Cell-penetrating Alphabody protein scaffolds for intracellular drug targeting.

The therapeutic scope of antibody and nonantibody protein scaffolds is still prohibitively limited against intracellular drug targets. Here, we demonstrate that the Alphabody scaffold can be engineered into a cell-penetrating protein antagonist against induced myeloid leukemia cell differentiation protein MCL-1, an intracellular target in cancer, by grafting the critical B-cell lymphoma 2 homology 3 helix of MCL-1 onto the Alphabody and tagging the scaffold's termini with designed cell-penetration polypeptides. Introduction of an albumin-binding moiety extended the serum half-life of the engineered Alphabody to therapeutically relevant levels, and administration thereof in mouse tumor xenografts based on myeloma cell lines reduced tumor burden. Crystal structures of such a designed Alphabody in complex with MCL-1 and serum albumin provided the structural blueprint of the applied design principles. Collectively, we provide proof of concept for the use of Alphabodies against intracellular disease mediators, which, to date, have remained in the realm of small-molecule therapeutics.

Reviving old protecting group chemistry for site-selective peptide-protein conjugation.

Methodologies to conjugate proteins to property-enhancing entities are highly sought after. We report a remarkably simple strategy for conjugating proteins bearing accessible cysteines to unprotected peptides containing a Cys(Scm) protecting group, which is introduced on-resin via a Cys(Acm) building block. The peptides employed for this proof of principle study are highly varied and structurally diverse, and undergo multiple on-resin decoration steps prior to conjugation. The methodology was applied to three different proteins, and proved to be efficient and site-selective. This twist on protecting group chemistry has led to a novel and generally applicable strategy for crossed-disulfide formation between proteins and peptides.

Structural basis of IL-23 antagonism by an Alphabody protein scaffold.

Protein scaffolds can provide a promising alternative to antibodies for various biomedical and biotechnological applications, including therapeutics. Here we describe the design and development of the Alphabody, a protein scaffold featuring a single-chain antiparallel triple-helix coiled-coil fold. We report affinity-matured Alphabodies with favourable physicochemical properties that can specifically neutralize human interleukin (IL)-23, a pivotal therapeutic target in autoimmune inflammatory diseases such as psoriasis and multiple sclerosis. The crystal structure of human IL-23 in complex with an affinity-matured Alphabody reveals how the variable interhelical groove of the scaffold uniquely targets a large epitope on the p19 subunit of IL-23 to harness fully the hydrophobic and hydrogen-bonding potential of tryptophan and tyrosine residues contributed by p19 and the Alphabody, respectively. Thus, Alphabodies are suitable for targeting protein-protein interfaces of therapeutic importance and can be tailored to interrogate desired design and binding-mode principles via efficient selection and affinity-maturation strategies.

Quantitative analysis of the interaction strength and dynamics of human IgG4 half molecules by native mass spectrometry.

Native mass spectrometry (MS) is a powerful technique for studying noncovalent protein-protein interactions. Here, native MS was employed to examine the noncovalent interactions involved in homodimerization of antibody half molecules (HL) in hinge-deleted human IgG4 (IgG4Δhinge). By analyzing the concentration dependence of the relative distribution of monomer HL and dimer (HL)(2) species, the apparent dissociation constant (K(D)) for this interaction was determined. In combination with site-directed mutagenesis, the relative contributions of residues at the CH3-CH3 interface to this interaction could be characterized and corresponding K(D) values quantified over a range of 10(-10)-10(-4) M. The critical importance of this noncovalent interaction in maintaining the intact dimeric structure was also proven for the full-length IgG4 backbone. Using time-resolved MS, the kinetics of the interaction could be measured, reflecting the dynamics of IgG4 HL exchange. Hence, native MS has provided a quantitative view of local structural features that define biological properties of IgG4.

Species-specific determinants in the IgG CH3 domain enable Fab-arm exchange by affecting the noncovalent CH3-CH3 interaction strength.

A distinctive feature of human IgG4 is its ability to recombine half molecules (H chain and attached L chain) through a dynamic process termed Fab-arm exchange, which results in bispecific Abs. It is becoming evident that the process of Fab-arm exchange is conserved in several mammalian species, and thereby represents a mechanism that impacts humoral immunity more generally than previously thought. In humans, Fab-arm exchange has been attributed to the IgG4 core-hinge sequence (226-CPSCP-230) in combination with unknown determinants in the third constant H chain domain (CH3). In this study, we investigated the role of the CH3 domain in the mechanism of Fab-arm exchange, and thus identified amino acid position 409 as the critical CH3 determinant in human IgG, with R409 resulting in exchange and K409 resulting in stable IgG. Interestingly, studies with IgG from various species showed that Fab-arm exchange could not be assigned to a common CH3 domain amino acid motif. Accordingly, in rhesus monkeys (Macaca mulatta), aa 405 was identified as the CH3 determinant responsible (in combination with 226-CPACP-230). Using native mass spectrometry, we demonstrated that the ability to exchange Fab-arms correlated with the CH3-CH3 dissociation constant. Species-specific adaptations in the CH3 domain thus enable Fab-arm exchange by affecting the inter-CH3 domain interaction strength. The redistribution of Ag-binding domains between molecules may constitute a general immunological and evolutionary advantage. The current insights impact our view of humoral immunity and should furthermore be considered in the design and evaluation of Ab-based studies and therapeutics.

[Immunogenicity of therapeutic antibodies]. / Immunogénicité de protéines d'intérêt thérapeutique : les anticorps monoclonaux thérapeutiques.

Unwanted immunogenicity, i.e., the developpement by patients of anti-drug antibodies is a significant problem with biologicals therapeutic reagents and can compromise clinical response. Over 20 antibodies currently on the market and over 100 drug candidates are currently in clinical trials; all therapeutic antibodies are showing some level of immunogenicity, and although it has been reduced with the advent of antibodies including human sequences, or even humanised antibodies, this concern will not be totally eradicated. Whereas the actual anti-drug response can only be addressed during clinical development or post-marketing, the industry and the regulatory instances are facing a challenge to develop accurate procedures for the assessment of immunogenicity related to antibody therapeutics, addressing both the likelihood and the severity of the drug-related immunogenicity. This review will discuss the multiple factors that can contribute to a potential immunogenicity of protein therapeutics patient/disease related, as well as related to the drug itself, and the strategies to identify anti-drug antibodies, both in clinical and non clinical assays.

Strategies for preclinical immunogenicity assessment of protein therapeutics.

The immunogenicity assessment of protein therapeutics has received significant attention, both from regulatory bodies and industry. With the advent of several industry white papers and the EMEA guidance on the clinical assessment of antidrug antibodies (ADAs), the immunogenicity screening framework has become better defined. Immunogenicity in many cases leads to the loss of efficacy of a drug, but can also lead to the production of severe adverse effects. A risk assessment should be conducted for every biotherapeutic under development to address the incidence rate of immunogenicity, as well as the severity of the adverse effects caused. Typically, immunogenicity is characterized by measuring the ADAs that occur in individuals exposed to a drug. Industry and academic research efforts are geared toward the assessment of potential immunogenicity in a preclinical setting, with the aim of predicting immunogenicity prior to clinical trials, and thereby reducing the attrition rate of drugs in clinical development. This article addresses the current status, strategies and challenges related to the predictive immunogenicity assessment of protein therapeutics.

Heterosubtypic neutralizing monoclonal antibodies cross-protective against H5N1 and H1N1 recovered from human IgM+ memory B cells.

BACKGROUND: The hemagglutinin (HA) glycoprotein is the principal target of protective humoral immune responses to influenza virus infections but such antibody responses only provide efficient protection against a narrow spectrum of HA antigenic variants within a given virus subtype. Avian influenza viruses such as H5N1 are currently panzootic and pose a pandemic threat. These viruses are antigenically diverse and protective strategies need to cross protect against diverse viral clades. Furthermore, there are 16 different HA subtypes and no certainty the next pandemic will be caused by an H5 subtype, thus it is important to develop prophylactic and therapeutic interventions that provide heterosubtypic protection. METHODS AND FINDINGS: Here we describe a panel of 13 monoclonal antibodies (mAbs) recovered from combinatorial display libraries that were constructed from human IgM(+) memory B cells of recent (seasonal) influenza vaccinees. The mAbs have broad heterosubtypic neutralizing activity against antigenically diverse H1, H2, H5, H6, H8 and H9 influenza subtypes. Restriction to variable heavy chain gene IGHV1-69 in the high affinity mAb panel was associated with binding to a conserved hydrophobic pocket in the stem domain of HA. The most potent antibody (CR6261) was protective in mice when given before and after lethal H5N1 or H1N1 challenge. CONCLUSIONS: The human monoclonal CR6261 described in this study could be developed for use as a broad spectrum agent for prophylaxis or treatment of human or avian influenza infections without prior strain characterization. Moreover, the CR6261 epitope could be applied in targeted vaccine strategies or in the design of novel antivirals. Finally our approach of screening the IgM(+) memory repertoire could be applied to identify conserved and functionally relevant targets on other rapidly evolving pathogens.

TNF: a moonlighting protein at the interface between cancer and infection.

The remarkable ability of TNF, especially in combination with Interferon-gamma or melphalan, to inhibit the growth of malignant tumor cells is so far unmatched. Unfortunately, its high systemic toxicity and hepatotoxicity prevent its systemic use in cancer patients. An elegant manner to circumvent this problem is the isolated limb and liver perfusion for the treatment of melanoma, soft tissue sarcoma and liver tumors, respectively, although the latter method can lead to a reversible hepatotoxicity. In order to allow also the treatment of other cancers with TNF, new strategies have to be developed that aim at sensitizing tumor cells to TNF and at reducing its systemic and liver toxicity, without losing its antitumor efficiency. Moreover, the lectin-like domain of TNF, which is spatially distinct from the receptor binding sites, could be useful in reducing cancer treatment-related pulmonary edema formation. This review will discuss some recent developments in these areas, which can lead to a renewed interest in TNF for the systemic treatment of cancer.

Non-immunized natural human heavy chain CDR3 repertoires allow the isolation of high affinity peptides mimicking a human influenza hemagglutinin epitope.

In this study we constructed two phage libraries displaying non-immunized natural human IgM derived HCDR3 repertoires. One library was structurally constrained by a Gly to Cys substitution at position 104 enabling the formation of a disulfide bridge with the Cys at position 92. Panning of these libraries on an anti-human influenza hemagglutinin (HA) antibody resulted in the selection of 16 different HCDR3 loops displaying different degrees of sequence homology with the HA epitope. The specificity of the HCDR3 loops recovered from the structurally constrained library was confirmed by competition assays using the HA epitope. Only one of these HCDR3 peptides contained Cys104. Structural analysis of these sequences revealed that the loss of Cys104 was associated with an increased preference for the formation of the type I beta-turn required for high affinity binding to the antibody. Affinity studies confirmed that the HCDR3 peptides containing the sequence YDVPDY and Gly104 had affinities in the nanomolar range (K(d)=7.6 nM) comparable to the HA epitope. These findings provided evidence that the recovered HCDR3 sequences may bind to their target in a conformation that is unreachable by the parental antibody from which the HCDR3 was derived. Furthermore, the isolation of target-specific and high affinity binders demonstrates the value of HCDR3 libraries as a source of 'biologically randomized' sequences of human origin for the identification of peptidic lead molecules.

Immunogenicity screening in protein drug development.

Most therapeutic proteins in clinical trials or on the market are, to a variable extent, immunogenic. Formation of antidrug antibodies poses a risk that should be assessed during drug development, as it possibly compromises drug safety and alters pharmacokinetics. The generation of these antibodies is critically dependent on the presence of T helper cell epitopes, which have classically been identified by in vitro methods using blood cells from human donors. As a novel development, in silico methods that identify T cell epitopes have been coming on line. These methods are relatively inexpensive and allow the mapping of epitopes from virtually all human leukocyte antigen molecules derived from a wide genetic background. In vitro assays remain important, but guided by in silico data they can focus on selected peptides and human leukocyte antigen haplotypes, thereby significantly reducing time and cost.

SABmark--a benchmark for sequence alignment that covers the entire known fold space.

The Sequence Alignment Benchmark (SABmark) provides sets of multiple alignment problems derived from the SCOP classification. These sets, Twilight Zone and Superfamilies, both cover the entire known fold space using sequences with very low to low, and low to intermediate similarity, respectively. In addition, each set has an alternate version in which unalignable but apparently similar sequences are added to each problem.

Anchor profiles of HLA-specific peptides: analysis by a novel affinity scoring method and experimental validation.

The study of intermolecular interactions is a fundamental research subject in biology. Here we report on the development of a quantitative structure-based affinity scoring method for peptide-protein complexes, named PepScope. The method operates on the basis of a highly specific force field function (CHARMM) that is applied to all-atom structural representations of peptide-receptor complexes. Peptide side-chain contributions to total affinity are scored after detailed rotameric sampling followed by controlled energy refinement. A de novo approach to estimate dehydration energies was developed, based on the simulation of individual amino acids in a solvent box filled with explicit water molecules. Transferability of the method was demonstrated by its application to the hydrophobic HLA-A2 and -A24 receptors, the polar HLA-A1, and the sterically ruled HLA-B7 receptor. A combined theoretical and experimental study on 39 anchor substitutions in FxSKQYMTx/HLA-A2 and -A24 complexes indicated a prediction accuracy of about two thirds of a log-unit in Kd. Analysis of free energy contributions identified a great role of desolvation and conformational strain effects in establishing a given specificity profile. Interestingly, the method rightly predicted that most anchor profiles are less specific than so far assumed. This suggests that many potential T-cell epitopes could be missed with current prediction methods. The results presented in this work may therefore significantly affect T-cell epitope discovery programs applied in the field of peptide vaccine development.

Align-m--a new algorithm for multiple alignment of highly divergent sequences.

MOTIVATION: Multiple alignment of highly divergent sequences is a challenging problem for which available programs tend to show poor performance. Generally, this is due to a scoring function that does not describe biological reality accurately enough or a heuristic that cannot explore solution space efficiently enough. In this respect, we present a new program, Align-m, that uses a non-progressive local approach to guide a global alignment. RESULTS: Two large test sets were used that represent the entire SCOP classification and cover sequence similarities between 0 and 50% identity. Performance was compared with the publicly available algorithms ClustalW, T-Coffee and DiAlign. In general, Align-m has comparable or slightly higher accuracy in terms of correctly aligned residues, especially for distantly related sequences. Importantly, it aligns much fewer residues incorrectly, with average differences of over 15% compared with some of the other algorithms. AVAILABILITY: Align-m and the test sets are available at http://bioinformatics.vub.ac.be

Consistency matrices: quantified structure alignments for sets of related proteins.

Comparing two remotely similar structures is a difficult problem: more often than not, resulting structure alignments will show ambiguities and a unique answer usually does not even exist. In addition, alignments in general have a limited information content because every aligned residue is considered equally important. To solve these issues to a certain extent, one can take the perspective of a whole group of similar structures and then evaluate common structural features. Here, we describe a consistency approach that, although not actually performing a multiple structure alignment, does produce the information that one would conceivably want from such an experiment: the key structural features of the group, e.g., a fold, which in this case are projected onto either a pair of proteins or a single protein. Both representations are useful for a number of applications, ranging from the detection of (partially) wrong structure alignments to protein structure classification and fold recognition. To demonstrate some of these applications, the procedure was applied to 195 SCOP folds containing a total of 1802 domains sharing very low sequence similarity.

Comparison of predicted scaffold-compatible sequence variation in the triple-hairpin structure of human imunodeficiency virus type 1 gp41 with patient data.

It has been proposed that the ectodomain of human immunodeficiency virus type 1 (HIV-1) gp41 (e-gp41), involved in HIV entry into the target cell, exists in at least two conformations, a pre-hairpin intermediate and a fusion-active hairpin structure. To obtain more information on the structure-sequence relationship in e-gp41, we performed in silico a full single-amino-acid substitution analysis, resulting in a Fold Compatible Database (FCD) for each conformation. The FCD contains for each residue position in a given protein a list of values assessing the energetic compatibility (ECO) of each of the 20 natural amino acids at that position. Our results suggest that FCD predictions are in good agreement with the sequence variation observed for well-validated e-gp41 sequences. The data show that at a minECO threshold value of 5 kcal/mol, about 90% of the observed patient sequence variation is encompassed by the FCD predictions. Some inconsistent FCD predictions at N-helix positions packing against residues of the C helix suggest that packing of both peptides may involve some flexibility and may be attributed to an altered orientation of the C-helical domain versus the N-helical region. The permissiveness of sequence variation in the C helices is in agreement with FCD predictions. Comparison of N-core and triple-hairpin FCDs suggests that the N helices may impose more constraints on sequence variation than the C helices. Although the observed sequences of e-gp41 contain many multiple mutations, our method, which is based on single-point mutations, can predict the natural sequence variability of e-gp41 very well.

Fast and accurate side-chain topology and energy refinement (FASTER) as a new method for protein structure optimization.

We have developed an original method for global optimization of protein side-chain conformations, called the Fast and Accurate Side-Chain Topology and Energy Refinement (FASTER) method. The method operates by systematically overcoming local minima of increasing order. Comparison of the FASTER results with those of the dead-end elimination (DEE) algorithm showed that both methods produce nearly identical results, but the FASTER algorithm is 100-1000 times faster than the DEE method and scales in a stable and favorable way as a function of protein size. We also show that low-order local minima may be almost as accurate as the global minimum when evaluated against experimentally determined structures. In addition, the new algorithm provides significant information about the conformational flexibility of individual side-chains. We observed that strictly rigid side-chains are concentrated mainly in the core of the protein, whereas highly flexible side-chains are found almost exclusively among solvent-oriented residues.