Therapeutic assessment of SEED: a new engineered antibody platform designed to generate mono- and bispecific antibodies.

The strand-exchange engineered domain (SEED) platform was designed to generate asymmetric and bispecific antibody-like molecules, a capability that expands therapeutic applications of natural antibodies. This new protein engineered platform is based on exchanging structurally related sequences of immunoglobulin within the conserved CH3 domains. Alternating sequences from human IgA and IgG in the SEED CH3 domains generate two asymmetric but complementary domains, designated AG and GA. The SEED design allows efficient generation of AG/GA heterodimers, while disfavoring homodimerization of AG and GA SEED CH3 domains. Using a clinically validated antibody (C225), we tested whether Fab derivatives constructed on the SEED platform retain desirable therapeutic antibody features such as in vitro and in vivo stability, favorable pharmacokinetics, ligand binding and effector functions including antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. In addition, we tested SEED with combinations of binder domains (scFv, VHH, Fab). Mono- and bivalent Fab-SEED fusions retain full binding affinity, have excellent biochemical and biophysical stability, and retain desirable antibody-like characteristics conferred by Fc domains. Furthermore, SEED is compatible with different combinations of Fab, scFv and VHH domains. Our assessment shows that the new SEED platform expands therapeutic applications of natural antibodies by generating heterodimeric Fc-analog proteins.

Ligand-induced structural transitions in ErbB receptor extracellular domains.

Crystallographic studies showed that epidermal growth factor (EGF) receptor activation involves major domain rearrangements. Without bound ligand, the extracellular region of the receptor (sEGFR) adopts a "tethered" configuration with its dimerization site occluded by apparently autoinhibitory intramolecular interactions. Ligand binding causes the receptor to become "extended," breaking the tether and exposing the dimerization site. Using small-angle X-ray scattering (SAXS), we confirm that the tethered and extended conformations are also adopted in solution, and we describe low-resolution molecular envelopes for an intact sEGFR dimer. We also use SAXS to monitor directly the transition from a tethered to extended configuration in the monomeric extracellular regions of ErbB3 and a dimerization-defective EGFR mutant. Finally, we show that mutating every intramolecular tether interaction in sEGFR does not greatly alter its conformation. These findings explain why tether mutants fail to activate EGF receptor and provide new insight into regulation of ErbB receptor conformation.

Invariant chain transmembrane domain trimerization: a step in MHC class II assembly.

The transmembrane (TM) domain of the major histocompatibility complex (MHC) class II-associated invariant chain (Ii) has long been implicated in both correct folding and function of the MHC class II complex. To function correctly, Ii must form a trimer, and the TM domain is one of the domains thought to stabilize the trimeric state. Specific mutations in the TM domain have been shown previously to disrupt MHC class II functions such as mature complex formation and antigen presentation, possibly due to disruption of Ii TM helix-helix interactions. Although this hypothesis has been reported several times in the literature, thus far no experimental measurements have been made to explore the relationship between TM domain structure and TM mutations that affect Ii function. We have applied biophysical and computational methods to study the folding and assembly of the Ii TM domain in isolation and find that the TM domain strongly self-associates. According to analytical ultracentrifugation analyses, the primary oligomeric state for this TM domain is a strongly associated trimer with a dissociation constant of approximately 120 nM in DPC micelles. We have also examined the effect of functionally important mutations of glutamine and threonine residues in the TM domain on its structure, providing results that now link the disruption of TM helix interactions to previously reported losses of Ii function.

Epidermal growth factor receptor dimerization and activation require ligand-induced conformational changes in the dimer interface.

Structural studies have shown that ligand-induced epidermal growth factor receptor (EGFR) dimerization involves major domain rearrangements that expose a critical dimerization arm. However, simply exposing this arm is not sufficient for receptor dimerization, suggesting that additional ligand-induced dimer contacts are required. To map these contributions to the dimer interface, we individually mutated each contact suggested by crystallographic studies and analyzed the effects on receptor dimerization, activation, and ligand binding. We find that domain II contributes >90% of the driving energy for dimerization of the extracellular region, with domain IV adding little. Within domain II, the dimerization arm forms much of the dimer interface, as expected. However, a loop from the sixth disulfide-bonded module (immediately C-terminal to the dimerization arm) also makes a critical contribution. Specific ligand-induced conformational changes in domain II are required for this loop to contribute to receptor dimerization, and we identify a set of ligand-induced intramolecular interactions that appear to be important in driving these changes, effectively "buttressing" the dimer interface. Our data also suggest that similar conformational changes may determine the specificity of ErbB receptor homo- versus heterodimerization.

Sequence context strongly modulates association of polar residues in transmembrane helices.

Polar residues are capable of mediating the association of membrane-embedded helices through the formation of side-chain/side-chain inter-helical hydrogen bonds. However, the extent to which native van der Waals packing of the residues surrounding the polar locus can enhance, or interfere with, the interaction of polar residues has not yet been studied. We examined the propensities of four polar residues (aspartic acid, asparagine, glutamic acid, and glutamine) to promote self-association of transmembrane (TM) domains in several biologically derived sequence environments, including (i). four naturally occurring TM domains that contain a Glu or Gln residue (Tnf5/CD40 ligand, C79a/Ig-alpha, C79b/Ig-beta, and Fut3/alpha-fucosyltransferase); and (ii). variants of bacteriophage M13 major coat protein TM segment with Asp and Asn at interfacial and non-interfacial positions. Self-association was quantified by the TOXCAT assay, which measures TM helix self-oligomerization in the Escherichia coli inner membrane. While an appropriately placed polar residue was found in several cases to significantly stabilize TM helix-helix interactions through the formation of an interhelical hydrogen bond, in other cases the strongly polar residues did not enhance the association of the two helices. Overall, these results suggest that an innate structural mechanism may operate to control non-specific association of membrane-embedded polar residues.

Motifs of serine and threonine can drive association of transmembrane helices.

Known sequence motifs containing key glycine residues can drive the homo-oligomerization of transmembrane helices. To find other motifs, a randomized library of transmembrane interfaces was generated in which glycine was omitted. The TOXCAT system, which measures transmembrane helix association in the Escherichia coli inner membrane, was used to select high-affinity homo-oligomerizing sequences in this library. The two most frequently occurring motifs were SxxSSxxT and SxxxSSxxT. Isosteric mutations of any one of the serine and threonine residues to non-polar residues abolished oligomerization, indicating that the interaction between these positions is specific and requires an extended motif of serine and threonine hydroxyl groups. Computational modeling of these sequences produced several chemically plausible structures that contain multiple hydrogen bonds between the serine and threonine residues. While single serine or threonine side-chains do not appear to promote helix association, motifs can drive strong and specific association through a cooperative network of interhelical hydrogen bonds.