA and engineering in FcRn binding with the generation of a polymeric IgG-IgA Fc fusion. While previous reports and the results presented in this study indicate that glycan-mediated clearance plays a major role for monomeric IgA, systemic clearance of polymeric IgA in mice is predominantly controlled by mechanisms other than glycan receptor clearance, such as pIgR-mediated transcytosis. The developed IgA platform now provides the potential to specifically target pIgR expressing tissues, while maintaining low systemic exposure.

Rapid identification of residues that influence antibody expression and thermostability is often needed to move promising therapeutics into the clinic. To establish a method that can assess small expression differences, we developed a Bacterial Antibody Display (BAD) system that overcomes previous limitations, enabling the use of full-length formats for antibody and antigen in a live cell setting. We designed a unique library of individual framework variants using natural diversity introduced by somatic hypermutation, and screened half-antibodies for increased expression using BAD. We successfully identify variants that dramatically improve expression yields and in vitro thermostability of two therapeutically relevant antibodies in E. coli and mammalian cells. While we study antibody expression, bacterial display can now be expanded to examine the processes of protein folding and translocation. Additionally, our natural library design strategy could be applied during antibody humanization and library design for in vitro display methods to maintain expression and formulation stability.

No abstract