HK1189238A - Erythrocyte-binding therapeutics - Google Patents

Description

Erythrocyte-binding therapeutic agent

Cross reference to related applications

This application claims priority from U.S. provisional application No.61/372,181, filed on 8/10 2010, and is incorporated herein by reference.

Technical Field

The technical field relates to medical compositions and uses of ligands or antibodies that bind red blood cells. Specific uses include immune tolerance (immunotolerisation), drug delivery, and cancer therapy.

Background

The clinical success or failure of a therapeutic drug can be predicted by its efficacy in affecting target tissues and organs, as well as its feasible delivery model. The optimal drug delivery platform is one that delivers and maintains a therapeutic agent payload at an optimal working concentration and delivers it to an optimal working cellular target while minimizing patient and professional caregiver intervention.

Summary of The Invention

Peptides have been found that specifically bind red blood cells (also known as erythrocytes). These peptide ligands bind specifically to erythrocytes even in the presence of other factors present in the blood. These ligands can be used in a variety of ways. One embodiment involves forming a molecular fusion of the ligand and the therapeutic agent. The ligand binds to red blood cells in the body, whereby the therapeutic agent attaches to and circulates with the red blood cells. Red blood cells circulate in the bloodstream for a long period of time, about 90 to 120 days in humans, and they approach many body compartments involved in disease (such as tumor vascular beds) and physiology (such as the liver and spleen). These characteristics can be exploited to make red blood cells useful for therapeutic agent delivery, e.g., for + extending the circulation of therapeutic agents in the blood.

Furthermore, unexpectedly and surprisingly, it has been found that these erythrocyte affinity ligands, or the approximating (able) antibodies, can be used to generate immune tolerance. In this embodiment, a molecular fusion is generated comprising a tolerogenic antigen and an erythrocyte affinity ligand. The fusion is injected or otherwise administered in sufficient quantity until tolerance is observed. In contrast, previous reports reported that attachment of antigens to the surface of erythrocytes produced immunological rejection.

Embodiments are also directed to treating cancer by embolizing (embolizing) tumors. Many antigens of tumors and/or tumor microvasculature are known. Antibodies that specifically bind to these antigens can be readily generated. Such tumor binding ligands are fused to ligands that bind red blood cells, i.e., antibody (or fragment thereof) or peptide ligand molecules. These fusions bind at the tumor site and also bind red blood cells, resulting in a block of blood supply to the tumor. These embodiments and others are described herein.

Brief Description of Drawings

FIG. 1 is a scatter plot of flow cytometry analysis of red blood cell binding of ERY1 phage.

FIG. 2 is a photographic mosaic of affinity pull-down (affinity pull-down) with soluble biotinylated ERY1 peptide: in panel a: streptavidin-hrppantern blots of eluted samples using ERY1 and mismatched peptides; in panel B; western blot against mouse GYPA using eluted samples of ERY1 peptide compared to red blood cell whole lysate.

FIG. 3 is a diagram of cell-bound groups.

FIG. 4 is a semilogarithmic graph of intravenous bolus of ERY1-MBP showing plasma MBP concentration and concentration versus time following intravenous administration of ERY1-MBP compared to MBP.

FIG. 5 is a semilogarithmic graph of subcutaneous bolus injection of ERY1-MBP showing plasma MBP concentration following subcutaneous administration; and concentration-time comparison of MBP to ERY 1-MBP.

FIG. 6 is a schematic of scFv engineering design; in panel a: linear representation of each scFv domain from N to C-terminus; in panel B: construction of a folded scFv; in panel C: construction of a folded scFv with a chemically conjugated ERY1 peptide. FIG. 6 includes the linker sequence GGGGS (SEQ ID NO:18) repeated four times.

FIG. 7 is a mosaic of histograms showing the percentage of cells bound to bacteria as determined by flow cytometry; in panel (a), peptides on the bacterial surface bound erythrocytes, but not epithelial 293T or endothelial HUVEC, except ERY 50; in panel (B), the peptides bound to various human samples, but not to mouse blood.

FIG. 8 shows experimental protocols and results for molecular fusions of ERY1 and Ovalbumin (OVA), where the ERY1-OVA fusion binds with high affinity to the equatorial periphery of mouse red blood cells; panel (a): schematic representation of conjugation of ERY1 peptide to Ovalbumin (OVA), resulting in binding to erythrocyte surface glycophorin-a; panel (b): binding of each OVA conjugate to an intermediate, characterized by flow cytometry; black solid histogram, ERY 1-OVA; hollow histogram, SMCC-OVA; dashed histogram, MIS-OVA; ERY1= erythrocyte-binding peptide WMVLPWLPGTLD (SEQ ID NO:1), MIS = mismatch peptide PLLTVGMDLWPW (SEQ ID NO:2), SMCC = succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid, for conjugating ERY1 to OVA; panel (c): ERY1-OVA pairEquilibrium binding of erythrocytes showing a low dissociation constant (R) for ERY1-OVA as determined by flow cytometry²=0.97, single site binding).

Fig. 9 shows the experimental protocol and results of binding and cycling of molecular fusions of antigen conjugated with ERY 1: the fusion, upon intravenous administration, biospecifically binds to circulating healthy red blood cells and to apoptotic (erythrocytic) red blood cells, inducing uptake by a specific subpopulation of antigen presenting cells; panel (a) OVA (grey solid histogram) and ERY1-OVA (black solid histogram) determined by flow cytometry were treated in vivo on red blood cells (CD 45) compared to non-injected mice (open histogram)^-) Population binding to leukocytes (CD 45)⁺) The population does not bind; panel (b): ERY1-OVA bound but OVA did not bind to apoptosis in circulating erythrocytes as determined by flow cytometry (annexin-V)⁺) Erythrocytes and healthy (annexin-V)^-) Red blood cells; panel (c): cell surface half-life of ERY1-OVA bound to circulating erythrocytes as determined by flow cytometry measurement of geometric mean fluorescence intensity (n =2, R)²=0.98, single-phase exponential decay); panel (d): time-dependent ERY1-OVA cell surface concentration at the administered dose of 150 μ g (n =2) was determined by ELISA.

FIG. 10 is a mosaic of several figures showing that erythrocyte binding does not alter hematological behavior; panel (a) hematocrit; panel (b) mean corpuscular volume, and panel (c) red blood cell hemoglobin content measured at different time points after administration of 10 μ g OVA (open circles) or ERY1-OVA (closed circles).

FIG. 11 is a bar graph of the results, where the ERY1 conjugated antigen biospecifically induced uptake of a specific subset of antigen presenting cells: panel (a) MHCII at 12 and 36 hours post injection compared to MIS-allophycocyanin⁺ CD11b^- CD11c⁺And MHCII⁺ CD8α⁺ CD11c⁺ CD205⁺Increased cellular uptake of ERY 1-allophycocyanin by splenic Dendritic Cells (DCs); panel (b) hepatocytes 36 hours post intravenous administration compared to MIS-allophycocyanin (CD 45)^- MHCII^- CD1d^-) And hepatic stellate cell (CD 45)^- MHCII⁺CD1d⁺) Rather than hepatic DC (CD 45)⁺ CD11c⁺) Or Kupffer cell (CD 45)⁺ MHCII⁺ F4/80⁺) Increased cellular uptake of ERY 1-allophycocyanin in the liver. (n =2, P ≦ 0.05, P ≦ 0.01, P ≦ 0.001). Data represent mean ± SE.

FIG. 12 is a mosaic of results showing that ERY1-OVA molecular fusion enhances antigen-specific OTI CD8⁺Cross-priming and apoptotic fate deletion proliferation of T cells in vivo (cross priming): panel (a) spleen OTI CD8 labeled with carboxyfluorescein succinimidyl ester (CFSE) at 5 days after intravenous administration of 10. mu.g ERY 1-glutathione-S-transferase (ERY1-GST, left panel), 10. mu.g OVA (middle panel), or 10. mu.g ERY1-OVA (right panel)⁺T cells (CD3 epsilon)⁺ CD8α⁺ CD45.2⁺) Proliferation of (4); panel (b) OTI CD8 from A⁺Dose-dependent quantitative proliferative populations of T cell proliferation, and identical 1 μ g dosing studies, data represent median ± min to max (n =5,. star.p ≦ 0.01,. # # P<0.01); small graph (c) OTICD8⁺T cell proliferation generation, which exhibited greater annexin-V after administration of ERY1-OVA (right panel) compared to OVA (middle panel) or ERY1-GST (left panel)⁺A population; panel (d) quantified annexin-V⁺ OTI CD8⁺T cell proliferation generation displaying ERY1-OVA induced OTICD8⁺T cell apoptosis, data represent mean ± SE (n = 5;. P)<0.0001). All data were determined by multiparameter flow cytometry.

FIG. 13 is a mosaic of results presented as a bar graph showing the molecular fusion of ERY1-OVA to OTI CD8⁺T cell proliferation is induced into an antigen experienced phenotype (antigen-experiential phenotype); panel (a) CD44 in spleen 5 days after administration of 1. mu.g OVA or 1. mu.g ERY1-OVA⁺ OTICD8⁺T cells (CD3 epsilon)⁺ CD8α⁺ CD45.2⁺ CD44⁺) (iv) quantification of (v) (. SP)<0.0001)；Panel (b) CD62L in spleen 5 days after administration of 1. mu.g OVA or 1. mu.g ERY1-OVA^- OTI CD8⁺T cells (CD3 epsilon)⁺ CD8α⁺ CD45.2⁺ CD62L^-) (ii) quantification of (i) (. P)<0.05); panel (c) CD44 in spleen 5 days after 10 μ g OVA or 10 μ g ERY1-OVA administration⁺ OTI CD8⁺T cells (CD3 epsilon)⁺ CD8α⁺CD45.2⁺ CD44⁺) (xvp = 0.0005); panel (d) CD62L in spleen 5 days after 10 μ g OVA or 10 μ g ERY1-OVA administration^- OTI CD8⁺T cells (CD3 epsilon)⁺ CD8α⁺ CD45.2⁺CD62L^-) (iv) quantification of (v) (. SP)<0.0001). Data represent mean ± SE, n = 5.

Figure 14 is a mosaic of results showing that erythrocyte binding induces resistance to antigen challenge: panel (a) OTI CD8⁺T cell adoptive transfer tolerance model showing experimental protocol for experimental and challenge and naive control group (n = 5); panel (b) OTI CD8⁺T cell population (CD3 epsilon)⁺ CD8α⁺ CD45.2⁺) Flow cytometry detection of (a); panel (c) CD45.1⁺OTI CD8 in draining lymph nodes (groin and popliteal) on day 4 post antigen challenge in mice⁺Quantification of T cell populations (. about.P)<0.01); panel (d) IFN γ expressing OTI CD8⁺Flow cytometry detection of T cells; panel (e) OTI CD8 expressing IFN γ in draining lymph nodes at day 4 post challenge and restimulation with SIINFEKL peptide (SEQ ID NO:3) antigen⁺T cells (. about.P)<0.01); panel (f) IFN γ concentration (. about.P.) in lymph node cell culture media at day 4 after restimulation with SIINFEKL peptide (SEQ ID NO:3) determined by ELISA<0.01); panel (g) concentration of IL-10 (. P.) in lymph node cell culture medium at day 4 after restimulation with OVA, determined according to ELISA<0.05). Data represent mean ± min to max; panel (h) OVA-specific serum IgG titers at day 19 (. P)<0.05), data represent mean ± SE; panel (i) EL4 thymoma (e.g7-OVA) tumor tolerance model expressing a combination of OTI and OVA, showing experimental protocols for experimental and control groups (n =4,3, respectively); panel (j) at day 5 after adoptive transferCirculating non-proliferative (passage 0) OTI CD8 in blood⁺Quantification of T cells; data represent median ± min to max (. about.p)<0.01); panel (k) growth profile of subcutaneously injected e.g. g7-OVA tumors 9 days after adoptive transfer of OTI, data representing mean ± SE (. sp.)/P<0.05)。

FIG. 15 is a bar graph showing how erythrocyte binding attenuates antigen-specific humoral responses in C57BL/6 mice. OVA-specific IgG assay in serum (P.ltoreq.0.05) 19 days after two administrations of 1. mu.g OVA or 1. mu.g ERY1-OVA in C57BL/6 mice 6 days apart.

FIG. 16 presents experimental results in which 8-arm PEG-ERY1 bound erythrocytes in vitro and in vivo; panel (a) 8-arm PEG-ERY1 (black solid histogram) bound to mouse red blood cells after in vitro incubation, whereas 8-arm PEG-MIS (gray solid histogram) or 8-arm PEG-pyridyl disulfide did not; panel (b) 8-arm PEG-ERY1 (black solid histogram) bound circulating red blood cells after intravenous injection, while 8-arm PEG-MIS (gray solid histogram) did not.

Fig. 17 presents experimental results depicting the erythrocyte surface half-lives of 8-arm PEG-ERY1 (solid ring) and 8-arm PEG-MIS (open box) as determined by flow cytometry.

Detailed Description

Described herein are peptides that specifically bind red blood cells. These are provided as peptide ligands having sequences that specifically bind red blood cells, or as antibodies or fragments thereof that provide specific binding to red blood cells. The peptides can be prepared as molecular fusions with therapeutic agents, tolerogenic antigens, or targeting peptides. Advantageously, the therapeutic agent, when part of the fusion, may have an extended circulating half-life in vivo. By using the fusion and selecting the antigen on the substance for which tolerance is desired, immunological tolerance can be developed. The fusion formed with the targeting peptide directs the fusion to a target, such as a tumor, where the red blood cell-binding ligand reduces or completely eliminates blood flow to the tumor by recruiting red blood cells to the target.

Thus, molecular design involving erythrocyte binding is taught to extend the circulating half-life of drugs, including protein drugs. The drug is formed as a conjugate, also known as a molecular fusion, e.g., a recombinant fusion or a chemical conjugate, with the erythrocyte-binding ligand. Molecular design for forming tolerance is also taught. Protein antigens sought to be tolerated are formed as conjugates with erythrocyte-binding ligands, such as recombinant fusions or chemical conjugates, including polymer or polymer micelle or polymer nanoparticle conjugates. Molecular design for tumor embolization is also taught. Forming the red blood cell binding ligand as a conjugate with a ligand of the tumor vasculature; targeting tumor vasculature, thus targeting erythrocyte binding within tumor vasculature.

Peptide sequences that specifically bind erythrocytes

Peptides have been found that specifically bind red blood cells. Example 1 describes the discovery of a peptide (ERY1) for specific binding to red blood cells. Example 8 describes the discovery of 6 peptides (ERY19, ERY59, ERY64, ERY123, ERY141 and ERY162) that specifically bind human red blood cells. One embodiment of the invention is a substantially pure polypeptide comprising the amino acid sequence of ERY1, or one of the human erythrocyte binding peptides, or a conservative substitution thereof, or a nucleic acid encoding the same. Such polypeptides specifically bind to red blood cells and are ligands of red blood cells. Ligand refers to the term of a chemical moiety that has specific binding to a target molecule. Target refers to a predetermined molecule, tissue, or location that the user intends to bind to the ligand. As such, targeted delivery to a tissue refers to the delivery of molecules or other materials (such as cells) to the intended target tissue. Thus, embodiments include molecules or compositions comprising at least one ligand disclosed herein for binding red blood cells. The binding activity of a polypeptide to red blood cells can be determined by following only the experimental protocol as described herein. Using such methods, the binding strength of a polypeptide variant to ERY1 or a human erythrocyte binding peptide under a given physiological condition can be determined, for example, by conservative substitutions, addition or removal of flanking groups, or by a sequence generated by changes or additions that serve to modulate the solubility of the sequence in aqueous solution.

As detailed in example 2, these peptide ligands bind to the surface of red blood cells without altering cell morphology and without cytoplasmic translocation. The ligands are distributed throughout the cell surface and are not clustered. Specific proteins can be identified as targets for ligands, such as glycophorin-A (GYPA) identified as targets for ERY-1 in example 3. ERY-1 is only reactive with mouse and rat species (example 4). Peptide ligands that specifically bind to human erythrocytes are specific for human erythrocytes and not for other species (example 9).

Instead of screening for purified erythrocyte surface proteins, naive peptide libraries containing whole erythrocytes were screened for affinity partners. Care was taken to minimize the number of unbound phage that escaped round elimination (round elimination) via the use of density gradient centrifugation and extensive washing. In addition, selection was stopped early in the screening process and clones were analyzed to prevent highly infectious phage clones from dominating in the population. The entire screening process was performed in the presence of high concentrations of serum albumin (50mg/mL) and 37 ℃ to reduce non-specific binding events and, perhaps more importantly, to select for peptides with favorable binding characteristics in serum. In the first set of experiments (example 1), clonal analysis revealed a phage clone (FIG. 1) displaying high affinity peptide WMVLPWLPGTLD (SEQ ID NO:1, referred to herein as ERY1) against the surface of mouse red blood cells. When searching for similarity using the BLAST algorithm in UniProt, no relevant protein sequence homology was identified for the entire peptide. Other experiments (example 8) identified binding ligands for human erythrocytes as shown in tables 1-2. The 6 sequences specifically bind human erythrocytes. The seventh sequence (called ERY50) binds to human erythrocytes, but also to epidermal/endothelial cells.

Table 1: peptide ligands that bind human red blood cells

The underlined sequence portion indicates the linker sequence.

Table 2: peptide ligands that bind mouse or human red blood cells

Not specific for erythrocytes

To mice

Embodiments of the invention include peptides that specifically bind to the surface of red blood cells. These sequences are not optimized for minimum length. Such optimization is within the skill in the art and may be implemented using the techniques described herein. For example, Kenrick et al (Protein Eng. Des. sel. (2010)23(1):9-17) screened from a library of 15 residues and then identified the minimum binding sequence of 7 residues in length. Getz (ACS chem.biol., 26/5/2011) identified a minimal binding domain as small as 5 residues in length. The erythrocyte-binding peptide may be present in a repeat of the same sequence, e.g., 2-20 repeats; the skilled artisan will readily appreciate that all ranges and values within the explicitly recited ranges are contemplated. Furthermore, the peptides may be present in a combination in which two or more different sequences are in the same peptide or form part of a single molecular fusion.

The number of contiguous residues that provide specific binding is expected to be about 4-12 residues. Thus, all peptides present in table 2 that are 4 contiguous residues in length, and for example all peptides of 5,6, 7, or 8 contiguous residues, have been disclosed. This number is based on the number of residues of other peptidic protein-binding ligands. Embodiments of the invention include the minimum length sequences of one of the sets of SEQ IDs that the erythrocytes herein (including table 1) bind. Thus, certain embodiments relate to a composition comprising a peptide, or an isolated (or purified) peptide, comprising several consecutive amino acid sequences of 4-12 consecutive amino acid residues of a sequence selected from the group consisting of: 11, 13, 14, 15, 16, 17, 1 and conservative substitutions thereof, wherein said sequences specifically bind erythrocytes. Alternatively, the number of contiguous residues may be selected from about 5 to about 18; the skilled person will readily appreciate that all ranges and values within the explicitly recited ranges are contemplated, e.g. 7,8, 9, 10, or 8 to 18. The red blood cell binding sequence can have, for example, conservative substitutions of at least one and no more than two amino acids of the sequence, or substitutions 1, 2, or 3, or substitutions 1-5. Furthermore, it is generally possible to effect the replacement of an L-amino acid in the sequence found with a D-amino acid, as in Giordano. In some embodiments, the peptide or composition may consist essentially of a sequence selected from the group consisting of seq id no:11, 13, 14, 15, 16, 17, 1. The peptide may be of limited length, e.g., having a number of residues from about 10 to about 100; all ranges and values within the explicitly recited ranges are contemplated as would occur to the skilled artisan, for example, from about 10 to about 50 or from about 15 to about 80. A peptide erythrocyte binding module can be provided comprising a peptide ligand having a dissociation constant of about 10 μ M to 0.1nM,

as determined by equilibrium binding measurements between the peptide and red blood cells; the skilled artisan will readily appreciate that all ranges and values within the explicitly recited ranges are contemplated, e.g., from about 1 μ M to about 1 nM. The peptide may further comprise a therapeutic agent. For example, the therapeutic agent can be a protein, biologic, antibody fragment, ScFv, or peptide. The peptide may further comprise a tolerogenic antigen, for example, a human protein (e.g., a blood factor such as factor VIII or factor IX), a protein with non-human glycosylation, a synthetic protein not naturally present in humans, a human food allergen, or a human autoimmune antigen for use in humans lacking the certain protein.

Others have sought peptide ligands that specifically bind to the surface of erythrocytes. One prior study attempted to discover red blood cell-binding peptides by using a novel bacterial surface displayed peptide library screening method (Hall, mitragotril et al, 2007). Their focus was to establish their novel bacterial peptide display system to screen naive libraries for peptides with affinity for erythrocytes and to use the peptides to attach 0.22 μm particles to erythrocytes. Although they reported the identification of several peptides that could accomplish this task, they did not characterize this binding phenomenon to a sufficient extent for practical considerations. They did not report the cell binding specificity of the peptide; the problem of which other cell types the peptide binds to is not addressed. They also do not report cell surface ligands for peptides. An electron micrograph taken of red blood cells labeled with peptide-functionalized 0.22 μm particles depicts red blood cells with a single particle cluster per cell. The vast majority of potential binding sites are expected to be widely distributed on the cell surface, and therefore the fact that all ligands tested are localized to small cellular regions indicates that these results are experimental artefacts. Such artifacts may be the result of a molar excess or other factors in labeling. Most importantly, no in vivo characterization of peptide-particle erythrocyte binding or pharmacokinetics was performed. Overall, the results described by Hall and coworkers do not suggest that peptide ligands to red blood cells can be used as tools to improve the pharmacokinetics of therapeutic agents or in other medical or therapeutic ways.

Polypeptides of different lengths may be suitably used for a particular application. In general, for a polypeptide containing a polypeptide ligand sequence, the polypeptide will exhibit specific binding if it has an opportunity (is available for) to interact with erythrocytes in vivo. Peptides with folding potential can be tested using the methods described herein. Thus, certain embodiments relate to polypeptides having a polypeptide ligand but which do not occur in nature, and certain other embodiments relate to polypeptides having a particular length, e.g., 6 to 3000 residues, or 12-1000, or 12-100, or 10-50 residues; the skilled artisan will readily appreciate that each and every numerical value and range within the explicitly recited limits is contemplated.

Certain embodiments provide a plurality of polypeptide sequences and/or purified or isolated polypeptides. Polypeptide refers to the term chain of amino acid residues, synthesized as a multimeric complex, independent of nucleic acids and/or carbohydrates, or other molecules, with post-translational modifications (e.g., phosphorylation or glycosylation) and/or complexation with other polypeptides. Thus, proteoglycans are also referred to herein as polypeptides. As used herein, a "functional polypeptide" is a polypeptide that is capable of facilitating the specified function. Polypeptides can be produced by a number of methods, many of which are well known in the art. For example, the polypeptide may be obtained by extraction (e.g., from isolated cells), by expression of a recombinant nucleic acid encoding the polypeptide, or by chemical synthesis. The polypeptide may be produced, for example, by recombinant techniques, and an expression vector encoding the polypeptide is introduced into a host cell (e.g., by transformation or transfection) to express the encoded polypeptide.

There are a number of conservative changes that can be made to the amino acid sequence without altering activity. These changes are referred to as conservative substitutions or mutations; that is, another amino acid may be substituted with an amino acid belonging to a group of amino acids having a specific size or characteristic. The substitutesheet of an amino acid sequence may be selected from other members of the class to which the amino acid belongs. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, methionine, and tyrosine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such changes are not expected to substantially affect the apparent molecular weight or isoelectric point as determined by polyacrylamide gel electrophoresis. Conservative substitutions also include the substitution of an optical isomer of the sequence for another, specifically, the substitution of a D amino acid for one or more residues of the sequence for an L amino acid. In addition, all amino acids in the sequence may undergo D-L (D to L) isomer substitutions. Exemplary conservative substitutions include, but are not limited to, those made with LysArg and vice versa to maintain a positive charge; substitution of Glu for Asp and vice versa to maintain negative charge; substitution of Ser for Thr to maintain free OH; and substitution of Asn with Gln to maintain free NH₂. Furthermore, in some cases, point mutations, deletions, and insertions of the polypeptide sequence or corresponding nucleic acid sequence can be made without loss of function of the polypeptide or nucleic acid fragment. Substitutions may include, for example, 1, 2, 3, or more residues. Amino acid residues described herein are abbreviated using a single letter amino acid designator or three letters. Abbreviations used herein are consistent with standard polypeptide nomenclature, j.biol.chem., (1969),243, 3552-3559. All amino acid residue sequences are presented herein in the left-right orientation in the conventional amino-terminal to carboxy-terminal direction.

In some cases, it may be desirable to determine the percent identity of a peptide to a sequence listed herein. In such cases, percent identity is measured in terms of the number of residues of the peptide or portion of the peptide. A polypeptide having, for example, 90% identity may also be part of a larger peptide.

The term "purified" as used herein in reference to a polypeptide refers to a polypeptide that has been chemically synthesized and is therefore substantially free of contamination by other polypeptides, or has been isolated or purified from most other cellular components with which it is naturally associated (e.g., other cellular proteins, polynucleotides, or cellular components). An example of a purified polypeptide is a polypeptide that does not contain at least 70% (by dry weight) of the proteins and naturally occurring organic molecules with which it is naturally associated. Thus, a preparation of a purified polypeptide may be, for example, at least 80%, at least 90%, or at least 99% (by dry weight) of the polypeptide. The polypeptide may also be engineered to contain a tag sequence (e.g., a polyhistidine tag, a myc tag, orTags) that allow the polypeptide to be easily purified or labeled (e.g., captured to an affinity matrix, visualized under a microscope). Thus, a purified composition comprising a polypeptide, unless otherwise indicated, refers to a purified polypeptide. Operation of the artThe term "isolated" indicates that the polypeptide or nucleic acid of the invention is not in its natural environment. Thus, an isolated product of the invention can be contained in a culture supernatant, partially enriched, produced from a heterologous source, cloned in a vector, or formulated with a vehicle, and the like.

The polypeptide may comprise chemical modifications: the term in this context refers to changes in the natural chemical structure of amino acids. Such modifications may be made to single strands or termini, for example to alter the amino terminus or the carboxy terminus. In some embodiments, modifications may be used to create chemical groups: it may be conveniently used to link polypeptides with other materials, or to attach therapeutic agents.

By specific binding, as that term is generally used in the field of biological technology, it is meant a molecule that binds a target with relatively high affinity as compared to non-target tissue, and generally involves a variety of non-covalent interactions, such as electrostatic interactions, van der waals interactions, hydrogen bonding, and the like. Specific binding interactions are characteristic of antibody-antigen binding, enzyme-substrate binding, and specific binding protein-receptor interactions, among others; while such molecules may sometimes bind to tissues other than their target, such binding is referred to as lacking specificity, not specific binding. In some cases, the peptide ERY1 and derivatives thereof and human erythrocyte-binding peptides and derivatives thereof may bind to non-erythrocytes, but it has been observed that such binding is non-specific, as evidenced by the much stronger binding of these peptides to erythrocytes than to other cells or proteins.

Thus, embodiments include ligands that specifically bind red blood cells and do not specifically bind other blood components, such as one or more of the following: blood proteins, albumin, fibronectin, platelets, leukocytes, essentially all components found in blood samples taken from a typical human. In the context of a blood sample, the term "substantially all" means that incidental components are normally present, but are excluded at very low concentrations, such that they do not effectively reduce the potency of the ligand that would otherwise be bioavailable.

Antibody peptides

In addition to peptides that bind red blood cells, proteins, particularly antibodies, and particularly single chain antibodies are provided herein. Techniques for generating antibodies to antigens are well known. In this context, the term "antigen" refers to a site recognized by the immune system of a host in response to the antigen. Antigen selection is known in the field of antibody production technology and the like. Embodiments include the use of these peptides in the molecular fusions and other methods provided herein. One of skill in reading this disclosure will be able to create antibodies that specifically bind red blood cells. Examples 15-17 relate to the production of antibodies or fragments thereof.

The term "peptide" is used interchangeably herein with the term "polypeptide". Antibodies and antibody fragments are peptides. The term "antibody fragment" refers to the portion of an antibody that retains the antigen-binding function of the antibody. Fragments may be generated as the name implies from a portion of a larger antibody, or may be synthesized de novo. Antibody fragments include, for example, single chain variable fragments (scFv). An scFv is a fusion protein in which the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin are linked by a linker peptide (e.g., about 10 to about 50 amino acids). The linker may connect the N-terminus of VH to the C-terminus of VL, or vice versa. The term "scFv" includes bivalent scFv, diabodies, triabodies (triabodies), tetrabodies (tetrabodies) and other combinations of antibody fragments. Antibodies have an antigen-binding portion called a paratope. The term "peptide ligand" refers to a peptide that is not part of a paratope.

Aptamers for specific binding to erythrocytes

In addition to peptide ligands that bind red blood cells, nucleotide aptamer ligands to components of the surface of red blood cells are taught. Thus, aptamers are generated and used as described herein for other erythrocyte binding modules. DNA and RNA aptamers can be used to provide non-covalent red blood cell binding. Since they are composed of only nucleotides, aptamers are promising biomolecular targeting modules because: screening methodologies are well established, they are easy to synthesize chemically, and cause limited toxicity and/or immunogenicity as a result of rapid clearance in vivo (Keefe, Pai et al, 2010). Furthermore, due to the non-canonical nature of nucleotide-target protein interactions, it is nearly impossible for any effective agonistic signaling to occur upon target binding in vivo, thus contributing to low immunogenicity and toxicity. Thus, many aptamer-based molecules are currently in human clinical trials for many clinical indications, including leukemia, macular degeneration, thrombosis, and type 2 diabetes (Keefe, Pai et al, 2010). Aptamers have also been used as targeting agents to deliver drug payloads to specific tissues in the body in applications such as cancer chemotherapy and fluorescence or radiology tumor detection techniques (Rockey, Huang et al, 2011; Savla, tartula et al, 2011).

Aptamers are oligonucleotides or peptides that bind to a specific target molecule. Aptamers are typically created by selecting them from a large random set of sequences to bind to a target of interest. Aptamers can be classified as DNA aptamers, RNA aptamers, or peptide aptamers. Nucleic acid aptamers are nucleic acid species that have been engineered to specifically bind to targets such as small molecules, proteins, nucleic acids, cells, tissues and organisms via repeated rounds of in vitro selection or Exponential Enrichment of the ligand phylogenetic Evolution (SELEX) method (Archemix, Cambridge, MA, USA) (Sampson, 2003). Peptide aptamers typically have short peptide variable domains attached to a protein scaffold at both ends. Peptide aptamers are proteins designed to interfere with other protein interactions inside the cell. They consist of a variable peptide loop attached at both ends to a protein scaffold. This dual structural constraint greatly improves the binding affinity of peptide aptamers, even comparable to antibodies. Variable loop lengths are typically made up of about 10 to about 20 amino acids, while scaffolds are well soluble and dense proteins. For example, the bacterial protein thioredoxin-a is a scaffold protein in which a variable loop is inserted into a reductive active site (which in the wild-type protein is the-Cys-Gly-Pro-Cys-loop) and two cysteine side chains are capable of forming a disulfide bridge.

Some techniques for generating aptamers are detailed in Lu et al, Chem Rev2009:109(5):1948-1998, and also US 7,892,734, US 7,811,809, US2010/0129820, US 2009/0149656, US 2006/0127929, and US 2007/0111222. Example 19 further details materials and methods for making and using aptamers for use in embodiments disclosed herein.

Molecular fusions

A molecular fusion can be formed between the first peptide erythrocyte-binding ligand and the second peptide. The fusion comprises peptides conjugated directly or indirectly to each other. The peptides may be conjugated to each other directly or indirectly via a linker. The linker may be a peptide, a polymer, an aptamer, a nucleic acid, or a particle. For example, the particle may be a microparticle, a nanoparticle, a polymersome (polymersome), a liposome, or a micelle. For example, the polymer may be natural, synthetic, linear, or branched. Fusion proteins comprising a first peptide and a second peptide are examples of molecular fusions of peptides, where the fusion proteins comprise the peptides directly linked to each other or have an intervening linker sequence and/or other sequences at one or both ends. Conjugation to the linker may be via a covalent bond. Other bonds include ionic bonds. The methods include preparing a molecular fusion or a composition comprising a molecular fusion, wherein the molecular fusion comprises a peptide that specifically binds red blood cells and a therapeutic agent, tolerizing antigen, or other substance.

The term "molecular fusion", or the term "conjugated", refers to direct or indirect binding by chemical bonds (including covalent, electrostatic ionic, charge-charge). Conjugation forms a unit that is maintained by chemical bonding. Direct conjugation refers to chemical bonding to an agent, with or without an intermediate linker or chemical group. Indirect conjugation refers to chemical attachment to a support. The carrier may encapsulate the agent mostly, such as a polymersome, liposome or micelle or some type of nanoparticle, or have the agent on its surface, such as a metallic nanoparticle or bead, or both, e.g., contain a portion of the agent inside and a portion of the agent on the outside. The carrier may also encapsulate antigens for immune tolerance. For example, polymersomes, liposomes, or particles can be made that encapsulate the antigen. The term "encapsulate" means completely covering, substantially without any partial exposure, for example, a polymersome can be made that encapsulates an antigen or agent. Examples of therapeutic agents are single chain variable fragments (scFv), antibody fragments, small molecule drugs, bioactive peptides, bioactive proteins, and bioactive biomolecules.

Conjugation may be achieved by covalently bonding the peptide to another molecule with or without a linker. The formation of such conjugates is within the skill of the artisan, and a variety of techniques for effecting conjugation are known, the selection of a particular technique being dependent on the material to be conjugated. Amino acids added to the polypeptide (C or N-terminus) that contain ionizable side chains (i.e., aspartic acid, glutamic acid, lysine, arginine, cysteine, histidine, or tyrosine) and are not included in the active portion of the polypeptide sequence in their unprotonated state as strong nucleophiles undergo various bioconjugation reactions with reactive groups attached to polymers (i.e., homo-or hetero-bifunctional PEGs) (e.g., Lutolf and Hubbell, Biomacromolecules 2003;4:713-22, Hermanann, Bioconjugate technologies, London. academic Press Ltd; 1996). In some embodiments, a soluble polymer linker is used, and may be administered to the patient in a pharmaceutically acceptable form. Alternatively, the drug may be encapsulated in a polymersome or vehicle or covalently attached to a peptide ligand.

One embodiment is a conjugate of a non-protein therapeutic and a peptide ligand, antibody fragment, or aptamer specific for red blood cells. The use of the erythrocyte-binding peptide methodology is not limited to polypeptide therapeutics; it can be converted into other pharmaceutical formulations such as small molecules and polymeric particles. The short circulating half-life and poor bioavailability have long influenced the in vivo efficacy of small molecules and their use in medicine. Polymeric micelles and nanoparticles represent a relatively more recent generation of drug classes, but their pharmacokinetic behavior is still poorly understood, for reasons including a high rate of removal via the action of the reticuloendothelial system (Moghimi and Szebeni, 2003). The erythrocyte binding design can be extended to these and other drug types to increase their circulating half-life and clinical efficacy.

The conjugate may comprise a particle. The erythrocyte-binding peptide may be attached to the particle. The antigen, agent, or other substance may be in or on the particle. Examples of nanoparticles, micelles, and other particles can be found, for example, in US 2008/0031899, US 2010/0055189, US 2010/0003338, which applications are incorporated herein by reference for all purposes, including combining them with ligands as listed herein; in case of conflict, however, the present specification will control. Examples 11 and 12 describe in detail the creation of certain particles.

Depending on the polydispersity produced by the method of preparation, nanoparticles may be prepared in the form of a collection of particles having diameters of about 10nm to about 200nm (including all ranges and values between well-defined boundaries, e.g., about 20 to about 200, and about 20 to about 40, to about 70, or to about 100 nm). Various nanoparticle systems may be utilized, such as those formed from copolymers of poly (ethylene glycol) and poly (lactic acid), those formed from copolymers of poly (ethylene oxide) and poly (beta-amino ester), and those formed from proteins such as serum albumin. Other nanoparticle systems are known to those skilled in the art. Devalapally et al, Cancer Chemother Pharmacol, 07-25-06, Langer et al, International journal of pharmaceuticals, 257: 169-.

Larger particles incorporating cartilage tissue binding ligands exceeding about 200nm average diameter, referred to herein as microparticles, can also be prepared as they begin to approach the micrometer scale and fall within the limits of optical resolution. For example, certain techniques for generating microparticles are listed in U.S. Pat. Nos.5,227,165,6,022,564,6,090,925, and 6,224,794.

To utilize targeting ability to functionalize nanoparticles, targeting polypeptides need to be associated with the particles, for example by covalent binding using bioconjugation techniques, where the selection of a particular technique depends on the particle or nanoparticle or other construct to which the polypeptide is to be attached. In general, many bioconjugation techniques for attaching peptides to other materials are well known, and the most suitable technique can be selected for a particular material. For example, other amino acids may be attached to the polypeptide sequence, such as cysteine in the case of attaching the polypeptide to a thiol-reactive molecule.

Example 18 details the creation of a multimeric branched polymer comprising a red blood cell specific binding module. To create a multimeric molecule capable of displaying multiple different biologically reactive molecules, a commercial 8-arm PEG dendrimer (dendrimer) was chemically modified to contain reactive groups to facilitate the conjugation reaction. The 8-arm PEG-pyridyl disulfide contains a pyridyl disulfide group that readily reacts with thiolates or esters from small molecules and/or cysteine-containing peptides or proteins, creating a disulfide bond between the attached bioactive moiety and the 8-arm PEG scaffold. The multimeric configuration of 8-arm PEG allows conjugation of different peptides or molecules to the scaffold, thus creating a heterofunctionalized biomolecule with multiple activities depending on the moiety to which it is attached. A heterofunctionalized fluorescent 8-arm PEG construct capable of binding red blood cells in vitro (fig. 16A) and in vivo (fig. 16B) was created. This binding was specific for the ERY1 peptide sequence, as conjugates containing non-specific MIS peptides showed little or no binding to erythrocytes. In vivo binding was long lived because fluorescent 8-arm PEG-ERY1-ALEXAFLUOR647 was detected on circulating red blood cells 5 hours after intravenous administration and exhibited a cell surface half-life of 2.2 hours (fig. 17). To demonstrate tolerance induction in an autoimmune diabetic mouse model, 8-arm PEG conjugated to both ERY1 and the diabetes antigen chromogranin-a (cra) was created. The modular nature of the 8-arm PEG-pyridyl disulfide scaffold makes it possible to co-conjugate different thiol-containing molecules by sequential addition of a stoichiometrically upper-limited number of molecules.

The molecular fusion may comprise a polymer. The polymer may be branched or linear. The molecular fusion may comprise a dendrimer. Generally, soluble hydrophilic biocompatible polymers can be used, such that the conjugate is soluble and bioavailable after introduction into a patient. Examples of soluble polymers are polyvinyl alcohols, polyethyleneimines, and polyethylene glycols (which term includes polyethylene oxide) having a molecular weight of at least 100, 400, or 100-400,000 (all ranges and values between these explicit values are contemplated). Solubility in this context means a solubility of at least 1 gram per liter in water or physiological saline. Domains of biodegradable polymers may also be used, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polycaprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydropyrans, and polynitrile acrylates.

In some embodiments, polypeptide-polymer associations (e.g., polymers) are prepared and introduced into the body in the form of a purified composition in a pharmaceutically acceptable state, or with pharmaceutically acceptable excipients. For example, the site of introduction may be systemic, or at a tissue or graft site.

The skilled person can use techniques known in the art to prepare fusion proteins. Embodiments include preparing fusion proteins, isolating them, and administering them in a pharmaceutically acceptable form with or without other agents, e.g., an interleukin in combination with TGF- β. Embodiments include vectors and methods for transfecting cells, thereby engineering cells to produce fusion proteins in vivo, wherein the cells are transfected in vitro, ex vivo, or in vivo, and the cells are members of or distinct from a tissue graft. The following U.S. patent applications are hereby incorporated by reference for all purposes, including the purpose of generating fusion proteins, in case of conflict the present specification shall control: 5227293,5358857,5885808,5948639,5994104,6512103,6562347,6905688,7175988,7704943, US 2002/0004037, US 2005/0053579, US 2005/0203022, US 2005/0250936, US 2009/0324538.

For example, embodiments of the molecular fusion include a molecular fusion comprising a tolerogenic antigen and an erythrocyte-binding moiety that specifically binds to erythrocytes in a patient, thereby linking the antigen to the erythrocytes, wherein the molecular fusion is administered in an amount effective to produce immune tolerance to a substance comprising the tolerogenic antigen. For example, embodiments include compositions comprising an erythrocyte binding module that specifically binds erythrocytes linked to a support selected from the group consisting of: polymers, branched polymers, and particles, wherein the carrier is linked to a therapeutic agent. For example, the particle may be a microparticle, a nanoparticle, a polymersome, a liposome, or a micelle. The red blood cell binding module may comprise a peptide comprising at least 5 consecutive amino acid residues of a sequence selected from the group consisting of: 11, 13, 14, 15, 16, 17, 1 and conservative substitutions thereof, wherein said sequences specifically bind erythrocytes. The erythrocyte-binding moiety may comprise an antibody, an antibody fragment, an aptamer, an scFv, or a peptide ligand. Embodiments of the molecular fusion include an erythrocyte-binding moiety and a tolerogenic antigen, antibody fragment, ScFv, small molecule drug, particle, protein, peptide, or aptamer.

Erythrocyte-binding ligands for improved pharmacokinetics

Since many drugs are delivered systemically to the blood circulation, the answer to the problem of effective drug delivery often focuses on maintaining the drug in the blood for a longer period of time. Thus, the development of long-circulating (long half-life) therapeutics that maintain bioavailability in the blood for long periods of time would represent a new generation of drugs engineered for efficacy, safety, and economic feasibility, among other purposes.

Embodiments of the invention include molecular fusions of erythrocyte-binding peptides with therapeutic agents. Molecular fusions between peptides that specifically bind red blood cells and therapeutic agents or other substances provide agents/substances with extended circulation time (in vivo blood circulation half-life). Examples 5 and 6 provide working examples thereof. For example, the extension may be about a 1.5-fold to 20-fold increase in serum half-life, and the skilled artisan will readily appreciate that all ranges and values within the explicitly stated ranges are contemplated, e.g., about 3-fold or about 6-fold or about 3-fold to about 6-fold.

For example, molecular fusions can be achieved by recombinant addition of peptides, or by chemical conjugation of peptides to reactive sites on therapeutic or related molecules or particles. Since solid phase peptide synthesis can be used to synthesize high yields of pure peptides with different terminal reactive groups, there are a variety of conjugation strategies for attaching peptides to therapeutic agents. Although this functionalization approach varies depending on the recombinant method used for the protein, it is believed that the effect (erythrocyte binding, which results in an increased circulation half-life) is the same.

One embodiment of the invention relates to the use of short peptide ligands that specifically bind red blood cells as a means for improving the pharmacokinetic parameters of a therapeutic agent to functionalize the therapeutic agent. This half-life extension methodology takes into account key parameters in therapeutic drug design, namely manufacturing simplicity, modularity (modulation), and the regulatory ability to extend the effect. Proteins are readily altered at the amino acid level to contain new or altered functionality using standard recombinant DNA techniques. Generally, relying on the use of shorter peptide domains for functionality is preferred over the use of larger polypeptide domains for reasons including ease of manufacture, proper folding into a functional therapeutic protein, and minimal biophysical changes to the original therapeutic agent itself. A polypeptide, such as ERY1 (a human erythrocyte-binding ligand), or an antibody or fragment thereof, can be engineered to specifically bind erythrocytes, and can be conjugated to a therapeutic agent to extend bioavailability (e.g., as measured by the circulatory half-life of the agent).

The results reported herein provide an opportunity to generate molecular fusions to improve pharmacokinetic parameters of therapeutic agents such as insulin, pramlintide acetate (pramlintide acetate), growth hormone, insulin-like growth factor-1, erythropoietin, type 1 alpha interferon, interferon alpha 2a, interferon alpha 2b, interferon beta 1a, interferon beta 1b, interferon gamma 1b, beta-glucocerebrosidase, adenosine deaminase, granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor, interleukin 1, interleukin 2, interleukin 11, factor VIIa, factor VIII, factor IX, exenatide (exenatide), L-asparaginase, labyrinase (rasburiase), tumor necrosis factor receptor, and enfuvirtide (enfurtide).

Attempts by others to implement passive half-life improvement methods have focused on increasing the apparent hydrodynamic radius of the drug. The glomerular filtration device of the kidney is the primary site in the body where blood components are filtered. The main determinant of filtration is the hydrodynamic radius of the molecules in the blood; smaller molecules (<80kDa) are filtered out of the blood to a higher degree than larger molecules. Researchers have utilized this generalized rule to modify drugs to exhibit larger hydrodynamic radii and therefore longer half-lives, primarily by chemical conjugation with high molecular weight water-soluble polymers such as polyethylene glycol (PEG). This approach has achieved significant success in many pegylated protein and small molecule therapeutics currently in clinical supply (Pasut and Veronese,2009; fisherburn, 2008). While effective in extending the circulatory half-life in many cases, particularly as the hydrodynamic radius of the graft or fusion is increased (Gao, Liu et al, 2009), these approaches present challenges in making and maintaining biological effector functions. Heterogeneity in the conjugation reaction may lead to complex product mixtures with different biological activities, mainly due to the use of site-non-specific chemistry. Extensive biochemical characterization is often performed after an accurate purification process to retain a homogeneous therapeutic product (Huang, Gough et al, 2009; Bailon, Palleroni et al, 2001; Dhalluin, Ross et al, 2005). Furthermore, attaching large moieties (such as branched PEG) to reactive regions of proteins can lead to a decrease in receptor affinity (fisherburn, 2008).

Other work by others has provided that therapeutic proteins bind albumin to achieve prolonged drug circulation (Dennis,2002; Walker, Dunlevy et al, 2010). In view of the same general principles described above with respect to renal filtration, Dennis and colleagues proposed the hypothesis that increasing the apparent size of a therapeutic agent by engineering it to bind another protein in the blood (such as serum albumin) would reduce the rate of drug clearance. In this way, the drug will only acquire a larger molecular size after administration to the bloodstream. Addition of affinity matured serum albumin binding peptides to antibody fragments extended their circulation time 24-fold in mice (Dennis, 2002). This approach, while effective, is complicated by the kinetics of albumin recycling at the neonatal Fc receptor (FcRn) and the use of cysteine-constrained cyclic peptides to achieve functionality. Walker and colleagues confirmed the contribution of Dennis in 2002 as a result of conferring serum albumin affinity to proteins to prolong their half-life. The method described by Walker and colleagues involves the recombinant addition of large antibody fragments to protein drugs, which can cause structural and manufacturing complications. The Dennis and Walker approaches, while ingenious and efficient, are complicated by the use of complex loop or large domains to achieve functionality. The peptides discovered by Dennis and colleagues, while exhibiting high affinity for albumin, require physical constraints to properly form the loop structure prior to use. The method of Walker's fusion of larger antibody fragments is a more cumbersome approach and may not be suitable for proteins that already have a complex folding structure or are expressed in low yields.

Single chain antibody

One embodiment of the invention is a molecular fusion of an scFv to a peptide that specifically binds red blood cells. scFv can be used as a therapeutic agent, and its combination with an erythrocyte-binding peptide can be used to extend its circulating half-life and enable its entry into a body compartment. Recombinant antibodies and recombinant antibody fragments have potential as therapeutic agents in the bioproduct industry (sheeridan, 2010).

Single chain variable fragment (scFv) antibody fragments contain the entire antigen-binding domain of a full-length IgG, but lack the hinge and constant fragment regions (Maynard and Georgiou, 2000). Recombinant construction of scFv involves fusion of the variable heavy chain (V) with a short polypeptide structure_H) Domains and variable light chains (V)_L) A domain consisting of a tandem repeat of glycine and serine (e.g., (GGGGS)₄)(SEQ ID NO: 18). Although the simplicity of scfvs is attractive for therapeutic applications, its major drawback is the short circulating half-life they exhibit due to their relatively small molecular weight of 26-28kDa (Weisser and Hall, 2009).

Since the glycine-serine linker commonly used in scFv design is essentially non-functional, but exists as a physical bridge to ensure the correct V_H-V_LFolding, and thus linker domains exhibiting the function of binding red blood cells in blood were tested herein. Thus, the engineered scFv can be multifunctional and bispecific, via V_H-V_LThe domain displays affinity for its native antigen and in its linker domain displays affinity for red blood cells. In binding to erythrocytes, engineered scfvs will exhibit a longer circulating half-life, as has been demonstrated for another model protein with this same functionality. The scFv antibody fragment may have a linker as described herein, or other linkers may be provided as known to those of skill in the art. An alternative embodiment provides for engineering the free cysteine group in the linker region of the scFv and using this cysteine thiol to attach the erythrocyte-binding ligand by chemical conjugation.

The scFv antibody was engineered as detailed in example 7. The design of engineered scFv antibodies focuses on the linker domain length, and the importance of the arrangement (spacing) of the erythrocyte-binding peptides. Since the wild type variant is designed to have (GGGGS)₄Linker (SEQ ID NO:18) and verified antigen binding, the subsequent mutants were designed as linkers with a minimum linker length of 20 amino acids (FIG. 6A). Since the linker domain can regulate the correct folding of the scFv into its correct tertiary structure (fig. 6B), two mutants containing ERY1 were designed. The REP mutant contains an ERY1 peptide centered in the linker domain, flanked by the correct number of Gly and Ser residues to maintain the parent 20 amino acid linker length. Where possible, the hydrophobic nature of the ERY1 peptide is not linear, but is clustered into shorter assembly domains, where the linker length of the REP is shorter and thus may prevent correct foldingAnd (5) stacking. For this reason, INS mutants were designed to contain the ERY1 peptide added to the center of the parental linker domain and extended the linker to 32 amino acids. Since the ERY1 peptide was found to have a free N-terminus, it was unknown whether its presence in the constrained polypeptide conformation would achieve red blood cell binding. To cope with this potential behavior, scFv variants were created by chemical conjugation to a synthetic ERY1 peptide, such that the N-terminus of this peptide was free, while the C-terminus was conjugated to the scFv (fig. 6C).

In this way, the number of erythrocyte-binding peptides, and accordingly the erythrocyte-binding capacity of the scFv, can be adjusted in a stoichiometric manner during the entire conjugation reaction. Thus, ScFv can be engineered to comprise an erythrocyte-binding peptide as taught herein. Embodiments include scfvs comprising a number of ligands ranging from 1 to 20; the skilled person will readily appreciate that all ranges and values within the explicitly specified ranges are contemplated, e.g. 2-6.

Embodiments include conjugation of scFv to tolerogenic antigen to generate molecular fusions that induce tolerance, e.g., as in example 17, example 17 describes details for creating tolerance, exemplified by creating tolerance to OVA attached to scFv. Example 17 also details materials and methods for generating scFv protein constructs recombinantly fused to an immunologically recognized epitope of an antigen. This scFv is intended for red blood cell recognition. The antigen is an antigen as described herein, e.g. a tolerogenic antigen. The working examples reported herein describe the results of using a murine model, using murine TER119 scFv as the antibody domain in the construct. TER119 is an antibody that binds mouse red blood cells. The TER119 antibody domain can be replaced with other antibody domains, such as domains directed against red blood cells in humans or other animals. For example, the 10F7 antibody domain can be used to create an antibody-antigen construct capable of binding to human red blood cells. Additional fusions of scFv from Ter-119 were prepared with three different antigens (as reported in example 17), including the MHC-I immunodominant epitope of OVA, the chromogranin-A mimotope (mimetope)1040-p31, and proinsulin.

Embodiments include scfvs that bind tumor markers and block blood flow to tumors, as described in examples 10 and 13. For example, the scFv can bind a tumor marker, and further be part of a molecular fusion with an erythrocyte-binding peptide. These conjugates can also be used to treat cancer by blocking blood flow to tumors.

Binding of erythrocytes to specific sites, such as tumor vasculature

In addition to extending the half-life of the drug, the ability of engineered therapeutic agents to bind red blood cells is also useful for the purpose of selectively binding and localizing red blood cells to specific sites in the body. In the treatment of solid tumors, trans-arterial chemoembolization (TACE) can be used to limit the blood supply to the tumor, thereby preventing it from obtaining the nutrients needed for growth. TACE treatment involves surgical insertion of polymeric solid particles upstream of the tumor blood supply. When the microparticles reach the tumor vascular bed, they are physically trapped in the vascular network, thereby creating an obstruction to the tumor blood supply (Vogl, Naguib et al, 2009).

According to the TACE theme, one embodiment herein is the use of autologous red blood cells circulating in the blood as natural microparticles for tumor embolization by engineering tumor-homing (tumor-homing) therapeutics to contain red blood cell-binding peptides. In this way, the therapeutic agent localizes to the tumor vascular bed and recruits passing red blood cells to bind to the blood vessels, thereby restricting and blocking blood flow to the tumor mass. Such treatments are less invasive than classical TACE: the drug would only be injected intravenously and would use the natural red blood cells already present in the blood as embolic particles. The term "tumor binding" or "tumor homing" refers to peptides that bind to components on tumor cells in the tumor vasculature accessible from the blood compartment.

The discovery of specific tumor-homing therapeutics is known in the field of cancer research. An example of bioactive targeting of tumors relies on binding to protein markers specifically expressed in the tumor environment. These include, but are not limited to: RGD-directed integrins (directed integrins), aminopeptidase-A and-N, endosialin, cell surface nucleolin, cell surface annexin-1, cell surface p32/gC1q receptor, cell surface reticulin-1, fibronectin EDA and EDB, interleukin 11 receptor alpha, tenascin-C, endoglin/CD 105, BST-2, galectin-1, VCAM-1, fibrin, and tissue factor receptor. (Fonstatti, Nicolay et al, 2010; Dienst, Grunow et al, 2005; Ruoslahti, Bhatia et al, 2010; Thijssen, Postel et al, 2006; Schlemann, Roesli et al, 2010; Brack, Silaci et al, 2006; Rybak, Roesli et al, 2007). Therapeutic agents targeting any of these molecules may be carriers that carry the red blood cell-binding peptide to the tumor vasculature to cause specific occlusion.

One embodiment is a first ligand that specifically binds red blood cells conjugated to a second ligand that specifically binds to cancerous cells or tumor vasculature or a component of tumor vasculature, such as a protein in the subendothelial membrane (which is partially exposed to blood in the tumor) or a protein on the surface of tumor endothelial cells. The ligand may be part of a pharmaceutically acceptable composition that is introduced into the patient, for example into the bloodstream. The ligand binds red blood cells, while the tumor-homing ligand binds to the tumor or a site at or near the tumor vasculature, or to cancerous cells. The red blood cells accumulate at the target site and block the target site from contact with nutrients, such as by embolizing blood vessels. Whereas embolization is mechanical, it will be rapid, as determined by the physical size of the red blood cells.

Solid tumors are largely dependent on their vascular supply, and biomolecular and material therapeutics have been developed to block the growth of or block the flow of their vascular supply. One embodiment is a biomolecule formulation or biomolecule-nanoparticle formulation for systemic injection to rapidly occlude solid tumor vasculature in a primary tumor or metastatic tumor at a known or unknown location.

Attempts have been made to address tumor embolization in a variety of ways, including using particle and biomolecule based approaches. Particles of biomaterial, including those produced from polyvinyl alcohol, have a diameter greater than the tumor microvasculature, e.g., 50-500 microns, and have been developed for clinical use in transcatheter arterial embolization, or TACE (Maluccio, Covey et al, 2008). One parallel approach involves loading the interior of the particles with chemotherapeutic agents for slow release in transarterial chemoembolization (TACE), primarily for the treatment of hepatocellular carcinoma (gadalleta and Ranieri, 2010). In both cases, when particles are injected into the arterial circulation (usually under radiographic guidance by a radiotherapist involved in the intervention), these particles can flow into the tumor vasculature and occlude them, blocking the blood flow (Maluccio, Covey et al, 2008). With these local methods only tumors that are directly targeted by placement of the catheter are treated, while other tumors, such as metastases at known or unknown locations, are not treated because the particles are not easily targeted in the blood vessels. More recently, biomolecular approaches have been explored, such as using bispecific antibodies that recognize both thrombotic factors and tumor vascular endothelial markers not present in normal vasculature. Upon specific binding to tumor vasculature, the antibodies accumulate and initiate blood clot formation within the tumor vasculature to block them; this effect is only induced when the antibody targets a tumor (Huang, Molema et al, 1997). These biomolecular approaches have the benefit of: targeting both primary and secondary tumors by intravenous infusion if specific tumor vessel signatures can be identified; they have the disadvantage of not providing rapid mechanical occlusion of the tumor.

Embodiments of the invention include a method of embolizing a tumor in a patient comprising administering to the patient a composition comprising an erythrocyte-binding module coupled to a targeting module, wherein the targeting module is an antibody, antibody fragment, or peptide directed against a target selected from the group consisting of a tumor and tumor microvasculature, and wherein the erythrocyte-binding module comprises a peptide, antibody fragment, or aptamer that specifically binds erythrocytes. For example, the peptide may be a sequence as listed herein.

Antigen-specific immunological tolerance

In addition to improving the pharmacokinetic behavior of therapeutic agents, it has been discovered that erythrocyte affinity can be used in methods of creating antigen-specific tolerance. Certain embodiments are set forth in the examples.

Example 14 details how tolerance was created in a mouse animal model that predicts human behavior. Briefly, the mouse erythrocyte-binding peptide ERY1 was found. The test antigen Ovalbumin (OVA) was used to generate molecular fusions of ERY 1. The fusion specifically binds to red blood cells in vivo, and does not bind to other molecules, including those in the blood or vascular system. A long circulating half-life is observed. It was observed that red blood cell binding of ERY1-OVA resulted in efficient cross-presentation of OVA MHC I immunodominant epitopes (SIINFEKL) by Antigen Presenting Cells (APCs) and corresponding cross-priming of reactive T cells. ERY1-OVA induced much higher numbers of annexin-V than OVA induced⁺Proliferative OT-I CD8⁺T cells (fig. 12d), suggesting an apoptotic fate that eventually leads to clonal deletion. Using the established OT-I challenge-tolerance model (Liu, Iyoda et al, 2002) (fig. 14a), it was demonstrated that ERY1-OVA can prevent subsequent immune responses to vaccine-mediated antigen challenge even with very strong bacterially derived adjuvants. Intravenous administration of ERY1-OVA resulted in draining lymph nodes (FIG. 4; gated in FIG. 14 b) and OT-I CD8 in the spleen, compared to mice administered unmodified OVA prior to antigen challenge with LPS (FIG. 14c)⁺The depth of the T cell population decreased, demonstrating deletion tolerance (deletion tolerance). This potent clonal deletion exhibited in mice administered ERY1-OVA supports the early OT-I CD8⁺The observation that T cell cross-priming was enhanced (fig. 12) also shows that cross-priming occurs without APC presentation of costimulatory molecules, resulting in deletion tolerance. Intravenous administration of ERY1-OVA reduced OVA-specific serum IgG levels by 39.8-fold 19 days after the first antigen administration compared to OVA-treated mice (fig. 15). To further demonstrate induction of antigen-specific immune tolerance, the OT-I challenge-tolerance model was combined with an OVA expressing tumor transplantation model (fig. 14) to obtainWith favorable results. The results detailed in this example indicate that ERY1-OVA induce antigen-specific immune tolerance to red blood cell binding. This was shown both in response to strong adjuvant challenge and implanted cell grafts expressing xeno-antigens (xeno-antigen). Furthermore, tolerance is via interaction with antigens present on circulating red blood cells resulting in reactive CD8⁺By functional inactivation and deletion of T cells, independent of direct CD4⁺T cell regulation. These detailed experiments with ERY1, a mouse erythrocyte-binding peptide, may predict similar results in humans with human erythrocyte-binding peptides (several of which are taught herein). In addition, conjugates with other erythrocyte-binding ligands, such as antibodies, antibody fragments, or aptamers, can be used to produce similar results under conditions where peptide ligands have been shown to be effective.

In contrast, previous reports have described creation of immune rejection by attaching antigens to the surface of red blood cells, thereby generating vaccines, and other reports have created vaccines using antigens encapsulated within red blood cells. For example, when the antigen is encapsulated within red blood cells, a Vaccine is thereby produced (Murray et al, Vaccine 24:6129-6139 (2006)). Alternatively, antigens conjugated to the surface of erythrocytes are immunogenic and have been proposed as vaccines (Chiarantini et al, Vaccine15(3): 276-. These references show that the erythrocyte delivery method gives an immune response as good as that obtained with a normal vaccine with adjuvant. Others have reported that induction of tolerance requires intracellular placement of erythrocytes, as in patent application WO2011/051346, which also teaches several means of altering the surface of erythrocytes to enhance clearance by kupffer cells in the liver. This application also teaches binding of antibodies to erythrocyte surface proteins such as glycophorin A, but the purpose is to generate immune complexes on erythrocytes to enhance their clearance by Kupffer cells.

The embodiments listed herein provide a method of generating immune tolerance comprising administering a composition comprising a molecular fusion comprising a tolerogenic antigen and a red blood cell-binding moiety that specifically binds to red blood cells in a patient, thereby linking the antigen to the red blood cells, wherein the molecular fusion is administered in an amount effective to generate immune tolerance to a substance comprising the tolerogenic antigen. The red blood cells and the patient may be untreated to cause other changes to the red blood cells and may be free of changes other than red blood cell cross-linking, chemical covalent conjugation, coating, and specific binding to the peptide. The molecular fusion may comprise, or consist of, an erythrocyte-binding moiety covalently bonded directly to an antigen. The molecular fusion may comprise an erythrocyte-binding moiety attached to a particle, which is attached to an antigen. The particles may include microparticles, nanoparticles, liposomes, polymersomes, or micelles. The tolerogenic antigen may comprise a therapeutic protein, for example, a portion of a blood factor administered to a patient suffering from a deficiency in the production of the blood factor. Embodiments include examples wherein: the patient is a human and the tolerogenic antigen is a human protein that is genetically deficient in the patient; wherein the patient is a human and the tolerogenic antigen comprises a portion of a non-human protein; wherein the patient is a human and the tolerogenic antigen comprises a portion of the engineered therapeutic protein that does not naturally occur in humans; wherein the patient is a human and the tolerogenic antigen comprises a portion of a protein comprising non-human glycosylation; wherein the tolerogenic antigen comprises a portion of a human autoimmune disease protein; wherein the tolerogenic antigen is an antigen in an allograft transplant; wherein the tolerogenic antigen comprises a portion of a substance selected from the group consisting of: human food; and/or wherein the red blood cell binding module is selected from the group consisting of: peptides, antibodies, and antibody fragments. Embodiments include tolerizing materials and methods wherein the red blood cell binding module comprises a peptide comprising at least 5 contiguous amino acid residues of a sequence selected from the group consisting of: 11, 13, 14, 15, 16, 17, 1 and conservative substitutions thereof, wherein the sequence specifically binds erythrocytes.

The molecular fusion can be selected to place the antigen on the inside or outside of the red blood cell. Without being limited to a specific mechanism of action, the following theory is proposed. In humans, about 1% of erythrocytes are apoptotic (apoptotic) and are cleared daily, and a large number of cells and their proteins are processed in such a way that tolerance to the erythrocyte autoantigens is maintained. The same tolerogenic response can also be elicited via antigen engineered to bind erythrocytes using ERY1 peptide or a human erythrocyte-binding peptide, an erythrocyte-binding single chain antibody or antibody, an erythrocyte-binding aptamer, or another erythrocyte-binding agent. Given that the prior art method developed by Miller and colleagues (see above) is cumbersome because it requires donor splenocytes to be harvested and reacted for reapplication, our non-covalent erythrocyte binding method provides a simpler alternative. Since ERY 1-erythrocyte or human erythrocyte-binding peptide-erythrocyte or other affinity biomolecule (e.g., single chain antibody, or aptamer) interactions occur spontaneously upon introduction of an antigen conjugate or fusion in vivo, binding occurs in situ simply by injection administration of the engineered antigen.

In some cases, the tolerogenic antigen is derived from a therapeutic protein for which tolerance is desired. Examples are wild-type protein drugs to which the patient does not establish central tolerance due to a defect in the protein, e.g. human factor VIII or factor IX; or a non-human protein drug for use in humans. Other examples are protein drugs that are glycosylated in a non-human form as a result of their preparation, or engineered protein drugs that have, for example, non-native sequences that can elicit an unwanted immune response. Examples of tolerogenic antigens that are engineered therapeutic proteins not naturally occurring in the human body include human proteins having engineered mutations, e.g., mutations that improve pharmacological characteristics. Examples of tolerogenic antigens comprising non-human glycosylation include proteins produced in yeast or insect cells.

Embodiments include administering a protein at a certain frequency X or dose Y and also administering an antigen from the protein at a lesser frequency and/or dose (e.g., a frequency of no more than 0.2X or a dose of no more than 0.2Y); the skilled person will readily appreciate that all ranges and values within the explicitly specified ranges are contemplated, e.g. 0.01 or 005X or some range in between.

Embodiments include selecting a tolerogenic antigen from a protein administered to a protein deficient human. By deficient is meant that the patient receiving the protein does not naturally produce enough of the protein. In addition, the protein may be a protein that is genetically deficient in the patient. Such proteins include, for example, antithrombin-III, protein C, factor VIII, factor IX, growth hormone, auxin, insulin, pramlintide acetate, meccasemin (IGF-1), β -glucocerebrosidase, alglucosidase- α, Laronidase (α -L-iduronidase), Idursuphase (iduronate-2-sulfatase), Galsulphase, acagosidase- β (α -galactosidase), α -1 protease inhibitors, and albumin.

Embodiments include selecting tolerogenic antigens from non-human proteins. Examples of such proteins include adenosine deaminase, pancreatic lipase, pancreatic amylase, lactase, botulinum toxin type A, botulinum toxin type B, collagenase, hyaluronidase, papain, L-asparaginase, labyrinase, lepirudin, streptokinase, anistreplase (anistreplase) (anistreponemal activator complex of anisyl plasminogen), antithymotrypsin, Crotalus multivalent immune Fab, digoxin immune serum Fab, L-arginase, and L-methioninase.

Embodiments include selecting a tolerogenic antigen from human allograft transplantation antigens. Examples of such antigens are the subunits of various MHC class I and MHC class II haplotype proteins, as well as minor blood group antigens (minor blood group antigens), including single amino acid polymorphisms on RhCE, Kell, Kidd, Duffy and Ss.

In some cases, a tolerogenic antigen is an autoantigen against which a patient has developed an autoimmune response or is likely to develop an autoimmune response. Examples are proinsulin (diabetes), collagen (rheumatoid arthritis), myelin basic protein (multiple sclerosis). StoreThere are many proteins that are human autoimmune proteins (the term refers to various autoimmune diseases in which one or more disease-causing proteins are known or can be established by routine testing). Embodiments include testing patients to identify autoimmune proteins and to create antigens for use in molecular fusions and to create immunological tolerance to proteins. Embodiments include, or select antigens from, one or more of the following proteins. In type 1 diabetes, several major antigens have been identified: insulin, proinsulin, preproinsulin, glutamic acid decarboxylase-65 (GAD-65), GAD-67, insulinoma-associated protein 2(IA-2), and insulinoma-associated protein 2 beta (IA-2 beta); other antigens include ICA69, ICA12(SOX-13), carboxypeptidase H, Imogen38, GLIMA38, chromogranin-A, HSP-60, carboxypeptidase E, peripherin, glucose transporter 2, hepatoma-gut-pancreas/pancreas associated protein, S100 beta, glial fibrillary acidic protein, regenogen II, pancreatic duodenum homeobox 1, myotonic dystrophy (dystrophia myotonica) kinase, islet-specific glucose-6-phosphatase catalytic subunit-related protein, and SST G protein-coupled receptors 1-5. In autoimmune thyroid diseases, including hashimoto's thyroiditis and graves' disease, the major antigens include Thyroglobulin (TG), Thyroid Peroxidase (TPO) and Thyroid Stimulating Hormone Receptor (TSHR); other antigens include sodium iodine symporter (NIS) and megalin. In thyroid-related eye diseases and skin diseases, the antigen is the insulin-like growth factor 1 receptor, in addition to thyroid self-antigens (including TSHR). In hypoparathyroidism, the primary antigen is the calcium sensitive receptor. In Addison's (Addison) disease, the major antigens include 21-hydroxylase, 17 α -hydroxylase, and P450 side chain cleaving enzyme (P450 scc); other antigens include ACTH receptor, P450c21, and P450c 17. In premature ovarian failure, the major antigens include the FSH receptor and α -enolase. In autoimmune pituitary inflammation, or autoimmune diseases of the pituitary, major antigens include pituitary ligand-specific protein factors (PGSF)1a and 2; another antigen is type 2 iodothyronine deiodinase. In the hard hair of multiple hairMajor antigens in chemolysis include myelin basic protein, myelin oligodendrocyte glycoprotein, and proteolipid protein. In rheumatoid arthritis, the major antigen is collagen II. In immunogastritis (immunogastritis), the major antigen is H⁺,K⁺-an ATPase. In pernicious anemia (pernicious angelis), the major antigen is the intrinsic factor. In celiac disease, the major antigens are tissue transglutaminase and gliadin. In vitiligo, the major antigens are tyrosinase, and tyrosinase-related proteins 1 and 2. In myasthenia gravis, the major antigen is the acetylcholine receptor. In pemphigus vulgaris and variants, the major antigens are desmoglein (desmoglein)3, 1 and 4; other antigens include pemphigus annexin, desmocollin (desmocollin), plakoglobin (plakoglobin), periplakin (perplakin), desmoplakin (desmoplakin), and acetylcholine receptors. In bullous pemphigoid, the major antigens include BP180 and BP 230; other antigens include reticulin and laminin 5. In dermatitis herpetiformis of Dulin, the major antigens include endomysial and tissue transglutaminase. In acquired epidermolysis bullosa (epidermolysis bullosa acquisia), the primary antigen is collagen VII. In systemic sclerosis (systemic sclerosis), the major antigens include matrix metalloproteinases 1 and 3, collagen-specific chaperone heat shock protein 47, fibrillin-1, and PDGF receptor; other antigens include Scl-70, U1 RNP, Th/To, Ku, Jo1, NAG-2, centromere protein, topoisomerase I, nucleolin, RNA polymerases I, II and III, PM-Slc, fibrin, and B23. In mixed connective tissue disease, the major antigen is U1 snRNP. In Sjogren's syndrome, the major antigens are the nuclear antigens SS-A and SS-B; other antigens include fodrin, poly (ADP-ribose) polymerase and topoisomerase. In systemic lupus erythematosus, the major antigens include nucleoproteins, including SS-A, high mobility group box 1(HMGB1), nucleosomes, histone proteins, and double stranded dnA. In Goodpasture's syndrome, the major antigens include glomerular basement membrane proteins, including collagen IV. In rheumatic heart disease, the main antigen is heartDirty myosin. Other autoantigens disclosed in autoimmune polyadenylation syndrome type 1 include aromatic L-amino acid decarboxylase, histidine decarboxylase, cysteine sulfinate decarboxylase, tryptophan hydroxylase, tyrosine hydroxylase, phenylalanine hydroxylase, liver P450 cytochrome P4501A2 and 2A6, SOX-9, SOX-10, calcium sensing receptor protein, and interferon type 1 interferon alpha, beta, and omega.

In some cases, the tolerogenic antigen is a foreign antigen against which the patient has developed an unwanted immune response. An example is a food antigen. Embodiments include testing a patient to identify foreign antigens and creating a molecular fusion comprising the antigens and treating the patient to develop immune tolerance to the antigens or food. Examples of such foods and/or antigens are provided. Examples are from peanuts: conglycinin (Ara h1), allergen II (Ara h2), peanut agglutinin, lupin conglutinin (Ara h 6); from apple: 31kda major allergen/disease resistance protein homologue (Mal D2), lipid transporter precursor (Mal D3), major allergen Mal D1.03D (Mal D1); from milk: alpha-lactalbumin (ALA), lactoferrin; from kiwi (kiwi): actinidin (actidin) (Act c1, Act d 1), phytocystatin (phytocystatin), thaumatin (thaumatin) -like protein (Act d 2), Kiwellin (Act d 5); from mustard: 2S albumin (Sin a 1), 11S globulin (Sin a 2), lipid transporter (Sin a 3), arrestin (Sin a 4); from celery: arrestin (Api g 4), high molecular weight glycoprotein (Api g 5); from shrimp: pen a1 allergen (Pen a 1), allergen Pen m 2(Pen m2), tropomyosin rapid isoform (fast isoform); from wheat and/or other grains: high molecular weight glutenin, low molecular weight glutenin, alpha and gamma-gliadin, hordein, secalin, avenin; from a strawberry: major strawberry allergies Fra a 1-E (Fra a 1), from banana: profilin (Mus xp 1).

Many protein drugs used in human and veterinary medicine induce immune responses, pose a risk to patients, and limit the efficacy of the drug. This may occur for human proteins that have been engineered, for use in patients with congenital defects in protein production, and for non-human proteins. It may be advantageous to tolerize the recipient to these protein drugs prior to initial administration, and it may be advantageous to tolerize the recipient to these protein drugs after initial administration and development of an immune response. In autoimmune patients, the autoantigens against which autoimmunity is formed are known. In these cases, it may be advantageous to tolerize the subject at risk prior to development of autoimmunity, and it may be advantageous to tolerize the subject at the time or after the development of the biomolecule indicative of initial autoimmunity. For example, in type 1 diabetes, there is an immunological indication of autoimmunity prior to the onset of clinical conditions of extensive destruction of beta cells and glucose homeostasis in the pancreas. It may be advantageous to tolerize a subject after detection of these immunological indicators prior to the onset of a clinical condition.

Recent work undertaken by Miller and colleagues has shown that ex vivo covalently conjugated antigens and allogeneic splenocytes produce antigen-specific immune tolerance when administered intravenously to mice (Godsel, Wang et al, 2001; Luo, Pothoven et al, 2008). The method involves harvesting donor spleen antigen presenting cells and chemically reacting them in an amine-carboxylic acid crosslinking reaction procedure. This technique has been shown to be effective in inducing antigen-specific tolerance in mouse models of multiple sclerosis (Godsel, Wang et al, 2001), newly developed diabetes type 1 (Fife, Guleria et al, 2006), and allogeneic islet transplants (Luo, Pothoven et al, 2008). Although the exact mechanism responsible for the tolerogenic response is unknown, it has been suggested that one major condition involves antigen presentation without expression of costimulatory molecules on apoptotic antigen-coupled cells (Miller, Turley et al, 2007). It has also been considered to encapsulate antigens within erythrocyte ghosts, process erythrocytes ex vivo, and reinject them, as in WO 2011/051346.

Administration of

Many of the embodiments of the invention listed herein describe compositions that can be administered to human or other animal patients. Embodiments of the invention include, for example, molecular fusions, fusion proteins, peptide ligands, antibodies, scfvs and combinations thereof that recognize antigens on red blood cells or tumors or tumor vasculature. These compositions can be prepared as pharmaceutically acceptable compositions and with suitable pharmaceutically acceptable carriers or excipients.

The composition that binds red blood cells can be specific for binding red blood cells. This specificity provides the possibility of in vivo binding of the composition to red blood cells, as well as an alternative ex vivo process. Thus, the composition may be injected directly into the vascular system of a patient. An alternative is injection into tissue (e.g. muscle), skin, or subcutaneously for subsequent contact and uptake by red blood cells.

The embodiments as described herein can be delivered using a pharmaceutically acceptable carrier or excipient. Excipient refers to an inert substance used as a diluent or vehicle for a therapeutic agent. Pharmaceutically acceptable carriers are generally used with the compounds, thereby making the compounds useful in therapy or as products. Generally, for any substance, a pharmaceutically acceptable carrier is a material that is combined with the substance to be delivered to the animal. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. In some cases, the support is necessary for delivery, e.g., to dissolve insoluble compounds for liquid delivery; buffering to control the pH of the material to maintain its activity; or a diluent to prevent loss of material from the storage vessel. However, in other cases, the carrier is for convenience, e.g., a liquid for more convenient application. Pharmaceutically acceptable salts of the compounds described herein can be synthesized according to methods known to those skilled in the art. Thus, the pharmaceutically acceptable compositions are highly purified, and thus free of contaminants, biocompatible and non-toxic, and further comprise a carrier, salt, or excipient suitable for administration to a patient. In the case of water as a carrier, the water is highly purified and treated to be free of contaminants, such as endotoxins.

Generally, the compounds described herein are administered in admixture with a suitable pharmaceutical diluent, excipient, bulking agent or carrier (referred to herein as a pharmaceutically acceptable carrier, or carrier) suitably selected with respect to the intended form of administration and in accordance with conventional pharmaceutical practice. As such, the deliverable compound can be prepared in a form suitable for oral, rectal, topical, intravenous, intraarterial, or parenteral administration. The carrier comprises a solid or a liquid, and the type of carrier is selected based on the type of application used. Suitable binders, lubricants, disintegrants, colorants, fragrances, flow-inducing agents (flow-inducing agents), and fusing agents (fusing agents) may be included as carriers, for example for pills. For example, the active ingredient may be combined with oral, non-toxic, pharmaceutically acceptable, inert carriers such as lactose, gelatin, agar, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like. The compounds can be administered orally in solid dosage forms such as capsules, tablets, and powders, or in liquid dosage forms such as elixirs, syrups, and suspensions. The active compounds may also be administered parenterally in sterile liquid dosage forms. Buffers may also be used to achieve a biological pH or osmolality.

Examples

Example 1: screening of erythrocyte-binding peptides Using mouse erythrocytes

A12 amino acid peptide phage library of PhD naive, available from New England Biolabs (NEB), was used in the selection. In each round of screening, 10 are put¹¹One input phage was incubated with mouse red blood cells in PBS with 50mg/mL BSA (PBSA-50). After 1 hour at 37C, unbound phage were removed by centrifugation in PERCOLL (GE Life sciences) at 1500g for 15 minutes. A subsequent dissociation step was performed in PBSA-50 to remove low affinity binding phage. Increase dissociation duration and temperature in subsequent screening runs to increaseThe stringency of the selection process. In round 1, phage binding was followed by a2 minute dissociation step at room temperature, followed by washing and elution. In round 2, phage binding was followed by 10 min dissociation at 37 ℃. In rounds 3 and 4, two separate and sequential dissociation steps were performed at 37 ℃:10 and 15 minutes after each other in round 3 and 10 and 30 minutes after each other in round 4. The erythrocyte-bound phage were eluted with 0.2M glycine pH 2.2 for 10 minutes and the solution was neutralized with 0.15 volumes of 1M Tris, pH 9.1. High affinity phage clones in the library were substantially enriched after applying 4 rounds of selection against whole red blood cells as shown by flow cytometry. Infectivity or phage formation units were calculated by standard titration (titering) techniques. Phage samples were serially diluted into fresh LB medium and 10 μ Ι _ phage dilution was added to 200 μ Ι _, log-early ER2738 e.coli (NEB). After 3 minutes of incubation at room temperature, the solution was added to 3mL of top agar, mixed, and poured onto LB plates containing IPTG and XGal. After overnight incubation at 37 ℃, blue colonies were counted as phage forming units (pfu).

Example 2: characterization of binding to mouse erythrocytes

As a result: microscopy confirmed that ERY1 phage bound to the surface of red blood cells without altering cell morphology and without cytoplasmic translocation. The fluorescence and phase contrast images again show the red blood cell binding capacity of the ERY1 phage relative to the unselected library. High resolution confocal imaging revealed that ERY1 phage were distributed throughout the cell surface (as opposed to clustered at a single location) and preferentially bound to the equatorial periphery of the cell surface, and that binding was uniform between red blood cells (fig. 1).

The method comprises the following steps: for all samples, 10 will be used¹¹Each input phage was incubated with mouse red blood cells in PBS-50. After 1 hour at 37 ℃ unbound phage were removed by centrifugation at 200g for 3 minutes. For regular fluorescence microscopy samples, cells were incubated for 1 hour at room temperature with anti-M13 coat protein-PE antibody (Santa Cruz Biotechnology) diluted 1:20 in PBSA-5.Cells were spun at 200g for 3 min, resuspended in 10. mu.L of Hard-set mounting medium (Hard-set) VECTASHIELD, smeared onto a microscope slide, covered with a coverslip, and visualized. For confocal microscope samples, cells were incubated with rabbit anti-fd phage (Sigma) and anti-rabbit ALEXAFLUOR conjugate (Invitrogen).

Example 3: characterization of molecular targets binding to mouse erythrocytes

As a result: to search for molecular targets of the ERY1 peptide, affinity pull-down (pull-down) technique using biotinylated soluble peptides was employed; this method reveals glycophorin-A (GYPA) as a ligand for ERY1 on the erythrocyte membrane. When whole red blood cells were incubated with the ERY1 peptide functionalized with biotin and a photoactivatable crosslinker, a 28kDa protein was detected conjugated to the peptide-biotin complex by streptavidin Western blotting (FIG. 2A). The reaction lysate was washed well and purified using streptavidin magnetic beads to ensure that no unlabeled protein from the red blood cell lysate remained. As expected, the mismatch peptide could not be conjugated to any erythrocyte protein. Mismatch peptide PLLTVGMDLWPW (SEQ ID NO:2) was designed to contain the same amino acid residues as ERY1 and is consistent with its hydrophobicity profile. Evidence of the apparent size of interacting proteins suggests that several smaller, single-transmembrane proteins are possible ligands, i.e., glycophorins. anti-GYPAWestern blots of the same purified samples from the cross-linking reaction confirmed that the candidate biotinylated protein was indeed GYPA (fig. 2B).

Co-localization of ERY1 phage with GYPA was analyzed by high resolution confocal microscopy. GYPA is naturally expressed and is presented as part of a complex composed of several membrane and cytoskeletal proteins (Mohandas and Gallagher, 2008). This was visually evident in GYPA staining, with inconsistent labeling seen at the periphery of the equator of the cell. Labeling with ERY1 phage produced very similar staining profiles. An overlap factor of up to 0.97 in the co-localization assay supports the conclusion that ERY1 phage and anti-GYPA bind the same protein. GYPA clustering was also seen in erythrocytes labeled with library phage, but there was no phage binding and thus no apparent co-localization.

The method comprises the following steps: ERY1 (H) was synthesized using standard solid phase f-moc chemistry on TGR resin₂N-WMVLPWLPGTLDGGSGCRG-CONH₂) (SEQ ID NO:19) and mismatches (H)₂N-PLLTVGMDLWPWGGSGCRG-CONH₂) (SEQ ID NO:20) peptide. The peptide was cleaved from the resin in 95% trifluoroacetic acid, 2.5% ethanedithiol, 2.5% water and precipitated in ice-cold diethyl ether. Purification was performed on Waters preparative HPLC-MS using a C18 reverse phase column.

ERY1 and the mismatch peptide were conjugated to Mts-Atf-biotin (Thermo Scientific), as suggested by the manufacturer. Briefly, peptides were dissolved in PBS/DMF and reacted with 1.05 equivalents of Mts-atf-biotin at 4C overnight. After the reaction was clarified by centrifugation, biotinylated peptide was incubated with red blood cells in PBSA-50 for 1 hour at 37C, the cells were washed twice in fresh PBS and UV-irradiated at 365nm for 8 minutes at room temperature. Cells were lysed by sonication and lysates were purified using streptavidin-coated magnetic beads (Invitrogen). The eluate was run on SDS-PAGE and transferred to PVDF membrane and immunoblotted with streptavidin-HRP (R & D Systems) or anti-mouse GYPA.

Example 4: binding or lack of binding of ERY1 to other mouse cells and erythrocytes from other species was characterized.

As a result: flow cytometry screening of a panel of inter-species cell lines showed that the ERY1 phage was specific for mouse and rat erythrocytes with no measurable binding to mouse leukocytes or human cells (fig. 3). These data suggest that specific membrane proteins that act as ligands for ERY1 are only present in erythroid cells, but not in myeloid or lymphoid cell lineages. Furthermore, this validates a screening method using, as a target, freshly separated blood which has hardly been purified beforehand other than centrifugation.

The method comprises the following steps: to determine phage binding, approximately 10 was used at 37C¹⁰Individual phage particles to tag 5x10 in PBSA-50⁵And 1 hour for each cell. After centrifugation at 200g for 4 minutes, the cells were resuspended in PBSA-5 and anti-phage-PE was added at 1:20 dilution for 1 hour at room temperature. After the final spin/wash cycle, cells were resuspended in PBSA-5 and analyzed on a flow cytometer.

Example 5: characterization of intravenous pharmacokinetics with model proteins

As a result: to characterize the effect of the ERY1 peptide on protein pharmacokinetics, we expressed the model protein Maltose Binding Protein (MBP) as an N-terminal fusion with ERY1 peptide (ERY 1-MBP). After intravenous administration, the ERY1-MBP variant exhibited prolonged circulation relative to the wild-type protein (fig. 4). Blood samples taken at the time points immediately after injection confirmed that the initial concentrations were the same in both formulations, and thus the doses. Beginning 4 hours after intravenous injection, ERY1-MBP cleared from the circulation at a statistically significantly slower rate than non-binding wild-type MBP.

Compared to wild-type MBP, ERY1-MBP demonstrated a 3.28-fold increase in serum half-life (for the single compartment model) to 6.39-fold (for the two compartment model) and a 2.14-fold decrease in clearance. Concentrations gave half-lives of 0.92 hours and 3.02 hours for wild type and ERY1 variants, respectively, using a standard one-compartment pharmacokinetic model. Data were also accurately fitted to a two-compartment model (R)²≧ 0.98), alpha and beta half-lives of 0.41 hours and 1.11 hours, and 2.62 hours and 3.17 hours, respectively, were obtained for the wild-type and the ERY1 variants. Thus, an increase in half-life can be expected with human erythrocyte-binding peptides and other erythrocyte-binding ligands as taught herein.

The method comprises the following steps: the cloned, replicative form of M13KE DNA was extracted using a standard plasmid isolation kit. The resulting plasmid was digested with Acc651 and EagI to obtain the gIII fusion gene, which was then ligated into the same site in pMAL-pIII to produce a plasmid referred to herein as pMAL-ERY 1. The sequence-verified clones were expressed in BL21 E.coli. Briefly, medium log phase BL21 cultures were induced with IPTG at a final concentration of 0.3mM for 3 hours at 37C. With 20mM Tris, 20% sucrose, 2mMEDTA osmotic shock treatment for 10 min, followed by 5mM MgSO₄Second treatment at 4 ℃ for 15 min, which allows periplasmic expression of the MBP fusion to separate from the cell debris. Purification of the fusion protein was performed on amylose Sepharose and purity was analyzed by SDS-PAGE.

All animal procedures were previously approved by the Swiss Vaud Veterinary Office. Under anesthesia with ketamine/xylazine, the mouse tails were incubated in 42 ℃ water and 150 μ g of protein was injected directly into the tail vein in a volume of 100 μ L. Care was taken to ensure that mice were maintained at 37 ℃ under anesthesia. Blood was withdrawn through a small scalpel incision at the tail root and diluted 10-fold in PBSA-5, 10mM EDTA and stored at-20C until further analysis. Blood samples were analyzed for MBP concentration by sandwich ELISA. Briefly, monoclonal mouse anti-MBP was used as the capture antibody, polyclonal rabbit anti-MBP as the primary antibody, and goat anti-rabbit HRP as the secondary antibody. Data were analyzed in PRISM4 using equation 1 and equation 2 using standard pharmacokinetic compartment analysis.

Equation 1: standard one-compartment model

A=A₀e^-Kt

Where A is the amount of free drug in the body at time t and A0 is the initial amount of drug at time 0.

Equation 2: standard two-compartment model

A=ae^-αt+be^-βt

Where A is the amount of free drug in the central compartment at time t.

Example 6: characterization of subcutaneous pharmacokinetics with model proteins

As a result: after extravascular administration, the ERY1-MBP variant exhibited prolonged circulation relative to the wild-type protein (fig. 5). Blood samples taken at the time points immediately after injection confirmed that the initial concentrations were the same in both formulations, and thus the doses. A similar trend in the increase in blood levels of ERY1-MBP was seen to continue throughout the entire experimental period following subcutaneous injection. Analysis of blood concentrations revealed that the ERY1-MBP variant showed an increase in bioavailability of 1.67 compared to wild-type MBP. Thus, half-life extension is possible with human erythrocyte-binding peptides and other erythrocyte-binding ligands as taught herein.

The method comprises the following steps: the cloned, replicative form of M13KE DNA was extracted using a standard plasmid isolation kit. The resulting plasmid was digested with Acc651 and EagI to obtain the gIII fusion gene, which was then ligated into the same site in pMAL-pIII to produce a plasmid referred to herein as pMAL-ERY 1. The sequence-verified clones were expressed in BL21 E.coli. Briefly, medium log phase BL21 cultures were induced with IPTG at a final concentration of 0.3mM for 3 hours at 37C. Osmotic shock treatment with 20mM Tris, 20% sucrose, 2mM EDTA for 10 min, followed by 5mM MgSO₄Second treatment at 4 ℃ for 15 min, which allows periplasmic expression of the MBP fusion to separate from the cell debris. Purification of the fusion protein was performed on amylose Sepharose and the purity was analyzed by SDS-PAGE.

All animal procedures were previously approved by the Swiss Waals veterinary office. Under anesthesia with isoflurane, 150 μ g of protein was injected directly into the dorsal skin of mice in a volume of 100 μ L. Care was taken to ensure that the mice remained at 37C under anesthesia. Blood was withdrawn through a small scalpel incision at the tail root and diluted 10-fold in PBSA-5, 10mM EDTA and stored at-20C until further analysis. Blood samples were analyzed for MBP concentration by sandwich ELISA. Briefly, monoclonal mouse anti-MBP was used as the capture antibody, polyclonal rabbit anti-MBP as the primary antibody, and goat anti-rabbit HRP as the secondary antibody. Data were analyzed in Prism4 using standard pharmacokinetic compartment analysis using equation 3.

Equation 3: bioavailability of

Where AUC is the area under the plasma concentration versus time plot, s.c. is subcutaneous, and i.v. is intravenous.

Example 7: engineering linker domains of scFv antibodies

The method comprises the following steps: genes encoding scFv fragments against the extracellular domain a of fibronectin were custom synthesized from DNA2.0(Menlo Park, CA, USA):

5 'ATGGCAAGCATGACCGGTGGCCAACAAATGGGTACGGAAGTGCAACTGCTGGAGTCTGGCGGTGGCCTGGTTCAGCCGGGTGGCAGCTTGCGCCTGAGCTGTGCGGCGTCTGGCTTCACCTTTAGCGTCATGAAAATGAGCTGGGTTCGCCAGGCACCAGGTAAAGGCCTGGAGTGGGTGTCGGCAATCAGCGGTTCCGGTGGTAGCACCTATTACGCTGACAGCGTGAAAGGCCGTTTTACGATTTCGCGTGATAACAGCAAGAACACGCTGTACTTGCAAATGAATAGCCTGCGTGCAGAGGACACGGCAGTGTACTATTGTGCGAAGAGCACTCACCTGTACTTGTTTGATTACTGGGGTCAAGGCACCCTGGTTACCGTTAGCAGCGGCGGTGGTGGCTCCGGTGGTGGTGGTAGCGGTGGCGGTGGTTCTGGTGGTGGCGGCTCTGAAATTGTCCTGACTCAGAGCCCTGGCACGCTGAGCCTGAGCCCGGGTGAGCGCGCGACGCTGAGCTGCCGTGCGAGCCAGTCCGTTAGCAACGCGTTCCTGGCTTGGTATCAACAGAAACCGGGTCAGGCCCCTCGCCTGCTGATTTACGGTGCCAGCTCCCGTGCGACGGGCATCCCGGACCGTTTTTCCGGCTCCGGTAGCGGCACCGACTTCACCCTGACCATCAGCCGCCTGGAGCCGGAGGATTTCGCGGTGTATTACTGCCAGCAAATGCGTGGCCGTCCGCCGACCTTCGGTCAGGGTACCAAGGTCGAGATTAAGGCTGCGGCCGAACAGAAACTGATCAGCGAAGAAGATTTGAATGGTGCCGCG-3' (SEQ ID NO: 21). To construct an expression plasmid containing wild-type scFv, the gene was PCR amplified using primers SK01 and SK02, with the addition of HindIII (5 ' end) and XhoI (3 ' end) restriction sites, and two stop codons at the 3 ' end. To construct a REP mutant scFv containing the ERY1 peptide in the linker region of the scFv, overlap extension PCR was used. A gene fragment comprising the 5' half of the scFv followed by an ERY1 gene fragment was created by PCR using primers SK01 and SK 03. A gene fragment comprising the 3' half of the ERY1 gene fragment (complementary to the preceding fragment) followed by the scFv was created by PCR using primers SK02 and SK 04. The gene fragments were purified using standard kits (ZymoResearch, Orange, CA, USA) after agarose electrophoresis and the two fragments were fused using PCR. Final amplification PCR was performed using SK01 and SK02 primers to create the correct restriction sites and stop codons. Construction of INS mutant scFv was performed in exactly the same manner as the REP mutant, except that primer SK05 was used in place of SK03, and SK06 was used in place of SK 04. Each of the finally completed scFv gene products was digested with HindIII and XhoI (NEB, Ipswich, MA, USA) and ligated into the same site on the pSecTagA mammalian expression plasmid (Invitrogen, Carlsbad, Calif., USA).

The sequence-verified clones were amplified and their plasmid DNA was purified for expression in Human Embryonic Kidney (HEK)293T cells. The expression plasmid contains an N-terminal signal sequence for secretion of the recombinant protein of interest into the culture medium. After 7 days of expression, cells were pelleted by centrifugation, the medium was harvested and the scFv was purified using size exclusion chromatography on a SUPERDEX 75 column (GE Life Sciences, Piscataway, NJ, USA).

An ERY1 peptide containing a C-terminal cysteine was conjugated to a wild-type scFv using succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC, CAS #64987-85-5, Thermo Scientific) as a cross-linker. SMCC was dissolved in dimethylformamide and added to scFv in Phosphate Buffered Saline (PBS) in 30-fold molar excess. After 2 hours at 4C, the reaction was desalted on a zebasini desalting column (Thermo Scientific) and the product was reacted with ERY1 peptide (peptide 5 molar excess). After 2 hours at 4C, the reaction system was dialyzed against PBS in a 10kDa MWCO dialysis tube at 4C for 2 days. The conjugated scFv were analyzed by SDS-PAGE, Western blotting, and MALDI.

Example 8: screening of erythrocyte-binding peptides with human erythrocytes

As a result: to select 7 novel peptides that bind to human erythrocytes, an e.coli surface display library was used. The screening process was performed with 4C in high concentration serum albumin (50mg/mL) in the washed whole blood to reduce non-specific binding to leukocytes. First, the peptide library was enriched for 3 rounds by incubation with blood followed by extensive washing and density gradient centrifugation to carefully separate the bacterially bound red blood cells from the other cells. Subsequently, the bacterial plasmid encoding the selected peptide is transformed into bacteria expressing the green fluorescent protein variant. This allows the red blood cell-bound green bacteria to be sorted by high throughput FACS and the individual recovered bacterial clones are assayed for binding to red blood cells using cytometry. 7 unique erythrocyte-binding peptides were identified, as shown in Table 1. These peptides do not contain consensus motifs, nor do they find relevant protein sequence homology when analyzed against known proteins using the BLAST algorithm in UniProt.

The method comprises the following steps: the e.coli surface display consists of more than a billion different bacteria, each displaying approximately 1000 copies of a random 15-mer peptide on the N-terminus of the scaffold protein eCPX, a circularly permuted variant of outer membrane protein X (Rice and Daugherty, 2008). For the first three selection cycles, co-sedimentation was used followed by one round of FACS to select for bacterial binding to human erythrocytes (Dane, Chan et al, 2006). Freezing equal parts of 10¹¹Cells containing the eCPX surface display library were thawed and cultured overnight at 37 ℃ in Luria Bertani (LB) medium supplemented with 34 μ g/mL chloramphenicol (Cm) and 0.2% D- (+) -glucose. Will be thinThe bacteria were subcultured at 1:50 for 3 hours in LB supplemented with Cm and induced with 0.02% L- (+) -arabinose for 1 hour. Human blood (type B) from healthy donors was washed twice with 5% HSA,2% FBS (HFS) in PBS, resuspended in conical tubes, and mixed with 10¹¹The bacterial cells were co-incubated for 1 hour at 4 ℃ on inverted shake flasks. The cell suspension was centrifuged at 500g for 5 minutes and the supernatant was freed of unbound bacteria. Erythrocytes were washed three times in 50mL HFS and resuspended in LB to culture bound bacteria overnight. Recovered bacterial clones were counted by plating on LB agar plates supplemented with Cm. For the second and third rounds, 10 was added, respectively⁸And 5x10⁷The bacteria were washed once as above and red blood cells were separated using a gradient of 70% Percoll (GE Life sciences) at 1000g10 min. For flow cytometry sorting, plasmids of selected eCPX library populations were extracted from bacterial cells using a zypyy miniprep kit. Subsequently, these plasmids were transformed into E.coli MC1061/pLAC22Grn1 for inducible GFP expression. GFP expression was induced with 1mM IPTG for 2 hours followed by surface expression of the peptide with 0.02% L- (+) -arabinose for 1 hour, both at 37C. Sample preparation for FACS was performed using similar techniques to those described above, and a fluorescent third round population of bound red blood cells was sorted using facsaria (bd biosciences).

Example 9: characterization of binding to human erythrocytes

As a result: to characterize the selected peptides that bind to human erythrocytes, binding assays were performed on bacteria displaying each peptide with multiple cell types. Six of the seven peptides (ERY19, ERY59, ERY64, ERY123, ERY141, and ERY162) specifically bound human erythrocytes compared to binding to human epithelial 293T cells and human endothelial HUVECs (fig. 7A). In addition, the peptides bound to human blood types a and B, but not to mouse blood (fig. 7B), indicating that these peptides are specific for human blood, but do not rely on common blood group antigens. Peptides were synthesized using standard solid phase f-moc chemistry, conjugated to nanoparticles, and analyzed for binding to various cell types, as above. Binding to the surface of erythrocytes was investigated using both microscopy and flow cytometry.

The method comprises the following steps: to characterize specificity, individually sequenced clones were analyzed for binding to human erythrocytes (type a and B), mouse erythrocytes, HEK293T cells, and HUVECs using flow cytometry. For binding assays, 10 will be used⁶Mammalian cells at 4C and 5X10⁷The bacteria were co-incubated for 1 hour, followed by three washes in HFS (5% HSA in PBS, 2% FBS) and then scanned on AccuriA 6. The percentage of cells bound with green bacteria was calculated using FLOWJO software.

Example 10: engineering linker domains of scFv antibodies

An engineered scFv against the tumor vascular marker fibronectin EDA (EDA) can be prepared in the form of a fusion with a peptide that specifically binds human erythrocytes. Multiple or each peptide from example 8 was inserted (GGGGS)₄(SEQ ID NO:18) linker region, or equivalent region (similar to the two mutants designed to contain ERY 1); thus, peptides ERY19, ERY50, ERY59, ERY64, ERY123, ERY141, ERY162 were added to replace ERY1 in the REP and INS mutant sequences (fig. 6A). These constructs inserted into the linker region may affect red blood cell binding due to the discovery that the human ERY peptide is tethered to the N-terminus of the scaffold protein eCPX. To address this, scFv variants were created by chemical conjugation to synthetic human ERY peptides (similar to ERY1) (fig. 6C). This would allow the addition of an optimal number of ERY peptides to the svFv, alone or in combination, to stimulate red blood cell binding.

Example 11: characterization of pharmacokinetics and biodistribution of polymeric nanoparticles and micelles

Invention laboratories have previously developed a number of polymer-based nanoparticles and micelles for drug delivery and immunomodulation. This technique is robust because it allows thiol-containing molecules to be easily site-specifically conjugated to nanoparticles in a quantifiable manner (van der Vlies, O' Neil et al, 2010). This laboratory has also developed micellar formulations that display multiple chemical groups on a single micelle, and formulations capable of controlled delivery of hydrophobic drugs (O' Neil, van der Vlies et al, 2009; Velluto, Demurtas et al, 2008). This laboratory has also explored the use of its nanoparticle technology as modulators of immune responses, as they target antigen presenting cells in lymph nodes (Reddy, Rehor et al, 2006; Reddy, van der Vlies et al, 2007). Micelle and particle technologies combined with the materials and methods herein include US 2008/0031899, US 2010/0055189, and US 2010/0003338, herein incorporated by reference.

The addition of either ERY1 peptide or human erythrocyte-binding peptide to these nanoparticles and micelle platforms improved their pharmacokinetic behavior, thereby enhancing their performance as circulating drug carriers. Conjugation to any nanoparticle or micellar variant of ERY1 or human erythrocyte-binding peptide can be carried out by a variety of reaction schemes, and conjugation of the detection molecule to the final product can be achieved using orthogonal chemistry. Due to the presence of ERY1 or the human erythrocyte-binding peptide group, confirmation of erythrocyte-binding nanoparticles or micelles could be verified by flow cytometry and microscopy, and further confirmation by in vivo characterization could be performed by quantitatively detecting the molecule at different time points after administration in mice.

Example 12: engineering polymeric nanoparticles and micelles to occlude tumor vasculature

Dual specifically engineered polymer nanoparticles and micelles can be designed and prepared for both red blood cells and tumor vascular markers, which can cause aggregation events of red blood cells in the tumor vascular bed and specifically block their blood supply. Several tumor targeting markers can be evaluated and utilized, including modified scFv against fibronectin EDA containing cysteine in the linker region, fibrinogen binding peptides containing the GPRP peptide motif, and truncated tissue factor fusion proteins with engineered cysteine or biotin, respectively, to allow attachment to particles. These tumor targeting ligands can be combined with erythrocyte-binding peptides or glycophorin a scFv on nanoparticles and micelles in an optimal ratio to achieve dual targeting; multiple ligands can be attached to the particle via disulfide linkages or avidin-biotin interactions. To confirm, mouse tumor cells can be injected into the dorsal skin of the mouse using a standard mouse solid tumor model and allowed to grow for a predetermined period of time, at which point the mouse is administered nanoparticles or micelles. The dosage and treatment regimen may be determined after characterizing the pharmacokinetics of the therapeutic agent. To further confirm, tumor volumes can be compared between treatment groups at different time points after treatment to assess the potential of therapeutic agents to block further tumor mass growth. Further confirmation of erythrocyte-mediated blockade of tumor vasculature can be assessed by perfusion experiments in live tumor-bearing mice. A positive correlation between the affinity of the therapeutic agent for red blood cells and tumor vessel occlusion was observed.

Example 13: engineering scFv antibodies to occlude tumor vasculature

The engineered scFv specific for the tumor vascular marker EDA and for erythrocytes can cause aggregation events of erythrocytes in the tumor vascular bed and specifically block their blood supply. Modified scfvs against EDA include human ERY binding peptides as fusions in linker regions or as conjugates with scfvs. Mouse tumor cells can be injected into the dorsal skin of a mouse using a standard mouse solid tumor model and allowed to grow for a predetermined period of time, at which point the mouse is administered nanoparticles or micelles. The dosage and treatment regimen may be determined after characterizing the pharmacokinetics of the therapeutic agent. At various time points after treatment, tumor volumes can be compared between treatment groups to assess the potential of therapeutic agents to block further tumor mass growth. Confirmation of erythrocyte-mediated blockade of tumor vasculature can be assessed by perfusion experiments in live tumor-bearing mice. The affinity of the therapeutic agent for red blood cells can be correlated with tumor vessel occlusion.

Example 14: inducing antigen-specific immunological tolerance via non-covalent binding of erythrocytes to antigen conjugated with ERY1 peptide or antigen conjugated with human erythrocyte-binding peptide

To obtain a strong and specific biophysical binding of antigen to erythrocytes, we used a synthetic 12 amino acid peptide (ERY1) which we found specific by phage displayBind to mouse glycophorin-A (Kontos and Hubbell, 2010). In this investigation, the model antigen OVA was used together with a transgenic mouse strain (OT-I) of CD8⁺The T cell population expresses a T cell receptor specific for the MHC I OVA immunodominant peptide SIINFEKL (SEQ ID NO: 3). The ERY1 peptide was chemically conjugated to OVA to create OVA variants (ERY1-OVA) that bound mouse red blood cells with high affinity and specificity (fig. 8 a). High resolution confocal microscopy confirmed previous observations regarding ERY1 binding (Kontos and Hubbell,2010), i.e. localization to the periphery of the cell membrane equator, without intracellular translocation of ERY 1-conjugated proteins. ERY 1-mediated binding to glycophorin-a was sequence specific, as OVA variants conjugated with a mismatch peptide (MIS-OVA) containing the same amino acids as ERY1, but scrambled in primary sequence exhibited negligible binding (fig. 8 b). OVA conjugated only with the cross-linking molecule used to conjugate the peptide did not exhibit any measurable affinity for erythrocytes, confirming that ERY1-OVA binding is the result of non-covalent interactions between ERY1 peptide and glycophorin-a on the surface of erythrocytes. Furthermore, ERY1-OVA was specific for erythrocytes with high affinity, exhibiting an antibody-like dissociation constant (K) of 6.2. + -. 1.3nM_d) As determined by equilibrium binding measurements (fig. 8 c).

Binding of ERY1-OVA to circulating red blood cells in vivo was confirmed after intravenous administration in mice. Whole blood samples collected 30 minutes after injection of 150 μ g OVA or ERY1-OVA confirmed the specific erythrocyte binding capacity of ERY1-OVA, even in a complex heterogeneous environment of the blood and vascular system (fig. 9 a). Consistent with glycophorin-A binding, ERY1-OVA binds to erythrocytes (CD 45)^-) But not leukocytes (CD 45)⁺). ERY1-OVA binding was not preferred with respect to the apoptotic state of erythrocytes, for annexin-V⁺Population and annexin-V^- CD45^-Both populations were strongly bound (fig. 9 b). Pharmacokinetic studies of OVA conjugates demonstrated that ERY1-OVA red blood cell binding was long in vivo, exhibiting a cell surface half-life of 17.2 hours (fig. 9 c). ERY1-OVA remained bound to red blood cells for up to 72 hours after administration; during this time period, about 13% of the red blood cellsIs cleared in mice (Khandelwal and Saxena, 2006). In vivo quantification of erythrocyte-bound ERY1-OVA showed every 10⁶A relatively high load of 0.174 ± 0.005ng OVA per erythrocyte.

To rule out any potential physiological effects of OVA loading on erythrocyte function, hematological parameters were characterized at different time points after intravenous administration of ERY1-OVA or OVA. Red blood cell binding of ERY1-OVA did not elicit detectable differences in hematocrit, hematocrit (corpuscle volume), or hemoglobin content of blood cells compared to OVA administration (fig. 10). These results indicate that glycophorin-A mediated binding of erythrocytes to antigens did not alter their haematological parameters.

To reveal cellular targets of erythrocyte-bound antigen after administration, mice were injected intravenously with highly fluorescent allophycocyanin protein conjugated to either ERY1(ERY 1-allophycocyanin) or MIS peptide (MIS-allophycocyanin). Flow cytometric analysis of splenic DC populations 12 and 36 hours post-administration showed MHCII compared to MIS-allophycocyanin⁺ CD11b^- CD11c⁺The uptake of ERY 1-allophycocyanin by DC was enhanced by 9.4-fold, but MHCII⁺ CD11b⁺ CD11c⁺The uptake of ERY 1-allophycocyanin and MIS-allophycocyanin by DCs was similar (fig. 11 a). In addition, MHCII was found⁺ CD8α⁺ CD11c⁺ CD205⁺Spleen DCs ingested ERY 1-allophycocyanin to a 3.0-fold greater extent than MIS-allophycocyanin, although absolute amounts were significantly lower than other DC populations in the spleen. Antigen vs. non-activated and CD8 alpha⁺ CD205⁺Such targeting of splenic DCs may potentiate the tolerogenic potential of erythrocyte binding, as these populations have been widely implicated in apoptotic cell-driven tolerogenicity (Ferguson, Choi et al, 2011; Yamazaki, Dudziak et al, 2008). In the liver, ERY 1-allophycocyanin also greatly enhanced hepatocytes compared to MIS-allophycocyanin (CD 45)^- MHCII^-CD1d^-(ii) a 78.4 fold) and hepatic stellate cells (CD 45)^- MHCII⁺ CD1d⁺(ii) a 60.6 fold) uptake (fig. 11 b); both populations have been reported as triggering CD8⁺T cell deletion tolerant antigen presentationCells (Holz, Warren et al, 2010; Ichikawa, Mucida et al, 2011; Thomson and Knolle, 2010). Interestingly, in hepatic DC (CD 45)⁺ CD11c⁺) Or Kupffer cell (CD 45)⁺ MHCII⁺ F4/80⁺) No such uptake was seen (which served as a member of the reticuloendothelial system that helped clear red blood cells and complement-coated particles). The increased uptake of erythrocyte-bound antigen by tolerogenic splenic DC and hepatocyte populations suggests the potential for a complex interconnected mechanism of antigen-specific T cell deletion driven by non-lymphoid hepatocyte and classical splenocyte cell dialogues.

It was observed that erythrocyte binding of ERY1-OVA resulted in efficient cross-presentation of OVA MHC I immunodominant epitope (SIINFEKL) (SEQ ID NO:3) by APC and corresponding cross-priming of reactive T cells. CFSE-labeled OT-I CD8⁺Adoptive transfer of T cells (CD45.2+) into CD45.1⁺In mice. Against OT-I CD8 within 5 days after intravenous administration of 10. mu.g OVA, 10. mu.g ERY1-OVA, or 10. mu.g of the irrelevant erythrocyte binding antigen ERY 1-glutathione-S-transferase (ERY1-GST)⁺Proliferation of T cells was measured. OT-I CD8 in comparison to OVA⁺T cell proliferation (as measured by flow cytometry, as determined by dilution of fluor CFSE (FIG. 12a)) was significantly enhanced in mice administered ERY1-OVA (FIG. 12b), indicating increased erythrocyte binding compared to soluble antigen-specific CD8⁺T cell cross-priming. Similar results were also obtained by administering a 10-fold lower antigen dose of 1 μ g, indicating induction of OT-I CD8 by erythrocyte-bound antigen⁺The kinetics of T cell proliferative efficacy are broad. The results on cross-presentation and cross-initialization are consistent with other studies on tolerogenic antigen presentation on MHC I by APCs that phagocytose antigens from apoptotic cells (Albert, Pearce et al, 1998; Green, Ferguson et al, 2009).

To distinguish T cells expanded to a functional effector phenotype from those expanded and deleted, proliferative OT-I CD8 was analyzed⁺annexin-V of T cells served as a marker for apoptosis and thus deletion (fig. 12 c). ERY1-OVA induced by much higher numbers than OVAannexin-V⁺Proliferating OT-ICD8⁺T cells (fig. 12d), suggesting an apoptotic fate that eventually leads to clonal deletion. Same proliferative OT-I CD8 induced by ERY1-OVA administration⁺T cells exhibited the phenotype experienced by the antigen at both 1 and 10 μ g doses, displaying up-regulated CD44 and down-regulated CD62L (fig. 13). Proliferative CD8⁺This phenotype of T cells is consistent with other reported OT-I adoptive transfer models, in which regulated antigen-specific T cell receptor engagement by APC fails to induce an inflammatory response (burst, Rich et al, 2009).

Using an established OT-I challenge-tolerance model (Liu, Iyoda et al, 2002) (fig. 14a), it was demonstrated that ERY1-OVA can prevent subsequent immune responses to vaccine-mediated antigen challenge even with very strong bacterially derived adjuvants. To induce tolerance, we are dealing with OT-I CD8⁺(CD45.2⁺) Adoptive transfer of T cells to CD45.1⁺Mice were administered 10 μ g OVA or ERY1-OVA intravenously 1 and 6 days later. After an additional 9 days to allow possible deletion of the transferred T cells, we then challenged the recipient mice by intradermal injection with OVA adjuvanted with Lipopolysaccharide (LPS). We characterized draining lymph nodes and splenocytes and their inflammatory response on day 4 post challenge to determine if deletion actually occurred.

Intravenous administration of ERY1-OVA resulted in draining lymph nodes (FIG. 4; gated in FIG. 14 b) and OT-ICD8 in the spleen, compared to mice administered unmodified OVA prior to antigen challenge with LPS (FIG. 14c)⁺The depth of the T cell population was reduced, demonstrating deletion tolerance. Draining lymph nodes from mice treated with ERY1-OVA contained more than 11-fold less OT-I CD8 than OVA-treated mice⁺T cells, and 39-fold less than challenge control mice that did not receive intravenous injection of antigen; the response in splenocytes was similar. This potent clonal deletion exhibited in mice administered ERY1-OVA supports previous work on OT-I CD8⁺The observation that T cell cross-priming was enhanced (fig. 12) and shows that cross-priming occurs without APC presentation of costimulatory molecules, leading to deletion tolerance.

To further evaluate the immune response following antigen challenge, OT-I CD8 was used⁺Interferon-gamma (IFN γ) expression by T cells to characterize the inflammatory properties of resident lymph nodes and splenocytes (fig. 14 d). After challenge with OVA and LPS, the lymph nodes of mice previously treated with ERY1-OVA contained 53-fold fewer IFN γ expressing cells compared to challenge control mice (not previously receiving antigen), and more than 19-fold fewer IFN γ expressing cells compared to mice previously treated with an equivalent dose of OVA (fig. 14e), demonstrating the importance of erythrocyte binding in tolerogenic protection against challenge; the response in splenocytes was similar. In addition, small OT-I CD8 present in lymph nodes and spleen of mice previously treated with ERY1-OVA⁺In the T cell population, the percentage of IFN γ expressed was lower, suggesting clonal inactivation (clonal inactivation). Furthermore, the magnitude of total IFN γ levels produced by isolated cells from draining lymph nodes after SIINFEKL restimulation was substantially reduced in mice previously treated with ERY1-OVA (fig. 14f), with erythrocyte binding reducing IFN γ levels by 16-fold (compared to OVA administration) and more than 115-fold (compared to challenge controls). Notably, the inhibitory phenomenon was also associated with down-regulated interleukin-10 (IL-10) expression, as lymph node cells from mice previously treated with ERY1-OVA expressed 38% and 50% less IL-10 than mice previously treated with OVA and challenge control mice, respectively (fig. 14 g). IL-10 is generally thought of as being mediated by regulatory CD4 in the context of APC-T cell communication⁺T cells express cytokines to suppress the Th1 response (Darrah, Hegde et al, 2010; Lee and Kim,2007), and IL-10 expression is not necessary for desensitization to challenge. Similarly, IL-10 downregulation has been proposed in combination with CD8⁺T cell mediated tolerogenicity has been implicated (Fife, Guleria et al, 2006; Arneboldi, Roth-Walter et al, 2009; Saint-Lu, Tourdot et al, 2009). Erythrocyte binding also substantially attenuated the humoral immune response against the antigen, since mice treated with ERY1-OVA exhibited 100-fold lower antigen-specific serum IgG titers compared to mice treated with soluble OVA (fig. 14 h). In non-adoptive transfer C57BL/6(CD 45.2)⁺) Class in mice with reduced OVA-specific IgG titres due to erythrocyte bindingThe likelihood decreases. Mice treated with either ERY1-OVA after two intravenous administrations of either OVA or ERY1-OVA 7 days apart exhibited 39.8-fold lower OVA-specific serum IgG levels 19 days after the first antigen administration (fig. 15). This apparent reduction in B cell activation following red blood cell ligation of antigen supports the current hypothesis for non-inflammatory antigen presentation during tolerance induction (Miller, Turley et al, 2007; Green, Ferguson et al, 2009; Mueller, 2010).

To further confirm the induction of antigen-specific immune tolerance, the OT-I challenge-tolerance model was combined with an OVA expressing tumor graft model (fig. 14I). Similar to previous experimental design, through adoptive transfer of OT-I CD8⁺Mice were tolerized by two intravenous administrations of either 10 μ g ERY1-OVA or 10 μ g OVA after T cells. Significant T cell deletion was detected 5 days after the first antigen administration, as mice injected with ERY1-OVA contained 2.9-fold less non-proliferative (passage 0) OT-I CD8 in the blood⁺T cells (fig. 14 j). To determine proliferative OT-I CD8 in the absence of exogenously administered strong adjuvant⁺Functional responsiveness of T cells, OVA expressing EL-4 thymoma cells (e.g. g7-OVA) were injected intradermally into the back skin of mice 9 days after adoptive transfer. To assess the tolerogenic potency of the erythrocyte-bound antigen, tumor-bearing mice were challenged with LPS-adjuvanted OVA 6 days after tumor transplantation, using similar doses and schedules as the challenge-tolerance model. Robust tumor growth was continuously observed in mice treated with ERY1-OVA until 8 days post tumor transplantation (FIG. 14k) as compared to OVA-treated or naive control mice, confirming ERY 1-OVA-driven OT-I CD8⁺T cell proliferation induces functional immune non-responsiveness to OVA. Arrest of tumor size at steady state 8 days post-transplant may suggest that OT-I CD8 has not experienced ERY1-OVA driven deletion tolerance⁺T cell remnants.

Animal(s) production

All animal procedures were previously approved by the Swiss veterinary authority. Female C57BL/6 mice (Charles River) 8-12 weeks old were used for in vivo binding studies and as e.g. g7-OVA tumor hosts. Mixing C57BL/6-Tg (Tcratcrb))1100Mjb (OT-I) mice (Jackson Labs) were bred in EPFL animals houses and splenocytes isolated at 6-12 weeks of age using females. Using 8-12 week old female B6.SJL-Ptprc^aPepc^bthe/Boy (CD45.1) mouse (Charles River) was used as recipient host for OT-I CD8⁺Adoptive transfer of T cells and tolerance induction studies.

Peptide design and Synthesis

ERY1 (H) was synthesized using standard solid phase f-moc chemistry on an automated liquid phase processor (CHEMSPEED) using TGR resin (Nova Biochem)₂N-WMVLPWLPGTLDGGSGCRG-CONH₂) (SEQ ID NO:19) and mismatches (H)₂N-PLLTVGMDLWPWGGSGCRG-CONH₂) (SEQ ID NO:20) peptide. The underlined sequence is the ERY 112 mer sequence, which we previously found by phage display to be a mouse glycophorin-a conjugate (Kontos and Hubbell, 2010). The GGSG region serves as a linker to the cysteine residues for conjugation; flanking arginine residues serve to lower the pKa and thus increase the reactivity of cysteine residues (Lutolf, Tirelli et al, 2001). The peptide was cleaved from the resin in 95% trifluoroacetic acid, 2.5% ethanedithiol, 2.5% water over 3 hours and precipitated in ice-cold ether. Purification was performed on preparative HPLC-MS (Waters) using a C18 reverse phase column (PerSpective Biosystems).

ERY 1-antigen conjugation

10 molar equivalents of succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC, CAS #64987-85-5, Thermo Scientific) dissolved in dimethylformamide were reacted at room temperature with 5mg/mL endotoxin free (<1 EU/mg) OVA (Hyglos GmbH) in PBS for 1 hour. After desalting on a 2ml zeba desalting spin column (Thermo Scientific), 10 equivalents of either ERY1 or MIS peptide dissolved in 3M guanidine hydrochloride was added and allowed to react at room temperature for 2 hours. The conjugate was desalted using a 2mL Zeba desalting spin column, sterile filtered 0.2 μm, dispensed into working aliquots and stored at-20C. Protein concentration was determined by BCA assay (Thermo Scientific). This protocol results in conjugation of cysteine side chains on the peptide to lysine side chains on the antigen. glutathione-S-transferase (GST) was expressed in BL21 E.coli and purified using standard glutathione affinity chromatography. Endotoxin removal on the column was performed by extensive Triton-X114(Sigma Aldrich) washing and confirmed using THP-1 XBlue cells (InvivoGen). The same reaction protocol was used to conjugate ERY1 with GST. Maleimide-activated allophycocyanin (Innova Biosciences) was solubilized in PBS and conjugated to ERY1 or MIS, as described above.

Microscopy for erythrocyte binding

Mix 5x10⁵Freshly isolated mouse erythrocytes were exposed to 100nM ERY1-OVA or OVA in PBS containing 10mg/mL BSA at 37C for 1 hour. After centrifugation and washing, cells were labeled with goat anti-mouse glycophorin-A (Santa Cruz) and rabbit anti-OVA (AbD SEROTEC) at 1:200 dilution for 20 minutes on ice. After centrifugation and washing, cells were labeled with 1:200 ALEXAFLUOR488 anti-goat igg (invitrogen) and ALEXAFLUOR546 anti-rabbit igg (invitrogen) on ice for 20 minutes. After the final spin/wash cycle, cells were Hard mounted (Hard set) and imaged on a Zeiss LSM700 inverted confocal microscope with 63x oil immersion objective. Image analysis was performed in imagej (nih), and the same processing was performed on both images.

In vivo binding and biodistribution

Mu.g of ERY1-OVA or OVA in a volume of 100. mu.L of 0.9% saline (B.Braun) were injected intravenously into the tails of 8-12 week old female C57BL/6 mice under isoflurane anesthesia. Care was taken to ensure that the mice were kept at 37C with a heating pad during the experiment. At predetermined time points, 5 μ L of blood was collected from a small incision on the tail, diluted 100-fold into PBS containing 10mM EDTA, washed three times with PBS containing 10mg/mL BSA, and analyzed for OVA content by flow cytometry and ELISA. OVA was quantified by sandwich ELISA, where capture was performed using a mouse monoclonal anti-OVA antibody (Sigma), detection was performed using a polyclonal rabbit anti-OVA antibody (AbD SEROTEC), final detection was performed using a goat anti-rabbit IgG-HRP antibody (BioRad), followed by TMB substrate (GE Life Sciences). Hematological characterization was performed on the ADVIVA 2120 hematology system (Siemens). Erythrocyte-bound ERY1-GST was detected by incubating labeled cells with goat anti-GST (ge healthcare Life sciences), followed by incubation with AlexaFluor488 donkey anti-goat (Invitrogen), and analyzed by flow cytometry. For biodistribution studies, 20 μ g of either ERY1-APC or MIS-APC were injected intravenously into the tail vein of 8-12 week old female C57BL/6 mice, as described above. Mice were sacrificed at predetermined time points and spleen, blood and liver were removed. Each organ was digested with collagenase d (roche) and homogenized to obtain single cell suspensions for flow cytometry staining.

Adoptive transfer of T cells

Isolation of a magnetic bead negative selection kit (Miltenyi Biotec) from OT-I (CD 45.2) Using CD8 following the manufacturer's instructions⁺) Mouse spleen CD8⁺T cells. Freshly isolated CD8⁺OT-I cells were resuspended in PBS and labeled with 1. mu.M carboxyfluorescein succinimidyl ester (CFSE, Invitrogen) for 6 minutes at room temperature, and the reaction was quenched with an equal volume of IMDM (Gibco) with 10% FBS for 1 minute. Cells were washed, counted, and resuspended in pure IMDM prior to injection. Mix 3x10⁶A CFSE labeled CD8⁺OT-I cells intravenous injection into recipient CD45.1⁺In the tail vein of the mice. For short-term proliferation studies, 10 μ g of ERY1-OVA or OVA in a volume of 100 μ L was injected 24 hours after adoptive transfer. Splenocytes were harvested 5 days after antigen administration and stained for analysis by flow cytometry.

OT-I tolerance and challenge model

Mix 3x10⁵CFSE-labeled OT-I CD8⁺T cell injection into CD45.1⁺In the recipient mice, as described above. Mice were administered 10 μ g of ERY1-OVA or OVA in 100 μ L saline intravenously to the tail vein 1 and 6 days after adoptive transfer. 15 days after adoptive transfer, mice were challenged intradermally with 5 μ g OVA in 25 μ L and 25ng ultrapure E.coli LPS (InvivoGen) per hind leg footpad (Hock method, total dose 10 μ g OVA and 50ng LPS). Mice were sacrificed on day 4 post challenge andspleen and draining lymph node cells were isolated for restimulation. For flow cytometry analysis of intracellular cytokines, cells were restimulated in the presence of 1mg/mL OVA or 1 μ g/mL SIINFEKL (SEQ ID NO:3) peptide (Genscript) for 3 hours. Brefeldin (Brefeldin) -a (Sigma,5 μ g/mL) was added and restimulation continued for 3 hours, after which staining and flow cytometry analysis. For ELISA measurements of secreted factors, cells were restimulated in the presence of 100. mu.g/mL OVA or 1. mu.g/mL SIINFEKL (SEQ ID NO:3) peptide for 4 days. Cells were spun and media was collected for ELISA analysis using IFN γ and IL-10 Ready-Set-Go kit (eBiosciences) according to the manufacturer's instructions. OVA-specific serum IgG was detected by incubation of different dilutions of mouse blood on OVA-coated plates, followed by final incubation with goat anti-mouse IgG-HRP (southern Biotech).

OT-I E.G7-OVA tolerance model

1x10 as described above⁶CFSE-labeled OT-I CD8⁺T cells were injected into 8-12 week old female C57BL/6 mice. Mice were administered 10 μ g of ERY1-OVA or 10 μ g of OVA in 100 μ L saline intravenously in the tail vein 1 and 6 days after adoptive transfer. Blood was collected 5 days after adoptive transfer to characterize OT-I CD8 by flow cytometry⁺T cells proliferate. OVA-expressing EL-4 thymoma cells (E.G7-OVA, ATCC CRL-2113) were cultured according to ATCC guidelines. Briefly, cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 10mM HEPES, 1mM sodium pyruvate, 0.05mM β -mercaptoethanol, 1% puromycin/streptomycin (Invitrogen Gibco), and 0.4mg/mL G418(PAA Laboratories). Immediately prior to injection, cells were expanded in medium without G418 and resuspended in hbss (gibco) after harvest. 9 days after adoptive transfer, mice were anesthetized with isoflurane, the dorsal zone was debrided, and an intrascapular intradermal injection of 1x10 was made⁶G7-OVA cells. Tumor size was measured every 24 hours with a digital caliper 4 days after transplantation of g7-OVA, and tumor volume was calculated as ellipsoid (V = (pi/6) l · w · h), where V is the volume of the tumor, l is the length, w is the width, and h is the height). 15 days after adoptive transfer, 25 μMice were challenged intradermally at each foreleg footpad with 5 μ g OVA and 25ng of ultrapure e.coli LPS (invivogen) in L (total dose 10 μ g OVA and 50ng LPS).

Antibodies and flow cytometry

Flow cytometry used the following anti-mouse antibodies: CD1d Pacific Blue, CD3 ε PerCP-Cy5.5, CD8 α PE-Cy7, CD11b PE-Cy7, CD11c Pacific Blue, biotinylated CD45, CD45.2 Pacific Blue, CD45 Pacific Blue, IFN γ -APC, CD8 α APC-eF780, CD44PE-Cy5.5, CD62L PE, CD205 PE-Cy7, F4/80 PE, I-A/I-E MHCII FITC (all from eBioScience), and immobilized live/dead dyes (Invitron), annexin-V-Cy 5 labeling kit (BioVision), streptavidin Pacific Orange (Invitrogen), and anti-OVA-FITC (Abcam). Samples were analyzed on a CyAn ADP flow cytometer (Beckman Coulter). Cells were first washed with PBS, stained with live/dead dye on ice for 20 minutes, blocked with 24G2 hybridoma medium on ice for 20 minutes, surface stained on ice for 20 minutes, fixed in 2% paraformaldehyde on ice for 20 minutes, stained intracellularly in ice for 45 minutes in the presence of 0.5% saponin, followed by final washing, and then analyzed. For apoptotic staining, annexin-V-Cy 5 was added for 5 min and then analyzed. For CD45 staining, cells were stained with streptavidin pacifiic Orange on ice for 20 minutes, washed, and analyzed.

By means of particles

ERY1 peptide has also been implemented in the form of nanoparticles conjugated to both ERY1 peptide and tolerogenic antigen to develop tolerance.

To form a conjugate of ERY1 with a polymeric nanoparticle (which is also conjugated to a peptide or protein antigen), a stoichiometric amount of each component may be added sequentially to control the conjugation transformation. To form nanoparticles conjugated with both OVA and ERY1 or the mismatch peptide, the peptide was first dissolved in 3M aqueous guanidine hydrochloride solution and 0.5 equivalents were added to the nanoparticles containing the thiol-reactive pyridyl disulfide group. Absorbance measurements were made at 343nm to monitor reaction conversion, as the reaction produced non-reactive pyrimidine-2-thione species with high absorbance at this wavelength. After 2 hours at room temperature, the absorbance at 343nm had stabilized, OVA was dissolved in 3M aqueous guanidine hydrochloride solution and added to the nanoparticle solution in 2-fold molar excess. After 2 hours at room temperature, the absorbance at 343nm had stabilized again to a higher value and the concentration of both peptide and OVA in the solution was calculated. The bifunctional nanoparticles were purified from the unreacted components by gel filtration on a Sepharose CL6B packed column. Each 0.5mL fraction was analyzed for the presence of proteins and/or peptides by means of fluorescamine and the nanoparticle size was assessed by Dynamic Light Scattering (DLS).

If the antigens do not contain any free thiol groups to carry out the reaction, they can be introduced by recombinant DNA techniques to create mutants which can then be expressed recombinantly and purified. Alternatively, amine-carboxylic acid crosslinking may be performed between the nanoparticles and the antigen using 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC).

To form a conjugate of ERY1 with a polymeric micelle (which is also conjugated to a peptide or protein antigen), a reaction similar to that described for polymeric nanoparticles may be used. Micelles can be formed that contain functional groups that are desirable for a suitable conjugation scheme. Given that our nanoparticles and micelles can be synthesized to contain many different chemical group functionalisations, there are many possibilities for conjugation schemes that can be employed in creating nanoparticle/micelle-antigen-ERY 1 complexes.

Example 15: development of antibodies and antibody fragments that bind mouse and/or human red blood cells

As an alternative to non-covalent binding of erythrocytes, erythrocyte-binding antibodies can also be used to induce antigen-specific immunological tolerance. Antibodies displaying high affinity for red blood cell surface proteins can be isolated by screening antibody libraries using prior art display platforms including, but not limited to, phage display, yeast and e. After the discovery of new erythrocyte-binding antibodies, similar biochemical characterization of binding can be assessed as performed for the ERY1 peptide. To create higher affinity mutants with improved binding characteristics, antibody fragments found to bind red blood cells from the initial library screen were affinity matured. Novel libraries are created from parental binding sequences using standard recombinant DNA techniques, such as error-prone PCR and site-directed mutagenesis. The affinity maturation library is then displayed using a prior art display platform as described above to obtain additional antibody fragments with enhanced affinity for erythrocytes as compared to the parent binding sequence.

Affinity maturation was also performed on existing antibodies that bind mouse erythrocytes or human erythrocytes. The binding sites of the rat monoclonal TER-119 clone antibody (Kina et al, Br J Haematol,2000) to mouse red blood cells have yet to be fully determined, but due to its specificity it has been commonly used to remove red blood cells from heterogeneous cell populations. Affinity maturation was performed on the TER-119 antibody (either as a full-length antibody or as an antibody fragment such as scFv) to discover new antibodies with higher affinity for mouse erythrocytes. The mouse monoclonal 10F7 clone antibody (Langlois et al, J Immunol 1984) binds human blood plasma glycoprotein-A on the surface of human red blood cells. Affinity maturation was performed on the 10F7 antibody (either as a full length antibody or as an antibody fragment such as scFv) to find new antibodies with higher affinity for human erythrocytes.

To determine the primary sequence of the TER-119 antibody, we cloned the specifically isolated cDNA of the antibody from the TER-119 hybridoma into a plasmid that allows for easy sequencing of the gene fragment. A set of specific primers is used to perform an antibody fragment PCR amplification process that allows for the amplification of multiple variable domains of a gene segment (Krebber et al, 1997; Reddy et al, 2010). The DNA sequence of the antibody domain allows us to determine the variable regions of the heavy and light chains of the TER-119 IgG antibody. To construct an scFv version of TER-119 IgG, we used assembly pcr (assembly pcr) to create the following genes, which were composed: the variable heavy chain of TER-119, followed by (Gly-Gly-Gly-Gly-Ser)₄(SEQ ID NO:18) linker followed by the variable light chain of TER-119.

Standard reverse transcriptase PCR (RT-PCR) was performed on mRNA from the TER-119 hybridoma clone using Superscript III first strand synthesis system (Invitrogen) to make complementary DNA (cDNA) for that clone. Then, PCR was performed to specifically amplify the DNA sequences of the Variable Heavy (VH) and Variable Light (VL) regions of the antibody using the following set of primers:

the amplified VH and VL genes were then digested with restriction endonucleases (NcoI and NotI for VL, NdeI and HindIII for VH), and the gene fragments were purified after agarose electrophoresis using standard kits (ZymoResearch, Orange, CA, USA) and ligated into the cloning plasmid pMAZ 360. Plasmids containing either the VH or VL genes were sequenced and a new gene was constructed using assembly PCR to create the TER-119scFv sequence:

5’-GAGGTGAAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAACCTGGGGGGTCTCTGAAACTCTCCTGTGTAGCCTCAGGATTCACTTTCAGGGACCACTGGATGAATTGGGTCCGGCAGGCTCCCGGAAAGACCATGGAGTGGATTGGAGATATTAGACCTGATGGCAGTGACACAAACTATGCACCATCTGTGAGGAATAGATTCACAATCTCCAGAGACAATGCCAGGAGCATCCTGTACCTGCAGATGAGCAATATGAGATCTGATTACACAGCCACTTATTACTGTGTTAGAGACTCACCTACCCGGGCTGGGCTTATGGATGCCTGGGGTCAAGGAACCTCAGTCACTGTCTCCTCAGCCGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGATCCGACATTCAGATGACGCAGTCTCCTTCAGTCCTGTCTGCATCTGTGGGAGACAGAGTCACTCTCAACTGCAAAGCAAGTCAGAATATTAACAAGTACTTAAACTGGTATCAGCAAAAGCTTGGAGAAGCTCCCAAAGTCCTGATATATAATACAAACAATTTGCAAACGGGCATCCCATCAAGGTTCAGTGGCAGTGGATCTGGTACAGATTTCACACTCACCATCAGTAGCCTGCAGCCTGAAGATTTTGCCACATATTTCTGCTTTCAGCATTATACTTGGCCCACGTTTGGAGGTGGGACCAAGCTGGAAATCAAACGTACT-3' (SEQ ID NO:76) encoding the TER-119 clone VH domain located N-terminal to the translated protein, followed by (Gly-Gly-Gly-Gly-Ser)₄(SEQ ID NO:18) linker domain followed by the TER-119 clone VL domain at the C-terminus of the translated protein. The TER-119cDNA was amplified with primers SK07 and SK08 (specific for the VH region) and SK09 and SK10 (specific for the VL region) to construct the TER-119scFv gene:

each of the finally completed scFv gene products was digested with SfiI and XhoI (NEB, Ipswich, MA, USA) and ligated into the same site on the pSecTagA mammalian expression plasmid (Invitrogen, Carlsbad, CA, USA).

This gene was commercially synthesized and obtained from DNA2.0 (menlopack, CA, USA) for affinity maturation of the 10F7 scFv that binds human blood group glycoprotein-a, and the sequence was as follows:

5’-GTTATTACTCGCGGCCCAGCCGGCCATGGCGGCGCAGGTGAAACTGCAGCAGAGCGGCGCGGAACTGGTGAAACCGGGCGCGAGCGTGAAACTGAGCTGCAAAGCGAGCGGCTATACCTTTAACAGCTATTTTATGCATTGGATGAAACAGCGCCCGGTGCAGGGCCTGGAATGGATTGGCATGATTCGCCCGAACGGCGGCACCACCGATTATAACGAAAAATTTAAAAACAAAGCGACCCTGACCGTGGATAAAAGCAGCAACACCGCGTATATGCAGCTGAACAGCCTGACCAGCGGCGATAGCGCGGTGTATTATTGCGCGCGCTGGGAAGGCAGCTATTATGCGCTGGATTATTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGATATTGAACTGACCCAGAGCCCGGCGATTATGAGCGCGACCCTGGGCGAAAAAGTGACCATGACCTGCCGCGCGAGCAGCAACGTGAAATATATGTATTGGTATCAGCAGAAAAGCGGCGCGAGCCCGAAACTGTGGATTTATTATACCAGCAACCTGGCGAGCGGCGTGCCGGGCCGCTTTAGCGGCAGCGGCAGCGGCACCAGCTATAGCCTGACCATTAGCAGCGTGGAAGCGGAAGATGCGGCGACCTATTATTGCCAGCAGTTTACCAGCAGCCCGTATACCTTTGGCGGCGGCACCAAACTGGAAATTAAACGCGCGGCGGCGGCCTCGGGGGCCGAGGGCGGCGGTTCT-3’(SEQ ID NO:81)。

similar affinity maturation using the recombinant DNA technology described above for TER-119 was performed on the 10F7 gene to obtain a library of mutants, providing conditions for screening for enhanced binding to human erythrocytes.

Example 16: antigen-specific immunological tolerance is induced via non-covalent binding of erythrocytes to antigen coupled with antibodies.

Standard crosslinking reactions can be used to conjugate the antibody to the antigen, as mentioned in example 14 and elsewhere herein. The purified antibody-antigen conjugates will exhibit induction of tolerance to antigens in standard mouse models of type 1 diabetes, multiple sclerosis, islet transplantation, and OVA model antigens.

To demonstrate induction of tolerance to OVA, mice can be administered either intravenously or extravascularly an OVA-antibody conjugate or an OVA-nanoparticle-antibody conjugate. At a predetermined number of days post-administration, mice were sacrificed and lymph nodes, spleen, and blood were harvested for analysis. Splenocytes and lymph node derived cells were plated and re-stimulated with OVA and/or SIINFEKL peptides ex vivo for 3 days and measured for down-regulation of IFN γ, IL-17a, IL-2, and IL-4 expression, and TGF- β 1 up-regulation by ELISA, all as evidence of established tolerance. Intracellular staining of splenocytes and lymph node derived cells was performed using flow cytometry after 6 hours of ex vivo restimulation with OVA and/or SIINFEKL peptides for IFN γ, IL-17a, IL-2 and IL-4. In addition, flow cytometry was used to characterize the expression profiles of CD4, CD8, and regulatory T cells from lymph nodes, spleen, and blood-derived cells. In addition, blood samples were taken from mice at different time points to measure humoral antibody responses against OVA antigens. Ex vivo restimulation variant experiments were performed to determine if systemic tolerance had been established. Mice were administered either OVA-antibody conjugates or OVA-antibody-nanoparticle conjugates, as described above, 9 days later with additional OVA in the presence of adjuvants (lipopolysaccharide, complete freund's adjuvant, alum, etc.), and splenocytes were evaluated for responsiveness to OVA antigen by ELISA and/or flow cytometry, as described above. OVA-antibody conjugates and/or OVA-antibody-nanoparticle formulations can render splenocytes unresponsive to a second challenge with OVA and adjuvant, an effectively established method to demonstrate systemic tolerance. Similar in vivo challenge experiments can be performed with transgenic cell lines after the initial administration of OVA-antibody conjugates and/or OVA-antibody-nanoparticle formulations as further demonstration of tolerance, such as adoptive transfer with OT-I T cells, similar to the study detailed in example 14. To demonstrate immune tolerance of therapeutic molecules in autoimmune or deimmunized mouse models, similar antibody conjugates can be generated against the relevant antigens as described herein for OVA.

Example 17: non-covalent binding of antigens via fusion of erythrocytes with single-chain antibodies induces antigen-specific immunological tolerance

Single chain antibody fragments (scFv) can be used as non-covalent binding agents for erythrocytes. scFv exhibiting high affinity for red blood cell surface proteins can be isolated by screening scFv libraries using prior art display platforms, as discussed in example 13. After the discovery of new erythrocyte-binding antibody fragments, similar biochemical characterization of binding, as performed with ERY1 peptide, was evaluated. Since scFv has one polypeptide chain, it is fused to the antigen in a site-specific recombination manner using standard recombinant DNA techniques. Depending on the nature of the antigen fusion partner, the scFv is fused to the N-or C-terminus of the antigen to create bifunctional protein species. Where the Major Histocompatibility Complex (MHC) peptide recognition sequence is known for the antigen, a peptide is also inserted into the linker domain of the scFv, thus creating a novel bifunctional antibody/antigen construct that contains the natural terminus of the scFv.

To demonstrate tolerance induction to OVA, mice can be administered either intravenously or extravascularly OVA-scFv or OVA-nanoparticle-scFv conjugates. At a predetermined number of days post-administration, mice were sacrificed and lymph nodes, spleen, and blood were harvested for analysis. Spleen cells and lymph node-derived cells are plated, re-stimulated with OVA and/or SIINFEKL peptide (SEQ ID NO:3) ex vivo for 3 days, and their down-regulation of IFN γ, IL-17a, IL-2, and IL-4 expression, as well as up-regulation of TGF-. beta.1, measured, for example, by ELISA, are evidence of established tolerance. After 6 hours of ex vivo restimulation with OVA and/or SIINFEKL peptide (SEQ ID NO:3), intracellular staining of IFN γ, IL-17a, IL-2 and IL-4 was performed on splenocyte and lymph node derived cells using flow cytometry. In addition, flow cytometry can be used to characterize the expression profiles of CD4, CD8, and regulatory T cells from lymph nodes, spleen, and blood-derived cells. In addition, blood samples were taken from mice at different time points to measure humoral antibody responses against OVA antigens. Ex vivo restimulation variant experiments were performed to determine if systemic tolerance had been established. Mice were administered OVA-scFv or OVA-nanoparticle-scFv, as described above, 9 days later with OVA in the presence of adjuvants (lipopolysaccharide, complete freund's adjuvant, alum, etc.), and splenocytes were assessed for responsiveness to OVA antigen by ELISA and/or flow cytometry, as described above. The OVA-scFv and/or OVA-scFv-nanoparticle formulations made splenocytes non-responsive to a second challenge with OVA and adjuvant, thereby demonstrating an effective establishment of systemic tolerance. Following initial administration of the OVA-scFv and/or OVA-scFv-nanoparticle formulations, similar in vivo challenge experiments can be performed with transgenic cell lines to demonstrate tolerance, such as adoptive transfer with OT-I T cells, similar to the study detailed in example 14. To demonstrate immune tolerance of therapeutic molecules in autoimmune or de-immunized mouse models, similar scFv fusions can be generated to the relevant antigens, as described herein for OVA.

Antibody constructs that bind both mouse red blood cells and display MHC-I immunodominant epitopes of OVA (SGLEQLESIINFEKL) (SEQ ID NO:82) were created using standard recombinant DNA techniques. Using overlap extension PCR, we first created a DNA fragment encoding the terminal 3 ' domain (comprising SGLEQLESIINFEKL (SEQ ID NO:82) peptide) and the overlapping 5 ' domain complementary to the 3 ' end of the TER119 sequence. This DNA fragment was used as a reverse primer, along with a complementary forward 5' primer, in a standard PCR to create the entire DNA fragment encoding TER119-SGLEQLESIINFEKL (SEQ ID NO: 82):

5’-GAGGTGAAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAACCTGGGGGGTCTCTGAAACTCTCCTGTGTAGCCTCAGGATTCACTTTCAGGGACCACTGGATGAATTGGGTCCGGCAGGCTCCCGGAAAGACCATGGAGTGGATTGGAGATATTAGACCTGATGGCAGTGACACAAACTATGCACCATCTGTGAGGAATAGATTCACAATCTCCAGAGACAATGCCAGGAGCATCCTGTACCTGCAGATGAGCAATATGAGATCTGATTACACAGCCACTTATTACTGTGTTAGAGACTCACCTACCCGGGCTGGGCTTATGGATGCCTGGGGTCAAGGAACCTCAGTCACTGTCTCCTCAGCCGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGATCCGACATTCAGATGACGCAGTCTCCTTCAGTCCTGTCTGCATCTGTGGGAGACAGAGTCACTCTCAACTGCAAAGCAAGTCAGAATATTAACAAGTACTTAAACTGGTATCAGCAAAAGCTTGGAGAAGCTCCCAAAGTCCTGATATATAATACAAACAATTTGCAAACGGGCATCCCATCAAGGTTCAGTGGCAGTGGATCTGGTACAGATTTCACACTCACCATCAGTAGCCTGCAGCCTGAAGATTTTGCCACATATTTCTGCTTTCAGCATTATACTTGGCCCACGTTTGGAGGTGGGACCAAGCTGGAAATCAAACGTACTCATCATCACCATCATCACGGTGGCGGTTCTGGCCTGGAGCAGCTGGAGTCTATTATTAATTTCGAAAAACT G-3' (SEQ ID NO: 83). The underlined sequence indicates the gene segment encoding SGLEQLESIINFEKL. The DNA fragment is inserted into mammalian and prokaryotic expression vectors for recombinant expression.

Standard recombinant DNA techniques were used to create antibody constructs that both bind mouse red blood cells and display the chromogranin-A mimotope 1040-p31(YVRPLWVRME) (SEQ ID NO: 84). A DNA fragment encoding the terminal 3 ' domain comprising the YVRPLWVRME (SEQ ID NO:84) peptide and an overlapping 5 ' domain complementary to the 3 ' end of the TER119 sequence was created using overlap extension PCR. This DNA fragment was used as a primer, along with a complementary forward 5' primer, in standard PCR to create the entire DNA fragment encoding TER 119-YVRPLWVRME:

5’-GAGGTGAAGCTGCAGGAGTCAGGAGGAGGCTTGGTGCAACCTGGGGGGTCTCTGAAACTCTCCTGTGTAGCCTCAGGATTCACTTTCAGGGACCACTGGATGAATTGGGTCCGGCAGGCTCCCGGAAAGACCATGGAGTGGATTGGGGATATTAGACCTGATGGCAGTGACACAAACTATGCACCATCTGTGAGGAATAGATTCACAATCTCCAGAGACAATACCAGGAGCATCCTGTACCTGCAGATGGGCAATATGAGATCTGATTACACAGCCACTTATTACTGTGTTAGAGACTCACCTACCCGGGCTGGGCTTATGGATGCCTGGGGTCAAGGAACCTCAGTCACTGTCTCCTCAGCCGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGATCCGACATTCAGATGACGCAGTCTCCTTCAGTCCTGTCTGCATCTGTGGGAGACAGAGTCACTCTCAACTGCAAAGCAAGTCAGAATATTAACAAGTACTTAAACCGGTATCAGCAAAAGCTTGGAGAAGCTCCCAAAGTCCTGGTATATAATACAAACAATTTGCAAACGGGCATCCCATCAAGGTTCAGTGGCAGTGGATCTGGCACAGATTTCACACTCACCATCAGTAGCCTGCAGCCTGAAGATTTTGCCACATATTTCTGCTTTCAGCATTATACTTGGCCCACGTTTGGAGGTGTGACCAAGCTGGAAATCAAACGTACTCATCATCACCATCATCACGGTGGCGGTTATGTCAGACCTCTGTGGGTCAGAATGGAA-3' (SEQ ID NO: 85). The underlined sequence indicates the gene segment encoding the mimotope (YVRPLWVRME) (SEQ ID NO:84) of chromogranin-A (1040-p 31). The DNA fragment is inserted into mammalian and prokaryotic expression vectors for recombinant expression.

Standard recombinant DNA techniques were used to create antibody constructs that both bind mouse erythrocytes and display mouse proinsulin, a major diabetic autoantigen in NOD mice. Using overlap extension PCR, we first created a DNA fragment that encodes the terminal 3 ' domain (comprising the entire proinsulin protein), and the overlapping 5 ' domain that is complementary to the 3 ' end of the TER119 sequence. This DNA fragment was used as a primer in standard PCR, along with a complementary forward 5' primer to create the entire DNA fragment encoding TER 119-proinsulin:

5’-GAGGTGAAGCTGCAGGAGTCAGGAGGAGGCTTGGTGCAACCTGGGGGGTCTCTGAAACTCTCCTGTGTAGCCTCAGGATTCACTTTCAGGGACCACTGGATGAATTGGGTCCGGCAGGCTCCCGGAAAGACCATGGAGTGGATTGGAGATATTAGACCTGATGGCAGTGACACAAACTATGCACCATCTGTGAGGAATAGATTCACAATCTCCAGAGACAATGCCAGGAGCATCCTGTACCTGCAGATGAGCAATATGAGATCTGATTACACAGCCACTTATTACTGTGTTAGAGACTCACCTACCCGGGCTGGGCTTATGGATGCCTGGGGTCAAGGAACCTCAGTCACTGTCTCCTCAGCCGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGATCCGACATTCAGATGACGCAGTCTCCTTCAGTCCTGTCTGCATCTGTGGGAGACAGAGTCACTCTCAACTGCAAAGCAAGTCAGAATATTAACAAGTACTTAAACTGGTATCAGCAAAAGCTTGGAGAAGCTCCCAAAGTCCTGATATATAATACAAACAATTTGCAAACGGGCATCCCATCAAGGTTCAGTGGCAGTGGATCTGGTACAGATTTCACACTCACCATCAGTAGCCTGCAGCCTGAAGATTTTGCCACATATTTCTGCTCTCAGCATTATACTTGGCCCACGTTTGATGGTGGGACCAAGCTGGAAATCAAACGTACTCATCATCACCATCATCACGGTGGCGGTTTTGTGAAACAGCATCTGTGCGGTCCGCATCTGGTGGAAGC GCTGTATCTGGTGTGCGGCGAACGTGGCTTTTTTTATACCCCGAAAAGC CGTCGTGAAGTGGAAGATCCGCAGGTGGAACAGCTGGAACTGGGCGG CAGCCCGGGTGATCTGCAGACCCTGGCCCTGGAAGTGGCGCGTCAGA AACGTGGCATTGTGGATCAGTGCTGCACCAGCATTTGCAGCCTGTATCA GCTGGAAAACTATTACAAC-3' (SEQ ID NO: 86). The underlined sequence indicates the proinsulin gene segment of the construct. The DNA fragment is inserted into mammalian and prokaryotic expression vectors for recombinant expression.

Example 18: synthesis of branched polymers comprising erythrocyte-binding ligands and other functions

To synthesize 8-arm PEG-thioacetate, 8-arm PEG-OH (Nektar) was dissolved in toluene and reacted with 10 equivalents of triethylamine (Sigma Aldrich, CAS #121-44-8) and 10 equivalents of methanesulfonyl chloride (Sigma Aldrich, CAS #124-63-0) under argon at room temperature for 18 hours. The residue was filtered off, the filtrate was concentrated under reduced pressure, dissolved in Dimethylformamide (DMF), and 10 equivalents of potassium thioacetate (Sigma Aldrich, CAS #10387-40-3) were added. After 18 hours at room temperature, the residue is filtered off, the filtrate is concentrated under reduced pressure and precipitated in diethyl ether. The precipitate was filtered and dried under reduced pressure to obtain the final product.

To synthesize 8-arm PEG-pyridyl disulfide, 8-arm PEG-thioacetate was dissolved in Dimethylformamide (DMF) and deprotected with 1.05 equivalents of sodium methanolate (Sigma Aldrich, CAS #124-41-4) in a Schlenk tube under argon at room temperature for 1 hour. To reduce the deprotected thiol to the thiolate, 2 equivalents of tris (2-carboxyethyl) phosphine hydrochloride (TCEP, Thermoscientific, CAS #51805-45-9) and 2 equivalents of distilled water were added to the solution. After 2 hours at room temperature, 12 equivalents of 2, 2' -dithiodipyridine (Aldrithiol-2, SigmaAldrich, CAS #2127-03-9) were added and the solution was stirred at room temperature for 24 hours. The reaction mixture was then dialyzed against 5L of distilled water in an MWCO 3,500Da dialysis tube for 48 hours, during which the distilled water was changed 4 times. The pyridyl disulfide loaded onto 8-arm PEG was quantified by reduction in 25mM TCEP in 100mM HEPES, pH8.0, and UV-visible spectroscopy was measured at 343nm to monitor for the presence of a pyrimidine-2-thione leaving group.

To synthesize 8-arm PEG-pyridyl disulfide-ALEXAFULUOR 647, 8-arm PEG-thioacetate was dissolved in DMF and deprotected with 1.05 equivalents of sodium methoxide (Sigma Aldrich, CAS #124-41-4) in a Schlenk tube under argon at room temperature for 1 hour. To reduce the deprotected thiol to thiolate, 2 equivalents of tris (2-carboxyethyl) phosphine hydrochloride (TCEP, Thermo Scientific, CAS #51805-45-9) and an equal volume of 100mM HEPES pH8.0 were added to the solution. After 2 hours at room temperature, 0.125 equivalents (equivalent to 1 arm in 8 strips) of AlexaFluor 647-C2-maleimide (Invitrogen) were added to the solution. After 2 hours at room temperature, 12 equivalents of 2, 2' -dithiodipyridine (Aldrithiol-2, Sigma Aldrich, CAS #2127-03-9) were added and the solution was stirred at room temperature for 24 hours. The reaction mixture was then dialyzed against 5L of distilled water in an MWCO 3,500Da dialysis tube for 48 hours, during which the distilled water was changed 4 times. The pyridyl disulfide loaded onto 8-arm PEG was quantified by reduction in 25mM TCEP in 100mM HEPES, pH8.0, and UV-visible spectroscopy was measured at 343nm to monitor for the presence of a pyrimidine-2-thione leaving group.

Thiol-containing peptides were conjugated to 8-arm PEG-pyridyl disulfide by adding a stoichiometric amount of the peptide dissolved in 3M aqueous guanidine hydrochloride (Sigma Aldrich, CAS #50-01-10) to an aqueous solution of 8-arm PEG-pyridyl disulfide at room temperature. The reaction conversion was monitored by quantifying the presence of the pyrimidine-2-thione leaving group by measuring the UV-visible spectrum at 343 nm. If more than one molecule is to be conjugated to the 8-arm PEG-pyridyl disulfide, the reaction process is repeated with the new molecule in the same pot. Once conjugation was complete, the reaction mixture was desalted on a ZEBASPIN desalting column (Thermo Scientific) and the purified product was stored under suitable sterile conditions.

For the 8-arm PEG-ERY1/MIS-SIINFEKL conjugate (SIINFEKL: SEQ ID NO:3), induction of tolerance to OVA can be demonstrated by administering it intravenously or extravascularly to mice. When human specific ligands are used, this test will also indicate induction of tolerance in humans. In such demonstrations, mice were sacrificed a predetermined number of days after administration, and lymph nodes, spleen, and blood were harvested for analysis. Splenocytes and lymph node derived cells were plated and re-stimulated with OVA and/or SIINFEKL (SEQ ID NO:3) peptides ex vivo for 3 days and measured for down-regulation of IFN γ, IL-17a, IL-2, and IL-4 expression, as well as up-regulation of TGF-. beta.1 by ELISA, as evidence of established tolerance. Intracellular staining of IFN γ, IL-17a, IL-2 and IL-4 was performed using flow cytometry on splenocyte and lymph node derived cells after 6 hours of ex vivo restimulation with OVA and/or SIINFEKL (SEQ ID NO:3) peptides. In addition, flow cytometry was used to characterize the expression profiles of CD4, CD8, and regulatory T cells from lymph nodes, spleen, and blood-derived cells. In addition, blood samples were taken from mice at different time points to measure humoral antibody responses against OVA antigens. Ex vivo restimulation variant experiments were performed to determine if systemic tolerance had been established. Mice were administered with 8-arm PEG-ERY1/MIS-SIINFEKL conjugate (SIINFEKL: SEQ ID NO:3), as described above, followed by administration of OVA in the presence of adjuvants (lipopolysaccharide, complete Freund's adjuvant, alum, etc.) after 9 days, and splenocytes were evaluated for responsiveness to OVA antigen by ELISA and/or flow cytometry, as described above. The 8-arm PEG-ERY1/MIS-SIINFEKL conjugate (SIINFEKL: SEQ ID NO:3) formulation rendered splenocytes unresponsive to a second challenge with OVA and adjuvant, an effectively established method to demonstrate systemic tolerance. After the initial administration of the 8-arm PEG-ERY1/MIS-SIINFEKL conjugate formulation (SIINFEKL: SEQ ID NO:3), similar in vivo challenge experiments can be performed with transgenic cell lines to further demonstrate tolerance, such as adoptive transfer with OT-IT cells, similar to the study detailed in example 14. To demonstrate immune tolerance of therapeutic molecules in autoimmune or de-immunized mouse models, similar 8-arm PEG constructs can be generated for the relevant antigens as described herein for SIINFEKL (SEQ ID NO: 3).

Example 19: induction of antigen-specific immunological tolerance via non-covalent binding of erythrocytes to aptamer-conjugated antigens

Other non-antibody bioaffinity reagents can be used to perform methods to measure their ability to induce immunological tolerance by non-covalent erythrocyte binding. Screening for other protein-based affinity modules, such as designed ankyrin repeat proteins (DARPins) (Steiner, Forrer et al, 2008), designed armadillo (armadillo) repeat proteins (Parmeggiani, pelalin et al, 2008), fibronectin domains (Hackel, Kapila et al, 2008), and cysteine-knot (knottin) affinity scaffolds (Silverman, Levin et al, 2009) exhibited binding affinity to red blood cells.

Library screening was performed using a well-established exponential enrichment of ligand phylogenetic (SELEX) method (Archemix, Cambridge, MA, USA) (Sampson,2003) to find DNA/RNA aptamers with high affinity for erythrocytes. After new DNA/RNA sequences have been discovered that bind red blood cells with high affinity, they are chemically synthesized to contain additional chemically reactive groups at their 3 'or 5' ends for conjugation to antigens and/or polymeric micelles/nanoparticles. For example, chemically synthesized aptamers do contain reactive NH₂A group conjugated to a COOH group present on the nanoparticle or antigen or nanoparticle-antigen complex via EDC/NHS conjugation chemistry to create a single bioconjugate consisting of a red blood cell binding aptamer and an antigen or antigen-nanoparticle. Various chemical conjugation techniques were attempted by varying the orthogonal reactive groups and conjugation schemes for both aptamers, antigens, and/or antigen-nanoparticles.

To demonstrate tolerance induction against OVA, mice were administered either intravenously or extravascularly OVA-aptamer or OVA-nanoparticle-aptamer conjugates. At a predetermined number of days post-administration, mice were sacrificed and lymph nodes, spleen, and blood were harvested for analysis. Splenocytes and lymph node derived cells were plated and re-stimulated with OVA and/or SIINFEKL peptide (SEQ ID NO:3) ex vivo for 3 days and measured for down-regulation of IFN γ, IL-17a, IL-2, and IL-4 expression, as well as up-regulation of TGF-. beta.1 by ELISA, as evidence of established tolerance. Intracellular staining of IFN γ, IL-17a, IL-2 and IL-4 was performed using flow cytometry on splenocyte and lymph node derived cells after 6 hours of ex vivo restimulation with OVA and/or SIINFEKL (SEQ ID NO:3) peptides. In addition, flow cytometry was used to characterize the expression profiles of CD4, CD8, and regulatory T cells from lymph nodes, spleen, and blood-derived cells. In addition, blood samples were taken from mice at different time points to measure humoral antibody responses against OVA antigens. Ex vivo restimulation variant experiments were performed to determine if systemic tolerance had been established. Mice were administered OVA-antibody or OVA-antibody-nanoparticle conjugates as described above, 9 days later with OVA in the presence of adjuvants (lipopolysaccharide, complete freund's adjuvant, alum, etc.) and splenocytes were assessed for responsiveness to OVA antigen by ELISA and/or flow cytometry as described above. We expect our OVA-antibody and/or OVA-antibody-nanoparticle formulations to render splenocytes non-responsive to a second challenge with OVA and adjuvant, thus demonstrating effective establishment of systemic tolerance. After the initial administration of our OVA-aptamer and/or OVA-aptamer-nanoparticle formulations, similar in vivo challenge experiments were performed with transgenic cell lines to demonstrate tolerance, such as adoptive transfer with OT-I T cells, similar to the study detailed in example 14. To demonstrate immune tolerance of therapeutic molecules in autoimmune or deimmunized mouse models, similar aptamer constructs can be generated against the relevant antigens, as described herein for OVA.

Further disclosure

Various embodiments of the present invention are described. One embodiment is an isolated peptide comprising at least 5 contiguous amino acid residues of a sequence selected from the group consisting of seq id no:11, 13, 14, 15, 16, 17, 1 and conservative substitutions thereof, wherein said sequences specifically bind erythrocytes. One embodiment is a peptide having one or more residues with D to L substitutions or conservative substitutions with at least one and no more than two amino acids in a sequence selected from the group consisting of: 11, 13, 14, 15, 16, 17 and 1. One embodiment is a peptide having at least 5 consecutive amino acid residues of a sequence selected from the group consisting of: 11, 13, 14, 15, 16, 17, 1 and conservative substitutions thereof, wherein said sequences specifically bind erythrocytes. For example, the peptide may have a number of residues from about 10 to about 80. The peptide may further comprise a therapeutic agent, for example selected from the group consisting of: insulin, pramlintide acetate, growth hormone, insulin-like growth factor-1, erythropoietin, type 1 alpha interferon, interferon alpha 2a, interferon alpha 2b, interferon beta 1a, interferon beta 1b, interferon gamma 1b, beta-glucocerebrosidase, adenosine deaminase, granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor, interleukin 1, interleukin 2, interleukin 11, factor VIIa, factor VIII, factor IX, exenatide, L-asparaginase, labyrinase, tumor necrosis factor receptor, and enfuvirdine. The peptide may further comprise a member of the group: antibodies, antibody fragments, and single chain antigen binding domains (ScFv). The peptide may further comprise a tolerogenic antigen, for example, selected from the group consisting of: proteins defective in genetic diseases, proteins with non-human glycosylation, non-human proteins, synthetic proteins not naturally occurring in humans, human food antigens, human transplantation antigens, and human autoimmune antigens. The peptide may have one or more sequences that specifically bind to red blood cells, the sequences may be repeats of the same sequence or a mixture of various sequences that effect such binding.

One embodiment is a method of generating immune tolerance comprising administering a composition comprising a molecular fusion comprising a tolerogenic antigen and an erythrocyte-binding moiety that specifically binds to an erythrocyte in the patient and thereby links the antigen to the erythrocyte, wherein the molecular fusion is administered in an amount effective to generate immune tolerance to a substance comprising the tolerogenic antigen. One embodiment is the method, wherein the molecular fusion is comprised of at least one erythrocyte-binding moiety directly covalently bonded to an antigen: for example a fusion protein comprising a moiety and an antigen. One embodiment is the method, wherein the molecular fusion comprises at least one erythrocyte-binding moiety attached to a particle that is attached to or contains an antigen, e.g., wherein the particle is selected from the group consisting of: microparticles, nanoparticles, liposomes, polymersomes, and micelles. One embodiment is the case wherein the tolerogenic antigen comprises a portion of a therapeutic protein, e.g., the protein comprises factor VIII or factor IX. One embodiment is the case wherein the tolerogenic antigen comprises a portion of a non-human protein. One embodiment is the case where the protein comprises adenosine deaminase, L-asparaginase, labyrinase, antithymocyte globulin, L-arginase, and L-methioninase. One embodiment is the method, wherein the patient is a human and the tolerogenic antigen comprises a portion of a protein that does not occur in nature. One embodiment is where the patient is a human and the tolerogenic antigen comprises glycans containing non-human glycosylated proteins. One embodiment is the case wherein the tolerogenic antigen comprises at least a portion of a human transplantation antigen. One embodiment is the case wherein the tolerogenic antigen comprises a portion of a human autoimmune disease protein, for example selected from the group consisting of: preproinsulin, proinsulin, insulin, GAD65, GAD67, IA-2 β, thyroglobulin, thyroid peroxidase, thyroid stimulating hormone receptor, myelin basic protein, myelin oligodendrocyte glycoprotein, proteolipid protein, collagen II, collagen IV, acetylcholine receptor, matrix metalloproteins 1 and 3, chaperone heat shock protein 47, microfibril-1, PDGF receptor α, PDGF receptor β, and nucleoprotein SS-A. One embodiment is the case wherein the tolerogenic antigen comprises a portion of a human food, for example selected from the group consisting of: chaperonin (Ara h1), allergen II (Ara h2), peanut agglutinin (Ara h6), alpha-lactalbumin (ALA), lactoferrin, gluten, low molecular weight gluten, alpha and gamma-gliadins, hordein, secalin, and avenin. One embodiment is the case where the red blood cell binding module is selected from the group consisting of: peptide ligands, antibodies, antibody fragments, and single chain antigen binding domains (ScFv). One embodiment is the case where the scFv comprises some or all of 10F7, e.g., one or more of the higher affinity variants of a light chain of 10F7 and/or a heavy chain of 10F7 and/or a light chain of 10F7 and/or a heavy chain of 10F 7. One embodiment is the method, wherein the red blood cell binding moiety comprises a peptide ligand comprising at least 5 contiguous amino acid residues of a sequence selected from the group consisting of seq id no:11, 13, 14, 15, 16, 17, 1 and conservative substitutions thereof, wherein said sequences specifically bind erythrocytes.

One embodiment is a composition comprising a molecular fusion comprising a tolerogenic antigen and a red blood cell-binding moiety that specifically binds to red blood cells in the patient and thereby links the antigen to the red blood cells. An example is the case where the erythrocyte binding moiety is covalently bonded to the antigen. Another example is where the molecular fusion comprises an erythrocyte-binding moiety attached to a particle, which is attached to an antigen, such as a microparticle, nanoparticle, liposome, polymersome, or micelle. Examples of tolerogenic antigens are: a portion of a therapeutic protein, a portion of a non-human protein, a portion of a protein not naturally found in humans (including an intact portion, i.e., whole), a glycan comprising a non-human glycosylated protein, a portion of a human autoimmune antigen, a portion of a human food. One embodiment is a composition, wherein the red blood cell binding moiety is selected from the group consisting of: peptide ligands, antibodies, antibody fragments, and single chain antigen binding domains (ScFv), e.g., all or part of 10F 7. The red blood cell binding module may comprise a peptide ligand comprising at least 5 consecutive amino acid residues of a sequence selected from the group consisting of: 11, 13, 14, 15, 16, 17, 1 and conservative substitutions thereof, wherein said sequences specifically bind erythrocytes. The red blood cell binding moiety may be one that comprises a peptide ligand having a dissociation constant of about 10 μ M to 0.1nM, as determined by equilibrium binding measurement between the peptide and red blood cells.

Another example is a composition comprising a red blood cell binding module that specifically binds red blood cells linked to an entity selected from the group consisting of: synthetic polymers, branched synthetic polymers, and particles. For example, the particles can be microparticles, nanoparticles, liposomes, polymersomes, and micelles. The composition may further comprise a tolerogenic antigen, a therapeutic agent, or a tumor homing ligand.

Embodiments include a method of embolizing a tumor in a patient, comprising: administering to the patient a composition comprising a molecular fusion of an erythrocyte-binding moiety and a tumor-homing ligand, or a medicament comprising the composition, wherein the tumor-homing ligand is an antibody, an antibody fragment, a single-chain antigen-binding domain (ScFv), or a peptide ligand targeted to specifically bind a target selected from the group consisting of a tumor and tumor vasculature, and wherein the erythrocyte-binding moiety comprises a peptide ligand, an antibody fragment, a ScFv, or an aptamer that specifically binds erythrocytes. Examples of tumor-homing ligands are aminopeptidase-A, aminopeptidase-N, endosialin, cell surface nucleolin, cell surface annexin-1, cell surface p32/gC1q receptor, cell surface reticulin-1, fibronectin EDA, fibronectin EDB, interleukin 11 receptor alpha, tenascin-C, endoglin/CD 105, BST-2, galectin-1, VCAM-1, fibrin and tissue factor receptor. For example, the red blood cell module may comprise a peptide ligand, scFv, or antibody or fragment.

One embodiment is a single-chain antigen-binding domain (scFv) comprising a peptide ligand that specifically binds red blood cells. The peptide may be attached to the scFv or disposed in a linker moiety. One or more peptide ligands may be included.

* * *

All patent applications, patents, and publications mentioned herein are hereby incorporated by reference for all purposes; in case of conflict, the present specification will control.

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Claims (68)

1. An isolated peptide comprising at least 5 contiguous amino acid residues of a sequence selected from the group consisting of: 11, 13, 14, 15, 16, 17, 1 and conservative substitutions thereof, wherein the sequence specifically binds erythrocytes.

2. The peptide of claim 1, wherein the sequence has one or more residues with D-L substitutions or conservative substitutions with at least one and no more than two amino acids in a sequence selected from the group consisting of: 11, 13, 14, 15, 16, 17, and 1.

3. The peptide of claim 1, consisting essentially of a sequence selected from the group consisting of: 11, 13, 14, 15, 16, 17, and 1.

4. The peptide of claim 1, further comprising at least one of the group consisting of:

at least 5 contiguous amino acid residues of a sequence selected from the group consisting of: 11, 13, 14, 15, 16, 17, 1 and conservative substitutions thereof, wherein said sequences specifically bind erythrocytes.

5. The peptide of claim 1, having a number of residues from about 10 to about 80.

6. The peptide of claim 1, further comprising a therapeutic agent.

7. The peptide of claim 6, wherein the therapeutic agent is selected from the group consisting of: insulin, pramlintide acetate, growth hormone, insulin-like growth factor-1, erythropoietin, type 1 alpha interferon, interferon alpha 2a, interferon alpha 2b, interferon beta 1a, interferon beta 1b, interferon gamma 1b, beta-glucocerebrosidase, adenosine deaminase, granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor, interleukin 1, interleukin 2, interleukin 11, factor VIIa, factor VIII, factor IX, exenatide (exenatide), L-asparaginase, labyrinase (rasburicase), tumor necrosis factor receptor, and enfuvirtide.

8. The peptide of claim 1, further comprising a member of the group consisting of: antibodies, antibody fragments, and single chain antigen binding domains (ScFv).

9. The peptide of claim 1, further comprising a tolerogenic antigen.

10. The peptide of claim 9, wherein the tolerogenic antigen is selected from the group consisting of: proteins deficient for genetic diseases, proteins with non-human glycosylation, non-human proteins, synthetic proteins not naturally occurring in humans, human food antigens, human transplantation antigens, and human autoimmune antigens.

11. The peptide of claim 10, comprising a plurality of sequences that specifically bind red blood cells.

12. A method of producing immune tolerance comprising administering a composition comprising a molecular fusion comprising a tolerogenic antigen and a red blood cell-binding moiety that specifically binds to red blood cells in the patient and thereby links the antigen and the red blood cells, wherein the molecular fusion is administered in an amount effective to produce immune tolerance to a substance comprising the tolerogenic antigen.

13. The method of claim 12 wherein the molecular fusion consists of at least one erythrocyte-binding moiety covalently bonded directly to the antigen.

14. The method of claim 12 wherein the molecular fusion comprises at least one erythrocyte-binding moiety attached to a particle that is attached to or contains the antigen.

15. The method of claim 14, wherein the particles are selected from the group consisting of: microparticles, nanoparticles, liposomes, polymersomes, and micelles.

16. The method of claim 12, wherein the tolerogenic antigen comprises a portion of a therapeutic protein.

17. The method of claim 16, wherein the protein comprises factor VIII or factor IX.

18. The method of claim 12, wherein the patient is a human and the tolerogenic antigen comprises a portion of a non-human protein.

19. The method of claim 18, wherein the protein comprises adenosine deaminase, L-asparaginase, labyrinase, antithymocyte globulin, L-arginase, and L-methioninase.

20. The method of claim 12, wherein the patient is a human and the tolerogenic antigen comprises a portion of a protein not found in nature.

21. The method of claim 12, wherein the patient is a human and the tolerogenic antigen comprises glycans containing non-human glycosylated proteins.

22. The method of claim 12, wherein the tolerogenic antigen comprises at least a portion of a human transplantation antigen.

23. The method of claim 12, wherein the tolerogenic antigen comprises a portion of a human autoimmune disease protein.

24. The method of claim 23, wherein the human autoimmune disease protein is selected from the group consisting of: preproinsulin, proinsulin, insulin, GAD65, GAD67, IA-2 β, thyroglobulin, thyroid peroxidase, thyroid stimulating hormone receptor, myelin basic protein, myelin oligodendrocyte glycoprotein, proteolipid protein, collagen II, collagen IV, acetylcholine receptor, matrix metalloproteins 1 and 3, chaperone heat shock protein 47, microfibril-1, PDGF receptor α, PDGF receptor β, and nucleoprotein SS-A.

25. The method of claim 12, wherein the tolerogenic antigen comprises a portion of human food.

26. The method of claim 25, wherein the portion of human food is selected from the group consisting of: chaperonin (Ara h1), allergen II (Ara h2), peanut agglutinin (Ara h6), alpha-lactalbumin (ALA), lactoferrin, gluten, low molecular weight gluten, alpha-and gamma-gliadins, hordeins, secalins, and avenin.

27. The method of claim 12, wherein the red blood cell binding moiety is selected from the group consisting of: peptide ligands, antibodies, antibody fragments, and single chain antigen binding domains (ScFv).

28. The method of claim 27, wherein the red blood cell binding moiety comprises an scFv.

29. The method of claim 28, wherein the scFv comprises a light chain of 10F7 or a higher affinity variant of 10F7 light chain.

30. The method of claim 12, wherein the red blood cell binding moiety comprises a peptide ligand comprising at least 5 contiguous amino acid residues of a sequence selected from the group consisting of seq id no:11, 13, 14, 15, 16, 17, 1 and conservative substitutions thereof, wherein the sequence specifically binds erythrocytes.

31. A composition, comprising:

a molecular fusion comprising a tolerogenic antigen and an erythrocyte-binding moiety that specifically binds to erythrocytes in the patient and thereby links the antigen to the erythrocytes.

32. The composition of claim 31, wherein the red blood cell binding moiety is covalently bonded to the antigen.

33. The composition of claim 31, wherein the molecular fusion comprises the red blood cell-binding moiety attached to a particle that is attached to the antigen.

34. The composition of claim 33, wherein the particle comprises a microparticle, a nanoparticle, a liposome, a polymersome, or a micelle.

35. The composition of claim 31, wherein the tolerogenic antigen comprises a portion of a therapeutic protein.

36. The composition of claim 31, wherein the patient is a human and the tolerogenic antigen comprises a portion of a non-human protein.

37. The composition of claim 31, wherein the patient is a human and the tolerogenic antigen comprises a portion of a protein not present in a human.

38. The composition of claim 31, wherein the patient is a human and the tolerogenic antigen comprises glycans containing non-human glycosylated proteins.

39. The composition of claim 31, wherein the tolerogenic antigen comprises a portion of a human autoimmune antigen.

40. The composition of claim 31, wherein the tolerogenic antigen comprises a portion of human food.

41. The composition of claim 31, wherein the red blood cell binding moiety is selected from the group consisting of: peptide ligands, antibodies, antibody fragments, and single chain antigen binding domains (ScFv).

42. The composition of claim 41, wherein the red blood cell binding moiety comprises an scFv.

43. The composition of claim 42, wherein the scFv comprises a light chain of 10F7 or a higher affinity variant of 10F7 light chain.

44. The composition of claim 31, wherein the red blood cell binding moiety comprises a peptide ligand comprising at least 5 contiguous amino acid residues of a sequence selected from the group consisting of seq id no:11, 13, 14, 15, 16, 17, 1 and conservative substitutions thereof, wherein said sequences specifically bind erythrocytes.

45. The composition of claim 31, wherein the red blood cell binding moiety comprises a peptide ligand having a dissociation constant of about 10 μ Μ -0.1nM, as determined by equilibrium binding measurements between the peptide and red blood cells.

46. A composition, comprising:

an erythrocyte-binding module that specifically binds to an erythrocyte linked to an entity selected from the group consisting of: synthetic polymers, branched synthetic polymers, and particles.

47. The composition of claim 46, wherein the particles are selected from the group consisting of: microparticles, nanoparticles, liposomes, polymersomes, and micelles.

48. The composition of claim 46, further comprising a tolerogenic antigen.

49. The composition of claim 46, wherein said red blood cell binding moiety comprises a peptide ligand comprising at least 5 contiguous amino acid residues of a sequence selected from the group consisting of SEQ ID NO:11, 13, 14, 15, 16, 17, 1 and conservative substitutions thereof, wherein said sequences specifically bind erythrocytes.

50. The composition of claim 46, further comprising a therapeutic agent.

51. The composition of claim 50, wherein the therapeutic agent is selected from the group consisting of: antibodies, antibody fragments, single chain antigen binding domains (ScFv), small molecule drugs, and peptides.

52. The composition of claim 46, further comprising a tumor-homing ligand attached to said entity.

53. A method of embolizing a tumor in a patient, comprising:

administering to the patient a composition comprising a molecular fusion of an erythrocyte-binding moiety and a tumor-homing ligand,

wherein the tumor-homing ligand is an antibody, antibody fragment, single-chain antigen-binding domain (ScFv), or peptide ligand targeted to specifically bind a target selected from the group consisting of tumor and tumor vasculature, and

wherein the red blood cell binding moiety comprises a peptide ligand, antibody fragment, scFv, or aptamer that specifically binds red blood cells.

54. The method of claim 53, wherein said tumor-homing ligand is selected from the group consisting of: aminopeptidase-A, aminopeptidase-N, endosialin, cell surface nucleolin, cell surface annexin-1, cell surface p32/gC1q receptor, cell surface reticulin-1, fibronectin EDA, fibronectin EDB, interleukin 11 receptor alpha, tenascin-C, endoglin/CD 105, BST-2, galectin-1, VCAM-1, fibrin and tissue factor receptor.

55. The method of claim 53, wherein said red blood cell binding moiety comprises a peptide ligand comprising at least 5 contiguous amino acid residues of a sequence selected from the group consisting of SEQ ID NO:11, 13, 14, 15, 16, 17, 1 and conservative substitutions thereof, wherein the sequence specifically binds erythrocytes.

56. The method of claim 53, wherein the red blood cell binding moiety comprises an scFv.

57. The method of claim 53, wherein said red blood cell binding moiety comprises an antibody or antibody fragment.

58. The method of claim 53, comprising injecting the composition into the vascular system of the patient.

59. The method of claim 53, wherein the molecular fusion comprises an entity selected from the group consisting of: particles, peptides, and synthetic polymers, wherein the entity is linked to the red blood cell binding moiety and the tumor homing ligand.

60. A medicament for embolizing a tumor in a patient, comprising:

a composition comprising a molecular fusion of an erythrocyte binding moiety and a tumor-homing ligand,

wherein the tumor-homing ligand is an antibody, antibody fragment, single-chain antigen-binding domain (ScFv), or peptide ligand targeted to specifically bind a target selected from the group consisting of tumors and tumor microvasculature, and

wherein the erythrocyte-binding moiety comprises a peptide ligand, an antibody fragment, an ScFv, or an aptamer that specifically binds erythrocytes.

61. The medicament of claim 60, wherein the tumor-homing ligand is selected from the group consisting of: aminopeptidase-A, aminopeptidase-N, endosialin, cell surface nucleolin, cell surface annexin-1, cell surface p32/gC1q receptor, cell surface reticulin-1, fibronectin EDA, fibronectin EDB, interleukin 11 receptor alpha, tenascin-C, endoglin/CD 105, BST-2, galectin-1, VCAM-1, fibrin and tissue factor receptor.

62. The medicament of claim 60, wherein the red blood cell binding moiety comprises a peptide ligand comprising at least 5 contiguous amino acid residues of a sequence selected from the group consisting of SEQ ID NO:11, 13, 14, 15, 16, 17, 1 and conservative substitutions thereof, wherein the sequence specifically binds erythrocytes.

63. The medicament of claim 60, wherein the erythrocyte binding moiety comprises an scFv.

64. The medicament of claim 60, wherein the red blood cell binding moiety comprises an antibody or antibody fragment.

65. The medicament of claim 60, wherein the molecular fusion comprises an entity selected from the group consisting of: particles, peptides, and synthetic polymers, wherein the entity links the red blood cell binding moiety to the tumor homing ligand.

66. A single-chain antigen-binding domain (scFv) comprising a peptide ligand that specifically binds red blood cells.

67. The scFv of claim 66, wherein the peptide ligand is disposed in a linker portion of the scFv.

68. The scFv of claim 66 comprising a plurality of peptide ligands that specifically bind red blood cells.