The immune system protects animals from injury by unwanted foreign organisms, such as viruses, bacteria and parasites. The immune system functions in two basic ways. Innate immunity is a basic protection found even in a very simple animal that simply attacks and kills general types of foreign organisms or foreign materials. Acquired immunity is a much more complicated form of protection in which the body responds to foreign organisms that it has encountered and defended against before.
Acquired immunity forms the basis for many modern medical treatments, particularly acquired treatments like vaccines. If the immune system is first taught to respond to a particular foreign organism or even an aberrant part of the body itself, such as cancer cells, acquired immunity allows the body to attack those unwanted organisms or cells very efficiently. However, in order to prevent the body from learning to attack many harmless things in the environment, acquired immunity requires very particular circumstances before an organism or cell is recognized as dangerous and specifically targeted for destruction.
In one type of acquired immunity, special immune system cells called antigen presenting cells (APCs) have to engulf the unwanted organism or cells, process it into components called antigens, then display those antigens on their surface in special antigen present proteins. Only then can the immune system's attack cells learn to recognize the antigens and attack the unwanted organisms or cells that contain those antigens. This process is further regulated by the need for certain chemicals, often called cytokines (which include chemoattractants and chemokines) to be present for various events to take place. For example, APCs are often found throughout the body and are only recruited specifically to the area where antigens are present by certain chemoattractant chemokines.
Immune-modulating agents, such as vaccines, often fail to cause an acquired immune response, or cause only a weak response, because they do not trigger enough elements of the complicated system used to acquire immunity. For example, it is often useful to use viral DNA as an antigen or to otherwise use DNA to cause the production of antigens in an area where it is injected. Naked DNA vaccines and DNA antigen-loaded microparticles, however, often fail to induce a significant immune response when administered intramuscularly. This is largely due to the fact that significant numbers of APCs are not recruited to the injection site.
One approach to increasing vaccine effectiveness is to co-administer another composition called an adjuvant. The adjuvant is usually something recognized by most immune systems as an unwanted invader. The body therefore begins to fight the adjuvant and in the process looks for new antigens in the area. However, the effectiveness of adjuvants is limited by the fact that the immune system is somewhat engaged in fighting the adjuvant and is not solely focused on the vaccine antigens. Further, many adjuvants trigger such a strong response they cause a great deal of swelling and pain near the injection site and can actually be dangerous to individuals who have a strong immune response to the adjuvant.
Chemokines have also been previously injected with antigens to try to improve vaccination. However, chemokines rapidly leave the administration site and are substantially gone within 24 hours of injection. Although this problem initially seems remediable by repeated injection of chemokines, such daily injections have also proved unsuccessful in at least some studies.
Currently, microparticles have been used to induce an immune response in animals, but without significant success. In particular, microparticles that have been surface functionalized to facilitate uptake by APCs and release from phagosomes in the APCs after uptake have been produced. These microparticles have contained both antigen and chemokines These microparticles have suffered from loss of proteins during formation, inactivation of the proteins after the microparticle is formed, and poor burst release of the proteins. Further, chemokine proteins such as MIP-3 and MCP-1 that need to act on the surface of APCs are useless after the microparticle containing them has been taken up by an APC and cannot help recruit more APCs to the administration site.
The present disclosure generally relates to an immune modulating composition and associated methods. More particularly, the present disclosure relates to an immune modulating composition comprising a hydrogel containing at least two different biomolecules and associated methods.
In one embodiment, the present disclosure provides an immune-modulating composition comprising a hydrogel-forming polymer, an immune-modulating biomolecule operable to recruit or retain an immune cell, and an antigen-related biomolecule.
In another embodiment, the present disclosure provides a method of providing an antigen to an antigen presenting cell in an animal by administering to the animal at an administration site an immune-modulating composition as described above. Next, one forms a hydrogel in situ from the hydrogel-forming polymer, then recruits at least one antigen presenting cell to the administration site using the immune-modulating biomolecule, and finally inducing phagocytosis of the at least one antigen-related biomolecule by the antigen presenting cell.
Some embodiments of the disclosure may achieve one or more of the following advantages:
One of ordinary skill in the art will recognize that not all embodiments may achieve all advantages and some embodiments may achieve different advantages.
A more complete understanding of embodiments of the disclosure will be apparent from the detailed description taken in conjunction with the accompanying drawings which describe various embodiments of the disclosure.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
While the present disclosure is susceptible to various modifications and alternative forms, specific example embodiments have been shown in the figures and are described in more detail below. It should be understood, however, that the description of specific example embodiments is not intended to limit the invention to the particular forms disclosed, but on the contrary, this disclosure is to cover all modifications and equivalents as illustrated, in part, by the appended claims.
The current disclosure relates to immune-modulating compositions and methods of using them. In one embodiment, an immune-modulating composition of the present disclosure comprises a hydrogel-forming polymer, an immune-modulating biomolecule, and a antigen-related biomolecule. In general, immune-modulating biomolecules may recruit or help retain immune cells, such as antigen presenting cells (APC) in the area where the immune-modulating biomolecule is located. Additionally, antigen-related biomolecules may induce a specific response, such as an antigen specific response, in the recruited immune cells. The immune-modulating composition may contain multiple different molecules of each type. Other types of biomolecules, such as biomolecules able to increase antigen presentation or the efficiency of attack cell reaction to the presented antigen, may also be included. In one embodiment, the antigen-related biomolecule may be in a microparticle to modulate the timing of its release.
Immune-modulating biomolecules suitable for use in the present disclosure may be a cytokine, such as a chemokine or a chemoattractant. For example, in some embodiments, it may be a chemokine able to attract and/or retain APCs. Target APCs may include any APC involved in inducing an acquired immune response, particularly an acquired immune response to the antigen-related biomolecule. Specific APCs that may be targeted include Langerhans cells and dendritic cells, such as myeloid dendritic cells. In some embodiments, immature APCs may be targeted for recruitment. Examples of suitable chemokines may include, but are not limited to, Macrophage Inflammatory Protein 3α (MIP3α), Monocyte Chemotactic Protein-1 (MCP-1), MIP1α, MIP1β, Secondary Lymphoid Tissue Chemokine (SLC), N-formyl-methionyl-leucyl-phenylalanine (fMLP), IL-8, Regulated on Activation Normal T Cell Expressed and Secreted (RANTES, also known as Chemokine (C—C motif) Ligand 5 or CCL5), and stromal cell-derived factor-1 (SDF-1), or any combinations of these and other factors. In specific embodiments, an immune-modulating composition of the present may contain two or more or three or more types of immune-modulating biomolecules.
In a specific embodiment, an immune-modulating biomolecule may be a molecule, such as a protein or peptide, that is normally rapidly removed when injected into a tissue, for example by diffusion or degradation. The hydrogel may slow this movement of the biomolecule or release more of it over time to allow for a longer period during which the amount of the immune-modulating biomolecule is elevated near the administration site. For the example, the hydrogel may release any immune-modulating biomolecule in such a manner as to create a sustained gradient over a few days. This sustained gradient may increase both the number of immature APCs at the administration site and/or the duration of their presence.
Antigen-related biomolecules suitable for use in the present disclosure may be any type of biomolecule linked to an agent that ultimately triggers an immune response. For example, it might be the agent itself or something that metabolizes the agent or causes it to be produced. In examples where the agent is an antigen, the second type of biomolecule may be the antigen, a nucleic acid containing the antigen, or a protein or other molecule cleaved or modified in an antigen presenting cell to produce the antigen. In particular embodiments, the second type of biomolecule may be an antigen able to induce a vaccinating immune response, such as any currently used vaccine antigens. The second type of biomolecule may also be an antigen derived from a cancer cell.
In some embodiments, an antigen-related biomolecule may be included in a microparticle. Microparticles may improve uptake of an antigen-related biomolecule because they are often readily taken up by APCs. The synthetic nature of microparticles as well as their size (microns), which is similar to that of many pathogens, may facilitate this uptake by APCs. In some embodiments, microparticles suitable for use in the present disclosure may be cationic in order to enhance delivery of their cargos to the cytoplasm by buffering the phagosomes in which they end up after being taken up by the APCs. Microparticles may also persist at the administration site longer when present in a hydrogel than if simply injected or otherwise administered.
Microparticles in certain embodiments may be made from synthetic polymers like polyesters, polyanhydrides, polycaprolactone, natural polymers like hyaluronic acid, chitosan, alginate, dextran, as well as lipid based materials like phosphatidyl choline, and the like.
In some embodiments, one or more different types of antigen-related biomolecules may be included in an immune-modulating composition of the present disclosure. For example, in one embodiment, two different types of nucleic acids may be included. In some embodiments where microparticles are used, the one or more different antigen-related biomolecules may be both included in the same microparticle or they may be in separate microparticles. There may be advantages to including both in one microparticle so that an APC need to potentially only take up one microparticle to present the antigen.
Additional biomolecules that are neither immune-modulating biomolecules nor antigen-related biomolecules may also be included in an immune-modulating composition of the present disclosure. For example, chemokines that recruit attack immune cells or facilitate their recognition of antigens on APCs may be included. siRNA that downregulates various proteins in the APCs may also be included as this downregulation facilitates the overall desired immune response. Similarly, plasmids or other nucleic acids encoding proteins or peptides that facilitate the overall desired immune response may also be included. For example, the production of IL-10 by APCs may be decreased or increased to induce either a TH-1 type immune response (more effective against intracellular pathogens such as viruses) or a TH-2 type immune response (more effective against extracellular pathogens such as most bacteria).
These additional biomolecules may be included in an immune-modulating composition alone or they may also be part of any microparticles. The most appropriate location for any additional biomolecules may be determined by when they need to be released and where they need to go to be effective. If the additional biomolecules need to cause a particular effect within the APCs, inclusion in microparticles may be more effective. As in the case of different examples of the antigen-related biomolecules, the additional biomolecules may be in separate microparticles or combined in microparticles with other molecules.
As mentioned previously, an immune-modulating composition of the present disclosure comprises a hydrogel forming polymer. In some embodiments, a hydrogel forming polymer may crosslink once administered and form a hydrogel only after an additional ingredient is added or conditions are altered to match administration-site conditions, such as temperature or pH. Use of a hydrogel forming polymer may facilitate administration of the hydrogel. Hydrogels formed after administration may be referred to as in-situ crosslinkable hydrogels. In example embodiments, the hydrogel may be subject to hydrolytic degradation under physiological conditions normally present at the administration site. The hydrogel may also be made of biocompatible materials such as a biocompatible polymer.
In specific embodiments using in-situ crosslinkable hydrogels, particularly those administered by intramuscular injection, a hydrogel forming polymer suitable for use may include, but are not limited to, a vinyl sulfone, an acryl-derivatized polysaccharide, a thiol-derivatized polysaccharide, an acryl-derivatized polyethyleneglycol, a thiol-derivatized polyethyleneglycol, and any a combination thereof. In situ polymerization may allow a high loading capacity of an immune modulating biomolecule and may allow more than one different type to be used.
The chemical composition, polymer concentration, degree of crosslinking and other properties of a hydrogel of the present disclosure may be varied to influence the rate of degradation and thus the rate of release for various components. In specific embodiments, an immune-modulating composition of the present disclosure may be injected into a patient and form a hydrogel within about forty to sixty seconds after injection. Longer times for hydrogel crosslinking may also be suitable, so long as inappropriate amounts of biomolecules are not lost prior to hydrogel formation.
According to one very particular embodiment, an immune-modulating composition may comprise a hydrogel forming polymer capable of crosslinking in-situ and comprising chemokines to attract immature dendritic cells, such as MIP3α, as well as antigen-loaded microparticles.
In another embodiment, a hydrogel may be formed prior to administration or it may be administered in an uncrosslinked form. If administered in an uncrosslinked form, it may then crosslink at physiological temperature and pH. In various examples, the crosslinked or uncrosslinked hydrogel may be administered intramuscularly, subcutaneously, or intradermally.
After crosslinking, any immune-modulating biomolecule, such as a chemokine, may be released from the hydrogel in a sustained fashion to recruit and/or retain APCs at the site of administration. These APCs may then take up (phagocytose) an antigen-related biomolecule, which may be a microparticle, ultimately triggering the presentation of antigens by the APCs and the recognition of those antigens by immune system attack cells. The antigen-related biomolecules may become more available as the hydrogel degrades, for example over the course of three to four days. The APCs may take up the antigen-related biomolecule when it is released from the hydrogel, after entering the hydrogel, or both.
In particular embodiments, the hydrogel may degrade over three to four days, allowing synchronization between APC recruitment and availability of the antigen-related biomolecule.
The immune-modulating compositions and related methods of this disclosure may be used for a variety of purposes. For example, they may be used as vaccines or for delivering multiple growth factors to the body at different release rates. Overall the immune-modulating compositions may allow ready substitution of specific co-administered agents. For example, any chemokine may be used depending on the immunological requirements of the system such as the administration site, the antigen, and the target immune cells. The immune-modulating compositions may also be used for multi-modal delivery of various biomolecule such as CpG oligos, interleukins, various proteins and the like.
The present disclosure may be better understood through reference to the following examples. These examples are included to describe exemplary embodiments only and should not be interpreted to encompass the entire breadth of the invention.
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Similarly, although less effective two and five days after transfection, the microparticles were nevertheless similarly effective in decreasing IL-10 expression as IL-10 siRNA delivered using the traditional siPORT Amine system. Significant decreases in IL-10 expression were not seen in untreated cells or cells treated with microspheres containing scrambled siRNA. (
IL-4 levels were increased in mice that received microparticles lacking the siRNA, but were low in mice receiving the DNA/siRNA microparticles, naked DNA, or PBS. This indicates a significant divergence towards TH1 type immune response (as indicated by high IFNγ levels and low IL-4 levels) thereby confirming the immuno-modulatory effect of the formulation. (
Synthesis of dextran vinylsulfone (DextranVS) with ethyl spacer was performed as mentioned earlier using N,N′-dicyclohexyl-carbodiimide and 4-(Dimethylamino)pyridinium 4-toluenesulfonate (DPTS) catalyst. DPTS was synthesized by dissolving 5 g of pTSA monohydrate in 100 ml Tetrahydrofuran (THF). 4-(dimethylamino)-pyridine (DMAP, 99%) at one molar equivalent to pTSA was added to this mixture and filtered to obtain precipitate which was further dissolved in dichloromethane and recrystallized using a rotary vacuum evaporator. Dextran vinyl sulfone ester synthesis was performed by adding 16.425 g DVS in 90 ml of inert nitrogen saturated DMSO followed by drop wise addition of 0.75 g 3-MPA to it under continuous stirring (molar ratio of 3-MPA to DVS was 1:20). The reaction was continued for 4 hrs at room temperature in the dark. The reaction was performed in the dark to avoid any photo-crosslinking of vinyl sulfone moieties. 5 g or 2.5 g Dextran was dissolved in 30 ml DMSO and solution of 2.17 g DCC and 0.32 g DPTS in 30 ml DMSO was added to it drop-wise and stirred until clear solution was obtained. DPTS is a weak acidic catalyst and enhances the reaction efficacy of DCC. Finally, the mixture was added to DVS/MPA solution in the dark and the reaction was allowed to proceed for 24 hrs at room temperature. After the completion of reaction, N,N-dicyclohexylurea (DCU) salt was filtered using a vacuum filter and the product was recovered by precipitation in 1000 ml of ice cold 100% ethanol. The precipitate was separated from residual ethanol through centrifugation at 3000 rpm for 15 min followed by vacuum drying. Precipitate was re-dissolved in at least 100 ml of de-ionized water (pH adjusted to 7.8) and vortexed to obtain a clear solution. Finally, unreacted polymer was removed through ultra filtration using Amicon filter (MWCO 10000 Da, Millipore) and the viscous product was lyophilized to remove water, analyzed using NMR.
To systematically study the immune response arising from various formulations, microparticle with or without encapsulated IL10 siRNA were administered intramuscularly in Balb/c mice (
Granzyme B levels in target cells were measured to assess the CTL activity by T cells. Purified CD4+ and CD8+ T cells were co-incubated with A20 murine B cell Lymphoma tumor cells at 20:1 E:T ratio for two hours. Flow cytometry based measurement of Granzyme B activity inside target cells provides an early time point sensitive, quantitative assessment of T cell-mediated cytotoxicity. The results indicated that CD4+ cell mediated Granzyme B response was essentially low in mice immunized with naked DNA or microparticles delivering DNA with or without IL10 siRNA. Even bolus supplement of chemokine failed to boost up any CTL response. On the other hand, fast degrading DS2 hydrogels showed stronger Granzyme B positive response as indicated in the double positive quadrants of each plot (
Although the present disclosure has been described in several embodiments, a myriad of changes, variations, alterations, transformations, and modifications may be suggested to one skilled in the art, and it is intended that the present disclosure encompass such changes, variations, alterations, transformations, and modifications as falling within the spirit and scope of the appended claims.
This application claims priority to U.S. Provisional Application No. 61/171,663, filed Apr. 22, 2009, which is incorporated herein by reference.
This disclosure was developed at least in part using funding from the National Institutes of Health (NIH R21A1064179-01). The U.S. government has certain rights in the invention.
Number | Date | Country | |
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61171663 | Apr 2009 | US |