Composition and methods for controlled recruitment of analytes

Abstract

This invention is in the field of implantable polymer scaffolds. In particular, provided herein are methods, compositions, and uses of an implantable scaffold with a controlled structure allowing for precise recruitment and enrichment of analytes. For example, the methods and use relate to an efficient system of fabricating an implantable scaffold with preloaded ratios of conjugated polymers of disease-specific antigens to recruit and enrich disease-specific T-cells for diagnostic, therapeutic and research applications.

Description

[0001] COMPOSITION AND METHODS FOR CONTROLLED RECRUITMENT OF ANALYTES

[0002] CROSS REFERENCE TO RELATED APPLICATION

[0003] This application claims priority to U.S. Provisional Patent Application Ser. No. 63 / 661,180, filed June 18, 2024, and the disclosure of which is herein incorporated by reference in in its entirety.

[0004] SEQUENCE LISTING

[0005] The text of the computer readable sequence listing filed herewith, titled “43168- 601_SEQUENCE_LISTING”, created June 18, 2025, having a file size of 4,539 bytes, is hereby incorporated by reference in its entirety.

[0006] STATEMENT REGARDING FEDERAL FUNDING

[0007] This invention was made with government support under EB028840 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0008] FIELD

[0009] This invention is in the field of implantable polymer scaffolds. In particular, provided herein are methods, compositions, and uses of an implantable scaffold with a controlled structure allowing for precise recruitment and enrichment of analytes. For example, the method and compositions relate to an efficient system of fabricating an implantable scaffold with preloaded ratios of conjugated polymers of disease-specific antigens to recruit and enrich disease-specific T-cells for diagnostic, therapeutic and research applications.

[0010] BACKGROUND

[0011] Various radiologic and laboratory tests for analytes of interest are performed for diagnosis, screening, disease staging, forensic analysis, pregnancy testing, drug testing, therapeutic decisions, and research analysis. Disease-specific analytes can be difficult to isolate and collect due to their rarity, difficulty in accessing their habitats, or a combination thereof. These difficulties can necessitate numerous, sometimes invasive, tests to capture the specific disease analyte in time and space. Numerous tests increase the cost of analysis and delay the results. Furthermore, tests may be futile despite successful capture of analytes because of the inefficiencies of analyte expansion associated with ex vivo manipulations (Yixin Li, Roger J Kurlander, Comparison of Anti-CD3 and Anti-CD-Coated Beads with Soluble Anti-CD3 for Expanding Human T cells: Differing Impact on CD8 T Cell Phonotype and Responsiveness to Restimulation, J. Translational Med. 2010; 8: 104, herein incorporated by reference in its entirety). For example, the diagnosis of a fluctuating autoimmune disease such as multiple sclerosis may require a plurality of diagnostic tests including magnetic resonance imaging (MRI), and sampling methods including blood draws and spinal taps for collecting samples of blood and cerebral spinal fluid. The MRI does not provide any analyte samples, and the sampling techniques are invasive and also limited by providing samples that are indicative only of that particular moment in time. Furthermore, the sampling techniques are inefficient due to the difficulty in analyte expansion with ex vivo manipulations.

[0012] Porous biodegradable scaffolds are actively being explored as embodiments for detecting specific analytes in specific diseases. Existing systems include fabricating porous polymer scaffolds that are implanted to induce cell ingrowth and vascularization to form vascularized inflammatory tissue that will extravasate immune cells into the newly formed tissue. Attempts to capture disease-relevant innate and adaptive immune cells with biopsies of the newly formed tissue remains limited by how efficacious the cell ingrowth and vascularization is in forming new tissue along with its recruitment of desired immune cells. The unpredictable results of these types of systems produce insufficient recruitment of the desired analytes.

[0013] A need exists for precision-controlled loading of antigens into a scaffold to efficiently recruit and enrich desired analytes to help solve these inefficient techniques.

[0014] SUMMARY

[0015] Provided herein are methods, compositions, and uses of an implantable scaffold with a controlled structure allowing for precise recruitment and enrichment of analytes. In particular, provided herein are the methods, compositions, and uses related to an efficient system of fabricating an implantable scaffold with preloaded ratios of conjugated polymers of disease-specific antigens to recruit and enrich disease-specific T-cells for diagnostic, therapeutic and research applications. In some embodiments, the invention comprises methods, compositions, or systems for forming an implantable scaffold with controlled ratios of conjugated monomers to unconjugated monomers.

[0016] In some embodiments, methods comprise of conjugating biodegradable backbone monomers of a structure polymer group consisting of poly(lactic acid) (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLG or PLGA), poly(vinyl alcohol) (PVA), poly(caprolactone) (PCL), polyethylene glycol) (PEG), poly(ethylene oxide), or copolymers, or blends thereof, with one, two, or more moieties.

[0017] In some embodiments, the moieties that are conjugated to the polymer backbone can be one or more multiple moieties (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20-25, 20-30, 30-35, 30-40, 40-45, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, or ranges therebetween). The different moieties are conjugated to the polymer backbone in moiety backbone ratios of various combinations.

[0018] In some embodiments, a conjugated biodegradable polymer comprises one or more biological or chemical moieties. The present invention is not limited by the nature of the chemical or biological moieties. Such moieties include, but are not limited to, peptides, proteins, nucleic acid molecules, small molecule drugs, lipids, carbohydrates, cells, cell components, and the like.

[0019] In some embodiments, a conjugated biodegradable polymer is conjugated to a functional moiety. A functional moiety of this invention may be an antigen, polypeptide, polynucleotide, carbohydrate, glycolipid, or other molecule isolated from a biological source, or it may be a chemically synthesized small molecule, polymer, or derivative of a biological material, providing it has the ability to recruit desired analytes.

[0020] In some embodiments, the conjugated biodegradable polymer and the conjugated partner (e.g., antigen) are coupled with the use of polyfunctional coupling reagents (e.g., bifunctional coupling reagents). The selection of the polyfunctional coupling reagent depends on the functional groups present on the conjugated polymer and the conjugate partner (e.g., antigen).

[0021] In some embodiments, the biodegradable polymer, or a portion thereof, is configured for various release rates of biological or chemical agents. In some embodiments, the release provides release of biologically active amounts of the agent over a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85- 90, 90-95, 95-100, 100-105, 105-110, 110-112, 112-115 115-120, 120-125, 125-130, 130-135, 135-140, 140-145, 145-150, 150-155, 155-160, 160-165, 165-170, 170-175, 175-180 days, etc., or any number of days therein and therebetween).

[0022] In some embodiments, polymers have thereon or therein pharmaceutical agents, DNA, RNA, extracellular matrix proteins, exendin-4, etc. In certain embodiments of this invention, the antigen is a single isolated or recombinantly produced molecule. Examples of such antigens are most polynucleotide antigens, and some carbohydrate antigens (such as blood group antigens).

[0023] In some embodiments, the conjugated antigen may or may not be the same as the target antigen, which is the antigen present or to be placed in the scaffold being treated which is a target for the disease- or condition-specific analyte.

[0024] In some embodiments, the antigen is expressed or denoted in a specific disease that is targeted for research, diagnosis, monitoring, or treatment options. Specific diseases can include, but are not limited to, autoimmune diseases and conditions such as multiple sclerosis, diabetes, rheumatoid arthritis, lupus, Crohn’s disease, inflammatory diseases or conditions, virus infections, cancers, or any other condition with an antigen-specific component.

[0025] In some embodiments, the conjugated antigen is the model peptide-antigen Ovalbumin(323-339) (OVA323-339) (amino acid sequence ISQAVHAAHAEINEAGR (SEQ ID NO: 1)), Ovalbumin(257-264) (OVA257-264) (amino acid sequence SIINFEKL (SEQ ID NO:2)), Ovalbumin(321-339) (OVA321-339) (amino acid sequence LKISQAVHAAHAEINEAGR (SEQ ID NO:3)). In some embodiments, the conjugated antigen is a disease specific antigen such as a multiple sclerosis-specific antigen, including the myelin proteolipid protein(139-151) (PLPi39-i5i)(amino acid sequence HCLGKWLGHPDKF (SEQ ID NO:4)).

[0026] In some embodiments, antigens and / or functional moieties are selected to work in a cooperative manner, for example, by providing antigens and adjuvants for vaccination.

[0027] In some embodiments, the method comprises forming a solution of the conjugated biodegradable polymer with unconjugated, biodegradable or non-biodegradable, polymer. In some embodiments, the conjugated polymer is contacted with a solution in the presence of an unconjugated polymer at predetermined mixing ratios. In some embodiments, the unconjugated polymer is PLG (poly(lactide-co-glycolic acid)), but can also include poly(lactic acid) (PLA), polyglycolide (PGA), poly(caprolactone) (PCL), poly(vinyl alcohol) (PVA), poly(ethylene glycol) (PEG), poly(ethylene oxide), poly(ethylene oxide)-co- poly(propylene oxide) block copolymers (poloxamers, meroxapols), poloxamines, polyanhydrides, polyorthoesters, poly(hydroxy acids), polydioxanones, polycarbonates, polyaminocarbonates, poly(vinyl pyrrolidone), poly(ethyl oxazoline), carboxymethyl cellulose, hydroxyalkylated celluloses such as hydroxyethyl cellulose and methylhydroxypropyl cellulose, and natural polymers such as nucleic acids, polypeptides, polysaccharides or carbohydrates such as polysucrose, hyaluronic acid, dextran and similar derivatives thereof, heparan sulfate, chondroitin sulfate, heparin, or alginate, and proteins including without limitation gelatin, collagen, albumin, or ovalbumin, or copolymers, or blends thereof.

[0028] In some embodiments, the composition of an implantable scaffold is fabricated with the solution mixture with controlled ratios of desired moieties in order to recruit desired analytes.

[0029] In some embodiments, the precise mixture of conjugated and unconjugated polymers is fabricated into scaffolds by a solvent casting / particulate leaching technique. In one embodiment, the mixture is cast into a mold of various sizes, thicknesses, and shapes. The mold may also be amended to the desired implantable space of the subject. The scaffold may be amended for implantation by biopsy, cutting, shearing, tearing, folding, heating, breaking, or some other force.

[0030] In some embodiments, the target analyte is disease-specific molecular biomarkers found in the blood including blood’s cellular fractions, serum and plasma, cerebral or spinal fluid, urine, ejaculate and prostatic secretions, tissue or other biological environments. In some embodiments, the blood molecular biomarkers targeted for recruitment consist of circulating tumor cells, cell-free circulating DNA, erythrocytes, leukocytes, platelets, fibrinogen and other clotting factors, proteins including albumins and globulins, glucose, simple and complex carbohydrates, electrolytes, lipids, nucleic acids, and hormones including compositions and components thereof.

[0031] In some embodiments, such analytes are immunological cell lines that are specific to certain diseases and conditions. In some embodiments, the immune cell is selected from the group consisting of a T cell, a natural killer (NK) cell, an NK T cell, a macrophage, a tumor infiltrating lymphocyte (TIL), a tumor infiltrating NK cell (TINK), and a marrow infiltrating lymphocyte (MIL). In some embodiments, the cell is a stem cell. In some embodiments the cell is a hematopoietic cell. In some embodiments, the cell is an engineered cell. In certain embodiments, the engineered cell includes, but is not limited to, cells derived from adipose tissue, skin tissue, muscle tissue, blood, bone marrow, nerve tissue, liver tissue, pancreatic tissue, cartilage tissue, lung tissue, intestinal tissue, spleen tissue, lymph node tissue, ovarian tissue, testicular tissue, umbilical cord tissue, placental tissue, synthetic and biomimetic scaffolds, and other derivatives thereof. In some embodiments, the cell can be any cell type. In certain embodiments, the cell type can be prokaryotic cells, eukaryotic cells, humanspecific cells, immune cells, stem cells, cancer cells, microbial cells, specialized cells, and any derivatives thereof. In some embodiments, methods comprise of analyzing specific biological diseases or conditions by enriching and procuring the scaffold’s recruited analytes without inducing systemic or local disease stimulation.

[0032] BRIEF DESCRIPTION OF THE FIGURES

[0033] FIG. 1 depicts schematics of antigen-conjugated scaffolds that promote enrichment of antigen-specific cell populations. Panel A shows that when antigen-loaded scaffolds are implanted, disease-relevant immune cells traffic to the scaffold site and become enriched upon recognition of target-antigens. Panel B shows that when un-loaded scaffolds are implanted, decreased antigen-specific CD4+ T-cell enrichment is observed due to a loss in antigen signaling.

[0034] FIGS. 2A-2E show the synthesis of PLG-antigen conjugates and their efficiencies. FIG. 2A shows the schematic of the carbodiimide crosslinking chemistry that was used to conjugate antigen OVA323-339 (amino acid sequence ISQAVHAAHAEINEAGR, (SEQ ID NO: 1)) to 7.5 kDa PLG, displaying an example of where the conjugation of other antigens such as OVA257-264, OVA321-339, and PLP139-151, takes effect. FIGS. 2B shows nuclear magnetic resonance (' H-NMR) spectrum of PLG, PLP139-151, PLG-PLP139-151 measured in dimethyl sulfoxide-d6 (referenced at 2.5 ppm) and the coupling efficiency of PLP139-151 to PLG which was calculated by comparing the integration values of the leucine peak present at 0.8 ppm in PLP139-151 to the methylene peak present at 5.2 ppm in PLG. FIG. 2C shows ' H-NMR spectrum of PLG, OVA323-339, and PLG-OVA323-339 measured in dimethyl sulfoxide-d6 (referenced at 2.5 ppm) and the coupling efficiency of OVA323-339 to PLG which was calculated by comparing the integration values of the overlapping valine and isoleucine proton peaks present at 0.8 ppm in OVA323-339 to the methylene proton peak present at 5.2 ppm in PLG. FIG. 2D shows a table displaying the calculated results of PLG-OVA323-339 having a 96.65% coupling efficiency, and the results of PLG- PLP139-151 having an 81.39% coupling efficiency. FIG. 2E is a table of further examples of 'H-NMR calculated coupling efficiencies, including a panel of OVA peptide antigens conjugated to PLG by carbodiimide crosslinking, showing consistently high coupling efficiency of PLG-OVA323-339.

[0035] FIGS. 3A-3F depict the scaffold fabrication techniques and results. FIG. 3A shows a schematic illustrating the scaffold fabrication process with ratios of conjugated 7.5 kDa PLG mixed with unconjugated 7.5 kDa PLG and 80 kDa PLG, then dissolved in chloroform at 13% w / v to create polymer mixtures with known peptide loading which were furthered mixed with sodium chloride crystals which were sieved to the 250-425 pm range at a ratio of 3.9 g NaCl per 1 mL of polymer mixture, and then cast in a cylindrical mold overnight for solvent evaporation, followed by salt leaching and biopsy punching to yield porous scaffolds. FIG. 3B shows a SEM image of a 2.2 pg OVA323-339 / mg PLG-loaded scaffold showing a cylindrical scaffold with pores throughout, and FIG. 3C shows a SEM image of a 4 pg PLPi39-i5i / mg PLG-loaded cylindrical scaffold with pores throughout. FIG. 3D presents resulting porosity percentages and pore volumes of blank scaffolds and scaffolds loaded with various concentrations of OVA323-339. FIG. 3E shows the results of quantifying the total antigen loading for each OVA peptide across a range of antigen concentrations using CBQCA assays. FIG. 3F shows results of quantifying the total disease-specific antigen PLP139-151 loading.

[0036] FIGS. 4A-4C show how carbodiimide crosslinking allows for sustained release of antigens. FIG. 4A depicts the quantification of antigen release for various concentrations of OVA257-264-conjugated scaffolds and OVA32i-339-conjugated scaffolds for 3 weeks. FIG. 4B shows a graph depicting the curve of the cumulative PLP release from the scaffold as the polymer degrades, revealing sustained PLP release through 112 days. FIG. 4C depicts the quantification of antigen release for the various concentrations of PLPi39-isi-conjugated scaffolds for 3 weeks.

[0037] FIGS. 5A-5C show the induction of antigen presentation in vitro with loaded scaffolds co-cultured with cell lines. FIG. 5 A shows a schematic describing antigen presentation by a B-cell in the context of MHC, thereby activating the T-cell to produce the cytokine IL-2, an indicator of T-cell proliferation. FIG. 5B shows IL-2 production and concentration after 30,000 DO-11.10 T-cells and A20 B-cells were cultured in the presence model antigen OVA323-339 in cell media, scaffolds, or soluble antigen for 24 hours by showing the results of antigen loading in units of pg of PLP / mg PLG (negative control) or pg of OVA / mg PLG, while soluble OVA is in units of pg / mL OVA. Error bars are the SEM of 9 replicates for cell media and scaffolds and SEM of 9 replicates for soluble OVA. Error bars may be smaller than symbols. Statistical significance was evaluated using a one-way ANOVA (*** = P<0.001, **** = P<0.0001). FIG. 5C shows how the media conditioned with OVA323-339- conjugated scaffolds or soluble OVA323-339 peptide led to significantly more IL-2 production than media conditioned with the negative control PLPi39-isi-conjugated scaffolds, indicating that the peptide that was released retained biological activity.

[0038] FIGS. 6A-6E show the induction of antigen presentation in vitro with loaded scaffolds co-cultured with splenocytes, with FIGS. 6A-6B representing OVA-reactive splenocytes and FIGS. 6C-6D representing PLP -reactive splenocytes. FIG. 6A shows that when A20 and DO- 11.10 cells were directly co-cultured with antigen-conjugated scaffolds or soluble OVA 323-339 for 24 hours, the cells were activated by antigen loading, and as the concentration of antigen loaded in each scaffold increased, the concentration of IL-2 secreted increased, indicating dose dependency. Due to the low levels of stimulation in response to 0.04 and 0.4 OVA 323- 339-congjugated scaffolds, these conditions were excluded from the rest of the study. FIG. 6B further shows that this T-cell activation (IL-2 secretion) included proliferation in response to the antigen-conjugated scaffolds by showing the flow cytometry results of co-culturing CFSE-stained splenocytes from OT-II mice with antigen-conjugated scaffolds or soluble antigen for 72 hours. The vertical line indicates the end of the parent population signal. FIG 6C shows a schematic of an in vitro experiment procedure with scaffolds being co-cultured for 24 hours with 1 million PLP -reactive splenocytes from PLPi39-i5i-immunized mice. FIG. 6D also shows the results of the IL-2 production and concentration after the splenocytes were harvested and cultured thereby illustrating a graph of various antigen loadings of scaffolds in units of pg of PLP / mg PLG, as well as a blank scaffold. Error bars may be smaller than the symbols. Statistical significance was evaluated using a one-way ANOVA (**** = P<0.0001). FIG. 6E shows that 4 pg PLPi39-i5i / mg PLG- and 20 pg PLPi39-i5i / mg PLG-conjugated scaffolds induced significantly more T-cell activation than the blank scaffold, as evidenced by an increase in IL-2 secretion.

[0039] FIGS. 7A-7B show cell trafficking in vivo with GFP experimental procedures. FIG. 7A shows a schematic timeline of the in vivo GFP experimental procedure starting with the immunization of GFP+ mice with OVA323-339 and Complete Freund’s Adjuvant (CFA) to the co-culturing of the scaffolds, to the intravenous adoptive transfer of GFP-labeled cells and subcutaneous implantation of 4 OVA-loaded scaffolds, 1 PLP -loaded scaffold, and 1 blank scaffold in mice, followed by the harvest and analysis with flow cytometry. FIG. 7B shows graphs of the results of the flow cytometry of each scaffold by measuring the total number of CD4+ GFP+ cells and the CD4+ GFP+ percent of CD45+. CD4 GFP+ represents OVA- specific CD4+ T-cells (* = 0.01<P<0.05) (** = O.OKPO.OOl). FIGS. 8A-8C show cell trafficking in vivo with passive induction EAE experiments. FIG. 8A shows a schematic timeline of the in vivo EAE induction experimental procedure starting with the immunization of mice with PLP139-151 in CFA, to the co-culturing of the spleens and lymph nodes, to the intravenous adoptive transfer of 12,000,000 CFSE-labeled autoreactive T-cells and subcutaneous implantation of scaffolds, to the harvest and analysis with flow cytometry. One group of mice received 6 implants consisting of 1 blank scaffold, 1 OVA-loaded scaffold and 4 PLP -loaded scaffolds and the other group of mice was the control group with no scaffolds implanted. FIG. 8B shows a graph of the daily disease scoring which shows that the loaded scaffolds do not impact disease onset. FIG. 8C shows graphs of the total number and percentage of antigen-specific CD4+ T-cells in scaffolds that were quantified using flow cytometry. Antigen-specific CD4+ T-cells were gated on Live+ CD45+ CD4+ Tetramer+ cells. Antigen loadings are represented in units of pg PLP / mg PLG or pg OVA / mg PLG. Error bars are the SEM of 5 replicated and may be smaller than symbols. Statistical significance was evaluated using a two-way ANOVA (* = P<0.05).

[0040] FIGS. 9A-9F show the enrichment of disease-relevant CD4 T-cells in an in vivo active induction EAE model using antigen-conjugated scaffolds. FIG. 9A models how disease was actively induced in SJL / J mice through immunization, followed by subcutaneous scaffold implantation. FIG. 9B illustrates the clinical scores of disease for the control and scaffold groups of mice. The clinical scale ranges from 0 (normal mouse) to 5 (death by EAE), with intermediate scores of 1 (limp tail or hind limb weakness), 2 (both limp tail and hind limb weakness), 3 (partial hind limb paralysis), and 4 (total hind limb paralysis). FIG. 9C shows the analyzed results of the harvested scaffolds, pooled inguinal lymph nodes, and spleens from the immunized SJL / J mice for IL-2 secretion in response to 10 pg / mL PLP139-151 stimulation via ELISPOT. FIG. 9D shows the quantification of the total number of IL-2+ T- cell spots in FIG. 9C found using an ELISPOT analyzer. FIG. 9E shows the total number of CD4 T-cells across implants, pooled inguinal lymph nodes, and spleens found using flow cytometry. FIG. 9F shows that the frequency of PLPi39-i5i-reactive T-cells was determined by normalizing the number of spot-forming cells in FIG. 9D to the total number of CD4 T-cells in FIG. 9E. Error bars are the SEM of 3-8 replicates. Statistical analysis was evaluated via one-way ANOVA (*p<0.05, **p<0.01, ****p<0.0001).

[0041] DETAILED DESCRIPTION OF THE INVENTION Embodiments of this disclosure provide methods, processes, systems, and compositions related to precise and efficient recruiting and enriching of disease-specific biomolecules with an implantable scaffold. In particular, in some embodiments, the present disclosure pertains to implantable porous scaffolds that efficiently recruit or retain diseasespecific analytes, by crosslinking one or more moiety to one or more biodegradable polymers to form polymer-moiety conjugates, combining the polymer-moiety conjugates with unconjugated polymers at predetermined mixing ratios, and fabricating the mixture into an implantable scaffold. In accordance with these embodiments, this present description demonstrates the efficiency of disease-specific T cells recruitment and enrichment by implantable scaffolds when the biodegradable polymers are conjugated with disease-specific antigens prior to scaffold fabrication. As described further herein, the implantable porous scaffolds of the present disclosure yielded activation of antigen-specific cells in vitro when antigen-loaded scaffolds were co-cultured with either antigen-specific cell lines or splenocytes, and enrichment in vivo when antigen-loaded scaffolds were implanted in mice that had antigen-specific cells transferred to them. Embodiments of this disclosure demonstrate a method for precise loading of peptide antigens, sustained antigen release, and dose-dependent antigen-specific cell activation and enrichment.

[0042] Definitions

[0043] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities, and plural terms shall include the singular.

[0044] As noted herein, the disclosed embodiments have been presented for illustrative purposes only and are not limited. Other embodiments are possible and are covered by the disclosure, which will be apparent from the teachings contained herein. Thus, the breadth and scope of the disclosure should not be limited by any of the described embodiments but should be defined only in accordance with claims supported by the present disclosure and their equivalents. Moreover, embodiments of the subject disclosure may include a process, method, system, composition, device, apparatus, or kit which may further include any and all elements from any other disclosed processes, methods, systems, compositions, devises, apparatuses, or kits including any and all elements corresponding to controlled structures of polymer scaffolds. In other words, elements from one or another disclosed embodiment may be interchangeable with elements from other disclosed embodiments. Moreover, some further embodiments may be realized by combining one and / or another feature disclosed herein with processes, methods, systems, compositions, devices, apparatuses, kits and one or more features thereof, disclosed in materials incorporated by reference. In addition, one or more features or elements of disclosed embodiments may be removed and still result in patentable subject matter (and thus, resulting in yet more embodiments of the subject disclosure). Furthermore, some embodiments correspond to processes, methods, systems, compositions, devices, apparatuses and kits which specifically lack one and / or another element, structure and / or steps (as applicable), as compared to teachings of the prior art, and therefore represent patentable subject matter and are distinguishable therefrom (i.e. claims directed to such embodiments may contain negative limitations to note the lack of one or more features prior art teachings).

[0045] As used herein, “a,” “an,” and “the” include plural reference unless the context clearly indicates otherwise.

[0046] For the recitation of numeric ranges herein, each intervening number therebetween with the same degree of precision is explicitly contemplated. For example, for the range of 6- 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.8, 6.9, and 7.0 are explicitly contemplated. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed the “less than or equal to 10” as well as “greater than or equal to 10” is also disclosed.

[0047] It is also understood that throughout the application, data is provided in a number of different formats, and that this data represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point “15” are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15.

[0048] As used herein, “disease,” “pathologic condition,” and “condition” are used interchangeably, unless indicated otherwise herein, to describe a deviation from the condition regarded as normal or average for members of a species or group (e.g. humans), and which is detrimental to an affected individual under conditions that are not inimical to the majority of individuals of that species or group. Such a deviation can manifest as a state, signs, and / or symptoms (e.g. diarrhea, nausea, fever, pain, blisters, boils, rash, immune suppression, inflammation, etc.) that are associated with any impairment of the normal state of a subject or of any of its organs or tissues that interrupts or modifies the performance of normal functions. A disease or pathological condition may be caused by or result from contact with a microorganism (e.g. a pathogen or other infective agent (e.g. a virus or bacteria)), may be responsive to environmental factors (e.g. malnutrition, industrial hazards, and / or climate), may be responsive to an inherent or latent defect in the organism (e.g. genetic anomalies), or to combinations of these and other factors.

[0049] As used herein, “subject” refers to any animal (e.g. mammal, reptiles, avians, amphibians), including, but not limited to, humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms “subject” and “patient” are used interchangeably herein in reference to a human subject, unless indicated otherwise.

[0050] As used herein, “sample” is used in its broadest sense. In one sense, it is meant to include a specimen or culture obtained from any source as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include, but are not limited to, urine, secretions, or blood products such as plasma, serum, interstitial fluid, and the like. “Sample” may also refer to cells, cell lysates or purified forms of the enzymes, peptides, and / or polypeptides described herein (e.g., a purified protein sample). The term sample may also include purified samples, such as purified protein samples. Such examples are not, however, to be construed as limiting the sample types applicable to the present invention.

[0051] As used herein, “protein” is used synonymously with “peptide,” “polypeptide,” or “peptide fragment.”

[0052] As used herein, “cancer” means a disease or condition involving unregulated and abnormal cell growth. Non-limiting exemplary cancers herein include multiple myeloma, leukemia, pancreatic cancer, breast cancer, colorectal cancer, cachexia, melanoma, cervical cancer, ovarian cancer, lymphoma, gastrointestinal, lung cancer, prostate cancer, renal cell carcinoma, metastatic kidney cancer, solid tumors, non-small cell lung carcinoma, nonHodgkin's lymphoma, bladder cancer, oral cancer, myeloproliferative neoplasm, B-cell lymphoproliferative disease, and plasma cell leukemia.

[0053] As used herein, “comprise” (or variations thereof), “contain” (or variations thereof), “have” (or variations thereof), or “include” (or variations thereof), are not intended to be limiting, are inclusive or open-ended and do not exclude additional, unrecited additives, components, integers, elements, or method steps. For example, a process, method, system, composition, kit, or apparatus that comprises a list of features is not necessarily limited only to those features but may include other features not expressly listed or inherent to such process, method, system, composition, device, apparatus, or kit.

[0054] As used herein, “loading” refers to the amount of antigen per amount of structure polymer. Loading can be expressed as micrograms (pg) of antigen per milligram (mg) of polymer.

[0055] As used herein, “biomarker” is any measurable characteristic that indicates the presence or absence of disease or the biological response to a stimulus, typically an exposure or intervention than can be used clinically for detecting or measuring the disease in terms of a subject’s susceptibility and risk, diagnosis, monitoring, prognostication, predictive determinations, pharmacodynamic and treatment response, and / or safety.

[0056] Methods of Making

[0057] The methods disclosed herein provide one or more advantages, for example, providing a consistent method for preparing a polymer scaffold having precise moiety- conjugated polymers fabricated into implantable scaffolds thereto, providing control over molecular functionalization of the scaffolds, and providing scaffolds with localized molecules. Further, the disclosed methods allow for attaching a broad range of molecules to the surface of the scaffold through the moiety-conjugated structure polymer, allowing for use in a wide range of applications. Thus, the methods herein can provide specialized polymer scaffolds having one or more advantages such as a providing a known quantity of an antigen- conjugated polymer homogeneously throughout the scaffold and / or providing a polymer structure having functional groups purposely distributed spatially throughout the polymer structure. Non-limiting examples of applications include presenting moieties to recruit disease-specific analytes allowing for the detection and study of a variety of inflammatory diseases, autoimmunity, cancers, viral infections, and other pathologic conditions.

[0058] Antigen Conjugation

[0059] In some embodiments, the moiety-conjugated polymers consist of backbone monomers selected from the group consisting of poly(lactic acid) (PLA), polyglycolide (PGA), poly(caprolactone) (PCL), poly(lactide-co-glycolide) (PLG or PLGA), poly(vinyl alcohol) (PVA), polyethylene glycol) (PEG), poly(ethylene oxide), poly(ethylene oxide)-co- poly(propylene oxide) block copolymers (poloxamers, meroxapols), poloxamines, polyanhydrides, polyorthoesters, poly(hydroxy acids), polydioxanones, polycarbonates, polyaminocarbonates, poly(vinyl pyrrolidone), poly(ethyl oxazoline), carboxymethyl cellulose, hydroxyalkylated celluloses such as hydroxyethyl cellulose and methylhydroxypropyl cellulose, and natural polymers such as nucleic acids, polypeptides, polysaccharides or carbohydrates such as polysucrose, hyaluronic acid, dextran and similar derivatives thereof, heparan sulfate, chondroitin sulfate, heparin, or alginate, and proteins including without limitation gelatin, collagen, albumin, or ovalbumin, or copolymers, or blends thereof. Embodiments of the present disclosure selected monomers from the PLG group. In one aspect, a monomer detailed above is derivatized to include a functional group that is mutually reactive with a carboxyl functional group. In one aspect, a monomer detailed above is derivatized to include an amine functional group. As described further herein, embodiments of the present disclosure consist of backbone monomers selected from the group consisting of poly(lactide-co-glycolide) (PLG or PLGA). In some embodiments, antigens may be conjugated using a cleavable linker. Use of cleavable linkers permits conditional release of antigens when presented with cleaving conditions (e.g., contact with a cleaving enzyme, light-cleavable, heat-cleavable, pH-cleavable, etc.).

[0060] In some embodiments of the invention, the biodegradable polymers contain copolymers. These co-polymers may have varying molar ratio. Suitable co-polymer ratio of modified polymers may be 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 81 : 19, 82:18, 83: 17, 84: 16, 85: 15, 86: 14, 87: 13, 88: 12, 89: 11, 90: 10, 91 :9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2, 99: 1, or 100:0. As described further herein, embodiments of the present disclosure consist of a co-polymer made from D,L-lactide and glycolide in a 75:25 ratio. In another embodiment, the co-polymer may be periodical, statistical, linear, branched (including star, brush, or comb co-polymers) co-polymers. In some embodiments, the copolymers ratio may be, but not limited to, polystyrene: poly (vinyl carboxylate) / 80:20, polystyrene:poly(vinyl carboxylate) / 90: 10, poly(vinyl carboxylate):polystyrene / 80:20, poly(vinyl carboxylate):polystyrene / 90: 10, polylactic acid:polyglycolic acid / 50:50, polylactic acid:polyglycolic acid / 80:20, or polylactic acid:polyglycolic acid / 90: 10.

[0061] In some embodiments, polymers or co-polymers comprise variable molecular weights including molecular weight ranges that are considered low, medium, or high. Molecular weights ranges include all ranges produced by manufacturers. Ranges can include 1-200 kilodaltons (kDa). Commercial ranges can include, but not limited to, 1-5 kDa, 5-10 kDa, 1- 10 kDa, 5-15 kDa, 10-15 kDa, 15-20 kDa, 10-20 kDa, 15-25 kDa, 20-25 kDa, 25-30 kDa, SO- 35 kDa, 35-40 kDa, 40-45 kDa, 45-50 kDa, 50-55 kDa, 55-60 kDa, 60-65 kDa, 65-70 kDa, 70-75 kDa, 75-80 kDa, 75-85 kDa, 80-85 kDa, 85-90 kDa, 90-95 kDa, 95-100 kDa, 100-105 kDa, 100-110 kDa, 100-120 kDa, 100-130 kDa, 110-166 kDa, 66-107 kDa, 65-105 kDa, 23- 37 kDa, 37-52 kDa, 52-69 kDa, 69-87 kDa, 87-106 kDa, 5-20 kDa, 37-84 kDa, 65-85 kDa, 76-130 kDa, 110-166 kDa. In some embodiments, the present disclosure uses a low molecular weight considered at a range of 5-10 kDa and a high molecular weight considered at a range of 75-85 kDa.

[0062] The present disclosure provides conjugates of biodegradable polymers. The polymer is typically a chemical species containing a plurality of repeating units that are bonded to each other. A polymer may contain more than one different repeating unit. The repeating unit typically derives from polymerization of a monomer. A copolymer specifically refers to a polymer containing two or more structurally different repeating units. The different repeating units of a polymer may be randomly ordered in the polymer chain, or the same repeating units may be grouped into contiguous blocks in the polymer. When there are contiguous blocks of the two or more repeating units in a polymer, the polymer is a block co-polymer. In certain embodiments, the polymer is a graft co-polymer.

[0063] Suitable biodegradable polymer for conjugation comprises a functional group for reaction. The biodegradable polymer described herein can be chemically modified by reactions to introduce a desired terminal functional group and use ester terminated high molecular weight polymers and acid terminated low molecular weight polymers. Terminal functional groups include among others, carboxyl, hydroxyl, thiol, amine, azide, alkyne, alkene, ketone, phenol, halide, imidazole, guanidinium, carboxylate, or phosphate groups. These functional groups can be introduced at the terminus of the biodegradable polymer herein employing well known chemical methods.

[0064] The functional groups can be employed to further conjugate the biodegradable polymer with moieties, such as other chemical species, including other polymers, other oligomers, peptides, proteins, small molecules, carbohydrates, antibodies, nucleic acids, and / or aptamers.

[0065] In some embodiments, the biodegradable polymer may be conjugated with one or more moieties. In some embodiments, polymers are conjugated to multiple moieties (e.g.2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20-25, 20-30, 30-35, 30-40, 40-45, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, or ranges therebetween).

[0066] In some embodiments, the different moieties are conjugated to the polymer backbone in ratios of 1, 2 or more, 3 or more, 4 or more, in ratios of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 moiety backbone. More than one moieties conjugated to each polymer backbone can also be in various ratios, including 1:1, 1:1:1, 1 : 1 : 1 : 1, 1:2:1, 1:2:2: 1, 1:2: 1:2, 1:2:3, 1:2:2, 1:2:3:, 1:3:3, 3:1:3, 3:2:3, 1:3:1, 2:3:2, 1 :3: 1 :3, 2:3:2:3, 1 :4: 1 :4:, 1:2, 1:3, 1:4, 1:4.5, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1;45, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 2:3, 2:5, 2:7, 2:9, 2:11, 2:13, 2:15, 2:17, 2:19, 2:25, 2:35, 2:45, 2:55, 2:65, 2:75, 2:85, 2:95, 3:4, 3:5, 3:7, 3:8, 3:10, 3:11, 3:13, 3:14, 3:17, 3:19, 3:20, 3:22, 3:23, 3:25, 3:26, 3:28, 3:29, 3:35, 3:40, 3:50, 3:55, 3:65, 3:70, 3:80, 3:85, 3:95, 3:100, 4:5, 4:7, 4:9, 4:11, 4:13, 4:15, 4:17, 4:19, 4:21, 4:23, 4:25, 4:27, 4:29, 4:35, 4:45, 4:55, 4:65, 4:75, 4:85, 4:95, 5:6, 5:7, 5:8, 5:9, 5:11, 5:12, 5:13, 5:14, 5:16, 5:17, 5:18, 5:19, or ranges therebetween and combinations thereof.

[0067] In some embodiments, the moiety is conjugated to the polymer with different methods of chemistry, including but not limited to carbodiimide crosslinking chemistry, encapsulated chemistry, click chemistry, and / or bonding chemistry.

[0068] In some embodiments, the biodegradable polymer comprises a carboxyl group, which can be conjugated to an amino-containing conjugate partner with use of carbodiimide crosslinking chemistry. Carbodiimide reagents are used as coupling reagents for reaction of carboxylic acids towards amide or ester formation. Examples of carbodiimide reagents include EDCI (l-ethyl-3-(3-dimethylaminopropyl)carbodiimide, or EDC, or ECDI), DCC (N,N'-dicyclohexylcarbodiimide), and DIC (N,N'-diisopropylcarbodiimide). In addition to the coupling reagent, activation reagents may be used to facilitate the reaction. Examples of activating reagents include hydroxysuccinimide. Reaction of carbodiimide-activated carboxylic acid on a biodegradable polymer with an amino-containing conjugate partner can produce an amide bond connecting the polymer and conjugate partner.

[0069] In some embodiments, the biodegradable polymer and the conjugate partner (e.g., antigen) are coupled with the use of polyfunctional coupling reagents (e.g., bifunctional coupling reagents). The selection of the polyfunctional coupling reagent depends on the functional groups present on the biodegradable polymer and the conjugate partner (e.g., antigen).

[0070] The groups of the polyfunctional coupling reagent can independently include a carboxyl -reactive group, carbonyl -reactive group, an amine-reactive group, a thiol -reactive group or a photo-reactive group, but are not the same (e.g., orthogonal). Examples of carboxyl -reactive groups include hydrazine derivatives and amines. Examples of carbonylreactive groups include aldehyde- and ketone-reactive groups like hydrazine derivatives and amines. Examples of amine-reactive groups include active esters such as NHS or sulfo-NHS, isothiocyanates, isocyanates, acyl azides, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, anhydrides and the like. Examples of thiolreactive groups include non-polymerizable Michael acceptors, haloacetyl groups (such as iodoacetyl), alkyl halides, maleimides, aziridines, acryloyl groups, vinyl sulfones, benzoquinones, aromatic groups that can undergo nucleophilic substitution such as fluorobenzene groups (such as tetra and pentafluorobenzene groups), and disulfide groups such as pyridyl disulfide groups and thiols activated with Ellman's reagent. Examples of photo-reactive groups include aryl azide and halogenated aryl azides. Additional examples of each of these types of groups will be apparent to those skilled in the art. Further examples and information regarding reaction conditions and methods for exchanging one type of reactive group for another are provided in Hermanson, “Bioconjugate Techniques,” Academic Press, San Diego, 1996, which is incorporated by reference herein.

[0071] Conjugates such as ethylene carbodiimide (EDCI), hexamethylene diisocyanate, propyleneglycol di-glycidylether which contain 2 epoxy residues, and epichlorohydrin may be used for fixation of peptides or proteins to the polymer. Without being bound by theory, EDCI is suspected of chemically coupling the protein / peptides to the polymer via catalysis of peptide bond formation between free amino and free carboxyl groups.

[0072] In some embodiments, the antigen peptides and proteins are bound to the polymer via a covalent chemical bond. For example, a reactive component near the C-terminus of the antigen (e.g., the C-terminal carboxyl group, or a hydroxyl, thiol, or amine group from an amino acid side chain) may be conjugated directly to a reactive component of the polymer (e.g., a hydroxyl or carboxyl group of a PLA or PGA polymer, a terminal amine or carboxyl group of a dendrimer, or a hydroxyl, carboxyl or phosphate group of a phospholipid) by direct chemical reaction. Alternatively, there may be a conjugating moiety which covalently conjugates to both the antigen peptides and proteins and the polymer, thereby linking them together.

[0073] Reactive carboxyl groups of a biodegradable polymer may be joined to free amines (e.g., from Lys residues) on the antigen peptide or protein, by reacting them with, for example, l-ethyl-3-[3,9-dimethyl aminopropyl]carbodiimide hydrochloride (EDC) or N- hydroxysuccinimide ester (NETS). Similarly, the same chemistry may be used to conjugate free amines on the surface of a carrier polymer with free carboxyls (e.g., from the C- terminus, or from Asp or Glu residues) on the antigen peptide or protein. Alternatively, free amine groups on a polymer may be covalently bound to antigen peptides and proteins, or antigen peptide or protein fusion proteins, using sulfo-SIAB chemistry, essentially as described by Arano et al. (1991) Chem. 2:71-6.

[0074] In some embodiments, the loading of antigen is about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1, 1.5, 2, 2.5 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 pg antigen / mg polymer and all values in between. In some embodiments, the loading of antigen is about 0.10 to about 0.50 pg antigen / mg polymer. In some embodiments, the loading of antigen is about 0.50 to about 2 pg antigen / mg polymer. In some embodiments, the loading of antigen is about 2 to about 4 pg antigen / mg polymer. In some embodiments, the loading of antigen is about 4 to about 8 pg antigen / mg polymer. In some embodiments, the loading of antigen is about 8 to about 16 pg antigen / mg polymer. In some embodiments, the loading of antigen is about 16 to about 20 pg antigen / mg polymer. In some embodiments, the loading of antigen is about 20 to about 30 pg antigen / mg polymer. In some embodiments, the loading of antigen is about 30 to about 40 pg antigen / mg polymer. In some embodiments, the loading of antigen is about 40 to about 50 pg antigen / mg polymer. In some embodiments, the loading of antigen is about 50 to about 60 pg antigen / mg polymer. In some embodiments, the loading of antigen is about 60 to about 70 pg antigen / mg polymer. In some embodiments, the loading of antigen is about 70 pg antigen or more / mg polymer.

[0075] In some embodiments, conjugation of a nucleic acid moiety to a platform molecule can be effected in any number of ways, typically involving one or more crosslinking agents and functional groups on the nucleic acid moiety and platform molecule. Linking groups are added to platforms using standard synthetic chemistry techniques. Linking groups can be added to nucleic acid moieties using standard synthetic techniques. The practitioner has a number of choices for nucleic acid antigens used in the combination of this invention. The nucleic acid antigen present in the combination contributes to the specificity of the response that is induced.

[0076] In some embodiments, a biodegradable polymer is conjugated to a functional moiety. A functional moiety of this invention may be a peptide, polypeptide, protein, antigen, polynucleotide, carbohydrate, glycolipid, nucleic acid, or other molecule isolated from a biological source, or it may be a chemically synthesized small molecule, polymer, or derivative of a biological material, providing it has the ability to recruit desired analytes.

[0077] In some embodiments, a biodegradable polymer is covalently coupled to one or more peptides, polypeptides, and / or proteins including antigens. The practitioner has a number of choices for antigens used in the combinations of this invention. The antigen present in the combination contributes to the specificity of the response that is induced. In some embodiments, a conjugated carrier polymer (e.g., PLG carrier), such as those described herein, are effective to recruit antigen-specific analytes while inducing other analytes that induce antigen-specific tolerance to prevent the onset of an immune related disease (such as EAE in a mouse model) and / or diminish the severity of a pre-existing immune related disease. In some embodiments, the compositions and methods of the present invention can recruit T-cells and cause T-cells to undertake early events associated with T-cell activation, but do not allow T-cells to acquire effector function.

[0078] In some embodiments, a biodegradable polymer comprises one or more biological or chemical agents adhered to, adsorbed on, encapsulated within, and / or contained throughout the polymer. In some embodiments, a chemical or biological agent is encapsulated in and / or contained throughout the polymer. The present invention is not limited by the nature of the chemical or biological agents. Such agents include, but are not limited to, peptides, proteins, nucleic acid molecules, small molecule drugs, lipids, carbohydrates, cells, cell components, and the like. In some embodiments, agents are configured for specific release rates. For example, a first agent may release over a period of hours while a second agent releases over a longer period of time (e.g., days, weeks, months, etc.). In some embodiments, the carrier polymer, or a portion thereof, is configured for slow-release of biological or chemical agents. In some embodiments, the biodegradable polymer, or a portion thereof, is configured for various release rates of biological or chemical agents. In some embodiments, the release provides release of biologically active amounts of the agent or agents over a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, or any number of hours therein and therebetween. In some embodiments, the release provides release of biologically active amounts of the agent or agents over a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-100, 100-105, 105-110, 110-112, 112-115 115-120, 120-125, 125-130, 130-135, 135-140, 140-145, 145-150, 150-155, 155-160, 160-165, 165-170, 170-175, 175-180 days, etc., or any number of days therein and therebetween).

[0079] Encapsulation of the antigen, biological, and / or chemical agents within the polymer of the invention has several advantages. First, the encapsulation provides a slower cytokine response. Second, when using multiple antigens, biological, and / or chemical agents, encapsulation removes the competition between these various molecules that might occur if the agents were attached to the surface of the polymer. Third, encapsulation allows more antigens, biological, and / or chemical agents to be incorporated within the polymer. Finally, encapsulation allows for easier use of complex protein antigens or organ homogenates. The encapsulation of the antigen, biological and / or chemical agents in the polymers may be performed by any method known in the art. In one embodiment, the polypeptide antigens are encapsulated in by a double-emulsion process. In a further embodiment, the polypeptide antigens are water soluble. In some embodiments, the polypeptide antigens are encapsulated by a single-emulsion process. In other embodiments, the polypeptide antigens are more hydrophobic.

[0080] In some embodiments, a biodegradable polymer comprises thereon or therein cells or other biological or chemical agents. Where cells are employed, the conjugated polymer carriers are not limited to a particular type of cells. In some embodiments, conjugated polymer carriers have thereon or therein pharmaceutical agents, DNA, RNA, extracellular matrix proteins, exendin-4, etc. In certain embodiments of this invention, the antigen is a single isolated or recombinantly produced molecule. For treating conditions where the target antigen is disseminated to various locations in the host, it is generally useful that the antigen be immunologically related to the target antigen. Examples of such antigens are most polynucleotide antigens, and some carbohydrate antigens (such as blood group antigens).

[0081] Any suitable antigens may find use within the scope of the present invention. In some embodiments, the conjugated antigen may or may not be the same as the target antigen, which is the antigen present or to be placed in the scaffold being treated which is a target for the disease- or condition-specific analyte and for the unwanted immunological response.

[0082] Where the target antigen is preferentially expressed on a particular organ, cell, or tissue type, the invention includes the option of using an antigen which is identical with or immunologically related to the target antigen. In some embodiments, there is also the additional option of using an antigen which is a bystander for the target. This is an antigen which may not be immunologically related to the target antigen but is preferentially expressed in a tissue where the target antigen is expressed. A working theory as to the effectiveness of bystander suppression is that suppression is an active cell-mediated process that down-regulates the effector arm of the immune response at the target cells. Through an interactive or cytokine-mediated mechanism, the localized suppressor cells then down- regulate effector cells (or inducers of effector cells), regardless of what they are reactive against. If the effector cells are specific for a target different from the conjugated antigen, then the result is a bystander effect. Reference to International Patent Publication WO 93 / 16724 will provide further elaboration of the bystander reaction and a list of tolerogenic peptides having this effect. An implication of bystander theory is that one of ordinary skill need not identify or isolate a particular target antigen against which tolerance is desired in order to practice the present invention. The invention only needs to be able to obtain at least one molecule preferentially expressed at the scaffold for use as a functional antigen.

[0083] In some embodiments, the antigen is not in the same form as expressed in the individual being treated but is a fragment or derivative thereof. Antigens of this invention include peptides based on a molecule of the appropriate specificity but adapted by fragmentation, residue substitution, labeling, conjugation, and / or fusion with peptides having other functional properties. The adaptation may be performed for any desirable purposes, including but not limited to the elimination of any undesirable property, such as toxicity or immunogenicity, or stimulation of the tolerogenic arm of the immune response. Terms such as insulin peptide, collagen peptide, and myelin basic protein peptide, as used herein, refer not only to the intact subunit, but also to allotypic and synthetic variants, fragments, fusion peptides, conjugates, and other derivatives that contain a region that is homologous (preferably 70% identical, more preferably 80% identical and even more preferably 90% identical at the amino acid level) to at least 10 and preferably 20 consecutive amino acids of the respective molecule for which it is an analog, wherein the homologous region of the derivative shares with the respective parent molecule an ability to recruit the target analytes. In certain embodiments of this invention, two, three, or a higher plurality of functional antigens may be used. It may be desirable to implement these embodiments when there is a plurality of target antigens, or to provide a plurality of bystanders for the target. For example, natural antigens of multiples sclerosis can be mixed for recruitment of multiple sclerosis specific T-cells through cell-enhancing cytokines, along with antigens that will recruit suppressor cells to control stimulating-cytokines of disease effector cells. It may also be desirable to provide a cocktail of antigens to cover several possible alternative targets. For example, a mixture of allergens may serve as functional antigen for the treatment of atopy.

[0084] Functional antigens can be prepared by a number of techniques known in the art, depending on the nature of the molecule. Polynucleotide, polypeptide, and carbohydrate antigens can be isolated from cells of the species to be studied in which they are enriched. Short peptides are conveniently prepared by amino acid synthesis. Longer proteins of known sequence can be prepared by synthesizing an encoding sequence or PCR-amplifying an encoding sequence from a natural source or vector and then expressing the encoding sequence in a suitable bacterial or eukaryotic host cell.

[0085] In certain embodiments of this invention, the combination comprises a complex mixture of antigens obtained from a cell or tissue, one or more of which plays the role of a functional antigen. The antigens may be in the form of whole cells, either intact or treated with a fixative such as formaldehyde, glutaraldehyde, or alcohol. The antigens may be in the form of a cell lysate, created by detergent solubilization or mechanical rupture of cells or tissue, followed by clarification. The antigens may also be obtained by subcellular fractionation, particularly an enrichment of plasma membrane by techniques such as differential centrifugation, optionally followed by detergent solubilization and dialysis. Other separation techniques are also suitable, such as affinity or ion exchange chromatography of solubilized membrane proteins.

[0086] In some embodiments, the antigenic peptide or protein is an autoantigen, an alloantigen or a transplantation antigen. In other embodiments, the autoantigen is selected from the group consisting of myelin basic protein, collagen or fragments thereof, DNA, nuclear and nucleolar proteins, mitochondrial proteins and pancreatic P-cell proteins. In some embodiments, the peptide or protein is a proteolipid protein, neurofilament protein, glial fibrillary acidic protein, leptin, brain-derived neurotrophic factor, chitinase-3 -like protein 1, C-X-C motif chemokine 13, or osteopontin.

[0087] In some embodiments, the polymers of the invention are covalently coupled to antigens comprising one or more epitopes associated with allergies, autoimmune diseases, infectious diseases, and / or inflammatory diseases or disorders. The antigens may comprise one or more copies of an epitope. In some embodiments, the antigens comprise a single epitope associated with one disease or disorder. In some embodiments, the antigens comprise more than one epitope associated with the same disease or disorder. In some embodiments, the antigens comprise more than one epitope associated with different diseases or disorders. In some embodiments, the antigens comprise one or more epitopes associated with one or more allergies. In some embodiments, the antigens comprise one or more epitopes associated with multiple sclerosis, type 1 diabetes, Celiac's disease, and / or inflammatory bowel disease, including Crohn's disease or ulcerative colitis. In some embodiments, the epitopes are from myelin basic protein, proteolipid protein, myelin oligodendrocyte glycoprotein, aquaporin, myelin associated glycoprotein, aB-crystallin, insulin, glutamic acid decarboxylase, gliadin, the a3 chain of type IV collagen, or fragments, homologs, or isoforms thereof. In some embodiments, the epitopes are from gluten, including from gliadin and / or glutenin.

[0088] Scaffold Fabrication

[0089] In some embodiments, the scaffolds of the invention are made by adding a composition comprising the conjugated polymer to a solution of an unconjugated biodegradable or non-biodegradable polymer. In some embodiments, the conjugated polymer is contacted with a solution in the presence of an unconjugated polymer at predetermined mixing ratios. In some embodiments, the unconjugated polymer comprise, consist essentially of, or consist of poly(lactic acid) (PLA), polyglycolide (PGA), poly(caprolactone) (PCL), poly(lactide-co-glycolide) (PLG or PLGA), poly(vinyl alcohol) (PVA), poly(ethylene glycol) (PEG), poly(ethylene oxide), poly(ethylene oxide)-co-poly(propylene oxide) block copolymers (poloxamers, meroxapols), poloxamines, polyanhydrides, polyorthoesters, poly(hydroxy acids), polydioxanones, polycarbonates, polyaminocarbonates, poly(vinyl pyrrolidone), poly(ethyl oxazoline), carboxymethyl cellulose, hydroxyalkylated celluloses such as hydroxyethyl cellulose and methylhydroxypropyl cellulose, and natural polymers such as nucleic acids, polypeptides, polysaccharides or carbohydrates such as polysucrose, hyaluronic acid, dextran and similar derivatives thereof, heparan sulfate, chondroitin sulfate, heparin, or alginate, and proteins including without limitation gelatin, collagen, albumin, or ovalbumin, or copolymers, or blends thereof. In some embodiments, as described further herein, the unconjugated polymer is PLG (poly(lactide-co-glycolic acid)).

[0090] In some embodiments of the invention, the unconjugated polymers contain copolymers. These co-polymers may have varying molar ratio. Suitable co-polymer ratio of modified polymers may be 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 81 : 19, 82:18, 83: 17, 84: 16, 85: 15, 86: 14, 87: 13, 88: 12, 89: 11, 90: 10, 91 :9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2, 99: 1, or 100:0. As described further herein, embodiments of the present disclosure comprise a co-polymer made from D,L-lactide and glycolide in a 75:25 ratio. In another embodiment, the co-polymer may be periodical, statistical, linear, branched (including star, brush, or comb co-polymers) co-polymers. In some embodiments, the copolymers ratio may be, but not limited to, polystyrene: poly (vinyl carboxylate) / 80:20, polystyrene:poly(vinyl carboxylate) / 90: 10, poly(vinyl carboxylate):polystyrene / 80:20, poly(vinyl carboxylate):polystyrene / 90: 10, polylactic acid:polyglycolic acid / 50:50, polylactic acid:polyglycolic acid / 80:20, or polylactic acid:polyglycolic acid / 90: 10.

[0091] In some embodiments, polymers or co-polymers comprise variable molecular weights including molecular weight ranges that are considered low, medium, or high. Molecular weights ranges include all ranges produced by manufacturers and definitions of low, medium or high molecule weights are those known in the art. Ranges can include 1-200 kilodaltons (kDa). Commercial ranges can include, but not limited to, 1-5 kDa, 5-10 kDa, 1-10 kDa, 5-15 kDa, 10-15 kDa, 15-20 kDa, 10-20 kDa, 15-25 kDa, 20-25 kDa, 25-30 kDa, 30-35 kDa, 35- 40 kDa, 40-45 kDa, 45-50 kDa, 50-55 kDa, 55-60 kDa, 60-65 kDa, 65-70 kDa, 70-75 kDa, 75-80 kDa, 75-85 kDa, 80-85 kDa, 85-90 kDa, 90-95 kDa, 95-100 kDa, 100-105 kDa, 100- 110 kDa, 100-120 kDa, 100-130 kDa, 110-166 kDa, 66-107 kDa, 65-105 kDa, 23-37 kDa, 37-52 kDa, 52-69 kDa, 69-87 kDa, 87-106 kDa, 5-20 kDa, 37-84 kDa, 65-85 kDa, 76-130 kDa, 110-166 kDa. As described further herein, embodiments of the present disclosure use a low molecular weight considered at a range of 5-10 kDa and a high molecular weight considered at a range of 75-85 kDa.

[0092] In some embodiments, the conjugated polymer within a range of molecular weight is contacted with a solution in the presence of an unconjugated polymer within a range of molecular weight at predetermined mixing ratios. As described further herein, embodiments of the present disclosure determined ratios by combining low molecular weight conjugated PLG with high molecular weight unconjugated PLG. In some embodiments, ratios are determined by combining low molecular weight conjugated polymer with high, medium, or low molecular weight unconjugated polymer. In some embodiments, ratios are determined by combining medium molecular weight conjugated polymer with high, medium, or low molecular weight unconjugated polymer. In some embodiments, ratios are determined by combining high molecular weight conjugated polymer with high, medium, or low molecule unconjugated polymer. In some embodiments, the ratios are determined by a combination of different weighted conjugated polymers combined with a combination of different weighted unconjugated polymers. The scaffold could comprise 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 1-10, 10- 15, 15-25, 25-50, 50-75, 75-100% low molecule weighted polymer; 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 1-10, 10-15, 15-25, 25-50, 50-75, 75-100% medium weighted polymer; and / or 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 1-10, 10-15, 15-25, 25-50, 50-75, 75-100% high weighted polymer.

[0093] In some embodiments, the solution contacting the conjugated polymer and the unconjugated polymer may have a basic pH. Suitable basic pH for the basic solution includes 7.1, 7.5, 8.0, 8.5, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, and 13.5. The solution may also be made of any suitable base and its conjugate. In some embodiments of the invention, the solution may include, without limitation, sodium bicarbonate, potassium bicarbonate, lithium bicarbonate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, or lithium dihydrogen phosphate and conjugates thereof.

[0094] In some embodiments, the polymer scaffold can include fusing together a plurality of polymers or a plurality of microparticles or microspheres. Methods of fusing together a plurality of polymers or a plurality of microparticles or microspheres are known in the art, for example, by solvent evaporation or by subcritical CO2 sintering. In particular, the scaffolds can be formed by a solvent casting approach wherein the polymer is dissolved in a solvent and mixed with a salt, with subsequent removal of the solvent, and leaching of the salt to form a porous scaffold. Alternatively, scaffolds can be formed by mixing the polymers with a salt, pressing the mixture into a KBr die, and foaming in CO2 at 750 psi, followed by removal of the salt to form a porous scaffold. The salt used to prepare the polymer scaffold may be any water-soluble salt, including but not limited to chloride, bromide, and iodide alkali metal salts. The salt can be included in any amount to achieve the desired porosity. For example, the polymer to salt weight ratio can be in a range of about 1 :5 to about 1 : 100, about 1 :5 to about 1 :80, about 1 : 10 to about 1 :60, about 1 : 15 to about 1 :50, about 1 :20 to about 1 :40, or about 1 :25 to about 1 :35. Other solvents include, but are not limited to, chloroform, chlorinated hydrocarbons, dichloromethane, propylene carbonate, acetone, tetrahydrofuran, nonhalogenated solvents such as ethylene carbonate, dimethyl sulfoxide, dimethyl formamide, hexane, propanol, methanol, acetic acid, isoamyl alcohol, phenol, ethyl formate, ethyl acetate, or combinations thereof. In some embodiments, the precise mixture of conjugated and unconjugated polymers is fabricated into scaffolds by a solvent casting / particulate leaching technique. One embodiment dissolved a precise ration of conjugated and unconjugated polymer mixture in chloroform at about 13% w / v to create a polymer mixture with known polymer loading. Other w / v percentage concentrations that may be used include, but are not limited to, about: 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%,

[0095] 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,

[0096] 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,

[0097] 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,

[0098] 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. Other solvents include, but are not limited to, other chlorinated hydrocarbons, di chloromethane, propylene carbonate, acetone, tetrahydrofuran, nonhalogenated solvents such as ethylene carbonate, dimethyl sulfoxide, dimethyl formamide, hexane, propanol, methanol, acetic acid, isoamyl alcohol, phenol, ethyl formate, ethyl acetate, or combinations thereof. In some embodiments, the dissolved mixture of known polymer loading is mixed with sodium chloride crystals of various size ranges. One embodiment used ranges of sodium chloride crystals in the range of about 250-425 pm. In some embodiments ranges of sodium chloride crystals used may vary from about 10 pm to 700 pm, including, but not limited to ranges about 10 pm to 50 pm, 20 pm to 50 pm, 20 pm to 100 pm, 20 pm to 150 pm, 75 pm to 150 pm, 100 pm to 150 pm, 150 pm to 200 pm, 200 pm to 300 pm, 200 pm to 400 pm, 250 pm to 500 pm, 300 pm to 600 pm, 350 pm to 700 pm. In some embodiments, the casting of the scaffold mixture of known and crystals are mixed at a ratio of g NaCl per mL of polymer. One embodiment used a ratio of 3.9 g NaCl / mL of polymer. In some embodiments, the ratios of g NaCl / mL polymer is about 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1, 1.5, 2, 2.5 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and all values in between.

[0099] In one embodiment, the mixture is cast in a cylindrical mold. In some embodiments, the mixture can be casted into a variety of regular shapes, including, but not limited to sphere, cube, cuboid, cone, tetrahedron, square pyramid, hexagonal pyramid, triangular prism, octahedron, pentagonal prism, hexagonal prism, dodecahedron, ellipsoid, icosahedron. Additionally, the scaffold can be fabricated in molds that are irregularly shaped. Scaffold thickness can range from 0.5 mm to 50 cm, including 0.5 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5 mm, 5.5 mm, 6 mm, 6.5 mm, 7 mm, 7.5 mm, 8 mm, 8.5 mm, 9 mm, 9.5 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, 15 mm, 16 mm, 17 mm, 18 mm, 19 mm, 20 mm, 30 mm, 40 mm, 50 mm, 60 mm, 70 mm, 80 mm, 90 mm, 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, 10 cm, 15 cm, 20 cm, 25 cm, 30 cm, 40 cm, 50 cm, all values in between. Regular or irregular shaped molds can have a surface area of between 1 mm2to 2500 cm2. The mold may also include one that is amendable to the desired implantable space of the subject. The scaffold may be amended for implantation by biopsy, cutting, shearing, tearing, folding, heating, breaking, or some other force. The shape of the mold is not indicative of the shape of an explanted scaffold that morphed after implantation. Implanted scaffolds may change shape based on temperature and composition of the implantable space.

[0100] In some embodiments, the mold is left in the solvent for solvent evaporation for about 1-96 hours. In some embodiments, it may be left for about 8, 10, 12, 15, 20, 24, 36, 48, 60, 72, 84 or 96 hours.

[0101] In some embodiments, the scaffold structure may contain pores which are microporous or macroporous. Pore shapes and sizes may vary. Scaffold may include pore sizes that are approximately 5 pm, 10 pm, 20 pm, 25 pm, 30 pm, 40 pm, 50 pm, 60 pm, 70 pm, 75 pm, 80 pm, 90 pm, 100 pm, 150 pm, 175 pm, 200 pm, 225 pm, 250 pm, 275 pm, 300 pm, 325 pm, 350 pm, 375 pm, 400 pm, 425 pm, 450 pm, 475 pm, 500 pm, 525 pm, 550 pm, 575 pm, 600 pm, 625 pm, 650 pm, 675 pm, 700 pm, 725 pm, 750 pm, 775 pm, 800 pm, 825 pm, 850 pm, 875 pm, 900 pm, 925 pm, 950 pm, 975 pm, 1000 pm, 1250 pm, 1500 pm, 1750 pm, 2000 pm, and all values in between. The pattern of the pores is optionally homogeneous, heterogenous, aligned, repeating, or random. In some embodiments, the pores make up approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% of the scaffold volume.

[0102] In some embodiments, the carrier or a portion thereof is configured to be sufficiently porous to permit ingrowth of cells into the pores. The size of the pores may be selected for particular cell types of interest and / or for the amount of ingrowth desired.

[0103] Methods of Use

[0104] Analyte Recruitment

[0105] Embodiments of the present disclosure recruit target analytes that are direct or indirect molecular biomarkers of biological diseases of, for example, the autoimmune disease multiple sclerosis. In some embodiments of the present disclosure, target analytes are molecular biomarkers of other autoimmune diseases, inflammatory diseases, cancers, viral infections, and other pathologic diseases. In some embodiments, the target analyte is diseasespecific molecular biomarkers found in the blood, including blood’s cellular fractions, serum and plasma, urine, ejaculate and prostatic secretions, and tissue. In some embodiments, the blood molecular biomarkers targeted for recruitment consist of circulating tumor cells, cell- free circulating DNA, erythrocytes, leukocytes, platelets, fibrinogen and other clotting factors, proteins including albumins and globulins, glucose, simple and complex carbohydrates, electrolytes, lipids, nucleic acids, and hormones including compositions and components thereof.

[0106] In some embodiments, the target analyte is a cell from a commercial (e.g., immortal) cell line or a primary cell line. In some embodiments, the target cell is an immune cell. In some embodiments, the immune cell is selected from the group consisting of a T cell, a natural killer (NK) cell, an NK T cell, a macrophage, a tumor infiltrating lymphocyte (TIL), a tumor infiltrating NK cell (TINK), and a marrow infiltrating lymphocyte (MIL). In some embodiments, the cell is a stem cell. In some embodiments the cell is a hematopoietic cell. In some embodiments, the cell is an engineered cell. In certain embodiments, the engineered cell includes, but is not limited to, cells derived from adipose tissue, skin tissue, muscle tissue, blood, bone marrow, nerve tissue, liver tissue, pancreatic tissue, cartilage tissue, lung tissue, intestinal tissue, spleen tissue, lymph node tissue, ovarian tissue, testicular tissue, umbilical cord tissue, placental tissue, synthetic and biomimetic scaffolds, and other derivatives thereof. In some embodiments, the cell can be any cell type. In certain embodiments, the cell type can be prokaryotic cells, eukaryotic cells, human-specific cells, immune cells, stem cells, cancer cells, microbial cells, specialized cells, and any derivatives thereof.

[0107] As described further herein, the present disclosure provides methods of recruiting commercial DO-11.10 T-cell hybridoma line which specifically recognizes and responds to the model antigen OVA323-339 through the commercial A20 line of B cells who present the scaffold OVA323 -339 antigen and recruit the T-cells. The present disclosure provides methods of confirming T-cell activation by measuring production of cytokine IL-2 ELISA concentrations. In addition, the present disclosure provides methods of recruiting T-cells which specifically recognize and respond to the multiple sclerosis antigen PLP139-151 on splenocytes by co-culturing the splenocytes with the scaffold PLP139-151. The present disclosure provides methods of confirming the activation of the T-cells from the interactions by measuring production of cytokine IL-2 ELISA concentrations. As described further herein, the present disclosure provides methods of recruiting antigen-specific cells in vivo with OVA323-339 -loaded scaffolds that were implanted in mice. Through adoptive transfer, the mice were injected with GFP-labeled OVA-specific cells. The scaffolds were explanted after seven days and flow cytometry was used to quantify the OVA- specific cell enrichment. In some embodiments, the explantation of scaffolds can be after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50 days, followed by flow cytometry to quantify the OVA-specific cell enrichment.

[0108] As described further herein, the present disclosure provides methods of recruiting antigen-specific cells in vivo with PLPi39-isi-loaded scaffolds that were implanted in mice without impacting disease progression. Through adoptive transfer, the mice were injected with CFSE-labeled PLP-specific SJL / J splenocytes. In some embodiments, the explantation of scaffolds can be after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50 days, followed by flow cytometry to quantify the CD4+ T-cell enrichment identified by a measurement of the degree of CFSE dye dilution in CD4+ APC+ CFSE+ cell populations. Disease scoring was performed verifying a lack of impact on disease progression by the methods. In some embodiments, the quantification of T-cell enrichment is measured with a ELISpot analyzer.

[0109] In some embodiments, the target analyte is proteins, peptides, polypeptides, and components thereof. In some embodiments, recruitment of protein-based biomarkers includes particular proteins or isoforms. Furthermore, in some embodiments, the target is the post- translational modifications of proteins by phosphorylation, methylation, glycosylation, ubiquitination, acetylation, and lipidation. Proteins care cleaved by proteases and smaller peptides are cleaved by peptidases found in blood and urine. In some embodiments, the pattern of cleavage can be a biomarker of certain cancers.

[0110] In some embodiments, the target analyte is glycosylation of proteins and lipids. Different forms of protein glycosylation include N-linked (glycan attached to the nitrogen of asparagine) and O-linked (glycan attached to the oxygen of threonine and serine). In some embodiments, indicative components of the amount, size and type of glycosylation are targeted. In some embodiments, glycoproteins are targeted for urologic biomarkers including alpha-fetoprotein, prostate-specific antigen, and human chorionic gonadotropin. In some embodiments, glycolipids and glycosaminoglycans (mucopolysaccharides) are targeted for recruitment. In some embodiments, the target analyte are lipids or components thereof, including, but not limited to glycerophospholipids, glycerolipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, and polyketides.

[0111] In some embodiments the target analyte is nucleic acid. As would be recognized by one of ordinary skill in the art based on the present disclosure, the target nucleic acid can be any nucleic acid. In some embodiments, the target nucleic acid comprises RNA. In other embodiments, the target nucleic acid comprises DNA, including any derivatives or variants thereof. In some embodiments, the target nucleic acid is DNA that encodes an RNA. In some embodiments, the target analyte is a specific type of RNA including mRNA, rRNA, tRNA, snRNA, snoRNA, miRNA, siRNA, IncRNA, shRNA, or asRNA. In some embodiments, the target analytes is an enzyme that cleaves RNA including ribonuclease P or ribonuclease MRP.

[0112] Indications of Use

[0113] Embodiments of the present disclosure include detecting and monitoring of diseasespecific analytes recruited to the scaffolds over the course of disease progression. Embodiments of the present disclosure also include verification and / or monitoring of vaccine efficacy. In accordance with these embodiments, and as described further herein, the method includes implanting a scaffold within or adjacent to a target tissue to recruit desired diseasespecific analytes.

[0114] In some embodiments of the method, target tissue is tumor tissue. In some embodiments of the method, the target tissue is solid tumor tissue. In some embodiments of the method, the target tissue comprises at least one of lung tissue, bone tissue, skin tissue, breast tissue, muscle tissue, nerve tissue, brain tissue, lymph tissue, prostate tissue, bladder tissue, stomach tissue, intestinal tissue, uterine tissue, ovarian tissue, liver tissue, adipose tissue, cartilaginous tissue, thyroid tissue, and / or pancreatic tissue. In some embodiments of the method, the subject has been diagnosed with a disease or condition. In some embodiments of the method, the disease or condition comprises cancer.

[0115] In some embodiments of the method, the target analyte comprise one or more immune cells selected from the group consisting of a T cell, a B cell, a natural killer (NK) cell, an NK T cell, a macrophage, a dendritic cell, a tumor infiltrating lymphocyte (TIL), a tumor infiltrating NK cell (TINK), and a marrow infiltrating lymphocyte (MIL). In some embodiments of the method, the one or more immune cells are biomarkers of disease diagnosis, prognosis, susceptibility and risk, monitoring, predictive determinations, pharmacodynamic and treatment response, and / or safety. In certain embodiments, the individual suffers from a disorder associated with unwanted immune activation, such as autoimmune disease and inflammatory disease. An individual having an autoimmune disease or inflammatory disease is an individual with specific immune cells as biomarkers to be recruited, retained, and / or analyzed.

[0116] One embodiment of this disclosure is the individual suffers from unwanted immune activation in the autoimmune disease multiple sclerosis where autoreactive T cells orchestrate cellular and humoral effectors, which attack myelin, oligodendrocytes (ODC), and neurons. The signals for T-cell activation come from the T-cell receptors and their recognition of antigens presented by MHC molecules on the surface of antigen-presenting cells (APC) which secrete co-stimulatory molecules such as cytokines, chemokine, and eicosanoids.

[0117] In certain embodiments, the individual suffers from a disorder associated with unwanted immune activation, including, but not limited to, rheumatoid arthritis, type 1 diabetes, Sjogren syndrome, systemic lupus erythematosus, Hashimoto thyroiditis, psoriasis, Grave’s disease, inflammatory bowel disease, myasthenia gravis, celiac disease, Addison’s disease, scleroderma autoimmune vasculitis, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, giant cell myocarditis, mixed connective tissue disease, anti- NMDA receptor encephalitis, pernicious anemia, colitis, atherosclerosis, asthma, sinusitis, vasculitis, fatty liver disease, endometriosis, type 2 diabetes mellitus, obesity, Alzeheimer’s disease, Parkinson’s disease. In some embodiments, the inflammatory disease is caused by a viral infection, bacterial infection, genetic mutation, cancer or environmental exposure.

[0118] In certain embodiments, analyte recruitment is induced in the individual who suffers from a disorder associated with unwanted immune activation, such as allergic disease or condition, allergy and asthma. Analyte recruitment can be induced in such an individual, for example, by particles complexed with the specific foods (e.g. peanut proteins, etc.), injected substances (e.g. bee venom proteins, etc.), or inhaled substances (e.g. ragweed pollen proteins, pet dander proteins, etc.) which elicit the allergic reaction.

[0119] In some embodiments, reduction or elimination of chronic anti -rejection therapies may be achieved by the recruitment of specific antigens for analysis to direct treatment in an individual who is a candidate for a transplant from a non-identical twin suffering from rejection of the engrafted cells, tissues, or organs, as the engrafted antigens are foreign to the recipient. In another example, many autoimmune diseases are characterized by a cellular immune response to an endogenous or self-antigen. Recruitment of the immune system to the endogenous antigen is desirable to detect and study the specific analytes of the disease. In some embodiments, recruitment of an individual’s immunological analytes after an industrial pollutant or chemical, such as may be encountered on-the-job, presents a hazard of an immune response. Recruitment of the individual’s immunological analytes to the chemical, in particular in the form of the chemical reacted with the individual’s endogenous proteins, may be desirable to analyze, treat or prevent the occupational development of an immune response.

[0120] Notably, even in diseases where the pathogenic autoantigen is unknown, in some embodiments, the pathogenic autoantigen along with bystander suppression may be induced using a controlled ratio of antigen-loaded scaffold placed in the anatomical vicinity. For example, autoantibodies to collagen are observed in rheumatoid arthritis and, accordingly, a collagen-encoding gene may be utilized as the antigen-expressing gene module in rheumatoid arthritis (see e.g. Choy (2000) Curr Opin Investig Drugs 1 :58-62).

[0121] In other embodiments, auto-antibodies directed against myelin oligodendrocyte glycoprotein (MOG) are observed in autoimmune encephalomyelitis and in many other CNS diseases as well as multiple sclerosis (see e.g. Iglesias et al. (2001) Glia 36:22-34). Accordingly, use of MOG antigen expressing constructs in the invention allows for detection of multiple sclerosis as well as related autoimmune disorders of the central nervous system in presymptomatic stages, or in subjects with hereditary propensity for the diseases.

[0122] Still other examples of embodiments, include recruitment of individual candidate autoantigens for use in treating autoimmune disease include: pancreatic beta-cell antigens, insulin and GAD to treat insulin-dependent diabetes mellitus; collagen type 11, human cartilage gp 39 (HCgp39) and gpl30-RAPS for use in treating rheumatoid arthritis; myelin basic protein (MBP), proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG, see above) to detect multiple sclerosis; fibrillarin, and small nucleolar protein (snoRNP) to detect scleroderma; thyroid stimulating factor receptor (TSH-R) for detecting Graves' disease; nuclear antigens, histones, glycoprotein gp70 and ribosomal proteins for use in detecting systemic lupus erythematosus; pyruvate dehydrogenase dihydrolipoamide acetyltransferase (PCD-E2) for use in detecting primary biliary cirrhosis; hair follicle antigens for detecting alopecia areata; and human tropomyosin isoform 5 (hTM5) for use in detecting ulcerative colitis.

[0123] EXAMPLES

[0124] It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the present disclosure described herein are readily applicable and appreciable and may be made using suitable equivalents without departing from the scope of the present disclosure or the aspects and embodiments disclosed herein. The following examples are provided to further illustrate and clarify the detailed description of the disclosure, but the examples are merely intended to illustrate some aspects and embodiments of the disclosure and should not be conceived as limiting to the scope of this disclosure.

[0125] The precise loaded antigen-conjugated scaffold system was first developed with baseline model ovalbumin antigens and then applied to autoimmune disease-relevant peptides.

[0126] Example 1: Antigen Conjugation and Quantification

[0127] Antigen-conjugated polymers were generated by using carbodiimide crosslinking chemistry to modify 7.5 kDa carboxyl -terminated poly(lactide-co-gly colic acid) (PLG), and other low molecular weight (5-10 kDa) acid terminated PLG (PolySciTech). Modifications were generated by performing antigen conjugations with model antigens OVA323-339, OVA321- 339, and OVA257-264, and proteolipid protein, PLP139-151, a known target of autoreactive cells in multiple sclerosis (MS) (GenScript). PLG was dissolved in dimethyl sulfoxide (DMSO) at a total polymer concentration of 2% weight per volume (w / v) and set to stir. Reactions to PLG were calculated as molar ratios to PLG, denoted as “x to PLG.” l-Ethyl-3- (dimethylaminopropyl)carbodiimide (EDC, lOx to PLG) (Sigma) was dissolved in DMSO at 2% w / v and added dropwise to the stirring PLG solution. N-hydroxysuccinimide (NHS, lOx to PLG) (Sigma) was dissolved in DMSO at 1% w / v and added dropwise to the stirring PLG solution and allowed to react for 15 minutes. Then peptide antigen (2x to PLG) was dissolved in DMSO at 2% w / v. Triethylamine (TEA, 5x to PLG) (Sigma) was added dropwise to the peptide antigen solution. The peptide antigen and TEA solution was added dropwise to the stirring PLG solution and allowed to react overnight at room temperature. The resulting antigen-conjugated polymer was purified via dialysis using 3500 MW cutoff snakeskin tubing (Fisher) against 3.5 L of distilled water over 8 hours. The distilled water was replaced a total of 7 times, and the resulting purified polymer was collected, frozen, and lyophilized overnight. The EDC-NHS chemical reaction crosslinked carboxyl groups of the PLG to primary amines in the presence of OVA323-339 thereby producing PLG- OVA323-339 (FIG. 2A). The EDC-NHS chemical reaction crosslinked carboxyl groups of the PLG to primary amines in the presence of PLP139-151 thereby producing PLG- PLP139-151. Conjugations were verified and coupling efficiencies were quantified using nuclear magnetic resonance (' H-NMR) using deuterated dimethyl sulfoxide (DMSO-d6) as the solvent.

[0128] The 'H-NMR spectrum of PLG, PLP139-151, and PLG- PLP139-151 was measured in DMS0-d6 (referenced at 2.5 parts per million (ppm)). The coupling efficiency was determined by comparing the integration values of the leucine peak present at 0.8 ppm in PLP 139-151 to the methylene peak present at 5.2 ppm in PLG (FIG. 2B). In one example the method of antigen conjugation of PLG to PLP139-151 yielded a coupling efficiency of 91.14%.

[0129] The 'H-NMR spectrum of PLG, OVA323-339, PLG-OVA323-339 was measured in DMSO-d6 (referenced at 2.5 ppm). The coupling efficiency was determined by comparing the integration values of the valine and isoleucine proton peaks present at 0.8 ppm in OVA323- 339 to the methylene proton peak present at 5.2 ppm in PLG (FIG. 2C).

[0130] The 'H-NMR spectrum of PLG, OVA321-339, PLG-OVA321-339 was measured in DMSO-d6 (referenced at 2.5 ppm). The coupling efficiency was determined by comparing the integration values of the overlapping valine, leucine and isoleucine proton peaks present at 0.8 ppm in OVA32i-339to the methylene proton peak present at 5.2 ppm in PLG.

[0131] The 'H-NMR spectrum of PLG, OVA257-264, PLG-OVA257-264 was measured in DMSO-d6 (referenced at 2.5 ppm). The coupling efficiency was determined by comparing the integration values of the overlapping leucine and isoleucine proton peaks present at 0.8 ppm in OVA257-264to the methylene proton peak present at 5.2 ppm in PLG.

[0132] In one example, the method of antigen conjugation of PLG-OVA323-339 resulted in a 5.65% coupling efficiency, and the method of antigen conjugation of PLG- PLP139-151 resulted in an 81.39% coupling efficiency (FIG. 2D). In another example, the method of antigen conjugation of PLG to model ovalbumin antigens resulted in PLG-OVA323-339, PLG- OVA321-339, and PLG-OVA257 having coupling efficiencies of 97.22%, 72.06%, and 91.67% respectively (FIG. 2E).

[0133] Example 2: Antigen-Conjugated Scaffold Fabrication

[0134] Porous PLG scaffolds were fabricated using solvent casting and salt leaching techniques. Ratios of low molecular weight conjugated PLG were combined with high molecular weight (75-85 kDa) ester terminated 75:25 PLG (PolySciTech) and dissolved in chloroform at a total polymer concentration of 13% w / v. For blank scaffolds, 10% of the total PLG mass comprised low molecular weight PLG, with the rest being high molecular weight PLG. In one example, ratios of conjugated 7.5 kDa PLG made in Example 1 and 7.5 kDa / 80 kDa unconjugated polymer (PolySciTech) were dissolved in chloroform at a total polymer concentration of 13% w / v to fabricate scaffolds loaded with controlled amounts of antigen (FIG. 3 A). This polymer mixture was mixed with 250-425 pm sieved sodium chloride (NaCl) at a ratio of 3.9 g NaCl / mL PLG mixture and cast into a cylindrical mold overnight to allow for chloroform evaporation (FIG. 3 A). The resulting disks (41.5 mm diameter and 3 mm thick) were leached in MilliQ water to dissolve the NaCl, resulting in porous bulk antigen- conjugated PLG scaffolds (FIG. 3 A). Then using a 5 mm diameter biopsy punch, the bulk scaffold was punched into smaller sections to generate scaffolds sized for implantation (5 mm wide and 3 mm thick) (FIG. 3 A). High-resolution analytical SEM imaging performed with the TESCAN MIRA3 FEG-SEM analyzed the scaffolds and their pores at 46x magnification and 79x magnification (FIG. 3B-3C). Scaffolds were lyophilized, sterilized in ethanol, and stored at -80°C until use.

[0135] This scaffold manufacturing process yielded uniform, porous scaffolds with consistent porosity and pore volume, independent of the type and concentration of peptide used (FIG. 3D). The tunability of the scaffold system is demonstrated by ratiometrically combining conjugated and nonconjugated PLG to fabricate loaded scaffolds with a range of precise concentrations of antigens. Scaffolds were fabricated with a loaded range of antigen concentrations including 0.04 pg antigen / mg PLG, 0.4 pg antigen / mg PLG, 2.2 pg antigen / mg PLG, 4 pg antigen / mg PLG, 20 pg antigen / mg PLG, and 40 pg antigen / mg PLG. The total peptide loading for each antigen at each scaffold concentration was quantified using CBQCA assays known in the art. Consistently, the presence of PLG polymer did not interfere with the CBQCA assay, and the fluorescent output increased as the concentration of peptide antigen in each scaffold increased (FIG. 3E-3F).

[0136] Example 3: Antigen-Conjugated Porous Scaffold Peptide Release Kinetics Evaluation

[0137] Antigen-conjugated porous scaffolds fabricated in Example 1 and Example 2 release antigens slowly as the polymer degrades. The peptide release kinetics were evaluated with CBQCA assays. Consistent with previous findings, the signal of the CBQCA assay for OVA323-339 was low, leading to the inability to generate a release curve, but replacing it for ones that included a lysine — OVA257-264 and OVA321-339 — increased the sensitivity of the assay, allowing for peptide release detection (FIG. 4A). The higher concentration scaffolds exhibited sustained release over time, with less than 10% of antigen released after 3 weeks for both peptides. The release curve of the PLP139-151 antigen resulted in a sustained peptide release through 112 days (FIG. 4B). Similar to the model OVA peptides, the amount of PLP139-151 released increased as the antigen concentration increased and after 3 weeks, had 10% or less released for each concentration (FIG. 4C).

[0138] Example 4: In Vitro Interactions of Loaded Scaffold Co-Cultured with Cell Lines

[0139] Loaded antigen-conjugated scaffolds induce antigen presentation. To characterize these interactions between scaffolds and cells in vitro, antigen-loaded scaffolds were cocultured with antigen-specific cell lines. The DO- 11.10 T-cell hybridoma line was used, which specifically recognizes and responds to the OVA323-339 peptide. In addition, the A20 B- cell line was used to present the antigen to the T-cell (FIG. 5A). After 30,000 DO-11.10 T- cells and A20 B-cells were co-cultured with cell media, with soluble OVA in a 0.5 pg / mL concentration, and with scaffolds with antigen loadings of 40 pg PLPi39-i5i / mg PLG (negative control antigen), 0.04 pg OVA323-339 / mg PLG, 0.4 pg OVA323-339 / mg PLG, 2.2 pg OVA323-339 / mg PLG, 4 pg OVA323-339 / mg PLG, 20 pg OVA323-339 / mg PLG, 40 pg OVA323- 339 / mg PLG, for 24 hours (FIG. 5B). Upon antigen recognition and costimulatory signaling, T-cells were activated and thereby produced IL-2, a cytokine that drives T-cell proliferation. After allowing 24 hours of co-culture interactions, the samples were evaluated via ELISA (Thermo Fisher) for the presence of IL-2 (FIG. 5B). The BioTek Cytation 5 Imaging Reader (Agilent) and Microsoft Excel were used to process the samples and statistical analysis (oneway ANOVA) was completed in GraphPad Prism. The media conditioned with OVA323-339- conjugated scaffolds or soluble OVA323-339 peptide repeatedly led to significantly more IL-2 cytokine production than media conditioned with negative-control PLPi39-i5i-Conjugated scaffolds, indicating that the peptide that was released retained biological activity (FIG. 5C).

[0140] Example 5: In Vitro Interactions of Loaded Scaffold Co-Cultured with Splenocytes

[0141] Because cells cultured with soluble peptides, 4, 20, or 40 pg OVA323-339 / mg PLG- conjugated scaffolds led to significantly greater IL-2 production than control scaffolds, and low levels of stimulation were found in response to 0.04 and 0.4 pg OVA323-339 / mg PLG- conjugated scaffolds, the 0.04 and 0.4 concentrations were excluded from further studying including in vitro interaction of loaded scaffolds co-cultured with splenocytes (FIG. 6A-6B). In one example involving OVA-reactive splenocytes, CFSE-stained splenocytes from OT-II mice were co-cultured with antigen-conjugated scaffolds or soluble antigen. After 48-72 hours, cell supernatants were collected and antigen-specific proliferation of CD4+ T-cells were measured with flow cytometry via CFSE dye dilution (FIG. 6B).

[0142] In one example involving PLP139-151 splenocytes, female C57BL / 6J (Jackson) mice aged 5-6 weeks were anesthetized via inhalation of 2.5-4% isoflurane (MWI) and immunized with 4 mg / mL PLP139-151 in Complete Freund’s Adjuvant (Thermo Fisher), CFA, via subcutaneous injection. After 10 days, the mice were euthanized via CO2 asphyxiation and cervical dislocation and spleens were harvested. Spleens were passed through a 70-pm cell strainer (Thermo Fisher), and red blood cells were lysed with ACK buffer (Thermo Fisher), generating single-cell suspensions (FIG. 6C).

[0143] In one example, 1,000,000 splenocytes / well were cultured in vitro either in the presence of scaffolds loaded with different controlled concentrations of PLP139-151 or a 0.5 pg / mL soluble PLP139-151 dose for 24 hours. The different concentrations of demonstrated were 0.04 pg PLPi39-i5i / mg PLG, 0.4 pg PLPi39-i5i / mg PLG, 2.2 pg PLPi39-i5i / mg PLG, and 4 pg PLPi39-i5i / mg PLG, and after allowing 24 hours of co-culture interactions, the samples were evaluated via ELISA (Thermo Fisher) for the presence of IL-2 (FIG. 6D). The BioTek Cytation 5 Imaging Reader (Agilent) and Microsoft Excel were used to process the samples and statistical analysis (one-way ANOVA) was completed in GraphPad Prism.

[0144] In another example, antigen-specific primary cells were seeded onto PLP139-151- conjugated scaffolds where SJL / J mice were immunized to generate PLP -reactive splenocytes, followed by isolation of spleens and lymph nodes. Isolated PLP-reactive cells were co-cultures with PLP139-151 -conjugated scaffolds, soluble PLP139-151, or blank scaffolds for 24 hours. For this example, a range of PLPi39-i5i-conjugated scaffold doses were chosen based on previous results, thereby excluding doses that resulted in little stimulation or low antigen-specific cell enrichment (FIG. 6E).

[0145] Example 6: Cell-Trafficking to Implanted Scaffolds In Vivo with GFP-Labeled OVA323- 339-Specific Splenocytes

[0146] Female GFP+ mice (Jackson) aged 5-6 weeks were anesthetized via inhalation of 2.5- 4% isoflurane and immunized with 4 mg / mL OVA323-339 in Complete Freund’s Adjuvant, CFA (BD Difco), via subcutaneous injection. After 10 days, the mice were euthanized via CO2 asphyxiation and cervical dislocation and spleens were harvested. Spleens were passed through a 70-pm cell strainer (Thermo Fisher), and red blood cells were lysed with ACK buffer (Thermo Fisher), generating single-cell suspensions. RPMI 1640 media (Sigma) was prepared and supplemented to reach the final concentrations of 10% FBS, 4mM L-glutamine, lx penicillin-streptomycin, lx MEM non-essential amino acids, lx sodium pyruvate and 10 mM HEPES solutions. Splenocytes were then cultured at 2-3 million cells / cm2in the supplemented RPMI for 72 hours at 37°C in the presence of 50 pg / mL OVA323-339 and 25 ng / mL rIL-12 (Peprotech) to promote expansion of antigen-specific cell populations (FIG. 7A).

[0147] Female C57BL / 6J (Jackson) mice aged 5-6 weeks received adoptive transfers and subcutaneous scaffold implants on the same day and were monitored for 7 days after surgery to ensure health and well-being. The method of adoptive transfers of antigen-specific cells was exemplified with female C57BL / 6J (Jackson) mice aged 5-6 weeks receiving adoptive transfers of 9,000,000 cells in 200 pL of sterile PBS via lateral tail vein injection. The mice received adoptive transfers of GFP-labeled OVA323-339-specific splenocytes (FIG. 7A). The method of scaffold implantation was exemplified with female C57BL / 6J (Jackson) mice aged 5-6 weeks being anesthetized via inhalation of 2.5-4% isoflurane and administered 5 mg / kg of carprofen (MWI) subcutaneously as a preemptive analgesic. The surgeries were performed on a sterile field with sterilized surgical tools. The upper back of each mouse was shaved and sterilized for surgery with povidone iodine and ethanol wipes. After surgical preparation, each mouse received a 1 cm incision along the midline of the upper back and subcutaneous pockets for scaffold implants were made with blunt dissection. Each mouse received between 6 implants for each experiment and the skin was closed with surgical clips (Reflex 7 mm, Durect Corporation) (FIG. 7A). The mice received another dose of 5 mg / kg carprofen the day after surgery and were monitored daily for 7 days for signs of infection or pain. After 7 days, the surgical clips were removed as needed to facilitate proper tissue healing.

[0148] At the experimental endpoints, the mice were euthanized via CO2 asphyxiation and cervical dislocation and spleens and scaffolds were harvested. Spleens were passed through a 70-pm cell strainer (Thermo Fisher), and red blood cells were lysed with ACK buffer (Thermo Fisher), generating single-cell suspensions. Scaffolds were harvested, minced by hand, enzymatically degraded with 0.26 U / mL liberase (Sigma) for 30 minutes, and filtered through a 70-pm cell strainer to generate single-cell suspensions (FIG. 7A).

[0149] Cells were collected and processed for flow cytometric analysis on the Bio-Rad ZE5 Cell Analyzer. All samples were treated with Zombie Violet live / dead stain, followed by antiCD 16 / 32 to block nonspecific staining, as well as antibodies for CD45, CD4, CD8a, and CD 19 (Biolegend). After all the staining was complete, the samples were fixed in 1% paraformaldehyde for 45 minutes. The scaffold samples were then resuspended in precision counting beads to assist in normalizing between samples (Biolegend). Antigen-specific cells from donor GFP+ mice were identified as GFP+. All gating and cell population analysis was done using FlowJo software (FIG. 7B).

[0150] Example 7: Cell-Trafficking to Implanted Scaffolds In vivo with CFSE-Labeled SJL / J Splenocytes

[0151] Female SJL / J mice (Jackson) mice aged 6 weeks were anesthetized via inhalation of 2.5-4% isoflurane (MWI) and immunized with 2 mg / mL PLP139-151 in Complete Freund’s Adjuvant, CFA (BD Difco), via subcutaneous injection. After 10 days, the mice were euthanized via CO2 asphyxiation and cervical dislocation and spleens and inguinal, axillary, and brachial lymph nodes were harvested. Spleens and lymph nodes were passed through a 70-pm cell strainer (Thermo Fisher) and red blood cells were lysed with ACK buffer (Thermo Fisher) to generate a single-cell suspension. RPMI 1640 media (Sigma) was prepared and supplemented to reach the final concentrations of 10% FBS, 4mM L-glutamine, lx penicillinstreptomycin, lx MEM non-essential amino acids, lx sodium pyruvate and 10 mM HEPES solutions. Splenocytes were then cultured at 2-3 million cells / cm2in the supplemented RPMI for 72 hours at 37°C in the presence of 20 pg / mL PLP139-151 to promote expansion of PLP- specific cell populations (FIG. 8A).

[0152] After 72 hours of culturing, the SJL / J splenocytes were harvested and labeled with CFSE dye immediately prior to adoptive transfer. CFSE dye dilutions were detected via flow cytometry as a measurement of PLP-specific CD4+ T-cell proliferation (FIG. 8 A).

[0153] For EAE induction and scaffold implantation methods, on day 0, female SJL / J mice aged 8 weeks received adoptive transfers of 12,000,000 CFSE-labeled PLP-specific SJL / J splenocytes in 200 pL of sterile PBS via lateral tail vein injection (FIG. 8A). Following adoptive transfer, a subset of mice received subcutaneous scaffold implants (FIG. 8A). The implantation method included anesthetizing the mice via inhalation of 2.5-4% isoflurane and the administration of eye drops and 5 mg / kg carprofen subcutaneously as a preemptive analgesic. The surgeries were performed on a sterile field with sterile surgical tools. The upper backs of the mice were shaved and sterilized with povidone iodine and ethanol wipes. The mice then received a 1 cm incision along the midline of the upper back and tweezers were used to create subcutaneous pockets. Each mouse received a total of 6 implants, including 1 blank scaffold, 4 concentrations of PLP139-151 scaffolds, and an OVA323-339-loaded scaffold. After the scaffolds were set, the skin was closed with surgical clips (Reflex 7 mm, Durect Corporation). Another subset of mice was the control-disease group who received the same surgery prep, followed by sham surgeries with no implants. The day after surgery, mice received a second dose of 5 mg / kg carprofen. The mice were monitored daily until the experimental endpoints for signs of infection, pain, or disease. After 7 days, the surgical clips were removed as needed to facilitate proper tissue healing.

[0154] Disease scoring began on day 5, where the mice were scored daily using the established grading system for clinical assessment of EAE. The clinical scale ranges from 0 (normal mouse) to 5 (death by EAE), with intermediate scores of 1 (limp tail or hind limb weakness), 2 (both limp tail and hind limb weakness), 3 (partial hind limb paralysis), and 4 (total hind limb paralysis). For scoring, the mice were observed while walking across wire cage tops for signs of limp tails, repeated slipping between rungs, and the ability to use hind limbs for walking. The disease scores were then compared between mice with and without implants to assess any impact implants have on disease course and severity (FIG. 8B).

[0155] Tetramers were utilized to assist in fluorescent labeling PLP-specific CD4+ T-cells for analysis via flow cytometry. MHC II restricted tetramers provided by the NIH Tetramer Core Facility were used. Tetramers used included PLP139-151 and Human CLIP87-101 as a negative control.

[0156] Flow cytometric analysis was performed on the extracted scaffold and harvested spleen samples at the experimental endpoints using the Bio-Rad ZE5 Cell Analyzer. (FIG. 8A). All the samples were treated with Zombie Violet live / dead stain, followed by antiCD 16 / 32 to block nonspecific staining, as well as antibodies for CD45, CD4, CD8a, and CD 19 (Biolegend). APC-labeled PLP139-151 tetramers were included to stain the PLP -reactive CD4+ T-cells (NIH). The samples were fixed in 1% paraformaldehyde for 45 minutes, followed by resuspension in FACS buffer (all controls and spleen samples) or precision counting beads (scaffold samples) for performance on the Bio-Rad ZE5 Cell Analyzer. For analysis, the antigen-specific CD4+ T-cells were identified as CD4+ APC+ and the degree of proliferation was measured based on the degree of CFSE dye dilution in CD4+ APC+ CFSE+ cell populations (FIG. 8C). All gating and cell population analysis was done using the FlowJo software.

[0157] Example 8: Disease-Relevant CD4 T-cell Enrichment In vivo EAE Model

[0158] Female SJL / J mice aged 6 weeks were immunized with a mixture of 2 mg / mL PLP139- i5i and Complete Freund’s Adjuvant (Freund’s Incomplete Adjuvant (BD Difco) with 4mg / mL heat-killed M. tuberculosis H37 Ra (BD Difco)). Mice were injected with 33 pL of immunization solution in three subcutaneous locations — at the base of the tail and hind legs — totaling 100 pL per mouse. Following immunization, the mice were anesthetized via inhalation of 2.5-4% isoflurane (MWI) and administered ophthalmic lubricant (MWI) and 5 mg / kg carprofen (MWI) subcutaneously as a preemptive analgesic. A subset of immunized mice received subcutaneous scaffold implants. In these mice, the dorsa were shaved and prepared for surgery with povidone iodine and ethanol. Mice received an incision measuring approximately 1 cm along the midline of the dorsum and subcutaneous pockets for each of the three implants were made by blunt dissection. The incision was closed using surgical clips (Relex 7 mm, Durect Corporation). Mice received another dose of 5 mg / kg carprofen one day after surgery and were monitored daily for signs of infection or pain. For these experiments, mice that received implants received a 4 pg PLPi39-i5i / mg PLG implant, a 4 pg O VA323-339 / mg PLG implant, and one blank PLG implant. The control disease cohort of mice received sham surgeries with no scaffold implants. The mice were then clinically assessed and then further analyzed with ELISpot and flow cytometry (FIG. 9A).

[0159] Starting on day 5, mice were scored daily using the established grading system for clinical assessment of EAE. The clinical scale ranges from 0 (normal mouse) to 5 (death by EAE), with intermediate scores of 1 (limp tail or hind limb weakness), 2 (both limp tail and hind limb weakness), 3 (partial hind limb paralysis), and 4 (total hind limb paralysis). The results of the clinical disease scoring showed that the presence of antigen-conjugated scaffold implants did not impact the onset of disease (FIG. 9B).

[0160] On day 11, ELISpot analysis was used to quantify antigen-specific T-cells. Some of the mice were euthanized via CO2 asphyxiation and cervical dislocation and inguinal lymph nodes, spleens, and scaffolds were harvested and placed in PBS on ice. Inguinal lymph nodes and spleens were passed through a 70-pm cell strainer and red blood cells were lysed with ACK buffer (Thermo) to generate single-cell suspensions. Scaffolds were harvested, minced with a scalpel, enzymatically degraded with 0.26 wU / mL liberase (Sigma) for 30 minutes, and filtered through a 70-mm cell strainer to generate single-cell suspensions. PLP-specific CD4 T-cell enrichment in lymph nodes, spleens, and scaffolds were analyzed via IL-2 ELISpot (BD Biosciences) according to the manufacturer’s directions with the following specification: entire scaffold samples and 500,000 cells / mL lymph node and spleen samples were seeded into ELISpot wells and stimulated with 10 pg / mL PLP139-151 for 24 hours (FIG. 9C). Spots were enumerated in the Rogel Cancer Center Immunology Core at the University of Michigan. The number of spot-forming cells across conditions were compared to the 4 pg PLPi39-i5i / mg PLG implant using a one-way ANOVA (n = 5). The total number of IL-2 T- cell spots — representing the number of disease-relevant cells — from the ELISpot analyzer were then quantified (FIG. 9D).

[0161] On day 13, flow cytometry analysis was utilized. Mice were euthanized via CO2 asphyxiation and cervical dislocation and inguinal lymph nodes, spleens, and scaffolds were harvested and placed in PBS on ice. Inguinal lymph nodes and spleens were passed through a 70-pm cell strainer and red blood cells were lysed with ACK buffer (Thermo) to generate single-cell suspensions. Scaffolds were harvested, minced with a scalpel, enzymatically degraded with 0.26 wU / mL liberase (Sigma) for 30 minutes, and filtered through a 70-pm cell strainer to generate single-cell suspensions. Isolated cell suspensions were blocked with anti-CD16 / 32 (clone 93, Biolegend). Cells were stained with Zombie Violet Fixable Viability Kit (Biolegend), AF700 anti-CD45 (clone QA17A26, Biolegend), and PE-Cy7 anti-CD4 (clone GK1.5, Biolegend). Prepared samples were run on the Bio-Rad ZE5 Cell Analyzer in the University of Michigan Flow Cytometry Core and data was analyzed using FlowJo software. The number of CD45+ CD4+ cells across conditions were compared to the 4 pg PLPi39-i5i / mg PLG implant using a one-way ANOVA (n = 5). The total number of CD4 T-cells (CD45+ cells and CD4+ cells) that were isolated from the scaffolds, lymph nodes, and spleens were quantified (FIG. 9E).

[0162] The ratio of antigen-specific CD4 T-cells to total T-cells found in the scaffolds, lymph nodes, and spleens were calculated by dividing the number of spot-forming cells from the ELISpot analysis by the total number of CD4 T-cells from the flow cytometry analysis, resulting in the number of spots per T-cell. The number of spots per T-cell were compared to the 4 pg PLP139-151 PLG implant using a one-way ANOVA (n = 3-5). The data shows that the controlled ratios of PLP-loaded implants are recruiting a greater ratio of antigen-specific to total CD4 T-cells, resulting in successful enrichment of antigen-specific cells (FIG. 9E).

Claims

CLAIMSWhat is claimed is:

1. A method of making a conjugated scaffold, comprising: forming a synthetic scaffold with controlled ratios of conjugated monomers to unconjugated monomers.

2. The method of claim 1, wherein the conjugated monomers comprise a biodegradable polymer conjugated to a moiety.

3. The method of claim 1, wherein the conjugated monomers comprise a plurality of biodegradable polymers conjugated to two or more different moieties.

4. The method of claim 3, wherein a ratio of biodegradable polymers conjugated to a first moiety is greater than 1:1.

5. The method of claim 3, wherein a ratio of biodegradable polymers conjugated to a first moiety is less than 1:1.

6. The method of claim 2, wherein the moiety is a protein.

7. The method of claim 2, wherein the moiety is a peptide-antigen.

8. The method of claim 7, wherein the peptide-antigen is expressed or denoted in an autoimmune disease or condition (AID), inflammatory disease or condition, virus infection, cancer, or any other antigen-specific condition.

9. The method of claim 8, wherein the AID is multiple sclerosis, diabetes, rheumatoid arthritis, lupus, Crohn’s disease, or any other autoimmune disease or condition.

10. The method of claim 7, wherein the peptide-antigen is a myelin-derived antigen.

11. The method of claim 10, wherein the myelin-derived antigen is a myelin basic protein (MBP), a proteolipid protein (PLP), a myelin oligodendrocyte glycoprotein (MOG), or aB -cry stallin.

12. The method of claim 11, wherein the PLP is a proteolipid protein(139-151) (PLP 139-151).

13. The method of claim 2, wherein the conjugated biodegradable polymer comprises a polylactide acid (PLA) polymer, a poly(lactide-co-glycolide) (PLG) polymer, a poly(lactide-co-glycolide acid (PLGA), or a polycaprolactone (PCL) polymer.

14. The method of claim 2, wherein the conjugated biodegradable polymer is a poly(lactide- co-glycolide) (PLG).

15. The method of claim 14, wherein the conjugated PLG comprises a specified molecular weight conjugated by carbodiimide crosslinking.

16. The method of claim 15, wherein the specified weight is 7500 Da PLG.

17. The method of claim 1, wherein the unconjugated polymer is a biodegradable polymer comprises a polylactide acid (PLA) polymer, a poly(lactide-co-glycolide) (PLG) polymer, poly(lactide-co-glycolide acid) (PLGA) polymer, or polycaprolactone (PCL) polymer.

18. The method of claim 1, wherein the unconjugated polymer is a poly(lactide-co- glycolide)(PLG).

19. The method of claim 18, wherein the unconjugated PLG comprises a specified molecular weight.

20. The method of claim 19, wherein the specified molecular weighed unconjugated PLG is mixed with the components of claim 15.

21. The method of claim 18, wherein the PLG comprises of a mixture of PLG polymers of different molecular weights.

22. The method of claim 21, wherein the mixture is combined with the components of claim23. The method of claim 21, wherein the plurality of molecular weights is in a mixture of 7500 Da PLG and 80,000 Da PLG.

24. The method of claim 23, wherein the mixture of 7500 Da PLG and 80,000 Da PLG is combined with the components of claim 15.

25. The method of claim 23, wherein the mixture of Da PLG and 80,000 Da PLG is combined with the components of claim 16.

26. The method of claim 22, wherein the mixture is a controlled ratio configured to recruit desired biological analytes.

27. The method of claim 26, wherein the biological analytes comprise immunological cell lines.

28. The method of claim 27, wherein the immunological cell lines are expressed or denoted in an autoimmune disease or condition (AID), inflammatory disease or condition, cancer, virus infection, or any other antigen-specific condition.

29. The method of claim 28, wherein the AID is multiple sclerosis (MS), diabetes, rheumatoid arthritis, lupus, Crohn’s disease, or any other autoimmune condition.

30. The method of claim 26, wherein the mixture of controlled ratios is fabricated into porous scaffolds.

31. A conjugated scaffold made by the method of any of claims 1 to 30.

32. A method of analyzing biological diseases or conditions in a subject, comprising: implanting a preloaded conjugated scaffold of claim 31 into a subject and recruiting or retaining an analyte from said subject into said conjugated scaffold for analyte local enrichment without systemic stimulation.

33. The method of claim 32, wherein the analyte local enrichment produces a localized sample of desired analytes capable of being procured by biopsy, explantation, or methods of sample retrieval.

34. The method of claim 33, wherein the procured sample is used for detection or monitoring of diseases and conditions with an analyte-specific component.

35. The method of claim 33, wherein the procured sample is used for verification of vaccine efficacy.

36. The method of claim 32, wherein the recruited or retained analyte is a DNA, RNA, protein, T-cell, B-cell, or combination thereof.

37. The method of claim 36, wherein the T-cells, B-cells, or a combination thereof produce cytokines.

38. The method of claim 37, wherein the cytokines develop enrichment of desired analytes.

39. The method of claim 38, wherein the enrichment of desired analytes produces a localized sample of desired cells capable of being procured by biopsy, explantation, or other methods of sample retrieval.

40. The method of claim 39, wherein the procured sample is used for detection or monitoring of conditions with an analyte-specific component.

41. The method of claim 40, wherein the condition with an analyte-specific component is an antigen-specific component.

42. The method of claim 39, wherein the procured sample is used for verification of vaccine efficacy.

43. The method of claim 36, wherein the recruited or retained DNA or RNA is procured for verification of vaccine efficacy.

44. The method of claim 32, wherein the subject has an autoimmune disease or condition (AID), inflammatory disease or condition, virus infection, cancer, or any other antigenspecific condition.

45. The method of claim 44, wherein the AID is multiple sclerosis (MS), diabetes, rheumatoid arthritis, lupus, Crohn’s disease, or any other autoimmune disease or condition.

46. The method of claim 45, wherein the said MS condition has specific T-cells.

47. The method of claim 46, wherein the specific T-cells in said MS are recruited, retained, or enriched under the conditions of the preloaded scaffold comprising conjugated myelin- derived antigen.

48. The method of claim 47, wherein the myelin-derived antigen is a proteolipid protein(139- 151)(PLPi39 -151).

49. The method of claim 47, wherein the recruitment, retainment, or enrichment of MS specific T-cells by the preloaded scaffold with conjugated myelin-derived antigens is under conditions that antigen presenting cells (APCs) present the myelin-derived antigens in the context of a major histocompatibility complex (MHC) to T-cells.

50. The method of claim 47, wherein the T-cells produce a localized sample of desired cells capable of being procured by biopsy, explantation, or other methods of sample retrieval.

51. The method of claim 50, wherein the procured samples are used for detection and monitoring of conditions with an antigen-specific component.

52. The method of claim 50, wherein the procured samples are used for verification of vaccine efficacy.

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