A Novel Fusion of ALT-803 (Interleukin (IL)-15 Superagonist) with an Antibody Demonstrates Antigen-specific Antitumor Responses.

IL-15 and its receptor α (IL-15Rα) are co-expressed on antigen-presenting cells, allowing transpresentation of IL-15 to immune cells bearing IL-2RßγC and stimulation of effector immune responses. We reported previously that the high-affinity interactions between an IL-15 superagonist (IL-15N72D) and the extracellular IL-15Rα sushi domain (IL-15RαSu) could be exploited to create a functional scaffold for the design of multivalent disease-targeted complexes. The IL-15N72D·IL-15RαSuFc complex, also known as ALT-803, is a multimeric complex constructed by fusing IL-15N72D·IL-15RαSu to the Fc domain of IgG1. ALT-803 is an IL-15 superagonist complex that has been developed as a potent antitumor immunotherapeutic agent and is in clinical trials. Here we describe the creation of a novel fusion molecule, 2B8T2M, using the ALT-803 scaffold fused to four single chains of the tumor-targeting monoclonal antibody rituximab. This molecule displays trispecific binding activity through its recognition of the CD20 molecule on tumor cells, stimulation via IL-2RßγC displayed on immune effector cells, and binding to Fcγ receptors on natural killer cells and macrophages. 2B8T2M activates natural killer cells to enhance antibody-dependent cellular cytotoxicity, mediates complement-dependent cytotoxicity, and induces apoptosis of B-lymphoma cells. Compared with rituximab, 2B8T2M exhibits significantly stronger antitumor activity in a xenograft SCID mouse model and depletes B cells in cynomolgus monkeys more efficiently. Thus, ALT-803 can be modified as a functional scaffold for creating multispecific, targeted IL-15-based immunotherapeutic agents and may serve as a novel platform to improve the antitumor activity and clinical efficacy of therapeutic antibodies.

Therapeutic administration of IL-15 superagonist complex ALT-803 leads to long-term survival and durable antitumor immune response in a murine glioblastoma model.

Glioblastoma is the most aggressive primary central nervous system malignancy with a poor prognosis in patients. Despite the need for better treatments against glioblastoma, very little progress has been made in discovering new therapies that exhibit superior survival benefit than the standard of care. Immunotherapy has been shown to be a promising treatment modality that could help improve clinical outcomes of glioblastoma patients by assisting the immune system to overcome the immunosuppressive tumor environment. Interleukin-15 (IL-15), a cytokine shown to activate several effector components of the immune system, may serve as an excellent immunotherapeutic candidate for the treatment of glioblastoma. Thus, we evaluated the efficacy of an IL-15 superagonist complex (IL-15N72D:IL-15RαSu-Fc; also known as ALT-803) in a murine GL261-luc glioblastoma model. We show that ALT-803, as a single treatment as well as in combination with anti-PD-1 antibody or stereotactic radiosurgery, exhibits a robust antitumor immune response resulting in a prolonged survival including complete remission in tumor bearing mice. In addition, ALT-803 treatment results in long-term immune memory against glioblastoma tumor rechallenge. Flow cytometric analysis of tumor infiltrating immune cells shows that ALT-803 leads to increased percentage of CD8+-cell infiltration, but not the NK cells, and IFN-γ production into the tumor microenvironment. Cell depletion studies, in accordance with the flow cytometric results, show that the ALT-803 therapeutic effect is dependent on CD4+ and CD8+ cells. These results provide a rationale for evaluating the therapeutic activity of ALT-803 against glioblastoma in the clinical setting.

IL-15:IL-15 receptor alpha superagonist complex: high-level co-expression in recombinant mammalian cells, purification and characterization.

IL-15, a promising cytokine for treating cancer and viral diseases, is presented in trans by the IL-15 receptor (IL-15R) alpha-chain to the IL-15Rßγc complex displayed on the surface of T cells and natural killer (NK) cells. We previously reported that an asparagine to aspartic acid substitution at amino acid 72 (N72D) of IL-15 provides a 4-5-fold increase in biological activity compared to the native molecule. In this report, we describe Chinese hamster ovary (CHO) cell expression of a soluble complex (IL-15 N72D:IL-15RαSu/Fc) consisting of the IL-15 N72D superagonist and a dimeric IL-15Rα sushi domain-IgG1 Fc fusion protein. A simple but readily scalable affinity and ion exchange chromatography method was developed to highly purify the complex having both IL-15 binding sites fully occupied. The immunostimulatory effects of this complex were confirmed using cell proliferation assays. Treatment of mice with a single intravenous dose of IL-15N72D:IL-15RαSu/Fc resulted in a significant increase in CD8+ T cells and NK cells that was not observed following IL-15 treatment. Pharmacokinetic analysis indicated that the complex has a 25-h half-life in mice which is considerably longer than <40-min half-life of IL-15. Thus, the enhanced activity of the IL-15N72D:IL-15RαSu/Fc complex is likely the result of the increased binding activity of IL-15N72D to IL-15Rßγc, optimized cytokine trans-presentation by the IL-15RαSu domain, the dimeric nature of the cytokine domain and its increased in vivo half-life compared to IL-15. These findings indicate that this IL-15 superagonist complex could serve as a superior immunostimulatory therapeutic agent.

Novel human interleukin-15 agonists.

IL-15 is an immunostimulatory cytokine trans-presented with the IL-15 receptor alpha-chain to the shared IL-2/IL-15Rbeta and common gamma-chains displayed on the surface of T cells and NK cells. To further define the functionally important regions of this cytokine, activity and binding studies were conducted on human IL-15 muteins generated by site-directed mutagenesis. Amino acid substitutions of the asparagine residue at position 72, which is located at the end of helix C, were found to provide both partial agonist and superagonist activity, with various nonconservative substitutions providing enhanced activity. Particularly, the N72D substitution provided a 4-5-fold increase in biological activity of the IL-15 mutein compared with the native molecule based on proliferation assays with cells bearing human IL-15Rbeta and common gamma-chains. The IL-15N72D mutein exhibited superagonist activity through improved binding ability to the human IL-15Rbeta-chain. However, the enhanced potency of IL-15N72D was not observed with cells expressing the mouse IL-15Ralpha-IL-15Rbeta-gamma(c) complex, suggesting that this effect is specific to the human IL-15 receptor. The enhanced biological activity of IL-15N72D was associated with more intense phosphorylation of Jak1 and Stat5 and better anti-apoptotic activity compared with the wild-type IL-15. IL-15N72D superagonist activity was also preserved when linked to a single-chain TCR domain to generate a tumor-specific fusion protein. Thus, the human IL-15 superagonist muteins and fusions may create opportunities to construct more efficacious immunotherapeutic agents with clinical utility.

Distinct molecular and cellular contributions to stabilizing selectin-mediated rolling under flow.

Leukocytes roll on selectins at nearly constant velocities over a wide range of wall shear stresses. Ligand-coupled microspheres roll faster on selectins and detach quickly as wall shear stress is increased. To examine whether the superior performance of leukocytes reflects molecular features of native ligands or cellular properties that favor selectin-mediated rolling, we coupled structurally defined selectin ligands to microspheres or K562 cells and compared their rolling on P-selectin. Microspheres bearing soluble P-selectin glycoprotein ligand (sPSGL)-1 or 2-glycosulfopeptide (GSP)-6, a GSP modeled after the NH2-terminal P-selectin-binding region of PSGL-1, rolled equivalently but unstably on P-selectin. K562 cells displaying randomly coupled 2-GSP-6 also rolled unstably. In contrast, K562 cells bearing randomly coupled sPSGL-1 or 2-GSP-6 targeted to a membrane-distal region of the presumed glycocalyx rolled more like leukocytes: rolling steps were more uniform and shear resistant, and rolling velocities tended to plateau as wall shear stress was increased. K562 cells treated with paraformaldehyde or methyl-beta-cyclodextrin before ligand coupling were less deformable and rolled unstably like microspheres. Cells treated with cytochalasin D were more deformable, further resisted detachment, and rolled slowly despite increases in wall shear stress. Thus, stable, shear-resistant rolling requires cellular properties that optimize selectin-ligand interactions.

Characterization of an antibody scFv that recognizes fibrillar insulin and beta-amyloid using atomic force microscopy.

Fibrillar amyloid is the hallmark feature of many protein aggregation diseases, such as Alzheimer's and Parkinson's diseases. A monoclonal single-chain variable fragment (scFv) targeting insulin fibrils was isolated using phage display technology and an atomic force microscopy (AFM) mica substrate. Specific targeting of the scFv to insulin fibrils but not monomers or other small oligomeric forms, under similar conditions, was demonstrated both by enzyme-linked immunosorbent assays and AFM recognition imaging. The scFv also recognizes beta-amyloid fibrils, a hallmark feature of Alzheimer's disease. The results suggest that the isolated scFv possibly targets a shared fibrillar motif-probably the cross-beta-sheet characteristic of amyloid fibrils. The techniques outlined here provide additional tools to further study the process of fibril formation. The scFvs isolated can have potential use as diagnostic or therapeutic reagents for protein aggregation diseases.

Tissue factor-specific ultra-bright SERRS nanostars for Raman detection of pulmonary micrometastases.

Here we demonstrate a novel application of 'surface enhanced resonance Raman scattering nanoparticles' (SERRS NPs) for imaging breast cancer lung metastases with much higher precision than currently feasible. A breast cancer lung metastasis mouse model was established by intravenous injection of LM2 cells. These mice were intravenously administered SERRS NPs conjugated with ALT-836, an anti-tissue factor (TF) monoclonal antibody, and subjected to Raman imaging to visualize the expression of TF both in vivo and ex vivo. Raman imaging indicated marked uptake of αTF-SERRS-NPs by the lung metastases compared to isotype and blocking controls. Conversely, little uptake of αTF-SERRS-NPs was observed in the lungs of healthy mice. Successful detection and delineation of pulmonary micrometastatic lesions as small as 200 µm, corroborated by histology, immunohistochemistry, and bioluminescence imaging confirmed the suitability of both TF as a target and αTF-SERRS-NPs as an effective contrast agent for imaging breast cancer lung metastases. Further advancements of this technique in the form of Raman endoscopes coupled with ultrabright SERRS NPs developed in this work could lead to minimally invasive detection and resection of lung metastases.

Identification and repair of positive binding antibodies containing randomly generated amber codons from synthetic phage display libraries.

Phage display technology allows for the rapid isolation and characterization of monoclonal antibodies that have vast potential for therapeutic and diagnostic applications. However, the panning process, which utilizes a host strain that suppresses termination by the amber codon, has an inherent bias toward clones containing randomly generated amber stop codons, complicating identification of positive binding antibodies when the antibody genes are finally expressed in a nonsupressor host. Here, we perform biopanning against a Histone 2A peptide using streptavidin- or anti-biotin-coated beads. After four rounds, a dominant clone is characterized but contains a spurious amber stop codon. A protocol is given that readily corrects the amber codon, allowing for soluble antibody production once the phagemid is transformed into a nonsuppressor bacterial strain. This work also highlights the ability to isolate antibodies against a protein antigen by using only a small peptide (15 amino acids) representing a portion of the antigen.

Efficacy and mechanism-of-action of a novel superagonist interleukin-15: interleukin-15 receptor αSu/Fc fusion complex in syngeneic murine models of multiple myeloma.

ALT-803, a complex of an interleukin (IL)-15 superagonist mutant and a dimeric IL-15 receptor αSu/Fc fusion protein, was found to exhibit significantly stronger in vivo biologic activity on NK and T cells than IL-15. In this study, we show that a single dose of ALT-803, but not IL-15 alone, eliminated well-established 5T33P and MOPC-315P myeloma cells in the bone marrow of tumor-bearing mice. ALT-803 treatment also significantly prolonged survival of myeloma-bearing mice and provided resistance to rechallenge with the same tumor cells through a CD8(+) T-cell-dependent mechanism. ALT-803 treatment stimulated CD8(+) T cells to secrete large amounts of IFN-γ and promoted rapid expansion of CD8(+)CD44(high) memory T cells in vivo. These memory CD8(+) T cells exhibited ALT-803-mediated upregulation of NKG2D (KLRK1) but not PD-1 (PDCD1) or CD25 (IL2RA) on their cell surfaces. ALT-803-activated CD8(+) memory T cells also exhibited nonspecific cytotoxicity against myeloma and other tumor cells in vitro, whereas IFN-γ had no direct effect on myeloma cell growth. ALT-803 lost its antimyeloma activity in tumor-bearing IFN-γ knockout mice but retained the ability to promote CD8(+)CD44(high) memory T-cell proliferation, indicating that ALT-803-mediated stimulation of CD8(+)CD44(high) memory T cells is IFN-γ-independent. Thus, besides well-known IL-15 biologic functions in host immunity, this study shows that IL-15-based ALT-803 could activate CD8(+)CD44(high) memory T cells to acquire a unique innate-like phenotype and secrete IFN-γ for nonspecific tumor cell killing. This unique immunomodulatory property of ALT-803 strongly supports its clinical development as a novel immunotherapeutic agent against cancer and viral infections.

Two-dimensional kinetics regulation of alphaLbeta2-ICAM-1 interaction by conformational changes of the alphaL-inserted domain.

The leukocyte integrin alphaLbeta2 mediates cell adhesion and migration during inflammatory and immune responses. Ligand binding of alphaLbeta2 is regulated by or induces conformational changes in the inserted (I) domain. By using a micropipette, we measured the conformational regulation of two-dimensional (2D) binding affinity and the kinetics of cell-bound intercellular adhesion molecule-1 interacting with alphaLbeta2 or isolated I domain expressed on K562 cells. Locking the I domain into open and intermediate conformations with a disulfide bond increased the affinities by approximately 8000- and approximately 30-fold, respectively, from the locked closed conformation, which has similar affinity as the wild-type I domain. Most surprisingly, the 2D affinity increases were due mostly to the 2D on-rate increases, as the 2D off-rates only decreased by severalfold. The wild-type alphaLbeta2, but not its I domain in isolation, could be up-regulated by Mn2+ or Mg2+ to have high affinities and on-rates. Locking the I domain in any of the three conformations abolished the ability of divalent cations to regulate 2D affinity. These results indicate that a downward displacement of the I domain C-terminal helix, induced by conformational changes of other domains of the alphaLbeta2, is required for affinity and on-rate up-regulation.

Membrane tethers formed from blood cells with available area and determination of their adhesion energy.

Fundamental to all mammalian cells is the adherence of the lipid bilayer membrane to the underlying membrane associated cytoskeleton. To investigate this adhesion, we physically detach the lipid membrane from the cell by mechanically forming membrane tethers. For the most part these have been tethers formed from either neutrophils or red cells. Here we do a simple thermodynamic analysis of the tether formation process using the entire cell, including tether, as the control volume. For a neutrophil, we show that the total adhesion energy per unit area between lipid membrane and cytoskeleton depends on the square of the tether force. For a flaccid red cell, we show that the total adhesion energy minus the tension in the spectrin cytoskeleton depends also on the square of the tether force. Finally, we discuss briefly the viscous flow of membrane. Using published data we calculate and compare values for the various adhesion energies and viscosities.

Experimental studies of membrane tethers formed from human neutrophils.

Membrane tethers (thin, cylindrical pieces of membrane) have been implicated in the rolling of neutrophils along the endothelium. In our studies, these tethers were formed from passive, stimulated (0.1 microM fMLP), and osmotically swollen (170-180 mOsm) human neutrophils; as well as neutrophils treated with 0.3 microM latrunculin A to disrupt the cytoskeleton. This tether formation was accomplished by micropipette suction of latex beads coated with antibodies to proteins on the neutrophil membrane surface. From plots of force versus velocity for the tether formation process, we calculated adhesion energies per unit area of the lipid membrane to the cytoskeleton and the viscous resistance (effective viscosity) that occurs during the formation of these tethers at finite velocity. Most of the properties of the neutrophil were altered once it had been treated as described above. We were also able to show mechanical reversibility of membrane tethers, as well as an unexpected formation rate at "high" tether forces. Since membrane tethers have been implicated in the rolling of neutrophils, then the changes in tether formation may ultimately alter how these cells roll.

Forces required to initiate membrane tether extrusion from cell surface depend on cell type but not on the surface molecule.

When a cell adhered to another cell or substratum via surface proteins is forced to detach, lipid membrane tethers are often extruded from the cell surface before the protein bond dissociates. For example, during the inflammatory reaction leukocytes roll on the surface of activated endothelial cells. The rolling adhesion is mediated by interactions of selectins with their ligands, e.g., P-selectin glycoprotein ligand (PSGL)-1, which extrudes membrane tethers from the surfaces of both leukocytes and endothelial cells. Membrane tether extrusion has been suggested to regulate leukocyte rolling. Here we examine several factors that may affect forces required to initiate membrane tethers, or initial tether force. It was found that initial tether forces were similar regardless of the presence or absence of the cytoplasmic tail of P-selectin and regardless of whether the tethers were extruded via binding to PSGL-1 or Fcy receptors. Initial tether forces were found to depend on the cell types tested and were greatly reduced by treatment of latrunculin A, which inhibits actin polymerization. These data provide additional insights to the control of membrane tether extrusion, which should be taken into account when cellular functions such as rolling where tether extrusion plays a regulatory role are compared using different cell types expressing the same molecule.