HK40069974A - Compositions and methods related to scavanger particles - Google Patents

Description

Compositions and methods relating to particle removal

The present application is a divisional application of an invention patent application having a chinese patent application No. 201680049849.5 entitled "composition and method relating to particle removal" filed on year 2018, month 06, 29, filed as PCT international application PCT/US2016/040022, filed 2016, month 06, 29, and entered the chinese national stage at year 2018, month 02, 27.

Priority

Priority of this application is claimed for U.S. provisional patent application No. 62/186,838 filed on 30/6/2015, U.S. provisional patent application No. 62/198,519 filed on 29/7/2015, U.S. provisional patent application No. 62/198,541 filed on 29/7/2015, U.S. provisional patent application No. 62/236,507 filed on 2/10/2015, and U.S. provisional patent application No. 62/319,092 filed on 6/4/2016, each of which is incorporated herein by reference in its entirety.

Technical Field

The present invention relates to compositions and methods relating to particle removal.

Background

Many anticancer therapies available clinically or under development involve the ability to stimulate the immune system to recognize or destroy cancer, or both. The most prominent three are anti-checkpoint inhibitors from the Potentilla-Stokes (Bristol-Myers Squibb)(Iplilimumab), available from Merck(Pembrolizumab, formerly Raolizumab (lambrolizumab)). However, these and other pathways involve a net up-regulation of the immune system of the subject, inducing potentially severe symptoms like autoimmune disorders and/or other significant side effects.

There is a need in the art for more effective pharmacological approaches to overcome cancer, particularly metastatic cancer, without disturbing the ability of the subject to avoid autoimmunity. The present disclosure provides, among other things, methods and compositions based on alternative approaches to combat cancer using the subject's autoimmune system, including de-inhibition of the tumor microenvironment, i.e., weakening the tumor's defense system, without stimulating immune cells.

Disclosure of Invention

The present disclosure provides, among other things, compositions that bind to and inhibit the biological activity of biomolecules (particularly soluble biomolecules) and pharmaceutical compositions thereof. The present disclosure also provides for applications in which the compositions are useful. For example, the compositions described herein are useful for inhibiting proliferation, growth, and/or survival of cells (e.g., cancer cells). Furthermore, the compositions described herein are useful for the prevention and/or treatment of aging, metabolic disorders, and neurodegenerative diseases. In another embodiment, the compositions described herein can be useful for binding to and neutralizing toxins (e.g., animal, bacterial, and/or plant toxins), viruses, or other foreign compounds in the circulation of a subject.

Drawings

FIG. 1 depicts an exemplary embodiment of a soluble form of particles that bind to TNF receptor (sTNF-R). The particles are about 1 cubic micron. The interior surface of the particle includes an immobilized TNF agent that is capable of binding to and sequestering (clearing) the sTNF-R target from its natural ligand, thereby inhibiting interactions between the sTNF-R target and other proteins and cells. The inner surface of the particle defines a boundary that includes a void space.

Fig. 2 depicts an exemplary embodiment of a particle comprising a soluble form of a TNF agent that binds to a TNF receptor (sTNF-R) target. The three particles shown in FIG. 2 are depicted as molecules that have bound 0, 3, or 10 sTNF-R targets. Although the TNF agent and sTNF-R target are not shown to scale, the circular particles have a diameter of about 175 nm. The interior surface of the particle contains an immobilized TNF agent that is capable of binding to and sequestering (clearing) the sTNF-R target from its natural ligand, thereby inhibiting interactions between the sTNF-R target and other proteins and cells. The interior of the annular particle includes a void space.

Fig. 3 depicts an exemplary embodiment of a particle comprising protrusions. The particles on the left side of the figure are octahedra with a core with longest dimension of 100 to 150 nm. The particles on the right side of the figure are icosahedrons with cores having longest dimensions of 200 to 300 nm. Each particle also includes a molecular bulge directed outward from the apex of the core polyhedral structure. Particles are depicted as including agents shown in dark gray, and some particles are depicted as having bound a target (e.g., a biomolecule) that is shown in light gray and identified as 0 or 3 "traps. These protrusions act as "cell reflectors" that inhibit the interaction between the target of the agent bound to the particle and the cell surface. The representations of particles, projections, agents and bound targets in fig. 3 are not necessarily shown to scale.

Fig. 4 consists of two pictures, labeled pictures (a) and (B). Panel (a) depicts submicron particle packing within a particle comprising a core submicron particle and a protective submicron particle, wherein each submicron particle is substantially spherical and approximately the same size. However, the particles may comprise submicron particles of different shapes and/or sizes. Furthermore, the submicron particles appear to be packed in a hexagonal pattern; however, the submicron particles can be randomly packed or packed into other geometries. Panel (B) depicts (i) a "capture ligand" (i.e., an agent) immobilized on the surface of the core subparticle, (ii) a target (e.g., a biomolecule) that specifically binds to the agent, and (iii) the target within the fluid-filled pore space of the particle. Panel (B) does not depict protective submicron particles. The relative sizes of the subparticles, capture ligands, targets, and pore spaces in fig. 4 are not necessarily shown to scale.

Fig. 5 consists of four pictures, labeled pictures (a), (B), (C), and (D). Each picture depicts submicron particles of particles, with the core submicron particles appearing gray and the protective submicron particles appearing white. Each particle comprises 55 core subparticles. Pictures (a) and (B) depict views of the particles that are orthogonal to the views depicted in pictures (C) and (D). Pictures (a) and (C) depict only core subparticles, and pictures (B) and (D) depict core subparticles and some protective subparticles. The intact particles, including the core subparticle and the protective subparticle, are preferably covered by at least one layer of protective subparticle, which is not fully shown in any of the figures. In fig. 5, each of the core subparticles and the protective subparticles are substantially spherical and approximately the same size; however, the submicron particles within the particles may differ in shape and/or size. Furthermore, the submicron particles of FIG. 5 are shown packed in a hexagonal pattern; however, submicron particles of particles may be packed into other geometries, or they may be randomly packed. The relative sizes of the subparticles, capture ligands, targets, and pore spaces in fig. 5 are not necessarily shown to scale. In particular, the length of the linker connecting the various subparticles may be adjusted to allow more or less pore space between the subparticles.

Fig. 6 consists of 6 pictures (labeled pictures (a), (B), (C), (D), (E), and (F)). Each picture depicts a view of a substantially two-dimensional particle. In each picture, circles depict the agent immobilized on the surface of the particles. The substantially two-dimensional particles may include "void spaces," for example, between the arms of a cross or star. Panel (a) depicts a "top view" of a particle comprising a cross shape, and panel (B) depicts an orthogonal "side view" of the same cross-shaped particle. The "cross" of picture (a) is a "substantially two-dimensional shape" and the orthogonal "side view" is the third dimension that does not contain a two-dimensional shape. The "side view" shows that a substantially two-dimensional particle may comprise different surfaces, i.e. "inner surface" (on which the agent is immobilized) (black) and "outer surface" (which is substantially free of agent) (i.e. "outer surface"). The different surfaces may comprise different materials, for example, the particles may be layered, or the different surfaces may be prepared, for example, by masking one surface while the other surface is crosslinked to the agent or coating molecules. Depending on the size of the particles and the nature of the agent and target, the cruciform will inhibit interactions between the bound target (e.g., biomolecule) and other proteins or cells to varying degrees. The geometry of the particles may be adjusted, for example, to further inhibit such interactions. Panel (C) depicts particles comprising a hexagram geometry that may inhibit interactions between bound targets and other proteins or cells to a greater extent than the cross-shaped particles of panel (a). Panel (D) depicts triangular stars that may only minimally inhibit the interaction between the bound target and other proteins or cells. Nevertheless, particles comprising a triangular star geometry may be modified to inhibit interactions between bound targets and other proteins or cells to a greater extent. For example, panel (E) depicts particles comprising a triangular star geometry wherein material substantially free of pharmaceutical agent surrounds the particles, and panel (F) depicts particles comprising a triangular star geometry (i.e., comprising four triangular stars) having an outer surface that is substantially free of pharmaceutical agent.

Detailed Description

The present disclosure features compositions and methods for sequestering a soluble biomolecule from its natural environment, e.g., thereby inhibiting the biological activity of the soluble biomolecule. For example, the present disclosure provides a particle or a plurality of particles having a surface comprising an agent that selectively binds to a soluble biomolecule (e.g., immobilized on the surface of the particle). Once bound by the agent, the soluble biomolecule is sequestered by the particle such that the soluble biomolecule has a reduced ability (e.g., a substantially reduced ability or inability) to interact with other natural binding partners of the soluble biomolecule. Thus, the soluble biomolecules become inert.

I. Biological molecules

Soluble biomolecules are typically the first member of a specific binding pair. As used herein, a "binding partner," "specific binding partner," or "member of a specific binding pair" generally includes any member of a pair of binding members that bind to each other with a substantial degree of affinity and specificity. A pair of binding partners can bind to each other to a large extent excluding other components of the sample At least a majority or at least substantially all, and/or may have a thickness of less than about 10^-4、10^-5、10^-6、10^-7Or 10^-8Dissociation constants of M and the like. A pair of binding partners may "fit" together in a predetermined manner that relies on multiple atomic interactions to cooperatively increase specificity and affinity. The binding partners may be derived from biological systems (e.g., receptor-ligand interactions), chemical interactions, and/or by molecular imprinting techniques, among others. Exemplary corresponding pairs of binding partners, also referred to as specific binding pairs, having arbitrary and interchangeable designations "first" and "second" are given in table 1.

The term "biomolecule" as used herein refers to any molecule that can exert an effect on a living body. In some embodiments, the biomolecule is an atom, such as lithium or lead (e.g., the biomolecule may be a metal cation). In some embodiments, the biomolecule is not an atom or metal ion. For example, the biomolecule may be a molecule, such as an organic compound or an inorganic compound. In some embodiments, the biomolecule is a drug, such as warfarin or dabigatran etexilate. The biomolecule may be a psychoactive drug, such as diacetylmorphine. The biomolecule may be a poison, toxin or venom. The biomolecule may be an allergen. The biomolecule may be a carcinogen. The biomolecule may be a chemical weapon agent, such as a neurological agent. The biomolecule may be a molecule endogenous to the organism, such as a hormone, cytokine, neurotransmitter, soluble extracellular receptor, antibody or soluble matrix protein. The biomolecule may be a peptide, polypeptide, protein, nucleic acid, carbohydrate or sugar. Biomolecules may include peptides, polypeptides, proteins, nucleic acids, carbohydrates or sugars. The biomolecule may be a misfolded protein. The biomolecule may be an amyloid protein or a soluble precursor of an amyloid protein. "polypeptide," "peptide," and "protein" are used interchangeably and mean any peptide-linked chain of amino acids, regardless of length or post-translational modification. The biomolecule may be a lipid, a steroid or cholesterol. The biomolecule may comprise a lipid, a steroid or cholesterol. The biomolecule may be circulating free nucleic acid, such as circulating free RNA. The biomolecule may be a microrna (mirna).

The biomolecule may be a biomolecule secreted by a cell (e.g., a mammalian cell). The biomolecule may be an extracellular region of a membrane protein that is susceptible to cleavage into a soluble form. The biomolecule may be a cytosolic biomolecule. For example, the biomolecule may be a cytosolic biomolecule that is released in vivo following apoptosis, or the particle may be used in an in vitro method, wherein the cytosolic biomolecule is free in solution.

In certain preferred embodiments, the biomolecule is a soluble biomolecule. In certain preferred embodiments, the target is a soluble biomolecule. Nonetheless, the particles can target biomolecules that are not solutes in aqueous solution, and/or biomolecules that do not interact with binding partners on the cell surface. For example, the particles can specifically bind biomolecules associated with protein aggregates (e.g., amyloid or prion aggregates). Such particles may provide therapeutic benefits by disaggregating aggregates (e.g., by shifting thermodynamic equilibrium away from an aggregated state) and/or by segregating aggregates (e.g., to inhibit further aggregation and/or to allow clearance of bound aggregates). Similarly, the particles may specifically bind to crystalline calcium or hydroxyapatite. Similarly, the particle may specifically bind to a biomolecule associated with a virus or cell (e.g., a bacterial, protozoan, fungal, or yeast cell), for example, where the biomolecule is not a solute in an aqueous solution, but the biomolecule is partitioned into a membrane, cell wall, or capsid. Thus, the particle may sequester pathogenic viruses or cells, thereby attenuating the pathogenicity of the virus or cell. The particles may specifically bind to biomolecules associated with extracellular vesicles (e.g., extranuclear particles, exosomes, shedding vesicles or apoptotic bodies). The particles may specifically bind to low density lipoprotein, for example, to sequester low density lipoprotein particles.

The biomolecule may be a ligand for a cell surface receptor. The ligand may be a naturally occurring ligand or a synthetic ligand. The ligand may be a natural ligand for the receptor (e.g., a ligand produced in vivo by the subject) or a non-natural ligand (e.g., a ligand introduced into the subject, such as a virus or drug). The biomolecule may be a ligand for a cytosolic receptor or a nuclear receptor.

TABLE 1 examples of specific binding pairs

Tumor cells are known to protect themselves from host immune surveillance by shedding soluble forms of cytokine receptors that bind to cytokines produced by immune cells in the tumor microenvironment. For example, cancer cells shed soluble forms of the TNF receptor and other cytokine receptors, such as the IL-2 receptor and TRAIL receptor. These soluble receptors confer a growth advantage on cancer cells by mitigating the pro-apoptotic effects of cells on TNF α, IL-2, and TRAIL. Karpatova et al reported that the excretion of the 67kD laminin receptor by human cancer cells could increase tumor invasion and metastasis (J Cell Biochem 60(2): 226-. Thus, the particles described herein can be designed to eliminate soluble forms of cell surface receptor proteins, e.g., for the treatment of cancer.

Thus, in some embodiments, the cell surface receptor protein is expressed by the cancer cell, and/or the cell surface receptor protein is a protein that is excreted by the cancer cell in a soluble form of the cell surface receptor protein. In some embodiments, the cell surface receptor protein induces apoptosis (e.g., death receptor) when activated. In some embodiments, the cell surface receptor protein is a Tumor Necrosis Factor Receptor (TNFR) protein (e.g., TNFR-1 or TNFR-2). In some embodiments, the cell surface receptor protein is a Fas receptor protein. In some embodiments, the cell surface receptor protein is a TNF-related apoptosis ligand receptor (TRAILR) protein, a 4-1BB receptor protein, a CD30 protein, an EDA receptor protein, an HVEM protein, a lymphotoxin beta receptor protein, a DR3 protein, or a TWEAK receptor protein. In some embodiments, the cell surface receptor protein is an interleukin receptor protein, e.g., an IL-2 receptor protein. It is to be understood that in such embodiments, the target soluble biomolecule may be a soluble form of a cell surface receptor, e.g., excreted from a cancer cell.

In some embodiments, the biomolecule is soluble Tim3 ("T cell Ig mucin 3"). Soluble Tim3 (sttim 3) has been implicated in autoimmune diseases and cancer, and elevated sttim 3 is associated with HIV infection. The association of galectin 9 ("Gal 9") and potentially other ligands with Tim3 (which is said to associate Tim3 heterodimerically with CEACAM 1) results in the suppression of T cell responses, and the co-blockade of Tim3 and CEACAM1 results in an anti-tumor immune response. Accordingly, the biomolecule may be a natural ligand of setim 3 or setim 3 (e.g., Tim3L or Gal 9). The biomolecule may be a soluble isoform of CEACAM 1. In this way, the particles can be adapted to clear sttim 3 without inhibiting the interaction between Gal9 and membrane bound Tim3(mTim 3). Similarly, the agent may be sttim 3, an antibody selective for sttim 3 (or an antigen-binding portion thereof), or a ligand of Tim 3. The agent may be a natural ligand of CEACAM1 (such as Gal9 or a variant thereof) or an antibody selective for CEACAM1 or a soluble isoform thereof. Any of the foregoing particles can be used, for example, in methods of treating cancer, methods of treating HIV infection, and methods of treating autoimmune diseases (e.g., graft versus host disease).

In some embodiments, the biomolecule may be Gal9 (galectin 9). The particle may comprise an agent selective for Gal9 (e.g., a natural ligand of Gal9 (e.g., Tim3) or a variant thereof), or an antibody selective for Gal 9. In this way, the particles can be adapted to scavenge Gal9 without inhibiting the interaction of membrane-bound Gal9(mGal9) with membrane-bound Tim3(mTim 3). In some embodiments, the biomolecule may be a soluble isoform of CEACAM1 ("sCEACAM 1"). The agent may be a natural ligand of CEACAM1 (e.g. Gal9) or a variant thereof, or an antibody selective for CEACAM1 or a soluble isoform of CEACAM 1.

In some embodiments, the biomolecule is soluble CTLA 4. Soluble CTLA4 ("sCTLA 4") has been implicated in cancer, and antibodies effective against sCTLA4 but not against membrane-bound CTLA4 ("mCTLA 4") are effective in animal models of cancer. In some embodiments, the biomolecule is sCTLA 4. The agent may be a natural ligand of CTLA4 (e.g. soluble B7-1 or soluble B7-2) or a variant thereof, or an antibody selective for CTLA4 (e.g. yiprimumab or Ticilimumab). In this way, the particles may be adapted to clear sCTLA4 without inhibiting the interaction between the ligands and mCTLA 4. Thus, sCTLA4 can be removed from the tumor microenvironment ("TME") and/or the external circulation of TME, while leaving mCTLA4 free for interaction as part of the normal immune response. Particles targeting sCTLA4 can be used, for example, in methods of treating cancer.

Soluble PD-1 ("sPD 1") has been implicated in autoimmune diseases such as rheumatoid arthritis. Excess sPD1 may disturb the balance between PD1 and its ligands PD-L1 and PD-L2, leading to autoimmunity. Thus, the biomolecule may be sPD 1. The agent may be a natural ligand of sPD1 (e.g., PD-L1, PD-L2) or a variant thereof, or an antibody selective for PD1 (e.g., a PD1 blocking drug, e.g., nivolumab (nivolumab), pidilizumab (pidilizumab), or pembrolizumab (pembrolizumab))). Thus, the particles may be suitable for scavenging sPD1 without inhibiting the interaction of PD-L1 or PD-L2 with membrane-bound PD 1. Such particles may be used, for example, in methods of treating autoimmune diseases (e.g., arthritis).

LAG3 is a T cell surface receptor that, when bound by its ligand, causes inhibition. Soluble forms of LAG3 ("sLAG 3") are associated with autoimmunity, for example, in type I diabetes and other autoimmune diseases. The biomolecule may be sLAG 3. The agent may be a natural ligand of sLAG3 or a variant thereof, or an antibody selective for sLAG 3. Thus, the particles may be adapted to clear sLAG3 without inhibiting the interaction between ligands and membrane-bound LAG 3. Such particles may be used, for example, in methods of treating autoimmune diseases (such as type I diabetes).

The biomolecule may be TNF α. The agent can include an anti-TNF α antibody (e.g., infliximab, adalimumab, cerolizumab, afilomab, nerrimumab, nerelilimumab, or golimumab), or the agent can include an antigen-binding portion of an anti-TNF α antibody. The agent may be etanercept (etanercept). The agent may be a soluble receptor for TNF α (sTNF-R or a variant thereof). Particles targeting TNF α may be particularly useful for treating or preventing various autoimmune diseases such as ankylosing spondylitis, crohn's disease, hidradenitis suppurativa, psoriasis, plaque psoriasis, psoriatic arthritis, refractory asthma, juvenile idiopathic arthritis, ulcerative colitis, and rheumatoid arthritis. Particles that target TNF α can also be useful for treating or preventing alzheimer's disease, cardiovascular disease, type II diabetes, muscular dystrophy, and obesity, among other diseases and conditions.

The biomolecule may be β 2 microglobulin (B2M). The agent may be an anti-B2M antibody. Particles targeting B2M can be useful for treating or preventing memory loss, cognitive decline, peripheral artery disease, dialysis-related amyloidosis, chronic lymphocytic leukemia, multiple myeloma, and lymphoma, among other diseases and conditions.

The biomolecule may be CCL2 (chemokine (C-C motif) ligand 2). The agent may be an anti-CCL 2 antibody. Particles targeting CCL2 may be useful for treating or preventing alzheimer's disease, atherosclerosis, ischemia (e.g., ischemic stroke), epilepsy, multiple sclerosis, psoriasis, rheumatoid arthritis, glomerulonephritis, and traumatic brain injury, among other diseases and conditions.

The biomolecule may be CCL11(C-C motif chemokine 11; eotaxin 1). The agent may be an anti-CCL 11 antibody. Particles targeting CCL11 may be useful for treating or preventing memory loss and cognitive decline, among other diseases and conditions.

The biomolecule may be CCL 19. The agent may be an anti-CCL 19 antibody. Particles targeting CCL19 may be useful for treating or preventing aging and cognitive decline, among other diseases and conditions.

The biomolecule may be interferon gamma (INF gamma). The agent may include an anti-INF γ antibody (e.g., aryltuzumab (Fontolizumab)) or a soluble INF γ receptor (sINF γ R). The biomolecule may be a soluble INF gamma receptor. The agent may comprise INF γ or an anti-sINF γ R antibody. Particles targeting interferon gamma may be particularly useful for treating or preventing autoimmune diseases such as crohn's disease, rheumatoid arthritis, and psoriasis, among other diseases and conditions.

The biomolecule may be clusterin (e.g., secreted clusterin, isoform 2). The agent may comprise an anti-clusterin antibody, or an antigen binding portion thereof. The clusterin-targeted particles can be useful for treating or preventing cancer (e.g., head and neck cancer, renal cell carcinoma, colorectal cancer, endometrial cancer, ovarian cancer, breast cancer, prostate cancer, pancreatic cancer, lung cancer, hepatocellular carcinoma, or melanoma), renal disease (e.g., nephrotic cystinosis, fanconi syndrome, glomerulonephritis, atherosclerosis, and myocardial infarction, among other diseases and conditions.

The biomolecule may be high mobility group protein B1(HMGB 1). The agent may comprise an anti-HMGB 1 antibody or antigen binding portion thereof. The biomolecule may be a heat shock protein (e.g., HSP60, HSP70, HSP 90). The agent may comprise an anti-HSP antibody or antigen binding portion thereof. The biomolecule may be a peroxiredoxin (e.g., peroxiredoxin 1 or peroxiredoxin 2). The agent may comprise an anti-peroxiredoxin antibody or antigen binding portion thereof.

The agent can be an extracellular portion of a scavenger receptor, such as a class a scavenger receptor (e.g., SCARA1 (macrophage scavenger receptor 1; MSR 1; CD204), SCARA2 (macrophage receptor; MARCO), SCARA3, SCARA4(COLEC12), SCARA5), class B scavenger receptor (e.g., SCARB1, SCARB2, SCARB3(CD36)), CD68, mucin, or gelike oxidized low density lipoprotein receptor-1 (LOX-1).

The biomolecule may be insulin-like growth factor 1(IGF-1) or insulin-like growth factor binding proteins (e.g., IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, IGFBP-6). The agent can be insulin-like growth factor 1(IGF-1) or insulin-like growth factor binding proteins (e.g., IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, IGFBP-6). The agent can be an antibody or antigen-binding portion thereof that selectively binds to insulin-like growth factor 1(IGF-1) or an insulin-like growth factor binding protein (e.g., IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, IGFBP-6).

The agent may be an antibody that selectively binds to an extracellular epitope of CD63, CD9, or CD 81. Particles targeting CD63, CD9, and/or CD81 may be particularly useful for clearing extracellular vesicles (such as extranuclear granules, exosomes, shedding vesicles, or apoptotic bodies). Particles that clear various extracellular vesicles may be particularly useful for treating or preventing cancer (e.g., cancer with disease progression associated with vesicle excretion).

The biomolecule may be CXCL1, CXCL2, CXCL3, CXCL4, CXCL4L 4, CXCL4, CCL3L 4, CCL4L 4, CCL4, XCL4, xc 3655, CCL 362012, jney, or nio 4, see (yok, yie, yo). An agent can include an antibody (or an antigen-binding portion thereof) that specifically binds CXCL1, CXCL2, CXCL3, CXCL4, CXCL4L 4, CXCL4, CCL3L 4, CCL4L 4, CCL4, XCL4, or CCL 4.

The biomolecule may be interleukin 1, interleukin 1 α, interleukin 1 β, interleukin 2, interleukin 3, interleukin 4, interleukin 5, interleukin 6, interleukin 7, interleukin 8, interleukin 9, interleukin 10, interleukin 11, interleukin 12, interleukin 13, interleukin 14, interleukin 15, interleukin 16, interleukin 17, interleukin 18, interleukin 19, interleukin 20, interleukin 21, interleukin 22, interleukin 23, interleukin 24, interleukin 25, interleukin 26, interleukin 27, interleukin 28, interleukin 29, interleukin 30, interleukin 31, interleukin 32, interleukin 33, interleukin 35, or interleukin 36. The agent may comprise an antibody (or antigen-binding portion thereof) that specifically binds interleukin 1, interleukin 1 alpha, interleukin 1 beta, interleukin 2, interleukin 3, interleukin 4, interleukin 5, interleukin 6, interleukin 7, interleukin 8, interleukin 9, interleukin 10, interleukin 11, interleukin 12, interleukin 13, interleukin 14, interleukin 15, interleukin 16, interleukin 17, interleukin 18, interleukin 19, interleukin 20, interleukin 21, interleukin 22, interleukin 23, interleukin 24, interleukin 25, interleukin 26, interleukin 27, interleukin 28, interleukin 29, interleukin 30, interleukin 31, interleukin 32, interleukin 33, interleukin 35, or interleukin 36. The agent may include a soluble interleukin-2 receptor, a soluble interleukin-3 receptor, a soluble interleukin-4 receptor, a soluble interleukin-5 receptor, a soluble interleukin-6 receptor, a soluble interleukin-7 receptor, a soluble interleukin-9 receptor, a soluble interleukin-10 receptor, a soluble interleukin-11 receptor, a soluble interleukin-12 receptor, a soluble interleukin-13 receptor, a soluble interleukin-15 receptor, a soluble interleukin-20 receptor, a soluble interleukin-21 receptor, a soluble interleukin-22 receptor, a soluble interleukin-23 receptor, a soluble interleukin-27 receptor, or a soluble interleukin-28 receptor. The agent may be soluble ST2, said soluble ST2 binding interleukin 33.

The biomolecule may be a soluble interleukin-2 receptor, a soluble interleukin-3 receptor, a soluble interleukin-4 receptor, a soluble interleukin-5 receptor, a soluble interleukin-6 receptor, a soluble interleukin-7 receptor, a soluble interleukin-9 receptor, a soluble interleukin-10 receptor, a soluble interleukin-11 receptor, a soluble interleukin-12 receptor, a soluble interleukin-13 receptor, a soluble interleukin-15 receptor, a soluble interleukin-20 receptor, a soluble interleukin-21 receptor, a soluble interleukin-22 receptor, a soluble interleukin-23 receptor, a soluble interleukin-27 receptor, or a soluble interleukin-28 receptor. The agent can include an antibody (or antigen-binding portion thereof) that specifically binds to a soluble interleukin-2 receptor, a soluble interleukin-3 receptor, a soluble interleukin-4 receptor, a soluble interleukin-5 receptor, a soluble interleukin-6 receptor, a soluble interleukin-7 receptor, a soluble interleukin-9 receptor, a soluble interleukin-10 receptor, a soluble interleukin-11 receptor, a soluble interleukin-12 receptor, a soluble interleukin-13 receptor, a soluble interleukin-15 receptor, a soluble interleukin-20 receptor, a soluble interleukin-21 receptor, a soluble interleukin-22 receptor, a soluble interleukin-23 receptor, a soluble interleukin-27 receptor, or a soluble interleukin-28 receptor. The agent may be interleukin 2, interleukin 3, interleukin 4, interleukin 5, interleukin 6, interleukin 7, interleukin 9, interleukin 10, interleukin 11, interleukin 12, interleukin 13, interleukin 15, interleukin 20, interleukin 21, interleukin 22, interleukin 23, interleukin 27, or interleukin 28.

The biomolecule may be epinephrine, norepinephrine, melatonin, serotonin, potassium triiodide, adenines, or thyroxine. The biomolecule may be a prostaglandin (e.g., prostacyclin I2(PGI2), prostaglandin E2(PGE2), prostaglandin F2 a (PGF2 a)), a leukotriene, a prostacyclin, or a thromboxane. The biomolecule may be testosterone, Dehydroepiandrosterone (DHEA), androstenedione, Dihydrotestosterone (DHT), aldosterone, estrone, estradiol, estriol, progesterone, cortisol, calcitriol, or calcifediol.

The biomolecule may be amylin, adiponectin, adrenocorticotropic hormone, angiotensinogen, angiotensin I, angiotensin II, antidiuretic hormone (vasopressin), apelin peptide, atrial natriuretic peptide, brain natriuretic peptide, calcitonin, chemokine, cholecystokinin, corticotropin releasing hormone, cortistatin, enkephalin, endothelin, erythropoietin, follicle stimulating hormone, galanin, gastric inhibitory peptide, gastrin, ghrelin, glucagon-like polypeptide-1, gonadotropin releasing hormone, growth hormone releasing hormone, hepcidin, human chorionic gonadotropin, human placental lactogen, growth hormone, inhibin, insulin-like growth factors (growth regulators, e.g., IGF-I), leptin, liptropin, luteinizing hormone, Melanocyte stimulating hormone, motilin, orexin, oxytocin, pancreatic polypeptide, parathyroid hormone, pituitary adenylate cyclase activating peptide, prolactin releasing hormone, relaxin, renin, secretin, somatostatin, thrombopoietin, thyroid-stimulating hormone (thyrotropin), thyrotropin releasing hormone or vasoactive intestinal peptide. The agent can include an antibody (or antigen-binding portion thereof) that specifically binds to amylin, adiponectin, corticotropin, apelin, angiotensinogen, angiotensin I, angiotensin II, antidiuretic hormone (vasopressin), atrial natriuretic peptide, brain natriuretic peptide, calcitonin, chemokine, cholecystokinin, corticotropin-releasing hormone, cortistatin, enkephalin, endothelin, erythropoietin, follicle stimulating hormone, galanin, gastric inhibitory peptide, gastrin, ghrelin, glucagon-like polypeptide-1, gonadotropin-releasing hormone, growth hormone-releasing hormone, hepcidin, chorionic gonadotropin, human placental lactogen, growth hormone, inhibin, insulin-like growth factor (growth regulin, for example, IGF-I), leptin, liptropin, luteinizing hormone, melanocyte-stimulating hormone, motilin, orexin, oxytocin, pancreatic polypeptide, parathyroid hormone, pituitary adenylate cyclase activating peptide, prolactin-releasing hormone, relaxin, renin, secretin, somatostatin, thrombopoietin, thyroid-stimulating hormone (thyrotropin), thyrotropin-releasing hormone or vasoactive intestinal peptide.

The biomolecule may be vascular endothelial growth factor-A (VEGF-A). The agent may include an antibody that specifically binds VEGF-a (e.g., bevacizumab or bruuzumab), or an antigen binding portion thereof (e.g., ranibizumab). For example, the agent may be aflibercept. Particles targeting VEGF-a can be particularly useful for treating or preventing, among other conditions and diseases, macular degeneration (e.g., wet macular degeneration), proliferative diabetic retinopathy, neovascular glaucoma, macular edema, cancer (e.g., colorectal, lung, prostate, breast, kidney, brain), bronchial asthma, diabetes, ischemic cardiomyopathy, and myocardial ischemia.

The biomolecule may be a soluble vascular endothelial growth factor receptor, such as soluble vascular endothelial growth factor receptor 1 (soluble VEGFR-1), soluble vascular endothelial growth factor receptor 2 (soluble VEGFR-2), or soluble vascular endothelial growth factor receptor 3 (soluble VEGFR-3). The agent can be an antibody or antigen-binding portion thereof that selectively binds to a soluble VEGF receptor (e.g., eculizumab, or ramucirumab). The agent may be a ligand for a VEGF receptor (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D or Placental Growth Factor (PGF)). Particles that target soluble VEGF receptors may be particularly useful for treating or preventing cancer, among other diseases and conditions.

The biomolecule may be a member of the epidermal growth factor family, such as Epidermal Growth Factor (EGF), heparin-binding epidermal growth factor-like growth factor (HB-EGF), transforming growth factor-alpha (TGF-alpha), Amphiregulin (AR), epithelial regulatory protein (EPR), epithelial mitosis protein antibodies, Betacellulin (BTC), neuregulin-1 (NRG1), neuregulin-2 (NRG2), neuregulin-3 (NRG3), or neuregulin-4 (NRG 4). The agent may be an antibody or antigen-binding portion thereof that selectively binds EGF, HB-EGF, TGF- α, AR, EPR, an epithelial mitotic protein antibody, BTC, NRG1, NRG2, NRG3, or NRG 4. The agent may comprise a soluble EGF receptor (such as soluble EGF receptor, soluble HER2, or soluble HER 3). Particles that target members of the epidermal growth factor family may be particularly useful for treating or preventing cancer, among other conditions and diseases.

The biomolecule may be a soluble epidermal growth factor receptor (EGF receptor) (e.g. soluble EGF receptor, soluble human epidermal growth factor receptor 2 (soluble HER2) or soluble human epidermal growth factor receptor 3 (soluble HER 3)). The agent may be an antibody or antigen-binding portion thereof that selectively binds to a soluble EGF receptor, such as cetuximab (cetuximab), futuximab (futuximab), agoutimab (imgatuzumab), matuzumab (matuzumab), nimotuzumab (necitumumab), nimotuzumab (nimotuzumab), panlizumab (panitumumab), zalutumumab (zalutumumab), duriguzumab (duligumab), patritumab (patritumab), ertussumab (ertumaxomab), pertuzumab (pertuzumab), or trastuzumab (trastuzumab). The agent may be a ligand of an EGF receptor (e.g., an EGF family member as described above). Particles that target soluble EGF receptors may be particularly useful for treating or preventing cancer, among other diseases and conditions.

The biomolecule may be an IgE antibody. The agent may include an anti-IgE antibody, such as omalizumab or talizumab, or an antigen-binding portion thereof. The agent may be an extracellular portion of fcsri. Particles targeting IgE antibodies may be particularly useful for treating chronic idiopathic urticaria and allergic asthma, among other conditions and diseases.

The biomolecule may be proprotein convertase subtilisin kexin 9(PCSK 9). The agent may be an anti-PCSK 9 antibody (e.g., alirocumab, lodelcizumab, lausetuzumab, or elvucizumab) or an antigen-binding portion thereof. Particles targeting PCSK9 may be particularly useful for treating or preventing hypercholesterolemia, atherosclerosis, ischemia, and myocardial infarction, among other conditions and diseases.

The biomolecule may be adrenomedullin, brain derived neurotrophic factor, erythropoietin, fibroblast growth factor, liver cancer derived growth factor, glucose-6-phosphate isomerase, keratinocyte growth factor, macrophage migration inhibitory factor, neurotrophic factor (nerve growth factor, brain derived neurotrophic factor, neurotrophic factor-3, neurotrophic factor-4), platelet derived growth factor, stem cell factor, thrombopoietin, T cell growth factor, vascular endothelial growth factor (VEGF-A, VEGF-B, VEGF-C, VEGF-D, Placental Growth Factor (PGF)) or renalase. The agent can include an antibody or antigen-binding portion thereof that selectively binds to adrenomedullin, brain-derived neurotrophic factor, erythropoietin, fibroblast growth factor, liver cancer-derived growth factor, glucose-6-phosphate isomerase, keratinocyte growth factor, macrophage migration inhibitory factor, neurotrophic factor (nerve growth factor, brain-derived neurotrophic factor, neurotrophic factor-3, neurotrophic factor-4), platelet-derived growth factor, stem cell factor, thrombopoietin, T-cell growth factor, vascular endothelial growth factor (VEGF-A, VEGF-B, VEGF-C, VEGF-D, Placental Growth Factor (PGF)), or renalase.

The biomolecule may be soluble tropomyosin receptor kinase B (soluble TrkB). The agent may be an anti-TrkB antibody or antigen-binding portion thereof. The biomolecule may be soluble tropomyosin receptor kinase a (soluble TrkA). The agent may be an anti-TrkA antibody or antigen-binding portion thereof. The agent may be a brain-derived neurotrophic factor.

The biomolecule may be angiogenin (e.g., angiogenin 1, angiogenin 2, angiogenin 3, or angiogenin 4) or an angiogenin-like protein (e.g., angiogenin-like 1, angiogenin-like 2, angiogenin-like 3, angiogenin-like 4, angiogenin-like 5, angiogenin-like 6, or angiogenin-like 7). The agent can be an antibody that selectively binds to angiogenin (e.g., angiogenin 1, angiogenin 2, angiogenin 3, or angiogenin 4) or an angiogenin-like protein (e.g., angiogenin-like 1, angiogenin-like 2, angiogenin-like 3, angiogenin-like 4, angiogenin-like 5, angiogenin-like 6, or angiogenin-like 7).

The biomolecule may be a hedgehog protein (e.g., sonic hedgehog). The agent may be an antibody that selectively binds to hedgehog protein. The hedgehog-targeted particles may be particularly useful for treating or preventing cancer (such as pancreatic cancer, cerebellar tumors, and medulloblastoma), among other conditions and diseases.

The biomolecule may be a soluble Human Leukocyte Antigen (HLA) protein (e.g., soluble HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, HLA-F or HLA-G (see, e.g., Bassani-Sternberg, M.et al., Proceedings National Academy Sciences USA 107(44):18769 (2010)). The agent may be an antibody that selectively binds to a soluble Human Leukocyte Antigen (HLA) protein. The agent may be a soluble killer cell immunoglobulin-like receptor. Particles targeting soluble HLA can be particularly useful for treating or preventing cancer, among other diseases and conditions.

The biomolecule may be a soluble UL16 binding protein isoform (e.g., soluble RAET1(ULBP 1; RAET1E2), soluble RAET1H (ULBP2), soluble RAET1N (ULBP3), soluble RAET1E (ULBP4), soluble RAET1G (ULBP5), or soluble RAET1L (ULBP 6)). The agent can be an antibody, or antigen-binding portion thereof, that specifically binds to a soluble UL 16-binding protein isoform. The agent may be a soluble NKG2D receptor (see, e.g., PCT patent application publication No. WO2006/024367, which is incorporated herein by reference in its entirety).

The biomolecule may be soluble MIC-A or soluble MIC-B (see, e.g., Groh, V.et al., Nature419(6908):734 (2002)). The agent may be an anti-MIC-A antibody or an anti-MIC-B antibody, or an antigen-binding portion of either antibody. The agent may be a soluble NKG2D receptor (see, e.g., PCT patent application publication No. WO2006/024367, which is incorporated herein by reference in its entirety).

The agent can be a soluble natural cytotoxic receptor (see, e.g., Jarahian, M.et al. plos Pathologens 7(8): e1002195 (2011)).

The biomolecule may be soluble C-type lectin domain family 2 member D (soluble CLEC 2D; soluble lectin-like transcript-1 (LLT1)) (see, e.g., Chalan, p.et al, PloS One 10(7): e0132436 (2015)). The agent can be an antibody that selectively binds to soluble LLT 1. Particles targeting soluble LLT1 can be particularly useful for treating or preventing autoimmune diseases (such as arthritis rheumatoid arthritis), among other diseases and conditions.

The biomolecule may be soluble CD16 (see, e.g., Hoover, R.G., J Clinical Investigation 95:241 (1995)). The agent may be an antibody that selectively binds soluble CD 16. Particles targeting soluble CD16 may be particularly useful for treating or preventing cancer, among other diseases and conditions.

The biomolecule may be plasminogen activator inhibitor-1 (PAI-1), plasminogen activator inhibitor-2 (PAI-2), tissue-type plasminogen activator, urokinase, plasminogen, thrombin, or alpha 2-macroglobulin. The agent can be an antibody that selectively binds to plasminogen activator inhibitor-1 (PAI-1), plasminogen activator inhibitor-2 (PAI-2), tissue-type plasminogen activator, urokinase, plasminogen, thrombin, or alpha 2-macroglobulin.

The biomolecule may be factor XII, factor XIIa, factor XI, factor XIa, factor IX, factor IXa, factor X, factor Xa, factor VII, factor VIIa, factor XIII, factor XIIIa, factor V, prothrombin, thrombin, von willebrand factor, thromboxane a2, fibrinogen or fibrin. The agent can be an antibody that selectively binds to factor XII, factor XIIa, factor XI, factor XIa, factor IX, factor IXa, factor X, factor Xa, factor VII, factor VIIa, factor XIII, factor XIIIa, factor V, prothrombin, thrombin, von willebrand factor, thromboxane a2, fibrinogen, or fibrin.

The biomolecule may be a serine protease inhibitor (e.g., alpha 1-antitrypsin, antitrypsin-related protein, alpha 1-antichymotrypsin, human kallikrein binding protein, protein C inhibitor, cortin transfer protein, thyroxine-binding globulin, angiotensinogen, centerin (GCET1), protein Z-related protease inhibitor, serine protease inhibitor, antithrombin, heparin cofactor II, plasminogen activator inhibitor 1, glial derived linker (proteinase nexin I), pigment epithelial cell derived factor, alpha 2-antifibrinolysin, complement 1-inhibitor, neurogenic serine protease inhibitor, plasminogen activator inhibitor, 2SERPINA1, or SERPINA 2). The agent may comprise an antibody, or antigen-binding portion thereof, that selectively binds to a serine protease inhibitor.

The biomolecule may be soluble ST 2. The agent may be interleukin 33 or an antibody that specifically binds soluble ST2 (or a fragment thereof). Particles targeting soluble ST2 may be particularly useful for treating or preventing heart disease, myocardial infarction, acute coronary syndrome, and heart failure, among other diseases and conditions.

The biomolecule may be myostatin (growth differentiation factor 8 (GDF-8)). The agent may be an anti-myostatin antibody (e.g., semulumab or trevoguemab). The agent may be an activin receptor or myostatin binding portion thereof, for example, the agent may be a soluble activin type IIB receptor. Particles that target myostatin can be particularly useful for treating muscular dystrophy, cachexia, sarcopenia, and various forms of muscle loss (e.g., zero gravity muscle loss), among other diseases and conditions.

The biomolecule may be ghrelin. The agent may be an anti-ghrelin antibody. Particles targeted to ghrelin can be particularly useful for treating or preventing obesity, prader-willi syndrome, addiction, alcoholism, and leptin resistance (e.g., genetic leptin resistance).

The biomolecule may be sLR11 (soluble SOLL 1; soluble SOLLA 1). The agent may be an anti-sLR 11 antibody. sLR 11-targeted particles can be particularly useful for treating or preventing obesity, among other diseases and conditions.

The biomolecule may be TGF-beta (transforming growth factor beta, e.g. TGF-beta 1)TGF-. beta.2 or TGF-. beta.3). The agent may be an anti-TGF- β antibody (such as non-hematoxylin mab (fresolimumab), lerdellimumab (lerdellimumab) or mettelumumab). The agent may comprise a TGF-beta binding domain of a TGF-beta receptor. The agent may be LTBP₁(in-tissue transforming growth factor beta binding protein 1), 14-3-3-protein epsilon (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activating protein, epsilon; YWHAE) or eukaryotic translation initiation factor 3 subunit I (EIF3I), each of which binds to TGF-beta. Particles targeting TGF- β may be particularly useful for treating or preventing scleroderma, idiopathic pulmonary fibrosis, kidney disease, focal segmental glomerulosclerosis, keratoconus, marfan's syndrome, alzheimer's disease, cognitive decline, traumatic brain injury, muscle atrophy, and cancer (e.g., renal tumors and melanoma), among other diseases and conditions.

The biomolecule may be Wnt (e.g., Wnt1, Wnt2, Wnt2B, Wnt3, Wnt3A, Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, Wnt8A, Wnt8B, Wnt9A, Wnt9B, Wnt10A, Wnt10B, Wnt11, or Wnt 16). The agent may be an anti-Wnt antibody. Wnt-targeted particles can be particularly useful for treating or preventing obesity, type II diabetes, atherosclerosis, calcified aortic stenosis, heart attack, heart failure, stroke, and cancer (e.g., breast cancer, colorectal cancer, esophageal cancer, melanoma, prostate cancer, lung cancer, non-small cell lung cancer, mesothelioma, sarcoma, glioblastoma, or ovarian cancer), among other diseases and conditions.

The biomolecule may be a soluble Notch ligand (e.g., soluble Jagged1, soluble Jagged2, soluble Delta-like ligand 1(DLL1), soluble Delta-like ligand 3(DLL3), and Delta-like ligand 4(DLL 4)). The agent can be an anti-Notch ligand antibody, such as daclizumab (democizumab) or denosumab (enoticumab), or a soluble Notch receptor (e.g., soluble Notch1, Notch2, Notch3, or Notch4), or a variant thereof. Particles targeting soluble Notch ligands can be particularly useful for treating or preventing atherosclerosis, calcified aortic valve stenosis, heart attack, heart failure, stroke, and cancer (e.g., breast cancer, pancreatic cancer, renal cell carcinoma, non-small cell lung cancer, and solid tumors), among other diseases and conditions.

The biomolecule can be a soluble Notch receptor (e.g., soluble Notch1, Notch2, Notch3, or Notch 4). The agent may be an anti-Notch receptor antibody such as tarrituximab (tarextumab) or brentuzumab (bronticuzumab) or a soluble Notch ligand. Particles targeting soluble Notch receptors can be particularly useful for treating or preventing atherosclerosis, calcified aortic valve stenosis, heart attack, heart failure, stroke, and cancer (e.g., breast cancer, pancreatic cancer, renal cell carcinoma, non-small cell lung cancer, and solid tumors), among other diseases and conditions.

The target may be hydroxyapatite or calcium (e.g., crystalline calcium). The agent may be a chelating agent such as ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), Sodium Thiosulfate (STS), inositol hexaphosphate or citric acid. Particles targeting hydroxyapatite or calcium may be particularly useful for treating or preventing atherosclerosis, calcified aortic valve stenosis, and calcified tendonitis, among other diseases and conditions.

In some embodiments, the biomolecule is an autoantibody. Autoantibodies are antibodies produced by a subject that specifically bind to an antigen produced by the subject. Autoantibodies are associated with many different disease states, including lupus. Furthermore, the induction of new autoantibodies may be associated with therapeutic intervention, for example, causing drug-induced lupus. Thus, a composition comprising a plurality of particles, wherein the plurality of particles comprises an agent that selectively binds one or more autoantibodies, can be used, for example, in a method of treating or preventing lupus (e.g., drug-induced lupus). The biomolecule may be, for example, a double-stranded DNA autoantibody or an anti-nuclear autoantibody.

The particles that target autoantibodies may include an agent that is an antigen of the autoantibody.

The biomolecule may be an anti-beta adrenoceptor autoantibody or an anti-M2 muscarinic receptor autoantibody, for example, for use in the prevention or treatment of idiopathic dilated cardiomyopathy. In particular, particles targeting anti-beta adrenoreceptor autoantibodies or anti-M2 muscarinic receptor autoantibodies can be administered to subjects with chagas' disease, which are associated with induction of such autoantibodies (see, e.g., Herda, l.r.et al., Br J Pharmacol 166(3)847 (2012)). The biomolecule may be an anti-alpha-1-adrenergic receptor autoantibody, for example, for use in the treatment or prevention of Hypertension (see, e.g., Luther, h.p. et al, Hypertension 29(2):678 (1997)). The biomolecule may be an anti-muscarinic type 3 receptor autoantibody, e.g. for use in the treatment or prevention of sjogren's syndrome (see, e.g., Lee, b.h.et al, PloS One 8(1): e53113 (2013)).

For example, autoantibodies directed against hormones and cytokines can buffer their concentration by reversibly binding to them to control the concentration of free active substances. Deviations from healthy autoantibody levels can lead to disease caused by loss of cytokine or hormone homeostasis. For example, anti-IFN γ autoantibodies can induce disseminated nontuberculous mycobacterial infection, anti-IL-17 autoantibodies have been associated with the development of chronic mucosal candidiasis, and anti-IL-6 autoantibodies have been associated with severe staphylococcal or streptococcal infection. Autoantibodies to the ghrelin can modulate the effective concentration of ghrelin available for binding to ghrelin receptor GHSR 1.

In some embodiments, the biomolecule is an autoantibody. For example, the autoantibody may be an anti-IFN γ, anti-IL-17, anti-IL-6 or anti-ghrelin autoantibody. In some embodiments, the agent is a natural ligand of the autoantibody (e.g., an antigen targeted by the autoantibody). For example, the agent may be IFN γ, IL-17, IL-6 or ghrelin. In some embodiments, the invention relates to methods of treating patients suffering from cytokine dysregulation diseases (e.g., autoimmune diseases). In some embodiments, the invention relates to methods of treating a patient suffering from a metabolic disorder (e.g., obesity).

Activin binding to activin type IIB receptor ActRIIB causes muscle atrophy in models of cachexia. Excessive activin levels in serum are associated with muscle atrophy and fibrosis in cachexia models, which can be reversed by antibodies that block activin a and B/ActRIIB signaling, and elevated activin levels are found in the serum of cancer patients. Sarcopenia is a progressive condition of loss of muscle mass in aging and is also associated with excessive activin signaling. Thus, the biomolecule may be activin (e.g., activin a or activin B). The agent may be a natural ligand for activin (e.g., an activin receptor protein (e.g., ActRIIB) or a variant thereof) or an anti-activin antibody. The agent may be myostatin. In some embodiments, the invention relates to methods of treating a patient suffering from a muscle wasting disease (such as cachexia or sarcopenia).

Those skilled in the art will also appreciate that the particles described herein are also useful for clearing a wider variety of targets whose biological activity may be, for example, undesirable. For example, the particles may be designed to bind to a component of the viral capsid or envelope, thereby isolating the virus from the blood of the subject. In some embodiments, the particles can be designed to bind and sequester toxins (e.g., bacterial toxins, plant toxins, and animal toxins (such as one or more components of snake venom)) in the circulation of a subject. In some embodiments, the particles can be designed to bind to and isolate a small molecule (e.g., a psychoactive drug or a small molecule toxin) from the circulation of a subject. In such embodiments, the particles may be useful for removing toxins from the body (e.g., after being bitten by a snake or insect). In some embodiments, the particles can be used to treat, prevent, delay the onset of, or reduce the severity of anaphylactic shock (e.g., by scavenging antigens that elicit an anaphylactic immune response) in a subject.

In some embodiments, the target is associated with a virus (e.g., a viral structural protein (such as a viral capsid or viral envelope protein) that is bound by an agent). In such embodiments, the particles can be used as an antiviral therapy, e.g., for a subject infected with or at risk of being infected with a virus. The virus may be an enveloped virus or a non-enveloped virus.

In some embodiments, canSoluble biomolecules are small or large molecules. In some embodiments, the longest dimension of the soluble biomolecule is no greater than 600nm (e.g., less than 550nm, 500nm, 450nm, 400nm, 350nm, 300nm, 250nm, 200nm, 150nm, 100nm, 50nm, or 25 nm). For example, a biomolecule may have a molecular weight of aboutTo a molecular radius of about 1 μm, such asTo about 100nm, aboutTo about 20nm, from about 1nm to about 1 μm, from about 1nm to about 100nm, or from about 1nm to about 20 nm. The biomolecule may have about 3amu to about 10⁷amu molecular weight, e.g., from about 100amu to about 10⁷amu, about 3amu to about 10⁶amu, about 3amu to about 10⁵amu, from about 100amu to about 10⁶amu or about 400amu to about 10⁶amu. The biomolecule may have a molecular weight of about 10⁵amu to about 10⁷Molecular weight of amu.

The terms "specific binding," "specifically binds," "selective binding," "selectively binds," and similar grammatical terms, as used herein, refer to two molecules that form a complex that is relatively stable under physiological conditions. Typically, when combined with a constant (k)_a) Higher than 10⁶M^-1s^-1Binding is considered specific. Thus, the first member of a specific binding pair may be at least (or greater than) 10 ⁶M^-1s^-1(e.g., at least or greater than 10)⁷M^-1s^-1、10⁸M^-1s^-1、10⁹M^{- 1}s^-1、10¹⁰M^-1s^-1、10¹¹M^-1s^-1、10¹²M^-1s^-1、10¹³M^-1s^-1、10¹⁴M^-1s^-1Or 10¹⁵M^-1s^-1Or higher) k_aSpecifically binds to the second member of the binding pair. In some embodiments, the selective interaction has a value less than or equal to 10^-3s^-1(e.g., 8X 10)^-4s^-1、5×10^-4s^-1、2×10^-4s^-1、10^-4s^-1Or 10^-5s^-1) Dissociation constant (k) of_d)。

Specific binding does not refer to an interaction driven primarily by non-specific electrostatic interactions or non-specific hydrophobic interactions, which may have favorable binding constants. For example, a negatively charged nucleic acid can bind to a cationic particle with a favorable binding constant, independent of specific interactions, and such binding is not "specific binding" as defined herein. Similarly, lipids can bind to hydrophobic particles with favorable binding constants, without relying on specific interactions, and such binding is not "specific binding" as defined herein.

In some embodiments, the biomolecule and the particle have the same charge at physiological pH (-7.4). For example, the biomolecule may have a negative charge and the particle may have a negative charge, or the biomolecule may have a positive charge and the particle may have a positive charge. In some embodiments, the biomolecule and the particle have opposite charges at physiological pH. For example, the biomolecule may have a positive charge and the particle may have a negative charge, or the biomolecule may have a negative charge and the particle may have a positive charge. In some embodiments, the biomolecule has a neutral charge at physiological pH and/or the particle has a neutral charge at physiological pH.

The biomolecule may have an isoelectric point of about 0 to about 14. The nucleic acid has an isoelectric point of about 4 to about 7, and thus, the biomolecule can have an isoelectric point of about 4 to about 7. Proteins typically have an isoelectric point of about 4 to about 10, and thus, biomolecules may have an isoelectric point of about 4 to about 10. However, unmodified peptides and proteins may have isoelectric points ranging from about 2.5 (based on aspartic acid; pI-2.8) to about 11 (based on arginine; pI-11), although proteins having isoelectric points outside this range are known. Accordingly, the biomolecule may have an isoelectric point ranging from about 2.5 to about 11. The soluble extracellular portion of secreted and membrane proteins typically has a slight negative charge at physiological pH, and thus, biomolecules can have an isoelectric point of about 4 to about 7 (e.g., about 4 to about 6). The biomolecule may have an isoelectric point of about 0 to about 4, about 2 to about 6, about 4 to about 8, about 6 to about 10, about 8 to about 12, or about 10 to about 14. The biomolecule has an isoelectric point of about 0 to about 2, about 1 to about 3, about 2 to about 4, about 3 to about 5, about 4 to about 6, about 5 to about 7, about 6 to about 8, about 7 to about 9, about 8 to about 10, about 9 to about 11, about 10 to about 12, about 11 to about 13, or about 12 to about 14.

In some embodiments, the selective interaction has less than 10^-8M、10^-9M、10^-10M、10^-11M or 10^{- 12}K of M_D. Equilibrium constant K_DIs the ratio of the kinetic rate constants, i.e. k_d/k_a. In some embodiments, the selective interaction has less than 1 × 10^-9K of M_D。

As used herein, the term "interaction," when referring to an interaction between two molecules, refers to the physical contact (e.g., binding) of the molecules to each other. Typically, such interaction results in the activity of one or both of the molecules (which produces a biological effect). Inhibition of such interactions results in disruption of the activity of one or more molecules involved in the interaction.

As used herein, the term "inhibit" and grammatical equivalents thereof refer to a reduction, limitation, and/or blocking of a particular action, function, or interaction. In one embodiment, the term refers to reducing the level of a given output or parameter to an amount that is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or less than the amount in a corresponding control (e.g., the background level of interaction between two members of a specific binding pair). The reduced level of a given output or parameter need not (although it may) mean the absolute absence of the output or parameter. The present invention does not require and is not limited to methods that eliminate the output or parameter altogether. The substantial inhibition can be, for example, an inhibition of at least 50% (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more) of the interaction between two biomolecules (e.g., the first and second members of a binding pair).

Methods for detecting interactions or measuring the affinity of one biomolecule for another are known in the art. For example, the binding of two biomolecules may be performed using a variety of techniques (such as, but not limited to, Biofilm Layer Interference (BLI), Western blotting, dot blotting, Surface Plasmon Resonance (SPR), enzyme-linked immunosorbent assay (ELISA)),OrAssays or mass spectrometry based methods) are detected and/or quantified.

In some embodiments, binding may be determined using any SPR-based assay known in the art for characterizing kinetic parameters of an interaction of two biomolecules. Any commercially available SPR instrument including, but not Limited to, BIAcore Instruments (Biacore AB; Uppsala, Sweden), lAsys Instruments (Affinity Sensors; Franklin, Mass.), IBIS systems (Windsor Scientific Limited; Berkshire, England), SPR-CELLIA systems (Nippon Laser and Electronics Lab; North Hai Japan), and SPR detectors Spreeta (Texas Instruments; Dallas, Tex.) may be used in the methods described herein. (see, e.g., Mullett et al, Methods22:77-91 (2000); Dong et al, Reviews in Mol Biotech 82:303-323 (2002); Fivash et al, Curr Opin Biotechnol 9:97-101 (1998); and Rich et al, Curr Opin Biotechnol 11:54-61 (2000)).

In some embodiments, the biomolecular interaction between two biomolecules can be determined using BLI on Octet (ForteBio Inc.). BLI is a label-free optical analysis technique that senses binding between a ligand immobilized on the biosensor tip and an analyte in solution by measuring changes in the thickness of a protein layer on the biosensor tip in real time.

In some embodiments, an alphascreen (perkinelmer) assay may be used to characterize the binding of two biomolecules. The acronym ALPHA stands for Amplified chemiluminescent affinity Homogeneous Assay (Amplified Luminescent reagent Assay). AlphaScreen is a bead-based affinity assay that senses binding between molecules attached to donor and acceptor beads by measuring the signal generated by energy transfer between the donor and acceptor beads. (see, e.g., Eglen et al, Curr Chem Genomics 1:2-10 (2008)).

In some embodiments of the present invention, the substrate is,the (PerkinElmer) assay can be used to characterize the binding of two biomolecules. AlphaLISA was adapted from the AlphaScreen assay described above to contain europium-containing acceptor beads and used as a replacement for the traditional ELISA assay. (see, e.g., Eglen et al, Curr Chem Genomics 1:2-10 (2008)).

A wide variety of immunoassay techniques may be used, including competitive or non-competitive immunoassays. The term "immunoassay" encompasses techniques including, but not limited to, flow cytometry, FACS, Enzyme Immunoassay (EIA) (such as Enzyme Multiplex Immunoassay Technique (EMIT), enzyme linked immunosorbent assay (ELISA), IgM antibody capture ELISA (mac ELISA), and Microparticle Enzyme Immunoassay (MEIA)), and Capillary Electrophoresis Immunoassay (CEIA), Radioimmunoassay (RIA), immunoradiometric assay (IRMA), Fluorescence Polarization Immunoassay (FPIA), and chemiluminescence assay (CL). Such immunoassays may be automated, if desired. Immunoassays can also be used in conjunction with laser-induced fluorescence. Liposomal immunoassays (e.g., flow injection liposome immunoassays and liposome immunosensors) are also suitable for use in the present invention. Furthermore, nephelometry (where, for example, the formation of a biomolecular complex results in increased light scattering which is converted to a peak rate signal as a function of marker concentration) is suitable for use in the method of the invention. In a preferred embodiment of the invention, the incubation products are detected by ELISA, RIA, Fluorescent Immunoassay (FIA) or Soluble Particle Immunoassay (SPIA).

In some embodiments, the binding of two biomolecules can be determined using thermal denaturation methods involving Differential Scanning Fluorimetry (DSF) and Differential Static Light Scattering (DSLS).

In some embodiments, the binding of two biomolecules can be determined using mass spectrometry-based methods such AS, but not limited to, affinity selection coupled to a mass spectrometry (AS-MS) platform. This is a label-free method in which the protein and test compound are incubated, unbound molecules are washed away, and the protein-ligand complex is analyzed by MS for ligand identification after the decomplexation step.

In some embodiments, a detectably labeled protein (e.g., a radiolabeled (e.g.,³²P、³⁵S、¹⁴c or³H) Fluorescently labeled (e.g., FITC) or enzymatically labeled biomolecules), the binding of two biomolecules is quantified by immunoassay or by chromatographic detection.

In some embodiments, the present invention contemplates the use of fluorescence polarization assays and Fluorescence Resonance Energy Transfer (FRET) assays to measure, directly or indirectly, the degree of interaction between two biomolecules.

Particles II

As used herein, the term "particle" refers to a small mass that may include any material (such as alumina, metal (e.g., gold or platinum), glass, silica, latex, plastic, agarose, polyacrylamide, methacrylate, or any polymeric material) and may be of any size or shape. In some embodiments, the particle or particles comprise silicon. (see, e.g., international patent application publication numbers WO 2013/011764, WO 2013/029278, and WO 2014/151381, and U.S. patent application publication number 2014/0271886, the disclosures of each of which are incorporated herein by reference in their entireties). In some embodiments, the granules comprise or consist of starch (see, e.g., international patent application publication No. WO 2010/084088). In some embodiments, the particle or particles are composed of nucleic acids (e.g., naturally occurring or non-naturally occurring nucleic acids). Methods for preparing such nucleic acid-based microstructures are known in the art and are described, for example, in Douglas et al, Nucl Acids Res 37(15):5001-5006 (2009); douglas et al, Nature 459(7245), 414-428 (2009); voigt et al, Nat Nanotechnol 5(3): 200-; and Endo et al, Curr Protoc Nucleic Acid Chem Chapter 12(Unit 12.8) (2011).

In a preferred embodiment, the particles are insoluble in aqueous solution (e.g., the particles may be insoluble in water, serum, plasma, extracellular fluid, and/or interstitial fluid). For example, particles can be separated from an aqueous solution of a cell suspension by, for example, centrifuging the solution including the particles at a speed sufficient to separate the cells of the cell suspension from the aqueous solution. However, the particles may readily be present as a suspension in the aqueous solution, for example, a slight shaking or swirling of a plurality of particles in the aqueous solution is sufficient to suspend the particles in the solution. In some embodiments, the particle is not a hydrogel. In some embodiments, the particles do not comprise a hydrogel. In some embodiments, the particles do not comprise a polymer.

The particles are preferably large enough to bind to more than one biomolecule and inhibit the interaction of more than one bound biomolecule with a binding partner. For example, the particles may be about 50nm to about 10 μm. The size of the particles may be 1 μm to 5 μm, 1.2 μm to 4 μm, 1.5 μm to 4 μm, or 2 μm to 4 μm.

Particles having a size of less than 300nm (such as less than 200nm or less than 150nm) are preferred for applications in which the particles are intended to enter and/or exit the vasculature of a subject (such as particles that can be administered by subcutaneous injection). However, for methods in which the particles are not intended to enter the vasculature, larger particles are also well suited for subcutaneous injection. Particles having a size of about 1 μm to about 5 μm are preferred for applications in which the particles are intended to circulate within the vasculature of a subject (e.g., after intravenous administration). Particles having a size greater than 5 μm are preferred for applications in which the particles are intended to reside at the location where they are implanted (e.g., within or adjacent to a tumor); however, particles smaller than 5 μm may also be suitable for implantation. Particles of any size may be used for in vitro applications.

This document also features collection of particles. In some embodiments, the plurality of particles has a narrow or broad polydispersity. As used herein, "polydispersity" refers to the size range of particles within a particular population of particles. That is, an extremely polydisperse population may involve particles having, say, an average size of 1 μm, with individual particles ranging from 0.1 μm to 4 μm. In some embodiments, "narrow polydispersity" is preferred. That is, given a particular average particle size, it is preferred herein that individual particles in the population differ from the average particle size by no more than ± 20%, preferably no more than ± 15%, and most preferably no more than ± 10% in the present application. More specifically, the population of particles preferably has an average particle size of about 0.5 μm to about 2 μm, more preferably from about 0.8 to about 1.5 μm in the present application. Thus, if an average particle size of 1 μm is selected, the individual particles in the population will most preferably be in the range of from about 0.8 μm to about 1.2 μm. In some embodiments, the population of particles has an average particle size of about 0.3 μm to about 1 μm, for example, about 0.4 μm to about 0.9 μm, about 0.5 μm to about 0.9 μm, about 0.4 μm to about 0.8 μm, about 0.5 μm to about 0.7 μm, about 0.3 μm to about 0.9 μm, or about 0.3 μm to about 0.7 μm. In some embodiments, the population of particles has an average particle size of about 1 μm to about 10 μm (e.g., about 1.1 μm to about 4.8 μm, about 1.2 μm to about 4.6 μm, about 1.4 μm to about 4.4 μm, about 1.6 μm to about 4.2 μm, about 1.8 μm to about 4.0 μm, or about 2.0 μm to about 3.8 μm).

In some embodiments, the disclosure features some or more particles having a determined average particle size. As used herein, the "average particle size" is obtained by measuring the size of individual particles, then dividing by the total number of particles. Determination of average particle size is well known in the art. Typically, the longest average linear dimension of the particles is no greater than 4 μm. In some embodiments, the longest average dimension of the particle is no greater than 3.9 μm (e.g., no greater than 3.8 μm, 3.7 μm, 3.6 μm, 3.5 μm, 3.4 μm, 3.3 μm, 3.2 μm, 3.1 μm, 3.0 μm, 2.9 μm, 2.8 μm, 2.7 μm, 2.6 μm, 2.5 μm, 2.4 μm, 2.3 μm, 2.2 μm, 2.1 μm, 2 μm, 1.9 μm, 1.8 μm, 1.7 μm, 1.6 μm, 1.5 μm, 1.4 μm, 1.3 μm, 1.2 μm, 1.1 μm, or 1 μm). In some embodiments, the longest average linear dimension of the particles is no greater than 2.5 μm, 2 μm, 1.5 μm, or 1.25 μm. In some embodiments, the particles have a longest average linear dimension of at least 1 μm, but no greater than 4 μm. In some embodiments, the particles have a longest average linear dimension of at least 1 μm, but no greater than 2 μm. In some embodiments, the particles have a longest average linear dimension of at least 1 μm, but no greater than 1.5 μm. In some embodiments, the longest average dimension of the particle is at least 0.5 μm (e.g., at least 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1 μm, 1.1 μm, 1.2 μm, 1.3 μm, 1.4 μm, or 1.5 μm), but not greater than 4 μm (e.g., not greater than 3.9 μm, 3.8 μm, 3.7 μm, 3.6 μm, 3.5 μm, 3.4 μm, 3.3 μm, 3.2 μm, 3.1 μm, 3.0 μm, 2.9 μm, 2.8 μm, 2.7 μm, 2.6 μm, 2.5 μm, 2.4 μm, 2.3 μm, 2.2 μm, 2.1 μm, 2 μm, 1.9 μm, 1.8 μm, 1.7 μm, or 1.6 μm).

In some embodiments, the particle is a nanoparticle. In some embodiments, the longest average linear dimension of the particle is no greater than 900nm (e.g., 850nm, 800nm, 750nm, 700nm, 650nm, 600nm, 550nm, 500nm, 450nm, 400nm, 350nm, 300nm, 250nm, 200nm, or 150 nm). In some embodiments, the particles are shaped and sized to circulate in the blood or vasculature (e.g., arteries, veins, and capillaries) of a subject (e.g., a human subject). Exemplary particle designs are set forth in fig. 1 through 6.

In some embodiments, the longest dimension of the particle is from about 50nm to about 5 μm (such as from about 100nm to about 4.5 μm, from about 200nm to about 4 μm, from about 300nm to about 3.5 μm, from about 300nm to about μm, or from about 400nm to about 3 μm). In some embodiments, the shortest dimension of the particle is at least about 300nm (such as from about 300nm to about 4 μm or from about 400nm to about 3 μm).

In some embodiments, the plurality of particles are polyhedral, e.g., cubic. In some embodiments, the plurality of particles are spherical. In some embodiments, any of the particles described herein can be porous. Such porous particles include an outer surface and a plurality of inner surfaces of the pores of the particle. The pharmaceutical agent may, for example, be immobilized on the plurality of inner surfaces. In some embodiments, the plurality of pores have a cross-sectional dimension of at least 50 nm. In some embodiments, the plurality of pores have a cross-sectional dimension of at least 100 nm. Porous nanoparticles have been described, for example, in U.S. patent application publication nos. 20140199352, 20080277346, and 20040105821, the disclosure of each of which is incorporated herein by reference in its entirety. Spherical particles have been described, for example, in U.S. patent nos. 8,778,830 and 8,586,096, each of which is incorporated herein by reference.

In some embodiments, the spherical particle may further comprise two intersecting ridges extending from the spherical surface of the particle, wherein the longest dimension of each structure is no greater than 4 μ ι η (e.g., no greater than 3.9 μ ι η, 3.8 μ ι η, 3.7 μ ι η, 3.6 μ ι η, 3.5 μ ι η, 3.4 μ ι η, 3.3 μ ι η, 3.2 μ ι η, 3.1 μ ι η, 3.0 μ ι η, 2.9 μ ι η, 2.8 μ ι η, 2.7 μ ι η, 2.6 μ ι η, 2.5 μ ι η, 2.4 μ ι η, 2.3 μ ι η, 2.1 μ ι η, 2 μ ι η, 1.9 μ ι η, 1.8 μ ι η, 1.7 μ ι η, 1.6 μ ι η, 1.5 μ ι η, 1.4 μ ι η, 1.3 μ ι η, 1.2 μ ι η ι, 1 μ ι η ι, or 1 μ ι η ι), and wherein the ridges are oriented in size and wherein: (i) to inhibit binding of the agent immobilized on the surface of the spherical particle to or activation of a cell surface receptor protein, and/or (ii) to inhibit interaction of the soluble biomolecule with the second member of the specific binding pair when the soluble biomolecule is bound to the agent, wherein the soluble biomolecule is the first member of the specific binding pair.

In some embodiments, the plurality of particles are annular. In such embodiments, the agent may be immobilized on the inner peripheral surface of the particle (e.g., around the hole, see fig. 2). In some embodiments, the particles have a diameter of no greater than 4 μm (e.g., 3.9 μm, 3.8 μm, 3.7 μm, 3.6 μm, 3.5 μm, 3.4 μm, 3.3 μm, 3.2 μm, 3.1 μm, 3.0 μm, 2.9 μm, 2.8 μm, 2.7 μm, 2.6 μm, 2.5 μm, 2.4 μm, 2.3 μm, 2.2 μm, 2.1 μm, 2 μm, 1.9 μm, 1.8 μm, 1.7 μm, 1.6 μm, 1.5 μm, 1.4 μm, 1.3 μm, 1.2 μm, 1.1 μm, or 1 μm). In some embodiments, the particle is no greater than 900nm in diameter (e.g., 850nm, 800nm, 750nm, 700nm, 650nm, 600nm, 550nm, 500nm, 450nm, 400nm, 350nm, 300nm, 200nm, or 150 nm).

In some embodiments, the particles described herein are dendritic. For example, Du et al, Small 11(4):392- > 413 (2015); siegwart, D.J.et al, Proceedings National Academy Sciences USA 108(32):12996 (2011); such particles are described in U.S. patent nos. 5,814,272 and 7,932,311 and U.S. patent application publication No.20040166166, the disclosure of each of which is incorporated herein by reference. As detailed below, in some embodiments, the geometry of the dendritic particle is such that the agent immobilized on the inner surface of the particle has a reduced or substantially reduced ability to interact with biomolecules on the cell surface, and/or the soluble biomolecules bound to the particle by the agent have a reduced or substantially reduced ability to interact with their cognate ligands (second member of a specific binding pair).

In some embodiments, the plurality of particles are polyhedral, e.g., octahedral or icosahedral (see, e.g., fig. 3), whether regular or irregular. The particles may include at least one protrusion from at least one of the vertices of the particle (see, e.g., fig. 3). The particles may comprise more than one (e.g. 2,3, 4, 5, 6, 7 or 8 or more) protrusion from the apex of the particle. Such projections may be, for example, sized and/or oriented: (i) to inhibit binding of the agent immobilized on the surface of the spherical particle to or activation of a cell surface receptor protein and/or (ii) to inhibit interaction of the soluble biomolecule with the second member of the specific binding pair when the soluble biomolecule is bound to the agent, wherein the biomolecule is the first member of the specific binding pair.

The particles may include a void space, referred to herein as a "void" or "voids". Pores are spaces in a particle that are filled with a fluid (e.g., a liquid (which may include biomolecules) or a gas (such as when the particle is dry)) or with empty spaces (e.g., when the particle is under vacuum, such as after lyophilization). The pore volume of the particle may comprise, for example, the pore volume of the particle and/or the internal volume of the hollow core/shell particle, lumen of the tube, annulus or ring.

In some embodiments, for example, when the particles are located in the vasculature of a subject, the particles are configured such that plasma can freely enter and/or exit the pore space of the particles. In some embodiments, for example, when the particles are located in the vasculature of a subject, the particles are configured such that serum can freely enter and/or exit the pore space of the particles. In a preferred embodiment, the particles are configured such that blood cells cannot enter the pore space of the particles. In some embodiments, the particles are configured such that platelets cannot enter the pore space of the particles. However, for example, when the particle is configured for use in vitro or when the particle is configured to bind to a virus, bacteria, protist, fungus or yeast cell or other large target (such as a target from about 100nm to about 2 μm in size), the particle may allow platelets to enter its pore space.

In some embodiments, the particles are configured such that extracellular fluid may freely enter and/or exit the pore space of the particles. In some embodiments, the particles are configured such that interstitial fluid may freely enter and/or exit the pore space of the particles. In some embodiments, the particles are configured such that cerebrospinal fluid may freely enter and/or exit the void space of the particles.

The volume of pore space in the particle is preferably large enough to accommodate more than one biomolecule, e.g., the total pore volume of the particle is preferably large enough to accommodate each biomolecule bound to the particle. However, the pores may be smaller than the total volume of each bound biomolecule, as long as the particles are capable of inhibiting the interaction between each bound biomolecule and the second building block comprising the binding pair of each biomolecule. For example, a particle may only need to sequester the binding sites of a biomolecule to inhibit interaction between the biomolecule and the second member of the binding pair, and such a particle may contain a pore volume that accommodates the binding site of each biomolecule, but allows other portions of one or more biomolecules to protrude outward from the pore space.

In some embodiments, the particles may comprise from about 5% to about 95% void space. Particles comprising projections may comprise little or no void space, for example, because the projections may inhibit interaction between the bound biomolecule and the second member of the binding pair. Particles comprising a tube may comprise a large amount of void space, for example, because the tube may comprise a large internal volume relative to the thickness of the tube wall. However, the pore volume of particles having similar geometries may include different amounts of pore volume, e.g., a tube including walls of the same thickness may vary widely in pore volume percentage depending on the tube diameter.

The particles can include 0% to about 40% void space, about 20% to about 60% void space, about 40% to about 80% void space, or about 60% to 100% void space. The particle may comprise 0% to about 20% void space, about 10% to about 30% void space, about 20% to about 40% void space, about 30% to about 50% void space, about 40% to about 60% void space, about 50% to about 70% void space, about 60% to about 80% void space, about 70% to about 90% void space, or about 80% to 100% void space. The particle may comprise 0% to about 10% void space, about 5% to about 15% void space, about 10% to about 20% void space, about 15% to about 25% void space, about 20% to about 30% void space, about 25% to about 35% void space, about 30% to about 40% void space, about 35% to about 45% void space, about 40% to about 50% void space, about 45% to about 55% void space, about 50% to about 60% void space, about 55% to about 65% void space, about 60% to about 70% void space, about 65% to about 75% void space, about 10% to about 25% void space, about 10% to about 20% void space, about 25% to about 35% void space, about 30% to about 40% void space, about 35% to about 45% void space, about 60% to about 75% void space, or a combination of the foregoing particle, About 70% to about 80% void space, about 75% to about 85% void space, about 80% to about 90% void space, about 85% to about 95% void space, or about 90% to 100% void space.

The particles may include a neutral charge at physiological pH (e.g., -7.4). The particles may include a slight negative charge or a slight positive charge at physiological pH. The surface (e.g., outer surface) of the particle may include a slight negative charge or a slight positive charge at physiological pH. In preferred embodiments, the surface (e.g., outer surface) of the particle comprises a slight negative or neutral charge at physiological pH. The particles may have an isoelectric point of from about 5 to about 9, preferably from about 6 to about 8. The particles comprising nucleic acids can have an isoelectric point of about 4 to about 7. In some embodiments, the particles have an isoelectric point of less than 7.4, i.e., such that the particles have a net negative charge at physiological pH. For example, the isoelectric point of the particles can be about 6.0 to about 7.4 (e.g., about 6.4 to about 7.4). Particles that include a net negative charge at physiological pH are less likely to interact with eukaryotic cells (e.g., mammalian cells) because eukaryotic cells typically include a cell membrane with a net negative charge. The particles preferably do not include sufficient charge (and/or charge density) to participate in non-specific interactions with other charged molecules.

Particles comprising pores

In some embodiments, the material (e.g., silicon) used to prepare the particles may have a porosity of about 40% to about 95% (such as about 60% to about 80%). As used herein, porosity is a measure of the void space in a material and is the fraction of the void volume in the total volume of the material. In certain embodiments, the support material has a porosity of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or even at least about 90%. In particular embodiments, the porosity is greater than about 40%, such as greater than about 50%, greater than about 60%, or even greater than about 70%.

In certain embodiments, the agent is distributed to pores at a depth of at least about 0.005 μm, at least 0.05 μm, at least about 0.1 μm, at least about 0.2 μm, at least about 0.3 μm, at least about 0.4 μm, at least about 0.5 μm, at least about 0.6 μm, or at least about 0.7 μm from the surface of the material. In certain embodiments, the pharmaceutical agent is substantially uniformly distributed in the pores of the carrier material.

The pharmaceutical agent may be loaded into the particle to a depth measured as a ratio of the total width of the particle. In certain embodiments, the agent is distributed to at least about 10% of the depth of the entry particle, to at least about 20% of the depth of the entry particle, to at least about 30% of the depth of the entry particle, to at least about 40% of the depth of the entry particle, to at least about 50% of the depth of the entry particle, or to at least about 60% of the depth of the entry particle.

Methods for immobilizing agents on porous particles are known, including methods for immobilizing agents to a first surface of a particle and immobilizing different molecules (e.g., coatings) to a second surface of a particle (see, e.g., Cauda, v.et al., j.am.chem.soc.131(32): 11361-. Further, such methods are generally applicable to the manufacture of any of the particles described herein.

The pore size may be preselected to the size characteristics of the agent and the target biomolecule to control the release of the biomolecule. Typically, too small a pore size hinders loading of the agent and/or binding of the biomolecule. For example, the average pore size of the material may be selected from larger pores (e.g., 15nm to 40nm) for high molecular weight molecules (e.g., 200,000-500,000amu) and smaller pores (e.g., 2nm to 10nm) for lower molecular weight molecules (e.g., 10,000-50,0000 amu). For example, an average pore size of about 6nm in diameter may be suitable for molecules having a molecular weight of about 14,000 to 15,000amu (e.g., about 14,700 amu). Molecules having a molecular weight of about 45,000 to 50,000amu (e.g., about 48,000amu) may be selected for an average pore size of about 10nm in diameter. Molecules having a molecular weight of about 150,000nm may be selected with an average pore size of about 25-30nm in diameter.

The pore size may be preselected to accommodate the molecular radius of the agent or biomolecule. For example, an average pore size of about 25nm to about 40nm in diameter may be suitable for molecules having a maximum molecular radius of from about 6nm to about 8 nm. The molecular radius may be calculated by any suitable method, such as by using the physical dimensions of the molecule based on X-ray crystallographic data or using hydrodynamic radii representing the solution-state dimensions of the molecule. Since solution state calculations depend on the nature of the solution from which the calculations are made, some measurements may prefer to use the physical linearity of the molecules based on X-ray crystallographic data. As used herein, the maximum molecular radius reflects half of the maximum linear dimension of the therapeutic agent.

In certain embodiments, the average pore size is selected to limit aggregation of molecules (e.g., proteins) within the pores. It would be advantageous to prevent aggregation of biomolecules (such as proteins) in the carrier material, as aggregation is believed to hinder the controlled release of molecules into biological systems. Thus, a pore that allows, for example, only one biomolecule to enter the pore at any one time will be preferred over a pore that allows multiple biomolecules to enter the pore together and aggregate within the pore due to the relationship between pore size and size of the biomolecules. In certain embodiments, multiple biomolecules may be loaded into a well, but due to the depth of the well, proteins distributed throughout the depth of the well will aggregate to a lesser extent.

Particles comprising at least one tube

In some embodiments, the particle comprises at least one tube. In preferred embodiments, at least one tube comprises one open end or two open ends.

The term "tube" refers to a three-dimensional shape having a length along an axis (e.g., a one-dimensional axis in cartesian space) and an internal cavity, lumen, aperture, or reservoir along the length of the shape. In some embodiments, the vertical cross-sections along the axis of the tube have substantially the same shape and/or size. As used in relation to a tube, the term "cross-section" refers to a two-dimensional cross-section perpendicular to the axis of the tube. The larger structure may comprise a tube. For example, the syringe includes a tube, but the tube does not include a syringe plunger. The particles or other objects may comprise more than one tube. For example, the syringe may include two tubes corresponding to a syringe needle and syringe barrel, or to parallel barrels of a dual syringe (e.g., for epoxy compositions).

The tube may have a diameter that is the average length of line segments perpendicular to the axis of the tube, where each line segment is defined by two points on the outer surface of the tube. The tube may have a width and a height, wherein the width of the tube is the longest line segment defined by two points on the outer surface of the tube that is perpendicular to the axis of the tube, and the height of the tube is the line segment defined by two points on the outer surface of the tube that is perpendicular to the axis of the tube and the line segment defining the width of the tube.

The tube may have an inner diameter that is the average length of line segments perpendicular to the axis of the tube, where each line segment is bounded by two points on the inner surface of the tube. The tube may have an inner width and an inner height, wherein the inner width of the tube is the longest line segment defined by two points on the outer surface of the tube that is perpendicular to the axis of the tube, and the inner height of the tube is the line segment defined by two points on the outer surface of the tube that is perpendicular to the axis of the tube and the line segment defining the width of the tube.

The tube may be substantially cylindrical. The tube may have a substantially circular cross-section. The cross-section of the tube may be oval (e.g., circular).

The cross-section of the tube may be polygonal (e.g. regular polygonal). The cross-section of the tube may be triangular (e.g. equilateral). The cross-section of the tube may be quadrilateral (e.g. regular quadrilateral, rectangular or square). The cross-section of the tube may be pentagonal (e.g., regular pentagonal). The cross-section of the tube may be hexagonal (e.g. regular hexagonal). The tube can be a triangular tube, a square tube, a pentagonal tube, a hexagonal tube, a heptagonal tube or an octagonal tube.

The length of the tube may be about 5nm to about 5 μm (such as about 5nm to about 4 μm, about 5nm to about 3 μm, about 5nm to about 2 μm, or about 5nm to about 1 μm). The length of the tube may be about 50nm to about 5 μm (such as about 50nm to about 4 μm, about 50nm to about 3 μm, about 50nm to about 2 μm, or about 50nm to about 1 μm). The length of the tube may be about 100nm to about 5 μm (such as about 100nm to about 4 μm, about 100nm to about 3 μm, about 100nm to about 2 μm, or about 100nm to about 1 μm). The length of the tube may be about 300nm to about 5 μm (such as about 300nm to about 4 μm, about 300nm to about 3 μm, about 300nm to about 2 μm, or about 300nm to about 1 μm). The length of the tube may be about 500nm to about 5 μm (such as about 500nm to about 4 μm, about 500nm to about 3 μm, about 500nm to about 2 μm, or about 500nm to about 1 μm).

The diameter, width, and/or height of the tube can be about 5nm to about 5 μm (such as about 5nm to about 4 μm, about 5nm to about 3 μm, about 5nm to about 2 μm, about 5nm to about 1 μm, about 5nm to about 900nm, about 5nm to about 800nm, about 5nm to about 700nm, about 5nm to about 600nm, about 5nm to about 500nm, about 5nm to about 400nm, about 5nm to about 300nm, about 5nm to about 200nm, or about 5nm to about 100 nm). The diameter, width, and/or height of the tube can be about 50nm to about 5 μm (such as about 50nm to about 4 μm, about 50nm to about 3 μm, about 50nm to about 2 μm, about 50nm to about 1 μm, about 50nm to about 900nm, about 50nm to about 800nm, about 50nm to about 700nm, about 50nm to about 600nm, about 50nm to about 500nm, about 50nm to about 400nm, about 50nm to about 300nm, about 50nm to about 200nm, or about 50nm to about 100 nm).

The inner diameter, inner width and/or inner height of the tube is preferably large enough to accommodate the agent and the biomolecule. The internal diameter, internal width, and/or internal height of the tube are preferably small enough to inhibit a cell from entering the interior of the tube (e.g., a nucleated eukaryotic cell (such as a nucleated human cell or a diploid human cell)). The internal diameter, internal width, and/or internal height of the tube may be about 5nm to about 4 μm (such as about 5nm to about 3 μm, about 5nm to about 2 μm, about 5nm to about 1 μm, about 5nm to about 900nm, about 5nm to about 800nm, about 5nm to about 700nm, about 5nm to about 600nm, about 5nm to about 500nm, about 5nm to about 400nm, about 5nm to about 300nm, about 5nm to about 200nm, or about 5nm to about 100 nm). The internal diameter, internal width, and/or internal height of the tube may be about 20nm to about 4 μm (such as about 20nm to about 3 μm, about 20nm to about 2 μm, about 20nm to about 1 μm, about 20nm to about 900nm, about 20nm to about 800nm, about 20nm to about 700nm, about 20nm to about 600nm, about 20nm to about 500nm, about 20nm to about 400nm, about 20nm to about 300nm, about 20nm to about 200nm, or about 20nm to about 100 nm). The internal diameter, internal width, and/or internal height of the tube may be about 40nm to about 4 μm (such as about 40nm to about 3 μm, about 40nm to about 2 μm, about 40nm to about 1 μm, about 40nm to about 900nm, about 40nm to about 800nm, about 40nm to about 700nm, about 40nm to about 600nm, about 40nm to about 500nm, about 40nm to about 400nm, about 40nm to about 300nm, about 40nm to about 200nm, or about 40nm to about 100 nm).

In certain preferred embodiments, the particles comprise a plurality of tubes. Each tube of the plurality of tubes may be substantially parallel. In some embodiments, at least two tubes of the plurality of tubes are not parallel. In some embodiments, none of the plurality of tubes are parallel. The tubes may be arranged in configurations other than parallel to distribute the openings to the tubes on different faces of the particles, or to allow the particles to tumble in a flow (e.g., laminar or turbulent).

The plurality of tubes may be arranged in a grid or bundle.

The plurality of tubes may be arranged in a polyhedron (e.g., a regular polyhedron). The plurality of tubes may be arranged in a tetrahedron (e.g., a regular tetrahedron). The plurality of tubes may be arranged in a hexahedron (e.g., a cube, a cuboid, or a cube). The plurality of tubes may be arranged in an octahedron (e.g., a regular octahedron). The plurality of tubes may be arranged in a dodecahedron (e.g., a regular dodecahedron). The plurality of tubes may be arranged in an icosahedron (e.g., a regular icosahedron). In some embodiments, each edge of the polyhedron is defined by a single tube. In some embodiments, less than each edge of the polyhedron is defined by a single tube (e.g., when each of the tubes are substantially parallel).

The plurality of tubes may be arranged in a pyramid (e.g., a triangular pyramid, an oblique square pyramid, a rectangular pyramid, a square pyramid, a pentagonal pyramid, a hexagonal pyramid, a seven pyramid, or an eight pyramid). The plurality of tubes may be arranged in a right pyramid or an oblique pyramid. In some embodiments, each edge of the pyramid is defined by a single tube. In some embodiments, less than each edge of the pyramid is defined by a single tube (e.g., when each of the tubes are substantially parallel).

The plurality of tubes may be arranged in a prism (e.g., a triangular prism, a rectangular prism, a square prism, a pentagonal prism, a hexagonal prism, a heptagonal prism, or an octagonal prism). The plurality of tubes may be arranged in a right prism, a truncated prism, or a truncated prism. In some embodiments, each edge of the prism is defined by a single tube. In some embodiments, less than each edge of the prism is defined by a single tube (e.g., when each of the tubes are substantially parallel).

The plurality of tubes may be arranged in a configuration having a length, a width, and a height, wherein no single degree of linearity is more than 5 times greater than any other degree of linearity. For example, the plurality of tubes may be arranged in a configuration in which no single degree of linearity is more than 4 times greater than any other degree of linearity, or no single degree of linearity is more than 3 times greater than any other degree of linearity. Such a configuration is advantageous for, for example, intravenous administration of particles, as elliptical particles may not flow well in the bloodstream of a patient.

The plurality of tubes may be arranged in a configuration having a length and a diameter, wherein the length of the configuration is no more than 5 times its diameter. For the plurality of tubes may be arranged in a configuration wherein the length of the configuration is no more than 4 times its diameter, or the length of the configuration is no more than 3 times its diameter. Such a configuration is advantageous for, for example, intravenous administration of particles, as elliptical particles may not flow well in the bloodstream of a patient.

The particles may comprise 1 to 500 tubes (e.g. 1 to 100 tubes). The particle may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 330, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 50, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 tubes.

The plurality of tubes may include 1 to 500 tubes (e.g., 1 to 100 tubes). The plurality of tubes may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 330, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 50, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 tubes.

Each tube of the plurality of tubes may have the same length, or different tubes of the plurality of tubes may have different lengths. The average length of the tubes may be about 5nm to about 5 μm (such as about 5nm to about 4 μm, about 5nm to about 3 μm, about 5nm to about 2 μm, or about 5nm to about 1 μm). The average length of the tubes may be about 50nm to about 5 μm (such as about 50nm to about 4 μm, about 50nm to about 3 μm, about 50nm to about 2 μm, or about 50nm to about 1 μm). The average length of the tubes may be about 100nm to about 5 μm (such as about 100nm to about 4 μm, about 100nm to about 3 μm, about 100nm to about 2 μm, or about 100nm to about 1 μm). The average length of the tubes may be about 300nm to about 5 μm (such as about 300nm to about 4 μm, about 300nm to about 3 μm, about 300nm to about 2 μm, or about 300nm to about 1 μm). The average length of the tubes may be about 500nm to about 5 μm (such as about 500nm to about 4 μm, about 500nm to about 3 μm, about 500nm to about 2 μm, or about 500nm to about 1 μm).

Each tube of the plurality of tubes may have the same diameter, width, and/or height, or different tubes of the plurality of tubes may have different diameters, widths, and/or heights. The average diameter, width, and/or height of the tubes can be from about 5nm to about 5 μm (such as from about 5nm to about 4 μm, from about 5nm to about 3 μm, from about 5nm to about 2 μm, from about 5nm to about 1 μm, from about 5nm to about 900nm, from about 5nm to about 800nm, from about 5nm to about 700nm, from about 5nm to about 600nm, from about 5nm to about 500nm, from about 5nm to about 400nm, from about 5nm to about 300nm, from about 5nm to about 200nm, or from about 5nm to about 100 nm). The average diameter, width, and/or height of the tubes can be from about 50nm to about 5 μm (such as from about 50nm to about 4 μm, from about 50nm to about 3 μm, from about 50nm to about 2 μm, from about 50nm to about 1 μm, from about 50nm to about 900nm, from about 50nm to about 800nm, from about 50nm to about 700nm, from about 50nm to about 600nm, from about 50nm to about 500nm, from about 50nm to about 400nm, from about 50nm to about 300nm, from about 50nm to about 200nm, or from about 50nm to about 100 nm).

Each tube of the plurality of tubes may have the same inner diameter, inner width, and/or inner height, or different tubes of the plurality of tubes may have different inner diameters, widths, and/or heights. The average internal diameter, internal width, and/or internal height of the tube may be about 5nm to about 4 μm (such as about 5nm to about 3 μm, about 5nm to about 2 μm, about 5nm to about 1 μm, about 5nm to about 900nm, about 5nm to about 800nm, about 5nm to about 700nm, about 5nm to about 600nm, about 5nm to about 500nm, about 5nm to about 400nm, about 5nm to about 300nm, about 5nm to about 200nm, or about 5nm to about 100 nm). The average internal diameter, internal width, and/or internal height of the tube may be about 20nm to about 4 μm (such as about 20nm to about 3 μm, about 20nm to about 2 μm, about 20nm to about 1 μm, about 20nm to about 900nm, about 20nm to about 800nm, about 20nm to about 700nm, about 20nm to about 600nm, about 20nm to about 500nm, about 20nm to about 400nm, about 20nm to about 300nm, about 20nm to about 200nm, or about 20nm to about 100 nm). The average internal diameter, internal width, and/or internal height of the tube may be about 40nm to about 4 μm (such as about 40nm to about 3 μm, about 40nm to about 2 μm, about 40nm to about 1 μm, about 40nm to about 900nm, about 40nm to about 800nm, about 40nm to about 700nm, about 40nm to about 600nm, about 40nm to about 500nm, about 40nm to about 400nm, about 40nm to about 300nm, about 40nm to about 200nm, or about 40nm to about 100 nm).

The tube may comprise, for example, a polymer. The polymer may be a naturally occurring polymer or a synthetic polymer. The polymer can be, for example, a nucleic acid (e.g., DNA) or a protein.

Particles comprising a DNA backbone

In some embodiments, the particles comprise a DNA scaffold, e.g., the particles can comprise a DNA origami scaffold (see, e.g., U.S. patent nos. 8,554,489 and 7,842,793; U.S. patent application publication nos. 2013/0224859 and 2010/0216978; and PCT patent application publication No. 2014/170898, each of which is incorporated herein by reference).

The particle may comprise a DNA scaffold, and the DNA scaffold may comprise at least one tube or a plurality of tubes as described herein. For example, the DNA scaffold can include at least one substantially hexagonal tube (see, e.g., U.S. patent application publication No. 2013/0224859, which is incorporated herein by reference).

The DNA scaffold may comprise a honeycomb or a grid (e.g., a hexagonal grid or a square grid) (see, e.g., U.S. patent No. 8,554,489, which is incorporated herein by reference).

In some embodiments, the particle comprises a DNA scaffold, and the DNA scaffold does not comprise a tube. For example, the DNA scaffold may comprise a three-dimensional shape (e.g., a polyhedron), and the agent may be immobilized in an interior surface of the shape.

The DNA scaffold may comprise a polyhedron (e.g., a regular polyhedron). The DNA scaffold may comprise a tetrahedron (e.g., a regular tetrahedron). The DNA scaffold may comprise a hexahedron (e.g., a cube, cuboid, or cube). The DNA backbone may comprise octahedra (e.g., regular octahedra). The DNA scaffold may comprise a dodecahedron (e.g., a regular dodecahedron). The DNA scaffold may comprise an icosahedron (e.g., a regular icosahedron).

The DNA scaffold may comprise a pyramid (e.g., a triangular pyramid, an oblique square pyramid, a rectangular pyramid, a square pyramid, a pentagonal pyramid, a hexagonal pyramid, a seven-pyramid, or an eight-pyramid). The DNA scaffold may comprise a right pyramid or an oblique pyramid.

The DNA scaffold may comprise a prism (e.g., a triangular prism, a rectangular prism, a square prism, a pentagonal prism, a hexagonal prism, a heptagonal prism, or an octagonal prism). The DNA scaffold may comprise a right prism, a truncated prism, or a beveled prism.

The DNA scaffold may include a length, width, and height, wherein no single dimension is more than 5 times greater than any other dimension. For example, no single degree of linearity may be more than 4 times greater than any other degree of linearity, or no single degree of linearity may be more than 3 times greater than any other degree of linearity. Such a configuration is advantageous for, for example, intravenous administration of particles, as elliptical particles may not flow well in the bloodstream of a patient.

In some embodiments, the agent is immobilized on a DNA scaffold. In some embodiments, the agent is bound to a nucleic acid comprising a nucleotide sequence that is complementary to a nucleotide sequence on the DNA backbone, i.e., the nucleotide sequence has at least about 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the reverse complement of the nucleotide sequence of the DNA backbone. Thus, by hybridizing nucleic acids to the DNA backbone, the agent can be immobilized on the surface of the particle.

Particles comprising a shield

The particles can include a core subparticle and a shield, for example, wherein the shield inhibits a biomolecule bound to the core subparticle from interacting with a molecule on the surface of the cell. The shield may include a plurality of shield components. The core subparticle may comprise silicon dioxide. For example, the core subparticle may comprise a silica surface. The core subparticle may comprise gold, silicon, or a polymer. For example, the core subparticle may comprise gold, silicon, or a polymer surface.

A particle comprising an inner core subparticle and having a shield comprising a plurality of shield components attached to the core subparticle may comprise a core subparticle comprising a silica surface (such as a solid silica subparticle, a porous silica subparticle, or a silica nanoshell having a non-silica interior). The core subparticle may comprise a non-silica core material (e.g., silicon or gold) coated with silica. The shield component may be in the form of shield subparticles smaller than the core subparticles (e.g., nanospheres) and may comprise silicon dioxide or a different material (e.g., gold or a polymer). The materials of the core subparticle and the surface of the shield component may be selected to be different to allow for different coupling chemistries to be used to couple additional components or substances to the surface. As described herein, the core subparticle may comprise a surface moiety having a reactive group, and the shield component may comprise a functional group capable of reacting with the reactive group to form a covalent bond between the surface of the core subparticle and the surface of the shield component or subparticle.

The pharmaceutical agent may be provided on the surface of the core subparticle, but to a lesser extent, or preferably not at all, on the surface of the shield component. For example, an agent can be attached to the surface of the silica core subparticle (e.g., having a gold surface instead of a silica surface) by preferentially (or exclusively) forming bonds (e.g., ionic, covalent, or electrostatic interactions) with the silica core subparticle but not with the shield subparticle.

In some embodiments, such particles may include a silica core, such as a substantially spherical silica core, and a shield on a surface of the silica core including a plurality of gold nanoparticles having a cross-sectional dimension less than a cross-sectional dimension of the core (e.g., a diameter of the core). The gold nanoparticles may be substantially spherical. The core subparticle may be solid and non-porous, or may have a porous surface. For example, as described in U.S. Pat. Nos. 6,344,272, Sadtler and Wei, chem.Comm.1604-5 (2002); the formation of silica cores and gold nanoparticles on cores can be achieved as described in Meuhlig et al, ACS Nano,5(8): 6586-. For example, gold nanoparticles can be adsorbed on amine-coated silica cores, or can be attached to silica cores having thiol groups coupled to the silica surface, which are then bound to the gold surface of the gold nanoparticles, by means of electrostatic attraction.

A linker group may be provided between the silica and thiol groups of the core subparticle comprising silica for attaching the shield component to the core subparticle. The linker may have a length selected to set a maximum distance between the silica surface and the thiol group (or, when a thiol is attached to the gold surface, between the silica surface and the gold surface). In this way, the distance between the surface of the silicon dioxide subparticle and the gold subparticle may vary over a range of distances, thereby possibly allowing for a greater number of connections (e.g., because more gold subparticles may be packed at a greater distance from the core silicon dioxide subparticle), and/or enhancing the association between the silicon dioxide subparticle and the gold subparticle (e.g., because at shorter distances, more connections from the surface of the silicon dioxide subparticle may be able to interact with the same gold subparticle, thereby enhancing the association). The linker may comprise an alkylene chain, the length of which may be selected to alter the distance between the surface of the core subparticle and the shield subparticle.

The core subparticle may have a cross-sectional dimension (e.g., diameter of a spherical subparticle or a cylindrical subparticle) of 50nm to 4 μm (e.g., 50nm to 200nm, 100nm to 500nm, 200nm to 1 μm, or 500nm to 4 μm).

The particles may be assembled from a range of core subparticle diameters and shield subparticle diameters. The available surface area of the core subparticle for clearance of the biomolecule may depend on the diameter of the shield subparticle and the effective height above the surface of the core subparticle (including the effective range above the surface of any linker between the surface and the capture agent) required for binding of the target/agent complex to the surface.

The amount of agent that can be bound to the core subparticle can be calculated based on the surface area of the subparticle. Similarly, the number of target biomolecules that can be bound to the core subparticle can be calculated in a similar manner. Such calculations may, for example, be confirmed by in vitro studies of protein binding and may be used to predict the dose of particles that may be required to clear a selected number of target biomolecules (or, in some embodiments, an effective dose of particles or formulations containing the particles to remove some or reduce the concentration of target biomolecules from a system (e.g., an in vitro system) or from circulation of a patient being treated for a disease).

The particles may comprise 0.01 μm²To 50 μm ²(e.g., 0.01 μm)²To 0.1 μm²、0.05μm²To 0.5 μm²、0.1μm²To 1.0 μm²、0.5μm²To 5 μm²、1.0μm²To 10 μm²、5μm²To 25 μm²Or 10 μm²To 50 μm²) The available surface area for capturing the target. For a selected loading of agent per unit area of the surface of the core subparticle, based on the diameter of the core subparticle and the shield subparticle, a maximum dose of the particle may be established that is suitable for scavenging a desired amountA target biomolecule.

The cross-sectional dimension (e.g., diameter) of the shield subparticle may be a multiple of the cross-sectional dimension (e.g., diameter) of the core subparticle. The multiple may be, for example, 0.01 to 0.5 (e.g., 0.02 to 0.2, such as 0.05 to 0.1).

In order for the target biomolecule to be in effective proximity to the agent, the target must be able to diffuse between the shield components to access the agent on the surface of the core subparticle. For example, targets of less than 100kDa (e.g., sTNF-R1/2) have a size that can readily diffuse between protective spheres that are 40nm or greater in diameter. For smaller guard spheres, the effective pore length between spheres is short, and therefore guard spheres smaller than 40nm are also less likely to impede diffusion.

Particles comprising submicron particles

In some embodiments, the particle may comprise a core subparticle and a plurality of protective subparticles. The particle may comprise a shield, and the shield may comprise a plurality of protective subparticles. The agent can be immobilized on the surface of the core subparticle, for example, wherein the surface of the core subparticle is an internal surface. For example, when a biomolecule is bound to a particle, the plurality of protective subparticles may be configured to inhibit interaction of the biomolecule with the second member of the specific binding pair. For example, the plurality of protective subparticles may be configured to inhibit the interaction of the biomolecule with a cell (e.g., a mammalian cell) when the biomolecule is bound to the particle.

The protective subparticles may define an outer surface. In a preferred embodiment, the agent is not immobilized on the surface of the protective subparticle.

The core subparticle is preferably large enough to bind to more than one molecule of the agent. For example, the core subparticle may be about 20nm to about 4 μm in size (e.g., about 50nm to about 2 μm in size). The size of the core subparticle may be about 100nm to about 1000nm, about 100nm to about 800nm, about 100nm to about 600nm, about 100nm to about 400nm, about 100nm to about 200nm, about 200nm to about 1000nm, about 200nm to about 800nm, about 200nm to about 600nm, about 200nm to about 400nm, about 400nm to about 1000nm, about 400nm to about 800nm, about 400nm to about 600nm, about 600nm to about 1000nm, or about 600nm to about 800 nm. The size of the core subparticle may be about 100nm to about 4 μm, 100nm to about 3 μm, 100nm to about 2 μm, about 200nm to about 4 μm, 200nm to about 3 μm, 200nm to about 2 μm, about 400nm to about 4 μm, 400nm to about 3 μm, 400nm to about 2 μm, about 600nm to about 4 μm, 600nm to about 3 μm, 600nm to about 2 μm, about 800nm to about 4 μm, 800nm to about 3 μm, or 800nm to about 2 μm.

The core subparticle may comprise metal, gold, alumina, glass, silica, silicon, starch, agarose, latex, plastic, polyacrylamide, methacrylate, polymer, or nucleic acid. In some embodiments, the core subparticle comprises silicon (e.g., porous silicon).

The core subparticle may be any shape (e.g., cubic, pyramidal, conical, spherical, cylindrical, disk, tetrahedral, hexahedral, octahedral, dodecahedral, or icosahedral), or the core subparticle may not have a defined shape.

The particles may comprise 1 core subparticle. For example, the core subparticle may be a particle of U.S. patent No. 7,368,295 or 8,920,625 (each of which is incorporated herein by reference in its entirety), which is further incorporated into a plurality of protective subparticles.

The particle may comprise a plurality of core subparticles, such as 2 to 300 core subparticles, 2 to 200 core subparticles, 2 to 150 core subparticles, 2 to 100 core subparticles, 2 to 80 core subparticles, or 2 to 42 core subparticles (see, e.g., fig. 4 and 5). In embodiments where the particle comprises a plurality of core subparticles, each of the core subparticles is preferably substantially spherical. Particles comprising a plurality of spherical core subparticles allow for porosity, thereby allowing soluble biomolecules to diffuse through the interior of the particle. However, various other shapes of core subparticles may allow for porosity. A particle comprising a plurality of core subparticles may comprise core subparticles of different shapes and sizes.

The particles may comprise from 1 to about 10⁶One core submicron particle, 1 to about 10⁵CoreSubmicron particle, 1 to about 10⁴A core subparticle, 1 to about 1000 core subparticles, 1 to about 100 core subparticles, or 1 to about 10 core subparticles. The particles may comprise from 2 to about 10⁶One core submicron particle, 2 to about 10⁵One core submicron particle, 2 to about 10⁴A core subparticle, 2 to about 1000 core subparticles, 2 to about 100 core subparticles, or 2 to about 10 core subparticles. The particles may comprise from about 10 to about 10⁶About 10 to about 10 core submicron particles⁵About 10 to about 10 core submicron particles⁴A core subparticle, about 10 to about 1000 core subparticles, or about 10 to about 100 core subparticles.

The core subparticles of the plurality of core subparticles may be linked by a linker (e.g., a covalent linker). For example, each core subparticle of the plurality of core subparticles may be connected to another core subparticle through a linker.

The core subparticle may comprise pores, i.e., the core subparticle may be porous.

The protective subparticle may comprise metal, gold, alumina, glass, silica, silicon, starch, agarose, latex, plastic, polyacrylamide, methacrylate, polymer, or nucleic acid. Some of the protective subparticles are preferentially bound to the core subparticle by a linker (e.g., a covalent linker). However, the protective subparticles may be associated with one or more core subparticles without any covalent attachment. The protected subparticle may be bound to another protected subparticle via a linker (e.g., via a covalent linker). For example, the protective subparticle may form a network or network around the core subparticle, thereby isolating the core subparticle within the particle.

In some embodiments, each protective subparticle of the plurality of protective subparticles is bound to the core subparticle by a linker (e.g., a covalent linker). In some embodiments, some of the plurality of protective subparticles are bound to the core subparticle, and each of the plurality of protective subparticles that are not directly bound to the core subparticle are bound to the protective subparticle, i.e., such that each of the plurality of protective subparticles is directly or indirectly bound to the core subparticle. Thus, a particle may comprise a single layer of protective subparticles (e.g., wherein substantially all of the protective subparticles are directly bound to one or more core subparticles), or a particle may comprise more than one layer of protective subparticles (e.g., wherein a substantial portion of the protective subparticles are indirectly bound to one or more core subparticles through a direct connection to other protective subparticles).

In some embodiments, the particles comprise a first layer of protective subparticles (which comprise the first material) and a second layer of protective subparticles (which comprise the second material). For example, the first material may include silicon dioxide or silicon, and the second material may include gold. For example, the particles can be assembled by attaching a subparticle of a first layer of subparticles to one or more core subparticles and then attaching a subparticle of a second layer of subparticles to the first layer of subparticles. The subparticles of the second layer may comprise a surface similar to the one or more core subparticles, e.g., thereby allowing the subparticles of the first layer to be attached to the one or more core subparticles and the subparticles of the second layer using similar chemistry.

The particles may be assembled using a layer-by-layer method. For example, the particles may be formed by first linking a plurality of core subparticles. The plurality of core subparticles may be substantially homogeneous, e.g., such that the linking molecule may crosslink the core subparticle. The plurality of subparticles may include at least two types of subparticles, for example, having different shapes, sizes, and/or surfaces that allow for desired features within the particle, such as pores. After connecting the plurality of core subparticles, the plurality of protective subparticles may be connected to the plurality of core subparticles. After attaching the plurality of protective subparticles to the core subparticle, a second plurality of protective subparticles may be attached to the plurality of protective subparticles. However, the particles can be assembled in many different ways, and many different layer-by-layer strategies can be employed depending on the desired properties of the particles and the desired chemistry for connecting the subparticles.

Methods for crosslinking submicron particles are known, including methods for crosslinking submicron particles that include antibodies for use in vivo (see, e.g., Cheng, K.et al, ACS Appl Mater Interfaces 2(9): 2489-2495 (2010), which is incorporated herein by reference in its entirety). Such methods may be suitable for producing particles as described herein, for example, by simply varying the relative sizes of the submicron particles.

The protective subparticles may be about 10nm to about 4 μm in size (e.g., about 10nm to about 1 μm in size or about 20nm to about 500nm in size). The protective subparticle may have a size of about 10nm to about 200nm, 10nm to about 100nm, about 10nm to about 80nm, about 10nm to about 60nm, about 10nm to about 40nm, about 10nm to about 20nm, 20nm to about 200nm, about 20nm to about 100nm, about 20nm to about 80nm, about 20nm to about 60nm, about 20nm to about 40nm, 30nm to about 200nm, about 40nm to about 100nm, about 40nm to about 80nm, about 40nm to about 60nm, 60nm to about 200nm, about 60nm to about 100nm, or about 60nm to about 80 nm. The protective subparticle may have a size of about 100nm to about 1000nm, about 100nm to about 800nm, about 100nm to about 600nm, about 100nm to about 400nm, about 100nm to about 200nm, about 200nm to about 1000nm, about 200nm to about 800nm, about 200nm to about 600nm, about 200nm to about 400nm, about 400nm to about 1000nm, about 400nm to about 800nm, about 400nm to about 600nm, about 600nm to about 1000nm, or about 600nm to about 800 nm. The protective subparticle may have a size of about 100nm to about 4 μm, about 100nm to about 3 μm, about 100nm to about 2 μm, about 200nm to about 4 μm, about 200nm to about 3 μm, about 200nm to about 2 μm, about 400nm to about 4 μm, about 400nm to about 3 μm, about 400nm to about 2 μm, about 600nm to about 4 μm, about 600nm to about 3 μm, about 600nm to about 2 μm, about 800nm to about 4 μm, about 800nm to about 3 μm, or about 800nm to about 2 μm.

The particles may comprise from 1 to about 10⁶Protective submicron particles, about 4 to about 10⁶Protective submicron particles, about 10 to about 10⁶Protective submicron particles, 1 to about 10⁵Protective submicron particles, about 4 to about 10⁵Protective submicron particles, about 10 to about 10⁵1 protective submicron particleTo about 10⁴Protective submicron particles, about 4 to about 10⁴Protective submicron particles, about 10 to about 10⁴A plurality of protective subparticles, 1 to about 1000 protective subparticles, about 4 to about 1000 protective subparticles, about 10 to about 1000 protective subparticles, 1 to about 100 protective subparticles, about 4 to about 100 protective subparticles, or about 10 to about 100 protective subparticles.

The core subparticle and the protective subparticle may or may not have similar or identical shapes, sizes, and compositions. Nonetheless, the core subparticle is different from the protective subparticle in that (1) the agent can be immobilized on the core subparticle and the agent is preferentially not immobilized on the protective subparticle, and (2) the core subparticle is preferentially located inside the particle and the protective subparticle can be present on the outer surface of the particle.

Substantially two-dimensional particles

The particles may be two-dimensional in shape. For example, the particles may be circular, annular, cross-shaped, fishbone-shaped, oval, triangular, square, pentagonal, hexagonal, heptagonal, octagonal, or star-shaped. The particles may be star-shaped, and the star-shape may be a concave hexagon, a concave octagon, a concave decagon, or a concave dodecagon. The shape may be a regular shape or an irregular shape. An embodiment of a substantially two-dimensional particle is shown in fig. 6.

In some embodiments, the particle comprises a first side, a second side, and an edge. The first side and the second side may be substantially the same shape. The first side and the second side may include a length and a width. The edge may define a height, which is the distance between the first side and the second side. The width and length can be at least 4 times greater than the height (e.g., 4 to 1000 times greater, 6 to 100 times greater, 8 to 75 times greater, or 10 to 50 times greater than the height). The width and/or length may be greater than 0.2 times to about 20 times the height.

The edge may include one or more concave or recessed portions. The medicament may be bonded to the concave or recessed portion of the rim. The recessed portion is a portion in which the perimeter of the particle includes two adjacent perimeter portions that make an outer angle therebetween of greater than 270 degrees, such as either side of the point of the star shape. In this way, the capture agent can be protected from contact with the membrane of the cell contacting the particle.

In some embodiments, the first side and/or the second side are substantially planar. In some embodiments, the first side and/or the second side comprises a concave or concave portion.

In some embodiments, the particles are in the form of substantially flat stars, e.g., with recessed portions between the dots. The star may have 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more points. The particles may include regular sides or irregular sides.

In some embodiments, the particles are in the form of a cross or fishbone shape, e.g., comprising a stem having arms extending outwardly from the stem on each side to define a concave surface portion between the arms. The cruciform or fishbone arm may further include lateral projections.

The concave edges or the cross-shaped or fishbone-shaped arms between the points of the star preferably extend a distance from the line of the junction point so that the cell membrane cannot be deformed between the points in order to come into contact with the edges. For example, the number of points and the angle between them may determine the depth of the concave edge portion between the points.

Particles suitable for use in the present invention may be formed by nanofabrication (e.g. by nanoimprinting or nano-molding). For example, particles can be produced by the PRINT ("Non-invasive template micro-printing technology" (Particle transfer In Non-printing) ") process (see, e.g., International patent application WO 2007/024323; Perry, J.L.et al., Acc Chem Res.44(10):990-998(2011), each of which is incorporated herein by reference). The particles may be produced by photolithography using known methods.

In some embodiments, the agent may be immobilized on the edge of the particle and not immobilized, or to a lesser extent on the first and second sides of the particle.

In some implementationsIn the protocol, the desired surface area of each particle was 0.2 μm²To 25 μm²Within the range of (1). Therefore, the area of the protected edge portion of the particle that can be manufactured by nano-molding is within a desired range.

IX. medicine

In some embodiments, the agent immobilized on the surface of the particle is a small molecule, a macrocyclic compound, a polypeptide, a peptidomimetic compound, an aptamer (aptamer), a nucleic acid, or a nucleic acid analog. As used herein, "small molecule" refers to an agent having a molecular weight of less than about 6kDa and most preferably less than about 2.5 kDa. Many pharmaceutical companies have extensive chemical and/or biological mixture libraries, including small molecule arrays, typically including fungal, bacterial or algal extracts, which can be screened with any assay applied. The present application contemplates, among other things, the use of small chemical libraries, peptide libraries, or collections of natural products. Tan et al describe a library of over two million synthetic compounds that is compatible with miniaturized cell-based assays (J Am Chem Soc 120:8565-8566 (1998)).

The peptidomimetic can be a compound in which at least a portion of the subject polypeptide is modified and the three-dimensional structure of the peptidomimetic remains substantially the same as the three-dimensional structure of the subject polypeptide. The peptidomimetic can be an analog of a subject polypeptide of the disclosure, which analog is itself a polypeptide that contains one or more substitutions or other modifications within the subject polypeptide sequence. Alternatively, at least a portion of the subject polypeptide sequence may be replaced with a non-peptide structure such that the three-dimensional structure of the subject polypeptide is substantially preserved. In other words, one, two or three amino acid residues within the subject polypeptide sequence may be replaced by a non-peptide structure. In addition, other peptide portions of the subject polypeptide may (but need not) be replaced with non-peptide structures. Peptidomimetics (both peptide and non-peptidyl analogs) may have improved properties (e.g., reduced proteolysis, increased retention, or increased bioavailability). Peptoids generally have improved oral availability, which makes them particularly suitable for treating humans or animals. It should be noted that peptidomimetics may or may not have similar two-dimensional chemical structures, but have common three-dimensional structural features and geometries. Each peptidomimetic may also have one or more unique additional binding elements.

Aptamers are short oligonucleotide sequences that can be used to recognize and specifically bind to virtually any molecule (including cell surface proteins). The exponential enrichment ligand system evolution (SELEX) process is powerful and can be used to easily identify such aptamers. Aptamers can be prepared for a wide range of important proteins (such as growth factors and cell surface antigens) for therapeutic and diagnostic purposes. These oligonucleotides bind their target with similar affinity and specificity as antibodies (see, e.g., Ulrich (2006) Handb Exp Pharmacol173:305-326)。

The agent can be an antibody or antigen-binding portion thereof (i.e., an antibody fragment), wherein the antibody or antigen-binding portion thereof specifically binds to a target (e.g., a soluble biomolecule). The agent can include an antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof specifically binds to a target (e.g., a soluble biomolecule). The term "antibody" refers to all antibodies comprising antibodies of different isotypes, such as IgM, IgG, IgA, IgD and IgE antibodies. The term "antibody" encompasses polyclonal, monoclonal, chimeric or chimeric antibodies, humanized antibodies, primate antibodies, deimmunized antibodies, and fully human antibodies. Antibodies can be prepared or derived from any of a variety of species, for example, mammals, such as humans, non-human primates (e.g., chimpanzees, baboons, or chimpanzees), horses, cows, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice. The antibody may be a purified or recombinant antibody.

The terms "antibody fragment," "biomolecule-binding fragment," "antigen-binding portion of an antibody," and similar terms refer to a fragment of an antibody that retains the ability to bind to a target antigen. Such fragments include, for example, single chain antibodies, single chain Fv fragments (scFv), Fd fragments, Fab 'fragments or F (ab')₂And (3) fragment. scFv fragments are single polypeptide chains, said single polypeptideThe peptide chain comprises both the heavy and light chain variable regions of the antibody from which the scFv was derived. In addition, intrabodies, minibodies, triabodies, and diabodies are also included in the definition of antibodies and are compatible for use with the Methods described herein (see, e.g., Todorovska et al, J Immunol Methods248(1):47-66(2001), Hudson and Kort J Immunol Methods 231(1): 177-. Bispecific antibodies (including DVD-Ig antibodies) are also encompassed by the term "antibody". Bispecific antibodies are monoclonal antibodies, preferably of human or humanized origin, having binding specificity for at least two different antigens.

As used herein, the term "antibody" also encompasses, for example, single domain antibodies, such as camelized (camelized) single domain antibodies. See, e.g., Muydermans et al, Trends Biochem Sci 26: 230-; nuttall et al, Curr Pharm Biotech 1:253-263 (2000); reichmann et al, J Immunol Meth231:25-38 (1999); PCT application publication nos. WO 94/04678 and WO 94/25591; and U.S. patent nos. 6,005,079, 6,015,695, and 7,794,981, which are all incorporated herein by reference in their entirety. In some embodiments, the present disclosure provides single domain antibodies comprising two VH domains with modifications such that a single domain antibody is formed.

In some embodiments, the agent is a non-antibody scaffold protein. Typically, these proteins are obtained by combinatorial chemistry-based modulation of pre-existing ligands or antigen binding proteins. For example, the human transferrin binding site for the human transferrin receptor can be modified using combinatorial chemistry to create different libraries of transferrin variants, some of which have acquired affinity for different antigens (see Ali et al, J Biol Chem 274: 24066-. The portion of human transferrin not involved in binding to the receptor remains unchanged and serves as a scaffold (framework regions resembling antibodies) to present different binding sites. The library is then screened as a library of antibodies against the target antigen of interest to identify those variants that have the best selectivity and affinity for the target antigen. Non-antibody scaffold proteins, while functionally similar to antibodies, have many advantages over antibodies, including, among others, enhanced solubility and tissue penetration, lower manufacturing costs, and ease of conjugation to other molecules of interest (see Hey et al, TRENDS Biotechnol23(10):514-522 (2005)).

Those skilled in the art will appreciate that the scaffold portion of a non-antibody scaffold protein may comprise, for example, all or part of: s. aureus protein A Z domain, human transferrin, human tenth fibronectin type III domain, kunitz-type human trypsin inhibitor domain, human CTLA-4, anchoring repeat protein, human lipocalin, human crystallin, human ubiquitin or trypsin inhibitor from Ecballium elaterium (E.elaterium) (see Hey et al, TRENDS Biotechnol 23(10): 514-.

In some embodiments, the agent is a natural ligand for the target biomolecule. For example, the agent may be a cytokine. As used herein, the term "cytokine" refers to any secreted polypeptide that affects the function of a cell and is a molecule that modulates interactions between cells in the immune, inflammatory, or hematopoietic response. Cytokines include, but are not limited to, monokines and lymphokines, regardless of which cell produces them. For example, monokines are generally produced and secreted by monocytes (e.g., macrophages and/or mononuclear leukocytes). However, many other cells also produce monokines, such as natural killer cells, fibroblasts, basophils, neutrophils, endothelial cells, brain astrocytes, bone marrow stromal cells, epidermal keratinocytes and B-lymphocytes. Lymphokines are generally produced by lymphocytes. Examples of cytokines include, but are not limited to, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-a), and tumor necrosis factor beta (TNF- β).

In some embodiments, the agent is a Tumor Necrosis Factor (TNF) family ligand, e.g., a TNF family ligand selected from TNF α, TNF β, Fas ligand, lymphotoxin α, lymphotoxin β, 4-1BB ligand, CD30 ligand, EDA-a1, LIGHT (TNFSF14), TNF-like ligand (TLA1), a weak inducer of TNF-related apoptosis (TWEAK), and TNF-related apoptosis-inducing ligand (TRAIL). The agent may be a CD40 ligand, CD27 ligand, OX40 ligand, B cell activator (BAFF; TNFSF 13B; BLYS), xenoprotein A (EDA), activation-induced TNFR family receptor ligand (AITRL), Vascular Endothelial Growth Inhibitor (VEGI), proliferation-induced ligand (APRIL), or nuclear factor kappa-B receptor activator ligand (RANKL). In some embodiments, the target is TNF α, TNF β, Fas ligand, lymphotoxin α, lymphotoxin β, 4-1BB ligand, CD30 ligand, EDA-A1, LIGHT, TL1A, TWEAK, TRAIL, CD40 ligand, CD27 ligand, OX40 ligand, B cell activating factor (BAFF; TNFSF 13B; BLYS), exotrotein A (EDA), activation-induced TNFR family receptor ligand (AITRL), Vascular Endothelial Growth Inhibitor (VEGI), proliferation-inducing ligand (APRIL), or nuclear factor kappa-B receptor activating factor ligand (RANKL).

In some embodiments, the agent is a viral protein or a portion thereof that specifically binds to a target (e.g., a soluble form of a membrane protein). In some embodiments, the agent is vTNF, which is a protein capable of specifically binding TNF that is not encoded by the genome of an organism comprising TNF and a TNF receptor. vTNF comprises TNF binding proteins from viruses such as poxviruses (e.g., yatapoxvirus (e.g., yabayavirus, tenuapox virus, and yabayonu oncovirus); vaccinia virus; myxoma virus; and murine poxvirus)) and retroviruses (e.g., simian foamy virus). For example, vTNF may be CrmB, CrmC, CrmD or CrmE of vaccinia virus, M-T2 of myxoma virus, S-T2 of simian foamy virus, vCD30 of vaccinia virus or TPV2L of tenuipox virus. In some embodiments, the agent is E6 or E7 of human papilloma virus, which binds to TNFR1 or TRAILR2 ortholog, CAR1 of avian sarcoma leukemia virus that binds to TNFR.

In some embodiments, the agent is a variant of a natural ligand of the target biomolecule, for example a variant of an interleukin polypeptide, such as variant IL-2 or variant TNF α. According to some embodiments of the invention, the variant may contain one or more amino acid substitutions, deletions or insertions. Substitutions may be conservative or non-conservative. As used herein, the term "conservative substitution" refers to the replacement of an amino acid that occurs in the native sequence in a given polypeptide with a naturally or non-naturally occurring amino acid that has similar steric properties. In the case where the side chain of the natural amino acid to be substituted is polar or hydrophobic, a conservative substitution should be with a naturally occurring or non-naturally occurring amino acid that is also polar or hydrophobic, and optionally has the same or similar steric properties as the side chain of the amino acid being substituted. Conservative substitutions typically include substitutions in the following groups: glycine and alanine; valine, isoleucine and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine, and threonine; lysine, histidine and arginine; and phenylalanine and tyrosine. One letter amino acid is abbreviated as follows: alanine (a); arginine (R); asparagine (N); aspartic acid (D); cysteine (C); glycine (G); glutamine (Q); glutamic acid (E); histidine (H); isoleucine (I); leucine (L); lysine (K); methionine (M); phenylalanine (F); proline (P); serine (S); threonine (T); tryptophan (W); tyrosine (Y) and valine (V). Variants also include fragments of the full-length wild-type natural ligand and fragments comprising one or more amino acid substitutions, insertions, or deletions relative to the wild-type full-length natural ligand from which the fragment is derived.

As used herein, the phrase "non-conservative substitution" refers to the replacement of an amino acid present in the parent sequence by another naturally or non-naturally occurring amino acid having different electrochemical and/or steric properties. Thus, the side chain of a substituted amino acid may be significantly larger (or smaller) than the side chain of the substituted natural amino acid and/or a functional group that may have significantly different electronic properties than the substituted amino acid.

In some embodiments, the variant polypeptide comprises at least 2 (e.g., 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, or more than 100) amino acid substitutions, deletions, or insertions relative to the wild-type full-length polypeptide from which it is derived. In some embodiments, the variant polypeptide comprises no more than 150 (e.g., no more than 145, 140, 135, 130, 125, 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2) amino acid substitutions, deletions, or insertions relative to the wild-type full-length polypeptide from which it is derived.

In some embodiments, a variant polypeptide (e.g., a variant IL-2 or TNF α polypeptide) retains at least 10 (e.g., at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100)% of the ability of a wild-type full-length polypeptide from which it is derived to bind to a target biomolecule (e.g., a member of a specific binding pair, wherein the wild-type full-length polypeptide is a member of the specific binding pair). In some embodiments, the variant polypeptide will have an affinity for the target biomolecule that is greater than the wild-type full-length polypeptide from which the variant is derived. For example, in some embodiments, the variant polypeptide has an affinity for the target biomolecule that is 2(3, 4, 5, 10, 20, 30, 40, 50, 100, 200, 500, or even 1000) times greater than the wild-type full-length polypeptide from which the variant polypeptide is derived. Methods for detecting or measuring the interaction between two proteins are known in the art and described above.

In some embodiments, the wild-type full-length natural ligand modulates the activity of a cell surface receptor. Thus, a variant of a natural ligand may have an increased or decreased ability to modulate the activity of the receptor relative to the activity of the wild-type natural ligand. For example, in some embodiments, a variant polypeptide has less than 90 (e.g., 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or less than 5)% of the ability of the full-length wild-type polypeptide from which it is derived to activate a cell surface receptor protein. In some embodiments, the variant polypeptide does not activate the receptor to which it binds.

Such exemplary variant polypeptides are known in the art. For example, international patent application publication No. WO 2012/085891 describes TNF family ligand variants with reduced trimerization capacity and thus reduced capacity to activate TNF family receptors (see also U.S. patent application publication No. US 2014/0096274, which is incorporated herein by reference). However, variant TNF ligands retain the ability to bind to TNF family receptors. Suitable methods for comparing activity between a variant and a wild-type natural ligand are known in the art.

In some embodiments, the soluble biomolecule is a ligand for a cell surface receptor, for example, a cytokine or chemokine (e.g., MCP-1/CCL2, CCL5, CCL11, CCL12, or CCL19), such as any known in the art or described herein. In some embodiments, the ligand is a Tumor Necrosis Factor (TNF) family ligand or variant thereof. In some embodiments, the TNF family ligand is TNF α or a variant thereof. In some embodiments, the TNF family ligand is Fas ligand, lymphotoxin alpha, lymphotoxin beta, 4-1BB ligand, CD30 ligand, EDA-a1, LIGHT, TL1A, TWEAK, TNF beta, TRAIL, or a variant of any of the foregoing. In some embodiments, the ligand is a TGF β superfamily ligand or variant thereof, e.g., activin a, activin B, anti-mullerian hormone, growth differentiation factor (e.g., GDF1 or GDF11), Bone Morphogenic Protein (BMP), inhibin (e.g., inhibin α, inhibin β), left and right asymmetric developmental factor (lefty), persephin, nodal, neurotrophic factor, TGF β 1, TGF β 2, TGF β 3, or myostatin. In some embodiments, the ligand is a hormone (e.g., a peptide hormone), such as ghrelin.

In some embodiments, the soluble biomolecule is haptoglobin or beta-2 microglobulin.

In some embodiments, the soluble biomolecule is one identified in table 2.

TABLE 2 exemplary soluble biomolecules and/or agents

"AD" refers to an autoimmune disorder and/or an inflammatory disorder. "OA" refers to osteoarthritis.

In some embodiments, the agent can bind (e.g., specifically bind) to a biomolecule selected from the group consisting of: TNF alpha, TNF beta, soluble TNF receptor, soluble TNFR-1, soluble TNFR-2, lymphotoxin alpha, lymphotoxin beta, 4-1BB ligand, CD30 ligand, EDA-A1, LIGHT, TL1A, TWEAK, TRAIL, soluble TRAIL receptor, IL-1, soluble IL-1 receptor, IL-1A, soluble IL-1A receptor, IL-1B, soluble IL-1B receptor, IL-2, soluble IL-2 receptor, IL-5, soluble IL-5 receptor, IL-6, soluble IL-6 receptor, IL-8, IL-10, soluble IL-10 receptor, CXCL1, CXCL8, CXCL9, CXCL10, CX3CL1, FAS ligand, soluble death receptor-3, soluble death receptor-4, soluble death-5, soluble receptor-6, soluble IL-6 receptor, IL-8, IL-10, CXCL1, CXCL10, CXCL 353, FAS ligand, soluble death receptor-3, soluble death receptor-4, A weak inducer of TNF-related apoptosis, MMP1, MMP2, MMP3, MMP9, MMP10, MMP12, CD28, a soluble member of the B7 family, soluble CD80/B7-1, soluble CD86/B7-2, soluble CTLA 7, soluble PD-L7, soluble PD-1, soluble Tim 7, Tim3 7, galectin 3, galectin 9, soluble CEACAM 7, soluble LAG 7, TGF- β 1, TGF- β 2, TGF- β 3, antimilux, neublastin, Glial Derived Neurotrophic Factor (GDNF), bone morphogenetic proteins (e.g., BMP7, BMP3 7, BMP7, GDF α growth inhibitors such as GDF, GDF7, GDF α growth inhibitors such as, GDF, 7, inhibin beta A, B, C, E), bilateral asymmetric developmental factor, nodal, neurotrophic factor, persephin, myostatin, ghrelin, sLR11, CCL2, CCL5, CCL11, CCL12, CCL19, interferon alpha, interferon beta, interferon gamma, clusterin, VEGF-A, granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), prostaglandin E2, hepatocyte growth factor, nerve growth factor, sclerostin, complement C5, angiopoietin 2, angiopoietin 3, PCSK9, beta amyloid, activin A, activin B, beta 2 microglobulin, soluble NOTCH1, soluble NOTCH2, soluble NOTCH3, soluble NOTCH4, soluble Jagged1, soluble Jagged2, soluble DLL1, soluble DLL3, soluble DLL4, conjugated globin, fibrinogen alpha chain, Corticotropin releasing factor, corticotropin releasing factor type 1, corticotropin releasing factor type 2, urocortin 1, urocortin 2, urocortin 3, CD47, anti-interferon gamma autoantibodies, anti-interleukin 6 autoantibodies, anti-interleukin 17 autoantibodies, anti-ghrelin autoantibodies, wnt, indoleamine 2, 3-dioxygenase, C-reactive protein, HIV-1gp120, endotoxin, ricin toxin, Clostridium perfringens (Clostridium perfringens) epsilon toxin, staphylococcal (Staphylococcus) enterotoxin B toxin, and botulinum toxin.

In some embodiments, the agent may comprise an antibody (or antigen-binding portion thereof) that specifically binds to: TNF alpha, TNF beta, soluble TNF receptor, soluble TNFR-1, soluble TNFR-2, lymphotoxin alpha, lymphotoxin beta, 4-1BB ligand, CD30 ligand, EDA-A1, LIGHT, TL1A, TWEAK, TRAIL, soluble TRAIL receptor, IL-1, soluble IL-1 receptor, IL-1A, soluble IL-1A receptor, IL-1B, soluble IL-1B receptor, IL-2, soluble IL-2 receptor, IL-5, soluble IL-5 receptor, IL-6, soluble IL-6 receptor, IL-8, IL-10, soluble IL-10 receptor, CXCL1, CXCL8, CXCL9, CXCL10, CX3CL1, FAS ligand, soluble death receptor-3, soluble death receptor-4, soluble death-5, soluble receptor-6, soluble IL-6 receptor, IL-8, IL-10, CXCL1, CXCL10, CXCL 353, FAS ligand, soluble death receptor-3, soluble death receptor-4, A weak inducer of TNF-related apoptosis, MMP1, MMP2, MMP3, MMP9, MMP10, MMP12, CD28, a soluble member of the B7 family, soluble CD80/B7-1, soluble CD86/B7-2, soluble CTLA 7, soluble PD-L7, soluble PD-1, soluble Tim 7, Tim3 7, galectin 3, galectin 9, soluble CEACAM 7, soluble LAG 7, TGF- β 1, TGF- β 2, TGF- β 3, antimilux, neublastin, Glial Derived Neurotrophic Factor (GDNF), bone morphogenetic proteins (e.g., BMP7, BMP3 7, BMP7, GDF α growth inhibitors such as GDF, GDF7, GDF α growth inhibitors such as, GDF, 7, inhibin beta A, B, C, E), bilateral asymmetric developmental factor, nodal, neurotrophic factor, persephin, myostatin, ghrelin, sLR11, CCL2, CCL5, CCL11, CCL12, CCL19, interferon alpha, interferon beta, interferon gamma, clusterin, VEGF-A, granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), prostaglandin E2, hepatocyte growth factor, nerve growth factor, sclerostin, complement C5, angiopoietin 2, angiopoietin 3, PCSK9, beta amyloid, activin A, activin B, beta 2 microglobulin, soluble NOTCH1, soluble NOTCH2, soluble NOTCH3, soluble NOTCH4, soluble Jagged1, soluble Jagged2, soluble DLL1, soluble DLL3, soluble DLL4, conjugated globin, fibrinogen alpha chain, Corticotropin releasing factor, corticotropin releasing factor type 1, corticotropin releasing factor type 2, urocortin 1, urocortin 2, urocortin 3, CD47, anti-interferon gamma autoantibodies, anti-interleukin 6 autoantibodies, anti-interleukin 17 autoantibodies, anti-ghrelin autoantibodies, wnt, indoleamine 2, 3-dioxygenase, C-reactive protein, HIV-1gp120, endotoxin, ricin toxin, Clostridium perfringens epsilon toxin, staphylococcal enterotoxin B toxin, or botulinum toxin.

The pharmaceutical agent may include Yiprimumab (ipilimumab), pembrolizumab (pembrolizumab), nivolumab (nivolumab), infliximab (infliximab), adalimumab (adalimumab), certolizumab (certolizumab) (e.g., certolizumab (certolizumab pegol)), golimumab (golimumab), etanercept (etanercept), semuzumab (statiumab), nonhemazumab (fresolimumab), metrilizumab (metelimumab), daclizumab (cancicluzumab), tarrituximab (tarexizumab), brontizumab (brontituzumab), merimab (metolizumab), retezumab (urelizumab), brazzumab (brazzumab), granuzumab (brazzumab), merizumab (netuzumab), brazzumab (urelizumab), brazzumab (uralizumab), brazzumab (brazzumab), brazzumab (brazizumab), brazilizumab), brazizumab (brazilizumab), brazizumab (brazilizumab), brazilizumab (brazilizumab), brazilizumab (brazilizumab), brazilizumab (brazilizumab), brazilizumab (brazilizumab), brazilizumab (brazilizumab), brazilizumab (brazilizumab), brazilizumab (brazilizumab), brazilizumab (brazilizumab), braziliz, Bruxizumab (brolizumab), ranibizumab (ranibizumab), aflibercept (aflibercept), alexan (actoxumab), esimumab (elsimimomab), situximab (siltuximab), aleximab (afleimimomab), nerrimumab (nerelimomab), ulilizumab (ozolarizumab), patulizumab (patulizumab), resiuzumab (sikuzumab), omalizumab (omab), omalizumab (omalizumab), adoniuzumab (aducanumab), bamipeuzumab (bapineuzumab), kreinzerumab (crenetizumab), griseolub (crenetitum), more lutetium (ganumumab), penuzumab (ponuzumab), perizumab (sunuzumab), ranibizizumab (ranibizizumab), ranibizizumab (grimazumab), ranibizumab (rituximab), ranibizumab (ezumab), ranibizumab (olelizumab), ranibizizumab (perizumab), ranibizizumab (soulizumab), ranibizizumab (zelizumab), ranibizizumab (perilizumab), ranibizizumab), perilizumab), ranibizizumab (perizumab), perizumab (perizemab), perizemazumab), perizemab), peritricitable (perizemazumab (perize), perizemazumab), perizemazumab (perize), perizorubizumab (perize), perizemazumab (perizemab), perize), perizemajimab), perizemakivu (perizemazumab (perizemab (perizobium (perize), perizemab), perizemazumab (perizemab), trastuzumab (perizemab), trastuzumab (perizemab), trastuzumab (perituzumab), trastuzumab (perizorubimab), trastuzumab (perituzumab), and (perituzumab), trastuzumab (perituzumab), trastuzumab (perituzumab), and (perituzumab), and (perituzumab), trastuzumab (rituximab (perituzumab), trastuzumab (perituzumab), and (perituzumab), trastuzumab), pertuzumab (necitumumab), nimotuzumab (nimotuzumab), palivizumab (panitumumab), ramucirumab (ramucirumab), zalutumumab (zalutumumab), durigumab (duliguzumab), pertuzumab (patritumumab), ertuzumab (ertuzumab), pertuzumab (pertuzumab), trastuzumab (trastuzumab), aleurozumab (alirocumab), amrubizumab (anlukinumab), dirigizumab (diridavuzumab), drouzumab (dupiriumab), duruzumab (duvituzumab), eculizumab (epuizumab), epucirumab (edutab), epritumomab (edutab), efuzumab (edemakumakumakumab), epritumomab (epuzumab), epritumomab (edutab), epritumomab (epuzumab), epritumomab (efuzumab), epratuzumab (efuzumab), epritumomab (perituzumab (perifukumakub), epritumomab (peritub), epritumomab (perituzumab), epritumomab (perituzumab), irutemab (duvituzumab), irutemab (perifujikuvitub), irutemab (perifujikumakumakumakumakumakub), epritumomab (perifujikumakumakub), irkumakumakumakumakumab (perifukumakub), epritumomab (perifukumakumakumakumakub), epuzumab), epritumomab (perifukumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakub), epuz-tekumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumakumaku, Non-revampa (filivumab), veliverumab (fletikumab), floribuzumab (formalumab), furaluzumab (foravirumab), franumab (furraniumab), faliximab (galitumab), ganeitab (ganitumab), gardottanuzumab (gevokizumab), fuselkumab (fusekumab), idavizumab (idarubizumab), imazerumab (imalizumab), inolimumab (inolimumab), linumumab (inolimumab), cetuximab (irauzumab), bivizumab (ixekizumab), lepriuzumab (irkuzumab), laburnumab (labiumikumab), labelizumab (labelizumab), linkumab (leprikumab), rituzumab), dolugumab (leprikulizumab), ritukumab (olelizumab), griffolizumab (olelizumab), griffuzumab (griffuzumab), griffuluzumab (griffusab), griffusab (olezumab), griffusamibevacizumab), griffusab (olezumab), griffusab (olelizumab), griffusab (olezumab), griffusalib (griffusab), griffusab (olezumab), griffusamikavakumab), griffusab (olezumab), griffusab (griffusab), griffusab (olezumab), griffusamikavakumab (griffusab), griffusamikavakumab (griffusab), griffusamikavakumab), griffusab (griffusamikavakumab), griffusab), griffusamikavakumab (griffusamikavakumab), griffusamikavakumab (griffusab), griffusamikavakumab (griffusamikavakumab), griffusame (griffusab), griffusame (griffia, griffusame), griffusame (griffusame, griffia, griffusame, griffia, gri, Ottai (orthoumab), pargyloximab (pagibaximab), palivizumab (palivizumab), pannocumab (panobacumumab), paclobutrazumab (paclobulizumab), perleclizumab (perlizumab), perlazurizumab (pidilizumab), pidilizumab (pidilizumab), pexelizumab (pexelizumab), pristuximab (pristoxiximab), kunklizumab (basizumab), rexalizumab (radretuzumab), rexalizumab (rafilvirumab), raloxizumab (raxinbacumab), regalizumab (regavirumab), rayleigh mab (reslizumab), rituzumab (rituzumab), roseuzumab (raluzumab), roseuzumab (romuzumab), rexizumab (rexalizumab), rituximab (resluruzumab), rituximab (rituximab), rituximab (roxuzumab), roseuzumab (roxuzumab), sexib (seiralizumab), sexib (seqiuzumab), sexib (sexib), sexizumab (seqiuzumab), sexib (sexib), sexitub), sexizumab (sexib), sexituzumab (sexi, Tanezumab, tefrazumab (tefibuzumab), TGN1412, tiujiuzumab (tiltrakizumab), tiujiuzumab (tildakizumab), TNX-650, tosatsuzumab (tosatoxumab), trilojikumab (tralokinab), tremelimumab (tremelimumab), trevogurumab, tuvelumab (tuvirumab), umbuzumab (urtuzumab), vatuzumab (vatuzumab), or an antigen-binding portion of any of the foregoing.

In some embodiments, the agent comprises TNF α, TNF β, soluble TNF receptor, soluble TNFR-1, soluble TNFR-2, vTNF, lymphotoxin α, lymphotoxin β, 4-1BB ligand, CD30 ligand, EDA-A1, LIGHT, TL1A, TWEAK, TRAIL, soluble TRAIL receptor, IL-1, soluble IL-1 receptor, IL-1A, soluble IL-1A receptor, IL-1B, soluble IL-1B receptor, IL-2, soluble IL-2 receptor, IL-5, soluble IL-5 receptor, IL-6, soluble IL-6 receptor, IL-8, IL-10, soluble IL-10 receptor, CXCL1, CXCL8, CXCL9, CXCL10, CX3CL1, FAS ligand, soluble death receptor-3, soluble death receptor-4, vTNF, lymphotoxin α, lymphotoxin β, 4-1BB ligand, CD30 ligand, EDA-A-B ligand, soluble IL-2 receptor, LIGHT, TL1, soluble IL-2 receptor, soluble IL-1, soluble IL-6 receptor, soluble IL-5 receptor, soluble IL-6 receptor, soluble IL-8, soluble IL-10 receptor, CXCL1, soluble IL-10, and a pharmaceutically acceptable salt thereof, Soluble death receptor-5, a weak inducer of TNF-related apoptosis, MMP1, a soluble building block of the CD 1, B1 family, soluble CD 1/B1-1, soluble CD 1/B1-2, soluble CTLA 1, soluble PD-L1, soluble PD-1, soluble Tim 1, Tim3 1, galectin 3, galectin 9, soluble CEACAM1, soluble LAG 1, TGF- β 1, TGF- β 2, TGF- β 3, CCL1, activin, a, activin B, soluble NOTCH1, soluble ch1, soluble jaggch 1, soluble Jagged1, soluble DLL1, or soluble binding protein.

In some embodiments, each particle comprises a plurality of agents. A plurality of saidThe agent may comprise from 10 to about 10⁹Portions, e.g. about 10³To about 10⁷Portion medicament or about 10⁴To about 10⁶And (5) preparing the medicament.

Method for producing antibodies

As described above, in some embodiments, the agent immobilized on the surface of the particle or particles is an antibody or antigen-binding fragment thereof. Antibodies can be obtained by methods known in the art. For example, a mammal (e.g., a mouse, hamster, or rabbit) may be immunized with an immunogenic form of a biomolecule (e.g., soluble TNFR, toxin, or viral protein). Alternatively, immunization can occur by using nucleic acids that express biomolecules (e.g., soluble proteins) in vivo, thereby generating the immunogenic response observed. Techniques for conferring immunogenicity to a protein or peptide include conjugation to a carrier or other techniques known in the art. For example, a peptidyl moiety of a polypeptide of the invention may be administered in the presence of an adjuvant. The progress of the immunization can be monitored by measuring the antibody titer in plasma or serum. A standard ELISA or other immunoassay may be used with the immunogen as the antigen to assess the concentration of the antibody.

After immunization, antisera reactive with the polypeptides of the invention can be obtained, and if desired, polyclonal antibodies isolated from the sera. To produce monoclonal antibodies, antibody-producing cells (lymphocytes) can be harvested from an immunized animal and fused by standard somatic cell fusion procedures with immortalized cells (e.g., myeloma cells) to produce hybridoma cells. Such techniques are well known in the art and include, for example, hybridoma technology (originally developed by Kohler and Milstein ((1975) Nature,256: 495-497)), such as the human B-cell hybridoma technology (Kozbar et al, (1983) Immunology Today,4:72) and the EBV hybridoma technology for the production of human Monoclonal Antibodies (Cole et al, (1985) Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc.pp.77-96). Hybridoma cells can be immunochemically screened for the production of antibodies and isolated monoclonal antibodies that specifically react with the polypeptides of the invention.

Localization of agents on particles XI

In some embodiments, the geometry of the particles is such that the immobilized agent has a reduced or substantially reduced ability to interact with biomolecules on the surface of a cell (e.g., an immune cell, blood cell, or lymphocyte). An immobilized agent can have less than 50% (e.g., 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%) of the ability to bind to a biomolecule on the surface of a cell relative to a free, soluble form of the agent. For example, in some embodiments, TNF α or IL-2 immobilized on the surface of a particle described herein has less than 50 (e.g., 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1)% of the ability of free TNF α or IL-2 to bind to TNF α receptors or IL-2 receptors on the surface of a cell.

In some embodiments, the soluble biomolecule bound to the particle has a reduced or substantially reduced ability to interact with its cognate ligand (the second member of the specific binding pair). The biomolecule may be bound to the particle by means of an agent. A biomolecule bound to a particle can have less than 50% (e.g., 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%) of its ability to interact with its cognate ligand relative to the ability of the biomolecule to be unbound. For example, soluble TNFR bound to a particle described herein has the ability of less than 50 (e.g., 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1)% of free soluble TNFR to interact with free TNF α. In another embodiment, soluble virions bound to particles described herein have an ability of less than 50 (e.g., 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1)% of free virions to interact with their cognate cell surface receptor(s) and infect cells.

In some embodiments, the agent can be immobilized on the inner surface of the particle (e.g., the inner surface of the pores or tubes of the porous particle). In some embodiments, the agent may be immobilized on the outer surface of the particle, but is sterically hindered from interacting with the cell surface by one or more protrusions from the particle. In some embodiments, e.g., a ring-shaped particle, the agent is immobilized on the inner surface of the particle such that the agent has a reduced or substantially reduced ability to interact with biomolecules on the cell surface, and/or the soluble biomolecules bound to the particle by the agent have a reduced or substantially reduced ability to interact with their cognate ligands (second member of a specific binding pair).

Exemplary particle geometries that can reduce or substantially reduce the interaction of an agent with a biomolecule on the cell surface, or the interaction between a biomolecule bound to a particle and its cognate ligand, are listed in fig. 1-6 and described herein.

Scavenger and coating XII

In some embodiments, the particles comprise a scavenger. The scavenger may facilitate clearance of the particle through biological pathways, such as through excretion in the urine, degradation, excretion through the hepatobiliary pathway, and/or phagocytosis.

For example, the particles may comprise a reservoir, wherein the reservoir comprises a scavenger. The reservoir may be a hole or void in the body, for example, a void in a porous silicon body.

For particles comprising pores, the reservoir may be a pore, or the reservoir may be larger or smaller than the average pore size. The reservoir may consist of a recess (e.g., a shallow recess) in the granule, wherein the width or diameter of the recess is greater than the width or diameter of the average pore size. The width or diameter of the reservoir may be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 110, 120, 130, 140, 150, 175, 200, 250, 300, 400, or even about 500 times greater than the width or diameter of the average pore size. The width or diameter of the reservoir may be from about 2 to about 10 times, such as from about 2 to about 8 or from about 2 to about 6 times the width or diameter of the average pore size. The width or diameter of the reservoir may be about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 110, 120, 130, 140, 150, 175, 200, 250, 300, 400, or even about 500 times greater than the width or diameter of the average pore size.

For particles comprising a DNA scaffold, the reservoir may be an internal region of the DNA scaffold. The reservoir (e.g., interior region) may be inaccessible to the cell, e.g., the DNA scaffold may be constructed such that the scaffold sterically hinders the cell from entering the interior region. In some embodiments, the reservoir (e.g., the interior region) is inaccessible to the extracellular protein, e.g., the DNA scaffold can be constructed such that the scaffold sterically hinders entry of the extracellular protein into the reservoir. The reservoir (e.g., interior region) may be inaccessible to the antibody. However, the DNA scaffold may allow the reservoir (e.g., the interior region) to become accessible to cells and/or extracellular proteins after a predetermined period of time. For example, the DNA scaffold may include a biodegradable wall that can degrade (e.g., by hydrolysis) after a predetermined period of time, thereby exposing the clearing agent to cells and/or extracellular proteins. The DNA scaffold can include biodegradable latches that can degrade (e.g., by hydrolysis) after a predetermined period of time, thereby allowing the DNA scaffold to undergo a conformational change, thereby exposing the scavenger to cells and/or extracellular proteins (see, e.g., PCT patent application publication No. WO2014/170899, which is incorporated herein by reference). Similarly, the DNA scaffold may include reservoirs including and opening as described below.

The reservoir may comprise an opening. The opening may be covered by a lid or member, thereby inhibiting interaction between the clearing agent and the cell and/or extracellular protein (e.g., antibody). The cap or member may comprise a polymer, such as a biodegradable polymer. The cover or member may degrade (e.g., by hydrolysis) after a predetermined period of time, thereby exposing the clearing agent to cells and/or extracellular proteins. The cap or member may degrade (e.g., biodegrade) upon exposure to a biological fluid (e.g., plasma or extracellular fluid) for about 1 day to about 5 years (e.g., about 1 day to about 4 years, about 1 day to about 3 years, or about 1 day to about 1 year).

The predetermined period of time may be a period of time in which the particles are in a liquid (e.g., an aqueous liquid). The predetermined period of time may be a period of time during which the particles are retained in vivo (e.g., exposure to biological fluids, pH, enzymes, and/or temperature). The predetermined period of time may be determined, at least in part, by binding the particle to the biomolecule. For example, the particles can be configured such that binding of the biomolecule exposes the scavenger to cells and/or extracellular proteins (see, e.g., PCT patent application publication No. WO2014/170899, which is incorporated herein by reference). The predetermined period of time may be from about 1 day to about 5 years (e.g., from about 1 day to about 3 years or from about 1 day to about 1 year).

Exemplary materials suitable for use as a lid or membrane are described in U.S. patent No. 7,918,842, which is incorporated herein by reference. Typically, these materials degrade or dissolve by enzymatic hydrolysis or exposure to water in vivo or in vitro or by surface or volume erosion. Representative synthetic biodegradable polymers comprise: poly (amides), such as poly (amino acids) and poly (peptides); poly (esters) such as poly (lactic acid), poly (glycolic acid), poly (lactic-co-glycolic acid), and poly (caprolactone); poly (anhydrides); poly (ortho esters); poly (carbonate); and chemical derivatives thereof (substitution, addition of chemical groups such as alkyl, alkylene, hydroxylation, oxidation, and other modifications routinely made by those skilled in the art), copolymers thereof, and mixtures thereof. Other polymers that may be used for the cover or film include: poly (ethers) such as poly (ethylene oxide), poly (ethylene glycol), and poly (tetrahydrofuran); vinyl polymers-poly (acrylates) and poly (methacrylates) such as methyl, ethyl, other alkyl, hydroxyethyl methacrylate, acrylic and methacrylic acids, and others such as poly (vinyl alcohol), poly (vinyl pyrrolidone), and poly (vinyl acetate); poly (urethane); cellulose and its derivatives such as alkyl, hydroxyalkyl, ether, ester, nitrocellulose and various cellulose acetates; poly (siloxane); and any chemical derivatives thereof (substitution, addition of chemical groups, e.g., alkyl, alkylene, hydroxylation, oxidation, and other modifications routinely made by those skilled in the art), copolymers thereof, and mixtures thereof. In certain embodiments, the reservoir lid is formed from one or more crosslinked polymers (e.g., crosslinked polyvinyl alcohol).

In some embodiments, the particles comprise a coating. In some embodiments, the coating includes a scavenger. The coating may shield the scavenger.

The particle may comprise a first surface and a second surface; the agent may be immobilized on the first surface; and the coating may cover at least a portion of the second surface. The first surface may be an interior or inner surface, e.g., the first surface may be oriented such that the agent has a reduced ability to bind to molecules on the surface of the cell. Examples of internal or inner surfaces include a bore, an inner wall of a reservoir or tube, an annular inner peripheral surface, or a concave void. Other embodiments of the interior surface or the inner surface comprise the outer surface of the particle, wherein the outer surface is protected from interaction with the cells by one or more protrusions. The second surface may be an exterior or outer surface, for example, the second surface may be oriented such that the coating may interact with the cells. In some embodiments, the particles can include one or more core subparticles and a plurality of protective subparticles. The particle may comprise a shield, and the shield may comprise a plurality of protective subparticles. The first surface may be a surface of one or more core particles and the second surface may be a surface of a protective subparticle.

The coating may inhibit interactions between the particles, e.g., the coating may reduce the tendency of the particles to form aggregates. The coating may inhibit particle-cell interaction, for example, by presenting a biologically inert surface. The coating may inhibit non-specific interactions with extracellular molecules, e.g., non-specific adsorption of biomolecules. The coating may inhibit specific interactions with cells or extracellular molecules, for example, the coating may disfavor or delay excretion or phagocytosis of the particle. The coating may target the particle for excretion or phagocytosis. A coating (e.g., a second coating) that can mask the targeted particles for excretion or phagocytosis is applied, e.g., to promote the particles in the bloodstream to remain in place for a predetermined amount of time.

The coating may comprise a plurality of elongated coating molecules bound at one end to the surface of the particle. The coating may inhibit interaction between the biomolecule bound to the particle and the second member comprising a specific binding pair for the biomolecule. The coating may inhibit interaction between biomolecules bound to the particles and cells. The agent may be oriented on the particle relative to the coating such that the agent has a reduced ability to bind to molecules on the cell surface. The agent may be oriented on the particle relative to the coating such that the agent has a reduced ability to bind to a target on the cell surface. The agent may be oriented on the particle relative to the coating such that the coating sterically inhibits the agent from binding to molecules on the cell surface. The agent may be oriented on the particle such that the coating sterically inhibits the agent from binding to the target on the cell surface. The coating may be oriented on the particle such that the agent of the particle has a reduced ability to bind to molecules on the cell surface. The coating may reduce the ability of the agent of the particle to activate the cell surface receptor protein relative to the ability of the natural ligand of the cell surface receptor protein.

The particle may include a second coating, for example, where the second coating is comprised of a second plurality of coating molecules. The particle may comprise a second plurality of coating molecules. The second coating and/or the second plurality of coating molecules may reduce in vivo clearance of the particles, for example, by masking the coating and/or the plurality of coating molecules. The second coating and/or the second plurality of coating molecules may be biodegradable, for example, by exposing the coating and/or the plurality of coating molecules to cells and/or extracellular proteins after a predetermined period of time. The second coating and/or the second plurality of coating molecules may comprise a biodegradable polymer, for example each molecule of the second plurality of coating molecules may comprise a biodegradable polymer. The second coating and/or second plurality of coating molecules may include CD47 that inhibits phagocytosis.

In some embodiments, the particle comprises a first surface (e.g., an interior surface) and a second surface (e.g., an exterior surface or an exterior surface); the agent is immobilized on the first surface; and the coating covers at least a portion of the second surface. The orientation of the first surface may reduce the ability of the agent to interact with molecules on the surface of the cell. The orientation of the second surface may allow for interaction between the coating and the cells, extracellular molecules, and/or different particles. The "interaction" between the coating and the cell, extracellular molecule and/or different particle may be a weak, neutral or adverse interaction, e.g., adverse to the stable binding of the particle to the cell, extracellular molecule or other particle. Alternatively, the interaction between the coating and the cells and/or extracellular molecules may be a specific or designed interaction, e.g., to facilitate clearance of the particles by biological pathways (e.g., phagocytosis). In certain preferred embodiments, the second surface is substantially free of pharmaceutical agent. In certain preferred embodiments, the first surface is substantially free of a coating. In certain preferred embodiments, the coating covers substantially all of the second surface.

In some embodiments, the particle comprises a first surface (e.g., an interior surface) and a second surface (e.g., an exterior surface or an exterior surface); the agent is immobilized on the first surface and the second surface; and the coating covers at least a portion of the second surface. In such embodiments, the coating (and/or the second coating) may inhibit interaction between the agent and molecules on the cell surface. In certain preferred embodiments, the coating covers substantially all of the second surface.

In some embodiments, the particle comprises a first surface (e.g., an interior surface) and a second surface (e.g., an exterior surface or an exterior surface); the agent is immobilized on the first surface; and the coating covers at least a portion of the first surface and at least a portion of the second surface. In such embodiments, the coating preferably does not affect the ability of the agent to specifically bind to the biomolecule. In certain preferred embodiments, the coating covers substantially all of the second surface.

In some embodiments, the particle comprises a surface; the agent is immobilized on the surface; and the coating covers at least a portion of the surface. In such embodiments, the coating may not affect the ability of the agent to specifically bind to the biomolecule. The coating may allow some of the agents to specifically bind to the biomolecule and inhibit interaction between some of the agents and the biomolecule. The coating may inhibit interaction between the agent and molecules on the cell surface. In certain preferred embodiments, the coating covers substantially all of the surface.

In some embodiments, the particle comprises a coating covering at least a portion of the second surface and a second coating covering at least a portion (e.g., substantially all) of the coating on the second surface. In such embodiments, the coating may comprise a scavenger (e.g., an "excretion-inducing compound") to target the particle for excretion or phagocytosis. Such a coating may include beta-cyclodextrin. The second coating can include a material (e.g., a second plurality of coating molecules) to inhibit interaction with cells and/or to inhibit non-specific interaction with extracellular molecules (e.g., non-specific adsorption of biomolecules). The second coating may be biodegradable, for example, by exposing the coating on the second surface to cells and/or extracellular proteins after a predetermined period of time. For example, in a particle comprising one or more core subparticles and a plurality of protective subparticles, wherein the capture agent is immobilized on the surface (i.e., the first surface) of the one or more core subparticles, at least a portion of the surface (i.e., the second surface) of the protective subparticles comprises a coating (e.g., a coating comprising a scavenger or a coating comprising a material) to inhibit interaction with a cell and/or inhibit non-specific interaction with an extracellular molecule.

The coating may comprise coating molecules, for example, the coating may consist of a plurality of coating molecules, or the coating may consist of a population of coating molecules. As used herein, the terms "plurality of coating molecules" and "population of coating molecules" each refer to a coating. However, the term "coating" may refer to additional compositions, such as hydrogels. The coating molecule may be a scavenger (and thus, the scavenger may be a coating molecule).

The particles may comprise a plurality of coating molecules. The particle may include a surface and a plurality of agents immobilized on the surface, and at least one molecule of the plurality of coating molecules may be bound to the surface. For example, all or substantially all of the plurality of coating molecules may be bound to the surface.

The particle may include a surface and a second surface, wherein at least one molecule of the plurality of agents and the plurality of coating molecules immobilized on the surface may be bound to the second surface. For example, all or substantially all of the plurality of coating molecules may be bound to the second surface. In some embodiments, some of the plurality of coating molecules are bound to the surface and some of the plurality of coating molecules are bound to the second surface.

In some embodiments, the coating molecule increases the clearance of the particle in vivo. For example, the coating molecules may include pathogen-associated molecular patterns.

In some embodiments, the particles described herein have a coating that includes an excretion-inducing compound that facilitates removal of the particle from circulation, e.g., via the kidney, liver/intestine (e.g., via bile), or phagocytosis (e.g., by antigen presenting cells). The plurality of coating molecules may be a plurality of excretion-inducing compounds. For example, in embodiments in which the particles are annular, the inner peripheral surface (e.g., the first surface) can include an immobilized agent and the outer surface (e.g., the second surface) can include a compound that induces clearance of the particles, e.g., by the kidney, liver, or macrophages. In some embodiments, the excretion-inducing compound is programmed. That is, the compound may be covered with a coating that degrades (e.g., by the action of enzymes, hydrolysis, or gradual dissolution) over time (e.g., a predetermined amount of time), eventually exposing the excretion-inducing compound or other feature that increases clearance. The coating may degrade after exposure to a biological fluid (e.g., plasma or extracellular fluid) for about 1 day to about 5 years (e.g., about 1 day to about 3 years or about 1 day to about 1 year). Thus, in vivo retention of the particles can be modified and/or controlled.

The coating may include an organic polymer, such as polyethylene glycol (PEG). The organic polymer may be attached to the particle, for example, to the surface of the particle. The organic polymer may comprise PEG, polylactate, polylactic acid, a sugar, a lipid, polyglutamic acid, polyglycolic acid (PGA), polylactic acid (PLA), poly (lactic-co-glycolic acid) (PLGA), polyvinyl acetate (PVA), and combinations thereof. In certain embodiments, the particles are covalently conjugated to PEG, which blocks adsorption of serum proteins, facilitates efficient urinary excretion and reduces aggregation of the particles (see, e.g., Burns et al, Nano Letters,9(1): 442-.

In one embodiment, the coating includes at least one hydrophilic moiety, e.g.,polymer of type (having the general formula HO (C)₂H₄O)_a(-C₃H₆O)_b(C₂H₄O)_aH) The nonionic polyoxyethylene-polyoxypropylene block copolymer of (a), triblock copolymer poly (ethylene glycol-b- (DL-lactic acid-co-glycolic acid) -b-ethylene glycol) (PEG-PLGA-PEG), diblock copolymer polycaprolactone-PEG (PCL-PEG), poly (vinylidene fluoride) -PEG (PVDF-PEG), poly (lactic acid-co-PEG) (PLA-PEG), poly (methyl methacrylate) -PEG (PMMA-PEG), and the like. In embodiments having such moieties, the hydrophilic moiety is a PEG moiety, such as: [ methoxy (polyoxyethylene) propyl group ]Trimethoxysilanes (e.g. CH)₃(OC₂H₄)_6-9(CH₂)OSi(OCH₃)₃) , [ methoxy (polyoxyethylene) propyl group]Dimethoxysilanes (e.g. CH)₃(OC₂H₄)_6-9(CH₂)OSi(OCH₃)₂) Or [ methoxy (polyoxyethylene) propyl ]]Methoxysilanes (e.g. CH)₃(OC₂H₄)_6-9(CH₂)OSi(OCH₃)). Suitable coatings are described, for example, in U.S. patent application publication No. 2011/0028662 (which is incorporated herein by reference).

The coating may comprise a polyhydroxyl polymer, such as a natural polymer or a hydroxyl-containing polymer comprising a polyhydroxyl polymer, a polysaccharide, a carbohydrate, a polyol, a polyvinyl alcohol, a polyamino acid (such as polyserine), or other polymers (such as 2- (hydroxyethyl) methacrylate), or combinations thereof. In some embodiments, the polyhydroxypolymer is a polysaccharide. Polysaccharides include mannan, pullulan, maltodextrin, starch, cellulose and cellulose derivatives, gums, xanthan gum, locust bean gum or pectin, combinations thereof (see, e.g., U.S. patent application publication No. 2013/0337070, which is incorporated herein by reference).

In some embodiments, the coating comprises a zwitterionic polymer (see, e.g., U.S. patent application publication Nos. 2014/0235803, 2014/0147387, 2013/0196450, and 2012/0141797; and U.S. patent No. 8,574,549, each of which is incorporated herein by reference).

Other suitable coatings include poly-alpha-hydroxy acids (including polylactic or polylactide, polyglycolic or polyglycolide), poly-beta-hydroxy acids (such as polyhydroxybutyrate or polyhydroxyvalerate), epoxy polymers (including polyethylene oxide (PEO)), polyvinyl alcohols, polyesters, polyorthoesters, polyesteramides, and polyphosphoesters-polyurethanes. Examples of degradable polyesters include: poly (hydroxyalkanoates) comprising poly (lactic acid) or (polylactide, PLA), poly (glycolic acid) or Polyglycolide (PGA), poly (3-hydroxybutyrate), poly (4-hydroxybutyrate), poly (3-hydroxyvalerate) and poly (caprolactone) or poly (valerolactone). Examples of polyoxaesters include poly (alkylene oxalates), such as poly (vinyl oxalate), and polyoxaesters containing amide groups. Other suitable coating materials include polyethers, ether-ester copolymers (copoly (ether-ester)), and polycarbonates, the polyethers including polyethylene glycol. Examples of biodegradable polycarbonates include poly (ortho carbonates), poly (imino carbonates), poly (alkyl carbonates) such as poly (trimethylene carbonate), poly (1, 3-dioxan-2-one), poly (p-dioxanone), poly (6, 6-dimethyl-1, 4-dioxan-2-one), poly (1, 4-dioxacycloheptan-2-one), and poly (1, 5-dioxacycloheptan-2-one). Suitable biodegradable coatings may also comprise polyanhydrides, polyimines (e.g. poly (ethylenimine) (PEI)), polyamides (comprising poly-N- (2-hydroxypropyl) -methacrylamide), poly (amino acids) (comprising polylysine (e.g. poly-L-lysine) or polyglutamic acid (e.g. poly-L-glutamic acid)), polyphosphazenes (e.g. poly (phenoxy-co-carboxyphenoxyphosphazene), polyorganophosphazenes, polycyanoacrylates and polyalkylcyanoacrylates (comprising polybutylcyanoacrylate), polyisocyanates and polyvinylpyrrolidone.

The chain length of the polymer coating molecule can be from about 1 to about 100 monomer units, such as from about 4 to about 25 units.

The particles may be coated with naturally occurring polymers including fibrin, fibrinogen, elastin, casein, collagen, chitosan, extracellular matrix (ECM), carrageenan, chondroitin, pectin, alginate, alginic acid, albumin, dextrin, dextran, gelatin, mannitol, n-halamine, polysaccharides, poly-1, 4-glucan, starch, hydroxyethyl starch (HES), dialdehyde starch, glycogen, amylase, hydroxyethyl amylase, amylopectin, glucose-glycans, fatty acids (and esters thereof), hyaluronic acid, protamine, polyaspartic acid, polyglutamic acid, D-mannuronic acid, L-gulonic acid, zein and other prolamines, alginic acid, guar gum, and choline phosphate, and copolymers and derivatives thereof. The coating may also include modified polysaccharides such as cellulose, chitin, dextran, starch, hydroxyethyl starch, polygluconate, hyaluronic acid, and elafin hydrochloride, as well as copolymers and derivatives thereof.

The particles may be coated with a hydrogel. For example, the hydrogel may be formed using a base polymer selected from any suitable polymer, such as a poly (hydroxyalkyl (meth) acrylate), a polyester, a poly (meth) acrylamide, a poly (vinylpyrrolidone), or a polyvinyl alcohol. The crosslinking agent may be one or more of a peroxide, sulfur dichloride, a metal oxide, selenium, tellurium, a diamine, a diisocyanate, an alkylbenzene disulfide, tetraethylthiuram disulfide, 4' -dithiomorpholine, p-quinine dioxime, and tetrachloro-p-benzoquinone. In addition, boronic acid-containing polymers may be incorporated into the hydrogel, with optional photopolymerizable groups.

In certain preferred embodiments, the coating comprises a material approved for use by the U.S. Food and Drug Administration (FDA). These FDA approved materials include polyglycolic acid (PGA), polylactic acid (PLA), poly (saccharide) lactic acid complex 910 (comprising glycolide in a 9:1 ratio per unit lactide, also known as VICRYL @^TM) Polygluconate (comprising glycolide in a ratio of 9:1 per trimethylene carbonate unit, also known as MAXON^TM) And Polydioxanone (PDS).

Attachment of the coating to the particle may be achieved by covalent or non-covalent bonds, such as by ionic, hydrogen, hydrophobic, coordination, adhesive, or physical absorption or interaction.

Conventional nanoparticle coating methods include dry and wet methods. The dry method comprises the following steps: (a) physical vapor deposition (Zhang, y.et al. solid State commun.115:51(2000)), (b) plasma treatment (Shi, d.et al, appl.phys.lett.78:1243 (2001); Vollath, d.et al, j.nanoparticle res.1:235(1999)), (c) chemical vapor deposition (Takeo, o.et al, j.mater.chem.8:1323(1998)), and (d) pyrolysis of polymeric or non-polymeric organic materials for in situ precipitation of nanoparticles within a matrix (sgravo, v.m.et al, j.mater sci.28:6437 (1993)). The wet process for coating particles comprises: (a) sol-gel processes and (b) emulsification and solvent evaporation processes (Cohen, h.et al, Gene ther.7:1896 (2000); Hrkach, j.s.et al, Biomaterials 18:27 (1997); Wang, d.et al, j.control.rel.57:9 (1999)). The coating may be applied by electroplating, spraying, dip coating, sputtering, chemical vapor deposition or physical vapor deposition. In addition, methods of coating various nanoparticles with polysaccharides are known in the art (see, e.g., U.S. patent No. 8,685,538 and U.S. patent application publication No. 2013/0323182, each of which is incorporated herein by reference).

In some embodiments, the particles may be adapted to facilitate clearance by renal excretion. Renal clearance in subjects with normal renal function typically requires particles with at least one dimension less than 15nm (see, e.g., Choi, h.s., et al, Nat Biotechnol 25(1):1165 (2007); Longmire, m.et al, Nanomedicine 3(5):703 (2008)). However, larger particles may be excreted in urine. For embodiments in which the particles are too large for renal clearance, the particles may then be cleared after degradation to a smaller size in vivo.

In some embodiments, the particles may be adapted to facilitate clearance by hepatobiliary excretion. The Mononuclear Phagocytic System (MPS) of kupffer cells contained in the liver involves hepatic uptake and subsequent biliary excretion of nanoparticles. Certain sizes and surface properties of nanoparticles are known to increase MPS uptake in the liver (see Choi et al, j.dispersion sci.tech.24(3/4): 475-. For example, increasing the hydrophobicity of particles is known to increase the uptake of MPS. Thus, one of ordinary skill in the art can select particles having certain properties to modulate biliary excretion. The hepatobiliary system allows the excretion of particles that are slightly larger (e.g., 10nm to 20nm) than can be excreted through the renal system. For embodiments in which the particles are too large for hepatobiliary excretion, the particles may be cleared after degradation to smaller sizes in vivo. In such embodiments, a coating that facilitates clearance by hepatobiliary excretion may cover a portion of the inner surface of the particle such that the coating becomes exposed after the particle degrades. The particles may include a plurality of coating molecules, e.g., hydrophobic molecules, covering a portion of the surface. The surface may be exposed after the particles degrade, allowing for the removal of degraded particles.

In some embodiments, the particles are adapted to facilitate clearance by phagocytosis. For example, the particles may include a scavenger, wherein the scavenger includes a pathogen-associated molecular pattern, e.g., for recognition by macrophages. Pathogen-associated molecular patterns (PAMPs) comprise unmethylated CpG DNA (bacterial), double-stranded RNA (viral), lipopolysaccharides (bacterial), peptidoglycans (bacterial), lipoarabinomannans (bacterial), zymosan (yeast), mycoplasma lipoproteins (e.g., MALP-2 (bacterial)), flagellins (bacterial), poly (inosinic-cytidylic) acids (bacterial), lipoteichoic acids (bacterial), and imidazoquinolines (synthetic). In preferred embodiments, the PAMP scavenger is masked such that the macrophage does not engulf the particle prior to binding of the particle to the one or more targets. For example, the PAMP scavenger may be masked by any of the above-described coatings (e.g., a polymeric coating, such as a biodegradable polymeric coating). Macrophages can engulf 20 μm large particles (see, e.g., Cannon, G.J., and Swanson, J.A., J.cell Science101: 907-. In some embodiments, a scavenger that facilitates clearance by phagocytosis may cover a portion of the inner surface of the particle such that the scavenger becomes exposed upon degradation of the particle. The particles may include a plurality of scavengers, e.g., PAMPs, covering a portion of the surface. The surface may be exposed after the particles degrade, allowing for the removal of degraded particles. The scavenger may cover a portion of the surface that overlaps with the surface comprising the pharmaceutical agent. The scavenger (e.g., PAMPs) may elicit an immune response against the particle, e.g., after degradation of the second coating or after degradation of the particle.

In some embodiments, the immune response to the scavenger (e.g., PAMPs) may exceed the immune response to the agent and/or agent/biomolecule complex, thereby inhibiting or delaying the onset of the immune response to the agent and/or agent/biomolecule complex. For example, degradation of the particles may expose both the scavenger and the agent (and/or agent/biomolecule complex) to leukocytes. PAMP scavengers may allow for rapid clearance of degraded particles by macrophages, thereby delaying an immune response (e.g., a B cell mediated immune response) against the agent and/or agent/biomolecule complex.

The scavenger may be calreticulin which induces phagocytosis.

In certain preferred embodiments, the coating molecules comprise nucleic acids, e.g., for hybridization with the coating molecules into particles comprising a DNA backbone. For example, a particle can include a nucleic acid and a coating molecule, wherein the coating molecule includes a complementary nucleic acid that can hybridize to the nucleic acid, thereby forming a bond (i.e., hydrogen bond) between the coating molecule and the particle. The nucleic acid may comprise a nucleotide sequence and the complementary nucleic acid may comprise a complementary nucleotide sequence, for example, wherein the nucleotide sequence has at least 95%, 96%, 97%, 98%, or 99% sequence identity to the reverse complement of the complementary nucleotide sequence. The nucleotide sequence may have 100% sequence identity to the reverse complement of the complementary nucleotide sequence.

Preferably, the melting temperature of the nucleic acid and complementary nucleic acid in the physiological fluid (e.g., blood) is greater than body temperature (e.g., body temperature of a subject such as a human or mouse). For example, the melting temperature of the nucleic acid and complementary nucleic acid in the physiological fluid is preferably greater than 37 ℃ (e.g., greater than about 38 ℃, greater than about 39 ℃, greater than about 40 ℃, greater than about 41 ℃, greater than about 42 ℃, greater than about 43 ℃, greater than about 44 ℃, or greater than about 45 ℃). The melting temperature of the nucleic acid and complementary nucleic acids can be from about 37 ℃ to about 120 ℃ (such as from about 38 ℃ to about 120 ℃, from about 39 ℃ to about 120 ℃, from about 40 ℃ to about 120 ℃, from about 41 ℃ to about 120 ℃, from about 42 ℃ to about 120 ℃, from about 43 ℃ to about 120 ℃, from about 44 ℃ to about 120 ℃, from about 45 ℃ to about 120 ℃, from about 46 ℃ to about 120 ℃, from about 47 ℃ to about 120 ℃, from about 48 ℃ to about 120 ℃, from about 49 ℃ to about 120 ℃, from about 50 ℃ to about 120 ℃, from about 38 ℃ to about 100 ℃, from about 39 ℃ to about 100 ℃, from about 40 ℃ to about 100 ℃, from about 41 ℃ to about 100 ℃, from about 42 ℃ to about 100 ℃, from about 43 ℃ to about 100 ℃, from about 44 ℃ to about 100 ℃, from about 45 ℃ to about 100 ℃, from about 46 ℃ to about 100 ℃, from about 47 ℃ to about 100 ℃, from about 48 ℃ to about 100 ℃, from about 49 ℃ to about 100 ℃, or from about 50 ℃ to about 100 ℃).

The length of the nucleic acid of the reactive group, the nucleotide sequence of the reactive group, the complementary nucleic acid and the complementary nucleotide sequence is preferably greater than 9 nucleotides. The nucleic acid of the reactive group, the nucleotide sequence of the reactive group, the complementary nucleic acid and the complementary nucleotide sequence may be greater than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides in length. The length of the nucleic acid of the reactive group, the nucleotide sequence of the reactive group, the complementary nucleic acid, and the complementary nucleotide sequence can be from about 10 nucleotides to about 100 nucleotides, such as from about 11 nucleotides to about 80 nucleotides, from about 12 nucleotides to about 60 nucleotides, from about 13 nucleotides to about 50 nucleotides, from about 14 nucleotides to about 40 nucleotides, from about 15 nucleotides to about 30 nucleotides, or from about 16 nucleotides to about 25 nucleotides. The GC content of the nucleic acids, nucleotide sequences, complementary nucleic acids, and complementary nucleotide sequences can be about 10% to about 100%, such as about 40% to about 100%, about 45% to about 100%, about 50% to about 100%, about 55% to about 100%, about 40% to about 95%, about 45% to about 90%, about 50% to about 85%, or about 55% to about 80%.

In some embodiments, the particles can be cleared by the organism within about 1 day to about 5 years (e.g., about 1 day to about 3 years, or about 1 day to about 1 year).

Application method XIII

The present disclosure contemplates that the compositions described herein (e.g., any of the particles or plurality of particles described generally or specifically herein) can be administered to cells and tissues in vitro and/or in vivo. In vivo administration includes administration to an animal model of a disease, such as an animal model of cancer, or to a subject in need thereof. Suitable cells, tissues or subjects include animals, such as companion animals, livestock, zoo animals, endangered species, rare animals, non-human primates, and humans. Exemplary companion animals include dogs and cats.

For in vitro delivery, e.g., to and/or around cells or tissues in culture, the composition may be added to the culture medium, e.g., contacting the microenvironment or contacting soluble material in the culture medium or contacting the cells or even permeabilizing the cells. The desired active site affects the delivery mechanism and manner for administering the composition (e.g., the particles described herein).

For delivery in vivo, such as to cells or tissues in vivo (including microenvironments to cells and tissues) and/or to a subject in need thereof, many methods of administration are contemplated. The particular method may be selected based on the particulate composition and the particular application and patient. Various delivery systems are known and can be used to administer the agents of the present disclosure. Any such method may be used to administer any of the agents described herein. Methods of introduction may be enteral or parenteral, including, but not limited to, intradermal, intramuscular, intraperitoneal, intramyocardial, intravenous, subcutaneous, pulmonary, intranasal, intraocular, epidural, and oral routes. The compositions of the present disclosure may be administered by any convenient route, such as by infusion or bolus injection (bolus injection), by absorption through epithelial or cutaneous mucosal linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered with (simultaneously or sequentially) other biologically active agents. Administration may be systemic or topical.

In certain embodiments, the composition is administered intravenously, such as by bolus injection or infusion. In certain embodiments, the composition is administered orally, subcutaneously, intramuscularly or intraperitoneally.

In certain embodiments, it may be desirable to administer a composition of the present disclosure locally to an area in need of treatment (e.g., a tumor site, such as by injection into a tumor).

The liver is a common site of metastasis. Thus, in certain embodiments, the delivery of the compositions described herein is to the liver. For example, an intravenous catheter may be placed in the hepatic portal vein to deliver the agents of the present disclosure to the liver. Other methods of delivery via the hepatic portal vein are also contemplated.

In certain embodiments, the compositions of the present disclosure are administered by intravenous infusion. In certain embodiments, the composition is infused over a period of at least 10 minutes, at least 15 minutes, at least 20 minutes, or at least 30 minutes. In other embodiments, the agent is infused over a period of at least 60 minutes, 90 minutes, or 120 minutes. Regardless of the infusion time period, the present disclosure contemplates that, in certain embodiments, each infusion is part of an overall treatment plan in which the agent is administered according to a regular schedule (e.g., weekly, monthly, etc.) for a period of time. However, in other embodiments, the composition is delivered by bolus injection, e.g., as part of an overall treatment plan in which the agent is administered according to a regular schedule for a period of time.

For any of the foregoing, it is contemplated that the compositions of the present disclosure (comprising one agent or a combination of two or more such agents) can be administered in vitro or in vivo via any suitable route or method. The composition may be administered as part of a treatment regimen, wherein the composition is administered one or more times, including according to a particular schedule. In addition, it is contemplated that the compositions of the present disclosure will be formulated to be suitable for the route of administration and the particular application. The present disclosure contemplates any combination of the aforementioned features, as well as combinations with any aspects and embodiments of the disclosure described herein.

The foregoing applies to any composition (e.g., particle or particles) of the present disclosure used alone or in combination and for any of the methods described herein. The present disclosure specifically contemplates any combination of features of such compositions, and methods of the present disclosure with features describing various pharmaceutical compositions and routes of administration described in this section and below.

XIV. pharmaceutical composition

In certain embodiments, the subject particle or particles of the present disclosure are formulated with a pharmaceutically acceptable carrier. One or more compositions (e.g., including a particle or particles described herein) can be administered alone or as a component of a pharmaceutical formulation (composition). As described herein, any of the compositions of the present disclosure described generally or specifically herein can be formulated. In certain embodiments, the composition comprises two or more particles of the present disclosure or particles of the present disclosure formulated with a second therapeutic agent.

The compositions of the present disclosure may be formulated for administration of human or veterinary medicine in any convenient manner. Wetting agents, emulsifiers and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, mold release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition.

The subject particle or particle formulations comprise, for example, those suitable for oral, nasal, topical, parenteral, rectal, and/or intravaginal administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect.

In certain embodiments, the methods of making these formulations or compositions comprise combining one or more particles and a carrier, and optionally one or more accessory ingredients. In general, the formulations can be prepared with liquid carriers or finely divided solid carriers or both, and the product shaped, if desired.

Formulations for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, typically sucrose and acacia or tragacanth), powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base such as gelatin and glycerin, or sucrose and acacia) and/or as a mouthwash, and the like, each containing a predetermined amount of the particles of the present disclosure. Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitol esters, microcrystalline cellulose, aluminum hydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, etc.), one or more compositions of the present disclosure may be mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or calcium hydrogen phosphate, and/or any of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption promoters, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents such as kaolin and bentonite clays; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof; and (10) a colorant. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredients, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitol, and mixtures thereof. In addition to inert diluents, the oral compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

In certain embodiments, the methods of the present disclosure comprise topical administration to the skin or mucosa (such as those on the cervix and vagina). The topical formulations may also contain one or more of a wide variety of adjuvants known to be effective as skin or stratum corneum penetration enhancers. Examples of these are 2-pyrrolidone, N-methyl-2-pyrrolidone, dimethylacetamide, dimethylformamide, propylene glycol, methanol or isopropanol, dimethyl sulfoxide and azone. Additional agents may also be included to make the formulation cosmetically acceptable. Examples of these are fats, waxes, oils, dyes, fragrances, preservatives, stabilizers and surfactants. Keratolytic agents (such as those known in the art) may also be included. Examples are salicylic acid and sulphur. Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, drug patches and inhalants. The subject agents of the present disclosure may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required. In addition to the subject medicaments of the present disclosure, ointments, pastes, creams and gels may contain excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof. Powders and sprays can contain, in addition to the subject agents of the present disclosure, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate, and polyamide powder, or mixtures of these substances. Sprays can additionally contain conventional propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

Pharmaceutical compositions suitable for parenteral administration may include combinations of one or more of the compositions of the present disclosure with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood or suspension or thickening agent of the intended recipient. Examples of suitable aqueous and nonaqueous carriers that can be used in the pharmaceutical compositions of the present disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants (such as preserving, wetting, emulsifying, and dispersing agents). Prevention of microbial activity can be ensured by inclusion of various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol sorbic acid, and the like). It may also be desirable to include isotonic agents (e.g., sugars, sodium chloride, etc.) in the composition. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.

Injectable, pharmaceutically durable forms are prepared by forming a microencapsule matrix of one or more particles in a biodegradable polymer, such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the nature of the particular polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Injectable formulations with durable drug properties are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.

In preferred embodiments, the compositions of the present disclosure are formulated according to conventional procedures as pharmaceutical compositions suitable for intravenous administration to humans or animals (such as companion animals). If desired, the composition may also contain a solubilizing agent and a local anesthetic (such as lidocaine) to relieve pain at the site of injection. When the composition is administered by infusion, the composition can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients may be mixed prior to administration.

In another embodiment, a composition described herein (e.g., a particle or a plurality of particles) is formulated for subcutaneous, intraperitoneal, or intramuscular administration to a human or animal (such as a companion animal).

In certain embodiments, the agents and particles of the present disclosure are formulated for local delivery to a tumor, such as delivery for intratumoral injection.

In certain embodiments, the compositions are intended for topical administration to the liver via the hepatic portal vein, and the agents and particles may be formulated accordingly.

In certain embodiments, a particular formulation is suitable for use where delivery is via more than one route. Thus, for example, formulations suitable for intravenous infusion may also be suitable for delivery via the hepatic portal vein. However, in other embodiments, the formulation is suitable for use with one delivery route, but not a second delivery route.

The amount of the agent or particle of the present disclosure can be determined by standard clinical or laboratory techniques, which amount will be effective to treat a condition (such as cancer), and/or will be effective to neutralize soluble TNFR, and/or will be effective to reduce the amount of soluble TNFR or the TNF α binding activity of soluble TNFR, particularly soluble TNFR present in the tumor microenvironment and optionally in plasma, and/or will be effective to inhibit tumor cell proliferation, growth or survival in vitro or in vivo. In addition, in vitro assays may optionally be used to help determine the optimal dosage range. The precise dose to be used in the formulation will also depend on the route of administration and the severity of the condition, and should be decided according to the judgment of the physician and the circumstances of each subject. An effective dose for administration to a human or animal can be extrapolated from dose-response curves from in vitro or animal model test systems.

In certain embodiments, the compositions (including pharmaceutical formulations) of the present disclosure are non-pyrogenic. In other words, in certain embodiments, the composition is substantially pyrogen-free. In one embodiment, the formulations of the present disclosure are pyrogen-free formulations that are substantially free of endotoxins and/or associated pyrogen species. Endotoxins comprise toxins that are confined within the microorganism and are released only when the microorganism is ruptured or killed. Pyrogen substances also comprise fever-induced heat-stable substances (glycoproteins) from the outer membrane of bacteria and other microorganisms. If administered to humans, these substances can cause fever, hypotension and shock. Due to potentially harmful effects, even small amounts of endotoxins must be removed from a pharmaceutical solution administered intravenously. The food and drug administration ("FDA") has set an upper limit of 5 Endotoxin Units (EU) per kilogram body weight per dose over an hour for intravenous drug use (U.S. pharmacopoeia committee, pharmacopoeia forum 26(1):223 (2000)). Even small amounts of harmful and dangerous endotoxins can be dangerous when the therapeutic protein is administered at relatively large doses and/or over extended periods of time (e.g., as for the entire life of a patient). In certain embodiments, the endotoxin and pyrogen concentration in the composition is less than 10EU/mg, or less than 5EU/mg, or less than 1EU/mg, or less than 0.1EU/mg, or less than 0.01EU/mg, or less than 0.001 EU/mg.

The foregoing applies to any of the agents, compositions, and methods of the present disclosure described herein. The present disclosure specifically contemplates any combination of the features of the agents, compositions, and methods of the present disclosure described herein (alone or in combination) with the features described for the various pharmaceutical compositions and routes of administration described in this section and above.

The present disclosure provides many general and specific examples of agents and classes of agents suitable for use in the methods of the present disclosure ("agents of the present disclosure"). The present disclosure contemplates that any such agent or class of agents may be formulated for in vitro or in vivo administration as described herein.

Furthermore, in certain embodiments, the present disclosure contemplates compositions, including pharmaceutical compositions, comprising any of the agents of the present disclosure described herein formulated with one or more pharmaceutically acceptable carriers and/or excipients. Such compositions may be described using any of the functional and/or structural features of the agents of the present disclosure provided herein. Any such composition or pharmaceutical composition may be used in vivo or in vitro in any of the methods of the present disclosure.

Similarly, the present disclosure contemplates isolated or purified agents of the present disclosure. The agents of the present disclosure described based on any functional and/or structural feature of the agents described herein can be provided as isolated agents or purified agents. Such isolated or purified agents have many uses in vitro or in vivo, including use in any of the in vitro or in vivo methods described herein.

XV. application

The compositions described herein (e.g., particles and pharmaceutical compositions thereof) are useful in a variety of diagnostic and therapeutic applications. For example, the particles described herein may be used to treat cancer, detoxify a subject, or treat a viral or bacterial infection.

Therapeutic applications include the administration of one or more compositions described herein to a subject (e.g., a human subject) using a variety of methods depending in part on the route of administration. The route may be, for example, intravenous injection or Infusion (IV), subcutaneous injection (SC), Intraperitoneal (IP) injection, or intramuscular Injection (IM).

Administration can be achieved, for example, by local infusion, injection, or via an implant. The implant may be a porous, non-porous or gel-like material comprising a membrane, such as a silicone rubber membrane or a fiber. The implant can be configured to continuously or periodically release the composition to a subject (see, e.g., U.S. patent application publication No. 2008/0241223; U.S. patent nos. 5,501,856, 5,164,188, 4,863,457 and 3,710,795; EP488401 and EP430539, the disclosures of each of which are incorporated herein by reference in their entirety). The composition can be delivered to a subject by an implantable device based on, for example, a diffusive, erodible, or convective system (e.g., osmotic pump, biodegradable implant, electro-diffusion system, electro-osmotic system, vapor pressure pump, electrolytic pump, effervescent pump, piezoelectric pump, corrosion-based system, or electromechanical system).

As used herein, in an in vivo setting, the term "effective amount" or "therapeutically effective amount" means a dose sufficient to treat, inhibit or alleviate one or more symptoms of the disorder being treated or to otherwise provide a desired pharmacological and/or physiological effect (e.g., modulate (e.g., enhance) an immune response to an antigen). The precise dosage will vary depending on a variety of factors, such as the subject's variables (e.g., age, immune system health, etc.), the disease, and the ongoing treatment.

In some aspects, the invention relates to methods of treating or preventing a disease or condition in a patient by administering to the patient a composition comprising nanoparticles as described herein. In some embodiments, the present invention relates to methods of reducing the concentration of a biomolecule in a patient, such as the concentration of a biomolecule in a bodily fluid (e.g., blood and/or extracellular fluid) of a patient, by administering a composition comprising nanoparticles as described herein to the patient.

As used herein, a mammal can be a human, a non-human primate (e.g., monkey, baboon, or chimpanzee), a horse, cow, pig, sheep, goat, dog, cat, rabbit, guinea pig, gerbil, hamster, rat, or mouse. In some embodiments, the mammal is an infant (e.g., a human infant). In certain preferred embodiments, the subject is a human.

As used herein, a subject mammal that is "in need of prevention", "in need of treatment", or "in need thereof" refers to a subject mammal that would reasonably benefit from the treatment administered at the discretion of a suitable medical practitioner (e.g., a doctor, nurse, or nurse practitioner in the case of a human; veterinarian in the case of a non-human mammal).

The term "preventing" is art-recognized and is well understood in the art when used in connection with a condition and comprises administration of a composition that reduces the frequency of, or delays the onset of, symptoms of a physical condition in a mammal of a subject relative to a subject that does not receive the composition.

Suitable human dosages of any of the compositions described herein can also be evaluated in, for example, a phase I dose escalation study. See, e.g., van Gurp et al, Am J transfer 8(8):1711-1718 (2008); hanouska et al, Clin Cancer Res 13(2, part 1):523-531 (2007); and Hetherington et al, analytical Agents and Chemotherapy 50(10) 3499-3500 (2006).

The method can further include measuring a concentration of the biomolecule of interest in the subject (e.g., in serum of blood of the subject) prior to administering the composition comprising the plurality of biomolecule-targeting particles to the subject. The method can further include, for example, calculating the number of particles administered to the subject based on the concentration of the biomolecule in the subject (e.g., in the serum of the subject's blood) and/or the height, weight, and/or age of the subject.

Toxicity and therapeutic efficacy of such compositions can be determined by known pharmaceutical procedures in cell cultures or experimental animals (e.g., animal models of cancer, toxicity, or infection). These procedures can be used, for example, to determine LD₅₀(dose lethal to 50% of the population) andED₅₀(a therapeutically effective dose in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and the therapeutic index can be expressed as the ratio LD₅₀/ED₅₀. Agents that exhibit high therapeutic indices are preferred. While compositions exhibiting toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of the affected tissue and minimizes potential damage to normal cells, thereby reducing side effects.

Data obtained from cell culture assays and animal studies can be used to formulate a range of dosages for use in humans. The dosage of such compositions is generally within the range of circulating concentrations of the compositions comprising ED with little or no toxicity₅₀. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The therapeutically effective dose can be estimated initially from cell culture assays. The dose can be formulated in animal models to achieve a circulating plasma concentration range comprising the IC as determined in cell culture ₅₀(i.e., the concentration of antibody that achieves half-maximal inhibition of symptoms). Such information can be used to more accurately determine useful doses in humans. Plasma concentrations can be measured, for example, by High Performance Liquid Chromatography (HPLC). In some embodiments, for example, where local administration is desired, cell culture or animal models can be used to determine the dose required to achieve a therapeutically effective concentration within the local site.

In some embodiments of any of the methods described herein, the particles can be co-administered to the mammal with one or more additional therapeutic agents (e.g., therapeutic agents for treating infection or treating cancer).

In some embodiments, the particles and additional therapeutic agent may be administered to the mammal using different administration uses. For example, the additional therapeutic agent may be administered subcutaneously or intramuscularly, and the particles may be administered intravenously.

In some embodiments, the methods of the invention comprise measuring the concentration of a biomolecule in a subject. For example, the method may comprise measuring the concentration of the biomolecule in the blood of the subject. The method may further comprise administering to the subject a composition comprising a plurality of particles targeting the biomolecule (i.e., a plurality of particles comprising an agent that selectively binds to the biomolecule as described herein). The measuring step may allow for an appropriate dosage of the particles. Thus, the measuring step may be performed prior to application of the composition. Nevertheless, the measuring step may be performed after administration of the composition, for example, to assess the efficacy of the composition. The method may further comprise administering to the subject a second or subsequent dose of a composition comprising a plurality of particles, e.g., if permitted according to the measured biomolecule concentration. In this way, the concentration of the biomolecule can be titrated, for example, by repeatedly measuring the concentration of the biomolecule in the subject and administering the composition at different doses or rates. Similarly, the number of particles administered to a subject can be titrated against the concentration of biomolecules targeted by the particles.

For example, titrating the concentration of a biomolecule in a subject or the number of particles administered to a subject can be particularly useful when the biomolecule brings a detrimental local effect (e.g., in a tumor) but has a beneficial systemic effect. Thus, a plurality of particles may be inserted into or adjacent to a location in a patient to bind biomolecules at the location, and the systemic concentration of the biomolecules may be monitored to determine whether additional particles may be safely administered to the patient.

It may also be useful to titrate the concentration of the biomolecule in the subject or the number of particles administered to the subject, for example, to maintain the concentration of the biomolecule within a predetermined range. The predetermined range may be a range associated with a health state, for example, where the subject overproduces a biomolecule, or the predetermined range may be a therapeutic range. Such titrations may be particularly useful in methods of treating diseases caused by excessive hormone secretion. For example, the particles may comprise an agent that binds to a biomolecule growth hormone, e.g. for use in a method of treating acromegaly or gigantism, and such particles may be titrated to ensure growth hormone levels remain within a healthy range. The particles may include an agent that binds to the biomolecule thyroxine and/or triiodothyronine, for example, for use in a method of treating hyperthyroidism, and such particles may be titrated to ensure that the level of thyroxine and/or triiodothyronine remains within a healthy range. The particles may comprise an agent that binds to the biomolecule adrenocorticotropic hormone or cortisol, for example, for use in a method of treating cushing's disease, and such particles may be titrated to ensure that the level of adrenocorticotropic hormone and/or cortisol remains within a healthy range. Examples of therapeutic ranges include titration of a clotting factor (e.g., factor VIII, factor IX, or factor XI) to a range that inhibits blood clotting for a period of time. Such a range may be lower than normal healthy concentrations, while a therapeutic range may be useful, for example, to inhibit thrombosis or ischemia in certain patients.

Xvi. adoptive cell transfer therapy

The methods may comprise administering a composition comprising a plurality of particles described herein to a subject that has received adoptive cell transfer therapy (ACT). The methods may comprise administering a composition comprising a plurality of particles described herein to a subject who may benefit from adoptive cell transfer therapy. The methods can also include, for example, administering an adoptive cell transfer therapy to the subject before, after, or concurrently with administering the composition comprising the plurality of particles.

Adoptive cell transfer therapy can include administering a composition including lymphocytes to a subject. The lymphocytes can be T lymphocytes (i.e., T cells), such as Tumor Infiltrating Lymphocytes (TILs). In a preferred embodiment, the lymphocytes are T lymphocytes, such as tumor infiltrating lymphocytes. A composition comprising lymphocytes can be substantially free of cells that are not lymphocytes, e.g., a composition can be substantially free of cells and cell debris derived from myeloid progenitor cells (e.g., erythrocytes, mast cells, basophils, neutrophils, eosinophils, monocytes, macrophages, megakaryocytes, platelets). A composition comprising lymphocytes can be substantially free of cells that are not T cells, e.g., a composition can be substantially free of natural killer cells, B cells, and/or plasma cells. A composition comprising lymphocytes can comprise cells, wherein the cells consist essentially of T cells. The composition comprising lymphocytes can be substantially free of cells that are not tumor infiltrating lymphocytes. A composition comprising lymphocytes can comprise tumor infiltrating lymphocytes. A composition comprising lymphocytes can comprise cells, wherein the cells consist essentially of tumor infiltrating lymphocytes.

Compositions comprising lymphocytes can comprise recombinant lymphocytes, for example, wherein the lymphocytes comprise exogenous nucleic acids. For example, the lymphocytes can include a Chimeric Antigen Receptor (CAR). Similarly, lymphocytes may include gene knockouts, e.g., which reduce the risk of graft-versus-host immune responses or host-versus-graft immune responses (e.g., for non-autografts, such as allografts). In some embodiments, a composition comprising lymphocytes can comprise recombinant T cells (e.g., recombinant tumor infiltrating lymphocytes), for example, the lymphocytes can be recombinant T cells (e.g., recombinant tumor infiltrating lymphocytes).

Adoptive cell transfer therapies may include autografting or non-autografting (e.g., xenotransplantation).

The subject may have received adoptive cell transfer therapy about 1 year prior to administration of the composition to the subject (e.g., about 6 months, about 5 months, about 4 months, about 3 months, about 2 months, about 1 month, about 4 weeks, about 3 weeks, about 2 weeks, about 14 days, about 13 days, about 12 days, about 11 days, about 10 days, about 9 days, about 8 days, about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or 1 day prior to administration of the composition to the subject). The method can include administering a composition comprising a plurality of particles to the subject less than about 1 year after administering the composition comprising lymphocytes to the subject (e.g., less than about 6 months, about 5 months, about 4 months, about 3 months, about 2 months, about 1 month, about 4 weeks, about 3 weeks, about 2 weeks, about 14 days, about 13 days, about 12 days, about 11 days, about 10 days, about 9 days, about 8 days, about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or 1 day after administering the composition comprising lymphocytes to the subject). The method can include administering a composition comprising a plurality of particles to the subject within about 1 year of administering the composition comprising lymphocytes to the subject (e.g., within about 6 months, about 5 months, about 4 months, about 3 months, about 2 months, about 1 month, about 4 weeks, about 3 weeks, about 2 weeks, about 14 days, about 13 days, about 12 days, about 11 days, about 10 days, about 9 days, about 8 days, about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or about 1 day of administering the composition comprising lymphocytes to the subject).

Adoptive cell transfer therapies can be particularly effective in patients with tumors (e.g., cervical cancer, breast cancer, lymphoma, leukemia, chronic lymphocytic leukemia, follicular lymphoma, large cell lymphoma, lymphoblastic leukemia, myeloid leukemia, multiple myeloma, cholangiocarcinoma, colorectal cancer, neuroblastoma, lung cancer, sarcoma, synovial sarcoma, or melanoma). Nonetheless, adoptive cell transfer therapies can be useful for treating other diseases, such as severe or life-threatening infections (e.g., HIV).

Use of tumor-associated selection

In some embodiments, the particles described herein can be useful for treating a subject having cancer. Exemplary agents useful in the particle compositions described herein and/or soluble biomolecules that can be scavenged by such particles are described herein (e.g., table 2) and are known in the art. For example, particles capable of clearing stnfrs, MMP2, MMP9, sIL-2R, sIL-1 receptor, and the like, are useful for treating cancer and/or enhancing immune responses to cancer by reducing immune disinhibition.

The immunosuppressive pathway of immunotherapy is based in part on the concept that many cancer patients are generally immunologically competent overall, but their immune system is locally suppressed in the microenvironment of their tumor. If this suppression of the immune system is mitigated by administering the particles of the present disclosure, the patient's own immune system may act on the tumor. Thus, in certain embodiments, the particles of the present disclosure provide an immunotherapeutic approach without over-stimulating the patient's immune system by adding exogenous active cytokines intended to bind to cell surface receptors to elicit an immune response, and/or without otherwise over-stimulating the patient's immune system.

Without being bound by theory, because cancer patients are generally immunologically competent, the ability of lymphocytes to recognize tumor antigens is generally unaffected by tumors. Thus, lymphocytes are attracted (as they do to any abnormal cell clusters) into the tumor microenvironment, at which point cytokines and cytotoxic factors (such as tumor necrosis factor (TNF, e.g., TNF α, the major cytotoxic "shock" of the immune system)) are cleaved from the lymphocytes into the microenvironment. In the case of virally infected cells, rather than cancer cells, TNF (e.g., TNF α) will occupy (engage) the TNF receptor (TNFR) on the surface of the infected cells, resulting in rapid destruction by apoptosis or oxidative stress, depending on whether the R1 or R2 type receptors for TNF are occupied. In other words, in the absence of a normal immune response stimulated by the presence of a tumor and/or tumor antigen, lymphocyte-deployed TNF will be available to bind to cell surface TNF receptors (R1 and/or R2 receptors) as part of increasing the immune response. Even in the case of tumors, lymphocytes are deployed to the tumor site.

However, many types of cancer cells and other abnormal cell types (e.g., virus-infected cells) behave differently in that they overproduce TNF receptors (both types) and shed them to form a cloud (cloud) around the tumor. Thus, the microenvironment of cancer cells and/or tumors contains a large number of soluble TNF receptors. Without being bound by theory, the concentration of soluble TNF receptors in the tumor microenvironment exceeds the level of TNF receptors found in the microenvironment of healthy cells (e.g., healthy cells of the same tissue type). Additionally or alternatively, for cancer cells, the rate and extent of TNF receptor excretion is greater than from healthy cells. Furthermore, without being bound by theory, in certain embodiments, the concentration of soluble TNF receptors found in the plasma of cancer patients may be higher than in healthy patients (health patents).

Regardless of the mechanism, in this model, these shed soluble TNF receptors bind to TNF released endogenously by recruited lymphocytes, neutralize the endogenous TNF and effectively create a peritumoral immune privileged bleb (bubble) within which the tumor continues to grow and shed additional TNF receptors. In other words, the shed soluble TNF receptors take up TNF α produced endogenously by lymphocytes and prevent or inhibit TNF binding to cell surface TNF receptors on cancer cells. This reduces or eliminates TNF available to bind cell surface TNF receptors on cancer cells. Soluble TNF receptors substantially outperform TNF α binding to TNF α, thus reducing the activity of TNF, such as TNF α for binding to cell surface TNF receptors.

This can be accomplished similarly in the case of IL-2 and an excreted soluble IL-2 receptor.

In some embodiments, the biomolecule is a toxin released by the cancer cell upon apoptosis.

The present disclosure provides pharmacological approaches that can be deployed systemically or locally to mitigate suppression of the immune system (e.g., immune de-suppression) by shedding receptors in cancer. The present disclosure provides methods and compositions for reducing the amount and/or activity (e.g., neutralizing activity) of soluble TNF receptors and/or soluble IL-2 receptors (or any other soluble biomolecules that cause immune disinhibition), such as in the microenvironment of cancer cells and tumors. Without being bound by theory, reducing the amount and/or activity of, for example, a soluble TNF receptor (e.g., as in a tumor microenvironment) can be used as part of a method of inhibiting proliferation, growth, or survival of a cell (e.g., a cancer cell). In certain embodiments, it can be used to inhibit the survival of cells (e.g., cancer cells). Exemplary methods and agents are described herein.

Regulatory T cells (TREGs) can secrete the same ligands as cancer cells as a way to suppress immune responses to avoid autoimmune diseases caused by, for example, over-activated T cells or prolonged T cell function. For example, CD80/B7-1 and CD86/B7-2 bind to CTLA-4 receptors on T cells and inhibit T cell activity. The particles described herein do not block CTLA-4 receptors, but may be designed to clear CD80/B7-1 and/or CD 86/B7-2. Likewise, the particles described herein can be designed to clear other immune checkpoint inhibitors (such as PD-L1), for example, using particles that include the PD-1 receptor. Such particulate compositions provide several benefits compared to other approaches to stimulating the immune system to treat cancer.

The target may be soluble PD-L2, for example, to inhibit the interaction between soluble PD-L2 and PD 1. The agent may be PD 1. Inhibition of the interaction between soluble PD-L2 and PD1 may allow PD1 to bind to the membrane-bound form of PD-L2, thereby favoring apoptosis of cancer cells. The target may be soluble PD 1. The agent may be a ligand of PD1 (e.g., PD-L2, soluble PD-L2, or variants thereof) or an anti-PD 1 antibody (e.g., nivolumab or pembrolizumab). Particles that target PD1 (i.e., soluble PD1) and its ligands, among other diseases and conditions, can be particularly useful for treating autoimmune diseases.

The target may be soluble CTLA4, for example, to inhibit interaction between B7-1 or B7-2 and soluble CTLA 4. The agent may be a ligand of CTLA4 (e.g. soluble B7-1, soluble B7-2 or variants thereof) or an anti-CTLA 4 antibody (e.g. lypima (ipilimumab) or tremelimumab (tremelimumab)). Inhibition of the interaction between B7-1 or B7-2 and soluble CTLA4 may allow B7-1 or B7-2 to bind to CD28 on T cells, thereby facilitating activation of T cells. Particles targeting CTLA4 (i.e., soluble CTLA4) can be particularly useful for treating melanoma and lung cancer (such as non-small cell lung cancer), among other diseases and conditions.

The agent may be a protein that specifically binds adenosine (e.g., an adenosine binding portion of an adenosine receptor). The target may be adenosine. Adenosine-targeting particles can be particularly useful for treating solid tumors, and such particles can be injected into solid tumors, for example, to inhibit adenosine signaling within the tumor microenvironment.

The agent may be an osteoprotegerin or a ligand binding portion thereof, e.g., a ligand for selectively binding osteoprotegerin. Particles of ligands targeting osteoprotegerin may be particularly useful for treating cancer (e.g., breast cancer), among other diseases and conditions.

In some embodiments, the subject is a subject having, suspected of having, or at risk of developing cancer. In some embodiments, the subject is a subject having, suspected of having, or at risk of developing an autoimmune disease.

As used herein, a subject "at risk for developing" cancer is a subject having one or more (e.g., two, three, four, five, six, seven, eight, or more) risk factors for developing cancer. For example, a subject at risk of developing cancer may have a predisposition to develop cancer (i.e., a genetic predisposition to develop cancer, such as a tumor suppressor gene (e.g., a mutation in BRCA1, p53, RB, or APC) or have been exposed to conditions that may lead to a condition). Thus, a subject may be a "at risk of developing cancer" subject when the subject has been exposed to mutagenic or carcinogenic concentrations of certain compounds (e.g., carcinogenic compounds in cigarette smoke, such as acrolein, arsenic, benzene, benzanthracene, benzopyrene, polonium 210 (radon), urethane, or vinyl chloride). In addition, a subject may be "at risk of developing cancer" when the subject has been exposed to, for example, a large dose of ultraviolet light or X-ray radiation, or is exposed to (e.g., infected by) a tumor-causing/tumor-associated virus (e.g., papillomavirus), epstein barr virus, hepatitis b virus, or human T-cell leukemia lymphoma virus. Cancer is a group of diseases or disorders characterized by uncontrolled cell division and its ability to spread by invasion, either by direct growth into adjacent tissues or by implantation into distant sites by distant metastasis (where cancer cells are transported through the bloodstream or lymphatic system). Cancer can affect people of all ages, but risk tends to increase with age. The type of cancer can include, for example, lung cancer, breast cancer, colon cancer, pancreatic cancer, kidney cancer, stomach cancer, liver cancer, bone cancer, hematological cancer, cancer of neural tissue (e.g., glioblastoma, such as glioblastoma multiforme), melanoma, thyroid cancer, ovarian cancer, testicular cancer, prostate cancer, cervical cancer, vaginal cancer, or bladder cancer. In certain preferred embodiments, the patient (or subject) has brain, endometrial, prostate, renal, or squamous cell carcinoma (e.g., head and neck squamous cell carcinoma), each of which is particularly sensitive to extracellular biological molecules that may exacerbate the disease.

Similarly, a subject at risk of developing an infection is a subject with one or more risk factors that increase the likelihood of exposure to a pathogenic microorganism.

A subject "suspected of having" a cancer or infection is a subject having one or more symptoms of cancer or infection. It is understood that a subject at risk of developing or suspected of having cancer or infection does not encompass all subjects within the species of interest.

In some embodiments, the method comprises determining whether the subject has cancer.

Use of a selection associated with inflammatory and autoimmune disorders

In some embodiments, the particles described herein can be used to treat inflammatory and/or autoimmune disorders. Exemplary agents useful in the particle compositions described herein and/or soluble biomolecules that can be scavenged by such particles are described herein (e.g., table 2) and are known in the art. For example, particles capable of clearing cytokines (e.g., TNF α or interleukins such as IL-2, IL-6 or IL-1) or chemokines (e.g., CXCL8 or CXCL1) can be useful for treating a wide variety of autoimmune and/or inflammatory disorders.

The agent may be soluble CD28 or a ligand binding portion thereof, e.g., a ligand for selectively binding CD28 (such as soluble B7 (e.g., soluble B7-1 or soluble B7-2)). The agent may be galiximab. The target may be a ligand of CD28 (e.g., soluble B7). Particles of ligands targeting CD28 can be particularly useful for preventing or treating lupus (e.g., systemic lupus erythematosus), among other diseases and conditions.

The agent may be an anti-B7-H4 antibody, e.g., for selectively binding soluble B7-H4. The target may be soluble B7-H4. Particles targeting soluble B7-H4 can be particularly useful for treating arthritis (such as rheumatoid arthritis and juvenile idiopathic arthritis), among other diseases and conditions.

The agent may be soluble CD278 (inducible costimulator; "ICOS") or a ligand-binding portion thereof, e.g., a ligand for selectively binding CD278 (e.g., ICOSL (inducible costimulator ligand; CD 275)). The target may be a ligand for CD278 (e.g., ICOSL). Particles targeting CD278 ligands may be particularly useful for preventing or treating lupus (e.g., systemic lupus erythematosus), among other diseases and conditions.

The agent may be an anti-CD 275 antibody, e.g., for selectively binding CD275 (inducible costimulatory factor ligand; "ICOSL"). The target may be CD 275. Particles targeting CD275 may be particularly useful for preventing or treating lupus (e.g., systemic lupus erythematosus), among other diseases and conditions.

The agent may be an anti-CD 40L antibody, such as daprolizumab (daprolizumab), lullizumab (ruplizumab), or tollizumab (toralizumab), for example, for selectively binding CD40L (CD40 ligand; CD 154). The target may be CD 40L. Particles targeting CD40L may be particularly useful for preventing or treating lupus (such as systemic lupus erythematosus), arthritis (such as rheumatoid arthritis, collagen-induced arthritis, and juvenile idiopathic arthritis), and sjogren's syndrome, among other diseases and conditions.

The agent may be soluble CD134(OX40) or a ligand binding portion thereof, e.g., a ligand for selectively binding CD134 (e.g., CD252(OX40 ligand; "OX 40L")). The target may be a ligand for CD134 (e.g., CD 252). Particles targeting CD134 ligands can be particularly useful for preventing or treating lupus (e.g., lupus nephritis), its symptoms (e.g., glomerulonephritis), and systemic sclerosis, among other diseases and conditions.

The agent can be 4-1BB (CD137) or a ligand binding portion thereof, e.g., a ligand for selectively binding 4-1BB (e.g., a soluble 4-1BB ligand (soluble 4-1 BBL)). The target may be a ligand for 4-1BB (e.g., a soluble 4-1BB ligand). Particles of ligands targeting 4-1BB can be particularly useful for preventing or treating lupus (e.g., systemic lupus erythematosus) and arthritis (e.g., rheumatoid arthritis), among other diseases and conditions.

The agent can be a 4-1BB ligand, e.g., for selectively binding soluble 4-1BB (soluble CD 137). The agent can be an anti-4-1 BB antibody (e.g., Urelumab). The target may be soluble 4-1 BB. Particles targeting soluble 4-1BB may be particularly useful for preventing or treating arthritis (such as rheumatoid arthritis), among other diseases and conditions, including cancer. In some embodiments, the inflammatory disorder can be, for example, acute disseminated encephalomyelitis; edison's disease; ankylosing spondylitis; antiphospholipid antibody syndrome; autoimmune hemolytic anemia; autoimmune hepatitis; autoimmune inner ear disease; bullous pemphigoid; chagas disease; chronic obstructive pulmonary disease; celiac disease; dermatomyositis, diabetes type 1; type 2 diabetes; endometriosis; goodpasture's syndrome; graves' disease; acute febrile polyneuritis; hashimoto's disease; idiopathic thrombocytopenic purpura; interstitial cystitis; systemic Lupus Erythematosus (SLE); metabolic syndrome; multiple sclerosis; myasthenia gravis; myocarditis; narcolepsy; obesity; pemphigus vulgaris; pernicious anemia; polymyositis; primary biliary cirrhosis; rheumatoid arthritis; schizophrenia; scleroderma; sjogren's syndrome; vasculitis; vitiligo; wegener's granulomatosis; allergic rhinitis; prostate cancer; non-small cell lung cancer; ovarian cancer; breast cancer; melanoma; gastric cancer; colorectal cancer; brain cancer; metastatic bone lesions; pancreatic cancer; lymphoma; nasal polyps; gastrointestinal cancer; ulcerative colitis; crohn's disease; collagenous colitis; lymphocytic colitis; ischemic colitis; metastatic colitis; behcet's syndrome; infectious colitis; indeterminate colitis; inflammatory liver disease, endotoxic shock, rheumatoid spondylitis, ankylosing spondylitis, gouty arthritis, polymyalgia rheumatica, alzheimer's disease, parkinson's disease, epilepsy, aids, dementia, asthma, adult respiratory distress syndrome, bronchitis, cystic fibrosis, acute leukocyte-mediated lung injury, distal proctitis, wegener's granulomatosis, fibromyalgia, bronchitis, cystic fibrosis, uveitis, conjunctivitis, psoriasis, eczema, dermatitis, smooth muscle cell proliferation disorders, meningitis, shingles, encephalitis, nephritis, tuberculosis, retinitis, atopic dermatitis, pancreatitis, periodontitis, coagulation necrosis, liquification necrosis, fibrosis-like necrosis, hyperacute transplant rejection, acute transplant rejection, chronic transplant rejection, acute graft-versus-host disease, chronic inflammatory bowel disease, rheumatoid arthritis, epilepsy, aids, rheumatoid arthritis, fibromyalgia, chronic inflammatory bowel disease, rheumatoid arthritis, fibromyalgia, rheumatoid arthritis, and psoriasis, Or a combination of any of the foregoing. In some embodiments, the autoimmune or inflammatory disorder can be, for example, colitis, multiple sclerosis, arthritis, rheumatoid arthritis, osteoarthritis, juvenile arthritis, psoriatic arthritis, acute pancreatitis, chronic pancreatitis, diabetes, insulin-dependent diabetes mellitus (IDDM or type I diabetes), insulitis, inflammatory bowel disease, crohn's disease, ulcerative colitis, autoimmune hemolytic syndrome, autoimmune hepatitis, autoimmune neuropathy, autoimmune ovarian failure, autoimmune orchitis, autoimmune thrombocytopenia, reactive arthritis, ankylosing spondylitis, silicon transplants associated with autoimmune diseases, sjogren's syndrome, Systemic Lupus Erythematosus (SLE), vasculitis syndrome (e.g., giant cell arteritis, behcet's disease, and wegener's granulomatosis), Vitiligo, a secondary hematological manifestation of an autoimmune disease (e.g., anemia), drug-induced autoimmunity, hashimoto's thyroiditis, hypophysitis, idiopathic thrombocytopenic purpura, metal-induced autoimmunity, myasthenia gravis, pemphigus, autoimmune hearing loss (e.g., meniere's disease), goodpasture's syndrome, graves' disease, HIV-associated autoimmune syndrome, and/or acute febrile polyneuritis.

In some embodiments, the autoimmune or inflammatory disorder is a hypersensitivity reaction. As used herein, "hypersensitivity" refers to an undesired immune system response. Hypersensitivity reactions fall into four categories. Type I hypersensitivity reactions include allergies (e.g., atopy, anaphylaxis, or asthma). Type II hypersensitivity reactions are cytotoxic/antibody mediated (e.g., autoimmune hemolytic anemia, thrombocytopenia, fetal erythroblastosis, or goodpasture's syndrome). Type III is an immune complex disease (e.g., seropathy, abis response, or SLE). Type IV is delayed-type hypersensitivity (DTH), a cell-mediated immune memory response and is independent of antibodies (e.g., contact dermatitis, tuberculin skin test, or chronic transplant rejection). As used herein, "allergy" refers to a disorder characterized by excessive activation of mast cells and basophils by IgE. In some cases, excessive activation of mast cells and basophils by IgE leads to (partial or total) inflammatory responses. In some cases, the inflammatory response is local. In some cases, the inflammatory response results in narrowing of the airway (i.e., bronchoconstriction). In some cases, the inflammatory response results in inflammation in the nose (i.e., rhinitis). In some cases, the inflammatory response is systemic (i.e., allergic reaction).

In some embodiments, the method comprises determining whether the subject has an autoimmune disease.

Use of pathogen and toxin related selection

In some embodiments, the particles described herein can be designed to bind to a microorganism (e.g., a virus or a bacterium) or a component of a microorganism (e.g., an endotoxin). Thus, the particles described herein can be useful for treating, for example, infectious diseases (e.g., viral infectious diseases, including HPV, HBV, Hepatitis C Virus (HCV), retrovirus (e.g., human immunodeficiency virus (HIV-1 and HIV-2)), herpes virus (e.g., EB virus (EBV)), Cytomegalovirus (CMV), HSV-1 and HSV-2), and influenza virus Streptococcus, Toxoplasma and Vibrio cholerae. Exemplary species include Neisseria gonorrhoeae, Mycobacterium tuberculosis, Candida albicans, Candida tropicalis, Trichomonas vaginalis, Haemophilus vaginalis, Streptococcus B, Mycoplasma hominis (Microplasma hominis), Haemophilus ducreyi, granuloma inguinalis, lymphogranuloma venenatum, Treponema pallidum, Brucella abortus, Brucella equi, Brucella suis, Brucella canicola canis, Campylobacter fetus subspecies, Leptospira pomonella, Listeria monocytogenes, Brucella ovis, Chlamydia psittaci, Trichomonas fetalis, Toxoplasma gondii (Toxoplasma gondii), Escherichia coli, Actinobacillus foenii, Salmonella ovis, Salmonella abortus, Pseudomonas aeruginosa, Corynebacterium parvus, Corynebacterium pyogenes, Mycobacterium ovis, Mycoplasma bovis, Aspergillus fumigatus, Trypanosoma recited in, Trypanosoma cruzi, Babesia caballi disease, clostridium tetani, clostridium botulinum; or a fungus, such as, for example, Paenibacillus brasiliensis; or other pathogens, such as plasmodium falciparum. Also included are National Institute for Allergy and Infectious Disease (NIAID) priority pathogens. These comprise class a agents such as smallpox (smallpox), bacillus anthracis (anthrax), yersinia pestis (plague), clostridium botulinum toxin (botulism), francisella tularensis (tularemia), filoviruses (ebola hemorrhagic fever, marburg hemorrhagic fever), arenavirus (lassa fever)), huining fever (argentine hemorrhagic fever), and related viruses; class B agents, such as Bertoni-Rickettsia (Q fever), Brucella (Brucella disease), Pseudomonas rhinocerotis (melittis equines), alpha-virus (Venezuelan encephalomyelitis, eastern and western equine encephalomyelitis), ricin toxin from Ricinus communis (castor seeds), Clostridium perfringens epsilon toxin; staphylococcal enterotoxin B, Salmonella, Shigella dysenteriae, Escherichia coli strain O157H 7, Vibrio cholerae, Cryptosporidium; class C agents, such as nipah virus, hantaan virus, tick-borne hemorrhagic fever virus, tick-borne encephalitis virus, yellow fever, and multi-drug resistant tuberculosis; helminths, such as the genera schistosoma and cestode; and protozoa, such as leishmania (e.g., leishmania mexicana) and plasmodium.

The target may be a viral protein. The viral protein may be from arbovirus, adenovirus, alpha-virus, arenavirus, astrovirus, BK virus, bunyavirus, calicivirus, herpes simplex virus type 1, Colorado tick fever virus, coronavirus, coxsackievirus, Crimean-Congo hemorrhagic fever virus, cytomegalovirus, dengue virus, Ebola virus, echinovirus, echovirus, enterovirus, EB virus, flavivirus, foot and mouth disease virus, hantavirus, hepatitis A, hepatitis B, hepatitis C, herpes simplex virus type I, herpes simplex virus type II, human herpesvirus, human immunodeficiency virus type I (HIV-1), human immunodeficiency virus type II (HIV-II), human papillomavirus, human T cell leukemia virus type I, human T cell leukemia virus type II, influenza virus, Japanese encephalitis virus, JC virus, junin virus, lentivirus, equine papova virus, marburg virus, measles virus, mumps virus, narcoleus virus, norovirus, circovirus, orthomyxovirus, papilloma virus, papova virus, parainfluenza virus, paramyxovirus, parvovirus, picornavirus, poliovirus, polyoma virus, poxvirus, rabies virus, reovirus, respiratory syncytial virus, rhinovirus, rotavirus, rubella virus, fizzas such as virus, smallpox virus, togavirus, toscana virus, varicella zoster virus, west nile virus or yellow fever virus. The viral protein may be, for example, a viral capsid protein or a viral envelope protein.

The target may be a bacterial protein or a bacterial cell wall component. For example, the bacterial protein or cell wall component is from Actinomyces israeli, Bacillus anthracis, Bacillus cereus, Bacteroides fragilis, Bartonella henryi, Bartonella pentandra, Bordetella pertussis, Borrelia burgdorferi, Borrelia gariae, Borrelia avermitis, Borrelia regressiva, Brucella abortus, Brucella canicola, Brucella equina, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetanus, Corynebacterium diphtheriae, Escherichia canis, Escherichia coli, enterococcus faecalis, enterococcus faecium, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Haemophilus vaginalis, helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Leptobacterium interrogans, Leptobacterium, and its, Leptospira santolinella, Leptospira westermani, Leptospira interorum, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Nocardia asteroides, Rickettsia rickettsii, Salmonella typhimurium, Shigella sonnei, Shigella dysenteriae, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus viridis, Treponema pallidum, Urea urealyticum, Vibrio cholerae, Yersinia pestis, Yersinia enterocolitica or Yersinia pseudotuberculosis.

The target may be a yeast or fungal protein or a component of a yeast or fungal cell wall. For example, the yeast or fungal protein or cell wall component may be from Pholiopsis variabilis, Aspergillus clavatus, Aspergillus flavus, Aspergillus fumigatus, Rana frog-producing faecalis, Candida albicans, Candida glabrata, Candida guilliermondii, Candida krusei, Candida viticola, Candida parapsilosis, Candida tropicalis, Candida stellatoides, Candida virginosa, Aureobasidium, Cryptococcus albicans, Cryptococcus gatheiiensis, Cryptococcus laurentii, cryptococcus neoformans, Microsporosis enteroencephalitis, Enterocytosis bivalens, Extra Callicarpa, Blastomyces tigrinus, Byssochlamys euonymus, Geotrichum candidum, Histoplasma capsulatum, Mucor umbellatus, Mucor hiemalis, Paphiospora brasiliensis, Pneumocystis carinii, Pneumocystis jeikeii, Pseudomyces poensis boydii, nosespodopsis sibiricus, Rhodotorula mucilaginosa, Stachybotrys charitis, Tocopora racemosa, or Rhizopus oryzae.

The target may be a protozoan protein. The protozoan protein may be from Cryptosporidium, Giardia intestinalis, Giardia lamblia, Leishmania aegypti, Leishmania braziliana, Leishmania donovani, Leishmania infantis, Leishmania macroleprae, Leishmania mexicana, Leishmania tropica, Plasmodium chrysi, Plasmodium falciparum, Plasmodium glaucens, Plasmodium hyorhynchophora, Plasmodium faecium, Trichomonas vaginalis, Trichomonas foetidus, Trypanosoma brucei, Trypanosoma cruzi, Trypanosoma malariae, Trypanosoma evansi, Trypanosoma leyi, Trypanosoma lewisi, Trypanosoma spicum pernici, Trypanosoma cruzi or Trypanosoma mobilis.

The target may be a toxin (e.g., a bacterial toxin, a plant toxin, or an animal toxin). The toxin can be, for example, melittin, dianthotoxin, tetrodotoxin, chlorotoxin, tetanus toxoid, bungarotoxin, clostridium botulinum toxin, ricin, clostridium perfringens epsilon toxin, staphylococcal enterotoxin B, or endotoxin.

The target can be a bacterial cell surface lipopolysaccharide, a lipopolysaccharide binding protein, a lipoteichoic acid, a bacterial lipoprotein, a bacterial peptidoglycan, a fatty arabinomannan, a bacterial flagellin (e.g., flagellin), an arrestin, HSP70, a zymosan, a double stranded RNA, a bacterial ribosomal RNA, or a DNA comprising an unmethylated CpG.

In some aspects, the invention relates to a method of treating or preventing an infection caused by a pathogen, the method comprising administering to a subject a composition comprising a plurality of particles as described herein. In some embodiments, the particle comprises an agent that specifically binds to a biomolecule of or produced by the pathogen. In some embodiments, the particle comprises an agent that specifically binds to a biomolecule of the subject (e.g., a biomolecule produced by the subject), such as a cytokine or a peroxiredoxin enzyme (e.g., peroxiredoxin 1 or peroxiredoxin 2). For example, a method can include administering to a subject a composition including a plurality of particles that selectively bind TNF α, interleukin 1, interleukin 6, interleukin 8, interleukin 12, interferon γ, macrophage migration inhibitory factor, GM-CSF, and/or a blood coagulation factor, e.g., to treat or prevent sepsis associated with an infection caused by a pathogen. In some embodiments, the method is a method of treating or preventing sepsis, e.g., the method comprises administering to a subject a composition comprising a plurality of particles as described herein.

The target may be acetaminophen (acetaminophen). The agent may be an antibody, or antigen-binding portion thereof, that specifically binds acetaminophen. Particles targeting acetaminophen can be particularly useful for treating or preventing acetaminophen toxicity.

XX. application of selection related to diet and metabolism

In some embodiments, the particles described herein can be used to treat obesity, eating disorders, reduce body weight, promote a healthy diet, or reduce appetite in a subject. For example, in some embodiments, particles comprising an agent that binds to ghrelin (e.g., an antibody or a soluble form of ghrelin receptor (GHSR)) can be administered to a subject (e.g., an overweight or obese subject) to reduce appetite in the subject, treat obesity or obesity-related disorders, or metabolic disorders.

As used herein, a metabolic disorder may be any disorder related to metabolism, and examples include, but are not limited to, obesity, central obesity, insulin resistance, glucose intolerance, abnormal glycogen metabolism, type II diabetes, hyperlipidemia, hypoalbuminemia, hypertriglyceridemia, metabolic syndrome, syndrome X, fatty liver disease, polycystic ovary syndrome, and acanthosis nigricans.

"obesity" refers to a condition in which the body weight of a mammal exceeds medically recommended limits by at least about 20%, based on age and bone size. "obesity" is characterized by hypertrophy and hyperplasia of adipocytes. "obesity" can be characterized by the presence of one or more obesity-associated phenotypes including, for example, increased body weight (as measured, for example, by body mass index or "BMI"), altered anthropometry, basal metabolic rate, or total energy expenditure, slow energy balanceDestructive, increased fat mass (e.g., as determined by DEXA (DEXA fat mass percent)), altered maximum oxygen consumption (VO)₂) High fat oxidation, high relative rest rate, glucose resistance, hyperlipidemia, insulin resistance and hyperglycemia. See also, for example, Hopkinson et al, Am J Clin Nutr 65(2):432-8(1997) and button et al, Am J Clin Nutr 69(2): 299-. An "overweight" individual typically has a Body Mass Index (BMI) between 25 and 30. An "obese" individual or an individual suffering from "obesity" is typically an individual with a BMI of 30 or greater. Obesity may or may not be associated with insulin resistance.

An "obesity-related disease" or "obesity-related disorder" or "obesity-related condition" (all used interchangeably) refers to a disease, disorder or condition associated with obesity, and/or caused directly or indirectly by obesity. "obesity-related diseases" or "obesity-related disorders" or "obesity-related conditions" include, but are not limited to, coronary artery disease/cardiovascular disease, hypertension, cerebrovascular disease, stroke, peripheral vascular disease, insulin resistance, glucose intolerance, diabetes, hyperglycemia, hyperlipidemia, dyslipidemia, hypercholesterolemia, hypertriglyceridemia, hyperinsulinemia, atherosclerosis, cell proliferation and endothelial dysfunction, diabetic dyslipidemia, HIV-related lipodystrophy, peripheral vascular disease, cholesterol stones, cancer, menstrual abnormalities, infertility, polycystic ovary, osteoarthritis, sleep apnea, metabolic syndrome (syndrome X), type II diabetes, diabetic complications (including diabetic neuropathy, nephropathy, retinopathy, cataracts, diabetes mellitus complications, diabetes mellitus, and conditions of a condition of a patient, a patient of a patient, a patient of a patient, a patient, Heart failure, inflammation, thrombosis, congestive heart failure) and any other cardiovascular disease associated with obesity or an overweight condition, and/or obesity-associated asthma, airway and lung diseases.

In another aspect, the disclosure features a method for increasing muscle mass or muscle strength in a subject in need thereof, the method comprising administering one or more compositions described herein to the subject in an amount sufficient to increase muscle mass or muscle strength in the subject. For example, particles comprising an agent (e.g., an antibody or a soluble activin receptor) that binds to myostatin can be administered to a subject to increase muscle mass.

In some embodiments, the subject is a subject having a muscle disease (e.g., a muscle wasting disease).

As used herein, muscle wasting diseases include diseases or conditions where muscle wasting is one of the major symptoms, such as muscular dystrophy, spinal cord injury, neurodegenerative diseases, anorexia, sarcopenia, cachexia, muscle wasting due to immobilization, prolonged bed rest or weight loss, and the like, as well as diseases where an abnormally high fat to muscle ratio is implicated in a disease or pre-disease state (e.g., type II diabetes or syndrome X).

Skeletal muscle atrophy occurs in the muscles of adult animals due to lack of use, aging, hunger, and due to a variety of diseases, disorders, and conditions (such as sepsis, muscular dystrophy, aids, aging, and cancer). Muscle loss is generally characterized by a reduction in protein content, strength production, fatigue resistance, and muscle fiber diameter. These reductions can be attributed to a decrease in protein synthesis and an increase in protein degradation. Muscle wasting and related conditions to which the compositions and methods of the invention are directed include any condition in which enhancing muscle growth or reducing muscle wasting produces a therapeutic or other desired outcome. Conditions include muscular dystrophy, sarcopenia, cachexia, diabetes, and improvements in muscle mass (where such improvements are moral and desirable, for example, in food animals).

As mentioned above, one type of muscle wasting disease is muscular dystrophy. These are a heterogeneous group of neuromuscular disorders that comprise the most common types, Duchenne Muscular Dystrophy (DMD), multiple types of limb-girdle muscular dystrophy (LGMD) and other Congenital Muscular Dystrophies (CMD). Progressive muscle damage and loss, tissue inflammation, and replacement of healthy muscle with fibrous and adipose tissue lead to muscle atrophy in muscular dystrophy. Extreme muscle loss is one of the most prominent signs of the disease and leads to complications and symptoms, including death.

Sarcopenia is an age-related loss of muscle mass, strength and function. It begins in the fourth decade of life and accelerates after about the age of 75 years. Sarcopenia can be caused by a number of factors, including lack of physical exercise, motor unit remodeling, reduced hormone levels, and reduced protein synthesis. All of these, except for lack of physical exercise, may be subject to genetic control, where genetic regulation may be useful. For example, the rate of muscle protein synthesis and protein breakdown affects sarcopenia. The balance of protein synthesis and breakdown determines the protein content in the body. Studies consistently report that the muscle protein synthesis rate is lower in the elderly compared to young adults. By way of example, a reduction in muscle protein catabolism affected by gene regulation can result in the slowing or reversal of muscle mass loss.

Use of selection related to aging and neurodegenerative diseases

In some embodiments, the compositions described herein are useful for promoting healthy aging in a subject. For example, particles comprising an agent (e.g., a soluble form of an antibody or receptor) capable of binding to any of TGF β 1, CCL11, MCP-1/CCL2, β -2 microglobulin, GDF-8/myostatin, or binding globin, can be used to promote healthy aging in a subject, extend the lifespan of a subject, prevent or delay the onset of an age-related disease in a subject, or treat a subject suffering from an age-related disease. In some embodiments, particles comprising an agent that binds to TGF β 1 can be used to enhance/promote neurogenesis and/or muscle regeneration in a subject (e.g., an elderly subject). In some embodiments, the age-related disease is cardiovascular disease. In some embodiments, the age-related disease is a bone loss disorder. In some embodiments, the age-related disease is a neuromuscular disorder. In some embodiments, the age-related disease is a neurodegenerative disease or a cognitive disorder. In some embodiments, the age-related disease is a metabolic disorder. In some embodiments, the age-related disorder is sarcopenia, osteoarthritis, chronic fatigue syndrome, alzheimer's disease, senile dementia, mild cognitive impairment due to aging, schizophrenia, parkinson's disease, huntington's disease, pick's disease, creutzfeldt-jakob disease, stroke, central nervous system brain aging, age-related cognitive decline, prediabetes, diabetes, obesity, osteoporosis, coronary artery disease, cerebrovascular disease, heart attack, stroke, peripheral artery disease, aortic valve disease, stroke, lewy body disease, Amyotrophic Lateral Sclerosis (ALS), mild cognitive impairment, pre-dementia, progressive subcortical collagen hyperplasia, progressive supranuclear palsy, thalamic syndrome, hereditary aphasia, myoclonic epilepsy, Macular degeneration or cataracts.

The biomolecule may be alpha-synuclein, tau, amyloid precursor protein or beta amyloid. For example, the method can include administering a composition including a plurality of particles to a subject having alzheimer's disease, and the particles can include an agent that specifically binds beta amyloid (e.g., soluble beta amyloid and/or beta amyloid aggregates). The biomolecule may be a β 40 or a β 42. The agent may comprise aducairumab, basilizumab, clenbuteromab, alemtuzumab, perlizumab, solanesol mab, or an antigen-binding portion of any of the foregoing. Similarly, the method can include administering a composition including a plurality of particles to a subject having alzheimer's disease, and the particles can include an agent that specifically binds τ.

The biomolecule may be TDP-43 or FUS. The biomolecule may be a prion. The biomolecule may be PrP^ScSoluble PrP protein or PrP aggregates.

Xxii. selective diagnostic applications

The particles described herein are also useful as diagnostic agents, or in conjunction with diagnostic tools or devices. For example, the particles described herein can be coupled to a detection device that monitors the concentration of a given soluble ligand of interest. For example, a nanochannel in a detection device lined with an agent (e.g., a first member of a binding pair) can detect (e.g., in a blood sample) or monitor (e.g., as an implanted device in a subject) the concentration of a soluble biomolecule (e.g., a second member of a binding pair). Such detectors may be useful, for example, to determine the effectiveness of the particles described herein (to scavenge soluble biomolecules) or to determine/adjust the appropriate dose of the particle composition (e.g., increase the dose or dose frequency to more effectively scavenge soluble biomolecules).

In some embodiments, the particles and detection devices described herein are integrated and used as "micro-or" nano-sealing devices "(see, e.g., Sabek et al, Lab Chip 13(18):3675-3688 (2013)). The nano-sealing device is characterized, for example, by the ability of nanochannel diagnostics to provide an accurate quantitative measurement of the concentration of soluble biomolecules in a biological fluid of a subject in which the nano-sealing device is implanted. The nanoseal device is further characterized by a means (e.g., a nanosyringe) that releases particles that are capable of scavenging biomolecules, for example, when the concentration of biomolecules in the biological fluid reaches a set threshold concentration. Given that thousands of nanochannels may be deployed in nail-sized implantable biochips, a micro-or nano-seal may be designed to monitor many different soluble biomolecules and release multiple types of therapeutic particles.

XXIII. selected in vitro uses

In some aspects, the invention relates to a method for removing a biomolecule from a composition, the method comprising contacting the composition with a particle described herein. Such methods are particularly useful for scientific research. For example, it is relatively easy to add biomolecules to a solution, whereas it is somewhat more challenging to remove specific biomolecules from a solution.

Current techniques for removing biomolecules from solutions include, for example, binding biomolecules to particles (e.g., agarose beads), and then physically separating the beads from the solution. The particles described herein can sequester biomolecules in a composition, thereby inhibiting interactions with other components (e.g., cells) of the composition, without the need to physically separate the particles from the composition.

The particles may comprise a fluorophore. The particles may be magnetic or paramagnetic, or the particles may comprise magnetic or paramagnetic subparticles or components that allow the particles to be attracted to a magnetic field.

The method can comprise contacting a composition with a particle described herein, wherein the composition is a cell culture. For example, the cell culture may be a bacterial cell culture or a tissue culture. Such methods may be useful, for example, for removing secreted proteins from cell cultures or removing contaminants from cell cultures.

The method can comprise contacting a composition with a particle described herein, wherein the composition is a cell lysate. The cell lysate may be a prokaryotic cell lysate or a eukaryotic cell lysate. Such methods, for example, can be useful for inhibiting the activity of a target biomolecule.

The above methods may be particularly useful for assessing the function of a biomolecule of interest in a particular system. For example, a biomolecule may be introduced into a system (e.g., a tissue culture) to assess the effect of the biomolecule on the system (e.g., cell proliferation or cell death), and using the particles as described herein, the biomolecule may be depleted from a similar system to assess the effect of a lack of the biomolecule on the system.

In some aspects, the invention relates to a method for expanding or differentiating a cell population, the method comprising contacting a composition comprising a cell population with a plurality of particles as described herein. The plurality of particles may be depleted of one or more molecules that favor an alternative differentiation pathway that competes with a desired differentiation pathway. Thus, the methods can facilitate differentiation of a cell population into a desired cell type relative to an alternative cell type. The methods can further comprise contacting the composition with a cytokine (e.g., as described herein). The method can further comprise contacting the composition with one or more of a chemokine, an interleukin, a growth factor, a wnt family protein, a tumor necrosis factor, and/or a hormone (e.g., as described herein).

The cell population may include stem cells. The cell population may include adult stem cells or embryonic stem cells. The cell population can include induced stem cells (e.g., induced pluripotent stem cells). The cell population may include progenitor cells, precursor cells, blasts, unipotent cells, pluripotent stem cells, multipotent stem cells, and/or intermediate progenitor cells. The cell population may include meiocytes. The cell population may include hematopoietic stem cells, mammary stem cells, intestinal stem cells, mesenchymal stem cells, endothelial stem cells, neural stem cells, olfactory adult stem cells, neural crest stem cells, or testicular cells. The cell population may include satellite cells, oligodendrocyte progenitor cells, thymocytes, hemangioblasts, bone marrow stromal cells, pancreatic progenitor cells, endothelial progenitor cells, or melanoblasts. The cell population may include totipotent hematopoietic stem cells, common myeloid progenitor cells, myeloblasts, promonocytes, monocytes, common lymphoid progenitor cells, lymphoblasts, prolymphocytes, and/or small lymphocytes.

In some embodiments, the present invention relates to a method for differentiating cells, the method comprising contacting a composition comprising cells with a plurality of particles as described herein. The plurality of particles may be depleted of one or more molecules that favor an alternative differentiation pathway that competes with a desired differentiation pathway. Thus, the methods can facilitate differentiation of cells into desired cell types relative to alternative cell types. The methods can further comprise contacting the composition with a cytokine (e.g., as described herein). The methods can further comprise contacting the composition with one or more of a chemokine, an interleukin, a growth factor, a wnt family protein, and/or a tumor necrosis factor (e.g., as described herein).

The cells may be stem cells. The cells may be adult stem cells or embryonic stem cells. The cell may be an induced stem cell (e.g., an induced pluripotent stem cell). The cells may be progenitor cells, precursor cells, blasts, unipotent cells, pluripotent stem cells, multipotent stem cells, and/or intermediate progenitor cells. The cell may be a meiocyte. The cell may be a hematopoietic stem cell, a mammary stem cell, an intestinal stem cell, a mesenchymal stem cell, an endothelial stem cell, a neural stem cell, an olfactory adult stem cell, a neural crest stem cell, or a testicular cell. The cell may be a satellite cell, an oligodendrocyte progenitor cell, a thymocyte, a hemangioblast, a bone marrow stromal cell, a pancreatic progenitor cell, an endothelial progenitor cell, or a melanoblast. The cells may be totipotent hematopoietic stem cells, common myeloid progenitor cells, myeloblasts, promonocytes, monocytes, common lymphoid progenitor cells, lymphoblasts, prolymphocytes, and/or small lymphocytes.

Xxiv. kit for administering a medicament

In certain embodiments, the present disclosure also provides a pharmaceutical package or kit comprising one or more containers filled with at least one composition (e.g., particle or particles) of the present disclosure. Optionally associated with such container or containers may be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects (a) approval by the manufacturing, use or sale agency for human administration, (b) instructions for use, or both.

In certain embodiments, the kit comprises additional materials to facilitate delivery of the subject agents. For example, the kit may comprise one or more of a catheter, tube, infusion bag, syringe, and the like. In certain embodiments, the composition (e.g., including particles described herein) is packaged in lyophilized form, and the kit comprises at least two containers: a container comprising the lyophilized composition and a container comprising an appropriate amount of water, buffer, or other liquid suitable for reconstituting the lyophilized material.

The foregoing applies to any of the compositions and methods described herein. The present disclosure specifically contemplates any combination of features of such compositions and methods (alone or in combination) with features used to describe the various kits described in this section.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Preferred methods and materials are described herein, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the disclosed methods and compositions. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

The present disclosure contemplates all combinations of any of the foregoing aspects and embodiments, as well as combinations with any of the embodiments set forth in the specific embodiments and examples. These and other aspects of the present disclosure will be further understood upon consideration of the following examples, which are intended to illustrate certain specific embodiments of the present disclosure, but are not intended to limit the scope of the present disclosure, which is defined by the claims.

Example

Example 1-methods for treating cancer

The human patient is identified by a medical practitioner as having a cancer that shed soluble TNFR or soluble IL-2R (e.g., lung, colon, breast, brain, liver, pancreatic, skin, or hematological cancer). The patient is administered a composition comprising particles (described herein) that bind to and sequester soluble TNFR or IL-2R in an amount effective to treat cancer. Optionally, the patient is administered a "maintenance dose" of the composition to maintain inhibition of the effect of soluble TNFR or IL-2R, thereby continuing to enhance immune surveillance of the cancer in the patient.

Example 2 method of detoxifying human

There are human patients with the symptoms of toxicity associated with botulinum toxin. The patient is administered a composition comprising particles (described herein) that bind to and sequester soluble botulinum toxin in an amount effective to ameliorate one or more symptoms associated with toxicity.

Example 3 methods for treating viral infections

The human patient is identified by a medical practitioner as having an HIV-1 infection. The patient is administered a composition comprising particles (described herein) that bind to and sequester soluble HIV-1 virions in an amount effective to reduce viral titer in the patient's circulation. The patient is administered a "maintenance dose" of the composition to maintain a reduction in the titer of HIV-1 virions, thereby inhibiting infection in the patient, and reducing the likelihood of transmission of the virus to another.

Example 4 method for producing silicon particles

Porous silicon disks were fabricated with dimensions of 1000nm x 400nm and 1000nm x 800nm and variable pore sizes. The size and morphology of the discs and the hole diameter were characterized by scanning electron microscopy. Gold nanoparticles (Au) were deposited in the pores of the porous silicon disc. Tumor Necrosis Factor (TNF) is conjugated to the surface of gold nanoparticles through coordinate covalent bonds. Ligand density and TNF-Au binding stability were evaluated.

Example 5 Process for making Polymer particles

Poly (lactide-co-glycolide) (PLGA) particles were prepared from an emulsion. The size and morphology of the PLGA particles were characterized by scanning electron microscopy, atomic force microscopy and transmission electron microscopy. The particles are coated with quaternary ammonium beta-cyclodextrin for macrophage recruitment (i.e., phagocytosis). The coating was verified by atomic force microscopy and transmission electron microscopy. The cladding density and uniformity were characterized by transmission electron microscopy and dynamic light scattering.

The beta-cyclodextrin coated PLGA particles were incubated with macrophages and phagocytosis was monitored by fluorescence microscopy and by flow cytometry.

The beta-cyclodextrin coated PLGA particles are coated with a mixture of polyethylene glycol (PEG) and thiol moieties to allow for opsonization and evasion of macrophage uptake, as well as binding to other particles. The uniformity and density of the PEG and thiol coatings were characterized by atomic force microscopy. Coating stability is characterized by incubating the particles in the medium for different periods of time. As described above, the escape and uptake of particles was monitored at different time points by incubating the particles with macrophages.

PLGA particles are coated with Tumor Necrosis Factor (TNF), and the particles combine by disulfide bonds to form a "sponge" comprising TNF on the interior surface of the sponge. The outer surface (i.e., outer surface) of the sponge is optionally blocked with particles that do not include TNF to prevent interaction between the TNF of the sponge and the cells.

Example 6 pharmacokinetics of Polymer-based particles

The sponge of example 5 (i.e., including the "sponge" of example 5 (e.g., 10)³To 10¹²Sponge) by intravenous or intratumoral administration to mouse models of primary and metastatic cancers as well as to healthy controls. By identifying LD per route of administration ₅₀To determine the toxicity of the sponge. Sponge half-life was determined by monitoring sponge plasma concentrations for each route of administration by LC/MS and ICP. The biodistribution of the sponge was determined by taking a biopsy of the mice and analyzing the sponge tissue and its components by LC/MS, ICP and confocal microscopy.

Example 7 efficacy of Polymer-based particles

The sponge of example 5 (i.e., including the "sponge" of example 5 (e.g., 10)³To 10¹²Sponge) was administered to mice that included MDA-MB-231 or 4T1 xenografts. The MDA-MB-231 model was used to assess the reduction in tumor size and growth, and the 4T1 model was used to assess the inhibition of metastasis. Sponges were intratumorally administered to MDA-MB-231 mice once a week for 6 weeks, and body weight and tumor size were monitored periodically. Sponges were administered intravenously to 4T1 mice once a week for 6 weeks, and the number of metastases was monitored.

Example 8 pharmacokinetics and efficacy of silicon/gold-based particles

The experiments of examples 6 and 7 were repeated with the porous silicon particles of example 5.

While the disclosure has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the disclosure. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process step or steps, to the objective, spirit and scope of the present disclosure. All such modifications are intended to be within the scope of this disclosure.

Claims (113)

1. A particle having at least one surface and an agent immobilized on the surface, wherein:

the agent selectively binds to a target that is a first member of a specific binding pair; and is

Binding of the target to the particle inhibits interaction of the target with the second member of the specific binding pair.

2. A particle comprising a surface and an agent immobilized on the surface, wherein:

the agent is capable of selectively binding to a target; and is

Binding of the agent to the target inhibits interaction between the target and the cell.

3. The particle of claim 1 or 2, wherein the particle is shaped and sized to circulate in the vasculature of a subject.

4. The particle of any one of the preceding claims, wherein the particle is greater than 1 μ ι η.

5. The particle of any one of the preceding claims, wherein the longest dimension of the particle is not greater than about 5 μ ι η.

6. The particle of any one of the preceding claims, wherein the particle has a minimum dimension of at least about 300 nm.

7. The particle of any one of the preceding claims, further comprising a plurality of coating molecules.

8. The particle of claim 7, wherein:

the particle comprises an interior surface and an exterior surface;

the agent is immobilized on the inner surface and the outer surface;

the plurality of coating molecules are bound to the exterior surface; and is

The coating molecules inhibit the interaction between the agent and molecules on the cell surface.

9. The particle of claim 7 or 8, wherein the plurality of coating molecules increases clearance of the particle in vivo.

10. The particle of claim 9, wherein the plurality of coating molecules increases clearance of the particle by phagocytosis, renal clearance, or hepatobiliary clearance.

11. The particle of claim 7 or 8, wherein the plurality of coating molecules reduces clearance of the particle in vivo.

12. The particle of claim 7 or 8, wherein the plurality of coating molecules inhibit interaction between the agent and a cell or extracellular protein.

13. The particle of any one of claims 7 to 12, wherein the plurality of coating molecules comprises a polymer.

14. The particle of any one of claims 7-13, wherein the plurality of coating molecules are biodegradable.

15. The particle of any one of the preceding claims, wherein the particle is dendritic.

16. The particle of any one of the preceding claims, wherein:

the particles are porous;

the surface comprises an outer surface and an inner surface; and is

The inner surface is comprised of the inner walls of the pores of the particles.

17. The particle of claim 16, wherein the agent is immobilized on the inner surface.

18. The particle of claim 16 or 17, wherein a plurality of pores have a cross-sectional dimension of at least 50 nm.

19. The particle of any one of claims 16 to 18, wherein the particle has a porosity of about 40% to about 95%.

20. The particle of any one of claims 16 to 19, wherein the particle comprises metal, gold, alumina, glass, silica, silicon, starch, agarose, latex, plastic, polyacrylamide, methacrylate, a polymer, or a nucleic acid.

21. The particle of claim 20, wherein the particle comprises porous silicon.

22. The particle of any one of the preceding claims, wherein the particle is substantially cubic, pyramidal, conical, spherical, tetrahedral, hexahedral, octahedral, dodecahedral or icosahedral.

23. The particle of any one of the preceding claims, wherein the particle comprises one or more outwardly facing protrusions.

24. The particle of claim 23, wherein the particle comprises more than one outward-facing protrusion.

25. The particle of any one of the preceding claims, wherein the particle comprises:

one or more vertices; and

one or more outward-facing protrusions directed outward from at least one of the vertices of the particle.

26. The particle of any one of claims 23 to 25, wherein one or more protrusions are sized and oriented to inhibit: (i) the agent immobilized on the surface of the particle binds to or activates a cell surface receptor protein and/or (ii) an interaction of the target with a second member of a specific binding pair when the target is bound to the agent, wherein the target is a first member of the specific binding pair.

27. The particle of any one of the preceding claims, wherein the particle comprises two intersecting ridges extending from the surface of the particle, and the ridges are sized and oriented to inhibit: (i) the agent immobilized on the surface of the particle binds to or activates a cell surface receptor protein and/or (ii) an interaction of the target with a second member of a specific binding pair when the target is bound to the agent, wherein the target is a first member of the specific binding pair.

28. The particle of any one of the preceding claims, wherein the particle comprises a tube.

29. The particle of claim 28, wherein the agent is immobilized on an inner surface of the tube.

30. The particle of claim 28 or 29, wherein the tube comprises at least one open end.

31. The particle of any one of claims 28 to 30, wherein the tube is a cylindrical tube, triangular tube, square tube, pentagonal tube, hexagonal tube, heptagonal tube, octagonal tube, or irregularly shaped tube.

32. The particle of any one of claims 28 to 31, wherein the particle comprises more than one tube.

33. The particle of claim 32, wherein the particle comprises a grid defined by a plurality of tubes.

34. The particle of any one of claims 28 to 33, wherein the tube comprises a protein, a nucleic acid, or a polymer.

35. The particle of any one of claims 1 to 22, wherein:

the particles comprise a core subparticle and a plurality of protective subparticles; and is

The agent is immobilized on the core subparticle.

36. The particle of claim 35, wherein said core subparticle has a size of about 100nm to about 2 μ ι η.

37. The particle of claim 35 or claim 36, wherein the protective subparticle has a size of about 10nm to about 1 μ ι η.

38. The particle of any one of claims 35 to 37, wherein the particle comprises 4 to 10⁶And (3) protecting submicron particles.

39. The particle of any one of claims 35 to 38, wherein the particle comprises more than one core subparticle.

40. The particle of any one of claims 1 to 14, wherein the particle is a two-dimensional shape.

41. The particle of claim 40, wherein the shape is a circle, a cross, a fish bone, an ellipse, a triangle, a square, a pentagon, a hexagon, a heptagon, an octagon, or a star.

42. The particle of any one of the preceding claims, wherein the agent is oriented on the particle such that the agent has a reduced ability to bind to molecules on the surface of a cell.

43. The particle of claim 42, wherein the agent is oriented on the particle such that the agent has a reduced ability to bind to a target on the surface of a cell.

44. The particle of any one of the preceding claims, wherein the agent is oriented on the particle such that binding of the agent to molecules on the cell surface is spatially inhibited.

45. The particle of claim 44, wherein the agent is oriented on the particle such that binding of the agent to a target on the surface of a cell is spatially inhibited.

46. The particle of any one of the preceding claims, wherein the surface is oriented such that the agent has a reduced ability to bind to molecules on the surface of a cell.

47. The particle of any one of the preceding claims, wherein the agent has a reduced ability to activate a cell surface receptor protein relative to the ability of a natural ligand of the cell surface receptor protein.

48. The particle of claim 47, wherein the agent does not activate the cell surface receptor protein.

49. The particle of any one of claims 1 to 48, wherein the particle comprises void space.

50. The particle of any one of claims 1 to 49, wherein the particle has an isoelectric point of about 5 to about 9.

51. The particle of any one of claims 1-50, wherein the target is a viral protein.

52. The particle of claim 51, wherein the viral protein is from an arbovirus, an adenovirus, an alpha-virus, an arenavirus, an astrovirus, a BK virus, a buna virus, a calicivirus, a herpes simplex virus type 1, a Colorado tick-fever virus, a coronavirus, a coxsackievirus, a Crimean-Congo hemorrhagic fever virus, a cytomegalovirus, a dengue virus, an ebola virus, an echinovirus, an echovirus, an enterovirus, an EB virus, a flavivirus, a foot and mouth disease virus, a hantavirus, hepatitis A, hepatitis B, hepatitis C, a herpes simplex virus type I, a herpes simplex virus type II, a human herpes virus, a human immunodeficiency virus type I (HIV-1), a human immunodeficiency virus type II (HIV-II), a human papilloma virus, a human T cell leukemia virus type I, a human T cell leukemia virus type II, a human T cell leukemia virus, a virus type II, a human T cell leukemia virus, a virus, influenza virus, japanese encephalitis virus, JC virus, junin virus, lentivirus, equine papova virus, marburg virus, measles virus, mumps virus, nelus virus, norovirus, circovirus, orthomyxovirus, papilloma virus, papova virus, parainfluenza virus, paramyxovirus, parvovirus, picornavirus, poliovirus, polyoma virus, poxvirus, rabies virus, reovirus, respiratory syncytial virus, rhinovirus, rotavirus, rubella virus, fizzy virus, smallpox virus, togavirus, toscarnavirus, varicella zoster virus, west nile virus or yellow fever virus.

53. The particle of claim 51 or 52, wherein the viral protein is a viral capsid protein or a viral envelope protein.

54. The particle of any one of claims 1 to 50, wherein the target is a bacterial protein or a component of a bacterial cell wall.

55. The particle of claim 54, wherein the bacterial protein or cell wall component is from Actinomyces evansi, Bacillus anthracis, Bacillus cereus, Bacteroides fragilis, Bartonella henryi, Bartonella pentahelioica, Bordetella pertussis, Borrelia burgdorferi, Borrelia garinii, Borrelia arabidopsis, Borrelia regressive, Brucella abortus, Brucella canicola, Brucella equina, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Escherichia canis, Rickettsia, enterococcus faecalis, enterococcus faecium, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Haemophilus vaginalis, helicobacter pylori, Klebsiella pneumoniae, Haemophila influenzae, Brucella, Legionella pneumophila, leptospira interrogans, leptospira eriodictyona, leptospira westernella, leptospira vittae, listeria monocytogenes, mycobacterium leprae, mycobacterium tuberculosis, mycobacterium ulcerosa, mycoplasma pneumoniae, neisseria gonorrhoeae, neisseria meningitidis, pseudomonas aeruginosa, nocardia astrata, rickettsia rickettsii, salmonella typhimurium, shigella sonnei, shigella dysenteriae, staphylococcus aureus, staphylococcus epidermidis, staphylococcus saprophyticus, streptococcus agalactiae, streptococcus pneumoniae, streptococcus pyogenes, streptococcus viridans, treponema pallidum, ureaplasma urealyticum, vibrio cholerae, yersinia pestis, yersinia enterocolitica or yersinia pseudotuberculosis.

56. The particle of any one of claims 1 to 50, wherein the target is a yeast or fungal protein or a component of a yeast or fungal cell wall.

57. The particle of claim 56, wherein the yeast or fungal protein or cell wall component is from Pholiopsis variabilis, Aspergillus clavatus, Aspergillus flavus, Aspergillus fumigatus, Rana frog-producing molds, Candida albicans, Candida glabrata, Candida guilliermondii, Candida krusei, Candida viticola, Candida parapsilosis, Candida tropicalis, Candida stellatoides, Candida vellosis, Aureobasidium coronarium, Isosporum, Cryptococcus albicans, Cryptococcus gardnii, Cryptococcus laurentii, Cryptococcus neoformans, Microsporum encephalides, Enterobacter bifida, Exophiala quarriensis, Gekkoniella compacta, Blastomyces euonymus, Geotrichum umbellate, Mucor hiemalis, Paenibacillus brasiliensis, Pseudomyces boisiae, Pseudomyces boidinii, Sporomyces occidentalis, Rhodotorula mucilaginosa, Rhodotorula glutinis, Rhodotorula glutinosa, Candida parapsilosis, Stachybotrys chartarum, Torulopsis racemosa or Rhizopus oryzae.

58. The particle of any one of claims 1-50, wherein the target is a protozoan protein.

59. The particle of claim 58, wherein the protozoan protein is from Cryptosporidium, Giardia intestinalis, Giardia lamblia, Leishmania aegypti, Leishmania braziliana, Leishmania donovani, Leishmania infantis, Leishmania major, Leishmania mexicana, Leishmania tropica, Plasmodium falciparum, Plasmodium gandrum, Plasmodium hyoscyami, Plasmodium odontophyllum, Trichomonas gallinarum, Trichomonas vaginalis, Trichomonas fetalis, Trypanosoma brucei, Trypanosoma cruzi, Trypanosoma malacoporia, Trypanosoma evansi, Trypanosoma lewisi, Trypanosoma cruzi or Trypanosoma mobilis.

60. The particle of any one of claims 1-50, wherein the target is a toxin.

61. The particle of claim 60, wherein the toxin is a bacterial toxin, a plant toxin, or an animal toxin.

62. The particle of claim 60 or 61, wherein the toxin is melittin, bilateral dinoflagellate toxin, tetrodotoxin, chlorotoxin, tetanus toxoid, bungarotoxin, clostridium botulinum toxin, ricin, clostridium perfringens epsilon toxin, staphylococcal enterotoxin B, or endotoxin.

63. The particle of any one of claims 1 to 50, wherein the target is a poison, venom, allergen, carcinogen, psychoactive drug, or agent of a chemical weapon.

64. The particle of any one of claims 1-50, wherein the target is selected from TNF α, TNF β, soluble TNF receptor, soluble TNFR-1, soluble TNFR-2, lymphotoxin α, lymphotoxin β, 4-1BB ligand, CD30 ligand, EDA-A1, LIGHT, TL1A, TWEAK, TRAIL, soluble TRAIL receptor, IL-1, soluble IL-1 receptor, IL-1A, soluble IL-1A receptor, IL-1B, soluble IL-1B receptor, IL-2, soluble IL-2 receptor, IL-5, soluble IL-5 receptor, IL-6, soluble IL-6 receptor, IL-8, IL-10, soluble IL-10 receptor, CXCL1, CXCL8, CXCL9, CXCL10, CX3CL1, FAS ligand, soluble death receptor-3, soluble TNFR-1, soluble IL-1, soluble IL-2, IL-2, IL-2, and CXCL10, CXCL1, and CXCL1, Soluble death receptor-4, soluble death receptor-5, TNF-related weak inducer of apoptosis, MMP1, MMP2, MMP3, MMP9, MMP10, MMP12, CD28, soluble member of the B7 family, soluble CD80/B7-1, soluble CD86/B7-2, soluble CTLA4, soluble PD-L1, soluble PD-1, soluble Tim3, Tim3L, galectin 3, galectin 9, soluble CEACAM1, soluble LAG3, TGF- β 1, TGF- β 2, TGF- β 3, antimiluxel, neublastin, glial derived neurotrophic factor, bone morphogenic protein (e.g., 2, BMP3, BMP3B, BMP4, BMP5, BMP6, GDF 72, e.g., TGF- β 4, TGF- β 3, TGF- β 1, TGF- β 2, TGF- β 3, BMP6, and BMP6, GDF7, GDF8, GDF9, GDF10, GDF11, GDF15), inhibin α, inhibin β (e.g., inhibin β A, B, C, E), bilateral asymmetric developmental factors, nodal, neurotrophic factors, persephin, myostatin, ghrelin, sLR11, CCL2, CCL5, CCL11, CCL12, CCL19, interferon α, interferon β, interferon γ, clusterin, VEGF-a, granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), prostaglandin E2, hepatocyte growth factor, nerve growth factor, sclerostin, complement C5, angiopoietin 2, angiopoietin 3, PCSK9, beta amyloid, activin a, activin B, beta 2 microglobulin, soluble ch1, soluble ch2, soluble NOTCH3, soluble NOTCH4, binding globin, fibrinogen α chain protein, Corticotropin releasing factor, corticotropin releasing factor type 1, corticotropin releasing factor type 2, urocortin 1, urocortin 2, urocortin 3, CD47, anti-interferon gamma autoantibodies, anti-interleukin 6 autoantibodies, anti-interleukin 17 autoantibodies, anti-ghrelin autoantibodies, wnt, indoleamine 2, 3-dioxygenase, C-reactive protein, and HIV-1gp 120.

65. The particle of any one of claims 1-50 and 64, wherein the agent comprises an antibody or antigen-binding portion of the antibody that specifically binds avidly to TNF α, TNF β, soluble TNF receptor, soluble TNFR-1, soluble TNFR-2, lymphotoxin α, lymphotoxin β, 4-1BB ligand, CD30 ligand, EDA-A1, LIGHT, TL1A, TWEAK, TRAIL, soluble TRAIL receptor, IL-1, soluble IL-1 receptor, IL-1A, soluble IL-1A receptor, IL-1B, soluble IL-1B receptor, IL-2, soluble IL-2 receptor, IL-5, soluble IL-5 receptor, IL-6, soluble IL-6 receptor, soluble IL-1A receptor, soluble IL-1B, soluble IL-2 receptor, soluble IL-5 receptor, soluble IL-6 receptor, soluble TNF α, or an antigen-binding portion of the antibody, IL-8, IL-10, soluble IL-10 receptor, CXCL1, CXCL8, CXCL9, CXCL10, CX3CL1, FAS ligand, soluble death receptor-3, soluble death receptor-4, soluble death receptor-5, weak inducer of TNF-related apoptosis, MMP1, MMP2, MMP3, MMP9, MMP10, MMP12, CD28, soluble member of the B7 family, soluble CD80/B7-1, soluble CD86/B7-2, soluble CTLA4, soluble PD-L1, soluble PD-1, soluble Tim3, Tim3L, galectin 3, galectin 9, soluble CEACAM1, soluble LAG3, TGF-beta 1, TGF-beta 2, TGF-beta 3, antimicrotubulin, neublastin, glial cell-derived neurotrophic factor, bone morphogenic protein (e.g. BMP2, BMP-3, and optionally, BMP3, BMP3B, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, BMP10, BMP11, BMP12, BMP13, BMP15), growth differentiation factors (e.g., GDF1, GDF2, GDF3, GDF3A, GDF5, GDF6, GDF7, GDF8, GDF9, GDF10, GDF11, GDF15), inhibin α, inhibin β (e.g., inhibin β A, B, C, E), bilateral asymmetric developmental factors, nodal, neurotrophic factors, persephin, myostatin, ghrelin, sLR11, CCL2, CCL5, CCL11, interferon α, interferon β, interferon γ, clusterin, VEGF-A, granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage-stimulating factor (CSF), granulocyte stimulating factor (GM-11), angiopoietin, PCSFC 11, PCS 11, angiopoietin, PCS 11, amyloid 11, PCS 11, amyloid receptor-C3, amyloid- β, amyloid-C protein, amyloid-C3, amyloid- β, amyloid-C protein, and amyloid-C protein, Activin a, activin B, β 2 microglobulin, soluble NOTCH1, soluble NOTCH2, soluble NOTCH3, soluble NOTCH4, haptoglobin, fibrinogen α chain, corticotropin releasing factor type 1, corticotropin releasing factor type 2, urocortin 1, urocortin 2, urocortin 3, CD47, anti-interferon γ autoantibodies, anti-interleukin 6 autoantibodies, anti-interleukin 17 autoantibodies, anti-ghrelin autoantibodies, wnt, indoleamine 2, 3-dioxygenase, C-reactive protein, and HIV-1gp 120.

66. The particle of any one of claims 1-50 and 64, wherein the agent comprises TNF α, TNF β, soluble TNF receptor, soluble TNFR-1, soluble TNFR-2, vTNF, lymphotoxin α, lymphotoxin β, 4-1BB ligand, CD30 ligand, EDA-A1, LIGHT, TL1A, TWEAK, TRAIL, soluble TRAIL receptor, IL-1, soluble IL-1 receptor, IL-1A, soluble IL-1A receptor, IL-1B, soluble IL-1B receptor, IL-2, soluble IL-2 receptor, IL-5, soluble IL-5 receptor, IL-6, soluble IL-6 receptor, IL-8, IL-10, soluble IL-10 receptor, CXCL1, CXCL8, CXCL9, CXCL10, CX3CL1, FAS ligand, Soluble death receptor-3, soluble death receptor-4, soluble death receptor-5, weak inducer of TNF-related apoptosis, MMP1, MMP2, MMP3, MMP9, MMP10, MMP12, CD28, soluble member of the B7 family, soluble CD80/B7-1, soluble CD86/B7-2, soluble CTLA4, soluble PD-L1, soluble PD-1, soluble Tim3, Tim3L, galectin 3, galectin 9, soluble CEACAM1, soluble LAG3, TGF- β, TGF- β 1, TGF- β 2, TGF- β 3, sLR11, CCL2, CCL5, CCL11, CCL12, CCL19, activin a, activin B, soluble NOTCH1, soluble NOTCH2, soluble NOTCH3, soluble NOTCH4, soluble Jagged1, soluble Jagged2, soluble DLL1, soluble DLL3, soluble DLL4, or binding globin.

67. The particle of any one of claims 1-50, 64, and 65, wherein the pharmaceutical agent comprises Yiprimumab, pembrolizumab, Natuzumab, infliximab, adalimumab, certolizumab, golimumab, etanercept, Setarizumab, Caesalizumab, Mettiumumab, Deslizumab, Tarituzumab, Blumetuzumab, Meporlizumab, Urelumab, Carnaclizumab, daclizumab, belimumab, Desuzumab, Ekulizumab, Tolizumab, Attributuzumab, Urisuzumab, palivizumab, bevacizumab, Brucelizumab, ranibizumab, Abepristeritumumab, Acrituximab, Exiguzumab, Setuximab, Afuimumab, Neriumuzumab, Ullizumab, Pasteuzumab, Rituzumab, Ouzumab, Duranibizumab, Adeniumumab, Adeniumu-Cib, Ekulizumab, Bapituzumab, clenbuteromab, Rituzumab, Perlizumab, Sulan lizumab, Dapirolizumab, Lulizumab, Torilizumab, Eleutlizumab, Pelizumab, Cetuximab, Fuchizumab, Ekumazumab, Engolizumab, matuzumab, Nexituzumab, Netuzumab, Parlizumab, Remauzumab, Zalumumab, Duroguguzumab, Pasteur mAb, Ertuzumab, Epotuzumab, pertuzumab, trastuzumab, Alikumazumab, Anluzumab, Devuzumab, Dupirozumab, Epukuzumab, Epibuzumab, Epibuluzumab, Ebazumab, Ibrumazumab, Enobuzumab, Ennolukuzumab, Evovizumab, Evvizumab, Evviuzumab, Evzeuzumab, Eviuzumab, Evitiukumab, Evovizumab, Evovivizumab, and Vivuvix, (ii) Fisheritumomab, nonfruizumab, Fulikumaumab, Furaluzumab, Franmumab, faliximab, Ganitatumab, Galfatuzumab, Fujiuzumab, Edareuzumab, Immunuzumab, Cetuzumab, Hikauzumab, Muparvizumab, Lejinlizumab, Lolizumab, Ledellizumab, Lysimazumab, Rievilizumab, Ridgelizumab, Ludwizumab, Ridellizumab, Maparlimumab, Motaclizumab, Nanoluumab, Nembuuzumab, Cesivimumab, Otuximab, Orozezumab, Oukulizumab, Ouguzumab, Pageximab, Palixizumab, Panoliuzumab, Panuuzumab, Pakauzumab, Perlacuzumab, Pesuzulizumab, Pertuzumab, Deviuzumab, Rekumazekumazekumazemazemazemav, Raukumazemazemazu, Revituzumab, Rituzumab, Ritumazemazemazemazemazemazemazemazemazemav, Leitumumab, Lexumazu, Ralceuzumab, raloxizumab, reggavir mab, rayleigh mab, rituximab, lomustizumab, rolizumab, saruzumab, scuzucchinumab, stechizumab, sevivimab, sifacimumab, cetuximab, sovizumab, tebeluumab, taclizumab, talilizumab, talnicuzumab, temozuzumab, TGN1412, tikituzumab, tegafuzumab, TNX-650, tosatsutuzumab, tralojiumumab, tremelimumab, trevelumab, tuviruzumab, ubuzumab, vatuzumab, valnussezumab, or an antigen-binding portion of any of the foregoing.

68. The particle of any one of the preceding claims, wherein the target is a soluble biomolecule.

69. The particle of any one of the preceding claims, wherein the target is:

a target as described anywhere above;

a biomolecule as described anywhere above;

a soluble biomolecule as described anywhere above; or

An antigen of an antibody as described anywhere above.

70. The particle of any one of the preceding claims, wherein:

the agent is an agent as described anywhere above;

the agent comprises an antibody, wherein the antibody is described anywhere above;

the agent comprises an antigen binding portion of an antibody, wherein the antibody is described anywhere above; or

The agent comprises an antibody or an antigen-binding portion of the antibody that specifically binds to a target, biomolecule, or soluble biomolecule, wherein the target, biomolecule, or soluble biomolecule is described anywhere above.

71. The particle of any one of the preceding claims, wherein the longest dimension of the particle is not greater than about 1 μ ι η.

72. The particle of any one of the preceding claims, wherein:

the target is a soluble biomolecule;

the soluble biomolecule is in the form of a cell surface receptor protein; and is

The agent is oriented on the particle such that binding or activation of the agent to the cell surface receptor protein on the cell surface is spatially inhibited.

73. A particle having at least one surface and an agent immobilized on the surface, wherein:

the agent selectively binds to a soluble biomolecule;

the soluble biomolecule is in the form of a cell surface receptor protein; and is

The agent is oriented on the particle such that binding or activation of the agent to the cell surface receptor protein on the cell surface is spatially inhibited.

74. The particle of any one of the preceding claims, wherein the agent is a ligand for a cell surface receptor protein.

75. The particle of claim 74, wherein the agent is a natural ligand of the cell surface receptor protein.

76. The particle of any one of claims 72-75, wherein the cell surface receptor protein is expressed by a cancer cell.

77. The particle of any one of claims 72-76, wherein the cell surface receptor protein is a protein that is excreted by cancer cells in a soluble form of the cell surface receptor protein.

78. The particle of any one of claims 72-77, wherein the cell surface receptor protein induces apoptosis when activated on the cell surface.

79. The particle of any one of claims 72-78, wherein the cell surface receptor protein is a Tumor Necrosis Factor Receptor (TNFR) protein.

80. The particle of any one of claims 72-78, wherein the cell surface receptor protein is a Fas receptor protein.

81. The particle of any one of claims 72-78, wherein the cell surface receptor protein is a TNF-related apoptosis ligand receptor (TRAILR) protein, a 4-1BB receptor protein, a CD30 protein, an EDA receptor protein, an HVEM protein, a lymphotoxin beta receptor protein, a DR3 protein, or a TWEAK receptor protein.

82. The particle of any one of claims 72-81, wherein the agent comprises a Tumor Necrosis Factor (TNF) family ligand or a variant of a Tumor Necrosis Factor (TNF) family ligand.

83. The particle of claim 82, wherein the TNF family ligand is TNF α.

84. The particle of claim 82, wherein the TNF family ligand is selected from the group consisting of Fas ligand, lymphotoxin alpha, lymphotoxin beta, 4-1BB ligand, CD30 ligand, EDA-A1, LIGHT, TLA1, TWEAK, TNF beta, and TRAIL.

85. The particle of any one of claims 72-78, wherein the cell surface receptor protein is an interleukin receptor protein.

86. The particle of claim 85, wherein the interleukin receptor protein is an IL-2 receptor protein.

87. The particle of claim 85 or 86, wherein the agent is an interleukin protein or a variant of an interleukin protein.

88. The particle of claim 87, wherein the interleukin protein is an IL-2 protein.

89. A plurality of particles according to any preceding claim.

90. The plurality of particles of claim 89, wherein the average particle size is greater than 1 μm.

91. The plurality of particles of claim 89, wherein the average particle size is from 1 μm to 5 μm.

92. A method for treating a subject having cancer, the method comprising administering to the subject a plurality of particles of any one of claims 89 to 91, wherein:

the cancer comprises cells that shed a soluble form of at least one cell surface receptor protein; and is

The plurality of particles inhibits the biological activity of the excreted soluble form of the at least one cell surface receptor protein, thereby treating the cancer.

93. The method of claim 92, wherein the cancer cells shed a soluble form of the TNF receptor.

94. The method of claim 93, wherein each particle in the plurality of particles comprises an agent comprising a TNF α polypeptide or a variant of a TNF α polypeptide.

95. The method of claim 92, wherein the cancer cells shed a soluble form of the IL-2 receptor.

96. The method of claim 95, wherein each particle of the plurality of particles comprises an agent comprising an IL-2 polypeptide or a variant of an IL-2 polypeptide.

97. The method of any one of claims 92-96, wherein the subject has received adoptive cell transfer therapy (ACT).

98. The method of any one of claims 92 to 97, further comprising administering adoptive cell transfer therapy to the subject.

99. The method of claim 97 or 98, wherein the adoptive cell transfer therapy is administering a composition comprising lymphocytes to the subject.

100. The method of claim 99, wherein the lymphocytes are Tumor Infiltrating Lymphocytes (TILs).

101. The method of claim 99 or 100, wherein the lymphocyte comprises a Chimeric Antigen Receptor (CAR).

102. A method for treating a subject having an autoimmune disease, the method comprising administering to the subject a plurality of particles of any one of claims 89 to 91.

103. The method of claim 102, wherein the target is interleukin 1A, interleukin 1B, interleukin 2, interleukin 5, interleukin 6, interleukin 8, tumor necrosis factor alpha, fas ligand, TNF-related apoptosis inducing ligand, CXCL8, CXCL1, CD80/B7-1, CD86/B7-2, or PD-L1.

104. A method for treating a subject having a neurodegenerative disease, the method comprising administering to the subject a plurality of particles of any one of claims 89 to 91.

105. The method of claim 104, wherein the target is amyloid beta.

106. A method of promoting healthy aging in a subject, the method comprising administering to the subject a plurality of particles of any one of claims 89 to 91.

107. The method of claim 106, wherein the target is TGF- β 1, CCL11, MCP-1/CCL2, β -2 microglobulin, GDF-8/myostatin, or haptoglobin.

108. A method for treating a metabolic disorder in a subject, the method comprising administering to the subject a plurality of particles of any one of claims 89 to 91.

109. The method of claim 108, wherein the target is ghrelin, an anti-ghrelin autoantibody, or cortisol.

110. A method for increasing muscle mass in a subject, the method comprising administering to the subject a plurality of particles of any one of claims 89 to 91.

111. The method of claim 110, wherein the target is myostatin or TGF- β 1.

112. The method of any one of claims 92-111, wherein the subject is a mammal.

113. The method of claim 112, wherein the subject is a human.