Triglycerides (TG) are required for fatty acid transport and storage and are essential for human health. Angiopoietin-like-protein 8 (ANGPTL8) has previously been shown to form a complex with ANGPTL3 that increases circulating TG by potently inhibiting LPL. We also recently showed that the TG-lowering apolipoprotein A5 (ApoA5) decreases TG levels by suppressing ANGPTL3/8-mediated LPL inhibition. To understand how LPL binds ANGPTL3/8 and ApoA5 blocks this interaction, we used hydrogen-deuterium exchange mass-spectrometry and molecular modeling to map binding sites of LPL and ApoA5 on ANGPTL3/8. Remarkably, we found that LPL and ApoA5 both bound a unique ANGPTL3/8 epitope consisting of N-terminal regions of ANGPTL3 and ANGPTL8 that are unmasked upon formation of the ANGPTL3/8 complex. We further used ANGPTL3/8 as an immunogen to develop an antibody targeting this same epitope. After refocusing on antibodies that bound ANGPTL3/8, as opposed to ANGPTL3 or ANGPTL8 alone, we utilized bio-layer interferometry to select an antibody exhibiting high-affinity binding to the desired epitope. We revealed an ANGPTL3/8 leucine zipper-like motif within the anti-ANGPTL3/8 epitope, the LPL-inhibitory region, and the ApoA5-interacting region, suggesting the mechanism by which ApoA5 lowers TG is via competition with LPL for the same ANGPTL3/8-binding site. Supporting this hypothesis, we demonstrate that the anti-ANGPTL3/8 antibody potently blocked ANGPTL3/8-mediated LPL inhibition in vitro and dramatically lowered TG levels in vivo. Together, these data show that an anti-ANGPTL3/8 antibody targeting the same leucine zipper-containing epitope recognized by LPL and ApoA5 markedly decreases TG by suppressing ANGPTL3/8-mediated LPL inhibition.

Hundreds of target specific peptides are routinely discovered by peptide display platforms. However, due to the high cost of peptide synthesis only a limited number of peptides are chemically made for further analysis. Here we describe an accurate and cost effective method to bin peptides on-phage based on binding region(s), without any requirement for peptide or protein synthesis. This approach, which integrates phage and yeast display platforms, requires display of target and its alanine variants on yeast. Flow cytometry was used to detect binding of peptides on-phage to the target on yeast. Once hits were identified, they were synthesized to confirm their binding region(s) by HDX (Hydrogen deuterium exchange) and crystallography. Moreover, we have successfully shown that this approach can be implemented as part of a panning process to deplete non-functional peptides. This technique can be applied to any target that can be successfully displayed on yeast; it narrows down the number of peptides requiring synthesis; and its utilization during selection results in enrichment of peptide population against defined binding regions on the target.

Recombinant T cell receptors (TCRs) can be used to redirect naïve T cells to eliminate virally infected or cancerous cells; however, they are plagued by low stability and uneven expression. Here, we use molecular modeling to identify mutations in the TCR constant domains (Cα/Cβ) that increase the unfolding temperature of Cα/Cβ by 20 °C, improve the expression of four separate α/β TCRs by 3- to 10-fold, and improve the assembly and stability of TCRs with poor intrinsic stability. The stabilizing mutations rescue the expression of TCRs destabilized through variable domain mutation. The improved stability and folding of the TCRs reduces glycosylation, perhaps through conformational stabilization that restricts access to N-linked glycosylation enzymes. The Cα/Cβ mutations enables antibody-like expression and assembly of well-behaved bispecific molecules that combine an anti-CD3 antibody with the stabilized TCR. These TCR/CD3 bispecifics can redirect T cells to kill tumor cells with target HLA/peptide on their surfaces in vitro.

Interest in the development of bi- or multispecific antibody (BsAbs)-based biotherapeutics is growing rapidly due to their inherent ability to interact with many targets simultaneously, thereby potentially protracting their functionality relative to monoclonal antibodies (mAbs). Biophysical property assays have been used to improve the probability of clinical success for various mAb therapeutics; however, there is a paucity of such data for BsAbs. This work evaluates a fusion of an IgG with an isolated protein domain (deemed ECD) and serves to understand how molecular architecture influences biophysical and biochemical properties and, in turn, how these relate to drug disposition. The biophysical characteristics of the molecules (charge, nonspecific binding, FcRn and Fcγ receptor interactions, thermal stability, structure-dynamics, and hydrophobic properties) indicated preferred orientations of ECD and IgG, which supported better pharmacokinetic outcomes. In certain instances, in which ECD-IgG configurations led to suboptimal biophysical behavior in the form of increased hydrophobicity and global ECD instability, drug clearance was found to be increased by ≥2-fold, driven by endothelial cell-based association/clearance mechanisms in the liver, kidneys, and spleen. Improvements in the pharmacokinetic properties were afforded by positional modulation of ECD that was able to bring the disposition characteristics in line with those of the parental mAb. The findings provide some pragmatic, broadly applicable strategies and guidance for the design considerations and evaluation of ECD-BsAb constructs. Additional studies, delineating the precise interactions involved in the clearance of the ECD-BsAb constructs, remain an opportunistic area for improving their in vivo kinetic properties.

IgG bispecific antibodies (BsAbs) represent one of the preferred formats for bispecific antibody therapeutics due to their native‐like IgG properties and their monovalent binding to each target. Most reported studies utilized transient expression in HEK293 cells to produce BsAbs. However, the expression of biotherapeutic molecules using stable CHO cell lines is commonly used for biopharmaceutical manufacturing. Unfortunately, limited information is available in the scientific literature on the expression of BsAbs in CHO cell lines. In this study we describe an alternative approach to express the multiple components of IgG BsAbs using a single plasmid vector (quad vector). This single plasmid vector contains both heavy chain genes and both light chain genes required for the expression and assembly of the IgG BsAb, along with a selectable marker. We expressed, purified, and characterized four different IgG BsAbs or “hetero‐mAbs” using transient CHO expression and stable CHO minipools. Transient CHO titers ranged from 90 to 160 mg/L. Stable CHO titers ranged from 0.4 to 2.3 g/L. Following a simple Protein A purification step, the percentage of correctly paired BsAbs ranged from 74% to 98% as determined by mass spectrometry. We also found that information generated from transient CHO expression was similar to information generated using stable CHO minipools. In conclusion, the quad vector approach represents a simple, but effective, alternative approach for the generation of IgG BsAbs in both transient CHO and stable CHO expression systems. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:469–477, 2017

Glycoprofiling recombinant proteins expressed and secreted from mammalian cells is key to understanding their interactions with glycoprotein receptors in vivo. Recently, recombinant T cell receptors (TCRs) are being considered as therapeutic moieties. Here we present a mass spectrometry based protocol with a "bottom up" approach to characterize glycosylation in recombinant fusion proteins with α/β TCR constant domains expressed in mammalian cells. The protocol focuses on using peptide mass mapping and mass spectrometry for N-linked glycan profiling, including analyses of site occupancy, glycan heterogeneity, and possible glycan compositions and structures.

A challenge in the structure-based design of specificity is modeling the negative states, i.e., the complexes that you do not want to form. This is a difficult problem because mutations predicted to destabilize the negative state might be accommodated by small conformational rearrangements. To overcome this challenge, we employ an iterative strategy that cycles between sequence design and protein docking in order to build up an ensemble of alternative negative state conformations for use in specificity prediction. We have applied our technique to the design of heterodimeric CH3 interfaces in the Fc region of antibodies. Combining computationally and rationally designed mutations produced unique designs with heterodimer purities greater than 90%. Asymmetric Fc crystallization was able to resolve the interface mutations; the heterodimer structures confirmed that the interfaces formed as designed. With these CH3 mutations, and those made at the heavy-/light-chain interface, we demonstrate one-step synthesis of four fully IgG-bispecific antibodies.

A myriad of innovative bispecific antibody (BsAb) platforms have been reported. Most require significant protein engineering to be viable from a development and manufacturing perspective. Single-chain variable fragments (scFvs) and diabodies that consist only of antibody variable domains have been used as building blocks for making BsAbs for decades. The drawback with Fv-only moieties is that they lack the native-like interactions with CH1/CL domains that make antibody Fab regions stable and soluble. Here, we utilize a redesigned Fab interface to explore 2 novel Fab-based BsAbs platforms. The redesigned Fab interface designs limit heavy and light chain mixing when 2 Fabs are co-expressed simultaneously, thus allowing the use of 2 different Fabs within a BsAb construct without the requirement of one or more scFvs. We describe the stability and activity of a HER2×HER2 IgG-Fab BsAb, and compare its biophysical and activity properties with those of an IgG-scFv that utilizes the variable domains of the same parental antibodies. We also generated an EGFR × CD3 tandem Fab protein with a similar format to a tandem scFv (otherwise known as a bispecific T cell engager or BiTE). We show that the Fab-based BsAbs have superior biophysical properties compared to the scFv-based BsAbs. Additionally, the Fab-based BsAbs do not simply recapitulate the activity of their scFv counterparts, but are shown to possess unique biological activity.

Immunoglobulins and T cell receptors (TCRs) share common sequences and structures. With the goal of creating novel bispecific antibodies (BsAbs), we generated chimeric molecules, denoted IgG_TCRs, where the Fv regions of several antibodies were fused to the constant domains of the α/β TCR. Replacing CH1 with Cα and CL with Cβ, respectively, was essential for achieving at least partial heavy chain/light chain assembly. Further optimization of the linker regions between the variable and constant domains, as well as replacement of the large FG loop of Cβ with a canonical β-turn, was necessary to consistently obtain full heavy chain/light chain assembly. The optimized IgG_TCR molecules were evaluated biophysically and shown to maintain the binding properties of their parental antibodies. A few BsAbs were generated by co-expressing native Fabs and IgG_TCR Fabs within the same molecular construct. We demonstrate that the IgG_TCR designs steered each of the light chains within the constructs to specifically pair with their cognate heavy chain counterparts. We did find that even with complete constant domain specificity between the CH1/CL and Cα/Cβ domains of the Fabs, strong variable domain interactions can dominate the pairing specificity and induce some mispairing. Overall, the IgG_TCR designs described here are a first step toward the generation of novel BsAbs that may be directed toward the treatment of multi-faceted and complex diseases.

Kinase recruitment to membrane receptors is essential for signal transduction. However, the underlying regulatory mechanisms are poorly understood. We investigated how conformational changes control T cell receptor (TCR) association and activity of the kinase Zap70. Structural analysis showed that TCR binding or phosphorylation of Zap70 triggers a transition from a closed, autoinhibited conformation to an open conformation. Using Zap70 mutants with defined conformations, we found that TCR dwell times controlled Zap70 activity. The closed conformation minimized TCR dwell times and thereby prevented activation by membrane-associated kinases. Parallel recruitment of coreceptor-associated Lck kinase to the TCR ensured Zap70 phosphorylation and stabilized Zap70 TCR binding. Our study suggests that the dynamics of cytosolic enzyme recruitment to the plasma membrane regulate the activity and function of receptors lacking intrinsic catalytic activity.