RU2773380C2 - Nanoparticle compositions for long-term therapy - Google Patents

Abstract

FIELD: pharmaceutics; chemistry.

SUBSTANCE: invention relates to the field of pharmaceutics and chemistry, namely to a pharmaceutical composition for the treatment of primary biliary cirrhosis in a subject, containing a complex of a nanoparticle, the core of which is related to a number of antigen-MHC class II (hereinafter – pMHCII) complexes. The core contains iron oxide, and its diameter is from 1 nm to 25 nm. Antigen is PDC-E2 or its fragment capable of inducing an immune response. The number of related pMHCII per the surface area of the nanoparticle is from 0.4 pMHC/100 nm² to 12 pMHC/100 nm². pMHCII are related to a maleimide-functionalized end of a polyethylene glycol (hereinafter – PEG) linker, the molecular weight of which is less than 5 kDa. A non-maleimide-functionalized end of polyethylene glycol (PEG) linker is related to the core surface. In this case, the complex differentiates an activated T-cell or a memory T-cell to a cell TR1 producing IL-10. The nanoparticle complex is present in the amount sufficient for the treatment of primary biliary cirrhosis in a subject.

EFFECT: treatment of primary biliary cirrhosis in a subject due to the capability of pMHC, on the nanoparticle core, of initiating the formation of TR1 cells suppressing an antigen-specific response, with regulated density of pMHC on the nanoparticle core.

9 cl, 22 dwg, 24 tbl, 3 ex

Description

Link to related patent applications

This application claims priority under 35 U.S.C. § 119(e). U.S. Provisional Applications Nos. 62/157933, 62/273953, and 62/296032 filed May 6, 2015, December 31, 2015, and February 16, 2016, respectively, the contents of each of which are hereby incorporated by reference in their entirety.

Background of the Invention

Throughout and within the scope of the present invention, technical and patent publications are indicated by an identifying reference or Arabic numerals. Full bibliographic references corresponding to Arabic numerals are in the description before the claims. The descriptions of all references cited herein are incorporated herein by reference for a more complete description of the prior art to which the present invention belongs.

Abnormal functioning of the immune system is involved in the pathogenesis or exacerbation of a wide range of diseases. Although there is a wide range of immunotherapeutic agents, their action is often associated with non-targeted action due to the lack of targeted specificity and/or adverse side effects.

Thus, for these disorders, there is a need to find safe and effective therapeutic agents. The present invention satisfies this need and also provides related benefits.

The essence of the invention

The present invention provides a nanodrug, which in one aspect is a complex comprising a nanoparticle core associated with a plurality of disease-associated antigen-MHC complexes (abbreviated herein as "rMHC" or "rMHC complexes") that are suitable for propagation and differentiating T cell populations; and treating the disease when administered to an individual in an effective amount. The core of the nanoparticle contains a number of compositions or components, as described in more detail herein. In certain aspects, the nanoparticle core diameter is a diameter selected from the group of about 1 nm to about 100 nm; about 1 nm to about 75 nm; from about 1 nm to about 50 nm; about 1 nm to about 25 nm; about 1 nm to about 25 nm; from about 5 nm to about 100 nm; from about 5 nm to about 50 nm; or about 5 nm to about 25 nm, or about 15 nm to about 25 nm, or about 20 nm. In certain embodiments, the nanoparticle core diameter is about 25 nm to about 60 nm, or about 25 nm to about 50 nm, or about 20 nm to about 40 nm, or about 15 nm to about 50 nm, or about 15 nm to about 40 nm, or about 15 nm to about 35 nm, or about 15 nm to about 30 nm, or about 15 nm to about 25 nm, or alternatively about 15 nm, or about 20 nm, or about 25 nm, or about 30 nm, or about 35 nm, or about 40 nm.

In accordance with certain aspects, the amount of pMHC per nanoparticle core (referred to herein as the "valence" of the nanoparticle nanoparticle complex) can range from about 1 pMHC complex per nanoparticle core to about 6000 pMHC complexes per nanoparticle core, or alternatively, from about 10:1 to about 6000:1, or alternatively from about 11:1 to about 6000:1, or alternatively from about 12:1 to about 6000:1, or alternatively at least 2: 1, or alternatively at least 8:1, or alternatively at least 9:1, or alternatively at least 10:1, or alternatively at least 11:1, or alternatively, at least 12:1. In certain aspects, the amount of pMHC per nanoparticle core is from about 10:1 to about 6000:1, or from about 20:1 to about 5500:1, or alternatively from about 10:1 to about 5000:1, or , alternatively from about 10:1 to about 4000:1, or alternatively from about 10:1 to about 3500:1, or alternatively from about 10:1 to about 3000:1, or alternatively from about 10:1 to about 2500:1, or alternatively about 10:1 to about 2000:1, or alternatively about 10:1 to about 1500:1, or alternatively about 10:1 to about 1000 :1, or alternatively about 10:1 to about 500:1, or alternatively about 10:1 to about 100:1, or alternatively about 20:1 to about 50:1, or, alternatively, from about 25:1 to about 60: one; alternatively about 30:1 to about 50:1, or alternatively about 35:1 to about 45:1, or alternatively about 40:1.

In certain aspects, the nanoparticle core has a specific valence per core surface area, also referred to herein as "density". In these aspects, the pMHC density per nanoparticle is from about 0.025 pMHC/100 nm ² to about 100 pMHC/100 nm ² of the nanoparticle core surface area, or alternatively, from about 0.406 pMHC/100 nm ² to about 50 pMHC/100 nm ² ; or, alternatively, from about 0.05 pMHC/100 nm ² to about 25 pMHC/100 nm ² . In accordance with certain aspects, the pMHC density per nanoparticle is from about 0.4 pMHC/100 nm ² to about 25 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 20 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 15 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 14 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 13 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 12 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 11.6 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 11.5 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 11 pMHC/100 nm ² , or from about 0.4 pMHC /100 nm ² to about 10 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 9 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 8 pMHC/100 nm ² , or about 0.4 pMHC/100 nm ² to about 7 pMHC/100 nm ² , or about 0.4 pMHC/100 nm ² to about 6 pMHC/100 nm ² , or about 0.4 pMHC/100 nm ² up to about 5 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 4 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 3 pMHC/100 nm ² , or about 0.4 pMHC/100 nm ² to about 2.5 pMHC/100 nm ² , or about 0.4 pMHC/100 nm ² to about 2 pMHC/100 nm ² , or about 0.4 pMHC/100 nm ² to about 1.5 pMHC/100 nm ² .

In another aspect, the pMHC density on the nanoparticles can be from about 0.22 pMHC/100 nm ² to about 10 pMHC/100 nm ² , or from about 0.22 pMHC/100 nm ² to about 9 pMHC/100 nm ² . or from about 0.22 pMHC/100 nm ² to about 8 pMHC/100 nm ² , or from about 0.22 pMHC/100 nm ² to about 7 pMHC/100 nm ² , or from about 0.22 pMHC/100 nm ² to about 6 pMHC/100 nm ² , or about 0.22 pMHC/100 nm ² to about 5 pMHC/100 nm ² , or about 0.22 pMHC/100 nm ² to about 4 pMHC/100 nm ² , or about 0.22 pMHC/100 nm ² to about 3 pMHC/100 nm ² , or about 0.22 pMHC/100 nm ² to about 2 pMHC/100 nm ² , or about 0.22 pMHC/100 nm ² to about 1.5 pMHC/100 nm ² . In certain aspects, the pMHC density on a nanoparticle may be from about 0.22 pMHC/100 nm ² to about 10 pMHC/100 nm ² or 0.24 pMHC/100 nm ² to about 9 pMHC/100 nm ² , or from about 0.26 pMHC/100 nm ² to about 8 pMHC/100 nm ² , or about 0.28 pMHC/100 nm ² to about 7 pMHC/100 nm ² , or about 0.24 pMHC/100 nm ² to about 4 pMHC/100 nm ² , or from about 0.5 pMHC/100 nm ² to about 3 pMHC/100 nm ² , or from about 0.6 pMHC/100 nm ² to about 1.5 pMHC/100 nm ² . In a further aspect, the pMHC density on the nanoparticle may be from about 0.4 pMHC/100 nm ² to about 1.3 pMHC/100 nm ² , or alternatively, from about 0.5 pMHC/100 nm ² to about 0.9 pMHC/100 nm ² , or alternatively from about 0.6 pMHC/100 nm ² to about 0.8 pMHC/100 nm ² .

According to certain embodiments, the pMHC density on the nanoparticle may be approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 .0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2 , 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3 .5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7 , 4.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7 .0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2 , 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9 .5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7 , 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9 or 12 .0 pMHC/100 nm ² . In accordance with specific embodiments, the pMHC density on the nanoparticle may be from about 0.4 pMHC/100 nm ² to about 1.5 pMHC/100 nm ² or from about 0.4 pMHC/100 nm ² to about 6 pMHC/100 nm ² or from about 0.4 pMHC/100 nm ² to about 12 pMHC/100 nm ² .

In yet another aspect, the density of pMHC on a nanoparticle as defined herein may be from about 0.4 pMHC/100 nm ² to about 1.3 pMHC/100 nm ² , or alternatively from about 0.5 pMHC/100 nm ² up to about 0.9 pMHC/100 nm ² , or alternatively, from about 0.6 pMHC/100 nm ² to about 0.8 pMHC/100 nm ² , and further, where the nanoparticle core diameter is from about 25 nm to about 60 nm, or about 25 nm to about 50 nm, or about 20 nm to about 40 nm, or about 15 nm to about 50 nm, or about 15 nm to about 40 nm, or about 15 nm to about 35 nm, or about 15 nm to about 30 nm, or about 15 nm to about 25 nm, or alternatively about 15 nm, or about 20 nm, or about 25 nm, or about 30 nm, or about 35 nm , or p approximately 40 nm.

In accordance with certain aspects, the nanoparticle core further comprises a number of costimulatory molecules, costimulatory antibodies, inhibitory receptor blocking antibodies, and/or a number of cytokines associated with the nanoparticle core.

Thus, certain aspects of the invention relate to a complex containing or alternatively substantially consisting of, or even more consisting of, nanoparticle cores associated with a number of pMHC complexes, where the nanoparticle cores optionally additionally contain or, moreover, consist of or alternatively , moreover, essentially consist of one or more costimulatory molecules and/or one or more cytokines associated with the core of the nanoparticle. In these compositions containing a number of complexes, the pMHC complexes on each nanoparticle core are the same or different from each other; and/or the MHC of the pMHC complexes on each nanoparticle core are the same or different from each other; and/or cytokines on each core of the nanoparticle are the same or different from each other; and/or the costimulatory molecules on each nanoparticle core are the same or different from each other; and/or the diameters of the cores of the nanoparticles are the same or different from each other; and/or the valency of the pMHC complexes on each nanoparticle core are the same or different from each other; and/or the density of the pMHC complexes on each core of the nanoparticle is the same or different for different complexes; and/or the valency of the costimulatory molecules on each nanoparticle core is the same or different for different molecules; and/or the valency of cytokines on each nanoparticle core is the same or different for different cytokines.

In accordance with certain aspects, compositions are provided herein containing a number of the complexes provided herein. According to certain embodiments, the compositions further comprise a carrier, optionally a pharmaceutical carrier. In accordance with certain embodiments, the compositions provided herein may optionally contain one or more nanoparticle cores associated with one or more of the costimulatory molecules and/or cytokines. Thus, in accordance with certain embodiments, the compositions comprise, or alternatively, substantially consist of, or even more so, consist of: 1) a series of nanoparticle cores associated with a series of antigen-MHC complexes, wherein at least one portion of the nanoparticle cores additionally contains one or more costimulatory molecules and/or one or more cytokines, and the second part of the nanoparticle cores does not additionally contain costimulatory molecules and/or cytokines, and 2) a number of nanoparticle cores associated with one or more of the costimulatory molecules and/or cytokines.

Additional aspects of the invention relate to disease-specific antigens, MHCs, and combinations thereof optimized for the treatment or prevention of diseases in human and animal patients.

The present invention also relates to compositions and methods of using any of the above complexes or compositions, each of which is optionally combined with a carrier, for example, a pharmaceutically acceptable carrier.

The present invention also relates to methods for differentiating or initiating the formation of type 1 regulatory T cells (T _R 1 ) in a dose-independent rMHC manner. Applicant has found that the density of rMHC at the core of the nanoparticle regulates the ability of rMHC at the core of the nanoparticle to initiate the formation of T _R 1 cells in a dose-independent manner, although the dose of rMHC regulates the level of proliferation of T _R 1 cells in a manner that is independent of the density of rMHC. Applicant has found that the rMHC density threshold and the independent effect of rMHC density versus dose on T _R 1 cell formation versus propagation are unexpected results that could not be expected based on the general knowledge of immunology in the art. These methods require contacting (in vitro or in vivo) recognizing T cells with an effective amount of pMHC-NP or a composition as described herein. In certain aspects, density-dependent methods refer to activated or memory T cells differentiating into appropriate IL-10-producing T _R 1 cells, optionally carrying CD49b and/or Lag3 markers, and/or B cells differentiating into regulatory B cells by contacting activated or memory T cells with an effective amount of the complex or composition described herein. In certain embodiments, the differentiated T _R 1 cell binds to a B cell, thereby allowing the B cell to differentiate into a regulatory B cell. In accordance with certain aspects of the methods, bringing into contact is carried out in vitro or in vivo. In accordance with certain embodiments, the pMHC-NP or composition comprising a series of pMHC-NPs comprises a pMHC-NP with an average nanoparticle core diameter of about 25 nm to about 60 nm, or about 25 nm to about 50 nm, or about 20 nm to about 40 nm, or about 15 nm to about 50 nm, or about 15 nm to about 40 nm, or about 15 nm to about 35 nm, or about 15 nm to about 30 nm, or about 15 nm to about 25 nm, or alternatively about 15 nm, or about 20 nm, or about 25 nm, or about 30 nm, or about 35 nm, or about 40 nm. In accordance with certain aspects, the core of the nanoparticle further comprises an outer coating or layer, where the average diameter of the core and the outer layer is from about 30 nm to about 75 nm, or from about 30 nm to about 70 nm, or from about 30 nm to about 60 nm , or about 30 nm to about 50 nm, or about 40 nm. In certain aspects, the average pMHC density on a nanoparticle is from about 0.4 pMHC/100 nm ² to about 12 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 11.6 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 11.5 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 11 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 10 pMHC/100 nm ² , or about 0.4 pMHC/100 nm ² to about 9 pMHC/100 nm ² , or about 0.4 pMHC/100 nm ² to about 8 pMHC/ 100 nm ² , or about 0.4 pMHC/100 nm ² to about 7 pMHC/100 nm ² , or about 0.4 pMHC/100 nm ² to about 6 pMHC/100 nm ² , or about 0.4 pMHC/100 nm ² to about 5 pMHC/100 nm ² , or about 0.4 pMHC/100 nm ² to about 4 pMHC/100 nm ² , or about 0.4 pMHC/100 nm ² to about 3 pMHC/ 100 nm ² , or from about 0.4 pMHC/100 nm ² to about 2.5 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 2 pMHC/100 nm ² , or from about 0, 4 pMHC/100 nm ² to about 1.5 pMHC/100 nm ² .

Additional aspects of the invention relate to methods for treating or preventing appropriate diseases or pathological conditions, as described herein, by administering an effective amount of pMHC-NP, as described herein. Methods for detecting the presence and effectiveness of treatment with pMHC-NP complexes and compositions are also described, as described herein.

Brief description of the drawings

The following drawings form part of the present description and are included to further demonstrate certain aspects of the present invention. The invention can be better understood by referring to one or more of these drawings in combination with the detailed description of specific embodiments provided herein.

Figures 1A-1B are schematic representations of NP complexes. Figure 1A is a schematic representation of a single chain pMHC class I expression construct (upper) and a representative flow cytometry profile of the binding of the corresponding pMHC tetramer (labeled with fluorochrome) to CD8 ⁺ T cells. Figure 1B is a schematic diagram showing the linkers and the two-dimensional structure of the NP complexes. As can be seen, one NP can contain the same antigen in complex with the nanoparticle core through different chemical linkers.

Figure 2 shows the structure of a typical class II pMHC monomer (upper) and a characteristic FACS profile of CD4 ⁺ T cells stained with the corresponding pMHC tetramer or left unstained.

The figure 3 shows the chemical structure of the grafted tree poly-L-lysine generation 3 (DGL G3).

Figure 4 shows the synthesis of a grafted arboreal poly-L-lysine G3 functionalized with PEG-azido (DGLN).

The figure 5 shows the synthesis of pMHC-DGLN.

The figure 6 shows the analysis of conjugates pMHC-DGLN in native and denaturing PAGE.

Figure 7 shows the analysis of AFM V7CHO-DGLN.

Figures 8A-8B show that V7CHO-DGLN has significant agonist properties in CD8 ⁺ recognizing T cells.

Figures 9A-9N show pMHC-NPs corresponding to T1D or EAE stimulating the proliferation of T _R 1 -like suppressor CD4 ⁺ T cells in vivo. Figures 9A and 9B show the tetramer staining profiles (Figure 9A) and the percentage of tetramer ⁺ CD4 ⁺ T cells (Figure 9B). Data are consistent with pre-diabetic NOD females treated for 5 weeks (blood: n=5, 8, and 6; spleen: n=5, 18, and 6, respectively). Tetr., tetramer. Figure 9C shows tetramer staining of CD4 ⁺ T cells in the spleens of treated or untreated NOD Foxp3-eGFP mice. Figure 9D shows that tetramer ⁺ CD4 ⁺ T cells from 2.5mi/IA ^g7 -NP treated mice exhibit a T _R 1 -like phenotype. Figure 9E shows the incidence of diabetes in NOD scid hosts with recovered T cells transfused with CD4+ T cells from various donors ±2.5 mi/IA ^g7 -NP (n=11, 5, 7 and 6 from top). Figure 9F shows the percentage of tetramer ⁺ CD4 ⁺ T cells in treated or untreated 2.5mi/IA ^g7 -NP NOD scid hosts (n=4-5 per group). Figure 9G shows the frequency of disease reversal in diabetic mice treated with pMHC-NP (n=9, 7, 7, 7 from top left to right) or IGRP _4-22 peptide (Burton, BR et al. (2014) Nature Commun. 5:4741-4747) and IGRP peptide _4-22 -NP complex (n=9). Figure 9H shows the percentage of tetramer ⁺ CD4 ⁺ T cells in early onset diabetic mice in response to 2.5mi/IA ^g7 -NP therapy and age-matched non-diabetic controls (n=8, 6, 2 and 7 left) . The figure 91 shows the indices of insulitis (n=6, 4, 3 and 6 on the left). Below illustrative images. Figures 9J-9M show C57BL/6 mice immunized with PMOG _35-55 . Figure 9J shows the EAE scores of the treated mice, day 14 (n=4 each). Figure 9K shows the EAE scores of the treated mice on day 21 (n=10, 7 and 3 on top). Figure 9L shows the percentage of tetramer ⁺ CD4 ⁺ T cells in the spleen and blood of mice in figures 9J and 9K (n=13, 14 and 5 top). Figure 9M shows characteristic flow cytometry profiles of CD4 ⁺ T cells from the mice described in Figures 9J and 9K. Figure 9N shows characteristic microglial IBA1 staining and relative rank indices in the cerebellums of the mice described in Figure 9K (n=4-5). P values were calculated by Mann-Whitney U-test, log-rank test (Mantel-Cox) or by two-way ANOVA. Error rates, standard error of the mean.

Brief description of the tables

Table 1. Functionalized PEG linkers.

Table 2. Codons.

Tables 3A and 3B. Transcription profile of pMHC-NP-expanded CD4 ⁺ T cells. (A) qPCR-RT for a panel of 384 immunological markers in 2.5mi/IA ^g7+ tetramer CD4 ⁺ T cells compared to 2.5mi/IA ^g7- tetramer CD4 ⁺ T cells sorted in NOD mice treated with 10 doses of 2.5mi/IA ^g7 -NP starting at 10-15 weeks of age (n=3 and 4 samples, respectively). Cells were stimulated in vitro with anti-CD3/anti-CD28 mAbs coated Dynabeads prior to RNA preparation. The panel shows the most significant differences. (B) qPCR-RT for 8 associated with T _R 1 markers, including markers that are not presented in the primer set used in (A). The data correspond to four additional samples of CD4 ⁺ T cells with the 2.5mi/IA ^g7+ tetramer and seven additional samples of CD4 ⁺ T cells with the 2.5mi/IA ^{g7- tetramer} .

Table 4A, 4B and 4C. Human T1D donors and outcome of pMHC-NP therapy in NSG hosts with transplanted PBMCs.

Table 5 is an illustrative list of cancer-associated antigens for use in the present invention.

Table 6 is an illustrative list of diabetes-associated antigens for use in the present invention.

Table 7 is an illustrative list of multiple sclerosis-associated antigens for use in the present invention.

Table 8 is an illustrative list of celiac disease associated antigens for use in the present invention.

Table 9 is an illustrative list of primary biliary cirrhosis-associated antigens for use in the present invention.

Table 10 is an illustrative list of pemphigus foliaceus-associated antigens and pemphigus vulgaris-associated antigens for use in the present invention.

Table 11 is an illustrative list of antigens associated with neuromyelitis optic nerve spectrum disorder for use in the present invention.

Table 12 is an illustrative list of allergic asthma-associated antigens for use in the present invention.

Table 13 is an illustrative list of inflammatory bowel disease-associated antigens for use in the present invention.

Table 14 is an illustrative list of systemic lupus erythematosus-associated antigens for use in the present invention.

Table 15 is an illustrative list of atherosclerosis-associated antigens for use in the present invention.

Table 16 is an illustrative list of chronic obstructive pulmonary disease-associated antigens and emphysema-associated antigens for use in the present invention.

Table 17 is an illustrative list of psoriasis-associated antigens for use in the present invention.

Table 18 is an illustrative list of autoimmune hepatitis associated antigens for use in the present invention.

Table 19 is an illustrative list of uveitis-associated antigens for use in the present invention.

Table 20 is an illustrative list of Sjogren's Syndrome-associated antigens for use in the present invention.

Table 21 is an illustrative list of scleroderma-associated antigens for use in the present invention.

Table 22 is an illustrative list of antiphospholipid syndrome associated antigens for use in the present invention.

Table 23 is an illustrative list of ANCA-associated vasculitis-associated antigens for use in the present invention.

Table 24 is an illustrative list of stiffness syndrome-associated antigens for use in the present invention.

Detailed description of the invention

It should be understood that the present invention is not limited to the specific embodiments described, as these may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the present invention is only defined by the appended claims.

It should be noted that, as used herein and in the appended claims, singular forms include an indication of the plural, unless the context clearly dictates otherwise. Thus, for example, the indication of "excipient" includes a number of excipients. The term "at least one" means one or more.

Throughout this application, the term "approximately" is used to indicate that the value includes the standard deviation of errors for the device or method used to determine the value. The term "about", when used before a numerical designation, for example, temperature, time, amount and concentration, including range, means approximate values that may vary by (+) or (-) 10%, 5% or 1%.

Definitions

Unless otherwise defined, all technical and scientific terms used herein have the same meanings as are commonly known to those skilled in the art to which the present invention belongs. As used herein, the following terms have the following meanings.

As used herein, the term "comprising" or "comprises" is intended to mean that compositions and methods include the enumerated elements, without excluding others. When used to define compositions and methods, "substantially consisting of" should mean the exclusion of other elements of any significant combination significance for the stated purpose. Thus, as defined herein, a composition substantially composed of elements does not exclude other materials or steps that do not significantly affect the essential and novel feature(s) of the claimed invention, such as compositions for the treatment or prevention of disseminated sclerosis. "Consisting of" means the exclusion of more than trace amounts of other ingredients and essential steps of the method. Embodiments defined by each of these transitional terms are within the scope of the present invention.

The terms "inhibition", "reduction" or "prevention" or any variation of these terms, when used in the claims and/or in the description, include any measurable reduction or complete inhibition to achieve the desired result.

By "biocompatible" is meant that the components of the delivery system do not cause tissue damage or damage to the human biological system. To impart biocompatibility, polymers and excipients are preferably used for which there is experience of safe treatment in humans or have GRAS (generally recognized as safe) status. By biocompatibility is meant that the ingredients and excipients used in the composition are ultimately "bioabsorbed" or excreted from the body without adverse effects on the body. For a composition to be biocompatible and considered non-toxic, it must not be toxic to cells. Similarly, the term "bioabsorbable" refers to nanoparticles derived from materials that are bioabsorbed in vivo over a period of time such that long-term accumulation of the material in the patient does not occur. In a specific embodiment, the biocompatible nanoparticle is bioabsorbed over a period of less than 2 years, preferably less than 1 year, and even more preferably less than 6 months. The bioabsorption rate depends on the particle size, the material used and other factors widely known to those skilled in the art. A mixture of bioabsorbable biocompatible materials can be used to form the nanoparticles used in the present invention. In one embodiment, iron oxide and a biocompatible bioabsorbable polymer can be combined. For example, iron oxide and PGLA can be combined to form nanoparticles.

As used herein, the term "dendrimer" refers to a re-branching molecule, also referred to as arborol or a cascade molecule. In relation to the synthesis of nanoparticles, the term "dendrimer core" refers to the use of a dendrimer as the central component of a nanoparticle so that it forms the basis of the structure of the nanoparticle. In accordance with certain embodiments, the core of the nanoparticle described herein contains a dendrimer.

As used herein, the term "polymer micelle" refers to an amphiphilic structure that contains a hydrophobic core and a hydrophilic shell, which can be obtained from block copolymers. In relation to synthesis, the synthesis of nanoparticles, the term "polymer micelle core" refers to the use of a polymer micelle as the central component of a nanoparticle so that it forms the backbone of the nanoparticle structure. In accordance with certain embodiments, the core of the nanoparticle described herein contains a polymeric micelle.

Antigen-MHC-nanoparticle complex ("NP complex" or "complex" or pMHC-NP or "nanoparticle complex") refers to the presentation of a peptide, carbohydrate, lipid or other antigenic site, fragment or epitope of an antigenic molecule or protein (i.e. , autologous peptide or self-antigen) on a surface, such as the core of a nanoparticle.

"Nanoparticle core" is the substrate of the nanoparticle, which may or may not contain layers or coatings. The nanoparticle complex contains a core with at least an antigen-MHC complex associated with the core.

"Density" in terms of pMHC content per nanoparticle is calculated as the surface area of the core of the nanoparticle with or without outer layers, which may also contain linkers. The surface area is the total available surface area of the structure being used. In one aspect, when a PEG linker is used, it can increase the overall diameter of the nanoparticle core by about 20 nm ^{2 of} the nanoparticle, which accordingly increases the surface area of the total available surface area of the nanoparticle. In other words, it represents the final surface area of the nanoparticle without the addition of one or more of the pMHC, costimulatory molecules, and/or cytokines.

As used herein, "antigen" refers to all, part, fragment or region of a molecule that can induce an immune response in an individual or the expansion of immunocytes, preferably T or B cells.

The term "alkyl" refers to monovalent saturated aliphatic hydrocarbon groups containing from 1 to 10 carbon atoms (i.e., C ₁ -C ₁₀ -alkyl), or from 1 to 6 carbon atoms (i.e., C ₁ - C ₆ -alkyl), or from 1 to 4 carbon atoms. This term includes, by way of example, linear and branched hydrocarbon groups such as methyl (CH ₃ -), ethyl (CH ₃ CH ₂ -), n-propyl (CH ₃ CH ₂ CH ₂ -), isopropyl ((CH ₃ ) ₂ CH-), n-butyl (CH ₃ CH ₂ CH ₂ CH ₂ -), isobutyl ((CH ₃ ) ₂ CHCH ₂ -), sec-butyl ((CH ₃ )(CH ₃ CH ₂ )CH-), tert -butyl ((CH ₃ ) ₃ C-), n-pentyl (CH ₃ CH ₂ CH ₂ CH ₂ CH ₂ -) and neopentyl ((CH ₃ ) ₃ CCH ₂ -).

The term "alkoxy" refers to -O-alkyl.

A "mimetic" is an analog of a given ligand, or a peptide, wherein the analog is substantially similar to the ligand. "Substantially similar" means that the analog has a binding profile similar to the ligand, except that the mimetic contains one or more functional groups or modifications that together are less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, or less than about 5% of the molecular weight of the ligand.

"Immunocytes" include, for example, leukocytes (leukocytes) which are derived from hematopoietic stem cells (HSC) produced in the bone marrow, lymphocytes (T cells, B cells, natural killer (NK) cells) and derived from myeloid lineage. cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells). As used herein, the term "B cell" refers to a specific type of lymphocytes active in the humoral immunity of the adaptive immune system. B cells predominantly function by producing antibodies, serving as antigen presenting cells, releasing cytokines, and developing into memory B cells after being activated by interaction with an antigen. B cells differ from other lymphocytes, such as T cells, by the presence of a B cell receptor on the cell surface. As used herein, the term "T cell" refers to a type of lymphocyte that matures in the thymus. T cells play an important role in cellular immunity and differ from other lymphocytes such as B cells by the presence of a T cell receptor on the cell surface. T cells can be isolated or obtained from a commercially available source. "T cell" includes all types of CD3-expressing immunocytes, including T helper cells (CD4 ⁺ cells), cytotoxic T cells (CD8 ⁺ cells), natural killer T cells, regulatory T cells ( _Treg ) and gamma delta T cells. "Cytotoxic cell" includes CD8 ⁺ T cells, natural killer (NK) cells and neutrophils that are capable of mediating a cytotoxic response.

As used herein, the term "effector T cells" refers to T cells that can specifically bind an antigen and mediate an immune response (effector function) without the need for further differentiation. Examples of effector T cells include CTL, TH1 cells, TH2 cells, memory effector cells, and T helper cells. Unlike effector T cells, naive T cells did not encounter their specific antigen:MHC complex and did not respond to it by proliferation and differentiation into effector T cells. Effector T cells can be quiescent (in the G0 phase of the cell cycle) or activated (proliferating).

The term "antipathogenic autoreactive T cell" refers to a T cell with antipathogenic properties (ie, T cells that prevent autoimmune diseases such as MS, MS related diseases or disorders, or prediabetes). These T cells may include anti-inflammatory T cells, central memory T cells, effector memory T cells, memory T cells, low avidity T cells, T helper cells, autoregulatory T cells, cytotoxic T cells, natural killer T cells, regulatory T cells, TR1 cells, suppressor T cells, CD4 ⁺ T cells, CD8 ⁺ T cells, and the like.

The term "anti-inflammatory T cell" refers to a T cell that promotes an anti-inflammatory response. The anti-inflammatory function of a T cell can be mediated through the production and/or secretion of anti-inflammatory proteins, cytokines, chemokines, and the like. Anti-inflammatory proteins are also designed to turn on anti-proliferative signals that suppress the immune response. Anti-inflammatory proteins include IL-4, IL-10, IL-13, IL-21, IL-23, IL-27, IFN-α, TGF-β, IL-1ra, G-CSF and soluble TNF and IL-6 receptors .

The term "differentiated" refers to the time when the development of a first type cell into a second type cell is induced. In certain embodiments, a recognition T cell is differentiated into a T _R 1 regulatory cell. In certain embodiments, an activated T cell is differentiated into a T _R 1 cell. In accordance with certain embodiments, a memory T cell is differentiated into a T _R 1 cell. In accordance with certain embodiments, the B cell is differentiated into a regulatory B cell.

As used herein, "protrusion into a cavity" refers to a polypeptidyl structure requiring a protrusion (or "protrusion") on the interacting surface of the first polypeptide and a corresponding depression (or "depression") on the interacting surface of the second polypeptide such that the protrusion can be located in deepening so as to ensure heteromultimeric formation. The protrusions are constructed by replacing amino acids with small side chains on the interacting surface of the first polypeptide with amino acids with large side chains (eg, phenylalanine or tyrosine). On the interacting surface of the second polypeptide, dimples of identical or protrusion-like dimensions are obtained by replacing amino acids with large side chains with amino acids with small side chains (eg, alanine or threonine). The protrusions and recesses can be obtained by synthetic methods, such as substitution of nucleic acids encoding polypeptides, or by peptide synthesis, by conventional methods used by a person skilled in the art. In certain embodiments, the interaction surface of the first polypeptide is on the Fc domain of the first polypeptide and the interaction surface of the second polypeptide is on the Fc domain of the second polypeptide. Tab-to-pit heteromultimers and methods for their preparation and use are described in US Pat. Nos. 5,731,168; 5807706; 5821333; 7642228; 7695936; 8216805 and 8679785, which are all incorporated herein by reference in their entirety

As used herein, "alpha-MHC-Fc/beta-MHC-Fc" refers to a heterodimer containing a first polypeptide and a second polypeptide, wherein the first polypeptide contains an MHC class II α-chain and an antibody Fc domain; the second polypeptide contains the MHC class II β-chain and the Fc domain of the antibody. For alpha-MHC-Fc/beta-MHC-Fc with ridge to cleft, it is additionally required that the Fc domains of each polypeptide interact with each other by the complementary arrangement of a protrusion on one of the Fc domains in a corresponding recess on the other Fc domain.

The term "isolated" means separated from cellular constituents and other components with which a polynucleotide, peptide, polypeptide, protein, antibody, or fragment(s) thereof is normally associated in nature. For example, with respect to a polynucleotide, an isolated polynucleotide is a polynucleotide that is separated from the 5' and 3' sequences with which it is normally associated on a chromosome. As will be appreciated by those skilled in the art, non-natural polynucleotide, peptide, polypeptide, protein, antibody, or fragment(s) thereof do not require "isolation" to separate them from their natural counterparts. In addition, "concentrated", "isolated", or "diluted" polynucleotide, peptide, polypeptide, protein, antibody, or fragment(s) thereof differ from their natural counterparts in that the concentration or number of molecules in a given volume is more "concentrated" or less "separated" than the concentration or number of molecules of their natural counterparts. A polynucleotide, peptide, polypeptide, protein, antibody, or fragment(s) thereof, which differs from their natural counterparts in their primary sequence or, for example, in their glycosylation profile, do not need to be present in their isolated forms, as they differ from their natural analogues by their primary sequence or, alternatively, by another characteristic such as their glycosylation profile. A mammalian cell, such as a T cell, is isolated if it has been removed from the anatomical site where it resides in the body.

A "self-reactive T cell" is a T cell that recognizes a "self antigen", which is a molecule produced by the same individual and present in the same individual who carries the T cell.

A "pathogenic T cell" is a T cell that is harmful to an individual carrying the T cell. While the non-pathogenic T cell is essentially not harmful to the individual, and the anti-pathogenic T cell reduces, smoothes, inhibits or eliminates the harm of the pathogenic T cell.

As used herein, the term regulatory B cells (" _Breg ") means cells that are responsible for anti-inflammatory action, which is characterized by the expression of CD1d, CD5 and the secretion of IL-10. B _reg is also identified by Tim-1 expression and can be induced by Tim-1 ligation to provide tolerance. It has been shown that the ability to exist as _Breg is due to a variety of stimulating factors, such as Toll-like receptors, CD40 ligand, and others. However, the complete characterization of _Breg continues. _Regs also express high levels of CD25, CD86 and TGF-β. This subpopulation of B cells is able to suppress Th1 proliferation, thus helping to maintain self-tolerance. Potentiation of _Breg function should be the goal of many immunomodulatory drugs, which will contribute to better control of autoimmune diseases. See for example: ncbi.nlm.nih.gov/pubmed/23707422, accessed October 31, 2013.

Regulatory T cells type 1 (T _R 1) are a subpopulation of CD4 ⁺ T cells with regulatory properties, and they are able to suppress the antigen-specific immune response in vitro and in vivo. These T _R 1 cells are identified by their unique profile of cytokine production and high levels of IL-10 and TGF-beta, but not IL-4 or IL-2. IL-10 and TGF-beta produced by these cells in vitro mediate the inhibition of primary naive T cells. There is also evidence that T _R cells exist in vivo, and the presence of high-producing IL-10 CD4(+) T cells has been reported in patients with severe combined immunodeficiency receiving allogeneic stem cell transplants. T _R 1 cells are involved in the regulation of peripheral tolerance, and they can potentially be used as cell therapy to modulate the immune response in vivo. See for example: ncbi.nlm.nih.gov/pubmed/10887343, accessed October 31, 2013.

T _R 1 cells are defined by their ability to produce IL-10 and TGF-beta at high levels. Multi-antigen specific T _R 1 cells are generated in vivo but can also differentiate from naive CD4 ⁺ T cells in the presence of IL-10 in vitro. T _R 1 cells have a low proliferative potential, which can be overcome by the action of IL-15. T _R 1 cells suppress the response of naive T cells and type 1 or 2 memory helper T cells through the production of IL-10 and TGF-beta. Further characterization of T _R 1 cells at the molecular level will determine their mechanism of action and clarify their relationship with other subsets of T _R cells. Predictions can be made about the use of T _R 1 cells to identify new targets for the development of new therapeutic agents and as cell therapy to modulate peripheral tolerance. See, for example, ncbi.nlm.nih.gov/pubmed/11722624, accessed 31 October 2013.

An "effective amount" is an amount sufficient to achieve a given goal, non-limiting examples of which include: initiating an immune response, modulating an immune response, suppressing an inflammatory response, and modulating the activity of T cells or populations of T cells. In one aspect, an effective amount is an amount that acts to achieve a specified therapeutic goal, for example, a therapeutically effective amount. As detailed herein, the effective amount or dose depends on the purpose and composition, and can be determined in accordance with the present description.

An effective amount of the therapeutic composition is determined based on the intended purpose. The term "dosage unit" or "dosage" refers to physically discrete units suitable for use in an individual, where each unit contains a predetermined amount of the composition calculated to elicit the desired response described above in connection with its administration, i.e. appropriate route and treatment regimen. The amount administered, in terms of number of drugs and unit dose, depends on the desired result and/or protection. The exact amounts of the composition also depend on the judgment of the practitioner and are individual to each individual. Factors influencing dose include the individual's physical and clinical condition, the route of administration, the intended goal of treatment (symptom relief versus cure), and the potency, stability, and toxicity of the particular composition. Once formulated, the solutions are administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easy to administer in a variety of dosage forms, such as the types of injectable solutions described above.

"MHC multimer" as used herein means a complex of two or more, typically four, up to about fifty or more, MHC monomers.

As used herein, "multimeric complex" refers to a complex between a population of target cells and one or more rMHC complexes, where the MHC protein of the rMHC complex includes the multimeric form of the MHC protein. In certain embodiments, the multimeric form of the MHC protein includes a dimer or trimer.

As used herein, the phrase "immune response" refers to the development of a cell-mediated response (mediated by antigen-specific T cells or their secretion products). The cellular immune response is elicited by the presentation of polypeptide epitopes in association with MHC class I or class II molecules, with the treatment or prevention of viral infection, the expansion of antigen-specific _Breg , TC1, CD4 ⁺ T-helper cells and/or CD8 ⁺ cytotoxic T-cells and/ or disease-generated autoregulatory T cells and "memory" B cells. The response may also include the activation of other components. In certain aspects, the term "immune response" can be used to include the formation of an immunocyte regulatory network. Thus, the term "regulatory network formation" may refer to an immune response elicited such that immunocytes, preferably T cells, more preferably regulatory T cells, initiate further differentiation of other immunocytes, such as, but not limited to, B cells or antigen-presenting cells, non-limiting examples of which include dendritic cells, monocytes, and macrophages. In accordance with certain embodiments, the formation of a regulatory network includes B cells differentiating into regulatory B cells; In accordance with certain embodiments, the formation of a regulatory network includes the formation of tolerogenic antigen-presenting cells.

By "nanosphere", "NP", or "nanoparticle", as used herein, is meant a small, discrete particle that is optionally administered in the singular or plural to an individual, cell sample, or tissue sample. In accordance with certain embodiments, the term "nanoparticle" as used herein includes any layers around the core of the nanoparticle. In accordance with certain embodiments, the shape of the nanoparticles is essentially spherical. In accordance with certain embodiments, the nanoparticle is not a liposome or a viral particle. According to further embodiments, the nanoparticle is comprised of any suitable material, for example, a solid, a solid core, a metal, a dendrimer, a polymer micelle, a metal or protein oxide, or a fragment thereof, or combinations thereof. As used herein, the term "substantially spherical" means that the shape of the particles does not deviate from a sphere by more than about 10%. Various known antigenic or peptide complexes of the invention can be applied to the particles. The size of the nanoparticles of the present invention ranges from about 1 nm to about 1 µm, and preferably from about 1 nm to about 500 nm, or alternatively from about 1 nm to about 100 nm, or alternatively from about 1 nm up to about 50 nm, or alternatively, from about 5 nm to about 100 nm, and In certain aspects, refers to the average or median diameter of a plurality of nanoparticles when a plurality of nanoparticles is given. Smaller nano-sized particles can be obtained, for example, by a fractionation process where larger particles are allowed to settle in an aqueous solution. The top of the solution is then separated by methods known to those skilled in the art. This upper part is enriched with smaller particles. The method can be repeated until the desired average size is obtained. The term "nanostructure" is generally used to describe structures smaller than about 1 micron.

As used herein, the terms "inflammatory response" and "inflammation" refer to the complex biological response of an individual's vascular tissue to noxious stimuli, such as pathogens, damaged cells, or irritants, and includes the secretion of cytokines, and more particularly pro-inflammatory cytokines, i.e. . cytokines, which are predominantly produced by activated immunocytes and which are involved in the development of inflammatory reactions. Illustrative pro-inflammatory cytokines include, but are not limited to, IL-1, IL-6, IL-10, TNF-α, IL-17, IL21, IL23, IL27, and TGF-β. Exemplary types of inflammation include acute inflammation and chronic inflammation. Acute inflammation means a short-term process characterized by the classic signs of inflammation (swelling, redness, pain, heat and loss of function) due to infiltration of tissues by plasma and leukocytes. Typically, acute inflammation occurs when a damaging stimulus is present and ceases once the stimulus is removed, destroyed, or protected by scarring (fibrosis). Chronic inflammation means a condition characterized by simultaneous active inflammation, tissue destruction and repair attempts. Chronic inflammation is not characterized by the classic signs of acute inflammation listed above. Instead, chronically inflamed tissue is characterized by infiltration by mononuclear immunocytes (monocytes, macrophages, lymphocytes, and plasma cells), tissue destruction, and attempts at healing, which includes angiogenesis and fibrosis. Inflammation in the context of the present invention can be inhibited by affecting and in particular by inhibiting one of the events that form a complex biological response associated with inflammation in an individual.

As used herein, "CD49b" or "cluster of differentiation 49b" is a protein that is the alpha subunit of integrin and makes up approximately one half of the alpha2beta1 integrin duplex. In humans, CD49b codes for the CD49b gene. CD49b can be present on a wide range of cell types, including T cells, natural killer cells, fibroblasts, and platelets. In certain embodiments, the T cell comprises a T _R 1 cell. In certain embodiments, expression of CD49b identifies a T _R 1 cell. available, for example, from a vendor such as BioLegend.

As used herein, "Lag3" or "lymphocyte activation gene 3" or "CD223" or "differentiation cluster 223" is a protein encoded by the Lag3 gene and belonging to the immunoglobulin (Ig) superfamily. Lag3 is a cell surface protein that is expressed on many cell types, including T cells, natural killer cells, B cells, and plasmacytoid dendritic cells. In certain embodiments, the T cell comprises a T _R 1 cell. In certain embodiments, expression of Lag3 identifies a T _R 1 cell. available, for example, from a vendor such as BioLegend.

As used herein, the term "disease-associated" antigen means an antigen or fragment thereof selected for the treatment of a specified disease and involved in a pathological process. For example, a diabetes-associated antigen is an antigen or fragment thereof which, when present, elicits an immune response that serves to treat diabetes; thus, a diabetes-associated antigen conferring such an effect is of choice for the treatment of diabetes. The multiple sclerosis (MS) associated antigen has been selected for the treatment of MS. Diabetes-associated antigen cannot be chosen for the treatment of MS. Similarly, an autoimmunity-associated antigen is an antigen that is associated with an autoimmune disease and that cannot be chosen to treat a disorder or disease other than autoimmunity, such as cancer. Non-limiting illustrative disease-associated antigens are described herein, and, in addition, such antigens can be determined for a specific disease based on technologies, mechanisms, and methods described in the literature.

"Autoimmune disease or disorder" includes diseases or disorders occurring in and directed against an individual's own tissues or organs, or their manifestations or conditions caused by them. In one embodiment, the term refers to a condition that results from or is exacerbated by the production of T cells that react with normal body tissues and antigens. Non-limiting examples of autoimmune diseases or disorders include arthritis (rheumatoid arthritis such as acute arthritis, chronic rheumatoid arthritis, gouty arthritis, acute gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis , Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, vertebral arthritis and juvenile onset rheumatoid arthritis, osteoarthritis, chronic progressive arthritis, arthritis deformans, primary chronic polyarthritis, reactive arthritis and ankylosing spondylitis), inflammatory hyperproliferative skin diseases, psoriasis such as plaque psoriasis, guttate psoriasis, pustular psoriasis and nail psoriasis, atopic diseases including atopic diseases such as hay fever and Job's syndrome, dermatitis including contact dermatitis, chronic conta contact dermatitis, flaky dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, nummular dermatitis, seborrheic dermatitis, nonspecific dermatitis, primary irritant contact dermatitis and atopic dermatitis, x-linked hyper-IgM syndrome, allergic intraocular inflammatory diseases, such as chronic allergic urticaria and chronic idiopathic urticaria including chronic autoimmune urticaria, myositis, polymyositis/dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic scleroderma), sclerosis such as systemic sclerosis, multiple sclerosis (MS) such as spino-optic PC, primary progressive PC (PPMS) and relapsing-remitting PC (RRMS), progressive systemic sclerosis, atherosclerosis, atherosclerosis, disseminated sclerosis, ataxic sclerosis, neuromyelitis optic nerve with spectral disorder (NMO, also known as Devic's disease or Devick's syndrome), inflammatory bowel disease (IBD) (eg, Crohn's disease, autoimmune-mediated gastrointestinal diseases, colitis such as ulcerative colitis, microscopic colitis, collagenous colitis, polyposis colitis, necrotizing enterocolitis and transmural colitis, and autoimmune inflammatory bowel disease) , inflammatory bowel disease, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, respiratory distress syndrome including adult respiratory distress syndrome or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the choroid, iritis, choroiditis, autoimmune haematological disorder, rheumatoid spondylitis, rheumatoid synovitis, hereditary angioedema, cranial nerve damage as in meningitis, herpes pregnancy, pregnancy pemphigoid, scrotal pruritus, autoimmune premature ovarian failure, severe hearing loss due to autoimmune r IgE-mediated disorders such as anaphylaxis and allergic and atopic rhinitis, encephalitis such as Rasmussen's encephalitis and limbic and/or brainstem encephalitis, uveitis such as anterior uveitis, acute anterior uveitis, granulomatous uveitis, non-granulomatous uveitis , phacoantigenic uveitis, posterior uveitis or autoimmune uveitis, glomerulonephritis (GN) with and without nephrotic syndrome such as chronic or acute glomerulonephritis such as primary GN, immune-mediated GN, membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membranous proliferative GN (MPGN), including type I and type II and rapidly progressive GN, proliferative nephritis, autoimmune polyglandular endocrine insufficiency, balanitis, including herpes zoster plasmacytic balanitis, balanoposthitis, annular centrifugal erythema, persistent dyschromic erythema, erythema multiforme, annulare granuloma, lichen lichen, lichen sclerosus, lichen simplex chronicus, lichen styloid, lichen planus, lamellar ichthyosis, epidermolytic hyperkeratosis, premalignant keratosis, pyoderma gangrenosum, allergic pathological conditions and responses, allergic reaction, eczema, including allergic or atopic eczema, asteatotic eczema , dyshidrotic eczema and vesicular palmoplantar eczema, asthma such as bronchial asthma and autoimmune asthma, pathological conditions including T-cell infiltration and types of chronic inflammatory response, types of immune response to foreign antigens, such as antigens of blood groups A-B -O fetus in pregnancy, chronic pulmonary inflammatory diseases, autoimmune myocarditis, leukocyte adhesion deficiency, lupus, including lupus nephritis, lupus encephalitis, pediatric lupus, non-renal lupus, extra-renal lupus, discoid lupus and discoid lupus erythematosus, lupus with alopecia, systemic lupus erythematosus (SLE) such as cutaneous SLE or subacute cutaneous SLE, neonatal lupus erythematosus (NLE) and disseminated lupus erythematosus, type I diabetes, type II diabetes, latent adult autoimmune diabetes (or type 1.5 diabetes) . Also included are types of immune response associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, sarcoidosis, granulomatosis, including lymphogranulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitis, including vasculitis, large vessel vasculitis (including polymyalgia rheumatica and giant cell (Takayasu) arteritis). ), medium vessel vasculitis (including Kawasaki syndrome and polyarteritis nodosa/periarteritis nodosa), microscopic polyarteritis, immunovasculitis, CNS vasculitis, cutaneous vasculitis, allergic vasculitis, necrotizing vasculitis such as systemic necrotizing vasculitis, and ANCA-associated vasculitis such as vasculitis or syndrome Churg-Strauss (CSS) and ANCA-associated small vessel vasculitis, temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs-positive anemia, Diamond-Blackfan anemia, hemolytic anemia, or immune hemolytic anemia, including autoimmune hemolytic anemia (AIHA), Addison's disease, autoimmune neutropenia, pancytopenia, leukopenia, diseases including diapedesis of leukocytes, inflammatory disorders of the CNS, Alzheimer's disease, Parkinson's disease, multiple organ failure syndrome, for example, secondary to septicemia, trauma or bleeding mediated by antigen- disease antibody, antiglomerular basement membrane disease, antiphospholipid antibody syndrome, antiphospholipid syndrome, allergic neuritis, Behçet's disease/syndrome, Castleman's syndrome, Goodpasture's syndrome, Raynaud's syndrome, Sjögren's syndrome, Stevens-Johnson syndrome, pemphigoid such as bullous pemphigoid and cutaneous pemphigoid, pemphigus (including pemphigus vulgaris, pemphigus exfoliative, mucosal membrane vesicular pemphigoid, and pemphigus erythematosus), autoimmune polyendocrinopathy, Reiter's disease or syndrome, thermal injury, preeclampsia, immune complex metabolism disorder such as immune complex nephritis, antibody-mediated nephritis, polyneuropathy, chronic neuropathy such as polyneuropathy involving IgM or IgM mediated neuropathy, autoimmune or immune-mediated thrombocytopenia such as idiopathic thrombocytopenic purpura (ITP), including chronic or acute ITP, acquired thrombocytopenic purpura, scleritis, such as idiopathic keratoscleritis, episcleritis, autoimmune diseases of the testes and ovaries, including autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases, including thyroiditis, such as autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis), or subacute, thyroid diseases thyroid, idiopathic hypothyroidism, Grave's disease, polyglandular syndromes such as autoimmune polyglandular syndromes (or polyglandular endocrinopathic syndromes), paraneoplastic syndromes, including neurological parane oplastic syndromes such as myasthenic Lambert-Eaton syndrome or Eaton-Lambert syndrome, muscle stiffness syndrome, encephalomyelitis such as allergic encephalomyelitis and experimental allergic encephalomyelitis (EAE), myasthenia gravis such as thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opso-myoclonal syndrome (OMS) and sensory neuropathy, multifocal motor neuropathy, Sheehan syndrome, autoimmune hepatitis, chronic hepatitis, lupus hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphoid interstitial pneumonitis (LIP), bronchiolitis obliterans (no graft) depending on NSIP, Guillain-Barré syndrome, Berger's disease (IgA nephropathy), IgA idiopathic nephropathy, IgA linear dermatosis, acute febrile neutrophilic dermatosis, subcorneal pustular dermatosis, transient acantholytic dermatosis, cirrhosis, t such as primary biliary cirrhosis and pneumocirrhosis, autoimmune enteropathic syndrome, celiac disease, celiac disease (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune ear diseases such as autoimmune inner ear disease (AIED), autoimmune hearing loss, polychondria such as refractory or relapsing polychondria, pulmonary alveolar proteinosis, Cogan's syndrome/non-syphilitic interstitial keratitis, Bell's palsy, Sweet's disease/syndrome, autoimmune rosacea, herpes zoster-associated pain, amyloidosis, non-malignant lymphocytosis, primary lymphocytosis, which includes monoclonal B-cell lymphocytosis (eg, benign monoclonal gammopathy and monoclonal gammopathy of uncertain origin, MGUS), peripheral neuropathy, paraneoplastic syndrome, channelopathies such as epilepsy, migraine, arrhythmias, muscle disorders, deafness, blindness, periodic paralysis, and CNS channelopathies, autism, inflammatory myopathy, focal or segmental or focal segmental glomerulosclerosis (FSGS), endocrine ophthalmopathy, uve retinitis, chorioretinitis, autoimmune liver disease, fibromyalgia, multiple endocrine insufficiency, Schmidt's syndrome, adrenalitis, gastric atrophy, presenile dementia, demyelinating diseases such as autoimmune demyelinating diseases and chronic inflammatory demyelinating polyneuropathy, Dressler's syndrome, alopecia areata, alopecia generalized, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dyskinesia, sclerodactyly and telangiectasia), male and female autoimmune infertility, for example, due to the presence of antibodies to spermatozoa, mixed connective tissue diseases, Chagas disease, rheumatic fever, habitual miscarriage, farmer's lung, erythema multiforme, postcardiotomy syndrome , Cushing's syndrome, poultry farmer's lung, allergic granulomatous vasculitis, benign lymphocytic vasculitis, Alport's syndrome, alveolitis such as allergic alveolitis and fibrosing alveolitis, interstitial lego disease, transfusion reaction, leprosy, malaria, parasitic diseases such as leishmaniasis, trypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Sumpter's syndrome, Kaplan's syndrome, dengue fever, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial pulmonary fibrosis, pulmonary fibrosis , idiopathic pulmonary fibrosis, cystic fibrosis (cystic fibrosis), endophthalmitis, persistent erythema elevated, fetal erythroblastosis, eosinophilic fasciitis, Schulmann's syndrome, Felty's syndrome, filariasis, cyclitis such as chronic cyclitis, heterochronic cyclitis, iridocyclitis (acute or chronic), or Fuchs cyclitis , Henoch-Schonlein purpura, human immunodeficiency virus (HIV) infection, SCID, acquired immunodeficiency syndrome (AIDS), echovirus infection, sepsis, endotoxemia, pancreatitis, thyrotoxicosis, parvovirus infection, rubella virus infection, post-vaccination syndromes, congenital rubella infection, Epstein-Barr virus infection, epidemic arotitis, Evan's syndrome, autoimmune gonadal insufficiency, Sydenham's chorea, poststreptococcal nephritis, thromboangiitis obliterans, thyrotoxicosis, spinal cord tabes, choroiditis, polymyalgia giant cell, chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathic nephritic syndrome and reperfusion injury after ischemia, transplant organ reperfusion, retinal autoimmunity, joint inflammation, bronchitis, chronic obstructive airway/pulmonary disease, silicosis, aphthae, aphthous stomatitis, arteriosclerotic disorders, aspermatogenesis, autoimmune hemolysis, Beck's disease, cryoglobulinemia, Dupuytren's contracture, phacoanaphylactic endophthalmitis, allergic enteritis, erythema nodosum leprosy, idiopathic facial paralysis, chronic fatigue syndrome, rheumatic fever, Hamman-Rich disease, sen soneural hearing loss, paroxysmal hemoglobinuria, hypogonadism, regional ileitis, leukopenia, infectious mononucleosis, transverse myelitis, primary idiopathic myxedema, nephrosis, sympathetic ocular inflammation, granulomatous orchitis, pancreatitis, acute polyradiculitis, pyoderma gangrenosum, de Quervain's thyroiditis, acquired atrophy of the spleen, non-malignant thymoma, vitiligo, toxic shock syndrome, food poisoning, pathological conditions including T-cell infiltration, leukocyte adhesion deficiency, immune response associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, diseases including diapedesis of leukocytes, multiple organ failure syndrome , antigen-antibody complex-mediated diseases, disease with the formation of antibodies to the glomerular basement membrane, allergic neuritis, autoimmune polyendocrinopathy, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic thalmia, rheumatism, mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine insufficiency, autoimmune polyglandular syndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH), cardiomyopathy such as dilated cardiomyopathy, epidermolysis bullosa acquired (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent or non-purulent sinusitis, acute or chronic sinusitis, sinusitis of the ethmoid, frontal, maxillary, or sphenoid bones, eosinophil-related disorders such as eosinophilia, eosinophilia with pulmonary infiltration, eosinophilia-myalgia syndrome, Leffler, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonic aspergillosis, aspergilloma or granulomas containing eosinophils, anaphylaxis, seronegative spondyloarthritis, polyendocrine autoimmune diseases, sclerosing cholangitis, chronic scleral candidiasis, episclera, mucosal and skin disorders, Bruton's syndrome, neonatal transient hypogammaglobulinemia, Wiskott-Aldrich syndrome, ataxia-telangiectasia syndrome, angiectasia, autoimmune disorders associated with collagen disease, rheumatism, neurological diseases, lymphadenitis, decreased blood pressure response, vascular dysfunction, tissue damage, cardiovascular ischemia, hyperalgesia, renal ischemia, cerebral ischemia and diseases accompanying vascularization, disorders associated with allergic hypersensitivity, glomerulonephritis, reperfusion injuries, disorders associated with reperfusion after ischemia, reperfusion injuries of myocardial or other tissues, lymphomatous tracheobronchitis, inflammatory dermatoses, dermatoses with acute inflammatory component, multiple organ failure, bullous disease, renal cortical necrosis, acute purulent meningitis or other inflammatory disorders of the central nervous system, inflammatory eye disorders and orbital syndromes, granulocyte transfusion-associated syndromes, cytokine-induced toxicity, narcolepsy, acute severe inflammation, chronic intractable inflammation, pyelitis, intra-arterial hyperplasia, peptic ulcer, valvulitis, emphysema, alopecia areata, adipose tissue inflammation/diabetes type II, obesity-associated adipose inflammation tissue/insulin resistance and endometriosis.

In accordance with certain embodiments, autoimmune disorders or diseases may include, but are not limited to, type I and type II diabetes mellitus, prediabetes, transplant rejection, multiple sclerosis, multiple sclerosis-related disorders, premature ovarian failure, scleroderma, Sjögren's disease/syndrome, lupus , vitiligo, alopecia (baldness), polyglandular insufficiency, Grave's disease, hypothyroidism, polymyositis, pemphigus, Crohn's disease, colitis, autoimmune hepatitis, hypopituitarism, myocarditis, Addison's disease, autoimmune skin diseases, uveitis, pernicious anemia, hypoparathyroidism and / or rheumatoid arthritis . Other indications of interest include, but are not limited to, asthma, allergic asthma, primary biliary cirrhosis, cirrhosis, neuromyelitis of the optic nerve with spectrum disorder (Devic's disease, spino-optic multiple sclerosis (OSMS)), pemphigus vulgaris, inflammatory bowel disease (IBD), arthritis, rheumatoid arthritis, systemic lupus erythematosus (SLE), celiac disease, psoriasis, autoimmune cardiomyopathy, idiopathic dilated cardiomyopathy (IDCM), myasthenia gravis, uveitis, ankylosing spondylitis, immune-mediated myopathies, prostate cancer, antiphospholipid syndrome (ANCA+), atherosclerosis, dermatomyositis, chronic obstructive pulmonary disease (COPD), emphysema, spinal cord injury, traumatic injury, tobacco-induced lung destruction, ANCA-associated vasculitis, psoriasis, sclerosing cholangitis, primary sclerosing cholangitis, and central and peripheral nerve disease systems.

In accordance with certain embodiments, autoimmune disorders or diseases may include, but are not limited to, diabetes, multiple sclerosis, celiac disease, primary biliary cirrhosis, pemphigus, pemphigus foliaceus, pemphigus vulgaris, optic neuromyelitis with spectrum disorder, arthritis (including rheumatoid arthritis), allergic asthma, inflammatory bowel disease (including Crohn's disease and ulcerative colitis), systemic lupus erythematosus, atherosclerosis, chronic obstructive pulmonary disease, emphysema, psoriasis, autoimmune hepatitis, uveitis, Sjögren's syndrome, scleroderma, antiphospholipid syndrome, ANCA-associated vasculitis, and musculoskeletal syndrome stiffness.

As used herein, the term "adipose tissue inflammation/type II diabetes" refers to inflammation of adipose tissue found in an individual suffering from type II diabetes. Inflammation of adipose tissue contributes to the development of insulin resistance in an individual.

As used herein, the term "obesity-associated adipose tissue inflammation/insulin resistance" refers to adipose tissue inflammation found in an obese individual. Inflammation of adipose tissue contributes to insulin resistance in an individual, thus increasing the likelihood that inflammation of adipose tissue will lead to the pathogenesis of type II diabetes.

As used herein, the term "canonical sequence" refers to a protein sequence used as a reference for amino acid numbering in the absence of further guidance in the specification or prior art. As will be apparent to those skilled in the art, the ends of antigenic fragments relative to the reference sequence on which the fragment is mapped may vary. Thus, it should be understood that, unless specifically stated otherwise, the fragment identifies the approximate ends.

As used herein, "PPI" refers to all isoforms, variants and fragments thereof of the protein associated with the name "preproinsulin", the biologically inactive precursor of the biologically active endocrine hormone insulin, or its biological equivalent. The length of the canonical sequence of the PPI isoform is 110 amino acids:

As used herein, "IGRP" refers to all isoforms, variants, and fragments thereof of the protein associated with the name "glucose-6-phosphatase-specific islet-specific catalytic subunit related protein" or "glucose-6-phosphatase 2", the major autoantigen for autoimmune type 1 diabetes, or its biological equivalent. The length of the canonical IGRP sequence is 355 amino acids:

As used herein, "GAD" refers to all isoforms, variants, and fragments thereof of the protein associated with the name "glutamic acid decarboxylase", the diabetes-associated antigen, or its biological equivalent. GAD may optionally refer to GAD1, GAD2, GAD65, GAD67, or any other diabetes-associated glutamic acid decarboxylase. The length of the canonical sequence of the GAD2 isoform is 585 amino acids, and this sequence is given below in this document:

As used herein, "periphery" refers to all isoforms, variants, and fragments thereof of the protein associated with that name, or its biological equivalent. A non-limiting illustrative sequence of human peripherin associated with UniProt accession number P41219 is provided below herein:

As used herein, "aGlia" refers to all isoforms, variants, and fragments thereof of a protein associated with the name "alpha/beta-gliadin" derived from a member of the wheat family or other celiac-associated allergen, or its biological equivalent. A non-limiting exemplary sequence of wheat-expressed alpha-gliadin associated with GenBank accession number CAA10257.1 is:

Another non-limiting exemplary sequence of alpha-gliadin expressed in wheat is provided below herein:

As used herein, "PDC-E2" refers to all isoforms, variants, and fragments thereof of the protein associated with the name "dihydrolipoamide-S-acetyltransferase" or "DLAT", the primary biliary cirrhosis autoantigen, or its biological equivalent. The length of the canonical sequence of PDC-E2 is 647 amino acids, and this sequence is given below in this document:

As used herein, "insulin" refers to all isoforms, variants, and fragments thereof of the protein associated with this name, or its biological equivalent. A non-limiting exemplary human insulin sequence associated with UniProt accession number P01308 is provided below in this document:

As used herein, "DG1EC2" refers to all isoforms, variants and fragments thereof of the protein associated with the name "desmosomal glycoprotein 1" or its biological equivalent. The length of the canonical sequence of DG1EC2 is 1054 amino acids and is given below in this document:

As used herein, "DG3" refers to all isoforms, variants and fragments thereof of the protein associated with the name "desmoglein 3" or its biological equivalent. A non-limiting illustrative sequence of human desmoglein 3 associated with UniProt accession number P32926-1 is provided below herein:

As used herein, "AQP4" refers to all isoforms, variants and fragments thereof of the protein associated with the name "aquaporin 4", belonging to the aquaporin family of integral membrane proteins that allow water to pass through the cell membrane and is the primary autoimmune target in neuromyelitis. optic nerve with spectral disorder, or its biological equivalent. The length of the canonical sequence of AQP4 is 323 amino acids and is given below in this document:

As used herein, "PLP" refers to all isoforms, variants, and fragments thereof of the protein associated with the name "myelin proteolipid protein" or its biological equivalent. A non-limiting illustrative human myelin proteolipid protein sequence associated with UniProt Accession P60201 is provided herein below:

As used herein, "MOG" refers to all isoforms, variants and fragments thereof of the protein associated with the name "myelin oligonocyte glycoprotein" or its biological equivalent. A non-limiting illustrative sequence of human myelin oligonedrocyte glycoprotein associated with UniProt accession number Q16653 is provided below herein:

As used herein, "MBP" refers to all isoforms, variants, and fragments thereof of the protein associated with the name "myelin basic protein" or its biological equivalent. A non-limiting illustrative human myelin basic protein sequence associated with UniProt accession number P02686 is provided below herein:

As used herein, "CII" refers to all isoforms, variants and fragments thereof of the protein associated with the name "native type II collagen", a high molecular weight fibrillar molecule involved in chronic polyarthritis, or its biological equivalent. A non-limiting illustrative human collagen II consensus sequence is provided herein below:

Below in this document is another non-limiting illustrative sequence of collagen II, mice:

As used herein, "DERP1" refers to all isoforms, variants and fragments thereof of the protein associated with the name "p1 Dermatophagoides pteronyssius" and known to cause an allergic reaction in humans, or its biological equivalent. A non-limiting exemplary DERP1 consensus sequence is provided below in this document:

As used herein, "DERP2" refers to all isoforms, variants, and fragments thereof of the protein associated with the name "Dermatophagoides pteronyssius p2" and known to cause an allergic reaction in humans, or its biological equivalent. A non-limiting exemplary DERP2 consensus sequence is provided below in this document:

As used herein, "OVA" refers to all isoforms, variants, and fragments thereof of the protein associated with the name "ovalbumin" for use in generating an allergic response in mice, or its biological equivalent.

As used herein, "BacInt" or "bacteroid integrase" refers to all isoforms, variants, and fragments thereof of the protein associated with this name, or its biological equivalent. The length of the canonical sequence of BacInt is 406 amino acids and is presented below in this document:

As used herein, "CBir", "Fla-X", and/or "Fla-2" refers to all isoforms, variants, and fragments thereof of a protein associated with one or more bacterial flagellins involved in colitis, or a biological equivalent thereof. . A non-limiting illustrative sequence of Fla-X is provided below in this document:

Below in this document is a non-limiting exemplary sequence of Fla-2:

As used herein, "YIDX" refers to all isoforms, variants and fragments thereof of the protein associated with this name, which is of bacterial origin and is involved in the pathogenesis of immune-mediated diseases, or its biological equivalent. A non-limiting exemplary YIDX sequence is provided below in this document:

Below in this document is another non-limiting illustrative YIDX sequence:

As used herein, "AChR" refers to all isoforms, variants, and fragments thereof of the protein associated with that name, or its biological equivalent.

Below is a non-limiting exemplary acetylcholine receptor sequence associated with UniProt accession number Q13702-1:

Below is a non-limiting exemplary acetylcholine receptor sequence associated with UniProt accession number Q04844-1:

Below is a non-limiting exemplary acetylcholine receptor sequence associated with UniProt Accession P02708-1:

Below is a non-limiting exemplary acetylcholine receptor sequence associated with UniProt Accession P07510-1:

Below is a non-limiting exemplary acetylcholine receptor sequence associated with UniProt Accession P11230-1:

Below is a non-limiting exemplary acetylcholine receptor sequence associated with UniProt accession number Q07001-1:

As used herein, "thyroid peroxidase" refers to all isoforms, variants and fragments thereof of the protein associated with this name, or its biological equivalent. A non-limiting illustrative sequence of human thyroid peroxidase associated with UniProt accession number P07202 is provided below in this document:

As used herein, "thyroid receptor" refers to all isoforms, variants and fragments thereof of the protein associated with this name, or its biological equivalent. In certain embodiments, "thyroid receptor" includes "thyroid stimulating hormone receptor". A non-limiting exemplary thyroid stimulating hormone receptor sequence associated with UniProt accession number P16473-1 is provided herein below:

Below is a non-limiting exemplary thyroid stimulating hormone receptor sequence associated with UniProt accession number Q59GA2-1:

Below is a non-limiting exemplary thyroid stimulating hormone receptor sequence associated with UniProt Accession B4E0H2-1:

As used herein, "phospholipid antigen" refers to all isoforms, variants and fragments thereof of the protein associated with this name, or its biological equivalent. One non-limiting example of a phospholipid antigen is "beta2-glycoprotein I", the sequence of which is given herein below:

As used herein, "H4" refers to all isoforms, variants, and fragments thereof of the protein associated with the name "Histone H4" or its biological equivalent. The canonical H4 sequence is given below in this document:

As used herein, "H2B" refers to all isoforms, variants, and fragments thereof of the protein associated with the name "H2B histone" or its biological equivalent. The canonical H2B sequence is given below in this document:

As used herein, "H1" refers to all isoforms, variants, and fragments thereof of the protein associated with the name "histone H1" or its biological equivalent. The canonical H1 sequence is given below in this document:

As used herein, "ApoB" refers to all isoforms, variants, and fragments thereof of the protein associated with the name "apolipoprotein B" or its biological equivalent. The canonical sequence of ApoB is given below in this document:

As used herein, "ApoE" refers to all isoforms, variants, and fragments thereof of the protein associated with the name "apolipoprotein E" or its biological equivalent. A non-limiting exemplary human apoE sequence associated with UniProt accession number P02649 is provided herein below:

As used herein, "NMDAR" refers to all isoforms, variants, and fragments thereof of the protein associated with the name "N-methyl-D-aspartate receptor" or its biological equivalent. A non-limiting exemplary N-methyl-D-aspartate receptor sequence associated with UniProt accession number Q13224-1 is provided herein below:

As used herein, "voltage-gated potassium channel" generally refers to a transmembrane channel specific for potassium and sensitive to changes in cell membrane potential voltage. During an action potential, these channels play a key role in returning a depolarized cell to its resting state. A non-limiting exemplary voltage-gated potassium channel sequence associated with UniProt accession number P22459-1 is provided below herein:

As used herein, "elastin" refers to all isoforms, variants and fragments thereof of the protein associated with this name, or its biological equivalent. The length of the canonical sequence of elastin is 786 amino acids and is given below in this document:

As used herein, "IRBP" refers to all isoforms, variants, and fragments thereof of the protein associated with the name "interphotoreceptor retinol-binding protein" or its biological equivalent.

As used herein, "human retinal arrestin S antigen" refers to all isoforms, variants, and fragments thereof of the protein associated with this name, or its biological equivalent. One of the non-limiting illustrative sequences is given below in this document:

As used herein, "myosin" refers to all isoforms, variants, and fragments thereof of the protein associated with this name, or its biological equivalent. A non-limiting exemplary myosin sequence associated with UniProt accession number P35580-1 is provided below in this document:

Below in this document is another non-limiting illustrative sequence of myosin:

As used herein, "CD1d-binding lipid antigens" generally refers to lipid antigens that bind to the non-classical MHC CD1d.

As used herein, "HSP" refers to all isoforms, variants, and fragments thereof of the protein associated with the name "heat shock protein" or its biological equivalent. In certain embodiments, heat shock proteins include heat shock protein 60. A non-limiting illustrative sequence of heat shock protein 60 associated with UniProt accession number P10809-1 is provided herein below:

Multiple sclerosis (PC), also known as "multiple sclerosis" or "allergic encephalomyelitis". MS is an inflammatory disease in which the lipid-containing myelin sheath around the axons of the brain and spinal cord is damaged, leading to demyelination and scarring, as well as a wide range of manifestations and symptoms. Disorders associated with multiple sclerosis include, for example, neuromyelitis of the optic nerve with spectrum disorder (NMO), uveitis, neuropathic pain, and the like.

"Oligonedrocytic myelin glycoprotein" (MOG) is a glycoprotein believed to be important in the myelination of nerves in the central nervous system (CNS). In humans, this protein codes for the MOG gene. It is thought to serve as a necessary "adhesion molecule" to maintain the structural integrity of the myelin sheath and is known to develop on the oligodendrocyte at a late stage. GenBank accession numbers NM_001008228.2 and NP_001008229.1 are mRNA and protein sequences encoded by the MOG gene, respectively. The sequences associated with each of these GenBank access numbers are included by reference for all purposes.

As used herein, the terms "cancer" and "malignant" represent or describe a physiological condition in mammals that is typically characterized by unregulated cell growth. Non-limiting examples of malignant tumors include carcinoma, lymphoma, blastoma, sarcoma, and leukemia and their metastases. "Metastasis" means the transfer of disease-producing organisms or malignant cells to other parts of the body via blood or lymphatic vessels or membrane surfaces. Non-limiting examples of such malignancies include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung carcinoma, peritoneal cancer, hepatic cell carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, cancer of the female vulva, thyroid cancer, hepatic carcinoma and various types of head and neck cancer.

As used herein, the term "diabetes" refers to various disorders of carbohydrate metabolism caused by a combination of hereditary and environmental factors and is typically characterized by impaired insulin secretion or utilization, excess urine production, excess blood and urine sugar, and thirst, hunger and weight loss. Diabetes is divided into type 1 diabetes and type 2 diabetes. An established animal model for the study and treatment of diabetes is non-obese diabetic ("NOD") mice. Type 1 diabetes (T1D) in mice is associated with autoreactive CD8 ⁺ T cells. Non-obese (NOD) mice develop a form of T1D that is very similar to human T1D, resulting from the selective destruction of pancreatic β-cells by T-cells that recognize a growing list of self-antigens. Although T1D initiation certainly requires input from CD4 ⁺ cells, there is strong evidence that T1D is dependent on CD8 ⁺ T cells. A significant fraction of islet-associated CD8 ⁺ cells in NOD mice have been found to use CDR3-invariant Vα17-Jα42 ⁺ TCRs, referred to as "8.3-TCR-like". These cells, which recognize the NRP-A7 mimotope (detected using combinatorial peptide libraries) in the context of the MHC K ^d molecule, are already a significant component of the earliest CD8 ⁺ cell infiltrates in NOD mouse islets, cause diabetes, and are targeted by a catalytic subunit-related peptide. islet-specific glucose-6-phosphatase protein (IGRP), a protein of unknown function. Typically, CD8 ⁺ cells recognizing this peptide (IGRP _206-214 , similar to NRP-A7) are often found in the circulation (>1/200 CD8 ⁺ cells). Moreover, the progression of insulitis to diabetes in NOD mice is invariably accompanied by a cyclic increase in the circulating population of CD8 ⁺ cells responsive to IGRP _206-214 and maturation of the avidity of its islet-associated equivalent. It has recently been shown that islet-associated CD8 ⁺ cells in NOD mice recognize multiple IGRP epitopes, implying that IGRP is the dominant autoantigen for CD8 ⁺ cells, at least in T1D mice. Islet-associated CD8 ⁺ NOD cells, in particular those detected early in the pathological process, also recognize the insulin epitope (Ins B _15-23 ).

As used herein, the term "prediabetes" means the asymptomatic period preceding the diabetic condition, characterized by subclinical damage to beta cells, when normal plasma glucose levels are detected in a patient. It is also characterized by the presence of islet cell autoantibodies (ICA) and, when clinical symptoms are close, is accompanied by impaired glucose tolerance.

As used herein, the term "multiple sclerosis-associated disorders" refers to disorders that are collectively identified in predisposition to MS or in MS. Non-limiting examples of such disorders include optic neuromyelitis with spectrum disorder (NMO), uveitis, sclerosis with neuropathic pain, atherosclerosis, disseminated sclerosis, systemic sclerosis, spino-optic PC, primary progressive PC (PPMS) and relapsing-remitting PC (RRMS), progressive systemic sclerosis and ataxic sclerosis.

The terms "epitope" and "antigenic determinant" are used interchangeably to refer to a site on an antigen that is responded to or recognized by B and/or T cells. B-cell epitopes can be formed from contiguous amino acids or non-contiguous amino acids adjacent due to protein tertiary folding. Typically, epitopes formed from contiguous amino acids are retained after exposure to denaturing solvents, while epitopes formed by tertiary folding are generally lost upon treatment with denaturing solvents. Typically, an epitope comprises at least 3, and more often, at least 5 or 8-20 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of epitopes include, for example, X-ray diffraction crystallography and 2D nuclear magnetic resonance. See, for example, Glenn E. Morris, Epitope Mapping Protocols (1996). T cells recognize continuous epitopes of approximately nine amino acids for CD8 ⁺ cells or approximately 13-15 amino acids for CD4 ⁺ cells. T cells that recognize a given epitope can be identified by in vitro assays that detect antigen-dependent proliferation as determined by ³ H-thymidine incorporation by primed T cells in response to the epitope (Burke et al., J. Inf. Dis., 170:1110-1119, 1994), by antigen-dependent cytotoxic activity (cytotoxic T-lymphocyte assay, Tigges et al., J. Immunol., 156(10):3901-3910, 1996) or by cytokine secretion . The presence of a cell-mediated immune response can be determined by proliferation assays (CD4 ⁺ T cells) or by CTL assays (cytotoxic T lymphocytes).

Optionally, the antigen, or preferably the epitope of the antigen, can be chemically conjugated or expressed as a fusion protein with other proteins such as MHC and MHC-related proteins.

As used herein, the terms "patient" and "individual" are used interchangeably and mean a mammal. In accordance with certain embodiments, the patient is a human. In other embodiments, the patient is a mammal in need of veterinary treatment or is a mammal commonly used in laboratories. In certain embodiments, the mammal is a mouse, rat, monkey, dog, cat, cow, horse, or sheep.

As used herein, the term "polynucleotide" refers to a nucleic acid molecule that is either recombinant or isolated from a total genomic nucleic acid. The term "polynucleotide" includes oligonucleotides (nucleic acids of 100 residues or less), recombinant vectors, including, for example, plasmids, cosmids, phages, viruses, and the like. In certain aspects, polynucleotides include regulatory sequences substantially isolated from naturally occurring genes or protein coding sequences containing them. Polynucleotides can be RNA, DNA, analogs thereof, or combinations thereof. A nucleic acid encoding all or part of a polypeptide may comprise a contiguous nucleic acid sequence encoding all or part of such a polypeptide with the following lengths: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 , 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370 , 380, 390, 400, 410, 420, 430, 440, 441, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610 , 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860 , 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1095, 1095 , 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 9000, 10000 or more nucleotides, nucleosides or base pairs. It is also contemplated that a particular polypeptide of a given species may be encoded by nucleic acids containing natural variations that contain slightly different nucleic acid sequences but nevertheless encode the same or substantially similar protein, polypeptide or peptide.

A polynucleotide consists of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T) and uracil (U) instead of thymine when the polynucleotide is RNA. Thus, the term "polynucleotide sequence" is a literal representation of a polynucleotide molecule. This letter representation can be entered into databases on a computer with a central processing unit and used for bioinformatics applications such as functional genomics and homology search.

As used herein, the term "isolated" or "recombinant" in relation to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNA or RNA, respectively, that are present in the natural source of macromolecules, as well as from polypeptides. The term "isolated or recombinant nucleic acid" is intended to include nucleic acid fragments that are not naturally occurring as fragments and cannot be found in the natural state. The term "isolated" is also used herein to refer to polynucleotides, polypeptides, and proteins that are separated from other cellular proteins and is intended to include purified and recombinant polypeptides. In other embodiments, the term "isolated or recombinant" means separated from constituents, cellular or otherwise, with which a cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof are normally associated in nature. For example, an isolated cell is a cell that is isolated from a tissue or cells with a different phenotype or genotype. An isolated polynucleotide is isolated from 3' and 5' contiguous nucleotides with which it is normally associated in nature or its natural environment, eg, on a chromosome. As will be appreciated by those skilled in the art, non-natural polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof do not require "isolation" to distinguish them from their natural equivalents.

A polynucleotide or region of a polynucleotide (or a polypeptide or region of a polypeptide) with a certain percentage (e.g., 80%, 85%, 90%, or 95%) of "sequence identity" with another sequence means that, after alignment, that percentage of bases (or amino acids) when compared two sequences is the same. Alignment can be performed and percent homology or sequence identity can be determined using software products known in the art, such as those described in Current Protocols in Molecular Biology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18 , Table 7.7.1. For alignment, the default parameters are preferably used. One of the alignment programs is BLAST, which uses default parameters. Specifically, certain programs are BLASTN and BLASTP, which use the following default parameters: genetic code = standard; filter = missing; chain = both; threshold = 60; waiting = 10; matrix=BLOSUM62; description = 50 sequences; sort = HIGH SCORE; databases = non-degenerate, GenBank + EMBL + DDBJ + PDB + GenBank translated CDS + SwissProtein + SPupdate + PIR. More detailed descriptions of these programs can be found at the following Internet addresses: ncbi.nlm.nih.gov/cgi-bin/BLAST.

Without express description and unless otherwise indicated, when the present invention relates to an antigen, polypeptide, protein, polynucleotide or antibody, the scope of the present invention implies the presence of their equivalents or biological equivalents. As used herein, the term "its biological equivalent" is considered to be synonymous with "its equivalent" with respect to the parent antigen, protein, antibody, fragment, polypeptide, or nucleic acid, and means an antigen, protein, antibody, fragment, polypeptide, or nucleic acid with minimal homology while maintaining the desired structure or functionality. Unless specifically stated herein, any polynucleotide, polypeptide or protein referred to herein is also intended to include their equivalents. In one aspect, an equivalent polynucleotide is a polynucleotide that hybridizes under stringent conditions to a polynucleotide or complementary polynucleotide as described herein for use in the described methods. In another aspect, equivalent antibody or antigen-binding polypeptide means an antibody or antigen-binding polypeptide that binds at least 70%, or alternatively at least 75%, or alternatively at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95% or greater affinity of the reference antibody or antigen-binding fragment. In another aspect, their equivalent competes with an antibody or antigen-binding fragment for binding to their antigen in a competitive ELISA assay. In another aspect, equivalent means at least about 80% homology or identity, and alternatively at least about 85%, or alternatively at least about 90%, or alternatively at least about 95%, or , alternatively, 98% homology or identity and exhibits substantially equivalent biological activity to that of the reference protein, polypeptide, or nucleic acid.

"Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized due to the formation of hydrogen bonds between the bases of the nucleotide residues. Hydrogen bonding can occur by Watson-Crick base pairing, Hoogsteen bonding, or any other sequence-specific manner. The complex may contain two strands forming a duplex structure, three or more strands forming a multistrand complex, one self-hybridizing strand, or any combination of these. The hybridization reaction may be a step in a more complex process such as the initiation of a polymerase chain (PC) reaction or the enzymatic cleavage of a polynucleotide by a ribozyme.

Examples of stringent hybridization conditions include: incubation temperatures from about 25°C to about 37°C; hybridization buffer concentrations from about 6x SSC to about 10x SSC; formamide concentrations from about 0% to about 25%; and wash solutions from about 4x SSC to about 8x SSC. Examples of moderate hybridization conditions include: incubation temperatures from about 40°C to about 50°C; buffer concentrations from about 9x SSC to about 2x SSC; formamide concentrations from about 30% to about 50%; and wash solutions from about 5x SSC to about 2x SSC. Examples of high stringency conditions include: incubation temperatures from about 55°C to about 68°C; buffer concentrations from about 1x SSC to about 0.1x SSC; formamide concentrations from about 55% to about 75%; and wash solutions of about 1x SSC, 0.1x SSC, or they may be deionized water. Typically, hybridization incubation times are from 5 minutes to 24 hours, with 1, 2, or more wash steps, and wash incubation times are approximately 1, 2, or 15 minutes. SSC is 0.15 M NaCl and 15 mm citrate buffer. It should be understood that equivalents to SSC using other buffer systems may be used.

"Homology" or "identity" or "similarity" refers to the sequence similarity of two peptides or two nucleic acid molecules. Homology can be determined by comparing positions within each of the sequences, which can be aligned for comparison purposes. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of sequence homology is a function of the number of matching or homologous positions in the sequences. The identity of the "unrelated" or "non-homologous" sequence with any of the sequences of the present invention is less than 40%, or alternatively, less than 25%.

"Homology" or "identity" or "similarity" can also refer to two nucleic acid molecules that hybridize under stringent conditions.

As used herein, the terms "treat", "treatment", and the like. used herein to denote obtaining the desired pharmacological and/or physiological action. The effect of partially or completely curing a disorder and/or an adverse effect associated with the disorder may be therapeutic. In one aspect, treatment means reducing the severity of the signs of the disease according to the established scale.

As used herein, the term "treat" or "treatment" as it relates to oncology means any treatment of a disease or condition or associated disorder in a patient, including suppression of the disease or condition, i.e. stopping or suppressing the development of clinical symptoms such as cachexia in malignant tumors; and/or mitigation of the disease or condition, ie. providing regression of clinical symptoms, for example, an increase in overall life expectancy or a decrease in tumor burden.

In certain aspects, the term "treatment" refers to improving clinical outcomes. The term "clinical outcome" refers to any clinical observation or definition relating to a patient's response to treatment. Non-limiting examples of clinical outcomes include tumor response (TR), overall survival (OS), progression-free survival (PFS), disease-free survival, time to tumor recurrence (TTR), time to tumor progression (TTR), relative risk ( RR), toxicity or side effect. "Total life expectancy" (OS) means the extension of life expectancy compared to intact or untreated individuals or patients. "Progression-free life" (PFS) or "pre-tumor progression period" (TTP) means the length of time during and after treatment when the cancer does not grow. Progression-free survival includes the period of time when patients show a complete response or partial response, as well as the period of time when the course of the disease in patients is stable. As used herein and as defined by the National Cancer Institute, "tumor recurrence" is a recurrent (returning) cancer, typically after a period of time in which cancer has not been detected. A malignant tumor can recur in the same area as the original (primary) tumor or in another part of the body. It is also called recurrent malignancy. "Time to tumor recurrence" (TTR) is defined as the period from the date of cancer diagnosis to the date of first recurrence, death, or to last contact if the patient did not show any tumor recurrence at the time of last contact. If the patient did not relapse, then TTR was assessed by the time of death or the last examination. "Relative risk" (RR) in statistics and mathematical epidemiology refers to the risk of an event (or development of a disease) relative to treatment. The relative risk is the ratio of the probabilities of an event occurring in the treated group and the untreated group.

The term "composition" is intended to mean a combination of an active agent and another compound or composition, either inert (eg, detectable agent or label) or active, such as an adjuvant. In accordance with certain embodiments, the composition does not contain an adjuvant.

The term "pharmaceutical composition" is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

The term "functionally equivalent codon" is used herein to refer to codons that code for the same amino acid, such as the six codons for arginine or serine, and also refers to codons that code for biologically equivalent amino acids (see Table 2).

As used herein, "protein" or "polypeptide" or "peptide" refers to a molecule containing at least five amino acid residues.

Other objects, characteristics and advantages of the present invention will be apparent from the following detailed description. Additional definitions are also provided there. However, it should be understood that the detailed description and specific examples, pointing to specific embodiments of the invention, are given by way of illustration only, since various changes and modifications will be apparent to those skilled in the art from this detailed description within the meaning and scope of the invention.

Descriptive Embodiments

Autoimmune diseases such as type 1 diabetes (T1D), multiple sclerosis and rheumatoid arthritis are the result of a chronic autoimmune response that involves T cells and B cells recognizing many antigenic epitopes from incompletely defined lists of autoantigens (Santamaria, R. (2010 ) Immunity 32:437-445; Babbe, H. et al. (2000) J. Exp. Med. 192:393-404; Firestein, G. S. (2003) Nature 423:356-361). At present, elimination or suppression of all polyclonal autoreactive specific T cells (known and unknown) for any particular autoimmune disorder is not possible without compromising systemic immunity.

As an alternative therapeutic approach, the adoptive transfer of polyclonal FOXP3 ⁺ CD4 ⁺ CD25 ⁺ regulatory T cells ( _Treg ) expanded ex vivo has been proposed (Sakaguchi, S. et al. (2006) Immunol. Rev. 212:8-27). The clinical application of translation of this approach is hampered by potential adverse immunosuppression, the lack of effective strategies for expanding antigen-specific _Treg cells in vitro, and the instability of FOXP3 ⁺ _Treg cell lines (Zhou, X. et al. (2009) Nature Immunol. 10:1000-1007; Komatsu , N. et al. (2014) Nature Med. 20:62-68; Bailey-Bucktrout, SL et al. (2013) Immunity 39:949-962). Another regulatory subpopulation of T cells recently used to treat human inflammatory diseases are T _R 1 FOXP3 ^- CD4 ⁺ CD25 ^- T cells that produce the cytokines IL-10 and IL-21 and express the surface markers CD49b and LAG-3 and factor c-Maf 8 transcriptions (McLarnon, A. (2012) Nature Rev. Gastroenterol. Hepatol. 9:559; Desreumaux, P. et al. (2012) Gastroenterology 143:1207-1217; Roncarolo, MG et al. (2011) Immunol Rev. 241:145-163). However, as with FOXP3+ T _reg cells, there are no pharmacological approaches by which self-antigen or disease-specific T _R 1-like cells can be propagated in vivo.

Thus, regulatory T cells have potential as targets for therapeutic intervention in autoimmunity, but approaches that can expand antigen-specific regulatory T cells in vivo are not currently available. Applicant herein demonstrates that systemic delivery of nanoparticles coated with autoimmune disease-associated peptides linked to major histocompatibility complex class II (pMHCII) molecules stimulates the formation and proliferation of antigen-specific regulatory type 1 CD4 ⁺ T cells (T _R 1 )-like cells in various mouse models, including mice humanized with patient lymphocytes, resulting in regression of chronic autoimmune symptoms. The ten pMHCII-based nanodrugs show similar biological effects regardless of genetic environment, proportion of the recognizing T cell population, or MHC restriction. These nanodrugs stimulate the differentiation of disease-primed autoreactive T cells into T _R 1-like cells, which in turn suppress autoantigen-loaded antigen-presenting cells and direct the differentiation of recognizing B cells into disease-suppressing regulatory B cells without compromising systemic immunity. Thus, pMHCII-based nanodrugs represent a novel class of drugs potentially useful in treating a wide range of autoimmune disease states in a disease-specific manner.

The Applicant has previously found that systemic delivery of nanoparticles (NPs) coated with T1D-associated class I pMHC complexes (pMHC-NPs) can retard the progression of T1D by expanding CD8 ⁺ T cell subpopulations with regulatory potential but a common memory cell-like phenotype (Tsai, S. et al (2010) Immunity 32:568-580). Since nanoparticles can be coated with various class I rMHC complexes, Applicant believes that pMHC-NP therapy can use the natural regulatory negative feedback loop, whereby long-term exposure to the self-antigen (and exposure to pMHC-NP) can initiate the differentiation of auto-reactive T cells. into progeny regulatory T cells. Based on this, Applicant predicted and demonstrated in the present invention that NPs coated with disease-associated pMHCII complexes can lead to the proliferation of disease-specific regulatory CD4 ⁺ T cells in vivo.

The present invention builds on these initial observations by providing pMHC-NPs, compositions and methods for their preparation, and their uses.

Substrates/Particles

By “particle,” “nanoparticle,” “microparticle,” “granule,” “microsphere,” and their grammatical equivalents, as used herein, are small, discrete particles that can be administered to an individual. In accordance with certain embodiments, the shape of the particles is essentially spherical. As used herein, the term "substantially spherical" means that the shape of the particles does not deviate from a sphere by more than about 10%. Various complexes with known antigens or peptides according to the invention can be applied to the particles.

The pMHC-NP nanoparticle core contains or substantially consists or even more of a core, e.g., solid core, metal core, dendrimer core, polymeric micelle nanoparticle core, nanorod, fullerene, nanoshell, core-shell, protein-based nanostructures or nanostructures based on lipids. In certain aspects, the nanoparticle core is bioabsorbable and/or biodegradable. In certain aspects, a nanoparticle core is a dendrimer nanoparticle core comprising, or alternatively, substantially consisting of, or even more consisting of, a highly branched, tree-like macromolecule growing from the middle. In additional aspects, the nanoparticle core of the dendrimer may comprise, or alternatively be substantially, or even more composed of, a poly(amidoamine) based dendrimer or a poly-L-lysine based dendrimer. In certain aspects, the nanoparticle core is a polymeric micelle core comprising, or alternatively substantially consisting of, or even more composed of, an amphiphilic block copolymer, arranged in a nanoscale core-shell structure. In additional aspects, the polymeric micelle core comprises, or alternatively, substantially consists of, or even more consists of, a polymeric micelle made using a polyethylene glycol-diastearoylphosphatidylethanolamine block copolymer. In an additional aspect, the core of the nanoparticle contains, or, alternatively, consists to a large extent, or even more consists of a metal. According to another aspect, the nanoparticle core is not a liposome. Additional examples of core materials include, but are not limited to, standard and specialty glasses, silicon dioxide, polystyrene, polyester, polycarbonate, acrylic polymers, polyacrylamide, polyacrylonitrile, polyamide, fluoropolymers, silicone, various types of cellulose, metals (e.g., iron, gold, silver ), minerals (eg ruby), nanoparticles (eg gold nanoparticles, colloidal particles, metal oxides, metal sulfides, metal selenides and magnetic materials such as iron oxide) and mixtures thereof. In certain embodiments, the core of the iron oxide nanoparticle contains iron (II, III) oxide. Depending on the properties required, the core may be of a homogeneous composition or of two or more material classes. In certain aspects, metal nanoparticles are used. These metallic particles or nanoparticles can be formed from Au, Pt, Pd, Cu, Ag, Co, Fe, Ni, M _n , Sm, Nd, Pr, Gd, Ti, Zr, Si and In, precursors, their binary alloys, their ternary alloys and their intermetallic compounds. See US Pat. No. 6,712,997 incorporated herein by reference in its entirety. In accordance with certain embodiments, the compositions and layers of the core (described below) may vary, provided that the nanoparticles are biocompatible and bioabsorbable. Depending on the properties required, the core may be of a homogeneous composition or of two or more material classes. In certain aspects, metal nanospheres are used. These metal nanoparticles can be formed from Fe, Ca, Ga, and the like. In accordance with certain embodiments, the nanoparticle contains, or alternatively consists to a large extent, or even more consists of a core containing a metal or metal oxide, such as gold or iron oxide.

Typically, the particles consist of a substantially spherical core and optionally one or more layers or coatings. The core may vary in size and composition as described herein. In addition to the core, the particle may contain one or more layers to provide the functionality required for the required applications. The thickness of the layers, if present, may vary depending on the requirements of a particular application. For example, the layers can impart suitable optical properties.

Layers can also provide chemical or biological functionality, referred to herein as chemically active or biologically active layers. Typically, these layers are applied to the outer surface of the particles and can provide pMHC-NP functionality. Typically, the thickness of the layer or layers can range from about 0.001 micrometer (1 nanometer) to about 10 micrometers or more (depending on the desired particle diameter), or from about 1 nm to 5 nm, or alternatively from about 1 nm. up to about 10 nm, or alternatively, from about 1 nm to about 40 nm, or from about 15 nm to about 25 nm, or about 20 nm and be in the ranges between them.

The layer or coating may contain, or alternatively be composed to a large extent, or even more so, of a biodegradable sugar or other polymer. Non-limiting examples of biodegradable layers include dextran; poly(ethylene glycol); poly(ethylene oxide); mannitol; poly(esters) based on polylactide (PLA), polyglycolide (PGA), polycaprolactone (PCL); poly(hydroxyalkanoate) class PHB-PHV; and other modified poly(saccharides) such as starch, cellulose and chitosan. In addition, the nanoparticle may include a layer with suitable surfaces for attaching chemical functional groups for chemical bonding or bonding sites.

Layers on nanoparticles can be obtained in a number of ways known to experts in this field. Examples include sol-gel chemistry methods, for example, as described in Iler, Chemistry of Silica, John Wiley & Sons, 1979; Brinker and Scherer, Sol-gel Science, Academic Press, (1990). Additional methods for producing layers on nanoparticles include surface chemistry and encapsulation methods, for example, as described in Partch and Brown, J. Adhesion, 67:259-276, 1998; Pekarek et al., Nature, 367:258, (1994); Hanprasopwattana, Langmuir, 12:3173-3179, (1996); Davies, Advanced Materials, 10:1264-1270, (1998); and in their links. Evaporative deposition methods can also be used; see, for example, Golman and Shinohara, Trends Chem. Engin., 6:1-6, (2000) and US Pat. No. 6,387,498. Other approaches include layer-by-layer self-aggregation methods, for example, as described in Sukhorukov et al., Polymers Adv. Tech., 9(10-11):759-767, (1998); Caruso et al., Macromolecules, 32(7):2317-2328, (1998); Caruso et al., J. Amer. Chem. Soc, 121(25):6039-6046, (1999); US Pat. No. 6,103,379 and references cited therein.

In certain aspects, a nanoparticle core is a nanoparticle core of a dendrimer containing, or alternatively consisting largely of, or even more of, a highly branched, tree-like macromolecule growing from the middle. In additional aspects, the dendrimeric nanoparticle may comprise, or alternatively, substantially consist of, or even more so, consist of a poly(amidoamine) based dendrimer or a poly-L-lysine based dendrimer. In certain aspects, the nanoparticle core is a polymeric micelle core comprising, or alternatively substantially consisting of, or even more composed of, an amphiphilic block copolymer, arranged in a nanoscale core-shell structure. In additional aspects, the polymeric micelle core may comprise, or alternatively, substantially consist of, or even more so, consist of a polymeric micelle made using a polyethylene glycol-diastearoylphosphatidylethanolamine block copolymer. The dendrimer core or polymeric micelle core may further comprise an outer coating or layer as described herein.

In accordance with certain embodiments, specific methods for synthesizing nanoparticles with dendrimers or nanoparticles with a core of nanoparticles from a dendrimer may require extraction of metal ions into the dendrimers and then chemical reduction to obtain almost monodisperse in terms of particle size with sizes less than 3 nm, for example, the method described in Crooks et al., "Synthesis, Characterization, and Applications of Dendrimer-Encapsulated Nanoparticles". The Journal of Physical Chemistry B (109): 692-704 (2005), where the resulting dendrimer core component serves not only as a matrix for preparing the nanoparticle, but also to stabilize the nanoparticle, allowing adjustment of solubility, and provides a means for immobilization nanoparticles on a solid substrate.

The size of the core of the nanoparticle may be in the range from about 1 nm to about 1 μm. In accordance with certain embodiments, the nanoparticle core diameter is less than about 1 micron. In other embodiments, the nanoparticle core diameter is less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, or less than about 50 nm. According to additional embodiments, the nanoparticle core diameter is from about 1 nm to about 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 40 nm, 50 nm, 75 nm, or 100 nm. In accordance with specific embodiments, the nanoparticle core diameter is from about 1 nm to about 100 nm; about 1 nm to about 75 nm; from about 1 nm to about 50 nm; about 1 nm to about 25 nm; about 1 nm to about 25 nm; from about 5 nm to about 100 nm; from about 5 nm to about 50 nm; or about 5 nm to about 25 nm, or about 15 nm to about 25 nm, or about 20 nm. In certain embodiments, the nanoparticle core diameter is about 25 nm to about 60 nm, or about 25 nm to about 50 nm, or about 20 nm to about 40 nm, or about 15 nm to about 50 nm, or about 15 nm to about 40 nm, or about 15 nm to about 35 nm, or about 15 nm to about 30 nm, or about 15 nm to about 25 nm, or alternatively about 15 nm, or about 20 nm, or about 25 nm, or about 30 nm, or about 35 nm, or about 40 nm.

The pMHC-NP diameter, with or without a layer, can range from about 5 nm to about 1 μm. In certain embodiments, the diameter of the pMHC-NP complex is less than about 1 μm, or alternatively, less than 100 nm. In other embodiments, the diameter of the pMHC-NP complex is less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, or less than about 50 nm. According to further embodiments, the complex diameter is from about 5 nm or 10 nm to about 50 nm, or from about 5 nm to about 75 nm, or from about 5 nm to about 50 nm, or from about 5 nm to about 60 nm. , or about 10 nm to about 50 nm, or about 10 nm to about 60 nm, or about 10 nm to about 70 nm, or about 10 nm to about 75 nm, or about 20 nm to about 50 nm , or about 20 nm to about 60 nm, or about 20 nm to about 70 nm, or about 20 nm to about 75 nm, or about 30 nm to about 50 nm, or about 30 nm to about 60 nm , or about 30 nm to about 70 nm, or about 30 nm to about 75 nm, or in one aspect about 55 nm. In certain embodiments, the diameter of the pMHC-NP complex is about 35 nm to about 60 nm, or about 35 nm to about 70 nm, or about 35 nm to about 75 nm. In one aspect, the diameter of the pMHC-NP complex is from about 30 nm to about 50 nm.

Antigen-MHC complexes

The nanoparticle complexes of the present invention comprise a nanoparticle core, with or without a layer, associated with an antigen-MHC complex (pMHC). The choice of antigen depends on the disease or condition being treated, as discussed above. An individual polypeptide (eg, MHC) and antigenic (eg, peptide) components form a complex via covalent or non-covalent bonding (eg, via hydrogen bonding, ionic bonding, or hydrophobic bonding). The preparation of such complexes may require varying degrees of processing, and such methods are well known in the literature. In certain aspects, the antigenic components can be linked to the cavity site of the MHC component non-covalently, for example by mixing the MHC and the antigenic components; this is based on the natural binding affinity of the MHC and the antigen. Alternatively, in certain aspects, the MHC component can be covalently coupled to the antigenic component by standard methods, as non-limiting examples, by methods such as the introduction of known binding agents or photoaffinity labeling (See, for example, Hall et al., Biochemistry 24:5702- 5711 (1985)). In certain aspects, an antigenic component may be operably linked to an MHC component via peptide bonds or other methods described in the literature, including, but not limited to, linkage via carbohydrate groups on glycoproteins, including, for example, alpha and/or beta carbohydrate groups. chains. According to specific embodiments, the antigenic component can be linked to the N-terminus or C-terminus of the corresponding MHC molecule. Alternatively, according to certain embodiments, the MHC complex can be formed recombinantly by inserting the sequence encoding the antigenic component into the sequence encoding the MHC, such that both retain their functional properties.

Several antigen-MHC complexes can bind to one nanoparticle core; these complexes, MHC and/or antigens may be the same or different from each other.

Valence is defined as the number of pMHC complexes per nanoparticle core. In accordance with certain embodiments, the valence of a nanoparticle can range from about 1 pMHC complex per nanoparticle core to about 6000 pMHC complexes per nanoparticle core, or alternatively from about 10:1 to about 6000:1, or alternatively, about 11:1 to about 6000:1, or alternatively, about 12:1 to about 6000:1, or alternatively at least 2:1, or alternatively at least 8:1, or, alternatively at least 9:1, or alternatively at least 10:1, or alternatively at least 11:1, or alternatively at least 12:1.

In certain aspects, the valence is from about 10:1 to about 6000:1, or from about 20:1 to about 5500:1, or alternatively from about 10:1 to about 5000:1, or alternatively about 10:1 to about 4000:1, or alternatively about 10:1 to about 3500:1, or alternatively about 10:1 to about 3000:1, or alternatively about 10:1 to about 2500:1, or alternatively about 10:1 to about 2000:1, or alternatively about 10:1 to about 1500:1, or alternatively about 10:1 to about 1000:1, or , alternatively from about 10:1 to about 500:1, or alternatively from about 10:1 to about 100:1, or alternatively from about 20:1 to about 50:1, or alternatively from about 25:1 to about 60:1; alternatively about 30:1 to about 50:1, or alternatively about 35:1 to about 45:1, or alternatively about 40:1.

Applicant has found that the density of pMHC on the nanoparticle regulates the ability of pMHC-NP to initiate or modify the formation of T _R 1 cells in a dose-independent manner. Density is calculated as the number of complexes per unit surface area of a nanoparticle. The surface area of a nanoparticle can be determined with or without layers, including but not limited to linkers that conjugate the pMHC complex to the nanoparticle. For the purpose of density calculation, the corresponding surface area value is based on the final diameter of the particle structure without the pMHC complex, with or without an outer layer on the core of the nanoparticle.

The present document defines and describes that the density of pMHC complexes on a nanoparticle contributes to a beneficial therapeutic effect in a dose-independent manner. Thus, as described herein, a nanoparticle may have a specific pMHC density in the range of about 0.01 pMHC, or alternatively, 0.025 pMHC molecules per 100 nm ² surface area of the nanoparticle, including the layer or complex, assuming at least 2 molecules MHC, or alternatively at least 8, or alternatively at least 9, or alternatively at least 10, or alternatively at least 11, or alternatively at least 12 pMHC molecules forming complex with a nanoparticle up to about 100 pMHC molecules per 100 nm ² surface area. In one aspect, the pMHC density on the nanoparticle is from about 0.05 pMHC per 100 nm ² to about 76 pMHC/100 nm ² , or alternatively, from 0.1 pMHC/100 nm ² to about 50 pMHC/100 nm ² . or alternatively from about 0.3 pMHC/100 nm ² to about 25 pMHC/100 nm ² , or alternatively from about 0.35 pMHC/100 nm ² to about 25 pMHC/100 nm ² , or alternatively, from about 0.4 pMHC/100 nm ² to about 50 pMHC/100 nm ² , or alternatively from about 0.4 pMHC/100 nm ² to about 25 pMHC/100 nm ² , or alternatively from about 0, 4 ^pMHC /100 nm2 to about 20 pMHC/100 nm2, 0.4 ^pMHC /100 nm2 to about 10 ^pMHC /100 nm2, 0.4 ^pMHC /100 nm2 to about ^{5 pMHC} /100 nm2, or, alternatively, from about 0.5 pMHC/100 nm ² to about 20 pMHC/100 nm ² , or alternatively, from about 0.5 pMHC/100 nm ² to about 10 pMHC/100 nm ² , or, alternatively, appr. preferably from 0.6 pMHC/100 nm ² to about 20 pMHC/100 nm ² , or alternatively from about 1.0 pMHC/100 nm ² to about 20 pMHC/100 nm ² , or alternatively from about 10 pMHC /100 nm ² to about 20 pMHC/100 nm ² , or alternatively at least about 0.4, or alternatively at least about 0.406, or alternatively at least about 0.5, or alternatively , at least about 1.0, or alternatively at least about 5.0, or alternatively at least about 10.0, or alternatively at least about 15.0 pMHC/100 nm ² , or alternatively less than about 76 pMHC/100 nm ² , or alternatively less than about 50 pMHC/100 nm ² , or alternatively less than about 47.75 pMHC/100 nm ² , or alternatively less than about 25 pMHC/100 nm ² , or alternatively less than about 20 pMHC/100 nm ² .

In accordance with certain embodiments, the pMHC density per nanoparticle is from about 0.4 pMHC/100 nm ² to about 25 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 20 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 15 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 14 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 13 pMHC/100 nm ² , or about 0.4 pMHC/100 nm ² to about 12 pMHC/100 nm ² , or about 0.4 pMHC/100 nm ² to about 11.6 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 11.5 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 11 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 10 pMHC/100 nm ² , or about 0.4 pMHC/100 nm ² to about 9 pMHC/100 nm ² , or about 0.4 pMHC/100 nm ² to about 8 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 7 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 6 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 5 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 4 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 3 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 2.5 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 2 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 1.5 pMHC/100 nm ² .

According to additional embodiments, the pMHC density on the nanoparticles can be from about 0.22 pMHC/100 nm ² to about 10 pMHC/100 nm ² , or from about 0.22 pMHC/100 nm ² to about 9 pMHC/100 nm ² , or from about 0.22 pMHC/100 nm ² to about 8 pMHC/100 nm ² , or from about 0.22 pMHC/100 nm ² to about 7 pMHC/100 nm ² , or from about 0.22 pMHC/100 nm ² to about 6 pMHC/100 nm ² , or from about 0.22 pMHC/100 nm ² to about 5 pMHC/100 nm ² , or from about 0.22 pMHC/100 nm ² to about 4 pMHC/100 nm ² , or from about 0.22 pMHC/100 nm ² to about 3 pMHC/100 nm ² , or from about 0.22 pMHC/100 nm ² to about 2 pMHC/100 nm ² , or from about 0.22 pMHC/100 nm ² to about 1.5 pMHC/100 nm ² . In certain aspects, the pMHC density on a nanoparticle may be from about 0.22 pMHC/100 nm ² to about 10 pMHC/100 nm ² or 0.24 pMHC/100 nm ² to about 9 pMHC/100 nm ² , or from about 0.26 pMHC/100 nm ² to about 8 pMHC/100 nm ² , or about 0.28 pMHC/100 nm ² to about 7 pMHC/100 nm ² , or about 0.24 pMHC/100 nm ² to about 4 pMHC/100 nm ² , or from about 0.5 pMHC/100 nm ² to about 3 pMHC/100 nm ² , or from about 0.6 pMHC/100 nm ² to about 1.5 pMHC/100 nm ² . In accordance with certain embodiments, the pMHC density on the nanoparticle may be from about 0.4 pMHC/100 nm ² to about 1.3 pMHC/100 nm ² , or alternatively, from about 0.5 pMHC/100 nm ² to about 0 .9 pMHC/100 nm ² , or alternatively from about 0.6 pMHC/100 nm ² to about 0.8 pMHC/100 nm ² .

In accordance with additional embodiments, the density of the nanoparticle may be approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 , 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2 .3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5 , 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4 .8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0 , 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8 .3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5 , 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10 .8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9 or 12.0 pMHC/100 nm ² . In accordance with specific embodiments, the density of the nanoparticle is from about 0.4 pMHC/100 nm ² to about 1.5 pMHC/100 nm ² or from about 0.4 pMHC/100 nm ² to about 6 pMHC/100 nm ² or about from 0.4 pMHC/100 nm ² to about 11 pMHC/100 nm ² .

In accordance with certain aspects, provided herein is a complex comprising a core of a nanoparticle, wherein a plurality of disease-associated antigen-MHC complexes (pMHC) are associated with the core; where the core diameter is from about 15 nm to about 25 nm and where the density of pMHC on the nanoparticle is from about 0.4 pMHC/100 nm ² to about 6 pMHC/100 nm ² surface area of the nanoparticle. In accordance with certain embodiments, the complex further comprises an outer layer on the nanoparticle core, wherein the pMHC complex is bonded to the nanoparticle core and/or outer layer, and wherein the diameter of the nanoparticle core and outer layer is between about 35 nm and about 45 nm.

As used herein, the term "operably linked" or "coated" refers to a situation where a single polypeptide (eg, MHC) and antigenic (eg, peptide) components are combined to form an active complex prior to binding to a target site, such as an immunocyte. . This includes the situation where the individual polypeptide components of the complex are synthesized or recombinantly expressed and then isolated and combined to form the complex prior to administration to the individual in vitro; a situation where a chimeric or fusion polypeptide is synthesized or recombinantly expressed (ie, each of the individual protein components of the complex is placed in a single polypeptide chain) as an intact complex. Typically, polypeptide complexes are added to nanoparticles to produce nanoparticles with adsorbed or bound polypeptide complexes with a molecular to nanoparticle ratio of about, at least about, or no more than about 0.1, 0.5, 1, 3, 5, 7, 10, 15, 20, 25, 30, 35, 40, 50, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500 or more: 1, typically 0.1:1, 1:1 to 50:1 or 300:1, and ranges in between where ratios provide for selected endpoints in each of the ranges. The content of polypeptides in a nanoparticle can be determined by standard methods.

MHC molecules

As used herein and unless specifically indicated, the term MHC within the rMHC complex means a classic or non-classical MHC class I protein and/or a classic or non-classical MHC class II protein, any of the HLA DR, HLA DQ, HLA DP, HLA- A, HLA-B, HLA-C, HLA-E, CD1d, or fragments or biological equivalents thereof, double or single chain constructs, dimers (Fc fusions), tetramers, multimeric forms, and polymeric forms of MHCI or MHCII. In accordance with certain embodiments, the pMHC may be a single stranded construct. In accordance with certain embodiments, the pMHC may be a double stranded construct.

In certain embodiments, the MHC protein may be a dimer or multimer.

In certain embodiments, the MHC protein may include a ridge-to-trough based heterodimer or multimer of alpha-MHC-Fc/beta-MHC-Fc.

As stated above, a "protrusion into a cavity" is a polypeptidyl architecture requiring a protrusion (or "protrusion") on the interacting surface of the first polypeptide and a corresponding recess (or "cavity") on the interacting surface of the second polypeptide such that the protrusion can be located in the recess so to allow the formation of a heteromultimer. The protrusions are constructed by replacing amino acids with small side chains on the interacting surface of the first polypeptide with amino acids with large side chains (eg, phenylalanine or tyrosine). On the interacting surface of the second polypeptide, dimples of identical or protrusion-like dimensions are obtained by replacing amino acids with large side chains with amino acids with small side chains (eg, alanine or threonine). The protrusions and recesses can be obtained by synthetic methods, such as substitution of nucleic acids encoding polypeptides, or by peptide synthesis, by conventional methods used by a person skilled in the art. In certain embodiments, the interaction surface of the first polypeptide is on the Fc domain of the first polypeptide and the interaction surface of the second polypeptide is on the Fc domain of the second polypeptide.

As indicated above, "alpha-MHC-Fc/eta-MHC-Fc" is a heterodimer containing a first polypeptide and a second polypeptide, where the first polypeptide contains the α-chain MHC class II and the Fc domain of the antibody; the second polypeptide contains the MHC class II β-chain and the Fc domain of the antibody. The bump-to-bottom alpha-MHC-Fc/beta-MHC-Fc further requires that the Fc domains of each polypeptide interact with each other by a complementary arrangement of a protrusion on one Fc domain to a corresponding recess on the other Fc domain.

In accordance with certain embodiments of the invention, a particular antigen is identified and presented in an antigen-MHC-nanoparticle complex in the context of an appropriate class I or II MHC polypeptide. Presentation of antigens to T cells is mediated by two different classes of molecules, MHC class I (MHC-I) and MHC class II (MHC-II), which use different antigen processing pathways. Peptides derived from intracellular antigens are presented to CD8 ⁺ T cells by MHC class I molecules, which are expressed on virtually all cells, while peptides derived from extracellular antigens are presented to CD4 ⁺ T cells by MHC-II molecules. However, there are certain exceptions to this dichotomy. Several studies have shown that peptides derived from endocytosed particles or soluble proteins are presented on MHC-I molecules on macrophages as well as on dendritic cells. In certain aspects, an individual's genetic makeup can be assessed to determine which MHC polypeptide to use for a particular patient and particular set of peptides. In accordance with certain embodiments, the MHC class I component may comprise, substantially consist of, or alternatively still more consist of all or part of the HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G or CD1. In embodiments where the MHC component is an MHC class II component, the MHC class II component may comprise, substantially consist of, or alternatively still more consist of all or part of HLA-DR, HLA-DQ, or HLA-DP. According to certain embodiments, the MHC may comprise HLA DRB1, HLA DRB3, HLA DRB4, HLA DRB5, HLA DQB1, HLA DQA1, IA ^g7 , I-Ab, I-Ad, HLA-DQ, HLA-DP, HLA-A, HLA-B, HLA-C, HLA-E, or CD1d.

Non-classical MHC molecules are also contemplated for use in the MHC complexes of the invention. In certain embodiments, non-classical MHC molecules are non-polymorphic, conserved across species, and contain narrow, deep, hydrophobic ligand-binding cavities. These binding cavities can present glycolipids and phospholipids to natural killer T (NKT) cells. NKT cells are a unique population of lymphocytes that co-express NK cell markers and a semi-invariant T cell receptor (TCR). They are involved in the regulation of the immune response associated with a wide range of diseases.

As indicated above, the term "MHC", when used in relation to the human MHC, can be used interchangeably with the term "human leukocyte antigen" (HLA); thus, MHC refers to all HLA subtypes, including but not limited to the classic MHC genes described above: HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR, and all of them. variants, isoforms, isotypes and other biological equivalents.

MHC for use in the present invention can be obtained, isolated or purified by methods known in the art. General protocols for producing MHC include, but are not limited to, steps such as electrophoresis or other charge or size based separation methods, biotinylation or other labeling and purification methods, or transfection and induction of vector constructs expressing MHC proteins. Purified animal antibodies are also available from commercial sources, including retailers such as eBioscience, Biolegend, or Tonbo Biosciences.

In certain embodiments, the MHC of antigen-MHC complexes can be classical MHCI, non-classical MHCI, classical MHCII, non-classical MHCII, Fc dimers, MHC tetramers, or polymeric forms of MHC. In accordance with certain embodiments, MHC multimers are prepared by methods well documented in the art, see, for example, Bakker et al. "MHC Multimer Technology: Current Status and Future Prospects", Current Opinion in Immunology, Vol. 17, no. 4 pp. 428-433 (2005) and references cited therein. Non-limiting exemplary methods include using a biotinylating agent, such as streptavidin or avidin, to bind the MHC monomers, producing a multimeric structure with this agent as a scaffold. Specifically, MHC dimers can alternatively be obtained by fusion with antibody constant regions or Fc regions; this can be done by operable linkage directly or via a linker, such as a cysteine linker.

Components of costimulatory molecules

According to certain aspects, the NP further comprises, or alternatively, substantially consists of, or even more than that, consists of at least one co-stimulatory molecule. Costimulatory molecules are molecules that in vivo produce a secondary signal that serves to activate naïve T cells into antigen-specific T cells capable of mount an immune response to cells bearing said specific antigen. The present invention is not limited to any particular costimulatory molecule. Various costimulatory molecules are well known in the art. Certain non-limiting examples of costimulatory molecules are 4-IBBL, OX40L, CD40, IL-15/IL-15Ra, CD28, CD80, CD86, CD30L, and ICOSL. Only one specific costimulatory molecule may be associated with one nanoparticle, or several costimulatory molecules may be associated with one nanoparticle. In certain embodiments, the costimulatory molecule is a protein, such as an antibody, capable of acting as a costimulatory receptor agonist on a T cell. In this case, the antibody is capable of inducing a co-stimulatory signal, which is necessary to activate naive T cells and induce an immune response in an antigen-specific manner. Additionally or alternatively, as used herein, the term "costimulatory molecule" can also mean an agent capable of generating a costimulatory signal by agonistically acting on native costimulatory signaling molecules, e.g., an anti-CD28 antibody or CD28 ligand, that elicits a costimulatory CD28 response.

According to specific embodiments, the co-stimulatory molecules of the present invention may be any one or more of the following molecules: B7-1/CD80, BTLA, B7-2/CD86, CD28, B7-H1/PD-L1, CTLA-4, B7-H2, GI ₂ 4/VISTA/B7-H5, B7-H3, ICOS, B7-H4, PD-1, B7-H6, PD-L2/B7-DC, B7-H7, PDCD6, LILRA3/CD85e, LILRB2/CD85d/ILT4, LILRA4/CD85g/ILT7, LILRB3/CD85a/ILT5, LILRB1/CD85j/ILT2, LILRB4/CD85k/ILT3, 4-1BB/TNFRSF9/CD137, GITR/TNFSF18 ligand, 4-1BB/TNFSF9 ligand , HVEM/TNFRSF14, BAFF/BLyS/TNFSF13B, LIGHT/TNFSF14, BAFFR/TNFRSF13C, lymphotoxin-alpha/TNF-beta, CD27/TNFRSF7, OX40/TNFRSF4, CD27/TNFSF7 ligand, OX40/TNFSF4 ligand, CD30/TNFRSF8, RELT /TNFRSF19L, ligand CD30/TNFSF8, TACI/TNFRSF13B, CD40/TNFRSF5, TL1A/TNFSF15, CD40 ligand/TNFSF5, TNF-alpha, DR3/TNFRSF25, TNFRII/TNFRSF1 B, GITR/TNFRSF18, 2B4/CD244/SLAMF4, CD84/ SLAMF5, BLAME/SLAMF8, CD229/SLAMF3, CD2, CRACC/SLAMF7, CD2F-10/SLAMF9, NTB-A/SLAMF6, CD48/SLAMF2, SLAM/CD150, CD58/LFA-3, CD7, DPPIV/CD26, CD96, EphB6 , CD160, integrin alpha 4 beta 1, CD200, integrin alpha 4 beta 7/LPAM-1, CD300a/LMIR1, LAG-3, CRTAM, TIM-1/KIM-1/HAVCR, DAP12, TIM-4, dectin-1 /CLEC7A, TSLP R, ICOSL and/or their biological equivalents.

The costimulatory molecule can be coupled to the nanoparticle in the same manner as the pMHC complex. In one embodiment of the present invention, the co-stimulatory molecule and the antigen/MHC complex are separately bound to the nanoparticle. In another embodiment of the invention, a complex of the co-stimulatory molecule and the pMHC complex is first obtained, and then their complex with the nanoparticle is obtained. Several costimulatory molecules can be bound to a nanoparticle; they may be several identical costimulatory molecules or several different costimulatory molecules. Typically, polypeptide complexes are added to nanoparticles to produce nanoparticles with adsorbed or bound polypeptide complexes with a ratio of costimulatory molecules:number of nanoparticles from about 1 to 6000 molecules per nanoparticle, or alternatively at least about or not more than about 0.1, 0.5, 1, 10, 100, 500, 1000, 2000, 3000, 4000, 5000, 6000 or more: 1, and in ranges in between, typically from about 0.1:1 to about 50:1. In another aspect, the ratio of co-stimulatory molecule to pMHC complex may be about 0.1, 0.5, 1, 2, 5, 10, 50 or more to 1, preferably a ratio of co-stimulatory molecule: pMHC complex of 1:1, 1: 2, 1:9, 1:10, 1:100, 2:1, 9:1, 10:1 or 100:1. Similarly, the density of costimulatory molecules relative to the surface area of the nanoparticle can be calculated using the same relative formula as for pMHC complexes. In certain embodiments, the density of the costimulatory molecule per unit surface area of the nanoparticle is from about 0.0022 costimulatory molecules/100 nm ² to about 13.26 costimulatory molecules/100 nm ² . In accordance with certain embodiments, the density range of the costimulatory molecules may be the same as or different from the density range of the pMHC complexes.

According to certain embodiments, where the nanoparticle contains one or more costimulatory molecules and does not contain the pMHC complex, the density of costimulatory molecules on the nanoparticle is about 0.2 costimulatory molecules/100 nm ² to about 6.5 costimulatory molecules/100 nm ² , or about 0.2 co-stimulatory molecules/100 nm ² to about 6 co-stimulatory molecules/100 nm ² , or from about 0.2 co-stimulatory molecules/100 nm ² to about 5.8 co-stimulatory molecules/100 nm ² , or from about 0, 2 co-stimulatory molecules/100 nm ² to about 5.75 co-stimulatory molecules/100 nm ² , or from about 0.2 co-stimulatory molecules/100 nm ² to about 5.5 co-stimulatory molecules/100 nm ² , or from about 0.2 co-stimulatory molecules / 100 nm ² to about 5 co-stimulatory molecules / 100 nm ² , or from about 0.2 co-stimulatory molecules / 100 nm ² to about 4.5 co-stimulatory mo molecules/100 nm ² , or from about 0.2 co-stimulatory molecules/100 nm ² to about 4 co-stimulatory molecules/100 nm ² , or from about 0.2 co-stimulatory molecules/100 nm ² to about 3.5 co-stimulatory molecules/100 nm ² , or from about 0.2 co-stimulatory molecules/100 nm ² to about 3 co-stimulatory molecules/100 nm ² , or from about 0.2 co-stimulatory molecules/100 nm ² to about 2.5 co-stimulatory molecules/100 nm ² , or approximately 0.2 co-stimulatory molecules/100 nm ² to about 2 co-stimulatory molecules/100 nm ² , or about 0.2 co-stimulatory molecules/100 nm ² to about 1.5 co-stimulatory molecules/100 nm ² , or about 0.2 co-stimulatory molecules/100 nm ² to about 1.25 co-stimulatory molecules/100 nm ² , or from about 0.2 co-stimulatory molecules/100 nm ² to about 1 co-stimulatory molecules/100 nm ² , or from about 0.2 co-stimulatory molecules/100 nm ² to approximately 0.75 costimulatory molecules/100 nm ² .

In another aspect, the density of co-stimulatory molecules on a nanoparticle may be from about 0.11 co-stimulatory molecules/100 nm ² to about 5 co-stimulatory molecules/100 nm ² , or from about 0.11 co-stimulatory molecules/100 nm ² to about 4.5 costimulatory molecules/100 ^nm2 , or from about 0.11 costimulatory molecules/100 ^nm2 to about 4 costimulatory molecules/100 ^nm2 , or from about 0.11 costimulatory molecules/100 ^nm2 to about 3.5 costimulatory molecules/100 nm ² , or from about 0.11 co-stimulatory molecules/100 nm ² to about 3 co-stimulatory molecules/100 nm ² , or from about 0.11 co-stimulatory molecules/100 nm ² to about 2.5 co-stimulatory molecules/100 nm ² , or approximately 0.11 costimulatory molecules/100 nm2 to approximately ² costimulatory molecules/100 ^nm2 , or approximately 0.11 costimulatory molecules/100 ^nm2 to approximately 1.5 costimulatory costimulatory molecules/100 nm2, or from about 0.11 costimulatory molecules/100 nm2 to about ^{1 pMHC} /100 ^nm2 , or from about 0.11 costimulatory molecules/100 ^nm2 to about 0.75 costimulatory molecules/100 nm2 ² . In certain aspects, the density of co-stimulatory molecules on the nanoparticle core is from about 0.11 co-stimulatory molecules/100 nm ² to about 5 co-stimulatory molecules/100 nm ² or 0.12 co-stimulatory molecules/100 nm ² to about 4.5 co-stimulatory molecules/ 100 nm ² , or from about 0.13 co-stimulatory molecules/100 nm ² to about 4 co-stimulatory molecules/100 nm ² , or from about 0.14 co-stimulatory molecules/100 nm ² to about 3.5 co-stimulatory molecules/100 nm ² , or from about 0.12 co-stimulatory molecules/100 nm ² to about 2 co-stimulatory molecules/100 nm ² , or from about 0.25 co-stimulatory molecules/100 nm ² to about 1.5 co-stimulatory molecules/100 nm ² , or from about 0 .3 co-stimulatory molecules/100 nm ² to about 0.75 co-stimulatory molecules/100 nm ² . In a further aspect, the density of co-stimulatory molecules on the nanoparticle core is from about 0.2 co-stimulatory molecules/100 nm ² to about 0.65 co-stimulatory molecules/100 nm ² , or alternatively from about 0.25 co-stimulatory molecules/100 nm ² to about 0.45 co-stimulatory molecules/100 nm ² , or alternatively from about 0.3 co-stimulatory molecules/100 nm ² to about 0.4 co-stimulatory molecules/100 nm ² .

According to certain embodiments where the nanoparticle comprises a pMHC complex and one or more costimulatory molecules, the density of costimulatory molecules on the nanoparticle is from about 0.4 costimulatory molecules/100 nm ² to about 13 costimulatory molecules/100 nm ² , or from about 0, 4 co-stimulatory molecules/100 nm ² to about 12 co-stimulatory molecules/100 nm ² , or from about 0.4 co-stimulatory molecules/100 nm ² to about 11.6 co-stimulatory molecules/100 nm ² , or from about 0.4 co-stimulatory molecules/ 100 nm ² to about 11.5 co-stimulatory molecules/100 nm ² , or from about 0.4 co-stimulatory molecules/100 nm ² to about 11 co-stimulatory molecules/100 nm ² , or from about 0.4 co-stimulatory molecules/100 nm ² to approximately 10 co-stimulatory molecules/100 nm ² , or approximately 0.4 co-stimulatory molecules/100 nm ² to approximately 9 co-stimulatory molecules/100 nm ² , or from about 0.4 co-stimulatory molecules/100 nm ² to about 8 co-stimulatory molecules/100 nm ² , or from about 0.4 co-stimulatory molecules/100 nm ² to about 7 co-stimulatory molecules/100 nm ² , or from about 0, 4 co-stimulatory molecules/100 nm ² to about 6 co-stimulatory molecules/100 nm ² , or from about 0.4 co-stimulatory molecules/100 nm ² to about 5 co-stimulatory molecules/100 nm ² , or from about 0.4 co-stimulatory molecules/100 nm ² to about 4 co-stimulatory molecules/100 nm ² , or about 0.4 co-stimulatory molecules/100 nm ² to about 3 co-stimulatory molecules/100 nm ² , or about 0.4 co-stimulatory molecules/100 nm ² to about 2.5 co-stimulatory molecules/100 nm ² , or from about 0.4 co-stimulatory molecules/100 nm ² to about 2 co-stimulatory molecules/100 nm ² , or from about 0.4 co-stimulatory molecules/100 nm ² to about 1.5 k stimulating molecules/100 nm ² .

In another aspect, the density of co-stimulatory molecules on a nanoparticle may be from about 0.22 co-stimulatory molecules/100 nm ² to about 10 co-stimulatory molecules/100 nm ² , or from about 0.22 co-stimulatory molecules/100 nm ² to about 9 co-stimulatory molecules /100 nm ² , or from about 0.22 co-stimulatory molecules/100 nm ² to about 8 co-stimulatory molecules/100 nm ² , or from about 0.22 co-stimulatory molecules/100 nm ² to about 7 co-stimulatory molecules/100 nm ² , or about 0.22 co-stimulatory molecules/100 nm ² to about 6 co-stimulatory molecules/100 nm ² , or from about 0.22 co-stimulatory molecules/100 nm ² to about 5 co-stimulatory molecules/100 nm ² , or from about 0.22 co-stimulatory molecules molecules/100 ^nm2 to about 4 costimulatory molecules/100 ^nm2 , or from about 0.22 costimulatory molecules/100 ^nm2 to about 3 costimulatory moles ecul/100 nm ² , or approximately 0.22 co-stimulatory molecules/100 nm ² to approximately 2 pMHC/100 nm ² , or approximately 0.22 co-stimulatory molecules/100 nm ² to approximately 1.5 co-stimulatory molecules/100 nm ² . In certain aspects, the density of co-stimulatory molecules on the nanoparticle core is from about 0.22 co-stimulatory molecules/100 nm ² to about 10 co-stimulatory molecules/100 nm ² or 0.24 co-stimulatory molecules/100 nm ² to about 9 co-stimulatory molecules/100 nm ² , or from about 0.26 co-stimulatory molecules/100 nm ² to about 8 co-stimulatory molecules/100 nm ² , or from about 0.28 co-stimulatory molecules/100 nm ² to about 7 co-stimulatory molecules/100 nm ² , or from about 0 .24 costimulatory molecules/100 ^nm2 to about 4 costimulatory molecules/100 ^nm2 , or from about 0.5 costimulatory molecules/100 ^nm2 to about 3 costimulatory molecules/100 ^nm2 , or from about 0.6 costimulatory molecules/100 nm ² to about 1.5 costimulatory molecules/100 nm ² . In a further aspect, the density of co-stimulatory molecules on a nanoparticle is from about 0.4 co-stimulatory molecules/100 nm ² to about 1.3 co-stimulatory molecules/100 nm ² , or alternatively from about 0.5 co-stimulatory molecules/100 nm ² to about 0 .9 co-stimulatory molecules/100 nm ² , or alternatively from about 0.6 co-stimulatory molecules/100 nm ² to about 0.8 co-stimulatory molecules/100 nm ² .

Cytokines

In accordance with a certain aspect, the NPs further comprise, or alternatively, consist largely of, or even more consist of, at least one cytokine molecule. As used herein, the term "cytokine" includes small molecular weight proteins secreted by various cells of the immune system that act as signaling molecules to regulate a wide range of biological processes in the body at the molecular and cellular levels. "Cytokines" includes individual immunomodulatory proteins that fall into the classes of lymphokines, interleukins, or chemokines.

Non-limiting examples are described herein: for example, IL-1A and IL-1B are two separate members of the human interleukin-1 (IL-1) family. Mature IL-1A is an 18 kDa protein, also known as fibroblast activating factor (FAF), lymphocyte activating factor (LAF), B cell activating factor (BAF), leukocyte endogenous mediator (LEM), etc. IL-4 is a cytokine that induces T-helper 2 (Th2) cell differentiation and is closely related to and has similar functions to IL-13. IL-5 is produced by Th2 cells and mast cells. Its action is to stimulate the growth of B cells and increase their secretion of immunoglobulin. It is also involved in the activation of eosinophils. IL-6 is an interleukin that acts as a pro-inflammatory or anti-inflammatory cytokine. It is secreted by T cells and macrophages to stimulate an immune response to injury or other tissue damage, leading to inflammation. IL-6 is also produced by muscles in response to muscle contraction. IL-8 is a chemokine produced by macrophages and other cell types such as epithelial cells and endothelial cells and acts as an important immune response mediator in the innate immune response. IL-12 is involved in the differentiation of naive T cells into T helper (Th1 or Th2) cells. As a heterodimeric cytokine, IL-12 is formed after two subunits encoded by two different genes, IL-12A (p35) and IL-12B (p40), dimerize after protein synthesis. IL-12p70 indicates this heterodimeric composition. IL-13, a cytokine secreted by many cell types, especially Th2 cells, is an important mediator of allergic inflammation and disease. IL-17 is a cytokine produced by T helper cells and induced by IL-23 that results in destructive tissue damage in delayed-type reactions. IL-17 functions as a pro-inflammatory cytokine that responds to invasion of the immune system by extracellular pathogens and induces destruction of the pathogen's cellular matrix. IP-10, or interferon-inducible gamma protein 10, also known as C-X-C motif chemokine 10 (CXCL10) or small inducible cytokine B10. As a small cytokine belonging to the CXC family of chemokines, IP-10 is secreted by several cell types (including monocytes, endothelial cells and fibroblasts) in response to IFN-γ. Macrophage inflammatory proteins (MIPs) belong to the chemokine family. There are two major forms of human MIP, M1P-1α and MIP-1β, which are also known as chemokine ligands (C-C motif) 3 (CCL3) and CCL4, respectively. Both produce macrophages after stimulation with bacterial endotoxins. Granulocyte colony stimulating factor (G-CSF or GCSF), also known as colony stimulating factor 3 (CSF 3), is a hormonal colony stimulating factor. G-CSF is a glycoprotein, growth factor and cytokine produced by a number of different tissues to stimulate the bone marrow to produce granulocytes and stem cells. G-CSF also stimulates the survival, proliferation, differentiation, and function of neutrophil progenitors and mature neutrophils. Epidermal growth factor or EGF is a growth factor that plays an important role in the regulation of cell growth, proliferation and differentiation by binding with high affinity to its EGFR receptor. Vascular endothelial growth factor (VEGF) is a family of growth factors that are important signaling proteins involved in vasculogenesis (de novo formation of the embryonic circulatory system) and angiogenesis (growth of blood vessels from a pre-existing vascular system).

The cytokine or cytokines can be bound to the nanoparticle in the same manner as the pMHC complex. In one embodiment of the present invention, the cytokine or cytokines and the pMHC complex are separately bound to the nanoparticle. In another embodiment of the invention, a complex of a cytokine molecule or molecules of cytokines and a pMHC complex is first obtained, and then their complex with a nanoparticle is obtained. Several cytokines can be bound to a nanoparticle; may be several molecules of the same cytokines or different cytokines.

In accordance with certain embodiments, the cytokine is complexed with an anti-cytokine antibody to form a cytokine/anti-cytokine antibody complex, which is then complexed with the nanoparticle. In accordance with certain embodiments, the cytokine/anti-cytokine antibody complex includes, but is not limited to, IL-2/anti-IL-2 antibody complexes. The IL-2/anti-IL-2 antibody complexes may have agonist properties or antagonist properties.

According to certain embodiments, a cytokine-cytokine receptor complex is formed to form a cytokine/cytokine receptor complex, which is then complexed to the nanoparticle. According to certain embodiments, the cytokine/cytokine receptor complex includes, but is not limited to, IL15/IL-15Ra and/or IL-1/IL-2Ra. In certain embodiments, the IL15/IL-15Ra complex can function as a T cell costimulator.

Typically, polypeptide complexes are added to nanoparticles to produce nanoparticles with adsorbed or bound polypeptide complexes with a ratio of cytokines:number of nanoparticles from about 1 to 5999 molecules per nanoparticle, or alternatively, at least about or not more than about 0.1, 0 .5, 1, 10, 100, 500, 1000, 2000, 3000, 4000, 5000, 6000 or more: 1, and in ranges between them, for example, from about 0.1:1 to about 50:1. In other aspects, the ratio of cytokine to antigen/MHC complex may be about 0.1, 0.5, 1, 2, 5, 10, 50 or more to 1, preferably a 1:1 cytokine:antigen/MHC complex ratio is obtained. , 1:2, 1:9, 1:10, 1:100, 2:1, 9:1, 10:1 or 100:1. Similarly, the density of cytokines relative to the surface area of the nanoparticle can be calculated using the same relative formula as for antigen/MHC complexes. According to certain embodiments, the density of cytokines per unit surface area of the nanoparticle is from about 0.0022 cytokines/100 nm ² to about 13.26 cytokines/100 nm ² . In accordance with certain embodiments, the range of cytokine densities may be the same as or different from the density range of antigen/MHC complexes.

Antigenic components

Certain aspects of the invention include methods and compositions relating to antigenic compositions comprising regions, fragments, or epitopes of polypeptides, peptides, nucleic acids, carbohydrates, lipids, and other molecules that elicit or induce an antigenic response, generally referred to as antigens. In particular, it is possible to identify and use in the preparation of the peptide-MHC/nanoparticle complex described herein, self-antigens or antigenic regions or fragments of such self-antigens that lead to cell destruction by an autoimmune response.

While specific examples of antigens and antigenic components are described herein, the invention is not limited to them. Unless specifically stated otherwise, the present invention includes equivalents of isolated or purified polypeptide antigens that contain, or substantially consist of, or even more, consist of amino acid sequences as described herein, or polypeptides with at least about 80 % sequence identity, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 98% sequence identity with amino acid sequences of antigens, or polypeptides encoded by polynucleotides with approximately 80% sequence identity, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95%, or , alternatively, with at least 98% sequence identity with a polynucleotide encoding the amino acid sequence of an antigen on, or its complementary polynucleotide, or a polypeptide encoded by a polynucleotide that, under medium to high stringency conditions, hybridizes to a polynucleotide encoding the amino acid sequence of an antigen, or its complementary polynucleotide. Also provided are isolated and purified polynucleotides encoding the antigenic polypeptides described herein, or amino acids with at least about 80% sequence identity with them, or alternatively at least 85%, or alternatively at least 90 %, or alternatively, at least 95%, or alternatively, at least 98% sequence identity with the described sequences, or equivalents, or polynucleotides that hybridize under stringent conditions with these polynucleotides, their equivalents, or their complementary polynucleotides and isolated or purified polypeptides encoded by those polynucleotides. Polypeptides and polynucleotides can be combined with non-natural substances with which they are not naturally associated, such as carriers, pharmaceutically acceptable carriers, vectors, and MHC molecules.

Modified peptides and their equivalents

Antigenic polypeptides, proteins and fragments thereof can be modified by various deletions, insertions and/or substitutions of amino acids. In accordance with specific embodiments, the modified polypeptides and/or peptides are capable of modulating an immune response in an individual. As used herein, "protein" or "polypeptide" or "peptide" refers to a molecule containing at least five amino acid residues. In certain embodiments, a wild-type variant of the protein or peptide is used, however, in many embodiments, a modified protein or polypeptide is used to form the peptide/MHC/nanoparticle complex. The peptide/MHC/nanoparticle complex can be used to stimulate an immune response and/or to modify the immune system's T cell population (eg, retrain the immune system). The terms described above may be used interchangeably in this document. "Modified protein" or "modified polypeptide" or "modified peptide" refers to a protein or polypeptide whose chemical structure, in particular the amino acid sequence, is changed from the wild-type protein or polypeptide. In certain embodiments, the modified protein or polypeptide or peptide has at least one activity or function modified (with the understanding that proteins or polypeptides or peptides may have multiple activities or functions). It is specifically believed that a modified protein or polypeptide or peptide can be altered in one activity or function while retaining other wild-type activities or functions, such as immunogenicity or the ability to interact with other cells of the immune system in the context of the MHC/ nanoparticle.

In accordance with certain embodiments, the length of the protein or polypeptide (wild-type or modified), including any complex of the protein or peptide of interest and in particular the MHC/peptide fusion, can be 5, 6, 7, 8, 9 as non-limiting examples , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 , 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84 , 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 . , 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500 amino molecules or more, including any range or value, conclusion of them or their derivatives. In certain aspects, 5, 6, 7, 8, 9, 10 or more contiguous amino acids, including derivatives thereof, and fragments of self antigens, such as the amino acid sequences described and referenced herein, can be used as antigens. It is envisaged that polypeptides can be modified by shortening, making them shorter than their corresponding wild-type form, but they can also be modified by fusion or conjugation to a heterologous protein sequence with a specific function (for example, for presentation as a protein complex, to enhance immunogenicity and etc.).

As used herein, "aminomolecule" refers to any amino acid, amino acid derivative, or amino acid mimetic known in the art. In certain embodiments, the residues of the protein molecule are consecutive without any non-amino molecule interrupting the sequence of amino residues. In other embodiments, the implementation of the sequence may contain one or more non-amino molecules. In accordance with specific embodiments, the sequence of residues of a protein molecule may be interrupted by one or more non-amino molecules.

Thus, the term "protein composition" includes amino molecule sequences containing at least one of the 20 standard amino acids from naturally synthesized proteins, or at least one modified or non-standard amino acid.

Protein compositions can be prepared by any method known to those skilled in the art, including (i) expression of proteins, polypeptides, or peptides by standard molecular biology techniques, (ii) isolation of protein compounds from natural sources, or (iii) chemical synthesis of protein compounds. Nucleotide as well as protein, polypeptide and peptide sequences for various genes have been previously described and can be found in recognized computerized databases. Examples of such databases are the GenBank and GenPept databases of the US National Center for Biotechnology Information (website ncbi.nlm.nih.gov/). The coding regions of these genes, in whole or in part, can be amplified and/or expressed by the methods described herein or as known to those skilled in the art.

Variants of the amino acid sequences of autoantigenic epitopes and other polypeptides of these compositions may be variants with substitutions, insertions or deletions. Modifications in the polypeptide of the invention compared to wild type may affect 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 20 4, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 39 6, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496, 497, 498, 499, 500 or more non-contiguous or contiguous amino acids of a peptide or polypeptide. For use in the methods of the invention, peptides or polypeptides are provided that elicit an autoimmune response, and especially an abnormal autoimmune response.

Typically, deletion variants lack one or more residues of the natural or wild-type amino acid sequence. You can remove individual residues or you can remove several consecutive amino acids. To obtain a truncated protein, a stop codon can be introduced into the nucleic acid coding sequence (via substitution or insertion). Typically, insertion mutants involve the addition of material to the non-terminal region of the polypeptide. This may include the insertion of one or more residues. You can also get proteins with terminal additions, called fused proteins.

Typically, substitutional variants involve the substitution of one amino acid for another at one or more regions of the protein, and they can be designed to modulate one or more properties of the polypeptide, with or without loss of other functions or properties. The substitutions may be conservative, ie. one amino acid is replaced by an amino acid with a similar shape and charge. Conservative substitutions are well known in the art and include, for example, substitutions of: alanine for serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan for tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Alternatively, the substitutions may be non-conservative, such that the function or activity of the polypeptide or peptide is affected, such as avidity or affinity for the cellular receptor(s). Typically, non-conservative substitutions involve substituting a residue for a residue that is chemically dissimilar, such as substituting a polar or charged amino acid for a nonpolar or uncharged amino acid and vice versa.

The proteins of the invention may be recombinant or synthesized in vitro. Alternatively, the recombinant protein can be isolated from bacteria or other host cells.

The term "functionally equivalent codon" is used herein to refer to codons that code for the same amino acid, such as the six codons for arginine or serine, and also refers to codons that code for biologically equivalent amino acids (see Table 2).

It is also to be understood that amino acid and nucleic acid sequences may contain additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' nucleic acid sequences, respectively, and yet remain relevant as indicated in one of the sequences described herein, provided that the sequence satisfies the criteria specified above, including retention of the biological activity of the protein (eg, immunogenicity). The addition of terminal sequences is particularly applicable to nucleic acid sequences which may, for example, contain various non-coding sequences flanking any of the 5' or 3' regions of the coding region.

Disease-associated antigens

Nanoparticles are useful in therapeutic methods as described herein. The pMHC complex from pMHC-NP is selected for use based on the disease being treated. For example, a diabetes-associated antigen is an antigen or fragment thereof that is expressed in a cell, tissue, or organ affected by the autoimmune disease and that, when the cell, tissue, or organ is damaged by the autoimmune response, is attacked by the immune system, even if the antigen is not is a factor initiating the pathological process or a key participant in its pathogenesis, and when it is presented, an immune response is formed, which serves to treat diabetes; thus, a diabetes-associated antigen meeting this definition is selected for the treatment of diabetes. For the treatment of MS, a PC-associated antigen is chosen. The diabetes-associated antigen is not of choice for the treatment of MS. Non-limiting illustrative disease-associated antigens are described herein, and such antigens can further be determined for a particular disease by methods well described in the literature.

Non-limiting examples of diseases of interest include, but are not limited to, asthma, type I and type II diabetes mellitus, prediabetes, multiple sclerosis, peripheral neuropathy, allergic asthma, primary biliary cirrhosis, cirrhosis of the liver, neuromyelitis of the optic nerve with spectral disorder associated with autoantibodies. neurological syndromes such as stiffness syndrome, autoimmune encephalitis, narcolepsy, pemphigus vulgaris, exfoliative pemphigus, psoriasis, Sjögren's disease/syndrome, inflammatory bowel disease (IBD), arthritis, rheumatoid arthritis, systemic lupus erythematosus (SLE), scleroderma, associated with ANCA vasculitis, Goodpasture's syndrome, Kawasaki disease, celiac disease, autoimmune cardiomyopathy, idiopathic dilated cardiomyopathy (IDCM), myasthenia gravis, autoimmune uveitis, ankylosing spondylitis, Grave's disease, immune-mediated myopathies, antiphospholipid syndrome (ANCA+), atherosclerosis, autism immune hepatitis, sclerosing cholangitis, primary sclerosing cholangitis, dermatomyositis, chronic obstructive pulmonary disease, spinal cord injury, traumatic injury, tobacco-induced lung destruction, emphysema, pemphigus, uveitis, any other relevant malignancy, and/or diseases of the central and peripheral nervous systems.

Cancer/tumor-associated antigens

In certain aspects, a disease-associated antigen is a cancer-associated antigen. In additional aspects, the cancer is a carcinoma, sarcoma, myeloma, leukemia, lymphoma, and/or metastases of mixed types of these or other cancers. Illustrative cancer or tumor associated antigens include, by way of non-limiting examples, the antigens listed in Table 5 below.

Other cancer-associated antigens include those tabulated in the online database http://cancerimmunity.org/peptide/ and incorporated herein by reference, last cited May 6, 2015.

Antigens associated with autoimmune diseases

In certain aspects, the disease-associated antigen contained in the antigen-MHC complex is selected from an autoimmune disease-associated antigen, an inflammation-associated antigen, or an allergic disease-associated antigen. In additional aspects, the immune inflammation-associated antigen is one or more antigens selected from the group of asthma-associated antigen, diabetes-associated antigen, prediabetes-associated antigen, multiple sclerosis-associated antigen, allergic asthma-associated antigen, primary biliary cirrhosis-associated antigen cirrhosis-associated antigen associated with neuromyelitis of the optic nerve with spectral disorder (Devic's disease, NMO) antigen associated with autoimmune encephalitis antigen associated with autoantibody-mediated neurological syndrome antigen associated with muscle stiffness syndrome antigen associated with paraneoplastic syndrome antigen, antigens associated with other diseases of the central and peripheral nervous systems, an antigen associated with pemphigus vulgaris, an antigen associated with inflamed severe bowel disease (IBD) antigen, Crohn's disease-associated antigen, ulcerative colitis-associated antigen, arthritis-associated antigen, rheumatoid arthritis-associated antigen, systemic lupus erythematosus (SLE) antigen, celiac disease-associated antigen, psoriasis-associated alopecia areata associated antigen alopecia areata associated antigen autoimmune cardiomyopathy associated antigen idiopathic dilated cardiomyopathy (IDCM) associated antigen myasthenia gravis associated antigen uveitis associated antigen ankylosing spondylitis associated antigen ankylosing spondylitis associated antigen Grave's disease antigen associated with Hashimoto's thyroiditis antigen associated with immune-mediated myopathies antigen associated with antiphospholipid syndrome (ANCA+) antigen atherosclerosis-associated antigen, scleroderma-associated antigen, autoimmune hepatitis-associated antigen, dermatomyositis-associated antigen, chronic obstructive pulmonary disease-associated antigen, spinal cord injury-associated antigen, traumatic injury-associated antigen, tobacco-induced lung destruction chronic obstructive pulmonary disease (COPD)-associated antigen, emphysema-associated antigen, sclerosing cholangitis-associated antigen, peripheral neuropathy-associated antigen, narcolepsy-associated antigen, Goodpasture syndrome-associated antigen, and Kawasaki disease-associated antigen associated with with autoimmune uveitis colitis-associated antigen, emphysema-associated antigen, pemphigus-associated antigen, association pemphigus foliaceus-associated antigen, arthritis-associated antigen, Sjögren's syndrome-associated antigen, ANCA-associated vasculitis-associated antigen, primary sclerosing cholangitis-associated antigen, type II adipose inflammation/diabetes-associated antigen, or obesity-associated adipose inflammation tissue/insulin resistance antigens.

In certain aspects, the disease-associated antigen is derived from one or more of the group: PPI, IGRP, GAD, peripheral, aGlia, PDC-E2, insulin, DG1EC2, DG3, AQP4, PLP, MOG, MBP, CII, DERP1, DERP2 , OVA, BacInt, CBir, Fla-X, Fla-2, YIDX, AChR, thyroid peroxidase, thyroid receptor, phospholipid antigen, H4, H2B, H1, DNA, ApoB, ApoE, NMDAR, voltage-gated potassium channel, elastin, arrestin, myeloperoxidase PERM_HUMAN, myeloblastin PRTN3_HUMAN, cytochrome P450 2D6 CP2D6_HUMAN, O-phosphoseryl-tRNA(Sec)-selenentransferase SPCS_HUMAN, antimicrobial peptide cathelicidin CAMP_HUMAN, DNA topoisomerase I, CENP-C, beta-2-glycoprotein 1 APOH_SS-HUMAN, ribonucleoprotein 1 APOH_SS-HUMAN, ribonucleoprotein 1 /Ro 60 kDa RO60_HUMAN, lupus protein La LA_HUMAN, IRBP, myosin, CD1d-binding lipid antigens, Cap18, CP2D6, SPCS, RO60, RO52, LA, APO, MPO, PRTN3, or HSP.

In certain embodiments, the disease-associated antigen is:

a) a diabetes-associated antigen and is derived from an antigen selected from one or more of the group: preproinsulin (PPI), islet-specific glucose-6-phosphatase (IGRP), glutamate decarboxylase (GAD), islet cell autoantigen 2 (ICA2) , insulin, proinsulin, or a fragment or equivalent of any of them;

b) a multiple sclerosis-associated antigen, and is derived from an antigen selected from one or more of the group: myelin basic protein, myelin-associated glycoprotein, oligodendrocyte myelin protein, proteolipid protein, oligodendrocyte myelin oligoprotein, oligodendrocyte myelin-associated basic protein, specific for oligodendrocytes, a protein, heat shock proteins, oligodendrocyte-specific proteins, NOGO A, glycoprotein Rho, peripheral myelin 22 protein, 3'-phosphodiesterase of 2'3'-cyclic nucleotides, or a fragment or equivalent of any of these;

c) an antigen associated with celiac disease and is derived from gliadin or a fragment or equivalent thereof;

d) an antigen associated with primary biliary cirrhosis and is derived from PDC-E2 or a fragment or equivalent thereof;

e) a pemphigus foliaceus-associated antigen and/or a pemphigus vulgaris-associated antigen, and is derived from an antigen selected from one or more of the group: DG1, DG3, or a fragment or equivalent of any of these;

f) an antigen associated with neuromyelitis of the optic nerve with spectral disorder, and it is derived from AQP4 or a fragment or equivalent thereof;

g) an arthritis-associated antigen and is derived from an antigen selected from one or more of the group: heat shock proteins, immunoglobulin-binding protein, heterogeneous nuclear RNPs, annex V, calpastatin, type II collagen, glucose-6-phosphate isomerase, elongation factor human cartilage gp39, mannose-binding lectin, citrullinated vimentin, type II collagen, fibrinogen, alpha-enolase, antibodies to carbamylated proteins (anti-CarP antibodies), peptidylarginine deiminase type 4 (PAD4), BRAF, fibrinogen gamma chain, inter-alpha heavy chain trypsin inhibitor H1, alpha-1 antitrypsin, C1 plasma protease inhibitor, gelsolin, alpha-1-B glycoprotein, ceruloplasmin, inter-alpha trypsin inhibitor H4 heavy chain, complement factor H, alpha-2 macroglobulin, serum amyloid, C-reactive protein, serum albumin, fibrinogen beta chain, serotransferrin, alpha-2-glycoprotein HS, vimentin, complement component C3, or a fragment or equivalent of any of these;

h) an allergic asthma-associated antigen and is derived from an antigen selected from one or more of the group: DERP1, DERP2, or a fragment or equivalent of any of these;

i) an inflammatory bowel disease associated antigen and is derived from an antigen selected from one or more of the group: flagellin, Fla-2, Fla-X, YIDX, bacteroid integrase, or a fragment or equivalent of any of these;

j) an antigen associated with systemic lupus erythematosus, and it is derived from an antigen selected from one or more of the group: double-stranded (ds)DNA, ribonucleoprotein (RNP), Smith (Sm), Sjögren's syndrome associated antigen A (SS-A) /Ro, associated with Sjögren's syndrome antigen B (SS-B)/La, RO60, R052, histones or a fragment or equivalent of any of them;

k) an atherosclerosis-associated antigen and is derived from an antigen selected from one or more of the group: ApoB, ApoE, or a fragment or equivalent of any of these;

l) a COPD-associated antigen and/or an emphysema-associated antigen, and is derived from elastin or a fragment or equivalent thereof;

m) a psoriasis-associated antigen and is derived from an antigen selected from one or more of the group: Cap 18, ADMTSL5, ATL5, or a fragment or equivalent of any of these;

n) an autoimmune hepatitis associated antigen and is derived from an antigen selected from one or more of the group: CYP2D6, SLA, or a fragment or equivalent of any of these;

o) an antigen associated with uveitis and is derived from arrestin or a fragment or equivalent thereof;

p) an antigen associated with Sjögren's syndrome and is derived from an antigen selected from one or more of the group: (SS-A)/Ro, (SS-B)/La, MR3, RO60, RO52, or a fragment or equivalent of any of them ;

q) a scleroderma-associated antigen and is derived from an antigen selected from one or more of the group: CENP-C, TOP 1, RNA polymerase III, or a fragment or equivalent of any of these;

d) an antiphospholipid syndrome associated antigen and is derived from APO or a fragment or equivalent thereof;

s) an ANCA-associated vasculitis-associated antigen and is derived from an antigen selected from one or more of the group: MPO, PRTN3, or a fragment or equivalent of any of these; or

t) a stiffness syndrome-associated antigen and is derived from GAD or a fragment or equivalent thereof.

Diabetes-Associated Antigens

Diabetes-associated antigens include, but are not limited to, antigens derived from PPI, IGRP, GAD, islet cell autoantigen 2 (ICA2), and/or insulin. Non-limiting examples of self-reactive diabetes-associated antigenic peptides include the peptides listed in Table 6 below, in addition to the peptides and proteins described in US Publication 2005/0202032, incorporated herein by reference in its entirety, as well as equivalents and/ or combinations of each.

PC-associated antigens

The antigens of the invention include those associated with multiple sclerosis. Such antigens include, for example, those described in US Patent Application Publication No. 2012/0077686 and antigens derived from myelin basic protein, myelin associated glycoprotein, oligodendrocyte myelin protein, proteolipid protein, oligodendrocyte myelin oligoprotein, oligodendrocyte myelin basic associated protein. , oligodendrocyte-specific protein, heat shock proteins, oligodendrocyte-specific NOGO A proteins, Po glycoprotein, myelin 22 peripheral protein, or 2'3'-cyclic nucleotide 3'-phosphodiesterase. In certain embodiments, the antigen is derived from myelin oligonedrocyte glycoprotein (MOG).

In additional aspects, peptide antigens for the treatment of MS and MS-related disorders include, as non-limiting examples, the antigens listed in Table 7, as well as equivalents and/or combinations of each of them:

Celiac Disease Associated (CD) Antigens

Associated with celiac disease antigens, as non-limiting examples, include antigens derived from gliadin. In accordance with certain embodiments, non-limiting types of gliadin include alpha/beta gliadin, γ-gliadin, or ω-gliadin. Other non-limiting illustrative celiac disease associated antigens include the antigens listed in Table 8, as well as equivalents and/or combinations of each.

Antigens associated with primary biliary cirrhosis (PBC)

Primary biliary cirrhosis-associated antigens include, but are not limited to, antigens derived from PDC-E2. Non-limiting examples of illustrative antigens include the antigens listed in Table 9, as well as equivalents and/or combinations of each.

Pemphigus foliaceus (PF) and Pemphigus vulgaris (PV) associated antigens

PF and PV associated antigens include, but are not limited to, antigens derived from desmoglein 3 (DG3) and/or desmoglein 1 (DG1). Non-limiting examples include the antigens listed in Table 10 as well as equivalents and/or combinations of each.

Optic neuromyelitis with spectrum disorder (NMO) associated antigens

Non-limiting examples of NMO-associated antigens include antigens derived from AQP4 or aquaporin 4. Non-limiting examples include the antigens listed in Table 11, as well as equivalents and/or combinations of each.

Arthritis-associated antigens

Arthritis-associated antigens include, but are not limited to, antigens derived from heat shock proteins, immunoglobulin-binding protein, heterogeneous nuclear RNPs, annexin V, calpastatin, type II collagen, glucose-6-phosphate isomerase, human cartilage elongation factor gp39, mannose-binding lectin, citrullinated vimentin, type II collagen, fibrinogen, alpha-enolase, antibodies to carbamylated proteins (anti-CarP), peptidylarginine deiminases type 4 (PAD4), BRAF, fibrinogen gamma chain, heavy chain inter-alpha trypsin inhibitor H1, alpha-1 -antitrypsin, C1 plasma protease inhibitor, gelsolin, alpha-1-B glycoprotein, ceruloplasmin, heavy chain inter-alpha trypsin inhibitor H4, complement factor H, alpha-2-macroglobulin, serum amyloid, C-reactive protein, serum albumin, beta chain fibrinogen, serotransferrin, alpha-2-glycoprotein HS, vimentin, complement component C3, or a fragment or equivalent of any of them.

Antigens associated with allergic asthma

Allergic asthma-associated antigens include, but are not limited to, antigens derived from DERP1 and DERP2. Non-limiting examples include the antigens listed in Table 12 as well as equivalents and/or combinations of each.

Antigens associated with inflammatory bowel disease

Inflammatory bowel disease-associated antigens include, but are not limited to, Crohn's disease-associated antigens and ulcerative colitis-associated antigens. In certain embodiments, inflammatory bowel disease-associated antigens include, but are not limited to, antigens derived from bacteroid integrase, flagellin, flagellin 2 (Fla-2/Fla-X), or uncharacterized E. coli protein (YIDX). Non-limiting examples include the antigens listed in Table 13 as well as equivalents and/or combinations of each.

Systemic lupus erythematosus (SLE) associated antigens

SLE-associated antigens include, but are not limited to, antigens derived from H4, H2 B, H1', dsDNA, RNP, Smith (Sm), Sjogren's Syndrome-Associated Antigen A (SS-A)/Ro, Sjogren's Syndrome-Associated Antigen B (SS-B)/La and/or histones. In accordance with certain embodiments, SS-A includes RO60 and RO52 as non-limiting examples. In accordance with certain embodiments, histones include, but are not limited to, H4, H2 B, H1'. Non-limiting examples include the antigens listed in Table 14 as well as equivalents and/or combinations of each.

Antigens associated with atherosclerosis

Atherosclerosis-associated antigens include, but are not limited to, antigens derived from apolipoprotein B (ApoB) or apolipoprotein E (ApoE). Non-limiting examples include the antigens listed in Table 15 as well as equivalents and/or combinations of each.

Antigens associated with chronic obstructive pulmonary disease (COPD) and/or emphysema

Antigens associated with COPD and/or emphysema include, but are not limited to, antigens derived from elastin. Non-limiting examples include the antigens listed in Table 16 as well as equivalents and/or combinations of each.

Antigens associated with psoriasis

Psoriasis-associated antigens include, but are not limited to, the antigens listed in Table 17 below, as well as equivalents and/or combinations thereof. Other non-limiting exemplary psoriasis-associated antigens can be derived from human adamis-like protein 5 (ATL5), cathelicidin antimicrobial peptide (CAP 18), and/or ADAMTS-like protein 5 (ADMTSL5).

Antigens associated with autoimmune hepatitis

Associated with autoimmune hepatitis antigens, as non-limiting examples, include the antigens described in Table 18 below, as well as equivalents and/or combinations thereof. Other non-limiting exemplary autoimmune hepatitis associated antigens can be derived from microsomal cytochrome P450IID6 (CYP2D6) and/or soluble liver antigen (SLA).

Antigens associated with uveitis

Associated with uveitis antigens, as non-limiting examples, include the antigens described in Table 19 below, as well as equivalents and/or combinations thereof. Other non-limiting exemplary uveitis-associated antigens can be derived from arrestin, human retinal S antigen, and/or interphotoreceptor retinol-binding protein (IRBP).

Antigens associated with Sjögren's syndrome

Antigens associated with Sjögren's Syndrome include, by way of non-limiting examples, the antigens described in Table 20 below, as well as equivalents and/or combinations thereof. Other non-limiting exemplary Sjögren syndrome associated antigens can be derived from (SS-A)/Ro, (SS-B)/La, RO60, RO52 and/or muscarinic receptor 3 (MR3).

Antigens associated with scleroderma

Associated with scleroderma antigens, as non-limiting examples, include the antigens described in Table 21 below, as well as equivalents and/or combinations thereof. Non-limiting illustrative scleroderma-associated antigens can be derived from centromeric autoantigenic protein C (CENP-C), DNA topoisomerase I (TOP1), and/or RNA polymerase III.

Antiphospholipid syndrome associated antigens

Associated with antiphospholipid syndrome antigens, as non-limiting examples, include the antigens described in Table 22 below, as well as equivalents and/or combinations thereof. Non-limiting illustrative antiphospholipid syndrome associated antigens can be derived from beta-2-glycoprotein 1 (BG2P1 or APO).

Antigens associated with ANCA-associated vasculitis

ANCA-associated vasculitis-associated antigens include, but are not limited to, the antigens described in Table 23 below, as well as equivalents and/or combinations thereof. Non-limiting exemplary ANCA-associated vasculitis-associated antigens can be derived from myeloperoxidase (MPO), proteinase (PRTN3), or bacterial permeability enhancing factor (BPI).

Antigens associated with stiffness syndrome

The antigens associated with stiffness syndrome include, by way of non-limiting examples, the antigens described in Table 24 below, as well as equivalents and/or combinations thereof. Non-limiting exemplary stiffness syndrome-associated antigens can be derived from glutamate decarboxylase (GAD). In accordance with certain embodiments, GAD includes GAD65 as non-limiting examples.

It is envisaged that in the compositions of the invention, the total composition contains from about 0.001 mg to about 10 mg of total protein per ml. Thus, the concentration of protein in the composition may be about, at least about, or not a maximum of about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0 .7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5 , 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 50, 100 mcg/mL or mg/mL or more ( or any range derived from them). Of which approximately, at least approximately, or maximum approximately 1, 2, 3, 4, 5.6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% may be a peptide/MHC/nanoparticle complex.

The present disclosure provides for the administration of a peptide/MHC/nanoparticle complex to diagnose, treat, or prevent the development of a disease or condition associated with an autoimmune response or cancer.

In addition, US Pat. No. 4,554,101 (Kopr), incorporated herein by reference, describes the identification and generation of epitopes from primary amino acid sequences based on hydrophilicity. Using the methods described in Knorr, a person skilled in the art can identify potential epitopes in an amino acid sequence and confirm their immunogenicity. There are also many scientific publications devoted to the prediction of secondary structure and identification of epitopes based on the analysis of amino acid sequences (Chou & Fasman, 1974a,b; 1978a,b; 1979). If desired, any of these can be used to supplement the description of Knorr in US Pat. No. 4,554,101.

Other antigenic components

Molecules other than peptides can be used as antigens or antigenic fragments in complex with MHC molecules. Such molecules include, but are not limited to, carbohydrates, lipids, small molecules, and the like. Carbohydrates are major components of the outer surface of many cells. Certain carbohydrates are characteristic of different stages of differentiation and very often these carbohydrates are recognized by specific antibodies. Expression of specific carbohydrates may be limited to specific cell types. It has been shown that the response of autoantibodies to endometrial and serum antigens is a common feature of endometriosis. Serum autoantibody response in endometriosis has been described for a number of previously identified antigens, including Geremans-Schmid 2-glycoprotein and carbonic anhydrase, which is specific for a carbohydrate epitope.

Non-limiting, exemplary antigen-MHC complexes

In accordance with certain embodiments, specific combinations of antigen and MHC can be optimized for the treatment of a particular disease. Non-limiting examples include, but are not limited to, the following examples:

For the treatment of type I diabetes, the pMHC complex antigen can be derived from the group antigens: PPI _76-90(K88S) , IGRP _13-25 , GAD _555-567 , GAD _{555-567(5571)} , IGRP _23-35 , B _24-C36 , PPI _76-90 or a fragment or equivalent of any of them, and the MHC of the pMHC complex contains all or part of a polypeptide from the group: HLA-DRB1*0401/DRA, HLA-DRB1*0301/DRA or a fragment or equivalent of any of them.

In certain embodiments, the pMHC complex antigen comprises:

a) a diabetes-associated antigen and is derived from an antigen selected from one or more of the group: preproinsulin (PPI), islet-specific glucose-6-phosphatase (IGRP), glutamate decarboxylase (GAD), islet cell autoantigen 2 (ICA2) , insulin, proinsulin, or a fragment or equivalent of any of them;

b) a multiple sclerosis-associated antigen, and is derived from an antigen selected from one or more of the group: myelin basic protein, myelin-associated glycoprotein, oligodendrocyte myelin protein, proteolipid protein, oligodendrocyte myelin oligoprotein, oligodendrocyte myelin-associated basic protein, specific for oligodendrocytes, protein, heat shock proteins, oligodendrocyte-specific proteins, NOGO A, glycoprotein Rho, peripheral myelin 22 protein, 3'-phosphodiesterase of 2'3'-cyclic nucleotides, or a fragment or equivalent of any of these;

c) an antigen associated with celiac disease and is derived from gliadin or a fragment or equivalent thereof;

d) an antigen associated with primary biliary cirrhosis and is derived from PDC-E2 or a fragment or equivalent thereof;

e) a pemphigus foliaceus-associated antigen and/or a pemphigus vulgaris-associated antigen, and is derived from an antigen selected from one or more of the group: DG1, DG3, or a fragment or equivalent of any of these;

f) an antigen associated with neuromyelitis of the optic nerve with spectral disorder, and it is derived from AQP4 or a fragment or equivalent thereof;

g) an arthritis-associated antigen and is derived from an antigen selected from one or more of the group: heat shock proteins, immunoglobulin-binding protein, heterogeneous nuclear RNPs, annexin V, calpastatin, type II collagen, glucose-6-phosphate isomerase, elongation factor human cartilage gp39, mannose-binding lectin, citrullinated vimentin, type II collagen, fibrinogen, alpha-enolase, antibodies to carbamylated proteins (anti-CarP antibodies), peptidylarginine deiminase type 4 (PAD4), BRAF, fibrinogen gamma chain, inter-alpha heavy chain trypsin inhibitor H1, alpha-1 antitrypsin, C1 plasma protease inhibitor, gelsolin, alpha-1-B glycoprotein, ceruloplasmin, inter-alpha trypsin inhibitor H4 heavy chain, complement factor H, alpha-2 macroglobulin, serum amyloid, C-reactive protein, serum albumin, fibrinogen beta chain, serotransferrin, alpha-2-glycoprotein HS, vimentin, complement component C3, or a fragment or equivalent of any of these;

h) an allergic asthma-associated antigen and is derived from an antigen selected from one or more of the group: DERP1, DERP2, or a fragment or equivalent of any of these;

i) an inflammatory bowel disease associated antigen and is derived from an antigen selected from one or more of the group: Flagellin, Fla-2, Fla-X, YIDX, bacteroid integrase, or a fragment or equivalent of any of these;

j) an antigen associated with systemic lupus erythematosus, and it is derived from an antigen selected from one or more of the group: double-stranded (ds)DNA, ribonucleoprotein (RNP), Smith (Sm), Sjögren's syndrome associated antigen A (SS-A) /Ro, Sjögren's syndrome-associated antigen B (SS-B)/La, RO60, RO52, histones or a fragment or equivalent of any of them;

k) an atherosclerosis-associated antigen and is derived from an antigen selected from one or more of the group: ApoB, ApoE, or a fragment or equivalent of any of these;

l) a COPD-associated antigen and/or an emphysema-associated antigen, and is derived from elastin or a fragment or equivalent thereof;

m) a psoriasis-associated antigen and is derived from an antigen selected from one or more of the group: Cap18, ADMTSL5, ATL5, or a fragment or equivalent of any of these;

n) an autoimmune hepatitis associated antigen and is derived from an antigen selected from one or more of the group: CYP2D6, SLA, or a fragment or equivalent of any of these;

o) an antigen associated with uveitis and is derived from arrestin or a fragment or equivalent thereof;

p) an antigen associated with Sjögren's syndrome and is derived from an antigen selected from one or more of the group: (SS-A)/Ro, (SS-B)/La, MR3, RO60, RO52, or a fragment or equivalent of any of them ;

q) a scleroderma-associated antigen and is derived from an antigen selected from one or more of the group: CENP-C, TOP 1, RNA polymerase III, or a fragment or equivalent of any of these;

r) an antiphospholipid syndrome associated antigen and is derived from APO or a fragment or equivalent thereof;

s) an ANCA-associated vasculitis-associated antigen and is derived from an antigen selected from one or more of the group: MPO, PRTN3, or a fragment or equivalent of any of these; or

t) a stiffness syndrome-associated antigen and is derived from GAD or a fragment or equivalent thereof.

In certain embodiments, the rMHC complex MHC protein comprises all or part of a classic MHC class I protein, a non-classical MHC class I protein, a classic MHC class II protein, a non-classical MHC class II protein, MHC dimers (Fc fusions), MHC tetramers, or polymeric a form of the MHC protein, wherein the MHC protein optionally comprises a "trough-to-trough" heterodimer or multimer alpha-MHC-Fc/beta-MHC-Fc.

In accordance with certain embodiments, the rMHC complex MHC protein comprises all or part of a polypeptide from the group: HLA DR, HLA DQ, HLA DP, HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G , CD1d or fragment or equivalent of any of them.

In certain embodiments, the rMHC complex MHC protein comprises all or part of a polypeptide from the group: HLA-DR, HLA-DQ, HLA-DP, or a fragment or equivalent of any of these.

In accordance with certain embodiments, the MHC protein of the rMHC complex contains all or part of a polypeptide from the group: HLA-DRB1/DRA, HLA-DRB3/DRA, HLA-DRB4/DRA, HLA-DRB5/DRA, HLA-DQA1/HLA-DQB1, HLA-DPB1/HLA-DPA1 or fragment or equivalent of any of them.

In accordance with certain aspects, the rMHC complex contains:

a) a diabetes associated antigen derived from an antigen selected from one or more of the group: hInsB _10-18 , hIGRP _228-236 , hIGRP _265-273 , IGRP _206-214 , hIGRP _206-214 , NRP-A7, NRP- I4, NRP-V7, YAI/D ^b , INS B _15-23 , PPI _{76-90 (K88S)} , IGRP _13-25 , GAD _555-567 , GAD _{555-567(557I)} , IGRP _23-35 , B ₂₄ -С ₃₆ , PPI _76-90 , INS-I9, TUM, G6Pase, proinsulin _L2-10 , proinsulin _L3-11 , proinsulin _L6-14 , proinsulin _B5-14 , proinsulin _B10-18 , proinsulin _B14-22 , proinsulin _{B15- 24} , proinsulin _B17-25 , proinsulin _B18-27 , proinsulin _B20-27 , proinsulin _B21-29 , proinsulin _B25-C1 , proinsulin _B27-C5 , proinsulin _C20-28 , proinsulin _C25-33 , proinsulin _C29-A5 , proinsulin _{A1- 10} , proinsulin _A2-10, proinsulin _A12-20 , or a fragment or equivalent of any of them;

b) a multiple sclerosis-associated antigen derived from an antigen selected from one or more of the group: MOG _35-55 , MOG _36-55 , MAG _287-295 , MAG _509-517 , MAG _556-564 , MBP _110-118 , MOG _114-122 , MOG _166-175 , MOG _172-180 , MOG _179-188 , MOG _188-196 , MOG _181-189 , MOG _205-214 , PLP _80-88 , MAG _287-295 , MAG _509-517 , MAG _556-564 , MOG _97-109 MOG _{97-109(E107S)} , MBP _89-101 , PLP _175-192 , PLP _94-108 , MBP _86-98 , PLP _54-68 , PLP _249-263 , MOG _{156- 170} , MOG _201-215 , MOG _38-52 , MOG _203-217 , PLP _250-264 , MPB _13-32 , MPB _83-99 , MPB _111-129 , MPB _146-170 , MOG _223-237 , MOG _{6- 20} , PLP _88-102 , PLP _139-154 , or a fragment or equivalent of any of them;

c) an antigen associated with celiac disease derived from an antigen selected from one or more of the group: aGlia _57-68 , aGlia _62-72 , aGlia _217-229 or a fragment or equivalent of any of them;

d) primary biliary cirrhosis associated antigen derived from an antigen selected from one or more of the group: PDC-E2 _122-135 , PDC-E2 _249-262 , PDC-E2 _249-263 , PDC-E2 _629-643 , PDC -E2 _72-86 , PDC-E2 _353-367 , PDC-E2 _422-436 , PDC-E2 _629-643 , PDC-E2 _80-94 , PDC-E2 _353-367 , PDC-E2 _535-549 or fragment or the equivalent of any of them;

e) a pemphigus foliaceus-associated antigen and/or a pemphigus vulgaris-associated antigen, each derived from an antigen selected from one or more of the group: _{DG1 216-229} , DG3 _97-111 , DG3 _251-265 , DG3 _441-455 , DG3 _351-365 , DG3 _453-467 , _{DG3 540-554} , _{DG3 280-294} , _{DG3 326-340} , _{DG3 367-381} , _{DG1 206-222} , _{DG1 363-377} , DG1 _3-17 , DG1 _192-206 , _{DG1 326-340} , DG1 _1-15 , DG1 _35-49 , _{DG1 325-339} or a fragment or equivalent of any of them;

f) an optic neuromyelitis-associated antigen derived from an antigen selected from one or more of the group: AQP4 _129-143 , AQP4 _284-298 , AQP4 _63-76 , AQP4 _129-143 , AQP4 _39-53 or a fragment or the equivalent of any of them;

g) an allergic asthma associated antigen derived from an antigen selected from one or more of the group: DERP1 _16-30 , DERP1 _171-185 , DERP1 _110-124 , DERP-2 _26-40 , DERP-2 _107-121 or a fragment or the equivalent of any of them;

h) an inflammatory bowel disease associated antigen derived from an antigen selected from one or more of the group: bacteroid integrase antigen _183-197 , bacteroid integrase antigen _146-160 , bacteroid integrase antigen _175-189 , bacteroid integrase antigen _1-15 , antigen bacteroid integrase _183-197 , bacteroid integrase antigen _30-44 , bacteroid integrase antigen _70-84 , bacteroid integrase antigen _337-351 , bacteroid integrase antigen _171-185 , bacteroid integrase antigen _4-18 , bacteroid integrase antigen _256-270 , Fla- 2/Fla-X _366-380 , Fla-2/Fla-X _164-178 , Fla-2/Fla-X _261-275 , Fla-2/Fla-X _1-15 , Fla-2/Fla-X _{51 -65} Fla-2/Fla-X _269-283 Fla-2/Fla-X _4-18 Fla-2/Fla-X _271-285 YIDX _78-92 YIDX _93-107 YIDX _98-112 , YIDX _23-37 , YIDX _78-92 , YIDX _195-209 , YIDX _22-36 , YIDX _90-94 , YIDX _101-115 or a fragment or equivalent of any of them;

i) an antigen associated with systemic lupus erythematosus derived from an antigen selected from one or more of the group: H4 _71-94 , H4 _74-88 , H4 _76-90 , H4 _75-89 , H4 _78-92 , H4 _80-94 , H2B _10-24 , H2B _16-30 , H1' _22-42 , H1' _27-41 or a fragment or equivalent of any of them;

j) an atherosclerosis-associated antigen derived from an antigen selected from one or more of the group: ApoB _3501-3516 , ApoB _1952-1966 , ApoB _978-993 , ApoB _3498-3513 , ApoB _210A , ApoB _210B , ApoB _210C or a fragment, or the equivalent of any of them;

k) a COPD-associated antigen and/or an emphysema-associated antigen, each derived from an antigen selected from one or more of the group: elastin _89-103 , elastin _698-712 , elastin _8-22 , elastin _94-108 , elastin _13-27 , elastin _695-709 , elastin _563-577 , elastin _558-572 , elastin _698-712 , elastin _566-580 , elastin _645-659 , or a fragment or equivalent of any of them;

l) a psoriasis-associated antigen derived from an antigen selected from one or more of the group: Cap18 _64-78 , Cap18 _34-48 , Cap18 _47-61 , Cap18 _151-165 , Cap18 _149-163 , Cap18 _152-166 , Cap18 _{ADMTSL5 267-281 ADMTSL5 372-386 ADMTSL5 289-303} ADMTSL5 _396-410 ADMTSL5 _{433-447 ADMTSL5 142-156} ADMTSL5 236 _ADMTSL5 236 _-315 , ADMTSL5 _203-217 , ADMTSL5 _404-418 , or fragment or equivalent of any of them;

m) _{autoimmune hepatitis associated antigen derived} from an _antigen selected from one or more of the group: ₂₁₃ , CYP2D6 _450-464 , CYP2D6 _301-315 , CYP2D6 _452-466 , CYP2D6 _59-73 , CYP2D6 _130-144 , CYP2D6 _193-212 , CYP2D6 _305-324 , CYP2D6 _131-145 , CYP2D6 _{216-230-230-230 238- 252} , CYP2D6 _199-213 , CYP2D6 _235-252 , CYP2D6 _293-307 , CYP2D6 _381-395 , CYP2D6 _429-443 , SLA _334-348 , SLA _196-210 , SLA _115-129 , SLA _{37 . 197} SLA _317-331 SLA _171-185 SLA _417-431 SLA _359-373 SLA _215-229 SLA _{111-125 SLA 110-124 SLA 299-313 63} , SLA _119-133 , SLA _260-274 , SLA _26-40 , SLA _86-100 , SLA _331-345 , or a fragment or equivalent of any of them;

n) a uveitis associated antigen derived from an antigen selected from one or more of the group: arrestin _199-213 , arrestin _77-91 , arrestin _250-264 , arrestin _172-186 , arrestin _354-368 , arrestin _239-253 , arrestin _102-116 , arrestin _59-73 , arrestin _280-294 , arrestin _291-306 , arrestin _195-209 , arrestin _200-214 or fragment or equivalent of any of them;

o) an _antigen associated with _{Sjögren 's syndrome} , _derived from an _antigen selected from one or more of the group: _{RO60426-440 , RO60267-281 , RO60178-192 , RO60358-372 _ _ _} LA _241-255 , LA _101-115 , LA _153-167 , LA _178-192 , LA _19-33 , LA _37-51 , LA _133-147 , LA _50-64 , LA _32-46 , LA _153-167 , LA _83-97 , LA _136-150 , LA _297-311 , LA _59-73 , LA _151-165 , LA _86-100 , LA _154-168 or a fragment or equivalent of any of them;

p) a scleroderma-associated antigen derived from an antigen selected from one or more of the group: TOP1 _346-360 , TOP1 _420-434 , TOP1 _750-764 , TOP1 _419-433 , TOP1 _591-605 , TOP1 _695-709 , TOP1 _305-319 , TOP1 _346-360 , TOP1 _419-433 , TOP1 _425-439 , TOP1 _614-628 , CENP-C _297-311 , CENP-C _857-871 , CENP-C _887-901 , CENP-C _{212- 226} , CENP-C _643-657 , CENP-C _{832-846 ,} CENP-C _{167-181 ,} CENP- _{C 246-260} , CENP-C _847-861 or fragment or equivalent of any of them;

q) an antiphospholipid syndrome associated antigen derived from an antigen selected from one or more of the group: APO _235-249 , APO _306-320 , APO _237-251 , APO _295-309 , APO _28-42 , APOH _173-187 , APOH _264-278 , APO _295-309 , APO _49-63 , APO _269-283 , APO _295-309 , APO _321-355 , APO _322-336 , APO _324-338 or a fragment or equivalent of any of them;

r) an ANCA-associated vasculitis-associated antigen derived from an antigen selected from one or more of the group: MPO _506-520 , MPO _302-316 , MPO _7-21 , MPO _689-703 , MPO _248-262 , MPO _{444- 458} MPO _513-527 MPO _97-111 MPO _616-630 MPO _462-476 MPO _617-631 MPO _714-728 PRTN3 _44-58 PRTN3 _234-248 PRTN3 _59-73 PRTN3 _{117 131} , PRTN3 _164-178 , PRTN3 _71-85 , PRTN3 _241-255 , PRTN3 _59-73 , PRTN3 _183-197 , PRTN3 _62-76 , PRTN3 _118-132 , PRTN3 _239-253 or a fragment or equivalent of any of them; or

s) a stiffness syndrome-associated antigen derived from an antigen selected from one or more of the group: GAD _212-226 , GAD _555-569 , GAD _297-311 or a fragment or equivalent of any of them.

In accordance with certain aspects, the rMHC complex contains:

a) a diabetes associated antigen derived from an antigen selected from one or more of the group: hInsB _10-18 , hIGRP _228-236 , hIGRP _265-273 , IGRP _206-214 , hIGRP _206-214 , NRP-A7, NRP- I4, NRP-V7, YAI/D ^b , INS B _15-23 , PPI _76-90(K88S) , IGRP _13-25 , GAD _555-567 , GAD _{555-567(557I)} , IGRP _23-35 , B ₂₄ -C ₃₆ , PPI _76-90 , INS-19, TUM, G6Pase, proinsulin _L2-10 , proinsulin _L3-11 , proinsulin _L6-14 , proinsulin _B5-14 , proinsulin _B10-18 , proinsulin _B14-22 , proinsulin _{B15- 24} , proinsulin _B17-25 , proinsulin _B18-27 , proinsulin _B20-27 , proinsulin _B21-29 , proinsulin _B25-C1 , proinsulin _B27-C5 , proinsulin _C20-28 , proinsulin _C25-33 , proinsulin _C29-A5 , proinsulin _{A1- 10} , proinsulin _A2-10 , proinsulin _A12-20 , or a fragment or equivalent of any of them, and the MHC protein of the pMHC complex contains all or part of HLA-DR, or a fragment or equivalent thereof;

b) a multiple sclerosis-associated antigen derived from an antigen selected from one or more of the group: MOG _35-55 , MOG _36-55 , MAG _287-295 , MAG _509-517 , MAG _556-564 , MBP _110-118 , MOG _114-122 , MOG _166-175 , MOG _172-180 , MOG _179-188 , MOG _188-196 , MOG _181-189 , MOG _205-214 , PLP _80-88 , MAG _287-295 , MAG _509-517 , MAG _556-564 , MOG _97-109 MOG _{97-109(E107S)} , MBP _89-101 , PLP _175-192 , PLP _94-108 , MBP _86-98 , PLP _54-68 , PLP _249-263 , MOG _{156- 170} , MOG _201-215 , MOG _38-52 , MOG _203-217 , PLP _250-264 , MPB _13-32 , MPB _83-99 , MPB _111-129 , MPB _146-170 , MOG _223-237 , MOG _{6- 20} , PLP _88-102 , PLP _139-154 , or a fragment or equivalent of any of them, and the MYC protein of the pMHC complex contains all or part of HLA-DR, or a fragment or equivalent thereof;

c) a celiac disease associated antigen derived from an antigen selected from one or more of the group: aGlia _57-68 , aGlia _62-72 , aGlia _217-229 or a fragment or equivalent of any of them, and the MHC protein of the pMHC complex contains all or part of HLA-DQ or fragment or equivalent;

d) primary biliary cirrhosis associated antigen derived from an antigen selected from one or more of the group: PDC-E2 _122-135 , PDC-E2 _249-262 , PDC-E2 _249-263 , PDC-E2 _629-643 , PDC -E2 _72-86 , PDC-E2 _353-367 , PDC-E2 _422-436 , PDC-E2 _629-643 , PDC-E2 _80-94 , PDC-E2 _353-367 , PDC-E2 _535-549 or fragment or the equivalent of any of them, and the MHC protein of the pMHC complex contains all or part of HLA-DR, or a fragment or equivalent thereof;

e) a pemphigus foliaceus-associated antigen and/or a pemphigus vulgaris-associated antigen, each derived from an antigen selected from one or more of the group: _{DG1 216-229} , DG3 _97-111 , DG3 _251-265 , DG3 _441-455 , DG3 _351-365 , DG3 _453-467 , _{DG3 540-554} , _{DG3 280-294} , _{DG3 326-340} , _{DG3 367-381} , _{DG1 206-222} , _{DG1 363-377} , DG1 _3-17 , DG1 _192-206 , _{DG1 326-340} , DG ¹ _1-15 , DG1 _35-49 , _{DG1 325-339} , or a fragment or equivalent of any of them, and the MHC protein of the rMHC complex contains all or part of HLA-DR, or a fragment or equivalent thereof;

f) an optic neuromyelitis-associated antigen derived from an antigen selected from one or more of the group: AQP4 _129-143 , AQP4 _284-298 , AQP4 _63-76 , AQP4 _129-143 , AQP4 _39-53 or a fragment or an equivalent of any of them, and the MHC protein of the pMHC complex contains all or part of HLA-DR, or a fragment or equivalent thereof;

g) an allergic asthma associated antigen derived from an antigen selected from one or more of the group: DERP1 _16-30 , DERP1 _171-185 , DERP1 _110-124 , DERP-2 _26-40 , DERP-2 _107-121 or a fragment or an equivalent of any of them, and the MHC protein of the pMHC complex contains all or part of a polypeptide from the group: HLA-DR, HLA-DP, or a fragment or equivalent of any of them;

h) an inflammatory bowel disease associated antigen derived from an antigen selected from one or more of the group: bacteroid integrase antigen _183-197 , bacteroid integrase antigen _146-160 , bacteroid integrase antigen _175-189 , bacteroid integrase antigen _1-15 , antigen bacteroid integrase _183-197 , bacteroid integrase antigen _30-44 , bacteroid integrase antigen _70-84 , bacteroid integrase antigen _337-351 , bacteroid integrase antigen _171-185 , bacteroid integrase antigen _4-18 , bacteroid integrase antigen _256-270 , Fla- 2/Fla-X _366-380 , Fla-2/Fla-X _164-178 , Fla-2/Fla-X _261-275 , Fla-2/Fla-X _1-15 , Fla-2/Fla-X _{51 -65} Fla-2/Fla-X _269-283 Fla-2/Fla-X _4-18 Fla-2/Fla-X _271-285 YIDX _78-92 YIDX _93-107 YIDX _98-112 , YIDX _23-37 , YIDX _78-92 , YIDX _195-209 , YIDX _22-36 , YIDX _80-94 , YIDX _101-115 or a fragment or equivalent of any of them, and the MHC protein of the rMHC complex contains all or part of HLA-DR or a fragment or equivalent thereof;

i) an antigen associated with systemic lupus erythematosus derived from an antigen selected from one or more of the group: H4 _71-94 , H4 _74-88 , H4 _76-90 , H4 _75-89 , H4 _78-92 , H4 _80-94 , H2B _10-24 , H2B _16-30 , H1' _22-42 , H1' _27-41 or a fragment or equivalent of any of them, and the MHC protein of the pMHC complex contains all or part of a polypeptide from the group: IA _d , HLA-DR or fragment or equivalent of any of them;

j) an atherosclerosis-associated antigen derived from an antigen selected from one or more of the group: ApoB _3501-3516 , ApoB _1952-1966 , ApoB _978-993 , ApoB _3498-3513 , ApoB _210A , ApoB _210B , ApoB _210C or a fragment, or the equivalent of any of them, and the MHC protein of the pMHC complex contains all or part of IA _b or a fragment or equivalent thereof;

k) a COPD-associated antigen and/or an emphysema-associated antigen, each derived from an antigen selected from one or more of the group: elastin _89-103 , elastin _698-712 , elastin _8-22 , elastin _94-108 , elastin _13-27 , elastin _695-709 , elastin _563-577 , elastin _558-572 , elastin _698-712 , elastin _566-580 , elastin _645-659 , or a fragment or equivalent of any of them, and the MHC protein of the pMHC complex contains all or part HLA-DR or fragment or equivalent;

l) a psoriasis associated antigen derived from an antigen selected from one or more of the group: Cap18 _64-78 , Cap18 _34-48 , Cap18 _47-61 , Cap18 _151-165 , Cap18 _149-163 , Cap18 _152-166 , Cap18 _{ADMTSL5 267-281 ADMTSL5 372-386 ADMTSL5 289-303} ADMTSL5 _{396-410 ADMTSL5 433-447 ADMTSL5 132-156 -315} , ADMTSL5 _203-217 , ADMTSL5 _404-418 or a fragment or equivalent of any of them, and the MHC protein of the pMHC complex contains all or part of HLA-DR or a fragment or equivalent thereof;

m) an autoimmune hepatitis associated antigen derived from an antigen selected from one or more of the group: CYP2D6 _193-207 , CYP2D6 _76-90 , CYP2D6 _293-307 , CYP2D6 _313-332 , CYP2D6 _393-412 , CYP2D6 _199-213 , CYP2D6 _450-464 , CYP2D6 _301-315 , CYP2D6 _452-466 , CYP2D6 _59-73 , CYP2D6 _130-144 , CYP2D6 193-212, CYP2D6 305-324, CYP2D6 _131-145 , CYP2D6 _216-230 , CYP2D6 _238-252 , CYP2D6 _238-252 , CYP2D6 _238-252 , CYP2D6 _199-213 , CYP2D6 _235-252 , CYP2D6 _293-307 , CYP2D6 _381-395 , CYP2D6 _429-443 , SLA _334-348 , SLA _196-210 , SLA _115-129 , _{SLA 373-386} SLA _317-331 , SLA _171-185 , SLA _417-431 , SLA _359-373 , SLA _215-229 , SLA _111-125 , SLA _110-124 , SLA _299-313 , SLA _342-356 , SLA _49-63 , SLA _119-133 , SLA _260-274 , SLA _26-40 , SLA _86-100 , SLA _331-345 , or a fragment or equivalent of any of them, and the MHC protein of the pMHC complex contains all or part of HLA-DR or a fragment or equivalent thereof;

n) a uveitis associated antigen derived from an antigen selected from one or more of the group: arrestin _199-213 , arrestin _77-91 , arrestin _250-264 , arrestin _172-186 , arrestin _354-368 , arrestin _239-253 , arrestin _102-116 , arrestin _59-73 , arrestin _280-294 , arrestin _291-306 , arrestin _195-209 , arrestin _200-214 or a fragment or equivalent of any of them, and the MHC protein of the rMHC complex contains all or part of HLA-DR or its fragment or equivalent;

o) an _antigen associated with _{Sjögren 's syndrome derived} from an _antigen selected from one or more of the group: _{RO60426-440 , RO60267-281 , RO60178-192 , RO60358-372 _ _ _} LA _241-255 , LA _101-115 , LA _153-167 , LA _178-192 , LA _19-33 , LA _37-51 , LA _133-147 , LA _50-64 , LA _32-46 , LA _153-167 , LA _83-97 , LA _136-150 , LA _297-311 , LA _59-73 , LA _151-165 , LA _86-100 , LA _154-168 or a fragment or equivalent of any of them, and the MHC protein of the rMHC complex contains all or part of a polypeptide from the group: HLA-DR, HLA-DP or a fragment or equivalent of any of them;

p) a scleroderma-associated antigen derived from an antigen selected from one or more of the group: TOP1 _346-360 , TOP1 _420-434 , TOP1 _750-764 , TOP1 _419-433 , TOP1 _591-605 , TOP1 _695-709 , TOP1 _305-319 , TOP1 _346-360 , TOP1 _419-433 , TOP1 _425-439 , TOP1 _614-628 , CENP-C _297-311 , CENP-C _857-871 , CENP-C _887-901 , CENP-C _{212- 226} , CENP-C _643-657 , CENP-C _{832-846 ,} CENP-C _{167-181 ,} CENP- _{C 246-260} , CENP-C _847-861 , or a fragment or equivalent of any of them, and the MHC protein of the pMHC complex contains all or part of HLA-DR, or a fragment or equivalent thereof;

q) an antiphospholipid syndrome associated antigen derived from an antigen selected from one or more of the group: APO _235-249 , APO _306-320 , APO _237-251 , APO _295-309 , APO _28-42 , APO _173-187 , APO _264-278 , APO _295-309 , APO _49-63 , APO _269-283 , APO _295-309 , APO _321-355 , APO _322-336 , APO _324-338 or fragment or equivalent of any of them, and MHC protein the rMHC complex contains all or part of HLA-DR or a fragment or equivalent thereof;

r) an ANCA-associated vasculitis-associated antigen derived from an antigen selected from one or more of the group: MPO _506-520 , MPO _302-316 , MPO _7-21 , MPO _689-703 , MPO _248-262 , MPO _{444- 458} MPO _513-527 MPO _97-111 MPO _616-630 MPO _462-476 MPO _617-631 MPO _714-728 PRTN3 _44-58 PRTN3 _234-248 PRTN3 _59-73 PRTN3 _{117 . _ _ _ _ _ _ _ _} the MHC protein of the rMHC complex contains all or part of HLA-DR, or a fragment or equivalent thereof; or

s) a stiffness syndrome-associated antigen derived from an antigen selected from one or more of the group: GAD _212-226 , GAD _555-569 , GAD _297-311 , and the MHC protein of the pMHC complex contains all or part of a polypeptide from the group: HLA -DR, HLA-DQ, or fragment or equivalent of any of them.

In accordance with certain aspects, the rMHC complex is intended for the treatment of:

a) type I diabetes, and the rMHC complex is selected from the group

PPI _76-90(K88S) -HLA-DRB1*0401/DRA, IGRP _13-25 -HLA.DRB1*0301/DRA, GAD _555-567 -HLA-DRB1*0401/DRA, GAD _{555-567(557I)} - HLA-DRB1*0401/DRA, IGRP _23-35 -HLA-DRB1*0401/DRA, B ₂₄ -C ₃₆ -HLA-DRB1*0301/DRA or PPI _76-90 -HLA-DRB1*0401/DRA;

b) multiple sclerosis, and the rMHC complex is selected from the group: MBP _86-98 -HLA-DRB1*1501/DRA, MBP _89-101 -HLA-DRB5*0101/DRA, MOG _38-52 -HLA-DRB4*0101/DRA , MOG _{97-109(E107S)} -HLA-DRB1*0401/DRA, MOG _203-217 -HLA-DRB3*0101/DRA, PLP _54-68 -HLA-DRB3*0101/DRA, PLP _94-108 -HLA- DRB1*0301/DRA, PLP _250-264 -HLA-DRB4*0101/DRA, MPB _13-32 -HLA-DRB5*0101/DRA, MPB _83-99 -HLA-DRB5*0101/DRA, MPB _111-129 - HLA-DRB5*0101/DRA, MPB _146-170 -HLA-DRB5*0101/DRA, MOG _223-237 -HLA-DRB3*0202/DRA, MOG _6-20 -HLA-DRB5*0101/DRA, PLP _{88- 102} -HLA-DRB3*0202/DRA or PLP _139-154 -HLA-DRB5*0101/DRA;

c) celiac disease, and the rMHC complex is selected from the group: aGlia _57-68 -HLA-DQA1*0501/HLA-DQB1*0201, aGlia _62-72 -HLA-DQA1*0501/HLA-DQB1*0201, aGlia _217-229 -HLA-DQA1*0501/HLA-DQB1*0302 or aGlia _217-229 -HLA.DQA1*03/HLA-DQB1*0302;

d) primary biliary cirrhosis, and the rMHC complex is selected from the group: PDC-E2 _122-135 -HLA-DRB4*0101/DRA, PDC-E2 _249-262 -HLA-DRB4*0101/DRA, PDC-E2 _249-263 - HLA-DRB1*0801/DRA, PDC-E2 _629-643 -HLA-DRB1*0801/DRA, PDC-E2 _72-86 -HLA-DRB3*0202/DRA, PDC-E2 _353-367 -HLA-DRB3*0202 /DRA, PDC-E2 _422-436 -HLA-DRB3*0202/DRA, PDC-E2 _629-643 -HLA-DRB4*0101/DRA, PDC-E2 _80-94 -HLA-DRB5*0101/DRA, PDC- E2 _353-367 -HLA-DRB5*0101/DRA or PDC-E2 _535-549 -HLA-DRB5*0101/DRA, mPDC-E2 _166-181 -IA _g7 or mPDC-E2 _82-96 -IA _g7 ;

e) pemphigus foliaceus and/or pemphigus vulgaris, and the rMHC complex is selected from the group: _{DG1 216-229} -HLA-DRB 1*0101/DRA, _{DG1 216-229} -HLA-DRB1*0102/DRA, DG3 _97-111 -HLA -DRB1*0402/DRA, DG3 _251-265 -HLA-DRB1*0402/DRA, DG3 _251-265 -HLA-DRB1*0401/DRA, DG3 _441-455 -HLA-DRB1*0402/DRA, DG3 _351-365 -HLA-DRB3*0202/DRA, DG3 _453-467 -HLA-DRB3*0202/DRA, DG3 _540-554 -HLA-DRB3*0202/DRA, DG3 _280-294 -HLA-DRB4*0101/DRA, DG3 _{326 -340} -HLA-DRB4*0101/DRA, DG3 _367-381 -HLA-DRB4*0101/DRA, DG3 _13-27 -HLA-DRB5*0101/DRA, DG3 _323-337 -HLA-DRB5*0101/DRA, DG3 _438-452 -HLA-DRB5*0101/DRA, _{DG1 48-62} -HLA-DRB3*0202/DRA, _{DG1 206-222} -HLA-DRB3*0202/DRA, _{DG1 363-377} -HLA-DRB3*0202/ DRA, DG1 _3-17 -HLA-DRB4*0101/DRA, DG1 _192-206 -HLA-DRB4*0101/DRA, _{DG1 326-340} -HLA-DRB4*0101/DRA, DG1 _1-15 -HLA-DRB5* 0101/DRA, DG1 _35-49 -HLA-DRB5*0101/DRA or _{DG1 325-339} -HLA-DRB5*0101/DRA;

f) neuromyelitis of the optic nerve with spectral disorder, and the rMHC complex is selected from the group: AQP4 _129-143 -HLA-DRB1*0101/DRA, AQP4 _284-298 -HLA-DRB1*0301/DRA, AQP4 _63-76 -HLA-DRB1 *0301/DRA, AQP4 _129-143 -HLA-DRB1*0401/DRA or AQP4 _39-53 -HLA-DRB1*1501/DRA;

g) allergic asthma, and the rMHC complex is selected from the group: DERP-1 _16-30 -HLA-DRB1*0101/DRA, DERP-1 _16-30 -HLA-DRB1*1501/DRA, DERP _1171-185 -HLA-DRB1 *1501/DRA, DERP-1 _110-124 -HLA-DPB1*0401/DRA, DERP-2 _26-40 -HLA-DRB1*0101/DRA; DERP-2 _26-40 -HLA-DRB1*1501/DRA or DERP-2 _107-121 -HLA-DRB1*0301/DRA;

h) inflammatory bowel disease, and the rMHC complex is selected from the group: bacteroid integrase antigen _183-197 -HLA-DRB3*0101/DRA, bacteroid integrase antigen _146-160 -HLA-DRB3*0101/DRA, bacteroid integrase antigen _175-189 - HLA-DRB3*0101/DRA, bacteroid integrase antigen _1-15 -HLA-DRB5*0101/DRA, bacteroid integrase antigen _183-197 -HLA-DRB5*0101/DRA, bacteroid integrase antigen _183-197 - HLA-DRB3*0101 /DRA, bacteroid integrase antigen _30-44 -HLA-DRB5*0101/DRA, bacteroid integrase antigen _70-84 -HLA-DRB4*0101/DRA, bacteroid integrase antigen _337-351 -HLA-DRB4*0101/DRA, integrase antigen bacteroid _171-185 -HLA-DRB4*0101/DRA, bacteroid integrase antigen _4-18 -HLA-DRB3*0202/DRA, bacteroid integrase antigen _171-185 -HLA-DRB3*0202/DRA, bacteroid integrase antigen _256-270 - HLA-DRB3*0202/DRA, Fla-2/Fla-X _366-380 -HLA-DRB3*0101/DRA, Fla-2/Fla-X _164-178 -HLA-DRB3*0101/DRA, Fla-2/ Fla-X _261-275 -HLA-DRB5*0101/DRA, Fla-2/Fla-X _1-15 -HLA-DRB5*0101/DRA, Fla-2/Fla-X _51-65 -HLA -DRB4*0101/DRA, Fla-2/Fla-X _269-283 -HLA-DRB4*0101/DRA, Fla-2/Fla-X _4-18 -HLA-DRB3*0202/DRA, Fla-2/Fla -X _261-275 -HLA-DRB3*0202/DRA, Fla-2/Fla-X _271-285 -HLA-DRB3*0202/DRA, YIDX _78-92 -HLA-DRB3*0101/DRA, YIDX _78-92 -HLA-DRB4*0101/DRA, YIDX _93-107 -HLA-DRB3*0101/DRA, YIDX _98-112 -HLA-DRB5*0101/DRA, YIDX _23-37 -HLA-DRB5*0101/DRA, YIDX _{78 -92} -HLA-DRB4*0101/DRA, YIDX _195-209 -HLA-DRB4*0101/DRA, YIDX _22-36 -HLA-DRB3*0202/DRA, YIDX _80-94 -HLA-DRB3*0202/DRA or YIDX _101-115 -HLA-DRB3*0202/DRA;

i) COPD and/or emphysema and rMHC complex selected from the group: elastin _89-103 -HLA-DRB3*0101/DRA, elastin _698-712 -HLA-DRB5*0101/DRA, elastin _8-22 -HLA-DRB5* 0101/DRA, elastin _94-108 -HLA-DRB5*0101/DRA, elastin _13-27 -HLA-DRB4*0101/DRA, elastin _695-709 -HLA-DRB4*0101/DRA, elastin _563-577 -HLA- DRB4*0101/DRA, elastin _558-572 -HLA-DRB4*0101/DRA, elastin _698-712 -HLA-DRB5*0101/DRA, elastin _566-580 -HLA-DRB3*0202/DRA or elastin _645-659 - HLA-DRB3*0202/DRA;

j) psoriasis, and the rMHC complex is selected from the group: Cap18 _64-78 -HLA-DRB3*0101/DRA, Cap18 _34-48 -HLA-DRB3*0101/DRA, Cap18 _47-61 -HLA-DRB3*0101/DRA, Cap18 _151-165 -HLA-DRB4*0101/DRA, Cap18 _149-163 -HLA-DRB5*0101/DRA, Cap18 _152-166 -HLA-DRB5*0101/DRA, Cap18 _131-145 -HLA-DRB5*0101/ DRA, Cap18 _24-38 -HLA-DRB3*0202/DRA, ADMTSL5 _245-259 -HLA-DRB3*0101/DRA, ADMTSL5 _267-281 -HLA-DRB3*0101/DRA, ADMTSL5 _372-386 -HLA-DRB3* 0101/DRA, ADMTSL5 _289-303 -HLA-DRB4*0101/DRA, ADMTSL5 _396-410 -HLA-DRB4*0101/DRA, ADMTSL5 _433-447 -HLA-DRB4*0101/DRA, ADMTSL5 _142-156 -HLA- DRB5*0101/DRA, ADMTSL5 _236-250 -HLA-DRB5*0101/DRA, ADMTSL5 _301-315 -HLA-DRB5*0101/DRA, ADMTSL5 _203-217 -HLA-DRB3*0202/DRA, ADMTSL5 _404-418 - HLA-DRB3*0202/DRA or ADMTSL5 _433-447 -HLA-DRB3*0202/DRA;

k) autoimmune hepatitis, and the rMHC complex is selected from the group: CYP2D6 _193-207 -HLA-DRB1*0301/DRA, CYP2D6 _76-90 -HLA-DRB1*0301/DRA, CYP2D6 _293-307 -HLA-DRB1*0301/DRA , CYP2D6 _313-332 -HLA-DRB1*0301/DRA, CYP2D6 _393-412 -HLA-DRB1*0301/DRA, CYP2D6 _199-213 -HLA-DRB1*0401/DRA, CYP2D6 _450-464 -HLA-DRB1*0401 /DRA, CYP2D6 _301-315 -HLA-DRB1*0401/DRA, CYP2D6 _452-466 -HLA-DRB1*0701/DRA, CYP2D6 _59-73 -HLA-DRB1*0701/DRA, CYP2D6 _130-144 -HLA-DRB1 *0701/DRA, CYP2D6 _193-212 -HLA-DRB1*0701/DRA, CYP2D6 _305-324 -HLA-DRB1*0701/DRA, CYP2D6 _131-145 -HLA-DRB3*0202/DRA, CYP2D6 _216-230 -HLA -DRB3*0202/DRA, CYP2D6 _238-252 -HLA-DRB3*0202/DRA, CYP2D6 _199-213 -HLA-DRB4*0101/DRA, CYP2D6 _235-252 -HLA-DRB4*0101/DRA, CYP2D6 _293-307 -HLA-DRB4*0101/DRA, CYP2D6 _238-252 -HLA-DRB5*0101/DRA, CYP2D6 _381-395 -HLA-DRB5*0101/DRA, CYP2D6 _429-443 -HLA-DRB5*0101/DRA, SLA _{334 -348} -HLA-DRB1*0301/DRA, SLA _196-210 -HLA-DRB1*0301/DRA, SLA _115-129 -HLA-DRB1*0301/DRA, SLA _373-386 -HLA-DRB1*0301/DRA, SLA _186-197 -HLA-DRB1*0301/DRA, SLA _317-331 -HLA-DRB1*0401/DRA, SLA _171-185 -HLA-DRB1*0401/DRA, SLA _417-431 -HLA-DRB1*0401/DRA, SLA _359-373 -HLA-DRB1*0701/DRA, SLA _{215 -229} -HLA-DRB1*0701/DRA, SLA _111-125 -HLA-DRB1*0701/DRA, SLA _110-124 -HLA-DRB3*0202/DRA, SLA _299-313 -HLA-DRB3*0202/DRA, SLA _342-356 -HLA-DRB3*0202/DRA, SLA _49-63 -HLA-DRB4*0101/DRA, SLA _119-133 -HLA-DRB4*0101/DRA, SLA _260-274 -HLA-DRB4*0101/ DRA, SLA _26-40 -HLA-DRB5*0101/DRA, SLA _86-100 -HLA-DRB5*0101/DRA or SLA _331-345 -HLA-DRB5*0101/DRA;

l) uveitis, and the rMHC complex is selected from the group: arrestin _199-213 -HLA-DRB3*0101/DRA, arrestin _77-91 -HLA-DRB3*0101/DRA, arrestin _250-264 -HLA-DRB3*0101/DRA, arrestin _172-186 -HLA-DRB4*0101/DRA, arrestin _354-368 -HLA-DRB4*0101/DRA, arrestin _239-253 -HLA-DRB4*0101/DRA, arrestin _102-116 -HLA-DRB5*0101/ DRA, arrestin _59-73 -HLA-DRB5*0101, arrestin _280-294 -HLA-DRB5*0101, arrestin _291-306 -HLA-DRB1*0301/DRA, arrestin _195-209 -HLA-DRB3*0202/DRA, arrestin _199-213 -HLA-DRB3*0202/DRA or arrestin _200-214 -HLA-DRB3*0202/DRA;

m) Sjögren's syndrome, and the rMHC complex is selected from the group: RO60 _127-141 -HLA-DRB1*0301/DRA, RO60 _523-537 -HLA-DRB1*0301/DRA, RO60 _243-257 -HLA-DRB1*0301/DRA RO60 _484-498 -HLA-DRB3*0101/DRA, RO60 _347-361 -HLA-DRB3*0101/DRA, RO60 _{369-383 -HLA} -DRB3*0101/DRA /DRA, RO60 _267-281 -HLA-DRB4*0101/DRA, RO60 _178-192 -HLA-DRB4*0101/DRA, RO60 _358-372 -HLA-DRB5*0101/DRA, RO60 _358-372 -HLA-DRB4 *0101/DRA, RO60 _221-235 -HLA-DRB5*0101/DRA, RO60 _221-235 -HLA-DRB4*0101/DRA, RO60 _318-332 -HLA-DRB5*0101/DRA, RO60 _318-332 -HLA -DRB4*0101/DRA, RO60 _407-421 -HLA-DRB4*0101/DRA, RO60 _407-421 -HLA-DQA1*0501/HLA-DQB1*0201, RO60 _459-473 -HLA-DRB4*0101/DRA, RO60 _459-473 -HLA-DQA1*0501/HLA-DQB1*0201, RO60 _318-332 -HLA-DQA1*0501/HLA-DQB1*0201, RO60 _51-65 -HLA-DRB3*0202/DRA, RO60 _{312- 326} -HLA-DRB3*0202/DRA, RO60 _347-361 -HLA-DRB3*0202/DRA, LA _241-255 -HLA-DRB1*0301/DRA, LA _101-115 -HLA-DRB1*0301/DRA, LA _153-167 -HLA-DRB1*0301/DRA, LA _178-192 -HLA-DRB3*0101/DRA, LA _19-33 -HLA-DRB3*0101/DRA, LA _{3 7-51} -HLA-DRB3*0101/DRA, LA _133-147 -HLA-DRB4*0101/DRA, LA _50-64 -HLA-DRB4*0101/DRA, LA _32-46 -HLA-DRB4*0101/DRA , LA _153-167 -HLA-DRB5*0101/DRA, LA _83-97 -HLA-DRB5*0101/DRA, LA _136-150 -HLA-DRB5*0101/DRA, LA _297-311 -HLA-DQA1*0501 /HLA-DQB1*0201, LA _59-73 -HLA-DQA1*0501/HLA-DQB1*0201, LA _59-73 -HLA-DRB4*0101/DRA, LA _151-165 -HLA-DQA1*0501/HLA- DQB1*0201, LA _151-165 -HLA-DRB4*0101/DRA, LA _297-311 -HLA-DRB4*0101/DRA, LA _50-64 -HLA-DRB3*0202/DRA, LA _86-100 -HLA- DRB3*0202/DRA or LA _154-168 -HLA-DRB3*0202/DRA;

n) scleroderma, and the rMHC complex is selected from the group: TOP1 _346-360 -HLA-DRB3*0101/DRA, TOP1 _420-434 -HLA-DRB3*0101/DRA, TOP1 _750-764 -HLA-DRB3*0101/DRA, TOP1 _419-433 -HLA-DRB4*0101/DRA, TOP1 _591-605 -HLA-DRB4*0101/DRA, TOP1 _695-709 -HLA-DRB4*0101/DRA, TOP1 _305-319 -HLA-DRB5*0101/ DRA, TOP1 _346-360 -HLA-DRB5*0101/DRA, TOP1 _419-433 -HLA-DRB5*0101/DRA, TOP1 _420-434 -HLA-DRB3*0202/DRA, TOP1 _425-439 -HLA-DRB3* 0202/DRA, TOP1 _614-628 -HLA-DRB3*0202/DRA, CENP-C _297-311 -HLA-DRB3*0101/DRA, CENP-C _857-871 -HLA-DRB3*0101/DRA, CENP-C _887-901 -HLA-DRB3*0101/DRA, CENP-C _212-226 -HLA-DRB4*0101/DRA, CENP-C _643-657 -HLA-DRB4*0101/DRA, CENP-C _832-846 -HLA -DRB4*0101/DRA, CENP-C _167-181 -HLA-DRB5*0101/DRA, CENP-C _246-260 -HLA-DRB5*0101/DRA, CENP-C _846-860 -HLA-DRB5*0101/ DRA, CENP-C _149-163 -HLA-DRB3*0202/DRA, CENP-C _833-847 -HLA-DRB3*0202/DRA or CENP-C _847-861 -HLA-DRB3*0202/DRA;

o) antiphospholipid syndrome, and the rMHC complex is selected from the group: ARON _235-249 -HLA-DRB3*0101/DRA, APOH _306-320 -HLA-DRB3*0101/DRA, APOH _237-251 -HLA-DRB3*0101/DRA _{APOH 295-309 -HLA} - _DRB3 *0101/DRA /DRA, APOH _295-309 -HLA-DRB4*0101/DRA, APOH _49-63 -HLA-DRB5*0101/DRA, APOH _269-283 -HLA-DRB5*0101/DRA, APOH _295-309 -HLA-DRB5 *0101/DRA, APOH _321-355 -HLA-DRB3*0202/DRA, APOH _322-336 -HLA-DRB3*0202/DRA or APOH _324-338 -HLA-DRB3*0202/DRA;

p) ANCA-associated vasculitis and the rMHC complex is selected from the group: MPO _506-520 -HLA-DRB3*0101/DRA, MPO _302-316 -HLA-DRB3*0101/DRA, MPO _7-21 -HLA-DRB3*0101 /DRA, MPO _689-703 -HLA-DRB4*0101/DRA, MPO _248-262 -HLA-DRB4*0101/DRA, MPO _444-458 -HLA-DRB4*0101/DRA, MPO _513-527 -HLA-DRB5 *0101/DRA, MPO _97-111 -HLA-DRB5*0101/DRA, MPO _616-630 -HLA-DRB5*0101/DRA, MPO _462-476 -HLA-DRB3*0202/DRA, MPO _617-631 -HLA -DRB3*0202/DRA, MPO _714-728 -HLA-DRB3*0202/DRA, PRTN3 _44-58 -HLA-DRB3*0101/DRA, PRTN3 _234-248 -HLA-DRB3*0101/DRA, PRTN3 _59-73 -HLA-DRB3*-0101/DRA, PRTN3 _59-73 -HLA-DRB5*0101/DRA, PRTN3 _117-131 -HLA-DRB4*0101/DRA, PRTN3 _164-178 - HLA-DRB4*0101/DRA, PRTN3 _71-85 -HLA-DRB4*0101/DRA, PRTN3 _241-255 -HLA-DRB5*0101/DRA, PRTN3 _183-197 -HLA-DRB5*0101/DRA, PRTN3 _62-76 -HLA-DRB3*0202/DRA , PRTN3 _118-132 -HLA-DRB3*0202/DRA or PRTN3 _239-253 -HLA-DRB3*0202/DRA; or

q) stiffness syndrome, and the rMHC complex is selected from the group: GAD _212-226 -HLA-DRB1*0801/DRA, GAD _555-569 -HLA-DRB1*0801/DRA or GAD _297-311 -HLA-DRB1*0301/ DRA.

In accordance with certain aspects, the rMHC complex is intended for the treatment of:

a) type I diabetes, and the rMHC complex is selected from the group

PPI _76-90(K88S) -HLA-DRB1*0401/DRA, IGRP _13-25 -HLA.DRB1*0301/DRA, GAD _555-567 -HLA-DRB1*0401/DRA, GAD _{555-567(557I)} - HLA-DRB1*0401/DRA, IGRP _23-35 -HLA-DRB1*0401/DRA or PPI _76-90 -HLA-DRB1*0401/DRA;

b) multiple sclerosis, and the rMHC complex is selected from the group: MBP _86-98 -HLA-DRB1*1501/DRA, MBP _89-101 -HLA-DRB5*0101/DRA, MOG _38-52 -HLA-DRB4*0101/DRA , MOG _{97-109(E107S)} -HLA-DRB1*0401/DRA, MOG _203-217 -HLA-DRB3*0101/DRA, PLP _54-68 -HLA-DRB3*0101/DRA, PLP _94-108 -HLA- DRB1*0301/DRA, PLP _250-264 -HLA-DRB4*0101/DRA, MPB _13-32 -HLA-DRB5*0101/DRA, MPB _83-99 -HLA-DRB5*0101/DRA, MPB _111-129 - HLA-DRB5*0101/DRA, MPB _146-170 -HLA-DRB5*0101/DRA, MOG _223-237 -HLA-DRB3*0202/DRA, MOG _6-20 -HLA-DRB5*0101/DRA, PLP _{88- 102} -HLA-DRB3*0202/DRA or PLP _139-154 -HLA-DRB5*0101/DRA;

c) celiac disease, and the rMHC complex is selected from the group: aGlia _57-68 -HLA-DQA1*0501/HLA-DQB1*0201, aGlia _62-72 -HLA-DQA1*0501/HLA-DQB1*0201 or aGlia _217-229 -HLA-DQA1*0501/HLA-DQB1*0302;

d) primary biliary cirrhosis, and the rMHC complex is selected from the group: PDC-E2 _122-135 -HLA-DRB4*0101/DRA, PDC-E2 _249-262 -HLA-DRB4*0101/DRA, PDC-E2 _249-263 - HLA-DRB1*0801/DRA, PDC-E2 _629-643 -HLA-DRB1*0801/DRA, PDC-E2 _72-86 -HLA-DRB3*0202/DRA, PDC-E2 _353-367 -HLA-DRB3*0202 /DRA, PDC-E2 _422-436 -HLA-DRB3*0202/DRA, PDC-E2 _629-643 -HLA-DRB4*0101/DRA, PDC-E2 _80-94 -HLA-DRB5*0101/DRA, PDC- E2 _353-367 -HLA-DRB5*0101/DRA or PDC-E2 _535-549 -HLA-DRB5*0101/DRA;

e) pemphigus foliaceus and/or pemphigus vulgaris, and the rMHC complex is selected from the group: _{DG1 216-229} -HLA-DRB1*0101/DRA, DG3 _97-111 -HLA-DRB1*0402/DRA, DG3 _251-265 -HLA- DRB1*0401/DRA, DG3 _441-455 -HLA-DRB1*0402/DRA, DG3 _351-365 -HLA-DRB3*0202/DRA, DG3 _453-467 -HLA-DRB3*0202/DRA, DG3 _540-554 - HLA-DRB3*0202/DRA, DG3 _280-294 -HLA-DRB4*0101/DRA, DG3 _326-340 -HLA-DRB4*0101/DRA, DG3 _367-381 -HLA-DRB4*0101/DRA, DG3 _{13- 27} -HLA-DRB5*0101/DRA, DG3 _323-337 -HLA-DRB5*0101/DRA, DG3 _438-452 -HLA-DRB5*0101/DRA, _{DG1 48-62} -HLA-DRB3*0202/DRA, DG1 _206-222 -HLA-DRB3*0202/DRA, _{DG1 363-377} -HLA-DRB3*0202/DRA, DG1 _3-17 -HLA-DRB4*0101/DRA, DG1 _192-206 -HLA-DRB4*0101/DRA , _{DG1 326-340} -HLA-DRB4*0101/DRA, DG1 _1-15 -HLA-DRB5*0101/DRA, DG1 _35-49 -HLA-DRB5*0101/DRA or _{DG1 325-339} -HLA-DRB5*0101 /DRA;

f) neuromyelitis of the optic nerve with spectral disorder, and the rMHC complex is selected from the group: AQP4 _284-298 -HLA-DRB1*0301/DRA, AQP4 _63-76 -HLA-DRB1*0301/DRA, AQP4 _129-143 -HLA-DRB1 *0401/DRA or AQP4 _39-53 -HLA-DRB14501/DRA;

g) allergic asthma, and the rMHC complex is selected from the group: DERP-1 _16-30 -HLA-DRB1*0101/DRA, DERP-1 _16-30 -HLA-DRB1*1501/DRA, DERP _1171-185 -HLA-DRB1 *1501/DRA, DERP-1 _110-124 -HLA-DPB1*0401/DRA, DERP-2 _26-40 -HLA-DRB1*0101/DRA; DERP-2 _26-40 -HLA-DRB1*1501/DRA or DERP-2 _107-121 -HLA-DRB1*0301/DRA;

h) inflammatory bowel disease, and the rMHC complex is selected from the group: bacteroid integrase antigen _1-15 -HLA-DRB5*0101/DRA, bacteroid integrase antigen _183-197 -HLA-DRB3*0101/DRA, bacteroid integrase antigen _70-84 - HLA-DRB4*0101/DRA, bacteroid integrase antigen _4-18 -HLA-DRB3*0202/DRA, bacteroid integrase antigen _171-185 -HLA-DRB3*0202/VKA, bacteroid integrase antigen _256-270 -HLA-DRB3*0202 /DRA, Fla-2/Fla-X _366-380 -HLA-DRB3*0101/DRA, Fla-2/Fla-X _261-275 -HLA-DRB3*0101/DRA, Fla-2/Fla-X _{51- 65} -HLA-DRB4*0101/DRA, Fla-2/Fla-X _4-18 -HLA-DRB3*0202/DRA, Fla-2/Fla-X _26l-275 -HLA-DRB5*0202/DRA, Fla- 2/Fla-X _271-285 -HLA-DRB3*0202/DRA, YIDX _78-92 -HLA-DRB3*0101/DRA, YIDX _78-92 -HLA-DRB4*0101/DRA, YIDX _98-112 -HLA- DRB5*0101/DRA, YIDX _22-36 -HLA-DRB3*0202/DRA, YIDX _80-94 -HLA-DRB3*0202/DRA or YIDX _101-115 -HLA-DRB3*0202/DRA;

i) emphysema, and the rMHC complex is selected from the group: elastin _89-103 -HLA-DRB3*0101/DRA, elastin _698-712 -HLA-DRB5*0101/DRA, elastin _558-572 -HLA-DRB4*0101/DRA, elastin _566-580 -HLA-DRB3*0202/DRA or elastin _645-659 -HLA-DRB3*0202/DRA;

j) psoriasis, and the rMHC complex is selected from the group: Cap18 _64-78 -HLA-DRB3*0101/DRA, Cap 18 _34-48 -HLA-DRB3*0101/DRA, Cap18 _47-61 -HLA-DRB3*0101/DRA , Cap18 _151-165 -HLA-DRB4*0101/DRA, Cap18 _149-163 -HLA-DRB5*0101/DRA, Cap18 _152-166 -HLA-DRB5*0101/DRA, Cap18 _131-145 -HLA-DRB5*0101 /DRA, Cap18 _24-38 -HLA-DRB3*0202/DRA, ADMTSL5 _245-259 -HLA-DRB3*0101/DRA, ADMTSL5 _267-281 -HLA-DRB3*0101/DRA, ADMTSL5 _372-386 -HLA-DRB3 *0101/DRA, ADMTSL5 _289-303 -HLA-DRB4*0101/DRA, ADMTSL5 _396-410 -HLA-DRB4*0101/DRA, ADMTSL5 _433-447 -HLA-DRB4*0101/DRA, ADMTSL5 _142-156 -HLA -DRB5*0101/DRA, ADMTSL5 _236-250 -HLA-DRB5*0101/DRA, ADMTSL5 _301-315 -HLA-DRB5*0101/DRA, ADMTSL5 _203-217 -HLA-DRB3*0202/DRA, ADMTSL5 _404-418 -HLA-DRB3*0202/DRA or ADMTSL5 _433-447 -HLA-DRB3*0202/DRA;

k) autoimmune hepatitis, and the rMHC complex is selected from the group: CYP2D6 _193-207 -HLA-DRB1*0301/DRA, CYP2D6 _76-90 -HLA-DRB1*0301/DRA, CYP2D6 _293-307 -HLA-DRB1*0301/DRA , CYP2D6 _313-332 -HLA-DRB1*0301/DRA, CYP2D6 _393-412 -HLA-DRB1*0301/DRA, CYP2D6 _199-213 -HLA-DRB1*0401/DRA, CYP2D6 _450-464 -HLA-DRB1*0401 /DRA, CYP2D6 _301-315 -HLA-DRB1*0401/DRA, CYP2D6 _452-466 -HLA-DRB1*0701/DRA, CYP2D6 _59-73 -HLA-DRB1*0701/DRA, CYP2D6 _130-144 -HLA-DRB1 *0701/DRA, CYP2D6 _193-212 -HLA-DRB1*0701/DRA, CYP2D6 _305-324 -HLA-DRB1*0701/DRA, CYP2D6 _131-145 -HLA-DRB3*0202/DRA, CYP2D6 _216-230 -HLA -DRB3*0202/DRA, CYP2D6 _238-252 -HLA-DRB3*0202/DRA, CYP2D6 _199-213 -HLA-DRB4*0101/DRA, CYP2D6 _235-252 -HLA-DRB4*0101/DRA, CYP2D6 _293-307 -HLA-DRB4*0101/DRA, CYP2D6 _238-252 -HLA-DRB5*0101/DRA, CYP2D6 _381-395 -HLA-DRB5*0101/DRA, CYP2D6 _429-443 -HLA-DRB5*0101/DRA, SLA _{334 -348} -HLA-DRB1*0301/DRA, SLA _196-210 -HLA-DRB1*0301/DRA, SLA _115-129 -HLA-DRB1*0301/DRA, SLA _373-386 -HLA-DRB1*0301/DRA, SLA _186-197 -HLA-DRB1*0301/DRA, SLA _317-331 - HLA-DRB1*0401/DRA, SLA _171-185 -HLA-DRB1*0401/DRA, SLA _417-431 -HLA-DRB1*0401/DRA, SLA _359-373 -HLA-DRB1*0701/DRA, SLA _{215- 229} -HLA-DRB1*0701/DRA, SLA _111-125 -HLA-DRB1*0701/DRA, SLA _110-124 -HLA-DRB3*0202/DRA, SLA _299-313 -HLA-DRB3*0202/DRA, SLA _342-356 -HLA-DRB3*0202/DRA, SLA _49-63 -HLA-DRB4*0101/DRA, SLA _119-133 -HLA-DRB4*0101/DRA, SLA _260-274 -HLA-DRB4*0101/DRA , SLA _26-40 -HLA-DRB5*0101/DRA, SLA _86-100 -HLA-DRB5*0101/DRA or SLA _331-345 -HLA-DRB5*0101/DRA;

l) uveitis, and the rMHC complex is selected from the group: arrestin _199-213 -HLA-DRB3*0101/DRA, arrestin _77-91 -HLA-DRB3*0101/DRA, arrestin _250-264 -HLA-DRB3*0101/DRA, arrestin _172-186 -HLA-DRB4*0101/DRA, arrestin _354-368 -HLA-DRB4*0101/DRA, arrestin _239-253 -HLA-DRB4*0101/DRA, arrestin _102-116 -HLA-DRB5*0101/ DRA, arrestin _59-73 -HLA-DRB5*0101, arrestin _280-294 -HLA-DRB5*0101, arrestin _291-306 -HLA-DRB1*0301/DRA, arrestin _195-209 -HLA-DRB3*0202/DRA, arrestin _199-213 -HLA-DRB3*0202/DRA or arrestin _200-214 -HLA-DRB3*0202/DRA;

m) Sjögren's syndrome, and the rMHC complex is selected from the group: RO60 _127-141 -HLA-DRB1*0301/DRA, RO60 _523-537 -HLA-DRB1*0301/DRA, RO60 _243-257 -HLA-DRB1*0301/DRA RO60 _484-498 -HLA-DRB3*0101/DRA, RO60 _347-361 -HLA-DRB3*0101/DRA, RO60 _{369-383 -HLA} -DRB3*0101/DRA /DRA, RO60 _267-281 -HLA-DRB4*0101/DRA, RO60 _178-192 -HLA-DRB4*0101/DRA, RO60 _358-372 -HLA-DRB5*0101/DRA, RO60 _221-235 -HLA-DRB5 *0101/DRA, RO60 _318-332 -HLA-DRB5*0101/DRA, RO60 _51-65 -HLA-DRB3*0202/DRA, RO60 _312-326 -HLA-DRB3*0202/DRA, RO60 _347-361 -HLA -DRB3*0202/DRA, LA _241-255 -HLA-DRB1*0301/DRA, LA _101-115 -HLA-DRB1*0301/DRA, LA _153-167 -HLA-DRB1*0301/DRA, LA _178-192 -HLA-DRB3*0101/DRA, LA _19-33 -HLA-DRB3*0101/DRA, LA _37-51 -HLA-DRB3*0101/DRA, LA _133-147 -HLA-DRB4*0101/DRA, LA _{50 -64} -HLA-DRB4*0101/DRA, LA _32-46 -HLA-DRB4*0101/DRA, LA _153-167 -HLA-DRB5*0101/DRA, LA _83-97 -HLA-DRB5*0101/DRA, LA _136-150 -HLA-DRB5*0101/DRA, LA _50-64 -HLA-DRB3*0202/DRA, LA _86-100 -HLA-DRB3*0202/DRA or LA _154-168 -HLA-DRB3*0202/ DR A;

n) scleroderma, and the pMHC complex is selected from the group: TOP1 _346-360 -HLA-DRB3*0101/DRA, TOP1 _420-434 -HLA-DRB3*0101/DRA, TOP1 _750-764 -HLA-DRB3*0101/DRA, TOP1 _419-433 -HLA-DRB4*0101/DRA, TOP1 _591-605 -HLA-DRB4*0101/DRA, TOP1 _695-709 -HLA-DRB4*0101/DRA, TOP1 _305-319 -HLA-DRB5*0101/ DRA, TOP1 _346-360 -HLA-DRB5*0101/DRA, TOP1 _419-433 -HLA-DRB5*0101/DRA, TOP1 _420-434 -HLA-DRB3*0202/DRA, TOP1 _425-439 -HLA-DRB3* 0202/DRA, TOP1 _614-628 -HLA-DRB3*0202/DRA, CENP-C _297-311 -HLA-DRB3*0101/DRA, CENP-C _857-871 -HLA-DRB3*0101, CENP-C _{887- 901} -HLA-DRB3*0101, CENP-C _212-226 -HLA-DRB4*0101/DRA, CENP-C _643-657 -HLA-DRB4*0101/DRA, CENP-C _832-846 -HLA-DRB4*0101 /DRA, CENP-C _167-181 -HLA-DRB5*0101/DRA, CENP-C _246-260 -HLA-DRB5*0101/DRA, CENP-C _846-860 -HLA-DRB5*0101/DRA, CENP- C _149-163 -HLA-DRB3*0202/DRA, CENP-C _833-847 -HLA-DRB3*0202/DRA or CENP-C _847-861 -HLA-DRB3*0202/DRA;

o) antiphospholipid syndrome, and the pMHC complex is selected from the group: ARON _235-249 -HLA-DRB3*0101/DRA, APOH _306-320 -HLA-DRB3*0101/DRA, APOH _237-251 -HLA-DRB3*0101/DRA _{APOH 295-309 -HLA} - _DRB3 *0101/DRA /DRA, APOH _295-309 -HLA-DRB4*0101/DRA, APOH _49-63 -HLA-DRB5*0101/DRA, APOH _269-283 -HLA-DRB5*0101/DRA, APOH _295-309 -HLA-DRB5 *0101/DRA, APOH _321-355 -HLA-DRB3*0202/DRA, APOH _322-336 -HLA-DRB3*0202/DRA or APOH _324-338 -HLA-DRB3*0202/DRA;

p) ANCA-associated vasculitis, and the pMHC complex is selected from the group: MPO _506-520 -HLA-DRB3*0101/DRA, MPO _302-316 -HLA-DRB3*0101/DRA, MPO _7-21 -HLA-DRB3*0101 /DRA, MPO _689-703 -HLA-DRB4*0101/DRA, MPO _248-262 -HLA-DRB4*0101/DRA, MPO _444-458 -HLA-DRB4*0101/DRA, MPO _513-527 -HLA-DRB5 *0101/DRA, MPO _97-111 -HLA-DRB5*0101/DRA, MPO _616-630 -HLA-DRB5*0101/DRA, MPO _462-476 -HLA-DRB3*0202/DRA, MPO _617-631 -HLA -DRB3*0202/DRA, MPO _714-728 -HLA-DRB3*0202/DRA, PRTN3 _44-58 -HLA-DRB3*0101/DRA, PRTN3 _234-248 -HLA-DRB3*0101/DRA, PRTN3 _59-73 -HLADRB3*0101/DRA, PRTN3 _59-73 -HLA-DRB5*0101/DRA, PRTN3 _117-131 -HLA-DRB4*0101/DRA, PRTN3 _164-178 -HLA-DRB4*0101/DRA, PRTN3 _71-85 -HLA-DRB4*0101/DRA, PRTN3 _241-255 -HLA-DRB5*0101/DRA, PRTN3 _183-197 -HLA-DRB5*0101/DRA, PRTN3 _62-76 -HLA-DRB3*0202/DRA, PRTN3 _{118 -132} -HLA-DRB3*0202/DRA or PRTN3 _239-253 -HLA-DRB3*0202/DRA; or

q) stiffness syndrome, and the rMHC complex is selected from the group: GAD _212-226 -HLA-DRB1*0801/DRA, GAD _555-569 -HLA-DRB1*0801/DRA or GAD _297-311 -HLA-DRB1*0301/ DRA.

It is also possible to optimize the choice of co-stimulatory molecules or molecules to bind to the pMHC/NP complex in a similar manner, and this mainly depends on the nature of the immunocyte population that is to be differentiated or expanded. For example, if the goal is to expand and differentiate regulatory T cell populations, appropriate combinations may include, but are not limited to, co-stimulatory molecules and cytokines such as IL15-IL15Ra, IL-2, IL-10, IL-35, ICOS-L, complex IL2/mAb to IL2, TGF-beta, IL-21, ITE or ICOSL. In contrast, in accordance with certain embodiments, such as in certain types of cancers, the expansion and/or differentiation of T cells with a regulatory phenotype may not be a required response. Thus, alternative costimulatory molecules and cytokines can be optimized for a particular treatment.

Methods for obtaining nanoparticles and complexes

MHC and nanoparticles can be obtained in a number of ways. The following is purely illustrative.

WPC

Two illustrative methods are provided for preparing class I MHC complexes. The first involves the refolding of MHC class I heavy and light chains that are expressed in bacteria in the presence of the peptide, followed by purification by gel filtration and anion exchange chromatography as described in the literature (Garboczi, DN et al. (1992) Proc Natl. Acad Sci USA 89:3429-3433 Altman, JD et al (1996) Science 274:94-96). The second involves the expression of MHC class I complexes at high levels in lentivirus-transduced CHO FreeStyle cells as single-stranded constructs in which the peptide coding sequence, MHC class I light and heavy chains are sequentially linked by flexible GS linkers (Yu, YY et al. (2002) J Immunol. 168:3145-3149) followed by a C-terminal linker encoding a BirA region, a 6×His tag ending in free Cys. Secreted proteins are isolated from culture supernatants using nickel columns and anion exchange chromatography and used directly for NP coating or biotinylated to prepare pMHC tetramers using fluorochrome-conjugated streptavidin. Tetramers generated using the appropriate single chain pMHC complexes encoding the IGRP _206-214 autoantigenic peptide or its NRP-V7 mimetic bind efficiently to recognizing monoclonal autoreactive CD8 ⁺ T cells, but not to their polyclonal variants as determined by flow cytometry.

Recombinant class II rMHC monomers can be isolated from Drosophila SC2 cells transfected with constructs encoding I-Aβ and I-Aα chains bearing c-Jun or c-Fos leucine zippers, respectively, and BirA and 6×His tags, as previously described ( Stratmann, T. et al (2000) J. Immunol 165:3214-3225, Stratmann, T. et al (2003) J. Clin Invest 112:3214-3225). Since the yields of this approach are generally low and time consuming, Applicant developed an expression system in FreeStyle CHO cells transduced with lentiviruses encoding a monocistronic transcript in which the peptide chains of the IAβ and IAα complex are separated by the P2A ribosome hopping sequence (Holst, J. et al (2006) Nat Protoc 1:406-417). As with the class I single strand pMHC constructs described above, a linker encoding the BirA region, a 6xHis tag, and free Cys is added to the C-terminus of the construct. Class II self-assembling rMHC complexes are isolated from cell culture supernatants by nickel chromatography followed by anion exchange chromatography and used for loading onto NPs or treated for biotinylation and formation of tetramers as described above. Class II rMHC tetramers produced using the appropriate class II rMHC complex encoding the 2.5mi autoantigenic peptide bind specifically and efficiently to recognizing monoclonal autoreactive CD4 ⁺ T cells as determined by flow cytometry.

PE-conjugated tetramers can be prepared using biotinylated pMHC monomers as described (Stratmann, T. et al. (2000) J. Immunol. 165:3214-3225; Stratmann, T. et al. (2003) J. Clin. Invest 112:3214-3225 Amrani, A. et al (2000) Nature 406:739-742). Peripheral blood mononuclear cells, splenocytes, and CD8 ⁺ or CD4 ⁺ lymph node T cells can be stained with tetramer (5 µg/mL) in FACS buffer (0.1% sodium azide and 1% FBS in PBS) for 1 hour at 4° C, wash and incubate with FITC-conjugated anti-CD8α or anti-CD4 antibody (5 µg/mL) and PerCP-conjugated anti-B220 antibody (2 µg/mL; as a blank window) for 30 min at 4°C. Cells are washed, fixed in 1% PFA/PBS and analyzed by FACS.

NP synthesis

Nanoparticles can be formed by contacting an aqueous phase containing co-stimulatory molecule(s), a pMHC complex and/or a cytokine, and a polymeric and non-aqueous phase, followed by evaporation of the non-aqueous phase, allowing the particles to bind from the aqueous phase, as described in US Pat. Nos. 4,589,330 or 4,818,542 Certain polymers for such preparation are natural or synthetic copolymers or polymers which include gelatin agar, starch, arabinogalactan, albumin, collagen, polyglycolic acid, polylactic acid, glycolide-L(-)-lactide, poly(exilon-caprolactone, poly( epsilon-caprolactone-CO-lactic acid), poly(epsilon-caprolactone-CO-glycolic acid), poly(β-hydroxybutyric acid), poly(ethylene oxide), polyethylene, poly(alkyl-2-cyanoacrylate), poly(hydroxyethyl methacrylate ), polyamides, poly(amino acids), poly(2-hydroxyethyl-DL-aspartamide), poly(urea ester), poly(L-phenylalanine/ethylene glycol/1,6-diisocyanatohexane) and poly(methyl methacrylate). Certain polymers are polyesters such as polyglycolic acid, polylactic acid, glycolide-L(-)-lactide-poly(exilon-caprolactone), poly(epsilon-caprolactone-CO-lactic acid) and poly(epsilon-caprolactone-CO -glycolic acid). Suitable solvents for dissolving the polymer include: water, hexafluoroisopropanol, methylene chloride, tetrahydrofuran, hexane, benzene, or hexafluoroacetone sesquihydrate.

Gold nanoparticles (GNPs) are synthesized using the chemical reduction of gold chloride with sodium citrate as described (Perrault, SD et al. (2009) Nano Lett 9:1909-1915). Briefly, 2 ml of 1% HAuCl ₄ (Sigma Aldrich) is added to 100 ml of H ₂ O with vigorous stirring and the solution is heated in an oil bath. Six (for 14 nm GNP) or two (for 40 nm GNP) ml of 1% Na citrate are added to the boiling HAuCl ₄ solution, stirred for an additional 10 min, and then cooled to room temperature. GNP is stabilized by adding 1 μM thiol-PEG linkers (Nanocs, MA) functionalized with -COOH or -NH ₂ groups as MHC acceptors. PEGylated GNPs are washed with water to remove free thiol-PEG, concentrated and stored in water for further analysis. The NP density is determined by spectrophotometry and calculated according to Beer's law.

It is also possible to obtain NP from iron oxide of the SFP series (IONP SFP) by thermal destruction of iron acetate in organic solvents in the presence of surfactants, then converting the solvent into aqueous buffers through pegylation (Xie, J. et al. (2007) Adv Mater 19 :3163; Xie, J. et al. (2006) Pure Appl Chem. 78:1003-1014; Xu, C. et al. (2007) Polymer International 56:821-826). Briefly, 2 mM Fe(AcAc) ₃ (Sigma Aldrich, Oakville, ON) is dissolved in 10 ml of a mixture of benzyl ether and oleylamine and heated to 100°C for 1 hour followed by 300°C for 2 hours with boiling with a reflux condenser under the protection of a nitrogen blanket. Synthesized NP precipitated by adding ethanol and resuspended in hexane. For IONP pegylation, 100 mg of various 3.5 kDa DPA-PEG linkers (Jenkem Tech USA) are dissolved in a mixture of CHCl ₃ and HCON(CH ₃ ) ₂ (dimethylformamide (DMF)). Then, the NP solution (20 mg Fe) was added to the DPA-PEG solution and stirred for 4 hours at room temperature. PEGylated NP SFPs are precipitated overnight by adding hexane and then resuspended in water. Traces of aggregates are removed by high speed centrifugation (20,000×g, 30 min) and monodisperse NP SFPs are stored in water for further characterization and conjugation with pMHC. The iron concentration in the IONP products is determined by spectrophotometry at A410 in 2H HCL. Based on the molecular structure and diameter of NP SFP (Fe ₃ O ₄ ; 8 ± 1 nm diameter) (Xie, J. et al. (2007) Adv Mater 19:3163; Xie, J. et al. (2006) Pure Appl. Chem. 78:1003-1014), the Applicant calculates that SFP solutions containing 1 mg of iron contain 5×10 ¹⁴ NP.

Nanoparticles can also be produced by thermal destruction or heating of the nanoparticle precursor. In one embodiment, the nanoparticle is a metal or metal oxide nanoparticle. In one embodiment, the nanoparticle is an iron oxide nanoparticle. In one embodiment, the nanoparticle is a gold nanoparticle. In one embodiment, provided herein are nanoparticles produced in accordance with the technology of the present invention. In one embodiment, provided herein is a process for producing iron oxide nanoparticles, comprising a thermal degradation reaction of iron acetylacetonate. In one embodiment, the resulting iron oxide nanoparticles are water soluble. In one aspect, the iron oxide nanoparticles are suitable for protein conjugation. In one embodiment, the method includes a one-step thermal destruction reaction.

In one aspect, thermal degradation occurs in the presence of functionalized PEG molecules. Certain non-limiting examples of functionalized PEG linkers are shown in Table 1.

In one aspect, thermal destruction includes heating iron acetylacetonate. In one embodiment, thermal degradation comprises heating iron acetylacetonate in the presence of functionalized PEG molecules. In one embodiment, thermal degradation comprises heating iron acetylacetonate in the presence of benzyl ether and functionalized PEG molecules.

Practically, in one embodiment, functionalized PEG molecules are used as reducing agents and as surfactants. The method for producing nanoparticles provided herein simplifies and improves conventional methods that use surfactants that are difficult to replace, or cannot be completely replaced by PEG molecules, making the particles water soluble. Generally, surfactants can be expensive (eg phospholipids) or toxic (eg oleic acid or oleylamine). In another aspect, the nanoparticle production process virtually eliminates the need for conventional surfactants, thus achieving a high degree of molecular purity and water solubility.

In one embodiment, thermal degradation includes ferrous acetylacetonate and benzyl ether, and in the absence of conventional surfactants other than those used herein.

In one embodiment, the temperature for thermal breakdown is from about 80°C to about 300°C, or from about 80°C to about 200°C, or from about 80°C to about 150°C, or from about 100°C. C to about 250°C, or from about 100°C to about 200°C, or from about 150°C to about 250°C, or from about 150°C to about 250°C. In one embodiment, thermal destruction is carried out over a period of about 1 to about 2 hours.

In one of the embodiments, the method for producing nanoparticles from iron oxide includes a purification step, for example, using an LS Miltenyi Biotec magnetic column.

In one embodiment, the nanoparticles are stable at approximately 4° C. in phosphate buffered saline (PBS) without any detectable degradation or aggregation. In one embodiment, the nanoparticles are stable for at least 6 months.

In one aspect, provided herein is a process for preparing nanoparticle complexes, comprising contacting pMHC with iron oxide nanoparticles provided herein. In practice, pMHC encodes at its C-terminus a cysteine that can react with a maleimide group in functionalized PEG at about pH 6.2 to about pH 6.5 for about 12 to about 14 hours.

In one aspect, the method for preparing nanoparticle complexes includes a purification step, for example, using an LS Miltenyi Biotec magnetic column.

Coating of nanoparticles

In certain aspects, the antigen-MHC complex and/or cytokine and/or co-stimulatory molecule can be linked to the nanoparticle core by one or more of covalent binding, non-covalent binding, or cross-linking, and optionally linked via a linker. In additional aspects, the size of the linker may be less than 5 kDa, and it is optionally a polyethylene glycol. In aspects involving a linker or linkers, the linkers on the same core of the nanoparticle may be the same or different from each other.

The following methods can be used to bind the substrate or particles with antigen-MHC complexes and/or cytokines and/or costimulatory molecules.

Coupling can be carried out by chemically modifying the substrate or particle, which typically includes providing surface "functional groups" wherein said functional groups are capable of binding to an MHC complex, and/or linking an optionally chemically modified surface or particle surface with a covalently or non-covalently bound by so-called "linker molecules", followed by the reaction of the MHC or the complex of the MHC with the resulting particles.

The term "linker molecule" or "linker" means a substance capable of binding to a substrate or particle and also capable of binding to an MHC complex.

The term "functional groups" as used herein above is not limited to reactive chemical groups that form covalent bonds, but also includes chemical groups that provide ionic interactions or hydrogen bonds with MHC complexes. In addition, it should be noted that a strict distinction between "functional groups" obtained on the surface and linker molecules bearing "functional groups" is not possible, since sometimes surface modification requires small linker molecules, such as ethylene glycol, to react with the surface of the particle.

The functional groups or linker molecules carrying them can be selected from amino groups, carbonic acid groups, thiols, thioethers, disulfides, guanidino, hydroxyl groups, amino groups, vincial diols, aldehydes, alpha haloacetyl groups, organic mercury compounds, ester groups, acid halides, complex acid thioesters, acid anhydrides, isocyanates, isothiocyanates, sulfonic halides, imidoesters, diazoacetates, diazonium salts, 1,2-diketones, phosphonic acids, phosphate esters, sulfonic acids, azolides, imidazoles, indoles, N-maleimides, alpha- beta-unsaturated carbonyl compounds, aryl halides or derivatives thereof.

Non-limiting examples of other higher molecular weight linker molecules are nucleic acid molecules, polymers, copolymers, polymerizable binders, silica, proteins, and chain molecules with an opposite surface polarity to the substrate or particle. Nucleic acids can bind to affinity molecules, which themselves contain nucleic acid molecules, via a complementary sequence to the linker molecule.

In accordance with certain embodiments, the linker molecule contains polyethylene glycol. In accordance with certain embodiments, the linker molecule contains polyethylene glycol and maleimide. According to certain embodiments, the polyethylene glycol contains one or more of C ₁ -C ₃ alkoxy, -R ¹⁰ NHC(O)R-, -R ¹⁰ C(O)NHR-, -R ¹⁰ OC(O)R-, -R ¹⁰ C(O)OR-, where each R is independently H or C ₁ -C ₆ -alkyl, and where each R ¹⁰ is independently a bond or C ₁ -C ₆ -alkyl.

Examples of polymerizable binders include diacetylene, styrene butadiene, vinyl acetate, acrylate, acrylamide, vinyl compounds, styrene, silica, boron oxide, phosphorus oxide, borates, pyrrole, polypyrrole and phosphates.

rMHC complexes can be coupled to nanoparticles in a variety of ways, one non-limiting example includes conjugation to NPs produced with PEG linkers bearing distal -NH ₂ or -COOH groups, which can be accomplished by forming amide bonds in the presence of 1-ethyl-3-[ hydrochloride 3-dimethylaminopropyl]carbodiimide (EDC). NPs with -COOH groups are first dissolved in 20 mM MES buffer, pH 5.5. N-hydroxysulfosuccinimide sodium salt (Sulpha-NHS, Thermo scientific, Waltham, MA, final concentration 10 mM) and EDC (Thermo scientific, Waltham, MA, final concentration 1 mM) are added to the NP solution. After 20 minutes of stirring at room temperature, the NP solution was added dropwise to a solution containing pMHC monomers dissolved in 20 mM borate buffer (pH 8.2). The mixture is stirred for an additional 4 hours. For conjugation of MHC with functionalized NH ₂ NP complexes pMHC first dissolved in 20 mm MES buffer, pH 5.5, containing 100 mm NaCl. Sulpha-NHS (10 mM) and EDC (5 mM) are then added to the MHC solution. The activated MHC molecules are then added to a solution of NP in 20 mM borate buffer (pH 8.2) and stirred for 4 hours at room temperature.

For conjugation of MHC to maleimide-functionalized NPs, pMHC complexes are first incubated with tributylphosphine (TBP, 1 mM) for 4 hours at room temperature. NPs in 40 mM phosphate buffer pH 6.0 containing 2 mM EDTA, 150 mM NaCl are then mixed with pMHC designed to contain a free C-terminal Cys residue and incubated overnight at room temperature. The MHCs of the pMHC complexes are covalently linked to NP by forming a carbon-sulfide bond between the maleimide groups and the Cys residue.

Click chemistry can be used to conjugate pMHC or avidin with azide-functionalized NPs. For this reaction, MHC or avidin molecules are first incubated with dibenzocyclooctyl reagent (DBCO, Click chemistry Tools, Scottdale, AZ) for 2 hours at room temperature. Free DBCO molecules can be removed by dialysis overnight. The MHC-or avidin-DBCO conjugates are then incubated with SFP-Z for 2 hours, resulting in the formation of triazole bonds between the pMHC or avidin molecules and NP.

Unconjugated pMHC complexes in various MHC-NP conjugation reactions can be removed by extensive dialysis by methods known in the art. A non-limiting example is dialysis against PBS, pH 7.4, at 4° C. through 300 kDa molecular weight separating membranes (Spectrum labs). Alternatively, pMHC-conjugated IONPs can be purified by magnetic separation. Conjugated NPs can be concentrated by ultrafiltration through Amicon Ultra-15 blocks (MWCO 100 kDa) and stored in PBS.

The surface of the substrate or particle can be chemically modified, for example by linking phosphonic acid derivatives with functional reactive groups. One example of these phosphonic acids or ester derivatives of phosphonic acids is imino-bis(methylenephosphono)carbonic acid, which can be synthesized according to the "Mannich-Medritzer" reaction. This coupling reaction can be carried out with the substrate or particle, either directly from the preparation process or after pre-treatment (eg with trimethylsilyl bromide). In the first case, a phosphonic acid derivative (ester), for example, can replace components of the reaction medium that are still bound to the surface. This substitution can be enhanced at elevated temperatures. On the other hand, trimethylsilyl bromide is believed to dealkylate phosphorus-based complexing agents containing alkyl groups, thus forming new binding sites for the phosphonic acid derivative (ester). The phosphonic acid derivative (ester) or linked linker molecules may provide the same functional groups as above. A further example of surface treatment of a substrate or particle includes heating in a diol such as ethylene glycol. It should be noted that this treatment may be redundant if the synthesis has already been carried out in a diol. Under these conditions, the directly obtained synthesis product probably already provides the necessary functional groups. However, this treatment is applicable to a substrate or particle that has been prepared in N- or P-containing complexing agents. If such a substrate or particle is post-treated with ethylene glycol, the ingredients of the reaction medium (eg the complexing agent) still bound to the surface may be displaced with a diol and/or may be dealkylated.

Also, N-containing complexing agents still bound to the surface of the particle can be replaced by derivatives of primary amines with a second functional group. The surface of the substrate or particle can also be coated with silica. Silicon dioxide provides a relatively simple chemical conjugation of organic molecules, as silicon dioxide readily reacts with organic linkers such as triethoxysilane or chlorosilane. The particle surface can also be coated with homo- or copolymers. Examples of polymerizable binders are: N-(3-aminopropyl)-3-mercaptobenzamidine, 3-(trimethoxysilyl)propylhydrazide and 3-(trimethoxysilyl)propylmaleimide. Other non-limiting examples of polymerizable binders are listed herein. These binders may be used singly or in combination depending on the type of copolymer to be coated.

Another method of surface modification that can be used with substrates or particles containing oxidized transition metal compounds is the conversion of oxidized transition metal compounds with chlorine gas or organic chlorinating agents to the corresponding oxychlorides. These oxychlorides are capable of reacting with nucleophiles, such as hydroxy or amino groups, which are often present in biomolecules. This method allows for direct conjugation to proteins, for example via the amino groups of the side chains of lysine. Protein conjugation after surface modification with oxychlorides can also be carried out using a bifunctional linker such as maleimidopropionic acid hydrazide.

For non-covalent binding methods, chain molecules with opposite polarity relative to the substrate or particle surface are particularly suitable. Examples of linker molecules that can be non-covalently bonded to the core/shell of nanoparticles include anionic, cationic, or zwitterionic surfactants, acidic or basic proteins, polyamines, polyamides, polysulfones, or polycarboxylic acids. The necessary linkage can be provided by a hydrophobic interaction between the substrate or particle and the amphiphilic reagent with a functional reactive group, especially suitable chain molecules with an amphiphilic character, such as phospholipids or derivatized polysaccharides, which can be crosslinked to each other. The adsorption of these molecules on the surface can be carried out by co-incubation. The binding of an affinity molecule and a substrate or particle can also be based on non-covalent self-organizing bonds. One of their examples includes simple detector probes with biotin as a linker molecule and associated with avidin or streptavidin molecules.

Protocols for functional group coupling reactions with biological molecules can be found in the literature, for example, in "Bioconjugate Techniques" (Greg T. Hermanson, Academic Press 1996). A biological molecule (eg, an MHC molecule or a derivative thereof) can be coupled to the linker molecule, covalently or non-covalently, by standard organic chemistry methods such as oxidation, halogenation, alkylation, acylation, addition, substitution, or amidation. These methods of linking a covalently or non-covalently linked linker molecule can be used before or after the linker molecule is bound to the substrate or particle. In addition, by means of incubation it is possible to carry out direct binding of molecules to a suitably pretreated substrate or particles (eg via trimethylsilyl bromide) which, as a result of this pretreatment, provide a modified surface (eg a higher charge or a polar surface).

Pharmaceutical compositions and administration

Provided herein are pharmaceutical compositions useful in the treatment and prevention of diseases. The compositions contain, or, alternatively, consist to a large extent, or even more so, consist of complexes of nanoparticles, as described herein, and a carrier.

The compositions can be used to induce or modify an immune response to a disease-associated antigen, such as a polypeptide, peptide, carbohydrate, lipid, or other molecule or molecule fragment, and against an advanced condition or disease caused by such an autoimmune response or cancer.

In general, compositions of the invention may be administered parenterally, by injection, for example intravenously, subcutaneously or intramuscularly. Additional formulations suitable for other routes of administration include oral formulations. Oral formulations include such commonly used excipients as, for example, pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharinate, cellulose, magnesium carbonate, and the like. These compositions are prepared in the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain from about 10% to about 95% active ingredient, preferably from about 25% to about 70%. The preparation of aqueous compositions containing an antigen-MHC-nanoparticle complex that modifies the immune status of an individual will be understood by those skilled in the art in light of the present invention. In accordance with certain embodiments, the composition may be inhaled (eg, US Pat. No. 6,651,655, which is specifically incorporated by reference in its entirety). In one embodiment, the antigen-MHC-nanoparticle complex is administered systemically. In accordance with specific embodiments, the pMHC-NP complex or compositions containing a variety of pMHC-NP complexes can be administered intravenously.

In general, the compositions of the invention are administered in a manner compatible with the dosage formulation and in such amount as will be therapeutically effective and will modify immunity. The amount administered will depend on the individual being treated. The exact amounts of the active ingredient required for administration are at the discretion of the practitioner. However, suitable dosage ranges are on the order of ten to several hundred nanograms or micrograms of the antigen/MHC/nanoparticle complex per administration. Suitable regimens for initial administration and repeated administrations also vary but are characterized by initial administration followed by subsequent administrations.

Methods of application can vary widely. Any conventional means of administering vaccines is applicable. These are believed to include oral administration of a physiologically acceptable solid base or in a physiologically acceptable dispersion, parenteral route, injection route, and the like. The dose of the antigen/MHC/nanoparticle complex depends on the route of administration and varies according to the size and health of the individual.

In many cases, multiple administrations of the peptide/MHC/nanoparticle complex are required, about, at least about, or no more than about 3, 4, 5, 6, 7, 8, 9, 10 or more administrations. The intervals between injections normally range from 1, 2, 3, 4, 5, 6 or 7 days to twelve weeks, usually from one to two weeks. To maintain the state of the immune system, periodic secondary injections are required at intervals of a day, twice a week, once a week, once every two weeks, once a month, or 0.1, 0.2, 0.3, 0.4, 0.5 , 1, 2, 3.4 or 5 years, usually two years. After the course of administration, autoreactive immune response assays can be performed, recognizing T _R 1 cells and T cell activity.

In certain aspects, a single dose of the rMHC complex without including the nanoparticle core and any outer layer is about 0.001 mg/kg to about 2.0 mg/kg, or about 0.001 mg/kg to about 1.5 mg/kg, or about 0.001 mg/kg to about 1.4 mg/kg, or about 0.001 mg/kg to about 1.3 mg/kg, or about 0.001 mg/kg to about 1.2 mg/kg, or about 0.001 mg/kg to about 1.1 mg/kg, or about 0.001 mg/kg to about 1.0 mg/kg. In certain embodiments, a single dose is about 0.004 mg/kg to about 1.014 mg/kg, or about 0.02 mg/kg to about 0.811 mg/kg, or about 0.041 mg/kg to about 0.608 mg/kg. kg, or about 0.061 mg/kg to about 0.507 mg/kg, or about 0.081 mg/kg to about 0.405 mg/kg, or about 0.121 mg/kg to about 0.324 mg/kg, or about 0.162 mg/kg kg to about 0.243 mg/kg. In certain embodiments, a single dose is about 0.004 mg/kg to about 1.015 mg/kg, or about 0.004 mg/kg to about 1.0 mg/kg, or about 0.004 mg/kg to about 0.9 mg/kg, or about 0.004 mg/kg to about 0.8 mg/kg, or about 0.004 mg/kg to about 0.7 mg/kg, or about 0.004 mg/kg to about 0.6 mg/kg kg, or about 0.004 mg/kg to about 0.5 mg/kg, or about 0.004 mg/kg to about 0.4 mg/kg, or about 0.004 mg/kg to about 0.3 mg/kg, or about 0.004 mg/kg to about 0.2 mg/kg, or about 0.004 mg/kg to about 0.1 mg/kg.

Combined treatment

Compositions and related methods of the present invention, in particular the administration of an antigen/MHC/nanoparticle complex, can also be used in combination with the administration of traditional therapeutic agents. These include, but are not limited to, the administration of immunosuppressive or modulating therapeutics or treatments. Non-limiting examples of certain disease-associated treatments include Avonex (interferon beta-1a), betaseron (interferon beta-1b), copaxone (glatiramer acetate), novantrone (mitoxantrone), rebif (interferon beta-1a), tisarbi (natalizumab), gilenia (fingolimod), glatiramer, steroids, cytoxan, imuran, baclofen, deep brain stimulation, ampira (dalfampridine), acupuncture, and physiotherapy. In the treatment of cancer, additional chemotherapeutic agents, radiation, or surgery may be added to enhance the therapeutic response of the described compositions and methods.

In one aspect, it is contemplated that the antigen/MHC/nanoparticle complex is used in conjunction with cytokine treatment. Alternatively, administration of the antigen/MHC/nanoparticle complex may precede or follow other treatments at intervals ranging from a few minutes to several weeks. In certain embodiments where other agents and/or antigen/MHC/nanoparticle complexes are administered separately, it should be ensured that no significant time elapses between each delivery point so that the agent and antigen/MHC/nanoparticle complex can still exert predominantly combined effect on the individual. In such cases, it is contemplated that both agents may be administered within about 12-24 hours of each other, and more preferably within about 6-12 hours of each other. However, in certain situations it may be desirable to significantly increase the period of time for administration, when between the respective administrations passes from several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6 , 7 or 8).

Various combinations can be used, for example, when the introduction of the antigen/MHC/nanoparticle complex is "A" and the additional agent is "B":

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B

B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A

B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

Administration of the peptide-MHC complex compositions of the present invention to a patient/individual follows the general protocols for administering such compounds, taking into account toxicity, if any. It is believed that treatment cycles can be repeated as needed. It is also contemplated that various standard therapeutic agents such as fluid replacement can be used in combination with the described treatment.

Pharmaceutical carriers and formulations

In accordance with certain embodiments, pharmaceutical compositions are administered to an individual. Various aspects of the present invention include administering to an individual an effective amount of an antigen/MHC/nanoparticle complex composition. In addition, such compositions can be administered in combination with immune system modifiers. Typically, such compositions are dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

The phrases "pharmaceutically acceptable" or "pharmacologically acceptable" refer to molecular structures and compositions that, when administered to an animal or human, do not lead to harmful, allergic or other undesirable reactions. As used herein, a "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such carriers and agents for pharmaceutically active substances is well known in the art. Unless any conventional carriers or agents are incompatible with the active ingredients, their use in immunogenic and therapeutic compositions is contemplated.

The active compounds of the present invention can be formulated for parenteral administration, for example formulated for injection via intravenous, intramuscular, subcutaneous or even intraperitoneal routes. The preparation of an aqueous composition containing an antigen/MHC/nanoparticle complex that modifies the immune status of an individual will be understood by those skilled in the art in light of the present invention. Typically, such compositions can be prepared as injectables, liquid solutions or suspensions; solid forms suitable for the preparation of solutions or suspensions after the addition of a liquid prior to injection; and preparations can also be emulsified.

Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations comprising sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for immediate preparations of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that it can be easily injected. It must be stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi.

The compositions can be formulated in neutral form or in salt form. Pharmaceutically acceptable salts, including acid addition salts (formed with the free amino groups of the protein), are formed with inorganic acids such as, for example, hydrochloric or phosphoric acid, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. It is also possible to form salts formed with free carboxyl groups from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or iron hydroxides, and organic bases, such as isopropylamine, trimethylamine, histidine, procaine, and the like.

The carrier may also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg glycerol, propylene glycol and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. The necessary fluidity can be maintained, for example, using a coating such as lecithin, maintaining the required particle size in the case of a dispersion, and using surfactants. Prevention of the action of microorganisms can be provided by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases it is preferable to include isotonicity agents such as sugars or sodium chloride. Prolonged absorption of injectable compositions can be achieved by the use of absorption delaying agents in the compositions, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by adding the active compounds in the required amount of the appropriate solvent with the various other ingredients listed above, as appropriate, followed by sterilization. Sterilization of the solution is carried out in such a way as not to reduce the therapeutic properties of the antigen-MHC-nanoparticle complex. Typically, dispersions are prepared by adding the various sterilized active ingredients to a sterile vehicle which contains the basic dispersion medium and the other required ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, certain methods of preparation are vacuum drying and lyophilization methods, which provide a powder of the active ingredient with any desired additional ingredient from their previously sterilized solutions. One such solution sterilization method is sterile filtration, however, the present invention is intended to include any sterilization method that does not significantly reduce the therapeutic properties of the antigen-MHC-nanoparticle complexes. Sterilization methods involving intense heat and pressure, such as autoclaving, can damage the tertiary structure of the complex, thus significantly reducing the therapeutic properties of the antigen-MHC-nanoparticle complexes.

As a rule, the introduction of the compositions of the present invention is carried out by any conventional route. As non-limiting examples, they include orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intranasal, or intravenous injection. In accordance with certain embodiments, the vaccine composition may be inhaled (eg, US Pat. No. 6,651,655, which is specifically incorporated by reference).

An effective amount of a therapeutic or prophylactic composition is determined based on the intended purpose. The term "unit dose" or "dosage" refers to physically discrete units suitable for use in an individual, where each unit contains a predetermined amount of the composition, calculated to provide the desired response described above with respect to its administration, for example, the appropriate route and regimen treatment. The amount to be administered, in relation to the amount of therapeutic agents and the dosage unit, depends on the desired result and/or protection. The exact amounts of the composition also depend on the judgment of the practitioner and are specific to each individual. Factors influencing dose include the individual's physical and clinical condition, the route of administration, the intended goal of treatment (reduction of symptoms versus cure), and the potency, stability, and toxicity of the particular composition. Once formulated, the solutions are administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easy to administer in a variety of dosage forms, such as the types of injectable solutions described above. A typical dosing regimen in a mouse model includes administration of 1 μg - 50 μg total pMHC (applied to NP) and 1 μg - 50 μg total iron per dose, which can be converted to a specific dosage unit in humans. In accordance with certain embodiments, the dosage may range from about 0.1 µg to about 400 µg. However, it should be understood that the amount of rMHC per dose may range from only 0.1 μg to 100 mg. By way of example, in a 60 kg human patient, the amount of rMHC per dose may range from 0.24 mg to 12 mg, with the understanding that this corresponds to the 1 μg to 50 μg discussed above. Also, as stated above, this dose may be varied to correspond to an amount of 0.1 µg to 100 mg as described above, corresponding to an equivalent human dose of 0.0004 mg/kg to 405.4 mg/kg and fluctuates between them depending on the patient undergoing treatment, his condition and other parameters at the discretion of the attending physician.

Administration in vitro or ex vivo

As used herein, the term in vitro administration refers to manipulations performed on cells isolated from or outside the individual, including but not limited to cells in culture. The term ex vivo administration refers to cells that have been treated in vitro and then administered to an individual. The term in vivo administration includes all manipulations performed on an individual, including administration.

In accordance with certain aspects of the present invention, the compositions can be administered in vitro, ex vivo or in vivo. In certain in vitro embodiments, autologous T cells are incubated with the compositions of the present invention. The cells are then used for in vitro analysis or, alternatively, for ex vivo administration.

Obtaining protein components

The present specification describes polypeptides, peptides and proteins for use in various embodiments of the present invention. For example, specific peptides and complexes thereof are evaluated for their ability to induce or modulate an immune response. In accordance with specific embodiments, all or part of the peptides or proteins of the invention can also be synthesized by conventional methods in solution or on a solid support. Various automatic synthesizers are commercially available and can be used according to known protocols.

Alternatively, recombinant DNA technology can be used where the nucleotide sequence that encodes the peptide of the invention is inserted into an expression vector, transformed or transfected into an appropriate host cell, and cultured under conditions suitable for expression.

One of the embodiments of the invention includes the use of gene transfer proteins in cells, including microorganisms, to obtain proteins. The gene of interest for the protein can be transferred to appropriate host cells, followed by culturing the cells under suitable conditions. A nucleic acid encoding virtually any polypeptide can be used. The preparation of recombinant expression vectors and the elements included therein are known to those skilled in the art and are briefly described herein. Examples of mammalian host cell lines include, but are not limited to, Vero and HeLa cells, other B and T cell lines such as CEM, 721, 221, H9, Jurkat, Raji, and Chinese Hamster Ovary cell lines, W138 cells, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK. In addition, a host cell line can be selected that modulates the expression of the inserted sequences or that modifies and processes the gene product in the desired manner. Such modifications (eg, glycosylation) and processing (eg, cleavage) of protein products may be important for protein function. Various host cells contain characteristic and specific mechanisms for post-translational processing and modification of proteins. Appropriate cell lines or host systems can be selected to ensure correct modification and processing of the expressed foreign protein.

A number of selection systems can be used, including, but not limited to, the HSV thymidine kinase, hypoxanthine-guanine phosphoribosyl transferase, and adenine phosphoribosyl transferase genes in tk-, hgprt-, or aprt- cells, respectively. Also, resistance to antimetabolites can be used as a basis for selection: dhfr, which confers resistance to trimethoprim and methotrexate; gpt, which confers resistance to mycophenolic acid; neo, which confers resistance to the aminoglycoside G418; and hygro, which confers resistance to hygromycin.

Nucleic acids

The present invention may include recombinant polynucleotides encoding proteins, polypeptides, peptides according to the invention. Included and can be used to generate peptide/MHC complexes of nucleic acid sequences for self antigens and MHC molecules for presentation of self antigens.

As used herein, the term "polynucleotide" refers to a nucleic acid molecule that is recombinant or isolated from a common genomic nucleic acid. The term "polynucleotide" includes oligonucleotides (nucleic acids of 100 residues or less), recombinant vectors, including, for example, plasmids, cosmids, phages, viruses, and the like. In certain aspects, polynucleotides include regulatory sequences substantially separate from natural genes or protein-coding sequences. Polynucleotides can be RNA, DNA, analogs thereof, or combinations thereof.

In this regard, the term "gene", "polynucleotide", or "nucleic acid" is used to refer to nucleic acids encoding proteins, polypeptides, or peptides (including any sequences necessary for correct transcription, post-translational modification, or localization). As understood by those skilled in the art, the term includes genomic sequences, expression cassettes, cDNA sequences, and small engineered nucleic acid regions that express or can be adapted to express proteins, polypeptides, domains, peptides, fusion proteins, and mutants. Nucleic acids encoding all or parts of the polypeptides may contain contiguous sequences of nucleic acids encoding all or parts of such polypeptides with the following lengths: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 , 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370 ,380,390,400,410,420,430,440,441,450,460,470,480,490,500,510,520,530,540,550,560,570,580,590,600,610 , 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860 . . It is also contemplated that a particular polypeptide of a given species may be encoded by nucleic acids containing naturally occurring variations that contain slightly different nucleic acid sequences but nevertheless encode the same or substantially similar protein, polypeptide, or peptide.

In accordance with specific embodiments, the invention relates to isolated nucleic acid regions and recombinant vectors containing nucleic acid sequences that encode a self-antigen and/or an MHC molecule. The term "recombinant" may be used in conjunction with a polypeptide or the name of a particular polypeptide, and generally refers to a polypeptide derived from a nucleic acid molecule that has been processed in vitro or is a replication product of such a molecule.

The nucleic acid regions used in the present invention, regardless of the length of the coding sequence itself, can be combined with other nucleic acid sequences such as promoters, polyadenylation signals, additional restriction enzyme recognition regions, multiple cloning regions, other coding regions, and the like. so that their total length can vary greatly. Thus, it is envisaged that a nucleic acid fragment of almost any length can be used, with an overall length preferably limited by the ease of obtaining and using the required recombinant nucleic acid processing protocol. In some cases, a nucleic acid sequence may encode a polypeptide sequence along with additional heterologous coding sequences, for example, to provide purification, transport, secretion, post-translational modification of the polypeptide, or for beneficial therapeutic effects such as targeting or efficacy. A coding tag or other heterologous polypeptide sequence may be added to the modified polypeptide coding sequence, where "heterologous" refers to a polypeptide that is not the same as the modified polypeptide.

Methods of treatment

Medical and diagnostic methods are also provided. In one aspect, a method is provided for allowing the formation, expansion, and recruitment of immunocytes, including, but not limited to, effector cells, regulatory B cells, and/or T _R 1 cells (e.g., T _R 1 and CD4 ⁺ cells) or CD8 ⁺ β-cells, in an antigen-specific manner in an individual in need thereof, comprising, or alternatively, substantially consisting of, or even more consisting of, administering to the individual an effective amount of an NP complex or composition as described herein.

The present invention also relates to methods for differentiating or initiating the formation of type 1 regulatory T cells (T _R 1 ) in a dose-independent manner in a rMHC manner. Applicant has found that the density of rMHC at the core of the nanoparticle regulates the ability of rMHC at the core of the nanoparticle to initiate the formation of T _R 1 cells in a dose-independent manner, while the dose of rMHC regulates the amount of proliferation of T _R 1 cells in a manner that is independent of the density of rMHC. Applicant has found that the rMHC density threshold and the independent effect of rMHC density versus dose on T _R 1 cell formation versus propagation are unexpected findings that could not be assumed on the basis of conventional knowledge in the field of immunology. These methods require contact of recognizing T cells with an effective amount of pMHC-NP or pMHC-NP composition as described herein. In certain aspects, density dependent methods refer to activated or memory T cells differentiating into IL-10 producing T _R 1 recognition cells, optionally carrying CD49b and/or Lag3 markers, and/or B cells. that differentiates into regulatory B cells upon contact of activated T cells or memory T cells with an effective amount of the complex or composition described herein. In certain embodiments, a differentiated regulatory T cell binds to a B cell, thereby allowing the B cell to differentiate into a regulatory B cell. According to certain aspects of the methods, contacting is carried out in vitro or in vivo.

Thus, aspects of the invention relate to a method of differentiating or initiating the formation of T _R 1 cells in a dose-independent manner of rMHC, comprising contacting recognizing T cells with effective amounts of the complex or composition described herein. In accordance with certain aspects, bringing into contact can be carried out in vitro or in vivo. In certain aspects, the methods relate to activated or memory T cells differentiating into IL-10 producing T _R 1 cells, optionally expressing CD49b and/or Lag3 markers, including bringing the activated or memory T cells to contact with effective amounts of the complex or composition described herein. Based on correlation with the appropriate cell type for each disease, the appropriate optimized MHC/NP complex and optionally a co-stimulatory molecule and/or cytokine are also administered.

In view of the above, the Applicant provides a method for differentiating activated or memory T cells into IL-10 producing T _R 1 cells, optionally expressing CD49b and/or Lag3 markers, and/or differentiating B cells into regulatory B cells, comprising , or alternatively consisting of, or even more consisting of, contacting activated or memory T cells with an effective amount of a complex or composition as described herein. Bringing into contact can be carried out in vitro or in vivo. In accordance with certain embodiments, the pMHC-NP or composition comprising a plurality of pMHC-NPs comprises pMHC-NPs with an average nanoparticle core diameter of about 25 nm to about 60 nm, or about 25 nm to about 50 nm, or about 20 nm to about 50 nm. about 40 nm, or about 15 nm to about 50 nm, or about 15 nm to about 40 nm, or about 15 nm to about 35 nm, or about 15 nm to about 30 nm, or about 15 nm to about 25 nm, or alternatively about 15 nm, or about 20 nm, or about 25 nm, or about 30 nm, or about 35 nm, or about 40 nm. In accordance with certain aspects, the core of the nanoparticle further comprises an outer coating or layer, where the diameter of the core and the outer layer have an average diameter of from about 30 nm to about 75 nm, or from about 30 nm to about 70 nm, or from about 30 nm to about 60 nm, or about 30 nm to about 50 nm, or about 40 nm. In certain aspects, the average pMHC density on a nanoparticle is from about 0.4 pMHC/100 nm ² to about 12 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 11.6 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 11.5 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 11 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 10 pMHC/100 nm ² , or about 0.4 pMHC/100 nm ² to about 9 pMHC/100 nm ² , or about 0.4 pMHC/100 nm ² to about 8 pMHC/ 100 nm ² , or about 0.4 pMHC/100 nm ² to about 7 pMHC/100 nm ² , or about 0.4 pMHC/100 nm ² to about 6 pMHC/100 nm ² , or about 0.4 pMHC/100 nm ² to about 5 pMHC/100 nm ² , or about 0.4 pMHC/100 nm ² to about 4 pMHC/100 nm ² , or about 0.4 pMHC/100 nm ² to about 3 pMHC/ 100 nm ² , or from about 0.4 pMHC/100 nm ² to about 2.5 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 2 pMHC/100 nm ² , or from about 0, 4 pMHC/100 nm ² to about 1.5 pMHC/100 nm ² .

Also provided are methods for differentiating activated or memory T cells into IL-10 producing T _R 1 cells, optionally expressing CD49b and/or Lag3 markers, and/or differentiating B cells into regulatory B cells, comprising, or alternatively consisting essentially of, or even more of, administering to an individual an effective amount of a complex or composition as described herein. As used herein, an individual may include an animal, mammal, mouse, cow, horse, dog, cat, sheep, or human. In accordance with certain embodiments, a pMHC-NP or a composition comprising a plurality of pMHC-NPs comprises a pMHC-NP with an average nanoparticle core diameter of about 25 nm to about 60 nm, or about 25 nm to about 50 nm, or about 20 nm to about 40 nm, or about 15 nm to about 50 nm, or about 15 nm to about 40 nm, or about 15 nm to about 35 nm, or about 15 nm to about 30 nm, or about 15 nm to about 25 nm, or alternatively about 15 nm, or about 20 nm, or about 25 nm, or about 30 nm, or about 35 nm, or about 40 nm. In accordance with certain aspects, the core of the nanoparticle further comprises an outer coating or layer, where the diameter of the core and the outer layer have an average diameter of from about 30 nm to about 75 nm, or from about 30 nm to about 70 nm, or from about 30 nm to about 60 nm, or about 30 nm to about 50 nm, or about 40 nm. In certain aspects, the average pMHC density on a nanoparticle is from about 0.4 pMHC/100 nm ² to about 12 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 11.6 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 11.5 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 11 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 10 pMHC/100 nm ² , or about 0.4 pMHC/100 nm ² to about 9 pMHC/100 nm ² , or about 0.4 pMHC/100 nm ² to about 8 pMHC/ 100 nm ² , or about 0.4 pMHC/100 nm ² to about 7 pMHC/100 nm ² , or about 0.4 pMHC/100 nm ² to about 6 pMHC/100 nm ² , or about 0.4 pMHC/100 nm ² to about 5 pMHC/100 nm ² , or about 0.4 pMHC/100 nm ² to about 4 pMHC/100 nm ² , or about 0.4 pMHC/100 nm ² to about 3 pMHC/ 100 nm ² , or from about 0.4 pMHC/100 nm ² to about 2.5 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 2 pMHC/100 nm ² , or from about 0, 4 pMHC/100 nm ² to about 1.5 pMHC/100 nm ² .

Provided herein are methods of treating an autoimmune disease or disorder in an individual in need thereof, comprising administering to the individual an effective amount of any of the complexes or compositions described herein, provided that the complexes and compositions do not contain costimulatory molecules.

Also provided herein are methods for treating a cancer or tumor and/or inhibiting tumor cell or tissue growth in an individual in need thereof, comprising administering an effective amount of any pMHC-NP complex with one or more co-stimulatory molecules.

Additional aspects provided herein include a nanoparticle complex with a pMHC density of about 0.4 pMHC/100 nm ² to about 12 pMHC/100 nm ² for use in initiating the differentiation of activated or memory T cells into IL-producing 10 T _R 1 cells optionally expressing CD49b and/or Lag3 markers. In accordance with certain embodiments, the pMHC-NP or a composition comprising a plurality of pMHC-NPs comprises a pMHC-NP with an average nanoparticle core diameter of about 25 nm to about 60 nm, or about 25 nm to about 50 nm, or about 20 nm to about 40 nm, or about 15 nm to about 50 nm, or about 15 nm to about 40 nm, or about 15 nm to about 35 nm, or about 15 nm to about 30 nm, or about 15 nm to about 25 nm, or alternatively about 15 nm, or about 20 nm, or about 25 nm, or about 30 nm, or about 35 nm, or about 40 nm. In accordance with certain aspects, the core of the nanoparticle further comprises an outer coating or layer, where the diameter of the core and the outer layer have an average diameter of from about 30 nm to about 75 nm, or from about 30 nm to about 70 nm, or from about 30 nm to about 60 nm, or about 30 nm to about 50 nm, or about 40 nm. In certain aspects, the average pMHC density on a nanoparticle is from about 0.4 pMHC/100 nm ² to about 12 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 11.6 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 11.5 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 11 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 10 pMHC/100 nm ² , or about 0.4 pMHC/100 nm ² to about 9 pMHC/100 nm ² , or about 0.4 pMHC/100 nm ² to about 8 pMHC/ 100 nm ² , or about 0.4 pMHC/100 nm ² to about 7 pMHC/100 nm ² , or about 0.4 pMHC/100 nm ² to about 6 pMHC/100 nm ² , or about 0.4 pMHC/100 nm ² to about 5 pMHC/100 nm ² , or about 0.4 pMHC/100 nm ² to about 4 pMHC/100 nm ² , or about 0.4 pMHC/100 nm ² to about 3 pMHC/ 100 nm ² , or from about 0.4 pMHC/100 nm ² to about 2.5 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 2 pMHC/100 nm ² , or from about 0, 4 pMHC/100 nm ² to about 1.5 pMHC/100 nm ² .

In one aspect, provided herein are methods for differentiating activated or memory T cells into IL-10 producing T _R 1 cells expressing a marker comprising CD49b and/or LAG3 and/or differentiating B cells into regulatory B- cells, wherein the method comprises contacting activated or memory T cells with an effective amount of a complex comprising: a nanoparticle core, wherein: a plurality of disease-associated antigen-MHC complexes (rMHC) are associated with the nanoparticle core; the core diameter is from about 15 nm to about 25 nm; and where the density pMHC on the nanoparticle is from about 0.4 pMHC/100 nm ² to about 12 pMHC/100 nm ² surface area of the nanoparticle. In accordance with certain embodiments, the nanoparticle core further comprises an outer layer on the core, wherein the pMHC complex is bonded to the nanoparticle core and/or outer layer, and wherein the combined diameter of the nanoparticle core and outer layer is between about 35 nm and about 45 nm. According to certain embodiments, the contacting is carried out in vitro or in vivo.

According to another aspect, provided herein is a nanoparticle complex for use in differentiation of activated or memory T cells into IL-10 producing T _R 1 cells expressing a marker comprising CD49b and/or LAG3 and/or B- cells into regulatory B cells, where the nanoparticle complex contains the core of the nanoparticle, where: a plurality of disease-associated antigen-MHC complexes (rMHC) are associated with the core of the nanoparticle; the core diameter is from about 15 nm to about 25 nm; and where the density pMHC on the nanoparticle is from about 0.4 pMHC/100 nm ² to about 12 pMHC/100 nm ² surface area of the nanoparticle.

According to another aspect, provided herein is a nanoparticle complex for use in differentiation of activated or memory T cells into IL-10 producing T _R 1 cells expressing a marker comprising CD49b and/or LAG3 and/or B- cells into regulatory B cells, where the nanoparticle complex comprises a nanoparticle core and an outer layer on the nanoparticle core, where: a plurality of disease-associated antigen-MHC complexes (pMHC) are associated with the nanoparticle core and/or outer layer; the combined diameter of the core and the outer layer is from about 25 nm to about 45 nm; and where the density pMHC on the nanoparticle is from about 0.4 pMHC/100 nm ² to about 12 pMHC/100 nm ² surface area of the nanoparticle.

In certain embodiments, the therapeutic effect comprises from about 0.1% to about 250% increase in the population of T _R 1 cells. In certain embodiments, the increase includes from about 0.1% to about 225%, or from about 0. 1% to about 200%, or about 0.1% to about 175%, or about 0.1% to about 150%, or about 0.1% to about 125%, or about 0.1% to about 100%, or from about 0.1% to about 75%, or from about 0.1% to about 50%, or from about 0.1% to about 25%, or from about 0.1% to about 20%, or about 0.1% to about 15%, or about 0.1% to about 10%, or about 0.1% to about 9%, or about 0.1% to about 8% , or from about 0.1% to about 7%, or and about 0.1% to about 6%, or about 0.1% to about 5%, or about 0.1% to about 4%, or about 0.1% to about 3%, or about 0.1% to about 2%, or about 0.1% to about 1%, or about 0.1% to about 0.9%, or about 0.1% to about 0.8%, or about 0.1% to about 0.7%, or about 0.1% to about 0.6%, or about 0.1% to about 0.5%, or about 0.1% to about 0.4%, or about 0.1% to about 0.3%, or about 0.1% to about 0.2% increase in T _R 1 cell population.

For therapeutic use, the following diseases can be combined with the following antigen-MHC complexes and compositions containing them:

In certain embodiments, the pMHC complex antigen comprises:

a) a diabetes-associated antigen and is derived from an antigen selected from one or more of the group: preproinsulin (PPI), islet-specific glucose-6-phosphatase (IGRP), glutamate decarboxylase (GAD), islet cell autoantigen 2 (ICA2) , insulin, proinsulin, or a fragment or equivalent of any of them;

b) a multiple sclerosis-associated antigen, and is derived from an antigen selected from one or more of the group: myelin basic protein, myelin-associated glycoprotein, oligodendrocyte myelin protein, proteolipid protein, oligodendrocyte myelin oligoprotein, oligodendrocyte myelin-associated basic protein, specific for oligodendrocytes, protein, heat shock proteins, oligodendrocyte-specific proteins, NOGO A, glycoprotein Rho, peripheral myelin 22 protein, 3'-phosphodiesterase of 2'3'-cyclic nucleotides, or a fragment or equivalent of any of these;

c) an antigen associated with celiac disease and is derived from gliadin or a fragment or equivalent thereof;

d) an antigen associated with primary biliary cirrhosis and is derived from PDC-E2 or a fragment or equivalent thereof;

e) a pemphigus foliaceus-associated antigen and/or a pemphigus vulgaris-associated antigen, and is derived from an antigen selected from one or more of the group: DG1, DG3, or a fragment or equivalent of any of these;

f) an antigen associated with neuromyelitis of the optic nerve with spectral disorder, and it is derived from AQP4 or a fragment or equivalent thereof;

g) an arthritis-associated antigen and is derived from an antigen selected from one or more of the group: heat shock proteins, immunoglobulin-binding protein, heterogeneous nuclear RNPs, annexin V, calpastatin, type II collagen, glucose-6-phosphate isomerase, elongation factor human cartilage gp39, mannose-binding lectin, citrullinated vimentin, type II collagen, fibrinogen, alpha-enolase, antibodies to carbamylated proteins (anti-CarP antibodies), peptidylarginine deiminase type 4 (PAD4), BRAF, fibrinogen gamma chain, inter-alpha heavy chain trypsin inhibitor H1, alpha-1 antitrypsin, C1 plasma protease inhibitor, gelsolin, alpha-1-B glycoprotein, ceruloplasmin, inter-alpha trypsin inhibitor H4 heavy chain, complement factor H, alpha-2 macroglobulin, serum amyloid, C-reactive protein, serum albumin, fibrinogen beta chain, serotransferrin, alpha-2-glycoprotein HS, vimentin, complement component C3, or a fragment or equivalent of any of these;

h) an allergic asthma-associated antigen and is derived from an antigen selected from one or more of the group: DERP1, DERP2, or a fragment or equivalent of any of these;

i) an inflammatory bowel disease associated antigen and is derived from an antigen selected from one or more of the group: Flagellin, Fla-2, Fla-X, YIDX, bacteroid integrase, or a fragment or equivalent of any of these;

j) an antigen associated with systemic lupus erythematosus, and it is derived from an antigen selected from one or more of the group: double-stranded (ds)DNA, ribonucleoprotein (RNP), Smith (Sm), Sjögren's syndrome associated antigen A (SS-A) /Ro, Sjögren's syndrome-associated antigen B (SS-B)/La, RO60, RO52, histones or a fragment or equivalent of any of them;

k) an atherosclerosis-associated antigen and is derived from an antigen selected from one or more of the group: ApoB, ApoE, or a fragment or equivalent of any of these;

l) a COPD-associated antigen and/or an emphysema-associated antigen, and is derived from elastin or a fragment or equivalent thereof;

m) a psoriasis-associated antigen and is derived from an antigen selected from one or more of the group: Cap18, ADMTSL5, ATL5, or a fragment or equivalent of any of these;

n) an autoimmune hepatitis associated antigen and is derived from an antigen selected from one or more of the group: CYP2D6, SLA, or a fragment or equivalent of any of these;

o) an antigen associated with uveitis and is derived from arrestin or a fragment or equivalent thereof;

p) an antigen associated with Sjögren's syndrome and is derived from an antigen selected from one or more of the group: (SS-A)/Ro, (SS-B)/La, MR3, RO60, RO52, or a fragment or equivalent of any of them ;

q) a scleroderma-associated antigen and is derived from an antigen selected from one or more of the group: CENP-C, TOP 1, RNA polymerase III, or a fragment or equivalent of any of these;

r) an antiphospholipid syndrome associated antigen and is derived from APO or a fragment or equivalent thereof;

s) an ANCA-associated vasculitis-associated antigen and is derived from an antigen selected from one or more of the group: MPO, PRTN3, or a fragment or equivalent of any of these; or

t) a stiffness syndrome-associated antigen and is derived from GAD or a fragment or equivalent thereof.

In certain embodiments, the rMHC complex MHC protein comprises all or part of a classic MHC class I protein, a non-classical MHC class I protein, a classic MHC class II protein, a non-classical MHC class II protein, MHC dimers (Fc fusions), MHC tetramers, or polymeric a form of the MHC protein, wherein the MHC protein optionally comprises a "trough-to-trough" heterodimer or multimer alpha-MHC-Fc/beta-MHC-Fc.

In accordance with certain embodiments, the rMHC complex MHC protein comprises all or part of a polypeptide from the group: HLA DR, HLA DQ, HLA DP, HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G , CD1d or fragment or equivalent of any of them.

In certain embodiments, the rMHC complex MHC protein comprises all or part of a polypeptide from the group: HLA-DR, HLA-DQ, HLA-DP, or a fragment or equivalent of any of these.

In accordance with certain embodiments, the MHC protein of the rMHC complex contains all or part of a polypeptide from the group: HLA-DRB1/DRA, HLA-DRB3/DRA, HLA-DRB4/DRA, HLA-DRB5/DRA, HLA-DQA1/HLA-DQB1, HLA-DPB1/HLA-DPA1 or fragment or equivalent of any of them.

In accordance with certain aspects, the rMHC complex contains:

a) a diabetes associated antigen derived from an antigen selected from one or more of the group: hInsB _10-18 , hIGRP _228-236 , hIGRP _265-273 , IGRP _206-214 , hIGRP _206-214 , NRP-A7, NRP- I4, NRP-V7, YAI/D ^b , INS B _15-23 , PPI _{76-90 (K88S)} , IGRP _13-25 , GAD _555-567 , GAD _{555-567(557I)} , IGRP _23-35 , B ₂₄ -C ₃₆ , PPI _76-90 , INS-I9, TUM, G6Pase, proinsulin _L2-10 , proinsulin _L3-11 , proinsulin _L6-14 , proinsulin _B5-14 , proinsulin _B10-18 , proinsulin _B14-22 , proinsulin _{B15- 24} , proinsulin _B17-25 , proinsulin _B18-27 , proinsulin _B20-27 , proinsulin _B21-29 , proinsulin _B25-C1 , proinsulin _B27-C5 , proinsulin _C20-28 , proinsulin _C25-33 , proinsulin _C29-A5 , proinsulin _{A1- 10} , proinsulin _A2-10 , proinsulin _A12-20 , or a fragment or equivalent of any of them;

b) a multiple sclerosis-associated antigen derived from an antigen selected from one or more of the group: MOG _35-55 , MOG _36-55 , MAG _287-295 , MAG _509-517 , MAG _556-564 , MBP _110-118 , MOG _114-122 , MOG _166-175 , MOG _172-180 , MOG _179-188 , MOG _188-196 , MOG _181-189 , MOG _205-214 , PLP _80-88 , MAG _287-295 , MAG _509-517 , MAG _556-564 , MOG _97-109 MOG _{97-109(E107S)} , MBP _89-101 , PLP _175-192 , PLP _94-108 , MBP _86-98 , PLP _54-68 , PLP _249-263 , MOG _{156- 170} , MOG _201-215 , MOG _38-52 , MOG _203-217 , PLP _250-264 , MPB _13-32 , MPB _83-99 , MPB _111-129 , MPB _146-170 , MOG _223-237 , MOG _{6- 20} , PLP _88-102 , PLP _139-154 , or a fragment or equivalent of any of them;

c) an antigen associated with celiac disease derived from an antigen selected from one or more of the group: aGlia _57-68 , aGlia _62-72 , aGlia _217-229 or a fragment or equivalent of any of them;

d) primary biliary cirrhosis associated antigen derived from an antigen selected from one or more of the group: PDC-E2 _122-135 , PDC-E2 _249-262 , PDC-E2 _249-263 , PDC-E2 _629-643 , PDC -E2 _72-86 , PDC-E2 _353-367 , PDC-E2 _422-436 , PDC-E2 _629-643 , PDC-E2 _80-94 , PDC-E2 _353-367 , PDC-E2 _535-549 or fragment or the equivalent of any of them;

e) a pemphigus foliaceus-associated antigen and/or a pemphigus vulgaris-associated antigen, each derived from an antigen selected from one or more of the group: _{DG1 216-229} , DG3 _97-111 , DG3 _251-265 , DG3 _441-455 , DG3 _351-365 , DG3 _453-467 , _{DG3 540-554} , _{DG3 280-294} , _{DG3 326-340} , _{DG3 367-381} , _{DG1 206-222} , _{DG1 363-377} , DG1 _3-17 , DG1 _192-206 , _{DG1 326-340} , DG1 _1-15 , DG1 _35-49 , _{DG1 325-339} or a fragment or equivalent of any of them;

f) an optic neuromyelitis-associated antigen derived from an antigen selected from one or more of the group: AQP4 _129-143 , AQP4 _284-298 , AQP4 _63-76 , AQP4 _129-143 , AQP4 _39-53 or a fragment or the equivalent of any of them;

g) an allergic asthma associated antigen derived from an antigen selected from one or more of the group: DERP1 _16-30 , DERP1 _171-185 , DERP1 _110-124 , DERP-2 _26-40 , DERP-2 _107-121 or a fragment or the equivalent of any of them;

h) an inflammatory bowel disease associated antigen derived from an antigen selected from one or more of the group: bacteroid integrase antigen _183-197 , bacteroid integrase antigen _146-160 , bacteroid integrase antigen _175-189 , bacteroid integrase antigen _1-15 , antigen bacteroid integrase _183-197 , bacteroid integrase antigen _30-44 , bacteroid integrase antigen _70-84 , bacteroid integrase antigen _337-351 , bacteroid integrase antigen _171-185 , bacteroid integrase antigen _4-18 , bacteroid integrase antigen _256-270 , Fla- 2/Fla-X _366-380 , Fla-2/Fla-X _164-178 , Fla-2/Fla-X _261-275 , Fla-2/Fla-X _1-15 , Fla-2/Fla-X _{51 -65} Fla-2/Fla-X _269-283 Fla-2/Fla-X _4-18 Fla-2/Fla-X _271-285 YIDX _78-92 YIDX _93-107 YIDX _98-112 , YIDX _23-37 , YIDX _78-92 , YIDX _195-209 , YIDX _22-36 , YIDX _80-94 , YIDX _101-115 or a fragment or equivalent of any of them;

i) an antigen associated with systemic lupus erythematosus derived from an antigen selected from one or more of the group: H4 _71-94 , H4 _74-88 , H4 _76-90 , H4 _75-89 , H4 _78-92 , H4 _80-94 , H2B _10-24 , H2B _16-30 , H1' _22-42 , H1' _27-41 or a fragment or equivalent of any of them;

j) an atherosclerosis-associated antigen derived from an antigen selected from one or more of the group: ApoB _3501-3516 , ApoB _1952-1966 , ApoB _978-993 , ApoB _3498-3513 , ApoB _210A , ApoB _210B , ApoB _210C or a fragment, or the equivalent of any of them;

k) a COPD-associated antigen and/or an emphysema-associated antigen, each derived from an antigen selected from one or more of the group: elastin _89-103 , elastin _698-712 , elastin _8-22 , elastin _94-108 , elastin _13-27 , elastin _695-709 , elastin _563-577 , elastin _558-572 , elastin _698-712 , elastin _566-580 , elastin _645-659 , or a fragment or equivalent of any of them;

l) a psoriasis-associated antigen derived from an antigen selected from one or more of the group: Cap18 _64-78 , Cap18 _34-48 , Cap18 _47-61 , Cap18 _151-165 , Cap18 _149-163 , Cap18 _152-166 , Cap18 _{ADMTSL5 267-281 ADMTSL5 372-386 ADMTSL5 289-303} ADMTSL5 _{396-410 ADMTSL5 433-447 ADMTSL5 132-156 -315} , ADMTSL5 _203-217 , ADMTSL5 _404-418 , or fragment or equivalent of any of them;

m) _{autoimmune hepatitis associated antigen derived} from an _antigen selected from one or more of the group: ₂₁₃ , CYP2D6 _450-464 , CYP2D6 _301-315 , CYP2D6 _452-466 , CYP2D6 _59-73 , CYP2D6 _130-144 , CYP2D6 _193-212 , CYP2D6 _305-324 , CYP2D6 _131-145 , CYP2D6 _{216-230-230-230 238- 252} , CYP2D6 _199-213 , CYP2D6 _235-252 , CYP2D6 _293-307 , CYP2D6 _381-395 , CYP2D6 _429-443 , SLA _334-348 , SLA _196-210 , SLA _115-129 , SLA _{37 . 197} SLA _317-331 SLA _171-185 SLA _417-431 SLA _359-373 SLA _215-229 SLA _{111-125 SLA 110-124 SLA 299-313 63} , SLA _119-133 , SLA _260-274 , SLA _26-40 , SLA _86-100 , SLA _331-345 , or a fragment or equivalent of any of them;

n) a uveitis associated antigen derived from an antigen selected from one or more of the group: arrestin _199-213 , arrestin _77-91 , arrestin _250-264 , arrestin _172-186 , arrestin _354-368 , arrestin _239-253 , arrestin _102-116 , arrestin _59-73 , arrestin _280-294 , arrestin _291-306 , arrestin _195-209 , arrestin _200-214 or fragment or equivalent of any of them;

o) an _antigen associated with _{Sjögren 's syndrome} , _derived from an _antigen selected from one or more of the group: _{RO60426-440 , RO60267-281 , RO60178-192 , RO60358-372 _ _ _} LA _241-255 , LA _101-115 , LA _153-167 , LA _178-192 , LA _19-33 , LA _37-51 , LA _133-147 , LA _50-64 , LA _32-46 , LA _153-167 , LA _83-97 , LA _136-150 , LA _297-311 , LA _59-73 , LA _151-165 , LA _86-100 , LA _154-168 or a fragment or equivalent of any of them;

p) a scleroderma-associated antigen derived from an antigen selected from one or more of the group: TOP1 _346-360 , TOP1 _420-434 , TOP1 _750-764 , TOP1 _419-433 , TOP1 _591-605 , TOP1 _695-709 , TOP1 _305-319 , TOP1 _346-360 , TOP1 _419-433 , TOP1 _425-439 , TOP1 _614-628 , CENP-C _297-311 , CENP-C _857-871 , CENP-C _887-901 , CENP-C _{212- 226} CENP-C _643-657 CENP-C _832-846 CENP-C _167-181 CENP-C _246-260 CENP-C _846-860 CENP-C _149-163 CENP-C _833-847 , CENP-C _847-861 or fragment or equivalent of any of them;

q) an antiphospholipid syndrome associated antigen derived from an antigen selected from one or more of the group: APO _235-249 , APO _306-320 , APO _237-251 , APO _295-309 , APO _28-42 , APOH _173-187 , APOH _264-278 , APO _295-309 , APO _49-63 , APO _269-283 , APO _295-309 , APO _321-355 , APO _322-336 , APO _324-338 or a fragment or equivalent of any of them;

r) an ANCA-associated vasculitis-associated antigen derived from an antigen selected from one or more of the group: MPO _506-520 , MPO _302-316 , MPO _7-21 , MPO _689-703 , MPO _248-262 , MPO _{444- 458} MPO _513-527 MPO _97-111 MPO _616-630 MPO _462-476 MPO _617-631 MPO _714-728 PRTN3 _44-58 PRTN3 _234-248 PRTN3 _59-73 PRTN3 _{117 131} , PRTN3 _164-178 , PRTN3 _71-85 , PRTN3 _241-255 , PRTN3 _59-73 , PRTN3 _183-197 , PRTN3 _62-76 , PRTN3 _118-132 , PRTN3 _239-253 or a fragment or equivalent of any of them; or

s) a stiffness syndrome-associated antigen derived from an antigen selected from one or more of the group: GAD _212-226 , GAD _555-569 , GAD _297-311 or a fragment or equivalent of any of them.

In accordance with certain aspects, the rMHC complex contains:

a) a diabetes associated antigen derived from an antigen selected from one or more of the group: hInsB _10-18 , hIGRP _228-236 , hIGRP _265-273 , IGRP _206-214 , hIGRP _206-214 , NRP-A7, NRP- I4, NRP-V7, YAI/D ^b , INS B _15-23 , PPI _{76-90 (K88S)} , IGRP _13-25 , GAD _555-567 , GAD _{555-567(557I)} , IGRP _23-35 , B ₂₄ -С ₃₆ , PPI _76-90 , INS-I9, TUM, G6Pase, proinsulin _L2-10 , proinsulin _L3-11 , proinsulin _L6-14 , proinsulin _B5-14 , proinsulin _B10-18 , proinsulin _B14-22 , proinsulin _{B15- 24} , proinsulin _B17-25 , proinsulin _B18-27 , proinsulin _B20-27 , proinsulin _B21-29 , proinsulin _B25-C1 , proinsulin _B27-C5 , proinsulin _C20-28 , proinsulin _C25-33 , proinsulin _C29-A5 , proinsulin _{A1- 10} , proinsulin _A2-10 , proinsulin _A12-20 , or a fragment or equivalent of any of them, and the MHC protein of the pMHC complex contains all or part of HLA-DR, or a fragment or equivalent thereof;

b) a multiple sclerosis-associated antigen derived from an antigen selected from one or more of the group: MOG _35-55 , MOG _36-55 , MAG _287-295 , MAG _509-517 , MAG _556-564 , MBP _110-118 , MOG _114-122 , MOG _166-175 , MOG _172-180 , MOG _179-188 , MOG _188-196 , MOG _181-189 , MOG _205-214 , PLP _80-88 , MAG _287-295 , MAG _509-517 , MAG _556-564 , MOG _97-109 MOG _{97-109(E107S)} , MBP _89-101 , PLP _175-192 , PLP _94-108 , MBP _86-98 , PLP _54-68 , PLP _249-263 , MOG _{156- 170} , MOG _201-215 , MOG _38-52 , MOG _203-217 , PLP _250-264 , MPB _13-32 , MPB _83-99 , MPB _111-129 , MPB _146-170 , MOG _223-237 , MOG _{6- 20} , PLP _88-102 , PLP _139-154 , or a fragment or equivalent of any of them, and the MHC protein of the pMHC complex contains all or part of HLA-DR, or a fragment or equivalent thereof;

c) a celiac disease associated antigen derived from an antigen selected from one or more of the group: aGlia _57-68 , aGlia _62-72 , aGlia _217-229 or a fragment or equivalent of any of them, and the MHC protein of the pMHC complex contains all or part of HLA-DQ or fragment or equivalent;

d) primary biliary cirrhosis associated antigen derived from an antigen selected from one or more of the group: PDC-E2 _122-135 , PDC-E2 _249-262 , PDC-E2 _249-263 , PDC-E2 _629-643 , PDC -E2 _72-86 , PDC-E2 _353-367 , PDC-E2 _422-436 , PDC-E2 _629-643 , PDC-E2 _80-94 , PDC-E2 _353-367 , PDC-E2 _535-549 or fragment or the equivalent of any of them, and the MHC protein of the pMHC complex contains all or part of HLA-DR, or a fragment or equivalent thereof;

e) a pemphigus foliaceus-associated antigen and/or a pemphigus vulgaris-associated antigen, each derived from an antigen selected from one or more of the group: _{DG1 216-229} , DG3 _97-111 , DG3 _251-265 , DG3 _441-455 , DG3 _351-365 , DG3 _453-467 , _{DG3 540-554} , _{DG3 280-294} , _{DG3 326-340} , _{DG3 367-381} , _{DG1 206-222} , _{DG1 363-377} , DG _13-17 , DG1 _192-206 , _{DG1 326-340} , DG1 _1-15 , DG1 _35-49 , _{DG1 325-339} or fragment or equivalent of any of them, and protein The MHC of the rMHC complex contains all or part of HLA-DR, or a fragment or equivalent thereof;

f) an optic neuromyelitis-associated antigen derived from an antigen selected from one or more of the group: AQP4 _129-143 , AQP4 _284-298 , AQP4 _63-76 , AQP4 _129-143 , AQP4 _39-53 or a fragment or an equivalent of any of them, and the MHC protein of the pMHC complex contains all or part of HLA-DR, or a fragment or equivalent thereof;

g) an allergic asthma associated antigen derived from an antigen selected from one or more of the group: DERP1 _16-30 , DERP1 _171-185 , DERP1 _110-124 , DERP-2 _26-40 , DERP-2 _107-121 or a fragment or an equivalent of any of them, and the MHC protein of the pMHC complex contains all or part of a polypeptide from the group: HLA-DR, HLA-DP, or a fragment or equivalent of any of them;

h) an inflammatory bowel disease associated antigen derived from an antigen selected from one or more of the group: bacteroid integrase antigen _183-197 , bacteroid integrase antigen _146-160 , bacteroid integrase antigen _175-189 , bacteroid integrase antigen _1-15 , antigen bacteroid integrase _183-197 , bacteroid integrase antigen _30-44 , bacteroid integrase antigen _70-84 , bacteroid integrase antigen _337-351 , bacteroid integrase antigen _171-185 , bacteroid integrase antigen _4-18 , bacteroid integrase antigen _256-270 , Fla- 2/Fla-X _366-380 , Fla-2/Fla-X _164-178 , Fla-2/Fla-X _261-275 , Fla-2/Fla-X _1-15 , Fla-2/Fla-X _{51 -65} Fla-2/Fla-X _269-283 Fla-2/Fla-X _4-18 Fla-2/Fla-X _271-285 YIDX _78-92 YIDX _93-107 YIDX _98-112 , YIDX _23-37 , YIDX _78-92 , YIDX _195-209 , YIDX _22-36 , YIDX _80-94 , YIDX _101-115 , or a fragment or equivalent of any of them, and the MHC protein of the rMHC complex contains all or part of HLA-DR or a fragment or equivalent thereof;

i) an antigen associated with systemic lupus erythematosus derived from an antigen selected from one or more of the group: H4 _71-94 , H4 _74-88 , H4 _76-90 , H4 _75-89 , H4 _78-92 , H4 _80-94 , H2B _10-24 , H2B _16-30 , H1' _22-42 , H1' _27-41 or a fragment or equivalent of any of them, and the MHC protein of the pMHC complex contains all or part of a polypeptide from the group: IA _d , HLA-DR or fragment or equivalent of any of them;

j) an atherosclerosis-associated antigen derived from an antigen selected from one or more of the group: ApoB _3501-3516 , ApoB _1952-1966 , ApoB _978-993 , ApoB _3498-3513 , ApoB _210A , ApoB _210B , ApoB _210C or a fragment, or the equivalent of any of them, and the MHC protein of the pMHC complex contains all or part of IA _b or a fragment or equivalent thereof;

k) a COPD-associated antigen and/or an emphysema-associated antigen, each derived from an antigen selected from one or more of the group: elastin _89-103 , elastin _698-712 , elastin _8-22 , elastin _94-108 , elastin _13-27 , elastin _695-709 , elastin _563-577 , elastin _558-572 , elastin _698-712 , elastin _566-580 , elastin _645-659 , or a fragment or equivalent of any of them, and the MHC protein of the pMHC complex contains all or part HLA-DR or fragment or equivalent;

l) a psoriasis associated antigen derived from an antigen selected from one or more of the group: Cap18 _64-78 , Cap18 _34-48 , Cap18 _47-61 , Cap18 _151-165 , Cap18 _149-163 , Cap18 _152-166 , Cap18 _{ADMTSL5 267-281 ADMTSL5 372-386 ADMTSL5 289-303} ADMTSL5 _{396-410 ADMTSL5 433-447 ADMTSL5 132-156 -315} , ADMTSL5 _203-217 , ADMTSL5 _404-418 or a fragment or equivalent of any of them, and the MHC protein of the pMHC complex contains all or part of HLA-DR or a fragment or equivalent thereof;

m) an autoimmune hepatitis associated antigen derived from an antigen selected from one or more of the group: CYP2D6 _193-207 , CYP2D6 _76-90 , CYP2D6 _293-307 , CYP2D6 _313-332 , CYP2D6 _393-412 , CYP2D6 _199-213 , CYP2D6 _450-464 , CYP2D6 _301-315 , CYP2D6 _452-466 , CYP2D6 _59-73 , CYP2D6 _130-144 , CYP2D6 193-212, CYP2D6 305-324, CYP2D6 _131-145 , CYP2D6 _216-230 , CYP2D6 _238-252 , CYP2D6 _238-252 , CYP2D6 _238-252 , CYP2D6 _199-213 , CYP2D6 _235-252 , CYP2D6 _293-307 , CYP2D6 _381-395 , CYP2D6 _429-443 , SLA _334-348 , SLA _196-210 , SLA _115-129 , _{SLA 373-386} SLA _317-331 , SLA _171-l85 , SLA _417-431 , SLA _359-373 , SLA _215-229 , SLA _111-125 , SLA _110-124 , SLA _299-313 , SLA _342-356 , SLA _49-63 , SLA _119-133 , SLA _260-274 , SLA _26-40 , SLA _86-100 , SLA _331-345 , or a fragment or equivalent of any of them, and the MHC protein of the pMHC complex contains all or part of HLA-DR or a fragment or equivalent thereof;

n) a uveitis associated antigen derived from an antigen selected from one or more of the group: arrestin _199-213 , arrestin _77-91 , arrestin _250-264 , arrestin _172-186 , arrestin _354-368 , arrestin _239-253 , arrestin _102-116 , arrestin _59-73 , arrestin _280-294 , arrestin _291-306 , arrestin _195-209 , arrestin _200-214 or a fragment or equivalent of any of them, and the MHC protein of the rMHC complex contains all or part of HLA-DR or its fragment or equivalent;

o) an _antigen associated with _{Sjögren 's syndrome} , _derived from an _antigen selected from one or more of the group: _{RO60426-440 , RO60267-281 , RO60178-192 , RO60358-372 _ _ _} LA _241-255 , LA _101-115 , LA _153-167 , LA _178-192 , LA _19-33 , LA _37-51 , LA _133-147 , LA _50-64 , LA _32-46 , LA _153-167 , LA _83-97 , LA _136-150 , LA _297-311 , LA _59-73 , LA _151-165 , LA _86-100 , LA _154-168 or a fragment or equivalent of any of them, and the MHC protein of the rMHC complex contains all or part of a polypeptide from the group: HLA-DR, HLA-DP or a fragment or equivalent of any of them;

p) a scleroderma-associated antigen derived from an antigen selected from one or more of the group: TOP1 _346-360 , TOP1 _420-434 , TOP1 _750-764 , TOP1 _419-433 , TOP1 _591-605 , TOP1 _695-709 , TOP1 _305-319 , TOP1 _346-360 , TOP1 _419-433 , TOP1 _425-439 , TOP1 _614-628 , CENP-C _297-311 , CENP-C _857-871 , CENP-C _887-901 , CENP-C _{212- 226} , CENP-C _643-657 , CENP-C _{832-846 ,} CENP-C _{167-181 ,} CENP- _{C 246-260} , CENP-C _847-861 , or a fragment or equivalent of any of them, and the MHC protein of the pMHC complex contains all or part of HLA-DR, or a fragment or equivalent thereof;

q) an antiphospholipid syndrome associated antigen derived from an antigen selected from one or more of the group: APO _235-249 , APO _306-320 , APO _237-251 , APO _295-309 , APO _28-42 , APOH _173-187 , APOH _264-278 , APO _295-309 , APO _49-63 , APO _269-283 , APO _295-309 , APO _321-355 , APO _322-336 , APO _324-338 or fragment or equivalent of any of them, and MHC protein the rMHC complex contains all or part of HLA-DR or a fragment or equivalent thereof;

d) an ANCA-associated vasculitis-associated antigen derived from an antigen selected from one or more of the group: MPO _506-520 , MPO _302-316 , MPO _7-21 , MPO _689-703 , MPO _248-262 , MPO _{444- 458} MPO _513-527 MPO _97-111 MPO _616-630 MPO _462-476 MPO _617-631 MPO _714-728 PRTN3 _44-58 PRTN3 _234-248 PRTN3 _59-73 PRTN3 _{117 . _ _ _ _ _ _ _ _} the MHC protein of the rMHC complex contains all or part of HLA-DR, or a fragment or equivalent thereof; or

s) a stiffness syndrome-associated antigen derived from an antigen selected from one or more of the group: GAD _212-226 , GAD _555-569 , GAD _297-311 , and the MHC protein of the pMHC complex contains all or part of a polypeptide from the group: HLA -DR, HLA-DQ, or fragment or equivalent of any of them.

In accordance with certain aspects, the rMHC complex is intended for the treatment of:

a) type I diabetes, and the rMHC complex is selected from the group

PPI _76-90(K88S) -HLA-DRB1*0401/DRA, IGRP _13-25 -HLA-DRB1*0301/DRA, GAD _555-567 -HLA-DRB1*0401/DRA, GAD _{555-567(557I)} - HLA-DRB1*0401/DRA, IGRP _23-35 -HLA-DRB1*0401/DRA, B ₂₄ -C ₃₆ -HLA-DRB1*0301/DRA or PP1 _76-90 -HLA-DRB1*0401/DRA;

b) multiple sclerosis, and the rMHC complex is selected from the group: MBP _86-98 -HLA-DRB1*1501/DRA, MBP _89-101 -HLA-DRB5*0101/DRA, MOG _38-52 -HLA-DRB4*0101/DRA , MOG _{97-109(E107S)} -HLA-DRB1*0401/DRA, MOG _203-217 -HLA-DRB3*0101/DRA, PLP _54-68 -HLA-DRB3*0101/DRA, PLP _94-108 -HLA- DRB1*0301/DRA, PLP _250-264 -HLA-DRB4*0101/DRA, MPB _13-32 -HLA-DRB5*0101/DRA, MPB _83-99 -HLA-DRB5*0101/DRA, MPB _111-129 - HLA-DRB5*0101 /DRA, MPB _146-170 -HLA-DRB5*0101/DRA, MOG _223-237 -HLA-DRB3*0202/DRA, MOG _6-20 -HLA-DRB5*0101/DRA, PLP _{88- 102} -HLA-DRB3*0202/DRA or PLP _139-154 -HLA-DRB5*0101/DRA;

c) celiac disease, and the rMHC complex is selected from the group: aGlia _57-68 -HLA-DQA1*0501/HLA-DQB1*0201, aGlia _62-72 -HLA-DQA1*0501/HLA-DQB1*0201, aGlia _217-229 -HLA-DQA1*0501/HLA-DQB1*0302 or aGlia _217-229 -HLA-DQA1*03/HLA-DQB1*0302;

d) primary biliary cirrhosis, and the rMHC complex is selected from the group: PDC-E2 _122-135 -HLA-DRB4*0101/DRA, PDC-E2 _249-262 -HLA-DRB4*0101/DRA, PDC-E2 _249-263 - HLA-DRB1*0801/DRA, PDC-E2 _629-643 -HLA-DRB 1*0801/DRA, PDC-E2 _72-86 -HLA-DRB3*0202/DRA, PDC-E2 _353-367 -HLA-DRB3* 0202/DRA, PDC-E2 _422-436 -HLA-DRB3*0202/DRA, PDC-E2 _629-643 -HLA-DRB4*0101/DRA, PDC-E2 _80-94 -HLA-DRB5*0101/DRA, PDC -E2 _353-367 -HLA-DRB5*0101/DRA or PDC-E2 _535-549 -HLA-DRB5*0101/DRA, mPDC-E2 _166-181 -I-Ag ₇ or mPDC-E2 _82-96 -I- _Ag7 ;

e) pemphigus foliaceus and/or pemphigus vulgaris, and the rMHC complex is selected from the group: _{DG1 216-229} -HLA-DRB1*0101/DRA, _{DG1 216-229} -HLA-DRB1*0102/DRA, DG3 _97-111 -HLA- DRB1*0402/DRA, DG3 _251-265 -HLA-DRB1*0402/DRA, DG3 _251-265 -HLA-DRB1*0401/DRA, DG3 _441-455 -HLA-DRB1*0402/DRA, DG3 _351-365 - HLA-DRB3*0202/DRA, DG3 _453-467 -HLA-DRB3*0202/DRA, DG3 _540-554 -HLA-DRB3*0202/DRA, DG3 _280-294 -HLA-DRB4*0101/DRA, DG3 _{326- 340} -HLA-DRB4* 0101/DRA, DG3 _367-381 -HLA-DRB4*0101/DRA, DG3 _13-27 -HLA-DRB5*0101/DRA, DG3 _323-337 -HLA-DRB5*0101/DRA, DG3 _438-452 -HLA-DRB5*0101/DRA, _{DG1 48-62} -HLA-DRB3*0202/DRA, _{DG1 206-222} -HLA-DRB3*0202/DRA, _{DG1 363-377} -HLA-DRB3*0202/DRA , DG1 _3-17 -HLA-DRB4*0101/DRA, DG1 _192-206 -HLA-DRB4*0101/DRA, _{DG1 326-340} -HLA-DRB4*0101/DRA, DG1 _1-15 -HLA-DRB5*0101 /DRA, DG1 _35-49 -HLA-DRB5*0101/DRA or _{DG1 325-339} -HLA-DRB5*0101/DRA;

f) neuromyelitis of the optic nerve with spectral disorder, and the rMHC complex is selected from the group: AQP4 _129-143 -HLA-DRB1*0101/DRA, AQP4 _284-298 -HLA-DRB1*0301/DRA, AQP4 _63-76 -HLA-DRB1 *0301/DRA, AQP4 _129-143 -HLA-DRB1*0401/DRA or AQP4 _39-53 -HLA-DRB1*1501/DRA;

g) allergic asthma, and the rMHC complex is selected from the group: DERP-1 _16-30 -HLA-DRB1*0101/DRA, DERP-1 _16-30 -HLA-DRB1*1501/DRA, DERP _1171-185 -HLA-DRB1 *1501/DRA, DERP-1 _110-124 -HLA-DPB1*0401/DRA, DERP-2 _26-40 -HLA-DRB1*0101/DRA; DERP-2 _26-40 -HLA-DRB1*1501/DRA or DERP-2 _107-121 -HLA-DRB1*0301/DRA;

h) inflammatory bowel disease, and the rMHC complex is selected from the group: bacteroid integrase antigen _183-197 -HLA-DRB3*0101/DRA, bacteroid integrase antigen _146-160 -HLA-DRB3*0101/DRA, bacteroid integrase antigen _175-189 - HLA-DRB3*0101/DRA, bacteroid integrase antigen _1-15 -HLA-DRB5*0101/DRA, bacteroid integrase antigen _183-197 -HLA-DRB5*0101/DRA, bacteroid integrase antigen _183-197 -HLA-DRB3*0101 /DRA, bacteroid integrase antigen _30-44 -HLA-DRB5*0101/DRA, bacteroid integrase antigen _70-84 -HLA-DRB4*0101/DRA, bacteroid integrase antigen _337-351 -HLA-DRB4*0101/DRA, integrase antigen bacteroid _171-185 -HLA-DRB4*0101/DRA, bacteroid integrase antigen _4-18 -HLA-DRB3*0202/DRA, bacteroid integrase antigen _171-185 -HLA-DRB3*0202/DRA, bacteroid integrase antigen _256-270 - HLA-DRB3*0202/DRA, Fla-2/Fla-X _366-380 -HLA-DRB3*0101/DRA, Fla-2/Fla-X _164-178 -HLA-DRB3*0101/DRA, Fla-2/ Fla-X _261-275 -HLA-DRB5*0101/DRA, Fla-2/Fla-X _1-15 -HLA-DRB5*0101/DRA, Fla-2/Fla-X _51-65 -HLA- DRB4*0101/DRA, Fla-2/Fla-X _269-283 -HLA-DRB4*0101/DRA, Fla-2/Fla-X _4-18 -HLA-DRB3*0202/DRA, Fla-2/Fla- X _261-275 -HLA-DRB3*0202/DRA, Fla-2/Fla-X _271-285 -HLA-DRB3*0202DRA, YIDX _78-92 -HLA-DRB3*0101/DRA, YIDX _78-92 -HLA- DRB4*0101/DRA, YIDX _93-107 -HLA-DRB3*0101/DRA, YIDX _98-112 -HLA-DRB5*0101/DRA, YIDX _23-37 -HLA-DRB5*0101/DRA, YIDX _78-92 - HLA-DRB4*0101/DRA, YIDX _195-209 -HLA-DRB4*0101/DRA, YIDX _22-36 -HLA-DRB3*0202/DRA, YIDX _80-94 -HLA-DRB3*0202/DRA or YIDX _{101- 115} -HLA-DRB3*0202/DRA;

i) COPD and/or emphysema and rMHC complex selected from the group: elastin _89-103 -HLA-DRB3*0101/DRA, elastin _698-712 -HLA-DRB5*0101/DRA, elastin _8-22 -HLA-DRB5* 0101/DRA, elastin _94-108 -HLA-DRB5*0101/DRA, elastin _13-27 -HLA-DRB4*0101/DRA, elastin _695-709 -HLA-DRB4*0101DRA, elastin _563-577 -HLA-DRB4* 0101/DRA, elastin _558-572 -HLA-DRB4* 0101/DRA, elastin _698-712 -HLA-DRB5*0101/DRA, elastin _566-580 -HLA-DRB3*0202/DRA or elastin _645-659 -HLA- DRB3*0202/DRA;

j) psoriasis, and the rMHC complex is selected from the group: Cap18 _64-78 -HLA-DRB3*0101/DRA, Cap18 _34-48 -HLA-DRB3*0101/DRA, Cap18 _47-61 -HLA-DRB3*0101/DRA, Cap18 _151-165 -HLA-DRB4*0101/DRA, Cap18 _149-163 -HLA-DRB5*0101/DRA, Cap18 _152-166 -HLA-DRB5*0101/DRA, Cap18 _131-145 -HLA-DRB5*0101/ DRA, Cap _1824-38 -HLA-DRB3*0202/DRA, ADMTSL5 _245-259 -HLA-DRB3*0101/DRA, ADMTSL5 _267-281 -HLA-DRB3*0101/DRA, ADMTSL5 _372-386 -HLA-DRB3* 0101/DRA, ADMTSL5 _289-303 -HLA-DRB4*0101/DRA, ADMTSL5 _396-410 -HLA-DRB4*0101/DRA, ADMTSL5 _433-447 -HLA-DRB4*0101/DRA, ADMTSL5 _142-156 -HLA- DRB5*0101/DRA, ADMTSL5 _236-250 -HLA-DRB5*0101/DRA, ADMTSL5 _301-315 -HLA-DRB5*0101/DRA, ADMTSL5 _203-217 -HLA-DRB3*0202/DRA, ADMTSL5 _404-418 - HLA-DRB3*0202/DRA or ADMTSL5 _433-447 -HLA-DRB3*0202/DRA;

k) autoimmune hepatitis, and the rMHC complex is selected from the group: CYP2D6 _193-207 -HLA-DRB1*0301/DRA, CYP2D6 _76-90 -HLA-DRB1*0301/DRA, CYP2D6 _293-307 -HLA-DRB1*0301/DRA , CYP2D6 _313-332 -HLA-DRB1*0301/DRA, CYP2D6 _393-412 -HLA-DRB1*0301/DRA, CYP2D6 _199-213 -HLA-DRB1*0401/DRA, CYP2D6 _450-464 -HLA-DRB1*0401 /DRA, CYP2D6 _301-315 -HLA-DRB1*0401/DRA, CYP2D6 _452-466 -HLA-DRB1*0701/DRA, CYP2D6 _59-73 -HLA-DRB1*0701/DRA, CYP2D6 _130-144 -HLA-DRB1 *0701/DRA, CYP2D6 _193-212 -HLA-DRB1*0701/DRA, CYP2D6 _305-324 -HLA-DRB1*0701/DRA, CYP2D6 _131-145 -HLA-DRB3*0202/DRA, CYP2D6 _216-230 -HLA -DRB3*0202/DRA, CYP2D6 _238-252 -HLA-DRB3*0202/DRA, CYP2D6 _199-213 -HLA-DRB4*0101/DRA, CYP2D6 _235-252 -HLA-DRB4*0101/DRA, CYP2D6 _293-307 -HLA-DRB4*0101/DRA, CYP2D6 _238-252 -HLA-DRB5*0101/DRA, CYP2D6 _381-395 -HLA-DRB5*0101/DRA, CYP2D6 _429-443 -HLA-DRB5*0101/DRA, SLA _{334 -348} -HLA-DRB1*0301/DRA, SLA _196-210 -HLA-DRB1*0301/DRA, SLA _115-129 -HLA-DRB1*0301/DRA, SLA _373-386 -HLA-DRB1*0301/DRA, SLA _186-197 -HLA-DRB1*0301/DRA, SLA _317-331 - HLA-DRB1*0401/DRA, SLA _171-185 -HLA-DRB1*0401/DRA, SLA _417-431 -HLA-DRB1*0401/DRA, SLA _359-373 -HLA-DRB1*0701/DRA, SLA _{215- 229} -HLA-DRB1*0701/DRA, SLA _111-125 -HLA-DRB1*0701/DRA, SLA _110-124 -HLA-DRB3*0202/DRA, SLA _299-313 -HLA-DRB3*0202/DRA, SLA _342-356 -HLA-DRB3*0202/DRA, SLA _49-63 -HLA-DRB4*0101/DRA, SLA _119-133 -HLA-DRB4*0101/DRA, SLA _260-274 -HLA-DRB4*0101/DRA , SLA _26-40 -HLA-DRB5*0101/DRA, SLA _86-100 -HLA-DRB5*0101/DRA or SLA _331-345 -HLA-DRB5*0101/DRA;

l) uveitis, and the rMHC complex is selected from the group: arrestin _199-213 -HLA-DRB3*0101/DRA, arrestin _77-91 -HLA-DRB3*0101/DRA, arrestin _250-264 -HLA-DRB3*0101/DRA, arrestin _172-186 -HLA-DRB4*0101/DRA, arrestin _354-368 -HLA-DRB4*0101/DRA, arrestin _239-253 -HLA-DRB4*0101/DRA, arrestin _102-116 -HLA-DRB5*0101/ DRA, arrestin _59-73 -HLA-DRB5*0101, arrestin _280-294 -HLA-DRB5*0101, arrestin _291-306 -HLA-DRB1*0301/DRA, arrestin _195-209 -HLA-DRB3*0202/DRA, arrestin _199-213 -HLA-DRB3*0202/DRA or arrestin _200-214 -HLA-DRB3*0202/DRA;

m) Sjögren's syndrome, and the rMHC complex is selected from the group: RO60 _127-141 -HLA-DRB1*0301/DRA, RO60 _523-537 -HLA-DRB1*0301/DRA, RO60 _243-257 -HLA-DRB1*0301/DRA RO60 _484-498 -HLA-DRB3*0101/DRA, RO60 _347-361 -HLA-DRB3*0101/DRA, RO60 _{369-383 -HLA} -DRB3*0101/DRA /DRA, RO60 _267-281 -HLA-DRB4*0101/DRA, RO60 _178-192 -HLA-DRB4*0101/DRA, RO60 _358-372 -HLA-DRB5*0101/DRA, RO60 _358-372 -HLA-DRB4 *0101/DRA, RO60 _221-235 -HLA-DRB5*0101/DRA, RO60 _221-235 -HLA-DRB4*0101/DRA, RO60 _318-332 -HLA-DRB5*0101/DRA, RO60 _318-332 -HLA -DRB4*0101/DRA, RO60 _407-421 -HLA-DRB4*0101/DRA, RO60 _407-421 -HLA-DQA1*0501/HLA-DQB1*0201, RO60 _459-473 -HLA-DRB4*0101/DRA, RO60 _459-473 -HLA-DQA1*0501/HLA-DQB1*0201, RO60 _318-332 -HLA-DQA1*0501/HLA-DQB1*0201, RO60 _51-65 -HLA-DRB3*0202/DRA, RO60 _{312- 326} -HLA-DRB3*0202/DRA, RO60 _347-361 -HLA-DRB3*0202/DRA, LA _241-255 -HLA-DRB1*0301/DRA, LA _101-115 -HLA-DRB1*0301/DRA, LA _153-167 -HLA-DRB1*0301/DRA, LA _178-192 -HLA-DRB3*0101/DRA, LA _19-33 -HLA-DRB3*0101/DRA, LA _{37 -51} -HLA-DRB3*0101/DRA, LA _133-147 -HLA-DRB4*0101/DRA, LA _50-64 -HLA-DRB4*0101/DRA, LA _32-46 -HLA-DRB4*0101/DRA, LA _153-167 -HLA-DRB5*0101/DRA, LA _83-97 -HLA-DRB5*0101/DRA, LA _136-150 -HLA-DRB5*0101/DRA, LA _297-311 -HLA-DQA1*0501/ HLA-DQB1*0201, LA _59-73 -HLA-DQA1*0501/HLA-DQB1*0201, LA _59-73 -HLA-DRB4*0101/DRA, LA _151-165 -HLA-DQA1*0501/HLA-DQB1 *0201, LA _151-165 -HLA-DRB4*0101/DRA, LA _297-311 -HLA-DRB4*0101/DRA, LA _50-64 -HLA-DRB3*0202/DRA, LA _86-100 -HLA-DRB3 *0202/DRA or LA _154-168 -HLA-DRB3*0202/DRA;

n) scleroderma, and the rMHC complex is selected from the group: TOP1 _346-360 -HLA-DRB3*0101/DRA, TOP1 _420-434 -HLA-DRB3*0101/DRA, TOP1 _750-764 -HLA-DRB3*0101/DRA, TOP1 _419-433 -HLA-DRB4*0101/DRA, TOP1 _591-605 -HLA-DRB4*0101/DRA, TOP1 _695-709 -HLA-DRB4*0101/DRA, TOP1 _305-319 -HLA-DRB5*0101/ DRA, TOP1 _346-360 -HLA-DRB5*0101/DRA, TOP1 _419-433 -HLA-DRB5*0101/DRA, TOP1 _420-434 -HLA-DRB3*0202/DRA, TOP1 _425-439 -HLA-DRB3* 0202/DRA, TOP1 _614-628 -HLA-DRB3*0202/DRA, CENP-C _297-311 -HLA-DRB3*0101/DRA, CENP-C _857-871 -HLA-DRB3*0101/DRA, CENP-C _887-901 -HLA-DRB3*0101/DRA, CENP-C _212-226 -HLA-DRB4*0101/DRA, CENP-C _643-657 -HLA-DRB4*0101/DRA, CENP-C _832-846 -HLA -DRB4*0101/DRA, CENP-C _167-181 -HLA-DRB5*0101/DRA, CENP-C _246-260 -HLA-DRB5*0101/DRA, CENP-C _846-860 -HLA-DRB5*0101/ DRA, CENP-C _149-163 -HLA-DRB3*0202/DRA, CENP-C _833-847 -HLA-DRB3*0202/DRA or CENP-C _847-861 -HLA-DRB3*0202/DRA;

o) antiphospholipid syndrome, and the rMHC complex is selected from the group: ARON _235-249 -HLA-DRB3*0101/DRA, APOH _306-320 -HLA-DRB3*0101/DRA, APOH _237-251 -HLA-DRB3*0101/DRA _{APOH 295-309 -HLA} - _DRB3 *0101/DRA /DRA, APOH _295-309 -HLA-DRB4*0101/DRA, APOH _49-63 -HLA-DRB5*0101/DRA, APOH _269-283 -HLA-DRB5*0101/DRA, APOH _295-309 -HLA-DRB5 *0101/DRA, APOH _321-355 -HLA-DRB3*0202/DRA, APOH _322-336 -HLA-DRB3*0202/DRA or APOH _324-338 -HLA-DRB3*0202/DRA;

p) ANCA-associated vasculitis, and the rMHC complex is selected from the group: MPO _506-520 -HLA-DRB3*0101/DRA, MPO _302-316 -HLA-DRB3*0101/DRA, MPO _7-21 -HLA-DRB3*0101 /DRA, MPO _689-703 -HLA-DRB4*0101/DRA, MPO _248-262 -HLA-DRB4*0101/DRA, MPO _444-458 -HLA-DRB4*0101/DRA, MPO _513-527 -HLA-DRB5 *0101/DRA, MPO _97-111 -HLA-DRB5*0101/DRA, MPO _616-630 -HLA-DRB5*0101/DRA, MPO _462-476 -HLA-DRB3*0202/DRA, MPO _617-631 -HLA -DRB3*0202/DRA, MPO _714-728 -HLA-DRB3*0202/DRA, PRTN3 _44-58 -HLA-DRB3*0101/DRA, PRTN3 _234-248 -HLA-DRB3*0101/DRA, PRTN3 _59-73 -HLA DRB3*0101/DRA, PRTN3 _59-73 -HLA-DRB5*0101/DRA, PRTN3 _117-131 -HLA-DRB4*0101/DRA, PRTN3 _164-178 -HLA-DRB4*0101/DRA, PRTN3 _{71- 85} -HLA-DRB4*0101/DRA, PRTN3 _241-255 -HLA-DRB5*0101/DRA, PRTN3 _183-197 -HLA-DRB5*0101/DRA, PRTN3 _62-76 -HLA-DRB3*0202/DRA, PRTN3 _118-132 -HLA-DRB3*0202/DRA or PRTN3 _239-253 -HLA-DRB3*0202/DRA; or

q) stiffness syndrome, and the rMHC complex is selected from the group: GAD _212-226 -HLA-DRB1*0801/DRA, GAD _555-569 -HLA-DRB1*0801/DRA or GAD _297-311 -HLA-DRB1*0301/ DRA.

In accordance with certain aspects, the rMHC complex is intended for the treatment of:

a) type I diabetes, and the rMHC complex is selected from the group

PPI _76-90(K88S) -HLA-DRB1*0401/DRA, IGRP _13-25 -HLA-DRB1*0301/DRA, GAD _555-567 -HLA-DRB1*0401/DRA, GAD _{555-567(557I)} - HLA-DRB1*0401/DRA, IGRP _23-35 -HLA-DRB1*0401/DRA or PPI _76-90 -HLA-DRB1*0401/DRA;

b) multiple sclerosis, and the rMHC complex is selected from the group: MBP _86-98 -HLA-DRB1*1501/DRA, MBP _89-101 -HLA-DRB5*0101/DRA, MOG _38-52 -HLA-DRB4*0101/DRA , MOG _{97-109(E107S)} -HLA-DRB1*0401/DRA, MOG _203-217 -HLA-DRB3*0101/DRA, PLP _54-68 -HLA-DRB3*0101/DRA, PLP _94-108 -HLA- DRB1*0301/DRA, PLP _250-264 -HLA-DRB4*0101/DRA, MPB _13-32 -HLA-DRB5*0101/DRA, MPB _83-99 -HLA-DRB5*0101/DRA, MPB _111-129 - HLA-DRB5*0101/DRA, MPB _146-170 -HLA-DRB5*0101/DRA, MOG _223-237 -HLA-DRB3*0202/DRA, MOG _6-20 -HLA-DRB5*0101/DRA, PLP _{88- 102} -HLA-DRB3*0202/DRA or PLP _139-154 -HLA-DRB5*0101/DRA;

c) celiac disease, and the rMHC complex is selected from the group: aGlia _57-68 -HLA-DQA1*0501/HLA-DQB1*0201, aGlia _62-72 -HLA-DQA1*0501/HLA-DQB1*0201 or aGlia _217-229 -HLA-DQA1*0501/HLA-DQB1*0302;

d) primary biliary cirrhosis, and the rMHC complex is selected from the group: PDC-E2 _122-135 -HLA-DRB4*0101/DRA, PDC-E2 _249-262 -HLA-DRB4*0101/DRA, PDC-E2 _249-263 - HLA-DRB1*0801/DRA, PDC-E2 _629-643 -HLA-DRB1*0801/DRA, PDC-E2 _72-86 -HLA-DRB3*0202/DRA, PDC-E2 _353-367 -HLA-DRB3*0202 /DRA, PDC-E2 _422-436 -HLA-DRB3*0202/DRA, PDC-E2 _629-643 -HLA-DRB4*0101/DRA, PDC-E2 _80-94 -HLA-DRB5*0101/DRA, PDC- E2 _353-367 -HLA-DRB5*0101/DRA or PDC-E2 _535-549 -HLA-DRB5*0101/DRA;

e) pemphigus foliaceus and/or pemphigus vulgaris, and the rMHC complex is selected from the group: _{DG1 216-229} -HLA-DRB1*0101/DRA, DG3 _97-111 -HLA-DRB1*0402/DRA, DG3 _251-265 -HLA- DRB1*0401/DRA, DG3 _441-455 -HLA-DRB1*0402/DRA, DG3 _351-365 -HLA-DRB3*0202/DRA, DG3 _453-467 -HLA-DRB3*0202/DRA, DG3 _540-554 - HLA-DRB3*0202/DRA, DG3 _280-294 -HLA-DRB4*0101/DRA, DG3 _326-340 -HLA-DRB4*0101/DRA, DG3 _367-381 -HLA-DRB4*0101/DRA, DG3 _{13- 27} -HLA-DRB5*0101/DRA, DG3 _323-337 -HLA-DRB5*0101/DRA, DG3 _438-452 -HLA-DRB5*0101/DRA, _{DG1 48-62} -HLA-DRB3*0202/DRA, DG1 _206-222 -HLA-DRB3*0202/DRA, _{DG1 363-377} -HLA-DRB3*0202/DRA, DG1 _3-17 -HLA-DRB4*0101/DRA, DG1 _192-206 -HLA-DRB4*0101/DRA , _{DG1 326-340} -HLA-DRB4*0101/DRA, DG1 _1-15 -HLA-DRB5*0101/DRA, DG1 _35-49 -HLA-DRB5*0101/DRA or _{DG1 325-339} -HLA-DRB5*0101 /DRA;

f) neuromyelitis of the optic nerve with spectral disorder, and the rMHC complex is selected from the group: AQP4 _284-298 -HLA-DRB1*0301/DRA, AQP4 _63-76 -HLA-DRB1*0301/DRA, AQP4 _129-143 -HLA-DRB1 *0401/DRA or AQP4 _39-53 -HLA-DRB1*1501/DRA;

g) allergic asthma, and the rMHC complex is selected from the group: DERP-1 _16-30 -HLA-DRB1*0101/DRA, DERP-1 _16-30 -HLA-DRB1*1501/DRA, DERP _1171-185 -HLA-DRB1 *1501/DRA, DERP-1 _110-124 -HLA-DPB1*0401/DRA, DERP-2 _26-40 -HLA-DRB1*0101/DRA; DERP-2 _26-40 -HLA-DRB1*1501/DRA, or DERP-2 _107-121 -HLA-DRB1*0301/DRA;

h) inflammatory bowel disease, and the rMHC complex is selected from the group: bacteroid integrase antigen _1-15 -HLA-DRB5*0101/DRA, bacteroid integrase antigen _183-197 -HLA-DRB3*0101/DRA, bacteroid integrase antigen _70-84 - HLA-DRB4*0101/DRA, bacteroid integrase antigen _4-18 -HLA-DRB3*0202/DRA, bacteroid integrase antigen _171-185 -HLA-DRB3*0202/DRA, bacteroid integrase antigen _256-270 -HLA-DRB3*0202 /DRA, Fla-2/Fla-X _366-380 -HLA-DRB3*0101/DRA, Fla-2/Fla-X _261-275 -HLA-DRB5*0101/DRA, Fla-2/Fla-X _{51- 65} -HLA-DRB4*0101/DRA, Fla-2/Fla-X _4-18 -HLA-DRB3*0202/DRA, Fla-2/Fla-X _261-275 -HLA-DRB3*0202/DRA, Fla- 2/Fla-X _271-285 -HLA-DRB3*0202/DRA, YIDX _78-92 -HLA-DRB3*0101/DRA, YIDX _78-92 -HLA-DRB4*0101/DRA, YIDX _98-112 -HLA- DRB5*0101/DRA, YIDX _22-36 -HLA-DRB3*0202/DRA, YIDX _80-94 -HLA-DRB3*0202/DRA or YIDX _101-115 -HLA-DRB3*0202/DRA;

i) emphysema, and the rMHC complex is selected from the group: elastin _89-103 -HLA-DRB3*0101/DRA, elastin _698-712 -HLA-DRB5*0101/DRA, elastin _558-572 -HLA-DRB4*0101/DRA, elastin _566-580 -HLA-DRB3*0202/DRA, or elastin _645-659 -HLA-DRB3*0202/DRA;

j) psoriasis, and the rMHC complex is selected from the group: Cap18 _64-78 -HLA-DRB3*0101/DRA, Cap18 _34-48 -HLA-DRB3*0101/DRA, Cap18 _47-61 -HLA-DRB3*0101/DRA, Cap18 _151-165 -HLA-DRB4*0101/DRA, Cap18 _149-163 -HLA-DRB5*0101/DRA, Cap18 _152-166 -HLA-DRB5*0101/DRA, Cap18 _131-145 -HLA-DRB5*0101/ DRA, Cap _1824-38 -HLA-DRB3*0202/DRA, ADMTSL5 _245-259 -HLA-DRB3*0101/DRA, ADMTSL5 _267-281 -HLA-DRB3*0101/DRA, ADMTSL5 _372-386 -HLA-DRB3* 0101/DRA, ADMTSL5 _289-303 -HLA-DRB4*0101/DRA, ADMTSL5 _396-410 -HLA-DRB4*0101/DRA, ADMTSL5 _433-447 -HLA-DRB4*0101/DRA, ADMTSL5 _142-156 -HLA- DRB5*0101/DRA, ADMTSL5 _236-250 -HLA-DRB5*0101/DRA, ADMTSL5 _301-315 -HLA-DRB5*0101/DRA, ADMTSL5 _203-217 -HLA-DRB3*0202/DRA, ADMTSL5 _404-418 - HLA-DRB3*0202/DRA or ADMTSL5 _433-447 -HLA-DRB3*0202/DRA;

k) autoimmune hepatitis, and the rMHC complex is selected from the group: CYP2D6 _193-207 -HLA-DRB1*0301/DRA, CYP2D6 _76-90 -HLA-DRB1*0301/DRA, CYP2D6 _293-307 -HLA-DRB1*0301/DRA , CYP2D6 _313-332 -HLA-DRB1*0301/DRA, CYP2D6 _393-412 -HLA-DRB1*0301/DRA, CYP2D6 _199-213 -HLA-DRB1*0401/DRA, CYP2D6 _450-464 -HLA-DRB1*0401 /DRA, CYP2D6 _301-315 -HLA-DRB1*0401/DRA, CYP2D6 _452-466 -HLA-DRB1*0701/DRA, CYP2D6 _59-73 -HLA-DRB1*0701/DRA, CYP2D6 _130-144 -HLA-DRB1 *0701/DRA, CYP2D6 _193-212 -HLA-DRB1*0701/DRA, CYP2D6 _305-324 -HLA-DRB1*0701/DRA, CYP2D6 _131-145 -HLA-DRB3*0202/DRA, CYP2D6 _216-230 -HLA -DRB3*0202/DRA, CYP2D6 _238-252 -HLA-DRB3*0202/DRA, CYP2D6 _199-213 -HLA-DRB4*0101/DRA, CYP2D6 _235-252 -HLA-DRB4*0101/DRA, CYP2D6 _293-307 -HLA-DRB4*0101/DRA, CYP2D6 _238-252 -HLA-DRB5*0101/DRA, CYP2D6 _381-395 -HLA-DRB5*0101/DRA, CYP2D6 _429-443 -HLA-DRB5*0101/DRA, SLA _{334 -348} -HLA-DRB1*0301/DRA, SLA _196-210 -HLA-DRB1*0301/DRA, SLA _115-129 -HLA-DRB1*0301/DRA, SLA _373-386 -HLA-DRB1*0301/DRA, SLA _186-197 -HLA-DRB1*0301/DRA, SLA _317-331 - HLA-DRB1*0401/DRA, SLA _171-185 -HLA-DRB1*0401/DRA, SLA _417-431 -HLA-DRB1*0401/DRA, SLA _359-373 -HLA-DRB1*0701/DRA, SLA _{215- 229} -HLA-DRB1*0701/DRA, SLA _111-125 -HLA-DRB1*0701/DRA, SLA _110-124 -HLA-DRB3*0202/DRA, SLA _299-313 -HLA-DRB3*0202/DRA, SLA _342-356 -HLA-DRB3*0202/DRA, SLA _49-63 -HLA-DRB4*0101/DRA, SLA _119-133 -HLA-DRB4*0101/DRA, SLA _260-274 -HLA-DRB4*0101/DRA , SLA _26-40 -HLA-DRB5*0101/DRA, SLA _86-100 -HLA-DRB5*0101/DRA or SLA _331-345 -HLA-DRB5*0101/DRA;

l) uveitis, and the rMHC complex is selected from the group: arrestin _199-213 -HLA-DRB3*0101/DRA, arrestin _77-91 -HLA-DRB3*0101/DRA, arrestin _250-264 -HLA-DRB3*0101/DRA, arrestin _172-186 -HLA-DRB4*0101/DRA, arrestin _354-368 -HLA-DRB4*0101/DRA, arrestin _239-253 -HLA-DRB4*0101/DRA, arrestin _102-116 -HLA-DRB5*0101/ DRA, arrestin _59-73 -HLA-DRB5*0101, arrestin _280-294 -HLA-DRB5*0101, arrestin _291-306 -HLA-DRB1*0301/DRA, arrestin _195-209 -HLA-DRB3*0202/DRA, arrestin _199-213 -HLA-DRB3*0202/DRA, or arrestin _200-214 -HLA-DRB3*0202/DRA;

m) Sjögren's syndrome, and the rMHC complex is selected from the group: RO60 _127-141 -HLA-DRB1*0301/DRA, RO60 _523-537 -HLA-DRB1*0301/DRA, RO60 _243-257 -HLA-DRB1*0301/DRA RO60 _484-498 -HLA-DRB3*0101/DRA, RO60 _347-361 -HLA-DRB3*0101/DRA, RO60 _{369-383 -HLA} -DRB3*0101/DRA /DRA, RO60 _267-281 -HLA-DRB4*0101/DRA, RO60 _178-192 -HLA-DRB4*0101/DRA, RO60 _358-372 -HLA-DRB5*0101/DRA, RO60 _221-235 -HLA-DRB5 *0101/DRA, RO60 _318-332 -HLA-DRB5*0101/DRA, RO60 _51-65 -HLA-DRB3*0202/DRA, RO60 _312-326 -HLA-DRB3*0202/DRA, RO60 _347-361 -HLA -DRB3*0202/DRA, LA _241-255 -HLA-DRB1*0301/DRA, LA _101-115 -HLA-DRB1*0301/DRA, LA _153-167 -HLA-DRB1*0301/DRA, LA _178-192 -HLA-DRB3*0101/DRA, LA _19-33 -HLA-DRB3*0101/DRA, LA _37-51 -HLA-DRB3*0101/DRA, LA _133-147 -HLA-DRB4*0101/DRA, LA _{50 -64} -HLA-DRB4*0101/DRA, LA _32-46 -HLA-DRB4*0101/DRA, LA _153-167 -HLA-DRB5*0101/DRA, LA _83-97 -HLA-DRB5*0101/DRA, LA _136-150 -HLA-DRB5*0101/DRA, LA _50-64 -HLA-DRB3*0202/DRA, LA _86-100 -HLA-DRB3*0202/DRA or LA _154-168 -HLA-DRB3*0202/ DR A;

n) scleroderma, and the rMHC complex is selected from the group: TOP1 _346-360 -HLA-DRB3*0101/DRA, TOP1 _420-434 -HLA-DRB3*0101/DRA, TOP1 _750-764 -HLA-DRB3*0101/DRA, TOP1 _419-433 -HLA-DRB4*0101/DRA, TOP1 _591-605 -HLA-DRB4*0101/DRA, TOP1 _695-709 -HLA-DRB4*0101/DRA, TOP1 _305-319 -HLA-DRB5*0101/ DRA, TOP1 _346-360 -HLA-DRB5*0101/DRA, TOP1 _419-433 -HLA-DRB5*0101/DRA, TOP1 _420-434 -HLA-DRB3*0202/DRA, TOP1 _425-439 -HLA-DRB3* 0202/DRA, TOP1 _614-628 -HLA-DRB3*0202/DRA, CENP-C _297-311 -HLA-DRB3*0101/DRA, CENP-C _857-871 -HLA-DRB3*0101, CENP-C _{887- 901} -HLA-DRB3*0101, CENP-C _212-226 -HLA-DRB4*0101/DRA, CENP-C _643-657 -HLA-DRB4*0101/DRA, CENP-C _832-846 -HLA-DRB4*0101 /DRA, CENP-C _167-181 -HLA-DRB5*0101/DRA, CENP-C _246-260 -HLA-DRB5*0101/DRA, CENP-C _846-860 -HLA-DRB5*0101/DRA, CENP- C _149-163 -HLA-DRB3*0202/DRA, CENP-C _833-847 -HLA-DRB3*0202/DRA, or CENP-C _847-861 -HLA-DRB3*0202/DRA;

o) antiphospholipid syndrome, and the rMHC complex is selected from the group: ARON _235-249 -HLA-DRB3*0101/DRA, APOH _306-320 -HLA-DRB3*0101/DRA, APOH _237-251 -HLA-DRB3*0101/DRA , APOH _295-309 -HLA-DRB3*0101/DRA, APOH _28-42 -HLA-DRB4*0101/DRA, ARON _173-187 -HLA-DRB4*0101/DRA, ARON _264-278 -HLA-DRB4*0101 /DRA, ARON _295-309 -HLA-DRB4*0101/DRA, APOH _49-63 -HLA-DRB5*0101/DRA, APOH _269-283 -HLA-DRB5*0101/DRA, APOH _295-309 -HLA-DRB5 *0101/DRA, APOH _321-355 -HLA-DRB3*0202/DRA, APOH _322-336 -HLA-DRB3*0202/DRA or APOH _324-338 -HLA-DRB3*0202/DRA;

p) ANCA-associated vasculitis, and the rMHC complex is selected from the group: MPO _506-520 -HLA-DRB3*0101/DRA, MPO _302-316 -HLA-DRB3*0101/DRA, MPO _7-21 -HLA-DRB3*0101 /DRA, MPO _689-703 -HLA-DRB4*0101/DRA, MPO _248-262 -HLA-DRB4*0101/DRA, MPO _444-458 -HLA-DRB4*0101/DRA, MPO _513-527 -HLA-DRB5 *0101/DRA, MPO _97-111 -HLA-DRB5*0101/DRA, MPO _616-630 -HLA-DRB5*0101/DRA, MPO _462-476 -HLA-DRB3*0202/DRA, MPO _617-631 -HLA -DRB3*0202/DRA, MPO _714-728 -HLA-DRB3*0202/DRA, PRTN3 _44-58 -HLA-DRB3*0101/DRA, PRTN3 _234-248 -HLA-DRB3*0101/DRA, PRTN3 _59-73 -HLA DRB3*0101/DRA, PRTN3 _59-73 -HLA-DRB5*0101/DRA, PRTN3 _117-131 -HLA-DRB4*0101/DRA, PRTN3 _164-178 -HLA-DRB4*0101/DRA, PRTM3 _{71- 85} -HLA-DRB4*0101/DRA, PRTN3 _241-255 -HLA-DRB5*0101/DRA, PRTN3 _183-197 -HLA-DRB5*0101/DRA,PRTN3 _62-76 -HLA-DRB3*0202/DRA, PRTN3 _118-132 -HLA-DRB3*0202/DRA or PRTN3 _239-253 -HLA-DRB3*0202/DRA; or

q) stiffness syndrome, and the rMHC complex is selected from the group: GAD _212-226 -HLA-DRB1*0801/DRA, GAD _555-569 -HLA-DRB1*0801/DRA or GAD _297-311 -HLA-DRB1*0301/ DRA.

In accordance with certain aspects, provided herein are methods for treating Type I diabetes in an individual in need thereof, comprising administering an effective amount of a complex or composition described herein, wherein the complex and composition may comprise one or more nanoparticle cores associated with a plurality of rMHC complexes. where the rMHC complex antigen is a diabetes-associated antigen, wherein the rMHC complex MHC protein comprises an MHC class II protein, where the nanoparticle core diameter is from about 1 nm to about 100 nm, and where the rMHC density on the nanoparticle core is from about 0.4 pMHC/100 nm ² to about 11.6 pMHC/100 nm ² . In accordance with certain embodiments, the core diameter of the nanoparticle is from about 1 nm to about 75 nm; from about 1 nm to about 50 nm; about 1 nm to about 25 nm; about 1 nm to about 25 nm; from about 5 nm to about 100 nm; from about 5 nm to about 50 nm; or about 5 nm to about 25 nm, or about 15 nm to about 25 nm, or about 20 nm. In certain embodiments, the nanoparticle core diameter is about 25 nm to about 60 nm, or about 25 nm to about 50 nm, or about 20 nm to about 40 nm, or about 15 nm to about 50 nm, or about 15 nm to about 40 nm, or about 15 nm to about 35 nm, or about 15 nm to about 30 nm, or about 15 nm to about 25 nm, or alternatively about 15 nm, or about 20 nm, or about 25 nm, or about 30 nm, or about 35 nm, or about 40 nm. In accordance with certain embodiments, the pMHC density at the core of the nanoparticle is from about 0.4 pMHC/100 nm ² to about 11.6 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 11.0 pMHC /100 nm ² , or from about 0.4 pMHC/100 nm ² to about 10 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 9 pMHC/100 nm ² , or from about 0, 4 pMHC/100 nm ² to about 8 pMHC/100 nm ² , or about 0.4 pMHC/100 nm ² to about 7 pMHC/100 nm ² , or about 0.4 pMHC/100 nm ² to about 6 pMHC /100 nm ² , or from about 0.4 pMHC/100 nm ² to about 5 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 4 pMHC/100 nm ² , or from about 0, 4 pMHC/100 nm ² to about 3 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about 2 pMHC/100 nm ² , or from about 0.4 pMHC/100 nm ² to about lno 1.5 pMHC / 100 nm ² . In accordance with certain embodiments, the pMHC density on the core of the nanoparticle is from about 0.4 pMHC/100 nm ² to about 6 pMHC/100 nm ² or from about 0.4 pMHC/100 nm ² to about 1.5 pMHC/100 nm ² . In certain embodiments, the pMHC complex comprises an antigen derived from one or more of IGRP or PPI. In certain embodiments, the pMHC complex comprises an antigen selected from one or more of the group: IGRP _13-25 , PPI _76-90 or PPI _76-90(K88S) . In accordance with certain embodiments, the rMHC complex contains HLA-DR. In accordance with certain embodiments, the rMHC complex comprises HLA-DR/DRA. In certain embodiments, the rMHC complex comprises, or alternatively consists of, or even more substantially consists of, one or more of IGRP _13-25 -HLA-DRB1*0301/DRA, PPI _76-90 -HLA-DRB1* 0401/DRA or PPI _76-90(K88S) -HLA-DRB1*0401/DRA.

Methods for determining and monitoring therapy are known in the art and are briefly described herein. For in vitro delivery, administration is by bringing the composition into contact with tissue or cells in any suitable manner, for example, by incorporation into a cell or tissue culture medium, and is useful as a screen to determine the suitability of a therapy for an individual or to screen alternative therapeutic agents for use. as a substitute or in combination with the described compositions. When administered in vivo, administration is by systemic or local administration. Prior to administration to a human, in vivo methods can be performed in a non-human animal to screen for alternative therapeutics for use as a substitute or in combination with the described compositions. In a human or non-human mammal, they are also useful in the treatment of a disease or disorder.

These methods require the administration of an effective amount of an antigen/MHC complex operably linked to the nanoparticles as described herein above, which may optionally further comprise, alternatively consist largely of, or even more so, consist of co-stimulatory molecules and/or cytokines associated the same nanoparticle. Target diseases and associated antigens are described above herein.

Details regarding in vitro and in vivo administration methods are described above in this document.

The present invention also relates to the use of NP complexes for the preparation of medicaments for the treatment and/or prevention of diseases and disorders as described herein.

Therapy monitoring and T cell detection

Certain aspects of the present invention relate to methods for detecting and/or monitoring a population of immunocytes, preferably T cells, comprising administering a labeled antigen-MHC complex when a pMHC-NP or composition is administered to an individual as described herein.

In accordance with certain aspects, provided herein are methods for detecting a population of T _R 1 cells and/or effector T cells in an antigen-specific manner in an individual who has received the complex or composition described herein. The method comprises, alternatively consists of, or even more essentially consists of bringing a sample containing suspected T _R 1 cells into contact with an effective amount of a labeled pMHC complex to form a multimeric complex, and detecting any multimeric complex, thereby detecting a population of T _R 1 cells. In certain embodiments, the method further comprises, alternatively further consists of, or even more essentially consists of, staining any population of T cells using a labeled multimeric complex. In accordance with certain embodiments, the detection step of the T _R 1 cell population comprises flow cytometry to detect any multimeric complex. In accordance with certain embodiments, the method further comprises, or alternatively consists of, or even more essentially consists of, administering the complex or composition to the individual.

In accordance with certain aspects, provided herein are methods for detecting a population of T _R 1 cells and/or effector T cells in an antigen-specific manner in an individual who has received the complex or composition described herein. The method includes, alternatively consists of, or even more essentially consists of any of the following assays: cytokine ELISPOT assay, multimer-guided epitope assay, or multimer precipitation assay. In accordance with certain embodiments, the method further comprises, alternatively further consists of, or even more essentially consists of administering the complex or composition described herein.

In accordance with other aspects, provided herein are methods for monitoring the expansion of a population of antigen-specific T _R 1 and/or effector T cells in an individual. The method comprises, alternatively consists of, or even more essentially consists of: a) administering to an individual an effective amount of a complex or composition described herein, wherein a disease-associated rMHC is selected to propagate the antigen-specific T _R 1 and/or effector T cells in the complex antigen; b) isolating a suitable sample from an individual suspected of having a population; c) contacting the sample with an effective amount of the labeled pMHC complex to form a multimeric complex, and detecting any multimeric complex; and d) quantifying antigen-specific T _R 1 and/or effector T cell populations. According to certain embodiments, the method further comprises, alternatively further consists of, or even more essentially consists of, staining any multimeric complex. In certain embodiments, the step of quantifying antigen-specific T _R 1 and/or effector T cells comprises flow cytometry and/or ELISA. In accordance with certain embodiments, the method further comprises, alternatively further consists of, or even more essentially consists of administering the complex or composition described herein.

There are many types of immunoassays that can be used. Immunological assays included in the present invention include, by way of non-limiting examples, the assays described in US Pat. No. 4,367,110 (two monoclonal antibody sandwich assay) and US Pat. Other assays include labeled ligand immunoprecipitation and immunocytochemistry, in vitro and in vivo.

One way to quantify the number of circulating antigen-specific immunocytes is by tetramer analysis. In this assay, a specific epitope is associated with synthetic multimeric forms of fluorescently labeled MHC molecules. Since immunocytes recognize antigens in the form of short peptides associated with MHC molecules, cells with the appropriate T cell receptor bind to labeled tetramers and can be quantified by flow cytometry. Although this method is less time consuming than the ELISPOT assay, only binding and not function is determined in the multimer assay. Not all cells that bind a particular antigen are necessarily activated. However, a correlation between ELISPOT assays, multimers and cytotoxicity has been demonstrated.

Typically, immunoassays are binding assays. Certain immunoassays are known in the art, including various types of enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), or bead-based assays such as Luminex® technology. Also particularly useful is immunohistochemical detection using tissue sections.

In one example of an ELISA, the antibodies or antigens are immobilized on a selected surface, such as a well in a polystyrene microtiter plate, a test strip, or a column carrier. The test composition, presumably containing the desired antigen or antibody, such as a clinical specimen, is then added to the wells. After binding and washing to remove non-specifically bound immune complexes, bound antigen or antibody can be detected. Typically, detection is carried out by adding another antibody specific for the desired antigen or antibody that is bound to the label to be detected. This type of ELISA is known as "sandwich ELISA". Detection can also be carried out by adding a second antibody specific for the desired antigen, followed by the addition of a third antibody with binding affinity for the second antibody, where the third antibody is bound to a detectable label. Variations in ELISA methods are known to those skilled in the art.

Competitive ELISAs are also possible, in which test samples compete for binding with known amounts of labeled antigens or antibodies. The number of reactive molecules in an unknown sample is determined by mixing the sample with known labeled molecules before or during incubation with coated wells. The presence of reactive molecules in the sample reduces the number of labeled molecules available to bind to the wells and thus reduces the final signal.

Regardless of the format used, ELISA has certain common characteristics such as coating, incubation or binding, washing to remove non-specifically bound molecules, and detection of bound immune complexes.

The antigen or antibodies can also be bound to a solid support, such as in the form of a plate, bead, test strip, membrane, or column matrix, and the assay sample is applied to the immobilized antigen or antibody. When a plate is coated with an antigen or antibody, typically the wells of the plate are incubated with a solution of the antigen or antibody overnight or for a specified period. The wells of the plate are then washed to remove incompletely adsorbed material. Any remaining accessible well surfaces are then "coated" with non-specific proteins that are antigenically neutral to the antisera being tested. These include bovine serum albumin (BSA), casein, and powdered milk solutions. The coating makes it possible to block sites of non-specific adsorption on the immobilizing surface and, thus, reduces the background caused by non-specific binding of antiserum on the surface.

As a rule, in ELISA, secondary or tertiary detection means are used, and not a direct method. Thus, after binding of the antigen or antibody to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is brought into contact with the clinical or biological specimen under test under conditions that allow efficient formation of immune complexes (antigen/antibody). A labeled secondary binding ligand or antibody, or a secondary binding ligand or antibody in combination with a labeled tertiary antibody or third binding ligand, is then required for the detection of immune complexes.

In addition, flow cytometry can be used to detect and quantify specific cell subtypes according to cell surface markers. Common means of detection and quantification by flow cytometry include the use of fluorescently labeled beads that bind to cell surface markers specific for each of the immunocyte subtypes, such as CD4-specific beads, to select for CD4 ⁺ T cells, and so on.

Sets

Also provided herein are kits containing complexes of nanoparticles as described herein or compositions as described herein for diagnostic, prognostic, or therapeutic use. Additional reagents and/or instructions may be provided as needed.

Examples

The following examples are provided to illustrate various embodiments of the invention and are not intended to limit the present invention in any way. One of ordinary skill in the art will readily appreciate that the present invention is well adapted to accomplish the objects and achieve the stated objects and advantages, as well as the objects, objects and advantages arising from this document. The present examples, together with the methods described herein, are presently a presentation of embodiments and are illustrative, and are not intended to limit the scope of the invention. Those skilled in the art will recognize variations therein and other uses that are included in the gist of the invention as defined by the scope of the claims.

Example 1 Polymeric and Dendrimeric Nanoparticle Cores for Autoimmunity and Immunity

The great antigenic complexity of autoimmune diseases and other chronic inflammatory processes, including allergies, is a barrier to developing strategies that could rid the immune system of auto- or allergenic activity without impairing systemic immunity; modern systemic immunosuppressive approaches impair immunity to infections and malignant tumors.

Thus, in one aspect, the present invention found that the systemic delivery of nanoparticles (NPs) coated with autoimmune or allergic disease-associated peptide-major histocompatibility complex (pMHC) class II molecules initiates in vivo proliferation of CD4 ⁺ recognizing T cells. -regulatory type 1 (T _R 1) in various disease models and in various genetic environments, leading to the resolution of various autoimmune or allergic processes, including spontaneous type 1 diabetes, experimental autoimmune encephalomyelitis, and house dust mite-induced asthma. These nanodrugs stimulate the differentiation of disease-primed autoreactive T cell progenitors into disease-suppressing T _R 1 cells, which then continue to suppress the response of autoreactive and allergen-specific T cells in diseased tissues by targeting autoantigenic or allergen-loaded antigen-presenting cells (APCs), with it does not react with unloaded APCs elsewhere in the body. Disease suppression does not reduce the host's ability to clear viral infections or produce an antibody response to conventional vaccines; mediated by local secretion of IL-10 and TGF-beta in response to these interactions recognizing T _R 1-APC; and involves profound inhibition of the ability of local (but not distal) APCs to secrete pro-inflammatory cytokines and activate other T cells. In addition, proliferating TR 1 cells have been shown to initiate in vivo differentiation of recognizing _B lymphocytes into IL-10 producing regulatory B cells, which contribute to the significant therapeutic activity of this therapeutic platform. Importantly, the examples demonstrate that appropriate human type 1 diabetes nanodrugs can expand human T _R 1 cells transplanted with patient-derived peripheral blood mononuclear cells in NSG mice, demonstrating the translational potential of this approach. Thus, class II rMHC-based nanodrugs may represent a long-awaited antigen-specific therapy for autoimmune and allergic inflammation. Similar results can be achieved with class I rMHC-based nanodrugs to expand appropriate T cell populations.

It has been determined that the therapeutic properties of these nanodrugs are predominantly a function of MHC density (intermolecular distance). Mathematical modeling of experimental data suggests that for any given rMHC valency, small but densely coated NPs have superior biological and therapeutic activity.

In one aspect, excellent results have been demonstrated for an NP core diameter of approximately 8-12 nm. The MHC binding capacity of the PEGylated iron oxide NP corresponds to ≈55 pMHC per NP with a hydrodynamic diameter of 68 nm.

In the preparation of MHC-based nanodrugs using third-generation poly-L-lysine-based dendrimers (DGL; 7 nm), this limitation is overcome. The ordered structure of pMHC-accepting PEGs on these compounds increases the ligand-binding capacity (thus molecular density) by several times (52 pMHC on a 19 nm pMHC-DGLN with a hydrodynamic diameter compared to 55 pMHC on a pMHC-IONP with a diameter of 68 nm, which leads to a multiple increase in the density of rMHC, a critical parameter of biological activity).

Dendrimers are highly branched macromolecules with a tree structure, with branches growing from the core. They are well known for their three-dimensional, monodisperse, highly branched macromolecular nanoscopic architecture with a number of reactive end groups. These properties, in addition to many other biomedical applications, make dendrimers popular tools for drug, peptide, and gene delivery.

Well-studied dendrimers mainly bear primary amino groups on the branched surface, for example, dendrimers based on poly-(amidoamine) (PAMAM) and poly-L-lysine (PLL). These dendrimers are water soluble at physiological pH due to the presence of charged NH ₃ ⁺ end groups. However, PAMAM cationic dendrimers exhibit bioincompatibility, biodegradability, and a positive association with cytotoxicity, which limits their wide application in vitro and in vivo.

Cationic PLLs are promising new candidates due to their biodegradability properties. A previous study demonstrated that free lysine and larger molecules (non-dendrimeric) appeared in plasma 1 hour after dosing L-lysine coated dendrimers, indicating rapid degradation of PLL in vivo (Bailey-Bucktrout, S.L. et al. (2013) Immunity 39 :949-962). However, rapid degradation is not a positive effect to maintain an effective therapeutic level. Fortunately, fully PEGylated PLL dendrimers have been reported to have a greater ability to increase plasma stability and circulation time while completely masking the positive charge on the surface. PLL-based dendrimers have already been used in the design of drug delivery systems. Kaminskas and colleagues conjugated methotrexate (MTX) to a range of pegylated PLL dendrimers and demonstrated their potential as long-term circulating vectors for delivery and tumor-targeting of hydrophobic drugs. Other investigators have covalently linked camptothecin (CPT) to pegylated dendrimers from PLL and have demonstrated significantly increased lifespan in tumor-bearing mice compared to free CPT. However, most of the PLL-based dendrimers used were synthesized by the researchers themselves. The structures of these PLL-based dendrimers were not exactly the same, which greatly limits the prevalence of these dendrimers.

Grafted tree poly-L-lysine (DGL), a type of PLL-based dendrimer, is a new commercially available product. It is 100% L-lysine, biodegradable, monodisperse and well defined, having the basic properties of PLL based dendrimers. Current research is focused on the suitability of DGL for drug or gene delivery. To the knowledge of the Applicant, DGL has never before been used in the field of presenting rMHC to T cells in the circulation. In the present study, DGL generation 3 (G3) (123 amino groups, 7 nm) was used as a scaffold for presentation of rMHC and assessment of immunological activity.

Preparation, purification and characterization of pMHC-PEG-DGL

In the present study, DGL G3 with 123 amino groups was chosen as the vector material. Its surface is coated with a heterobifunctional crosslinking agent, NHS-PEG ₄ -azido (M _W 388 g/mol) through a specific reaction between primary amino groups and an activated NHS ester. The heterobifunctional PEG, maleimide-PEG-alkyne (Mal-PEG-alkyne, M _W 2000) can be conjugated to the pMHC molecule via a thiol-maleimide reaction. The free alkyne at the end of pMHC-PEG conjugates can be reacted with azide-coated DGLs via click chemistry. The resulting NPs were purified by gel filtration to remove unconjugated pMHC. Significant changes in DGL charge before and after coating can be observed by Z-potential and agarose gel electrophoresis. The resulting NPs can be characterized by DLS, Z-potential, SDS-page and TEM.

Grafted arborescent poly-L-lysine generation 3 (DGL G3)

In another study, generation 3 grafted arborescent poly-L-lysine (DGL G3) was purchased from COLCOM in France. DGL G3 is a synthetic polymer with a core structure of nine equivalent dendrons. The core is a linear poly-L-lysine with an average of eight monomers. Each dendron looks like traditional Tam-type dendrons synthesized from Merrifield resins. DGL G3 is a non-immunogenic carrier with a molecular weight of 22 kDa and 123 terminal primary amino groups (-NH ₂ ) for functionalization and conjugation (figure 3).

Synthesis of grafted tree poly-L-lisinazido (-N ₃ ) ("DGLN")

DGL was first functionalized with N-hydroxysuccinimide-PEG ₄ -azido (NHS-PEG ₄ -azido, M _W 388.37, available from Conju-Probe, Canada) to 1) allow pMHC conjugation and 2) neutralize the positive surface charge of non-functionalized DGLs.

Functionalization of the DGL surface was performed using a heterobifunctional crosslinker, NHS-PEG ₄ -azido. The NHS activated ester readily reacts with primary amino groups on DGL under mild water conditions. Approximately 1 mg of DGL-NH ₂ was dissolved in PBS, at a pH of approximately 8.0. Approximately 4.3 mg of NHS-PEG ₄ -N ₃ (-NH ₂ :PEG ₄ =1:2, mol:mol) was added to the solution and the reaction was carried out at room temperature for approximately 2 hours. After the reaction, DGL-N ₃ was washed three times by ultrafiltration (threshold M _W 3000) with PBS at a pH of approximately 7.4 to remove unreacted NHS-PEG ₄ -N ₃ (figure 4).

Conjugation of rMHC with DGLN (Fig. 5)

For conjugation of pMHC monomers to the DGLN surface, a single chain NRP-V7/K ^d construct designed to encode a C-terminal Cys (-SH) was first pegylated and generated in CHO cells (designated as V7CHO-Cys). Briefly, 3.5 ml of a solution of V7CHO-Cys (3.58 mg/ml) in PBS pH 7.4 was mixed with 24 µl of 500 mM EDTA, 375 µl of 1M NaCl, 500 µl of 200 mM PB buffer and 1.625 ml of water ETF . Then, 4 mg of Maleimide-PEG _2k -alkyne was added to the mixture (the final volume of the reaction mixture was 6.0 ml) and left to react overnight at room temperature. Then, the reaction solution was subjected to dialysis against PBS pH 7.4 at 4°C for 48 hours.

The V7CHO-PEG _2k -alkyne solution was then concentrated to a final volume of 3.5 ml in PBS pH 7.4 under nitrogen and 60 μl DGLN (5 mg/ml in PBS), 150 μl ascorbic acid (50 mM in PBS) and 175 μl of Cu-TBTA, which was left to react for 24 hours at room temperature. After the reaction, the nanoparticles were purified by ultrafiltration (threshold M _W 100 kDa) against PBS pH 7.4, 6 times.

Biochemical and biophysical analyzes of conjugates

The conjugates were analyzed by native and denaturing (SDS) PAGE. Figure 6 shows the presence of a prominent Coomassie blue stain under native PAGE conditions (two left panels), with detection of pMHC-conjugated DLGNs, in the absence of free (non-conjugated) V7CHO monomer. Electrophoresis of these compounds under denaturing/reducing conditions revealed the release of V7CHO from NP, confirming the successful conjugation of pMHC to DLGN.

The biophysical properties of the pMHC-DGLN compound were then determined using atomic force microscopy (AFM) (FIG. 7). Briefly, the NP solution was layered on mica and observed with AFM. V7CHO-DGLN showed a spherical conformation with an average diameter of 19.95±0.25 nm (AFM measurements). NPs were well distributed with monodispersity. Polydispersity index (PI) and hydrodynamic diameter in aqueous solution were tested by DLS. The concentration used to prepare the sample for AFM was 4 μg DGLN/ml (equivalent to 4.38×10 ¹³ NP/ml). The analysis was performed using 5 μl of this solution (≈2.19×10 ¹¹ NP) on a scanning area of 1 μm×1 μm.

Bradford's analysis showed that the pMHC content of the compound described above corresponded to 52 pMHC monomers per NP.

Finally, to determine whether this compound has agonist activity against T-recognizing cells, its ability to initiate IFNγ secretion by NRP-V7/K ^d specific CD8 ⁺ T cells isolated from 8.3-TCR transgenic NOD mice was determined. Briefly, 8.3-CD8 ⁺ T cells were cultured in the presence of free V7CHO protein, pegylated V7CHO, V7CHO-DGLN, or DGLN for 48 hours. Then the content of IFN-γ in the supernatants was determined by ELISA. Figures 8A-8B show that V7CHO-DGLN and, to a much lesser extent, pegylated V7CHO, had very high concentration-dependent agonistic activity against these T cells, demonstrating the functional properties of these compounds.

Preparation, purification and characterization of pMHC-PEG-DSPE micelles.

Amphiphilic block copolymers assemble into nanoscale core-shell structures, polymeric micelles, which have been of significant interest for drug delivery with poor water solubility. Poly(ethylene glycol)-distearoylphosphatidylethanolamine (PEG-DSPE) block copolymers are safe, biocompatible, and approved by the Food and Drug Administration for clinical use. DSPE-PEG, among others, has been widely used to produce liposomes, polymeric nanoparticles, polymeric hybrid nanoparticles, and solid lipid nanoparticles. Amphiphilic copolymers are nanostructures consisting of a hydrophobic core (DSPE) and a hydrophilic shell (PEG). The core-shell structure can encapsulate and carry poorly water-soluble drugs by accumulating them in the DSPE core, and the PEG shell reduces in vivo clearance and plasma protein adsorption. Thus, the use of DSPE-PEG to form nanostructures can prolong circulation in the body. Most importantly, the critical micelle concentration (CMC) of DSPE-PEG is extremely low (10 ^-5 M). This property leads to certain positive functions of formulated micelles, such as greater solubility of hydrophobic drugs and greater thermodynamic stability when diluted with a large volume of blood after intravenous administration.

DSPE-PEG-maleimide (DSPE-PEG-Mal) was chosen as the copolymer for applying pMHC to the surface of polymeric micelles. DSPE-PEG polymeric micelles are prepared by the solvent evaporation method as described in Vakil, R. et al. (2008) Mol. Pharm. 5:98-104 and Musacchio, T. et al. (2009) Mol. Pharm. 6:468-479. Briefly, DSPE-PEG-Mal was dissolved in methanol in a round bottom flask. The organic solvent mixture was evaporated under high vacuum to give a thin film of copolymers. This film was further dried under vacuum overnight to remove any traces of remaining solvents. The dry polymer film was then dissolved in PBS pH 7.4 to self-assemble into micelles with maleimide groups on the surface. pMHC can be conjugated on the surface of micelles by a specific thiol-maleimide reaction. The resulting NPs were purified by gel filtration to remove unconjugated pMHC. The resulting NPs can then be characterized by DLS, Z-potential, SDS-page and TEM.

Example 2

Propagation of disease-specific T _R 1 cells

Applicant has treated diabetic non-obese (NOD) mice and NOD Foxp3-eGFP mice expressing enhanced green fluorescent protein (eGFP) under the control of the mouse Foxp3 promoter) with bare or pMHC-coated nanoparticles, 2.5mi/IA ^g7 (Stratmann, T. et al. (2003) J. Clin. Invest. 112:902-914), recognized by the diabetogenic BDC2.5-specific T cell receptor (TCR), or 2.5mi/IA ^g7 monomers. Nanoparticles coated with 2.5 mi/IA ^g7 induced the expansion of CD4 ⁺ recognizing T cells in the blood and spleen of all mice (FIGS. 9A, 9B). These cells had a memory cell-like (CD44 ^hi CD62L ^low ) FOXP3 ^- like T _R 1 phenotype, expressing ICOS, latent-associated TGF-β, and the T _R 1 markers CD49b and LAG-3 (FIGS. 9C, 9D). A similar result was observed in mice treated with 2.5mi/IA ^g7 -NP after removal of CD4 ⁺ CD25 ⁺ T cells (figure 14A). Unlike their tetramer analogs ^, these cells, in response to dendritic cells (DC) pulsed with the 2.5mi peptide, proliferated and secreted IL-10 and, to a lesser extent, IFNγ, but not IL-2, IL-4, or IL-17 ( figure 14B). Real-time reverse transcription (RT)-PCR assays confirmed the T _R 1 -like phenotype of these cells (Tables 3A-3B).

To determine the ability of pMHCII-NP to directly initiate the T _R 1 marker and IL-10 expression on CD4 ⁺ T cells, Applicant cultured naive and pre-activated anti-CD3 and anti-CD28 monoclonal antibodies (mAb) tetramer 2.5mi/IA ^g7+ CD4 ⁺ T- NOD BDC2.5-TCR-Foxp3-eGFP transgenic mice or NOD Il11 ^GFP mice (carrying an eGFP insert at the Il10 locus) (Kamanaka, M. et al. (2006) Immunity 25:941-952) in the presence of 2.5mi/IA ^g7 -NP, peptide 2.5mi or monomer 2.5mi/IA ^g7 . Naive T cells did not express either CD49b or LAG-3 even after incubation with 2.5mi/IA ^g7 -NP, 2.5mi/IA ^g7 monomer or 2.5mi peptide (Fig. 14C, 14D). However, pre-activated T cells upregulated both markers as well as eGFP (IL-10) only in response to 2.5 mi/IA ^g7 -NP (Fig. 14D, 14E). Consistent with this, IL-10 expression in NOD Il10 ^GFP mice treated with 2.5mi/IA ^g7 -NP was largely restricted to the CD49b ⁺ LAG-3 ⁺ CD4 ⁺ subset (Figure 14F).

In vitro, tetramer ⁺ CD4 ⁺ T cells of pMHC-NP-treated mice suppressed the proliferation of non-recognizing (glucose-6-phosphatase islet-specific protein (IGRP) catalytic subunit ^- related or Gp33-specific LCMV) CD8 ⁺ T cells in response to activated peptides DC, dependent on IL-10 and TGF-β manner (figure 14G). In vivo, spleen CD4 ⁺ T cells derived from pMHC-NP-treated donors suppressed the development of diabetes in scid hosts with reconstituted NOD T cells (also known as NOD Prkdc ^scid ) (Figure 9E), an effect that was enhanced by treatment pMHC-NP hosts (FIGS. 9E, 9F).

Applicant then investigated the ability of 2.5mi/IA ^g7 -NPs or NPs coated with IGRP _4-22 /IA ^g7 or IGRP _128-145 /IA ^g7 to target subdominant populations of autoreactive CD4 ⁺ T cells (Mukherjee, R. et al. (2005) J. Immunol. 174:5306-5315) restore normoglycemia in diabetic NOD mice. In contrast to mice treated with nanoparticles coated with hen egg protein lysozyme (HEL) _14-22 /IA ^g7 , 90-100% of mice treated with nanoparticles coated with 2.5mi/IA ^g7 , IGRP _4-22 /IA ^g7 or IGRP _{128 -145} /IA ^g7 , stable normoglycemia was restored (FIGS. 9G, 14H) and systemic expansion of T _R 1 like-recognizing T cells was shown (FIGS. 1H, 15A-15G). Treatment with this peptide (Burton, BR et al. (2014) Nature Commun. 5:4741-4747) or peptide-coated nanoparticles but without MHC could not reproduce either of these effects (Fig. 9G, 14H, 15H). Termination of treatment resulted in a loss of normoglycemic state in 25-60% of mice (FIG. 14I) in association with loss of tetramer ⁺ CD4 ⁺ T cell populations (FIGS. 9H, 15A). Animals that maintained normoglycemia showed normal postprandial serum insulin levels, fasting glucose tolerance (FIGS. 14J-14M), and reduced insulitis (FIG. 9I). In addition, their pancreatic lymph nodes (PLNs) could not support the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-specific IGRP _206-214 /K ^d CD8 ⁺ T cells in vivo (FIG. 14N).

Applicant then tested the ability of nanoparticles coated with myelin oligonedrocyte glycoprotein (pMOG) _38-49 /IA ^b to slow the progression of pMOG _35-55 induced experimental autoimmune encephalomyelitis (EAE, multiple sclerosis model) in C57BL/6 mice. pMOG _38-49 /IA ^b NP therapy slowed disease progression when administered on day 14 post-immunization and restored motor function in paralyzed mice when administered on day 21 (Fig. 9J, 9K). These effects were reflected in weight gain and were associated with systemic proliferation of T _R 1 -like recognizing T cells, decreased numbers of activated macrophages/microgolia in the cerebellum, fewer inflammatory foci and areas of demyelination in the cerebellar white matter, and reduced spinal cord demyelination (Fig. 9L- 9N, 16A-16F). Similar therapeutic effects were observed in HLA-DR4-IE transgenic C57BL/6 IAb ^null mice (MHCII knockout mice expressing a transgenic MHCII fusion molecule consisting of the human HLA-DR4 peptide-binding domain and the mouse IE membrane-proximal domain (DR4-IE)) immunized with human (h) proteolipid protein (hPLP) _{peptides 75-192} or hMOG _97-108 and treated with hPLP _175-192 /DR4-IE or hMOG _97-108 /DR4-IE-NP after the development of paralysis (Fig. 17A- 17D).

Specificity for diseases and organs

Studies in another autoimmune disease model, collagen-induced arthritis (CIA), demonstrated that nanoparticles exposed to mouse collagen (mCII) _259-273 /DR4-IE could reduce joint inflammation in HLA-DR4-IE transgenic mice C57BL/10.M with arthritis in association with systemic proliferation of T _R 1 -like recognizing T cells (FIGS. 10A-10E, 1E). In contrast, nanoparticles coated with hMOG _97-108 /DR4-IE complexes had no effect (FIGS. 10A-10C).

To further investigate the disease specificity of pMHC-NP therapy, Applicant induced EAE in HLA-DR4-IE transgenic C57BL/6 IAb ^null mice by _{hPLP 175-192} immunization and treated diseased mice with the hPLP _175-192 /DR4-IE-NP complex ( positive control), uncoated nanoparticles (negative control), EAE-associated hMOG _97-108 /DR4-IE-NP complex (which exhibited a peptide from a CNS autoantigen other than that used to induce disease) or CIA-associated mCII _259-273 / DR4-IE-NP. While hMOG _97-108 /DR4-IE-NP inhibited EAE as effectively as the positive control, mCII _259-273 /DR4-IE-NP showed no therapeutic activity (FIGS. 10F, 17F, 17G). Here, therapeutic activity is also associated with systemic expansion of T _R 1 -like recognizing T cells (FIGS. 10G, 10H). Administration of mCII _259-273 peptide (Burton, BR et al. (2014) Nature Commun. 5:4741-4747) or mCII _259-273 peptide coated microparticles (MP) (Getts, DR et al. (2012) Nature Biotechnol. 30 :1217-1224) HLA-DR4-IE transgenic C57BL/10.M arthritis mice (FIGS. 10A-10D, 17E) or hMOG _97-108 peptide, hMOG _97-108 /DR4-IE monomers or coated hMOG _{97- 108} nanoparticles or microparticles in HLA-DR4-IE transgenic C57BL/6 IAb ^null mice did not result in expansion of T _R 1 -like recognizing cells or in disease retardation (FIGS. 10F, 10G, 17F-17I). Thus, the biological and therapeutic effects of pMHCII-NP are disease specific and distinct from the pathogenic role of epitopes (disease initiation versus downstream autoantigenic targets), suggesting that these compounds act on pre-activated autoreactive T cells and can promote the expansion of Tk1-like cells from rare populations of T-cell progenitors.

Soluble mediators

Blockade of IL-10, TGF-β and IL-21R (but not IFNγ) abolished the anti-diabetogenic properties of 2.5mi/IA ^g7 -NP or IGRP _4-22 /IA ^g7 -NP (FIGS. 11A, 18A). With the exception of blockade of IL-21R (which is known to inhibit CD8 ⁺ T cell activation), these interventions also abolished the suppression of self-antigen cross-presentation by pMHC-NP-expanded T _R 1 like T cells in PLN (Fig. 18B). , 18C, 19). Studies using NOD Ifng ^-/- and NOD Il10 ^-/- mice with diabetes revealed that the development of T _R 1 progenitors and/or T _R 1 like cells that proliferate in response to therapy requires IFNγ in addition to IL-10 ( Fig. 18D-18F, 19). mAbs to IL-10, TGF-β and IL-21R also abolished the anti-encephalitogenic activity of hPLP _175-192 /DR4-IE-NP in HLA-DR4-IE transgenic C57BL/6 IAb ^null mice (Fig. 18G-18J). Immunized pMOG _35-55 C57BL/6 Il27r ^-/- mice responded to pMOG _33-49 /IA ^b -NP like wild-type individuals (FIGS. 9J-9N, 18K-18N). Thus, IFNγ and IL-10, but not IL-27, are required for development of pMHC-NP-induced T _R 1 -like cells (Pot, C. et al. (2009) J. Immunol. 183:797-801); and autoreactive T _R 1 like T cells use IL-10, TGF-β, and IL-21 (but not IFNγ) to suppress the disease.

Downstream Effectors and Network Formation

PLNs (but not mesenteric lymph nodes (MLNs) or splenic lymph nodes) of pMHC-NP-treated NOD mice contain increased numbers of B cells compared to PLNs of non-T1D-associated pMHCII-NP-treated mice (Figure 18O). Studies of mice treated with a range of doses of pMHC-NP revealed that population sizes of B cells and T _R 1 like cells in PLN (but not in spleen) were correlated (FIG. 18P). Unlike their counterparts in the spleen or MLN, the B cells in the PLN of these mice cannot efficiently present the peptide to ex vivo CD4 ⁺ T cells (FIG. 18Q). In addition, these cells produced IL-10 in response to lipopolysaccharide (LPS) (Figure 18R), suggesting that pMHC-NP-induced T _R 1 like cells can initiate the formation and expansion of regulatory B (B _reg ) cells in PLN. In fact, 2.5mi sensitized B cells, but not DCs, proliferated in 2.5mi/IA ^g7 -NP treated hosts within a week of transfer (FIGS. 18S, 18T).

To further investigate this phenomenon, Applicant transfused NOD Il10 ^GFP spleen B cells that were sensitized with 2.5mi or a negative control peptide (GPI _282-292 ) treated with 2.5mi/IA ^g7 -NP to NOD or NOD Il10 ^-/- hosts. Seven days later, hosts were analyzed for IL-10 (eGFP ⁺ ) producing CD5 ⁺ CD1d ^high B cells. NOD (but not NOD Il10 ^-/- ) mice treated with 2.5mi/IA ^g7 -NP effectively specifically induced Breg cells from _2.5mi -sensitized B cells, and blockade of IL-21R, but not IL-10 or TGF-β suppressed this effect (FIGS. 11B, 11C, 18U).

In vitro, PLN B cells of 2.5 min/IA ^g7 -NP-treated mice had a moderate inhibitory effect on the proliferative activity of BDC2.5 CD4 ⁺ T cells in response to peptide-sensitized DCs (FIG. 18V). In vivo, these B cells suppressed the development of diabetes in reconstituted T cell NOD scid hosts compared to PLN B cells of control mice (FIG. 11D). Co-transfer of PLN B cells and all or tetramer 2.5mi/IA ^g7+ CD4 ⁺ spleen T cells derived from 2.5mi/IA ^g7 -NP treated mice resulted in >95% suppression compared to PLN B cells with tetramer ^- CD4 ⁺ T cells derived from or without 2.5mi/IA ^g7 -NP mice, with or without CD4 ⁺ T cells with MLN B cells derived from 2.5mi/IA ^g7 -NP mice (≈40%) them, or with CD4 ⁺ T cells obtained from untreated or treated control NP mice (0%) (figure 11E), indicating a synergistic effect. Consistently, treatment of newly diabetic NOD mice with B cell-depleting anti-CD20 mAb abolished the anti-diabetogenic activity of 2.5mi/IA ^g7 -NP (FIGS. 11A, 18X).

Comparison of cytokine and chemokine profiles of CD11b ⁺ cells derived from PLN or MLN treated with pMHC-NP NOD mice additionally revealed that CD11b ⁺ cells from PLN produced lower levels of the pro-inflammatory mediators IL-3, IL-17, IL-6, IFNγ, CXCL9, and CXCL10 in response to LPS than their MLN counterparts (Figure 11F). Importantly, the effect of pMHC-NP therapy on antigen-presenting cells (APC) from draining lymph nodes was not associated with a deterioration in systemic immunity, as pMHC-NP-treated NOD mice abolished acute viral infection and produced antibodies to exogenous antigens as efficiently as untreated mice. mice (FIGS. 11G, 11H).

T cells that encountered antigen as targets

The memory cell-like phenotype and increased expression of T-bet mRNA in proliferating T _R 1 like cells, together with the inability of pMHC-NP to stimulate proliferation of recognizing T _R 1 like cells in undiseased or NOD Ifng ^-/- mice, suggests that pMHC-NPs stimulate propagating pre-existing TR1 cells that are derived from antigen encountered precursors and/or initiate differentiation of antigen encountered T _H 1 cells into T _R 1 like cells. In fact, although diabetic NOD G6pc2 ^−/− mice (which lack IGRP) responded to 2.5mi/IA ^g7 -NP like wild-type NOD mice, they did not respond to IGRP _4-22 /IA ^g7 -NP (Figure 12A, 12B ). In vitro, 2.5mi/IA ^g7 -NP initiated CD49b and LAG-3 expression and upregulation of c-maf, Il21, Il10, and Lag3 mRNA only in activated anti-CD3 and anti-CD28 mAbs, but not in naïve CD4 ⁺ BDC2 T cells .5 (FIGS. 12C, 14D).

To further investigate this phenomenon, Applicant transfused naïve (CD44 ^low CD62L ^hi ) or memory cell-like (CD44 ^hi CD62L ^low ) BDC2.5 CD4 ⁺ T cells to hosts of the NOD.Thy1 ^a cogen line and determined the change in their expression of LAG-3 proteins. and CD49b and mRNA c-maf, Il21, Il10, Ifng, Lag3 and Cd49b, after 2.5mi/IA ^g7 -NP therapy and in the absence of therapy. Of note, memory T cells of untreated pMHC-NP hosts expressed approximately one hundred times higher levels of c-maf and Il21 mRNA and, to a lesser extent, Lag3 and Cd49b, but not Il10, than their naive counterparts (FIG. 12D). This is consistent with the observed Il21 demethylation and c-Maf/IL-10 and IL-21 expression ability of effector/memory cells from CD4 ⁺ T cells (Pot, C. et al. (2009) J. Immunol. 183:797 -801; Spensieri, F. et al. (2013) Proc. Natl Acad. Sci. USA 110:14330-14335; Hale, JS et al. (2013) Immunity 38:805-817; Sato, K. et al. (2011) J. Biol. Chem. 286:14963-14971; Saraiva, M. et al. (2009) Immunity 31:209-219), suggesting that the memory T cell population is enriched in uncommitted T _R 1 precursors, expressing a balanced T _R 1 transcription program. Notably, while pMHC-NP therapy only upregulates Lag3 mRNA and, to a lesser extent, LAG-3 protein expression in naïve BDC2.5 CD4 ⁺ T cells, it stimulates upregulation of Il10 and LAG-3 mRNA and CD49b protein and CD4 proliferation. ⁺ BDC2.5 memory T cells (Fig. 12D-12F). Similar results were observed using eGFP ^- (FOXP3 ^- ) CD4 ⁺ memory cells BDC2.5, obtained from mice NOD Foxp3-eGFP BDC2.5 (figure 18Y).

This effect on antigen-encountered T cells was accompanied by the acquisition of antidiabetogenic properties: while pMHC-NP therapy provided 100% protection against diabetes in NOD scid hosts with recovered T cells bearing BDC2.5 memory T cells, therapy in hosts receiving naïve BDC2.5 T cells was not significant (Figure 12G). Thus, pMHC-NP therapy stimulates differentiation (and proliferation) of c-Maf-expressing CD4 ⁺ antigen-encountered T cells into T _R 1 progeny.

Translation potential

Applicant determined the ability of human T1D-associated pMHCII-NPs to stimulate the proliferation of T _R 1 -like recognizing T cells in NOD scid Il2rg ^-/- (NSG) hosts with reconstituted peripheral blood mononuclear cells (PBMCs) of T1D patients (Table 2). Development of the initial assay focused on NSG hosts with reconstituted PBMCs of five DRB1*0401 ⁺ early-onset T1D patients treated with nanoparticles coated with human glutamic acid decarboxylase-65 (GAD65) _555-567 (F557I)/DR4 or preproinsulin (PPI) complexes76 -90(K88S)/DR4 (FIGS. 13A, 13B, Table 2). Applicant then repeated these experiments using NSG hosts with reconstituted PBMCs of 7 DRB1*0301 ⁺ T1D patients and a third T1D-associated pMHC-NP (hIGRP13-25/DR3-NP) administered at an increased dose. Applicant observed tetramer ⁺ CD49b ⁺ LAG-3 ⁺ CD4 ⁺ T cell proliferation in spleen and/or PLN (endogenous murine (m)IGRP _13-25 highly homologous to hIGRP _13-25 ) of all seven pMHC-NP-treated mice and none from unrelated controls (FIGS. 13C, 13D, 20A and Table 2). The mean percentage and numbers of tetramer ⁺ CB4 ⁺ T cells in IGRP13-25/DR3-NP treated mice were significantly greater than in untreated mice of the same litter (Figure 13D) and Il10 mRNA was expressed (Figure 13E). This response could not be induced by the peptide or peptide-coated nanoparticles or microparticles (FIGS. 13D, 20B and Table 2).

PLN treated with pMHC-NP mice containing an increased percentage of tetramer ⁺ CB4 ⁺ T cells had increased cell saturation (figure 13F). In addition, correlations were observed between the number of tetramers ⁺ CB4 ⁺ PLN T cells and the percentage and absolute number of human B cells in PLN, and PLN B cells, in contrast to their splenic counterparts, produced IL-10 in response to LPS (Fig. 13G-13I), which suggests the formation and/or recruitment of B _reg . No such response was observed in NSG mice with repaired hPBMCs of patients treated with peptide or peptide-NP/MP (FIG. 13F).

Discussion

Applicant has demonstrated that systemic therapy with nanoparticles coated with autoimmune disease-associated rMHC class II complexes initiates the proliferation of T _R 1 -like recognizing CD4 ⁺ T cells, restores normoglycemia in spontaneously diabetic mice and motor function in paralyzed EAE mice, and eliminates swelling and joint destruction in mice with arthritis without compromising systemic immunity. Applicant has demonstrated that this result is unrelated to genetic environment and type of autoimmune disease, and can be repeated with ten different autoimmune disease-associated human or mouse pMHC-NP types. The cell surface phenotype, cytokine secretion profile, transcription profile, and function of T _R 1 -like cell populations propagated by pMHCII-based nanodrugs are consistent with those described for T _R 1 -like CD4 ⁺ T cells in mice and are very similar to T _R 1 -like cells derived from healthy subjects. individuals and patients with autoimmune diseases (Gagliani, N. et al. (2013) Nature Med. 19:739-746). Applicant has demonstrated the key roles of pre-stimulation with autoantigens and the ability to express IFNγ and IL-10 in the ontogeny of autoreactive T _R 1 _{cells .} met antigen CD4 ⁺ T cells among the APC and independent of IL-27 in a way, followed by systemic reproduction. Need for IFNγ, T _H 1 T-bet transcription factor expression, c-Maf/IL-10 and IL-21 expression ability of effector CD4 ⁺ T cells and CD4 ⁺ memory T cells (Pot, C. et al. ( 2009) J. Immunol 183:797-801; Spensieri, F. et al. (2013) Proc. Natl Acad. Sci. USA 110:14330-14335; Hale, JS et al. (2013) Immunity 38:805- 817) and the ability of pMHCII-NP to convert T cells primed by active immunization into T _R 1 suppressors suggest that these T _R 1 precursors are T _H1 effector/memory cells. Applicant identified the mechanism of action and identified a cascade of cellular interactions occurring after pMHC-NP-like expansion of T _R 1 cells, including the formation of B _reg cells, which coordinatedly leads to the elimination of inflammation in an antigen-dependent but antigen-non-specific manner (Figure 21).

Taken together, Applicant's data support the contention that any particular rMHC involved in a given autoimmune disease can be used to slow the complex autoimmune response through this approach. Consistent with this assumption, the 20 pMHCI/II-based nanodrugs tested to date have shown similar efficacy, regardless of antigen level, effect, or role in the pathological process. Neither pMHC monomers, peptides, nor peptide-coated nanoparticles/microparticles stimulate the formation/proliferation of T _R 1 recognition cells from the polyclonal T cell repertoire or promote regression in the chronic T1D, CIA, or EAE models tested in these studies. Thus, rMHC-based nanodrugs represent a new class of autoimmunity therapeutic agents capable of abolishing the cellular and antigen complex autoimmune response in a disease- and organ-specific manner without compromising systemic immunity.

Ways

NOD/Ltj mice, NOD scid, BDC2.5-NOD, NOD Il10 ^-/- , C57BL/6, C57BL/6 Il27r ^-/- , C57BL/10.M, NOD Foxp3-egfp mice and NOD scid Il2rg mice ^{-/- (} NSG) purchased from Jackson Lab. NOD Ifng ^-/- mice and transgenic NOD mice with LCMV-specific Gp33 TCRs were purchased from D. Serreze (Jackson Lab). HLA-DR4-IE transgenic C57BL/6 IAb ^null mice were purchased from Taconic Farms, NOD Il10 ^GFP (brindle) mice were generated by backcrossing the Il10 ^GFP allele of C57BL/6 Il10 ^GFP mice (Jackson Lab) with the original NOD/Ltj for 10 generations. 8.3-NOD and NOD G6pc2 ^-/- mice are described elsewhere (Verdaguer, J. et al. (1997) J. Exp. Med. 186:1663-1676; Wang, J. et al. (2010) Proc. Natl Acad. Sci. USA 107:9317-9322). These studies are approved by the animal welfare committees of the respective institutions. Statistical methods for predetermining the size of the samples were not used.

Antibodies, tetramer staining and flow cytometry. Anti-CD4 (RM4-5), CD8α (53-6.7), B220 (RA36B2), CD62L (MEL-1), CD69 (H1.2F3), CD44 (IM7) mAb conjugated to FITC, PE, PerCP or biotin and CD49b (DX5) mice and streptavidin-PerCP were purchased from BD Pharmingen. An anti-LAG-3 (C9B7W) mouse antibody was purchased from eBioscience. An antibody to latent associated TGF-β (TW7-16B4) was purchased from BioLegend. PE-conjugated class II pMHC tetramers were prepared using biotinylated pMHC monomers. Peripheral blood mononuclear cells, splenocytes, lymph node and bone marrow CD4 ⁺ T cells were incubated with avidin for 15 minutes at room temperature and stained with tetramer (5 µg ml ^- 1) in FACS buffer (0.05% sodium azide and 1% FBS in PBS) for 30-120 minutes at 4°C or 37°C depending on the tetramer, washed and incubated with FITC-conjugated anti-CD4 antibody (5 µg ml ^- 1) and PerCP-conjugated anti-B220 antibody (2 µg ml ^- 1; as a "nonsense" channel) for 30 minutes at 4°C. Cells were washed, fixed in 1% paraformaldehyde (PFA) in PBS and analyzed using FACScan, FACSaria or BD LSRII flow cytometers. For other phenotype assays, single cell suspensions were stained with pMHC tetramers and antibodies diluted 1:100 in FACS buffer (all used at 4°C except for anti-LAG-3 antibody which was used at 37°C), washed, fixed in 1% PFA and analyzed by FACS. All phenotypic staining was performed in the presence of an anti-CD16/CD32 mAb (2.4G2; BD Pharmingen) to block Fc receptors. Analysis was performed using FlowJo software.

NSG transplanted human T cells were analyzed using the following mAbs: FITC-conjugated anti-CD4 mAb (OCT4, BioLegend), APC-conjugated anti-CD 19 mAb (HIB19, BD Pharmingen), PerCP-conjugated goat anti-LAG-3 IgG polyclonal antibody (R&D Systems), biotin-conjugated anti-CD49b mAb (AK7, Pierce Antibodies, Thermo Scientific) and EF450-conjugated streptavidin (eBioscience). Briefly, splenocytes and pancreatic lymph node cells were incubated with avidin (0.25 mg ml ^-1 in FACS buffer) for 30 minutes at room temperature, washed and stained with tetramer (5 μg ml ^-1 ) for 1 hour at 37°C, washed and incubated with FITC-conjugated anti-CD4 antibodies (2/100), APC-conjugated anti-CD 19 antibodies (5/100; used as "nonsense" channel), PerCP-conjugated anti-LAG-3 antibodies ( 8/100) and biotin-conjugated antibodies to CD49b (4/100) at 4°C for 45 minutes. After washing, cells were incubated with EF450-conjugated streptavidin for 30 minutes at 4°C, washed, fixed in 1% PFA in PBS, and cells in the hCD4 ⁺ /hCD19 ^- window were analyzed using FACSCanto II (BD Bioscience).

Peptides and rMHC. Unless otherwise indicated, class II recombinant rMHC monomers were isolated from culture supernatants of induced Drosophila SC2 cells transfected with constructs encoding I-Aβ and I-Aα chains bearing c-Jun or c-Fos leucine zippers, respectively, and BirA and 6× labels. His. In these constructs, the peptide coding sequence is linked to the N-terminus of the I-Aβ chain via a flexible Gly-Ser linker as described (Stratmann, T. et al. (2003) J. Clin. Invest. 112:902-914). GAD65 _{555(557I)-567} /DR4, PPI _76-90(88S) /DR4, and IGRP _13-25 /DR3 monomers were generated by loading the respective peptides onto DR4 and DR3 complexes isolated from induced SC2 cell supernatants as described (Yang, J et al (2006) J Immunol 176:2781-2789). Other constructs (encoding 2.5mi/IA ^g7 , pMOG _35-55 /IA ^b , hMOG _97-108 /DR4-IE, hPLP _175-192 /DR4-IE and mCII _259-273 /DR4-IE) were isolated from supernatants of Chinese hamster (CHO) transduced with lentiviruses encoding a monocistronic transcript in which the peptide-MHCβ and MHCα complex chains are separated by a P2A ribosome hopping sequence (Hoist, J. et al. (2006) Nature Protocols 1:406-417). These monomers were designed to encode a BirA region, a 6x His tag, and a free Cys at the C-terminus of the construct. Class II self-assembling pMHC complexes were purified by nickel chromatography and used for loading onto nanoparticles or treated for biotinylation and formation of tetramers as described above. Epitopes encoded in other monomer constructs described herein include: 2.5mi (AHHPIWARMDA) (Stratmann, T. et al. (2003) J. Clin. Invest. 112:902-914); IGRP _128-145 (TAALSYTISRMEESSVTL) and IGRP _4-22 (LHRSGVLIIHHLQEDYRTY) (15); HEL _14-22 (RHGLDNYRG); GAD65 _{555(557I)-567} (NFIRMVISNPAAT) (Reijonen, H. et al. (2002) Diabetes 51:1375-1382); PPI _76-90 (88S) (SLQPLALEGSLQSRG) (Yang, J. et al. (2008) J. Autoimmun. 31:30-41); IGRP _13-25 (QHLQKDYRAYYTF) (Yang, J. et al. (2006) J. Immunol. 176:2781-2789); pMOG _38-49 (GWYRSPFSRVVH); hMOG _97-108 (TCFFRDHSYQEE); hPLP _175-192 (YIYFNTWTTCQSIAFPSK) and mCII _259-273 (GIAGFKGDQGPKGET). Peptides IGRP _4-22 , IGRP _128-145 and GPI _282-292 (LSIALHVGFDH) or 2.5mi, pMOG _35-55 (MEVGWYRSPFSRVVHLYRNGK), pMOG _38-49 , hMOG _97-108 and hPLP _{175-192 were} purchased from Sigma Genosys, Mimotopes or genscript.

Synthesis and purification of nanoparticles, pMHC-NP, peptide-NP and peptide-MP. Applicant applied pMHC to cross-linked dextran-coated NPs or NPs with pegylated iron oxide (CLIO- or PFM-NPs, respectively). Briefly, CLIO-NP was treated with ammonia to give amino groups (NH ₂ ). Avidin was oxidized with sodium periodate and added to amino-NP. Additional incubation with sodium cyanoborohydride was used to obtain a stable covalent bond. Finally, biotinylated monomers were added to the nanoparticles at a molar ratio of 4 mol biotin/mol avidin (Moore, A. et al. (2004) Diabetes 53:1459-1466). PFM-NP was obtained by thermal destruction of Fe(acac) ₃ in the presence of 2 kDa methoxy polyethylene glycol maleimide (Singha, S. et al., unpublished data). NPs were purified using magnetic (MACS) columns (Miltenyi Biotec) or IMag cell separation system (BD Biosciences). With a conjugate of pMHC or free peptide with PFM-NP, the authors incubated pMHC or peptide bearing free C-terminal Cys with nanoparticles in 40 mM phosphate buffer, pH 6.0, containing 2 mM EDTA, 150 mM NaCl overnight at room temperature. PMHC-conjugated nanoparticles were separated from free pMHC or peptide using magnetic columns, sterilized by filtration through 0.2 μm filters and stored in water or PBS at 4°C. Quality control was performed using transmission electron microscopy, dynamic light scattering, and native and denaturing gel electrophoresis. pMHC or peptide content was determined using a variety of approaches, including Bradford analysis (Thermo Scientific), denaturing SDS-PAGE, amino acid analysis (HPLC-based quantification of 17 different amino acids in hydrolysable pMHC-NP preparations), or dot-ELISA (Singha, S et al., unpublished data).

Peptide-coated microparticles were prepared using 500 nm diameter carboxylated polystyrene beads from Polysciences (Warrington, RA) as previously described (Getts, DR et al. (2012) Nature Biotechnol. 30:1217-1224). The peptides were conjugated to polystyrene beads by carbodiimide chemistry according to the manufacturer's instructions. Briefly, Applicant incubated 250 μl PSB (containing ≈9 x 10 ¹¹ beads) with 250 μg peptide in 0.1 M MES buffer, pH 5.0 at room temperature with gentle shaking in the presence of 1 mg EDC for 2 hours. Peptide-conjugated polystyrene beads were washed with PBS to remove unconjugated peptides and analyzed using native and denaturing PAGE for serial dilutions of unconjugated peptide and control microparticles.

Therapy with pMHC-NP and peptide or peptide-NP complexes in NOD mice. Experiments in prediabetic NOD mice involved iv treatment of groups of 10 week old female mice with 7.5 μg pMHC-NP or equivalent amounts of soluble pMHC monomers or uncoated nanoparticles twice weekly for 5 consecutive weeks. Experiments in diabetic mice included monitoring the development of diabetes in groups of female NOD/Ltj, NOD G6pc2 ^-/- , NOD Il10 ^-/- or NOD Ifng ^-/- mice at 10 weeks of age by measuring blood glucose levels with Accucheck strips (Roche) twice a week. Mice with two consecutive determinations of >11 mM were considered diabetic and treated twice weekly with 7.5 μg pMHC-NP, nanoparticles delivering the molecular equivalent of the peptide, or free peptide (8 μg per dose) (Burton, BR et al. (2014) Nature Commun. 5:4741-4747) until stable normoglycemia (defined as 8 consecutive measurements <11 mM) or until hyperglycemia is declared irreversible (3 determinations >25 mM). In Figures 9G, 12B, and 14H, mice were randomized into treatment groups with 2.5mi/IA ^g7 -NP or HEL _14-22 /IA ^g7 -NP (Figure 9G) or 2.5mi/IA ^g7 -NP or IGRP _4-22 /IA ^g7 -NP (figure 12B). Figure 9G in separate groups of mice tested IGRP _4-22 /IA ^g7 and IGRP _128-145 /I ^- A ^g7 . Mice treated with the peptide or peptide-NP complex (FIG. 9G) were randomly assigned to either treatment group within the same experiment. In vivo cytokine neutralization experiments included administration of an anti-CD20 mAb (5D2, gift of A. Chan, Genentech; three doses of 250 μg IV on day 0-2 relative to the onset of hyperglycemia) or 500 μg HRPN (rIgG1), IFNγ (R4-6A2 ), IL-10 (JES5-2A5), TGF-β (1D11) or IL-21R (4A9) (BioXcell) i/p twice a week for 2 weeks followed by 200 mcg per dose for 3 additional weeks. Mice were randomly assigned to treatment with cytokine-blocking mAb (IFNγ, IL-10, TGFβ) or anti-rat HRPN IgG1. mAb to CD20 and to IL21R were tested in separate groups of mice with diabetes (figure 11A). Animals were assessed daily for glycosuria (corresponding to >16 mM blood glucose) and s.c. injected with human insulin Isophane (1 IU per day) if the test was positive. After completion of treatment, NOD mice were checked for relapses of hyperglycemia up to 50 weeks of age.

Therapy with peptide, pMHC, pMHC-NP, peptide-NP or peptide-MP complexes in EAE. Female mice C57BL/6, C57BL/6 Il27r ^-/- or transgenic for HLA-DR4-IE C57BL/6 IAb ^null at the age of six to eight weeks were immunized with 150 μg of PMOG _35-55 or hMOG _97-108 or hPLP _{175 -192} , respectively, s.c. in CFA at the base of the tail, under isoflurane anesthesia. Mice were intravenously injected with 300 ng pertussis toxin on days 0 and 3. Mice were weighed and assessed daily starting on day 10 post-immunization. The rating system used is published elsewhere 30 and was plotted against a 5-point scale. When most mice showed signs of full-blown disease (day 14) or when they reached the maximum disease index (day 21), mice were divided into treatment groups, synchronized by mass and mean disease index, and treated twice a week with coated pMHC and uncoated nanoparticles. identical amounts of pMHC monomer, peptide-coated nanoparticles (at an equivalent dose of peptide), free peptide (8 μg per IV or SC dose) (Burton, BR et al. (2014) Nature Commun. 5:4741-4747), peptide-conjugated microparticles (15 µg peptide per dose) (Getts, DR et al. (2012) Nature Biotechnol. 30:1217-1224) or unconjugated microparticles for 5 weeks. Mice were randomized into pMHC-NP treatment groups (one or two different types depending on the experiment, as described in Figures 9J-9N, 10F-10H, 16A-16F, 17A-17I), uncoated nanoparticles, or no treatment. Peptide, peptide-MP, peptide-NP, pMHC monomers and uncoated microparticles were tested together; mice were randomized into each treatment group as the mice reached the indicated disease index (FIGS. 10F, 17F-17I). An additional control group was treated with a single dose of conjugated peptide-microparticles (FIG. 17H). Cytokine and cytokine receptor mAb blocking studies (FIG. 18G) included random assignment of mice to each treatment group.

Peptide, pMHC-NP, or peptide-MP complex therapy for CIA. Bovine collagen II (bCII) dissolved in 0.05M acetic acid at 2 mg ml ^-1 was emulsified in CFA (v/v) containing 4 mg ml ^-1 killed Mycobacterium tuberculosis (H37Ra). HLA-DR4-IE transgenic C57BL/10.M mice aged eight to twelve weeks were intradermally immunized at the base of the tail with 100 μg bCII in CFA and boosted with 100 μg bCII in IFA on days 14 and 28. The size of all four paws was determined with using a compass before immunization (day 0) and daily after the onset of the disease. Disease progression was defined as the percentage increase in joint swelling relative to day 0. When this value reached 130%, mice were split into different treatment groups and treated intravenously twice a week for 5 weeks with pMHC-NPs coated with Cys (non-pMHC-conjugated) NPs. (25 μg rMHC per pMHCNP or iron equivalent for Cys NP conjugated), free peptide (8 μg per s.c. dose) (Burton, BR et al. (2014) Nature Commun. 5:4741-4747) or conjugated with MP peptide (15 μg of peptide per dose) (Getts, DR et al. (2012) Nature Biotechnol. 30:1217-1224). Mice were randomized into different treatment groups with pMHC-NP or uncoated nanoparticles or peptide or peptide-MP, respectively (Figure 10A and Figure 14-21). Mice were also assessed for clinical signs of disease up to a maximum clinical index of 12 as described elsewhere (Leavenworth, JW et al. (2013) J. Clin. Invest. 123:1382-1389).

Therapy with peptide, pMHC-NP, peptide-NP and peptide-MP complexes in NSG hosts with reconstituted human PBMCs. CD8 ⁺ T cells were isolated from PBMCs in new or newly diagnosed T1D patients with HLA-DRB1* 401 ⁺ or -DRB1*0301 ⁺ (included with informed consent, approved by the Institutional Review Board at Hospital Clinic) using coated anti-CD8 mAb magnetic granules (Miltenyi Biotech) and iv were injected (2 x 10 ⁷ ) to 8-10 week old NSG hosts. Mice were treated with pMHC-NP at the indicated doses coated with NP peptide (at an equivalent dose of peptide), peptide alone (8 μg per sc dose) (Burton, BR et al. (2014) Nature Commun. 5:4741-4747) or peptide-conjugated microparticles (15 μg peptide per dose) (Getts, DR et al. (2012) Nature Biotechnol. 30:1217-1224) starting on day 5 after PBMC transfusion twice a week for 5 consecutive weeks or left untreated. Individual patient samples were treated separately and injected into two (for pMHC-NP and peptide-NP complex experiments) or three different mice (for peptide and peptide-MP complex experiments); one or two of the two to three hosts used in each of these experiments were treated and the others were left untreated (Tables 4A-4C). Therapy-induced expansion of CD4 ⁺ recognizing T cells was determined in PLN and/or spleen as described above. HLA genotype, sex, age, months after diagnosis, and type of pMHC-NP tested for each patient are shown in Tables 4A-4C.

Testing tolerance to intraperitoneal glucose. Animals were left fasted overnight and/p stimulated with 2 mg kg ^-1 D-glucose. Blood glucose analysis was performed from the tail vein using a glucometer at various time points before and after glucose stimulation. Serum insulin levels were determined using the Mouse Ultrasensitive Insulin ELISA Kit (ALPCO).

Assessment of systemic cellular and humoral immunity. To evaluate cellular response, pMHC-NP-treated and untreated female mice were intravenously injected with 2×10 ⁶ plaque-forming units (PFU) of recombinant vaccinia virus (rVV). Groups of mice were sacrificed on days 4 and 14 post-injection and treated with pMHC tetramer staining and rVV titer determination. Briefly, ovaries were weighed, homogenized using a pestle in 300 μl RPMI-1640 containing 10% FBS, freeze-thawed 3 times followed by 3 sonication cycles (20 seconds each). Serial dilutions of lysates were added to confluent cultures of BSC-1 cells in 6-well plates, incubated at 37°C for 2 hours, washed twice with PBS, and cultured in DMEM10. On day 2, the supernatants were removed and the cell layers were stained with crystal violet to reveal plaques.

To evaluate humoral immunity, pMHC-NP-treated and untreated mice were i/p immunized with 100 μg DNP-KLH (Alpha Diagnostic International) in CFA. After 3 weeks, an identical secondary injection was performed. Mice were sacrificed after 10 days. Anti-DNP antibody titers were determined by diluting serum samples in PBS containing 0.05% Tween 20. Anti-DNP antibodies were determined semi-quantitatively using an anti-DNP Ig ELISA kit (Alpha Diagnostic International) according to the manufacturer's instructions.

Cytokine proliferation and secretion assays. CD4 ⁺ T cells of pMHC-NP treated mice from peripheral lymphoid organs were enriched using the BD Imag enrichment kit, stained with pMHC tetramers as described above, and sorted by flow cytometry. For memory cell and naïve BDC2.5 CD4 ⁺ T cell assays, cells were enriched using Stem Cell Technologies stem cell enrichment kit, antibody stained, and sorted. FACS-sorted cells (2-3×10 ⁴ ) were co-cultured with bone marrow-derived DCs (2×10 ⁴ ) pulsed with 2 μg ml ^-1 peptide. After 48 hours, supernatants were harvested for cytokine measurement with Luminex and cells were pulsed with 1 microCi (μCi) ( ³ H)-thymidine and harvested 24 hours later to determine thymidine incorporation in triplicate.

To determine the ability of pMHC-NP therapy to stimulate the formation of IL-10 secreting B cells in the PLN of PBMC transplanted NSG hosts, Applicant stained PLN and spleen cell suspensions from each of the mice with anti-hCD4-FITC antibodies, anti-hCD19-APC antibodies, and tetramer-PE, as described above, and sorted B cells by flow cytometry (FACSAria-BD Biosciences). Sorted B cells of each organ were stimulated with LPS (1 μg ml ^- 1, Sigma) for 24 hours in RPMI-1640 supplemented with 10% human AB serum. The content of IL-10 in the supernatants was determined in duplicate by Meso Scale technology using the V-PLEX Custom Human Cytokine for hIL-10 (Meso Scale Discovery). Data were normalized to spleen B cell values and reported as fold change.

Isolation and stimulation of CD11b ⁺ cells from PLN and MLN in vitro. CD11b ⁺ LN cells were obtained by digestion with collagenase D (1.25 μg ml ^-1 ) and DNase I (0.1 μg ml ^-1 ) for 15 min at 37°C, followed by purification with coated anti-CD11b mAb (BD Imag) magnetic granules. Cells were stimulated with LPS (2 μg ml ^-1 ) for 3 days and supernatants were analyzed for cytokine content using Luminex multiplex cytokine assay.

In vitro suppression assays. FACS sorted positive or tetramer negative 2.5mi/IA ^g7 cells (2×10 ⁴ ) were co-cultured with bone marrow-derived DCs (2×10 ⁴ ) pulsed with 2 μg ml ^-1 "suppressive" (2.5mi or GPI _{282- 292} ) and response peptides (gp33 or NRP-V7). The response cells were CD8 ⁺ T cells (2×10 ⁴ ) isolated from 8.3-NOD mice or LCMV-Gp33-specific TCR NOD mice transgenic using BD-Imag beads. These cells were labeled with CFSE (5 μM) and added to DC cultures in duplicate or triplicate. After 48 hours, the dilution of CFSE in responding cells was determined by FACS. In other experiments, rIgG, anti-IFNγ antibodies, anti-IL10 antibodies, or anti-TGF-β antibodies (all 10 µg ml ^- 1) or the IDO inhibitor, 1-methyltryptophan (1-MT; 400 μM).

Suppression of cross-presentation in vivo. For cross-presentation assays in non-transgenic mice, Applicant transfused CFSE-labeled 8.3-CD8 ⁺ reporter cells (5-10×10 ⁶ ) into untreated or pMHC-NP-treated mice and determined the dilution of CFSE in host lymphoid organs within a period of 7 days post-transfer.

Adoptive transfer of suppression. CD4 ⁺ or CD8 ⁺ T cells from the spleen (10 ⁷ ) of untreated mice or mice treated with 10 doses of 2.5 mi/IA ^g7 -NP or uncoated nanoparticles were transplanted into NOD scid females at 5-10 weeks of age. Hosts were transfused 2×10 ⁷ with a mixture of CD4 ⁺ or CD8 ⁺ spleen T cells isolated from NOD female donors 24 hours later. The owners were observed to develop diabetes for at least 90 days after the transfer (figure 9E). In another experiment, hosts were treated twice a week with 2.5 mi/IA ^g7 -NP (Figure 9E). In other experiments (Figure 11E), female NOD scid aged 5-6 weeks with restored CD4 ⁺ or CD8 ⁺ T cells were transfused with 5×10 ⁵ CD19 ⁺ cells isolated from PLN mice treated with 10 doses of uncoated or coated 2.5mi/ IA ^g7 NP within the previous 5 weeks (figure 11D). B cells were isolated using the EasySep Mouse CD19-positive selection Kit II (StemCell Technologies). Other groups, studied separately (figure 11E), were injected with CD19 ⁺ cells (5×10 ⁵ ) PLN or MLN and total CD4 ⁺ T cells of the spleen (10 ⁷ ) or tetramer 2.5mi/IA ^g7+ (2×10 ⁵ ) or tetramer ^- CD4 ⁺ T cells (10 ⁷ ) obtained from treated 2.5mi/IA ^g7 -NP donors. Hosts were randomly assigned to groups with each of the transfusions and co-monitored for the development of diabetes. Figure 11E includes data from the respective groups described in Figures 9E and 11D. Isolation of 2.5mi/IA ^g7+ tetramer and 2.5mi/IA ^g7- tetramer cells from total spleen CD4 ⁺ T cells of 2.5mi/IA ^g7 -NP treated mice was performed using anti-PE mAb coated microbeads and MACS LD columns (Miltenyi Biotec).

B cell proliferation and induction of _Breg in vivo and suppression of _Breg in vitro. To isolate spleen DCs, spleens were digested in collagenase D and DNase for 15 minutes at 37° C. and DCs were isolated using anti-CD11c mAb coated magnetic beads (MACS). Cells were pulsed with 10 μg ml ^-1 peptides 2.5mi or GPI _282-292 for 2 hours at 37°C and labeled with CFSE (0.5 μm) or PKH26 (2 μm), respectively. Labeled cells (5-10×10 ⁶ ; mixed 1:1) were injected intravenously into pMHC-NP treated or untreated NOD mice. Three days later, Applicant compared the ratios of CFSE ⁺ cells and PKH26 ⁺ cells in the spleens of different hosts by means of FACS. Similar experiments were performed using pulsed peptide-labeled spleen B cells isolated from female donor mice using anti-B220 mAb-coated magnetic beads (MACS).

For in vivo Breg induction assays, mouse NOD _Il10 ^GFP (tiger) B cells were enriched using a CD19 enrichment kit (Stem Cell Technologies) and pulsed with 2.5mi or GPI _{282-292 peptides} (10 μg ml ^-1 ) in for 2 hours at 37°C. Peptide-pulsed B cells were washed twice with PBS, labeled with PKH26, and transplanted (1×10 ⁶ ) into pMHC-NP treated or untreated mice. After 7 days, the hosts were sacrificed and their spleens were labeled with anti-B220-APC antibodies and biotinylated anti-CD1d or anti-CD5 mAb and streptavidin-PerCP. PKH26 ⁺ cells were analyzed for the presence of eGFP ⁺ CD1d ^high or CD5 ⁺ cells by flow cytometry.

To determine the role of cytokines produced by T _R 1 in the formation of B _reg (figure 18U), the applicant repeated the experiments described above, but using 3×10 ⁶ B cells and hosts treated with 250 or 500 μg (introduced i/p daily starting from day - 3 to day 6 relative to B cell transfer) mAb to HRPN (rIgG1), to IL-10 (JES5-2A5), to TGFβ (1D11) or to IL-21R (4A9) (BioXcell). Hosts were randomly assigned to each antibody treatment group and co-examined.

To determine the ability of T _R 1 induced _Breg cells to suppress antigen-induced T cell activation in vitro, Applicant isolated CD19 ⁺ B cells from PLN age-matched untreated NOD mice or NOD mice treated with 10 doses of 2.5mi/IA ^g7 -NPs and cultured these cells with LPS (10 μg ml ^-1 ) overnight. Applicant then cultured these cells (2×10 ⁴ ) with pulsed 2.5mi peptide (0.1 μg ml ^-1 ) bone marrow-derived DC (2×10 ⁴ ) and CFSE-labeled BDC2.5 CD4 ⁺ cells (4× ¹⁰⁴ ). After 3 days, the dilution of CFSE in CD4 ⁺ cells was determined.

Depletion of CD25 ⁺ CD4 ⁺ T _reg . NOD i/p mice were treated with 500 µg of anti-CD25 antibody (PC61.5.3, BioXcell) 3 times per week starting at 8 weeks of age, followed by 10 pMHC-NP injections twice a week starting at 10 weeks of age. The mean depletion of CD4 ⁺ CD25 ⁺ and FOXP3-eGFP ⁺ CD4 ⁺ T cells was 90% and 70%, respectively.

Histology. Tissues were fixed in 10% formalin and embedded in paraffin, H&E stained pancreas were assessed for insulitis as described (Verdaguer, J. et al. (1997) J. Exp. Med. 186:1663-1676). Briefly, insulitis was scored as: 0, no; 1, with peri-insulitis; 2, with infiltration involving < 25% of the islets; 3, when covering 25-50% of the islets; and 4, when covering > 50% of the islets.

Spinal cord and brain tissues were fixed in 10% buffered formalin for a minimum period of 24 hours, embedded in paraffin, and sectioned at 6 μm. Sections from paraffin-embedded tissues were deparaffinized and subjected to antigen demasking by evaporating the sections in 10 mM sodium citrate buffer (pH 6.0) for 20 min and cooling at room temperature for 20 min. For immunohistochemistry, sections were fixed with 10% formalin and treated with 3% H ₂ O ₂ in methanol at -20°C. Sections were permeated with 0.25% Triton-X 100 and blocked with skim milk blocking solution. Rabbit anti-IBA1 antibodies (Wako, 1:500) or rat anti-MBP antibodies (Abcam) were incubated at 4°C overnight followed by appropriate biotinylation with secondary antibodies (1:500), avidin-biotin complex and 3,3'-diaminobenzidine . Sections were counterstained with hematoxylin and eosin, dehydrated stepwise with ethanol, and mounted with acritol. For histological staining of myelin, sections were fixed with 10% formalin or deparaffinized, dehydrated stepwise with ethanol, and incubated with 0.2% Luxol fast blue in 95% ethanol at 65°C. Sections were stained in 0.05% lithium carbonate, counterstained with hematoxylin and eosin, and mounted with acritol. The cerebellum was imaged using an Olympus bright-field microscope. Inflammatory lesions (dense nucleated cell clusters or perivascular muffs with appropriate demyelination) were counted and sized using ImageJ software. To quantify the relative intensity of IBA1, observers blindly ranked the images from highest to lowest intensity.

The knee joints of bCII immunized mice were fixed in 4% buffered formalin overnight and decalcified using 14% EDTA for 3 weeks. Decalcified limbs were embedded in paraffin, sectioned at 8 µm, and stained with hematoxylin and eosin to assess infiltration and pannus formation on a 5-point scale, where 5 corresponds to erosive arthritis with severe infiltration and pannus covering 60% of the joint space. Proteoglycan depletion at the joint surface of the tibia and femur was assessed by the loss of safranin-O staining intensity. To do this, the sections were deparaffinized, hydrated and stained with hematoxylin, followed by staining with 0.05% aqueous fast green for 5 minutes. Sections were fixed with 1% acetic acid and stained with 0.1% aqueous safranin-O for 2 minutes, dehydrated stepwise with ethanol, cleared with xylene, and mounted using DPX. The assessment was performed on a scale from 0 to 3 corresponding: 0, at 0% depletion, 1, at low (<25%), 2, moderate (25-50%) and 3, severe (>50%). Articular cartilage destruction included the presence of dead chondrocytes (empty lacunae) and was rated on a scale of 3 (0, no empty lacunae; 3, total absence of chondrocytes in the articular cartilage/severe joint erosion).

Isolation of CNS-infiltrating lymphocytes. Mice were anesthetized with ketamiomn-xylazine and perfused with PBS through the left ventricle of the heart. The brain and spinal cord were isolated by hand, but cut into small fragments and digested with a solution of collagenase D (1.25 μg ml ^-1 ) and DNase I (1% w/v) in HBSS for 30 minutes at 37°C. Cleaved CNS was passed through a 70 μm cell filter. Cells were resuspended in DMEM (supplemented with 2% FBS and 10 mM HEPES) and 100% percoll (to a final concentration of percoll 30%). The solution was layered on 65% percoll and centrifuged at 380 g for 30 minutes at room temperature. Before further analyses, the layer of mononuclear cells located in the interphase was washed with RPMI.

Quantitative RT-PCR. RNA was isolated from 2.5mi/IA ^g7+ tetramer or tetramer ^{-CD4 +} T cells sorted from 2.5mi/IA ^g7 -NP treated NOD mice and stimulated in vitro with CD3/CD28 mAb-coated DynaBeads.

Each sample tetramer ⁺ , corresponded to cells collected from 2-3 mice. RNA was reverse transcribed and cDNA was loaded onto Mouse Immunology 384 StellArray qPCR plates (Bar Harbor BioTechnology) using 2× SYBR Green Master Mix (Applied Biosystems). The plate was reacted in an Applied Biosystems 7900HT real-time PCR device and raw data was analyzed using the Global Pattern Recognition (GPR) analysis tool (http://www.gene-quantification.com/qpcr-array.html) . mRNA isolated from additional samples was subjected to RT-qPCR using primers specific for IL-21 (forward: 5'-TCATCATTGACCTCGTGGCCC-3'; reverse: 5'-ATCGTACTTCTCCACTTGCAATCC-3'), IL-10 (forward: 5'-CTTGCACTACCAAAGCCACA-3'; reverse: 5'-GTTATTGTCTTCCCGGCTGT-3'), c-Maf (forward: 5'-AGCAGTTGGTGACCATGTCG-3'; reverse: 5'-TGGAGATCTCCTGCTTGAGG-3'), IFN-γ (forward: 5' -TGAACGCTACACACTGCA TCTTGG-3'; reverse: 5'-CGACTCCTTTTCCGCTTCCTGAG-3'), LAG-3 (direct: 5'-TCCCAAATCCTTCGGGTTAC-3'; reverse: 5'-GAGCTAGACTCTGCGGCGTA-3'), CD49b (direct: 5'- CCGGGTGCTACAAAGTCAT-3'; reverse: 5'-GTCGGCCACATTGAAAAAGT-3'), aryl hydrocarbon receptor (forward: 5'-CGTCCCTGCATCCCACTACTT-3'; reverse: 5'-GGACATGGCCCCAGCATAG-3'), and ICOS (forward: 5'-TGACCCACCTCCTTTTCAAG- 3'; reverse: 5'-TTAGGGTCATGCACACTGGA-3').

pMHC-NP-induced upregulation of T _R 1 transcripts in activated in vitro CD4 ⁺ T cells was performed by culturing naive eGFP ^- BDC2.5-CD4 ⁺ T cells of BDC2.5 NOD Foxp3-eGFP mice (CD62L ^hi FOXP3 ^- eGFP ^- ; 1.5×10 ⁶ ml ^- 1) with mAb-coated anti-CD3/anti-CD28 microparticles (1 bead per cell) for three days in the absence of APC followed by one day of cultivation of re-isolated (without microparticles) CD4 ⁺ T cells in rhIL -2 (30 IU ml ^-1 ) and 6 days of cultivation with peptide 2.5mi (10 μg ml ^-1) , monomers 2.5mi/IA ^g7 (25 μg rMHC per ml), 2.5mi/IA ^g7 -NP (25 μg rMHC per ml and 50 µg ml ^- 1 iron) or unconjugated nanoparticles (50 µg iron per ml). Relative gene expression was calculated using unstimulated cultures as controls.

pMHC-NP-induced upregulation of T _R 1 transcripts in naive BDC2.5 CD4 ⁺ T cells compared to memory cells in vivo was performed by transfusion of naive eGFP-CD4 ⁺ T cells (CD44 ^med CD62L ^hi ) or eGFP-CD4 ⁺ T -memory cells (CD44 ^hi CD62L ^low ) of BDC2.5-TCR transgenic NOD mice or NOD Foxp3-eGFP (Thy1 ^b+ ) mice (1-1.5×10 ⁶ cells per host) to NOD.Thy1 ^a hosts and treating the hosts with four doses of 2.5mi/IA ^g7 -NP for two weeks or leaving them untreated. Two and a half weeks later, Thy1 ^b+ CD4 ⁺ host T cells were sorted and stimulated with anti-CD3 and anti-CD28 coated DynaBeads magnetic beads for 3 days, followed by mRNA isolation and RT-qPCR using primers specific for c-Maf, IL- 21, IL-10, IFNγ, LAG-3 and CD49b.

To compare IL-10 mRNA levels in tetramer ⁺ CD4 ⁺ T cells versus tetramer ^- CD4 ⁺ T cells of pMHC-NP treated NSG hosts transplanted with PBMC, Applicant stained splenocytes with anti-hCD4-FITC, anti-hCD19-APC and tetramer complex. -PE as described above and cell sorted tetramer ⁺ and tetramer ^- individual hosts by FACS (FACSAria-BD Biosciences). Sorted cells were cultured for 72 hours in RPMI-1640 containing 10% human AB serum in the presence of DynaBeads with human CD3/CD28 T-activator (LifeTechnologies) using a cell to bead ratio of 1:1. Total RNA from cell pellets was reverse transcribed using a double reverse transcriptase/lysing solution containing 5 mM DTT, 2 U ml ^-1 RNase, 500 mM dNTP, 10 U ml ^-1 Superscript reverse transcriptase (Invitrogen, LifeTechnologies), 100 mg ml ^-1 bovine serum albumin, 1% Triton X-100, 25 ng ml ^-1 oligo-dT (Invitrogen), 0.5 nM spermidine and 1× First Strant buffer (Invitrogen) in 20 µl for 60 minutes at 50° C and 15 minutes at 70°C. We then mixed 1 µl cDNA reaction volume with 12.5 µl Power SyBRGreen PCR ready mix solution (Applied Biosystems) and loaded into a real-time PCR device (7900HT, Applied Biosystems) using the following primers: β-actin ( direct: 5'-CTGGAACGGTGAAGGTGACA-3'; reverse: 5'-AAGGGACTTCCTGTAACAATGCA-3'), IL-10 (direct: 5'-AAGACCCAGACATCAAGGCG-3'; reverse: 5'-AATCGATGACAGCGCCGTAG-3').

Statistical analyses. The sample sizes described in the legends of the figures correspond to the numbers of individual mice tested (no repetitions) and the data presented correspond to the pooled data from different experiments. Data were compared by Student's t-test, Mann-Whitney U-test, chi-square test, log-rank test (Mantel-Cox), Pearson correlation, or two-way ANOVA. Statistical significance was considered at P<0.05.

Example 3 Effects of valence and density of pMHC in vivo.

The Applicant then experimentally tested the predictions of the mathematical model by comparing the proliferation properties of Treg cells of various NP preparations with PF-M (≈20 nm) and SFP-Z (≈8 nm) coated with pMHC _2.5mi /IA ^g7 , which stimulated the proliferation of recognition regulatory Type 1 CD4 ⁺ T cells. Comparison of the Treg proliferating properties of 7 different _2.5mi -IA ^g7 -PF-M preparations with 29-59 pMHC/NP demonstrated a clear dose-dependent effect of rMHC within individual preparations, as well as no significant effect rMHC valency in batches (Figure 22A). However, it is important that studies using smaller preparations of 2.5mi-IA ^g7 -SFP-Z with 22-44 _pMHC /NP show a significantly stronger T re proliferating effect at all doses tested (0.75-1 µg, 7 .5-10 μg and 25 μg total pMHC/dose) than 2.5mi-IA ^g7 -PF-M particles with 29-45 pMHC/NP (Figure 22B). These results were further confirmed by generating 11 nm diameter PF-M NPs and testing the ability of their 2.5mi-IA ^g7 coated analogs to expand T _R 1 recognizing T cells in vivo. Notably, 11 nm diameter PF-M NPs delivering 7.5 µg of total pMHC at 15 pMHC/NP propagated TR1 recognition cells up to 1.6±0.3% of total spleen CD4 ⁺ T cells, values comparable to a series of SFP NPs delivering 7.5 µg of 22-44 pMHC/NP.

As stated above, Applicants have demonstrated that autoreactive CD4 ⁺ memory T cells express a balanced T-regulatory type 1 (T _R 1) transcriptional program and export to the cell surface LAG3 rather than CD49b (T _R 1 markers). Since 2.5mi-IA ^g7 -NP therapy initiates IL-10 expression and upregulation of CD49b on balanced T _R 1 memory T cells, thus allowing them to be converted into stable T _R 1 cells, Applicant wondered if the efficacy was also regulated. these processes by the pMHC density on the NP surface. Notably, a statistically significant correlation was observed between 2.5mi/IA ^g7 density (but not total rMHC dose) and upregulation of CD49b (but not LAG3) expression on T _R 1 -like CD4 ⁺ T cells that proliferate in wild-type NOD mice in response to for 2.5mi/IA ^g7 -NP therapy; this effect peaked at ≈0.012 pMHC/nm ² (FIG. 22C). Taken together, these results support the thesis that rMHC density is a critical parameter in the development of rMHC-based nanodrugs.

This effect of rMHC density on biological activity has also been observed in vivo; an increase in rMHC density resulted in increased expression of the T _R 1 cell marker CD49b in pMHC-NP-treated mice, suggesting that _rMHC density is responsible for promoting Treg proliferation. Although the total dose of rMHC was associated with the Treg proliferating properties of these _nanodrugs , it had only a minor effect on this phenotype, suggesting that rMHC density and _rMHC dose play different roles in mediating Treg conversion and expansion, respectively.

Equivalents

It should be understood that although the present invention is specifically described by way of certain embodiments and optional features, modifications, improvements, and variations of the invention described herein can be made by those skilled in the art, and that such modifications, improvements, and variations are considered to be within the scope of this inventions. The materials, methods, and examples provided herein are characteristics of certain embodiments, are illustrative, and are not intended as limitations on the scope of the invention.

The invention is described herein in a broad and general sense. Each of the narrower components and sub-general groupings included in the main invention also forms part of the invention. This includes a general description of the invention with the condition or negative limitation of removing any of the subject matter from the genus, whether or not the excluded material is specifically described herein.

In addition, when features or aspects of the invention are described in terms of Markush groups, those skilled in the art will appreciate that the invention is also so described in relation to any individual members or subgroups of members of the Markush group.

The use of the term "or" in the claims is used to mean "and/or" unless explicitly stated to indicate only alternatives or alternatives are mutually exclusive, although the invention supports a definition that refers only to alternatives and "and/or".

As used in the present description and claims, the words "comprising" (and any form of content such as "comprise" and "comprises"), "having" (and any form of possession such as "possess" and "possesses"), "comprising" (and any form of inclusion such as "includes" and "include") are inclusive or open-ended and do not exclude additional, non-listed elements or method steps.

Throughout this specification, various publications, patents, and published patent specifications are cited by means of an identifying citation. All publications, patent applications, patents, and other references herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In the event of a conflict, the present description, including definitions, shall govern.

* The result is considered (+) if more than the average + 3 S.E. is stained with the control tetramer. samples

* The result is considered (+) if more than the average + 3 S.E. correspond to samples obtained from NSG hosts with reconstituted PBMCs, but not untreated pMHC-NPs.

**PLN samples with increased IGRP 13-25/DR3 tetramer+ cells were significantly enlarged and had increased cell saturation compared to samples obtained from treated mice with no such increase (P=0.048) or from untreated mice (P=0.036 ). There was a statistically significant correlation between % tetramer+ cells and total PLN cells (r2=0.455; P=0.032)

* The result is considered (+) if more than the average + 3 S.E. correspond to samples obtained from NSG hosts with reconstituted PBMCs, but not untreated.

Links

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Claims (14)

1. Pharmaceutical composition for the treatment of primary biliary cirrhosis in a subject, containing a complex of nanoparticles, the core of which is associated with the number of antigen-MHC class II complexes (pMHCII), where the core contains iron oxide and its diameter is from 1 nm to 25 nm; where the antigen is PDC-E2 or a fragment thereof capable of inducing an immune response; where the number of bound pMHCII per nanoparticle surface area is from 0.4 pMHC/100 nm ² to 12 pMHC/100 nm ² , where the pMHCIIs are linked to the maleimide-functionalized end of a polyethylene glycol (PEG) linker having a molecular weight less than 5 kDa, where the non-maleimide-functionalized end of the polyethylene glycol (PEG) linker is linked to the surface of the core, the complex differentiating an activated or memory T cell into a TR1 cell , producing IL-10, and wherein the nanoparticle complex is contained in an amount sufficient to treat primary biliary cirrhosis in a subject. 2. The pharmaceutical composition according to claim 1, wherein the MHCII molecule of the plurality of antigen-pMHCII complexes contains HLA DR, HLA DQ, HLA DP, or a fragment thereof. 3. Pharmaceutical composition according to claim 2, where the MHCII molecule of the plurality of antigen-pMHCII complexes contains HLA-DRB1/DRA, HLA-DRB3/DRA, HLA-DRB4/DRA, HLA-DRB5/DRA, HLA-DQA1/HLA-DQB1, HLA-DPB1/HLA-DPA1 or a fragment thereof. 4. Pharmaceutical composition according to claim 1, wherein the complex further comprises an outer layer. 5. The pharmaceutical composition according to claim 1, wherein the valency of the plurality of antigen-pMHCII complexes per core is between 10:1 and 100:1. 6. Pharmaceutical composition according to claim 1, wherein the nanoparticle core diameter is from 5 nm to 25 nm. 7. The pharmaceutical composition according to claim 1, wherein the amount of bound pMHCII per nanoparticle surface area is from 0.4 pMHCII/100 nm ² to 6 pMHCII/100 nm ² . 8. The pharmaceutical composition of claim 1, wherein the pMHCII is covalently linked to the core by forming a carbon-sulfur bond between the thiol (-SH) group of the cysteine residue added to the carboxyl end of the pMHCII and the maleimide. 9. The pharmaceutical composition according to claim 1, wherein the IL-10 producing TR1 cell expresses CD49b.

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