Self-assembling proteins that are phagocytosed

Abstract

The present invention relates to self-assembling proteins that can be used to deliver cargo proteins to phagocytes, and the subsequent delivery of these self-assembling proteins by phagocytes and their use at disease sites. In particular, the present invention relates to phagocytes containing polyhedrin protein crystals, which themselves encapsulate therapeutic and / or diagnostic proteins. The present invention also provides methods of treatment 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 of these self-assembling proteins and their utility by phagocytes at disease sites. In particular, the present invention relates to phagocytes containing, inter alia, polyhedrin protein crystals, which themselves encapsulate therapeutic and / or diagnostic proteins. The present invention also provides methods of therapy using these cells. [Background technology]

[0002] Proteins, including antibodies, growth factors, hormones, enzymes, and vaccines, can be used for treatment. 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 require high doses that can be rapidly cleared and can have significant toxic side effects on non-target cells. For primary tumors, it may be possible to inject proteins directly into the tumor mass; however, this approach remains challenging due to protein instability, particularly in the case of growth factors. While treatment via direct injection into the tumor mass may be feasible, delivery of growth factors and other proteins to widespread disease remains virtually unachievable.

[0003] Phagocytes engulf microscopic particles through an active phagocytic process. A wide variety of different phagocyte types exist in mammals. Phagocytes are important for the immune system's response to infection and disease. For example, macrophages are specialized cells of the innate and adaptive immune systems that play important roles in defense against foreign pathogens, wound healing, and regulating tissue homeostasis. In response to subtle changes in their environment, macrophages undergo activation, also referred to as "polarization," toward a pro-inflammatory M1 phenotype or an anti-inflammatory M2 phenotype. Activation can be transient and non-binary, with a high level of cellular plasticity.

[0004] Stimuli such as IFN-γ, LPS, and GM-CSF result 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) and the release of reactive oxygen and nitrogen intermediates (the "classical" pathogens that kill Th1 responses). In contrast, M2 macrophages stimulated by the cytokines IL-4 and IL-13 secrete anti-inflammatory cytokines (e.g., IL-10 and TGF-β) and chemokines (e.g., CCL17 and CCL22).

[0005] When polarized macrophages encounter foreign bodies, such as microorganisms or other pathogens, cellular uptake is initiated. Once phagocytosed, innate acidic phagolysosomes efficiently degrade biological materials, usually containing proteins. Fragments of the surface proteins of the degraded pathogens are then 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 contribute to the development of diseases such as solid cancers (van Dalen et al., 2019). For example, tumor-associated macrophages (TAMs) typically express an M2 phenotype (alternatively activated subset) and exert immunosuppressive and tumor-promoting functions. Reprogramming such cells to an M1 phenotype (classically activated subset) may suppress the pro-tumor phenotype and reverse anti-tumor immunity.

[0007] Given their close association with disease and their ability to infiltrate diseased tissues, their ease of collection for ex vivo manipulation, and their binary status, it has been proposed that (1) the phenotype of phagocytes can be modulated to influence disease prognosis, and (2) phagocytes can be used as "Trojan horses" to deliver drugs preferentially or specifically to diseased areas, targeting other cells while reducing toxic side effects to non-target tissues (Choi et al., 2014). 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, attempts to use them as a Trojan horse strategy to treat diseases have failed due to the vulnerability of therapeutic cargo proteins. Successful delivery of therapeutic cargo proteins using phagocytes such as macrophages faces numerous challenges, including toxicity to phagocytes, metabolic changes, uptake efficiency, and cargo digestion.

[0009] It is an aim of some embodiments of the present invention to alleviate some of the problems identified in the prior art. Summary of the Invention

[0010] The present invention relates to the use of polyhedrin protein crystals (e.g., PODS® protein crystals) to deliver cargo proteins via phagocytic cells. PODS® are micron-scale cubic co-crystals that can contain stabilized recombinant protein cargo that is released in a sustained manner under the activity of proteases. Certain aspects of the present invention relate to the discovery that such crystals are efficiently endocytosed into those cells and, unexpectedly, secrete their cargo in a manner that retains the biological activity of the cargo and also modulates the behavior of nearby cells.

[0011] Certain aspects of the present invention relate to the discovery that polyhedrin protein crystals are efficiently taken up by macrophage cells and that, unexpectedly, proteins incorporated into these crystals are then released from those cells in a biologically active form. Furthermore, uptake of these crystals does not affect macrophage cell viability or interfere with important macrophage functions such as following chemotactic signals secreted by tumor cells or migrating through narrow spaces, both of which are important for the efficient delivery of therapeutic cargo to disease sites.

[0012] Certain aspects of the present invention relate to improved methods for delivering therapeutic cargo proteins to disease sites by infiltrating phagocytic cells such as macrophages to enhance efficacy and reduce off-target side effects. Another aspect of the present invention relates to methods for delivering diagnostic cargo proteins to disease sites by infiltrating phagocytic cells such as macrophages to image the disease.

[0013] Therefore, the present invention provides - phagocytes containing one or more polyhedrin protein crystals, which crystals encapsulate one or more cargo proteins; 1 x 10 as described herein 5 a population containing more than phagocytes, - a package comprising a phagocyte (or a population thereof) as described herein, - a method for producing a phagocyte (or a population thereof) as described herein, comprising: (a) contacting one or more phagocytes with polyhedrin protein crystals; and (b) culturing the phagocytes under conditions that induce phagocytosis. A method comprising: - a pharmaceutical composition comprising (a) a phagocyte (or population thereof) as described herein, comprising one or more therapeutic agents; and (b) a pharmaceutically acceptable carrier or diluent a pharmaceutical composition comprising a pharmaceutical composition as described herein for use in medicine, a pharmaceutical composition as described herein for use in the treatment of solid cancer, and - a method for imaging a solid tumor, comprising: (a) administering a phagocyte (or population thereof) as described herein, wherein the phagocyte comprises one or more detectable agents; and (b) detecting the detectable agent, thereby imaging at least a portion of the cancer. [Brief explanation of the drawings]

[0014] Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0015] [Figure 1] Phagocytosis of PODS® protein crystals by professional phagocytes is shown. THP-1 monocytes were incubated with PODS® crystals containing enhanced green fluorescent protein (eGFP) for 24 hours. Cells were then imaged in a) brightfield mode (A), fluorescence mode (B), and composite image (C). Or b-d) brightfield mode (A), DAPI staining of nuclei (B), fluorescence mode (C), and composite image (D). The composite image shows multiple cuboidal PODS® eGFP crystals phagocytosed by live monocytes. [Figure 2] Phagocytosis of PODS® protein crystals by various types of professional phagocytes is shown. THP-1 monocytes were first differentiated into M0 macrophages (a), and then further differentiated into M1 (b) or M2 (c) macrophages. Differentiated cell types were cultured with PODS® eGFP for 24 hours and subsequently imaged in bright field (a) and fluorescence mode (b), respectively. [Figure 3]Still images from a time-lapse video observing the phagocytosis of PODS® eGFP crystals are shown. Differentiated THP-1 cells (M0 macrophages) were incubated with PODS® eGFP crystals for 48 hours. Images were captured at a rate of 1 frame per minute in brightfield and fluorescence modes. A) Brightfield tracking of two individual monocytes (black ovals) within the field of view over time, as indicated by the frame numbers, reveals the mobility of M0 macrophages and the phagocytosis of several PODS® crystals along their migratory path. B) Three corresponding still images from the time-lapse video in both brightfield and fluorescence imaging modes (the positions of the tracked M0 macrophages are marked (black and white ovals, respectively)). Once internalized, the initially static PODS® eGFP particles move to follow the trajectory of the migrating monocyte. [Figure 4] Phagocytosis of PODS® protein crystals by various types of non-professional phagocytes. Cells were cultured with PODS® eGFP for 24 hours and then imaged live (a) or fixed (b). a) Phagocytosis of mouse fibroblasts (NIH-3T3) was imaged in brightfield mode (A) and fluorescence mode (B), respectively, after a 24-hour incubation period. [Figure 5] Phagocytosis analysis is shown. PODS® Empty cells were attached to wells and incubated with professional phagocytes (monocytes or M1 macrophages) for 24 hours. Subsequently, the cells were fixed and exposed first to anti-polyhedrin primary antibody, followed by anti-rabbit FITC secondary antibody. Micrographs of monocytes (a) and M1 macrophages (b) with phagocytosed PODS® Empty cells were taken in brightfield and fluorescence modes. c) b) Immunohistochemistry (IHC) images of phagocytosed PODS® eGFP cells within chondrocytes. PODS® eGFP cells were attached to the surface of wells and incubated with chondrocytes for 24 hours. The cells were then fixed, and slides were stained for the cytoskeleton with actin (A) or for nuclei with DAPI (B). Fluorescent signals from PODS® eGFP (C) and composite (D) are shown. [Figure 6] Figure 1 shows the proliferation of THP-1 monocytes stimulated by PODS® protein crystals. PODS® were seeded as a monolayer directly onto the wells of a 24-well plate or into a 24-well tissue culture insert. Monocytes were then seeded into the wells containing the monolayer of PODS® protein crystals, or into a single well of a 24-well plate, or into an empty insert as a baseline control. a) Representative bright-field micrographs of monocyte cultures with PODS® seeded as a monolayer in wells on days 0 (I) and 4 (II). Micrographs of the experiment using PODS® in a TC insert show only cells in the wells on both days 0 (III) and 4 (IV). b) After 4 days of incubation, total cell number was assessed using a colorimetric assay (OrangU, Cell Guidance Systems). The horizontal line represents THP-1 cells without further supplementation, the solid gray bars represent the results of experiments using PODS® as a monolayer, and the dotted bars show the results for PODS® in TC inserts. Error bars indicate standard deviation from the mean. [Figure 7] Macrophage viability after PODS® incorporation is shown. M0, M1, and M2 macrophages were incubated with PODS® Empty or PODS® FGF-10 (1:5 and 1:15) in 96-well TC plates for 24 hours. The medium was then changed, and cell viability was measured 48 hours (A) and 96 hours (B) after PODS® incorporation using a colorimetric assay (Orangu™, Cell Guidance Systems). [Figure 8]Motility of PODS® IL-2-loaded macrophages. Motility of empty macrophages (A) and PODS® IL-2-loaded macrophages (10 PODS® / cell) (B) in complete growth medium was monitored using a live-cell imaging system. Images were taken every 2 minutes over 24 hours and analyzed using Image J's manual tracker and chemotaxis and migration tools (Ibidi). 25 cells per condition were tracked, and migration trajectories were visualized as rose plots (each line represents the trajectory of an individual cell over 24 hours). Track positions were normalized so that each trajectory originated from the (0,0) coordinate. [Figure 9] Directional migration of PODS® IL-2-loaded macrophages is shown. Empty macrophages (A) and PODS® IL-2-loaded macrophages were transferred to Chemotaxis μ-Slides (Ibidi) and tested for their ability to follow a chemoattractant gradient generated by 0-10% BCS. Images were taken every minute for 12 hours and analyzed using the Image J manual tracker utility and chemotaxis and migration tools (Ibidi). 26 cells per condition were tracked, and migration trajectories were visualized as rose plots (each line represents the trajectory of an individual cell over 12 hours). Trajectory positions were normalized so that each trajectory originated from the (0,0) coordinate. Trajectories of cells that migrated toward the 10% BCS origin are shown in black (top), while trajectories of cells that migrated away from the BCS origin are shown in red (bottom). [Figure 10] Figure 1 shows the migration of PODS® eGFP-loaded bone marrow-derived mouse macrophages. BMDMs were incubated with PODS® eGFP for 24 hours and then monitored using a live-cell imaging system at 1 frame per minute. The white dashed circle tracks the migration and retraction of macrophages over 21 frames, which equates to 21 minutes. [Figure 11]Figure 1 shows directed migration of PODS® eGFP-loaded macrophages through 8 μm pores. PODS® eGFP-loaded macrophages (5 PODS® / cell) in serum-free medium were transferred to 24-well inserts with 8 μm pores. The bottom wells were filled with RPMI-1640 alone (A) or RPMI-1640 supplemented with 10% FBS (B). The top image shows the insert after 24 hours of incubation, and the bottom image shows the bottom well. [Figure 12] PODS® eGFP-loaded macrophages track chemical signals from a melanoma cell line through an 8 μm pore. PODS® eGFP-loaded macrophages (5 PODS® / cell) in complete growth medium were transferred to a 24-well insert with 8 μm pores. The wells were filled with complete growth medium (A) or medium conditioned with A375 cells for 3 days (B). The top image is a representative image showing the number of macrophages that migrated into the well after a subsequent 24-hour incubation. The bottom image shows a magnified view. [Figure 13] Endogenous secretion of IL-6 is shown. Media from THP-1, M0, M1, and M2 cells was collected after polarization and tested for the presence of IL-6 by ELISA (A). The same cells were then washed and incubated with PODS® FGF-2 in complete growth medium for 24 hours. The media was collected and tested for the presence of IL-6 by ELISA (B). [Figure 14] Figure 1 shows the release of IL-6 from PODS® IL-6-loaded macrophages. (A) M0 cells were loaded with 5, 10, or 20 PODS® IL-6 or PODS® FGF-2 per cell. Cells were washed and incubated in complete growth medium for 4 days. The medium was collected and tested for the presence of IL-6. (B) As before, the same amount of PODS® IL-6 was spun down into 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. [Figure 15]Figure 1 shows the bioactivity of FGF-2 released from PODS® FGF-2-loaded macrophages. Non-loaded and FGF-2-loaded macrophages (M0, M1, and M2 at 10 PODS® / cell) in TC inserts were incubated with FGF-2-responsive NIH-3T3 cells under serum-free conditions for 4 days. NIH-3T3 cell proliferation was measured using a colorimetric assay (Orangu™, Cell Guidance Systems). DETAILED DESCRIPTION OF THE INVENTION

[0016] The practice of embodiments of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, pharmaceutical formulation, pharmacology and medicine, which are within the skill of those in the art.

[0017] The most common chemical methods are described in Comprehensive Heterocyclic Chemistry IF (Katritzky et al., 1996, Publishers, Pergamon Press), Comprehensive Organic Functional Group Transformations( Katritzky et al., 1995, Publisher, Pergamon Press), Comprehensive Organic Synthesis( Trost et al,.1991, Publisher, Pergamon), Heterocyclic Chemistry (Joule et al. Publisher, Chapman & Hall), Protective Groups in It has been published in Organic Synthesis (Greene et al., 1999, Publisher: Wiley-Interscience) and Protecting Groups (Kocienski et al., 1994).

[0018] The most common molecular biology method is described by Sambrook et al. Molecular Cloning,A Laboratory Manual(2001)Cold Harbor-Laboratory Press,Cold Spring Harbor, NY or Ausubel et al., It is published in Current Protocols in Molecular Biology, published by John Wiley and Sons, NY (1990).

[0019] The most common method of drug formulation is Preformulation and Formulation (2nd ed., Mark Gibson ed.) and Pharmaceutical Excipients: Properties, Functionality and Applications in Research and Industry( It is published in "The Nature of the Brain" (ed. Otilia MY Koo, Publisher: Wiley).

[0020] The most common pharmacological methods are described in A Textbook of Clinical Pharmacology and Therapeutics (5th edition, publisher, Arnold Hodder).

[0021] The most common method for prescribing, dispensing and administering medicines is the British National Formulary 72(BMJ Publishing Group Ltd and the Royal Pharmaceutical Society).

[0022] 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 this disclosure 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 of skill in the art with general dictionaries of many of the terms used in this disclosure. For chemical terms, those of skill in the art can refer to the International Union of Pure and Applied Chemistry (IUPAC).

[0023] Units, prefixes, and symbols are written in the form accepted by the International System of Units (SI). Numeric ranges are inclusive of the numbers defining the range.

[0024] phagocytic cells The present invention provides one or more phagocytes. Typically, the phagocytes are mammalian cells. For example, the phagocytes may be mouse 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.

[0025] In certain embodiments, the phagocytes of the present invention are derived from monocytes. For example, the phagocytes may be derived from an immortalized monocyte-like cell line (e.g., THP-1 ATCC).

[0026] In certain embodiments, pre-existing phagocytes are isolated from resident or induced monocytes in body cavity lavage fluid (alveoli, peritoneal cavity) (Zhang et al, 2008).

[0027] In certain embodiments, the phagocytes of the present invention are obtained by differentiating blood-derived monocytes or monocytes extracted from bone marrow, for example, phagocytes may be obtained from peripheral blood mononuclear cells (PBMCs).

[0028] In certain embodiments, monocytes can be differentiated by supplementing the culture medium with signaling molecules such as growth factors or cytokines. For example, the culture medium can be supplemented with macrophage colony-stimulating factor (M-CSF). The cells can be incubated with additional immune stimuli such as LPS and / or interferon gamma (IFN-γ). The use of such stimuli can induce distinct polarizations that mimic in vivo phenotypes (Mosser et al., 2008).

[0029] In certain embodiments, phagocytes may contain cargo proteins as described herein that are secreted to affect the behavior of other cells without any effect on the phagocyte itself.

[0030] In certain embodiments, the phagocytes of the present invention are autologous phagocytes, that is, they originate from the subject being treated, which may reduce the risk of immune rejection.

[0031] Methods for generating large numbers of phagocytes for autologous cell therapy are available in the art. Typically, phagocytes, such as macrophages, are generated from progenitor cells. For example, such progenitor cells can be established by overexpressing Hoxb8 in medium supplemented with GM-CSF (Redecke et al., 2013). This results in rapidly proliferating clonal cells. Removal of Hoxb8 activity allows the precursors to differentiate into phagocytes (e.g., macrophages).

[0032] The present invention also provides populations of two or more phagocytes. Any number of cells may be present in the population. For example, the population may be comprised of about 10 5 , 10 6 , 10 7 or 10 8 In certain embodiments, the population may comprise more than 10 phagocytes. 9 , 10 10 , 10 11 or 10 12 The population comprises phagocytes, which is preferably an allogeneic and / or autologous population.

[0033] Phagocytic cells may be isolated, substantially isolated, purified, or substantially purified. Phagocytic cells are isolated or purified when they are completely free of any other components, such as culture medium or other cells or cell types. Phagocytic cells are substantially isolated when they are mixed with a carrier or diluent, such as culture medium, that does not interfere with the intended use.

[0034] Phagocytic cells can be isolated by any suitable technique. For example, phagocytes can be isolated by leukapheresis and / or elutriation (Faradji et al., 1994; Andreeson et al., 2000). Phagocytes can be isolated by the method of (Beck et al., 1990). Typically, phagocytes are isolated from the peripheral blood of a subject as described herein. Phagocytes (e.g., monocytes) can then be recovered from the blood and isolated using a combination of leukapheresis and elutriation.

[0035] As discussed in detail below, phagocytes are treated ex vivo, particularly loaded with polyhedrin protein crystals (which themselves encapsulate one or more cargo proteins as described herein) and used therapeutically in the methods of the invention.

[0036] In certain embodiments, the phagocytes are provided in frozen aliquots with substances such as DMSO to facilitate survival during freezing. Such frozen cells are typically thawed and then placed in a maintenance or administration buffer or medium.

[0037] In certain embodiments, the phagocytes are provided in a package. For example, the package may protect the cells from damage during collection, cell culture, or shipping. Suitable packages include, for example, Teflon bags.

[0038] Polyhedrin protein crystals The phagocytes of the present invention contain one or more polyhedrin protein crystals (e.g., PODS® protein crystals). Typically, the crystals are derived from a virus, such as a cypovirus or a baculovirus. For example, the crystals can be derived from a silkworm cypovirus.

[0039] In certain embodiments, polyhedrin protein crystals are produced in cells in which the polyhedrin protein 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 called the active protein or cargo protein. Typically, the promoter is a polyhedrin promoter. Typically, the crystals are produced in insect cells. For example, the crystals can be produced using Spodoptera frugiperda 9 (sf9) cells.

[0040] In certain embodiments, cargo proteins are tagged with a short peptide sequence that binds the cargo protein to the growing polyhedrin crystal. As the crystal continues to grow, the cargo protein is enveloped. In this way, the crystal encapsulates one or more cargo proteins.

[0041] In certain embodiments, the polyhedrin protein comprises the silkworm cypovirus polyhedrin sequence set forth in SEQ ID NO: 1. Alternatively, the polyhedrin protein can 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: 1 based on amino acid identity over the entire sequence of SEQ ID NO: 1.

[0042] In certain embodiments, one or more cargo proteins comprise a polyhedrin-binding tag selected from the H1-tag sequence set forth 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 across the entire sequence of SEQ ID NO: 2.

[0043] In certain embodiments, the one or more cargo proteins comprise a polyhedrin-binding tag selected from the VP3-tag sequence set forth 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 sequence of SEQ ID NO: 3, based on amino acid identity across the entire sequence of SEQ ID NO: 3.

[0044] In certain embodiments, one or more cargo proteins comprise 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 sequence of SEQ ID NO: 4, based on amino acid identity over the entire sequence of SEQ ID NO: 4.

[0045] In certain embodiments, the crystals are internalized within the phagocyte (e.g., inside the phagocyte). For example, they may be located internally within the phagosome or phagolysosome of the cell, or within the cytoplasm or nucleus, rather than externally on the cell membrane.

[0046] In certain embodiments, intact crystals can secrete cargo proteins from within phagocytic cells. In other words, the cargo protein can be released from the crystal and retain its native conformation and / or function. Typically, the cargo protein is released by the action of intracellular proteases (e.g., matrix metalloproteases).

[0047] In certain embodiments, cargo proteins have a desired phenotypic effect on the phagocytes into which they are secreted, for example, the cargo protein may be able to repolarize the phagocyte (e.g., toward an M1 macrophage phenotype).

[0048] In certain embodiments, the cargo protein is released from the phagocytic cell over a sustained period of time. For example, the cargo protein may be released from the cell across the cell membrane. Unexpectedly, the cargo protein can alter the behavior of neighboring cells that would not themselves contain the crystals. In certain embodiments, the cargo protein is released from the cell via exosomes. These exosomes can be collected for therapeutic or research purposes.

[0049] Typically, the cargo protein is released in a sustained manner for a period longer than the time required for the phagocytes to reach and / or infiltrate the intended target, for example, the cargo protein may be released in a sustained manner from the phagocytes for at least 2 days, 5 days, 7 days, 10 days, 14 days, 20 days, 21 days, 1 month or more.

[0050] Typically, crystals are regular arrays of polyhedrin proteins assembled into a crystal lattice. They can be cubic or any other shape. The crystals of the present invention are typically isolated from cells, such as insect cells, infected with a virus (typically a baculovirus or cypovirus). They are typically smaller than 10 microns, e.g., 0.1 to 6 microns (measured as the largest distance within the crystal between different faces or the largest length of a side). They can be treated to modify their shape and / or reduce their size before use.

[0051] In certain embodiments, the crystals are isolated from the cells, for example, by centrifugation. Typically, the crystals are prepared in an aqueous suspension. For example, the concentration of the crystal suspension is 10 per milliliter of volume. 4 More than 10 crystals, usually 10 4 ~10 7 Crystals / ml volume, preferably 5 x 10 4 ~5×10 6 or 10 5 ~10 6 The crystals can be applied (contacted) to the culture system / culture medium at these concentrations. 5 ~10 7 crystals / microliter, e.g. 3 x 10 6 It can be used therapeutically in concentrations of crystals per milliliter.

[0052] Advantageously, the crystals are highly stable and rigid. They can be attached (e.g., dried onto a solid support). Typically, high levels of biological activity are maintained after storage in protease-free solution at 37°C for 6 months.

[0053] cargo protein Polyhedrin protein crystals as described herein encapsulate one or more cargo proteins.

[0054] Any type of cargo protein can be used. The phagocyte can contain polyhedrin protein crystals containing one type of cargo protein. Alternatively, the phagocyte can contain polyhedrin protein crystals containing different types of cargo proteins (e.g., a combination of cargo proteins). For example, the phagocyte can contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different cargo proteins.

[0055] As further described herein, the cargo protein typically includes a polyhedrin binding tag. The cargo protein is typically secreted intact from the crystal. The cargo protein is typically released sustainedly from the phagocyte. The cargo protein may have a desired phenotypic effect within the phagocyte into which it is secreted and / or on neighboring cells.

[0056] In embodiments in which more than one cargo protein is present, each protein may have the same polyhedrin binding tag, or each protein may have a different polyhedrin binding tag.

[0057] In certain embodiments, the crystals are coated with an agent that retards the release of the polyhedrin and cargo protein.

[0058] 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.

[0059] In certain embodiments, one or more therapeutic agents can reprogram M2 tumor-associated macrophages (TAMS), inflammatory monocytes, and / or myeloid-derived suppressor cells (MDSCs) into M1 macrophage cells. The ability of a therapeutic agent to reprogram the production of M2 cells can be measured, for example, by identifying and / or quantifying the expression of markers such as those described herein.

[0060] In certain embodiments, the one or more therapeutic agents activate or inhibit STAT1. They activate Notch, promote the synthesis of ROS and / or the release of NO, promote AKt1 kinase activity, activate NF-κB signaling, or inhibit JNK activity or CSFR1.

[0061] In certain embodiments, the one or more therapeutic agents include one or more Th-1-associated cytokines and / or TLR ligands.

[0062] In certain embodiments, one or more therapeutic agents can stimulate the production of M1 macrophage cells. The ability of a therapeutic agent to stimulate the production of M1 macrophage cells can be measured, for example, by identifying and / or quantifying the expression of markers within a macrophage population after contact with the therapeutic agent. As illustrated in Table 1, different subsets of macrophages (humans) have different phenotypes.

[0063] [Table 1]

[0064] In certain embodiments, the one or more therapeutic agents are selected from any one or more of the proteins listed in Table 1.

[0065] In certain embodiments, the one or more therapeutic agents are selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the proteins listed in Table 1.

[0066] In certain embodiments, the one or more therapeutic agents are selected from the group consisting of 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, C D86, CD68, IL-1, IL-1 beta, 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.

[0067] 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.

[0068] In certain embodiments, the cargo protein comprises one or more detectable agents. For example, the cargo protein may comprise a fluorescent and / or bioluminescent label, an enzyme label, a chemiluminescent labeling group, or a biotinyl group. Radioisotopes or radionuclides 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 fluorophores or FITC, and enzyme labels may include horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase.

[0069] Typically, the detectable agent is a fluorescent label such as GFP ("green fluorescent protein"). For example, the detectable agent can be enhanced GFP (eGFP). Alternatively, the detectable agent can be a bioluminescent label such as luciferase.

[0070] phagocytosis The phagocytes of the present invention may be collected or produced by any suitable method.

[0071] In certain embodiments, phagocytes are contacted with polyhedrin protein crystals. Typically, phagocytes (e.g., monocytes) are contacted with the crystals. Such cells may then be unaffected by the crystals or may differentiate or transdifferentiate (e.g., into M1 macrophages) after contact with the crystals.

[0072] In another embodiment, the crystals may be contacted with phagocytes after they have been cultured and / or differentiated.

[0073] In certain embodiments, the crystals contain a 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).

[0074] In certain embodiments, the crystals are attached to a solid support before contacting with phagocytic cells. Examples of suitable solid supports include, but are not limited to, slides, membranes, and tissue culture plates. Microcarriers may also be used. The crystals are maintained in a non-dried form, and can be centrifuged and / or dried on the support, for example, immediately before contacting with phagocytic cells.

[0075] In another embodiment, the crystals are pre-mixed with the phagocytic cells. Typically, the crystals are pre-mixed with the phagocytic cells for up to 12, 24, 36, 48 hours or more.

[0076] Phagocytic cells can be cultured under any conditions that induce phagocytosis, including the methods further described herein. Phagocytosis of crystals by cells can be measured by any suitable immunohistochemistry (IHC) method. In certain embodiments, the crystals themselves can contain cargo (e.g., detectable agents such as GFP and / or luciferase), allowing phagocytosis by cells to be verified (e.g., by fluorescence microscopy).

[0077] In certain embodiments, the phagocytes may be cultured in complete medium, typically supplemented with PMA. Typically, the phagocytes are cultured for up to 12, 24, 36, 48 hours or more.

[0078] As an example, phagocytes can be cultured at 37°C at a seeding density of 0.5-1.0e6 / ml 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 Glutamax, and optionally supplemented with 20 ng / mL M-CSF).

[0079] In certain embodiments, phagocytes can be differentiated into polarized cells that mimic in vivo phenotypes (e.g., cytotoxic phenotypes). For example, the culture medium can be supplemented with additional immune stimulants, such as LPS and / or IFN-γ, which induce the production of M1 macrophages. Typically, adding 100 ng / ml LPS or 20 ng / ml IFN-γ to complete medium induces the production of M1 macrophages after 12 hours of culture in complete medium.

[0080] In certain embodiments, phagocytes may not be affected by ingested crystals containing proteins such as IL-2 that target other cells within the affected tissue.

[0081] In certain embodiments, the method further comprises formulating the one or more phagocytes using any suitable method, including those further described herein. Typically, the one or more phagocytes are formulated for delivery via an injection route.

[0082] Pharmaceutical Compositions The present invention provides pharmaceutical compositions comprising (a) a phagocyte of the invention or a population of phagocytes of the invention, and (b) a pharmaceutically acceptable carrier or diluent. Typically, the phagocytes of the pharmaceutical composition comprise any one or more therapeutic agents as described herein.

[0083] The compositions of the present invention can be formulated using any suitable method. Formulation of cells using standard pharmaceutically acceptable carriers and / or excipients can be carried out using conventional methods in the pharmaceutical field. The exact quality of the formulation depends on several factors, including the cells to be administered and the desired route of administration. A suitable type of formulation is Remington's Pharmaceutical Sciences, 19 th Edition, Mack Publishing Company, Eastern Pennsylvania, USA.

[0084] 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 administration routes. 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 include, for example, water, saline, dextrose, glycerol, etc., and combinations thereof.

[0085] Typically, the composition is formulated for delivery by the injectable route.

[0086] In certain embodiments, the composition is used in combination with one or more other therapies intended to treat the same subject. Combination means that the therapies can be administered to the subject simultaneously, either in a combined form or separately. The therapies can be administered to the subject separately as part of the same therapeutic regimen, or can be administered sequentially. For example, the composition of the present invention can be used in combination with another therapy intended to treat cancer (e.g., solid cancer) in the subject.

[0087] In certain embodiments, treatment with one or more chemotherapeutic agents may be combined with the pharmaceutical compositions of the present invention. Typically, the chemotherapeutic agents are selected from dacarbazine (DTIC), temozolomide, Nab-paclitaxel, paclitaxel, carmustine (BCNU), cisplatin, carboplatin, vinblastine, or any combination thereof.

[0088] In certain embodiments, treatment with one or more biologics may be used in combination with the pharmaceutical compositions of the present invention. For example, the biologic may be a growth factor / cytokine / chemokine, such as IL-2. In certain embodiments, the biologic is a checkpoint inhibitor (e.g., anti-PD, PD-L1, and / or CTLA4 therapy). For example, the biologic may be selected from ipilumab, nivolumab, atezolizumab, pembrolizumab, and / or any combination thereof. For example, the biologic may be selected from nivolumab and ipilimumab. In some embodiments, the biologic is a B-Raf inhibitor, such as vemurafenib and / or dabrafenib. In certain embodiments, the biologic is nivolumab and / or ipilimumab, dabrafenib and / or trametinib, vemurafenib and / or cobimetinib, and / or any combination thereof.

[0089] cell therapy The present invention further provides a phagocyte, population, or pharmaceutical composition as described herein for use in therapy or medicine. Thus, the present invention includes administering the phagocyte to prevent or treat any disease. The phagocyte may be used in the manufacture of a medicament for preventing or treating any disease.

[0090] The present invention provides phagocytes for use in methods for therapeutically treating the human or animal body. For example, the therapeutic target may be any cell, extracellular vesicle, and / or free protein in the tumor microenvironment. As used herein, the term "tumor microenvironment" is understood to include the environment surrounding a solid tumor, including surrounding blood vessels, immune cells, fibroblasts, signaling molecules, and the extracellular matrix (ECM).

[0091] 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.

[0092] In certain embodiments, the present invention involves administering to a subject an immunologically effective number of phagocytes of the present invention loaded with polyhedrin protein crystals to deliver one or more cargo proteins to a desired site. For example, the phagocytes can be used to deliver one or more cargo proteins directly to a disease site (e.g., cancer).

[0093] The present invention provides a method of delivering any one or more cargo proteins as described herein to a disease site, the method comprising administering to a subject a pharmaceutical composition of the present invention.

[0094] 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 modulated as described herein. Typically, adjacent or nearby phagocytes (e.g., macrophages) at the disease site can be modulated. Alternatively, the therapeutic target of the cargo protein can be another type of immune cell, such as a T cell, dendritic cell, stromal cell, cancer cell, etc.

[0095] In certain embodiments, the present invention relates to the use of phagocytes to stimulate the production of M1 macrophage cells. In one embodiment, stimulating the production of M1 macrophages and / or reprogramming M2-associated TAMs, MDSCs, and inflammatory M1 macrophages into M1 macrophage cells is useful for clearing solid cancers (e.g., tumors) from a subject.

[0096] In certain embodiments, the disease is associated with M2 macrophage activation, as further described herein. One skilled in the art would be able to determine whether a particular disease is associated with M2 macrophage activation. For example, one skilled in the art would be able to determine the phenotype / score of macrophages at the disease site using markers such as those listed in Table 1.

[0097] In certain embodiments, the disease is cancer. For example, the cancer may be associated with M2 macrophage activation and / or solid tumors.

[0098] In certain embodiments, the phagocytes are derived from breast cancer, brain cancer, bone cancer, bone marrow cancer, skin cancer, colon cancer, liver cancer, colorectal cancer, prostate cancer, stomach cancer, stomach ( The present invention may be used to treat or prevent solid cancers selected from: gastric cancer, ovarian cancer, oral cancer, esophageal cancer, pancreatic cancer, gallbladder cancer, lung cancer, thyroid cancer, endometrial cancer, head and neck cancer, renal cancer, bladder cancer and / or glioma.

[0099] In certain embodiments, administration of phagocytes acts to increase the proportion of M1 macrophages at the disease site, e.g., the number of M1 macrophages can increase by 1.1x, 1.2x, 1.3x, 1.4x, 1.5x, 2x, 3x, 10x, 15x, 20x, 100x, 1000x, or more.

[0100] In certain embodiments, administration of phagocytes acts to reduce the proportion of M2 macrophages at the disease site, e.g., the number of M2 macrophages can be reduced by 1.1x, 1.2x, 1.3x, 1.4x, 1.5x, 2x, 3x, 10x, 15x, 20x, 100x, 1000x, or more.

[0101] In certain embodiments, administration of phagocytes acts to reduce angiogenesis, invasion, metastasis, growth and / or cell proliferation of the disease.

[0102] 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, juvenile, or adult subject. Typically, the subject has, is susceptible to, or is at risk for the relevant disease. For example, the subject can have cancer, such as a solid tumor, or can have a genetic predisposition to developing cancer.

[0103] The phagocytes of the present invention can be administered in a manner compatible with the dosage formulation and in such amount as to be immunologically, prophylactically, and / or therapeutically effective. The amount administered can depend on the subject being treated, capacity of the subject's immune system to respond to the antigen, and the degree of response desired.

[0104] Precise amounts of phagocyte / pharmaceutical composition required to be administered depend on the judgment of the practitioner and may be peculiar to each subject.

[0105] 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 For reference, the number of cells of the present invention administered may be 10 5 ~10 9 , preferably 10 6 ~10 8 Any of the specific numbers discussed above for the populations of the present invention can be administered. Typically, one or more doses of phagocytes are administered to a subject.

[0106] When cells are administered or present, a culture medium may be present to promote cell survival. In some cases, the cells of the present invention may be provided as frozen aliquots, and substances such as DMSO may be present to promote survival during freezing. Such frozen cells are typically placed in a maintenance or administration buffer or medium after thawing.

[0107] In certain embodiments, the present invention provides autologous cell therapy.

[0108] In certain embodiments, one or more phagocytes are derived from peripheral blood as described herein, for example, the phagocytes are derived from monocytes and can be cultured, expanded, and / or differentiated using methods known in the art, including those described herein.

[0109] In certain embodiments, phagocytes are contacted with polyhedrin protein crystals under conditions that induce phagocytosis, as described herein. For example, the crystals can be contacted with undifferentiated phagocytes (e.g., monocytes). Such cells can then be cultured and differentiated into macrophages (e.g., M1 macrophages) after contact with the crystals. Alternatively, the phagocytes can be cultured and / or differentiated before contacting them with the crystals.

[0110] In certain embodiments, phagocytes are cultured and / or differentiated in vitro using methods known in the art, including those described herein.

[0111] In certain embodiments, the phagocytes are isolated and / or formulated with a pharmaceutically acceptable carrier or diluent using methods known in the art, including those described herein.

[0112] In certain embodiments, the phagocytic cells are returned to the subject.

[0113] In the methods of autologous cell therapy described herein, the cargo protein is typically one or more therapeutic agents as described herein.

[0114] In the methods of autologous cell therapy described herein, the disease is usually one or more diseases as described herein.

[0115] Those skilled in the art will recognize that additional modes of administration, dosages of therapeutic agents, and treatment regimens can be determined by the treating physician according to methods known in the art.

[0116] Imaging The present invention also provides methods for imaging and / or characterizing disease. The disease can be any disease as described herein, such as any solid cancer described herein.

[0117] In certain embodiments, the methods of imaging and / or characterizing disease comprise administering to a subject a phagocyte (or population thereof) of the invention. Any phagocyte (or population thereof) as described herein may be administered to a subject.

[0118] In certain embodiments, the subject is a mouse or a human as described herein.

[0119] In certain embodiments, the phagocyte comprises one or more detectable agents as described herein. For example, the one or more detectable agents can be fluorescent agents (e.g., GFP) or luminescent agents (e.g., luciferase).

[0120] In certain embodiments, the method further comprises imaging and / or characterizing at least a portion of the diseased tissue (e.g., a solid tumor) by detecting the detectable agent. Any suitable method for detecting the detectable agent may be used, including methods as further described herein.

[0121] Example Example 1 - Phagocytosis of PODS® Protein Crystals by Professional Phagocytes We investigated whether PODS® protein crystals could be engulfed by professional phagocytes, such as monocytes, neutrophils, and macrophages. PODS® eGFP crystals were deposited as a monolayer on the wells of a 12-well plate, and then undifferentiated THP-1 cells (monocytes) were seeded on top of the crystals. In a different setup, PODS® eGFP was briefly mixed with monocytes, and then the mixture was uniformly plated on the wells. After 24 hours of incubation, the cells were stained and imaged. In both experimental setups, multiple PODS® proteins were successfully internalized by monocytes (Figures 1a–d). In some cases, more than 35 individual crystals were ingested by a single cell. Phagocytosis of the crystalline protein depot was independent of the embedded cargo protein, as different PODS® crystals, either protein-free (PODS® Empty) or containing growth factors, cytokines, chemokines, or fluorescent proteins, were all efficiently internalized.

[0122] THP-1 monocytes were differentiated into M0 macrophages by incubating them in complete medium supplemented with PMA (Genin et al., 2015). (Del Arosa et al., 2016). After 24 hours of incubation in differentiation medium, the cells began to adhere and change their morphology, as well as their cytokine secretion patterns, such as increased secretion of activin A, demonstrating differentiation into M0 macrophages. Subsequently, M0 macrophages were further differentiated into M1 or M2 macrophages, and PODS® eGFP protein crystals were added to each of the M0, M1, and M2 cell cultures. The cultures were further incubated, and phagocytosis of PODS® crystals was observed after 48 hours (Figures 2a–c). M0 and M2 phagocytes ingested almost all PODS® protein crystals, demonstrating similar levels of phagocytosis (Figures 2a and 2c, respectively), whereas M1 macrophages were less efficient at ingesting particles (Figure 2b). Our observations suggest that the efficiency of PODS® crystal uptake is M2 > M0 > M1.

[0123] M0 macrophages were also assessed for their mobility and therefore viability during and after phagocytosis. After adding PODS® eGFP protein crystals, M0 cultures were monitored for 48 hours using both brightfield and fluorescent channels on a real-time cell history recorder at a rate of 1 frame per minute (Figures 3a-3b). Still images from the time-lapse recording demonstrated that many M0 macrophages were mobile during and after phagocytosis of PODS® eGFP and therefore healthy. Successful phagocytosis was detected by observing the change in position of green fluorescent PODS® crystals from a stationary (i.e., surface-attached) state to a mobile state after uptake by phagocytic cells. Furthermore, differentiated macrophages, i.e., M0, M1, and M2, are typically adherent when healthy, and no cell detachment was observed during the incubation period, even after many PODS® protein crystals had been incorporated into each of them.

[0124] Example 2 - Phagocytosis of PODS® Protein Crystals by Non-Professional Phagocytes Next, we determined whether the shape and size of PODS® protein crystals could equally contribute to phagocytosis by nonprofessional phagocytes (e.g., fibroblasts, osteochondrogenic cells, lymphocytes, erythrocytes, epithelial cells, myoblasts, or myocytes). PODS® eGFP was again seeded as a dense monolayer in the wells of a 96-well plate and allowed to dry. Fibroblasts or chondrocytes were then seeded on top of the attached crystals and cultured for up to 48 hours. Similar to macrophages, most of the seeded PODS® protein crystals were efficiently taken up by both fibroblasts (Figure 4a) and chondrocytes (Figure 4b), forming intracellular aggregates; few protein crystals were found in the intercellular spaces. Similar to differentiated macrophages, fibroblasts and chondrocytes are adherent cells that detach when they become unhealthy or undergo apoptosis. However, after ingesting PODS® crystals, the cells remained attached to the surface and continued to grow to overconfluence.

[0125] We next determined whether PODS® was ingested rather than merely attached to the macrophage surface. Phagocytosis was demonstrated by incubating undifferentiated THP-1 monocytes or M1 macrophages with PODS® Empty (pure polyhedrin protein crystals, no cargo) for 24 hours and then fixing the cells to make the cell membrane impermeable to the antibody. The cells were then subjected to an anti-polyhedrin primary antibody, which recognizes only PODS® crystals that were not phagocytosed but adhered to the cell surface or remained attached to the TC plastic surface. Crystals recognized by this primary antibody were visualized using a FITC secondary antibody and a fluorescent microscope. When THP-1 monocytes were observed using brightfield microscopy, nearly all cells had ingested multiple PODS® crystals (Figure 5a). When switched to the fluorescent channel, an estimated 90% of the crystals did not have equal fluorescent signal compared to the brightfield image. This provided clear evidence that the polyhedrin crystals were located within THP-1 cells, as PODS® was not recognized by this antibody pair. In the case of M1 macrophages, different levels of phagocytosis were observed, with more protein crystals found outside the cell boundary. Consequently, these PODS® crystals were clearly visible in the fluorescence channel because they were easily recognized by the antibody pair (Figure 5b). Finally, phagocytosis in nonprofessional phagocytes could be demonstrated by first incubating chondrocytes with PODS® eGFP for 24 hours. The cells were then fixed, stained for cytoskeleton and nuclei, and imaged using confocal microscopy. Focal plane scanning revealed that the cytoskeleton, nucleus, and PODS® eGFP crystals were all present in the same focal plane (Figure 5c).

[0126] Example 3 - PODS® exhibits specific biological activity after phagocytosis To assess whether endocytosed PODS® growth factors (PODS® GF) still exhibit the specific bioactivity of the embedded cargo, proliferation assays were performed on undifferentiated monocytes (THP-1) using PODS® GM-CSF and PODS® SCF in two different configurations. In the first configuration, PODS® GF was seeded into the wells of a 24-well plate as a fairly uniform monolayer of protein crystals and allowed to adhere to the TC surface. Monocytes were then seeded on the same surface (Fig. 6a, I) and cultured for 4 days. In this configuration, PODS® GF crystals were readily phagocytosed (Fig. 6a, II). In contrast, in the second configuration, in which PODS® GF crystals were allowed to adhere to the porous membrane of the TC insert and monocytes were then seeded into the wells, PODS® and cells were physically separated, preventing phagocytosis (Fig. 6a, III). Throughout this experiment, only cells were present on the surface of the wells, preventing them from ingesting the protein crystals (Fig. 6a, IV). After 4 days of culture, the final cell number was assessed using a colorimetric assay. Regardless of whether the PODS® crystals were phagocytosed (Fig. 6b, solid gray and dotted bars, respectively), each growth factor cargo exhibited growth-stimulating activity, as the number of cells present was significantly increased compared with measurements for unsupplemented cells (Fig. 6b, horizontal line). This demonstrates that phagocytosed PODS® protein crystals exhibit specific biological activity and, therefore, that macrophages can be used in a Trojan horse strategy to deliver protein depots for sustained release.

[0127] Materials and Methods Synthesis of PODS® Crystals All PODS® 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 to an H1 integration tag (US8554493B2). Briefly, standard Spodoptera frugiperda 9 (Sf9) cells were co-transduced with baculovirus (BV) DNA and transfer DNA using TransIT®-Insect (Mirus Bio). The resulting infectious BVs were collected and then plaque-purified to isolate single recombinant BVs. Isolated plaques were first screened, and positive BVs were then collected, expanded, and finally used to infect large-scale Sf9 cell cultures to generate PODS® crystals. Crystals were then collected and purified by lysing Sf9 cells with successive sonication and PBS washes. Finally, the purified PODS® was sterility tested, lyophilized, and used in experiments. 4 One PODS® crystal is approximately functionally equivalent in terms of biological activity to 1 ng of many standard growth factors, cytokines, or chemokines (Matsumoto et al. 2015).

[0128] Cell culture and differentiation Suspended monocytes (THP-1, ATCC) were cultured in an undifferentiated state in RPMI-1640 medium supplemented with 2 mM L-glutamine and 10% FBS (complete medium). For M0 differentiation, THP-1 cells were centrifuged, and the culture supernatant was replaced with fresh complete medium supplemented with 100 ng / ml phorbol 12-myristate-13-acetate (PMA, Sigma P8139). Cells were seeded and cultured for up to 12 days, with medium changes every 3–4 days. After 24–48 h, M0 macrophages were further differentiated into M1 or M2 macrophages as described [Genin et al., 2015; Del Arosa et al., 2016]. Briefly, the M0 differentiation medium was removed, and 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 further incubated for up to 10 days.

[0129] phagocytosis PODS® crystals were either centrifuged onto tissue culture (TC) plates, dried by centrifugation onto TC plates, or premixed with phagocytes for up to 48 hours before plating. Phagocytes were then seeded as needed and subsequently cultured in complete medium. Intracellular phagocytosis was monitored using a real-time cell history recorder (JuLi stage, This was verified by monitoring with a fluorochrome-independent immunoassay (FISH) (NanoEnTek Inc.) or by routine immunohistochemistry (IHC) staining and techniques.

[0130] To specifically stain polyhedrin protein, cells were fixed with 10% formalin for 10 minutes and then blocked in 1% PBS-BSA for 1 hour. Subsequently, an in-house developed rabbit anti-polyhedrin antibody (diluted 1:1000 in 1% PBS-BSA) was incubated on the cells for 1 hour, followed by three washes with 1% PBS-BSA. Finally, an anti-rabbit FITC secondary antibody (diluted 1:1000 in 1% PBS-BSA) was incubated for 1 hour, followed by three washes with 1% PBS-BSA, followed by three washes with ProLong Diamond Fluorescent Lamp ... Mounting was performed with Antifade Mountant (ThermoFisher Scientific).

[0131] Proliferation assay PODS® GM-CSF or PODS® SCF (1.8 × 10 each) 6 PODS® protein crystals were seeded directly onto wells of a 24-well plate as a monolayer of crystals or into 24-well TC inserts (1 μm pore size, Corning Falcon). Subsequently, 4,000 monocytes were seeded into wells containing a monolayer of PODS® protein crystals, into inserts containing PODS® protein crystals, or into empty inserts as baseline controls. Cultures were then incubated for 4 days, and final cell numbers were assessed using a colorimetric assay (OrangU, Cell Guidance Systems).

[0132] Consideration The potential utility of self-assembling proteins in phagocyte-mediated strategies for treating disease is now demonstrated. This utility was demonstrated by polyhedrin crystals produced using the PODS® Expression System, which is based on the polyhedrin protein derived from the silkworm cypovirus. Advantageously, the following is demonstrated herein: (1) PODS® crystals are efficiently taken up by both professional and non-professional phagocytes. (2) Cells remain healthy after ingestion of PODS® crystals, and polyhedrin protein alone has no detectable effect on phagocyte phenotype. (3) When PODS® crystals contain mitogenic cytokines, the proliferation rate of phagocytes increases significantly, indicating that the cargo protein is released intact into the cells and can interact with receptors. (4) Cargo proteins are secreted by PODS® crystals. (5) Co-cultured cells that did not ingest PODS® crystals themselves alter the phenotype of the co-cultured cells by responding to cargo proteins secreted by phagocytes that ingested PODS® crystals.

[0133] Phagocytes are intrinsically involved in the pathogenesis of diseases and tissue remodeling. Macrophages, in particular, are attractive as targets for therapeutic agents and delivery agents due to their ability to infiltrate diseased tissues.

[0134] Significant advances in understanding the role of the immune system and phagocytes in disease pathogenesis have led to the development of new, effective treatments. Proteins, including cytokines, have shown therapeutic potential, and many have received market approval, primarily for applications that allow repeated administration via systemic delivery. With a few notable exceptions, such as IL-2 for treating melanoma and renal cell carcinoma, attempts to use proteins to treat disease directly or through Trojan horse strategies have been confounded by high toxicity to non-target cells and the fragility of proteins.

[0135] Given the importance of phagocytes in disease and the therapeutic potential of cytokines and other vulnerable proteins, drug delivery devices that can specifically and sustainably affect phagocytes provide a platform technology for protein-based treatment strategies.

[0136] PODS® crystals exhibit exceptional stability. For example, they maintain high levels of biological activity after storage for 6 months at 37°C in protease-free solution. Furthermore, PODS® crystals possess an optimal size range (0.2-6 microns), shape (cubic), and elasticity (rigid). PODS® crystals are shown herein to be efficiently taken up by a wide range of professional and non-professional phagocytes.

[0137] Another notable feature of PODS® crystals is their dynamic release mechanism, which requires a protease. It has previously been shown that many matrix metalloproteinases can mediate this release. Unexpectedly, it is shown herein that PODS® crystals persist for weeks once taken up by phagocytes, that the ingested phagocytes remain healthy, and that cytokines released by PODS® crystals continue to exert a proliferative effect on their cell population. Furthermore, it is demonstrated herein that cytokines are released from phagocytes and that the released cytokines can modulate the phenotype of neighboring cells that did not take up the crystals.

[0138] Example 4 - PODS® "Trojan Horse" Approach PODS® are micron-scale cubic co-crystals containing stabilized recombinant protein cargo that are released in a sustained manner under the activity of proteases. PODS® are the ideal size, shape, and consistency for efficient phagocytosis. However, for the PODS® Trojan Horse approach to be viable as a delivery tool for therapeutic proteins, several criteria must be met. These include: (1) Monocytes and macrophages must be able to phagocytose micron-sized PODS® without dramatically altering their viability and metabolism. (2) Phagocytosis of micron-sized PODS® should not alter the ability or motility of macrophages, including their ability to sense chemicals and navigate small blood vessels and capillaries. (3) Intracellular degradation of endocytosed PODS® protein crystals must release intact, biologically active cargo protein. (4) PODS®-filled phagocytes that release cargo proteins must be able to affect the behavior of neighboring cells.

[0139] It is demonstrated herein that cubic PODS® crystals, which are micron-sized, rigid protein crystals, can be efficiently phagocytosed by professional and non-professional phagocytes. Furthermore, growth factors secreted from phagocytosed PODS® can still induce specific biological activities, such as proliferation, in the phagocytes that ingest them.

[0140] Unexpectedly, it is further demonstrated herein that PODS® crystals meet all of the above-listed criteria necessary for therapeutic strategies that employ phagocytes as Trojan horses by modulating the function of macrophages themselves or by orchestrating the behavior of other cells.

[0141] result Considering the cytotoxic effects of various types of nanoparticles on macrophages in previous studies (Lanone et al., 2009; Akter et al., 2018; Feng et al., 2018), we investigated the viability of M0, M1, and M2 macrophages after the uptake of PODS® crystals. In this experiment, THP-1 monocytes were differentiated into M0 macrophages by incubating the cells in complete medium supplemented with PMA. The M0 cells were then polarized into M1 and M2 types of 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 PODS® crystals. Cells were incubated with empty (no cargo protein) or PODS® FGF-10 for 24 hours and tested for viability 48 and 96 hours after uptake (Figures 7a and 7b). Results from the WST-8 assay demonstrated no reduction in cell viability compared to untreated cells, indicating that PODS® particles were nontoxic at the median dose range of up to 15 PODS / cell.

[0142] Next, we investigated whether macrophage migration triggered by chemotactic signals is regulated by PODS phagocytosis. To quantify potential changes due to PODS® uptake into macrophages, M0 cells were incubated with PODS® IL-2 for 24 hours and then observed under normal growth conditions for an additional 24 hours using a live-cell imaging system. Subsequently, migration of unloaded and PODS® IL-2-loaded cells was first monitored using the manual tracker function in Image J and then by imaging. Using Ibidi's chemotaxis and migration tool, 25 cells were tracked over 720 frames (Figures 8a and 8b, respectively). Trajectories were compared for both migration distance and randomness of migration, and no detectable differences were found between the migration of cells with and without PODS®. To test the ability of PODS® IL-2-loaded M0 cells to follow a chemotaxis gradient, the experiment was repeated with minor modifications. After PODS® incorporation, cells were placed in serum-free medium and transferred to a chemotaxis slide containing two reservoirs. Reservoir 1 was filled with medium containing 10% serum, which acts as a chemoattractant, and reservoir 2 was filled with serum-free medium. The migration of unloaded and PODS® IL-2-loaded cells was examined by tracking 26 cells each as described above (Figures 9a and 9b, respectively). Analysis of this tracking data revealed that PODS® crystal incorporation did not impair the directed migration of macrophages toward the source of the chemoattractant.

[0143] To assess whether primary cells phagocytose PODS® crystals as efficiently as THP-1 cells, monocytes and macrophages were isolated from the tibiae of C57BL / 6 mice and cultured for 5 days. These cells were then incubated with PODS® eGFP for 24 hours and subsequently monitored with a live-cell imaging system for another 24 hours. Mouse primary cells were more mobile than the human monocytic cell line. They phagocytosed PODS® crystals with equally high efficiency and were also able to ingest similar numbers of PODS® crystals. Figure 10 shows four frames in which the loaded cell (dashed circle) can be seen extending forward to retrieve and ingest another PODS® crystal to the left.

[0144] Having demonstrated that PODS®-loaded macrophages were still able to respond to and track chemotactic signals, we assessed whether loaded cells retained another important macrophage characteristic: their ability to traverse narrow spaces. M0 cells were incubated with PODS®-eGFP for 24 hours and then placed in serum-free medium and transferred to 24-well tissue culture inserts with 8-μm pores, a diameter similar to that of small blood vessels. Regular maintenance medium containing 10% serum was used as a chemoattractant in the bottom well (Fig. 11b). A second well without serum as a chemoattractant served as a control to demonstrate that cells in the bottom well actively migrated downward and did not randomly pass through the pores or simply sink due to gravity (Fig. 11a). After an additional 24 hours of incubation, both experimental chambers were analyzed by bright-field and fluorescence microscopy. The large difference in cell numbers in wells with and without chemoattractant demonstrated that loaded M0 cells were not only able to pass through the narrow pores despite their PODS® cargo, but also actively migrated through the 8 μm pores toward the chemotactic origin.

[0145] Importantly, because macrophages are actively recruited by chemical signals from solid tumors (Dandenkar et al., 2011), we used the same cell chamber described above to test whether culture supernatants from cancer cell lines could attract PODS® eGFP-loaded macrophages. A375 cells, a human melanoma cell line, were grown in 10% serum-containing growth medium for 3 days. The culture supernatant was then used as a chemoattractant in the bottom well (Figure 12b). In a second cell chamber, culture supernatant containing 10% serum in the bottom well served as a control to measure the level of non-directional macrophage migration in the absence of factors secreted by A375 cells (Figure 12a). Subsequent microscopic imaging demonstrated that PODS® eGFP-loaded M0 cells were specifically attracted by the culture supernatants from the cancer cell line and migrated in a directed manner through the 8 μm pore toward the chemotactic origin.

[0146] Monocyte activation and polarization toward an M1-like phenotype upon contact with foreign materials are key components of the innate immune system. To test whether contact and uptake of PODS® crystals by monocytes and macrophages influences their polarization state, we examined the secretion of IL-6, a marker for M1 polarization, before and after PODS® uptake (Figure 13). First, we confirmed that IL-6 can indeed be used as a surrogate for polarization state. We showed that M1 cells elevated IL-6 levels in the cell culture medium to over 4 ng / ml immediately after polarization, whereas neither unactivated THP-1 cells nor M0 or M1 cells elevated IL-6 levels above background levels. The polarization medium was then changed to medium containing PODS® FGF-2, and phagocytic uptake was allowed to proceed for 24 hours. During this incubation period, both loaded and unloaded M1 cells increased their IL-6 concentrations to 200 pg / ml. There was no change in the level of the polarization marker IL-6 between M1 cells that had incorporated PODS® crystals and those that had not been incubated with PODS® crystals, indicating that PODS® crystal incorporation did not alter the polarization state of M1 cells. Similarly, PODS® crystal incorporation did not induce IL-6 secretion in M0 and M2 cells, indicating that PODS® crystal phagocytosis does not alter the polarization state of either macrophage type.

[0147] To test whether ingested PODS® cargo proteins are released from macrophages into the medium, M0 cells were incubated with PODS® IL-6 for 24 hours, washed twice, and then incubated for an additional 4 days in regular growth medium without additional PODS®. The medium was then collected and analyzed by ELISA for the presence of IL-6 (Figure 14a). Surprisingly, not only was the cargo protein IL-6 detected in the cell culture medium, but the level of IL-6 was dose-dependent. The more PODS® IL-6 loaded onto M0 cells, the greater the amount detected in the medium after 4 days. For comparison, the amount of IL-6 released from a similar number of unphagocytosed (naked) PODS® IL-6 cells was also measured (Figure 14b). The amount of IL-6 released from naked PODS® was up to 10-fold greater than that from PODS® ingested by macrophages. As an additional control, MO cells were similarly incubated with PODS® FGF-2 and analyzed for IL-6 levels in the medium. Not only were IL-6 levels below background (<10 pg / ml), but they did not change in a dose-dependent manner.

[0148] To determine whether the PODS® cargo proteins released by macrophages are bioactive, we performed a proliferation assay using NIH-3T3 cells responsive to FGF-2 protein. NIH-3T3 cells were seeded in regular growth medium in 24-well plates and grown for 24 hours, after which the medium was replaced with serum-free culture supernatant. Macrophages were incubated with PODS® FGF-2 for 24 hours (10 PODS® / cell), placed in NIH-3T3 serum-free culture supernatant, and seeded into 24-well TC inserts. Cells were then co-incubated for an additional 4 days, and the number of NIH-3T3 cells was assessed by performing a WST-8 assay (Figure 15). Co-incubation of all types of macrophages loaded with PODS® FGF-2 confirmed the growth of FGF-2-responsive NIH-3T3 cells in serum-free conditions compared to empty macrophages (blue bars) and cells alone (green horizontal lines).

[0149] Consideration We investigated the potential utility of encapsulated proteins delivered by macrophages as a novel therapeutic drug delivery strategy, particularly polyhedrin crystals produced using the PODS® expression system, which is based on the polyhedrin protein derived from the silkworm cypovirus.

[0150] Selective targeting of drugs to diseased tissues, particularly cancer, is needed to reduce off-target toxicity and increase efficacy. The use of cells as a therapeutic delivery system, also described as a Trojan horse strategy, has been discussed since the late 1970s. Developing effective particles that contain drugs and allow macrophage function and subsequent release has been challenging, especially for protein drugs such as cytokines. Non-phagocytic cells also have the potential for Trojan horse delivery. However, although research is ongoing, no cell-based delivery system has yet been approved for clinical use. At least two therapeutics that use red blood cells as drug vehicles are currently undergoing phase III testing (NCT02770807, NCT03665441). A distinct advantage of macrophages over other cellular delivery systems is their unique disease-homing ability. Macrophages are directly attracted to solid tumors and other diseases, serving as the most important defenses in inflammation and as part of the foreign body response.

[0151] We demonstrate herein that phagocytosed PODS® proteins are nontoxic to multiple types of macrophages, even at high particle numbers. Furthermore, the directed migration of macrophages loaded with PODS® crystals toward chemoattractants is not impaired. Crucially, loaded macrophages are still able to change their cell morphology and migrate through narrow spaces, as evidenced by their active passage through 8-μm-diameter pores. Following phagocytosis, we demonstrate that proteins encapsulated in PODS® crystals are released from macrophages. The released cargo proteins were reliably detected in a dose-dependent manner using ELISA in macrophage cultures conditioned with PODS®-containing macrophages at as few as five PODS® crystals per macrophage. Surprisingly, the released proteins were bioactive, as verified by the proliferation of FGF-2-responsive cell lines upon co-incubation with PODS®-FGF-2-loaded macrophages. Therefore, a PODS-based macrophage-mediated Trojan horse strategy for drug delivery to cancer and other diseases is developed.

[0152] Materials and Methods Synthesis of PODS® Crystals All PODS® proteins were synthesized as previously described ( All constructs were fused to an H1 integration tag (Metcalf et al., 2008). Briefly, standard Spodoptera frugiperda 9 (Sf9) cells were co-transfected with baculovirus (BV) DNA and transfer DNA using TransIT®-Insect (Mirus Bio). The resulting infectious BVs were collected and then plaque-purified to isolate single recombinant BVs. The isolated plaques were first screened, and positive BVs were then collected, expanded, and finally used to infect large-scale Sf9 cell cultures to produce PODS® crystals. The crystals were then harvested and purified by lysing the Sf9 cells with successive sonication and PBS washes. Finally, the purified PODS® was sterility tested and lyophilized before use in experiments. While equivalence is dependent on the circumstances, 1.5 × 10 4 One PODS® crystal is approximately functionally equivalent in terms of bioactivity to 1 ng of many standard growth factors, cytokines, or chemokines ( Matsumoto et al. 2015).

[0153] Cell culture and differentiation Suspended monocytes (THP-1, Public Health England) THP-1 cells (Culture Collection) were cultured in an undifferentiated state in RPMI-1640 (A10491, Gibco®) (complete medium) supplemented with 10% BCS (30-2030, ATCC). For M0 differentiation, THP-1 cells were centrifuged, and the culture supernatant was treated with 100 ng / ml phorbol 12-myristate-13-acetate (PMA, The medium was replaced with fresh complete medium supplemented with Sigma P8139. After 48 hours, M0 macrophages were further differentiated into M1 or M2 macrophages as described (Rios de la Rosa et al., 2017; Genin et al., 2015). Briefly, the M0 differentiation medium was removed, and 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 incubated for up to 48 hours. A375 cells (ATCC) were cultured in DMEM (41966, Gibco®) supplemented with 10% FBS (F7524, SIGMA). Cells were grown at a density of 2e4 cells / cm. 2 Cells were seeded at a density of 2e5 cells / ml and passaged twice a week. TF-1 cells (ATCC) were cultured in RPMI-1640 supplemented with 2ng / ml GM-CSF and 10% FBS. Cells were seeded at a density of 2e5 cells / ml and passaged twice a week.

[0154] phagocytosis PODS® crystals were either centrifuged onto tissue culture (TC) plates, dried by centrifugation onto TC plates, or premixed with phagocytes for up to 48 hours before plating. Phagocytes were then seeded as needed and subsequently cultured in complete medium. Intracellular phagocytosis was monitored using a real-time cell history recorder (JuLi stage, This was verified by monitoring with a NanoEnTek Inc.

[0155] Viability assay To test macrophage viability after PODS® uptake, THP-1 cells were seeded at a density of 2e5 cells / ml and differentiated and polarized as described above. Addition of 1e6 or 3e6 PODS® Empty and PODS® FGF-10 per ml to non-polarized M0 and polarized M1 and M2 macrophages resulted in an average of 5 or 15 PODS® / cell, respectively. Cells were then incubated for 48 and 96 hours and analyzed using the WST-8 assay ( Orangu™ (Cell Guidance Systems) was performed to measure cell viability.

[0156] Movement Tracking THP-1 cells were seeded in 24-well plates at a density of 2e5 cells / ml and differentiated into M0 macrophages as described above. M0 cells were then incubated with PODS® IL-2 in complete medium for 24 hours. The medium was then refreshed and the cells were recorded on a real-time cell history recorder (JuLi stage, NanoEnTek). Cells were recorded using a fluorochrome platelet analyzer (Fiberglass-based Imaging Device, Inc.). Images were taken every 2 minutes for 24 hours, and then Analysis was performed using Image J's manual tracker and chemotaxis and migration tools (Ibidi).

[0157] chemotaxis THP-1 cells were seeded in T25 TC flasks at a density of 2e5 cells / ml and differentiated into M0 macrophages as described above. M0 cells were then incubated with PODS® IL-2 in complete medium for 24 hours. M0 macrophages were subsequently washed with PBS and then detached using enzyme-free Cell Dissociation Buffer (Gibco®). Cells were then centrifuged, counted again, and cultured at a density of 2e6 cells / ml in serum-free RPMI-1640 medium. The cells were seeded onto Chemotaxis μ-Slides (Ibidi). 10% FBS was used as a chemoattractant. Cells within the observation area were recorded using a real-time cell history recorder (JuLi stage, NanoEnTek Inc.). Images were captured once per minute for 12 hours and then analyzed using the manual tracker and chemotaxis / migration tools in Image J (Ibidi).

[0158] Directional migration THP-1 cells were seeded in T25 TC flasks at a density of 2e5 cells / ml and differentiated into M0 macrophages as described above. M0 cells were then incubated with 5e5 PODS® eGFP cells per mL in complete medium (2.5® PODS / cell) for 24 hours. M0 macrophages were subsequently washed with PBS and then detached in enzyme-free Cell Dissociation Buffer (Cell Dissociation Buffer, The cells were detached using a Gibco® filter. The cells were centrifuged, counted again, and seeded into 24-well inserts at a density of 1e6 cells / ml in serum-free RPMI-1640 medium or RPMI-1640 medium containing 10% FBS. The bottom wells were then filled with serum-free medium, 10% FBS-containing medium, or 3-day culture supernatant of A375 cells. After 24 hours of migration, microscopic images of the bottom wells and inserts were taken.

[0159] Endogenous IL-6 secretion THP-1 cells were seeded in 24-well plates at a density of 2e5 cells / ml and differentiated and polarized as described above. The polarization culture supernatant was then collected and stored at -20°C for later analysis. Macrophages were then incubated with 2e6 PODS® FGF-2 per ml in complete medium (10 PODS® / cell) for 24 hours. The medium was then collected and stored at -20°C. Medium samples were tested for the presence of IL-6 by ELISA (DY206, R&D systems) according to the manufacturer's protocol.

[0160] IL-6 release from PODS® IL-6-loaded macrophages THP-1 cells were seeded in 96-well plates at a density of 2e5 cells / ml and differentiated into M0 macrophages. The M0 cells were then incubated with 1e6, 2e6, or 3e6 PODS® IL-6 cells per ml in complete medium for 24 hours (resulting in 5, 10, or 30 PODS® cells per cell). The cells were then washed twice with PBS, fresh complete medium was added, and the cells were incubated for 4 days. Additionally, the same number of naked PODS® IL-6 cells were added to wells of the 96-well plate and spun down at 3000 g for 25 minutes. The PBS was removed, and the plate was allowed to dry in a laminar flow hood before being replenished with complete growth medium for 4 days. After incubation, the medium was collected and subsequently tested by IL-6 ELISA (DY206, R&D Systems) according to the manufacturer's protocol.

[0161] Functional assays THP-1 cells were seeded in 6-well plates at a density of 2e5 cells / ml and differentiated and polarized as described above. Macrophages were then incubated with 2e6 PODS® FGF-2 per ml in complete medium for 24 hours (10 PODS® / cell). Subsequently, macrophages were washed with PBS and then detached using enzyme-free Cell Dissociation Buffer (Gibco®). Cells were then centrifuged, counted again, and seeded in serum-free TF-1 culture supernatant at a density of 2e6 cells / ml into 24-well inserts. TF-1 cells were grown in complete growth medium at a density of 3e4 cells / cm in 24-well plates. 2 After 1 day of growth, the medium was changed to RPMI-1640 supplemented with 0.5% BCS, and the inserts containing macrophages were added. The cells were co-incubated for 4 days and then analyzed by WST-8 assay ( Orangu™ (Cell Guidance Systems) was performed to measure the viability of TF-1 cells.

[0162] Derivation of primary monocytes from mouse tibia Murine bone marrow-derived monocytes were isolated according to Wagner et al. (2014). Briefly, tibiae from three C57BL / 6 mice were prepared for 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 with warmed medium (M199 supplemented with 10% FBS and 1% penicillin / streptomycin) using a 28G needle and a 1 ml syringe. The bone flow-through was collected and filtered through a 70 μm cell strainer. The cell suspension was centrifuged at 200 × g for 10 min at RT. The pellet was washed with 25 ml of medium and centrifuged again. The cells were then seeded at 0.5 e6 cells / ml into 6-well ultra-low attachment plates. The cells were cultured for 5 days before use.

[0163] Collectively, these results demonstrate the utility of self-assembling proteins and PODS® crystals, particularly in modulating the phenotype of phagocytes and neighboring cells. These findings make it possible to develop effective therapeutic approaches for disease treatment and tissue remodeling, as described herein.

[0164] The reader's attention is drawn to all articles and documents related to this application that have been filed contemporaneously or prior to this specification and that are open to public inspection herewith, the contents of all such articles and documents being incorporated herein by reference.

[0165] References - Andersen et al. Long-Lasting Complete Responses in Patients with Metastatic Melanoma after Adoptive Cell Therapy with Tumor-Infiltrating Lymphocytes and an Attenuated IL2 Regimen (2016) DOI: 10.1158 / 1078-0432.CCR-15-1879. - Andreeson et al. Cancer Res. 1990;50:7450-7456. - Choi et al. (2007) A Cellular Trojan Horse for Delivery of Therapeutic Nanoparticles into Tumors Nanotechnology letters (12):3759-65. - van Dalen et al. (2019) Molecular repolarization of tumor-associated macrophages Molecules 2019, 24, 9 DOI 10.3390. - Faradji et al, Journal of immunological methods. 1994;174:297-309. - Jiang et al. (2016) Role of IL-2 in cancer immunotherapy. Oncoimmunology. 5(6): e1163462. - Matsumoto et al. J Biomater Appl 2015;30:193-200. - Mosser et al, Current protocols in Immunology. 2008 14:14:12. - Redecke et al., Nature Methods. 2013; 10: 795-803. - Zhang et al, Current protocols in Immunology. 2008 14:14: 11. - Xu, F. et al. Membrane-wrapped nanoparticles probe divergent roles of GM3 and phosphatidylserine in lipid-mediated viral entry pathways. Proc. Natl. Acad. Sci. 115, E9041-E9050 (2018). - 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). - Rios de la Rosa, J. M., Tirella, A., Gennari, A., Stratford, I. J. & Tirelli, N. The CD44-Mediated Uptake of Hyaluronic Acid-Based Carriers in Macrophages. Adv. Healthc. Mater. 6, (2017). - Nishishita, N. et al. The use of leukemia inhibitory factor immobilized on virus-derived polyhedra to support the proliferation of mouse embryonic and induced pluripotent stem cells. Biomaterials 32, 3555-3563 (2011). - Matsumoto, G. et al. Bone regeneration by polyhedral microcrystals from silkworm virus. Sci. Rep. 2, 935 (2012). - Lanone, S. et al. Comparative toxicity of 24 manufactured nanoparticles in human alveolar epithelial and macrophage cell lines. Part. Fibre Toxicol. 6, 14 (2009). - Akter, M. et al. A systematic review on silver nanoparticles-induced cytotoxicity: Physicochemical properties and perspectives. J. Adv. Res. 9, 1-16 (2018). - Feng, Q. et al. Uptake, distribution, clearance, and toxicity of iron oxide nanoparticles with different sizes and coatings. Sci. Rep. 8, 2082 (2018). - Dandekar, R. C., Kingaonkar, A. V. & Dhabekar, G. S. Role of macrophages in malignancy. Ann. Maxillofac. Surg. 1, 150-154 (2011). - Metcalf, P. et al. Viral polyhedra complexes and methods of use. (2008). - Genin, M., Clement, F., Fattaccioli, A., Raes, M. & Michiels, C. M1 and M2 macrophages derived from THP-1 cells differentially modulate the response of cancer cells to etoposide. BMC Cancer 15, 577 (2015). - Wagner, M. et al. Isolation and intravenous injection of murine bone marrow derived monocytes. J. Vis. Exp. JoVE (2014) doi:10.3791 / 52347.

Claims

1. A phagocyte comprising one or more polyhedrin protein crystals, said crystals encapsulating one or more cargo proteins, said cargo proteins comprising one or more therapeutic agents, wherein the phagocyte is a professional cell.

2. (a) the crystals are internalized within the phagocyte; (b) the crystals are capable of secreting the cargo protein intact within the phagocyte; and / or (c) the cargo protein is capable of being released sustainedly from the phagocyte; Optionally, the cell is a mammalian cell, and further optionally, the mammalian cell is a human cell. The phagocyte of claim 1.

3. the crystals are smaller than 10 microns and / or substantially cubic in shape; optionally (a) the polyhedrin protein comprises the polyhedrin sequence of the silkworm cypovirus set forth in SEQ ID NO: 1; or (b) the cargo protein is (a) an H1-tag sequence as shown in SEQ ID NO: 2; (b) the VP3-tag sequence shown in SEQ ID NO: 3, or (c) a PH tag sequence shown in SEQ ID NO: 4; comprising one or more polyhedrin binding tag sequences selected from A phagocyte according to claim 1 or 2.

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

5. The phagocyte according to any one of claims 1 to 4, wherein the phagocyte is derived from the peripheral blood of a subject.

6. The 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.

7. (a) the one or more therapeutic agents are capable of stimulating the production of M1 macrophage cells; 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 macrophage cells; optionally, the one or more therapeutic agents are growth factors, cytokines, enzymes, signaling molecules, hormones, antibodies, and / or vaccines; Further optionally, said one or more therapeutic agents include: (a) Th-1-associated 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-1 beta, 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; The phagocyte described in claim 6.

8. A phagocyte described in any one of claims 1 to 7, wherein the cargo protein comprises one or more detectable agents, optionally wherein the one or more detectable agents are fluorescent and / or bioluminescent agents.

9. 1×10 phagocytes according to any one of claims 1 to 8 ５ A population comprising more than one individual, optionally wherein the population is an autologous population.

10. A phagocyte described in any one of claims 1 to 8, or a population described in claim 9, wherein the phagocytes are further contained in a package to protect the cells from damage during collection, cell culture, or transportation.

11. A method for producing a population of phagocytes according to any one of claims 1 to 8 or phagocytes according to claim 9, comprising: (a) contacting the one or more phagocytes with polyhedrin protein crystals; and (b) culturing the phagocytes under conditions that induce phagocytosis; optionally (i) attaching said polyhedrin protein crystals to a solid support prior to contacting said phagocytes; or (ii) mixing the phagocytes with polyhedrin protein crystals before culturing the phagocytes; and optionally (c) isolating said one or more phagocytes; and / or (d) formulating said one or more phagocyte cells for delivery by injection; The method further comprises:

12. (a) the phagocyte cells of any one of claims 1 to 8 or the population of claim 9, wherein the phagocyte cells comprise one or more therapeutic agents; and (b) a pharmaceutically acceptable carrier or diluent 10. A pharmaceutical composition comprising:

13. 13. A pharmaceutical composition according to claim 12 for use in medicine.

14. 13. The pharmaceutical composition of claim 12 for use in a method for treating and / or preventing solid cancer in a subject.

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