[go: up one dir, main page]
More Web Proxy on the site http://driver.im/

WO2024182722A1 - Therapeutic nanoparticles for solid organ immune acceptance - Google Patents

Therapeutic nanoparticles for solid organ immune acceptance Download PDF

Info

Publication number
WO2024182722A1
WO2024182722A1 PCT/US2024/018111 US2024018111W WO2024182722A1 WO 2024182722 A1 WO2024182722 A1 WO 2024182722A1 US 2024018111 W US2024018111 W US 2024018111W WO 2024182722 A1 WO2024182722 A1 WO 2024182722A1
Authority
WO
WIPO (PCT)
Prior art keywords
fasl
nanoparticle
biotin
patient
anchors
Prior art date
Application number
PCT/US2024/018111
Other languages
French (fr)
Inventor
Andres J. Garcia
Stephen W. Linderman
Juan Medina
Haval Shirwan
Susan Napier Thomas
Esma S. Yolcu
Original Assignee
Georgia Tech Research Corporation
The Curators Of The University Of Missouri
Emory University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Georgia Tech Research Corporation, The Curators Of The University Of Missouri, Emory University filed Critical Georgia Tech Research Corporation
Publication of WO2024182722A1 publication Critical patent/WO2024182722A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A61K47/551—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds one of the codrug's components being a vitamin, e.g. niacinamide, vitamin B3, cobalamin, vitamin B12, folate, vitamin A or retinoic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6935—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/525—Tumour necrosis factor [TNF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/22—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a Strep-tag

Definitions

  • the invention is directed to compositions for the controlled delivery of immunomodulating agents.
  • the compositions are useful to selectively deliver agents to target tissues and organs, including lymph nodes.
  • Organ transplantation is a life-saving therapeutic strategy for patients with end-stage organ failure, but rates of significant morbidity and graft failure associated with immunosuppression-mediated toxicities persist as significant limitations.
  • a critical unmet need for improving long-term transplant outcomes is the lack of effective immunomodulatory approaches that reduce the need for chronic immunosuppression.
  • Figure 1 depicts a schematic of process useful to prepare particles of the disclosure.
  • Figure 2A depicts a chromatogram for SA-AlexaFluor488 (green) incubation with biotinylated NPs tagged with AlexaFluor647 (red). Co-localization of peaks for NPs and SA- AlexaFluor488 demonstrates capture of SA onto NPs.
  • Figure 2B depicts normalized loading efficiency of SA-FasL on NPs measured using anti-FasL antibody.
  • Figure 2C depicts binding capacity of biotinylated NPs over time.
  • Figure 3 A depicts a bioactivity assay evaluating apoptosis of A20 cells demonstrating equivalent activity between SA-FasL captured on biotinylated NPs and soluble SA-FasL. Residual activity on non-biotinylated NPs is attributed to incomplete washing/removal of soluble SA-FasL (one-way ANOVA; ****P ⁇ 0.0001).
  • Figure 3B depicts a dose-response curve of A20 cell apoptosis versus total SA-FasL delivered on biotin-NPs (ng SA-FasL/mL media).
  • Figure 4 depicts dynamic light scattering measurement of nanoparticle (NP) diameter by intensity, for nonbiotinylated NPs (black) and biotinylated (blue) NPs.
  • Figure 6A depicts IVIS images of draining inguinal LN (dLN), contralateral nondraining LN (ndLN), liver, spleen, and kidneys extracted from mice injected subcutaneously near inguinal LN with AF750-SA-FasL and AF647-NPs; organs were harvested one, three, and seven days after injections, demonstrating localization of SA-FasL and NPs to targeted, draining LNs, compared to contralateral non-draining LNs (n>4 per timepoint).
  • Figure 6B depicts SA-FasL signal intensity values of the inguinal draining LN in mice injected with SA- FasL NPs were significantly higher than that of their contralateral non-draining LN for all timepoints; this same signal was significantly higher than that of both inguinal LNs in mice injected with sol. SA-FasL for all time points.
  • Figure 6C depicts targeted delivery of NPs is confirmed by NP signal intensity values that are significantly higher than all other harvested organs across all timepoints (mean ⁇ SE, N>4; two-way ANOVA).
  • Figure 7 depicts fluorescence intensity and absorbance of conjugated nanoparticles vs unconjugated nanoparticles vs. unconjugated SA-FasL complex, demonstrating tethering of SA-FasL to nanoparticles.
  • Figure 7 (middle panel) depicts the molar ratio of biotin: SA-FasL vs. the amount of captured SA-FasL.
  • Figure 7 (right panel) depicts the increased degree of apoptosis achieved with SA-FasL conjugated nanoparticles vs. individual nanoparticles and free SA-FasL, showing that SA-FasL tethered onto nanoparticles retain high bioactivity.
  • Figure 8 depicts images and quantification of near IR signal for SA-FasL in organs/tissues explanted at different time points for mice receiving SA-FasL nanoparticles or soluble SA-FasL injected subcutaneously in the vicinity of the inguinal (draining) lymph node. Images and quantification demonstrate increased in vivo residence time and localization of SA-FasL to draining lymph node when delivered by nanoparticles compared to soluble SA-FasL. Significant higher levels and residence time is observed for draining lymph nodes compared to non-draining lymph nodes and major organs.
  • Figure 9 depicts images and quantification of near IR signal for nanoparticles in organs/tissues explanted at different time points for mice receiving nanoparticles injected subcutaneously in the vicinity of the inguinal (draining) lymph node. Images and quantification demonstrate increased in vivo residence time and localization of nanoparticles to draining lymph node compared to non-draining lymph nodes and major organs.
  • Figure 10 depicts the increased Treg/Teff ratios produced by SA-FasL nanoparticles vs. control SA-nanoparticles complexes and saline control (no nanoparticles). Mice treated with SA-FasL-NPs exhibit higher Treg:Teff ratios in dLNs.
  • Figure 11 the reduced off-target effects produced by SA-FasL nanoparticles vs. control SA-nanoparticles complexes and saline control (no nanoparticles).
  • SA-FasL nanoparticles do not induce off-target effects on other LN resident cells nor on splenocytes in the periphery.
  • the word “comprise” and variations of the word, such as “comprising” and “comprises,” means “including but not limited to,” and is not intended to exclude, for example, other additives, components, integers or steps.
  • “Exemplary” means “an example of’ and is not intended to convey an indication of a preferred or ideal embodiment. “Such as” is not used in a restrictive sense, but for explanatory purposes.
  • biotin includes biotin-containing moieties that are able to bind to surfaces, such as cell surfaces, such as NHS-biotin and EZ-LinkTM Sulfo-NHS-LC-Biotin (Pierce). Biotin and protein-reactive forms of biotin are available commercially.
  • Streptavidin fragments or avidin fragments which retain substantial binding activity for biotin, such as at least 50% or more of the binding affinity of native streptavidin or avidin, respectively, also may be used.
  • Such fragments include “core streptavidin” (“CSA”), a truncated version of the full-length streptavidin polypeptide which may include streptavidin residues 13-138, 14-138, 13-139 or 14-139.
  • CSA core streptavidin
  • Other truncated forms of streptavidin and avidin that retain strong binding to biotin also may be used. See, e.g.
  • mutants with reduced immunogenicity such as mutants mutated by site -directed mutagenesis to remove potential T cell epitopes or lymphocyte epitopes
  • mutants of avidin and core forms of avidin which retain substantial biotin binding activity or increased biotin binding activity also may be used.
  • the terms “avidin” and “streptavidin” encompass fragments, mutants and core forms of these molecules.
  • Avidin and streptavidin are available from commercial suppliers. Moreover, the nucleic acid sequences encoding streptavidin and avidin and the streptavidin and avidin amino acid sequences are known. See, e.g., GenBank Accession Nos. X65082; X03591; NM 205320; X05343; Z21611; and Z21554.
  • FasL refers to the Fas ligand.
  • FasL moiety means at least the apoptosis-inducing moiety of FasL and is inclusive of the Fas ligand itself.
  • the FasL moiety comprises or consists of the extracellular domain of FasL.
  • the FasL moiety comprises or consists of a matrix metalloproteinase (MMP) resistant FasL protein.
  • MMP matrix metalloproteinase
  • the matrix metalloproteinase (MMP) resistant FasL protein is a form of FasL in which the extracellular domain of FasL lacks MMP sensitive sites. See Yolcu et al., Immunity 17: 795-808 (2002).
  • the immunomodulatory complex is SA-PD1 or SA-CXCL12.
  • SA-FasL conjugate is a FasL-streptavidin conjugate, a FasL-avidin conjugate, a FasL-streptavidin fragment conjugate, a FasL-avidin fragment conjugate, or a chimeric protein comprising a FasL moiety and a streptavidin or avidin moiety, as defined herein.
  • compositions including a SA-FasL complex conjugated to a nanoparticle.
  • the SA-FasL complex is a streptavidin-FasL complex.
  • the SA-FasL complex is an avidin-FasL complex.
  • the SA-FasL complex is a streptavidin fragment-FasL complex.
  • the SA-FasL complex is an avidin fragment-FasL complex.
  • nanoparticles conjugated to a SA-FasL complex through a biotin anchor are disclosed herein.
  • a key feature is the physical diameter of the nanoparticle that enhances the uptake and residence time in lymph nodes and lymphatics.
  • the nanoparticle comprises polypropylene sulfide.
  • the polypropylene sulfide has a MWn from 5,000-1,000,000 g/mol, from 5,000-100,000 g/mol, from 5,000-50,000 g/mol, from 5,000-25,000 g/mol, from 10, GOO- 25, 000 g/mol, from 10,000-50,000 g/mol, from 25,000-100,000 g/mol, from 50,000-250,000 g/mol, from 100,000-250,000 g/mol, from 100,000-500,000 g/mol, from 250,000-500,000 g/mol, from 250,000-750,000 g/mol, or from 500,000-1,000,000 g/mol.
  • the nanoparticle includes the reaction product of a mixture including propylene sulfide and a thiol-containing core.
  • the mixture can further include additional polymers, for example pluronics, i.e., poloxamers which are block ethylene glycol/propylene glycol/ethylene glycol polymers.
  • the poloxamers can include a mixture of hydroxyl-terminated poloxamer and carboxy-terminated poloxamer.
  • the nanoparticle can include the reaction product of propylene sulfide and a thiol-containing core having 2, 3, 4, 5, or 6 thiol groups.
  • the nanoparticle can include the reaction product of propylene sulfide and a thiol-containing core like pentaerythritol tetrakis (3 -mercaptopropionate) or 1,2,3 propanetriol.
  • the nanoparticle has a PDI from 0.5-2.0, from 0.5-1.5, from 0.5-1, from 0.75-1.25, from 1-1.25, from 1-1.5, from 1.25-1.5, from 1.25-1.75, or from 1.5 to 1.75.
  • nanoparticle has a particle size from 0.5-1,000 nm, from 0.5-500 nm, from 0.5-250 nm, from 0.5-100 nm, from 0.5-50 nm, from 10-50 nm, from 10- 100 nm, from 25-50 nm, from 25-75 nm, from 50-150 nm, from 100-250 nm, from 100-500 nm, from 250-500 nm, from 250-750 nm, or from 500-1,000 nm.
  • the nanoparticle has a particle size from 10-100 nm.
  • the nanoparticle has a particle size from 10-50 nm.
  • the nanoparticle has a particle size from 50-100 nm.
  • the nanoparticle is covalently bound to a plurality of biotin anchors.
  • the nanoparticle includes from 2-1,000 biotin anchors, from 2-500 biotin anchors, from 2-250 biotin anchors, from 2-100 biotin anchors, from 2-50 biotin anchors, from 2-25 biotin anchors, from 2-10 biotin anchors, from 5-10 SA- biotin anchors, from 10-25 biotin anchors, from 10-50 biotin anchors, from 50-100 biotin anchors, from 100-250 biotin anchors, or from 250-500 SA- biotin anchors.
  • the nanoparticle is covalently bound to a plurality of biotin anchors through a polyethylene glycol linker.
  • the polyethylene linker includes from 2-100 ethylene glycol units, from 2-50 ethylene glycol units, from 2-10 ethylene glycol units, from 5-25 ethylene glycol units, from 10-50 ethylene glycol units, from 25-100 ethylene glycol units, from 25-75 ethylene glycol units, or from 50-100 ethylene glycol units.
  • the polyethylene glycol linker is covalently bonded to the biotin via a disulfide bond.
  • a terminus of the polyethylene glycol is functionalized with a thiol group and covalently bonded to the biotin.
  • polyethylene glycol linker can be covalently bonded to the biotin via the terminal carboxy group present in biotin.
  • Polyethylene glycol can be directly bonded to biotin via an ester, while in some implementations one terminus of the polyethylene glycol can be functionalized with a primary amine and covalently bonded to biotin via an amide.
  • one terminus of the polyethylene glycol can be functionalized with a thiol group and covalently bonded to the nanoparticle via a disulfide bond.
  • the nanoparticle may be functionalized with reactive compounds like pyridyl disulfide cysteamine (via pendant carboxy groups on the surface of the nanoparticle) and then subsequently reacted with a thiol-functionalized polyethylene glycol.
  • the thiol-functionalized polyethylene glycol may include a biotin residue at the other terminus (as described above) or a biotin residue may be introduced subsequent to the reaction of the thiol-functionalized polyethylene glycol with the nanoparticle.
  • the SA-FasL complex is a FasL moiety conjugated to streptavidin. In some implementations the SA-FasL complex is a chimeric protein including a FasL moiety and a streptavidin or avidin moiety.
  • the nanoparticle is conjugated to 2-1,000 SA-FasL complexes, from 2-500 SA-FasL complexes, from 2-250 SA-FasL complexes, from 2-100 SA-FasL complexes, from 2-50 SA-FasL complexes, from 2-25 SA-FasL complexes, from 2- 10 SA-FasL complexes, from 5-10 SA-FasL complexes, from 10-25 SA-FasL complexes, from 10-50 SA-FasL complexes, from 50-100 SA-FasL complexes, from 100-250 SA-FasL complexes, or from 250-500 SA-FasL complexes.
  • compositions including the SA-FasL nanoparticles disclosed herein and a pharmaceutically acceptable carrier.
  • a “pharmaceutical acceptable carrier” refers to a collection of excipients that can be used as a vehicle or diluent that is unreactive with the nanoparticle complex.
  • the carrier can include water and can include additional pharmaceutical excipients well known in the art, including preservatives, pH adjusting agents, tonicity agents, and the like.
  • the pharmaceutically acceptable carrier is suitable for administration by intravenous, subcutaneous, or intraperitoneal injection.
  • compositions can be administered to a subject to deliver FasL to subjects in need thereof.
  • the compositions are formulated for parenteral administration, for example intravenous or subcutaneous injection.
  • the compositions may be directly implanted in or around transplanted tissue or lymph, for example lymph nodes.
  • compositions can include one or more additional therapeutic agents, such as an immunosuppressant drug.
  • immunosuppressant drugs include rapamycin, cyclophosamide busulfan, fludarabine, methotrexate, sulfasalazine, hydroxychloroquine, azathioprine, tocilizumab, etanercept, adalimumab, anakinra, abatacept, rituximab, certolizumab, golimumab, cyclosporine, dexamethasone, methylprednisolone, prednisone, tacrolimus and triamcinolone.
  • the immunosuppressant drug is rapamycin.
  • the additional therapeutic agent is associated with the nanoparticle, for example conjugated to the nanoparticle via a covalent linker or entrapped in and/or around the nanoparticle via non- covalent interactions.
  • the additional therapeutic agent is in the compositions without being associated with the nanoparticle.
  • the additional therapeutic agent can be dissolved or dispersed in a solvent vehicle.
  • FasL is the only active agent in the composition.
  • the method is for preventing or reducing the risks of rejection of tissue or solid organs grafts in transplantation and related procedures.
  • the nanoparticles can be administered to a patient in advance of receiving a transplanted organ or tissue.
  • the nanoparticles can be administered subsequent to transplant to reduce the likelihood of rejection.
  • the nanoparticles can be administered to the patient during the transplant procedure.
  • methods of inducing immunosuppression in a subject in need thereof comprising administering to the subject a FasL moiety in an amount effective to induce immune tolerance.
  • administering a FasL moiety in conjunction with a transplant or graft procedure may induce specific immune tolerance to transplanted or grafted tissues.
  • methods of inducing specific immune tolerance in a subject in need thereof comprising administering to the subject a FasL moiety in an amount effective to induce immune tolerance in the subject.
  • graft refers to a donor tissue or solid organ that is administered to a subject in need thereof.
  • Types of graft tissues and solid organs include skin, heart, kidney, pancreas, lung, liver, etc., depending on the condition being treated.
  • the FasL moiety induces specific immune tolerance to the graft tissue and solid organ.
  • the nanoparticles are used to increase acceptance and/or reduce rejection of a transplanted graft cell.
  • the graft cell is selected from PBMCs, bone marrow cells, hematopoietic stem cells, stem cells, mesenchymal stem cells, dendritic cells, dendritic cells pulsed with autoantigens, human beta cell products, and splenocytes.
  • the graft cell may be pancreatic islet cells.
  • the compositions can be embedded within the graft cell prior to transplantation.
  • the FasL moiety is administered with an additional therapeutic agent, such as an immunosuppressive drug, such as rapamycin or any of the others mentioned above.
  • an immunosuppressive drug such as rapamycin or any of the others mentioned above.
  • the FasL moiety and immunosuppressive drug may be formulated together (e.g., the nanoparticles may comprise the immunosuppressive drug), or they may be administered in separate compositions, simultaneously or sequentially in any order.
  • a shorter course and/or reduced dosage of immunosuppressive drug may be required than when no FasL moiety is administered.
  • the nanoparticles can be used to selectively delivery FasL to the lymph system, for example to lymph nodes.
  • the nanoparticles can be used to prevent transplant rejection by delivering tolerance-inducing FasL to the lymph system, for example lymph nodes.
  • the nanoparticles may be administered subcutaneously to tissues adjacent to the lymph, for example lymph nodes.
  • the nanoparticles may be administered intravenously leading to accumulation in the lymph, for example lymph nodes.
  • FasL nanoparticles can include a therapeutic drug, for example an immunosuppressant drug, wherein the nanoparticles provide controlled release of the drug. Controlled release may be obtained from passive diffusion of a drug entrapped within the nanoparticles. Controlled release may also be achieved by controlled breaking of a covalent tether conjugating the drug to the nanoparticle. In some embodiments, FasL nanoparticles that comprise an immunosuppressive drug provide controlled release of the drug within the transplant microenvironment or in the lymph system, for example the lymph nodes.
  • a therapeutic drug for example an immunosuppressant drug
  • administering a FasL moiety as described herein with an immunosuppressive drug achieves a synergistic immunosuppressive effect.
  • the immunosuppressive drug such as rapamycin
  • the immunosuppressive drug works in synergy with FasL to specifically eliminate pathogenic T effector cells while expanding T regulatory cells, thereby tipping the balance of immune response towards protection.
  • administering a FasL moiety as described herein with an immunosuppressive drug does not impair the systemic immune response, and may increase the ratio of T regulatory cells to T helper cells.
  • the SA-FasL capture efficiency was determined to be ⁇ 99% in solution yielding ⁇ 8- 10 SA-FasL molecules per NP (Fig. 2B). Importantly, the capture efficiency of biotinylated NPs was sustained for at least 25 days, an important consideration for clinical translatability (Fig. 2C). Given that NP size drives the targeted delivery towards draining LNs, it was important that biotinylation did not impact NP size significantly. Using dynamic light scattering, we confirmed that the synthesized NPs achieved the desired ⁇ 30 nm diameter with high uniformity ( Figure 4)
  • the bioactivity of SA-FasL-coated NPs was assessed by culturing with apoptosissensitive cells. 1,000,000 Fas apoptosis-sensitive A20 cells (ATCC) were co-incubated with 1) biotinylated nanoparticles presenting SA-FasL, 2) unbiotinylated nanoparticles that were incubated with SA-FasL, 3) SA-FasL (not conjugated to NPs), or 4) no SA-FasL in 1.0 mL media.
  • A20 cells A20 cells
  • SA-FasL-presenting NPs maintained full bioactivity relative to soluble SA-FasL, measured in vitro via an apoptosis assay in A20 cells (Fig. 3 A). Residual apoptotic activity from non-biotinylated NPs incubated with SA-FasL is attributed to incomplete washing and removal of SA-FasL. SA-FasL delivered on biotinylated NPs induced apoptosis in a dosedependent manner (Fig. 3B).
  • SA-FasL was labelled with DyLight 750 NHS Ester (Thermo Scientific) and subsequently purified.
  • NPs encapsulating Al exaFluor 647 were provided by the Thomas lab at Georgia Tech and reacted with AF750 SA-FasL for one hours. Labelled AF647 NPs and AF750 SA-FasL were confirmed to have undetectable signal overlap prior to undertaking biotracking studies.
  • mice were injected subcutaneously near the inguinal LNs with 1) Reacted AF750 SA- FasL + AF647 biotin-NPs, or 2) AF750 SA-FasL in the absence of NPs. At specific timepoints, mice were imaged using a Perkin-Elmer IVIS imaging system. A different cohort of mice were sacrificed on day 1, 2, or 3 and their organs were harvested and imaged via IVIS.
  • SA-FasL was fluorescently labelled with a near infrared fluorophore, coated over AlexaFluor 647-encapsulating biotinylated NPs and injected subcutaneously near the right inguinal LN.
  • Control mice received the same amount of labelled SA-FasL without NP delivery vehicles (Sol. SA-FasL).
  • SA-FasL delivered on the surface of biotinylated NPs exhibited sustained and localized signal at the injection site, while soluble SA-FasL was rapidly cleared (Figure 5 A).
  • NP delivery increased in vivo SA-FasL residence time by ⁇ 40-fold compared to soluble delivery (half-life 100 h vs. 2.6 h, p ⁇ 0.0001).
  • relevant organs draining LNs, contralateral LN, liver, spleen, kidneys
  • mice injected with labelled SA-FasL with or without labelled biotin NPs were harvested at DI, D3, and D7 and imaged immediately (Fig. 6A).
  • compositions and methods of the appended claims are not limited in scope by the specific compositions and methods described herein, which are intended as illustrations of a few aspects of the claims and any compositions and methods that are functionally equivalent are intended to fall within the scope of the claims.
  • Various modifications of the compositions and methods in addition to those shown and described herein are intended to fall within the scope of the appended claims.
  • other combinations of the compositions and method steps also are intended to fall within the scope of the appended claims, even if not specifically recited.
  • a combination of steps, elements, components, or constituents may be explicitly mentioned herein or less, however, other combinations of steps, elements, components, and constituents are included, even though not explicitly stated.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Organic Chemistry (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Nanotechnology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Transplantation (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicinal Preparation (AREA)

Abstract

Disclosed are FasL presenting compositions. The compositions may be used to effect immunomodulation in a variety of contexts. The compositions may be used to decrease rates of transplant rejection.

Description

THERAPEUTIC NANOPARTICLES FOR SOLID ORGAN IMMUNE ACCEPTANCE
ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT
This invention was made with government support under grant number 7U01AI 132817-04 awarded by the National Institutes of Health. The government has certain rights in the invention.
CROSS-REFERENCE TO RELATED APPLICATION
This application claims the benefit of U.S. Provisional Application 63/487,755, filed March 1, 2023, the contents of which are hereby incorporated in its entirety.
FIELD OF THE INVENTION
The invention is directed to compositions for the controlled delivery of immunomodulating agents. The compositions are useful to selectively deliver agents to target tissues and organs, including lymph nodes.
BACKGROUND
Organ transplantation is a life-saving therapeutic strategy for patients with end-stage organ failure, but rates of significant morbidity and graft failure associated with immunosuppression-mediated toxicities persist as significant limitations. A critical unmet need for improving long-term transplant outcomes is the lack of effective immunomodulatory approaches that reduce the need for chronic immunosuppression.
There remains a need for improved compositions and methods for reducing morbidity and/or graft in transplant patients. There remains a need for improved compositions and methods that eliminate the need for chronic immunosuppression. There remains a need for improved compositions and methods for targeted and tunable delivery of immunomodulatory agents into lymph nodes as a means of achieving sustained survival of solid organ grafts (e.g., heart, kidney, lung, liver, pancreas) in the absence of chronic immunosuppression. There remains a need for improved compositions and methods for the delivery of FasL and other immunomodulatory agents to specific tissues in the body. There remains a need for improved compositions and methods for the delivery of FasL and other immunomodulatory agents to the lymph system. There remains a need for improved compositions and methods for the delivery of FasL and other immunomodulatory agents to the lymph nodes. BRIEF DESCRIPTION OF THE FIGURES
Figure 1 depicts a schematic of process useful to prepare particles of the disclosure.
Figure 2A depicts a chromatogram for SA-AlexaFluor488 (green) incubation with biotinylated NPs tagged with AlexaFluor647 (red). Co-localization of peaks for NPs and SA- AlexaFluor488 demonstrates capture of SA onto NPs. Figure 2B depicts normalized loading efficiency of SA-FasL on NPs measured using anti-FasL antibody. Figure 2C depicts binding capacity of biotinylated NPs over time.
Figure 3 A depicts a bioactivity assay evaluating apoptosis of A20 cells demonstrating equivalent activity between SA-FasL captured on biotinylated NPs and soluble SA-FasL. Residual activity on non-biotinylated NPs is attributed to incomplete washing/removal of soluble SA-FasL (one-way ANOVA; ****P<0.0001). Figure 3B depicts a dose-response curve of A20 cell apoptosis versus total SA-FasL delivered on biotin-NPs (ng SA-FasL/mL media).
Figure 4 depicts dynamic light scattering measurement of nanoparticle (NP) diameter by intensity, for nonbiotinylated NPs (black) and biotinylated (blue) NPs.
Figure 5 depicts IVIS imaging of AF750-SA-FasL and AF647-NPs injected subcutaneously near inguinal LN in mice (n=4). Signal intensity values over time were curve fit with one -phase decay to estimate half-life (t50). Longitudinal in vivo imaging using near IR labeled SA-FasL (750 channel) and nanoparticles (647 channel) injected subcutaneously in the vicinity of the inguinal lymph node of mice. Quantification of images of time demonstrates localization and increased in vivo resident time for SA-FasL in lymph when delivered via nanoparticles compared to soluble SA-FasL (top panels). Quantification of nanoparticle signal demonstrates retention at lymph node location (bottom panels).
Figure 6A depicts IVIS images of draining inguinal LN (dLN), contralateral nondraining LN (ndLN), liver, spleen, and kidneys extracted from mice injected subcutaneously near inguinal LN with AF750-SA-FasL and AF647-NPs; organs were harvested one, three, and seven days after injections, demonstrating localization of SA-FasL and NPs to targeted, draining LNs, compared to contralateral non-draining LNs (n>4 per timepoint). Figure 6B depicts SA-FasL signal intensity values of the inguinal draining LN in mice injected with SA- FasL NPs were significantly higher than that of their contralateral non-draining LN for all timepoints; this same signal was significantly higher than that of both inguinal LNs in mice injected with sol. SA-FasL for all time points. Figure 6C depicts targeted delivery of NPs is confirmed by NP signal intensity values that are significantly higher than all other harvested organs across all timepoints (mean ± SE, N>4; two-way ANOVA).
Figure 7 (left panel) depicts fluorescence intensity and absorbance of conjugated nanoparticles vs unconjugated nanoparticles vs. unconjugated SA-FasL complex, demonstrating tethering of SA-FasL to nanoparticles. Figure 7 (middle panel) depicts the molar ratio of biotin: SA-FasL vs. the amount of captured SA-FasL. Figure 7 (right panel) depicts the increased degree of apoptosis achieved with SA-FasL conjugated nanoparticles vs. individual nanoparticles and free SA-FasL, showing that SA-FasL tethered onto nanoparticles retain high bioactivity.
Figure 8 depicts images and quantification of near IR signal for SA-FasL in organs/tissues explanted at different time points for mice receiving SA-FasL nanoparticles or soluble SA-FasL injected subcutaneously in the vicinity of the inguinal (draining) lymph node. Images and quantification demonstrate increased in vivo residence time and localization of SA-FasL to draining lymph node when delivered by nanoparticles compared to soluble SA-FasL. Significant higher levels and residence time is observed for draining lymph nodes compared to non-draining lymph nodes and major organs.
Figure 9 depicts images and quantification of near IR signal for nanoparticles in organs/tissues explanted at different time points for mice receiving nanoparticles injected subcutaneously in the vicinity of the inguinal (draining) lymph node. Images and quantification demonstrate increased in vivo residence time and localization of nanoparticles to draining lymph node compared to non-draining lymph nodes and major organs.
Figure 10 depicts the increased Treg/Teff ratios produced by SA-FasL nanoparticles vs. control SA-nanoparticles complexes and saline control (no nanoparticles). Mice treated with SA-FasL-NPs exhibit higher Treg:Teff ratios in dLNs.
Figure 11 the reduced off-target effects produced by SA-FasL nanoparticles vs. control SA-nanoparticles complexes and saline control (no nanoparticles). SA-FasL nanoparticles do not induce off-target effects on other LN resident cells nor on splenocytes in the periphery.
DETAILED DESCRIPTION
Before the present methods and systems are disclosed and described, it is to be understood that the methods and systems are not limited to specific synthetic methods, specific components, or to particular compositions. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.
“Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.
Throughout the description and claims of this specification, the word “comprise” and variations of the word, such as “comprising” and “comprises,” means “including but not limited to,” and is not intended to exclude, for example, other additives, components, integers or steps. “Exemplary” means “an example of’ and is not intended to convey an indication of a preferred or ideal embodiment. “Such as” is not used in a restrictive sense, but for explanatory purposes.
As used herein “biotin” includes biotin-containing moieties that are able to bind to surfaces, such as cell surfaces, such as NHS-biotin and EZ-Link™ Sulfo-NHS-LC-Biotin (Pierce). Biotin and protein-reactive forms of biotin are available commercially.
Streptavidin fragments or avidin fragments which retain substantial binding activity for biotin, such as at least 50% or more of the binding affinity of native streptavidin or avidin, respectively, also may be used. Such fragments include “core streptavidin” (“CSA”), a truncated version of the full-length streptavidin polypeptide which may include streptavidin residues 13-138, 14-138, 13-139 or 14-139. See, e.g., Pahler et al., J Biol. Chem., 262: 13933-37 (1987). Other truncated forms of streptavidin and avidin that retain strong binding to biotin also may be used. See, e.g. Sano et al., J Blot Chem. 270(47): 28204-09 (1995) (describing core streptavidin variants 16-133 and 14-138) (U.S. Pat. No. 6,022,951). Mutants of streptavidin and core forms of streptavidin which retain substantial biotin binding activity or increased biotin binding activity also may be used. See, e.g., Chilkoti et al., Proc Natl Acad Sci, 92(5): 1754-58 (1995), Reznik et al., Nat Biotechnol, 14(8): 1007-11(1996). For example, mutants with reduced immunogenicity, such as mutants mutated by site -directed mutagenesis to remove potential T cell epitopes or lymphocyte epitopes, can be used. See Meyer et al., Protein Sei., 10: 491-503 (2001). Likewise, mutants of avidin and core forms of avidin which retain substantial biotin binding activity or increased biotin binding activity also may be used. See Hiller et al., J Biochem, 278: 573-85 (1991); and Livnah et al., Proc Natl Acad Sci USA 90: 5076-80 (1993). For convenience, in the discussion herein, the terms “avidin” and “streptavidin” (or “SA”) encompass fragments, mutants and core forms of these molecules.
Avidin and streptavidin are available from commercial suppliers. Moreover, the nucleic acid sequences encoding streptavidin and avidin and the streptavidin and avidin amino acid sequences are known. See, e.g., GenBank Accession Nos. X65082; X03591; NM 205320; X05343; Z21611; and Z21554.
As used herein, “FasL” refers to the Fas ligand. As used herein, “FasL moiety” means at least the apoptosis-inducing moiety of FasL and is inclusive of the Fas ligand itself. In some embodiments, the FasL moiety comprises or consists of the extracellular domain of FasL. In some embodiments, the FasL moiety comprises or consists of a matrix metalloproteinase (MMP) resistant FasL protein. As used herein, the matrix metalloproteinase (MMP) resistant FasL protein is a form of FasL in which the extracellular domain of FasL lacks MMP sensitive sites. See Yolcu et al., Immunity 17: 795-808 (2002). In some implementations the immunomodulatory complex is SA-PD1 or SA-CXCL12.
As used herein a “SA-FasL conjugate” is a FasL-streptavidin conjugate, a FasL-avidin conjugate, a FasL-streptavidin fragment conjugate, a FasL-avidin fragment conjugate, or a chimeric protein comprising a FasL moiety and a streptavidin or avidin moiety, as defined herein.
We have previously reported the construction of a chimeric form of FasL with streptavidin (SA), SA-FasL, in which the extracellular domain of FasL, lacking MMP sensitive sites, was cloned C-terminal to SA, which is useful as an effective immunomodulatory agent. See Yolcu et al., Immunity 17, 795-808 (2002). This protein exists as tetramers and oligomers with robust apoptotic activity on Fas-expressing cells. Importantly, pancreatic islets, modified with biotin attached to the cell surface followed by engineering with SA-FasL, acquired an immune privileged status and survived indefinitely in the absence of chronic immunosuppression in an allogeneic transplant murine model. See Yolcu et al., J Immunol 187, 5901-5909 (2011).
Disclosed herein are compositions including a SA-FasL complex conjugated to a nanoparticle. In certain implementations the SA-FasL complex is a streptavidin-FasL complex. In certain implementations the SA-FasL complex is an avidin-FasL complex. In certain implementations the SA-FasL complex is a streptavidin fragment-FasL complex. In certain implementations the SA-FasL complex is an avidin fragment-FasL complex.
Disclosed herein are nanoparticles conjugated to a SA-FasL complex through a biotin anchor. A key feature is the physical diameter of the nanoparticle that enhances the uptake and residence time in lymph nodes and lymphatics.
In certain implementations, the nanoparticle comprises polypropylene sulfide. In certain implementations the polypropylene sulfide has a MWn from 5,000-1,000,000 g/mol, from 5,000-100,000 g/mol, from 5,000-50,000 g/mol, from 5,000-25,000 g/mol, from 10, GOO- 25, 000 g/mol, from 10,000-50,000 g/mol, from 25,000-100,000 g/mol, from 50,000-250,000 g/mol, from 100,000-250,000 g/mol, from 100,000-500,000 g/mol, from 250,000-500,000 g/mol, from 250,000-750,000 g/mol, or from 500,000-1,000,000 g/mol.
In certain implementations the nanoparticle includes the reaction product of a mixture including propylene sulfide and a thiol-containing core. The mixture can further include additional polymers, for example pluronics, i.e., poloxamers which are block ethylene glycol/propylene glycol/ethylene glycol polymers. The poloxamers can include a mixture of hydroxyl-terminated poloxamer and carboxy-terminated poloxamer. In some implementations the nanoparticle can include the reaction product of propylene sulfide and a thiol-containing core having 2, 3, 4, 5, or 6 thiol groups. In some implementations the nanoparticle can include the reaction product of propylene sulfide and a thiol-containing core like pentaerythritol tetrakis (3 -mercaptopropionate) or 1,2,3 propanetriol.
In certain implementations the nanoparticle has a PDI from 0.5-2.0, from 0.5-1.5, from 0.5-1, from 0.75-1.25, from 1-1.25, from 1-1.5, from 1.25-1.5, from 1.25-1.75, or from 1.5 to 1.75.
In certain implementations nanoparticle has a particle size from 0.5-1,000 nm, from 0.5-500 nm, from 0.5-250 nm, from 0.5-100 nm, from 0.5-50 nm, from 10-50 nm, from 10- 100 nm, from 25-50 nm, from 25-75 nm, from 50-150 nm, from 100-250 nm, from 100-500 nm, from 250-500 nm, from 250-750 nm, or from 500-1,000 nm. In certain implementations, the nanoparticle has a particle size from 10-100 nm. In certain preferred implementations, the nanoparticle has a particle size from 10-50 nm. In certain preferred implementations the nanoparticle has a particle size from 50-100 nm.
In certain implementations the nanoparticle is covalently bound to a plurality of biotin anchors.
In certain implementations the nanoparticle includes from 2-1,000 biotin anchors, from 2-500 biotin anchors, from 2-250 biotin anchors, from 2-100 biotin anchors, from 2-50 biotin anchors, from 2-25 biotin anchors, from 2-10 biotin anchors, from 5-10 SA- biotin anchors, from 10-25 biotin anchors, from 10-50 biotin anchors, from 50-100 biotin anchors, from 100-250 biotin anchors, or from 250-500 SA- biotin anchors.
In certain implementations the nanoparticle is covalently bound to a plurality of biotin anchors through a polyethylene glycol linker. In certain implementations, the polyethylene linker includes from 2-100 ethylene glycol units, from 2-50 ethylene glycol units, from 2-10 ethylene glycol units, from 5-25 ethylene glycol units, from 10-50 ethylene glycol units, from 25-100 ethylene glycol units, from 25-75 ethylene glycol units, or from 50-100 ethylene glycol units.
In certain implementations the polyethylene glycol linker is covalently bonded to the biotin via a disulfide bond. In such implementation a terminus of the polyethylene glycol is functionalized with a thiol group and covalently bonded to the biotin.
In some implementations the polyethylene glycol linker can be covalently bonded to the biotin via the terminal carboxy group present in biotin. Polyethylene glycol can be directly bonded to biotin via an ester, while in some implementations one terminus of the polyethylene glycol can be functionalized with a primary amine and covalently bonded to biotin via an amide.
In certain implementations, one terminus of the polyethylene glycol can be functionalized with a thiol group and covalently bonded to the nanoparticle via a disulfide bond. The nanoparticle may be functionalized with reactive compounds like pyridyl disulfide cysteamine (via pendant carboxy groups on the surface of the nanoparticle) and then subsequently reacted with a thiol-functionalized polyethylene glycol. The thiol-functionalized polyethylene glycol may include a biotin residue at the other terminus (as described above) or a biotin residue may be introduced subsequent to the reaction of the thiol-functionalized polyethylene glycol with the nanoparticle.
In certain implementations the SA-FasL complex is a FasL moiety conjugated to streptavidin. In some implementations the SA-FasL complex is a chimeric protein including a FasL moiety and a streptavidin or avidin moiety.
In certain implementations the nanoparticle is conjugated to 2-1,000 SA-FasL complexes, from 2-500 SA-FasL complexes, from 2-250 SA-FasL complexes, from 2-100 SA-FasL complexes, from 2-50 SA-FasL complexes, from 2-25 SA-FasL complexes, from 2- 10 SA-FasL complexes, from 5-10 SA-FasL complexes, from 10-25 SA-FasL complexes, from 10-50 SA-FasL complexes, from 50-100 SA-FasL complexes, from 100-250 SA-FasL complexes, or from 250-500 SA-FasL complexes. Also disclosed herein are compositions including the SA-FasL nanoparticles disclosed herein and a pharmaceutically acceptable carrier. A “pharmaceutical acceptable carrier” refers to a collection of excipients that can be used as a vehicle or diluent that is unreactive with the nanoparticle complex. The carrier can include water and can include additional pharmaceutical excipients well known in the art, including preservatives, pH adjusting agents, tonicity agents, and the like. In some embodiments, the pharmaceutically acceptable carrier is suitable for administration by intravenous, subcutaneous, or intraperitoneal injection.
The compositions can be administered to a subject to deliver FasL to subjects in need thereof. In certain implementations, the compositions are formulated for parenteral administration, for example intravenous or subcutaneous injection. In certain implementations the compositions may be directly implanted in or around transplanted tissue or lymph, for example lymph nodes.
In certain implementations the compositions can include one or more additional therapeutic agents, such as an immunosuppressant drug. Examples of suitable immunosuppressant drugs include rapamycin, cyclophosamide busulfan, fludarabine, methotrexate, sulfasalazine, hydroxychloroquine, azathioprine, tocilizumab, etanercept, adalimumab, anakinra, abatacept, rituximab, certolizumab, golimumab, cyclosporine, dexamethasone, methylprednisolone, prednisone, tacrolimus and triamcinolone. In some embodiments, the immunosuppressant drug is rapamycin. In certain implementations, the additional therapeutic agent is associated with the nanoparticle, for example conjugated to the nanoparticle via a covalent linker or entrapped in and/or around the nanoparticle via non- covalent interactions. In some implementations, the additional therapeutic agent is in the compositions without being associated with the nanoparticle. For example, the additional therapeutic agent can be dissolved or dispersed in a solvent vehicle. In some implementations FasL is the only active agent in the composition.
In accordance with some embodiments, there are provided methods of effecting immunomodulation comprising administering to a subject in need thereof a nanoparticle as described herein. In accordance with some embodiments, the method is for preventing or reducing the risks of rejection of tissue or solid organs grafts in transplantation and related procedures. In certain implementations, the nanoparticles can be administered to a patient in advance of receiving a transplanted organ or tissue. In certain implementations the nanoparticles can be administered subsequent to transplant to reduce the likelihood of rejection. In certain implementations the nanoparticles can be administered to the patient during the transplant procedure.
In accordance with some embodiments, there are provided methods of inducing immunosuppression in a subject in need thereof comprising administering to the subject a FasL moiety in an amount effective to induce immune tolerance. For example, administering a FasL moiety in conjunction with a transplant or graft procedure may induce specific immune tolerance to transplanted or grafted tissues. Thus, in accordance with some embodiments, there are provided methods of inducing specific immune tolerance in a subject in need thereof comprising administering to the subject a FasL moiety in an amount effective to induce immune tolerance in the subject.
As used herein “graft” refers to a donor tissue or solid organ that is administered to a subject in need thereof. Types of graft tissues and solid organs include skin, heart, kidney, pancreas, lung, liver, etc., depending on the condition being treated. In accordance with these methods the FasL moiety induces specific immune tolerance to the graft tissue and solid organ.
In certain implementations the nanoparticles are used to increase acceptance and/or reduce rejection of a transplanted graft cell. In some embodiments, the graft cell is selected from PBMCs, bone marrow cells, hematopoietic stem cells, stem cells, mesenchymal stem cells, dendritic cells, dendritic cells pulsed with autoantigens, human beta cell products, and splenocytes. When the subject is in need of the treatment or prevention of type 1 diabetes, the graft cell may be pancreatic islet cells. In some implementations the compositions can be embedded within the graft cell prior to transplantation.
In some embodiments the FasL moiety is administered with an additional therapeutic agent, such as an immunosuppressive drug, such as rapamycin or any of the others mentioned above. In such embodiments, the FasL moiety and immunosuppressive drug may be formulated together (e.g., the nanoparticles may comprise the immunosuppressive drug), or they may be administered in separate compositions, simultaneously or sequentially in any order. In some embodiments, a shorter course and/or reduced dosage of immunosuppressive drug may be required than when no FasL moiety is administered.
In certain implementations the nanoparticles can be used to selectively delivery FasL to the lymph system, for example to lymph nodes. The nanoparticles can be used to prevent transplant rejection by delivering tolerance-inducing FasL to the lymph system, for example lymph nodes. In some implementations the nanoparticles may be administered subcutaneously to tissues adjacent to the lymph, for example lymph nodes. In some implementations, the nanoparticles may be administered intravenously leading to accumulation in the lymph, for example lymph nodes.
In some embodiments, FasL nanoparticles can include a therapeutic drug, for example an immunosuppressant drug, wherein the nanoparticles provide controlled release of the drug. Controlled release may be obtained from passive diffusion of a drug entrapped within the nanoparticles. Controlled release may also be achieved by controlled breaking of a covalent tether conjugating the drug to the nanoparticle. In some embodiments, FasL nanoparticles that comprise an immunosuppressive drug provide controlled release of the drug within the transplant microenvironment or in the lymph system, for example the lymph nodes.
In some embodiments, administering a FasL moiety as described herein with an immunosuppressive drug achieves a synergistic immunosuppressive effect. For example, in some embodiments, the immunosuppressive drug (such as rapamycin) works in synergy with FasL to specifically eliminate pathogenic T effector cells while expanding T regulatory cells, thereby tipping the balance of immune response towards protection.
In some embodiments, administering a FasL moiety as described herein with an immunosuppressive drug does not impair the systemic immune response, and may increase the ratio of T regulatory cells to T helper cells.
EXAMPLES
The following examples are for the purpose of illustration of the invention only and are not intended to limit the scope of the present invention in any manner whatsoever.
Example 1: Synthesis of biotinylated nanoparticles
An emulsion of propylene sulfide, carboxylate -pluronic and hydroxyl-pluronic was made and then added to a four-arm thiol initiator, along with a base, to initiate polymerization. Once polymerization was complete, the particles were cross-linked by exposure to air. These carboxylate -nanoparticles (NPs) were reacted with pyridyl disulfide cysteamine to yield thiol-reactive NPs, and then reacted with a biotin-PEG-thiol (MW 1 kDa, Nanocs Inc.) to yield biotinylated NPs. Excess biotin-PEG-thiol was removed using column chromatography. Purified biotinylated NPs were incubated with SA-FasL for 1 hour to yield SA-FasL-coated nanoparticles (Figure 1).
The SA-FasL capture efficiency was determined to be ~99% in solution yielding ~8- 10 SA-FasL molecules per NP (Fig. 2B). Importantly, the capture efficiency of biotinylated NPs was sustained for at least 25 days, an important consideration for clinical translatability (Fig. 2C). Given that NP size drives the targeted delivery towards draining LNs, it was important that biotinylation did not impact NP size significantly. Using dynamic light scattering, we confirmed that the synthesized NPs achieved the desired ~30 nm diameter with high uniformity (Figure 4)
The bioactivity of SA-FasL-coated NPs was assessed by culturing with apoptosissensitive cells. 1,000,000 Fas apoptosis-sensitive A20 cells (ATCC) were co-incubated with 1) biotinylated nanoparticles presenting SA-FasL, 2) unbiotinylated nanoparticles that were incubated with SA-FasL, 3) SA-FasL (not conjugated to NPs), or 4) no SA-FasL in 1.0 mL media. After 18 h of incubation in a 5% CO2 incubator, the cells were stained with markers of early and late apoptosis (APC annexin V and propidium iodide, BD Biosciences). Samples were analysed by flow cytometry.
SA-FasL-presenting NPs maintained full bioactivity relative to soluble SA-FasL, measured in vitro via an apoptosis assay in A20 cells (Fig. 3 A). Residual apoptotic activity from non-biotinylated NPs incubated with SA-FasL is attributed to incomplete washing and removal of SA-FasL. SA-FasL delivered on biotinylated NPs induced apoptosis in a dosedependent manner (Fig. 3B).
To assess SA-FasL persistence and release from biotin-NPs in vivo, SA-FasL was labelled with DyLight 750 NHS Ester (Thermo Scientific) and subsequently purified. NPs encapsulating Al exaFluor 647 were provided by the Thomas lab at Georgia Tech and reacted with AF750 SA-FasL for one hours. Labelled AF647 NPs and AF750 SA-FasL were confirmed to have undetectable signal overlap prior to undertaking biotracking studies.
Mice were injected subcutaneously near the inguinal LNs with 1) Reacted AF750 SA- FasL + AF647 biotin-NPs, or 2) AF750 SA-FasL in the absence of NPs. At specific timepoints, mice were imaged using a Perkin-Elmer IVIS imaging system. A different cohort of mice were sacrificed on day 1, 2, or 3 and their organs were harvested and imaged via IVIS.
To determine the kinetics and localized nature of SA-FasL release from NPs in vivo, SA-FasL was fluorescently labelled with a near infrared fluorophore, coated over AlexaFluor 647-encapsulating biotinylated NPs and injected subcutaneously near the right inguinal LN. Control mice received the same amount of labelled SA-FasL without NP delivery vehicles (Sol. SA-FasL). SA-FasL delivered on the surface of biotinylated NPs exhibited sustained and localized signal at the injection site, while soluble SA-FasL was rapidly cleared (Figure 5 A). NP delivery increased in vivo SA-FasL residence time by ~40-fold compared to soluble delivery (half-life 100 h vs. 2.6 h, p<0.0001). To ensure that SA-FasL delivery was indeed localized to draining LNs, relevant organs (draining LNs, contralateral LN, liver, spleen, kidneys), from mice injected with labelled SA-FasL with or without labelled biotin NPs, were harvested at DI, D3, and D7 and imaged immediately (Fig. 6A). Signal from draining LNs was significantly higher than that of non-draining LNs for all timepoints, demonstrating SA-FasL delivery on NPs colocalize to the targeted draining LNs but not non-draining LNs (Fig. 6B). Furthermore, localization of SA-FasL to LNs is not observed in mice injected with sol. SA-FasL in the absence of biotin NPs - demonstrating that biotinylated NPs are necessary for targeted SA-FasL delivery to LNs. Trace signal of labelled SA-FasL is observed in the liver and kidneys beyond DI for both groups. With regards to NPs, there is a significantly higher amount present in the draining LNs compared to that of non-draining LNs, liver, spleen, and kidneys (Fig. 6C).
The compositions and methods of the appended claims are not limited in scope by the specific compositions and methods described herein, which are intended as illustrations of a few aspects of the claims and any compositions and methods that are functionally equivalent are intended to fall within the scope of the claims. Various modifications of the compositions and methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative compositions and method steps disclosed herein are specifically described, other combinations of the compositions and method steps also are intended to fall within the scope of the appended claims, even if not specifically recited. Thus, a combination of steps, elements, components, or constituents may be explicitly mentioned herein or less, however, other combinations of steps, elements, components, and constituents are included, even though not explicitly stated. The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of’ and “consisting of’ can be used in place of “comprising” and “including” to provide for more specific embodiments of the invention and are also disclosed. Other than in the examples, or where otherwise noted, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood at the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, to be construed in light of the number of significant digits and ordinary rounding approaches.

Claims

CLAIMS What is claimed is:
1. A nanoparticle conjugated to a SA-FasL complex through a biotin anchor.
2. The nanoparticle according to claim 1, wherein the nanoparticle comprises polypropylene sulfide and a poloxamer.
3. The nanoparticle according to claim 1, wherein the nanoparticle comprises polypropylene sulfide and a carboxy-terminated poloxamer.
4. The nanoparticle according to claim 2, wherein the polypropylene sulfide has a MWn from 5,000-1,000,000 g/mol, from 5,000-100,000 g/mol, from 5,000-50,000 g/mol, from 5,000-25,000 g/mol, from 10,000-25,000 g/mol, from 10,000-50,000 g/mol, from 25,000- 100,000 g/mol, from 50,000-250,000 g/mol, from 100,000-250,000 g/mol, from 100,000- 500,000 g/mol, from 250,000-500,000 g/mol, from 250,000-750,000 g/mol, or from 500,000-1,000,000 g/mol.
5. The nanoparticle according to any of claims 1-4, wherein the nanoparticle has a PDI from 0.5-2.0, from 0.5-1.5, from 0.5-1, from 0.75-1.25, from 1-1.25, from 1-1.5, from 1.25-1.5, from 1.25-1.75, or from 1.5 to 1.75.
6. The nanoparticle according to any of claims 1-4, wherein the nanoparticle has an average particle size from 0.5-1,000 nm, from 0.5-500 nm, from 0.5-250 nm, from 0.5-100 nm, from 0.5-50 nm, from 10-50 nm, from 10-100 nm, from 25-50 nm, from 25-75 nm, from 50-150 nm, from 100-250 nm, from 100-500 nm, from 250-500 nm, from 250-750 nm, or from 500-1,000 nm.
7. The nanoparticle according to any of claims 1-4, wherein the nanoparticle has an average particle size from 10-50 nm.
8. The nanoparticle according to any of claims 1-4, wherein the nanoparticle is covalently bound to a plurality of biotin anchors.
9. The nanoparticle according to claim 8, wherein the nanoparticle comprises from 2-1,000 biotin anchors, from 2-1,000 biotin anchors, from 2-500 biotin anchors, from 2-250 biotin anchors, from 2-100 biotin anchors, from 2-50 biotin anchors, from 2-25 biotin anchors, from 2-10 biotin anchors, from 5-10 SA- biotin anchors, from 10-25 biotin anchors, from 10-50 biotin anchors, from 50-100 biotin anchors, from 100-250 biotin anchors, or from 250-500 SA- biotin anchors.
10. The nanoparticle according to claim 8, wherein the nanoparticle comprises from 2-25 biotin anchors.
11. The nanoparticle according to claim 8, wherein the nanoparticle is covalently bound to a plurality of biotin anchors through a polyethylene glycol linker.
12. The nanoparticle according to any of claims 8-11, wherein the polyethylene glycol linker is covalently bonded to the biotin via a disulfide bond.
13. The nanoparticle according to any of claims 8-11, wherein the polyethylene linked comprising from 2-100 ethylene glycol units, from 2-50 ethylene glycol units, from 2-10 ethylene glycol units, from 5-25 ethylene glycol units, from 10-50 ethylene glycol units, from 25-100 ethylene glycol units, from 25-75 ethylene glycol units, or from 50-100 ethylene glycol units.
14. The nanoparticle according to any of claims 1-3 wherein the SA-FasL complex is a FasL moiety conjugated to streptavidin.
15. The nanoparticle according to any of claims 1-3 wherein the SA-FasL complex is a chimeric protein comprising a FasL moiety and a streptavidin or avidin moiety.
16. The nanoparticle according to any of claims 1-3, wherein the nanoparticle is conjugated to 2-1,000 SA-FasL complexes, from 2-500 SA-FasL complexes, from 2-250 SA-FasL complexes, from 2-100 SA-FasL complexes, from 2-50 SA-FasL complexes, from 2-25 SA-FasL complexes, from 2-10 SA-FasL complexes, from 5-10 SA-FasL complexes, from 10-25 SA-FasL complexes, from 10-50 SA-FasL complexes, from 50-100 SA-FasL complexes, from 100-250 SA-FasL complexes, or from 250-500 SA-FasL complexes.
17. The nanoparticle according to any of claims 1-3, wherein the nanoparticle is conjugated to 2-25 SA-FasL complexes.
18. A pharmaceutical composition comprising the nanoparticle according to any preceding claim and at least one pharmaceutically acceptable excipient.
19. A method of providing immunomodulation in a patient in need thereof, comprising administering to a patient in need thereof the nanoparticle according to any preceding claim.
20. The method according to any preceding claim, wherein the patient is a transplant patient.
21. The method according to any preceding claim, wherein the patient is a heart transplant patient, a kidney transplant patient, a lung transplant patient, a uterus transplant patient, a thymus transplant patient, a corneae transplant patient, a bone transplant patient, a liver transplant patient, a skin transplant patient, a bone marrow transplant patient, a bone graft patient, a bone marrow graft patient, a skin graft patient, a blood vessel graft patient, a stomach transplant patient, a penis transplant patient, a testes transplant patient, an intestine transplant patient, a limb transplant patient, an islet cell transplant patient, or a pancreas transplant patient.
22. The method according to any preceding claim, wherein the patient is a multi-organ transplant patient, receiving further comprising administering a graft in combination with the FASL presenting composition.
23. The method according to any preceding claim, comprising administering the nanoparticle to the patient before transplant surgery, during transplant surgery, after transplant surgery, or a combination thereof.
24. The method according to any preceding claim, comprising administering an immunosuppressant drug to the patient.
25. A method of delivering SA-FasL to a patient in need thereof, comprising administering to the patient the nanoparticle according to any preceding claim.
26. The method of any preceding claim, wherein the SA-FasL is selectively delivered to the lymph nodes.
27. A method of preparing the nanoparticle according to any preceding claim, comprising combining a biotin-expressing nanoparticle with a SA-FasL complex.
28. The method according to any preceding claim, wherein the biotin-expressing nanoparticles is prepared by covalently bonding biotin to a biotin-receiving nanoparticle.
29. The method according to any preceding claim, wherein the biotin is covalently bonded to the nanoparticle through a disulfide bond.
30. The method according to any preceding claim, wherein the biotin-receiving nanoparticle comprises pyridine disulfide groups.
31. A nanoparticle prepared by the method of any of claims 27-30.
PCT/US2024/018111 2023-03-01 2024-03-01 Therapeutic nanoparticles for solid organ immune acceptance WO2024182722A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363487755P 2023-03-01 2023-03-01
US63/487,755 2023-03-01

Publications (1)

Publication Number Publication Date
WO2024182722A1 true WO2024182722A1 (en) 2024-09-06

Family

ID=92590966

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/018111 WO2024182722A1 (en) 2023-03-01 2024-03-01 Therapeutic nanoparticles for solid organ immune acceptance

Country Status (1)

Country Link
WO (1) WO2024182722A1 (en)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7368295B2 (en) * 2001-08-31 2008-05-06 Fraunhofer-Gesellschaft Zur Foderung Der Angewandten Forschung E.V. Nanoparticles comprising biologically active TNF which is immobilized on the same
WO2010085509A1 (en) * 2009-01-20 2010-07-29 Northwestern University Compositions and methods for induction of antigen-specific tolerance
WO2017003940A1 (en) * 2015-06-30 2017-01-05 Tarveda Therapeutics, Inc. Targeted conjugates and particles and formulations thereof
WO2018165547A1 (en) * 2017-03-10 2018-09-13 University Of Louisville Research Foundation, Inc. Fasl-engineered biomaterials with immunomodulatory function
US11547667B2 (en) * 2007-10-12 2023-01-10 Massachusetts Institute Of Technology Vaccine nanotechnology

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7368295B2 (en) * 2001-08-31 2008-05-06 Fraunhofer-Gesellschaft Zur Foderung Der Angewandten Forschung E.V. Nanoparticles comprising biologically active TNF which is immobilized on the same
US11547667B2 (en) * 2007-10-12 2023-01-10 Massachusetts Institute Of Technology Vaccine nanotechnology
WO2010085509A1 (en) * 2009-01-20 2010-07-29 Northwestern University Compositions and methods for induction of antigen-specific tolerance
WO2017003940A1 (en) * 2015-06-30 2017-01-05 Tarveda Therapeutics, Inc. Targeted conjugates and particles and formulations thereof
WO2018165547A1 (en) * 2017-03-10 2018-09-13 University Of Louisville Research Foundation, Inc. Fasl-engineered biomaterials with immunomodulatory function

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LEI JI, CORONEL MARÍA M., YOLCU ESMA S., DENG HONGPING, GRIMANY-NUNO ORLANDO, HUNCKLER MICHAEL D., ULKER VAHAP, YANG ZHIHONG, LEE : "FasL microgels induce immune acceptance of islet allografts in nonhuman primates", SCIENCE ADVANCES, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, US, vol. 8, no. 19, 13 May 2022 (2022-05-13), US , XP093210032, ISSN: 2375-2548, DOI: 10.1126/sciadv.abm9881 *
SKOUMAL ET AL.: "Localized immune tolerance from FasL-functionalized PLG scaffolds", BIOMATERIALS, vol. 192, 13 November 2018 (2018-11-13), pages 271 - 281, XP093140862, DOI: 10.1016/j.biomaterials.2018.11.015 *

Similar Documents

Publication Publication Date Title
US20230181752A1 (en) Multimeric elastin-like polypeptides
US7589169B2 (en) Compositions for treatment with glucagon-like peptide, and methods of making and using the same
US9061071B2 (en) Analogs of ShK toxin and their uses in selective inhibition of Kv1.3 potassium channels
US20090035257A1 (en) Devices, compositions and methods for the protection and repair of cells and tissues
WO2019039483A1 (en) Antibody-drug conjugate preparation and lyophilization for same
WO2017025889A1 (en) Polymeric nanoparticles with dec-205 ligand and co-encapsulating an antigen subject to an autoimmune response and a glucocorticoid receptor agonist
Guo et al. A novel elastin-like polypeptide drug carrier for cyclosporine A improves tear flow in a mouse model of Sjögren's syndrome
US20150209335A1 (en) Methods and small molecule therapeutics comprising fused elps
AU2019389807B2 (en) Combined treatment of primary central nervous system lymphoma
US20190290726A1 (en) Protein polymer fusions for subcutaneous delivery of small molecules
CZ20013270A3 (en) Antibody and chemokine constructs and their use for treating auto-immune diseases
CN110022886A (en) A kind of method and reagent for treating autoimmune disease and allergy
Říhová et al. Antibody-targeted cyclosporin A
WO2024182722A1 (en) Therapeutic nanoparticles for solid organ immune acceptance
WO2023246325A1 (en) Nanobody targeting cd73, nanobody-drug conjugate, preparation method therefor and use thereof
EP0835662A2 (en) A drug for the treatment of cancer
EP4201431A1 (en) Intermediate for preparing antibody-drug conjugate (adc), preparation method therefor, and use thereof
EP4233868A1 (en) Use of folic acid and folic acid-modified b cell lymphoma inducing b cell immune tolerance and targeting migm positive expression
US9782494B2 (en) Methods of using multilayer magnetic micelle compositions
AU2023268552A1 (en) Transient sirolimus with fasl microgels
WO1998056823A1 (en) Induction of immune tolerance by immunotoxin
JP2024511537A (en) Synthetic peptide shuttle vehicle bioconjugates for intracellular cargo delivery
KR20220030217A (en) improved vaccine formulation
US20180125791A1 (en) Polymer nanocapsules for protein delivery

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24764662

Country of ref document: EP

Kind code of ref document: A1