A sequence-specific DNA binding small molecule triggers the release of immunogenic signals and phagocytosis in a model of B-cell lymphoma.

Means to cause an immunogenic cell death could lead to significant insight into how cancer escapes immune control. In this study, we screened a library of five pyrrole-imidazole polyamides coding for different DNA sequences in a model of B-cell lymphoma for the upregulation of surface calreticulin, a pro-phagocytosis signal implicated in immunogenic cell death. We found that hairpin polyamide 1 triggers the release of the damage-associated molecular patterns calreticulin, ATP and HMGB1 in a slow necrotic-type cell death. Consistent with this signaling, we observed an increase in the rate of phagocytosis by macrophages after the cancer cells were exposed to polyamide 1. The DNA sequence preference of polyamide 1 is 5'-WGGGTW-3' (where W = A/T), indicated by the pairing rules and confirmed by the Bind-n-Seq method. The close correspondence of this sequence with the telomere-repeat sequence suggests a potential mechanism of action through ligand binding at the telomere. This study reveals a chemical means to trigger an inflammatory necrotic cell death in cancer cells.

Oral microbe-host interactions: influence of ß-glucans on gene expression of inflammatory cytokines and metabolome profile.

BACKGROUND: The aim of this study was to evaluate the effects of ß-glucan on the expression of inflammatory mediators and metabolomic profile of oral cells [keratinocytes (OBA-9) and fibroblasts (HGF-1) in a dual-chamber model] infected by Aggregatibacter actinomycetemcomitans. The periodontopathogen was applied and allowed to cross the top layer of cells (OBA-9) to reach the bottom layer of cells (HGF-1) and induce the synthesis of immune factors and cytokines in the host cells. ß-glucan (10 µg/mL or 20 µg/mL) were added, and the transcriptional factors and metabolites produced were quantified in the remaining cell layers and supernatant. RESULTS: The relative expression of interleukin (IL)-1-α and IL-18 genes in HGF-1 decreased with 10 µg/mL or 20 µg/mL of ß-glucan, where as the expression of PTGS-2 decreased only with 10 µg/mL. The expression of IL-1-α increased with 20 µg/mL and that of IL-18 increased with 10 µg/mL in OBA-9; the expression of BCL 2, EP 300, and PTGS-2 decreased with the higher dose of ß-glucan. The production of the metabolite 4-aminobutyric acid presented lower concentrations under 20 µg/mL, whereas the concentrations of 2-deoxytetronic acid NIST and oxalic acid decreased at both concentrations used. Acetophenone, benzoic acid, and pinitol presented reduced concentrations only when treated with 10 µg/mL of ß-glucan. CONCLUSIONS: Treatment with ß-glucans positively modulated the immune response and production of metabolites.

Controlled Multi-functionalization Facilitates Targeted Delivery of Nanoparticles to Cancer Cells.

A major objective of nanomedicine is to combine in a controlled manner multiple functional entities into a single nanoscale device to target particles with great spatial precision, thereby increasing the selectivity and potency of therapeutic drugs. A multifunctional nanoparticle is described for controlled conjugation of a cytotoxic drug, a cancer cell targeting ligand, and an imaging moiety. The approach is based on the chemical synthesis of polyethylene glycol that at one end is modified by a thioctic acid for controlled attachment to a gold core. The other end of the PEG polymers is modified by a hydrazine, amine, or dibenzocyclooctynol moiety for conjugation with functional entities having a ketone, activated ester, or azide moiety, respectively. The conjugation approach allowed the controlled attachment of doxorubicin through an acid-labile hydrazone linkage, an Alexa Fluor dye through an amide bond, and a glycan-based ligand for the cell surface receptor CD22 of B-cells using strain promoted azide-alkyne cycloaddition. The incorporation of the ligand for CD22 led to rapid entry of the nanoparticle by receptor-mediated endocytosis. Covalent attachment of doxorubicin via hydrazone linkage caused pH-responsive intracellular release of doxorubicin and significantly enhanced the cytotoxicity of nanoparticles. A remarkable 60-fold enhancement in cytotoxicity of CD22 (+) lymphoma cells was observed compared to non- targeted nanoparticles.

Intracellular delivery system for antibody-Peptide drug conjugates.

Antibodies armed with biologic drugs could greatly expand the therapeutic potential of antibody-drug conjugates for cancer therapy, broadening their application to disease targets currently limited by intracellular delivery barriers. Additional selectivity and new therapeutic approaches could be realized with intracellular protein drugs that more specifically target dysregulated pathways in hematologic cancers and other malignancies. A multifunctional polymeric delivery system for enhanced cytosolic delivery of protein drugs has been developed that incorporates endosomal-releasing activity, antibody targeting, and a biocompatible long-chain ethylene glycol component for optimized safety, pharmacokinetics, and tumor biodistribution. The pH-responsive polymeric micelle carrier, with an internalizing anti-CD22 monoclonal targeting antibody, effectively delivered a proapoptotic Bcl-2 interacting mediator (BIM) peptide drug that suppressed tumor growth for the duration of treatment and prolonged survival in a xenograft mouse model of human B-cell lymphoma. Antitumor drug activity was correlated with a mechanistic induction of the Bcl-2 pathway biomarker cleaved caspase-3 and a marked decrease in the Ki-67 proliferation biomarker. Broadening the intracellular target space by more effective delivery of protein/peptide drugs could expand the repertoire of antibody-drug conjugates to currently undruggable disease-specific targets and permit tailored drug strategies to stratified subpopulations and personalized medicines.

Expression and purification of full-length anti-apoptotic Bcl-2 using cell-free protein synthesis.

The anti-apoptotic B cell CLL/lymphoma-2 (Bcl-2) protein is a key player in the regulation of programmed cell death and is linked to various types of cancer and their resistance to drug treatment. Biophysical and structural studies of the full-length intact Bcl-2 have been hampered due to difficulties in expression and severe solubility problems, precluding isolation of this hydrophobic membrane protein. Therefore, previous work has so far mainly been carried out using structurally modified Bcl-2 variants, lacking the transmembrane region. Thus, biophysical information regarding the full-length protein is still missing. Here, a protocol is presented for expression and purification of preparative amounts of the full-length human isoform 2 of Bcl-2 (Bcl-2(2)). A batch-based cell-free expression system, using extract isolated from Escherichia coli (E. coli) was employed to produce recombinant protein encoded by an optimized gene sequence. Presence of polyoxyethylene-(20)-cetyl-ether (Brij-58) in the reaction mixture and subsequently in the immobilized metal-affinity purification steps was crucial to keep Bcl-2(2) soluble. The obtained yield was 0.25-0.3mg per ml of cell-free reaction. Far-UV circular dichroism (CD) spectroscopy confirmed the α-helical structure of the purified protein, characteristic for members of the Bcl-2 protein family.

Identification of a moderate affinity CD22 binding peptide and in vitro optimization of peptide-targeted nanoparticles for selective uptake by CD22+ B-cell malignancies.

B cell malignancies, such as B cell leukemia and lymphoma, have CD22 overexpression with ∼7% of patients. A short CD22 binding peptide (PV3) with a moderate affinity (Kd ∼ 9 µM) was identified by screening multiple peptide candidates determined through analysis of CD22-epratuzumab complex crystal structure. PV3 binding specificity was confirmed via competitive binding inhibition, then was used as the targeting moiety on CD22-targeted liposomal nanoparticle (TNPPV3) formulations. To maximize the potential therapeutic outcome of TNPPV3 formulation, nanoparticle design parameters, such as peptide hydrophilicity, ethylene glycol linker length, valency, and particle size were optimized for maximum selective cellular uptake by CD22+ malignant cancer cells. The effects of altering design parameters one at a time on TNP uptake were evaluated using flow cytometry, and the optimal parameters for TNPPV3 were determined to be 8% peptide density, EG18 linker, and 3 lysines of 100 nm nanoparticles. This optimally designed TNPPV3 achieved ∼4 and 40-fold enhancement of cellular uptake by CD22+ Raji cells over CD22- Jurkat and MOLT-4 cells, respectively, demonstrating selectivity for malignant cells with CD22 overexpression. Overall, this study establishes PV3 to be CD22 binding peptide with proven effectiveness as a targeting element. In future, the optimal TNPPV3 formulation will potentially achieve maximal in vivo therapeutic outcomes by efficiently targeting CD22+ blood cancer cells in vivo.

Different proteomic protein patterns in saliva of Sjögren's syndrome patients.

OBJECTIVE: To investigate the salivary protein profile in patients with Sjögren's syndrome (SS), and healthy control subjects. MATERIALS AND METHODS: Unstimulated whole saliva samples were collected from 16 age-matched females; eight healthy subjects and eight patients diagnosed with SS (six primary SS, one incomplete SS and one primary SS associated with B cell lymphoma). Proteins were extracted and separated individually by 2D sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Selected protein spots of interest were analysed by electrospray ionization--tandem mass spectrometry. Obtained data were searched against the Swiss-Prot and NCBI non-redundant protein databases using Mascot software. RESULTS: Two groups of patterns of protein expression were observed in the eight SS patients: a major group (six patients) with significant expression differences from the healthy subjects and the second group (two patients) with a pattern similar to the eight healthy subjects. CONCLUSION: In this preliminary study, protein expression differences were found between SS patients and healthy subjects. Individual analysis of SS patients exhibited two patterns of protein expression with no direct relation to the clinical, serological or histological severity of disease. This study emphasizes the difficulty of the present proteomic knowledge to diagnose and monitor the sequel of SS development.

Prophylactic anti-tumor effects in a B cell lymphoma model with DNA vaccines delivered on polyethylenimine (PEI) functionalized PLGA microparticles.

Idiotypic sequences, specific to the hypervariable regions of immunoglobulins expressed by malignant B cells offer a therapeutic target in B cell lymphoma. Efficient approaches have been described to clone a single chain fragment of the tumor immunoglobulin (Ig) comprising of heavy and light Ig chains (sFv) fused with proinflammatory chemokines. Tumor associated, poorly immunogenic self antigens encoded by plasmid DNA (pDNA) have been rendered immunogenic by chemokine fusion, thereby targeting to antigen presenting cells (APCs) which differentially express chemokine receptors. Here we present an injectable (parenteral) approach using synthetic polymer based cationic microparticle formulations for enhancing the potency of such chemokine/self antigen expressing plasmid construct. Branched and linear polyethyleneimine (PEI) were conjugated on poly (D, L lactide-co-glycolide) (PLGA) microparticles using carbodiimide chemistry followed by efficient loading of plasmid DNA. In addition to imparting significant buffering ability to these cationic microparticles, flow cytometry studies indicate that these DNA loaded microparticles significantly up regulate CD80 and MHC class II markers in phagocytic RAW264.7 cells, indicating intrinsic adjuvant effects. Intradermal injections in Balb/c mice with these formulations induced significant protection upon tumor challenge with 2.5 times the minimal lethal dose. Long term survival rates were significant (p < 0.05) in comparison with saline injected controls or blank microparticles. Further studies indicated that intramuscular delivery might provide better protection compared to intradermal injections and perform similar to gene gun mediated administration. We conclude, based on these promising in vivo results, that such surface-functionalized microparticles offer an attractive strategy to improve the potency of self antigen-based cancer DNA vaccines.

Internalizing antibodies are necessary for improved therapeutic efficacy of antibody-targeted liposomal drugs.

Direct experimental proof has been sought for the hypothesis that liposomal drugs targeted against internalizing epitopes (e.g., CD19) will have higher therapeutic efficacies than those targeted against noninternalizing epitopes (e.g., CD20). Anti-CD19-targeted liposomes were rapidly internalized into human B-lymphoma (Namalwa) cells, whereas those targeted with anti-CD20 were not internalized. Similar in vitro binding and cytotoxicity were observed for anti-CD19-targeted and anti-CD20-targeted liposomal formulations of doxorubicin (DXR). Therapeutic experiments were performed in severe combined immunodeficient mice inoculated i.v. with Namalwa cells. Administration of single i.v. doses of DXR-loaded anti-CD19-targeted liposomes resulted in significantly greater survival times than either DXR-loaded anti-CD20-targeted liposomes or nontargeted liposomes. The therapeutic advantage of targeting internalizing versus noninternalizing epitopes has been directly demonstrated.

In vitro and in vivo targeting of immunoliposomal doxorubicin to human B-cell lymphoma.

The ability to selectively target liposomal anticancer drugs via specific ligands against antigens expressed on malignant cells could improve the therapeutic effectiveness of the liposomal preparations as well as reduce adverse side effects associated with chemotherapy. Long-circulating formulations of liposomes containing lipid derivatives of poly(ethyleneglycol) [sterically stabilized liposomes (SLs)] have been described previously, and new techniques have recently been developed for coupling monoclonal antibodies (Abs) at the poly(ethyleneglycol) terminus of these liposomes. Ab-targeted SLs [immunoliposomes (SILs)] containing entrapped anticancer drugs are predicted to be useful in the treatment of hematological malignancies such as B-cell lymphomas or multiple myeloma, in which the target cells are present in the vasculature. The specific binding, in vitro cytotoxicity, and in vivo antineoplastic activity of doxorubicin (DXR) encapsulated in SILs coupled to monoclonal Ab anti-CD19 (SIL[anti-CD19]) were investigated against malignant B cells expressing CD19 surface antigens. Binding experiments with SIL[anti-CD19] resulted in a 3-fold higher association of the SILs with a human CD19+ B lymphoma cell line (Namalwa) in comparison with nontargeted SLs. Using flow cytometry, fluorescently labeled SIL[anti-CD19] bound to B cells with no recognition of T cells in a mixture of B cells and T cells in culture. Nontargeted SLs demonstrated significantly lower recognition of either B cells or T cells. Targeted DXR-SIL[anti-CD19] displayed a higher cytotoxicity to B cells relative to DXR entrapped in nontargeted SLs. Therapeutic experiments in severe combined immunodeficient mice implanted with Namalwa cells by the i.v. or i.p. routes resulted in significantly increased effectiveness of DXR-SIL[anti-CD19] compared to similar amounts of free DXR, DXR-SL (no Ab), or isotype-matched nonspecific Abs attached to DXR-SL. Single doses (3 mg/kg) of DXR-SIL[anti-CD19] administered i.v. resulted in a significantly improved therapeutic benefit, including some long-term survivors. From our results, we infer that targeted anti-CD19 liposomes containing the anticancer drug DXR may be selectively cytotoxic for B cells and may be useful in the selective elimination of circulating malignant B cells in vivo.

Integrin-specific hydrogels as adaptable tumor organoids for malignant B and T cells.

Non-Hodgkin lymphomas are a heterogeneous group of lymphoproliferative disorders of B and T cell origin that are treated with chemotherapy drugs with variable success rate that has virtually not changed over decades. Although new classes of chemotherapy-free epigenetic and metabolic drugs have emerged, durable responses to these conventional and new therapies are achieved in a fraction of cancer patients, with many individuals experiencing resistance to the drugs. The paucity in our understanding of what regulates the drug resistance phenotype and establishing a predictive indicator is, in great part, due to the lack of adequate ex vivo lymphoma models to accurately study the effect of microenvironmental cues in which malignant B and T cell lymphoma cells arise and reside. Unlike many other tumors, lymphomas have been neglected from biomaterials-based microenvironment engineering standpoint. In this study, we demonstrate that B and T cell lymphomas have different pro-survival integrin signaling requirements (αvß3 and α4ß1) and the presence of supporting follicular dendritic cells are critical for enhanced proliferation in three-dimensional (3D) microenvironments. We engineered adaptable 3D tumor organoids presenting adhesive peptides with distinct integrin specificities to B and T cell lymphoma cells that resulted in enhanced proliferation, clustering, and drug resistance to the chemotherapeutics and a new class of histone deacetylase inhibitor (HDACi), Panobinostat. In Diffuse Large B cell Lymphomas, the 3D microenvironment upregulated the expression level of B cell receptor (BCR), which supported the survival of B cell lymphomas through a tyrosine kinase Syk in the upstream BCR pathway. Our integrin specific ligand functionalized 3D organoids mimic a lymphoid neoplasm-like heterogeneous microenvironment that could, in the long term, change the understanding of the initiation and progression of hematological tumors, allow primary biospecimen analysis, provide prognostic values, and importantly, allow a faster and more rational screening and translation of therapeutic regimens.

Specific binding of sterically stabilized anti-B-cell immunoliposomes and cytotoxicity of entrapped doxorubicin.

Administration of doxorubicin (DXR) formulated in sterically stabilized liposomes, (SL) containing engrafted poly(ethylene glycol)-modified phosphatidylethanolamine (PEG-PE) on their surface, has been shown to increase the therapeutic index of the drug. A further improvement could be achieved through targeting of liposome-entrapped drug selectively to cancer cells. This paper describes the conjugation of the anti-B-cell lymphoma monoclonal antibody LL2 to the surface of DXR-loaded liposomes by use of a PEG-based heterobifunctional coupling agent. Competitive-binding ELISA of the resulting immunoliposomes (SIL) against the monoclonal anti-idiotype antibody, WN, indicated preserved immunological activity. The pH-sensitive probe, HPTS was used to study the binding of liposomes with target cells. The results showed a 3.8-fold increased cellular association of SIL compared to that of SL and an apparent internalization of SIL into low pH compartments. Addition of an excess of unconjugated free LL2 displaced about 72% of the HPTS-SIL association with cells. Experiments with 125I-labeled free and SIL-bound LL2 showed approximately 50% degradation for both preparations. In vitro MTT cytotoxicity tests against neoplastic B cells gave IC(50) values of 1.6, 2.9 and 0.35 microM for DXR-SIL, DXR-SL and free DXR, respectively. Leakage of drug from the liposomes apparently reduced the specificity of the cytotoxic action of DXR-SIL.

Milatuzumab-conjugated liposomes as targeted dexamethasone carriers for therapeutic delivery in CD74+ B-cell malignancies.

PURPOSE: Corticosteroids are widely used for the treatment of B-cell malignancies, including non-Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), and acute lymphoblastic leukemia; however, this class of drug is associated with undesirable off-target effects. Herein, we developed novel milatuzumab-conjugated liposomes as a targeted dexamethasone carrier for therapeutic delivery in CD74(+) B-cell malignancies and explored its effect against the disease. EXPERIMENTAL DESIGN: The targeting efficiency of milatuzumab-targeted liposomes to CD74(+) cells was evaluated in vitro. The effect of CD74-targeted liposomal dexamethasone was compared with free dexamethasone in primary CLL cells and cell lines in vitro. The therapeutic efficacy of CD74-targeted liposomal dexamethasone was evaluated in a Raji-severe combined immunodeficient (SCID) xenograft model in vivo. RESULTS: Milatuzumab-targeted liposomes promoted selective incorporation of carrier molecules into transformed CD74-positive B cells as compared with CD74-negative T-cells. The CD74-dexamethasone-targeted liposomes (CD74-IL-DEX) promoted and increased killing in CD74-positive tumor cells and primary CLL cells. Furthermore, the targeted drug liposomes showed enhanced therapeutic efficacy against a CD74-positive B-cell model as compared with free, or non-targeted, liposomal dexamethasone in SCID mice engrafted with Raji cells in vivo. CONCLUSIONS: These studies provide evidence and support for a potential use of CD74-targeted liposomal dexamethasone as a new therapy for B-cell malignancies.

Evaluation of a B-cell leukemia-lymphoma 2-specific radiolabeled peptide nucleic acid-peptide conjugate for scintigraphic detection of neoplastic lymphocytes in dogs with B-cell lymphoma.

OBJECTIVE: To evaluate use of a radiolabeled peptide nucleic acid-peptide conjugate (RaPP) targeting B-cell leukemia-lymphoma 2 (BCL2) mRNA for scintigraphic detection of neoplastic lymphocytes in dogs with B-cell lymphoma and to assess associations among RaPP uptake, time to tumor progression (TTP), and BCL2 mRNA expression. ANIMALS: 11 dogs with B-cell lymphoma and 1 clinically normal dog. PROCEDURES: Scintigraphic images were acquired 1 hour after IV injection of the RaPP. Regions of interest (ROIs) were drawn around lymph nodes, liver, and spleen; ROI intensity (relative to that of an equally sized region of muscle in the same image) was measured. Each ROI was also subjectively categorized as positive or negative for increased RaPP uptake. Expression of BCL2 mRNA was determined via quantitative reverse transcriptase PCR assay of a lymph node sample from dogs with lymphoma. Associations among imaging results, TTP, and BCL2 mRNA expression were evaluated. RESULTS: Increased RaPP uptake was detected in affected tissues of dogs with lymphoma. Dogs with superficial cervical lymph node ROIs categorized as negative (n = 8) for increased RaPP uptake had a significantly longer TTP than did dogs for which this ROI was considered positive (2). Measured intensity of mandibular and superficial cervical lymph node ROIs was negatively associated with TTP. Associations among BCL2 mRNA and ROI intensity or TTP were not significant. CONCLUSIONS AND CLINICAL RELEVANCE: Increased RaPP uptake at mandibular or superficial cervical lymph node ROIs may be a negative prognostic indicator in dogs with lymphoma. A larger investigation is needed to determine clinical value of the RaPP for disease detection and prognostication.