Targeting GD2-Positive Tumor Cells by Pegylated scFv Fragment-Drug Conjugates Carrying Maytansinoids DM1 and DM4.

Oligomerization of antibody fragments via modification with polyethylene glycol (pegylation) may alter their function and properties, leading to a multivalent interaction of the resulting constructs with the target antigen. In a recent study, we generated pegylated monomers and multimers of scFv fragments of GD2-specific antibodies using maleimide-thiol chemistry. Multimerization enhanced the antigen-binding properties and demonstrated a more efficient tumor uptake in a syngeneic GD2-positive mouse cancer model compared to monomeric antibody fragments, thereby providing a rationale for improving the therapeutic characteristics of GD2-specific antibody fragments. In this work, we obtained pegylated conjugates of scFv fragments of GD2-specific antibodies with maytansinoids DM1 or DM4 using tetravalent PEG-maleimide (PEG4). The protein products from the two-stage thiol-maleimide reaction resolved by gel electrophoresis indicated that pegylated scFv fragments constituted the predominant part of the protein bands, and most of the scFv formed pegylated monomers and dimers. The conjugates retained the ability to bind ganglioside GD2 comparable to that of the parental scFv fragment and to specifically interact with GD2-positive cells. Both induced significant inhibitory effects in the GD2-positive B78-D14 cell line, in contrast to the GD2-negative B16 cell line. The decrease in the B78-D14 cell viability when treated with scFv-PEG4-DM4 was more prominent than that for scFv-PEG4-DM1, and was characterized by a twofold lower half-maximal inhibitory concentration (IC50). Unlike the parental scFv fragment, the product of scFv and PEG4 conjugation (scFv-PEG4), consisting predominantly of pegylated scFv multimers and monomers, induced direct cell death in the GD2-positive B78-D14 cells. However, the potency of scFv-PEG4 was low in the selected concentration range, thus demonstrating that the cytotoxic effect of DM1 and DM4 within the antibody fragment-drug conjugates was primary. The suggested approach may contribute to development of novel configurations of antibody fragment-drug conjugates for cancer treatment.

Design of an aryne-platform for the synthesis of non-racemic heterocyclic allocolchicinoids.

A four-step semisynthetic approach towards a highly versatile allocolchicine-related chiral aryne intermediate starting from naturally occurring colchicine was developed, and some of its synthetic transformations were studied. The in situ generated benzyne intermediate afforded a number of non-racemic heterocyclic allocolchicinoids, which were shown to exhibit potent cytotoxicity towards COLO 357, OSA and Raji cells. The proposed methodology is attractive for the synthesis of libraries of new cytotoxic tubulin inhibitors.

Design and preparation of pH-sensitive cytotoxic liposomal formulations containing antitumor colchicine analogues for target release.

Herein, we describe the synthesis of pH-sensitive lipophilic colchicine prodrugs for liposomal bilayer inclusion, as well as preparation and characterization of presumably stealth PEGylated liposomes with above-mentioned prodrugs. These formulations liberate strongly cytotoxic colchicinoid derivatives selectively under slightly acidic tumor-associated conditions, ensuring tumor-targeted delivery of the compounds. The design of the prodrugs is addressed to pH-triggered release of active compounds in the slight acidic media, that corresponds to tumor microenvironment, while keeping sufficient stability of the whole formulation at physiological pH. Correlations between the structure of the conjugates, their hydrolytic stability, colloidal stability, ability of the prodrug retention in the lipid bilayer are described. Several formulations were found promising for further development and in vivo investigations.

Minibody-Based and scFv-Based Antibody Fragment-Drug Conjugates Selectively Eliminate GD2-Positive Tumor Cells.

Ganglioside GD2 is a well-established target expressed on multiple solid tumors, many of which are characterized by low treatment efficiency. Antibody-drug conjugates (ADCs) have demonstrated marked success in a number of solid tumors, and GD2-directed drug conjugates may also hold strong therapeutic potential. In a recent study, we showed that ADCs based on the approved antibody dinutuximab and the drugs monomethyl auristatin E (MMAE) or F (MMAF) manifested potent and selective cytotoxicity in a panel of tumor cell lines and strongly inhibited solid tumor growth in GD2-positive mouse cancer models. Here, we employed two different GD2-binding moieties-minibodies and scFv fragments that carry variable antibody domains identical to those of dinutuximab, and site-directly conjugated them to MMAE or MMAF by thiol-maleimide chemistry with drug-to-antibody ratios (DAR) of 2 and 1, respectively. Specific binding of the antibody fragment-drug conjugates (FDCs) to GD2 was confirmed in direct ELISA, flow cytometry, and confocal microscopy. Selective cytotoxic and cytostatic effects of the conjugates were observed in GD2-positive but not GD2-negative neuroblastoma and melanoma cell lines. Minibody-based FDCs demonstrated more pronounced cytotoxic effects and stronger antigen binding compared to scFv-based FDCs. The developed molecules may offer considerable practical benefit, since antibody fragment-drug conjugates are capable of enhancing therapeutic efficacy of ADCs by improving their pharmacokinetic characteristics and reducing side effects.

Chemokine Homeostasis in Healthy Volunteers and during Pancreatic and Colorectal Tumor Growth in Murine Models.

Chemokines are involved in the humoral regulation of body homeostasis. Changes in the blood level of chemokines were found in cancer, atherosclerosis, diabetes, and other systemic diseases. It is essential to distinguish the effects of co-morbid pathologies and cancer on the level of chemokines in the blood. We aimed to analyze, by multiplex cytometry, the levels of chemokines in the blood of healthy young volunteers as well as of intact mice and mice with CT26 colon and Pan02 pancreatic tumors. Two types of chemokines were identified both in human and murine plasmas: homeostatic ones, which were found in high concentrations (>100 pg/mL), and inducible ones, which can be undetectable or determined at very low levels (0−100 pg/mL). There was a high variability in the chemokine levels, both in healthy humans and mice. To analyze chemokine levels during tumor growth, C57BL/6 and BALB/c were inoculated with Pan02 or CT26 tumor cells, accordingly. The tumors significantly differed in the growth and the mortality of mice. However, the blood chemokine levels did not change in tumor-bearing mice until the very late stages. Taken collectively, blood chemokine level is highly variable and reflects in situ homeostasis. Care should be taken when considering chemokines as prognostic parameters or therapeutic targets in cancer.

Resistance of Human Liver Mesenchymal Stem Cells to FAS-Induced Cell Death.

Mesenchymal stem cells (MSCs) have a pronounced therapeutic potential in various pathological conditions. Though therapeutic effects of MSC transplantation have been studied for a long time, the underlying mechanisms are still not clear. It has been shown that transplanted MSCs are rapidly eliminated, presumably by apoptosis. As the mechanisms of MSC apoptosis are not fully understood, in the present work we analyzed MSC sensitivity to Fas-induced apoptosis using MSCs isolated from the biopsies of liver fibrosis patients (L-MSCs). The level of cell death was analyzed by flow cytometry in the propidium iodide test. The luminescent ATP assay was used to measure cellular ATP levels; and the mitochondrial membrane potential was assessed using the potential-dependent dye JC-1. We found that human L-MSCs were resistant to Fas-induced cell death over a wide range of FasL and anti-Fas mAb concentrations. At the same time, intrinsic death signal inducers CoCl2 and staurosporine caused apoptosis of L-MSCs in a dose-dependent manner. Despite the absence of Fas-induced cell death treatment of L-MSCs with low concentrations of FasL or anti-Fas mAb resulted in a cellular ATP level decrease, while high concentrations of the inducers caused a decline of the mitochondrial membrane potential. Pre-incubation of L-MSCs with the pro-inflammatory cytokine TNF-α did not promote L-MSC cell death. Our data indicate that human L-MSCs have increased resistance to receptor-mediated cell death even under inflammatory conditions.

Silicon-Gold Nanoparticles Affect Wharton's Jelly Phenotype and Secretome during Tri-Lineage Differentiation.

Multiple studies have demonstrated that various nanoparticles (NPs) stimulate osteogenic differentiation of mesenchymal stem cells (MSCs) and inhibit adipogenic ones. The mechanisms of these effects are not determined. The aim of this paper was to estimate Wharton's Jelly MSCs phenotype and humoral factor production during tri-lineage differentiation per se and in the presence of silicon-gold NPs. Silicon (SiNPs), gold (AuNPs), and 10% Au-doped Si nanoparticles (SiAuNPs) were synthesized by laser ablation, characterized, and studied in MSC cultures before and during differentiation. Humoral factor production (n = 41) was analyzed by Luminex technology. NPs were nontoxic, did not induce ROS production, and stimulated G-CSF, GM-CSF, VEGF, CXCL1 (GRO) production in four day MSC cultures. During MSC differentiation, all NPs stimulated CD13 and CD90 expression in osteogenic cultures. MSC differentiation resulted in a decrease in multiple humoral factor production to day 14 of incubation. NPs did not significantly affect the production in chondrogenic cultures and stimulated it in both osteogenic and adipogenic ones. The major difference in the protein production between osteogenic and adipogenic MSC cultures in the presence of NPs was VEGF level, which was unaffected in osteogenic cells and 4-9 times increased in adipogenic ones. The effects of NPs decreased in a row AuNPs > SiAuNPs > SiNPs. Taken collectively, high expression of CD13 and CD90 by MSCs and critical level of VEGF production can, at least, partially explain the stimulatory effect of NPs on MSC osteogenic differentiation.

Synthesis, Molecular Docking, In Vitro and In Vivo Studies of Novel Dimorpholinoquinazoline-Based Potential Inhibitors of PI3K/Akt/mTOR Pathway.

A (series) range of potential dimorpholinoquinazoline-based inhibitors of the PI3K/Akt/mTOR cascade was synthesized. Several compounds exhibited cytotoxicity towards a panel of cancer cell lines in the low and sub-micromolar range. Compound 7c with the highest activity and moderate selectivity towards MCF7 cells which express the mutant type of PI3K was also tested for the ability to inhibit PI3K-(signaling pathway) downstream effectors and associated proteins. Compound 7c inhibited the phosphorylation of Akt, mTOR, and S6K at 125-250 nM. It also triggered PARP1 cleavage, ROS production, and cell death via several mechanisms. Inhibition of PI3Kα was observed at a concentration of 7b 50 µM and of 7c 500 µM and higher, that can indicate minority PI3Kα as a target among other kinases in the titled cascade for 7c. In vivo studies demonstrated an inhibition of tumor growth in the colorectal tumor model. According to the docking studies, the replacement of the triazine core in gedatolisib (8) by a quinazoline fragment, and incorporation of a (hetero)aromatic unit connected with the carbamide group via a flexible spacer, can result in more selective inhibition of the PI3Kα isoform.

Gausemycins†A,B: Cyclic Lipoglycopeptides from Streptomyces sp.*.

We report a novel family of natural lipoglycopeptides produced by Streptomyces sp. INA-Ac-5812. Two major components of the mixture, named gausemycins A and B, were isolated, and their structures were elucidated. The compounds are cyclic peptides with a unique peptide core and several remarkable structural features, including unusual positions of d-amino acids, lack of the Ca2+ -binding Asp-X-Asp-Gly (DXDG) motif, tyrosine glycosylation with arabinose, presence of 2-amino-4-hydroxy-4-phenylbutyric acid (Ahpb) and chlorinated kynurenine (ClKyn), and N-acylation of the ornithine side chain. Gausemycins have pronounced activity against Gram-positive bacteria. Mechanistic studies highlight significant differences compared to known glyco- and lipopeptides. Gausemycins exhibit only slight Ca2+ -dependence of activity and induce no pore formation at low concentrations. Moreover, there is no detectable accumulation of cell wall biosynthesis precursors under treatment with gausemycins.

Phospholipidic Colchicinoids as Promising Prodrugs Incorporated into Enzyme-Responsive Liposomes: Chemical, Biophysical, and Enzymological Aspects.

Enzyme-responsive liposomes release their cargo in response to pathologically increased levels of enzymes at the target site. We report herein an assembly of phospholipase A2-responsive liposomes based on colchicinoid lipid prodrugs incorporated into lipid bilayer of the nanosized vesicles. The liposomes were constructed to addresses two important issues: (i) the lipid prodrugs were designed to fit the structure of the enzyme binding site; and (ii) the concept of lateral pressure profile was used to design lipid prodrugs that introduce almost no distortions into the lipid bilayer packing, thus ensuring that corresponding liposomes are stable. The colchicinoid agents exhibit antiproliferative activity in subnanomolar range of concentrations.

Recognition of arylmethylidene derivatives of imidazothiazolotriazinones as novel tubulin polymerization inhibitors.

Two series of arylmethylidene derivatives of imidazothiazolotriazinone differing in the structure of the imidazothiazolotriazine fragment were synthesized and their antiproliferative activity and effect on tubulin polymerization were evaluated. Some of the synthesized derivatives showed a significant antiproliferative effect, among which (Z)-7-(2,4-dichlorobenzylidene)-1,3-diethyl-1,3a,4,9a-tetrahydroimidazo[4,5-e]thiazolo[2,3-c][1,2,4]triazine-2,8(3H,7H)-dione 2n exhibited the highest antiproliferative activity. The GI50 values of the compound against 56 of the 58 cell lines were 19.4-87.8 nM; against the remaining 2 cell lines, they were 0.544-1.29 µM. Moreover, further mechanism analysis demonstrated that 2n caused G2/M arrest, induced cell apoptosis in K562 cells and blocked tubulin polymerization in the same way as colchicine.

Targeted Delivery of HSP70 to Tumor Cells via Supramolecular Complex Based on HER2-Specific DARPin9_29 and the Barnase:Barstar Pair.

(1) Background: We have previously shown that the use of an artificial supramolecular two-component system based on chimeric recombinant proteins 4D5scFv-barnase and barstar-heat shock protein 70 KDa (HSP70) allows targeted delivery of HSP70 to the surface of tumor cells bearing HER2/neu antigen. In this work, we studied the possibility to using DARPin9_29-barnase as the first targeting module recognizing HER2/neu-antigen in the HSP70 delivery system. (2) Methods: The effect of the developed systems for HSP70 delivery to human carcinomas SK-BR-3 and BT474 cells hyperexpressing HER2/neu on the activation of cytotoxic effectors of the immune cells was studied in vitro. (3) Results: The results obtained by confocal microscopy and cytofluorimetric analysis confirmed the binding of HSP70 or its fragment HSP70-16 on the surface of the treated cells. In response to the delivery of HSP70 to tumor cells, we observed an increase in the cytolytic activity of different cytotoxic effector immune cells from human peripheral blood. (4) Conclusions: Targeted modification of the tumor cell surface with molecular structures recognized by cytotoxic effectors of the immune system is among new promising approaches to antitumor immunotherapy.

Design, Synthesis and In Vitro Biological Activity of Novel C-7 Methylene Congeners of Furanoallocolchicinoids.

A series of novel heterocyclic colchicine derivatives bearing a C-7 methylene fragment were synthesized via Wittig, Horner-Wadsworth-Emmons and Nenajdenko-Shastin olefination approaches. The in vitro biological activities of the most promising compounds were investigated using MTT assays and cell cycle analyses. Compounds with an electron withdrawing group on the methylene fragment exhibited substantial antiproliferative activity towards COLO-357, BxPC-3, HaCaT, PANC-1 and A549 cell lines. The spatial orientation of the substituent at the double bond significantly influenced its biological activity.

Polyvinylpyrrolidone-Alginate Film Barriers for Abdominal Surgery: Anti-Adhesion Effect in Murine Model.

Surgical operations on the peritoneum are often associated with the formation of adhesions, which can interfere with the normal functioning of the internal organs. The effectiveness of existing barrier materials is relatively low. In this work, the effectiveness of soluble alginate-polyvinylpyrrolidone (PVP-Alg) and non-soluble Ca ion cross-linked (PVP-Alg-Ca) films in preventing these adhesions was evaluated. Experiments in vivo were performed on mice via mechanical injury to the adjacent peritoneum wall and the caecum, followed by the application of PVP-Alg or PVP-Alg-Ca films to the injured area. After 7 days, samples from the peritoneal wall and caecum were analyzed using histology and quantitative polymerase chain reaction (qPCR). It was shown that the expression of genes responsible for adhesion formation in the caecum in the PVP-Alg group was comparable to that in the control group, while in the PVP-Alg-Ca group, it increased by 5-10 times. These results were consistent with the histology: in the PVP-Alg group, the adhesions did not form, while in the PVP-Alg-Ca group, the adhesions corresponded to five points on the adhesion scale. Therefore, the formation of intraperitoneal adhesions can be effectively prevented by non-crosslinked, biodegradable PVP-Alg films, whereas cross-linked, not biodegradable PVP-Alg-Ca films cause inflammation and adhesion formation.

Probing tricarbocyanine dyes for targeted delivery of anthracyclines.

Along with bright fluorescence in the near-IR range, heptamethine carbocyanine dyes possess affinity to cancer cells. Thus, these dyes could be utilized as fluorescent labels and vectors for drug delivery as covalent conjugates with cytotoxic compounds. To test the properties, structure-activity relationship, and scope of such conjugates, we synthesized drug-dye dyads of tricarbocyanine dyes with anthracycline drug daunorubicin. We used hydrophilic zwitterionic and hydrophobic positively charged benzoindoline-benzothiazole-based heptamethine dyes as terminal alkyne derivatives and N-acylated or oxime-linked daunorubicin as azido-derivatives. These two alkynes and two azides were coupled to each other by Cu-catalyzed Huisgen-Meldal-Sharpless cycloaddition (click reaction) to afford four conjugates. Molecules based on hydrophobic dyes possess submicromolar cytotoxicity to HCT116 cells. Cytotoxicity, cell penetration, intracellular distribution, apoptosis induction and the effect of antioxidants on toxicity were evaluated. The results show that the structure of the cyanine-anthracycline conjugate (hydrophilicity/hydrophobicity, charge, linker, attachment site) is important for its biological activity, thus, expansion of the chemical space of such conjugates could provide new molecular research tools for diagnostics and therapy.

Asymmetric depth-filtration: A versatile and scalable method for high-yield isolation of extracellular vesicles with low contamination.

We developed a novel asymmetric depth filtration (DF) approach to isolate extracellular vesicles (EVs) from biological fluids that outperforms ultracentrifugation and size-exclusion chromatography in purity and yield of isolated EVs. By these metrics, a single-step DF matches or exceeds the performance of multistep protocols with dedicated purification procedures in the isolation of plasma EVs. We demonstrate the selective transit and capture of biological nanoparticles in asymmetric pores by size and elasticity, low surface binding to the filtration medium, and the ability to cleanse EVs held by the filter before their recovery with the reversed flow all contribute to the achieved purity and yield of preparations. We further demonstrate the method's versatility by applying it to isolate EVs from different biofluids (plasma, urine, and cell culture growth medium). The DF workflow is simple, fast, and inexpensive. Only standard laboratory equipment is required for its implementation, making DF suitable for low-resource and point-of-use locations. The method may be used for EV isolation from small biological samples in diagnostic and treatment guidance applications. It can also be scaled up to harvest therapeutic EVs from large volumes of cell culture medium.

Therapeutic efficacy of antibody-drug conjugates targeting GD2-positive tumors.

BACKGROUND: Both ganglioside GD2-targeted immunotherapy and antibody-drug conjugates (ADCs) have demonstrated clinical success as solid tumor therapies in recent years, yet no research has been carried out to develop anti-GD2 ADCs against solid tumors. This is the first study to analyze cytotoxic activity of clinically relevant anti-GD2 ADCs in a wide panel of cell lines with varying GD2 expression and their effects in mouse models of GD2-positive solid cancer. METHODS: Anti-GD2 ADCs were generated based on the GD2-specific antibody ch14.18 approved for the treatment of neuroblastoma and commonly used drugs monomethyl auristatin E (MMAE) or F (MMAF), conjugated via a cleavable linker by thiol-maleimide chemistry. The antibody was produced in a mammalian expression system, and its specific binding to GD2 was analyzed. Antigen-binding properties and biodistribution of the ADCs in mice were studied in comparison with the parent antibody. Cytotoxic effects of the ADCs were evaluated in a wide panel of GD2-positive and GD2-negative tumor cell lines of neuroblastoma, glioma, sarcoma, melanoma, and breast cancer. Their antitumor effects were studied in the B78-D14 melanoma and EL-4 lymphoma syngeneic mouse models. RESULTS: The ch14.18-MMAE and ch14.18-MMAF ADCs retained antigen-binding properties of the parent antibody. Direct dependence of the cytotoxic effect on the level of GD2 expression was observed in cell lines of different origin for both ADCs, with IC50 below 1 nM for the cells with high GD2 expression and no cytotoxic effect for GD2-negative cells. Within the analyzed cell lines, ch14.18-MMAF was more effective in the cells overexpressing GD2, while ch14.18-MMAE had more prominent activity in the cells expressing low GD2 levels. The ADCs had a similar biodistribution profile in the B78-D14 melanoma model compared with the parent antibody, reaching 7.7% ID/g in the tumor at 48 hours postinjection. The average tumor size in groups treated with ch14.18-MMAE or ch14.18-MMAF was 2.6 times and 3.8 times smaller, respectively, compared with the control group. Antitumor effects of the anti-GD2 ADCs were also confirmed in the EL-4 lymphoma model. CONCLUSION: These findings validate the potential of ADCs targeting ganglioside GD2 in treating multiple GD2-expressing solid tumors.

Allocolchicinoids bearing a Michael acceptor fragment for possible irreversible binding of tubulin.

We describe an attempt to apply the concept of covalent binding towards the highly active allocolchicinoids selected on the basis of SAR analysis of previously synthesized molecules. To achieve the irreversible binding of the agent to the cysteine residues of the colchicine site of tubulin protein, we synthesized a number of new allocolchicinoids bearing the acceptor moiety. Some of the new derivatives possess cytotoxic activity against COLO-357, BxPC-3, HaCaT, and HEK293 cell lines in a low nanomolar range of concentrations. A substoichiometric mode of microtubule assembly inhibition was demonstrated. The most active compounds possess close to colchicine general toxicity on mice.

Discovery of dihydrofuranoallocolchicinoids - Highly potent antimitotic agents with low acute toxicity.

Two series of heterocyclic colchicinoids bearing ß-methylenedihydrofuran or 2H-pyran-2-one fragments were synthesized by the intramolecular Heck reaction. Methylenedihydrofuran compounds 9a and 9h were found to be the most cytotoxic among currently known colchicinoids, exhibiting outstanding antiproliferative activity on tumor cell lines in picomolar (0.01-2.1 nM) range of concentrations. Compound 9a potently and substoichiometrically inhibits microtubule formation in vitro, being an order of magnitude more active in this assay than colchicine. Derivatives 9a and 9h revealed relatively low acute toxicity in mice (LD50 ≥ 10 mg/kg i.v.). The X-Ray structure of colchicinoid 9a bound to tubulin confirmed interaction of this compound with the colchicine binding site of tubulin.

Rapamycin delays growth of Wnt-1 tumors in spite of suppression of host immunity.

BACKGROUND: Rapamycin, an inhibitor of mammalian target of Rapamycin (mTOR), is an immunosuppressive agent that has anti-proliferative effects on some tumors. However, the role of Rapamycin-induced immune suppression on tumor progression has not been examined. METHODS: We developed a transplantation model for generation of mammary tumors in syngeneic recipients that can be used to address the role of the immune system on tumor progression. We examined the effect of Rapamycin on the immune system and growth of MMTV-driven Wnt-1 mammary tumors which were transplanted into irradiated and bone marrow-reconstituted, or naïve mice. RESULTS: Rapamycin induced severe immunosuppression and significantly delayed the growth of Wnt-1 tumors. T cell depletion in spleen and thymus and reduction in T cell cytokine secretion were evident within 7 days of therapy. By day 20, splenic but not thymic T cell counts, and cytokine secretion recovered. We determined whether adoptive T cell therapy enhances the anti-cancer effect using ex vivo generated Rapamycin-resistant T cells. However, T cell transfer during Rapamycin therapy did not improve the outcome relative to drug therapy alone. Thus, we could not confirm that suppression of T cell immunity contributes to tumor growth in this model. Consistent with suppression of the mTOR pathway, decreased 4E-BP1, p70 S6-kinase, and S6 protein phosphorylation correlated with a decrease in Wnt-1 tumor cell proliferation. CONCLUSION: Rapamycin has a direct anti-tumor effect on Wnt-1 breast cancer in vivo that involves inhibition of the mTOR pathway at doses that also suppress host immune responses.