T cell engaging bispecific antibodies targeting CD33 IgV and IgC domains for the treatment of acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) remains one of the most challenging hematological malignancies. Despite progress in therapeutics, majority of patients succumb to this neoplasm. CD33 is a proven therapeutic target, given its expression on most AML cells. Almost all anti-CD33 antibodies target the membrane distal immunoglobulin V (IgV) domain of the CD33 extracellular domain. METHODS: In this manuscript, we present data on three bispecific antibodies (BsAbs) against the CD33 IgV and membrane proximal immunoglobulin C (IgC) domains. We use in vitro binding and cytotoxicity assays to show the effect of these BsAbs on AML cell lines. We also use immunodeficient mice-bearing leukemias from cell lines and patient-derived xenografts to show the effect of these BsAbs in vivo. RESULTS: In vitro, the IgV-targeting BsAb had higher binding to AML cell lines using flow cytometry and delivered more potent cytotoxicity in T-cell-dependent cytotoxicity assays; importantly, the IgC domain-targeting outperformed the IgV domain-targeting BsAb in medullary and extramedullary leukemia animal models. CONCLUSIONS: These data support further clinical development of this BsAb for first-in-human phase I clinical trial.

Intrapulmonary inoculation of multicellular spheroids to construct an orthotopic lung cancer xenograft model that mimics four clinical stages of non-small cell lung cancer.

INTRODUCTION: Lung cancer leads in mortality among all types of cancer in US and Non-small cell lung cancer (NSCLC) is the major type of lung cancer. Mice models of lung cancer based on subcutaneous or orthotopic inoculation of cancer cell suspension do not adequately mimic the progression of lung cancer in clinic. METHODS: A549-iRFP cells (human NSCLC adenocarcinoma) were cultured to form multicellular spheroids (MCS), which were then inoculated intrapulmonarily into male athymic nude mice. The xenograft cancer development was monitored by in vivo fluorescent imaging and validated by open-chest anatomy, ex vivo fluorescent imaging, and histological studies. RESULTS: The newly developed orthotopic xenograft model of lung cancer simulated all four clinical stages of NSCLC progression over one month: Stage 1) localized tumor at the inoculation site, Stage 2) multiple tumor nodules or larger tumor nodule on the same side of the lung, Stage 3) cancer growth on heart surface, and Stage 4) metastatic cancer on both sides of the lung. The model yielded high rates of postoperative survival (100%) and parenchymal tumor establishment (88.9%). The roughness of the inoculated MCS associated negatively with the time needed to develop metastatic cancer (p = .0299). DISCUSSION: This new orthotopic xenograft model of NSCLC would facilitate the development of medications to treat lung cancer.

Lipid-Coated, pH-Sensitive Magnesium Phosphate Particles for Intracellular Protein Delivery.

PURPOSE: To develop cationic lipid-coated magnesium phosphate nanoparticles (LPP) for intracellular catalase (CAT) delivery. METHODS: Magnesium phosphate nanoparticles (MgP NP) were prepared by micro-emulsion precipitation and mixed with catalase-loaded cationic liposomes (DOTAP/cholesterol) to yield LPP formulation of catalase (LPP-CAT). The size and ζ-potential of LPP-CAT were measured by dynamic light scattering. The pH-sensitivity of LPP-CAT was determined by monitoring their degradation of hydrogen peroxide (H2O2) and their morphologies under transmission electron microscopy (TEM) at pH 7.4 and 5.5. The ability of LPP-CAT to protect MCF-7 cells against hydrogen peroxide was measured by MTS assay. ROS levels in EA.hy926 cells were measured after treatment with LPP-CAT. RESULTS: LPP-CAT were successfully prepared and carried an average diameter of <300 nm and ζ -potential of about +40 mV. At pH 5.5, LPP-CAT degraded H2O2 almost 4-fold as fast as pH 7.4 and displayed drastic morphological changes of an osmotic explosion. LPP-CAT protected MCF-7 cells from lethal level of exogenous H2O2 and significantly lowered the ROS levels in EA.hy926 cells. A lipid with a pH-sensitive conformational switch (flipid) further enhanced the protein delivery of LPP-CAT. CONCLUSION: LPP represents a promising nano-system for intracellular protein delivery.