Hydrogel-like particles, methods and uses thereof

Abstract

The present disclosure relates to a composition for the use in the fields of cancer, immunotherapy and biotechnology. Particularly it relates to the field of gellan gum hydrogel-like particles for artificial antigen presentation in immunotherapy.

Claims

1. A composition comprising: a hydrogel-like particle comprising a substance selected from the group consisting of: gellan gum, hyaluronan, pectin, and mixtures thereof; a biotin binding affinity protein bound to the hydrogel-like particle; a biotinylated antibody bound to the biotin binding affinity protein; wherein the biotinylated antibody is able to bind and target an antigen.

2. The composition of claim 1, wherein the hydrogel-like particle encapsulates an active ingredient.

3. The composition of claim 2, wherein the substance of the hydrogel-like particle is gellan gum.

4. The composition of claim 3, wherein the biotin binding affinity protein is avidin, neutravidin, streptavidin or a combination thereof.

5. (canceled)

6. The composition of claim 1, wherein the hydrogel-like particle is a gellan gum nanoparticle and wherein the composition comprises: 5-60% w.sub.gellan gum/w.sub.total of the gellan gum nanoparticle; 1-20% w.sub.antibody/w.sub.total of the biotinylated antibody.

7. The composition of claim 2, wherein the active ingredient is a cytokine selected from the group consisting of IL-2, IL-12, IL-15, IL-17, and IL-23.

8. (canceled)

9. (canceled)

10. The composition of claim 1, according to any one of the previous claims comprising: 0.00001-1% w.sub.protein/w.sub.total of biotin binding affinity protein.

11. (canceled)

12. The composition of claim 1, comprising: 10-50% w.sub.active ingredient/w.sub.total of active ingredient.

13. (canceled)

14. The composition of claim 1, wherein the biotinylated antibody is α-CD3 and/or α-CD28.

15. (canceled)

16. The composition of claim 3, according to any one of the previous claims wherein the size of gellan gum particle is from 100-200 nm.

17. (canceled)

18. (canceled)

19. (canceled)

20. (canceled)

21. The composition of claim 1, wherein the composition is suitable for the treatment of solid tumours.

22. The composition of claim 1, wherein the composition is suitable for the treatment of breast cancer, lymphomas, brain cancer, kidneys cancer, liver cancer, lung cancer, or pancreatic cancer.

23. The composition of claim 1, wherein the particles are nanoparticles.

24. An artificial antigen presenting platform comprising the composition of claim 1.

25. A method to obtain the composition of claim 3, comprising: mixing gellan gum particles with a buffer; activating groups of water-soluble carbodiimide/organic compound; adding a biotin binding affinity protein to the previous solution, so that it adheres to the surface of the activated gellan gum particles; adding biotinylated antibodies to previous solution/dispersion; and incubating the solution/dispersion obtained in the previous step at a temperature between around 0° C.-10° C.

26. The method of claim 25, further comprising: washing the gellan gum particles to remove the excess and unreacted chemical agents; and washing to remove the excess of/unreacted biotin binding affinity protein.

27. The method of claim 25, wherein the biotin binding affinity protein is neutravidin, streptavidin or a combination thereof.

28. (canceled)

29. The method of claim 25, wherein the groups of water-soluble carbodiimide/organic compound are 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)/N-hydroxysuccinimide (NHS) groups.

30. The method of claim 25, wherein the biotinylated antibody is α-CD3 and/or α-CD28 antibodies.

31. (canceled)

32. The method of claim 25, further comprising the step of removing unbound antibodies.

33. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of invention.

[0051] FIG. 1—Schematic illustration representing the process to generate the gellan gum-based particles through an emulsion procedure. Low-acyl gellan gum powder (1) is dissolved at 90° C. for 20 min (2) to give origin to GG solution (w.sub.1) at a predefined concentration (3). Temperature of the dissolved GG is then decreased and added to Chlorophorm and Span80 (O) (4). The mixture is then added in a dropwise manner to a PVA solution (W.sub.2) (5) giving origin to W.sub.1/O/W.sub.2 (6) and afterwards added dropwise to CaCl.sub.2) (7). At this point the solution is crosslinked giving origin to GG nanosphere hydrogels (8) which are then freeze dried (9) originating dried polymeric GG spongy-like hydrogel particles (10).

[0052] FIG. 2—Size distribution analysis of GG particles. A) Particle size analysis using Malvern Zetasizer, Nano ZS Series. B) Scanning transmission electron microscopy (STEM) for GG particles.

[0053] FIG. 3—Cytotoxicity in primary human dermal fibroblast (hDFbs) cells. A) Dose-response effect of GG nanoparticles on hDFbs. The cells were exposed for 72 h with different particle doses (2, 20, 100 and 200 μg). MTS values were corrected for DNA determination and normalized to the control subset. Data is reported as means±standard deviation of 3 experiments. B) Morphological analysis of hDFbs cells which were plated at 10×10.sup.3 cells/well in a 48-well plate and photographed 72 h after plating (magnification ×10).

[0054] FIG. 4—Off-the-shelf stability of GG particles. A) Water uptake profile of dried GG particles over 4 days of immersion in PBS. B) Release profile of BSA from GG nanoparticles C) GG particle stability in different solvents analysed with a Malvern Zetasizer, Nano ZS Series, (I) Average size (II) Polydispersity Index of particles. Data are presented as means±standard error, n=3.

[0055] FIG. 5—Schematic illustration regarding the fabrication of GG-based aAPCS modified with either functional grade α-CD3 and α-CD28 or biotinylated α-CD3 and α-CD28. GG particles (1) are resuspended in MES buffer 50 mM pH=6.5 (2). A mixture of EDC/NHS in then added to promote terminal group activation (3). Excess and unreacted EDC/NHS is washed out by repetitive centrifugation (4). Neutravidin is then conjugated to the particle surface (5). The particles are then washed again by repetitive centrifugation to remove unreacted Neutravidin (6). Antibodies are then incubated O.N at 4° C. with either functional grade or biotinylated α-CD3/CD28 antibodies (7). Unbound antibodies are removed by several centrifugation washes (8) and functional artificial antigen presenting cells are achieved (9).

[0056] FIG. 6—Surface functionalization of GG particles. A) Neutravidin density on the surface of GG particles after chemical binding through EDC/NHS. BI) Characterization of the amount of either functional grade or biotinylated antibodies bound to the surface of GG particles by densitometric analysis. 611) Surface functionalization of GG particles with aCD3 antibody and biotinylated aCD3 were measured by SDS-PAGE. M, molecular mass standards (in kDa; from top to bottom): 250, 130, 100, 70, 55, 35, 25, 15 and 10. 100 μg of GG particles both control (lane 1) or modified (lanes 2-5 and 9-11) or were prepared and heated at 60° C. for 30 min. GG particles were reacted either 500 μg (lanes 2-3) or 1 mg (lanes 4-5) of Neutravidin®. Each condition was respectively incubated with both 10 μg and 20 μg of biotinylated antibody. Note that lanes 2-5 present the 16 kDa subunit of the Neutravidin® protein. A calibration curve of the biotinylated antibody of 0.5 μg, 1 μg and 2 μg was performed (lanes 6-8). The direct binding of the antibody to the particles was also performed (lanes 9 and 10) and again both 10 μg and 20 μg of the antibody were tested. A calibration curve of the standard antibody was equally performed 0.5 μg, 1 μg and 2 μg (lanes 11-13).

[0057] FIG. 7—Murine splenocytes were labelled with 5 μM of CFSE for 20 min at 37° C. and then stimulated with both anti-CD3 and anti-CD28 GG particles in a ratio of 1:1 for a period of 7 days. After this period cells were labelled and gated for anti-CD4 and analyzed on a BD FACSAria™ III. Results are presented as the percentage of cells in the final population that have divided. Data are presented as means±standard error.

[0058] FIG. 8—Il-2 production by murine splenocytes when stimulated over 7 days with both anti-CD3 and anti-CD28 GG particles in a ratio of 1:1. Control (CTRL) corresponds to the conditions in which unmodified particles were used to stimulate the cell cultures. Results are presented as the percentage of cells in the final population that have divided. Data are presented as means±standard error.

[0059] FIG. 9—GG particles tethered to fluorescein probe and visualized under a fluorescent microscope (magnification ×10).

DETAILED DESCRIPTION

[0060] The present disclosure relates to a method to produce submicron GG hydrogel-like particles through an emulsion protocol at the nanoscale, where the nanoparticles may be loaded with cytokines and coated with recombinant molecules in order to prime and expand tumor-specific T-cell responses. This will allow not only the expansion of ongoing T-cell responses but also to modulate their function by means of preventing the development of their “terminal differentiation” or “exhaustion”.

[0061] In order to produce such described particles, we used a double emulsion protocol as exemplified in (FIG. 1).

[0062] In an embodiment, upon in depth characterization we could confirm the production of particles within the nanometer range in terms of dimension, with and a Z-average (d.Math.nm) of 133.6 (FIG. 2).

[0063] In an embodiment, the biocompatibility and cytotoxicity of the produced GG nanoparticles was also evaluated by means of metabolic assays and cell morphology analysis (FIG. 3). GG particles did not show any effect on regular metabolic behavior or cell morphology.

[0064] In an embodiment, the capability of the produced nanoparticles to share similar properties to GG spongy-like hydrogels regarding the high percentage of water uptake after freeze drying was analyzed. To that end, the freeze-dried GG nanoparticles were immersed in H.sub.2O (rehydration step). A rapid weight gain due to the water uptake was observed, with levels between 2700%-3200% (FIG. 4).

[0065] Taking this into consideration, the underlying possibility to be able to uptake biologically relevant molecules in an easy fashion and proceed with their controlled release was present. Freeze-dried GG particles were therefore soaked with 10 μg BSA/mg of particles overnight and release profile was assessed over a period of 3 days where we could observe an initial burst release followed by a steady release of up to 2 μg (FIG. 4B).

[0066] In an embodiment, it was evaluated both the binding potential of neutravidin to the system of the present disclosure as an intermediate for the binding of biotinylated antibodies as well as the grafting of functional antibodies to the system per se (FIG. 6).

[0067] In an embodiment, the functionality of the produced anti-CD3 and anti-CD28 to trigger T-cell proliferation was assessed by in vitro CFSE assays with murine freshly isolated splenocytes. Results are shown in FIG. 7.

[0068] Additionally, to the surface conjugation of biologically relevant molecules or the entrapment by soaking of small molecules for latter release, the aforementioned nanoparticles may be used for tracking by means of conjugating a fluorescent dye to the GG structure previous particle fabrication as can be seen in FIG. 8.

[0069] In an embodiment, once the antibody tethering to the GG particles was confirmed, in vitro studies regarding particle and cell interactions were performed. The percentage of CD4+ murine spleen cells which bound to functionalized α-CD3 particles can be shown in the table below (Table 1).

TABLE-US-00001 TABLE 1 Interaction of functionalized anti-CD3 GG nanoparticles with murine CD4.sup.+ T cells. Results are presented as percentage of cells in the CD4.sup.+ population that showed labelling specific for the modified GG nanoparticles. Data are presented as means ± standard error, n = 2. Quantity of nanoparticles % of CD4.sup.+ cells positive Days of Stimuli Experimental conditions added (μg) for nanoparticles 1 GG nanoparticles CTL 1 1.7 ± 0.42 10 1.9 ± 0.65 50 1.7 ± 0.68 100 1.7 ± 0.60 GG nanoparticles α-CD3 1 1.6 ± 0.45 10 2.23 ± 0.39  50 2.6 ± 1.10 100 5.8 ± 1.79 GG nanoparticles NaV α-CD3 1 2.1 ± 0.52 10 4.9 ± 0.96 50 10.3 ± 3.81  100 18.1 ± 6.25  5 GG nanoparticles CTL 1 1.5 ± 1.08 10 1.6 ± 1.39 50 1.4 ± 0.99 100   2 ± 1.72 GG nanoparticles α-CD3 1 2.7 ± 2.05 10 1.9 ± 1.30 50 2.3 ± 1.47 100 2.5 ± 1.38 GG nanoparticles NaV α-CD3 1 1.6 ± 0.93 10 2.8 ± 1.42 50 6.4 ± 3.23 100 10.6 ± 3.03 

[0070] Upon confirmation that these novel particles would withhold the properties of GG freeze dried hydrogels, it was evaluated their potential as an artificial antigen presenting platform. For this effect the GG nanoparticles were functionalized as presented in the scheme represented in FIG. 5.

[0071] The key aspects of the present disclosure are: [0072] Nanoparticles produced from gellan gum through a double emulsion; [0073] Tethering of functional biomolecules (e.g. anti-CD3, anti-CD28, MHCI/MHCII, anti-PD-L1, others); [0074] Encapsulation of small molecules after production by soaking (e.g. IL-2, IL-12, IL-17, IL-23, anti-hypoxia agents, chemotherapeutics, biologics or mixtures thereof); [0075] Conjugation of a fluorescent probe which allows for tracking; [0076] In one system resides an off the shelf approach for artificial antigen presentation, as well as, combination therapy.

[0077] In an embodiment for improved results, the measured diameter of the particle can be between 500-8000 Å, preferably 1000-3500 Å.

[0078] In an embodiment, the composition may comprise: [0079] 5-60% m/m of a GG nanoparticle, preferably 20-40% m/m; [0080] 10-50% m/m of a cytokine (IL-2, IL-12, IL-15, IL-17, IL-23), preferably 10-40% m/m; [0081] 1-20% m/m of an antibody, preferably 10-20% m/m.

[0082] In an embodiment, the fluorescent probe may be selected from DAPI, FITC, RITC, fluorescein-amine, fluorescein, near-infrared dyes or mixtures thereof.

[0083] In an embodiment, the linker between the functional group of the polysaccharide and of the biomolecule may be through EDC/NHS or Avidin/Biotin.

[0084] In an embodiment, the core nanoparticle should comprise of low-acyl GG, but may also be selected from various negatively charged polysaccharides, such as, alginate, hyaluronan, pectin, or mixture thereof.

[0085] In a particular embodiment, the gellan gum particle is a hydrogel-like particle.

[0086] The term “comprising” whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0087] It will be appreciated by those of ordinary skill in the art that unless otherwise indicated herein, the particular sequence of steps described is illustrative only and can be varied without departing from the disclosure. Thus, unless otherwise stated the steps described are so unordered meaning that, when possible, the steps can be performed in any convenient or desirable order.

[0088] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof.

[0089] The above described embodiments are combinable.

[0090] The following claims further set out particular embodiments of the disclosure.

REFERENCES

[0091] 1. Agnihotri, S. A., Jawalkar, S. S. & Aminabhavi, T. M., 2006. Controlled release of cephalexin through gellan gum beads: Effect of formulation parameters on entrapment efficiency, size, and drug release. European Journal of Pharmaceutics and Biopharmaceutics, 63(3), pp. 249-261. Available at: http://linkinghub.elsevier.com/retrieve/pii/S0939641106000373 [Accessed Aug. 16, 2017]. [0092] 2. Ahuja, M., Yadav, M. & Kumar, S., 2010. Application of response surface methodology to formulation of ionotropically gelled gum cordia/gellan beads. Carbohydrate Polymers, 80(1), pp. 161-167. Available at: http://linkinghub.elsevier.com/retrieve/pii/S0144861709006377 [Accessed Aug. 16, 2017]. [0093] 3. Narkar, M., Sher, P. & Pawar, A., 2010. Stomach-Specific Controlled Release Gellan Beads of Acid-Soluble Drug Prepared by Ionotropic Gelation Method. AAPS PharmSciTech, 11(1), pp. 267-277. Available at: https://doi.org/10.1208/s12249-010-9384-1. [0094] 4. da Silva, L. P. et al., 2014. Engineering cell-adhesive gellan gum spongy-like hydrogels for regenerative medicine purposes. Acta Biomaterialia, 10(11), pp. 4787-4797. Available at: http://linkinghub.elsevier.com/retrieve/pii/S1742706114003031 [Accessed Aug. 16, 2017]. [0095] 5. da Silva, L. P. et al., 2017. Stem Cell-Containing Hyaluronic Acid-Based Spongy Hydrogels for Integrated Diabetic Wound Healing. Journal of Investigative Dermatology, 137(7), pp. 1541-1551. Available at: http://linkinghub.elsevier.com/retrieve/pii/S0022202X17311612 [Accessed Aug. 17, 2017].