Inhibition of NETosis by a Nuclear-Penetrating Anti-DNA Autoantibody.

Nuclear-penetrating anti-DNA autoantibodies have therapeutic potential as delivery agents and in targeting DNA and the DNA damage response (DDR). Derivatives of such Abs have advanced to human testing in genetic disease and are in preparation for oncology clinical trials. DNA release associated with neutrophil extracellular traps (NETs) contributes to immunity, inflammation, and the pathophysiology of multiple diseases. The DDR contributes to mechanisms of NETosis, and we hypothesize that anti-DNA autoantibodies that localize into live cell nuclei and inhibit DNA repair will suppress release of NETs by activated neutrophils. In the current study we evaluated the impact of a nuclear-penetrating anti-DNA autoantibody that interferes with the DDR on decondensation and release of DNA and NETs by activated human granulocyte-like differentiated PLB-985 cells and neutrophils isolated from C57BL/6 mice. The response of cells pretreated with control or autoantibody to subsequent stimulators of NETosis, including PMA and the calcium ionophore ionomycin, was evaluated by DAPI and SYTOX Green stains, measurement of DNA release, analysis of histone citrullination by Western blot, or visualization of NETs by immunostaining and confocal fluorescence microscopy. Autoantibody treatment of the cells yielded significant inhibition of NADPH oxidase-dependent and independent NETosis. These findings establish the concept of nuclear-penetrating anti-DNA autoantibodies as modulators of neutrophil biology with potential for use in strategies to suppress NETosis.

ENT2 facilitates brain endothelial cell penetration and blood-brain barrier transport by a tumor-targeting anti-DNA autoantibody.

The blood-brain barrier (BBB) prevents antibodies from penetrating the CNS and limits conventional antibody-based approaches to brain tumors. We now show that ENT2, a transporter that regulates nucleoside flux at the BBB, may offer an unexpected path to circumventing this barrier to allow targeting of brain tumors with an anti-DNA autoantibody. Deoxymab-1 (DX1) is a DNA-damaging autoantibody that localizes to tumors and is synthetically lethal to cancer cells with defects in the DNA damage response. We found that DX1 penetrated brain endothelial cells and crossed the BBB, and mechanistic studies identify ENT2 as the key transporter. In efficacy studies, DX1 crosses the BBB to suppress orthotopic glioblastoma and breast cancer brain metastases. ENT2-linked transport of autoantibodies across the BBB has potential to be exploited in brain tumor immunotherapy, and its discovery raises hypotheses on actionable mechanisms of CNS penetration by neurotoxic autoantibodies in CNS lupus.

Re-engineering and evaluation of anti-DNA autoantibody 3E10 for therapeutic applications.

A key challenge in the development of novel chemotherapeutics is the design of molecules capable of selective toxicity to cancer cells. Antibodies have greater target specificity compared to small molecule drugs, but most are unable to penetrate cells, and predominantly target extracellular antigens. A nuclear-penetrating anti-DNA autoantibody isolated from the MRL/lpr lupus mouse model, 3E10, preferentially localizes to tumors, inhibits DNA repair, and selectively kills cancer cells with defects in DNA repair. A murine divalent single chain variable fragment of 3E10 with mutations for improved DNA binding affinity, 3E10 (D31N) di-scFv, has previously been produced in P. pastoris and yielded promising pre-clinical findings, but is unsuitable for clinical testing. The present study reports the design, expression and testing of a panel of humanized 3E10 (D31N) di-scFvs, some of which contain CDR substitution. These variants were expressed in a modified CHO system and evaluated for their physicochemical attributes and ability to penetrate nuclei to selectively cause DNA damage accumulation in and kill cancer cells with DNA repair defects. Secondary structure was conserved and most variants retained the key characteristics of the murine 3E10 (D31N) di-scFv produced in P. pastoris. Moreover, several variants with CDR substitutions outperformed the murine prototype. In conclusion, we have designed several humanized variants of 3E10 (D31N) di-scFv that have potential for application as monotherapy or conjugates for targeted nuclear drug delivery.

A GRP78-Directed Monoclonal Antibody Recaptures Response in Refractory Multiple Myeloma with Extramedullary Involvement.

PURPOSE: Glucose-regulated protein (GRP) 78 is overexpressed in multiple myeloma, and both its surface expression and its biologic significance as key sensor of the unfolded protein response make GRP78 an ideal candidate for immunotherapeutic intervention. The monoclonal antibody PAT-SM6 targets surface GRP78 and leads to disease stabilization when used as single agent in a clinical trial. In this article, we evaluated expression of GRP78 in relapsed-refractory disease and explored PAT-SM6 therapy in combination regimens. EXPERIMENTAL DESIGN: GRP78 expression was immunohistochemically analyzed during disease progression and development of drug resistance throughout different stages of multiple myeloma. Activity of PAT-SM6 was evaluated in combination with anti-multiple myeloma agents lenalidomide, bortezomib, and dexamethasone in vitro Finally, we report on a multiple myeloma patient with relapsed-refractory disease treated with PAT-SM6 in combination with bortezomib and lenalidomide. RESULTS: Although sGRP78 expression was present at all stages, it increased with disease progression and was even strongly elevated in patients with drug-resistant and extramedullary disease. Pretreatment with dexamethasone as well as dual combination of PAT-SM6/lenalidomide further increased sGRP78 expression and consecutively showed synergistic anti-multiple myeloma effects with PAT-SM6 in proliferation assays. As proof of concept, a 62-year-old male with triple resistant multiple myeloma treated with PAT-SM6, bortezomib, and lenalidomide experienced partial remission of both intra- and extramedullary lesions. CONCLUSIONS: PAT-SM6 therapy in combination regimens showed efficacy in relapsed-refractory multiple myeloma. Clin Cancer Res; 22(17); 4341-9. ©2016 AACR.

GRP78-directed immunotherapy in relapsed or refractory multiple myeloma - results from a phase 1 trial with the monoclonal immunoglobulin M antibody PAT-SM6.

UNLABELLED: The primary objective of this phase 1 study was to evaluate the safety and tolerability of the anti-glucose regulated protein 78 monoclonal immunoglobulin M antibody PAT-SM6 in subjects with relapsed or refractory multiple myeloma. Twelve heavily pretreated patients received four intravenous infusions of PAT-SM6 at doses of 0.3, 1, 3, and 6 mg/kg within 2 weeks. Efficacy, pharmacokinetics and immunogenicity were followed up until the end of the trial (day 36). In addition, immune cell patterns in peripheral blood were assessed by flow cytometry and glucose regulated protein 78 expression status was evaluated in bone marrow specimens by immunohistochemistry and flow cytometry at screening. All doses administered were found to be safe and well tolerated; the maximum tolerated dose was not reached. The most common treatment emergent adverse event was leukopenia (grades 1 and 2) in eight out of the 12 multiple myeloma patients. Pharmacokinetic analysis demonstrated dose-proportional increases in drug serum concentration. The terminal half-life ranged from 5.86 to 8.41 h, the apparent volume of distribution ranged from 101 to 150 mL/kg, and clearance ranged from 8.11 to 16.1 mL/h/kg. All patients showed glucose regulated protein 78 surface expression on multiple myeloma cells. Four out of the 12 patients (33.3 %) had stable disease, according to the International Myeloma Working Group criteria, after PAT-SM6 treatment across the doses 1, 3 and 6 mg/kg. In summary, single-agent PAT-SM6 was well tolerated with modest clinical activity in relapsed or refractory multiple myeloma. Further trials exploring the combination of PAT-SM6 with existing myeloma therapies are planned. TRIAL REGISTRATION: clinicaltrials.gov identifier: NCT01727778.

Antibody-functionalized porous silicon nanoparticles for vectorization of hydrophobic drugs.

We describe the preparation of biodegradable porous silicon nanoparticles (pSiNP) functionalized with cancer cell targeting antibodies and loaded with the hydrophobic anti-cancer drug camptothecin. Orientated immobilization of the antibody on the pSiNP is achieved using novel semicarbazide based bioconjugate chemistry. To demonstrate the generality of this targeting approach, the three antibodies MLR2, mAb528 and Rituximab are used, which target neuroblastoma, glioblastoma and B lymphoma cells, respectively. Successful targeting is demonstrated by means of flow cytometry and immunocytochemistry both with cell lines and primary cells. Cell viability assays after incubation with pSiNPs show selective killing of cells expressing the receptor corresponding to the antibody attached on the pSiNP.

The microtubule depolymerizing agent CYT997 causes extensive ablation of tumor vasculature in vivo.

The orally active microtubule-disrupting agent (S)-1-ethyl-3-(2-methoxy-4-(5-methyl-4-((1-(pyridin-3-yl)butyl)amino)pyrimidin-2-yl)phenyl)urea (CYT997), reported previously by us (Bioorg Med Chem Lett 19:4639-4642, 2009; Mol Cancer Ther 8:3036-3045, 2009), is potently cytotoxic to a variety of cancer cell lines in vitro and shows antitumor activity in vivo. In addition to its cytotoxic activity, CYT997 possesses antivascular effects on tumor vasculature. To further characterize the vascular disrupting activity of CYT997 in terms of dose and temporal effects, we studied the activity of the compound on endothelial cells in vitro and on tumor blood flow in vivo by using a variety of techniques. In vitro, CYT997 is shown to potently inhibit the proliferation of vascular endothelial growth factor-stimulated human umbilical vein endothelial cells (IC(50) 3.7 ± 1.8 nM) and cause significant morphological changes at 100 nM, including membrane blebbing. Using the method of corrosion casting visualized with scanning electron microscopy, a single dose of CYT997 (7.5 mg/kg i.p.) in a metastatic cancer model was shown to cause destruction of tumor microvasculature in metastatic lesions. Furthermore, repeat dosing of CYT997 at 10 mg/kg and above (intraperitoneally, b.i.d.) was shown to effectively inhibit development of liver metastases. The time and dose dependence of the antivascular effects were studied in a DLD-1 colon adenocarcinoma xenograft model using the fluorescent dye Hoechst 33342. CYT997 demonstrated rapid and dose-dependent vascular shutdown, which persists for more than 24 h after a single oral dose. Together, the data demonstrate that CYT997 possesses potent antivascular activity and support continuing development of this promising compound.

Interaction of the exported malaria protein Pf332 with the red blood cell membrane skeleton.

Intra-erythrocytic Plasmodium falciparum malaria parasites synthesize and export numerous proteins into the red blood cell (RBC) cytosol, where some bind to the RBC membrane skeleton. These interactions are responsible for the altered antigenic, morphological and functional properties of parasite-infected red blood cells (IRBCs). Plasmodium falciparum protein 332 (Pf332) is a large parasite protein that associates with the membrane skeleton and who's function has recently been elucidated. Using recombinant fragments of Pf332 in in vitro interaction assays, we have localised the specific domain within Pf332 that binds to the RBC membrane skeleton to an 86 residue sequence proximal to the C-terminus of Pf332. We have shown that this region partakes in a specific and saturable interaction with actin (K(d)=0.60 microM) but has no detectable affinity for spectrin. The only exported malaria protein previously known to bind to actin is PfEMP3 but here we demonstrate that there is no competition for actin-binding between PfEMP3 and Pf332, suggesting that they bind to different target sequences in actin.

Plasmodium falciparum: genetic and immunogenic characterisation of the rhoptry neck protein PfRON4.

The Apicomplexan parasites Toxoplasma and Plasmodium, respectively, cause toxoplasmosis and malaria in humans and although they invade different host cells they share largely conserved invasion mechanisms. Plasmodium falciparum merozoite invasion of red blood cells results from a series of co-ordinated events that comprise attachment of the merozoite, its re-orientation, release of the contents of the invasion-related apical organelles (the rhoptries and micronemes) followed by active propulsion of the merozoite into the cell via an actin-myosin motor. During this process, a tight junction between the parasite and red blood cell plasma membranes is formed and recent studies have identified rhoptry neck proteins, including PfRON4, that are specifically associated with the tight junction during invasion. Here, we report the structure of the gene that encodes PfRON4 and its apparent limited diversity amongst geographically diverse P. falciparum isolates. We also report that PfRON4 protein sequences elicit immunogenic responses in natural human malaria infections.

Interactions of Plasmodium falciparum erythrocyte membrane protein 3 with the red blood cell membrane skeleton.

Plasmodium falciparum parasites express and traffick numerous proteins into the red blood cell (RBC), where some associate specifically with the membrane skeleton. Importantly, these interactions underlie the major alterations to the modified structural and functional properties of the parasite-infected RBC. P. falciparum Erythrocyte Membrane Protein 3 (PfEMP3) is one such parasite protein that is found in association with the membrane skeleton. Using recombinant PfEMP3 proteins in vitro, we have identified the region of PfEMP3 that binds to the RBC membrane skeleton, specifically to spectrin and actin. Kinetic studies revealed that residues 38-97 of PfEMP3 bound to purified spectrin with moderately high affinity (K(D(kin))=8.5 x 10(-8) M). Subsequent deletion mapping analysis further defined the binding domain to a 14-residue sequence (IFEIRLKRSLAQVL; K(D(kin))=3.8 x 10(-7) M). Interestingly, this same domain also bound to F-actin in a specific and saturable manner. These interactions are of physiological relevance as evidenced by the binding of this region to the membrane skeleton of inside-out RBCs and when introduced into resealed RBCs. Identification of a 14-residue region of PfEMP3 that binds to both spectrin and actin provides insight into the potential function of PfEMP3 in P. falciparum-infected RBCs.