Humanization of a strategic CD3 epitope enables evaluation of clinical T-cell engagers in a fully immunocompetent in vivo model.

T-cell engagers (TCEs) are a growing class of biotherapeutics being investigated in the clinic for treatment of a variety of hematological and solid tumor indications. However, preclinical evaluation of TCEs in vivo has been mostly limited to xenograft tumor models in human T-cell reconstituted immunodeficient mice, which have a number of limitations. To explore the efficacy of human TCEs in fully immunocompetent hosts, we developed a knock-in mouse model (hCD3E-epi) in which a 5-residue N-terminal fragment of murine CD3-epsilon was replaced with an 11-residue stretch from the human sequence that encodes for a common epitope recognized by anti-human CD3E antibodies in the clinic. T cells from hCD3E-epi mice underwent normal thymic development and could be efficiently activated upon crosslinking of the T-cell receptor with anti-human CD3E antibodies in vitro. Furthermore, a TCE targeting human CD3E and murine CD20 induced robust T-cell redirected killing of murine CD20-positive B cells in ex vivo hCD3E-epi splenocyte cultures, and also depleted nearly 100% of peripheral B cells for up to 7 days following in vivo administration. These results highlight the utility of this novel mouse model for exploring the efficacy of human TCEs in vivo, and suggest a useful tool for evaluating TCEs in combination with immuno-oncology/non-immuno-oncology agents against heme and solid tumor targets in hosts with a fully intact immune system.

Improved therapeutic index of an acidic pH-selective antibody.

Although therapeutically efficacious, ipilimumab can exhibit dose-limiting toxicity that prevents maximal efficacious clinical outcomes and can lead to discontinuation of treatment. We hypothesized that an acidic pH-selective ipilimumab (pH Ipi), which preferentially and reversibly targets the acidic tumor microenvironment over the neutral periphery, may have a more favorable therapeutic index. While ipilimumab has pH-independent CTLA-4 affinity, pH Ipi variants have been engineered to have up to 50-fold enhanced affinity to CTLA-4 at pH 6.0 compared to pH 7.4. In hCTLA-4 knock-in mice, these variants have maintained anti-tumor activity and reduced peripheral activation, a surrogate marker for toxicity. pH-sensitive therapeutic antibodies may be a differentiating paradigm and a novel modality for enhanced tumor targeting and improved safety profiles.

Antibody blockade of CD96 by distinct molecular mechanisms.

The molecular interactions of mouse CD96 to CD155 ligand and to two surrogate antibodies have been investigated. Biophysical and structural studies demonstrate that CD96 forms a homodimer but assembles as 1:1 heterodimeric complexes with CD155 or with one of the surrogate antibodies, which compete for the same binding interface. In comparison, the other surrogate antibody binds across the mouse CD96 dimer and recognizes a quaternary epitope spanning both protomers to block exposure of the ligand-binding site. This study reveals different blocking mechanisms and modalities of these two antibodies and may provide insight into the functional effects of antibodies against CD96.

Tumor burden limits bispecific antibody efficacy through T cell exhaustion averted by concurrent cytotoxic therapy.

BCMA-CD3-targeting bispecific antibodies (BsAb) are a recently developed immunotherapy class which shows potent tumor killing activity in multiple myeloma (MM). Here, we investigated a murine BCMA-CD3-targeting BsAb in the immunocompetent Vk*MYC and its IMiD-sensitive derivative Vk*MYChCRBN models of MM. The BCMA-CD3 BsAb was safe and efficacious in a subset of mice, but failed in those with high-tumor burden, consistent with clinical reports of BsAb in leukemia. The combination of BCMA-CD3 BsAb with pomalidomide expanded lytic T cells and improved activity even in IMiD resistant high-tumor burden cases. Yet, survival was only marginally extended due to acute toxicity and T cell exhaustion, which impaired T cell persistence. In contrast, the combination with cyclophosphamide was safe and allowed for a tempered pro-inflammatory response associated with long-lasting complete remission. Concurrent cytotoxic therapy with BsAb actually improved T cell persistence and function, offering a promising approach to patients with a large tumor burden.

Structures of mouse and human GITR-GITRL complexes reveal unique TNF superfamily interactions.

Glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) and GITR ligand (GITRL) are members of the tumor necrosis superfamily that play a role in immune cell signaling, activation, and survival. GITR is a therapeutic target for directly activating effector CD4 and CD8 T cells, or depleting GITR-expressing regulatory T cells (Tregs), thereby promoting anti-tumor immune responses. GITR activation through its native ligand is important for understanding immune signaling, but GITR structure has not been reported. Here we present structures of human and mouse GITR receptors bound to their cognate ligands. Both species share a receptor-ligand interface and receptor-receptor interface; the unique C-terminal receptor-receptor enables higher order structures on the membrane. Human GITR-GITRL has potential to form a hexameric network of membrane complexes, while murine GITR-GITRL complex forms a linear chain due to dimeric interactions. Mutations at the receptor-receptor interface in human GITR reduce cell signaling with in vitro ligand binding assays and minimize higher order membrane structures when bound by fluorescently labeled ligand in cell imaging experiments.

Integrated Approach for Characterizing Bispecific Antibody/Antigens Complexes and Mapping Binding Epitopes with SEC/MALS, Native Mass Spectrometry, and Protein Footprinting.

Bispecific antibodies (BsAbs), with a unique mechanism of recognizing two different epitopes or antigens, have shown potential in various therapeutic areas. Molecular characterization of BsAbs' epitopes not only allows for detailed understanding of their mechanism of actions but also guides the design and selection of drug candidate molecules. In this study, we illustrate the practical utility of an integrated approach, including size exclusion chromatography with multiangle light scattering and native mass spectrometry (MS) for the biophysical characterization of complex formation of a BsAb with two target antigens, cluster of differentiation 3 (CD3) and B-cell maturation antigen (BCMA). MS-based protein footprinting strategies, including hydrogen/deuterium exchange MS, fast photochemical oxidation of proteins, and carboxyl group footprinting with glycine ethyl ester, were further applied to determine BsAb's binding epitopes. This combination approach provides molecular details on the binding mechanisms of BsAb to the two distinct antigens with rapid output and high resolution.

Design and characterization of mouse IgG1 and IgG2a bispecific antibodies for use in syngeneic models.

The development of antibody therapeutics relies on animal models that accurately recapitulate disease biology. Syngeneic mouse models are increasingly used with new molecules to capture the biology of complex cancers and disease states, and to provide insight into the role of the immune system. The establishment of syngeneic mouse models requires the ability to generate surrogate mouse counterparts to antibodies designed for humans. In the field of bispecific antibodies, there remains a dearth of technologies available to generate native IgG-like mouse bispecific antibodies. Thus, we engineered a simple co-expression system for one-step purification of intact mouse IgG1 and IgG2a bispecific antibodies from any antibody pair. We demonstrated proof of concept with CD3/CD20 bispecific antibodies, which highlighted both the quality and efficacy of materials generated by this technology.

A comparative systems analysis of polysaccharide-elicited responses in Neurospora crassa reveals carbon source-specific cellular adaptations.

Filamentous fungi are powerful producers of hydrolytic enzymes for the deconstruction of plant cell wall polysaccharides. However, the central question of how these sugars are perceived in the context of the complex cell wall matrix remains largely elusive. To address this question in a systematic fashion we performed an extensive comparative systems analysis of how the model filamentous fungus Neurospora crassa responds to the three main cell wall polysaccharides: pectin, hemicellulose and cellulose. We found the pectic response to be largely independent of the cellulolytic one with some overlap to hemicellulose, and in its extent surprisingly high, suggesting advantages for the fungus beyond being a mere carbon source. Our approach furthermore allowed us to identify carbon source-specific adaptations, such as the induction of the unfolded protein response on cellulose, and a commonly induced set of 29 genes likely involved in carbon scouting. Moreover, by hierarchical clustering we generated a coexpression matrix useful for the discovery of new components involved in polysaccharide utilization. This is exemplified by the identification of lat-1, which we demonstrate to encode for the physiologically relevant arabinose transporter in Neurospora. The analyses presented here are an important step towards understanding fungal degradation processes of complex biomass.

Structural basis for alternating access of a eukaryotic calcium/proton exchanger.

Eukaryotic Ca(2+) regulation involves sequestration into intracellular organelles, and expeditious Ca(2+) release into the cytosol is a hallmark of key signalling transduction pathways. Bulk removal of Ca(2+) after such signalling events is accomplished by members of the Ca(2+):cation (CaCA) superfamily. The CaCA superfamily includes the Na(+)/Ca(2+) (NCX) and Ca(2+)/H(+) (CAX) antiporters, and in mammals the NCX and related proteins constitute families SLC8 and SLC24, and are responsible for the re-establishment of Ca(2+) resting potential in muscle cells, neuronal signalling and Ca(2+) reabsorption in the kidney. The CAX family members maintain cytosolic Ca(2+) homeostasis in plants and fungi during steep rises in intracellular Ca(2+) due to environmental changes, or following signal transduction caused by events such as hyperosmotic shock, hormone response and response to mating pheromones. The cytosol-facing conformations within the CaCA superfamily are unknown, and the transport mechanism remains speculative. Here we determine a crystal structure of the Saccharomyces cerevisiae vacuolar Ca(2+)/H(+) exchanger (Vcx1) at 2.3 Å resolution in a cytosol-facing, substrate-bound conformation. Vcx1 is the first structure, to our knowledge, within the CAX family, and it describes the key cytosol-facing conformation of the CaCA superfamily, providing the structural basis for a novel alternating access mechanism by which the CaCA superfamily performs high-throughput Ca(2+) transport across membranes.

Crystal structure of a eukaryotic phosphate transporter.

Phosphate is crucial for structural and metabolic needs, including nucleotide and lipid synthesis, signalling and chemical energy storage. Proton-coupled transporters of the major facilitator superfamily (MFS) are essential for phosphate uptake in plants and fungi, and also have a function in sensing external phosphate levels as transceptors. Here we report the 2.9 Å structure of a fungal (Piriformospora indica) high-affinity phosphate transporter, PiPT, in an inward-facing occluded state, with bound phosphate visible in the membrane-buried binding site. The structure indicates both proton and phosphate exit pathways and suggests a modified asymmetrical 'rocker-switch' mechanism of phosphate transport. PiPT is related to several human transporter families, most notably the organic cation and anion transporters of the solute carrier family (SLC22), which are implicated in cancer-drug resistance. We modelled representative cation and anion SLC22 transporters based on the PiPT structure to surmise the structural basis for substrate binding and charge selectivity in this important family. The PiPT structure demonstrates and expands on principles of substrate transport by the MFS transporters and illuminates principles of phosphate uptake in particular.