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The T-cell receptor (TCR) initiates T-lymphocyte activation, but the mechanism of TCR activation remains uncertain. Here, we present cryogenic electron microscopy structures for the unliganded and human leukocyte antigen (HLA)-bound human TCR–CD3 complex in nanodiscs that provide a native-like lipid environment. Distinct from the open and extended conformation seen in detergent, the unliganded TCR–CD3 in nanodiscs adopts two related closed and compacted conformations that represent its physiologic resting state in vivo. By contrast, the HLA-bound complex adopts the open and extended conformation, and conformation-locking disulfide mutants show that ectodomain opening is necessary for maximal ligand-dependent T-cell activation. These structures also reveal conformation-dependent protein–lipid and glycan–glycan interactions within the TCR. Together, these results establish allosteric conformational change during TCR activation, reveal avenues for immunotherapeutic engineering, and highlight the importance of native-like lipid environments for membrane protein structure determination. The T-cell receptor (TCR) activation mechanism has remained uncertain. Here, the authors present molecular structures for the apo and ligand-bound human TCR–CD3 complex in lipid nanodiscs, revealing large conformational changes during activation.

The use of T cells reactive with intracellular tumor-associated or tumor-specific antigens has been a promising strategy for cancer immunotherapies in the past three decades, but the approach has been constrained by a limited understanding of the T cell receptor’s (TCR) complex functions and specificities. Newer TCR and T cell-based approaches are in development, including engineered adoptive T cells with enhanced TCR affinities, TCR mimic antibodies, and T cell-redirecting bispecific agents. These new therapeutic modalities are exciting opportunities by which TCR recognition can be further exploited for therapeutic benefit. In this review we summarize the development of TCR-based therapeutic strategies and focus on balancing efficacy and potency versus specificity, and hence, possible toxicity, of these powerful therapeutic modalities.

BackgroundCertain phosphorylated peptides are differentially presented by major histocompatibility complex (MHC) molecules on cancer cells characterized by aberrant phosphorylation. Phosphopeptides presented in complex with the human leukocyte antigen HLA-A*02:01 provide a stability advantage over their non-phosphorylated counterparts. This stability is thought to contribute to enhanced immunogenicity. Whether tumor-associated phosphopeptides presented by other common alleles exhibit immunogenicity and structural characteristics similar to those presented by A*02:01 is unclear. Therefore, we determined the identity, structural features, and immunogenicity of phosphopeptides presented by the prevalent alleles HLA-A*03:01, HLA-A*11:01, HLA-C*07:01, and HLA-C*07:02.MethodsWe isolated peptide-MHC complexes by immunoprecipitation from 11 healthy and neoplastic tissue samples using mass spectrometry, and then combined the resulting data with public immunopeptidomics data sets to assemble a curated set of phosphopeptides presented by 96 samples spanning 20 distinct healthy and neoplastic tissue types. We determined the biochemical features of selected phosphopeptides by in vitro binding assays and in silico docking, and their immunogenicity by analyzing healthy donor T cells for phosphopeptide-specific multimer binding and cytokine production.ResultsWe identified a subset of phosphopeptides presented by HLA-A*03:01, A*11:01, C*07:01 and C*07:02 on multiple tumor types, particularly lymphomas and leukemias, but not healthy tissues. These phosphopeptides are products of genes essential to lymphoma and leukemia survival. The presented phosphopeptides generally exhibited similar or worse binding to A*03:01 than their non-phosphorylated counterparts. HLA-C*07:01 generally presented phosphopeptides but not their unmodified counterparts. Phosphopeptide binding to HLA-C*07:01 was dependent on B-pocket interactions that were absent in HLA-C*07:02. While HLA-A*02:01 and HLA-A*11:01 phosphopeptide-specific T cells could be readily detected in an autologous setting even when the non-phosphorylated peptide was co-presented, HLA-A*03:01 or HLA-C*07:01 phosphopeptides were repeatedly non-immunogenic, requiring use of allogeneic T cells to induce phosphopeptide-specific T cells.ConclusionsPhosphopeptides presented by multiple alleles that are differentially expressed on tumors constitute tumor-specific antigens that could be targeted for cancer immunotherapy, but the immunogenicity of such phosphopeptides is not a general feature. In particular, phosphopeptides presented by HLA-A*02:01 and A*11:01 exhibit consistent immunogenicity, while phosphopeptides presented by HLA-A*03:01 and C*07:01, although appropriately presented, are not immunogenic. Thus, to address an expanded patient population, phosphopeptide-targeted immunotherapies should be wary of allele-specific differences.

Phosphopeptides derived from dysregulated protein phosphorylation in cancer cells can be processed and presented by MHC class I and class II molecules and, therefore, represent an untapped class of tumor-specific antigens that could be used as widely expressed \"public\" cancer neoantigens (NeoAgs). We generated a TCR mimic (TCRm) mAb, 6B1, specific for a phosphopeptide derived from insulin receptor substrate 2 (pIRS2) presented by HLA-A*02:01. The pIRS2 epitope's presentation by HLA-A*02:01 was confirmed by mass spectrometry. The TCRm 6B1 specifically bound to pIRS2/HLA-A2 complex on tumor cell lines that expressed pIRS2 in the context of HLA-A*02:01. Bispecific mAbs engaging CD3 of T cells were able to kill tumor cell lines in a pIRS2- and HLA-A*02:01-restricted manner. Structure modeling shows a prerequisite for an arginine or lysine at the first position to bind mAb. Therefore, 6B1 could recognize phosphopeptides derived from various phosphorylated proteins with similar amino acid compositions. This raised the possibility that a TCRm specific for the pIRS2/HLA-A2 complex could target a range of phosphopeptides presented by HLA-A*02:01 in various tumor cells. This is the first TCRm mAb to our knowledge targeting a phosphopeptide/MHC class I complex; the potential of this class of agents for clinical applications warrants further investigation.

Cancer immunotherapies are capable of inducing durable remissions in refractory diseases. Immunotherapeutic development offers opportunities for target selection and candidate discovery while posing challenges for therapeutic design and biomarker identification. This thesis aims to discover novel targets, develop new modalities, and determine biomarkers of response to immunotherapies. The first chapter summarizes clinical experiences with tumor antigen-specific T cells with demonstrated potential to induce tumor regressions. The second chapter explores the presentation and immunogenicity of phosphopeptide tumor antigens presented by common MHC alleles. The third chapter presents a method for identifying antigen-specific T cell receptors (TCRs) to expedite the discovery of cellular therapy candidates. The fourth chapter presents the preclinical development of an immunocytokine for treating AML, employing protein engineering, immune cell assays, and immunoPET to select a lead candidate. The fifth chapter presents a computational pipeline for screening TCR mimic antibodies (TCRm) for crossreactivity, providing exemplary analysis on a TCRm targeting the HLA-A2/pIRS2 phosphoantigen. The final chapter introduces a scoring algorithm to stratify patients receiving immune checkpoint therapies based on antigen presentation. These new methodologies, predictive tools, and therapeutic strategies could enhance the next generation of cancer immunotherapies.

Purpose Immune cells are capable of eliminating leukemic cells, as evidenced by outcomes in hematopoietic cell transplantation (HCT). However, patients who fail induction therapy will not benefit from HCT due to their minimal residual disease (MRD) status. Thus, we aimed to develop an immunomodulatory agent to reduce MRD by activating immune effector cells in the presence of leukaemia cells via a novel fusion protein that chimerises two clinically tolerated biologics: a CD33 antibody and the IL15Ra/IL15 complex (CD33xIL15). Methods We generated a set of CD33xIL15 fusion protein constructs with varying configurations and identified those with the best in vitro AML-binding, T cell activation, and NK cell potentiation. Using 89 Zr-immunoPET imaging we then evaluated the biodistribution and in vivo tumour retention of the most favourable CD33xIL15 constructs in an AML xenograft model. Ex vivo biodistribution studies were used to confirm the pharmacokinetics of the constructs. Results Two of the generated fusion proteins, CD33xIL15 (N72D) and CD33xIL15wt, demonstrated optimal in vitro behaviour and were further evaluated in vivo. These studies revealed that the CD33xIL15wt candidate was capable of being retained in the tumour for as long as its parental CD33 antibody, Lintuzumab (13.9 ± 3.1%ID/g vs 18.6 ± 1.1%ID/g at 120 h). Conclusion This work demonstrates that CD33xIL15 fusion proteins are capable of targeting leukemic cells and stimulating local T cells in vitro and of concentrating in the tumour in AML xenografts. It also highlights the importance of 89 Zr-immunoPET to guide the development and selection of tumour-targeted antibody-cytokine fusion proteins. Graphical Abstract

Certain phosphorylated peptides are differentially presented by MHC molecules on cancer cells characterized by aberrant phosphorylation. Phosphopeptides presented in complex with the human leukocyte antigen HLA-A*02:01 provide a stability advantage over their nonphosphorylated counterparts. This stability is thought to contribute to enhanced immunogenicity. Whether tumor-associated phosphopeptides presented by other common alleles exhibit immunogenicity and structural characteristics similar to those presented by A*02:01 is unclear. Therefore, we determined the identity, structural features, and immunogenicity of phosphopeptides presented by the prevalent alleles HLA-A*03:01, -A*11:01, -C*07:01, and -C*07:02. We isolated peptide-MHC complexes by immunoprecipitation from 10 healthy and neoplastic tissue samples using mass spectrometry, and then combined the resulting data with public immunopeptidomics datasets to assemble a curated set of phosphopeptides presented by 20 distinct healthy and neoplastic tissue types. We determined the biochemical features of selected phosphopeptides by in vitro binding assays and in silico docking, and their immunogenicity by analyzing healthy donor T cells for phosphopeptide-specific multimer binding and cytokine production. We identified a subset of phosphopeptides presented by HLA-A*03:01, A*11:01, C*07:01 and C*07:02 on multiple tumor types, particularly lymphomas and leukemias, but not healthy tissues. These phosphopeptides are products of genes essential to lymphoma and leukemia survival. The presented phosphopeptide generally exhibited similar or worse binding to A*03:01 than their nonphosphorylated counterparts. HLA-C*07:01 generally presented phosphopeptides but not their unmodified counterparts. Phosphopeptide binding to HLA-C*07:01 was dependent on B-pocket interactions that were absent in HLA-C*07:02. While HLA-A*02:01 and -A*11:01 phosphopeptide-specific T cells could be readily detected in an autologous setting even when the nonphosphorylated peptide was co-presented, HLA-A*03:01 or -C*07:01 phosphopeptides were repeatedly nonimmunogenic, requiring use of allogeneic T cells to induce phosphopeptide-specific T cells. Phosphopeptides presented by multiple alleles that are differentially expressed on tumors constitute tumor-specific antigens that could be targeted for cancer immunotherapy, but the immunogenicity of such phosphopeptides is not a general feature. In particular, phosphopeptides presented by HLA-A*02:01 and A*11:01 exhibit consistent immunogenicity. Whereas phosphopeptides presented by HLA-A*03:01 and C*07:01, while appropriately presented, are not immunogenic. Thus, to address an expanded patient population, phosphopeptide-targeted immunotherapies should be wary of allele-specific differences.Competing Interest StatementDAS is on a board of, or has equity in, or income from: Lantheus, Sellas, Iovance, Pfizer, Actinium Pharmaceuticals, Inc., OncoPep, Repertoire, Sapience, and Eureka Therapeutics. TD is a consultant for Eureka Therapeutics. MGK is a consultant to Ardigen. RJO declares consultancy, research support, and royalties from Atara Biotherapeutics. All other authors declare no competing financial interests.Footnotes* Diction changes and funding acknowledgments update.

Immune cells are capable of eliminating leukemic cells, as evidenced by outcomes in hematopoietic cell transplantation (HCT). However, patients who fail induction therapy will not benefit from HCT due to their minimal residual disease (MRD) status. Thus, we aimed to develop an immunomodulatory agent to reduce MRD by activating immune effector cells in the presence of leukaemia cells via a novel fusion protein that chimerises two clinically tolerated biologics: a CD33 antibody and the IL15Ra/IL15 complex (CD33xIL15). We generated a set of CD33xIL15 fusion protein constructs with varying configurations and identified those with the best in vitro AML-binding, T cell activation, and NK cell potentiation. Using Zr-immunoPET imaging we then evaluated the biodistribution and in vivo tumour retention of the most favourable CD33xIL15 constructs in an AML xenograft model. Ex vivo biodistribution studies were used to confirm the pharmacokinetics of the constructs. Two of the generated fusion proteins, CD33xIL15 (N72D) and CD33xIL15wt, demonstrated optimal in vitro behaviour and were further evaluated in vivo. These studies revealed that the CD33xIL15wt candidate was capable of being retained in the tumour for as long as its parental CD33 antibody, Lintuzumab (13.9 ± 3.1%ID/g vs 18.6 ± 1.1%ID/g at 120 h). This work demonstrates that CD33xIL15 fusion proteins are capable of targeting leukemic cells and stimulating local T cells in vitro and of concentrating in the tumour in AML xenografts. It also highlights the importance of Zr-immunoPET to guide the development and selection of tumour-targeted antibody-cytokine fusion proteins.

The T-cell receptor (TCR) initiates T-lymphocyte activation, but mechanistic questions remain( ). Here, we present cryogenic electron microscopy structures for the unliganded and human leukocyte antigen (HLA)-bound human TCR-CD3 complex in nanodiscs that provide a native-like lipid environment. Distinct from the \"open and extended\" conformation seen in detergent( ), the unliganded TCR-CD3 in nanodiscs adopts two related \"closed and compacted\" conformations that represent its physiologic resting state . By contrast, the HLA-bound complex adopts the open and extended conformation, and conformation-locking disulfide mutants show that ectodomain opening is necessary for maximal ligand-dependent T-cell activation. Together, these results reveal allosteric conformational change during TCR activation and highlight the importance of native-like lipid environments for membrane protein structure determination.