LOADED CELL POPULATIONS, METHODS OF PREPARATION, AND METHODS OF USE THEREOF

RELATED APPLICATION

[001] The present PCT application claims the benefit of United States Provisional Application No. 63/298,454, filed January 11, 2022, the content of which is hereby incorporated by reference in its entirety.

FIELD OF THE DISCLOSURE

[002] The present disclosure relates to loaded leukocyte lineage cells and methods of making and using said cells and in particular to loaded neutrophil lineage cells and methods of making and using thereof.

BACKGROUND OF THE DISCLOSURE

[003] Cell- mediated drug delivery systems using circulating hematopoietic cells are of interest.

[004] Neutrophils, or polymorphonuclear leukocytes, represent the largest population of white blood cells in the human body (Rosales, 2018). Produced in the bone marrow and a critical component of innate immunity, this dynamic cell type migrates to sites of damage or inflammation following the release of chemoattractants or microbial signals (Mortaz et al., 2018; Schmidt et al., 2011). Neutrophils can phagocytose, generate reactive oxygen species, release microbicidal molecules (granulation) and/or produce extracellular fibers (Neutrophil Extracellular Traps, NETs) to block, degrade, and kill pathogens (Brinkmann et al., 2004; Kaplan and Radic, 2012). They also participate in adaptive immunity by secreting cytokines to recruit and activate additional leukocytes, engage in antigen presentation to stimulate dendritic cells (DC) and influence their maturation (Li et al., 2019).

[005] Xue et al., utilized purified mouse neutrophils as a therapeutic delivery tool to attempt to treat a surgically resected model of glioblastoma. The purified neutrophils were packaged with paclitaxel encapsulated cationic liposomes (Xue et al., 2017; US Patent 11,085,022).

[006] Ju et al, describe a neoadjuvant chemotherapy based on Abraxane/ human neutrophils (NEs) cytopharmaceuticals with radiotherapy (Ju et al, 2019).

[007] Loading cells with an appropriate therapeutic and for example, loading to a high or desired concentration is one of the challenges to utilizing cells as drug delivery systems.

SUMMARY OF THE INVENTION

[008] The following paragraphs are intended to introduce the reader to the more detailed description that follows and not to define or limit the claimed subject matter of the present disclosure. [009] Described herein are cell populations comprising leukocyte lineage cells loaded with an agent such as a biologic or a synthetic therapeutic or an imaging agent, as well as methods of making and using said loaded cell populations. Markers have been identified and are used to prepare cell populations that comprise for example an enhanced level of agent.

[010] One aspect of the present disclosure is a method for loading leukocyte lineage cells, comprising enriching, from a population of cells, leukocyte lineage cells expressing one or more, CD16+ and/or CDllb+ and loading an agent into or onto said enriched leukocyte lineage cells. Said leukocyte lineage cells can be CD15+ or CD15-.

[Oil] In an embodiment, the leukocyte lineage cells are neutrophil lineage cells.

[012] Another aspect of the present disclosure is a method for loading neutrophil lineage cells with an agent, optionally a biologic or synthetic therapeutic or an imaging agent, the method comprising enriching, from a population of leukocyte lineage cells, CD16+ and/or CDllb+ neutrophil lineage cells, and loading the agent, optionally the imaging agent, the biologic or the synthetic therapeutic into or onto the enriched CD16+ and/or CDllb+ neutrophil lineage cells.

[013] A further aspect of the present disclosure is a method of delivering an agent to a subject in need thereof, the method comprising enriching, from a population of neutrophil lineage cells, CD16+ and/or CDllb+ neutrophil lineage cells, loading the agent into or onto the enriched CD16+ and/or CDllb+ neutrophil lineage cells, thereby generating a population of agent-loaded leukocyte lineage cells, optionally neutrophil lineage cells, and delivering the agent-loaded leukocyte lineage cells to the subject.

[014] Another aspect of the present disclosure is a cell population comprising leukocyte lineage cells expressing one or more of CD16+ and/or CDllb+ loaded with an agent, optionally an imaging agent, biologic or synthetic therapeutic. The cells can for example be prepared using a method as described herein. In an embodiment, the leukocyte lineage cells are neutrophil lineage cells.

[015] Yet another aspect of the present disclosure is a composition comprising isolated leukocyte lineage cells, optionally neutrophil lineage cells, expressing at least one of from CDllb and CD16 or a combination thereof, and an agent loaded into or onto at least a portion of said leukocyte lineage cells, optionally neutrophil lineage cells. Such cells comprising an agent loaded into or onto at least a portion of said cells, can be referred to as loaded leukocyte lineage cells or if neutrophil lineage cells, loaded neutrophil lineage cells.

[016] A further aspect of the present disclosure is a drug delivery system comprising a cell population or composition described herein. [017] Another aspect of the present disclosure is a use of the cell population, or a composition as described herein, for delivering an agent, optionally a biologic or therapeutic or imaging agent, to a subject in need thereof.

[018] In an embodiment, the leukocyte lineage cells are derived from progenitor cells in vivo, optionally induced pluripotent stem cells (iPSCs).

[019] In various embodiments, the agent is encapsulated, optionally by a lipid, optionally the agent is encapsulated by or formulated as polymeric nanoparticles.

[020] Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the disclosure are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

DESCRIPTION OF THE DRAWINGS

[021] For a better understanding of the described embodiments and to show more clearly how they may be carried into effect, reference will now be made, by way of example, to the accompanying drawings in which:

[022] Fig. 1 depicts flow cytometry analysis demonstrating neutrophil subset differences in liposome uptake. Neutrophils were loaded with liposomes labelled with Genhance™680. Histograms show the percentage of cells within a specific subset that were loaded by liposomes. It was observed that liposome uptake was greater in CD34 CD15 ⁺ CDllb ⁺ CD16 ⁺ and CD34 CD15 ⁺ CDllb ⁺ CD16 ^_ cells than CD34" CD15 ⁺ CDllb CD16 ^_ cells. It was also observed that in non-neutrophil CD34 CD15" cells (Fig IB), CDllb ⁺ CD16 ⁺ and CDllb CD16 ⁺ subsets showed greater liposome uptake than CDllb CD16 ^_ cells.

[023] Fig. 2 is a series of graphs that shows loading of liposomes fluorescently labelled with DiL (Encapsula) in Neutrophils under different incubation conditions.

[024] Fig. 3 is a series of flow cytometry histograms that shows uptake of liposome-encapsulated doxorubicin in neutrophils. Drug loaded neutrophils were washed with PBS, doxorubicin was detected by flow cytometry. When cells were loaded with 25 ug of encapsulated doxorubicin, 93% of cells were observed to contain doxorubicin.

[025] Fig. 4A is a schematic of an experimental overview of the injection protocol used. Fig. 4B is a series of images that shows the migration of fluorescently labelled neutrophils to a site of inflammation. Specifically, the images show that fluorescent signal is detected in LPS-injected ears of animals injected with labelled neutrophils. Control animals treated with either i) cells in the absence of LPS, or ii) LPS in the absence of cells showed no significant fluorescent signal in ears.

[026] Fig 5A is a schematic description of the experimental design. Fig 5B depicts ex vivo imaged brains 24 hours after neutrophil injected. Fluorescent signal in the brain is only detected in animals injected with both LPS and labelled cells.

[027] The drawings included herewith are for illustrating various examples of articles, methods, and apparatuses of the teaching of the present specification and are not intended to limit the scope of what is taught in any way.

DETAILED DESCRIPTION OF THE DISCLOSURE

[028] Various apparatuses, methods and compositions are described below to provide an example of an embodiment of each claimed invention. No embodiment described below limits any claimed invention and any claimed invention may cover apparatuses and methods that differ from those described below. The claimed inventions are not limited to apparatuses, methods and compositions having all of the features of any one apparatus, method or composition described below or to features common to multiple or all of the apparatuses, methods or compositions described below. It is possible that an apparatus, method or composition described below is not an embodiment of any claimed invention. Any invention disclosed in an apparatus, method or composition described below that is not claimed in this document may be the subject matter of another protective instrument, for example, a continuing patent application, and the applicant(s), inventor(s) and/or owner(s) do not intend to abandon, disclaim, or dedicate to the public any such invention by its disclosure in this document.

[029] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

Definitions

[030] The term "active pharmaceutical ingredient" or API refers to one or more molecular entities that has a desired pharmacologic and/or therapeutic activity. An API may be comprised of a small molecule of synthetic or natural origin, or a protein or fragment thereof, therapeutic antibody or binding fragment thereof, or other biologic or chemical entity that is known to, or suspected of, having pharmacologic activity.

[031] The term "agent" as used herein refers to one or more molecular entities and includes but is not limited to biologic or synthetic therapeutics and imaging agents, including small molecules, metabolites, macromolecules such as a polypeptides or fragments thereof, for example ligands or antibodies or binding fragments thereof, polynucleotides etc. The agent may be natural e.g., a biologic, or synthetic. The agent can for example be an active pharmaceutical ingredient (API) or an imaging agent that comprises a detectable label or emits a detectable signal. The agent may be encapsulated, for example in a lipid, for example in a liposome.

[032] The term "antibody" as used herein but not limited to is intended to include monoclonal antibodies including chimeric and humanized monoclonal antibodies, polyclonal antibodies, humanized antibodies, human antibodies, and chimeric antibodies. Bispecific antibodies, including bispecific monoclonal antibodies such as Bi-specific T-cell engagers (BiTEs), which are a class of artificial bispecific monoclonal antibodies, are also included. The antibody may be from recombinant sources and/or produced in transgenic animals. The term "antibody fragment" as used herein is intended to include Fab, Fab', F(ab') ₂ , scFv, dsFv, ds-scFv, minibodies, diabodies, and multimers thereof as well as bispecific antibody fragments. Antibodies can be fragmented using conventional techniques. For example, F(ab') ₂ fragments can be generated by treating the antibody with pepsin. The resulting F(ab') ₂ fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab') ₂ , scFv, dsFv, ds-scFv, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques. The skilled person can readily recognize that a suitable antibody for the invention is any antibody useful for detecting biomarkers described herein in any detection method described herein. For example, useful antibodies include antibodies that specifically bind to a biomarker of the invention described herein.

[033] The term "biologic" as used herein refers to but is not limited to a product manufactured in, extracted from, or semi-synthesized from biological sources. The biologic can for example a polypeptide, a ligand, immunogen or an antibody or fragment thereof, a nucleic acid molecule, optionally comprised in a vector, other macromolecule, or cellular fraction. Biologies can be isolated from natural sources such as human, animal including insect, plants or microorganisms.

[034] The term "cell" as used herein refers to a single cell or a plurality of cells. [035] The term "oligonucleotide" used interchangeably with "polynucleotide" and "nucleic acid" as used herein but is not limited to a nucleic acid molecule comprising, a sequence of nucleotide or nucleoside monomers (two or more) consisting of naturally and non-naturally occurring bases, sugars, and intersugar (backbone) linkages, and includes single-stranded and double-stranded molecules, RNA and DNA, as well as hybrids thereof. Oligonucleotides may be long (e.g., greater than 1000 monomers and up to 10K monomers), medium sized (e.g., between and inclusive of 200 and 1000 nucleotides) or short for example less than 200 monomers, 100 monomers, 50 monomers, including non-naturally occurring monomers. The term "oligonucleotide" includes, for example, single stranded DNA (ssDNA), genomic DNA (gDNA), complementary DNA (cDNA, reverse transcribed from an RNA), messenger RNA (mRNA), "antisense oligonucleotides" including shRNA and siRNA antisense oligonucleotides and "miRNA" as well as oligonucleotide analogues such as "morpholino oligonucleotides", "phosphorothioate oligonucleotides", or any oligonucleotide or analog thereof known to one of skill in the art as well as hybrid sequences.

[036] The term "encapsulating" as used herein refers to entrapping of an agent (e.g., an API) in or within a secondary material, typically inert, optionally a matrix, membrane, coating or shell, optionally within a cavity of, incorporated in the matrix of or tethered on a surfaced of the secondary material. Various encapsulation technologies can be used including for example emulsification, liposomes, electrospray, solid particles and structured lipid carriers.

[037] The term "leukocyte lineage cell" refers to a hematopoietic cell that is, will or can differentiate to a leukocyte, which include lymphocytic lineage cells including B lymphocytes and T lymphocytes, natural killer cells, granulocytic lineage cells including eosinophil, basophil and neutrophil lineage cells and monocytic lineage cells including monocytes and macrophages. Progenitor cells and/or mature forms are contemplated.

[038] A "polymeric nanoparticle" as used herein refers to, but is not limited to, a structure having one or more dimensions at the nanometer level, for example between the lower and upper dimensions of 0.5 nm and 1000 nm and may include, for example, nanocapsules, nanotubes, nanospheres and/or other nanostructures. Nanotubes may include structures having geometries resembling, but not limited to, tubes, solid rods, whiskers, and rhomboids. Said particles can be loaded with one or more agents including one or more actives, with said agents entrapped within a cavity, within a layer of polymer or surface absorbed onto the polymeric core. [039] The term "neutrophil lineage cell" as used herein defines a class of cells comprising granulocyte- committed progenitor cells and their differentiated and mature forms, including but not limited to myeloblasts, promyelocytes, myelocytes, metamyelocytes, eosinophils, basophils, banded neutrophils and segmented neutrophils.

[040] As used herein, the terms "peptide," "polypeptide," and "protein" refer to any chain of two or more natural or unnatural amino acid residues, regardless of post-translational modifications (e.g., glycosylation or phosphorylation). The polypeptides incorporated into the biphasic vesicles of the disclosure can include for example from 3 to 3500 natural or unnatural amino acid residues. Included are proteins that are a single polypeptide chain and multisubunit proteins (e.g. composed of 2 or more polypeptides).

[041] The term "synthetic therapeutic" as used herein means any synthesized molecule and can include naturally occurring molecules that are synthesized, including small molecules, macromolecules such as nucleic acid molecules, siRNA molecules and peptides using non-cell based methods e.g. cell free assays, chemical synthesis schemes, automated synthesizers and the like.

[042] The term "subject" as used herein includes all members of the animal kingdom including mammals, and suitably refers to humans. Thus the methods and uses of the present application are applicable to both human therapy and cosmetic applications and veterinary applications.

[043] The term "treating" or "treatment" as used herein and as is well understood in the art, means an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results include, but are not limited to alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total), whether detectable or undetectable. "Treating" and "treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. "Treating" and "treatment" as used herein also include prophylactic treatment. For example, a subject with a skin disease, disorder or condition can be treated to prevent progression. Treatment methods comprise administering to a subject a therapeutically effective amount of one or more of the compounds of the disclosure and optionally consist of a single administration, or alternatively comprise a series of administrations. [044] In understanding the scope of the present disclosure, the term "comprising" and its derivatives,

(such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "include" and "includes") or "containing" (and any form of containing, such as "contain" and "contains"), as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, "including", "having" and their derivatives.

[045] The term "consisting of" and its derivatives, as used herein, are intended to be closed ended terms that specify the presence of stated features, elements, components, groups, integers, and/or steps, and also exclude the presence of other unstated features, elements, components, groups, integers and/or steps.

[046] The term "consisting essentially of", as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and/or steps as well as those that do not materially affect the basic and novel characteristic(s) of these features, elements, components, groups, integers, and/or steps.

[047] The term "and/or" as used herein means that the listed items are present, or used, individually or in combination. In effect, this term means that "at least one of" or "one or more" of the listed items is used or present. That is, "X and/or Y" is intended to mean X or Y or both, for example. As a further example, "X, Y, and/or Z" is intended to mean X or Y or Z or any combination thereof.

[048] In embodiments comprising an "additional" or "second" component, such as an additional or second compound, the second component as used herein is chemically different from the other components or first component.

[049] Further, terms of degree such as "substantially", "about" and "approximately" as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies.

[050] More specifically, the term "about" means plus or minus 0.1 to 50%, 5-50%, or 10-40%, 10-20%, 10%-15%, preferably 5-10%, most preferably about 5% of the number to which reference is being made. [051] As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural references unless the content clearly dictates otherwise. Thus, for example, a composition containing "a compound" includes a mixture of two or more compounds. It should also be noted that the term "or" is generally employed in its sense including "and/or" unless the content clearly dictates otherwise.

[052] The terms "an embodiment," "embodiment," "embodiments," "the embodiment," "the embodiments," "one or more embodiments," "some embodiments," and "one embodiment" mean "one or more (but not all) embodiments of the present invention(s)," unless expressly specified otherwise.

[053] The definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art.

[054] The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about."

[055] Further, the definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art. For example, in the following passages, different aspects of the disclosure are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[056] For ranges described herein, subranges are also contemplated, for example every, 0.1 increment there between. For example, if the range is 80% to about 90%, also contemplated are 80.1% to about 90%, 80% to about 89.9%, 80.1% to about 89.9% and the like.

General Description of the Disclosure

[057] One aspect of the present disclosure is a method for loading of leukocyte lineage cells, comprising enriching, from a population of cells, leukocyte lineage cells expressing one or more of CD16 and/or CDllb, and loading an agent into or onto the enriched leukocyte lineage cells. [058] As demonstrated herein, leukocyte lineage cells such as neutrophils expressing one or more CD16 and/or CDllb, show for example enhanced drug loading. Enhanced drug loading is desirable, for example since fewer cells can be used to deliver a particular dose.

[059] The population of cells of the present disclosure from which the leukocyte lineage cell population is enriched may come from different sources. In some embodiments, the population of cells is obtained from a patient. In these embodiments, populations of cells are withdrawn from a patient using known methods such as a blood draw, and a population of leukocyte lineage cells belonging to the subject is enriched from the same.

[060] Alternatively, the subject may be a donor. In these embodiments, the donor's cell population may be expanded and cultivated prior to enrichment. The population of leukocyte lineage cells is then enriched from said population, and may be further cultivated, expanded, or stored, or used in accordance with the present teachings.

[061] In some embodiments, the population of cells may be obtained from a cell bank, or otherwise commercially sourced.

[062] In some embodiments, the leukocyte lineage cells are prepared from stem or progenitor cells including for example pluripotent stem cells. Methods for making neutrophil lineage cells are known in the art (see for example Majumder et al. (2020), STAR Protocols, 1, 100075; Trump et al., (2019), Stem Cells Trans Med, 8:557-567; Dannenmann et al. (2020), Methods Mol Biol, 2115:471-483.; and Pittermann et al., (2017) Blood Adv l(14):903-914.) Leukocyte lineage cells such as neutrophil lineage cells can also be prepared from blood stems cells such as circulating or cord blood stem cells (see for example Jie et al., (2017) PLos One 12(7): e0180832, Kuhikar et al., (2021) Stem Cell Res, 50: 102150; Timmins et al. (2009), Biotech and Bioeng. 104 (4):832-840.

[063] The cells enriched from the population of cells may be any one leukocyte lineage including neutrophils, macrophage, monocytes.

[064] In some embodiments, the leukocyte lineage cells are neutrophil lineage cells. The population of cells can be enriched for neutrophil lineage cells and one or more of CDllb and CD16 or a combination thereof.

[065] As indicated above, these cells may be enriched from different sources. They may also be derived from stem or progenitor cells.

[066] Like leukocyte lineage cells, the neutrophil lineage cells can be loaded with an agent, optionally a biologic or synthetic therapeutic, the method comprising enriching, from a population of neutrophil lineage cells, CD16+ and/or CDllb+ neutrophil lineage cells, and loading the agent, optionally the imaging agent, the biologic or the synthetic therapeutic into or onto the enriched CD16+ and/or CDllb+ neutrophil lineage cells.

[067] The loading of the agent may comprise incubating the enriched CD16+ and/or CDllb+ neutrophil lineage cells with the agent.

[068] The agent to be loaded into the leukocyte lineage cells may comprise, but is not limited to, , a biologic or a synthetic therapeutic.

[069] The imaging agent comprise a detectable label such as a fluorescent bioluminescent or radioactive label. The imaging agent can for example be or comprise a contrast or metallic agent. The label can for example be conjugated to an agent or the label can be encapsulated with an agent. Other labels can also be used.

[070] The biologic or synthetic therapeutic can for example be a polypeptide, such as an antibody, immunogen or ligand. The polypeptide can be a cytokine or hormone, or a protein, as well as a fragment or subunit thereof. It can in some embodiments be a nucleic acid molecule. The nucleic acid molecule can be comprised in a vector, optionally a plasmid or other expression vector. Alternatively, the nucleic acid molecule can be naked or linear DNA or RNA or a hybrid thereof. The nucleic acid molecule can be an antisense molecule, optionally an antisense oligonucleotide, mRNA, siRNA or shRNA.

[071] In some embodiments, the nucleic acid is or comprises RNA, mRNA, DNA or a hybrid thereof.

[072] The synthetic therapeutic is not particularly limited and can include combinations of actives or active and non-active ingredients.

[073] The agent to be loaded into or onto the leukocyte lineage cells, optionally the biologic or synthetic therapeutic may be encapsulated. Alternatively, the agent may be loaded in its unaltered form. Where the agent is encapsulated, it may be encapsulated in or formulated as a nanoparticle, optionally a polymeric nanoparticle.

[074] Said nanoparticle may be, but is not limited to, a lipid-based nanoparticle, optionally a liposome, a lipid nanoparticle or an emulsion. Alternatively, the nanoparticle may be a polymeric nanoparticle, optionally a nanocapsule or nanosphere, such as a polymersome, micelle, or dendrimer.

[075] Alternatively, the nanoparticle may be composed of amphiphilic cyclodextrins. Alternatively, the nanoparticle may be an inorganic nanoparticle, optionally a ceramic, silica, silicon, silver, gold, iron oxide nanoparticle, a salt nanoparticle (such as calcium phosphate) or a nanoparticle composed of another biocompatible compound. [076] The nanoparticle may be porous and the agent, optionally the imaging agent, the biologic or synthetic therapeutic may be absorbed into or onto the nanoparticle. The nanoparticle may be formed as a porous nanoparticle, or it may have porosity imparted to it through the use of a reagent, such as CTAB. [077] The encapsulation may be through any methods known in the art, for example as described in Li et al., (2020). The therapeutic may be encapsulated in, for example, polymer nanoparticles, polymer microparticles, lipid nanoparticles, liposomes, exosomes, magnetic/paramagnetic particles, carbon nanotubes (CNT), nanoemulsions, gold nanoparticles, dendrimers, micelles, ceramic particles, silica particles, calcium sulfate particles, or cyclodextrins.

[078] In an embodiment, the polymeric nanoparticles may comprise one or more polymers selected from the group comprising poly (a-hydroxyacids) including poly (D,L-lactide-co-glycolide)(PLGA), poly D,L- lactic acid (PDLLA), polyethyleneimine (PEI), polylactic or polyglcolic acids, poly-lactide poly-glycolide copolymers, and poly-lactide poly-glycolide polyethylene glycol copolymers, polyethylene glycol (PEG), polyesters, poly (e-caprolactone), poly (3-hydroxy-butyrate), poly (ortho esters), polyanhydrides, poly (sebacic anhydride) (PSA), poly (amino acids), poly (pseudo amino acids), polyphosphazenes, polyphosphates, polyethylene glycol polypropylene block co-polymers for example that sold under the trade mark Pluronics™, elastin, chitin, chitosan, fibrin, fibrinogen, polysaccharides (including pectins), alginates, peptides, polypeptides or proteins, copolymers prepared from the monomers of any of these polymers, random blends of these polymers, any suitable polymer and mixtures or combinations thereof. [079] In an embodiment, the agent, optionally the biologic synthetic therapeutic or imaging agent, may be encapsulated in lipid such as a liposome. The lipid or liposomes may comprise one or more neutral lipids, cholesterol and one or more PEGylated lipids. Example neutral lipids include but are not limited to phosphatidylcholine (PC) lipids, for example dipalmitoylphosphatidylcholine (DPPC). Example PEGylated lipids include, but are not limited to, PEGylated phosphatidylethanolamine (PEG-PE) lipids, such as PEGylated Distearoylphosphoethanolamine (PEG-DSPE). In an embodiment, the liposomal carrier comprises (in molar %) about 40% to about 60% of the lipid, about 20% to about 40% of the cholesterol and about 0 to about 5% of the PEGylated lipid. In an embodiment, the liposomal carrier comprises the one or more lipids, cholesterol and one or more PEGylated lipids at a molar percent ratio of about 55:40:5. Liposome compositions, their synthesis and use can for example be as described in Ahmed, KS et al, 2019. [080] The agent may be conjugated to at least one carrier. Said carrier may comprise, but is not limited to, a protein, such as an albumin, a lipid, or a polymer. Said carrier-compound conjugate may be formulated as or part of nanoparticles or aggregates prior to incubation with the leukocyte lineage cell. Said carrier-compound conjugate may be incubated with the leukocyte lineage cell without further processing.

[081] The agent may be loaded into and/or onto the leukocyte lineage cell by incubating the cells with the agent. For example, the liposome encapsulated agent may be loaded by shaking incubation or static incubation as shown for example in Fig. 2.

[082] The agent or the nanoparticle (e.g., the encapsulated agent) may be loaded into an internal cavity of said neutrophil lineage cells.

[083] The agent may, alternatively, be loaded onto a surface of said leukocyte lineage cells, optionally neutrophil lineage cells. In this embodiment, the agent, or a nanoparticle containing said agent, may react with reactive groups on the surface of the cell membrane of said neutrophil lineage cells, forming chemical bonds wherein the agent becomes attached to the cell membrane. Alternatively, the agent or nanoparticle may be non-covalently associated.

[084] The agent or the nanoparticle (e.g., the encapsulated agent) may be loaded into an internal cavity of said leukocyte lineage cells and also onto a surface of said leukocyte lineage cells.

[085] In some embodiments, multiple agents or actives may be used in combination without departing from the present teachings. Said multiple agents may be separately encapsulated, or may optionally be loaded into or onto the leukocyte lineage cells according to different loading techniques described herein. In some embodiments, a first encapsulated agent may be loaded into the leukocyte lineage cells, and at least a second agent may be integrated into the leukocyte lineage cell membrane, lineage Also provides is a cell population comprising leukocyte lineage cells expressing one or more of CD16 and/or CDllb loaded with an agent, optionally an imaging agent, biologic or synthetic therapeutic, prepared as described herein.

[086] The leukocyte lineage cells may be neutrophil lineage cells expressing markers CD16+ and/or CDllb+, prepared by any of the methods described above. The leukocyte lineage cell population may be prepared from stem cells, optionally from pluripotent stem cells.

[087] The leukocyte lineage cell population may comprise a target purity. Such minimum ortarget purity levels may comprise, but are not limited to, 80%, at least 90%, at least 95%, at least 96%, at least 97% or at least 98% or more of the cell population.

[088] Another aspect of the present disclosure is a composition comprising isolated leukocyte lineage cells, optionally neutrophil lineage cells, expressing at least one CDllb and CD16 or a combination thereof, and an agent loaded into or onto at least a portion of said neutrophil lineage cells. The composition can comprise a pharmaceutically acceptable diluent such as cell media, phosphate buffered saline or a carrier such as serum. In another embodiment, the composition can comprise a cyroprotectant for example glycerol, DMSO, ethylene glycol and/or polymers. Examples of commercially available cryoprotectant solutions include CryoStor® CS10, Stem-cellbanker®, CryoNovo™ P24, and PentaHibe®.The loaded leukocyte lineage cells, optionally the neutrophil lineage cells, can be used to deliver the agent.

[089] Accordingly, also provided in a further aspect of the present disclosure is a method of delivering an agent to a subject in need thereof, the method comprising enriching, from a population of neutrophil lineage cells, CD16+ and/or CDllb+ neutrophil lineage cells, loading the agent into or onto the enriched CD16+ and/or CDllb+ neutrophil lineage cells, thereby generating a population of agent-loaded neutrophil lineage cells, and delivering the agent-loaded neutrophils to the subject.

[090] The delivering may comprise intravenous injection of the agent-loaded neutrophil lineage cells. The delivering may comprise intraperitoneal or intratumoral injection.

[091] Depending on the agent loaded into the leukocyte lineage cells, the subject may be in need of imaging or treatment.

[092] Also provided herein in some embodiments, is a use of the cell population described herein, or a composition as described herein, or the loaded leukocyte lineage cells described herein for delivering an agent, optionally a biologic or therapeutic, to a subject in need thereof.

[093] In some embodiments, the method, use, cell population, leukocyte lineage cells or a composition comprising any thereof, is one wherein the agent is selected from antineoplastic agents, thrombolytic agents, anti-inflammatory drugs, antibacterial drugs or antiviral drugs

[094] In some embodiments, the subject in need thereof is afflicted with a cancer. The cancer may be a solid tumor including ovarian cancer, glioblastoma, pancreatic cancer, liver cancer or osteosarcoma. Any cancer or tumor that shows signs of inflammation (e.g., immunologically "hot" tumor, for example with T cell infiltration) can be targeted.

[095] In other embodiments, the subject in need thereof is afflicted with an inflammatory disease.

[096] Inflammatory disease may include multiple sclerosis, inflammatory bowel disease, lupus, or rheumatoid arthritis. In an embodiment, the subject in need thereof is afflicted with a viral or bacterial infection.

[0100] The above disclosure generally describes the present disclosure. While the above description describes features of example embodiments, it will be appreciated that some features and/or functions of the described embodiments are susceptible to modification without departing from the spirit and principles of operation of the described embodiments. For example, the various characteristics which are described by means of the represented embodiments or examples may be selectively combined with each other. Accordingly, what has been described above is intended to be illustrative of the claimed concept and non-limiting. It will be understood by persons skilled in the art that other variants and modifications may be made without departing from the scope of the invention as defined in the claims appended hereto. The scope of the claims should not be limited by the preferred embodiments and examples, but should be given the broadest interpretation consistent with the description as a whole.

[097] The teachings disclosed herein will be better understood in light of the following examples. It is understood that the examples are not intended to in any way limit the scope of the present disclosure. The following examples provide additional enablement for persons of ordinary skill in the art to put the present teachings into practice. Changes in form and substitution of equivalents are contemplated as circumstances might suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

Examples

Example 1: Methods

[098] In Example 1, the methods used in Examples 2 and 3 are described.

Neutrophil Culture

[099] Hematopoietic progenitor cells expanded from cord blood CD34 ⁺ cells were used to seed neutrophil differentiation cultures at a density of l.OxlO ⁵ cells/mL. Neutrophil differentiation media consisted of StemSpan-ACF™ (Stem Cell Technologies) supplemented with 2 nM L-Glutamine (Thermo), 100 ng/mL G-CSF, 100 ng/mL SCF, 100 ng/mL TPO (Biotechne). After seeding, cells were incubated at 37C for 5 days with gentle agitation. At Day 5 of culture, an equal volume of fresh neutrophil differentiation was added to cultures, and this was repeated every 48 hours until Day 13-15 of differentiation.

Liposome uptake by neutrophils

[100]

5xl0 ⁵ human neutrophils were incubated with liposomes labelled with Genhance™680 (Perkin Elmer), for 4 hours at 37 °C. After incubation, cells were washed twice with PBS, and analyzed by flow cytometry for expression of CD34, CD16, CDllb and CD15. Viable cells successfully targeted by liposomes were identified by detecting Genhance™680 signal by excitation with a 640 nm laser and670/10 filters (Fig 1). Cells were also targeted with liposomes containing Di L (Encapsula) to test the effect of different conditions on the efficiency of liposome uptake (Fig 2) Similarly, neutrophils were loaded with liposome- encapsulated doxorubicin (Doxoves™, Formumax). Uptake of doxorubicin by neutrophils was monitored by flow cytometry by excitation with a 488 nm laser and detection with a 530/30 filter.

Flow Cytometry

[101] Cells were prepared and stained for flow cytometry analysis using standard protocols. Combinations of the following antibodies were used as appropriate: CDllb-FITC, CD15-PE, CD34-APC, CD16-AF700, CXCR2-PE-Cy7. After staining with antibodies, cells were stained with 7AAD, and 7AAD" cells were identified as viable. Cells were analyzed on a LSRFortessa™ (BD Biosciences).

Migration to inflammatory site in vivo

[102] To demonstrate that neutrophils will migrate to a site of inflammation in vivo, athymic nude mice were injected either intracranially with 10 ug of LPS; or intradermally into the ear with 50 ug of LPS. 24 hours later, cryopreserved neutrophils were thawed and incubated with 10 ug/mL of Xenolight DiR™ (Perkin Elmer) for 25 minutes at 37°C and washed twice with PBS. Labelled neutrophils were administered to animals by tail vein injection. Mice were injected with doses of CD15 ⁺ labelled neutrophils ranging between 4xl0 ⁵ and 3.5xl0 ⁶ per animal. Mice were monitored by fluorescence microscopy, and after 24 hours, animals were euthanized, and brains and ears were isolated and imaged ex vivo using a c720 nm filter.

Example 2: Particle Loading in Neutrophils

[103] After loading with fluorescent liposomes, cells were analyzed by flow cytometry. CD34-CD15+ cells were further analyzed for expression of CD16 and CDllb (Figure 1A). Within each gated subset, cells positive for liposomes were identified based on fluorescence in the APC channel. Histograms represent Liposome positive cells as a percentage of each cell subset. CD34 CD15 ⁺ CDllb ⁺ CD16 ⁺ and CD34" CD15 ⁺ CDllb ⁺ CD16 ^_ cells showed greater liposome uptake than less mature CD34 CD15 ⁺ CDllb CD16 ^_ cells. Furthermore, CD34-CD15+CDllb-CD16+ cells also showed greater uptake than CD34 CD15 ⁺ CDllb CD16 ^_ cells. Similarly, in non-neutrophil CD34 CD15" cells (Figure IB), CDllb ⁺ CD16 ⁺ and CDllb CD16 ⁺ cells showed greater uptake than CDllb CD16 ^_ cells, indicating that these cell surface markers can be utilized to identify specific cell populations with greater propensity for agent uptake.

Example 3: Inflammatory Targeting

[104] Fluorescently labelled neutrophils were observed to migrate to sites of transdermal injection of LPS. In 5/5 animals (Figure 4), 24 hours after cell injection, neutrophils were detected in the LPS-injected ear, but not the control ear. When LPS was injected intracranially, neutrophils were detected in the brains of LPS injected animals. No fluorescent signal was detected in the brains of control animals that were injected with i) LPS with no cells or ii) cells with no LPS (Figure 5).

Example 4: Non-neutrophils

[105] CD56 ^dim or CD56 ^bright natural killer lineage cells are generated from induced pluripotent stem or hematopoietic stem cells or isolated from blood and a CD16+ and/or CDllb+ fraction is purified and loaded with an encapsulated agent by incubating the encapsulated agent with the cells for at least 1 hour. The agent can be an API.

[106] Loaded CD16+ and/or CDllb+ natural killer cells comprising an API are administered to a subject comprising cancer with an inflammatory component.

Example 5: Incorporation of Doxorubicin-loaded FE3O4 nanoparticles into mesoporous silica

[107] Doxorubicin is integrated into hydrophobic magnetic Fe ₃ O ₄ nanoparticles in mesoporous silica spheres, using cetyl- trimethylammonium bromide (CTAB) surfactant as a porogen for the formation of mesostructure. The particles are then incubated with CDllb+ and/or CD16+ isolated neutrophil lineage cells, optionally CD34- CD15+ CDllb+ CD16+ neutrophils for at least 1 hour, optionally 2, 3 or 4 hours. After incubation, cells are washed twice with sterile PBS.

Example 6: Gadolinum-containing nanoparticles conjugated to neutrophil membrane.

[108] A gadolinium-loaded bovine serum albumin nanoparticle is conjugated to the neutrophil membrane directly by using a sulfhydryl group on the external surface of the nanoparticle. The sulfhydryl group, upon incubation of the nanoparticles with neutrophils, conjugates with disulfide groups on the surface of the neutrophil membrane, for example as described in Qiu et al, 2019.

Example 7: Abraxane nanoparticle loading into neutrophils.

[109] Abraxane, a form of paclitaxel bound to albumin, is used to prepare nanoparticles that are then incubated with neutrophils to form abraxane-loaded neutrophils, [as described in Ju et al (2019).

[110] Although the disclosure has been described with reference to certain specific embodiments, various modifications thereof will be apparent to those skilled in the art. Any drawings provided herein are solely for the purpose of illustrating various aspects of the disclosure and are not intended to be drawn to scale or to limit the disclosure in any way. Any examples provided herein are included solely for the purpose of illustrating the disclosure and are not intended to limit the disclosure in any way. The scope of the claims appended hereto should not be limited by the preferred embodiments set forth in the above description but should be given the broadest interpretation consistent with the present specification as a whole. The disclosures of all prior art recited herein are incorporated herein by reference in their entirety. [111] While the present application has been described with reference to examples, it is to be understood that the scope of the claims should not be limited by the embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.

[112] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Where a term in the present application is found to be defined differently in a document incorporated herein by reference, the definition provided herein is to serve as the definition for the term.

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US Patent 11,085,022