Advertisement

T-cell Receptor-optimized Peptide Skewing of the T-cell Repertoire Can Enhance Antigen Targeting*

  • Julia Ekeruche-Makinde
    Footnotes
    Affiliations
    Institute of Infection and Immunity, Cardiff University School of Medicine, Henry Wellcome Building, Heath Park, Cardiff CF14 4XN, Wales, United Kingdom
    Search for articles by this author
  • Mathew Clement
    Footnotes
    Affiliations
    Institute of Infection and Immunity, Cardiff University School of Medicine, Henry Wellcome Building, Heath Park, Cardiff CF14 4XN, Wales, United Kingdom
    Search for articles by this author
  • David K. Cole
    Footnotes
    Affiliations
    Institute of Infection and Immunity, Cardiff University School of Medicine, Henry Wellcome Building, Heath Park, Cardiff CF14 4XN, Wales, United Kingdom
    Search for articles by this author
  • Emily S.J. Edwards
    Affiliations
    Institute of Infection and Immunity, Cardiff University School of Medicine, Henry Wellcome Building, Heath Park, Cardiff CF14 4XN, Wales, United Kingdom
    Search for articles by this author
  • Kristin Ladell
    Affiliations
    Institute of Infection and Immunity, Cardiff University School of Medicine, Henry Wellcome Building, Heath Park, Cardiff CF14 4XN, Wales, United Kingdom
    Search for articles by this author
  • John J. Miles
    Affiliations
    Institute of Infection and Immunity, Cardiff University School of Medicine, Henry Wellcome Building, Heath Park, Cardiff CF14 4XN, Wales, United Kingdom

    Australian Centre for Vaccine Development, Human Immunity Laboratory, Queensland Institute of Medical Research, Brisbane 4029, Australia
    Search for articles by this author
  • Katherine K. Matthews
    Affiliations
    Institute of Infection and Immunity, Cardiff University School of Medicine, Henry Wellcome Building, Heath Park, Cardiff CF14 4XN, Wales, United Kingdom
    Search for articles by this author
  • Anna Fuller
    Affiliations
    Institute of Infection and Immunity, Cardiff University School of Medicine, Henry Wellcome Building, Heath Park, Cardiff CF14 4XN, Wales, United Kingdom
    Search for articles by this author
  • Katy A. Lloyd
    Affiliations
    Institute of Infection and Immunity, Cardiff University School of Medicine, Henry Wellcome Building, Heath Park, Cardiff CF14 4XN, Wales, United Kingdom
    Search for articles by this author
  • Florian Madura
    Affiliations
    Institute of Infection and Immunity, Cardiff University School of Medicine, Henry Wellcome Building, Heath Park, Cardiff CF14 4XN, Wales, United Kingdom
    Search for articles by this author
  • Garry M. Dolton
    Affiliations
    Institute of Infection and Immunity, Cardiff University School of Medicine, Henry Wellcome Building, Heath Park, Cardiff CF14 4XN, Wales, United Kingdom
    Search for articles by this author
  • Johanne Pentier
    Affiliations
    Institute of Infection and Immunity, Cardiff University School of Medicine, Henry Wellcome Building, Heath Park, Cardiff CF14 4XN, Wales, United Kingdom
    Search for articles by this author
  • Anna Lissina
    Affiliations
    Institute of Infection and Immunity, Cardiff University School of Medicine, Henry Wellcome Building, Heath Park, Cardiff CF14 4XN, Wales, United Kingdom
    Search for articles by this author
  • Emma Gostick
    Affiliations
    Institute of Infection and Immunity, Cardiff University School of Medicine, Henry Wellcome Building, Heath Park, Cardiff CF14 4XN, Wales, United Kingdom
    Search for articles by this author
  • Tiffany K. Baxter
    Affiliations
    Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556
    Search for articles by this author
  • Brian M. Baker
    Affiliations
    Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556
    Search for articles by this author
  • Pierre J. Rizkallah
    Affiliations
    Institute of Infection and Immunity, Cardiff University School of Medicine, Henry Wellcome Building, Heath Park, Cardiff CF14 4XN, Wales, United Kingdom
    Search for articles by this author
  • David A. Price
    Affiliations
    Institute of Infection and Immunity, Cardiff University School of Medicine, Henry Wellcome Building, Heath Park, Cardiff CF14 4XN, Wales, United Kingdom
    Search for articles by this author
  • Linda Wooldridge
    Footnotes
    Affiliations
    Institute of Infection and Immunity, Cardiff University School of Medicine, Henry Wellcome Building, Heath Park, Cardiff CF14 4XN, Wales, United Kingdom
    Search for articles by this author
  • Andrew K. Sewell
    Correspondence
    To whom correspondence should be addressed. Tel.: 44-29-2068-7055; Fax: 44-29-2068-7007
    Footnotes
    Affiliations
    Institute of Infection and Immunity, Cardiff University School of Medicine, Henry Wellcome Building, Heath Park, Cardiff CF14 4XN, Wales, United Kingdom
    Search for articles by this author
  • Author Footnotes
    * This work was supported by the Biotechnology and Biological Sciences Research Council (Grant BB/H001085/1), Wellcome Trust Grants WT086716 (to A. K. S.), WT095767 (to D. K. C.), and WT079848MA (to L. W.), and National Institutes of Health Grant GM067079 (to B. M. B.). Fellowship support was received from the Research Councils UK (to P. J. R.), Wales Office of Research and Development (to J. J. M.), and The Medical Research Council (to D. A. P.).
    This article contains supplemental Tables S1 and S2 and Figs. S1–S4.
    1 These authors contributed equally to this work.
Open AccessPublished:September 05, 2012DOI:https://doi.org/10.1074/jbc.M112.386409
      Altered peptide antigens that enhance T-cell immunogenicity have been used to improve peptide-based vaccination for a range of diseases. Although this strategy can prime T-cell responses of greater magnitude, the efficacy of constituent T-cell clonotypes within the primed population can be poor. To overcome this limitation, we isolated a CD8+ T-cell clone (MEL5) with an enhanced ability to recognize the HLA A*0201-Melan A27–35 (HLA A*0201-AAGIGILTV) antigen expressed on the surface of malignant melanoma cells. We used combinatorial peptide library screening to design an optimal peptide sequence that enhanced functional activation of the MEL5 clone, but not other CD8+ T-cell clones that recognized HLA A*0201-AAGIGILTV poorly. Structural analysis revealed the potential for new contacts between the MEL5 T-cell receptor and the optimized peptide. Furthermore, the optimized peptide was able to prime CD8+ T-cell populations in peripheral blood mononuclear cell isolates from multiple HLA A*0201+ individuals that were capable of efficient HLA A*0201+ melanoma cell destruction. This proof-of-concept study demonstrates that it is possible to design altered peptide antigens for the selection of superior T-cell clonotypes with enhanced antigen recognition properties.

      Introduction

      The key aim of vaccination is to establish populations of memory T-cells and B-cells expressing antigen receptors that provide a rapid, robust targeted immune response. Antigen receptors on B-cells undergo affinity maturation through a process of somatic hypermutation that allows evolution toward more effective responses over time. In contrast, the T-cell receptor (TCR)
      The abbreviations used are: TCR
      T-cell receptor
      TOP
      TCR-optimized peptide
      TOPSORT
      TCR-optimized peptide skewing of the repertoire of T-cells
      APL
      altered peptide ligand
      pMHC
      peptide-MHC
      pMHCI
      peptide-MHC class I
      CEA
      carcinoembryonic antigen
      PE
      phycoerythrin
      CPL
      combinatorial peptide library
      MIP-1β
      macrophage inflammatory protein-1β.
      is fixed on individual T-cell clonotypes.
      Antigen-specific T-cells bearing heterodimeric αβ TCRs play a pivotal role in adaptive immunity to pathogens and cellular malignancies by recognizing short peptide fragments bound to major histocompatibility complex (MHC) molecules on the target cell surface (
      • Davis M.M.
      • Bjorkman P.J.
      T-cell antigen receptor genes and T-cell recognition.
      ,
      • Garcia K.C.
      • Teyton L.
      • Wilson I.A.
      Structural basis of T cell recognition.
      ). It is estimated that there are <108 αβ TCRs in the human naive T-cell pool (
      • Arstila T.P.
      • Casrouge A.
      • Baron V.
      • Even J.
      • Kanellopoulos J.
      • Kourilsky P.
      A direct estimate of the human αβ T cell receptor diversity.
      ), a number that is dwarfed by the immense array of potential antigenic peptides that could be encountered (
      • Mason D.
      A very high level of cross-reactivity is an essential feature of the T-cell receptor.
      ). Evolution appears to have solved this conundrum by endowing T-cells with an extremely high degree of cross-reactivity (
      • Mason D.
      A very high level of cross-reactivity is an essential feature of the T-cell receptor.
      ,
      • Wooldridge L.
      • Ekeruche-Makinde J.
      • van den Berg H.A.
      • Skowera A.
      • Miles J.J.
      • Tan M.P.
      • Dolton G.
      • Clement M.
      • Llewellyn-Lacey S.
      • Price D.A.
      • Peakman M.
      • Sewell A.K.
      A single autoimmune T cell receptor recognizes more than a million different peptides.
      ,
      • Sewell A.K.
      Why must T cells be cross-reactive?.
      ). A corollary of this hypothesis is that multiple TCRs can recognize individual peptide-MHC (pMHC) antigens. Indeed, there is now evidence that antigen-specific T-cell populations, although frequently skewed toward dominant clonotypes present at high frequency, can be highly polyclonal (
      • Casanova J.L.
      • Maryanski J.L.
      Antigen-selected T-cell receptor diversity and self-nonself homology.
      ,
      • Maryanski J.L.
      • Attuil V.
      • Bucher P.
      • Walker P.R.
      A quantitative, single-cell PCR analysis of an antigen-specific TCR repertoire selected during an in vivo CD8 response: direct evidence for a wide range of clone sizes with uniform tissue distribution.
      ,
      • Price D.A.
      • Brenchley J.M.
      • Ruff L.E.
      • Betts M.R.
      • Hill B.J.
      • Roederer M.
      • Koup R.A.
      • Migueles S.A.
      • Gostick E.
      • Wooldridge L.
      • Sewell A.K.
      • Connors M.
      • Douek D.C.
      Avidity for antigen shapes clonal dominance in CD8+ T cell populations specific for persistent DNA viruses.
      ).
      To date, vaccine strategies have aimed to induce the largest T-cell responses possible without specific consideration of the individual clonotypes that constitute each response. However, emerging evidence suggests that the quality of a T-cell response, determined at the clonotypic level, might be more important than quantity, defined as the overall magnitude of a specific T-cell population (
      • Appay V.
      • Douek D.C.
      • Price D.A.
      CD8+ T cell efficacy in vaccination and disease.
      ,
      • Bangham C.R.
      CTL quality and the control of human retroviral infections.
      ,
      • Seder R.A.
      • Darrah P.A.
      • Roederer M.
      T-cell quality in memory and protection: implications for vaccine design.
      ). For example, clonotype usage within two different codominant simian immunodeficiency virus-specific CD8+ T-cell responses in Mamu A*01+ rhesus macaques can determine patterns of viral escape by antigen mutation during acute infection and act as a molecular signature of subsequent disease course (
      • Price D.A.
      • West S.M.
      • Betts M.R.
      • Ruff L.E.
      • Brenchley J.M.
      • Ambrozak D.R.
      • Edghill-Smith Y.
      • Kuroda M.J.
      • Bogdan D.
      • Kunstman K.
      • Letvin N.L.
      • Franchini G.
      • Wolinsky S.M.
      • Koup R.A.
      • Douek D.C.
      T cell receptor recognition motifs govern immune escape patterns in acute SIV infection.
      ,
      • Price D.A.
      • Asher T.E.
      • Wilson N.A.
      • Nason M.C.
      • Brenchley J.M.
      • Metzler I.S.
      • Venturi V.
      • Gostick E.
      • Chattopadhyay P.K.
      • Roederer M.
      • Davenport M.P.
      • Watkins D.I.
      • Douek D.C.
      Public clonotype usage identifies protective Gag-specific CD8+ T cell responses in SIV infection.
      ). Adoptive transfer experiments further underscore the fact that T-cell clonotypes recognizing the same antigen should not be viewed on an equal basis as their individual sensitivity to antigen density at the target cell surface is a critical determinant of in vivo efficacy (
      • Alexander-Miller M.A.
      • Leggatt G.R.
      • Berzofsky J.A.
      Selective expansion of high- or low-avidity cytotoxic T lymphocytes and efficacy for adoptive immunotherapy.
      ). Thus, it seems that the efficacy of an antigen-specific T-cell response and its clonotypic architecture are inexorably linked. Consequently, the best vaccines should aim to induce the most effective T-cell clonotypes.
      In many cases, the most effective T-cells are those that recognize target cells bearing low densities of cognate pMHC on their surface (
      • Appay V.
      • Douek D.C.
      • Price D.A.
      CD8+ T cell efficacy in vaccination and disease.
      ). Consistent with early studies using altered peptide ligands (APLs) with weak binding affinities (
      • Matsui K.
      • Boniface J.J.
      • Steffner P.
      • Reay P.A.
      • Davis M.M.
      Kinetics of T-cell receptor binding to peptide/I-Ek complexes: correlation of the dissociation rate with T-cell responsiveness.
      ), our own experiments using biophysically defined T-cell antigens indicate that the best T-cell agonists are those that engage the TCR with the highest affinities and for the longest dwell times (
      • Bridgeman J.S.
      • Sewell A.K.
      • Miles J.J.
      • Price D.A.
      • Cole D.K.
      Structural and biophysical determinants of αβ T-cell antigen recognition.
      ,
      • Laugel B.
      • van den Berg H.A.
      • Gostick E.
      • Cole D.K.
      • Wooldridge L.
      • Boulter J.
      • Milicic A.
      • Price D.A.
      • Sewell A.K.
      Different T cell receptor affinity thresholds and CD8 coreceptor dependence govern cytotoxic T lymphocyte activation and tetramer binding properties.
      ). Similarly, transduction experiments using TCRs with different affinities for defined natural antigens (
      • Varela-Rohena A.
      • Molloy P.E.
      • Dunn S.M.
      • Li Y.
      • Suhoski M.M.
      • Carroll R.G.
      • Milicic A.
      • Mahon T.
      • Sutton D.H.
      • Laugel B.
      • Moysey R.
      • Cameron B.J.
      • Vuidepot A.
      • Purbhoo M.A.
      • Cole D.K.
      • Phillips R.E.
      • June C.H.
      • Jakobsen B.K.
      • Sewell A.K.
      • Riley J.L.
      Control of HIV-1 immune escape by CD8 T cells expressing enhanced T-cell receptor.
      ) also show, across multiple systems, that T-cells expressing higher affinity TCRs are more sensitive to antigen (
      • Bridgeman J.S.
      • Sewell A.K.
      • Miles J.J.
      • Price D.A.
      • Cole D.K.
      Structural and biophysical determinants of αβ T-cell antigen recognition.
      ). Thus, TCRs with relatively high affinities are preferable when a high level of sensitivity to low antigen density is required. This feature becomes all the more important in the case of neoplastic targets as pMHC class I (pMHCI) antigen copy number at the cell surface is often very low (
      • Liddy N.
      • Bossi G.
      • Adams K.J.
      • Lissina A.
      • Mahon T.M.
      • Hassan N.J.
      • Gavarret J.
      • Bianchi F.C.
      • Pumphrey N.J.
      • Ladell K.
      • Gostick E.
      • Sewell A.K.
      • Lissin N.M.
      • Harwood N.E.
      • Molloy P.E.
      • Li Y.
      • Cameron B.J.
      • Sami M.
      • Baston E.E.
      • Todorov P.T.
      • Paston S.J.
      • Dennis R.E.
      • Harper J.V.
      • Dunn S.M.
      • Ashfield R.
      • Johnson A.
      • McGrath Y.
      • Plesa G.
      • June C.H.
      • Kalos M.
      • Price D.A.
      • Vuidepot A.
      • Williams D.D.
      • Sutton D.H.
      • Jakobsen B.K.
      Monoclonal TCR-redirected tumor cell killing.
      ). In addition, TCRs that recognize “self” tumor-associated antigens strongly are likely to be culled during thymic selection; as a consequence, antitumor TCRs bind their cognate pMHC antigens relatively weakly when compared with pathogen-specific TCRs (
      • Cole D.K.
      • Pumphrey N.J.
      • Boulter J.M.
      • Sami M.
      • Bell J.I.
      • Gostick E.
      • Price D.A.
      • Gao G.F.
      • Sewell A.K.
      • Jakobsen B.K.
      Human TCR-binding affinity is governed by MHC class restriction.
      ). The importance of clonotypic composition within antigen-specific T-cell populations should therefore be taken into consideration when attempting to design optimal vaccine strategies that aim to elicit effective T-cell immunity.
      Here, we examined a system that has been popular for therapeutic vaccination and adoptive T-cell transfer for the treatment of malignant melanoma, the leading cause of skin cancer-related deaths worldwide. Melanoma immunotherapy efforts have largely focused on an 18-kDa melanocyte-specific transmembrane protein called melanoma antigen A (Melan A), also termed primary melanoma antigen recognized by T-cells (MART-1) (
      • Coulie P.G.
      • Brichard V.
      • Van Pel A.
      • Wölfel T.
      • Schneider J.
      • Traversari C.
      • Mattei S.
      • De Plaen E.
      • Lurquin C.
      • Szikora J.P.
      • Renauld J.C.
      • Boon T.
      A new gene coding for a differentiation antigen recognized by autologous cytolytic T lymphocytes on HLA-A2 melanomas.
      ,
      • Kawakami Y.
      • Eliyahu S.
      • Delgado C.H.
      • Robbins P.F.
      • Rivoltini L.
      • Topalian S.L.
      • Miki T.
      • Rosenberg S.A.
      Cloning of the gene coding for a shared human melanoma antigen recognized by autologous T cells infiltrating into tumor.
      ), which represents a good candidate target antigen because it is expressed by ∼90% of melanomas (
      • Coulie P.G.
      • Brichard V.
      • Van Pel A.
      • Wölfel T.
      • Schneider J.
      • Traversari C.
      • Mattei S.
      • De Plaen E.
      • Lurquin C.
      • Szikora J.P.
      • Renauld J.C.
      • Boon T.
      A new gene coding for a differentiation antigen recognized by autologous cytolytic T lymphocytes on HLA-A2 melanomas.
      ,
      • Kawakami Y.
      • Eliyahu S.
      • Delgado C.H.
      • Robbins P.F.
      • Rivoltini L.
      • Topalian S.L.
      • Miki T.
      • Rosenberg S.A.
      Cloning of the gene coding for a shared human melanoma antigen recognized by autologous T cells infiltrating into tumor.
      ). HLA A*0201-restricted Melan A-specific CD8+ T-cells derived from melanoma patients primarily recognize the Melan A27–35 (AAGIGILTV) and Melan A26–35 (EAAGIGILTV) peptides (
      • Kawakami Y.
      • Eliyahu S.
      • Sakaguchi K.
      • Robbins P.F.
      • Rivoltini L.
      • Yannelli J.R.
      • Appella E.
      • Rosenberg S.A.
      Identification of the immunodominant peptides of the MART-1 human melanoma antigen recognized by the majority of HLA-A2-restricted tumor infiltrating lymphocytes.
      ,
      • Romero P.
      • Gervois N.
      • Schneider J.
      • Escobar P.
      • Valmori D.
      • Pannetier C.
      • Steinle A.
      • Wolfel T.
      • Lienard D.
      • Brichard V.
      • van Pel A.
      • Jotereau F.
      • Cerottini J.C.
      Cytolytic T lymphocyte recognition of the immunodominant HLA-A*0201-restricted Melan-A/MART-1 antigenic peptide in melanoma.
      ). The AAGIGILTV peptide is the primary epitope expressed on the surface of tumor-derived cells (
      • Skipper J.C.
      • Gulden P.H.
      • Hendrickson R.C.
      • Harthun N.
      • Caldwell J.A.
      • Shabanowitz J.
      • Engelhard V.H.
      • Hunt D.F.
      • Slingluff Jr., C.L.
      Mass-spectrometric evaluation of HLA-A*0201-associated peptides identifies dominant naturally processed forms of CTL epitopes from MART-1 and gp100.
      ). There is a large HLA A*0201-restricted Melan A-specific naive T-cell pool available for manipulation in both melanoma patients and healthy donors (
      • Dutoit V.
      • Rubio-Godoy V.
      • Pittet M.J.
      • Zippelius A.
      • Dietrich P.Y.
      • Legal F.A.
      • Guillaume P.
      • Romero P.
      • Cerottini J.C.
      • Houghten R.A.
      • Pinilla C.
      • Valmori D.
      Degeneracy of antigen recognition as the molecular basis for the high frequency of naive A2/Melan-A peptide multimer+ CD8+ T cells in humans.
      ,
      • Zippelius A.
      • Pittet M.J.
      • Batard P.
      • Rufer N.
      • de Smedt M.
      • Guillaume P.
      • Ellefsen K.
      • Valmori D.
      • Liénard D.
      • Plum J.
      • MacDonald H.R.
      • Speiser D.E.
      • Cerottini J.C.
      • Romero P.
      Thymic selection generates a large T cell pool recognizing a self-peptide in humans.
      ). However, the natural antigens AAGIGILTV and EAAGIGILTV are poorly immunogenic (
      • Valmori D.
      • Fonteneau J.F.
      • Lizana C.M.
      • Gervois N.
      • Liénard D.
      • Rimoldi D.
      • Jongeneel V.
      • Jotereau F.
      • Cerottini J.C.
      • Romero P.
      Enhanced generation of specific tumor-reactive CTL in vitro by selected Melan-A/MART-1 immunodominant peptide analogues.
      ) and, consequently, this system has become widely used to champion the use of MHC anchor-modified “heteroclitic” peptides. Indeed, the heteroclitic peptide ELAGIGILTV has been used in many published studies and several vaccination trials (
      • Bins A.
      • Mallo H.
      • Sein J.
      • van den Bogaard C.
      • Nooijen W.
      • Vyth-Dreese F.
      • Nuijen B.
      • de Gast G.C.
      • Haanen J.B.
      Phase I clinical study with multiple peptide vaccines in combination with tetanus toxoid and GM-CSF in advanced-stage HLA-A*0201-positive melanoma patients.
      ,
      • Chen Q.
      • Jackson H.
      • Shackleton M.
      • Parente P.
      • Hopkins W.
      • Sturrock S.
      • MacGregor D.
      • Maraskovsky E.
      • Tai T.Y.
      • Dimopoulos N.
      • Masterman K.A.
      • Luke T.
      • Davis I.D.
      • Chen W.
      • Cebon J.
      Characterization of antigen-specific CD8+ T lymphocyte responses in skin and peripheral blood following intradermal peptide vaccination.
      ,
      • Jäger E.
      • Höhn H.
      • Necker A.
      • Förster R.
      • Karbach J.
      • Freitag K.
      • Neukirch C.
      • Castelli C.
      • Salter R.D.
      • Knuth A.
      • Maeurer M.J.
      Peptide-specific CD8+ T-cell evolution in vivo: response to peptide vaccination with Melan-A/MART-1.
      ,
      • Speiser D.E.
      • Baumgaertner P.
      • Voelter V.
      • Devevre E.
      • Barbey C.
      • Rufer N.
      • Romero P.
      Unmodified self antigen triggers human CD8 T cells with stronger tumor reactivity than altered antigen.
      ). The ELAGIGILTV peptide variant binds to HLA A*0201 with increased stability when compared with the two natural peptides and exhibits heightened immunogenicity (
      • Valmori D.
      • Fonteneau J.F.
      • Lizana C.M.
      • Gervois N.
      • Liénard D.
      • Rimoldi D.
      • Jongeneel V.
      • Jotereau F.
      • Cerottini J.C.
      • Romero P.
      Enhanced generation of specific tumor-reactive CTL in vitro by selected Melan-A/MART-1 immunodominant peptide analogues.
      ,
      • Cole D.K.
      • Edwards E.S.
      • Wynn K.K.
      • Clement M.
      • Miles J.J.
      • Ladell K.
      • Ekeruche J.
      • Gostick E.
      • Adams K.J.
      • Skowera A.
      • Peakman M.
      • Wooldridge L.
      • Price D.A.
      • Sewell A.K.
      Modification of MHC anchor residues generates heteroclitic peptides that alter TCR binding and T cell recognition.
      ,
      • Miles K.M.
      • Miles J.J.
      • Madura F.
      • Sewell A.K.
      • Cole D.K.
      Real time detection of peptide-MHC dissociation reveals that improvement of primary MHC-binding residues can have a minimal, or no, effect on stability.
      ). The shorter LAGIGILTV peptide variant adopts a similar bulged confirmation to ELAGIGILTV in the HLA A*0201 binding groove (
      • Borbulevych O.Y.
      • Insaidoo F.K.
      • Baxter T.K.
      • Powell Jr., D.J.
      • Johnson L.A.
      • Restifo N.P.
      • Baker B.M.
      Structures of MART-126/27-35 Peptide/HLA-A2 complexes reveal a remarkable disconnect between antigen structural homology and T cell recognition.
      ) and is also more immunogenic than its natural AAGIGILTV counterpart (
      • Rivoltini L.
      • Squarcina P.
      • Loftus D.J.
      • Castelli C.
      • Tarsini P.
      • Mazzocchi A.
      • Rini F.
      • Viggiano V.
      • Belli F.
      • Parmiani G.
      A superagonist variant of peptide MART1/Melan A27–35 elicits anti-melanoma CD8+ T cells with enhanced functional characteristics: implication for more effective immunotherapy.
      ). We have recently demonstrated that TCRs specific for Melan A26–35 can distinguish between the heteroclitic ELAGIGILTV variant and the natural EAAGIGILTV peptide sequence (
      • Cole D.K.
      • Edwards E.S.
      • Wynn K.K.
      • Clement M.
      • Miles J.J.
      • Ladell K.
      • Ekeruche J.
      • Gostick E.
      • Adams K.J.
      • Skowera A.
      • Peakman M.
      • Wooldridge L.
      • Price D.A.
      • Sewell A.K.
      Modification of MHC anchor residues generates heteroclitic peptides that alter TCR binding and T cell recognition.
      ). Furthermore, the ELAGIGILTV variant primes CD8+ T-cells in vitro that bear different TCRs when compared with those primed in parallel with the natural EAAGIGILTV peptide (
      • Cole D.K.
      • Edwards E.S.
      • Wynn K.K.
      • Clement M.
      • Miles J.J.
      • Ladell K.
      • Ekeruche J.
      • Gostick E.
      • Adams K.J.
      • Skowera A.
      • Peakman M.
      • Wooldridge L.
      • Price D.A.
      • Sewell A.K.
      Modification of MHC anchor residues generates heteroclitic peptides that alter TCR binding and T cell recognition.
      ) despite the fact that HLA A*0201-ELAGIGILTV and HLA A*0201-EAAGIGILTV adopt similar unligated structures (
      • Borbulevych O.Y.
      • Insaidoo F.K.
      • Baxter T.K.
      • Powell Jr., D.J.
      • Johnson L.A.
      • Restifo N.P.
      • Baker B.M.
      Structures of MART-126/27-35 Peptide/HLA-A2 complexes reveal a remarkable disconnect between antigen structural homology and T cell recognition.
      ). Indeed, recent work by Speiser et al. (
      • Speiser D.E.
      • Baumgaertner P.
      • Voelter V.
      • Devevre E.
      • Barbey C.
      • Rufer N.
      • Romero P.
      Unmodified self antigen triggers human CD8 T cells with stronger tumor reactivity than altered antigen.
      ) has shown that vaccination with the natural decapeptide (EAAGIGILTV) induces T-cells with superior lytic activity against tumor cells when compared with those induced by the commonly used heteroclitic ELAGIGILTV variant. Thus, the use of anchor-modified heteroclitic peptides requires careful re-evaluation to ensure that T-cells with the best specificity and sensitivity for the intended target are elicited (
      • Cole D.K.
      • Edwards E.S.
      • Wynn K.K.
      • Clement M.
      • Miles J.J.
      • Ladell K.
      • Ekeruche J.
      • Gostick E.
      • Adams K.J.
      • Skowera A.
      • Peakman M.
      • Wooldridge L.
      • Price D.A.
      • Sewell A.K.
      Modification of MHC anchor residues generates heteroclitic peptides that alter TCR binding and T cell recognition.
      ).
      The TCR repertoire of HLA A*0201-restricted Melan A-specific CD8+ T-cell populations is extremely diverse (
      • Cole D.K.
      • Edwards E.S.
      • Wynn K.K.
      • Clement M.
      • Miles J.J.
      • Ladell K.
      • Ekeruche J.
      • Gostick E.
      • Adams K.J.
      • Skowera A.
      • Peakman M.
      • Wooldridge L.
      • Price D.A.
      • Sewell A.K.
      Modification of MHC anchor residues generates heteroclitic peptides that alter TCR binding and T cell recognition.
      ,
      • Cole D.J.
      • Wilson M.C.
      • Rivoltini L.
      • Custer M.
      • Nishimura M.I.
      T-cell receptor repertoire in matched MART-1 peptide-stimulated peripheral blood lymphocytes and tumor-infiltrating lymphocytes.
      ,
      • Dietrich P.Y.
      • Le Gal F.A.
      • Dutoit V.
      • Pittet M.J.
      • Trautman L.
      • Zippelius A.
      • Cognet I.
      • Widmer V.
      • Walker P.R.
      • Michielin O.
      • Guillaume P.
      • Connerotte T.
      • Jotereau F.
      • Coulie P.G.
      • Romero P.
      • Cerottini J.C.
      • Bonneville M.
      • Valmori D.
      Prevalent role of TCR α-chain in the selection of the preimmune repertoire specific for a human tumor-associated self-antigen.
      ,
      • Valmori D.
      • Dutoit V.
      • Liénard D.
      • Lejeune F.
      • Speiser D.
      • Rimoldi D.
      • Cerundolo V.
      • Dietrich P.Y.
      • Cerottini J.C.
      • Romero P.
      Tetramer-guided analysis of TCR β-chain usage reveals a large repertoire of melan-A-specific CD8+ T cells in melanoma patients.
      ) and encompasses a spectrum of antigen recognition properties that influences the ability of individual T-cells to recognize the naturally expressed Melan A epitopes (
      • Valmori D.
      • Gervois N.
      • Rimoldi D.
      • Fonteneau J.F.
      • Bonelo A.
      • Liénard D.
      • Rivoltini L.
      • Jotereau F.
      • Cerottini J.C.
      • Romero P.
      Diversity of the fine specificity displayed by HLA-A*0201-restricted CTL specific for the immunodominant Melan-A/MART-1 antigenic peptide.
      ). To design an analog peptide that stimulates functionally superior T-cell clonotypes, we studied multiple Melan A-specific CD8+ T-cell clones and selected a candidate, MEL5, that recognized the dominant natural epitope, AAGIGILTV, at the lowest antigen density (
      • Cole D.K.
      • Edwards E.S.
      • Wynn K.K.
      • Clement M.
      • Miles J.J.
      • Ladell K.
      • Ekeruche J.
      • Gostick E.
      • Adams K.J.
      • Skowera A.
      • Peakman M.
      • Wooldridge L.
      • Price D.A.
      • Sewell A.K.
      Modification of MHC anchor residues generates heteroclitic peptides that alter TCR binding and T cell recognition.
      ). Unlike other Melan A-specific TCRs, the MEL5 TCR bound the natural melanoma antigen with a dissociation constant (KD) of ∼14 μm; this is one of the strongest affinities for TCR interactions with natural self tumor antigens (
      • Cole D.K.
      • Pumphrey N.J.
      • Boulter J.M.
      • Sami M.
      • Bell J.I.
      • Gostick E.
      • Price D.A.
      • Gao G.F.
      • Sewell A.K.
      • Jakobsen B.K.
      Human TCR-binding affinity is governed by MHC class restriction.
      ). We hypothesized that CD8+ T-cells with identical or similar specificities to MEL5 might represent the most desirable targets for optimal therapeutic vaccination (
      • Cole D.K.
      • Edwards E.S.
      • Wynn K.K.
      • Clement M.
      • Miles J.J.
      • Ladell K.
      • Ekeruche J.
      • Gostick E.
      • Adams K.J.
      • Skowera A.
      • Peakman M.
      • Wooldridge L.
      • Price D.A.
      • Sewell A.K.
      Modification of MHC anchor residues generates heteroclitic peptides that alter TCR binding and T cell recognition.
      ). Accordingly, we used this model system to design and test a methodology for priming high quality Melan A-specific CD8+ T-cells from the human naive T-cell pool.

      DISCUSSION

      It is possible to expand and maintain large numbers of functionally active tumor-specific CD8+ T-cells from the peripheral blood of cancer patients and healthy donors by repeated peptide-based vaccination. Despite this achievement, however, objective clinical response rates in peptide vaccine trials to date are low (<3%) and not significantly higher than spontaneous remission rates (
      • Rosenberg S.A.
      • Yang J.C.
      • Schwartzentruber D.J.
      • Hwu P.
      • Marincola F.M.
      • Topalian S.L.
      • Restifo N.P.
      • Dudley M.E.
      • Schwarz S.L.
      • Spiess P.J.
      • Wunderlich J.R.
      • Parkhurst M.R.
      • Kawakami Y.
      • Seipp C.A.
      • Einhorn J.H.
      • White D.E.
      Immunologic and therapeutic evaluation of a synthetic peptide vaccine for the treatment of patients with metastatic melanoma.
      ). One possible reason for this general lack of success is the inadvertent induction of ineffective CD8+ T-cell clonotypes with low levels of sensitivity for the natural antigenic target that are unable to control tumor growth in vivo.
      A popular approach for developing peptide candidates for vaccination is to introduce substitutions at MHC anchor residue positions that enhance the stability of MHC binding and, as a result, immunogenicity in vivo. The majority of tumor-derived HLA A*0201-restricted peptides do not contain an ideal MHC binding consensus, and the use of anchor-modified heteroclitic peptides in these systems is widespread (
      • Cole D.K.
      • Edwards E.S.
      • Wynn K.K.
      • Clement M.
      • Miles J.J.
      • Ladell K.
      • Ekeruche J.
      • Gostick E.
      • Adams K.J.
      • Skowera A.
      • Peakman M.
      • Wooldridge L.
      • Price D.A.
      • Sewell A.K.
      Modification of MHC anchor residues generates heteroclitic peptides that alter TCR binding and T cell recognition.
      ). We have recently shown that TCRs can differentiate between natural and anchor-modified heteroclitic peptides, enabling T-cells to exhibit a strong preference for either type of antigen (
      • Cole D.K.
      • Edwards E.S.
      • Wynn K.K.
      • Clement M.
      • Miles J.J.
      • Ladell K.
      • Ekeruche J.
      • Gostick E.
      • Adams K.J.
      • Skowera A.
      • Peakman M.
      • Wooldridge L.
      • Price D.A.
      • Sewell A.K.
      Modification of MHC anchor residues generates heteroclitic peptides that alter TCR binding and T cell recognition.
      ). Accordingly, MHC anchor-modified heteroclitic peptides can induce T-cell populations that are clonotypically distinct from those induced by natural tumor epitopes (
      • Cole D.K.
      • Edwards E.S.
      • Wynn K.K.
      • Clement M.
      • Miles J.J.
      • Ladell K.
      • Ekeruche J.
      • Gostick E.
      • Adams K.J.
      • Skowera A.
      • Peakman M.
      • Wooldridge L.
      • Price D.A.
      • Sewell A.K.
      Modification of MHC anchor residues generates heteroclitic peptides that alter TCR binding and T cell recognition.
      ,
      • Wieckowski S.
      • Baumgaertner P.
      • Corthesy P.
      • Voelter V.
      • Romero P.
      • Speiser D.E.
      • Rufer N.
      Fine structural variations of αβ TCRs selected by vaccination with natural versus altered self-antigen in melanoma patients.
      ). As a result, T-cells primed with MHC anchor-modified peptides can exhibit poor cross-recognition of the naturally occurring tumor antigen (
      • Speiser D.E.
      • Baumgaertner P.
      • Voelter V.
      • Devevre E.
      • Barbey C.
      • Rufer N.
      • Romero P.
      Unmodified self antigen triggers human CD8 T cells with stronger tumor reactivity than altered antigen.
      ,
      • Cole D.K.
      • Edwards E.S.
      • Wynn K.K.
      • Clement M.
      • Miles J.J.
      • Ladell K.
      • Ekeruche J.
      • Gostick E.
      • Adams K.J.
      • Skowera A.
      • Peakman M.
      • Wooldridge L.
      • Price D.A.
      • Sewell A.K.
      Modification of MHC anchor residues generates heteroclitic peptides that alter TCR binding and T cell recognition.
      ). Thus, vaccination with MHC anchor-modified peptides may elicit T-cells that exhibit suboptimal recognition of the intended natural antigen and, consequently, impaired functional attributes in vivo (
      • Cole D.K.
      • Edwards E.S.
      • Wynn K.K.
      • Clement M.
      • Miles J.J.
      • Ladell K.
      • Ekeruche J.
      • Gostick E.
      • Adams K.J.
      • Skowera A.
      • Peakman M.
      • Wooldridge L.
      • Price D.A.
      • Sewell A.K.
      Modification of MHC anchor residues generates heteroclitic peptides that alter TCR binding and T cell recognition.
      ). It is therefore important that the T-cell clonotypes induced by any APL-based immune intervention are carefully evaluated after ex vivo priming to ensure efficacy prior to studies in vivo.
      An alternative approach to improve T-cell epitopes from tumor-associated antigens is to enhance their engagement with cognate TCRs. This approach has the advantage that the resultant APLs are highly likely to be viewed as “nonself” and may therefore have an increased chance of breaking immune tolerance. However, the use of any APL also runs the risk of inducing clonotypes that are ineffective at recognizing the native sequence. Several previous studies have attempted this approach and are therefore relevant to the current discussion. Walden and colleagues (
      • Linnemann T.
      • Tumenjargal S.
      • Gellrich S.
      • Wiesmüller K.
      • Kaltoft K.
      • Sterry W.
      • Walden P.
      Mimotopes for tumor-specific T lymphocytes in human cancer determined with combinatorial peptide libraries.
      ) demonstrated that CPL scans can be used to generate mimotopes for tumor-reactive CD8+ T-cells when the natural antigen is unknown. A CPL-selected mimotope that activated a cutaneous T-cell lymphoma-reactive HLA B*08-restricted CD8+ T-cell clone was able to induce populations of T-cells that killed tumor cells in vitro (
      • Linnemann T.
      • Tumenjargal S.
      • Gellrich S.
      • Wiesmüller K.
      • Kaltoft K.
      • Sterry W.
      • Walden P.
      Mimotopes for tumor-specific T lymphocytes in human cancer determined with combinatorial peptide libraries.
      ). Vaccination of two HLA B*08+ cutaneous T-cell lymphoma patients with this mimotope induced initial tumor regression in both cases (
      • Tumenjargal S.
      • Gellrich S.
      • Linnemann T.
      • Muche J.M.
      • Lukowsky A.
      • Audring H.
      • Wiesmüller K.H.
      • Sterry W.
      • Walden P.
      Anti-tumor immune responses and tumor regression induced with mimotopes of a tumor-associated T cell epitope.
      ). As the natural antigen recognized by these HLA B*08-restricted tumor-reactive CD8+ T-cells remains unknown, it has not been possible to compare the response generated by the native peptide with that of the mimotope in this system. Nevertheless, these studies serve as a good demonstration of how TOPs can be used in a clinical setting. Other studies have attempted to use APL of known epitopes to generate better immune responses. For example, one multi-institutional effort used CPL screening of CD8+ T-cell lines to generate an optimal mimotope of the HLA A*0201-restricted HIV-1 p24 Gag-derived peptide TLNAWVKV (
      • Schaubert K.L.
      • Price D.A.
      • Salkowitz J.R.
      • Sewell A.K.
      • Sidney J.
      • Asher T.E.
      • Blondelle S.E.
      • Adams S.
      • Marincola F.M.
      • Joseph A.
      • Sette A.
      • Douek D.C.
      • Ayyavoo V.
      • Storkus W.
      • Leung M.Y.
      • Ng H.L.
      • Yang O.O.
      • Goldstein H.
      • Wilson D.B.
      • Kan-Mitchell J.
      Generation of robust CD8+ T-cell responses against subdominant epitopes in conserved regions of HIV-1 by repertoire mining with mimotopes.
      ). This mimotope failed to mobilize clonotypes that were more efficient at recognizing natural targets (
      • Schaubert K.L.
      • Price D.A.
      • Salkowitz J.R.
      • Sewell A.K.
      • Sidney J.
      • Asher T.E.
      • Blondelle S.E.
      • Adams S.
      • Marincola F.M.
      • Joseph A.
      • Sette A.
      • Douek D.C.
      • Ayyavoo V.
      • Storkus W.
      • Leung M.Y.
      • Ng H.L.
      • Yang O.O.
      • Goldstein H.
      • Wilson D.B.
      • Kan-Mitchell J.
      Generation of robust CD8+ T-cell responses against subdominant epitopes in conserved regions of HIV-1 by repertoire mining with mimotopes.
      ). A similar approach has been attempted to improve CD8+ T-cell responses to human carcinoembryonic antigen (CEA). CEA is expressed in the majority of colonic, rectal, gastric, and pancreatic tumors (
      • Muraro R.
      • Wunderlich D.
      • Thor A.
      • Lundy J.
      • Noguchi P.
      • Cunningham R.
      • Schlom J.
      Definition by monoclonal antibodies of a repertoire of epitopes on carcinoembryonic antigen differentially expressed in human colon carcinomas versus normal adult tissues.
      ), 70% of lung carcinomas (
      • Vincent R.G.
      • Chu T.M.
      • Lane W.W.
      • Gutierrez A.C.
      • Stegemann P.J.
      • Madajewicz S.
      Carcinoembryonic antigen as a monitor of successful surgical resection in 130 patients with carcinoma of the lung.
      ), and 50% of breast carcinomas (
      • Steward A.M.
      • Nixon D.
      • Zamcheck N.
      • Aisenberg A.
      Carcinoembryonic antigen in breast cancer patients: serum levels and disease progress.
      ), and there has been interest in a CEA-derived, HLA A*0201-restricted epitope (YLSGANLNL) known as CAP1 (
      • Zaremba S.
      • Barzaga E.
      • Zhu M.
      • Soares N.
      • Tsang K.Y.
      • Schlom J.
      Identification of an enhancer agonist cytotoxic T lymphocyte peptide from human carcinoembryonic antigen.
      ). Substitution of CAP1 at position 6, a TCR contact residue, with aspartic acid to generate the APL YLSGADLNL enhanced CAP1-specific CD8+ T-cell recognition in amplified lines by >100-fold when compared with the native CAP1 peptide (
      • Zaremba S.
      • Barzaga E.
      • Zhu M.
      • Soares N.
      • Tsang K.Y.
      • Schlom J.
      Identification of an enhancer agonist cytotoxic T lymphocyte peptide from human carcinoembryonic antigen.
      ). This superagonist variant, designated CAP1-6D, induced substantially larger responses in vitro when compared with the native sequence (
      • Zaremba S.
      • Barzaga E.
      • Zhu M.
      • Soares N.
      • Tsang K.Y.
      • Schlom J.
      Identification of an enhancer agonist cytotoxic T lymphocyte peptide from human carcinoembryonic antigen.
      ). Furthermore, CAP1-6D-generated CD8+ T-cell lines killed CEA-expressing human tumor lines efficiently (
      • Zaremba S.
      • Barzaga E.
      • Zhu M.
      • Soares N.
      • Tsang K.Y.
      • Schlom J.
      Identification of an enhancer agonist cytotoxic T lymphocyte peptide from human carcinoembryonic antigen.
      ). Subsequent studies in this system, however, demonstrated that the predominant CD8+ T-cells primed with the CAP-6D peptide displayed restricted TCR usage and lower levels of sensitivity for the natural antigen (
      • Hou Y.
      • Kavanagh B.
      • Fong L.
      Distinct CD8+ T cell repertoires primed with agonist and native peptides derived from a tumor-associated antigen.
      ). Thus, previous studies with TOPs have failed to improve either the functional qualities of the selected clonotypes or the overall efficacy of the T-cell response in comparison with the native antigen (
      • Speiser D.E.
      • Baumgaertner P.
      • Voelter V.
      • Devevre E.
      • Barbey C.
      • Rufer N.
      • Romero P.
      Unmodified self antigen triggers human CD8 T cells with stronger tumor reactivity than altered antigen.
      ,
      • Schaubert K.L.
      • Price D.A.
      • Salkowitz J.R.
      • Sewell A.K.
      • Sidney J.
      • Asher T.E.
      • Blondelle S.E.
      • Adams S.
      • Marincola F.M.
      • Joseph A.
      • Sette A.
      • Douek D.C.
      • Ayyavoo V.
      • Storkus W.
      • Leung M.Y.
      • Ng H.L.
      • Yang O.O.
      • Goldstein H.
      • Wilson D.B.
      • Kan-Mitchell J.
      Generation of robust CD8+ T-cell responses against subdominant epitopes in conserved regions of HIV-1 by repertoire mining with mimotopes.
      ).
      Unlike the process described herein, however, these previous approaches did not use improved ligands for the best T-cell clonotype. The optimized HIV-1 p24 Gag-derived peptide used by Kan-Mitchell and colleagues (
      • Schaubert K.L.
      • Price D.A.
      • Salkowitz J.R.
      • Sewell A.K.
      • Sidney J.
      • Asher T.E.
      • Blondelle S.E.
      • Adams S.
      • Marincola F.M.
      • Joseph A.
      • Sette A.
      • Douek D.C.
      • Ayyavoo V.
      • Storkus W.
      • Leung M.Y.
      • Ng H.L.
      • Yang O.O.
      • Goldstein H.
      • Wilson D.B.
      • Kan-Mitchell J.
      Generation of robust CD8+ T-cell responses against subdominant epitopes in conserved regions of HIV-1 by repertoire mining with mimotopes.
      ) was selected because it exhibited improved recognition by multiple polyclonal CD8+ T-cell lines. This approach does not optimize a ligand for a specific and beneficial functional phenotype, and so is equally likely to improve recognition by good and poor clonotypes alike. Such “universal” optimization may also combine with the selection of APL-specific clonotypes that recognize the natural sequence poorly to ensure that although the magnitude of the APL-induced response might far exceed that generated by the natural antigen, the overall quality of the mobilized T-cell population with respect to recognition of the intended natural target will be inferior.
      The approach adopted here incorporates some fundamental differences to the attempts that preceded it. Most notably, we started by selecting a CD8+ T-cell clonotype with superior natural target antigen recognition properties. In this way, we hoped to skew the expanded CD8+ T-cell population toward clonotypes with optimal antitumor efficacy. We describe this approach as “TCR-optimized peptide skewing of the repertoire of T-cells” (TOPSORT). The primary natural Melan A-derived antigen on the surface of melanoma cells, AAGIGILTV (
      • Skipper J.C.
      • Gulden P.H.
      • Hendrickson R.C.
      • Harthun N.
      • Caldwell J.A.
      • Shabanowitz J.
      • Engelhard V.H.
      • Hunt D.F.
      • Slingluff Jr., C.L.
      Mass-spectrometric evaluation of HLA-A*0201-associated peptides identifies dominant naturally processed forms of CTL epitopes from MART-1 and gp100.
      ), is an extremely poor immunogen. Accordingly, the highly immunogenic MHC anchor-modified peptide ELAGIGILTV has been widely adopted in the clinic (
      • Bins A.
      • Mallo H.
      • Sein J.
      • van den Bogaard C.
      • Nooijen W.
      • Vyth-Dreese F.
      • Nuijen B.
      • de Gast G.C.
      • Haanen J.B.
      Phase I clinical study with multiple peptide vaccines in combination with tetanus toxoid and GM-CSF in advanced-stage HLA-A*0201-positive melanoma patients.
      ,
      • Chen Q.
      • Jackson H.
      • Shackleton M.
      • Parente P.
      • Hopkins W.
      • Sturrock S.
      • MacGregor D.
      • Maraskovsky E.
      • Tai T.Y.
      • Dimopoulos N.
      • Masterman K.A.
      • Luke T.
      • Davis I.D.
      • Chen W.
      • Cebon J.
      Characterization of antigen-specific CD8+ T lymphocyte responses in skin and peripheral blood following intradermal peptide vaccination.
      ,
      • Jäger E.
      • Höhn H.
      • Necker A.
      • Förster R.
      • Karbach J.
      • Freitag K.
      • Neukirch C.
      • Castelli C.
      • Salter R.D.
      • Knuth A.
      • Maeurer M.J.
      Peptide-specific CD8+ T-cell evolution in vivo: response to peptide vaccination with Melan-A/MART-1.
      ,
      • Speiser D.E.
      • Baumgaertner P.
      • Voelter V.
      • Devevre E.
      • Barbey C.
      • Rufer N.
      • Romero P.
      Unmodified self antigen triggers human CD8 T cells with stronger tumor reactivity than altered antigen.
      ). This approach has failed, however, and it has become apparent that HLA A*0201-ELAGIGILTV is recognized differently by TCRs when compared with the natural antigen (
      • Cole D.K.
      • Edwards E.S.
      • Wynn K.K.
      • Clement M.
      • Miles J.J.
      • Ladell K.
      • Ekeruche J.
      • Gostick E.
      • Adams K.J.
      • Skowera A.
      • Peakman M.
      • Wooldridge L.
      • Price D.A.
      • Sewell A.K.
      Modification of MHC anchor residues generates heteroclitic peptides that alter TCR binding and T cell recognition.
      ). Consequently, HLA A*0201-ELAGIGILTV can induce populations of T-cells that bear TCRs with poor reactivity against the natural antigen (
      • Cole D.K.
      • Edwards E.S.
      • Wynn K.K.
      • Clement M.
      • Miles J.J.
      • Ladell K.
      • Ekeruche J.
      • Gostick E.
      • Adams K.J.
      • Skowera A.
      • Peakman M.
      • Wooldridge L.
      • Price D.A.
      • Sewell A.K.
      Modification of MHC anchor residues generates heteroclitic peptides that alter TCR binding and T cell recognition.
      ). The naive Melan A-specific T-cell population is characterized by a highly diverse TCR repertoire and, as a result, exhibits a large degree of functional diversity. We selected a Melan A-specific CD8+ T-cell clone (MEL5) that displayed potent recognition of the natural tumor epitopes EAAGIGILTV and AAGIGILTV and then used CPL scan technology to identify analog peptides that exhibited improved recognition by this clonotype. The CPL scan data revealed that MEL5 exhibited a preference for phenylalanine, threonine, and isoleucine at positions 1, 3, and 8 of the antigenic peptide, respectively. Insertion of the triple mutation into the EAAGIGILTV peptide sequence produced the FATGIGIITV peptide. Biophysical data demonstrated that HLA A*0201-FATGIGIITV bound the MEL5 TCR with an affinity >10-fold stronger than that measured for the alternative MEL187.c5 TCR. These findings were confirmed with cell surface pMHCI tetramer staining experiments. As a consequence, the FATGIGIITV peptide was a poor stimulator of the MEL187.c5 clone, which preferentially recognizes ELAGIGILTV, yet potently stimulated the MEL5 clone, which preferentially recognizes the natural peptide antigens. Indeed, the MEL5 TCR engaged the dominant natural antigen (HLA A*0201-AAGIGILTV) with a relatively high affinity (KD ∼14 μm), which explained the antitumor efficacy of the MEL5 clone. The magnitude of the HLA A*0201-EAAGIGILTV tetramer+ CD8+ T-cell population primed by FATGIGIITV exceeded that primed by ELAGIGILTV in 6/10 donors tested and was comparable or reduced in 4/10 donors. Furthermore, FATGIGIITV-primed CD8+ T-cells were clonotypically distinct from those induced by the ELAGIGILTV peptide and were capable of enhanced melanoma cell killing in 36% of donors. Notably, clonotypic analysis has revealed that the MEL5 TCR β-chain (Vβ 20.1) was not ubiquitously expressed in all donors (
      • Pittet M.J.
      • Gati A.
      • Le Gal F.A.
      • Bioley G.
      • Guillaume P.
      • de Smedt M.
      • Plum J.
      • Speiser D.E.
      • Cerottini J.C.
      • Dietrich P.Y.
      • Romero P.
      • Zippelius A.
      Ex vivo characterization of allo-MHC-restricted T cells specific for a single MHC-peptide complex.
      ). Therefore, although FATGIGIITV improved antigen recognition by the Melan A-sensitive MEL5 clone, this clonotype might be absent in some donors, which likely explains why this approach was not universally superior when compared with priming with ELAGIGILTV. Thus, the availability of highly shared, or public, clonotypes may dictate the overall applicability of TOPSORT (
      • Venturi V.
      • Price D.A.
      • Douek D.C.
      • Davenport M.P.
      The molecular basis for public T-cell responses?.
      ). It is not known whether there is an effective public clonotype for the HLA A*0201-AAGIGILTV antigen.
      The observation that the FATGIGIITV peptide elicits CD8+ T-cell populations with a different clonotypic architecture when compared with ELAGIGILTV raises the possibility that some unwanted or autoreactive T-cell clones might be selected. This seems unlikely, however, because the primed clonotypes in both cases will have been through the same thymic editing process. Combined with peripheral tolerance mechanisms, there is therefore no a priori reason to suspect that FATGIGIITV-primed autologous T-cells will be more deleterious than autologous T-cells primed with other natural or heteroclitic peptides.
      The TOPSORT process used to generate the FATGIGIITV sequence selected in this study differs in a number of key aspects from previous studies. These differences include: (i) identification of a high quality T-cell clonotype from the polyclonal population induced by the natural antigen; (ii) selection of a TOP superagonist for this clonotype; (iii) elimination of mutations that favor both “good” and “bad” clonotypes; and (iv) verification that superior functional qualities are induced in TOP-primed responses relative to those primed by the natural antigen. The work described here represents a proof-of-concept study for the use of TOPSORT, although confirmation of efficacy in vivo is still required. Nonetheless, combined with emerging next generation sequencing strategies for comprehensive TCR repertoire analysis, it can be envisaged that this approach might enable the rational design of individualized peptide-based vaccines.

      Acknowledgments

      We thank the staff at Diamond Light Source for providing facilities and support.

      REFERENCES

        • Davis M.M.
        • Bjorkman P.J.
        T-cell antigen receptor genes and T-cell recognition.
        Nature. 1988; 334: 395-402
        • Garcia K.C.
        • Teyton L.
        • Wilson I.A.
        Structural basis of T cell recognition.
        Annu. Rev. Immunol. 1999; 17: 369-397
        • Arstila T.P.
        • Casrouge A.
        • Baron V.
        • Even J.
        • Kanellopoulos J.
        • Kourilsky P.
        A direct estimate of the human αβ T cell receptor diversity.
        Science. 1999; 286: 958-961
        • Mason D.
        A very high level of cross-reactivity is an essential feature of the T-cell receptor.
        Immunol. Today. 1998; 19: 395-404
        • Wooldridge L.
        • Ekeruche-Makinde J.
        • van den Berg H.A.
        • Skowera A.
        • Miles J.J.
        • Tan M.P.
        • Dolton G.
        • Clement M.
        • Llewellyn-Lacey S.
        • Price D.A.
        • Peakman M.
        • Sewell A.K.
        A single autoimmune T cell receptor recognizes more than a million different peptides.
        J. Biol. Chem. 2012; 287: 1168-1177
        • Sewell A.K.
        Why must T cells be cross-reactive?.
        Nat. Rev. Immunol. 2012; 12: 669-677
        • Casanova J.L.
        • Maryanski J.L.
        Antigen-selected T-cell receptor diversity and self-nonself homology.
        Immunol. Today. 1993; 14: 391-394
        • Maryanski J.L.
        • Attuil V.
        • Bucher P.
        • Walker P.R.
        A quantitative, single-cell PCR analysis of an antigen-specific TCR repertoire selected during an in vivo CD8 response: direct evidence for a wide range of clone sizes with uniform tissue distribution.
        Mol. Immunol. 1999; 36: 745-753
        • Price D.A.
        • Brenchley J.M.
        • Ruff L.E.
        • Betts M.R.
        • Hill B.J.
        • Roederer M.
        • Koup R.A.
        • Migueles S.A.
        • Gostick E.
        • Wooldridge L.
        • Sewell A.K.
        • Connors M.
        • Douek D.C.
        Avidity for antigen shapes clonal dominance in CD8+ T cell populations specific for persistent DNA viruses.
        J. Exp. Med. 2005; 202: 1349-1361
        • Appay V.
        • Douek D.C.
        • Price D.A.
        CD8+ T cell efficacy in vaccination and disease.
        Nat. Med. 2008; 14: 623-628
        • Bangham C.R.
        CTL quality and the control of human retroviral infections.
        Eur. J. Immunol. 2009; 39: 1700-1712
        • Seder R.A.
        • Darrah P.A.
        • Roederer M.
        T-cell quality in memory and protection: implications for vaccine design.
        Nat. Rev. Immunol. 2008; 8: 247-258
        • Price D.A.
        • West S.M.
        • Betts M.R.
        • Ruff L.E.
        • Brenchley J.M.
        • Ambrozak D.R.
        • Edghill-Smith Y.
        • Kuroda M.J.
        • Bogdan D.
        • Kunstman K.
        • Letvin N.L.
        • Franchini G.
        • Wolinsky S.M.
        • Koup R.A.
        • Douek D.C.
        T cell receptor recognition motifs govern immune escape patterns in acute SIV infection.
        Immunity. 2004; 21: 793-803
        • Price D.A.
        • Asher T.E.
        • Wilson N.A.
        • Nason M.C.
        • Brenchley J.M.
        • Metzler I.S.
        • Venturi V.
        • Gostick E.
        • Chattopadhyay P.K.
        • Roederer M.
        • Davenport M.P.
        • Watkins D.I.
        • Douek D.C.
        Public clonotype usage identifies protective Gag-specific CD8+ T cell responses in SIV infection.
        J. Exp. Med. 2009; 206: 923-936
        • Alexander-Miller M.A.
        • Leggatt G.R.
        • Berzofsky J.A.
        Selective expansion of high- or low-avidity cytotoxic T lymphocytes and efficacy for adoptive immunotherapy.
        Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 4102-4107
        • Matsui K.
        • Boniface J.J.
        • Steffner P.
        • Reay P.A.
        • Davis M.M.
        Kinetics of T-cell receptor binding to peptide/I-Ek complexes: correlation of the dissociation rate with T-cell responsiveness.
        Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 12862-12866
        • Bridgeman J.S.
        • Sewell A.K.
        • Miles J.J.
        • Price D.A.
        • Cole D.K.
        Structural and biophysical determinants of αβ T-cell antigen recognition.
        Immunology. 2012; 135: 9-18
        • Laugel B.
        • van den Berg H.A.
        • Gostick E.
        • Cole D.K.
        • Wooldridge L.
        • Boulter J.
        • Milicic A.
        • Price D.A.
        • Sewell A.K.
        Different T cell receptor affinity thresholds and CD8 coreceptor dependence govern cytotoxic T lymphocyte activation and tetramer binding properties.
        J. Biol. Chem. 2007; 282: 23799-23810
        • Varela-Rohena A.
        • Molloy P.E.
        • Dunn S.M.
        • Li Y.
        • Suhoski M.M.
        • Carroll R.G.
        • Milicic A.
        • Mahon T.
        • Sutton D.H.
        • Laugel B.
        • Moysey R.
        • Cameron B.J.
        • Vuidepot A.
        • Purbhoo M.A.
        • Cole D.K.
        • Phillips R.E.
        • June C.H.
        • Jakobsen B.K.
        • Sewell A.K.
        • Riley J.L.
        Control of HIV-1 immune escape by CD8 T cells expressing enhanced T-cell receptor.
        Nat. Med. 2008; 14: 1390-1395
        • Liddy N.
        • Bossi G.
        • Adams K.J.
        • Lissina A.
        • Mahon T.M.
        • Hassan N.J.
        • Gavarret J.
        • Bianchi F.C.
        • Pumphrey N.J.
        • Ladell K.
        • Gostick E.
        • Sewell A.K.
        • Lissin N.M.
        • Harwood N.E.
        • Molloy P.E.
        • Li Y.
        • Cameron B.J.
        • Sami M.
        • Baston E.E.
        • Todorov P.T.
        • Paston S.J.
        • Dennis R.E.
        • Harper J.V.
        • Dunn S.M.
        • Ashfield R.
        • Johnson A.
        • McGrath Y.
        • Plesa G.
        • June C.H.
        • Kalos M.
        • Price D.A.
        • Vuidepot A.
        • Williams D.D.
        • Sutton D.H.
        • Jakobsen B.K.
        Monoclonal TCR-redirected tumor cell killing.
        Nat. Med. 2012; 18: 980-987
        • Cole D.K.
        • Pumphrey N.J.
        • Boulter J.M.
        • Sami M.
        • Bell J.I.
        • Gostick E.
        • Price D.A.
        • Gao G.F.
        • Sewell A.K.
        • Jakobsen B.K.
        Human TCR-binding affinity is governed by MHC class restriction.
        J. Immunol. 2007; 178: 5727-5734
        • Coulie P.G.
        • Brichard V.
        • Van Pel A.
        • Wölfel T.
        • Schneider J.
        • Traversari C.
        • Mattei S.
        • De Plaen E.
        • Lurquin C.
        • Szikora J.P.
        • Renauld J.C.
        • Boon T.
        A new gene coding for a differentiation antigen recognized by autologous cytolytic T lymphocytes on HLA-A2 melanomas.
        J. Exp. Med. 1994; 180: 35-42
        • Kawakami Y.
        • Eliyahu S.
        • Delgado C.H.
        • Robbins P.F.
        • Rivoltini L.
        • Topalian S.L.
        • Miki T.
        • Rosenberg S.A.
        Cloning of the gene coding for a shared human melanoma antigen recognized by autologous T cells infiltrating into tumor.
        Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 3515-3519
        • Kawakami Y.
        • Eliyahu S.
        • Sakaguchi K.
        • Robbins P.F.
        • Rivoltini L.
        • Yannelli J.R.
        • Appella E.
        • Rosenberg S.A.
        Identification of the immunodominant peptides of the MART-1 human melanoma antigen recognized by the majority of HLA-A2-restricted tumor infiltrating lymphocytes.
        J. Exp. Med. 1994; 180: 347-352
        • Romero P.
        • Gervois N.
        • Schneider J.
        • Escobar P.
        • Valmori D.
        • Pannetier C.
        • Steinle A.
        • Wolfel T.
        • Lienard D.
        • Brichard V.
        • van Pel A.
        • Jotereau F.
        • Cerottini J.C.
        Cytolytic T lymphocyte recognition of the immunodominant HLA-A*0201-restricted Melan-A/MART-1 antigenic peptide in melanoma.
        J. Immunol. 1997; 159: 2366-2374
        • Skipper J.C.
        • Gulden P.H.
        • Hendrickson R.C.
        • Harthun N.
        • Caldwell J.A.
        • Shabanowitz J.
        • Engelhard V.H.
        • Hunt D.F.
        • Slingluff Jr., C.L.
        Mass-spectrometric evaluation of HLA-A*0201-associated peptides identifies dominant naturally processed forms of CTL epitopes from MART-1 and gp100.
        Int. J. Cancer. 1999; 82: 669-677
        • Dutoit V.
        • Rubio-Godoy V.
        • Pittet M.J.
        • Zippelius A.
        • Dietrich P.Y.
        • Legal F.A.
        • Guillaume P.
        • Romero P.
        • Cerottini J.C.
        • Houghten R.A.
        • Pinilla C.
        • Valmori D.
        Degeneracy of antigen recognition as the molecular basis for the high frequency of naive A2/Melan-A peptide multimer+ CD8+ T cells in humans.
        J. Exp. Med. 2002; 196: 207-216
        • Zippelius A.
        • Pittet M.J.
        • Batard P.
        • Rufer N.
        • de Smedt M.
        • Guillaume P.
        • Ellefsen K.
        • Valmori D.
        • Liénard D.
        • Plum J.
        • MacDonald H.R.
        • Speiser D.E.
        • Cerottini J.C.
        • Romero P.
        Thymic selection generates a large T cell pool recognizing a self-peptide in humans.
        J. Exp. Med. 2002; 195: 485-494
        • Valmori D.
        • Fonteneau J.F.
        • Lizana C.M.
        • Gervois N.
        • Liénard D.
        • Rimoldi D.
        • Jongeneel V.
        • Jotereau F.
        • Cerottini J.C.
        • Romero P.
        Enhanced generation of specific tumor-reactive CTL in vitro by selected Melan-A/MART-1 immunodominant peptide analogues.
        J. Immunol. 1998; 160: 1750-1758
        • Bins A.
        • Mallo H.
        • Sein J.
        • van den Bogaard C.
        • Nooijen W.
        • Vyth-Dreese F.
        • Nuijen B.
        • de Gast G.C.
        • Haanen J.B.
        Phase I clinical study with multiple peptide vaccines in combination with tetanus toxoid and GM-CSF in advanced-stage HLA-A*0201-positive melanoma patients.
        J. Immunother. 2007; 30: 234-239
        • Chen Q.
        • Jackson H.
        • Shackleton M.
        • Parente P.
        • Hopkins W.
        • Sturrock S.
        • MacGregor D.
        • Maraskovsky E.
        • Tai T.Y.
        • Dimopoulos N.
        • Masterman K.A.
        • Luke T.
        • Davis I.D.
        • Chen W.
        • Cebon J.
        Characterization of antigen-specific CD8+ T lymphocyte responses in skin and peripheral blood following intradermal peptide vaccination.
        Cancer Immun. 2005; 5: 5
        • Jäger E.
        • Höhn H.
        • Necker A.
        • Förster R.
        • Karbach J.
        • Freitag K.
        • Neukirch C.
        • Castelli C.
        • Salter R.D.
        • Knuth A.
        • Maeurer M.J.
        Peptide-specific CD8+ T-cell evolution in vivo: response to peptide vaccination with Melan-A/MART-1.
        Int. J. Cancer. 2002; 98: 376-388
        • Speiser D.E.
        • Baumgaertner P.
        • Voelter V.
        • Devevre E.
        • Barbey C.
        • Rufer N.
        • Romero P.
        Unmodified self antigen triggers human CD8 T cells with stronger tumor reactivity than altered antigen.
        Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 3849-3854
        • Cole D.K.
        • Edwards E.S.
        • Wynn K.K.
        • Clement M.
        • Miles J.J.
        • Ladell K.
        • Ekeruche J.
        • Gostick E.
        • Adams K.J.
        • Skowera A.
        • Peakman M.
        • Wooldridge L.
        • Price D.A.
        • Sewell A.K.
        Modification of MHC anchor residues generates heteroclitic peptides that alter TCR binding and T cell recognition.
        J. Immunol. 2010; 185: 2600-2610
        • Miles K.M.
        • Miles J.J.
        • Madura F.
        • Sewell A.K.
        • Cole D.K.
        Real time detection of peptide-MHC dissociation reveals that improvement of primary MHC-binding residues can have a minimal, or no, effect on stability.
        Mol. Immunol. 2011; 48: 728-732
        • Borbulevych O.Y.
        • Insaidoo F.K.
        • Baxter T.K.
        • Powell Jr., D.J.
        • Johnson L.A.
        • Restifo N.P.
        • Baker B.M.
        Structures of MART-126/27-35 Peptide/HLA-A2 complexes reveal a remarkable disconnect between antigen structural homology and T cell recognition.
        J. Mol. Biol. 2007; 372: 1123-1136
        • Rivoltini L.
        • Squarcina P.
        • Loftus D.J.
        • Castelli C.
        • Tarsini P.
        • Mazzocchi A.
        • Rini F.
        • Viggiano V.
        • Belli F.
        • Parmiani G.
        A superagonist variant of peptide MART1/Melan A27–35 elicits anti-melanoma CD8+ T cells with enhanced functional characteristics: implication for more effective immunotherapy.
        Cancer Res. 1999; 59: 301-306
        • Cole D.J.
        • Wilson M.C.
        • Rivoltini L.
        • Custer M.
        • Nishimura M.I.
        T-cell receptor repertoire in matched MART-1 peptide-stimulated peripheral blood lymphocytes and tumor-infiltrating lymphocytes.
        Cancer Res. 1997; 57: 5320-5327
        • Dietrich P.Y.
        • Le Gal F.A.
        • Dutoit V.
        • Pittet M.J.
        • Trautman L.
        • Zippelius A.
        • Cognet I.
        • Widmer V.
        • Walker P.R.
        • Michielin O.
        • Guillaume P.
        • Connerotte T.
        • Jotereau F.
        • Coulie P.G.
        • Romero P.
        • Cerottini J.C.
        • Bonneville M.
        • Valmori D.
        Prevalent role of TCR α-chain in the selection of the preimmune repertoire specific for a human tumor-associated self-antigen.
        J. Immunol. 2003; 170: 5103-5109
        • Valmori D.
        • Dutoit V.
        • Liénard D.
        • Lejeune F.
        • Speiser D.
        • Rimoldi D.
        • Cerundolo V.
        • Dietrich P.Y.
        • Cerottini J.C.
        • Romero P.
        Tetramer-guided analysis of TCR β-chain usage reveals a large repertoire of melan-A-specific CD8+ T cells in melanoma patients.
        J. Immunol. 2000; 165: 533-538
        • Valmori D.
        • Gervois N.
        • Rimoldi D.
        • Fonteneau J.F.
        • Bonelo A.
        • Liénard D.
        • Rivoltini L.
        • Jotereau F.
        • Cerottini J.C.
        • Romero P.
        Diversity of the fine specificity displayed by HLA-A*0201-restricted CTL specific for the immunodominant Melan-A/MART-1 antigenic peptide.
        J. Immunol. 1998; 161: 6956-6962
        • Johnson L.A.
        • Heemskerk B.
        • Powell Jr., D.J.
        • Cohen C.J.
        • Morgan R.A.
        • Dudley M.E.
        • Robbins P.F.
        • Rosenberg S.A.
        Gene transfer of tumor-reactive TCR confers both high avidity and tumor reactivity to nonreactive peripheral blood mononuclear cells and tumor-infiltrating lymphocytes.
        J. Immunol. 2006; 177: 6548-6559
        • Topalian S.L.
        • Solomon D.
        • Rosenberg S.A.
        Tumor-specific cytolysis by lymphocytes infiltrating human melanomas.
        J. Immunol. 1989; 142: 3714-3725
        • Wooldridge L.
        • Lissina A.
        • Vernazza J.
        • Gostick E.
        • Laugel B.
        • Hutchinson S.L.
        • Mirza F.
        • Dunbar P.R.
        • Boulter J.M.
        • Glick M.
        • Cerundolo V.
        • van den Berg H.A.
        • Price D.A.
        • Sewell A.K.
        Enhanced immunogenicity of CTL antigens through mutation of the CD8 binding MHC class I invariant region.
        Eur. J. Immunol. 2007; 37: 1323-1333
        • Kan-Mitchell J.
        • Bajcz M.
        • Schaubert K.L.
        • Price D.A.
        • Brenchley J.M.
        • Asher T.E.
        • Douek D.C.
        • Ng H.L.
        • Yang O.O.
        • Rinaldo Jr., C.R.
        • Benito J.M.
        • Bisikirska B.
        • Hegde R.
        • Marincola F.M.
        • Boggiano C.
        • Wilson D.
        • Abrams J.
        • Blondelle S.E.
        • Wilson D.B.
        Degeneracy and repertoire of the human HIV-1 Gag p1777–85 CTL response.
        J. Immunol. 2006; 176: 6690-6701
        • Wilson D.B.
        • Wilson D.H.
        • Schroder K.
        • Pinilla C.
        • Blondelle S.
        • Houghten R.A.
        • Garcia K.C.
        Specificity and degeneracy of T cells.
        Mol. Immunol. 2004; 40: 1047-1055
        • Wooldridge L.
        • Laugel B.
        • Ekeruche J.
        • Clement M.
        • van den Berg H.A.
        • Price D.A.
        • Sewell A.K.
        CD8 controls T cell cross-reactivity.
        J. Immunol. 2010; 185: 4625-4632
        • Cole D.K.
        • Dunn S.M.
        • Sami M.
        • Boulter J.M.
        • Jakobsen B.K.
        • Sewell A.K.
        T cell receptor engagement of peptide-major histocompatibility complex class I does not modify CD8 binding.
        Mol. Immunol. 2008; 45: 2700-2709
        • Wooldridge L.
        • van den Berg H.A.
        • Glick M.
        • Gostick E.
        • Laugel B.
        • Hutchinson S.L.
        • Milicic A.
        • Brenchley J.M.
        • Douek D.C.
        • Price D.A.
        • Sewell A.K.
        Interaction between the CD8 coreceptor and major histocompatibility complex class I stabilizes T cell receptor-antigen complexes at the cell surface.
        J. Biol. Chem. 2005; 280: 27491-27501
        • Cole D.K.
        • Yuan F.
        • Rizkallah P.J.
        • Miles J.J.
        • Gostick E.
        • Price D.A.
        • Gao G.F.
        • Jakobsen B.K.
        • Sewell A.K.
        Germ line-governed recognition of a cancer epitope by an immunodominant human T-cell receptor.
        J. Biol. Chem. 2009; 284: 27281-27289
        • Cole D.K.
        • Rizkallah P.J.
        • Boulter J.M.
        • Sami M.
        • Vuidepot A.L.
        • Glick M.
        • Gao F.
        • Bell J.I.
        • Jakobsen B.K.
        • Gao G.F.
        Computational design and crystal structure of an enhanced affinity mutant human CD8 αα coreceptor.
        Proteins. 2007; 67: 65-74
        • Khan A.R.
        • Baker B.M.
        • Ghosh P.
        • Biddison W.E.
        • Wiley D.C.
        The structure and stability of an HLA-A*0201/octameric tax peptide complex with an empty conserved peptide-N-terminal binding site.
        J. Immunol. 2000; 164: 6398-6405
        • Leslie A.
        Joint CCP4 and ESF-EACMB Newsletter. Collaborative Computational Project Number 4, Oxon, UK1992
        • Collaborative Computational Project, Number 4
        The CCP4 suite: programs for protein crystallography.
        Acta Crystallogr. D Biol. Crystallogr. 1994; 50: 760-763
        • McCoy A.J.
        • Grosse-Kunstleve R.W.
        • Storoni L.C.
        • Read R.J.
        Likelihood-enhanced fast translation functions.
        Acta Crystallogr. D Biol. Crystallogr. 2005; 61: 458-464
        • Emsley P.
        • Cowtan K.
        Coot: model-building tools for molecular graphics.
        Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132
        • Murshudov G.N.
        • Vagin A.A.
        • Dodson E.J.
        Refinement of macromolecular structures by the maximum-likelihood method.
        Acta Crystallogr. D Biol. Crystallogr. 1997; 53: 240-255
        • DeLano W.L.
        The PyMOL Molecular Graphics System. Schrödinger, LLC, New York2010 (version 1.3r1)
        • Speiser D.E.
        • Liénard D.
        • Pittet M.J.
        • Batard P.
        • Rimoldi D.
        • Guillaume P.
        • Cerottini J.C.
        • Romero P.
        In vivo activation of melanoma-specific CD8+ T cells by endogenous tumor antigen and peptide vaccines: a comparison to virus-specific T cells.
        Eur. J. Immunol. 2002; 32: 731-741
        • Ayyoub M.
        • Zippelius A.
        • Pittet M.J.
        • Rimoldi D.
        • Valmori D.
        • Cerottini J.C.
        • Romero P.
        • Lejeune F.
        • Liénard D.
        • Speiser D.E.
        Activation of human melanoma reactive CD8+ T cells by vaccination with an immunogenic peptide analog derived from Melan-A/melanoma antigen recognized by T cells-1.
        Clin. Cancer Res. 2003; 9: 669-677
        • Derré L.
        • Ferber M.
        • Touvrey C.
        • Devevre E.
        • Zoete V.
        • Leimgruber A.
        • Romero P.
        • Michielin O.
        • Lévy F.
        • Speiser D.E.
        A novel population of human melanoma-specific CD8 T cells recognizes Melan-AMART-1 immunodominant nonapeptide but not the corresponding decapeptide.
        J. Immunol. 2007; 179: 7635-7645
        • Hunt D.F.
        • Henderson R.A.
        • Shabanowitz J.
        • Sakaguchi K.
        • Michel H.
        • Sevilir N.
        • Cox A.L.
        • Appella E.
        • Engelhard V.H.
        Characterization of peptides bound to the class I MHC molecule HLA-A2.1 by mass spectrometry.
        Science. 1992; 255: 1261-1263
        • Parker K.C.
        • Bednarek M.A.
        • Hull L.K.
        • Utz U.
        • Cunningham B.
        • Zweerink H.J.
        • Biddison W.E.
        • Coligan J.E.
        Sequence motifs important for peptide binding to the human MHC class I molecule, HLA-A2.
        J. Immunol. 1992; 149: 3580-3587
        • Borbulevych O.Y.
        • Do P.
        • Baker B.M.
        Structures of native and affinity-enhanced WT1 epitopes bound to HLA-A*0201: implications for WT1-based cancer therapeutics.
        Mol. Immunol. 2010; 47: 2519-2524
        • Rosenberg S.A.
        • Yang J.C.
        • Schwartzentruber D.J.
        • Hwu P.
        • Marincola F.M.
        • Topalian S.L.
        • Restifo N.P.
        • Dudley M.E.
        • Schwarz S.L.
        • Spiess P.J.
        • Wunderlich J.R.
        • Parkhurst M.R.
        • Kawakami Y.
        • Seipp C.A.
        • Einhorn J.H.
        • White D.E.
        Immunologic and therapeutic evaluation of a synthetic peptide vaccine for the treatment of patients with metastatic melanoma.
        Nat. Med. 1998; 4: 321-327
        • Wieckowski S.
        • Baumgaertner P.
        • Corthesy P.
        • Voelter V.
        • Romero P.
        • Speiser D.E.
        • Rufer N.
        Fine structural variations of αβ TCRs selected by vaccination with natural versus altered self-antigen in melanoma patients.
        J. Immunol. 2009; 183: 5397-5406
        • Linnemann T.
        • Tumenjargal S.
        • Gellrich S.
        • Wiesmüller K.
        • Kaltoft K.
        • Sterry W.
        • Walden P.
        Mimotopes for tumor-specific T lymphocytes in human cancer determined with combinatorial peptide libraries.
        Eur. J. Immunol. 2001; 31: 156-165
        • Tumenjargal S.
        • Gellrich S.
        • Linnemann T.
        • Muche J.M.
        • Lukowsky A.
        • Audring H.
        • Wiesmüller K.H.
        • Sterry W.
        • Walden P.
        Anti-tumor immune responses and tumor regression induced with mimotopes of a tumor-associated T cell epitope.
        Eur. J. Immunol. 2003; 33: 3175-3185
        • Schaubert K.L.
        • Price D.A.
        • Salkowitz J.R.
        • Sewell A.K.
        • Sidney J.
        • Asher T.E.
        • Blondelle S.E.
        • Adams S.
        • Marincola F.M.
        • Joseph A.
        • Sette A.
        • Douek D.C.
        • Ayyavoo V.
        • Storkus W.
        • Leung M.Y.
        • Ng H.L.
        • Yang O.O.
        • Goldstein H.
        • Wilson D.B.
        • Kan-Mitchell J.
        Generation of robust CD8+ T-cell responses against subdominant epitopes in conserved regions of HIV-1 by repertoire mining with mimotopes.
        Eur. J. Immunol. 2010; 40: 1950-1962
        • Muraro R.
        • Wunderlich D.
        • Thor A.
        • Lundy J.
        • Noguchi P.
        • Cunningham R.
        • Schlom J.
        Definition by monoclonal antibodies of a repertoire of epitopes on carcinoembryonic antigen differentially expressed in human colon carcinomas versus normal adult tissues.
        Cancer Res. 1985; 45: 5769-5780
        • Vincent R.G.
        • Chu T.M.
        • Lane W.W.
        • Gutierrez A.C.
        • Stegemann P.J.
        • Madajewicz S.
        Carcinoembryonic antigen as a monitor of successful surgical resection in 130 patients with carcinoma of the lung.
        J. Thorac. Cardiovasc. Surg. 1978; 75: 734-739
        • Steward A.M.
        • Nixon D.
        • Zamcheck N.
        • Aisenberg A.
        Carcinoembryonic antigen in breast cancer patients: serum levels and disease progress.
        Cancer. 1974; 33: 1246-1252
        • Zaremba S.
        • Barzaga E.
        • Zhu M.
        • Soares N.
        • Tsang K.Y.
        • Schlom J.
        Identification of an enhancer agonist cytotoxic T lymphocyte peptide from human carcinoembryonic antigen.
        Cancer Res. 1997; 57: 4570-4577
        • Hou Y.
        • Kavanagh B.
        • Fong L.
        Distinct CD8+ T cell repertoires primed with agonist and native peptides derived from a tumor-associated antigen.
        J. Immunol. 2008; 180: 1526-1534
        • Pittet M.J.
        • Gati A.
        • Le Gal F.A.
        • Bioley G.
        • Guillaume P.
        • de Smedt M.
        • Plum J.
        • Speiser D.E.
        • Cerottini J.C.
        • Dietrich P.Y.
        • Romero P.
        • Zippelius A.
        Ex vivo characterization of allo-MHC-restricted T cells specific for a single MHC-peptide complex.
        J. Immunol. 2006; 176: 2330-2336
        • Venturi V.
        • Price D.A.
        • Douek D.C.
        • Davenport M.P.
        The molecular basis for public T-cell responses?.
        Nat. Rev. Immunol. 2008; 8: 231-238