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1.
J Immunother Cancer ; 11(5)2023 05.
Article in English | MEDLINE | ID: mdl-37258040

ABSTRACT

BACKGROUND: Treatment of some blood cancers with T cells that express a chimeric antigen receptor (CAR) against CD19 have shown remarkable results. In contrast, CAR-T cell efficacy against solid tumors has been difficult to achieve. METHODS: To examine the potential of CAR-T cell treatments against ovarian cancers, we used the mouse ovarian cancer cell line ID8 in an intraperitoneal model that exhibits disseminated solid tumors in female C57BL/6J mice. The CAR contained a single-chain Fv from antibody 237 which recognizes a Tn-glycopeptide-antigen expressed by ID8 due to aberrant O-linked glycosylation in the absence of the transferase-dependent chaperone Cosmc. The efficacy of four Tn-dependent CARs with varying affinity to Tn antigen, and each containing CD28/CD3ζ cytoplasmic domains, were compared in vitro and in vivo in this study. RESULTS: In line with many observations about the impact of aberrant O-linked glycosylation, the ID8Cosmc knock-out (ID8Cosmc-KO) exhibited more rapid tumor progression compared with wild-type ID8. Despite the enhanced tumor growth in vivo, 237 CAR and a mutant with 30-fold higher affinity, but not CARs with lower affinity, controlled advanced ID8Cosmc-KO tumors. Tumor regression could be achieved with a single intravenous dose of the CARs, but intraperitoneal administration was even more effective. The CAR-T cells persisted over a period of months, allowing CAR-treated mice to delay tumor growth in a re-challenge setting. The most effective CARs exhibited the highest affinity for antigen. Antitumor effects observed in vivo were associated with increased numbers of T cells and macrophages, and higher levels of cleaved caspase-3, in the tumor microenvironment. Notably, the least therapeutically effective CAR mediated tonic signaling leading to antigen-independent cytokine expression and it had higher levels of the immunosuppressive cytokine interleukin10. CONCLUSION: The findings support the development of affinity-optimized CAR-T cells as a potential treatment for established ovarian cancer, with the most effective CARs mediating a distinct pattern of inflammatory cytokine release in vitro. Importantly, the most potent Tn-dependent CAR-T cells showed no evidence of toxicity in tumor-bearing mice in a syngeneic, immunocompetent system.


Subject(s)
Ovarian Neoplasms , Receptors, Chimeric Antigen , Humans , Female , Mice , Animals , Receptors, Chimeric Antigen/genetics , Receptors, Chimeric Antigen/metabolism , T-Lymphocytes , Immunotherapy, Adoptive/methods , Xenograft Model Antitumor Assays , Mice, Inbred C57BL , Cytokines/metabolism , Tumor Microenvironment
2.
J Immunother Cancer ; 11(2)2023 02.
Article in English | MEDLINE | ID: mdl-36808076

ABSTRACT

BACKGROUND: Adoptive cellular therapies with chimeric antigen receptor T cells have revolutionized the treatment of some malignancies but have shown limited efficacy in solid tumors such as glioblastoma and face a scarcity of safe therapeutic targets. As an alternative, T cell receptor (TCR)-engineered cellular therapy against tumor-specific neoantigens has generated significant excitement, but there exist no preclinical systems to rigorously model this approach in glioblastoma. METHODS: We employed single-cell PCR to isolate a TCR specific for the Imp3D81N neoantigen (mImp3) previously identified within the murine glioblastoma model GL261. This TCR was used to generate the Mutant Imp3-Specific TCR TransgenIC (MISTIC) mouse in which all CD8 T cells are specific for mImp3. The therapeutic efficacy of neoantigen-specific T cells was assessed through a model of cellular therapy consisting of the transfer of activated MISTIC T cells and interleukin 2 into lymphodepleted tumor-bearing mice. We employed flow cytometry, single-cell RNA sequencing, and whole-exome and RNA sequencing to examine the factors underlying treatment response. RESULTS: We isolated and characterized the 3×1.1C TCR that displayed a high affinity for mImp3 but no wild-type cross-reactivity. To provide a source of mImp3-specific T cells, we generated the MISTIC mouse. In a model of adoptive cellular therapy, the infusion of activated MISTIC T cells resulted in rapid intratumoral infiltration and profound antitumor effects with long-term cures in a majority of GL261-bearing mice. The subset of mice that did not respond to the adoptive cell therapy showed evidence of retained neoantigen expression but intratumoral MISTIC T cell dysfunction. The efficacy of MISTIC T cell therapy was lost in mice bearing a tumor with heterogeneous mImp3 expression, showcasing the barriers to targeted therapy in polyclonal human tumors. CONCLUSIONS: We generated and characterized the first TCR transgenic against an endogenous neoantigen within a preclinical glioma model and demonstrated the therapeutic potential of adoptively transferred neoantigen-specific T cells. The MISTIC mouse provides a powerful novel platform for basic and translational studies of antitumor T-cell responses in glioblastoma.


Subject(s)
Glioblastoma , Immunotherapy, Adoptive , Mice , Humans , Animals , Antigens, Neoplasm , CD8-Positive T-Lymphocytes , Receptors, Antigen, T-Cell
3.
Nat Commun ; 13(1): 3207, 2022 06 09.
Article in English | MEDLINE | ID: mdl-35680861

ABSTRACT

In Fall 2020, universities saw extensive transmission of SARS-CoV-2 among their populations, threatening health of the university and surrounding communities, and viability of in-person instruction. Here we report a case study at the University of Illinois at Urbana-Champaign, where a multimodal "SHIELD: Target, Test, and Tell" program, with other non-pharmaceutical interventions, was employed to keep classrooms and laboratories open. The program included epidemiological modeling and surveillance, fast/frequent testing using a novel low-cost and scalable saliva-based RT-qPCR assay for SARS-CoV-2 that bypasses RNA extraction, called covidSHIELD, and digital tools for communication and compliance. In Fall 2020, we performed >1,000,000 covidSHIELD tests, positivity rates remained low, we had zero COVID-19-related hospitalizations or deaths amongst our university community, and mortality in the surrounding Champaign County was reduced more than 4-fold relative to expected. This case study shows that fast/frequent testing and other interventions mitigated transmission of SARS-CoV-2 at a large public university.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/epidemiology , COVID-19/prevention & control , COVID-19 Testing , Humans , SARS-CoV-2/genetics , Sensitivity and Specificity , Universities
4.
Methods Mol Biol ; 2491: 117-142, 2022.
Article in English | MEDLINE | ID: mdl-35482188

ABSTRACT

Protein engineering using display platforms such as yeast display and phage display has allowed discovery of proteins with therapeutic and industrial applications. Antibodies and T cell receptors developed for therapeutic applications are often engineered by constructing libraries of mutations in loops of five to ten residues called complementarity determining regions that are in proximity to the antigen. In the past decade, deep mutational scanning has become a powerful tool in a protein engineer's toolbox, as it allows one to compare the impact of all 20 amino acids at each position, across the length of the protein. Thus, a single experiment can provide a sequence-activity landscape with information about hotspots or suboptimal binding sites in the original proteins. These residues or regions may be overlooked by engineering methods that are driven solely by structures or directed evolution of error-prone PCR libraries. Here, we describe experimental methods to engineer proteins by combining yeast display and deep mutational scanning mutagenesis, using T cell receptors as an example.


Subject(s)
Protein Engineering , Saccharomyces cerevisiae , Mutagenesis , Mutation , Protein Engineering/methods , Receptors, Antigen, T-Cell/genetics , Saccharomyces cerevisiae/genetics
5.
Biochemistry ; 59(43): 4163-4175, 2020 11 03.
Article in English | MEDLINE | ID: mdl-33074657

ABSTRACT

T cell receptors (TCRs) orchestrate cellular immunity by recognizing peptides presented by a range of major histocompatibility complex (MHC) proteins. Naturally occurring TCRs bind the composite peptide/MHC surface, recognizing peptides that are structurally and chemically compatible with the TCR binding site. Here we describe a molecularly evolved TCR variant that binds the human class I MHC protein HLA-A2 independent of the bound peptide, achieved by a drastic perturbation of the TCR binding geometry that places the molecule far from the peptide binding groove. This unique geometry is unsupportive of normal T cell signaling. A substantial divergence between affinity measurements in solution and in two dimensions between proximal cell membranes leads us to attribute the lack of signaling to steric hindrance that limits binding in the confines of a cell-cell interface. Our results provide an example of how receptor binding geometry can impact T cell function and provide further support for the view that germline-encoded residues in TCR binding loops evolved to drive productive TCR recognition and signaling.


Subject(s)
Receptors, Antigen, T-Cell/metabolism , Binding Sites , HLA-A Antigens/metabolism , Humans , Major Histocompatibility Complex/genetics , Major Histocompatibility Complex/physiology , Protein Binding , Protein Conformation
6.
Science ; 369(6508): 1261-1265, 2020 09 04.
Article in English | MEDLINE | ID: mdl-32753553

ABSTRACT

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds angiotensin-converting enzyme 2 (ACE2) on host cells to initiate entry, and soluble ACE2 is a therapeutic candidate that neutralizes infection by acting as a decoy. By using deep mutagenesis, mutations in ACE2 that increase S binding are found across the interaction surface, in the asparagine 90-glycosylation motif and at buried sites. The mutational landscape provides a blueprint for understanding the specificity of the interaction between ACE2 and S and for engineering high-affinity decoy receptors. Combining mutations gives ACE2 variants with affinities that rival those of monoclonal antibodies. A stable dimeric variant shows potent SARS-CoV-2 and -1 neutralization in vitro. The engineered receptor is catalytically active, and its close similarity with the native receptor may limit the potential for viral escape.


Subject(s)
Betacoronavirus/metabolism , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , Protein Engineering , Receptors, Virus/genetics , Receptors, Virus/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Substitution , Angiotensin-Converting Enzyme 2 , Binding Sites , Binding, Competitive , Cell Line , Humans , Models, Molecular , Mutagenesis , Mutation , Peptidyl-Dipeptidase A/chemistry , Protein Binding , Protein Interaction Domains and Motifs , Protein Multimerization , Receptors, Coronavirus , Receptors, Virus/chemistry , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry
7.
Proc Natl Acad Sci U S A ; 117(26): 15148-15159, 2020 06 30.
Article in English | MEDLINE | ID: mdl-32541028

ABSTRACT

The potency of adoptive T cell therapies targeting the cell surface antigen CD19 has been demonstrated in hematopoietic cancers. It has been difficult to identify appropriate targets in nonhematopoietic tumors, but one class of antigens that have shown promise is aberrant O-glycoprotein epitopes. It has long been known that dysregulated synthesis of O-linked (threonine or serine) sugars occurs in many cancers, and that this can lead to the expression of cell surface proteins containing O-glycans comprised of a single N-acetylgalactosamine (GalNAc, known as Tn antigen) rather than the normally extended carbohydrate. Previously, we used the scFv fragment of antibody 237 as a chimeric antigen receptor (CAR) to mediate recognition of mouse tumor cells that bear its cognate Tn-glycopeptide epitope in podoplanin, also called OTS8. Guided by the structure of the 237 Fab:Tn-OTS8-glycopeptide complex, here we conducted a deep mutational scan showing that residues flanking the Tn-glycan contributed significant binding energy to the interaction. Design of 237-scFv libraries in the yeast display system allowed us to isolate scFv variants with higher affinity for Tn-OTS8. Selection with a noncognate human antigen, Tn-MUC1, yielded scFv variants that were broadly reactive with multiple Tn-glycoproteins. When configured as CARs, engineered T cells expressing these scFv variants showed improved activity against mouse and human cancer cell lines defective in O-linked glycosylation. This strategy provides CARs with Tn-peptide specificities, all based on a single scFv scaffold, that allows the same CAR to be tested for toxicity in mice and efficacy against mouse and human tumors.


Subject(s)
Antigens, Tumor-Associated, Carbohydrate/immunology , Receptors, Chimeric Antigen/metabolism , T-Lymphocytes/physiology , Amino Acid Sequence , Animals , Antibodies , Cell Line, Tumor , Directed Molecular Evolution , Epitopes/genetics , Humans , Mice , Models, Molecular , Mutation , Protein Conformation , Receptors, Chimeric Antigen/genetics
8.
Neuro Oncol ; 22(9): 1276-1288, 2020 09 29.
Article in English | MEDLINE | ID: mdl-32133512

ABSTRACT

BACKGROUND: Although clinical trials testing immunotherapies in glioblastoma (GBM) have yielded mixed results, new strategies targeting tumor-specific somatic coding mutations, termed "neoantigens," represent promising therapeutic approaches. We characterized the microenvironment and neoantigen landscape of the aggressive CT2A GBM model in order to develop a platform to test combination checkpoint blockade and neoantigen vaccination. METHODS: Flow cytometric analysis was performed on intracranial CT2A and GL261 tumor-infiltrating lymphocytes (TILs). Whole-exome DNA and RNA sequencing of the CT2A murine GBM was employed to identify expressed, somatic mutations. Predicted neoantigens were identified using the pVAC-seq software suite, and top-ranking candidates were screened for reactivity by interferon-gamma enzyme linked immunospot assays. Survival analysis was performed comparing neoantigen vaccination, anti-programmed cell death ligand 1 (αPD-L1), or combination therapy. RESULTS: Compared with the GL261 model, CT2A exhibited immunologic features consistent with human GBM including reduced αPD-L1 sensitivity and hypofunctional TILs. Of the 29 CT2A neoantigens screened, we identified neoantigen-specific CD8+ T-cell responses in the intracranial TIL and draining lymph nodes to two H2-Kb restricted (Epb4H471L and Pomgnt1R497L) and one H2-Db restricted neoantigen (Plin2G332R). Survival analysis showed that therapeutic neoantigen vaccination with Epb4H471L, Pomgnt1R497L, and Plin2G332R, in combination with αPD-L1 treatment was superior to αPD-L1 alone. CONCLUSIONS: We identified endogenous neoantigen specific CD8+ T cells within an αPD-L1 resistant murine GBM and show that neoantigen vaccination significantly augments survival benefit in combination with αPD-L1 treatment. These observations provide important preclinical correlates for GBM immunotherapy trials and support further investigation into the effects of multimodal immunotherapeutic interventions on antiglioma immunity. KEY POINTS: 1. Neoantigen vaccines combined with checkpoint blockade may be promising treatments.2. CT2A tumors exhibit features of human GBM microenvironments.3. Differential scanning fluorimetry assays may complement in silico neoantigen prediction tools.


Subject(s)
Glioblastoma , Animals , Antigens, Neoplasm , CD8-Positive T-Lymphocytes , Glioblastoma/therapy , Humans , Immunotherapy , Lymphocytes, Tumor-Infiltrating , Mice , Tumor Microenvironment , Vaccines, Combined
10.
JCI Insight ; 4(21)2019 11 01.
Article in English | MEDLINE | ID: mdl-31672936

ABSTRACT

Human cancer cells were eradicated by adoptive transfer of T cells transduced with a chimeric antigen receptor (CAR) made from an antibody (237Ab) that is highly specific for the murine Tn-glycosylated podoplanin (Tn-PDPN). The objectives were to determine the specificity of these CAR-transduced T (CART) cells and the mechanism for the absence of relapse. We show that although the 237Ab bound only to cell lines expressing murine Tn-PDPN, the 237Ab-derived 237CART cells lysed multiple different human and murine cancers not predicted by the 237Ab binding. Nevertheless, the 237CART cell reactivities remained cancer specific because all recognitions were dependent on the Tn glycosylation that resulted from COSMC mutations that were not present in normal tissues. While Tn was required for the recognition by 237CART, Tn alone was not sufficient for 237CART cell activation. Activation of 237CART cells required peptide backbone recognition but tolerated substitutions of up to 5 of the 7 amino acid residues in the motif recognized by 237Ab. Together, these findings demonstrate what we believe is a new principle whereby simultaneous recognition of multiple independent Tn-glycopeptide antigens on a cancer cell makes tumor escape due to antigen loss unlikely.


Subject(s)
Antigens, Neoplasm/immunology , Neoplasms/immunology , Receptors, Chimeric Antigen/immunology , Adoptive Transfer , Animals , Antigens, Tumor-Associated, Carbohydrate/immunology , Cell Line , Glycosylation , Humans , Membrane Glycoproteins/immunology , Mice , Neoplasms/pathology
11.
Crit Rev Immunol ; 39(2): 105-122, 2019.
Article in English | MEDLINE | ID: mdl-31679251

ABSTRACT

The past decade has seen enormous progress in cancer immunotherapy. Checkpoint inhibitors are a class of immunotherapy that act to recruit endogenous T cells of a patient's immune system against cancer-associated peptide- MHC antigens. In this process, mutated antigenic peptides referred to as neoantigens often serve as the target on cancer cells that are recognized by the T cell receptor (TCR) on endogenous T cells. Another successful immunotherapy has involved adoptive T cell therapy, where therapeutic doses of T cells expressing a gene for an anti-cancer receptor are delivered to a patient. This approach has been used primarily against hematopoietic cancers using synthetic receptors called chimeric antigen receptors (CARs). CARs typically contain an antibody fragment (single-chain Fv, scFv) against a cancer cell surface antigen such as the B cell molecule CD19. While therapeutic CARs (and full antibodies) target antigens expressed on cell surfaces, TCRs can target a much larger array of intracellular proteins by binding to any cellular peptide associated with an MHC product. These cancer targets include self-peptides from aberrantly expressed/overexpressed proteins or neoantigens. In this review, we discuss the use of TCRs in adoptive T cell therapy and their target antigens. We focus on two properties that impact sensitivity, potency, and possible toxic cross-reactivity of TCR-mediated therapy: (1) the affinity of the TCR for the target antigen, and (2) the density of the target antigen. Finally, we provide a comprehensive listing of the current clinical trials that involve TCRs in adoptive T cell cancer therapy.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Immunotherapy, Adoptive/methods , Neoplasms/therapy , Receptors, Antigen, T-Cell/genetics , Receptors, Chimeric Antigen/genetics , Animals , Antigens, Neoplasm/immunology , CD8-Positive T-Lymphocytes/transplantation , Cytotoxicity, Immunologic , Genetic Engineering , Humans , Neoplasms/immunology
12.
Cancer Immunol Res ; 7(12): 2025-2035, 2019 12.
Article in English | MEDLINE | ID: mdl-31548259

ABSTRACT

Despite progress in adoptive T-cell therapies, the identification of targets remains a challenge. Although chimeric antigen receptors recognize cell-surface antigens, T-cell receptors (TCR) have the advantage that they can target the array of intracellular proteins by binding to peptides associated with major histocompatibility complex (MHC) products (pepMHC). Although hundreds of cancer-associated peptides have been reported, it remains difficult to identify effective TCRs against each pepMHC complex. Conventional approaches require isolation of antigen-specific CD8+ T cells, followed by TCRαß gene isolation and validation. To bypass this process, we used directed evolution to engineer TCRs with desired peptide specificity. Here, we compared the activity and cross-reactivity of two affinity-matured TCRs (T1 and RD1) with distinct origins. T1-TCR was isolated from a melanoma-reactive T-cell line specific for MART-1/HLA-A2, whereas RD1-TCR was derived de novo against MART-1/HLA-A2 by in vitro engineering. Despite their distinct origins, both TCRs exhibited similar peptide fine specificities, focused on the center of the MART-1 peptide. In CD4+ T cells, both TCRs mediated activity against MART-1 presented by HLA-A2. However, in CD8+ T cells, T1, but not RD1, demonstrated cross-reactivity with endogenous peptide/HLA-A2 complexes. Based on the fine specificity of these and other MART-1 binding TCRs, we conducted bioinformatics scans to identify structurally similar self-peptides in the human proteome. We showed that the T1-TCR cross-reacted with many of these self-peptides, whereas the RD1-TCR was rarely cross-reactive. Thus, TCRs such as RD1, generated de novo against cancer antigens, can serve as an alternative to TCRs generated from T-cell clones.


Subject(s)
Antigens, Neoplasm/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Peptides/immunology , Receptors, Antigen, T-Cell/immunology , Animals , Cell Line, Tumor , Cross Reactions , Humans , Mice, Transgenic
13.
Proc Natl Acad Sci U S A ; 116(8): 3136-3145, 2019 02 19.
Article in English | MEDLINE | ID: mdl-30728302

ABSTRACT

Successful efforts to activate T cells capable of recognizing weak cancer-associated self-antigens have employed altered peptide antigens to activate T cell responses capable of cross-reacting on native tumor-associated self. A limitation of this approach is the requirement for detailed knowledge about the altered self-peptide ligands used in these vaccines. In the current study we considered allorecognition as an approach for activating CTL capable of recognizing weak or self-antigens in the context of self-MHC. Nonself antigen-presenting molecules typically contain polymorphisms that influence interactions with the bound peptide and TCR interface. Recognition of these nonself structures results in peptide-dependent alloimmunity. Alloreactive T cells target their inducing alloantigens as well as third-party alloantigens but generally fail to target self-antigens. Certain residues located on the alpha-1/2 domains of class I antigen-presenting molecules primarily interface with TCR. These residues are more conserved within and across species than are residues that determine peptide antigen binding properties. Class I variants designed with amino acid substitutions at key positions within the conserved helical structures are shown to provide strong activating signals to alloreactive CD8 T cells while avoiding changes in naturally bound peptide ligands. Importantly, CTL activated in this manner can break self-tolerance by reacting to self-peptides presented by native MHC. The ability to activate self-tolerant T cells capable of cross-reacting on self-peptide-MHC in vivo represents an approach for inducing autoimmunity, with possible application in cancer vaccines.


Subject(s)
Antigen Presentation/immunology , Cytotoxicity, Immunologic , Histocompatibility Antigens Class I/immunology , T-Lymphocytes, Cytotoxic/immunology , Amino Acid Sequence/genetics , Animals , CD8-Positive T-Lymphocytes/immunology , Humans , Immune Tolerance , Ligands , Lymphocyte Activation/immunology , Mice , Peptides/genetics , Peptides/immunology , Tumor Necrosis Factor Receptor Superfamily, Member 7/immunology
14.
Cancer Immunol Res ; 7(1): 50-61, 2019 01.
Article in English | MEDLINE | ID: mdl-30425106

ABSTRACT

Mutated peptides (neoantigens) from a patient's cancer genome can serve as targets for T-cell immunity, but identifying which peptides can be presented by an MHC molecule and elicit T cells has been difficult. Although algorithms that predict MHC binding exist, they are not yet able to distinguish experimental differences in half-lives of the complexes (an immunologically relevant parameter, referred to here as kinetic stability). Improvement in determining actual neoantigen peptide/MHC stability could be important, as only a small fraction of peptides in most current vaccines are capable of eliciting CD8+ T-cell responses. Here, we used a rapid, high-throughput method to experimentally determine peptide/HLA thermal stability on a scale that will be necessary for analysis of neoantigens from thousands of patients. The method combined the use of UV-cleavable peptide/HLA class I complexes and differential scanning fluorimetry to determine the Tm values of neoantigen complexes. Measured Tm values were accurate and reproducible and were directly proportional to the half-lives of the complexes. Analysis of known HLA-A2-restricted immunogenic peptides showed that Tm values better correlated with immunogenicity than algorithm-predicted binding affinities. We propose that temperature stability information can be used as a guide for the selection of neoantigens in cancer vaccines in order to focus attention on those mutated peptides with the highest probability of being expressed on the cell surface.


Subject(s)
Antigens, Neoplasm/immunology , HLA-A2 Antigen/immunology , Peptides/immunology , Antigens, Neoplasm/chemistry , HLA-A2 Antigen/chemistry , High-Throughput Screening Assays , Peptides/chemistry , Protein Stability , Temperature
15.
J Biol Chem ; 293(5): 1820-1834, 2018 02 02.
Article in English | MEDLINE | ID: mdl-29229779

ABSTRACT

Most affinity-maturation campaigns for antibodies and T-cell receptors (TCRs) operate on the residues at the binding site, located within the loops known as complementarity-determining regions (CDRs). Accordingly, mutations in contact residues, or so-called "second shell" residues, that increase affinity are typically identified by directed evolution involving combinatorial libraries. To determine the impact of residues located at a distance from the binding site, here we used single-codon libraries of both CDR and non-CDR residues to generate a deep mutational scan of a human TCR against the cancer antigen MART-1·HLA-A2. Non-CDR residues included those at the interface of the TCR variable domains (Vα and Vß) and surface-exposed framework residues. Mutational analyses showed that both Vα/Vß interface and CDR residues were important in maintaining binding to MART-1·HLA-A2, probably due to either structural requirements for proper Vα/Vß association or direct contact with the ligand. More surprisingly, many Vα/Vß interface substitutions yielded improved binding to MART-1·HLA-A2. To further explore this finding, we constructed interface libraries and selected them for improved stability or affinity. Among the variants identified, one conservative substitution (F45ßY) was most prevalent. Further analysis of F45ßY showed that it enhanced thermostability and increased affinity by 60-fold. Thus, introducing a single hydroxyl group at the Vα/Vß interface, at a significant distance from the TCR·peptide·MHC-binding site, remarkably affected ligand binding. The variant retained a high degree of specificity for MART-1·HLA-A2, indicating that our approach provides a general strategy for engineering improvements in either soluble or cell-based TCRs for therapeutic purposes.


Subject(s)
Complementarity Determining Regions/chemistry , HLA-A2 Antigen/chemistry , MART-1 Antigen/chemistry , Binding Sites , Complementarity Determining Regions/genetics , Complementarity Determining Regions/immunology , HLA-A2 Antigen/genetics , HLA-A2 Antigen/immunology , Humans , MART-1 Antigen/genetics , MART-1 Antigen/immunology , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Saccharomyces cerevisiae
16.
J Immunol ; 200(3): 1088-1100, 2018 02 01.
Article in English | MEDLINE | ID: mdl-29288199

ABSTRACT

Adoptive T cell therapies have achieved significant clinical responses, especially in hematopoietic cancers. Two types of receptor systems have been used to redirect the activity of T cells, normal heterodimeric TCRs or synthetic chimeric Ag receptors (CARs). TCRs recognize peptide-HLA complexes whereas CARs typically use an Ab-derived single-chain fragments variable that recognizes cancer-associated cell-surface Ags. Although both receptors mediate diverse effector functions, a quantitative comparison of the sensitivity and signaling capacity of TCRs and CARs has been limited due to their differences in affinities and ligands. In this study we describe their direct comparison by using TCRs that could be formatted either as conventional αß heterodimers, or as single-chain fragments variable constructs linked to CD3ζ and CD28 signaling domains or to CD3ζ alone. Two high-affinity TCRs (KD values of ∼50 and 250 nM) against MART1/HLA-A2 or WT1/HLA-A2 were used, allowing MART1 or WT1 peptide titrations to easily assess the impact of Ag density. Although CARs were expressed at higher surface levels than TCRs, they were 10-100-fold less sensitive, even in the absence of the CD8 coreceptor. Mathematical modeling demonstrated that lower CAR sensitivity could be attributed to less efficient signaling kinetics. Furthermore, reduced cytokine secretion observed at high Ag density for both TCRs and CARs suggested a role for negative regulators in both systems. Interestingly, at high Ag density, CARs also mediated greater maximal release of some cytokines, such as IL-2 and IL-6. These results have implications for the next-generation design of receptors used in adoptive T cell therapies.


Subject(s)
Antibody Affinity/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , MART-1 Antigen/immunology , Receptors, Antigen, T-Cell/immunology , WT1 Proteins/immunology , Antigens, Tumor-Associated, Carbohydrate/immunology , HLA Antigens/immunology , Humans , Lymphocyte Activation/immunology , Mutant Chimeric Proteins/immunology
17.
Nat Biotechnol ; 35(12): 1188-1195, 2017 Dec.
Article in English | MEDLINE | ID: mdl-29106410

ABSTRACT

Many promising targets for T-cell-based cancer immunotherapies are self-antigens. During thymic selection, T cells bearing T cell receptors (TCRs) with high affinity for self-antigen are eliminated. The affinity of the remaining low-avidity TCRs can be improved to increase their antitumor efficacy, but conventional saturation mutagenesis approaches are labor intensive, and the resulting TCRs may be cross-reactive. Here we describe the in vitro maturation and selection of mouse and human T cells on antigen-expressing feeder cells to develop higher-affinity TCRs. The approach takes advantage of natural Tcrb gene rearrangement to generate diversity in the length and composition of CDR3ß. In vitro differentiation of progenitors transduced with a known Tcra gene in the presence of antigen drives differentiation of cells with a distinct agonist-selected phenotype. We purified these cells to generate TCRß chain libraries pre-enriched for target antigen specificity. Several TCRß chains paired with a transgenic TCRα chain to produce a TCR with higher affinity than the parental TCR for target antigen, without evidence of cross-reactivity.


Subject(s)
Autoantigens/metabolism , Cell Differentiation/genetics , Precursor Cells, T-Lymphoid , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/metabolism , Animals , Cell Line , Flow Cytometry , Genes, T-Cell Receptor beta/genetics , Humans , Mice , Precursor Cells, T-Lymphoid/cytology , Precursor Cells, T-Lymphoid/metabolism , Protein Binding
18.
F1000Res ; 52016.
Article in English | MEDLINE | ID: mdl-27703664

ABSTRACT

Adoptive T-cell therapies have shown exceptional promise in the treatment of cancer, especially B-cell malignancies. Two distinct strategies have been used to redirect the activity of ex vivo engineered T cells. In one case, the well-known ability of the T-cell receptor (TCR) to recognize a specific peptide bound to a major histocompatibility complex molecule has been exploited by introducing a TCR against a cancer-associated peptide/human leukocyte antigen complex. In the other strategy, synthetic constructs called chimeric antigen receptors (CARs) that contain antibody variable domains (single-chain fragments variable) and signaling domains have been introduced into T cells. Whereas many reviews have described these two approaches, this review focuses on a few recent advances of significant interest. The early success of CARs has been followed by questions about optimal configurations of these synthetic constructs, especially for efficacy against solid tumors. Among the many features that are important, the dimensions and stoichiometries of CAR/antigen complexes at the synapse have recently begun to be appreciated. In TCR-mediated approaches, recent evidence that mutated peptides (neoantigens) serve as targets for endogenous T-cell responses suggests that these neoantigens may also provide new opportunities for adoptive T-cell therapies with TCRs.

19.
J Biol Chem ; 291(47): 24566-24578, 2016 Nov 18.
Article in English | MEDLINE | ID: mdl-27681597

ABSTRACT

Proteins are often engineered to have higher affinity for their ligands to achieve therapeutic benefit. For example, many studies have used phage or yeast display libraries of mutants within complementarity-determining regions to affinity mature antibodies and T cell receptors (TCRs). However, these approaches do not allow rapid assessment or evolution across the entire interface. By combining directed evolution with deep sequencing, it is now possible to generate sequence fitness landscapes that survey the impact of every amino acid substitution across the entire protein-protein interface. Here we used the results of deep mutational scans of a TCR-peptide-MHC interaction to guide mutational strategies. The approach yielded stable TCRs with affinity increases of >200-fold. The substitutions with the greatest enrichments based on the deep sequencing were validated to have higher affinity and could be combined to yield additional improvements. We also conducted in silico binding analyses for every substitution to compare them with the fitness landscape. Computational modeling did not effectively predict the impacts of mutations distal to the interface and did not account for yeast display results that depended on combinations of affinity and protein stability. However, computation accurately predicted affinity changes for mutations within or near the interface, highlighting the complementary strengths of computational modeling and yeast surface display coupled with deep mutational scanning for engineering high affinity TCRs.


Subject(s)
Computer Simulation , HLA-A2 Antigen/chemistry , Models, Molecular , Peptides/chemistry , Receptors, Antigen, T-Cell/chemistry , HLA-A2 Antigen/genetics , HLA-A2 Antigen/immunology , Humans , Mutagenesis , Peptides/genetics , Peptides/immunology , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/immunology
20.
Neurol Neuroimmunol Neuroinflamm ; 3(5): e276, 2016 Oct.
Article in English | MEDLINE | ID: mdl-27648463

ABSTRACT

Neuromyelitis optica (NMO) and spectrum disorder (NMO/SD) represent a vexing process and its clinical variants appear to have at their pathogenic core the loss of immune tolerance to the aquaporin-4 water channel protein. This process results in a characteristic pattern of astrocyte dysfunction, loss, and demyelination that predominantly affects the spinal cord and optic nerves. Although several empirical therapies are currently used in the treatment of NMO/SD, none has been proven effective in prospective, adequately powered, randomized trials. Furthermore, most of the current therapies subject patients to long-term immunologic suppression that can cause serious infections and development of cancers. The following is the first of a 2-part description of several key immune mechanisms in NMO/SD that might be amenable to therapeutic restoration of immune tolerance. It is intended to provide a roadmap for how potential immune tolerance restorative techniques might be applied to patients with NMO/SD. This initial installment provides a background rationale underlying attempts at immune tolerization. It provides specific examples of innovative approaches that have emerged recently as a consequence of technical advances. In several autoimmune diseases, these strategies have been reduced to practice. Therefore, in theory, the identification of aquaporin-4 as the dominant autoantigen makes NMO/SD an ideal candidate for the development of tolerizing therapies or cures for this increasingly recognized disease.

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