Antibody Specific for the Peptide·Major Histocompatibility Complex

Abstract

Antibodies recognizing peptide bound to a major histocompatability complex (MHC) protein usually have a higher affinity for the composite peptide⅐MHC (pMHC) ligand than T cell receptors (TCR) with the same specificity. Because the solvent-accessible peptide area constitutes only a small portion of the contacting pMHC surface, we hypothesized that the contribution of the MHC moiety to the TCR-pMHC complex stability is limited, ensuring a small increment of the binding energy delivered by the peptide to be distinguishable by the TCR or the peptide-specific antibody. This suggests that the gain in affinity of the antibody-pMHC interaction can be achieved through an increase in the on-rate without a significant change in the off-rate of the interaction. To test the hypothesis, we have analyzed the binding of an ovalbumin peptide (pOV8) and its variants associated with soluble H-2K b protein to the 25-D1.16 monoclonal antibody and compared it with the binding of the same pMHC complexes to the OT-1 TCR. This comparison revealed a substantially higher on-rate of the antibody-pMHC interaction compared with the TCR-pMHC interaction. In contrast, both the antibody and the TCR-pMHC complexes exhibited comparably fast off-rates. Sequencing of the 25-D1.16 V H and V L genes showed that they have very few somatic mutations and those occur mainly in framework regions. We propose that the above features constitute a signature of the recognition of MHC-bound peptide antigens by TCR and TCR-like antibodies, which could explain why the latter are rarely produced in vivo.T cell antigen-specific receptors (TCR) 1 must recognize major histocompatibility complex (MHC) protein and at the same time discriminate between many different peptides bound to that MHC. Approximately 80% of the solvent-accessible area of the peptide is buried in the MHC binding groove (1), limiting the amount of energy that a peptide can contribute to the TCR-pMHC interaction. To ensure TCR specificity for the MHC-bound peptide, the amount of binding energy coming from the MHC moiety must be restricted, allowing the peptide contribution of the composite ligand to be distinguishable by the TCR. In accord with this idea, it has been found that only two to three residues of an MHC class I protein mediate critical TCR-MHC contacts (2, 3). In addition, experimentally measured values of the free energy for various TCR-pMHC interactions (4 -8) are significantly lower than the free energy of a typical protein-protein interaction (9, 10). However, it has been shown that the TCR intrinsic affinity can be significantly increased without the loss of its specificity (11). The increase in the TCR affinity was mainly the result of the on-rate, not the off-rate, indicating that limited activation energy of the dissociation phase of the TCR-pMHC reaction is required to preserve the peptide specificity. This led us to suggest that, similar to the TCR with enhanced affinity, the apparent increase in the binding energy of pMHC-specific antibodies is achieved through a faster on-rat...

How a T Cell Receptor-like Antibody Recognizes Major Histocompatibility Complex-bound Peptide

“…16 G-agarose (2). Fab fragments of the antibody were isolated by papain digestion, followed by anion-exchange chromatography on a Mono Q column (GE Healthcare).…”

“…Fab fragments of the antibody were isolated by papain digestion, followed by anion-exchange chromatography on a Mono Q column (GE Healthcare). ectodomain including a synthetic C-terminal His 6 tag and mouse ␤ 2 -microglobulin (␤ 2 m) were expressed in Schneider (S2) cells as described previously (2). S2 cells that were stably transfected with plasmids containing H-2K b , ␤ 2 m, and neomycin resistance genes were expanded in Sf-900 II SFM serumfree medium (Invitrogen) and grown to a density of 1.4 -2.0 ϫ 10 7 /ml.…”

“…S2 cells that were stably transfected with plasmids containing H-2K b , ␤ 2 m, and neomycin resistance genes were expanded in Sf-900 II SFM serumfree medium (Invitrogen) and grown to a density of 1.4 -2.0 ϫ 10 7 /ml. H-2K b expression was induced by 1 M CuSO 4 for 72 h, and soluble H-2K b molecules were isolated from the culture supernatant as described (2). Purity of the isolated H-2K b was confirmed by SDS-PAGE.…”

TCR-like antibody drug conjugates mediate killing of tumor cells with low peptide/HLA targets

“…To date, several reports have analyzed the structural binding properties between TCR-like antibodies and cognate peptide/MHC ligands, although these studies only assessed similarities/differences to T cell receptor-MHC engagement. [40][41][42][43] Our data introduce a previously unpublished aspect of TCR-like antibody binding, but the mechanism behind how TCR-like antibody docking influences ADC function is currently unknown and a focus of study in our laboratory. Certainly, it has been previously recognized that ADC candidacy depends to a large degree on antibody internalization kinetics (following surface protein interaction) 44 since internalization rates vary even among antibody clones generated against the same target.…”

Visualization of p53264–272/HLA-A*0201 Complexes Naturally Presented on Tumor Cell Surface by a Multimeric Soluble Single-Chain T Cell Receptor

“…These Abs could prove useful as staining reagents to visualize or quantify peptide/MHC complexes on the surface of APCs. However, detailed studies comparing the peptide/MHC recognition of a panel of such Abs and TCRs reveal a number of important differences, suggesting that the TCRs and Abs bind to overlapping but nonidentical components on the peptide/MHC complex (37,49,50). For example, the MHC component appears to play a more significant role in Ab recognition than what is seen in TCR to peptide/MHC interactions.…”