MHC‐peptide‐specific antibodies reveal inefficient presentation of an HLA‐A*0201‐restricted, Melan‐A‐derived peptide after active intracellular processing

Abstract

MHC-peptide-specific Fab antibodies binding to HLA-A*0201 complexes presenting the wild-type EAAGIGILTV (EAA) or analogue Melan-A 10-mer ELAGIGILTV (ELA) peptide were generated to study efficacy of peptide processing and presentation. None of the selected Fab antibodies detected the naturally processed EAA/HLA-A*0201 complex on melanoma tumor cells, confirming the known low peptide number on the cell surface. To study the effect of peptide presentation and processing in more detail, genes coding for the A27L-mutated Melan-A protein or the processed ELA peptide were introduced into HLA-A*0201 + B cells by infection with the respective recombinant vaccinia virus construct producing equimolar amounts of GFP-ubiquitin directly linked to the fragment of interest. Correlating GFP expression to actual numbers of peptide presented, 110-260 ELA peptides had to be synthesized to be presented by a single MHC class I antigen-peptide complex. This number increased 10-to 20-fold when ELA peptide presentation from the A27L-mutated full length Melan-A protein was studied, since 1600-5200 GFP molecules needed to be synthesized for the detection of one ELA peptide. Our results indicate that peptide processing rather than presentation is the rate-limiting step in our experimental setting and is much more ineffective for Melan-A than has been previously shown for other MHC class I-restricted epitopes.

Specific Increase in Potency via Structure-Based Design of a TCR

“…In general Mart-1 expression levels on melanoma cell lines (such as Gmel) range from low to moderate levels (99). Also, Renner and colleagues showed that endogenous antigen processing of the decamer (Melan-A) protein or its A27L-mutated variant results in inefficient presentation of these peptides both wt and 27L mutant by HLA-A2-positive tumor cell lines (98). Therefore, since the Gmel melanoma cell line in Fig.…”

Inactivation of protein‐phosphatase 2A causing hyperphosphorylation of autoantigenic paraprotein targets in MGUS/MM is due to an exchange of its regulatory subunits

“…For selection of antibodies specific for wtP-7 or pP-7, a phagemid library expressing a large non-immune, semi-synthetic human Fab repertoire, containing 1.45 3 10 10 different antibody fragments, was used. 14 Selection was done as described by Held et al 15 Two peptides were used for selection: for wtP-7 Biot-SGGGGSRGTGALLLRGSL-LASGRAPRRA; for pP-7 Biot-SGGGGSRGTGALLLRG(pS)LLAS-GRAPRRA. Specifically bound phages were eluted and used to infect E. coli strain TG1 (30 min at 37 C), and bacteria were grown overnight at 30 C on agar.…”

Differential presentation of tumor antigen‐derived epitopes by MHC‐class I and antigen‐positive tumor cells

“…30 For detection of SSX2 103-111 /HLA-A*0201 complexes after endogenous processing a ubiquitin/protein/reference (UPR)-based pEGFP vector was used. 31 The vectors encoded for EGFP/ Ubiquitin/Melan-A [26][27][28][29][30][31][32][33][34][35] or EGFP/ubiquitin/SSX2 103-111 fusion protein. In eukaryotic cells, the resulting fusion protein is cleaved co-translationally at the junction between EGFP-ubiquitin and the peptide of interest by ubiquitin-specific proteases.…”

“…We used several antigen presenting cells to demonstrate that the Fab-VIE1 could bind to the specific MHC-peptide complex not only in its recombinant soluble form, but also in the native form expressed on the cell surface. Soluble Fab-VIE1 reacted with SSX2 103-111 peptide loaded T2 cells and HLA-A*0201 1 EBV-transformed lymphoblastoid cell line, but not with cells loaded with one of 6 different HLA-A*0201-binding peptides, SSX2 [41][42][43][44][45][46][47][48][49] , NY-ESO-1 157-165 , influenza matrix protein 58-66 , gp100 209-217 , Melan-A [26][27][28][29][30][31][32][33][34][35] and Carboanhydrase-IV 254-262 , respectively. No binding of Fab-VIE1 to the HLA-A*0201 2 EBV-transformed B-lymphocytes was observed, neither when pulsed with the SSX2 103-111 peptide nor with control peptides (Fig.…”

“…Both melanoma cell lines became positive upon peptide pulsing, but at equal peptide concentrations the increase in fluores- cence intensity was lower than that observed on T2 cells or EBVtransformed B-lymphocytes. Next, we transfected HLA-A*0201 1 EBV-transformed B-lymphocytes with a vector encoding 2 antigenic HLA-A*0201-restricted peptides, Melan-A [26][27][28][29][30][31][32][33][34][35] and SSX2 103-111 , respectively. The peptides were expressed as a fusion protein EGFP/ubiquitin/peptide.…”