Self-assembling proteins

Abstract

The present invention relates to self-assembling proteins that can be used to deliver cargo proteins to phagocytes, and the subsequent delivery and utilization of these self-assembling proteins by phagocytes at disease sites. In particular, the present invention relates to phagocytes comprising polyhedrin crystals, wherein the crystals themselves encapsulate therapeutic and / or diagnostic proteins. The present invention also provides therapies using these cells.

Description

Technical Field

[0001] The present invention relates to self-assembling proteins that can be used to deliver cargo proteins to phagocytes, and the subsequent delivery and utilization of these self-assembling proteins by phagocytes at disease sites. In particular, the present invention particularly relates to phagocytes comprising polyhedral protein crystals, wherein the crystals themselves encapsulate therapeutic and / or diagnostic proteins. The present invention also provides therapies using these cells. Background Art

[0002] Proteins, including antibodies, growth factors, hormones, enzymes, and vaccines, can be used therapeutically. For example, growth factors such as IL-2 have been shown to have therapeutic value in cancer (Andersen et al., 2016). However, when injected systemically, proteins are rapidly cleared and the required doses can have significant toxic side effects on non-target cells. For primary tumors, proteins can be injected directly into the tumor mass, but the instability of proteins, especially growth factors, still poses a challenge to this approach. Although treatment by direct injection into the tumor mass is feasible, this approach is not practical for delivering growth factors and other proteins to any widespread disease.

[0003] Phagocytes engulf tiny particles through a process of active phagocytosis. There are many different types of phagocytes in mammals. Phagocytes are key to the immune system's response to infection and disease. For example, macrophages are specialized cells of the innate and adaptive immune systems that play a key role in defending against foreign pathogens, healing wounds, and regulating tissue homeostasis. Macrophages are activated (also called "polarized") to become either a pro-inflammatory M1 or anti-inflammatory M2 phenotype in response to subtle changes in the environment. Activation can be transient and non-binary, with a high level of cellular plasticity.

[0004] Stimulation with IFN-γ, LPS, and GM-CSF, for example, results in polarization toward an M1 phenotype characterized by the secretion of proinflammatory cytokines (e.g., IL-6, IL-1β, and IL-12) and chemokines (e.g., CCL5, CCL10, and CXL9), as well as the release of reactive oxygen and nitrogen intermediates—the “classic” pathogen-killing Th1 response. In contrast, M2 macrophages secrete anti-inflammatory cytokines (e.g., IL-10 and TGF-β) and chemokines (e.g., CCL17 and CCL22) upon stimulation with the cytokines IL-4 and IL-13.

[0005] When polarized macrophages encounter foreign objects such as microorganisms or other pathogens, phagocytosis begins. Once engulfed, the innate acidic phagolysosomes usually efficiently degrade biological materials including proteins. Subsequently, the surface protein fragments of the degraded pathogens are presented on the surface of the macrophage for recognition by other cells of the immune system.

[0006] Phagocytes can also infiltrate diseased tissues. However, phagocytes can be subverted and actively promote disease, such as solid cancer (van Dalen et al., 2019). For example, tumor-associated macrophages (TAMs) typically express an M2 phenotype (or activated subset), which causes them to perform immunosuppressive and tumor-promoting functions. Reprogramming these cells to an M1 phenotype (a classically activated subset) can suppress their pro-cancerous phenotype and unleash anti-tumor immunity.

[0007] Given the close association of phagocytes with disease and their ability to infiltrate diseased tissues, their ease of harvesting for ex vivo manipulation, and their alternative states, it has been proposed that (1) the phenotype of phagocytes can be modulated to influence disease outcomes and (2) phagocytes serve as “Trojan horses” for preferential or specific drug delivery to disease areas, targeting other cells while minimizing toxic side effects to non-target tissues ( Choi et al., 2007 ).

[0008] To date, several strategies have been developed to repurpose monocytes and macrophages to deliver drugs to sites of infection, inflammation, or solid tumors (Xu et al., 2018; Pang et al., 2017; Rosa et al., 2017). However, the fragility of therapeutic cargo proteins has hampered attempts to use them as Trojan Horse strategies to treat diseases. There are many challenges in successfully delivering therapeutic cargo proteins using phagocytes such as macrophages, such as toxicity to phagocytes, alterations in their metabolism, uptake and digestion efficiency of the cargo, etc.

[0009] It is an object of some embodiments of the present invention to alleviate some of the problems found in the prior art.

[0010] Summary of Certain Embodiments of the Invention

[0011] The present invention relates to polyhedrin crystals (e.g. The application of protein crystals) to deliver cargo proteins through phagocytes. The invention relates to the discovery that such crystals are efficiently engulfed by cells and unexpectedly secrete their cargo in a manner that retains the cargo's biological activity and modulates the behavior of nearby cells.

[0012] Certain aspects of the invention relate to the discovery that polyhedrin crystals are efficiently taken up by macrophages and, contrary to expectation, proteins incorporated into these crystals are subsequently released from the cells in a biologically active form. Furthermore, uptake of these crystals neither affects macrophage viability nor interferes with key macrophage functions, such as tracking chemotactic signals secreted by tumor cells or migrating through confined spaces, both of which are important for the efficient delivery of therapeutic cargo to disease sites.

[0013] Certain aspects of the invention relate to improved methods for delivering therapeutic cargo proteins to disease sites by infiltrating phagocytes such as macrophages to increase drug efficacy and reduce off-target side effects. Alternative aspects of the invention relate to methods for delivering diagnostic cargo proteins to disease sites by infiltrating phagocytes such as macrophages to image the disease.

[0014] Therefore, the present invention provides:

[0015] a phagocytic cell comprising one or more polyhedrin crystals, wherein the crystals encapsulate one or more cargo proteins;

[0016] - a substance containing more than 1×10 5 a population of phagocytes as described herein;

[0017] - A package comprising the phagocyte (or a population thereof) described herein;

[0018] - A method for producing a phagocyte (or a population thereof) as described herein, comprising:

[0019] (a) contacting one or more phagocytes with polyhedrin crystals; and

[0020] (b) culturing phagocytes under conditions that induce phagocytosis;

[0021] - A pharmaceutical composition comprising:

[0022] (a) a phagocytic cell (or a population thereof) as described herein, wherein the phagocytic cell comprises one or more therapeutic agents; and

[0023] (b) a pharmaceutically acceptable carrier or diluent;

[0024] - Use of the pharmaceutical composition as described herein in medicine;

[0025] - Use of a pharmaceutical composition as described herein in the treatment of solid cancers; and

[0026] - A method for imaging a solid cancer, comprising:

[0027] (a) administering a phagocyte (or a population thereof) as described herein, wherein the phagocyte comprises one or more detectable agents; and

[0028] (b) detecting the detectable agent, thereby imaging at least a portion of the cancer.

[0029] Detailed Description of Certain Embodiments

[0030] Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0031] Figure 1 shows Protein crystals are engulfed by professional phagocytes. THP-1 monocytes were incubated with cells expressing enhanced green fluorescent protein (eGFP). The crystals were incubated together for 24 hours. The cells were then imaged in the following modes: a) bright field mode (A), fluorescence mode (B), composite image (C); or bd) bright field mode (A), nuclei stained with DAPI (B), fluorescence mode (C), composite image (D). Composite shows multiple eGFP crystals were phagocytosed by living monocytes.

[0032] Figure 2 shows Protein crystals are engulfed by various types of professional phagocytes. THP-1 monocytes first differentiate into M0 macrophages (a), and then further differentiate into M1 (B) or M2 (C) macrophages. eGFP were co-cultured for 24 hours and subsequently imaged in bright field mode (A) and fluorescence mode (B).

[0033] Figure 3 shows the observation Still images from a time-lapse video of phagocytosis of eGFP crystals. Differentiated THP-1 cells (M0 macrophages) and eGFP crystals for 48 hours. Images were acquired at 1 frame / min in both brightfield and fluorescence modes. A) Brightfield tracking of two individual monocytes (black ovals) in the field of view over time, as indicated by the frame number, reveals the motility of M0 macrophages and their targeting of several Phagocytosis of crystals. B) Three corresponding still images of the time-lapse video, both in bright field mode and fluorescence imaging mode, where the position of the tracked M0 macrophage is marked (black and white ovals, respectively). eGFP particles will move once internalized.

[0034] Figure 4 shows Protein crystals are engulfed by various types of non-professional phagocytes. Cells were incubated with PODSeGFP for 24 hours and subsequently imaged live (a) or fixed (b). a) Phagocytosis of mouse fibroblasts (NIH-3T3) was imaged in bright field (A) and fluorescence mode (B) after 24 hours of incubation.

[0035] Figure 5 shows the analysis of phagocytosis. Empty cells were attached to the wells and incubated with professional phagocytes (monocytes or M1 macrophages) for 24 hours. The cells were then fixed and exposed first to anti-polyhedrin primary antibody and then to anti-rabbit FITC secondary antibody. Phagocytosis was imaged in bright field and fluorescence mode. Micrographs of empty monocytes (a) and M1 macrophages (b). c) b) chondrocyte phagocytosis Immunohistochemistry (IHC) images of eGFP. eGFP was attached to the surface of the wells and incubated with chondrocytes for 24 hours. The cells were then fixed and the slides were stained with actin for the cytoskeleton (A) or DAPI for the nuclei (B); Fluorescence signal of eGFP (C); and composite image (D).

[0036] Figure 6 shows the Protein crystals stimulate the proliferation of THP-1 monocytes. As a single layer directly seeded into the wells of a 24-well plate or into a 24-well tissue culture insert. Subsequently, the monocytes were seeded into wells containing the monolayer. Protein crystals; or protein crystals; or blank inserts as baseline controls. a) On day 0 (I) and day 4 (II), monolayers were seeded in wells Representative bright field micrographs of mononuclear cell cultures from TC inserts Micrographs of the experiment show only cells in the wells at day 0 (III) and day 4 (IV). b) After 4 days of incubation, total cell number was assessed using a colorimetric assay (OrangU, Cell Guidance Systems). Horizontal lines represent THP-1 cells without further addition; solid grey bars represent cells with As a monolayer experimental result; the dashed line column shows the The error bars represent the standard deviation of the mean.

[0037] Figure 7 Shown macrophage uptake M0, M1 and M2 macrophages were mixed with Empty or FGF-10 (1:5 and 1:15) was incubated in 96-well TC plates for 24 h. Subsequently, the medium was replaced and the colorimetric assay (Orangu TM , Cell Guidance Systems) measure uptake Cell viability after 48 hours (A) and 96 hours (B).

[0038] Figure 8 Shows the load The activity of macrophages loaded with IL-2 was monitored using a live cell imaging system. / cell) (B) activity. Images were taken every 2 minutes for 24 hours and analyzed using the manual tracker of Image J and the chemotaxis and migration tool (Ibidi). 25 cells were tracked under each condition and their migration tracks were visualized as rose diagrams, where each line represents the track of a single cell over 24 hours. Track positions were normalized so that each track started from the (0,0) coordinate.

[0039] Fig. 9 Shows the load Directed migration of macrophages induced by IL-2. Empty (A) and loaded Macrophages of IL-2 are transferred to chemotaxis μ-slides (Ibidi), and their ability to track the gradient chemokine produced by 0-10% BCS is tested. Images are taken every 1 minute in 12 hours, and analyzed using the manual tracker of Image J and the chemotaxis and migration tool (Ibidi). Track 26 cells under each condition, and visualize their migration tracks as rose diagrams, wherein each line represents the track of a single cell in 12 hours. Track position normalization makes each track start from (0,0) coordinates. Cell tracks migrating to 10% BCS source show black (up), and cell tracks migrating away from BCS source show red (down).

[0040] Fig.10 Shows the load Activity of bone marrow-derived mouse macrophages expressing eGFP. eGFP for 24 hours and then monitored using a live cell imaging system at 1 frame / min. The white dotted circles indicate the movement and retraction of macrophages in 21 frames equivalent to 21 minutes.

[0041] Fig.11 Shows the load Directed migration of eGFP macrophages through 8 μm pores. eGFP macrophages (5 / cell) were transferred into a 24-well insert with 8 μm pores. The bottom wells were filled with RPMI-1640 alone (A) or RPMI-1640 supplemented with 10% FBS (B). The upper panel shows the insert and the lower panel shows the bottom well after 24 hours of incubation.

[0042] Fig.12 Shows the load Macrophages expressing eGFP were followed by chemical signals from a melanoma cell line through an 8 μm pore. eGFP macrophages (5 / cell) were transferred into a 24-well insert with 8 μm pores. The wells were filled with complete growth medium (A) or medium treated with A375 cells for 3 days (B). The top image is representative and shows the number of macrophages that migrated into the wells after a subsequent 24-hour incubation; the lower image shows a local magnification.

[0043] Fig.13 Endogenous secretion of IL-6 is shown. After polarization, the culture medium of THP-1, M0, M1 and M2 was collected and tested for the presence of IL-6 by ELISA (A). The same cells were then washed and incubated in complete growth medium with FGF-2 for 24 hours. The culture medium was collected and analyzed by ELISA for the presence of IL-6 (B).

[0044] Fig.14 Shows the load from IL-6 released from IL-6-expressing macrophages. (A) M0 cells were loaded with 5, 10, or 20 IL-6 or FGF-2. The cells were washed and incubated in complete growth medium for 4 days. The culture medium was collected and tested for the presence of IL-6. (B) The same amount of IL-6 was centrifuged to the bottom of a 96-well plate and incubated in complete growth medium for 4 days. The medium was collected and IL-6 levels were measured by ELISA.

[0045] Fig.15 Shows the load from Bioactivity of FGF-2 released from FGF-2-loaded macrophages. Macrophages (M0, M1, and M2, 10 / cell) and incubated with FGF-2-responsive NIH-3T3 cells for 4 days. TM , Cell Guidance Systems) was used to measure the proliferation of NIH-3T3 cells.

[0046] The practice of the embodiments of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, pharmaceutical preparation, pharmacology, and medicine, within the skill of those skilled in the art.

[0047] Most conventional chemistry techniques can be found in Comprehensive Heterocyclic Chemistry IF (Katritzky et al., 1996, published by Pergamon Press); Comprehensive Organic Functional Group Transformations (Katritzky et al., 1995, published by Pergamon Press); Comprehensive Organic Synthesis (Trost et al., 1991, published by Pergamon); Heterocyclic Chemistry (Joule et al., published by Chapman & Hall); Protective Groups in Organic Synthesis (Greene et al., 1999, published by Wiley-Interscience); and Protecting Groups (Kocienski et al., 1994).

[0048] Most conventional molecular biology techniques can be found in Sambrook et al., Molecular Cloning, A Laboratory Manual (2001) Cold Harbor-Laboratory Press, Cold Spring Harbor, NY or Ausubel et al., Current Protocols in Molecular Biology (1990), published by John Wiley and Sons, NY.

[0049] Most conventional pharmaceutical preparation techniques can be found in Pharmaceutical Preformulation and Formulation (2 ndEdition edited by Mark Gibson) and Pharmaceutical Excipients: Properties, Functionality and Applications in Research and Industry (edited by Otilia M YKoo, published by Wiley).

[0050] Most conventional pharmacology techniques can be found in A Textbook of Clinical Pharmacology and Therapeutics (5 th Edition, Arnold Hodder).

[0051] Most general techniques for prescribing, dispensing and administering drugs can be found in British National Formulary 72 (published jointly by BMJ Publishing Group Ltd and the Royal Pharmaceutical Society).

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2 nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3 rd Ed., Academic Press and Oxford University Press provide those skilled in the art with a general dictionary of many of the terms used in this disclosure. For chemical terms, the skilled artisan can refer to the International Union of Pure and Applied Chemistry (IUPAC).

[0053] Units, prefixes and symbols are expressed in the form recognized by the International System of Units (SI). Numerical ranges are inclusive of the numbers defining the range.

[0054] Macrophages

[0055] The present invention provides one or more phagocytes. Typically, the phagocytes are mammalian cells. For example, the phagocytes can be mouse cells or human cells. The phagocytes of the present invention include professional phagocytes, such as monocytes, macrophages and glial cells. The phagocytes of the present invention also include non-professional phagocytes, such as chondrocytes, fibroblasts, erythrocytes, lymphocytes, epithelial cells, myoblasts and muscle cells. Typically, the phagocytes of the present invention are macrophages.

[0056] In certain embodiments, the phagocytes of the present invention are obtained from monocytes. For example, phagocytes can be obtained from immortalized monocyte-like cell lines (eg, THP-1 ATCC).

[0057] In certain embodiments, pre-existing phagocytes are isolated from body cavity washes (alveolar, peritoneal) of resident or induced monocytes (Zhang et al., 2008).

[0058] In certain embodiments, the phagocytes of the present invention are obtained by differentiating monocytes from blood or extracting monocytes from bone marrow. For example, phagocytes can be obtained from peripheral blood mononuclear cells (PBMC).

[0059] In certain embodiments, monocytes can be differentiated by adding growth factors or signaling molecules such as cytokines to the culture medium. For example, the culture medium can be supplemented with macrophage colony stimulating factor (M-CSF). Cells can also be incubated with additional immunostimulants such as LPS and / or interferon gamma (IFN-γ). The use of such stimulants may induce different polarizations that mimic in vivo phenotypes (Mosser et al., 2008).

[0060] In certain embodiments, phagocytes may comprise cargo proteins as described herein that do not have any effect on the phagocyte itself but are secreted to affect the behavior of other cells.

[0061] In certain embodiments, the phagocytes of the invention are autologous. In other words, the phagocytes can be derived from the subject to be treated, thereby reducing the risk of immune rejection.

[0062] Large numbers of phagocytes are produced using techniques available in the art for autologous cell-based therapy. Typically, phagocytes such as macrophages are produced from progenitor cells. For example, such progenitor cells can be established by overexpressing Hoxb8 in a culture medium supplemented with GM-CSF (Redecke et al., 2013). This results in rapidly proliferating clonogenic cells. Removal of Hoxb8 activity can cause progenitor cells to differentiate in phagocytes (e.g., macrophages).

[0063] The present invention also provides a population of two or more phagocytes. Any number of cells may be present in the population. For example, the population may contain more than about 10 5 , 10 6 , 10 7 or 10 8 In certain embodiments, the population comprises at least 10 9 , 10 10 , 10 11 or 10 12 The population is preferably homogenous and / or autologous.

[0064] The phagocytes may be isolated, substantially isolated, purified, or substantially purified. The phagocytes are isolated or purified if they are completely free of any other components, such as culture medium, other cells, or other cell types. The phagocytes are substantially isolated if they are mixed with a carrier or diluent, such as culture medium, which does not interfere with their intended use.

[0065] Any suitable technique can be used to isolate phagocytes. For example, phagocytes can be isolated by leukapheresis and / or elutriation (Faradji et al., 1994; Andreeson et al., 1990). Typically, phagocytes are isolated from the peripheral blood of a subject as described herein. Phagocytes (e.g., monocytes) can then be collected from the blood and isolated by leukapheresis and elutriation.

[0066] As discussed in more detail below, phagocytes are processed ex vivo. Specifically, phagocytes are loaded with polyhedrin crystals (which themselves encapsulate one or more cargo proteins, as described herein) and used in the methods of the invention for treatment.

[0067] In certain embodiments, phagocytic cells are provided in frozen aliquots, and substances such as DMSO may be present to promote survival during freezing. Such frozen cells will typically be thawed and then placed in a buffer or culture medium for maintenance or administration.

[0068] In certain embodiments, the phagocytes are provided in a packaged form. For example, the package can protect the cells from damage during collection, cell culture or transportation. Suitable packages include, for example, Teflon bags, etc.

[0069] Polyhedrin crystals

[0070] The phagocytic cells of the present invention contain one or more polyhedrin crystals (e.g. Protein crystals). Typically, crystals are derived from viruses, such as cytoplasmic polyhedrosis virus and baculovirus. For example, crystals can be derived from Bombyx mori cytoplasmic polyhedrosis virus.

[0071] In certain embodiments, polyhedrin crystals are produced in cells where polyhedrin is expressed at high levels under the control of a promoter. When a second protein is co-expressed and incorporated into the crystal, a co-crystal is formed. This second protein is referred to as an active protein or cargo protein. Typically, the promoter is a polyhedrin promoter. Typically, crystals are produced in insect cells. For example, fall armyworm 9 (sf9) (Spodoptera frugiperda) cells can be used to produce crystals.

[0072] In certain embodiments, a cargo protein is tagged with a short peptide sequence, which allows the cargo protein to bind to a growing polyhedrin crystal. As the crystal continues to grow, the cargo protein is encapsulated. Thus, the crystal encapsulates one or more cargo proteins.

[0073] In certain embodiments, the polyhedron protein comprises the Bombyx mori cytoplasmic polyhedrosis virus protein sequence shown in SEQ ID NO: 1. Alternatively, the polyhedron protein may comprise a sequence having at least about 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology to SEQ ID NO: 1 based on the amino acid identity of the entire SEQ ID NO: 1 sequence.

[0074] In certain embodiments, one or more cargo proteins comprises a polyhedrin binding tag selected from the H1 tag sequence shown in SEQ ID NO: 2. Alternatively, the polyhedrin binding tag may comprise a sequence having at least about 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology to the sequence of SEQ ID NO: 2 based on amino acid identity throughout the sequence of SEQ ID NO: 2.

[0075] In certain embodiments, one or more cargo proteins comprises a polyhedrin binding tag selected from the VP3 tag sequence shown in SEQ ID NO: 3. Alternatively, the polyhedrin binding tag may comprise a sequence having at least about 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology to the SEQ ID NO: 3 sequence based on amino acid identity throughout the SEQ ID NO: 3 sequence.

[0076] In certain embodiments, one or more cargo proteins comprises a polyhedrin binding tag selected from the PH tag sequence set forth in SEQ ID NO: 4. Alternatively, the polyhedrin binding tag may comprise a sequence having at least about 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology to the SEQ ID NO: 4 sequence based on amino acid identity throughout the SEQ ID NO: 4 sequence.

[0077] In certain embodiments, the crystal is internalized within (eg, inside) a phagocytic cell. For example, the crystal can be located inside a phagosome or phagolysosome, or in the cytoplasm or nucleus, rather than outside of the cell membrane.

[0078] In certain embodiments, the intact crystals are capable of secreting cargo proteins from phagocytic cells. In other words, the cargo proteins can be released from the crystals and retain their native conformation and / or function. Typically, the cargo proteins are released by the action of intracellular proteases (e.g., matrix metalloproteinases).

[0079] In certain embodiments, the cargo proteins have a desired phenotypic effect on the phagocytes that secrete them. For example, the cargo protein may be able to repolarize phagocytes (e.g., to an M1 macrophage phenotype).

[0080] In some embodiments, the cargo protein is released from the phagocytic cell over a sustained period of time. For example, the cargo protein can be released from the cell across the cell membrane. Unexpectedly, the cargo protein can change the behavior of neighboring cells, which themselves may not contain any crystals. In some embodiments, the cargo protein is released from the cell via exosomes. These exosomes can be used for therapeutic or research applications.

[0081] Typically, the cargo protein is released over a longer period of time than the time required for the phagocyte to reach and / or penetrate its intended target. For example, the cargo protein can be released from the phagocyte over a period of at least 2 days, 5 days, 7 days, 10 days, 14 days, 20 days, 21 days, 1 month or more.

[0082] Typically, crystals are regular arrays of polyhedral proteins assembled in a lattice. Crystals can be cubes or any other shape. Crystals of the present invention are typically separated from cells (e.g., insect cells) infected by viruses (e.g., baculoviruses or plasma polyhedrosis viruses). Crystals are typically less than 10 microns, e.g., 0.1 to 6 microns (measured as the maximum distance between different surfaces inside the crystal or the maximum length of the edge). Crystals can be processed to change their shape and / or reduce their size before use.

[0083] In certain embodiments, the crystals are separated from the cells, for example, by centrifugation. Typically, the crystals are prepared in an aqueous suspension. For example, the concentration of the crystal suspension may exceed 10 μg / mL. 4 crystals, usually 10 per ml volume 4 Up to 10 7 crystals, preferably 5×10 per ml volume 4 Up to 5×10 6 or 10 5 Up to 10 6 crystals. The crystals can be applied to (contacted with) the culture system / medium at these concentrations. These can be 10 per microliter 5 Up to 10 7 The concentration of crystals used for treatment is, for example, 3 × 10 per ml 6 A crystal.

[0084] Advantageously, the crystals are highly stable and rigid. The crystals can be attached (eg, dehydrated to a solid support). Typically, the crystals retain a high level of biological activity after storage for 6 months at 37°C in a protease-free solution.

[0085] Cargo protein

[0086] The polyhedrin crystals described herein encapsulate one or more cargo proteins.

[0087] Any type of cargo protein can be used. The phagocytes can contain polyhedrin crystals containing a single type of cargo protein. Alternatively, the phagocytes can contain polyhedrin crystals containing different types of cargo proteins (e.g., a combination of cargo proteins). For example, the phagocytes can contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different cargo proteins.

[0088] As further described herein, cargo proteins typically comprise a polyhedrin binding tag. Cargo proteins are typically secreted from intact crystals. Cargo proteins are typically continuously released from phagocytic cells. Cargo proteins can cause desired phenotypic effects in the phagocytic cells that secrete them and / or in neighboring cells.

[0089] In embodiments where more than one cargo protein is present, each protein may have the same polyhedrin binding tag. Alternatively, each protein may have a different polyhedrin binding tag.

[0090] In certain embodiments, the crystals are coated with agents that delay the release of the polyhedrin and cargo protein.

[0091] In certain embodiments, the cargo protein comprises one or more therapeutic agents. For example, the cargo protein may comprise one or more different antibodies, immunogens, enzymes, signaling proteins, hormones, growth factors and / or cytokines.

[0092] In certain embodiments, one or more therapeutic agents are capable of reprogramming M2 tumor-associated macrophages (TAMs), inflammatory monocytes, and / or myeloid-derived suppressor cells (MDSCs) into M1 macrophages. The ability of a therapeutic agent to produce reprogrammed M2 cells can be determined, for example, by the expression of identification and / or quantitative markers described herein.

[0093] In certain embodiments, the one or more therapeutic agents activate STAT1, activate Notch, promote ROS synthesis and / or NO release, promote AKt1 kinase activity, activate NF-κB signaling, or inhibit JNK activity or CSFR1.

[0094] In certain embodiments, the one or more therapeutic agents comprise one or more Th-1 associated cytokines and / or TLR ligands.

[0095] In certain embodiments, one or more therapeutic agents are capable of stimulating the generation of M1 macrophages. The ability of a therapeutic agent to stimulate the generation of M1 macrophages can be determined, for example, by identifying and / or quantifying the expression of markers in a macrophage population after contact with the therapeutic agent. As shown in Table 1, different macrophage subsets (human) have different phenotypes.

[0096]

[0097]

[0098] Table 1, adapted from Roszer et al., 2015 and Duluc et al., 2007

[0099] In certain embodiments, the one or more therapeutic agents are selected from any one or more of the proteins listed in Table 1.

[0100] In certain embodiments, the one or more therapeutic agents are selected from one, two, three, four, five, six, seven, eight, nine, ten or more proteins listed in Table 1.

[0101] In certain embodiments, the one or more therapeutic agents are selected from granulocyte-macrophage colony stimulating factor (GM-CSF), CSF-2, CSF-1 (M-CSF), CSF-3 (G-CSF), interferon gamma (IFN-γ), TNF-α, IFN-β, IL-6, IL-12, IL-15, IL-15R, IL-21, IL-23, MHC-II, IL-1R, TLR2, TLR4, CD80, CD86, CD68, IL-1, IL-1β, IL-2, CCL2, CCL3, CCL4, CCL5, CCL8, CCL9, CCL10, CCL11, CXCL8, CXCL9, CXCL10, CXCL16, stem cell factor (SCF), histidine-rich glycoprotein, paclitaxel, SOCS3, activin A, BtK, iNOS and / or NOS2.

[0102] In certain embodiments, the one or more therapeutic agents include IFN-γ, GM-CSF, and / or TNFα. Typically, the one or more therapeutic agents include GM-CSF.

[0103] In certain embodiments, the cargo protein comprises one or more detectable agents. For example, the cargo protein may comprise a fluorescent and / or bioluminescent marker, an enzyme marker, a chemiluminescent marker, or a biotin group. A radioisotope or radionuclide may include 3 H. 14 C. 15 N. 35 S. 90 Y. 99 Tc, m In, 125 1. 131 I. Fluorescent labels may include rhodamine, lanthanide phosphors or FITC, and enzyme labels may include horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase.

[0104] Typically, the detectable agent is a fluorescent marker, such as GFP ("green fluorescent protein"). For example, the detectable agent can be enhanced green fluorescent protein (eGFP). Alternatively, the detectable agent can be a bioluminescent marker, such as luciferase.

[0105] Phagocytosis

[0106] The phagocytes of the present invention may be harvested or produced by any suitable method.

[0107] In certain embodiments, phagocytic cells are contacted with polyhedrin crystals. Typically, phagocytic cells (e.g., monocytes) are contacted with the crystals. Such cells may be unaffected by the crystals, or may differentiate or transdifferentiate (e.g., become M1 macrophages) after contact with the crystals.

[0108] In alternative embodiments, the crystals may be contacted with the phagocytic cells following culture and / or differentiation.

[0109] In certain embodiments, the crystals contain cargo (e.g., GM-CSF) that can stimulate the production of macrophages (e.g., M1 macrophages). Additionally or alternatively, the culture medium can be supplemented with one or more agents that stimulate differentiation (e.g., LPS, IFN-γ, GM-CSF).

[0110] In certain embodiments, the crystals are attached to a solid support prior to contacting the phagocytes. Examples of suitable solid supports include, but are not limited to, slides, membranes, and tissue culture plates. Microcarriers may also be used. The crystals may be maintained in a non-dried form, for example, by centrifugation and / or dehydration onto a support shortly before contacting the phagocytes.

[0111] In alternative embodiments, the crystals are premixed with the phagocytes. Typically, the crystals are premixed with the phagocytes for up to 12, 24, 36, 48 hours or longer.

[0112] Phagocytic cells can be cultured under any conditions that induce phagocytosis, including methods further described herein. The phagocytosis of crystals to cells can be determined by any suitable immunohistochemistry (IHC) technique. In certain embodiments, the crystals themselves can include cargo (e.g., detectable agents, such as GFP and / or luciferase) that allow phagocytosis to enter cells to be verified (e.g., by fluorescence microscopy).

[0113] In certain embodiments, phagocytes can be cultured in complete medium. Typically, PMA is added to the complete medium. Typically, phagocytes are cultured for 12, 24, 36, 48 hours or longer.

[0114] For example, phagocytes can be cultured in complete medium (e.g., DMEM supplemented with 4.5 g / L D-glucose, L-glutamine, 10% heat-inactivated FBS, 100 U / mL penicillin, and 100 μg / mL glutamine (Glutamax), optionally supplemented with 20 ng / mL M-CSF) at a seeding density of 0.5-1.0e6 / ml at 37°C.

[0115] In certain embodiments, phagocytes can be differentiated into polarizations that mimic in vivo phenotypes (e.g., cytotoxic phenotypes). For example, the culture medium can be supplemented with additional immunostimulants such as LPS and / or IFN-γ to induce M1 macrophage production. Typically, after 12 hours of culture in complete medium, 100 ng / ml LPS or 20 ng / ml IFN-γ is added to the complete medium to induce M1 macrophage production.

[0116] In certain embodiments, phagocytes may be unaffected by ingested crystals containing proteins, such as IL-2, that target other cells in the diseased tissue.

[0117] In certain embodiments, the method further comprises formulating the one or more phagocytic cells using any suitable method, including those further described herein. Typically, the one or more phagocytic cells are formulated for delivery via an injectable route.

[0118] Pharmaceutical composition

[0119] The present invention provides a pharmaceutical composition comprising (a) a phagocyte of the present invention or a population of phagocytes of the present invention, and (b) a pharmaceutically acceptable carrier or diluent. Typically, the phagocytes of the pharmaceutical composition comprise any one or more therapeutic agents described herein.

[0120] The compositions of the present invention may be formulated by any suitable method. Formulation of cells with standard pharmaceutically acceptable carriers and / or excipients may be carried out using conventional methods of the pharmaceutical art. The exact nature of the formulation will depend on several factors, including the cells to be administered and the desired route of administration. Suitable types of formulations are described in Remington's Pharmaceutical Sciences, 19 th It is fully described in , Edition, Mack Publishing Company, Eastern Pennsylvania, USA.

[0121] The compositions of the present invention can be administered by any route. Suitable routes include, but are not limited to, intravenous, intramuscular, intraperitoneal or other suitable routes of administration. The compositions can be prepared with a physiologically acceptable carrier or diluent. Typically, such compositions are prepared as a liquid suspension of cells. The cells can be mixed with a pharmaceutically acceptable excipient that is compatible with the active ingredient. Suitable excipients are, for example, water, saline, glucose, glycerol, etc. and combinations thereof.

[0122] Typically, the composition is formulated for delivery by injection.

[0123] In certain embodiments, the composition is used in combination with one or more other therapies intended to treat the same subject. By combination, this means that the treatment can be given to the subject simultaneously in a combined or separate form. As part of the same treatment regimen, the treatment can be given to the subject individually or sequentially. For example, the composition of the present invention can be used in combination with another therapy intended to treat a cancer (e.g., solid cancer) of the subject.

[0124] In certain embodiments, treatment with one or more chemotherapeutic agents can be combined with the pharmaceutical composition of the present invention. Typically, the chemotherapeutic agent is selected from dacarbazine (DTIC), temozolomide, nataxel, paclitaxel, carmustine (BCNU), cisplatin, carboplatin, vinblastine or any combination thereof.

[0125] In certain embodiments, treatment with one or more biological agents can be combined with the pharmaceutical composition of the present invention. For example, a biological agent can be a growth factor / cytokine / chemokine, such as IL-2. In certain embodiments, a biological agent is a checkpoint inhibitor (e.g., anti-PD, PD-L1 and / or CTLA4 therapy). For example, a biological agent can be selected from ipilimumab, nivolumab, atezolizumab, pembrolizumab and / or any combination thereof. For example, a biological agent can be selected from nivolumab and ipilimumab. In some embodiments, a biological agent is a B-Raf inhibitor, such as vemurafenib and / or dabrafenib. In certain embodiments, a biological agent is nivolumab and / or ipilimumab; dabrafenib and / or trametinib; vemurafenib and / or cobimetinib; and / or any combination thereof.

[0126] Cell therapy

[0127] The present invention also provides phagocytes, populations or pharmaceutical compositions for use in treatment or medicine as described herein. Therefore, the present invention includes administering phagocytes to prevent or treat any disease. The phagocytes can be used to prepare a medicine for preventing or treating any disease.

[0128] The present invention provides phagocytes for use in methods for treating a human or animal body by therapy. For example, the therapeutic target can be any cell, extracellular vesicle and / or free protein in the tumor microenvironment. In this article, the term "tumor microenvironment" is understood to include the environment surrounding a solid cancer, including surrounding blood vessels, immune cells, fibroblasts, signaling molecules and extracellular matrix (ECM).

[0129] In certain embodiments, the therapeutic target of the protein released by macrophages is another type of immune cell, such as a T cell or a dendritic cell. The target may also be a stromal cell or a cancer cell.

[0130] In certain embodiments, the present invention includes administering to a subject an immunologically effective amount of phagocytes of the present invention, which are loaded with polyhedrin crystals, thereby delivering one or more cargo proteins to a desired site. For example, phagocytes are used to deliver one or more cargo proteins directly to a disease site (e.g., cancer).

[0131] The present invention provides a method of delivering any one or more cargo proteins described herein to a disease site, comprising administering a pharmaceutical composition of the present invention to a subject.

[0132] In certain embodiments, cells and / or tissues are remodeled at the disease site. For example, any cell, extracellular vesicle, or free protein in the tumor microenvironment can be regulated as described herein. Typically, adjacent or neighboring phagocytes (e.g., macrophages) can be regulated at the disease site. Alternatively, the therapeutic target of the cargo protein can be another type of immune cell, such as a T cell, a dendritic cell, a stromal cell, a cancer cell, etc.

[0133] In certain embodiments, the present invention relates to the use of phagocytic cells to stimulate the production of M1 macrophages. In one embodiment, stimulating the production of M1 macrophages and / or reprogramming M2-related TAMs, MDSCs, inflammatory M1 macrophages into M1 macrophages will help to remove solid cancers (e.g., tumors) in subjects.

[0134] In certain embodiments, as further described herein, the disease is associated with M2 macrophage activation. A skilled person can determine whether a particular disease is associated with M2 macrophage activation. For example, a skilled person will be able to phenotype / score macrophages at the disease site using markers such as those described in Table 1.

[0135] In certain embodiments, the disease is cancer. For example, the cancer may be associated with M2 macrophage activation and / or solid cancer.

[0136] In certain embodiments, the phagocytes can be used to treat or prevent a solid cancer selected from the group consisting of breast, brain, bone, bone marrow, skin, colon, liver, colorectal, prostate, stomach, gastric, ovarian, oral, esophageal, pancreatic, gallbladder, lung, thyroid, endometrial, head and neck, kidney, bladder and / or glioma.

[0137] In certain embodiments, administration of phagocytes acts to increase the proportion of M1 macrophages at the disease site. For example, the number of M1 macrophages can be increased by 1.1×, 1.2×, 1.3×, 1.4×, 1.5×, 2×, 3×, 10×, 15×, 20×, 100×, 1000× or more.

[0138] In certain embodiments, administration of phagocytes acts to reduce the proportion of M2 macrophages at the disease site. For example, the number of M2 macrophages can be reduced by 1.1×, 1.2×, 1.3×, 1.4×, 1.5×, 2×, 3×, 10×, 15×, 20×, 100×, 1000× or more.

[0139] In certain embodiments, administration of phagocytes acts to reduce angiogenesis, invasion, metastasis, growth, and / or cell proliferation of a disease.

[0140] The phagocytes of the present invention can be administered to any suitable subject. For example, the subject can be a mouse, sheep, cow, pig, horse, or any other mammal. The subject can be a human. The subject can be an infant, a teenager, or an adult. Typically, the subject suffers from, is susceptible to, or is at risk of suffering from a related disease. For example, the subject may suffer from or be genetically susceptible to cancer, such as a solid cancer.

[0141] The phagocytes of the present invention can be administered in a manner compatible with the dosage form, and the dosage will have an immune, preventive and / or therapeutic effect. The amount administered may depend on the subject to be treated, the ability of the subject's immune system to respond to the antigen, and the degree of response required.

[0142] The exact amount of phagocytes / drug composition required to be administered may depend on the judgment of the physician and may vary for each subject.

[0143] Any suitable number of cells can be administered to a subject. For example, at least or about 10 5 , 10 6 , 10 7 , 10 8 , 10 9 As a guide, the number of cells of the invention to be administered may be 10 5 Up to 10 9 , preferably 10 6 Up to 10 8 Administration may be with reference to any of the specific numbers discussed above in the context of the present invention. Typically, one or more doses of phagocytes are administered to a subject.

[0144] In the case of administration or presence of cells, a culture medium may be present to promote survival of the cells. In some cases, the cells of the present invention may be provided in frozen aliquots, and a substance such as DMSO may be present to promote survival during freezing. Such frozen cells will typically be thawed and then placed in a buffer or culture medium for maintenance or administration.

[0145] In certain embodiments, the present invention provides autologous cell therapy.

[0146] In certain embodiments, one or more phagocytic cells are derived from peripheral blood as described herein. For example, phagocytic cells can be derived from monocytes and cultured, proliferated and / or differentiated using methods known in the art, including those described herein.

[0147] In certain embodiments, phagocytic cells are contacted with polyhedrin crystals under conditions that induce phagocytosis as described herein. For example, the crystals can be contacted with undifferentiated phagocytic cells (e.g., monocytes). After contact with the crystals, such cells can be cultured and differentiated into macrophages (e.g., M1 macrophages). Alternatively, the crystals can be contacted with phagocytic cells after culture and / or differentiation.

[0148] In certain embodiments, phagocytes are cultured and / or differentiated in vitro using methods known in the art, including those described herein.

[0149] In certain embodiments, phagocytes are isolated and / or formulated with a pharmaceutically acceptable carrier or diluent using methods known in the art, including those described herein.

[0150] In certain embodiments, phagocytes are re-administered to the subject.

[0151] In the autologous cell therapy methods described herein, the cargo protein is typically one or more therapeutic agents described herein.

[0152] In the autologous cell therapy methods described herein, the disease is typically one or more of the diseases described herein.

[0153] Those skilled in the art can predict that the treating physician can determine further administration modes, dosages of therapeutic agents and treatment regimens according to methods known in the art.

[0154] Imaging

[0155] The present invention also provides a method of imaging and / or characterizing a disease. The disease may be any disease described herein, such as any solid cancer described herein.

[0156] In certain embodiments, a method of imaging and / or characterizing a disease comprises administering to a subject a phagocyte (or a population thereof) of the invention.Any phagocyte (or a population thereof) described herein may be administered to a subject.

[0157] In certain embodiments, the subject is a mouse or a human as described herein.

[0158] In certain embodiments, the phagocytic cells comprise one or more detectable agents described herein. For example, the one or more detectable agents can be fluorescent agents (eg, GFP) or luminescent agents (eg, luciferase).

[0159] In certain embodiments, the method further comprises detecting the detectable agent, thereby imaging and / or characterizing at least a portion of the diseased tissue (eg, solid cancer).Any suitable method can be used to detect the detectable agent, including the methods further described herein. DETAILED DESCRIPTION

[0160] Example 1 - Phagocytosis by professional phagocytes Protein crystals

[0161] We investigated whether professional phagocytes such as monocytes, neutrophils, and macrophages can engulf Protein crystals. eGFP was attached as a monolayer to the wells of a 12-well plate, and undifferentiated THP-1 cells (monocytes) were then seeded on top of the crystals. eGFP was briefly mixed with monocytes before the mixture was evenly plated on the wells. After a 24-hour incubation period, the cells were stained and imaged. In either experimental setup, monocytes successfully uptake multiple protein (Fig. 1a-d). In some cases, a single cell could take up more than 35 individual crystals. Phagocytosis of the crystallized protein pool was independent of the embedded cargo protein because the absence of protein ( Empty), growth factors, cytokines, chemokines or fluorescent proteins The crystals were taken up efficiently.

[0162] THP-1 monocytes were differentiated into M0 macrophages by incubating the cells in complete medium supplemented with PMA (Genin et al., 2015; Del Arosa et al., 2016). After 24 h of incubation in differentiation medium, the cells began to attach and change their morphology as well as their cytokine secretion pattern, such as increased activin A secretion, indicating differentiation into M0 macrophages. Subsequently, M0 macrophages were further differentiated into M1 or M2 macrophages and eGFP protein crystals were added to the M0, M1, and M2 cell cultures. The cultures were further incubated and observed after 48 hours. Although M0 and M2 phagocytic cells showed similar levels of phagocytosis, almost all protein crystals (Figure 2a and c, respectively), but the particles were taken up less efficiently by M1 macrophages (Figure 2b). The efficiency of the crystal is M2>M0>M1.

[0163] The motility and viability of M0 macrophages during and after phagocytosis were also assessed. After the addition of PODSeGFP protein crystals, M0 cultures were monitored for 48 hours using a real-time cell history recorder at a rate of 1 frame / min in both bright field and fluorescence channels (Figures 3a-3b). Still images from the time-lapse recordings showed that During and after eGFP phagocytosis, many M0 macrophages are active and therefore healthy. This can be seen by observing the uptake of green fluorescence by phagocytic cells. Successful phagocytosis is detected by its position change from static (i.e., surface adherent) to mobile. In addition, differentiated macrophages, i.e., M0, M1, and M2, are usually adherent when healthy and take up many cells per cell during culture. No cell detachment was observed after the protein crystals.

[0164] Example 2 - Phagocytosis by non-professional phagocytes Protein crystals

[0165] Next, determine Do the shape and size of protein crystals also facilitate phagocytosis by non-professional phagocytes, such as fibroblasts, osteochondrocytes, lymphocytes, erythrocytes, epithelial cells, myoblasts, or muscle cells? eGFP was seeded as a dense monolayer into the wells of a 96-well plate and dehydrated. Subsequently, fibroblasts or chondrocytes were seeded on the adherent crystals and cultured for up to 48 hours. As with macrophages, most of the seeded Protein crystals were efficiently taken up by fibroblasts (Figure 4a) and chondrocytes (Figure 4b), accumulated inside the cells, and very few protein crystals could be found in the spaces between cells. Similar to differentiated macrophages, fibroblasts and chondrocytes are adherent cells that detach when unhealthy or apoptotic. After forming protein crystals, cells remained attached to the surface and continued to proliferate to a hyperconfluent state.

[0166] Next, determine have been taken up by the cells, rather than simply attached to the surface of macrophages. Empty (pure polyhedrin crystals, no cargo) were incubated with the cells for 24 hours, and then the cells were fixed to make their membranes impermeable to the antibody, demonstrating phagocytosis. The cells were then exposed to the anti-polyhedrin primary antibody, which recognizes only the cargo that has not been phagocytosed and is attached to the cell surface or is still attached to the TC plastic surface. Crystals. Crystals recognized by the primary antibody were observed using FITC secondary antibodies and fluorescence microscopy. When THP-1 monocytes were observed using bright field microscopy, almost all cells took up multiple After switching to the fluorescence channel, an estimated 90% of the crystals had no equivalent fluorescence signal compared to the bright field image. This provided clear evidence that the polyhedrin crystals were located within THP-1 cells, as the antibody pair could not recognize In the case of M1 macrophages, different levels of phagocytosis can be observed, where more protein crystals can be found outside the cell borders. Therefore, the antibody pair readily recognizes these crystals, which are clearly visible in the fluorescence channel (Figure 5b). eGFP for 24 hours to demonstrate phagocytosis by non-professional phagocytes. The cells were then fixed, stained for cytoskeleton and nucleus, and imaged using confocal microscopy. The eGFP crystals were all in the same focal plane (Figure 5c).

[0167] Example 3 - After phagocytosis Shows specific biological activity

[0168] To assess phagocytosis Growth Factors Whether GF still exhibits specific biological activity of the encapsulated cargo, using GM-CSF and SCF was tested on undifferentiated monocytic cells (THP-1) in two different configurations. GFs were seeded as dense monolayers of protein crystals in the wells of a 24-well plate and adhered to the TC surface. Monocytes were then seeded on the same surface (Fig. 6a, I) and cultured for 4 days. GF crystals were easily engulfed (Fig. 6a, II). In contrast, in the second setting, GF crystals adhere to the porous membrane of the TC insert, and then monocytes are seeded in the wells and physically separated. and cells, thereby preventing phagocytosis (Figure 6a, III). Throughout the experiment, only cells were on the surface of the wells and therefore could not take up protein crystals (Figure 6a, IV). After 4 days of culture, the final cell number was assessed using a colorimetric method. Regardless of whether phagocytosis crystals (Fig. 6b, solid grey bars and dashed lines, respectively), each growth factor cargo showed proliferation stimulatory activity, as the number of cells in their presence increased significantly compared to measurements of cells without addition (Fig. 6b, horizontal lines). This suggests that the engulfed Protein crystals can exhibit specific biological activities, and thus macrophages can be used in a Trojan Horse strategy to deliver protein depots for sustained release.

[0169] Materials and methods

[0170] Crystal synthesis

[0171] All Proteins were synthesized as previously described (Nishishita et al., Biomaterials 2011; 32: 3555–3563; Matsumoto et al., Sci Rep 2012; 2: 935). All constructs were fused with the H1 incorporation tag (US8554493B2). Briefly, -Insect (Mirus Bio) Baculovirus (BV) DNA and transfer DNA are co-transfected into standard Spodoptera frugiperda 9 (Sf9) cells. The infectious BV produced is collected and then plaque purified to isolate single recombinant BV. Isolated plaques are first screened, then positive BVs are collected, amplified and finally used to infect large-scale Sf9 cell cultures to produce Subsequently, the crystals were collected and purified by lysing Sf9 cells through several rounds of sonication and PBS washing. Finally, the purified Sterility testing and lyophilization are performed. Although equivalence depends on the specific situation, in terms of biological activity, 1.5×10 4 Crystals are roughly functionally equivalent to 1 ng of many standard growth factors, cytokines, or chemokines (Matsumoto et al., 2015).

[0172] Cell culture and differentiation

[0173] Undifferentiated culture of mononuclear suspension cells (THP-1, ATCC) in RPMI-1640 supplemented with 2 mm L-glutamine and 10% FBS (complete medium). For M0 differentiation, THP-1 was centrifuged and the conditioned medium was replaced with fresh complete medium supplemented with 100 ng / ml phorbol 12-myristate-13-acetate (PMA, Sigma P8139). Cells were inoculated and cultured for up to 12 days, with the medium replaced every 3-4 days. After 24-48 hours, M0 macrophages were further differentiated into M1 or M2 macrophages as described in [Genin et al. 2015, Del Arosa et al. 2016]. In brief, the M0 differentiation medium was removed and the adherent M0 cells were washed twice with serum-free medium. For further differentiation, complete medium supplemented with 100 ng / ml LPS and 20 ng / ml IFN-γ (M1) or 20 ng / ml IL-4 and 20 ng / ml IL-13 (M2) was added to the adherent M0 cells and then cultured for another 10 days.

[0174] Phagocytosis

[0175] Will Crystals were centrifuged onto tissue culture (TC) plates; or centrifuged and dehydrated on TC plates; or premixed with phagocytes for up to 48 hours before plating. Phagocytes were then inoculated when needed and subsequently cultured in complete medium. Phagocytosis was demonstrated using a real-time cell history recorder (JuLi stage, NanoEnTek Inc.) or commonly used immunohistochemistry (IHC) staining and techniques.

[0176] For specific staining of polyhedrin, cells were fixed with 10% formalin for 10 minutes and then blocked in 1% PBS-BSA for 1 hour. Subsequently, proprietary rabbit anti-polyhedrin (1:1000 dilution in 1% PBS-BSA) was incubated on cells for 1 hour, followed by three washes with 1% PBS-BSA. Finally, anti-rabbit FITC secondary antibody (1:1000 dilution in 1% PBS-BSA) was incubated for 1 hour and washed three times with 1% PBS-BSA before mounting in ProLong Diamond Antifade mounting medium (ThermoFisher Scientific).

[0177] Proliferation assay

[0178] Will GM-CSF or SCF (1.8×10 6 ) were seeded directly into the wells of a 24-well plate as a single layer or into a 24-well TC insert (1 μm pore size, Corning Falcon). Subsequently, 4000 monocytes were seeded into the wells containing the monolayer. Protein crystals; or containing The cultures were then incubated for 4 days and the final cell number was assessed using a colorimetric assay (OrangU, Cell Guidance Systems).

[0179] discuss

[0180] This application demonstrates the potential use of self-assembling proteins in phagocyte-mediated strategies for treating diseases. The polyhedrin crystals produced by the expression system demonstrate this use. Advantageously, it is demonstrated herein that:

[0181] (1) Crystals are taken up efficiently by both professional and nonprofessional phagocytes;

[0182] (2) Intake Post-crystal cells remained healthy, and polyhedrin alone had no detectable effect on the phenotype of PCs;

[0183] (3) When When the crystals contained mitogenic cytokines, the proliferation rate of phagocytes increased significantly, indicating that the cargo protein was released intact inside the cell and was able to interact with its receptor;

[0184] (4) Cargo protein Crystal secretion;

[0185] (5) No intake The crystal co-culture cells themselves have an effect on the uptake of The crystal phagocytes respond to the cargo proteins secreted by the phagocytes, thereby changing the phenotype of the co-cultured cells.

[0186] Phagocytes are closely associated with disease etiology and tissue remodeling. In particular, the ability of macrophages to infiltrate diseased tissues makes them attractive targets for therapy and also as agents for drug delivery.

[0187] Significant progress has been made in understanding the role of the immune system and phagocytes in disease development, leading to the development of effective new therapies. Proteins, including cytokines, have demonstrated therapeutic potential, and many have received marketing approval, primarily for applications where repeated dosing is possible via systemic administration. With some notable exceptions, such as IL-2 for the treatment of melanoma and renal cell carcinoma, the fragility of proteins has hampered attempts to use them directly or in Trojan Horse strategies to treat disease, despite significant toxicity to non-target cells.

[0188] Given the importance of phagocytes in disease and the therapeutic potential of cytokines and other vulnerable proteins, drug delivery devices that can maintain specific effects on phagocytes provide a technology platform for protein-based therapeutic strategies.

[0189] The crystals exhibit outstanding stability. For example, after storage in a protease-free solution at 37°C for 6 months, they still retain high levels of biological activity. The crystals have an optimal size range (0.2-6 microns), shape (cubic), and elasticity (rigidity). Crystals are efficiently taken up by a wide range of professional and non-professional phagocytes.

[0190] Another striking feature of crystals is their dynamic release mechanism that requires proteases. It has been previously shown that many matrix metalloproteinases can mediate this release. Unexpectedly, this paper shows that once taken up by phagocytes, Crystals persist for several weeks, and the ingesting phagocytes remain healthy. The cytokines released by the crystals continue to have a proliferative effect on the cell population. Furthermore, we demonstrate that cytokines are released from phagocytic cells and that the released cytokines are able to modulate the phenotype of neighboring cells of the non-ingested macrophages.

[0191] Example 4- The Trojan Horse Approach

[0192] Micrometer-sized cubic co-crystals containing a stable recombinant protein cargo that can be released continuously under the activity of proteases. have an ideal size, shape, and hardness to facilitate efficient phagocytosis. For a Trojan Horse approach to become a viable tool for therapeutic protein delivery, several criteria must be met. These include:

[0193] (1) Monocytes and macrophages need to tolerate micron-sized phagocytosis of mitochondria without significantly altering their viability and metabolism;

[0194] (2) Swallowing micron-sized should not alter the capacity and motility characteristics of macrophages, including chemosensing and the ability to cross small vessels and capillaries;

[0195] (3) Devouring Intracellular degradation of protein crystals must result in the release of intact and biologically active cargo protein; and

[0196] (4) Release of cargo protein The phagocytes should be able to influence the behavior of neighboring cells.

[0197] This paper proves that the cubic Crystals are rigid micrometer-sized protein crystals that can be efficiently engulfed by both professional and non-professional phagocytes. Growth factors secreted from the cells can still trigger their specific biological activities, such as the proliferation of phagocytes that ingest them.

[0198] Contrary to expectations, this paper further demonstrates that The crystals meet all of the criteria required for a therapeutic strategy using phagocytes as Trojan horses, either by modulating the function of the macrophages themselves or coordinating the behavior of other cells.

[0199] result

[0200] Given the cytotoxic effects of different types of nanoparticles on macrophages in previous studies (Lanone et al. 2009, Akter et al. 2018, Feng et al. 2018), After crystallization, the viability of M0, M1, and M2 was examined. In this experiment, THP-1 monocytes were differentiated into M0 macrophages by incubating the cells in complete medium supplemented with PMA. M0 cells were then polarized into M1 and M2 macrophages by incubating the cells in complete medium supplemented with IFN-γ and LPS or IL-4 and IL-13, respectively. Subsequently, the cells were incubated with different numbers of Empty (no cargo protein) or FGF-10 for 24 h, and their viability was tested at 48 and 96 h after uptake ( Figure 7 a and b). The results of the WST-8 assay showed that cell viability was not reduced compared with untreated cells, indicating that The particles were non-toxic over a median dose range up to 15 PODS / cell.

[0201] We next examined whether the movement of macrophages triggered by chemotactic signals was affected by To quantify the phagocytosis of potential changes caused by the separation of M0 cells with The cells were incubated with IL-2 for 24 h and then observed for another 24 h under normal growth conditions using a live cell imaging system. Subsequently, the unloaded and loaded cells were analyzed by tracking each of 25 cells in 720 frames. The movement of cells induced by IL-2 was determined first using the manual tracker function of Image J and then using the chemotaxis and migration tools of Ibidi (see Figure 8 a and b). Loaded and unloaded trajectories when comparing migration distance and activity randomness No differences were detected in the activity of the cells. The ability of M0 cells to follow a chemotactic gradient of IL-2 was repeated with slight changes: After that, the cells were placed in serum-free medium and transferred to a chemotaxis slide with two reservoirs. Reservoir 1 contained medium containing 10% serum, which served as a chemoattractant, and reservoir 2 contained serum-free medium. Unloaded and loaded cells were studied by tracking 26 cells as described above. IL-2 cell motility (respectively Fig. 9 a and b). Analysis of this tracking data shows that Uptake of crystals did not impair the directional migration of macrophages toward the chemoattractant source.

[0202] To evaluate the effects of primary cells on To determine whether phagocytosis of crystals was equally efficient, monocytes and macrophages were isolated from the tibiae of C57BL / 6 mice and cultured for 5 days. eGFP for 24 hours and then monitored for another 24 hours using a live cell imaging system. Compared to the human monocytic cell line, the mouse primary cells were more active. They phagocytosed the Crystals, which can also take in similar amounts Crystal. Fig.10 Four frames are shown in which a loaded cell (dashed circle) can be seen reaching forward to collect and ingest another cell to its left. Crystal.

[0203] It has been proven that the load The loaded macrophages were still able to respond to and follow chemotactic signals, and here we assessed whether the loaded cells retained their ability to cross narrow spaces, another important characteristic of macrophages. eGFP for 24 h and then placed in serum-free medium and transferred to 24-well tissue culture inserts containing 8 μm pores, which are similar in diameter to small blood vessels. Regular maintenance medium containing 10% serum was used as a chemoattractant in the bottom wells ( Fig.11 b). A second well without any serum as a chemoattractant served as a control to show that the cells in the bottom well had actively migrated downwards, rather than randomly moving across the well or simply settling under gravity ( Fig.11 a) After a further 24 h of incubation, both labs were analyzed by bright field and fluorescence microscopy. The number of cells in the wells with and without the chemoattractant showed a large difference, indicating that despite the presence of Cargo-loaded M0 cells were not only able to pass through narrow pores but also actively migrate through 8 μm pores to the chemotactic source.

[0204] Importantly, macrophages are actively recruited by chemical cues from solid tumors (Dandenkar et al. 2011), so we tested whether conditioned media from cancer cell lines could attract the cargo using the same cell chambers as described above. eGFP macrophages. A375 cells, a human malignant melanoma cell line, were grown in growth medium containing 10% serum for 3 days. The conditioned medium was then used as a chemoattractant in the bottom wells ( Fig.12 b) In the second cell chamber, the medium without 10% serum in the bottom well served as a control to determine the level of non-directed macrophage motility in the absence of factors secreted by A375 cells ( Fig.12 a) Subsequent microscopic examination showed that the load The eGFP M0 cells were specifically attracted by the conditioned medium of the cancer cell line and migrated directionally through the 8 μm pores toward the chemotactic source.

[0205] The activation and polarization of monocytes toward an M1-like phenotype upon contact with foreign substances is a key element of the innate immune system. We tested whether contact and uptake of the crystals affect their polarization state. Secretion of IL-6 before and after ingestion, IL-6 is a marker of M1 polarization ( Fig.13 ). First, it was confirmed that IL-6 can indeed represent the polarization state, showing that M1 cells increased IL-6 concentration levels in the cell culture medium to more than 4 ng / ml immediately after polarization, while non-activated THP-1 cells and M0 and M1 cells did not increase IL-6 concentrations above background levels ( Fig.13 a). Then the polarization medium was replaced with FGF-2-containing medium was added and phagocytic uptake was allowed to proceed for 24 hours. During this incubation time, both loaded and unloaded M1 cells increased IL-6 concentrations to 200 pg / ml. M1 cells of the crystal and not Levels of the polarization marker IL-6 remained unchanged among crystal-incubated M1 cells. Crystal ingestion did not alter the polarization state of M1 cells. Crystal uptake did not induce IL-6 secretion from M0 and M2 cells, suggesting that phagocytosis Crystals did not alter the polarization state of any macrophage type.

[0206] To test the intake Whether the cargo proteins are released from macrophages into the culture medium, M0 cells and The cells were incubated with IL-6 for 24 h, washed twice, and then incubated with The cells were cultured in normal growth medium for another 4 days. The culture medium was then collected and analyzed by ELISA for the presence of IL-6 ( Fig.14 a). Surprisingly, not only was the cargo protein IL-6 detected in the cell culture medium, but the level of IL-6 was also dose-dependent: the more IL-6 was loaded into M0 cells, the more was detected in the culture medium after 4 days. As a comparison, we also measured a similar amount of non-phagocytic (naked) The amount of IL-6 released by Fig.14 b) From naked The amount of IL-6 released is greater than the amount taken up by macrophages As a further control, M0 cells were incubated with FGF-2 was incubated together and the IL-6 level in the culture medium was analyzed. Not only was the IL-6 level lower than the background (<10 pg / ml), but its level also did not change in a dose-dependent manner.

[0207] To study the release of macrophages To determine whether the cargo protein was biologically active, a proliferation assay was performed using NIH-3T3 cells that respond to FGF-2 protein. NIH-3T3 cells were seeded in a 24-well plate in normal growth medium and, after 24 hours of growth, the medium was replaced with serum-free conditioned medium. FGF-2 (10 / cell) for 24 hours, placed in NIH-3T3 serum-free conditioned medium, and seeded into 24-well TC inserts. Then, the cells were co-incubated for another 4 days, and the number of NIH-3T3 cells was assessed by performing the WST-8 assay ( Fig.15 ). Compared with empty macrophages (blue bars) and cells only (horizontal green line), all types of loading Co-incubation of macrophages with FGF-2 supported the growth of FGF-2-responsive NIH-3T3 cells under serum-free conditions.

[0208] discuss

[0209] The potential use of packaging proteins delivered by macrophages as a novel therapeutic drug delivery strategy has been explored. In particular, the use of polyhedrin based on the polyhedrin from Bombyx mori cytoplasmic polyhedrosis virus has been investigated. Use of polyhedrin crystals produced by expression systems.

[0210] Drugs need to be selectively targeted to diseased tissues (particularly cancer) to reduce the level of off-target toxicity and improve efficacy. The use of cells as therapeutic delivery systems, also known as the Trojan Horse strategy, has been discussed since the late 1970s. The development of effective particles containing drugs makes the function and subsequent release of macrophages a challenge, especially for protein drugs such as cytokines. Non-phagocytic cells may also be used for Trojan Horses. Although still under investigation, there are currently at least two therapies using red blood cells as drug carriers in Phase III trials (NCT02770807, NCT03665441), but to date, no cell-based delivery system has been approved for clinical use. A clear advantage of macrophages over other cell delivery systems is their inherent disease homing ability. As the first line defender of inflammation and as part of the foreign body response, they are directly attracted to the side of solid tumors and other diseases.

[0211] This paper proves that the swallowed The protein is nontoxic to many types of macrophages, even at high particle counts. Directed migration of crystal-loaded macrophages toward the chemoattractant was not impaired. Crucially, loaded macrophages were still able to change their cell morphology to pass through narrow spaces, as evidenced by their ability to actively pass through pores as small as 8 μm in diameter. The released cargo protein was reliably detected in a dose-dependent manner using ELISA in macrophage culture media containing as few as 5 More surprisingly, the released proteins showed biological activity, as shown by the FGF-2 responsive cell lines and the loading Therefore, we developed a PODS-based macrophage-mediated Trojan Horse strategy for drug delivery to cancer and other diseases.

[0212] Materials and methods

[0213] Crystal synthesis

[0214] All Protein synthesis was as described previously (Nishihita et al. 2011, Matsumo et al. 2012). All constructs were fused to the H1 incorporation tag (Metcalf et al. 2008). -Insect (MirusBio) Baculovirus (BV) DNA and transfer DNA are co-transfected into standard Spodoptera frugiperda 9 (Sf9) cells. The resulting infectious BV is collected and then plaque purified to isolate single recombinant BV. Isolated plaques are first screened, then positive BVs are collected, amplified and ultimately used to infect large-scale Sf9 cell cultures to produce Subsequently, the crystals were collected and purified by lysing Sf9 cells through several rounds of sonication and PBS washing. Finally, the purified Sterility testing and lyophilization are performed. Although equivalence depends on the specific situation, in terms of biological activity, 1.5×10 4 Crystals are roughly functionally equivalent to 1 ng of many standard growth factors, cytokines, or chemokines (Matsumoto et al., 2015).

[0215] Cell culture and differentiation

[0216] Undifferentiated mononuclear suspension cells (THP-1, ATCC) were cultured in RPMI-1640 (A10491, Gibco) supplemented with 10% BCS (30-2030, ATCC) (complete medium). For M0 differentiation, THP-1 cells were centrifuged and the conditioned medium was replaced with fresh complete medium supplemented with 100 ng / ml phorbol 12-myristate-13-acetate (PMA, Sigma P8139). After 48 hours, M0 macrophages were further differentiated into M1 or M2 macrophages as described in (Rios de la Rosa et al., 2017, Genin et al., 2015). Briefly, the M0 differentiation medium was removed and the adherent M0 cells were washed twice with serum-free medium. For further differentiation, complete medium supplemented with 100 ng / ml LPS and 20 ng / ml IFN-γ (M1) or 20 ng / ml IL-4 and 20 ng / ml IL-13 (M2) was added to adherent M0 cells and then cultured for another 48 hours. A375 cells (ATCC) were cultured in DMEM (41966, Gibco) containing 10% FBS (F7524, SIGMA). 2e4 cells / cm 2 Cells were seeded at a density of 2e5 cells / ml and passaged every 3 days. TF-1 cells (ATCC) were cultured in RPMI-1640 containing 2 ng / ml GM-CSF and 10% FBS. Cells were seeded at a density of 2e5 cells / ml and passaged twice a week.

[0217] Phagocytosis

[0218] Will Crystals were centrifuged onto tissue culture (TC) plates; or centrifuged and dehydrated on TC plates; or premixed with phagocytes for up to 48 hours before plating. Phagocytes were then inoculated when needed and subsequently cultured in complete medium. Phagocytosis was demonstrated by monitoring with a real-time cell history recorder (JuLi stage, NanoEnTek Inc.).

[0219] Viability analysis

[0220] For testing To assess the viability of macrophages after uptake, THP-1 cells were seeded at a density of 2e5 cells / ml and differentiated and polarized as described above. Empty and Addition of FGF-10 to unpolarized M0 and polarized M1 and M2 macrophages produced an average of 5 or 15 The cells were then incubated for 48 and 96 hours and analyzed by WST-8 assay (Orangu TM , Cell Guidance Systems) to determine cell viability.

[0221] Activity Tracking

[0222] THP-1 cells were seeded at a density of 2e5 cells / ml in 24-well plates and differentiated into M0 macrophages as described above. IL-2 was incubated with complete medium for 24 hours. The medium was then renewed and cells were recorded using a real-time cell history recorder (JuLi stage, NanoEnTek Inc.). Images were taken every 2 minutes for 24 hours and analyzed using the manual tracker and chemotaxis and migration tools (Ibidi) of Image J.

[0223] Chemotaxis

[0224] THP-1 cells were seeded at a density of 2e5 cells / ml in T25 TC flasks and differentiated into M0 macrophages as described above. M0 cells were then IL-2 was incubated with the cells in complete medium for 24 hours. Subsequently, the M0 macrophages were washed with PBS and then lysed with enzyme-free detachment buffer (cell dissociation buffer, ) was separated. The cells were centrifuged, recounted and seeded in serum-free RPMI-1640 medium at a density of 2e6 cells / ml and placed in chemotaxis μ-slides (Ibidi). 10% FBS was used as a chemoattractant. A real-time cell history recorder (JuLi stage, NanoEnTek Inc.) was used to record cells in the observation area. Images were taken every minute for 12 hours and then analyzed using the manual tracker of Image J and the chemotaxis and migration tool (Ibidi).

[0225] Directed Migration

[0226] THP-1 cells were seeded at a density of 2e5 cells / ml in T25 TC flasks and differentiated into M0 macrophages as described above. M0 cells were then incubated with 5e5 eGFP was incubated in complete medium for 24 h ( PODS / cell). Subsequently, the M0 macrophages were washed with PBS and then lysed with enzyme-free detachment buffer (cell dissociation buffer, ) was isolated. The cells were centrifuged, recounted, and seeded at a density of 1e6 cells / ml in serum-free or RPMI-1640 medium containing 10% FBS into 24-well inserts. The bottom wells were then filled with serum-free medium, medium containing 10% FBS, or 3-day conditioned medium from A375 cells. Microscope images of the bottom wells and inserts were taken after 24 hours of migration.

[0227] Endogenous IL-6 secretion

[0228] THP-1 cells were seeded at a density of 2e5 cells / ml in 24-well plates and differentiated and polarized as described above. The conditioned polarization medium was then collected and stored at -20°C for later analysis. Macrophages were incubated with FGF-2 for 24 hours (10 / cell). The culture medium was then collected and stored at -20°C. The presence of IL-6 in the culture medium samples was tested by ELISA (DY206, R&D Systems) according to the manufacturer's protocol.

[0229] From the load IL-6 release from macrophages

[0230] THP-1 cells were seeded at a density of 2e5 cells / ml in 96-well plates and differentiated into M0 macrophages. M0 cells were then incubated with 1e6, 2e6, or 3e6 IL-6 was incubated in complete medium for 24 h (producing 5, 10 or 30 / cell). The cells were then washed twice with PBS, fresh complete medium was added, and the cells were incubated for 4 days. IL-6 was added to the wells of a 96-well plate and centrifuged at 3000 g for 25 minutes. PBS was removed, the plate was dehydrated in a laminar flow hood, and then complete growth medium was added for 4 days. After incubation, the culture medium was collected and subsequently tested by IL-6 ELISA (DY206, R&D Systems) according to the manufacturer's protocol.

[0231] Functional analysis

[0232] THP-1 cells were seeded at a density of 2e5 cells / ml in 6-well plates and differentiated and polarized as described above. Macrophages were incubated with FGF-2 in complete medium for 24 h (10 / cell). Subsequently, the macrophages were washed with PBS and then with enzyme-free detachment buffer (cell dissociation buffer, ) were isolated. The cells were centrifuged, recounted, and seeded at a density of 2e6 cells / ml in serum-free TF-1 conditioned medium in 24-well inserts. TF-1 cells were grown at 3e4 cells / cm in full growth medium. 2 After one day of growth, the medium was changed to RPMI-1640 supplemented with 0.5% BCS and the macrophage inserts were added. The cells were incubated for 4 days and the WST-8 assay (Orangu TM , Cell Guidance system) was used to measure the viability of TF-1 cells.

[0233] Primary monocytes from mouse tibia

[0234] Murine bone marrow-derived mononuclear cells were isolated according to Wagner et al. 2014. Briefly, tibiae from three C57BL / 6 mice were prepared for cell collection. After washing once with 96% ethanol and twice with PBS, the distal end of each bone was cut with fine scissors, and the bones were flushed through warm culture medium (M199 supplemented with 10% FBS and 1% penicillin / streptomycin) using a 28-G needle and a 1 ml syringe. The effluent from the bones was collected and filtered through a 70 μm cell strainer. The cell suspension was centrifuged at 200 × g for 10 minutes at room temperature. The pellet was washed with 25 ml of culture medium and centrifuged again. The cells were then seeded in 6-well ultra-low attachment plates at 0.5e6 cells / ml. The cells were cultured for 5 days before the experiment.

[0235] Together, these results demonstrate that self-assembling proteins and The utility of crystals, in particular, to modulate the phenotype of phagocytes and neighboring cells, has allowed the development of effective therapies for the treatment of diseases and tissue remodeling as described herein.

[0236] The reader is advised that all papers and documents filed concurrently with this application or prior to this specification and which are open to public inspection with this specification, and the contents of all such papers and documents, are incorporated herein by reference.

[0237] References

[0238] -Andersen et al.Long-Lasting Complete Responses in Patients withMetastatic Melanoma after Adoptive Cell Therapy with Tumor-InfiltratingLymphocytes and an Attenuated IL2 Regimen(2016)DOI:10.1158 / 1078-0432.CCR-15-1879.

[0239] -Andreeson et al.Cancer Res.1990;50:7450–7456.

[0240] -Choi et al.(2007)ACellular Trojan Horse for Delivery of TherapeuticNanoparticles into Tumors Nanotechnology letters(12):3759-65.

[0241] -van Dalen et al.(2019)Molecular repolarization of tumor-associatedmacrophages Molecules 2019,24,9 DOI 10.3390.

[0242] -Faradji et al,Journal of immunological methods.1994;174:297–309.

[0243] -Jiang et al.(2016)Role of IL-2 in cancerimmunotherapy.Oncoimmunology.5(6):e1163462.

[0244] -Matsumoto et al.J Biomater Appl 2015;30:193–200.

[0245] -Mosser et al,Current protocols in Immunology.2008 14:14:12.

[0246] -Redecke et al.,Nature Methods.2013;10:795-803.

[0247] -Zhang et al,Current protocols in Immunology.2008 14:14:11.

[0248] -Xu,F.et al.Membrane-wrapped nanoparticles probe divergent roles ofGM3 and phosphatidylserine in lipid-mediated viral entrypathways.Proc.Natl.Acad.Sci.115,E9041–E9050(2018).

[0249] -Pang,L.et al.A novel strategy to achieve effective drug delivery:exploit cells as carrier combined with nanoparticles.Drug Deliv.24,83–91(2017).

[0250] -Rios de la Rosa,J.M.,Tirella,A.,Gennari,A.,Stratford,I.J.&Tirelli,N.The CD44-Mediated Uptake of Hyaluronic Acid-Based Carriers inMacrophages.Adv.Healthc.Mater.6,(2017).

[0251] -Nishishita,N.et al.The use of leukemia inhibitory factor immobilizedon virus-derived polyhedra to support the proliferation of mouse embryonicand induced pluripotent stem cells.Biomaterials 32,3555–3563(2011).

[0252] -Matsumoto,G.et al.Bone regeneration by polyhedral microcrystals fromsilkworm virus.Sci.Rep.2,935(2012).

[0253] -Lanone,S.et al.Comparative toxicity of 24 manufactured nanoparticlesin human alveolar epithelial and macrophage cell lines.Part.Fibre Toxicol.6,14(2009).

[0254] -Akter,M.et al.A systematic review on silver nanoparticles-inducedcytotoxicity:Physicochemical properties and perspectives.J.Adv.Res.9,1–16(2018).

[0255] -Feng,Q.et al.Uptake,distribution,clearance,and toxicity of ironoxide nanoparticles with different sizes and coatings.Sci.Rep.8,2082(2018).

[0256] -Dandekar,R.C.,Kingaonkar,A.V.&Dhabekar,G.S.Role of macrophages in malignancy.Ann.Maxillofac.Surg.1,150–154(2011).

[0257] -Metcalf,P.et al.Viral polyhedra complexes and methods of use.(2008).

[0258] -Genin, M., Clement, F., Fattaccioli, A., Raes, M. & Michiels, C. M1 andM2 macrophages derived from THP-1 cells differentially modulate the response of cancer cells to etoposide. BMC Cancer 15, 577 (2015).

[0259] -Wagner, M.et al.Isolation and intravenous injection of murine bonemarrow derived monocytes.J.Vis.Exp.JoVE(2014)doi:10.3791 / 52347. SEQUENCE LISTING <110> Cell Guidance Systems Ltd. <120> Self-assembling proteins <130> P22112483WP <150> GB 1917381.4 <151> 2019-11-28 <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 250 <212> PRT <213> silkworm <400> 1 Met Ile Met Ala Asp Val Ala Gly Thr Ser Asn Arg Asp Phe Arg Gly 1 5 10 15 Arg Glu Gln Arg Leu Phe Asn Ser Glu Gln Tyr Asn Tyr Asn Ser Ser 20 25 30 Leu Asn Gly Glu Val Ser Val Trp Val Tyr Ala Tyr Tyr Ser Asp Gly 35 40 45 Ser Val Leu Val Ile Asn Lys Asn Ser Gln Tyr Lys Val Gly Ile Ser 50 55 60 Glu Thr Phe Lys Ala Leu Lys Glu Tyr Arg Glu Gly Gln His Asn Asp 65 70 75 80 Ser Tyr Asp Glu Tyr Glu Val Asn Gln Ser Ile Tyr Tyr Pro Asn Gly 85 90 95 Gly Asp Ala Arg Lys Phe His Ser Asn Ala Lys Pro Arg Ala Ile Gln 100 105 110 Ile Ile Phe Ser Pro Ser Val Asn Val Arg Thr Ile Lys Met Ala Lys 115 120 125 Gly Asn Ala Val Ser Val Pro Asp Glu Tyr Leu Gln Arg Ser His Pro 130 135 140 Trp Glu Ala Thr Gly Ile Lys Tyr Arg Lys Ile Lys Arg Asp Gly Glu 145 150 155 160 Ile Val Gly Tyr Ser His Tyr Phe Glu Leu Pro His Glu Tyr Asn Ser 165 170 175 Ile Ser Leu Ala Val Ser Gly Val His Lys Asn Pro Ser Ser Tyr Asn 180 185 190 Val Gly Ser Ala His Asn Val Met Asp Val Phe Gln Ser Cys Asp Leu 195 200 205 Ala Leu Arg Phe Cys Asn Arg Tyr Trp Ala Glu Leu Glu Leu Val Asn 210 215 220 His Tyr Ile Ser Pro Asn Ala Tyr Pro Tyr Leu Asp Ile Asn Asn His 225 230 235 240 Ser Tyr Gly Val Ala Leu Ser Asn Arg Gln 245 250 <210> 2 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> H1-tag <400> 2 Met Ala Asp Val Ala Gly Thr Ser Asn Arg Asp Phe Arg Gly Arg Glu 1 5 10 15 Gln Arg Leu Phe Asn Ser Glu Gln Tyr Asn Tyr Asn Asn Ser 20 25 30 <210> 3 <211> 53 <212> PRT <213> Artificial Sequence <220> <223> VP3-tag <400> 3 Ala Phe Asn Ser Gln Arg Arg Ser His Thr Leu Arg Leu Leu Gly Pro 1 5 10 15 Phe Gln Tyr Phe Asn Phe Ser Glu Thr Asp Arg Gly His Pro Leu Phe 20 25 30 Arg Leu Pro Leu Lys Tyr Pro Ser Lys Ala Ile Pro Ala Asp Glu Leu 35 40 45 Ile Asp Asn Leu His 50 <210> 4 <211> 235 <212> PRT <213> Artificial Sequence <220> <223> PH label <400> 4 Gly Arg Glu Gln Arg Leu Phe Asn Ser Glu Gln Tyr Asn Tyr Asn Ser 1 5 10 15 Ser Leu Asn Gly Glu Val Ser Val Trp Val Tyr Ala Tyr Tyr Ser Asp 20 25 30 Gly Ser Val Leu Val Ile Asn Lys Asn Ser Gln Tyr Lys Val Gly Ile 35 40 45 Ser Glu Thr Phe Lys Ala Leu Lys Glu Tyr Arg Glu Gly Gln His Asn 50 55 60 Asp Ser Tyr Asp Glu Tyr Glu Val Asn Gln Ser Ile Tyr Tyr Pro Asn 65 70 75 80 Gly Gly Asp Ala Arg Lys Phe His Ser Asn Ala Lys Pro Arg Ala Ile 85 90 95 Gln Ile Ile Phe Ser Pro Ser Val Asn Val Arg Thr Ile Lys Met Ala 100 105 110 Lys Gly Asn Ala Val Ser Val Pro Asp Glu Tyr Leu Gln Arg Ser His 115 120 125 Pro Trp Glu Ala Thr Gly Ile Lys Tyr Arg Lys Ile Lys Arg Asp Gly 130 135 140 Glu Ile Val Gly Tyr Ser His Tyr Phe Glu Leu Pro His Glu Tyr Asn 145 150 155 160 Ser Ile Ser Leu Ala Val Ser Gly Val His Lys Asn Pro Ser Ser Tyr 165 170 175 Asn Val Gly Ser Ala His Asn Val Met Asp Val Phe Gln Ser Cys Asp 180 185 190 Leu Ala Leu Arg Phe Cys Asn Arg Tyr Trp Ala Glu Leu Glu Leu Val 195 200 205 Asn His Tyr Ile Ser Pro Asn Ala Tyr Pro Tyr Leu Asp Ile Asn Asn 210 215 220 His Ser Tyr Gly Val Ala Leu Ser Asn Arg Gln 225 230 235

Claims

1. A professional phagocyte comprising one or more internalized polyhedrin crystals, wherein the crystals encapsulate one or more therapeutic agents; The polyhedron protein is the polyhedron protein of Bombyx mori cytoplasmic polyhedrosis virus shown in SEQ ID NO: 1; The professional phagocytes are monocytes or macrophages.

2. The professional phagocyte of claim 1, wherein: (a) the crystal is capable of secreting the therapeutic agent intact within the professional phagocyte; and / or (b) The therapeutic agent is capable of sustained release from the professional phagocytes.

3. The professional phagocyte of claim 1 or 2, wherein the cell is a mammalian cell.

4. The professional phagocyte of claim 3, wherein the mammalian cell is a human cell.

5. The professional phagocyte of claim 1, wherein the crystals are smaller than 10 microns and / or are substantially cubic in shape.

6. The professional phagocyte of claim 1, wherein the therapeutic agent comprises one or more polyhedrin binding tag sequences selected from the group consisting of: (a) the H1 tag sequence shown in SEQ ID NO: 2; (b) the VP3 tag sequence as shown in SEQ ID NO: 3; or (c) The PH tag sequence shown in SEQ ID NO:

4.

7. The professional phagocyte of claim 1, wherein the professional phagocyte is a macrophage.

8. The professional phagocyte of any one of claims 1-7, wherein the professional phagocyte is derived from the peripheral blood of a subject.

9. The professional phagocyte of claim 1, wherein the one or more therapeutic agents are capable of targeting cells, extracellular vesicles, or free proteins in the tumor microenvironment.

10. The professional phagocyte of claim 9, wherein: (a) the one or more therapeutic agents are capable of stimulating the production of M1 macrophages; and / or (b) the one or more therapeutic agents are capable of reprogramming M2 tumor-associated macrophages (TAMs), inflammatory monocytes and / or myeloid-derived suppressor cells (MDSCs) into M1 macrophages.

11. The professional phagocyte of claim 10, wherein the one or more therapeutic agents are growth factors, cytokines, enzymes, signaling molecules, hormones, antibodies and / or vaccines.

12. The professional phagocyte of claim 11, wherein the one or more therapeutic agents are selected from: (a) Th-1 related cytokines and / or TLR ligands; and / or (b) granulocyte-macrophage colony-stimulating factor (GM-CSF), CSF-2, CSF-1 (M-CSF), CSF-3 (G-CSF), interferon gamma (IFN-γ), TNF-α, IFN-β, IL-6, IL-12, IL-15, IL-21, IL-23, MHC-II, IL-1R, TLR2, TLR4, CD80, CD86, CD68, IL-1, IL-1β, IL-2, CCL2, CCL3, CCL4, CCL5, CCL8, CCL9, CCL10, CCL11, CXCL8, CXCL9, CXCL10, CXCL16, stem cell factor (SCF), histidine-rich glycoprotein, paclitaxel, SOCS3, activin A, BtK, iNOS and / or NOS2.

13. A population comprising more than 1×10 5 A professional phagocyte as claimed in any one of claims 1 to 12.

14. The population of claim 13, wherein the population is autologous.

15. A package comprising the professional phagocytes as claimed in any one of claims 1 to 12 or the population as claimed in claim 13 or 14.

16. A method for producing a professional phagocyte as claimed in any one of claims 1 to 12 or a population as claimed in claim 13 or 14, comprising: (a) contacting one or more professional phagocytes with polyhedrin crystals; and (b) culturing the professional phagocytes under conditions that induce phagocytosis.

17. The method of claim 16, wherein: (i) the polyhedrin crystals are attached to a solid support prior to contacting the professional phagocytes; or (ii) prior to culturing the professional phagocytes, mixing the professional phagocytes with polyhedrin crystals.

18. The method of claim 16 or 17, further comprising: (c) isolating one or more professional phagocytes; and / or (d) formulating one or more professional phagocytes for delivery via an injectable route.

19. A pharmaceutical composition comprising: (a) a professional phagocyte as claimed in any one of claims 1 to 12 or a population as claimed in claim 13 or 14, wherein the professional phagocyte comprises one or more therapeutic agents; and (b) a pharmaceutically acceptable carrier or diluent.

20. Use of the pharmaceutical composition according to claim 19 in the preparation of a medicament for treating and / or preventing a disease in a subject.

21. The use according to claim 20, wherein the disease is a solid cancer.

22. The use according to claim 20 or 21, wherein: (a) the professional phagocytes are derived from the peripheral blood of a subject; (b) contacting the professional phagocytes with polyhedrin crystals under conditions that induce phagocytosis; (c) culturing the professional phagocytes in vitro; and / or (d) returning the professional phagocytes to the subject.

Images