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Summary Bispecific antibodies (Bispecifics) demonstrate exceptional clinical potential to address some of the most complex diseases. However, Bispecific production in a single cell often requires the correct pairing of multiple polypeptide chains for desired assembly. This is a considerable hurdle that hinders the development of many immunoglobulin G (IgG)-like bispecific formats. Our approach focuses on the rational engineering of charged residues to facilitate the chain pairing of distinct heavy chains (HC). Here, we deploy structure-guided protein design to engineer charge pair mutations (CPMs) placed in the CH3-CH3′ interface of the fragment crystallizable (Fc) region of an antibody (Ab) to correctly steer heavy chain pairing. When used in combination with our stable effector functionless 2 (SEFL2.2) technology, we observed high pairing efficiency without significant losses in expression yields. Furthermore, we investigate the relationship between CPMs and the sequence diversity in the parental antibodies, proposing a rational strategy to deploy these engineering technologies., Graphical abstract, Highlights • Crystal structures unveil molecular basis of SEFL2.2 and CPM technologies • Next gen structure-guided design of CPMs to steer HC-HC pairing • Top CPMs show high pairing efficiency and optimal expression and stability • Balancing CPM charge distribution minimizes impact of sequence diversity, Biochemistry; Bioengineering; Biomolecular engineering; Structural biology

Therapeutic proteins including antibodies and Fc-fusion proteins undergo a large number of chemical modifications during cell culture, purification, storage and in human circulation. They are also exposed to harsh conditions during stress studies, including elevated temperature, extremes of pH, forced oxidation, physiological pH, UV light to assess the possible degradation pathways and suitability of methods for detecting them. Some of these modifications are located on residues in binding regions, leading to loss of binding and potency and classified as critical quality attributes. Currently, criticality of modifications is assessed by a laborious process of collecting antibody fractions from the soft chromatography techniques ion exchange and hydrophobic interaction chromatography and characterizing the fractions one-by-one for potency and chemical modifications. Here, we describe a method for large-scale, parallel identification of all critical chemical modifications in one experiment. In the first step, the antibody is stressed by one or several stress methods. It is then mixed with target protein and separated by size-exclusion chromatography (SEC) on bound antibody-target complex and unbound antibody. Peptide mapping of fractions and statistical analysis are performed to identify modifications on amino acid residues that affect binding. To identify the modifications leading to slight decreases in binding, competitive SEC of antibody and antigen mixtures was developed and described in a companion study by Shi et al, where target protein is provided at lower level, below the stoichiometry. The newly described method was successfully correlated to crystallography for assessing criticality of chemical modifications and paratope mapping. It is more sensitive to low-level modifications, better streamlined and platform ready.

Hepatitis C virus (HCV) is a major cause of liver disease, affecting over 2% of the world's population. The HCV envelope glycoproteins E1 and E2 mediate viral entry, with E2 being the main target of neutralizing antibody responses. Structural investigations of E2 have produced templates for vaccine design, including the conserved CD81 receptor-binding site (CD81bs) that is a key target of broadly neutralizing antibodies (bNAbs). Unfortunately, immunization with recombinant E2 and E1E2 rarely elicits sufficient levels of bNAbs for protection. To understand the challenges for eliciting bNAb responses against the CD81bs, we investigated the E2 CD81bs by electron microscopy (EM), hydrogen-deuterium exchange (HDX), molecular dynamics (MD), and calorimetry. By EM, we observed that HCV1, a bNAb recognizing the N-terminal region of the CD81bs, bound a soluble E2 core construct from multiple angles of approach, suggesting components of the CD81bs are flexible. HDX of multiple E2 constructs consistently indicated the entire CD81bs was flexible relative to the rest of the E2 protein, which was further confirmed by MD simulations. However, E2 has a high melting temperature of 84.8 °C, which is more akin to proteins from thermophilic organisms. Thus, recombinant E2 is a highly stable protein overall, but with an exceptionally flexible CD81bs. Such flexibility may promote induction of nonneutralizing antibodies over bNAbs to E2 CD81bs, underscoring the necessity of rigidifying this antigenic region as a target for rational vaccine design.

VRC01-class antibodies neutralize diverse HIV-1 strains by targeting the conserved CD4-binding site. Despite extensive investigations, crucial events in the early stage of VRC01 development remain elusive. We demonstrated how VRC01-class antibodies emerged in a Chinese donor by antigen-specific single B cell sorting, structural and functional studies, and longitudinal antibody and virus repertoire analyses. A monoclonal antibody DRVIA7 with modest neutralizing breadth was isolated that displayed a subset of VRC01 signatures. X-ray and EM structures revealed a VRC01-like angle of approach, but less favorable interactions between the DRVIA7 light-chain CDR1 and the N terminus with N276 and V5 glycans of gp120. Although the DRVIA7 lineage was unable to acquire broad neutralization, longitudinal analysis revealed a repertoire-encoded VRC01 light-chain CDR3 signature and VRC01-like neutralizing heavy-chain precursors that rapidly matured within 2 years. Thus, light chain accommodation of the glycan shield should be taken into account in vaccine design targeting this conserved site of vulnerability.

Summary: Calcitonin-gene-related peptide (CGRP) plays a key role in migraine pathophysiology. Aimovig (erenumab; erenumab-aooe in the United States) is the only US Food and Drug Administration (FDA)-approved monoclonal antibody (mAb) therapy against the CGRP receptor (CGRPR) for the prevention of migraine. Aimovig is also the first FDA-approved mAb against a G-protein-coupled receptor (GPCR). Here, we report the architecture and functional attributes of erenumab critical for its potent antagonism against CGRPR. The crystal structure of erenumab in complex with CGRPR reveals a direct ligand-blocking mechanism, enabled by a remarkable 21-residue-long complementary determining region (CDR)-H3 loop, which adopts a tyrosine-rich helix-turn tip and projects into the deep interface of the calcitonin receptor-like receptor (CLR) and RAMP1 subunits of CGRPR. Furthermore, erenumab engages with residues specific to CLR and RAMP1, providing the molecular basis for its exquisite selectivity. Such structural insights reveal the drug action mechanism of erenumab and shed light on developing antibody therapeutics targeting GPCRs. : Migraine is a neurological condition that affects more than 10% of the world population, and it is related to aberrant CGRPR signaling. Garces et al. describe the molecular mechanism by which erenumab, the first human antibody approved by the FDA for migraine prevention, specifically targets CGRPR pathway, preventing migraine. Keywords: CGRPR, erenumab, migraine, monoclonal antibodies, GPCR, CDR-H3 loop, Aimovig

The HIV-1 envelope gp160 glycoprotein (Env) is a trimer of gp120 and gp41 heterodimers that mediates cell entry and is the primary target of the humoral immune response. Broadly neutralizing antibodies (bNAbs) to HIV-1 have revealed multiple epitopes or sites of vulnerability, but mapping of most of these sites is incomplete owing to a paucity of structural information on the full epitope in the context of the Env trimer. Here, a crystal structure of the soluble BG505 SOSIP gp140 trimer at 4.6 Å resolution with the bNAbs 8ANC195 and PGT128 reveals additional interactions in comparison to previous antibody–gp120 structures. For 8ANC195, in addition to previously documented interactions with gp120, a substantial interface with gp41 is now elucidated that includes extensive interactions with the N637 glycan. Surprisingly, removal of the N637 glycan did not impact 8ANC195 affinity, suggesting that the antibody has evolved to accommodate this glycan without loss of binding energy. PGT128 indirectly affects the N262 glycan by a domino effect, in which PGT128 binds to the N301 glycan, which in turn interacts with and repositions the N262 glycan, thereby illustrating the important role of neighboring glycans on epitope conformation and stability. Comparisons with other Env trimer and gp120 structures support an induced conformation for glycan N262, suggesting that the glycan shield is allosterically modified upon PGT128 binding. These complete epitopes of two broadly neutralizing antibodies on the Env trimer can now be exploited for HIV-1 vaccine design.

SummaryThe HIV envelope glycoprotein (Env) is densely covered with self-glycans that should help shield it from recognition by the human immune system. Here, we examine how a particularly potent family of broadly neutralizing antibodies (Abs) has evolved common and distinct structural features to counter the glycan shield and interact with both glycan and protein components of HIV Env. The inferred germline antibody already harbors potential binding pockets for a glycan and a short protein segment. Affinity maturation then leads to divergent evolutionary branches that either focus on a single glycan and protein segment (e.g., Ab PGT124) or engage multiple glycans (e.g., Abs PGT121–123). Furthermore, other surrounding glycans are avoided by selecting an appropriate initial antibody shape that prevents steric hindrance. Such molecular recognition lessons are important for engineering proteins that can recognize or accommodate glycans.

International audience; The high-mannose patch on HIV Env is a preferred target for broadly neutralizing antibodies (bnAbs), but to date, no vaccination regimen has elicited bnAbs against this region. Here, we present the development of a bnAb lineage targeting the high-mannose patch in an HIV-1 subtype-C-infected donor from sub-Saharan Africa. The Abs first acquired autologous neutralization, then gradually matured to achieve breadth. One Ab neutralized >47% of HIV-1 strains with only ∼11% somatic hypermutation and no insertions or deletions. By sequencing autologous env, we determined key residues that triggered the lineage and participated in Ab-Env coevolution. Next-generation sequencing of the Ab repertoire showed an early expansive diversification of the lineage followed by independent maturation of individual limbs, several of them developing notable breadth and potency. Overall, the findings are encouraging from a vaccine standpoint and suggest immunization strategies mimicking the evolution of the entire high-mannose patch and promoting maturation of multiple diverse Ab pathways.

SummaryThe high-mannose patch on the HIV-1 envelope (Env) glycoprotein is the epicenter for binding of the potent broadly neutralizing PGT121 family of antibodies, but strategies for generating such antibodies by vaccination have not been defined. We generated structures of inferred antibody intermediates by X-ray crystallography and electron microscopy to elucidate the molecular events that occurred during evolution of this family. Binding analyses revealed that affinity maturation was primarily focused on avoiding, accommodating, or binding the N137 glycan. The overall antibody approach angle to Env was defined very early in the maturation process, yet some variation evolved in the PGT121 family branches that led to differences in glycan specificities in their respective epitopes. Furthermore, we determined a crystal structure of the recombinant BG505 SOSIP.664 HIV-1 trimer with a PGT121 family member at 3.0 Å that, in concert with these antibody intermediate structures, provides insights to advance design of HIV vaccine candidates.

The high-mannose patch of human immunodeficiency virus (HIV) envelope (Env) elicits broadly neutralizing antibodies (bnAbs) during natural infection relatively frequently, and consequently, this region has become a major target of vaccine design. However, it has also become clear that antibody recognition of the region is complex due, at least in part, to variability in neighboring loops and glycans critical to the epitopes. bnAbs against this region have some shared features and some distinguishing features that are crucial to understand in order to design optimal immunogens that can induce different classes of bnAbs against this region. Here, we compare two branches of a single antibody lineage, in which all members recognize the high-mannose patch. One branch (prototype bnAb PGT128) has a 6-amino-acid insertion in CDRH2 that is crucial for broad neutralization. Antibodies in this branch appear to favor a glycan site at N332 on gp120, and somatic hypermutation is required to accommodate the neighboring V1 loop glycans and glycan heterogeneity. The other branch (prototype bnAb PGT130) lacks the CDRH2 insertion. Antibodies in this branch are noticeably effective at neutralizing viruses with an alternate N334 glycan site but are less able to accommodate glycan heterogeneity. We identify a new somatic variant within this branch that is predominantly dependent on N334. The crystal structure of PGT130 offers insight into differences from PGT128. We conclude that different immunogens may be required to elicit bnAbs that have the optimal characteristics of the two branches of the lineage described. IMPORTANCE Development of an HIV vaccine is of vital importance for prevention of new infections, and it is thought that elicitation of HIV bnAbs will be an important component of an effective vaccine. Increasingly, bnAbs that bind to the cluster of high-mannose glycans on the HIV envelope glycoprotein, gp120, are being highlighted as important templates for vaccine design. In particular, bnAbs from IAVI donor 36 (PGT125 to PGT131) have been shown to be extremely broad and potent. Combination of these bnAbs enhanced neutralization breadth considerably, suggesting that an optimal immunogen should elicit several antibodies from this family. Here we study the evolution of this antibody family to inform immunogen design. We identify two classes of bnAbs that differ in their recognition of the high-mannose patch and show that different immunogens may be required to elicit these different classes.

15 p.-2 tab.-8 fig., In order to maintain proper cellular function, the metabolism of the bacterial microbiota presents several mechanisms oriented to keep a correctly balanced amino acid pool. Central components of these mechanisms are enzymes with alanine transaminase activity, pyridoxal 59-phosphate-dependent enzymes that interconvert alanine and pyruvate, thereby allowing the precise control of alanine and glutamate concentrations, two of the most abundant amino acids in the cellular amino acid pool. Here we report the 2.11-A° crystal structure of full-length AlaA from the model organism Escherichia coli, a major bacterial alanine aminotransferase, and compare its overall structure and active site composition with detailed atomic models of two other bacterial enzymes capable of catalyzing this reaction in vivo, AlaC and valine-pyruvate aminotransferase (AvtA). Apart from a narrow entry channel to the active site, a feature of this new crystal structure is the role of an active site loop that closes in upon binding of substrate-mimicking molecules, and which has only been previously reported in a plant enzyme. Comparison of the available structures indicates that beyond superficial differences, alanine aminotransferases of diverse phylogenetic origins share a universal reaction mechanism that depends on an array of highly conserved amino acid residues and is similarly regulated by various unrelated motifs. Despite this unifying mechanism and regulation, growth competition experiments demonstrate that AlaA, AlaC and AvtA are not freely exchangeable in vivo, suggesting that their functional repertoire is not completely redundant thus providing an explanation for their independent evolutionary conservation., This work was supported by the Spanish Ministry of Economy and Competitiveness (grants PET2008_0101, BIO2009-11184 and BFU2010-22260-C02-02 to MCV and BFU2008-02372/BMC, CSD 2006-23 and BFU2011-22588 to MC), the Generalitat de Catalunya (grant SGR2009-1309 to MC), the European Commission (Framework Programme 7 (FP7) projects ComplexINC No. 279039 to MCV), and the US National Institutes of Health (grant 1-R01-GM58560 to KER and AJR).

et al., The ulaG gene, located in the ula regulon, is crucial for the catabolism of l-ascorbate under anaerobic conditions and it has been proposed to encode for the putative l-ascorbate-6-P lactonase. The ulaG gene is widespread among eubacteria, including human commensal and pathogenic genera such as Escherichia, Shigella, Klebsiella and Salmonella. Here, we report the three-dimensional structures of the apoenzyme and Mn2+ holoenzyme of UlaG from E. coli to 2.6 Å resolution, determined using single-wavelength anomalous diffraction phasing and molecular replacement, respectively. The structures reveal a highly specialized metallo-β-lactamase-like fold derived from an ancient structural template that was involved in RNA maturation and DNA repair. This fold has a novel quaternary architecture consisting of a hexameric ring formed by a trimer of UlaG dimers. A mononuclear Mn2+-binding site resides at the core of the active site, which displays micromolar affinity for Mn2+ and a distorted trigonal bipyramidal coordination. The active site Mn2+ ion can be replaced by Co2+ or Zn2+, but not by Fe3+. We further show that the Mn2+ or Co2+-loaded enzyme exhibits lactonase activity towards l-ascorbate 6-P, thereby providing the first direct evidence of its catalytic role in the l-ascorbate catabolic pathway. Guided by the structural homology, we show that UlaG is able to cleave phosphodiester linkages in cyclic nucleotides, suggesting that the conservation of the fold and of the key catalytic residues allows for the evolutionary acquisition of substrate specificity for novel but related substrates. © 2010 Elsevier Ltd., Este trabajo fue financiado por el Ministerio de España de Ciencia y Educación (MEC) (concesiones BFU 2006-15573 para M.C.V., BFU 2007-63090/BMC para L.B., y BFU2008-02372 y BFU2005-24123-E para M.C.), por la Generalitat de Catalunya (concesión 2009-SGR-1309 para M.C.) y por la EU (Proyectos Spine2-Complexes LSHG-2006-031220 y 3D-Repertoire LSHG-CT-2005-512028).

3 páginas, 3 figuras, 1 tabla -- PGS nros. 36-38, UlaG, the putative l-ascorbate-6-phosphate lactonase encoded by the ulaG gene from the utilization of l-ascorbate regulon in Escherichia coli, has been cloned, overexpressed, purified using standard chromatographic techniques and crystallized. Crystals were obtained by sitting-drop vapour diffusion at 293 K. Preliminary X-ray diffraction analysis revealed that the UlaG crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 104.52, b = 180.69, c = 112.88 Å, β = 103.26°. The asymmetric unit is expected to contain six copies of UlaG, with a corresponding volume per protein weight of 2.16 Å3 Da−1 and a solvent content of 43%, This work was supported by grants from the Spanish Ministry of Science and Education (MEC): BFU 2006-15573 to MCV, BFU 2004-03586/BMC to JB and GEN2003-20642 to MC. FG was the recipient of a predoctoral fellowship from the Generalitat de Catalunya. FJF was supported by an I3P postdoctoral contract from MEC

Volume 89, no. 2, p. [1105–1118][1], 2015. Page 1116, Acknowledgments: The following sentence should appear at the end of the second paragraph. A portion of this work was supported by an American Foundation for AIDS Research Mathilde Krim Fellowship in Basic Biomedical Research (L.K.). [1]: /