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1. Therapeutic high affinity T cell receptor targeting a KRASG12D cancer neoantigen

Author

Poole, Andrew, Karuppiah, Vijaykumar, Hartt, Annabelle, Haidar, Jaafar N., Moureau, Sylvie, Dobrzycki, Tomasz, Hayes, Conor, Rowley, Christopher, Dias, Jorge, Harper, Stephen, Barnbrook, Keir, Hock, Miriam, Coles, Charlotte, Yang, Wei, Aleksic, Milos, Lin, Aimee Bence, Robinson, Ross, Dukes, Joe D., Liddy, Nathaniel, Van der Kamp, Marc, Plowman, Gregory D., Vuidepot, Annelise, Cole, David K., Whale, Andrew D., and Chillakuri, Chandramouli

Published
2022
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2. Specificity of bispecific T cell receptors and antibodies targeting peptide-HLA

Author

Holland, Christopher J., Crean, Rory M., Pentier, Johanne M., de WeAngharad Lloyd, Ben, Srikannathasan, Velupillai, Lissin, Nikolai, Lloyd, Katy A., Blicher, Thomas H., Conroy, Paul J., Hock, Miriam, Pengelly, Robert J., Spinner, Thomas E., Cameron, Brian, Potter, Elizabeth A., Jeyanthan, Anitha, Molloy, Peter E., Sami, Malkit, Aleksic, Milos, Liddy, Nathaniel, Robinson, Ross A., Harper, Stephen, Lepore, Marco, Pudney, Chris R., van der Kamp, Marc W., Rizkallah, Pierre J., Jakobsen, Bent K., and Cole, Annelise Vuidepoand David K.

Subjects
Peptides -- Health aspects, Antibodies -- Health aspects, Antigenic determinants -- Health aspects, HLA antigens -- Health aspects, T cells -- Health aspects, Antigens, Cancer treatment, Cancer, Reagents, Tumors, Health care industry
Abstract

Tumor-associated peptide-human leukocyte antigen complexes (pHLAs) represent the largest pool of cell surface-expressed cancer-specific epitopes, making them attractive targets for cancer therapies. Soluble bispecific molecules that incorporate an anti-CD3 effector function are being developed to redirect T cells against these targets using 2 different approaches. The first achieves pHLA recognition via affinity-enhanced versions of natural TCRs (e.g., immune-mobilizing monoclonal T cell receptors against cancer [ImmTAC] molecules), whereas the second harnesses an antibody-based format (TCR-mimic antibodies). For both classes of reagent, target specificity is vital, considering the vast universe of potential pHLA molecules that can be presented on healthy cells. Here, we made use of structural, biochemical, and computational approaches to investigate the molecular rules underpinning the reactivity patterns of pHLA-targeting bispecifics. We demonstrate that affinity-enhanced TCRs engage pHLA using a comparatively broad and balanced energetic footprint, with interactions distributed over several HLA and peptide side chains. As ImmTAC molecules, these TCRs also retained a greater degree of pHLA selectivity, with less off-target activity in cellular assays. Conversely, TCR-mimic antibodies tended to exhibit binding modes focused more toward hot spots on the HLA surface and exhibited a greater degree of crossreactivity. Our findings extend our understanding of the basic principles that underpin pHLA selectivity and exemplify a number of molecular approaches that can be used to probe the specificity of pHLA-targeting molecules, aiding the development of future reagents., Introduction The ability to selectively target tumor-specific antigens holds great promise for the development of specific cancer treatments, but their identification remains a key challenge. Peptide fragments presented on the [...]

Published
2020
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3. Molecular engineering of high affinity T-cell receptors for bispecific therapeutics

Author

Liddy, Nathaniel

Subjects
615.5, RM Therapeutics. Pharmacology
Abstract

Cytotoxic T lymphocytes are able to identify malignant cells by scanning for aberrant peptides presented on cell surface human leukocyte antigen (HLA) Class I molecules by virtue of an antigen binding receptor called the T-cell receptor (TCR). Peptides presented by HLA Class I complexes represent the largest array of tumour associated antigens (TAAs) and are therefore ideal targets for immunotherapeutic reagents. Cancer patients frequently mount T-cell responses to tumour-specific antigens, but these are in most cases ineffective at clearing the tumour. This is in part due to the low affinity of TCRs for self-antigens coupled with low-level expression of target peptides on the surface of cancer cells. To harness the exquisite antigen recognition property of TCRs for use as potential therapeutic proteins, the principal goal of this thesis was to generate ultra-high affinity TCRs against three clinically relevant HLA Class I melanoma-specific epitopes, including peptides derived from Melan-A/MART-1(26-35), gp100(280-288) and MAGE-A3(168-176). TCRs are membrane-bound disulphide (ds)-linked heterodimers consisting of an alpha and a beta chain. Each chain comprises three hypervariable or complementarity-determining region (CDR) loops, which assemble to form the antigen binding domains. As a general rule the CDR3 loops, and to a lesser extent the CDR1 loops, contact the peptide bound in the HLA groove and as such specificity is largely attributable to the CDR3 loops. The remaining CDR loops interact with the HLA surface and not the bound peptide. Each CDR loop was mutagenised using degenerative NNK oligonucleotides and expressed on the surface of bacteriophage as fusions to the phage coat protein pIII. Through a Darwinian process of in vitro evolution using pHLA ligand as the target molecule, mutated TCRs with improved affinity for pHLA were identified. TCRs engineered by phage display were produced as soluble ds-linked proteins and the contribution to affinity of each mutated CDR was measured by surface plasmon resonance (SPR). Using a combinatorial strategy, individual mutated CDRs were spliced into the same TCR molecule in a stepwise manner to further increase binding affinity. The final combination of mutated CDRs was shown to bind their cognate pHLA antigen with substantially improved KD values of 18 pM (Melan-A/MART-1(26-35)), 11 pM (gp100(280-288)) and 58 pM (MAGE-A3(168-176)), representing an increase over the wild-type TCR of approximately 1.8 million-fold, 1.7 million-fold and 3.7 million-fold respectively. In addition, having discovered an off-target binding profile for the high affinity MAGE-A3 TCR, the phage display methodologies were explored to 5 reestablish the specificity of this molecule. These results are significant because this has provided a platform on which, for the first time, to make TCR-based therapeutics. For example, the affinity enhanced gp100 TCR is currently undergoing clinical evaluation in a Phase I/II trial.

Published
2013

5. Therapeutic high affinity T cell receptor targeting a KRASG12D cancer neoantigen.

Author

Poole, Andrew, Karuppiah, Vijaykumar, Hartt, Annabelle, Haidar, Jaafar N., Moureau, Sylvie, Dobrzycki, Tomasz, Hayes, Conor, Rowley, Christopher, Dias, Jorge, Harper, Stephen, Barnbrook, Keir, Hock, Miriam, Coles, Charlotte, Yang, Wei, Aleksic, Milos, Lin, Aimee Bence, Robinson, Ross, Dukes, Joe D., Liddy, Nathaniel, and Van der Kamp, Marc

Subjects
T cell receptors, MOLECULAR dynamics, SOMATIC mutation, BISPECIFIC antibodies, PEPTIDES, CARCINOGENESIS
Abstract

Neoantigens derived from somatic mutations are specific to cancer cells and are ideal targets for cancer immunotherapy. KRAS is the most frequently mutated oncogene and drives the pathogenesis of several cancers. Here we show the identification and development of an affinity-enhanced T cell receptor (TCR) that recognizes a peptide derived from the most common KRAS mutant, KRASG12D, presented in the context of HLA-A*11:01. The affinity of the engineered TCR is increased by over one million-fold yet fully able to distinguish KRASG12D over KRASWT. While crystal structures reveal few discernible differences in TCR interactions with KRASWT versus KRASG12D, thermodynamic analysis and molecular dynamics simulations reveal that TCR specificity is driven by differences in indirect electrostatic interactions. The affinity enhanced TCR, fused to a humanized anti-CD3 scFv, enables selective killing of cancer cells expressing KRASG12D. Our work thus reveals a molecular mechanism that drives TCR selectivity and describes a soluble bispecific molecule with therapeutic potential against cancers harboring a common shared neoantigen. Cancers often harbor mutations in genes encoding important regulatory proteins, but therapeutic targeting of these molecules proves difficult due to their high structural similarity to their non-mutated counterpart. Here authors show the engineering of T cell engaging bispecific protein able to selectively target cancer cells with a high-frequency mutation in the KRAS oncogene. [ABSTRACT FROM AUTHOR]

Published
2022
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6. Monoclonal TCR-redirected tumor cell killing

Author

Liddy, Nathaniel, Bossi, Giovanna, Adams, Katherine J., Lissina, Anna, Mahon, Tara M., Hassan, Namir J., Avarret, Jessie G., Bianchi, Frayne C., Pumphrey, Nicholas J., Ladell, Kristin, Gostick, Emma, Lissin, Nikolai M., Harwood, Naomi E., Molloy, Peter E., Li, Yi, Cameron, Brian J., Sami, Malkit, Baston, Emma E., Todorov, Penio T., Dennis, Rebecca E., Harper, Jane V., Dunn, Steve M., Ashfield, Rebecca, Johnson, Andy, McGrath, Yvonne, Plesa, Gabriela, June, Carl H., Kalos, Michael, Price, David A., Williams, Daniel D., Sutton, Deborah H., and Jakobsen, Bent K.

Subjects
T cells -- Receptors, Antigen receptors, T cell -- Research -- Analysis, Cancer cells -- Research -- Analysis, Biological sciences, Health
Abstract

T cell immunity can potentially eradicate malignant cells and lead to clinical remission in a minority of patients with cancer. In the majority of these individuals, however, there is a failure of the specific T cell receptor (TCR)-mediated immune recognition and activation process. Here we describe the engineering and characterization of new reagentstermed immune-mobilizing monoclonal TCRs against cancer (ImmTACs). Four such ImmTACs, each comprising a distinct tumor-associated epitope-specific monoclonal TCR with picomolar affinity fused to a humanized cluster of differentiation 3 (CD3)-specific single-chain antibody fragment (scFv), effectively redirected T cells to kill cancer cells expressing extremely low surface epitope densities. Furthermore, these reagents potently suppressed tumor growth in vivo. Thus, ImmTACs overcome immune tolerance to cancer and represent a new approach to tumor immunotherapy., Harnessing the power of adaptive immunity to combat cancer has been a long-term goal of translational immunotherapy. However, although many avenues in this field have been explored over the past [...]

Published
2012
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8. TCR-induced alteration of primary MHC peptide anchor residue

Author

Madura, Florian, Rizkallah, Pierre J., Legut, Mateusz, Holland, Christopher J., Fuller, Anna, Bulek, Anna, Schauenburg, Andrea J., Trimby, Andrew, Hopkins, Jade R., Wells, Stephen A., Godkin, Andrew, Miles, John J., Sami, Malkit, Li, Yi, Liddy, Nathaniel, Jakobsen, Bent K., Loveridge, E. Joel, Cole, David K., and Sewell, Andrew K.

Abstract

The HLA‐A*02:01‐restricted decapeptide EAAGIGILTV, derived from melanoma antigen recognized by T‐cells‐1 (MART‐1) protein, represents one of the best‐studied tumor associated T‐cell epitopes, but clinical results targeting this peptide have been disappointing. This limitation may reflect the dominance of the nonapeptide, AAGIGILTV, at the melanoma cell surface. The decapeptide and nonapeptide are presented in distinct conformations by HLA‐A*02:01 and TCRs from clinically relevant T‐cell clones recognize the nonapeptide poorly. Here, we studied the MEL5 TCR that potently recognizes the nonapeptide. The structure of the MEL5‐HLA‐A*02:01‐AAGIGILTV complex revealed an induced fit mechanism of antigen recognition involving altered peptide–MHC anchoring. This “flexing” at the TCR–peptide–MHC interface to accommodate the peptide antigen explains previously observed incongruences in this well‐studied system and has important implications for future therapeutic approaches. Finally, this study expands upon the mechanisms by which molecular plasticity can influence antigen recognition by T cells.

Published
2019

10. Abstract 4872: Developing high affinity, soluble T cell receptors for the treatment of cancer

Author

Knox, Andrew, primary, Chester, Fiona, additional, Bianchi, Frayne, additional, Bailey, Sarah, additional, Bouard, Lucie, additional, Liddy, Nathaniel, additional, Bossi, Giovanna, additional, Harper, Jane, additional, Dukes, Joseph, additional, Paston, Samantha, additional, Mahon, Tara, additional, Gavarret, Jessie, additional, Molloy, Peter, additional, Sami, Malkit, additional, Baston, Emma, additional, Cameron, Brian, additional, Powlesland, Alex, additional, Todorov, Penio, additional, Johnson, Andrew, additional, Ebner, Martin, additional, McGrath, Yvonne, additional, Hassan, Namir, additional, Vuidepot, Annelise, additional, and Jakobsen, Bent, additional

Published
2016
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11. Abstract 4873: ImmTACs re-direct the immune system efficiently to eradicate cancer

Author

Bossi, Giovanna, primary, Kenefeck, Rupert, additional, Caroline Boudousquie, Caroline, additional, Harper, Jane, additional, Dukes, Joseph, additional, Liddy, Nathaniel, additional, Paston, Samantha, additional, Mahon, Tara, additional, Molloy, Peter, additional, Sami, Malkit, additional, Baston, Emma, additional, Cameron, Brian, additional, Vuidepot, Annelise, additional, Hassan, Namir, additional, and Jakobsen, Bent K., additional

Published
2016
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12. Different affinity windows for virus and cancer-specific T-cell receptors – implications for therapeutic strategies

Author

Aleksic, Milos, Liddy, Nathaniel, Molloy, Peter E, Pumphrey, Nick, Vuidepot, Annelise, Chang, Kyong-Mi, and Jakobsen, Bent K

Subjects
Antigen Presentation, Neoplasms, T-Lymphocytes, HLA-A2 Antigen, Receptors, Antigen, T-Cell, Humans, chemical and pharmacologic phenomena, Thymus Gland, Peptides, Cancer Vaccines, Article, Neoplasm Proteins
Abstract

T-cell destiny during thymic selection depends on the affinity of the T-cell receptor (TCR) for autologous peptide ligands presented in the context of MHC molecules. This is a delicately balanced process; robust binding leads to negative selection, yet some affinity for the antigen complex is required for positive selection. All TCRs of the resulting repertoire thus have some intrinsic affinity for an MHC type presenting an assortment of peptides. Generally, TCR affinities of peripheral T cells will be low towards self-derived peptides, as these would have been presented during thymic selection, whereas, by serendipity, binding to pathogen-derived peptides which are encountered de novo could be stronger. A crucial question in assessing immunotherapeutic strategies for cancer is whether natural TCR repertoires have the capacity for efficiently recognizing tumor associated peptide antigens (TAPAs). Here, we report a comprehensive comparison of TCR affinities to a range of HLA-A2 presented antigens. TCRs which bind viral antigens (VAs) fall within a strikingly higher affinity range than those which bind cancer-related antigens. This difference may be one of the key explanations for tumor immune escape and for the deficiencies of T-cell vaccines against cancer.

Published
2012

13. ImmTACs: bi-specific TCR-anti-CD3 fusions for targeted tumour killing

Author

Donnellan, Zoe, primary, Bossi, Giovanna, additional, Harper, Jane, additional, Dukes, Joseph, additional, Liddy, Nathaniel, additional, Paston, Samantha, additional, Mahon, Tara, additional, Molloy, Peter, additional, Sami, Malkit, additional, Baston, Emma, additional, Cameron, Brian, additional, Johnson, Andrew, additional, Vuidepot, Annelise, additional, Hassan, Namir, additional, and Jakobsen, Bent, additional

Published
2015
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14. Abstract 670: ImmTACs: Bi-specific TCR-anti-CD3 fusions for potent re-directed killing of cancer cells

Author

Bossi, Giovanna, primary, Baker, Debbie, additional, Adams, Katherine, additional, Harper, Jane, additional, Dukes, Joseph, additional, Liddy, Nathaniel, additional, Paston, Samantha, additional, McGrath, Yvonne, additional, Mahon, Tara, additional, Molloy, Pater, additional, Sami, Malkit, additional, Baston, Emma, additional, Cameron, Brian, additional, Johnson, Andrew, additional, Vuidepot, Annelise, additional, Hassan, Namir, additional, and Jakobsen, Bent, additional

Published
2014
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15. Abstract 2900: IMCgp100: A novel bi-specific biologic for the treatment of malignant melanoma

Author

Hassan, Namir J., primary, Bossi, Giovanna, additional, Baker, Debbie, additional, Adams, Katherine, additional, Harper, Jane, additional, Dukes, Joseph, additional, Liddy, Nathaniel, additional, Paston, Samantha, additional, McGrath, Yvonne, additional, Mahon, Tara, additional, Molloy, Peter, additional, Sami, Malkit, additional, Baston, Emma, additional, Cameron, Brian, additional, Johnson, Andrew, additional, Vuidepot, Annelise, additional, Linette, Gerry, additional, Kalos, Michael, additional, June, Carl, additional, and Jakobsen, Bent, additional

Published
2014
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16. T-cell Receptor (TCR)-Peptide Specificity Overrides Affinity-enhancing TCR-Major Histocompatibility Complex Interactions

Author

Cole, David K., primary, Miles, Kim M., additional, Madura, Florian, additional, Holland, Christopher J., additional, Schauenburg, Andrea J.A., additional, Godkin, Andrew J., additional, Bulek, Anna M., additional, Fuller, Anna, additional, Akpovwa, Hephzibah J.E., additional, Pymm, Phillip G., additional, Liddy, Nathaniel, additional, Sami, Malkit, additional, Li, Yi, additional, Rizkallah, Pierre J., additional, Jakobsen, Bent K., additional, and Sewell, Andrew K., additional

Published
2014
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17. T-cell Receptor Specificity Maintained by Altered Thermodynamics

Author

Madura, Florian, primary, Rizkallah, Pierre J., additional, Miles, Kim M., additional, Holland, Christopher J., additional, Bulek, Anna M., additional, Fuller, Anna, additional, Schauenburg, Andrea J.A., additional, Miles, John J., additional, Liddy, Nathaniel, additional, Sami, Malkit, additional, Li, Yi, additional, Hossain, Moushumi, additional, Baker, Brian M., additional, Jakobsen, Bent K., additional, Sewell, Andrew K., additional, and Cole, David K., additional

Published
2013
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19. Abstract 3525: IMCgp100: A novel bi-specific biologic for the treatment of malignant melanoma

Author

Hassan, Namir J., primary, Adams, Katherine, additional, Bossi, Giovanna, additional, Harper, Jane, additional, Buisson, Sandrine, additional, Paston, Samantha, additional, Liddy, Nathaniel, additional, Ashfield, Rebecca, additional, Gavarret, Jessie, additional, Bianchi, Frayne, additional, Baston, Emma, additional, Baker, Debbie, additional, Ladell, Kristin, additional, Sewell, Andrew, additional, Li, Yi, additional, Cameron, Brian, additional, Johnson, Andrew, additional, Vuidepot, Annelise, additional, Todorov, Penio, additional, Kalos, Michael, additional, June, Carl, additional, Karakousis, Giorgos, additional, Linette, Gerry, additional, Price, David A., additional, Williams, Daniel, additional, McGrath, Yvonne, additional, and Jakobsen, Bent K., additional

Published
2012
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20. Abstract 3528: IMCmage1: A novel bi-specific biologic re-directing T cells to kill MAGE-A3/A6 presenting cancers

Author

Hassan, Namir J., primary, Baker, Debbie, additional, Harper, Jane, additional, Adams, Katherine, additional, Bossi, Giovanna, additional, Liddy, Nathaniel, additional, Buisson, Sandrine, additional, Paston, Samantha, additional, Gavarret, Jessie, additional, Bianchi, Frayne, additional, Baston, Emma, additional, Legg, Alison, additional, Ashfield, Rebecca, additional, McGrath, Yvonne, additional, Li, Yi, additional, Williams, Daniel, additional, Cameron, Brian, additional, Johnson, Andrew, additional, Vuidepot, Annelise, additional, Todorov, Penio, additional, Roberts, Pamela, additional, Hatton, Chris, additional, Kalos, Michael, additional, June, Carl, additional, and Jakobsen, Bent K., additional

Published
2012
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21. Abstract 4744: Soluble, high affinity TCRs fused to anti-CD3 redirect T cells to kill cancer cells presenting MAGE-A3 and NY-ESO antigens

Author

Harper, Jane V., primary, Hassan, Namir J., additional, Liddy, Nathaniel, additional, Mahon, Tara, additional, Bossi, Giovanna, additional, Adams, Katherine, additional, Gavarret, Jessie, additional, Bianchi, Frayne, additional, Lissin, Nikolai, additional, Molloy, Peter, additional, Li, Yi, additional, Pumphrey, Nick, additional, Cameron, Brian, additional, Sami, Malkit, additional, Baston, Emma, additional, Ashfield, Rebecca, additional, Johnson, Andrew, additional, Vuidepot, Annelise, additional, McGrath, Yvonne, additional, Todorov, Penio, additional, Paston, Samantha, additional, Aleksic, Milos, additional, Legg, Alison, additional, Wright, Debbie, additional, Dennis, Rebecca, additional, Sewell, Andrew K., additional, Price, David A., additional, Harwood, Naomi, additional, Lissina, Anna, additional, Gostik, Emma, additional, June, Carl, additional, Kalos, Michael, additional, Pleas, Gabriela, additional, Sutton, Deborah, additional, Williams, Daniel, additional, and Jakobsen, Bent, additional

Published
2011
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22. Abstract 1787: ImmTACs: Bi-functional reagents for redirected tumour cell killing

Author

Paston, Samantha J., primary, Ashfield, Rebecca, additional, Liddy, Nathaniel, additional, Bossi, Giovanna, additional, Adams, Katherine, additional, McCormack, Emmet, additional, Lissina, Anna, additional, Mahon, Tara, additional, Hassan, Namir, additional, Gavarret, Jessie, additional, Bianchi, Frayne, additional, Pumphrey, Nick, additional, Ladell, Kristin, additional, Gostick, Emma, additional, Sewell, Andrew K., additional, Lissin, Nikolai, additional, Molloy, Peter, additional, Li, Yi, additional, Cameron, Brian, additional, Sami, Malkit, additional, Baston, Emma, additional, Harper, Jane, additional, Johnson, Andrew, additional, Vuidepot, Annelise, additional, Todorov, Penio, additional, Dennis, Rebecca, additional, Kalos, Michael, additional, June, Carl, additional, Price, David A., additional, Williams, Daniel, additional, and Jakobsen, Bent K., additional

Published
2011
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23. Abstract 5616: Soluble, high affinity TCRs fused to anti-CD3 redirect T cells to kill cancer cells presenting MAGE-A3 and NY-ESO antigens

Author

Hassan, Namir J., primary, Liddy, Nathaniel, additional, Mahon, Tara, additional, Bossi, Giovanna, additional, Adams, Katherine, additional, Gavarret, Jessie, additional, Bianchi, Frayne, additional, Lissin, Nikolai, additional, Molloy, Peter, additional, Li, Yi, additional, Pumphrey, Nick, additional, Cameron, Brian, additional, Sami, Malkit, additional, Baston, Emma, additional, Harper, Jane, additional, Ashfield, Rebecca, additional, Johnson, Andrew, additional, Vuidepot, Annelise, additional, McGrath, Yvonne, additional, Todorov, Penio, additional, Paston, Samantha, additional, Dennis, Rebecca, additional, Sewell, Andrew K., additional, Price, David A., additional, Harwood, Naomi, additional, Lissina, Anna, additional, Gostick, Emma, additional, June, Carl, additional, Kalos, Michael, additional, Plesa, Gabriela, additional, Sutton, Deborah, additional, Williams, Daniel, additional, and Jakobsen, Bent K., additional

Published
2010
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24. The HLA A*0201–restricted hTERT540–548 peptide is not detected on tumor cells by a CTL clone or a high-affinity T-cell receptor

Author

Purbhoo, Marco A., primary, Li, Yi, additional, Sutton, Deborah H., additional, Brewer, Joanna E., additional, Gostick, Emma, additional, Bossi, Giovanna, additional, Laugel, Bruno, additional, Moysey, Ruth, additional, Baston, Emma, additional, Liddy, Nathaniel, additional, Cameron, Brian, additional, Bennett, Alan D., additional, Ashfield, Rebecca, additional, Milicic, Anita, additional, Price, David A., additional, Classon, Brendan J., additional, Sewell, Andrew K., additional, and Jakobsen, Bent K., additional

Published
2007
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27. Directed evolution of human T-cell receptors with picomolar affinities by phage display.

Author

Yi Li, Moysey, Ruth, Molloy, Peter E, Vuidepot, Anne-Lise, Mahon, Tara, Baston, Emma, Dunn, Steven, Liddy, Nathaniel, Jacob, Jansen, Jakobsen, Bent K, and Boulter, Jonathan M

Subjects
T cells, CELL membranes, HLA histocompatibility antigens, CELL receptors, BIOMOLECULES, BIOLOGICAL membranes
Abstract

Peptides derived from almost all proteins, including disease-associated proteins, can be presented on the cell surface as peptide-human leukocyte antigen (pHLA) complexes. T cells specifically recognize pHLA with their clonally rearranged T-cell receptors (TCRs), whose natural affinities are limited to~1-100µM. Here we describe the display of ten different human TCRs on the surface of bacteriophage, stabilized by a nonnative interchain disulfide bond. We report the directed evolution of high-affinity TCRs specific for two different pHLAs: the human T-cell lymphotropic virus type 1 (HTLV-1) tax11-19 peptide-HLA-A*0201 complex and the NY-ESO-1157-165 tumor-associated peptide antigen-HLA-A*0201 complex, with affinities of up to 2.5 nM and 26 pM, respectively, and we demonstrate their high specificity and sensitivity for targeting of cell-surface pHLAs. [ABSTRACT FROM AUTHOR]

Published
2005
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28. TCR‐induced alteration of primary MHC peptide anchor residue

Author

Madura, Florian, Rizkallah, Pierre J., Legut, Mateusz, Holland, Christopher J., Fuller, Anna, Bulek, Anna, Schauenburg, Andrea J., Trimby, Andrew, Hopkins, Jade R., Wells, Stephen A., Godkin, Andrew, Miles, John J., Sami, Malkit, Li, Yi, Liddy, Nathaniel, Jakobsen, Bent K., Loveridge, E. Joel, Cole, David K., and Sewell, Andrew K.

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29. IMCGP100: A Novel BI-SPECIFIC Biologic Immunotherapy for the Treatment of Malignant Melanoma

Author

Bossi, Giovanna, Hassan, Namir, Adams, Katherine, Harper, Jane, Buisson, Sandrine, Paston, Samantha, Liddy, Nathaniel, Ashfield, Rebecca, Bianchi, Frayne, Baston, Emma, Sewell, Andrew K., Li, Yi, Cameron, Brian, Andrew Sewell, Vuidepot, Annelise, Todorov, Penio, Kalos, Michael, June, Carl, Karakousis, Giorgos, Linette, Gerry, Price, David A., Williams, Daniel, Mcgrath, Yvonne, and Jakobsen, Bent K.

30. Molecular engineering of high affinity T-cell receptors for bispecific therapeutics

Author

Liddy, Nathaniel

Subjects
RM, chemical and pharmacologic phenomena
Abstract

Cytotoxic T lymphocytes are able to identify malignant cells by scanning for aberrant peptides presented on cell surface human leukocyte antigen (HLA) Class I molecules by virtue of an antigen binding receptor called the T-cell receptor (TCR). Peptides presented by HLA Class I complexes represent the largest array of tumour associated antigens (TAAs) and are therefore ideal targets for immunotherapeutic reagents. Cancer patients frequently mount T-cell responses to tumour-specific antigens, but these are in most cases ineffective at clearing the tumour. This is in part due to the low affinity of TCRs for self-antigens coupled with low-level expression of target peptides on the surface of cancer cells. To harness the exquisite antigen recognition property of TCRs for use as potential therapeutic proteins, the principal goal of this thesis was to generate ultra-high affinity TCRs against three clinically relevant HLA Class I melanoma-specific epitopes, including peptides derived from Melan-A/MART-1(26-35), gp100(280-288) and MAGE-A3(168-176). TCRs are membrane-bound disulphide (ds)-linked heterodimers consisting of an alpha and a beta chain. Each chain comprises three hypervariable or complementarity-determining region (CDR) loops, which assemble to form the antigen binding domains. As a general rule the CDR3 loops, and to a lesser extent the CDR1 loops, contact the peptide bound in the HLA groove and as such specificity is largely attributable to the CDR3 loops. The remaining CDR loops interact with the HLA surface and not the bound peptide. Each CDR loop was mutagenised using degenerative NNK oligonucleotides and expressed on the surface of bacteriophage as fusions to the phage coat protein pIII. Through a Darwinian process of in vitro evolution using pHLA ligand as the target molecule, mutated TCRs with improved affinity for pHLA were identified. TCRs engineered by phage display were produced as soluble ds-linked proteins and the contribution to affinity of each mutated CDR was measured by surface plasmon resonance (SPR). Using a combinatorial strategy, individual mutated CDRs were spliced into the same TCR molecule in a stepwise manner to further increase binding affinity. The final combination of mutated CDRs was shown to bind their cognate pHLA antigen with substantially improved KD values of 18 pM (Melan-A/MART-1(26-35)), 11 pM (gp100(280-288)) and 58 pM (MAGE-A3(168-176)), representing an increase over the wild-type TCR of approximately 1.8 million-fold, 1.7 million-fold and 3.7 million-fold respectively. In addition, having discovered an off-target binding profile for the high affinity MAGE-A3 TCR, the phage display methodologies were explored to\ud 5\ud reestablish the specificity of this molecule. These results are significant because this has provided a platform on which, for the first time, to make TCR-based therapeutics. For example, the affinity enhanced gp100 TCR is currently undergoing clinical evaluation in a Phase I/II trial.

31. Molecular engineering of high affinity T-cell receptors for bispecific therapeutics

Author

Liddy, Nathaniel and Liddy, Nathaniel

Abstract

Cytotoxic T lymphocytes are able to identify malignant cells by scanning for aberrant peptides presented on cell surface human leukocyte antigen (HLA) Class I molecules by virtue of an antigen binding receptor called the T-cell receptor (TCR). Peptides presented by HLA Class I complexes represent the largest array of tumour associated antigens (TAAs) and are therefore ideal targets for immunotherapeutic reagents. Cancer patients frequently mount T-cell responses to tumour-specific antigens, but these are in most cases ineffective at clearing the tumour. This is in part due to the low affinity of TCRs for self-antigens coupled with low-level expression of target peptides on the surface of cancer cells. To harness the exquisite antigen recognition property of TCRs for use as potential therapeutic proteins, the principal goal of this thesis was to generate ultra-high affinity TCRs against three clinically relevant HLA Class I melanoma-specific epitopes, including peptides derived from Melan-A/MART-1(26-35), gp100(280-288) and MAGE-A3(168-176). TCRs are membrane-bound disulphide (ds)-linked heterodimers consisting of an alpha and a beta chain. Each chain comprises three hypervariable or complementarity-determining region (CDR) loops, which assemble to form the antigen binding domains. As a general rule the CDR3 loops, and to a lesser extent the CDR1 loops, contact the peptide bound in the HLA groove and as such specificity is largely attributable to the CDR3 loops. The remaining CDR loops interact with the HLA surface and not the bound peptide. Each CDR loop was mutagenised using degenerative NNK oligonucleotides and expressed on the surface of bacteriophage as fusions to the phage coat protein pIII. Through a Darwinian process of in vitro evolution using pHLA ligand as the target molecule, mutated TCRs with improved affinity for pHLA were identified. TCRs engineered by phage display were produced as soluble ds-linked proteins and the contribution to affinity of each mu

32. Molecular engineering of high affinity T-cell receptors for bispecific therapeutics

Author

Liddy, Nathaniel and Liddy, Nathaniel

Abstract

Cytotoxic T lymphocytes are able to identify malignant cells by scanning for aberrant peptides presented on cell surface human leukocyte antigen (HLA) Class I molecules by virtue of an antigen binding receptor called the T-cell receptor (TCR). Peptides presented by HLA Class I complexes represent the largest array of tumour associated antigens (TAAs) and are therefore ideal targets for immunotherapeutic reagents. Cancer patients frequently mount T-cell responses to tumour-specific antigens, but these are in most cases ineffective at clearing the tumour. This is in part due to the low affinity of TCRs for self-antigens coupled with low-level expression of target peptides on the surface of cancer cells. To harness the exquisite antigen recognition property of TCRs for use as potential therapeutic proteins, the principal goal of this thesis was to generate ultra-high affinity TCRs against three clinically relevant HLA Class I melanoma-specific epitopes, including peptides derived from Melan-A/MART-1(26-35), gp100(280-288) and MAGE-A3(168-176). TCRs are membrane-bound disulphide (ds)-linked heterodimers consisting of an alpha and a beta chain. Each chain comprises three hypervariable or complementarity-determining region (CDR) loops, which assemble to form the antigen binding domains. As a general rule the CDR3 loops, and to a lesser extent the CDR1 loops, contact the peptide bound in the HLA groove and as such specificity is largely attributable to the CDR3 loops. The remaining CDR loops interact with the HLA surface and not the bound peptide. Each CDR loop was mutagenised using degenerative NNK oligonucleotides and expressed on the surface of bacteriophage as fusions to the phage coat protein pIII. Through a Darwinian process of in vitro evolution using pHLA ligand as the target molecule, mutated TCRs with improved affinity for pHLA were identified. TCRs engineered by phage display were produced as soluble ds-linked proteins and the contribution to affinity of each mu

33. TCR-induced alteration of primary MHC peptide anchor residue.

Author

Madura F, Rizkallah PJ, Legut M, Holland CJ, Fuller A, Bulek A, Schauenburg AJ, Trimby A, Hopkins JR, Wells SA, Godkin A, Miles JJ, Sami M, Li Y, Liddy N, Jakobsen BK, Loveridge EJ, Cole DK, and Sewell AK

Subjects
Amino Acids, Antigen Presentation, Binding Sites, Cells, Cultured, Clone Cells, HLA-A2 Antigen chemistry, HLA-A2 Antigen metabolism, Humans, Lymphocyte Activation, MART-1 Antigen chemistry, Melanoma therapy, Peptides chemistry, Protein Binding, Protein Conformation, Receptors, Antigen, T-Cell genetics, T-Lymphocytes transplantation, Immunodominant Epitopes metabolism, Immunotherapy, Adoptive methods, MART-1 Antigen metabolism, Melanoma immunology, Peptides metabolism, Receptors, Antigen, T-Cell metabolism, T-Lymphocytes immunology
Abstract

The HLA-A*02:01-restricted decapeptide EAAGIGILTV, derived from melanoma antigen recognized by T-cells-1 (MART-1) protein, represents one of the best-studied tumor associated T-cell epitopes, but clinical results targeting this peptide have been disappointing. This limitation may reflect the dominance of the nonapeptide, AAGIGILTV, at the melanoma cell surface. The decapeptide and nonapeptide are presented in distinct conformations by HLA-A*02:01 and TCRs from clinically relevant T-cell clones recognize the nonapeptide poorly. Here, we studied the MEL5 TCR that potently recognizes the nonapeptide. The structure of the MEL5-HLA-A*02:01-AAGIGILTV complex revealed an induced fit mechanism of antigen recognition involving altered peptide-MHC anchoring. This "flexing" at the TCR-peptide-MHC interface to accommodate the peptide antigen explains previously observed incongruences in this well-studied system and has important implications for future therapeutic approaches. Finally, this study expands upon the mechanisms by which molecular plasticity can influence antigen recognition by T cells., (© 2019 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Published
2019
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