We determined the crystal structures of the T cell receptor (TCR)-like antibody 25-D1.16 Fab fragment bound to a complex of SIINFEKL peptide from ovalbumin and the H-2K b molecule. Remarkably, this antibody directly "reads" the structure of the major histocompatibility complex (MHC)-bound peptide, employing the canonical diagonal binding mode utilized by most TCRs. This is in marked contrast with another TCR-like antibody, Hyb3, bound to melanoma peptide MAGE-A1 in association with HLA-A1 MHC class I. Hyb3 assumes a non-canonical orientation over its cognate peptide-MHC and appears to recognize a conformational epitope in which the MHC contribution is dominant. We conclude that TCR-like antibodies can recognize MHC-bound peptide via two different mechanisms: one is similar to that exploited by the preponderance of TCRs and the other requires a non-canonical antibody orientation over the peptide-MHC complex.B cell receptor and its soluble analog, i.e. antibody molecules, typically recognize native antigens. However, some antibodies can recognize major histocompatibility complex (MHC) 4 -bound peptides and have been termed T cell receptor (TCR)-like antibodies. They have been derived either from large libraries containing diverse fragments encoding variable antibody regions or in the course of immunization of laboratory animals. The latter approach has proved to be less productive, suggesting that under natural conditions, TCR-like antibodies are rather rare. Because these antibodies offer attractive opportunities to track and measure particular peptide-MHC (pMHC) complexes on live cells in vitro and in vivo, a growing number of TCR-like antibodies are being developed. The molecular basis for their specificity is poorly understood, however.The TCR-like antibody 25-D1.16 has been elicited in response to immunization of mice with a whole cell bearing pOV8-K b complexes (1). This antibody has been shown to discriminate pOV8-K b from other pMHC complexes on the cell surface and has been widely used to study various aspects of processing and presentation of MHC class I (MHC-I)-restricted epitopes to cytotoxic T lymphocytes. We determined the complete primary structure of this antibody and compared its parameters of binding to pOV8-K b with those of a TCR recognizing the same ligand. This analysis led us to conclude that antibody 25-D1.16 indeed behaves like a TCR (2).Here, we report the crystal structure of 25-D1.16 Fab bound to soluble pOV8-K b protein. 25-D1.16 interacts with amino acid residues in conserved MHC positions that are believed to mediate the canonical TCR orientation of all TCR-pMHC structures studied thus far (3). Such a diagonal orientation facilitates direct contacts between both CDR3 loops of the Fab fragment and the K b -bound peptide side chains, allowing the antibody to "read" the structure of MHC-bound peptide in the same way as a TCR. Because 25-D1.16 was raised without direct contribution of CD8, which could influence TCR binding to pMHC, but still utilizes the same conserved positions to con...

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...

We have utilized soluble HIV Gag-specific T-cell receptor (TCR) D3 with low affinity and TCRlike antibody 25-D1.16 recognizing its natural peptide-MHC (pMHC) ligand with high affinity to determine how affinity and off-rate of the receptor-pMHC interactions affect the sensitivity of pMHC detection on the cell surface. We found that with soluble TCR cognate pMHCs can be detected only at relatively high cell surface densities when the TCR was oligomerized using either Strepavidin or quantum dots (QD) scaffolds. While the higher affinity probe led to a greater sensitivity of pMHC detection, monomers and oligomers of the probe showed essentially the same detection limit, which is restricted by the sensitivity of standard flow cytometry technique. We have also shown that imaging of QD/TCR specifically bound to cognate pMHC on the cell surface yielded a very bright fluorescent signal that can enhance the sensitivity of viral peptide detection on infected cells. Keywordssoluble TCR oligomers; detection of cognate pMHC on the cell surface; quantum dot/TCR conjugates