WO2024197098A2 - Local conditioning of lymph nodes with diffusion-limited particles for in vivo car therapy - Google Patents

Abstract

Provided herein are composition comprising microparticles; wherein each of the microparticles comprises a nucleic acid encoding, for example, a Chimeric Antigen Receptor (CAR).

Description

LOCAL CONDITIONING OF LYMPH NODES WITH DIFFUSION-LIMITED PARTICLES FOR IN VIVO CAR THERAPY Related Applications

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. provisional patent application, U.S.S.N. 63/491,282, filed March 20, 2023, the entire contents of which are incorporated herein by reference.

Background

[0002] A Chimeric Antigen Receptor (CAR) is a synthetic transmembrane protein comprising an extracellular antigen recognition domain (e.g., an antibody single-chain variable fragment), a transmembrane domain, and an intracellular signaling domain (e.g., a T cell signaling domain, e.g., CD3-zeta). When, by artificial means, the CAR is expressed in or by a first cell (e.g., a T cell), the CAR directs the first cell to kill a second cell, such as a cancer cell, wherein the second cell expresses a surface antigen that is, by design, recognized by the CAR’s extracellular antigen recognition domain. The cell modified to express the CAR, for example, a T cell (such as a CAR T cell), can be administered to a patient to kill tumor cells or other pathogenic cells. Toward this end, CARs have been developed with extracellular antigen recognition domains that specifically bind surface antigens (markers), such as CD19, BCMA, EGFR/HER, CD22, mesothelin, CD123, CD20, PD1, and CD30. CAR-expressing cells, e.g., CAR T cells, have been developed to treat hematologic malignancies, solid tumors, and non-cancerous conditions, such as autoimmune disease. See, for example, Alnefaie et al., “Chimeric Antigen Receptor T-Cells: an overview of concepts, applications, limitations, and proposed solutions” Front. Bioeng. Biotechnol. 2022;

10:797440 (doi 10.3389); and U.S. Patent 10,934,337, each of which is incorporated herein by reference. Summary

[0003] Nevertheless, there remains a need to transfect cells in situ in the lymph node with a nucleic acid that encodes a protein, such as therapeutic protein (e.g., a CAR for in vivo CAR therapy). The present disclosure provides supporting studies and clinical products for in vivo therapy (e.g., CAR therapy) leveraging intra-lymph node injection to access and condition these unique immune niches without the need for systemic infusion or exposure of either therapy-encoding nucleic acids or cell products (e.g., CAR proteins and/or CAR T-cells). The concept relies on delivery of diffusion limited particles (e.g., nanoparticles, microparticles) loaded with a nucleic acid encoding the protein of interest (e.g. therapeutic protein), such as mRNA encoding CARs, as well as transfection aids, such that the particles are retained in the lymph node for local and sustained transfection.

[0004] Thus, in one aspect of the present disclosure, provided is a nanoparticle (NP) comprising a nucleic acid, wherein the NP is capable of delivering the nucleic acid to cells after being injected into a lymph node of a subject.

[0005] In some embodiments, the NP delivers the nucleic acid to cells after being retained in the lymph node of a subject after injection.

[0006] In certain embodiments, the nucleic acid encodes a chimeric antigen receptor (CAR) [0007] In some embodiments, the NP of the present disclosure further comprises at least one transfection agent. In some embodiments, the at least one transfection agent is ApoE. In certain embodiments, the at least one transfection agent is CpG. In some embodiments, the at least one transfection agent increases the delivery mRNA to the cells in the lymph node. In certain embodiments, the at least one transfection agent increases expression of the construct. [0008] In some embodiments, the NP of the present disclosure further comprises a T-cell activating factor. In certain embodiments, the T-cell activating factor is CD3/CD28 or OKT. In some embodiments, the T-cell activating factor is a mitogen or superantigen. In certain embodiments, the mitogen or superantigen is phorbol 12-myristate 13-acetate (PMA), ionomycin, and/or PHA (Phytohaemagglutinin P).

[0009] In some embodiments, the NP of the present disclosure further comprises a molecular adjuvant. In certain embodiments, the molecular adjuvant is an innate ligand. In some embodiments, the innate ligand is TLR, RIG, and/or STING.

[0010] In certain embodiments, the NP further comprises at least one lymph node binding moiety. In some embodiments, the lymph node binding moiety binds RANKL, sLeX, PNAd, GL7, CLDN11, EFNB2, ACKR4, TNFRSF9, CCL20, CXCL5, GlyCAM-1, CD34, Ptx3, CD209/Marco, SPNS2, CSF-1, CCL21, CCL19, CCL25, or CCL27.

[0011] In certain embodiments, the nucleic acid encodes a CAR protein with an extracellular antigen-binding domain. In some embodiments, the extracellular antigenbinding domain binds BCMA. In certain embodiments, the extracellular antigen-binding domain binds CD- 19.

[0012] In some embodiments, the nucleic acid is an mRNA.

[0013] In certain embodiments, the NP comprises 250-500 ng of mRNA.

[0014] In some embodiments, the NP is a lipid nanoparticle (LNP).

[0015] In certain embodiments, the LNP comprises ionizable lipids, stabilizing lipids, cationic lipids, neutral lipids, or any combination thereof.

[0016] In some embodiments, the NP is a polymer nanoparticle.

[0017] In certain embodiments, the polymer nanoparticles comprise cationic polymers.

[0018] In some embodiments, the concentration of NP in the lymph node is greater than the concentration in any other tissue of the subject. In certain embodiments, the concentration of NP in the lymph node is greater than the systemic biodistribution in the subject.

[0019] In another aspect, provided is a nanoparticle composition comprising a plurality of the NPs described herein. [0020] In yet another aspect of the present disclosure, provided is a microparticle (MP) loaded with at least one nanoparticle described herein.

[0021] In one aspect, provided herein is a microparticle (MP) loaded with a nucleic acid, wherein the microparticle is capable of delivering the nucleic acid to cells after being injected into a lymph node of a subject. In certain embodiments, the MP is also loaded with at least one nanoparticle. In some embodiments, the MP is further mixed with at least one nanoparticle in solution.

[0022] In certain embodiments, the MP delivers the nucleic acid to cells after being retained in the lymph node of a subject after injection.

[0023] In some embodiments, the nucleic acid encodes a chimeric antigen receptor (CAR) [0024] In certain embodiments, the MP is also loaded with at least one transfection agent. In some embodiments, the MP is further mixed with at least one transfection agent in solution. In certain embodiments, the at least one transfection agent is ApoE. In some embodiments, the at least one transfection agent is CpG. In certain embodiments, the at least one transfection agent increases the delivery mRNA to the cells in the lymph node. In some embodiments, the at least one transfection agent increases expression of the construct.

[0025] In certain embodiments, the MP is also loaded with a T-cell activating factor. In some embodiments, the MP is further mixed with a T-cell activating factor in solution. In certain embodiments, the T-cell activating factor is CD3/CD28 or OKT. In some embodiments, the T-cell activating factor is a mitogen or superantigen. In certain embodiments, the mitogen or superantigen is phorbol 12-myristate 13-acetate (PMA), ionomycin, and/or PHA (Phytohaemagglutinin P).

[0026] In some embodiments, the MP is also loaded with a molecular adjuvant. In certain embodiments, the MP is further mixed with a molecular adjuvant in solution. In some embodiments, the molecular adjuvant is an innate ligand. In certain embodiments, the innate ligand is TLR, RIG, and/or STING. [0027] In some embodiments, the MP is also loaded with at least one lymph node binding moiety. In certain embodiments, the MP is further mixed with at least one lymph node binding moiety in solution. In some embodiments, the at least one lymph node binding moiety binds RANKL, sLeX, PNAd, GL7, CLDN11, EFNB2, ACKR4, TNFRSF9, CCL20, CXCL5, GlyCAM-1, CD34, Ptx3, CD209/Marco, SPNS2, CSF-1, CCL21, CCL19, CCL25, or CCL27.

[0028] In certain embodiments, the nucleic acid encodes a CAR protein with an extracellular antigen-binding domain. In some embodiments, the extracellular antigenbinding domain binds BCMA. In certain embodiments, the extracellular antigen-binding domain binds CD- 19.

[0029] In some embodiments, the nucleic acid is an mRNA.

[0030] In certain embodiments, the MP comprises 250-500 ng of mRNA.

[0031] In certain embodiments, the MP comprise a degradable polymer.

[0032] In certain embodiments, the degradable polymer comprises one or more of ester bonds, amide bonds, glycosidic bonds, phosphodiester bonds, ether bonds, disulfide bonds, peptide bonds, urethane bonds, carbonate bonds, thioester bonds, and orthoester bonds. [0033] In some embodiments, the degradable polymer is poly(lactide-co-glycolide) (PLGA).

[0034] In certain embodiments, the concentration of MP in the lymph node is greater than the concentration in any other tissue of the subject. In some embodiments, the concentration of MP in the lymph node is greater than the systemic biodistribution in the subject.

[0035] In certain embodiments, the microparticle is 1-100 microns in size. In some embodiments, the microparticle is 2-8 microns in size.

[0036] In another aspect of the present disclosure, provided is a microparticle composition comprising a plurality of microparticles described herein. [0037] In one aspect, provided is a pharmaceutical composition comprising a plurality of the NPs disclosed herein, the nanoparticle composition of the present disclosure, a plurality of microparticles described herein, or the microparticle composition of the present disclosure, and a pharmaceutically acceptable excipient.

[0038] In another aspect, the present disclosure provides a method of transfecting cells with a nucleic acid, the method comprising contacting the cells with a plurality of the NPs of any the present disclosure, the nanoparticle composition defined herein, a plurality of microparticles of the present disclosure, the microparticle composition claim described herein, or the disclosed pharmaceutical composition.

[0039] In some embodiments, the contacting is conducted in vivo. In certain embodiments, the cells are located in a lymph node of a subject. In some embodiments, the cell is transiently located in the lymph node of a subject. In certain embodiments, the cells are immune cells. In some embodiments, the cells are lymphocytes. In certain embodiments, the cells are T-cells. In some embodiments, the T-cells are CD8+ T cells. In certain embodiments, the T-cells are CD4+ T cells.

[0040] In some embodiments, the plurality of nanoparticles, the nanoparticle composition, the plurality of microparticles, the microparticle composition, or pharmaceutical composition is administered into the lymph node of a subject.

[0041] In certain embodiments, the plurality of nanoparticles, the nanoparticle composition, the plurality of microparticles, the microparticle composition, or pharmaceutical composition is administered into the lymph node of a subject by injection.

[0042] In one aspect, provide by the present disclosure is a method of treating disease in a subject in need thereof, the method comprising administering to the subject a plurality of the NPs described herein, the nanoparticle composition defined herein, a plurality of microparticles described herein, the microparticle composition of the present disclosure, or the provided pharmaceutical composition. [0043] In certain embodiments, the disease is a proliferative disease, an autoimmune disease, or an inflammatory disease. In some embodiments, the proliferative disease is cancer. In certain embodiments, the cancer is multiple myeloma. In some embodiments, the autoimmune disease is myasthenia gravis.

[0044] In certain embodiments, the plurality of nanoparticles, the nanoparticle composition, the plurality of microparticles, the microparticle composition, or pharmaceutical composition is administered by injection.

[0045] In some embodiments, the plurality of nanoparticles, the nanoparticle composition, the plurality of microparticles, the microparticle composition, or pharmaceutical composition is administered directly into a lymph node.

[0046] In certain embodiments, the plurality of nanoparticles, the nanoparticle composition, the plurality of microparticles, the microparticle composition, or pharmaceutical composition is administered directly into at least two lymph nodes.

[0047] In some embodiments, the method is characterized by an increase in cytokine production in the cells transfected with the nucleic acid. In one aspect of the disclosure, the methods of the disclosure are characterized by an increase or modulation in cytokine production in the cells transfected with the nucleic acid. The cytokines that are increased can included one or more of the following: interferon gamma (IFNy), APRIL, BAFF, CD40 Ligand, Fit- 3 Ligand, G-CSF, GM-CSF, Granzyme B, GRO alpha, GRO beta, IFN-a2, IFN-P, IFN-Y, IL- la, IL-ip, IL- Ira, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9„ IL- 10, IL- 12 p70, IL-13, IL-15 IL-17A, IL-17E, IL-21, IL-23, IL-24, IL-27, IL-28, IL-29, IL-33, IP-10, MCP-1, MIP-1 alpha, MIP-1 beta, MIP-3 alpha, MIP-3 beta, RANTES, TGF-a, TNF-a, TNF-P, and TRAIL. However, this list is not meant to be exhaustive. The one or more cytokines that would be increased or modulated could depend on the formulation of the NPs and MPs of the present disclosure. In certain embodiments, the cytokine is interferon-gamma. [0048] In another aspect, the present disclosure provides a method of inducing cytokine expression in the cells of a subject, the method comprising administering to the lymph nodes of a subject a plurality of the NPs defined herein, the nanoparticle composition described herein, a plurality of microparticles described herein, the microparticle composition described herein, or the pharmaceutical composition described herein.

[0049] In certain embodiments, the plurality of the LNPs, the nanoparticle composition, the plurality of microparticle, the microparticle composition, or the pharmaceutical composition, is administered to a lymph node in the subject.

[0050] In certain embodiments, the cytokine is interferon-gamma.

[0051] In one aspect, provided herein is the use of the plurality of the NPs described herein, the nanoparticle composition described herein, the plurality of microparticles described herein, the microparticle composition described herein, or the pharmaceutical composition of claim described herein for transfecting cells in a lymph node with a nucleic acid.

[0052] In one aspect, provided herein is the use of the plurality of the NPs described herein, the nanoparticle composition described herein, the plurality of described herein, the microparticle composition described herein, or the pharmaceutical composition described herein for treating a disease in a subject.

[0053] In another aspect, provided herein is the use of the LNPs of the plurality of the NPs described herein, the nanoparticle composition described herein, the plurality of microparticles described herein, the microparticle composition described herein, or the pharmaceutical composition described herein for inducing cytokine expression.

[0054] In one aspect, the present disclosure provides a medicament for administration into a lymph node, the medicament comprising the plurality of the NPs describe herein, the nanoparticle composition described herein, the plurality of microparticles described herein, the microparticle composition described herein, or the pharmaceutical composition described herein. [0055] In another aspect of the present disclosure, provided is a kit comprising the plurality of the NPs described herein, the nanoparticle composition described herein, the plurality of microparticles described herein, the microparticle composition described herein, or the pharmaceutical composition described herein.

Brief Description of the Drawings

[0056] FIG. 1A shows fluorescence imaging of C57BL/6 mice 0, 1, and 2 days after intralymph node injection of LNPs containing IVT mRNA encoding firefly luciferase and anti- BCMA CAR and formulated with apolipoprotein E (ApoE) and activating factors.

[0057] FIG. IB shows quantification of fluorescence intensity (FLI) as a measure of total flux of C57BL/6 mice 0, 1, and 2 days after intra-lymph node injection of LNPs containing IVT mRNA encoding firefly luciferase and anti-BCMA CAR and formulated with ApoE and activating factors.

[0058] FIG. 1C shows quantification of bioluminescence (BLI) as a measure of total flux of C57BL/6 mice 0, 1, and 2 days after intra-lymph node injection of LNPs containing IVT mRNA encoding firefly luciferase and anti-BCMA CAR and formulated with ApoE and activating factors.

[0059] FIG. 2 shows a schematic of Firefly Luciferase and Anti-BCMA CAR bicistronic RNA with CAR ORF in the second position (construct 818, top) or first position (construct 811, bottom).

[0060] FIG. 3A shows relative light units (RLU) and mean fluorescence intensity (MFI) of activated CD8+ T cells 24 hours after transfection with 818 RNA (FLuc-anti-BCMA CAR fusion).

[0061] FIG. 3B shows RLU and MFI of activated CD8+ T cells 24 hours after transfection with 811 RNA (anti-BCMA-CAR-Fluc fusion). [0062] FIG. 4A shows bioluminescence images of C57BL/6 mice 0.5, 6, and 24 hours after intra- lymph node injection of LNPs containing 818 or 811 fusion constructs, with ApoE and activating factors.

[0063] FIG. 4B shows the quantification of BLI of C57BL/6 mice 0.5, 6, and 24 hours after intra- lymph node injection of LNPs containing 818 or 811 fusion constructs and formulated with ApoE and activating factors.

[0064] FIG. 5A shows bioluminescence images of C57BL/6 mice 0.5, 6, and 24 hours after intra- lymph node injection of various indicated constructs and particles.

[0065] FIG. 5B shows quantification of BLI of C57BL/6 mice 0.5, 6, and 24 hours after intra- lymph node injection of various indicated constructs and particles.

[0066] FIG. 6A shows fluorescence imaging of C57BL/6 mice before dosing, 30 minutes, 24 hours, and 48 hours after intra-lymph node injection of fluorescently labeled microparticles (MPs) containing IVT mRNA encoding anti-BCMA CAR.

[0067] FIG. 6B shows quantification of fluorescence intensity (FLI) as a measure of total flux of C57BL/6 mice 0, 0.5, 24, and 48 hours after intra-lymph node injection of fluorescently labeled MPs containing IVT mRNA encoding anti-BCMA CAR and formulated with ApoE and activating factors.

[0068] FIG. 7A shows a schematic diagram of IVT mRNA encoding anti-BCMA CAR and formulated with ApoE and activating factors.

[0069] FIG. 7B shows a schematic diagram of IVT mRNA encoding firefly luciferase.

[0070] FIG. 8 shows flow cytometric plots of CAR expression on lymphocytes isolated from mice administered with LNPs encoding anti-BCMA CAR and formulated with ApoE and activating factors. Definitions

[0071] As used herein and in the claims, the singular forms “a,” “an,” and “the” include the singular and the plural reference unless the context clearly indicates otherwise. Thus, for example, a reference to “an agent” includes a single agent and a plurality of such agents.

[0072] By the term “AChr” is meant “acetylcholine receptor.”

[0073] By the term “APC” is meant “antigen presenting cells.”

[0074] By the term “ApoE” is meant “Apolipoprotein E.”

[0075] By the term “BCMA” is meant “B cell maturation antigen.”

[0076] By the term “CAR” is meant “chimeric antigen receptor.”

[0077] By the term “CDN” is meant “cyclic dinucleotides.”

[0078] By the term “DCM” is meant “dichloromethane.”

[0079] By the term “DSPE-PEG” is meant “l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[amino(p(ethyleneglycol)-2k.”

[0080] By the term “EP” is meant “electroporation.”

[0081] By the term “IVIS” is meant “in vivo imaging system.”

[0082] By the term “LEC” is meant “lymphatic endothelial cells.”

[0083] By the term “LN” is meant “lymph node” (LN).”

[0084] By the term “MG” is meant “myasthenia gravis.”

[0085] By the term “MM” is meant “multiple myeloma.”

[0086] By the term “MOG” is meant “myelin oligodendrocyte glycoprotein.”

[0087] As used herein, cytokines are signaling molecules secreted by immune cells in response to stimuli. Exemplary cytokines include, but are not limited to: interferon gamma (IFNy), APRIL, BAFF, CD40 Ligand, Flt-3 Ligand, G-CSF, GM-CSF, Granzyme B, GRO alpha, GRO beta, IFN-a2, IFN-P, IFN-Y, IL- la, IL-ip, IL- Ira, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9„ IL-10, IL-12 p70, IL-13, IL-15 IL-17A, IL-17E, IL-21, IL-23, IL-24, IL-27, IL-28, IL-29, IL-33, IP- 10, MCP-1, MIP-1 alpha, MIP-1 beta, MIP-3 alpha, MIP-3 beta,

RANTES, TGE-a, TNE-a, TNE-p, and TRAIL.

[0088] As used herein, particles include polymeric particles, single-emulsion particles, double-emulsion particles, coacervates, liposomes, microparticles, nanoparticles, macroscopic particles, pellets, crystals, aggregates, composites, pulverized, milled or otherwise disrupted matrices, and cross-linked protein or polysaccharide particles, each of which have an average characteristic dimension of about less than about 1 mm and at least 1 nm, where the characteristic dimension, or “critical dimension,” of the particle is the smallest cross-sectional dimension of the particle. A particle may be composed of a single substance or multiple substances. In certain embodiments, the particle is not a viral particle. In other embodiments, the particle is not a liposome. In certain embodiments, the particle is not a micelle. In certain embodiments, the particle is substantially solid throughout. In certain embodiments, the particle is a nanoparticle. In certain embodiments, the particle is a lipid nanoparticle. In certain embodiments, the particle is a microparticle.

[0089] By the term “NP” is meant “nanoparticle.” As used herein, nanoparticles refer to a particles having an average (e.g., mean) dimension (e.g., diameter) of between about 1 nanometer (nm) and about 1 micrometer (pm) (e.g., between about 1 nm and about 300 nm, between about 1 nm and about 100 nm, between about 1 nm and about 30 nm, between about 1 nm and about 10 nm, or between about 1 nm and about 3 nm), inclusive. Nanoparticles described herein can be constructed using a NanoAssemblr Spark Instrument (# NIS0001) and lipids and buffers from the Precision Nanosystems (GenVoy-ILM T Cell Kit). However, nanoparticles can be constructed using any means that result in particles of the correct dimensions. Exemplary nanoparticles include but are not limited to lipid nanoparticles, liposomes, polymer nanoparticles, dendrimer nanoparticles, polymersomes, polymeric micelles, micelles, mesoporous or silica nanoparticles among other degradable nanoparticles. [0090] By the term “LNP” is meant “lipid nanoparticle.” The term “lipid nanoparticle” refers to a lipid composition having a typically spherical structure with an average diameter between 10 and 1000 nanometers. In some formulations, lipid nanoparticles comprise ionizable and/or cationic lipids, helper lipids, sterols, and stabilization lipids.

Lipid nanoparticles known in the art that are suitable for encapsulating nucleic acids, such as mRNA, are contemplated for use in the present disclosure. In certain embodiments, lipid nanoparticles can be assembled from lipid mixtures using microfluidic mixing as known to those skilled in the art.

[0091] By the term “MP” is meant “microparticle.” The term “microparticle” refers to a particle having an average (e.g., mean) dimension (e.g., diameter) of between about 1 micrometer (pm) and about 1000 micrometers (pm) (e.g., between about 1 pm and about 300 pm, between about 1 pm and about 100 pm, between about 1 pm and about 30 pm, between about 1 pm and about 10 pm, or between about 1 pm and about 3 pm), inclusive. Microparticles described herein can be constructed using a double emulsion solvent evaporation process with degradable poly(lactide-co-glycolide) (PLGA) as a degradable polymer carrier in the organic phase, mRNA in the aqueous phase, and poly(vinyl alcohol) in the secondary aqueous continuous phase. A lipophilic dye (DiD) can be used to label hydrophobic structures during synthesis to fluorescently label the MP depots. Additional degradable polymers in the MP may comprise ester bonds, amide bonds, glycosidic bonds, phosphodiester bonds, ether bonds, disulfide bonds, urethane bonds, carbonate bonds, thioester bonds, or orthoester bonds. However, microparticles can be constructed using any means that result in particle of the correct dimensions. Microparticles can, but need not, contain LNPs.

[0092] By the term “ODN” is meant “oligonucleotide.”

[0093] By the term “PLGA” is meant “polylactic-co-glycolic acid.” [0094] By the term “PBAE” is meant “poly(beta-amino ester).” [0095] By the term “PI” is meant “propidium iodide.”

[0096] By the term “TLR” is meant “toll-like receptor.”

[0097] A “protein,” “peptide,” or “polypeptide” comprises a polymer of amino acid residues linked together by peptide bonds. The term refers to proteins, polypeptides, and peptides of any size, structure, or function. Typically, a protein will be at least three amino acids long. A protein may refer to an individual protein or a collection of proteins. Inventive proteins preferably contain only natural amino acids, although non-natural amino acids (z.e., compounds that do not occur in nature but that can be incorporated into a polypeptide chain) and/or amino acid analogs as are known in the art may alternatively be employed. Also, one or more of the amino acids in a protein may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation or functionalization, or other modification. A protein may also be a single molecule or may be a multi-molecular complex. A protein may be a fragment of a naturally occurring protein or peptide. A protein may be naturally occurring, recombinant, synthetic, or any combination of these.

[0098] As used herein, the term “activating factor" refers to a composition which induces activation of immune cells. Exemplary activating factors include but are not limited to anti- CD3/CD28, OKT, or superagonists and mitogens (e.g., phorbol 12-myristate 13-acetate (PMA), ionomycin, phytohaemagglutinin P (PHA)).

[0099] An antibody (interchangeably used in plural form) is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. As used herein, the term “antibody” encompasses not only intact (e.g., full-length) polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (such as Fab, Fab', F(ab')2, Fv), single chain (scFv), mutants thereof, fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies, nanobodies, linear antibodies, single chain antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. The term “antibody” refers to any immunoglobulin (Ig) molecule comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art. Non-limiting embodiments thereof are discussed below.

[00100] In a full-length antibody, each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (ER). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG 1, IgG2, IgG 3, IgG4, IgAl and IgA2) or subclass.

[00101] The term “antigen-binding portion” of an antibody (or simply “antibody portion”), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., BCMA). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Such antibody embodiments may also be bispecific, dual specific, or multi-specific formats; specifically binding to two or more different antigens. Multispecific, dual specific, and bispecific antibody constructs are well known in the art and described and characterized in Kontermann (ed.), Bispecific Antibodies, Springer, NY (2011), and Spiess et al., Mol. Immunol. 67(2):96-106 (2015).

[00102] Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546, Winter et al., PCT publication WO 90/05144 Al herein incorporated by reference), which comprises a single variable domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VE and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VE and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term “antigenbinding portion” of an antibody. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VE domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123). Such antibody binding portions are known in the art (Kontermann and Dubel eds., Antibody Engineering (2001) Springer- Verlag. New York. 790 pp. (ISBN 3-540-41354-5). Exemplary antigen-binding domains may target CD19, BCMA, EGFR/HER, CD22, mesothelin, CD 123, CD20, PD1, CD30, BAFF-R, CD20, CD22, CD30, CD37, CD38, CD70, CD79B, CD123, CD138, GPRC5D, LMP1, ROR1, SLAMF7, CD32B, CD70, CD72, CD133, FcpR, Siglec-6, or TSLPR, among others.

[00103] The term “synthetic antibody” as used herein, refers an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a viral vector. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.

[00104] In some embodiments, the term “antigen” or “Ag” as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from RNA or recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein.

Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present disclosure includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.

[00105] The term “tumor antigen” as used herein refers to a molecule (typically a protein, carbohydrate or lipid) that is expressed on the surface of a cancer cell, either entirely or as a fragment (e.g., MHC/peptide), and which is useful for the preferential targeting of a pharmacological agent to the cancer cell. In some embodiments, a tumor antigen is a marker expressed by both normal cells and cancer cells, e.g., a lineage marker, e.g., CD19 on B cells. In some embodiments, a tumor antigen is a cell surface molecule that is overexpressed in a cancer cell in comparison to a normal cell. In some embodiments, a tumor antigen is a cell surface molecule that is inappropriately synthesized in the cancer cell, for instance, a molecule that contains deletions, additions or mutations in comparison to the molecule expressed on a normal cell. In some embodiments, a tumor antigen will be expressed exclusively on the cell surface of a cancer cell, entirely or as a fragment (e.g., MHC/peptide), and not synthesized or expressed on the surface of a normal cell. Examples of tumor antigens include but are not limited to BCMA, CD19, EGFR/HER, CD22, mesothelin, CD123, CD20, PD1, and CD30

[00106] The term “anti-tumor effect” as used herein, refers to a biological effect which can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or amelioration of various physiological symptoms associated with the cancerous condition. An “anti-tumor effect” can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the present disclosure in prevention of the occurrence of tumor in the first place. [00107] An “autoimmune disease” refers to a disease arising from an inappropriate immune response of the body of a subject against substances and tissues normally present in the body. In other words, the immune system mistakes some part of the body as a pathogen and attacks its own cells. An autoimmune disease can be mediated by an autoantibody, i.e., an antibody produced by an individual that recognizes an antigen of that individual’s own cells or tissue(s). This may be restricted to certain organs (e.g., in autoimmune thyroiditis) or involve a particular tissue in different places (e.g., Goodpasture’s disease which may affect the basement membrane in both the lung and kidney). The treatment of autoimmune diseases is typically with immunosuppression, e.g., medications which decrease the immune response. Exemplary autoimmune diseases include, but are not limited to, myasthenia gravis, systemic lupus erythematosus (SLE), rheumatoid arthritis, blistering skin diseases, e.g., pemphigus, psoriasis, inflammatory bowel disease, celiac sprue, pernicious anemia, idiopathic thrombocytopenia purpura, sceleroderma, Graves disease, Sjogren syndrome, Goodpasture syndrome, multiple sclerosis, type 1 diabetes, glomerulonephritis, necrotizing vasculitis, lymphadenitis, peri-arteritis nodosa, systemic lupus erythematosis, psoriatic arthritis, ulcerative colitis, systemic sclerosis, dermatomyositis/polymyositis, anti-phospholipid antibody syndrome, pemphigus vulgaris, ANCA-associated vasculitis (e.g., Wegener’s granulomatosis, microscopic polyangiitis), uveitis, Sjogren’s syndrome, Crohn’s disease, Reiter’s syndrome, ankylosing spondylitis, Lyme disease, Guillain-Barre syndrome, Hashimoto’s thyroiditis, cardiomyopathy, idiopathic inflammatory myositis, neuromyelitis optica, chronic inflammatory demyelinating polyneuropathy (CIDP), inclusion body myositis, autoimmune encephalitis, Lambert-Eaton syndrome, stiff person syndrome, Opsoclonus-myoclonus syndrome, antiphospholipid syndrome, immune thrombocytopenia (ITP), pure red cell aplasia, autoimmune hemolytic anemia, Cold agglutinin disease, Evans syndrome, autoimmune neutropeni, IgA nephropathy, lupus nephritis, membranous nephropathy, ANCA-associated vasculitis, Juvenile SLE, IgA Vasculitis, autoimmune thyroiditis, juvenile dermatomyositis, juvenile idiopathic arthritis, scleroderma, bullous pemphigoid, pemphigus vulgaris, discoid lupus erythematosus, alopecia areata, vitiligo, systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, ankylosing spondylitis, mixed connective tissue disease, polymyalgia rheumatica, Sjogren syndrome, thyroid eye disease, ocular cicatricial pemphigoid, autoimmune retinitis, autoimmune uveitis, Churg-Strauss syndrome, egener’s granulomatosis, sarcoidosis, interstitial lung disease, autoimmune hepatitis, autoimmune pancreatitis, Crohn’s disease, ulcerative colitis, celiac disease, eosinophilic gastroenteritis, microscopic colitis, primary biliary cholangitis, and primary sclerosing cholangitis.

[00108] Immune disorders, such as auto-immune disorders, include, but are not limited to, arthritis (including rheumatoid arthritis, spondyloarthopathies, gouty arthritis, degenerative joint diseases such as osteoarthritis, systemic lupus erythematosus, Sjogren’s syndrome, ankylosing spondylitis, undifferentiated spondylitis, Behcet’s disease, haemolytic autoimmune anaemias, multiple sclerosis, amyotrophic lateral sclerosis, amylosis, acute painful shoulder, psoriatic, and juvenile arthritis), asthma, atherosclerosis, osteoporosis, bronchitis, tendonitis, bursitis, skin condition (e.g., psoriasis, eczema, bums, dermatitis, pruritus (itch)), enuresis, eosinophilic disease, gastrointestinal disorder (e.g. , selected from peptic ulcers, regional enteritis, diverticulitis, gastrointestinal bleeding, eosinophilic gastrointestinal disorders (e.g., eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic colitis), gastritis, diarrhea, gastroesophageal reflux disease (GORD, or its synonym GERD), inflammatory bowel disease (IBD) (e.g., Crohn’s disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischaemic colitis, diversion colitis, Behcet’s syndrome, indeterminate colitis) and inflammatory bowel syndrome (IBS)), and disorders ameliorated by a gastroprokinetic agent (e.g., ileus, postoperative ileus and ileus during sepsis; gastroesophageal reflux disease (GORD, or its synonym GERD); eosinophilic esophagitis, gastroparesis such as diabetic gastroparesis; food intolerances and food allergies and other functional bowel disorders, such as non-ulcerative dyspepsia (NUD) and noncardiac chest pain (NCCP, including costo-chondritis)).

[00109] In certain embodiments, the inflammatory disorder and/or the immune disorder is a gastrointestinal disorder. In some embodiments, the gastrointestinal disorder is selected from gastrointestinal disorder (e.g., selected from peptic ulcers, regional enteritis, diverticulitis, gastrointestinal bleeding, eosinophilic gastrointestinal disorders (e.g., eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic colitis), gastritis, diarrhea, gastroesophageal reflux disease (GORD, or its synonym GERD), inflammatory bowel disease (IBD) (e.g., Crohn’s disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischaemic colitis, diversion colitis, Behcet’s syndrome, indeterminate colitis) and inflammatory bowel syndrome (IBS)). In certain embodiments, the gastrointestinal disorder is inflammatory bowel disease (IBD).

[00110] In certain embodiments, the inflammatory condition and/or immune disorder is a skin condition. In some embodiments, the skin condition is pruritus (itch), psoriasis, eczema, bums or dermatitis. In certain embodiments, the skin condition is psoriasis. In certain embodiments, the skin condition is pruritis.

[00111] The term “autologous” as used herein, is meant to refer to any material derived from the same individual to which it is later to be re-introduced into the individual.

[00112] The term “allogeneic” as used herein refers to a graft derived from a different animal of the same species. “Xenogeneic” refers to a graft derived from an animal of a different species.

[00113] A “proliferative disease” refers to a disease that occurs due to abnormal growth or extension by the multiplication of cells (Walker, Cambridge Dictionary of Biology, Cambridge University Press: Cambridge, UK, 1990). A proliferative disease may be associated with: 1) the pathological proliferation of normally quiescent cells; 2) the pathological migration of cells from their normal location (e.g., metastasis of neoplastic cells); 3) the pathological expression of proteolytic enzymes such as the matrix metalloproteinases (e.g., collagenases, gelatinases, and elastases); or 4) the pathological angiogenesis as in proliferative retinopathy and tumor metastasis. Exemplary proliferative diseases include cancers (z.e., “malignant neoplasms”), benign neoplasms, angiogenesis, inflammatory diseases, and autoimmune diseases.

[00114] The term “angiogenesis” refers to the physiological process through which new blood vessels form from pre-existing vessels. Angiogenesis is distinct from vasculogenesis, which is the de novo formation of endothelial cells from mesoderm cell precursors. The first vessels in a developing embryo form through vasculogenesis, after which angiogenesis is responsible for most blood vessel growth during normal or abnormal development. Angiogenesis is a vital process in growth and development, as well as in wound healing and in the formation of granulation tissue. However, angiogenesis is also a fundamental step in the transition of tumors from a benign state to a malignant one, leading to the use of angiogenesis inhibitors in the treatment of cancer. Angiogenesis may be chemically stimulated by angiogenic proteins, such as growth factors (e.g., VEGF). “Pathological angiogenesis” refers to abnormal (e.g., excessive or insufficient) angiogenesis that amounts to and/or is associated with a disease.

[00115] The terms “neoplasm” and “tumor” are used herein interchangeably and refer to an abnormal mass of tissue wherein the growth of the mass surpasses and is not coordinated with the growth of a normal tissue. A neoplasm or tumor may be “benign” or “malignant,” depending on the following characteristics: degree of cellular differentiation (including morphology and functionality), rate of growth, local invasion, and metastasis. A “benign neoplasm” is generally well differentiated, has characteristically slower growth than a malignant neoplasm, and remains localized to the site of origin. In addition, a benign neoplasm does not have the capacity to infiltrate, invade, or metastasize to distant sites. Exemplary benign neoplasms include, but are not limited to, lipoma, chondroma, adenomas, acrochordon, senile angiomas, seborrheic keratoses, lentigos, and sebaceous hyperplasias. In some cases, certain “benign” tumors may later give rise to malignant neoplasms, which may result from additional genetic changes in a subpopulation of the tumor’s neoplastic cells, and these tumors are referred to as “pre-malignant neoplasms.” An exemplary pre-malignant neoplasm is a teratoma. In contrast, a “malignant neoplasm” is generally poorly differentiated (anaplasia) and has characteristically rapid growth accompanied by progressive infiltration, invasion, and destruction of the surrounding tissue. Furthermore, a malignant neoplasm generally has the capacity to metastasize to distant sites. The term “metastasis,” “metastatic,” or “metastasize” refers to the spread or migration of cancerous cells from a primary or original tumor to another organ or tissue and is typically identifiable by the presence of a “secondary tumor” or “secondary cell mass” of the tissue type of the primary or original tumor and not of that of the organ or tissue in which the secondary (metastatic) tumor is located. For example, a prostate cancer that has migrated to bone is said to be metastasized prostate cancer and includes cancerous prostate cancer cells growing in bone tissue.

[00116] The term “cancer” refers to a class of diseases characterized by the development of abnormal cells that proliferate uncontrollably and have the ability to infiltrate and destroy normal body tissues. See e.g., Stedman’s Medical Dictionary, 25th ed.; Hensyl ed.; Williams & Wilkins: Philadelphia, 1990. Exemplary cancers include, but are not limited to, acoustic neuroma; adenocarcinoma; adrenal gland cancer; anal cancer; angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma); appendix cancer; benign monoclonal gammopathy; biliary cancer (e.g., cholangiocarcinoma); bladder cancer; breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast); brain cancer (e.g., meningioma, glioblastomas, glioma (e.g., astrocytoma, oligodendroglioma), medulloblastoma); bronchus cancer; carcinoid tumor; cervical cancer (e.g., cervical adenocarcinoma); choriocarcinoma; chordoma; craniopharyngioma; colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma); connective tissue cancer; epithelial carcinoma; ependymoma; endotheliosarcoma (e.g., Kaposi’s sarcoma, multiple idiopathic hemorrhagic sarcoma); endometrial cancer (e.g., uterine cancer, uterine sarcoma); esophageal cancer (e.g., adenocarcinoma of the esophagus, Barrett’s adenocarcinoma); Ewing’s sarcoma; ocular cancer (e.g., intraocular melanoma, retinoblastoma); familiar hypereosinophilia; gall bladder cancer; gastric cancer (e.g., stomach adenocarcinoma); gastrointestinal stromal tumor (GIST); germ cell cancer; head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma), throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)); hematopoietic cancers (e.g., leukemia such as acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chronic lymphocytic leukemia (CLL) (e.g., B- cell CLL, T-cell CLL)); lymphoma such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma), follicular lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas (e.g., mucosa-associated lymphoid tissue (MALT) lymphomas, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (z.e., Waldenstrom’s macroglobulinemia), hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B -lymphoblastic lymphoma and primary central nervous system (CNS) lymphoma; and T-cell NHL such as precursor T-lymphoblastic lymphoma/leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g., mycosis fungoides, Sezary syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, and anaplastic large cell lymphoma); a mixture of one or more leukemia/lymphoma as described above; and multiple myeloma (MM)), heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease); hemangioblastoma; hypopharynx cancer; inflammatory myofibroblastic tumors; immunocytic amyloidosis; kidney cancer (e.g., nephroblastoma a.k.a. Wilms’ tumor, renal cell carcinoma); liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma); lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung); leiomyosarcoma (LMS); mastocytosis (e.g., systemic mastocytosis); muscle cancer; myelodysplastic syndrome (MDS); mesothelioma; myeloproliferative disorder (MPD) (e.g., polycythemia vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)); neuroblastoma; neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis); neuroendocrine cancer (e.g., gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoid tumor); osteosarcoma (e.g., bone cancer); ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma); papillary adenocarcinoma; pancreatic cancer (e.g., pancreatic andenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors); penile cancer (e.g., Paget’s disease of the penis and scrotum); pinealoma; primitive neuroectodermal tumor (PNT); plasma cell neoplasia; paraneoplastic syndromes; intraepithelial neoplasms; prostate cancer (e.g., prostate adenocarcinoma); rectal cancer; rhabdomyosarcoma; salivary gland cancer; skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)); small bowel cancer (e.g., appendix cancer); soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma); sebaceous gland carcinoma; small intestine cancer; sweat gland carcinoma; synovioma; testicular cancer (e.g., seminoma, testicular embryonal carcinoma); thyroid cancer (e.g., papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer); urethral cancer; vaginal cancer; and vulvar cancer (e.g., Paget’s disease of the vulva). [00117] Exemplary hematological cancers include but are not limited to monoclonal B-cell lymphocytosis, chronic lymphocytic leukaemia/small lymphocytic lymphoma, hairy cell leukemia, splenic marginal zone lymphoma, splenic diffuse red pulp small B-cell lymphoma, splenic B-cell lymphoma/leukaemia with prominent nucleoli, lymphoplasmacytic lymphoma, marginal zone lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, NOS T-cell/histiocyte-rich large B-cell lymphoma, diffuse large B-cell lymphoma/ high grade B-cell lymphoma with MYC and BCL2 rearrangements, ALK- positive large B-cell lymphoma, large B-cell lymphoma with IRF4 rearrangement, high-grade B-cell lymphoma with l lq aberrations, lymphomatoid granulomatosis, EBV-positive diffuse large B-cell lymphoma, diffuse large B-cell lymphoma associated with chronic inflammation, fibrin-associated large B-cell lymphoma, fluid overload-associated large B-cell lymphoma, plasmablastic lymphoma, primary large B-cell lymphoma of immune-privileged sites, primary cutaneous diffuse large B-cell lymphoma leg type, intravascular large B-cell lymphoma, primary mediastinal large B-cell lymphoma, mediastinal grey zone lymphoma, high-grade B-cell lymphoma NOS, Burkitt lymphoma, primary effusion lymphoma, KSHV/HHV8-positive diffuse large B-cell lymphoma, KSHV/HHV8-positive germinotropic lymphoproliferative disorder, Hodgkin lymphoma, cold agglutinin disease, IgM monoclonal gammopathy of undetermined significance, Non-IgM monoclonal gammopathy of undetermined significance, monoclonal gammopathy of renal significance, immunoglobulin- related (AL) amyloidosis, monoclonal immunoglobulin deposition disease, heavy chain diseases, mu heavy chain disease, gamma heavy chain disease, alpha heavy chain disease, plasma cell neoplasms, plasmacytoma, plasma cell myeloma, and multiple myeloma.

[00118] Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. In some embodiments, the cancer is a cancer that expresses BCMA.

Exemplary cancers that express BCMA include multiple myeloma, Hodgkin lymphoma, non¬

Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), and glioblastoma. In some embodiments, cancer refers to multiple myeloma. Multiple myeloma is a cancer of plasma cells. Multiple myeloma can be diagnosed with blood tests (serum protein electrophoresis, serum free kappa/lambda light chain assay), bone marrow examination, urine protein electrophoresis, and/or X-rays of commonly involved bones. In some embodiments, cancer refers to Hodgkin’s lymphoma (HL). HL is a cancer of B cells.

[00119] An “effective amount” of a nanoparticle and/or microparticle described herein refers to an amount sufficient to elicit the desired biological response, for example, an effective amount to transfect cells in the lymph node in situ with mRNA within the nanoparticle and/or microparticle. An effective amount of a nanoparticle and/or microparticle described herein may vary depending on such factors as the desired biological endpoint, severity of side effects, disease, or disorder, the identity, pharmacokinetics, and pharmacodynamics of the particular nanoparticle and/or microparticle, the condition being treated, the mode, route, and desired or required frequency of administration, the species, age and health or general condition of the subject. In certain embodiments, an effective amount is a therapeutically effective amount. In certain embodiments, an effective amount is a prophylactic treatment. In certain embodiments, an effective amount is the amount of a nanoparticle and/or microparticle described herein in a single injection or dose. In certain embodiments, an effective amount is the combined amounts of a nanoparticle and/or microparticle described herein in multiple injections or doses. In certain embodiments, the desired dosage is delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage is delivered using multiple injections or administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more injections).

[00120] It will be appreciated that dose ranges of the injections as described herein provide guidance for the administration of provided nanoparticle and/or microparticle to a subject, such as an adult. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.

[00121] As used herein, the term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue or system.

[00122] “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide. [00123] The term “immunoglobulin” or “Ig,” as used herein is as a class of proteins, which function as antibodies, and the term has it usual meaning in the art.

[00124] ‘ ‘Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[00125] The terms “nucleic acid” or “nucleic acid sequence”, “nucleic acid molecule”, “nucleic acid fragment” or “polynucleotide” may be used interchangeably. The terms “polynucleotide”, “nucleotide sequence”, “nucleic acid”, “nucleic acid molecule”, “nucleic acid sequence”, and “oligonucleotide” refer to a series of nucleotide bases (also called “nucleotides”) in DNA and RNA and mean any chain of two or more nucleotides. The polynucleotides can be chimeric mixtures or derivatives or modified versions thereof, singlestranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, its hybridization parameters, etc. A nucleotide sequence typically carries genetic information, including the information used by cellular machinery to make proteins and enzymes. These terms include double- or single-stranded genomic and cDNA, RNA, any synthetic and genetically manipulated polynucleotide, and both sense and antisense polynucleotides. This includes single- and double- stranded molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA hybrids, as well as “protein nucleic acids” (PNAs) formed by conjugating bases to an amino acid backbone. This also includes nucleic acids containing carbohydrate or lipids. Exemplary DNAs include single-stranded DNA (ssDNA), double- stranded DNA (dsDNA), plasmid DNA (pDNA), genomic DNA (gDNA), complementary DNA (cDNA), antisense DNA, chloroplast DNA (ctDNA or cpDNA), micro satellite DNA, mitochondrial DNA (mtDNA or mDNA), kinetoplast DNA (kDNA), provirus, lysogen, repetitive DNA, satellite DNA, and viral DNA. Exemplary RNAs include single- stranded RNA (ssRNA), double-stranded RNA (dsRNA), small interfering RNA (siRNA), messenger RNA (mRNA), precursor messenger RNA (pre-mRNA), small hairpin RNA or short hairpin RNA (shRNA), microRNA (miRNA), guide RNA (gRNA), transfer RNA (tRNA), antisense RNA (asRNA), heterogeneous nuclear RNA (hnRNA), coding RNA, non-coding RNA (ncRNA), long non-coding RNA (long ncRNA or IncRNA), satellite RNA, viral satellite RNA, signal recognition particle RNA, small cytoplasmic RNA, small nuclear RNA (snRNA), ribosomal RNA (rRNA), Piwi- interacting RNA (piRNA), polyinosinic acid, ribozyme, flexizyme, small nucleolar RNA (snoRNA), spliced leader RNA, viral RNA, and viral satellite RNA.

[00126] Polynucleotides described herein may be synthesized by standard methods known in the art, e.g., by use of an automated DNA synthesizer (such as those that are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein et al., Nucl. Acids Res., 16, 3209,

(1988), methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci. U.S.A. 85, 7448-7451, (1988)). RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences. Such DNA sequences may be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines. Vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells. Expression of the sequence encoding the antisense RNA can be by any promoter known in the art to act in mammalian, preferably human, cells. Such promoters can be inducible or constitutive. Any type of plasmid, cosmid, yeast artificial chromosome, or viral vector can be used to prepare the recombinant DNA construct that can be introduced directly into the tissue site.

[00127] The polynucleotides may be flanked by natural regulatory (expression control) sequences or may be associated with heterologous sequences, including promoters, internal ribosome entry sites (IRES) and other ribosome binding site sequences, enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, introns, 5'- and 3 '-noncoding regions, and the like. The nucleic acids may also be modified by many means known in the art. Non-limiting examples of such modifications include methylation, “caps”, substitution of one or more of the naturally occurring nucleotides with an analog, and intemucleotide modifications, such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.). Polynucleotides may contain one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc.), and alkylators. The polynucleotides may be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage. Furthermore, the polynucleotides herein may also be modified with a label capable of providing a detectable signal, either directly or indirectly. Exemplary labels include radioisotopes, fluorescent molecules, isotopes (e.g., radioactive isotopes), biotin, and the like.

[00128] The term “mRNA” or “mRNA molecule” refers to messenger RNA, or the RNA that serves as a template for protein synthesis in a cell. The sequence of a strand of mRNA is based on the sequence of a complementary strand of DNA comprising a sequence coding for the protein to be synthesized.

[00129] Unless otherwise specified, a “nucleotide sequence or nucleic acid encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some versions contain an intron(s).

[00130] By the term “modulate” or “modulating,” as used herein, is meant mediating a detectable increase or decrease in the level of a response compared with the level of a response in the absence of a treatment, and/or compared with the level of a response in an otherwise identical situation. The term encompasses perturbing and/or affecting a native signal or response thereby mediating a beneficial effect.

[00131] The terms “patient,” “subject,” and “individual” are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In some embodiments, the patient, subject or individual is a human. Other examples include dogs, cats, mice, rats, and transgenic species thereof. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a rodent. In some embodiments, the subject is a sheep, a goat, a cattle, a cat, or a dog. In some embodiments, the subject is a vertebrate, an amphibian, a reptile, a fish, an insect, a fly, or a nematode. In some embodiments, the subject is a research animal. In some embodiments, the subject is genetically engineered, e.g., a genetically engineered non-human subject. The subject may be of either sex and at any stage of development. In some embodiments, the subject has cancer (e.g., multiple myeloma). In other embodiments, the subject is a healthy volunteer.

[00132] The term “specifically binds” or “specific for”, as used herein with respect to an antigen recognition domain, e.g., an antibody, e.g., an scFv, means a protein or domain thereof that recognizes a specific antigen (or surface marker) but does not substantially recognize or bind other molecules in a sample (or, in certain contexts, in the body of an individual). For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species, but such cross-species reactivity does not itself alter the classification of an antibody as specific. An antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms “specific binding” or “specifically binding,” refers to the interaction of an antibody, a protein (or a domain thereof), or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.

[00133] As used herein, unless otherwise clear from the context, the term “surface marker” means an antigen or other molecular moiety present on the surface of a cell to which a CAR can specifically bind. Examples of useful surface markers BCMA, CD 19, EGFR/HER, CD22, mesothelin, CD 123, CD20, PD1, and CD30. A tumor antigen, which is an antigen specific or relatively specific to a cancerous cell, can serve as surface marker. Many (but not all) surface markers are membrane-bound proteins or domains thereof, which can include glycosylation and other post-translational modifications.

[00134] As used herein, unless otherwise clear from the context, the terms “target” and derivatives such as “target cell surface marker” refer to a surface marker or a cell, tissue, or tumor that is specifically bound by a CAR. In such cases where the target refers to a cell, tissue, or type of tumor, such cell, tissue, or tumor typically expresses (i.e., displays) a surface marker that is specifically bound by a CAR. Thus, as used herein, a “target cell” refers to a cell that is specifically bound by a particular CAR or CAR-expressing cell, e.g., a CAR T cell.

[00135] The term “therapeutic” as used herein means a treatment and/or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state. [00136] The term “therapeutically effective amount” as used herein, refers to the amount of the composition/particle that will elicit the biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms, signs, or causes of the condition, and/or enhances the therapeutic efficacy of another therapeutic agent. A therapeutically effective amount means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the condition. In some embodiments, the therapeutically effective amount provides a therapeutic benefit in the treatment of a condition or to delay or minimize one or more symptoms associated with the condition. In some embodiments, the therapeutically effective amount is sufficient to prevent development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the composition/particle, the disease and its severity and the age, weight, etc., of the subject to be treated. A therapeutically effective amount does not need to be an amount required for clinical efficacy. In certain embodiments, a therapeutically effective amount is an amount sufficient for CAR expression in lymph nodes. In certain embodiments, a therapeutically effective amount is an amount sufficient for treating proliferative disease, autoimmune disease, or inflammatory disease.

[00137] As used herein, the terms “treatment,” “treat,” and “treating” refer to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed and/or after a disease has been diagnosed. In other embodiments, treatment may be administered in the absence of symptoms, e.g., to prevent or delay onset of a symptom or inhibit onset or progression of a disease. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to prevent or delay their recurrence.

[00138] The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[00139] A “transfection agent” increases the delivery mRNA to the target cells and/or increase expression of the construct. Exemplary transfection agents include but are not limited to ApoE or CpG.

[00140] A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.

[00141] A “subject” to which administration is contemplated refers to a human (z.e., male or female of any age group, e.g., pediatric subject (e.g., infant, child, or adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) or non-human animal. In certain embodiments, the non-human animal is a mammal (e.g., primate (e.g., cynomolgus monkey or rhesus monkey), commercially relevant mammal (e.g., cattle, pig, horse, sheep, goat, cat, or dog), or bird (e.g., commercially relevant bird, such as chicken, duck, goose, or turkey)). In certain embodiments, the non-human animal is a fish, reptile, or amphibian. The non-human animal may be a male or female at any stage of development. The non-human animal may be a transgenic animal or genetically engineered animal. The term “patient” refers to a human subject in need of treatment of a disease.

[00142] The term “biological sample” refers to any sample including tissue samples (such as tissue sections and needle biopsies of a tissue); cell samples (e.g., cytological smears (such as Pap or blood smears) or samples of cells obtained by microdissection); samples of whole organisms (such as samples of yeasts or bacteria); or cell fractions, fragments or organelles (such as obtained by lysing cells and separating the components thereof by centrifugation or otherwise). Other examples of biological samples include blood, serum, urine, semen, fecal matter, cerebrospinal fluid, interstitial fluid, mucous, tears, sweat, pus, biopsied tissue (e.g., obtained by a surgical biopsy or needle biopsy), nipple aspirates, milk, vaginal fluid, saliva, swabs (such as buccal swabs), or any material containing biomolecules that is derived from a first biological sample. [00143] The term “target tissue” refers to any biological tissue of a subject (e.g., lymph node) or a part thereof, including blood and/or lymph vessels, which is the object to which a compound, particle, and/or composition of the present disclosure is delivered. A target tissue may be an abnormal or unhealthy tissue, which may need to be treated. A target tissue may also be a normal or healthy tissue that is under a higher-than-normal risk of becoming abnormal or unhealthy, which may need to be prevented. In certain embodiments, the target tissue is the liver. In certain embodiments, the target tissue is the lung. A “non-target tissue” is any biological tissue of a subject (including a group of cells, a body part, or an organ) or a part thereof, including blood and/or lymph vessels, which is not a target tissue.

[00144] The term “administer,” “administering,” or “administration” refers to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing the particle described herein, or a composition thereof, in or on a subject.

[00145] The terms “condition,” “disease,” and “disorder” are used interchangeably.

[00146] An “effective amount” of a composition, particle, or plurality of particles described herein refers to an amount sufficient to elicit the desired biological response. An effective amount of a composition, particle, or plurality of particles described herein may vary depending on such factors as the desired biological endpoint, severity of side effects, disease, or disorder, the identity, pharmacokinetics, and pharmacodynamics of the particular composition, particle, or plurality of particles, the condition being treated, the mode, route, and desired or required frequency of administration, the species, age and health or general condition of the subject. In certain embodiments, an effective amount is a therapeutically effective amount. In certain embodiments, an effective amount is a prophylactic treatment. In certain embodiments, an effective amount is the amount of a composition, particle, or plurality of particles described herein in a single dose. In certain embodiments, an effective amount is the combined amounts of a co composition, particle, or plurality of particles described herein in multiple doses. In certain embodiments, the desired dosage is delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage is delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations).

[00147] It will be appreciated that dose ranges as described herein provide guidance for the administration by injection of provided pharmaceutical compositions to an adult. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.

[00148] The terms “agent”, “therapeutic agent”, or “pharmaceutical agent” are used herein to refer to any substance, compound (e.g., molecule), supramolecular complex, material, or combination or mixture thereof. A compound may be any agent that can be represented by a chemical formula, chemical structure, or sequence. Example of agents, include, e.g., small molecules, polypeptides, nucleic acids (e.g., RNAi agents, antisense oligonucleotide, aptamers), lipids, polysaccharides, etc. In general, agents may be obtained using any suitable method known in the art. The ordinary skilled artisan will select an appropriate method based, e.g., on the nature of the agent. An agent may be at least partly purified. In some embodiments, an agent may be provided as part of a composition, which may contain, e.g., a counter-ion, aqueous or non-aqueous diluent or carrier, buffer, preservative, or other ingredient, in addition to the agent, in various embodiments. In some embodiments, an agent may be provided as a salt, ester, hydrate, or solvate. In some embodiments, an agent is cell- permeable, e.g., within the range of typical agents that are taken up by cells and acts intracellularly, e.g., within mammalian cells, to produce a biological effect.

[00149] A “prophylactically effective amount” of a composition, particle, or plurality of particles described herein is an amount sufficient to prevent a condition, or one or more symptoms associated with the condition or prevent its recurrence. A prophylactically effective amount of a compound means an amount of a therapeutic agent, alone or in combination with other agents, which provides a prophylactic benefit in the prevention of the condition. The term “prophylactically effective amount” can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent.

[00150] The term “prevent,” “preventing,” or “prevention” refers to a prophylactic treatment of a subject who is not and was not with a disease but is at risk of developing the disease or who was with a disease, is not with the disease, but is at risk of regression of the disease. In certain embodiments, the subject is at a higher risk of developing the disease or at a higher risk of regression of the disease than an average healthy member of a population.

Detailed Description of Certain Embodiments

Overall CAR Structure

[00151] The overall design of CAR proteins is known in the art. See, for example, Alnefaie, supra, and U.S. Patent 10,934,337, the entire contents of which are hereby incorporated by reference. In some embodiments, a CAR of the present invention comprises an extracellular antigen-binding domain specific for a particular surface antigen (marker) (e.g., CD19, BCMA, EGFR/HER, CD22, mesothelin, CD 123, CD20, PD1, CD30, BAFF-R, CD20, CD22, CD30, CD37, CD38, CD70, CD79B, CD123, CD138, GPRC5D, LMP1, ROR1, SLAMF7, CD32B, CD70, CD72, CD133, FcpR, Siglec-6, TSLPR), a transmembrane domain, and an intracellular (T-cell signaling) domain of the present disclosure, as described herein. The CARs of the present disclosure also comprise an intracellular domain which can contain a CD3-zeta intracellular domain. Optionally, the intracellular domain can further comprise a CD8-alpha protein, a CD28 protein, an FcR gamma protein, a CD27 protein, an 0X40 protein, a 4- IBB protein, a CD30 protein, a CD40 protein, PD-1 protein, an ICOSprotein, an LFA-1 protein, a CD2 protein, a CD7 protein, a LIGHT protein, an NKG2C protein, a B7 H3 protein or other costimulatory domains known for use in CARs, and combinations thereof. [00152] Generally, for purposes of CAR construction, wherein a class of suitable extracellular (antigen-binding) domains is known and a separate class of suitable intracellular domains is known, any particular species of such extracellular domain can be combined (via a transmembrane domain) with any particular species of such intracellular domain, to obtain an operable CAR.

[00153] Without further elaboration, it is believed that one skilled in the art can, based on the disclosure, utilize the present disclosure to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.

Particle composition

[00154] In one aspect of the present disclosure, provided is a nanoparticle (NP) comprising a nucleic acid, wherein the NP is capable of delivering the nucleic acid to cells after being injected into a lymph node of a subject.

[00155] In some embodiments, the NP delivers the nucleic acid to cells after being retained in the lymph node of a subject after injection.

[00156] In certain embodiments, the nucleic acid encodes a chimeric antigen receptor (CAR) [00157] In some embodiments, the NP of the present disclosure further comprises at least one transfection agent. In some embodiments, the transfection agent comprises ApoeE or CpG. In some embodiments, the at least one transfection agent is ApoE. In certain embodiments, the at least one transfection agent is CpG. In some embodiments, the at least one transfection agent increases the delivery mRNA to the cells in the lymph node. In certain embodiments, the at least one transfection agent increases expression of the construct. [00158] In some embodiments, the NP of the present disclosure further comprises an activating factor. In certain embodiments, the NP comprises an activating factor. In some embodiments, the activating factor activates immune cells. In certain embodiments, the immune cell is a T-cell. In certain embodiments, the immune cell is a myeloid cell. In certain embodiments, the immune cell is a lymphocyte. In certain embodiments, the immune cell is a NK cell. In certain embodiments, the immune cell is a B-cell. In certain embodiments, the immune cell is an antigen presenting cell. In certain embodiments, the immune cell is a dendritic cell. In certain embodiments, the immune cell is a macrophage. In certain embodiments, the immune cell is a monocyte. In certain embodiments, the immune cell is a lymphatic endothelial cell.

[00159] In some embodiments, the NP of the present disclosure further comprises a T-cell activating factor. In certain embodiments, the activating factor is CD3/CD28, OKT, phorbol-

12-myristate-13-acetate (PMA), ionomycin, phytohaemagglutinin P (PHA-P), or other superagonists or mitogens. In certain embodiments, the T-cell activating factor is CD3/CD28 or OKT. In certain embodiments, the activating factor is CD3/CD28. In certain embodiments, the activating factor is OKT. In some embodiments, the T-cell activating factor is a mitogen or superantigen. In certain embodiments, the mitogen or superantigen is phorbol 12-myristate

13-acetate (PMA), ionomycin, and/or PHA (Phytohaemagglutinin P).

[00160] In certain embodiments, the lipid nanoparticle comprises an additional agent. In some embodiments, the additional agent enhanced cell update or processing of the nanoparticle. In certain embodiments, the additional agent is a molecular adjuvant. In some embodiments, the NP of the present disclosure further comprises a molecular adjuvant. In certain embodiments, the molecular adjuvant is an innate ligand. In some embodiments, the innate ligand is TLR, RIG, and/or STING.

[00161] In certain embodiments, the NP further comprises at least one lymph node binding moiety. In some embodiments, the lymph node binding moiety binds RANKL, sLeX, PNAd, GL7, CLDN11, EFNB2, ACKR4, TNFRSF9, CCL20, CXCL5, GlyCAM-1, CD34, Ptx3, CD209/Marco, SPNS2, CSF-1, CCL21, CCL19, CCL25, or CCL27.

[00162] In certain embodiments, the lymph node binding moiety is covalently bound to the microparticle. In some embodiments, the lymph node binding moiety is covalently bound to the microparticle by N-hydroxysuccinimide (NHS) chemistry, carbodiimide coupling chemistry, click chemistry, maleimide chemistry, or other conjugation strategies known in the art.

[00163] In certain embodiments, the nucleic acid encodes a CAR protein with an extracellular antigen-binding domain. In some embodiments, the extracellular antigenbinding domain binds CD19, BCMA, EGFR/HER, CD22, mesothelin, CD123, CD20, PD1, CD30, BAFF-R, CD20, CD22, CD30, CD37, CD38, CD70, CD79B, CD123, CD138, GPRC5D, LMP1, ROR1, SLAMF7, CD32B, CD70, CD72, CD133, FcpR, Siglec-6, or TSLPR. In some embodiments, the extracellular antigen-binding domain binds BCMA. In certain embodiments, the extracellular antigen-binding domain binds CD-19.

[00164] In some embodiments, the nucleic acid is RNA. In some embodiments, the RNA is mRNA. In some embodiments, the nucleic acid is DNA.

[00165] In some embodiments, the NP comprises between 100 and 1000 ng of mRNA. In certain embodiments, the NP comprises 250-500 ng of mRNA. In certain embodiments, the NP comprises between 10 and 50 ng mRNA. In certain embodiments, the NP comprises between 50 and 100 ng mRNA. In certain embodiments, the NP comprises between 100 and 250 ng mRNA. In certain embodiments, the NP comprises between 10 and 250 ng mRNA. In certain embodiments, the NP comprises between 250 and 500 ng mRNA. In certain embodiments, the NP comprises between 500 and 1000 ng mRNA.

[00166] In some embodiments, the NP comprises a lipid nanoparticle, liposome, polymer nanoparticle, dendrimer nanoparticle, polymersome, polymeric micelle, micelle, mesoporous or silica nanoparticle. In some embodiments, the nucleic acid of the present disclosure is in (e.g., formulated as) a lipid composition, such as a composition comprising a lipid nanoparticle, a liposome, a lipoplex and/or a microparticle. In some embodiments the particle comprises a plurality of particles.

[00167] In some embodiments, the NP is a lipid nanoparticle (LNP). In some embodiments the lipid nanoparticle comprises at least one lipid. In some embodiments, the lipid nanoparticle comprises at least two lipids. In some embodiments, the lipid nanoparticle comprises at least three lipids. In some embodiments, the lipid nanoparticle comprises at least four lipids.

[00168] In certain embodiments, the LNP comprises one or more ionizable lipids, stabilizing lipids, cationic lipids, helper lipids, or any combination thereof.

[00169] In some embodiments, the LNP comprises ionizable lipids, stabilizing lipids, cationic lipids, helper lipids, and a sterol.

[00170] In some embodiments, the LNP comprises at least one ionizable lipid, at least one stabilizing lipid, at least one cationic lipid, at least one helper lipid, and at least one sterol. [00171] In some embodiments, the LNP comprises at least one ionizable lipid, at least one stabilizing lipid, at least one helper lipid, and at least one sterol.

[00172] In some embodiments, the LNP comprises at least one stabilizing lipid, at least one cationic lipid, at least one helper lipid, and at least one sterol.

[00173] In some embodiments, the ionizable lipid is a cationic lipid. In some embodiments, the cationic lipid is an ionizable lipid.

[00189] In some embodiments, the LNP comprises 30-60 mol % ionizable and/or cationic lipids, 1-20 mol % helper lipids, 20-50 mol % sterol, and 0.1-5 mol % stabilization lipid. [00190] In some embodiments, the LNP comprises 40-50 mol % ionizable and/or cationic lipids, 5-15 mol % helper lipids, 30-40 mol % sterol, and 0.5-3 mol % stabilization lipid. In some embodiments, the LNP comprises 30-40 mol % ionizable lipids. In some embodiments, the LNP comprises 30-40 mol % cationic lipids. In some embodiments, the LNP comprises 40-50 mol % ionizable lipids. In some embodiments, the LNP comprises 40-50 mol % cationic lipids. In some embodiments, the LNP comprises 50-60 mol % ionizable lipids. In some embodiments, the LNP comprises 50-60 mol % cationic lipids. In some embodiments, the LNP comprises 1-10 mol % helper lipids. In some embodiments, the LNP comprises 5-15 mol % helper lipids. In some embodiments, the LNP comprises 20-50 mol % sterol. In some embodiments, the LNP comprises 20-30 mol % sterol. In some embodiments, the LNP comprises 30-50 mol % sterol. In some embodiments, the LNP comprises 40-50 mol % sterol. In some embodiments, the LNP comprises 0.1-5 mol % stabilization lipid. In some embodiments, the LNP comprises less than 5 mol % stabilization lipid.

[00191] As used herein, “ionizable lipid’ refers to a lipid which have a tunable charge based on the pH of the local environment. As used herein “cationic lipids” refers to lipids which bear a positive change. Exemplary ionizable or cationic lipids include but are not limited to ALC-0315, CKK-E12, DLin-MC3-DMA, SM-102, OF-02, A6, A18-Iso5-2DC18, 9A1P9, 98N12-5, C12-200, 7C1, GO-14, L319, 304013, 306-O12B, OF-Deg-Lin, 3060iio, FTT5, 1,2- dioleoyl-3-trimethylammonium-propane (DOTAP), 1 ,2-dioleoyl-3-trimethylammonium propane (DOTMA), l-[2-dioleoyloxy)ethyl]-2-oleyl-3(2-hydroxyethyl)imidazolinium (DOTIM), orpolyethylenimine (PEI), or poly(beta-amino esters) (PBAE). In some embodiments, the ionizable lipid is positively charged.

[00192] As used herein, the term “stabilizing lipid” includes sterols, PEG-lipids, or phospholipids. Incorporation of stabilizing lipids in a lipid nanoparticle may help mitigate aggregation of other lipids in the particle, stabilizing lipids can be selected from the group including but not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof. In some embodiments, the stabilizing lipid is a sterol. As defined herein, “sterols” are a subgroup of steroids consisting of steroid alcohols.

In certain embodiments, the stabilizing lipid is a steroid. In certain embodiments, the structural lipid is cholesterol. In certain embodiments, the stabilizing lipid is an analog of cholesterol. In certain embodiments, the stabilizing lipid is alpha- tocopherol.

[00193] As used herein, the term “PEG-lipid” or “PEG-modified lipid” refers to polyethylene glycol (PEG) -modified lipids. Non-limiting examples of PEG-lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG- CerC14 or PEG-CerC20), PEG-modified dialkylamines, and PEG-modified 1,2- diacyloxypropan-3-amines. Such lipids are also referred to as PEGylated lipids. For example, a PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid. In some embodiments, the PEG-lipid includes, but not limited to 1,2- dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero- 3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEGDAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-1, 2- dimyristyloxlpropyl-3-amine (PEG-c-DMA). In some embodiments, the PEG-modified lipid is PEG-DMG, PEG-c-DOMG (also referred to as PEG-DOMG), PEG-DSG, and/or PEG- DPG.

[00194] In some embodiments, the lipid moiety of the PEG-lipids includes those having lengths of from about Ci4 to about C22, preferably from about C14 to about C16. In some embodiments, a PEG moiety has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons.

[00195] The lipid component of a lipid nanoparticle composition may include one or more molecules comprising polyethylene glycol, such as PEG or PEG-modified lipids. Such species may be alternately referred to as PEGylated lipids. A PEG lipid is a lipid modified with polyethylene glycol. A PEG lipid may be selected from the non-limiting group including PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids,

PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, a PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid. [00196] As used herein, the term “helper lipid” PEG-lipids, or phospholipids. In some embodiments, the helper lipid comprises a PEG-lipid. In some embodiments, the helper lipid comprises a phospholipid. In some embodiments, a phospholipid of the disclosure comprises l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), l,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2- dimyristoyl-sn-gly cero-phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn- glycero-phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), l,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2 cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1 -hexadecyl- sn- glycero-3-phosphocholine (C16 Lyso PC), l,2-dilinolenoyl-sn-glycero-3-phosphocholine,l,2- diarachidonoyl-sn-glycero-3-phosphocholine, l,2-didocosahexaenoyl-sn-glycero-3- phosphocholine, l,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinoleoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1 ,2- diarachidonoyl-sn-glycero-3-phosphoethanolamine, l,2-didocosahexaenoyl-sn-glycero-3- phosphoethanolamine, l,2-dioleoyl-sn-glycero-3-phospho-rac-(l -glycerol) sodium salt (DOPG), sphingomyelin, or mixtures thereof.

[00197] In some embodiments, the NP is a polymer nanoparticle. In certain embodiments, the polymer nanoparticles comprise cationic polymers.

[00198] In some embodiments, the concentration of NP in the lymph node is greater than the concentration in any other tissue of the subject. In certain embodiments, the concentration of

NP in the lymph node is greater than the systemic biodistribution in the subject. In some embodiments, the nanoparticle is 1-999 nm in size. In some embodiments, the nanoparticle is 10-500 nm in size. In some embodiments, the nanoparticle is 10-250 nm in size. In some embodiments, the nanoparticle is 100-200 nm in size. In some embodiments, the nanoparticle is about 200 nm in size.

[00199] In another aspect, provided is a nanoparticle composition comprising a plurality of the NPs described herein.

[00200] In yet another aspect of the present disclosure, provided is a microparticle (MP) loaded with at least one nanoparticle described herein.

[00201] In one aspect, provided herein is a microparticle (MP) loaded with a nucleic acid. In another aspect, provided herein is a microparticle (MP) loaded with a nucleic acid and at least one nanoparticle, wherein the microparticle is capable of delivering the nucleic acid to cells after being injected into a lymph node of a subject.

[00202] In certain embodiments, the MP is also loaded with at least one nanoparticle. In another embodiments, the MP is mixed with at least one nanoparticle in solution. In certain embodiments, the MP delivers the nucleic acid to cells after being retained in the lymph node of a subject after injection.

[00203] In some embodiments, the nucleic acid encodes a protein. In some embodiments, the nucleic acid encodes a therapeutic protein. In some embodiments, the nucleic acid encodes a chimeric antigen receptor (CAR).

[00204] In certain embodiments, the MP is also loaded with at least one transfection agent. In certain embodiments, the MP comprises at least one transfection agent. In some embodiments, the MP is further mixed with at least one transfection agent in solution. In some embodiments, the transfection agent comprises ApoeE or CpG. In certain embodiments, the at least one transfection agent is ApoE. In some embodiments, the at least one transfection agent is CpG. In certain embodiments, the at least one transfection agent increases the delivery mRNA to the cells in the lymph node. In some embodiments, the at least one transfection agent increases expression of the construct. [00205] In certain embodiments, the MP is also loaded with an activating factor. In certain embodiments, the activating factor activated an immune cell. In certain embodiments, the immune cell is a myeloid cell. In certain embodiments, the immune cell is a lymphocyte. In certain embodiments, the immune cell is a NK cell. In certain embodiments, the immune cell is a B-cell. In certain embodiments, the immune cell is an antigen presenting cell. In certain embodiments, the immune cell is a dendritic cell. In certain embodiments, the immune cell is a macrophage. In certain embodiments, the immune cell is a monocyte. In certain embodiments, the immune cell is a lymphatic endothelial cell.

[00206] In certain embodiments, the MP is loaded with a T-cell activating factor. In some embodiments, the MP is further mixed with a T-cell activating factor in solution. In certain embodiments, the T-cell activating factor is CD3/CD28 or OKT. In certain embodiments, the T-cell activating factor is CD3/CD28. In certain embodiments, the T-cell activating factor is OKT. In some embodiments, the T-cell activating factor is a mitogen or superantigen. In certain embodiments, the mitogen or superantigen is phorbol 12-myristate 13-acetate (PMA), ionomycin, and/or PHA (Phytohaemagglutinin P).

[00207] In some embodiments, the MP is also loaded with a molecular adjuvant. In certain embodiments, the MP is further mixed with a molecular adjuvant in solution. In some embodiments, the molecular adjuvant is an innate ligand. In certain embodiments, the innate ligand is TLR, RIG, and/or STING.

[00208] In some embodiments, the MP is also loaded with at least one lymph node binding moiety. In certain embodiments, the MP is further mixed with at least one lymph node binding moiety in solution. In some embodiments, the at least one lymph node binding moiety binds RANKL, sLeX, PNAd, GL7, CLDN11, EFNB2, ACKR4, TNFRSF9, CCL20, CXCL5, GlyCAM-1, CD34, Ptx3, CD209/Marco, SPNS2, CSF-1, CCL21, CCL19, CCL25, or CCL27. In certain embodiments, the lymph node binding moiety is covalently bound to the microparticle. In some embodiments, the lymph node binding moiety is covalently bound to the microparticle by N-hydroxysuccinimide (NHS) chemistry, carbodiimide coupling chemistry, click chemistry, maleimide chemistry, or other conjugation strategies known in the art.

[00209] In certain embodiments, the nucleic acid encodes a CAR protein with an extracellular antigen-binding domain. In certain embodiments, the extracellular antigenbinding domain binds CD19, BCMA, EGFR/HER, CD22, mesothelin, CD123, CD20, PD1, CD30, BAFF-R, CD20, CD22, CD30, CD37, CD38, CD70, CD79B, CD123, CD138, GPRC5D, LMP1, ROR1, SLAMF7, CD32B, CD70, CD72, CD133, FcpR, Siglec-6, or TSLPR. In some embodiments, the extracellular antigen-binding domain binds BCMA. In certain embodiments, the extracellular antigen-binding domain binds CD-19.

[00174] In some embodiments, the MP comprises between 100 and 1000 ng of mRNA. In certain embodiments, the MP comprises 250-500 ng of mRNA. In certain embodiments, the MP comprises between 10 and 50 ng mRNA. In certain embodiments, the MP comprises between 50 and 100 ng mRNA. In certain embodiments, the MP comprises between 100 and 250 ng mRNA. In certain embodiments, the MP comprises between 10 and 250 ng mRNA. In certain embodiments, the MP comprises between 250 and 500 ng mRNA. In certain embodiments, the MP comprises between 500 and 1000 ng mRNA.

[00210] In certain embodiments, the MP comprise a degradable polymer.

[00211] In certain embodiments, the degradable polymer comprises one or more of ester bonds, amide bonds, glycosidic bonds, phosphodiester bonds, ether bonds, disulfide bonds, peptide bonds, urethane bonds, carbonate bonds, thioester bonds, orthoester bonds, or a combination thereof. In some embodiments, the degradable polymer is poly(lactide-co- glycolide) (PLGA). In some embodiments, the microparticle further comprises polyvinyl alcohol. In some embodiments, the microparticle further comprises a lipid. In certain embodiments, the microparticle comprises a plurality of nanoparticles described herein. [00212] In certain embodiments, the concentration of MP in the lymph node is greater than the concentration in any other tissue of the subject. In some embodiments, the concentration of MP in the lymph node is greater than the systemic biodistribution in the subject.

[00213] In some embodiments, the microparticle is 1-100 microns in size. In some embodiments, the microparticle is 1-50 microns in size. In some embodiments, the microparticle is 1-10 microns in size. In some embodiments, the microparticle is 2-8 microns in size.

[00214] In another aspect of the present disclosure, provided is a microparticle composition comprising a plurality of microparticles described herein.

Methods

[00215] In another aspect, the present disclosure provides a method of transfecting cells with a nucleic acid, the method comprising contacting the cells with a plurality of the NPs of any the present disclosure, the nanoparticle composition defined herein, a plurality of microparticles of the present disclosure, the microparticle composition claim described herein, or the disclosed pharmaceutical composition.

[00216] In some embodiments, the contacting is conducted in vivo. In some embodiments, the contacting is conducted in situ in a lymph node. In certain embodiments, the cells are located in a lymph node of a subject. In some embodiments, the cell is transiently located in the lymph node of a subject. In certain embodiments, the cells are immune cells. In certain embodiments, the immune cell is a myeloid cell. In certain embodiments, the immune cell is a lymphocyte. In certain embodiments, the immune cell is a NK cell. In certain embodiments, the immune cell is a B-cell. In certain embodiments, the immune cell is an antigen presenting cell. In certain embodiments, the immune cell is a dendritic cell. In certain embodiments, the immune cell is a macrophage. In certain embodiments, the immune cell is a monocyte. In certain embodiments, the immune cell is a lymphatic endothelial cell. [00217] In some embodiments, the cells are lymphocytes. In certain embodiments, the cells are T-cells. In some embodiments, the T-cells are CD8+ T cells. In certain embodiments, the T-cells are CD4+ T cells.

[00218] In some embodiments, the plurality of nanoparticles, the nanoparticle composition, the plurality of microparticles, the microparticle composition, or pharmaceutical composition is administered into the lymph node of a subject.

[00219] In certain embodiments, the plurality of nanoparticles, the nanoparticle composition, the plurality of microparticles, the microparticle composition, or pharmaceutical composition is administered into the lymph node of a subject by injection. In certain embodiments, the plurality of nanoparticles, the nanoparticle composition, the plurality of microparticles, the microparticle composition, or pharmaceutical composition is administered into the lymph node of a subject by intravenous administration.

[00220] In one aspect, provide by the present disclosure is a method of treating disease in a subject in need thereof, the method comprising administering to the subject a plurality of the NPs described herein, the nanoparticle composition defined herein, a plurality of microparticles described herein, the microparticle composition of the present disclosure, or the provided pharmaceutical composition. In some embodiments, the method comprises a administering to the subject a plurality of the NPs described herein, the nanoparticle composition defined herein, a plurality of microparticles described herein, the microparticle composition of the present disclosure, or the provided pharmaceutical composition by injection.

[00221] In certain embodiments, the disease is a proliferative disease, an autoimmune disease, or an inflammatory disease.

[00175] In some embodiments, the proliferative disease is cancer. In some embodiments, the cancer is monoclonal B-cell lymphocytosis, chronic lymphocytic leukemia/small lymphocytic lymphoma, hairy cell leukemia, splenic marginal zone lymphoma, splenic diffuse red pulp small B-cell lymphoma, splenic B-cell lymphoma/ leukemia with prominent nucleoli, lymphoplasmacytic lymphoma, marginal zone lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, NOS T-cell/histiocyte-rich large B- cell lymphoma, diffuse large B-cell lymphoma/ high grade B-cell lymphoma with MYC and BCL2 rearrangements, ALK-positive large B-cell lymphoma, large B-cell lymphoma with IRF4 rearrangement, high-grade B-cell lymphoma with l lq aberrations, lymphomatoid granulomatosis, EBV-positive diffuse large B-cell lymphoma, diffuse large B-cell lymphoma associated with chronic inflammation, fibrin-associated large B-cell lymphoma, fluid overload-associated large B-cell lymphoma, plasmablastic lymphoma, primary large B-cell lymphoma of immune-privileged sites, primary cutaneous diffuse large B-cell lymphoma leg type, intravascular large B-cell lymphoma, primary mediastinal large B-cell lymphoma, mediastinal grey zone lymphoma, high-grade B-cell lymphoma NOS, Burkitt lymphoma, primary effusion lymphoma, KSHV/HHV8-positive diffuse large B-cell lymphoma, KSHV/HHV8-positive germinotropic lymphoproliferative disorder, Hodgkin lymphoma, cold agglutinin disease, IgM monoclonal gammopathy of undetermined significance, Non-IgM monoclonal gammopathy of undetermined significance, monoclonal gammopathy of renal significance, immunoglobulin-related (AL) amyloidosis, monoclonal immunoglobulin deposition disease, heavy chain diseases, mu heavy chain disease, gamma heavy chain disease, alpha heavy chain disease, plasma cell neoplasms, plasmacytoma, plasma cell myeloma, or multiple myeloma. In certain embodiments, the cancer is multiple myeloma. [00222] In certain embodiments, the autoimmune diseases is myasthenia gravis, systemic lupus erythematosus (SLE), rheumatoid arthritis, blistering skin diseases, e.g., pemphigus, psoriasis, inflammatory bowel disease, celiac sprue, pernicious anemia, idiopathic thrombocytopenia purpura, sceleroderma, Graves disease, Sjogren syndrome, Goodpasture syndrome, multiple sclerosis, type 1 diabetes, glomerulonephritis, necrotizing vasculitis, lymphadenitis, peri-arteritis nodosa, systemic lupus erythematosis, psoriatic arthritis, ulcerative colitis, systemic sclerosis, dermatomyositis/polymyositis, anti-phospholipid antibody syndrome, pemphigus vulgaris, ANCA-associated vasculitis (e.g., Wegener’s granulomatosis, microscopic polyangiitis), uveitis, Sjogren’s syndrome, Crohn’s disease, Reiter’s syndrome, ankylosing spondylitis, Lyme disease, Guillain-Barre syndrome, Hashimoto’s thyroiditis, cardiomyopathy, idiopathic inflammatory myositis, neuromyelitis optica, chronic inflammatory demyelinating polyneuropathy (CIDP), inclusion body myositis, autoimmune encephalitis, Lambert-Eaton syndrome, stiff person syndrome, Opsoclonus-myoclonus syndrome, antiphospholipid syndrome, immune thrombocytopenia (ITP), pure red cell aplasia, autoimmune hemolytic anemia, Cold agglutinin disease, Evans syndrome, autoimmune neutropeni, IgA nephropathy, lupus nephritis, membranous nephropathy, ANCA-associated vasculitis, Juvenile SLE, IgA Vasculitis, autoimmune thyroiditis, juvenile dermatomyositis, juvenile idiopathic arthritis, scleroderma, bullous pemphigoid, pemphigus vulgaris, discoid lupus erythematosus, alopecia areata, vitiligo, systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, ankylosing spondylitis, mixed connective tissue disease, polymyalgia rheumatica, Sjogren syndrome, thyroid eye disease, ocular cicatricial pemphigoid, autoimmune retinitis, autoimmune uveitis, Churg-Strauss syndrome, egener’s granulomatosis, sarcoidosis, interstitial lung disease, autoimmune hepatitis, autoimmune pancreatitis, Crohn’s disease, ulcerative colitis, celiac disease, eosinophilic gastroenteritis, microscopic colitis, primary biliary cholangitis, or primary sclerosing cholangitis. In some embodiments, the autoimmune disease is myasthenia gravis.

[00223] In certain embodiments, the plurality of nanoparticles, the nanoparticle composition, the plurality of microparticles, the microparticle composition, or pharmaceutical composition is administered by injection. [00224] In some embodiments, the plurality of nanoparticles, the nanoparticle composition, the plurality of microparticles, the microparticle composition, or pharmaceutical composition is administered directly into a lymph node.

[00225] In certain embodiments, the plurality of nanoparticles, the nanoparticle composition, the plurality of microparticles, the microparticle composition, or pharmaceutical composition is administered directly into at least two lymph nodes.

[00226] In some embodiments, the method is characterized by an increase in cytokine production in the cells transfected with the nucleic acid. In one aspect of the disclosure, the methods of the disclosure are characterized by an increase or modulation in cytokine production in the cells transfected with the nucleic acid. The cytokines that are increased or modulated can included one or more of the following: interferon gamma (IFNy), APRIL, BAFF, CD40 Ligand, Fit- 3 Ligand, G-CSF, GM-CSF, Granzyme B, GRO alpha, GRO beta, IFN-a2, IFN-P, IFN-Y, IL-la, IL-ip, IL- Ira, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9„ IL-10, IL-12 p70, IL-13, IL-15 IL-17A, IL-17E, IL-21, IL-23, IL-24, IL-27, IL-28, IL-29, IL- 33, IP- 10, MCP-1, MIP-1 alpha, MIP-1 beta, MIP-3 alpha, MIP-3 beta, RANTES, TGF-a, TNF-a, TNF-P, and TRAIL. However, this list is not meant to be exhaustive. The one or more cytokines that would be increased or modulated could depend on the formulation of the NPs and MPs of the present disclosure. In certain embodiments, the cytokine is interferongamma.

[00227] In another aspect, the present disclosure provides a method of inducing cytokine expression in the cells of a subject, the method comprising administering to the lymph nodes of a subject a plurality of the NPs defined herein, the nanoparticle composition described herein, a plurality of microparticles described herein, the microparticle composition described herein, or the pharmaceutical composition described herein.

[00228] In certain embodiments, the plurality of the LNPs, the nanoparticle composition, the plurality of microparticle, the microparticle composition, or the pharmaceutical composition, is administered to a lymph node in the subject. In certain embodiments, the cytokine is interferon gamma (IFNy), APRIL, BAFF, CD40 Ligand, Fit- 3 Ligand, G-CSF, GM-CSF, Granzyme B, GRO alpha, GRO beta, IFN-a2, IFN-P, IFN-Y, IL-la, IL-ip, IL-lra, IL-2, IL- 3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9„ IL-10, IL-12 p70, IL-13, IL-15 IL-17A, IL-17E, IL-21, IL-23, IL-24, IL-27, IL-28, IL-29, IL-33, IP- 10, MCP-1, MIP-1 alpha, MIP-1 beta, MIP-3 alpha, MIP-3 beta, RANTES, TGF-a, TNF-a, TNF-P, and TRAIL. In certain embodiments, the cytokine is interferon-gamma.

[00229] In one aspect, provided herein is the use of the plurality of the NPs described herein, the nanoparticle composition described herein, the plurality of microparticles described herein, the microparticle composition described herein, or the pharmaceutical composition of claim described herein for transfecting cells in a lymph node with a nucleic acid.

[00230] In one aspect, provided herein is the use of the plurality of the NPs described herein, the nanoparticle composition described herein, the plurality of described herein, the microparticle composition described herein, or the pharmaceutical composition described herein for treating a disease in a subject.

[00231] In another aspect, provided herein is the use of the LNPs of the plurality of the NPs described herein, the nanoparticle composition described herein, the plurality of microparticles described herein, the microparticle composition described herein, or the pharmaceutical composition described herein for inducing cytokine expression.

[00232] In one aspect, the present disclosure provides a medicament for administration into a lymph node, the medicament comprising the plurality of the NPs describe herein, the nanoparticle composition described herein, the plurality of microparticles described herein, the microparticle composition described herein, or the pharmaceutical composition described herein. Pharmaceutical Compositions

[00233] The present disclosure provides pharmaceutical compositions comprising nanoparticles and microparticles disclosed herein, and optionally pharmaceutically acceptable excipients. In one aspect of the present disclosure, provided is a kit comprising the plurality of the NPs described herein, the nanoparticle composition described herein, the plurality of microparticles described herein, the microparticle composition described herein, or the pharmaceutical composition described herein.

[00234] In certain embodiments, the pharmaceutical composition described herein comprises nanoparticles and/or microparticles disclosed herein, and a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition described herein comprises a plurality of nanoparticles and/or microparticles disclosed herein, and pharmaceutically acceptable excipients.

[00235] In certain embodiments, the nanoparticles and/or microparticles described herein is provided in an effective amount in the pharmaceutical composition. In certain embodiments, the plurality of nanoparticles and/or microparticles described herein is provided in an effective amount in the pharmaceutical composition. In certain embodiments, the effective amount is a therapeutically effective amount. In certain embodiments, the effective amount is a prophy tactically effective amount. In certain embodiments, the effective amount is an amount effective for treating a proliferative disease in a subject in need thereof. In certain embodiments, the effective amount is an amount effective for preventing a proliferative disease in a subject in need thereof. In certain embodiments, the effective amount is an amount effective for treating a hematological disease in a subject in need thereof. In certain embodiments, the effective amount is an amount effective for preventing a hematological disease in a subject in need thereof. In certain embodiments, the effective amount is an amount effective for treating a neurological disease in a subject in need thereof. In certain embodiments, the effective amount is an amount effective for preventing a neurological disease in a subject in need thereof. In certain embodiments, the effective amount is an amount effective for treating a in a painful condition subject in need thereof. In certain embodiments, the effective amount is an amount effective for preventing a painful condition in a subject in need thereof. In certain embodiments, the effective amount is an amount effective for treating a psychiatric disorder in a subject in need thereof. In certain embodiments, the effective amount is an amount effective for preventing a psychiatric disorder in a subject in need thereof. In certain embodiments, the effective amount is an amount effective for treating a metabolic disorder in a subject in need thereof. In certain embodiments, the effective amount is an amount effective for preventing a metabolic disorder in a subject in need thereof. In certain embodiments, the effective amount is an amount effective for reducing the risk of developing a disease (e.g., proliferative disease, hematological disease, neurological disease, painful condition, psychiatric disorder, or metabolic disorder) in a subject in need thereof. In certain embodiments, the effective amount is an amount effective for inhibiting the activity (e.g., aberrant activity, such as increased activity) of a target in a subject or cell.

[00236] In certain embodiments, the subject is an animal. The animal may be of either sex and may be at any stage of development. In certain embodiments, the subject described herein is a human. In certain embodiments, the subject is a non-human animal. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a non-human mammal. In certain embodiments, the subject is a domesticated animal, such as a dog, cat, cow, pig, horse, sheep, or goat. In certain embodiments, the subject is a companion animal, such as a dog or cat. In certain embodiments, the subject is a livestock animal, such as a cow, pig, horse, sheep, or goat. In certain embodiments, the subject is a zoo animal. In another embodiment, the subject is a research animal, such as a rodent (e.g., mouse, rat), dog, pig, or non-human primate. In certain embodiments, the animal is a genetically engineered animal. In certain embodiments, the animal is a transgenic animal (e.g., transgenic mice and transgenic pigs). In certain embodiments, the subject is a fish or reptile.

[00237] In certain embodiments, the cell is present in vitro. In certain embodiments, the cell is present ex vivo. In certain embodiments, the cell is present in vivo.

[00238] Pharmaceutical compositions described herein can be prepared by any method known in the art of pharmaceutics. In general, such preparatory methods include bringing the nanoparticle and/or microparticle described herein (z.e., the “active ingredient”) into association with a carrier or excipient, and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping, and/or packaging the product into a desired single- or multi-dose unit. Additionally, such preparatory methods include bringing the plurality of nanoparticles or microparticles described herein (z.e., the “active ingredient”) into association with a carrier or excipient, and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping, and/or packaging the product into a desired single- or multi-dose unit.

[00239] Pharmaceutical compositions can be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. A “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage, such as one-half or one-third of such a dosage.

[00240] The exact amount of a composition required to achieve an effective amount will vary from subject to subject, depending, for example, on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular composition, mode of administration, and the like. An effective amount may be included in a single dose (e.g., single oral dose) or multiple doses (e.g., multiple oral doses). In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, any two doses of the multiple doses include different or substantially the same amounts of a composition described herein. In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is three doses a day, two doses a day, one dose a day, one dose every other day, one dose every third day, one dose every week, one dose every two weeks, one dose every three weeks, or one dose every four weeks. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is one dose per day. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is two doses per day. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is three doses per day. In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, the duration between the first dose and last dose of the multiple doses is one day, two days, four days, one week, two weeks, three weeks, one month, two months, three months, four months, six months, nine months, one year, two years, three years, four years, five years, seven years, ten years, fifteen years, twenty years, or the lifetime of the subject, tissue, or cell. In certain embodiments, the duration between the first dose and last dose of the multiple doses is three months, six months, or one year. In certain embodiments, the duration between the first dose and last dose of the multiple doses is the lifetime of the subject, tissue, or cell. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.1 pg and 1 pg, between 0.001 mg and 0.01 mg, between 0.01 mg and 0.1 mg, between 0.1 mg and 1 mg, between 1 mg and 3 mg, between 3 mg and 10 mg, between 10 mg and 30 mg, between 30 mg and 100 mg, between 100 mg and 300 mg, between 300 mg and 1,000 mg, or between 1 g and

10 g, inclusive, of a composition described herein. In certain embodiments, a dose described herein includes independently between 1 mg and 3 mg, inclusive, of a composition described herein. In certain embodiments, a dose described herein includes independently between 3 mg and 10 mg, inclusive, of a composition described herein. In certain embodiments, a dose described herein includes independently between 10 mg and 30 mg, inclusive, of a composition described herein. In certain embodiments, a dose described herein includes independently between 30 mg and 100 mg, inclusive, of a composition described herein. [00241] Dose ranges as described herein provide guidance for the administration of provided pharmaceutical compositions to an adult. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.

[00242] A pharmaceutical composition, as described herein, can be administered in combination with one or more additional pharmaceutical agents (e.g., therapeutically and/or prophylactically active agents). The pharmaceutical compositions can be administered in combination with additional pharmaceutical agents that improve their activity (e.g., activity (e.g., potency and/or efficacy) in treating a disease in a subject in need thereof, in preventing a disease in a subject in need thereof, in reducing the risk to develop a disease in a subject in need thereof, and/or in inhibiting the activity of a target in a subject or cell), improve bioavailability, improve safety, reduce drug resistance, reduce and/or modify metabolism, inhibit excretion, and/or modify distribution in a subject or cell. It will also be appreciated that the therapy employed may achieve a desired effect for the same disorder, and/or it may achieve different effects. In certain embodiments, a pharmaceutical composition described herein including a particle or plurality of particles described herein and an additional pharmaceutical agent shows a synergistic effect that is absent in a pharmaceutical composition including one of the particles and the additional pharmaceutical agent, but not both. In some embodiments, the additional pharmaceutical agent achieves a desired effect for the same disorder. In some embodiments, the additional pharmaceutical agent achieves different effects. [00243] The pharmaceutical composition can be administered concurrently with, prior to, or subsequent to one or more additional pharmaceutical agents, which may be useful as, e.g., combination therapies. Pharmaceutical agents include therapeutically active agents. Pharmaceutical agents also include prophylactically active agents. Pharmaceutical agents include small organic molecules such as drug compounds (e.g., compounds approved for human or veterinary use by the U.S. Food and Drug Administration as provided in the Code of Federal Regulations (CFR)), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins, and cells. In certain embodiments, the additional pharmaceutical agent is a pharmaceutical agent useful for treating and/or preventing a disease (e.g., proliferative disease, hematological disease, neurological disease, painful condition, psychiatric disorder, or metabolic disorder). Each additional pharmaceutical agent may be administered at a dose and/or on a time schedule determined for that pharmaceutical agent. The additional pharmaceutical agents may also be administered together with each other and/or with the composition described herein in a single dose or composition or administered separately in different doses or compositions. The particular combination to employ in a regimen will take into account compatibility of the nanoparticle, microparticle, or plurality of either described herein with the additional pharmaceutical agent(s) and/or the desired therapeutic and/or prophylactic effect to be achieved. In general, it is expected that the additional pharmaceutical agent(s) in combination be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually.

[00244] The additional pharmaceutical agents include, but are not limited to, antiproliferative agents, anti-cancer agents, anti-angiogenesis agents, steroidal or non-steroidal anti-inflammatory agents, immunosuppressants, anti-bacterial agents, anti-viral agents, cardiovascular agents, cholesterol-lowering agents, anti-diabetic agents, anti-allergic agents, contraceptive agents, pain-relieving agents, anesthetics, anti-coagulants, inhibitors of an enzyme, steroidal agents, steroidal or antihistamine, antigens, vaccines, antibodies, decongestant, sedatives, opioids, analgesics, anti-pyretic s, hormones, and prostaglandins. In certain embodiments, the additional pharmaceutical agent is an anti-proliferative agent. In certain embodiments, the additional pharmaceutical agent is an anti-cancer agent. In certain embodiments, the additional pharmaceutical agent is an anti-viral agent. In certain embodiments, the additional pharmaceutical agent is an binder or inhibitor of a protein kinase. In certain embodiments, the additional pharmaceutical agent is selected from the group consisting of epigenetic or transcriptional modulators (e.g., DNA methyltransferase inhibitors, histone deacetylase inhibitors (HD AC inhibitors), lysine methyltransferase inhibitors), antimitotic drugs (e.g., taxanes and vinca alkaloids), hormone receptor modulators (e.g., estrogen receptor modulators and androgen receptor modulators), cell signaling pathway inhibitors (e.g., tyrosine protein kinase inhibitors), modulators of protein stability (e.g., proteasome inhibitors), Hsp90 inhibitors, glucocorticoids, all-trans retinoic acids, and other agents that promote differentiation. In certain embodiments, the compounds described herein or pharmaceutical compositions can be administered in combination with an anti-cancer therapy including, but not limited to, surgery, radiation therapy, transplantation (e.g., stem cell transplantation, bone marrow transplantation), immunotherapy, and chemotherapy. Additional pharmaceutical agents include small organic molecules such as drug compounds (e.g., compounds approved by the US Food and Drug Administration as provided in the Code of Federal Regulations (CFR)), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins and cells. [00245] Also encompassed by the disclosure are kits (e.g., pharmaceutical packs). The kits provided may comprise a pharmaceutical composition or compound described herein and a container (e.g., a vial, ampule, bottle, syringe, and/or dispenser package, or other suitable container). In some embodiments, provided kits may optionally further include a second container comprising a pharmaceutical excipient for dilution or suspension of a pharmaceutical composition or compound described herein. In some embodiments, the pharmaceutical composition or compound described herein provided in the first container and the second container are combined to form one unit dosage form.

[00246] Thus, in one aspect, provided are kits including a first container comprising a compound or pharmaceutical composition described herein. In certain embodiments, the kits are useful for treating a disease (e.g., proliferative disease, hematological disease, neurological disease, painful condition, psychiatric disorder, or metabolic disorder) in a subject in need thereof. In certain embodiments, the kits are useful for preventing a disease (e.g., proliferative disease, hematological disease, neurological disease, painful condition, psychiatric disorder, or metabolic disorder) in a subject in need thereof. In certain embodiments, the kits are useful for reducing the risk of developing a disease (e.g., proliferative disease, hematological disease, neurological disease, painful condition, psychiatric disorder, or metabolic disorder) in a subject in need thereof. In certain embodiments, the kits are useful for inhibiting the activity (e.g., aberrant activity, such as increased activity) of a target in a subject or cell.

[00247] In certain embodiments, a kit described herein further includes instructions for using the kit. A kit described herein may also include information as required by a regulatory agency such as the U.S. Food and Drug Administration (FDA). In certain embodiments, the information included in the kits is prescribing information. In certain embodiments, the kits and instructions provide for treating a disease (e.g., proliferative disease, hematological disease, neurological disease, painful condition, psychiatric disorder, or metabolic disorder) in a subject in need thereof. In certain embodiments, the kits and instructions provide for preventing a disease (e.g., proliferative disease, hematological disease, neurological disease, painful condition, psychiatric disorder, or metabolic disorder) in a subject in need thereof. In certain embodiments, the kits and instructions provide for reducing the risk of developing a disease (e.g., proliferative disease, hematological disease, neurological disease, painful condition, psychiatric disorder, or metabolic disorder) in a subject in need thereof. In certain embodiments, the kits and instructions provide for inhibiting the activity (e.g., aberrant activity, such as increased activity) of a target in a subject or cell. A kit described herein may include one or more additional pharmaceutical agents described herein as a separate composition.

EXAMPLES

[00248] In order that the present disclosure described herein may be more fully understood, the following examples are set forth. The synthetic examples described in this application are offered to illustrate the compounds and methods provided herein and are not to be construed in any way as limiting their scope.

[00249] 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 the present disclosure pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used. Example 1. in vivo biodistribution/expression of anti-BCMA CAR; LNP

[00250] This example describes a method to localize anti-BCMA CAR mRNA to lymph node’s (LN’s) in mice using intra-LN delivery of LNPs (lipid nanoparticle’s), resulting in delivery to and expression of the CAR in LN-resident cells and cells migrating through LNs. Transfection of these cells using this method was achieved by intra-LN injection of mRNA- encoding LNPs formed via microfluidics. LNPs were prepared using an automated microfluidics system (NanoAssemblr, Precision Nanosystems) according to the manufacturer’s instructions. The mRNA-containing LNPs were characterized using a series of physicochemical assays, along with functional in vitro tests in primary human T cells. For example, 10 pg of mRNA encoding an anti-BCMA CAR was tested. mRNA was prepared in lOx RNA Buffer 1 (Precision Nanosystems # 1000681) and water to yield 35 pl of mRNA solution. 32 pL of mRNA solution and 16 pL of lipid mixture (Precision Nanosystems #1000701) were transferred to a synthesis cartridge, which was inserted into the instrument. The synthesis run was then carried out per the manufacturer’s instructions, and the resulting LNPs were collected from the instrument cartridge and transferred to microcentrifuge tubes. The LNPs were diluted 2x with Buffer 2 (Precision Nanosystems # 1000682). mRNA loading in the LNPs was measured using a RiboGreen assay and the concentration was 52 ng/pL of mRNA.

[00251] To assess the ability of the LNPs to transfect human T cells with the CAR-encoding mRNA, lymphocytes were obtained from whole blood of a healthy human donor. From these lymphocytes, CD8⁺ T cells were positively selected by use of paramagnetic microbeads conjugated to an anti-CD8 antibody. This yielded cells that were approximately 95% CD8⁺ T cells and 95% viable. These enriched CD8⁺ T cells were expanded by incubation at 37 °C with 5% CO2 in the presence of anti-CD3 antibody (clone OKT3), IL-7, and IL-15 for 3 days. The cells were then resuspended in complete T cell media (X-Vivo 15, Lonza + lx Gibco GlutaMax) with 0.5% human AB serum (ImmunoCult™-XF, Stem Cell Technologies). On day 3 of activation, cells were re-plated at a density of 0.5 M cells/mL in a volume of 200 pL/well containing 0.1 ug/mL ApoE. The transfection was carried out by addition of the formulated LNP volumes corresponding to mRNA doses of 250 ng or 500 ng of mRNA (i.e., 5 pL or 10 pF). After 6 hours, 12 hours, and 24 hours, the cells were washed to remove excess nucleic acid and particles, then returned to culture in complete medium with 5% human AB serum.

[00252] At 24 hours, 48 hours, and 72 hours after addition of the LNPs, CAR T cells obtained from this process were assessed for viability and expression of anti-BCMA CAR protein, and BCMA binding. Viability, CAR expression, and BCMA binding were determined by flow cytometry on a Guava® EasyCyte® 12HT Flow cytometer (Luminex). To assess viability, a sample of the CAR T cells were mixed with PI and acridine orange and analyzed by fluorescence microscopy using a Nexcelom Auto 2000 cytometer. To determine CAR protein expression and BCMA binding, a sample of the CAR T cells were incubated with 0.4 pg/mL of allophycocyanin (APC)-conjugated BCMA (Recombinant Human TNFRSF17 protein, Fc- tagged, APC labeled; Creative Biomart, Shirley, NY). CAR expression was assessed on the flow cytometer with electronic gating on fluorescence in the red channel to detect presence or absence of emission from BCMA- APC on CAR-positive and CAR-negative cells. BCMA binding was determined by measuring the intensity of fluorescence in the red channel to determine the relative quantity of BCMA- APC bound to labelled CAR-positive cells.

Controls for these studies included negative control (non-CAR) CD8⁺ T cells generated by exposure to media with free mRNA lacking ENPs.

LNPs (Precision) containing anti-BCMA CAR mRNA efficiently transfect human T cells.

[00253] As shown in Tables 1-3, viability modestly decreased with increasing transfection periods using the ENPs. This level was lower relative to free mRNA controls, but cells recovered in all cases as the analysis time increased from 24 hours - 72 hours. Viability was generally insensitive to the dosing range tested, though a few samples showed a modest trend of decreased viability at the higher dose. After 24 hours, the transfection levels associated with LNPs trended higher as a function of both mRNA dose and transfection period. The frequencies of cells expressing the anti-BCMA CAR ranged from 86.8% ± 1.8% to 96.9% ± 0.1%. Analogous results for dose and transfection period were observed at 48 hours and 72 hours, though the frequency of transfected cells decreased with analysis time. At 48 hours and 72 hours, respectively, the frequency of cells expressing anti-BCMA CAR ranged from 73.6% ± 2.1% to 97.7% ± 0.2% and 56.3% ± 2.9% to 88.9% ± 0.1%. Together these data suggest LNPs can be used to efficiently transfect primary human T cells with mRNA encoding anti-BCMA CARs, leading to CAR expression with suitable viability.

Table 1: anti-BCMA CAR expression and T cell viability 24 hours after transfection initiation

mean ± SD (n = 2), *n = 1

Table 2: anti-BCMA CAR expression and T cell viability 48 hours after transfection initiation

mean ± SD (n = 2), *n = 1

Table 3: anti-BCMA CAR expression and T cell viability 72 hours after transfection initiation

mean ± SD (n = 2), *n = 1

[00254] Anti-BCMA CAR⁺ T cells generated using LNPs as described were assessed in vitro to validate the LNPs by confirming the ability to kill BCMA⁺ myeloma (tumor) cells and secrete target-induced cytokines. 50,000 CAR⁺ T cells generated from each culture condition (i.e., dose, transfection period, culture time) were co-incubated with 100,000 MM1S tumor cells in wells of a 96-well V-bottom plate; these cells express BCMA. Additional cultures of CAR T cells alone (without target cells) were prepared as a control. Following 24 hours of incubation at 37°C, tissue culture supernatants and cells were harvested. CAR expression is analyzed by staining cells with CD8-BV421 antibody, PI and BCMA-APC. Viable CD8⁺ T cells were identified by exclusion of dead cells staining with PI (near-infrared fluorescence) and selection of CD8⁺ cells with blue fluorescence off the violet laser. CAR expression on total viable CD8⁺ T cells is quantitated by intensity of red fluorescence off the red laser. Signaling of the CAR is evaluated by analysis of IFNy production in the tissue culture supernatant by specific ELISA.

[00255] The capacity of the remaining expressed CAR protein to signal is determined by a secondary culture. The secondary culture is set up by co-culturing pre-exposed CAR T cells with MM1S-GFP tumor cells. Aliquots of 50,000 MM1S-GFP tumor cells were placed in wells of a 96-well plate. CAR T cells from the primary cell culture were washed to remove residual components of the media. Between 1,500 to 50,000 washed CAR T cells were added to each well to obtain specific effector:target ratios (i.e., ratios of CAR T cells to BCMA⁺ myeloma cells) that were between 1:1 and 1:32. Following 24 hours - 72 hours of incubation, PI was used to stain dead cells. Viable target cells were identified by expression of GFP (green fluorescence off the blue laser) and exclusion of PI, and cell density was determined by flow cytometry. The degree of myeloma cell killing by the CAR T cells was calculated by comparison to the number of myeloma cells in concurrent control wells that do not contain CAR T cells. Signaling was further evaluated by analysis of IFNy production in the supernatant using specific ELISA.

CAR T cells transfected using LNPs (Precision) detect target and produce IFNy.

[00256] Supernatants of co-cultures between the CAR T cells and BCMA⁺ MM IS multiple myeloma were analyzed for expression of IFNy by ELISA. This analysis revealed IFNy levels correlating with the level of BCMA CAR expression. The highest levels were observed during co-culture with T cells exhibiting the highest degree of BCMA expression as a function of dose and transfection period. Since expression was greatest in LNP-transfected cells 24 hours after transfection, co-cultures with these cells generated the greatest IFNy levels.

CAR T cells transfected using LNPs (Precision) efficiently kill MM1S target cells.

[00257] The T cells above exposed to MM IS co-culture (“pre-exposed”) were then cultured in a secondary culture. Cytotoxicity was evaluated by co-culture at defined effector:target ratios. These analyses result in BCMA-dependent cell killing and cytokine (IFNy) production that correlates with BCMA CAR expression on T cells resulting from each transfection strategy. Thus, CAR T cells exhibiting the highest degree of BCMA expression drive the most efficient target cell killing and cytokine production. As with the IFNy assay above, killing was greatest in LNP-transfected cells 24 hours after transfection. Taken together, these in vitro analyses suggest that LNPs transfect primary human T cells, leading to anti-BCMA CAR expression in T cells that respond to and efficiently kill cells expressing the target. [00258] The validated anti-BCMA CAR mRNA LNPs were next used in an method to show the ability of diffusion-limited particles injected intra-LN in mice to i) retain anti-BCMA CAR mRNA in LNs, ii) deliver this mRNA to cells in LNs and migrating through LNs, and iii) drive subsequent CAR expression in these cells. For these assessments, mRNA encoding anti-BCMA CAR was covalently labeled with Cy5 fluorescent label per the manufacturer’s instructions (Minis Bio). A control of soluble (labeled) mRNA encoding anti-BCMA CAR was also prepared. Next, the inguinal LN injection site was identified in C57BL/6J mice (female, 4-6 weeks), by removing the hair surrounding the ventral inguinal region with a mild depilatory cream. Mice were then injected subcutaneously at the tail base with 10 pL of a 0.1% (w/v) solution of tracer dye (Evan’s Blue, VWR). One day after tracer dye injection, the left inguinal LN was visually identified through accumulation of the tracer dye. Using a 31- gauge insulin syringe, the left inguinal LN was injected (N=5 mice/cohort) with 10 pL of solution containing each mRNA particle formulation or the soluble mRNA control formulation. For all formulations, the final injected mRNA dose in the injected volume was 100 ng, 1 |jg, and 10 |jg. The mRNA dose was used as a basis for determining particle dose in conjunction with mRNA mass loading levels in each particle formulation. For control cohorts, the same formulations were instead injected i) intra-muscularly (z.m.) at the left thigh (25 pL, or ii) subcutaneously (s.c.) at the left side of the tail-base (50 pL). At 4 hours, 24 hours, 48 hours, 96 hours, 1 week, 2 weeks, 4, weeks, 6 weeks, 8 weeks, 10 weeks, and 12 weeks cohorts were imaged by IVIS. Guided by IVIS imaging, three replicate sets of cohorts were euthanized at 4 hours, 24 hours, and 96 hours, then the injected LN (i.e., left inguinal), uninjected LNs, spleen, major organs, and peripheral blood were isolated. Flow cytometry was used to assess cell populations of interest for i) viability (PI), ii) morphology (FSC/SSC), iii) presence of the injected mRNA, iv) expression of the anti-BCMA CAR, and v) T cell phenotypic profiles. For the flow cytometry analysis, tissues were isolated and processed to single cell suspensions. The following cell compartments were stained: T cells (CD3), B cells (B220), and APCs (cdllc, dendritic cells; F4/80, macrophages).

LNPs injected intra-LN localized and retained anti-BCMA CAR mRNA in LNs and induced anti-BCMA CAR expression in LN resident cells and lymphocytes migrating through LNs.

[00259] After 4 hours, analysis of the left inguinal LN by IVIS and flow cytometry on the LN source cells revealed increased levels of each cell type associated with Cy5 signal in the LNPs cohorts, and to a lesser extent, cohorts receiving free mRNA. Conversely, in the right inguinal LNs and all other tissues, Cy5 signal was absent. At 24 hours and 96 hours, Cy5 signal was absent from all tissues in mice treated with soluble mRNA. In contrast, all particle formulations exhibited clear Cy5 fluorescence in the treated LN at both 24 hours and 96 hours; signal was absent in other sites. Fluorescence for all particle groups decreased from 24 hours to 96 hours. Expression of anti-BCMA mRNA correlated with these results for the particle groups and was absent for soluble mRNA cohorts. Thus, for particle groups, after 4 hours, 24, hours, and 96 hours, antibody staining and flow cytometry revealed clear anti- BCMA CAR expression in cells in the left inguinal LN. No detectable levels of CAR expression are expected in uninjected LNs or other tissues. Expression was present in common LN resident cell compartments, including APCs, LECs, T cells, and B cells. In blood, a moderate but significant frequency of lymphocytes showed expression of the CAR at all three time points; for all tissue types, the particle-driven expression was higher at 96 hours relative to 24 hours and 4 hours. For all animals receiving s.c. or i.m. injections, no Cy5 signal was detected, nor was any expression of anti-BCMA CAR measured. Taken together, these data suggest an method using intra-LN injection of anti-BCMA CAR mRNA LNPs to achieve superior retention and uptake of mRNA in LN-resident cells, and subsequent expression of the anti-BCMA CAR. Additionally, the study revealed the ability to localize these processes to specific LNs. The examples describe an approach to intra-LN injection of particles loaded with CAR-encoding mRNA to drive CAR expression in LN-resident cells and circulating lymphocytes that could not be achieved using free mRNA.

Example 2. in vivo biodistribution/expression of anti-BCMA CAR; LNP/ApoE

[00260] This example describes a method to enhance transfection of LN-resident cells with anti-BCMA CAR mRNA after LN injection by preparing LNPs integrating both the mRNA and ApoE. The ApoE is incorporated directly in LNPs synthesized using microfluidics (NanoAssemblr, Precision Nanosystems) - instead of adding this molecule to culture, while maintaining the transfection capabilities of these LNPs. This example is analogous to Example 1, but 5 pL of ApoE solution is added to each lipid mixture prior to the LNP synthesis run. Thus, for the anti-BCMA CAR mRNA loading level described in Example 1, a replicate synthesis run is carried out for ApoE inputs of 0.1 pg, 0.5 pg, 2.5 pg, and 25 pg.

Incorporation of ApoE is measured using microBCA protein assay on an aliquot of each LNP synthesis product. The LNPs are then used to transfect T cells without addition of soluble ApoE to culture media and compared to LNPs prepared as in Example 1, where soluble ApoE was added to culture media. The in vivo biodistribution studies are also carried out in an analogous manner, comparing mRNA retention in LNs and CAR expression between mRNA LNPs prepared as in Example 1 against LNPs integrating ApoE along with the mRNA at equivalent mRNA doses.

ApoE incorporated in LNPs is expected to enhance local mRNA delivery and retention in LNs, and in vivo expression of anti-BCMA CAR.

[00261] Using the approach above, these examples are expected to reveal anti-BCMA CAR expression in vitro for all LNPs containing ApoE. Higher inputs of ApoE are expected to increase ApoE loading in the particles and the corresponding transfection. With respect to target- induced cytokines and MM IS cell killing, it is anticipated that CAR expression induced by ApoE-containing LNPs will be similar relative to equivalent levels of expression induced by LNPs cultured with soluble ApoE. It is expected that the ApoE-containing LNPs will be well tolerated, as determined by viability measurements. During in vivo studies, LNPs integrating ApoE are expected to significantly increase retention of Cy5-labeled mRNA at all timepoints relative to the LNPs lacking ApoE, and this signal is expected to localize to the treated LNs. Correspondingly, increased expression of anti-BCMA CAR in the treated LNs and in circulating lymphocytes relative to LNPs lacking ApoE at each dose and analysis time is anticipated. This example is expected to be consistent across both IVIS and flow cytometry measurements. These examples are expected to support inclusion of ApoE in CAR mRNA- containing LNPs as a method to further enhance delivery and local transfection of LN- resident cells and cells migrating through LNs.

Example 3. in vivo biodistribution/expression of anti-BCMA CAR; LNP/ApoE/CpG

[00262] This example describes a method to further enhance expression of anti-BCMA CAR in LN-resident cells after LN injection by preparing LNPs integrating an activating adjuvant signal, CpG. CpG is incorporated directly in LNPs synthesized using microfluidics (NanoAssemblr, Precision Nanosystems) using a study procedure analogous to Example 2 (i.e., LNPs with ApoE); however, a mass of CpG equivalent to 5% and 10% of the mass of mRNA is added to the mRNA mixture solution prior to transfer to the instrument cartridge. Thus, for the anti-BCMA CAR mRNA loading level described in Example 1, replicate synthesis runs are carried out with 5% and 10% CpG mass input. Incorporation of CpG is measured using Cy3 labeling and fluorescence detection. LNPs (all with ApoE) are then used to transfect T cells and are compared to LNPs prepared as in Example 2 that lack CpG. The in vivo biodistribution studies are also carried out in an analogous manner, comparing mRNA retention in LNs and CAR expression between mRNA LNPs prepared as in Example 2 (i.e. with ApoE) against LNPs integrating CpG along with the mRNA (and ApoE) at equivalent mRNA doses.

CpG incorporation in LNPs is expected to enhance local transfection and anti-BCMA CAR expression in LNs.

[00263] Using the approach above, these examples are expected to reveal anti-BCMA CAR expression for all LNPs containing CpG. It is expected that higher inputs of CpG increase CpG loading, but do not increase transfection levels. However, with respect to target-induced cytokines and MM IS cell killing, CAR expression induced by CpG-containing LNPs is expected to increase relative to equivalent levels of expression induced by LNPs lacking CpG. It is expected that the CpG-containing LNPs will be well tolerated, as determined by viability measurements. During in vivo studies, it is anticipated that LNPs integrating CpG will not impact retention of Cy5-labeled mRNA, but will significantly increase expression of anti-BCMA CAR in the treated LNs and in circulating lymphocytes relative to LNPs lacking CpG at each dose and analysis time. These examples are expected to support inclusion of

CpG in CAR mRNA-containing LNPs (with ApoE) as a method to enhance expression of anti-BCMA CAR during transfection of LN-resident cells and cells migrating through LNs. Example 4. in vivo biodistribution/expression of anti-BCMA CAR; LNP in depots

[00264] This example describes a method to enhance local transfection of LN-resident cells and cells migrating through LNs using LNPs loaded in diffusion-limited MP depots; these depots are too large to drain from LNs, thus maximizing local retention and delivery of anti- BCMA CAR mRNA after intra-LN injection. A mass of LNPs -prepared and validated as in Example 1 - containing 500 pg of anti-BCMA CAR mRNA is prepared in 500 pL of aqueous buffer. To load the LNPs in MPs, a polymer-containing organic phase is prepared using 80 mg PLGA with a 50=50 lactide: glycolide ratio dissolved in 5 mL dichloromethane. The 500 pL of mRNA-containing LNPs is added to the polymer/lipid-containing organic phase while sonicating on ice for 30 seconds at 12 W. The resulting water/oil emulsion is homogenized to form a double emulsion (water/oil/water) and create the MPs. This involves adding the primary emulsion to 40 mL distilled water over a period of 1 minute while homogenizing at 12,000 rpm. The emulsification is performed for a total of 3 minutes, and solvent is removed by stirring overnight. After solvent removal, particles are collected by centrifugation at 4 °C, washed 3x with distilled water, and resuspended in buffer for immediate use. The resulting MPs loaded with the LNPs containing mRNA are assessed for i) mRNA loading ii) particle size and polydispersity using dynamic light scattering, iii) charge by surface potential measurement, and iv) morphology using scanning electron microscopy. For mRNA loading, a known particle mass is hydrolyzed in 0.2 M NaOH or dissolved in DCM to enable UV-visible absorbance measurements (mRNA). Release of the CAR-encoding mRNA is assessed by incubation of known particle masses in buffer at defined temperatures, sampling supernatants over time for up to 3 months; this allows quantification of nucleic acid concentration and construction of kinetic release curves. These studies are expected to reveal that LNPs loaded with anti-BCMA CAR mRNA can be loaded into MPs depots, releasing the RNA as the MPs degrade. This creates a platform for exploiting diffusion-limited retention in LNs for in vivo CAR therapy by drawing on larger scale MP depots.

LN injection of LNPs in MPs is anticipated to confer greater retention and CAR expression than LNPs alone.

[00265] The LNPs loaded in diffusion-limited MPs are used to demonstrate a method for superior i) retention of anti-BCMA CAR mRNA in LNs, ii) delivery of mRNA to cells in LNs and migrating through LNs, and iii) subsequent CAR expression in mice. This is achieved by comparing LNPs and LNPs loaded in MPs - containing equivalent mRNA doses - using the in vivo biodistribution design in Example 1. This example is expected to reveal that LNPs loaded in MP maximize mRNA signal in treated LNs at all doses and time points, with higher levels compared to LNPs not loaded in MP depots. Correspondingly, it is anticipated that relative to LNPs, LNPs loaded in MP depots will show increased anti-BCMA CAR expression within the treated LN and in circulating lymphocytes at all time points. For both formulations, the signal is expected to be absent in untreated LNs and other tissues.

Taken together, this would suggest a platform for locally conditioning LNs for delivery and expression of CAR-encoding mRNA at levels that cannot be achieved with LNPs alone.

Example 5. in vivo biodistribution/expression of anti-BCMA CAR; LNP/ApoE in depots [00266] This example describes a method to further enhance the performance of anti-BCMA CAR mRNA LNPs loaded into MPs by using LNPs from Example 2 that integrate ApoE.

These LNPs are loaded into diffusion-limited depots as in Example 4, then compared against LNPs (without ApoE) loaded in MPs using the in vivo biodistribution study design in

Example 1. mRNA LNPs with ApoE loaded in MP depots are expected to enhance retention and

CAR expression.

[00267] Using the approach above, it is anticipated that LNPs integrating ApoE and loaded in MPs will significantly increase retention of Cy5-labeled mRNA at multiple timepoints relative to the LNPs without ApoE loaded into MPs; in both cases it is expected that signal is localized to the treated LNs. Correspondingly, it is expected that ApoE LNPs loaded in MPs will show increased expression of anti-BCMA CAR in the treated LNs and in circulating lymphocytes relative to the LNPs (without ApoE) loaded in MPs at each dose and analysis time. These results are expected to be consistent across both IVIS and flow cytometry measurements. In conclusion, these examples are expected to support inclusion of ApoE in LNPs loaded in MP depots as a method to further enhance delivery and local transfection of LN-resident cells and cells migrating through LNs.

Example 6. in vivo biodistribution/expression of anti-BCMA CAR; LNP/ApoE/CpG in depots

[00268] This example describes a method to further enhance expression of anti-BCMA CAR in LN-resident cells after LN injection using MP depots loaded with LNPs integrating an activating adjuvant signal, CpG. CpG is incorporated directly in LNPs as described in Example 3 (i.e., with ApoE). These LNPs are loaded into diffusion-limited depots as in Example 4, then compared against LNPs (with ApoE, but without CpG) loaded in MPs using the in vivo biodistribution study in Example 1.

CpG incorporation in LNPs loaded in MPs are expected to enhance local transfection and anti-BCMA CAR expression in LNs.

[00269] Using the approach above, MPs containing LNPs integrating CpG (and ApoE) are not expected to impact retention of Cy5-labeled mRNA. However, at multiple analysis times and doses, these MPs are expected to show increased expression of anti-BCMA CAR in the treated LNs and in circulating lymphocytes relative to MPs containing LNPs (with ApoE) that lack CpG. In conclusion, these examples are expected to support inclusion of CpG in CAR mRNA-containing LNPs (with ApoE) loaded in MPs as a method to enhance expression of anti-BCMA CAR during transfection of LN-resident cells and cells migrating through LNs.

Example 7. Efficacy with anti-BCMA CAR; one vs. multiple LNs; Examples 1-6 [00270] This example describes a strategy to control tumor burden in mice by intra-LN injection of the distinct particle-based formulations described in Examples 1-6, each containing mRNA encoding anti-BCMA CAR. The study design includes determination of the extent to which these formulations control tumor burden irrespective of whether the total dose is administered to a single LN or split between or among multiple LNs. mRNA encoding anti-BCMA CAR mRNA is prepared using the best formulation from each of Examples 1-6, as selected based on the highest frequency of BCMA CAR expression in treated LNs 24 hours after intra-LN injection in each example. Thus, the example delineates the relative performance of i) LNPs without ApoE, ii) with ApoE, iii) with ApoE/CpG, and iv - vi) each of these LNPs loaded in MP depots. NOD-scid-gamma (NSG) mice are inoculated with 2 million MMlS-fluc human multiple myeloma tumor cells s.c. in the central scruff. Tumor burden is monitored by serial bioluminescence imaging. On Day 5 mice are randomized, then one set of cohorts receives an intra-LN injection of the total dose (lx) in the left inguinal LN. Another set of cohorts receives 0.5x of each dose in each of the left inguinal LN and right inguinal LN. Control cohort includes mice receiving vehicle or free mRNA in the right and left inguinal LN, and cohorts administered the best performing formulation via the s.c. and i.m. Tumor burden is measured daily to confirm reduced burden over time against vehicle. Anti-tumor response is expected to be maximized by intra-LN delivery of LNPs

(ApoE/CpG) loaded in MPs

[00271] All six LNP formulations are expected to reduce tumor burden, but LNPs loaded in MPs are expected to outperform LNPs not loaded in LNPs. It is expected that the relative level of efficacy will decrease from MPs/LNPs (ApoE/CpG), MPs/LNPs (ApoE), MP/LNPs, LNPs (ApoE/CpG), LNPs (ApoE), and LNPs. It is expected that injecting the free RNA and vehicle controls intra-LN will not confer any significant effects, nor would particles administered by other routes. Reduction in tumor burden is expected to be equivalent, irrespective of whether the total dose is administered to a single LN or split between two nodes. Taken together, it is anticipated that intra-LN injection of LNPs in MPs particles will provide efficacy in a humanized cancer model - without constraint on the number of LNs over which the dose is distributed. Further, the study is expected to confirm that intra-LN injection is required for efficacy, as is formulation of the CAR-encoding mRNA into particles. This study is also expected to support flexibility in selecting and dosing easily accessible LNs.

Example 8. Efficacy with anti-BCMA CAR; tumor draining vs. contra-lateral LN;

Examples 1-6

[00272] The example describes a method showing intra-LN injection of LNPs in MPs containing anti-BCMA mRNA controls tumor burden, even when the LN treated is nontumor draining. The same six mRNA-encoding anti-BCMA CAR particles are prepared as in Example 9. NOD-scid-gamma (NSG) mice are inoculated with 1 million MMlS-fluc human multiple myeloma tumor cells s.c. in the left flank. Tumor burden is monitored by serial bioluminescence imaging. On Day 5 mice are randomized, then each cohort receives an intra- LN injection in the right inguinal LN. Another set of cohorts receives injections in the left inguinal LN. Control cohorts include mice receiving vehicle or free mRNA in the right or left inguinal LN.

Anti-tumor therapy using intra-LN delivery of LNPs (ApoE/CpG) loaded in MPs is expected to be an agonist to treated LN location.

[00273] All six LNP formulations are expected to reduce tumor burden, but LNPs loaded in MPs are expected to outperform LNPs not loaded in LNPs. The relative level of efficacy is expected to decrease from MPs/LNPs (ApoE/CpG), MPs/LNPs (ApoE), MP/LNPs, LNPs (ApoE/CpG), LNPs (ApoE), and LNPs. The free RNA and vehicle controls injected intra-LN are not expected to not confer any significant effects, nor would particles administered by other routes. Reduction in tumor burden is expected, irrespective of whether the treated LN is proximal or distal to the tumor. Taken together, this example is anticipated to show that intra- LN injection of particles provides efficacy in a humanized cancer model without a constraint on which LN is treated relative to the tumor site. This example is expected to support flexibility for future human therapies in which tumor locations are unclear or widespread.

Example 9. in vivo biodistribution/expression of anti-CD19 CAR; LNP

[00274] This example describes a method to localize anti-CD19 CAR mRNA to LNs in mice using intra-LN delivery of LNPs, resulting in delivery to and expression of the CAR in LN- resident cells and cells migrating through LNs. The six types of LNPs are prepared as described in Examples 1-3, but the mRNA instead encodes an anti-CD19 CAR. The in vitro methods described in Example 1 are carried out, replacing MM1S+ cells for Raji cells that express CD19. For these co-culture studies, Raji cells exposed to all LNP designs (i.e. LNP, LNP w/ ApoE, LNP with ApoE and CpG) transfect the T cells with the anti-CD19 CAR. The greatest expression is expected at higher mRNA doses and longer transfection periods. The formulations are expected to be well tolerated and generated T cells are expected to be capable of both binding the target (i.e., CD19) and killing Raji cells expressing the target. After in vitro validation, each of these three LNP designs containing anti-CD19 CAR mRNA is also loaded in MP depots, as in Examples 4-6 (but with anti-CD19 CAR mRNA). Thus, a total of six designs are created containing mRNA encoding anti-CD19 CAR: i) LNPs, ii) LNPs (ApoE), iii) LNPs (CpG), iv) LNPs in MPs, v) LNPs (ApoE) in MPs, and vi) LNPs (ApoE/CpG) in MPs. Together, these examples are expected to show the robustness of the approach to transfect primary human T cells with mRNA encoding CARs of different sequences.

Expression of anti-CD19 CAR is expected to be maximized using intra-LN delivery of LNPs (ApoE/CpG) loaded in MPs.

[00275] Next, the set of six particle designs above are used in an approach for the superior ability of diffusion-limited particles injected intra-LN to provide retention of anti-CD19 CAR mRNA in LNs, deliver this cargo to cells in LNs and migrating through LNs, and to drive subsequent CAR expression in mice. Except for the substituted particle formulations containing mRNA encoding anti-CD19 CAR, the study design, read-out, and outcomes are analogous to the in vivo biodistribution study of Example 1. All of the particle formulations are expected to provide sustained retention of mRNA in the treated LN, and significant expression of the CAR in treated LNs and circulating T cells at all timepoints are assayed. As with the anti-BCMA CAR studies, the relative performance of these parameters is expected to decrease from MPs/LNPs (ApoE/CpG), MPs/LNPs (ApoE), MP/LNPs, LNPs (ApoE/CpG), LNPs (ApoE), and LNPs. In all cases, both LN delivery and mRNA loading in particles are useful for CAR expression, as soluble mRNA are not expected to lead to expression, nor is administration of particles by other injection routes. Thus, the example is expected to show the robustness of the approach for delivery and expression of mRNA encoding CARs using diffusion-limited particles introduced to LNs. Example 10. Efficacy with anti-CD19 CAR; tumor draining vs. contra-lateral LN; Examples 9-14

[00276] The example describes a method showing intra-LN injection of LNPs in MPs containing anti-CCl 9 mRNA controls tumor burden, even when the LN treated is non-tumor draining. The same six mRNA-encoding anti-CD19 CAR particles are prepared as in Example 9. The study details are otherwise analogous to that of Example 8 but using a known animal model suitable to facilitate testing of mRNA encoding the anti-CD19 CAR. Mice of this model are inoculated with corresponding tumor cells s.c. in the left flank. Tumor burden is monitored by serial bioluminescence imaging. On Day 5 mice are randomized, then each cohort receives an intra-LN injection in the right inguinal LN. Another set of cohorts receive injections in the left inguinal LN. Control cohorts include mice receiving vehicle or free mRNA in the right or left inguinal LN.

Anti-tumor therapy using intra-LN delivery of LNPs (ApoE/CpG) loaded in MPs is expected to be an agonist to treated LN location.

[00277] All six LNPs formulations are expected to reduce tumor burden, but LNPs loaded in MPs are expected to outperform LNPs not loaded in LNPs. The relative level of efficacy is expected to decrease from MPs/LNPs (ApoE/CpG), MPs/LNPs (ApoE), MP/LNPs, LNPs (ApoE/CpG), LNPs (ApoE), and LNPs. It is expected that injecting the free RNA and vehicle controls intra-LN id would not confer any significant effects, nor would particles administered by other routes. Reduction in tumor burden is expected, irrespective of whether the treated LN is proximal or distal to the tumor. Taken together, this example is expected to show that intra-LN injection of particles provides significant efficacy in a humanized cancer model without a constraint on which LN is treated relative to the tumor site. This example is expected to support flexibility for future human therapies in which tumor locations are unclear or widespread. Example 11. Human example in MM

[00278] This example describes a strategy to eradicate myeloma cells and treat disease in patients with MM using intra-LN injection of diffusion-limited MPs containing NPs loaded with ApoE, CpG, and mRNA encoding anti-BCMA CAR. Patients with MM are prepared with lymphodepleting chemotherapy or no conditioning therapy then receive 100 pg of mRNA, formulated in the LNPs (w/ mRNA/ApoE/CpG) loaded in MP depots. The injected LNs are identified using ultrasound guidance, MRI guidance, superficial surgery access, or other methods. The dose is administered to one or more LNs at one or more access sites, based on ease of access and other patient- specific factors. The procedure is performed as an outpatient procedure by an interventional radiologist or imaging technician. Serum M-protein levels, free light chains of the MM-related immunoglobulin, soluble serum BCMA levels, peripheral blood CAR⁺ T cell counts, serum cytokine levels (e.g., IFN-y, IL-2, IL- 10), and bone marrow biopsies are analyzed at 2, 4, 8, 12 and 24 weeks after treatment. CAR T cells generated in vivo through intra-LN injection of these particles are expected to effectively eradicate the myeloma, as measured by reduction of serum M-protein levels, free light chains of the MM-related immunoglobulin, soluble serum BCMA levels, and MM cells in bone marrow biopsies.

Example 12. Human example in MG

[00279] This example describes a strategy to control autoimmune disease using intra-LN injection of diffusion-limited MPs containing NPs loaded with ApoE, CpG, and mRNA encoding anti-BCMA CAR. Patients with MG are prepared with lymphodepleting chemotherapy or no conditioning therapy then receive 100 pg of mRNA, formulated in the LNPs (w/ mRNA/ApoE/CpG) loaded in MP depots. The injected LNs are identified using ultrasound guidance, MRI guidance, superficial surgery access, or other methods. The dose is administered to one or more LNs at one or more access sites, based on ease of access and other patient-specific factors. The procedure is performed as an outpatient procedure by an interventional radiologist or imaging technician. Anti-autoantigen antibodies (e.g. anti-AChR or anti-MUSK), soluble serum BCMA levels, peripheral blood CAR+ T cell counts, serum cytokine levels (e.g. TNF, IL-6, IL-2, IFN-y, IL- 10), and clinical assessment of disease such as the MG Activities of Daily Living Scale (MG-ADL) are assessed in patients at 2, 4, 8, 12, 24, and 52 weeks after treatment. CAR T cells generated in vivo through intra-LN injection of particles are expected to effectively control the autoimmune disease as measured by reduction of auto-antibody levels, reduction in circulating cytokine concentrations and reduced clinical manifestations of disease as measured by decreases in the MG-ADL or other clinical score.

Example 13.

[00280] The following are examples of specific molecules or moieties, e.g., markers, expressed in or on lymph nodes (e.g., cell surfaces or extracellular components of lymph nodes), for which one or more specific binders (e.g., antibodies, binding proteins, or ligands) can be integrated into one or more nanoparticles of the present disclosure or above Examples to improve the binding, concentration, residency time, pharmacokinetics, pharmacodynamics, and/or effect of the nanoparticles: RANKL; sLeX; PNAd; GL7; CLDN11; EFNB2; ACKR4; TNFRSF9; CCL20; CXCL5; GlyCAM-1 and CD34; Ptx3; CD209/Marco; SPNS2; CSF-1 ; CCL21 ; CCL19: CCL25; CCL27.

[00281] Specific binders, e.g., monoclonal antibodies, against each of the aforementioned markers are known in the art, and methods of integrating one or more such binders into LNPs are known in the art. Example 14.

[00282] This example describes a method to localize anti-BCMA CAR mRNA to lymph nodes (LNs) in mice using intra-LN delivery of lipid nanoparticles (LNPs), resulting in delivery to and expression of the CAR in LN-resident cells and cells migrating through LNs. The mRNA-containing LNPs were characterized using a series of physicochemical assays, along with functional in vitro tests in primary human T cells. LNPs were prepared using an in vitro transcribed mRNA encoding an anti-BCMA CAR (SEQ ID NO :1) and lipids from the Precision Nanosystems GenVoy-ILM T Cell Kit. 11 pg of mRNA was prepared in lOx Formulation Buffer 1 (Precision Nanosystems # 1000681) and water to yield 35 pl of mRNA solution and combined with 48 pL of Formulation Buffer 2 (Precision Nanosystems 1000682), and 16 pF of lipid mixture (Precision Nanosystems #1000680). The mixture was transferred to three different wells of a ENP synthesis cartridge, which was then inserted into the Precision Nanosystems NanoAssemblr Spark Instrument (# NIS0001). Synthesis was performed and the resulting ENPs were collected from the instrument cartridge and transferred to microcentrifuge tubes. The ENPs were diluted 2x with Formulation Buffer 2 (Precision Nanosystems # 1000682). mRNA loading in the ENPs was measured using a RiboGreen assay.

[00283] Human T cells were isolated by paramagnetic selection and activated with anti- CD3/anti-CD28 microspheres in the presence of IE- 15 and IL-7 for three days. As a comparator, non-activated human T cells were isolated. LNP mRNA formulations were mixed with ApoE, and in some instances with CpG oligodeoxynucleotide, prior to use. 100,000 activated or non-activated T cells were incubated with LNP formulations and cells were cultured for a period of 24 hours. Following incubation, the CAR Expression was analyzed according to the method described in Example 1. Table 4 shows the results of this analysis. Activated and non-activated T cells were robustly transfected with mRNA encoding anti-BCMA CAR using the LNP vector. [00284] Activated T cells transfected using the anti-BCMA mRNA LNP or control cells were cocultured with MM1S-GFP multiple myeloma cells overnight and cytotoxicity was evaluated by flow cytometry (Table 5). Transfection of activated T cells with mRNA LNP resulted in T cells that showed high levels of cytotoxicity (98% at 1:2 effector: target ratio). Cytotoxicity was specific to introduction of the CAR as negative control activated T cells showed < 20% cytotoxicity against the MM1S-GFP cell line. Cytotoxic effector function was observed at high levels in the presence and absence of additional immunomodulatory cues (CpG).

[00285] In summary, mRNA LNPs armed with mRNA encoding an anti-BCMA CAR provided potent expression of CAR in human T cells and provided cytotoxic effector function against BCMA+ target cells.

Table 4. Expression of anti-BCMA CAR on human T cells using an anti-BCMA CAR mRNA LNP

Table 4: Expression of anti-BCMA CAR on T cells in vitro using LNPs: Human T cells were isolated by paramagnetic selection and activated with anti-CD3/anti-CD28 microspheres in the presence of IL- 15 and IL-7 for three days. As a comparator, nonactivated human T cells were isolated. 100,000 activated or non-activated T cells were incubated with LNP vectors containing mRNA encoding anti-BCMA CAR in the presence of ApoE. Certain formulations included CpG. Cells were cultured for a period of 24 hours and CAR Expression was analyzed according to the method described in Example 1. Activated T cells electroporated with the same anti-BCMA CAR or water were used as positive and negative controls, respectively.

Table 5. Cytotoxicity of Activated T cells expressing anti-BCMA CAR from mRNA-LNP against BCMA⁺ MM1S-GFP tumor cells

Table 5: BCMA⁺ MM1S-GFP tumor cell killing by anti-BCMA CAR LNP transfected Activated T cells: Activated T cells transfected using the anti-BCMA mRNA LNP or control cells were cocultured with MM1S-GFP multiple myeloma cells overnight and cytotoxicity was evaluated by flow cytometry.

Example 15.

[00286] This example provides a method to perform controlled transfection by intra-LN delivery of synthetic particles, including LNPs (LNPs).

[00287] LNPs were prepared by methods described in Example 1 using individual IVT mRNAs encoding firefly luciferase (SEQ ID NO: 3) and anti-BCMA CAR (SEQ ID NO: 2) . The firefly luciferase mRNA was conjugated to the fluorescent dye Cy5. LNPs were formulated containing all mRNAs along with CpG oligodeoxynucleotide and ApoE. LNPs were injected into the inguinal LNs of C57BL/6 mice. After administration, fluorescence (FLI) and bioluminescence (BLI) were measured at Day 0, Day 1, and Day 2 by IVIS (Perkin Elmer).

[00288] One day after administration, LNP showed Cy5 FLI and luciferase BLI that was concentrated in the inguinal LN (FIG. 1A, center). mRNA was localized and retained in the LNs at day 2 - as visualized by Cy5, indicating persistence of labelled mRNA in the LN. BLI detected high levels of expression of the firefly luciferase mRNA (>1,500,000 photons/second) that required formulation of the mRNA with LNPs. In particular, IVT mRNA administered in a formulation that only provided activating factors and ApoE showed <10% of the luciferase expression (FIG. 1C).

[00289] In summary, intra- LN administration of LNP containing IVT mRNA encoding firefly luciferase and anti-BCMA CAR led to localized sequestration of mRNA and drove expression of mRNA in the LN. Particular formulation was required to achieve this outcome.

Example 16.

[00290] This example provides a method to perform controlled transfection of LN cells, including lymphocytes through intra-LN delivery of particles, including LNPs.

[00291] LNPs were prepared as detailed in Example 14. Single IVT mRNA constructs, 818 (SEQ ID NO: 6) and 811 (SEQ ID NO: 5) were designed that encoded both firefly luciferase and anti-BCMA CAR that were expressed from a single open reading frame using a linking T2A site (FIG. 2). Two fusion constructs were designed in such a way that in the 818 construct, the leading nucleic acid sequence encoded luciferase (followed by anti-BCMA CAR), whereas the in the 811 construct, the leading nucleic acid sequence encoded anti- BCMA CAR (followed by luciferase). In vitro analysis of the IVT mRNA constructs showed robust expression of both anti-BCMA CAR and luciferase from each fusion construct (FIGs. 3A-3B). IVT mRNA constructs were loaded in LNPs formulated as detailed in Example 14. Physicochemical properties, including size and concentration, were characterized as reported in Table 6. In the case of both constructs, the resulting LNPs exhibited uniform, reproducible properties.

[00292] Particles were injected into the inguinal LNs of C57BL/6 mice. At 30 minutes, 6 hours and 24 hours following administration, BLI was measured by IVIS (Perkin Elmer). BLI confirmed luciferase expression in the LNs of mice administered with IVT mRNALNP that were prepared using either 818 mRNA or 811 mRNA (FIG. 4A). In contrast, signal was not observed in the absence of LNP administration. Signal was observed at high levels from 30 minutes through 24 hours (FIG. 4B). These results were further confirmed upon excision of LNs and direct imaging, revealing clear expression in the LNs (FIG. 4A) consistent with the levels visualized in living animals (FIG. 4A)

[00293] Therefore, since expression was confirmed with luciferase in either position of the construct-and these constructs were fusions — the data indicated intra-LN delivery of IVT mRNA loaded in LNPs conferred sustained expression of luciferase and anti-BCMA CAR in

LN-resident cells.

Table 6. Physicochemical characterization of LNPs

Table 6: Physicochemical property characterization of LNPs formulated with 818 mRNA or 811 mRNA. Shown are measurements obtained using tunable resistive pulse sensing (TRPS) to measure size and concentration of a sample on a per-particle basis, or dynamic light scattering to measure surface/zeta potential or hydrodynamic diameter. Example 17.

[00294] This example provides a method to perform controlled transfection of lymphocytes by intra-LN delivery of nanoparticles, including LNPs and microparticle (MP) depots.

[00295] LNPs were prepared as detailed in Example 14. The 818 IVT mRNA fusion construct — encoding both luciferase and BCMA — described in Example 16 was used to formulate LNPs. IVT mRNA constructs were loaded in LNPs formulated in the presence or absence of ApoE and activating factors. Synthesis and characterization were carried out as described in Example 14 and Example 16. Particle formulations were injected into the inguinal LNs of C57BL/6 mice. Following administration, BLI was measured by IVIS (Perkin Elmer) at 30 minutes, 6 hours, and 24 hours post-administration.

[00296] BLI confirmed expression from the fusion construct in the LNs of mice administered with IVT mRNA LNP containing 818 mRNA (FIGs. 5A-5B). For all LNP formulations, expression was measured at 30 minutes post-administration and persisted beyond 24 hours. The addition of factors — including ApoE and T cell-activating factors — in the formulation led to substantial increases in mRNA expression compared with IVT mRNA LNP alone (a 6.7- fold increase in expression using ApoE/CD3/CD28, and a 12-fold increase in expression using ApoE/OKT3 at 6 hours after administration). In contrast to the LNP formulations, IVT mRNA alone or formulated with APOE and activating factors did not generate detectable expression at 30 minutes, with minimal expression at the other time points.

[00297] Therefore, intra-LN delivery of IVT mRNA LNP conferred sustained expression and functional activity of single mRNA constructs that encoded anti-BCMA CAR. Expression of the cargo mRNA was enhanced by modification of LNP through additional formulation components including ApoE and T cell-activating factors. Example 18.

[00298] This example provides a method to achieve diffusion-limited retention of MP depots loaded with mRNA encoding anti-BCMA CAR.

[00299] IVT mRNA encoding an anti-BCMA CAR (SEQ ID NO: 6) was prepared using a double emulsion solvent evaporation process with degradable poly(lactide-co-glycolide) (PLGA) as a degradable polymer carrier in the organic phase, mRNA in the aqueous phase, and poly(vinyl alcohol) in the secondary aqueous continuous phase. A lipophilic dye (DiD) that that labels hydrophobic structures was added during synthesis to fluorescently label the MP depots. The resulting MPs exhibited diameters of ~3 pm.

[00300] The MP depot formulations were injected into the inguinal LNs of C57BL/6 mice. Following administration to LNs, FLI was measured by IVIS (Perkin Elmer) at 30 minutes, 24 hours, and 48 hours. FLI revealed MP formulations in LNs immediately after administration from 30 minutes and continuing for at least 48 hours after administration (FIGs. 6A-6B).

[00301] Therefore, intra- LN delivery of MPs depots loaded with IVT mRNA encoding anti- BCMA CAR enabled sustained retention of particles in LNs. This local retention is expected to provide direct control over sustained delivery of CAR or other protein cargo encoded by IVT mRNA in the particles.

Example 19.

[00302] This example provides a method to perform controlled expression of CAR in lymphocytes by intra-LN delivery of nanoparticles, including LNPs.

[00303] LNPs containing IVT mRNA encoding an anti-BCMA CAR were prepared as detailed in Example 14 using IVT mRNA encoding anti-BCMA CAR (SEQ ID NO: 2) and firefly luciferase (SEQ ID NO: 3) (FIGs. 7A-7B). LNPs were formulated in the presence of ApoE and T cell activating factors. LNPs were characterized as in Example 16. [00304] Particle formulations were injected into the inguinal LNs of C57BL/6 mice. At 24 hours following administration, mice were sacrificed and inguinal LNs were harvested. Single cell suspensions were generated by mechanical dissociation, and expression of anti- BCMA CAR among LN-resident cell populations was analyzed by flow cytometry after surface antigen staining.

[00305] Anti-BCMA CAR expression was evaluated on lymphocytes through staining with recombinant BCMA-PE conjugate. Cell surface CD8, CD3, CD45, and CD 11b were evaluated using specific fluorochrome-conjugated antibodies. Staining of LNs from mice treated with PBS showed low background levels of staining for the BCMA-PE stain. In contrast, lymphocytes in LNs of mice treated with anti-BCMA CAR mRNA formulated with LNPs, ApoE, and activating factors exhibited increased recombinant BCMA-PE staining (FIG. 8. This indicated specific introduction of the IVT mRNA encoding the anti-BCMA CAR and expression of protein on subsets of lymphocytes including CD3+CD8+ T cells (FIG. 8).

[00306] Thus, intra- LN delivery provided efficient introduction of mRNA encoding anti- BCMA CAR in vivo. Expression of mRNA CAR by intra-LN particle administration was detectable on CD3+CD8+ T cells. The intra-LN administration method is therefore expected to provide a robust method of generation of CAR T cells in vivo.

[00307] All publications, patents, patent applications, publication, and database entries (e.g., sequence database entries) mentioned herein, e.g., in the Background, Summary, Detailed Description, Examples, and/or References sections, are hereby incorporated by reference in their entirety as if each individual publication, patent, patent application, publication, and database entry was specifically and individually incorporated herein by reference. In case of conflict, the present application, including any definitions herein, will control. Equivalents and Scope

[00308] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the embodiments described herein. The scope of the present disclosure is not intended to be limited to the above description, but rather is as set forth in the appended claims.

[00309] Articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between two or more members of a group are considered satisfied if one, more than one, or all of the group members are present, unless indicated to the contrary or otherwise evident from the context. The disclosure of a group that includes “or” between two or more group members provides embodiments in which exactly one member of the group is present, embodiments in which two or more members of the group are present, and embodiments in which all of the group members are present. For purposes of brevity those embodiments have not been individually spelled out herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed. [00310] It is to be understood that the present disclosure encompasses all variations, combinations, and permutations in which one or more limitation, element, clause, or descriptive term, from one or more of the claims or from one or more relevant portion of the description, is introduced into another claim. For example, a claim that is dependent on another claim can be modified to include one or more of the limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is to be understood that methods of making or using the composition according to any of the methods of making or using disclosed herein or according to methods known in the art, if any, are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. [00311] Where elements are presented as lists, e.g., in Markush group format, it is to be understood that every possible subgroup of the elements is also disclosed, and that any element or subgroup of elements can be removed from the group. It is also noted that the term “comprising” is intended to be open and permits the inclusion of additional elements or steps. It should be understood that, in general, where an embodiment, product, or method is referred to as comprising particular elements, features, or steps, embodiments, products, or methods that consist, or consist essentially of, such elements, features, or steps, are provided as well. For purposes of brevity those embodiments have not been individually spelled out herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.

[00312] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in some embodiments, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. For purposes of brevity, the values in each range have not been individually spelled out herein, but it will be understood that each of these values is provided herein and may be specifically claimed or disclaimed. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

[00313] In addition, it is to be understood that any particular embodiment of the present invention may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and/or methods of the present disclosure, can be excluded from any one or more claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein.

EMBODIMENTS

[00286] The following embodiments are within the scope of the present disclosure.

1. A composition comprising a plurality of microparticles; wherein each of the microparticles is between 1 and 100 microns, inclusive; and wherein each of the microparticles comprises a nucleic acid encoding a Chimeric Antigen Receptor (CAR).

2. The composition of embodiment 1; wherein each of the microparticles comprises a molecule that specifically binds to a lymph node; and wherein each of the microparticles comprises a nucleic acid encoding a Chimeric Antigen Receptor (CAR).

3. A composition comprising a plurality of microparticles; wherein each of the microparticles is between 1 and 100 microns, inclusive; wherein each of the microparticles comprises a molecule that specifically binds to a lymph node; and wherein each of the microparticles comprises a nucleic acid encoding a Chimeric Antigen Receptor (CAR).

4. A medicament for administration into a lymph node, the medicament comprising: a plurality of microparticles; wherein each of the microparticles is between 1 and 100 microns, inclusive; and wherein each of the microparticles comprises a nucleic acid encoding a Chimeric Antigen Receptor (CAR).

5. A medicament for administration into a lymph node, the medicament comprising: a plurality of microparticles; wherein each of the microparticles comprises a molecule that specifically binds to a lymph node; and wherein each of the microparticles comprises a nucleic acid encoding a Chimeric Antigen Receptor (CAR).

6. A medicament for administration into a lymph node, the medicament comprising: a plurality of microparticles; wherein each of the microparticles is between 1 and 100 microns, inclusive; and wherein each of the microparticles comprises a molecule that specifically binds to a lymph node.

7. A method for treating disease in a patient in need thereof, the method comprising administering to an individual in need thereof an effective amount of a composition comprising: a plurality of microparticles; wherein each of the microparticles is between 1 and 100 microns, inclusive; and wherein each of the microparticles comprises a nucleic acid encoding a Chimeric Antigen Receptor (CAR).

8. A method for treating disease in a patient in need thereof, the method comprising administering to an individual in need thereof an effective amount of a composition comprising: a plurality of microparticles; wherein each of the microparticles comprises a molecule that specifically binds to a lymph node; and wherein each of the microparticles comprises a nucleic acid encoding a Chimeric Antigen Receptor (CAR).

9. A method for treating disease in a patient in need thereof, the method comprising administering to an individual in need thereof an effective amount of a composition comprising: a plurality of microparticles; wherein each of the microparticles is between 1 and 100 microns, inclusive; wherein each of the microparticles comprises a molecule that specifically binds to a lymph node; and wherein each of the microparticles comprises a nucleic acid encoding a Chimeric Antigen Receptor (CAR).

Sequences

[00314] The following amino acid (AA) or nucleotide (nt) sequences are referenced herein. The nucleic acid sequences SEQ ID NOs: 1-6 encode the corresponding amino acid sequences SEQ ID NOs: 7-12. Except where otherwise noted, nucleic acid sequences set forth below and in the instant application may recite “T”s in a representative cDNA sequence but where the sequence represents RNA, the “T”s would be substituted for “U”s. Thus, any of the DNAs disclosed and identified by a particular sequence herein also discloses the corresponding RNA sequence where each “T” of the DNA sequence is substituted with “U”: SEQ ID NO : 1 (nt, Anti- BCMA CAR)

Claims

Claims

1. A nanoparticle (NP) comprising a nucleic acid, wherein the NP is capable of delivering the nucleic acid to cells after being injected into a lymph node of a subject.

2. The NP of claim 1, wherein the NP delivers the nucleic acid to cells after being retained in the lymph node of a subject after injection.

3. The NP of claim 1 or 2, wherein the nucleic acid encodes a chimeric antigen receptor (CAR)

4. The NP of any one of claims 1-3 further comprising at least one transfection agent.

5. The NP of claim 4, wherein the at least one transfection agent is ApoE.

6. The NP of claim 4 or 5, wherein the at least one transfection agent is CpG.

7. The NP of any one of claims 4-6, wherein the at least one transfection agent increases the delivery mRNA to the cells in the lymph node.

8. The NP of any one of claims 4-7, wherein the at least one transfection agent increases expression of the construct.

9. The NP of any one of claims 1-8 further comprising a T-cell activating factor.

10. The NP of claim 9, wherein the T-cell activating factor is CD3/CD28 or OKT.

11. The NP of claim 9, wherein the T-cell activating factor is a mitogen or superantigen.

12. The NP of claim 11, wherein the mitogen or superantigen is phorbol 12-myristate 13-acetate (PMA), ionomycin, and/or PHA (Phytohaemagglutinin P).

13. The NP of any one of claims 1-12 further comprising a molecular adjuvant.

14. The NP of claims 13, wherein the molecular adjuvant is an innate ligand.

15. The NP of claims 14, wherein the innate ligand is TLR, RIG, and/or STING.

16. The NP of claims 1-15 further comprising at least one lymph node binding moiety.

17. The NP of claim 16, wherein the lymph node binding moiety binds RANKL, sLeX, PNAd, GL7, CLDN11, EFNB2, ACKR4, TNFRSF9, CCL20, CXCL5, GlyCAM-1, CD34, Ptx3, CD209/Marco, SPNS2, CSF-1, CCL21, CCL19, CCL25, or CCL27.

18. The NP of any one of claims 1-17, wherein the nucleic acid encodes a CAR protein with an extracellular antigen-binding domain.

19. The NP of claim 18, wherein the extracellular antigen-binding domain binds BCMA.

20. The NP of claim 18, wherein the extracellular antigen-binding domain binds CD-19.

21. The NP of any one of claims 1-20, wherein the nucleic acid is an mRNA.

22. The NP of claim 21, wherein the NP comprises 250-500 ng of mRNA.

23. The NP of claim 1-22, wherein the NP is a lipid nanoparticle (LNP).

24. The NP of claim 23, wherein the LNP comprises ionizable lipids, stabilizing lipids, cationic lipids, helper lipids, sterol, or any combination thereof.

25. The NP of claim 1-22, wherein the NP is a polymer nanoparticle.

26. The NP of claim 25, wherein the polymer nanoparticles comprise cationic polymers.

27. The NP of any one of claims 1-26, wherein the concentration of NP in the lymph node is greater than the concentration in any other tissue of the subject.

28. The NP of any one of claims 1-27, wherein the concentration of NP in the lymph node is greater than the systemic biodistribution in the subject.

29. A nanoparticle composition comprising a plurality of the NPs of any one of claims 1-28.

30. A microparticle (MP) loaded with at least one nanoparticle of any one of claims 1- 29.

31. A microparticle (MP) loaded with a nucleic acid, wherein the microparticle is capable of delivering the nucleic acid to cells after being injected into a lymph node of a subject.

32. The MP of claim 31, wherein the MP is also loaded with at least one nanoparticle.

33. The MP of claim 31, wherein the MP is further mixed with at least one nanoparticle in solution.

34. The MP of any of claims 31-33, wherein the MP delivers the nucleic acid to cells after being retained in the lymph node of a subject after injection.

35. The MP of any of claims 31-34, wherein the nucleic acid encodes a chimeric antigen receptor (CAR)

36. The MP of any one of claims 31-35, wherein the MP is also loaded with at least one transfection agent.

37. The MP of any one of claims 31-35, wherein the MP is further mixed with at least one transfection agent in solution.

38. The MP of claim 36 or 37, wherein the at least one transfection agent is ApoE.

39. The MP of claim 36 or 37, wherein the at least one transfection agent is CpG.

40. The MP of any one of claims 36-39, wherein the at least one transfection agent increases the delivery mRNA to the cells in the lymph node.

41. The MP of any one of claims 36-40, wherein the at least one transfection agent increases expression of the construct.

42. The MP of any one of claims 31-41, wherein the MP is also loaded with a T-cell activating factor.

43. The MP of any one of claims 31-41, wherein the MP is further mixed with a T-cell activating factor in solution.

44. The MP of claim 42 or 43, wherein the T-cell activating factor is CD3/CD28 or OKT.

45. The MP of claim 42 or 43, wherein the T-cell activating factor is a mitogen or superantigen.

46. The MP of claim 45, wherein the mitogen or superantigen is phorbol 12-myristate 13-acetate (PMA), ionomycin, and/or PHA (Phytohaemagglutinin P).

47. The MP of any one of claims 31-46, wherein the MP is also loaded with a molecular adjuvant.

48. The MP of any one of claims 31-46, wherein the MP is further mixed with a molecular adjuvant in solution.

49. The MP of claims 47 or 48, wherein the molecular adjuvant is an innate ligand.

50. The MP of claims 49, wherein the innate ligand is TLR, RIG, and/or STING.

51. The MP of claims 31-50, wherein the MP is also loaded with at least one lymph node binding moiety.

52. The MP of claims 31-50, wherein the MP is further mixed with at least one lymph node binding moiety in solution.

53. The MP of claim 51 or 52, wherein the at least one lymph node binding moiety binds RANKL, sLeX, PNAd, GL7, CLDN11, EFNB2, ACKR4, TNFRSF9, CCL20, CXCL5, GlyCAM-1, CD34, Ptx3, CD209/Marco, SPNS2, CSF-1, CCL21, CCL19, CCL25, or CCL27.

54. The MP of any one of claims 31-53, wherein the nucleic acid encodes a CAR protein with an extracellular antigen-binding domain.

55. The MP of claim 54, wherein the extracellular antigen-binding domain binds BCMA.

56. The MP of claim 54, wherein the extracellular antigen-binding domain binds CD- 19.

57. The MP of any one of claims 31-56, wherein the nucleic acid is an mRNA.

58. The MP of claim 57, wherein the MP comprises 250-500 ng of mRNA.

59. The MP of claim 31-58, wherein the MP comprise a degradable polymer.

60. The MP of claim 59, wherein the degradable polymer comprises one or more of ester bonds, amide bonds, glycosidic bonds, phosphodiester bonds, ether bonds, disulfide bonds, peptide bonds, urethane bonds, carbonate bonds, thioester bonds, and orthoester bonds.

61. The MP of claim 60, wherein the degradable polymer is poly(lactide-co-glycolide) (PLGA).

62. The MP of any one of claims 31-61, wherein the concentration of MP in the lymph node is greater than the concentration in any other tissue of the subject.

63. The MP of any one of claims 31-62, wherein the concentration of MP in the lymph node is greater than the systemic biodistribution in the subject.

64. The microparticle of any one of claims 31-63, wherein the microparticle is 1-100 microns in size.

65. The microparticle of any one of claims 31-63, wherein the microparticle is 2-8 microns in size.

66. A microparticle composition comprising a plurality of microparticles of any one of claims 30-65.

67. A pharmaceutical composition comprising a plurality of the NPs of any one of claims 1-28, the nanoparticle composition of claim 29, a plurality of microparticles of any one of claims 30-65, or the microparticle composition claim 66, and a pharmaceutically acceptable excipient.

68. A method of transfecting cells with a nucleic acid, the method comprising contacting the cells with a plurality of the NPs of any one of claims 1-28, the nanoparticle composition of claim 29, a plurality of microparticles of any one of claims 30-65, the microparticle composition claim 66, or the pharmaceutical composition of claim 67.

69. The method of claim 68, wherein the contacting is conducted in vivo.

70. The method of claim 68 or 69, wherein the cells are located in a lymph node of a subject.

71. The method of any one of claims 68-70, wherein the cell is transiently located in the lymph node of a subject.

72. The method of any one of claims 68-71, wherein the cells are immune cells.

73. The method of any one of claims 68-72, wherein the cells are lymphocytes.

74. The method of any one of claims 68-72, wherein the cells are T-cells.

75. The method of claim 74, wherein the T-cells are CD8+ T cells.

76. The method of claim 74, wherein the T-cells are CD4+ T cells.

77. The method of any one of claims 68-76, wherein the plurality of nanoparticles, the nanoparticle composition, the plurality of microparticles, the microparticle composition, or pharmaceutical composition is administered into the lymph node of a subject.

78. The method of any one of claims 68-77, wherein the plurality of nanoparticles, the nanoparticle composition, the plurality of microparticles, the microparticle composition, or pharmaceutical composition is administered into the lymph node of a subject by injection.

79. A method of treating disease in a subject in need thereof, the method comprising administering to the subject a plurality of the NPs of any one of claims 1-28, the nanoparticle composition of claim 29, a plurality of microparticles of any one of claims 30-65, the microparticle composition claim 66, or the pharmaceutical composition of claim 67.

80. The method of claim 79, wherein the disease is a proliferative disease, an autoimmune disease, or an inflammatory disease.

81. The method of claim 80, wherein the proliferative disease is cancer.

82. The method of claim 81, wherein the cancer is multiple myeloma.

83. The method of claim 80, wherein the autoimmune disease is myasthenia gravis.

84. The method of any one of claims 79-83, wherein the plurality of nanoparticles, the nanoparticle composition, the plurality of microparticles, the microparticle composition, or pharmaceutical composition is administered by injection.

85. The method of any one of claims 79-84, wherein the plurality of nanoparticles, the nanoparticle composition, the plurality of microparticles, the microparticle composition, or pharmaceutical composition is administered directly into a lymph node.

86. The method of claim 85, wherein the plurality of nanoparticles, the nanoparticle composition, the plurality of microparticles, the microparticle composition, or pharmaceutical composition is administered directly into at least two lymph nodes.

87. The method of any one of claims 79-86, wherein the method is characterized by an increase in cytokine production in the cells transfected with the nucleic acid.

88. The method of claim 87, wherein the cytokine is interferon-gamma.

89. A method of inducing cytokine expression in the cells of a subject, the method comprising administering to the lymph nodes of a subject a plurality of the NPs of any one of claims 1-28, the nanoparticle composition of claim 29, a plurality of microparticles of any one of claims 30-65, the microparticle composition claim 66, or the pharmaceutical composition of claim 67.

90. The method of claim 89, wherein the plurality of the LNPs, the nanoparticle composition, the plurality of microparticle, the microparticle composition, or the pharmaceutical composition, is administered to a lymph node in the subject.

91. The method of claim 89 or 90, wherein the cytokine is interferon-gamma.

92. Use of the plurality of the NPs of any one of claims 1-28, the nanoparticle composition of claim 29, the plurality of microparticles of any one of claims 30-65, the microparticle composition claim 66, or the pharmaceutical composition of claim 67 for transfecting cells in a lymph node with a nucleic acid.

93. Use of the plurality of the NPs of any one of claims 1-28, the nanoparticle composition of claim 29, the plurality of microparticles of any one of claims 30-65, the microparticle composition claim 66, or the pharmaceutical composition of claim 67 for treating a disease in a subject.

94. Use of the LNPs of the plurality of the NPs of any one of claims 1-28, the nanoparticle composition of claim 29, the plurality of microparticles of any one of claims 30- 65, the microparticle composition claim 66, or the pharmaceutical composition of claim 67 for inducing cytokine expression.

95. A medicament for administration into a lymph node, the medicament comprising the plurality of the NPs of any one of claims 1-28, the nanoparticle composition of claim 29, the plurality of microparticles of any one of claims 30-65, the microparticle composition claim

66, or the pharmaceutical composition of claim 67.

96. A kit comprising the plurality of the NPs of any one of claims 1-28, the nanoparticle composition of claim 29, the plurality of microparticles of any one of claims 30-65, the microparticle composition claim 66, or the pharmaceutical composition of claim 67.