ABSTRACT
Clinical applications of CAR-T cells are limited by the scarcity of tumor-specific targets and are often afflicted with the same on-target/off-tumor toxicities that plague other cancer treatments. A new promising strategy to enforce tumor selectivity is the use of logic-gated, two-receptor systems. One well-described application is termed Tmod™, which originally utilized a blocking inhibitory receptor directed towards HLA-I target antigens to create a protective NOT gate. Here we show that the function of Tmod blockers targeting non-HLA-I antigens is dependent on the height of the blocker antigen and is generally compatible with small, membrane-proximal targets. We compensate for this apparent limitation by incorporating modular hinge units to artificially extend or retract the ligand-binding domains relative to the effector cell surface, thereby modulating Tmod activator and blocker function. By accounting for structural differences between activator and blocker targets, we developed a set of simple geometric parameters for Tmod receptor design that enables targeting of blocker antigens beyond HLA-I, thereby broadening the applications of logic-gated cell therapies.
Subject(s)
Neoplasms , T-Lymphocytes , Humans , Antigens/metabolismABSTRACT
Innovative cell-based therapies are important new weapons in the fight against difficult-to-treat cancers. One promising strategy involves cell therapies equipped with multiple receptors to integrate signals from more than one antigen. We developed a specific embodiment of this approach called Tmod, a two-receptor system that combines activating and inhibitory inputs to distinguish between tumor and normal cells. The selectivity of Tmod is enforced by the inhibitory receptor (blocker) that recognizes an antigen, such as an HLA allele, whose expression is absent from tumors because of loss of heterozygosity. Although unwanted cross-reactivity of the blocker likely reduces efficacy rather than safety, it is important to verify the blocker's specificity. We have tested an A∗02-directed blocker derived from the PA2.1 mouse antibody as a safety mechanism paired with a mesothelin-specific activating CAR in our Tmod construct. We solved the crystal structure of humanized PA2.1 Fab in complex with HLA-A∗02 to determine its binding epitope, which was used to bioinformatically select specific class I HLA alleles to test the blocker's functional specificity in vitro. We found that this A∗02-directed blocker is highly specific for its cognate antigen, with only one cross-reactive allele (A∗69) capable of triggering comparable function.
ABSTRACT
Immune cells that are engineered with receptors to integrate signals from multiple antigens offer a promising route to achieve the elusive property of therapeutic selectivity in cancer patients. Several types of multi-signal integrators have been described, among them mechanisms that pair activating and inhibitory receptors which are termed NOT gates by analogy to logical operations performed by machines. Here we review one such NOT-gated signal integrator called the Tmod system which is being developed for patients with solid tumors. Coupled with rigorous selection for patients with defined lesions in their tumor genomes (loss of heterozygosity), the Tmod approach presents an unusual opportunity to create truly selective therapies for certain cancer patients. Several of these agents are advancing toward the clinic, supported by a large body of quantitative preclinical data.
Subject(s)
Immunotherapy , Neoplasms , Humans , Immunotherapy, Adoptive , Neoplasms/therapyABSTRACT
BACKGROUND: Mesothelin (MSLN) is a classic tumor-associated antigen that is expressed in lung cancer and many other solid tumors. However, MSLN is also expressed in normal mesothelium which creates a significant risk of serious inflammation for MSLN-directed therapeutics. We have developed a dual-receptor (Tmod™) system that exploits the difference between tumor and normal tissue in a subset of patients with defined heterozygous gene loss (LOH) in their tumors. METHODS: T cells engineered with the MSLN CAR Tmod construct described here contain (1) a novel MSLN-activated CAR and (2) an HLA-A*02-gated inhibitory receptor (blocker). A*02 binding is intended to override T-cell cytotoxicity, even in the presence of MSLN. The Tmod system is designed to treat heterozygous HLA class I patients, selected for HLA LOH. When A*02 is absent from tumors selected for LOH, the MSLN Tmod cells are predicted to mediate potent killing of the MSLN(+)A*02(-) malignant cells. RESULTS: The sensitivity of the MSLN Tmod cells is comparable with a benchmark MSLN CAR-T that was active but toxic in the clinic. Unlike MSLN CAR-T cells, the Tmod system robustly protects surrogate "normal" cells even in mixed-cell populations in vitro and in a xenograft model. The MSLN CAR can also be paired with other HLA class I blockers, supporting extension of the approach to patients beyond A*02 heterozygotes. CONCLUSIONS: The Tmod mechanism exemplified by the MSLN CAR Tmod construct provides an alternative route to leverage solid-tumor antigens such as MSLN in safer, more effective ways than previously possible.
Subject(s)
HLA-A2 Antigen/genetics , Immunotherapy, Adoptive/methods , Mesothelin/immunology , Neoplasms/therapy , Receptors, Chimeric Antigen/immunology , Animals , Cell Line, Tumor , Female , HLA-A2 Antigen/immunology , Humans , Loss of Heterozygosity , Mice , T-Lymphocytes/immunology , Xenograft Model Antitumor AssaysABSTRACT
Neoantigens are among the most intriguing potential immuno-oncology targets because, unlike many cancer targets that are expressed on normal tissues, they are by definition restricted to cancer cells. Medicines directed at common neoantigens such as mutant KRAS are especially interesting because they may offer the convenience and cost of an off-the-shelf therapy. However, all common KRAS mutations produce proteins that differ from the wild type at a single amino acid, creating challenges for molecular discrimination. We have undertaken an effort to optimize single-chain variable fragments (scFv) against peptide/major histocompatibility antigen complexes composed of HLA-A*11 and either G12V- or G12D-mutant KRAS peptides. These scFvs could in principle be used in chimeric antigen receptor (CAR) T-cell therapies for selected patients whose tumors bear either of these mutations. Here we show that optimization of such CARs involves a trade-off between potency and selectivity. We further show that targeting this family without high selectivity engenders risks of cross-reactivity against other members of the G-protein family to which KRAS belongs. Significance: We report an effort to generate high potency, selective CARs directed at mutant KRAS peptides. Although the heavily optimized CARs maintain high selectivity against wild-type KRAS, they lose selectivity against other KRAS-related peptides derived from human proteins. To our knowledge, this work is the first to examine the trade-off between potency and selectivity with regard to KRAS pMHC-directed CARs, illustrating the challenge to achieve both sufficient potency and high selectivity.
Subject(s)
Neoplasms , Receptors, Chimeric Antigen , Single-Chain Antibodies , Humans , Receptors, Chimeric Antigen/genetics , Proto-Oncogene Proteins p21(ras)/genetics , Immunotherapy, Adoptive , Single-Chain Antibodies/geneticsABSTRACT
We designed variant human TCRs composed of the full-length TCRα/ß or extracellular and transmembrane domains of the associated CD3 subunits fused to polypeptides derived from proteins thought to either enhance or inhibit normal T cell function. First, we showed that the C termini of both the TCR α- and ß-chains can accommodate specific additional sequences, without abrogating complex formation or acute sensitivity of the receptor. Replacement of ITAMs with ITIM-containing intracellular domains inverted the TCR signal (i.e., created a ligand-dependent inhibitory receptor). The normal signaling function of the CD3 complex was transferable to the TCR by eliminating all CD3 ITAMs and grafting three to six ITAMs onto the C termini of the α/ß-chains, with no effect on acute sensitivity. The observation that TCR variants of such diverse C-terminal composition can fold and function as signaling receptors demonstrates substantial structural and functional malleability of TCRs. These results add to knowledge about TCR structure-function with regard to acute signaling and may provide a route to use TCRs in different ways for T cell therapy.
Subject(s)
Receptor-CD3 Complex, Antigen, T-Cell/immunology , Receptors, Antigen, T-Cell/metabolism , Signal Transduction , T-Lymphocytes/metabolism , Carrier Proteins/metabolism , Humans , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell, alpha-beta/genetics , T-Lymphocytes/cytology , T-Lymphocytes/immunologyABSTRACT
We describe an approach to cancer therapy based on exploitation of common losses of genetic material in tumor cells (loss of heterozygosity) (Basilion et al., 1999; Beroukhim et al., 2010). This therapeutic concept addresses the fundamental problem of discrimination between tumor and normal cells and can be applied in principle to the large majority of tumors. It utilizes modular activator/blocker elements that integrate signals related to the presence and absence of ligands displayed on the cell surface (Fedorov et al., 2013). We show that the targeting system works robustly in vitro and in a mouse cancer model where absence of the HLA-A*02 allele releases a brake on engineered T cells activated by the CD19 surface antigen. This therapeutic approach potentially opens a route toward a large, new source of cancer targets.
Subject(s)
Loss of Heterozygosity/immunology , Neoplasms/immunology , T-Lymphocytes/immunology , Alleles , Animals , Antigens, CD19/immunology , Cell Line, Tumor , Female , HLA-A Antigens/immunology , Humans , Jurkat Cells , Ligands , Mice , Mice, Inbred NODABSTRACT
Chimeric antigen receptors (CARs) and their parent signaling molecule, the T cell receptor (TCR), are fascinating proteins of increasing relevance to disease therapy. Here we use a collection of 1221 pMHC-directed CAR constructs representing 10 pMHC targets to study aspects of CAR structure-activity relationships (SAR), with particular focus on the extracellular and transmembrane structural components. These experiments that involve pMHC targets whose number/cell can be manipulated by peptide dosing in vitro enable systematic analysis of the SAR of CARs in carefully controlled experimental situations (Harris and Kranz, 2016). We find that CARs tolerate a wide range of structural variation, with the ligand-binding domains (LBDs) dominating the SAR of CAR antigen sensitivity. Notwithstanding the critical role of the LBD, CAR antigen-binding on the cell surface, measured by pMHC tetramer staining, is not an effective predictor of functional sensitivity. These results have important implications for the design and testing of CARs aimed toward the clinic.
Subject(s)
HLA-A Antigens/immunology , Receptors, Chimeric Antigen/metabolism , Signal Transduction/immunology , T-Lymphocytes/immunology , Binding Sites/immunology , HLA-A Antigens/metabolism , Humans , Jurkat Cells , Ligands , MCF-7 Cells , Protein Domains/immunology , Protein Multimerization/immunology , Receptors, Chimeric Antigen/immunology , Structure-Activity Relationship , T-Lymphocytes/metabolismABSTRACT
The endocrine hormone FGF21 has attracted considerable interest as a potential therapeutic for treating diabetes and obesity. As an alternative to the native cytokine, we generated bispecific Avimer polypeptides that bind with high affinity and specificity to one of the receptor and coreceptor pairs used by FGF21, FGFR1c and ß-Klotho. These Avimers exhibit FGF21-like activity in in vitro assays with potency greater than FGF21. In a study conducted in obese male cynomolgus monkeys, animals treated with an FGFR1c/ß-Klotho bispecific Avimer showed improved metabolic parameters and reduced body weight comparable to the effects seen with FGF21. These results not only demonstrate the essential roles of FGFR1c and ß-Klotho in mediating the metabolic effects of FGF21, they also describe a first bispecific activator of this unique receptor complex and provide validation for a novel therapeutic approach to target this potentially important pathway for treating diabetes and obesity.
Subject(s)
Anti-Obesity Agents/pharmacology , Fibroblast Growth Factors/physiology , Membrane Proteins/antagonists & inhibitors , Obesity/drug therapy , Peptides/pharmacology , Receptor, Fibroblast Growth Factor, Type 1/antagonists & inhibitors , Amino Acid Sequence , Animals , Anti-Obesity Agents/pharmacokinetics , Binding Sites , Binding, Competitive , Body Weight/drug effects , Cell Line , Drug Evaluation, Preclinical , Fibroblast Growth Factors/chemistry , Insulin/blood , Klotho Proteins , Macaca fascicularis , Male , Membrane Proteins/biosynthesis , Mice , Molecular Mimicry , Molecular Sequence Data , Obesity/blood , Peptides/pharmacokinetics , Protein Binding , Rats , Receptor, Fibroblast Growth Factor, Type 4/chemistry , Recombinant Fusion Proteins/antagonists & inhibitors , Recombinant Fusion Proteins/biosynthesis , Serum Albumin/pharmacokinetics , Serum Albumin/pharmacology , Signal Transduction , Triglycerides/bloodABSTRACT
There are currently two Food and Drug Administration-approved classes of biologic agents that target tumor necrosis factor-alpha (TNF-alpha): anti-TNF monoclonal antibodies (mAbs) (adalimumab and infliximab), and soluble TNF receptors (etanercept). This study examined the ability of the TNF antagonists to: (1) bind various polymorphic variants of cell surface-expressed Fc receptors (FcgammaRs) and the complement component C1q, and (2) mediate Ab-dependent cellular cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) killing of cells expressing membrane-bound TNF (mTNF) in vitro. Both mAbs and the soluble TNF receptor demonstrated low-level binding to the activating receptors FcgammaRI, FcgammaRIIa, and FcgammaRIIIa, and the inhibitory receptor FcgammaRIIb, in the absence of exogenous TNF. However, upon addition of TNF, the mAbs, but not etanercept, showed significantly increased binding, in particular to the FcgammaRII and FcgammaRIII receptors. Infliximab and adalimumab induced ADCC much more potently than etanercept. In the presence of TNF, both mAbs bound C1q in in vitro assays, but etanercept did not bind C1q under any conditions. Infliximab and adalimumab also induced CDC in cells expressing mTNF more potently than etanercept. Differences in the ability to bind ligand and mediate cell death may account for the differences in efficacy and safety of TNF antagonists.
Subject(s)
Antibodies, Monoclonal/metabolism , Immunoglobulin G/metabolism , Receptors, Tumor Necrosis Factor/metabolism , Tumor Necrosis Factor-alpha , Adalimumab , Antibodies, Monoclonal, Humanized , Cell Line , Complement C1q/metabolism , Cytotoxicity, Immunologic , Etanercept , Humans , Infliximab , Receptors, IgG/metabolism , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/metabolismABSTRACT
The exocyst is an evolutionarily conserved multiprotein complex required for the targeting and docking of post-Golgi vesicles to the plasma membrane. Through its interactions with a variety of proteins, including small GTPases, the exocyst is thought to integrate signals from the cell and signal that vesicles arriving at the plasma membrane are ready for fusion. Here we describe the three-dimensional crystal structure of one of the components of the exocyst, Exo70p, from Saccharomyces cerevisiae at 3.5A resolution. Exo70p binds the small GTPase Rho3p in a GTP-dependent manner with an equilibrium dissociation constant of approximately 70 microM. Exo70p is an extended rod approximately 155 angstroms in length composed principally of alpha helices, and is a novel fold. The structure provides a first view of the Exo70 protein family and provides a framework to study the molecular function of this exocyst component.
Subject(s)
Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/cytology , Amino Acid Sequence , Animals , Conserved Sequence , Crystallography, X-Ray , Guanosine Triphosphate/pharmacology , Humans , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Folding , Protein Structure, Quaternary , Protein Structure, Tertiary , Saccharomyces cerevisiae Proteins/genetics , Sequence Alignment , Sequence Homology, Amino Acid , Vesicular Transport Proteins , rho GTP-Binding Proteins/genetics , rho GTP-Binding Proteins/metabolismABSTRACT
Ferritins are iron storage proteins made of 24 subunits forming a hollow spherical shell. Vertebrate ferritins contain varying ratios of heavy (H) and light (L) chains; however, known ferritin structures include only one type of chain and have octahedral symmetry. Here, we report the 1.9A structure of a secreted insect ferritin from Trichoplusia ni, which reveals equal numbers of H and L chains arranged with tetrahedral symmetry. The H/L-chain interface includes complementary features responsible for ordered assembly of the subunits. The H chain contains a ferroxidase active site resembling that of vertebrate H chains with an endogenous, bound iron atom. The L chain lacks the residues that form a putative iron core nucleation site in vertebrate L chains. Instead, a possible nucleation site is observed at the L chain 3-fold pore. The structure also reveals inter- and intrasubunit disulfide bonds, mostly in the extended N-terminal regions unique to insect ferritins. The symmetrical arrangement of H and L chains and the disulfide crosslinks reflect adaptations of insect ferritin to its role as a secreted protein.
Subject(s)
Ferritins/chemistry , Amino Acid Sequence , Animals , Computer Simulation , Cystine/chemistry , Cystine/genetics , Ferric Compounds , Ferritins/genetics , Ferritins/isolation & purification , Ferritins/metabolism , Humans , Models, Molecular , Molecular Sequence Data , Moths/metabolism , Protein Structure, Secondary , Protein Structure, TertiaryABSTRACT
During human immunodeficiency virus entry, gp41 undergoes a series of conformational changes that induce membrane fusion. Immediately prior to fusion, gp41 exists in a prehairpin intermediate in which the N- and C-peptide regions of gp41 are exposed. Rearrangement of this intermediate into a six-helix bundle composed of a trimeric coiled coil from the N-peptide region (N-trimer) surrounded by three peptides from the C-peptide region provides the driving force for membrane fusion, whereas prevention of six-helix bundle formation inhibits viral entry. Because of its central role in mediating viral entry, the N-trimer region of gp41 is a key vaccine target. Extensive efforts to discover potent and broadly neutralizing antibodies (Abs) against the N-trimer region have, thus far, been unsuccessful. In this study, we attached a potent C-peptide inhibitor that binds to the N-trimer region to cargo proteins of various sizes to examine the steric accessibility of the N-trimer during fusion. These inhibitors show a progressive loss of potency with increasing cargo size. Extension of the cargo/C-peptide linker partially restores inhibitory potency. These results demonstrate that the human immunodeficiency virus defends its critical hairpin-forming machinery by steric exclusion of large proteins and may explain the current dearth of neutralizing Abs against the N-trimer. In contrast, previous results suggest the C-peptide region is freely accessible during fusion, demonstrating that the N- and C-peptide regions are in structurally distinct environments. Based on these results, we also propose new strategies for the generation of neutralizing Abs that overcome this steric block.
Subject(s)
HIV Envelope Protein gp41/chemistry , HIV Envelope Protein gp41/physiology , Antibodies, Monoclonal/chemistry , Cell Fusion , Dimerization , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Humans , Inhibitory Concentration 50 , Models, Biological , Peptides/chemistry , Plasmids/metabolism , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Proteins/chemistry , Stereoisomerism , Surface Plasmon Resonance , Time Factors , Ultraviolet RaysABSTRACT
Streptococcus pneumoniae (pneumococcus) remains a significant health threat worldwide, especially to the young and old. While some of the biomolecules involved in pneumococcal pathogenesis are known and understood in mechanistic terms, little is known about the molecular details of bacterium/host interactions. We report here the solution structure of the 'repeated' adhesion domains (domains R1 and R2) of the principal pneumococcal adhesin, choline binding protein A (CbpA). Further, we provide insights into the mechanism by which CbpA binds its human receptor, polymeric immunoglobulin receptor (pIgR). The R domains, comprised of 12 imperfect copies of the leucine zipper heptad motif, adopt a unique 3-alpha-helix, raft-like structure. Each pair of alpha-helices is antiparallel and conserved residues in the loop between Helices 1 and 2 exhibit a novel 'tyrosine fork' structure that is involved in binding pIgR. This and other structural features that we show are conserved in most pneumococcal strains appear to generally play an important role in bacterial adhesion to pIgR. Interestingly, pneumococcus is the only bacterium known to adhere to and invade human cells by binding to pIgR.
Subject(s)
Bacterial Proteins/chemistry , Protein Conformation , Amino Acid Sequence , Animals , Bacterial Adhesion , Bacterial Proteins/classification , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cell Line , Crystallography, X-Ray , Humans , Models, Molecular , Molecular Sequence Data , Nasal Mucosa/metabolism , Nasopharynx/anatomy & histology , Nuclear Magnetic Resonance, Biomolecular , Phylogeny , Protein Binding , Receptors, Polymeric Immunoglobulin/metabolism , Sequence Alignment , Streptococcus pneumoniae/geneticsABSTRACT
The polymeric immunoglobulin receptor (pIgR) is a type I transmembrane protein that delivers dimeric IgA (dIgA) and pentameric IgM to mucosal secretions. Here, we report the 1.9 A resolution X-ray crystal structure of the N-terminal domain of human pIgR, which binds dIgA in the absence of other pIgR domains with an equilibrium dissociation constant of 300 nM. The structure of pIgR domain 1 reveals a folding topology similar to immunoglobulin variable domains, but with differences in the counterparts of the complementarity determining regions (CDRs), including a helical turn in CDR1 and a CDR3 loop that points away from the other CDRs. The unusual CDR3 loop position prevents dimerization analogous to the pairing of antibody variable heavy and variable light domains. The pIgR domain 1 structure allows interpretation of previous mutagenesis results and structure-based comparisons between pIgR and other IgA receptors.