Danièle Cantacuzene

Anti-Tumor Immunity Provided by a Synthetic Multiple Antigenic Glycopeptide Displaying a Tri-Tn Glycotope

In many cancer cells the alteration of glycosylation processes leads to the expression of cryptic carbohydrate moieties, which make them good targets for immune intervention. Identification of cancer-associated glycotopes as well as progress in chemical synthesis have opened up the way for the development of fully synthetic immunogens that can induce anti-saccharide immune responses. Here, we synthesized a dendrimeric multiple antigenic glycopeptide (MAG) containing the Tn Ag O-linked to a CD4+ T cell epitope. This MAG is based on three consecutive Tn moieties (tri-Tn) corresponding to the glycotope recognized by an mAb (MLS 128) produced against the LS180 colon carcinoma cell line. The Abs induced by this MAG recognized murine and human tumor cell lines expressing the Tn Ag. Prophylactic vaccination using MAG provided protection of mice against tumor challenge. When used in active specific immunotherapy, the MAG carrying the tri-Tn glycotope was much more efficient than the mono-Tn analogue in promoting the survival of tumor-bearing mice. Furthermore, in active specific immunotherapy, a linear glycopeptide carrying two copies of the tri-Tn glycotope was shown to be poorly efficient compared with the dendrimeric MAG. Therefore, both the clustering of carbohydrate Ags and the way they are displayed seem to be important parameters for stimulating efficient anti-saccharide immune responses.

Analysis of the fine specificity of Tn-binding proteins using synthetic glycopeptide epitopes and a biosensor based on surface plasmon resonance spectroscopy

Using synthetic Tn (GalNAc‐O‐Ser/Thr) glycopeptide models and a biosensor based on surface plasmon resonance spectroscopy we have determined that isolectin B4 from Vicia villosa (VVLB4) binds to one Tn determinant whereas the anti‐Tn monoclonal antibodies 83D4 and MLS128 require at least two Tn residues for recognition. When an unglycosylated amino acid is introduced between the Tn residues, both antibodies do not bind. MLS128 affinity was higher on a glycopeptide with three consecutive Tn residues. These results indicate that Tn residues organized in clusters are essential for the binding of these antibodies and indicate a different Tn recognition pattern for VVLB4.

Condensation of perfluoroalkyl iodides with unsaturated nitrogen compounds

Condensation of enamines with perfluoroalkyl iodides yields α-perfluoroalkyl ketones. An intermediate charge-transfer complex between an iminium iodide and the perfluoroalkyl iodide has been detected. The reaction has been extended to α-alkoxy-enamines, ynamines, and pyrroles. It is rapid and uncatalysed for many simple enamines; others require u.v. light.

Synthesis of amino acid esters by papain

Abstract A wide range of N-Boc-amino add esters were synthesized from N-Boc-amino acids and alcohol using papain as catalyst. Suitable biphasic reaction mixtures were found for most amino acids to achieve high yield of ester synthesis. With N-Boc-L-aspartic and glutamic adds only the a carboxyl group is esterified, without racemisation.

Enzymic synthesis of galactopyranosyl-l-serine derivatives using galactosidases

Although the physiological role of the carbohydrate moieties in biologically active glycoproteins is still a subject of investigation, it is known that sugars extend the biological half-life of these molecules. Furthermore, the attachment of a sugar residue to a peptide sequence could improve the affinity of the molecule for its receptor. For this reason, several glycosylated enkephalins have been synthesisedlm3. Chemical syntheses of glycopeptides are well developed”. However, because carbohydrates contain multiple hydroxyl groups of similar reactivity, the chemical methods involved in regioselective synthesis require numerous protection and deprotection steps. In addition, stereospecific reactions that give the desired (r or /I) anomer are often difficult. Many enzymes are now available commercially and their routine use in synthesis5 is becoming accepted. Most glycosidases can transfer the glycosyl moiety of a substrate to acceptors other than water and, although they are less selective than glycosyl transferases, they have been used in the synthesis of glycosides’.‘. For example, D-Dgalactosidase catalyses transgalactosidation with monosaccharides, oligosaccharides, alkanols8~‘0, and phenols”. The reactions of E. c&p-D-galactosidase in the presence of various acceptors have been investigated, as have the structural requirements and reactivity”. Alkyl galactosides, lactose, and raffinose can be used as glycosyl donors with various primary alcohols as acceptors for the synthesis of dior tri-saccharide glycosides”-“. We now report on the use of lactose and P-D-galactosidase for the formation of 3-O-/?-D-galactopyranosyl-L-serine @‘-Gal-Ser). p-D-Galactosidase does not induce the synthesis of /I-Gal-Ser from lactose and serine, and no condensation occurs with the N-urethane derivative of the amino acid or the amino acid ester. Both the amino and the carboxyl groups of the amino acid have to be protected for transgalactosidation to take place. The choice of the amino blocking group is important. The best yields (15%) for transgalactosidation were observed with serine methyl esters N-protected by a tertbutoxycarbonyl group. With N-benzyloxycarbonylserine methyl ester, the yield was

Production and functional characterization of two mouse/human chimeric antibodies with specificity for the tumor-associated Tn-antigen

In this work, we have constructed two functional mouse/human chimeric antibodies (IgMkappa and IgG1kappa isotypes) by inserting genomic DNA fragments encoding VH and Vkappa variable regions of the murine monoclonal antibody IgMK-83D4 into mammalian expression vectors containing human mu, gamma1, and kappa constant exons, and by transfecting them into the nonsecreting mouse myeloma X-63 cell line. In previous works, we have demonstrated that 83D4 murine mAb reacts with Tn determinant (GalNAcalpha-O-Ser/Thr) expressed in 90% of breast, ovary, and colon carcinomas. Both expressed chimeric antibodies were purified from the transfected cell line supernatant by affinity chromatography, and their reactivities against Tn antigen were confirmed by ELISA on asialo ovine submaxilar mucin and immunofluorescence studies on MCF-7 breast carcinoma cell line. We have demonstrated by gel filtration chromatography, that the principal secreted forms were monomers for IgG1kappa and pentamers for IgMkappa. The binding affinities of these chimeric antibodies against synthetic Tn glycopeptides, were evaluated by surface plasmon resonance showing an affinity constant similar to that of 83D4 native antibody for IgMkappa and a lower affinity constant for IgG1kappa chimeric antibody. On the other hand, the replacement of mouse C regions with human C regions confers both chimeric antibodies the ability to activate human complement. These mouse/human chimeric antibodies should be much less immunogenic and could play an important role in the lysis of tumor cell expressing Tn-antigen. Therefore, these anti-Tn chimeric antibodies could be considered as potential tools for human in vivo studies.

Enzymatic synthesis of β-galactosyldipeptides and of β-1,3-digalactosylserine derivatives using β-galactosidase

Abstract The transgalactosidation from lactose to dipeptides has been achieved using β-galactosidase from E. Coli as catalyst. Two series of dipeptides have been studied, each of them containing a serine residue. The best condensations occur when serine is at the N-terminal end of the dipeptide. Mild hydrolysis of the ester group of the labile glycosyl-dipeptide derivatives has been achieved using subtilisin. We also describe the condensation of lactose with β-galactosyl serine to give β-1,3-digalactosyl-serine derivatives.

Optimization of the papain catalyzed esterification of animo acids by alcohols and diols

Abstract Esterification of Boc-Alanine and Boc-Aspartic acids by alcohols C n H 2n+1 OH and dio1s HO(CH 2 ) n OH with imnobilized papain (XAD-7 or Sepharose) is discussed. Great improvement is obtained for the esterification of Boc-Ala-OH if papain is entrapped in XAD-7. For example no esterification is observed with 1-decanol if free papain is used whereas a 55% yield is obtained with papain immobilized on XAD-7. Esterification of Boc-Asp-OH with dio1s has been achieved with papain immobilized on Sepharose. In the case of ethyleneglycol no condensation could be observed with free papain or papain on XAD-7 whereas a 40% yield of esterification was obtained with papain on Sepharose.

Stereospecific Chemoenzymatic systhesis of galactopyranosyl-L-serine

Abstract The transglycosylation from raffinose and lactose to Aloc-Ser-OMe is catalyzed respectively by α and β galactosidases. The simplicity of the enzymatic, the stereospecificity of the condensations in one-pot reactions and the ease of purification give the method value for large scale preparation of β-linked derivatives. The protective groups of the serine residue can be cleaved under mild conditions: the ester group has been removed quantitatively by papain catalyzed hydrolysis and the Aloc group by a Pd (0) hydrostannolytic cleavage.

Enzymatic synthesis of some O-β-d-digalactosyl glycopeptides, using β-d-galactosidase

Abstract Disaccharide-peptide conjugates were obtained in yields of 30–50% from o -nitrophenyl β- d -galactopyranoside by employing β- d -galactosidase from E. coli as catalyst. Two series of β- d -galactosyldipeptides were examined as galactosyl acceptors. They both contain an l -serine residue β-linked to the anomeric carbon of galactose. In the first series, serine is in the N-terminal position of the dipeptide; in the second series, serine is in the C-terminal position. The second amino acid is l -alanine or glycine. Some of our substrates gave a high yield of β-(1 → 3)-digalactosyldipeptide derivatives and all gave very little of the β-(1 → 6) regioisomer. The conditions and the limitations of the transgalactosylation reaction are discussed.