Emma Baston

Directed evolution of human T-cell receptors with picomolar affinities by phage display

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 ∼1–100 μM. 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.

Monoclonal TCR-redirected tumor cell killing

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 reagents termed 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.

Directed evolution of human T cell receptor CDR2 residues by phage display dramatically enhances affinity for cognate peptide-MHC without increasing apparent cross-reactivity.

The mammalian α/β T cell receptor (TCR) repertoire plays a pivotal role in adaptive immunity by recognizing short, processed, peptide antigens bound in the context of a highly diverse family of cell‐surface major histocompatibility complexes (pMHCs). Despite the extensive TCR–MHC interaction surface, peptide‐independent cross‐reactivity of native TCRs is generally avoided through cell‐mediated selection of molecules with low inherent affinity for MHC. Here we show that, contrary to expectations, the germ line‐encoded complementarity determining regions (CDRs) of human TCRs, namely the CDR2s, which appear to contact only the MHC surface and not the bound peptide, can be engineered to yield soluble low nanomolar affinity ligands that retain a surprisingly high degree of specificity for the cognate pMHC target. Structural investigation of one such CDR2 mutant implicates shape complementarity of the mutant CDR2 contact interfaces as being a key determinant of the increased affinity. Our results suggest that manipulation of germ line CDR2 loops may provide a useful route to the production of high‐affinity TCRs with therapeutic and diagnostic potential.

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

Tumor-associated human telomerase reverse transcriptase (hTERT) is expressed in >85% of human tumors but not in most normal cells. As a result, this antigen has received considerable attention from those interested in cancer immunotherapy. Specifically, there has been strong interest in MHC class I–associated peptides derived from hTERT because these are expressed on the cell surface and thus may enable the targeting of tumor cells. Much of this interest has focused on peptide 540–548, ILAKFLHWL, which was predicted to exhibit the strongest binding to the common HLA A*0201 presenting molecule. The hTERT540–548 peptide is currently being assessed in therapeutic vaccination trials; however, there is controversy surrounding whether it is naturally processed and presented on the surface of neoplastic cells. Here, we generate two highly sensitive reagents to assess the presentation of hTERT540–548 on tumor cells: (a) a CD8+ CTL clone, and (b) a recombinant T-cell receptor (TCR) that binds with picomolar affinity and a half-life exceeding 14 h. This TCR enables the identification of individual HLA A2-hTERT540–548 complexes on the cell surface. The use of both this TCR and the highly antigen-sensitive CTL clone shows that the hTERT540–548 peptide cannot be detected on the surface of tumor cells, indicating that this peptide is not a naturally presented epitope. We propose that, in future, rigorous methods must be applied for the validation of peptide epitopes used for clinical applications. [Mol Cancer Ther 2007;6(7):2081–91]

Direct molecular mimicry enables off-target cardiovascular toxicity by an enhanced affinity TCR designed for cancer immunotherapy.

Natural T-cell responses generally lack the potency to eradicate cancer. Enhanced affinity T-cell receptors (TCRs) provide an ideal approach to target cancer cells, with emerging clinical data showing significant promise. Nevertheless, the risk of off target reactivity remains a key concern, as exemplified in a recent clinical report describing fatal cardiac toxicity, following administration of MAGE-A3 specific TCR-engineered T-cells, mediated through cross-reactivity with an unrelated epitope from the Titin protein presented on cardiac tissue. Here, we investigated the structural mechanism enabling TCR cross-recognition of MAGE-A3 and Titin, and applied the resulting data to rationally design mutants with improved antigen discrimination, providing a proof-of-concept strategy for altering the fine specificity of a TCR towards an intended target antigen. This study represents the first example of direct molecular mimicry leading to clinically relevant fatal toxicity, mediated by a modified enhanced affinity TCR designed for cancer immunotherapy. Furthermore, these data demonstrate that self-antigens that are expressed at high levels on healthy tissue should be treated with extreme caution when designing immuno-therapeutics.

High affinity soluble ILT2 receptor: a potent inhibitor of CD8 + T cell activation

Using directed mutagenesis and phage display on a soluble fragment of the human immunoglobulin super-family receptor ILT2 (synonyms: LIR1, MIR7, CD85j), we have selected a range of mutants with binding affinities enhanced by up to 168,000-fold towards the conserved region of major histocompatibility complex (MHC) class I molecules. Produced in a dimeric form, either by chemical cross-linking with bivalent polyethylene glycol (PEG) derivatives or as a genetic fusion with human IgG Fc-fragment, the mutants exhibited a further increase in ligand-binding strength due to the avidity effect, with resident half-times (t1/2) on the surface of MHC I-positive cells of many hours. The novel compounds antagonized the interaction of CD8 co-receptor with MHC I in vitro without affecting the peptide-specific binding of T-cell receptors (TCRs). In both cytokine-release assays and cell-killing experiments the engineered receptors inhibited the activation of CD8+ cytotoxic T lymphocytes (CTLs) in the presence of their target cells, with subnanomolar potency and in a dose-dependent manner. As a selective inhibitor of CD8+ CTL responses, the engineered high affinity ILT2 receptor presents a new tool for studying the activation mechanism of different subsets of CTLs and could have potential for the development of novel autoimmunity therapies.

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

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Despite significant advances in the treatment of metastatic melanoma, long-term remission for the majority of patients remains elusive. Kinase inhibitors provide potent but short-term responses for a significant proportion of patients and immunotherapy elicits long-term responses with the prospect of cure, but only in a minority. IMCgp100 is a novel bi-specific immunotherapy comprising a soluble, affinity enhanced, T cell receptor (TCR) specific for the melanoma-associated antigen gp100, fused to an anti-CD3 specific antibody fragment (scFv). The engineered TCR portion of the drug targets and binds the gp100 peptide 280-288 antigen, which is over-expressed and presented by HLA-A2 on the surface of melanoma cells. The anti-CD3 scFv portion captures and redirects T cells to kill the melanoma cells, while normal antigen negative tissues are unaffected. Here, we present data which provides the foundation for the clinical observations. In vitro, IMCgp100 is demonstrated to potently redirect T cells from late stage cancer patients to target melanoma tumors exhibiting HLA-down regulation, even in the presence of high numbers of regulatory T cells. Target cell killing is observed within hours and is specific for gp100. In addition killing is associated with the release of various pro-inflammatory cytokines and chemokines as well as cross-presentation of gp100 and other melanoma-associated antigens by dendritic cells. Thus, IMCgp100 demonstrates the potential to elicit potent short-term responses and trigger longer-term anti melanoma activity in vivo. IMCgp100 is undergoing Phase I clinical testing in patients with advanced malignant melanoma; with the maximum tolerated dose having been established. The drug is well tolerated with evidence of tumor shrinkage. Analyses of serum samples obtained from patients on the trial demonstrate T cell mobilisation and transient drug mediated increases in various cytokines and chemokines, some of which are reported to play a key role in anti-melanoma responses. These data support the potential of IMCgp100 as an effective treatment for malignant melanoma. Citation Format: Namir J. Hassan, Giovanna Bossi, Debbie Baker, Katherine Adams, Jane Harper, Joseph Dukes, Nathaniel Liddy, Samantha Paston, Yvonne McGrath, Tara Mahon, Peter Molloy, Malkit Sami, Emma Baston, Brian Cameron, Andrew Johnson, Annelise Vuidepot, Gerry Linette, Michael Kalos, Carl June, Bent Jakobsen. IMCgp100: A novel bi-specific biologic for the treatment of malignant melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2900. doi:10.1158/1538-7445.AM2014-2900

Abstract 4873: ImmTACs re-direct the immune system efficiently to eradicate cancer

Immunotherapy strategies that are able to induce T cell infiltration into tumors and activate a cytotoxic T cell response have the potential to destroy the tumor. Although T cells can mediate clearance of a tumor, thymic selection and the suppressive microenvironment limit their effectiveness. To overcome poor tumor immunogenicity, we have developed a unique platform that enables the generation of ImmTACs (Immune-mobilising monoclonal TCRs Against Cancer); these are comprised of an affinity enhanced TCR specific for a cancer antigen fused to an anti-CD3 scFv. The TCR end targets and binds MHC class I/peptide complex displayed on cancerous cells while the anti-CD3 scFv end engages polyclonal T cells to mediate a potent anti-tumor response. The most advanced ImmTAC, IMCgp100, targets the HLA-A2/gp100280-288 epitope presented by melanoma cells. IMCgp100 is currently in a Phase I/IIa clinical trial for advance malignant melanoma and is showing promising clinical efficacy in some patients. Here we report a series of in vitro experiments evaluating IMCgp100 mechanism of action. IMCgp100 is able to redirect T cells from healthy donors or from melanoma patients to destroy cancer cells and secrete a range of inflammatory cytokines and chemokines associated with T cell trafficking into tumors; some of these cytokines also induce upregulation of inhibitory pathway molecules. We show that effector memory cells in the CD8+ and CD4+ T cell compartments are very efficient in eliminating melanoma cells and in expanding upon IMCgp100 engagement. The effects of combining IMCgp100 with agents that relieve the suppression imposed by immune check-point molecules have also been investigated. Citation Format: Giovanna Bossi, Rupert Kenefeck, Caroline Caroline Boudousquie, Jane Harper, Joseph Dukes, Nathaniel Liddy, Samantha Paston, Tara Mahon, Peter Molloy, Malkit Sami, Emma Baston, Brian Cameron, Annelise Vuidepot, Namir Hassan, Bent K. Jakobsen. ImmTACs re-direct the immune system efficiently to eradicate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4873.

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

Immunotherapeutic strategies that drive activation of cytotoxic T cells possess significant potential to eradicate tumours. Whereas monoclonal antibodies are restricted to targeting secreted or cell surface proteins, T cell receptors (TCRs) are able to recognise a wider range of targets. This is achieved through binding to short peptide fragments derived from proteins that are degraded intracellularly and presented at the cell surface by human leukocyte antigens (HLAs). Natural cancer specific TCRs however, have weak affinities and cancer cells often develop escape mechanisms to avoid destruction by T cells. To overcome this, we have developed Immune mobilising monoclonal TCRs Against Cancer (ImmTACs); a new class of soluble bi-specific molecules comprising affinity-enhanced, monoclonal T cell receptors (mTCRs) fused to an anti-CD3 scFv. ImmTACs target peptides presented by HLA, and through the anti-CD3 effector, re-direct cytotoxic T cells to achieve highly specific and potent tumour cell killing. At Immunocore, we have developed an integrated in-house process for the generation of ImmTACs and here describe the critical engineering steps involved. T-cell clones that specifically recognise validated cancer antigens are isolated from peripheral blood lymphocytes and the TCR-encoding sequences are identified by RACE. To confirm antigen binding, TCR a and â chains are expressed as inclusion bodies in bacteria, co-refolded in vitro, and their binding to the target peptide:HLA tested by Surface Plasmon Resonance (SPR). The affinity of the TCR is then enhanced up to a million-fold through directed evolution, utilising phage display. Individual mutants are screened by SPR and combined to generate ImmTACs with pM affinities (KD) and binding half-lives of many hours. A range of biochemical and cellular assays are then performed to assess the potency and specificity of each ImmTAC generated. This process has been successfully applied to produce ImmTACs for a wide range of targets, demonstrating the robustness of the platform. Our lead candidate, IMCgp100, is undergoing Phase IIa clinical trials in patients with advanced malignant melanoma. This reagent, which specifically targets the gp100 (280-288) peptide presented by HLA-A2 on melanoma cells, is well tolerated and shows very promising therapeutic potential. Citation Format: Andrew Knox, Fiona Chester, Frayne Bianchi, Sarah Bailey, Lucie Bouard, Nathaniel Liddy, Giovanna Bossi, Jane Harper, Joseph Dukes, Samantha Paston, Tara Mahon, Jessie Gavarret, Peter Molloy, Malkit Sami, Emma Baston, Brian Cameron, Alex Powlesland, Penio Todorov, Andrew Johnson, Martin Ebner, Yvonne McGrath, Namir Hassan, Annelise Vuidepot, Bent Jakobsen. Developing high affinity, soluble T cell receptors for the treatment of cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4872.

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

Meeting abstracts Tumour-specific T cells have the potential to eradicate cancer. However anti-tumour immunity is limited by low TCR affinities, MHC downregulation by cancer cells and an immunosuppressive tumour microenvironment. As most tumour-associated antigens are auto-antigens, tumour specific