WO2024050388A2 - Hybrid nanoparticles for inducing immune tolerance - Google Patents

Abstract

The present invention provides hybrid nanoparticle compositions for inducing B cell and T cell immune tolerance. The hybrid nanoparticles contain a polymeric nanoparticle core encapsulating an immunomodulatory agent, and a lipid monolayer coating the nanoparticle core and also displaying both a ligand for a B cell Siglec receptor and a target antigen (e.g., an autoantigen or self-antigen). The invention also provides methods for inducing immune tolerance to a specific target antigen, and methods for preventing and treating autoimmune diseases mediated by or associated with a specific target antigen.

Description

HYBRID NANOPARTICLES FOR INDUCING IMMUNE TOLERANCE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The subject patent application claims the benefit of priority to US Provisional Patent Application No. 63/402,335 (filed August 30, 2022; now pending). The full disclosure of the priority application is incorporated herein by reference in its entirety and for all purposes.

STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under All 32790 and AI050143 awarded by the National Institutes of Health. The government has certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] Autoimmune diseases affect tens of millions of people just in the United States alone. There are over 80 different autoimmune disorders, with rheumatoid arthritis, thyroid diseases and type 1 diabetes being the most common. Although in most cases it is possible to manage the debilitating symptoms of autoimmune diseases, they cannot be typically cured. Current standards of care include broad immunosuppressants and low-dose chemotherapeutic drugs. These, however, can compromise the overall immune system and increase the risk of opportunistic infections and other health complications.

[0004] The pathogenesis of autoimmune diseases is mediated by B and T cells that escape central tolerance and react against self-antigens. As a consequence, the immune system of the affected individual attacks healthy tissues that express the autoantigen and causes chronic inflammation. In many autoimmune diseases, autoantibodies against a self-antigen cause disease pathology. For example, in Graves’ disease inflammation is caused by antibodies to the thyroid-stimulating hormone (TSH) receptor, and in some patients with rheumatoid arthritis antibodies that target citrullinated proteins induce inflamed joint tissue. Other well documented autoimmune disorders based on a single antigen include pemphigus, the skin blister disease, with autoantibodies to desmoglein 1 and 3, and thrombotic thrombocytopenic purpura (TTP) with autoantibodies to the protease AdamTS13 leading to a severe deficiency in von Willibrands factor. The fact that patients with these diseases benefit from treatment with the B cell depleting antibody rituximab is viewed as direct evidence for the pathology mediated by autoantibodies.

[0005] There is a strong need for alternative methods for inducing B and T cell tolerance to self-antigens and for treating autoimmune diseases. The present invention is directed to addressing such unmet needs in the art.

SUMMARY OF THE INVENTION

[0006] In one aspect, the invention provides lipid-polymer hybrid nanoparticles.

These nanoparticles (NP) contain (1) a polymeric nanoparticle core that encapsulates an immunomodulatory agent, and (2) a lipid monolayer coating the nanoparticle core and displaying on its surface both (i) a ligand for an inhibitory B cell Siglec receptor conjugated to a lipid and (ii) a target antigen conjugated to a lipid. Typically, the displayed target antigen is a polypeptide. In some embodiments, the target antigen is conjugated to the lipid monolayer after formation of the lipid monolayer. In some preferred embodiments, the displayed target antigen is associated with or implicated in an autoimmune disease. For example, the displayed target antigen can be a self-antigen or an autoantigen.

[0007] In some embodiments, the lipid for conjugating to the target antigen is functionalized with a coupling moiety. In some of these embodiments, the employed coupling moiety is maleimide. In some embodiments, the target antigen is modified to contain a functional group that is reactive with the coupling moiety prior to being conjugated to the lipid. In some of these embodiments, the functional group on the modified target antigen is a thiol group, and the target antigen is conjugated to the lipid via thiol-maleimide chemistry.

[0008] In some hybrid nanoparticles of the invention, the nanoparticle core is formed of poly(lactic-co-glycolic acid) (PLGA). In various embodiments, the employed immunomodulatory agent is rapamycin (RAPA), a rapamycin derivative, a mTOR inhibitor, a corticosteroid, an antimetabolite agent, a calcineurin inhibitor, a steroid, Azathioprine, Methotrexate, Mycophenolate mofetil, Mycophenolic acid, Sirolimus, Fingolimod (FTY720), or Manitimus. In some of these embodiments, the amount of RAPA loaded in the nanoparticle core is at least about 2.5%, 5%, 7.5% or 10% (wt/wt) of the hybrid nanoparticle. In some embodiments, the ratio of RAPA and PLGA for forming the nanoparticle core is about at least 20% (wt/wt).

[0009] In some embodiments, the ligand for the inhibitory B cell Siglec receptor is coupled to the lipid prior to formation of the lipid monolayer. In some preferred embodiments, the hybrid nanoparticles contain a ligand that bind to B cell Siglec CD22. In some of these hybrid nanoparticles, the employed ligand for CD22 is a monosaccharide sialic acid derivative. In some other embodiments, the employed ligand for CD22 is a trisaccharide glycan.

[0010] In some hybrid nanoparticles of the invention, the ligand and the target antigen are each conjugated to a phospholipid or derivative thereof. In some of these embodiments, the employed phospholipid is DSPE-PEG2000. In some embodiments, the lipid monolayer is formed with the phospholipid and a mixture of unmodified lipids. In some of these embodiments, the mixture of unmodified lipids contain lecithin. In some hybrid nanoparticles of the invention, the lipid monolayer is formed with about 25% of DSPE-PEG2000 and about 75% of lecithin. In some of these embodiments, the 25% of DSPE-PEG2000 contains about 1% to about 15% of B cell binding ligand- conjugated DSPE-PEG2000, about 0.01% to about 2% of maleimide-functionalized DSPE-PEG2000, and about 0% to about 24% of unmodified DSPE-PEG2000. In a specific embodiments, the 25% of DSPE-PEG2000 contains about 10% of B cell binding ligand-conjugated DSPE-PEG2000, about 0.025% of maleimide-functionalized DSPE-PEG2000, and about 15% of unmodified DSPE-PEG2000.

[0011] In a related aspect, the invention provides methods for producing a lipidpolymer hybrid nanoparticle displaying a target antigen. The method entail (1) providing a drug mixture containing an immunomodulatory agent and a nanoparticle polymer, wherein the nanoparticle polymer is capable of self-assembling to form a nanoparticle core that encapsulates the immunomodulatory agent, (2) providing a lipid composition containing a mixture of lipids for forming a lipid monolayer that coats the nanoparticle core, wherein the mixture of lipids contain a phospholipid that is conjugated to a ligand of CD22, a phospholipid that is functionalized with a coupling moiety for conjugating to the target antigen, and one or more unmodified lipid molecules, (3) adding the drug mixture in organic solvent to the aqueous phase of the lipid composition to form a lipid coated nanoparticle core that displays the CD22 ligand and the coupling moiety, and (4) conjugating the target antigen to the a lipid coated nanoparticle core, wherein the target antigen is modified to contain a functional group that reacts with the coupling moiety.

[0012] In some of the methods, the lipid coated nanoparticle core is formed via nanoprecipitation and nanoparticle self-assembly. In some methods, the employed immunomodulatory agent is rapamycin (RAPA). In some methods, the nanoparticle polymer is poly(lactic-co-glycolic acid) (PLGA), and the phospholipid is DSPE. In some of these methods, the employed phospholipid for conjugating to CD22 ligand and the target antigen is DSPE-PEG2000. In some embodiments, the employed one or more unmodified lipid molecules contain lecithin. In some methods, the employed CD22 ligand is a monosaccharide sialic acid derivative. In some other methods, the employed CD22 ligand (CD22L) is a trisaccharide glycan. In some methods of the invention, the employed coupling moiety is maleimide, and the employed functional group on the target antigen is thiol. In some preferred methods of the invention, the immunomodulatory agent/nanoparticle polymer ratio and/or the lecithin/phospholipids ratio is optimized.

[0013] In another aspect, the invention provides methods for inducing immune tolerance to a target antigen in a subject. These methods involve administering to the subject a pharmaceutical composition that contains a therapeutic effective amount of the hybrid nanoparticle described herein. Some of these methods are directed to inducing immune tolerance to an autoantigen or a self-antigen. Some preferred methods are directed to inducing immune tolerance in human subjects.

[0014] In still another aspect, the invention provides methods for preventing, treating or ameliorating symptoms of an autoimmune disease in a subject. These therapeutic methods entail administering to the subject a pharmaceutical composition that contains a therapeutic effective amount of the hybrid nanoparticle described herein. In these methods, the targeted autoimmune disease is mediated by or associated with the target antigen displayed by the hybrid nanoparticle. Typically, the target antigen is an autoantigen or a self-antigen.

[0015] A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification and claims. DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 shows assembly of hybrid nanoparticles displaying a CD22 ligand(CD22L) and antigen on a lipid monolayer encapsulating a PLGA core with rapamycin (R). (A) Schematic illustration of nanoprecipitation and self-assembly of hybrid nanoparticles composed of a polymeric PLGA core encapsulating rapamycin (RAPA) coated with a lipid monolayer displaying CD22L and maleimide functional groups. (B) TEM image of the PLGA core of hybrid nanoparticle. (C) Conjugation of SPDP -modified ovalbumin (OVA) antigen to maleimide functionalized CD22L-NP(R) nanoparticles via thiol-maleimide chemistry. (D) Agarose gel electrophoresis (0.3%) of the CD22L-NP(R) (4% DSPE-PEG2000-maleimide) (1) incubated with OVA-SH (2) before (3) and after (4) purification from the OVA-SH excess. Gel was stained with Simply Blue.

[0017] Figure 2 shows characterization of PLGA nanoparticles. (A) TEM images of CD22L-NP(R)-OVA (1), NP(R)-OVA (2) and NP-OVA (3). Scale bar= 500 nm. (B) Size distribution of PLGA core diameters analyzed from TEM images. (C) Table summarizing size (DLS, TEM) and RAPA loading/encapsulation in set of three different nanoparticles. (D) Both CD22L-NP(R)-OVA (1) and non-targeted NP(R)- OVA (2) encapsulate the same amount of RAPA. Graphs represent how much RAPA [pg] is encapsulated in 1 mg of NPs, or (E) RAPA encapsulation yield defined as the weight ratio of the encapsulated drug to the initial drug input. Statistical analyses were performed using one-way ANOVA followed by the Tukey post-test (***P < 0.0002). [0018] Figure 3 shows that CD22 ligand on tolerogenic nanoparticles CD22L- NP(R)-OVA mediates binding to B cells. (A) Schematic illustration of DSPE- PEG2000- Alexa Fluor 647 labeled CD22L-NP(R)-OVA binding to B cells via CD22 receptor. Insert: agarose gel (0.3%) image of CD22L-NP(R)-OVA stained with SimplyBlue (a) and scanned for fluorescence to detect AlexaFluor 647 (b). (B) Flow cytometry analysis revealed increased binding of CD22L -targeted CD22L-NP-OVA nanopanicles (blue) to ( TH O 14122 B cells from mouse splenocytes compared to nontargeted NP-OVA nanoparticles (pink). (C) Quantification of the flow data illustrated as a median fluorescence intensity [a.u.J Statistical analyses were performed using one-way ANOVA followed by the Tukey post-test (***p < 0.0002).

[0019] Figure 4 shows biodistribution and blood circulation half-life of hybrid

NPs. (A) Ex vivo fluorescence images of main organs at 24 h post-injection of DSPE- PEG2000-Cy7 labeled CD22-NP-OVA (1 ) and NP-OVA (2). Abbreviations: LNs- lymph nodes; K- kidney; S- spleen; Lu-lungs; Li-liver; H-heart. (B) Corresponding total radiant efficiency of the images. Insert, represents image of spleens with significantly higher accumulation of CD22L -NP-OVA (1) versus NP-OVA (2) 24h after injection. Data are presented as the mean ± SEM. (C) Measured fluorescence intensity of CD22L- NP-OVA and NP-OVA (2) in the blood of WT mice 1.5, 3, 6 and 24h post-injection. (D) Corresponding change of NPs concentration over time calculated from the radiant efficiency of the images. Data are presented as the mean ± SEM. The curve indicates an exponential decay curve fit to the data (n = 3 mice per time point)

[0020] Figure 5 shows that tolerogenic CD22L-NP(R)-OVA nanoparticles suppress development of anti-OVA IgGl in naive mice. (A) Outline of the experimental design. Naive C57BL/6J WT mice (6-8 weeks old) received two doses (z.v. of nanoparticles 2 weeks apart to deliver 50 pg of RAPA per injection. On day 28, animals were challenged with OVA/ Alum (i.p.) and two weeks later (day 42) animals were bled and sacrificed. (B) Treatment with CD22L-NP(R)-OVA (blue) reduces production of serum anti-OVA IgGl levels. Anti-OVA IgG titers from naive mice (grey, n = 5), CD22L-NP(R)-OVA treated (blue, n=7), NP(R)-OVA treated (green, n=7) and NP-OVA treated (pink, n=7) mice measured before challenge on day 27 or two weeks after the challenge on day 42 determined by ELISA. Statistical analysis was performed using one-way ANOVA (Kruskal -Wallis followed by Dunn’s multiple comparison test, ***P < 0.0002, **P < 0.0021, *P < 0.0332)

[0021] Figure 6 shows that tolerogenic nanoparticle treatment delays onset of arthritis and reduces serum anti-GPI antibody titers in K/BxN mice. (A) Outline of the experimental design. Twenty-one day old K/BxN mice received four doses (z.v. injections) of NP(R)-GPI or CD22L-NP(R)-GPI every 5 days (at age 21, 26, 31, and 36 days) containing 1 mg nanoparticles with 100 pg of RAPA per injection. Untreated K/BxN mice are used as controls. Some animals from each group were sacrificed for tissue harvesting (hind paws-histology; spleens-immune cell analysis) when they reached 42 days of age. The remaining animals were imaged using the IVIS and maintained for the duration of the study. Mice were bled every week to assess serum anti-GPI IgGl levels, and ankle joints were measured three times per week. (B) Ankle joint measurements are shown over time as a percent of mice with no joint swelling over 4 mm. CD22L-NP(R)-GPI delayed onset of arthritis and cured 33% of animals. Untreated K/BxN (grey, n=13), CD22L-NP(R)-GPI treated (blue, n=22), and NP(R)- GPI treated (pink, n=16). Mice were considered to be at the study end point when one or more joint(s) measured 4 mm. Statistical analysis was performed using a log-rank Mantel-Cox test

< 0.0001). Results are combined from two independent experiments. (C) Treatment of arthritic mice with tolerogenic CD22L-NP(R)-GPI reduces production of serum anti-GPI IgGl levels. Anti-GPI IgG titers from untreated (grey, n =9), CD22L-NP(R)-GPI treated (blue, n=10), and NP(R)-GPI treated (pink, n=9) K/BxN mice measured by ELISA every week to day 81. (D) Anti-GPI IgGl titers of CD22L-NP(R)-GPI treated survivors (300 days old) are very low and comparable to healthy 21 -day old K/BxN mice.

[0022] Figure 7 shows that tolerogenic CD22L-NP(R)-GPI nanoparticles reduce inflammation in hind paw joints of K/BxN mice. Inflammation was assessed in the hind paws of K/BxN mice 24h after being injected with ProSense750EX using IVIS. (A) Examples of fluorescence of paws of healthy 21 day old K/BxN mice (orange) compared with hind paws of 42 day old untreated diseased mice (gray) or mice treated with either CD22L-NP(R)-GPI (blue) or NP(R)-GPI (pink). (B) Quantification of results for paws from 5-6 mice of each group represented as total radiant efficiency of ProSense 750EX for IVIS datasets. All data represent the mean ± SD. Statistical analysis was performed by one-way ANOVA followed by the Tukey post-test (***p < 0.0002, **P < 0.0021, *P < 0.0332).

[0023] Figure 8 shows that CD22L-NP(R)-GPI nanoparticles protect K/BxN mice from joint damage. (A) Comparison of ankle joint histology of healthy, and diseased and nanoparticle treated K/BxN mice from representative paraffin-embedded sections stained with Safranin O/Fast Green and hematoxylin. Joint sections from ‘healthy’ 21- day old K/BxN mice (1) (upper left) are characterized by the maintenance of Safranin O cartilage staining green*), a healthy bone matrix (green**), and synovial tissue lacking infiltrating immune cells. As they age, untreated 42-day old K/BxN mice (2) develop arthritis and their joint sections exhibit severe loss of cartilage, as judged by a loss of Safranin O staining (red *), bone resorption (red **), and inflamed synovium with leukocyte invasion (red ***). The histology of the sections from 42 day old mice treated with NP(R)-GPI (4) appear no different than the untreated mice, while those treated with CD22L-NP(R)-GPI (3) appear equivalent to the 21 day healthy mice. Images are representative of three mice for each condition. Scale bars 200pm. (B) Quantification of histological score for joint inflammation, cartilage damage, and bone resorption using criteria described in supporting information. (C) A representative paraffin-embedded ankle section from a 300 day old disease free mouse treated with CD22L-NP(R)-GPI (5). (D) The quantification of stained sections using combined histological scores from all groups including the 300 day old disease free mice. All data represent the mean ± SEM. Statistical analyses were performed using one-way ANOVA followed by the Tukey post-test

< 0.0001).

[0024] Figure 9 shows that tolerogenic nanoparticles increase the level of Tregs and reduce the level of antibody producing plasma cells. (A) Representative scatter plots of CD4⁺Foxp3⁺ Tregs gated on CD9O.2⁺CD4⁺CD1 Ic'CDl lb'B220'live cells for untreated, NP(R)-GPI treated, and CD22L-NP(R)-GPI treated arthritic mice measured on day 42 of the study. (B) Quantification of Foxp3⁺ Tregs. (C) Representative scatter plots of CD138^highB220^interplasma cells gated on CD19^lowlive cells for untreated, NP(R)-GPI treated, and CD22L-NP(R)-GPI treated arthritic mice measured on day 42 of the study. (D) Quantification of antibody secreting plasma cells. All data represent the mean ± SEM and were combined from two independent experiments. Statistical analyses were performed using one-way ANOVA followed by the Tukey post-test (****P < 0.0001).

DETAILED DESCRIPTION

I. Overview

[0025] Autoimmune diseases affect over 4% of the world’s population. Treatments are generally palliative or use broad spectrum immunosuppressants to reduce symptoms and disease progression. In some diseases antibodies generated to a single autoantigen are the major cause of pathogenic inflammation, suggesting that treatments to induce tolerance to the autoantigen could be therapeutic.

[0026] The subject invention is derived in part from the present inventors’ studies to develop hybrid nanoparticles (NPs) that induce tolerance in both T cells and B cells. As detailed herein, the inventors developed lipid-polymer hybrid nanoparticles that contain a lipid monolayer encapsulating a PLGA nanoparticle core that is loaded with rapamycin (RAPA). Rapamycin is a known immunomodulatory agent that promotes development of regulatory T cells (Tregs). Other than the nanoparticle core, the lipid monolayer of the hybrid nanoparticle displays a protein antigen and a ligand of the B cell inhibitory co-receptor CD22 (CD22L), which act together to suppress activation of B cells recognizing the antigen. As exemplification, the inventors demonstrated that the hybrid NPs decorated with ovalbumin (OVA) elicit tolerance to OVA in naive mice, as judged by low OVA-specific antibody titers after the challenge. Further, in a mouse model of rheumatoid arthritis (RA) caused by B and T cell dependent responses to the self-antigen glucose-6-phosphate-isom erase (GPI), the inventors found that GPI hybrid nanoparticles delay development of disease, with some treated mice remaining arthritis- free for 300 days. It was shown that the mechanism of protection involves tolerance induction in both T and B cells, as evidenced by increased numbers of regulatory T cells, and suppression of anti-GPI antibodies and antibody secreting plasma cells. The therapeutic effect of the hybrid nanoparticles displaying the CD22 ligand and GPI is evident from the reduction in joint swelling, in vivo imaging of inflamed tissues, and joint histology. Importantly, the inventors also observed that the therapeutic effect requires co-presentation of the GPI antigen with CD22 ligand and the encapsulated RAPA, since nanoparticles missing either CD22L or RAPA have little impact on disease progression. Results from these studies demonstrated the potential of the versatile hybrid nanoparticle platform described herein for inducing immune tolerance to self-antigens and for suppressing autoimmune diseases

[0027] In accordance with these studies, the invention accordingly provides lipidpolymer hybrid nanoparticles as described herein, as well as therapeutic applications of such compositions. The therapeutic applications include, e.g., methods of inducing B cell and T cell immune tolerance against target antigens (e.g., self-antigens and autoantigens), and methods of suppressing immune responses to antigens that induce unwanted immune responses in various autoimmune diseases or allergies to food or environmental allergens.

[0028] The following sections provide more detailed guidance for practicing the invention.

II. Definitions

[0029] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains. The following references provide one of skill with a general definition of many of the terms used in this invention: Academic Press Dictionary of Science and Technology, Morris (Ed.), Academic Press (1^st ed., 1992); Oxford Dictionary of Biochemistry and Molecular Biology, Smith et al. (Eds.), Oxford University Press (revised ed., 2000); Encyclopaedic Dictionary of Chemistry, Kumar (Ed.), Anmol Publications Pvt. Ltd. (2002); Dictionary of Microbiology and Molecular Biology, Singleton et al. (Eds.), John Wiley & Sons (3^rd ed., 2002); Dictionary of Chemistry, Hunt (Ed.), Routledge (1^st ed., 1999); Dictionary of Pharmaceutical Medicine, Nahler (Ed.), Springer-Verlag Telos (1994); Dictionary of Organic Chemistry, Kumar and Anandand (Eds.), Anmol Publications Pvt. Ltd. (2002); and 4 Dictionary of Biology (Oxford Paperback Reference) , Martin and Hine (Eds.), Oxford University Press (4^th ed., 2000). Further clarifications of some of these terms as they apply specifically to this invention are provided herein.

[0030] The term "agent" includes any substance, molecule, element, compound, entity, or a combination thereof. It includes, but is not limited to, e.g., protein, polypeptide, small organic molecule, polysaccharide, polynucleotide, and the like. It can be a natural product, a synthetic compound, or a chemical compound, or a combination of two or more substances. Unless otherwise specified, the terms “agent”, “substance”, and “compound” are used interchangeably herein.

[0031] The term "analog" or “derivative” is used herein to refer to a molecule that structurally resembles a reference molecule (e.g., a known Siglec ligand) but which has been modified in a targeted and controlled manner, by replacing a specific substituent of the reference molecule with an alternate substituent. Compared to the reference molecule, an analog would be expected, by one skilled in the art, to exhibit the same, similar, or improved utility. For example, many analogs or derivatives of sialic acids (modified sialic acids) can be used in the practice of the invention, as described in detail below. Synthesis and screening of analogs to identify variants of known compounds having improved traits (such as higher binding affinity for a target molecule) is an approach that is well known in pharmaceutical chemistry.

[0032] The term antigen broadly refers to a molecule that can be recognized by the immune system. It encompasses proteins, polypeptides, polysaccharides, small molecule haptens, nucleic acids, as well as lipid-linked antigens (polypeptide- or polysaccharide-linked lipids.

[0033] Alloantigens refer to antigens present in some but not all individuals of the same species, as those in different human blood groups. They elicit immune responses when introduced into a genetically different individual of the same species. Antibodies produced in response to alloantigens are called alloantibodies. The majority of alloantigens are glycoproteins (e g. MHC, MNS antigens), sialoglycoproteins (e.g. CD43), oligosaccharides (ABO(H), Secretor, Lewis, Li and P), sialo-oligosaccharides (sialyl-Lewis^x or sialyl-Lewis^a) or proteins (Rh).

[0034] Self-antigens are by convention antigens in the body of an individual. They are antigens that the immune system derives from the body it is protecting. For example, a heart cell, a liver cell, and a kidney cell would all contain self-antigens. A healthy immune system tolerates self-antigens and does not attack them. In some autoimmune diseases, autoantibodies can be generated against such self-antigens including cellular proteins, peptides, enzyme complexes, ribonucleoprotein complexes, DNA, and post-translationally modified antigens.

[0035] T cell-dependent or T-dependent antigens refer to antigens which require T cell assistance in eliciting antibody production by B cells. Structurally these antigens are characterized by multiple antigenic determinants. Proteins or polypeptides are typical examples of T-dependent antigens that contain antigenic determinants for both B and T cells. With a T-dependent antigen, the first signal comes from antigen cross linking of the B cell receptor (BCR) and the second signal comes from co-stimulation provided by a T cell. T-dependent antigens contain antigenic peptides that stimulate the T cell. Upon ligation of the BCR, the B cell processes the antigen, releasing antigenic peptides that are presented on B cell Class II MHC to a special subtype of T cell called a Th2 cell. The Th2 cell then secretes potent cytokines that activate the B cell. These cytokines trigger B cell proliferation, induce the B cells to produce antibodies of different classes and with increased affinity, and ultimately differentiate into antibody producing plasma cells.

[0036] T cell-independent or T-independent (TI) antigens are antigens which can directly stimulate the B cells to elicit an antibody response, do not contain proteins, and cannot induce T helper cell. Often, T-independent antigens have polymeric structures, e.g., the same antigenic determinant repeated many times. Examples of T-independent antigens include small molecule haptens, nucleic acids, carbohydrates and polysaccharides.

[0037] 1, 2-Distearoyl-sn-glycero-3-phosphoethanoiamine-Poly(ethylene glycol)

(DSPE- PEG) is a widely used phospholipids-polymer conjugate in drug delivery applications. It is a biocompatible, biodegradable and amphiphilic material that can also be functionalized with various biomolecules for specific functions.

[0038] As used herein, immune tolerance (or simply “tolerance”) is the process by which the immune system does not attack an antigen. It occurs in three forms: central tolerance, peripheral tolerance and acquired tolerance. Tolerance can be either “natural” or “self tolerance”, where the body does not mount an immune response to selfantigens, or “induced tolerance”, where tolerance to antigens can be created by manipulating the immune system. When tolerance is induced, the body cannot produce an immune response to the antigen. Mechanisms of tolerance and tolerance induction are complex and poorly understood. As is well known in the art (see, e.g., Basten et al., Curr. Opinion Immunol. 22:566-574, 2010), known variables in the generation of tolerance include the differentiation stage of the B cell when antigen is presented, the type of antigen, and the involvement of T cells and other leukocytes in production of cytokines and cofactors. Thus, suppression of B cell activation cannot be equated with immune tolerance. For example, while B cell activation can be inhibited by crosslinking CD22 to the BCR, the selective silencing of B cells does not indicate induction of tolerance. See, e.g., Nikolova et al., Autoimmunity Rev. 9:775-779, 2010; Mihaylova et al., Mol. Immunol. 47: 123-130, 2009; and Courtney et al., Proc. Natl. Acad. Sci. 106:2500-2505, 2009.

[0039] A “hybrid nanoparticle composition” or “lipid-polymer hybrid nanoparticle” as used herein refers to a lipid-polymer complex that contains (1) a polymer based nanoparticle that encapsulates an immunomodulatory agent (e.g., rapamycin or derivative) and (2) a lipid monolayer that coats the nanoparticle core. The lipid monolayer that coats the nanoparticle core displays on or incorporates into the lipid moiety both (1) a glycan ligand that binds to a sialic acid binding Ig like lectin (Siglec) co-receptor (e.g., CD22) on B cells and (2) a target antigen (e.g., an autoantigen or self-antigen) implicated in an autoimmune disease. Preferably, the ligand for the B Siglec is integrated into the lipid monolayer of the hybrid nanoparticle. The target antigen is typically also integrated into the lipid component of the hybrid nanoparticle.

[0040] Siglecs, short for sialic acid binding Ig-like lectins, are cell surface receptors and members of the immunoglobulin superfamily (IgSF) that recognize sugars. Their ability to recognize carbohydrates using an immunoglobulin domain places them in the group of I-type (Ig-type) lectins. They are transmembrane proteins that contain an N-terminal V-like immunoglobulin (IgV) domain that binds sialic acid and a variable number of C2-type Ig (IgC2) domains. The first described Siglec is sialoadhesin (Siglec- 1/CD 169) that is a lectin-like adhesion molecule on macrophages. Other Siglecs were later added to this family, including CD22 (Siglec-2) and Siglec- G/10 (i.e., human Siglec-10 and mouse Siglec-G), which are expressed on B cells and have an important role in regulating their adhesion and activation, CD33 (Siglec-3) and myelin-associated glycoprotein (MAG/Siglec-4). Several additional Siglecs (Siglecs 5- 12) have been identified in humans that are highly similar in structure to CD33 so are collectively referred to as ‘CD33-related Siglecs’. These Siglecs are expressed on human NK cells, B cells, and/or monocytes. CD33-related Siglecs all have two conserved immunoreceptor tyrosine-based inhibitory motif (ITIM)-like motifs in their cytoplasmic tails suggesting their involvement in cellular activation. Detailed description of Siglecs is provided in the literature, e.g., Crocker et al., Nat. Rev. Immunol. 7:255-66, 2007; Crocker et al., Immunol. 103: 137-45, 2001; Angata et al., Mol. Diversity 10:555-566, 2006; and Hoffman et al., Nat. Immunol. 8:695-704, 2007. [0041] Glycan ligands of Siglecs refer to compounds which specifically recognize one or more Siglecs and which comprise homo- or heteropolymers of monosaccharide residues. In addition to glycan sequences, the Siglec glycan ligands can also contain pegylated lipid moiety connected to the glycan via a linker. Some embodiments of invention utilize glycan ligands that bind to B cell Siglecs, as detailed below. There are many B cell Siglec targeting glycan ligands that have been reported in the literature. See, e.g., Paulson et al., WO 2007/056525; and Blixt et al., J. Am. Chem. Soc. 130:6680-1, 2008; Rillahan et al., Chem Sci 5: 2398-406, 2014; and Rillahan et al., Angew Chem Int Ed Engl 51 : 11014-18, 2012.

[0042] Administration "in conjunction with" one or more other therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

[0043] Sialic acids refer to a class of alpha-keto acid sugars with a nine-carbon backbone. Based on different modifications of the C5 position, sialic acids are classified in three basic forms: Neu5Ac, Neu5Gc, and KDN. Besides the C5 position, single or multiple substitutions can occur at the hydroxyl groups at C4, C7, C8, and C9 positions (substituted by acetyl, sulfate, methyl, lactyl, phosphate, etc.). Sialic acids are typically negatively charged molecules which are commonly presented as the terminal residues in glycans of the glycoconjugates on eukaryotic cell surface or as components of capsular polysaccharides or lipooligosaccharides of some pathogenic bacteria. Unless otherwise noted, sialic acids and modified sialic acids as used herein are monosaccharides. Unless otherwise noted, the terms “modified sialic acids” and “monosaccharide sialic acid derivatives” are used interchangeably herein.

[0044] As used herein, sialosides refer to sialic acid-containing carbohydrates and derivatives. Unless otherwise noted, they include monosaccharide compounds (e.g., modified sialic acids described herein), di saccharide compounds, tri saccharides and etc. [0045] The term “subject” refers to any animal classified as a mammal, e.g., human and non-human mammals. Examples of non-human animals include dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, and etc. Unless otherwise noted, the terms “patient” or “subject” are used herein interchangeably. Preferably, the subject is human.

[0046] The term “treating” or “alleviating” includes the administration of compounds or agents to a subject to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease (e.g., an autoimmune disease), alleviating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder. Subjects in need of treatment include those already suffering from the disease or disorder as well as those being at risk of developing the disorder. Treatment may be prophylactic (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease.

III. Hybrid tolerogenic nanoparticles for inducing tolerance

[0047] The present invention provides lipid-polymer hybrid nanoparticle compositions for inducing B and T cell tolerance and suppressing immune response to specific target antigens. Typically, the hybrid nanoparticles contain a polymeric nanoparticle core encapsulated by a lipid monolayer. Inside the nanoparticle core is loaded with an immunomodulatory agent. Various immunomodulatory agents can be employed in the practice of the invention. As exemplified herein, one of such agents is rapamycin (aka RAPA or sirolimus), a drug that promotes development of regulatory T cells (Tregs). Other immunomodulatory agents suitable for the invention include, but not limited to, rapamycin derivatives and a number of other small molecule drugs that suppress T cell mediated immunity. Examples of these agents include, e.g., rapamycin derivative everolimus, Azathioprine, Methotrexate, Mycophenolate mofetil, Mycophenolic acid, Sirolimus, Steroids, Tacrolimus, Fingolimod (FTY720), Corticosteroids (e.g., Prednisone), other mTOR inhibitors (e.g., SRL and EVL), Antimetabolite agents (e.g., Azathioprine, Methotrexate and MPA), Calcineurin inhibitors (e.g., Cyclosporine A and Tacrolimus), and Manitimus (FK778). See, e.g., Baroja-Mazo et al., World J. Transplant 6(1): 183-192, 2016; Meneghini et al., Best Pract. Res. Clin. Gastroenterol. 54-55, 101757, 2021; and Getts et al., Immunotherapy 3(7): 853-870, 2011.

[0048] The lipid monolayer of the hybrid nanoparticles of the invention displays (e.g., via chemical conjugation or coupling) a ligand or binding moiety conjugated to the lipid monolayer specifically binds to an inhibitory Siglec receptor on B cells, e.g., CD22 (aka Siglec 2) or Siglec-G/10 (i.e., human Siglec-10 and mouse Siglec-G). In addition to the B-cell targeting ligand, the lipid monolayer also displays one or more target antigens against which immune tolerance is desired. In some preferred embodiments, the target antigen conjugated to the lipid monolayer is an autoantigen or self-antigen.

[0049] The nanoparticle core inside the hybrid nanoparticles of the invention is typically composed of a hydrophobic polymeric shell that encapsulates an immunomodulatory agent such as rapamycin. In general, the polymeric shell is formed with a biodegradable polymer compound. Biodegradable polymer materials are natural or synthetic in origin and are degraded in vivo, either enzymatically or non- enzymatically or both, to produce biocompatible, toxicologically safe by-products. The by-products are further eliminated by the normal metabolic pathways. A number of such materials have been used in or as adjuncts in controlled drug delivery. These include, e.g., (1) synthetic biodegradable polymers, which includes relatively hydrophobic materials such as the a-hydroxy acids (a family that includes poly lactic- co-glycolic acid, PLGA), polyanhydrides, and others, and (2) naturally occurring polymers, such as complex sugars (hyaluronan, chitosan) and inorganics (hydroxyapatite).

[0050] In some preferred embodiments of the invention, the hydrophobic polymeric shell is formed with PLGA as exemplified herein. Polyester PLGA is a copolymer of poly lactic acid (PLA) and poly glycolic acid (PGA). It is the best known biomaterial available for drug delivery with respect to design and performance. Poly lactic acid contains an asymmetric a-carbon which is typically described as the D or L form in classical stereochemical terms and sometimes as R and S form, respectively. The enantiomeric forms of the polymer PLA are poly D-lactic acid (PDLA) and poly L- lactic acid (PLLA). PLGA is generally an acronym for poly D,L-lactic-co-glycolic acid where D- and L- lactic acid forms are in equal ratio. PLGA can be processed into almost any shape and size, and can encapsulate molecules of virtually any size. It is soluble in wide range of common solvents including chlorinated solvents, tetrahydofuran, acetone or ethyl acetate. In water, PLGA biodegrades by hydrolysis of its ester linkages.

[0051] The nanoparticle core also contains an encapsulated immunomodulatory agent beyond the hydrophobic polymeric shell. As noted above, a number of immunomodulatory agents can be used in the invention. In some preferred embodiments, the immunomodulatory agent encapsulated inside the nanoparticle core is rapamycin, as exemplified herein. Rapamycin encapsulated inside the hydrophobic nanoparticle core is a drug that is known to promote development of regulatory T cells (Tregs). It inhibits the mTOR signaling pathway, and has been shown to induce tolerance to a variety of antigens. In the hybrid nanoparticles of the invention, the nanoparticle core is loaded with a therapeutically effective amount of the immunomodulatory agent such as rapamycin.

[0052] The lipid monolayer that coats the hydrophobic nanoparticle core is typically a vesicular structure of a water soluble particle obtained by aggregating amphipathic molecules such as lipids. It can be made with any suitable lipid material that can self-assemble under appropriate condition to form a lipid monolayer. To display a specific B cell binding ligand on the lipid monolayer, the ligand is conjugated to a lipid molecule (e.g., a pegylated phospholipid molecule such as DSPE-PEG) included in the lipid material for forming the lipid monolayer. Similarly, to allow display of the target antigen on the lipid monolayer, a coupling moiety for attaching the antigen is conjugated to a lipid molecule, which can be the same or different from the lipid molecule for displaying the inhibitory B cell Siglec binding ligand. In addition to these two modified lipid molecules the lipid material for forming the lipid monolayer typically also contain unconjugated and unmodified lipid molecules. The unconjugated and unmodified lipid molecules can be the same lipid molecule (e.g., a phospholipid such as DSPE-PEG) for attaching the B cell Siglec ligand or the target antigen. The unconjugated and unmodified lipids for forming the lipid monolayer can also be a mixture of different lipids or phospholipids such as lecithin. Lecithin contains a mixture of lipids from soybean, including triglycerides, phospholipids, and glycolipids. Lecithin phospholipids include, e.g., phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine, and phosphatidic acid. The unconjugated and unmodified lipids used for forming the lipid monolayer can also contain one or more synthetic phospholipids. As a specific exemplification, the lipid monolayer of the hybrid nanoparticles of the invention can be formed with lecithin and N-(Methylpolyoxy ethylene oxycarbonyl)-l,2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE-PEG2000), plus the B cell binding ligand-modified DSPE-PEG2000 and maleimide-functionalized DSPE-PEG2000. Other than DSPE- PEG2000, additional phospholipids that may be employed as the key component of the lipid monolayer include, e.g., distearoyl phosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), dioleylphosphatidylcholine (DOPC) and dioleylphosphatidyl ethanolamine (DOPE), sphingoglycolipid and glyceroglycolipid. These phospholipids and derivatives thereof may be used alone or in combination to form the lipid monolayer.

[0053] In some preferred embodiments, the B cell Siglec ligand or binding moiety displayed on the lipid monolayer of the hybrid nanoparticles of the invention specifically binds to the inhibitory Siglec B-cell receptor CD22. In some other embodiments, the B cell targeting ligand can be one that specifically recognizes Siglec 10/G. CD22 is an inhibitory receptor that negatively regulates B cell receptor signaling, and may also be involved in the cell-cell interactions and localization of B-cells in lymphoid tissues. CD22 from a number of species are known in the art, e.g., human CD22 and mouse CD22. While ligands that bind to CD22 of any species may be used in the invention, they are preferably ligands that bind human CD22. Any CD22 ligand known in the art that can be linked to and displayed on the lipid monolayer can be employed in the construction of the hybrid nanoparticles of the invention. See, e.g., Paulson et al., WO 2007/056525; Chen et al., Blood 115:4778-86, 2010; Blixt et al., J. Am. Chem. Soc. 130:6680-1,2008; Kumari et al., Virol. J. 4:42, 2007; and Kimura et al., J. Biol. Chem. 282:32200-7, 2007. Additional B cell Siglec ligands that can be employed int he practice of the invention are described in, e.g., Rillahan et al., Chem Sci 5: 2398-406, 2014; Rillahan et al., Angew Chem Int Ed Engl 51 : 11014-18, 2012; Mesh et al., ChemMedChem 7: 134-43, 2012; Abdu-Allah et al., Bioorg Med Chem 19: 1966-71, 2011; Abdu-Allah et al., Bioorg Med Chem Lett 19: 5573-75, 2009; Magesh et al., Curr Med Chem 18: 3537-50, 2011; Matsubara et al., Front Immunol 9: 820, 2018; Prescher et al., ACS Chem Biol 9: 1444-50, 2014; Prescher et al., Chembiochem 18: 1216-25, 2017; Zaccai et al., Structure 11 : 557-67, 2003.

[0054] In some embodiments, the B cell Siglec ligand in the hybrid nanoparticles of the invention is a monosaccharide derivative of sialic acid. A number of such sialic acid derivatives that bind to B cell Siglecs are known in the art, which can all be employed in the practice of the invention. For example, Mesch et al. (Mesh et al., ChemMedChem 7: 134-143, 2012) described a group of sialoside based CD22 ligands. These compounds contain hydrophobic substituents at the 2-, 5-, and 9-positions of the sialic acid scaffold. Zaccai et al. (Structure 11 : 557-567, 2003) described a group of sialoside CD22 ligands that contain aromatic ring substitutions for the glycerol group in the sialic acid scaffold. Specific examples of the compounds include Me-a-9-A- benzoyl-amino-9-deoxy-Neu5Ac (BENZ), Me-a-9-A^r-(napthyl-2-carbonyl)-amino-9- deoxy-Neu5Ac (NAP), and Me-a-9- V-(biphenyl-4-carbonyl)-amino-9-deoxy-Neu5Ac (BIP) (R.B. and S Geis, in preparation). Some other modified sialic acids or sialic acid derivatives that function as human CD22 ligands are described in Prescher et al., Chembiochem 18, 1216-1225, 2017; and Prescher et al., ACS Chem Biol 9: 1444-50, 2014. These include sialic acid derivatives with a triazole moiety replacing the natural glycoside oxygen atom, and BPC-sialosides that have combined modification at positions 2, 4, and 9 or at positions 2, 3, 4, and 9 of the sialic acid scaffold.

[0055] In some embodiments, the B cell Siglec ligand used in the hybrid nanoparticles of the invention is a glycan ligand with a 3-sugar scaffold. In some embodiments, the employed glycan ligand is a synthetic glycan ligand for CD22. Examples of synthetic CD22 ligands include mouse CD22 ligand 6’- BPANeuGc exemplified herein, and human CD22 ligands 9-N-biphenylcarboxyl-NeuAca2-6Gaipi- 4GlcNAc (6’-BPCNeuAc) and 9-N-biphenylcarboxyl-NeuAca2-3Gaipi-4GlcNAc (3’- BPCNeuAc). In some other embodiments, the employed CD22 glycan ligand is a natural glycan ligand of human CD22. Examples of such ligands include NeuAca2- 6Gaipi-4GlcNAc and NeuAca2-6Gaipi-4(6-sulfo)GlcNAc, which can be used for targeting an antigen to human B cells. Also suitable for the invention are other specific glycan ligands for human CD22 or Siglec-10 are described in the art, e.g., Blixt et al., J. Am. Chem. Soc. 130:6680-1, 2008; and Paulson et al., WO 2007/056525. Similarly, the invention can employ additional mouse CD22 ligands known in the art, e.g., NeuGca2- 6Gaipi-4GlcNAc (NeuGc), and NeuGca2-3Gaipi-4GlcNAc. In some embodiments, the employed B cell Siglec binding ligand used in the hybrid nanoparticles of the invention is a glycan ligand that can bind to all the 4 known B-cell Siglects in human and mouse (i.e., human CD22, Siglec-10, mouse CD22 and Siglec-G). Examples of such B cell Siglec ligands include a number of sialoside analogues described in, e.g., Rillahan et al., Angew Chem Int Ed Engl 51 : 11014-18, 2012.

[0056] Other than the B cell Siglec ligand, at least one specific antigen against which immune tolerance is desired is also conjugated to the lipid monolayer of the hybrid nanoparticles of the invention. More detailed description of the target antigens suitable for the invention and specific exemplifications are provided herein.

[0057] Construction of the hybrid nanoparticles of the invention can be performed with the methods described herein, as well as some standard techniques known in the art. In some embodiments, the hybrid nanoparticles are synthesized in accordance with the specific optimized protocols exemplified herein. For example, the phospholipid components of the lipid monolayer (e.g., phospholipid DSPE-PEG2000) can be linked to a CD22 ligand compound (e.g., 6’- BPANeuGc) or functionalized with a coupling moiety (e.g., maleimide) for conjugating the target antigen. Phospholipid (e.g., DSPE- PEG2000) that is functionalized with a coupling moiety (e.g., maleimide) can be readily synthesized via standard protocols of organic chemistry or obtained from commercial sources. Conjugation of the CD22 ligand to the phospholipid can be performed as described herein or in the literature, e.g., Macauley et al., J. Clin. Invest. 123:3074-83, 2013; and Chen et al., Blood 115:4778-86, 2010. A mixture of lecithin (or other unmodified phospholipids) and the modified phospholipid molecules (the phospholipid that is linked to the CD22 ligand, and the phospholipid functionalized with the coupling moiety) are then used for forming the lipid monolayer that encapsulates the nanoparticle core to be formed with the biodegradable polymer and the immunomodulatory agent (e.g., rapamycin). As exemplified herein, the rapamycin drug and the hydrophobic polymer compound such as PLGA can be mixed at proper ratios, and dissolved in organic solvent to the aqueous phase of the lipid mixture. Under appropriate conditions (e.g., heated to around 68°C), the PLGA polymer will readily precipitate to form a nanoparticle hydrophobic core encapsulating rapamycin. As the nanoparticles form, the lipids self-assemble around the hydrophobic nanoparticle core to form a lipid monolayer displaying the CD22 ligand and the coupling moiety (e.g., a maleimide functional handle). As exemplified herein, the target antigen (e.g., OVA or GPI) can be modified with a suitable linker, e.g., a SPDP heterobifunctional linker, to provide a functional group (e.g., thiol group) that can react with the coupling moiety (e.g., maleimide) on the phospholipid. The lipid coated nanoparticle is then subject to attachment of one or more target antigens thus modified. Upon completion of the construction, the hybrid nanoparticles can be further concentrated and purified to remove excess rapamycin and residual organic solvent, e.g., by ultrafiltration.

[0058] The invention also provides methods for producing hybrid lipid-polymer nanoparticles described herein, which are suitable for inducing immune tolerance against a specific antigen. As described herein, these methods involve the use of optimized parameters so that the nanoparticles are reproducible and size-controlled. As exemplified herein, the methods can employ an optimized lipid to polymer ratio. For example, a total lipid to the polymer (e.g., PLGA) weight ratio of about 10% to about 20% (e.g., around 13.6% as exemplified) is used. In some embodiments, the ratio of the immunomodulatory agent to the polymer in the nanoparticle core is also optimized. For example, the RAPA to the polymer (e.g., PLGA) weight ratio is at least about 10% or 15%. Preferably, the ratio is at least higher than about 20%, 25%, 30%, 35%, 40%, 45% or even higher. As demonstrated herein, a RAPA/PLGA input lower than 20% (wt/wt) may result in a decreased drug loading and could be insufficient to encapsulate a therapeutic dose of the immunomodulatory agent (e.g., rapamycin) for tolerance induction.

[0059] In addition to an optimal lipid/polymer ratio and/or an optimal immunomodulatory agent/polymer ratio, the invention also involves the use of an optimized ratio of the different lipid components for forming the lipid monolayer (e.g., lecithin, DSPE-PEG2000 and its derivatives), as well as an optimized ratio of aqueous phase to organic phase volume in the synthesis mixture, as exemplified herein. In some embodiments, the lipid monolayer of the hybrid nanoparticle can be formed with about 25% of DSPE-PEG2000 and about 75% of lecithin. In some of these embodiments, the 25% DSPE-PEG2000 include the ligand-conjugated DSPE-PEG2000 and coupling moiety-functionalized DSPE-PEG2000, as well as unmodified DSPE-PEG2000, if any. In various embodiments, the 25% DSPE-PEG2000 can be composed of about 1% to about 15% of B cell binding ligand-conjugated DSPE-PEG2000, about 0.01% to about 2% of maleimide-functionalized DSPE-PEG2000, and about 0% to about 24% of unmodified DSPE-PEG2000. In some embodiments, the 25% DSPE-PEG2000 can be composed of about 10% of B cell binding ligand-conjugated DSPE-PEG2000, about 0.025% of maleimide-functionalized DSPE-PEG2000, and about 15% of unmodified DSPE-PEG2000.

[0060] In addition to the methods and procedures exemplified herein, various methods routinely used by the skilled artisans for preparing biodegradable polymeric nanoparticles and nanoparticles may also be adapted and employed in the present invention. These include, e.g., the methods described in Gagliardi et al., Front. Pharmacol., 12: 601626, 2021; Srivastava et al., ACS Chem. Biol. 16: 1985-93, 2021; Karlsson et al., Annu. Rev. Chem. Biomol. Eng. 9: 105-127, 2018; Maldonado et al., Proc. Natl. Acad. Sci. USA 112: E156-E165, 2015; Kelly et al., Gastroenterol. 161 : 66- 80, 2021; Kishimoto et al., Nat. Nanotechnol. 11 : 890-9, 2016; Guerrero-Cazares et al., ACS Nano 8: 5141-53, 2014; Kumari et al., Colloids Surf., B: Biointerfaces 75: 1-18, 2010; Tenchov et al., ACS Nano 15: 16982-7015, 2021; Guimaraes et al., Int. J. Pharmaceutics 601 : 120571, 2021; Gao et al., J. Mater. Chem. B Mater. Biol. Med. 1(48): 10.1039/C3TB21238F, 2013; Chen et al., Blood 115:4778-86, 2010; and Nanoparticle Technology, vol. 1, 2^nd edition (by Gregory Gregoriadis (CRC Press, Boca Raton, Ann Arbor, London, Tokyo), Chapter 4, pp 67-80, Chapter 10, pp 167-184 and Chapter 17, pp 261-276 (1993)) can be used.

IV. Target antigens for inducing immune tolerance

[0061] The invention provides hybrid nanoparticle compositions and methods for inducing systemic tolerance to specific antigens. Various target antigens may be used in preparing the hybrid nanoparticles for inducing immune tolerance as disclosed herein. In some preferred embodiments, the target antigens are T cell-dependent antigens (e.g., a polypeptide antigen). In some of these embodiments, the employed target antigen is one that is involved in an undesired immune reaction or response. In some embodiments, the employed target antigen is any antigen to which an individual may be at risk of developing an undesired immune reaction or response. In some embodiments, the hybrid nanoparticles of the invention are directed to inducing immune tolerance against autoantigens or self-antigens, alloantigens or allergens.

[0062] In various embodiments, target antigens of different chemical nature can be used in the hybrid nanoparticles of the invention. They include polypeptides or proteins, haptens, carbohydrates, nucleic acids, peptides, polyethylene glycol, lipids (e.g., sterols excluding cholesterol, fatty acids, and phospholipids), polysaccharides, and gangliosides. The various target antigens suitable for practicing the present invention may be isolated from their source using purification techniques known in the art or, more conveniently, may be produced using recombinant methods. For example, the antigens can be obtained through a number of methods known in the art, including isolation and synthesis using chemical and enzymatic methods. In some embodiments, the target antigens can be derived from infectious agents. These antigens may be obtained using methods known in the art, for example, from native viral or bacterial extracts, from cells infected with the infectious agent, from purified polypeptides, from recombinantly produced polypeptides and/or as synthetic peptides.

[0063] Some embodiments of the invention are directed to hybrid nanoparticles intended for inducing immune tolerance to various autoantigens. Autoantigens are known for a number of autoimmune diseases. For example, Grave's disease is characterized by production of autoantibodies to the thyroid-stimulating hormone receptor of the thyroid gland, Hashimoto's thyroiditis by autoantibodies and T cells to thyroid antigens (e.g., thyroid peroxidase), and type I diabetes by T cells and autoantibodies to P cell antigens (e.g., glutamic acid decarboxylase and insulin). Other examples of autoantigens involved in autoimmune diseases include, but are not limited to, cytochrome P450 antigens in Addison's disease, myelin proteins (e.g., myelin basic protein) in MS, uveal antigens in uveitis, gastric parietal cell antigens (e.g., H⁺/ATPase, intrinsic factor) in pernicious anemia, transglutaminase in gluten enteropathy, myocardial cell proteins (e.g., myosin) in myocarditis and rheumatic heart disease, platelet antigens (e.g., GP Hb/IIIa) in idiopathic thrombocytopenic purpura, red blood cell membrane proteins in autoimmune hemolytic anemia, neutrophil membrane proteins in autoimmune neutropenia, basement membrane antigens (e.g., type IV collagen .alpha.3 chain) in Goodpasture's disease, intrahepatic bile duct/mitochondrial antigens (e.g., 2-oxoacid dehydrogenase complexes) for primary biliary cirrhosis, hepatocyte antigens (e.g., cytochrome P450, 206) for autoimmune hepatitis, acetylcholine receptors for myasthenia gravis, and desmogleins for pemphigus and other bullous diseases.

[0064] Some embodiments of the invention are directed to hybrid nanoparticles intended for inducing tolerance against protein antigens that are normally self-antigens, but which certain individuals lack owing to genetic deficiency and to which unwanted immune reactions occur upon replacement therapy. Examples of such antigens include blood coagulation factors VIII and IX in subjects with hemophilia A and B (see, e.g., van Helden et al., Haemophilia. 16:35-43, 2010; and DiMichele, Br J Haematol. 138:305-15, 2007), alpha-L-iduronidase in subjects with Hurler syndrome (see, e.g., Kakavanos et al., FEBS Lett. 580:87-92, 2006), and adenosine deaminase in subjects with adult-type adenosine deaminase deficiency (see, e.g., Bax et al., Eur. J. Haematol. 79:338-48, 2007).

[0065] Some embodiments of the invention are directed to hybrid nanoparticles for inducing immune tolerance to allergens. Any allergen can be employed in the practice of the invention. For example, various allergens from food are suitable for practice of the invention. Example of such allergens include peanut allergen (e.g., Ara h I or Ara h II); walnut allergen (e.g., Jug r I); brazil nut allergen (e.g., albumin); shrimp allergen (e.g., Pen a I); egg allergen (e.g., ovomucoid); milk allergen (e.g., bovine P- lactoglobin); wheat gluten antigen (e.g., gliadin); and fish allergen (e.g., parvalbumins). In some embodiments, the employed allergen is a latex allergen such as Hev b 7 (Sowka et al., Eur. J. Biochem. 255:213-219, 1998). In addition to food allergens, other types of allergens can also be used in the practice of the invention. Examples of such allergens including, but not limited to, ragweed pollen allergen Antigen E (Amb a I) (Rafinar et al., J. Biol. Chem. 266: 1229-1236, 1991), grass allergen Lol p 1 (Tamborini et al., Eur. J. Biochem. 249:886-894, 1997), major dust mite allergens Der pl and Der PII (Chua et al., J. Exp. Med 167: 175-182, 1988; Chua et al., Int. Arch. Allergy Appl. Immunol. 91 : 124-129, 1990), domestic cat allergen Fel d I (Rogers et al., Mol. Immunol. 30:559-568, 1993), white birch pollen Bet vl (Breiteneder et al., EMBO J. 8: 1935-1938, 1989), Japanese cedar allergens Cry j 1 and Cry j 2 (Kingetsu et al., Immunol. 99:625-629, 2000), and protein antigens from other tree pollen (Elsayed et al., Scand. J. Clin. Lab. Invest. Suppl. 204: 17-31, 1991). Also suitable for the invention are protein antigens from grass pollen and known allergens from trees, including allergens from birch, juniper and Japanese cedar. [0066] In some embodiments, the employed target antigen in the hybrid nanoparticles is an alloantigen. Alloantigens are generally cellular antigens that vary in structure among individual members of a single species. Alloantigens from one individual can be recognized as foreign antigens by other members of the same species and are often the basis for graft rejection reactions. Examples of alloantigens include, but are not limited to major histocompatability complex (MHC) class I and class II antigens, minor histocompatability antigens, certain tissue-specific antigens, endothelial glycoproteins such as blood group antigens, and carbohydrate determinants.

V. Inducing immune tolerance with hybrid nanoparticles in therapeutic applications [0067] The invention provides methods and therapeutic uses for suppressing undesired immune responses and/or inducing immune tolerance to a specific antigen (“target antigen”). The hybrid nanoparticles described herein can be used for treating or preventing various diseases or disorders which are associated with or mediated by an undesired immune response or immune activation. In general, the methods are directed to inducing immune tolerance in a human subject by using the hybrid nanoparticles disclosed herein to suppress immune responses to a specific antigen (e.g., a polypeptide antigen) by B cells, T cells and/or other leukocytes (e.g., monocytes and macrophage) in a subject. The antigen can be any antigen against which an immune tolerance is desired. In some embodiments, the specific antigen is a polypeptide, e.g., a T cell-dependent antigen. In some preferred embodiments, the target antigen displayed on the hybrid nanoparticle is an autoantigen or a self-antigen.

[0068] In various embodiments, the hybrid nanoparticles can display an antigen recognized by B cells either in vitro or in vivo. Preferably, the hybrid nanoparticle displaying both the B cell Siglec ligand and the specific antigen is administered to a subject in vivo. By inducing immune tolerance and suppressing undesired immune response, the methods and compositions described herein find uses in the treatment of various diseases and disorders. In any of these applications, the hybrid nanoparticles disclosed herein can be used alone or administered in conjunction with other known drugs in the treatment of a specific disease or condition. The invention further provides for a pharmaceutical combination (e.g., a kit) for carrying out these therapeutic applications. Such pharmaceutical combination can contain a hybrid nanoparticle disclosed herein, in free form or in a composition, an optional co-agent or carrier, as well as instructions for administration of the agents.

[0069] In some preferred embodiments, the therapeutic methods are directed to suppressing immune responses to a specific autoantigen or a self-antigen by B cells, T cells and/or other leukocytes (e.g., monocytes and macrophage) in a subject in need of treatment or prevention of relevant immune disorders. Such disorders include, e.g., autoimmune diseases, allergies, asthma, graft-versus-host reactions or graft rejection reaction. In some of these embodiments, the hybrid nanoparticles of the invention are employed for preventing the development, and treating or ameliorating the symptoms of an autoimmune disease. An representative list of autoimmune diseases and the target autoantigens suitable for these methods are shown in Table 1.

Table 1. Selected human autoimmune diseases caused by known autoantigen(s)

[0070] To practice the therapeutic methods of the invention, hybrid nanoparticles displaying one or more target antigens implicated in immune disorders (especially autoimmune diseases) can be produced as described herein. In various embodiments, the displayed target antigen can be any of the autoantigens or self-antigens described herein or otherwise well known in the art. The pathogenesis of autoimmune diseases is mediated by B and T cells that escape central tolerance and react against self-antigens. As a consequence, the immune system of the affected individual attacks healthy tissues that express the autoantigen and causes chronic inflammation. As a desirable alternative to depletion of all B cells, the lipid-polymer hybrid nanoparticles of the invention can be employed for inducing immune tolerance to specific antigens. They can be used to treat autoimmune diseases involving a plethora of self-antigens and autoantigens. For example, Hashimoto's thyroiditis is caused by antibodies to thyroid peroxidase. In Graves' disease, inflammation is caused by antibodies to the thyroid-stimulating hormone (TSH) receptor, and in some patients with rheumatoid arthritis antibodies that target citrullinated proteins induce inflamed joint tissue. Other well documented autoimmune disorders based on a single antigen include pemphigus, the skin blister disease, with autoantibodies to desmoglein 1 and 3, and thrombotic thrombocytopenic purpura (TTP) with autoantibodies to the protease AdamTS13 leading to a severe deficiency in von Willibrands factor. Hybrid nanoparticles displaying these target antigens can be readily developed and employed to induce immune tolerance and to treat these diseases and other autoimmune disorders.

[0071] The hybrid nanoparticles described herein can be administered alone or as a component of pharmaceutical compositions. Pharmaceutical compositions of the invention comprise an effective amount of the hybrid nanoparticle formulated with at least one pharmaceutically acceptable carrier. Pharmaceutical compositions of the invention can be prepared and administered to a subject by any methods well known in the art of pharmacy. See, e.g., Goodman & Gilman's The Pharmacological Bases of Therapeutics, Hardman et al., eds., McGraw-Hill Professional (10^th ed., 2001); Remington: The Science and Practice of Pharmacy, Gennaro, ed., Lippincott Williams & Wilkins (20^th ed., 2003); and Pharmaceutical Dosage Forms and Drug Delivery Systems, Ansel et al. (eds.), Lippincott Williams & Wilkins (7^th ed., 1999). In addition, the pharmaceutical compositions of the invention may also be formulated to include other medically useful drugs or biological agents.

[0072] In some preferred embodiments, the hybrid nanoparticles are used for in vivo applications. In these applications, the nanoparticle complexes set forth herein can be administered to a subject in need of treatment according to protocols already well established in the art. The hybrid nanoparticle can be administered alone or in combination with a carrier in an appropriate pharmaceutical composition. Typically, a therapeutically effective amount of the hybrid nanoparticle is combined with a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is any carrier known or established in the art. Exemplary pharmaceutically acceptable carriers include sterile pyrogen-free water and sterile pyrogen-free saline solution. Other forms of pharmaceutically acceptable carriers that can be utilized for the present invention include binders, disintegrants, surfactants, absorption accelerators, moisture retention agents, absorbers, lubricants, fillers, extenders, moisture imparting agents, preservatives, stabilizers, emulsifiers, solubilizing agents, salts which control osmotic pressure, diluting agents such as buffers and excipients. These are optionally selected and used depending on the unit dosage of the resulting formulation.

[0073] A therapeutically effective amount of the antigen varies depending upon the disorder that a subject is afflicted with, other known factors of the subject such as age, weight, etc., and thus must be determined empirically in each case. This empirical determination can be made by routine experimentation. Typically, though, the nanoparticle components may be used at a ratio of about 200: 1 w/w, e.g., 100-300: 1 w/w, compared to the antigen delivered. A typical therapeutic dose of the nanoparticle composition is about 5-100 mg per dose, e.g., 10 mg per dose. For any given condition or disease, one can prepare a suitable hybrid nanoparticle which contains an appropriate amount of B cell Siglec targeting ligand and an appropriate antigen in accordance with the present disclosure and knowledge well known in the art , e.g., Springhouse, Physician's Drug Handbook, Lippincott Williams & Wilkins (12^th edition, 2007).

[0074] For in vivo applications, the hybrid nanoparticles can be administered to the patient by any customary administration route, e.g., orally, parenterally or by inhalation. As shown in the Example below, a nanoparticle co-displaying an antigen and a B cell Siglec targeting ligand can be administered to a subject by intravenous injection. In some other embodiments, the nanoparticle complex can be administered to a subject intravascularly. A nanoparticle useful for intravascular administration can be a small unilamellar nanoparticle, or may be a nanoparticle comprising PEG-2000 as exemplified herein. When the composition is parenterally administered, the form of the drug includes injectable agents (liquid agents, suspensions) used for intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection and intraperitoneal injection, liquid agents, suspensions, emulsions and dripping agents.

[0075] In some other embodiments, the hybrid nanoparticle is administered orally to a subject. In these embodiments, a form of the drug includes solid formulations such as tablets, coated tablets, powdered agents, granules, capsules and pills, liquid formulations such as liquid agents (e.g., eye drops, nose drops), suspension, emulsion and syrup, inhales such as aerosol agents, atomizers and nebulizers, and nanoparticle inclusion agents. In still some other embodiments, the nanoparticle composition is administered by inhalation to the respiratory tract of a patient to target the trachea and/or the lung of a subject. In these embodiments, a commercially available nebulizer may be used to deliver a therapeutic dose of the nanoparticle complex in the form of an aerosol.

[0076] The invention also provides kits useful in therapeutic applications of the compositions and methods disclosed herein. Typically, the kits of the invention contain one or more hybrid nanoparticles described herein. The kits can further comprise a suitable set of instructions, generally written instructions, relating to the use of the compounds for inducing immune tolerance to a specific antigen present in the compounds. The hybrid nanoparticle can be present in the kits in any convenient and appropriate packaging. The instructions in the kits generally contain information as to dosage, dosing schedule, and route of administration for the intended method of use. The containers of kits may be unit doses, bulk packages (e.g., multi -dose packages) or sub-unit doses. Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0077] In some embodiments, kits of the invention contain materials for production of a hybrid nanoparticle described herein. The materials include a polymeric compound for self-assembling into a nanoparticle core and lipid components for forming the outer lipid layer, as well as one or more specific antigens (e.g., an autoantigen or self-antigen) and a ligand for an inhibitory B cell Siglec (e.g., a CD22 ligand). Generally, these kits contain separate containers of the structural components, the target antigens and the B cell Siglec ligands from which a hybrid nanoparticle can be made. Additional regents required for generating the hybrid nanoparticle can also be provided in the kits. The B cell Siglec targeting ligands and the antigens are preferably supplied in a form which allows formation of complexes upon mixing of the structural components and other reagents with the supplied B cell Siglec targeting ligand and antigen.

EXAMPLES

[0078] The following examples are offered to illustrate, but not to limit the present invention.

Example 1 Design, assembly and characterization of hybrid nanoparticles [0079] Our hybrid nanoparticle design is based on the nanoprecipitation and selfassembly method reported by the laboratory of Farokhzad²⁴. We formulated rapamycin encapsulating lipid- polymer hybrid nanoparticles decorated with a surface CD22 glycan ligand (CD22L) and a protein antigen (Figure 1, A). We used hydrophobic, ester terminated PLGA to form the polymeric core of the NPs and lecithin and pegylated phospholipids (DSPE-PEG2000, DSPE-PEG2000-CD22L and DSPE-PEG2000- maleimide) to coat the core with a lipid monolayer (Table 1). Most materials used have been well validated in the clinic: PLGA, rapamycin and DSPE-PEG are approved by the Food and Drug Administration (FDA) for medical application²⁵'²⁷. Soy lecithin is also regarded as safe for human use and is often utilized in a variety of foods as an emulsifier and stabilizer²⁸. [0080] Briefly, nanoparticles are formed by adding mixture of PLGA and rapamycin dissolved in organic solvent to the aqueous phase of lecithin and pegylated phospholipids heated to 68°C. The PLGA polymer precipitates to form a nanoparticle hydrophobic core encapsulating rapamycin, NP(R). As the nanoparticles form, lipids self-assemble around the NP(R) core to form a lipid monolayer displaying a PEG shell carrying CD22 ligand, CD22L-NP(R), and a maleimide functional handle for further attachment of protein antigen (Figure 1, A). The mixture is stirred for 2h at room temperature, the organic solvent partially evaporates, and CD22L-NP(R) solidifies. Nanoparticles are then concentrated and purified from the excess rapamycin and residual organic solvent by ultrafiltration using centrifugal filtration devices (molecular- weight cutoff of lOka).

[0081] The maleimide functionalized nanoparticles are now ready for conjugation to protein. For the optimization of nanoparticle formulation and protein attachment we chose ovalbumin (OVA) as a model antigen. As shown on Figure 1C, OVA is first modified with succinimidyl 3-(2-pyridyldithio)propionate (SPDP) heterobifunctional linker and reduced with 1,4-dithiothreitol (25 mM) to afford OVA-SH. The stoichiometry of the linker to protein is kept low to prevent crosslinking of nanoparticles in the next step. Purified protein is directly added to the nanoparticle solution for thio-maleimide coupling. After overnight reaction, nanoparticles are sonicated and purified by ultrafiltration to afford the final CD22L-NP(R)-OVA nanoparticles.

[0082] Analysis of the OVA protein attachment was achieved by agarose gel electrophoresis (Figure ID). CD22L-NP(R)-OVA nanoparticles showed a slightly different mobility towards a positive electrode compared to CD22L-NP(R)-maleimide. The gel image confirmed the global negative charge of nanoparticles at neutral pH and showed successful separation/purification of the nanoparticles from the excess OVA by ultrafiltration, as manifested by the disappearance of the OVA-SPDP band from the gel (Figure ID, line 3 and 4). Overall, this two-step strategy for hybrid nanoparticle synthesis allows for an efficient coupling of intact protein to the surface of PLGA NPs and offers an opportunity for surface functionalization of biologically relevant molecules (e.g., for targeting) and encapsulated hydrophobic drugs in a single nanoparticle. [0083] To characterize the size of the PLGA core of the nanoparticles they were imaged by transmission electron microscopy (TEM) with negative staining by uranyl acetate which does not visualize the lipid monolayer (Figure 2A)²⁴. The images showed spherical particles in the 80-nm size range and a good size distribution (Figure 2B). NPs sizes measured by dynamic light scattering were 90.6 nm (PDI 0.099), 92.0 nm (PDI 0.092), and 93.7 nm (PDI 0.090) for CD22L-NP(R)-OVA, NP(R)-OVA, and NP- OVA, respectively (Figure 2C). We optimized several parameters for the NPs synthesis, such as total lipid to PLGA weight ratio which resulted in generation of reproducible, size-controlled nanoparticles (summarized in Table 2). To determine whether the same drug loading was achieved for CD22L-NP(R)-OVA and NP(R)-OVA, nanoparticles were lyophilized, and RAPA content was measured by RP-HPLC (Figure 2, C-E). The final drug loading based on a 20% RAPA to PLGA ratio was slightly above 100 pg per 1 mg of both nanoparticles, CD22L-NP(R)-OVA (105.3 ± 3.3 pg) and NP(R)-OVA (106.8± 1.4 pg) (Figure 2D-E). That corresponded to an encapsulation yield of -50% for both particle types (53.4% and 52.6%, respectively). RAPA/PLGA input lower than 20% (wt/wt) resulted in a decreased drug loading and was insufficient to encapsulate a therapeutic dose of rapamycin for tolerance induction in mice.¹⁴

Table 2. Optimized formulation used for the synthesis of hybrid NPs.

[0084] The encapsulated RAPA was released at the same rate in vitro as measured for both CD22-NP(R)-OVA and NP(R)-OVA. It took approximately 24h at 37°C to release -50% of drug and another 24h to almost completely empty the core. That suggests that CD22 ligand did not influence the encapsulation yield of RAPA nor the rate in which the drug was released compared to non-targeted NP(R)-OVA. When stored at 4-8° C for 21 days, there was no significant change in the hydrodynamic size of CD22-NP(R)-OVA, NP(R)-OVA, and NP-OVA as demonstrated by DLS. However, after 14 days we noticed that part of RAPA leaked out from the PLGA core. Accordingly, when the nanoparticles were stored at 4° C we used them within a week. Long-term storage could be achieved by lyophilization of nanoparticles with 3% sucrose. Sucrose acts as a cryoprotectant and enables stability of freeze-dried nanoparticles that maintain their size after reconstruction in PBS²⁹.

Example 2 Binding of nanoparticles to CD22 on murine B cells [0085] Formulating the targeted nanoparticles to contain 10 mol% of DSPE- PEG2000-CD22L results in a highly multivalent display of the sialic acid ligands of CD22. Thus, we anticipated that the CD22L-decorated hybrid nanoparticles would increase binding to murine B cells since CD22 is well documented to by highly and selectively expressed on B cells.³⁰ To test this directly we fluorescently labeled the CD22L-NP(R)-OVA and non-targeted NP(R)-OVA nanoparticles using 0.15 mol% of DSPE-PEG2000-AlexaFluor 647, incubated them with murine splenocytes (Figure 3 A), and performed flow cytometric analysis.

[0086] After gating on live, CD19⁺CD22⁺ B cells, we observed that the high affinity CD22 glycan ligands significantly increased binding of CD22L-NP(R)-OVA to B cells compared to non-targeted NPs, confirming that CD22L-DSPE-PEG2000 is accessible and binds to inhibitory co-receptor CD22 on murine B cells (Figure 3B-C). CD22L-NP(R)-OVA containing a lower percentage of CD22L-DSPE-PEG2000 (3 mol%) did not bind well to B cells in vitro, whereas the higher percentage of CD22L- DSPE-PEG2000 (15 mol%) resulted in unstable nanoparticles that precipitate after the final wash and addition of OVA protein. Thus, 10% of CD22L-DSPE-PEG2000 was established as the optimal amount to efficiently deliver CD22L-NP(R)-OVA to B cells and maintain a stable nanoparticle formulation.

Example 3 Tissue distribution of nanoparticles with and without CD22L [0087] It is well documented that the size of spherical nanoparticles (sNPs) strongly influences their organ distribution.³¹ Non-targeted nanoparticles of 80- 200 nm NPs distribute primarily to lung, spleen, and liver.³² However, since CD22L decorated nanoparticles preferentially bind to CD22 on B cells (Figure 3) we wanted to determine the extent to which the CD22L might alter tissue distribution. [0088] To assess biodistribution of CD22L-NP(R)-OVA and non-targeted NP(R)- OVA they were formulated with 1% DSPE-PEG2000-Cy7 and injected i.v. to C57BL/6J WT mice. After 24h-post injection, mice were sacrificed and their major organs were dissected and visualized using an in vivo imaging system, IVIS (Figure 4A). The fluorescence signals were recorded from mouse liver, kidney and spleen, with lower signal in the lungs (Figure 4 A-B). Notably, accumulation of targeted CD22L- NP-OVA was significantly higher than NP-OVA in the spleen 24h-post injection (Figure 4B). The increased distribution of CD22L-NP-OVA in spleen is consistent with targeting to CD22 containing B cells which represent 44-58% of the cellular content of spleen.

[0089] To assess NP clearance from peripheral blood, the fluorescent particles were injected i.v. and mice were bled via the retro-orbital sinus at 1.5, 3, 6 and 24h post-injection. Blood was placed in wells of a black ELISA plate and subjected to fluorescence imaging to quantitate the particles present in the blood as shown in Figure 4, C-D. The exponential decay analysis (Figure 4D) show that the presence of the CD22 ligand has no appreciable impact on clearance of the particles, with CD22-NP-OVA and NP-OVA both exhibiting a half-life of approximately 2 hours 30 minutes, and with both nanoparticles being undetectable at 24h. Thus, while the CD22 ligand does influence accumulation in the spleen, presumably as a result of B cells uptake mediated by CD22 (Figure 4, B), the capacity of this targeted clearance has minimal impact on the overall biodistribution and pharmacokinetics of the particles in tissues and blood respectively.

Example 4 Evaluation of immunogenicity of OVA-NPs in naive mice [0090] Next, we investigated the immunogenicity of OVA containing hybrid nanoparticles in naive animals. C57BL/6J WT mice were administered two doses of hybrid nanoparticles containing 0.05 % mol OVA (day 0 and 14) to deliver 0.5 mg of NPs per intravenous injection (Figure 5). The molar percentage of OVA in the particles was optimized for suppression of antibody production by the CD22L. Four groups of mice were compared. Three groups were treated with hybrid nanoparticles (CD22L- NP(R)-OVA, NP(R)-OVA and NP-OVA) and one group had no treatment (Naive). For nanoparticles with RAPA the dose contained 50 pg per injection. Two weeks after the last treatment (day 28), mice were challenged by intraperitoneal administration of OVA emulsified in alum adjuvant (OVA/ Alum) and sacrificed on day 42. To compare the treatment effects between all four groups, animals were bled on day 27 (before challenge) and day 42 (after the challenge) and anti-OVA IgGl antibody titers were measured by ELISA (Figure 5, A-B).

[0091] As shown in Figure 5, B, two weeks after the last treatment (day 27), groups treated with non-targeted NP(R)-OVA (green) and NP-OVA (pink), had high anti-OVA serum titers. Notably, in this context with the highly multivalent OVA nanoparticles, the presence of RAPA in NP-(R)-OVA had no apparent suppression of anti-OVA IgGl production. In contrast, in the group treated with CD22L-NP(R)-OVA (blue) only a few mice had detectable titers above the naive animals.

[0092] Following challenge with OVA/alum, anti-OVA antibody titers in the NP- OVA and NP-(R)-OVA groups were boosted significantly while titers in the CD22L- NP(R)-OVA group remained low but were detectable over the naive mice. The results demonstrate the importance of the CD22 ligand in suppression of antibody production and induction of tolerance to challenge with antigen.

[0093] By increasing the RAPA dose to 100 pg per injection and three weekly injections of CD22L-NP(R)-OVA stronger suppression of anti-OVA IgGl was seen in most mice, with one outlier (n=5). Accordingly, we chose 100 pg rapamycin as the preferred dose for subsequent studies.

Example 5 CD22L-NP(R)-GPI delays onset of inflammatory arthritis and suppresses GPI antibody production in K/BxN mice

[0094] The K/BxN model of rheumatoid arthritis was chosen to test our hybrid nanoparticle platform in a disease model since both T and B cells are required for disease^{33, 34}. At the age of 4-5 weeks, K/BxN transgenic mice develop spontaneous autoimmune arthritis with joint remodeling, synovitis, leukocyte invasion and profound cartilage and bone erosion^{33, 35, 36}. All these features, as well as other immunological abnormalities, including autoantibody production are common to human RA.

Autoantibodies generated in K/BxN mice are directed to the ubiquitously expressed self-antigen, glucose-6-phosphate isomerase (GPI), a cytosolic glycolytic enzyme that interconverts glucose-6-phosphate and fructose-6-phosphate. The key component of the K/BxN model is the transgenic T cell receptor (KRN) that recognizes a GPI peptide bound on the MHC class II antigen-presenting cells (APCs). Once T cells are activated, they interact with B cells through TCR: A^g7-MHC class II molecules and provide help to anti-GPI B cells, which differentiate into the plasma cells that produce arthritogenic anti-GPI IgG. It is well documented that transfer of anti-GPI to healthy wild type mice induces arthritic disease³⁷. So, both B and T cells play a critical role in disease pathology.

[0095] Based on the known etiology of the glucose-6-phosphate isomerase (GPI) as the autoantigen we produced CD22L-NP(R)-GPI and NP(R)-GPI hybrid nanoparticles to assess the potential to control both T and B cells responses to GPI and delay or prevent disease onset in the K/BxN mice. K/BxN mice (21 days old) were administered four doses of CD22L-NP(R)-GPI or NP(R)-GPI on days 0, 5, 10, and 15 of the study comprising 1 mg of NPs containing 100 pg of RAPA per injection (z.v.). Animals were monitored for disease progression by measuring hind paw thickness three times per week and anti-GPI IgGl titers every week (Figure 6, A). Mice were considered to have arthritis when 1 or more joint(s) measured 4 mm indicative of severe swelling. Diseased animals were kept on pain medication and euthanized at 84 days. [0096] As shown in Figure 6, B, untreated K/BxN mice started to develop arthritis by 30-35 days old, with the average age of arthritis onset being 42-44 days. There was no significant difference between untreated and NP(R)-GPI treated K/BxN mice. In contrast, disease onset in the CD22L-NP(R)-GPI treated group was significantly delayed. None had disease at 44 days old. For those that did get arthritis, the average age of onset was 64 days, 28 days after treatment ended. As described below, some animals in each group were sacrificed at 42 days for comparative analysis of paw inflammation (IVIS), ankle joint histology and splenic plasma cell/regulatory T cell populations. Remarkably, 33% of CD22L-NP(R)-GPI treated mice remained disease- free at the end of the 300-day study, demonstrating profound impact on the progression of disease in this autoimmune arthritis mode.

[0097] This study demonstrated that hybrid nanoparticles with both CD22L and RAPA suppressed disease progression while those bearing RAPA alone had no significant impact. Next, to test if the hybrid nanoparticles with CD22L alone would be protective we conducted a study including an additional group with nanoparticles containing CD22L and GPI but no RAPA (CD22L-NP-GPI). We found that treatment with CD22L-NP-GPI on K/BxN mice had no impact on disease progression, showing no difference in disease onset relative to the untreated or NP(R)-GPI treated mice. Taken together the results show that all components of the CD22L-NP(R)-GPI hybrid particles are required for slowing disease progression in the K/BxN mice.

[0098] Not surprisingly disease onset of the treated mice strongly correlated with the production of GPI antibodies. As shown in Figure 6, C, untreated K/BxN mice (gray) and NP(R)-GPI treated animals (pink) developed high anti-GPI titers whereas mice treated with CD22L-NP(R)-GPI that had delayed onset of disease exhibited significantly lower anti-GPI IgGl antibody levels. Notably, the titers in this group included animals that acquired disease that were kept in the study until day 84. Evaluation of the disease free mice at day 300 revealed a more dramatic difference. As shown in Figure 6, D, the mice treated with CD22L-NP(R)-GPI exhibited levels of anti- GPI antibodies not statistically different from the disease free mice at 21 days and nearly 10,000 fold lower than the untreated mice at 42 days. The results show that CD22L-NP(R)-GPI treatment suppressed production of GPI-specific antibody, which is documented as a major cause of inflammatory arthritis in these mice³⁴.

Example 6 Quantification of inflammation in treated mice

[0099] As a quantitative measure of joint inflammation in hind paws of the mice in vivo we used the ProSense®750 EX fluorescent probe coupled with the in vivo imaging system (IVIS). ProSense® is optically silent in its inactivated state but becomes highly fluorescent when activated by cysteine proteases, cathepsins³⁸. Cathepsins are induced by immune cells in response to pro-inflammatory cytokines and their increased activity in arthritic mice leads to cartilage and bone destruction³⁹. We chose day 42 of the study for this analysis since at this time all the CD22L-NP(R)-GPI treated mice were considered arthritis free while all untreated and NP(R)-GPI treated animals had swollen paws and about 50% had reached the 4 mm threshold set for full arthritis.

[00100] Results from mice 24h post-injection of ProSense®750 EX are shown in Figure 7. Relative to the young disease free mice (21 days) there is strong fluorescence in the joints of the hind paws of untreated or NP(R)-GPI treated mice (Figure 7, A-B) indicative of infiltrating inflammatory cells and cathepsin activity in the joint space. However, in mice treated with CD22L-NP(R)-GPI the fluorescence is comparable to young healthy K/BxN mice. The results are consistent with the tolerogenic nanoparticles inducing a global reduction in joint inflammation. Example 7 Assessment of joint histology in healthy, untreated, and nanoparticle treated mice

[00101] To further characterize disease progression, we assessed joint histology of the untreated and treated mice. Ankle sections were stained with Safranin O/Fast Green/hematoxylin and scored 0-5 for inflammation, cartilage damage, and bone resorption according to the criteria described in Banda et al. ⁴⁰. As shown in representative tissue sections for healthy 21 day and untreated 42 day old mice (Figure 8, A), the ankle joints of 42 day old mice undergo profound loss of Safranin O cartilage staining and heavy infiltration of leukocytes in the inflamed synovial space.

[00102] The histology of mice treated with NP(R)-GPI also showed severe inflammation and damaged joint tissue, but the joint section of the mice treated with CD22L-NP(R)-GPI exhibited healthy cartilage and synovial spaces with little or no leukocyte infiltration. These changes are reflected in the scoring of the sections for all three mice (Figure 8, B). Overall, the histological data supports and complements the in vivo imaging results and demonstrate that treatment of CD22L-NP(R)-GPI delayed arthritis progression in K/BxN mice.

[00103] We also assessed the histology of the joints of three of the long term ‘survivor/cured’ mice that showed no ankle swelling or GPI antibodies at 300 days. As shown in the representative hind joint section of one of these mice (Figure 8, C), there is no evidence of joint inflammation. The total histological score of the cured mice is compared with those of the other four groups in Figure 8, D. This score is the sum of the scores of individual features (inflammation, cartilage damage and bone resorption) with a maximum value of 15. Histological data shows that on average, the ‘cured’ K/BxN mice maintained normal bone matrix, had little to no infiltration of immune cells into the synovium, and little to no loss of cartilage (Figure 8, C). The total histological score given to these cured mice are significantly lower than untreated K/BxN mice (42 days old) or mice treated with NP(R)-GPI (Figure 8, D). Indeed, there is no significant difference between histological scores of cured K/BxN mice compared to young, healthy K/BxN mice (21 days old).

Example 8 Tolerogenic CD22L-NP(R)-GPI hybrid nanoparticles induce Tregs and reduce plasma cells in K/BxN mice [00104] The hybrid nanoparticles were designed to suppress both the B and T cell arms of the immune response to GPI. To gain further insights into the mechanism by which CD22L-NP(R)-GPI delays/prevents the onset of arthritis in K/BxN mice, we sacrificed untreated and nanoparticle treated animals on day 42, to assess T regulatory cells (Tregs) and plasma cells.

[00105] The tolerogenic nanoparticles are formulated with rapamycin (RAPA) since RAPA containing PLGA nanoparticles in combination with soluble antigen have been previously shown to induce T regulatory cells (Tregs)¹². Tregs when activated produce cytokines that suppress T effector cells. They are generated during normal thymocyte differenti tion (natural Tregs) or they are induced from naive Foxp3“CD4⁺ T cells in the periphery (induced Tregs) when exposed to antigen in a tolerogenic environment. Most regulatory T cells are characterized by expression of the X -linked transcription factor forkhead box p3 (Foxp3) that is a critical for their development, function, and homeostasis. Regulatory T cells are fundamental regulators of autoimmunity⁴¹. The key mechanism by which they can limit excessive immune responses is by actively suppressing conventional T cell function and potential autoreactive T cells. In the context of the K/BxN model, mice deficient in Foxp3 regulatory T cells develop more aggressive arthritis with early disease onset.^{42, 43} This is consistent with the view that Tregs dampen CD4+ T cell help for B lymphocytes to lower the levels of anti-GPI autoantibodies.⁴⁴

[00106] As shown in Figure 9, A-B, the level of Tregs was assessed in splenocytes from untreated and hybrid nanoparticles treated 42 day old mice. The CD22L-NP(R)- GPI treated K/BxN mice group exhibited a nearly 20% increase in the percentage of Foxp3⁺ Tregs (51.2%) among CD4⁺ T cells compared to the untreated K/BxN (27.6%) or NP(R)-GPI (34.5%) treated mice. The higher level of Tregs in mice treated with CD22L-NP(R)-GPI is consistent with a role for Tregs suppressing arthritis in these animals. We further looked at the increase of Tregs over time in untreated and CD22L- NP(R)-GPI treated K/BxN mice. The percentage of Foxp3“CD4⁺ cells from untreated 21 day old K/BxN mice (9.4 ± 2.5%) is similar to what is observed in wild type C57BL/6J mice. As these animals age, the population of Foxp3‘^tCD4 cells increases until day 28 when it plateaus (29.5 ± 4.1%) and remains unchanged for the course of the study. In contrast CD22L-NP(R)-GPI treated mice exhibit consistently higher Tregs relative to untreated mice rising to 46.0 % at day 35 and peaking at 53% on day 42. Since K/BxN transgenic mice develop spontaneous autoimmune arthritis between 28- 35 days of age, we suggest that the enriched Foxp3 ’ CD4⁺ population in mice treated with CD22L-NP(R)-GPI contributes to delyaing disease onset by reducing CD4⁺ T cell help promoting activated B cells to become plasma cells that produce GPI-specific autoantibodies.

[00107] Since we assumed that rapamycin would be the major driver of expanded Tregs, it is somewhat surprising that there was no difference in Tregs in untreated mice and mice treated with NP(R)-GPI. There are several reasons why the addition of CD22L to the hybrid particles enhances Treg production. Since the CD22L-NP(R)-GPI nanoparticles were more strongly targeted to B cells in spleen (Figure 4, A-B) antigen presenting cells in spleen (or lymph nodes) may be exposed to higher local RAPA concentrations.

[00108] We also looked at the impact of treatments on antibody secreting plasma cells (PCs) that represent the terminal differentiation step of mature B lymphocytes (Figure 9, C-D). In K/BxN mice, PCs are well known to secrete large amounts of autoantibodies directed against GPI and contribute to disease manifestation^{33, 34}. High expression of CD138 and loss of CD 19 is a hallmark of murine plasma cells identification that we used as a gating strategy^{45, 46}. As shown in Figure 9, B, there was significant reduction in PCs in mice treated with CD22L-NP(R)-GPI (0.46%) as compared to untreated (1.48%) and NP(R)-GPI (1.61%) treated mice. This is consistent with the anti-GPI IgGl titers measured earlier (Figure 6, C-D) and the expected CD22 mediated suppression and activation of B cells that recognize GPI³⁰.

[00109] In summary we provide evidence that our tolerogenic hybrid nanoparticles delay and prevent development of arthritis in K/BxN mice by mechanisms involving B and T cell mediated immune tolerance. Animals treated with CD22L-NP(R)-GPI had increased populations of regulatory T cells and decreased levels of plasma cells and anti-GPI antibodies. The histological sections of K/BxN joints and IVIS imaging of their ankles clearly visualized therapeutic effect of CD22L-NP(R)-GPI. Animals treated with CD22L-NP-GPI lacking rapamycin or NP(R)-GPI lacking CD22L, developed arthritis at the same time as untreated mice. That confirmed that all three components of hybrid nanoparticles, including CD22L, RAPA and GPI are required for induction of T and B cells tolerance, slowing disease progression in the K/BxN mice. Example 9 Exemplified methods and materials

[00110] Materials. A poly(D,L-lactide-co-glycolide) PLGA polymer (ester terminated Resomer® RG 505), comprising a 50:50 mix of lactide to glycolide copolymers in molecular weight range of 54,000-69,000 was purchased from Sigma Aldrich (St. Louis, MO). N-(Methylpolyoxyethylene oxycarbonyl)-l,2-distearoyl-sn- glycero-3 -phosphoethanolamine, sodium salt (DSPE-PEG2000), N-[(3-Maleimide-l- oxopropyl)aminopropyl polyethyleneglycol -carbamyl] distearoylphosphatidylethanolamine (DSPE-PEG2000-maleimide) and ( 3-(N- succinimidyloxyglutaryl)aminopropyl, polyethyleneglycol-carbamyl distearoylphosphatidyl-ethanolamine (DSPE-PEG2000-NHS) were purchased from NOF America Corporation. Refined lecithin and rapamycin (RAPA) were purchased from Alfa Aesar™ and Biotang Incorporation (Boston, MA) respectively.

Heterobifunctional crosslinker succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and Alum was purchased from Thermo Scientific Pierce. The model antigen, ovalbumin (OVA) was bought from Worthington Biochemical. Recombinant mouse glucose-6- phosphate isomerase (GPI) was expressed in house using an E. coli (pGEX-4T-3-gpi) expression vector kindly gifted by Diane Mathis and Christophe Benoist (Harvard Medical School). Cyanine7-NHS ester was purchased from Lumiprobe Corp and Alexa Fluor 647 NHS ester was purchased from Invitrogen. Anti-IgGl mouse-horseradish peroxidase (HRP) conjugate was purchased from Santa Cruz Biotechnology Inc. The 3, 3', 5, 5'- tetramethylbenzidine (TMB) substrate kit was purchased from BD Biosciences (San Jose, CA). ProSense 750 EX imaging probe was purchased from PerkinElmer. CD4 (BV421), CD90.2 (FITC), CD3 (PE), B220 (APC_ eFluor® 780), CD11c (APC_ eFluor® 780), CD1 lb (APC_ eFluor® 780), B220 (BV421), B220 (BV605), CD19 (BV605), CD19 (BV421), CD138 (PE), streptavidin (PE_Cy7) were purchased from BioLegend (San Diego, CA). Foxp3/Transcription Factor Staining Buffer Set and Foxp3 (APC) were purchased from eBioscienceTM.

[00111] Instruments. High performance liquid chromatography (HPLC): Agilent Technologies 1260 Infinity HPLC equipped with a multiple wavelength detector (UV- vis). Liquid chromatography mass spectrometer (LC/MS): Agilent Technologies 6125 Single Quadrupole mass spectrometer coupled to an Agilent 1260 LC stack, complete with DAD. Capable of scanning from m/z=10-2000. Transmission Electron Microscopy (TEM): samples were analyzed at 80kV with a ThermoFisher Talos L120C transmission electron microscope and images were acquired with a CETA 16M CMOS camera. Dynamic light scattering (DLS): DynaPro® NanoStar® from Wyatt Technology (Santa Barbara, CA) Gel imaging system: BioRad ChemiDoc Imaging system. Flow cytometry data was collected using the following instruments: 5 laser (355, 405, 488, 561, and 640 nm) Bio-Rad ZE5 cell analyzer (Yeti A), 4 laser (405, 488, 561, and 640 nm) Bio-Rad ZE5 cell analyzer (Yeti B), and 5 laser (355, 405, 488, 561, and 640 nm) BD LSR. II. In vivo imaging system (IVIS): Perkin Elmer Lumina S5. [00112] Animals. Wild-type C57BL/6J mice were obtained from the rodent breeding colony of The Scripps Research Institute (TSRI). K/BxN mice were produced through the breeding of KRN and Ag7 mice provided by the late Dr. Kerri Mowen (The Scripps Research Institute) with permission from Dr. Diane Mathis and Dr. Christophe Benoist (Harvard Medical School). All experimental procedures involving mice in this work were approved by the Institutional Animal Care and Use Committee of The Scripps Research Institute (La Jolla, CA).

[00113] NPs synthesis: Hybrid NPs were synthesized from PLGA polymer, soybean lecithin and DSPE-PEG2000 lipids (DSPE-PEGm, DSPE-PEG-CD22L, DSPE-PEG- maleimide) using a modified nanoprecipitation method combined with self-assembly of nanoparticles^{24, 47}. Briefly, PLGA (2 mg mL'¹) and RAPA (10 mg mL'¹ ) were dissolved in acetonitrile and combined at 20% of RAPA to polymer ratio (wt/wt). Lecithin and DSPE-PEG lipids were dissolved in 4% ethanol and heated to 68°C for 3 minutes constantly stirring. PLGA/RAPA mixture was added drop wise into aqueous phase containing lipids and the resulting solution was vortexed for another 3 minutes at RT. The nanoparticles were allowed to self-assemble for 2h at RT with continuous stirring and then were passed through a sterile 0.45-mm syringe filter. The NPs were centrifuged at 3000g using Amicon Ultra- 15 centrifugal filter units (molecular weight cutoff, 10 kD; Sigma-Aldrich) and washed twice with ice-cold water. NPs were resuspended in water (5 mg mL'¹) followed by sterile PBS (lOx) directly before protein conjugation.

[00114] Conjugation of protein to NPs (OVA or GPI): NPs in PBS (5 mg mL'¹) were mixed with 1.8% (wt/wt) of reduced protein-SPDP conjugate (1 mg mL'¹) with gentle shaking overnight at 4°C. Resulting protein-NPs mixture was purified from the excess protein using Amicon Ultra-4 centrifugal filters (Millipore, Billerica, MA) with a 100 kDa molecular cut-off. After 4 washings with ice-cold water at 4°C and 2600g, NPs were collected and kept in water till further use at 4°C.

[00115] Agarose gel electrophoresis of NPs (before and after OVA addition) was performed using 0.3% agarose gel at 120 V for 45 min. Gel was stained with SimplyBlue.

[00116] Negative stain transmission electron microscopy (TEM) Carbon-coated copper grids (400 mesh) were glow-discharged and 10 pL of each sample was adsorbed for 2 minutes. Excess sample was wicked away and grids were negatively stained with 2% uranyl acetate for 2 minutes. Excess stain was wicked away and the grids were allowed to dry. Samples were analyzed at 80kV with a ThermoFisher Talos L120C transmission electron microscope and images were acquired with a CETA 16M CMOS camera.

[00117] Drug loading and encapsulation yield: drug loading (weight of encapsulated RAPA in total weight of NPs) and encapsulation yield (weight of encapsulated RAPA to weight of initial RAPA input) was determined using HPLC. Briefly, 0.25 mL of NPs solution at a concentration of 5 mg/mL in water was lyophilized and dissolved in acetonitrile to release the rapamycin from their PLGA core. The resulting free RAPA content was assayed using Agilent Technologies 1260 Infinity HPLC equipped with C18 column (150x 4.6 mm, 5p; ProSphere™ 100; Alltech). Absorbance was measured by a UV-vis detector at 278 nm and RAPA retention time of 14.9 min was recorded. Mobile phase: water (+0.05% TFA; A)/ acetonitrile (+0.05% TFA; B). Method (0 min: 100% A; 2 min: 95% A; 12 min: 0% A; 18 min: 0% A; Iml/min).

[00118] Dynamic light scattering (DLS) was performed at a fixed scattering angle of 90° on a DynaPro® NanoStar® from Wyatt Technology (Santa Barbara, CA). Before the measurements NPs were suspended at 0.5 mg/mL in water and sonicated. For size comparison of CD22L-NP(R)-OVA, NP(R)-OVA and NP-OVA, three independent samples were measured and average particle sizes were obtained.

[00119] In vitro binding assay: Six- to eight-week-old C57BL/6J mice were sacrificed and their spleens harvested. Spleens were mechanically digested into a single cell suspension, filtered through a 40 pm cell strainer to remove cell aggregates and centrifuged (350 g, 5 min). Then erythrocytes were lysed via exposure to a solution of 150 mM NH₄C1, 1 mM NaHCCh, and 1 ,27mM EDTA for 3-5 min at RT. After quenching with complete glutamine-supplemented RPMI-1640 containing 10% heat inactivated FBS, 100 U/mL penicillin and 100-pg/mL streptomycin, splenocytes were filtered again and centrifuged (350 g, 5 min.). Cells were resuspended in a complete RPMI medium to a final concentration of 4*10⁶ cells/well (80 pL/well) and were pulsed with fluorescently labeled CD22L-NP-OVA or NP-OVA containing 0.15% of DSPE- PEG2000-AF647 in PBS (2 mg/mL). After Ih of incubation (37°C, 5% CO2), cells were carefully washed with PBS and resuspended in FACS buffer (2% FBS, 2.5 mM EDTA, 25mM HEPES pH=7 in HBSS) containing CD16/32 (Fc-Block, dilution 1 : 100) at 4°C for 10 min. Subsequently, samples were incubated with CD19 (FITC; 1 :200 dilution) and CD22 (PE; 1 :200 dilution) monoclonal antibodies (for 30 min at 4°C in the dark. As a control, an unstained sample was also prepared. Cells were washed twice with FACS buffer, resuspended in FACS buffer containing PI, and vortexed before each measurement. Data were analyzed using FlowJo analysis software. For flow cytometry, the same gating was applied for all experimental conditions within one experiment.

[00120] Biodistribution of hybrid nanoparticles and their clearance from the blood circulation Six- to eight-week-old C57BL/6J mice were administrated intravenously with 200 pL of DSPE-PEG2000-Cy7 (1%) labeled CD22L-NP-OVA or NP-OVA in PBS (5 mg/mL). PBS-injected mice were used as fluorescence negative controls. For the assessment of NP-clearance from the blood circulation, mice were bled via the retro-orbital sinus at 5 various time points: 1.5, 3, 6 and 24h post-injection. To study the biodistribution of NPs, mice were humanely euthanized using carbon dioxide overdose and cervical dislocation after 3, 24 and 48h post-injection. Major organs, including lymph nodes, liver, spleen, kidney, heart, and lungs were harvested from the experimental and control mice. The organs and blood were subjected to fluorescence imaging using a IVIS Lumia S5 imaging system (Perkin Elmer) with excitation at 756 nm and emission at 779 nm. Fluorescence imaging data of total radiant efficiency in the region of interest (ROI) was quantify by the Living Image® software (Perkin Elmer). [00121] Use of CD22L-NP(R)-OVA in a naive system (C57BL/6J): On day 0 and 14, three groups of six- to eight-week-old C57BL/6J mice received lateral vein injections of CD22L-NP(R)-OVA, NP(R)-OVA, and NP-OVA to deliver 50 pg of RAPA and 0.5 mg of NPs. Untreated animals were included as controls. Two weeks later, animals were challenged with OVA (100 pg) and Alum (100 pL) via i.p. injection and sacrificed on day 42. To follow the anti-OVA IgGl responses, animals were bled via retro-orbital sinus while under anesthesia before the challenge (day 27) and two weeks after the challenge (day 42).

[00122] Use of CD22L-NP(R)-GPI for a delay and treatment of a murine arthritis model (K/BxN): K/BxN mice, prior to onset of arthritis, at 21 days of age, were treated by i.v. injections with CD22L-NP(R)-GPI and NP(R)-GPI to deliver 100 pg of RAPA and 1 mg of NPs per injection. Treatments were given on days 0, 5, 10 and 15. Untreated K/BxN mice were also included as controls. Animals were bled via retro- orbital sinus after isoflurane inhalation anesthesia every 7 days to control anti-GPI IgG titers. 42-days old K/BxN mice were split into 3 groups. First group was injected with ProSense750EX for in vivo imaging, the second group was sacrificed for tissue harvesting (spleens for the mechanistic studies and the hind paws for the histology) and the remaining third group was kept in the study to follow the swelling data and the survival curve. The endpoint for arthritic mice was assigned to 84 days whereas the survivors were kept till day 300.

[00123] IgGl antibody titers: The levels of anti-OVA or anti-GPI IgGl titers were measured by ELISA. Assay microplates were coated with protein (10 pg/ml in PBS) and incubated overnight at 4 °C. Plates were washed with tris-buffered saline containing 0.1% Tween® 20 detergent (TBS-T) and blocked for an hour with 1% BSA in PBS (blocking buffer). Serial dilutions of serum sample in blocking buffer were placed onto the plate and incubated at RT for 1 h. After washing with TBS-T, plates were incubated with anti -IgGl mouse-horseradish peroxidase (HRP) conjugate (1 :2000 dilution) at RT for 1 h, washed and developed with TMB substrate (75 pL/well). After 15 min plates were quenched with 2M H₂SO4 (75 pL/well) and the absorbance at 450 nm was measured using a Synergy Hl microplate reader (BioTek). Anti-IgGl titers were calculated with Prism (GraphPad Software) by applying a standard four-parameter IC50 function.

[00124] Swelling data and the survival curve: Ankle thickness of the hind paws of K/BxN mice was measured three times per week (starting when mice were 21 days old) in millimeters (mm) at die widest point with digital calipers (Coie-Pamier, 1433 light weight carbon fiber). Mice were considered sick when at least one ankle was above 4 mm and marked on the survival curve.

[00125] Cathepsin activity assessed by in vivo imaging system using ProSense 750EX: On day 21 of the study, CD22L-NP(R)-GPI and NP(R)-GPI treated K/BxN mice were injected intravenously with 100 pL of commercially available ProSense 750 EX imaging probe (PerkinElmer) prepared according to the manufacturer's instructions. As controls, untreated K/BxN and young, healthy K/BxN mice were also included.

Animals were imaged 24 hours post-injection using the IVIS S5 (PerkinElmer) imaging system with excitation at 750 nm and emission at 770 nm. Mice were held under isoflurane inhalation anesthesia for the duration of the scan, and the imaging chamber was heated to 37°C. Fluorescence imaging data of total radiant efficiency in the region of interest (ROI) was quantify by the Living Image® software (Perkin Elmer).

[00126] Histology: Mouse ankles were fixed for 5 days in zinc buffered formalin (Anatech Ltd), decalcified for 9 days in Shandon TBD-2, dehydrated by passage through increasing concentrations of EtOH, transferred to xylene, and paraffin- embedded. Tissue sections (4 pm) were mounted on glass slides, deparaffinized using Pro-Par Clearant (Anatech Ltd), and rehydrated in successive baths of absolute EtOH, decreasing concentrations of EtOH, and finally distilled water. Slides were stained in filtered hematoxylin (Ricca Chemical Company 353532) for 4 min and then washed in deionized water, acid alcohol (0.5% HC1 in 70% EtOH), deionized water, blued in Scott’s Water (0.2% w/v sodium bicarbonate, 2% w/v magnesium sulfate), and finally deionized water. Slides were then immersed in 0.2% Fast Green FCF (MP Biomedicals 211922) for 6 min, washed in 1% glacial acetic acid, and stained with 0.003% Safranin O (Acros Organics 146640250) for 4 min. After staining, tissues were subsequently dehydrated as described above and cover slipped using Refrax mounting medium (Anatech Ltd). Slides were scanned with a Leica SCN400 slide scanner, and images were captured using QuPath (v 0.2.3). Histological scores were given using scoring criteria described in supporting information.

[00127] Intracellular staining of Foxp3+ regulatory T cells: K/BxN mice, 42 days old, treated four times with either CD22L-NP(R)-GPI or NP(R)-GPI (day 0, 5, 10, 15) were sacrificed, and their spleens were harvested. Untreated K/BxN were also included as controls. Single cell suspensions of murine splenocytes were obtained via mechanical digestion of spleens followed by erythrocyte lysis (see in vitro binding assay for details). After erythrocytes lysis, cell suspension was filtered, centrifuged (350 g, 5 min) and washed twice with PBS. Staining of cells was carried out in 96-well plates on ice in a final volume of 100 pl. First, cells were stained with a solution of

FVD eFluor® 780 (1 : 1000) for 30 min at 4°C in the dark. Splenocytes were centrifuged (350 g, 5 min) and resuspended in FACS buffer containing CD16/32 (Fc-Block, dilution 1 : 100) blocking antibody at 4°C for 10 min. Subsequently, samples were stained with an antibody mixture: CD4 (BV421), CD90.2 (FITC), CD3 (PE), B220/CD1 Ic/CDl lb (APC_ eFluor® 780) for 30 min at 4°C in the dark. After the final wash of cells, the supernatant was discarded, and pellet was pulse vortex until complete dissociation. Cells were resuspended in 200 pL of Foxp3 Fixation/Permeabilization working solution prepared according to the manufacturer’s instructions (eBioscience™ Foxp3 /Transcription Factor Staining Buffer Set) and incubated for 16h at 2-8°C in the dark. Samples were centrifuged (600g, 5 min), washed and resuspended in 100 pL of Permeabilization buffer. After blocking with 2% mouse serum for 15 min at room temperature, Foxp3 (APC; 1/25) antibody was added and incubated for an hour at room temperature in the dark. Cells were washed with Permeabilization buffer, centrifuged (600g, 5min) and resuspended in FACS buffer. Before the flow cytometry analysis, each sample was filtered through a cell-strainer cap placed on the round-bottom flow tube (Falcon, Corning Science; Mexico).

[00128] Plasma cell staining and analysis by flow cytometry: Single cell suspensions of murine splenocytes from 42 day old K/BxN mice treated with either CD22L-NP(R)-GPI or NP(R)-GPI were obtained via mechanical digestion of spleens followed by erythrocyte lysis (see in vitro binding assay for details). After erythrocytes lysis, cell suspension was filtered, centrifuged (350 g, 5 min) and resuspended in FACS buffer containing CD16/32 (Fc-Block, dilution 1 : 100) at 4°C for 10 min. Cells were washed, centrifuged and stained with B220 (BV421; 1/200), CD138 (PE; 1/100), CD19 (BV605; 1/200) for 30 min at 4°C in the dark. After the incubation cells were centrifuged (350 g, 5 min), washed twice with PBS and stained with FVD eFluor® 780 (1 :1000) for 30 min at 4°C in the dark. Splenocytes were centrifuged (350 g, 5 min), washed FACS buffer and fixed using Fixation/Permeabilization working solution 20 min at room temperature in the dark. Before analysis, cells were centrifuged (600 g, 5 min) and resuspended in FACS buffer.

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***

[00129] The invention thus has been disclosed broadly and illustrated in reference to representative embodiments described above. It is understood that various modifications can be made to the present invention without departing from the spirit and scope thereof. It is further noted that all publications, patents and patent applications cited herein are hereby expressly incorporated by reference in their entirety and for all purposes as if each is individually so denoted. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.

Claims

WHAT IS CLAIMED IS:

1. A lipid-polymer hybrid nanoparticle, comprising (1) a polymeric nanoparticle core that encapsulates an immunomodulatory agent, and (2) a lipid monolayer coating the nanoparticle core and displaying on its surface both (i) a ligand for a B cell Siglec receptor conjugated to a lipid and (ii) a target antigen conjugated to a lipid.

2. The hybrid nanoparticle of claim 1, wherein the target antigen is a polypeptide.

3. The hybrid nanoparticle of claim 1, wherein the target antigen is conjugated to the lipid monolayer after formation of the lipid monolayer.

4. The hybrid nanoparticle of claim 1, wherein the target antigen is associated with or implicated in an autoimmune disease.

5. The hybrid nanoparticle of claim 4, wherein the target antigen is a self-antigen or an autoantigen.

6. The hybrid nanoparticle of claim 1, wherein the lipid for conjugating to the target antigen is functionalized with a coupling moiety.

7. The hybrid nanoparticle of claim 6, wherein the coupling moiety is maleimide.

8. The hybrid nanoparticle of claim 6, wherein the target antigen is modified to contain a functional group that is reactive with the coupling moiety prior to being conjugated to the lipid.

9. The hybrid nanoparticle of claim 8, wherein the functional group on the modified target antigen is a thiol group, and wherein the target antigen is conjugated to the lipid via thiol-maleimide chemistry.

10. The hybrid nanoparticle of claim 1, wherein the nanoparticle core is formed of poly(lactic-co-glycolic acid) (PLGA).

11. The hybrid nanoparticle of claim 10, where the immunomodulatory agent is rapamycin (RAPA), a rapamycin derivative, a mTOR inhibitor, a corticosteroid, an antimetabolite agent, a calcineurin inhibitor, a steroid, Azathioprine, Methotrexate, Mycophenolate mofetil, Mycophenolic acid, Sirolimus, Fingolimod (FTY720), or Manitimus.

12. The hybrid nanoparticle of claim 11, wherein the amount of RAPA loaded in the core is at least about 2.5%, 5%, 7.5% or 10% (wt/wt) of the hybrid nanoparticle.

13. The hybrid nanoparticle of claim 11, wherein the ratio of RAPA and PLGA for forming the nanoparticle core is about at least 20% (wt/wt).

14. The hybrid nanoparticle of claim 1, wherein the ligand for a B cell Siglec receptor is coupled to the lipid prior to formation of the lipid monolayer.

15. The hybrid nanoparticle of claim 1, wherein the B cell Siglec receptor is CD22 or Siglec-G/10.

16. The hybrid nanoparticle of claim 15, wherein the ligand for CD22 is a monosaccharide sialic acid derivative.

17. The hybrid nanoparticle of claim 15, wherein the ligand for CD22 is a disaccharide glycan.

18. The hybrid nanoparticle of claim 15, wherein the ligand for CD22 is a trisaccharide glycan.

19. The hybrid nanoparticle of claim 1, wherein the ligand and the target antigen are each conjugated to a phospholipid or derivative thereof.

20. The hybrid nanoparticle of claim 19, wherein the phospholipid is DSPE-PEG2000.

21. The hybrid nanoparticle of claim 19, wherein the lipid monolayer is formed with the phospholipid and a mixture of unmodified lipids.

22. The hybrid nanoparticle of claim 21, wherein the mixture of unmodified lipids comprises lecithin.

23. The hybrid nanoparticle of claim 21, wherein the lipid monolayer is formed with about 25% of DSPE-PEG2000 and about 75% of lecithin.

24. The hybrid nanoparticle of claim 23, wherein the 25% of DSPE- PEG2000 comprises about 1% to about 15% of B cell binding ligand-conjugated DSPE-PEG2000, about 0.01% to about 2% of maleimide-functionalized DSPE- PEG2000, and about 0% to about 24% of unmodified DSPE-PEG2000.

25. The hybrid nanoparticle of claim 23, wherein the 25% of DSPE- PEG2000 comprises about 10% of B cell binding ligand-conjugated DSPE-PEG2000, about 0.025% of maleimide-functionalized DSPE-PEG2000, and about 15% of unmodified DSPE-PEG2000.

26. A method of producing a lipid-polymer hybrid nanoparticle displaying a target antigen, comprising (1) providing a drug mixture containing an immunomodulatory agent and a nanoparticle polymer, wherein the nanoparticle polymer is capable of self-assembling to form a nanoparticle core that encapsulates the immunomodulatory agent, (2) providing a lipid composition containing a mixture of lipids for forming a lipid monolayer that coats the nanoparticle core, wherein the mixture of lipids comprises a phospholipid that is conjugated to a ligand of CD22, a phospholipid that is functionalized with a coupling moiety for conjugating to the target antigen, and one or more unmodified lipid molecules, (3) adding the drug mixture in organic solvent to the aqueous phase of the lipid composition to form a lipid coated nanoparticle core that displays the CD22 ligand and the coupling moiety, and (4) conjugating the target antigen to the a lipid coated nanoparticle core, wherein the target antigen is modified to contain a functional group that reacts with the coupling moiety.

27. The method of claim 26, wherein the lipid coated nanoparticle core is formed via nanoprecipitation and nanoparticle self-assembly.

28. The method of claim 26, wherein the immunomodulatory agent is rapamycin (RAPA).

29. The method of claim 26, wherein the nanoparticle polymer is poly(lactic-co-glycolic acid) (PLGA), and the phospholipid is DSPE.

30. The method of claim 26, wherein the phospholipid for conjugating to CD22 ligand and the target antigen is DSPE-PEG2000.

31. The method of claim 26, wherein the one or more unmodified lipid molecules comprise lecithin.

32. The method of claim 26, wherein the CD22 ligand is a monosaccharide sialic acid derivative or a trisaccharide glycan.

33. The method of claim 26, wherein the coupling moiety is maleimide, and the functional group on the target antigen is thiol.

34. The method of claim 31, wherein the immunomodulatory agent/nanoparticle polymer ratio and/or the lecithin/phospholipids ratio is optimized.

35. A method for inducing immune tolerance to a target antigen in a subject, comprising administering to the subject a pharmaceutical composition comprising a therapeutic effective amount of the hybrid nanoparticle of claim 1; thereby inducing immune tolerance against the target antigen in the subject.

36. The method of claim 35, wherein the target antigen is an autoantigen or a self-antigen.

37. The method of claim 35, wherein the subject is a human.

38. A method for preventing, treating or ameliorating symptoms of an autoimmune disease in a subject, comprising administering to the subject a pharmaceutical composition comprising a therapeutic effective amount of the hybrid nanoparticle of claim 1, wherein the autoimmune disease is mediated by or associated with the target antigen; thereby preventing, treating or ameliorating symptoms of the autoimmune disease in the subject.

39. The method of claim 38, wherein the target antigen is an autoantigen or a self-antigen.