Nanobody (VHH) conjugates and their uses

Abstract

This invention provides compositions containing VHH conjugates and their use in treating diseases. [Solution] (i) A conjugate comprising a single-domain antibody (VHH) conjugated with an antigen and an anti-inflammatory agent, wherein the VHH conjugates the single-domain antibody (VHH) to a surface protein on an antigen-presenting cell (APC); or (ii) A first conjugate comprising VHH conjugated to an antigen, wherein the first VHH and the second VHH bind to one or more surface proteins on an antigen-presenting cell (APC), and a second conjugate comprising the second VHH conjugated to an anti-inflammatory agent, A composition containing the following is provided.

Description

[Technical Field]

[0001] Related applications This application claims the benefits under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 033,710, filed 2 June 2020, entitled “NANOBODY (VHH) CONJUGATES AND USES THERE OF”, and U.S. Provisional Application No. 63 / 154,455, filed 26 February 2021, entitled “NANOBODY (VHH) CONJUGATES AND USES THERE OF”. The entire contents of each of these are incorporated into this application by reference.

[0002] Federal funding research This invention was made with government assistance under P01DK011794, awarded by the National Institutes of Health. The government has certain rights to this invention. [Background technology]

[0003] Approximately 10% of the human population suffers from autoimmune conditions accompanied by symptoms ranging from mild to life-threatening. Current treatments for autoimmune diseases involve general immunosuppression, which dulls the response to all kinds of antigens. This exposes patients to an increased risk of infection and possibly even malignant pathogens. [Overview of the project]

[0004] This disclosure provides compositions comprising one or more conjugates, in several aspects, each comprising a single-domain antibody fragment (nanobody / VHH) conjugated to an antigen and / or drug (e.g., an anti-inflammatory or pro-inflammatory drug), the VHH binding to a surface protein on an antigen-presenting cell (APC). In some aspects, the antigen and drug (e.g., an anti-inflammatory or pro-inflammatory drug) are conjugated to the same VHH. In some aspects, the antigen and drug (e.g., an anti-inflammatory or pro-inflammatory drug) are conjugated to two VHHs.

[0005] The conjugates described herein involve antigen-presenting cells (APCs), which can lead to tolerance in non-inflammatory states, but the involvement of APCs in inflammatory states can induce a strong immune response to foreign antigens. Surprisingly, in this application, it has been found that the compositions of the disclosure are significantly more effective in inducing immune tolerance and alleviating symptoms of the target autoimmune disease compared to administration of VHH-antigen alone when the antigen is an autoantigen and the agent is an anti-inflammatory agent. Similarly, the compositions of the disclosure are significantly more effective in inducing an immune response to an antigen and / or pathogen compared to administration of VHH-antigen alone when the antigen is from a pathogen and the agent is a pro-inflammatory agent.

[0006] Some aspects of this disclosure are: (i) A conjugate containing a single-domain antibody (VHH) conjugated with an antigen and an anti-inflammatory agent, in which the VHH binds to a surface protein on an antigen-presenting cell (APC); or (ii) A first conjugate comprising VHH conjugated to an antigen, wherein the first VHH and the second VHH bind to one or more surface proteins on an antigen-presenting cell (APC), and a second conjugate comprising the second VHH conjugated to an anti-inflammatory agent, The present invention provides a composition comprising the above. In some embodiments, the surface protein on the APC is selected from the group consisting of MHCII, CD11c, DEC205, DC-SIGN, CLEC9a, CD103, CX3CR1, CD1a, and F4 / 80. In some embodiments, the targeting moiety may be replaced by a native or synthetic polypeptide, including but not limited to peptide fragments, single-stranded fragment variable regions (scFv), diabodies, Fab, or similar formats.

[0007] In some embodiments, the composition comprises a conjugate containing VHH conjugated to an antigen and an anti-inflammatory agent, wherein the VHH binds to MHCII. In some embodiments, the composition comprises a first conjugate containing a first VHH conjugated to an antigen and a second conjugate containing a second VHH conjugated to an anti-inflammatory agent, wherein both the first and second VHH bind to MHCII. In some embodiments, the VHH comprises the amino acid sequence of SEQ ID NO: 1. In further embodiments, the VHH conjugated to an antigen or anti-inflammatory agent may have the format of a DNA or RNA molecule encoding a predetermined conjugate.

[0008] In some embodiments, the MHCII-binding VHH further includes a saltase recognition sequence at its N-terminus or C-terminus. In some embodiments, the saltase recognition sequence includes the amino acid sequence LPETG (SEQ ID NO: 29). In some embodiments, the saltase recognition sequence includes the amino acid sequence LPETGG (SEQ ID NO: 43). In some embodiments, the anti-inflammatory agent or antigen is conjugated to the VHH via the saltase recognition sequence. In some embodiments, the anti-inflammatory agent further includes a hydrolyzable or non-hydrolyzable linker. In further embodiments, the conjugate is produced by means of gene fusion, other ligation enzymes (e.g., buterase, OaAEP1, subtiligase, etc.), or chemical methods (e.g., N-terminal modification using 2-pyridinecarbaldehyde (2-PCA), etc.).

[0009] In some embodiments, the composition comprises a conjugate containing a single-domain antibody (VHH) conjugated to an antigen and an anti-inflammatory agent, the VHH binding to CD11c. In some embodiments, the composition comprises a first conjugate containing a first VHH conjugated to an antigen and a second conjugate containing a second VHH conjugated to an anti-inflammatory agent, both of which bind to CD11c. In some embodiments, the VHH comprises the amino acid sequence of SEQ ID NO: 2. In further embodiments, the VHH conjugated to an antigen or anti-inflammatory agent may have the format of a DNA or RNA molecule encoding a predetermined adduct.

[0010] In some embodiments, the CD11c-binding VHH further includes a saltase recognition sequence at its N-terminus or C-terminus. In some embodiments, the saltase recognition sequence includes the amino acid sequence LPETG (SEQ ID NO: 29). In some embodiments, the saltase recognition sequence includes the amino acid sequence LPETGG (SEQ ID NO: 43). In some embodiments, the anti-inflammatory agent or antigen is conjugated to the VHH via the saltase recognition sequence. In some embodiments, the anti-inflammatory agent further includes a hydrolyzable or non-hydrolyzable linker. In further embodiments, the conjugate is produced by means of gene fusion, other ligation enzymes (e.g., buterase, OaAEP1, subtiligase, etc.), or chemical methods (e.g., N-terminal modification using 2-pyridinecarbaldehyde (2-PCA), etc.).

[0011] In some embodiments, the composition comprises a first conjugate containing a first VHH conjugated to an antigen and a second conjugate containing a second VHH conjugated to an anti-inflammatory agent, wherein the first and second VHHs bind to different surface proteins on the APC. In some embodiments, the first VHH binds to MHCII and the second VHH binds to CD11c. In some embodiments, the first VHH binds to DEC205 and the second VHH binds to MHCII.

[0012] In some embodiments, the anti-inflammatory agent is a steroidal anti-inflammatory agent, selected from the group consisting of dexamethasone, prednisone, prednisolone, triamcinolone, methylprednisolone, and betamethasone. In some embodiments, the anti-inflammatory agent is a nonsteroidal anti-inflammatory agent, selected from the group consisting of aspirin, celecoxib, diclofenac, ibuprofen, ketoprofen, naproxen, oxaprozin, piroxicam, cyclosporine A, and calcitriol. In some embodiments, the anti-inflammatory agent is an anti-inflammatory cytokine, selected from the group consisting of IL-10, IL-35, IL-4, IL-11, IL-13, and TGFβ.

[0013] In some embodiments, the antigen comprises polypeptides, polysaccharides, carbohydrates, lipids, nucleic acids, or combinations thereof. In some embodiments, the antigen is an autoantigen. In some embodiments, the autoantigen is selected from myelin oligodendrocyte glycoprotein, myelin proteolipidoprotein, citrullinated fibrinogen, insulin, chromogranin A, glutamate decarboxylase 65 kilodalton isoform (GAD65), desmoglein 1 (DSG1), desmoglein 3 (DSG3), acetylcholine receptor (AChR), muscle-specific tyrosine kinase (MuSK), and ribonucleoprotein. In some embodiments, the antigen comprises proteins used in protein replacement therapy or gene therapy. In some embodiments, the antigen is selected from factor IX, factor VIII, insulin, and AAV-derived proteins.

[0014] Other aspects of this disclosure provide methods comprising administering compositions described herein to subjects requiring such administration. In some embodiments, the administered composition comprises (i) a conjugate comprising a single-domain antibody (VHH) conjugated to an antigen and an anti-inflammatory agent, wherein the VHH binds to a surface protein on an antigen-presenting cell (APC); or (ii) a second conjugate comprising a first conjugate comprising a VHH conjugated to an antigen and a second VHH conjugated to an anti-inflammatory agent, wherein the first VHH and the second VHH bind to one or more surface proteins on an antigen-presenting cell (APC). In some embodiments, the method is for inducing immune tolerance to an antigen. In some embodiments, the method is for treating autoimmune diseases. In some embodiments, the autoimmune diseases are selected from the group consisting of autoimmune encephalomyelitis, multiple sclerosis, type 1 diabetes mellitus, pemphigus vulgaris, myasthenia gravis, lupus, celiac disease, and inflammatory bowel disease (IBD). In some embodiments, the administration is intravenous. In some embodiments, the subject is human.

[0015] Other aspects of this disclosure are: (i) A conjugate containing a single-domain antibody (VHH) conjugated with an antigen and a pro-inflammatory agent, in which the VHH binds to a surface protein on an antigen-presenting cell (APC); or (ii) A first conjugate comprising VHH conjugated to an antigen, wherein the first VHH and the second VHH bind to one or more surface proteins on an antigen-presenting cell (APC), and a second conjugate comprising the second VHH conjugated to a pro-inflammatory agent, The present invention provides a composition comprising the above. In some embodiments, the surface protein on the APC is selected from the group consisting of MHCII, CD11c, DEC205, DC-SIGN, CLEC9a, CD103, CX3CR1, CD1a, and F4 / 80. In some embodiments, the targeting moiety may be replaced by a native or synthetic polypeptide, including but not limited to peptide fragments, single-stranded fragment variable regions (scFv), diabodies, Fab, or similar formats.

[0016] In some embodiments, the composition comprises a conjugate containing a single-domain antibody (VHH) conjugated to an antigen and a pro-inflammatory agent, wherein the VHH binds to MHCII. In some embodiments, the composition comprises a first conjugate containing a first VHH conjugated to an antigen and a second conjugate containing a second VHH conjugated to a pro-inflammatory agent, wherein both the first and second VHH bind to MHCII. In some embodiments, the VHH comprises the amino acid sequence of SEQ ID NO: 1. In further embodiments, the VHH conjugated to an antigen or pro-inflammatory agent may have the format of a DNA or RNA molecule encoding a predetermined conjugate.

[0017] In some embodiments, the MHCII-binding VHH further includes a saltase-recognition sequence at its N-terminus or C-terminus. In some embodiments, the saltase-recognition sequence includes the amino acid sequence LPETG (SEQ ID NO: 29). In some embodiments, the saltase-recognition sequence includes the amino acid sequence LPETGG (SEQ ID NO: 43). In some embodiments, the pro-inflammatory agent or antigen is conjugated to the VHH via the saltase-recognition sequence. In some embodiments, the pro-inflammatory agent further includes a hydrolyzable or non-hydrolyzable linker. In further embodiments, the conjugate is produced by means of gene fusion, other ligation enzymes (e.g., buterase, OaAEP1, subtiligase, etc.), or chemical methods (e.g., N-terminal modification using 2-pyridinecarbaldehyde (2-PCA), etc.).

[0018] In some embodiments, the composition comprises a conjugate containing a single-domain antibody (VHH) conjugated to an antigen and a pro-inflammatory agent, the VHH binding to CD11c. In some embodiments, the composition comprises a first conjugate containing a first VHH conjugated to an antigen and a second conjugate containing a second VHH conjugated to a pro-inflammatory agent, both of which bind to CD11c. In some embodiments, the VHH comprises the amino acid sequence of SEQ ID NO: 2. In further embodiments, the VHH conjugated to an antigen or pro-inflammatory agent may have the format of a DNA or RNA molecule encoding a predetermined conjugate.

[0019] In some embodiments, the CD11c-binding VHH further includes a saltase recognition sequence at its N-terminus or C-terminus. In some embodiments, the saltase recognition sequence includes the amino acid sequence LPETG (SEQ ID NO: 29). In some embodiments, the saltase recognition sequence includes the amino acid sequence LPETGG (SEQ ID NO: 43). In some embodiments, the pro-inflammatory agent or antigen is conjugated to VHH via the saltase recognition sequence. In some embodiments, the pro-inflammatory agent further includes a hydrolyzable or non-hydrolyzable linker. In further embodiments, the conjugate is produced by means of gene fusion, other ligation enzymes (e.g., buterase, OaAEP1, subtiligase, etc.), or chemical methods (e.g., N-terminal modification using 2-pyridinecarbaldehyde (2-PCA), etc.).

[0020] In some embodiments, the composition comprises a first conjugate containing a first VHH conjugated to an antigen and a second conjugate containing a second VHH conjugated to a pro-inflammatory agent, wherein the first and second VHHs bind to different surface proteins on APV. In some embodiments, the first VHH binds to MHCII and the second VHH binds to CD11c. In some embodiments, the first VHH binds to DEC205 and the second VHH binds to MHCII.

[0021] In some embodiments, the pro-inflammatory agent is selected from the group consisting of: TLR9 agonists, LPS, HMGB1 protein, IL2, IL12, and CD40L.

[0022] In some embodiments, the antigen comprises polypeptides, polysaccharides, carbohydrates, lipids, nucleic acids, or combinations thereof. In some embodiments, the antigen is derived from a microbial pathogen. In some embodiments, the microbial pathogen is mycobacteria, bacteria, fungi, viruses, parasites, or prions. In some embodiments, the antigen comprises the SARS-CoV-2 spike protein.

[0023] In some embodiments, the antigen is a tumor antigen.

[0024] In some embodiments, the composition is a vaccine composition.

[0025] Other aspects of this disclosure provide methods comprising administering compositions described herein to subjects requiring such methods. In some embodiments, a composition comprises (i) a conjugate comprising a single-domain antibody (VHH) conjugated to an antigen and a pro-inflammatory agent, wherein the VHH binds to a surface protein on an antigen-presenting cell (APC); or (ii) a first conjugate comprising a VHH conjugated to an antigen and a second conjugate comprising a second VHH conjugated to a pro-inflammatory agent, wherein the first VHH and the second VHH bind to one or more surface proteins on an antigen-presenting cell (APC). In some embodiments, the method is for inducing an immune response to an antigen. In some embodiments, the antigen is from a microbial pathogen and the method is for treating an infection caused by the pathogen. In some embodiments, the method is therapeutic or prophylactic. In some embodiments, the antigen is a tumor antigen and the method is for treating cancer.

[0026] In some embodiments, administration is intravenous. In some embodiments, the subject is human.

[0027] The above summary is intended to illustrate, in an unlimited manner, some aspects, advantages, features, and uses of the technology disclosed herein. Other aspects, advantages, features, and uses of the technology disclosed herein will become apparent from the detailed description, drawings, examples, and claims. [Brief explanation of the drawing]

[0028] The accompanying drawings are not intended to be drawn to a fixed scale. In the drawings, each identical or nearly identical component illustrated in various figures is represented by the same type of number. For clarity purposes, not every component may be marked in every drawing. In the drawings: [Figure 1]A single dose of VHHMHCII-MOG35-55 provides sustained protection from EAE. (Figure 1A) Schematic diagram of nanobody C-terminal saltase labeling with antigen peptide containing GGG. (Figure 1B) LC-MS of purified VHHMHCII and VHHMHCII-antigen adducts. (Figures 1C-E) Mean disease scores of mice that received prophylactic treatment with VHH-peptide as directed in 3 (Figure 1C), 2 (Figure 1D), and 1 dose (single or multiple) (Figure 1E). Disease scores: 1, tail drooping; 2, partial hindlimb paralysis; 3, complete hindlimb paralysis; 4, complete hind and partial forelimb paralysis; and 5, mortality. **p<0.01, repeated measures two-way ANOVA. (Figure 1F) Flow cytometry of Th1 and Th17CD4+ lymphocytes in the spinal cord collected at the endpoint of mice that received 1 dose of VHH-antigen. The frequency of FoxP3+CD4+ regulatory T cells is also indicated. Data are shown as mean + / - SEM. ns not significant; *p<0.05, **p<0.01, ***p<0.001, unpaired t-test with Holm-Sidak adjustment. Representative (Figure 1G) H&E and (Figure 1H) Luxor Fast Blue staining of spinal cord sections from mice receiving a single dose of VHH-antigen adduct. Scale bar 100 μm. (Figure 1I) Mean disease scores of mice treated prophylactically with VHH-peptide at 60, 30, and 7 days prior to EAE induction. (Figure 1J) Mean clinical scores of VHHMHCII-MOG35-55 recipients subjected to multiple loadings with MOG / CFA / PTX and MOG / IFA / PTX. *p<0.05, **p<0.01, repeated measures two-way ANOVA. [Figure 2]Splenic CD11c+ dendritic cells are responsible for inducing VHHMHCII-MOG35-55 tolerance by enhancing antigen presentation. (Figure 2A) In vivo distribution of VHHMHCII. VHHMHCII-Alexa647 was intravenously injected into MHCII-GFP mice. 1.5 hours after injection, spleens, whole blood, and inguinal lymph nodes (iLNs) were collected and analyzed by flow cytometry. (Figure 2B) Mean clinical scores of mice that received splenocytes and peripheral blood mononuclear cells (PBMCs) from mice treated with VHHMHCII-OVA323-339 or VHHMHCII-MOG35-55. (Figure 2C) Mean clinical scores of mice prophylactically treated with VHHMHCII-OVA323-339 or VHHMHCII-MOG35-55 after depletion of the indicated cell subset, preceding induction of EAE. (Figure 2D) Mean disease score of mice treated with the indicated VHH-antigen. (Figure 2E) LC-MS of purified VHHMHCII-MOG17-78. (Figure 2F) Mean disease score of mice treated prophylactically with VHH-peptide. ***p<0.001, repeated measures two-way ANOVA. [Figure 3]VHHMHCII-MOG35-55 upregulates co-inhibitory receptors on MOG35-55-specific CD4 T cells. (Figure 3A) Congenically marked CD45.1 mice received 2D2 CD4 T cells of CellTrace Violet-labeled CD45.2 one day prior to infusion with VHH-antigen. The number of 2D2 CD4 T cells in the spleen, blood, and inguinal lymph nodes (iLNs) was determined by flow cytometry. (Figure 3B) Violet Trace dilution indicates proliferation of 2D2 T cells. (Figure 3C) In a separate experiment, on day 3 after infusion, the spleen was collected and CD45.2+CD4+TCRa3.2+TCRb11+ cells were sorted according to the number of divisions they had undergone and then processed for transcriptome analysis by RNA-seq. A volcanic plot of RNA-seq data compares 2D2 CD4 T cells from mice treated with VHHMHCII-MOG35-55 after three divisions (div3) with 2D2 CD4 T cells recovered from mice treated with VHHMHCII-OVA323-339. (Figure 3D) Heatmap showing co-inhibitory receptor expression on 2D2 CD4+ T cells. (Figure 3E) CellTrace Violet dilution reflects the proliferation of 2D2 T cells on day 3. VHHMHCII-MOG35-55 administration leads to a distinctive pattern of phenotypic markers in 2D2 CD4 T cells. Representative flow images are shown. The mean fluorescence intensity (MFI) of each marker is plotted as mean + / - SEM. *p<0.05, **p<0.01, ***p<0.001, unpaired t-test with Holm-Sidak adjustment. (Figure 3F) Mean disease scores of mice prophylactically treated with VHHMHCII-OVA323-339 or VHHMHCII-MOG35-55 for indicated genetic background; ***p<0.001, repeated measures two-way ANOVA. (Figure 3G) CD45.1 mice receiving 2D2 CD4 T cells of CD45.2 were administered VHH-antigen infusion with emulsified MOG35-55 in CFA on day 10. Spleen, blood, and iLN were collected at day 5.Unlike 2D2 T cells from mice injected with VHHMHCII-OVA323-339, 2D2 T cells from mice treated with VHHMHCII-MOG35-55 did not respond. Data are shown as mean + / - SEM; ***p<0.001, unpaired t-test with Holm-Sidak adjustment. [Figure 4] Tolerance mediated by the VHH-antigen is antigen-specific. (Figure 4A) Blood glucose levels of individual mice treated with VHH-antigen or saline to monitor T1D progression. Mice are considered hyperglycemic when glucose levels >260 mg / dL. (Figure 4B) Representative H&E staining of pancreatic sections from mice treated with a single dose of VHH-antigen. Scale bar 100 μm. (Figure 4C) Mean paw pad thickness of Balb / c mice treated with VHH-antigen to assess rheumatoid arthritis progression. (Figure 4D) Representative toluidine blue staining of joint sections from mice treated with a single dose of VHH-antigen. Scale bar 100 μm. (Figure 4E) Mice (CD45.1+CD8+OTI T cells) received allotypic marked CD45.2+CD8+OTI T cells one day prior to injection of VHHMHCII-ORF8604-612 or VHHMHCII-OVA257-264 (OTI peptide). Mice were loaded with emulsified OTI peptide in CFA on day 10. Spleen, iLN, and blood were collected after 5 days and analyzed by flow cytometry. (Figure 4F) Splenocytes were cultured for 3 days in complete RPMI supplemented with OTI peptide. Supernatant was collected and IFNγ production was measured by ELISA. Antibodies against OB1 peptide (Figure 4G) and OVA protein (Figure 4H) were measured by ELISA in serum collected from C57BL / 6J recipients who received three consecutive injections of physiological saline, VHHMHCII-OB1, or equimolar amounts of free OVA. Data are shown as mean + / - SEM. ns are not significant; *p<0.05, **p<0.01, ***p<0.001, unpaired t-test with Holm-Sidak adjustment. [Figure 5]Therapeutic efficacy of VHHMHCII-antigen adducts. (Figure 5A) Mean disease score of mice treated with a single dose of VHHMHCII-MOG35-55 when animals reached a disease score of 1 (tail drooping). ~40% (7 / 16) of mice died (†) attributable to cytokine storm. (Figure 5B) Structure of GGG-DEX and LC-MS of purified VHHMHCII-DEX. (Figure 5C) Serum levels of TNFα and IL-6 in EAE mice treated with VHH-antigen with or without co-administration of VHHMHCII-DEX. (Figures 5D-F) Mean and individual disease scores of a mouse cohort treated with doses of VHH-peptide + / -VHHMHCII-DEX on the day mice reached a disease score of 1 (Figure 5D), 2 (Figure 5E), or 3 (Figure 5F). ***p<0.001, repeated measures two-way analysis of variance (ANOVA). [Figure 6] Efficacy of anti-human MHCIIVHH (VHHhMHCII) antigen adducts. (Figure 6A) LC-MS of purified VHHhMHCII constructs. VHHhMHCII recognizes all human HLA-DR products with the exception of DRB3*01. (Figure 6B) Efficacy of VHHhMHCII in a mouse EAE model. (Figure 6C) VHHhMHCII-citrullinated fibrinogen (CitFib) adduct. CitFib is a citrullinated fibrinogen peptide having fibrinogen alpha chain amino acids 79-91 with citrullinated R84. [Figure 7] VHHMHCII-mediated tolerance is primarily provided by CD11c+APC. (Figures 7A and 7B) Flow cytometry analysis of APC subsets in blood, spleen, and iLN targeted by the VHHMHCII-Alexa647 adduct. (Figure 7C) Mean clinical scores of mice that received splenocytes from mice that received VHHMHCII-MOG35-55 or VHHMHCII-OVA323-339. (Figure 7D) Mean clinical scores of mice that received VHHMHCII-MOG35-55 in which various of their immune cell subsets were depleted. (Figure 7E) Mean clinical scores of mice that received VHHMHCII-MOG35-55 or other VHH-MOG35-55. [Figure 8] LC-MS of purified VHHMHCII and VHH-antigen constructs. VHHMHCII and VHH-antigen constructs were purified and analyzed by liquid chromatography-mass spectrometry (LC-MS) to verify their purity and identity. [Figure 9] Spinal cord CD4+ lymphocyte infiltration correlates with disease status. Individual clinical scores for each mouse that received prophylactic treatment with VHH-peptide as indicated by 3 (Figure 9A), 2 (Figure 9C), and 1 dose (single or multiple) (Figure 9E). Clinical scores: 1, tail drooping; 2, partial hindlimb paralysis; 3, complete hindlimb paralysis; 4, complete hind and partial forelimb paralysis; and 5, morbid. Flow cytometry analysis of Th1 and Th17 infiltrating CD4+ lymphocytes in the spinal cord at endpoints in mice that received 3 (Figure 9B) and 2 (Figure 9D) doses of VHH-antigen. Frequencies of FoxP3+CD4+ regulatory T cells are also indicated. Data are shown as mean + / - SEM. ns not significant; *p<0.05, **p<0.01, ***p<0.001, unpaired t-test with Holm-Sidak adjustment. [Figure 10] Prophylactic treatment with VHHMHCII-MOG35-55 confers reduced CD4+ lymphocyte infiltration. (Figure 10A) Individual clinical scores of mice treated prophylactically with VHH-peptide at -60, -30, and -7 days prior to EAE induction. Clinical scores: 1, tail drooping; 2, partial hindlimb paralysis; 3, complete hindlimb paralysis; 4, complete hind and partial forelimb paralysis; and 5, mortality. (Figure 10B) Flow cytometry analysis of Th1 and Th17 infiltrating CD4+ lymphocytes in the spinal cord at the endpoint of mice that received one dose of VHH-antigen at indicated time points. The frequency of FoxP3+CD4+ regulatory T cells is also indicated. Data are shown as mean + / - SEM. ns not significant; *p<0.05, **p<0.01, ***p<0.001, unpaired t-test with Holm-Sidak adjustment. [Figure 11]Treatment with a single dose of VHHMHCII-MOG35-55 prevents signs of disease from subsequent loading. (Figure 11A) Flow cytometry analysis of infiltrating CD4+ lymphocytes in the spinal cord of mice treated with a single dose of VHH-antigen, followed by exposure to multiple EAE loadings, at the endpoint. Data are shown as mean + / - SEM. (Figure 11B) Representative H&E and Luxor Fast Blue staining of spinal cord sections from these mice. Scale bar 100 μm. [Figure 12] In vitro characterization of VHH fluorophores. (Figure 12A) Coomassie and fluorescence Western blot of unmodified and modified VHH with Alexa647 produced by sol-tagging, i.e., VHHMHCII-Alexa647 and VHH control-Alexa647. (Figure 12B) Flow cytometry analysis of splenocytes from MHCII-GFP mice indicates a positive correlation between VHHMHCII binding and MHCII expression. [Figure 13] In vivo distribution of VHHMHCII. VHHMHCII-Alexa647 was intravenously injected into MHCII-GFP mice. 1.5 hours after injection, the spleen was removed and analyzed by flow cytometry. A subpopulation of spleen GFP+Alexa647+APC was further dissected. cDCs (classical DCs); pDCs (plasmacytoid DCs). [Figure 14] Only intravenous administration of VHHMHCII-MOG35-55 provides significant protection from EAE. Determination of whether the mode of delivery affects VHHMHCII-MOG35-55-mediated protection in EAE. Mean clinical score of mice treated prophylactically with VHH-peptides administered intravenously, intraperitoneally, or subcutaneously. Clinical score: 1, tail drooping; 2, partial hindlimb paralysis; 3, complete hindlimb paralysis; 4, complete hind and partial forelimb paralysis; and 5, mortality. ***p<0.01, repeated measures two-way ANOVA. [Figure 15]VHHMHCII-MOG35-55 treated splenocytes provide the most effective protection from EAE. (Figure 15A) Individual clinical scores of mice that received splenocytes and peripheral blood mononuclear cells (PBMCs) from mice treated with VHHMHCII-OVA323-339 or VHHMHCII-MOG35-55. Clinical scores: 1, tail drooping; 2, partial hindlimb paralysis; 3, complete hindlimb paralysis; 4, complete hind and partial forelimb paralysis; and 5, mortality. ***p<0.001, repeated measures two-way ANOVA. Composition of transferred splenocytes (Figure 15B) and PBMCs (Figure 15C) from the experimental setup of (Figure 15A). [Figure 16] Depletion of selected cell subsets indicated cell types that supported antigen-specific tolerance mediated by VHHMHCII. (Figure 16A) Individual clinical scores of mice treated with VHHMHCII-OVA323-339 or prophylactic treatment with VHHMHCII-MOG35-55, based on the depletion of the indicated cell subset. To deplete CD8+ T cells, mice were intraperitoneally injected 400 μg twice weekly, starting 2 weeks prior to VHH-antigen administration and continuing throughout the EAE observation period. Macrophages were depleted by every other day ip injections of 300 μg of anti-CSF1R, starting 2 weeks prior to VHH-antigen administration and continuing throughout the EAE observation period. Finally, to deplete DCs, a single dose of 100 ng of DTX was administered to CD11c-DTR mice 2 days prior to VHH-antigen administration (ip). (Figure 16B) Flow cytometry confirmed the depletion of CD8+ T cells, macrophages, and DCs one day prior to VHH- antigen administration. [Figure 17] VHH adducts that primarily recognize dendritic cells provide a moderate level of protection from EAEs. Individual clinical scores of mice treated with the specified VHH antigen. Clinical score: 1, tail drooping; 2, partial hindlimb paralysis; 3, complete hindlimb paralysis; 4, complete hind and partial forelimb paralysis; and 5, mortality. ***p<0.001, repeated measures two-way ANOVA. [Figure 18]VHHMHCII-MOG35-55 confers protection from EAE in dendritic cell-independent Batf3- / - mice. Mean clinical scores of wild-type C57BL6 / J or Batf3- / - mice (mice lacking CD8a+DC) treated with the specified VHH- antigen. Clinical score: 1, tail drooping; 2, partial hindlimb paralysis; 3, complete hindlimb paralysis; 4, complete hind and partial forelimb paralysis; and 5, mortality. ***p<0.001, repeated measures two-way ANOVA. [Figure 19] Imaging of CD4+ cells after treatment with VHHMHCII-MOG35-55. Non-invasive positron emission tomography (PET)-CT imaging of adoptively transferred 2D2 CD4 T cells in Rag1- / - mice. Briefly, 2D2 CD4 T cells were adoptively transferred into Rag1- / - mice, and VHH- antigen was administered 1 day later. On days 3 and 10, 89Zr-labeled PEGylated anti-CD4scFV was injected to track the in vivo distribution of 2D2 CD4 T cells throughout the recipient mice. [Figure 20] RNA-seq analysis of 2D2 CD4 T cell populations after treatment with VHHMHCII-MOG35-55. (Figure 20A) CellTrace Violet-labeled 2D2 CD4 T cells were adopted into congenically marked CD45.1 mice one day prior to infusion with VHHMHCII-OVA323-339 or VHHMHCII-MOG35-55. On day 3 after infusion, spleens were collected, and CD45.2+CD4+TCRa3.2+TCRb11+ cells were sorted as indicated and processed for bulk transcriptome analysis by RNA-seq. (Figure 20B) Principal component plot of shaded RNA-seq data by FACS-sorted populations. (Figure 20C) Heatmap showing some transcriptional features of CD4+ T cells. Genetic ontology analysis of the top 500 genes that were upregulated (Figure 20D) and downregulated (Figure 20E) in 2D2 CD4T cells from mice treated with VHHMHCII-MOG35-55 after three divisions (div3), compared to 2D2 CD4T cells from mice treated with VHHMHCII-OVA323-339. [Figure 21] Expression of phenotypic markers in 2D2 CD4 T cells after treatment with VHHMHCII-MOG35-55. CellTrace Violet-labeled 2D2 CD4 T cells were adopted into congenically marked CD45.1 mice one day prior to infusion of VHHMHCII-MOG35-55, VHHMHCII-OVA323-339, or equimolar MOG35-55 peptide in the presence of poly(I:C / anti-CD40) as an adjuvant. On day 3 after infusion, spleens were collected and analyzed by flow cytometry. CellTrace Violet dilution indicates proliferation of 2D2 T cells on day 3. VHHMHCII-MOG35-55 administration leads to a distinctive pattern of phenotypic markers in 2D2 CD4 T cells. Representative flow images are shown, with the mean fluorescence intensity (MFI) of each marker plotted as mean + / - SEM. *p<0.05, ***p<0.001, unpaired t-test adjusted for Holm-Sidak. [Figure 22]Regulatory T cells are required for protection from EAEs conferred by treatment with VHHMHCII-MOG35-55. (Figure 22A) Mean clinical scores of mice treated prophylactically with VHH-peptide with or without regulatory T cell (Treg) depletion. Tregs were depleted in FoxP3-DTR mice by ip injection of 1 μg DTX three times prior to treatment on days -9, -8, and -1, followed by weekly ip injections of 1 μg until the endpoint. Clinical scores: 1, tail drooping; 2, partial hindlimb paralysis; 3, complete hindlimb paralysis; 4, complete hind and partial forelimb paralysis; and 5, mortality. ***p<0.001, repeated measures two-way ANOVA. (Figure 22B) Flow cytometry confirmation of FoxP3+ Treg cell depletion one day prior to VHH-antigen administration. (Figure 22C) 2D2 CD4 T cells were adopted into congenically marked CD45.1 mice one day prior to infusion with VHH-antigen. On day 3, the mice were further loaded with emulsified MOG35-55 in CFA. Spleens and iLNs were collected at day 7. 2D2 T cells from mice infused with VHHMHCII-MOG35-55 did not proliferate as effectively as 2D2 T cells from mice treated with VHHMHCII-OVA323-339. Data are shown as mean + / - SEM. *p<0.05, ***p<0.001, unpaired t-test with Holm-Sidak adjustment. (Figure 22D) 2D2 T cells showed an increase in FoxP3+ cells. Data are shown as mean + / - SEM. **p<0.01, unpaired t-test with Holm-Sidak adjustment. [Figure 23]Treatment with VHHMHCII-p31 may prevent type 1 diabetes (T1D). (Figure 23A) Schematic diagram of prophylactic T1D treatment on day 1 after adoptive transfer of activated BDC2.5 splenocytes. Percentage of overall euglycemic levels in the data in Figure 3C. p<0.001, log-rank test. (Figure 23B) Flow cytometry analysis of infiltrating BDC2.5CD4+ T cells in a specified organ 14 days after adoptive transfer of BDC2.5 splenocytes. Data are shown as mean + / - SEM. ns not significant; *p<0.05, ***p<0.001, unpaired t-test with Holm-Sidak adjustment. (Figure 23C) Schematic diagram of semi-therapeutic T1D treatment on day 5 after adoptive transfer of activated BDC2.5 splenocytes. Blood glucose levels were measured to monitor T1D progression. Mice were considered to have diabetes when their glucose levels were >250 mg / dL. [Figure 24] The N-terminal glycine of insulin immediately acts as a saltase nucleophile. A schematic diagram indicating the N-terminal glycine residue of insulin that can act as a saltase nucleophile, and FC-MS analysis of the produced VHHMHCII-insulin adduct. [Figure 25] Treatment with VHHMHCII-OVA323-339 may reduce the severity of RA. (Figure 25A) Individual foot thickness of mice treated with VHH-antigen to assess RA progression. (Figure 25B) Representative image of mouse foot at 3 days after thermoaggregated ovalbumin (HAO) loading. (Figure 25C) Th1 response of popliteal lymph node-derived splenocytes harvested at the endpoint (7 days after HAO loading). Data are shown as mean + / - SEM. *p<0.05, unpaired t-test adjusted for Folm-Sidak. Anti-ovalbumin (Figure 25D) and anti-OVA323-339 (Figure 25E) antibody responses from mice described in (Figure 25A). Data are shown as mean + / - SEM. *p<0.05, unpaired t-test adjusted for Folm-Sidak. [Figure 26]Mice administered VHHMHCII-MOG35-55 concurrently with the initial symptoms of EAE exhibit heterogeneous outcomes. Individual clinical scores of mice treated with doses of VHHMHCII-MOG35-55 on the day the mouse reached a clinical score of 1. Clinical scores: 1, tail drooping; 2, partial hindlimb paralysis; 3, complete hindlimb paralysis; 4, complete hind and partial forelimb paralysis; and 5, mortally ill. ~40% (7 / 16) of mice were found dead (†) and attributed to cytokine storm. [Figure 27] Synthesis of dexamethasone (DEX) with GGG adduct. The schematic diagram shows the step in which the VHHMHCII-dexamethasone adduct is produced. [Figure 28] Concurrent treatment with VHHMHCII-DEX reduces CD4+ T cell infiltration of the spinal cord. Clinical scores: 1, tail drooping; 2, partial hindlimb paralysis; 3, complete hindlimb paralysis; 4, complete hind and partial forelimb paralysis; and 5, mortality. Flow cytometry analysis of Th1 and Th17 infiltrating CD4+ lymphocytes in the spinal cord at each mouse endpoint. The frequency of FoxP3+CD4+ regulatory T cells is also indicated. Data are shown as mean + / - SEM. *p<0.05, **p<0.01, ***p<0.001, unpaired t-test with Holm-Sidak adjustment. [Figure 29] Concurrent treatment with free dexamethasone requires substantially higher doses than VHHMHCII-DEX. Individual clinical scores of mice treated with doses of VHHMHCII-MOG35-55 in the presence of 0.5 μg DEX (Figure 29A) or 100 μg DEX (Figure 29B) on the day the mouse reached a clinical score of 1. Clinical scores: 1, tail drooping; 2, partial hindlimb paralysis; 3, complete hindlimb paralysis; 4, complete hind and partial forelimb paralysis; and 5, mortality. 50% (2 / 4) of the mice were found dead when they received only 0.5 μg DEX (†) and attributed to a cytokine storm. [Figure 30]Schematic diagram of the conjugation process by sol-tagging. The diagram outlines the steps for a maleimide and copper-free click chemistry sol-tagging approach. [Figure 31] VHHMHCII-spike RBD immunization induces high-titer, durable anti-spike RBD antibodies that neutralize pseudotyped VSV-SARS-CoV-2. (Figure 31A) Design of VHHMHCII-spike RBD. (Figure 31B) Coomasiegel of the produced VHHMHCII, spike RBD, and VHHMHCII-spike RBD fusion product. (Figure 31C) Immunization scheme: C57BL / 6J mice were vaccinated intraperitoneally and serum was collected as directed: pre-immunization serum was collected 3 days prior to immunization. Blood was collected from the first dose of immunization to day 32 and day 150. (Figure 31D) Humoral response of serum from immunized mice was evaluated by ELISA for anti-spike RBD IgG (n=4 / group). (Figure 31E) IgM, IgA, IgG1, IgG2b. (Figure 31F) Neutralization data of pseudotyped VSV by SARS-CoV-2 spike glycoprotein. [Figure 32] Immunization of mice with a single dose of the VHHMHCII-spike RBD fusion rapidly induces a strong T cell response to spike RBD. (Figure 32A) Immunization scheme: C57BL / 6J mice were intraperitoneally vaccinated and spleens were harvested for T cell assay. (Figure 32B) Schematic diagram of spike RBD amino acid sequences and peptides created for ELISPOT analysis. (Figure 32C) ELISPOT analysis of spike RBD-specific T cells in mice vaccinated with adjuvant alone, spike RBD + adjuvant, or VHHMHCII-spike RBD + adjuvant. Spike RBD was truncated to a 15-mer peptide with a 10-amino acid overlap and directed as peptides 1-53. (Figure 32D) Cytokine secretion from spleen cells on day 3 after culturing with the directed peptides. (Figure 32E) Flow cytometry analysis of spleen cells after 6 hours of incubation with or without peptide mixture (peptides 42+47+48+49). [Figure 33] Two doses of VHHMHCII-spike RBD vaccine were sufficient to produce persistent neutralizing antibody titers against multiple variants of SARS-CoV-2. (Figure 33A) The dynamics of the humoral response of serum in immunized mice were evaluated by ELISA for anti-spike RBDIgG (n=4 / group). (Figure 33B) IgM, IgA, IgG1, IgG2b. (Figure 33C) Antibody titers in immunized mice against spike RBD protein with K417T, E484K, and N501Y mutations. (Figure 33D) Neutralization data for VSV pseudotyped by SARS-CoV-2 spike glycoprotein Wuhan+D418G and other variants. [Figure 34] The VHHMHCII-spike RBD adduct induces a strong antibody response regardless of delivery mode, storage conditions, lyophilization, and the suboptimal immunization of aging mice. (Figure 34A) Time series of immunization. (Figure 34B) Anti-spike RBD IgG, IgM, IgA, IgG1, IgG2b in serum of mice immunized using different delivery modes. (Figure 34C) Anti-spike RBD IgG, IgM, IgA, IgG1, IgG2b in serum of mice immunized using different formulation storage conditions. (Figure 34D) Efficacy of antibody production in aging mice. Antibody titers were evaluated by ELISA (n=4 / group). [Modes for carrying out the invention]

[0029] Detailed description This disclosure provides, in several aspects, a composition comprising one or more conjugates (also referred to in examples and figures as “adducts”) comprising a single-domain antibody fragment (nanobody / VHH) conjugated with an antigen (e.g., an antigen for which immune tolerance is required, e.g., an autoantigen or an exogenous enzyme used for therapeutic purposes) and / or an anti-inflammatory agent, a method of using such a composition to induce immune tolerance to an antigen, and a method of using such a composition to treat an autoimmune disease. In some embodiments, the composition comprises a conjugate comprising VHH conjugated with an antigen (e.g., an antigen for which immune tolerance is required) and an anti-inflammatory agent, wherein the VHH conjugates to a surface protein on an antigen-presenting cell (APC). In some embodiments, the composition comprises a first conjugate containing a first VHH conjugated with an antigen (e.g., an antigen for which immune tolerance is required) and a second conjugate containing a second VHH conjugated with an anti-inflammatory agent, wherein the first and second VHHs bind to one or more surface proteins on an antigen-presenting cell (APC).

[0030] Other aspects of this disclosure provide compositions comprising one or more conjugates comprising VHH conjugated to an antigen (e.g., an antigen against which an immune response is required, e.g., an antigen from a pathogen or a tumor antigen) and / or a pro-inflammatory agent, against which VHH binds to a surface protein on an antigen-presenting cell (APC); methods of using such compositions to induce an immune response to an antigen; and methods of using such compositions to treat infections (e.g., caused by a pathogen) and cancer. In some embodiments, the composition comprises a conjugate comprising VHH conjugated to an antigen (e.g., an antigen against which an immune response is required, e.g., an antigen from a pathogen or a tumor antigen) and a pro-inflammatory agent, where the VHH binds to a surface protein on an antigen-presenting cell (APC). In some embodiments, the composition comprises a first conjugate containing a first VHH conjugated with an antigen (e.g., an antigen against which an immune response is required, e.g., an antigen from a pathogen or a tumor antigen) and a second conjugate containing a second VHH conjugated with a pro-inflammatory agent, wherein the first and second VHHs bind to one or more surface proteins on an antigen-presenting cell (APC).

[0031] VHH and Conjugate The conjugates of this disclosure include single-domain antibodies (also known as nanobodies or VHHs). As used herein, “single-domain antibody fragment,” “nanobody,” or “VHH” refers to an antibody fragment consisting of a single monomeric variable antibody domain. It is known that camels produce heavy-chain-only antibodies (as described, for example, in Hamers-Casterman et al., 1992, incorporated herein by reference). Single-domain variable fragments of these heavy-chain-only antibodies are referred to as VHHs or nanobodies. VHHs hold an immunoglobulin fold shared by the antibody and bind to their targets using three hypervariable loops, CDR1, CDR2, and CDR3. Many VHHs bind to their targets with affinity similar to conventional full-size antibodies but possess other superior properties. Therefore, VHHs are an attractive tool for biological research and therapeutic use. VHHs are typically between 10 and 15 kDa in size and can be recombinantly expressed in high yield in both the cytosol and periplasm of E. coli. VHHs can bind to their targets in the mammalian cytosol. VHH fragments (e.g., nanobodies®) are recombinant antigen-specific single-domain variable fragments derived from camel heavy chain antibodies. Although small, VHH fragments retain the full antigen-binding capacity of the complete antibody. VHHs are small in size, highly soluble, stable, and have a large set of accessible epitopes compared to conventional antibodies. Since no reformatting is required, they can also be readily used as extracellular target-binding portions of chimeric receptors as described in this application.

[0032] In some embodiments, the VHH used in the conjugates described herein binds to surface proteins on antigen-presenting cells (APCs). “Antigen-presenting cells (APCs)” refer to cells that present antigens complexed with major histocompatibility complexes (MHCs) on their surface, a process known as antigen presentation. T cells can recognize these complexes using their T cell receptors (TCRs). Almost all cell types can present antigens in some way; they are found in various tissue types. As used herein, the term “antigen-presenting cells” refers to professional antigen-presenting cells, without limitation, including macrophages, B cells, and dendritic cells. Antigen-presenting cells play a crucial role in effective adaptive immune responses because both cytotoxic and helper T cell functions are APC-dependent. Antigen presentation enables the specificity of adaptive immunity and can contribute to immune responses against both intracellular and extracellular pathogens. It is also involved in defense against tumors. Some cancer therapies involve the creation of artificial APCs to prime the adaptive immune system to target malignant cells. In addition, as described, for example, in Best et al., Front Immunol. 2015; 6: 360, which is incorporated into this application by reference, APCs also play a role in immune tolerance by presenting autoantigens to T cells.

[0033] In addition to MHC family proteins, other specialized signaling molecules on the surface of both APCs and T cells are also required for antigen presentation. In some embodiments, the conjugates described herein include VHHs that bind to proteins on the surface of APCs and are therefore involved in APCs. An unspecified example of surface proteins on APCs that can be targeted by VHHs on the conjugates described herein is, without limitation: major histocompatibility complex II (MHCII), integrin alpha X (CD11c), lymphocyte antigen 75 (DEC205, also known as CD205), dendritic cell-specific ICAM-3 grabbing nonintegrin 1 (DC-SIGN), C-type lectin domain-containing 9A (CLEC9a), integrin alpha E (CD103), C-X3-C motif chemokine receptor 1 (CX3CR1), differentiation antigen group 1a (CD1a), and EGF-like module-containing mucin-like hormone receptor-like 1 (F4 / 80, also known as EMR1).

[0034] In some embodiments, the VHH on the conjugate described herein binds to one surface protein on the APC (e.g., MHCII, CD11c, DEC205, DC-SIGN, CLEC9a, CD103, CX3CR1, CD1a, or F4 / 80, without limitation). In some embodiments, the VHH on the conjugate described herein is bispecific or multispecific. In some embodiments, the VHH on the conjugate described herein binds to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) surface proteins on the APC. Any known VHH that binds to a surface protein on the APC may be used in accordance with this disclosure.

[0035] In some embodiments, VHH binds to MHCII. Examples of MHCII-binding VHH are described, for example, in US Patent No. US9751945, which is incorporated herein by reference. Amino acid sequences of examples of MHCII-binding VHH are provided in Table 1.

[0036] In some embodiments, the VHH on the conjugate described herein includes an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 99%) identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the VHH on the conjugate described herein includes an amino acid sequence that is 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the VHH on the conjugate described herein includes the amino acid sequence of SEQ ID NO: 1.

[0037] In some embodiments, VHH binds to CD11c. Examples of CD11c-binding VHH are described, for example, in Bannas et al., Front Immunol. 2017; 8: 1603, which is incorporated herein by reference. Amino acid sequences of examples of CD11c-binding VHH are provided in Table 1.

[0038] In some embodiments, the VHH on the conjugate described herein includes an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 99%) identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the VHH on the conjugate described herein includes an amino acid sequence that is 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the VHH on the conjugate described herein includes the amino acid sequence of SEQ ID NO: 2.

[0039] [Table 1]

[0040] In some embodiments, any one of the VHHs on the conjugate described herein includes an additional sequence, such as a saltase recognition sequence (as described, for example, in US Patent No. US9751945, incorporated herein by reference). Enzymes identified as “saltase” from Gram-positive bacteria cleave proteins and translocate them to proteoglycan moieties on intact cell walls. Saltases isolated from Staphylococcus aureus include saltase A (SrtA) and saltase B (SrtB).

[0041] In some embodiments, the recognition sequence of the saltase may further include one or more additional amino acids, for example, at its N-terminus or C-terminus. For example, one or more amino acids (e.g., up to five amino acids) having the identity of amino acids found immediately at the N-terminus or C-terminus of a five-amino acid recognition sequence on a naturally occurring saltase substrate may be incorporated. Such additional amino acids may provide a context that improves the recognition of the recognition motif.

[0042] Saltases have been classified into four classes, designated A, B, C, and D, based on sequence alignment and phylogenetic analysis of 61 saltases from Gram-positive bacterial genomes (Dramsi S, Trieu-Cuot P, Bierne H, Sorting sortases: a nomenclature proposal for the various sortases of Gram-positive bacteria. Res Microbiol. 156(3):289-97, 2005). These classes correspond to the following subfamilies into which saltases have also been classified by Comfort and Clubb (Comfort D, Clubb R T. A comparative genome analysis identifies distinct sorting pathways in gram-positive bacteria. Infect Immun., 72(5):2710-22, 2004): Class A (subfamily 1), Class B (subfamily 2), Class C (subfamily 3), and Class D (subfamilies 4 and 5). The above references disclose numerous saltases and recognition motifs. Pallen, MJ; See also Lam, AC; Antonio, M.; Dunbar, K. TRENDS in Microbiology, 2001, 9(3), 97-101. Those skilled in the art will be able to immediately assign the saltase to the correct class based on its sequence and / or other characteristics, e.g., Drami, et al., as described above. The term “saltase A” is used in this application to refer to a class A saltase of any particular bacterial species, usually designated as SrtA, e.g., SrtA from S. aureus. Similarly, “saltase B” is used in this application to refer to a class B saltase of any particular bacterial species, usually designated as SrtB, e.g., SrtB from S. aureus.

[0043] In some embodiments, the saltase used to produce the conjugate described herein is saltase A (SrtA). SrtA recognizes the motif LPXTG (SEQ ID NO: 25), and common recognition motifs include, for example, LPKTG (SEQ ID NO: 26), LPATG (SEQ ID NO: 27), and LPNTG (SEQ ID NO: 28). In some embodiments, LPETG (SEQ ID NO: 29) is used. However, motifs belonging to outside this consensus may also be recognized. For example, in some embodiments, the motif includes "A" rather than "T" at position 4, for example LPXAG (SEQ ID NO: 30), for example LPNAG (SEQ ID NO: 31). In some embodiments, the motif includes "A" rather than "G" at position 5, for example LPXTA (SEQ ID NO: 32), for example LPNTA (SEQ ID NO: 33). In some embodiments, the motif includes "G" rather than "P" at position 2, for example LGXTG (SEQ ID NO: 34), for example LGATG (SEQ ID NO: 35). In some embodiments, the motif includes "I" rather than "L" at position 1, e.g., IPXTG (SEQ ID NO: 36), e.g., IPNTG (SEQ ID NO: 37), or IPETG (SEQ ID NO: 38).

[0044] In some embodiments, the saltase used to produce the conjugates described herein is saltase B (SrtB), e.g., saltase B of S. aureus, B. anthrosis, or L. monocytogenes. The motif recognized by class B saltase (SrtB) is often attributed to the consensus sequence NPXTX (SEQ ID NO: 39), e.g., NP[Q / K]-[T / s]-[N / G / s], e.g., NPQTN (SEQ ID NO: 40) or NPKTG (SEQ ID NO: 41). For example, saltase B of S. aureus or B. anthrosis cleaves the NPQTN (SEQ ID NO: 40) or NPKTG (SEQ ID NO: 41) motif of IsdC in their respective bacteria (see, e.g., Marraffini, L. and Schneewind, O., Journal of Bacteriology, 189(17), p. 6425-6436, 2007). Other recognition motifs found on putative substrates of class B sortases include NSKTA (SEQ ID NO: 44), NPQTG (SEQ ID NO: 45), NAKTN (SEQ ID NO: 46), and NPQSS (SEQ ID NO: 47). For example, SrtB from L. monocytogenes recognizes certain motifs lacking P at position 2 and / or Q or K at position 3, such as NAKTN (SEQ ID NO: 46) and NPQSS (SEQ ID NO: 47) (Mariscotti JF, Garcia-Del Portillo F, Pucciarelli M G. The listeria monocytogenes sortase-B recognizes varied amino acids at position two of the sorting motif. J Biol Chem. 2009 Jan. 7. [Epub ahead of print]).

[0045] In some embodiments, the saltase used to produce the conjugate described herein is a class C saltase. The class C saltase may utilize LPXTG (SEQ ID NO: 25) as a recognition motif.

[0046] In some embodiments, the saltase is a class D saltase. This class of saltases is expected to recognize motifs having the consensus sequence NA-[E / A / S / H]-TG (Comfort D, above). Class D saltases have been found, for example, in Streptomyces spp., Corynebacterium spp., Troferima whipperi, Thermobifida fusca, and Bifidobacterium longum. LPXTA (SEQ ID NO: 32) or LAXTG (SEQ ID NO: 48) may serve as recognition sequences for class D saltases of subfamilies 4 and 5, respectively (subfamily 4 and subfamily 5 enzymes process the motifs LPXTA (SEQ ID NO: 32) and LAXTG (SEQ ID NO: 48), respectively). For example, the class D saltase B. anthracis saltase C has been shown to specifically cleave the LPNTA (SEQ ID NO: 33) motif of B. anthracis BasI and BasH (Marrafini, above).

[0047] In some embodiments, naturally occurring saltase variants may be used. Such variants may be produced by processes such as directed evolution and site-directed modification. For example, a variant of S. aureus saltase A has been identified that exhibits up to a 140-fold increase in LPETG (SEQ ID NO: 29) coupling activity compared to the starting wild-type enzyme (Chen, I., et al., PNAS 108(28): 11399-11404, 2011). In some embodiments, the saltase variant contains one or more of the following substitutions relative to wild-type S. aureus SrtA: P94S or P94R, D160N, D165A, K190E, and K196T mutations. An exemplary wild-type S. aureus SrtA sequence (gene ID: 1125243, NCBI RefSeq Acc. No. NP_375640) is shown below: [ka] (Sequence ID 3)

[0048] For example, as described in US9751945 incorporated herein by reference, saltase tagging may be used to install a reactive chemical moiety (e.g., a click chemistry handle) onto VHH. The click chemistry handle may be used to conjugate VHH to other agents (e.g., antigens, anti-inflammatory agents, and / or pro-inflammatory agents). In some embodiments, the saltase recognition sequence is located at the N-terminus of VHH. In some embodiments, the saltase recognition sequence is located at the C-terminus of VHH.

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[43] AJ Inglis, S. Sinwell, MH Stenzel, C. Barner-Kowollik, Angew. Chem. 2009, 121, 2447-2450; Angew. Chem. Int. Ed. 2009, 48, 2411-2414. All references cited above are incorporated by reference to the disclosure of click chemistry handles suitable for protein installations according to the progressive concepts and methods provided herein.

[0050] Other tags may also be added to VHH by sol-tagging. Examples of suitable tags include, without limitation, amino acids, peptides, proteins, nucleic acids, polynucleotides, sugars, carbohydrates, polymers, lipids, fatty acids, and small molecules. Other suitable tags will be obvious to those skilled in the art. The present invention is not limited in this aspect. In some embodiments, tags include sequences useful for purifying, expressing, solubilizing, and / or detecting polypeptides. In some embodiments, tags may perform multiple functions. Tags are often relatively small, for example, ranging in length from a few amino acids to up to about 100 amino acids. In some embodiments, tags are longer than 100 amino acids, for example, up to about 500 amino acids, or more. In some embodiments, to name a few examples, tags include His6, HA, TAP, Myc, Flag, or GST tags. In some embodiments, tags include solubility-enhancing tags (e.g., SUMO tags, NUSA tags, SNUT tags, Strep tags, or monomeric variants of the Ocr protein of bacteriophage T7). See, for example, Esposito D and Chatterjee D K. Curr Opin Biotechnol.; 17(4):353-8 (2006). In some embodiments, the tag is cleavable such that it can be removed by, for example, a protease. In some embodiments, this is achieved by incorporating a protease cleavage site on the tag that is adjacent to or linked to the functional portion of the tag, for example. Exemplary proteases include, for example, thrombin, TEV protease, factor Xa, and prescission protease. In some embodiments, "self-cleaving" tags are used. See, for example, PCT / US05 / 05763.

[0051] The conjugates described herein comprise VHH conjugated into a second molecule. In some embodiments, VHH comprises a saltase recognition motif and is conjugated into a second molecule by click chemistry. In some embodiments, the conjugate of the disclosure comprises VHH conjugated into one molecule. In some embodiments, the one molecule conjugated into VHH is an antigen. In some embodiments, the one molecule conjugated into VHH is an anti-inflammatory or pro-inflammatory agent. In some embodiments, the conjugate of the disclosure comprises VHH conjugated into two molecules. In some embodiments, the conjugate of the disclosure comprises VHH conjugated into an antigen to which an immune response is required (e.g., an antigen from a pathogen or a tumor antigen) and an anti-inflammatory agent. In some embodiments, the conjugate of the disclosure comprises VHH conjugated into an antigen to which immune tolerance is required (e.g., an autoantigen or an exogenous enzyme used for therapeutic purposes) and a pro-inflammatory agent. Examples of methods for conjugating two molecules into VHH are shown in Figures 30A-30C.

[0052] In some embodiments, an anti-inflammatory or pro-inflammatory agent is conjugated to the saltase-recognizing motif of VHH via a linker. In some embodiments, the linker is a non-hydrolyzable linker (i.e., non-cleavable). Not limited examples of non-hydrolyzable linkers include N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), maleimidomethylcyclohexane-1-carboxylate (MCC), maleimidocaproyl (MC), and their derivatives. In some embodiments, the linker is a hydrolyzable linker (i.e., cleavable). Examples of hydrolyzable linkers, though not limited to them, include hydrazones, hydrazides, disulfides, 4-(4'-acetylphenoxy)butanoic acid (AcBut), N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP) and N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB), valine-citrulline (VC), valine-alanine (VA), phenylalanine-lysine (FK), and their derivatives. Hydrolyzable linkers can be self-immolating linkers (i.e., autocleaving), such as pH-sensitive linkers (e.g., hydrazones). For example, a pH-sensitive linker may be used to release an anti-inflammatory or pro-inflammatory agent conjugated to VHH when, for example, VHH is delivered to a desired destination (e.g., an APC or its intracellular compartment) due to a shift in the acidity of the physiological environment. In some embodiments, the linker is an auto-hydrolyzable hydrazone linker, as shown in Figure 27. Additional linkers suitable for use in the methods described herein are well known to those skilled in the art and include, but are not limited to, those described in Jain, N., Smith, SW, Ghone, S., & Tomczuk, B. Pharm Res, 2015, 32(11), 3526-3540 and Lu, J., Jiang, F., Lu, A., & Zhang, G. Int J Mol Sci, 2016, 17(4), 561. Both of these are incorporated herein by reference.

[0053] In some embodiments, the compositions described herein comprise a conjugate containing VHH conjugated with an antigen (e.g., an antigen for which immune tolerance is required) and an anti-inflammatory agent (e.g., dexamethasone), wherein the VHH binds to MHCII (e.g., VHH having the amino acid sequence of SEQ ID NO: 1). In some embodiments, the compositions described herein comprise a conjugate containing an autoantigen and an anti-inflammatory agent (e.g., dexamethasone), wherein the VHH binds to MHCII (e.g., VHH having the amino acid sequence of SEQ ID NO: 1). Any one of the autoantigens described herein may be used. In some embodiments, the autoantigen is myelin oligodendrocyte glycoprotein (MOG) or a fragment thereof (e.g., amino acids 35-55 of the MOG protein). In some embodiments, the autoantigen is citrullinated fibrinogen. In some embodiments, the autoantigen is insulin. In some embodiments, the composition described herein comprises a conjugate containing VHH conjugated to a protein used in protein replacement therapy or gene therapy (e.g., an enzyme, e.g., factor IX or factor VIII, or an adeno-associated virus (AAV)-derived protein) and an anti-inflammatory agent (e.g., dexamethasone), wherein the VHH binds to MHCII (e.g., VHH having the amino acid sequence of SEQ ID NO: 1).

[0054] In some embodiments, the composition comprises a first conjugate containing a first VHH conjugated to an antigen (e.g., an antigen for which immune tolerance is required) and a second conjugate containing a second VHH conjugated to an anti-inflammatory agent (e.g., dexamethasone), wherein both the first and second VHHs bind to MHCII (e.g., VHH having the amino acid sequence of SEQ ID NO: 1). In some embodiments, the composition comprises a first conjugate containing a first VHH conjugated to an autoantigen and a second conjugate containing a second VHH conjugated to an anti-inflammatory agent (e.g., dexamethasone), wherein both the first and second VHHs bind to MHCII (e.g., VHH having the amino acid sequence of SEQ ID NO: 1). Any one of the autoantigens described herein may be used. In some embodiments, the autoantigen is myelin oligodendrocyte glycoprotein (MOG) or a fragment thereof (e.g., amino acids 35-55 of the MOG protein). In some embodiments, the autoantigen is citrullinated fibrinogen. In some embodiments, the autoantigen is insulin. In some embodiments, the composition comprises a first conjugate containing a first VHH conjugated to a protein used in protein replacement therapy or gene therapy (e.g., an enzyme, e.g., factor IX or factor VIII or an adeno-associated virus (AAV)-derived protein), and a second conjugate containing a second VHH conjugated to an anti-inflammatory agent (e.g., dexamethasone), wherein both the first and second VHHs bind to MHCII (e.g., VHH having the amino acid sequence of SEQ ID NO: 1).

[0055] In some embodiments, the compositions described herein comprise a conjugate comprising VHH conjugated with an antigen (e.g., an antigen for which immune tolerance is required) and an anti-inflammatory agent (e.g., dexamethasone), wherein the VHH binds to CD11c (e.g., VHH having the amino acid sequence of SEQ ID NO: 2). In some embodiments, the compositions described herein comprise a conjugate comprising VHH conjugated with an autoantigen and an anti-inflammatory agent (e.g., dexamethasone), wherein the VHH binds to CD11c (e.g., VHH having the amino acid sequence of SEQ ID NO: 2). Any one of the autoantigens described herein may be used. In some embodiments, the autoantigen is myelin oligodendrocyte glycoprotein (MOG) or a fragment thereof (e.g., amino acids 35-55 of the MOG protein). In some embodiments, the autoantigen is citrullinated fibrinogen. In some embodiments, the autoantigen is insulin. In some embodiments, the composition described herein comprises a conjugate containing VHH conjugated to a protein used in protein replacement therapy or gene therapy (e.g., an enzyme, e.g., factor IX or factor VIII, or an adeno-associated virus (AAV)-derived protein) and an anti-inflammatory agent (e.g., dexamethasone), wherein the VHH binds to CD11c (e.g., VHH having the amino acid sequence of SEQ ID NO: 2).

[0056] In some embodiments, the composition comprises a first conjugate containing a first VHH conjugated to an antigen (e.g., an antigen for which immune tolerance is required) and a second conjugate containing a second VHH conjugated to an anti-inflammatory agent (e.g., dexamethasone), wherein both the first and second VHHs bind to CD11c (e.g., a VHH having the amino acid sequence of SEQ ID NO: 2). In some embodiments, the composition comprises a first conjugate containing a first VHH conjugated to an autoantigen and a second conjugate containing a second VHH conjugated to an anti-inflammatory agent (e.g., dexamethasone), wherein both the first and second VHHs bind to CD11c (e.g., a VHH having the amino acid sequence of SEQ ID NO: 2). Any one of the autoantigens described herein may be used. In some embodiments, the autoantigen is myelin oligodendrocyte glycoprotein (MOG) or a fragment thereof (e.g., amino acids 35-55 of the MOG protein). In some embodiments, the autoantigen is citrullinated fibrinogen. In some embodiments, the autoantigen is insulin. In some embodiments, the composition comprises a first conjugate containing a first VHH conjugated to a protein used in protein replacement therapy or gene therapy (e.g., an enzyme, e.g., factor IX or factor VIII, or an adeno-associated virus (AAV)-derived protein), and a second conjugate containing a second VHH conjugated to an anti-inflammatory agent (e.g., dexamethasone), wherein both the first and second VHHs bind to CD11c (e.g., VHH having the amino acid sequence of SEQ ID NO: 2).

[0057] In some embodiments, the composition comprises a first conjugate containing a first VHH conjugated to an antigen (e.g., an antigen for which immune tolerance is required) and a second conjugate containing a second VHH conjugated to an anti-inflammatory agent (e.g., dexamethasone), wherein the first and second VHHs bind to different surface proteins on the APC. In some embodiments, the composition comprises a first conjugate containing a first VHH conjugated to an antigen (e.g., an antigen for which immune tolerance is required) and a second conjugate containing a second VHH conjugated to an anti-inflammatory agent (e.g., dexamethasone), wherein the first VHH binds to MHCII (e.g., VHH having the amino acid sequence of SEQ ID NO: 1) and the second VHH binds to CD11c (e.g., VHH having the amino acid sequence of SEQ ID NO: 2). In some embodiments, the composition comprises a first conjugate containing a first VHH conjugated to an antigen (e.g., an antigen for which immune tolerance is required) and a second conjugate containing a second VHH conjugated to an anti-inflammatory agent (e.g., dexamethasone), wherein the first VHH binds to DEC205 and the second VHH binds to MHCII (e.g., a VHH having the amino acid sequence of SEQ ID NO: 1). In some embodiments, the composition comprises a first conjugate containing a first VHH conjugated to an autoantigen and a second conjugate containing a second VHH conjugated to an anti-inflammatory agent (e.g., dexamethasone), wherein the first VHH binds to MHCII (e.g., a VHH having the amino acid sequence of SEQ ID NO: 1) and the second VHH binds to CD11c (e.g., a VHH having the amino acid sequence of SEQ ID NO: 2). Any one of the autoantigens described herein may be used. In some embodiments, the autoantigen is myelin oligodendrocyte glycoprotein (MOG) or a fragment thereof (e.g., amino acids 35-55 of the MOG protein). In some embodiments, the autoantigen is citrullinated fibrinogen. In some embodiments, the autoantigen is insulin.In some embodiments, the composition comprises a first conjugate containing a first VHH conjugated to a protein used in protein replacement therapy or gene therapy (e.g., an enzyme, e.g., factor IX or factor VIII, or an adeno-associated virus (AAV)-derived protein) and a second conjugate containing a second VHH conjugated to an anti-inflammatory agent (e.g., dexamethasone), wherein the first VHH binds to DEC205 and the second VHH binds to MHCII (e.g., a VHH having the amino acid sequence of SEQ ID NO: 1).

[0058] In some embodiments, the compositions described herein comprise a conjugate containing VHH conjugated with an antigen (e.g., an antigen against which an immune response is required) and a pro-inflammatory agent, wherein the VHH binds to MHCII (e.g., VHH having the amino acid sequence of SEQ ID NO: 1). In some embodiments, the compositions described herein comprise a conjugate containing VHH conjugated with an antigen from a pathogen (e.g., SARS-CoV-2 protein, e.g., spike protein) and a pro-inflammatory agent (e.g., IL2), wherein the VHH binds to MHCII (e.g., VHH having the amino acid sequence of SEQ ID NO: 1). Any one of the antigens from pathogens described herein may be used. In some embodiments, the compositions described herein comprise a conjugate containing a tumor antigen and a pro-inflammatory agent (e.g., IL2), wherein the VHH binds to MHCII (e.g., VHH having the amino acid sequence of SEQ ID NO: 1). Any one of the tumor antigens described herein may be used.

[0059] In some embodiments, the composition comprises a first conjugate containing a first VHH conjugated to an antigen (e.g., an antigen against which an immune response is required) and a second conjugate containing a second VHH conjugated to a pro-inflammatory agent, wherein both the first and second VHHs bind to MHCII (e.g., a VHH having the amino acid sequence of SEQ ID NO: 1). In some embodiments, the composition comprises a first conjugate containing a first VHH conjugated to an antigen from a pathogen (e.g., SARS-CoV-2 protein, e.g., spike protein) and a second conjugate containing a second VHH conjugated to a pro-inflammatory agent (e.g., IL2), wherein both the first and second VHHs bind to MHCII (e.g., a VHH having the amino acid sequence of SEQ ID NO: 1). Any one of the antigens from pathogens described in this application may be used. In some embodiments, the composition comprises a first conjugate containing a first VHH conjugated to a tumor antigen and a second conjugate containing a second VHH conjugated to a pro-inflammatory agent (e.g., IL2), wherein both the first and second VHHs bind to MHCII (e.g., a VHH having the amino acid sequence of SEQ ID NO: 1). Any one of the tumor antigens described herein may be used.

[0060] In some embodiments, the compositions described herein comprise a conjugate containing VHH conjugated with an antigen (e.g., an antigen against which an immune response is required) and a pro-inflammatory agent, wherein the VHH binds to CD11c (e.g., VHH having the amino acid sequence of SEQ ID NO: 2). In some embodiments, the compositions described herein comprise a conjugate containing VHH conjugated with an antigen from a pathogen (e.g., SARS-CoV-2 protein, e.g., spike protein) and a pro-inflammatory agent (e.g., IL2), wherein the VHH binds to CD11c (e.g., VHH having the amino acid sequence of SEQ ID NO: 2). Any one of the antigens from pathogens described herein may be used. In some embodiments, the compositions described herein comprise a conjugate containing a tumor antigen and a pro-inflammatory agent (e.g., IL2), wherein the VHH binds to CD11c (e.g., VHH having the amino acid sequence of SEQ ID NO: 2).

[0061] In some embodiments, the composition comprises a first conjugate containing a first VHH conjugated to an antigen (e.g., an antigen against which an immune response is required) and a second conjugate containing a second VHH conjugated to a pro-inflammatory agent, wherein both the first and second VHHs bind to CD11c (e.g., a VHH having the amino acid sequence of SEQ ID NO: 2). In some embodiments, the composition comprises a first conjugate containing a first VHH conjugated to an antigen from a pathogen (e.g., SARS-CoV-2 protein, e.g., spike protein) and a second conjugate containing a second VHH conjugated to a pro-inflammatory agent (e.g., IL2), wherein both the first and second VHHs bind to CD11c (e.g., a VHH having the amino acid sequence of SEQ ID NO: 2). Any one of the antigens from pathogens described in this application may be used. In some embodiments, the composition comprises a first conjugate containing a first VHH conjugated to a tumor antigen and a second conjugate containing a second VHH conjugated to a pro-inflammatory agent (e.g., IL2), wherein both the first and second VHHs bind to CD11c (e.g., a VHH having the amino acid sequence of SEQ ID NO: 2). Any one of the tumor antigens described herein may be used.

[0062] In some embodiments, the composition comprises a first conjugate containing a first VHH conjugated to an antigen (e.g., an antigen against which an immune response is required) and a second conjugate containing a second VHH conjugated to a pro-inflammatory agent, wherein the first and second VHHs bind to different surface proteins on the APC. In some embodiments, the composition comprises a first conjugate containing a first VHH conjugated to an antigen (e.g., an antigen against which an immune response is required) and a second conjugate containing a second VHH conjugated to a pro-inflammatory agent (e.g., IL2), wherein the first VHH binds to MHCII (e.g., VHH having the amino acid sequence of SEQ ID NO: 1) and the second VHH binds to CD11c (e.g., VHH having the amino acid sequence of SEQ ID NO: 2). In some embodiments, the composition comprises a first conjugate containing a first VHH conjugated to an antigen (e.g., an antigen against which an immune response is required) and a second conjugate containing a second VHH conjugated to a pro-inflammatory agent (e.g., IL2), wherein the first VHH binds to DEC205 and the second VHH binds to MHCII (e.g., a VHH having the amino acid sequence of SEQ ID NO: 1). In some embodiments, the composition comprises a first conjugate containing a first VHH conjugated to an antigen from a pathogen (e.g., SARS-CoV-2 protein, e.g., spike protein) and a second conjugate containing a second VHH conjugated to a pro-inflammatory agent (e.g., IL2), wherein the first VHH binds to MHCII (e.g., a VHH having the amino acid sequence of SEQ ID NO: 1) and the second VHH binds to CD11c (e.g., a VHH having the amino acid sequence of SEQ ID NO: 2). Any one of the antigens from the pathogens described in this application may be used. In some embodiments, the composition comprises a first conjugate containing a first VHH conjugated to a tumor antigen and a second conjugate containing a second VHH conjugated to a pro-inflammatory agent (e.g., IL2), wherein the first VHH is bound to DEC205 and the second VHH is bound to MHCII (e.g., a VHH having the amino acid sequence of SEQ ID NO: 1).

[0063] antigen As used in this application, "antigen" refers to a molecule that induces an immune response in a target. The target antigen may be, for example, a polypeptide, polysaccharide, carbohydrate, lipid, nucleic acid, or a combination thereof, or may include such a molecule. The antigen may occur naturally or be synthesized.

[0064] In some embodiments, the antigen is an antigen for which immune tolerance is required. In some embodiments, such an antigen is a self-antigen (also called an "autoantigen") or a drug capable of initiating or enhancing an autoimmune response that causes an autoimmune disease. Therefore, it is desirable to induce immune tolerance to such self-antigens. In some embodiments, the compositions described herein are used to induce immune tolerance to self-antigens (e.g., antigen-specific immune tolerance). Induction of immune tolerance (e.g., antigen-specific immune tolerance) reduces the antigen-specific immune response to the antigen, which in some embodiments reduces the severity of autoimmune diseases.

[0065] In some embodiments, the autoantigens used in accordance with this disclosure are selected from the group consisting of myelin oligodendrocyte glycoprotein (MOG), myelin proteolipidoprotein, citrullinated fibrinogen, insulin, chromogranin A, GAD65, desmoglein 1 (DSG1) and desmoglein 3 (DSG3), acetylcholine receptor (AChR), muscle-specific tyrosine kinase (MuSK), and ribonucleoprotein.

[0066] In some embodiments, the autoantigen comprises myelin oligodendrocyte glycoprotein (MOG) or an antigenic fragment thereof. Myelin oligodendrocyte glycoprotein (MOG) is a surface protein embedded in the membrane of the central nervous system (CNS) myelin sheath. Antibodies targeting MOG have been consistently found in the serum of patients with autoimmune diseases such as acquired inflammatory demyelinating disorders of the CNS (as described, e.g., Nessier et al., EBioMedicine. 2019 Oct; 48: 18-19, incorporated herein by reference). Autoimmune diseases associated with MOG antibodies include, without limitation, acute disseminated encephalomyelitis (ADEM), optic neuritis (ON), transverse myelitis, and brainstem encephalitis. In some embodiments, the autoantigen of the compositions described herein is full-length MOG. In some embodiments, the autoantigen of the compositions described herein comprises a MOG fragment (e.g., amino acids 35-55 of MOG, MEVGWYRSPFSRVVHLYRNGK (SEQ ID NO: 49)).

[0067] In some embodiments, the autoantigen comprises fibrinogen or its antigenic fragment. Fibrinogen (blood clotting factor I) is a major player in thrombus formation; it is cleaved by thrombin to form fibrin, which is the most abundant component of blood clots. Fibrinogen plays a crucial role in blood coagulation and cardiovascular disease (CVD). In addition, fibrinogen is a pro-inflammatory factor in autoimmune and inflammatory diseases, such as rheumatoid arthritis, vasculitis, inflammatory bowel disease, multiple sclerosis, chronic obstructive pulmonary disease, renal impairment, and post-transplant fibrosis, as well as in some types of cancer (as described, for example, in Arbustini et al., Circulation. 2013;128:1276-1280, incorporated herein by reference). In some embodiments, the autoantigen is citrullinated fibrinogen. In some embodiments, the autoantigen of the compositions described herein comprises a fibrinogen fragment (amino acids 79-91 of citrullinated fibrinogen, QDFTNCitINKLKNS (SEQ ID NO: 50)). Anti-citrullinated protein antibodies (ACPAs) are specifically and frequently detected in the serum of patients with rheumatoid arthritis (as described, e.g., Takizawa et al., Ann Rheum Dis. 2006 Aug; 65(8): 1013-1020).

[0068] In some embodiments, the autoantigen comprises myelin proteolipide proteins or antigenic fragments thereof. For example, as described in Tuohy et al., Neurochem Res. 1994 Aug;19(8):935-44, incorporated herein by reference, myelin proteolipide proteins have been shown to be involved in autoimmune demyelinating diseases.

[0069] In some embodiments, the autoantigen comprises insulin or an antigenic fragment thereof. In some embodiments, the autoantigen comprises the insulin alpha chain GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 51). In some embodiments, the autoantigen comprises the insulin beta chain FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 52). Insulin is involved in rare autoimmune diseases, including insulin autoimmune syndrome and type B insulin resistance (as described, for example, in Censi et al., Ann Transl Med. 2018 Sep; 6(17): 335, which is incorporated herein by reference).

[0070] In some embodiments, the autoantigen comprises chromogranin A or an antigenic fragment thereof. Chromogranin A is associated with autoimmune gastritis (for example, as described by Peracchi et al., European Journal of Endocrinology (2005) 152 443–448, incorporated herein by reference).

[0071] In some embodiments, the autoantigen comprises glutamate decarboxylase 65 kilodalton isoform (GAD65) or its antigenic fragment, which is known to be associated with autoimmune diseases of the central nervous system, neuroautoimmune diseases, type 1 diabetes, autoimmune thyroid diseases, and pernicious anemia (as described, for example, in McKeon et al., Muscle Nerve. 2017 Jul;56(l):15-27, which is incorporated herein by reference).

[0072] In some embodiments, the autoantigen comprises desmoglein 1 (DSG1) and / or desmoglein 3 (DSG3) or its antigenic fragment. DSG1 and DSG3 are involved in cutaneous autoimmune diseases, as described, for example, in Amagai et al., Proc Jpn Acad Ser B Phys Biol Sci. 2010;86(5):524-37, which is incorporated herein by reference.

[0073] In some embodiments, the autoantigen comprises an acetylcholine receptor (AChR) or an antigenic fragment thereof. An autoimmune response mediated by an antibody against the acetylcholine receptor causes myasthenia gravis, as described, for example, in Lindstrom et al., J Neurobiol. 2002 Dec;53(4):656-65, which is incorporated herein by reference.

[0074] In some embodiments, the autoantigen comprises muscle-specific tyrosine kinase (MuSK) or its antigenic fragment. MuSK has been shown to be involved in neuromuscular junction autoimmune diseases, as described, for example, in Vincent et al., Curr Opin Neurol. 2005 Oct;18(5):519-25, which is incorporated herein by reference.

[0075] In some embodiments, the autoantigen comprises a ribonucleoprotein or an antigenic fragment thereof. Ribonucleoproteins are involved in autoimmune diseases such as systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD), as described, for example, in Whittingham et al., Aust NZJ Med. 1983 Dec;13(6):565-70; and Newkirk et al., Arthritis Research & Therapy volume 3, Article number: 253 (2001), which are incorporated herein by reference.

[0076] Other, not limited to, examples of such autoimmune antigens and associated autoimmune diseases include: pancreatic beta cell antigen, insulin, and GAD for treating insulin-dependent diabetes mellitus (Type 1 diabetes); collagen type 11, human cartilage gp39 (HCgp39), and gp130-RAPS for use in treating rheumatoid arthritis; myelin basic protein (MBP) and proteolipid protein (PLP) for treating multiple sclerosis; fibrillarin and nucleolar low molecular weight protein (snoRNP) for treating scleroderma; and Graves' disease. This includes thyroid-stimulating factor receptor (TSH-R) for use in treating systemic lupus erythematosus; nuclear antigens, histones, glycoprotein gp70, and ribosomal proteins for use in treating systemic lupus erythematosus; pyruvate dehydrogenase / dehydrolipoamide acetyltransferase (PCD-E2) for use in treating primary biliary cirrhosis; hair follicle antigens for use in treating alopecia areata; and human tropomyosin isoform 5 (hTM5) for use in treating ulcerative colitis. These examples are not meant to be limiting. Those skilled in the art will have the ability to identify autoimmune antigens associated with the autoimmune disease of interest.

[0077] In some embodiments, the antigens include proteins used in protein replacement therapy or gene therapy, such as, without limitation, factor IX, factor VIII, insulin, and AAV-derived proteins. These examples are not intended to be limiting. Those skilled in the art have the ability to identify target proteins used in protein replacement therapy or gene therapy. Inducing immune tolerance to these proteins reduces the destruction of the proteins by the immune system, leading to longer-lasting therapeutic effects.

[0078] In some embodiments, the antigens used in accordance with this disclosure are antigens against which an immune response is required. For example, in some embodiments, such antigens include polypeptides or peptides that are naturally produced and / or encoded by genes of pathogens, infected cells, or neoplastic cells (e.g., cancer cells). In some embodiments, the antigen is produced or encoded by genes of viruses, bacteria, fungi, or parasites, which in some embodiments are pathogenic factors. In some embodiments, the pathogen is intracellular for at least part of its life cycle. In some embodiments, the pathogen is extracellular. It will be understood that antigens originating from a particular source may, in some embodiments, be isolated or produced from such source by any appropriate means (e.g., recombinantly, synthetically, etc.) for the purpose of, for example, identifying, producing, testing, or using antibodies against the antigen. Antigens may be modified, for example, by conjugatement to another molecule or entity (e.g., an adjuvant), chemical or physical modification, etc. In some embodiments, the antigen is an envelope protein, capsid protein, secretory protein, structural protein, cell wall protein or polysaccharide, outer shell protein or polysaccharide, or enzyme. In some embodiments, the antigen is a toxin, such as a bacterial toxin.

[0079] In some embodiments, the antigen is a viral antigen. Exemplary viruses include, for example, SARS-CoV-2, retroviridae (e.g., lentivirus, e.g., human immunodeficiency virus, e.g., HIV-I); caliciviridae (e.g., strains that cause gastroenteritis); togaviridae (e.g., equine encephalitis virus, rubella virus); flaviviridae (e.g., dengue virus, encephalitis virus, yellow fever virus, hepatitis C virus); coronavirusidae (e.g., coronavirus); rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus); filoviridae (e.g., Ebola virus); paramyxoviridae (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus); orthomyxoviridae (e.g., influenza virus); bunyaviridae (e.g., Hantan virus, Bunga virus) This includes viruses, phleboviruses, and nairoviruses; Arenaviridae (hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviruses, and rotaviruses); Birnaviridae; Hepadnaviridae (hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae (papillomaviruses, polyomaviruses); Adenoviridae, Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella-zoster virus, cytomegalovirus (CMV), EBV, KSV); Poxviridae (smallpox virus, vaccinia virus, poxvirus); and Picornaviridae (e.g., poliovirus, hepatitis A virus; enteroviruses, anthropococcyte virus, rhinovirus, echovirus). In some embodiments, the antigen includes betacoronavirus proteins, e.g., spike proteins (e.g., full-length or receptor-binding domain (RBD)), envelope proteins, membrane proteins, or nucleocapsid proteins. In some embodiments, the antigen includes SARS-CoV (e.g., SARS-CoV-1 or SARS-CoV-2) proteins, such as spike proteins (e.g., full-length or receptor-binding domain (RBD)), envelope proteins, membrane proteins, or nucleocapsid proteins. Examples of beta-coronavirus proteins that can be used as antigens in accordance with this disclosure are provided in Table 2.

[0080] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0081] In some aspects, the antigen is a bacterial antigen. Exemplary bacteria include, for example, Helicobacter pylori, Borrelia burgdorferi, Legionella pneumophila, Mycobacteria (e.g., M. tubercurosis, M. avium, M. intracellulare, M. kansasi, M. goldone), Staphylococcus aureus, Neisseria gonorea, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (Villidance group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic sp.), and Stre This includes Ptococcus pneumoniae, Campylobacter sp., Enterococcus sp., Chlamydia sp., Haemophilus influenzae, Bacillus anthracis, Corynebacterium diphtheriae, Erytheperothrix luciopatiensis, Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasteurella murtocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidum, Treponema pertenuum, Leptospira, Actinomyces israeri, and Francisella tularensis.

[0082] In some aspects, the antigen is a fungal antigen. Exemplary fungi include, for example, Aspergillus, e.g., Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger; Blastomyces, e.g., Blastomyces dermatichidis; Candida, e.g., Candida albicans, Candida glabrata, Candida gilliermondii, Candida crusei, Candida parapsilosis, Candida tropicalis; Coccidioides, e.g., Coccidioides imitis; Cryptococcus, e.g., Cryptococcus neoformans; Epidermophyton; Fusarium; Histoplasma, e.g., Histoplasma capsulatum; Malassezia, e.g., Malassezia furfur, Microsporum, Mucor; Paracoccidioides, e.g., Paracoccidioides brasiliensis; Penicillium, e.g., Penicillium This group includes Marnefei, Pichia (e.g., Pichia anomala, Pichia gilliermondii), Pneumocystis (e.g., Pneumocystis carinii), Pseudoalescheria (e.g., Pseudoalescheria boisii), Rhizopus (e.g., Rhizopus oryzae), Rhodotorula (e.g., Rhodotorula rubra), Skedosporium (e.g., Skedosporium apiospermum), Schizophyllum (e.g., Schizophyllum commune), Sporotrix (e.g., Sporotrix schenkyi), Trichophyton (e.g., Trichophyton mentagrophytes, Trichophyton rubrum, Trichophyton belcosum, Trichophyton violaceum), Trichophyton asahi, Trichophyton ctaneum, Trichophyton inkin, and Trichophyton mucoides.

[0083] In some embodiments, the antigen is derived from a parasite. Exemplary parasites include, for example, parasites of the genus Plasmodium (e.g., Plasmodium falciparum, P. vivax, P. oval, and P. malariae), Trypanosoma, Toxoplasma (e.g., Toxoplasma gondii), Leishmania (e.g., Leishmania major), Cystosoma, or Cryptosporidium. In some embodiments, the parasite is a protozoan. In some embodiments, the parasite belongs to the phylum Apicomplexa. In some embodiments, the parasite is extracellular for at least part of its life cycle. Examples include nematodes, trematodes, and tapeworms. In some embodiments, the antigen is intended from Ascaris or Tricris. In various embodiments, the antigen may originate from any component of the parasite. In some embodiments, the antigen may originate from any stage of the parasite's life cycle, for example, any stage of the parasite occurring in an infected organism such as a mammal or bird. In some embodiments, the antigen is derived from the eggs of a parasite or a substance secreted by the parasite.

[0084] In some embodiments, an antigen is a tumor antigen. Generally, a tumor antigen can be any antigenic substance produced by tumor cells (e.g., tumorogenic cells, or in some embodiments, tumor stromal cells, e.g., tumor-associated cells, e.g., cancer-associated fibroblasts). In some embodiments, a tumor antigen is a molecule (or a portion thereof) that is differentially expressed by tumor cells compared to non-tumor cells. Tumor antigens may include, for example, proteins that are normally produced in very small quantities but expressed in larger quantities by tumor cells, proteins that are normally produced at certain stages of development, proteins whose structure (e.g., sequence or post-translational modifications) is altered in tumor cells due to mutation, or normal proteins that are (under normal conditions) isolated from the immune system. Tumor antigens may be useful, for example, for identifying or detecting tumor cells (e.g., for diagnostic purposes and / or for monitoring subjects treated for tumors, e.g., to test for recurrence) and / or for targeting various drugs (e.g., therapeutic agents) to tumor cells. For example, in some embodiments, chimeric antibodies are provided, comprising antibodies of antibody fragments conjugated by click chemistry to a therapeutic agent, such as a cytotoxic agent, and bound to a tumor antigen. In some embodiments, the tumor antigen is a mutated gene, such as an oncogene or mutated tumor suppressor gene expression product, an overexpressed or abnormally expressed cellular protein, an antigen encoded by an oncogenic virus (e.g., HBV; HCV; herpesvirus family members, such as EBV, KSV; papillomavirus, etc.), or a carcinoembryonic antigen. Carcinoembryonic antigens are normally produced in the early stages of embryonic development and disappear mostly or completely by the time the immune system is fully developed. Examples include alpha-fetoprotein (AFP, e.g., found in germ cell tumors and hepatocellular carcinoma) and carcinoembryonic antigen (CEA, e.g., found in intestinal cancer and occasionally in lung or breast cancer). Tyrosinase is an example of a protein that is normally produced in very low quantities, but whose production is greatly increased in certain tumor cells (e.g., melanoma cells).Other exemplary tumor antigens include, for example, CA-125 (e.g., found in ovarian cancer); MUC-1 (e.g., found in breast cancer); epithelial tumor antigens (e.g., found in breast cancer); melanoma-associated antigens (MAGE; e.g., found in malignant melanoma); and prostatic acid phosphatase (PAP; found in prostate cancer). In some embodiments, tumor antigens are exposed at least partially on the cell surface of tumor cells. In some embodiments, tumor antigens include abnormally modified polypeptides or lipids, such as abnormally modified cell surface glycolipids or glycoproteins. It will be understood that tumor antigens may be expressed by a subset of a particular type of tumor and / or by a subset of tumor cells.

[0085] In some aspects, tumor antigens include: MAGE family members, NY-ESO-1, tyrosinase, Melan-A / MART-1, prostate cancer antigen, Her-2 / neu, survivor, telomerase, WT1, CEA, gp100, Pme117, mammoglobin-A, NY-BR-1, ERBB2, OA1, PAP, RAB38 / NY-MEL-1, TRP-1 / gp75, TRP-2, CD33, BAGE-1, D393-CD20n, and others. Ikurin-A1, GAGE-1, GAGE-2, GAGE-8, GnTVf, HERV-K-MEL, KK-LC-1, KM-HN-1, LAGE-1, LY6K, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12, MAGE-C1, MAGE-C2, Mucin K, NA88-A, SAGE, sp17, SSX-2, SSX-4, Survival g, TAG-1, TAG-3, TRAG-3, XAGE-1b, BCR-AB1, Adipophylline, AIM-2, ALDH1A1, BCLX(L), BING-4, CALCA, CD45, CD274, CPSF, Cyclin D1, DKK1, ENAH, EpCAM, EphA3, EZH2, FGF5, Glypican-3, G250, HER-2, HLA-DOB, Hepsin, IDO1, IGF2B3, IL12R Alpha-2, Intestinal Carboxylesterase, Alpha- The group is selected from the following: fetoprotein, kallikrein 4, KIF20A, lengusine, M-CSF, M-CSP, mdm-2, Meloe, midkine, MMP-2, MMP-7, MUC1, MUC5AC, p53, PAX5, PBF, PRAME, PSMA, RAGE-1, RGS5, RhoC, RNF43, RU2AS, secerine1, SOX10, STEAP1, telomerase, TPBG, mesothelin, Ax1, and VEGF.

[0086] In some embodiments, the antigen is a whole cell, a whole parasite, a whole virus, a whole bacterium, or a whole nanoparticle, exosome, or microparticle containing one or more antigens. In one example, VHH may be conjugated to beta-islet cells and delivered in a non-inflammatory state to induce beta-islet cell tolerance in the course of organ or tissue replacement therapy. In yet another example, VHH may be conjugated to a parasite and delivered in an inflammatory state to induce a strong immune response to multiple parasitic antigens at once.

[0087] Anti-inflammatory drugs and pro-inflammatory drugs As shown in this application, a conjugate containing VHH conjugated to an antigen for which immune tolerance is required (e.g., an autoantigen) is more effective in inducing antigen-specific immune tolerance to the autoantigen when administered to subjects in a non-inflammatory state. In some embodiments, the non-inflammatory state is provided by attaching an anti-inflammatory agent to the same conjugate containing VHH and the antigen. In some embodiments, the non-inflammatory state is provided by co-administering VHH conjugated to an autoantigen in addition to VHH conjugated to an anti-inflammatory agent.

[0088] An "anti-inflammatory agent" is a substance that reduces inflammation in the body. Anti-inflammatory agents block certain substances in the body that cause inflammation. Any anti-inflammatory agent known in the art may be used in accordance with this disclosure. In some embodiments, the anti-inflammatory agent is a steroidal anti-inflammatory agent. In some embodiments, the steroidal anti-inflammatory agent is selected from the group consisting of: dexamethasone, prednisone, prednisolone, triamcinolone, methylprednisolone, and betamethasone. In some embodiments, the anti-inflammatory agent is a non-steroidal anti-inflammatory agent. In some embodiments, the non-steroidal anti-inflammatory agent is selected from the group consisting of: aspirin, celecoxib, diclofenac, ibuprofen, ketoprofen, naproxen, oxaprozin, piroxicam, cyclosporine A, and calcitriol. In some embodiments, the anti-inflammatory agent used in accordance with this disclosure is dexamethasone.

[0089] In some embodiments, anti-inflammatory agents are anti-inflammatory cytokines. "Anti-inflammatory cytokines" refer to cytokines that inhibit the synthesis of IL-1, tumor necrosis factor (TNF), and other major pro-inflammatory cytokines, and reduce the inflammatory response. In some embodiments, anti-inflammatory cytokines are selected from the group consisting of IL-10, IL-35, IL-4, IL-11, IL-13, and TGFβ.

[0090] This disclosure also provides that, in other respects, a conjugate containing VHH conjugated to an antigen (e.g., an antigen from a pathogen or a tumor antigen) against which an immune response is required is more effective in inducing an antigen-specific immune response to the antigen when administered to a subject in an inflammatory state. In some embodiments, an inflammatory state is provided by attaching a pro-inflammatory agent to the same conjugate containing VHH and the antigen. In some embodiments, a non-inflammatory state is provided by co-administering VHH conjugated to an antigen in addition to VHH conjugated to an antigen. In some embodiments, the pro-inflammatory agent is selected from the group consisting of: TLR9 agonists (e.g., CpG-ODN), LPS, HMGB1 protein, IL2, IL12, and CD40L. In some embodiments, the pro-inflammatory agent is IL2.

[0091] Treatment method Several aspects of this disclosure provide a method for subjects requiring it: (i) a conjugate comprising VHH conjugated with an antigen (e.g., autoantigen) to which immune tolerance is required, and an anti-inflammatory agent, wherein VHH conjugates to a surface protein (e.g., MHCII or CD11c) on an APC; or (ii) a first conjugate comprising VHH conjugated to an antigen (e.g., autoantigen) to which immune tolerance is required, and a second conjugate comprising VHH conjugated with an anti-inflammatory agent, wherein the first and second VHHs conjugate to one or more (e.g., the same or different) surface proteins on an APC. In some embodiments, when the two VHHs are administered, they are administered (e.g., sequentially) as the same composition or as different compositions. In some embodiments, the method is for inducing immune tolerance to an antigen. In some embodiments, the method is for treating autoimmune diseases.

[0092] An "autoimmune disease" is a disorder that causes abnormal overactivity of the immune system, leading to attacks and damage of its own tissues. Examples of autoimmune diseases, not limited to: rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), myasthenia gravis (MG), Graves' disease, idiopathic thrombocytopenic purpura (ITP), Guillain-Barré syndrome, autoimmune myocarditis, membranous glomerulonephritis, type 1 or type 2 diabetes, juvenile-onset diabetes, multiple sclerosis, Raynaud's syndrome, autoimmune thyroiditis, gastritis, celiac disease, vitiligo, hepatitis, primary biliary cirrhosis, inflammatory bowel disease, spondyloarthritis, and experimental diseases. Autoimmune encephalomyelitis, immune neutropenia, and immune responses associated with cytokine-mediated delayed-type hypersensitivity reactions, T lymphocytes typically found in tuberculosis, sarcoidosis, and polymyositis, polyarteritis, cutaneous vasculitis, pemphigus (e.g., pemphigus vulgaris, pemphigus foliaceus, or paraneoplastic pemphigus), bullous pemphigoid, Goodpasture syndrome, Kawasaki disease, systemic scleroderma, antiphospholipid syndrome, and Sjögren's syndrome. In some embodiments, autoimmune diseases are selected from the group consisting of: multiple sclerosis, type II diabetes mellitus, pemphigus vulgaris, myasthenia gravis, lupus, celiac disease, and inflammatory bowel disease (IBD). In some embodiments, autoimmune diseases are selected from the group consisting of: autoimmune encephalomyelitis, acute disseminated encephalomyelitis (ADEM), optic neuritis (ON), transverse myelitis, and brainstem encephalitis, rheumatoid arthritis, vasculitis, inflammatory bowel disease, multiple sclerosis, chronic obstructive pulmonary disease, renal impairment, post-transplant fibrosis, and several types of cancer; autoimmune demyelinating diseases, insulin autoimmune syndrome, type B insulin resistance, autoimmune gastritis, autoimmune diseases of the central nervous system, neuroautoimmune diseases, type 1 diabetes mellitus, autoimmune thyroid disease, pernicious anemia, cutaneous autoimmune diseases, myasthenia gravis, and neuromuscular junction autoimmune diseases. Different autoantigens may be used as conjugates to treat different autoimmune diseases. Those skilled in the art have the ability to identify appropriate autoantigens to be used.

[0093] Other aspects of this disclosure provide a method for subjects requiring it: (i) a conjugate comprising VHH conjugated to an antigen (e.g., an antigen from a pathogen or a tumor antigen) against which an immune response is required, and a pro-inflammatory agent, wherein VHH conjugates to a surface protein (e.g., MHCII or CD11c) on an APC; or (ii) a first conjugate comprising VHH conjugated to an antigen (e.g., an antigen from a pathogen or a tumor antigen) against which an immune response is required, wherein the first VHH and the second VHH conjugate to one or more (e.g., the same or different) surface proteins on an APC, and a second conjugate comprising the second VHH conjugated to a pro-inflammatory agent. In some embodiments, when the two VHHs are administered, they are administered as the same composition or as different compositions (e.g., sequentially). In some embodiments, the method is for inducing an immune response to an antigen. In some embodiments, the method is for treating an infection caused by a pathogen (e.g., a microbial pathogen, e.g., as described herein). In some embodiments, the method is for treating cancer.

[0094] Cancer can be primary or metastatic. Cancers include adult and pediatric acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, anal cancer, appendiceal cancer, astrocytoma, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, biliary tract cancer, osteosarcoma, fibrous histiocytoma, brain cancer, brainstem glioma, cerebellar astrocytoma, malignant glioma, glioblastoma, ependymoma, medulloblastoma, supratentorial primitive neuroectoderm tumor, hypothalamic glioma, breast cancer, male breast cancer, bronchial adenoma, Burkitt lymphoma, carcinoid tumor, carcinoma of unknown origin, central nervous system lymphoma, cerebellar astrocytoma, malignant glioma, cervical cancer, childhood cancer, and chronic lymphoma. Passive leukemia, chronic myeloid leukemia, acute lymphoblastic and myeloid leukemia, chronic myeloproliferative disorders, colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer, ependymoma, esophageal cancer, Ewing family tumors, extracranial germ cell tumors, extragonadal germ cell tumors, extrahepatic cholangiocarcinoma, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinal stromal tumors, extracranial germ cell tumors, extragonadal germ cell tumors, ovarian germ cell tumors, gestational trophoblastic neoplasm, glioma, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, Hodgkin lymphoma, non-Hodgkin lymphoma, hypopharyngeal cancer, optic nerve hypothalamic glioma, intraocular melanoma M, pancreatic islet cell tumor, Kaposi's sarcoma, kidney cancer, renal cell carcinoma, laryngeal cancer, oral and lip cancer, small cell lung cancer, non-small cell lung cancer, primary central nervous system lymphoma, Waldenström macroglobulinemia, malignant fibrous histiocytoma, medulloblastoma, melanoma, Merkel cell carcinoma, malignant mesothelioma, cervical squamous cell carcinoma, multiple endocrine neoplasia, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myeloproliferative disorder, chronic myeloproliferative disorder, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oropharyngeal cancer, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal gland blastoma and supratentorial primordial cancer This includes, but is not limited to, transectodermal tumors, pituitary cancer, plasma cell neoplasms, pleuroblastoma, prostate cancer, rectal cancer, rhabdomyosarcoma, salivary gland cancer, soft tissue sarcoma, uterine sarcoma, Sézary syndrome, non-melanoma skin cancer, small intestine cancer, squamous cell carcinoma, cervical squamous cell carcinoma, supratentorial primitive neuroectodermal tumor, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma, trophoblastic tumor, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, choriocarcinoma, hematopoietic neoplasms, adult T-cell leukemia, lymphoma, lymphocytic lymphoma, stromal tumors and germ cell tumors, or Wilms' tumor.In some aspects, cancer is lung cancer, breast cancer, prostate cancer, colorectal cancer, stomach cancer, liver cancer, pancreatic cancer, brain and central nervous system cancer, skin cancer, ovarian cancer, leukemia, endometrial cancer, bone, cartilage, and soft tissue sarcomas, lymphoma, neuroblastoma, nephroblastoma, retinoblastoma, or gonadal germ cell tumors.

[0095] In its broadest sense, the term “treatment” or “to treat” refers to both therapeutic and preventive treatment. If the subject requiring treatment has a disease (e.g., autoimmune disease, infection, or cancer), “to treat a condition” means to improve, reduce, or eliminate one or more symptoms or the severity of the disease associated with it, or to prevent any further progression of the disease. If the subject requiring treatment is at risk of having a disease (e.g., infection or cancer), “to treat a subject” means to reduce the subject’s risk of having an infection or cancer, or to prevent the subject from developing an infection or cancer.

[0096] The subjects are humans or vertebrates or mammals, including but not limited to rodents such as rats or mice, dogs, cats, horses, cattle, pigs, sheep, goats, turkeys, chickens, and primates such as monkeys. The methods of this disclosure are useful for treating subjects that require them.

[0097] In some embodiments, the compositions described herein are pharmaceutical compositions. Pharmaceutically, the compositions that may be used in accordance with this disclosure may be administered directly to a subject or to a subject in need in a therapeutically effective dose. The term “therapeutably effective dose” means the amount necessary or sufficient to achieve the desired biological effect. For example, a therapeutically effective dose of a composition relating to this disclosure may be sufficient to improve one or more symptoms of a target disease (e.g., autoimmune disease, infection, or cancer). Combined with the teachings provided herein, by selecting from a variety of active compounds and weighing factors such as potency, relative bioavailability, patient weight, severity of adverse side effects, and preferred mode of administration, effective prophylactic or therapeutic treatment regimens can be designed that are entirely effective in treating a particular subject without causing substantial toxicity. The effective dose for any particular application may vary depending on factors such as the disease or condition to be treated, the specific pharmaceutically effective composition to be administered, the size of the subject, or the severity of the disease or condition. Those skilled in the art can empirically determine the effective amount of a particular therapeutic compound related to this disclosure without requiring excessive experimental work.

[0098] The target doses of the compositions described herein for delivery are typically in the range of about 0.1 μg to 10 mg per dose, which may be given daily, weekly, or monthly, or at any other time in between, depending on the application. In some embodiments, a single dose is administered during an intensive reinforcement or re-reinforcement period. Doses for these purposes may range from about 10 μg to 5 mg per dose, most typically from about 100 μg to 1 mg, with 2 to 4 doses spaced, for example, over several days or weeks, or at more distant intervals. However, in some embodiments, parenteral doses for these purposes may be used in a range of 5 to 10,000 times higher than the typical doses described above.

[0099] In some embodiments, the compositions disclosed herein are administered in doses between approximately 1 and 10 mg / kg of mammalian body weight. In other embodiments, the compositions disclosed herein are administered in doses between approximately 0.001 and 1 mg / kg of mammalian body weight. In yet another embodiment, the compositions disclosed herein are administered in doses between approximately 10-100 ng / kg, 100-500 ng / kg, 500 ng / kg-1 mg / kg, or 1-5 mg / kg of mammalian body weight, or any of these individual doses.

[0100] The compositions of this disclosure are administered as pharmaceutically acceptable solutions, which may conventionally contain pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, and optionally other therapeutic components.

[0101] For therapeutic use, an effective amount of the composition relating to this disclosure may be administered to a target by any mode of delivery of the therapeutic agent or compound to a desired surface, such as mucosa, injection into cancer, or systemically. Administration of the pharmaceutical compositions of this disclosure may be achieved by any means known to those skilled in the art. Preferred routes of administration include, but are not limited to, oral, parenteral, intravenous, intramuscular, nasal, sublingual, tracheal, inhalation, intraocular, vaginal, rectal, and intraventricular. In some embodiments, the composition is administered intravenously (e.g., by injection or infusion).

[0102] The pharmaceutical compositions of this disclosure may be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion, when systemic delivery is desired. Formulations for injection may be presented in unit dosage forms, for example, as ampoules or multi-dose containers with additional preservatives. The compositions may take the form of suspensions, solutions, or emulsions in an oil-based or aqueous base, and may contain formulation agents, such as suspensions, stabilizers, and / or dispersants.

[0103] Pharmaceutical formulations for parenteral administration comprise aqueous solutions of the active compound in a water-soluble form. In addition, suspensions of the active compound can be prepared as suitable oil-based injection suspensions. Suitable lipophilic solvents or bases include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound to enable the preparation of highly concentrated solutions.

[0104] In addition to the formulations described above, the compositions may also be formulated as depot preparations. Such sustained-release formulations may be formulated with suitable polymer-based or hydrophobic materials (e.g., as emulsions in acceptable oils) or ion-exchange resins, or as poorly soluble derivatives, such as poorly soluble salts.

[0105] The composition may also contain a suitable solid or gel phase support or excipient. Examples of such supports or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers, such as polyethylene glycol.

[0106] Suitable liquid or solid pharmaceutical preparation forms include, for example, aqueous or saline solutions for inhalation, microencapsulation, cocreation, coating on gold nanoparticles, containment in liposomes, nebulization, aerosols, pellets for skin implantation, or drying on sharp objects to be scratched into the skin. Pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops, or preparations having a prolonged release of the active compound, in which excipients and additives and / or auxiliary agents, such as disintegrants, binders, coatings, leavening agents, lubricants, fragrances, sweeteners, or solubilizers, are commonly used as described above. Pharmaceutical compositions are suitable for use in various drug delivery systems. For a brief review of methods for drug delivery, see Langer, Science 249:1527–1533, 1990, which is incorporated herein by reference.

[0107] The compositions of this disclosure and optionally other therapeutic agents may be administered neat or in the form of pharmaceutically acceptable salts. When used in medicine, the salts should be pharmaceutically acceptable, but pharmaceutically unacceptable salts may be conveniently used to prepare pharmaceutically acceptable salts. Such salts include, but are not limited to, those prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, p-toluenesulfonic acid, tartaric acid, citric acid, methanesulfonic acid, formic acid, malonic acid, succinic acid, naphthalene-2-sulfonic acid, and benzenesulfonic acid. Such salts may also be prepared as alkali metal or alkaline earth salts, such as sodium, potassium, or calcium salts of a carboxylic acid group.

[0108] Suitable buffering agents include: acetic acid and salts (1-2% w / v); citric acid and salts (1-3% w / v); boric acid and salts (0.5-2.5% w / v); and phosphoric acid and salts (0.8-2% w / v). Suitable preservatives include benzalkonium chloride (0.003-0.03% w / v); chlorobutanol (0.3-0.9% w / v); parabens (0.01-0.25% w / v); and thimerosal (0.004-0.02% w / v).

[0109] The pharmaceutical compositions of this disclosure contain an effective amount of the therapeutic compound of this disclosure, optionally encapsulated in a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" means one or more suitable solid or liquid fillers, diluents, or encapsulating materials that are suitable for administration to humans or other vertebrates. The term "carrier" refers to a natural or synthetic organic or inorganic component that, when combined with the active ingredient, facilitates application. The components of the pharmaceutical compositions may also be mixed with and with the compounds of this disclosure in such a manner that there are no interactions that would substantially impair the desired pharmaceutical efficacy.

[0110] The pharmaceutical compositions of this disclosure may be delivered together with other therapeutic agents for treating diseases (e.g., autoimmune diseases, infections, or cancer). [Examples]

[0111] Example 1. Manipulating the modularity of single-domain antibodies that target MHC class II to protect against autoimmune diseases. Autoimmunity arises from the recognition of autoantigens by components of the adaptive immune system. This explains the link in most autoimmune diseases to specific allele variants of class II MHC products that present harmful autoantigens. To treat autoimmune diseases, the induction of antigen-specific tolerance would be a highly desirable goal. Regardless of the pathology, antigen-presenting cells (APCs) are essential for disease induction, and conversely, APCs can be tolerogenic if they encounter antigens in a non-inflammatory state. In this application, nanobodies that recognize class II MHC products present on all APCs are described, which can be enzymatically conjugated to autoantigens such as myelin oligodendrocyte glycoprotein (MOG) fragments in experimental autoimmune encephalitis (EAE), a preclinical model of autoimmune disease. Administration of these adducts in a non-inflammatory state provides long-lasting protection from EAE. Similar adducts prevented hyperglycemia and rheumatoid arthritis in a mouse model of rapid type 1 diabetes. Not only were autoantigens conjugated into nanobodies, but dexamethasone derivatives were also attached via cleavable hydrazone linkers. Co-administration of class II MHC-specific nanobodies containing MOG peptides with the same nanobodies modified with cleavable dexamethasone derivatives halted disease progression in already symptomatic animals. While the precise target cell population responsible for the therapeutic effect remains unidentified, aside from the fact that these must be class II MHC-positive cells, these findings have practical utility. The application of such antibody-drug conjugates in various inflammatory conditions should be considered a promising treatment option.

[0112] Introduction Approximately 10% of the human population suffers from autoimmune conditions accompanied by symptoms ranging from mild to life-threatening. Only in selected cases is there a clear explanation for how the disease begins. Cancer immunotherapy using checkpoint blocking is highly successful against a select set of malignancies, but it carries the risk of inducing autoimmunity by releasing the brakes on immune homeostasis. It is a clear example of an experimental trigger that exposes the presence of harmful, self-reactive cells that were suppressed until checkpoint blocking was applied.

[0113] Current treatments for autoimmune diseases encompass general immunosuppression, which dulls the response to all kinds of antigens. This exposes patients to an increased risk of infection and possibly even malignancies. Since autoimmune diseases are often organ-specific, the immune components include antigen-specific elements, either as triggers, targets, or some combination of the two. This is perhaps best illustrated by the various preclinical models of autoimmunity in which pathology can be induced by the administration of a defined antigen under appropriate stimulating conditions. In certain human autoimmune diseases, the antigens that induce and are recognized in the course of the autoimmune response are known. Examples include islet antigens in the case of type 1 diabetes, myelin sheath components in multiple sclerosis, and citrullinated antigens in the case of arthritis.

[0114] Nanoparticles composed of peptide-loaded MHC products have been used to induce forms of tolerance constrained to both class I and class II MHCs. A further notable example is the ability of erythrocytes modified with autoantigens to induce a state of severe antigen unresponsiveness. This trait is attributed to their exceptional turnover rate compared to other cell types and the need to eliminate erythrocyte remnants without triggering an inflammatory response. The phenomenon of eliminating the tolerogenicity of cellular remnants is not limited to erythrocytes, as infusion of chemically modified apoptotic peripheral blood lymphocytes can also reduce autoimmune responses.

[0115] This invention reports the development and characterization of alpaca-derived single-domain antibody fragments (nanobodies / VHHs) that recognize class II MHC molecules. These nanobodies target all class II MHC-positive cells, including antigen-presenting cells (APCs). One-tenth the size of conventional immunoglobulins, the small size of the nanobodies ensures excellent tissue penetration and rapid clearance from circulation. This makes VHHs an ideal base for targeted delivery of desired payloads, such as antigen peptides or small molecule drugs. Furthermore, a manipulation strategy using the S. aureus-derived transpeptidase, saltase A, was established. This enabled site-specific modification of these VHHs at their C-terminus. Class II MHC-specific nanobodies modified by saltase were used as imaging agents for positron emission tomography. The results were consistent with a short circulating half-life paired with excellent targeting properties. These methods also allowed for the installation of a wide range of antigens involved in infectious and autoimmune diseases. There is a broad consensus that while antigen-presenting cells can induce tolerance in non-inflammatory states, administration in inflammatory states, for example, in the presence of an adjuvant, can induce a strong protective response to foreign antigens. Antigen valency, aggregation state, and dose are additional parameters that can swing the pendulum from toxicogen to immunogen. The distribution and dynamics of diverse sets of APCs in different anatomical sites pose a challenge to identifying the relevant toxicogenic APCs in vivo. The aim of this study was not to pinpoint specific APC (sub)sets responsible for tolerance induction, but rather to demonstrate the effectiveness of using VHH to target class II MHC-positive cell populations in different contexts, including the targeted delivery of immunosuppressive small molecule dexamethasone. While the clinical use of self-peptide-loaded purified dendritic cells is documented, there may be practical merits in avoiding cell-based therapy when purely protein-based preparations can be administered for the same purpose.Indeed, the findings indicate that the combination of class II MHCVHH-peptide adducts with the same VHH conjugated to dexamethasone is remarkably effective in inhibiting the progression of EAE in animals with clear signs of the disease.

[0116] method VHH expression and endotoxin removal E. coli from WK6 containing the plasmid encoding the corresponding VHH were grown at 37°C with Terrific Broth plus ampicillin until the middle of the log phase and induced overnight at 30°C with 1 mM IPTG. Bacteria were harvested by centrifugation at 5,000 × g for 15 minutes at 4°C, then resuspended in 25 mL of 1 × TES buffer (200 mM Tris, pH 8, 0.65 mM EDTA, 0.5 M sucrose) per liter of culture and incubated at 4°C for 1 hour with agitation. The resuspended cells were then subjected to osmotic shock with a 1:4 dilution in 0.25 × TES buffer and incubated overnight at 4°C. The periplasm fraction was isolated by centrifugation at 5,000 × g for 30 minutes at 4°C, and then loaded onto Ni-NTA (Qiagen) in 50 mM Tris, pH 8, 150 mM NaCl, and 10 mM imidazole. The protein was eluted with 50 mM Tris, pH 8, 150 mM NaCl, 500 mM imidazole, and 10% glycerol, and then loaded onto a Superdex 7510 / 300 column in 50 mM Tris, pH 8, 150 mM NaCl, and 10% glycerol. The peak fraction was collected and recombined with Ni-NTA to deplete LPS (<2 IU / mg). The bound VHH was washed with 40 column volumes of PBS + 0.1% TritonX-114 and eluted with 2.5 column volumes of endotoxin-free PBS (Teknova) containing 500 mM imidazole. Imidazole was removed using a PD10 column (GE Healthcare) eluted with LPS-free PBS. Recombinant VHH purity was evaluated by SDS / PAGE and LC-MS.

[0117] Chemical synthesis of GGG antigen, GGG-Cy5, and GGG-DEX Peptides were synthesized on 2-chlorotrityl resin (ChemImpex) according to a standard solid-phase peptide synthesis (SPPS) protocol, or ordered on GenScript. For GGG-Cy5, GGGC (SEQ ID NO: 61) (7.0 mg, 24 μmol) was dissolved in DMSO (Sigma Aldrich) (400 μL) and added to cyanine 5-maleimide (Lumiprobe) (5.0 mg, 7.8 μmol). The resulting mixture was gently stirred at room temperature until LC-MS analysis showed no remaining starting material. The ligated product was then purified by RP-HPLC and lyophilized. GGG-Cy5:C 47 H 62 The calculated LC-MS value for N8O8S2[M+H]+ was 898.44, while the measured value was 898.56. The resulting powder was stored at 4°C.

[0118] In the GGG-Dexamethasone (DEX) reaction, in the first reaction, dexamethasone (Sigma Aldrich) (25 mg, 64 μmol) and N-β-maleimidepropionate hydrazide (ThermoFisher) (40 mg, 135 μmol) were dissolved in 3.0 mL of dry MeOH (Sigma Aldrich), and one drop of TFA was added to the solution. The resulting mixture was stirred overnight at room temperature. Then, the MeOH was evaporated, the precipitate was dissolved in DMSO (1.0 mL), purified by RP-HPLC, and lyophilized. DEX-Maleimide:C 29 H 37 The calculated LC-MS value for FN3O7[M+H]+ was 558.26, while the experimental value was 558.32. The resulting powder was stored at -20°C. In the second reaction, DEX-maleimide (20 mg, 36 μmol) and GGGC (SEQ ID NO: 61) (21 mg, 72 μmol) were dissolved in 5% 0.1 M NaHCO3 (1.0 mL) in DMSO. The resulting mixture was stirred at room temperature until the reaction was complete. Once the starting materials were used up, the reaction was purified directly by RP-HPLC and lyophilized. GGG-DEX:C38 H 53 FN7O 12 The calculated LC-MS value for S[M+H]+ was 850.35, while the experimental value was 850.21. The resulting peptide was stored at -20°C and redissolved in PBS at an appropriate concentration before salter-gel ligation.

[0119] C-terminal sol tagging of VHH or GFP using the GGG-containing portion (using LPETGG (SEQ ID NO: 43)) The sol-tagging reaction was carried out in a 1 mL mixture containing Tris-HCl (50 mM, pH 7.5), CaCl2 (10 mM), NaCl (150 mM), triglycine-containing probe (500 μM), GGG-containing probe (100 μM), and 5M-soltase A (5 μM). After incubation at 4 °C with stirring for 1.5 hours, unreacted VHH and 5M-SrtA were removed by adsorption onto Ni-NTA agarose beads. The unbound fraction was concentrated, and excess nucleophile was filtered using an Amicon 3,000 kDaMWCO filtration unit (Millipore). The reaction product was analyzed for purity by LC-MS and stored at -80 °C.

[0120] mouse All animals were received at the animal facility of Boston Children's Hospital (BCH) and maintained according to protocols approved by the BCH Animal Experimentation Committee. C57BL / 6J (CD45.2+), B6.SJL-Ptprc (CD45.1+), NOD / SCID, BALB / c, B6 / 2D2, NOD / BDC2.5, Balbc / DO11.10, CD11c-DTR, μMT- / -, Batf3- / -, LAG3- / -, and FoxP3-DTR mice were either purchased from Jackson Laboratory or bred in-house. MHCII-GFP and PD1- / - mice were bred in-house. OTI Rag2- / - and HLA-DR4-IE- transgenic C57BL / 6IAb null mice were purchased from Taconic.

[0121] Flow cytometry analysis Cells were harvested from the spleen, lymph nodes, or other organs and dispersed in RPMI1640 via a 40-micron cell strainer using the back of a 1 mL syringe plunger. The cell mixture was subjected to hypotonic lysis (NH4Cl) to remove erythrocytes, washed twice with FACS buffer (2 mM EDTA and 1% FBS in PBS), and resuspended in FACS buffer containing the corresponding fluorescent dye-conjugated antibody. All staining was performed in the dark at 4°C for 30 minutes by Fc block with a 1:100 dilution. Samples were washed twice with FACS buffer before further analysis. All flow data were acquired by a FACS Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star).

[0122] The antibodies used in this study are listed in Table 3.

[0123] [Table 3-1] [Table 3-2]

[0124] Experimental autoimmune encephalomyelitis (EAE) model using C57BL / 6J mice Female C57BL / 6 mice (10-12 weeks old) or other mouse strains with a C57BL / 6J genetic background, in the Hooke Kit: CFA with MOG 35-55Immunized (Hooke laboratories) according to the manufacturer's instructions with the emulsion and PTX in PBS. Mice were scored daily starting on the 7th day after immunization by investigators blinded to the individual mouse experimental treatments. Mice were randomly assigned to different experimental treatments and co-housed to eliminate cage-to-cage variation. All treatments were performed in at least 3 mice and in at least 2 independent experiments as indicated in the figure legend. All animals were included in the analysis. Clinical scores were defined as follows: 1, tail droop; 2, partial hind limb paralysis; 3, complete hind limb paralysis; 4, complete hind and partial forelimb paralysis; and 5, moribund. Easy access to wet food and water was provided to experimental mice during disease progression. Unless otherwise indicated, for prophylactic treatment, 20 μg of VHH-antigen conjugated with sortag was administered intravenously 7 days prior to the induction of EAE. For therapeutic treatment, 20 μg of VHH MHCII -OVA 323-339 , VHH MHCII -MOG 35-55 , or 20 μg of VHH MHCII -MOG 35-55 mixed with 20 μg of VHH MHCII-DEX was administered on the day of EAE. At this time, the mice exhibited symptoms defined as clinical scores of 1, 2, and 3 as instructed. On day 30 after EAE induction, or when the mice reached clinical score 4, the mice were sacrificed by asphyxiation and then perfused with 5 mM EDTA in PBS. Spinal cord was isolated, fixed with 10% (wt / vol) formalin solution (Sigma), embedded in paraffin, sectioned to 20 μm, and stained with H&E or Luxor Fast Blue (Harvard Medical School Rodent Histology Core Facility). Stained sections were imaged at 4× and 10× magnification. Isolation of immune cells infiltrating the spinal cord was performed by homogenizing the spinal cord, followed by 38% Percoll (Sigma) gradient separation (100% Percoll is 1.123 g / mL). Isolated cells were plated in 48-well plates and treated with 50 ng / mL PMA (Sigma) and 500 ng / mL ionomycin (Sigma) in complete RPMI medium at 37°C for 2 hours, followed by the addition of 10 μg / mL monensin (Sigma) and incubation for another 2 hours. The cells were then surface-stained, fixed, and permeabilized using the Foxp3 / transcription factor staining buffer set (ThermoFisher Scientific, 00-5523-00) according to the manufacturer's protocol. Intracellular and Foxp3 staining were performed according to the manufacturer's protocol, and the cell samples were then used for flow cytometry.

[0125] For cytokine storm analysis, on the first day these EAE mice reached a clinical score of 3, blood samples were taken with 20 μg of VHH. MHCII -MOG 35-55 , VHH MHCII -OVA 323-339 , or 20 μg of VHH MHCII -MOG 35-55 +20μg VHH MHCIIBlood was collected 5 hours after therapeutic treatment with -DEX. Blood was collected in an EDTA-containing tube, and plasma was isolated by repeated centrifugation (500g, 5min, 4°C). Plasma was stored at -80°C until further analysis of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). TNF-α (ThermoFisher, 88-7324-22) and IL-6 (ThermoFisher, 88-7064-22) ELISA was performed according to the manufacturer's protocol.

[0126] Cell subset depletion CD8 T cells were depleted by intraperitoneal administration of 400 μg of anti-CD8α depletion antibody (clone 2.43, BioXCell) twice a week, starting two weeks prior to prophylactic treatment with VHH-antigen and continuing throughout the EAE observation period. Macrophage subsets were eliminated by injection of 300 μg of anti-CSF1R (clone AFS98, BioXCell) every other day, starting two weeks prior to prophylactic treatment and continuing until the end of the experimental setup. To deplete DCs, 100 ng of DTX(Sigma) was intraperitoneally administered to CD11c-DTR mice two days prior to VHH-antigen administration. To deplete Tregs, FoxP3-DTR mice were intraperitoneally injected with 1 μg of DTX(Sigma) three times on days -9, -8, and -1 prior to prophylactic treatment with VHH-antigen, and then weekly until the end of the observation period. Cell depletion was confirmed by flow cytometry of PBMCs or spleen cells.

[0127] 2D2 CD4 T cell adoptive transfer and loading CD4 T cells derived from the spleen and iLN of 2D2 mice were enriched by negative selection using magnetic beads (Miltenyi Biotec, 130-104-453) and labeled with Violet CellTrace (ThermoFisher Scientific, C34571) according to the manufacturer's protocol. 500,000 of these 2D2 CD4+ T cells were transferred into CD45.1+ mice with 20 μg of VHH. MHCII -OVA 323-339, 20 μg VHH MHCII -MOG 35-55 , equimolar MOG 35-55 Peptide, or 100 μg of MOG 35-55 Peptide infusion was performed on the day after adoptive transfer, mixed with 25 μg of anti-CD40 (SouthernBiotech) and 50 μg of poly-I:C (Sigma) as adjuvants. On days 3, 5, and 10, mice were sacrificed, and spleen, iLN, and blood were collected and analyzed by flow cytometry. Some 2D2 T cell adoptive transfer mice also received 100 μg of MOG in CFA on day 3 or 10. 35-55 The substance was applied subcutaneously. Mice were sacrificed after 7 or 5 days as instructed in each experimental setup. Spleens, iLN, and blood were harvested and analyzed by flow cytometry.

[0128] RNA-seq of 2D2 CD4 T cells Cells were sorted and lysed in RLT lysis buffer (Qiagen) supplemented with β-mercaptoethanol. RNA was isolated using the RNeasy microkit (Qiagen) according to the manufacturer's protocol. 20 ng of RNA was used as input to a modified SMART-seq2 protocol. The resulting libraries were validated using a high-sensitivity DNA chip on a Bioanalyzer 2100 system (Agilent), and then libraries were prepared using the Nextera XT kit (Illumina) and custom index primers according to the manufacturer's protocol. The final libraries were quantified using the Qubit dsDNA HS assay kit (Invitrogen) and a high-sensitivity DNA chip on a Bioanalyzer 2100 system (Agilent). All libraries were sequenced using the Nextseq High Output Cartridge kit and Nextseq 500 sequencer (Illumina). The sequenced libraries were demultiplexed using the bcl2fastq program, and the resulting Fastq data were trimmed and cropped using Trimmomatic. Alignment to the mouse mm10 reference genome and gene expression counts were performed using Kallisto. Principal component analysis (PCA) was performed in R. DEseq2 was used to test differential gene expression and differential chromatin accessibility at individual loci from our RNA-seq data. Volcano plots and heatmaps were generated using Python 3.6 with NumPy 1.12.1 and Matplotlib 2.2.2. For functional analysis, Gorilla (a gene ontology enrichment analysis and visualization tool) was used to identify enriched gene ontology (GO) terms in upregulated and downregulated subsets of the top 500 most differentially expressed genes.

[0129] Type 1 diabetes (T1D) model in NOD / SCID mice Spleens and inguinal lymph nodes were harvested from 7-9 week old BDC2.5 mice. Cells were resuspended in complete RPMI (RPMI with 2 mM glutaMAX, 10 mM HEPES, non-essential amino acids, 1 mM sodium pyruvate, 55 μM β-mercaptoethanol, and 10% heat-inactivated FBS) supplemented with 0.5 μM p31 peptide (BDC2.5 mimotope, GenScript), and plated in tissue culture dishes at 1 million cells / mL. After 4 days, cells were harvested, washed twice, and resuspended in PBS. 5 million cells were adopted into 9-12 week old female NOD.SCID mice by post-orbital injection. Physiological saline and 20 μg VHH were added. MHCII -p31, or VHH MHCII -MOG 35-55 The mice were infused with the solution one or five days later as instructed. Blood glucose levels were measured every other day for two weeks and weekly for up to one to two months. Mice were considered to have diabetes if their blood glucose levels exceeded 260 mg / dL over the following two weeks, as measured using the Active meter (Accu-Chek) (range 20-600 mg / dL) on the corresponding Aviva Plus test strip (Accu-Check).

[0130] Mice were sacrificed by asphyxiation at the 2-month endpoint or when blood glucose levels exceeded 600 mg / dL over the following two weeks. The pancreas was fixed for further immunohistochemical analysis, i.e., H&E staining (Harvard Medical School Rodent Histology Core Facility). In a separate cohort of mice, the spleen, inguinal / pancreatic lymph nodes, and pancreas were harvested for flow cytometry analysis on day 14 after adoptive transfer.

[0131] Rheumatoid arthritis (RA) model in BALB / c mice Spleens and lymph nodes were collected from DO11.10 mice. CD4+ T cells from these mice were enriched by negative selection using magnetic beads (Miltenyi Biotec, 130-104-453). APCs were obtained by irradiating DO11.10 splenocytes at 2000 rad. Differentiation of these naive CD4 T cells into the Th1 phenotype was induced by culturing them as follows: 200,000 CD4+ T cells and 2 million APCs in 0.3 μM OVA 323-339 Cells were co-cultured for 3 days in a complete RPMI containing (GenScript), 5 ng / mL IL12 (PeproTech), and 10 μg / mL anti-IL4 mAb (R&D Systems). Cells were then harvested, washed, and counted. A total of 2 million Th1 DO11.10 T cells were intravenously injected into BALB / c recipients. One day after T cell transfer, recipients were subcutaneously immunized with 100 μg of OVA in CFA (Sigma-Aldrich). On day 11, heat-aggregated OVA (HOA) was injected into the left paw pad of mice, and paw pad thickness was measured daily until day 18. Mice were then sacrificed, their paw pads were removed, fixed in 10% (wt / vol) formalin solution (Sigma), embedded in paraffin, sectioned to 20 μm, and stained with toluidine blue (Harvard Medical School Rodent Histology Core Facility). Stained sections were imaged at 4× and 10× magnification. Popliteal lymph nodes were also collected, and cells were restimulated in vitro with 1 mg / mL OVA in complete RPMI for 3 days for IFN-γ production. IFN-γ was measured using the mouse IFN-γ ELISA set (BD Biosciences, 555138) according to the manufacturer's protocol. Serum was also collected for ELISA assays at the D18 endpoint to test for anti-OVA and anti-OVA 323-339 Antibody response was measured. 96-well plates were treated with 10 μg / mL OVA or GFP-OVA. 323-339 (GGG-OVA 323-339The plates were coated overnight in PBS at 4°C with the GFP-LPETGG (sequence number 43) protein (produced by sol-tagging) and incubated in blocking buffer (0.05% Tween 20 + 2% BSA in PBS) before adding serum samples. Incubation with the tested serum was 3 hours at room temperature. The plates were washed four times with PBS and incubated with goat anti-mouse IgG-HRP (Southern Biotech) at a ratio of 1:10,000 in blocking buffer for 1 hour, and developed with 3,3',5,5'-tetramethylbenzidine (TMB) liquid substrate reagent (Sigma). The reaction was stopped with 1 M HCl and the absorbance was read at 450 nm.

[0132] CD8 T cell adoptive transfer and loading of OTI Spleen and lymph nodes in OTI Rag2 - / - Collected from mice. OTI Rag2 - / - CD8+ T cells were enriched by negative selection using magnetic beads (Miltenyi Biotec, 130-095-236) and labeled with Violet CellTrace according to the manufacturer's protocol. 500,000 CD8+ T cells were intravenously transferred into CD45.1+ mice. 20 μg of VHH MHCII -OTI or VHH MHCII -ORF8 infusion was administered on the day following adoptive transfer. Mice were loaded with 25 μg of OTI peptide in CFA(Sigma) on day 10, and then sacrificed 5 days later for analysis. Spleens, iLN, and blood were harvested, and splenocytes were analyzed by flow cytometry.

[0133] Two million splenocytes were plated in 96-well round-bottom plates and treated with a cell stimulation mixture (eBioscience) and brefelzin A (eBioscience) for three days at 37°C in a complete RPMI [RPMI1640, 10% (vol / vol) heat-inactivated FBS, 50 μM β-mercaptoethanol, 100 U / mL Pen / Strep, 1× Gibco MEM non-essential amino acid solution (Life Technologies), 1 mM sodium pyruvate, 1 mM HEPES]. The supernatant was collected and used for ELISA to measure interferon-gamma (IFNγ) production. IFNγ was measured using a mouse IFN-γ ELISA set (BD Biosciences, 555138) according to the manufacturer's protocol.

[0134] VHH MHCII - Repeated infusion of OB1 OB1 is a 17-mer B-cell epitope derived from OVA. 20 μg of VHH was administered to C57BL6 / J recipient mice. MHCII -OB1, equimolar amounts of OVA protein, or PBS were intravenously injected on day 0. Subsequent boosts were performed on days 7 and 14. Serum samples were collected before immunization and 7 days after the final boost. For OVA-specific and OB1 peptide-specific ELISAs, 96-well plates were coated overnight at 4°C in PBS with 10 μg / mL of OVA or GFP-OB1 protein and incubated in blocking buffer (0.05% Tween 20 + 2% BSA in PBS) before adding serum samples. Incubation with the tested serum was 3 hours at room temperature. Plates were washed four times with PBS and incubated with goat anti-mouse IgG-HRP (Southern Biotech) at 1:10,000 in blocking buffer for 1 hour, and developed with 3,3',5,5'-tetramethylbenzidine (TMB) liquid substrate reagent (Sigma). The reaction was stopped with 1M HCl, and the absorbance was measured at 450nm.

[0135] EAE model in HLA-DR4-IE-transgenic C57BL / 6IAb null mouse DR4-IE mice were given 400 μg of emulsified human PLP in CFA. 175-192 (hPLP 175-192 Mice were subcutaneously immunized with ) . Mice also received 300 ng of pertussis toxin intravenously on days 0 and 3. On day 7, mice received 400 μg of emulsified hPLP in incomplete Freund's adjuvant (IFA). 175-192 A second subcutaneous boost was administered by [method]. Mice were weighed and scored daily starting on day 7 after immunization. The clinical scoring system was implemented similarly to the EAE model of C56BL / 6J mice. On day 1, when mice reached a clinical score of 3, 20 μg of anti-human MHCIIVHH(VHH) with an unrelated peptide control was administered. hMHCII ) or 20 μg of VHH hMHCII -hPLP 175-192 Either one, 20 μg of VHH hMHCII - It was administered intravenously after being mixed with DEX. Spinal flow cytometry results were described above.

[0136] statistical methods All data represented at least two independent experiments. All statistical analyses were performed using Prism 6. The statistical methods used are indicated in the corresponding legend for each figure. Statistically significant differences are indicated by asterisks as follows: *p<0.05; **p<0.01; ***p<0.001.

[0137] result VHH MHCII -MOG 35-55 A single dose provides durable protection against the induction of experimental autoimmune encephalomyelitis (EAE). In this application, IA b and IA d Alpaca-derived single-domain antibodies that recognize a broad range of mouse class II MHC molecules (i.e., VHH) MHCIIThe creation and characterization of VHH were described. This VHH was engineered to possess the saltase recognition motif LPETGG (SEQ ID NO: 43) to allow site-specific ligation to antigen peptides and small molecules modified by at least one preferably exposed glycine residue(s) (Figure 1A). Antigen peptides conjugated to VHH in this manner are listed in Table 4. The purified VHH-peptide adducts were characterized by LC-MS (Figures 1B and 8) to verify their identity, homogeneity, and purity.

[0138] [Table 4]

[0139] MOG in an inflammatory state, i.e., in the presence of complete Freund's adjuvant (CFA) and pertussis toxin (PTX) 35-55 Immunization of C57BL / 6 mice with induced experimental autoimmune encephalitis (EAE) with multiple sclerosis-like conditions within 10-14 days. MOG delivered to MHCII+APCs in a non-inflammatory state. 35-55 Prior administration of was predicted to interfere with the induction of EAE. To determine possible doses of VHH-peptide adducts that could interfere with the onset and severity of symptoms, 20 μg of VHH MHCII -MOG 35-55 Three doses of the adduct were administered intravenously (iv) seven days prior to disease induction. This treatment completely suppressed the induction of EAE, but the same amount of VHH conjugated to an unrelated peptide was suppressed. MHCII (VHH MHCII -OVA 323-339 ) or unrelated specificity VHH(VHH GFP MOG connected to ) 35-55 Mice treated with the peptide progressed to EAE (Figure 1C). 20 μg of VHH MHCII -MOG 35-55Even a single injection achieved complete protection (Figures 1D and 1E). Therefore, this dose was used in all subsequent experiments. Flow cytometry of CD4+ lymphocyte infiltrates recovered from the spinal cords of diseased mice 15–18 days after immunization and protected mice 30 days after EAE induction was consistent with the observed disease score: diseased mice showed a significant influx of IL17 and IFNγ-producing CD4+ T cells and some Foxp3 + CD4 + Regulatory T cells were shown (Figure 1F and 9A-9E). VHH preceded the induction of EAE. MHCII -MOG 35-55 H&E and Luxor Fast Blue staining of spinal cord sections from mice treated with VHH showed preservation of myelination and less immune cell infiltration, unlike samples from animals that progressed to EAE (Figure 1G and 1H). The results suggest that VHH administered prior to the induction of EAE is beneficial. MHCII -MOG 35-55 It was indicated that a single 20 μg dose of the adduct is sufficient to prevent the onset of the disease.

[0140] VHH MHCII -MOG 35-55 To explore the durability of protection induced by VHH, MHCII -MOG 35-55 MOG 35-55 The / CFA / PTX cocktail was administered 1 or 2 months prior to induction of EAEs. Delayed onset of EAEs was observed, even if not complete suppression (Figures 1I, 10A, and 10B). Free VHH was estimated to be <0.5 hours. MHCII -MOG 35-55 Despite its short cyclic half-life, VHH MHCII -MOG 35-55 This provides extended protection. To explore the degree of resistance to EAE, protected mice were subjected to MOG in the presence of PTX 37 days after the initial EAE exposure. 35-55The second administration of / CFA was reloaded. Despite this second highly inflammatory load, once protected, the mice did not show signs of developing EAE (Figures 1J, 11A, and 11B). Therefore, the tolerance induced by a single dose of VHH MHCII -MOG 35-55 provides sustained protection even weeks after its administration.

[0141] Spleen CD11c+ DC are APCs associated with the induction of antigen-specific tolerance To explore the possible mechanisms of induction mediated by tolerogenic VHH MHCII , VHH MHCII -Alexa647 (Figures 12A and 12B) was generated and injected into MHCII-GFP mice (i.v.) to follow the in vivo distribution of VHH MHCII -Alexa647. These mice have a targeted gene replacement encoding an I-A b -GFP fusion. It replaces the endogenous I-A b locus, ensuring that all class II MHC+ cells express GFP. 1.5 hours after injection, VHH MHCII -Alexa647 is captured by splenic and circulating MHCII-GFP+ cell populations (Figures 2A and 13). The fluorescent VHH MHCII adducts were captured by B cells and DC subsets including splenic CD8a+ DC, CD4 - classical DC (cDC), and CD4+ cDC, rather than plasmacytoid DC (Figure 13).

[0142] VHH MHCII -MOG 35-55 Intravenous injection, rather than subcutaneous or intraperitoneal injection, protected from the induction of EAE (Figure 14). This hinted at the role of the spleen or bloodstream as the site of tolerance induction. Thus, 20 μg of VHH MHCII -MOG 35-55 (Figures 2B and 15A - 15C) was injected into mice (i.v.), and 1 week later, splenocytes and whole blood were harvested as a source of donor cells. Then, naive mice were given VHH MHCII -MOG35-55 Received 20 million unfractionated splenocytes or peripheral blood mononuclear cells (PBMCs) from treated animals. One day after cell transfer, MOG 35-55 +PTX in CFA was administered to induce EAE (Figs. 2B and 15A-15C). VHH MHCII -MOG 35-55 There was a significant reduction in the mean clinical EAE score in mice that received splenocytes from VHH MHCII -MOG 35-55 -treated mice (Figs. 2B and 15A-15C). Macrophages and CD8 T cells were depleted in vivo by administration of the corresponding depletion antibodies: anti-CFS1R antibody and anti-CD8α antibody, respectively (Figs. 2C, 16A, and 16B). To deplete DCs, diphtheria toxin (DTX) was administered to CD11c-DTR (diphtheria toxin receptor) mice (Figs. 2C, 16A, and 16B). To examine the possible involvement of B cells, VHH MHCII -MOG 35-55 was administered to μMt mice lacking B cells. Only the depletion of CD11C+ DCs reduced the protective measures provided by VHH 35-55 -MOG CD11c -MOG 35-55 combinations provided a moderate level of protection from EAE induction (Figs. 2D and 17), consistent with the results from the depletion of CD11c+ cells. VHH MHCII -MOG 35-55 -treated Batf3- / - mice remained resistant to EAE induction. Thus, in this context, CD8α+ DCs do not apparently contribute to the set of tolerogenic APCs (Fig. 18).

[0143] More VHH MHCIITo determine whether delivery by VHH could similarly induce leniency, MHCII -MOG 17-78 This was created and used to treat mice 7 days prior to the load. VHH MHCII -MOG 17-78 This also protected against the induction of EAE (Figures 2E and 2F).

[0144] VHH MHCII -MOG 35-55 The administration of MOG 35-55 This induces a surge in the proliferation of specific CD4+ T cells, followed by depletion. VHH against T cells with defined antigen specificity MHCII -MOG 35-55 To investigate the impact of the adduct, 2D2TCR transgenic mice were used in IA b -MOG 35-55 Monoclonal CD4 that recognizes complex + It was used as a source of T cells. Congenically marked Violet CellTrace-labeled 2D2 CD45.2+CD4+ T cells were transferred into CD45.1 recipients, and then, after 1 day, VHH MHCII -Peptide adducts were injected (iv). The number of 2D2 cells in the spleen, inguinal lymph nodes (iLN), and blood was tracked for 10 days. The absolute number of 2D2 cells recovered from the spleen, iLN, and blood, as well as the number of CD4+ cells determined by whole-body imaging using non-invasive positron emission tomography (PET) imaging, were used to determine the VHH MHCII -MOG 35-55 In mice treated with VHH, 2D2 CD4+ T cells underwent an initial surge of expansion, followed by a contraction 5 days after injection (Figures 3A and 19). This disappearance occurred after several cell divisions, because all recovered 2D2 CD4 T cells had experienced the antigen and were dividing as shown by Violet CellTrace dilution (Figure 3B). MHCII -MOG 35-55 MOG in equimolar amounts with respect to the adduct 35-55 Delivery of VHH resulted in division of at most ~5% of 2D2 T cells. Therefore, VHHMHCII Antigen delivery mediated by [antigen delivery agent] clearly enhances its presentation (Figure 3B).

[0145] CD4 T cells of MOG-specific 2D2 upregulate co-inhibitory receptors upon administration of VHH MHCII -MOG 35-55 To confirm these results, the transcriptome of recipient 2D2 T cells of VHH -MOG was examined. CD4 T cells of 2D2 at different division stages were sorted (Figure 3B) and RNAseq analysis was performed. Injection of VHH MHCII -MOG 35-55 upregulates co-inhibitory receptor transcripts and negative regulatory transcription factors. LAG3 transcripts are prominent in both magnitude and significance (Figures 3C, 3D, and 20A - 20E). At the protein level, these 2D2 T cells also showed higher levels of apoptosis and exhaustion markers such as PD1 and LAG3 but not Tim3, Fas / CD95, or LAP (Figures 3E and 21). On the 3rd day after injection, CD4 T cells of 2D2 in VHH MHCII -MOG 35-55 recipients, although rarely, remained CD44+ and did not downregulate CD62F (Figure 21). When LAG3- / - mice were treated with a single dose of VHH MHCII -MOG 35-55 and then challenged with EAE induction, there was a significant delay but protection was lost, while PD1- / - mice were still tolerized by VHH MHCII -MOG 35-55 (Figure 3F). Deletion of LAG3 in 2D2 TCR transgenic mice leads to spontaneous EAE. Since both activated effector T cells and Tregs express LAG3, it was evaluated whether the increase in regulatory T cells contributes to the tolerance imposed by VHH MHCII -MOG 35-55 MHCII -MOG 35-55 To evaluate whether the increase in regulatory T cells contributes to the tolerance imposed by VHH

[0146] VHH MHCII -MOG 35-55The administration of MOG 35-55 It induces specific regulatory CD4T cells. VHH MHCII -MOG 35-55 To uncover the role of regulatory T cells in tolerance mediated by , Tregs were eliminated in Foxp3-DTR mice by DTX administration (Figures 22A-22D). Treated mice lost Tregs and were no longer protected from EAEs, and VHH MHCII -MOG 35-55 It demonstrated the contribution of tolerance imposed by (Figures 22A-22D). VHH MHCII -MOG 35-55 The administration of FoxP3+MOG 35-55 It increases the number of specific Tregs (Figures 22A-22D). In addition to the increase in the number of Tregs, the expression of fatigue markers is also VHH. MHCII -MOG 35-55 It increased with the administration of [the substance]. Finally, mice that received 2D2 T cells were given MOG 35-55 / CFA was loaded on the 10th day. VHH MHCII -MOG 35-55 The 2D2 T cells of the mice that received the treatment did not respond, but VHH MHCII -OVA 323-339 T cells in 2D2 mice injected with VHH proliferated robustly (Figure 3G). MHCII -MOG 35-55 This clearly demonstrates the antigen specificity of tolerance induction.

[0147] VHH MHCII - Antigen adducts also act in an antigen-specific manner in other models of autoimmunity. Next, we tested the ability of VHH-antigen adducts to interfere with other autoimmune conditions. In type 1 diabetes (T1D), we used an aggressive BDC2.5 T cell adoptive transfer model that mimics the destruction of β cells mediated by autoreactive T cells. Transgenic CD4 T cells with the BDC2.5 T cell receptor recognize pancreatic β cells and can be activated ex vivo by the mimotope p31. In NOD / SCID mice, such activated BDC2.5 T cells induce hyperglycemia within 8 days after transfer. MHCIIThe cells were conjugated (Figure 8). NOD / SCID mice that received activated BDC2.5 splenocytes were given saline and 20 μg of VHH after 1 day. MHCII -MOG 35-55 , or 20 μg of VHH MHCII -Mice treated with either saline or p31 developed hyperglycemia by day 8 after transfer (Figure 4A). MHCII Only mice treated with -p31 maintained normal blood glucose levels throughout the experimental period (Figures 4A and 23A-23C). VHH MHCII -p31 treated mice had fewer BDC2.5 CD4 T cells in their pancreas and secondary lymphoid organs (Figures 23A-23C). The pancreatic islets of protected mice remained intact (Figure 4B). VHH MHCII -p31 was also administered to mice on day 5 after transfer of BDC2.5 T cells activated by this gene, and a mild protective effect was observed (Figure 22C). Whole insulin protein also showed a mild protective effect. MHCII It was attached to (Figure 24).

[0148] Arthritis is present in BALB / c recipients, OVA 323-339 Th1 DO11.10 T cells that recognize the ex vivo activation of the DO11.10 T cells can be induced by intravenous transfer, followed by paw-plantar injection of OVA / CFA emulsion 1 day later and loading with thermoaggregated ovalbumin (HAO) 10 days later (Figure 4C). Mice were then monitored for the development of arthritis by measuring paw-plantar thickness and by histological evaluation on day 7 after HAO loading. MHCII -OVA 323-339 Prior administration of reduced arthritis caused by exposure to ovalbumin, but VHH MHCII -MOG 35-55 It had no effect (Figures 4C and 25A-25E). VHH MHCII -OVA 323-339 Mice treated with VHH also showed fewer signs of cartilage destruction (Figure 4D). MHCII -OVA 323-339Immune cells obtained from the popliteal lymph nodes of mice treated with OVA did not produce IFNγ when stimulated ex vivo with OVA (Figures 25A-25E). Perhaps not surprising, VHH MHCII -OVA 323-339 Serum from mice treated with [the agent] showed lower levels of anti-OVA and anti-OVA [the agent]. 323-339 They also possessed IgG1 antibodies (Figures 25A-25E).

[0149] These combined results are VHH MHCII - We have confirmed the ability of antigen adducts to reduce the damage inflicted by activated, self-reactive CD4 T cells. The underlying mechanism(s) should be conserved among mouse MHC haplotypes.

[0150] VHH MHCII - Antigen adducts also suppress CD8-mediated T and B cell responses. CD8 T cell response is VHH MHCII - To determine whether it is affected by the administration of antigen adducts, OVA-derived CD8 T cell epitope SIINFEKL(H-2K b OTI peptides constrained by VHH MHCII It was attached to (Figure 8). The mouse then used congenically marked OTI T cells, followed by VHH MHCII -OTI or VHH MHCII - ORF8 injections (with or without adjuvant) were administered one day later (Figure 4E). The ORF8 epitope derived from MCMV was H-2 b In mice, it was recognized by CD8 T cells and served as a control. Reloading of recipients with OVA / CFA on day 10 after transfer did not activate any remaining OTI T cells (Figure 4F). B cell response was VHH MHCII -To explore whether the administration of antigen adducts has a similar effect, VHH MHCII The cells were modified with a B cell-specific OVA-derived epitope (OB1) (Figure 8). VHH was then applied to C57BL / 6J recipients. MHCIIThree consecutive injections of OBI did not induce an IgG antibody response to either intact OVA protein or OB1 peptide (Figures 4G and 4H), however, mice that received equimolar amounts of free OVA protein immediately produced such antibodies.

[0151] VHH MHCII -MOG 35-55 and VHH MHCII - Simultaneous delivery of dexamethasone increases therapeutic efficacy. Furthermore, regarding EAE, VHH was administered to mice that were already symptomatic. MHCII -MOG 35-55 The impact of administration was explored. VHH was administered to mice that developed a clinical score of 1 (tail drooping). MHCII -MOG 35-55 The injection stopped the progression of EAE in 9 out of 16 mice (Figures 5A and 26). The overall condition of the remaining 7 mice was, seemingly unrelated to EAE, VHH MHCII -MOG 35-55 The condition rapidly worsened after the injection (e.g., tremors; reduced activity). In fact, VHH MHCII -MOG 35-55 Approximately 40% of the mice that received the treatment died on the day after infusion, without correlation to the clinical scores of mice prior to the injection. This was due to a cytokine storm induced by targeted delivery of the antigen to an already inflamed environment, as indicated by elevated levels of IL-6 and TNFα (Figure 5C).

[0152] The polyclonal nature of the evoked T cell response and the rather superficial clinical scoring system imply heterogeneity of the diseased cohort. This is why VHH MHCII -MOG 35-55 This could explain why not all animals that received the treatment responded similarly. Then, we investigated whether it might be possible to avoid cytokine storms by co-delivering immunosuppressive drugs via autohydrolyzable hydrazone linkers. MHCII Dexamethasone, an immunosuppressive corticosteroid attached to a cytometer, was delivered to class II MHC+ cells (VHH).MHCII -DEX; Figures 5B and 27). 20 μg of VHH MHCII -MOG 35-55 and 20 μg of VHH MHCII Mice receiving the combination dose of -DEX survived and returned to lower clinical EAE clinical scores without obvious side effects (Figure 5D). The improvement in clinical scores was reflected in a reduction of infiltrating CD4 T cells in the spinal cord (Figure 28). The observed benefit was VHH MHCII -The equivalent of DEX in the form of a DEX adduct was required at most 0.5 μg. On the other hand, free DEX provided protection only when administered at a 200-fold higher dose of 100 μg in an inoculation (Figures 29A and 29B). The therapeutic range extended to animals with EAE scores of 2 or 3, and all of these were treated by halting disease progression without side effects, resulting in VHH MHCII -MOG 35-55 and VHH MHCII - Responded to co-administration of DEX. Affected mice further showed a significant improvement in disease score (Figures 5E, 5F, and 28). Surprisingly, VHH MHCII The route of administration is important because only intravenous delivery of -DEX, and not subcutaneous or intraperitoneal delivery, can provide prophylactic protection (Figure 14).

[0153] Anti-human MHCIIVHH(VHH) in humanized mouse models of autoimmune diseases hMHCII )- Antigen adduct VHH(VHH) recognizes a wide range of human class II MHC molecules. hMHCII We developed a VHH. This VHH was prepared in a format ready for immediate use in saltase and modified with several autoantigens of human origin (Figure 6A-6C).

[0154] Human MOG 97-108 Peptide (TCFFRDHSYQEE (SEQ ID NO: 53)), hPLP 175-192 Peptide (YIYFNTWTTCQSIAFPSK (SEQ ID NO: 42)), and DEX to VHH hMHCIIThe adducts were attached to (Figure 6B). The effectiveness of these simultaneously delivered adducts was demonstrated by HLA-DR4-IE-transgenic C57BL / 6IAb, which expresses a transgenic hybrid MHC-II molecule composed of the peptide-binding domain of human HLA-DR4 and the membrane-proximal domain of mouse IE (DR4-IE), instead of mouse MHC-II. ヌル Tested in mice. VHH hMHCII -MOG 97-108 VHH reduced the EAE clinical score in mice 20 days after administration (n=1, Figure 6B). hMHCII -OVA 323-339 We used as a negative control (n=2, Figure 6B).

[0155] In RA patients, a high frequency of autoantibodies targets post-translationally modified antigens such as fibrinogen with modified arginine residues in citrulline. Therefore, VHH hMHCII Citrullinated Fibα 79-91 Modified by (QDFTNCitINKLKNS (SEQ ID NO: 50), Figure 6C). This illustrates the flexibility of a chemical-enzymatic approach, which, unlike genetic methods, immediately allows for the incorporation of non-native or post-translational modified amino acids in a site-specific manner.

[0156] VHH MHCII To explore the mechanisms of tolerance induction mediated by VHH, MHCII - Build Alexa647, VHH MHCII The in vivo distribution of the adduct was tracked. 20 μg of VHH MHCII -Alexa647 was administered intravenously to class II MHC-GFP mice. 1.5 hours after injection, VHH MHCII -The majority of Alexa647 cells were captured in vivo by a population of spleen MHCII-GFP+ cells (Figure 7A). VHH MHCII The adducts were delivered to multiple subsets of DCs, including spleen CD8α+ DCs, CD4-negative classical DCs, and CD4+ classical DCs (Figure 7B).

[0157] In the EAE model, VHHMHCII -MOG 35-55 Intravenous injection, rather than subcutaneous or intraperitoneal injection, provided protection from the induction of EAE (Figure 14). Then, 20 mg of VHH MHCII -MOG 35-55 The drug was injected into mice (iv), and one week later, their splenocytes were harvested, and a total of 20 million splenocytes were transferred into a cohort of recipient mice. EAE was induced one day after transfer (Figure 7C). There was a significant reduction in the mean clinical EAE score, and even unfractionated splenocytes showed VHH MHCII -MOG 35-55 We demonstrated that tolerance mediated by this gene is induced (Figure 7C). Depletion experiments were performed to target a subset of spleen APCs. B cells, macrophages, and dendritic cells were depleted by administering the corresponding depletion agents, anti-CD20 antibody, anti-CFS1R antibody, and diphtheria toxin (DTX), to CD11c-DTR (diphtheria toxin receptor) mice (Figure 7D). Three different VHHs were identified that likely target different but overlapping subsets of APCs: CD11b (mostly present on macrophages), CD11c (mostly present on dendritic cells), and Igk (on B cells). These VHHs were expressed in a format readily available for use with saltase, and GGG-MOG was used with saltase. 35-55 Site-specific labeling was performed using VHH (Figure 8). CD11c -MOG 35-55 Only provided a moderate level of protection from EAE induction. This suggested a role for CD11c+ cells as toxicogenic APCs (Figure 7E).

[0158] Consideration Induction of antigen-specific tolerance is an ambitious goal in the treatment of autoimmune diseases. This is a particularly high hurdle to clear when considering the pathology and the presence of pre-existing autoimmunity in the diagnosis. Autoimmune destruction of target cells is already underway before symptoms appear. Therefore, treatment must address not only pre-existing autoimmunity but also the possibility of epitope spreading beyond the initial injury. Unless a predisposed population can be uniquely identified, and the risk of inducing unnecessary side effects is relatively low, any type of prophylactic treatment will have limited value.

[0159] In addition to suppressing inflammation, extensive immunosuppression is a bulwark in treating autoimmune conditions, which are often accompanied by an increased risk of infectious disease. While antibiotic treatment can at least partially mitigate this drawback, the search for more targeted approaches to blunt undesirable immune responses remains a priority. Most autoimmune diseases are mediated by T cells; professional antigen-presenting cells are involved in T cell activation. When antigen-presenting cells (APCs) take up antigens in an inflammatory environment, upregulation of costimulatory molecules and production of an appropriate mix of cytokines contribute to T cell activation. Tolerogenic dendritic cells lack such costimulatory signals, and consequently, antigen presentation in non-inflammatory states promotes unresponsiveness or tolerance. This concept has driven the search for tolerogenic dendritic cells. Dendritic cells can be subdivided into subsets with unique functional capabilities. For example, the ability to participate in antigen cross-presentation is a characteristic attributed almost entirely to the DC1 subset. The identification of surface receptors involved in antigen uptake has led to the identification of DEC205, DC-SIGN, and Clec9a, which are particularly relevant to the entry of antigens into the cross-presentation pathway. While these receptors are primarily sought after as strong inducers of desirable immune responses, such as antitumor responses, their ability to induce regulatory T cells as a means of reducing unnecessary responses is considered equally important.

[0160] This rather narrow focus on dendritic cells casts a shadow over previous work targeting antigens to class II MHC products expressed on all antigen-presenting cells by creating anti-class II MHC antibodies conjugated to autoantigens. Ultimately, the class II MHC peptide complex is the battle command for the CD4 T cell compartment. For this reason, the strategy of delivering autoantigens to class II MHC-positive cells under a non-inflammatory state is effective, even though it does not distinguish between various APC subsets. Ideally, from both manufacturing and application standpoint, the intervention should be antigen-specific and as simple as possible.

[0161] This data is from MOG that recognizes Class II MHC products. 35-55 We have established that modified VHH can protect mice from the induction of EAE. A single injection of 10 micrograms of the adduct provided protection that lasted for at least two months after administration of the nanobody-peptide adduct. The same VHH in animals already exhibiting symptoms of EAE (score 1, 2, or 3) MHCII -MOG 35-55 Administration of adducts halted progression and even partially reduced the severity of symptoms. When animals with EAE symptoms were treated, only a subset responded, while the rest showed rapid exacerbation, which could be attributed to a cytokine storm, followed by death. In symptomatic animals, an inflammatory environment was already present, and VHH to APC MHCII -MOG 35-55 The delivery of the adduct only added fuel to the fire. To overcome this acute response, VHH MHCII -Dexamethasone adducts were delivered simultaneously. This dramatically improved survival without causing death.

[0162] Administration of nanobody-peptide adducts in the presence of anti-CD40 and poly(dIdC) adjuvants significantly enhanced the antibody response to them. Administration in the context of chronic inflammatory responses was VHH MHCII -This would only be possible if appropriate countermeasures are available, as in the case of dexamethasone adducts.

[0163] The pharmacokinetic properties of nanobodies make them attractive for the construction of antibody-drug conjugates (ADCs). Nanobodies have a much shorter circulatory half-life than full-size antibodies, and thus minimize systemic exposure to toxic compounds. Their targeting properties are excellent, ensuring that once at a site, a suicide linker will release the payload at the predominantly intended site. ADCs based on full-size immunoglobulins circulate continuously for up to several weeks, releasing the payload directly into the bloodstream through hydrolysis of the linker to which the drug is attached. Thus, VHH MHCII - dexamethasone adducts have the desired excellent targeting properties, as verified by non-invasive imaging, short circulatory half-life, and ease of modification. VHH MHCII The cellular targets recognized by VHH encompass all class II MHC-positive cells. Even if the APCs that are responsible for inducing tolerance and eliciting the cytokine storm are different, the class II MHC-based targeting approach will self-evidently cover both. Nanobody-drug adducts have not yet found a wide range of applications of their full-size counterparts, but these data indicate that this is an opportunity that should not be overlooked.

[0164] Regarding the mechanism underlying the remarkable ability of anti-class II nanobodies to induce tolerance to the attached payload, many possibilities can be excluded based on the responses seen in knockout mice or depletion of certain sets of cells. Whether a single type of APC is tolerogenic when targeted in a non-inflammatory state and can elicit a strong response when the antigen is encountered in an inflammatory environment remains unknown.

[0165] Example 2. VHH MHCII - antigen fusion protein. Single-domain antibody fragments (nanobodies or VHH) that bind to MHC class II antigens (VHH MHCII) was isolated and characterized by nanomolar affinity. To adapt this vaccine platform for SARS-CoV-2, VHH MHCII A recombinant protein consisting of a fusion between the SARS-CoV-2 receptor binding domain and the SARS-CoV-2 receptor binding domain was created (VHH MHCII -spike RBD (Figures 31A-31B).

[0166] VHH MHCII -spike RBD To confirm its immunogenicity, C57BL / 6J mice were spiked with 20ug of adjuvant (polydIdC and anti-CD40 monoclonal antibody). RBD VHH with adjuvant MHCII -spike RBD Alternatively, the animals were primed intraperitoneally with the adjuvant alone, and subsequently boosted with the same vaccine postpriming as directed (Figure 31C). Serum was collected from all animals on days 32 and 150, and IgG titers were recombinant SARS-CoV-2 spike titers. RBD This was determined by ELISA (Figure 31D). VHH MHCII -spike RBD Immunization by the fusion resulted in spikes exhibiting various immune responses. RBD In comparison, it consistently produces antigen-specific IgG with high titers. Unsurprisingly, the titer of circulating IgG drops after the 32-day sample, but even at 150 days, it spikes. RBD The immediately detectable titer for VHH MHCII -spike RBD or spikes RBD It persists in all mice that received the treatment. Even on day 150, VHH MHCII -spike RBD The antibody titers of mice that received the fusion still show spikes. RBD This surpasses that of the cohort alone. Predictably, serum or preimmune serum obtained from mice that received only the adjuvant showed a significant anti-spike. RBDNo antibody production was observed. Immunoglobulin subclass analysis revealed evidence of class switching, as high levels of IgA, IgG1, and IgG2b were detected 32 days after the initial dose (Figure 31E). VHH MHCII -spike RBD A particularly prominent, stronger IgA response evoked by the fusion is observed, which would provide important mucosal protection against respiratory tract infections. A much stronger IgG1 response corresponding to complement-mediated lysis in opsonized cells is also evident.

[0167] Next, the functional correlation of serological responses was evaluated by assaying the serum neutralizing capacity against pseudotyped vesicular stomatitis virus (VSV) by the SARS-CoV-2 spike glycoprotein. MHCII -spike RBD Serum obtained from mice immunized by the fusion agent is spike RBD It outperformed those from mice immunized only with (Figure 31F). The latter showed considerable inter-mouse variability, but VHH MHCII -spike RBD The response from mice that received the test was both stronger and more consistent across individual mice, highlighting the importance of direct targeting of antigen-presenting cells (APCs). This is consistent with the level of humoral immune response detected.

[0168] A robust CD8+ T cell response is crucial for the clearance of virus-infected cells. Therefore, mice were subjected to VHH in the presence of adjuvants. MHCII -spike RBD or spikes RBD Cells were immunized with either single dose (Figure 32A). After one week, splenocytes were harvested and the ELISpot assay was performed to identify peptides that could induce IFNγ production in vitro as a surrogate measure of a specific T cell response. Using overlapping 15-mer peptides, five peptides (42, 47, 48, 49, and 50) that induced a strong response were identified (Figures 32B-32D).RBD Mice immunized by VHH only allowed some of the peptides to be absorbed. MHCII -spike RBD It recognizes with a much weaker signal than immunized cells and directs a smaller number of cells to secrete IFNγ. These results also direct at least two stimulant regions. Interestingly, peptides 47-50 spike outside of known mutations in circulating SARS-CoV-2 variants. RBD Belongs to the domain. Selected spikes RBD Cytokine secretion assays for IFNγ, IF6, IF2, and TNFα by co-culturing peptides (42, 47, 48, 49, and 50) with splenocytes were performed at VHH MHCII -spike RBD This further supports the excellent T cell response induced by (Figure 32E).

[0169] To distinguish CD4+ and CD8+ T cells as sources of IFNγ, flow cytometry assays were performed, followed by intracellular cytokine staining. Most inflammatory cytokines were observed to arise from the CD8+ T cell response based on the incubation of splenocytes with a mixture of peptides 42, 47, 48, and 49 (Figure 32E). Therefore, VHH MHCII - Antigen adducts enhance cross-presentation and spike RBD This can induce an effective CD8+ T cell response against [the target].

[0170] Furthermore, a strong CD8+ T cell response is observed with only a single immunization, occurring within 7 days after immunization. This is VHH MHCII -spike RBD This strongly suggests that this can provide protective immunity against SARS-CoV-2 infection, because the immunized cohort demonstrates a relatively rapid T-cell response while waiting for a slower humoral response to appear. Together, these data point to the superiority of directly targeting APCs via class II MHC.

[0171] While immunization with more than three doses is likely impractical on the scale of the SARS-CoV-2 (COVID-19) pandemic, a strong CD8+ T cell response may still be possible with single-dose immunization. Therefore, if animals are VHH MHCII -spike RBD Experiments were conducted in which participants received two sequential doses (Figure 33A). Serum immunoglobulins were tracked on days 7, 14, and 21 after immunization (Figure 33A). VHH MHCII -spike RBD Total IgG levels in the cohort peaked on day 7 after the second dose, and these levels persisted until day 21. VHH MHCII -spike RBD Animals that received single doses and adjuvants of the preparation were VHH at all time points. MHCII -spike RBD It demonstrated lower efficacy than a double dose (Figure 33A). Isotype switching was also investigated (Figure 33B). Then, serum from immunized animals was used with spikes containing K417T, E484K, and N501Y mutations. RBD The recognition of this was tested. The serum obtained on the 14th day contained this variant spike. RBD It efficiently recognized (Figure 33C). Therefore, serum from these animals was tested for neutralization of pseudotyped VSV with a diverse set of spike variants. VHH MHCII -spike RBD Serum from mice immunized with two doses effectively neutralized all tested variants (Figure 33D).

[0172] The experiments described in Figures 31-33 all relied on intraperitoneal delivery of the vaccine preparation. For human use, intramuscular delivery is preferred. Needle-free approaches, such as intranasal delivery, would be a highly desirable alternative to injection. Therefore, we investigated whether different delivery routes would lead to different levels of antibody production when administered in two doses with a two-week interval (Figure 34A). VHH MHCII -spike RBDThe preparation was delivered intraperitoneally (ip), intramuscularly (im), or intranasally (in). ip and im delivery induced an IgA response, but intranasal delivery did not (Figure 34B). However, serum IgG production appeared to be independent of the vaccine delivery route. All three delivery routes produced similar levels of total anti-spike. RBD It produced IgG (Figure 34B).

[0173] Then, VHH MHCII -spike RBD We explored whether the vaccine preparations could withstand room temperature storage and freeze-drying, and whether they could produce a final "dried" product at room temperature without loss of potency. All storage methods tested produced comparable levels of total IgG, in addition to other Ig isotypes previously observed (Figure 34C).

[0174] Another key consideration is VHH MHCII -spike RBD The question is whether the vaccine will work well for all age groups, especially for older individuals. Therefore, VHH MHCII -spike RBD The vaccine was tested in aging mice (72 weeks old, equivalent to the age of humans 56-69 years), and it spiked RBD A robust total antibody response to [the target] was demonstrated (Figure 34D).

[0175] In this Application, all publications, patents, patent applications, publications, and database entries (e.g., sequence database entries) mentioned in, for example, the sections on background, summary, detailed description, examples, and / or references are incorporated hereby by reference in whole, as if each individual publication, patent, patent application, publication, and database entry were incorporated hereby by reference specifically and individually. In case of inconsistency, the Application that incorporates either definition here shall prevail.

[0176] Equivalents and range Those skilled in the art will recognize, or at best verify, many equivalents of the embodiments described herein by conventional experimental procedures. The scope of this disclosure is not intended to be limited to the foregoing, but rather as submitted in the attached claims.

[0177] Conversely, unless otherwise indicated or evident from the context, articles such as “a,” “an,” and “the” can mean one or more than one. Conversely, unless otherwise indicated or evident from the context, a claim or statement containing “or” between two or more members of a group is considered satisfied if one, more than one, or all of the members of the group are present. Disclosures of groups containing “or” between two or more members of a group provide embodiments of the presence of exactly one member of the group, more than one member of the group, and all of the members of the group. For the sake of brevity, these embodiments are not written out individually in this application, but it will be understood that each of these embodiments is provided in this application and can be specifically claimed or disclaimed.

[0178] It should be understood that this disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, closures, or descriptive terms from one or more claims or applicable parts of the specification are introduced into another claim. For example, a claim dependent on another claim may be modified to encompass one or more limitations found in any other claim dependent on the same original claim. Furthermore, where a claim describes a composition, it should be understood that this encompasses methods of making or using the composition according to any of the methods disclosed herein, or according to any method known in the art, unless otherwise indicated or it would be obvious to a person skilled in the art that otherwise, or that it would result in a contradiction or inconsistency.

[0179] Where elements are presented as a list, for example in Markush group format, it should be understood that all possible subgroups of the elements are also disclosed, and that any element or subgroup of an element may be removed from the group. The term “includes” is intended to be open, and it should also be noted that it allows for the inclusion of additional elements or steps. Generally, where an aspect, product, or method is said to include a particular element, feature, or step, it should be understood that an aspect, product, or method consisting of or essentially comprising such element, feature, or step is also provided. For the sake of brevity, these aspects are not individually listed in this application, but it should be understood that each of these aspects is provided in this application and can be specifically claimed or disclaimed.

[0180] Where a range is given, the endpoints are encompassed. Furthermore, unless otherwise indicated or otherwise obvious from the context and / or the understanding of a person skilled in the art, the values ​​expressed as a range may, in some embodiments, take any specific value within the claimed range down to one-tenth of the lower limit unit of the range, unless the context clearly states otherwise. For the sake of brevity, the values ​​within each range are not individually listed in this application, but it will be understood that each of these values ​​is provided in this application and may be specifically claimed or disclaimed. Unless otherwise indicated or otherwise obvious from the context and / or the understanding of a person skilled in the art, the values ​​expressed as a range may take any subrange of a given range, and the endpoints of subranges are expressed with the same precision as one-tenth of the lower limit unit of the range.

[0181] Where a website is provided, the URL address is provided as non-browser executable code with the period of each web address enclosed in parentheses. The actual web address does not contain parentheses.

[0182] In addition, it should be understood that any particular aspect of this disclosure may be expressly excluded from any one or more claims. Where a scope is given, any value within that scope may be expressly excluded from any one or more claims. Any aspect, element, feature, application, or aspect of any composition and / or method of this disclosure may be excluded from any one or more claims. For the sake of brevity, not all aspects in which one or more elements, features, applications, or aspects are excluded are expressly submitted in this application.

Claims

1. (i) A conjugate containing a single-domain antibody (VHH) conjugated with an antigen and an anti-inflammatory agent, wherein the VHH conjugates the VHH to a surface protein on an antigen-presenting cell (APC); or (ii) A first conjugate comprising VHH conjugated to an antigen, wherein the first VHH and the second VHH bind to one or more surface proteins on an antigen-presenting cell (APC), and a second conjugate comprising the second VHH conjugated to an anti-inflammatory agent, A composition containing the following:

2. The composition according to claim 1, wherein the surface protein on the APC is selected from the group consisting of MHCII, CD11c, DEC205, DC-SIGN, CLEC9a, CD103, CX3CR1, CD1a, and F4 / 80.

3. The composition according to claim 2, wherein the composition comprises a conjugate containing VHH conjugated to an antigen and an anti-inflammatory agent, the VHH being bound to MHCII.

4. The composition according to claim 2, comprising a first conjugate containing a first VHH conjugated to an antigen and a second conjugate containing a second VHH conjugated to an anti-inflammatory agent, wherein both the first VHH and the second VHH are bound to MHCII.

5. The composition according to claim 3 or claim 4, wherein VHH comprises the amino acid sequence of SEQ ID NO:

1.

6. The composition according to any one of claims 1 to 5, wherein VHH further comprises a saltase recognition sequence at its N-terminus or C-terminus.

7. The composition according to claim 6, wherein the saltase recognition sequence comprises LPETG (SEQ ID NO: 29), and optionally comprises LPETGG (SEQ ID NO: 43).

8. The composition according to claim 6 or 7, wherein an anti-inflammatory agent or antigen is conjugated to VHH via a saltase recognition sequence.

9. The composition according to any one of claims 1 to 8, wherein the anti-inflammatory agent further comprises a hydrolyzable or non-hydrolyzable linker.

10. The composition according to claim 2, wherein the composition comprises a conjugate containing a single-domain antibody (VHH) conjugated to an antigen and an anti-inflammatory agent, the VHH being bound to CD11c.

11. The composition according to claim 2, wherein the composition comprises a first conjugate containing a first VHH conjugated to an antigen and a second conjugate containing a second VHH conjugated to an anti-inflammatory agent, and both the first VHH and the second VHH are bound to CD11c.

12. The composition according to claim 10 or claim 11, wherein VHH comprises the amino acid sequence of SEQ ID NO:

2.

13. The composition according to any one of claims 10 to 12, wherein VHH further comprises a saltase recognition sequence at its N-terminus or C-terminus.

14. The composition according to claim 13, wherein the saltase recognition sequence comprises LPETG (SEQ ID NO: 29), and optionally comprises LPETGG (SEQ ID NO: 43).

15. The composition according to claim 13 or claim 14, wherein an anti-inflammatory agent or antigen is conjugated to VHH via a saltase recognition sequence.

16. The composition according to any one of claims 10 to 15, wherein the anti-inflammatory agent further comprises a hydrolyzable or non-hydrolyzable linker.

17. The composition according to claim 2, comprising a first conjugate containing a first VHH conjugated to an antigen and a second conjugate containing a second VHH conjugated to an anti-inflammatory agent, wherein the first VHH and the second VHH bind to different surface proteins on the APC.

18. The composition according to claim 17, wherein the first VHH is bonded to MHCII and the second VHH is bonded to CD11c.

19. The composition according to claim 17, wherein the first VHH is bonded to DEC205 and the second VHH is bonded to MHCII.

20. The composition according to any one of claims 1 to 19, wherein the anti-inflammatory agent is a steroidal anti-inflammatory agent selected from the group consisting of dexamethasone, prednisone, prednisolone, triamcinolone, methylprednisolone, and betamethasone.

21. The composition according to any one of claims 1 to 19, wherein the anti-inflammatory agent is a nonsteroidal anti-inflammatory agent selected from the group consisting of aspirin, celecoxib, diclofenac, ibuprofen, ketoprofen, naproxen, oxaprozin, piroxicam, cyclosporine A, and calcitriol.

22. The composition according to any one of claims 1 to 19, wherein the anti-inflammatory agent is an anti-inflammatory cytokine selected from the group consisting of IL-10, IL-35, IL-4, IL-11, IL-13, and TGFβ.

23. The composition according to any one of claims 1 to 22, wherein the antigen comprises a polypeptide, polysaccharide, carbohydrate, lipid, nucleic acid, or a combination thereof.

24. The composition according to any one of claims 1 to 23, wherein the antigen is a self-antigen.

25. The composition according to claim 24, wherein the autoantigen is selected from myelin oligodendrocyte glycoprotein, myelin proteolipidoprotein, citrullinated fibrinogen, insulin, chromogranin A, glutamate decarboxylase 65 kilodalton isoform (GAD65), desmoglein 1 (DSG1), desmoglein 3 (DSG3), acetylcholine receptor (AChR), muscle-specific tyrosine kinase (MuSK), and ribonucleoprotein.

26. The composition according to claim 23, wherein the antigen comprises a protein used in protein replacement therapy or gene therapy.

27. The composition according to claim 26, wherein the antigen is selected from factor IX, factor VIII, insulin, and AAV-derived protein.

28. A method comprising administering the composition according to any one of claims 1 to 27 to a subject requiring it.

29. A method for inducing immune tolerance to an antigen, comprising administering the composition according to any one of claims 1 to 27 to a subject requiring such tolerance.

30. A method for treating an autoimmune disease, comprising administering the composition according to any one of claims 1 to 25 to a subject in need thereof.

31. The method according to claim 30, wherein the autoimmune disease is selected from the group consisting of autoimmune encephalomyelitis, multiple sclerosis, type 1 diabetes mellitus, pemphigus vulgaris, myasthenia gravis, lupus, celiac disease, and inflammatory bowel disease (IBD).

32. The method according to any one of claims 28 to 31, wherein the administration is intravenous.

33. The method according to any one of claims 28 to 32, wherein the subject is a human.

34. (i) a conjugate containing a single-domain antibody (VHH) conjugated to an antigen and a pro-inflammatory agent, wherein the VHH conjugates to a surface protein on an antigen-presenting cell (APC); or (ii) A first conjugate comprising VHH conjugated to an antigen, wherein the first VHH and the second VHH bind to one or more surface proteins on an antigen-presenting cell (APC), and a second conjugate comprising the second VHH conjugated to a pro-inflammatory agent, A composition containing the following:

35. The composition according to claim 34, wherein the surface protein on the APC is selected from the group consisting of MHCII, CD11c, DEC205, DC-SIGN, CLEC9a, CD103, CX3CR1, CD1a, and F4 / 80.

36. The composition according to claim 35, wherein the composition comprises a conjugate containing a single-domain antibody (VHH) conjugated to an antigen and a pro-inflammatory agent, the VHH being bound to MHCII.

37. The composition according to claim 35, comprising a first conjugate containing a first VHH conjugated to an antigen and a second conjugate containing a second VHH conjugated to a pro-inflammatory agent, wherein both the first VHH and the second VHH are bound to MHCII.

38. The composition according to claim 36 or claim 37, wherein VHH comprises the amino acid sequence of SEQ ID NO:

1.

39. The composition according to any one of claims 34 to 38, wherein VHH further comprises a saltase recognition sequence at its N-terminus or C-terminus.

40. The composition according to claim 39, wherein the saltase recognition sequence comprises LPETG (SEQ ID NO: 29), and optionally comprises LPETGG (SEQ ID NO: 43).

41. The composition according to claim 39 or 40, wherein a pro-inflammatory agent or antigen is conjugated to VHH via a saltase recognition sequence.

42. The composition according to any one of claims 34 to 41, wherein the pro-inflammatory agent further comprises a hydrolyzable or non-hydrolyzable linker.

43. The composition according to claim 35, wherein the composition comprises a conjugate containing a single-domain antibody (VHH) conjugated to an antigen and a pro-inflammatory agent, the VHH being bound to CD11c.

44. The composition according to claim 35, comprising a first conjugate containing a first VHH conjugated to an antigen and a second conjugate containing a second VHH conjugated to a pro-inflammatory agent, wherein both the first VHH and the second VHH are bound to CD11c.

45. The composition according to claim 43 or claim 44, wherein VHH comprises the amino acid sequence of SEQ ID NO:

2.

46. The composition according to any one of claims 43 to 45, wherein VHH further comprises a saltase recognition sequence at its N-terminus or C-terminus.

47. The composition according to claim 46, wherein the saltase recognition sequence comprises LPETG (SEQ ID NO: 29), and optionally comprises LPETGG (SEQ ID NO: 43).

48. The composition according to claim 46 or claim 47, wherein a pro-inflammatory agent or antigen is conjugated to VHH via a saltase recognition sequence.

49. The composition according to any one of claims 43 to 48, wherein the pro-inflammatory agent further comprises a hydrolyzable or non-hydrolyzable linker.

50. The composition according to claim 35, comprising a first conjugate containing a first VHH conjugated to an antigen and a second conjugate containing a second VHH conjugated to a pro-inflammatory agent, wherein the first VHH and the second VHH bind to different surface proteins on the APC.

51. The composition according to claim 50, wherein the first VHH is bonded to MHCII and the second VHH is bonded to CD11c.

52. The composition according to claim 50, wherein the first VHH is bonded to DEC205 and the second VHH is bonded to MHCII.

53. The composition according to any one of claims 34 to 52, wherein the pro-inflammatory agent is selected from the group consisting of: TLR9 agonists, LPS, HMGB1 protein, IL2, IL12, and CD40L.

54. The composition according to any one of claims 34 to 53, wherein the antigen comprises a polypeptide, polysaccharide, carbohydrate, lipid, nucleic acid, or a combination thereof.

55. The composition according to any one of claims 34 to 54, wherein the antigen is derived from a microbial pathogen.

56. The composition according to claim 55, wherein the microbial pathogen is mycobacteria, bacteria, fungi, viruses, parasites, or prions.

57. The composition according to any one of claims 34 to 56, wherein the antigen comprises the SARS-CoV-2 spike protein.

58. The composition according to any one of claims 34 to 54, wherein the antigen is a tumor antigen.

59. The composition according to any one of claims 34 to 58, wherein the composition is a vaccine composition.

60. A method comprising administering the composition according to any one of claims 34 to 59 to a subject requiring it.

61. A method for inducing an immune response to an antigen, comprising administering the composition according to any one of claims 34 to 59 to a subject requiring such treatment.

62. A method for treating an infection caused by a pathogen, the method comprising administering to a subject in need thereof a composition according to any one of claims 34 to 59, wherein the antigen is from a microbial pathogen.

63. The method according to claim 62, wherein the method is therapeutic or preventive.

64. A method for treating cancer, the method comprising administering a composition according to any one of claims 34 to 59 to a subject in need thereof, wherein the antigen is a tumor antigen.

65. The method according to any one of claims 60 to 64, wherein the administration is intravenous.

66. The method according to any one of claims 60 to 65, wherein the subject is a human.

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