EP3113760A1 - Applying mechanical force by remote-controlled rotation - Google Patents

Abstract

Dynamic magnetic field (DMF) treatment to direct movement, and specifically rotation, of magnetic particles associated with a target structure, so that mechanical force is applied to the target structure. DMF treatment directs rotation about the axis in individual magnetic particles (e.g., superparamagnetic nanoparticles ), effecting permeabilization and/or other disruption of membranes (e.g., cell membranes and/or intracellular membranes). Cell death is remotely induced by application of mechanical force through rotational movement of magnetic particles (specifically superparamagnetic iron oxide nanoparticles, "SPIONs") exposed to DMF treatment. Particles were specifically targeted to lysosomal membrane structures, and were individually induced through application of DMF treatment to rotate about their axes. The resulting mechanical force disrupted (e.g., permeabilized) the lysosomal membranes, releasing lysosomal enzymes and triggering apoptosis, all without heating the particles (or their surroundings).

Description

APPLYING MECHANICAL FORCE BY REMOTE-CONTROLLED ROTATION

Cross Reference To Related Applications

[0001] This application claims the benefit of U.S. Provisional Application No.

61/947,830, filed March 4, 2014, the contents of which are hereby incorporated by reference herein in their entirety.

Background

[0002] There is a need for systems that can exert targeted mechanical force. The present disclosure describes such a system, including providing an explicit exemplification of application of the provided system to achieve directed disruption of lysosomes in certain cells, leading to cell death from apoptosis.

Summary

[0003] The present invention provides the ability to remotely control movement of certain magnetic particles remotely and with high precision, with far-reaching implications in many technology areas. Among other things, the present invention demonstrates use of a dynamic magnetic field (DMF) generator that can induce rotational movement of certain magnetic particles. Particular surprising and advantageous features of the described technology include that it can be used to rotate individual particles, and moreover can rotate such particles about their own axis. Additional or alternative surprising and advantageous features include that the present invention provides for manipulation of magnetic particles in a magnetic field without significant heat generation (e.g., without heating the particles). [0004] The invention particularly demonstrates that use of magnetic particles designed and/or constructed to specifically bind to a target structure is surprisingly effective, even as compared with use of otherwise comparable (e.g., otherwise identical) magnetic particles that lack such specific binding, for exertion of mechanical force on the target structure.

[0005] Specifically exemplified herein is remote induction of cell death by application of mechanical force through rotational movement of magnetic particles (specifically

superparamagnetic iron oxide nanoparticles, "SPIONs") exposed to DMF treatment. As exemplified, particles were specifically targeted to lysosomal membrane structures, and were individually induced through application of DMF treatment to rotate about their axes. The resulting mechanical force disrupted (e.g., permeabilized) the lysosomal membranes, releasing lysosomal enzymes and triggering apoptosis, all without heating the particles (or their surroundings).

[0006] The Examples included herein demonstrate, among other things, that shear forces created by the generation of oscillatory torques (incomplete rotation) of magnetic particles (e.g., superparamagnetic nanoparticles such as SPIONs) specifically bound to lysosomal membranes causes membrane permeabilization, and furthermore leads to extravasation of lysosomal contents into the cytoplasm. Still further, the present exemplification demonstrates that such application of shear forces to lysosomal membranes can induce apoptosis.

[0007] Still further, the exemplification provided herein explicitly demonstrates that use of specifically targeted magnetic particles (e.g., superparamagnetic nanoparticles such as SPIONs covalently conjugated to specific binding agents that target lysosomal ligands, in particular to antibodies targeting the lysosomal protein marker LAMPl (LAMPl-SPION)) shows surprisingly superior effect in application of mechanical force as described herein. The present exemplification specifically demonstrates, for example, that remote activation of slow rotation of LAMPl-SPIONs significantly improved the efficacy of cellular internalization of the

nanoparticles. LAMPl-SPIONs then preferentially accumulated along the membrane in lysosomes in both rat insulinoma tumor cells and human pancreatic beta-cells, presumably due to binding of LAMPl-SPIONs to endogenous LAMP1. Further activation of torques by the LAMPl-SPIONs bound to lysosomes resulted in rapid decrease in size and number of lysosomes. Without wishing to be bound by any particular theory, it is contemplated that such rapid decrease are attributable to tearing of the lysosomal membrane by the shear force of the rotationally activated LAMPl-SPIONs. Regardless of mechanism, the present exemplification demonstrates that such remote activation resulted in an increased expression of early and late apoptotic markers and impaired cell growth. Findings described herein suggest, among other things, that DMF treatment of lysosome -targeted nanoparticles offers a non-invasive tool to induce apoptosis remotely.

[0008] The present disclosure demonstrates, among other things, that use of DMF treatment with appropriate magnetic particles (e.g., with superparamagnetic nanoparticles), and particularly with specifically-targeted such particles, can apply mechanical force in a controlled and effective manner, for use in a wide range of applications, specifically including biomedical applications. In some embodiments, provided technologies are utilized to induce apoptosis or otherwise to achieve cell death or destruction, including of tumor cells, malignant cells, or otherwise aberrantly proliferating cells.

[0009] One particular advantage of technologies described herein is that mechanical force is applied, and appropriate results achieved, without significant generation of heat. Thus, for example, in some embodiments, the present invention provides compositions and methods that achieve selective cell destruction without significant generation of heat. In particular, provided technologies apply mechanical force, and in some embodiments, achieve selective cell destruction, without generating heat sufficient to result in off-target cell damage.

Description of the Drawing

[0010] Fig. 1 presents a schematic depiction of exemplified use of LAMPl-SPIONs with

DMF to disrupt lysosomal membranes and induce apoptosis. Additional supporting information, including Movie 1 : Illustrating the rotational control of magnetic microparticles achieved with a DMF device; Movie 2: Illustrating the rotational control of magnetic nanoparticles (300 nm diameter) achieved with a DMF device; and Movie 3 : Dynamic confocal microscopy study illustrating the real-time destruction of lysosomes via DMF and LAMPl-SPIONs, is available free of charge via the Internet at http://pubs.acs.org.

[0011] Figs. 2A-B illustrate DMF controlled rotation of magnetic nanoparticles. Fig. 2A shows schematic representation of a DMF generator utilized in the present Exemplification. The device controls rotation and movement of magnetic nanoparticles (e.g., SPIONs) with a low frequency (10-40 Hz) field. In contrast to reported alternating magnetic field generators, the DMF generator causes the nanoparticles to rotate around their own axis. Fig. 2B illustrates principle of use of the DMF generator and remote induction of apoptosis. As depicted in Fig. 2B, when targeted nanoparticles (LAMPl-SPIONs) first come into contact with cell membranes, their internalization can be enhanced by activation of slow nanoparticle rotation. This causes rotational motion (rolling) of the nanoparticles across the cell membrane and eventually internalization. Once internalized, the LAMPl-SPIONs enter lysosomes and bind to the lysosomal membrane. When the DMF is activated at this point, the nanoparticles start to rotate and the resulting shear forces cause injury to the lysosomal membrane. This in turn causes leakage of the lysosomal contents into the cytoplasm, leading to a decrease in its pH and subsequently apoptosis.

[0012] Fig. 3, comprised of Panels A-D, demonstrates DMF-induced rotation of magnetic particles. In order to enable better visualization of the effect of the DMF on magnetic particles under the microscope, larger micrometer sized magnetic beads (diameter 5.8 μιη) were used. A dish containing beads in a physiologic salt solution (Krebs buffer) was placed in the vicinity of the DMF device. Once the DMF was switched on, the beads started to rotate around their own axis, which also caused a slow directional movement of the beads across the floor of the dish. The beads completed a rotation of 360° in seconds (time depends on the viscosity of the liquid) between the Fig. 3 A and Fig. 3D. The speed of rotation can be controlled by varying the frequency setting on the DMF device and in this experiment was varied between 5-15 Hz. Movie 1, noted above, depicts this rolling.

[0013] Fig. 4, comprised of Panels A-D, demonstrates the loading of magnetic nanoparticle into lysosomes in INS-1 cells. Fig. 4A shows confocal imaging of SPIONs location in INS-1 cells. The SPIONs conjugated with the fluorescent dye TRITC (upper left image) were incubated with living cells in a static magnetic field for 5 minutes. Thereafter the cells were treated by DMF with 20 Hz, for 20 min. and then confocal microscopy images obtained (upper middle image). Plasma membrane and early endosomes were stained with CellMask (upper right image); nuclei and lysosomes were stained with Hoechst 32580 (lower left image) and

LysoTracker Green (green, lower middle image), respectively. The squares in the merge stack (lower right image) indicate SPIONs located in the lysosomes. Scale bars = 2 μιη. Fig. 4B shows statistical analysis of SPION co-localization with LysoTracker Green and CellMask under same conditions as in Fig. 4A. Fig. 4C shows co-localization analysis of lysosomes with SPION and CellMask under same conditions as Fig. 4A. Fig. 4D demonstrates loading efficiency of LAMP1 antibody conjugated SPIONs (LAMPl-SPION) increased under condition with the DMF treatment. The loading efficiency is calculated by the ratio of TRITC fluorescence intensity (lighter greyscale values shown in the merged image) over nuclear intensity. The data was collected from three independent experiments. The "*" symbol indicates p<0.05, and symbol indicates p<0.001.

[0014] Fig 5, comprised of Panels A-D, demonstrates that DMF treatment decreases intracellular lysosomes and the pH in LAMPl-SPION loaded INS-1 cells. Fig. 5A, shows cells (in the lower images) treated by DMF (20 Hz, 20 min.), and lysosomes (in all four images) stained with LysoTracker Green. Scale bars = 5 μιη. Fig. 5B shows the mean intensity of fluorescence measured under the various different conditions shown in Fig. 5A. Fig. 5C shows representative confocal images indicating the intracellular pH value using an acidotropic probe, LysoSensor Green DND 189 in INS-1 cells. Scale bars = 5 μιη. Fig. 5D shows the mean intensity of fluorescence measured under the various different conditions shown in Fig. 5C. The data were collected from five experiments with at least 6 cells under each condition. The symbol indicates p<0.01, and symbol indicates p<0.001.

[0015] Fig. 6 is a temperature plot demonstrating that DMF-field induced SPION rotation does not increase temperature. A dish with 100 nm SPIONs (10 mg/ml) in water and a control dish containing water only, each containing a temperature probe, were placed

simultaneously on the DMF device. The dishes were then subjected to the DMF field for 20 min. at 20 Hz. No significant change in temperature was observed in either the dish with the SPIONs (thicker, darker curve) or the control dish (thinner, lighter curve). [0016] Fig 7, comprised of Panels A-F, demonstrates that DMF treatment disrupts lysosomes in human pancreatic beta cells after loading with LAMPl-SPIONs. Fig. 7A shows immunostaining of human islet beta cells with or without DMF treatment. Lysosomes were stained with the anti-LAMPl antibody (two left images), SPIONs with TRITC (two left-center images) and islet beta-cells with an anti-insulin antibody (two right-center images), respectively. Scale bars = 2 μιη. Fig. 7B shows the SPIONs located in the membrane of a lysosome in which an intensity profile (right plot) was derived along the horizontal line shown in the stained image (left image). Fig. 7C shows the SPIONs located in the membrane of a lysosome, after treatment with DMF. An intensity profile (right plot) was also derived based on the horizontal line shown in the corresponding stained image (left). Fig. 7D illustrates differences in size distribution of lysosomes before (grey bars) and after (black bars) DMF treatment. Fig. 7E illustrates average sizes of lysosomes without and with DMF treatment. The symbol indicates p<0.001. Fig.

7F shows transmission electron microscopy (TEM) images of the intracellular distribution of SPIONs in INS-1 cells. Images on the bottom are magnified versions of the areas indicated with white boxes (shown in the top images). While without DMF treatment the LAMPl-SPIONs are clustered in vesicular structures, their distribution is scattered throughout the cytosol after DMF treatment.

[0017] Fig 8, comprised of Panels A-D, demonstrate DMF treatment-induced apoptosis in LAMP1-SPION loaded cells. Fig. 8A shows INS-1 cells treated with DMF for 20 min. at 20 Hz and stained with the nuclear marker Hoechst (shown as grey values in the left image and center-left image), the apoptosis marker annexin V (lighter grey values in the right image) and 7- AAD (lighter grey values in the right image and center-right image). Scale bars = 5 μιη. After 6 hours of incubation (5% C0₂, 37 °C), early (Fig. 8B) and late (Fig. 8C) stage apoptosis were detected by percentage ₀f ^FAnnexm V( _AAD) _{χ100 num}¾_{er 0}f _annexmy _and 7-AAD positive cells to

'' Hoechst 34580

the number of Hoechst stained cells. As demonstrated, DMF caused significant increase in apoptosis in LAMPl-SPION-loaded cells compared to when loading was done using

conventional SPIONs. Each treatment was conducted with 28 cells. The "*" symbol indicates p<0.05. Fig. 8D shows decrease of the rate of cell growth in LAMP1-SPION loaded INS-1 cells. Cells were treated with DMF (20 Hz, 20 minutes) once/day. Data are from 3 independent experiments and represent mean values ±S.E.M. The symbol indicates p<0.001.

Definitions

[0018] Administration: As used herein, the term "administration" refers to the administration of a composition to a subject or system. Administration to an animal subject (e.g., to a human) may be by any appropriate route. For example, in some embodiments, administration may be bronchial (including by bronchial instillation), buccal, enteral, interdermal, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including by intratracheal instillation), transdermal, vaginal and vitreal.

[0019] Affinity : As is known in the art, "affinity" is a measure of the tightness with a particular ligand binds to its partner. Affinities can be measured in different ways. In some embodiments, affinity is measured by a quantitative assay. In some such embodiments, binding partner concentration may be fixed to be in excess of ligand concentration so as to mimic physiological conditions. Alternatively or additionally, in some embodiments, binding partner concentration and/or ligand concentration may be varied. In some such embodiments, affinity may be compared to a reference under comparable conditions (e.g., concentrations).

[0020] Agent : The term "agent" as used herein may refer to a compound or entity of any chemical class including, for example, polypeptides, nucleic acids, saccharides, lipids, small molecules, metals, or combinations thereof. In some embodiments, an agent is or comprises a natural product in that it is found in and/or is obtained from nature. In some embodiments, an agent is or comprises one or more entities that is man-made in that it is designed, engineered, and/or produced through action of the hand of man and/or is not found in nature. In some embodiments, an agent may be utilized in isolated or pure form; in some embodiments, an agent may be utilized in crude form. In some embodiments, potential agents are provided as collections or libraries, for example that may be screened to identify or characterize active agents within them. Some particular embodiments of agents that may be utilized in accordance with the present invention include small molecules, antibodies, antibody fragments, aptamers, nucleic acids (e.g., siRNAs, shRNAs, DNA/RNA hybrids, antisense oligonucleotides, ribozymes), peptides, peptide mimetics, etc. In some embodiments, an agent is or comprises a polymer. In some embodiments, an agent is not a polymer and/or is substantially free of any polymer. In some embodiments, an agent contains at least one polymeric moiety. In some embodiments, an agent lacks or is substantially free of any polymeric moiety.

[0021] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to humans, at any stage of development. In some embodiments, "animal" refers to non-human animals, at any stage of development. In some embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and/or worms. In some embodiments, an animal may be a transgenic animal, genetically-engineered animal, and/or a clone.

[0022] Antibody agent: As used herein, the term "antibody agent" refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide with immunoglobulin structural elements sufficient to confer specific binding.

Suitable antibody agents include, but are not limited to, human antibodies, primatized antibodies, chimeric antibodies, bi-specific antibodies, humanized antibodies, conjugated antibodies {i.e., antibodies conjugated or fused to other proteins, radiolabels, cytotoxins), Small Modular ImmunoPharmaceuticals ("SMIPs™ ), single chain antibodies, cameloid antibodies, and antibody fragments. As used herein, the term "antibody agent" also includes intact monoclonal antibodies, polyclonal antibodies, single domain antibodies (e.g., shark single domain antibodies (e.g., IgNAR or fragments thereof)), multispecific antibodies {e.g. bi-specific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. In some embodiments, the term encompasses stapled peptides. In some embodiments, the term encompasses one or more antibody-like binding peptidomimetics. In some embodiments, the term encompasses one or more antibody-like binding scaffold proteins. In come embodiments, the term encompasses monobodies or adnectins. In many embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as a complementarity determining region (CDR); in some embodiments an antibody agent is or comprises a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and/or at least one light chain CDR) that is substantially identical to one found in a reference antibody. In some embodiments an included CDR is substantially identical to a reference CDR in that it is either identical in sequence or contains between 1-5 amino acid substitutions as compared with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR . In some embodiments an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, an antibody agent is a polypeptide protein having a binding domain which is homologous or largely homologous to an immunoglobulin-binding domain.

[0023] Antibody: As is known in the art, an "antibody" is an immunoglobulin that binds specifically to a particular antigen. The term encompasses immunoglobulins that are naturally produced in that they are generated by an organism reacting to the antigen, and also those that are synthetically produced or engineered. An antibody may be monoclonal or polyclonal. An antibody may be a member of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, and IgD. A typical immunoglobulin (antibody) structural unit as understood in the art, is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (approximately 25 kD) and one "heavy" chain (approximately 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms "variable light chain"(VL) and "variable heavy chain" (VH) refer to these light and heavy chains respectively. Each variable region is further subdivided into hypervariable (HV) and framework (FR) regions. The hypervariable regions comprise three areas of hypervariability sequence called complementarity determining regions (CDR 1, CDR 2 and CDR 3), separated by four framework regions (FRl, FR2, FR2, and FR4) which form a beta-sheet structure and serve as a scaffold to hold the HV regions in position. The C-terminus of each heavy and light chain defines a constant region consisting of one domain for the light chain (CL) and three for the heavy chain (CHI, CH2 and CH3). In some embodiments, the term "full length" is used in reference to an antibody to mean that it contains two heavy chains and two light chains, optionally associated by disulfide bonds as occurs with naturally-produced antibodies. In some embodiments, an antibody is produced by a cell. In some embodiments, an antibody is produced by chemical synthesis. In some embodiments, an antibody is derived from a mammal. In some embodiments, an antibody is derived from an animal such as, but not limited to, mouse, rat, horse, pig, or goat. In some embodiments, an antibody is produced using a recombinant cell culture system. In some embodiments, an antibody may be a purified antibody (for example, by immune-affinity chromatography). In some embodiments, an antibody may be a human antibody. In some embodiments, an antibody may be a humanized antibody (antibody from non- human species whose protein sequences have been modified to increase their similarity to antibody variants produced naturally in humans). In some embodiments, an antibody may be a chimeric antibody (antibody made by combining genetic material from a non-human source, e.g., mouse, rat, horse, or pig, with genetic material from humans).

[0024] Antibody fragment: As used herein, an "antibody fragment" includes a portion of an intact antibody, such as, for example, the antigen-binding or variable region of an antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; triabodies;

tetrabodies; linear antibodies; single-chain antibody molecules; and multi specific antibodies formed from antibody fragments. For example, antibody fragments include isolated fragments, "Fv" fragments, consisting of the variable regions of the heavy and light chains, recombinant single chain polypeptide molecules in which light and heavy chain variable regions are connected by a peptide linker ("ScFv proteins"), and minimal recognition units consisting of the amino acid residues that mimic the hypervariable region. In many embodiments, an antibody fragment contains sufficient sequence of the parent antibody of which it is a fragment that it binds to the same antigen as does the parent antibody; in some embodiments, a fragment binds to the antigen with a comparable affinity to that of the parent antibody and/or competes with the parent antibody for binding to the antigen. Examples of antigen binding fragments of an antibody include, but are not limited to, Fab fragment, Fab' fragment, F(ab')2 fragment, scFv fragment, Fv fragment, dsFv diabody, dAb fragment, Fd' fragment, Fd fragment, and an isolated complementarity determining region (CDR) region. An antigen binding fragment of an antibody may be produced by any means. For example, an antigen binding fragment of an antibody may be enzymatically or chemically produced by fragmentation of an intact antibody and/or it may be recombinantly produced from a gene encoding the partial antibody sequence. Alternatively or additionally, antigen binding fragment of an antibody may be wholly or partially synthetically produced. An antigen binding fragment of an antibody may optionally comprise a single chain antibody fragment. Alternatively or additionally, an antigen binding fragment of an antibody may comprise multiple chains which are linked together, for example, by disulfide linkages. An antigen binding fragment of an antibody may optionally comprise a multimolecular complex. A functional antibody fragment typically comprises at least about 50 amino acids and more typically comprises at least about 200 amino acids.

[0025] Antigen : An "antigen" is a molecule or entity that i) elicits an immune response; and/or (ii) is specifically bound by a T cell receptor (e.g., when presented by an MHC molecule) or an antibody (e.g., produced by a B cell), for example when exposed or administered to an organism. In some embodiments, an antigen elicits a humoral response (e.g., including production of antigen-specific antibodies) in an organism; alternatively or additionally, in some embodiments, an antigen elicits a cellular response (e.g., involving T-cells whose receptors specifically interact with the antigen) in an organism. It will be appreciated by those skilled in the art that a particular antigen may elicit an immune response in one or several members of a target organism (e.g., mice, rabbits, primates, humans), but not in all members of the target organism species. In some embodiments, an antigen elicits an immune response in at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the members of a target organism species. In some embodiments, an antigen binds to an antibody and/or T cell receptor, and may or may not induce a particular physiological response in an organism. In some embodiments, for example, an antigen may bind to an antibody and/or to a T cell receptor in vitro, whether or not such an interaction occurs in vivo. In general, an antigen may be or include any chemical entity such as, for example, a small molecule, a nucleic acid, a polypeptide, a carbohydrate, a lipid, a polymer [in some embodiments other than a biologic polymer (e.g., other than a nucleic acid or amino acid polymer)] etc. In some embodiments, an antigen is or comprises a polypeptide. In some embodiments, an antigen is or comprises a glycan. Those of ordinary skill in the art will appreciate that, in general, an antigen may be provided or utilized in isolated or pure form, or alternatively may be provided in crude form (e.g., together with other materials, for example in an extract such as a cellular extract or other relatively crude preparation of an antigen-containing source). In some embodiments. In some embodiments, an antigen is or comprises a recombinant antigen. In some embodiments, an antigen is or comprises a polypeptide or portion thereof. In some embodiments, an antigen is associated with (e.g., expressed by) an infectious agent. In some embodiments, an antigen is associated with cancer (e.g., with tumor cells and/or metastases).

[0026] Approximately: As used herein, the terms "approximately" and "about" are each intended to encompass normal statistical variation as would be understood by those of ordinary skill in the art as appropriate to the relevant context. In certain embodiments, the terms

"approximately" or "about" each refer to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of a stated value, unless otherwise stated or otherwise evident from the context (e.g., where such number would exceed 100% of a possible value).

[0027] Aptamer: As used herein, the term "aptamer" refers to a macromolecule composed of nucleic acid (e.g., R A, DNA) that binds tightly to a specific molecular target (e.g., an umbrella topology glycan). A particular aptamer may be described by a linear nucleotide sequence and is typically about 15-60 nucleotides in length. Without wishing to be bound by any theory, it is contemplated that the chain of nucleotides in an aptamer form intramolecular interactions that fold the molecule into a complex three-dimensional shape, and this three- dimensional shape allows the aptamer to bind tightly to the surface of its target molecule. Given the extraordinary diversity of molecular shapes that exist within the universe of all possible nucleotide sequences, aptamers may be obtained for a wide array of molecular targets, including proteins and small molecules. In addition to high specificity, aptamers typically have very high affinities for their targets (e.g., affinities in the picomolar to low nanomolar range for proteins). In many embodiments, aptamers are chemically stable and can be boiled or frozen without loss of activity. Because they are synthetic molecules, aptamers are amenable to a variety of modifications, which can optimize their function for particular applications. For example, aptamers can be modified to dramatically reduce their sensitivity to degradation by enzymes in the blood for use in in vivo applications. In addition, aptamers can be modified to alter their biodistribution or plasma residence time.

[0028] Associated with: Two events or entities are "associated" with one another, as that term is used herein, if the presence, level and/or form of one is correlated with that of the other. For example, a particular entity (e.g., polypeptide) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and/or form correlates with incidence of and/or susceptibility of the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically "associated" with one another if they interact, directly or indirectly, so that they are and remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non- covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.

[0029] Binding: It will be understood that the term "binding", as used herein, typically refers to a non-covalent association between or among two or more entities. "Direct" binding involves physical contact between entities or moieties; indirect binding involves physical interaction by way of physical contact with one or more intermediate entities. Binding between two or more entities can typically be assessed in any of a variety of contexts - including where interacting entities or moieties are studied in isolation or in the context of more complex systems (e.g., while covalently or otherwise associated with a carrier entity and/or in a biological system or cell).

[0030] Binding agent: In general, the term "binding agent" is used herein to refer to any entity that binds to a target of interest as described herein. In many embodiments, a binding agent of interest is one that binds specifically with its target in that it discriminates its target from other potential binding partners in a particular interaction contact. In general, a binding agent may be or comprise an entity of any chemical class (e.g., polymer, non-polymer, small molecule, polypeptide, carbohydrate, lipid, nucleic acid, etc.). In some embodiments, a binding agent is a single chemical entity. In some embodiments, a binding agent is a complex of two or more discrete chemical entities associated with one another under relevant conditions by non-covalent interactions. For example, those skilled in the art will appreciate that in some embodiments, a binding agent may comprise a "generic" binding moiety (e.g., one of biotin/avidin/streptaviding and/or a class-specific antibody) and a "specific" binding moiety (e.g., an antibody or aptamers with a particular molecular target) that is linked to the partner of the generic biding moiety. In some embodiments, such an approach can permit modular assembly of multiple binding agents through linkage of different specific binding moieties with the same generic binding poiety partner. In some embodiments, binding agents are or comprise polypeptides (including, e.g., antibodies or antibody fragments). In some embodiments, binding agents are or comprise small molecules. In some embodiments, binding agents are or comprise nucleic acids. In some embodiments, binding agents are aptamers. In some embodiments, binding agents are polymers; in some embodiments, binding agents are not polymers. In some embodiments, binding agents are non-polymeric in that they lack polymeric moieties. In some embodiments, binding agents are or comprise carbohydrates. In some embodiments, binding agents are or comprise lectins. In some embodiments, binding agents are or comprise peptidomimetics. In some embodiments, binding agents are or comprise scaffold proteins. In some embodiments, binding agents are or comprise mimotopes. In some embodiments, binding agents are or comprise stapled peptides. In certain embodiments, binding agents are or comprise nucleic acids, such as DNA or R A.

[0031] Characteristic portion : As used herein, the term "characteristic portion" is used, in the broadest sense, to refer to a portion of a substance whose presence (or absence) correlates with presence (or absence) of a particular feature, attribute, or activity of the substance. In some embodiments, a characteristic portion of a substance is a portion that is found in the substance and in related substances that share the particular feature, attribute or activity, but not in those that do not share the particular feature, attribute or activity. In certain embodiments, a characteristic portion shares at least one functional characteristic with the intact substance. For example, in some embodiments, a "characteristic portion" of a protein or polypeptide is one that contains a continuous stretch of amino acids, or a collection of continuous stretches of amino acids, that together are characteristic of a protein or polypeptide. In some embodiments, each such continuous stretch generally contains at least 2, 5, 10, 15, 20, 50, or more amino acids. In general, a characteristic portion of a substance (e.g., of a protein, antibody, etc.) is one that, in addition to the sequence and/or structural identity specified above, shares at least one functional characteristic with the relevant intact substance. In some embodiments, a characteristic portion may be biologically active.

[0032] Combination therapy: As used herein, the term "combination therapy" refers to those situations in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents). In some embodiments, two or more agents may be administered simultaneously; in some embodiments, such agents may be administered sequentially; in some embodiments, such agents are administered in overlapping dosing regimens.

[0033] Comparable: The term "comparable", as used herein, refers to two or more agents, entities, situations, sets of conditions, etc. that may not be identical to one another but that are sufficiently similar to permit comparison therebetween so that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc. to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.

[0034] Corresponding to : As used herein, the term "corresponding to" is often used to designate a structural element or moiety in an agent of interest that shares a position (e.g., in three-dimensional space or relative to another element or moiety) with one present in an appropriate reference agent. For example, in some embodiments, the term is used to refer to position/identity of a residue in a polymer, such as an amino acid residue in a polypeptide or a nucleotide residue in a nucleic acid. Those of ordinary skill will appreciate that, for purposes of simplicity, residues in such a polymer are often designated using a canonical numbering system based on a reference related polymer, so that a residue in a first polymer "corresponding to" a residue at position 190 in the reference polymer, for example, need not actually be the 190^th residue in the first polymer but rather corresponds to the residue found at the 190^th position in the reference polymer; those of ordinary skill in the art readily appreciate how to identify "corresponding" amino acids, including through use of one or more commercially-available algorithms specifically designed for polymer sequence comparisons.

[0035] Designed: As used herein, the term "designed" refers to an agent (i) whose structure is or was selected by the hand of man; (ii) that is produced by a process requiring the hand of man; and/or (iii) that is distinct from natural substances and other known agents. [0036] Detection entity : The term "detection entity" as used herein refers to any element, molecule, functional group, compound, fragment or moiety that is detectable. In some embodiments, a detection entity is provided or utilized alone. In some embodiments, a detection entity is provided and/or utilized in association with (e.g., joined to) another agent. Examples of detection entities include, but are not limited to: various ligands, radionuclides (e.g., ³H, ¹⁴C, ¹⁸F,

specific exemplary fluorescent dyes, see below), chemiluminescent agents (such as, for example, acridinum esters, stabilized dioxetanes, and the like), bioluminescent agents, spectrally resolvable inorganic fluorescent semiconductors nanocrystals (i.e., quantum dots), metal nanoparticles (e.g., gold, silver, copper, platinum, etc.) nanoclusters, paramagnetic metal ions, enzymes (for specific examples of enzymes, see below), colorimetric labels (such as, for example, dyes, colloidal gold, and the like), biotin, dioxigenin, haptens, and proteins for which antisera or monoclonal antibodies are available.

[0037] Determine: Many methodologies described herein include a step of

"determining". Those of ordinary skill in the art, reading the present specification, will appreciate that such "determining" can utilize or be accomplished through use of any of a variety of techniques available to those skilled in the art, including for example specific techniques explicitly referred to herein. In some embodiments, determining involves manipulation of a physical sample. In some embodiments, determining involves consideration and/or manipulation of data or information, for example utilizing a computer or other processing unit adapted to perform a relevant analysis. In some embodiments, determining involves receiving relevant information and/or materials from a source. In some embodiments, determining involves comparing one or more features of a sample or entity to a comparable reference. [0038] Diagnostic information: As used herein, "diagnostic information" or

"information for use in diagnosis" is information that is useful in determining whether a patient has a disease, disorder or condition and/or in classifying a disease, disorder or condition into a phenotypic category or any category having significance with regard to prognosis of a disease, disorder or condition, or likely response to treatment (either treatment in general or any particular treatment) of a disease, disorder or condition. Similarly, "diagnosis" refers to providing any type of diagnostic information, including, but not limited to, whether a subject is likely to have or develop a disease, disorder or condition (such as cancer), state, staging or characteristic of a disease, disorder or condition as manifested in the subject, information related to the nature or classification of a tumor, information related to prognosis and/or information useful in selecting an appropriate treatment. Selection of treatment may include the choice of a particular therapeutic (e.g., chemotherapeutic) agent or other treatment modality such as surgery, radiation, etc., a choice about whether to withhold or deliver therapy, a choice relating to dosing regimen (e.g., frequency or level of one or more doses of a particular therapeutic agent or combination of therapeutic agents), etc.

[0039] Dosage form: As used herein, the term "dosage form" refers to a physically discrete unit of an active agent (e.g., a therapeutic or diagnostic agent) for administration to a subject. Each unit contains a predetermined quantity of active agent. In some embodiments, such quantity is a unit dosage amount (or a whole fraction thereof) appropriate for administration in accordance with a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e., with a therapeutic dosing regimen). Those of ordinary skill in the art appreciate that the total amount of a therapeutic composition or agent administered to a particular subject is determined by one or more attending physicians and may involve administration of multiple dosage forms.

[0040] Dosing regimen: As used herein, the term "dosing regimen" refers to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a

recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).

[0041] Engineered: In general, the term "engineered" refers to the aspect of having been manipulated by the hand of man. For example, a polynucleotide is considered to be

"engineered" when two or more sequences, that are not linked together in that order in nature, are manipulated by the hand of man to be directly linked to one another in the engineered polynucleotide. For example, in some embodiments of the present invention, an engineered polynucleotide comprises a regulatory sequence that is found in nature in operative association with a first coding sequence but not in operative association with a second coding sequence, is linked by the hand of man so that it is operatively associated with the second coding sequence. Comparably, a cell or organism is considered to be "engineered" if it has been manipulated so that its genetic information is altered (e.g., new genetic material not previously present has been introduced, for example by transformation, mating, somatic hybridization, transfection, transduction, or other mechanism, or previously present genetic material is altered or removed, for example by substitution or deletion mutation, or by mating protocols). As is common practice and is understood by those in the art, progeny of an engineered polynucleotide or cell are typically still referred to as "engineered" even though the actual manipulation was performed on a prior entity.

[0042] High affinity binding: The term "high affinity binding", as used herein refers to a high degree of tightness with which a particular ligand binds to its partner. Affinities can be measured by any available method, including those known in the art. Those skilled in the art will be aware of affinities that are appropriately considered to be "high" in a particular context. In some embodiments, high affinity binding may be characterized by preferentially binding to a particular target when in the presence of alternative potential (e.g., competitive) targets, particularly when such competitive targets are present in excess relative to the target of interest. In some embodiments, high affinity binding may be characterized by relatively rapid on-rate and/or slow off-rate.

[0043] Isolated: As used herein, the term "isolated" refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) designed, produced, prepared, and/or manufactured by the hand of man. Isolated substances and/or entities may be separated from about 10%, about 20%, about 30%>, about 40%>, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were initially associated. In some embodiments, isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, a substance may still be considered "isolated" or even "pure", after having been combined with certain other components such as, for example, one or more carriers or excipients (e.g., buffer, solvent, water, etc.); in such embodiments, percent isolation or purity of the substance is calculated without including such carriers or excipients. In some embodiments, isolation involves or requires disruption of covalent bonds (e.g., to isolate a polypeptide domain from a longer polypeptide and/or to isolate a nucleotide sequence element from a longer oligonucleotide or nucleic acid).

[0044] Low affinity binding: The term "low affinity binding", as used herein refers to a low degree of tightness with which a particular ligand binds to its partner. As described herein, affinities can be measured by any available method, including methods known in the art. Those skilled in the art will be aware of affinities that are appropriately considered to be "low" in a particular context. In some embodiments, low affinity binding may be characterized by failure to discriminate among potential (e.g., competitive) targets, particularly when they are present at comparable levels. In some embodiments, low affinity binding may be characterized by relatively slow on-rate and/or rapid off-rate. [0045] Marker: A marker, as used herein, refers to an entity or moiety whose presence or level is a characteristic of a particular state or event. In some embodiments, presence or level of a particular marker may be characteristic of presence or stage of a disease, disorder, or condition. To give but one example, in some embodiments, the term refers to a gene expression product that is characteristic of a particular tumor, tumor subclass, stage of tumor, etc.

Alternatively or additionally, in some embodiments, a presence or level of a particular marker correlates with activity (or activity level) of a particular signaling pathway, for example that may be characteristic of a particular class of tumors. The statistical significance of the presence or absence of a marker may vary depending upon the particular marker. In some embodiments, detection of a marker is highly specific in that it reflects a high probability that the tumor is of a particular subclass. Such specificity may come at the cost of sensitivity (i.e., a negative result may occur even if the tumor is a tumor that would be expected to express the marker).

Conversely, markers with a high degree of sensitivity may be less specific that those with lower sensitivity. According to the present invention a useful marker need not distinguish tumors of a particular subclass with 100% accuracy.

[0046] Nucleic acid: As used herein, the term "nucleic acid," in its broadest sense, refers to any compound and/or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and/or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage. As will be clear from context, in some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and/or nucleosides); in some embodiments, "nucleic acid" refers to an

oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, a "nucleic acid" is or comprises R A; in some embodiments, a "nucleic acid" is or comprises DNA. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments, a nucleic acid is, comprises, or consists of one or more "peptide nucleic acids", which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention. Alternatively or additionally, in some embodiments, a nucleic acid has one or more phosphorothioate and/or 5'-N-phosphoramidite linkages rather than phosphodiester bonds. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxy cytidine). In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2- aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5- methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5- bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8- oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a nucleic acid comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or protein. In some embodiments, a nucleic acid includes one or more introns. In some embodiments, nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long.

[0047] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity;

intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.

[0048] Pharmaceutically acceptable: The term "pharmaceutically acceptable" as used herein, refers to agents that, within the scope of sound medical judgment, are suitable for use in contact with tissues of human beings and/or animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[0049] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and/or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations.

[0050] Polypeptide: The term "polypeptide", as used herein, generally has its art- recognized meaning of a polymer of at least three amino acids, linked to one another by peptide bonds. In some embodiments, a polypeptide may include at least 3-5 amino acids, each of which is attached to others by way of at least one peptide bond. Those of ordinary skill in the art will appreciate that polypeptides sometimes include "non-natural" amino acids or other entities that nonetheless are capable of integrating into a polypeptide chain. Those of ordinary skill in the art understand that protein sequences generally tolerate some substitution without destroying activity. Thus, in some embodiments, any polypeptide that retains activity and shares at least about 30-40% overall sequence identity, often greater than about 50%, 60%>, 70%>, or 80%>, and further usually including at least one region of much higher identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99% in one or more highly conserved regions, usually

encompassing at least 3-4 and often up to 20 or more amino acids, with another polypeptide (e.g., a reference polypeptide) is considered to be of the same class as that other polypeptide. In some embodiments, polypeptides of a particular class all show significant sequence homology or identity with a relevant reference polypeptide. In many embodiments, such member also shares significant activity with the reference polypeptide. For example, in some embodiments, a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%), and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and/or includes at least one region (i.e., a conserved region, often including a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region usually encompasses at least 3-4 and often up to 20 or more amino acids; in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids. In some embodiments, a polypeptide may contain L-amino acids, D-amino acids, or both. In some embodiments, a polypeptide may contain any of a variety of amino acid modifications or analogs known in the art. For example, in some embodiments, a polypeptide may include one more modifications such as, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, a polypeptide may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and/or combinations thereof. The term "peptide" is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, proteins are antibodies, antibody fragments, biologically active portions thereof, and/or characteristic portions thereof.

[0051] Prevention : The term "prevention", as used herein, refers to a delay of onset, and/or reduction in frequency and/or severity of one or more symptoms of a particular disease, disorder or condition. In some embodiments, prevention is assessed on a population basis such that an agent is considered to "prevent" a particular disease, disorder or condition if a statistically significant decrease in the development, frequency, and/or intensity of one or more symptoms of the disease, disorder or condition is observed in a population susceptible to the disease, disorder, or condition

[0052] Prognostic and predictive information: As used herein, the terms "prognostic information" and "predictive information" are used to refer to any information that may be used to indicate any aspect of the course of a disease or condition either in the absence or presence of treatment. Such information may include, but is not limited to, the average life expectancy of a patient, the likelihood that a patient will survive for a given amount of time (e.g., 6 months, 1 year, 5 years, etc.), the likelihood that a patient will be cured of a disease, the likelihood that a patient's disease will respond to a particular therapy (wherein response may be defined in any of a variety of ways). Prognostic and predictive information are included within the broad category of diagnostic information. [0053] Protein: As used herein, the term "protein" refers to a polypeptide {i.e., a string of at least two amino acids linked to one another by peptide bonds). Proteins may include moieties other than amino acids {e.g., may be glycoproteins, proteoglycans, etc.) and/or may be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a

"protein" can be a complete polypeptide chain as produced by a cell (with or without a signal sequence), or can be a characteristic portion thereof. Those of ordinary skill will appreciate that a protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means. Polypeptides may contain L-amino acids, D- amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, proteins are antibodies, antibody fragments, biologically active portions thereof, and/or characteristic portions thereof.

[0054] Reference: The term "reference" is often used herein to describe a standard or control agent, individual, population, sample, sequence or value against which an agent, individual, population, sample, sequence or value of interest is compared. In some

embodiments, a reference agent, individual, population, sample, sequence or value is tested and/or determined substantially simultaneously with the testing or determination of the agent, individual, population, sample, sequence or value of interest. In some embodiments, a reference agent, individual, population, sample, sequence or value is a historical reference, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference agent, individual, population, sample, sequence or value is determined or characterized under conditions comparable to those utilized to determine or characterize the agent, individual, population, sample, sequence or value of interest.

[0055] Response: As used herein, a response to treatment may refer to any beneficial alteration in a subject's condition that occurs as a result of or correlates with treatment. Such alteration may include stabilization of the condition (e.g., prevention of deterioration that would have taken place in the absence of the treatment), amelioration of symptoms of the condition, and/or improvement in the prospects for cure of the condition, etc. It may refer to a subject's response or to a tumor's response. Tumor or subject response may be measured according to a wide variety of criteria, including clinical criteria and objective criteria. Techniques for assessing response include, but are not limited to, clinical examination, positron emission tomatography, chest X-ray CT scan, MRI, ultrasound, endoscopy, laparoscopy, presence or level of tumor markers in a sample obtained from a subject, cytology, and/or histology. Many of these techniques attempt to determine the size of a tumor or otherwise determine the total tumor burden. Methods and guidelines for assessing response to treatment are discussed in Therasse et. al, "New guidelines to evaluate the response to treatment in solid tumors", European

Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada, J. Natl. Cancer Inst., 2000, 92(3):205-216. The exact response criteria can be selected in any appropriate manner, provided that when comparing groups of tumors and/or patients, the groups to be compared are assessed based on the same or comparable criteria for determining response rate. One of ordinary skill in the art will be able to select appropriate criteria. [0056] Risk: As will be understood from context, a "risk" of a disease, disorder or condition is a degree of likelihood that a particular individual will develop the disease, disorder, or condition. In some embodiments, risk is expressed as a percentage. In some embodiments, risk is from 0,1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 100%. In some embodiments risk is expressed as a risk relative to a risk associated with a reference sample or group of reference samples. In some embodiments, a reference sample or group of reference samples have a known risk of a disease, disorder, or condition. In some embodiments a reference sample or group of reference samples are from individuals comparable to a particular individual. In some embodiments, relative risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

[0057] Sample: As used herein, the term "sample" typically refers to a biological sample obtained or derived from a source of interest, as described herein. In some embodiments, a source of interest comprises an organism, such as an animal or human. In some embodiments, a biological sample is or comprises biological tissue or fluid. In some embodiments, a biological sample may be or comprise bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy samples; cell-containing body fluids; free floating nucleic acids; sputum; saliva; urine;

cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph; gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; washings or lavages such as a ductal lavages or broncheoalveolar lavages; aspirates; scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces, other body fluids, secretions, and/or excretions; and/or cells therefrom, etc. In some embodiments, a biological sample is or comprises cells obtained from an individual. In some embodiments, obtained cells are or include cells from an individual from whom the sample is obtained. In some embodiments, a sample is a "primary sample" obtained directly from a source of interest by any appropriate means. For example, in some embodiments, a primary biological sample is obtained by methods selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of body fluid (e.g., blood, lymph, feces etc.), etc. In some embodiments, as will be clear from context, the term "sample" refers to a preparation that is obtained by processing (e.g., by removing one or more components of and/or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane. Such a "processed sample" may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to techniques such as amplification or reverse transcription of m NA, isolation and/or purification of certain components, etc.

[0058] Small molecule: As used herein, the term "small molecule" means a low molecular weight organic and/or inorganic compound. In general, a "small molecule" is a molecule that is less than about 5 kilodaltons (kD) in size. In some embodiments, a small molecule is less than about 4 kD, 3 kD, about 2 kD, or about 1 kD. In some embodiments, the small molecule is less than about 800 daltons (D), about 600 D, about 500 D, about 400 D, about 300 D, about 200 D, or about 100 D. In some embodiments, a small molecule is less than about 2000 g/mol, less than about 1500 g/mol, less than about 1000 g/mol, less than about 800 g/mol, or less than about 500 g/mol. In some embodiments, a small molecule is not a polymer. In some embodiments, a small molecule does not include a polymeric moiety. In some embodiments, a small molecule is not a protein or polypeptide (e.g., is not an oligopeptide or peptide). In some embodiments, a small molecule is not a polynucleotide (e.g., is not an oligonucleotide). In some embodiments, a small molecule is not a polysaccharide. In some embodiments, a small molecule does not comprise a polysaccharide (e.g., is not a glycoprotein, proteoglycan, glycolipid, etc.). In some embodiments, a small molecule is not a lipid. In some embodiments, a small molecule is a modulating agent. In some embodiments, a small molecule is biologically active. In some embodiments, a small molecule is detectable (e.g., comprises at least one detectable moiety). In some embodiments, a small molecule is a therapeutic.

[0059] Specific binding: As used herein, the terms "specific binding" or "specific for" or "specific to" refer to an interaction (typically non-covalent) between a target entity (e.g., a target protein or polypeptide) and a binding agent (e.g., an antibody, such as a provided antibody). As will be understood by those of ordinary skill, an interaction is considered to be "specific" if it is favored in the presence of alternative interactions. In many embodiments, an interaction is typically dependent upon the presence of a particular structural feature of the target molecule such as an antigenic determinant or epitope recognized by the binding molecule. For example, if an antibody is specific for epitope A, the presence of a polypeptide containing epitope A or the presence of free unlabeled A in a reaction containing both free labeled A and the antibody thereto, will reduce the amount of labeled A that binds to the antibody. It is to be understood that specificity need not be absolute. For example, it is well known in the art that numerous antibodies cross-react with other epitopes in addition to those present in the target molecule. Such cross-reactivity may be acceptable depending upon the application for which the antibody is to be used. In particular embodiments, an antibody specific for receptor tyrosine kinases has less than 10% cross-reactivity with receptor tyrosine kinase bound to protease inhibitors (e.g., ACT). One of ordinary skill in the art will be able to select antibodies having a sufficient degree of specificity to perform appropriately in any given application (e.g., for detection of a target molecule, for therapeutic purposes, etc.). Specificity may be evaluated in the context of additional factors such as the affinity of the binding molecule for the target molecule versus the affinity of the binding molecule for other targets (e.g., competitors). If a binding molecule exhibits a high affinity for a target molecule that it is desired to detect and low affinity for non-target molecules, the antibody will likely be an acceptable reagent for immunodiagnostic purposes. Once the specificity of a binding molecule is established in one or more contexts, it may be employed in other, preferably similar, contexts without necessarily reevaluating its specificity.

[0060] Specific: The term "specific", when used herein with reference to an agent or entity having an activity, is understood by those skilled in the art to mean that the agent or entity discriminates between potential targets or states. For example, an agent is said to bind

"specifically" to its target if it binds preferentially with that target in the presence of competing alternative targets. In some embodiments, the agent or entity does not detectably bind to the competing alternative target under conditions of binding to its target. In some embodiments, the agent or entity binds with higher on-rate, lower off-rate, increased affinity, decreased

dissociation, and/or increased stability to its target as compared with the competing alternative target(s).

[0061] Stage of cancer: As used herein, the term "stage of cancer" refers to a qualitative or quantitative assessment of the level of advancement of a cancer. Criteria used to determine the stage of a cancer include, but are not limited to, the size of the tumor and the extent of metastases (e.g., localized or distant).

[0062] Subject: By "subject" is meant a mammal (e.g., a human, in some embodiments including prenatal human forms). In some embodiments, a subject is suffering from a relevant disease, disorder or condition. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. In some embodiments, a subject is an individual to whom therapy is administered.

[0063] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0064] Suffering from: An individual who is "suffering from" a disease, disorder, or condition has been diagnosed with and/or exhibits or has exhibited one or more symptoms or characteristics of the disease, disorder, or condition.

[0065] Susceptible to: An individual who is "susceptible to" a disease, disorder, or condition {e.g., influenza) is at risk for developing the disease, disorder, or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition does not display any symptoms of the disease, disorder, or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition has not been diagnosed with the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition is an individual who has been exposed to conditions associated with development of the disease, disorder, or condition. In some embodiments, a risk of developing a disease, disorder, and/or condition is a population-based risk (e.g., family members of individuals suffering from the disease, disorder, or condition).

[0066] Symptoms are reduced: According to the present invention, "symptoms are reduced" when one or more symptoms of a particular disease, disorder or condition is reduced in magnitude {e.g., intensity, severity, etc.) or frequency. For purposes of clarity, a delay in the onset of a particular symptom is considered one form of reducing the frequency of that symptom.

[0067] Therapeutic agent: As used herein, the phrase "therapeutic agent" refers to any agent that elicits a desired pharmacological effect when administered to an organism. In some embodiments, an agent is considered to be a therapeutic agent if it demonstrates a statistically significant effect across an appropriate population. In some embodiments, the appropriate population may be a population of model organisms. In some embodiments, an appropriate population may be defined by various criteria, such as a certain age group, gender, genetic background, preexisting clinical conditions, etc. In some embodiments, a therapeutic agent is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition.

[0068] Therapeutic regimen : A "therapeutic regimen", as that term is used herein, refers to a dosing regimen whose administration across a relevant population is correlated with a desired or beneficial therapeutic outcome.

[0069] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" refers to an amount of a therapeutic protein (e.g., receptor tyrosine kinases antibody) which confers a therapeutic effect on the treated subject, at a reasonable benefit/risk ratio applicable to any medical treatment. The therapeutic effect may be objective (i.e., measurable by some test or marker) or subjective (i.e., subject gives an indication of or feels an effect). In particular, the "therapeutically effective amount" refers to an amount of a therapeutic protein or composition effective to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect, such as by ameliorating symptoms associated with the disease, preventing or delaying the onset of the disease, and/or also lessening the severity or frequency of symptoms of the disease. A therapeutically effective amount is commonly administered in a dosing regimen that may comprise multiple unit doses. For any particular therapeutic protein, a therapeutically effective amount (and/or an appropriate unit dose within an effective dosing regimen) may vary, for example, depending on route of

administration, on combination with other pharmaceutical agents. Also, the specific

therapeutically effective amount (and/or unit dose) for any particular patient may depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific pharmaceutical agent employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and/or rate of excretion or metabolism of the specific fusion protein employed; the duration of the treatment; and like factors as is well known in the medical arts.

[0070] Treatment: As used herein, the term "treatment" (also "treat" or "treating") refers to any administration of a substance {e.g., provided compositions) that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, and/or condition {e.g., influenza). Such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition. Alternatively or additionally, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and/or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and/or condition.

[0071] Unit dose: The expression "unit dose" as used herein refers to an amount administered as a single dose and/or in a physically discrete unit of a pharmaceutical

composition. In many embodiments, a unit dose contains a predetermined quantity of an active agent. In some embodiments, a unit dose contains an entire single dose of the agent. In some embodiments, more than one unit dose is administered to achieve a total single dose. In some embodiments, administration of multiple unit doses is required, or expected to be required, in order to achieve an intended effect. A unit dose may be, for example, a volume of liquid (e.g., an acceptable carrier) containing a predetermined quantity of one or more therapeutic agents, a predetermined amount of one or more therapeutic agents in solid form, a sustained release formulation or drug delivery device containing a predetermined amount of one or more therapeutic agents, etc. It will be appreciated that a unit dose may be present in a formulation that includes any of a variety of components in addition to the therapeutic agent(s). For example, acceptable carriers (e.g., pharmaceutically acceptable carriers), diluents, stabilizers, buffers, preservatives, etc., may be included as described infra. It will be appreciated by those skilled in the art, in many embodiments, a total appropriate daily dosage of a particular therapeutic agent may comprise a portion, or a plurality, of unit doses, and may be decided, for example, by the attending physician within the scope of sound medical judgment. In some embodiments, the specific effective dose level for any particular subject or organism may depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of specific active compound employed; specific composition employed; age, body weight, general health, sex and diet of the subject; time of administration, and rate of excretion of the specific active compound employed; duration of the treatment; drugs and/or additional therapies used in combination or coincidental with specific compound(s) employed, and like factors well known in the medical arts.

Description of Certain Embodiments

[0072] As described herein, the present invention provides technologies that utilize certain magnetic particles, and particularly utilize targeted such particles, to apply mechanical force to a target structure, through application of dynamic magnetic field (DMF) treatment. In particular embodiments, the present invention provides technologies for applying mechanical force to cellular membranes and/or to intracellular membranes. In some embodiments, the present invention provides technologies for disrupting or otherwise damaging or injuring (e.g., permeabilizing) cellular membranes and/or intracellular membranes. In some embodiments, the present invention provides technologies for inducing or promoting apoptosis and/or cell death or destruction. In some embodiments, the present invention provides technologies for inducing and/or stimulating apoptosis and/or cell death or destruction in malignant (e.g., cancer, tumor, and/or metastatic) cells.

[0073] Among other advantages, provided technologies can achieve application of mechanical force, and particularly can achieve specific application of mechanical force (i.e., to a particular, e.g., preselected, site of interest) without generation of heat. Provided technologies show a variety of advantages as compared with other strategies for inducing and/or stimulating apoptosis and/or cell death or destruction, including those that utilize magnetic particles, many of which generate heat, with undesirable effect(s).

Dynamic Magnetic Field Treatment

[0074] Provided technologies utilize magnetic particles (e.g., superparamagnetic, ferromagnetic, or ferrimagnetic particles, particularly nanoparticles) that respond to application of dynamic magnetic field (DMF) treatment.

[0075] Certain such particles, superparamagnetic iron oxide nanoparticles ("SPIONs"), have found wide-spread applications in the biomedical field spanning in vitro diagnostic tests such as nanosensors, in vivo imaging and therapies such as magnetic fluid hyperthermia or drug delivery. Recent investigations have also explored the capability of controlling the position or temperature of magnetic nanoparticles (e.g., SPIONs) within cells and tissues by remote application of magnetic fields. So far, this possibility has been investigated using permanent magnets that set nanoparticles in a longitudinal motion, using alternating magnetic fields, or through rotating permanent magnets outside of the tissues of interest. In the latter scenario, the nanoparticles describe circular motions, but do not individually rotate around their own axis. The combination of alternating magnetic fields and magnetic nanoparticles allows one to transform energy into forces or heat. Hyperthermia is used as an adjunctive treatment in cancer therapy; here, high-frequency alternating (but not moving) magnetic fields in the KHz/MHz range have been used to kill cancer cells loaded with magnetic nanoparticles through thermal induction. However, such treatment is not without risks particularly near thermally sensitive structures such as the gut or gallbladder if nanoparticles are injected systemically, as the heat induction cannot be controlled spatially with high precision and could cause tissue necrosis. Therefore, in contrast to thermal ablation systems, ambient temperature increases greater than 46°C are not desirable for purposes of remote controlling apoptosis with magnetic fields.

[0076] Fundamentally different from prior studies using high frequency alternating magnetic fields that cause apoptosis via heat induction, the present disclosure describes a principle of controlling particle rotation and inducing apoptosis via mechanical forces exerted on membranes by targeted particles. Specifically, the present disclosure utilizes dynamic magnetic field (DMF) treatment to induce and precisely control the rotation of magnetic nanoparticles around their own axis. DMF treatment creates a dynamic force field, which is converted inside the particle into a magnetic flux field B, which operates on a particle with a magnetic Moment M and a moment of inertia I. The field generates a torque τ equal to τ = μ(Β) χ B . This approach enables for the first time the ability to induce rotation of individual magnetic particles around their own axis, and furthermore allows precise control of the rotation speed. The present invention therefore provides technologies that permit specific application of mechanical force via remote triggering of individual magnetic particle rotation about its own axis. Among other advantages, the present invention achieves such rotation, and therefore such application of mechanical force, without generating heat (e.g., without heating the particle).

[0077] The present disclosure specifically exemplifies induction of this kind of rotation in certain magnetic particles (e.g., superparamagnetic nanoparticles) bound to a target structure, and demonstrates that it can be used to remotely apply mechanical force to a target structure. The present disclosure particularly demonstrates that such magnetic particles can be targeted to an intracellular site, internalized into cells, and bound to the targeted site. Furthermore, the present disclosure demonstrates that subsequent remote activation of a dynamic magnetic field causes the bound particles to exert mechanical force on the target structure. The particular context exemplified here represents a specific biological application; those skilled in the art will appreciate that the same principle should enable many other new applications in the fields of nanotechnology and nanomedicine.

[0078] In some embodiments, provided technologies do not utilize high-frequency (e.g., above the Hz range, for example in the kHz [sometimes considered to be medium- frequency], MHz, or GHz range) magnetic field oscillations. In many embodiments, provided technologies utilize low-frequency (e.g., within the Hz range) oscillations.

[0079] In many embodiments, the present invention applies a DMF treatment (e.g., utilizes a DMF device, for example such as that described in German patent no. DE 10 2005 030 986) that does not heat the particles. In some embodiments, the present invention applies a DMF treatment that does not aggregate the particles. One feature of the present invention is that, in many embodiments, it applies a DMF treatment that rotates individual particles about their axes, without also necessarily moving the particles in a direction (e.g., in a circulatory motion) or moving them in bulk. Such individual rotation about a particle's own axis, as provided herein, is particularly and surprisingly useful in the application of mechanical force, especially to structures such as cell membranes or intracellular membranes, for example as exemplified herein.

Particles

[0080] Particles for use in accordance with the present invention include any particles responsive to DMF as described herein so that their movement, and particularly their rotation about their axis, can be specifically controlled. [0081] In certain embodiments, the present invention utilizes magnetic particles with a high, positive magnetic susceptibility, including for example, superparamagnetic, paramagnetic, ferromagnetic, or ferrimagnetic particles.

[0082] In certain embodiments, the present invention utilizes nanoparticles (i.e., particles whose longest dimension is below 1 um). In certain embodiments, the present invention utilizes nanoparticles whose longest dimension is less than about 300 nm; on some embodiments such nanoparticles are particularly useful for delivery into or onto cells. In certain embodiments, the present invention utilizes nanoparticles whose longest dimension is less than about 200 nm. In certain embodiments, the present invention utilizes nanoparticles whose longest dimension is less than about 100 nm. In certain embodiments, the present invention utilizes nanoparticles whose size is within a range of about 10 - about 500 nm, or within a range of about 10, about 20, about 30, about 40 or about 50 nm to about 100 nm, about 200 nm, or about 300 nm.

[0083] Those of ordinary skill in the art, reading the present specification, would immediately be aware of a variety of appropriate particles, many of which are commercially available, for use in accordance with the present invention. Exemplified herein are

superparamagnetic iron oxide nanoparticles (SPIONs); those of ordinary skill in the art will readily appreciate the extent to which exemplified results can be expected to be generalizable to other particular nanoparticle formats. Relevant considerations include, for example, that larger magnetic moments can be generated with larger particles, but such can also generate unwanted heat. Additionally, some particles (e.g., certain ferromagnetic particles) may tend to aggregate, which could be undesirable particularly in certain biological contexts. Those skilled in the art will appreciate that certain available coating or other technologies may be utilized in some embodiments to adjust particle characteristics (e.g., to reduce accumulation) if desired. [0084] In many embodiments, useful magnetic particles have a metal or metal oxide core

(e.g., iron oxide), and particularly a ferromagnetic metal oxide core, optionally associated or coated with one or more inorganic (e.g., silica, gold) or organic (e.g., polypeptides, small molecule, glycan, lipid and/or nucleic acid) components.

[0085] In some embodiments, useful magnetic particles are characterized in that they lose their magnetism when not exposed to an external magnetic field.

[0086] As noted above, the present specification specifically exemplifies use of superparamagnetic nanoparticles, and particularly superparamegnetic iron oxide nanoparticles (e.g., SPIONs; see Hofmann-Amtenbrink et al "Superparamagnetic Nanoparticles for Biomedical Applications" in Nano structured Materials for Biomedical Applications, 2009). In some embodiments, utilized SPIONs comprise a single ferrimagnetic unit (elementary cell). In some embodiments one elementary cell includes 8 e ⁺ ions. Use of such particles permits precise computation of parameters such as magnetic energy, torque, and/or mechanical forces, as may not be readily feasible with certain larger particles, for example with changing internal structures (e.g., Bloch walls).

[0087] Among other things, the present disclosure establishes that use of specifically targeted DMF-responsive magnetic particles (i.e., magnetic particles associated with a targeting agent that specifically binds to a target structure - e.g., to a target entity or moiety in or on a target structure of interest) shows surprising advantages, even as compared with use of otherwise identical magnetic particles not associated with the targeting agent. Those skilled in the art will be aware of a wide variety of appropriate targeting agents for use in accordance with the present invention, as appropriate for particular target entities or moieties of interest. [0088] In some embodiments, a targeting agent is or comprises a polypeptide, a small molecule, a glycan, a lipid, and/or a nucleic acid. In some particular embodiments, a targeting agent is or comprises an antibody agent. In some embodiments, a targeting agent is or comprises an antibody or antigen-binding portion thereof.

Target Structures

[0089] Those skilled in the art, reading the present disclosure, will immediately appreciate that provided technologies are useful to direct appropriate magnetic particles to any of a variety of target structures, for application of mechanical force thereto. For many biomedical applications, target structures of particular interest are cells or cell structures. In some particular embodiments, target structures are cell membranes or intracellular membranes. In some embodiments, target structures are organelles, for example selected from the group consisting of lysosomes, the endoplasmic reticulum (ER), the golgi apparatus, the mitochondria.

[0090] In some embodiments, a target structure, entity, or moiety is or comprises a surface marker. In some embodiments, a surface marker is or comprises a receptor, an enzyme, a channel, etc.

[0091] In some embodiments, a target structure, entity or moiety is or comprises an intracellular target. In some embodiments, a target structure, entity or moiety is or comprises a marker specifically associated with a disease, disorder, or condition (e.g., with an infectious disease, with cancer, etc.). In some embodiments, a target structure, entity, or moiety is or comprises a surface marker is or comprises component of transcription machinery, a splicosome, or a ribosome. [0092] The present disclosure particularly exemplifies targeting of superparamagnetic nanoparticles to lysosomal membranes, and application of DMF treatment so that individual superparamagnetic particles rotate about their axes and exert mechanical force on the lysosomal membrane. In some embodiments, superparamagnetic nanoparticles are targeted to one or more lysosomal membrane components such as, for example, LAMP-1 (CD 107a), LAMP-2

(CD 107b), or LAMP-3 (CD63).

Applied Forces

[0093] In some embodiments, application of DMF to magnetic particles as described herein exerts a force on the target structure that is within the range of about 10^"9 (i.e., nN range) to about 10^~12 (i.e., pN range); in some embodiments within the range of about 10^"10 to 10^~12 Newtons.

[0094] In some embodiments, application of DMF to magnetic particles as described herein exerts torque on the target structure within the range of about 10^"18 Nm - 10^"20 Nm. In some embodiments, the torque is about 10^"18 Nm.

[0095] In some embodiments, magnetic particles are delivered to a target structure in accordance with the present invention so that they interact with the target structure at a density sufficient to apply a desired force across an appropriate area.

[0096] As described herein, application of DMF treatment to appropriate magnetic particles bound to lysosomes at a density within the range of about 1-60 nanoparticles, on average, per lysosome, is sufficient to achieve lysosomal disruption and/or permeabilization. In some embodiments, magnetic particles are bound to lysosomes at an average density of about 30 nanoparticles/lysosome. Applications

[0097] In some embodiments, application of DMF to magnetic particles as described herein is utilized to disrupt or otherwise damage or injure (e.g., permeabilize) the target structure (e.g., a biological membrane). In some particular embodiments, the technology described herein is utilized to induce apoptosis in cells. Advantageously, such disruption (e.g., permeabilization) and/or induction can be achieved without heating.

[0098] In particular embodiments, the inventive technology is utilized to kill cancer cells.

The cytosol of normal human cells has a neutral pH value (pH about 7). The surrounding of normal human cells in the body has a pH about 7.1, also approximately neutral. Lysosomes, by contrast, have an acidic pH (pH about 5); lysosomal enzymes are typically only active at such low pH. The cytosol of cancerous cells, like that of normal human cells, has a neutral pH value, but their surroundings have an acidic pH. In some embodiments, the present invention provides technologies to apply mechanical force to lysosomal membranes within cancerous cells, so that lysosomal enzymes are released into the cytosol, from which they are pumped out of the cells. In accordance with certain embodiments of the present invention, such released lysosomal enzymes actively become activated in the low-pH extracellular milieu that surrounds cancerous cells. They therefore digest materials, including cancer cells, within that surrounding milieu. Without wishing to be bound by any particular theory, it is contemplated that such released lysosomal enzymes will become inactivated once cancerous tissue is destroyed, as normal tissues have a neutral pH milieu. Exemplification

Example 1:

[0099] The present Example demonstrates that use of DMF to induces rotation as described herein in targeted superparamagnetic iron oxide nanoparticles (SPIONs) can be used to remotely activate apoptosis. Specifically, the present Example demonstrates that SPIONs conjugated with LAMP1 (Lysosomal-associated membrane protein 1) antibodies (LAMP1- SPION) internalize into cells and bind to lysosomal membranes. This Example further demonstrates that subsequent remote activation of the dynamic magnetic field causes mechanical disruption and/or permeabilization of lysosomes, which leads to apoptosis via extravasation of lysosomal contents into the cytoplasm and a decrease of intracellular pH. As those skilled in the art will appreciate, certain examples of SPIONs conjugated with LAMP1 antibodies are described in Gruttner et al, "Synthesis and Antibody Conjugation of Magnetic Nanoparticles with Improved Specific Power Absorption Rates for Alternating Magnetic Field Cancer

Therapy," Journal Magnetism and Magnetic Materials 2007.

RESULTS

Dynamic magnetic field stimulation results in rotation of individual nanoparticles

[00100] A DMF generator as described in German Patent No: DE 10 2005 030 986 was utilized to control directional movement and self-centered rolling. Fig. 2A shows a schematic representation of a DMF generator utilized in the present Exemplification. The device controls rotation and movement of magnetic nanoparticles (e.g., SPIONs) with a low frequency (10-40 Hz) field. The DMF generator causes the nanoparticles to rotate around their own axis. To demonstrate the pattern of the particle movement, the rotation of larger magnetic beads of different sizes (5.8, 1, 0.5 and 0.3 μηι diameter) was first monitored by filming them in a cell culture dish under a microscope. Once the DMF is switched on, the beads start to rotate around their own axis, which also causes a slow directional movement of the beads across the floor of the dish, as shown in Figs. 3A-D. The present study clearly demonstrated that the applied DMF treatment enables a self-turning of magnetic particles. The speed of rotation can be controlled by varying the frequency setting on the DMF device. This observation suggested that the DMF could be used to contrive a virus-like interaction between the SPIONs and the cell surface, which in turn could enhance internalization of the SPIONs into the cytosol. Once the SPIONs have internalized into the intracellular compartments, e.g. endosomes or lysosomes, the loaded SPIONs can be operated non-invasively by DMF to regulate the cellular compartmental activities and further cell functions, as for example, shown in Fig. 2B.

Dynamic magnetic field stimulation enhances uptake of superparamagnetic nanoparticles

[00101] Internalization of SPIONs into living cells was reported previously. In the present study, the internalization efficiency in the absence or presence of DMF stimulation is first evaluated. To monitor the process of SPION internalization, the present study used fluorescently labeled (TRITC) 100-nm SPIONs and incubated them with rat insulinoma cells (INS-1). 300-nm SPIONs were also tested, but they exhibited markedly lower loading efficiency. The present study then applied a DMF field for 20 min. at a frequency of 20 Hz, before staining the cells using the plasma membrane marker CellMask, the lysosomal marker LysoTracker Green and the nuclear marker Hoechst 34580. The cells were then imaged by live confocal microscopy, which demonstrated that the majority of the SPIONs were loaded into the lysosomes after 20 min. of DMF treatment, as shown in Fig. 4A. Fig. 4B shows 71.2±3.8% of SPIONs colocalized with the lysosomal marker LysoTracker Green, while only 18.2±2.2% of SPIONs colocalized with the plasma membrane and early endosome probe CellMask. Conversely, Fig. 4C shows 91 ±8.7% of the LysoTracker Green fluorescence appeared in conjunction with SPIONs, which means that nearly all lysosomes contained several loaded SPIONs.

[00102] Next the present study conjugated SPIONs with an antibody against the lysosomal membrane protein, LAMP1 (LAMPl-SPION). The loading efficiencies between SPION and LAMPl-SPION were compared in order to evaluate if the conjugation of the LAMP1 antibody enhanced internalization and loading into the lysosomes. LAMPl-SPION nanoparticles were more efficiently loaded into the lysosomes than the unconjugated SPIONs, and the loading efficiency after 20 min. Fig. 4D shows the loading efficiencies of DMF treatment averaged 13.3±2.3% and 21.2±2.4% for SPIONs and LAMP 1 -SPIONs, respectively.

Dynamic magnetic field stimulation can injure lysosomes via antibody-conjugated superparamagnetic nanoparticles

[00103] To evaluate whether DMF treatment has the potential to injure lysosomes in

LAMPl-SPION-loaded cells, the present study visualized the lysosome compartment with the marker LysoTracker Green. After DMF-facilitated loading of the LAMPl-SPION nanoparticles, the cells were cultured at 37 °C for 40 min to allow binding of the antibody paratope on the nanoparticles to LAMP1 in the lysosomal membrane. After that culture period, any remaining LAMPl-SPION nanoparticles outside the cells were removed by washing, before subjecting the cells to DMF treatment (20 Hz) for 20 minutes.

[00104] The capability of the DMF treatment to disrupt and/or permeabilize the compartments of the lysosomes was evaluated by assessing changes in LysoTracker Green fluorescence intensity. Indeed, in LAMPl-SPION-loaded cells, the DMF treatment significantly decreased LysoTracker Green fluorescence by 75% as compared to cells loaded with

conventional SPIONs without the LAMP1 antibody (483.6±84.2 A.U. vs. 120.3±20.9 A.U. for SPION- and LAMPl-SPION-treated cells, respectively, as shown in Figs. 5A and 5B. To confirm these findings, the present study next used the acidotropic probe (pKa=5.2) LysoSensor Green DND 189 as described in Eto et al., "Glucose Metabolism and Glutamate Analog Acutely Alkalinize Ph of Insulin Secretory Vesicles of Pancreatic Beta-Cells," American Journal of Physiology Endocrinology and Metabolism 2003. The rationale for this experiment was that disruption of lysosomes would reduce the volume of the very acidic compartments in the cell and lead to a decrease in LysoSensor Green fluorescence. The results are shown in Figs. 5C and 5D.

[00105] Prior to DMF treatment, the present study found no difference in fluorescence intensity between SPION- and LAMPl-SPION-loaded cells. DMF treatment had no effect in SPION-loaded cells. In contrast, in LAMPl-SPION-treated cells fiuorescence intensity dropped (769.5±82.5 A.U./cell vs. 368.4±69.6 A.U./cell in SPION- vs. LAMP1-SPION treated cells, respectively; P<0.001). In light of the low frequency of the alternating magnetic fields utilized herein, heat induction, which is common if not ubiquitous when high frequency alternating magnetic fields are utilized, was not expected to occur. The present study confirmed the absence of heat induction by monitoring temperature runs in phantoms. These experiments showed that no significant change in temperature was caused by the DMF induced magnetic field, as shown in the temperature plot of Fig. 6. Taken together, these results suggest that the remote application of the DMF treatment causes permeabilization of lysosomal compartments, induced via the torque of the membrane-bound LAMPl-SPIONs. Effects of dynamic magnetic field stimulation of superparamagnetic nanoparticles in human primary cells

[00106] To monitor the LAMP 1 -SPION loaded lysosomes, SPIONs are covalently attached to both LAMPl antibodies and the fluorescence marker TRITC (TRITC -LAMP 1 - SPION). To further validate the possibility to translate the present findings to clinical settings, the present study used isolated primary cells from human pancreatic islets and seeded on glass bottom petri dishes. Then TPJTC-LAMPl -SPIONs were added to cell culture medium, followed by DMF treatment or no treatment, and finally the cells were fixed for confocal microscopy.

[00107] Human primary islet cells contained larger lysosomes than INS-1 cells as assessed by TRITC fluorescence (as shown in Fig. 7A, upper images). To assess co-localization of LAMPl and TRITC-LAMPl -SPION, LAMPl was detected by indirect immunocytochemistry using a Cy2 -tagged secondary antibody. In cells not exposed to DMF treatment, LAMPl and TRITC-LAMPl -SPION co-localized by 49.9±9.2%, which was not significantly affected by DMF treatment (54.8±9.5%) (Data not shown). These results indicate that the bonds between LAMPl and TRITC remain stable during DMF treatment. Interestingly, the TRITC-LAMPl - SPIONs loaded into the cells mainly appeared around the boundaries of structures, which are likely to represent a location in the lysosomal membrane, as for example shown in Fig. 7B. However, after a second round of DMF treatment, most of the LAMPl was apparently separated from the TRITC-LAMPl -SPIONs (as shown in Fig. 7A, bottom images) and the SPIONs aggregated tightly (as shown in Fig. 7C).

[00108] DMF treatment also led to a marked downward shift in the distribution of lysosomal sizes, as for example shown in Fig. 7D, and accordingly the average size of lysosomes decreased from 1.77± 0.06 μιη (n=141) to 0.79±0.05 μιη (n=105) after DMF treatment, as shown in Fig. 7E. These results suggest that the second round of DMF treatment in cells loaded with TRITC-LAMP1 -SPIONs results in a certain degree of damage to the lysosome membrane. However, a potential alternative explanation could be that the detachment of TRITC-LAMP1- SPIONs occurs from the lysosome membrane without disruption which can lead to the particles aggregating in the center of the still intact lysosomes.

[00109] To address this possibility, the subcellular locations of the LAMP 1 -SPIONs were further identified by transmission electrical microscopy (TEM). The TEM images clearly showed that LAMP1-SPION particles had accumulated within intracellular compartments after loading into cells (as shown in Fig. 7F, left). In contrast, after the second round of DMF treatment, the LAMP 1 -SPIONs were scattered throughout the cells (as shown in Fig. 7F, right). These results demonstrate that DMF-induced rotational movement of LAMP 1 -SPIONs are also capable of disrupting lysosomal membranes in human primary cells.

Consequences of DMF-mediated disruption of lysosomes

[00110] Disruption of lysosomes has previously been reported to activate apoptotic reactions. To determine whether DMF treatment can elicit apoptosis in LAMP 1 -SPION-loaded cells, the present study measured the extent of apoptosis in INS-1 cells with and without DMF. Annexin V and 7-AAD were used to indicate early and late stage apoptosis, respectively (as shown in Fig. 8A). After DMF treatment, early and late apoptosis in LAMP1-SPION loaded INS-1 cells significantly increased from 4.56±0.55% to 12.45±1.6% and from 0.73±0.17% to 1.31±0.16%, as evidenced by positive staining for Annexin V or 7-AAD, respectively (shown in Figs. 8B and 8C). Furthermore, the elevated rates of apoptosis also had consequences on cell proliferation during culture. A single 20-minute DMF treatment (20 Hz) in SPION-loaded cells had no significant effect on cell number during a 6-days culture period when compared to control cells. In contrast, the number of LAMP1-SPION loaded cells after DMF treatment was significantly (p < 0.001) lower from day 2 and onwards (as shown in Fig. 8D). These results indicate that the attack on lysosomes via DMF-activated lysosomal membrane -targeted SPIONs prompts apoptotic cell death and affects the growth of the cell population.

DISCUSSION

[00111] The present Example describes a novel biomedical platform based on a unique dynamic magnetic field generator, which in combination with superparamagnetic nanoparticles can be utilized for various new applications.

[00112] Several prior studies have investigated the effect of 'alternating magnetic fields' on magnetic nanoparticles. These studies generally used high-frequency (typically MHz range), or at least medium-frequency (e.g., kHz range), alternations in the magnetic field polarity, and observed that targeted iron oxide nanoparticles caused damage to cellular membranes leading to permeabilization. In most such studies, energy dissipated locally as heat by the iron oxide nanoparticles was thought to lead to (and therefore presumably to be required for) disruption of lipid bilayers. Huang et al., "Remote Control of Ion Channels and Neurons through Magnetic- Field Heating of Nanoparticles," Nature Nanotechnology 2010 demonstrated a temperature- induced change in fluorescence when a fluorochrome was attached to a magnetic nanoparticle and exposed to an alternating magnetic field, whereas no change was observed in free

fluorochromes.

[00113] In contrast to these reports using high-frequency alternating (but not dynamic) magnetic fields, the present DMF approach uses low-frequency (~ 10-20 Hz), dynamic {i.e. moving) magnetic fields of ~30 mT that uniquely induce rotation of every individual particle in the field around their own axis (as illustrated in Fig. 2B). The speed of this rotation (and its direction) can be controlled by varying the frequency setting on the device. For example, at lower frequencies nanoparticles can be prompted to roll over cell membranes, which may mimic the movement of viruses along cell membrane surfaces, as for example, described in Burckhardt et al., "Virus Movements on the Plasma Membrane Support Infection and Transmission between Cells,". PLoS Pathogen 2009, and increase the efficiency with which nanoparticles internalize into cells. Higher rotational speeds can be used in order to destroy particular targets via rotational shear forces without inducing unwanted thermal effects. Molecular simulations of lipid bilayers have shown that both incremental shear and tension can destabilize cell membranes, and that the energy that is required to cause such membrane damage can be achieved with rotating magnetic nanoparticles.

[00114] The present study has applied the DMF on LAMP1 antibody-conjugated superparamagnetic iron oxide nanoparticles to facilitate nanoparticle uptake into cells and to disrupt the lysosomal membrane as a means to induce cell apoptosis. The DMF approach described herein has at least two major advantages: 1) Nanoparticles can be rotated around their own axis, and 2) No significant heat is created.

[00115] Heat creation is the presumed mechanism of how high-frequency alternating magnetic fields cause damage to cell membranes as suggested in Ivkov, "Application of High Amplitude Alternating Magnetic Fields for Heat Induction of Nanoparticles Localized in Cancer," Clinical Cancer Research 2005. This however can potentially cause extensive and unspecific cellular necrosis. By contrast, the DMF technology described herein does not induce heating; when applied in cellular systems or organisms, therefore, temperature at the site can be maintained within the physiological temperature range (e.g., typically considered to be within a range of about 37 °C to about 42 °C). Technologies described herein achieve induction of apoptosis specifically in nanoparticle-loaded cells only. Apoptotic cells are removed in vivo by endogenous scavenger systems e.g. the innate immune system and macrophages. Tissue damage is thus limited to only the targeted cells, in contrast to procedures leading to supraphysiological temperatures and resultant necrosis, potentially sparing wide-spread acute inflammatory reactions.

[00116] In some embodiments, provided methodologies achieve a particularly high degree of specificity of nanoparticle-mediated intervention, by targeting of nanoparticles to a particular cell type and/or into a desired subcellular compartment is important. Among other things, in some embodiments, the provided technology platform represents an example of unique utilization of DMFs to target magnetic nanoparticles to specific intracellular compartments.

[00117] Previous reports have demonstrated the usefulness of magnetic nanoparticles for controlling activity of plasma membrane receptors or ion channels. Upon protracted stimulation of receptors or ion channels there is solid evidence for down-regulation of their activities. One important mechanism of such desensitization is internalization of receptors or ion channels to intracellular sites where they reside in an inactive standby pool. In the context of nanoparticle- mediated activation of receptors or ion channels this means that after activation, the number of the receptors/ion channels in plasma membrane decreases and the desired cellular signals and responses are blunted. Moreover, the magnetic nanoparticles themselves are internalized already after short incubation with live cells via the endocytotic pathway, as for example described in Gupta et al, "Cytotoxicity Suppression and Cellular Uptake Enhancement of Surface Modified Magnetic Nanoparticles," Biomaterials 2005 and Bogart et al., "Photothermal Microscopy of the Core of Dextran-Coated Iron Oxide Nanoparticles During Cell Uptake," ACS Nano 2012 .

[00118] In some embodiments, the present invention makes use of this property of internalization and facilitates the process by DMF treatment to accelerate delivery of

nanoparticles (e.g., as exemplified, of LAMP 1 antibody-attached nanoparticles) to intracellular compartments, and specifically to the lysosome. Following down the endocytotic pathway, the targeted nanoparticles enter the early- and late-endosomes and afterwards they should enter other compartments, e.g. ER, via recycling endosomes, or being removed from the lysosomes. In the lysosomes, the targeting agent associated with its nanoparticle recognizes and binds to its target moiety (e.g., LAMP1, which is highly expressed in the lysosomal membrane). In response to a low frequency DMF, as employed in the disclosure herein, the now bound nanoparticle generates dynamic forces strong enough to tear the lysosomal membrane, leading to destruction of the lysosome integrity, leakage of lysosomal enzymes and finally induction of apoptosis.

[00119] In INS-1 cells, the present study observed that the SPIONs mostly loaded into lysosomes after 20 minutes DMF treatment (Fig. 4A). This preferential lysosomal localization was also observed when SPIONs were located into primary human pancreatic islet cells (as shown in Fig. 7A). However, as the direction and extent of intracellular membrane trafficking may differ markedly between cell types, those of skill in the art will appreciate that adjustments to lysosome loading efficiency may be desirable when this technology is applied to other cell types. Those skilled in the art will appreciate that this technology can provide tools to regulate specific subcellular compartmental functions including not only the nuclei, but also

compartments such as the ER, Golgi apparatus and different types of endosomes along the intracellular membrane trafficking system. [00120] Those skilled in the art will also appreciate that adjustments (e.g., stronger fields etc.) may be desirable, when applying embodiments of provided technology to in vivo contexts. For example, the particular DMF field strength utilized in the present Example was about 30 mT, and may have a reach of up to about 1 cm, though such penetration was not required (reach of even 1 mm being sufficient in certain instances). Those skilled in the art will appreciate that stronger fields (e.g., potentially within the range of mT to T) may be required for certain in vivo applications, for example in order to reach deep organs, which may well involve penetration of 10 cm, 20 cm, 30 cm, 40 cm, or more. Regardless, ultimate clinical translation using presently disclosed low frequency DMF approach may be more straightforward than using high-frequency alternating fields, among other things because the DMF fields are not expected to cause nonspecific heating of tissues through induced eddy currents and should therefore have a better safety profile.

[00121] The achievement of rotational control on nanoparticles with the DMF method as described herein has many other potential applications beyond the model systems used here, in both biomedical and non-biological nanotechnology fields. To give but a few examples, magnetic actuation has been shown to control timing and drug release from vesicles containing iron oxide nanoparticles. An increase in permeability of lysosomal membranes not only can be used to promote apoptosis, such as through the release of proteolytic enzymes and increase in reactive oxygen species, but can also increase the efficacy of drugs trapped in lysosomes.

Because sequestration of drugs in lysosomes is responsible for up to 40% of whole tissue drug uptake, lysosomal drug trapping plays an important role in the development of tumor drug resistance. Thus, is some embodiments, provided DMF-mediated lysosomal membrane permeabilization technologies could be used to treat cancer drug resistance. CONCLUSIONS

[00122] In summary, using a unique dynamic magnetic field (DMF) generator the present exemplification can control rotational movements of superparamagnetic iron oxide nanoparticles (SPIONs) in solution. This rotational nanoparticle movement was applied for remote induction of cell death by injuring lysosomal membrane structures. SPIONs are covalently coated with antibodies targeting the lysosomal protein marker LAMPl (LAMPl-SPION). Remote activation of slow rotation of LAMPl -SPIONs with 20 Hz for 20 minutes, significantly improved the efficacy of cellular internalization of the nanoparticles. It is observed that 71.2=1=3.8% of LAMPl -SPIONs accumulated along the membrane in lysosomes in rat insulinoma tumor cells due to binding of LAMPl -SPIONs to endogenous LAMPl . Further activation of torque by the LAMPl -SPIONs bound to lysosomes resulted in rapid decrease in size and number of lysosomes, attributable to tearing of the lysosomal membrane by the shear force of the rotationally activated LAMPl -SPIONs. This remote activation resulted in increased cell apoptosis and impaired cell growth. The findings of the present disclosure suggest that DMF treatment of lysosome -targeted nanoparticles offers a non-invasive tool to induce apoptosis remotely, providing an important platform technology for a wide range of biomedical applications.

METHODS

[00123] Nanoparticle assembly. The protocol of conjugation of LAMPl antibodies to magnetic nanoparticles was described previously, for example, in Gruttner, "Synthesis and Antibody Conjugation of Magnetic Nanoparticles with Improved Specific Power Absorption Rates for Alternating Magnetic Field Cancer Therapy," Journal Magnetism and Magnetic Materials 2007. Briefly, SPION nanoparticles (Micromod, Germany) were amino- functionalized and the density of amino groups per mg of particles was determined. After washing, the other parts of amino groups were reacted with sulfo-SMCC in PBS-EDTA buffer to introduce maleimide groups on the particle surface. The monoclonal LAMP1 antibody (Abeam, UK) was purified with a G-25 column containing PBS/EDTA buffer to remove the glycerin and sodium azide. After purification the LAMP1 antibody was treated with imnothiolane solution to introduce the SH groups. After washing the SH-modified antibody with PBS-EDTA buffer in a G-25 column, the maleimide -modified particles were added. The particles were shaken for 1 h at room temperature. Then cystein was added to quench the remaining reactive sites. Finally the particles were purified with PBS buffer in magnetic columns in a high gradient magnetic field.

[00124] DMF device. The dynamic fields to control SPION rotation used in this study were created with a DMF generator (DM-01, Feldkraft and Stetter Elektronik, Germany). The device consists of an array of multiphase coil systems, where the coils are displaced against each other. The field can be altered in a highly dynamic fashion. A device-integrated digital controller regulates the frequency as well as the magnetic flux. The dynamic flux produces an electromagnetic gradient force F = (mV)^■ B , where m are the magnetic moments of the beads in total and B is the dynamic flux density field vector. This vector is established by the H field of the present device. The magnetic field strength generated by the DMF device used in this study is approximately as large as 30 mT (rms).

[00125] Live cell imaging. Cells were seeded onto glass coverslips. After DMF treatment, live images were acquired using a Zeiss 510 Meta confocal system with a x 40 water immersion objective (NA=1.2). SPION-TRITC was visualized by excitation at 543 nm and emitted light collected using a long-pass 560 nm filter. The pinhole was ~1 airy unit and the scanning frame was 512x512 pixels. The cellular location of SPION was stained with the plasma membrane marker CellMask (Invitrogen, USA), lysosome marker LysoTracker Green (Invitrogen, USA) and a marker for cell nuclei, Hoechst 34580 (Invitrogen, USA). Image analysis was performed with ZEN 2009 software (Zeiss, Germany). Co-localization was analyzed by a Pearson's efficiency methods within ZEN 2009. The SPION-TRITC co- localization of lysosomes was calculated by coefficient CSPION= ^{Plxelcolocallzed} χ 100. Likewise,

Pixel_{to ta}i

the LysoTracker-labeled lysosomes were counted by coefficient Ci_vsosome= ^{Plxelcolocallzed} χ 100.

^J > Pixeltotai

The coefficient is reported as a percentage from 0-100, with 0 meaning no co-localization and 100 meaning all pixel-pairs are co-localized. Since TRITC labelled SPIONs were loaded into all the INS-1 cells after DMF treatment, the present study used the fluorescence intensity of TRITC to indicate the amount of SPIONs in a cell after 3 times washing. The total SPION loading efficiency was calculated by percentage ₀f ^FAnnexm V^-AAD) _{χ100 num}¾_{er 0}f fluorescence intensity

'' Hoechst 34580

of TRITC to the intensity of Hoechst 34580 which reflects cell number in a field.

[00126] Transmission electron microscopy (TEM) imaging. The preparation of cells for TEM is now discussed. Briefly, INS-1 insulinoma tumor cells were treated with DMF, followed by fixation in 2.5% glutaraldehyde for 1 hour at 4 °C. The cells were then treated with 1% osmium tetroxide, dehydrated, and embedded in AGAR100 (Oxford instruments Nordiska AB, Stockholm) before being sliced in ultra-thin sections (70-90 nm). After slicing, the samples were placed on Cu grids and contrasted with uranyl acetate and lead citrate. The TEM images were obtained using a JEM 1230 electron microscope (Jeol-USA, Peabody, MA, USA).

[00127] Human islet cell Immunostaining. Human islets were provided by the

EXODIAB Human Tissue Lab and the Nordic Network of Clinical Islet Transplantation Programme (www.nordicislets.org). The islet cells were separated in a Ca²⁺-free solution at 37 °C for 10 minutes and treated with DMF after 12 hour culture on the cover slips centered dish (MatTek, Germany). Then the cells were fixed with 3% PFA-PIPES and 3% PFA-Na2B04 for 5 min. and 10 min., respectively, followed by permeabilization with 0.1% Triton-X 100 for 30 min. and blocked with 5% normal donkey serum in PBS for 15 minutes. Guinea pig sourced antibody against insulin (EuroProxima, Netherlands) was diluted in 5% block buffer and incubated overnight at 4 °C. Immunoreactivity was done using fluorescently labeled secondary antibodies: cy2 anti-guinea pig (1 :400) and cy5 anti-mouse (1 :400). The images of SPION- TRITC (EX: 543 nm), cy2-labeled insulin (EX: 488 nm) and cy5-labeled LAMP1 (EX: 633 nm) were visualized through three or four channels by a confocal system (Zeiss, Germany).

Lysosomal size (LAMP1 marked) was calculated using the profile function of the ZEN 2009 software based on the shapes of the fluorescence areas.

[00128] Intracellular pH measurement. INS-1 cells were seeded on the glass centered dish (MatTek, Germany), loaded with SPION or SPION-LAMPl and treated by DMF. Then the cells were incubated with 1 μΜ acidic indicator, LysoSensor Green DND 189 (Invitrogen, USA) for 30 min. in 37 °C. After incubation, the cells were washed out and the fluorescence images were acquired by confocal microscopy. The average fluorescence intensity per cell was measured using the ZEN 2009 software and further applied for quantitative analysis.

[00129] Cell apoptosis detection. FITC-Annexin V (51-65874X, BD, USA) and 7-AAD (51-68981E, BD, USA) were used to assess early and late apoptosis of INS-1 cells. After DMF treatment, the INS-1 cells were incubated with the dyes at 37 °C for 30 minutes. Images were acquired under identical conditions (using the same settings for pinhole (1 airy unit), exposure time, gain and scanning speed). Fluorescence intensity was analyzed by the ZEN 2009 software and early/late stage apoptosis was quantified by percentage of ^ ⁾ _xioo number of

Annexin V/7-AAD positive cells to the number of Hoechst 34580 stained cells. [00130] Proliferation. INS-1 cells were seeded on 24-well plates loaded with SPION or SPION-LAMP1 nanoparticles and treated by DMF once/day. Then the cell number was calculated by a plate cytometer after 6 hours of DMF treatment. Cells were counted once per day for 5 days.

[00131] Temperature measurements. A dish with 100 nm SPIONs (lOmg/ml) in water and a dish without the SPIONs in water as a control, each containing a temperature sensor (Radial leaded glass-encapsulated NTC thermistor, EPCOS Inc., Germany), were placed simultaneously on the DMF device. The dishes were then subjected to the DMF field for 20 minutes at 20 Hz and the temperature recorded in each dish.

[00132] Statistical analysis. The data was presented as average ± standard error of the mean (S.E.M.). Statistical comparison of paired-factors experiments was performed by Student's T-test and one-way analysis of variance (ANOVA) and the Friedman tests were performed for multi-factor experiments which have more than two group treatments in one experiment.

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Claims

What is claimed is:

1. A method of applying mechanical force to a target structure, the method comprising: exposing a target structure to magnetic particles so that the particles bind to the target structure;

applying dynamic magnetic field (DMF) treatment to the nanoparticles sufficient to cause rotation of the particles, so that mechanical force within the range of about nN to about fN is applied to the target structure but the particles are not significantly heated.

2. The method of claim 1, wherein the particles are or comprise nanoparticles.

3. The method of claim 1 or 2, wherein the particles are or comprise superparamagnetic nanoparticles.

4. The method of claim 3, wherein the particles are or comprise iron oxide nanoparticles ("SPIONs").

5. The method of claim 1, wherein the superparamagnetic nanoparticles are characterized by an iron oxide core.

6. The method of claim 1 or 5, wherein the particles are characterized in losing their magnetism when not exposed to an external magnetic field.

7. The method of claim 1 or 6, wherein the particles are characterized in maintaining their magnetism when not exposed to an external magnetic field.

8. The method of claim 1, wherein the superparamagnetic nanoparticles are associated with a targeting agent that specifically binds to the target structure.

9. The method of claim 8, wherein the targeting agent is covalently linked to the superparamagnetic nanoparticles.

10. The method of claim 8 or 9, wherein the targeting agent is or comprises an antibody agent.

11. The method of claim 8 or 9, wherein the targeting agent is or comprises a polypeptide, a small molecule, a glycan, a lipid, or a nucleic acid that specifically binds to a target moiety in or on the target structure.

12. The method of claim 11 , wherein the target moiety is or comprises a polypeptide.

13. The method of claim 11, wherein the target moiety is or comprises a glycan.

14. The method of claim 11 , wherein the target moiety is or comprises a nucleic acid.

15. The method of claim 1, wherein the target structure is or comprises a cell membrane.

16. The method of claim 15, wherein the target structure is or comprises a tumor-associated marker.

17. The method of claim 16, wherein the target structure is or comprises an ion channel.

18. The method of claim 1, wherein the target structure is or comprises an intracellular membrane.

19. The method of claim 18, where the target structure is or comprises a lysosomal membrane.

20. The method of claim 1, wherein the target structure is or comprises an intracellular entity.

21. The method of claim 20, wherein the intracellular entity is or comprises an organelle.

22. The method of claim 21 , wherein the organelle is selected from the group consisting of the endoplasmic reticulum (ER), the golgi apparatus, the mitochondria, and combinations thereof.

23. The method of claim 21 , wherein the intracellular entity is or comprises component of transcription machinery, a splicosome, or a ribosome.

24. The method of claim 20, wherein the target structure is or comprises a lysosomal membrane and the superparamagnetic nanoparticles are associated with a targeting agent that specifically binds to the target structure.

25. The method of claim 24, wherein the targeting agent is covalently linked to the

superparamagnetic nanoparticles.

26. The method of claim 25, wherein the targeting agent specifically binds to a target moiety on the surface of the lysosomal membrane.

27. The method of claim 26, wherein the target moiety is or comprises LAMP-1 (CD 107a), LAMP-2 (CD 107b), or LAMP-3 (CD63).

28. The method of claim 1, wherein the step of exposing comprises exposing the target structure to the superparamagnetic nanoparticles so that the nanoparticles bind to the target structure with a density sufficient to apply a desired force across a relevant area.

29. The method of claim 28, wherein the step of exposing comprises exposing the target structure to the superparamagnetic nanoparticles so that, on average, about 1 to about 60 superparamagnetic nanoparticles become bound to each lysosome.

30. The method of claim 29, wherein the step of exposing comprises exposing the target structure to the superparamagnetic nanoparticles so that, on average, about 10 to about 50 superparamagnetic nanoparticles become bound to each lysosome.

31. The method of claim 29, wherein the step of exposing comprises exposing the target structure to the superparamagnetic nanoparticles so that, on average, about 30 superparamagnetic nanoparticles become bound to each lysosome.

32. The method of claim 1, wherein the step of applying DMF treatment comprises applying a DMF field with a strength within the range of about mT to T.

33. The method of claim 1, wherein the step of applying DMF treatment comprises applying a DMF field with a reach within the range of about mm to about cm.

34. The method of claim 33, wherein the step of applying DMF treatment comprises applying a DMF field with a reach of at least 1 cm.

35. The method of claim 33, wherein the step of applying DMF treatment comprises applying a DMF field with a reach of at least 2-5 cm.

36. The method of claim 33, wherein the step of applying DMF treatment comprises applying a DMF field with a reach of at least 10 cm.

37. The method of claim 33, wherein the step of applying DMF treatment comprises applying a DMF field with a reach of more than 10 cm.

38. The method of claim 1 or 3, wherein the target structure is or comprises a tumor- associated entity.

39. The method of claim 38, wherein the tumor-associated entity is or comprises a cell- surface entity.

40. The method of claim 39, wherein the tumor-associated entity is or comprises and intracellular entity.