Expression patterns of ERVWE1/Syncytin-1 and other placentally expressed human endogenous retroviruses along the malignant transformation process of hydatidiform moles.

INTRODUCTION: Up to 20% of hydatidiform moles are followed by malignant transformation in gestational trophoblastic neoplasia and require chemotherapy. Syncytin-1 is involved in human placental morphogenesis and is also expressed in various cancers. We assessed the predictive value of the expression of Syncytin-1 and its interactants in the malignant transformation process of hydatidiform moles. METHODS: Syncytin-1 glycoprotein was localized by immunohistochemistry in hydatidiform moles, gestational trophoblastic neoplasia and control placentas. The transcription levels of its locus ERVWE1, its interaction partners (hASCT1, hASCT2, TLR4 and DC-SIGN) and two loci (ERVFRDE1 and ERV3) involved the expression of other placental envelopes were assessed by real-time PCR. RESULTS: Syncytin-1 glycoprotein was expressed in syncytiotrophoblast of hydatidiform moles with an apical enhancement when compared with normal placentas. Moles with further malignant transformation had a higher staining intensity of Syncytin-1 surface unit C-terminus but the transcription level of its locus ERVWE1 was not different from that of moles with further remission and normal placentas. hASCT1 and TLR4, showed lower transcription levels in complete moles when compared to normal placentas. ERVWE1, ERVFRDE1 and ERV3 transcription was down-regulated in hydatidiform moles and gestational trophoblastic neoplasia. CONCLUSIONS: Variations of Syncytin-1 protein localization and down-regulation of hASCT1 and TLR4 transcription are likely to reflect altered functions of Syncytin-1 in the premalignant context of complete moles. The reduced transcription in gestational trophoblastic diseases of ERVWE1, ERVFRDE1 and ERV3, which expression during normal pregnancy is differentially regulated by promoter region methylation, suggest a joint dysregulation mechanism in malignant context.

Crystal structure of human prostate-specific antigen in a sandwich antibody complex.

Human prostate-specific antigen (PSA or human kallikrein-related peptidase 3) present in small quantities in the sera of healthy men becomes elevated in prostate cancer (PCa) and other prostate disorders. The ability to identify the free PSA fraction associated with PCa could increase the reliability of the PSA diagnostic test. Here we present the crystal structure of human PSA from seminal fluid in a sandwich complex with two monoclonal antibodies (mAbs). MAb 5D5A5 captures total PSA with exceptionally high affinity, and mAb 5D3D11 selectively discriminates between free PSA subforms that are more abundant in sera from patients with PCa. Although the antigen is not of seric origin, several insights into cancer diagnosis can be discerned from this complex. MAb 5D3D11 recognizes a PSA conformation different from that previously reported. Interacting with the kallikrein loop, the PSA N-linked glycan attached to asparagine 61 is an uncommonly complex sialated triantennary chain. O-linked glycosylation is observed at threonine 125. The description of how PSA subforms in prostatic fluid can be discriminated using pairs of antibodies is a first step in the design of new strategies that are capable of real discrimination among PSA subforms, which will lead to the formulation of more reliable diagnostic tests. In a companion article [Muller, B. H., Savatier, A., L'Hostis, G., Costa, N., Bossus, M., Michel, S., et al. (2011). In vitro affinity maturation of an anti-PSA antibody for prostate cancer diagnostic assay. J. Mol. Biol.], we describe engineering efforts to improve the affinity of mAb 5D3D11, a first step towards such goal.

In vitro affinity maturation of an anti-PSA antibody for prostate cancer diagnostic assay.

Prostate-specific antigen (PSA) is a serum marker that is widely used for the diagnosis of prostatic diseases. Various subforms of free PSA, which are associated with prostate cancer differently, have been identified in sera. Thus, specific detection of certain subforms could permit discrimination between benign and malignant cases. Although the monoclonal antibody 5D3D11 displays the desired selectivity, its relative weak binding affinity prevents its development into an effective diagnostic tool. The directed-evolution strategy presented here succeeds in enhancing affinity and immunoassay sensitivity while maintaining selectivity. Starting without structural data, we constructed four independent phage-display single-chain variable fragment (scFv) libraries targeting hot spots from CDR-L1, H1, H2, and H3. Mutations derived from each library were combined, yielding further affinity gains. This constitutes the first demonstration of additivity for independently selected complementarity-determining region (CDR) hot-spot mutations. The X-ray structure of the Fab' 5D3D11-PSA complex (after it became available) inspired the design of two new libraries targeting CDR-L3 that resulted in other higher-affinity variants. Attempts at combining the new variants with previous ones did not result in further gains, suggesting that mutations from the two strategies provide alternative but noncomplementary solutions for affinity enhancement of 5D3D11. The results can be interpreted to provide a plausible explanation for the observed lack of additivity. Finally, with respect to the wild-type scFv, the best binders show an enhancement of sensitivity in sandwich immunoassay. Its ability to discriminate between prostate cancer sera and benign prostatic hyperplasia sera has now been confirmed through the dosage of 63 patients.

Fab'-induced folding of antigenic N-terminal peptides from intrinsically disordered HIV-1 Tat revealed by X-ray crystallography.

Tat, the transcriptional activator protein of human immunodeficiency virus type 1 (HIV-1), is critical for viral replication and is a potential HIV-1 vaccine candidate. This intrinsically disordered protein is present in the extracellular medium and is involved in the pathogenicity of HIV through its interaction with different cellular and viral biological partners. A monoclonal antibody termed 11H6H1, which is specific for the N-terminal region of Tat, was selected for a functional and structural study of the HIV-1 Tat protein. The equilibrium dissociation constants (K(d)) of Tat and Tat fragments complexed with 11H6H1 were estimated by competitive ELISA. Tat contains a single tryptophan residue, Trp11, located in the N-terminal region. We show that the substitution of Trp11 by a phenylalanine completely abolishes the binding of 11H6H1, whereas the transactivating activity of Tat is preserved. The epitope recognized by 11H6H1 was restricted to the 9-mer peptide P(6)KLEPWKHP(14) centered on Trp11. The crystal structures of this 9-mer peptide and of an overlapping 15-mer peptide were determined in complex with Fab' 11H6H1 at 2.4 Å and 2.1 Å resolution, respectively. Tat is intrinsically disordered and can undergo induced folding upon association with a biological partner. Our crystallographic study reveals that the two Tat peptides, which are lodged in the U-shaped groove of the Fab' antigen-binding site, adopt a standard type I ß-turn conformation. The central Trp11 that is critical for Fab' recognition is further stabilized by π-stacking interactions. The structural and biological consequences of this induced folding in HIV pathogenesis are discussed.

Crystal structure of a ternary complex between human prostate-specific antigen, its substrate acyl intermediate and an activating antibody.

Human prostate-specific antigen (PSA or KLK3) is an important marker for the diagnosis and management of prostate cancer. This is an androgen-regulated glycoprotein of the kallikrein-related protease family secreted by prostatic epithelial cells. Its physiological function is to cleave semenogelins in the seminal coagulum and its enzymatic activity is strongly modulated by zinc ions. Here we present the first crystal structure of human PSA in complex with monoclonal antibody (mAb) 8G8F5 that enhances its enzymatic activity. The mAb recognizes an epitope composed of five discontinuous segments including residues from the kallikrein loop and stabilizes PSA in an "open and active conformation" that accelerates catalysis. We also present the crystal structure of PSA in complex with both the mAb 8G8F5 and a fluorogenic substrate Mu-KGISSQY-AFC, derived from semenogelin I. By exploiting the inhibition of PSA by zinc ions, we were able to obtain a substrate acyl intermediate covalently linked to the catalytic serine, at pH 7.3 but not at pH 5.5. Moreover, the inhibition of PSA activity by zinc was found to be modulated by pH variations but not by the antibody binding. The correlation of the different data with the physiological conditions under which PSA can cleave semenogelins is discussed.

[Directed molecular evolution of antibodies]. / Evolution moléculaire dirigée d'anticorps.

Antibodies play a key role in the immune system, are characterized by a homogeneous overall structure and by their ability to interact with an almost unlimited number of compounds. Encoded by a fixed number of genes, they acquire their specificity and affinity of recognition after a succession of genetic recombination and molecular mutation processes. Since the pioneer works of Kohler and Milstein in 1975 describing the possibility of producing monoclonal antibodies with pre-determined specificity, the use of antibodies in the fields of research, diagnosis and therapy has never stopped increasing. Thus, about twenty monoclonal antibodies have yet been authorized to be used in human immunotherapy. However, a majority of these molecules have been engineered to bring them into line with their clinical use: chimerization, humanization, recombinant expression of single or fused fragments. Furthermore, the recent development of in vitro molecular evolution approaches now make it possible to engineer the affinity, the specificity as well as the stability of monoclonal antibodies. The potential of in vitro molecular evolution of antibodies will be illustrated through the example of the specificity improvement of an anti-progesterone antibody.

Crystal structure of the complex between the monomeric form of Toxoplasma gondii surface antigen 1 (SAG1) and a monoclonal antibody that mimics the human immune response.

Toxoplasma gondii, the intracellular parasite responsible for toxoplasmosis infects more than one-third of the world population and can be life-threatening for fetuses and immunocompromised patients. The surface protein SAG1 is an important immune target, which provides a strong immune response against the invasive tachyzoite while the other forms of the parasite, devoid of SAG1 at their surface, are multiplying. In addition to this role as a "hot spot" decoy, SAG1 is predicted to act as an adhesin during host-cell attachment through its binding to proteoglycans. To begin to understand the relationships between SAG1 epitopes and the ligand-binding site, we have solved the crystal structure of the monomeric form of T.gondii SAG1 complexed to a Fab derived from a monoclonal antibody raised against tachyzoite particles. This antibody competes strongly with human Toxoplasma-specific sera, suggesting that its epitope is part of an immunodominant region present on the surface of SAG1. The structure reveals that this conformational epitope, located within the SAG1 N-terminal domain, does not overlap with the proposed ligand-binding pocket. This study provides the first structural description of the monomeric form of SAG1, and significant insights into its dual role of adhesin and immune target during parasite infection.

Fine tuning of the specificity of an anti-progesterone antibody by first and second sphere residue engineering.

The specificity of anti-progesterone P15G12C12G11 antibody was improved by combination of in vitro scanning saturation mutagenesis and error-prone PCR. The most evolved mutant is able to discriminate against 5beta- or 5alpha-dihydroprogesterone, 23 and 15 times better than the starting antibody, while maintaining the affinity for progesterone that remains in the picomolar range. The high level of homology with anti-progesterone monoclonal antibody DB3 allowed the construction of three-dimensional models of P15G12C12G11 based on the structures of DB3 in complex with various steroids. These models together with binding data, derived from site-directed mutagenesis, were used to build a phage library in which five first sphere positions in complementarity-determining regions 2H and 3L were varied. Variants selected by an initial screening in competition against a large excess of 5beta- or 5alpha-dihydroprogesterone were characterized by a convergent amino acid signature different from that of the wild-type antibody and had lower cross-reactivity. Binding properties of this first set of mutants were further improved by the addition of second sphere mutations selected independently from an error-prone library. The three-dimensional models of the best variant show changes in the antigen binding site that explain well the increase in selectivity. The improvements are partly linked to a change in the canonical class of the light chain third hypervariable loop.

Crystal structure of a hydrophobic immunodominant antigenic site on hepatitis C virus core protein complexed to monoclonal antibody 19D9D6.

The first crystal structure of a complex between a hepatitis C virus (HCV) core protein-derived peptide (residues 13-40) and the Ab fragment of a murine mAb (19D9D6) has been solved, allowing determination of the recognized epitope and elucidation of its conformation. This Ab, raised against the first 120 residues of the core protein, recognizes core particles and strongly competes with anticore human Abs, suggesting that it is highly representative of the human anti-HCV core response. Its epitope lies within the first 45 aa of the protein, the major antigenic segment of core recognized both by murine and human Abs. Surprisingly, the recognized epitope (29-37: QIVGGVYLL) has an unusual preponderance of hydrophobic residues, some of which are buried in a small hydrophobic core in the nuclear magnetic resonance structure of the peptide (2-45) in solution, suggesting that the Ab may induce a structural rearrangement upon recognition. The flexibility may reside entirely within the Ag, since the Fab'-peptide complex structure at 2.34 A shows that the Ab binding site is hardly perturbed by complexation. Given that the recognized residues are unlikely to be solvent exposed, we are left with the interesting possibility that Ab-core interactions may take place in a nonaqueous environment.

Lipopeptide-based melanoma cancer vaccine induced a strong MART-27-35-cytotoxic T lymphocyte response in a preclinal study.

Identification of tumor antigens and their optimal antigenic peptides raised hopes for the development of peptide-based immunotherapeutic vaccine strategies for human melanoma, however. Synthetic peptides alone are not immunogenic enough, and adequate formulation is critical for elaboration of peptide vaccines. To improve formulation, we evaluated 2 lipopeptide constructs, both including HLA-A2-restricted MART 27-35-CD8+ T lymphocyte (CTL) epitope covalently linked to universal tetanus toxoid (TT) 830-843 helper T lymphocyte (HTL) epitope, in HLA-A2 transgenic mouse models that mimic human CTL responses in vivo. These 2 constructs only differed in the formulation of their lipid tail. We showed that lipopeptide constructs were strongly recognized, in vitro, by human MART 27-35 cytotoxic T cells derived from tumor-infiltrating lymphocytes. The transgenic Mice immunized with these 2 MART lipopeptide formulations containing covalently linked HTL-CTL epitopes induced strong MART 27-35 cytotoxic T cells. This CTL induction was critically dependent on the presence of the helper T lymphocyte epitope. These results also showed that a single palmitoyl-lysine chain is enough to assure immunogenicity of a given peptide and that the presence of a lipid tail bypass the need for adjuvant. These results support the selection of MART-lipopeptide melanoma vaccine for evaluation in a clinical trial.

Immunogenicity and antigenicity of the N-term repeat amino acid sequence of the Plasmodium falciparum P126 antigen

The P126 protein, a parasitosphorus vacuole antigen of Plasmodium falciparum has beenshoen to induce protective immunity in Saimiri and Aotus monkeys. In the present work we investigated its immunogenicity. Our results suggest that the N-term of P126 is poorly immunogenic and antibody response against the P126 could be under a MHC restricted control in C57BL/6(H-2b) mice, which could be problematic in ternms of a use of the P126 in a vaccine program. However, we observed that a synthetic peptide, copying the 6 octapeptide repeat corresponding to the N-term of the P126, induces an antibody response to the native molecule in C57BL/6 non-responder mice. Moreover, the vaccine-P126 recombinant induced anmtibodies against the N-term of the molecule in rabbits while the unprocessed P126 did not