Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an onco-embryonic antigen. Due to its expression on the cell surface of leukemia cells from patients with chronic lymphocytic leukemia (CLL), but not on normal B-cells or other post-partum tissues, ROR1 is an attractive candidate for targeted therapies. UC-961 is a first-in-class humanized monoclonal antibody (mAb) that binds the extracellular domain of ROR1. In this paper we outline some of the preclinical studies leading to an investigation new drug (IND) designation, enabling clinical studies in patients with CLL.

Toll like receptor 7 (TLR7) is located in the endosomal compartment of immune cells. Signaling through TLR7, mediated by the adaptor protein MyD88, stimulates the innate immune system and shapes adaptive immune responses. Previously, we characterized TLR7 ligands conjugated to protein, lipid or polyethylene glycol (PEG). Among the TLR7 ligand conjugates, the addition of PEG chains reduced the agonistic potency. PEGs are safe in humans and widely used for improvement of pharmacokinetics in existing biologics and some low molecular weight compounds. PEGylation could be a feasible method to alter the pharmacokinetics and pharmacodynamics of TLR7 ligands. In this study, we systematically studied the influence of PEG chain length on the in vitro and in vivo properties of potent TLR7 ligands. PEGylation increased solubility of the TLR7 ligands and modulated protein binding. Adding a 6-10 length PEG to the TLR7 ligand reduced its potency toward induction of interleukin (IL)-6 by murine macrophages in vitro and IL-6 and tumor necrosis factor (TNF) in vivo. However, PEGylation with 18 or longer chain restored, and even enhanced, the agonistic activity of the drug. In human peripheral blood mononuclear cells, similar effects of PEGylation were observed for secretion of proinflammatory cytokines, IL-6, IL-12, TNF-α, IL-1β and type 1 interferon, as well for B cell proliferation. In summary, these studies demonstrate that conjugation of PEG chains to a synthetic TLR ligand can impact its potency for cytokine induction depending on the size of the PEG moiety. Thus, PEGylation may be a feasible approach to regulate the pharmacological properties of TLR7 ligands.

Chronic lymphocytic leukemia (CLL) cells express high levels of CD44, a cell-surface glycoprotein receptor for hyaluronic acid. We found that a humanized mAb specific for CD44 (RG7356) was directly cytotoxic for leukemia B cells, but had little effect on normal B cells. Moreover, RG7356 could induce CLL cells that expressed the zeta-associated protein of 70 kDa (ZAP-70) to undergo caspasedependent apoptosis, independent of complement or cytotoxic effector cells. The cytotoxic effect of this mAb was not mitigated when the CLL cells were cocultured with mesenchymal stromal cells (MSCs) or hyaluronic acid or when they were stimulated via ligation of the B-cell receptor with anti-μ. RG7356 induced rapid internalization of CD44 on CLL cells at 37°C, resulting in reduced expression of ZAP-70, which we found was complexed with CD44. Administration of this mAb at a concentration of 1 mg/kg to immune-deficient mice engrafted with human CLL cells resulted in complete clearance of engrafted leukemia cells. These studies indicate that this mAb might have therapeutic activity, particularly in patients with CLL that express ZAP-70.cell survival | preclinical studies | animal model | antibody therapy B -cell chronic lymphocytic leukemia (CLL) is characterized by the clonal expansion of mature, antigen-stimulated CD5+/ CD23+ B cells in blood, secondary lymphoid tissues, and marrow (1). Most of the circulating CLL cells in patients are arrested in the G0/G1 phase of the cell cycle and express high levels of antiapoptotic proteins (2). CLL therefore has been characterized as a process of defective apoptosis, rather than increased proliferation. However, despite their apparent longevity in vivo, CLL cells undergo spontaneous and drug-induced apoptosis in vitro, unless rescued by monocyte-derived Nurse-like cells (NLCs), follicular dendritic cells, or mesenchymal stromal cells (MSCs) (3-6). Thus, it has been postulated that CLL cells receive survival signals from these accessory cells, which constitute part of the CLL B-cell microenvironment in secondary lymphoid tissues and marrow (6). These survival signals can inhibit spontaneous or drug-induced apoptosis, particularly for CLL cells that express unmutated Ig heavy-chain variable genes (IGHVs) and/or the zeta-associated protein of 70 kDa (ZAP-70), which typically is not expressed by normal B cells (7). Patients with leukemia cells that possess such characteristics typically have a relatively short interval from diagnosis to initial therapy compared with patients with CLL cells that express mutated IGHVs or that lack expression of .One of the survival signals received by leukemia cells may be mediated via CD44, a surface glycoprotein receptor for the nonsulfated glycosaminoglycan hyaluronic acid (HA), which typically is found in the microenvironment of lymphoid tissues (13). CLL cells express high levels of CD44, particularly those that express unmutated IGHVs and/or . Upon binding HA in the extracellular matrix, CD44 activates the phosphoinositol 3-kinase (PI3K)/AKT and MAPK/ERK ...

Sato-Kaneko et al.Protective Liposomal TLR4/7 Vaccine titers and cytokine levels. The combination adjuvant induced a greater diversity in B cell clonotypes of immunoglobulin heavy chain (IGH) genes in the draining lymph nodes and antibodies against a broad spectrum of HA epitopes encompassing HA head and stalk domains and with cross-reactivity against different subtypes of HA and NA. This novel combination liposomal adjuvant contributes to a more broadly protective vaccine while demonstrating an attractive safety profile.

Extracellular vesicles (EVs) are identified as mediators of intercellular communication and cellular regulation. In the immune system, EVs play a role in antigen presentation as a part of cellular communication. To enable drug discovery and characterization of compounds that affect EV biogenesis, function, and release in immune cells, we developed and characterized a reporter cell line that allows the quantitation of EVs shed into culture media in phenotypic high-throughput screen (HTS) format. Tetraspanins CD63 and CD9 were previously reported to be enriched in EVs; hence, a construct with dual reporters consisting of CD63-Turbo-luciferase (Tluc) and CD9-Emerald green fluorescent protein (EmGFP) was engineered. This construct was transduced into the human monocytic leukemia cell line, THP-1. Cells expressing the highest EmGFP were sorted by flow cytometry as single cell, and clonal pools were expanded under antibiotic selection pressure. After four passages, the green fluorescence dimmed, and EV biogenesis was then tracked by luciferase activity in culture supernatants. The Tluc activities of EVs shed from CD63Tluc-CD9EmGFP reporter cells in the culture supernatant positively correlated with the concentrations of released EVs measured by nanoparticle tracking analysis. To examine the potential for use in HTS, we first miniaturized the assay into a robotic 384-well plate format. A 2210 commercial compound library (Maybridge) was then screened twice on separate days, for the induction of extracellular luciferase activity. The screening data showed high reproducibility on days 1 and 2 (78.6%), a wide signal window, and an excellent Z′ factor (average of 2-day screen, 0.54). One hundred eighty-seven compounds showed a response ratio that was 3SD above the negative controls in both day 1 and 2 screens and were considered as hit candidates (approximately 10%). Twenty-two out of 40 re-tested compounds were validated. These results indicate that the performance of CD63Tluc-CD9EmGFP reporter cells is reliable, reproducible, robust, and feasible for HTS of compounds that regulate EV release by the immune cells.