JPWO2021195508A5 - - Google Patents

Description

Related applications

This application claims priority to U.S. Provisional Application No. 63/000,590, filed March 27, 2020, the entire contents of which are incorporated herein by reference.

(Sequence list)
This application contains a sequence listing submitted in ASCII format via EFS-Web, which is incorporated herein by reference in its entirety. The ASCII copy created on March 24, 2021 is named EPV0025WO_ST25.txt and is 14KB in size.

The present disclosure generally relates to novel T-regitope-blood component conjugates and modified polypeptides comprising a T-regitope, said modified peptides capable of reacting with blood components to form such T-regitope-blood component conjugates. It is. In embodiments, the T-regitope-blood component conjugate comprises a blood component that functions as a carrier protein (e.g., albumin) and further comprises one or more regulatory T cell epitopes (referred to as "T-regitopes"). Contains modified polypeptides. The polypeptide is modified by attaching to the polypeptide a reactive moiety capable of forming a bond (e.g., a covalent bond) with a reactive functionality on a blood component. . In embodiments, the modified polypeptide further comprises one or more antigenic peptides associated with immunogenicity in diabetes ("T1Dgen" peptides; e.g., PPI-derived peptides), which have a cleavage site (e.g., lysosomal cleavage). may be optionally separated from one or more T regitopes by a T-regitope. The present disclosure also relates to methods of using the T-regitope-blood component conjugates and modified polypeptides comprising the T-regitope in the treatment and prevention of autoimmune diseases such as type 1 diabetes.

Type 1 diabetes (T1D) is an autoimmune disease that affects 1.25 million Americans, is caused by the destruction of insulin-producing pancreatic islet cells, and is increasing at 2-3% per year worldwide, especially among children. ing. Autoimmune T1D is induced by environmental factors and is caused by antigen-specific regulatory T1D.
It is thought to be accelerated by defects in the regulation of T cell responses by cells (TRegs). In the absence of effective control, CD8+ and CD4+ autoreactive T cells target islet cell antigens presented by human leukocyte antigen (HLA) molecules. Progressive destruction of islet cells by T cells results in glucose intolerance and lifelong dependence on insulin replacement therapy.

Mitigating islet cell destruction and preserving islet cell function is believed to be critical to developing treatments for T1D. TReg protects islet cells from damage by T effector cells (Teff). Therefore, preservation, expansion, and activation of islet antigen-specific TRegs to restore tolerance is an important area of T1D research. Adoptive TReg therapy and monoclonal antibodies that promote TReg expansion have been investigated, but neither is antigen-specific and important efficacy endpoints have not been met. We propose that T-regitopes improve the outcome of antigen-specific therapy by adding natural TReg induction to the antigen specificity of selected PPI peptides. Therefore, there is a need in the art for compositions comprising such T regitopes and T1D-associated antigens (eg, peptides), and methods related to their preparation and use in the treatment of T1D.

Accordingly, it is an object of the present disclosure to provide novel T-regitope-blood component conjugates and modified polypeptides comprising a T-regitope, wherein said modified peptides react with blood components to provide such T-regitope-blood component conjugates. A conjugate can be formed. Conjugates of T-regitope-containing polypeptides and blood components such as albumin can be useful as carrier proteins for the T-regitope payload. The T-regitope-blood component conjugate extends the half-life of the T-regitope-containing engineered polypeptide in vivo, protects the T-regitope-containing engineered polypeptide from rapid proteolysis, and protects the T-regitope-containing engineered polypeptide from rapid proteolysis. protects against rapid clearance from the circulation and/or rapid renal excretion, allows wide distribution of the T resitope-blood component conjugate throughout the subject's body , and directs T to appropriate immune cells (such as macrophages and APCs). It aids in the delivery of a modified polypeptide containing a regitope, allowing the modified polypeptide containing a T regitope to be processed by the endocytic pathway of specific immune cells (such as macrophages and APCs), and allowing the modified polypeptide containing a T regitope to be processed by the endocytic pathway of specific immune cells (such as macrophages and APCs), and to allow T regitope-containing modified polypeptides to be processed by the endocytic pathway of specific immune cells (such as macrophages and APCs) and to be used as an antigen by said immune cells. Assists in the presentation of modified polypeptides containing resitopes.

Selective engagement and activation of endogenous TRegs (including, in embodiments, natural TRegs and/or adaptive TRegs) through the use of T-regitope-blood component conjugates and modified polypeptides comprising T-regitopes disclosed herein. conjugation is therapeutically valuable as a means of treatment for diseases or conditions characterized by the presence of an undesirable immune response, such as autoimmune diseases such as type 1 diabetes. Thus, the present disclosure also relates to methods of using the T-regitope-blood component conjugates and modified polypeptides comprising the T-regitope in the treatment and prevention of autoimmune diseases such as type 1 diabetes.

In embodiments, the T regitope-blood component conjugate comprises a blood component that acts as a carrier protein (e.g., albumin) and further comprises a modified polypeptide, said modified polypeptide comprising one or more regulatory T cell epitopes. (referred to as "T-resitope"). The modified polypeptide includes a reactive site attached to the polypeptide that is capable of forming a bond (eg, a covalent bond) with a reactive functional group on a blood component. In embodiments, the modified polypeptide further comprises one or more antigenic peptides associated with immunogenicity in diabetes ("T1Dgen"peptides; e.g., PPI-derived peptides), which optionally include a linker and/or cleavage. It may be separated from one or more T regitopes by a site (eg, a lysosomal cleavage site). T-resitope-blood component conjugates are disclosed in U.S. Pat. No. 6,849,714, U.S. Pat. No. 6,887,470, U.S. Pat.
, 256, 253, and U.S. Patent No. 7,307, 148 (incorporated herein by reference in its entirety), a polypeptide containing a T-regitope is synthesized by adding a reactive site to a polypeptide. A modified polypeptide may be conjugated to a peptide, and a bond then formed between a reactive site on the modified polypeptide and a reactive functional group on a blood component. In embodiments of the T-regitope-blood component conjugates and modified polypeptides comprising a T-regitope, the T-regitope-blood component conjugates and modified polypeptides comprising a T-regitope may be isolated, synthetic, or recombinant. good.

In embodiments, the blood component of the T-regitope-blood component conjugate is described in US Pat. No. 6,849,714, US Pat. No. 6,887,470, US Pat. No. 7,307,148, it can be either fixed or mobile. Fixed blood components are those blood components that do not move and are involved in tissues, membrane receptors, interstitial proteins, fibrin proteins, collagen, platelets, endothelial cells, epithelial cells and their associated membranes and membrane receptors, the body Included are cells, skeletal and smooth muscle cells, neuronal components, bone cells and osteoclasts, and all body tissues, especially those associated with the circulatory and lymphatic systems. A mobile blood component is a blood component that does not have a fixed seat for an extended period of time (generally less than 5 minutes, more usually less than 1 minute). These blood components are not membrane bound, exist in the blood for long periods of time, and are present in minimum concentrations of at least 0.1 μg/ml. Mobile blood components include serum albumin, transferrin, ferritin, and immunoglobulins such as IgM and IgG. The half-life of mobile blood components is at least about 12 hours. In embodiments of the T-regitope-blood component conjugate, the blood component is albumin, such as serum albumin, human serum albumin, recombinant albumin, and recombinant human serum albumin. Albumin is a preferred blood component because it contains an Fc neonatal binding domain that transports the T-regitope-albumin conjugate to appropriate cells such as macrophages and APCs. Additionally, albumin contains cysteine at amino acid 34 (Cys34), the amino acid position in the amino acid sequence of human serine albumin, and contains a free thiol with a pKa of about 5, which can serve as the preferred reactive functional group of albumin. Cys34 of albumin can form a stable thioester bond with maleimidopropionamide (MPA), which is a preferred reactive site for modified T resitope peptides.

In embodiments, the reactive functional group on the blood component of the T regitope-blood component conjugate or on the blood component capable of forming a conjugate with a modified polypeptide of the present disclosure is the reactive group on the modified therapeutic peptide. are groups on blood components, including mobile and immobilized proteins, that react to form covalent bonds. Such as disclosed in U.S. Pat. No. 6,849,714, U.S. Pat. The functionalities are typically hydroxyl groups for coupling with ester reactive groups, maleimide groups, imidate groups and thiol groups for coupling with thioester groups, activated carboxyl groups of reactive groups, phosphate groups or other Contains an amino group for bonding with any acyl group.

In embodiments, the modified polypeptide of the T-regitope-blood component conjugate and the modified polypeptide used to form the T-regitope-blood component conjugate include a reactive site attached to the polypeptide, The sexual moiety is one that is capable of forming a bond (eg, a covalent bond) with a reactive functional group on a blood component. In embodiments, the reactive group is capable of reacting with and forming a covalent bond with an amino group, hydroxyl group, or thiol group on a blood component. In embodiments, the reactive group is placed at a site such that when the modified polypeptide is bound to a blood component, the modified peptide retains a substantial proportion of the activity of the parent compound. In embodiments, the reactive moiety can be a succinimidyl group or a maleimide group. In embodiments, the reactive site may be attached to an amino acid located in a region of the polypeptide to be modified that has less therapeutic activity. In embodiments, the reactive site is attached to the amino terminal amino acid of the polypeptide to be modified. In embodiments, the reactive site is attached to the carboxy-terminal amino acid of the modified polypeptide. In embodiments, the reactive site is attached to an amino acid located between the amino terminal amino acid and the carboxy terminal amino acid of the modified polypeptide. In embodiments, a reactive group may be attached to a polypeptide (to be modified) via a linking group or optionally without the use of a linking group. Additionally, one or more additional amino acids (eg, one or more lysines) may be added to the polypeptide to facilitate attachment of reactive groups. A linking group is a chemical moiety that links or connects a reactive group of a blood component to a polypeptide containing one or more T-regitopes. The linking group is an alkyl group, an alkoxy group, an alkenyl group, an alkynyl group, or an amino group substituted with an alkyl group, a cycloalkyl group, a polycyclic group, an aryl group, a polyaryl group, a substituted aryl group, a heterocyclic group, and It may be one or more substituted heterocyclic groups. Linking groups may also be composed of polyethoxy amino acids such as AEA ((2-amino)ethoxyacetic acid), and a preferred linking group may be AEEA ([2-(2-amino)ethoxy]ethoxyacetic acid). In embodiments, the linking group may be comprised of a polyethylene glycol linker, such as, but not limited to, PEG2 or PEG12.

It should be understood that modified polypeptides may be administered in vivo such that conjugation with blood components occurs in vivo, or they may first be conjugated to blood components in vitro and obtained. Peptidase stabilizing polypeptides may be administered in vivo. Additionally, peptidases, as disclosed in U.S. Patent No. 6,849,714, U.S. Patent No. 6,887,470, U.S. Patent No. 7,256,253 and U.S. Patent No. 7,307,148, A stabilizing polypeptide is a modified polypeptide that is attached to a blood component through a covalent bond formed between a reactive group on the modified peptide and a functional group on the blood component, with or without a linking group. . Such reactions preferably involve covalently linking polypeptides modified with maleimide links (e.g., prepared from GMBS, MPA, or other maleimides) to thiol groups on mobile blood proteins such as serum albumin or IgG. established by. Peptidase-stabilized polypeptides are more stable in the presence of peptidases in vivo than non-stabilized peptides. Peptidase-stabilized therapeutic peptides generally can increase half-life by at least 10-50% compared to non-stabilized peptides of the same sequence. Peptidase stability is determined by comparing the half-life of an unmodified therapeutic peptide in serum or blood to the half-life of its counterpart modified therapeutic peptide in serum or blood. Half-life is determined by sampling serum or blood after administering modified and unmodified peptides and measuring the activity of the peptides. In addition to determining activity, the length of a therapeutic peptide can also be measured.

In embodiments, modified polypeptides of T-regitope-blood component conjugates, and modified polypeptides used to form T-regitope-blood component conjugates, include one or more T-regitopes (herein “TReg (sometimes referred to as "activated regulatory T cell epitopes", "T regitopes" or "T cell epitope polypeptides"). In embodiments, the one or more T resitopes of the modified polypeptide comprise, consist exclusively of, or consist essentially of one or more of SEQ ID NOs: 1-55 (and fragments and variants thereof). It consists of this (ra). The expression "consisting essentially of" means any of SEQ ID NOs: 1-55 (or a fragment thereof), as long as the T-regitope of the modified polypeptide according to the present disclosure does not significantly impair the activity of the peptide functioning as a T-regitope. In addition to the sequence according to the MHC ligand or variant), it is meant to include additional amino acids or residues that may be present at either terminus and/or side chain of the peptide, which do not necessarily form part of the peptide that functions as an MHC ligand. intend to do so. In embodiments, the T resitope of a modified polypeptide according to the present disclosure comprises, consists exclusively of, or consists essentially of one or more of the sequences SEQ ID NO: 1 and 28. In embodiments, the T resitope of a modified polypeptide according to the present disclosure comprises, consists exclusively of, or consists essentially of the amino acid sequence(s) of SEQ ID NO: 1. In embodiments, the T resitope of a modified polypeptide according to the present disclosure comprises, consists exclusively of, or consists essentially of the amino acid sequence(s) of SEQ ID NO: 28. In embodiments, the one or more T resitopes of the modified polypeptide optionally have one or more linkers adjacent to their N-terminus and/or C-terminus, which may be cleavage-sensitive sites. You can leave it there. In such modified polypeptides, two or more T regitopes may have cleavage sensitive sites between them. Alternatively, two or more T regitopes may be connected to each other directly or via a linker that is not a cleavage sensitive site. Polypeptides of the present disclosure may be isolated, synthetic, and/or recombinant and may include post-transcriptional modifications such as glycosylation, added chemical groups, and the like. In embodiments, the peptide or polypeptide may be in either neutral (uncharged) or salt form and may be free of modifications such as glycosylation, side chain oxidation, or phosphorylation, or May contain. In certain embodiments, the T resitope can be capped with an N-terminal acetyl group and/or a C-terminal amino group. In embodiments, one or more T resitopes included in the modified polypeptide can be capped with an N-terminal acetyl group and/or a C-terminal amino group.

In embodiments, the modified polypeptide of the T-regitope-blood component conjugate and the one or more T-regitopes of the modified polypeptide used to form the T-regitope-blood component conjugate are one or more of SEQ ID NO: 1 ~55 (and/or fragments or variants thereof) and optionally 1 to 12 additional sequences distributed in any proportion at the N-terminus and/or C-terminus of the polypeptides of SEQ ID NOS: 1 to 55. It has a sequence that contains, consists only of, or consists essentially of amino acids. In embodiments, the one or more T resitopes comprise, consist exclusively of, or consist essentially of a peptide or polypeptide having an amino acid sequence of SEQ ID NOs: 1-55. an amino acid sequence and optionally an extension of 1 to 12 amino acids distributed in any proportion at the N-terminus and/or C-terminus of said core amino acid sequence, and these adjacent amino acids (flanking amino acids) ) (flanking amino acids) are 1-12, 1-3, 2-4, 3-6, 1-10, 1-8, 1-6, 2-12, 2-10, 2- 8, 2-6, 3-12, 3-10, 3-8, 3-6, 4-12, 4-10, 4-8, 4-6, 5-12, 5-10, 5-8, 5-6, 6-12, 6-10, 6-8, 7-12, 7-10, 7-8, 8-12, 8-10, 9-12, 9-10 or 10-12, Adjacent amino acids may be distributed in any ratio relative to the C-terminus and the N-terminus (e.g., all adjacent amino acids may be added to one terminus, amino acids may be added equally to both termini, or the other (can also be added in any proportion). In embodiments, the one or more T resitopes comprise or only one or more peptides or polypeptides having an amino acid sequence of SEQ ID NOs: 1-55 (and/or fragments and variants thereof). a core sequence consisting of, or consisting essentially of, and optionally an extension of 1 to 12 amino acids distributed in any proportion at the N-terminus and/or C-terminus of said core amino acid sequence; The total number of these flanking amino acids is 1-12, 1-3, 2-4, 3-6, 1-10, 1-8, 1 ~6, 2~12, 2~10, 2~8, 2~6, 3~12, 3~10, 3~8, 3~6, 4~12, 4~10, 4~8, 4~6 , 5-12, 5-10, 5-8, 5-6, 6-12, 6-10, 6-8, 7-12, 7-10, 7-8, 8-12, 8-10, 9 ~12, 9-10 or 10-12, and adjacent amino acids may be distributed in any ratio to the C-terminus and N-terminus (e.g., all adjacent amino acids may be added to one terminus). (amino acids can be added equally to both termini, or in any other ratio), but a T-regitope with its flanking amino acids still has the same HLA as a T-regitope without its flanking amino acids. Can bind to molecules (i.e. retain MHC binding). In embodiments, the T-regitope with the contiguous amino acids is capable of binding to the same HLA molecule (i.e., retains MHC binding) as the T-regitope core sequence without the contiguous amino acids, and/or The same TCR specificity can be retained. In embodiments, the polypeptide with the contiguous amino acids binds to the same HLA molecules (i.e., retains MHC binding) and/or has the same TCR specificity as the polypeptide core sequence without the contiguous amino acids. and/or can retain T regitope activity. In some embodiments, the contiguous amino acid sequence is also present as a contiguous portion of a peptide or polypeptide contained therein in an endogenous protein, such as an IgG antibody. In embodiments, the extension(s) are provided to improve the biochemical properties of the peptide or polypeptide (e.g., without limitation, solubility or stability) or to improve the potential for efficient proteasomal processing of the peptide. can function and be designed to improve. In embodiments, the contiguous amino acid sequences described herein can function as MHC stabilizing regions. The use of longer peptides allows for endogenous processing by the cells of interest, which may lead to more effective antigen presentation and induction of T cell responses. In embodiments, the one or more T resitopes of the modified polypeptides have one or more linkers (optionally cleavage sensitive sites) adjacent to their N-terminus and/or C-terminus. may have. In such modified polypeptides, two or more T regitopes may have cleavage sensitive sites between them. Alternatively, two or more T-regitopes may be connected to each other directly or via a linker that is not a cleavage-sensitive site. Polypeptides may be isolated, synthetic, and/or recombinant. In embodiments, the modified polypeptide comprising one or more T-regitopes and/or the T-regitopes contained therein can be neutral (uncharged) or in salt form, and can be glycosylated, side chain oxidized, Alternatively, it may not contain or may contain modifications such as phosphorylation. In certain embodiments, a modified polypeptide comprising one or more T resitope peptides or polypeptides can be capped with an N-terminal acetyl group and/or a C-terminal amino group.

In embodiments, the modified polypeptide of the T regitope-blood component conjugate, and the modified polypeptide used to form the T regitope-blood component conjugate, is one or more T1Dgen peptides (e.g., PPI-derived peptides). further includes . In embodiments , the one or more T1Dgen peptides may optionally be separated from one or more T regitopes by a cleavage site (eg, a lysosomal cleavage site). In embodiments, the one or more T1Dgen peptides of the modified polypeptide comprise, consist solely of, or consist essentially of the amino acid sequences of SEQ ID NOs: 56-63 (and/or fragments or variants thereof). It consists of this (ra). The expression "consisting essentially of" means that the T1Dgen peptide of the modified polypeptide according to the present disclosure does not significantly impair the activity of the peptide to function as an antigen. In addition to the sequence according to any of It is intended that In embodiments, the one or more T1Dgen peptides comprise, consist exclusively of, or consist essentially of the amino acid sequence(s) of SEQ ID NO: 63. In embodiments, the one or more T1Dgen peptides of the modified polypeptides have one or more linkers (optionally cleavage sensitive sites) adjacent to their N-terminus and/or C-terminus. may have. In such modified polypeptides, two or more T1Dgen peptides may have a cleavage sensitive site between them. Alternatively, two or more T1Dgen peptides may be connected to each other directly or via a linker that is not a cleavage-sensitive site. Additionally, in embodiments, the T1Dgen peptide and the T regitope of the modified polypeptide may have a cleavage-sensitive site between them or may be connected to each other directly or via a linker that is not a cleavage-sensitive site. good.

In embodiments, the modified polypeptide of the T regitope-blood component conjugate, and the one or more T1Dgen peptides of the modified polypeptide used to form the T regitope-blood component conjugate, have SEQ ID NOs: 56-63 ( and/or fragments or variants thereof) and optionally 1 to 12 additional amino acids distributed in any proportion at the N-terminus and/or C-terminus of the polypeptides of SEQ ID NOs: 56-63. Contains, consists exclusively of, or consists essentially of (a). In embodiments, the one or more T1Dgen peptides comprise, consist exclusively of, or consist essentially of one or more peptides or polypeptides having the amino acid sequences of SEQ ID NOs: 56-63. etc.) and optionally an extension of 1 to 12 amino acids distributed in arbitrary proportions at the N-terminus and/or C-terminus of the core amino acid sequence, and these The total number of flanking amino acids is 1-12, 1-3, 2-4, 3-6, 1-10, 1-8, 1-6, 2-12, 2 ~10, 2-8, 2-6, 3-12, 3-10, 3-8, 3-6, 4-12, 4-10, 4-8, 4-6, 5-12, 5-10 , 5-8, 5-6, 6-12, 6-10, 6-8, 7-12, 7-10, 7-8, 8-12, 8-10, 9-12, 9-10 or 10 ~12, and adjacent amino acids may be distributed in any ratio relative to the C-terminus and N-terminus (e.g., all adjacent amino acids can also be added to one end, or amino acids can be added to both ends). or any other percentage). In embodiments, the one or more T1Dgen peptides include or contain one or more peptides or polypeptides having the amino acid sequence of SEQ ID NOs : 56-63 (and/or fragments and variants thereof). a core sequence consisting of, or consisting essentially of, and optionally an extension of 1 to 12 amino acids distributed in any proportion at the N-terminus and/or C-terminus of said core amino acid sequence; The total number of these flanking amino acids (flanking amino acids) is 1 to 12, 1 to 3, 2 to 4, 3 to 6, 1 to 10, 1 to 8, 1-6, 2-12, 2-10, 2-8, 2-6, 3-12, 3-10, 3-8, 3-6, 4-12, 4-10, 4-8, 4- 6, 5-12, 5-10, 5-8, 5-6, 6-12, 6-10, 6-8, 7-12, 7-10, 7-8, 8-12, 8-10, 9-12, 9-10 or 10-12, and the adjacent amino acids may be distributed in any ratio relative to the C-terminus and the N-terminus (e.g., all adjacent amino acids are added to one end). (amino acids can be added equally to both termini, or in any other ratio), but a T1Dgen peptide with its flanking amino acids is still the same as a T1Dgen peptide without its flanking amino acids. Can bind to HLA molecules (i.e. retain MHC binding). In embodiments, the T1Dgen peptide with the contiguous amino acids is still capable of binding to the same HLA molecule (i.e., retains MHC binding) as the T1Dgen peptide core sequence without the contiguous amino acids, and /or retain the same TCR specificity. In some embodiments, the flanking amino acid sequence is also present in an endogenous protein as a flanking region of the T1Dgen peptide contained therein. For example, the peptide or polypeptide comprises, consists exclusively of, or consists essentially of one or more peptides or polypeptides having the amino acid sequence of SEQ ID NOs: 56-63 (and/or fragments and variants thereof). a core sequence consisting of these(s) and optionally an extension of 1 to 12 amino acids distributed in any proportion at its N-terminus and/or C-terminus; The extension is that found in the amino acid sequence of preproinsulin adjacent to the amino acid sequences of SEQ ID NOs: 56-63. In embodiments, the extension(s) are for improving biochemical properties of the peptide or polypeptide (e.g., without limitation, solubility or stability) or for efficient proteasomal processing of the peptide. can be designed to function to improve the possibilities of In embodiments, the contiguous amino acid sequences described herein can function as MHC stabilizing regions. The use of longer peptides allows for endogenous processing by the cells of interest, which may lead to more effective antigen presentation and induction of T cell responses. In embodiments, the one or more T1Dgen peptides can be in neutral (uncharged) or salt form, and are free or free of modifications such as glycosylation, side chain oxidation, or phosphorylation. It can be either.

In embodiments, the present disclosure provides methods for forming T-regitope-blood component conjugates and/or T-regitope-blood component conjugates of the present disclosure, which in embodiments may be isolated, synthetic, or recombinant. The present invention relates to a nucleic acid (eg, DNA or RNA, including mRNA) encoding a modified polypeptide. In aspects, the present disclosure relates to expression cassettes, plasmids, expression vectors, recombinant viruses, or cells that include the nucleic acids described herein. In aspects, the present disclosure relates to cells or vaccines comprising vectors as described. In aspects, the present disclosure relates to cells comprising vectors of the present disclosure.

In aspects, the present disclosure provides compounds or compositions that include the disclosed T regitope-blood component conjugates and/or modified polypeptides used to form the T regitope-blood component conjugates ( as well as T regitope-blood component conjugates). Nucleic acids disclosed herein, including nucleic acids encoding modified polypeptides of regitope-blood component conjugates and modified polypeptides used to form T-regitope-blood component conjugates, expression cassettes, plasmids, expression vectors, recombinant viruses, cells), and pharmaceutical compositions or formulations comprising pharmaceutically acceptable carriers, excipients, and/or adjuvants.

In embodiments, the present disclosure provides methods for stimulating, inducing, and/or expanding regulatory T cells (in embodiments, endogenous TRegs, natural TRegs, and/or adaptive TRegs) in a subject in need thereof. a method of stimulating a T1D-associated autoimmune response in a subject in need thereof, comprising the disclosed T-regitope-blood component conjugates and/or T-regitope-blood component The present invention includes modified polypeptides used to form conjugates (as well as modified polypeptides of T-regitope-blood component conjugates and nucleic acids encoding modified polypeptides used to form T-regitope-blood component conjugates). a therapeutically effective amount of a nucleic acid disclosed herein, an expression cassette, a plasmid, an expression vector, a recombinant virus, a cell, and/or a pharmaceutical composition or formulation disclosed herein ). administering the compound or composition to the subject.

In aspects, the present disclosure provides a method of treating or preventing a medical condition in a subject in need thereof, comprising: a therapeutically effective amount of the disclosed T-regitope-blood component conjugates and/or T-regitope-blood components. The modified polypeptide used to form the component conjugate (as well as the modified polypeptide of the T-regitope-blood component conjugate and the nucleic acid encoding the modified polypeptide used to form the T-regitope-blood component conjugate) the nucleic acids disclosed herein, the expression cassettes, plasmids, expression vectors, recombinant viruses, cells, and/or pharmaceutical compositions or formulations disclosed herein). Regarding the method . In embodiments, the medical condition is selected from the group consisting of: allergy, autoimmune disease, transplant-related disorder, graft-versus-host disease, blood clotting disorder, enzyme or protein deficiency disorder, hemostatic disorder, cancer, infertility; and viral, bacterial or parasitic infections. In another embodiment, the condition is hemophilia A, B, or C. In aspects, the present disclosure relates to a method of treating or preventing type 1 diabetes in a subject in need thereof, comprising a therapeutically effective amount of a T-regitope-blood component conjugate and/or a T-regitope-blood component conjugate of the present disclosure. The method includes administering to a subject a modified polypeptide used to form a gate, or a pharmaceutical composition or formulation containing the same. In embodiments, the subject is a human.

In embodiments, the present disclosure provides for the use of regulatory T cells (e.g., endogenous TRegs (in embodiments, natural TRegs and/or adaptive TRegs) to suppress an autoimmune response in a subject in need thereof. )) in a subject in need thereof, comprising: administering a therapeutically effective amount of the disclosed T-regitope-blood component conjugates and/or T-regitope-blood component conjugates to a subject in need thereof; The present invention includes modified polypeptides used to form conjugates (as well as modified polypeptides of T-regitope-blood component conjugates and nucleic acids encoding modified polypeptides used to form T-regitope-blood component conjugates). the nucleic acids disclosed herein, the expression cassettes, plasmids, expression vectors, recombinant viruses, cells, and/or pharmaceutical compositions or formulations disclosed herein). In embodiments, the immune response is the result of one or more therapeutic treatments with at least one therapeutic protein, treatment with a vaccine , or treatment with at least one antigen. In embodiments, such administration shifts one or more antigen-presenting cells to a regulatory phenotype, shifts one or more dendritic cells to a regulatory phenotype, and shifts one or more dendritic cells to a regulatory phenotype. or reduce CD11c and HLA-DR expression in other antigen-presenting cells.

In aspects, the present disclosure relates to a method for expanding a population of regulatory T cells, the method comprising: (a) providing a biological sample from a subject; (b) ) isolating regulatory T cells from a biological sample; (c) obtaining a regulatory T cell composition in which the regulatory T cells increase in number and expand, thereby increasing the number of regulatory T cells in the biological sample; Used to form an effective amount of the disclosed T-regitope-blood component conjugates and/or T-regitope-blood component conjugates to isolated regulatory T cells under conditions that expand the cells. Nucleic acids disclosed herein, including nucleic acids encoding modified polypeptides used to form T-regitope-blood component conjugates (as well as modified polypeptides of T-regitope-blood component conjugates and modified polypeptides used to form T-regitope-blood component conjugates) , an expression cassette, a plasmid, an expression vector, a recombinant virus, a cell, and/or a pharmaceutical composition or formulation as disclosed herein; and, additionally, returning the sample to the subject in need of treatment.

In aspects, the present disclosure relates to a method for stimulating regulatory T cells in a biological sample, the method comprising: (a) providing a biological sample from a subject; (b) a biological sample; (c) conditions under which the regulatory T cells are stimulated to alter one or more biological functions, thereby stimulating the regulatory T cells in the biological sample; and an effective amount of the disclosed T regitope-blood component conjugates and/or modified polypeptides used to form the T regitope-blood component conjugates (and Nucleic acids disclosed herein comprising modified polypeptides of T-regitope-blood component conjugates and nucleic acids encoding modified polypeptides used to form T-regitope-blood component conjugates, disclosed herein (d) contacting said cells with a therapeutic agent (an expression cassette, plasmid, expression vector, recombinant virus, cell, and/or a pharmaceutical composition or formulation disclosed herein); It includes the process of returning it to the desired target.

In aspects, the present disclosure provides a method for suppressing/suppressing an immune response in a subject, comprising a therapeutically effective amount of a T-regitope-blood component conjugate and/or a T-regitope-blood component conjugate of the present disclosure. Modified polypeptides used to form T-regitope-blood component conjugates (as well as nucleic acids encoding modified polypeptides used to form T-regitope-blood component conjugates) a nucleic acid disclosed herein, an expression cassette, a plasmid, an expression vector, a recombinant virus, a cell, and/or a pharmaceutical composition or formulation disclosed herein); The composition administered with suppresses/suppresses the immune response. In embodiments, the administered composition suppresses/suppresses the innate immune response. In embodiments, the administered composition suppresses/suppresses an adaptive immune response. In embodiments, the administered composition suppresses/suppresses effector T cell responses. In embodiments, the administered composition suppresses/suppresses memory T cell responses. In embodiments, the administered composition suppresses/suppresses helper T cell responses. In embodiments, the administered composition suppresses/suppresses B cell responses. In embodiments, the administered composition suppresses/suppresses nkT cell (natural killer T cell) responses. In another aspect, administration of a T-regitope compound or composition of the present disclosure shifts one or more antigen-presenting cells toward a regulatory phenotype and shifts one or more dendritic cells toward a regulatory phenotype. and decrease CD11c and HLA-DR expression in dendritic cells or other antigen-presenting cells.

In aspects, the present disclosure relates to a method of suppressing an immune response, particularly an antigen-specific immune response in a subject, wherein the method comprises a therapeutically effective amount of the disclosed T-regitope-blood component conjugate and/or T-regitope. - a modified polypeptide used to form a blood component conjugate (and a T-regitope-modified polypeptide of a blood component conjugate and a nucleic acid encoding a modified polypeptide used to form a T-regitope-blood component conjugate) the nucleic acids disclosed herein, the expression cassettes disclosed herein, plasmids, expression vectors, recombinant viruses, cells, and/or pharmaceutical compositions or formulations disclosed herein), including: wherein the administered composition comprises endogenous TRegs (in some embodiments, natural TRegs and/or adaptive TRegs, and in some embodiments CD4 ⁺ /CD25 ⁺ /FoxP3 ⁺ regulatory T cells). or inhibiting activation of CD4 ⁺ T cells, proliferation of CD4 ⁺ and/or CD8 ⁺ T cells, and/or inhibiting activation or proliferation of β cells or nkT cells. be. In embodiments, modified polypeptides used to form T regitope-blood component conjugates and/or T regitope-blood component conjugates of the present disclosure (as well as modified polypeptides and T regitope-blood component conjugates) Nucleic acids disclosed herein, expression cassettes, plasmids, expression vectors, recombinant viruses disclosed herein, including nucleic acids encoding modified polypeptides used to form resitope-blood component conjugates. , cells, and/or pharmaceutical compositions or formulations disclosed herein) may be covalently, non-covalently, or mixed with a particular target antigen. In embodiments, the specific target antigen is T1Dgen included in the modified polypeptide. In embodiments, the modified polypeptides used to form the administered T-regitope-blood component conjugates and/or the T-regitope-blood component conjugates of the present disclosure (as well as the modified polypeptides of the T-regitope-blood component conjugates) Nucleic acids as disclosed herein, expression cassettes, plasmids, expression vectors as disclosed herein, including nucleic acids encoding modified polypeptides used to form peptides and T-regitope-blood component conjugates; When a recombinant virus, cell, and/or pharmaceutical composition or formulation disclosed herein) is covalently, non-covalently, or mixed with one or more specific target antigens, an immune response against the target antigens This results in attenuation of In embodiments, the one or more specific target antigens comprise, consist exclusively of, or consist essentially of one or more of the sequences SEQ ID NOs: 56-63 disclosed herein. (r), or consisting solely of, or consisting essentially of (r), one or more peptides associated with immunogenicity in diabetes (referred to as "T1Dgens") having a sequence consisting of (r) .

In embodiments, the target antigen can be a self protein or protein fragment. In embodiments, the target antigen may be an allogeneic protein or protein fragment. In embodiments, the target antigen may be a biological drug or a fragment thereof. In embodiments, the target antigen is preproinsulin or a fragment thereof. In embodiments, the target antigen comprises, consists solely of, or consists essentially of one or more of SEQ ID NOs: 56-63, or fragments and variants thereof, as described herein. ). In embodiments, the suppressive effect is mediated by natural TRegs. In aspects, the suppressive effect is mediated by adaptive TRegs. In embodiments, one or more T regitopes of the disclosed modified polypeptides suppress the innate immune response. In embodiments, one or more T regitopes of the disclosed modified polypeptides suppress the adaptive immune response. In embodiments, one or more T regitopes of the disclosed modified polypeptides suppress T helper cell responses. In embodiments, one or more T regitopes of the disclosed modified polypeptides suppress memory T cell responses. In embodiments, one or more T regitopes of the disclosed modified polypeptides suppress B cell responses. In embodiments, one or more T regitopes of the disclosed modified polypeptides suppress nkT cell responses.

In embodiments, the present disclosure relates to a kit for preventing or treating a medical condition, particularly for suppressing an immune response associated with type 1 diabetes in a subject, the kit comprising the presently disclosed T. One or more modified polypeptides of the present disclosure used to form regitope-blood component conjugates and/or T-regitope-blood component conjugates (as well as modified polypeptides of T-regitope-blood component conjugates and T-regitope-blood component conjugates). Nucleic acids disclosed herein, expression cassettes, plasmids, expression vectors, recombinant viruses, cells, and and/or pharmaceutical compositions or formulations disclosed herein). In embodiments, the kit may further include an effective amount of an antigen or therapeutic agent, such as a replacement protein or peptide.

The present disclosure may be better understood by referring to the following figures. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
FIG. 1 shows exemplary T-regitope-blood component conjugates of the present disclosure. HSA refers to human serum albumin. FIG. 2 depicts an exemplary T-regitope-blood component conjugate of the present disclosure. HSA refers to human serum albumin. Figure 3 shows the experimental design for the TTBSA assay to assess the efficacy of T-resitope-albumin delivery vehicles. Figure 4A shows the results of each of the numerous available T regitopes individually and in pairwise combinations on their potential to suppress CD4+ T cell proliferation in the TTBSA assay. Figure 4B shows the results of each of the numerous available T regitopes individually and in pairwise combinations on their potential to suppress CD4+ T cell proliferation in the TTBSA assay. Figure 5A shows the results of conjugating selected T regitopes for their potential to inhibit CD4+ T cell proliferation in the TTBSA assay. Figure 5B shows the results of conjugating selected T regitopes for their potential to inhibit CD4+ T cell proliferation in the TTBSA assay.

(overview)
The adaptive immune cascade begins when soluble protein antigens are taken up by antigen presenting cells (APCs) and processed through the class II antigen presentation pathway. Protein antigens in the class II antigen presentation pathway are degraded by various proteases present in the endoplasmic reticulum. Some of the resulting protein fragments are bound to class II MHC molecules. MHC molecules with bound peptides are transported to the cell surface where they are interrogated by CD4+ T cells. Peptide fragments that can bind MHC molecules and mediate cell-to-cell interactions between APCs and circulating T cells are called T cell epitopes. Recognition of these peptide-MHC complexes by CD4+ T cells can trigger either immune activation or immunosuppressive responses based on the responding T cell phenotype and the local cytokine/chemokine environment. Generally, binding of MHC/peptide complexes to T cell receptors (TCRs) of T effector cells causes activation and subsequent secretion of inflammatory cytokines such as IL-4 and IFN. On the other hand, activation of natural regulatory T cells (TReg) causes the expression of immunosuppressive cytokines such as IL-10 and TGF-β (Shevach E, (2002), Nat Rev Immunol, 2(6) :389-400). These cytokines act directly on nearby effector T cells, causing anergy and apoptosis in some cases. Additionally, regulatory cytokines and chemokines may convert effector T cells to a T regulatory phenotype. This process is referred to herein as "inducible" or "adaptive" tolerance. T cell epitopes that can bind to MHC molecules and engage and/or activate circulating endogenous TRegs (including, in embodiments, natural and/or adaptive TRegs) are referred to as T-regitopes. In aspects, the disclosed T regitopes are T cell epitope clusters that are epitopes that can bind to multiple MHC alleles and multiple TCRs.

The first self/non-self discrimination occurs in the thymus during neonatal development, where cortical and medullary epithelial cells express self protein epitopes specific for immature T cells. T cells that recognize self-antigens with high affinity are deleted, but self-reactive T cells with intermediate affinity escape deletion and can transform into natural regulatory T cells (TReg) cells. . These natural TReg cells are transported to the periphery and help control potential autoimmune reactions. Natural regulatory T cells are important components of immune regulation and self-tolerance.

Self-tolerance is controlled by complex interactions between T cells, B cells, cytokines and surface receptors. T-regulated immune responses balance T-effector immune responses to protein antigens (whether self or foreign). When the balance shifts towards autoreactivity, either due to an increase in the number and/or function of autoreactive T effector cells or a decrease in the number and/or function of regulatory T cells, it manifests as autoimmunity.

When mature T cells are activated via the T cell receptor in the presence of IL-10 and TGF-β, they typically receive a supply of bystander regulatory T cells and adopt an “adaptive” TReg phenotype. If converted, a second tolerance develops in the periphery. The role of these "adaptive" TReg cells may be to attenuate the immune response after successfully eliminating an invading pathogen, to control excessive inflammation due to allergic reactions, to control excessive inflammation due to low-level or chronic infections, or to provide beneficial Control of inflammatory responses to commensal bacteria and viruses is thought to be possible. “Adaptive” TRegs may also play a role in suppressing immune responses targeting human antibodies that have undergone somatic hypermutation (Chaudhry A et al. (2011), Immunity, 34(4):566- 78).

TReg cells also play a role in B cell tolerance. B cells express a single low affinity Fc receptor, FcyRIIB, on their cell surface (Ravetch JV et al. (1986), Science, 234(4777):718-25). This receptor contains an immunoreceptor tyrosine-based inhibition motif sequence (ITIM) in its cytoplasmic domain. Co-ligation of FcyRIIB and B cell receptor (BCR) by immune complexes induces tyrosine phosphorylation of ITIM, leading to recruitment of the inositol phosphatase, SHIP. SHIP suppresses BCR-triggered proliferation by preventing activation of MAP kinase, inhibits phagocytosis by dissociating Barton tyrosine kinase (Btk) from the cell membrane, and inhibits intracellular calcium influx. FcyRIIB can also induce apoptosis independently of ITIM. Homoassociation of FcyRIIB by IC promotes the association of Btk with the cell membrane, thereby triggering an apoptotic response (Pearse R, et al., (1999), Immunity, 10(6):753-60) . Expression of FcyRIIB is highly variable and cytokine dependent. IL-4 and IL-10 expressed by activated Th2 and TReg cells have been shown to act synergistically to increase the expression of FcyRIIB (Joshi T et al. (2006), Mol Immuno , 43(7):839-50), and thus can assist in suppressing humoral responses.

It is possible to utilize T-regitope-specific TReg cells to suppress unwanted immune responses, and it is also possible to induce adaptive TRegs against co-transferred proteins. This discovery has implications for the design of therapeutic regimens and antigen-specific therapies for transplantation, protein therapeutics, allergy, chronic infections, autoimmunity and vaccine design. Novel T-regitope-blood component conjugates and modified polypeptides comprising the T-regitopes of the present disclosure (as well as modified polypeptides of T-regitope-blood component conjugates and/or modifications used to form T-regitope-blood component conjugates) Nucleic acids disclosed herein, expression cassettes, plasmids, expression vectors, recombinant viruses, cells, and/or pharmaceutical compositions disclosed herein, including nucleic acids encoding polypeptides. When administered together with drugs, proteins, or allergens, effector immune responses can be suppressed and can be used to intentionally manipulate the immune system toward tolerance.

The T-regitope-blood component conjugates and modified polypeptides containing T-regitopes of the present disclosure are useful for selective engagement and activation of regulatory T cells. Certain endogenous TRegs (including, in embodiments, natural TRegs and/or adaptive TRegs) participate in the suppression of unwanted immune responses, activation, and Applicability is demonstrated herein. For example, certain human proteins circulating in the blood stream, such as immunoglobulins, have T cell epitopes associated with endogenous regulatory T cell (including in some embodiments natural and/or adaptive TRegs ) populations. including. During normal immune surveillance, these proteins are taken up and degraded by specialized APCs such as dendritic cells and macrophages. During the degradation process, some of the epitopes contained in these proteins bind to MHC molecules, are transported to the cell surface, and are presented to regulatory T cells. When activated by APCs, these cells release cytokines and chemokines that help suppress autoimmune responses that inhibit the function of extracellular proteins. In embodiments, the T regitope compositions of the present disclosure can be used to engage and activate pre-existing populations of regulatory T cells to suppress autoimmune responses associated with T1D. Suppression of the autoimmune response associated with T1D involves the suppression of CD8+ and/or CD4+ autoreactive T cells that target islet cell antigens presented by human leukocyte antigen (HLA) molecules, and the destruction of islet cells by T cells. Suppression, expansion and/or activation of islet cell antigen-specific TRegs, restoration of islet cell tolerance, and/or induction of tolerance to proteins associated with T1D and islet cells, such as preproinsulin.

The novel T-regitope-blood component conjugates and modified polypeptides comprising the T-regitope (as well as modified polypeptides of the T-regitope-blood component conjugates and/or modifications used to form the T-regitope-blood component conjugates) of the present disclosure. Nucleic acids disclosed herein, expression cassettes, plasmids, expression vectors, recombinant viruses, cells, and/or pharmaceutical compositions disclosed herein, including nucleic acids encoding polypeptides. It is shown herein that such compositions can be used to suppress a variety of undesirable immune responses. In its simplest form, systemic application of the T-regitope-blood component conjugates and modified polypeptides comprising the T-regitope of the present disclosure can be used to treat general immunosuppression useful in the control of severe autoimmune reactions, such as autoimmune T1D. It can be used as an agent.

In more controlled applications, the novel T-regitope-blood component conjugates and modified polypeptides containing T-regitopes of the present disclosure can be used to suppress local autoimmune responses. In targeting applications, such as can be accomplished by linking or incorporation of the disclosed T regitope-blood component conjugates and modified polypeptides comprising the T regitope to certain other T cell epitopes, the disclosed compositions It is possible to suppress highly specific immune responses to linked or mixed T-cell epitopes while leaving the immune system in balance. For example, a T-regitope-blood component conjugate and/or a modified polypeptide (both comprising an autoimmune antigen such as preproinsulin or insulin (e.g., one or more of SEQ ID NOs: 56-63) or only these(s)) The immune system can be trained to "tolerate" the co-delivered antigen, e.g. , this training can, for example, induce endogenous TRegs (including, in embodiments, natural TRegs and/or adaptive TRegs) and/or change the phenotype of responsive effector T cells to preproinsulin, insulin, or islet cells. and/or by converting to an adaptive regulatory T cell phenotype that can suppress immune responses targeting other proteins associated with T1D. Such immune reprogramming may reduce and/or eliminate immune responses targeting islet cells, insulin, preproinsulin, and other antigens associated with autoimmune responses in T1D, while leaving the immune system in balance. .

The T-regitopes of the present disclosure are derived from circulating extracellular proteins. To be useful, these T regitopes should be bona fide T cell epitopes (ie, capable of binding to both MHC molecules and the TCR). In embodiments, it is desirable that the T regitope be associated with a pre-existing population of regulatory T cells of sufficient size to have a therapeutic effect. T cell epitope clusters, which are epitopes capable of binding multiple MHC alleles and multiple TCRs, are key to meeting this latter qualification.

In their natural state, the T-regitopes of the present disclosure are capable of binding and activating endogenous TRegs (including, in embodiments, natural TRegs and/or adaptive TRegs), preventing or terminating an immune response. be. Furthermore, treatment with the T-regitope-blood component conjugates and modified polypeptides comprising the T-regitope of the present disclosure expands the corresponding endogenous TReg population (including, in embodiments, natural TRegs and/or adaptive TRegs) and or can be activated by a cognate peptide derived from preproinsulin to suppress effector responses targeting insulin or preproinsulin. The therapeutic methods of the present disclosure provide the following advantages.
1. Treatment with the T-regitope compositions of the present disclosure is highly antigen-specific (e.g., treatment with the T-regitope compositions corresponds to the corresponding endogenous TReg population (in multiple aspects) in a highly antigen-specific manner. (including natural TRegs and/or adaptive TRegs).
2. an efficient and less expensive treatment regimen when compared to current antigen-specific therapies for T1D; and 3. Prevention or treatment of T1D such that the subject maintains and/or acquires tolerance to glucose and/or does not need to rely on insulin replacement therapy.

In aspects, the present disclosure provides novel T regitope-blood component conjugates and T
A modified polypeptide is provided that includes a regitope, said modified peptide capable of reacting with a blood component to form such a T regitope-blood component conjugate. Conjugates of T-regitope-containing polypeptides and blood components such as albumin can be useful as carrier proteins for T-regitope payloads. The T-regitope-blood component conjugate extends the half-life of the T-regitope-containing engineered polypeptide in vivo, protects the T-regitope-containing engineered polypeptide from rapid proteolysis, and protects the T-regitope-containing engineered polypeptide from rapid proteolysis. It is possible to protect against rapid clearance from the circulation and/or rapid renal excretion and allow wide distribution of the T-regitope-blood component conjugate throughout the body of the subject, assisting delivery to appropriate immune cells (such as macrophages and APCs), allowing the modified polypeptide containing the T resitope to be processed by the endocytic pathway of specific immune cells (such as macrophages and APCs), and/or Alternatively, it assists the immune cells in presenting a modified polypeptide containing a T-regitope as an antigen.

(definition)
To further facilitate understanding of the present invention, a number of terms and phrases are defined below. Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. . Terms as defined in commonly used dictionaries should be construed to have meanings consistent with the relevant art and their meanings in the context of this disclosure, and are not expressly defined as such herein. It will be further understood that insofar as this is not to be construed in an idealized or overly formal sense.

Ranges provided herein are understood to be shorthand for all values within the range. For example, the range 1 to 25 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25, as well as all intervening decimal values between the aforementioned integers, such as 1, for example. Includes all decimal values between said integers such as 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9. With respect to subranges, "enclosed subranges" extending from either endpoint of the range are specifically contemplated. For example, enclosed subranges of the exemplary range 1-25 are 1-5, 1-10, 1-15, and 1-20 in one direction, or 25-20, 25-15, 25-10, and 25-5.

As used herein, the term " biological sample " is used to refer to any sample of tissue, cells, or secretions from an organism.

As used herein, the term "transplantation" refers to the removal of cells, tissues, or organs, referred to as "transplants" or "grafts," from a subject, or Refers to the process of putting them into (usually) different objects. The subject who performs the transplant is called the "donor," and the subject who receives the transplant is called the "recipient." An organ or graft transplanted between genetically different recipients of the same species is called an "allograft." A graft transplanted between subjects of different species is referred to as a "xenograft."

As used herein, the term "medical condition" includes, but is not limited to, any condition or disease manifested as one or more physical and/or psychological symptoms for which treatment and/or prevention is desirable. This includes previously and newly identified diseases and other disorders.

As used herein, the term " immune response " refers to lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules (antibodies) produced by these cells or the liver. selective damage or destruction of cancer cells, metastatic tumor cells, malignant melanoma, invading pathogens, pathogen-infected cells or tissues from the human body as a result of the cooperative action of , or elimination, or in the case of autoimmunity or pathological inflammation, the selective damage, destruction, or elimination of normal human cells or tissues. In embodiments, the immune response is a measurable cytotoxic T lymphocyte (CTL) response (e.g., to a virus expressing an immunogenic polypeptide) or a measurable B cell response, such as the production of antibodies (e.g., to a virus expressing an immunogenic polypeptide). , against immunogenic polypeptides). Those skilled in the art will appreciate various assays for determining whether an immune response has occurred to a peptide, polypeptide, or related composition, including the use of experiments and assays as disclosed in the Examples of the specification. It is publicly known. Various B and T lymphocyte assays include ELISA, EliSpot assay, cytotoxic T lymphocyte CTL assay, chromium release assay, proliferation assay using peripheral blood lymphocytes (PBL), tetramer assay and other cytokines. These are well known such as production assays. See Benjamini et al. (1991), incorporated herein by reference.

As used herein, T-regitope-blood component conjugates and modified polypeptides comprising a T-regitope of the present disclosure, wherein the modified peptides are capable of reacting with blood components to form such T-regitope-blood component conjugates. (as well as nucleic acids encoding modified polypeptides of T-regitope-blood component conjugates and/or modified polypeptides used to form T-regitope-blood component conjugates). "effective amount", "therapeutically effective amount" of a nucleic acid, expression cassette, plasmid, expression vector, recombinant virus, cell, and/or pharmaceutical composition or formulation disclosed herein); '' refers to an amount sufficient to achieve the desired therapeutic and/or prophylactic effect, eg, an amount that results in the prevention or reduction of symptoms associated with the disease being treated. The amount of a composition of the present disclosure administered to a subject will depend on the type and severity of the disease, as well as the characteristics of the individual, such as general health, age, sex, weight and tolerance to drugs. It will also depend on the extent, severity, and type of disease. One of ordinary skill in the art will be able to determine the appropriate dosage depending on these and other factors. Compositions of the invention can also be administered in combination with each other or with one or more additional therapeutic compounds.

As used herein, the terms "regulatory T cells,""Tregs," etc. refer to CD4+ and/or CD8+ effector T cells (Teff) through a variety of different mechanisms, including cell-cell contact and suppressive cytokine production. ) Refers to a subpopulation of T cells that suppress immune effector functions, including induction, proliferation, and/or inhibition or downregulation of cytokine production. In embodiments, CD4+ TRegs are characterized by the presence of certain cell surface markers including, but not limited to, CD4, CD25, and FoxP3. In embodiments, upon activation, CD4+ regulatory T cells secrete immunosuppressive cytokines and chemokines including, but not limited to, IL-10 and/or TGF-β. CD4+ Tregs can also exert immunosuppressive effects through direct killing of target cells, characterized by the expression upon activation of effector molecules including, but not limited to, granzyme B and perforin. In embodiments, CD8+TRegs are characterized by the presence of certain cell surface markers including, but not limited to, CD8, CD25, and, upon activation, FoxP3. In embodiments, upon activation, regulatory CD8+ T cells secrete immunosuppressive cytokines and chemokines including, but not limited to, IFNγ, IL-10, and/or TGF-β. In embodiments, CD8+ TRegs can also exert immunosuppressive effects through direct killing of target cells and through expression upon activation of effector molecules including, but not limited to, granzyme B and/or perforin. characterized.

As used herein, the term "T cell epitope" refers to a 7-30 amino acid long that specifically binds to human leukocyte antigen (HLA) molecules and interacts with a particular T cell receptor (TCR). Refers to an MHC ligand or protein determinant that can interact. As used herein, in the context of a T cell epitope that is known or determined (e.g., predicted) to engage a T cell, the terms "engage," "engage," etc. means that a T cell epitope, when bound to an MHC molecule (eg, a human leukocyte antigen (HLA) molecule), can interact with the TCR of a T cell and activate the T cell. Generally, T cell epitopes are linear and do not express specific three-dimensional characteristics. T cell epitopes are not affected by the presence of denaturing solvents. The ability to interact with T cell epitopes can be predicted by in silico methods (De Groot AS et al. (1997), AIDS Res Hum Retroviruses, 13(7):539-41; Schafer JR et al. (1998), Vaccine, 16(19):1880-4; De Groot AS et al., (2001), Vaccine, 19(31):4385-95; De Groot AR et al., (2003), Vaccine, 21(27-30):4486 -504, all of which are incorporated herein by reference in their entirety).

As used herein, the term "T cell epitope cluster" refers to a polypeptide containing about 4 to about 40 MHC binding motifs. In certain embodiments, the T cell epitope cluster has between about 5 and about 35 MHC binding motifs, between about 8 and about 30 MHC binding motifs, or between about 10 and about 20 MHC binding motifs. Contains motifs.

As used herein, the term "regulatory T cell epitope" ("T regitope") refers to the term "regulatory T cell epitope" ("T regitope") that elicits a tolerance response (Weber CA et al. (2009), Adv Drug Deliv, 61(11): 965-76 ), refers to a "T cell epitope" that is capable of binding to MHC molecules and engaging (ie, interacting with and activating) circulating endogenous TRegs (including, in embodiments, natural and/or adaptive TRegs). In embodiments, upon activation, CD4+ regulatory T cells secrete immunosuppressive cytokines and chemokines including, but not limited to, IL-10 and/or TGF-β. CD4+ Tregs may also exert immunosuppressive effects through direct killing of target cells and are characterized by the expression upon activation of effector molecules including, but not limited to, granzyme B and perforin, IL-10 and TGF. - leading to the expression of immunosuppressive cytokines including, but not limited to, TNF-α and TNF-α. In embodiments, upon activation, regulatory CD8+ T cells secrete immunosuppressive cytokines and chemokines, including, but not limited to, IFNγ, IL-10, and/or TGF-β. In embodiments, CD8+ TRegs can also exert immunosuppressive effects through direct killing of target cells, characterized by the expression upon activation of effector molecules including, but not limited to, granzyme B and/or perforin. It will be done. In aspects, the disclosed T regitopes are T cell epitope clusters that are epitopes capable of binding to multiple MHC alleles and multiple TCRs.

As used herein, the term "immunostimulatory T cell epitope polypeptide" refers to a molecule capable of inducing an immune response, such as a humoral, T cell-based, or innate immune response. In embodiments, the immunostimulatory T cell epitope polypeptide is human coagulation factor V or coagulation factor VIII.

As used herein, the term "B cell epitope" refers to a protein determinant that is capable of specifically binding an antibody. B cell epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that binding to the former is lost, but binding to the latter is not lost in the presence of denaturing solvents.

The term "subject" as used herein refers to any living organism against which an immune response is elicited. The term covers humans, non-human primates such as apes and monkeys such as chimpanzees, domestic animals such as cows, sheep, pigs, goats and horses, domestic mammals such as dogs and cats, mice, rats and guinea pigs. Laboratory animals, including, but not limited to, rodents. Note that this term does not indicate a specific age or gender. Therefore, it is intended for adults and newborns, as well as fetuses, whether male or female.

As used herein, the term "major histocompatibility complex (MHC)", "MHC molecule", "MHC protein" or "HLA protein" specifically refers to It can be understood to be a protein that can bind peptides representing T cell epitopes and transport them to the cell surface where they can be presented to specific cells, in particular cytotoxic T lymphocytes or T helper cells. The major histocompatibility complex in the genome contains gene regions whose gene products expressed at the cell surface are important for binding and presenting endogenous and/or foreign antigens and thus controlling immunological processes. . The major histocompatibility complex is divided into two groups of genes that encode different proteins, namely MHC class I molecules and MHC class II molecules. These two MHC classes of molecules are specialized for different antigen sources. MHC class I molecules present internally synthesized antigens, such as viral proteins and tumor antigens. MHC class II molecules present foreign protein antigens, such as bacterial products. The cell biology and expression patterns of the two MHC classes are adapted to their different roles. Class I MHC molecules consist of heavy and light chains, and if this peptide has the appropriate binding motif, it can bind peptides of about 8 to 11 amino acids, usually 9 to 10 amino acids, and induce cytotoxic T lymphocytes. Can be presented to the ball. The peptides bound by class I MHC molecules are derived from endogenous protein antigens. The heavy chain of class I MHC molecules is preferably a monomer of HLA-A, HLA-B or HLA-C, and the light chain is β-2-microglobulin. Class II MHC molecules consist of α and β chains and can bind peptides of approximately 12 to 25 amino acids and present them to T-helper cells if this peptide has an appropriate binding motif. . Peptides bound by class II MHC molecules are usually derived from extracellular, foreign protein antigens. The α and β chains are in particular HLA-DR, HLA-DQ and HLA-DP monomers.

As used herein, the term "MHC complex" refers to a protein complex that is capable of binding a specific repertoire of polypeptides known as HLA ligands and transporting said ligands to the cell surface.

As used herein, the term "MHC ligand" refers to a polypeptide that is capable of binding to one or more specific MHC alleles. The term "HLA ligand" is interchangeable with the term "MHC ligand." Cells expressing MHC/ligand complexes on their surface are termed "antigen presenting cells" (APCs). Similarly, as used herein, the term "MHC-binding peptide" refers to peptides that bind to MHC class I and/or MHC class II molecules. For MHC class I/peptide complexes, the binding peptide is typically 8-10 amino acids long, although longer or shorter peptides may be useful. For MHC class II/peptide complexes, the binding peptide is typically 10-25 amino acids long, especially 13-18 amino acids long, but longer and shorter peptides may also be effective.

As used herein, the term "T cell receptor" or "TCR" engages a specific repertoire of MHC/ligand complexes presented on the surface of cells such as antigen presenting cells (APCs). refers to a protein complex expressed by T cells that is capable of

As used herein, the term "MHC binding motif" refers to a pattern of amino acids in a protein sequence that predicts binding to a particular MHC allele.

As used herein, the term "EpiBar®" refers to a single nonameric frame that is predicted to bind at least four different HLA alleles. Cluster scores higher than 10 are considered significant. All scores in the top 5% are considered "hits".

As used herein, the term "immune synapse" refers to the protein complex formed by the simultaneous engagement of a given T cell epitope with both a cell surface MHC complex and a TCR.

The term "polypeptide" refers to a polymer of amino acids and does not imply a particular length. Thus, peptides, oligopeptides and proteins are included within the definition of polypeptide. As used herein, polypeptides are defined as being substantially free of cellular material when isolated from recombinant and non-recombinant cells, or chemical precursors when chemically synthesized. It is said to be "separated" or "purified" when it does not contain body or other chemicals. However, polypeptides (e.g., comprising, consisting solely of, or consisting essentially of one or more of SEQ ID NOs: 1-55 (and variants and fragments thereof disclosed herein)) ), or consisting solely of, or consisting essentially of, one or more of SEQ ID NOs: 56-63 (and variants and fragments thereof disclosed herein). ) and associated with immunogenicity in diabetes (referred to as “T1Dgens”) can be attached to, linked to, or inserted into another polypeptide (e.g., a heterologous peptide). However, it is not normally associated within the cell and is still "separated" or "purified."

As used herein, the term "pharmaceutically acceptable" means approved or approvable by a federal or state regulatory agency, or approved or approved by the United States Pharmacopeia for use in animals, including humans. means listed in another generally accepted pharmacopoeia.

As used herein, the term "pharmaceutically acceptable excipient, carrier, or diluent" and the like refers to a drug that can be administered to a subject with a drug, does not destroy its pharmacological activity, and does not destroy the drug's pharmacological activity. Refers to an excipient, carrier, or diluent that is nontoxic when administered in sufficient doses to deliver a therapeutic amount.

As used herein, "free thiol" refers to a thiol side chain of an optional amino acid in a polypeptide and/or protein, where the thiol contains a sulfhydryl group. For example, free thiols are not attached to the side chains of other amino acids through intramolecular or intermolecular disulfide bonds.

As used herein, "functional group" is a group on blood components, including mobile and fixed proteins, with which a reactive group on a modified therapeutic peptide reacts to form a covalent bond. . Functional groups include hydroxyl groups for coupling with ester-reactive groups; thiol groups for coupling with maleimide, imidate and thioester groups; activated carboxyl, phosphate or any other acyl groups on the reactive groups; It can contain an amino group for attachment.

As used herein, a "blood component" can be either fixed or mobile. Fixed blood components are non-mobile blood components that include tissues, membrane receptors, interstitial proteins, fibrin proteins, collagen, platelets, endothelial cells, epithelial cells and their associated membranes and membrane receptors, somatic cells. , skeletal and smooth muscle cells, neuronal components, bone cells and osteoclasts, and all body tissues, especially those associated with the circulatory and lymphatic systems. Mobile blood components are blood components that generally do not remain fixed for long periods of time (less than 5 minutes, more usually less than 1 minute). These blood components are not membrane bound, exist in the blood for long periods of time, and are present in minimum concentrations of at least 0.1 μg/ml. Mobile blood components include serum albumin, transferrin, ferritin, and immunoglobulins such as IgM and IgG. The half-life of mobile blood components may be at least about 12 hours.

As used herein, the term "special purpose computer program" refers to a computer program designed for a specific purpose; typically, to analyze a specific set of raw data and answer a specific scientific question. Refers to a program.

As used herein, the term "z-score" indicates how many standard deviations an element is from the mean. The z-score can be calculated from the following formula: z = (X-μ)/σ, where z is the z-score, X is the element value, μ is the population mean, and σ is the standard deviation. be.

As used herein, the singular forms "a," "an," and "the" include plural forms, including "at least one," unless the content clearly dictates otherwise. Intended. "Or" means "and/or". As used herein, the terms "and/or" and "one or more" include any and all combinations of the associated listed items. For example, with respect to "one or more of SEQ ID NOs: 1-55 of the present disclosure," the term "one or more" includes any and all combinations of SEQ ID NOs: 1-55. Additionally, the term "or a combination thereof" refers to a combination comprising at least one of the aforementioned elements.

The following abbreviations and/or acronyms are used throughout this application.
APC: Antigen-presenting cell CEF: Cytomegalovirus, Epstein-Barr virus and influenza virus CFSE: Carboxyfluorescein succinimidyl ester dye DMSO: Dimethyl sulfoxide DR Antibody: Antigen D-related antibody ELISA: Enzyme-linked immunosorbent assay FACS: Fluorescence Activated cell sorting
Fmoc: 9-Fluoronylmethoxycarbonyl FV: Human coagulation factor V FVIII: Human coagulation factor VIII HLA: Human leukocyte antigen HPLC: High performance liquid chromatography IVIG: Intravenous purified immunoglobulin G antibody MFI: Mean fluorescence index MHC : major histocompatibility complex PBMC: peripheral blood mononuclear cells PI: proliferation index RPMI: Rothwell Park Memorial Institute medium Teff: effector T cells Treg: regulatory T cells TT: tetanus toxoid UV: ultraviolet light

As used herein, a "variant" peptide or polypeptide (including a mutant T resitope) includes one or more substitutions, deletions, insertions, inversions, fusions, and/or truncations. The amino acid sequences may differ depending on the combination. In embodiments, the variant polypeptide may differ in amino acid sequence by one or more substitutions, deletions, insertions, inversions, fusions, and truncations, or any combination thereof, but the variant polypeptide In embodiments where the peptide or polypeptide comprises a T regitope, provided that it retains binding and/or TCR specificity, and/or retains regulatory T cell stimulatory or suppressive activity. subject to the following conditions.

The present disclosure also includes polypeptide fragments of the T resitopes and other peptides or polypeptides described herein. The present invention also encompasses fragments of T-regitopes and other peptide or polypeptide variants described herein, provided that said fragments and/or variants retain MHC binding and/or TCR specificity. and in some embodiments where the peptide or polypeptide comprises a T regitope and/or retains regulatory T cell stimulatory or suppressive activity.

An "isolated" peptide or polypeptide (e.g., an isolated TReg-activating regulatory T-cell epitope, T-regitope, or T-cell epitope polypeptide) is purified from a cell that naturally expresses it, or It can be purified from cells modified to do so (recombinant) or synthesized by known protein synthesis methods. In one embodiment, the T-regitope is produced by recombinant DNA or RNA technology. For example, a nucleic acid molecule encoding a T-regitope is cloned into an expression vector, the expression vector is introduced into a host cell, and the polypeptide is expressed in the host cell. The T-regitope can then be separated from the cells by an appropriate purification scheme using standard protein purification techniques.

For purposes of this disclosure, T resitopes and other peptides or polypeptides of the present disclosure include, for example, modified forms of naturally occurring amino acids such as the D stereoisomer; non-naturally occurring amino acids; ; and mimetics. Additionally, in embodiments, the T resitopes and other peptides or polypeptides of the present disclosure may include retro-inverso peptides, provided that the retro-inverso peptides or polypeptides of the present disclosure are , at least partially retains MHC binding and/or TCR specificity, in embodiments the peptide or polypeptide comprises a T regitope, and/or has regulatory T cell stimulatory or suppressive activity. .

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described. Other features, objects, and advantages of the disclosure will be apparent from the specification and claims. In this specification and the appended claims, the singular forms include plural references unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All references cited herein are the same as if the individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. To this extent, it is incorporated herein by reference in its entirety.

[T-regitope-blood component conjugate and modified polypeptide containing T-regitope]
An object of the present disclosure is to provide novel T-regitope-blood component conjugates and modified polypeptides comprising T-regitopes, wherein the modified peptides react with blood components to form such T-regitope-blood component conjugates. It is possible to form a gate. Conjugates of T-regitope-containing polypeptides and blood components such as albumin can be useful as carrier proteins for T-regitope payloads. The T-regitope-blood component conjugate extends the half-life of the T-regitope-containing engineered polypeptide in vivo, protects the T-regitope-containing engineered polypeptide from rapid proteolysis, and protects the T-regitope-containing engineered polypeptide from rapid proteolysis. protects against rapid clearance from the circulation and/or rapid renal excretion, allows wide distribution of the T resitope-blood component conjugate throughout the subject's body , and directs T to appropriate immune cells (such as macrophages and APCs). aids in the delivery of modified polypeptides containing T-regitopes, allows modified polypeptides containing T-regitopes to be processed by the endocytic pathway of specific immune cells (such as macrophages and APCs), and/or allows antigens to be processed by said immune cells. The purpose of this invention is to assist in the presentation of modified polypeptides containing the T regitope.

selective engagement of endogenous TRegs (including, in embodiments, natural TRegs and/or adaptive TRegs) and Activation is of therapeutic value as a means of treatment for diseases or conditions characterized by the presence of an undesirable immune response, such as autoimmune diseases such as type 1 diabetes. Thus, the present disclosure also relates to methods of using the T-regitope-blood component conjugates and modified polypeptides comprising the T-regitope in the treatment and prevention of autoimmune diseases such as type 1 diabetes.

In embodiments, the T regitope-blood component conjugate comprises a blood component that acts as a carrier protein (e.g., albumin) and further comprises a modified polypeptide, said modified polypeptide comprising one or more regulatory T cell epitopes. (referred to as "T-regitope"). The modified polypeptide includes a reactive site attached to the polypeptide that is capable of forming a bond (eg, a covalent bond) with a reactive functional group on a blood component. In embodiments, the modified polypeptide further comprises one or more antigenic peptides associated with immunogenicity in diabetes ("T1Dgen"peptides; e.g., PPI-derived peptides), which optionally include a linker and/or cleavage. It may be separated from one or more T regitopes by a site (eg, a lysosomal cleavage site). T-regitope-blood component conjugates may be formed by modifying a polypeptide containing a T-regitope, attaching a reactive site to this peptide to create a modified polypeptide, and then Bonds can be formed between reactive functional groups and reactive sites of modified polypeptides, which are described in U.S. Pat. No. 6,849,714, U.S. Pat. No. 256,253, and US Pat. No. 7,307,148, incorporated herein by reference in their entirety. In embodiments of the disclosed T-regitope-blood component conjugates and modified polypeptides comprising a T-regitope, the T-regitope-blood component conjugates and modified polypeptides comprising a T-regitope are isolated, synthesized, or recombinant. It's good to be there.

In embodiments, the blood component of the T-regitope-blood component conjugate is described in US Pat. No. 6,849,714, US Pat. No. 6,887,470, US Pat. No. 7,307,148, it can be either fixed or mobile. Fixed blood components are blood components that do not move, and include tissues, membrane receptors, interstitial proteins, fibrin proteins, collagen, platelets, endothelial cells, epithelial cells and their associated membranes and membrane receptors, somatic cells, and the skeleton. Included are muscle and smooth muscle cells, neuronal components, bone cells and osteoclasts, and all body tissues, especially those associated with the circulatory and lymphatic systems. Mobile blood components are blood components that generally do not remain in a fixed position for long periods of time (less than 5 minutes, more usually less than 1 minute). These blood components are not membrane bound, exist in the blood for long periods of time, and are present in minimum concentrations of at least 0.1 μg/ml. Mobile blood components include serum albumin, transferrin, ferritin, and immunoglobulins such as IgM and IgG. The half-life of mobile blood components is at least about 12 hours. In embodiments of the T-regitope-blood component conjugate, the blood component is albumin, such as serum albumin, human serum albumin, recombinant albumin, and recombinant human serum albumin. Albumin is a preferred blood component because it contains an Fc neonatal binding domain that transports the T-regitope-albumin conjugate to appropriate cells such as macrophages and APCs. Additionally, albumin contains cysteine at amino acid 34 (Cys34) (the amino acid position in the amino acid sequence of human serine albumin) and contains a free thiol with a pKa of approximately 5, which may function as a preferred reactive functional group of albumin. . Cys34 of albumin can form a stable thioester bond with maleimidopropionamide (MPA), which is a preferred reactive site for modified T resitope peptides.

In embodiments, the reactive functional group on the blood component of the T regitope-blood component conjugate or on the blood component capable of forming a conjugate with the disclosed modified polypeptides is reactive on the modified therapeutic peptide. Groups on blood components, including mobile and immobilized proteins, with which the groups react to form covalent bonds. Such as disclosed in U.S. Pat. No. 6,849,714, U.S. Pat. Functionalities typically include hydroxyl groups for coupling with ester reactive groups, thiol groups for coupling with maleimide, imidate and thioester groups, activated carboxyl on reactive groups, phosphate or any other acyl group. Contains an amino group for In embodiments, the reactive functional group of the blood component is an amino group, a hydroxyl group, or a thiol group. In embodiments, the reactive functional group of the blood component is a component of the side chain of an amino acid in the polypeptide and/or protein, and the reactive functional group is present near the surface of the polypeptide and/or protein. In embodiments, the reactive functional group of the blood component is a thiol group of a free cysteine residue of the proteinaceous blood component. In embodiments, the reactive functional group is the free thiol group of cysteine at amino acid 34 (Cys34) of serine albumin. In embodiments, the reactive functional group of the blood component is a thiol that has a pKa of about 5 in a physiological environment such as plasma. In embodiments, the reactive functional group of the blood component is a thiol that has a pKa of about 5.5 in a physiological environment such as plasma. In embodiments, the reactive functional group of the blood component is a thiol that has a pKa of 3-7 in a physiological environment such as plasma. In embodiments, the reactive functional group of the blood component is a thiolate anion. In embodiments, the reactive functional group is the thiolate anion of cysteine at amino acid 34 (Cys34) of serine albumin.

In embodiments, the modified polypeptide of the T-regitope-blood component conjugate and the modified polypeptide used to form the T-regitope-blood component conjugate comprises a reactive site attached to the polypeptide; The reactive moiety is capable of forming a bond (eg, a covalent bond) with a reactive functional group on a blood component. In embodiments, the reactive group is capable of reacting with and forming a covalent bond with an amino group, hydroxyl group, or thiol group on a blood component. In embodiments, the reactive group is placed at a site such that when the modified polypeptide is bound to a blood component, the modified peptide retains a substantial proportion of the activity of the parent compound. In embodiments, the reactive moiety can be a succinimidyl group or a maleimide group. In embodiments, the reactive site may be attached to an amino acid located in a region of the polypeptide to be modified that has less therapeutic activity. In embodiments, the reactive site is attached to the amino terminal amino acid of the polypeptide to be modified. In embodiments, the reactive site is attached to the carboxy-terminal amino acid of the modified polypeptide. In embodiments, the reactive site is attached to an amino acid located between the amino terminal amino acid and the carboxy terminal amino acid of the modified polypeptide. In embodiments, a reactive group may be attached to a polypeptide (to be modified) via a linking group or optionally without the use of a linking group. Additionally, one or more additional amino acids (eg, one or more lysines) may be added to the polypeptide to facilitate attachment of reactive groups. A linking group is a chemical moiety that links or connects a reactive group of a blood component to a polypeptide containing one or more T-regitopes. The linking group is an alkyl group, an alkoxy group, an alkenyl group, an alkynyl group, or an amino group substituted with an alkyl group, a cycloalkyl group, a polycyclic group, an aryl group, a polyaryl group, a substituted aryl group, a heterocyclic group, and It can consist of one or more substituted heterocyclic groups. The linking group may also be composed of polyethoxy amino acids such as AEA ((2-amino)ethoxyacetic acid) or the preferred linking group AEEA ([2-(2-amino)ethoxy]ethoxyacetic acid). In embodiments, the linking group may be comprised of a polyethylene glycol linker, such as, but not limited to, PEG2 or PEG12.

It should be understood that modified polypeptides may be administered in vivo such that conjugation with blood components occurs in vivo, or they may first be conjugated to blood components in vitro and obtained. Peptidase stabilizing polypeptides may be administered in vivo. Additionally, peptidases, as disclosed in U.S. Patent No. 6,849,714, U.S. Patent No. 6,887,470, U.S. Patent No. 7,256,253 and U.S. Patent No. 7,307,148, Stabilizing polypeptide refers to a modified peptide, with or without a linking group, that is conjugated to a blood component via a covalent bond formed between a reactive group on the modified peptide and a functional group on the blood component. Such reactions are preferably carried out by covalently linking polypeptides modified with maleimide linkages (e.g., prepared from GMBS, MPA or other maleimides) to thiol groups on mobile blood proteins such as serum albumin or IgG. Established. Peptidase-stabilized polypeptides are more stable in the presence of peptidases in vivo than non-stabilized peptides. Peptidase-stabilized therapeutic peptides generally have at least a 10-50% increase in half-life compared to non-stabilized peptides of the same sequence. Peptidase stability is determined by comparing the half-life of an unmodified therapeutic peptide in serum or blood to the half-life of a corresponding modified therapeutic peptide in serum or blood. Half-life is determined by sampling serum or blood after administering modified and unmodified peptides and measuring the activity of the peptides. In addition to determining activity, the length of a therapeutic peptide can also be measured.

In embodiments, the modified polypeptide of the T-regitope-blood component conjugate and the modified polypeptide used to form the T-regitope-blood component conjugate consist of peptides or polypeptide chains derived from a common human protein. one or more T-regitopes, which may be referred to herein as "Treg-activated T-cell epitopes,""T-regitopes," or "T-cell epitope polypeptides." The T-regitopes of the present disclosure are highly conserved among known variants of their source proteins (eg, present in greater than 10% of known variants). The T regitopes of the present disclosure include at least one putative T cell epitope identified by EpiMatrix® analysis. EpiMatrix® is a proprietary computer algorithm developed by EpiVax (Providence, RI) that is used to screen protein sequences to confirm the presence of putative T cell epitopes. The input sequence is parsed into nonamer frames, each frame overlapping the last frame by 8 amino acids. Each resulting frame contains eight common class II-HLA alleles (DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*0801, DRB1*1101, DRB1*1301, DRB1*1501) are scored for predicted binding affinity against a panel of The raw scores are normalized to the scores of a large sample of randomly generated peptides. As a result, a "Z" score is reported. In embodiments, any 9-mer peptide with an allele-specific EpiMatrix Z-score greater than 1.64 that is theoretically in the top 5% of any given sample is considered a putative T-cell epitope. .

Peptides containing clusters of putative T cell epitopes are more likely to be positive when validating in vitro and in vivo assays. The results of the initial EpiMatrix® analysis are further screened for the presence of putative T cell epitope "clusters" using a second proprietary algorithm known as the Clustimer® algorithm. The Clustimer® algorithm identifies subregions within any amino acid sequence that contain a statistically disproportionate number of putative T cell epitopes. A typical T cell epitope "cluster" is about 9 to about 30 amino acids in length, and considering multiple alleles and affinities for multiple nonamer frames, there are about 4 to about 40 putative T cell epitopes. can include epitopes. Each epitope cluster identified in the aggregated EpiMatrix® score is calculated by summing the scores of the putative T-cell epitopes and applying a correction based on the expected score of a randomly generated cluster of the same length as the candidate epitope cluster. An aggregate EpiMatrix® score is calculated by subtracting the coefficients. EpiMatrix® cluster scores greater than 10 are considered significant. In aspects, the T regitopes of the present disclosure include a number of putative T cell epitopes that form a pattern known as a T cell epitope cluster.

Many of the most reactive T cell epitope clusters contain a feature called "EpiBar®." As mentioned above, EpiBar® is a single nonameric frame that is predicted to be reactive to at least four different HLA alleles. In embodiments, the T-regitopes of the present disclosure can include one or more EpiBar®.

The JanusMatrix system (EpiVax, Providence, RI) is useful for screening peptide sequences for cross-conservation with the host proteome. JanusMatrix is an algorithm that predicts the likelihood of cross-reactivity between peptide clusters and the host genome or proteome based on the conservation of TCR-facing residues in their putative MHC ligands. The JanusMatrix algorithm first considers all predicted epitopes contained in a given protein sequence and divides each predicted epitope into its constituent agretopes and epitopes. Each sequence is then screened against a database of host proteins. Peptides with matching MHC-facing aggretopes (ie, the aggretopes of the input peptide and its host peptide are expected to bind to the same MHC allele) and exactly the same TCR-facing epitopes are returned. JanusMatrix homology scores suggest a bias towards immune tolerance. In the case of therapeutic proteins, cross-conservation of autologous human epitopes and therapeutic epitopes may increase the likelihood that such candidates will be tolerated by the human immune system. In the case of vaccines, cross-conservation between human and antigenic epitopes may indicate that such candidates utilize immune camouflage and thereby evade immune responses, resulting in ineffective vaccines. . If the host is, for example, a human, peptide clusters are screened against the human genome or proteome based on the conservation of TCR-facing residues in putative HLA ligands. The peptides are then scored using the JanusMatrix homology score. In embodiments, peptides with JanusMatrix homology scores greater than 3.0 exhibit high tolerance potential and, as such, may be highly useful T-regitopes of the present disclosure.

In embodiments, the T-regitopes of the present disclosure bind with at least moderate affinity to at least one, preferably two or more, common HLA class II molecules (e.g., in embodiments, bind to soluble HLA molecules). <1000 μM IC50, <500 μM IC50, <400 μM IC50, <300 μM IC50, or <200 μM IC50) in HLA-based binding assays. In embodiments, the T-regitopes of the present disclosure are capable of being presented on the cell surface by APCs in the context of at least one, and in other embodiments two or more, alleles of the HLA. In this situation, the T-regitope HLA complex has a TCR specific for the T-regitope HLA complex, and the endogenous TReg (in embodiments natural TReg and/or adaptive TReg). In embodiments, recognition of T regitope-HLA complexes can activate matched regulatory T cells to secrete regulatory cytokines and chemokines.

In embodiments, the one or more T resitopes of the modified polypeptide comprise, consist exclusively of, or consist essentially of one or more of SEQ ID NOs: 1-55 (and fragments and variants thereof). It has an array consisting of these (and others). The expression "consisting essentially of" means any of SEQ ID NOs: 1-55 (or a fragment thereof), as long as the T-regitope of the modified polypeptide according to the present disclosure does not significantly impair the activity of the peptide functioning as a T-regitope. In addition to the sequence according to the MHC ligand or variant), it is meant to include additional amino acids or residues that may be present at either terminus and/or side chain of the peptide, which do not necessarily form part of the peptide that functions as an MHC ligand. intend to do so. In embodiments, the peptides or polypeptides of the present disclosure may be in either neutral (uncharged) or salt form and are free of modifications such as glycosylation, side chain oxidation, or phosphorylation; It may be included or not. In certain embodiments, such polypeptides can be capped with an N-terminal acetyl group and/or a C-terminal amino group. In embodiments, the T resitope of a modified polypeptide according to the present disclosure comprises, consists exclusively of, or consists essentially of one or more of the sequences SEQ ID NO: 1 and 28. In embodiments, the T resitope of a modified polypeptide according to the present disclosure comprises, consists exclusively of, or consists essentially of the amino acid sequence(s) of SEQ ID NO: 1. In embodiments, the T resitope of a modified polypeptide according to the present disclosure comprises, consists exclusively of, or consists essentially of the amino acid sequence(s) of SEQ ID NO: 28. In embodiments, the one or more T resitopes of the modified polypeptides optionally have one or more linkers (which may be cleavage sensitive sites) adjacent to their N-terminus and/or C-terminus. may have. In such modified polypeptides, two or more T regitopes may have cleavage sensitive sites between them. Alternatively, two or more T regitopes may be connected to each other directly or via a linker that is not a cleavage sensitive site. In embodiments, modified polypeptides of T-regitope-blood component conjugates, and modified polypeptides used to form T-regitope-blood component conjugates, include one or more of the following regulatory T-regitopes (and fragments thereof, variants thereof, and fragments of such variants, provided that the fragments and/or variants retain MHC binding and TCR specificity.

Sequence number 1: EEQYNSTYRVVSVLTVLHQDW
Sequence number 2: PAVLQSSGLYSLSSVVTVPSSSLGTQ
Sequence number 3: PGLVRPSQTLSLTCT
SEQ ID NO: 4: GGLVQPGGSLRLSCAASGFTF
SEQ ID NO: 5: GGLVQPGRSLRLSCAASGFTF
Sequence number 6: GASVKVSCKASGYTF
Sequence number 7: WSWVRQPPGRGLEWI
Sequence number 8: WSWIRQPPGKGLEWI
Sequence number 9: MHWVRQAPGKGLEWV
Sequence number 10: MHWVRQAPGQGLEWM
Sequence number 11: VDTSKNQFSLRLSSVTAADTA
SEQ ID NO: 12: NTLYLQMNSLRAEDTAVYYCA
Sequence number 13: FQHWGQGTLVTVSS
SEQ ID NO: 14: FDLWGRGTLVTVSS
Sequence number 15: FDIWGQGTMVTVSS
Sequence number 16: FDYWGQGTLVTVSS
Sequence number 17: FDPWGQGTLVTVSS
Sequence number 18: MDVWGQGTLVTVSS
Sequence number 19: MDVWGQGTTVTVSS
SEQ ID NO: 20: LNNFYPREAKVQWKVDNALQSGNS
SEQ ID NO: 21: KVYACEVTHQGLSS
Sequence number 22: DIQMTQSPSSLSA
Sequence number 23: EIVLTQSFGTLSL
Sequence number 24: GDRVTITCRASQGIS
SEQ ID NO: 25: LAWYQQKPGKAPKL
Sequence number 26: LAWYQQKPGQAPRL
SEQ ID NO: 27: LLIYGASSRATGIPD
Sequence number 28: GTDFTLTISSLQPED
SEQ ID NO: 29: SEYELTQPPSVSVVS
SEQ ID NO: 30: GQSITISCTGTSSDV
Sequence number 31: VSWYQQHPGKAPKL
Sequence number 32: VHWYQQKPGQAPVL
Sequence number 33: VSWYQQLPGTAPKL
Sequence number 34: LMIYEVSNRPSGVPD
SEQ ID NO: 35: LKKYLYEIARRHPYFYAPE
SEQ ID NO: 36: APELLFFAKRYKAAFTECCQAA
Sequence number 37: HPDYSVVLLLRLAKTYETTLE
Sequence number 38: HPDYSVYLLLRLAKT
Sequence number 39: LLLRLAKTYETTLE
Sequence number 40: LGEYKFQNALLVRYTKKVPQVSTPT
SEQ ID NO: 41: PADVAIQLTFLRLMSTEASQNI
SEQ ID NO: 42: TGNLKKALLLQGSNEIEIR
Sequence number 43: DGDFYRADQPRSAPSL
SEQ ID NO: 44: SKEMATQLAFMRLLANYASQNITYH
Sequence number 45: VQHIQLLQKNVRAQLVDMK
SEQ ID NO: 46: GEFWLGNDYLHLLTQRQSVLRVE
Sequence number 47: QSGLYFIKPLKANQQFLVYCE
SEQ ID NO: 48: TEFWLGNEKIHLISTQSAIPY
SEQ ID NO: 49: NANFKFTDHLKYVMLPVADQDQCIR
SEQ ID NO: 50: GSEVVKRPRRYLYQWLGAPVPYPDPL
Sequence number 51: PCQWWRPTTTSTRCCT
SEQ ID NO: 52: PGEDFRMATLYSRTQTPRAELK
SEQ ID NO: 53: DGSLWRYRAGLAASLAGP
SEQ ID NO: 54: VTGVVLFRQLAPRAKLDAFFA
SEQ ID NO: 55: KASYLDCIRAIAANEADAV

In embodiments, the modified polypeptide of the T-regitope-blood component conjugate and the one or more T-regitopes of the modified polypeptide used to form the T-regitope-blood component conjugate are one or more of SEQ ID NO: 1 ~55 (and/or fragments or variants thereof) , consisting solely of, or consisting essentially of the sequence(s) , and optionally polypeptides of SEQ ID NOs: 1 to 55. 1 to 12 additional amino acids distributed in arbitrary proportions at the N-terminus and/or C-terminus of In embodiments, the one or more T resitopes comprise, consist exclusively of, or consist essentially of a peptide or polypeptide having an amino acid sequence of SEQ ID NOs: 1-55. an amino acid sequence and optionally an extension of 1 to 12 amino acids distributed in any proportion at the N-terminus and/or C-terminus of said core amino acid sequence, and these adjacent amino acids (flanking amino acids) ) (flanking amino acids) are 1-12, 1-3, 2-4, 3-6, 1-10, 1-8, 1-6, 2-12, 2-10, 2- 8, 2-6, 3-12, 3-10, 3-8, 3-6, 4-12, 4-10, 4-8, 4-6, 5-12, 5-10, 5-8, 5-6, 6-12, 6-10, 6-8, 7-12, 7-10, 7-8, 8-12, 8-10, 9-12, 9-10 or 10-12, Adjacent amino acids may be distributed in any ratio relative to the C-terminus and the N-terminus (e.g., all adjacent amino acids may be added to one terminus, amino acids may be added equally to both termini, or the other (can also be added in any proportion). In embodiments, the one or more T resitopes comprise or only one or more peptides or polypeptides having an amino acid sequence of SEQ ID NOs: 1-55 (and/or fragments and variants thereof). a core sequence consisting of, or consisting essentially of, and optionally an extension of 1 to 12 amino acids distributed in any proportion at the N-terminus and/or C-terminus of said core amino acid sequence; The total number of these flanking amino acids is 1-12, 1-3, 2-4, 3-6, 1-10, 1-8, 1 ~6, 2~12, 2~10, 2~8, 2~6, 3~12, 3~10, 3~8, 3~6, 4~12, 4~10, 4~8, 4~6 , 5-12, 5-10, 5-8, 5-6, 6-12, 6-10, 6-8, 7-12, 7-10, 7-8, 8-12, 8-10, 9 ~12, 9-10 or 10-12, and adjacent amino acids may be distributed in any ratio to the C-terminus and N-terminus (e.g., all adjacent amino acids may be added to one terminus). (amino acids can be added equally to both termini, or in any other ratio), but a T-regitope with its flanking amino acids still has the same HLA as a T-regitope without its flanking amino acids. Can bind to molecules (i.e. retain MHC binding). In embodiments, the T regitope with the contiguous amino acids binds to the same HLA molecule (i.e., retains MHC binding) and/or has the same TCR specificity as the T regitope core sequence without the contiguous amino acids. and/or regulatory T cell stimulatory or suppressive activity. In some embodiments, the adjacent amino acid sequence is also present in an endogenous protein, such as an IgG antibody, adjacent to a T regitope contained therein. In embodiments, the extension(s) are provided to improve the biochemical properties of the peptide or polypeptide (e.g., without limitation, solubility or stability) or to improve the potential for efficient proteasomal processing of the peptide. can function and be designed to improve. In embodiments, the contiguous amino acid sequences described herein can function as MHC stabilizing regions. The use of longer peptides allows for endogenous processing by the cells of interest, which may lead to more effective antigen presentation and induction of T cell responses. In embodiments, the one or more T resitopes of the modified polypeptides have one or more linkers (optionally cleavage sensitive sites) adjacent to their N-terminus and/or C-terminus. may have. In such modified polypeptides, two or more T regitopes may have cleavage sensitive sites between them. Alternatively, two or more T-regitopes may be connected to each other directly or via a linker that is not a cleavage-sensitive site. Additional linkers are described below. In embodiments, the modified polypeptide comprising one or more T-regitopes and/or the T- regitopes contained therein can be neutral (uncharged) or in salt form, including glycosylation, side chain oxidation. , or may contain or do not include modifications such as phosphorylation. In certain embodiments, a modified polypeptide comprising one or more T resitope peptides or polypeptides can be capped with an N-terminal acetyl group and/or a C-terminal amino group. In embodiments, one or more T resitopes included in the modified polypeptide can be capped with an N-terminal acetyl group and/or a C-terminal amino group.

In aspects, the present disclosure provides at least 75%, 80%, 85%, 90%, or 95% homology to any one of SEQ ID NOs: 1-55 (and/or fragments thereof). with respect to a polypeptide comprising an amino acid sequence, wherein said polypeptide still has the same HL
A molecule (ie, retains MHC binding), and/or retains the same TCR specificity, and/or retains regulatory T cell stimulatory or suppressive activity.

In embodiments, the modified polypeptide of the T regitope-blood component conjugate, and the modified polypeptide used to form the T regitope-blood component conjugate, is one or more T1Dgen peptides (e.g., PPI-derived peptides). further including. In embodiments, the one or more T1Dgen peptides may optionally be separated from one or more T regitopes by a cleavage site (eg, a lysosomal cleavage site). In embodiments, the one or more T1Dgen peptides of the modified polypeptide comprise, consist solely of, or consist essentially of the amino acid sequences of SEQ ID NOs: 56-63 (and/or fragments or variants thereof). It consists of these (ra). The expression "consisting essentially of" means that the T1Dgen peptide of the modified polypeptide according to the present disclosure, in addition to the sequence according to any of SEQ ID NOs: 56 to 63 (or a fragment or variant thereof), functions as an antigen. It is intended to include additional amino acids or residues that may be present at either terminus and/or side chain of the peptide, which do not necessarily form part of the peptide that functions as an MHC ligand, unless they significantly impair the activity of the MHC ligand. Ru. In embodiments, the one or more T1Dgen peptides comprise, consist exclusively of, or consist essentially of the amino acid sequence(s) of SEQ ID NO: 63. In embodiments, the one or more T1Dgen peptides of the modified polypeptides have one or more linkers (optionally cleavage sensitive sites) adjacent to their N-terminus and/or C-terminus. may have. In such modified polypeptides, two or more T1Dgen peptides may have a cleavage sensitive site between them. Alternatively, two or more T1Dgen peptides may be connected to each other directly or via a linker that is not a cleavage-sensitive site. Additionally, in embodiments, the T1Dgen peptide and the T regitope of the modified polypeptide may have a cleavage-sensitive site between them or may be connected to each other directly or via a linker that is not a cleavage-sensitive site. good. In embodiments, the modified polypeptide of the T-regitope-blood component conjugate, and the modified polypeptide used to form the T-regitope-blood component conjugate, comprises one or more of the following: T1Dgen, a fragment thereof, a fragment thereof; It includes variants and fragments of such variants.

Sequence number 56: LQPLALEGSLQKRGI
SEQ ID NO: 57: SHLVEALYLVCGERG
SEQ ID NO: 58: SHLVEALYLVCG
SEQ ID NO: 59: LCGSHLVEALYLVCG
Sequence number 60: AAAFVNQHLCGSHLV
SEQ ID NO: 61: GAGSLQPLALEGSLQKRGIV
SEQ ID NO: 62: GSLQPLALEGSLQKRGIV
Sequence number 63: RGFFYTPKTRREAEDLQV

In embodiments, the modified polypeptide of the T regitope-blood component conjugate, and the one or more T1Dgen peptides of the modified polypeptide used to form the T regitope-blood component conjugate, have SEQ ID NOs: 56-63 ( and/or fragments or variants thereof) and optionally 1 to 12 additional amino acids distributed in any proportion at the N-terminus and/or C-terminus of the polypeptides of SEQ ID NOs: 56-63. Contains, consists exclusively of, or consists essentially of (a). In embodiments, the one or more T1Dgen peptides comprise, consist exclusively of, or consist essentially of one or more peptides or polypeptides having the amino acid sequences of SEQ ID NOs: 56-63. etc.) and optionally an extension of 1 to 12 amino acids distributed in arbitrary proportions at the N-terminus and/or C-terminus of the core amino acid sequence, and these The total number of flanking amino acids is 1-12, 1-3, 2-4, 3-6, 1-10, 1-8, 1-6, 2-12,
2-10, 2-8, 2-6, 3-12, 3-10, 3-8, 3-6, 4-12, 4-10, 4-8, 4-6, 5-12, 5- 10, 5-8, 5-6, 6-12, 6-10, 6-8, 7-12, 7-10, 7-8, 8-12, 8-10, 9-12, 9-10 or 10 to 12, and adjacent amino acids may be distributed in any ratio to the C-terminus and N-terminus (e.g., all adjacent amino acids can also be added to one end, or amino acids can be added to both ends). (can also be added equal to the end or in any other proportion). In embodiments, the one or more T1Dgen peptides include or contain one or more peptides or polypeptides having the amino acid sequence of SEQ ID NOs : 56-63 (and/or fragments and variants thereof). a core sequence consisting of, or consisting essentially of, and optionally an extension of 1 to 12 amino acids distributed in any proportion at the N-terminus and/or C-terminus of said core amino acid sequence; The total number of these flanking amino acids (flanking amino acids) is 1 to 12, 1 to 3, 2 to 4, 3 to 6, 1 to 10, 1 to 8, 1-6, 2-12, 2-10, 2-8, 2-6, 3-12, 3-10, 3-8, 3-6, 4-12, 4-10, 4-8, 4- 6, 5-12, 5-10, 5-8, 5-6, 6-12, 6-10, 6-8, 7-12, 7-10, 7-8, 8-12, 8-10, 9-12, 9-10 or 10-12, and the adjacent amino acids may be distributed in any ratio relative to the C-terminus and the N-terminus (e.g., all adjacent amino acids are added to one end). (amino acids can be added equally to both termini, or in any other ratio), but a T1Dgen peptide with its flanking amino acids is still the same as a T1Dgen peptide without its flanking amino acids. Can bind to HLA molecules (i.e. retain MHC binding). In embodiments, the T1Dgen peptide with the contiguous amino acids is still capable of binding to the same HLA molecule (i.e., retains MHC binding) as the T1Dgen peptide core sequence without the contiguous amino acids, and /or retain the same TCR specificity. In some embodiments, the flanking amino acid sequence is also present in an endogenous protein as a flanking region of the T1Dgen peptide contained therein. For example, the peptide or polypeptide comprises, consists exclusively of, or consists essentially of one or more peptides or polypeptides having the amino acid sequence of SEQ ID NOs: 56-63 (and/or fragments and variants thereof). a core sequence consisting of these(s) and optionally an extension of 1 to 12 amino acids distributed in any proportion at its N-terminus and/or C-terminus; The extension is that found in the amino acid sequence of preproinsulin adjacent to the amino acid sequences of SEQ ID NOs: 56-63. In embodiments, the extension(s) are for improving biochemical properties of the peptide or polypeptide (e.g., without limitation, solubility or stability) or for efficient proteasomal processing of the peptide. can be designed to function to improve the possibilities of In embodiments, the contiguous amino acid sequences described herein can function as MHC stabilizing regions. The use of longer peptides allows for endogenous processing by the cells of interest, which may lead to more effective antigen presentation and induction of T cell responses. In embodiments, the one or more T1Dgen peptides can be in neutral (uncharged) or salt form, and are free or free of modifications such as glycosylation, side chain oxidation, or phosphorylation. It can be either.

The modified polypeptide of the T-regitope-blood component conjugate of the present disclosure and the method for producing the modified polypeptide used to form the T-regitope-blood component conjugate may vary widely depending on the nature of the various elements constituting the molecule. It's going to change. Synthetic procedures can be chosen to be simple, provide high yields, and allow highly purified and stable products. Typically, the reactive site is created as a final step; for example, in the case of carboxyl groups, esterification to form the active ester will be the final step in the synthesis.

In aspects, the present disclosure is disclosed in U.S. Pat. No. 6,849,714, U.S. Pat. No. 6,887,470, U.S. Pat. The present invention relates to modified polypeptides of T-regitope-blood component conjugates and methods of synthesizing modified polypeptides for use in forming T-regitope-blood component conjugates, as disclosed. In aspects, the method includes the following steps. In a first step, one or more T resitope sequences of a polypeptide can be synthesized essentially as disclosed herein; optionally, a polypeptide sequence can be synthesized essentially as disclosed herein; It additionally contains one or more T1Dgen sequences as shown in FIG. In the second step, if the polypeptide does not contain cysteine, the polypeptide can be synthesized from the carboxy-terminal amino acid and a reactive site can be added to the carboxy-terminal amino acid. Alternatively, a terminal lysine (or lysines) may be added to the carboxy-terminal amino acid and a reactive site may be added to the terminal lysine. In the third step, if the polypeptide contains only one cysteine, the cysteine is reacted with a protecting group before adding reactive sites to the amino acids in the less therapeutically active regions of the polypeptide. In the fourth step, if the polypeptide contains two cysteines as disulfide bridges, oxidize the two cysteines and transfer the reactive site to the amino-terminal amino acid, the carboxy-terminal amino acid, or between the carboxy-terminal amino acid and the amino-terminal amino acid of the polypeptide. It is added to the amino acid located at In the fifth step, if the polypeptide contains more than two cysteines as disulfide bridges, the cysteines are sequentially oxidized at the disulfide bridges and the peptide is purified before adding a reactive site to the carboxy-terminal amino acid.

In aspects, the present disclosure relates to methods of synthesizing T-regitope-blood component conjugates. In the first step, reactive maleimidopropionamide (MPA) is added via the N-terminal lysine on the polypeptide containing one or more T regitopes to create a modified polypeptide. In embodiments, one or more lysines are present on the N-terminus of the polypeptide, optionally on the N-terminus of a T regitope sequence selected from the group of SEQ ID NOs: 1-55 disclosed herein. exist. Optionally, a polyethylene glycol linker, such as PEG2 or PEG12, is present between the one or more lysines and the T-regitope sequence, or at the N-terminus of the T-regitope sequence. In embodiments, a lysosomal cleavage site, such as a cathepsin B site, optionally consisting of valine and citrulline (sequentially from N-terminus to C-terminus), is present between the PEG2 or PEG12 site and the T resitope sequence. Lysosomal cleavage sites (such as cathepsin B sites) can be incorporated to provide lysosomal protease sites and release T-regitopes into the lysosomal compartment. In embodiments, a lysosomal cleavage site (such as a cathepsin B site) is present to provide a lysosomal protease site, allowing the T-regitope to be released intracellularly, preferably within early endosomes. In a preferred embodiment, a lysosomal cleavage site (such as a cathepsin B site) is present to provide a lysosomal protease site, and the T retope can be isolated intracellularly, e.g., in membrane-enclosed vesicles (early endosomes, late endosomes, lysosomes). etc.) so that the T-regitope can be processed for antigen presentation. In embodiments, the T-regitope is presented as an antigen by an immune cell such as a macrophage or an antigen presenting cell, preferably as an MHC class II antigen. In embodiments, between the PEG2 or PEG12 moiety and the T-regitope sequence, and/or between the one or more T-regitopes, a lysosomal cleavage site, such as a cathepsin B site (optionally from valine and citrulline) (sequentially until)) exists. In embodiments, one or more T regitopes may be present on the construct, optionally closer to the C-terminus than the linker. In embodiments, one or more lysosomal cleavage sites are present between multiple T-regitopes (e.g., such that a single lysosomal cleavage site separates two T-regitopes, or one lysosomal cleavage site separates two T-regitopes). one and a second T regitope, such that another lysosomal cleavage site exists between the second and third T regitope, etc.). In embodiments, one or more T1Dgen peptides disclosed herein (e.g., PPI-derived peptides) may be present on the construct, optionally separated from one or more T regitopes by a lysosomal cleavage site. May be separated. In embodiments, one or more lysosomal cleavage sites are present between multiple T1Dgen peptides (e.g., such that a single lysosomal cleavage site separates two T1Dgen peptides, or one lysosomal cleavage site separates a second T1Dgen peptide). one and a second T1Dgen peptide, such that another lysosomal cleavage site exists between the second and third T1Dgen peptide, etc.). In embodiments, a norleucine (Nle) residue is added to the C resitope as a means of quantifying the amount of T regitope peptide incorporated into the final T regitope-blood component conjugate, e.g., for evaluation by mass spectrometry. Exists at the end. In embodiments, the C-terminus of the polypeptide is capped with a C-terminal amino group. In the second step, the modified polypeptide is covalently attached to a blood component, preferably serum albumin, in a 1:1 molar ratio using maleimide-based chemistry. The second step can be performed in vivo or ex vivo, as described further below and in the examples of this disclosure.

In embodiments, the formation of the T-regitope-blood component conjugate, when present in vivo, protects the T-regitope from rapid degradation by peptidases, rapid clearance from circulation, and/or rapid renal excretion. . In embodiments, the formation of a T-regitope-blood component conjugate significantly increases the half-life of the T-regitope in vivo. In embodiments, the formation of the T-regitope-blood component conjugate can widely distribute the T-regitope-blood component conjugate throughout the body of the subject. In embodiments, the T regitope-blood component conjugate does not cross the blood-brain barrier when present in the subject's plasma. In embodiments, the T-regitope-blood component conjugate assists in the delivery of the T-regitope to appropriate immune cells, such as macrophages and/or antigen presenting cells (APCs). In embodiments, upon delivery of the T-resitope to appropriate immune cells such as macrophages and/or APCs, the T-regitope is incorporated into membrane-bound vesicles, preferably vesicles in the endocytic pathway such as early endosomes, late endosomes, or lysosomes. be done. In embodiments, the T-regitope is presented as an MHC class II antigen once processed by appropriate immune cells such as macrophages and/or APCs.

In embodiments, the T-regitope in the T-regitope-blood component conjugate has an in vivo plasma half-life of up to 12 hours. In embodiments, the T-regitope in the T-regitope-blood component conjugate has an in vivo plasma half-life of up to 1 day. In embodiments, the T-regitope in the T-regitope-blood component conjugate has an in vivo plasma half-life of up to 40-48 hours. In embodiments, the T-regitope in the T-regitope-blood component conjugate has an in vivo plasma half-life of up to 60 hours. In embodiments, the T-regitope in the T-regitope-blood component conjugate has an in vivo plasma half-life of up to 15 days.

In embodiments, a modified polypeptide comprising one or more T-regitopes is administered to a subject, and upon administration, the modified polypeptide reacts in vivo with reactive functional groups of circulating blood components. In embodiments, the peptide is administered to a human subject and the blood component is human albumin, preferably circulating albumin of the human subject.

In embodiments, the modified polypeptide used to form the T regitope-blood component conjugate is capable of forming a bond ex vivo with a reactive functional group on the blood component, and the reaction of the modified polypeptide Upon formation of a bond between a reactive site and a reactive functional group on a blood component, U.S. Patent No. 6,849,714; and T-regitope-blood component conjugates are formed as disclosed in US Pat. No. 7,307,148. In embodiments, the modified polypeptides disclosed herein are configured to covalently bind to reactive functional groups of extracorporeal blood components. In embodiments, the blood component is albumin. In embodiments, the blood component is selected from the group of recombinant albumin, human recombinant albumin, and albumin from genomic sources.

In aspects, the present disclosure is disclosed in U.S. Patent No. 6,849,714; U.S. Patent No. 6,887,470; As disclosed, ex vivo methods for synthesizing modified T-regitope peptides and T-regitope-blood component conjugates are disclosed. In embodiments, the modified polypeptides disclosed herein are added to blood, serum, or saline containing human serum albumin to allow covalent bond formation between the modified therapeutic peptide and the blood component. . In embodiments, a polypeptide comprising one or more T resitopes disclosed herein is modified with a maleimide, which is reacted with serum albumin in saline. In embodiments, once the modified polypeptide has reacted with a blood component to form a T-regitope-blood component conjugate, the conjugate may be administered to the subject. In embodiments, after the modified polypeptide reacts with a blood component to form a conjugate, the conjugate may be separated from unconjugated blood components in the reaction mixture before the conjugate is administered to the subject. good. In embodiments, the conjugate is separated from the unconjugated blood in the reaction mixture by separating the substances based on differences in the strength of their hydrophobic interactions with hydrophobic ligands immobilized on an uncharged matrix. Can be separated from components. In embodiments, the uncharged matrix can be a hydrophobic solid support, including, but not limited to, columns containing hydrophobic resins such as octyl Sepharose, phenyl Sepharose, and butyl Sepharose. It is not something that will be done. In embodiments, this technique can be performed with a moderately high concentration of salt (approximately 1 M) in the starting buffer (promoted adsorption of salt). Elution is achieved by a linear or stepwise decrease in salt concentration. The type of ligand, degree of substitution, pH, and type and concentration of salts used in the adsorption step have a significant impact on the overall performance (e.g., selectivity and capacity) of the HIC matrix (hydrophobic interaction chromatography matrix). .

Solvent is one of the most important parameters affecting capacity and selectivity in HIC (hydrophobic interaction chromatography). In general, adsorption steps are more selective than desorption steps. Therefore, it is important to optimize the starting buffer with respect to pH, solvent type, salt type, and salt concentration. Addition of various "salting out" salts to the sample promotes ligand-protein interactions in HICs. Increasing the concentration of salt increases the amount of protein bound up to the point of protein precipitation. Different types of salts have different abilities to promote hydrophobic interactions.

Increasing the salting-out effect strengthens the hydrophobic interactions, and increasing the chaotropic effect weakens the hydrophobic interactions. Examples of salts with a high salting-out effect include the following in order from the highest salting-out effect to the lowest salting-out effect. Examples include PO ₄ ^3- , SO ₄ ^2- , CH ₃ COO ^- , Cl ^- , Br ^- , NO ₃ ^- , ClO ₄ ^- , I ^- , and SCN ^- . Examples of salts with a high chaotropic effect include the following in order from the highest to the lowest chaotropic effect. These include NH ₄ ⁺ , Rb ⁺ , K ⁺ , Na ⁺ , Cs ⁺ , Li ⁺ , Mg ²⁺ , and Ba ²⁺ . The most commonly used salts for HIC are ammonium sulfate ((NH ₄ ) ₂ SO ₄ ), sodium sulfate ((Na) ₂ SO ₄ )), magnesium sulfate (MgSO ₄ ), sodium chloride (NaCl), and potassium chloride (KCl). , and ammonium acetate (CH ₃ COONH ₄ ).

Protein binding to HIC adsorbents is facilitated by moderate to high concentrations of "salting out" salts, mostly due to preferential exclusion from native globular proteins, i.e., interaction of the salts with the protein surface. It also has a stabilizing effect on protein structure due to its thermodynamic disadvantage. The concentration of salt needs to be high enough (eg, 500-1000 mM) to promote ligand-protein interactions, but below concentrations that cause precipitation of proteins in the sample. In the case of albumin, the salt concentration needs to be 3M (mol/liter) or less. The principle of salting out is the increase in the surface tension of water due to salt (Melander and Horvath, 1977). Therefore, a compact structure is energetically advantageous due to the smaller protein-solution interfacial area. ^Under these _conditions , _a buffer _consisting ^{of e.g.} In a different manner, it exhibits a salting-out effect on essentially all of the conjugated albumins described here, thus allowing a consistent chromatographic separation between conjugated and non-conjugated albumins. Therefore, the concentration of salt required to promote interaction between the ligand and conjugated albumin will be lower than between the ligand and unconjugated albumin. This chromatographic separation determines (a) the sequence of the albumin (e.g., human, mouse, rat, etc.), (b) the source of the albumin (i.e., plasma-derived or recombinant), (c) the molecular weight of the conjugated modified T resitope, (d) the location of the reactive site within the structure of the molecule; (e) the peptide sequence or chemical structure of the molecule; and (f) the three-dimensional structure of the conjugate molecule (e.g., linear versus loop structure).

In embodiments, the aqueous buffer salt has sufficient salting-out effect. In embodiments, the salts may be phosphates, sulfates, and acetates to provide sufficient salting-out effect. In embodiments, the selection of buffer cations can be selected from the group consisting of, but not limited to, _NH4 ⁺ , Rb ⁺ , K ⁺ , Na ⁺ , Cs ⁺ , Li ⁺ , Mg2 ⁺ , and Ba2 ⁺ . In embodiments, the aqueous buffer may be selected from the group consisting of ammonium phosphate, ammonium sulfate, and magnesium phosphate. In embodiments, the pH of the buffer is between 3.0 and 9.0; more preferably between 6.0 and 8.0, and even more preferably the pH is 7.0. In embodiments, the buffer and hydrophobic solid support are at room temperature (about 25°C) or 4°C or between.

In embodiments, the T-regitope-blood component conjugates and modified polypeptides comprising a T-regitope of the present disclosure, wherein the modified peptide is capable of reacting with a blood component to form such a T-regitope-blood component conjugate. , can be purified to homogeneity, or can be partially purified. However, it will be appreciated that preparations in which such compositions are not purified to homogeneity are useful. An important feature is that the preparation allows the desired function of the T regitope and optionally T1Dgen even in the presence of significant amounts of other components. Accordingly, the present disclosure encompasses varying degrees of purity. In one embodiment, the phrase "substantially free of cellular material" means that the preparation of the T-regitope-blood component conjugate and the modified polypeptide comprising the T-regitope contains less than about 30% (by dry weight) Other proteins (e.g., contaminating proteins; "other proteins" does not include T1Dgen peptides or carrier proteins such as albumin), less than about 20% other proteins, less than about 10% other proteins, about 5% less than about 4% other proteins, less than about 3% other proteins, less than about 2% other proteins, less than about 1% other proteins, or any value or range therebetween. Including conditions involving other proteins.

In embodiments, when a T-regitope-blood component conjugate or a modified polypeptide comprising a T-regitope of the present disclosure is recombinantly produced, the T-regitope composition may be substantially free of culture medium; For example, the culture medium represents less than about 20%, less than about 10%, or less than about 5% of the volume of the preparation of the T regitope, T1Dgen, carrier protein, nucleic acid, or chimeric or fusion polypeptide formulation. The expression "substantially free of chemical precursors or other chemicals" refers to polypeptides, nucleic acids, or chimeric or including preparations of fusion polypeptides. The phrase "substantially free of chemical precursors or other chemicals" means, for example, a T resitope, a T1Dgen, a carrier protein having less than about 30% (by dry weight) of chemical precursors or other chemicals; A preparation of a nucleic acid or chimeric polypeptide can contain less than about 20% chemical precursors or other chemicals. less than about 10% chemical precursor or other chemical, less than about 5% chemical precursor or other chemical, less than about 4% chemical precursor or other chemical, less than about 3% chemical precursor or other chemicals, less than about 2% chemical precursors or other chemicals, or less than about 1% chemical precursors or other chemicals.

As used herein, two polypeptides (or regions of polypeptides) have an amino acid sequence of at least about 45-55%, usually at least about 70-75%, more usually at least about 80-85%, more usually Substantially homologous or identical if about 90% or more, and more usually 95% or more homologous or identical. To determine the percent homology or identity of two amino acid sequences, or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., the sequences of one polypeptide or nucleic acid molecule differ from those of the other). It is possible to introduce gaps for optimal alignment with polypeptides or nucleic acid molecules). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. Molecules are homologous at a position in one sequence if that position is occupied by the same amino acid residue or nucleotide as the corresponding position in the other sequence. As is known in the art, the percent identity between two sequences takes into account the number of gaps that need to be introduced for optimal alignment of the two sequences, and the length of each gap. It is also a function of the number of identical positions shared by the array. Sequence homology of polypeptides is typically determined using sequence analysis software. As used herein, amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity." In embodiments, the percent homology between two sequences is a function of the number of identical positions shared by the sequences (eg, the percent homology is equal to the number of identical positions/total number of positions x 100).

In embodiments, the present disclosure provides polypeptides that have a lower degree of identity but perform one or more of the same functions performed by the polypeptides of the present disclosure (e.g., T regitopes and T1Dgens disclosed herein). Also included are polypeptides with sufficient similarity such that any such variant retains MHC binding and/or TCR specificity. Similarity is determined by conservative amino acid substitutions. Such substitutions replace a given amino acid in a polypeptide with another amino acid having similar properties. Conservative substitutions are likely to be phenotypically silent. Typical conservative substitutions include substitution of aliphatic amino acids Ala, Val, Leu, and He, exchange of hydroxyl residues Ser and Thr, exchange of acidic residues Asp and Glu, and exchange of amide residues Asn and Gln. , exchange of base residues Lys and Arg, and exchange of aromatic residues Phe and Tyr. Guidance regarding which amino acid changes are likely to be phenotypically silent can be found (Bowie JU et al. (1990), Science, 247(4948):130610, which is incorporated herein by reference in its entirety. (incorporated into the book).

In embodiments, the variant polypeptide (e.g., variant T resitope or T1Dgen) has a modified amino acid sequence by one or more substitutions, deletions, insertions, inversions, fusions, and truncations, or any combination thereof. may be different. The variant polypeptide is fully functional (e.g., in a manner that retains MHC binding and/or TCR specificity, the variant polypeptide is a T regitope, and/or exhibits regulatory T cell stimulatory or suppressive activity). or may lack function in one or more activities. Fully functional variants typically contain only conservative mutations or mutations in non-critical residues or regions; this typically provides that MHC binding is retained. It is an MHC contact residue. Functional variants can also include similar amino acid substitutions that result in unchanged or insignificant changes in function (e.g., retaining MHC binding and/or TCR specificity and ensuring that the variant polypeptide has T in some embodiments that are resitopic and/or retain regulatory T cell stimulatory or suppressive activity). Alternatively, such substitutions may positively or negatively affect function to some extent. Non-functional variants typically include one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncations, or significant residues.
Including substitutions, insertions, inversions, or deletions of groups or critical regions ; in this case typically substitutions, insertions, inversions, or deletions in TCR contact residues. In embodiments, the variant and/or homologous T regitope polypeptides retain the desired regulatory T cell stimulatory or suppressive activity of the present disclosure. Alternatively, such substitutions may positively or negatively affect function to some extent. Non-functional variants typically include one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncations, or substitutions, insertions, inversions, or truncations of key residues or regions . or deletions; in this case typically substitutions, insertions, inversions, or deletions in TCR contact residues. In embodiments, a sequence (or core sequence) comprising, consisting solely of, or consisting essentially of one or more sequences of SEQ ID NO: 1-55 disclosed herein ) includes one or more conservative substitutions and, in embodiments , one or more amino acid residues that are not considered essential for the polypeptides of the disclosure to function. Non-conservative substitutions may be included (amino acid residues considered essential for function, eg, retain MHC binding and/or TCR specificity, and/or retain regulatory T cell stimulatory or suppressive activity ). For example, in embodiments, the sequence comprises, consists exclusively of, or consists essentially of one or more of the sequences SEQ ID NO: 1-55, or a fragment thereof disclosed herein. A variant polypeptide having a sequence (or core sequence) containing one or more conservative substitutions (and in some embodiments non-conservative substitutions) in one or more HLA contact residues, as long as HLA binding is retained. can include. MHC binding assays are well known in the art. In embodiments, such assays can include testing binding affinity for MHC class I and class II alleles in in vitro binding assays, such as those known in the art. Examples include, for example, soluble binding assays as disclosed in U.S. No. 7,884,184 or PCT/US2020/020089, both of which are incorporated herein by reference in their entirety. can be mentioned. Additionally, in embodiments, the sequence (or core sequence ) is free of mutations in one or more critical residues or regions, such as TCR contact residues.

In embodiments, a 9-mer identified epitope (a 9-mer fragment of one or more of SEQ ID NOs: 1-55 disclosed herein, a concatemeric peptide disclosed herein) that binds to an MHC class II molecule. TCR-binding epitopes (which can be referred to as TCR-binding residues, TCR-facing epitopes, TCR-facing residues or TCR contacts) of 2, 3, 5, 7 of the identified epitope (which can be a 9-mer fragment of and at position 8; a 9-mer identification epitope (a 9-mer fragment of one or more of SEQ ID NOs: 1-55 disclosed herein; The MHC-binding agretopes (which can be referred to as MHC contacts, MHC-opposing residues, MHC-binding residues or MHC-binding faces) of the position; both are counted from the amino terminus.

In embodiments, a 9-mer identified epitope (a 9-mer fragment of one or more of SEQ ID NOs: 1-55 disclosed herein, a 9-mer fragment of a concatemeric peptide) that binds to an MHC class I molecule; The TCR-binding epitopes ( obtained) are at positions 4, 5, 6, 7, and 8 of the identified epitope ; the nonamer-identified epitopes (disclosed herein The MHC-binding agretopes of one or more of SEQ ID NOs: 1-55, which can be nonamer fragments of concatemeric peptides, are at positions 1, 2, 3, and 9; both are amino-terminal It can be counted from

In embodiments, a decamer-identifying epitope (a decamer fragment of one or more of SEQ ID NOs: 1-55 disclosed herein, a decamer fragment of a concatemeric peptide, 1 or a 10-mer peptide comprising multiple 9-mer fragments of SEQ ID NOs: 1 to 55.
The TCR-binding epitopes (which may be A 10-mer peptide comprising one or more decamer fragments of SEQ ID NOs: 1 to 124, a 10- mer fragment of a concatemer peptide, and a 10-mer fragment of one or more SEQ ID NOs: 1 to 55 disclosed in The MHC-binding agretopes of (obtaining) are at positions 1, 2, 3, 9 and 10; both are counted from the amino terminus.

In embodiments, a 9-mer identified epitope (a 9-mer fragment of one or more of SEQ ID NOs: 1-55 disclosed herein, a 9-mer fragment of a concatemeric peptide) that binds to an MHC class II molecule; The TCR-binding epitope of the identified epitope is defined as a TCR-binding epitope at any combination of residues at positions 2, 3, 5, 7, and 8 of the identified epitope, including, but not limited to, positions 3, 5, 7, and 8. ; positions 2, 5, 7 and 8; positions 2, 3, 5 and 7); nonamer-identified epitopes (such as one or more SEQ ID NOS disclosed herein) that bind to MHC class I molecules; The MHC-binding agretope (which can be a 9-mer fragment of 1-55, a 9-mer fragment of a concatemeric peptide) is in a complementary plane to the TCR facing residue; both are counted from the amino terminus.

In embodiments, a 9-mer identified epitope (a 9-mer fragment of one or more of SEQ ID NOs: 1-55 disclosed herein, a 9-mer fragment of a concatemeric peptide) that binds to an MHC class I molecule; The TCR-binding epitopes of the identified epitope (obtaining) are determined at positions 4, 5, 6, 7, and 8 of the identified epitope; at positions 1, 4, 5, 6, 7, and 8; , and at position 8; an MHC-binding agretope of a 9-mer identification epitope (which can be a 9-mer fragment of one or more of SEQ ID NOs: 1-55 disclosed herein, a 9-mer fragment of a concatemeric peptide); are in the complementary plane to the TCR facing residue; both are counted from the amino terminus.

In embodiments, a decameric identification epitope (decameric fragment of one or more of SEQ ID NOs: 1-55 disclosed herein, a decameric fragment of a concatemeric peptide, 1 or a 10-mer peptide comprising multiple 9-mer fragments of SEQ ID NOs: 1-55). decamer identification epitopes (decamer fragments of one or more SEQ ID NOS: 1-55 disclosed herein, decamer fragments of concatemeric peptides, one or more SEQ ID NOS: The MHC-binding agretope (which can be a 10-mer peptide containing 1 to 55 9-mer fragments) is in a complementary plane to the TCR facing residue; both are counted from the amino terminus.

Based on the above, one or more nonamer and/or decamer epitopes are contained within the longer polypeptide and are predicted to bind one or more class I or class II MHC molecules and naturally occurring sequences. (e.g., position 1 of each set of bound nonamers and/or decamers is within e.g. 3 amino acids of each other), such epitopes are bound to form an epitope. It should be understood that clusters may form. In a given cluster, any given amino acid may be MHC-facing for a given 9-mer or 10-mer epitope and TCR-facing for another 9-mer epitope.

In embodiments, the present disclosure also includes T regitope-blood component conjugates and modified polypeptides comprising T regitopes , including polypeptides of the present disclosure and peptide fragments of T1Dgens . In embodiments, the present disclosure also encompasses fragments of the T regitopes and T1Dgens variants described herein. In embodiments, a fragment, as used herein, consists of at least about 9 contiguous amino acids. Useful fragments (and fragments of the polypeptides and concatemeric polypeptide variants described herein) have one or more biological activities, particularly MHC binding properties and/or TCR specificity, and/or regulation. This includes those that retain T cell stimulating activity or suppressing activity. Biologically active fragments include, for example, about 9, 10, 11, 12, 1, 14, 15, 16, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acid length, including any value or range therebetween. Fragments can be individual (not fused to other amino acids or polypeptides) or present within a larger polypeptide. Several fragments can be contained within a single large polypeptide. In embodiments, a fragment designed for expression in a host can have heterologous pre- and propolypeptide regions fused to the amino terminus of the polypeptide fragment and additional regions fused to the carboxyl terminus of the fragment.

In embodiments, the T resitopes and/or T1Dgens of the present disclosure can include alleles or sequence variants (“variants”) or analogs thereof, or include chemical modifications (e.g., pegylation, glycosylation). be able to. In embodiments, the variant retains the same function performed by the polypeptide encoded by the nucleic acid molecule of the present disclosure, in particular MHC binding and/or TCR specificity, and the variant polypeptide is a T-regitope. and/or retain regulatory T cell stimulatory or suppressive activity. In embodiments, the variant can provide enhanced binding to MHC molecules. In embodiments, the variant can provide enhanced binding to the TCR. In another example, the variant can result in decreased binding to MHC molecules and/or TCR. Also contemplated are variants that bind but do not allow signaling through the TCR.

Methods for producing polypeptides of the present disclosure will vary widely depending on the nature of the various elements that make up the molecule. For example, an isolated polypeptide can be purified from cells that naturally express it, purified from cells that have been modified to express it (recombinant), or purified using known protein synthesis methods. Can be synthesized. Synthetic procedures can be chosen to be simple, provide high yields, and allow highly purified and stable products. For example, polypeptides of the present disclosure can be produced either from the nucleic acids disclosed herein or through the use of standard molecular biology techniques such as recombinant techniques, mutagenesis, or other means known in the art. It can be manufactured by The isolated polypeptide can be purified from cells that naturally express it, purified from cells that have been modified to express it (recombinant), or purified using known protein synthesis techniques. Can be synthesized. In embodiments, polypeptides of the present disclosure are produced by recombinant DNA or RNA technology. In embodiments, polypeptides of the present disclosure can be produced by expression of recombinant nucleic acids of the present disclosure in a suitable host cell. For example, a nucleic acid molecule encoding a polypeptide is cloned into an expression cassette or expression vector, the expression cassette or expression vector is introduced into a host cell, and the polypeptide is expressed in the host cell. The polypeptide can then be separated from the cells by an appropriate purification scheme using standard protein purification techniques. Alternatively, polypeptides can be produced by a combination of ex vivo procedures such as protease digestion and purification. Additionally, polypeptides of the present disclosure can be produced using site-directed mutagenesis techniques, or other mutagenesis techniques known in the art (e.g., James A. Brannigan and Anthony J. Wilkinson). , 2002, Protein engineering 20
years on. Nature Reviews Molecular Cell Biology 3, 964-970; Turanli-Yildiz B. et al., 2012, Protein Engineering Methods and Applications, intechopen.com, incorporated herein by reference in its entirety).

In embodiments, a polypeptide can be purified to homogeneity, or can be partially purified. However, it will be appreciated that preparations in which the polypeptide is not purified to homogeneity are useful. An important feature is that the preparation allows the desired function of the composition even in the presence of significant amounts of other ingredients. Accordingly, the present disclosure encompasses various degrees of purity. In one embodiment, the phrase "substantially free of cellular material" means that the preparation of the polypeptide, concatemeric polypeptide, chimeric polypeptide or fusion polypeptide contains less than about 30% (by dry weight) other cellular material. of proteins (e.g., contaminating proteins), less than about 20% other proteins, less than about 10% other proteins, less than about 5% other proteins, less than about 4% other proteins, less than about 3% Other proteins, less than about 2% other proteins, less than about 1% other proteins, or any value or range therebetween.

In embodiments, when the polypeptides of the present disclosure are recombinantly produced, the compositions may also be substantially free of culture medium, e.g., the culture medium comprises about 20% of the volume of the polypeptide preparation. %, less than about 10%, or less than about 5%. The phrase "substantially free of chemical precursors or other chemicals" includes preparations of a polypeptide that are separated from chemical precursors or other chemicals involved in the synthesis of the polypeptide. "Substantially free of chemical precursors or other chemicals" means, for example, less than about 30% (by dry weight) of chemical precursors or other chemicals, less than about 20% of chemical precursors or other of chemicals, less than about 10% chemical precursors or other chemicals, less than about 5% chemical precursors or other chemicals, less than about 4% chemical precursors or other chemicals, less than about 3% of chemical precursors or other chemicals, less than about 2% chemical precursors or other chemicals, less than about 1% chemical precursors or other chemicals.

In aspects, the present disclosure also includes pharmaceutically acceptable salts of the polypeptides disclosed herein. "Pharmaceutically acceptable salt" means a salt that is pharmaceutically acceptable and retains the desired pharmacological activity of the parent peptide or polypeptide. As used herein, "pharmaceutically acceptable salts" refer to derivatives of the polypeptides of the disclosure, such compounds being modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include mineral or organic acid salts of basic residues such as amines, alkali salts or organic acid salts of acidic residues such as carboxylic acids, etc. It is not limited. Pharmaceutically acceptable salts include, for example, conventional non-toxic salts or quaternary ammonium salts formed from non-toxic inorganic or organic acids of the parent compound . For example, such conventional non-toxic salts include 2-acetoxybenzoic acid, 2-hydroxyethanesulfonic acid, acetic acid, ascorbic acid, benzenesulfonic acid, benzoic acid, bicarbonate, carbonic acid, citric acid, edetic acid, ethanedisulfonic acid. Acid, 1,2-ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, glycolyrsanilic acid, hexylresorcinic acid, hydrabamic acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, hydroxy Maleic acid, hydroxynaphthoic acid, isethionic acid, lactic acid, lactobionic acid, lauryl sulfonic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, napsylic acid, nitric acid, oxalic acid, pamoic acid, pantothenic acid, phenylacetic acid, phosphorus acids, polygalacturonic acid, propionic acid, salicylic acid, stearic acid, acetic acid, succinic acid, sulfamic acid, sulfanilic acid, sulfuric acid, tannic acid, tartaric acid, toluenesulfonic acid, and common amino acids such as glycine, alanine, Including, but not limited to, those derived from inorganic and organic acids selected from phenylalanine, arginine.

In embodiments, the T-regitope-blood component conjugates and/or modified polypeptides used to form the T-regitope-blood component conjugates of the present disclosure are in admixture with an antigen and/or a therapeutic protein. . Such compositions are useful in methods of inducing tolerance to an antigen or therapeutic protein in a subject in need thereof, where local delivery of the admixture with the antigen or therapeutic protein induces tolerance to the antigen in a subject. When delivered with appropriate excipients, tolerance to the antigen or therapeutic protein is induced. This combination can be administered with the T-regitope-blood component conjugate of the present disclosure and/or the modified polypeptide used to form the T-regitope-blood component conjugate, either covalently or non-covalently linked. or they may be administered as admixtures or as branched or chemically combined preparations. Such compositions are useful in methods of inducing tolerance to antigens, allergens, and/or therapeutic proteins such as, but not limited to, insulin and/or preproinsulin. For example, such compositions are useful in a subject in need thereof, and local delivery of an admixture with an antigen and/or therapeutic protein may induce tolerance to the antigen or therapeutic protein in the subject. and, when delivered with appropriate excipients, result in the induction of tolerance to the antigen or therapeutic protein. The combination may be administered either covalently or non-covalently associated with the T-regitope compositions of the present disclosure, or may be administered as an admixture.

[Nucleic acid]
In aspects, the present disclosure provides nucleic acids (e.g., , DNA (including but not limited to cDNA, RNA (such as mRNA), vector, virus or hybrids thereof, all of which may be isolated, synthetic or recombinant). In embodiments, the nucleic acid is , expression cassettes, plasmids, and expression vectors, or recombinant viruses, optionally comprising a nucleic acid, or an expression cassette, plasmid, expression vector, or recombinant virus. is contained within a cell, optionally a human cell or a non-human cell, and optionally said cell is transduced with a nucleic acid, or an expression cassette, plasmid, expression vector, or recombinant virus. Embodiments In , the cells contain an isolated, synthetic, or recombinant nucleic acid, expression cassette, plasmid, expression vector, or recombinant virus as disclosed herein; and/or as disclosed herein. Synthetic or recombinant chimeric or fusion polypeptide compositions, e.g., transformed to include therein one or more T-regitope-blood component conjugates and/or T-regitope-blood component conjugates of the present disclosure; transfected or otherwise engineered. In embodiments, the cell can be a mammalian cell, a bacterial cell, an insect cell, or a yeast cell. In embodiments, the nucleic acid molecules of the present disclosure , inserted into a vector and used, for example, as an expression vector or a gene therapy vector. Gene therapy vectors can be used, for example, by intravenous injection, local administration (U.S. Pat. No. 5,328,470) or stereotaxic injection (Chen SH et al., (1994), Proc Natl Acad Sci USA, 91(8):3054-7, herein incorporated by reference in their entirety) .Gene therapy vectors The pharmaceutical formulation can contain the gene therapy vector in an acceptable diluent or can consist of a sustained release matrix in which the gene delivery vehicle is embedded. Alternatively, the entire gene delivery vector can be If produced directly from recombinant cells, eg, retroviral vectors, the pharmaceutical formulation can include one or more cells that produce the gene delivery system. Such pharmaceutical compositions may be included in a container, pack, or dispenser together with instructions for administration . In aspects , the present disclosure relates to cells comprising vectors of the present disclosure. In embodiments, the cell can be a mammalian cell, a bacterial cell, an insect cell, or a yeast cell.

Nucleic acids of the present disclosure may be single-stranded or double-stranded DNA (including, but not limited to, cDNA) or RNA (including, but not limited to, mRNA). Nucleic acids may be, for example, DNA, cDNA, PNA, CNA, RNA, either single-stranded and/or double-stranded, or native or stabilized forms of polynucleotides as known in the art. Good too. Nucleic acids are typically DNA or RNA (including mRNA). Nucleic acids can be prepared using techniques well known in the art, such as synthesis, or cloning, or amplification of sequences encoding immunogenic polypeptides; synthesis, or cloning, or amplification of sequences encoding cell membrane address sequences; suitable vectors; and by ligation of the sequences and their cloning/amplification in cells. A nucleic acid ( RNA, DNA, vectors, viruses or hybrids thereof) can be isolated from various sources, genetically engineered, constructed, amplified, synthetically produced, and/or recombinantly expressed/produced. can be done. Recombinant polypeptides produced from these nucleic acids can be individually isolated or cloned and tested for the desired activity. Any recombinant expression system can be used, including in vitro, bacterial, fungal, mammalian, yeast, insect or plant cell expression systems. In embodiments, the nucleic acids provided herein are synthesized in vitro by well-known chemical synthesis techniques (e.g., Adams (1983) J. Am. Chem. Soc.
105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett. 22:1859; U.S. Patent No. 4,458,066, all of which are incorporated herein by reference in their entirety). Additionally, techniques for manipulation of nucleic acids provided herein, such as subcloning, labeled probes (e.g., random primer labeling with Klenow polymerase, nick translation, amplification), sequencing, hybridization, etc. and are well described in the patent literature (e.g., Sambrook, ed., Molecular Cloning: A Laboratory Manual (2ND Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); Current Protocols In Molecular Biology, Ausubel, ed. John Wiley & Sons, Inc., New York (1997); Laboratory Techniques In Biochemistry And Molecular Biology: Hybridization With Nucleic Acid Probes, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed., Elsevier, NY ( 1993), all of which are incorporated herein by reference in their entirety).

As mentioned above, nucleic acid molecules according to the present disclosure can be provided in the form of nucleic acid molecules themselves, such as naked nucleic acid molecules; plasmids, vectors; viruses or host cells, either of prokaryotic or eukaryotic origin. . Vectors include expression vectors containing the nucleic acid molecules of the invention. Expression vectors capable of expressing polypeptides can be prepared. Expression vectors for different cell types are well known in the art and can be selected without undue experimentation. Generally, RNA (eg, cDNA, or mRNA) is inserted into an expression vector, such as a plasmid, in the proper orientation and correct reading frame for expression. If desired, the DNA (e.g., cDNA, or RNA, including mRNA) may be linked to appropriate transcriptional and translational control nucleotide sequences recognized by the desired host (e.g., bacteria); Controls are generally available in expression vectors. This vector is then introduced into host bacteria for cloning using standard techniques. Vectors of the invention may include, for example, a transcription promoter and/or a transcription terminator, where the promoter is operably linked to the nucleic acid molecule and the nucleic acid molecule is operably linked to the transcription terminator. has been done. One or more peptides or polypeptides of the disclosure may be encoded by one expression vector. Such a nucleic acid molecule may serve as a vehicle for delivering the peptide/polypeptide in vivo to a subject in need thereof, eg, in the form of a DNA/RNA vaccine.

In embodiments, the vector may be a viral vector comprising a nucleic acid as defined above. Viral vectors can contain viruses of different types, such as swinepox, fowlpox, pseudorabies, Aujezky's virus, salmonella, vaccinia virus, BHV (bovine herpesvirus), HVT (turkish herpesvirus), adenovirus, TGEV (transmissible gastroenteritis coronavirus), erythrovirus, and SIV (simian immunodeficiency virus). Other expression systems and vectors can also be used, such as plasmids that replicate and/or deposit in yeast cells.

The present disclosure also relates to methods of preparing the T-regitope-blood component conjugates of the present disclosure and/or modified polypeptides used to form the T-regitope-blood component conjugates of the present disclosure, the methods comprising: culturing a host cell containing a nucleic acid or vector as defined above under conditions suitable for expression and used to form a T-regitope-blood component conjugate and/or a T-regitope-blood component conjugate. The method includes a step of recovering the modified polypeptide. As indicated above, proteins and peptides can be purified according to techniques known per se in the art.

[Pharmaceutical compositions and formulations]
In embodiments, modified polypeptides used to form T-regitope-blood component conjugates and/or T-regitope-blood component conjugates (as well as modified polypeptides and T-regitope-blood component conjugates of the present disclosure) Nucleic acids disclosed herein, and/or expression cassettes, plasmids, expression vectors disclosed herein, comprising nucleic acids encoding modified polypeptides used to form T-regitope-blood component conjugates. , recombinant viruses, cells) may be included in pharmaceutical compositions or formulations. In embodiments, a pharmaceutical composition or formulation generally comprises a T-regitope-blood component conjugate of the present disclosure and/or a modified polypeptide used to form a T-regitope-blood component conjugate and a pharmaceutically acceptable compound. carriers, excipients, and/or adjuvants. In embodiments, the pharmaceutical compositions or formulations of the present disclosure, depending on the route of administration, include diluents, adjuvants, lyophilization stabilizers, wetting or emulsifying agents, pH buffering agents, gelling or viscosity-enhancing additives, and preservatives. It may further include. In embodiments, the pharmaceutical composition is suitable for administration. Pharmaceutically acceptable carriers and/or excipients are determined, in part, by the particular composition being administered and by the particular method used to administer the composition. Accordingly, suitable formulations of pharmaceutical compositions for administering the T-resitope compositions of the present disclosure vary widely (e.g., Remington's Pharmaceutical Sciences, (18TH Ed, 1990), Mack Publishing Co., Easton, PA Publ. ref., which is incorporated herein by reference in its entirety). In embodiments, the pharmaceutical compositions are generally formulated to be sterile, substantially isotonic, and in full compliance with all Good Manufacturing Practice (GMP) regulations of the United States Food and Drug Administration.

"Pharmaceutically acceptable," "physiologically acceptable," and grammatical variations thereof to refer to compositions, carriers, excipients, and reagents are used interchangeably and the materials are indicates that it can be administered to or to a subject without producing undesirable physiological effects to the extent that it would inhibit For example, "pharmaceutically acceptable excipient" means an excipient that is generally safe, non-toxic, useful in the preparation of desirable pharmaceutical compositions, and that is useful for veterinary as well as human pharmaceutical uses. Also includes acceptable excipients. Such excipients can be solid, liquid, semi-solid, or gaseous in the case of aerosol compositions. One of ordinary skill in the art will be able to determine the appropriate timing, order of administration, and dosage for a particular T-regitope composition of the present disclosure. The dosage of the compounds and compositions of the present disclosure will depend on the species, breed, age, size, treatment history, and health condition of the animal (e.g., human) being treated, as well as the route of administration, e.g., subcutaneous, intradermal, oral, It will depend on intramuscular or intravenous administration. The compounds and compositions of this disclosure may be administered in a single dose or in multiple doses. The compounds and compositions of the present disclosure can be administered alone or together with one or more additional compositions, such as other porcine immunogenic or vaccine compositions, either simultaneously or sequentially. If the compositions are administered at different times, the administrations may be separate from each other or may overlap in time.

Examples of pharmaceutically acceptable carriers, excipients or diluents include demineralized or distilled water; saline; peanut oil, arachis oil, safflower oil, olive oil, cottonseed oil, corn oil, sesame oil or coconut oil. Plant-based oils such as; silicone oils containing polysiloxanes such as methylpolysiloxane, phenylpolysiloxane, and methylphenylpolysorpoxane; volatile silicones; mineral oils such as light liquid paraffin oil and heavy liquid paraffin oil; squalene; Cellulose derivatives such as methylcellulose, ethylcellulose, carboxymethylcellulose, carboxymethylcellulose sodium salt, or hydroxypropylmethylcellulose; lower alkanols such as ethanol or isopropanol; lower aralkanols, lower polyalkylene glycols or lower alkylene glycols, such as polyethylene glycol, polypropylene glycol , ethylene glycol, propylene glycol, 1,3-butylene glycol or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinylpyrrolidone; agar; carrageenan; gum tragacanth or gum acacia; and petrolatum. However, it is not limited to these. Typically, the carrier(s) will form 10% to 99.9% by weight of the vaccine composition and include sodium hydrogen phosphate, sodium dihydrogen phosphate, potassium hydrogen phosphate, dihydrogen phosphate. Buffering may be done by conventional methods using reagents known in the art such as potassium, mixtures thereof, and the like.

In embodiments, preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose, and 5% human serum albumin. Non-aqueous vehicles such as liposomes and fixed oils can also be used. The use of such media and compounds for pharmaceutically active substances is well known in the art. So long as any conventional vehicle or compound is incompatible with the T-regitope-blood component conjugates of the present disclosure and/or the modified polypeptide used to form the T-regitope-blood component conjugates, and as previously described. As such, their use in compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

Examples of adjuvants include oil-in-water emulsions, aluminum hydroxide (alum), immunostimulatory complexes, nonionic block polymers or copolymers, cytokines (IL-1, IL-2, IL-7, IFN-α, IFN -β, IFN-γ, etc.), saponin, monophospholipid A (MLA), muramyl dipeptide (MDP), etc. (but not limited to these). Other suitable adjuvants include, for example, potassium aluminum sulfate, thermostable or thermostable enterotoxin(s) isolated from Escherichia coli, cholera toxin or its B subunit, diphtheria toxin, tetanus toxin, pertussis toxin, Freund's incomplete or complete adjuvants, etc. Toxic adjuvants such as diphtheria toxin, tetanus toxin, pertussis toxin can be inactivated before use, for example by treatment with formaldehyde. Additional adjuvants include poly ICLC, 1018 ISS, aluminum salts, Amplivax, AS 15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, Immufact IMP321, IS Patch, ISS, ISCOMATRTX, Juvlmmune, Lipovac, MF59, Monophosphoryl Lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, These include, but are not limited to, ONTAK, PEPTEL, Vector Systems, PLGA microparticles, Resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF Trap, R848, Beta Glucan, Pam3Cys and Aquila QS21 stimulon. It's not a translation. In embodiments of the pharmaceutical compositions or vaccines disclosed herein, the adjuvant comprises poly ICLC. TLR9 agonist CpG and synthetic double-stranded RNA (dsRNA) TLR3 ligand polyICLC are two of the most promising vaccine adjuvants currently in clinical development. In preclinical studies, poly-ICLC appears to be the most potent TLR adjuvant compared to LPS and CpG.
This is likely due to the induction of inflammatory cytokines, lack of stimulation of IL-10, and maintenance of high levels of co-stimulatory molecules on DCs. Poly ICLC is a synthesized double-stranded RNA consisting of poly I chain and poly C chain with an average length of about 5000 nucleotides, and is resistant to heat denaturation and hydrolysis by serum nucleases due to the addition of polylysine and carboxymethylcellulose. It was stabilized by This compound activates the RNA helicase domains of PAMP family members TLR3 and MDA5, resulting in DC and natural killer (NK) cell activation and mixed production of type I interferons, cytokines, and chemokines.

Examples of lyophilization stabilizers may be carbohydrates such as sorbitol, mannitol, starch, sucrose, dextran or glucose, proteins such as albumin or casein, and derivatives thereof.

In embodiments, modified polypeptides used to form T-regitope-blood component conjugates and/or T-regitope-blood component conjugates of the present disclosure (as well as modified polypeptides and/or T-regitope-blood component conjugates) or a nucleic acid encoding a modified polypeptide used to form a T regitope-blood component conjugate, the expression cassettes, plasmids, expression vectors, assemblies disclosed herein, The recombinant viruses, cells, and/or pharmaceutical compositions or formulations disclosed herein are formulated to be compatible with their intended route of administration. The T-regitope-blood component conjugates and/or modified polypeptides used to form the T-regitope-blood component conjugates of the present disclosure may be administered parenterally, topically, intravenously, orally, subcutaneously, intraarterially, intradermally, It can be administered transdermally, rectally, intracranially, intrathecally, intraperitoneally, intranasally; vaginally; intramuscularly, or as an inhaler. In embodiments, the T-regitope-blood component conjugates and/or modified polypeptides used to form the T-regitope-blood component conjugates of the present disclosure can be injected directly into specific tissues where deposits have accumulated. For example, intracranial injection is possible. In other embodiments, intramuscular injection or intravenous infusion is used to administer the T-regitope-blood component conjugates and/or the modified polypeptides used to form the T-regitope-blood component conjugates of the present disclosure. I can do it. In some methods, the T-regitope-blood component conjugates and/or modified polypeptides used to form the T-regitope-blood component conjugates of the present disclosure are directly injected intracranially. In some methods, the modified polypeptides used to form the T-regitope-blood component conjugates and/or T-regitope-blood component conjugates of the present disclosure include, but are not limited to, the Medipad® device. administered as a sustained release composition or device (not available). In embodiments, the compounds and compositions of the present disclosure are administered intradermally , e.g., by using a microneedle patch as known in the art, or by using pulse needle-free technology (Pulse 50™). ) Micro Dose Injection System, Pulse Needle Free Systems;
Lenexa, KS, USA) is administered by using a commercially available needle-free hyperbaric device. In embodiments, the commercially available needle-free hyperbaric device (e.g., pulsed needle-free technology) provides one or more of the following advantages: non-invasiveness, reduced tissue trauma, reduced pain, and ease of administering the composition to a subject. Requiring small openings in the dermal layer for accumulation (e.g., only requiring microdermal openings), immediate dispersion of the composition, better absorption of the composition, increased dermal exposure of the composition, and/or or reducing the risk of sharps injury.

In embodiments, modified polypeptides used to form T-regitope-blood component conjugates and/or T-regitope-blood component conjugates of the present disclosure (as well as modified polypeptides and/or T-regitope-blood component conjugates) or a nucleic acid encoding a modified polypeptide used to form a T regitope-blood component conjugate; The modified viruses, cells, and/or pharmaceutical compositions or formulations disclosed herein may optionally be combined with other agents that are effective, at least in part, in the treatment of various medical conditions described herein. May be administered in combination. For example, for the treatment and/or prevention of T1D in a subject, the T-regitope-blood component conjugates and/or modified polypeptides used to form the T-regitope-blood component conjugates of the present disclosure may include insulin, etc. It can also be administered in combination with other drugs that provide temporary relief from T1D.

In embodiments, solutions or suspensions used for parenteral, intradermal, or subcutaneous applications can include, but are not limited to, the following ingredients: Sterile diluents such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents; Antimicrobial compounds such as benzyl alcohol or methylparaben; Antioxidants such as ascorbic acid or sodium bisulfite; ethylene diamine Chelating compounds such as tetraacetic acid (EDTA); buffers such as acetate, citrate or phosphate , and compounds for adjusting tonicity such as sodium chloride or dextrose may be included. pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Examples of excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, water, ethanol, DMSO, glycol, propylene, dry skim milk, and the like. The composition can also include pH buffering agents, and wetting or emulsifying agents.

In some embodiments, the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

In some embodiments, pharmaceutical compositions or formulations suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers are physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition should be sterile and fluid to the extent that easy syringability exists. It is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms such as bacteria and fungi. In embodiments, the T regitope preparation can include aggregates, fragments, degraders, and post-translational modifications, but to the extent that these impurities bind to HLA and present the same TCR face to homologous T cells. is expected to function in a similar manner to pure T resitopes. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols such as glycerol, propylene glycol, and liquid polyethylene glycol, and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of coating agents such as lecithin, by maintaining the required particle size in the case of dispersions, by the use of surfactants, and the like. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal compounds, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic compounds in the composition, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride. Prolonged absorption of the injectable compositions may be brought about by including in the composition a compound that delays absorption, for example, aluminum monostearate and gelatin.

In embodiments, the sterile injectable solution contains the T-regitope-blood component conjugates and/or modified polypeptides used to form the T-regitope-blood component conjugates (as well as the T-regitope-blood component conjugates). Nucleic acids disclosed herein, expression cassettes, plasmids disclosed herein, comprising nucleic acids encoding modified polypeptides and/or modified polypeptides used to form T-regitope-blood component conjugates. , expression vectors, recombinant viruses, cells, and/or pharmaceutical compositions or formulations disclosed herein) in the required amount in a suitable solvent, optionally with one or a combination of the ingredients listed above. , can be prepared by filter sterilization. Generally, dispersions are prepared by incorporating the binder into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation methods are vacuum drying and lyophilization, which yields a powder of the active ingredient plus any additional desired ingredients from its previously sterile-filtered solution. Additionally, the T-regitope-blood component conjugates and/or modified polypeptides used to form the T-regitope-blood component conjugates of the present disclosure are formulated to allow for sustained or pulsatile release of the active ingredient. It can be administered in the form of a depot injection or an implant preparation.

In embodiments, oral compositions generally include an inert diluent or an edible carrier and can be enclosed in gelatin capsules or compressed into tablets. In some embodiments, for the purpose of oral therapeutic administration, binding agents can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared with a liquid carrier for use as a mouthwash, in which the compound is applied orally and expectorated by swishing or swallowed. Pharmaceutically compatible binding compounds and/or adjuvant materials can be included as part of the composition. In embodiments, the tablets, pills, capsules, troches, etc. are made with the following ingredients: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose; a disintegrant such as alginic acid, Primogel or cornstarch. lubricants such as magnesium stearate or Stelotes; lubricants such as colloidal silicon dioxide; sweetening compounds such as sucrose or saccharin; or flavoring compounds such as peppermint, methyl salicylate or orange flavor, or similar. can include compounds with the properties of

For administration by inhalation, the T-regitope-blood component conjugates and/or the modified polypeptides used to form the T-regitope-blood component conjugates of the present disclosure may be combined with a suitable propellant, such as carbon dioxide. It can be delivered in the form of an aerosol spray from a pressurized container or dispenser containing a gas or a nebulizer.

In embodiments, systemic administration of T-regitope-blood component conjugates and/or modified polypeptides used to form T-regitope-blood component conjugates (as well as modified polypeptides of T-regitope-blood component conjugates) Nucleic acids, expression cassettes, plasmids, expressions disclosed herein, including nucleic acids encoding modified polypeptides used to form peptide and/or T-regitope-blood component conjugates; vectors, recombinant viruses, cells, and/or pharmaceutical compositions or formulations disclosed herein) can also be delivered by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be penetrated are used in the formulation. Such penetrants are generally known in the art and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the T-regitope-blood component conjugates and/or the modified polypeptides used to form the T-regitope-blood component conjugates of the present disclosure may be administered in a cream ointment, salve, They can be formulated into gels or creams and applied topically or through transdermal patch techniques, as generally known in the art.

In embodiments, the T-regitope-blood component conjugates and/or the modified polypeptides used to form the T-regitope-blood component conjugates of the present disclosure are formulated into suppositories (e.g., cocoa butter) for rectal delivery. and other glycerides) or in the form of a retention enema.

In embodiments, modified polypeptides used to form T-regitope-blood component conjugates and/or T-regitope-blood component conjugates of the present disclosure (as well as modified polypeptides and/or T-regitope-blood component conjugates) or a nucleic acid encoding a modified polypeptide used to form a T regitope-blood component conjugate, the expression cassettes, plasmids, expression vectors, assemblies disclosed herein, The modified viruses, cells, and/or pharmaceutical compositions or formulations disclosed herein) can be used to prepare T-resitope compositions for rapid elimination from the body, such as controlled release formulations, including implants and microencapsulated delivery systems. Prepared with a protective carrier. Biodegradable, biocompatible polymers can be used, such as, for example, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparing such formulations will be apparent to those skilled in the art. Materials are also available commercially, eg, from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies directed against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art (US
Pat. No. 4,522,811, herein incorporated by reference in its entirety). In embodiments, the T-regitope-blood component conjugates and/or modified polypeptides used to form the T-regitope-blood component conjugates of the present disclosure provide sustained release of the T-regitope composition to a desired site. It is possible to implant within or link to a biopolymer solid support that allows.

In some embodiments, it is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unit dosages for the subject being treated; each unit producing the desired therapeutic effect in association with the required pharmaceutical carrier. Contains a predetermined amount of binder calculated as follows. The specification of the dosage unit form of the present disclosure depends on the unique properties of the binding agent and the particular therapeutic effect to be achieved, as well as the T-regitope-blood component conjugates and/or T-regitope-blood component conjugates of the present disclosure for the treatment of the subject. It is defined by and directly dependent on the limitations inherent in the technology of formulation of the modified polypeptide used to form the conjugate.

[how to use]
Modified polypeptides used to form T-regitope-blood component conjugates and/or T-regitope-blood component conjugates ( as well as modified polypeptides of T-regitope-blood component conjugates and/or T-regitope-blood component conjugates of the present disclosure). Nucleic acids disclosed herein, expression cassettes, plasmids, expression vectors, recombinant viruses, cells disclosed herein, including nucleic acids encoding modified polypeptides used to form component conjugates; and/or pharmaceutical compositions or formulations disclosed herein; referred to in this section as "Related Compounds of the Disclosure"), stimulation of regulatory T cells with a corresponding endogenous TReg population (in embodiments, natural TRegs and/or adaptive TRegs) and, in some embodiments, result in one or more of the following cytokines and chemokines (IL-10, IL-35, TGF-β). , TNF α and MCP1). In embodiments, the stimulation increases the expression of IL-2Rα by the corresponding endogenous TReg population (including, in embodiments, natural TRegs and/or adaptive TRegs) and the scavenging of IL-2 to effector T cells. It is possible to bring about In further embodiments, the stimulation may result in an increase in perforin granzyme by a corresponding endogenous TReg population (including natural TRegs and/or adaptive TRegs in embodiments), such TReg populations being associated with T effector cells and It becomes possible to kill other immune stimulating cells. In further embodiments, such stimulation can result in the production of immunosuppressive adenosine by the corresponding endogenous TReg population (including natural and/or adaptive TRegs in embodiments). In other embodiments, such stimulation causes the corresponding endogenous TReg population (in embodiments including natural TRegs and/or adaptive TRegs) to bind and remove costimulation molecules on dendritic cells; The result can be suppression of dendritic cell function. Furthermore, in embodiments, such stimulation induces dendritic cells and other cell populations (e.g., endothelial cells (but not limited to) can result in TReg-induced upregulation of checkpoint molecules on. In additional aspects, such stimulation can result in TReg stimulation of regulatory B cells. Regulatory B cells (“B-Regs”) are cells involved in anti-inflammatory effects characterized by secretory expression of CD1d, CD5, and IL-10. B-Regs are also identified by Tim-1 expression and can promote tolerance through Tim-1 ligation. B-Reg competence has been shown to be driven by a number of stimulatory factors, including toll-like receptors and CD40-ligand, but a complete characterization of B-Reg is currently in progress. B-Reg also expresses high levels of CD25, CD86, and TGF-β. This increased secretion of regulatory cytokines and chemokines by regulatory T cells, as well as other activities mentioned above, are characteristic of regulatory T cells. In embodiments, regulatory T cells activated by the T regitope compositions of the present disclosure may express a CD4+CD25+FOXP3 phenotype. Regulatory T cells activated by the T-regitope-blood component conjugates and/or modified polypeptides used to form the T-regitope-blood component conjugates of the present disclosure may be antigen-specific Th1- or Th2-associated by a decrease in cytokine levels, primarily INF-γ, IL-4, and IL-5, and by a decrease in antigen-specific T effector cell proliferation and/or effector function as measured by CFSE dilution and/or cytolytic activity. Direct suppression of ex vivo T effector immune responses as measured. In embodiments, regulatory T cells activated by the T regitope-blood component conjugates and/or modified polypeptides used to form the T regitope-blood component conjugates of the present disclosure are antigen-specific Th1 - or Th2-related cytokine levels (measured by Elisa assay), antigen-specific T effector cell levels (measured by Eliaspot assay), cytolytic activity, and/or protein antigen antibody titers as measured by a decrease in directly suppresses the immune response of T effectors in vivo.

In embodiments, natural regulatory T cells activated by T regitope-blood component conjugates and/or modified polypeptides used to form T regitope-blood component conjugates of the present disclosure are adaptive TReg cells. stimulates the development of In embodiments, co-incubating peripheral T cells with a T regitope-blood component conjugate of the present disclosure and/or a modified polypeptide used to form a T regitope-blood component conjugate in the presence of antigen: Expansion of antigen-specific CD4+/CD25+ T cells occurs, upregulating the expression of the Foxp3 gene or Foxp3 protein in those cells, and suppressing the activation of antigen-specific T effector cells in vitro. In embodiments, the T-regitope-blood component conjugates and/or the modified polypeptides used to form the T-regitope-blood component conjugates of the present disclosure induce regulatory T1 (Tr1) cell activation and /or may result in expansion. Tr1 cells have strong immunosuppressive potential in several immune-mediated diseases (Roncarolo and Battaglia, 2007, Nat Rev Immunol 7, 585-598; Roncarolo et al., 2011, Immunol Rev
241, 145-163; Pot et al., 2011, Semin Immunol 23, 202-208). High level of IL-
10 secretion and granzyme B-mediated killing of myeloid antigen-presenting cells (APCs) are the main mechanisms of Tr1-mediated suppression (Groux et al., 1997, Nature 389, 737-742; Magnani et al., 2011 Eur J Immunol 41, 1652-1662). Tr1 cells are distinguished from T helper (T _H )1, T _H 2, and T _H 17 cells by their unique cytokine profile and regulatory functions. Tr1 cells have been shown to secrete higher levels of IL-10 than IL-4 and IL-17, the characteristic cytokines of T _H 2 and T _H 17 cells. Tr1 cells also secrete low levels of IL-2 and, depending on the local cytokine environment, can produce variable levels of IFN-γ, which together make up the T _H 1 major cytokine (Roncarolo et al. 2011, Immunol Rev 241, 145-163). FOXP3 is not a biomarker for Tr1 cells because its expression is low and transient upon activation. IL-10 producing Tr1 cells express ICOS (Haringer et al., 2009, J Exp Med 206, 1009-1017) and PD-1 (Akdis et al., 2004, J Exp Med 199, 1567-1575) However, these markers are not specific (Maynard et al., 2007, Nat Immunol 8, 931-941). CD49b, the α2 integrin subunit of very low activation antigen (VLA)-2, has been proposed as a marker for IL-10-producing T cells (Charbonnier et al., 2006, J Immunol 177, 3806-3813); It is also expressed by human T _H 17 cells (Boisvert et al., 2010, Eur.
J Immunol 40, 2710-2719). Furthermore, mouse CD49b+ T cells secrete IL-10 (Charbonnier et al., 2006, J Immunol 177, 3806-3813), but also inflammatory cytokines (Kassiotis et al., 2006, J Immunol 177, 968-975). Lymphocyte activation gene-3 (LAG-3), a CD4 homolog that binds with high affinity to MHC class II molecules, is a key component of murine IL-10-producing CD4 ⁺ T cells (Okamura et al., 2009, Proc Natl Acad Sci USA 106 , 13974-13979), but by activated effector T cells (Workman and Vignali, 2005, J Immunol 174, 688-695; Bettini et al., 2011, J Immunol 187, 3493-3498; Bruniquel et al.,
It is also expressed by FOXP3+ regulatory T cells (Tregs) (Camisaschi et al., 2010, J Immunol 184, 6545-6551). is also expressed. It was recently shown that human Tr1 cells express CD226 (DNAM-1) and are involved in specific killing of myeloid APCs (Magnani et al., 2011 Eur J Immunol 41, 1652-1662). In further embodiments, the T-regitope-blood component conjugates of the present disclosure and/or the modified polypeptides used to form the T-regitope-blood component conjugates (and related compounds of the present disclosure) induce TGF-β secretion. May lead to activation and/or expansion of Th3 cells, regulatory NKT cells, regulatory CD8+ T cells, double negative regulatory T cells. "Th3 cells" have the following phenotype CD4 ⁺ FoxP3 ⁺ , secrete high levels of TGF-β, predetermined amounts of IL-4 and IL-10 upon activation, and secrete IFN-γ or IL-2. refers to cells that cannot secrete These cells are TGF-β derived. "Regulatory NKT cells" refer to cells with the following phenotypes CD161 ⁺ CD56 ⁺ CD16 ⁺ and Vα24/Vβ11 TCR in a resting state. "Regulatory CD8+ T cells" refer to cells that have the following phenotype CD8 ⁺ CD122 ⁺ in a resting state and are capable of secreting high levels of IL-10 upon activation. "Double negative regulatory T cells" refer to cells that in the resting state have the following phenotype: TCRαβ ⁺ CD4 ⁻ CD8 ⁻ .

In embodiments, the T-regitope-blood component conjugates of the present disclosure and/or the modified polypeptides (and related compounds of the present disclosure) used to form the T-regitope-blood component conjugates are monoclonal antibodies, protein It is useful in modulating the immune response to therapeutic agents, autoantigens that promote autoimmune responses, allergens, transplanted tissues, and in other applications where tolerance is desirable.

In embodiments, the T-regitope of the disclosed T-regitope-blood component conjugate and/or the modified polypeptide used to form the T-regitope-blood component conjugate binds to an MHC class II molecule and binds to an MHC class II molecule. II molecules may engage the TCR and activate endogenous TRegs (including, in embodiments, natural TRegs and/or adaptive TRegs).

[Suppression of immune response in subjects that require it]
In aspects, the present disclosure provides therapeutically effective amounts of T-regitope-blood component conjugates or modified polypeptides (and related compounds of the present disclosure) used to form the T-regitope-blood component conjugates of the present disclosure . , a method of stimulating, inducing, and/or expanding regulatory T cells (in embodiments, endogenous TRegs, including natural TRegs and/or adaptive TRegs) in a subject by administering the same to a subject in need thereof. , as well as methods of suppressing immune responses in a subject in need thereof.

In aspects, the present disclosure provides therapeutically effective amounts of T-regitope-blood component conjugates or modified polypeptides (and related compounds of the present disclosure) used to form the T-regitope-blood component conjugates of the present disclosure . Regulatory T cells (e.g., endogenous TRegs (including, in embodiments, natural TRegs and/or adaptive TRegs)) are administered to a subject to suppress an immune response in a subject in need thereof. It relates to a method of stimulating and/or inducing. In embodiments, the immune response is the result of one or more therapeutic treatments with at least one therapeutic protein, treatment with a vaccine (particularly in the context of adverse events resulting from vaccination), or treatment with at least one antigen. be. In another aspect, administration of the T-regitope compositions of the present disclosure shifts one or more antigen-presenting cells to a regulatory phenotype, shifts one or more dendritic cells to a regulatory phenotype, and shifts one or more dendritic cells to a regulatory phenotype. Decrease expression of CD11c and HLA-DR in cells and/or other antigen-presenting cells

In aspects, the present disclosure provides a method for suppressing and/or suppressing an immune response in a subject, the method comprising: a therapeutically effective amount of a T-regitope-blood component conjugate or a T-regitope-blood component conjugate of the present disclosure. administering a modified polypeptide used to form a T-regitope-blood component conjugate (and related compounds of this disclosure) , wherein the T-regitope-blood component conjugate or modified polypeptide suppresses/depresses an immune response. In embodiments, the T regitope-blood component conjugate or modified polypeptide abrogates and/or suppresses the innate immune response. In embodiments, the T regitope-blood component conjugate or modified polypeptide abrogates and/or suppresses the adaptive immune response. In embodiments, the T regitope-blood component conjugate or modified polypeptide abrogates and/or suppresses effector T cell responses. In embodiments, the T regitope-blood component conjugate or modified polypeptide abrogates and/or suppresses memory T cell responses. In embodiments, the T regitope-blood component conjugate or modified polypeptide abrogates and/or suppresses helper T cell responses. In embodiments, the T regitope-blood component conjugate or modified polypeptide abrogates and/or suppresses B cell responses. In embodiments, the T regitope-blood component conjugate or modified polypeptide abrogates and/or suppresses nkT cell responses.

In aspects, the present invention provides methods for suppressing immune responses, particularly antigen-specific immune responses, in a subject through the administration of therapeutically effective amounts of T resitope-blood component conjugates or modified polypeptides (and related compounds of this disclosure). with respect to the method, wherein the T regitope-blood component conjugate or modified polypeptide comprises endogenous TRegs (in embodiments, natural TRegs and/or adaptive TRegs, and in embodiments CD4+/CD25+/FoxP3+ regulatory T cells), or suppresses activation of CD4+ T cells, suppresses proliferation of CD4+ and/or CD8+ T cells, and/or suppresses activation or proliferation of β cells or nkT cells. In aspects, the T of the present disclosure
The regitope-blood component conjugate or modified polypeptide (and related compounds of this disclosure) may be covalently, non-covalently, or miscible with a particular target antigen. In embodiments, the administered T-regitope-blood component conjugate or modified polypeptide of the present disclosure is covalently bound, non-covalently bound, or in admixture with a particular target antigen. , resulting in attenuation of the immune response to the target antigen.

In embodiments, the target antigen can be a self protein or protein fragment. In embodiments, the target antigen may be a cognate protein or protein fragment. In embodiments, the target antigen may be a biological drug or a fragment thereof. In embodiments, the target antigen is preproinsulin or a fragment thereof. In embodiments, the target antigen comprises, consists exclusively of, or consists essentially of one or more of SEQ ID NOs: 56-63. In embodiments, the suppressive effect is mediated by natural TRegs. In aspects, the suppressive effect is mediated by adaptive TRegs.

[Methods of preventing or treating medical conditions]
The present invention relates to, for example, a method of preventing or treating one or more medical conditions in a subject, the method comprising using a T-regitope-blood component conjugate or a T-regitope-blood component conjugate disclosed herein. administering the modified polypeptide used to form the modified polypeptide (and related compounds of this disclosure) and thereby preventing or treating a medical condition in the subject. In a preferred embodiment, the medical condition is type 1 diabetes.

Medical conditions include, for example, medical conditions such as primary immunodeficiency, immune-mediated thrombocytopenia, Kawasaki disease, hematopoietic stem cell transplantation in subjects over 20 years of age, chronic B-cell lymphocytic leukemia, and childhood HIV type 1. It could be an infection. Specific examples include: (hematology) aplastic anemia, pure red cell aplasia, Diamond-Blackfan anemia, autoimmune hemolytic anemia, neonatal hemolytic disease, acquired factor VIII inhibitors, Congenital von Willebrand disease immune-mediated neutropenia, platelet transfusion refractoriness, neonatal autoimmune thrombocytopenia, posttransfusion purpura, thrombotic thrombocytopenic purpura/hemolytic uremic syndrome. Infectious diseases, solid organ transplants, surgery, trauma, burns, HIV infection. (Neurology) Epilepsy/pediatric intractable Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuritis, myasthenia gravis, Lambert-Eaton myasthenic syndrome, multifocal motor neuropathy, multiple sclerosis, (obstetrics) Relapse Sexual pregnancy ulcer. (Respiratory) Asthma, chronic chest symptoms, rheumatism, rheumatoid arthritis (adult and juvenile), systemic lupus erythematosus, systemic vasculitis, dermatomyositis, polymyositis, inclusion body myositis, Wegener's granulomatosis; (other) address Noreukodystrophy, amyotrophic lateral sclerosis, Behçet syndrome, acute cardiomyopathy, chronic fatigue syndrome, congenital heart block, cystic fibrosis, autoimmune bullous dermatitis, diabetes, acute idiopathic autonomic imbalance Acute disseminated encephalomyelitis, endotoxemia, hemolytic transfusion reaction, hemophagocytic syndrome, acute lymphocytic leukemia, lower motor neuron syndrome, multiple myeloma, human T-cell lymphoblastic virus-1-associated myelopathy, nephric syndrome , membranous nephropathy, nephrotic syndrome, thyroid ophthalmopathy, opsoclonus-myoclonus, recurrent otitis media, paraneoplastic cerebellar degeneration, paraproteinemia, parvovirus infection (general), polyneuropathy, visceral hypertrophy, endocrine abnormality, M protein, and skin changes (POEMS) syndrome, progressive lumbosacral plexitis, Lyme radiculopathy, Rasmussen syndrome, Reiter syndrome, acute renal failure, thrombocytopenia (nonimmune), streptococcal toxicity These include shock syndrome, uveitis, and Vogt-Koyanagi-Harada syndrome.

In certain embodiments, the invention provides treatment for transplant related disorders such as allergies, autoimmune diseases, graft-versus-host disease, enzyme or protein deficiency disorders, hemostatic disorders (e.g. hemophilia A, B or C), cancer. (particularly tumor-related autoimmunity), infertility, or infections (viral, bacterial or parasitic). T-resitope compounds or compositions of the present disclosure may be used in combination with other proteins or compounds used to treat subjects with medical conditions to reduce adverse events or increase the effectiveness of co-administered compounds. I can do it.

In certain embodiments, the present disclosure relates to methods of treating autoimmune diseases, such as, for example, T1D. The T-regitope-blood component conjugates disclosed herein and/or the modified polypeptides used to form the T-regitope-blood component conjugates (and related compounds of the present disclosure) may be used to reduce adverse events. Alternatively, it can be used in combination with other proteins or compounds used in the treatment of subjects with medical conditions to increase the effectiveness of the co-administered compounds.

[Application to allergies]
Allergen-specific regulatory T cells play an important role in controlling the onset of allergies and asthma. Endogenous TRegs (including in embodiments natural TRegs and/or adaptive TRegs, and in embodiments CD4 ⁺ /CD25 ⁺ /FoxP3 ⁺ regulatory T cells) have been shown to suppress inappropriate immune responses involved in allergic diseases. has been done. A number of recent studies have shown that regulatory T cells play an important role in controlling the excessive development of helper T cell type 2- biased immune responses in susceptible individuals, not only in animal models but also in humans. It has been shown that Recent studies have shown that Tregs also suppress co-stimulation of T cells through secretion of TGF-β, IL-10, suggesting an important role for Tregs in controlling allergic diseases. Impaired proliferation of natural or adaptive regulatory T cells leads to the development of allergies, and treatment that induces allergen-specific Tregs is considered to be a radical treatment for allergies and asthma. One strategy for both the prevention and treatment of asthma is the induction of Tregs. Animals can be protected from developing asthma by immune stimulation leading to Th1 or Treg responses. Accordingly, the T-regitope compounds or compositions of the present disclosure are useful in methods for the prevention or treatment of allergy and/or asthma. Thus, in aspects, the present disclosure provides a method of preventing or treating allergy and/or asthma in a subject, comprising a therapeutically effective amount of a T-regitope-blood component conjugate of the present disclosure and/or the present invention. administering a modified polypeptide (and related compounds of the present disclosure) used to form a T-regitope-blood component conjugate as disclosed in Relating to a method, including.

[Application to transplantation]
The T-regitope-blood component conjugates disclosed herein and/or the modified polypeptides used to form the T-regitope-blood component conjugates (and related compounds of the present disclosure) enhance the immune response against donor cells. It is useful to induce tolerance during the transplantation process by promoting the development of specifically down-regulating cells. Induction of Ag-specific TReg cells to treat organ-specific autoimmunity is an important therapeutic development that circumvents general immunosuppression. In murine models of bone marrow transplantation, TRegs promote engraftment of donor bone marrow and reduce the incidence and severity of graft-versus-host disease without compromising graft-versus-tumor immunological effects. These findings, coupled with the observation that mouse and human TRegs share phenotypic and functional characteristics, raise the bar for using these cells to reduce complications associated with human hematopoietic cell transplantation. This led to further consideration. An imbalance between TRegs and effector T cells contributes to the development of graft-versus-host disease, but the mechanisms of immune regulation, especially the allorecognition properties of TRegs, their influence and interaction with other immune cells, and the sites of their suppressive activity, are unclear. Not well understood.

Accumulating evidence from both humans and experimental animal models implicates TRegs in the development of graft-versus-host disease (GVHD). The demonstration that TReg can separate GVHD and graft-versus-tumor (GVT) activity suggests the possibility of manipulating its immunosuppressive capacity to reduce GVHD without detrimentally affecting GVT efficacy. There is. A variety of T lymphocytes with suppressive abilities have been reported, but the two most distinctive subsets are naturally occurring TRegs generated within the adrenal cortex (natural TRegs) and inducible TRegs occurring in the periphery. be. Accordingly, the T-regitope-blood component conjugates disclosed herein and/or the modified polypeptides used to form the T-regitope-blood component conjugates (and related compounds of the present disclosure) may be used during the transplantation process. Useful in methods for inducing tolerance. Thus, in aspects, the present disclosure provides a method of inducing tolerance during a transplantation process in a subject, comprising a T-regitope-blood component conjugate and/or a T-regitope-blood component conjugate disclosed herein. A method comprising administering a therapeutically effective amount of a modified polypeptide (and related compounds of the present disclosure) used to form a conjugate, and inducing tolerance during a transplantation process in a subject by said step of administering. Regarding.

[Application as a tolerizing agent and application to autoimmunity]
In embodiments, the modified polypeptides (and related compounds of this disclosure) used to form the T-regitope-blood component conjugates and/or the T-regitope-blood component conjugates disclosed herein are: It can be used as a tolerizing agent for immunogenic compounds (protein therapeutics) (Weber CA et al. (2009), Adv Drug Deliv, 61(11):965-76). This discovery has implications for the design of protein therapeutics. Accordingly, immunogens in combination with the T-regitope-blood component conjugates disclosed herein and/or the modified polypeptides used to form the T-regitope-blood component conjugates (and related compounds of the present disclosure) Administration of therapeutic compounds, such as protein therapeutics such as antibodies (eg, monoclonal antibodies ) , autologous cytokines, or foreign proteins, suppresses deleterious T effector immune responses. In vivo, TRegs act through dendritic cells to limit the activation of autoreactive T cells, thereby inhibiting T cell differentiation and acquisition of effector functions. By limiting the supply of activated pathogenic cells, TRegs can prevent or slow the progression of autoimmune diseases. However, this defense mechanism appears to be insufficient in subjects with autoimmune diseases. This is probably due to the lack of TReg cells and/or the development and accumulation of TReg-resistant pathogenic T cells during the long course of the disease. Restoration of self-tolerance in these subjects may therefore require infusion of TRegs with increased ability to control ongoing tissue injury, as well as purging of pathogenic T cells. Organ-specific autoimmune conditions such as thyroiditis and insulin-dependent diabetes result from the disruption of this tolerance mechanism (Mudd PA et al. (2006), Scand J
Immunol, 64(3):211-8). Accordingly, the T-regitope-blood component conjugates disclosed herein and/or the modified polypeptides used to form the T-regitope-blood component conjugates (and related compounds of the present disclosure) may be used to combat autoimmunity. Useful in prophylactic or therapeutic methods. Thus, in aspects, the present disclosure provides a method of preventing or treating autoimmunity in a subject, comprising a T-regitope-blood component conjugate and/or a T-regitope-blood component conjugate disclosed herein. A method comprising administering a therapeutically effective amount of a modified polypeptide used to form a gate (and related compounds of the present disclosure) and preventing and/or treating autoimmunity in a subject by said step of administering. .

[Application to diabetes]
Type 1 (juvenile) diabetes is an organ-specific autoimmune disease caused by the destruction of insulin-producing pancreatic beta cells. In non-diabetic subjects, islet cell antigen-specific T cells are either deleted during thymic development or converted to regulatory T cells that actively suppress effector responses to islet cell antigens. These tolerance mechanisms are lacking in juvenile diabetic subjects and the NOD mouse model of juvenile diabetes. In the absence of tolerance mechanisms, islet cell antigens are presented by human leukocyte antigen (HLA) class I and II molecules and recognized by CD8(+) and CD4(+) autoreactive T cells. Destruction of islet cells by these autoreactive cells ultimately causes glucose intolerance. The combination of T regitopes and islet antigens activates endogenous regulatory T cells and converts existing antigen-specific effector T cells to a regulatory phenotype. In this way, the autoimmune response is redirected and antigen-specific adaptive tolerance is induced. Induction of antigen-specific tolerance can prevent ongoing β-cell destruction by modulating the autoimmune response to self-derived epitopes. Accordingly, the T-regitope-blood component conjugates disclosed herein and/or the modified polypeptides used to form the T-regitope-blood component conjugates (and related compounds of the present disclosure) may be useful for the prevention or treatment of diabetes. Useful in methods for treatment. Thus, in aspects, the present disclosure relates to a method of preventing or treating diabetes in a subject, the method comprising administering a therapeutically effective amount of a T-regitope-blood component conjugate or a T-regitope disclosed herein. - administering the modified polypeptide used to form the blood component conjugate (and related compounds of the present disclosure) and thereby preventing or treating diabetes in the subject.

[Application to hepatitis B (HBV) infection]
Chronic HBV is usually acquired (due to maternal-fetal infection) or may be a rare consequence of acute HBV infection in adults. Acute exacerbations of chronic hepatitis B (CH-B) are accompanied by increased cytotoxic T cell responses to hepatitis B core antigen and e antigen (HBcAg/HBeAg). A recent study used the SYFPEITHI T cell epitope mapping system to predict MHC class II-restricted epitope peptides from HBcAg and HbeAg (Feng IC et al., (2007), J Biomed Sci, 14(1):43-57 ). MHC class II tetramers (tetramers) using high-scoring peptides were constructed and used to measure TReg and CTL frequencies. The results showed that HBcAg-specific TReg cells decreased during the exacerbation phase, and HBcAg peptide-specific cytotoxic T cells increased. During the tolerance phase, FOXp3-expressing TReg cell clones were confirmed. These data suggest that HbcAgTRegT cell depletion is responsible for spontaneous exacerbation in the natural history of chronic hepatitis B virus infection. Accordingly, the T-regitope-blood component conjugates disclosed herein or the modified polypeptides used to form the T-regitope-blood component conjugates (and related compounds of the present disclosure) are useful for chronic hepatitis B virus infection. It is useful in methods for the prevention or treatment of. Thus, in aspects, the present disclosure provides a method of preventing or treating a viral infection (e.g., HBV infection) in a subject, comprising a T-regitope-blood component conjugate or a T-regitope disclosed herein. - administering a therapeutically effective amount of the modified polypeptide (and related compounds of this disclosure) used to form the blood component conjugate, said step of administering preventing and/or treating said viral infection in a subject; Relating to a method, including.

[Application to SLE]
TReg epitopes that play a role in systemic lupus erythematosus (SLE) or Sjögren's syndrome have been defined. Binding assays with soluble HLA class II molecules and molecular modeling experiments showed that this epitope behaves as a promiscuous epitope and binds to many human DR molecules. In contrast to normal T cells and T cells from subjects without autoimmune disease, PBMCs from 40% of randomly selected lupus subjects contained T cells that proliferated in response to peptide 131-151. Since T cell responses change when the ligand is changed, it was suggested that there are several T cell populations that respond to this peptide, and that Treg cells may be included among them. A regulatory T epitope has also been defined in Sjogren's syndrome. Accordingly, modified polypeptides used to form T-regitope-blood component conjugates or T-regitope-blood component conjugates disclosed herein (and related to compounds) are useful in methods for the prevention or treatment of SLE. Thus, in aspects, the present disclosure relates to a method of preventing or treating SLE in a subject, which method comprises administering a therapeutically effective amount of a T-regitope-blood component conjugate or as disclosed herein. administering a modified polypeptide used to form a T-regitope-blood component conjugate (and related compounds of the present disclosure) in combination with an SLE epitope, and thereby preventing and/or treating SLE in a subject. It includes the process of

[Application to autoimmune thyroiditis]
Autoimmune thyroiditis is a condition caused by the production of antibodies against one's own thyroid peroxidase and/or thyroglobulin, leading to gradual destruction of thyroid follicles. HLA DR5 is closely associated with this disease. Accordingly, the T-regitope-blood component conjugates or modifications used to form the T-regitope-blood component conjugates disclosed herein are administered in combination with thyroid peroxidase and/or thyroglobulin TSHR or a portion thereof. The polypeptides (and related compounds of this disclosure) are useful in methods for the prevention or treatment of autoimmune thyroiditis. Thus, in aspects, the present disclosure relates to a method of preventing or treating autoimmune thyroiditis in a subject, the method comprising a T-regitope-blood component conjugate or a T-regitope- administering a therapeutically effective amount of the modified polypeptide (and related compounds of this disclosure) used to form the blood component conjugate in combination with thyroid peroxidase and/or thyroglobulin TSHR or a portion thereof; preventing and/or treating autoimmune thyroiditis in a subject. In further embodiments, the T-regitope compositions of the present disclosure administered in combination with TSHR or other Graves' disease antigens or portions thereof are useful in methods for the prevention or treatment of Graves' disease. Graves' disease is an autoimmune disease characterized by antibodies against the autothyroid thyroid stimulating hormone receptor (TSHR), leading to hyperthyroidism, or an abnormally strong release of hormones from the thyroid gland. Several genetic factors influence the development of Graves' disease. Women are more associated with the disease than men, whites and Asians are at higher risk than blacks, and HLA DRB1 0301 is closely associated with the disease. Thus, in aspects, the present disclosure relates to a method of preventing or treating Grave's disease in a subject, which method comprises administering a therapeutically effective amount of a T resitope-blood component conjugate disclosed herein or administering the modified polypeptide (and related compounds of this disclosure) used to form the T-resitope-blood component conjugate in combination with the TSHR or other Grave's disease antigen or portion thereof; The method includes a step of preventing and/or treating a disease.

[kit]
The methods described herein can be used, for example, to modify the T-regitope-blood component conjugates disclosed herein or the modified polypeptides used to form the T-regitope-blood component conjugates (and related compounds of the present disclosure). ) and can be conveniently used, e.g., in a clinical setting, to treat a subject exhibiting symptoms or a family history of the pathologies described herein. . In one embodiment, the kit comprises a T-regitope-blood component conjugate or a T-regitope-blood component conjugate disclosed herein for treating a subject exhibiting symptoms or a family history of a medical condition described herein. Further includes instructions for use of the modified polypeptide (and related compounds of this disclosure) used to form the conjugate.

[Mode]

A first aspect is a T regitope-blood component conjugate comprising a blood component linked to a modified polypeptide, said modified polypeptide having a reactive site attached thereto;
The present invention relates to T regitope-blood component conjugates, wherein said modified polypeptide comprises one or more regulatory T cell epitopes.

In a second aspect, the one or more regulatory T cell epitopes are one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 55 , and/or fragments and variants thereof , and any Optionally, the T resitope-blood component conjugate according to aspect 1, consisting only of 1 to 12 additional amino acids distributed in any proportion on the N-terminus and/or C-terminus of the polypeptides of SEQ ID NOs: 1 to 55. Regarding gates.

In a third aspect, the one or more regulatory T cell epitopes are one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 55 , and/or fragments and variants thereof , and any T-resitope according to aspect 1, optionally consisting essentially of 1 to 12 additional amino acids distributed in any proportion on the N-terminus and/or C-terminus of the polypeptides of SEQ ID NOs: 1 to 55 - blood Concerning conjugates of ingredients.

In a fourth aspect, the one or more regulatory T cell epitopes are one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 55 , and/or fragments and variants thereof , and optionally and 1 to 12 additional amino acids distributed in any proportion on the N-terminus and/or C-terminus of the polypeptides of SEQ ID NOs: 1 to 55. Regarding.

A fifth aspect relates to the T regitope-blood component conjugate of aspect 1, wherein said one or more regulatory T cell epitopes comprises SEQ ID NO:1.

A sixth aspect relates to the T regitope-blood component conjugate of aspect 1, wherein said one or more regulatory T cell epitopes comprises SEQ ID NO:28.

A seventh aspect relates to the T-regitope-blood component conjugate according to any one of aspects 1 to 6, wherein the blood component is albumin.

An eighth aspect relates to the T-regitope-blood component conjugate of any one of aspects 1 to 6, wherein said blood component is human serum albumin.

A ninth aspect relates to the T regitope-blood component conjugate of any one of aspects 1 to 8, wherein the modified polypeptide further comprises a T1Dgen peptide.

A tenth aspect relates to a T resitope-blood component conjugate according to aspect 9, wherein the T1Dgen peptide comprises one or more amino sequences selected from the group consisting of SEQ ID NOs: 56-63.

An eleventh aspect relates to a T-regitope-blood component conjugate according to any one of aspects 1 to 10, wherein the reactive site is attached to the amino terminal amino acid of the modified polypeptide.

A twelfth aspect relates to the T-regitope-blood component conjugate of any one of aspects 1 to 10, wherein the reactive site is attached to the carboxy-terminal amino acid of the modified polypeptide.

A thirteenth aspect provides the T-resitope-blood component of any one of aspects 1 to 10, wherein the reactive site is attached to an amino acid located between the amino-terminal amino acid and the carboxy-terminal amino acid of the modified polypeptide. Concerning conjugates.

A fourteenth aspect relates to the T resitope-blood component conjugate of any one of aspects 1 to 13, wherein the reactive moiety is a succinimidyl group or a maleimide group.

A fifteenth aspect relates to the T resitope-blood component conjugate of any one of aspects 1 to 14, wherein the reactive moiety is a 3-maleimidopropionic acid moiety.

A sixteenth aspect relates to the T-regitope-blood component conjugate of any one of aspects 1 to 15, wherein the conjugation between the blood component and the modified polypeptide is a maleimide bond.

A seventeenth aspect relates to a modified polypeptide, said modified polypeptide having a reactive site attached thereto, said modified polypeptide comprising one or more regulatory T cell epitopes.

An eighteenth aspect relates to the modified polypeptide of aspect 17, wherein the one or more regulatory T cell epitopes are one or more selected from the group consisting of SEQ ID NOs: 1 to 55 , and/or fragments and variants thereof. and optionally 1 to 12 additional amino acids distributed in any proportion on the N-terminus and/or C - terminus of the polypeptides of SEQ ID NOs: 1-55.

A nineteenth aspect relates to the modified polypeptide of aspect 17, wherein the one or more regulatory T cell epitopes are one or more selected from the group consisting of SEQ ID NOs: 1 to 55 , and/or fragments and variants thereof. and optionally 1 to 12 additional amino acids distributed in any proportion on the N-terminus and/or C - terminus of the polypeptides of SEQ ID NOs: 1-55.

A 20th aspect relates to the modified polypeptide of aspect 17, wherein the one or more regulatory T cell epitopes are one or more selected from the group consisting of SEQ ID NOs: 1 to 55 , and/or fragments and variants thereof. and optionally 1 to 12 additional amino acids distributed in any proportion on the N-terminus and/or C - terminus of the polypeptides of SEQ ID NOs: 1-55.

A twenty-first aspect relates to the modified polypeptide of aspect 17, wherein said one or more regulatory T cell epitopes comprise SEQ ID NO:1.

A twenty-second aspect relates to the modified polypeptide of aspect 17, wherein said one or more regulatory T cell epitopes comprise SEQ ID NO:28.

A twenty-third aspect relates to a modified polypeptide according to any one of aspects 17 to 22, wherein said modified polypeptide further comprises a T1Dgen peptide.

A twenty-fourth aspect relates to the modified polypeptide of aspect 23, wherein said T1Dgen peptide comprises one or more amino sequences selected from the group consisting of SEQ ID NOs: 56-63.

A twenty-fifth aspect relates to the modified polypeptide of any one of aspects 17 to 24, wherein the reactive site is attached to the amino terminal amino acid of the modified polypeptide.

A twenty-sixth aspect relates to the modified polypeptide of any one of aspects 17 to 24, wherein the reactive site is attached to the carboxy-terminal amino acid of the modified polypeptide.

A twenty-seventh aspect relates to the modified polypeptide of any one of aspects 17 to 24, wherein the reactive site is attached to an amino acid located between the amino terminal amino acid and the carboxy terminal amino acid of the modified polypeptide.

A twenty-eighth aspect relates to the modified polypeptide of any one of aspects 17 to 27, wherein the reactive moiety is a succinimidyl group or a maleimide group.

A twenty-ninth aspect relates to the modified polypeptide of any one of aspects 17-28, wherein the reactive site is a 3-maleimidopropionic acid moiety.

A 30th aspect relates to a method for suppressing an autoimmune reaction characteristic of T1D in a subject in need thereof, and the method comprises administering to the subject the T resitope of any one of aspects 1 to 16. administering a blood component conjugate.

A 31st aspect relates to a method for suppressing an autoimmune reaction characteristic of T1D in a subject in need thereof, the method comprising the step of administering the modified polypeptide of any one of aspects 17 to 29 to the subject. including.

A thirty-second aspect relates to a method according to any one of aspects 30-31, wherein the administration shifts one or more antigen-presenting cells to a regulatory phenotype.

A thirty-third aspect relates to a method according to any one of aspects 30-31, wherein the administration shifts one or more dendritic cells to a regulatory phenotype.

A thirty-fourth aspect relates to a method according to any of aspects 30-31, wherein the regulatory phenotype is characterized by decreased CD11c and HLA-DR expression in dendritic cells or other antigen presenting cells.

A thirty-fifth aspect relates to a method according to any of aspects 30 to 31, wherein one or more T cells are shifted to a regulatory phenotype by administering a regulatory T cell epitope.

A thirty-sixth aspect relates to a method according to any of aspects 30-31, wherein CD4 ⁺ /CD25 ⁺ /FoxP3 ⁺ regulatory T cells are activated by administration of one or more regulatory T cell epitopes.

A thirty-seventh aspect provides that the administration causes an innate immune response, an adaptive immune response, an effector T cell response, a memory T cell response, a helper T cell response, a B cell response, an NKT cell response, or any combination thereof. 32. A method according to any of aspects 30-31, for suppressing a selected immune response.

A thirty-eighth aspect relates to a pharmaceutical composition comprising a T-regitope-blood component conjugate according to any one of aspects 1 to 16 and a carrier, excipient and/or adjuvant.

A 39th aspect is a pharmaceutical composition comprising a modified polypeptide capable of forming a T regitope-blood component conjugate according to any one of aspects 17 to 29, and a carrier, excipient, and/or adjuvant. Regarding.

The examples that follow are not to be construed as limiting the scope of the invention in any way. Numerous embodiments within the scope of the claims will be apparent to those skilled in the art in light of this disclosure.

(1) In silico identification of T resitope compositions

T cells specifically recognize epitopes presented by antigen presenting cells (APCs) in the context of MHC (major histocompatibility complex) class II molecules. These helper T cell epitopes can be expressed as linear sequences of 7 to 30 contiguous amino acids that fit into the MHC class II binding groove. Many computer algorithms have been developed and used to detect class II epitopes in protein molecules of various origins (DeGroot AS et al, (1997), AIDS Res Hum Retroviruses, 13(7):539 -41; Schafer JR et al., (1998), Vaccine,16(19):1880-4; De Groot AS et al., (2001), Vaccine, 19(31):4385-95; DeGroot AS et al., (2003), Vaccine, 21(27-30):4486-504). These "in silico" predictions of helper T cell epitopes are useful for vaccine design and therapeutic proteins, i.e. antibody-based drugs, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, engineered protein scaffolds, enzymes, It has been successfully applied to deimmunize growth factors, hormones, interferons, interleukins and thrombolytic agents (Dimitrov DS, (2012), Methods MolBiol, 899:1-26).

The EpiMatrix™ system (EpiVax, Providence, RI) is a set of prediction algorithms encoded in computer programs useful for predicting class I and class II HLA ligands and T cell epitopes. The EpiMatrix™ system uses a 20x9 coefficient matrix to model interactions between specific amino acids (20) and binding positions (9) within the HLA molecule. To identify putative T cell epitopes present in any input protein, the EpiMatrix™ system first parses the input protein to create nonamer frames, each frame overlapping the last frame by 8 amino acids. Each frame is then labeled with a human HLA molecule, typically DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*0801, DRB1*1101, DRB1*1301, and DRB1*1501 (Mack (2013), Tiss Antig, 81(4):194-203) for predicted affinity for one or more common alleles.
Briefly, for any 9-mer peptide, select coefficients from a prediction matrix using specific amino acid codes (one for each of the 20 endogenous amino acids) and relative binding positions (1-9). do. The individual coefficients are derived using a proprietary method similar to, but not identical to, the pocket profile method originally developed by Sturniolo (SturnioloT et al., 1999, NatBiotechnol, 17(6):555-61). The individual coefficients are then summed to produce a raw score. The EpiMatrix™ raw scores are then normalized with respect to the score distribution obtained from a very large set of randomly generated peptide sequences. The resulting "Z" scores are normally distributed and can be directly compared between alleles.

[EpiMatrix(TM) Peptide Scoring]
A peptide with a score of 1.64 or higher (approximately the top 5% of any peptide set) on the EpiMatrix™ "Z" scale was determined to be fairly likely to bind to the predicted MHC molecule. Peptides that score 2.32 or higher (top 1%) on the scale are extremely likely to bind, and most published T cell epitopes fall within this score range. Previous studies have also demonstrated that EpiMatrix™ accurately predicts published MHC ligands and T cell epitopes. (De Groot AS, Martin W. Reducing risk, improving outcomes: bioengineering less immunogenic protein
therapeutics. Clin Immunol. 2009 May;131(2):189-201.doi: 10.1016/j.clim.2009.01.009. Epub 2009 Mar 6., incorporated herein by reference in its entirety).

[Identification of promiscuous T cell epitope cluster]
Potential T cell epitopes are not randomly distributed throughout the protein sequence, but tend to "cluster". T-cell epitope "clusters" are 9 to about 30 amino acids in length, and can contain 4 to 40 binding motifs, considering affinities for multiple alleles and multiple frames. After epitope mapping, the result set generated by the EpiMatrix™ algorithm is screened for the presence of T cell epitope clusters and EpiBars™ using a proprietary algorithm known as Clustimer™. Briefly, the EpiMatrix™ scores for each nine-mer peptide analyzed are aggregated and matched against statistically derived thresholds. High scoring nonamers are then expanded one amino acid at a time. The scores of the extended sequences are re-aggregated and compared to the modified threshold. This process is repeated until the proposed expansion no longer improves the cluster's overall score. In embodiments, the T regitope(s) may be identified as a T cell epitope cluster by the Clustimer™ algorithm. In embodiments, the T cell epitope cluster may include a significant number of putative T cell epitopes and EpiBars™ that exhibit a high probability of MHC binding and T cell responsiveness.

[Test for cross-reactivity with host]
The JanusMatrix system (EpiVax, Providence, RI) is useful for screening peptide sequences for cross-conservation with the host proteome. JanusMatrix is an algorithm that predicts the likelihood of cross-reactivity between peptide clusters and the host genome or proteome based on the conservation of TCR-facing residues in their putative MHC ligands. The JanusMatrix algorithm first considers all predicted epitopes contained in a given protein sequence and divides each predicted epitope into its constituent agretopes and epitopes. Each sequence is then screened against a database of host proteins. Peptides with matching MHC-facing aggretopes (ie, the aggretopes of the input peptide and its host peptide are expected to bind to the same MHC allele) and exactly the same TCR-facing epitopes are returned. JanusMatrix homology scores suggest a bias towards immune tolerance. In the case of therapeutic proteins, cross-conservancy between autologous human epitopes and epitopes of therapeutic agents may increase the likelihood that such candidates will be tolerated by the human immune system. In the case of vaccines, cross-conservation between human and antigenic epitopes may indicate that such candidates utilize immune camouflage and thereby evade immune responses, resulting in ineffective vaccines. . If the host is, for example, a human, peptide clusters are screened against the human genome or proteome based on the conservation of TCR-facing residues in putative HLA ligands . The peptides are then scored using the JanusMatrix homology score. In embodiments, peptides with JanusMatrix homology scores greater than 3.0 exhibit a high potential for tolerance and, as such, are likely to be highly useful T-regitope compositions of the present disclosure. be. In embodiments, peptides with a JanusMatrix homology score of less than 2.0, less than 2.5, or less than 3.0 exhibit a low potential for tolerance and may be excluded from the T-regitope compositions of the present disclosure. good.

[Method for evaluating binding of T-regitope to soluble MHC]

(Synthesis of peptide)
The T resitopes of the present disclosure are produced by direct chemical synthesis or by recombinant methods (J
Sambrook et al., Molecular Cloning: A Laboratory Manual, (2ED, 1989), Cold Spring Harbor Laboratory Press, Cold Springs Harbor, NY (Publ), herein incorporated by reference in its entirety). Sample T resitopes are prepared using Fmoc-chemistry (9-fluorenylmethyloxycarbonyl group synthesis) under the guidance and direction of the inventors of the present invention at 21st Century Biochemicals (Marlborough, MA). In certain embodiments, the T resitope is capped with an N-terminal acetyl group and a C-terminal amino group. HPLC, mass spectrometry and UV scan (ensuring purity, mass and spectra, respectively) analysis of selected T resitopes typically shows greater than 80% purity.

Amino acid analysis is performed by a third party contractor (New England Peptide, Inc, Gardner, MA) to confirm the predicted composition.

Mass spectrometry and analytical HPLC analyzes were performed by a second independent contractor (21St Century Biochemicals, Inc., Marlboro, MA) to further confirm the composition of the T-resitope.

[HLA binding assay]
Binding activity is analyzed with EpiVax (Providence, RI). The binding assay used (Steere AC et al. (2006), J Exp Med, 2003(4):961-71) provides an indirect measure of peptide-MHC affinity. For example, soluble HLA molecules are loaded into a 96-well plate along with an unlabeled experimental T regitope and a labeled control peptide. Once the binding mixture reaches steady equilibrium (after 24 hours), HLA-T regitope complexes are captured on ELISA plates coated with anti-human DR antibodies and detected with europium-conjugated probes (PerkinElmer, Waltham, Mass.). Time-resolved fluorescence of bound labeled control peptides is assessed on a SpectraMax® M5 unit (Spectramax, Radnor, PA). Binding of the experimental T-regitope is expressed as percent inhibition of the labeled control peptide (experimental fluorescence/control fluorescence multiplied by 100). The percent inhibition value (over a range of molar concentrations) for each experimental T-regitope is used to calculate the concentration at which it inhibits 50% of the specific binding of the labeled control T-regitope, ie, the IC ₅₀ of the T-regitope. T-regitope is dissolved in DMSO. The diluted T-regitope can be mixed with the binding reagent in an aqueous buffer to obtain a final concentration of 100,000 nM to 100 nM . T- regitopes were assayed against a panel of five common class II HLA alleles: HLA-DRB1*0101, HLA-DRB1*0301, HLA-DRB1*0701, HLA-DRB1*1101, and HLA-DRB1*1501. be done. IC ₅₀ values for each T regitope/allele combination are derived from the inhibition rate of the labeled control peptide at each concentration using linear regression analysis.

In this assay , a T- regitope is considered to bind with very high affinity if it inhibits 50% of control peptide binding at a concentration of 100 nM or less, and 50% of control peptide binding at concentrations between 100 nM and 1,000 nM. % inhibition is considered high affinity, and 50% inhibition of control peptide binding at concentrations between 1,000 nM and 10,000 nM is considered intermediate affinity. Low affinity peptides inhibit 50% of control peptide binding at concentrations between 10,000 nM and 100,000 nM. Peptides that fail to inhibit 50% or more of control peptide binding at any concentration below 100,000 nM and exhibit no dose response are designated as non-binders (NB).

In this assay, a T-regitope is considered to bind with very high affinity if it inhibits 50% of control peptide binding at a concentration of 100 nM or less, and 50% of control peptide binding at concentrations between 100 nM and 1,000 nM. A control peptide is considered to bind with high affinity if it inhibits 50% of binding, and is considered to have intermediate affinity if it inhibits 50% of control peptide binding at concentrations between 1,000 nM and 10,000 nM. Low affinity peptides inhibit 50% of control peptide binding at concentrations between 10,000 nM and 100,000 nM. Peptides that do not inhibit at least 50% of control peptide binding at any concentration up to 100,000 nM and exhibit no dose response are considered non-binders (NB).

[Example 1. Characteristics of peptides due to binding to HLA class II molecules]

Soluble MHC binding assays are performed on the T regitopes of the present disclosure according to the methods described above. IC ₅₀ values (nM) are derived from 6-point inhibition curves.

EpiMatrix™ predictions, calculated _IC50 values, and classification of results are reported for each T regitope and HLA allele. Of particular interest are T regitope-allele combinations that are predicted to be cross-reactive between host proteomes. Of these T-regitope-allele interactions, 90% bound HLA, indicating that these T-regitopes elicit measurable responses in human PBMC assays. Based on these findings, T-regitopes are selected for further testing.

[Method for evaluating the phenotype of APC exposed to peptides]

Surface expression of class II HLA (HLA-DR) and CD86 by professional antigen presenting cells (APCs) is one way APCs modulate T cell responses. It has been previously demonstrated that expression of class II HLA surface markers is decreased in response to T regitopes, particularly the control T regitope 167 (21st Century Biochemicals, Marlboro, MA). Furthermore, decreased expression of the surface marker CD86 is positively correlated with improved Treg function (ZhengY et al., JImmunol, 2004, 172(5):2778-84). In this assay, candidate T-regitopes, including selected T-regitopes, are tested for their ability to down-regulate the expression of class II HLA and costimulatory molecule CD86 on the surface of pro-APCs, particularly dendritic cells.

T-regitopes are individually tested for modulatory potential using a proprietary APC phenotyping method previously developed at EpiVax (EpiVax, Providence, RI). Previously harvested frozen PBMCs are thawed in the usual manner and suspended in chRPMI. Under the direction and guidance of the inventors from EpiVax, HLA typing is performed by Hartford Hospital (Hartford, CT) on small extracted samples of cellular material provided by EpiVax. On assay day 0, ^0.5x10 cells were extracted and screened for the presence of the surface marker CD11c (a marker specific to dendritic cells) and analyzed by flow cytometry for the presence of the surface markers HLA-DR and CD86. Ru. The remaining cells were plated ( ^4.0x106 cells/mL in chRPMI+800ul medium) with buffer only ( negative control) , T resitope 167 (positive control) (21st Century Biochemicals, Marlboro, MA), Flu- Four selected peptides or positive and negative controls, including HA306-318 (negative control) (21ST Century Biochemicals, Marlboro, MA) and Ova323-339 (negative control) (21st Century Biochemicals, Marlboro, MA). Stimulate with one of them (50 μg/mL) . Plated cells are incubated at 37°C for 7 days. On day 7 of the assay, incubated cells are screened by flow cytometry for the presence of the surface marker CD11c. CD11c positive cells are then analyzed for the presence of surface markers HLA-DR and CD86. The experimental peptides will be tested on samples taken from five different human donors.

All whole blood samples used in the experiments were sourced from healthy donors under the IRB 07115 protocol (Clinical Partners, Johnston, RI) and were purified using conventional Ficoll® (GE Healthcare) separation gradients. (Noble PB and Cutts JH, Can Vet J, 1967, 8(5):110-11) or to filter leukocytes from whole blood obtained from healthy donors at the Rhode Island Blood Center (Providence). Leukopenia filters were obtained from A.D., Rhode Island). After the whole blood has passed through the filter, the filter is run in the opposite direction to force the collected white blood cells out of the filter. Leukocytes are then separated using a regular Ficoll separation gradient. The collected white blood cells are then stored frozen for future use. If necessary for use in the assay, frozen leukocytes are thawed using conventional methods.

Exposure to putative T regitopes on dendritic cell phenotype is measured by multiple means. First, a dot plot comparing the surface expression of CD11c and HLA-DR is created for each experimental condition. Dot plots for cells exposed to all control peptides and experimental peptides are overlaid with dot plots for control cells exposed to culture medium ( broth ) only. This superposition provides an effective way to visually observe the shift in HLA-DR distribution between T-regitope-stimulated cells and unstimulated CD11c-high cells. The observed shift in the distribution of HLA-DR is reported as a qualitative indicator. Next, the change in HLA-DR expression intensity is calculated for the CD11c-high segment of each dot plot. The rate of change in HLA-DR expression intensity is calculated by subtracting the MFI of HLA-DR expression in medium-exposed cells from the mean fluorescence index (MFI) of HLA-DR expression in peptide-exposed cells. divided by MFI and multiplied by 100 ( ^HLA-DR MFI _peptide - ^HLA-DR MFI _media / ^HLA-DR MFI _media *100)
or ( ^HLA-DR MFI _peptide - ^HLA-DR MFI _medium / ^HLA-DR MFI _medium x 100)
is equal to Compare the rate of change for each peptide stimulant. Next, the rate of change in the percentage of HLA-DR low expressing cells present in the CD11c high expression population relative to the medium control is calculated for each peptide. The rate of change in the percentage of HLA-DR-low cells is calculated by subtracting the percentage of HLA-DR-low in the medium-exposed cells from the percentage of HLA-DR-low in the peptide-exposed cells. Divide by the low ratio and multiply by 100 ( ^HLA-DR-low % _peptide - ^HLA-DR-low % _media / ^HLA-DR-low % _media *100)
or ( ^HLA-DR-low % _peptide - ^HLA-DR-low % _medium / ^HLA-DR-low % _medium x 100)
It is calculated so that In this assay, the observed negative changes in HLA-DRMFI and positive changes in the proportion of HLA-DR-low cells present in the CD11c-high expression population are associated with decreased HLA expression and toward a regulatory APC phenotype. Suggests a shift. The data are used to compare the % of HLA+ and HLA- peptide stimuli where vehicle is medium .

In a similar manner, we will also assess the effect of T-regitope exposure on the surface expression of CD86, a costimulation molecule known to promote T-cell activation. First, a dot plot comparing the surface expression of CD11c and CD86 is created for each experimental condition. The dot plots of cells exposed to all control and experimental T-regitopes are overlaid with the dot plots of control cells exposed to medium only. This superposition provides an effective way to visually observe the shift in CD86 distribution between T-regitope-stimulated cells and unstimulated CD11c-high expressing cells. The observed shift in the distribution of CD86 is reported as a qualitative indicator. Next, the change in intensity of CD86-high expression is calculated for the CD11c-high segment of each dotplot. The rate of change in the intensity of CD86-high expression is calculated as the mean fluorescence index (MFI) of CD86 expression for peptide-exposed cells minus the MFI of CD86-high expression for medium-exposed cells, which is the MFI of CD86 expression for medium-exposed cells. ( ^CD86-high MFI _peptide - ^CD86-high MFI _media / ^CD86-high MFI _media *100)
or ( ^CD86-high MFI _peptide - ^CD86-high MFI _medium / ^CD86-high MFI _medium x 100)
is equal to Next, the rate of change in the proportion of CD86 low expressing cells present in the CD11c high expressing population is calculated. The percent change in the proportion of CD86- low cells is the proportion of CD86- low to peptide-exposed cells minus the proportion of CD86- low to medium-exposed cells divided by the proportion of CD86- low to medium-exposed cells. Multiplied by 100 ( ^CD86-low %I _peptide - ^CD86-low % _media / ^CD86-low % _media *100)
or equals ( ^CD86-low % _peptide - ^CD86-low % _medium / ^CD86-low % _medium x 100). In this assay, the observed negative changes in CD86MFI and positive changes in the proportion of CD86-low cells present in the CD11c-high expression population indicate that CD86 expression is decreased and a shift toward a regulatory APC phenotype is observed. It means something.

[Example 2. Characterization of APC exposed to peptide]

Dendritic cell phenotyping assays can be performed on selected T regitopes according to the methods described above. Create dot plots corresponding to each experimental condition tested on each of the five human donors.

A series of dot plots were generated representing the surface expression of CD11 versus HLA-DR analyzed on day 7 of the assay for five donors in the presence of various peptide stimulants. A downward shift of the CD11c+/HLADR+ population was evident in samples treated with the strong T regitope compared to the media control, indicating an acquired regulatory phenotype. Although T-regitope 167 (positive control) and some of the other peptides react similarly, the observed shift is more pronounced with other more potent T-regitopes.

A series of dot plots were generated representing the surface expression of CD11c versus CD86 analyzed on day 7 of the assay for five donors in the presence of various peptide stimulants. An increase in CD86- low cells was seen in samples treated with the potent T regitope when compared to vehicle controls, indicating a shift towards an acquired regulatory phenotype. Dendritic cell phenotypic assays demonstrate that exposure to potent T regitopes reduces HLA-DR expression in all five subjects tested. Furthermore, in 4 out of 5 subjects, exposure to strong T regitopes increases the abundance of CD86-low in the CD11c-high cohort. Both trends indicate a shift towards an acquired regulatory phenotype.

[Method for evaluating the effect of peptides on proliferation of regulatory T cells]

Previous studies conducted by EpiVax (Providence, RI) showed that regulatory T cell proliferation was increased after exposure to known T regitopes, including the positive control T regitope 167 (21st Century Biochemicals, Marlboro, MA). This has been proven. In this assay, T-regitopes, including T-regitopes of the present disclosure, are tested for their ability to induce proliferation among CD4+CD25+FoxP3+ regulatory T cells. Previously collected frozen PBMCs are thawed and suspended in chRPMI (3.3x10 ⁶ cells/mL) prepared in a conventional manner. Cells were prepared using CFSE (Cat #: 65-0850-84, Affymet
rix, Santa Clara, CA) and plated at 300,000 cells per well. Plates are incubated overnight (37°C, 5% _CO2 ). On assay day 1, T resitope and control peptides are reconstituted in sterile DMSO to a final stock concentration of 20 mg/mL. Previous titration experiments performed with EpiVax (EpiVax, Providence, RI) showed that healthy control donors (Rhode Island Blood It has been demonstrated to induce measurable CD4+ effector memory T cell responses in PBMCs harvested from the Center for Disease Control and Prevention, Providence, RI). Tetanus toxoid stock (100 μg/mL) (Astarte Biologics, Bothell, WA) is diluted in adjusted chRPMI to give a working concentration of 1 μg/mL. The plated cells were mixed with 100 μL of conditioned chRPMI (negative control), 100 μL of tetanus toxoid solution (positive control) (Astarte Biologics, Bothell, WA), 100 μL of diluted 2991 μL tetanus toxoid solution + 9 μL of T resitope solution, Stimulate with either 100 μL diluted 2997 μL tetanus toxoid solution + 3 μL T resitope solution or 100 μL diluted 6998.2 μL tetanus toxoid solution + 1.8 μL T resitope solution. In parallel, control wells containing the same number of cells are incubated with a control peptide solution prepared in the same way as for the T-regitope solution. All plates are then incubated for an additional 6 days. On day 5 of the assay, 100 μL of supernatant is removed from each well and replaced with freshly prepared chRPMI.

Highly activated regulatory T cells are selected that exhibit elevated levels of FoxP3, CD25, granzyme B and proliferation . The gating strategy for highly activated regulatory T cells includes CD4+ T cells being gated on elevated CD25, granzyme B, FoxP3, and low CFSE (proliferation). The results of a representative assay without the addition of TT are compared to the results of a representative assay with the addition of 0.5 μg/ml TT.

[Example 3. T-regitopes strongly induce populations of highly proliferative and activated regulatory T cells]

Regulatory T cell proliferation assays are performed on the T regitopes of the present disclosure according to the methods described above. Gating on highly activated granzyme B positive CD4+ T cells (CD25+ granzyme B+) indicates that these subsets are composed of highly proliferative regulatory T cells (CFSE low FoxP3+). Strong T-regitopes increase the relative proportion of this population, while control peptides have no significant effect. This increase in the highly activated Granzyme B-positive regulatory T cell population correlates closely with the degree of effector T cell inhibition exhibited by different T regitopes from multiple donors, and the T regitope is associated with effector CD4+ cells. The relative number increases significantly only when it has an inhibitory effect on This suggests that cytotoxic regulatory T cells are involved in the potent T resitope inhibition mechanism. Overall, this data demonstrates that potent T resitopes strongly induce a population of highly proliferative activated regulatory T cells enriched in granzyme B.

[Method for evaluating peptide effects on proliferation of CD4+ effector T cells]

CD4+ effector memory T cells contained within the PBMC cell population are induced to proliferate in response to stimulation with known T cell epitopes. T-regitopes can bind multiple HLA molecules and induce a regulatory phenotype in exposed APCs (Clinical Partners, Johnston, RI). The results of competitive inhibition HLA binding assays provide an indirect measure of peptide-MHC affinity (Steere AC et al., J Exp Med, (2006), 203(4):961-71, incorporated herein by reference). (incorporated herein). Binding of the experimental T regitope is expressed as percent inhibition of the labeled control peptide (experimental fluorescence/control fluorescence multiplied by 100). The percent inhibition value (over a range of molar concentrations) for each experimental T-regitope is used to calculate the concentration at which it inhibits 50% of the specific binding of the labeled control peptide. This value is called _IC50 . HLA binding results and IC ₅₀ of T-regitope across five HLA-DR1 types indicate high affinity of binding of T-regitope across multiple HLA class II types.

The purpose of this experiment was to establish the ability of T regitopes to suppress the proliferation of antigen-stimulated CD4+ effector memory T cells, either by direct (TReg engagement and activation) or indirect (APC phenotype modulation) means. That's true. In the first study, a control peptide is used as a negative control. In subsequent studies, the performance of the T-regitope is compared to the homologous peptide from which it is derived.

Previously collected frozen PBMCs are thawed and suspended in chRPMI (3.3x10 ⁶ cells/mL) prepared in a conventional manner. Cells are stained with CFSE (Cat #: 65-0850-84, Affymetrix, Santa Clara, Calif.) and plated at 300,000 cells per well. Plates are incubated overnight (37°C, 5% _CO2 ). On assay day 1, T resitope peptides and control peptides are reconstituted in sterile DMSO to a final stock concentration of 20 mg/mL. Previous titration experiments performed with EpiVax (EpiVax, Providence, RI) showed that healthy control donors (Rhode Island Blood It has been demonstrated to induce measurable CD4+ effector memory T cell responses in PBMCs harvested from the Center for Disease Control and Prevention, Providence, RI). Tetanus toxoid stock (100 μg/mL) (Astarte Biologics, Bothell, WA) is diluted in adjusted chRPMI to give a working concentration of 1 μg/mL. The plated cells were mixed with 100 μL of conditioned chRPMI (negative control), 100 μL of tetanus toxoid solution (positive control) (Astarte Biologics, Bothell, WA), 100 μL of diluted 2991 μL tetanus toxoid solution + 9 μL of T resitope solution, Stimulate with either 100 μL diluted 2997 μL tetanus toxoid solution + 3 μL T resitope solution or 100 μL diluted 6998.2 μL tetanus toxoid solution + 1.8 μL T resitope solution. In parallel, control wells containing the same number of cells are incubated with a control peptide solution prepared in the same way as for the T-regitope solution. All plates are then incubated for an additional 6 days. On day 5 of the assay, 100 μL of supernatant is removed from each well and replaced with freshly prepared chRPMI.

On day 7 of the assay, remove the cells from the incubation. Cells are labeled for live/dead identification, for surface markers CD127, CCR7, CD4, CD45RA, and CD25, and for intracellular FoxP3. The stained cells are further prepared for FACS analysis in the usual manner. Cells are first gated to remove clumps and dead cells. Live cells are gated for CD4 T cells and all subsequent analyzes are performed on this population. The activated T effector population is identified as CD4+/CD25-high/FoxP3-intermediate (CD4+/CD25hi/FoxP3int). Parallel analysis of this identified T effector cell population shows that proliferation of this major population corresponds to CD45RA-low and CCR7-low effector memory T cells. The main proliferating population corresponds to the T effector memory phenotype (CD45RA-low/CCR7-low).

Proliferation of CD4+/CD25-high (CD4+/CD25 ^hi ) T cells was estimated from dilution of CFSE staining (Cat #: 65-0850-84, Affymetrix, Santa Clara, CA), and % proliferation was estimated from CFSE-low (CFSE ^lo ) determined by the group.

[Example 4A. T-regitope peptide suppresses proliferation and activation of CD4+ effector T cells]

Changes in the activation and proliferation of CD4+ effector cells when a proliferation stimulant (tetanus toxoid) is co-delivered with T resitopes can be measured to characterize the proliferative response of CD4+ T cells, which primarily consist of T effector memory cells.

T cell proliferation assays are performed on the T regitopes of the present disclosure according to the methods described above. Dot plots are generated corresponding to each experimental condition tested for activation and proliferation. Dot plots of the experimental conditions tested for activation show that T-resitope peptides stimulate the population of activated effector CD4+ T cells (CD4+/CD25-high/FoxP3-intermediate, expressed as CD4+/CD25hi/FoxP3int) in response to tetanus toxoid. was strongly inhibited in a dose-dependent manner, indicating that the control peptide had no appreciable effect. In the same experiment, the proliferation of T-regitope-activated CD4+ T cells (CFSE low cells) was strongly suppressed by the T-regitope peptide in a dose-dependent manner, whereas the control peptide had little effect. Tetanus toxoid stimulates a population of activated (CD25 high) CD4 T cells to proliferate (CFSE low), approximately 90% of which also proliferate activated cells. This highly activated cell population is actively suppressed by the T regitope peptide in a dose-dependent manner, but the control peptide has no significant suppressive effect on activated CD4 cells.

Furthermore, gating on CD4+ and CD4- live cells demonstrates that the suppressive effect of T regitopes on cell proliferation is more pronounced on the CD4+ population than on the CD4- population. T-regitopes inhibit CD4-T cell proliferation in a dose-dependent manner. Negative control peptides show little suppressive effect on the CD4- population.

[Example 4B. T resitope peptide and its homologous peptide suppress proliferation of CD4+ effector T cells]

Furthermore, the suppressive effects of T regitopes with homologous peptides from which the T regitopes are derived on CD4+ T cell effector memory responses to TT in normal donor PBMCs are compared, analyzed, and evaluated.

A study designed to evaluate the effect of T regitope peptides and homologous peptides on the recall response to TT of PBMCs derived from two normal donors is performed. The protocol used has been described above . T-regitope concentrations range from 5, 10, 15, and 20 μg/ml. T resitopes inhibit activation of CD4+ T cells and Teff proliferation in response to TT in a concentration-dependent manner. Activation of CD4+ T cells is reduced by 80-90% at 20 μg/ml (11 mM). Addition of homologous peptides together with TT inhibits both CD4+ T cell memory responses (CD4+ T cell activation and Teff proliferation) in a concentration-dependent manner. Activation of CD4+ T cells is reduced by 50-70% at 20 μg/ml (11 mM). T cell populations respond with greater than 5-20% inhibitory effects (for both CD4+ proliferation and Teff activation) for a given peptide concentration of either T-regitope peptides or homologous peptides over the concentration range tested . Peptide sequence similarities between T-regitopes and their homologs, together with their parallel T cell inhibitory functions, suggest that antigen-specific tolerance to the protein from which the T-regitope is derived may be useful in stimulating tolerance to replacement proteins of the homolog. This is proof that it is a reliable method. In this assay, both the T-regitope peptide and the homologous peptide inhibit CD4+ T cell proliferation and activate T effector T cells in a dose-dependent manner .

[Method for evaluating the effect of T resitope peptide on CD8+ effector T cells]

CD8+ effector memory T cells contained within the PBMC cell population are induced to proliferate in response to stimulation with known class I T cell epitopes. The T-regitope binds multiple HLA alleles and induces a regulatory phenotype in exposed APCs (Clinical Partners, Johnston, RI) in PBMCs collected from whole blood donors due to the gating strategy employed. It is possible to identify the APC fraction). The results of this assay demonstrate the ability of T-regitopes to suppress proliferation of antigen-stimulated CD8+ T effector memory T cells by either direct (TReg engagement and activation) or indirect (APC phenotype modulation) means. do.

T cell proliferation assays are performed using the T regitopes of the present disclosure according to the methods described above. PBMCs from two healthy donors are thawed and suspended in chRPMI (3.3x10 ⁶ cells/mL) prepared in the usual manner. Cells are stained with CFSE (Cat #: 65-0850-84, Affymetrix, Santa Clara, Calif.) and plated at 300,000 cells per well. Plates are incubated overnight (37°C, 5% _CO2 ). On assay day 1, T resitopes are reconstituted in sterile DMSO to a final stock concentration of 20 mg/mL. An intermediate solution of T resitope at 2x final concentration in chRPMI is prepared as described above. The final concentrations of T-regitope are tested at 2.5 μg/mL, 5 μg/mL, 10 μg/mL and 20 μg/mL. As CD8+ stimulating antigen, a CEF peptide pool consisting of 23 MHC class I-restricted viral epitopes from human cytomegalovirus, Epstein-Barr virus, and influenza virus is used. CEF peptide was added to the wells (2 μg/mL data shown), and the wells were treated with medium containing cells (control) or cells and 0 μg/mL, 1 μg/mL, 2 μg/mL, or 4 μg/mL . A medium with T- regitope is used. All plates are incubated for an additional 6 days. On day 5 of the assay, 100 μL of supernatant is removed from each well and replaced with freshly prepared chRPMI.

Stain for live/dead markers, extracellular markers CD4, CD8α, CD25, CD127, CD45RA, CCR7, and intracellular marker FoxP3 using conventional methods. After FACS analysis, cells are gated and aggregates and dead cells are removed. For live cell populations, CD8α and CD4 cells were gated separately and each population was assessed for proliferation (CFSElow population) or activation (CD25-high/FoxP3 low/medium: CD25-high/FoxP3) as described previously. low/intermediate).

[Example 5. T resitope peptide suppresses proliferation of CD8+ effector T cells]

The potential inhibition of CD8+ T cell responses by T regitope peptides is tested when PBMC from healthy donors are stimulated with a CEF peptide mixture. T-regitopes strongly inhibit CD8+ T cell proliferative responses to CEF peptides as well as CD8+ cell activation. The percentage of proliferating CD8+ T cells (CFSE low) and the percentage of activated CD8+ T effector cells (CD25hi FoxP3int/lo) decrease with increasing concentration of T-regitope, indicating that T-regitope has a suppressive effect on the CD8+ T-cell population. will also be demonstrated. In both cases, the T-regitope strongly suppresses the response in a dose-dependent manner.

(7) Preparation of T-resitope-blood component conjugate

The linkage between T-regitope and blood components (such as albumin) is useful as a carrier protein for the T-regitope payload. The T-regitope-blood component conjugates of the present disclosure extend the half-life of the T-regitope in vivo, protect the T-regitope from rapid proteolysis, and allow the T-regitope to be rapidly cleared from the circulation and/or rapidly renal. It protects against excretion, allows wide distribution of the T-regitope-blood component conjugate throughout the subject's body , aids in the delivery of T-regitope to appropriate immune cells (such as macrophages and and/or assist in the presentation of T-regitopes as antigens by said immune cells.

T-regitope-blood component conjugates are formed by modifying T -regitope peptides by adding reactive sites to the T-regitope peptide to create modified T-regitope peptides, and then as described in U.S. Pat. No. 849,714, U.S. Pat. No. 6,887,470, U.S. Pat. No. 7,256,253, and U.S. Pat. No. 7,307,148 . A bond is formed between the reactive site and the reactive functional group on the blood component. Albumin is a preferred blood component because it contains an Fc neonatal binding domain that transports the T-regitope-albumin conjugate to appropriate cells such as macrophages and APCs. Additionally, albumin contains cysteine at amino acid 34 (Cys34), the amino acid position in the amino acid sequence of human serine albumin, and contains a free thiol with a pKa of about 5, which can serve as the preferred reactive functional group of albumin. Cys34 of albumin can form a stable thioester bond with maleimidopropionamide (MPA), which is a preferred reactive site for modified T resitope peptides. The stable thioester bond between albumin and the MPA-modified T resitope peptide is not cleaved under physiological conditions.

The T-regitope peptide is preferably selected from SEQ ID NOs: 1-55. One or more lysines may be present on the N-terminus of the T-regitope peptide, for example added on the N-terminus of a peptide selected from SEQ ID NOs: 1-55. A linker, such as a polyethylene glycol linker (eg, PEG2 or PEG12), is present between the one or more lysines and the T-regitope sequence, or at the N-terminus of the T-regitope sequence. In embodiments, a lysosomal cleavage site, such as a cathepsin B site (a cleavage site optionally consisting of valine and citrulline (sequentially from N-terminus to C-terminus)) is located between the PEG2 moiety and the T resitope sequence, and/or at one or more It exists between the T resitopes. In embodiments, one or more T regitopes may be present on the construct, optionally closer to the C-terminus than the linker. In embodiments, one or more lysosomal cleavage sites are present between multiple T-regitopes (e.g., such that a single lysosomal cleavage site separates two T-regitopes, or one lysosomal cleavage site separates two T-regitopes). one and a second T regitope, such that another lysosomal cleavage site exists between the second and third T regitope, etc.). In embodiments, one or more antigenic peptides associated with immunogenicity in diabetes ("T1Dgen" peptides; e.g., PPI-derived peptides) may be present on the construct, optionally separated by one or more lysosomal cleavage sites. Separated from multiple T resitopes. In embodiments, one or more lysosomal cleavage sites are present between multiple T1Dgen peptides (e.g., such that a single lysosomal cleavage site separates two T1Dgen peptides, or one lysosomal cleavage site separates a second T1Dgen peptide). one and a second T1Dgen peptide, such that another lysosomal cleavage site exists between the second and third T1Dgen peptide, etc.). Maleimide-based chemistry may be used to covalently couple the modified T resitope peptide to a blood component, preferably serum albumin, in a 1:1 molar ratio. Linking modified T-regitope peptides to blood components can be performed in vivo or ex vivo.

Cathepsin B is the first reported member of the lysosomal cysteine protease family. Cathepsin B has both endopeptidase and exopeptidase activities, with the latter acting as a peptidyl dipeptidase. Cathepsin B may be incorporated into the T-regitope peptide design to facilitate proper cleavage of the T-regitope from albumin when it resides in the lysosomal compartment within antigen-presenting cells. Valine-citrulline is a cleavage site for cathepsin B and has previously been successfully used and FDA approved in antibody drug conjugates (eg, the monomethylauristatin E (MMAE) conjugate of brentuximab vedotin). The significance of incorporating this site is to provide a cleavage site that properly cleaves the T-regitope from human serum albumin for efficient MHC class II presentation after entering the APC.

[Example 6. Preparation of T resitope-albumin conjugate by ex vivo conjugation]

Standard Fmoc (9-fluorenylmethyloxycarbonyl group) solid phase peptide synthesis chemistry is used for peptide synthesis. Synthesis is performed on an Intavis™ MultiPep™ automated peptide synthesizer. Amino acids are added stepwise (from C-terminus to N-terminus, from right to left) to the growing peptide chain while bound to an insoluble polystyrene resin. Amino acid building blocks with the amino terminus protected with an Fmoc group can be synthesized with one or more condensing reagents (e.g., hexafluorophosphate azabenzotriazole tetramethyluronium (HATU), O-( 1H-6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethylammonium hexafluorophosphate (HCTU)) is coupled to the growing chain. Reaction byproducts from each addition are removed by solvent washes (6 times , dimethylformamide (DMF)). After each coupling and capping step, the Fmoc was removed by piperidine deprotection of the peptide resin (performed twice ; 20% vol/vol 0.1 M HOBt in DMF to suppress Asp dehydration) and the resin was dissolved in DMF for 6 Wash twice and add the next amino acid. A cathepsin B cleavage site is incorporated into the N-terminus of the T regitope sequence.

For the PEG2 construct (“PEG2” or “P2”), after the desired T resitope peptide is completed, a PEG2 moiety is added to the N-terminus, followed by 4 lysines to the N-terminus. PEG2 and lysine are incorporated to provide a docking region for cathepsin B. Additionally, PEG2 and lysine (via the primary amine on the lysine side chain) are expected to increase the solubility of the final construct. The composition of the PEG2 construct is shown in Table 1 (bottom).

For the PEG12 construct (“PEG12” or “P12”), two PEG6s are added after T resitope peptide synthesis. In this case, no lysine is added. By increasing the PEG length, a docking region for cathepsin B is secured and the solubility of the T regitope is improved. The composition of the PEG2 construct is shown in Table 2 (bottom).

A small amount of the peptide construct was then removed from the resin and treated with trifluoroacetic acid (TFA, 92.5% v/v) in the presence of TIS (triisopropylsilane, 5%) and water (2.5%). , cleave and deprotect the peptide sample by eliminating side chain protecting groups. Each crude linear peptide (approximately 3-5 mg) is purified by preparative reverse phase HPLC (Gilson) using a 20 mm x 50 mm, 5 μm, hydrosphere column (YMC C18 .5 μm ). Peptides were purified to greater than 90% purity (determined by analytical HPLC) and mass confirmed using an ABI-SCIEX QSTAR XL Pro Qo-TOF mass spectrometer prior to cathepsin B evaluation. The remaining peptides (PEG2-T regitope and PEG12-T regitope) are left on the resin for later addition of 3-maleimidopropionic acid (MPA).

Recombinant human cathepsin B (R&D Systems™ catalog 953-CY) is used to assess cleavage of the Val-Cit site incorporated into the T regitope peptide. The activity measurement protocol follows the recommendations of R&D Systems™ and is used with final measurement conditions of 0.01 μg rh cathepsin B and 10 μM peptide substrate. After incubating the purified peptide with cathepsin B (15 min at RT), Qstar XL
Peptides are evaluated by mass spec using Pro™. Cleavage of the PEG2 peptide was not successful, and it is determined that further changes in the cathepsin B protocol did not result in successful cleavage. For the PEG12 formulation, successful cleavage is demonstrated.

After assessing cleavage of the Val-Cit site by cathepsin B, a reactive site of 3-maleimidopropionic acid (MPA) is added to the N-terminus of the PEG2 and PEG12 peptides. Similar to amino acid building blocks, MPA is protected with an Fmoc group and attached to the growing chain after activation of the carboxylic acid terminus. The final MPA-T regitope construct was removed from the resin and the peptide sample was washed with trifluoroacetic acid (TFA, 92.5%) in the presence of TIS (triisopropylsilane, 5%) and water (2.5%). cleavage and deprotection by treatment with v/v). Each crude linear peptide (approximately 20 mg) is purified by preparative reverse phase HPLC (Gilson) using a 20 mm x 50 mm, 5 μm, hydrosphere column (YMC C18 .5 μm ). MPA peptides are purified to >90% purity (determined by analytical HPLC) and mass confirmed on an ABI-SCIEX QSTAR XL Pro™ Qo-TOF mass spectrometer (data not shown). A total of 15 mg of MPA-P2 and MPA-P12T regitopes are used for conjugation to rHSA (Albucult-Novozyme™) to finally form HSA-T regitope conjugates.

Ellman's reagent (5,5'-dithio-bis-[2-nitrobenzoic acid]) is used to estimate sulfhydryl groups in a sample by comparison with a standard curve of sulfhydryl group-containing compounds such as cysteine. Ru. The Ellman test is performed at multiple concentrations of rHSA (Sigma™, Albucult®) to ensure accuracy of the analysis. Ellman's Reagent (Sigma™), Sigma™ Lot RF-009 rHSA, is evaluated for free cysteine that may be available for conjugation with maleimide. As shown in Table 3 (bottom), it is estimated that 78% of rHSA contains free cysteine.

Peptides are solubilized in dH20, rHSA is added (15 mg/mL) and 100 mM phosphate buffer is added to bring the final pH to 8. Peptides are added in a 10-fold molar excess over HSA. The peptide/HSA is incubated for 2 hours at room temperature and then for approximately 24-30 hours at 4°C.

After the conjugation step, the HSA-conjugate is first dialyzed into PBS (pH 7.0) for 2 hours at room temperature and then replaced twice with fresh PBS for 18-24 hours at 4°C. This process removes excess peptide from the HSA and HSA-T resitope conjugate preparations.

Ellman's test is performed on each conjugate to demonstrate conjugation of the peptide through the rHSA free cysteine and determine the efficiency of conjugation in the reaction. HSA-conjugation preparations do not remove the reduced form of HSA (mercaptamine) inherent in existing preparations (approximately 22% of HAS before conjugation). The remaining unreacted HSA was determined to be 14% for the HSA-MPA_P2-T regitope construct, meaning 14% free cysteine remained after conjugation with maleimide-T regitope. Therefore, approximately 64% of the total rHSA preparation is reacted with the MPA_P2-T resitope peptide.

Additional T-regitope-albumin conjugates include those in FIGS. 1 and 2.

(8) Method for evaluating the influence of T resitope-blood component conjugate on immune cells

Maleimide-based chemistry may be utilized to covalently attach the T-regitope payload to recombinant HSA (rHSA) in a 1:1 stoichiometry. Maleimidopropionamide (MPA) forms a stable thiol ester bond with free Cys34 of HSA. HSA exploits the neonatal receptor (FcRn) recycling pathway, potentially increasing the half-life of bound payloads and reducing the need for repeated administration. It is also known that rHSA delivers the conjugated payload to lymph nodes where it is endocytosed by FcRn-expressing dendritic cells and other antigen-presenting cells.

EpiVax designed rHSA-T regitope conjugates that contain a cleavage site between the T regitopes. This cleavage site is specific for early endosomal proteases, which liberates the T resitope from the rHSA molecule and increases the efficiency of MHC class II presentation at the cell surface. The long history and proven methodology of this FDA-approved rHSA conjugation chemistry and its successful manufacturing history support its selection for the delivery of our T1D payload.

Once the T-regitope-blood component conjugate is formed, the T-regitope-blood component conjugate and the T-regitope peptide alone, for example, as described in Example 6 and Example (7), and in the detailed description. For example, the effectiveness in suppressing effector T cells and activating regulatory T cells and their proliferation can be evaluated. Additionally, T-regitope-blood component conjugates are evaluated for their ability to induce immune tolerance to specific antigens.

[Example 7. Evaluation of the inhibitory effect of T-resitope-albumin delivery vehicle]

To investigate the inhibitory effects of T-regitope delivery vehicles, healthy donor PBMCs were used in a tetanus toxoid bystander inhibition assay (TTBSA) to determine CD4 T cell proliferation, T cell activation, T effector and regulatory T cell frequencies. and determine the Treg/Teff ratio as shown in Figure 3.

Therefore, in order to examine the optimal combination of T-regitopes for clinical application, we will analyze the effects of combinations of T-regitopes that synergistically suppress effector T cell responses in vitro. To facilitate these comparisons, we developed a high-throughput in vitro assay using human donor peripheral blood mononuclear cells (PBMC). This assay, called the Tetanus Toxoid Bystander Suppression Assay (TTBSA), exploits the ability of Tregs to suppress tetanus-specific T memory cells induced in people with a history of tetanus toxoid (TT) vaccination. It is.

PBMCs are incubated on day 0 and stained with carboxyfluorescein succinimide ester (CFSE) dye. On day 1, culture medium, tetanus toxoid, and 8 μg/mL, 6 μg/mL, 24 μg/mL T resitope, or 10 μg/mL, 40 μg/mL, 100 μg/mL T resitope-albumin conjugate were added to the cells. stimulate. Tetanus toxoid is used at a final concentration of 0.5 μg/mL, and the concentration is methodically titrated and optimized to determine its ability to inhibit T-regitopes. A negative control containing only medium is also included. On day 7, add L/D cell population marker, extracellular stain, and intracellular stain. On the 8th day, a readout is performed. Cell sorting assays for analysis of activation markers (eg CFSE, CD25) and cell population markers (eg L/D, CD2c, CD4, FoxP3) are performed.

Incubation of TT with donor PBMC stimulates the expansion (proliferation) of T effector cells. T resitopes are added to PBMCs in vitro together with TT to activate CD25-high FoxP3-high regulatory T cells and suppress the proliferation of TT-specific T effector cells. T-regitopes significantly inhibit proliferation (measured by CFSE dilution) and activation (measured by CD25 expression) of CD4+ T effector cells in a dose-dependent manner, and slightly proliferate Tregs (CD25+/FoxP3+/CD127-low); This is suggested by the increased ratio of Treg/Teff cells. The reduction in effector T cell proliferation is a direct result of activation of regulatory T cells and/or conversion of TT-specific T effectors to Tregs, as supported by the induction of Tregs in vivo, for example. .

Using TTBSA, we will examine the possibility of suppressing CD4+ T cell proliferation using each of the many available T regitopes individually or in pairwise combinations. The most promising IgG-T regitope peptides are selected for further testing. One particular T-regitope, T-regitope 289 (SEQ ID NO: 1), is the single T-regitope with the most inhibitory activity in TTBSA compared to other single T-regitopes. By combining T-regitope 289 and T-regitope 084 (SEQ ID NO: 28), an even greater TT-specific T cell proliferation inhibitory effect is observed (see Figures 4A and B). Binding of T-regitope 289 and T-regitope 084 to rHSA improves efficacy in vitro (see Figures 5A and B).

Thus, using TTBSA, the HSA-T resitope conjugates of the present disclosure have been shown to inhibit CD4 T cell proliferation and activation and increase the ratio of Treg cells to Teff cells.

[Example 8. Evaluation of the efficacy of preformed conjugate HSA-T regitope therapeutics and maleimide-T regitope peptide therapeutics]

The effects of preformed HSA-T regitope conjugates and free maleimide-T regitope peptides on OVA immune responses are evaluated. The latter free maleimide peptide forms a conjugation in vivo with free Cys34 of the subject's endogenous HSA via the reactive maleimide group after injection. Using 5 mg of MPA-P2 and MPA-P12 as free-MPA-T resitopes, unconjugated HSA in the samples is accounted for by calculating the molar ratio of conjugated to unconjugated HSA.

Mice (female C57BL/6) are immunized with 50 mg ovalbumin (OVA) (subcutaneous injection) on day 0 (CFA) and day 14 (IFA). Pre-formed HSA conjugate treatments are co-administered with OVA in CFA on day 0. The test groups include OVA/HSA-P2-high and OCA/HSA-P2-low. One injection is 50 μg OVA, 800 μg HSA, and 825 μg HSA-P2H(high) conjugate ( approximately 20 μg T resitope). HSA-P2L(low) conjugate is 100 μg ( approximately 3.7 μg T regitope). The four control groups include PBS only, PBS/OVA, HSA/OVA, and T-regitope/OVA. The last group was included to evaluate the utility of free maleimide T resitope peptides, administered intravenously into the tail vein. Five mice are used per group.

Mice are sacrificed on day 17. At the time of sacrifice, blood from the heart and spleen are collected from each animal. IFNγ/IL2 fluorospot assay, IFNγ/IL17 fluorospot assay, CD4 T cell proliferation, and T cell characterization will be performed on OVA-stimulated splenocytes. PHA is used as a positive control stimulus for splenocyte assays. For PHA stimulation, all wells are confluent. Acceptance criteria are used that the SFC (spot-forming cells) after stimulation must be greater than 50 spots/10 ⁶ relative to the negative control (medium well) and must have a stimulation index greater than 2. In both the IFNγ/IL2 fluorospot assay and the IFNγ/IL17 fluorospot assay, treatment inhibits IFNγ production, with the HSA-only control group having less inhibition compared to the treated group.

For T cell proliferation and characterization assays, spleen cell samples were evaluated for induction of FoxP3 expression in TCR Tg cells and inhibition of OVA-specific T cell proliferation by CFSE dilution (response to OVA peptide in vitro). Ru. FoxP3+ Tre
To detect g, single cell suspensions of draining lymph nodes were incubated with 2.4G2 mAb (anti-CD16/32, ATCC) for 15 min to block FcR, followed by anti-CD3, It was stained with CD4, CD25, and anti-clonotype KJ1-26 at 4°C for 40 minutes. KJ1-26 is specific for the clonotyping TCR expressed in DO11.10 transgenic mice. Cells are then permeabilized, stained for FoxP3 nuclear expression, and subjected to FACS analysis on a Thermo Attune NxT Autosampler™. A CD4+ CD25+ FoxP3+ KJ1-26+ live cell gate population is established and the number and percentage of OVA-specific regulatory T cells compared to PBS or HSA alone is determined.

Antigen-specific T cell proliferation is assessed by CFSE dilution. Draining lymph nodes are harvested, stained with the cell proliferation dye CFSE, and single cell suspensions are prepared at 2x10 ⁶ cells/mL. Cells are added at 100 μL/well in 96-well plates in the presence of OVA323-339 (New England Peptide, Gardner, MA, USA) at a concentration of 10 μg/mL. Cells were stimulated for 72 hours and harvested for staining with CD3α, CD4, CD8, CD54RA, CCR7, CD25, CD127, IFNγ, HLA-II, CD69, CD154, IL-17, IL-21 for 40 minutes at 4°C. Ru. Cells are fixed, permeabilized, stained for FoxP3 expression, and analyzed by flow cytometry. OVA-specific KJ1-26+ CD4+ CD25+ FoxP3+ adaptive regulatory T cells are increased in mice treated with free maleimide-resitope and HSA-resitope conjugate compared to mice treated with rHSA. Free maleimide-T regitope and HSA-T regitope conjugate more effectively reduce OVA-specific proliferation of KJ1-26+ CD4+ T effector cells compared to rHSA alone.

Anti-OVA antibodies in serum from blood drawn on day 17 are assessed by ELISA, including serial dilution plots and standard ELISA to determine antibody concentration. Mice treated with HSA-conjugate and free maleimide have lower serum antibody titers compared to untreated, as shown by absorbance at different dilutions and comparison of absorbance on a standard curve.

(9) Method for evaluating the effectiveness of T resitope-blood component conjugates in the prevention and treatment of type 1 diabetes

The demonstration that T-regitopes induce antigen-specific tolerance to pancreatic islet antigens could fundamentally impact the field of diabetes treatment and obviate the need for insulin treatment in T1D; addition of HT-T1D Applications may include functional preservation after islet transplantation.

T-regitopes found in the Fc and variable domain frameworks of IgG bind multiple MHC class II molecules and are among the six natural TReg epitopes that suppress inflammatory responses to co-administered antigens (Ag) in vitro and in vivo. It is a gathering. Co-delivery of T-regitope and target antigen (Ag ) is the key to Ag specificity. The T resitopes contained in high-dose intravenous IgG therapy (IVIG) can activate CD4+CD25+FoxP3+ regulatory T cells, as seen in humans and mice. T resitopes may (partially) explain the efficacy of IVIG, which is widely used to treat autoimmune diseases. The mechanism of action of T-resitopes has been demonstrated in eight independent laboratories and in a wide range of models (Scott et al. (AI disease model), Najafian et al. (transplantation), Khoury and Elyaman (multiple sclerosis), Mingozzi and Hui, UniQure (inflammatory (intestinal diseases), Moingeon et al. (allergies)).

Early studies by EpiVax on T-regitopes pointed to the induction of antigen-specific tolerance to co-administered antigens. Published studies have shown that T regitope-specific natural TRegs (nTRegs) modulate T effector responses by inhibiting the activity of autoreactive effectors and/or by changing the Teff phenotype to aTRegs. It shows that. Based on this data, we hypothesized that when T-regitopes are processed and jointly presented with target Ags by the same APC, they activate a subset of circulating nTRegs and induce aTRegs. Ta. Recently, TReg transfer experiments in allergic models have reconfirmed the antigen specificity and efficacy of T regitope therapy.

T resitopes may provide a unifying mechanism for the disparate IVIG effects in mouse studies. The anti-inflammatory activity of IVIG has been attributed to blockade of Fc-γ receptors, neonatal Fc receptors (FcRn), or interaction with novel cell surface receptors DC-SIGN and DCIR. However, the induction of regulatory T cells by T regitopes better explains the IgG-induced T cell phenotypic changes and increase in aTReg after skin transplantation and IVIG treatment in EAE. T-regitopes also provide a new mechanism of action for IVIG in human autoimmune diseases. Engagement of TRegs by T-regitopes in IVIG may elucidate reports that IVIG induces expansion of TRegs in vivo and that IgG-derived peptides (similar to T-regitopes) have immunosuppressive effects.

T-regitopes have also significantly changed the field of monoclonal antibody (mAb) development. The presence of T regitopes in mAbs is associated with reduced immunogenicity in the clinic. T resitopes are eluted from Ag presenting cells pulsed with mAb. EpiVax has developed a risk assessment tool that predicts clinical immunogenicity of mAbs based on T-regitope adjustment scores .

HSA-T resitopes (HTs) have the potential to transform the treatment of numerous autoimmune diseases. Recombinant rHSA-T regitope pharmaceuticals can be produced on a large scale under GMP at Ajinomoto Co. (San Diego, Calif.).

By conjugating two highly effective T regitopes to a recombinant albumin delivery vehicle carrying a T1D targeting antigen (HT-T1D), we achieved more effective T1D antigen-specific tolerance than PPI antigen alone. We hypothesize that it has the potential to induce islet cell antigen-specific tolerance and halt islet cell destruction in mouse models of T1D.

In the HT-T1D therapy described in Example 9 below, T-regitopes SEQ ID NO: 1 and SEQ ID NO: 28 (as exemplary T-regitopes of this disclosure) are chemically coupled to rHSA with preproinsulin peptide (PPI) SEQ ID NO: 56. T-regitope-containing therapy will be administered before the onset of diabetes for dose-ranging studies and after the onset of diabetes for POC. Under the guidance of experienced immunologists and drug development experts, we validated the efficacy of HT-T1D treatment in an in vivo murine model under GLP conditions, scaled up production, and evaluated drug safety and toxicity. Apply established methods to support the shift to human treatment and prevention.

[Example 9. Antigen-specific tolerance by co-presentation with rHSA-T regitope conjugate protein in vivo]

Briefly, a recombinant rHSA-T regitope conjugate protein containing two T regitopes was administered to NOD mice in combination with a PPI peptide during the development of diabetes to test the effect of the combination therapy on diabetes progression. do. Diabetic animals are treated after confirmation of blood glucose levels of 200-350 mg/dl. It is expected that mice treated with rHSA with two T regitopes + PPI will be able to significantly control blood glucose over time compared to untreated or rHSA-treated control groups. The average blood glucose level from day 31 to day 49 is expected to be significantly lower in the rHSA group containing two T regitopes + PPI compared to the rHSA alone group. Severe diabetes (BG>600) or death is expected to occur less frequently or with a significant delay in the T resitope-conjugate-PPI drug therapy combination group peptides. Statistical differences in absolute BG values between groups are expected with a p value of less than 0.05. This study demonstrates that rHSA is an effective delivery vehicle for T-regitopes and that when co-administered with PPI peptides, the HT-T1D combination lowers blood glucose in NOD for up to >7 weeks.

[Example 10. Identification of optimal doses and treatment regimens for testing HT+T1D target antigens (PPIs) in in vivo models of T1D]

The dose of HT-T1D (including rHSA) tolerated by NOD mice is determined. Additionally, a determination will be made of which doses can prevent the development of diabetes in a two-dose regimen in the well-characterized NOD mouse model of spontaneous autoimmune diabetes. NOD mice are intolerant to human albumin, but fusion of the T resitope with rHSA in previous studies showed tolerance and significantly prevented the development of diabetes in T1D prevention and treatment studies. It turns out that it can be done.

Initially, the tolerability and initial efficacy of HT and HT-T1D conjugates will be tested in a four-arm pilot prophylaxis study in NOD lasting approximately 20-30 weeks. At week 8, NOD mice (female; 8 mice/group) were divided into four treatment groups, each group receiving control rHSA, HT (T regitope SEQ ID NO: 1 and 28) on days 0/1 and 14/15. (containing PPI peptide SEQ ID NO: 56) and HT-T1D (containing PPI peptide SEQ ID NO: 56). T-regitope is administered at 100 μg each for both HT and HT-T1D (200 μg total T-regitope). rHSA is administered in the same amount for the rHSA control group. Mice will be followed until 30 weeks to confirm the onset of diabetes. Cage-side observations are made daily, and non-fasting blood glucose levels, clinical status, and body weight are assessed twice weekly. Mice will be euthanized if their BG remains consistently above 600 (BG≧600 5 times), if they show excessive weight loss (more than 20% of their initial body weight), or if they appear pathological. Treatment groups include no treatment and rHSA control. Repeated injections of HT and HT-T1D are thought to be tolerated without significant side effects due to the presence of the TReg epitope, and both formulations are judged by the number of mice with a BG value of 250 mg/dL or less at the end of 30 weeks. It is expected to suppress the onset of diabetes.

We then performed multiple treatments of low (25 μg/T regitope; 50 μg total) and high doses (100 μg/T regitope; 200 μg total) of HT and HT-T1D in the same well-established prophylactic T1D model in NOD mice. A test will be conducted regarding. However, the parameters of this study have been modified according to the results of tolerability and initial efficacy studies (e.g., additional doses may be added or higher doses may not be well tolerated for NODs known to be responsive to rHSA). possible). From 8 weeks of age, NOD mice (female; 16 mice/group) were divided into 8 treatment groups, and each group was given 4 daily doses on days 0/1, 14/15, 28/29, and 42/43. receive an injection. Each group will be treated as indicated below and followed for the development of diabetes for 20 weeks (or more) . Conjugates containing T-regitopes are administered such that the lowest dose contains at least 25 μg of each T-regitope peptide, and rHSA is given as a negative control treatment. Blood sugar (BG) levels are measured and recorded twice a week for up to 20 weeks, and subjects are considered to have diabetes if their BG levels are 250 mg/dL or higher for two consecutive times. rHSA-PPI A23-C2 is compared to HT-T1D. In prevention studies, without treatment, at least 50% and up to 80% of NOD mice develop diabetes by 20 weeks. When administering HT-T1D in groups of 16 animals, we would expect to have 80% statistical power to observe a 50% difference (60% to 30% penetrance) in the NOD T1D prevention model. HT (rHSA-T resitope) administration is expected to suppress diabetes development in NOD mice compared to PBS or rHSA control-treated mice. Binding of T1D disease antigen PPI peptide to rHSA-T resitope conjugate (HT-T1D) is expected to control BG levels and the development of insulitis and significantly prevent the progression of diabetes. .

Overall, rHSA conjugate-T resitope with PPI peptide SEQ ID NO: 56 (HT-T1D) is expected to significantly prevent the development of diabetes at scalable doses and regimens in prevention studies in NOD mice.

[Example 11. Characterization of HT-T1D conjugate for scale-up and therapeutic research]

Large-scale manufacturing and characterization of research grade HT-T1D. Conjugate characterization included HPLC purification, percent conjugation evaluation, and HPLC in
May include validation of vitro peptide cleavage. We plan to demonstrate the efficacy of HT-T1D both in inducing adaptive tolerance in D011.10 mice and in preventing T1D in the NOD mouse model. Succeeded in producing 50g of GMP grade HT-T1D. Production of 50 g of GMP grade HT-T1D results.

[Example 12. Demonstration of the effect of HT-T1D conjugate in a human T1D treatment model]

HT-T1D is evaluated in a therapeutic in vivo study in diabetic NOD mice. The characteristics of HT-T1D as described above are tested in a therapeutic T1D model in NOD mice. NOD mice are a strain that spontaneously develops T1D and are selected because they recapitulate many features of human clinical disease and offer the possibility of testing HT-T1D in vivo. Once diabetes develops, NOD mice are assigned to the HT-T1D group or multiple control groups. Clinical parameters indicative of T1D progression (blood sugar levels, weight loss, mortality) are monitored over time as indicators of treatment effectiveness.

Starting at 9 weeks of age, a total of 246 NOD/ShiltJ female mice will be monitored for enrollment in the HT-T1D treatment study. Blood glucose levels are measured twice a week, and mice with blood sugar (BG) levels between 200 and 350 mg/dL are retested the next day. If BG values in this range are observed for two consecutive days, mice are immediately enrolled in the study and assigned to study groups in a rolling manner until each group contains 14 mice. Mice not used for testing will be euthanized. Mice enrolled in the study receive divided doses of the test substance (dosage determined as above) by subcutaneous injection on days 0/1, 14/15, 28/29, 42/43. The eight treatment groups included: untreated (PBS); rHSA; T-regitope peptide (SEQ ID NO: 1 and 28); rHSA-T-regitope (“HT”; T-regitope peptide SEQ ID NO: 1 and 28); rHSA-PPI (T-regitope peptide SEQ ID NO: 1 and 28; PPI peptide SEQ ID NO: 56); rHSA-Scrambled T-regitope-PPI (T-regitope peptide scrambled from SEQ ID NO: 1 and 28; PPI peptide SEQ ID NO: 56); and rHSA-T Regitope-PPI (“HT-T1D”; T resitope peptide SEQ ID NO: 1 and 28; PPI peptide SEQ ID NO: 56).

Clinical observations and body weight measurements are performed on a weekly basis. For injections given after study day 0, cageside observations will be made at 1, 2, and 4 hours post-injection to check for signs of treatment toxicity. Blood sugar levels and body weight are measured twice a week. Mice with hunched back, fluff, or weight loss of 20% or more are euthanized. For euthanized animals, blood is collected by cardiac puncture and PBMCs are isolated. Cells were labeled with antibodies against phenotypic and activation state markers (CD3, CD4, CD8, CD62L, CD45RA, CD25, CD127, FoxP3, IFNγ, CD69, IL-17 and IL-21) and fixed according to SOPs. Analyze with flow cytometer to determine CD8 and CD4 memory, effector and regulatory T cell status. The concentration of peptide C in serum is measured by ELISA method. Pancreases are harvested and fixed for later histological examination for lymphocytic infiltration and the presence of TRegs. The study will continue for 60 days after the start of dosing. At the end of the study, the remaining animals are sacrificed and tissue samples are collected for pathology.

In therapeutic trials, 100% of enrolled NOD mice develop diabetes in the absence of treatment. Treating 14 mice per group with HT-T1D provides a statistical power of 76.5% to observe a 50% difference (from 60% to 30% penetrance) in the NOD mouse model of treatment study. It is expected that there will be. For time series analysis, all available mouse BG measurements are aggregated. Mice that reach a test endpoint not defined by BG (molibund) are assigned the maximum BG level (600). Differences between groups and temporal changes in BG measurements at each time point were determined using (i) t test, (ii) Mann-Whitney U test, and (iii) nonparametric difference test using 1000 permutations. Evaluate using Comparisons of absolute BG values between two groups at each time point are considered statistically significant at p-value <0.05. Treatment with HT-T1D is expected to delay or reverse the onset of diabetes in NOD mice compared to PBS- or rHSA-treated control mice.

[Example 13. Evaluation of biodistribution, pharmacokinetics, and immunotoxicity of HT-T1D]

Immunotoxicity tests will be performed to confirm the antigen specificity and target immunomodulatory effects of HT-T1D. Evaluate the pharmacokinetics (PK) of HT-T1D using hFCRn/alb-/-/scid transgenic mice.

In vivo of rHSA-T1D labeled with IR or NIR dyes after administration by different routes (subcutaneous (SQ), intravenous (IV), intraperitoneal (IP), or intramuscular (IM)) into C57BL/6 mice. Evaluate the distribution. Mice are imaged with a LiCor Odyssey CLx infrared imaging system at different time points after injection. There are 6 mice per group for different injection routes. Labeled HT-T1D drugs are injected into mice by SC, IP, IV, IM, and the distribution at times 0, 0.5 h, 1 h, 3 h, 8 h, 24 h, and 48 h is analyzed. It was found that HT-T1D was mainly distributed in lymph nodes when administered subcutaneously, and distributed to the liver when administered IP, IV, or IM. SC administration is optimal.

In anticipation of conducting studies in humans, pharmacokinetic (PK) studies using human transgenic mice will be performed to define the effect of human FcRn binding on HT-T1D plasma levels. hFCRn/alb-/scid (JAX cat. No. 031644) is a mouse that has a knockout allele of the FcRnα chain, expresses the human FcRnα chain transgene under the control of the human FcRn promoter, and is albumin-deficient and immunodeficient. It is. These mice are a suitable model to test binding of HT-T1D conjugates to FcRn without competition with endogenous mouse serum albumin. The immunodeficiency prevents the mice from producing any anti-drug antibodies as these mice are intolerant to mouse and human serum albumin.

Mice, 8-10 weeks old, are assigned to two groups: 15 male mice and 15 female mice. Each group received a low dose (25 μg) or a high dose (25 μg) of HT-T1D SCs in PBS.
100 μg) can be received in the groin fold. On days 1, 4, 10, 14, and 21, 0.4 mL of whole blood from 3 males and 3 females per group was added to EDTA and 0.4 mL of whole blood was added to citrate anticoagulant. Collect (0.8 mL total of whole blood via cardiac puncture). The collected blood is centrifuged, plasma is collected and stored at -70°C, and the change in HT-T1D concentration is measured. This ELISA is performed using antibodies raised against rHSA. HT-T1D is expected to have an extended half-life in the serum of hFCRn/alb-/-/scid mice measured after SQ injection. These mice are the best model for rHSA half-life in humans.

The potential fulminant immunomodulatory effects of T resitope-induced adaptive tolerance on systemic immune responses to other antigens (immunotoxicity) were assessed by measuring the effects of HT on T cell and antibody responses to influenza vaccination. be done. To demonstrate the effect of HT on co-administered antigen in an in vivo mouse model, an OVA immunogenicity assay was developed in C57BL/6 mice in which the response to OVA antigen immunization was down-modulated by co-administration with HT. ing. To address whether HT-T1D immunotherapy reduces immune responses to other unrelated antigens, we investigated well-characterized murine memory T and B cell responses to the Fluzone influenza vaccine in C57BL/6 female mice in a three-arm study. Tests are conducted on its effect on. Arm A: Adjuvant (Montanide); Arm B: TFP in adjuvant (starting on day 21); Arm C: OVA in adjuvant (starting on day 21); Arm D: OVA+TFP in adjuvant (starting on day 21); Arm E: Fluzone in adjuvant (days 1 and 14 immunizations); Arm F: Fluzone + TFP in adjuvant (days 1 and 14 immunizations); Arm G: Fluzone in adjuvant (days 1 and 14 immunizations); Day 21 immunization), TFP in adjuvant (Day 21 immunization, opposite flank; Arm H: Fluzone + TFP in adjuvant (Day 21 immunization); Fluzone + TFP in adjuvant (Day 1 immunization) and 14 days), OVA in adjuvant (immunization day 21, contralateral flank); Arm I: fluzone in adjuvant (immunization days 1 and 14), OVA + TFP in adjuvant (immunization day 21); Day, Opposite Frank).

Fluzone (5 μg for children) is injected subcutaneously with adjuvant in the groin crease on days 1 and 14 (influenza vaccination). On day 21 , OVA (50 μg) in adjuvant is injected in the corresponding arm with or without HT. Animals are sacrificed on day 28 and spleens and serum are collected. Antibodies to OVA and influenza (Fluzone) are measured by ELISA, and T cell responses to OVA and influenza HA are evaluated as in Example 8. HT-T1D co-administered with OVA is expected to specifically downmodulate the response to this Ag while not affecting the memory immune response to fluzone. Furthermore, HT-T1D conjugates are expected to exhibit improved stability, extended half-life, and improved immune specificity of HT conjugates when co-administered with antigen.

[Example 14. Evaluation of HT-T1D conjugates in standard toxicity and safety tests]

Standard toxicity and safety testing will be performed, including genotoxicity testing, single-dose and repeated-dose toxicity testing, and standard safety testing. These studies will be conducted using the HT-T1D formulation to provide additional information for preclinical evaluation for Phase I clinical trials in humans.

In the pilot study, hFCRn/alb-/-/scid mice were divided into five groups, two males and two females. Administration is by a single dose of HT-T1D (subcutaneous injection), with each group receiving an increased dose based on the response of the previous group after 1 hour of observation. After administration, observe for 5 days.

For acute toxicity studies, hFCRn/alb-/-/scid mice are allocated into 5 groups of 6 males and 6 females (2 control + 3 treated). Administration is a single subcutaneous injection in PBS, followed by observation for 14 days. Monitor clinical symptoms daily and weight weekly. Hematology, coagulation, and blood chemistry tests are performed on all sacrificed animals. At the end of the study or in the event of unexpected death or abnormality, all tissues from each animal will be collected and cryopreserved.

For regulatory toxicity studies, hFCRn/alb-/-/scid mice were divided into 5 groups of 6 males and 6 females (2 control + 3 treated). Treatment is by subcutaneous injection administration of PBS once a week for two consecutive weeks (days 1 and 8). Clinical symptoms will be monitored daily. Body weight will be assessed at randomization, pre-treatment, and at termination. Macroscopic examination of all mice is performed on day 15. After euthanasia, select organ weights (pancreas, liver, lungs, heart, kidneys) are recorded for all mice.

For the 28-day toxicity study, hFCRn/alb-/-/scid mice were assigned to 4 groups of 9 males and 9 females and treated with PBS once weekly for 4 weeks (days 1, 8, 15 and 21). Treat with HT-T1D by subcutaneous injection administration . The test animals (6 males and 6 females in each group) are observed for an additional 7 days and 28 days. Convalescent animals (3 males and 3 females per group) are expected to survive until day 49. Clinical symptoms will be assessed daily. Body weight is measured before dosing and weekly during and at the end of dosing. Food intake is measured weekly. Blood samples are centrifuged and plasma/serum collected and stored at -70°C. For toxicokinetic animals, euthanize after the last hemorrhage without further investigation. All primary and convalescent mouse hematology, coagulation, blood chemistry, and urinalysis tests will be performed at termination. The bone marrow, pancreas, liver, lungs, heart and kidneys of all main study mice (control and high dose) will be harvested, weighed and preserved in fixative for histological examination.

In therapeutic T1D POC trials, minimal or no toxicity of HT-T1D is expected at the highest doses that significantly prevent diabetes. T-regitope and PPI are peptide sequences present in endogenous proteins and are therefore not expected to be toxic in either mice or humans. The safety and absence of acute or sustained toxicity of HT-T1D therapeutics will be demonstrated.

[Equivalent]

Although the invention has been described in relation to particular embodiments thereof, it will be understood that further modifications are possible. Furthermore, this application does not cover any modifications, uses or intended for adaptation.

Claims (39)

A T regitope-blood component conjugate comprising a blood component linked to a modified polypeptide, said modified polypeptide having a reactive site attached thereto, and wherein said modified polypeptide has one or more regulatory T regitope-blood component conjugates. T-regitope-blood component conjugates containing cellular epitopes. The one or more regulatory T cell epitopes are one or more amino acid sequences selected from the group consisting of SEQ ID NO: 1-55 , and/or fragments and variants thereof , and optionally SEQ ID NO: 1. The T-resitope-blood component conjugate of claim 1, consisting only of 1 to 12 additional amino acids distributed in arbitrary proportions on the N-terminus and/or C-terminus of the ~55 polypeptide. The one or more regulatory T cell epitopes are one or more amino acid sequences selected from the group consisting of SEQ ID NO: 1-55 , and/or fragments and variants thereof , and optionally SEQ ID NO: 1. The T-regitope-blood component conjugate of claim 1, consisting essentially of 1 to 12 additional amino acids distributed in any proportion on the N-terminus and/or C-terminus of the ~55 polypeptide. The one or more regulatory T cell epitopes are one or more amino acid sequences selected from the group consisting of SEQ ID NO: 1-55 , and/or fragments and variants thereof , and optionally SEQ ID NO: 1. 2. The T-resitope-blood component conjugate of claim 1, comprising 1 to 12 additional amino acids distributed in any proportion on the N-terminus and/or C-terminus of the ~55 polypeptide. 2. The T regitope-blood component conjugate of claim 1, wherein the one or more regulatory T cell epitopes comprises SEQ ID NO:1. 28. The T regitope-blood component conjugate of claim 1, wherein the one or more regulatory T cell epitopes comprises SEQ ID NO:28. T-regitope-blood component conjugate according to any one of claims 1 to 6, wherein the blood component is albumin. T-regitope-blood component conjugate according to any one of claims 1 to 6, wherein the blood component is human serum albumin. The T regitope-blood component conjugate according to any one of claims 1 to 8, wherein the modified polypeptide further comprises a T1Dgen peptide. 10. The T1Dgen peptide of claim 9, wherein the T1Dgen peptide comprises one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 56-63. T-regitope-blood component conjugate according to any one of claims 1 to 10, wherein the reactive site is attached to the amino terminal amino acid of the modified polypeptide. T-regitope-blood component conjugate according to any one of claims 1 to 10, wherein the reactive site is attached to the carboxy-terminal amino acid of the modified polypeptide. The T-regitope-blood component conjugate according to any one of claims 1 to 10, wherein the reactive site is attached to an amino acid located between the amino-terminal amino acid and the carboxy-terminal amino acid of the modified polypeptide. The T resitope-blood component conjugate according to any one of claims 1 to 13, wherein the reactive moiety is a succinimidyl group or a maleimide group. T-resitope-blood component conjugate according to any one of claims 1 to 14, wherein the reactive moiety is a 3-maleimidopropionic acid moiety. T-regitope-blood component conjugate according to any one of claims 1 to 15, wherein the conjugation between the blood component and the modified polypeptide is a maleimide bond. A modified polypeptide, said modified polypeptide having a reactive site attached thereto, said modified polypeptide comprising one or more regulatory T cell epitopes. The one or more regulatory T cell epitopes are one or more amino acid sequences selected from the group consisting of SEQ ID NO: 1-55 , and/or fragments and variants thereof , and optionally SEQ ID NO: 1. 18. The modified polypeptide according to claim 17, consisting only of 1 to 12 additional amino acids distributed in arbitrary proportions on the N-terminus and/or C-terminus of the ~55 polypeptide. The one or more regulatory T cell epitopes are one or more amino acid sequences selected from the group consisting of SEQ ID NO: 1-55 , and/or fragments and variants thereof , and optionally SEQ ID NO: 1. 18. The modified polypeptide of claim 17, consisting essentially of 1 to 12 additional amino acids distributed in any proportion on the N-terminus and/or C-terminus of the polypeptide. The one or more regulatory T cell epitopes are one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1-55 , and/or fragments and variants thereof , and optionally SEQ ID NOs: 1-55. 18. Modified polypeptide according to claim 17, comprising 1 to 12 additional amino acids distributed in any proportion on the N-terminus and/or C-terminus of the 55 polypeptide. 18. The modified polypeptide of claim 17, wherein the one or more regulatory T cell epitopes comprises SEQ ID NO:1. 18. The modified polypeptide of claim 17, wherein the one or more regulatory T cell epitopes comprises SEQ ID NO:28. The modified polypeptide according to any one of claims 17 to 22, wherein the modified polypeptide further comprises a T1Dgen peptide. 24. The modified polypeptide of claim 23, wherein the T1Dgen peptide comprises one or more amino sequences selected from the group consisting of SEQ ID NOs: 56-63. A modified polypeptide according to any one of claims 17 to 24, wherein the reactive site is attached to the amino terminal amino acid of the modified polypeptide. A modified polypeptide according to any one of claims 17 to 24, wherein the reactive site is attached to the carboxy-terminal amino acid of the modified polypeptide. 25. A modified polypeptide according to any one of claims 17 to 24, wherein the reactive site is attached to an amino acid located between the amino terminal amino acid and the carboxy terminal amino acid of the modified polypeptide. The modified polypeptide according to any one of claims 17 to 27, wherein the reactive site is a succinimidyl group or a maleimide group. The modified polypeptide according to any one of claims 17 to 28, wherein the reactive site is a 3-maleimidopropionic acid site. 17. A method for suppressing an autoimmune reaction characteristic of T1D in a subject in need thereof, the method comprising administering to the subject the T-regitope-blood component conjugate according to any one of claims 1 to 16. The method described above, comprising the steps of: A method for suppressing an autoimmune response characteristic of T1D in a subject in need thereof, the method comprising the step of administering to the subject the modified polypeptide according to any one of claims 17 to 29. . 32. The method of any one of claims 30-31, wherein the administration shifts one or more antigen presenting cells to a regulatory phenotype. 32. The method of any one of claims 30-31, wherein the administration shifts one or more dendritic cells to a regulatory phenotype. 32. The method of any one of claims 30-31, wherein the regulatory phenotype is characterized by decreased expression of CD11c and HLA-DR in dendritic cells or other antigen presenting cells. 32. The method of any one of claims 30 to 31, wherein the administration of a regulatory T cell epitope shifts one or more T cells to a regulatory phenotype. 32. The method of any one of claims 30-31, wherein CD4+/CD25+/FoxP3+ regulatory T cells are activated by administration of one or more regulatory T cell epitopes. The administration produces an immune response selected from the group consisting of an innate immune response, an adaptive immune response, an effector T cell response, a memory T cell response, a helper T cell response, a B cell response, an NKT cell response, or any combination thereof. 32. A method according to any one of claims 30 to 31, wherein the method inhibits. A pharmaceutical composition comprising a T-resitope-blood component conjugate according to any one of claims 1 to 16 and a carrier, excipient and/or adjuvant. A pharmaceutical composition comprising a modified polypeptide capable of forming a T-regitope-blood component conjugate according to any one of claims 17 to 29 and a carrier, excipient and/or adjuvant.