Structural interplay between germline interactions and adaptive recognition determines the bandwidth of TCR-peptide-MHC cross-reactivity.

The T cell antigen receptor (TCR)-peptide-major histocompatibility complex (MHC) interface is composed of conserved and diverse regions, yet the relative contribution of each in shaping recognition by T cells remains unclear. Here we isolated cross-reactive peptides with limited homology, which allowed us to compare the structural properties of nine peptides for a single TCR-MHC pair. The TCR's cross-reactivity was rooted in highly similar recognition of an apical 'hot-spot' position in the peptide with tolerance of sequence variation at ancillary positions. Furthermore, we found a striking structural convergence onto a germline-mediated interaction between the TCR CDR1α region and the MHC α2 helix in twelve TCR-peptide-MHC complexes. Our studies suggest that TCR-MHC germline-mediated constraints, together with a focus on a small peptide hot spot, might place limits on peptide antigen cross-reactivity.

Engineered CAR T Cells Targeting the Cancer-Associated Tn-Glycoform of the Membrane Mucin MUC1 Control Adenocarcinoma.

Genetically modified T cells expressing chimeric antigen receptors (CARs) demonstrate robust responses against lineage restricted, non-essential targets in hematologic cancers. However, in solid tumors, the full potential of CAR T cell therapy is limited by the availability of cell surface antigens with sufficient cancer-specific expression. The majority of CAR targets have been normal self-antigens on dispensable hematopoietic tissues or overexpressed shared antigens. Here, we established that abnormal self-antigens can serve as targets for tumor rejection. We developed a CAR that recognized cancer-associated Tn glycoform of MUC1, a neoantigen expressed in a variety of cancers. Anti-Tn-MUC1 CAR T cells demonstrated target-specific cytotoxicity and successfully controlled tumor growth in xenograft models of T cell leukemia and pancreatic cancer. These findings demonstrate the therapeutic efficacy of CAR T cells directed against Tn-MUC1 and present aberrantly glycosylated antigens as a novel class of targets for tumor therapy with engineered T cells.

Structure-guided engineering of the affinity and specificity of CARs against Tn-glycopeptides.

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.

Breaking tolerance with engineered class I antigen-presenting molecules.

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.

An Engineered T Cell Receptor Variant Realizes the Limits of Functional Binding Modes.

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.

T cell receptor signaling is limited by docking geometry to peptide-major histocompatibility complex.

T cell receptor (TCR) engagement of peptide-major histocompatibility complex (pMHC) is essential to adaptive immunity, but it is unknown whether TCR signaling responses are influenced by the binding topology of the TCR-peptide-MHC complex. We developed yeast-displayed pMHC libraries that enabled us to identify new peptide sequences reactive with a single TCR. Structural analysis showed that four peptides bound to the TCR with distinct 3D and 2D affinities using entirely different binding chemistries. Three of the peptides that shared a common docking mode, where key TCR-MHC germline interactions are preserved, induced TCR signaling. The fourth peptide failed to induce signaling and was recognized in a substantially different TCR-MHC binding mode that apparently exceeded geometric tolerances compatible with signaling. We suggest that the stereotypical TCR-MHC docking paradigm evolved from productive signaling geometries and that TCR signaling can be modulated by peptides that are recognized in alternative TCR-pMHC binding orientations.

T Cell Receptors for Gene Transfer in Adoptive T Cell Therapy.

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.

Comparison of T Cell Activities Mediated by Human TCRs and CARs That Use the Same Recognition Domains.

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.

Subtle changes at the variable domain interface of the T-cell receptor can strongly increase affinity.

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.

T cells use rafts for survival.

T cell homeostasis must be tightly controlled. In this issue of Immunity, Cho et al. (2010) describe results that begin to define the roles of the T cell receptor, self-peptide-MHC ligands, cytokines, and membrane rafts in this dynamic process.

Deep Mutational Scans as a Guide to Engineering High Affinity T Cell Receptor Interactions with Peptide-bound Major Histocompatibility Complex.

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.

TCR scanning of peptide/MHC through complementary matching of receptor and ligand molecular flexibility.

Although conformational changes in TCRs and peptide Ags presented by MHC protein (pMHC) molecules often occur upon binding, their relationship to intrinsic flexibility and role in ligand selectivity are poorly understood. In this study, we used nuclear magnetic resonance to study TCR-pMHC binding, examining recognition of the QL9/H-2L(d) complex by the 2C TCR. Although the majority of the CDR loops of the 2C TCR rigidify upon binding, the CDR3ß loop remains mobile within the TCR-pMHC interface. Remarkably, the region of the QL9 peptide that interfaces with CDR3ß is also mobile in the free pMHC and in the TCR-pMHC complex. Determination of conformational exchange kinetics revealed that the motions of CDR3ß and QL9 are closely matched. The matching of conformational exchange in the free proteins and its persistence in the complex enhances the thermodynamic and kinetic stability of the TCR-pMHC complex and provides a mechanism for facile binding. We thus propose that matching of structural fluctuations is a component of how TCRs scan among potential ligands for those that can bind with sufficient stability to enable T cell signaling.

Enhanced-affinity murine T-cell receptors for tumor/self-antigens can be safe in gene therapy despite surpassing the threshold for thymic selection.

Many of the most promising tumor antigens for T-cell-based cancer immunotherapies are unmodified self-antigens. Unfortunately, the avidity of T cells specific for these antigens is limited by central tolerance during T-cell development in the thymus, resulting in decreased anti-tumor efficacy of these T cells. One approach to overcoming this obstacle is to mutate T-cell receptor (TCR) genes from naturally occurring T cells to enhance the affinity for the target antigen. These enhanced-affinity TCRs can then be developed for use in TCR gene therapy. Although TCRs with significantly enhanced affinity have been generated using this approach, it is not clear whether these TCRs, which bypass the affinity limits imposed by negative selection, remain unresponsive to the low levels of self-antigen generally expressed by some normal tissues. Here we show that 2 variants of a high-affinity WT1-specific TCR with enhanced affinity for WT1 are safe and do not mediate autoimmune tissue infiltration or damage when transduced into peripheral CD8 T cells and transferred in vivo. However, if expressed in developing T cells and subjected to thymic selection, the same enhanced-affinity TCRs signal tolerance mechanisms in the thymus, resulting in T cells with attenuated antigen sensitivity in the periphery.

Endogenous viral antigen processing generates peptide-specific MHC class I cell-surface clusters.

Sensitivity is essential in CD8+ T-cell killing of virus-infected cells and tumor cells. Although the affinity of the T-cell receptor (TCR) for antigen is relatively low, the avidity of T cell-antigen-presenting cell interactions is greatly enhanced by increasing the valence of the interaction. It is known that TCRs cluster into protein islands after engaging their cognate antigen (peptides bound to MHC molecules). Here, we show that mouse K(b) class I molecules segregate into preformed, long-lasting (hours) clusters on the antigen-presenting cell surface based on their bound viral peptide. Peptide-specific K(b) clustering occurs when source antigens are expressed by vaccinia or vesicular stomatitis virus, either as proteasome-liberated precursors or free intracellular peptides. By contrast, K(b)-peptide complexes generated by incubating cells with synthetic peptides are extensively intermingled on the cell surface. Peptide-specific complex sorting is first detected in the Golgi complex, and compromised by removing the K(b) cytoplasmic tail. Peptide-specific clustering is associated with increased T-cell sensitivity: on a per-complex basis, endogenous SIINFEKL activates T cells more efficiently than synthetic SIINFEKL, and wild-type K(b) presents endogenous SIINFEKL more efficiently than tailless K(b). We propose that endogenous processing generates peptide-specific clusters of class I molecules to maximize the sensitivity and speed of T-cell immunosurveillance.

The same major histocompatibility complex polymorphism involved in control of HIV influences peptide binding in the mouse H-2Ld system.

Single-site polymorphisms in human class I major histocompatibility complex (MHC) products (HLA-B) have recently been shown to correlate with HIV disease progression or control. An identical single-site polymorphism (at residue 97) in the mouse class I product H-2L(d) influences stability of the complex. To gain insight into the human polymorphisms, here we examined peptide binding, stability, and structures of the corresponding L(d) polymorphisms, Trp(97) and Arg(97). Expression of L(d)W97 and L(d)R97 genes in a cell line that is antigen-processing competent showed that L(d)R97 was expressed at higher levels than L(d)W97, consistent with enhanced stability of self-peptide·L(d)R97 complexes. To further examine peptide-binding capacities of these two allelic variants, we used a high affinity pep-L(d) specific probe to quantitatively examine a collection of self- and foreign peptides that bind to L(d). L(d)R97 bound more effectively than L(d)W97 to most peptides, although L(d)W97 bound more effectively to two peptides. The results support the view that many self-peptides in the L(d) system (or the HLA-B system) would exhibit enhanced binding to Arg(97) alleles compared with Trp(97) alleles. Accordingly, the self-peptide·MHC-Arg(97) complexes would influence T-cell selection behavior, impacting the T-cell repertoire of these individuals, and could also impact peripheral T cell activity through effects of self-peptide·L(d) interacting with TCR and/or CD8. The structures of several peptide·L(d)R97 and peptide·L(d)W97 complexes provided a framework of how this single polymorphism could impact peptide binding.

A novel T cell receptor single-chain signaling complex mediates antigen-specific T cell activity and tumor control.

Adoptive transfer of genetically modified T cells to treat cancer has shown promise in several clinical trials. Two main strategies have been applied to redirect T cells against cancer: (1) introduction of a full-length T cell receptor (TCR) specific for a tumor-associated peptide-MHC, or (2) introduction of a chimeric antigen receptor, including an antibody fragment specific for a tumor cell surface antigen, linked intracellularly to T cell signaling domains. Each strategy has advantages and disadvantages for clinical applications. Here, we present data on the in vitro and in vivo effectiveness of a single-chain signaling receptor incorporating a TCR variable fragment as the targeting element (referred to as TCR-SCS). This receptor contained a single-chain TCR (Vα-linker-Vß) from a high-affinity TCR called m33, linked to the intracellular signaling domains of CD28 and CD3ζ. This format avoided mispairing with endogenous TCR chains and mediated specific T cell activity when expressed in either CD4 or CD8 T cells. TCR-SCS-transduced CD8-negative cells showed an intriguing sensitivity, compared to full-length TCRs, to higher densities of less stable pepMHC targets. T cells that expressed this peptide-specific receptor persisted in vivo, and exhibited polyfunctional responses. Growth of metastatic antigen-positive tumors was significantly inhibited by T cells that expressed this receptor, and tumor cells that escaped were antigen-loss variants. TCR-SCS receptors represent an alternative targeting receptor strategy that combines the advantages of single-chain expression, avoidance of TCR chain mispairing, and targeting of intracellular antigens presented in complex with MHC proteins.

Neutralization of staphylococcal enterotoxin B by soluble, high-affinity receptor antagonists.

Exotoxins of Staphylococcus aureus belong to a family of bacterial proteins that act as superantigens by activating a large subset of the T-cell population, causing massive release of inflammatory cytokines. This cascade can ultimately result in toxic shock syndrome and death. Therapeutics targeting the early stage of the pathogenic process, when the superantigen binds to its receptor, could limit the severity of disease. We engineered picomolar binding affinity agents to neutralize the potent toxin staphylococcal enterotoxin B (SEB). A single immunoglobulin-like domain of the T-cell receptor (variable region, Vbeta) was subjected to multiple rounds of directed evolution using yeast display. Soluble forms of the engineered Vbeta proteins produced in Escherichia coli were effective inhibitors of SEB-mediated T-cell activation and completely neutralized the lethal activity of SEB in animal models. These Vbeta proteins represent an easily produced potential treatment for diseases mediated by bacterial superantigens.

Induced sensitization of tumor stroma leads to eradication of established cancer by T cells.

Targeting cancer cells, as well as the nonmalignant stromal cells cross-presenting the tumor antigen (Ag), can lead to the complete destruction of well-established solid tumors by adoptively transferred Ag-specific cytotoxic T lymphocytes (CTLs). If, however, cancer cells express only low levels of the Ag, then stromal cells are not destroyed, and the tumor escapes as Ag loss variants. We show that treating well-established tumors expressing low levels of Ag with local irradiation or a chemotherapeutic drug causes sufficient release of Ag to sensitize stromal cells for destruction by CTLs. This was shown directly using high affinity T cell receptor tetramers for visualizing the transient appearance of tumor-specific peptide-MHC complexes on stromal cells. Maximum loading of tumor stroma with cancer Ag occurred 2 d after treatment and coincided with the optimal time for T cell transfer. Under these conditions, tumor rejection was complete. These findings may set the stage for developing rational clinical protocols for combining irradiation or chemotherapy with CTL therapy.

MHC-class I-restricted CD4 T cells: a nanomolar affinity TCR has improved anti-tumor efficacy in vivo compared to the micromolar wild-type TCR.

Clinical studies with immunotherapies for cancer, including adoptive cell transfers of T cells, have shown promising results. It is now widely believed that recruitment of CD4(+) helper T cells to the tumor would be favorable, as CD4(+) cells play a pivotal role in cytokine secretion as well as promoting the survival, proliferation, and effector functions of tumor-specific CD8(+) cytotoxic T lymphocytes. Genetically engineered high-affinity T-cell receptors (TCRs) can be introduced into CD4(+) helper T cells to redirect them to recognize MHC-class I-restricted antigens, but it is not clear what affinity of the TCR will be optimal in this approach. Here, we show that CD4(+) T cells expressing a high-affinity TCR (nanomolar K (d) value) against a class I tumor antigen mediated more effective tumor treatment than the wild-type affinity TCR (micromolar K (d) value). High-affinity TCRs in CD4(+) cells resulted in enhanced survival and long-term persistence of effector memory T cells in a melanoma tumor model. The results suggest that TCRs with nanomolar affinity could be advantageous for tumor targeting when expressed in CD4(+) T cells.

Novel platform for the detection of Staphylococcus aureus enterotoxin B in foods.

Staphylococcal contamination of food products and staphylococcal food-borne illnesses continue to be a problem worldwide. Screening of food for the presence of Staphylococcus aureus and/or enterotoxins using traditional methods is laborious. Reliable and rapid multiplex detection methods from a single food extract or culture supernatant would simplify testing. A fluorescence-based cytometric bead array was developed for the detection of staphylococcal enterotoxin B (SEB), using magnetic microspheres coupled with either an engineered, enterotoxin-specific Vß domain of the T-cell receptor (Vß-TCR) or polyclonal antibodies. The binding affinity of the Vß-TCR for SEB has been shown to be in the picomolar range, comparable to the best monoclonal antibodies. The coupled beads were validated with purified enterotoxins and tested in a variety of food matrices spiked with enterotoxins. The Vß-TCR or antibody was shown to specifically bind SEB in four different food matrices, including milk, mashed potatoes, vanilla pudding, and cooked chicken. The use of traditional polyclonal antibodies and Vß-TCR provides a redundant system that ensures accurate identification of the enterotoxin, and the use of labeled microspheres permits simultaneous testing of multiple enterotoxins from a single sample.