Glycosylation of recombinant rabbit immunoglobulins influences protease susceptibility as shown by comprehensive mass spectrometric glycan analysis.

Recombinant immunoglobulins (rIgGs) have become increasingly important as therapeutic agents and diagnostic tools in recent years. Genetic engineering allows the introduction of non-natural features such as the Sortase motif for site-directed labeling. In this study, the enzyme Sortase A (SrtA) was used for the proteolytic cleavage of rIgGs to produce their biotinylated Fab fragments by locating the cleavage site close to the hinge region. However, SrtA cleavage of engineered rabbit IgGs (rRb-IgGs) derived from human embryonic kidney (HEK) 293 cells showed significantly lower yields compared with their mouse counterparts. Nonrecombinant Rb-IgGs have N- and O-glycans, and the presence of O-glycans close to the hinge region of the rRb-IgGs might affect the susceptibility of these antibodies to SrtA cleavage. In addition, the glycosylation pattern of rIgGs differs depending on the host cell used for expression. Therefore, we analyzed the N- and O-glycans of various rRb-IgGs expressed in HEK293 cells, detecting and quantifying 13 different N-glycan and 3 different O-glycan structures. The distribution of the different detected glycoforms in our rRb-IgG N-glycan analysis is in agreement with previous studies on recombinant human IgG N-glycans, confirming the hypothesis that the host cell defines the glycosylation of the recombinant produced IgGs. O-glycosylation could be mapped onto the threonine residue within the hinge region sequence XPTCPPPX, as already described previously for nonrecombinant Rb-IgGs. Substitution of this threonine allowed an almost complete Fab fragment cleavage. Therefore, we could confirm the hypothesis that the O-glycans affect the SrtA activity, probably due to steric hindrance.

Sensitization of Tumors for Attack by Virus-Specific CD8+ T-Cells Through Antibody-Mediated Delivery of Immunogenic T-Cell Epitopes.

Anti-tumor immunity is limited by a number of factors including the lack of fully activated T-cells, insufficient antigenic stimulation and the immune-suppressive tumor microenvironment. We addressed these hurdles by developing a novel class of immunoconjugates, Antibody-Targeted Pathogen-derived Peptides (ATPPs), which were designed to efficiently deliver viral T-cell epitopes to tumors with the aim of redirecting virus-specific memory T-cells against the tumor. ATPPs were generated through covalent binding of mature MHC class I peptides to antibodies specific for cell surface-expressed tumor antigens that mediate immunoconjugate internalization. By means of a cleavable linker, the peptides are released in the endosomal compartment, from which they are loaded into MHC class I without the need for further processing. Pulsing of tumor cells with ATPPs was found to sensitize these for recognition by virus-specific CD8+ T-cells with much greater efficiency than exogenous loading with free peptides. Systemic injection of ATPPs into tumor-bearing mice enhanced the recruitment of virus-specific T-cells into the tumor and, when combined with immune checkpoint blockade, suppressed tumor growth. Our data thereby demonstrate the potential of ATPPs as a means of kick-starting the immune response against "cold" tumors and increasing the efficacy of checkpoint inhibitors.

Use of the 2-chlorotrityl chloride resin for microwave-assisted solid phase peptide synthesis.

A fast and efficient microwave (MW)-assisted solid-phase peptide synthesis protocol using the 2-chlorotrityl chloride resin and the Fmoc/tBu methodology, has been developed. The established protocol combines the advantages of MW irradiation and the acid labile 2-chlorotrityl chloride resin. The effect of temperature during the MW irradiation, the degree of resin substitution during the coupling of the first amino acids and the rate of racemization for each amino acid were evaluated. The suggested solid phase methodology is applicable for orthogonal peptide synthesis and for the synthesis of cyclic peptides.

Divergent and convergent synthesis of polymannosylated dibranched antigenic peptide of the immunodominant epitope MBP(83-99).

Multiple antigenic peptide (MAP) systems are dendrimeric structures bearing multiple copies of identical or different peptide epitopes, and they have been demonstrated to show enhanced immunogenicity. Herein, we report the direct (divergent) and indirect (convergent) synthesis, using contemporary synthetic approaches, of a di-branched antigenic peptide (di-BAP) containing the immunodominant epitope MBP(83-99), which is implicated in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). The direct synthesis (di-BAP 1) was performed using microwave irradiation. The indirect synthesis (di-BAP 2) was carried out performing an efficient chemoselective coupling reaction through the formation of a thioether bond. Both di-BAPs were conjugated to polysaccharide mannan since mannosylation is a promising technique to achieve modulation in immune response. The conjugation was achieved through free amino groups of both di-BAPs via the formation of Schiff bases. The mannan-conjugated di-BAPs were further evaluated in vivo in a prophylactic vaccination protocol, prior to EAE induction in Lewis rats.

Microwave-assisted solid-phase peptide synthesis of the 60-110 domain of human pleiotrophin on 2-chlorotrityl resin.

A fast and efficient microwave-assisted solid phase peptide synthesis (MW-SPPS) of a 51mer peptide, the main heparin-binding site (60-110) of human pleiotrophin (hPTN), using 2-chlorotrityl chloride resin (CLTR-Cl) following the 9-fluorenylmethyloxycarbonyl/tert-butyl (Fmoc/tBu) methodology and with the standard N,N'-diisopropylcarbodiimide/1-hydroxybenzotriazole (DIC/HOBt) coupling reagents, is described. An MW-SPPS protocol was for the first time successfully applied to the acid labile CLTR-Cl for the faster synthesis of long peptides (51mer peptide) and with an enhanced purity in comparison to conventional SPPS protocols. The synthesis of such long peptides is not trivial and it is generally achieved by recombinant techniques. The desired linear peptide was obtained in only 30 h of total processing time and in 51% crude yield, in which 60% was the purified product obtained with 99.4% purity. The synthesized peptide was purified by reversed phase high performance liquid chromatography (RP-HPLC) and identified by electrospray ionization mass spectrometry (ESI-MS). Then, the regioselective formation of the two disulfide bridges of hPTN 60-110 was successfully achieved by a two-step procedure, involving an oxidative folding step in dimethylsulfoxide (DMSO) to form the Cys(77)-Cys(109) bond, followed by iodine oxidation to form the Cys(67)-Cys(99) bond.

Design and synthesis of a cyclic double mutant peptide (cyclo(87-99)[A91,A96]MBP87-99) induces altered responses in mice after conjugation to mannan: implications in the immunotherapy of multiple sclerosis.

Altered peptide ligands that alter immune responses are a promising approach to the immunotherapy of multiple sclerosis. Cyclic peptides are of interest because the limited stability of linear peptides restricts their use in vivo. We designed and synthesized a cyclic double mutant peptide from MBP(87-99)-[cyclo(87-99)[A(91),A(96)]MBP(87-99)]. Immunization of mice, in CFA reduced Th1 responses. However, when conjugated to reduced mannan, a significant further reduction of Th1 responses and moderate Th2 responses were induced.

Putative bioactive conformations of amide linked cyclic myelin basic protein peptide analogues associated with experimental autoimmune encephalomyelitis.

The solution models of cyclo(87-99) MBP87-99, cyclo(87-99) [Ala91,96] MBP87-99, and cyclo(87-99) [Arg91, Ala96] MBP87-99 have been determined through 2D NMR spectroscopy in DMSO-d6. Chemical shift analysis has been performed in an attempt to elucidate structural changes occurring upon substitution of native residues. NMR-derived geometrical constraints have been used in order to calculate high-resolution conformers of the above peptides. Conformational analysis of the three synthetic analogues show that the bioactivity, or the lack of it, may possibly be due to the distinct local structure observed and the subsequent differences in the overall topology and exposed area after binding with Major Histocompatibility Complex II (MHC II). It is believed that an overall larger solvent accessible area blocks the approach and binding of the T-cell receptor (TCR) on the altered peptide ligand (APL)-MHC complex, whereas more compact structures do not occlude weak interactions with an approaching TCR and can cause Experimental Autoimmune Encephalomyelitis (EAE) antagonism. A pharmacophore model based on the structural data has been generated.

Design, synthesis, and molecular modeling of a novel amide-linked cyclic GnRH analogue cyclo(4-9)[Lys4,D-Trp6,Glu9]GnRH: stimulation of gonadotropin gene expression.

This report describes the rational design, synthesis, and pharmacological properties of an amide-linked cyclic analogue of gonadotropin-releasing hormone (GnRH) namely Cyclo(4-9)[Lys(4),d-Trp(6),Glu(9)]GnRH. The conformationally restricted analogue is characterized by reduced flexibility of the peptide strand due to the introduction of a beta-turn mimetic through 4,9 residue amide cyclization. The cyclic analogue was found to stimulate gonadotropin gene expression in the goldfish pituitary with similar potency compared to two native forms of GnRH. Simulation studies based on ROE connectivities in linear GnRH and potency of cyclic analogue supports the His(2), Trp(3), Tyr(5) clustering considered important for triggering receptor activation.