Hydrogel bead-based platform for high throughput screening of immunomodulatory peptides and design peptides for dendritic cell / macrophage reprogramming

Abstract

The subject invention relates to a novel bead-based platform that can be used to screen various immunologically active peptides with immunogenic or tolerogenic potential and to design peptide candidates and combinations thereof. More specifically, the subject invention uses peptides specified in parametric fashion to increase their efficiency of differentiation of immune cells.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 477,558, filed on December 28, 2022, the entire contents of which (including any tables, diagrams, or drawings) are incorporated herein by reference.

[0003] About Sequence Listing

[0004] The sequence listing of this application is named "HKUS185XPCT.xml", which was created on December 21, 2023, and has 13,184 bytes. The entire content of the sequence listing is incorporated herein by reference in its entirety. Background Art

[0005] The immune system regulates the progression of diseases including cancer, tissue degeneration and regeneration, tissue transplantation, aging, and autoimmune diseases. Immunomodulatory small molecule drugs such as cyclosporine A, cyclophosphamide, levamisole, imiquimod, thiocarbamates, and penicillamine have been synthesized in the art and are widely used in clinical settings to control infection and protect immune function. [1]-[4] . Despite this, these drugs can exhibit toxicity and adverse reactions, limiting their long-term use for the treatment of chronic diseases. On the other hand, naturally derived immunomodulatory proteins or peptides show higher biocompatibility, which suggests their potential in immunotherapy, particularly as adjuvants for vaccine development against complex diseases including cancer, autoimmune diseases and degenerative diseases. With the development of emerging sequencing technologies and data science, many new antigens and epitopes have been identified for use in subunit vaccines against complex diseases. However, clinical performance is largely limited by the poor immunogenicity / tolerogenicity of the antigens and their rapid clearance from the body before inducing potent and long-lasting immune responses and memory. Therefore, antigens are often co-delivered with adjuvants to improve their immune outcomes.

[0006] Many adjuvants are used in vaccine development; they can be naturally occurring or chemically engineered. A variety of naturally occurring immunologically active sources, including proteins and peptides, have been isolated from plants and microorganisms. Additionally, some marine-derived proteins exhibit immunomodulatory properties. [5][6]-[7] These proteins are classified as antimicrobial peptides (AMPs), host defense peptides (HDPs), or innate defense regulators (IDRs) (e.g., see the global website: dbaasp.org / home). These proteins exhibit enormous structural diversity.

[0007] Immunologically active proteins and peptides can be used to develop biomaterial-based adjuvants to enhance the immunogenicity or tolerogenicity of antigens in vaccine development and regenerative medicine. Despite this, the potential of immunologically active proteins in vaccine development remains untapped. Therefore, there remains a need for additional preparations and methods that can be used to effectively study the effects of various proteins and peptides on immune cell differentiation. Summary of the Invention

[0008] The present invention relates to a new bead-based platform that can be used to screen various immunogenic or tolerogenic potential immunoreactive peptides, designed peptide candidates, or combinations thereof. In some embodiments, the platform of this theme can be used to assess phenotypic changes of innate immune cells in vitro. In some embodiments, the bead-based platform includes hydrogel microparticles (HMPs) with variable size, stiffness, and degradability. In some embodiments, these parameters are fine-tuned to study the biophysical effects of peptides on immune cell differentiation.

[0009] In some embodiments, the bead-based platform of the present subject matter can be used for high-throughput construction of peptide and peptidomimetic libraries for new drug design, synthesis, and screening. In some embodiments, the bead-based platform of the present subject matter can be a solid phase for peptide binding and immunological activity screening. In some embodiments, the bead-based platform of the present subject matter can be used as a solid phase support for peptide immobilization, enzyme and substrate storage for high-throughput screening (HTS) analysis, and antigen delivery platform in vaccine formulations.

[0010] In some embodiments, the subject bead-based platform can be used to test the effects of different combinations of immunoreactive peptides, presentation ratios, and densities on the maturation phenotype of innate immune cells. In some embodiments, the subject bead-based platform includes four core elements: control of hydrogel core preparation, control of combinatorial peptide display, compatibility with high-throughput microfluidic workflows (e.g., barcoding and reading, multiplexing and sequencing, data analysis and interpretation), and compatibility with various HTS assays (e.g., cell-based HTS assays, affinity HTS assays, or catalytic HTS assays). Figure 1 ).

[0011] In some embodiments, the subject bead-based platform can be used as a vaccine delivery vehicle and reservoir for long-term release of antigens and subunits in vivo. In some embodiments, the subject bead-based platform can measure the host immune response for immunogenic activity or tolerance to the target antigen, thereby enabling the rapid development of various vaccine formulations with greater efficacy in immune protection and memory.

[0012] Innate immune cells such as dendritic cells (DCs) and macrophages have plasticity; they can be polarized into pro-inflammatory (immunogenic / M1) and anti-inflammatory (tolerogenic / M2) phenotypes. In some embodiments, the subject bead-based platform can be used to test peptides as adjuvants for the development of vaccines against complex diseases such as cancer, autoimmune diseases, and degenerative diseases by evaluating the effects of peptides on innate immune cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] This patent or patent application file contains at least one drawing drawn in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0014] Figure 1 The four elements of the proposed high-throughput screening workflow and their respective design parameters.

[0015] Figures 2A to 2E .HMP preparation and physical characterization. Figure 2A Droplet-based microfluidic synthesis of HMPs. Figure 2B SPG film extrusion by HMP. Figure 2C Microscopic images of HMPs prepared by different methods: bulk emulsion, microfluidics, filtration through a 50 μm cell strainer, and SPG extrusion. Images were taken at 20X magnification, scale bar 50 μm. The table shows the mean particle size ± standard deviation. Figure 2D Size distribution of HMP prepared by batch emulsification, and Figure 2E Size distribution of HMPs prepared by microfluidics, SPG extrusion, and filtration.

[0016] Figures 3A to 3D . Figure 3A Interaction of JAWSII and RAW264.7 with HMPs. JAWSII was co-cultured with FITC-conjugated HMPs (green) prepared by different methods. The HMPs had sizes ranging from 10 μm, 40 μm, and 100 μm. The cells were stained with Hoechst 33342 (blue).

[0017] The HMPs were stained with lysosomal tracer-red (red). Colocalization of HMPs and lysosomes produces a yellow signal. Images were captured using a fluorescence confocal microscope at 20X magnification. Scale bar, 50 μm. Figure 3B Flow cytometry was used to analyze the expression of surface markers such as CCR7 (lymph node homing marker) and CD40 (co-stimulatory marker) of JAWSII to detect the activation state of cells. The cells were cultured with HMPs of different sizes prepared at 30% wt / V for 96 hours, and Figure 3CCells were cultured with 40 μM HMP at different polymer concentrations for 96 h (N=4, *p<0.05 compared to other groups). Figure 3D RAW264.7 macrophages were cultured with 30% wt / V HMPs of different sizes. Flow cytometry was used to detect the M1 marker CCR7 and the M2 marker CD206 (N=4, *p<0.05 compared with 40 μm).

[0018] Figures 4A to 4H .Peptide type No.1( Figure 4A )、Peptide type No.2( Figure 4B )、Peptide type No.3( Figure 4C )、Peptide type No.4( Figure 4D )、Peptide type No.5( Figure 4E )、Peptide type No.6( Figure 4F )、Peptide type No.7( Figure 4G ) and peptide type No.8( Figure 4H )'s MALDI-TOF and chemical structure.

[0019] Figure 5 Chemical structures of peptide types 9 to 19. The dipeptide / amino acid regions are shaded in blue.

[0020] Figures 6A to 6H .Peptide type 9( Figure 5 A), peptide type 10 ( Figure 6B ), peptide type 11 ( Figure 6C ), peptide type 12 ( Figure 6D ), peptide type 13 ( Figure 6E ), peptide type 14 ( Figure 6F ), peptide type 15 ( Figure 6G ) and peptide type 16 ( Figure 6H ) of MALDI-TOF.

[0021] 7A to 7D .JAWSIINF-κB (Promega, Madison, WI) reporting system responses. Figure 7A The cell viability was determined using MTT assay. Figure 7B NF-κB signaling activity was detected by luminescence generated by the catalytic activity of luciferase (N=3, *p<0.05 compared to 0 for unmodified HMP). Figures 7C to 7D Load the merged cells into the microwells.

[0022] A brief description of the sequence

[0023] SEQ ID NO: 1: mLL-37 peptide

[0024] SEQ ID NO: 2: mlDR-1002 peptide

[0025] SEQ ID NO: 3: mQ peptide

[0026] SEQ ID NO:4:mTLR2-1 peptide

[0027] SEQ ID NO:5: C-SKKKK peptide

[0028] SEQ ID NO:6: C-GGCSKKKK peptide

[0029] SEQ ID NO:7: C-RGPPP peptide

[0030] SEQ ID NO:8:LL-37 peptide

[0031] SEQ ID NO:9:W1 site primer

[0032] SEQ ID NO: 10: PE1 site primer

[0033] SEQ ID NO: 11: Poly T region

[0034] SEQ ID NO: 12: Peptide linker

[0035] SEQ ID NO: 13: Peptide linker

[0036] SEQ ID NO: 14: Peptide linker DETAILED DESCRIPTION

[0037] The subject invention provides a novel bead-based platform that can be used as a vaccine delivery vehicle. In some embodiments, the subject bead-based platform can be used in methods for screening various immunogenic or tolerogenic potential immunoreactive peptides, designing peptide candidates, or combinations thereof by evaluating phenotypic changes in innate immune cells such as dendritic cells and macrophages in vitro.

[0038] Selected definition

[0039] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "include," "comprising," "containing," "having," "with," or variations thereof are used in the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term "comprising." The transitional terms / phrases (and any grammatical variations thereof) "comprising," "including," "consisting essentially of," and "consisting of" may be used interchangeably.

[0040] The phrase "consisting essentially of indicates that the claim covers embodiments including the specified materials or steps, as well as other materials and steps that do not materially affect the basic and novel characteristics of the claim.

[0041] The term "about" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, the error resulting from which depends in part on how the value is measured, i.e., the limitations of the measurement system. In the case of compositions containing a certain amount of ingredients for which the term "about" is used, these compositions contain the ingredients in that amount with a variation (error range) of 0% to 10% (X ± 10%) around the value. In other cases, the term "about" provides a variation (error range) of 0% to 10% (X ± 10%) around a given value. Clearly, this variation represents a range of 10% above or below a given value, such as X ± 1%, X ± 2%, X ± 3%, X ± 4%, X ± 5%, X ± 6%, X ± 7%, X ± 8%, X ± 9%, or X ± 10%.

[0042] In the present disclosure, scope is stated in abbreviated form to avoid having to state in detail and describe each value and all values ​​in the scope. Where appropriate, any suitable value in the scope can be selected as the upper limit, lower limit or end value of the scope. For example, scope 0.1 to 1.0 represents and includes end value 0.1 and 1.0 and intermediate value 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and all intermediate ranges included in 0.1 to 1.0, such as 0.2 to 0.5, 0.2 to 0.8 and 0.7 to 1.0, etc. It is conceivable that, in a range, there are the values ​​of at least two significant figures, such as, scope 5 to 10 represents between 5.0 and 10.0 and all values ​​between 5.00 and 10.00, including end value. When using scope herein, the combination and sub-combination (such as, the sub-range in the disclosed scope) and specific embodiments thereof are clearly included in scope.

[0043] As used herein, the term "nucleic acid" or "polynucleotide" refers to a polymer of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and its single-stranded or double-stranded form. Unless otherwise specified, the term encompasses those nucleic acids comprising known analogs of natural nucleotides, which have a binding performance similar to the nucleic acid mentioned and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a particular nucleic acid sequence also implies the inclusion of conservatively modified variants (e.g., degenerate codon substitutions), alleles, homologous sequences, single nucleotide polymorphisms (SNPs), and complementary sequences, as well as sequences specifically indicated. The term nucleic acid can be used interchangeably with gene, cDNA, and mRNA encoded by a gene.

[0044] The term "hybridizes to" means that the two sequences are sufficiently complementary to each other to allow hybridization between the two sequences. The sequences that hybridize to each other can be completely complementary, but can also have mismatches to a certain extent. Depending on the stringency of the hybridization, about 5% to about 20% mismatches between the two complementary sequences will allow hybridization between the two sequences. Generally, high stringency conditions have higher temperatures and lower salt concentrations, while low stringency conditions have lower temperatures and higher salt concentrations. High stringency conditions for hybridization are preferred.

[0045] In this application, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids. These terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, these terms encompass amino acid chains of any length, including full-length proteins, in which the amino acid residues are linked by covalent peptide bonds.

[0046] As used herein, the term "amino acid" refers to the standard nomenclature, with amino acid residues represented by either a three-letter or single-letter code as follows: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0047] As used herein, "isolated" or "purified" compounds are substantially free of other compounds. In some embodiments, the purified compound is at least 60% by weight (dry weight) of the target compound. Preferably, the preparation is at least 75% by weight, more preferably at least 90% by weight, and most preferably at least 99% by weight of the target compound. For example, the purified compound is a compound in which the desired compound is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99% or 100% by weight by weight. Purity is measured by any suitable standard method, for example, by column chromatography, thin layer chromatography or high performance liquid chromatography (HPLC) analysis.

[0048] "Decrease" refers to a negative change of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%.

[0049] By "increase" is meant a positive change of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%.

[0050] As used herein, "innate defense regulators" or "IDRs" are synthetic immunomodulatory analogs of natural host defense peptides.

[0051] As used herein, "antimicrobial peptides," "AMPs," "host defense peptides," or "HDPs" are small peptides that inhibit or reduce the growth of microorganisms, fungi, cancer cells, or viruses.

[0052] As used herein, "innate defense regulators" or "IDRs" are synthetic immunomodulatory peptides that may be analogs of natural host defense peptides.

[0053] As used herein, "adjuvant" refers to any substance that can enhance a subject's immune response to an antigen.

[0054] As used herein, "antigen" refers to any toxin or other foreign substance that can induce an immune response in a subject.

[0055] As used herein, the term "hydrogel" refers to a substance formed when (natural or synthetic) organic polymers are cross-linked by covalent, ionic, or hydrogen bonds to form a three-dimensional open lattice structure that traps water molecules to form a gel.

[0056] "Subject" refers to an animal such as a mammal, for example, a human. The methods described herein can be used for both humans and non-human animals. In some embodiments, the subject is a mammal (e.g., an animal model of a disease), and in some embodiments, the subject is a human. The terms "subject" and "patient" can be used interchangeably. The animal can be, for example, a human, pig, horse, goat, cat, mouse, rat, dog, ape, fish, chimpanzee, orangutan, guinea pig, hamster, cow, sheep, bird, chicken, and any other vertebrate or invertebrate (e.g., Drosophila spp.).

[0057] As used herein, the terms "treatment," "treating," "palliating," or "ameliorating" (and grammatical variations of these terms) are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results, including but not limited to a therapeutic benefit. A therapeutic benefit is achieved by eradicating or ameliorating one or more physiological symptoms associated with the underlying cancer, such that improvement is observed in the patient, although the patient may still be afflicted with the cancer.

[0058] As used herein, the term "cancer" refers to the presence of cells with abnormal growth characteristics, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rates, deregulated oncogenic signaling, and certain characteristic morphological features.

[0059] The term "effective amount" or "therapeutically effective amount" refers to the amount of peptide or protein present on the hydrogel microbeads described herein that is sufficient to affect the intended application, including but not limited to disease treatment. The therapeutically effective amount will vary depending on the intended application (in vitro or in vivo) or the subject and the disease condition being treated, such as the subject's weight and age, the severity of the disease condition, the mode of administration, etc., which can be readily determined by one of ordinary skill in the art. The term also applies to an amount that induces a specific response in target cells, such as innate immune cells. The specific amount will vary depending on the specific compound selected, the dosing regimen followed, whether it is administered in combination with other compounds, the time of administration, the tissue to which it is administered, and the physical delivery system that carries the compound.

[0060] In this document, descriptions of chemical groups listed in any definition of a variant include definitions of the variant as any single group or combination of the listed groups. In this document, descriptions of embodiments of a variant or situation include those embodiments as any single embodiment or in combination with any other embodiment or portion thereof.

[0061] Any composition or method provided herein can be combined with one or more of any other compositions and methods provided herein.

[0062] Other features and advantages of the invention will be apparent from the following description of preferred embodiments of the invention, and from the claims.

[0063] All references cited herein are incorporated by reference in their entirety.

[0064] Hydrogel bead-based platform

[0065] The hydrogel of this subject is formed by providing a chemically defined and universal support for peptides or proteins. The properties of the hydrogel can be changed by changing the polymer structure, polymer molecular weight and cross-linking reaction conditions. In some embodiments, the components of the hydrogel can be provided individually or in combination in a dry form such as a powder or a solution form. When cross-linking conditions are applied, a hydrogel is formed. For example, the precursor components of the hydrogel are dissolved in a liquid such as water, and the solution temperature is increased to induce cross-linking and form a hydrogel.

[0066] In some embodiments, the bead-based hydrogel platform of the present subject matter may comprise a polysaccharide, such as, for example, cyclodextrin, alginate, dextran, or any combination thereof; or a polyether, such as, for example, polyethylene glycol. In a preferred embodiment, the hydrogel comprises dextran. In some embodiments, each hydroxyl group may be chemically modified, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more hydroxyl groups of the polysaccharide may be chemically modified. In some embodiments, the polysaccharide or polyether is modified with a variety of functional groups, such as, for example, alkyl halides, carboxylates, alcohols, epoxides, or vinyl sulfone groups (-VS), and optionally subsequently modified with DTT to produce, for example, free thiol groups (-SH); modified with carbazate; or oxidized to aldehydes. In some embodiments, the degree of modification may be determined by, for example, 1 The degree of modification can be determined by H-NMR spectroscopy and / or colorimetry (e.g., Ellman analysis). In some embodiments, the degree of modification can be from about 1% to about 50%. In some embodiments, the degree of modification can be varied by varying the reaction time and / or by varying the amount of an oxidizing agent, such as hypochlorite, hydrogen peroxide, TEMPO-NaClO-NaBr, or NaIO4. In some embodiments, about 0.3 mmol to about 6.2 mmol of an oxidizing agent can be reacted per about 1 g of polysaccharide or polyether.

[0067] In some embodiments, the modified polysaccharide can be cross-linked to form a hydrogel polymer network using hydrogel precursor A and hydrogel precursor B. In some embodiments, hydrogel precursor A and hydrogel precursor B can be mixed in a ratio of about 1:1 and 1:10, about 1:1 and about 1:9, about 1:1 and about 1:8, about 1:1 and about 1:7, about 1:1 and about 1:6, about 1:1 and about 1:5, about 1:1 and about 1:4, about 1:1 and about 1:3, about 1:1 and about 1:2, about 1:1 and about 1:1.5. In some embodiments, hydrogel precursor B and hydrogel precursor A can be mixed in a ratio of about 1:1 and 1:10, about 1:1 and about 1:9, about 1:1 and about 1:8, about 1:1 and about 1:7, about 1:1 and about 1:6, about 1:1 and about 1:5, about 1:1 and about 1:4, about 1:1 and about 1:3, about 1:1 and about 1:2, about 1:1 and about 1:1.5. In some embodiments, hydrogel precursor A may be dextran modified with vinyl sulfone, and precursor B may be dextran modified with thiol groups (Table 1; row 1), hydrogel precursor A may be dextran modified with thiol groups, and hydrogel precursor B may be dextran modified with vinyl sulfone (Table 1, row 2); hydrogel precursor A may be oxidized dextran, and hydrogel precursor B may be dextran modified with carbazate groups (Table 1; row 3).

[0068] In some embodiments, the weight / volume concentration of the polysaccharide in the hydrogel microparticles can be from about 5.0% to about 50%, from about 7.5% to about 30%, or about 15%.

[0069] In some embodiments, the modified polysaccharide can be conjugated to at least one peptide, such as an IDR peptide or a muramyl peptide. In some embodiments, the peptide is LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES; SEQ ID NO: 8) or a derivative thereof, such as FKRIVQRIKDFLR (SEQ ID NO: 1); IDR-1002 (VQRWLIVWRIRK SEQ ID NO: 2); QHREDGS (SEQ ID NO: 3); CSKKKK (SEQ ID NO: 4); SKKKK (SEQ ID NO: 5); GGCSKKKK (SEQ ID NO: 6); RGPPP (SEQ ID NO: 7); GG; GA; GV; GL; GF; GC; GD; GK; S; T; or Y.

[0070] In some embodiments, the peptides have N-terminal modifications, such as, for example, maleimidocaproate, cysteine, alkyne, azide-modified peptides, or 3-[(3-acrylamidopropyl)dimethylammonium]propionate (ac). In some embodiments, the peptides have C-terminal modifications, such as, for example, formamide, ester, alcohol, amide, alkylamide, p-nitroanilide, cyclic amide, and dye modifications. In some embodiments, the side chain hydroxyl groups of the peptides can be modified with zwitterionic moieties, such as, for example, phosphorylcholine, sulfobetaine, carboxybetaine, or phosphoserine. In some embodiments, the peptide can be modified with a linker between the peptide and the peptide N-terminal linker, such as (e.g.): a flexible linker, such as (G)n, where n is the number of glycine residues (e.g., a GG linker), and n can be from 2 to 6; a rigid linker, such as (EAAK)n (SEQ ID NO: 12), where n can be from 1 to 5, or (XP)n, where n can be from 1 to 10; a cleavable linker, such as (e.g.) a disulfide or protease-sensitive sequence, such as (e.g.) GTAR (SEQ ID NO: 13) or DRIR (SEQ ID NO: 14); or C6(NH2-C5H 10-COOH). In some embodiments, peptides can be produced by automated microwave-assisted solid phase peptide synthesis (SPPS) or classical solution phase synthesis. In some embodiments, maleimidocaproate (m) can be added to the peptide during SPPS by condensation between primary amines and carboxylates, such as, for example, Fmoc deprotected peptides. In some embodiments, a cysteine ​​residue (Cys) can be added to the peptide during SPPS to provide a free thiol or amine for binding the peptide to a free vinyl sulfone group or aldehyde, respectively. In some embodiments, a cationic linker 3-[(3-acrylamidopropyl)dimethylammonium] propionate (ac) can be added to the N-terminus of the peptide by condensation between carboxylates and primary amine groups. In some embodiments, the hydroxyl side chain of an amino acid residue, such as Ser, Tyr, or Thr, can be coupled to Wang resin, Rink amide resin, or 2-chlorotrityl chloride (CTC) resin in a solvent such as dimethylformamide (DMF), dichloromethane (DCM), N-methyl-2-pyrrolidone (NMP), or dimethylacetamide (DMAc), and then a hydroxyl protecting group (e.g., tBoc on the amine of lysine or tryptophan, tBu on the -OH group of serine, threonine, or tyrosine; Trt on cysteine, histidine, asparagine, or glutamine; or Pbf on arginine) is added to the amino acid residue in an acid and solvent such as DMF, DCM, NMP, or DMAc, followed by addition of 2-chloro-1,3,2- dioxaphospholane 2-oxide, followed by the addition of N-[3-(dimethylamino)propyl]methacrylamide dissolved in acetonitrile, to modify the amino acid residue with a zwitterionic moiety, such as, for example, phosphorylcholine or sulfobetaine modified with 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propane-1-sulfonate, 4-[[2-(methacryloyloxy)ethyl]dimethylammonium]butane-1-sulfonate, 3-[[2-(acryloyloxy)ethyl]dimethylammonium]propane-1-sulfonate, 3-[(3-methacrylamidopropyl)dimethylammonium]propane-1-sulfonate, 4-[(3-methacrylamidopropyl)dimethylammonium]butane-1-sulfonate, 3-[(3-acrylamidopropyl)dimethylammonium]propane-1-sulfonate.

[0071] Preparation method of hydrogel

[0072] In some embodiments, hydrogel microparticles can be formed using hydrogel precursors by bulk emulsion, cell filtration, Shirasu porous glass (SPG) extrusion with a pore size of about 0.1 μm to about 50 μm, and / or microfluidics.

[0073] In some embodiments, batch emulsification, cell filtration and SPG extrusion are carried out in a water-in-oil emulsion to control the size and distribution of the resulting hydrogel microparticles. In some embodiments, the continuous phase (oil phase) comprises about 75% by volume to about 98% by volume, about 80% by volume to about 98% by volume, about 85% by volume to about 98% by volume, about 90% by volume to about 98% by volume, or about 95% by volume to about 98% by volume of n-heptane, mineral oil, cyclohexane or hexane and a surfactant, such as (e.g.) Span 80, Tween 80, saponin, Triton-X, sorbitan monooleate, polyethylene glycol or any combination thereof. In some embodiments, two surfactants can be used in a ratio of about 250: 1, about 200: 1, about 150: 1, about 100: 1, about 50: 1, about 25: 1, about 10: 1, about 3: 1, about 1: 1. In a preferred embodiment, the surfactant of the continuous phase comprises about 1.5% Span 80 and about 0.5% Tween 80. In some embodiments, the discontinuous phase (aqueous phase) comprises at least one hydrogel precursor prepared in a buffer, such as phosphate buffer and / or PBS, having a controlled pH of about 5.5 to about 8.0.

[0074] In some embodiments, continuous phase is used to carry out microfluidic assisted droplet synthesis.In some embodiments, continuous phase comprises about 75 volume % to about 98 volume %, about 80 volume % to about 98 volume %, about 85 volume % to about 98 volume %, about 90 volume % to about 98 volume % or about 95 volume % to about 98 volume % of n-heptane, mineral oil, cyclohexane or hexane and surfactant, such as (as) Span 80, Tween 80, saponin, Triton-X, sorbitan monooleate, polyoxyethylene or their combination in any.In some embodiments, can use two kinds of surfactants with the ratio of about 250: 1, about 200: 1, about 150: 1, about 100: 1, about 50: 1, about 25: 1, about 10: 1, about 1: 1. In preferred embodiments, the surfactant of the continuous phase comprises about 75% to about 95%, about 80% to about 95%, about 85% to about 95%, about 90% to about 95%, or about 95% by volume of mineral oil, n-heptane, cyclohexane, or hexane and a surfactant, such as, for example, Span 80, Tween 80, or a combination thereof. In some embodiments, two surfactants can be used in a ratio of about 250:1, about 200:1, about 150:1, about 100:1, about 50:1, about 25:1, about 10:1, about 1:1 to produce hydrogel microparticles. In some embodiments, the discontinuous phase comprises at least one hydrogel precursor in a solvent.

[0075] In some embodiments, when prepared using microfluidics, the average diameter of the resulting hydrogel microparticles is about 100.0 μm to about 200.0 μm, about 114.6 μm to about 159.2 μm, or about 136.9 μm. In some embodiments, when prepared using batch emulsification, the average diameter of the resulting hydrogel microparticles is about 0.1 μm to about 200.0 μm, about 0.9 μm to about 119.5 μm, or about 136.9 μm. In some embodiments, a cell strainer with a diameter of about 1 μm to about 200 μm can be used to prepare the hydrogel microparticles. In some embodiments, when prepared using batch emulsification and then filtered using a 50 μm cell strainer, the average diameter of the resulting hydrogel microparticles is about 10 μm to about 50.0 μm, about 17 μm to about 46.4 μm, or about 31.7 μm. In some embodiments, when prepared by extrusion with an SPG membrane, the resulting hydrogel microparticles have an average diameter of about 1 μm to about 15.0 μm, about 3.3 μm to about 9.7 μm, or about 6.0 μm.

[0076] How to use

[0077] The present invention relates to a new bead-based platform that can be used to screen various immunogenic or tolerogenic potential immunoreactive peptides, designed peptide candidates, or combinations thereof. In some embodiments, the platform of this theme can be used to assess phenotypic changes of innate immune cells in vitro. In some embodiments, the bead-based platform includes hydrogel microparticles (HMPs) with variable size, stiffness, and degradability. In some embodiments, these parameters are fine-tuned to study the biophysical effects of peptides on immune cell differentiation.

[0078] In some embodiments, HMPs can be co-cultured with innate immune cells, such as, for example, immature dendritic cell lines (e.g., JAWSII) or macrophage cell lines (e.g., RAW Blue TM , InvivoGen, San Diego, CA) to study the effects of HMP size and mechanical stiffness on cell-HMP interactions. In some embodiments, the interaction between HMPs and immune cells can be visualized using probes coupled to HMPs, such as fluorescent probes (e.g., fluorescein (FITC), rhodamine, cyanine dyes, BODIPY, or naphthalimide probes). In some embodiments, lysosomal and late endosomal compartments can be visualized.

[0079] In some embodiments, the activity of NFκB, CCR7, CD40, or any combination thereof can be measured in immune cells co-cultured with HMPs. In some embodiments, a peptide has immunogenic and / or tolerogenic activity when the NFκB activity, CCR7 activity, or CD40 activity is greater than the NFκB activity, CCR7 activity, or CD40 activity of immune cells not co-cultured with HMPs. In some embodiments, the NFκB activity, CCR7 activity, or CD40 activity is about 0.01% to about 500% greater than the NFκB activity, CCR7 activity, or CD40 activity of immune cells not co-cultured with HMPs.

[0080] In some embodiments, the bead-based platform of this theme can be used for high-throughput construction of peptide and peptidomimetic libraries for new drug design, synthesis and screening. In some embodiments, the combination of peptides and peptidomimetics is combined with residual functional groups on HMP by click chemistry. In some embodiments, the peptide density can be adjusted by changing the degree of modification of dextran or any other polysaccharide or polyether precursor before HMP formation. Coupling occurs in an aqueous solvent containing an HMP suspension and a peptide mixture. In some embodiments, unreacted peptides are removed by washing with excess solvent. In some embodiments, cargo loading is carried out during the HMP synthesis process and before peptide labeling, for example, (such as) enzyme substrates, antigens, nucleic acids for barcode encoding and reading.

[0081] In some embodiments, the subject bead-based platform can be a solid phase for peptide binding and immunological activity screening. In some embodiments, the subject bead-based platform can serve as a solid support for peptide immobilization, enzyme and substrate storage for high throughput screening (HTS) analysis, and antigen delivery platform (e.g., cell-based, affinity, or catalytic) in vaccine formulations ( Figure 1 In some embodiments, for cell-based activity, HMPs are co-cultured with primary cells and reporter cell lines such as RAW264.7, which have an NF-kB luciferase reporter gene, to detect cellular activity in response to peptide ligand and receptor activity. In some embodiments, for affinity, receptor and peptide binding, affinity can be measured by immunofluorescence analysis, affinity chromatography, surface plasmon resonance, and isothermal titration calorimetry. In some embodiments, the effect of peptide mixtures coupled to HMPs on the catalytic activity of enzymes from biological samples can also be measured.

[0082] In some embodiments, the subject bead-based platform can be used to test the effects of different combinations of immunoreactive peptides, presentation ratios, and densities on the maturation phenotype of innate immune cells. In some embodiments, immunoreactive peptides and combinations thereof can be immobilized to HMPs via click chemistry. In some embodiments, the presentation ratio depends on the peptide mix; however, the density of peptide presentation depends on the degree of modification of the dextran or any other polysaccharide or polyether precursor and the residual functional groups that are coupled to the peptides after HMP formation.

[0083] In some embodiments, the HMP can be co-cultured with cells. In some embodiments, an index primer can be added to the HMP and cells. The index primer can include a barcode sequence and can also include a sequence that hybridizes with the mRNA transcript of a gene in the cell. In some embodiments, the length of the index primer can be about 20 to about 30 nucleotides. Exemplary index primers include: W1 site: AAGGCGTCACAAGCAATCACTC (SEQ ID NO: 9) and PE1 site: GAAGAGCGTCGTGTAGGGAAAGAG (SEQ ID NO: 10). In some embodiments, a PolyT region such as TTTTTTTTTTTTTTTTTTTV (SEQ ID NO: 11) can be added to the index primer to hybridize with the poly A tail of the mRNA in the cell.

[0084] In some embodiments, a barcode sequence is a short unique sequence (about 4 to about 16 nucleotides) used to link sequencing data to a corresponding sample. In some embodiments, an mRNA transcript can be reverse transcribed to produce a cDNA sequence; the cDNA sequence can be amplified by polymerase chain reaction to produce an amplified cDNA sequence; and the amplified cDNA sequence can be sequenced.

[0085] In some embodiments, the platform based on beads of this theme can be used as a vaccine delivery vehicle and storage depot, for the long-term release of antigens and subunits in vivo. In some embodiments, the platform based on beads of this theme can measure the immunogenic activity for the target antigen or the host immune response of tolerance, so that various vaccine preparations with greater effectiveness in terms of immune protection and memory can be rapidly developed. In some embodiments, hydrogel and / or HMP vaccine can be administered to the subject by subcutaneous, intramuscular or intratumoral approach. In some embodiments, HMP can be administered more than once, for example (as) at least 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more times. In some embodiments, the dosage of HMP can be spaced at intervals of about 6 hours, about 12 hours, about 1 day, about 1 week, about 2 weeks, about 1 month, about 3 months, about 6 months, about 1 year, about 2 years, about 3 years, about 5 years or about 10 years. In some embodiments, for in vivo response monitoring, humoral immunity can be measured, for example, (e.g.) antigen-specific IgG1 and / or IgG2 levels in plasma can be measured. In some embodiments, systemic cytokine levels can be monitored after vaccination, for example, (e.g.) serum IL-6, IL-1β, IFN-γ, TNF-α, IL-10, and / or BAFF.

[0086] In some embodiments, cellular immunity (e.g., T cell populations and subtype profiles) can be measured in secondary lymphoid organs (such as, for example, lymph nodes and spleen). In some embodiments, the cytolytic activity of CD8-T and NK cells can also be assessed to detect vaccine efficacy. In some embodiments, professional antigen-presenting cells, such as, for example, dendritic cells, macrophages, microglia, and / or B cells, are targeted by the HMPs of the present invention.

[0087] In some embodiments, targets of the subject immunogenic HMP vaccines include cancers, solid tumors, and infectious diseases. In some embodiments, tolerogenic HMP vaccines can be used to combat autoimmune diseases, such as, for example, type 1 diabetes, rheumatoid arthritis, or multiple sclerosis.

[0088] In some embodiments, the above scheme can provide a method for preparing a vaccine delivery vector. In some embodiments, the subject invention provides a high throughput screening (HTS) system and workflow to screen and mine natural peptide derivatives or designed peptide ligands that exhibit immunogenicity / tolerogenicity properties suitable for vaccine delivery.

[0089] Materials and methods

[0090] Control of hydrogel cores

[0091] In this paper, we used dextran (DX) to prepare the HMP hydrogel core. DX is a bacterial polysaccharide composed of α-1,6-linked D-pyranose glucose residues. DX is widely used in biomedical applications due to its biocompatibility, low production cost, and versatility in chemical modification. The hydroxyl groups can be chemically modified with various functional groups for drug delivery device development. Hydrophilic polymers are able to encapsulate biomacromolecules for controlled release. As shown in Table 1, DX was modified with various functional groups such as vinyl sulfone groups (-VS), or subsequently modified with dithiothreitol (DTT) to generate free thiol groups (-SH), modified with carbazate, or oxidized to aldehydes. The degree of modification (DM) of DX can be expressed as 1 Quantification is performed by H-NMR spectroscopy and colorimetry (e.g., Ellman analysis). DM can be controlled by varying reaction conditions. [8][9] In short, the DM of VS can be adjusted by the reaction time and the degree of oxidation obtained by the amount of NaIO4.

[0092] Table 1. Hydrogel microparticle (HMP) formulations. Degree of modification (DM) of precursor A A Greater than DM B , thus providing excess linkers for peptide immobilization. VS: vinyl sulfone, SH: thiol, Cys: cysteine, L: lysine.

[0093]

[0094]

[0095] Subsequently, the same concentration of precursor A (DM A ) and precursor B (DM B ,DM B <DM A ) were mixed in a volume ratio of 1:1 to form a hydrogel polymer network by cross-linking. B The cross-linking density of the resulting hydrogel will determine the mechanical stiffness. The unreacted functional groups of precursor A will be the linkers for subsequent peptide binding. Therefore, the peptide density on HMP is about DM A -DM B To increase the surface peptide density, we can simply increase the DM A , fixed DM B To independently tune the HMP stiffness, we can vary the polymer concentrations of both Precursor A and Precursor B while fixing their DMs.

[0096] Different preparation methods were used to form HMPs in water-in-oil emulsions to control the size and distribution, e.g. Figures 2A to 2EAs shown. For the batch emulsification method, cell filtration method, and SPG extrusion method, the continuous phase (oil phase) consisted of 98% n-heptane, 1.5% Span 80, and 0.5% Tween 80; while the discontinuous phase (aqueous phase) was a hydrogel precursor prepared in a buffer with controlled pH. In contrast, the microfluidics-assisted droplet synthesis of HMP was previously reported.

[10] The continuous phase contained 98% mineral oil and 2% Span 80. Figures 2A to 2E The PDI of HMPs prepared by microfluidics is shown to be much lower than that of batch emulsification. The diameter of HMPs prepared by microfluidics was 136.9 ± 22.3 μm (100 μm for simplicity). The HMP mixture obtained by batch emulsification was filtered through a 50 μm cell strainer to separate smaller particles, and the measured value was 31.7 ± 14.7 μm (40 μm). In contrast, the average measured value of HMPs prepared by SPG extrusion was 6.0 ± 2.7 μm (10 μm).

[0097] HMPs were co-cultured with the immature dendritic cell line JAWSII to investigate the effects of HMP size and mechanical stiffness on cell-HMP interactions. During HMP synthesis, interactions were probed with FITC-labeled polymers (green), and cells were stained with a lysosomal tracer to visualize lysosomes and late endosomal compartments (red) present during HMP engulfment. For this experiment, DM was selected. A =DM B , so that there will be no reactive VS / SH / aldehyde groups on the HMP surface, which will not produce toxicity and affect cellular responses. In our screening system design, we need to induce low immune activity of HMP peptide supports. Figure 3A As shown, HMPs in the 40 μm size range (30% wt / V) appear to induce fewer cell-gel interactions, with less induction of lysosomal signals. In contrast, 10 μm HMPs were efficiently engulfed by JAWSII on day 3 after treatment, showing more colocalization of HMPs with lysosomes (yellow). 100 μm HMPs, on the other hand, were surrounded by activated cells. Figure 3B The activation phenotypes of JAWSII, CCR7 (lymph node homing marker) and CD40 (co-stimulatory marker) are also shown. Consistently, JAWSII co-cultured with 40 μM HMP showed the lowest activation effect of both markers compared to untreated cells, 0.7-fold for CCR7 and 1.05-fold for CD40.

[0098] During the preparation process, the mechanical strength of HMP can be adjusted by the concentration of hydrogel precursor when the polymer molecular weight and DM are fixed. The stiffness increases with the increase of polymer concentration. Figure 3CAs shown, cells cultured on the stiffest HMP (30% wt / V) showed the lowest activation effect compared to HMPs at polymer concentrations of 7.5% wt / V and 15% wt / V. Furthermore, we investigated the effect of HMP size on the response of the macrophage cell line RAW264.7. Similarly, 40 μm HMPs induced neither M1 nor M2 polarization. At 40 μm and 30% wt / V polymer concentrations, DX-based HMPs appeared to have no significant effect on the in vitro responses of dendritic cells and macrophages; therefore, these physical parameters can be used to construct microbeads for subsequent peptide immobilization and screening.

[0099] Peptide selection and synthesis; control of combinatorial peptide display

[0100] Tables 2 and 3 summarize the peptides synthesized for screening. Briefly, peptide types can be categorized as IDR peptides, dipeptides, and phosphocholine amino acids. The following section describes the selection, design principles, and synthesis protocols. Linkers were chemically added to the N-terminus or side chains of the peptide / amino acid candidates for coupling to the HMP.

[0101] Table 2. Peptide candidates with N-terminus modified with maleimidocaproate (m), cysteine ​​(Cys), or 3-[(3-acrylamidopropyl)dimethylammonium]propionate (ac) (A3279; TCI chemicals, Tokyo, Japan). MW: molecular weight, pI: isoelectric point.

[0102]

[0103] Table 3. Peptide candidates having side chain hydroxyl groups modified with zwitterionic phosphorylcholine moieties. MW: molecular weight, pI: isoelectric point.

[0104]

[0105] IDR peptide synthesis and display (No. 1 to No. 8)

[0106] Several studies have reported the immunomodulatory properties of IDR peptides. LL-37 is a human AMP that exhibits chemotactic and regulatory activities on innate immune cells, particularly dendritic cells. LL-37 has been used to promote wound healing and inhibit biofilm formation.

[11] Furthermore, intranasal immunization with LL-37-adjuvanted antigen elicited potent antigen-specific humoral responses mediated by systemic IgG and mucosal IgA, as well as cytotoxic T lymphocyte stimulation.

[12]

[13] This review has identified and summarized a list of LL-37-derived peptides and analogs with therapeutic functions.

[14] In this study, we selected FKRIVQRIKDFLR (SEQ ID NO: 1) (or FK16), which is one of the shortest peptide derivatives of LL-37 and has been shown to have antibacterial and anticancer activities in other studies.

[29] .

[0107] IDR-1002 (VQRWLIVWRIRK; SEQ ID NO: 2) is a synthetic cationic peptide (pI>14) that has been shown to inhibit bacterial infection by inducing chemokines on human PBMCs and enhancing leukocyte recruitment. In fact, IDR-1002 was selected from a library of synthetic derivatives of bovine antimicrobial peptides (bactenecin) that have the greatest potency in inducing chemokine-modulating activity on innate immune cells.

[16] In contrast, Q peptide (QHREDGS; SEQ ID NO: 3) exhibited both pro-inflammatory and anti-inflammatory responses on macrophages in vitro. Furthermore, Q peptide coating on chitosan-based hydrogels also demonstrated excellent wound healing responses in a diabetic mouse model and excellent tissue repair in a myocardial infarction model.

[17]

[18] The peptide is derived from angiopoietin-1, a ligand for integrins that helps maintain the barrier function of the endothelial cell wall and promotes vascular health and remodeling.

[19] -

[21] .

[0108] On the other hand, CSKKKK (SEQ ID NO: 4) is derived from Pam3CSK4. This synthetic lipopeptide is a potent TLR2 agonist, and its peptide head modulates the extent of ligand-receptor interaction.

[22] . Here, we added a C6 or Gly-Gly linker between the HMP and the N-terminus of the peptide to investigate the effect of the flexibility of the receptor-ligand interaction on cellular responses. RGPPP (SEQ ID NO:7) was isolated from the protein lysate of Pseudostellaria heterophylla, a plant used in traditional Chinese medicine. This herbal extract exhibited immunostimulatory activity and anti-tumor responses. In particular, the RGPPP (SEQ ID NO:7) peptide exhibited potent immunostimulatory responses in inducing TNF-α production in macrophages and upregulating TLR2 expression.

[0109] In this study, peptide types No. 1 to No. 8 were produced by automated microwave-assisted solid phase peptide synthesis (SPPS). Rink amide resin was used and HBTU / HOBt / DIPEA was used as a catalyst. In peptide types No. 1 to No. 4, maleimidohexanoate (m) was added to the Fmoc-deprotected peptide during SPPS via condensation between a primary amine and a carboxylate. The maleimide-terminated peptides were linked to HMPs with free thiols via efficient Michael addition. In contrast, in peptide types 9 to 6, a cysteine ​​residue (Cys) was added to the N-terminus of the peptide candidates during solid phase synthesis to obtain free thiols and amines for binding to HMPs with free VS or aldehydes, respectively. The final product was cleaved from the resin with TFA / EDT (95 / 5) and precipitated in cold ether. MALDI-TOF of peptide types such as Figures 4A to 4H shown.

[0110] Dipeptide Synthesis and Display (No.9 to No.16)

[0111] Muramyl dipeptide (MDP) is derived from the bacterial peptidoglycan cell wall. MDP is the smallest structural component that still retains immunostimulatory activity. MDP consists of a sugar unit linked to two amino acids. A typical MDP is made of N-acetylmuramic acid, the lactic acid moiety of which is linked to the N-terminus of a dipeptide of L-alanine and D-isoglutamine. MDP is a ligand for TLR2 and NOD-like receptors (NRs).

[23] MDP has been used as an adjuvant in several mucosal vaccine formulations.

[24] Several dipeptide variants have been introduced to improve adjuvant activity. In particular, studies have shown that replacing L-alanine with L-serine or D-glutamic acid with D-isoglutamine exhibits higher immunostimulatory activity.

[25] .

[0112] In this paper, we aimed to screen different combinations of dipeptides for immunomodulatory activity using an HMP-based screening platform. The dipeptide library was synthesized by SPPS (parallel synthesis) and the reactions are summarized in Scheme 1. The general structure of the dipeptide types of the first generation library is aG-X, as shown in Table 1. In our structural design, a cationic linker 3-[(3-acrylamidopropyl)dimethylammonium]propionate (ac) (a) was added to the N-terminus of the dipeptide via condensation between carboxylate and primary amine groups. The quaternary amine group of ac was used to improve cell-gel interactions and provide flexibility for ligand-receptor interactions, while the acrylamide group was used to bind to the HMP via thiol-acrylamide Michael addition. The N-terminal amino acid was fixed with Gly (as a simple analog of Ala), while the C-terminal amino acid was selected from Gly, Ala, Val, Leu, Phe, Cys, Asp or Lys (No. 9 to No. 16). The chemical structures of all aGX types are shown in Table 1. Figure 5 As shown. MALDI-TOF of dipeptide Figures 6A to 6H shown.

[0113] Scheme 1: Synthetic routes of peptide types 9 to 16.

[0114] The first amino acid variant (AA) was coupled to Fmoc-deprotected Wang resin on an automated microwave-assisted SPPS (4 equivalents of protected AA, 4 equivalents of 0.6 M HBTU, 0.5 M HOBt, 8 equivalents of 2 M DIPEA in DMF). 1. In a parallel synthesis, in a separate vial, Gly (G) was coupled to the Fmoc-deprotected AA using 4 equivalents of (Fmoc)-Gly-OH, 4 equivalents of 0.6 M HBTU, 0.5 M HOBt, 8 equivalents of 2 M DIPEA in DMF at room temperature overnight. 2. Fmoc deprotection was performed with 20% v / v piperidine in DMF at room temperature for 2 hours. The resin was washed with copious amounts of DMF (4 times) and DCM (4 times). 3. 4 equivalents of A3279 (3-[(3-acrylamidopropyl)dimethylammonium]propionate (ac)), 4 equivalents of DCC / HOBt in DMF at room temperature overnight. The resin was washed with DMF, DCM and methanol. 4. The final product was decoupled with TFA / TIPS / H2O (95 / 2.5 / 2.5) at room temperature for 2 hours and precipitated in cold ether / DCM.

[0115]

[0116] Scheme 2: Synthetic routes of peptide types 17 to 19.

[0117] 1. Bind the first amino acid variant (AA) to Rink amide resin (4 equivalents of protected AA, 4 equivalents of 0.6 M HBTU, 0.5 M HOBt, and 8 equivalents of 2 M DIPEA in DMF) using automated microwave-assisted SPPS. 2. Deprotect the tert-butyl group with H3PO4 (85% in water) in DMF at room temperature for 12 hours. Wash the resin with excess DMF. 3. Dilute 4 equivalents of 2-chloro-1,3,2-dioxaphospholane 2-oxide and 0.5 equivalents of triethylamine in ice-cold DMF and add dropwise to the resin reaction mixture over 30 minutes on ice. Stir at room temperature for 3 hours. 4. Dissolve 4 equivalents of N-[3-(dimethylamino)propyl]methacrylamide in acetonitrile and add to the reaction mixture. Stir overnight at 55°C in a silicone oil bath. Wash the resin with DMF, DCM, and then methanol. 5. The final product was cleaved with TFA / TIPS / H2O (95 / 2.5 / 2.5) and precipitated with cold ether / DCM.

[0118]

[0119]

[0120] All patents, patent applications, provisional applications, and publications referenced or cited herein, including all figures and tables thereof, are incorporated herein by reference in their entirety to the extent they are not inconsistent with the explicit teachings of this specification.

[0121] The following are examples illustrating the methods of practicing the present application. These examples should not be construed as limiting. Unless otherwise indicated, all percentages are by weight, and all solvent mixtures are by volume.

[0122] Example 1 - De novo synthesis of phosphocholine-amino acids (NPC-X, NO.17 to NO.19)

[0123] Lipoamino acids are commonly found in bacteria and algae. Their general structure consists of a hydrophobic hydrocarbon tail and a hydrophilic amino acid portion, usually Ser, Lys, Gly, or Orn.

[26] A library of synthetic lipoamino acid analogs has been synthesized and used as a nucleic acid delivery vehicle for targeted gene silencing in hepatocytes in vivo without reporting of severe systemic immune-related adverse events

[27] .

[0124] Nevertheless, it has been shown that lipoamino acids can induce different immune responses in vitro and in vivo. Cationic lipoamino acids are potential adjuvants for antigen / epitope vaccination. Compared with native peptides, the conjugation of lipid-oligomeric Lys to various antigenic peptides at the C-terminus greatly enhanced the epitope-specific humoral response.

[28] . In contrast, phosphatidylserine in mammalian cells has been associated with anti-inflammatory responses. During apoptosis, the native lipid appears on the plasma membrane and acts as an “eat me” signal to promote the phagocytic clearance of dead cells by macrophages while maintaining immune tolerance to self-antigens. Furthermore, in vitro results from studies constructing zwitterionic phosphatidylserine de novo showed that encapsulating hydrogels with zwitterionic entities can produce an anti-fouling response in terms of inhibiting protein and macrophage adhesion. Furthermore, zwitterionic lipoamino acids were able to inhibit the immunogenicity of uricase more effectively in vivo than unmodified lipoamino acids.

[29] .

[0125] We constructed zwitterionic phosphorylcholine-functionalized amino acids and screened them for immunomodulatory activity on a DX-based HMP support system. The reaction is summarized in Scheme 2. Briefly, amino acids containing hydroxyl side chains, Ser, Tyr, and Thr, were coupled to Wang resin in DMF. The hydroxyl protecting group, t-Bu, was then reacted with phosphoric acid (~85 vol%) in DMF at room temperature for 12 hours, as previously reported. The resin was washed with copious amounts of DMF, followed by the dropwise addition of triethylamine (0.5 equiv) and 2-chloro-1,3,2-dioxaphospholane 2-oxide (4 equiv, based on the hydroxyl groups on the amino acid) diluted in DMF. The reaction was maintained in an ice bath for 30 minutes and then stirred at room temperature for 3 hours. The resin was washed with copious amounts of DMF to remove excess soluble reactants, followed by the addition of N-[3-(dimethylamino)propyl]methacrylamide (4 equiv) dissolved in acetonitrile. The reaction proceeded in a silicone oil bath at 55°C overnight. Finally, the resin was washed with DMF, DCM, and then methanol. The final product (npc-X; where X represents an amino acid Ser, Tyr, or Thr) was cleaved with TFA / TIPS / H2O (95 / 2.5 / 2.5) and precipitated with cold ether / DCM. The chemical structure of npc-X is shown in Figure 5 shown.

[0126] Example 2 - Compatibility with (cell-based) high-throughput screening assays

[0127] DC and macrophage reporter cell lines were used to screen for NFκB signaling activity. NF-κB is a transcription factor that regulates a large number of genes involved in proinflammatory responses. Many PRRs, such as TLRs and CLRs, are conserved activators of NF-κB signaling. Therefore, we can detect and measure NFκB activity using a reporter system to infer peptide-receptor interactions (synergy, antagonism, additivity, or ineffective response). Figure 2BAs shown, DX HMPs (40 μM, 30% wt / v) exhibit low levels of immunogenic activation in JAWSII, which can be used to determine the baseline for M1 / M2 or immunogenicity / tolerogenicity induction in macrophages or JAWSII, respectively. For example, immunogenic or M1-polarizing peptides induce higher NFκB activity than naked HMPs. In contrast, tolerogenic or M2-polarizing peptides suppress background NFκB activity. The potent TLR4 agonists LPS and / or CpG (TLR9) will serve as positive controls for M1 / immunogenicity induction, while IL-10 will serve as a negative control for M2 / tolerogenicity induction.

[0128] RAW Blue TM This is a commercially available reporter cell line. In the presence of PRR agonists (except TLR5 and NOD1), endogenous production of NFκB and AP-1 can induce the reporter system to produce SEAP. TM SEAP levels were monitored by colorimetric analysis. JAWSII cells were transfected with the reporter system pNL3.2.NF-κB-RE [NlucP / NF-κB-RE / Hygro] (Promega, USA) to generate a DC reporter cell line. The cells were cultured for 72 hours and then treated with 10 ng / mL of the dipeptide candidates (No. 9 to No. 16). NFκB signaling activity can be reflected by luminescence due to luciferase enzymatic activity.

[0129] like Figure 7A As shown in Figure 2, the HMP-dipeptide conjugate exhibited good biocompatibility, with a survival rate of >80% as determined by MTT assay. Figure 7B We also observed that the dipeptide types could significantly induce NFκB activity in JAWSII compared to blank HMP (0) and untreated cells (NT). Type 9 (ac-GG) and type 14 (ac-GC) (10 ng / mL) could induce NF-κB activity by ~2-fold, which was comparable to 100 ng / mL LPS treatment as a positive control. In contrast, the remaining dipeptide candidates could induce NF-κB activity by >2-fold. In particular, dipeptide 13 (ac-GF) and dipeptide 16 (ac-GK) showed the highest response, indicating strong immunogenic adjuvant activity against JAWSII.

[0130] Example 3 - Compatibility with high-throughput screening workflows and barcoding

[0131] Both biomaterials and vaccine adjuvant development are rapidly evolving fields. To keep pace, researchers have employed high-throughput screening (HTS) systems to identify substrates and culture conditions suitable for the maturation phenotype of DCs and macrophages. Libraries of materials and adjuvants can be analyzed in vitro for their biological activity, in this case, their propensity to induce a tolerogenic / M2 or immunogenic / M1 phenotype. A major advantage of HTS is that it accelerates the biocharacterization of materials, guiding their design and optimization in an unbiased manner. Furthermore, HTS allows us to expand our analysis from a single readout of NFκB activity to a broad analysis of gene activity through sequencing and transcriptomics.

[0132] In our study design, solid-phase HMP-immobilized peptide candidates were well suited for HTS analysis. The workflow consisted of four main modules: 1) HMP-peptide library barcoding ("catalog indexing"), 2) associating barcodes with cellular responses, i.e., gene products (mRNA) ("barcode generation and readout"), 3) combining barcoded gene products for multiplex sequencing, and 4) data analysis and interpretation.

[0133] In the proposed workflow, beads are loaded into 96- or 384-well plates containing cells for parallel cellular analysis of various HMP-peptides. After treatment, cells are fixed for subsequent in-cell reverse transcription (RT), which has been optimized in other studies.

[29] We barcoded the mRNA products of well-specific cells with unique index primers corresponding to a given HMP-peptide perturbation at room temperature in the cell. To achieve this, unique index primers were loaded into the corresponding HMP-peptide during HMP preparation and released upon induced hydrogel degradation, as previously reported in other studies.

[30] The cells were combined and loaded into microwells (eg Figures 7C to 7D (as shown). The diameter and depth of the microwells are optimized to control cell loading, such as the number of cells per well and the number of wells containing cells. In addition, low-speed centrifugation is used to improve cell loading efficiency. Barcoded (complementary DNA-labeled) microbeads are then inoculated into each well to capture / call the corresponding barcoded RT product during cell lysis and DNA-DNA hybridization. The captured cDNA is then amplified by PCR. The PCR products are combined and sequenced. After sequencing, the transcriptome of a single cell is assembled by combining reads containing the same barcode combination.

[0134] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes based on the foregoing will occur to those skilled in the art, and that such modifications or changes will be included within the spirit and scope of this application and the scope of the appended claims. In addition, any element or limitation of any invention or embodiment thereof disclosed herein may be combined with any and / or all other elements or limitations disclosed herein (alone or in any combination) or any other invention or embodiment thereof, and all such combinations are included within the scope of the present invention and are not limited thereto.

[0135] Exemplary embodiments

[0136] Embodiment 1. A hydrogel microparticle comprising at least one polysaccharide and at least one peptide, wherein at least one peptide comprises an N-terminal linker or a side chain linker.

[0137] Embodiment 2. The hydrogel microparticles of embodiment 1, wherein the at least one polysaccharide is dextran.

[0138] Embodiment 3. The hydrogel microparticles of embodiment 1, wherein at least one peptide comprises the amino acid sequence FKRIVQRIKDFLR (SEQ ID NO: 1), VQRWLIVWRIRK (SEQ ID NO: 2), QHREDGS (SEQ ID NO: 3), CSKKKK (SEQ ID NO: 4), SKKKK (SEQ ID NO: 5), GGCSKKKK (SEQ ID NO: 6), RGPPP (SEQ ID NO: 7), GG, GA, GV, GL, GF, GC, GD, GK, S, T, or Y.

[0139] Embodiment 4. The hydrogel microparticles of embodiment 1, wherein the hydrogel microparticles have an average diameter of about 0.1 μm to about 200 μm.

[0140] Embodiment 5. The hydrogel microparticles according to embodiment 1, wherein the N-terminal linker is maleimidocaproate, cysteine, or 3-[(3-acrylamidopropyl)dimethylammonium]propionate.

[0141] Embodiment 6. The hydrogel microparticle of embodiment 1, wherein the side chain linker is a zwitterionic phosphorylcholine moiety.

[0142] Embodiment 7. The hydrogel microparticle of embodiment 1, wherein at least one peptide comprises an N-terminal linker and a linker between peptides.

[0143] Embodiment 8. The hydrogel microparticles according to embodiment 7, wherein the linker is C6 or Gly-Gly.

[0144] Embodiment 9. The hydrogel microparticles of embodiment 1, wherein the concentration of the at least one polysaccharide in the hydrogel microparticles is from about 5.0% to about 50%.

[0145] Embodiment 10. The hydrogel microparticles according to embodiment 1, further comprising a fluorescent probe.

[0146] Embodiment 11. The hydrogel microparticles according to embodiment 1, wherein the fluorescent probe is fluorescein.

[0147] Embodiment 12. A method for screening peptides having immunogenic activity or tolerogenic activity, the method comprising:

[0148] a) co-culturing the hydrogel described in embodiment 1 with immune cells;

[0149] b) measuring NFκb activity, CCR7 activity, CD40 activity, or any combination thereof of immune cells; and

[0150] c) The peptide is confirmed to have immunogenic activity when the NFκB activity, CCR7 activity or CD40 activity is at least about 0.01% to about 500% higher than the NFκB activity, CCR7 activity or CD40 activity of immune cells not co-cultured with the hydrogel of embodiment 1.

[0151] Embodiment 13. The method according to embodiment 12, wherein the immune cell is a dendritic cell line or a macrophage cell line.

[0152] Embodiment 14. A method for associating a barcode with gene expression in a cell, the method comprising:

[0153] a) co-culturing the hydrogel described in embodiment 1 with cells;

[0154] b) loading the hydrogel and cells with index primers, wherein the index primers comprise barcode sequences and hybridize to mRNA transcripts of genes in the cells;

[0155] c) reverse transcribing the mRNA transcript to generate a cDNA sequence;

[0156] d) amplifying the cDNA sequence to produce an amplified cDNA sequence; and

[0157] e) Sequencing the amplified cDNA sequence

[0158] References

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Claims

1. A hydrogel microparticle comprising at least one polysaccharide and at least one peptide, wherein the at least one peptide comprises an N-terminal linker or a side chain linker.

2. The hydrogel microparticles of claim 1, wherein the at least one polysaccharide is dextran.

3. The hydrogel microparticles of claim 1 , wherein the at least one peptide comprises the amino acid sequence FKRIVQRIKDFLR (SEQ ID NO: 1), VQRWLIVWRIRK (SEQ ID NO: 2), QHREDGS (SEQ ID NO: 3), CSKKKK (SEQ ID NO: 4), SKKKK (SEQ ID NO: 5), GGCSKKKK (SEQ ID NO: 6), RGPPP (SEQ ID NO: 7), GG, GA, GV, GL, GF, GC, GD, GK, S, T, or Y.

4. The hydrogel microparticles of claim 1, wherein the hydrogel microparticles have an average diameter of about 0.1 μm to about 200 μm. The hydrogel microparticles according to claim 1 , wherein the N-terminal linker is maleimidocaproate, cysteine, or 3-[(3-acrylamidopropyl)dimethylammonium]propionate.

6. The hydrogel microparticles of claim 1, wherein the side chain linker is a zwitterionic phosphorylcholine moiety.

7. The hydrogel microparticle of claim 1, wherein the at least one peptide comprises a linker between the N-terminal linker and the peptide. The hydrogel microparticles according to claim 7 , wherein the linker is C6 or Gly-Gly.

9. The hydrogel microparticles of claim 1, wherein the at least one polysaccharide is present in the hydrogel microparticles at a concentration of about 5.0% to about 50%.

10. The hydrogel microparticles according to claim 1, further comprising a fluorescent probe. The hydrogel microparticles according to claim 1 , wherein the fluorescent probe is fluorescein.

12. A method for screening a peptide having immunogenic activity or tolerogenic activity, the method comprising: a) co-culturing the hydrogel according to claim 1 with immune cells; b) measuring the NFκb activity, CCR7 activity, CD40 activity or any combination thereof of the immune cells; and c) when the NFκB activity, CCR7 activity or CD40 activity is at least about 0.01% to about 500% higher than the NFκB activity, CCR7 activity or CD40 activity of immune cells not co-cultured with the hydrogel of claim 1, the peptide is confirmed to have immunogenic activity. The method according to claim 12 , wherein the immune cell is a dendritic cell line or a macrophage cell line.

14. A method for associating a barcode with gene expression in a cell, the method comprising: a) co-culturing the hydrogel according to claim 1 with cells; b) loading the hydrogel and the cells with index primers, wherein the index primers comprise a barcode sequence and hybridize to mRNA transcripts of genes in the cells; c) reverse transcribing the mRNA transcript to generate a cDNA sequence; d) amplifying the cDNA sequence to produce an amplified cDNA sequence; as well as e) sequencing the amplified cDNA sequence.

Images