Distribución de la Tenencia de la Tierra
2.2 La reforma agraria y la ocupación habitacional de los ejidos
TCRs specific for cancer epitopes are generally characterised by low binding affinities (binding KDs high micromolar range) (Bridgeman et al., 2011). This lower binding affinity is thought to be a result of negative selection of T-‐cells that bear TCRs with higher affinity for self-‐ligands in the thymus. Since TCR affinity plays an important role in T-‐cell activation, the TCR affinity gap between anti-‐pathogen and anti-‐cancer T-‐cells leaves the latter at a distinct disadvantage and makes it more difficult to break self-‐tolerance to such antigens. One approach to enhance the T-‐cell response to tumour antigen-‐ derived peptides has been to immunize patients with altered peptide ligands that differ from the native sequence by a single, or multiple, amino acid residues (Lesterhuis et al., 2011). However, such ‘heteroclitic’ peptides with even single amino acid substitutions that are predicted to only contact the HLA can have unpredictable, yet important, effects on TCR engagement. Despite their extensive application in clinical trials as cancer vaccines, to date only a few X-‐ray structures of TCRs bound to cognate tumour antigens have been determined (Borbulevych et al., 2011; Chen, 2005; Deng et al., 2007; Madura et al., 2015). I have solved the first crystal structure of a HLA-‐A2 restricted gp100 peptide antigen bound to a cognate αβ TCR. In this chapter, I show how the PMEL17 TCR bound with a typical diagonal orientation over the central peptide residues, and mainly contacted residues 4, 7 and 8 of the YLE-‐9V peptide which protruded out of the HLA-‐A2 binding groove. Interestingly, the PMEL17 TCR was characterized by a binding affinity (KD) of 7.6 μM, a value that falls in the very high end of affinity ranges described so far for cancer TCR and pHLA interactions (Bridgeman et al., 2011; Aleksic et al., 2012). These observations suggest that healthy donors or melanoma patients may harbour T-‐cells bearing TCRs with reasonable affinity for some tumour associated antigens, which can be preferentially chosen for TCR-‐based applications. For example, gp100-‐specific ImmTACs (Immune-‐ mobilising monoclonal TCRs against cancer) are a new class of soluble bi-‐specific anti-‐tumour agents that combine a high-‐affinity TCR-‐based gp100 recognition domain with a T cell activation domain (Liddy et al., 2012; Bossi et al., 2014). IMCgp100 is being tested as a soluble drug and is showing partial or complete durable responses in Phase I/IIa trial in patients with advanced melanoma (Middleton et al., 2015).
In this chapter I also provide insight into YLE single amino acid contribution to TCR binding by performing an alanine scan mutagenesis across the peptide backbone with two different YLE-‐specific αβTCRs. Interestingly, both PMEL17 TCR and gp100 TCR were most sensitive to mutations at position 3 or 5 of the native YLE peptide sequence despite these TCRs being constructed from completely different Vα and Vβ genes. These results are supported by a recent study of YLE altered peptide ligands which described YLE-‐3A as a null agonist for a different TCR (Shaft et al., 2003; 2013). Overall, along with the two TCRs studied here, the sequences of further two distinct YLE-‐specific TCRs have been published, demonstrating diverse gene usage and different CDR3 loop sequences (Table 3.3). No structural data supporting these observations have been published to date.
Table 3.3. Alignment of TCR CDR3 regions of four gp100-‐specific TCRs
PMEL17, gp100, MPD (Schaft et al., 2003) and 296 (Schaft et al., 2003) gp100-‐specific TCR.
TCR CDR1α CDR2α CDR3α CDR1β CDR1β CDR1β
PMEL17 DSAIYN IQSSQRE CAVLSSGGSNYKLTFG SGHTA FQGTGA CASSFIGGTDTQYFG
gp100 TSINN IRSNERE CATDGDTPLVFG LNHDA SQIVND CASSIGGPYEQYFG
MPD KALYS LLKGGEQ CGTETNTGNQFYFG SGHDY FNNNVP CASSLGRYNEQFFG
296 DSASNY IRSNVGE CAASTSGGTSYGKLTFG MNHEY SMNVEV CASSLGSSYEQYFG
Interestingly, mutation in position 3 in the YLE peptide did not alter the conformation of the peptide backbone itself, but resulted in a ‘knock-‐on’ effect on the neighbouring residue Pro4 that completely abolished TCR binding and T-‐cell recognition. This can be explained by the fact that Pro4 was at the centre of a sizeable network of interactions (both vdW and hydrogen bonds) in the PMEL17-‐A2-‐YLE-‐ 9V structure. In addition, Position 3 in HLA-‐A2 restricted peptides is known to be a secondary anchor residue (Ruppert et al., 1993), in that it supports the exposed peptide bulge that is normally involved in TCR binding. By mutating the residue in position 3 with a smaller side chain, this support is lost causing a ‘molecular switch in the neighbour Pro4. A similar mechanism in an HIV-‐1 derived peptide, has recently been described by our group, with important implications for the immune control of HIV infection and patterns of viral escape mutants (Kløverpris et al., 2015). Additionally, the existence of a novel mode of flexible peptide presentation in a diabetes model has been demonstrated, showing the potential dynamic nature of the region surrounding the HLA F-‐pocket (Motozono et al., 2015; Borbulevych et al., 2009). Taken together, these studies support the notion that peptide-‐HLA interactions are more plastic and dynamic than previously appreciated, with obvious implications for immune recognition, epitope prediction and structural modelling.
Overall, the results presented here represent the first structural insight into TCR recognition of an important tumour antigen, targeted by many clinical therapies. They reveal that two very different TCRs share a similar pattern of specificity, demonstrated by their near identical sensitivity to different peptide modifications. Finally, I’ve shown that modification to peptide residues outside of the TCR binding motif can have unpredictable knock-‐on effects on adjacent peptide residues that abrogate TCR binding and T-‐cell recognition, highlighting that even conservative peptide substitutions can have unexpected consequences for T-‐cell recognition by different antigen-‐specific TCRs due to ‘knock-‐on’ structural changes in the HLA-‐bound peptide. Such ‘transmitted’ structural changes need to be taken into consideration when designing improved peptides for cancer vaccination. Given the growing evidence that plasticity at the TCR-‐pHLA interface can influence immune recognition, structural and biophysical studies of binding should be taken into account when attempting to design altered peptide ligands with improved immunogenicity.