An HLA-E-targeted TCR bispecific molecule redirects T cell immunity against Mycobacterium tuberculosis.

Peptides presented by HLA-E, a molecule with very limited polymorphism, represent attractive targets for T cell receptor (TCR)-based immunotherapies to circumvent the limitations imposed by the high polymorphism of classical HLA genes in the human population. Here, we describe a TCR-based bispecific molecule that potently and selectively binds HLA-E in complex with a peptide encoded by the inhA gene of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis in humans. We reveal the biophysical and structural bases underpinning the potency and specificity of this molecule and demonstrate its ability to redirect polyclonal T cells to target HLA-E-expressing cells transduced with mycobacterial inhA as well as primary cells infected with virulent Mtb. Additionally, we demonstrate elimination of Mtb-infected cells and reduction of intracellular Mtb growth. Our study suggests an approach to enhance host T cell immunity against Mtb and provides proof of principle for an innovative TCR-based therapeutic strategy overcoming HLA polymorphism and therefore applicable to a broader patient population.

An approved in vitro approach to preclinical safety and efficacy evaluation of engineered T cell receptor anti-CD3 bispecific (ImmTAC) molecules.

Robust preclinical testing is essential to predict clinical safety and efficacy and provide data to determine safe dose for first-in-man studies. There are a growing number of examples where the preclinical development of drugs failed to adequately predict clinical adverse events in part due to their assessment with inappropriate preclinical models. Preclinical investigations of T cell receptor (TCR)-based immunotherapies prove particularly challenging as these biologics are human-specific and thus the conventional testing in animal models is inadequate. As these molecules harness the full force of the immune system, and demonstrate tremendous potency, we set out to design a preclinical package that would ensure adequate evaluation of these therapeutics. Immune Mobilising Monoclonal TCR Against Cancer (ImmTAC) molecules are bi-specific biologics formed of an affinity-enhanced TCR fused to an anti-CD3 effector function. ImmTAC molecules are designed to activate human T lymphocytes and target peptides within the context of a human leukocyte antigen (HLA), thus require an intact human immune system and peptidome for suitable preclinical screening. Here we draw upon the preclinical testing of four ImmTAC molecules, including IMCgp100, the first ImmTAC molecule to reach the clinic, to present our comprehensive, informative and robust approach to in vitro preclinical efficacy and safety screening. This package comprises a broad range of cellular and molecular assays using human tissues and cultured cells to test efficacy, safety and specificity, and hence predict human responses in clinical trials. We propose that this entirely in vitro package offers a potential model to be applied to screening other TCR-based biologics.

Identification of a Titin-derived HLA-A1-presented peptide as a cross-reactive target for engineered MAGE A3-directed T cells.

MAGE A3, which belongs to the family of cancer-testis antigens, is an attractive target for adoptive therapy given its reactivation in various tumors and limited expression in normal tissues. We developed an affinity-enhanced T cell receptor (TCR) directed to a human leukocyte antigen (HLA)-A*01-restricted MAGE A3 antigen (EVDPIGHLY) for use in adoptive therapy. Extensive preclinical investigations revealed no off-target antigen recognition concerns; nonetheless, administration to patients of T cells expressing the affinity-enhanced MAGE A3 TCR resulted in a serious adverse event (SAE) and fatal toxicity against cardiac tissue. We present a description of the preclinical in vitro functional analysis of the MAGE A3 TCR, which failed to reveal any evidence of off-target activity, and a full analysis of the post-SAE in vitro investigations, which reveal cross-recognition of an off-target peptide. Using an amino acid scanning approach, a peptide from the muscle protein Titin (ESDPIVAQY) was identified as an alternative target for the MAGE A3 TCR and the most likely cause of in vivo toxicity. These results demonstrate that affinity-enhanced TCRs have considerable effector functions in vivo and highlight the potential safety concerns for TCR-engineered T cells. Strategies such as peptide scanning and the use of more complex cell cultures are recommended in preclinical studies to mitigate the risk of off-target toxicity in future clinical investigations.

Bi-specific TCR-anti CD3 redirected T-cell targeting of NY-ESO-1- and LAGE-1-positive tumors.

NY-ESO-1 and LAGE-1 are cancer testis antigens with an ideal profile for tumor immunotherapy, combining up-regulation in many cancer types with highly restricted expression in normal tissues and sharing a common HLA-A*0201 epitope, 157-165. Here, we present data to describe the specificity and anti-tumor activity of a bifunctional ImmTAC, comprising a soluble, high-affinity T-cell receptor (TCR) specific for NY-ESO-1157-165 fused to an anti-CD3 scFv. This reagent, ImmTAC-NYE, is shown to kill HLA-A2, antigen-positive tumor cell lines, and freshly isolated HLA-A2- and LAGE-1-positive NSCLC cells. Employing time-domain optical imaging, we demonstrate in vivo targeting of fluorescently labelled high-affinity NYESO-specific TCRs to HLA-A2-, NY-ESO-1157-165-positive tumors in xenografted mice. In vivo ImmTAC-NYE efficacy was tested in a tumor model in which human lymphocytes were stably co-engrafted into NSG mice harboring tumor xenografts; efficacy was observed in both tumor prevention and established tumor models using a GFP fluorescence readout. Quantitative RT-PCR was used to analyze the expression of both NY-ESO-1 and LAGE-1 antigens in 15 normal tissues, 5 cancer cell lines, 10 NSCLC, and 10 ovarian cancer samples. Overall, LAGE-1 RNA was expressed at a greater frequency and at higher levels than NY-ESO-1 in the tumor samples. These data support the clinical utility of ImmTAC-NYE as an immunotherapeutic agent for a variety of cancers.

Glycoproteomic characterization of carriers of the CD15/Lewisx epitope on Hodgkin's Reed-Sternberg cells.

BACKGROUND: The Lewisx trisaccharide, also referred to as the CD15 antigen, is a diagnostic marker used to distinguish Hodgkin's lymphoma from other lymphocytic cancers. However, the role of such fucosylated structures remains poorly understood, in part because carriers of Lewisx structures on Hodgkin's Reed-Sternberg cells have not been identified. METHODS: GalMBP, an engineered carbohydrate-recognition protein that binds selectively to oligosaccharides with paired terminal galactose and fucose residues, has been used in conjunction with proteomic and glycomic analysis to identify glycoprotein carriers of Lewisx and related glycan structures in multiple Hodgkin's Reed-Sternberg cell lines. RESULTS: Multiple glycoproteins that bind to GalMBP and carry CD15/Lewisx have been identified in a panel of six Reed-Sternberg cell lines. The most commonly identified Lewisx-bearing glycoproteins are CD98hc, which was found in all six cell lines tested, and intercellular adhesion molecule-1 and DEC-205, which were detected in five and four of the lines, respectively. Thus, several of the most prominent cell adhesion molecules on the lymphomas carry this characteristic glycan epitope. In addition, the Hodgkin's Reed-Sternberg cell lines can be grouped into subsets based on the presence or absence of less common Lewisx-bearing glycoproteins. CONCLUSIONS: CD98 and intercellular adhesion molecule-1 are major carriers of CD15/Lewisx on Reed-Sternberg cells. Binding of DC-SIGN and other glycan-specific receptors to the Lewisx epitopes on CD98 and intercellular adhesion molecule-1 may facilitate interaction of the lymphoma cells with lymphocytes and myeloid cells in lymph nodes.

Mouse LSECtin as a model for a human Ebola virus receptor.

The biochemical properties of mouse LSECtin, a glycan-binding receptor that is a member of the C-type lectin family found on sinusoidal endothelial cells, have been investigated. The C-type carbohydrate-recognition domain of mouse LSECtin, expressed in bacteria, has been used in solid-phase binding assays, and a tetramerized form has been used to probe a glycan array. In spite of sequence differences near the glycan-binding sites, the mouse receptor closely mimics the properties of the human receptor, showing high affinity binding to glycans bearing terminal GlcNAcß1-2Man motifs. Site-directed mutagenesis has been used to confirm that residues near the binding site that differ between the human and the mouse proteins do not affect this binding specificity. Mouse and human LSECtin have been shown to bind Ebola virus glycoprotein with equivalent affinities, and the GlcNAcß1-2Man disaccharide has been demonstrated to be an effective inhibitor of this interaction. These studies provide a basis for using mouse LSECtin, and knockout mice lacking this receptor, to model the biological properties of the human receptor.

Engineered carbohydrate-recognition domains for glycoproteomic analysis of cell surface glycosylation and ligands for glycan-binding receptors.

Modular calcium-dependent carbohydrate-recognition domains (CRDs) of mammalian glycan-binding receptors (C-type lectins), engineered to have novel glycan-binding selectivity, have been developed as tools for the study of glycans on cell surfaces. Structure-based specificity swapping between domains can be complemented by empirical characterization of ligand-binding specificity using glycan arrays. Both natural and modified CRDs can be used as probes for detecting and isolating glycoproteins that bear specific glycan epitopes and that act as target ligands for glycan-binding receptors. CRD-based affinity chromatography facilitates proteomic and glycomic analysis of such ligands.

Trimeric structure of langerin.

Langerin, an endocytic receptor of Langerhans cells, binds pathogens such as human immunodeficiency virus by recognition of surface glycoconjugates and mediates their internalization into Birbeck granules. Langerin has an extracellular region consisting of a C-type carbohydrate-recognition domain (CRD) and a neck region that stabilizes formation of trimers. As in many other C-type lectins, oligomerization is required for high affinity binding to glycan ligands and is also likely to be important for determining specificity. To facilitate structural analysis of the human langerin trimer, a truncated form of the extracellular region, consisting of part of the neck and the CRD, has been characterized. Like the full-length protein, truncated langerin exists as a stable trimer in solution. Glycan array screening with the trimeric fragment shows that high mannose oligosaccharides are the best ligands for langerin. Structural analysis of the trimeric fragment of langerin confirms that the neck region forms a coiled-coil of alpha-helices. Multiple interactions between the neck region and the CRDs make the trimer a rigid unit with the three CRDs in fixed positions and the primary sugar-binding sites separated by a distance of 42 A. The fixed orientation of the sugar-binding sites in the trimer is likely to place constraints on the ligands that can be bound by langerin.

Targeted glycoproteomic identification of cancer cell glycosylation.

GalMBP is a fragment of serum mannose-binding protein that has been modified to create a probe for galactose-containing ligands. Glycan array screening demonstrated that the carbohydrate-recognition domain of GalMBP selectively binds common groups of tumor-associated glycans, including Lewis-type structures and T antigen, suggesting that engineered glycan-binding proteins such as GalMBP represent novel tools for the characterization of glycoproteins bearing tumor-associated glycans. Blotting of cell extracts and membranes from MCF7 breast cancer cells with radiolabeled GalMBP was used to demonstrate that it binds to a selected set of high molecular weight glycoproteins that could be purified from MCF7 cells on an affinity column constructed with GalMBP. Proteomic and glycomic analysis of these glycoproteins by mass spectrometry showed that they are forms of CD98hc that bear glycans displaying heavily fucosylated termini, including Lewis(x) and Lewis(y) structures. The pool of ligands was found to include the target ligands for anti-CD15 antibodies, which are commonly used to detect Lewis(x) antigen on tumors, and for the endothelial scavenger receptor C-type lectin, which may be involved in tumor metastasis through interactions with this antigen. A survey of additional breast cancer cell lines reveals that there is wide variation in the types of glycosylation that lead to binding of GalMBP. Higher levels of binding are associated either with the presence of outer-arm fucosylated structures carried on a variety of different cell surface glycoproteins or with the presence of high levels of the mucin MUC1 bearing T antigen.

Prolectin, a glycan-binding receptor on dividing B cells in germinal centers.

Prolectin, a previously undescribed glycan-binding receptor, has been identified by re-screening of the human genome for genes encoding proteins containing potential C-type carbohydrate-recognition domains. Glycan array analysis revealed that the carbohydrate-recognition domain in the extracellular domain of the receptor binds glycans with terminal alpha-linked mannose or fucose residues. Prolectin expressed in fibroblasts is found at the cell surface, but unlike many glycan-binding receptors it does not mediate endocytosis of a neoglycoprotein ligand. However, compared with other known glycan-binding receptors, the receptor contains an unusually large intracellular domain that consists of multiple sequence motifs, including phosphorylated tyrosine residues, that allow it to interact with signaling molecules such as Grb2. Immunohistochemistry has been used to demonstrate that prolectin is expressed on a specialized population of proliferating B cells in germinal centers. Thus, this novel receptor has the potential to function in carbohydrate-mediated communication between cells in the germinal center.

Autonomous tetramerization domains in the glycan-binding receptors DC-SIGN and DC-SIGNR.

Multivalent binding of glycans on pathogens and on mammalian cells by the receptors DC-SIGN (CD209) and DC-SIGNR (L-SIGN, CD299) is dependent on correct disposition of the C-type carbohydrate-recognition domains projected at the C-terminal ends of necks at the cell surface. In the work reported here, neck domains of DC-SIGN and DC-SIGNR expressed in isolation are shown to form tetramers in the absence of the CRDs. Stability analysis indicates that interactions between the neck domains account fully for the stability of the tetrameric extracellular portions of the receptors. The neck domains are approximately 40% alpha-helical based on circular dichroism analysis. However, in contrast to other glycan-binding receptors in which fully helical neck regions are intimately associated with C-terminal C-type CRDs, the neck domains in DC-SIGN and DC-SIGNR act as autonomous tetramerization domains and the neck domains and CRDs are organized independently. Neck domains from polymorphic forms of DC-SIGNR that lack some of the repeat sequences show modestly reduced stability, but differences near the C-terminal end of the neck domains lead to significantly enhanced stability of DC-SIGNR tetramers compared to DC-SIGN.

A novel mechanism for LSECtin binding to Ebola virus surface glycoprotein through truncated glycans.

LSECtin is a member of the C-type lectin family of glycan-binding receptors that is expressed on sinusoidal endothelial cells of the liver and lymph nodes. To compare the sugar and pathogen binding properties of LSECtin with those of related but more extensively characterized receptors, such as DC-SIGN, a soluble fragment of LSECtin consisting of the C-terminal carbohydrate-recognition domain has been expressed in bacteria. A biotin-tagged version of the protein was also generated and complexed with streptavidin to create tetramers. These forms of the carbohydrate-recognition domain were used to probe a glycan array and to characterize binding to oligosaccharide and glycoprotein ligands. LSECtin binds with high selectivity to glycoproteins terminating in GlcNAcbeta1-2Man. The inhibition constant for this disaccharide is 3.5 microm, making it one of the best low molecular weight ligands known for any C-type lectin. As a result of the selective binding of this disaccharide unit, the receptor recognizes glycoproteins with a truncated complex and hybrid N-linked glycans on glycoproteins. Glycan analysis of the surface glycoprotein of Ebola virus reveals the presence of such truncated glycans, explaining the ability of LSECtin to facilitate infection by Ebola virus. High mannose glycans are also present on the viral glycoprotein, which explains why DC-SIGN also binds to this virus. Thus, multiple receptors interact with surface glycoproteins of enveloped viruses that bear different types of relatively poorly processed glycans.

Widely divergent biochemical properties of the complete set of mouse DC-SIGN-related proteins.

The mouse genome sequence has been examined to identify the complete set of proteins related to the human glycanbinding receptor, DC-SIGN. In addition to five SIGNR proteins previously described, a pseudogene, encoding a hypothetical SIGNR6, and a further two expressed proteins, SIGNR7 and SIGNR8, have been identified. The ligand-binding properties of these novel proteins and of the previously described mouse SIGNs have been systematically investigated in order to define the mouse proteins that most resemble human DC-SIGN and DC-SIGNR. Results from screening of a glycan array demonstrate that only mouse SIGNR3 shares with human DC-SIGN the ability to bind both high mannose and fucose-terminated glycans in this format and to mediate endocytosis. The finding that neither SIGNR1 nor SIGNR5 binds with high affinity to specific ligands in a large panel of mammalian glycans is consistent with the suggestion that these receptors bind surface polysaccharides on bacterial and fungal pathogens in a manner analogous to serum mannose-binding protein. The data also reveal that two of the mouse SIGNs have unusual binding specificities that have not been previously described for members of the C-type lectin family; the newly identified SIGNR7 binds preferentially to the 6-sulfo-sialyl Lewis(x) oligosaccharide, whereas SIGNR2 binds almost exclusively to glycans that bear terminal GlcNAc residues. The results presented demonstrate that the mouse homologs of DC-SIGN have a diverse set of ligand-binding and intracellular trafficking properties, some of which are distinct from the properties of any of the human receptors.