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Le potentiel thérapeutique des anticorps monoclonaux ne cesse de croître avec d’indiscutables succès. Mais le bénéfice clinique de leur utilisation peut être compromis par l’induction d’anticorps contre ces anticorps, réalisant une réponse de type ADA (anti-drug antibodies) bien connue pour d’autres agents médicamenteux. Si la conception d’anticorps chimères contenant des séquences humaines, humanisés voire totalement humains, a substantiellement réduit leur immunogénicité, ce risque ne pourra pas être entièrement éradiqué. Nous discutons dans cette revue les aspects spécifiques aux réactions induites par l’administration d’Acm, qu’ils soient inhérents au produit thérapeutique ou au patient ; nous évoquerons aussi le programme de gestion des risques mis en place par les instances réglementaires et notamment l’European Medicines Agency(EMEA), et dont le rôle est d’évaluer, chez le patient, mais aussi en amont lors des étapes de recherche et développement précédant la mise sur le marché du produit (démarche prédictive), non seulement la probabilité d’une réponse immune dirigée contre cette protéine mais aussi son intensité. Cela implique le développement de méthodes in silico, de tests in vitrodétectant l’activation T et finalement l’analyse fine des ADA à la recherche de l’épitope immunogène.

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 vitromethods using blood cells from human donors. As a novel development, in silicomethods 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 vitroassays remain important, but guided by in silicodata they can focus on selected peptides and human leukocyte antigen haplotypes, thereby significantly reducing time and cost.

ABSTRACTIt 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.

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. Proteins 2002;48:31–43. © 2002 Wiley‐Liss, Inc.

The identification of specific amino acid residues involved in protein-protein interaction is fundamental to understanding structure-function relationships. Supported by mathematical calculations, we designed a high-density mutagenesis procedure for the generation of a mutant library of which a limited number of random clones would suffice to exactly localize amino acid residues essential for a particular protein-protein interaction. This goal was achieved experimentally by consecutive cycles of DNA shuffling, under error prone conditions, each followed by exposure of the target protein on the surface of phages to screen and select for correctly folded, functional mutants. To validate the procedure, human plasminogen activator inhibitor 1 (PAI-1) was chosen, because its 3D structure is known, many experimental tools are available and it may serve as a model protein for structure-function studies of serine proteinases and their inhibitors (serpins). After five cycles of DNA shuffling and selection for t-PA binding, analysis of 27 randomly picked clones revealed that PAI-1 mutants contained an average of 9.1 amino acid substitutions distributed over 114 different positions, which were preferentially located at the surface of the protein. This limited collection of mutant PAI-1 preparations contained multiple mutants defective in binding to three out of four tested anti-PAI-1 monoclonal antibodies. Alignment of the nucleotide sequence of defective clones permitted assignment of single dominant amino acid residues for binding to each monoclonal antibody. The importance of these residues was confirmed by testing the properties of single point mutants. From the position of these amino acid residues in the 3D structure of PAI-1 and the effects of the corresponding monoclonal antibodies on t-PA-PAI-1 interaction, conclusions can be drawn with respect to this serpin-serine proteinase interaction.

Recombinant staphylokinase (SakSTAR) variants obtained by site-directed substitution with cysteine, in the core (lysine 96 [Lys96], Lys102, Lys109, and/or Lys135) or the NH2-terminal region that is released during activation of SakSTAR (serine 2 [Ser2] and/or Ser3), were derivatized with thiol-specific (ortho-pyridyl-disulfide or maleimide) polyethylene glycol (PEG) molecules with molecular weights of 5000 (P5), 10 000 (P10), or 20 000 (P20). The specific activities and thrombolytic potencies in human plasma were unaltered for most variants derivatized with PEG (PEGylates), but maleimide PEG derivatives had a better temperature stability profile. In hamsters, SakSTAR was cleared at 2.2 mL/min; variants with 1 P5 molecule were cleared 2-to 5-fold; variants with 2 P5 or 1 P10 molecules were cleared 10-to 30-fold; and variants with 1 P20 molecule were cleared 35-fold slower. A bolus injection induced dose-related lysis of a plasma clot, fibrin labeled with 125 iodine (125I-fibrin plasma clot), and injected into the jugular vein. A 50% clot lysis at 90 minutes required 110 μg/kg SakSTAR; 50 to 110 μg/kg of core-substitution derivatives with 1 P5; 25 μg/kg for NH2-terminal derivatives with 1 P5; 5 to 25 μg/kg with derivatives with 2 P5 or 1 P10; and 7 μg/kg with P20 derivatives. Core substitution with 1 or 2 P5 molecules did not significantly reduce the immunogenicity of SakSTAR in rabbits. Derivatization of staphylokinase with a single PEG molecule allows controllable reduction of the clearance while maintaining thrombolytic potency at a reduced dose. This indicates that mono-PEGylated staphylokinase variants may be used for single intravenous bolus injection.

Recombinant staphylokinase (SakSTAR) variants obtained by site-directed substitution with cysteine, in the core (lysine 96 [Lys96], Lys102, Lys109, and/or Lys135) or the NH2-terminal region that is released during activation of SakSTAR (serine 2 [Ser2] and/or Ser3), were derivatized with thiol-specific (ortho-pyridyl-disulfide or maleimide) polyethylene glycol (PEG) molecules with molecular weights of 5000 (P5), 10 000 (P10), or 20 000 (P20). The specific activities and thrombolytic potencies in human plasma were unaltered for most variants derivatized with PEG (PEGylates), but maleimide PEG derivatives had a better temperature stability profile. In hamsters, SakSTAR was cleared at 2.2 mL/min; variants with 1 P5 molecule were cleared 2-to 5-fold; variants with 2 P5 or 1 P10 molecules were cleared 10-to 30-fold; and variants with 1 P20 molecule were cleared 35-fold slower. A bolus injection induced dose-related lysis of a plasma clot, fibrin labeled with 125 iodine (125I-fibrin plasma clot), and injected into the jugular vein. A 50% clot lysis at 90 minutes required 110 μg/kg SakSTAR; 50 to 110 μg/kg of core-substitution derivatives with 1 P5; 25 μg/kg for NH2-terminal derivatives with 1 P5; 5 to 25 μg/kg with derivatives with 2 P5 or 1 P10; and 7 μg/kg with P20 derivatives. Core substitution with 1 or 2 P5 molecules did not significantly reduce the immunogenicity of SakSTAR in rabbits. Derivatization of staphylokinase with a single PEG molecule allows controllable reduction of the clearance while maintaining thrombolytic potency at a reduced dose. This indicates that mono-PEGylated staphylokinase variants may be used for single intravenous bolus injection.

We have engineered recombinant glucose isomerase (GI) from Actinoplanes missouriensis by site-directed mutagenesis to enhance its thermal stability in both the soluble and immobilized forms. Substitution of arginine for lysine at position 253, which lies at the dimer/dimer interface of the GI tetramer, produced the largest stabilization under model industrial conditions. We discuss our results in terms of a model in which chemical glycation of lysines by sugars in the industrial corn syrup substrate represents a major pathway of destabilization.

Docking algorithms play an important role in the process of rational drug design and in understanding the mechanism of molecular recognition. An important determinant for successful docking is the extent to which the configurational space (including conformational changes) of the li‐gand/receptor system is searched. Here we describe a new, combinatorial method for flexible docking of peptides to proteins that allows full rotation around all single bonds of the peptide ligand and around those of a large set of receptor side chains. We have simulated the binding of several viral peptides to murine major histocompatibility complex class I H‐2Kb. In addition, we have explored the limits of our method by simulating a complex between calmodulin and an 18‐residue long helical peptide from calmodulin‐dependent protein kinase IIα. The calculated peptide conformations generally matched well with the X‐ray structures. Essential information about local flexibility and about residues that are responsible for strong binding was obtained. We have frequently observed considerable side‐chain flexibility during the simulations, showing the need for a flexible treatment of the receptor. Our method may also be useful whenever the receptor side‐chain conformation is not available or uncertain, as illustrated by the docking of an H‐2Kbbinding nonapeptide to the receptor structure taken from an octapeptide/H‐2Kbcomplex.—Desmet, J., Wilson, I. A., Joniau, M., De Maeyer, M., Lasters, I. Computation of the binding of fully flexible peptides to proteins with flexible side chains. FASEB J11, 164‐172 (1997)

The dead-end elimination algorithm has proven to be a powerful tool in protein homology modeling since it allows one to determine rapidly the global minimum-energy conformation (GMEC) of an arbitrarily large collection of side chains, given fixed backbone coordinates. After introducing briefly the necessary background, we focus on logic arguments that increase the efficacy of the dead-end elimination process. Second, we present new theoretical considerations on the use of the dead-end elimination method as a tool to identify sequences that are compatible with a given scaffold structure. Third, we initiate a search for properties derived from the computed GMEC structure to predict whether a given sequence can be well packed in the core of a protein. Three properties will be considered: the nonbonded energy, the accessible surface area, and the extent by which the GMEC side-chain conformations deviate from a locally optimal conformation.

Four distinct areas of primary sequence conservation between known tumour necrosis factor and lymphotoxin polypeptides from various species can be recognized. When these amino acid sequences are highlighted in the three-dimensional structure, all are found in the same region, constituting the framework of the trimeric structure.

Using the tertiary structure of the globular domain of H5 (GH5) and based on an alternative sequence homology between GH5 and DNA-binding proteins containing the helix-turn-helix motif, a model for H5-DNA interaction is proposed. From molecular graphics it follows that helix II recognizes the major groove of the DNA, as does the second helix of the helix-turn-helix motif, while helix III makes minor groove contacts, in agreement with the hypothesis of Turnell et al. (FEBS letters 232, 263–268). In the resulting model GH5 makes contact with a full turn of DNA.

Four distinct areas of primary sequence conservation between known tumour necrosis factor and lymphotoxin polypeptides from various species can be recognized. When these amino acid sequences are highlighted in the three‐dimensional structure, all are found in the same region, constituting the framework of the trimeric structure.

Since most antibodies directed against protein antigens recognize epitopes composed of several discontinuous segments of the polypeptide chain, attempts to delineate the amino acids constituting these epitopes with the use of linear peptides have generally been unsuccessful. Here, a method is described based on error-prone PCR, phage display and negative selection, whereby amino acid residues constituting the functional epitope are identified in the context of the native protein. First a library of randomized antigen variants containing most single, double and triple amino acid mutants generated by single nucleotide substitutions is produced by error-prone PCR amplification of the DNA sequence encoding the protein antigen. The phage-displayed library is then negatively selected for epitope loss mutants by passing through an affinity matrix derivatized with a specific antibody and positively selected for retention of function.

The myoglobins of two trematodes, Paramphistomum epiclitumand Isoparorchis hypselobagri, were isolated to homogeneity. The native molecules are monomeric with Mr16,000-17,000 and pI 6.5-7.5. In each species, at least four different globin isoforms occur. Primary structure was determined at the protein level. The globin chains contain 147 amino acid residues. Although major determinants of the globin fold are conserved, characteristic substitutions are present. A Tyr residue occurs at the helical positions B10 and E7 (distal position). This is confirmed by NMR measurements (Zhang, W., Rashid, K. A., Haque, M., Siddiqi, A. H., Vinogradov, S. N., Moens, L. & La Mar, G. N. (1997) J. Biol. Chem.272, 3000-3006). A distal Tyr normally provokes oxidation of the iron atom and the inability to bind oxygen, whereas a Tyr-B10 is indicative for a high oxygen affinity. In contrast, trematode myoglobins are functional molecules with a high oxygen affinity. Molecular modeling predicts two possible positions for the aromatic ring of Tyr-E7: one being outside the heme pocket making it freely accessible to the ligand and one within the heme pocket potentially able to form a second hydrogen bond with the iron-bound oxygen. A hydrogen bond between Tyr-B10 and the bound oxygen as in the Ascarishemoglobin is predicted as well. The predicted structure may explain the high oxygen affinity of the trematode myoglobins.

Summary: 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. Availability: SABmark is available from http://bioinformatics.vub.ac.be Contact: ivwalle@vub.ac.be

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

Several authors, including ourselves, have reported the existence of chroma-tosomes with DNA size larger than 166 bp in bird erythrocyte chromatin. Itwas tempting to correlate this increased DNA size with the presence of histoneH5. In order to substantiate this hypothesis, we performed a micrococcal nuclease digestion kinetic on : chicken erythrocyte chromatin, either native, selectively depleted from H1, or from H1 and H5; and rat liver chromatin, either native or partially H1 depleted. The comparative analysis of the lengths of DNA in the chromatosome size region led to the following conclusions : denaturing gels clearly reveal a first discrete pause at 178 nucleotidesin H1 depleted chicken erythrocyte chromatin as well as in partially H1-depleted rat liver chromatin, before the material accumulates at thenext intermediate 166 nucleotide chromatosome pause. the generation of all discrete chromatosome+bands is critically dependenton low ionic strength conditions and low Ca++ concentrations during the digestion, suggesting it may result from the protection of DNA cleavage sitesby histone H5 or H1, C or N terminal domains.

The spatial organization of nucleosomes and linker DNA in dinudeosomes and oligonucleosomes of various chain lengths has been investigated through electric dichroism, birefringence and relaxation times measurements at low ionic strengths (0.5 to 2.2 mM). From the negative dichroism observed for all the samples, it is concluded that the nucleosome subunits in the oligonucleosome chain must lie with their disc planes closely parallel to the fibre axis. The large increase of the negative dichroism of dinudeosomes upon Hl removal is interpreted by the unwinding of the DNA tails and internucleosomal segment. All the samples displayed, under bipolar pulses, a predominantly induced orientation mechanism.