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.

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.

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.

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.

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.

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.

Opposite effects of endogenous peptide-MHC class I on T cell activity in the presence and absence of CD8.

Nonstimulatory or endogenous peptide-MHC (pepMHC) presented on the surfaces of APCs, either alone or alongside agonist pepMHC, plays various roles in T cell selection and activation. To examine these properties in more detail, we explored several model systems of TCR and pepMHC ligands with sufficient affinity to be activated in the absence of CD8. The TCRs had a range of affinities for agonist and nonstimulatory ligands and were restricted by MHC class I alleles with different properties. We observed CD8-independent antagonism from TCR-pepMHC interactions with very low affinities (e.g., K(D) = 300 µM). In addition, endogenous peptide-L(d) complexes on APCs antagonized activation of coreceptor (CD8)-negative 2C T cells even by the strong agonist QL9-L(d). In contrast, TCRs m33 and 3D-PYY, restricted by K(b) and D(b), respectively, did not show signs of antagonism by endogenous pepMHC in the absence of CD8. This did not appear to be an inherent difference in the ability of the TCRs to be antagonized, as altered peptide ligands could antagonize each TCR. In the presence of CD8, endogenous pepMHC ligands acted in some cases as coagonists. These results show that endogenous pepMHC molecules exhibit complex behavior in T cells, leading to either reduced activity (e.g., in cases of low coreceptor levels) or enhanced activity (e.g., in presence of coreceptor). The behavior may be influenced by the ability of different TCRs to recognize endogenous pepMHC but also perhaps by the inherent properties of the presenting MHC allele.

Interaction of streptavidin-based peptide-MHC oligomers (tetramers) with cell-surface TCRs.

The binding of oligomeric peptide-MHC (pMHC) complexes to cell surface TCR can be considered to approximate TCR-pMHC interactions at cell-cell interfaces. In this study, we analyzed the equilibrium binding of streptavidin-based pMHC oligomers (tetramers) and their dissociation kinetics from CD8(pos) T cells from 2C-TCR transgenic mice and from T cell hybridomas that expressed the 2C TCR or a high-affinity mutant (m33) of this TCR. Our results show that the tetramers did not come close to saturating cell-surface TCR (binding only 10-30% of cell-surface receptors), as is generally assumed in deriving affinity values (K(D)), in part because of dissociative losses from tetramer-stained cells. Guided by a kinetic model, the oligomer dissociation rate and equilibrium constants were seen to depend not only on monovalent association and dissociation rates (k(off) and k(on)), but also on a multivalent association rate (µ) and TCR cell-surface density. Our results suggest that dissociation rates could account for the recently described surprisingly high frequency of tetramer-negative, functionally competent T cells in some T cell responses.

ABBV-184: A Novel Survivin-specific TCR/CD3 Bispecific T-cell Engager is Active against Both Solid Tumor and Hematologic Malignancies.

CD3 bispecific T-cell engagers (TCE), comprised of a tumor-targeting domain linked to a CD3 binding domain, function by bridging target-positive tumors and CD3-expressing effector T cells enabling redirected T cell-mediated killing of tumor cells. Although the majority of CD3 bispecific molecules in clinical development incorporate tumor-targeting antibody-based binding domains, many tumor-associated antigens derive from intracellular proteins and are not accessible to targeting via antibody. Intracellular proteins processed into short peptide fragments and presented on the cell surface by MHC proteins are recognized by T-cell receptors (TCR) on the surface of T cells. Here we describe the generation and preclinical evaluation of ABBV-184, a novel TCR/anti-CD3 bispecific composed of a highly selective soluble TCR that binds a peptide derived from the oncogene survivin (BIRC5) bound to the class I MHC allele human leukocyte antigen (HLA)-A*02:01 expressed on tumor cells, linked to a specific binder to the CD3 receptor on T cells. ABBV-184 drives an optimal distance between T cell and target cell thereby enabling sensitive recognition of low-density peptide/MHC targets. Consistent with the expression profile of survivin across a broad range of both hematologic and solid tumors, treatment of acute myeloid leukemia (AML) and non-small cell lung cancer (NSCLC) cell lines with ABBV-184 results in T-cell activation, proliferation, and potent redirected cytotoxicity of HLA-A2-positive target cell lines, both in vitro and in vivo, including patient-derived AML samples. These results indicate that ABBV-184 is an attractive clinical candidate for the treatment of patients with AML and NSCLC.

Cutting edge: inhibitory effects of CD4 and CD8 on T cell activation induced by high-affinity noncognate ligands.

It has been proposed that MHC restriction during thymocyte selection is controlled by coreceptor (CD4 or CD8) sequestration of the signaling molecule Lck. We explored this model as a mechanism for preventing peripheral T cell activation due to non-MHC ligand cross-reactivities of TCRs. TCRs that have a range of affinities for a class I MHC ligand were transduced into a T cell hybridoma in the absence or presence of coreceptors. High and intermediate affinity TCRs (K(D) = 17 and 540 nM) did not require CD8 for T cell activity, but CD4 acted as a potent inhibitor of the intermediate affinity TCR. These and other findings support the view that even high-affinity TCR:ligand interactions can be influenced by coreceptor sequestration of Lck. Thus, CD4 and CD8 act as "coreceptor inhibitors" to maintain appropriate TCR-mediated MHC restriction in peripheral T cell activity.

The impact of TCR-binding properties and antigen presentation format on T cell responsiveness.

TCR interactions with cognate peptide-MHC (pepMHC) ligands are generally low affinity. This feature, together with the requirement for CD8/CD4 participation, has made it difficult to dissect relationships between TCR-binding parameters and T cell activation. Interpretations are further complicated when comparing different pepMHC, because these can vary greatly in stability. To examine the relationships between TCR-binding properties and T cell responses, in this study we characterized the interactions and activities mediated by a panel of TCRs that differed widely in their binding to the same pepMHC. Monovalent binding of soluble TCR was characterized by surface plasmon resonance, and T cell hybridomas that expressed these TCR, with or without CD8 coexpression, were tested for their binding of monomeric and oligomeric forms of the pepMHC and for subsequent responses (IL-2 release). The binding threshold for eliciting this response in the absence of CD8 (K(D) = 600 nM) exhibited a relatively sharp cutoff between full activity and no activity, consistent with a switchlike response to pepMHC on APCs. However, when the pepMHC was immobilized (plate bound), T cells with the lowest affinity TCRs (e.g., K(D) = 30 microM) responded, even in the absence of CD8, indicating that these TCR are signaling competent. Surprisingly, even cells that expressed high-affinity (K(D) = 16 nM) TCRs along with CD8 were unresponsive to oligomers in solution. The findings suggest that to drive downstream T cell responses, pepMHC must be presented in a form that supports formation of appropriate supramolecular clusters.

Distinct CDR3 conformations in TCRs determine the level of cross-reactivity for diverse antigens, but not the docking orientation.

T cells are known to cross-react with diverse peptide MHC Ags through their alphabeta TCR. To explore the basis of such cross-reactivity, we examined the 2C TCR that recognizes two structurally distinct ligands, SIY-K(b) and alloantigen QL9-L(d). In this study we characterized the cross-reactivity of several high-affinity 2C TCR variants that contained mutations only in the CDR3alpha loop. Two of the TCR lost their ability to cross-react with the reciprocal ligand (SIY-K(b)), whereas another TCR (m67) maintained reactivity with both ligands. Crystal structures of four of the TCRs in complex with QL9-L(d) showed that CDR1, CDR2, and CDR3beta conformations and docking orientations were remarkably similar. Although the CDR3alpha loop of TCR m67 conferred a 2000-fold higher affinity for SIY-K(b), the TCR maintained the same docking angle on QL9-L(d) as the 2C TCR. Thus, CDR3alpha dictated the affinity and level of cross-reactivity, yet it did so without affecting the conserved docking orientation.

T-cell receptor binding affinities and kinetics: impact on T-cell activity and specificity.

The interaction between the T-cell receptor (TCR) and its peptide-major histocompatibility complex (pepMHC) ligand plays a critical role in determining the activity and specificity of the T cell. The binding properties associated with these interactions have now been studied in many systems, providing a framework for a mechanistic understanding of the initial events that govern T-cell function. There have been various other reviews that have described the structural and biochemical features of TCR : pepMHC interactions. Here we provide an overview of four areas that directly impact our understanding of T-cell function, as viewed from the perspective of the TCR : pepMHC interaction: (1) relationships between T-cell activity and TCR : pepMHC binding parameters, (2) TCR affinity, avidity and clustering, (3) influence of coreceptors on pepMHC binding by TCRs and T-cell activity, and (4) impact of TCR binding affinity on antigenic peptide specificity.

Biosensor detection systems: engineering stable, high-affinity bioreceptors by yeast surface display.

Over the past two decades, the field of biosensors has been developing fast, portable, and convenient detection tools for various molecules of interest, both biological and environmental. Although much attention is paid to the transduction portion of the sensor, the actual bioreceptor that binds the ligand is equally critical. Tight, specific binding by the bioreceptor is required to detect low levels of the relevant ligand, and the bioreceptor must be stable enough to survive immobilization, storage, and in ideal cases, regeneration on the biosensing device. Often, naturally-occurring bioreceptors or antibodies that are specific for a ligand either express affinities that may be too low to detect useful levels, or the proteins are too unstable to be used and reused as a biosensor. Further engineering of these receptors can improve their utility. Here, we describe in detail the use of yeast surface display techniques to carry out directed evolution of bioreceptors to increase both the stability of the molecules and their affinity for the ligands. This powerful technique has enabled the production of stabilized, single-chain antibodies, T cell receptors, and other binding molecules that exhibit affinity increases for their ligands of up to 1 million-fold and expression of stable molecules in E. coli.

T-cell Receptors Engineered De Novo for Peptide Specificity Can Mediate Optimal T-cell Activity without Self Cross-Reactivity.

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.

Different thermodynamic binding mechanisms and peptide fine specificities associated with a panel of structurally similar high-affinity T cell receptors.

To understand the mechanisms that govern T cell receptor (TCR)-peptide MHC (pMHC) binding and the role that different regions of the TCR play in affinity and antigen specificity, we have studied the TCR from T cell clone 2C. High-affinity mutants of the 2C TCR that bind QL9-L(d) as a strong agonist were generated previously by site-directed mutagenesis of complementarity determining regions (CDRs) 1beta, 2alpha, 3alpha, or 3beta. We performed isothermal titration calorimetry to assess whether they use similar thermodynamic mechanisms to achieve high affinity for QL9-L(d). Four of the five TCRs examined bound to QL9-L(d) in an enthalpically driven, entropically unfavorable manner. In contrast, the high-affinity CDR1beta mutant resembled the wild-type 2C TCR interaction, with favorable entropy. To assess fine specificity, we measured the binding and kinetics of these mutants for both QL9-L(d) and a single amino acid peptide variant of QL9, called QL9-Y5-L(d). While 2C and most of the mutants had equal or higher affinity for the Y5 variant than for QL9, mutant CDR1beta exhibited 8-fold lower affinity for Y5 compared to QL9. To examine possible structural correlates of the thermodynamic and fine specificity signatures of the TCRs, the structure of unliganded QL9-L(d) was solved and compared to structures of the 2C TCR/QL9-L(d) complex and three high-affinity TCR/QL9-L(d) complexes. Our findings show that the QL9-L(d) complex does not undergo major conformational changes upon binding. Thus, subtle changes in individual CDRs account for the diverse thermodynamic and kinetic binding mechanisms and for the different peptide fine specificities.

Antagonism of HIV-specific CD4+ T cells by C-terminal truncation of a minimum epitope.

Antagonism of T cell responses by variants of the cognate peptide is a potential mechanism of viral escape from immune responses and may play a role in the ability of HIV to evade immune control. We show here a rarely described mechanism of antagonism by a peptide shorter than the minimum length epitope for an HIV p24-specific CD4+ T cell clone. The shorter antagonist peptide-MHC complex bound the T cell receptor (TCR), albeit with lower affinity than the full-length agonist peptide. Prior work showing the crystal structure of the peptide-MHC complex revealed a unique glycine hinge near the C-terminus of the agonist peptide, allowing the generation of full-length antagonist peptide lacking the hinge. These results confirm the dependence of productive TCR engagement on residues spilling out from the C-terminus of the MHC binding groove and show that partial engagement of the TCR with a truncated, low-affinity ligand can result in T cell antagonism.

TCR affinity for p/MHC formed by tumor antigens that are self-proteins: impact on efficacy and toxicity.

Recent studies have shown that the range of affinities of T cell receptors (TCRs) against non-mutated cancer peptide/class I complexes are lower than TCR affinities for foreign antigens. Raising the affinity of TCRs for optimal activity of CD8 T cells, and for recruitment of CD4 T cell activity against a class I antigen, provides opportunities for more robust adoptive T cell therapies. However, TCRs with enhanced affinities also risk increased reactivity with structurally related self-peptides, and off-target toxicities. Careful selection of tumor peptide antigens, in silico proteome screens, and in vitro peptide specificity assays will be important in the development of the most effective, safe TCR-based adoptive therapies.

A Multiplex Assay for Detection of Staphylococcal and Streptococcal Exotoxins.

Staphylococcal and streptococcal exotoxins, also known as superantigens, mediate a range of diseases including toxic shock syndrome, and they exacerbate skin, pulmonary and systemic infections caused by these organisms. When present in food sources they can cause enteric effects commonly known as food poisoning. A rapid, sensitive assay for the toxins would enable testing of clinical samples and improve surveillance of food sources. Here we developed a bead-based, two-color flow cytometry assay using single protein domains of the beta chain of T cell receptors engineered for high-affinity for staphylococcal (SEA, SEB and TSST-1) and streptococcal (SpeA and SpeC) toxins. Site-directed biotinylated forms of these high-affinity agents were used together with commercial, polyclonal, anti-toxin reagents to enable specific and sensitive detection with SD50 values of 400 pg/ml (SEA), 3 pg/ml (SEB), 25 pg/ml (TSST-1), 6 ng/ml (SpeA), and 100 pg/ml (SpeC). These sensitivities were in the range of 4- to 80-fold higher than achieved with standard ELISAs using the same reagents. A multiplex format of the assay showed reduced sensitivity due to higher noise associated with the use of multiple polyclonal agents, but the sensitivities were still well within the range necessary for detection in food sources or for rapid detection of toxins in culture supernatants. For example, the assay specifically detected toxins in supernatants derived from cultures of Staphylococcus aureus. Thus, these reagents can be used for simultaneous detection of the toxins in food sources or culture supernatants of potential pathogenic strains of Staphylococcus aureus and Streptococcus pyogenes.

Role of T cell receptor affinity in the efficacy and specificity of adoptive T cell therapies.

Over the last several years, there has been considerable progress in the treatment of cancer using gene modified adoptive T cell therapies. Two approaches have been used, one involving the introduction of a conventional αß T cell receptor (TCR) against a pepMHC cancer antigen, and the second involving introduction of a chimeric antigen receptor (CAR) consisting of a single-chain antibody as an Fv fragment linked to transmembrane and signaling domains. In this review, we focus on one aspect of TCR-mediated adoptive T cell therapies, the impact of the affinity of the αß TCR for the pepMHC cancer antigen on both efficacy and specificity. We discuss the advantages of higher-affinity TCRs in mediating potent activity of CD4 T cells. This is balanced with the potential disadvantage of higher-affinity TCRs in mediating greater self-reactivity against a wider range of structurally similar antigenic peptides, especially in synergy with the CD8 co-receptor. Both TCR affinity and target selection will influence potential safety issues. We suggest pre-clinical strategies that might be used to examine each TCR for possible on-target and off-target side effects due to self-reactivities, and to adjust TCR affinities accordingly.