Major histocompatibility complex peptides as therapeutic targets for autoimmune diseases

Abstract

The present invention relates, in part, to binding agents that specifically bind peptides associated with T cell-mediated disease, as well as related compositions and methods. These binding agents include those that specifically bind the peptide when bound to an MHC molecule, such as a MHC Class I and / or Class II molecule, and / or when a free peptide. In some embodiments, such binding agents do not bind the peptide epitope when comprised in a native protein. The binding agents provided herein include antibodies and antigen-binding fragments thereof as well as alternatives thereof. Methods of producing and / or selecting the binding agents are also provided.

Description

[0001] MAJOR HISTOCOMPATIBILITY COMPLEX PEPTIDES AS THERAPEUTIC TARGETS FOR AUTOIMMUNE DISEASES

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit under 5 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63 / 685,062 filed August 20, 2024, the entire contents of which are hereby incorporated by reference herein for all purposes.

[0004] FIELD OF THE INVENTION

[0005] The present invention relates, in part, to binding agents that specifically bind peptides associated with a T cell-mediated disease, such as autoimmune disease and cancer, as well as related compositions and methods. These binding agents include those that specifically bind peptides when bound to an MHC molecule (e.g., a MHC Class I and / or MHC Class II molecule) and / or when in the form of a free peptide. In some embodiments, such binding agents do not bind the peptide epitope when comprised in a native protein. The binding agents provided herein include antibodies and antigen-binding fragments thereof as well as antibody alternatives, such as RNA aptamers. Methods of producing and / or selecting the binding agents are also provided as are methods of inhibiting T cell binding, such as through administration, such as in the treatment of a T cell-mediated disease.

[0006] SUMMARY OF THE INVENTION

[0007] The specificity of each different T cell-mediated disease, such as autoimmune disease, is defined by its own unique peptide or set of peptides that is / are presented via the major histocompatibility complex (MHC) on the cell surface. Damage can occur after a T cell attaches via its receptor (TCR) to a peptide in a binding event. Type I diabetes is an example of how that mechanism can work to destroy pancreatic beta cells resulting in dramatically reduced insulin levels that define this disease and its symptoms. Similarly, there are disease processes where regulatory T cell activity is not advantageous, such as in cancer. Attachment of a binding agent, such as an antibody, to the MHC resident peptide mimics TCR binding to this same peptide occupied site and, thus, can block problematic interactions. The methods and compositions provided herein may be used for the inhibition of regulatory T cells, and such methods and compositions are provided herein in some aspects. To block these undesirable interactions, the binding agents provided herein can specifically bind the peptide when the peptide is in a complex with a MHC molecule, such as a Class I MHC or Class II MHC molecule, and / or as free peptide. As an example, antipeptide monoclonal antibodies were obtained against the insulin B 15-23 epitope peptide LYLVCGERG (SEQ ID NO: 1). T cells attach to the MHC -bound peptide on insulinproducing beta cells, eliminate them and thereby cause diabetes in both non-obese diabetic (NOD) mice and humans. Importantly, ELISA and native PAGE studies showed that the generated antibodies react exclusively with free peptide and did not cross-react with that same epitope on intact human insulin. It was found that the antibodies attached to the peptide bound within the MHC groove of LYLVCGERG-loaded MHC tetramers and consequently curtailed tetramer migration during native agarose gel electrophoresis. This retention was peptide-dependent, reversed by adding an excess of free peptide, and did not occur when MHC tetramers with an incorrect peptide were used.

[0008] A corresponding interaction was found when a LYLVCGERG-loaded biotinylated MHC monomer was tested in two streptavidin-based ELIS As. In both systems binding of an anti-peptide antibody to univalent monomer was peptide-dependent, reversed by adding an excess of free peptide, and was absent when MHC monomers with different irrelevant peptides were tested. Further studies using a live cell ELISA and confocal fluorescence microscopy indicated that the anti-peptide antibodies bound to LYLVCGERG-loaded surface MHC on P815 target cells but not to P815 cells that lack the peptide. The specificity of the ELISA interaction was verified by the absence of binding both to cells loaded with the incorrect peptide and to LYLVCGERG-treated cells in the presence of a large blocking excess of free peptide. Since anti-peptide antibodies bound to a peptide residing in the MHC I cleft, further investigations were designed to see if they could likewise access peptides presented within a MHC II groove. Anti-peptide antibodies bound to several MHC II biotinylated monomers that had their occupant peptides exchanged for the LYLVCGERG epitope peptide. Such findings allow for testing the effect of anti-peptide antibodies and other binding agents specific to peptides of T cell-mediated diseases for possible therapeutic impact such as in in vivo models (e.g., the NOD mouse). This and analogous ELISAs can facilitate screening for anti-peptide antibodies or other binding agents reactive with different MHC -bound peptides, and such ELISAs and other methods for selecting or screening for candidate binding agents are provided in some aspects. In one aspect of the invention any one of the binding agents as provided herein is provided. In one embodiment, the binding agent is any antibody such as any of the antibodies provided herein, or an antigen-binding fragment thereof. In one embodiment, the binding agent is any antibody alternative, such as an RNA aptamer. In one embodiment, the binding agent has any one or more or all of the features provided herein.

[0009] In one aspect of the invention a composition is provided that comprises any one of the binding agents as provided herein. In one embodiment, such a composition is any one of the compositions provided herein.

[0010] In one aspect a composition comprising a MHC monomer or tetramer complexed with a peptide associated with a T cell-mediated disease is provided. In one embodiment, such a composition is any one of the compositions provided herein.

[0011] In one aspect a method of producing a binding agent, such as an antibody, is provided. In one embodiment, the method is any one of the methods of producing a binding agent provided herein.

[0012] In one aspect a method of selecting or screening for a binding agent, such as an antibody, is provided. In one embodiment, the method is any one of the methods of screening provided herein.

[0013] Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention. This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used in this disclosure is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having,” “containing,” “involving,” and variations of thereof in this disclosure, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the content clearly dictates otherwise.

[0014] In embodiments of any of the compositions and methods provided herein, “comprising” may be replaced with “consisting essentially of’ or “consisting of.” The phrase “consisting essentially of’ is used herein to require the specified integer(s) or steps as well as those which do not materially affect the character or function of the claimed invention. As used herein, the term “consisting” is used to indicate the presence of the recited integer (e.g., a feature, element, characteristic, property, method / process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method / process steps or limitations) alone.

[0015] BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1 is a schematic showing the CD8+ cytotoxic T-lymphocyte (CTL) mediated destruction of pancreatic beta cells (PBCs) which occurs in the autoimmune disease diabetes. Beta-cell specific peptide epitopes (such as from insulin) are recognized by Class I major histocompatibility (MHC) molecules expressed on the surface of PBCs, where they are recognized by T cell receptors on the surface of a CD8+ CTL, leading to death of the PBCs.

[0017] FIG. 2 is a schematic showing a proposed anti-peptide blockade mechanism by which antibodies prevent CTL-mediated PBC destruction. Anti-epitope peptide antibodies compete with CTLs to bind epitopes presented on the surface of PBCs, thereby blocking their destruction. Anti-epitope antibodies can also bind with free peptide epitopes.

[0018] FIG. 3 shows representative peptide specificity of an 8 / 10 antibody against B9-23 (SHLVEALYLVCGERG (SEQ ID NO: 14)), an insulin B-chain peptide as verified by ELISA. Half-maximal attachment of the 8 / 10 antibody to the B9-23 epitope peptide (Bg.23Ab peptide) occurs at ~3xl0-11M antibody (Ab) concentration, while little to no binding to whole insulin (B9-23Ab insulin) occurs at 1X10'7M.

[0019] FIG. 4 shows retention of the B9-23 peptide and insulin by the 8 / 10 antibody and 15 / 6 antibody after native polyacrylamide gel electrophoresis separation at pH 8. Both the 8 / 10 and 15 / 6 antibodies retained the B9-23 peptide, but not human insulin. Well 1 corresponds to mouse monoclonal (MOPC-21) negative control with B9-23 (MOPC-21 + B9-23); Well 2 corresponds to B9-23 peptide only (B9-23); Well 3 corresponds to 15 / 6 antibody with B9-23 peptide (15 / 6 + B9-23); Well 4 corresponds to 8 / 10 antibody with B9-23 peptide (8 / 10 + B9-23); Well 5 corresponds to 8 / 10 antibody only (8 / 10 alone); Well 6 corresponds to MOPC-21 and insulin (MOPC-21 + insulin); Well 7 corresponds to insulin only (insulin); Well 8 corresponds to 15 / 6 antibody with insulin (15 / 6 + insulin); Well 9 corresponds to 8 / 10 antibody with insulin (8 / 10 + insulin); and Well 10 corresponds to 8 / 10 antibody alone.

[0020] FIG. 5 shows interactions between the B 15-23 peptide (“G9G”), its heteroclitic form (“G9V”), and the binding groove of the MHC Class I molecule, H-2K(d). FIG. 5A shows superposition of G9G, G9V, and the MHC H-2K(d) al domain. Arrows below the peptides indicate whether each residue is positioned away from the binding groove for potential TCR contact (up arrow), a primary or secondary anchor (down arrow), or in between (no arrow). FIG. 5C shows the extended conformation of the G9G peptide in the MHC binding groove. Modified from Motozono, C., et al. (2015). J Biol Chem, 290(31):18924-18933.

[0021] FIGs. 6A-6B are schematics showing H-2K(d), B 15-23 and streptavidin tetramer production from four biotinylated monomers synthesized using H-2K(d) with a B 15-23 peptide (SEQ ID NO: 1). FIG. 6A is a schematic showing biotinylated H-2K(d)-B 15-23 monomers bind to streptavidin to form an H-2K(d)-B 15-23 -strep tetramer attached to fluorescent phycoerythrin (PE). FIG. 6B is a schematic showing crystallography models of the molecules of FIG. 6A.

[0022] FIG. 7 is a schematic showing a possible structure comprising a bivalent anti-B 15-23 antibody (“Anti-LYLVCGERG”) binding to two univalent biotinylated H-2K(d)-B 15-23 monomers (as in FIGs. 6A-6B).

[0023] FIGs. 8A-8B are schematics showing possible complexes of anti-B 15-23 antibodies binding to a tetramer comprised of four H-2K(d)-B 15-23 monomers. FIG. 8A is a schematic showing complexing of four anti-B 15-23 antibodies binding to an H-2K(d)-Bi5-23-strep tetramer (as in FIGs. 6A-6B). FIG. 8B is a schematic showing cross-linking of H-2K(d)-Bi5-23-strep tetramers and multiple anti-B 15-23 antibodies. Because each antibody is bivalent and each tetramer is quadrivalent, cross-linking to form a lattice may occur.

[0024] FIGs. 9A and 9B show native horizontal agarose gel electrophoresis to detect formation of the structures shown in FIGs. 7, 8A, and 8B. Anti-B 15-23 monoclonal antibodies were mixed with homologous H-2K(d)-Bi5-23-strep tetramers (as in FIGs. 6A-6B). In FIG. 9A Well 1 corresponds to an H-2K(d)-B 15-23 -strep tetramer (“L-tet”) alone; Well 2 corresponds to L-tet with MOPC-21 (negative control); Well 3 corresponds to L-tet with the 15 / 6 antibody; Well 4 corresponds to L-tet with the 8 / 10 antibody; Well 5 corresponds to a AMQMLKETI (SEQ ID NO: 15) tetramer (A-tet; a negative control); Well 6 corresponds to A-tet with MOPC-21; Well 7 corresponds to A-tet with the 15 / 6 antibody; Well 8 corresponds to A-tet with the 8 / 10 antibody; Well 9 corresponds to L-tet with the 15 / 6 antibody, at 37°C for 1 hour; Well 10 corresponds to A-tet with the 15 / 6 antibody, at 37°C for 1 hour; and Well 11 corresponds to L-tet with MOPC-21, at 37°C for 1 hour. In FIG. 9B Wells 1-8 are the same as above; Well 9 corresponds to a tetramer of IYSTVASSL (SEQ ID NO: 25) (l-tet; a negative control) l-tet with MOPC-21; Well 10 corresponds to l-tet with the 15 / 6 antibody; Well 11 corresponds to I-tet with the 8 / 10 antibody. The gel electrophoresis was performed with a 0.5% SeaKem agarose gel, TBE buffer, pH 8 (FIG. 9A) or HM buffer pH 6 (FIG. 9B), and at 50 volts with cooling. Unstained gel at 366nm (left); Coomassie protein stain (right).

[0025] FIG. 10 is a schematic showing the peptide blockade experiment of FIG. 12. In an environment with an excess of free peptide, binding sites on the 8 / 10 and 15 / 6 antibodies quickly accumulate the B 15-23 peptides. When H-2K(d)-Bis-23-strep tetramers are added, no binding sites are available, and the tetramers move freely, as if in the absence of the antibody.

[0026] FIG. 11 shows native horizontal agarose gel electrophoresis in which H-2K(d)-B 15-23- strep tetramer and 15 / 6 antibody molar ratios were titrated. Well 1 corresponds to H-2K(d)- Bis-23-strep tetramer (“L-tet”) and 15 / 6 antibody (Ab) at a ratio of 1:0 (Tet / Ab 1 / 0); Well 2 corresponds to L-tet / Ab at a ratio of 1:0.2 (Tet / Ab 1 / 0.2); Well 3 corresponds to L-tet / Ab at a ratio of 1:0.8 (Tet / Ab 1 / 0.8); Well 4 corresponds to L-tet / Ab at a ratio of 1:3 (Tet / Ab 1 / 3); Well 5 corresponds to Tet / Ab at a ratio of 1:13 (Tet / Ab 1 / 13); Well 6 corresponds to Tet / Ab at a ratio of 1:53 (Tet / Ab 1 / 53); Well 7 corresponds to Tet / Ab at a ratio of 1:213 (Tet / Ab 1 / 213). The gel electrophoresis was performed with 0.5% SeaKem agarose gel, TBE buffer, pH 8, and at 50 volts with cooling. Unstained gel at 366nm (left); Coomassie protein stain (right).

[0027] FIG. 12 shows native horizontal agarose gel electrophoresis in which Well 1 corresponds to H-2K(d)-Bis-23-strep tetramers (“L-tet”); Well 2 corresponds to L-tet with a 35-fold excess of B15-23 peptide (L-tet + 35x Pep); Well 3 corresponds to L-tet with 15 / 6 antibodies; Well 4 corresponds to L-tet with 15 / 6 antibodies and 35-fold molar excess of B15- 23 peptide over antibody; Well 5 corresponds a 35-fold excess of B 15-23 peptide only; Well 6 corresponds to L-tet alone; Well 7 corresponds to L-tet with 166-fold excess of B15-23 peptide; Well 8 corresponds to L-tet with 15 / 6 antibodies; Well 9 corresponds to L-tet with 15 / 6 antibodies and 166-fold excess of B15-23 peptide; Well 10 corresponds to 166-fold excess of B 15-23 peptide. The gel electrophoresis was performed with a 0.5% SeaKem agarose gel, TBE buffer, pH 8, and at 50 volts with cooling. Unstained gel at 366nm (left); Coomassie protein stain (right).

[0028] FIGs. 13A-13D are schematics showing an ELISA protocol (“El”) to detect the specific reaction between the 15 / 6 and 8 / 10 antibodies (Anti-LYLVCGERG) and their complementary biotinylated H-2K(d)-B 15-23 monomer (“peptide”; left) vs a control H-2K(d) - AMQMLKETI (SEQ ID NO: 15) peptide biotinylated monomer (“wrong peptide”; right). These components are mixed, allowed to react and then added to separate wells of a streptavidin-coated ELISA plate FIG. 13A. FIG. 13B is a schematic showing that the 15 / 6 or 8 / 10 antibody and H-2K(d)-B 15-23 monomer complex binds to the streptavidin plate (left); while the H-2K(d)-AMQMLKETI monomer, but not the unbound 15 / 6 or 8 / 10 antibody binds to the plate (right); a subsequent wash thus removes those unattached antibodies, but not the bound antibody and H-2K(d)-B 15-23 monomer complex. FIG. 13C is a schematic showing the next step of the El ELISA protocol where a peroxidase-labelled anti-IgG reagent is added to the wells. Retained antibody :H-2K(d)-B 15-23 monomer complexes are tagged by the anti-IgG peroxidase reagent. A subsequent wash removes any unbound anti-IgG peroxidase reagent. FIG. 13D is a schematic showing the last step of the El ELISA protocol for detecting antibody specificity. In this step, the tagged H-2K(d)-B 15-23 monomer complexes produce a color, which can be measured to reveal the amount of bound antibody.

[0029] FIGs. 14A-14C is a schematic showing a second ELISA protocol (“E2”) for detecting the specific reaction between the anti-B 15-23 antibodies and H-2K(d)-B 15-23 biotinylated monomers in which monoclonal 15 / 6 or 8 / 10 or negative control MOPC-21 antibodies are first adsorbed to the ELISA plate which is then washed. FIG. 14A is a schematic showing the first step of the E2 ELISA protocol. H-2K(d)-B 15-23 monomers are added to the antibody- coated plate. FIG. 14B is a schematic showing the second step of the E2 ELISA protocol. H- 2K(d)-B 15-23 monomers bind to the adsorbed antibodies to form complexes and any unbound H-2K(d)-B 15-23 monomers are then washed out. FIG. 14C is a schematic showing the third step of the E2 ELISA protocol. Peroxidase-labelled streptavidin is added to the wells and bind to antibody -bound biotinylated H-2K(d)-B 15-23 monomers. After a wash to remove unbound streptavidin-peroxidase reagents, any bound reagent generates color.

[0030] FIGs. 15A-15C show the predicted peptide dependency of the binding of an anti- LYLVCGERG antibody- streptavidin HRP complex to MHC-LYLVCGERG sites presented on the P815 cell surface. FIG. 15A is a schematic showing a pre-assembled biotinylated antibody specific to a LYLVCGERG peptide (SEQ ID NO: 1) (Anti-LYLVCGERG-biotin) and complexed with a horseradish peroxidase-streptavidin conjugate (Streptavidin HRP). FIG. 15B is a schematic showing predicted absence of binding of the Anti- LYLVCGERG:Streptavidin HRP complex of FIG. 15A to MHC complexes on the surface of P815 cells lacking LYLVCGERG peptides. FIG. 15C is a schematic showing predicted binding of the Anti-LYLVCGERG:Streptavidin HRP complex of FIG. 15A to P815 cell membrane surface MHC complexes pre-loaded with LYLVCGERG peptides. FIGs. 16A-16B show direct fluorescence staining of LYLVCGERG peptide-loaded vs untreated P815 cells with a 15 / 6-fluorescein (FITC) conjugate as observed by confocal microscopy. Low magnification is shown in FIG. 16A; high magnification is shown in FIG. 16B. Phase contrast (left), fluorescence (right), untreated (top), peptide-loaded (bottom). Only the peptide-loaded cells show a characteristic ring of fluorescence around the cell circumference that is indicative of membrane staining.

[0031] FIG. 17A-17C shows structures of peptide LYLVCGERG. Fig. 17A shows a stick model of the LYLVCGERG peptide, in its extended linear version. Fig. 17B shows the LYLVCGERG peptide sequence in its conformation found within the insulin B-chain. Fig. 17C shows a 3D ribbon structure of insulin B-chain showing the positions of its LYLVCGERG amino acids.

[0032] FIG. 18A-18C shows structures of peptide LYLVCGERG within the MHC groove. Fig. 18A shows the extended, linear stick model of the LYLVCGERG peptide in MHC groove top ribbon view. Fig. 18B shows the stick model of the LYLVCGERG peptide in MHC groove top surface view. Fig. 18C shows a surface model of the LYLVCGERG peptide in MHC groove top surface view.

[0033] DESCRIPTION OF THE INVENTION

[0034] The MHC-peptide complex and its interaction with the TCR underlie T-cell specificity and their responses to both pathogens or, in the case of autoimmune disease, normal cells and tissues. Such peptides can be targets for therapeutic intervention such as for autoimmune diseases or cancer. Because these peptides usually reside within a shallow binding cleft of the MHC molecule it was important to confirm they are accessible while positioned within that site. In addition, it was important to confirm that binding agents, such as anti-peptide antibodies, can specifically seek out and sequester peptides while the peptide is bound to a MHC molecule. This was determined by using MHC tetramers and MHC monomers and living cells expressing MHC on their membrane. Provided herein are compositions and methods that comprise binding agents, such as antibodies, that specifically bind such peptides. Such binding agents can specifically bind the peptides as provided herein including when complexed with a MHC molecule, such as a MHC Class I and / or MHC Class II molecule. In some embodiments, the MHC molecule can be a MHC monomer or MHC tetramer as used herein or as provided herein. In some embodiments, the MHC molecule may be on a cell such as a cell membrane. In some embodiments, the MHC molecule may be a human MHC molecule. In other embodiments, the MHC molecule is a mouse MHC molecule. Methods of administering the binding agents provided herein are also provided. In such methods the binding agents can inhibit the binding of a T cell to a complex of a peptide and a MHC molecule.

[0035] Also provided herein are compositions comprising the peptide complexed with a MHC Class I or MHC Class II molecule such as a MHC monomer or MHC tetramer. Such compositions can be used to assess whether a candidate binding agent specifically binds the peptide in complex with a MHC molecule. Methods of determining the binding of candidate binding agents to such peptide-MHC monomer or peptide-MHC tetramer complexes are also provided. In some embodiments, the MHC molecule may be on a cell such as a cell membrane.

[0036] The binding agents provided herein in some embodiments specifically bind a peptide associated with a T cell-mediated disease. As used herein, a “T cell-mediated disease” is any disease, disorder or condition where T cell binding or activity causes or contributes to the onset of the disease, disorder or condition, to the progress of the disease, disorder or condition, or to one or more symptoms of the disease, disorder or condition. In one embodiment, the T cell-mediated disease is an autoimmune disease. “Autoimmune disease” is a disease in which the immune system fails to recognize a subject’s own organs, tissues or cells, and produces an immune response to attack those organs, tissues or cells as if they were foreign antigens. Autoimmune diseases are well known in the art; for example, as disclosed in The Encyclopedia of Autoimmune Diseases, Dana K. Cassell, Noel R. Rose, Infobase Publishing, 14 May 2014, incorporated by reference in its entirety as if fully disclosed herein. In one embodiment, the T cell-mediated disease is cancer. “Cancer” is a disease associated with undesirable or abnormal cell growth. Cancers are well known in the art.

[0037] As used herein, in some embodiments, the binding agents bind the peptide when complexed to a MHC molecule, such as a MHC Class I or MHC Class II molecule. In some embodiments, the binding agents do not bind the peptide associated with the T cell-mediated disease when comprised within a native protein. As used herein, “native protein” refers to a protein that is produced endogenously in a subject. The term is intended to encompass native forms to which the binding of a binding agent provided herein is undesirable. In one embodiment, the native protein is insulin.

[0038] The binding agents that specifically bind a peptide as provided here include antibodies (also referred to herein as “anti-peptide antibodies”). Antibodies in some embodiments are antibodies that specifically bind a peptide associated with a T cell-mediated disease, such as when complexed to a MHC molecule, such as a MHC Class I or MHC Class II molecule and / or as free peptide but, in some embodiments, not when comprised within a native protein.

[0039] As used herein, “specifically binds” refers to the preferential binding of a binding agent, such as an antibody or antigen-binding fragment thereof, to a peptide as provided herein over other peptides. In some embodiments the other antigens are other endogenous antigens. The antibody that specifically binds a peptide as provided herein can typically bind with an affinity that is greater by at least 25%, 50%, 100%, 150%, 200%, 300%, 400%, 500% or greater than its affinity for binding to other antigens. The antibodies provided can, in some embodiments, have an association constant of about 1X104M-1, 1X105M-1, 1X106M-1, 5xlO6M" 1X107M_1, 5xlO7M-1, 1X108M-1, 2xlO8M’1, 5X108M-1, 7.5xl08M-1, IxlO^’1, 2xl09M , 5X109M-1, 7.5X109M-1or 1 X 1010M_1. The antibodies provided can, in some embodiments, have an association constant of any one of such constants as provided herein. In one embodiment the antibodies provided have an association constant of at least 1X106M-1, 1X107M-1, 1X108M , 1X109M-1or 1 X 1010M_1. Anti-peptide antibodies include antibodies that specifically bind the peptide when complexed to a MHC molecule, such as a MHC Class I or MHC Class II molecule. In some of these embodiments, the antibodies do not bind or significantly bind the peptide associated with the T cell-mediated disease when comprised within a native protein. As used herein, “does not bind” refers to no measurable binding when assessed with routine methods in the art and which a skilled artisan would conclude that no binding occurred or occurs. “No significant binding” refers to some binding but at a level which a skilled artisan would conclude is not at a level that would be negatively impact the desired purpose or result.

[0040] As used herein, the term “antibody” refers to glycoproteins comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI , CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.

[0041] The term “antibody” is also intended to include antigen-binding fragments of the antibodies. The term “antigen-binding fragment” as used herein, refers to one or more portions of an antibody that retain the ability to specifically bind to an antigen (e.g., a peptide as provided herein). Examples of binding fragments encompassed within the term “antigenbinding fragment” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab’)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward el al., (1989) Nature 341:544-546) which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). These antibody fragments are obtained using conventional procedures, such as proteolytic fragmentation procedures, as described in J. Coding, Monoclonal Antibodies: Principles and Practice, pp 98-118 (N.Y. Academic Press 1983), which is hereby incorporated by reference, as well as by other techniques known to those with skill in the art. The fragments are screened for utility in the same manner as are intact antibodies.

[0042] The binding agents, such as antibodies, can be isolated binding agents, such as isolated antibodies. An “isolated antibody” refers to an antibody which is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to a peptide provided herein is substantially free of antibodies that specifically bind antigens other than the peptide) and / or other native cellular materials. An isolated antibody that specifically binds to a peptide may, however, have cross -reactivity to other peptide analogs or related antigens, e.g., from other species. Moreover, an isolated antibody may be substantially free of other cellular material and / or chemicals. In some embodiments, however, the isolated antibody specifically binds to a peptide as provided herein and does so with a specificity such that there is little to no cross-reactivity with the peptide when comprised in a native protein.

[0043] The isolated antibodies of the invention encompass various antibody isotypes, such as IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgAsec, IgD, IgE. As used herein, “isotype” refers to the antibody class (e.g., IgM or IgGl) that is encoded by heavy chain constant region genes. The antibodies can be full length or can include only an antigen-binding fragment such as the antibody constant and / or variable domain of IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgAsec, IgD or IgE or could consist of a Fab fragment, a F(ab')2 fragment, and a Fv fragment. The antibodies can be single chain antibodies (scFvs), comprising linked Vjq and VL domains and which retain the conformation and specific binding activity of the native idiotype of the antibody. Single chain antibodies are well known in the art and can be produced by standard methods (See, e.g., Alvarez et al., Hum. Gene Ther. 8: 229-242 (1997)). For example, recombinant methods can be used to generate scFvs, and such scFvs, can consist of a VL and VH joined by a synthetic peptide linker. The scFv antibodies can also be obtained as an antigen binding fragment of an antibody. In addition, scFv antibodies can be identified by screening against one or more antigens. For example, methods of selecting or screening are provided herein. Such methods can include the use of the MHC monomers or MHC tetramers provided herein. In some embodiments, the methods comprise any one of the methods provided herein, such as in the Examples.

[0044] The antibodies of the present invention can be monoclonal. The antibodies can be produced by a variety of techniques. Procedures for raising monoclonal antibodies are well known in the art. Methods for producing monoclonal antibodies are also provided in the Examples. The term “monoclonal antibody,” as used herein, refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody displays a single binding specificity and affinity for a particular epitope.

[0045] In other embodiments, the antibodies can be recombinant antibodies. The term “recombinant antibody”, as used herein, is intended to include antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (e.g., a mouse) that is transgenic for another species’ immunoglobulin genes, antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial antibody library, or antibodies prepared, expressed, created or isolated by any other means that involves splicing of immunoglobulin gene sequences to other DNA sequences.

[0046] In yet other embodiments, the antibodies can be chimeric or humanized antibodies. As used herein, the term “chimeric antibody” refers to an antibody, that combines the murine variable or hypervariable regions with the human constant region or constant and variable framework regions. As used herein, the term “humanized antibody” refers to an antibody that retains only the antigen-binding CDRs from the parent antibody in association with human framework regions (see, Waldmann, 1991, Science 252:1657). Such chimeric or humanized antibodies retaining binding specificity of a murine antibody are expected to have reduced immunogenicity when administered in vivo for diagnostic, prophylactic or therapeutic applications.

[0047] In certain embodiments, the antibodies are human antibodies. The term “human antibody,” as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term “human antibody,” as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse have been grafted onto human framework sequences (referred to herein as “humanized antibodies”).

[0048] Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e.g., U.S. patents 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and references cited therein, the contents of which are incorporated herein by reference. These animals have been genetically modified such that there is a functional deletion in the production of endogenous (e.g., murine) antibodies. The animals are further modified to contain all or a portion of the human germ-line immunoglobulin gene locus such that immunization of these animals results in the production of fully human antibodies to the antigen of interest. Following immunization of these mice (e.g., XenoMouse (Abgenix), HuMAb mice (Medarex / GenPharm)), monoclonal antibodies are prepared according to standard hybridoma technology. These monoclonal antibodies have human immunoglobulin amino acid sequences and therefore will not provoke human anti-mouse antibody (HAMA) responses when administered to humans.

[0049] The antibodies provided herein also include modified versions of the antibodies. Modified antibodies include antibody conjugates or genetic constructs thereof, where the antibodies are conjugated to other biomolecules (e.g., polyethylene glycol (PEG), albumin or albumin-binding peptide) that would, for example, prolong the serum half-life of an antipeptide antibody (e.g., a scFv antibody). In other words, these biomolecules can prolong the residence time of an agent in the body. A “biomolecule that prolongs the residence time of an agent in the body” is one which results in the presence of the agent in the body of a subject for a longer period of time or in a higher amount at a particular point in time than when the agent is administered without the biomolecule. An appended Cys residue can be used as a point of attachment for creating such antibody conjugates. Techniques for making such conjugates are known to those of ordinary skill in the art and are described herein in the Examples as well as in Smith et al., Bioconjugate Chem. 2001, 12, 750-756.

[0050] Any given anti-peptide antibody can also be modified by genetic engineering to achieve higher affinity (e.g., higher affinity to a peptide provided herein), bivalent binding or facilitated passage into the CNS (Pardridge, W.M., et al. 1991. J Pharmacol Exp Ther 259:66- 70.) For instance, mutagenic PCR can be used to evolve anti-peptide antibodies, such as scFv antibodies, toward higher binding affinities and greater functionality. Briefly, for molecular evolution, an anti-peptide scFv DNA is amplified using error-prone PCR to incorporate 3 to 7 point mutations / scFv. The material is then cloned into a surface expression vector using the endogenous homologous recombination system present in yeast. This allows mutated libraries of 1-10 x 106clones to be rapidly generated and screened. Mutagenic PCR can also be used to generate antibodies. Preferably, modified anti-peptide antibodies that are engineered to allow their passage into the CNS include scFv antibodies, diabodies or other anti-peptide antibody fragments. Any of the isolated anti-peptide scFv antibodies can be evolved into a higher affinity version to increase its therapeutic effectiveness (Boder, E.T., et al. 2000. Proc Natl Acad Sci USA 97:10701-10705.)

[0051] A person having ordinary skill in the art will be familiar with recent advances in alternatives to antibodies, such as aptamers (e.g., RNA aptamers), Spiegelmers and selective peptides. Spiegelmers are nucleic acids that are capable of binding specific target sites on molecules (similarly to antibodies). However, unlike traditional aptamers, Spiegelmers are designed to resist degradation by endogenous nucleases. This is because they are constructed from mirror image L-oligo nucleotides (Klussmann, et al., Nat. Biotechnol.14: 1121115,1996; Vater and Klussmann, Cor. Opin. Drug Discov. Devel.6: 253-261,2003). Thus, in an embodiment, the binding agent of any one of the methods or compositions provided herein is an alternative to an antibody, such as any one of the foregoing. The alternative may also be a DARpin, affimer, avimer, knottin, monobody or affinity clamp in any such embodiment. The alternative may also be a multicyclic peptide (T. Whalley, G. Dolton, P. E. Brown, A. Wall, L. Wooldridge, H. Van Den Berg, A. Fuller, J. R. Hopkins, M. D. Crowther, M. Attaf, R. R. Knight, D. K. Cole, M. Peakman, A. K. Sewell, B. Szomolay, GPU-Accelerated Discovery of Pathogen-Derived Molecular Mimics of a T-Cell Insulin Epitope. Front. Immunol. 11, 296 (2020)).

[0052] Binding agents can be produced with any of the peptides or peptide-containing compositions provided herein. Once produced, the binding agents that specifically bind as provided herein can be selected. Methods for selecting such binding agents are known in the art or provided herein, and examples of which are provided below in the Examples.

[0053] Methods for producing binding agents, such as antibodies, are provided herein. Such methods include administering a peptide or peptide-containing composition as described herein, such as peptide alone, with a carrier or adjuvant, or in a complex with a MHC molecule, such as a MHC monomer or tetramer, to a subject in an amount effective to generate binding agents, such as antibodies. In an embodiment, the peptide or peptide- containing composition as described herein is not in a complex with a MHC molecule, such as a MHC monomer or tetramer. In an embodiment, the peptide may be coupled to an immunogenic carrier protein, incorporated into a viral presentation particle or joined to a self- fibrilizing peptide. In one embodiment, the method further includes the administration of an adjuvant to the subject. The adjuvant can be administered prior to, concomitantly with or subsequent to the administration of the peptide or peptide-containing composition. Adjuvants, as provided herein, include complete Freunds and incomplete Freunds adjuvant. Other adjuvants, including those compatible for delivery to a human, are well known to those of ordinary skill in the art. The method can further comprise administering one or more booster doses of a peptide or peptide-containing composition, which can be the same or different from what was previously administered. In an embodiment of any one of the methods provided herein, instead of a peptide or peptide-containing compositions, a DNA or RNA vaccine may instead be used to actively elicit antibodies directed against the peptide provided herein. In such an embodiment, the DNA or RNA encodes any one of the peptides provided herein.

[0054] Vectors suitable for use in this context are well known to those of ordinary skill in the art. For example, plasmid vectors and viral vectors have been used as DNA vaccines for delivering antigen-encoding nucleic acids to cells in vivo. Plasmid vectors are particularly advantageous because they do not have the same safety concerns as with some of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a nucleic acid operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC / CMV, SV40, and pBlueScript. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids such as those used for DNA vaccines may be delivered by a variety of parenteral, mucosal and topical routes. For example, the plasmid DNA can be injected by intramuscular, intradermal, subcutaneous or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation. Further, DNA vaccines can be administered to a subject in conjunction with an adjuvant or additional therapeutic agent.

[0055] The administration of an adjuvant or additional therapeutic agent can occur prior to, concomitantly with or subsequent to the administration of the DNA vaccine.

[0056] In some embodiments the methods of administration of a peptide, peptide-containing composition or DNA or RNA vaccine further include determining whether or not anti-peptide antibodies that specifically bind the peptide when complexed to a MHC molecule and / or the peptide as free peptide are produced. Such a determination can include the harvesting of a sample that contains antibodies from the subject and assessing whether the sample includes one or more antibodies that specifically bind the peptide when complexed to a MHC molecule and / or as free peptide. Such determining can also comprise determining whether or not the antibodies specifically bind the peptide when complexed to a MHC molecule, such as a Class I or Class II MHC molecule, and / or determining whether or not the antibodies bind to the peptide when comprised within a native protein. Binding of peptides to antibodies can be determined using standard methodologies including surface plasmon resonance and ELISA assays. This as well as other methods for such assessment are well known to those of ordinary skill in the art and further examples of which are provided below in the Examples. The methods provided can further involve the steps of selecting compositions for administration that can generate the desired antibodies. In one embodiment, antibodies that specifically bind the peptide when complexed to a MHC molecule and / or free peptide are desired. In another embodiment, antibodies that do not bind or significantly bind the peptide when in a native protein are desired. Any one of the methods provided herein can further involve the steps of selecting compositions for administration that can generate the desired antibodies, which antibodies have any one or more or all of the foregoing features. As used herein, an “effective amount to generate anti-peptide antibodies” refers to the amount of a compound alone or in combination with an adjuvant or other antigen that can be used to generate anti-peptide antibodies as provided herein in the subject. In another embodiment, the effective amount is for specific binding to the peptide when complexed to a MHC molecule and / or as free peptide. In another embodiment the effective amount is an amount effective to inhibit T cell binding of the peptide when complexed to a MHC molecule. In yet another embodiment the effective amount is an amount effective to treat a T cell-mediated disease. “An effective amount for treating a T cell-mediated disease” is any amount of a compound alone or in combination that alleviates or eliminates any symptom of the T cell-mediated disease (e.g., autoimmune disease or cancer).

[0057] In an embodiment, the peptide used alone, with a carrier or adjuvant, or in a complex with a MHC molecule, such as a MHC monomer or tetramer, may be any one of the peptides provided herein. For example, the peptide may be a linear peptide sequence of insulin, such as B15-23, B9-23, B7-18 or B10-18 (E. Ciszak, G. D. Smith, Crystallographic Evidence for Dual Coordination Around Zinc in the T3R3 Human Insulin Hexamer. Biochemistry 33, 1512-1517 (1994); K. Kimura, T. Kawamura, S. Kadotani, H. Inada, S. Niihira, T. Yamano, Peptide- specific cytotoxicity of T lymphocytes against glutamic acid decarboxylase and insulin in type 1 diabetes mellitus. Diabetes Research and Clinical Practice 51, 173-179 (2001); G. G. M. Pinkse, O. H. M. Tysma, C. A. M. Bergen, M. G. D. Kester, F. Ossendorp, P. A. van Veelen, B. Keymeulen, D. Pipeleers, J. W. Drijfhout, B. O. Roep, Autoreactive CD8 T cells associated with beta cell destruction in type 1 diabetes. Proc Natl Acad Sci U S A 102, 18425-18430 (2005)). In an embodiment, the peptide comprises LYLVCGERG (SEQ ID NO: 1). In another embodiment, the peptide comprises HLVEALYLV (SEQ ID NO: 2). In another embodiment, the peptide comprises CHLVEALYLVCGERG (SEQ ID NO: 3) or CSHLVEALYLVCGERG (SEQ ID NO: 4). In an embodiment, the peptide comprises CGSHLVEALYLV (SEQ ID NO: 5). In an embodiment, the peptide has the sequence of any one of the peptides provided herein.

[0058] Other peptides may be any MHC I and MHC II peptides that are associated with a T cell-mediated disease. Such peptides include, but are not limited to, a citrulline peptide CSLNLXETNLDSL X = L-Citrulline (SEQ ID NO: 6), IGRP (islet- specific glucose-6- phosphatase catalytic subunit-related protein) peptides CVYLKTNVFL (SEQ ID NO: 7), VYLKTNVFLKC (SEQ ID NO: 8), and VLFGLGFAIKC (SEQ ID NO: 9), PPI (preproinsulin) peptides PPI18-27 CFLWESHPTQA (SEQ ID NO: 10) and PPI3-11 CLWMRFLPLL (SEQ ID NO: 11).

[0059] As another example, the peptide may be a linear peptide sequence of a peptide associated with multiple sclerosis (T. R. Petersen, E. Bettelli, J. Sidney, A. Sette, V. Kuchroo, B. T. Backstrdm, Characterization of MHC- and TCR-binding residues of the myelin oligodendrocyte glycoprotein 38-51 peptide. Eur J Immunol 34, 165-173 (2004); C. Massilamany, A. Gangaplara, T. Jia, C. Elowsky, G. Kang, J.-J. Riethoven, Q. Li, Y. Zhou, J. Reddy, Direct Staining with Major Histocompatibility Complex Class II Dextramers Permits Detection of Antigen-Specific, Autoreactive CD4 T Cells In Situ. PLoS ONE 9, e87519 (2014)). In an embodiment, the peptide is of myelin oligodendrocyte glycoprotein, such as MOG38-51. In another embodiment, the peptide comprises GWYRSPFSRVVHLY (SEQ ID NO: 12). In another embodiment, the peptide is of myelin proteolipid protein (PLP), such as PLP139 i5i. In an embodiment, the peptide comprises HSLGKWLGHPDKF (SEQ ID NO: 13).

[0060] In an embodiment, the peptide or peptide-containing composition is used to induce active immunity in a subject. Thus, in one embodiment of any one of the methods of producing an antibody as provided herein, the method comprises administration of the peptide or peptide-containing composition to the subject. In an embodiment, the subject has a T cell-mediated disease, such as an autoimmune disease or cancer. In such an embodiment, the subject may be a human. In another embodiment, the subject is one in which antibodies are produced and selected. Such a subject may be a non-human subject. The antibodies produced and selected may be used to adminster to a subject in which T cell inhibition is desired, such as a subject that has a T cell-mediated disease, such as an autoimmune disease or cancer.

[0061] As used herein, the term “subject” is intended to include humans and non-human animals, such as mice and non-human primates. Administration of the agents or compositions provided to mammals other than humans, e.g. for testing purposes or veterinary therapeutic purposes, can be carried out under conditions as described herein. As used herein, a “subject” also includes any individual who would benefit from the administration of any one of the agents provided herein. These subjects include those individuals who have an autoimmune disease or cancer. The subjects also include those with type I diabetes. The subjects also include those with any one of the diseases or conditions provided herein. The compositions of the agents provided herein can be administered to any such subjects. In addition, methods of their treatment through the administration of the compositions and agents as described herein are also provided.

[0062] The invention provides compositions comprising the agents provided herein. The compositions can include a physiologically or pharmaceutically acceptable carrier, excipient, or stabilizer mixed with the agent. Pharmaceutical compositions of the invention also can be administered in combination therapy, i.e., combined with other agents. As used herein, “pharmaceutically acceptable carrier” or “physiologically acceptable carrier” includes any and all salts, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In some embodiments, the carrier is suitable for intravenous, intramuscular, intraperitoneal, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the agent may be coated in a material to protect the agent from the action of acids and other natural conditions that may inactivate the agent.

[0063] When administered, the pharmaceutical preparations of the invention are applied in pharmaceutically-acceptable amounts and in pharmaceutically-acceptable compositions. The term “pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention. The term “pharmaceutically-acceptable carrier” as used herein also means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration into a human. The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being co-mingled with the agents of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy. The pharmaceutical compositions may contain suitable buffering agents, including: acetic acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt. The pharmaceutical compositions also may contain, optionally, suitable preservatives, such as: benzalkonium chloride; chlorobutanol; parabens and thimerosal. The pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. All methods include the step of bringing an agent into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.

[0064] Compositions suitable for parenteral administration conveniently comprise a sterile aqueous or non-aqueous preparation of an agent as provided herein, which is preferably isotonic with the blood of the recipient. This preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation also may be a sterile injectable solution or suspension in a nontoxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer’s solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or di-glycerides. In addition, fatty acids such as oleic acid may be used in the preparation of injectables. Carrier formulations suitable for oral, subcutaneous, intravenous, intramuscular, etc. administration can be found in Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.

[0065] The therapeutics of the invention can be administered by any conventional route, including injection or by gradual infusion over time. The administration may be, for example, mucosal (e.g., oral, buccal, nasal), intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, parenteral or intradermal. When agents, such as antibodies, are used therapeutically, preferred routes of administration include intravenous, intraperitoneal, intracranial, mucosal (e.g., oral, buccal, nasal), parenteral, intrapulmonary, by suppository or by local or targeted delivery. The agents in some embodiments can be administered intracerebrally by periodic bolus injection or by sustained infusion using a pump. Techniques for preparing aerosol delivery systems containing agents are well known to those of skill in the art. Generally, such systems should utilize components which will not significantly impair the biological properties of the agents, such as the binding capacity, for example paratope binding capacity, (see, for example, Sciarra and Cutie, “Aerosols,” in Remington’s Pharmaceutical Sciences, 18th edition, 1990, pp. 1694-1712; incorporated by reference). The compositions of the invention are administered in effective amounts. An “effective amount” is that amount of a composition as provided herein that alone, or together with further doses, adjuvants or additional therapeutic agents, produces the desired response. The effects of treatment can be monitored by routine methods. Such methods can be indirect or direct methods. For instance, the effects can be monitored by radioimmunoassay of agent levels in a subject or by assessing the levels of free peptide in a sample. Other assays will be known to one of ordinary skill in the art and can be employed for measuring the level of the response to treatment. Such amounts can depend, of course, on the severity of disease, the individual patient parameters including age, physical condition, size and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reason.

[0066] The pharmaceutical compositions used in the foregoing methods preferably are sterile and contain an effective amount of an agent for producing the desired response in a unit of weight or volume suitable for administration to a patient. The doses administered to a subject can be chosen in accordance with different parameters, in particular in accordance with the mode of administration used and the state of the subject. Other factors include the desired period of treatment. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits.

[0067] Based upon the composition, the dose can be delivered once, continuously, such as by continuous pump, or at periodic intervals. The periodic interval may be weekly, bi-weekly or monthly. The dosing can occur over the period of one month, two months, three months or more. Desired time intervals of multiple doses of a particular composition can be determined without undue experimentation by one skilled in the art.

[0068] In the event that the response in a subject is insufficient at such doses, higher doses (or effective higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. Other protocols for the administration of the compositions provided will be known to one of ordinary skill in the art, in which the dose amount, schedule of administration, sites of administration, mode of administration and the like vary from the foregoing.

[0069] The compositions of the present invention have in vitro and in vivo utilities. For example, the agents can be administered to a sample, e.g. in vitro or ex vivo, or in a subject, e.g., in vivo. In some embodiments, the agents are placed in contact with MHC complexed with a peptide as provided herein and, in an embodiment, T cell attachment and / or activity is reduced, inhibited or eliminated. Methods comprising placing any of the agents or compositions provided herein in contact with MHC complexed with any of the peptides provided herein to reduce, inhibit or eliminate T cell attachment and / or activity are provided.

[0070] As another example, the agents and compositions provided can be used in various screening methods. In one embodiment the screening method includes the step of contacting a candidate binding agent (e.g., one or more antibodies that are thought to possibly be able to specifically bind the peptide or peptide-containing composition or any of the alternatives as provided herein) with a composition comprising a peptide or peptide-containing composition and determining whether or not the candidate binding agent binds the peptide or peptide- containing composition as provided herein. The methods may also comprise determining whether or not the candidate binding agents specifically binds the peptide when complexed with MHC and / or as free peptide. The methods may also comprise determining whether or not the candidate binding agents specifically binds the peptide when comprised within a native protein. As used herein a “candidate binding agent” is any agent that could be screened in a method provided herein.

[0071] As an example, anti-peptide antibodies were selected based on their ability to bind a peptide as provided herein and were found to bind the peptide even when complexed to a MHC molecule (see Table 1). Methods are provided herein that comprise steps for determining binding to a peptide as provided herein when complexed to a MHC molecule and / or as free peptide. In an embodiment, the method comprises an ELISA assay wherein binding to a peptide when complexed to a MHC molecule, such as a MHC monomer or tetramer, is assessed (such as using a streptavidin ELISA plate). The specifics of an exemplary method is provided as follows where a candidate agent, in this case the 15 / 6 anti- peptide antibody, was added to 50pl media at a 20-100 pg / ml concentration range that is typically secreted into microtiter wells by monoclonal antibody producing hybridoma clones.

[0072] Any one of the methods provided herein can include any one or more or all of these steps.

[0073] Table 1. Hybridoma clones obtained, their isotype and reactivity with the B 15-23 peptide and monomer

[0074] Antibodies Bind to

[0075] Clone Isotype State B 15-23 H-2K(d)-B 15-23 Peptide Monomer

[0076] B 15-23 Peptide = LYLVCGERG H-2K(d)-B 15-23 = mouse MHC I monomer with LYLVCGERG

[0077] Table 1 summarizes the properties of several anti-peptide monoclonal antibodies that were obtained from a hybridoma cell fusion. Each clone was characterized with regard to its antibody isotype and its ability to bind the LYLVCGERG peptide (B 15-23) when either free or while bound within the major histocompatibility complex H-2K(d) binding groove.

[0078] Examples included within reveal that anti-LYLVCGERG monoclonal antibodies attach to this peptide while it resides within the groove of mouse MHC I (H-2K(d)), mouse MHC II (I- Ag7) and human HLA-A*24:02. Longer 14 residue peptides were recognized in the MHC II context provided they contained an appropriate LYLVCGERG epitope. Distinct anti- HLVEALYLV antibodies likewise bound to their complementary peptide as presented by the human HLA-A*02:01 complex. Accordingly, the same selection strategy can be used to screen for and select other binding agents that bind peptide within a complex with a MHC.

[0079] The present invention is further illustrated by the following Examples, which in no way should be construed as further limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference. EXAMPLES

[0080] Example 1. Antibodies against B9-23 are specific to B9-23 and do not bind insulin.

[0081] In this Example, antibodies against B9-23 (SHLVEALYLVCGERG (SEQ ID NO: 14)), an insulin B-chain peptide epitope, and its contained epitope, B 15-23 (LYLVCGERG; SEQ ID NO: 1), were elicited in Balb / c mice. B 15-23 has been shown to be involved in CTL- mediated pancreatic beta cell destruction (e.g., as in FIG. 1 and FIG. 2), and are implicated in Type 1 diabetes. CSHLVEALYLVCGERG (SEQ ID NO: 4) was synthesized and attached to PEGylated maleimide, KLH; bolded Cs indicate possible cysteine SH sites for attachment. Hybridomas were obtained by standard methods and screened for antibodies reactive with the B-chain peptide but not with intact insulin. Monoclonal antibodies were purified from the ascites on protein G columns using 4M MgCh for elution. A non-specific MOPC-21 control monoclonal antibody (MOPC-21) was purified from ascites in the same way.

[0082] The two selected pure anti-Bg-23 antibodies, an 8 / 10 antibody and a 15 / 6 antibody both (anti-LYLVCGERG), have an IgG2b, kappa isotype. The peptide specificity of these two purified anti-peptide monoclonal antibodies was verified by ELISA studies using the 3 peptides (B15-23, B9-23, and B10-18) and / or whole human insulin adsorbed to the microtiter plate. Biomolecular binding affinities and binding kinetics for the 15 / 6 and 8 / 10 antibody interactions with peptides was measured by label-free technology using interferometric reflectance imaging sensor technology. Analyses were performed both with the antibodies immobilized on the chip while flowing the peptides as well as with the peptides attached and flowing the antibodies. The kinetic parameters obtained for the immobile 15 / 6 and 8 / 10 antibodies using the flowed B 15-23 peptide analyte are shown below in Table 2. Both the 8 / 10 and 15 / 6 antibodies have almost the same specificity for the LYLVCGERG part of the CSHLVEALYLVCGERG (SEQ ID NO: 4) immunogen peptide and barely detectable reaction with the HLVEALYLV (SEQ ID NO: 2) peptide. Table 2 shows both binding strongly to LYLVCGERG. Residence times ( l / koff) spent by the B 15-23 peptide within the 15 / 6 and 8 / 10 antibody binding sites were 1.9 hours and 6.2 hours, respectively. The dissociation constant for the 15 / 6 and 8 / 10 monoclonal antibodies binding to LYLVCGERG was measured, Table 2. A MHC-I Binding Prediction tool (Birkir Reynisson, Bruno Alvarez, Sinu Paul, Bjoem Peters, Morten Nielsen. 2020. NetMHCpan-4.1 and NetMHCIIpan-4.0: improved predictions of MHC antigen presentation by concurrent motif deconvolution and integration of MS MHC eluted ligand data. Nucleic Acids Res. 48(W1):W449-W454. doi: 10.1093 / nar / gkaa379) was used to calculate KD ~1X10'5M for binding of this same peptide to mouse MHC H-2K(d). As a consequence of this KD differential, the stronger binding antibodies would be expected to gradually remove that critical target peptide from the MHC groove under equilibrium conditions.

[0083] MHC -peptide + Ab MHC + Ab-peptide

[0084] By doing so this would provide another mechanism for antibody-mediated prevention of T-cell destruction in addition to their aforementioned steric blocking action. In the absence of a MHC peptide the CD8+CTL would have no way to recognize and kill pancreatic beta cells.

[0085] Table 2. Kinetic Parameters of Immobile 15 / 6 Antibody and 8 / 10 Antibody and Flowed LYLVCGERG Peptide

[0086] A typical result is shown in FIG. 3, in which half-maximal attachment of the 8 / 10 antibody to the B9 23 peptide occurred at ~3x 10'11M, while little or no binding to insulin was seen at 1X10'7M. Both 15 / 6 and 8 / 10 bound equally well to the 69-23 and B 15-23 peptides but only weakly to Bio-is. The non-specific MOPC-21 negative control monoclonal antibody displayed no binding to either B9-23 or insulin. Finally, ELISA binding reactions of both the 8 / 10 and 15 / 6 antibodies was blocked via addition of excess free B9-23, but not by an excess of human insulin.

[0087] Further experiments revealed that both 8 / 10 and 15 / 6 antibodies retained the B9-23 peptide but not human insulin during PAGE separation using native pH 8 conditions (FIG 4). Well 2 shows the smeared migration of the B9-23 peptide; Well 1 shows that the non-specific MOPC-21 control antibody has no effect on its movement. In contrast, the peptide is entirely retained by the 15 / 6 antibody (Well 3) and 8 / 10 antibody (Well 4). Further, wells 6-9 indicate that insulin migrates as the same compact band regardless whether it is free (Well 7) or combined with MOPC-21 (Well 6), 15 / 6 (Well 8), or 8 / 10 (Well 9). These results confirm the ELISA conclusion that the 15 / 6 and 8 / 10 antibodies bind exclusively with the free peptide and do not cross-react with insulin. Example 2. Antibodies against B9-23 peptide epitopes are effective at binding monomers and tetramers of peptide-loaded MHC molecules.

[0088] Though blocking antibodies can interact with free B9-23 peptide epitopes, peptide loaded MHC complexes (MHC -peptide) are the direct target of CTLs in CTL-mediated beta cell killing. Specifically, the B15-23 (LYLVCGERG; SEQ ID NO: 1) insulin epitope peptide when attached to MHC has been shown to bind with T-cells, leading to diabetes-associated CTL-mediated pancreatic B cell destruction. Biotinylated monomers of H-2K(d) MHC with the B 15-23 peptide (H-2K(d)-B 15-23; FIGs. 6A, 6B) were synthesized and provided by the NIH. H-2K(d)-B 15-23 monomers formed tetramers in the presence of streptavidin with R- phycoerythrin (PE) for fluorescence detection. As it is possible that peptide-loaded MHC tetramers having peptide tetravalency increases the avidity of interaction with bivalent antibodies (e.g., 15 / 6 antibody, 8 / 10 antibody), both monomers and tetramers were used to demonstrate antibody specificity for peptide loaded MHC complexes.

[0089] CSHLVEALYLVCGERG (SEQ ID NO: 4) was used to immunize mice for hybridoma production and ELISA studies were conducted to assess reaction of both the 8 / 10 and 15 / 6 antibodies to the H-2K(d)-B 15-23 peptides. Both the 8 / 10 and 15 / 6 monoclonal antibodies react strongly with the H-2K(d)-B 15-23 peptide as they do with the B9-23 peptide.

[0090] One advantage to using multivalent H-2K(d)-B 15-23 tetramers is their ability to crosslink with antibodies to form larger complexes that improve the detection of their binding interaction. While the largest possible structure a bivalent antibody (e.g., 15 / 6) can form with the H-2K(d)-B 15-23 monomers is about 250kDa (as in FIG. 7), bivalent antibodies binding to tetramers can form larger complexes having a weight of about l,100kDa (FIG. 8A). Further interactions of these components can lead to the formation of even larger lattices without requiring additional reagents (FIG. 8B).

[0091] Native horizontal agarose gel electrophoresis was used in order to detect the migration of antibodies binding to H-2K(d)-B 15-23 tetramers and the larger aggregates possibly formed by multivalent interactions. 15 / 6 and 8 / 10 antibodies were mixed with homologous H-2K(d)- B 15-23 tetramers or a negative MHC-epitope tetramer control, H-2K(d)-AMQMLKETI. A MOPC-21 monoclonal antibody having no affinity for B 15-23 was used as a negative antibody control. Electrophoresis experiments were run at pH 8 (FIG. 9A) and pH 6 (FIG. 9B). The gels were examined unstained at 366 nm for the inherent phycoerythrin (PE) fluorescence of - l- the tetramer, then stained for protein to reveal the migration of both the tetramer and antibodies.

[0092] The 15 / 6 antibody was found to fully retain the complementary H-2K(d)-B 15-23 tetramer (FIG. 9A, 9B, Well 3) but not the mismatched H-2K(d)-AMQMLKETI tetramer (FIG. 9A, 9B, Well 7). Similarly, the 8 / 10 antibody partially retained the complementary H- 2K(d)-B 15-23 tetramer (FIG. 9A, 9B, Well 4) but not the H-2K(d)-AMQMLKETI tetramer (FIG. 9A, 9B, Well 8). Furthermore, binding of the 15 / 6 antibody to H-2K(d)-B 15-23 tetramer persisted at 37°C (FIG. 9A, 9B, Well 9), while the 15 / 6 antibody continued to not bind to H- 2K(d)-AMQMLKETI tetramer (FIG. 9A, 9B, Well 10). Likewise, FIG. 9B, Wells 9-11 show no binding of 15 / 6, 8 / 10 or MOPC-21 to a mismatched H-2K(d)-IYSTVASSL (SEQ ID NO: 25) tetramer. Finally, no retention was apparent in any wells using a MOPC-21 antibody, indicating that the affinity of 15 / 6 and 8 / 10 antibodies to H-2K(d)-B 15-23 tetramer is specific to the concurrent interaction of the antibodies with the MHC -bound B 15-23 peptide.

[0093] In a follow-up experiment, the amount of 15 / 6 antibody to H-2K(d)-B 15-23 tetramer was titrated in order to examine the dependency of tetramer migration on 15 / 6 antibody concentration. FIG. 11 shows results of this titration. Roughly 3-4-fold molar excess 15 / 6 antibody over H-2K(d)-B 15-23 tetramer (~2.8pg L-tet and l lpg antibody) was sufficient to partially stop tetramer migration (FIG. 11, Well 4), and increasing levels of antibodies fully stopped migration of the tetramer. Together, these results suggest the 15 / 6 antibody can effectively bind H-2K(d)-B 15-23 tetramer at relatively low abundance.

[0094] Example 3- The 15 / 6 antibody binds to the B 15-23 peptide, not the H-2K(d)-B 15-23 tetramer scaffold.

[0095] To further confirm the specificity of 15 / 6 antibody binding to the B 15-23 peptide itself, and not another portion of the H-2K(d)-B 15-23 tetramer, a peptide blocking experiment was performed. As shown in FIG. 12, a molar excess of free B 15-23 peptide over antibody was added to wells along with H-2K(d)-B 15-23 tetramers and the 15 / 6 antibody. In accordance with mass action this surplus of free B 15-23 peptide resulted in the preferential binding of the 15 / 6 antibodies to the free peptides rather than to the H-2K(d)-B 15-23 tetramer. This result affirms that the H-2K(d)-B 15-23 tetramer is retained during electrophoresis only because both the MHC and antibody simultaneously bind this B 15-23 peptide. This MHC-peptide-Ab ternary complex is analogous to the MHC-peptide-TCR ternary complex that leads to cell death (FIG. 1).

[0096] Native horizontal agarose gel electrophoresis was used in order to detect the migration of 15 / 6 antibodies binding to the H-2K(d)-B 15-23 tetramer, as described in Example 2. Inclusion of excess free B 15-23 peptide was found to not affect the migration of the H-2K(d)- B 15-23 tetramer in the absence of 15 / 6 antibodies (FIG. 12, Wells 2 and 7). However, in the presence of 15 / 6 antibody, H-2K(d)-B 15-23 tetramer retarded the migration of H-2K(d)-B 15-23 tetramer (FIG. 12, Wells 3 and 8); excess amounts of free B 15-23 peptide fully reversed retention of H-2K(d)-B 15-23 tetramer in a concentration dependent manner (FIG. 12, Wells 4 and 9). These results indicate that free B 15-23 peptide can block the interaction between 15 / 6 and the tetramer, thereby supporting the specificity of the 15 / 6 antibody to the B 15-23 peptide, and not another portion of H-2K(d)-B 15-23 tetramer.

[0097] Example 4- Alternative ELISA systems for detection of bivalent antibody binding to H- 2K(d)-B 15-23 monomer.

[0098] When applied to H-2K(d)-B 15-23 monomers (all such monomers here and throughout were biotinylated), the gel retention experiments of Example 3 were unsuccessful, possibly due to the size or univalency of the monomers. In this Example, two alternative methods of detecting bivalent antibody binding to peptide loaded MHC complexes were developed.

[0099] El ELISA Protocol

[0100] A first ELISA procedure (“El”) for detecting the specific reaction between the 15 / 6 antibody and its complementary H-2K(d)-B 15-23 monomer using a streptavidin-coated plate is shown in FIGs. 13A-13D. In the first step of this procedure, 15 / 6 antibodies and the H- 2K(d)-Bi5-23 monomer or negative control H-2K(d)-AMQMLKETI (SEQ ID NO: 15) monomer were mixed and allowed to interact in PBS. That allowed for the formation of a H- 2K(d)-Bi5-23-15 / 16 complex, while 15 / 6 and H-2K(d)-AMQMLKETI being mismatched should remain disengaged. Then 4% BSA / 2x PBS was added to each of the samples listed in Table 3 before loading them into separate wells of the streptavidin-coated ELISA plate (FIG. 13A). While H-2K(d)-B 15-23-15 / 6 complexes are expected to bind to streptavidin, only H- 2K(d)-AMQMLKETI monomers were expected to bind to the same, leaving the 15 / 6 antibody free, such that a wash would remove those uncoupled antibodies (FIG. 13B). Addition of anti-IgG peroxidase, which couples to remaining antibodies, will bind only to the H-2K(d)-B 15-23-15 / 6 complexes; any unbound anti-IgG peroxidase is removed in a subsequent wash (FIG. 13C). The binding of the secondary antibody ultimately produces a color reaction with its substrate (FIG. 13D). Those of skill in the art will appreciate how this procedure can be used to detect any binder (e.g., antibody) of any suitable peptide bound to a biotinylated MHC monomer.

[0101] Table 3 shows specificity for the 15 / 6 antibody against the H-2K(d)-B 15-23 monomer per the El ELISA procedure. In this experiment, detection of color (450nm) was determined from wells receiving the 15 / 6 antibody only, MOPC-21 only, H-2K(d)-B 15-23 monomer only, 15 / 6 antibody plus H-2K(d)-B 15-23 monomer, or MOPC-21 plus H-2K(d)-B 15-23 monomer. Neither 15 / 6 antibody nor MOPC-21 alone bound to streptavidin. Indeed, wells in which MOPC-21 was initially present (Wells 4 and 5) did not produce color (450nm), and only wells in which both H-2K(d)-B 15-23 monomer and the 15 / 6 antibody were both present (Well 2) showed successful binding. Accordingly, only H-2K(d)-B 15-23 monomers bound to 8 / 10 antibodies were analogously detected per this El ELISA protocol.

[0102] Table 3. El ELISA Showing H-2K(d)-Bis-23 Monomer Binding by 15 / 6 Antibody

[0103] In a related experiment, the specificity of the 15 / 6 antibody for B 15-23 peptide was tested by comparing detection of color (450nm) from wells containing 15 / 6 antibody plus H- 2K(d)-B 15-23 monomer in the presence of excess B 15-23 peptide, and / or excess HLVEALYLV (SEQ ID NO: 2) peptide, according to the El ELISA protocol. As shown in Table 4, the combination of both 15 / 6 antibody and H-2K(d)-B 15-23 monomer indicates formation of H- 2K(d)-B 15-23-15 / 6 complex (Well 4). It was also found that an excess of B15-23 peptide was sufficient to disrupt formation of 15 / 6-H-2K(d)-B 15-23 complexes when present before (Well 5) or after (Well 7) interaction of 15 / 6 antibodies and H-2K(d)-B 15-23 monomers. However, this disruptive effect was specific to an excess of B 15-23 peptide; an excess of the weak antibody-binding HLVEALYLV (SEQ ID NO: 2) peptide did not affect formation of 15 / 6-H- 2K(d)-B 15-23 complexes (Wells 6 and 8). Table 4. Excess B 15-23 Peptide both Prevents and Disrupts the Antibody / MHC Interaction

[0104] These results indicate that 1) the complex between the 15 / 6 antibody and the B 15-23 in the H-2K(d) groove is in a dynamic equilibrium. An excess of B 15-23 peptide not only prevents the binding of Ab and MHC but also disrupts the pre-formed MHC -peptide- Ab complex.

[0105] E2 ELISA Protocol

[0106] A second ELISA procedure (“E2”) for detecting the specific reaction between the 15 / 6 antibody and its complementary H-2K(d)-B 15-23 monomer uses 15 / 6 or 8 / 10 antibodies adsorbed to the plate, and a horseradish peroxidase-streptavidin conjugate is shown in FIGs. 14A-14C.

[0107] In the first step of this procedure, H-2K(d)-B 15-23 monomers are added to a plate with 15 / 6 or 8 / 10 antibodies adsorbed onto its surface (FIG. 14A). Addition of H-2K(d)-B 15-23 monomers is expected to result in binding to the adsorbed antibody, resulting in the formation of a H-2K(d)-B 15-23 -antibody complex (FIG. 14B). Finally, addition of a horseradish peroxidase-streptavidin conjugate is used to bind to the H-2K(d)-Bi5-23-antibody complex, thereby producing color for detection (FIG. 14C). Those of skill in the art will appreciate how this second ELISA procedure can be also used to detect any binder (e.g., antibody) of any suitable peptide bound to any biotinylated MHC monomer. Table 5 and Table 6 show confirmation of findings with the E2 ELISA protocol. Table 5 shows inhibition of the H-2K(d)-B 15-23- 15 / 6 complex formation by excess B15-23 peptide. Similarly, Table 6 confirms the findings of Table 3, in which H-2K(d)-B 15-23 monomer is found to bind to both anti-B 15-23 antibodies 15 / 6 and 8 / 10 (Wells 2 and 4) but not to the negative control MOPC-21 antibody (Well 3). In this experiment, the 8 / 10 antibody (Well 4) showed comparable binding to H-2K(d)-B 15-23 monomers; this finding is expected, as the 8 / 10 antibody also binds the B 15-23 as indicated in Example 1.

[0108] Table 5. E2 ELISA Showing Inhibition of 15 / 6-H-2K(d)-Bis-23 Complex Formation

[0109] Table 6. E2 ELISA Showing Selectivity for the 15 / 6 and 8 / 10 Anti-Bis-23 Antibodies

[0110] Both the El and E2 ELIS As allowed for further experiments testing the specificity of the 15 / 6 and 8 / 10 antibodies to H-2K(d)-B 15-23 monomer. Table 7 shows the results of two experiments in which H-2K(d)-B 15-23 monomer, H-2K(d)-SYIGSINNI peptide monomer (SEQ ID NO: 16), or H-2K(d)-AMQMLKETI monomer (SEQ ID NO: 15) were tested for their interaction with the 15 / 6 antibody according to both the El and E2 protocols. The H- 2K(d)-SYIGSINNI monomer and H-2K(d)-AMQMLKETI monomer served as “wrong peptide” controls; as expected, neither was detected regardless of 15 / 6 antibody presence or absence (Wells 3-6). Reactivity of the 15 / 6 antibody and H-2K(d)-B 15-23 monomer complex (Well 2) was confirmed by both El and E2 ELISA protocols.

[0111] Table 7. E2 ELISA Showing Specificity of 15 / 6 Antibody for B 15-23 Peptide in the MHC Monomer Groove

[0112] Finally, the ability of the El ELISA to detect antibodies reactive with peptide-loaded MHC complexes was assessed under condition anticipated during hybridoma cell screening for monoclonal antibodies that bind to peptides residing in their MHC groove. The 15 / 6 antibody was added to 50pl media at a 10-100 pg / ml concentration range to simulate hybridoma clone secretion into microtiter wells. Table 8 shows that El ELISA is capable of detecting low levels of complexed 15 / 6 antibody and H-2K(d)-B 15-23 monomer (Wells 2-5), without detecting uncomplexed 15 / 6 antibody (Wells 6 and 7).

[0113] Table 8. El ELISA Hybridoma Simulation to Selectively Screen for MHC-Peptide Binders

[0114] Overall, both the El and E2 ELISA protocols find that the 15 / 6 antibody binds H- 2K(d)-B 15-23 with high specificity. The El ELISA also provides a robust alternative method to screen for anti-peptide clones, as it exclusively selects antibodies with an ability to recognize peptides simultaneously attached to the MHC. These assays are quick and easy to perform, allowing for scaling to assay numerous clones.

[0115] Example 5- Live-cell assessment of 15 / 6 antibody binding to H-2K(d)-Bis-23.

[0116] P815 mouse mastocytoma cells were used as surrogates for pancreatic beta cells to demonstrate that 15 / 6 antibodies can react with peptide-loaded MHCs on the surface of live cells. The P815 mouse mastocytoma cell line expresses H-2K(d). When incubated with B15-23 peptide, H-2K(d)-B 15-23 can form and become a target for lysis by CTLs. If the 15 / 6 antibody indeed binds H-2K(d)-B 15-23 presented on the membrane of living cells, CTL-mediated destruction of P815 cells and by extension beta cells should be blocked. Thus, a live cell ELISA protocol (Jiang. W., et al. (2014). BMC Plant Biol, 14:244) was adapted to assess 15 / 6 antibody binding to H-2K(d)-B 15-23 on P815 cell surfaces.

[0117] Biotinylated 15 / 6 (15 / 6-biotin) antibodies were prepared so an 15 / 6-biotin:strep-HRP complex could be formed (FIG. 15A). This complex was then used as a single-step reagent to detect binding activity of the 15 / 6-biotin complex. MHC on P815 cells was either left unloaded as a negative control (Fig. 15B) or was pre-loaded with B 15-23 peptide for 1 hr at 37°C and then washed to serve as a target for the 15 / 6-biotin: strep-HRP complex, Fig. 15C, Table 9. Alternatively, the MHC on P815 cells was pre-loaded with the correct B15-23 peptide or a mismatched peptide that is non-reactive with the 15 / 6-biotin: strep-HRP complex, Table 10. Binding of the 15 / 6-biotin: strep-HRP complex was then tested in varying conditions. Blocking reagents (anti-Fc receptor antibodies, bovine serum albumin, and normal mouse serum) were used to suppress non-specific binding interactions.

[0118] Table 9 shows the peptide dependency for binding of the 15 / 6 antibody: streptavidin HRP complex to living P815 cells. Attachment of the complex required pre-loading cells with the peptide Well 1 versus Well 2. Adding a large excess of B 15-23 peptide competitively blocked the complex and abolished its binding to those preloaded cells, Well 3. Activity of 15 / 6-biotin binding of B 15-23 peptide-loaded MHCs on P815 cell surfaces was observed (Well 2).

[0119] Table 9. Live Cell ELISA Showing 15 / 6-biotin:strep-HRP Complex Binding to Peptide- loaded MHC on P815 Cells

[0120] Specificity of 15 / 6-biotin for the MHC-loaded B 15-23 peptide was demonstrated by incubating P815 cells with different amounts of either the B 15-23 peptide or a negative control GYKDGNEYI peptide (“G”; SEQ ID NO: 17) which is an epitope of Listeriolysin O (LLO91- 99) that is known to also bind to H-2K(d). Binding of 15 / 6-biotin to MHC-loaded G-peptide on P815 cells was not observed even at high levels, as shown in Table 10. Additionally, the live cell ELISA worked equally well when tested with P815 cells separated from potential dead cells or fragments using a Ficoll density centrifugation technique.

[0121] Table 10. Live Cell ELISA Showing Specificity for B15-23 Peptide on Loaded P815 Cells

[0122] Finally, direct fluorescent staining of peptide-loaded vs untreated live P815 cells was performed using 15 / 6 antibody-fluorescein (FITC) conjugates. As shown in the confocal micrographs FIG. 16A low magnification FIG. 16B high magnification, the B 15-23 peptide is required for attachment of the FITC-labeled 15 / 6 antibody to the cells. They displayed a ring of fluorescence around the cell circumference, indicative 5 of membrane staining. Overall, these findings with live P815 cells show that the anti-insulin B9-23 epitope antibodies (e.g., 15 / 6, 8 / 10) can be useful for blocking CTE-mediated pancreatic B-cell destruction.

[0123] Example 6- Antibody binding to peptides exchanged onto MHC II monomers.

[0124] Given the success of the antibodies described herein in binding to MHC I tetramers and monomers, it was next tested whether similar insulin B9-23 epitopes bound to MHC II molecules (I-A(g7)) could also be detected. Both El and E2 ELISA protocols were used to test the possibility that biotinylated I-A(g7) monomers made with B9-23 epitopes and other epitopes were accessible to anti-peptide antibodies. MHC II monomers made with the following peptides were tested: GYKDGNEYI peptide (“G”; SEQ ID NO: 17), VEALYLVCG (“V”; SEQ ID NO: 18), EALYLVCGE (“E”; SEQ ID NO: 19), HLVERLYLVAGEEG (“HA”; SEQ ID NO: 20), HLVERLYLVCGEEG (“HC”; SEQ ID NO: 21), PVSKMRMATPLLMQA (“P”; SEQ ID NO: 22); and HLVEALYLVCGERG (B10- 23; SEQ ID NO: 23).

[0125] Since this approach showed little or no antibody binding to those available MHC II bound peptides, they were exchanged with the B 15-23 epitope peptide prior to the assay. This interchange was accomplished by adding an excess of a given peptide to the monomer and incubating overnight at 37°C. These peptide-exchanged biotinylated MHC monomers were then bound to the streptavidin plate, which was subsequently washed to remove unbound peptide. Thereafter, the 15 / 6 antibody was added to attach to the newly immobilized MHC II- peptide combination, and the amount bound was quantified using an HRP-labeled anti-mouse IgG reagent, as shown in Table 11.

[0126] Table 11 also shows that MHC II monomers preloaded with partially matched V- peptide and E-peptide were insufficient for antibody binding but were able to bind to both I- A(g7) MHC II monomers after peptide exchange. Importantly, the H-2K(d)-B 15-23 monomer retained its antibody binding capacity when exchanged using excess B 15-23 peptide (Well 6) but lost it after replacement by the H-2K(d) reactive, but antibody mismatched, G-peptide (Well 7). When the H-2K(d)-G combination was assayed immediately after mixture, no exchange had occurred, showing that the interchange required incubation overnight at 37° C. The ELISA signal of the H-2K(d)-B 15-23 monomer was undiminished by overnight incubation at 37° C alone or when treated with excess Bio-is peptide, HLVEALYLV, that is non-reactive with both MHC H-2K(d) and the 15 / 6 antibody.

[0127] Table 11. ELISA Binding of 15 / 6 Antibodies to Peptide Loaded I-A(g7) Biotinylated Monomers

[0128] Similar results to those shown in Table 11 were obtained with the I-A(g7) HLVERLYLVAGEEG (SEQ ID NO: 20), I-A(g7) HLVERLYLVCGEEG (SEQ ID NO: 21) and I-A(g7) PVSKMRMATPLLMQA (SEQ ID NO: 22) biotinylated MHC II monomers. Namely, little or no antibody binding to those listed monomers prior to peptide exchange but substantial binding following substitution with LYLVCGERG.

[0129] The 15 / 6 and 8 / 10 complementary sequence LYLVCGERG is contained within the larger B 10-23 HLVEALYLVCGERG (SEQ ID NO: 23). But its exchange into I-A(g7) monomers was difficult because the peptide alone was capable of binding to the streptavidin ELISA plate. Accordingly, a much lower peptide concentration was used for exchange, and plating occurred in 10% dialyzed non-fat dry milk. Under these conditions, varying degrees of exchange were obtained but these were less than with the smaller B 15-23 peptide; results are shown in Table 12.

[0130] Table 12. Peptide exchange into MHC II Using B10-23

[0131] Bio-23 peptide HLVEALYLVCGERG

[0132] H-monomer = I-A(g7) HLVERLYLVCGEEG

[0133] E-monomer = I-A(g7) EALYLVCGE biotinylated monomer V-monomer = I-A(g7) VEALYLVCG biotinylated monomer

[0134] Neutravidin plates were washed 2x and then blocked Ihr with concentrated ST-01 (Surmodics). 0.5pl peptide, 1 pl monomer were used with final 5pl PBS and incubated overnight at 37°C. The results show that the 15 / 6 antibody binds to its homologous epitope on a large peptide in the MHC II groove. The 15 / 6 and 8 / 10 antibodies were found to have a strong preference for a positively charged R residue at that position 22. However, binding with that mismatched monomer was observed when the peptide was exchanged with the fully complementary B 10-23 peptide (Table 12).

[0135] Altogether these peptide exchange results further support the contention that peptidespecific antibodies can attach to a suitably matched resident peptide in both MHC I and MHC II binding grooves. These results also suggest that the same anti-peptide antibody (e.g., 15 / 6) could simultaneously inhibit the deleterious effects of MHC I dependent CD8 cells and MHC II dependent CD4 cells in various autoimmune diseases, such as Type I diabetes.

[0136] Methods and Materials

[0137] Most peptides were synthesized at the Tufts University Analytical Core Facility (Boston, MA). A Listeria monocytogenes Listeriolysin O peptide (GYKDGNEYI; SEQ ID NO: 17) was purchased from Anaspec (Fremont, CA). B 15-23 and Bio-is peptide solutions at 2.8 x 10'2M were made in a small volume of DW + 1 l 0.5M Tris, pH 8 with 0.5pl additions of 0.5 M NaOH until they were clear and at pH 8. Typically, Ipl of those peptide solutions were added to the reaction mix for inhibition experiments.

[0138] Multiple immunizations with complete and incomplete Freund's adjuvant were carried out to elicit antibodies in Balb / c mice, using 50 pg of a thioether conjugate of either the CSHLVEALYLVCGERG (SEQ ID NO: 4) peptide or the CGSHLVEALYLV (B7-18) peptide (SEQ ID NO: 5) and PEGylated maleimide KLH (Thermo Scientific; Waltham, MA). Antipeptide monoclonal antibodies were produced by hybridoma fusion of spleen and lymph node cells from a CSHLVEALYLVCGERG (SEQ ID NO: 4) immunized mouse. ELISAs were used to screen hybridoma supernatants for reaction with the peptide plus an absence of binding to intact human insulin.

[0139] Monoclonal antibodies were tested via ELISA using peptides or insulin adsorbed onto 4BX FLT polystyrene plates from Weber Scientific (Hamilton, NJ) at 100 pg / ml in PBS for 2 hours. Wells were subsequently blocked for 1 hr using 0.2% Tween / PBS. After a 2 hr incubation and wash steps with 0.05% Tween / PBS, a horseradish peroxidase-labeled antimouse IgG and TMB substrate (Surmodics; Eden Prairie, MN) were used to detect peptide- or insulin-bound antibodies. Except for the adsorption step, all ELISA plate incubations described herein were performed on a nutating rocker. Data were analyzed and graphed using GraphPad Prism, (GraphPad Software; Boston, MA).

[0140] Binding kinetics analyses were performed by axiVEND (Winter Garden, FL) using an iRiS MX- 100 kinetics system. Immobilization of peptides and antibodies onto the microarray chip was via amine-reactive copoly(DMA-NAS-MAPS). Both the 15 / 6 and 8 / 10 monoclonal antibodies were analyzed with the B 15-23 and B 10-23 peptides.

[0141] New England Immunotechnology provided the 15 / 6 and 8 / 10 anti-peptide monoclonal antibodies and the control MOPC-21 antibody from the DCTD Tumor Repository (Frederick, MD). All three were purified from ascites on separate Protein-G Sepharose (Lytic Solutions, Madison WI) columns eluted with 4M MgCh. An ISO-M6 cassette (Antagen Biosciences; Canton, MA) indicated that both antipeptide antibodies were IgG2b, kappa.

[0142] Native PAGE ligand retention experiments were performed on 4-20% MP TGX gels (BioRad; Hercules, CA) using a 25 mM Tris, 192 mM glycine running buffer and Focus Fast silver staining (G-Biosciences; St. Louis, MO). Each antibody (60pg = 4xlO'10moles) was mixed with an equal mole amount of either the B9-23 peptide or human insulin before separation on the gel to reveal any binding interactions. Horizontal native agarose gel electrophoresis was carried out on 0.5% SeaKem Gold agarose gels using either a pH 8 TBE buffer or a pH 6 100 mM histidine, 100 mM MES buffer (20) and run at 50 volts with cooling. The starting buffers contained Ficoll 400 (Dot Scientific Burton MI) to increase sample density and a bromophenol blue tracking dye.

[0143] NIH tetramers were supplied at 1.4 mg / ml (~2.7 x 10'6M). Unless otherwise indicated 2 pl / 2.8pg / 2.7 xlO'12moles of tetramer and 13.3pg / 8.9 x 10'11moles of antibody plus any other components in the same volume were mixed and an equal volume of starting buffer was added prior to electrophoresis on the native agarose gel.

[0144] Gels were examined unstained in a light box at 366 nm for the inherent R- phycoerythrin (PE) fluorescence of the tetramer. They were then stained for protein with Coomassie G-250 Bio-Safe (BioRad; Hercules, CA) to reveal the migration of both the tetramer and antibodies.

[0145] NIH biotinylated monomers were provided at 2mg / ml (4 x 10'5M) and Ipl containing 2pg / 4 x 10-11moles was used in all of the ELISA experiments. In the El ELISA protocol, the monomer was mixed with 6.7pg / 4.5 x 10'11moles of a monoclonal antibody plus any other components also in microliter volumes. All small volume reaction mixtures were made in 0.2 mL PCR tubes. The experimental and appropriate control reactions were then diluted with 100 pl of 4% BSA / 2xPBS and loaded into the wells of a Streptavidin-coated, SP-11 ELISA plate from ACROBiosystems (Newark, DE). The biotinylated monomer and any antibodies attached to it was allowed to bind to the plate for 1 hr at room temperature before washing 3- times with 0.05% Tween / PBS. A horseradish peroxidase-labeled anti-mouse IgG and then its TMB substrate were used to detect monomer-bound antibodies.

[0146] The E2 ELISA protocol used monoclonal antibodies at 100 pg / ml in PBS adsorbed to the ELISA plate as described above, but 4% BSA / 2xPBS served as the block. After 1 hr the wells were washed 3-times with 0.05% Tween / PBS. Biotinylated-monomers (Ipl) plus any other components were mixed in 0.2 mL PCR tubes, diluted with lOOpl 4% BSA / 2xPBS, added to the well and allowed to react with the plate-bound antibodies for 1 hr. After 3 washes with 0.05% Tween / PBS any antibody bound, biotinylated monomer was detected using a Streptavidin-horseradish peroxidase conjugate, STN-NH913 from ACROBiosystems (Newark DE) in 4% BSA / 2xPBS and then the TMB substrate.

[0147] The live-cell ELISA followed a described protocol but was modified to accommodate animal cells, which were centrifuged at 400g. The 15 / 6 monoclonal antibody was biotinylated using a Flexible Biotin Antibody Labeling Kit for Mouse IgG2b from Proteintech (Rosemont IL). This was mixed with a 5-fold molar excess (based on protein) of Streptavidin-Peroxidase Polymer, Ultrasensitive from Sigma (Saint Louis MO) to form the pre-assembled complex. P815 was obtained as an ascites from the DCTD Tumor Repository (Frederick, MD). Cultured P815 cells (107) in lOOpl media were incubated for 1 hr at 37° C with peptide and then washed using 1.5 ml of 4% BSA / PBS. Prior to and during the 1 hr exposure of cells to the complex, lOOpl of blocking agents which included a combination of 2pl anti-mouse CD16 / CD32 clone 2.4G2 from Bio X Cell (Lebanon, NH), 4% BSA and 10% normal mouse serum was used to suppress non-specific binding. Upon addition of Ipl complex / lOOpl cells the final antibody concentration was 3xl0'9M. Blocking used a final concentration of 2.8 x 10'3M peptide. All binding and wash operations were carried out at 4° C and centrifugation at 400g. After the final 3x washes, TMB was added and after development, acidification plus a final centrifugation at 3,000g the supernatant was read at 450nm.

[0148] Standard methods were used to attach fluorescein isothiocyanate, FITC (ApexBio; Houston, TX) to the 15 / 6 monoclonal antibody and isolate the conjugate by gel filtration. P815 cells were prepared blocked and handled as described for the live-cell ELISA but lOpl of the FITC conjugate was added instead of the HRP-complex. The labeled cells were examined at the Beth Israel Deaconess Medical Center Confocal Core Facility (Boston, MA) using a Zeiss LSM 880 Inverted Live-cell Laser Scanning Confocal Microscope.

[0149] Peptide exchange was accomplished by using Ipl (2 x 10'11moles) of biotinylated monomer plus 0.5pl (1.4 x 10'8moles) peptide or, as a control, no added peptide in 5pl PBS and incubating overnight at 37° C. These mixtures were diluted into lOOpl 4% BSA / 2xPBS or 10% dialyzed non-fat dry milk and then added to the streptavidin plate for Ihr. Wells were subsequently washed with 0.05% Tween / PBS to remove unbound peptide. Thereafter, the 15 / 6 antibody in 4% BSA / 2xPBS was added and after washing the amount bound was quantified using an HRP-labeled anti-mouse IgG reagent in 4% BSA / 2xPBS.

[0150] Example 7- Assessment of antibody binding to Bio-is peptide using B7-18 peptide ELISA.

[0151] Sera of mice that had been immunized and boosted with a B7-18, CGSHLVEALYLV peptide (SEQ ID NO: 5) thioether-linked to KLH, were tested by ELISA. Anti-peptide antibodies specific for Bio-is HLVEALYLV peptide were found. The data in Table 13 show the plate- adsorbed CGSHLVEALYLV peptide (SEQ ID NO: 5) reaction with antibodies in sera from mouse 1 and 2 following their immunization with a CSHLVEALYLV (SEQ ID NO: 24)-KLH conjugate. No peptide-binding antibodies were found in serum from non- immunized mice. The results indicate that mouse 1 produced more or higher affinity antibodies reactive with the adsorbed CGSHLVEALYLV peptide (SEQ ID NO: 5).

[0152] Table 14 shows the complete blockade of antibody binding to CSHLVEALYLV (SEQ ID NO: 24) by its smaller component Bio-is HLVEALYLV part. This demonstrates the antibody specificity for that clinically important human HLA 9-mer. The 15 / 6 and 8 / 10 monoclonal antibodies recognized the LYLVCGERG (SEQ ID NO: 1) B15-23 peptide and therefore showed little or no binding to Bio-is or B7-18.

[0153] Table 13. ELISA for Serum Antibodies Binding to the CGSHLVEALYLV (B7-18) Peptide

[0154] Table 14. Excess HLVEALYLV (BIO-18) Peptide Blockade of Antibody Binding to CGSHLVEALYLV (B7-18) Peptide

[0155] Example 8 - Detection of MHC-peptide reactive anti-peptide antibodies in sera from immunized mice

[0156] Sera of mice that had been immunized and boosted with CSHLVEALYLVCGERG (SEQ ID NO: 4) conjugated to KLH, were tested using the El ELISA. The data in Table 15 show the H-2K(d)-B 15-23 monomer reaction with sera from mouse 1 and 2 following their immunization. No monomer-binding antibodies were found in serum from non-immunized mice. The results indicate that mouse 1 produced more or higher affinity antibodies reactive with the monomer-bound LYLVCGERG peptide complex. Also, the data in Table 15 demonstrate that the El ELISA can be used to exclusively detect and monitor MHC -peptide reactive anti-peptide antibodies in sera of subjects immunized with an appropriate peptide immunogen.

[0157] Table 15. El ELISA Used to Detect MHC-peptide Reactive Antibodies in Serum of Peptide Immunized Mice

[0158] Example 9 - Results from further experiments

[0159] Table 16 shows the results from testing hybridoma cell media with added antibody using a monomer-based ELISA. The results indicate that the ELISA was capable of detecting low levels of anti-peptide monoclonal antibody that bind to a MHC-peptide monomer in a volume of hybridoma media usually sampled from a cell fusion microtiter well. Specificity for the Ab peptide-MHC ternary complex was ensured since no ELISA signal was observed in the absence of the added peptide bearing monomer. Accordingly, the same selection strategy can be used to screen for and select other binding agents that bind peptide within a complex with a MHC.

[0160] Table 16. Hybridoma Simulation to Selectively Screen for MHC-Peptide Binders

[0161] Media = Typical Hybridoma Cell Culture Media, 50pl

[0162] Antibody = 15 / 6 Anti-LYLVCGERG monoclonal antibody

[0163] Monomer = MHC I H-2K(d) LYLVCGERG biotinylated monomer Table 17 shows an antibody binding titration of B 15-23 peptide residing in the H-

[0164] 2K(d) binding groove. The results are indicative of a single specific and saturable binding site based on the law of mass action.

[0165] Table 17. 15 / 6 Ab Titration of B 15-23 Peptide Residing in the H-2K(d) Binding Groove

[0166] The El ELISA was used to detect and quantify monomer-peptide reactive monoclonal antibodies in the 55 / 3 clone cell culture media. Addition of known amounts of the 15 / 6 antibody allowed for estimation of the 55 / 3 antibody level in the media (e.g., 55 / 3 media high cell concentration ~ 4-5pg / ml and 55 / 3 media low cell concentration ~ Ipg / ml) (Table 18). This demonstrates the usefulness of that assay to exclusively select for clones that bind to a specific peptide while bound within its MHC groove.

[0167] Table 18. ELISA of H-2K(d)-Bis-23 Monomer Plus 55 / 3 Clone Media It has been established that anti-LYLVCGERG insulin peptide antibodies bind to this resident peptide in a mouse H-2K(d) MHC I cleft. Table 19 below shows that a 10-250-fold excess that B 15-23 peptide was used to produce a human HLA-A*24:02-B 15-23 monomer by replacing the Hep peptide. The data show that only the combination of monomer plus antibody (wells 5-8) gave a significant response above peptide alone. Both thel5 / 6 and 8 / 10 monoclonal antibodies bound to this clinically important peptide-loaded monomer demonstrating that those antibodies can access the B 15-23 peptide when it is presented within a human HLA-A*24:02 MHC I binding groove. That HLA-peptide system is thought to be related to the cytotoxic activity of CD8+ T lymphocytes against insulin in type 1 diabetes (K. Kimura, T. Kawamura, S. Kadotani, H. Inada, S. Niihira, T. Yamano, Peptide- specific cytotoxicity of T lymphocytes against glutamic acid decarboxylase and insulin in type 1 diabetes mellitus. Diabetes Research and Clinical Practice 51, 173-179 (2001)). As such it can be important for the design of a peptide antigen specific immunotherapy.

[0168] Table 19. ELISA Showing Anti-Bis-23 Binding to Peptide Exchanged HLA-A*24:02-Bis- 23 Monomer

[0169] HLA-A*24:02-Hep = HLA-A*24:02 SWPKFAVPNLGSGGSGGSGLEVLFQ

[0170] Likewise in Table 20 an additional similar system with importance to human diabetes, namely antibodies reactive with the insulin HLVEALYLV peptide (B7-18) in the human HLA-A*02:01 MHC I binding cleft, was explored (G. G. M. Pinkse, O. H. M. Tysma, C. A. M. Bergen, M. G. D. Kester, F. Ossendorp, P. A. van Veelen, B. Keymeulen, D. Pipeleers, J. W. Drijfhout, B. O. Roep, Autoreactive CD8 T cells associated with beta cell destruction in type 1 diabetes. Proc Natl Acad Sci U S A 102, 18425-18430 (2005)). Table 20. Reaction of Purified Anti-Ib is Serum IgG with the HLA-A*02:01 HLVEALYLV Monomer

[0171] 1 1 of concentrated Anti-Eh-ix mouse serum IgG Streptavidin Plate dilution buffer 4%BSA / 2xPBS

[0172] Purified anti-By-is IgG bound to the clinically important human H LA- A *02:0- B 10-18 (HLVEALYLV) monomer. Note this Bio-is peptide is different than the B 15-23 sequence recognized by the other monoclonal antibodies described and required immunizing mice with a new KLH-B7-18 immunogen. Binding data were acquired using purified IgG isolated from their pooled sera. These results with different antibodies and a new peptide target bound within the human HLA complex further expand the discoveries as provided herein regarding peptide accessibility in the MHC system.

[0173] Each of the foregoing patents, patent applications and references that are recited in this application are herein incorporated in their entirety by reference. Having described the presently preferred embodiments, and in accordance with the present invention, it is believed that other modifications, variations and changes will be suggested to those skilled in the art in view of the teachings set forth herein. It is, therefore, to be understood that all such variations, modifications, and changes are believed to fall within the scope of the present invention as defined by the appended claims.

Claims

1. We claim:CLAIMS1. A method of inhibiting T cell binding to a major histocompatibility complex (MHC) - peptide complex, wherein the peptide of the MHC-peptide complex is associated with a T cell-mediated disease, the method comprising: placing in contact with the MHC-peptide complex a composition comprising a pharmaceutically acceptable carrier and a means for specifically binding the peptide while complexed to the MHC molecule of the MCH-peptide complex.

2. The method of claim 1, wherein the MHC molecule is a MHC Class I molecule.

3. The method of claim 1, wherein the MHC molecule is a MHC Class II molecule.

4. The method of claim 2, wherein the T cell binding is CD8 T cell binding.

5. The method of claim 3, wherein the T cell binding is CD4 T cell binding.

6. The method of claim 5, wherein the CD4 T cell binding is regulatory T cell binding.

7. The method of any one of the preceding claims, wherein the T cell-mediated disease is an autoimmune disease.

8. The method of any one of the preceding claims, wherein the T cell-mediated disease is cancer.

9. The method of any one of the preceding claims, wherein the peptide is any one of the peptides provided herein.

10. The method of any one of the preceding claims, wherein the means is an antibody or antigen-binding portion thereof or any one of the alternatives provided herein.

11. The method of any one of the preceding claims, wherein the placing in contact is performed in vitro or in vivo, such as by administering the composition to a subject.

12. A method of producing an antibody that specifically binds a peptide of a MHC- peptide complex, wherein the peptide of the MHC-peptide complex is associated with a T cell-mediated disease, the method comprising administering to a subject the peptide.

13. The method of claim 12, wherein the peptide is coupled to a carrier.

14. The method of claim 13, wherein the carrier is keyhole limpet hemocyanin (KLH).

15. The method of claim 12, wherein the peptide is complexed to a MHC monomer or aMHC tetramer or MHC on the membrane of a cell.

16. The method of any one of claims 12-15, wherein the method further comprises administering an adjuvant to the subject.

17. The method of any one of claims 12-16, wherein the method further comprises selecting an antibody that specifically binds to the peptide when complexed to a MHC molecule and / or that specifically binds to the peptide as a free peptide, and, optionally, selecting an antibody that does not specifically bind to a native protein that comprises the peptide.

18. The method of any one of claims 12-17, wherein the MHC molecule is a MHC Class I and / or MHC Class II molecule.

19. The method of any one of claims 12-18, wherein the method further comprises testing the antibody for specific binding to the peptide when complexed to a MHC molecule and / or testing the antibody for specific binding to the peptide as a free peptide, and, optionally, testing the antibody for not specifically binding to a native protein that comprises the peptide.

20. The method of claim 19, wherein the testing of the antibody for specific binding comprises the any one or more or all of the steps of any one of such methods provided herein.

21. The method of any one of claims 12-20, wherein the peptide is any one of the peptides provided herein.

22. A method of selecting a binding agent, the method comprising: a) placing in contact with a MHC -peptide complex a candidate binding agent, wherein the peptide of the MHC-peptide complex is associated with a T cell-mediated disease; b) testing the candidate binding agent for specific binding to the peptide when complexed to the MHC molecule of the MHC-peptide complex and / or for specific binding to the peptide as a free peptide, and, optionally, testing the candidate binding agent for not specifically binding to a native protein that comprises the peptide; and c) selecting the binding agent that specifically binds the peptide when complexed to the MHC molecule of the MHC-peptide complex and / or that specifically binds to the peptide as a free peptide, and, optionally, selecting the binding agent that does not specifically bind to a native protein that comprises the peptide.

23. The method of claim 22, wherein the peptide is complexed to a MHC monomer or a MHC tetramer or MHC on the membrane of a cell.

24. The method of claim 22 or 23, wherein the MHC molecule is a MHC Class I and / or MHC Class II molecule.

25. The method of any one of claims 22-24, wherein the method is performed on a whole serum sample or hybridoma supernatant sample.

26. The method of any one of claims 22-25, wherein the peptide is any one of the peptides provided herein.

27. The method of any one of claims 22-26, wherein the testing of the candidate binding agent for specific binding comprises the steps of any one or more or all of such methods provided herein, such as any one of the ELISA methods provided herein.

28. The method of any one of claims 22-27, wherein the method comprises:mixing the candidate binding agent and the MHC-peptide complex, optionally wherein the MHC-peptide complex is labeled, such as with biotin; loading the mixture of the candidate binding agent and the MHC-peptide complex into well(s) of a plate for performing ELISA, such as a streptavidin-coated plate; removing any uncoupled candidate binding agent, such as by washing away any uncoupled binding agent; adding a reagent or set of reagents, such as anti-IgG peroxidase, that can be used to detect bound candidate binding agent that remains on the plate, optionally wherein unbound reagent or set of reagents are removed, such as by washing; and detecting the reagent or set of reagents.

29. The method of claim 28, wherein the reagent or set of reagents comprises anti-IgG peroxidase and / or a secondary binding antibody.

30. The method of any one of claims 22-27, wherein the method comprises adsorbing a candidate binding agent to a plate for performing ELISA; loading the MHC-peptide complex into well(s) of the plate, optionally wherein the MHC-peptide complex is labeled, such as with biotin; optionally removing any uncoupled MHC-peptide complex, such as by washing away any uncoupled MHC-peptide complex; adding a reagent or set of reagents, such as a horseradish peroxidase streptavidin conjugate, that can be used to detect bound MHC-peptide complex, optionally wherein unbound reagent or set of reagents are removed, such as by washing; and detecting the reagent or set of reagents.

31. The method of any one of the preceding claims, wherein the means, antibody or alternative or candidate binding agent does not significantly bind the peptide epitope when comprised in a native protein.

32. The method of claim 31, wherein the method further comprises selecting and / or testing the means, antibody or alternative or candidate binding agent for binding to the peptide epitope when comprised in a native protein.

33. The method of claim 31 or 32 wherein the native protein is insulin.

34. An antibody produced by the method of any one of claims 12-21, or an antigenbinding fragment thereof.

35. A composition comprising the antibody or antigen-binding fragment thereof of claim 34 and a pharmaceutically acceptable carrier.

36. A candidate binding agent selected in the method of any one of claims 22-33.

37. A composition comprising the candidate binding agent of claim 36 and a pharmaceutically acceptable carrier.

38. A binding agent as provided herein or a composition comprising the binding agent and a pharmaceutically acceptable carrier.

39. An antibody as provided herein or an antigen-binding fragment thereof or a composition comprising the antibody or antigen-binding fragment and a pharmaceutically acceptable carrier.

40. A MHC-peptide complex as provided herein, wherein the peptide is any one of the peptides provided herein and, optionally, wherein the MHC is a MHC Class I molecule, a MHC Class II molecule, a MHC monomer, a MHC tetramer or a MHC on the membrane of a cell, and wherein the MHC-peptide complex is isolated, recombinant and / or not a native MHC-peptide complex.

Images