EA041139B1 - COMPOSITIONS AND METHODS ASSOCIATED WITH CAPTURING PARTICLES - Google Patents

Description

Priority Statement

The present application claims the priority of US Provisional Application No. 62/186838, filed June 30, 2015; U.S. Provisional Application No. 62/198519, filed July 29, 2015; U.S. Provisional Application No. 62/198541, filed July 29, 2015; U.S. Provisional Application No. 62/236507, filed October 2, 2015; and U.S. Provisional Application No. 62/319092, filed April 6, 2016; each of which is included in its entirety in this description by reference.

State of the art

Many cancer drugs available clinically or in development stimulate the immune system's ability to recognize or destroy cancer, or both. Three of the best known drugs are checkpoint inhibitors such as Yervoy® or ipilimumab from Bristol-Myers Squibb. However, these agents and other approaches involve a general activation of the subject's immune system, inducing potentially dangerous symptoms such as autoimmune disorders and/or other significant side effects.

There is a need in the art for more effective pharmacological approaches to address the problem of treating cancers, in particular metastatic cancers, without compromising the subject's ability to avoid autoimmune reactions. In particular, the present invention provides methods and compositions based on alternative approaches involving the subject's own immune system against malignant tumors, including disinhibition of the tumor microenvironment, i. weakening the defense system of the tumor, rather than stimulating immune cells.

The essence of the invention

The invention provides, inter alia, compositions that bind and inhibit the biological activity of biomolecules, especially soluble molecules, as well as pharmaceutical compositions. In addition, a number of applications are provided herein in which such compositions are useful. For example, the composition described here can be used to inhibit the proliferation, growth and/or survival of cells, such as cancer cells. In addition, the compositions described herein are useful in the prevention and/or treatment of aging, metabolic disorders, and neurodegenerative diseases. In another example, the compositions described herein may be useful in binding and neutralizing toxins (eg, zootoxins, bacterial toxins, and/or plant toxins), viruses, or other foreign compounds, if present in the subject's systemic circulation.

Brief description of the figures

In FIG. 1 depicts an exemplary embodiment of a particle that binds to soluble forms of the TNF receptor (sTNF-R). The particle size is approximately one cubic micrometer. The inner surfaces of the particle contain an immobilized TNF agent that is able to bind to sTNF-R as a target and isolate (capture) it from its natural ligands, thereby inhibiting the interaction between the sTNF-R target and other proteins and cells. The inner surfaces of the particles are defined by the boundaries, within which the empty space is contained.

In FIG. 2 depicts an exemplary embodiment of a particle that contains a TNF agent that binds to soluble forms of the TNF receptor (sTNF-R) that it targets. These three particles, shown in Fig. 2 are depicted as having associations with 0, 3, or 10 sTNF-R target molecules. The annular particle has a diameter of approximately 175 nm, although the TNF agent and sTNF-R target are not shown to scale. The inner surfaces of the particles contain an immobilized TNF agent that is able to bind to the sTNF-R target and which is able to isolate (capture) it from its natural ligands, thereby inhibiting the interaction between the sTNF-R target and other proteins and cells. The interior of the annular particle contains empty space.

In FIG. 3 depicts an exemplary embodiment of a particle that contains protrusions. The particle on the left side of the figure is an octahedron (octahedron) with a core having the largest size from 100 to 150 nm. The particle on the right side of the figure is an icosahedron with a core having the largest size from 200 to 300 nm. Each particle additionally contains molecular protrusions extending outward from the tops of the polyhedral core structure. Particles are depicted as containing the agent, shown in dark gray, and some particles are depicted as being bound to a target (eg, biomolecule), shown in light gray, identified as 0 or 3 captures. The protrusions serve as cellular reflectors that inhibit interaction between the particle agent-bound target and cellular surfaces. Views of particles, protrusions, agent and associated target in FIG. 3 are not necessarily shown to scale.

Fig. 4 consists of two panels, referred to as panel (A) and panel (B). Panel (A) shows the packing of subparticles within a particle containing core subparticles and protective subparticles, where each

- 1 041139 the subparticle has an essentially spherical shape and approximately the same size. However, the particle may contain sub-particles of various shapes and/or sizes. In addition, the subparticles are shown as a pack with a hexagonal structure; however, the subparticles may be randomly packed or have other geometries. Panel (B) indicates (i) capture ligands (i.e. agents) that are immobilized on the surface of the subparticle core, (ii) targets (i.e. biomolecules) that specifically bind to the agent, and (iii) targets that are in liquid-filled voids of particles. Panel (B) does not show protective subparticles. The relative sizes of subparticles, capture ligands, targets, and voids shown in FIG. 4 are not necessarily shown to scale.

Fig. 5 consists of four panels, referred to as panels (A), (B), (C) and (D). Each panel shows the subparticles included in the structure of the particles, where the core subparticles are shown in gray, and the protective subparticles are shown in white. Each particle contains 55 core subparticles. Panels (A) and (B) show an orthogonal view of the particles shown in panels (C) and (D). Panels (A) and (C) show only core subparticles, while panels (B) and (D) show core subparticles and some protective subparticles. The entire particle containing all core subparticles and protective subparticles is preferably coated with at least one layer of protective subparticles, which is not shown in its entirety in the panels. In FIG. 5 each core subparticle and guard subparticle are substantially spherical in shape and approximately the same size; however, subparticles within a single particle may have different shapes and/or sizes. In addition, the subparticles shown in FIG. 5 shown as a package with a hexagonal structure; however, the subparticles may be packed with different geometries, or they may be randomly packed. The relative sizes of subparticles, capture ligands, targets, and voids in FIG. 5 are not necessarily shown to scale. In particular, the length of the linkers connecting the various subparticles can be adjusted in such a way as to provide more or less empty space between the subparticles.

Fig. 6 consists of four panels, referred to as panels (A), (B), (C), (D), (E) and (F). Each panel shows a substantially 2-dimensional particle. In each panel, the circles represent the agent that is immobilized on the surface of the particle. Essentially 2-dimensional particles may contain empty space, for example, between the rays of a star or a cross. Panel (A) shows a top view of a cross-shaped particle, and panel (B) shows an orthogonal side view of this cross-shaped particle. The cruciform shape in panel (A) is essentially a 2D shape, and the orthogonal side view is the third dimension that the 2D shape does not have. The side view shows that essentially 2-dimensional particles can contain different surfaces, i.e. an inner surface on which the agent is immobilized (black) and an outer surface (ie, outer surface) that is substantially free of the agent (grey). Different surfaces may contain different materials, for example, the particle may be lamellar, or different surfaces may be obtained, for example, by masking one surface while the other surface is cross-linked with a coating agent or molecule. Depending on the particle size and the nature of the agent and target, the cruciform particle will inhibit (inhibit) the interaction between the bound target (eg, biomolecule) and other proteins or cells to varying degrees. The geometry of the particle can be adjusted, for example, to further suppress such interactions. Panel (C) shows a particle with a 6-pointed pointed star geometry that can suppress interaction between the bound target and other proteins or cells to a greater extent than the cruciform particle shown in panel (A). Panel (D) shows a 3-ray star that can minimally inhibit interaction between the bound target and other proteins or cells. However, 3-star particles can be modified to suppress the interaction between the bound target and other proteins or cells to a greater extent. For example, panel (E) shows a particle with a 3-ray star geometry, where material that is substantially free of agent surrounds the particle, and panel (F) shows a particle containing elements with a 3-ray star geometry (i.e. . comprising four 3-ray stars) having an outer surface that is substantially free of the agent.

Detailed description

The invention relates to compositions and methods for isolating soluble biomolecules from their natural environment, for example, to thereby inhibit the biological activity of soluble biomolecules. For example, the invention provides a particle or plurality of particles with a surface containing an agent (eg, immobilized on the surface of the particle) that selectively binds to a soluble biomolecule. Once the soluble biomolecule binds to the agent, the particle isolates it, and in doing so, the ability of the soluble biomolecule (eg, significant limitation or lack of ability) to interact with other natural binding partners of the soluble biomolecule occurs. Thus, the soluble biomolecule becomes inert.

Biomolecule.

Typically, the soluble biomolecule is the first partner in a specific binding pair. As used herein, binding partner, specific partner

A specific binding or specific binding pair member generally includes any member of a pair of binding members that bind to each other with significant affinity and significant specificity. A pair of binding partners may, among other things, bind to each other to exclude from binding substantially at least most or at least substantially all of the other components present in the sample, and/or the pair may have a dissociation constant of less than about 10 ^-4 , 10 ^-5 , 10 ^-6 , 10 ^-7 or 10 ^-8 M. A pair of binding partners can match each other in a predictable manner, which is associated with a variety of atomic interactions that together increase binding specificity and affinity. Binding partners, among other methods, can be obtained from biological systems (eg, receptor-ligand interaction) based on chemical interactions and/or through molecular imprinting technology. Illustrative corresponding pairs of binding partners, also referred to as pairs of specific binding, are presented in table. 1 with arbitrary and interchangeable designations first and second.

As used herein, the term biomolecule refers to any molecule that can act in a living organism. In certain embodiments, the biomolecule is an atom, such as lithium or lead (in particular, the biomolecule may be a metal cation). In certain embodiments of the invention, the biomolecule is not a metal atom or ion. For example, the biomolecule may be a molecule such as an organic compound or an inorganic compound. In certain embodiments, the biomolecule is a drug such as warfarin or dabigatran. The biomolecule may be a psychoactive drug such as diacetylmorphine. The biomolecule can be a poison, a toxin, or a poison. The biomolecule may be an allergen. The biomolecule may be a carcinogen. The biomolecule may be a chemical weapon such as a nerve agent.

The biomolecule may be a molecule that is endogenous to the body, 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. The biomolecule may contain a peptide, polypeptide, protein, nucleic acid, carbohydrate or sugar. The biomolecule may be a misfolded protein. The biomolecule may be an amyloid or a soluble amyloid precursor. The terms polypeptide, peptide, and protein are used interchangeably herein and refer to any chain of amino acids linked to a peptide, regardless of length or post-translational modifications. The biomolecule can be a lipid, steroid or cholesterol. The biomolecule may contain a lipid, steroid or cholesterol. The biomolecule may be a circulating extracellular nucleic acid such as circulating extracellular RNA. The biomolecule may be a microRNA (miRNA).

The biomolecule may be a biomolecule that is secreted by a cell (eg, a mammalian cell).

The biomolecule may be the extracellular region of a membrane protein that, when cleaved, forms a soluble form. The biomolecule may be a cytosolic biomolecule. For example, the biomolecule may be a cytosolic biomolecule that is released in vivo during apoptosis, or when the particle is used in an in vitro method, the cytosolic biomolecule may be free in solution.

In some preferred embodiments of the invention, the biomolecule is a soluble biomolecule. In some preferred embodiments of the invention, the target is a soluble biomolecule. However, the particles can be targeted to target biomolecules that are not dissolved in aqueous solution and/or that do not interact with binding partners on the cell surface. For example, the particle may specifically bind to a biomolecule that is associated with a protein aggregate, such as an amyloid aggregate or a prion aggregate. Such particles can exert a therapeutic effect by disrupting the aggregate (eg, by shifting the thermodynamic equilibrium of stable aggregated states) and/or by sequestering the aggregate (eg, to inhibit further aggregation and/or to allow clearance of the associated aggregate). Similarly, the particle can specifically bind to a crystalline form of calcium or hydroxyapatite. Similarly, a particle can specifically bind to a biomolecule that is associated with a virus or cell, such as a bacterial cell, a protozoan cell, a fungal cell, or a yeast cell, for example, when the biomolecule is not dissolved in an aqueous solution, but when the biomolecule is part of a membrane, the cell wall or capsid. Thus, the particle can isolate the pathogenic virus or cell, thereby reducing the pathogenicity of the virus or cell. The particle can specifically bind to a biomolecule that is associated with an extracellular vesicle such as an ectosome, exosome, shedding vesicle, or apoptotic body. The particle can specifically bind to low density lipoprotein, for example

- 3 041139 measures for isolating low density lipoprotein particles.

The biomolecule may be a cell surface receptor ligand. The ligand may be a natural ligand or a synthetic ligand. The ligand may be a natural receptor ligand (eg, a ligand that is produced in the subject in vivo) or a non-natural ligand (eg, a ligand that enters or is administered to the subject, such as a virus or a drug). The biomolecule may be a ligand for a cytosolic receptor or a nuclear receptor.

Table 1 ____________ Examples of specific binding pairs ____________

First bonding partner Second bonding partner Cell surface receptor (eg, TNF receptor) Natural ligand (eg TNFa) Virus capsid or envelope protein (e.g. dr120 HIV-1) Appropriate cell receptor (eg CD4) Botulinum toxin synaptotagmin receptor II cell surface Soluble receptor (eg, soluble TNFR or soluble IL-2 receptor) natural ligand (for example, TNFa or IL-2)

Tumor cells are known to protect themselves from host immunological surveillance by releasing soluble forms of cytokine receptors into the environment that bind to cytokines produced by immune cells in the tumor microenvironment. For example, malignant cells release soluble forms of the TNF receptor and other cytokine receptors such as the IL-2 receptor and the TRAIL receptor into the medium. These soluble receptors confer a growth advantage on cancer cells by sparing the cells from the pro-apoptotic effects of TNFa, IL-2, and TRAIL. Karpatova et al. reported the release of the 67 kDa human laminin receptor by malignant cells into the medium, which may enhance tumor invasiveness and metastasis (J. Cell. Biochem., 60(2):226-234 (1996)). Thus, the particles described herein can be designed to capture soluble forms of cell surface receptor proteins, for example, for use in the treatment of cancer.

Thus, in certain embodiments, the cancer cell expresses a cell surface receptor protein and/or the cell surface receptor protein is a protein that the cancer cell releases into the environment as a soluble form of the cell surface receptor protein. In certain embodiments, a cell surface receptor protein, when activated, induces apoptosis (eg, a death receptor). In certain embodiments, the cell surface receptor protein is a tumor necrosis factor receptor (TNFR) protein (eg, TNFR-1 or TNFR-2). In certain embodiments, the cell surface receptor protein is a Fas receptor protein. In certain embodiments, the cell surface receptor protein is a TNF-related apoptosis-inducing 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 certain embodiments, the cell surface receptor protein is an interleukin receptor protein, such as an IL-2 receptor protein. It should be understood that in such embodiments, the soluble target biomolecule may be a soluble form of a cell surface receptor, which, for example, is released from a malignant cell.

In some embodiments, the biomolecule is soluble Tim3 (T cell Ig mucin 3). Soluble Tim3 (sTim3) is involved in autoimmune disease and cancer, and elevated levels of sTim3 are associated with HIV infection. Binding of the galectin 9 protein (Galectin 9 or Gal9) and other potential ligands to Tim3 into a heterodimeric associate with CEACAM1 leads to the inhibition of T cell reactions, and the joint blocking of Tim3 and CEACAM1 leads to an antitumor immune response. Accordingly, the biomolecules may be sTim3 or a natural ligand for sTim3 such as Tim3L or Gal9. The biomolecules may be soluble isoforms of CEACAM1. Thus, the particles can be adapted to capture sTim3 rather than inhibit interactions between Gal9 and membrane-bound Tim3 (mTim3). Similarly, the agent can be sTim3, an sTim3 selective antibody (or antigen-binding fragment thereof), or a Tim3 ligand. The agent may be a natural ligand for CEACAM1 (eg Gal9 or a variant thereof) or an antibody selective for CEACAM1 or a soluble isoform thereof. Any of

- 4 041139 the above particles can be used, for example, in methods of treating cancer, HIV infection and autoimmune disease such as graft-versus-host.

In some embodiments, the biomolecules may be galectin 9 (Gal9). The particle may contain a Gal9 selective agent, such as a natural Gal9 ligand, in particular Tim3, or a variant thereof, or a Gal9 selective antibody. Thus, the particles can be adapted to bind Gal9 rather than inhibit the interactions of membrane-bound Gal9 (mGal9) with membrane-bound Tim3 (mTim3). In some embodiments, the biomolecule may be a soluble isoform of CEACAM1 (sCEACAM1). The agent may be a natural ligand for sCEACAMl, such as Gal9 or a variant thereof, or an antibody selective for CEACAM1 or a soluble isoform of CEACAM1.

In some embodiments, the biomolecule is soluble CTLA4. Soluble CTLA4 (sCTLA4) has been implicated in cancer development, and antibodies active against SCTLA4 but not against membrane-bound CTLA4 (mCTLA4) are effective in animal models of cancer. In some embodiments, the biomolecule is SCTLA4. The agent may be a natural CTLA4 ligand such as soluble B7-1 or soluble B7-2 or variants thereof, or an antibody selective for CTLA4 such as ipilimumab or ticilimumab. Thus, the particles can be adapted to bind SCTLA4 without inhibiting the interaction between the ligands and mCTLA4. In this way, sCTLA4 can be removed from the tumor microenvironment (TME) and/or from circulation outside of the TME, leaving mCTLA4 free to interact as part of the normal immune response. Particles that target SCTLA4 can be used, for example, in methods of treating cancer.

Soluble PD-1 (SPDL) is involved in autoimmune diseases such as rheumatoid arthritis. Excess SPDL can disrupt the balance between PD1 and its PD-L1 and PD-L2 ligands, leading to an autoimmune reaction. Thus, the biomolecule may be SPDL. The agent may be a natural SPDL ligand, eg PD-L1, PD-L2, or variants thereof, or an antibody selective for PD1, such as a drug to block PD1, eg nivolumab, pidilizumab, or pembrolizumab (Keytruda®). Thus, the particle can be adapted to capture SPDL without inhibiting the interaction of PD-L1 or PD-L2 with membrane bound PD1. Such particles can be used, for example, in methods of treating autoimmune diseases such as arthritis.

LAG-3 is a T cell surface receptor which, when bound to its ligand, results in inhibition. Soluble forms of LAG-3 (sLAG3) have been correlated with autoimmunity, such as in type I diabetes and other autoimmune diseases. The biomolecule may be SLAG3. The agent may be a natural SLAG3 ligand, or a variant thereof, or an antibody selective for SLAG3. Thus, the particle can be adapted to capture sLAG3 without inhibiting interaction between ligands and with membrane-bound LAG3. Such particles can be used, for example, in methods of treating autoimmune diseases such as type I diabetes.

The biomolecule may be TNFa. The agent may contain an anti-TNFa antibody such as infliximab, adalimumab, certolizumab, afelimomab, nerelimomab, ororalizumab, or golimumab, or the agent may contain an antigen-binding fragment of an anti-TNFa antibody. The agent may be etanercept.

The agent may be a soluble TNFa receptor (sTNF-R or a variant thereof). TNFa-targeting particles may be particularly useful in the treatment or prevention of various autoimmune diseases such as ankylosing spondylitis, Crohn's disease, hidradenitis suppurativa, psoriasis, plaque psoriasis, psoriatic arthritis, intractable asthma, juvenile idiopathic arthritis, ulcerative colitis, and rheumatoid arthritis. TNFa-targeting particles may also be useful in the treatment or prevention of Alzheimer's disease, cardiovascular disease, type II diabetes, muscular dystrophy, and obesity, among other diseases and conditions.

The biomolecule may be β2 microglobulin (B2M). The agent may be an anti-B2M antibody. B2M-targeted particles may be useful in the treatment or prevention of memory loss, cognitive decline, peripheral arterial disease, dialysis-associated amyloidosis, chronic lymphocytic leukemia, multiple myeloma, and lymphoma, in addition to other diseases and conditions.

The biomolecule may be CCL2 (a chemokine ligand 2 with a C-C motif). The agent may be an anti-CCL2 antibody. Particles targeted to CCL2 may be useful in the treatment or prevention of Alzheimer's disease, atherosclerosis, myocardial ischemia (eg, ischemic stroke), epilepsy, multiple sclerosis, psoriasis, rheumatoid arthritis, glomerulonephritis, and traumatic brain injury, in addition to other diseases and conditions .

The biomolecule may be CCL11 (C-C motif chemokine 11; eotaxin 1). The agent may be an anti-CCLL11 antibody. Particles targeting CCL11 could be useful

- 5 041139 for the treatment or prevention of memory loss and cognitive decline in addition to other diseases and conditions.

The biomolecule may be CCL19. The agent may be an antibody against

CCL19. Particles targeting CCL19 may be useful in the treatment or prevention of aging and cognitive decline, in addition to other diseases and conditions.

The biomolecule may be interferon gamma (INFy). The agent may contain an anti-INFy antibody such as fontolizumab or a soluble INFy receptor (sINFyR). The biomolecule may be a soluble INFy receptor. The agent may contain INFy or an anti-sINFyR antibody. Interferon gamma-targeting particles may be particularly useful in the treatment or prevention of autoimmune diseases such as Crohn's disease, rheumatoid arthritis, and psoriasis, in addition to other diseases and conditions.

The biomolecule may be a clusterin (eg, secretory clusterin, isoform 2). The agent may comprise an anti-clusterin antibody or an antigen-binding fragment thereof. Clusterin-targeted particles may be useful in the treatment or prevention of cancer (e.g., neck cancer, renal cell cancer, colorectal cancer, endometrial cancer, ovarian cancer, breast cancer, prostate cancer, pancreatic cancer, lung cancer, hepatocellular cancer, or melanoma), kidney disease (eg, nephropathic cystinosis), Fanconi syndrome, glomerulonephritis, atherosclerosis, and myocardial infarction, in addition to other diseases and conditions.

The biomolecule may be a box high mobility group 1 (HMGB1) protein. The agent may contain an anti-HMGB1 antibody or an antigen-binding fragment thereof. The biomolecule may be a heat shock protein (eg HSP60, HSP70, HSP90). The agent may contain an anti-HSP antibody, or an antigen-binding portion thereof. The biomolecule may be a peroxiredoxin (eg, peroxiredoxin 1 or peroxiredoxin 2). The agent may contain an anti-peroxiredoxin antibody, or an antigen-binding fragment thereof.

The agent may be the extracellular portion of a phagocytic receptor (scavenger receptor), such as a class A phagocytic receptor (e.g., SCARA1 (macrophage phagocytic receptor 1; MSR1; CD204), SCARA2 (macrophage receptor; MARKO), CKARA3, SCARA4 (COLEC12), SCARA5) , class B phagocytic receptor (eg, SCARB1, SCARB2, SCARB3 (CD36)), CD68, mucin or lectin-like receptor-1 of oxidized LDL (LOX-1).

The biomolecule can be an insulin-like growth factor 1 (IGF-1), or an insulin-like growth factor binding protein (eg, IGFBP-1, IGFBP-2, IGFBP-3, iGfBP-4, IGFBP-5, IGFBP-6). The agent may be an insulin-like growth factor 1 (IGF-1) or an insulin-like growth factor binding protein (eg, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, IGFBP-6). The agent may be an antibody, or an antigen-binding fragment thereof, that selectively binds 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 an extracellular CD63, CD9 or CD81 epitope. Particles targeting CD63, CD9, and/or CD81 may be particularly useful for capturing extracellular vesicles such as an ectosome, exosome, shedding vesicle, or apoptotic body. Particles that capture various extracellular vesicles may be particularly useful in the treatment or prevention of cancer (eg, advanced cancer, which correlates with vesicle shedding).

The biomolecule may be CXCL1, CXCL2, CXCL3, CXCL4, CXCL4L1, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL16, CXCL17, CCL1, CCL2, CCL3, CCL3L1, CCL3L3, CCL4 CCL4L1, CCL4L2, CCL5, CCL7, CCL8, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, XCL1, XCL2 or CX3CL1 ( see, for example, Zlotnik A., Yoshie O., Immunity, 36(5):705 (2012)). The agent may contain an antibody (or antigen-binding fragment thereof) that specifically binds CXCL1, CXCL2, CXCL3, CXCL4, CXCL4L1, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL16, CXCL17, CCL1, CCL2, CCL3, CCL3L1, CCL3L3, CCL4, CCL4L1, CCL4L2, CCL5, CCL7, CCL8, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, XCL1, XCL2 or CX3CL1.

The biomolecule may be 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, interleukin, or 36. The agent may contain an antibody (or antigen-binding fragment 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, interleukin or 36. The agent may contain soluble interleukin 2 receptor, soluble interleukin 3 receptor, soluble interleukin 4 receptor, soluble interleukin 5 receptor, soluble interleukin 6 receptor, soluble interleukin 7 receptor, soluble interleukin receptor 9, Soluble Interleukin 10 Receptor, Soluble Interleukin 11 Receptor, Soluble Interleukin 12 Receptor, Soluble Interleukin 13 Receptor, Soluble Interleukin 15 Receptor, Soluble Interleukin 20 Receptor, Soluble Interleukin 21 Receptor, Soluble Interleukin 22 Receptor, Soluble Interleukin 23 Receptor, Soluble Interleukin Receptor 27 or a soluble interleukin 28 receptor. The agent may be a soluble ST2 that binds interleukin 33.

The biomolecule may be soluble interleukin 2 receptor, soluble interleukin 3 receptor, soluble interleukin 4 receptor, soluble interleukin 5 receptor, soluble interleukin 6 receptor, soluble interleukin 7 receptor, soluble interleukin 9 receptor, soluble interleukin 10 receptor, soluble interleukin 11 receptor, soluble interleukin 12 receptor, soluble interleukin 13 receptor, soluble interleukin 15 receptor, soluble interleukin 20 receptor, soluble interleukin 21 receptor, soluble interleukin 22 receptor, soluble interleukin 23 receptor, soluble interleukin 27 receptor, or soluble interleukin 28 receptor. The agent may contain an antibody (or its antigen-binding fragment) that specifically binds soluble interleukin 2 receptor, soluble interleukin 3 receptor, soluble interleukin 4 receptor, soluble interleukin 5 receptor, soluble interleukin receptor 6, Soluble Interleukin 7 Receptor, Soluble Interleukin 9 Receptor, Soluble Interleukin 10 Receptor, Soluble Interleukin 11 Receptor, Soluble Interleukin 12 Receptor, Soluble Interleukin 13 Receptor, Soluble Interleukin 15 Receptor, Soluble Interleukin 20 Receptor, Soluble Interleukin 21 Receptor, Soluble Interleukin Receptor 22, soluble interleukin 23 receptor, soluble interleukin 27 receptor, or 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, triiodothyronine, or thyroxine. The biomolecule can be a prostaglandin (eg, prostacyclin 12 (PGI2), prostaglandin E2 (PGE2), prostaglandin F2a (PGF2a)), 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 calcidiol.

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

The biomolecule may be vascular endothelial growth factor A (VEGF-A). The agent may contain an antibody that specifically binds VEGF-A, such as bevacizumab or brolucizumab, or an antigen-binding fragment thereof, such as ranibizumab. For example, the agent may be aflibercept. Particles that target VEGF-A may be particularly useful in the treatment or prevention of macular degeneration (eg, wet macular degeneration), proliferative diabetic retinopathy, neovascular glaucoma, macular edema, cancer (eg, colorectal cancer, lung cancer, cancer prostate, breast cancer, kidney cancer, brain cancer), bronchial asthma, diabetes mellitus, ischemic cardiomyopathy and myocardial ischemia, in addition to other conditions and diseases.

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 may be an antibody, or an antigen-binding fragment thereof, that selectively binds a soluble VEGF receptor, such as alacizumab, icrucumab, or ramucirumab. The agent may be a VEGF receptor ligand such as VEGF-A, VEGF-B, VEGF-C, VEGF-D or placental growth factor (PGF). Particles targeting soluble VEGF receptors may be particularly useful in the treatment or prevention of cancer, in addition to other conditions and diseases.

The biomolecule may be a member of the epidermal growth factor family and be epidermal growth factor (EGF), heparin-binding EGF-like growth factor (HB-EGF), transforming growth factor α (TGF-α), amphiregulin (AR), epiregulin (EPR), epigen, betacellulin (BTC), neuregulin-1 (NRG1), neuregulin-2 (NRG2), neuregulin-3 (NRG3), or neuregulin-4 (NRG4). The agent may be an antibody, or an antigen-binding fragment thereof, that selectively binds EGF, HB-EGF, TGF-α, AR, EPR, epigene, BTC, NRG1, NRG2, NRG3, or NRG4. The agent may contain a soluble EGF receptor, such as soluble EGF receptor, soluble HER2, or soluble HER3. Particles targeted to members of the epidermal growth factor family may be particularly useful in the treatment or prevention of cancer, in addition to other conditions and diseases.

The biomolecule may be a soluble epidermal growth factor receptor (EGF receptor), such as a soluble EGF receptor, a soluble human epidermal growth factor receptor 2 (soluble HER2), or a soluble human epidermal growth factor receptor 3 (soluble HER3). The agent may be an antibody or antigen-binding fragment thereof that selectively binds a soluble EGF receptor, such as cetuximab, futuximab, imgatuzumab, matuzumab, necitumumab, nimotuzumab, panitumumab, zalutumumab, duligotumab, patritumab, ertumaxomab, pertuzumab, or trastuzumab. The agent may be an EGF receptor ligand, such as a member of the EGF family, as described above. Particles targeting soluble EGF receptors may be particularly useful in the treatment or prevention of cancer, in addition to other conditions and diseases.

The biomolecule may be an IgE antibody. The agent may contain an anti-IgE antibody such as omalizumab or talizumab, or an antigen-binding fragment thereof. The agent may be the extracellular portion of the FcεRI. Particles that target an IgE antibody may be particularly useful in the treatment of chronic spontaneous urticaria and allergic asthma, in addition to other conditions and diseases.

The biomolecule may be a subtilisin/kexin type 9 (PCSK9) convertase proprotein. The agent may be an anti-PCSK9 antibody such as alirosumab, lodelcizumab, ralpancizumab, or evolocumab, or an antigen-binding fragment thereof. Particles targeted to PCSK9 may be particularly useful in the treatment or prevention of hypercholesterolemia, atherosclerosis, ischemia, and myocardial infarction, in addition to other conditions and diseases.

The biomolecule may be adrenomedullin, brain-derived neurotrophic factor, erythropoietin, fibroblast growth factor, hepatoma-derived growth factor, glucose-6-phosphate isomerase, keratinocyte growth factor, macrophage migration inhibition factor, neurotrophin (nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4), platelet-derived growth factor, stem cell growth 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 may contain an antibody or an antigen-binding fragment thereof that selectively binds adrenomedullin, brain-derived neurotrophic factor, erythropoietin, fibroblast growth factor, hepatoma-derived growth factor, glucose-6-phosphate isomerase, keratinocyte growth factor, macrophage inhibitory factor migration, neurotrophin (nerve growth factor, neurotrophic factor isolated from the brain

- 8 041139 brain, neurotrophin-3, neurotrophin-4), platelet growth factor, stem cell growth 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 a soluble tropomyosin kinase B receptor (soluble TrkB). The agent may be an anti-TrkB antitheo or an antigen-binding fragment thereof. The biomolecule may be a soluble tropomyosin kinase A receptor (soluble TrkA). The agent may be an anti-TrkA antibody or an antigen-binding fragment thereof. The agent may be a neurotrophic factor isolated from the brain.

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

The biomolecule may be a hedgehog protein (eg, a sonic hedgehog). The agent may be an antibody that selectively binds the hedgehog protein. Particles targeting the hedgehog protein may be particularly useful in the treatment or prevention of cancers such as pancreatic cancer, cerebellar cancer, and medulloblasts, in addition to other conditions and diseases.

The biomolecule may be a soluble human leukocyte antigen (HLA) (e.g., soluble HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, HLA-F, or HLA-G (see, e.g., BassaniSternberg, M et al., Proceedings National Academy Sciences, USA, 107(44):18769 (2010))). The agent may be an antibody that selectively binds soluble human leukocyte antigen (HLA). The agent may be a soluble protein receptor similar to killer cell immunoglobulin. Particles that target the soluble HLA antigen may be particularly useful in the treatment or prevention of cancer in addition to other diseases and conditions.

The biomolecule may be an isoform of a soluble UL16 binding protein (e.g., soluble RAET1 (ULBP1; RAET1E2), soluble RAET1H (ULBP2), soluble RAET1N (ULBP3), soluble RAET1E (ULBP4), soluble RAET1G (ULBP5), or soluble RAET1L (ULBP6) ). The agent may be an antibody that specifically binds an isoform of a soluble UL16 binding protein or an antigen-binding fragment thereof. The agent may be a soluble NKG2D receptor (see, for example, PCT Application Publication No. WO 2006/024367, which is incorporated herein in its entirety by reference).

The biomolecule can be soluble MIC-A or soluble MIC-B (see, for example, Groh, V. et al., Nature, 419(6908):734 (2002)). The agent may be an anti-MIC-A antibody, or an anti-MIC-B antibody, or an antigen-binding fragment of these antibodies. The agent may be a soluble NKG2D receptor (see, for example, PCT Application Publication No. WO 2006/024367, which is incorporated herein in its entirety by reference).

The agent may be a natural cytotoxicity soluble protein receptor (see, for example, Jarahian, M. et al., PloS Pathogens, 7(8): e1002195 (2011)).

The biomolecule may be the domain of a soluble C-type lectin family 2 member D (soluble CLEC2D; soluble lectin-like transcript-1 (LLT1)) (see, e.g., Chalan, P. et al., PloS One, 10(7): e0132436 (2015)). The agent may be an antibody that selectively binds soluble LLT1. Particles that target soluble LLT1 may be particularly useful in the treatment or prevention of autoimmune diseases such as rheumatoid arthritis, in addition to other diseases and conditions.

The biomolecule may be soluble CD16 (see, for example, Hoover, R. G., J. Clinical Investigation, 95:241 (1995)). The agent may be an antibody that selectively binds soluble CD16. Particles that target soluble CD16 may be particularly useful in the treatment or prevention of cancer in addition to other diseases and conditions.

The biomolecule may be a plasminogen activator inhibitor-1 (PAI-1), a plasminogen activator inhibitor-2 (PAI-2), tissue plasminogen activator, urokinase, plasminogen, thrombin, or a2-macroglobulin. The agent may be an antibody that selectively binds plasminogen activator inhibitor-1 (PAI-1), plasminogen activator inhibitor-2 (PAI-2), tissue plasminogen activator, urokinase, plasminogen, thrombin, or a2-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 may be an antibody that selectively binds Factor XII, Factor XIIa, Factor XI, Factor XIa, Factor IX, Factor IXa, 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 serpin (e.g. α1-antitrypsin, antitrypsin-related protein, α1-antichymotrypsin, callistatin, protein C inhibitor, transcortin, thyroxin-binding globulin, angiotensinogen, centerin (GCET1), protein Z-related protease inhibitor, vaspin, antithrombin, cofactor heparin II, plasminogen activator inhibitor 1, glial nexin (nexin I protease), epithelial pigment factor, a2-antiplasmin, complement-1 inhibitor, neuroserpin, plasminogen activator inhibitor, 2SERPINA1 or SERPINA2.The agent may contain an antibody that selectively binds serpin, or an antigen-binding fragment thereof.

The biomolecule may be soluble ST2. The agent may be interleukin 33 or an antibody that specifically binds soluble ST2 (or a fragment thereof).

Particles that target soluble ST2 may be particularly useful in the treatment or prevention of cardiovascular disease, myocardial infarction, acute coronary syndrome, and heart failure, in addition to other conditions and diseases.

The biomolecule may be myostatin (growth differentiation factor 8 (GDF-8)). The agent may be an anti-myostatin antibody such as stamulumab or anoractrumab. The agent may be an activin receptor or a myostatin-binding portion thereof, for example the agent may be a soluble activin type C receptor. like loss of muscle mass in zero gravity) in addition to other diseases and conditions.

The biomolecule may be ghrelin. The agent may be an anti-ghrelin antibody. Ghrelin-targeting particles may be particularly useful in the treatment or prevention of obesity, Prader-Willi syndrome, drug addiction, alcoholism, and leptin resistance (eg, genetic leptin resistance).

The biomolecule may be sLR11 (soluble SORL1; soluble SORLA; soluble SORLA1). The agent may be an anti-sLR11 antibody. Particles targeted to sLR11 may be particularly useful for the treatment or prevention of obesity in addition to other diseases and conditions.

The biomolecule may be TGF-β (transforming growth factor beta, eg TGF-βΕ TGF-e2 or TGF-e3). The agent may be an anti-TGF-β antibody such as fresolimumab, lerdelimumab, or metelimumab. The agent may contain the TGF-in-binding domain of the TGF-β receptor. The agent can be LTBP1 (latent transforming growth factor beta-binding protein 1), 14-3-3-epsilon protein (tyrosine-3-monooxygenase/tryptophan-5-monooxygenase activation protein epsilon, YWHAE), or eukaryotic translation initiation factor 3 subunit I (EIF3I). ), each of which binds to TGF-β. TGF-β targeting particles may be particularly useful for the treatment or prevention of scleroderma, idiopathic pulmonary fibrosis, kidney disease, focal segmental glomerulosclerosis, keratoconus, Marfan's syndrome, Alzheimer's disease, cognitive decline, traumatic brain injury, muscle wasting, and cancer (eg kidney cancer and melanoma) in addition to other conditions and diseases.

The biomolecule may be a Wnt (eg, Wnt1, Wnt2, Wnt2B, Wnt3, Wnt3A, Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, Wnt8A, Wnt8B, Wnt9A, Wnt9B, Wnt10A, Wnt10B, Wnt11 or Wnt16). The agent may be an anti-Wnt antibody. Wnt-targeted particles may be particularly useful in the treatment or prevention of obesity, type II diabetes mellitus, atherosclerosis, calcium aortic valve stenosis, heart attack, heart failure, stroke, and cancer (eg, breast cancer, colorectal cancer, esophageal cancer, melanoma , prostate cancer, lung cancer, non-small cell lung cancer, mesothelioma, sarcoma, glioblastoma or ovarian cancer) in addition to other diseases and conditions.

The biomolecule can be a soluble Notch ligand (eg, soluble Jagged1 protein, soluble Jagged2, soluble delta-like ligand 1 (DLL1), soluble delta-like ligand 3 (DLL3), and delta-like ligand 4 (DLL4)). The agent can be an anti-Notch ligand antibody, such as demcizumab or enoticumab, or a soluble Notch receptor (eg, soluble NOTCH1, NOTCH2, NOTCH3, or NOTCH4), or variants thereof. Particles targeting soluble Notch ligands may be particularly useful for the treatment or prevention of atherosclerosis, calcium aortic valve stenosis, heart attack, heart failure, stroke, and cancer (e.g., breast cancer, pancreatic cancer, kidney cancer, non-small cell lung cancer, and solid tumors) in addition to other diseases and conditions.

The biomolecule may be a soluble Notch receptor (eg, soluble NOTCH-1, NOTCH2, NOTCH3, or NOTCH4). The agent may be an anti-Notch receptor antibody such as tarextumab or brontistuzumab, or a soluble Notch ligand. Soluble Notch receptor-targeting particles may be particularly useful in the treatment or prevention of atherosclerosis, calcium aortic valve stenosis, heart attack, heart failure, stroke, and cancer (e.g., breast cancer, pancreatic cancer, kidney cancer, non-small cell lung cancer, and solid tumors) in addition to other diseases and conditions.

The target may be hydroxyapatite or calcium salts (eg, crystalline calcium salts). The agent may be a chelating agent such as ethylenediaminetetraacetic acid (EDTA), pentaacetic acid diethylenetriamine (DTPA), sodium thiosulfate (STS), inositol hexaphosphate, or citric acid. Particles targeted to hydroxyapatite or calcium salts may be particularly useful in the treatment or prevention of atherosclerosis, calcium aortic valve stenosis, and calcium tendonitis, in addition to other diseases and conditions.

In some embodiments, the biomolecule is an autoantibody. An autoantibody is an antibody produced by a subject that specifically binds an antigen produced by the subject. Autoantibodies are associated with many different disease states, including lupus. In addition, the induction of new autoantibodies may be associated with therapeutic intervention, for example, as a result of drug-induced lupus. Thus, a composition comprising a plurality of particles containing an agent that selectively binds one or more autoantibodies can be used, for example, in a method for treating or preventing lupus (eg, drug-induced lupus). The biomolecule can be, for example, a double-stranded DNA autoantibody or an antinuclear autoantibody.

An autoantibody-targeting particle may contain an agent that is an antigen for the autoantibody.

The biomolecule can be an anti-β-adrenergic receptor autoantibody or an anti-M2 muscarinic receptor autoantibody, for example, in the prevention or treatment of idiopathic dilated cardiomyopathy. In particular, particles that target an autoantibody against the beta adrenoreceptor or against the M2 muscarinic receptor can be administered to a subject with Chagas disease, which correlates with the induction of such autoantibodies (see, for example, Herda, L. R. et al., Br. J. Pharmacol ., 166(3):847 (2012)). The biomolecule may be an alpha-1 adrenergic receptor autoantibody, for example, in the treatment or prevention of hypertension (see, for example, Luther, H. P. et al., Hypertension, 29(2):678 (1997)). The biomolecule may be an autoantibody against muscarinic type 3 receptor used, for example, in the treatment or prevention of Sjögren's syndrome (see, for example, Lee, B.H. et al., PloS One, 8(1):e53113 (2013)).

Autoantibodies against hormones and cytokines can buffer the concentration of hormones and cytokines, for example by reversibly binding to them, controlling the concentration of free active species. Deviations from healthy levels of autoantibodies may contribute to diseases arising from loss of cytokines or hormonal homeostasis. For example, autoantibodies against IFNy can cause the spread of nontuberculous mycobacterial infections, autoantibodies against IL-17 are associated with the development of chronic mucosal candidiasis, and autoantibodies against IL-6 are associated with serious staphylococcal or streptococcal infections. Autoantibodies to the hunger hormone ghrelin can mediate the effective concentration of ghrelin available for binding to the GHSR1 ghrelin receptor.

In some embodiments, the biomolecule is an autoantibody. For example, the autoantibodies may be anti-IFNy, anti-IL-17, anti-IL-6, or anti-ghrelin autoantibodies. In some embodiments, the agent is a natural autoantibody ligand (eg, a target antigen for an autoantibody). Thus, for example, the agent may be IFNy, IL-17, IL-6, or ghrelin. In some embodiments, the present invention relates to a method of treating a patient with a disease associated with cytokine dysregulation, such as an autoimmune disease. In some embodiments, the present invention relates to a method of treating a patient with a metabolic disorder such as obesity.

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

It will also be appreciated by those skilled in the art that the particles described herein

- 11 041139 are also suitable for capturing a wide range of targets whose biological activity may, for example, be undesirable. For example, particles can be designed to bind to components of viral capsids or envelopes to thereby isolate the virus from the subject's blood. In certain embodiments, the particles can be designed to bind and isolate toxins (eg, bacterial toxins, plant toxins, and zootoxins, such as one or more snake venom components) in a subject's bloodstream. In certain embodiments, the particles can be designed to bind to and isolate small molecule compounds (eg, psychoactive drugs or small molecule toxins) from a subject's bloodstream. In such embodiments, the implementation of the particles may be useful for removing toxins from the body, for example, after snake or insect bites. In certain embodiments, the particles can be used to treat, prevent, delay the onset of, or reduce the severity of anaphylactic shock in a subject (eg, by capturing an antigen that elicits an anaphylactic immune response).

In certain embodiments, the target is associated with a virus and is, for example, a structural viral protein (such as a viral capsid or viral envelope protein) that binds the agent. In such embodiments, the particles are useful as antiviral therapeutics, for example, in a subject infected with a virus, or at risk of being infected with a virus. The virus may be an enveloped or non-enveloped virus.

In certain embodiments, the soluble biomolecule is a small molecule or a macromolecule. In certain embodiments, the largest size of the soluble biomolecule is less than 600 nm (eg, less than 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 50, or 25 nm). For example, the molecular radius of a biomolecule may be from about 1 A to about 1 µm, for example, from about 1 A to about 100 nm, from about 1 A to about 20 nm, from about 1 nm to about 1 µm, from about 1 nm to about 100 nm or from about 1 nm to about 20 nm. The molecular weight of the biomolecule can range from about 3 amu to about 3 amu. up to about 10 ⁷ amu, for example from about 100 amu. up to about 10 ⁷ a.m.u., from about 3 a.m.u. to about 106 a.m.u., from about 3 a.m.u. up to about 105 amu, from about 100 amu up to approximately 106 a.m.u. or from about 400 a.m.u. up to approximately 106 a.m.u. The biomolecule may have a molecular weight from about 105 amu. up to about 10 ⁷ a.m.u.

As used herein, the terms specific binding, specific binding, selective binding, selective binding, and similar phrases refer to two molecules forming a complex that is relatively stable under physiological conditions. As a rule, binding is considered specific when the association constant (k _a ) exceeds 106 Μ-1-s' ¹ . Thus, the first member of a specific binding pair can specifically bind to the second member of a binding pair with k _{a of} at least (or more) 106 Μ-1-c' ¹ (eg, at least or more than 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ , 10 ¹⁴ or 10 ¹⁵ M' ¹ -c' ¹ or more). In certain embodiments, the selective interaction dissociation constant (kd) is less than or equal to 10'3 s' ¹ (eg, 8x10'4, 5x10'4, 2x10'4, 10' ⁴ or 10'5 s' ¹ ).

Specific binding does not refer to interaction, which is primarily due to non-specific electrostatic interaction or non-specific hydrophobic interaction, which may have a desired association constant. Thus, for example, nucleic acids that are negatively charged can bind to a cationic particle with a fairly convenient association constant independent of specific interaction, and such binding is not specific binding as defined herein. Similarly, a lipid can bind to a hydrophobic particle with a convenient association constant independent of specific interaction, and such binding is not specific binding as defined here.

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

Biomolecules can have an isoelectric point in the range of about 0 to about 14. Nucleic acids have an isoelectric point in the range of about 4 to about 7, and thus a biomolecule can have an isoelectric point of about 4 to about 7. A protein typically has an isoelectric point about 4 to about 10, and thus a biomolecule can have an isoelectric point of about 4 to about 10. However, unmodified peptides and proteins can have an isoelectric point of about 4 to about 10.

- 12 041139 tric point ranging from about 2.5 (based on aspartate; pI~2.8) to about 11 (based on arginine; pI~11), although proteins with isoelectric points outside this range are known. Accordingly, a biomolecule may have an isoelectric point ranging from about 2.5 to about 11. Secreted proteins and soluble extracellular portions of membrane proteins typically have a slight negative charge at physiological pH, and thus a biomolecule can have an isoelectric point of about 4 to about 7, such as 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 may have an isoelectric point of about 0 to about 2, about 1 to about 3, about 2 to 4, about 3 to about 5, about 4 to about 6, about 4 to approximately 6, from 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 up to about 14.

In some embodiments, K _D selective interaction is less than 10' ⁸ , 10' ⁹ , 10' ¹⁰ , 10' ¹¹ or 10' ¹² M. The equilibrium constant K _D is the ratio of the kinetic rate constants of the reactions - k _d /k _a . In certain embodiments, the K _D of the selective interaction is less than 1x10'9 M.

As used herein, the term interaction, when referring to an interaction between two molecules, refers to the physical contact (eg, bonding) of molecules with each other. Typically, such an interaction results in activity (providing a biological effect) of one or both of these molecules. Inhibition of such an interaction leads to disruption of the activity of one or more molecules involved in this interaction.

As used herein, the term inhibition and its grammatical equivalents refer to the reduction, restriction and/or blocking of a particular action, function or interaction. In one embodiment, this term refers to reducing the level of a product or parameter to a value (for example, to a background level of interaction between two members of a pair with a specific binding) 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 of the corresponding control value. A reduced level of a product or parameter does not necessarily, although it can, mean the absolute absence of a product or parameter. The invention does not require and is not limited to methods that completely eliminate the product or reset the parameter. Significant inhibition may be, for example, at least 50% (for example, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more) of the inhibition of the interaction of two biomolecules (for example, the first and second members of the binding pair) .

Methods for detecting an interaction or determining the affinity of one biomolecule for another are known in the art. For example, the binding of two biomolecules can be detected and/or quantified by a number of methods such as, but not limited to, biolayer interferometry (BLI), Western blot, dot blot, surface plasmon resonance (SPR) method, enzyme-linked immunosorbent assay (ELISA), AlphaScreen® or AlphaLISA® assays or mass spectrometry based methods.

In some embodiments, binding can be analyzed using any SPR analysis based on methods known in the art to characterize the kinetic parameters of the interaction of two biomolecules. Any SPR analysis tool available on the market can be used in the methods described herein, including but not limited to the following tools: BIAcore kit (Biacore AB; Uppsala, Sweden); lAsys kit (Affinity Sensors; Franklin, Massachusetts); IBIS system (Windsor Scientific Limited; Berks, UK), SPRCELLIA system (Nippon Laser and Electronics Lab; Hokkaido, Japan), and SPR Detector Spreeta (Texas Instruments; Dallas, Texas) (see, e.g., Mullett et al., Methods 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, biomolecular interactions between two biomolecules can be analyzed using the BLI on an Octet system (ForteBio Inc.). BLI is a labelless optical analytical method that determines the binding of a ligand immobilized on a biosensor tip and an analyte in solution by measuring changes in the protein layer density on the biosensor tip in real time.

In some embodiments, AlphaScreen assays (PerkinElmer) can be used to characterize the binding of two biomolecules. The acronym ALPHA stands for Homogeneous Approach Luminescence Enhancement Assay. AlphaScreen is a bead-based proximity assay that determines the binding of molecules bound to donor and acceptor beads by measuring the signal produced by energy transfer between the donor and acceptor beads. (see, for example,

- 13 041139

Eglen et al., Curr. Chem. Genomics, 1:2-10 (2008)).

In some embodiments, AlphaLISA® assays (PerkinElmer) can be used to characterize the binding of two biomolecules. AlphaLISA is a modified form of the AlphaScreen assay described above, including acceptor beads containing europium, which is used as an alternative to traditional ELISA assays (see, for example, Eglen et al., Curr. Chem. Genomics, 1:2-10 (2008) ).

A number of immunoassay methods can be used, including competitive and non-competitive immunoassays. The term immunoassay encompasses methods including, but not limited to, flow cytometry, FACS, enzyme immunoassays (EIAs) such as enzyme enhanced immunoassay (EMIT) method, enzyme-linked immunosorbent assay (ELISA), IgM antibody capture ELISA (MAC ELISA), and enzyme immunoassay. microparticle assay (MEIA), as well as capillary electrophoresis immunoassays (CEIA), radioimmunoassays (RIA), quantitative radioimmunoassay (IRMA), fluorescence polarization immunoassays (FPIA), and chemiluminescence (CL) assays. If desired, such immunoassays can be automated. Immunological assays can also be used in combination with laser-induced fluorescence.

Also suitable for use in the present invention are liposomal immunoassays, such as injectable liposomal immunoassays and liposomal immunosensor assays. In addition, nephelometric assays are suitable for use in the methods of the present invention, in which, for example, the formation of complexes of biomolecules results in increased light scattering, which translates into a function of maximum signal intensity versus marker concentration. In a preferred embodiment of the present invention, incubation products are detected by ELISA, RIA, immunofluorescence assay (FIA) or soluble particle immunoassay (SPIA).

In some embodiments, the binding of two biomolecules can be assessed by thermal denaturation methods, including differential scanning fluorometry (DSF) and differential static light scattering (DSLS).

In some embodiments, the binding of two biomolecules can be assessed by methods based on mass spectrometry, such as, as non-limiting examples, based on affinity selection coupled with mass spectrometry (AS-MS). It is a labelless method in which the protein and test compound are incubated, unbound molecules are washed away, and then protein-ligand complexes are analyzed by MS with ligand identification after the decomplexing step.

In some embodiments, the binding of two biomolecules can be quantified using, for example, labeled with a detectable label, such as radiolabeled (eg, ³² P, ³⁵ S, ¹⁴ C, or ³ H), fluorescently labeled (eg, FITC), or enzymatically labeled biomolecules. , by immunoassay, or by chromatographic detection.

In some embodiments, the present invention relates to the use of fluorescence polarization assays and fluorescence resonance energy transfer (FRET) assays in determining, directly or indirectly, the degree of interaction between two biomolecules.

Particles.

As used herein, the term particle refers to a small mass that may contain a certain amount of material such as alumina, metal (such as gold or platinum), glass, silicon dioxide, latex, plastic, agarose, polyacrylamide, methacrylate, or any other polymeric material, and which can be of any size and shape. In some embodiments, the particle or particles contain silicon (see, for example, International Patent Application Publications WO 2013/011764, WO 2013/029278 and WO 2014/151381 and US Patent Application Publication No. 2014/0271886, the disclosures of which are incorporated herein by reference in their entirety. ). In some embodiments, the particles may contain or consist of starch (see, for example, international patent application publication WO 2010/084088). In some embodiments, the particle or particles are composed of a nucleic acid (eg, natural or non-naturally occurring nucleic acid). Methods for producing such nucleic acids based on microscopic structures 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):200-203 (2010); and Endo et al., Curr. Protocol. Nucleic. Acid Chem. Chapter 12 (Unit 12.8) (2011).

In preferred embodiments, the particle is insoluble in aqueous solution (eg, the particle may be insoluble in water, serum, plasma, extracellular fluid and/or interstitial fluid). For example, a particle can be separated from an aqueous solution by centrifuging the solution containing the particle, for example, at speeds that are sufficient to separate cells from the cell suspension in the aqueous solution of the cell suspension. However, the particles may also remain in suspension in the aqueous solution, for example, with slight agitation or agitation of the plurality of particles in the aqueous solution, which is sufficient to suspend the particles in the solution. In some embodiments, the particle is not a hydrogel. In some embodiments, the particle does not contain a hydrogel. In some embodiments, the particle does not contain a polymer.

Preferably, the particle is large enough to bind to more than one biomolecule and inhibit the interaction of more than one bound biomolecule with its binding partner. For example, the particle may have a size from about 50 nm to about 10 microns. The particle may have a size of 1 to 5 µm, 1.2 to 4 µm, 1.5 to 4 µm, or 2 to 4 µm.

Particles with sizes less than 300 nm, such as less than 200 nm or less than 150 nm, are preferred for applications in which the particles are intended to enter and/or exit from the vasculature of a subject, since the particles can be administered by subcutaneous injection. However, larger particles are also well suited for subcutaneous injection for methods in which the particles are not intended to enter the vasculature. Particle sizes from about 1 µm to about 5 µm are preferred for applications in which the particles are intended to circulate in the subject's vascular system, such as following intravenous administration. Particles larger than 5 microns may be preferred for applications in which the particles are intended to be held in place at their implantation site, eg within or near a tumor; however, particles smaller than 5 µm may also be suitable for implantation. Particles of any size can be used for in vitro use.

Particle systems are also provided here. In some embodiments, the plurality of particles has a narrow or broad polydispersity. As used herein, polydispersity refers to a measure of the range of particle sizes in a particular group of particles. For example, a very polydisperse system may contain particles with an average particle size of, for example, 1 µm, but the individual particles will have sizes ranging from 0.1 to 4 µm. In some embodiments, a narrow polydispersity is preferred. This means that for a particular average particle size according to the invention, it is preferable that the individual particles in the entire set of particles differ in size from the average particle size by no more than ±20%, preferably no more than ±15%, and most preferably for this case no more than ±10%. More specifically, the average particle size of the entire plurality of particles is preferably from about 0.5 µm to about 2 µm, and more preferably from about 0.8 µm to about 1.5 µm in this case. Thus, if an average particle size of 1 µm is chosen, it is most preferred that the individual particles in the plurality of particles have a size in the range of about 0.8 µm to about 1.2 µm. In some embodiments, the average particle size across the plurality of particles is from about 0.3 µm to about 1 µm, for example, from about 0.4 µm to about 0.9, from about 0.5 µm to about 0.9 µm, from 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 average particle size across the plurality of particles is from about 1 µm to about 10 µm, for example, from about 1.1 µm to about 4.8 µm, from about 1.2 µm to about 4.6 µm, from 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, particle systems or a plurality of particles having a certain average particle size are provided herein. As used herein, the term average particle size is obtained by measuring the size of all individual particles and then dividing by the total number of particles. The determination of the average particle size is well known in the art. As a rule, the largest average particle size is not more than 4 microns. In some embodiments, the largest average particle size does not exceed 3.9 microns (for example, not more than 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1 , 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1 .8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, or 1 µm). In some embodiments, the largest average particle size does not exceed 2.5, 2, 1.5, or 1.25 microns. In some embodiments, the largest average particle size is at least 1 micron, but not more than 4 microns. In some embodiments, the largest average particle size is at least 1 micron, but not more than 2 microns. In some embodiments, the largest average particle size is at least 1 micron, but not more than 1.5 microns. In some embodiments, the largest average particle size is at least 0.5 microns (for example, at least 0.6, 0.7, 0.8, 0.9, 1.0 1.1, 1.2, 1, 3, 1.4 or 1.5 µm), but not more than 4.0 µm (for example, not more than 3.9 3.8, 3.7, 3.6, 3.5, 3.4, 3.3 , 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2 , 1.9, 1.8, 1.7 or 1.6 µm).

In some embodiments, the particles are nanoparticles. In some embodiments, the largest average particle size is not more than 900 nm (for example, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 450, 400, 350, 300, 250, 200, or 150 nm) . In some embodiments, the particles are shaped and sized for circulation in the blood or vascular system (eg, arteries, veins, and capillaries)

- 15 041139 subject (for example, a person). The schematic structure of exemplary particles is shown in FIG. 1-6.

In some embodiments, the largest particle size is from about 50 nm to about 5 µm, for example, from about 100 nm to about 4.5 µm, from about 200 nm to about 4 µm, from about 300 nm to about 3.5 µm, or from about 400 nm to about 3 microns. In some embodiments, the smallest particle size is at least about 300 nm, such as from about 300 nm to about 4 microns, or from about 400 nm to about 3 microns.

In some embodiments, the particles in the plurality of particles are polyhedral structures, such as cubic. In some embodiments, the particles in the plurality of particles are spherical. In some embodiments, any of the particles described herein may be porous. Such porous particles have an outer surface and inner surfaces of the pores of the particles. The agent can be immobilized, for example, on internal surfaces. In some embodiments, the cross-sectional size of the plurality of pores is at least 50 nm. In some embodiments, the cross-sectional size of the plurality of pores is at least 100 nm. Porous nanoparticles are described, for example, in US Patent Application Publications US 2014/0199352, US 2008/0277346 and US 2004/0105821, the contents of each of which are hereby incorporated by reference in their entirety. Spherical particles are described, for example, in US patents US 8778830 and US 8586096, the content of each of them is also included here by reference.

In some embodiments, the spherical particles may further comprise two intersecting protrusions on the spherical surface of the particle, where the largest size of each of the structures is not more than 4 microns (for example, not more than 3.9, 3.8, 3.7, 3.6, 3, 5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1 or 1 µm ) and where the protrusions are located and oriented (i) to inhibit the binding or activation of the cell surface receptor protein by the agent immobilized on the surface of the spherical particle, and/or (ii) when the soluble biomolecule is associated with the agent, to inhibit the interaction of the soluble biomolecule and the second member of the pair specific binding, where the soluble biomolecule is the first participant.

In some embodiments, the particles in the plurality of particles are toroidal. In such embodiments, the agent may be immobilized on the inner annular surface of the particle (eg, around the hole; see Fig. 2). In some embodiments, the particle diameter is not more than 4 microns (for example, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3 .0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8 , 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, or 1 µm). In some embodiments, the particle diameter is not greater than 900 nm (eg, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 200, or 150 nm).

In some embodiments, the particles described herein are dendritic. Such particles are described, for example, in Du et al., Small, 11(4):392-413 (2015); Siegwart, D.J. et al., Proceedings National Academy Sciences, USA, 108(32):12996 (2011); US patents US 5814272 and US 7932311; and US Patent Application Publication US 2004/0166166, the descriptions of each of which are incorporated herein by reference. As will be shown below, in some embodiments, the geometry of the dendritic particles is such that the agent immobilized on the inner surface of the particle has a reduced or significantly reduced ability to interact with the biomolecule on the cell surface, and/or the soluble biomolecule associated with the particle by the agent has reduced or significantly reduced ability to interact with the ligand recognized by it (the second member of the specific binding pair).

In some embodiments, the particles in the plurality of particles are polyhedral, such as octahedral or icosahedral (see, for example, FIG. 3), regular or irregular. The particles may have at least one protrusion on at least one of the vertices (see, for example, Fig. 3). The particles may have more than one (eg, 2, 3, 4, 5, 6, 7, or 8 or more) ridges on their vertices. Such protrusions may be positioned and/or oriented, for example (i) to inhibit the binding or activation of a cell surface receptor protein by an agent immobilized on the surface of a spherical particle, and/or (ii) when a soluble biomolecule is bound to the agent, then to inhibit the interaction of the soluble a biomolecule and a second member of a specific binding pair in which the soluble biomolecule is the first member.

The particle may have an empty space, referred to as a void or voids in this description. A void is a space in a particle that is filled with a liquid (e.g. a liquid that may contain biomolecules or a gas, e.g. when the particle is in a dry state) or remains an empty space (e.g. when the particle is in a vacuum, e.g. after lyophilization). The void volume of the particle may include, for example, the volume of the pores of the particle and/or the volume of the interior of a hollow core/shell particle, the lumen volume of a tube, torus, or ring.

In some embodiments, the particle is configured such that blood plasma can freely enter and/or exit the void space of the particle, such as when the particle is in the vasculature of a subject. In some embodiments, the particle is configured such that blood plasma can freely enter and/or exit the void space of the particle, such as when the particle is in

- 16 041139 in the subject's vasculature. In preferred embodiments, the particle is configured such that blood cells cannot enter the empty space of the particle. In some embodiments, the particle is configured such that platelets cannot enter the empty space of the particle. However, the particle may allow platelets to enter the empty space, for example, when the particle is configured for in vitro use, or when the particle is configured to bind viruses, bacteria, protists, fungi, or yeast cells, or other large targets such as size from about 100 nm to about 2 microns.

In some embodiments, the particle is configured such that extracellular fluid can freely enter and/or exit the void space of the particle. In some embodiments, the particle is configured such that interstitial fluid can freely enter and/or exit the void space of the particle. In some embodiments, the particle is configured such that cerebrospinal fluid can freely enter and/or exit the empty space of the particle.

The volume of void space in the particles is preferably large enough to accommodate more than one biomolecule, for example, the total void volume of the particle is preferably large enough to accommodate each biomolecule that is associated with the particle. However, the void may be less than the total volume of each bound biomolecule as long as the particle has the ability to inhibit the interaction between each bound biomolecule and the second members of the binding pairs that comprise each biomolecule. For example, a particle may only need to isolate a binding site to a biomolecule to inhibit interaction between the biomolecule and the second binding member of the pair, such a particle may contain a void volume that accommodates the binding site of each biomolecule, but allows other parts of one or more biomolecules to remain outside. empty space.

In some embodiments, the particle may contain from about 5% to about 95% empty space. The particle containing the protrusions may contain little or no void space, for example, due to the fact that the protrusions can inhibit the interaction between the bound biomolecule and the second member of the binding pair. The particle containing the tube may contain a large number of voids, for example, because the tube may contain a large internal volume relative to the wall thickness of the tube. However, particles with similar geometries may have different void volumes; for example, tubes having walls of the same thickness may have significant differences in void volume fraction depending on the tube diameter.

The particle may contain from 0% to about 40% empty space, from about 20% to about 60% empty space, from about 40% to about 80% empty space, or from about 60% to 100% empty space. The particle may contain from 0% to about 20% empty space, from about 10% to about 30% empty space, from about 20% to about 40% empty space, from about 30% to about 50% empty space, from about 40% to about 60% empty space, from about 50% to about 70% empty space, from about 60% to about 80% empty space, from about 70% to about 90% empty space, or from about 80% to 100% empty space. The particle may contain from 0% to about 10% empty space, from about 5% to about 15% empty space, from about 10% to about 20% empty space, from about 15% to about 25% empty space, from about 10% to about 20% empty space, from about 15% to about 25% empty space, from about 10% to about 20% empty space, from about 15% to about 25% empty space, from about 10% to about 20% empty space , from about 15% to about 25% empty space, from about 20% to about 30% empty space, from about 25% to about 35% empty space, from about 30% to about 40% empty space, from about 35% to approximately 45% empty space, approximately 40% to approximately 50% empty space, from about 45% to about 55% empty, about 50% to about 60% empty, about 55% to about 65% empty, about 60% to about 70% empty, about 65% to about 75 % empty space, about 70% to about 80% empty space, about 75% to about 85% empty space, about 80% to about 90% empty space, about 85% to about 95% empty space, or about 90% to 100% empty space.

The particle may be neutrally charged at physiological pH (eg pH 7.4). The particle may have a slight negative or slight positive charge at physiological pH. The surface of the particle (for example, the outer surface) may have a slight negative or slight positive charge under physiological

- 17 041139 pH value. In preferred embodiments, the surface of the particle (eg, outer surface) has a slight negative or neutral charge at physiological pH. The isoelectric point of the particle may be from about 5 to about 9, preferably from about 6 to about 8. Particles containing nucleic acid may have an isoelectric point from about 4 to about 7. In some embodiments, the particles have an isoelectric point of less than 7.4, t .e. one in which the particle has a negative charge at physiological pH. For example, the particle may have an isoelectric point of about 6.0 to about 7.4, such as about 6.4 to about 7.4. A particle that has a full negative charge at physiological pH is less likely to interact with eukaryotic cells (eg, mammalian cells), since eukaryotic cells typically have cell membranes with a full negative charge. Preferably, the particle does not have sufficient charge (and/or charge density) to participate in non-specific interactions with other charged molecules.

Particles containing pores.

In some embodiments of the invention, the porosity of the material used to obtain particles (for example, silicon) may be from about 40% to about 95%, for example from about 60% to about 80%. As used herein, porosity is a measure of the void spaces in a material, and it is the percentage of void volume relative to the total volume of the material. In some embodiments, the porosity of the carrier material is 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 other 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 some embodiments, the agent is distributed to a pore depth from the material surface 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 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. In some embodiments, the agent is distributed substantially evenly throughout the pores of the carrier material.

The agent can be added to the particle up to a depth, which is defined as a ratio to the total particle size. In some embodiments, the agent is dispersed within the particle to a depth of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 60%.

Methods for immobilizing an agent on porous particles are known and include methods such as immobilizing the agent to the first surface of the particle and immobilizing another molecule (e.g., a coating) on the second surface of the particle (see, for example, Cauda, V. et al., J Am Chem Soc., 131(32):11361-11370 (2009), and Guan, B. et al., Langmuir, 27(1):328-334 (2011), each of which is included in its entirety. completeness in this description by reference). In addition, such methods are generally applied to the manufacture of any of the particles described in this application.

To control the release of the biomolecule, the pore size can be preset according to the spatial characteristics of the agent and the target biomolecule. Typically, pore sizes that are too small prevent agent loading and/or binding of the biomolecule. For example, the average pore diameter of the material can be selected from larger pores, such as 15 to 40 nm for large molecular weight molecules, such as 200,000-500,000 amu, and smaller pores, such as 2 to 10 nm for molecules with a lower molecular weight, for example 10,000-500,000 a.m.u. For example, average pore sizes of about 6 nm in diameter may be suitable for molecules with a molecular weight of about 14,000 to 15,000 amu, such as about 14,700 amu. Average pore sizes of about 10 nm in diameter can be selected for molecules with a molecular weight of about 45,000 to 50,000 amu, such as about 48,000 amu. Average pore sizes of about 25-30 nm in diameter can be selected for molecules with a molecular weight of about 150,000 nm.

The pore size can be preset to be adapted to the molecular radii of the agent or biomolecule. For example, average pore sizes of about 25 nm to about 40 nm in diameter may be suitable for molecules with a largest molecular radius of about 6 nm to about 8 nm. Molecular radii can be calculated in any suitable way, for example, using the physical dimensions of the molecule based on x-ray crystallography data, or using the hydrodynamic radius, which is the size of the molecule in the dissolved state. Since the calculation for the dissolved state depends on the characteristics of the solution for which the calculation is made,

- 18 041139 then for some measurements it may be preferable to use the physical dimensions of the molecule based on X-ray crystallography data. As used herein, the largest molecular radius reflects half of the largest size of the therapeutic agent.

In some embodiments, the average pore diameter of the particles is chosen to limit the aggregation of molecules, such as proteins, in the pore. Prevention of aggregation of biomolecules, such as proteins, in the carrier material may be advantageous, since aggregation is believed to prevent the controlled release of molecules into the biological system. Thus, a pore that, for example, due to the correspondence of its size and the size of a biomolecule, allows only one biomolecule to enter the pore at a time is preferable to a pore that allows several biomolecules to enter and aggregate within the pore at once. In some embodiments, several biomolecules can enter the pore of the particle, but due to the depth of the pore, proteins distributed throughout the depth of the pore aggregate to a lesser extent.

Particles containing at least one tube.

In some embodiments of the invention, the particle contains at least one tube. In preferred embodiments, at least one tube has one open end or two open ends.

The term tube refers to a three-dimensional shape having a length along an axis (i.e., a one-dimensional axis in Cartesian space) and an internal cavity, lumen, void, or free volume along its entire length. In some embodiments, perpendicular sections of the tube along the axis are substantially the same shape and/or size. The term cross-section, when used with respect to a tube, refers to a two-dimensional section of a tube perpendicular to its axis. The large structure may contain a tube. So, for example, a syringe contains a tube, but the tube does not contain a syringe plunger. The particle or other article may contain more than one tube. Thus, for example, the syringe may include two tubes corresponding to the syringe needle and syringe barrel, or parallel containers in a dual syringe (eg, as in a syringe used for epoxy compositions).

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

The tube may have an internal diameter which is the average length of segments perpendicular to the axis of the tube, where each segment is defined by two points on the inner surface of the tube. The tube may have an internal width and an internal height, where the internal width of the tube corresponds to the longest segment, which is defined by two points on the outer surface of the tube, perpendicular to the axis of the tube, and the internal height of the tube is the segment, defined by two points on the inner surface of the tube, which is perpendicular as to the axis of the tube, and to the line segment that determines 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 ellipsoidal, such as a circle.

The cross section of the tube may be polygonal, such as a regular polygon. The cross section of the tube may be triangular, such as an equilateral triangle. The cross section of the tube may be quadrilateral, such as a tetragon, rectangle or square. The cross section of the tube may be pentagonal, such as a regular pentagon. The cross section of the tube may be hexagonal, such as a regular hexagon. The tube may be a triangular tube, a square tube, a pentagonal tube, a hexagonal tube, a heptagonal tube, or an octahedral tube.

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

- 19 041139 from about 500 nm to about 2 µm, or from about 500 nm to about µm.

The diameter, width, and/or height of the tube may be from about 5 nm to about 5 µm, for example, from about 5 nm to about 4 µm, from about 5 nm to about 3 µm, from about 5 nm to about 2 µm, from about 5 nm to approximately 1 µm, approximately 5 nm to approximately 900 nm, approximately 5 nm to approximately 800 nm, approximately 5 nm to approximately 700 nm, approximately 5 nm to approximately 600 nm, approximately 5 nm to approximately 500 nm , from about 5 nm to about 400 nm, from about 5 nm to about 300 nm, from about 5 nm to about 200 nm, or from about 5 nm to about 100 nm. The diameter, width, and/or height of the tube may be from about 50 nm to about 5 µm, for example, from about 50 nm to about 4 µm, from about 50 nm to about 3 µm, from about 50 nm to about 2 µm, from about 50 nm to approximately 1 µm, approximately 50 nm to approximately 900 nm, approximately 50 nm to approximately 800 nm, approximately 50 nm to approximately 700 nm, approximately 50 nm to approximately 600 nm, approximately 50 nm to approximately 500 nm , from about 50 nm to about 400 nm, from about 50 nm to about 300 nm, from about 50 nm to about 200 nm, or about 50 nm to about 100 nm.

The inner diameter, inner width and/or inner height of the tube are preferably large enough to accommodate both the agent and the biomolecule. The inner diameter, inner width and/or inner height of the tube are advantageously small enough to prevent entry of a cell into the interior of the tube (eg, a nucleated eukaryotic cell such as a human nucleated cell or a human diploid cell). The inner diameter, inner width, and/or inner height of the tube may be from about 5 nm to about 4 µm, for example, from about 5 nm to about 3 µm, from about 5 nm to about 2 µm, from about 5 nm to about 1 µm, from about 5 nm to about 900 nm, from about 5 nm to about 800 nm, from about 5 nm to about 700 nm, from about 5 nm to about 600 nm, from about 5 nm to about 500 nm, from about 5 nm to about 400 nm, about 5 nm to about 300 nm, about 5 nm to about 200 nm, or about 5 nm to about 100 nm. The inner diameter, inner width and/or inner height of the tube may be from about 20 nm to about 4 µm, for example, from about 20 nm to about 3 µm, from about 20 nm to about 2 µm, from about 20 nm to about 1 µm, from about 20 nm to about 900 nm, from about 20 nm to about 800 nm, from about 20 nm to about 700 nm, from about 20 nm to about 600 nm, from about 20 nm to about 500 nm, from about 20 nm to about 400 nm, about 20 nm to about 300 nm, about 20 nm to about 200 nm, or about 20 nm to about 100 nm. The inner diameter, inner width and/or inner height of the tube may be from about 40 nm to about 4 µm, for example, from about 40 nm to about 3 µm, from about 40 nm to about 2 µm, from about 40 nm to about 1 µm, from about 40 nm to about 900 nm, from about 40 nm to about 800 nm, from about 40 nm to about 700 nm, from about 40 nm to about 600 nm, from about 40 nm to about 500 nm, from about 40 nm to about 400 nm, about 40 nm to about 300 nm, about 40 nm to about 200 nm, or about 40 nm to about 100 nm.

In some preferred embodiments, the particle contains a plurality of tubes. Each tube in the plurality of tubes may be substantially parallel to another tube. In some embodiments, at least two tubes in a plurality of tubes are not parallel. In some embodiments, none of the tubes in the plurality of tubes are parallel. The tubes may be arranged in a configuration other than parallel to each other, distributing the tube openings over different surfaces of the particles, or allowing the particles to move randomly in the flow (eg, laminar flow or turbulent flow).

The plurality of tubes may be configured as a lattice or tube assembly.

The plurality of tubes may have a polyhedral configuration, such as a regular polyhedron.

The plurality of tubes may have a tetrahedral configuration, such as a regular tetrahedron. The plurality of tubes may be hexagonal, such as cubic, rectangular, or cube-shaped. The plurality of tubes may have an octahedral configuration, such as a regular octahedron. The plurality of tubes may have a dodecahedral configuration, such as a regular dodecahedron. The plurality of tubes may have an icosahedral configuration, such as a regular icosahedron. In some embodiments, each edge of the polyhedron corresponds to one tube. In some embodiments, not every edge of the polyhedron corresponds to one tube (for example, when the tubes are substantially parallel to each other).

The plurality of tubes may have a pyramidal configuration, such as a triangular pyramid, a rhombic pyramid, a rectangular pyramid, a square pyramid, a pentagonal pyramid, a hexagonal pyramid, a heptagonal pyramid, or an octagonal pyramid. The plurality of tubes may be in a regular pyramid or slanted pyramid configuration. In some embodiments, each edge of the pyramid corresponds to one tube. In some embodiments, not every edge of the pyramid corresponds to one tube (eg, where the tubes are substantially parallel to each other).

The plurality of tubes may have a prism configuration, such as 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 configured as a straight prism, an oblique prism, or a truncated prism. In some embodiments, each edge of the prism corresponds to one tube. In some embodiments, not every edge is defined by a single tube. In some embodiments, not every edge of the prism corresponds to one tube (for example, when the tubes are substantially parallel to each other).

The plurality of tubes may have a structure configuration that has a length, width, and height where no dimension is more than 5 times any other dimension. For example, a plurality of tubes may be configured where no dimension is greater than 4 times any other dimension, or greater than 3 times any other dimension. Such configurations are favorable, for example, for intravenous administration of particles, since oblong particles cannot move well in the patient's bloodstream.

The plurality of tubes may have a structure configuration having a length and a diameter where the length of the configuration is no more than 5 times its diameter. For a plurality of tubes, a configuration may be used in which the length of the configuration is not more than 4 times its diameter, or the length of the configuration is not more than 3 times its diameter. Such configurations are favorable, for example, for intravenous administration of particles, since oblong particles cannot move well in the patient's bloodstream.

The particle may contain from 1 to 500 tubes, for example from 1 to 100 tubes. The particle can contain 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, such as 1 to 100 tubes. The plurality of pipes 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.

All tubes in a plurality of tubes may have the same length, or tubes in a plurality of tubes may have different lengths. The average tube length may be from about 5 nm to about 5 µm, for example, from about 5 nm to about 4 µm, from about 5 nm to about 3 µm, from about 5 nm to about 2 µm, or from about 5 nm to about 1 µm. . The average tube length may be from about 50 nm to about 5 µm, for example, from about 50 nm to about 4 µm, from about 50 nm to about 3 µm, from about 50 nm to about 2 µm, or from about 50 nm to about 1 µm. . The average tube length may be from about 100 nm to about 5 µm, for example, from about 100 nm to about 4 µm, from about 100 nm to about 3 µm, from about 100 nm to about 2 µm, or from about 100 nm to about 1 µm. . The average tube length may be from about 300 nm to about 5 µm, for example, from about 300 nm to about 4 µm, from about 300 nm to about 3 µm, from about 300 nm to about 2 µm, or from about 300 nm to about 1 µm. . The average tube length may be from about 500 nm to about 5 µm, for example, from about 500 nm to about 4 µm, from about 500 nm to about 3 µm, from about 500 nm to about 2 µm, or from about 500 nm to about 1 µm. .

All tubes in a plurality of tubes may have the same diameter, width and/or height, or tubes in a plurality of tubes may have a different diameter, width and/or height. The average diameter, width, and/or height of the tube may be from about 5 nm to about 5 µm, for example, from about 5 nm to about 4 µm, from about 5 nm to about 3 µm, from about 5 nm to about 2 µm, from about 5 nm to about 1 µm, about 5 nm to about 900 nm, about 5 nm to about 800 nm, about 5 nm to about 700 nm, about 5 nm to about 600 nm, about 5 nm to about 500 nm, from about 5 nm to about

- 21 041139

400 nm, about 5 nm to about 300 nm, about 5 nm to about 200 nm, or about 5 nm to about 100 nm. The average diameter, width, and/or height of the tube may be from about 50 nm to about 5 µm, for example, from about 50 nm to about 4 µm, from about 50 nm to about 3 µm, from about 50 nm to about 2 µm, from about 50 nm to about 1 µm, about 50 nm to about 900 nm, about 50 nm to about 800 nm, about 50 nm to about 700 nm, about 50 nm to about 600 nm, about 50 nm to about 500 nm, from about 50 nm to about 400 nm, from about 50 nm to about 300 nm, from about 50 nm to about 200 nm, or from about 50 nm to about 100 nm.

All tubes in a plurality of tubes may have the same inner diameter, inner width and/or inner height, or tubes in a plurality of tubes may have different inner diameters, widths and/or heights. The average inner diameter, inner width, and/or inner height of the tube may be from about 5 nm to about 4 µm, for example, from about 5 nm to about 3 µm, from about 5 nm to about 2 µm, from about 5 nm to about 1 µm, from about 5 nm to about 900 nm, from about 5 nm to about 800 nm, from about 5 nm to about 700 nm, from about 5 nm to about 600 nm, from about 5 nm to about 500 nm, from about 5 nm to about 400 nm, from about 5 nm to about 300 nm, from about 5 nm to about 200 nm, or about 5 nm to about 100 nm. The average inner diameter, inner width, and/or inner height of the tube may be from about 20 nm to about 4 µm, for example, from about 20 nm to about 3 µm, from about 20 nm to about 2 µm, from about 20 nm to about 1 µm, from about 20 nm to about 900 nm, from about 20 nm to about 800 nm, from about 20 nm to about 700 nm, from about 20 nm to about 600 nm, from about 20 nm to about 500 nm, from about 20 nm to about 400 nm, from about 20 nm to about 300 nm, from about 20 nm to about 200 nm, or about 20 nm to about 100 nm. The average inner diameter, inner width, and/or inner height of the tube may be from about 40 nm to about 4 µm, for example, from about 40 nm to about 3 µm, from about 40 nm to about 2 µm, from about 40 nm to about 1 µm, approximately 40 nm to approximately 900 nm, approximately 40 nm to approximately 800 nm, approximately 40 nm to approximately 700 nm, approximately 40 nm to approximately 600 nm, approximately 40 nm to approximately 500 nm, approximately 40 nm to about 400 nm, from about 40 nm to about 300 nm, from about 40 nm to about 200 nm, or from about 40 nm to about 100 nm.

The tube may include, for example, a polymer. The polymer may be a natural polymer or a synthetic polymer. The polymer may be, for example, a nucleic acid (eg DNA) or a protein.

Particles containing scaffold structures of DNA.

In some embodiments, the particle contains DNA scaffolds, for example, the particle may contain an origami DNA scaffold (see, for example, US Pat. PCT Application WO 2014/170898, each of which is incorporated herein by reference).

The particle may contain a DNA framework, and the DNA framework may comprise at least one tube or a plurality of tubes, as described herein. For example, a DNA scaffold may comprise at least one substantially hexagonal tube (see, for example, US Patent Application Publication US 2013/0224859, incorporated herein by reference).

The DNA framework may comprise honeycomb or lattice structures, such as a hexagonal lattice or square lattice (see, for example, US Pat. No. 8,554,489, incorporated herein by reference).

In some embodiments, the particle contains a DNA framework and the DNA framework does not contain tubes. For example, the DNA scaffold structure may have a three-dimensional shape, such as a polyhedral shape, and the agent may be immobilized on the inner surface of this structure.

The DNA framework structure may have a polyhedral structure, such as a regular polyhedron. The framework structure of DNA may have a tetrahedral structure, such as a regular tetrahedron. The scaffold structure of DNA may have a hexagonal structure, such as a cubic, rectangular, or cube-like structure. The framework structure of DNA may have an octahedral structure, such as a regular octahedron. The framework structure of DNA may have a dodecahedral structure, such as a regular dodecahedron. The framework structure of DNA can have a structure in

- 22 041139 in the form of an icosahedron, for example in the form of a regular icosahedron.

The DNA scaffold may be pyramidal, such as a triangular pyramid, a rhombic pyramid, a rectangular pyramid, a square pyramid, a pentagonal pyramid, a hexagonal pyramid, a heptagonal pyramid, or an octagonal pyramid. The framework structure of DNA can have a structure in the form of a regular pyramid or an oblique pyramid.

The DNA scaffold can be a prism structure, such as a triangular prism, a rectangular prism, a square prism, a pentagonal prism, a hexagonal prism, a heptagonal prism, or an octagonal prism. The scaffold structure of DNA can be a regular prism, an oblique prism, or a truncated prism.

The DNA framework can have a structure that has a length, width and height where no dimension is more than 5 times any other dimension. For example, no dimension is more than 4 times any other dimension, or more than 3 times any other dimension. Such configurations are favorable, for example, for intravenous administration of particles, since oblong particles cannot move well in the patient's bloodstream.

In some embodiments of the invention, the agent is immobilized in the scaffold structure of DNA. In some embodiments, the agent binds to a nucleic acid containing a nucleotide sequence that is complementary to the backbone DNA nucleotide sequence, i. the nucleotide sequence has at least about 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence having a reverse complementary nucleotide sequence to the backbone DNA. Thus, the agent can be immobilized on the surface of the particle by hybridization of the nucleic acid to scaffold DNA.

Particles containing protection.

The particle may include a subparticle in the form of a core with protection, where the protection, for example, inhibits biomolecules attached to the core subparticle from interacting with molecules on the surface of the cell. The security may comprise a plurality of security components. The core subparticle may contain silicon dioxide. For example, the core subparticle may have a silicon oxide surface. The core subparticle may contain gold, silicon, or a polymer. Thus, for example, a core subparticle may have a gold, silicon, or polymer surface.

A particle containing a core subparticle and an outer protection containing a plurality of protection components attached to the core of the subparticle may include a core subparticle having a silicon oxide surface, where such a subparticle consists of solid silicon dioxide, porous silicon dioxide, or is a nanoparticle with a shell of silicon dioxide , the inner part of which does not contain silicon. The core subparticle may contain a core material that is not silicon oxide, such as silicon or gold coated with silicon dioxide. The security components may be in the form of security subparticles smaller than core subparticles such as nanospheres and may include silicon oxide or other material such as gold or a polymer. The surface material of the core subparticle and the protection components can be selected from a variety of materials to allow them to enter into various chemical interactions that can be used to bind additional components or active substances to surfaces. The core subparticles may have a reactive group on their surface, and the protection components may contain a functional group capable of interacting with the reactive group to form a covalent bond between the surface of the core subparticles and the surface of the protection components, or with the surface of the protection subparticles, as described herein.

The agent may be present on the surface of the core subparticle, but to a lesser extent or preferably not always on the surface of the protection components. For example, an agent can be attached to the surface of a silica core subparticle via a bond (e.g., ionic, covalent bond, or electrostatic interaction) resulting in the formation of predominantly (or exclusively) silica core subparticles, but no protection subparticles, e.g. , as in the case of particles with a gold surface instead of a silicon oxide surface.

In some embodiments, such a particle may contain a silicon oxide core, for example, a substantially spherical silicon oxide core and protection, including a plurality of gold nanoparticles on the surface of the silicon oxide core, while the gold nanoparticles have a cross section smaller than the size of the cross section of the core, such as core diameter. These gold nanoparticles may be substantially spherical. The core of the subparticle may be solid and non-porous, or may have a porous surface. Obtaining a core from silicon oxide and obtaining cores with gold nanoparticles on their surface can be performed by the methods described in US patent US 6344272 and in publications Sadtler, Wei, Chem. Comm. 1604-5 (2002); and Meuhlig et al., ACS Nano, 5(8):6586-6592 (2011) (each of which is incorporated herein by reference in its entirety). For example, gold nanoparticles can be adsorbed onto an amine-coated silica core by electrostatic attraction, or they can be bonded to a silica core when the core contains thiol groups conjugated to the silica surface, which in turn bind - 23 041139 are called with the surface of gold nanoparticles.

A linker group can be used between the core silica of the silica-containing subparticle and the thiol groups to attach the protection components to the core subparticle. The linker may have a length chosen to establish a maximum distance between the silica surface and the thiol groups (or when the thiol is attached to the gold surface, between the silica surface and the gold surface). Thus, the distance between the surface of the silica subparticle and the gold subparticle can be varied within a range of some values, potentially allowing for more bonds (for example, due to the fact that more gold subparticles can be packed at a greater distance from the core of the gold subparticle). silica) and/or enhance bonding between the silica and gold subparticles (e.g., because at shorter distances, more bridges from the surface of the silica subparticle will be able to interact with the surface of the gold subparticles, enhancing association). The linker may contain an alkylene chain, the length of which may be chosen to vary the distance between the surface of the subparticle core and the protection of the subparticle.

The core subparticles may have a cross-sectional size, such as the diameter of a spherical or cylindrical subparticle, from 50 nm to 4 µm, such as 50 to 200 nm, 100 to 500 nm, 200 nm to 1 µm, or 500 nm to 4 µm.

The particles can be assembled from core subparticles with different diameters and different protection subparticle diameters. The available surface area of the core subparticle for capturing the biomolecule may depend on the diameter of the protective subparticles and the effective height of their location above the surface of the core of the subparticle, which is necessary for effective binding of the target/agent complex to the surface, including the surface above any linker, between the surface and the capture agent.

The amount of agent that can be associated with a core subparticle can be calculated based on the surface area of the subparticle. Similarly, the number of target biomolecules that can be associated with core subparticles can be calculated in the same way. Such calculations can be verified, for example, by studying protein binding under in vitro conditions, and they can be used to predict the dose of particles that may be needed to bind a selected number of target biomolecules of interest (or in some embodiments, the effective dose of particles or compositions, containing these particles, to remove or reduce the concentration of target biomolecules from a system, such as an in vitro system, or from a patient's bloodstream in the treatment of a disease).

The particle may have an available surface area for target capture from 0.01 to 50 µm ² , for example, from 0.01 to 0.1 µm ² , from 0.05 to 0.5 µm ² , from 0.1 to 1.0 µm ² , 0.5 to 5 µm ² , 1.0 to 10 µm ² , 5 to 25 µm ² , or 10 to 50 µm ² . For the chosen agent loading per unit core subparticle surface area, the maximum dose of particles can be set to be suitable to capture the required number of target biomolecules based on core subparticle diameters and protection.

The cross-sectional size, such as the diameter, of the protective subparticle may be a multiple of the cross-sectional size, such as the diameter, of the core of the particle. The multiplicity may be, for example, from 0.01 to 0.5, for example from 0.02 to 0.2, for example from 0.05 to 0.1.

For effective access of the target biomolecule to the agent, the target must be able to diffuse between the protection components in order to reach the agent on the surface of the core subparticle. Thus, for example, targets less than 100 kDa (eg, TNF-R1/2) are sized to easily diffuse between protective spheres of 40 nm or more in diameter. For smaller shield spheres, the effective pore length between spheres is small, and thus shield spheres smaller than 40 nm are unlikely to interfere with diffusion.

Particles containing subparticles.

In some embodiments, the particle may comprise a core subparticle and a plurality of protective subparticles. The particle may contain a protection, and this protection may include a plurality of protective subparticles. The agent can be immobilized on the surface of the core subparticle, for example, when the surface of the core subparticle is the inner surface. A plurality of protective subparticles may be configured to allow inhibition of the interaction of the biomolecule with the second member of the specific binding group, for example, when the biomolecule is bound to the particle. A plurality of protective subparticles may be configured to inhibit interaction between the biomolecule and a cell, such as a mammalian cell, for example, when the biomolecule is associated with the particle.

Protective subparticles may define the outer surface. In preferred embodiments of the present invention, the agent is not immobilized on the surface of the protective subparticles.

The core subparticle is preferably large enough to bind to more than one molecule of the agent. For example, the core subparticle may have a size of from about 20 nm to about 4 microns, such as from about 50 nm to about 2 microns. The core subparticle may have a size from about 100 nm to about 1000 nm, from about 100 nm to at

- 24 041139 about 800 nm, from about 100 nm to about 600 nm, from about 100 nm to about 400 nm, from about 100 nm to about 200 nm, from about 200 nm to about 1000 nm, from about 200 nm to about 800 nm, from about 200 nm to about 600 nm, from about 200 nm to about 400 nm, from about 400 nm to about 1000 nm, from about 400 nm to about 800 nm, from about 400 nm to about 600 nm, from about 600 nm to about 1000 nm or from about 600 nm to about 800 nm. The core subparticle may be about 100 nm to about 4 µm, about 100 nm to about 3 µm, about 100 nm to about 2 µm, about 200 nm to about 4 µm, about 200 nm to about 3 µm, from about 200 nm to about 2 µm, from about 400 nm to about 4 µm, from about 400 nm to about 3 µm, from about 400 nm to about 2 µm, from about 600 nm to about 4 µm, from about 600 nm to about 3 µm, about 600 nm to about 2 µm, about 800 nm to about 4 µm, about 800 nm to about 3 µm, or about 800 nm to about 2 µm.

The core subparticle may contain metal, gold, alumina, silicon oxide, glass, silicon, starch, agarose, latex, plastic, polyacrylamide, methacrylate, polymer, or nucleic acid. In some embodiments, the core subparticle contains silicon, such as porous silicon.

The core subparticle may be of any shape (eg, cubic, pyramidal, conical, spherical, cylindrical, disk-shaped, tetrahedral, hexahedral, octahedral, dodecahedral, or icosahedral) or the subparticle may lack a specific shape.

A particle may contain one core subparticle. Thus, for example, the core subparticle may be the one described in US Pat.

A particle may include 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, for example, Fig. 4 and 5). In some embodiments, the particle contains a plurality of core subparticles, each of which is predominantly substantially spherical. A particle containing a plurality of spherical core subparticles has voids, thereby allowing soluble biomolecules to diffuse through the interior of the particle. However, core subparticles having various other shapes may also have voids. A particle containing a plurality of core subparticles may contain core subparticles of various shapes and sizes.

The particle may contain 1 to about 106 core subparticles, 1 to about 105 core subparticles, 1 to about 10 ⁴ core subparticles, 1 to about 1000 core subparticles, 1 to about 100 core subparticles, or 1 to about 10 core subparticles kernels. The particle may contain 2 to about 106 core subparticles, 2 to about 105 core subparticles, 2 to about 10 ⁴ core subparticles, 2 to about 1000 core subparticles, 2 to about 100 core subparticles, or 2 to about 10 core subparticles kernels. The particle may contain from about 10 to about 106 core subparticles, from about 10 to about 105 core subparticles, from about 10 to about 10 ⁴ core subparticles, from about 10 to about 1000 core subparticles, or from about 10 to about 100 core subparticles.

The core subparticles in a plurality of core subparticles can be connected using a linker (eg, covalent linkers). For example, each core subparticle in a plurality of core subparticles can be connected to other core subparticles using a linker.

The core subparticle may contain pores, ie. the core subparticle may be porous.

The protective subparticle may contain metal, gold, alumina, silicon oxide, glass, silicon, starch, agarose, latex, plastic, polyacrylamide, methacrylate, polymer, or nucleic acid. Some protective subparticles are predominantly linked to the core subparticles with a linker, such as a covalent linker. However, the protective subparticles can be linked to one or more core subparticles without any covalent linker. Protective subparticles can be linked to other protective subparticles using linkers, such as covalent linkers. For example, the protective subparticles may form a bulk structure or network around the core subparticles, thereby keeping the core subparticles within the particle.

In some embodiments of the invention, each protective subparticle of a plurality of protective subparticles is linked to a core subparticle by a linker, such as a covalent linker. In some embodiments, some of the multiple security subparticles are associated with the core subparticle, and each of the multiple subparticles that are not directly associated with the core subparticle is associated with the security subparticle, i. such that each protective subparticle of the plurality of subparticles is directly or indirectly associated with a core subparticle. Thus, the particle may contain one layer of protective subparticles (for example, when essentially all of the protective subparticles are directly associated with one or more core subparticles), or

- 25 041139 particles may contain more than one layer of protective subparticles (for example, when a substantial part of the protective subparticles is indirectly associated with one or more core subparticles through direct bonds with other protective subparticles).

In some embodiments of the invention, the particle contains the first layer of protective subparticles containing the first material, and the second layer of protective subparticles contains the second material. For example, the first material may contain silicon oxide or silicon, and the second material may contain gold.

The particle can be assembled, for example, by associating subparticles of the first subparticle layer with one or more core subparticles, and then by associating subparticles of the second subparticle layer with the first subparticle layer. The second layer subparticles may include a similar surface as the core subparticles, thus allowing the first layer subparticles to bond to both the core subparticle(s) and the second layer subparticles using similar chemistry.

The particle can be collected using the layer-by-layer method. For example, a particle may be formed first by linking a plurality of core subparticles. The plurality of core subparticles may be substantially homogeneous, such that the molecule can crosslink all of the core subparticles, for example. The plurality of sub-particles may comprise at least two types of sub-particles, for example, with different shapes, sizes and/or surfaces, which provide the desired function, in particular the presence of voids within the particle. After linking the plurality of core subparticles, the plurality of protective subparticles may be associated with the plurality of core subparticles. After associating a plurality of protective sub-particles with core sub-particles, a second plurality of protective sub-particles may be associated with a plurality of protective sub-particles. However, particles can be assembled in other ways, and many different layer-by-layer strategies can be used depending on the desired properties of the particle and the desired chemicals used to bind the subparticles.

Subparticle crosslinking methods are known and include subparticle crosslinking methods that contain antibodies for in vivo use (see, for example, Cheng, K. et al., ACS Appl Mater Interfaces, 2(9):2489-2495 ( 2010), the contents of which are incorporated herein by reference in their entirety). Such methods can be adapted to obtain the particles described here, for example, by simply changing the relative sizes of the subparticles.

The protective subparticle may have a size of from about 10 nm to about 4 microns, such as from about 10 nm to about 1 micron, or from about 20 nm to about 500 nm. The protective subparticle may have a size of from about 10 nm to about 200 nm, from about 10 nm to about 100 nm, from about 10 nm to about 80 nm, from about 10 nm to about 60 nm, from about 10 nm to about 40 nm, from about 10 nm to about 20 nm, from about 20 nm to about 200 nm, from about 20 nm to about 100 nm, from about 20 nm to about 80 nm, from about 20 nm to about 60 nm, from about 20 nm to approximately 40 nm, approximately 30 nm to approximately 200 nm, approximately 40 nm to approximately 100 nm, approximately 40 nm to approximately 80 nm, approximately 40 nm to approximately 60 nm, approximately 60 nm to approximately 200 nm, from about 60 nm to about 100 nm, or from about 60 nm to about 80 nm. The protective subparticle may have a size of from about 100 nm to about 1000 nm, from about 100 nm to about 800 nm, from about 100 nm to about 600 nm, from about 100 nm to about 400 nm, from about 100 nm to about 200 nm, from about 200 nm to about 1000 nm, from about 200 nm to about 800 nm, from about 200 nm to about 600 nm, from about 200 nm to about 400 nm, from about 400 nm to about 1000 nm, from about 400 nm to about 800 nm, about 400 nm to about 600 nm, about 600 nm to about 1000 nm, or about 600 nm to about 800 nm. The protective subparticle may have a size of from about 100 nm to about 4 µm, from about 100 nm to about 3 µm, from about 100 nm to about 2 µm, from about 200 nm to about 4 µm, from about 200 nm to about 3 µm, from about 200 nm to about 2 µm, from about 400 nm to about 4 µm, from about 400 nm to about 3 µm, from about 400 nm to about 2 µm, from about 600 nm to about 4 µm, from about 600 nm to about 3 µm, about 600 nm to about 2 µm, about 800 nm to about 4 µm, about 800 nm to about 3 µm, or about 800 nm to about 2 µm.

The particle may contain from 1 to about 106 protective subparticles, from about 4 to about 106 protective subparticles, from about 10 to about 106 protective subparticles, from 1 to about 105 protective subparticles, from about 4 to about 105 protective subparticles, from about 10 to about 105 protective subparticles, from 1 to about 10 ⁴ protective subparticles, from about 4 to about 10 ⁴ protective subparticles, from about 10 to about 10 ⁴ protective subparticles, from 1 to about 1000 protective subparticles, from about 4 to about 1000 protective subparticles, from about 10 to about 1000 protective subparticles, from 1 to about 100 protective subparticles, from about 4 to about 100 protective subparticles, or from about 10 to about

100 protective sub-particles.

The core subparticle and the protective subparticle may or may not have similar or identical shapes, sizes and compositions. However, the core subparticle differs from the protective subparticle because (1) the agent can be immobilized on the core subparticle, while the agent is preferably not immobilized on the protective subparticle, and (2) the core subparticles are predominantly located in the interior of the particle, while the protective subparticles can present on the outer surface of the particle.

Essentially 2-dimensional particles.

The particle may have a 2-dimensional shape. For example, a particle can be a circle, ring, cross, herringbone, ellipse, triangle, square, pentagon, hexagon, heptagon, octagon, or star. The particle may be a star, and the star may be a concave hexagon, a concave octagon, a concave decagon, or a concave dodecagon. The shape may or may not be correct. Examples of substantially 2-dimensional particles are shown in FIG. 6.

In some embodiments, the particle comprises a first side, a second side, and an edge. The first and second sides may be substantially the same shape. The first side and the second side may have a length and a width. The edge may have a height that is the distance between the first side and the second side. The width and length may be at least 4 times the height, such as 4 to 1000 times the height, 6 to 100 times the height, 8 to 75 times the height, or 10 to 50 times the height. The width and/or length can be from 0.2 to about 20 times the height.

The edge may contain one or more concave or reentrant portions. The agent may be associated with concave or reentrant portions of the edge. The reentrant part is a part in which the particle perimeter contains two adjacent perimeter parts with an external angle between them that is greater than 270°, such as both sides of the rays of a star. Thus, the capture agent can be protected from contact with the cell membrane while in contact with 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 have a concave or reentrant portion.

In some embodiments, the particle is in the form of a substantially flat star, such as with reentrant regions between the arms. A star can have 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more rays. The particle may have regular sides or irregularly shaped sides.

In some embodiments, the particle is cross-shaped or herringbone-shaped, such as a stem with branches on each side of the stem with reentrant regions between the arms. Branches in a cross or herringbone may additionally contain side ledges.

The reentrant ribs between the arms of the star or branches of the cross or herringbone preferably extend a distance from the line connecting the arms so that the cell membrane cannot deform between the arms and come into contact with the edges. For example, the number of beams and the angles between them may determine the depth of the reentrant edge regions between the beams.

Particles suitable for use in the present invention can be obtained by nanotechnology, for example, using nanoprinting or nanoforming. For example, particles can be obtained using PRINT technology (Particle Replication In Non-wetting Templates; Replication of particles in non-wetting matrices): see, for example, international patent application WO 2007/024323 and Perry, J.L. et al., Acc. Chem. Res., 44(10):990-998 (2011), which are incorporated herein by reference. Particles can be obtained using photolithography using known methods.

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

In some embodiments of the invention, it is desirable when the surface area of the particles is in the range from 0.2 to 25 μm ² . Protected edge areas of the particles can be obtained by nanoforming in the desired range.

Agent.

In some embodiments, the agent immobilized on the surface of the particle is a small molecule compound, a macrocyclic compound, a polypeptide, a peptidomimetic compound, an aptamer, a nucleic acid, or a nucleic acid analog. As used herein, the term low molecular weight compound is intended to refer to an agent with a molecular weight of less than about 6 kDa, and most preferably less than about 2.5 kDa. Many pharmaceutical companies have extensive libraries of chemical and/or biological mixtures containing a variety of small molecule compounds, often fungal, bacterial or algal extracts, which can be screened using any of the assays described here. In particular, the description provides for the use of small chemical libraries, peptide libraries, or collections of natural products. Tan et al. describes a library of over two million synthetic compounds that is compatible with miniaturized cell assays (J. Am. Chem. Soc., 120:8565-8566 (1998)).

Peptidomimetics can be compounds in which at least a portion of the parent polypeptide is modified and the three-dimensional structure of the peptidomimetic remains substantially the same as that of the parent polypeptide. Peptidomimetics may be analogs of the parent polypeptide in properties, and they may be polypeptides containing one or more substitutions or other modifications in the parent polypeptide sequence. Alternatively, at least a portion of the sequence of the parent polypeptide may be replaced with a non-peptide structure in such a manner that the substantially three-dimensional structure of the parent polypeptide is retained. In other words, one, two or three amino acid residues in the sequence of the original polypeptide can be replaced by a non-peptide structure. In addition, a non-peptide structure may, but need not, replace other portions of the parent polypeptide. Peptidomimetics (peptide and non-peptide analogues) may have improved properties (eg, reduced proteolysis, increased duration of action, or increased bioavailability). In general, peptidomimetics have improved oral availability, making them particularly suitable for the treatment of humans or animals. It should be noted that peptidomimetics may or may not have a similar two-dimensional chemical structure, but have similar three-dimensional structural properties and geometry. Each peptidomimetic may additionally contain one or more unique additional binding elements.

Aptamers are short oligonucleotide sequences that can be used to recognize and specifically bind almost any molecule, including cell surface proteins. The Systematic Evolution of Ligands at Exponential Enrichment (SELEX) method is effective and can be used to rapidly identify such aptamers. Aptamers can be generated for a wide range of proteins important for therapy and diagnostics, such as growth factors and cell surface antigens. These oligonucleotides bind their targets with antibody-like affinities and specificities (see, for example, Ulrich Handb. Exp. Pharmacol., 173:305-326 (2006)).

The agent may be an antibody or an antigen-binding fragment thereof (ie, part of an antibody), wherein the antibody or antigen-binding fragment thereof specifically binds to a target (eg, a soluble biomolecule). The agent may comprise an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof specifically binds to a target (eg, a soluble biomolecule). The term antibody refers to whole antibodies, including antibodies of various isotypes such as IgM, IgG, IgA, IgD and IgE antibodies. The term antibody includes a polyclonal antibody, a monoclonal antibody, and a chimeric or chimeric antibody, a humanized antibody, a primatized antibody, a deimmunized antibody, and an entirely human antibody. The antibody can be obtained from or isolated from any of a variety of species, such as mammals such as humans, non-human primates (such as orangutan, baboon, or chimpanzee), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats and mice. The antibody may be a purified or recombinant antibody.

The term antibody fragment, biomolecule-binding fragment, antigen-binding antibody fragment, and similar terms refer to an antibody fragment that retains the ability to bind to a target antigen. Such fragments include, for example, a single chain antibody, a single chain Fv (scFv) fragment, an Fd fragment, a Fab fragment, a Fab' fragment, or an F(ab') ₂ fragment. An scFv fragment is a single polypeptide chain that contains the heavy and light chain variable regions of the antibody from which the scFv is derived. In addition, intraantibodies, minibodies, tribodies, and diabodies are also included in the definition of antibodies and suitable for use in the methods described herein (see, for example, Todorovska et al., J. Immunol. Methods, 248(1):47 -66 (2001), Hudson and Kortt J., Immunol Methods, 231(1):177-189 (1999), Poljak, Structure, 2(12):1121-1123 (1994), Rondon and Marasco, Annual Review of Microbiology, 51:257-283 (1997), the contents of which are incorporated herein by reference in their entirety). Also, the term antibody includes bispecific antibodies (including DVD-Ig antibodies).

Bispecific antibodies are monoclonal antibodies, preferably human or humanized, that specifically bind to at least two different antigens.

As used herein, the term antibody also encompasses, for example, single domain antibodies such as camelized single domain antibodies. See, for example, Muyldermans et al., Trends Biochem. Sci. 26:230-235 (2001); Nuttall et al., Curr. Pharm. Biotech. 1:253-263 (2000); Reichmann et al., J. Immunol. Meth., 231:25-38 (1999); PCT Application Publications WO 94/04678 and WO 94/25591 and US Patents US 6005079, US 6015695 and US 7794981, the contents of which are in full

- 28 041139 incorporated herein by reference. In some embodiments, the invention relates to single domain antibodies containing two VH domains with modifications that form single domain antibodies.

In some embodiments, the agent is a scaffold protein that is not an antibody. Typically, these proteins are obtained by adapting, based on combinatorial chemistry, preexisting ligand or antigen binding proteins. For example, using combinatorial chemistry in human transferrin, the binding site of the human transferrin receptor can be modified to generate a library of different transferrin variants, some of which have acquired affinity for various antigens (see Ali et al., J. Biol. Chem., 274:24066 -24073 (1999)). The part of human transferrin that is not involved in receptor binding remains unchanged and serves as a scaffold, similar to the framework regions of antibodies, presenting variants of binding sites. The libraries are then screened for the target antigen of interest, as an antibody library, to identify those variants that have optimal selectivity and the desired affinity for the target antigen. Non-antibody scaffold proteins, which have functions similar to antibodies, are believed to have a number of advantages over antibodies and, in particular, they have improved solubility and tissue penetration with less expensive production and ease of conjugation with other molecules of interest. (See Hey et al., TRENDS Biotechnol., 23(10):514-522) (2005).

One skilled in the art will appreciate that the framework portion of the non-antibody framework protein may include, for example, all or part of the S. aureus protein A Z-domain, human transferrin, human fibronectin type III tenth domain, human trypsin inhibitor Kunitz domain, CTLA- 4 human ankyrin repeat protein, human lipocalin, human crystallin, human ubiquitin, or E. elaterium trypsin inhibitor (see Hey et al., TRENDS Biotechnol., 23(10):514522 (2005)).

In some embodiments, the agent is a natural ligand of the target biomolecule. For example, the agent may be a cytokine. As used herein, the term cytokine refers to any secreted polypeptide that affects cell function and is a molecule that modulates interactions between cells in an immune, inflammatory, or hematopoietic response. Non-limiting examples of cytokines include monokines and lymphokines, regardless of which cells produce them. For example, a monokine is generally referred to in this way in view of its production and secretion by mononuclear cells such as macrophages and/or monocytes. However, monokines can also be produced by many other cells such as natural killer cells, fibroblasts, basophils, neutrophils, endothelial cells, brain astrocytes, bone marrow stromal cells, epidermal keratinocytes, and B lymphocytes. As a rule, lymphokines are so named because of their production by lymphocytes. Non-limiting examples of cytokines include interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-alpha (TNFa) and tumor necrosis factor beta (TNFe).

In some embodiments, the agent is a tumor necrosis factor (TNF) family ligand, e.g., the TNF family ligand is selected from TNFa, TNFe, Fas ligand, lymphotoxin, lymphotoxin alpha, lymphotoxin beta, 4-1BB ligand, CD30 ligand, EDA-A1, LIGHT (TNFSF14), TNF-like ligand 1A (TL1A), TNF-linked weak apoptosis inducer (TWEAK), and TNF-linked apoptosis-inducing ligand (TRAIL). The agent may be CD40 ligand, CD27 ligand, OX40 ligand, B cell activating factor (BAFF; TNFSF13B; BLYS), ectodysplasin A (EDA), activation-inducible TNFR family receptor ligand (AITRL), vascular endothelial growth inhibitor (VEGI) , a proliferation-inducing ligand (APRIL); or a nuclear factor-kappa-B activator receptor ligand (RANKL). In some embodiments, the target is TNFa, TNFe, Fas ligand, lymphotoxin, lymphotoxin alpha, lymphotoxin beta, 4-1BB ligand, CD30 ligand, EDA-A1, LIGHT (TNFSF14), TL1A, TWEAK, TRAIL, CD40 ligand, CD27 ligand, ligand OX40, B cell activating factor (BAFF; TNFSF13B; BLYS), ectodysplasin A (EDA), TNFR activation-inducible receptor family ligand (AITRL), vascular endothelial growth inhibitor (VEGI), proliferation-inducing ligand (APRIL), or activator receptor ligand nuclear factor kappa-B (RANKL).

In some embodiments, the agent is a viral protein, or a portion thereof, that specifically binds to a target (eg, 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 an organism's genome including TNF and TNF receptors. vTNF includes the TNF-binding proteins of viruses such as poxviruses (eg, Yatapoxvirus such as Yaba-like disease virus, Taparoch virus, and Yaba monkey tumor virus; vaccinia virus, myxoma virus, and mousepox virus) and retroviruses (eg, monkey foam virus). For example, vTNF can be vaccinia virus Crm B, Crm C, Crm D, or Crm E, myxoma virus M-T2, monkey foam virus S-T2, vaccinia virus VCD30, or Taparoch virus TPV2L. In some

- 29 041139 variants, the agent is the E6 or E7 protein of the human papillomavirus, which binds to

TNFR1 or an orthologue of TRAILR2, the avian sarcoma and leukemia virus CAR1 protein that binds to

TNFRs.

In some embodiments, the agent is a natural ligand variant of the target biomolecule, such as an interleukin polypeptide variant, such as an IL-2 variant or a TNFa variant. Options in accordance with embodiments of the invention may contain one or more substitutions, deletions or insertions of amino acids. Substitutions may be conservative or non-conservative. As used herein, the term conservative substitution refers to the replacement of an amino acid found in the natural sequence of a given polypeptide with a natural or non-natural amino acid with similar steric properties. When the side chain of the naturally occurring amino acid to be replaced is polar or hydrophobic, a conservative substitution is made using a naturally occurring amino acid or a non-natural amino acid that is also polar or hydrophobic and does not necessarily have the same or similar steric properties as the side chain in the amino acid being replaced. Typically, conservative substitutions include substitutions within 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. Single letter amino acid abbreviations are 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 full-length natural wild-type ligands, as well as fragments that contain one or more substitutions, insertions or deletions of amino acids, compared to the full-length natural wild-type ligand from which this fragment is derived.

As used herein, the phrase non-conservative substitutions refers to the replacement of an amino acid present in the original sequence with another natural or non-natural amino acid with different electrochemical and/or steric properties. Thus, the side chain of the substituting amino acid may be significantly larger (or smaller) than the side chain of the naturally occurring amino acid being replaced and/or may contain functional groups that are significantly different in electronic properties from the substituting amino acid.

In some embodiments, the polypeptide variant contains 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) substitutions, deletions or insertions of amino acids relative to the full-length wild-type polypeptide from which it is derived. In some embodiments, the variant polypeptide contains no more than 150 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) substitutions, deletions or insertions of amino acids, relative to the full-length wild-type polypeptide from which it is derived.

In some embodiments, a variant polypeptide (e.g., an IL-2 or TNFa polypeptide variant) 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 the full-length wild-type polypeptide from which it is derived to bind the target biomolecule (e.g., a member of a specific binding pair in which the full-length wild-type polypeptide is a member). In some embodiments, the variant polypeptide has greater affinity for the target biomolecule than the full-length wild-type polypeptide from which the variant is derived. For example, in some embodiments of the invention, a variant polypeptide has 2-fold (3, 4, 5, 10, 20, 30, 40, 50, 100, 200, 500, or even 1000-fold) greater affinity for the target biomolecule than the full-length wild-type polypeptide. the type from which the polypeptide variant is derived. Methods for detecting or determining the level of interaction between two proteins are known in the art and are described above.

In some embodiments, the wild-type full-length natural ligand modulates the activity of a cell surface receptor. Thus, natural ligand variants may have increased or decreased activity relative to wild-type natural ligand in terms of ability to modulate receptor activity. For example, in some embodiments, a polypeptide variant 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 the variant is derived to activate the cell surface receptor protein. In some embodiments, the variant polypeptide does not activate the receptor to which it binds.

Such exemplary polypeptide variants are known in the art. For example, international patent application publication WO 2012/085891 describes ligand variants of the TNF family with reduced ability to trimerize and thus with reduced ability to activate

- 30 041139 TNF family receptors (US Patent Application Publication US 2014/0096274, incorporated here by reference). Additional TNF ligand variants retain the ability to bind to TNF family receptors. Suitable methods for comparing the activity of variants and natural wild-type ligands are known in the art.

In some embodiments, the soluble biomolecule is a cell surface receptor ligand, such as a cytokine or chemokine (eg, MCP-1/CCL2, CCL5, CCL11, CCL12, or CCL19), i. any of those known in the art or described herein. In some embodiments, the ligand is a tumor necrosis factor (TNF) family ligand or a variant thereof. In some embodiments, the TNF family ligand is TNFa or a variant thereof. In some embodiments, the TNF family ligand is Fas ligand, lymphotoxin, lymphotoxin alpha, lymphotoxin beta, 4-1BB ligand, CD30 ligand, EDA-A1, LIGHT, TL1A, TWEAK, TNFp, TRAIL, or a variant of any of the above). In some embodiments, the ligand is a TGF-β superfamily ligand or a variant thereof, e.g., activin A, activin B, anti-Müllerian hormone, growth differentiation factor (e.g., GDF1 or GDF11), bone morphogenetic protein (BMP), inhibin (e.g., inhibin alpha, inhibin beta), lefty, persefin, nodal, neuroturin, TGF-β 1, TGF-e2, TGF-e3, or myostatin. In some embodiments, the ligand is a hormone (eg, 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 a molecule listed in Table. 2.

table 2

Illustrative Soluble Biomolecules and/or Agents

First member of a specific binding pair (soluble biomolecule or agent) Abbr. gene name Molecule class Associated Disease or Condition The second member of the specific binding pair tumor necrosis factor alpha TNF Cytokine AD, obesity, type II diabetes (T2D), disease Alzheimer's (AD) sTNF-R Soluble interleukin-2 receptor IL2RA Trap Cancer SIL-2R Ghrelin GHRL Hormone Obesity Ghrelin receptor (GHSR1); antibodies against ghrelin Soluble tumor necrosis factor-1 receptor TNFRSF1A Trap Cancer rTNF Soluble tumor necrosis factor-2 receptor TNFRSF1B Trap Cancer rTNF

- 31 041139

Transforming growth factor beta 1 LTBP1 growth factor Muscular degeneration, disdifferentiation Ligand 11 with C-C motif, or eosinophil chemotactic protein, eotaxin-1 CCL11 Cytokine Decreased neurogenesis and cognition Interleukin-2 IL2 Cytokine AD SIL-2R, briakinumab Interleukin-6 IL6 Cytokine AD SIL-6R, olokizumab, sarilumab, siltuximab Interleukin-8 CXCL8 Cytokine AD IL-8R Interleukin-1A ILIA Cytokine AD sIL-IRA Interleukin-1B IL1B Cytokine inflammation, diabetes canakinumab, gevokizumab Chemokin 10 s motif S-X-S 10 CXCL10 Chemokin Activation of the immune system CXCR3 Growth differentiation factor 8, or myostatin MSTN growth factor Sarcopenia Activin receptor (ActRIIB) Trap Receptor 3 FAS Trap Cancer FAS-L Soluble Death Receptor-4 TNFRSF10A Trap Cancer TRAIL Ri Soluble Death Receptor-5 TNFRSF10B Trap Cancer TRAIL-R2, drotizumab Fas ligand FAS LG Cytokine AD sDcR3 TNF-related apoptosis inducer ligand TNFSF10 Cytokine AD, T2D SDR4/5 Chemokine ligand 1 (C-X-C motif) (stimulating melanoma growth activity, Alpha) CXCL1 Chemokin aging/cancer Amyloid beta APP Fragment AD Anti-amyloid beta antibodies, such as aducanumab

-32041139

Microglobulin β2 V2M Protein Aging TNF-linked weak inducer of apoptosis TNFSF12 Cytokine TBD SDR3 Matrix metallopeptidase 1 (interstitial collagenase) MMP1 Protease aging/cancer Matrix metallopeptidase 2 (gelatinase A, gelatinase 72 kDa, collagenase 72 kDa type IV) MMP2 Protease OA/cancer Matrix metallopeptidase 3 (stromelysin 1, progelatinase) MMRP Protease aging/cancer Matrix metallopeptidase 9 (gelatinase B, gelatinase 92 kDa, collagenase 92 kDa type IV) MMP9 Protease aging/cancer Matrix metallopeptidase 10 (stromelizin 2) MMP10 Protease aging/cancer Matrix metallopeptidase 12 (macrophage elastase) MMP12 Protease aging/cancer Indolamine-2,3dioxygenase IDO1 Enzyme Cancer Homologous protein of 1 neurogenic notch protein locus NOTCH1 Cytokine Dysfunction stem cells Homologous protein 2 of the neurogenic locus of the notch protein NOTCH2 Cytokine Dysfunction stem cells Homologous protein 3 of the neurogenic locus of the notch protein NOTCH3 Cytokine Dysfunction stem cells Homologous protein 4 of the neurogenic locus of the notch protein NOTCH4 Cytokine Dysfunction stem cells

- 33 041139

Interleukin-5 IL5 Cytokine AD mepolizumab, reslizumab Soluble interleukin-5 receptor IL5RA Trap Cancer IL-5 Soluble receptor interleukin-6 IL6R Trap Cancer IL-6, tocilizumab Soluble interleukin-8 receptor CXCR1 Trap Cancer IL-8 Soluble interleukin-1A receptor IL1R1 Trap Cancer IL-1A C-reactive protein CRP Protein Marker of inflammation Haptoglobin HP Protein Heart diseases Soluble Death Receptor-3 TNFRSF25 Trap TWEAK CD47 CD47 Protein Cancer Thrombospondin -1; signal-regulating protein alpha

AD refers to autoimmune disorders and/or inflammatory disorders. OA refers to osteoarthritis.

In some embodiments, the agent may bind (eg, specifically bind) to a biomolecule selected from TNFa, TNFe, soluble TNF receptor, soluble TNFR-1, soluble TNFR-2, lymphotoxin, lymphotoxin alpha, lymphotoxin beta, 4-1BB ligand, CD30 ligand , EDAA1, LIGHT, TL1A, TWEAK, TRAIL, TRAIL soluble receptor, IL-1, soluble IL-1 receptor, IL-1A, soluble ILIA receptor, IL-1B, soluble IL-1B receptor, IL-2, soluble IL receptor -2, 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, TNF-coupled weak inducer of apoptosis, MMP1, MMP2, MMP3, MMP9, MMP10, MMP12, CD28, soluble B7 family member, soluble CD80/B7-1 , soluble CD86/B7-2, soluble CTLA4, soluble PD-L1, soluble PD-1, soluble Tim3, Tim3L, ha lectin 3, galectin 9, soluble CEACAM1, soluble LAG3, TGF-β, TGF-P1, TGF-P2, TGF-33, anti-Müllerian hormone, artemin, glial cell-derived neurotrophic factor (GDNF), bone morphogenetic protein (eg, BMP2, BMP3 , BMP3B, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, BMP10, BMP11, BMP12, BMP13, BMP15), growth differentiation factor (e.g. GDF1, GDF2, GDF3, GDF3A, GDF5, GDF6, GDF7, GDF8, GDF9, GDF10, GDF11, GDF15), inhibin alpha, inhibin beta (e.g. inhibin beta A, B, C, E), lefty, nodal, neuroturin, 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, amyloid beta, activin, activin A, activin B, microglobe ulin β2, soluble NOTCH1, soluble NOTCH2, soluble NOTCH3, soluble NOTCH4, soluble Jagged1, soluble Jagged2, soluble DLL1, soluble DLL3, soluble DLL4, haptoglobin, alpha chain fibrinogen, corticotropin releasing factor, 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, indolamine-2,3-dioxygenase, C-reactive protein , gp120 of HIV-1 virus, endotoxin, ricin toxin, epsilon toxin from Clostridium perfringens, enterotoxin B from Staphylococcus, and botulinum toxin.

In some embodiments, the agent may contain an antibody (or antigen-binding fragment thereof) that specifically binds to TNFa, TNFe, a soluble

- 34 041139 TNF rum, TNFR-1 soluble, TNFR-2 soluble, lymphotoxin, alpha lymphotoxin, beta lymphotoxin, 4-1BB ligand, CD30 ligand, EDA-A1, LIGHT, TL1A, TWEAK, TRAIL, TRAIL soluble 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, TNF-linked weak inducer of apoptosis, MMP1, MMP2, MMP3, MMP9, MMP10, MMP12, CD28, soluble B7 family member, 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-e1, TGF-e2, TGF-e3, anti-Mullerian hormone, artemin, glial cell neurotrophic factor (GDNF), bone morphogenetic protein (eg BMP2, BMP3, BMP3B, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, BMP10, BMP11, BMP12, BMP13, BMP15), growth differentiation factor (eg GDF1 , GDF2, GDF3, GDF3A, GDF5, GDF6, GDF7, GDF8, GDF9, GDF10, GDF11, GDF15), inhibin alfa, inhibin beta (eg, inhibin beta A, B, C, E), lefty, nodalom, neuroturin, 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, amyloid beta, activin, activin A, activin B, microglobulin β2, soluble NOTCH1, soluble NOTCH2, soluble NOTCH3, soluble NOTCH4, instant jagge d1, soluble Jagged2, soluble DLL1, soluble DLL3, soluble DLL4, haptoglobin, alpha chain fibrinogen, corticotropin releasing factor, corticotropin releasing factor type 1, corticotropin releasing factor type 2, urocortin 1, urocortin 2, urocortin 3, CD47, autoantibody anti-interferon-γ, anti-interleukin 6 autoantibody, anti-interleukin 17 autoantibody, anti-ghrelin autoantibody, Wnt, indolamine 2,3-dioxygenase, C-reactive protein, HIV-1 virus gp120, endotoxin, ricin toxin, epsilon toxin from Clostridium perfringens, enterotoxin B from Staphylococcus and botulinum toxin.

The agent may contain ipilimumab, pembrolizumab, nivolumab, infliximab, adalimumab, certolizumab (eg, certolizumab regol), golimumab, etanercept, stamulumab, fresolimumab, metelimumab, demcizumab, tarextumab, brontuktuzumab, mepolizumab, urelumumab, canakinumab, denosumumab, beldakimumab , тоцилизумаб, атлизумаб, устекинумаб, паливизумаб, адуканумаб, бевацизумаб, бролицизумаб, ранибизумаб, афлиберсепт, актоксумаб, элсилимомаб, силтуксимаб, афелимомаб, нерелимомаб, озорализумаб, патеклизумаб, сирукумаб, омализумаб, адуканумаб, бапинейзумаб, кренезумаб, гантенерумаб, понезумаб, соланезумаб, дапиролизумаб , раплизумаб, торализумаб, энотикумаб, алацизумаб, цетуксимаб, футуксимаб, икрукумаб, имгатузумаб, матузумаб, нецитумумаб, нимотузумаб, панитумумаб, рамуцирумаб, залутумумаб, дулиготумаб, патритумаб, эртумаксомаб, пертузумаб, трастузумаб, алирокумаб, анрукинзумаб, диридавумаб, дрозитумаб, дупилумаб, дузиджитумаб , eculizumab, edobacomab, efangumab, eldelumab, enobl итузумаб, энокизумаб, эвинакумаб, эволокумаб, эксбивирумаб, эксбивирумаб, фазинумаб, фельвизумаб, фезакинумаб, фиклатузумаб, фиривумаб, флетикумаб, форалумаб, форавирумаб, фулранумаб, фаликсимаб, ганитумаб, гевокизумаб, фузелкумаб, идаруцизумаб, ималумаб, инолимомаб, иратумумаб, иксекизумаб, лампализумаб, лебрикизумаб, ленцилумаб, лерделимумаб, лексатумумаб, либивирумаб, лигелизумаб, лоделцизумаб, лулизумаб, мапатумумаб, мотавизумаб, намилумаб, небакумаб, несвакумаб, обилтоксаксимаб, олокизумаб, ортикумаб, паджибаксимаб, паливизумаб, панобакумаб, пасколизумаб, перакизумаб, пидилизумаб, пекселизумаб, притоксаксимаб, куилизумаб, радретумаб, рафивирумаб, ралпанцизумаб, раксибакумаб, регавирумаб, реслизумаб, рилотумумаб, ромозозумаб, ронтализумаб, сарилумаб, секукинумаб, сетоксаксимаб, севирумаб, сифалимумаб, силтуксимаб, сувизумаб, табалумаб, такатузумаб, тализумаб, танезумаб, тефибазумаб, TGN1412, тилдракизумаб, тигатузумаб, TNX- 650, tozatoxumab, tralokinumab, tremelimumab, alarmrumab, tuvirumab, urtoxazumab, vantictumab, vanucizumab, or an antigen-binding fragment of any of the above antibodies.

In some embodiments, the agent comprises TNFa, TNFe, soluble TNF receptor, soluble TNFR-1, soluble TNFR-2, vTNF, lymphotoxin, 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 receptor-5, TNF-coupled weak inducer of apoptosis, MMP1, MMP2, MMP3, MMP9, MMP10, MMP12, CD28, soluble B7 family member, 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- βΤ TGF-e2, TGF-e3, sLR11, CCL2,

CCL5, CCL11, CCL12, CCL19, activin, activin A, activin B, soluble NOTCH1, soluble

NOTCH2, soluble NOTCH3, soluble NOTCH4, soluble Jagged1, soluble Jagged2, soluble DLL1, soluble DLL3, soluble DLL4, or haptoglobin.

In some embodiments, each particle contains a plurality of agents. A plurality of agents may contain from 10 to about 109 copies of the agent, for example, from about 103 to 10 ⁷ copies of the agent, or from about 10 ⁴ to about 10 ⁶ copies of the agent.

Methods for obtaining antibodies.

As indicated above, in some embodiments of the invention, the agents immobilized on the surface of the particle or particles are antibodies or antigen-binding fragments thereof. Antibodies can be obtained by methods known in the art. For example, a mammal, such as a mouse, hamster, or rabbit, can be immunized with an immunogenic form of a soluble biomolecule (eg, soluble TNFR, toxin, or viral protein). Alternatively, immunization can be carried out using a nucleic acid that, under in vivo conditions, allows the expression of a biomolecule (eg, a soluble protein) leading to an observable immune response. Methods for conferring immunogenicity on a protein or peptide include conjugation with carriers or other methods well known in the art. For example, the peptide portion of a polypeptide of the invention may be administered in the presence of an adjuvant. The progress of the immunization can be monitored by detecting antibody titers in plasma or serum. A standard ELISA assay or other immunoassays with an immunogen as antigen can be used to assess antibody concentration.

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

The location of the agent on the particle.

In some embodiments, the particle geometry is such that the immobilized agent has a reduced or substantially reduced ability to interact with a cell surface biomolecule, such as an immune cell, blood cells, or lymphocyte. The immobilized agent may have less than 50% (e.g., 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%) the ability to bind to the biomolecule on cell surface compared to the free soluble form of the agent. For example, in some embodiments, TNFa or IL-2 immobilized on the surface of the particle described here 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, compared to free TNFa or IL-2, to bind to the TNFa receptor or IL-2 receptor on the cell surface.

In some embodiments, the soluble biomolecule associated with the particle has a reduced or greatly reduced ability to interact with a cognate ligand (the second member of a specific binding pair). The biomolecule can be associated with the particle through an agent. A biomolecule bound to a particle may have less than 50% (e.g., 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%) the ability to interact with by its cognate ligand compared to the capacity of an unbound biomolecule. For example, the soluble TNFR associated with the particle described here 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 soluble TNFR to interact with free TNFa. In another example, the soluble virion associated with the particle described here 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 the free virion to interact with the cognate receptor (or receptors) on the cell surface and infect the cell.

In some embodiments of the invention, the agent may be immobilized on the inner surface of the particle (for example, in the pores of the porous particle or in the inner surface of the tube). In some embodiments, the agent may be immobilized on the outer surface of the particle, but its interaction with the cell surface is sterically excluded due to one or more protrusions of the particle. In some embodiments of the invention, for example, in the case of toroidal particles, the agent is immobilized on the inner surface of the particle in such a way that the agent has a reduced or significantly reduced ability to interact with a biomolecule on the cell surface and/or a soluble biomolecule associated with the particle, due to the fact that the agent has a reduced or significantly reduced ability to interact with a related ligand (the second member of the specific binding pair).

An exemplary particle geometry capable of reducing or substantially reducing the interaction of an agent with a biomolecule on the cell surface, or the interaction between a particle-bound biomolecule and its cognate ligand, is shown in FIG. 1-6 and described in this document.

Removal and coating agents.

In some embodiments of the invention, the particle contains an agent (tool) that promotes excretion. The excretion agent may assist in the elimination of the particles biologically, for example, via urinary excretion, degradation, hepatobiliary clearance, and/or phagocytosis.

For example, the particle may contain a reservoir, where the reservoir contains a release agent. The reservoir may be an opening or cavity in the body of the particle, such as a cavity in the body of a porous silicon particle.

For particles containing 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 in the body of the particle (for example, in the form of a small recess), where 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 approximately 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 the width or diameter of the average pore size. The width or diameter of the reservoir may be about 2-10 times the width or diameter of the average pore size, such as about 2-8 times or about 2-6 times. Tank width or diameter can be approximately 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 approximately 500 times the width or average pore diameter.

For particles containing scaffold DNA, the reservoir may be the interior of the DNA scaffold. The reservoir (eg, the inner region) may be inaccessible to cells, for example, the DNA scaffold may be designed in such a way that the scaffold sterically makes it difficult for cells to enter the inner region. In some embodiments, the reservoir (eg, the interior) is inaccessible to extracellular proteins, for example, the DNA scaffold can be designed such that the scaffold sterically impedes entry of extracellular proteins into the reservoir. The reservoir (eg, interior) may not be accessible to antibodies. However, the DNA scaffold may allow the reservoir (eg, internal region) to become available to cells and/or extracellular proteins after a predetermined period of time. For example, the DNA scaffold may comprise a biodegradable wall that can be degraded after a predetermined period of time (eg, by hydrolysis), thereby allowing the clearance agent to reach cells and/or extracellular proteins. The scaffold DNA may contain a biodegradable structural fixer that can be degraded after a predetermined period of time (eg, by hydrolysis), which allows the scaffold DNA to undergo a conformational change, thereby allowing the clearance agent to reach cells and/or extracellular proteins (see, e.g., , PCT Patent Application Publication WO 2014/170899, which is incorporated herein by reference). Similarly, scaffold DNA may contain a reservoir that contains a hole, as described below.

The reservoir may contain an opening. The opening may be closed with a lid or membrane, thereby inhibiting interaction between the excretion agent and cells and/or extracellular proteins (eg, antibody). The lid or membrane may contain a polymer, such as a biodegradable polymer. The lid or membrane can be destroyed (eg, by hydrolysis) after a predetermined period of time, thereby allowing access of the excretory agent to the cells and/or extracellular proteins. The lid or membrane can be degraded (e.g., by biodegradation) upon exposure to a biological fluid (e.g., blood plasma or extracellular fluid) for from about 1 day to about 5 years, for example, from about 1 day to about 4 years, from about 1 day to about 3 years or from about 1 day to about 1 year.

The predetermined period of time may be the period of time during which the particle is in the liquid (eg, in an aqueous liquid). The predetermined period of time may be the period the particle is exposed to in vivo conditions (eg, exposure to biological fluids, pH, enzymes, and/or temperature). A given period of time can be determined, at least in part, by binding the particle to the biomolecule. For example, the particle can be configured such that binding of the biomolecule results in the action of a release agent on cells and/or extracellular proteins (see, for example, PCT Patent Application Publication WO 2014/170899, which is incorporated herein by reference). The predetermined time period may be from approximately 1 day to

- 37 041139 about 5 years, for example from about 1 day to about 3 years, or from about 1 day to about 1 year.

Illustrative materials suitable for use as caps or membranes are described in US Pat. No. 7,918,842, which is incorporated herein by reference. Typically, these materials are degraded or dissolved by enzymatic hydrolysis or by exposure to water under in vivo or in vitro conditions, or by surface or bulk erosion. Illustrative synthetic, biodegradable polymers include poly(amides), such as poly(amino acids) and poly(peptides); poly(esters), such as poly(lactic acid), poly(glycolic acid), a copolymer of lactic and glycolic acids and poly(caprolactone); poly(anhydrides); poly(orthoesters); poly(carbonates) and their chemical derivatives (with substitution, addition of chemical groups such as alkyls, alkylenes, hydroxylation, oxidation, and other modifications generally carried out by those skilled in the art), copolymers, and mixtures thereof. Other polymers that can be used in closures or membranes include poly(ethers) such as poly(ethylene oxide), poly(ethylene glycol) and poly(tetramethylene oxide); 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(vinylpyrrolidone) and poly(vinyl acetate); poly(urethanes); cellulose and its derivatives such as alkyl, hydroxyalkyl, ethers, esters, nitrocellulose and various cellulose acetates; poly(siloxanes) and any of their chemical derivatives (with substitution, addition of chemical groups such as alkyls, alkylenes, hydroxylation, oxidation, and other modifications generally carried out by those skilled in the art), copolymers, and mixtures thereof. In some embodiments, the reservoir lid is made from one or more crosslinked polymers, such as crosslinked polyvinyl alcohol.

In some embodiments of the invention, the particle contains a coating. In some embodiments, the coating includes a release agent. The coating may mask the withdrawal agent.

The particle may include 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 internal surface, for example the first surface may be oriented so that the agent has a reduced ability to bind to a molecule on the cell surface. Examples of the inner surface include the inner walls of a pore, reservoir or tube, the inner annular surface of a toroid, or the cavity of a concave surface. Other examples of the inner surface include the outer surface of the particle, where the outer surface is protected from interaction with cells by one or more protrusions. The second surface may be an outer surface, for example the second surface may be oriented so that the coating can interact with the cell. In some embodiments, the particle may contain one or more core subparticles and a plurality of protective subparticles. The particle may contain a protection, and the protection may include a plurality of protective subparticles. The first surface may be the surface of one or more core particles and the second surface may be the surface of the protective subparticles.

The coating may prevent interactions between the particles, for example the coating may reduce the tendency of the particles to form aggregates. The coating may interfere with interactions between particles and cells, for example by providing a biologically inert surface. The coating can prevent non-specific interactions with extracellular molecules, for example by preventing non-specific adsorption of biomolecules. The coating may interfere with specific interactions with cells or extracellular molecules, for example the coating may prevent or delay the excretion or phagocytosis of particles. The coating can make the particle a target for excretion or phagocytosis. A coating or other characteristic (e.g., a clearance-inducing compound) that makes the particle a target for clearance or phagocytosis may be masked by a coating (e.g., a second coating) that delays clearance or phagocytosis of the particle, for example, to ensure that particles remain in the bloodstream for a given period of time.

The coating may include a plurality of elongated coating molecules connected at one end to the surface of the particle. The coating may prevent interaction between the particle-bound biomolecule and the second specific binding member of the pair, which includes the biomolecule. The coating may prevent interaction between the biomolecule associated with the particle and the cell. The agent can be oriented on the particle relative to the coating so that the agent has a reduced ability to bind to a molecule on the cell surface. The agent can be oriented on the particle relative to the coating so that the agent has a reduced ability to bind to a target on the cell surface. The agent can be oriented on the particle relative to the coating in such a way that the coating sterically hinders the binding of the agent to the molecule on the cell surface. The agent can be oriented on the particle in such a way that the coating sterically hinders the binding of the agent to the target on the cell surface. The coating may be oriented on the particle such that the agent in the particle has a reduced ability to bind to a molecule on the surface of the cell. The coating can reduce the ability of the agent in the particle

- 38 041139 to activate the cell surface receptor protein compared to the ability of the natural ligand of the cell surface receptor protein.

The particle may contain a second coating, where the second coating consists of a second plurality of coating molecules. The particle may contain a second plurality of coating molecules. The second coating and/or the second plurality of coating molecules may reduce the in vivo excretion of 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, allowing cells and/or extracellular proteins to be exposed to the coating and/or the plurality of coating molecules after a predetermined period of time. The second coating and/or the second plurality of coating molecules may contain a biodegradable polymer, for example, each molecule of the second plurality of coating molecules may contain a biodegradable polymer. The second coating and/or the second plurality of coating molecules may contain CD47, which inhibits phagocytosis.

In some embodiments, the implementation of the particle contains a first surface (for example, an inner surface) and a second surface (for example, an outer surface or an outer 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 cell surface.

The orientation of the second surface may provide interactions between the coating and cells, extracellular molecules and/or various particles. The interaction between the coating and cells, extracellular molecules and/or various particles can be a weak, neutral or unfavorable interaction, for example, preventing 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 planned interaction, for example, to facilitate the elimination of the particle by biological means, such as phagocytosis. In certain preferred embodiments, the second surface is substantially free of agent. In certain preferred embodiments, the first surface is substantially uncoated. In certain preferred embodiments, the coating covers substantially the entire second surface.

In some embodiments, the implementation of the particle contains a first surface (for example, an inner surface) and a second surface (for example, an outer surface or an outer 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 second coating) can inhibit interactions between the agent and molecules on the cell surface. In certain preferred embodiments of the invention, the coating covers substantially the entire second surface.

In some embodiments, the implementation of the particle contains a first surface (for example, an inner surface) and a second surface (for example, an outer surface or an outer 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 the entire second surface.

In some embodiments, the implementation of the particle contains a surface; the agent is immobilized on the surface; and the coating covers at least a portion of the surface. In such embodiments, the implementation of the coating may not affect the ability of the agent to specifically bind to the biomolecule. The coating may allow a certain proportion of the agent to specifically bind to the biomolecule and inhibit interactions between a certain proportion of the agent and the biomolecule. The coating can inhibit interactions between the agent and molecules on the cell surface. In certain preferred embodiments, the coating covers substantially the entire surface.

In some embodiments, the particle comprises a coating that covers at least a portion of the second surface and a second coating that covers at least a portion, such as substantially all, of the coating on the second surface. In such embodiments, the coating may contain a clearance agent, such as a clearance-inducing compound, which makes the particle a target for clearance or phagocytosis. Such a coating may contain beta-cyclodextrin. The second coating may contain a material, such as a second plurality of coating molecules, that inhibits interaction with cells and/or inhibits non-specific interactions with extracellular molecules, such as inhibiting non-specific adsorption of biomolecules. The second coating may be biodegradable, for example, allowing the surface to attack cells and/or extracellular proteins after a predetermined period of time. For example, for particles containing one or more core subparticles and a plurality of protective subparticles, where the capture agent is immobilized on the surface of the subparticles (subparticles) of the core (i.e., on the first surface), at least part of the surface of the protective subparticles (i.e. . second surface) contains a coating, for example a coating containing either

- 39 041139 a release agent or coating containing a material for inhibiting interaction with cells and/or inhibiting non-specific interaction with extracellular molecules.

The coating may contain coating molecules, for example a coating may be composed of a plurality of coating molecules, or a coating may be composed of a group of coating molecules. As used here, each of the terms set of coating molecules and the group of coating molecules refers to the coating. However, the term coating may refer to additional compositions such as a hydrogel. The coating molecule may be a release agent (and thus the release agent may be a coating molecule).

The particle may contain a plurality of coating molecules. The particle may contain a surface and a plurality of agents immobilized on the surface, and at least one molecule of the plurality of coating molecules may be associated with the surface. For example, all or substantially all of the molecules of a plurality of coating molecules may be associated with a surface.

The particle may contain a surface and a second surface, where a plurality of agents immobilized on the surface, and at least one molecule of the plurality of coating molecules may be associated with the second surface. For example, all or substantially all of the molecules of the plurality of coating molecules may be associated with the second surface. In some embodiments, certain molecules of the plurality of coating molecules are associated with a surface and certain molecules of the plurality of coating molecules are associated with a second surface.

In some embodiments, the coating molecules increase the release of particles under in vivo conditions. For example, the coating molecules may contain a pathogen-associated molecular structure.

In some embodiments, the particles described herein comprise a coating containing a excretion-inducing compound that promotes clearance of the particles from the circulation, e.g., by the kidneys, liver/intestines (e.g., via bile), or by phagocytosis (e.g., by antigen presenting cells). The plurality of coating molecules may be a plurality of excretion-inducing compounds. For example, in embodiments where the particles are toroidal, the inner annular surface (eg, the first surface) may contain an immobilized agent, and the outer surface (eg, the second surface) may contain a compound that induces the elimination of the particles, for example, by the kidneys, liver, or macrophages. In some embodiments, the excretion-inducing compound is programmable. Those. the compound may be coated with a coating that eventually breaks down over time (e.g., over a given period of time) (e.g., by enzyme action, hydrolysis, or gradual dissolution), making the excretion-inducing compound available or conferring another property that increases the rate of excretion . The coating can be degraded by exposure to a biological fluid (eg, blood plasma or extracellular fluid) over a period of from about 1 day to about 5 years, such as from about 1 day to about 3 years, or from about 1 day to about 1 year. Thus, the presence of particles in vivo can be modified and/or controlled.

The coating may contain an organic polymer such as polyethylene glycol (PEG). The organic polymer may be associated with the particle, for example associated with the surface of the particle. The organic polymer may include PEG, polylactate, polylactic acids, sugars, lipids, polyglutamic acid, polyglycolic acid (PGA), polylactic acid (PLA), lactic acid glycolic acid (PLGA), polyvinyl acetate (PVA), and combinations thereof. In some embodiments, the particle is covalently conjugated to PEG, which interferes with adsorption of serum proteins, promotes efficient urinary excretion, and reduces particle aggregation (see, for example, Burns et al., Nano Letters, 9(1):442-448 (2009) and US Patent Application Publications US 2013/0039848 and US 2014/0248210, each of which is incorporated here by reference).

In one of the embodiments of the invention, the coating contains at least one hydrophilic molecule, for example, Pluronics® type polymers (non-ionic polyoxyethylene polyoxypropylene block copolymers with the general formula HO(C ₂ H4O) _a (-C ₃ H ₆ O) _b (C ₂ H ₄ O) _a H), 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 one embodiment of the invention with such a molecule, the hydrophilic molecule is a PEG molecule such as [methoxy(polyethyleneoxy)propyl]trimethoxysilane (e.g. CH ₃ ( _{OC 2} H4) _6.9 (CH ₂ )OSi(OCH ₃ ) ₃ ), [methoxy(polyethyleneoxy ₎ propyl]dimethoxysilane (e.g. CH ₃ (OC ₂ H4) _6.9 (CH ₂ )OSi(OCH ₃ ) ₂ ) or [methoxy(polyethyleneoxy)propyl]monomethoxysilane (e.g. CH ₃ (OC ₂ H4) _6.9 (CH ₂ )OSi(OCH ₃ )) _. Suitable coatings are described, for example, in US Patent Application Publication US 2011/0028662 (incorporated here by reference).

The coating may include a polyhydroxylated polymer, such as a natural polymer or a hydroxyl-containing polymer, including polyhydroxylated polymers, polysaccharides, carbohydrates, polyols, polyvinyl alcohol, polyamino acids, such as polyserine, or other polymers, such as 2-(hydroxyethyl)methacrylate, or combinations thereof. In some embodiments, polyhydroxylated

- 40 041139 polymers are polysaccharides. The polysaccharides include mannan, pullulan, maltodextrin, various types of starch, cellulose and cellulose derivatives, gums, xanthan gum, locust bean gum or pectin, or combinations thereof (see, for example, US Patent Application Publication US 2013/0337070, incorporated here by links).

In some embodiments, the coating comprises a zwitterionic polymer (see, for example, US Patent Application Publications US 2014/0235803, US 2014/0147387, US 2013/0196450 and US 2012/0141797 and US Patent US 8574549, all of which are incorporated herein by reference. ).

Other suitable coatings include poly-alpha-hydroxy acids (including polylactic acid or polylactide, polyglycolic acid or polyglycolide), poly-beta-hydroxy acids (such as polyhydroxybutyrate or polyhydroxyvalerate), epoxy polymers (including polyethylene oxide (PEO)), polyvinyl alcohols, complex polyesters, polyorthoesters, polyamidoesters, polyesteramides, polyphosphoesters and polyphosphoetherurethanes. Examples of degradable polyesters include poly(hydroxyalkanoates), including poly(lactic acid) or (polylactide, PLA), poly(glycolic acid) or polyglycolide (PGA), poly(3-hydroxybutyrate), poly(4hydroxybutyrate), poly(3- hydroxyvalerate) and poly(caprolactone) or poly(valerolactone). Examples of polyoxyesters include poly(alkylene oxalates) such as poly(ethylene oxalate)) and polyoxyesters containing amido groups. Other suitable coating materials include polyethers, including polyglycols, copolymers of ethers and esters (copoly(ether-esters)) and polycarbonates. Examples of biodegradable polycarbonates include polyorthocarbonates, polyiminocarbonates, polyalkyl carbonates such as poly(trimethylene carbonate), poly(1,3-dioxan-2-one), poly(p-dioxanone), poly(6,6-dimethyl-1,4-dioxane -2one), poly(1,4-dioxepan-2-one) and poly(1,5-dioxepan-2-one). Also suitable biodegradable coatings may include polyanhydrides, polyimines (such as poly(ethyleneimine) (PEI)), polyamides (including poly-N-(2-hydroxypropyl)methacrylamide), poly(amino acids) (including polylysine such as polyL-lysine , or polyglutamic acid such as poly-L-glutamic acid), polyphosphazenes (such as poly(phenoxy-co-carboxylatophenoxyphosphazene), polyorganophosphazenes, polycyanoacrylates and polyalkylcyanoacrylates (including polybutylcyanoacrylate), polyisocyanates and polyvinyl pyrrolidones.

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

The particle may be coated with a natural polymer, including fibrin, fibrinogen, elastin, casein, collagens, chitosan, extracellular matrix (ECM), carrageenan, chondroitin, pectin, alginate, alginic acid, albumin, dextrin, dextrans, gelatin, mannitol, n-halamine , polysaccharides, poly-1,4 glucans, starch, hydroxyethyl starch (HES), dialdehyde starch, glycogen, amylase, hydroxyethyl amylase, amylopectin, glucose glycans, fatty acids (and their esters), hyaluronic acid, protamine, polyaspartic acid, polyglutamic acid, D- mannuronic acid, L-guluronic acid, zein and other prolamines, alginic acid, guar gum and phosphorylcholine, as well as their copolymers and derivatives. The coating may also contain a modified polysaccharide such as cellulose, chitin, dextran, starch, hydroxyethyl starch, polygluconate, hyaluronic acid and gelatin, as well as their copolymers and derivatives.

The particle may be coated with a hydrogel. The hydrogel can be prepared, for example, using a base polymer selected from any suitable polymers such as poly(hydroxyalkyl(meth)acrylates), polyesters, poly(meth)acrylamides, poly(vinylpyrrolidone) or polyvinyl alcohol. The crosslinking agent may be one or more of peroxides, sulfur, sulfur dichloride, metal oxides, selenium, tellurium, diamines, diisocyanates, alkylphenyl disulfides, tetraalkylthiuram disulfides, 4,4'-dithiomorpholine, p-quinine dioxime and tetrachloro-pbenzoquinone. Also, polymers containing boronic acid with optional photopolymerizable groups can be incorporated into hydrogels.

In some preferred embodiments of the invention, the coating contains a material that is approved for use by the U.S. Food and Drug Administration (FDA). These FDA-approved materials include polyglycolic acid (PGA), polylactic acid (PLA), polyglactin 910 (containing a 9:1 ratio of glycolide and lactide units and also known as vicryl (VICRYL™)), polyglyconate (containing glycolide and trimethylene carbonate units in a ratio of 9:1 and also known as Maxon (MAXON™)) and Polydioxanone (PDS).

The coating may be associated with the particle via a covalent bond or a non-covalent bond such as ionic bonding, hydrogen bonding, hydrophobic bonding, coordination, adhesion, or physical absorption or physical interaction.

Conventional methods for coating nanoparticles include dry and wet coating methods. Dry coating methods include (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

- 41 041139 al., J. Mater. Chem. 8:1323 (1998)); and (d) pyrolysis of polymeric or non-polymeric organic materials to deposit nanoparticles in situ in the matrix (Sglavo, W. M. et al., J. Mater Sci., 28:6437 (1993)). Wet coating methods for particles include (a) sol-gel methods; and (b) emulsification and solvent evaporation methods (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 can be applied by electroplating, spray coating, dip coating, sputtering, chemical vapor deposition, or physical vapor deposition. In addition, methods are known in the art for coating various nanoparticles with polysaccharides (see, for example, US Pat. No. 8,685,538 and US Patent Application Publication US 2013/0323182, each of which is hereby incorporated by reference).

In some embodiments of the invention, the particles can be adapted for their excretion through urinary excretion. Renal clearance in subjects with normal renal function generally requires at least one particle size less than 15 nm (see, for example, Choi, H.S., et al., Nat. Biotechnol., 25(1):1165 ( 2007); Longmire, M. et al., Nanomedicine, 3(5):703 (2008)). However, larger particles can also be excreted in the urine. In embodiments of the invention where the particle is too large to be excreted by the kidneys, the particle can be excreted after breaking it in vivo to a smaller size.

In some embodiments of the invention, the particles can be adapted for excretion via the hepatobiliary route of excretion. The mononuclear phagocyte system (MPS), which includes Kupffer cells in the liver, is involved in the capture in the liver and subsequent excretion of nanoparticles with bile. Certain size and surface properties of nanoparticles are known to increase MPS uptake in the liver (see Choi et al., J. of Dispersion Sci. Tech., 24(3/4):475-487 (2003); and Brannon-Peppas et al., J. Drug Delivery Sci. Tech., 14(4):257-264 (2004), each of which is incorporated by reference). For example, increased particle hydrophobicity is known to increase MPS uptake. Thus, to modulate biliary excretion, one skilled in the art can select particles with certain characteristics. The hepatobiliary system allows the excretion of particles that are slightly larger than the particles that can be excreted by the renal system (for example, from 10 to 20 nm). For embodiments of the invention where the particles are too large to be excreted by the hepatobiliary system, the particles can be excreted after breaking them in vivo to a smaller size. In such embodiments, a coating that promotes excretion by the hepatobiliary system may cover a portion of the interior surface of the particle such that the coating becomes available when the particle breaks. The particle may contain a plurality of coating molecules, such as hydrophobic molecules, which coat a portion of the surface. The surface can become accessible when the particle is broken, allowing the broken particle to be expelled.

In some embodiments of the invention, the particle is adapted for its removal through phagocytosis. For example, the particle may contain a clearance agent, where the clearance agent contains a pathogen-associated molecular structure, such as for recognition by macrophages. Pathogen-associated molecular structures (PAMPs) include unmethylated CpG DNA (bacterial), double-stranded RNA (viral), lipopolysaccharide (bacterial), peptidoglycan (bacterial), lipoarabinomannan (bacterial), zymosan (yeast), mycoplasmal lipoproteins such as MALP- 2 (bacterial), flagellin (bacterial), poly(inosinic-cytidylic) acid (bacterial), lipoteichoic acid (bacterial), and imidazoquinolines (synthetic). In preferred embodiments, the PAMP as a clearance agent is masked such that macrophages do not ingest the particle until the particle binds to one or more targets. For example, the PAMP may be masked by any of the above coatings (eg, a polymer coating such as a biodegradable polymer coating). Macrophages can ingest particles as small as 20 µm (see, e.g., Cannon, G.J. and Swanson, J.A., J. Cell Science, 101:907-913 (1992); Champion, J.A. et al., Pharm. Res., 25( 8):1815-1821 (2008)). In some embodiments, a phagocytosis-enhancing excretion agent may coat a portion of the interior surface of the particle such that once the particle is destroyed, the excretion agent becomes available. The particle may contain a plurality of release agents, such as PAMP, which coat a portion of the surface. The surface may become accessible after the destruction of the particle, allowing the destruction of the particles. The withdrawal agent may cover a portion of the surface that overlaps the surface containing the agent. The release agent (eg, PAMP) may elicit an immune response against the particle, for example, after the destruction of the second coating or after the destruction of the particle.

In some embodiments of the invention, the immune response directed against the agent for excretion (for example, PAMP) may exceed the immune response directed against the agent and/or the agent/biomolecule complex, inhibiting or delaying the onset of the immune response directed against the agent and/or the agent/biomolecule complex. /biomolecule. For example, disruption of the particle may result in the excretion agent and agent (and/or agent/biomolecule complex) being available for leukocyte action. A clearance agent, such as PAMP, can provide rapid clearance of disrupted particles by macrophages, thereby delaying the immune response (eg, B-42041139 cell-mediated immune response) against the agent and/or the agent/biomolecule complex.

The elimination agent may be calreticulin, which induces phagocytosis.

In some preferred embodiments of the invention, the coating molecule contains a nucleic acid, for example, for hybridization with a coating molecule on a particle containing scaffold DNA. For example, the particle may contain a nucleic acid and a coating molecule, where the coating molecule contains an additional nucleic acid that can hybridize with the nucleic acid, thereby forming a bond between the coating molecule and the particle (eg, hydrogen bonds). 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 complement of the complementary nucleotide sequence. A nucleotide sequence may have 100% sequence identity, i. e. be the reverse complement of the complementary nucleotide sequence.

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

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

In some embodiments, the body can excrete particles over a period of time from about 1 day to about 5 years, such as from about 1 day to about 3 years, or from about 1 day to about 1 year.

Methods of administration.

The description provides that the compositions described herein (eg, any of the particles described generally or specifically, or multiple particles described herein) can be administered to cells and tissues under in vitro and/or in vivo conditions. In vivo administration includes

- 43 041139 administration to an animal in an animal model of the disease, for example in an animal model of a malignant tumor, or administration to a subject in need thereof. Suitable cells, tissues or subjects include animals such as pets, livestock, zoo animals, endangered species, rare animals, non-human primates, and humans. Illustrative pets include dogs and cats.

For in vitro delivery, e.g. to cells or tissues in culture and/or their environment, compositions can be added to culture media, e.g. for contact with the microenvironment or for contact with soluble material in culture media or for contact with cells or even to enter cells. The delivery mechanism and means of administration of the composition (eg, the particles described herein) are influenced by the desired site of activity.

For in vivo delivery, for example, to cells or tissues in vivo (including the cell microenvironment and tissue) and/or a subject in need thereof, many administration routes are known. The specific method can be selected based on the composition of the particles, the specific destination and the patient. Various delivery systems are known and can be used to administer the agents of the invention. Any of these methods can be used to deliver any of the agents described herein. Routes of administration may be enteral or parenteral, including, but not limited to, intradermal, intramuscular, intraperitoneal, intramyocardial, intravenous, subcutaneous, pulmonary, intranasal, intraocular, epidural, and oral routes of administration. The composition of the invention can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucosal linings (eg, such as oral mucosa, rectal and intestinal mucosa, etc.), and the composition can be administered together (simultaneously or sequentially) with other biologically active agents. The introduction can be systemic or local.

In some embodiments of the invention, the composition is administered intravenously, for example, by bolus injection or infusion. In some embodiments of the invention, the composition is administered orally, subcutaneously, intramuscularly, or intraperitoneally.

In some embodiments of the invention, it may be desirable to administer the composition of the invention topically to the area being treated (eg, to the site of the tumor, in particular by injection into the tumor).

The site of metastasis is often the liver. Thus, in some embodiments of the invention, delivery of the composition described herein is to the liver. For example, to deliver an agent of the invention to the liver, a venous catheter may be placed in the portal vein of the liver. Other methods of delivery via the hepatic portal vein are also possible.

In some embodiments, the compositions of the invention are administered by intravenous infusion. In some embodiments, the composition is infused over a period of at least 10, at least 15, at least 20, or at least 30 minutes. In other embodiments, the infusion is carried out over a period of at least 60, 90, or 120 minutes. Regardless of the duration of the infusion, it is contemplated that, in some embodiments, each infusion is part of an overall treatment regimen where the agent is administered according to a standard schedule (eg, weekly, monthly, etc.) for a specified period of time. In other embodiments of the invention, the composition is delivered by bolus injection, for example, as part of a general treatment regimen, when the agent is administered in accordance with a standard schedule for a certain period of time.

For any of the above modes of administration, it is contemplated that the compositions of the invention (including one agent or a combination of two or more such agents) may be administered in vitro or in vivo by any suitable route or method. The compositions may be administered as part of a treatment regimen, when the composition is administered once or multiple times, including administration on a specific schedule. In addition, it is envisaged that the compositions of the invention, if necessary, are prepared in a form intended for a particular route and site of administration. The invention provides for the possibility of using any combination of the above features, as well as combinations with any of the aspects and embodiments of the invention described in this document.

All of the above applies to any of the compositions (eg, particle or multiple particles) of the invention, used alone or in combination, and used in any of the methods described herein. The invention specifically contemplates any combination of features of such compositions of the invention, a combination of compositions and methods with features described for various pharmaceutical compositions and routes of administration, as described in this section and below.

pharmaceutical compositions.

In some embodiments, the described particle or particles according to the invention are formulated with a pharmaceutically acceptable carrier. One or more compositions (eg, containing a particle or multiple particles described herein) can be administered separately or as a component of a pharmaceutical preparation (compositions). Any of the compositions according to the invention, in

- 44 041139 generally or specifically described herein can be prepared in the form as described herein. In some embodiments of the invention, the composition contains two or more particles according to the invention or a particle according to the invention together with a second therapeutic agent.

The composition of the invention may be formulated for administration by any convenient route for use in human medicine or for veterinary use. Wetting agents, emulsifiers and lubricants such as sodium lauryl sulfate and magnesium stearate may also be present in the compositions, as well as coloring agents, release control agents, coating agents, sweeteners, flavoring and flavoring agents, preservatives and antioxidants.

Formulations containing the particle or particles of the invention include, for example, oral, nasal, topical, parenteral, rectal and/or intravaginal preparations. The formulations may be presented in unit dosage form for ease of use and may be prepared by any of the methods well known in the art of pharmacy. The amount of active ingredient that can be combined with a carrier material to form a unit dosage form varies depending on the subject being treated and the particular route of administration. The amount of active ingredient that can be combined with a carrier material to form a unit dosage form, as a rule, is the amount of the compound that provides a therapeutic effect.

In some embodiments of the invention, methods for obtaining these preparations or compositions include the combination of one or more particles and a carrier, and optionally one or more additional ingredients. Typically, formulations can be prepared in a liquid carrier or a finely divided solid carrier, or both, and then, if desired, the product can be shaped into the desired shape.

Formulations for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored base, typically sucrose and gum arabic or tragacanth), powders, granules, or as a solution or suspension in 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 lozenges (using an inert base such as gelatin and glycerin, or such as sucrose and gum arabic), and/or in the form of washes for the oral cavity, etc., where each preparation contains a predetermined number of particles according to the invention. In addition to the active compounds, suspensions may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, 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 invention can be mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or calcium diphosphate, and/or with any of the following ingredients: (1) fillers or diluents such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binding agents such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or gum arabic; (3) humectants such as glycerin; (4) disintegrants such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) dissolution retarding agents such as paraffin; (6) absorption accelerators such as quaternary ammonium compounds; (7) wetting agents such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents such as kaolin and bentonite clay; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) dyes. In the case of capsules, tablets and pills, the pharmaceutical compositions may also contain buffering agents. Solid compositions of a similar type can also be used to fill soft and hard gelatin capsules using excipients such 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 ingredient, liquid dosage forms may contain inert diluents which are commonly used in the art such as water or other solvents, solubilizers and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (particularly cottonseed oil, peanut oil, corn, germ, olive, castor and sesame oils), glycerin, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitol, and mixtures thereof. In addition to inert diluents, oral compositions may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavors, colors, flavors and preservatives.

In some embodiments, the methods of the invention involve topical application to the skin or mucous membranes, such as those of the cervix and vagina. Topical formulations may additionally contain one or more of a wide range of

- 45 041139 range of compounds used as effective enhancers of absorption into the skin or into the stratum corneum. Examples of such compounds include 2-pyrrolidone, N-methyl-2-n-pyrrolidone, dimethylacetamide, dimethylformamide, propylene glycol, methyl or isopropyl alcohol, dimethyl sulfoxide and azone. In addition, to obtain cosmetic preparations, additional agents can be included in their composition. Examples of such additives include fats, waxes, oils, colorants, flavors, preservatives, stabilizers, and surfactants. Keratolytic agents such as keratolytics known in the art may also be included. Examples of keratolytics include salicylic acid and sulfur. Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalers. The agent of the invention may be mixed under sterile conditions with a pharmaceutically acceptable carrier and with any preservatives, buffers or propellants that may be required. Ointments, pastes, creams and gels, in addition to the agent according to the invention, may contain excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, organosilicon compounds, bentonites, silicic acid, talc, oxide zinc or mixtures thereof. Powders and sprays, in addition to the agent according to the invention, may contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates, polyamide powder or mixtures of these substances. Sprays may additionally contain standard propellants such as chlorofluorocarbons and volatile unsubstituted hydrocarbons such as butane and propane.

Pharmaceutical compositions suitable for parenteral administration may contain one or more of the compositions of the invention in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders, which can be reconstituted into sterile injectables shortly before use. solutions or dispersions, which may contain antioxidants, buffers, bacteriostatics, soluble components that provide isotonicity to the blood composition of the receiving recipient, or suspending agents or thickeners. Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the invention 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 injectables. organic esters such as ethyl oleate. The required fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifiers and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonicity agents such as sugars, sodium chloride, and the like in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be provided by the inclusion of absorption delaying agents such as aluminum monostearate and gelatin.

Depot injectable forms are prepared by forming microencapsulated matrices with one or more particles in biodegradable polymers 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 depot formulations are also prepared by encapsulating the drug substance in liposomes or microemulsions that are compatible with body tissues.

In a preferred embodiment, the compositions of the invention are prepared by standard methods as pharmaceutical compositions adapted for intravenous administration to humans or animals, such as pets. When necessary, the composition may also include a solubilizer and a local anesthetic such as lidocaine to reduce pain at the injection site. When the composition is to be administered by infusion, it may be placed in an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampoule of sterile water or saline for injection may be provided so that the ingredients may be mixed prior to administration.

In another embodiment, the compositions (eg, particles or particles) described herein are formulated for subcutaneous, intraperitoneal, or intramuscular administration to humans or animals, such as pets.

In some embodiments, the agents and particles of the invention are in a form for local delivery to a tumor, such as for delivery by intratumoral injection.

In some embodiments, the composition is for topical administration to the liver via the hepatic portal vein, and the agents and particles can be formulated into an appropriate formulation.

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In some embodiments, a particular drug is suitable for administration by more than one route. Thus, for example, a formulation suitable for intravenous infusion may also be suitable for delivery through the hepatic portal vein. However, in other embodiments of the invention, the drug is suitable for use in one route of administration, but not suitable for another route of administration.

An amount of an agent or particles of the invention that is effective in treating a pathological condition such as cancer and/or effective in neutralizing soluble TNFR and/or effective in reducing the amount of TNFa or its binding activity by soluble TNFR, in particular soluble TNFR present in the microenvironment tumor and optionally in plasma, and/or effective in inhibiting the proliferation, growth or survival of tumor cells in vitro or in vivo, can be determined by standard clinical or laboratory methods. In addition, in vitro assays may optionally be performed to assist in determining optimal dosage ranges. The exact dosage of the drug to be administered also depends on the route of administration and the severity of the patient's condition, and should be adjusted according to the judgment of the practitioner and the condition of each subject. Effective doses for administration to humans or animals can be extrapolated from dose-response curves obtained in in vitro test systems or animal models.

In some embodiments, pharmaceutical compositions of the invention are non-pyrogenic. In other words, in some embodiments, the compositions are substantially free of pyrogens. In one embodiment, the formulations of the invention are pyrogen-free formulations that are substantially free of endotoxins and/or related pyrogens. Endotoxins include toxins that are contained within microorganisms and are only released when the microorganisms are destroyed or killed. Pyrogens also include fever-inducing thermostable substances (glycoproteins) from the outer membrane of bacteria and other microorganisms. All of these substances, when administered to humans, can cause fever, pressure drop, and shock. Due to possible adverse effects, even small amounts of endotoxins must be removed from solutions of pharmaceutical drugs administered intravenously. The United States Pharmacopeial Convention for intravenous drugs has an upper limit of 5 endotoxin units (EE) per dose per kilogram of body weight over a period of one hour (The United States Pharmacopeial Convention). , Pharmacopeial Forum, 26(1):223 (2000)). When therapeutic proteins are administered at relatively high doses and/or over a long period of time (eg, such as for the lifetime of a patient), even small amounts of detrimental and harmful endotoxin can be harmful to the patient. In some specific embodiments, the concentrations of endotoxins and pyrogens in the composition are less than 10 EU/mg, or less than 5 EU/mg, or less than 1 EU/mg, or less than 0.1 EU/mg, or less than 0.01 EU/mg , or less than 0.001 EU/mg.

All of the above applies to any of the agents, compositions and methods of the invention described herein. The invention contemplates any combination of the characteristics of the agents of the invention, compositions and methods described herein (singly or in combination) with the characteristics described for the various pharmaceutical compositions and routes of administration described in this section and above.

The description provides many general and specific examples of agents and categories of agents suitable for use in the methods of the invention (agents of the invention). The invention contemplates that any of such agents or categories of agents may be formulated for in vitro or in vivo administration as described herein.

In addition, in some embodiments of the invention, compositions are provided, including pharmaceutical compositions containing any agent of the invention described herein, prepared with one or more pharmaceutically acceptable carriers and/or excipients. Such compositions can be described using any of the functional and/or structural characteristics of the agent of the invention, from among those given in this document. Any such compositions or pharmaceutical compositions can be used under in vitro or in vivo conditions in any of the methods of the invention.

Similarly, the invention provides an isolated or purified agent of the invention. An agent of the invention described in terms of any of the functional and/or structural characteristics of an agent disclosed herein may be provided as an isolated agent or a purified agent. Such isolated or purified agents have many in vitro or in vivo applications, including use in any of the in vitro or in vivo methods described herein.

Applications.

The compositions (eg, particles and their pharmaceutical compositions) described herein are suitable for many diagnostic and therapeutic applications. For example, the particles described herein can be used to treat cancer, detoxify a subject, or treat viral or bacterial infections.

Therapeutic uses include administering one or more of the compositions described herein to a subject, such as a human, in a variety of ways that depend in part on the route of administration. The route of administration may be, for example, intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneal (IP) injection or intramuscular injection (IM).

The introduction can be carried out, for example, by local infusion, injection or by means of an implant. The implant may be a porous, non-porous, or gelatinous material, including membranes such as sialastic membranes, or fibers. The implant can be configured to deliver the composition in a subject for sustained or intermittent release. See, for example, US Patent Application Publication US 2008/0241223; US patents US 5501856; US 5164188; US 4863457 and US 3710795; EP 488401 and EP 430539, the entire content of which is incorporated here by reference. The composition can be delivered to the subject via an implantable device based on, for example, diffusion, eroding, or convection systems, e.g., osmotic pumps, biodegradable implants, electrodiffusion systems, electroosmotic systems, vapor pressure pumps, electrolytic pumps, effluent pumps, piezoelectric pumps , erosion based systems or electromechanical systems.

As used herein, an effective amount or a therapeutically effective amount under in vivo conditions means a dose sufficient to treat, inhibit, or alleviate one or more symptoms of the disorder being treated, or otherwise provide the desired pharmacological and/or physiological effect, e.g., modulating (e.g., enhancing ) immune response to an antigen. The exact dose will vary depending on a number of factors such as subject-related variables (eg, age, immune system status, etc.), disease, and treatment being administered.

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

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

As used herein, a mammal in need of prevention, in need of treatment, or in need of it refers to a mammal that, at the discretion of a competent medical practitioner (e. mammals) can get an objective positive effect from the treatment received.

The term prevention is accepted in the art and widely used in relation to a pathological condition, and in this area it includes the introduction of a composition that reduces the frequency or delays the onset of symptoms of a medical condition in a mammal compared to a subject who has not received such a composition.

Suitable human doses of any of the compositions described herein can be further evaluated, for example, in phase I dose escalation studies. See, for example, van Gurp et al., Am. J. Transplantation, 8(8):1711-1718 (2008); Hanouska et al. Clin. cancer. Res., 13(2, part 1):523-531 (2007); and Hetherington et al., Antimicrobial Agents and Chemotherapy, 50(10):3499-3500 (2006).

The method may further include the step of measuring the concentration of the target biomolecule in the subject (eg, in the subject's serum) prior to administering to the subject a composition comprising a plurality of particles that target the biomolecules. The method may further include calculating the number of particles to be administered to the subject, for example, based on the concentration of biomolecules in the subject (eg, in the subject's blood serum) and/or the subject's height, weight, and/or age.

The toxicity and therapeutic efficacy of such compositions can be determined by known pharmaceutical methods in cell cultures or experimental animals (eg, animal models of cancer, toxicity, or infections). These methods can be used, for example, to determine LD ₅₀ (dose lethal in 50% of the population) and ED ₅₀ (dose therapeutically effective in 50% of the population). The toxic to therapeutic dose ratio is the therapeutic index and can be expressed as the ratio LD ₅₀ /ED ₅₀ . Agents that have a high therapeutic index are preferred. Compositions that have toxic side effects can be used, however care should be taken to develop delivery systems that deliver such compounds to the site of diseased tissue and to minimize potential damage to normal cells, thus reducing side effects.

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Data from cell culture assays and animal studies can be used to form a dosage range for use in humans. Typically, the dose of such compositions is in the range of circulating composition concentrations that includes the ED ₅₀ with little or no toxicity. The dose within this range may vary depending on the dosage form used and the route of administration. Initially, a therapeutically effective dose can be calculated based on cell culture assays. The dose can be adjusted in animal models to achieve a circulating plasma concentration range that includes the IC ₅₀ (eg, the concentration of antibody that provides half the maximum inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine appropriate doses in humans. Plasma concentrations can be determined, for example, by high performance liquid chromatography (HPLC). In some embodiments of the invention, for example, when local administration is desired, cell culture or animal modeling may be used to determine the dose required to achieve a therapeutically effective concentration at the local site.

In some embodiments of any of the methods described herein, the particles may be administered to a mammal in combination with one or more additional therapeutic agents/agents (eg, therapeutic agents for treating infection or treating cancer).

In some embodiments, the particles and additional therapeutic agent can be administered to a mammal using various routes of administration. For example, the additional therapeutic agent may be administered subcutaneously or intramuscularly, and the particles may be administered intravenously.

In some embodiments, the method of the invention comprises measuring the concentration of a biomolecule in a subject. For example, the method may include measuring the concentration of the biomolecule in the blood of the subject. The method may further include administering to the subject a composition comprising a plurality of particles that target the biomolecule (ie, a plurality of particles as described herein containing an agent that selectively binds to the biomolecule). The measuring step can ensure proper dosing of the particles. Thus, the measurement step can be performed prior to administration of the composition. However, a measurement step may be performed after administration of the composition, for example to evaluate the effectiveness of the composition. The method may further comprise administering to the subject a second or subsequent dose of the multiparticulate composition, eg, if warranted based on the measured concentration of the biomolecule. Thus, the concentration of a biomolecule can be titrated, for example, by iteratively measuring the concentration of biomolecules in a subject while administering the composition at various doses or levels. Similarly, the number of particles administered to a subject can be titrated against the concentration of the biomolecule targeted by the particles.

Titration of the concentration of the biomolecule in a subject, or the amount of particles administered to a subject, can be particularly useful, for example, when the biomolecule promotes deleterious local effects (eg, in a tumor) but has a beneficial systemic effect. Thus, a plurality of particles can be administered either at or near a specific location in a patient so that the biomolecules are localized at that location, and the systemic concentration of the biomolecules can be monitored to determine if additional particles can be safely administered to the subject.

Titration of the concentration of the biomolecule in the subject or the number of particles administered to the subject may also be useful, for example to maintain the concentration of the biomolecule within a predetermined range. The predetermined range may be a range that corresponds to a healthy state, such as in the case of an overproduction of biomolecules in a subject, or when the predetermined range is the therapeutic range. Such titration may be particularly useful in methods of treating diseases caused by excessive secretion of hormones. For example, the particle may contain an agent that binds to a growth hormone biomolecule, for example, for use in a method of treating acromegaly or gigantism, and such particles can be titrated to ensure that growth hormone levels remain within the range of a healthy patient. The particle may contain an agent that binds to the thyroxine and/or triiodothyronine biomolecule, for example for use in a method for treating hyperthyroidism, and such particles can be titrated to ensure that the thyroxine and/or triiodothyronine levels remain in the range of a healthy patient. The particle may contain an agent that binds to an adrenocorticotropic hormone or cortisol biomolecule, for example for use in a method of treating Cushing's disease, and such particles can be titrated to ensure that the levels of adrenocorticotropic hormone and/or cortisol remain in the range corresponding to that of a healthy patient. An example of a therapeutic range includes titration of a blood clotting factor such as factor VIII, factor IX or factor XI in a range that inhibits blood clotting for a certain period of time. Such a range may be below the normal concentration, the range corresponding to the level of a healthy patient, and yet the therapeutic range may be useful, for example, to inhibit thrombosis or ischemia in some patients.

Adoptive cell transfer therapy.

- 49 041139

The method may include administering a composition comprising a plurality of particles described herein to a subject who has received adoptive cell transfer therapy (ACT). The method may include administering a composition comprising a plurality of particles described herein to a subject who may benefit from adoptive cell transfer therapy. The method may further comprise administering adoptive cell transfer therapy to the subject before, after, or simultaneously with administration of the multiparticulate composition.

Adoptive cell transfer therapy may include administering to a subject a composition containing lymphocytes. The lymphocytes may be T lymphocytes (eg, T cells), such as tumor infiltrating lymphocytes (TILs). In preferred embodiments, the lymphocytes are T lymphocytes, such as tumor infiltrating lymphocytes. A composition containing lymphocytes may be substantially free of cells that are not lymphocytes, for example, the composition may be substantially free of cells and cell fragments derived from myeloid progenitor cells (e.g., erythrocytes, mast cells, basophils, neutrophils, eosinophils, monocytes, macrophages, megakaryocytes, platelets). A composition containing lymphocytes may be substantially free of cells that are not T cells, for example, the composition may be substantially free of natural killer cells, B cells and/or plasma cells. The composition containing lymphocytes may contain cells, where the cells consist mainly of T cells. A composition containing lymphocytes may be substantially free of cells that are not tumor infiltrating lymphocytes. The composition containing lymphocytes may contain tumor infiltrating lymphocytes. The composition containing lymphocytes may contain cells, where the cells consist mainly of tumor infiltrating lymphocytes.

The composition containing lymphocytes may contain recombinant lymphocytes, for example, where the lymphocytes contain exogenous nucleic acid. For example, lymphocytes may contain a chimeric antigen receptor (CAR). Similarly, lymphocytes may include a knockout gene, such as one that reduces the risk of graft-versus-host or host-versus-graft immune response (eg, in non-autologous transplantation, such as allogeneic transplantation). In some embodiments, the lymphocyte-containing composition may include recombinant T cells, such as recombinant tumor infiltrating lymphocytes, wherein, for example, the lymphocytes may be recombinant T cells, such as recombinant tumor infiltrating lymphocytes.

Adoptive cell transfer therapy may include autologous transplantation or non-autologous transplantation, such as allogeneic transplantation.

A subject may receive adoptive cell transfer therapy about 1 year prior to administration of a composition of the invention to the subject, e.g., about 6 months, about 5 months, about 4 months, about 3 months, about 2 months, about 1 month, approximately 4 weeks approximately 3 weeks approximately 2 weeks approximately 14 days approximately 13 days approximately 12 days approximately 11 days approximately 10 days approximately 9 days approximately 8 days about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or 1 day before administration of the composition of the invention to the subject. The method may include administering the multiparticulate composition to the subject less than about 1 year after administration of the lymphocyte-containing composition to the subject, e.g., less than about 6 months, about 5 months, about 4 months, about 3 months, after about 2 months, after about 1 month, after about 4 weeks, after about 3 weeks, after about 2 weeks, after about 14 days, after about 13 days, after about 12 days, after about 11 days, after about 10 days, approximately 9 days, approximately 8 days, approximately 7 days, approximately 6 days, approximately 5 days, approximately 4 days, approximately 3 days, approximately 2 days, or approximately 1 day after administration of the composition containing lymphocytes to the subject . The method may include administering the multiparticulate composition to the subject within about 1 year of administration of the lymphocyte composition to the subject, e.g., within about 6 months, about 5 months, about 4 months, about 3 months, about 2 months, about 1 month. , approximately 4 weeks, approximately 3 weeks, approximately 2 weeks, approximately 14 days, approximately 13 days, approximately 12 days, approximately 11 days, approximately 10 days, approximately 9 days, approximately 8 days, approximately 7 days, approximately 6 days, approximately 5 days, about 4 days, about 3 days, about 2 days, or within about 1 day after administration of a composition containing lymphocytes to a subject.

Adoptive cell transfer therapy may be particularly effective in patients who have neoplasms such as cervical cancer, breast cancer, lymphoma, leukemia,

- 50 041139 chronic lymphocytic leukemia, follicular lymphoma, large cell lymphoma, lymphoblastic leukemia, myeloid leukemia, multiple myeloma, bile duct cancer, colon cancer, neuroblastoma, lung cancer, sarcoma, synovial sarcoma or melanoma. However, adoptive cell transfer therapy may be useful for treating other diseases, such as serious or life-threatening infections (eg, HIV (HIV)).

Selected applications related to neoplasms.

In some embodiments, the particles described herein may be useful in the treatment of a subject with cancer. Exemplary agents useful in the particle compositions described herein and/or soluble biomolecules that can be captured by such particles are described herein (see, for example, Table 2) and are known in the art. For example, particles capable of capturing sTNFR, MMP2, MMP9, sIL-2R, sIL-1 receptor, and the like are useful in treating cancer and/or enhancing the immune response in cancer by facilitating the disinhibition of the immune system.

The immune system disinhibition approach used in immunotherapy is partly based on the concept that many cancer patients are generally immunocompetent overall, but their immune systems are locally inhibited in the tumor microenvironment. By reducing this inhibition of the immune system by administering the particles of the invention, the patient's own immune system can act on the tumor. Thus, in some embodiments, the particles of the invention provide a method of immunotherapy without the need to hyperstimulate the patient's immune system by adding exogenous active cytokines designed to bind to cell surface receptors to elicit an immune response, and/or without otherwise hyperstimulating the patient's immune system.

In practice, since cancer patients are generally immunocompetent, the ability of lymphocytes to recognize tumor antigens is generally not suppressed by the tumor. Thus, lymphocytes enter the tumor microenvironment as they would with any aberrant cell cluster, after which cytokines and cytotoxic factors such as tumor necrosis factor (TNF, such as TNFa, the immune system's major cytotoxic sword) are cleaved from the lymphocytes into microenvironment. If instead of malignant cells, the cells are infected with viruses, then TNF (such as TNFa) will bind to the TNF receptor (TNFR) on the surface of the infected cell, which will lead to their rapid destruction through apoptosis or oxidative stress, depending on the binding of TNF to the receptor type R1 or R2 . In other words, in the event of a normal immune response that is not stimulated by the presence of tumor and/or tumor antigens, TNF secreted by lymphocytes will be available to bind to TNF receptors (R1 and/or R2 type receptors) on the cell surface as part of the development of the immune response. Even in the case of a tumor, lymphocytes migrate to the site of the tumor.

However, many types of cancer cells behave differently than other types of aberrant cells, such as virus-infected cells, mainly in that they overproduce TNF receptors (both types) and release them into the tumor environment. Thus, the microenvironment of cancer cells and/or tumors contains certain amounts of soluble TNF receptors. Without being bound by any theory, it is clear that concentrations of soluble TNF receptors in the microenvironment of tumors are superior to levels observed in the microenvironment of healthy cells, such as normal tissue cells of the same type. Additionally or alternatively, the rate and amount of TNF receptor released in malignant cells is greater than in healthy cells. In addition, without being bound by any theory, it is clear that the levels of soluble TNF receptors found in the plasma of cancer patients may in some embodiments be higher than in healthy subjects.

Regardless of the mechanism of this model, these secreted soluble TNF receptors bind to TNF endogenously released by recruited lymphocytes, neutralize endogenous TNF, and effectively create a region of immunological privilege around the tumor in which the tumor continues to grow and secrete additional TNF receptors. In other words, secreted soluble TNF receptors take up TNFa endogenously produced by lymphocytes and prevent or inhibit the binding of this TNF to TNF receptors on the surface of cancer cells. This reduces or removes TNF available to bind to TNF receptors on the surface of cancer cells. Substantially soluble TNF receptors outcompete other TNFs for TNFa binding and thus reduce the activity of TNF, such as TNFa, to bind TNF receptors on the cell surface.

Similarly, the scenario described above can occur with IL-2 and isolated soluble IL-2 receptors.

In some embodiments, the biomolecule is a toxin released by cancer cells during apoptosis.

The present invention provides pharmacological approaches that can be used systemically or topically to reduce immune system suppression (e.g., to disinhibit

- 51 041139 of the immune system), caused by secreted receptors in cancer. The present invention 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 leading to immune system disinhibition), for example, in the microenvironment of cancer cells. and tumors. Without wishing to be bound by any theory, it is clear that reducing the number and/or activity of, for example, soluble TNF receptors (such as those in the tumor microenvironment) can be used as part of a method for inhibiting cell proliferation, growth, or survival, such as cancer cells. In some embodiments of the invention, this can be used to inhibit the survival of cells, such as cancer cells. Illustrative methods and agents are described here.

Regulatory T cells (TREGs), as a way to reduce the immune response to avoid, for example, an autoimmune disease caused by overactive T cells or long-term functioning of T cells, can secrete the same ligands as cancer cells. For example, CD80/B7-1 and CD86/B7-2 bind to the CTLA-4 receptor on T cells and inhibit T cell activity. Instead of blocking the CTLA-4 receptor, the particles described here can be designed to capture CD80/B7-1 and/or CD86/B7-2. Similarly, the particles described herein can be designed to capture other immune checkpoint inhibitors such as PD-1L, for example, using particles containing the PD-1 receptor. Such particle compositions provide a distinct advantage over other approaches to stimulating the immune system for cancer treatment.

The target may be soluble PD-L2, for example, to inhibit the interaction between soluble PD-L2 and PDL. The agent may be a PDL. Inhibition of the interaction between soluble PD-L2 and PDL may allow PDL to bind membrane-bound PD-L2 variants, thereby promoting cancer cell apoptosis. The target may be soluble PDL. The agent may be a PDL ligand such as PD-L2, soluble PD-L2 or variants thereof, or anti-PDL antibodies such as nivolumab or pembrolizumab. PDL-targeting particles (ie, soluble PDL) and their ligands may be particularly useful in the treatment of autoimmune disease in addition to other diseases and conditions.

The target may be soluble CTLA4, for example, to inhibit the interaction between B7-1 or B7-2 and soluble CTLA4. The agent may be a CTLA4 ligand such as soluble B7-1, soluble B7-2, or variants thereof, or anti-CTLA4 antibodies such as ipilimumab or 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 promoting T cell activation. Particles targeted to CTLA4 (ie, soluble CTLA4) may be particularly useful in the treatment of melanoma and lung cancer, such as non-small cell lung cancer, in addition to other diseases and conditions.

The agent may be a protein that specifically binds to adenosine, such as the adenosine-binding portion of the adenosine receptor. The target may be adenosine. Adenosine-targeting particles may be particularly useful in the treatment of solid tumors, and such particles can be introduced into a solid tumor, for example, to inhibit adenosine signaling within the tumor microenvironment.

The agent may be an osteoprotegerin or a ligand-binding portion thereof, for example for selective binding of osteoprotegerin ligands. Particles targeted to osteoprotegerin ligands may be particularly useful in the treatment of cancer, such as breast cancer, in addition to other diseases and conditions.

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

As used herein, a subject at risk of developing cancer is one with one or more (eg, two, three, four, five, six, seven, or eight or more) risk factors for developing cancer. For example, a subject at risk of developing cancer may have a predisposition to developing cancer (e.g., a genetic predisposition to developing cancer, such as a mutation in a tumor suppressor gene (e.g., a mutation in BRCA1, p53, RB, or APC) or the subject is exposed to conditions such as , which can lead to a pathological condition). Thus, a subject may be a subject at risk of developing cancer when the subject is exposed to mutagenic or carcinogenic concentrations of certain compounds (e.g., carcinogenic compounds in cigarette smoke such as acrolein, arsenic, benzene, benzo[a]anthracene, benzo[a] pyrene, polonium-210 (Radon), urethane or vinyl chloride). In addition, the subject may be at risk of developing cancer when the subject is exposed to, for example, high doses of ultraviolet light or X-rays, or is exposed to (for example, infection) by a tumor-causing or tumor-associated virus, such as papillomavirus,

- 52 041139 Epstein-Barr virus, hepatitis B virus or human T-cell leukemia-lymphoma virus. Cancers are a class of diseases or disorders characterized by the uncontrolled division of cells and their ability to spread through direct growth into neighboring tissue through invasion or penetration into distant sites through metastasis (when malignant cells are transported through the bloodstream or lymphatic system). Cancer can affect people of all ages, but the risk tends to increase with age. Types of cancers or malignancies may include, for example, lung cancer, breast cancer, colon cancer, pancreatic cancer, kidney cancer, gastric cancer, liver cancer, bone cancer, hematologic cancer, nervous tissue cancer (e.g., glioblastoma such as glioblastoma multiforme), melanoma, thyroid cancer, ovarian cancer, testicular cancer, prostate cancer, cervical cancer, vaginal cancer, or bladder cancer. In some preferred embodiments, the patient (or subject) has brain cancer, endometrial cancer, prostate cancer, kidney cancer, or squamous cell cancer (e.g., head and neck squamous cell carcinoma), each of which is particularly sensitive to extracellular biomolecules that can increase disease severity. .

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

A subject suspected of having cancer or infection is one who has one or more symptoms of cancer or infection. It should be understood that subjects at risk of developing or suspected of having cancer or infection do not include all subjects to be treated.

In some embodiments, the methods include determining whether a subject has cancer or an autoimmune disease.

Selected applications related to inflammatory disorders and autoimmune disorders.

In some embodiments of the present invention, the particles described herein can be used to treat an inflammatory disorder and/or an autoimmune disorder. Typical agents used in the compositions of the particles described here, and/or soluble biomolecules that can be captured by these particles, which are described here (see, for example, table. 2), are known in the art. For example, particles capable of capturing cytokines (eg, TNFa or interleukins such as IL-2, IL-6, or IL-1) or chemokines (eg, CXCL8 or CXCL1) may be useful in the treatment of various autoimmune and/or inflammatory conditions. diseases.

The agent may be soluble CD28 or a ligand-binding portion thereof, eg for selective binding of CD28 ligands such as soluble B7 (eg soluble B7-1 or soluble B7-2). The agent may be galiximab. The target may be a CD28 ligand such as soluble B7. Particles targeting CD28 ligands may be particularly useful in the prevention or treatment of lupus, such as systemic lupus erythematosus, in addition to other diseases and conditions.

The agent may be an anti-B7-H4 antibody, for example, to selectively bind soluble B7-H4. The target can be soluble B7-H4. Soluble B7-H4 targeting particles may be particularly useful in the treatment of arthritis such as rheumatoid arthritis and juvenile idiopathic arthritis, in addition to other diseases and conditions.

The agent may be a soluble CD278 (inducible costimulatory ligand; ICOS) or a ligand-binding portion thereof, for example for selective binding of CD278 ligands such as ICOSL (inducible costimulatory ligand; CD275). The target may be a CD278 ligand such as ICOSL. Particles targeting CD278 ligands may be particularly useful in the prevention or treatment of lupus, such as systemic lupus erythematosus, in addition to other diseases and conditions.

The agent may be an anti-CD275 antibody, eg for selective binding of CD275 (an inducible costimulatory ligand; ICOSL). The target may be CD275. Particles targeting CD275 may be particularly useful in the prevention or treatment of lupus, such as systemic lupus erythematosus, in addition to other diseases and conditions.

The agent may be an anti-CD40L antibody such as dapirolizumab, ruplizumab or toralizumab, for example to selectively bind CD40L (CD40 ligand; CD154). The target may be CD40L. Particles targeted to CD40L may be particularly useful for the prevention or treatment of lupus such as systemic lupus erythematosus, arthritis such as rheumatoid arthritis, collagen-induced arthritis and juvenile idiopathic arthritis, and Sjögren's syndrome, in addition to other diseases and conditions.

The agent may be soluble CD134 (OX40) or a ligand-binding portion thereof, eg for selective binding of CD134 ligands such as CD252 (OX40 ligand; OX40L). The target may be a CD134 ligand such as CD252. Particles targeting CD134 ligands can

- 53 041139 be particularly useful for the prevention or treatment of lupus, such as lupus nephritis, its symptoms, such as glomerulonephritis, and systemic sclerosis, in addition to other diseases and conditions.

The agent may be 4-1BB (CD137) or a ligand-binding portion thereof, for example for selective binding of 4-1BB ligands such as soluble 4-1BB ligand (soluble 4-1BBL). The target may be a 4-1BB ligand, such as a soluble 4-1BB ligand. Particles targeted to 4-1BB ligands may be particularly useful in the prevention or treatment of lupus, such as systemic lupus erythematosus, and arthritis, such as rheumatoid arthritis, in addition to other diseases and conditions.

The agent may be a 4-1BB ligand, for example, to selectively bind soluble 4-1BB (soluble CD137). The agent may be an anti-4-1BB antibody such as urelumab. The target can be soluble 4-1BB. Particles targeting soluble 4-1BB may be particularly useful in the prevention or treatment of arthritis, such as rheumatoid arthritis, in addition to other diseases and conditions, including cancer. In some embodiments, the inflammatory disease may be, for example, acute disseminated encephalomyelitis; Addison's disease; ankylosing spondylitis; antiphospholipid antibody syndrome; autoimmune hemolytic anemia; autoimmune hepatitis; autoimmune disease of the inner ear; bullous pemphigoid; Chagas disease; chronic obstructive pulmonary disease; celiac disease; dermatomyositis; diabetes mellitus type 1; diabetes mellitus type 2; endometriosis; Goodpasture's syndrome; Graves' disease; Guillain-Barré syndrome; Hashimoto's disease; idiopathic thrombocytopenic purpura; interstitial cystitis; systemic lupus erythematosus (SLE); metabolic syndrome, multiple sclerosis; myasthenia gravis; myocarditis, narcolepsy; obesity; ordinary pemphigus; pernicious anemia; polymyositis; primary biliary cirrhosis of the liver; rheumatoid arthritis; schizophrenia; scleroderma; Sjögren's syndrome; vasculitis; vitiligo; Wegener's granulomatosis; allergic rhinitis; prostate cancer; non-small cell lung cancer; ovarian cancer; mammary cancer; melanoma; stomach cancer; colorectal cancer; brain cancer; metastatic bone disease; pancreas cancer; lymphoma; polyps in the nasal cavity; gastrointestinal cancer; ulcerative colitis; Crohn's disease; collagen colitis; lymphocytic colitis; ischemic colitis; colitis in the disconnected gut; Behçet's syndrome; infectious colitis; indefinite 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 proliferation disorders, meningitis, herpes zoster, encephalitis, nephritis, tuberculosis, retinitis, atopic dermatitis, pancreatitis, periodontal gingivitis, coagulative necrosis, coliquational necrosis, fibrinoid necrosis, hyperacute graft rejection, acute graft rejection, chronic graft rejection, acute graft versus host disease, chronic graft versus host disease, or combinations of any of the above disorders and diseases. In some embodiments, the autoimmune or inflammatory disorder or disease is, 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 syndromes, autoimmune hepatitis, autoimmune neuropathies, autoimmune ovarian failure, autoimmune orchitis, autoimmune thrombocytopenia, reactive arthritis, ankylosing spondylitis, silicone implant related autoimmune disease , Sjögren's syndrome, systemic lupus erythematosus (SLE), vasculitis syndromes (eg, giant cell arteritis, Behcet's disease, and Wegener's granulomatosis), vitiligo, secondary hematologic manifestation of autoimmune diseases (eg, anemia), drug-induced autoimmune disorders hashimoto's thyroiditis, hypophysitis, idiopathic platelet purpura, metal-induced autoimmune disorders, myasthenia gravis, pemphigus, autoimmune deafness (eg, Meniere's disease), Goodpasture's syndrome, Graves' disease, HIV-related autoimmune syndromes, and/or Guillain-Barré disease.

In some embodiments, the autoimmune or inflammatory disorder is a hypersensitivity reaction. As used here, hypersensitivity refers to an unwanted response of the immune system. Hypersensitivity is divided into four categories. Type I hypersensitivity includes allergies (eg, atopy, anaphylaxis, or asthma). Type II hypersensitivity is mediated by cytotoxicity/antibodies (eg, autoimmune hemolytic anemia, thrombocytopenia, hemolytic anemia of the newborn, or Goodpasture's syndrome). Type III is an immune complex disease (eg, serum sickness, Arthus phenomenon, or SLE). Type IV is delayed type hypersensitivity (DTH),

- 54 041139 memory cell-mediated immune response and antibody response (eg contact dermatitis, tuberculin skin test or chronic transplant rejection). As used herein, allergy means a disorder characterized by excessive activation of mast cells and basophils by the action of IgE. In some cases, excessive IgE-mediated activation of mast cells and basophils results in a (partial or complete) inflammatory response. In some cases, the inflammatory response is local. In some cases, the inflammatory response leads to narrowing of the airways (eg, bronchospasm). In some cases, the inflammatory response leads to inflammation of the nose (eg, rhinitis). In some cases, the inflammatory response is systemic (eg, anaphylaxis).

In some embodiments, the methods include determining if the subject has an autoimmune disease.

Selected applications related to pathogenic organisms and toxins.

In some embodiments, the particles described herein can be designed to bind to microorganisms (eg, viruses or bacteria) or components of microorganisms, such as endotoxins. Thus, the particles described herein may be useful in the treatment of, for example, an infectious disease caused by a virus, including HPV, HBV, hepatitis C virus (HCV), retroviruses such as human immunodeficiency virus (HIV-1 and HIV-2) , herpes viruses such as Epstein-Barr virus (EBV), cytomegalovirus (CMV), HSV-1 and HSV-2, and influenza virus. Also included are bacterial, fungal, and other pathogenic infections caused by microorganisms such as Aspergillus, Brugia, Candida, Chlamydia, Coccidia, Cryptococcus, Dirofilaria, Gonococcus, Histoplasma, Leishmania, Mycobacterium, Mycoplasma, Paramecium, Pertussis, Plasmodium, Pneumococcus, Pneumocystis. , Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Toxoplasma and Vibrio cholerae. Illustrative species include Neisseria gonorrhea, Mycobacterium tuberculosis, Candida albicans, Candida tropicalis, Trichomonas vaginalis, Haemophilus vaginalis, Group B Streptococcus spp., Microplasma hominis, Hemophilus ducreyi, Granuloma inguinale, Lymphopathia venereum, Treponema pallidum, Brucella abortus. Brucella melitensis Brucella suis Brucella canis Campylobacter fetus Campylobacter fetus intestinalis Leptospira pomona Listeria monocytogenes Brucella ovis Chlamydia psittaci Trichomonas foetus Toxoplasma gondii Escherichia coli , Corynebacterium equi, Corynebacterium pyogenes, Actinobaccilus seminis, Mycoplasma bovigenitalium, Aspergillus fumigatus, Absidia ramosa, Trypanosoma equiperdum, Babesia caballi, Clostridium tetani, Clostridium botulinum; or fungi such as, for example, Paracoccidioides brasiliensis; or other pathogenic organisms such as Plasmodium falciparum. Also included are priority pathogens identified by the National Institute of Allergy and Infectious Diseases (NIAID). These include category A agents such as Variola Major (variola), Bacillus anthracis (anthrax), Yersinia pestis (plague), Clostridium botulinum toxin (botulism), Francisella tularensis (tularemia), filoviruses (Ebola haemorrhagic fever, Marburg), arenaviruses (Lassa (Lassa fever), Junin virus (Argentine hemorrhagic fever) and related viruses); category B agents such as Coxiella burnetti (Q fever), Brucella spp. (brucellosis), Burkholderia mallei (candida), alphaviruses ( Venezuelan encephalomyelitis, eastern and western equine encephalomyelitis), ricin toxin from Ricinus communis (castor bean), epsilon toxin from Clostridium perfringens; enterotoxin B from Staphylococcus, Salmonella spp., Shigella dysenteriae, Escherichia coli strain O157:H7, Vibrio cholerae, Cryptosporidium parvum; agents category C, such as Nipah virus, hantaviruses, tick-borne hemorrhagic fever viruses, tick-borne encephalitis viruses, yellow fever and multiresistant tuberculosis; helminths such as Schistosoma and Taenia; and protozoa such as Leishmania (eg L. mexicana) and Plasmodium.

The target may be a viral protein. Viral protein can be from arbovirus, adenovirus, alphavirus, arenaviruses, astrovirus, BK virus, bunyaviruses, calicivirus, marmoset herpes virus 1, Colorado tick fever virus, coronavirus, Coxsackie virus, Crimean Congo hemorrhagic fever virus, cytomegalovirus, dengue virus, virus Ebola, echinovirus, ECHO virus, enterovirus, Epstein-Barr virus, flavivirus, foot-and-mouth disease virus, hantavirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes simplex virus I, herpes simplex virus II, human herpes simplex virus, immunodeficiency virus human type I (HIV-1; HIV-1), human immunodeficiency virus type II (HIV-II; HIV-2), 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, lentiviruses, Machupo virus, Marburg virus, measles virus, mumps virus, Neapolitan fever virus, norovirus, virus a Norfolk, orbivirus, orthomyxvirus, papillomavirus, papovavirus, parainfluenza virus, paramyxvirus, parvovirus, picornavirus, poliovirus, polyomavirus, poxvirus, rabies virus, reovirus, respiratory syncytial virus, rhinovirus, rotavirus, rubella virus, sapovirus, poxvirus, toscanavirus, Tuscany 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

- 55 041139 locks.

The target may be a bacterial protein or a component of the bacterial cell wall. For example, the bacterial protein or cell wall component may be from Actinomyces israelii, Bacillus anthracis, Bacillus cereus, Bacteroides fragilis, Bartonella henselae, Bartonella Quintana, Bordetella pertussis, Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia recurrentis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diptheriae, Ehrlichia canis, Ehrlichia chaffeensis, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Francisella tularensis , Haemophilus influenzae, Haemophilus vaginalis, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Leptospira interrogans, Leptospira santarosai, Leptospira weilii, Leptospira noguchii, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulc erans, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Nocardia asteroides, Rickettsia rickettsii, Salmonella typhi, 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.

The target may be a protein from yeast or fungi, or a component of the cell wall of yeast or fungi. For example, a yeast or fungal protein or yeast or fungal cell wall component may be from Apophysomyces variabilis, Aspergillus clavatus, Aspergillus flavus, Aspergillus fumigatus, Basidiobolus ranarum, Candida albicans, Candida glabrata, Candida guilliermondii, Candida krusei, Candida lusitaniae, Candida parapsiiosis, Candida tropicalis, Candida stellatoidea, Candida viswanathii, Conidiobolus coronatus, Conidiobolus incongruous, Cryptococcus albidus, Cryptococcus gattii, Cryptococcus iaurentii, Cryptococcus neoformans, Encephalitozoon intestinalis, Enterocytozoon bieneusi, Exophiaia jeanselmei, Fonsecaea compacta, Fonsecaea pedrosoi, Geotrichum candidum, Histoplasma capsulatum, Lichtheimia corymbifera , Mucor indicus, Paracoccidioides brasiiiensis, Phialophora verrucosa, Pneumocystis carinii, Pneumocystis jirovecii, Pseudallescheria boydii, Rhinosporidium seeberi, Rhodotorula mucilaginosa, Stachybotrys chartarum, Syncephalastrum racemosum, or Rhizopus oryzae.

The target may be a toxin, such as a bacterial toxin, a plant toxin, or a zootoxin. The toxin may be, for example, melittin, brevetoxin, tetrodotoxin, chlorotoxin, tetanus toxin, bungarotoxin, botulinum toxin from Clostridium botulinum, ricin, epsilon toxin from Clostridium perfringen, enterotoxin B from Staphylococcus, or endotoxin.

The target can be bacterial cell wall lipopolysaccharide, lipopolysaccharide-binding protein, lipoteichoic acids, bacterial lipoprotein, bacterial peptidoglycan, lipoarabinomannan, bacterial flagella protein (e.g. flagellin), profilin, HSP70, zymosan, double-stranded RNA, bacterial ribosomal RNA or DNA containing unmethylated CpG.

In some aspects, the present invention provides a method for treating or preventing an infection caused by a pathogen, wherein the method comprises administering to a subject a composition comprising a plurality of particles as described herein. In some embodiments, the particle contains an agent that specifically binds to a pathogen biomolecule or a biomolecule derived from a pathogen. In some embodiments, the particle contains an agent that specifically binds to the subject's biomolecule (eg, when the biomolecule is produced by the subject), such as a cytokine or peroxiredoxin (eg, peroxiredoxin 1 or peroxiredoxin 2). For example, the method may include administering to the subject a composition comprising a plurality of particles that selectively bind to TNFa, interleukin 1, interleukin 6, interleukin 8, interleukin 12, interferon-gamma, macrophage migration inhibitory factor, GM-CSF, and/or blood clotting factor , for example, for the treatment or prevention of sepsis associated with an infection caused by a pathogen. In some embodiments of the invention, the method is a method of treating or preventing sepsis, where the method, for example, includes administering to the subject a composition containing a plurality of particles described here.

The target may be paracetamol (acetaminophen). The agent may be an antibody that specifically binds to paracetamol or an antigen-binding portion thereof. Particles that target paracetamol may be particularly useful in the treatment or prevention of paracetamol toxicity.

Selected applications related to nutrition and metabolism.

In some embodiments of the present invention, the particle described herein can be used to treat obesity, to treat an eating disorder, to reduce body weight, to promote a healthy diet, or to reduce appetite in a subject. For example, in some embodiments of the invention, particles containing an agent (for example, an antibody or soluble forms of the ghrelin receptor (GHSR)) that bind to ghrelin can be administered to a subject (for example, an overweight or obese subject) to reduce appetite in subject, treatment of obesity or a disorder associated with obesity or a violation of

- 56 041139 exchange of substances.

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

Obesity refers to a condition in which a mammal's body weight exceeds the medically recommended limits by at least about 20%, based on age and skeletal size. Obesity is characterized by fatty cell hypertrophy and hyperplasia. Obesity may be characterized by the presence of one or more phenotypes associated with obesity, including, for example, weight gain (as measured, for example, by body mass index or BMI), changes in anthropometry, basal rates of metabolic or total energy expenditure, chronic energy imbalance, increased fat mass as measured by e.g. DEXA (percentage of DEXA fat mass), altered maximal oxygen uptake (VO2), high fat oxidation, relatively high resting level, glucose tolerance, hyperlipidemia, insulin resistance and hyperglycemia . See. also, for example, Hopkinson et al., Am. J.Clin. Nutr., 65(2):432-8 (1997), and Butte et al., Am. J.Clin. Nutr., 69(2):299-307 (1999). Overweight individuals typically have a body mass index (BMI) between 25 and 30. Obese or obese individuals typically have a body mass index of 30 or higher. Obesity may or may not be associated with insulin resistance.

The terms obesity-related disease or obesity-related disorders or obesity-related conditions, which are used interchangeably, refer to a disease, disorder or condition that is related or associated directly or indirectly with obesity. Obesity-related diseases or obesity-related disorders or obesity-related conditions include, but are not limited to coronary heart disease/cardiovascular disease, hypertension, cerebrovascular disease, stroke, peripheral vascular disease, insulin resistance, impaired glucose tolerance, diabetes mellitus , hyperglycemia, hyperlipidemia, dyslipidemia, hypercholesterolemia, hypertriglyceridemia, hyperinsulinemia, atherosclerosis, endothelial cell proliferation and dysfunction, diabetic dyslipidemia, HIV-associated lipodystrophy, peripheral vascular disease, cholesterol gallstones, cancer, menstrual disorders, infertility, polycystic ovary, osteoarthritis, sleep apnea sleep, metabolic syndrome (syndrome X), type II diabetes, diabetic complications including diabetic neuropathies, nephropathies, retinopathy, cataracts, heart failure, inflammation, thrombosis, congestive heart failure, and any other some cardiovascular disease associated with obesity or overweight, and/or obesity associated asthma, respiratory and lung disorders.

In yet another aspect, the invention provides a method for increasing muscle mass or muscle strength in a subject in need thereof, wherein said method comprises administering to the subject one or more of the compositions described herein in an amount sufficient to increase muscle mass or muscle strength in the subject. Thus, for example, to increase muscle mass, a subject may be administered particles containing an agent (eg, an antibody or soluble activin receptor) that binds to myostatin.

In some embodiments, the subject is a subject having a muscle disorder (eg, a muscle wasting disorder).

Muscle wasting disorders referred to herein include disorders or conditions where muscle wasting is one of the main symptoms, such as muscular dystrophy, spinal cord injury, neurodegenerative diseases, anorexia, sarcopenia, cachexia, muscle atrophy due to immobilization, prolonged immobilization recumbency or weightlessness, and the like, as well as disorders in which an abnormally high ratio of fat to muscle mass is characteristic of a disease or a condition preceding the disease, such as type II diabetes or syndrome X.

Skeletal muscle atrophy occurs in adult animals as a result of insufficient muscle loading, aging, starvation, and as a consequence of various diseases, disorders, and conditions such as sepsis, muscular dystrophy, AIDS, aging, and cancer. Muscle loss is typically characterized by a decrease in protein content, a decrease in muscle strength, a decrease in fatigue resistance, and a decrease in muscle fiber diameter. This decrease can be associated with both a decrease in protein synthesis and an increase in protein degradation. Muscle wasting and related conditions that the composition and methods of the invention are intended to treat or prevent include any condition in which enhancement of muscle growth or reduction of muscle atrophy results in a therapeutic or other desired result. Such conditions include muscular dystrophy, sarcopenia, cachexia, diabetes mellitus, as well as the need to increase muscle mass, when such an increase is ethical and desirable, for example, for animals consumed in food.

- 57 041139

One class of disorders associated with muscle atrophy, as mentioned above, is dystrophy of various muscles. Such disorders represent a heterogeneous group of neuromuscular disorders that include, as the most common type, Duchenne muscular dystrophy (DMD), various types of limb muscular dystrophy (MD) (LGMD) and other congenital MD (CMD). Progressive muscle damage and loss of muscle mass, tissue inflammation, and replacement of healthy muscle mass with fibrous and adipose tissue lead to muscle atrophy in muscular dystrophy. Extreme loss of muscle mass is one of the most visible signs of the disease, and it leads to severe complications and symptoms, including death.

Sarcopenia is a disease characterized by age-related loss of muscle mass, strength and function. Sarcopenia begins in the fourth decade of life and accelerates after the age of approximately 75 years. Many factors, including physical inactivity, motor cell remodeling, decreased hormone levels, and decreased protein synthesis, can contribute to the development of sarcopenia. Except in the case of inactivity, they can all be genetically controlled, and gene modulation can be beneficial. For example, the rate of muscle protein synthesis and the rate of protein breakdown affect sarcopenia. The balance of protein synthesis and its destruction determines the protein content in the body. All of the studies that have been done strongly confirm that older people have lower levels of muscle protein synthesis than younger adults. The reduction of muscle protein catabolism is carried out, for example, by gene modulation, which can lead to a slowdown or reversal of muscle loss.

Selected applications related to aging and neurodegenerative diseases.

In some embodiments, the composition described herein is useful in promoting healthy aging in a subject. For example, particles containing an agent (eg, an antibody or a soluble form of a receptor) capable of binding to any of TGF-βΙ, CCL11, MCP-1/CCL2, beta-2-microglobulin, GDF-8/myostatin, or haptoglobin can be used to promote healthy aging in a subject, prolong active life in a subject, prevent or delay the development of age-related diseases in a subject, or treat a subject suffering from age-related disorders. In some embodiments of the invention, a particle containing a substance that binds to TGF-e1 can be used to increase/promote neurogenesis and/or muscle regeneration in a subject, for example, in an elderly subject. In some embodiments, the age-related disorder is a cardiovascular disease. In some embodiments, the age-related disorder is a bone loss disorder. In some embodiments, the age-related disorder is a neuromuscular disorder. In some embodiments, the age-related disorder is a neurodegenerative disorder or a cognitive disorder. In some embodiments, the age-related disorder 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, senile dementia associated with CNS and brain, age-related cognitive decline, pre-diabetic state, diabetes mellitus, obesity, osteoporosis, coronary heart disease, cerebrovascular disease, heart attack, stroke, peripheral arterial disease, aortic valve disease, stroke, Lewy body disease, amyotrophic lateral sclerosis (ALS ), mild cognitive impairment, predementia, dementia, progressive subcortical gliosis, progressive supranuclear palsy, thalamic degeneration syndrome, hereditary aphasia, myoclonic epilepsy, macular degeneration, or cataract.

The biomolecule may be alpha-synuclein, tau protein, amyloid precursor protein, or amyloid β. For example, the method may include administering the multiparticulate composition to a subject with Alzheimer's disease, and the particles may contain an agent that specifically binds to amyloid β (eg, soluble amyloid β and/or aggregates of amyloid β). The biomolecules may be Άβ40 or Άβ42. The agent may contain aducanumab, bapineuzumab, crenezumab, gantenerumab, ponezumab, solanezumab, or an antigen-binding portion of any of them. In addition, the method may include administering a composition comprising a plurality of particles to a subject with Alzheimer's disease, and the particles may contain an agent that specifically binds to the tau protein.

The biomolecule may be TDP-43 or FUS. The biomolecule may be a prion. The biomolecule can be a PrP ^Sc , a soluble PrP protein, or a PrP aggregate.

Selected diagnostic applications.

The particles described herein can also be used as diagnostic agents or in combination with a diagnostic tool or device. So, for example, the particles described here can be connected to a detection device that monitors the concentration of dan

- 58 041139 soluble ligand of interest. For example, a nanochannel in a detection device coated with an agent (for example, the first member of the binding pair) can detect (for example, in a blood sample) or monitor (for example, as an implantable device in the subject) the concentration of a soluble biomolecule (for example, the second member of the binding pair). ). Such a detector may be useful, for example, to determine the effectiveness of the particles described here (in the purification of a soluble biomolecule) or to determine/adjust the appropriate dose of the particle composition (for example, increasing the dose or frequency of dosages to more effectively capture the soluble biomolecule).

In some embodiments of the present invention, the particles and detection device described herein are integrated and function as microglands or nanoglands (see, for example, Sabek et al., Lab. Chip., 13(18):3675-3688 (2013)). Functions of a nanogland, such as nanochannel diagnostics, are able to provide an accurate quantification of the concentration of a soluble biomolecule in the body fluid of a subject implanted with a nanogland. In addition, the placement of a tool (eg, nano-syringe) in the nanogland will allow the release of particles capable of capturing biomolecules, for example, when the concentration of the biomolecule in the biological fluid reaches a predetermined threshold concentration. Whereas several thousand nanochannels can be present in a fingernail-sized implantable biochip, microglands or nanoglands can be engineered to control many different soluble biomolecules and release several types of therapeutic particles.

Selected in vitro applications.

In some aspects, the present invention relates to a method for removing a biomolecule from a composition, where the method includes contacting the composition with a particle described here. Such methods are especially useful for scientific research. For example, it is relatively easy to add a biomolecule to a solution, but removing a specific biomolecule from a solution is a rather complicated process.

Current methods for removing a biomolecule from solution include, for example, binding the biomolecule to a particle, such as Sepharose beads, and then physically separating the bead from the solution. The particles described herein can isolate the biomolecule in the composition, thereby inhibiting its interaction with other components of the composition (eg, a cell) without the need to physically separate the particle from the composition.

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

The method may include contacting the composition with the particle described here, where 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, to remove a secreted protein from a cell culture or to remove a contaminant from a cell culture.

The method may include contacting the composition with the particle described here, where the composition is a cell lysate. The cell lysate may be a prokaryotic or eukaryotic cell lysate. Such methods may be useful, for example, to inhibit the activity of a target biomolecule of interest.

The above methods may be particularly useful for evaluating the function of a biomolecule of interest in a particular system. For example, a biomolecule can be introduced into a system (eg, tissue culture) to evaluate the effect of the biomolecule on the system (eg, cell proliferation or cell death) and the biomolecules can be blocked or removed from such a system using the particle described herein to evaluate the effect of the absence of a biomolecule in the system.

In some aspects, the present invention relates to a method for the development or differentiation of a population of cells, where the method includes bringing into contact a composition containing a population of cells, with multiple particles described here. The plurality of particles may capture one or more molecules that promote an alternative cell differentiation pathway that competes with the desired differentiation pathway. Thus, the method can promote the differentiation of a population of cells into a desired cell type, in relation to an alternative way of differentiation into another cell type. The method may further include contacting the composition with a cytokine (eg, as described herein). The method may further include contacting the composition with one or more bioactive compounds such as a chemokine, interleukin, growth factor, Wnt family protein, tumor necrosis factor, and/or hormone (eg, as described herein).

The cell population may include stem cells. The cell population may include somatic stem cells or embryonic stem cells. The cell population may include induced stem cells, such as induced pluripotent stem cells. The cell population may include progenitor cells, differentiation progenitor cells, blast cells, unipotent cells, multipotent stem cells, pluripotent stem cells.

- 59 041139 cells and/or intermediate precursor cells. The cell population may include auxocytes. The cell population may comprise hematopoietic stem cells, breast stem cells, intestinal stem cells, mesenchymal stem cells, endothelial stem cells, neural stem cells, olfactory adult stem cells, medullary ridge stem cells, or testicular cells. The cell population may include satellite cells, oligodendrocyte, thymocyte, angioblast, bone marrow stromal, pancreatic, endothelial or melanoblast progenitor cells. The cell population may include multipotent hematopoietic stem cells, common myeloid progenitors, myeloblasts, monoblasts, promonocytes, monocytes, common lymphoid progenitors, lymphoblasts, prolymphocytes, and/or small lymphocytes.

In some embodiments, the invention relates to a method of differentiating a cell, where the method includes bringing into contact a composition containing cells with a plurality of particles described here. The plurality of particles may capture one or more molecules that promote an alternative differentiation pathway that competes with the desired differentiation pathway. Thus, the method may promote differentiation of cells into a desired cell type over an alternative pathway for differentiation into another cell type. The method may further include contacting the composition with a cytokine (eg, as described herein). The method may further include contacting the composition with one or more bioactive compounds such as a chemokine, interleukin, growth factor, Wnt family protein, and/or tumor necrosis factor (eg, as described herein).

The cell may be a stem cell. The cell may be a somatic stem cell or an embryonic stem cell. The cell may be an induced stem cell, such as an induced pluripotent stem cell. The cell may be a progenitor cell, a differentiation progenitor cell, a blast cell, a unipotent cell, a multipotent stem cell, a pluripotent stem cell, and/or an intermediate progenitor cell. The cell may be an auxocyte. 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 medullary ridge stem cell, or a testicular cell. The cell may be a satellite cell, an oligodendrocyte, thymocyte, angioblast, bone marrow stromal, pancreatic, endothelial or melanoblast progenitor cell. The cell may be a multipotent hematopoietic stem cell, a common myeloid progenitor cell, a myeloblast, a monoblast, a promonocyte, a monocyte, a common lymphoid progenitor cell, a lymphoblast, a prolymphocyte, and/or a small lymphocyte.

Kits for the introduction of the agent.

In some embodiments, the invention also provides a pharmaceutical kit or kit containing one or more containers filled with at least one composition (eg, particle or particles) according to the invention. Optionally, this container(s) may be accompanied by a notice in the form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, where the notice contains (a) the agency's approval of the manufacture, use, or sale for administration to a human, (b) instructions for use, or both.

In some embodiments, the kit contains additional materials to facilitate delivery of the agent to the subject. For example, the kit may contain one or more catheters, tubing, infusion bags, syringes, and the like. In some embodiments, the composition (for example, containing the particles described herein) is packaged in lyophilized form and the kit contains at least two containers: a container containing the lyophilized composition, and a container containing a suitable amount of water, buffer, or other liquid suitable for reconstituting the lyophilized material.

The foregoing applies to any of the compositions and for any of the methods described herein. The invention specifically contemplates any combination of the characteristics and features of such compositions and methods (singly or in combination) with the characteristics and features described for the various kits as set forth in this section.

Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those of ordinary skill in the art to which this invention pertains. The preferred methods and materials are described herein, although methods and materials similar or equivalent to those described herein may also be used in practice or when testing the methods and compositions described herein. All publications, patent applications, patents and other references cited herein are incorporated in their entirety by reference to these documents.

The invention contemplates any combination of all of the above aspects and embodiments, as well as combinations with any of the embodiments set forth in the Detailed Description and the Examples. These and other aspects of the present invention will be better understood after consideration of the following examples, which are intended to illustrate certain specific embodiments of the invention, but are not intended to limit its scope defined by the claims.

Examples

Example 1. Method for the treatment of cancer.

The GP detects a human patient for a cancer (eg, lung, colon, breast, brain, liver, pancreas, skin, or hematologic cancer) that secretes soluble TNFR or soluble IL-2R. The patient is administered a composition containing particles (described herein) that bind and isolate soluble TNFR or IL-2R in an amount effective to treat cancer. The patient is optionally administered maintenance doses of the composition to maintain inhibition of the action of soluble TNFR or IL-2R, and therapy is continued to enhance immunological control against cancer in the patient.

Example 2. Method for detoxifying a person.

The human patient exhibits symptoms of toxicity associated with botulinum toxin. The patient is administered a composition containing particles (described herein) that bind and sequester soluble botulinum toxin in an amount effective to reduce the severity of one or more symptoms associated with its toxicity.

Example 3. Method for the treatment of a viral infection.

The GP detects a human patient for HIV-1 infection. The patient is administered a composition containing particles (described herein) that bind and isolate soluble HIV-1 virions in an amount effective to reduce virus titers in the patient's circulation system. The patient is administered maintenance doses of the composition to maintain a decrease in titers of HIV-1 virions and thus suppress infection in the patient, as well as reduce the likelihood of transmitting the virus to another person.

Example 4. Method for obtaining silicon particles.

Get porous silicon disks with dimensions of 1000/400 and 1000/800 nm with different pore sizes. The size and morphology of the disks, as well as the pore diameters, are characterized by scanning electron microscopy. Gold (Au) particles are deposited in the pores of porous silicon disks. Tumor necrosis factors (TNF) are conjugated on the surface of gold nanoparticles via coordination covalent bonds. Then evaluate the density of the ligand and the stability of the binding of TNF to Au.

Example 5. Method for obtaining polymer particles.

By emulsification, lactide-glycolide copolymer (PLGA) particles are obtained. The size and morphology of PLGA particles are characterized by scanning electron microscopy, atomic force microscopy, and transmission electron microscopy. The particles are coated with quaternary ammonium functionalized betacyclodextrin for macrophage recruitment (eg, phagocytosis). The coating is checked by atomic force microscopy and transmission electron microscopy. The density and uniformity of the coating is assessed by transmission electron microscopy and dynamic light scattering.

Beta-cyclodextrin-coated PLGA particles are incubated with macrophages and phagocytosis is determined by fluorescence microscopy and flow cytometry.

Beta-cyclodextrin-coated PLGA particles are coated with a mixture of polyethylene glycol (PEG) and thiol molecules to prevent opsonization and avoid macrophage uptake and binding to other particles. The uniformity and density of the PEG and thiol coatings are characterized by atomic force microscopy. The stability of the coatings is characterized by incubating the particles in the media for various periods of time. Capture evasion and particle capture are determined at various time points by incubating the particles with macrophages as described above.

PLGA particles are coated with tumor necrosis factor (TNF) and the particles are combined via disulfide bonds to form a sponge containing TNF on the inner surface of the sponge. The outer surface (ie outer surface) of the sponge is optionally blocked with particles that do not contain TNF to prevent interaction between the TNF of the sponge and cells.

Example 6 Pharmacokinetics of polymer-based particles.

The sponge of Example 5 (i.e., the composition containing the sponges of Example 5, for example, from 103 to 10 ¹² sponges) is administered intravenously or intratumorally in a mouse model of primary and metastatic cancer, as well as healthy controls. Sponge toxicity is assessed by determining the LD ₅₀ for each route of administration. Sponge half-life is determined from plasma sponge concentration using LC/MS and ICP methods for each route of administration. Sponge biodistribution is determined by biopsying mice and analyzing the tissue for the presence of sponges and its components using LC/MS, ICP and confocal microscopy.

Example 7 Efficiency of polymer-based particles.

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Claims (18)

The sponge of Example 5 (ie, the composition containing the sponges of Example 5, in an amount of, for example, 10 ³ to 10 ¹² sponges) is administered to mice bearing MDA-MB-231 or 4T1 xenografts. The MDAMB-231 model is used to assess tumor size reduction and growth reduction, and the 4T1 model is used to assess metastasis inhibition. The sponge is injected into the tumor in mice with MDA-MB-231 once a week for 6 weeks and periodically determine the body weight and size of the tumors. In mice with 4T1, the sponge is injected intravenously once a week for 6 weeks and the number of metastases is determined. Example 8 Pharmacokinetics and Efficacy of Silicon/Gold Particles. The experiments described in examples 6 and 7 are repeated with the porous silicon particles of example 5. While the present invention has been described with reference to specific embodiments, it will be appreciated by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the spirit and scope of the invention. In addition, many modifications can be made to adapt specific conditions, materials, compositions, method steps or process steps according to the purpose, spirit and scope of the present invention. All such modifications are intended to be included within the scope of the present invention. CLAIM 1. A particle capable of capturing targets, comprising an agent immobilized on the surface of the particle, where said particle contains a metal, alumina, glass, silicon dioxide, silicon, or a polymer; an agent selectively binds to a target, said agent comprising (i) an antibody or a biomolecule-binding antibody fragment, (ii) a non-antibody scaffold protein, (iii) a nucleic acid or aptamer, (iv) an interleukin protein or a variant thereof, (v) a tumor necrosis factor (TNF) family ligand or variant thereof, (vi) a cytokine or chemokine, (vii) an enzyme, (viii) a cell surface receptor protein, (ix) a cell surface receptor protein ligand, or (x) a natural target ligand; and where said particle contains (a) a core subparticle and a plurality of protective subparticles, wherein said core subparticle and said protective subparticles each contain a metal, alumina, glass, silicon dioxide, silicon, or a polymer; the plurality of protective subparticles is composed of two or more protective subparticles; and the agent is immobilized on the core subparticle such that the protective subparticles inhibit interactions between the agent and molecules on the cell surface, or (b) an inner surface, an outer surface, and a plurality of coating molecules, where the plurality of coating molecules consists of two or more coating molecules; The plurality of coating molecules comprise the coating polymer; the agent is immobilized on the inner and outer surfaces; a plurality of coating molecules are associated with the outer surface; and the coating molecules inhibit interactions between the agent and molecules on the cell surface. 2. The particle according to claim 1, where (a) the agent is immobilized on the subparticle of the nucleus so that the protective subparticles inhibit interactions between the agent and the target on the cell surface; or (b) the coating molecules inhibit interactions between the agent and the target on the cell surface. 3. A particle according to any one of the preceding claims, wherein said particle contains a plurality of coating molecules. 4. The particle according to any one of paragraphs.1-3, where the specified particle contains many molecules of the coating; and said plurality of coating molecules sterically inhibit interactions between the agent and molecules on the cell surface. 5. Particle according to claim 3 or 4, wherein the plurality of coating molecules increase the excretion of the particle under in vivo conditions. 6. Particle according to claim 3 or 4, where a plurality of coating molecules reduces the release of the particle under conditions - 62 041139 in vivo. 7. Particle according to claim 3 or 4, wherein the plurality of coating molecules inhibit interaction between the agent and cells or extracellular proteins. 8. Particle according to any one of claims 3 to 7, wherein said coating polymer comprises polyethylene glycol (PEG), polylactate, polylactic acid, sugar, lipid, polyglutamic acid, polyglycolic acid (PGA), polylactic acid (PLA), lactic-glycolic copolymer acids (PLGA), polyvinyl acetate (PVA), polyamino acid, and combinations thereof. 9. Particle according to any one of claims 1 to 7, wherein the plurality of coating molecules are biodegradable. 10. Particle according to any of the preceding paragraphs, where the particle has a dendritic shape; the particle has a cubic, pyramidal, conical, spherical, tetrahedral, hexahedral, octahedral, dodecahedral or icosahedral shape; the particle has one or more protrusions facing outward; the particle contains two intersecting protrusions on the surface of the particle, the protrusions being sized and oriented to inhibit (i) the agent immobilized on the surface of the particle from binding or activating the cell surface receptor protein, and/or (ii) the interaction of the target and molecules and cells, when the target contacts the agent; and the particle contains a tube; or the particle is a 2-dimensional particle. 11. Particle according to any one of paragraphs.1-10, where the particle contains a subparticle core and many protective subparticles; the plurality of protective subparticles is composed of two or more protective subparticles; and the agent is immobilized on the core subparticle. 12. The particle of claim 11, wherein the core subparticle size is from about 100 nm to about 2 µm and the protective subparticle size is from about 10 nm to about 1 µm. 13. Particle according to any one of claims 11, 12, where the particle contains from 4 to 10 ⁶ protective subparticles. 14. A particle according to any one of claims 11-13, wherein the particle contains more than one core subparticle. 15. A particle according to any one of the preceding claims, wherein the agent has a reduced ability to activate the cell surface receptor protein compared to the ability of the natural ligand of the cell surface receptor protein. 16. The particle of claim 15 wherein the agent does not activate the cell surface receptor protein. 17. A particle according to any one of the preceding claims, wherein the target is a viral protein, a bacterial protein or a bacterial cell wall component, a yeast or fungal protein or a yeast or fungal cell wall component, a protozoan protein, a toxin, a poison, a poison, an allergen, a carcinogen , a psychoactive drug substance or a chemical weapon agent. 18. A particle according to any one of the preceding claims, wherein the target is selected from TNFa, TNFe, soluble TNF receptor, soluble TNFR-1, soluble TNFR-2, lymphotoxin, lymphotoxin alpha, lymphotoxin beta, 4-1BB ligand, CD30 ligand, EDA-A1 , LIGHT, TL1A, TWEAK, TRAIL, TRAIL soluble receptor, IL-1, soluble IL-1 receptor, IL-1A, soluble IL-1A receptor, IL-1B, soluble IL-1B receptor, IL-2, soluble IL receptor -2, 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, TNF-coupled weak inducer of apoptosis, MMP1, MMP2, MMP3, MMP9, MMP10, MMP12, CD28, soluble B7 family member, soluble CD80/B7-1 , Soluble CD86/B7-2, Soluble CTLA4, Soluble PD-L1, Soluble PD-1, Soluble Tim3, Tim3L, Galectin 3, Galectin 9, Soluble CE ACAM1, soluble LAG3, TGF-β, TGF-e1, TGF-e2, TGF-e3, anti-Mullerian hormone, artemin, glial cell-derived neurotrophic factor (GDNF), bone morphogenetic protein (e.g., BMP2, BMP3, BMP3B, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, BMP10, BMP11, BMP12, BMP13, BMP15), growth differentiation factor (e.g. GDF1, GDF2, GDF3, GDF3A, GDF5, GDF6, GDF7, GDF8, GDF9, GDF10, GDF11, GDF15), inhibin alfa, inhibin beta (eg, inhibin beta A, B, C, E), lefty, nodal, neuroturin, persephin, myostatin, ghrelin, sLR11, CCL2, CCL5, CCL11, CCL12, CCL19, interferon alfa, interferon beta, interferon gamma, clusterin, VEGF-A, granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GMCSF), prostaglandin E2, hepatocyte growth factor, nerve growth factor, sclerostin, complement C5, angiopoietin 2, angiopoietin 3, PCSK9, amyloid beta, activin, activin A, activin B, microglobulin β2, soluble NOTCH1, soluble th NOTCH2, soluble NOTCH3, soluble NOTCH4, haptoglobin, alpha chain fibrinogen, corticotropin releasing factor, corticotropin releasing factor type 1, corticotropin releasing factor type 2, urocorti- -